ail)p S. 1. Bill ?jtbrarg ?5nrtb (Taroliim ^tatp (Eollpnp coD.r >• s This book is due on the date indicated below and is subject to an overdue fine as posted at the Circulation Desk. r^.3„ >-'•<-' J«JiM 1 S 1979 n 26 19,3 DEC 2 4 1987 APR 2 1 mn i- THE CELL IN DEVELOPMENT AND INHERITANCE Columbia ^Snibcrsito Biological Scries. « EDITED BY HENRY FAIRFIELD OSBORN AND * EDMUND B. WILSON, 1. FROM THE CREEKS TO DARWIN. By Henry Fairfield Osborn, Sc.D. Princeton. 2. AMPHIOXUS AND THE ANCESTRY OF THE VERTEBRATES. By Arthur Willey. B.Sc. Lond. Univ. 3. FISHES. LIVINC AND FOSSIL. An Introductory Study. By Bashford Dean, Ph.D. Columbia. 4. THE CELL IN DEVELOPMENT AND INHERITANCE. By Edmund B. Wilson, Ph.D. J.H.U. 5. THE FOUNDATIONS OF ZOOLOGY. By William Keith Brooks. COLUMBIA UNIVERSITY BIOLOGICAL SERIES. IV THE CELL IN Development and Inheritance BY EDMUND B. WILSON, Ph.D. PROFESSOR OF ZOOLOGY, COLUMBIA UNIVERSITY SECOND EDITION REVISED AND ENLARGED " Natura nusquam magis est tota quam in minimis PLINY THE MACMILLAN COMPANY LONDON: MACMILLAN & CO., LTD. 3>3oV^vv\>A\— 1904 AN rights reserved Copyright, 1896, By the MACMILLAN COMPANY. Copyright, 1900, By the MACMILLAX COMPANY. Set up and electrotyped October, 1896. Reprinted September, 1897; September, 1898. New edition, revised, set up and electrotyped January, 1900; March, 1902 ; June, 1904. XorSuooti 39rc33 J. S. Gushing & Co. — Berwick & Smith Norwood, Mass. U. S. A. Co mo fxitnH THEODOR BOVERI 144370 PREFACE This volume is the outcome of a course of lectures, delivered at Columbia University in the winter of 1892-93, in which I endeavoured to give to an audience of general university students some account of recent advances in cellular biology, and more especially to trace the steps by which the problems of evolution have been reduced to problems of the cell. It was my first intention to publish these lectures in a simple and general form, in the hope of showing to wider circles how the varied and apparently heterogeneous cell- researches of the past twenty years have grown together in a coherent group, at the heart of which are a few elementary phe- nomena, and how these phenomena, easily intelligible even to those having no special knowledge of the subject, are related to the problems of development. Such a treatment was facilitated by the appearance, in 1893, of Oscar Hertwig's invaluable book on the cell, which brought together, in a form well designed for the use of special students, many of the more important results of modern cell-research. I am glad to acknowledge my debt to Hert- wig's book ; but it is proper to state that the present volume was fully sketched in its main outlines at the time the Zelle mid Gcwcbc appeared. Its completion was, however, long delayed by investiga- tions which I undertook in order to re-examine the history of the centrosomes in the fertilization of the ^gg, — a subject which had been thrown into such confusion by Fol's extraordinary account of the " Quadrille of Centres " in echinoderms that it seemed for a time impossible to form any definite conception of the cell in its relation to inheritance. By a fortunate coincidence the same task was inde- pendently undertaken, nearly at the same time, by several other investigators. The concordant results of these researches led to a decisive overthrow of Fol's conclusions, and the way was thus cleared for a return to the earlier and juster views founded by Hertwig, Strasburger, and Van Beneden, and so lucidly and forcibly developed by Boveri. The rapid advance of discovery in the mean time has made it seem desirable to amplify the original plan of the work, in order to render it useful to students as well as to more general readers ; and to this end it has been found necessary to go over a considerable vii Vlll PREFACE part of the ground already so well covered by Hertwig.i This book does not, however, in any manner aim to be a treatise on general histology, or to give an exhaustive account of the cell. It has rather been my endeavour to consider, within moderate limits, those features of the cell that seem more important and suggestive to the student of development, and in some measure to trace the steps by which our present knowledge has been acquired. A work thus limited neces- sarily shows many gaps ; and some of these, especially on the botani- cal side, are, I fear, but too obvious. On its historical side, too, the subject could be traced only in its main outlines, and to many investigators of whose results I have made use it has been impossible to do full justice. To the purely speculative side of the subject I do not desire to add more than is necessary to define some of the problems still to be solved ; for I am mindful of Blumenbach's remark that while Drelin- court rejected two hundred and sixty-two "groundless hypotheses" of development, ** nothing is more certain than that Drelincourt's own theory formed the two hundred and sixty-third." ^ i have no wish to add another to this list. And yet, even in a field where standpoints are so rapidly shifting and existing views are still so widely opposed, the conclusions of the individual observer may have a certain value if they point the way to further investigation of the facts. In this spirit I have endeavoured to examine some of the more important existing views, to trace them to their sources, and in some measure to give a critical estimate of their present standing, in the hope of finding suggestion for further research. Every writer on the cell must find himself under a heavy obliga- tion to the works of Van Beneden, Oscar Hertwig, Flemming, Stras- burger, and Boveri ; and to the last-named author I have a special sense of gratitude. I am much indebted to my former student, Mr. A. P. Mathews, for calling my attention to the importance of the recent work of physiological chemists in its bearing on the problems of synthetic metabolism. The views developed in Chap- ter VII. have been considerably influenced by his suggestions, and this subject will be more fully treated by him in a forthcoming work ; but I have endeavoured as far as possible to avoid anticipating his own special conclusions. Among many others to whom I am indebted for kindly suggestion and advice, I must particularly mention my ever helpful friend. Professor Henry F. Osborn, and Professors J. E. Humphrey, T. H. Morgan, and F. S. Lee. In copying so great a number of figures from the papers of other 1 Henneguy's Le(;ons sur la cellule is received, too late for further notice, as this volume is going through the press. ^ Allen Thomson. PREFACE ix investigators, I must make a virtue of necessity. Many of the facts could not possibly have been illustrated by new figures equal in value to those of special workers in the various branches of cytological research, even had the necessary material and time been available. But, apart from this, modern cytology extends over so much debatable ground that no general work of permanent value can be written that does not aim at an objective historical treatment of the subject; and I believe that to this end the results of investigators should as far as practicable be set forth by means of their original figures. Those for which no acknowledgment is made are original or taken from my own earlier papers. The arrangement of the literature lists is as follows. A general list of all the works referred to in the text is given at the end of the book (p. 449). These are arranged in alphabetical order, and are referred to in the text by name and date, according to Mark's con- venient system. In order, however, to indicate to students the more important references and partially to classify them, a short separate list is given at the end of each chapter. The chapter-lists include only a few selections from the general list, comprising especially works of a general character and those in which reviews of the special literature may be found. E. B. W. Columbia University, New York, July, 1896. PREFACE TO THE SECOND EDITION Since the appearance of the first edition of this work, in 1896, the aspect of some of the most important questions with which it deals has materially changed, most notably in case of those that are f ocussed in the centrosome and involve the phenomena of cell-division and fertilization. This has necessitated a complete revision of the book, many sections having been entirely rewritten, while minor changes have been made on almost every page. In its first form, the work was compressed within limits too nar- row for a sufficiently critical treatment of many disputed subjects. It has therefore been considerably enlarged, and upwards of fifty new illustrations have been added. The endeavour has, however, still been made to keep the book within moderate Hmits, even at some cost of comprehensiveness ; and the present edition aims no more than did the first to cover the whole vast field of cellular biology. Its limita- tions are, as before, especially apparent in the field of botanical cytology. Here progress has been so rapid that, apart from the dif- ficulty experienced by a zoologist in the attempt to maintain a due sense of proportion in reviewing the subject, an adequate treatment would have required a separate volume. I have therefore, for the most part, considered the cytology of plants in an incidental way, endeavouring only to bring the more important phenomena into rela- tion with those more fully considered in the case of animals. The steady and rapid expansion of the literature of the general subject renders increasingly difficult the historical form of treatment and the citation of specific authority in matters of detail. This plan has nevertheless still been followed as far as possible, despite the increased bulk of the book and the encumbrance of the text with references thus occasioned, in the hope that these disadvantages will be outweighed by increased usefulness of the work. I beg the reader to remember, however, that no approach to a complete history of cytology and experimental embryology could be attempted, save in a work of far greater proportions, and that it has been necessary XI xii PREFACE TO THE SECOXD EDITION to pass by, or dismiss with very brief mention, many works to which space would gladly have been given. Recent research has yielded many new results of high interest, conspicuous among them the outcome of experiments on cell-division, fertilization, and regeneration ; and they have cleared up many special problems. Broadly viewed, however, the recent advance of discovery has not, in the author's opinion, tended to simplify our conceptions of cell-life, but has rather led to an emphasized sense of the diversity and complexity of its problems. " One is sometimes tempted to con- clude," was recently remarked by a well-known embryologist, " that every S 327 328 CHAPTER VII Some Aspects of C-iLL-cHEMisTRV and Cell-phvsiology A. Chemical Relations of Nucleus ana Cytoplasm 1. The Proteids and their Allies 2. The Nuclein Series .... 3. Staining-reactions of the Nuclein Series 330 332 334 XVI TABLE OF COXTENTS B. Physiological Relations of Nucleus and Cytoplasm 1. Experiments on Unicellular Organisms 2. Position and Movements of the Nucleus 3. The Nucleus in Mitosis 4. The Nucleus in Fertilization 5. The Nucleus in Maturation C. The Centrosome ..... D. Summary and Conclusion Literature. VII PAGE 340 o:) I J3- 353 354 358 359 CHArrr.R viii Cell-division and Development A. Geometrical Relations of Cleavage-forms B. Promorphological Relations of Cleavage 1. Promorphology of the Ovum (^/) Polarity and the Egg-axis (<^) Axial Relations of the Primary Cleavage-planes (r) Other Promorphological Characters of the Ovum 2. Meaning of the Promorphology of the Ovum C. Cell-division and Growth ....... Literature, VIIL . . 362 37S 378 37S 379 382 3S4 394 CHAPTER IX Theories of Lmiekitance and Development A. The Theory of Germinal Localization . B. The Idioplasm Theory ..... C. Union of the Two Theories .... D. The Roux-Weismann Theory of Development E. Critique of the Roux-Weismann Theory F. On the Nature and Causes of Differentiation G. The Nucleus in Later Development H. The External Conditions of Development I. Development, Inheritance, and Metabolism . J. Preformation and Epigenesis. The Unknown Factor in Development Literature, IX. Glossary General Literature- list Lndex of Authors . Index of Subjects . 397 401 403 404 407 413 425 428 430 431 434 437 449 471 477 LIST OF FIGURES INTRODUCTION 1. Epidermis of larval salamander 2. Section of growing root-tip of the onion 3. Avioeba Proteus . . . . . 4. Cleavage of the ovum in Toxopneustes . 5. Diagram of inheritance . . PAGE 4 1 1 CHAPTER I 6. Diagram of a cell ......••• 7. Spermatogonia of salamander ...... Group of cells, showing cytoplasm, nucleus, and centrosomes Living cells of salamander larva, showing fibrillar structure . Alveolar or foam-structure of protoplasm, according to BiitschU Structure of protoplasm in the echinoderm e^ Aster-formation in alveolar protoplasm . Nuclei from the crypts of Lieberkiihn . Special forms of nuclei .... Scattered nucleus in Trachelocerca Scattered nucleus in Bacteria and Flagellata Ciliated cells . Cells of amphibian pancreas 8. 9. 10. II. 12. 13- 14. 15- 16. 17- 18. 19. 20. 21. 22. 2^. Nephridial cell of Clepsine Nerve-cell of frog . Diagram of dividing cell Diagrams of cell-polarity Centrosomes in epithelium and in blood-corpuscles 18 20 21 24 26 27 28 32 35 37 39 43 44 45 47 49 56 57 CHAPTER 11 24. Remak's scheme of cell-division 25. Diagram of the prophases of mitosis 26. Diagram of later phases of mitosis 27. Prophases in salamander-cells 28. Metaphase and anaphases in salamander-cells 29. Telophases in salamander-cells 30. Mid-body and cell-plate in cells of Umax 31. Middle phases of mitosis in Ascaris-Qgg% 32. Mitosis in Stypocaulon .... xvii 64 66 69 73 75 76 79 80 Si XVlll LIST OF FIGURES FIG. 2,2i- Mitosis in Erysiphe ....... 34. Mitosis in pollen-mother-cells of lily, according to Guignard 36. Mitosis in spore-cells of Eqiiisetiim 37. Heterotypical mitosis 38. Mitosis in Infusoria 39. Mitosis in Etiglypha 40. Mitosis in Euglena 41. Mitosis in Acanthocystis 42. Mitosis in Noctiliica 43. Mitosis in Paramaba 44. Mitosis in Actinospharium 45. Mitosis in Actinosphivriiim . 46. Pathological mitoses in cancer-cells 47. Pathological mitosis caused by poisons 48. \'an Beneden's account of astral systems in Ascaris 49. Leucocytes ..... 50. Pigment-cells .... 51. Heidenhain's model of mitosis 52. Mitosis in the egg of Toxopneusies 53. Pathological mitoses in polyspermy 54. Nuclei in the spireme-stage . 55. Early division of chromatin in Ascaris 56. Amitotic division .... PAGE 83 84 85 87 89 90 91 92 93 95 96 97 98 99 100 102 103 104 107 109 112 "3 "5 CHAPTER III 57. Volvox 58. Ovum of Toxopneustss . 59. Ovum of the cat .... 60. Ovum of Nereis .... 61. Germinal vesicles of Unio and Epeira 62. Insect-egg ..... 63. Micropyle in Argonauta 64. Germ-cells of Volvox 65. Diagram of the flagellate spermatozoon 66. Spermatozoa of fishes and amphibia 67. Spermatozoa of birds and other animals 68. Spermatozoa of mammals 69. Unusual forms of spermatozoa 70. Spermatozoids of Chara 71. Spermatozoids of various plants 72. Germ-cells of Cladonema 73. Primordial germ-cells of Ascaris . 74. Primordial germ-cells of Cyclops . 75. Ovarian ova and follicles of Helix 76. Egg and nurse-cells in Ophryotrocha 77. Ovarian eggs of insects . 78. Young ovarian eggs of various animals 79. Young ovarian eggs of birds and mammals 80. Ovarian eggs of spider, earthworm, ascidian, showing yolk-nucleus ^^2, 126 127 129 130 132 134 135 136 138 140 141 142 143 146 147 149 152 153 154 155 157 LIST OF^FIGURES XIX FIG. 8i. Ova.na.n eggs oi Limti/tis and Po/yzoniuf/i .... 82. Formation of the spermatozoon in Ajiasa .... 83. Transformation of the spermatids of the salamander 84. P'ormation of the spermatozoon in Salamandra and Amphinma 85. The same in Helix and in elasmobranchs .... 86. The same in mammals ....... 87. Formation of spermatozoids in cycads ..... 88. Formation of spermatozoids in cryptogams .... PAGE 159 162 164 166 168 169 ^IZ 174 CHAPTER IV 89. Fertilization of Physa . 90. Fertilization of Ascaris 91. Germ-nuclei of Nematodes , 92. Fertilization of the mouse . 93. Fertilization of Pterotrachea 94. Entrance and rotation of sperm-head in Toxopneustes 95. Conjugation of the germ-nuclei in Poxopnensies . 96. Diagrams of fertilization ..... 97. Fertilization of Nereis ...... 98. Fertilization of Cyclops ..... 99. Fertilization and persistence of centrosomes in Thalassema 100. Entrance of spermatozoon into the egg 101. Pathological polyspermy .... 102. Polar rings of Clepsine .... 103. Paths of the germ-nuclei in Toxopneustes 104. Fertilization of Myzostoma .... 105. Fertilization of Pihilaria .... 106. Penetration of the pollen-tube in angiosperms 107. Fertilization of the lily .... 108. Fertilization in Zamia .... 109. Diagram of conjugation in Infusoria 110. Qoxi]\xgzX\ox\ oi Pa ramie ciiim 111. Conjugation of Vorticella .... 112. Con]\xgz\.\on o( Noctiltcca .... 113. Co\\]\xg2ii\on oi Spirogyra .... 180 184 185 186 187 189 190 191 193 195 197 199 201 203 209 216 217 219 220 22 ? 225 226 227 228 CHAPTER V 114. Polar bodies in Toxopneustes 115. Genesis of the egg 116. Diagram of formation of polar bodies 117. VoXaxhoCixtsm. Ascaris 118. Genesis of the spermatozoon 119. Diagram of reduction in the male 120. Spermatogenesis of .'^5ke, and (jrew in the second lialf of the seventeenth century. Wolff, in the Theoria Cenerationis (1759), clearly recognized the "spheres" and "vesi- cles" composing the embryonic parts both of animals and of plants, though without grasping iheir real nature or mode of origin, and his conclusions were developed by the botanist Mirbel at the beginning of the i^resent century. Nearly at the same time (1805) Oken fore- shadowed the cell-theory in the form that it assumed with Schleiden and Schwann; but his conception of " Urschleim " and " Hlaschen " can hardly be regarded as more than a lucky guess. A still closer approximation to the truth is fuuntl in the works of 'ruri)in (1826), Meyen (1830), Raspail (1831), and Dutrochet (1837); '^"^^ these, like others of the same period, only paved the way for the real founders of the cell-theory. Among other immedi- ate predecessors f)r contemporaries of Schleiden and Schwann should be especially mentioned Robert Brown, Dujardin, Johannes Miiller, I'urkinje, Hugo von Mohl, Valentin, Unger, Nageli, and Henle. The significance of Schleiden's, and especially of Schwann's, work lies in the thorough and comprehensive way in which the problem was studied, the philosophic breadth with which the conclusions were developed, and the far-reaching influence which they exercised upon subsequent research. In this respect it is hardly too much to com- pare the Mikroikopische L'ntersiichnngcn with the Origin of Species. INTR OD UC TION 3 During the past thirty years the theory of organic descent has been shown, by an overwhelming mass of evidence, to be the only tenable conception of the origin of diverse living forms, however we mav conceive the causes of the process. While the study of general zoology and botany has systematically set forth the results, and in a measure the method, of organic evolution, the study of microscopical a X Fig i._ A portion of the epidermis of a larval salamander {Amblystoma) as seen in slightly oblique horizontal section, enlarged 550 diameters. Most of the cells are polygonal m form, con- tain large nuclei, and are connected by delicate protoplasmic bridges. Above v is a branched, dark pigment-cell that has crept up from the deeper layers and lies between the epidermal ceLs. Three of the latter are undergoing division, the earliest stage {sp,rcme) at a, ^J l-';»«^r/»-'f ("^"^"^ figure in the anaphase) at b, showing the chromosomes, and a final stage {telophase), showing fission of the cell-body, to the right. anatomy has shown us the nature of the material on which it has operated, demonstrating that the obvious characters ot plants and animals are but varving expressions of a subtle interior organization common to all. In its broader outlines the nature of this organiza- tion is now accurately determined; and the ''cell-theory," by which it is formulated, is, therefore, no longer of an inferential or hypo- ^ INTRODUCriOy thetical character, but a generalized statement of observed fact which may be outlined as follows : — * In all the higher forms of life, whether plants or animals, the body may be resolved into a vast host of minute structural units known as cells, out of which, directly or indirectly, every part is built (Figs. 1,2). The substance of the skin, of the brain, of the blood, of the bones or muscles or any other tissue, is not homogeneous, as it appears to the unaided eye, but is shown by the microscope to be an aercrretrate composed of innumerable minute bodies, as if it were a Fig. 2. — General view of cells in the growing root-tip of the onion, from a longitudinal section, enlarged 800 diameters. a. non-dividing cells, with chromatin-network and deeply stained nucleoli ; b. nuclei preparing for division (spireme-stage) ; ;; Fig. 3. — Amcela Proteus, an animal consisting of a single naked cell, x 280. (From Sedgwick and Wilson's Biology.) n. The nucleus; iv.v. water-vacuoles ; c.v. contractile vacuole ; f.v. food-vacuole, istic of only the higher forms of life. Among the lowest forms at the base of the series are an immense number of microscopic plants and animals, famiUar examples of which are the bacteria, diatoms, rhizo- pods, and Infusoria, in which the entire body consists of a single cell (Fig. 3), of the same general type as those which in the higher multi- cellular forms are associated to form one organic whole. Structurally, therefore, the multicellular body is in a certain sense comparable with a colony or aggregation of the lower one-celled forms.- This com- 1 The word protoplasm is due to Purkinje (1840), who applied it to the formative sub- stance of the animal embryo and compared it with the granular material of vegetable "cambium." It was afterward independently used by \\. von Mohl (1846) to designate the contents of the plant-cell. The full physiological signiticance of protoplasm, its identity with the "sarcode" (Dujardin) of the unicellular forms, and its essential similarity in plants and animals, was first clearly placed in evidence through the classical works of Max Schultze and De Bary, beside which should be placed the earlier works of Dujardin, L nger, Nageli, and Mohl, and that of Cohn, Huxley, Virchow, Leydig, Brucke, Kuhne, and Beale. 2 This comparison must be taken with some reservation, as will appear beyond. 6 IXTRODUCTIOX parison is not less suggestive to the physiologist than to the mor- phologist. In the one-celled forms all of the vital functions are performed by a single cell. In the multicellular forms, on the other hand, these functions are not ecjualh- i)erformed b)- all the cells, but are in varving degree distributed among them, the cells thus falling into physiological groups or tissues, each ot which is especially de- voted to the performance of a specific function. Thus arises the "physiological division of labour" through which alone the highest development of vital activity becomes possible ; and thus the cell becomes a unit, not merely of structure, but also of function. luich bodilv function, and even the life of the organism as a whole, may thus in one sense be regarded as a resultant arising through the inte- gration of a vast number of cell-activities ; and it cannot be adequately investigated without the study of the individual cell-activities that lie at its root.^ The foregoing conceptions, founded by Schwann, and skilfully developed by Kolliker, Siebold, Virchow, and Haeckel, gave an im- pulse to anatomical and physiological investigation the force of which could hardly be overestimated; yet they did not for many years measurably affect the more speculative side of biological inquiry. The Origin of Species, published in 1859, scarcely mentions it; nor, with the important exception of the theory of pangenesis, did Darwin attempt at any later period, to bring it into any very definite relation to his views. The initial impulse to the investigations that brought the cell-theory into definite contact with the evolution-theory was given nearly twenty years after the Origin of Species, by researches on the early history of the germ-cells and the fertilization of the ovum. Begun in 1873-74 by Auerbach, Fol, and Butschli, and eagerly followed up b\- Oscar Hertwig, Van Beneden, Strasburger, and a host of later workers, these investigations raised wholly new questions regarding the mechanism of development and the role of the cell in hereditary transmission. Through them it became for the first time clearly apparent that the general problems of embryology, heredity, and evolution are indissolubly bound up with those of cell- structure, and can only be fully apprehended in the light of cytologi- cal research. As the most significant step in this direction, we may re£:ard the identification of the cell-nucleus as the vehicle of inheri- 1 Cf. pp. 58-61. " It is to the cell that the study of every bodily function sooner or later drives us. In the muscle-cell lies the problem of the heart-beat and that of muscular con- traction ; in the gland-cell reside the causes of secretion ; in the epithelial cell, in the white blood-cell, lies the problem of the absorption of food, and the secrets of the mind are hidden in the ganglion-cell. ... If then physiology is not to rest content with the mere extension of our knowledge regarding the gross activities of the human body, if it would seek a real explanation of the fundamental phenomena of life, it can only attain its end through the study of cell-physiology'''' (Verworn, Alkemeine Fhysiologie, p. 53, 1895). INTRODUCTION y tance, made independently and almost simultaneously in 18S4-85 by -Oscar Hertwig, Strasburger, Kolliker, and Wcismann/ while nearly at the same time (1883) the splendid researches of Van Beneden on the early history of the animal Qgg opened possibilities of research into the finer details of cell-phenomena of which the early workers could hardly have dreamed. We can only appreciate the full historical significance of the new period thus inaugurated by a glance at the earlier history of opinion regarding embryological development and inheritance. To the modern student the germ is, in Huxley's words, simply a detached living por- tion of the substance of a preexisting living body ^ carrying with it a definite structural organization characteristic of the species. By the earlier embryologists, however, the matter was very differently re- garded ; for their views in regard to inheritance were vitiated by their acceptance of the Greek doctrine of the equivocal or spontaneous generation of life ; and even Harvey did not escape this pitfall, near as he came to the modern point of view. " The Qgg,'' he savs, " is the mid-passage or transition stage between parents and offspring, between those who are, or were, and those who are about to be ; it is the hinge or pivot upon which the whole generation of the bird revolves. The Qgg is the terminus from which all fowls, male and female, have sprung, and to which all their lives tend — it is the result which nature has proposed to herself in their being. And thus it comes that individuals in procreating their like for the sake of their species, endure forever. The egg, I say, is a period or por- tion of this eternity." ^ This passage appears at first sight to be a close approximation to the modern doctrine of s^erminal continuitv about which all theories of heredity are revolving. In point of fact, however, Harvey's view is only superficially similar to this doctrine ; for, as Huxley pointed out, it was obscured by his belief that the germ might arise *' spontaneously," or through the influence of a mysterious '' calidiun innaUmi,'' out of not-living matter."* Neither could Harvey, great physiologist and embryologist as he w^as, have had any adequate con- ception of the real nature of the ^gg and its morphological relation to 1 It must not be forgotten that Haeckel expressed the same view in 1866 — only, how- ever, as a speculation, since the data necessary to an inductive conclusion were not obtained until long afterward. "The internal nucleus provides for the transmission of hereditary characters, the external plasma on the other hand for accommodation i)« adaptation to the external world" {Gen. MorpJi., pp. 2S7-289). 2 Evolution in Biology, 1878; Science and Culture, p. 291. ^ De Generatione, 1651; Trans., p. 271. ^ Whitman, too, in a brilliant essay, has shown how far Harvey was from any real grasp of the law of cenetic continuitv. which is well characterized as the central fact of modern biology. Evolution and Epigenesis, Wood's HoU Biological Lectures, 1894. 8 JNTR OD UC TION the body of which it forms a part, since the cclkilar structure of Uving things was not comprehended until nearly two centuries later, the spermatozoon was still undiscovered, and the nature of fertilization was a subject of fantastic and baseless speculation. For a hundred years after Harvey's time embryologists sought in vain t(^ penetrate the mysteries enveloping the beginning of the individual life, and despite their failure the controversial writings of this period form one of the most interesting chapters in the history of biology. By the extreme " evolutionists " or " prceformationists " the egg was believed to contain an embryo fully formed in miniature, as the bud contains the flower or the chrysalis the butterfly. Development was to them merely the unfolding of that which already existed ; inheritance, the handing down from parent to child of an infinitesimal re])roduction of its own body. It was the service of Bonnet to push this concep- tion to its logical consequence, the theory of eiJiboitciiicjit or encase- ment, and thus to demonstrate the absurdity of its grosser forms, pointing out that if the egg contains a complete embryo, this must itself contain eggs for the next generation, these other eggs in their turn, and so ad infinitum, like an infinite series of boxes, one within another — hence the term cniboitemcnt. Bonnet himself renounced this doctrine in his later writings, and Caspar Friedrich Wolff ( 1759) led the way in a return to the teachings of Harvey, showing by pre- cise actual observation that the egg does not at first contain any formed embryo whatever ; that its structure is wholly different from that of the adult; that development is not a mere process of unfolding, but involves the continual formation, one after an- other, of new parts, previously non-existent as such. This is some- what as Harvey, himself following Aristotle, had conceived it — a process of cpigcncsis as opposed to evolution. Later researches established this conclusion as the very foundation of embryological science. But although the external nature of development was thus deter- mined, the actual structure of the egg and the mechanism of inheri- tance remained for nearly a century in the dark. It was reserved for Schwann (1839) and his immediate followers to recognize the fact, conclusively demonstrated by all later researches, that tJic egg is a cell having the same essential structure as other cells of the body. And thus the wonderful truth became manifest that a single cell may contain within its microscopic compass the sum-total of the heritage of the species. This conclusion first reached in the case of the female sex was soon afterward extended to the male as well. Since the time of Leeuwenhoek (1677) it had been known that the sperm or fertilizing fluid contained innumerable minute bodies endowed in nearly all cases with the power of active move- INTRODUCTION ment, and therefore regarded by the early observers as parasitic animalcules or infusoria, a view which gave rise to the name sperma- tozoa (sperm-animals) by which they are still generally known. ^ As long ago as 1786, however, it was shown by Spallanzani that the fertilizing power must lie in the spermatozoa, not in the liquid in which they swim, because the spermatic fluid loses its power when filtered. Two years after the appearance of Schwann's epoch-mak- ino- work Kolliker demonstrated (1841) that the spermatozoa arise directly from cells in the testis, and hence cannot be regarded as parasites, but are, like the ovum, derived from the parent-body. Not until 1865, however, was the final proof attained by Schweigger- Seidel and La Valette St. George that the spermatozoon contains not only a nucleus, as Kolliker believed, but also cytoplasm. It was thus shown to be, like the ^^,g, a single cell, peculiarly modified in structure, it is true, and of extraordinary minuteness, yet on the whole morphologically equivalent to other cells. A final step was taken ten years later (1875), when Oscar Hertwig established the all-important fact that fertilization of the egg is accomplished by its union with one spermatozoon, and one only. In sexual repro- duction, therefore, each sex contributes a single cell of its own body to the formation of the offspring, a fact which beautifully tallies with the conclusion of Darwin and Galton that the sexes play, on the whole, equal, though not identical parts in hereditary trans- mission. The ultimate problems of sex, fertilization, inheritance, and development were thus shown to be cell-problems. Meanwhile, during the years immediately following the announce- ment of the cell-theory, the attention of investigators was especially focussed upon the question : How do the cells of the body arise .? The origin of cells by the division of preexisting cells was clearly recognized by Hugo von Mohl in 1835, though the full significance of this epoch-making discovery was so obscured by the errrors of Schleiden and Schwann that its full significance was only perceived long afterward. The founders of the cell-theory were unfortunately led'to the conclusion that cells might arise in two different ways, viz. either by division or fission of a preexisting mother-cell, or by "Iree cell-formation," new cells arising in the latter case not from pre- existing ones, but by crystallizing, as it were, out of a formative or nutritive substance, termed the " cytoblastema " ; and they even beheved the latter process to be the usual and typical one. It was only after many years of painstaking research that " free cell- formation " was absolutely proved to be a myth, though many of iThe discovery of the spermatozoa is generally accredited to Ludwig Hamm. a pupil of Leeuwenhoek (1677). though Ilartsoeker afterward claimed the ment of havmg seen them as early as 1674 (Dr. Allen Thomson). 10 IXTRODUC TION Schwann's immediate followers threw doubts upon it,^ and as early as 1855 Virchow positively maintained the universality of cell-divi- sion, contending that ever}- cell is the offs})ring of a preexisting parent-cell, and summing up in the since famous aphorism, " oniuis celliila c Cillula.^''^ At the ]:)resent day this conclusion rests upon a foundation so firm that we arc justified in regarding it as a universal law of development. Now, if the cells of the body always arise by the division of pre- existing cells, all must be traceable back to the fertilized egg-cell as their common ancestor. Such is, in fact, the case in every plant and animal whose development is accurately known. The first step in development consists in the division of the <:.^^ into two j^arts, each of which is a cell, like the . ^^^-^-^'^lioiis cells showing the typical parts. N.c1;or2XrreS'„:,'f lli^e,,:^ 'o^J sa,an,a„de...,arva. T.o ce„,.oso„,es a, ,he rish. f?.'l' kT' ''T ''"'" ''''°P'='' ''>' ™'^"y' b"t not all. later writers eestio'n h ""I' "'"'t^'''"- '^-•'"S, however, at Flen,n,i„,'s u" gestion, been changed to ta,yop/as„r At the present time there fore, the word /;...^/.„,„ is used by some authors'(Hutsch i, H^ tw '" 22 GENERAL SKETCH OE THE CELL Kolliker, etc.) in its orif^inul narrower sense (equivalent to Stras-. burger's cytoplasm), while perhaps the majority of writers have accepted the terminology of Strasburger and Flemming. On the whole, the terms cytoplasin and kiDyop/asui seem too useful to be rejected, and, without attaching too much importance to them, they will be employed throughout the present work. It must not, how- ever, be supposed that either of the words denotes a single homo- geneous substance; for, as will soon appear, both cytoplasm and karyoplasm consist of several distinct elements. The nucleus is usually bounded by a definite membrane, and often appears to be a perfectly distinct vesicular body suspended in the cvtoplasm — a conclusion sustained by the fact that it may move actively through the latter, as often occurs in both vegetable and animal cells. Careful study of the nucleus during all its phases gives, however, reason to believe that its structural basis is similar to that of the cell-body ; and that during the course of cell-division, when the nuclear membrane usually disappears, cytoplasm and karyoplasm come into direct contmuity. Even in the resting cell there is good evidence that both the intranuclear and the extranuclear material may be structurally continuous with the nuclear membrane^ and among the Protozoa there are forms (some of the flagellates) in which no nuclear membrane can at any period be seen. For these and other reasons t/ic tcrtns ''nucleus^' and '' ccU-bQ,dy'' sJioiild probably be regarded as only topographical expressions denoting tzuo differentiated areas in a common structural basis. The terms karyoplasm and cytoplasm possess, however, a specific significance owing to the fact that there is on the whole a definite chemical contrast between the nuclear substance and that of the cell-body, the former being characterized by the abundance of a substance rich in phosphorus known as nuclein, while the latter contains no true nuclein and is especially rich in albuminous substances such as nucleo-albumins, albumins, globulins, and the like, which contain little or no phosphorus. Both morphologically and physiologically the differentiation of the active cell-substance into nucleus and cell-body must be regarded as a fundamental character of the cell because of its universal, or all but universal, occurrence, and because there is reason to believe that it is in some manner an expression of the dual aspect of the fundamental process of metabolism, constructive and destructive, that lies at the basis of cell life. The view has been widely held that a third essen- tial element is the centrosome, discovered by Flemming and Van Beneden in 1875-76, and since shown to exist in a large number of other cells (Figs. 7, 8). This is an extremely minute body which 1 Conklin ('97, i). Obst ('99), and some others have described a direct continuity in the resting cell between the intranuclear and extranuclear ineshworks. STRUCTURAL BASIS OF PROTOPLASM 23 is concerned in the process of cell-division and in the fertilization of the G.gg, and has been characterized as the " dynamic centre " of the cell. Whether it has such a significance, and whether it is in point of morphological persistence comparable with the nucleus, are ques- tions still sub judicCy which will be discussed elsewhere.^ B. Structural Basis of Protoplasm As ordinarily seen under moderate powers of the microscope, proto- plasm appears as a more or less vague granular substance which shows as a rule no definite structure organization. More precise examination under high powers, especially after treatment by suitable fixing and staining reagents, often reveals a highly complex structure in both nucleus and cytoplasm. Since the fundamental activities of protoplasm are everywhere of the same nature, investigators have naturally sought to discover a corresponding fundamental morpho- logical organization common to all forms of protoplasm and under- lying all of its special modifications. Up to the present time, however, these attempts have not resulted in any consensus of opinion as to whether such a common organization exists. In many forms of proto- plasm, both in hfe and after fixation by reagents, the basis of the structure is a more or less regular framework or niesJiwork, consisting of at least two substances. One of these forms the substance of the meshwork proper; the other, often called the ground-substance (also cell-sap, enchylema, hyaloplasma, paramitome, interfilar substance, etc.), 2 occupies the intervening spaces. To these two elements must be added minute, deeply staining granules or " microsomes " scattered along the branches of the meshwork, sometimes quite irregularly, sometimes with such regularity that the meshwork seems to be built of them. Besides the foregoing three elements, which we may pro- visionally regard as constituting the active substance, the protoplasm almost invariably contains various passive or metaplasmic substances in the form of larger granules, drops of liquid, crystalloid bodies, and the like. These bodies, which usually lie in the spaces of the mesh- work, are often difficult to distinguish from the microsomes lying in the meshw^ork proper — indeed, it is by no means certain that any adequate ground of distinction exists.^ From the time of Frommann and Arnold ('65-'67) onwards, most of the earlier observers regarded the meshwork as a fibrillar structure, either forming a continuous network or reticulum somewhat like the fibrous network of a sponge ("reticular theory " of Klein, Van Bene- den, Carnoy, Heitzmann), or consisting of disconnected threads, 1 Cf. pp. 304. 354. ' Q'- ^'l^^ssary. ^ Cf. p. 29. 24 GENERAL SKETCH OF THE CELL ••^ jD / [ TK IS ^ Fig- 9- — Living cells of salamander-larva. [Flemminc^..] A. Group of epidermal cells at different foci, showing protoplasmic bridges, nuclei, and cyto- plasmic fibrillae; the central cell with nucleus in the spireme-stage. B. Connective tissue cell. C. Epidermal cell in early mitosis (segmented spireme) surrounded by protoplasmic bridges. D. Dividing cell. E.F. Cartilage-ceils with cytoplasmic fibrillas (the latter somewhat exaggerated in the plate). STRUCTURAL BASIS OF PROTOPLASM 2$ whether simple or branching (''filar theory" of Flcmming), and the same view is widely held at the present time. The meshwork has received various names in accordance with this conception, among which may be mentioned reticulum, thrcad-ivork, spongioplasm, mitomc, filar subslance} all of which are still in use. Under this view the " granules " described by Schultze, Virchow and still earHer observers have been variously regarded as nodes of the network, optical sec- tions of the threads, or as actual granules (" microsomes ") suspended in the network as described above. Widely opposed to these views is the " alveolar theory " of Butschli, which has won an increasing number of adherents. Butschli regards protoplasm as having a foam-like alveolar structure ("W'aben- struktur"), nearly similar to that of an emulsion (Fig. lo), and he has shown in a series of beautiful experiments that artificial emul- sions, variously prepared, may show under the microscope a marvel- lously close resemblance to living protoplasm, and further that drops of oil-emulsion suspended in water may even exhibit amoeboid changes of form. To restate Biitschli's view, protoplasm consists of separate, closely crowded minute drops^ of a liquid alveolar substance suspended in a continuous interalvcolar substance, likewise liquid, but of different physical nature. The latter thus forms the walls of closed chambers or alveoli in which the alveolar drops lie, just as in a fine emulsion the emulsifying liquid surrounds the emulsified drops. The appear- ance of a network in protoplasm is illusory, being due to optical sec- tion of the interalvcolar walls or partitions as viewed at any given focus of the microscope. As thus seen, the walls themselves appear as fibres, while the "spaces of the network" are in like manner oi)ti- cal sections of the alveoli, the alveolar substance that fills them corresponding to the ''ground substance." As explained beyond/^ Butschli interprets in like manner the radiating systems or asters formed during cell-divison, the astral rays (usually considered as fibres) being regarded as merely the septa between radially arranged alveoH (Fig. lo). The two (three) general views above outlined may be designated respectively as thQ fibrillar (reticular or filar) and alveolar \.\\cox\c^ of protoplasmic structure ; and each of them has been believed by some of its adherents to be universally applicable to all forms of protoplasm. Beside them may be placed, as a third general view, Xh^ granular theory especially associated with the name of Altmann, by whom it has been most fully developed, though a number of earlier writers have held similar views. According to Altmann's view, which apart from its theoretical development approaches in 1 See Glossary. 2 Measuring on an average about .ooi mm. in diameter. ^ Cf. p. no. 26 GENERAL SKETCH OF THE CELL some respects that of Biitschli, protoplasm is compounded of innu- merable minute granules which alone form its essential active basis ; and while fibrillar or alveolar structures may occur, these are of only secondary importance. r 1 Fig. 10. — Alveolar or foam-structure of protoplasm, according to Biitschli. [BuTSCllLl.] A. Epidermal cell of the earthworm. B. Aster, attraction-sphere, and centrosome from sea- urchin egg. C. Intracapsular protoplasm of a radiolarian ( Thalassicolla) with vacuoles. D. Peripheral cytoplasm of sea-urchin egg. E. Artificial emulsion of olive-oil, sodium chloride, and water. It is impossible here adequately to review the many combinations and modifications of these views which different investigators have STRUCTURAL BASIS OF PROTOPLASM 27 made.^ On the whole, the present drift of opinion is toward the conclusion that none of the above interpretations has succeeded in the attempt to give a universal formula for protoplasmic structure ; and many recent observers have reached the conclusion, earlier advo- cated by Kolliker ('89), that the various types described above are connected by intermediate gradations and may be transformed one into another, in different phases of cell-activity. Unna ('95), for example, endeavours to show how an alveolar structure may pass into a sponge-like or reticular one by the breaking down of the inter- a .-. .' "■•.#^. i ''. .'*^. zer, "^ -•"t»*'/*'w"*-v'**-** ^> '*^m'l '. 005;^ ^.; ^ o:°Ao:„-D, o 0°b-,-?.-lio°voQ, 00. -o-.... o'o •""•" o" ■?.?o Fig. II.— ('?) Protoplasm of the egg of the sea-urchin {Toxopneustes) in section showing meshwork of microsomes; {b) protoplasm from a living star-fish egg {Astcrias) showing alveolar spheres with microsomes scattered between them ; {c) the same in a dying condition atler crush- ing the egg ; alveolar spheres fusing to form larger spheres ; {d) protoplasm from a young ovarian egg of the same. (All the figures magnified 1200 diameters.) alveolar walls. Flemming, for many years the foremost and most consistent advocate of the fibrillar theory, now admits that protoplasm may be fibrillar, alveolar, granular, or sensibly homogeneous,^ and that we cannot, therefore, regard any one of these types of structure as absolutely diagnostic of the living substance. In plant-cells Strasburger^ and a number of his pupils maintain that the "kino- plasm" (p. 322) or filar plasm, from which the spindle-fibres and astral rays are formed, is fibrillar, while the " trophoplasm " or alveolar plasm forming the main body of the cell is alveolar, the former, however, assuming the fibrillar structure, as a rule, only during the mitotic activity of the cell. My own long-continued studies on various forms of protoplasm have likewise led to the con- clusion that no universal formula for protoplasmic structure can be 1 For full discussion, with literature list, see Flemming, '82, '97. ^ '97. 2. and Butschli. '92, 2, '99. 2 '97, I, p. 260. 3 '95. '97, 3. '^8. 28 GENERAL SKETCH OF THE CELL given. ^ In that classical object, the echinoderm-egg, for example, it is easy to satisfy oneself, both in the living cell and in sections, that the protoplasm has a beautiful alveolar structure, exactly as described by Hutschli in the same object (Fig. 1 1 ). This structure is here, however, entirely of secondary origin ; for its genesis can be traced step by step during the growth of the ovarian eggs through the deposit of minute drops in a homogeneous basis, which ultimately gives rise to the interalveolar walls. In these same eggs the astral systems formed during their subsequent division (Fig. 12) are, I .. .■•.,••• .»•• ''/ '■. /' V"•• : " vr^t'-.- :••••. >-./•; *>'••-'"•.•< :••-..••..••-> Fig. 12. — Section of sea-urchin egg (Toxopficustcs), li minutes after entrance of the sperma- tozoon, showing alveoli and microsomes, sperm-nucleus, middle piece, and aster (about 2000 diameters). believe, no less certainly fibrillar ; and thus we see the protoplasm of the same cell passing successively through homogeneous, alveolar, and fibrillar phases, at different periods of growth and in different conditions of physiological activity. There is good reason to regard this as typical of protoplasm in general. BiJtschli's conclusions, based on researches so thorough, j^rolonged, and ingenious, are entitled to great weight ; yet it is impossible to resist the evidence that fibrillar and granular as well as alveolar structures are of wide occurrence ; and while each may be characteristic of certain kinds of 1 Wilson, '99. STRUCTURAL BASIS OF PROTOPLASM 29 cells, or of certain physiological conditions,^ none is common to all forms of protoplasm. If this position be well grounded, we must admit that the attempt to find in visible protoplasmic structure any adequate insight into its fundamental modes of physiological activity has thus far proved fruitless. We must rather seek the source of these activities in the ultramicroscopical organization, accepting the probability that apparently homogeneous protoplasm is a complex mixture of substances which may assume various forms of visible structure according to its modes of activity. Some of the theoretical speculations regarding the essential nature of that organization are discussed in Chapter VI., but one q2iasi-X\\(to- ' retical point must be here considered. Much discussion has been given to the (question as to which of the visible elements of the proto- plasm should be regarded as the "living" substance proper; and the diversity of opinion on this subject may be judged by the fact that although many of the earlier observers identified the "reticulum " as the living element, and the ground-substance as Ufeless, others, such as Leydig and Schafer, held exactly the reverse view, while Altmann insisted that only the " granules " were alive. Later discussions have shown the futility of this discussion, which is indeed largely a verbal one, turning as it does on the sense of the word "living." In practice we continually use the word "living" to denote various degrees of vital activity. Protoplasm deprived of nuclear matter has lost, wholly or in part, one of the most characteristic vital properties, namely, the power of synthetic metaboHsm ; yet we still speak of it as " living," since it still retains for a longer or shorter period such properties as irritability and the power of coordinated movement ; and, in like manner, various special elements of protoplasm may be termed " liv- ing " in a still more restricted sense. In its fullest meaning, however, the word "living" implies the existence of a group of cooperating activities more complex than those manifested by any one substance or structural element. I am therefore entirely in accord with the view urged by Sachs, Kolliker, Verworn, and other recent writers, that life can 'only be properly regarded as a property of the cell- system as a whole ; and the separate elements of the system would, with Sachs, better be designated as "active" or "passive," rather than as "living" or "lifeless." Thus regarded, the distinction 1 Thus the alveolar structure seems to be characteristic of Protozoa in general, and of the protoplasm of plant-cells when in the vegetative state, the fibrillar of nerve-cells and muscle-cells. The granular type is characteristic of some forms of leucocytes and gland- cells; but many of the granules in these cells are no doubt metaplasmic, and it is further very doubtful whether such a granular or "pseudo-alveolar" structure can be logically dis- tinguished from an alveolar (c/. Wilson, '99). In the pancreas-cell granular and hbr.llar structures alternate with the varying phases of secretory activity {r/. Mathews, '99). 30 GEXERAL SKETCH OF THE CELL between "protoplasmic" and " nictai:)lasmic " substances, while a real and necessary one, becomes after all one of degree. I believe that we are probably justified in regarding the continuous substance as the most constant and active element, and that which forms the fundamental basis of the system, transforming itself into granules, drops, fibrilla?, or networks in accordance with varying physiological needs. ^ Thus stated, the question as to the relative activity of the various elements becomes a real and important one. It now seems probable that the substance of the meshwork (fibrillar or interalveolar structure) is most active in the processes of cell-division, in contractile organs such as cilia and muscle-fibres, and in nerve-cells ; but the ground- substance, while apparently the most frequent seat of metaplasmic deposits, is certainly also the seat of active chemical changes. This subject has, however, not yet been sufficiently investigated. C. The Nucleus A fragment of a cell deprived of its nucleus may live for a consid- erable time and manifest the power of coordinated movement without perceptible impairment. Such a mass of protoplasm is, however, devoid of the powers of assimilation, growth, and repair, and sooner or later dies. In other words, those functions that involve destructive metabolism may continue for a time in the absence of the nucleus ; those that involve constructive metabolism cease with its removal. There is, therefore, strong reason to believe that the nucleus plays an essential part in the constructive metabolism of the cell, and through this is especially concerned with the formative processes involved in growth and development. For these and many other reasons, to be discussed hereafter, the nucleus is generally regarded as a controlling 1 Wilson, '99. Cf. Sachs ('92. '95), Kulliker ('97), Meyer ('96), and Kupffcr ('96) on energids. Sachs sharply distinguishes between the energid {r^\x(^^\x% and protoplasm), which forms a living unit, and the passive ^x^^ix^xA-prodticts, placing in the former the nucleus, nucleolus, general cytoplasm, centrosome and plastids (chloroplasts and leucoplasts), and in the latter the starch-grains, aleurone-crystals, and membrane. Meyer carries the analysis further, classifying the active energid-elemcnts m\.o protoplasmatic and alloplasmatic organs, the former (nucleus cytoplasm, chromatophores, and perhaps the centrosomes) arising only by division, the latter (cilia, and according to Kolliker, also the muscle- and nerve-fibriiiee) formed by differentiation from the protoplasmatic elements. The passive energid-products {ergastic structures or " formed material " of Beale) are formed as enclosures (starch-grains, etc.), or excretions (membranes). These general views arc accejited by Kolliker; but none of these writers has undertaken to show how " alloplasmatic "' structures are to be distinguished from metaplasmic or ergastic. I believe Sachs' view to be in principle not only true but of high utility. Practically, however, it involves us in considerable difficulty, unless the terminology adopted above — itself directly suggested by and nearly agreeing with the usage of Sachs and Kolliker — be employed. THE NUCLEUS 31 centre of cell-activity, and hence a primary factor in growth, develop- ment, and the transmission of specific qualities from cell to cell, and so from one generation to another. I, General Strncticre The cell-nucleus passes through two widely different phases, one of which is characteristic of cells in their ordinary or vegetative condi- tion, while the other only occurs during the complicated changes involved in cell-division. In the first phase, falsely characterized as the *' resting state," the nucleus usually appears as a rounded sac-like body surrounded by a distinct membrane and containing a conspicu- ous irregular network (Figs. 6, 7, 13), which is in some cases plainly visible in the living cell (Fig. 9). The form of the nucleus, though subject to variation, is on the whole singularly constant, and as a rule shows no very definite relation to that of the cell-body, though in elon- gated cells such as muscle-cells, in certain forms of parenchyma, and in epithelial cells (Fig. 49), the nucleus is itself often elongated. Typically spherical, it may, in certain cases, assume an irregular or amoeboid form, may break up into a group of more or less completely separated lobes (polymorphic nuclei. Fig. 49), sometimes forming an irregular ring (" ring-nuclei " of leucocytes, giant-cells, etc., Fig. 14. J)). It is usually very large in gland-cells and others that show a very active metabolism, and in such cases its surface is sometimes increased by the formation of complex branches ramifying through the cell (Fig. 14, E). ^ ^ These forms seem in general to be fairly constant in a given species of cell, but in a large number of cases the nucleus has been seen in the living cell (cartilage-cells, leucocytes, ova) to undergo more or less active changes of form, sometimes so marked as to merit the name of amoeboid (Fig. 77). Perhaps the most remarkable deviations from the usual type of nucleus occur among the unicellular forms. In the dil- ate Infusoria the nuclei are massive bodies of two kinds, viz. a large macrormcleiis and one or more smaller viicroujiclci, both of which arc present in the same cell, the former kind being generally regarded as the active nucleus, the latter as a reserve nucleus from which at cer- tain periods new macronuclei arise (p. 224). The macronuclei show a remarkable diversity of form and structure in different species. Still more interesting are the so-called scattered or distributed nuclei, de- scribed by Butschli in flagellates and Bacteria, by Gruber in certain rhizopods and Infusoria, and by several authors in the Cyanophyccx (Figs. 15, 16). The nuclear material is here apparently scattered through the cell in the form of numerous minute, deeply stained gran- ules, which, if this identification is correct, represent the most primi- 32 GENERAL SKETCH OF THE CELL tive known types of nucleus ; but this subject is still sub jiidice (p. 39). A transition from this condition to nuclei of the ordinary type appears to be L^iven in the nuclei of certain flagellates (e.g. CJii- Uwionas and Tracluhnotias), where the chromatin-granules are aggre- gated about a nucleolus-like body, but are not enclosed by a membrane.^ In considering the structure of the nucleus, as seen in sections, we must, as in the case of the cytoplasm, bear in mind the possibility, or rather probability, that some of the elements described may be coagulation - products ; for the nucleus is in life comj^osed of liquid or semi-liquid substance, and Albrecht ('99) has recently shown that nuclei isolated in the fresh condition will flow together to form a single body. Most of the main features of the nucleus, both in the resting and in the dividing phases, have, however, been seen in life (Fig. 9), and the principal danger of mistaking artifacts for normal structures re- lates to the finer elements, con- sidered beyond. In the ordinary forms of nuclei in their resting state the follow- ing structural elements may as a rule be distinguished (Figs. 6, 7, 10): — Fig. 13. — Two nuclei from the crypts of ^^ q^^e liuclcav DUmbraUC, a Lieberkiihn in the salamander. [Heidenhain.] ^^ -^ r i i t ^ n i • i well-defined delicate wall which The character of the chromatin-network • , i i (^aj/r/4r^wa//«) is accurately shown. The upper glVCS the UUClCUS a Sharp COUtOUr nucleus contains three plasmosomes or true -^^-^^ differentiates it clcarly from nucleoli; the lower, one. A few fine linin-threads . t . •> -r-^' {oxychromattn) are seen in the upper nucleus the SUrrOUuding Cytoplasm. ThlS running off from the chromatin-masses. The wall SOmctimCS StaiuS but VCiy clear spaces are occupied by the ground-sub- ^|i n] ^,^,| ^aU SCarCcly be dif- stance. n ./ ' j ferentiated from the outlying cytoplasm. In other and perhaps more frequent cases, it approaches in staining capacity the chromatin. b. The nuclear rcticuliiDi. This, the most essential part of the nucleus, forms an irregular branching network or reticulum which con- sists of two very different constituents. The first of these, forming the general protoplasmic basis of the nucleus, is a substance known as lini7i 1 Calkins, '98, i. THE NUCLEUS 33 (Schwarz), invisible until after treatment by reagents, which in sections shows a finely granular structure and stains like the cytoplasmic sub- stance, to which it is nearly related chemically (Figs. 7, 49). The second constituent, a deeply staining substance known as chromatiji (Flemming), is the nuclear substance /(^r excellence, for in many cases it appears to be the only element of the nucleus that is directly handed on by division from cell to cell, and it seems to have the power to pro- duce all the other elements. The chromatin often appears in the form of scattered granules and masses of differing size and form, which are embedded in and supported by the linin-substance (Figs. 7, 19). In some cases the entire chromatin-content of the nucleus appears to be condensed into a single mass which simulates a nucleolus ; for exam- ple, in Spirogyra and in various flagellates and rhizopods (e.g. Acti- jiospJicBviuni, Ai'cella) ; or there may be several such chromatin-masses, as in some of the Foraminifera and in Noctihica. More commonly the chromatin forms a more or less regular network intermingled with and more or less embedded in the linin, from which it is often hardly dis- tinguishable until the approach of mitosis, when a condensation of the chromatin-substance occurs. In contradistinction to the other nuclear elements, chromatin is nut acted upon, or is but slowly affected, by peptic digestion. It may thus be easily isolated for chemical analysis, which shows it to consist mainly of luiclein, i.e. a compound in varying proportions of a complex phosphorus-containing acid known as inicleinic acid, with albumi- nous bodies such as histon, protamin, or in some cases albumin itself.' Upon this, as will be show^n in Chapter VL, probably depends the pro- nounced staining capacity when treated with the so-called *' nuclear stains " {e.g. hsematoxylin, methyl-green, and the basic tar-colours gen- erally) from which chromatin takes its name. This capacity always increases as the nucleus prepares for division, reaching a climax in the spireme- and chromosome-stages, and it is also very marked in con- densed nuclei such as those of spermatozoa. These variations are almost certainly due to varying proportions in the constituents of the nuclein, the staining capacity standing in direct ratio to the amount of nucleinic acid. c. The nucleoli, one or more larger rounded or irregular bodies, suspended in the network, and staining intensely with many dyes. In some nuclei they are entirely absent. When present the nucleoli vary in number from one to five or more; and the number is otten inconstant in the same species of cell, and even varies in the same cell with varying physiological conditions. In the eggs of some animals, at certain periods of growth {e.g. lower vertebrates), the nucleus may contain hundreds of nucleoli. An interesting case is 1 See p. 334- D ^^ GENERAL SKETCH OF THE CELL that of the subcutaneous gland-cells of Pisciola, the nuclei of which contain in early phases of secretion but a single nucleolus. During growth of the cell the nucleolus fragments, finally giving rise to several hundred nucleoli which then appear to migrate out into the cytoplasm, leaving but a single nucleolus to repeat the cycle. ^ The bodies known as nucleoli are of at least two different kinds. The first of these, the so-called true nucleoli or phuinosomcs (Figs. 6, 8, B, 13), are of spherical form, and are shown by the staining reactions to differ widely from chromatin, being in general sharply stained by dyes which, like eosin, orange or acid fuchsin, stain the linin and the general cytoplasm. The plasmosomes sometimes seem to have no envelope, but in many cases {e.g. in leucocytes) are surrounded by a thin layer that stains Hke chromatin. Nucleoli of a quite different type are the *' net-knots " (Netzknoten), chromatin- nucleoli, or karyosojucs, which agree in staining reaction with chro- matin and are doubtless to be regarded as merely a portion of the chromatin-network (Figs. 8, 49). These are sometimes spherical, more often irregular (Fig. 8), and often are hardly to be distinguished, except in size, from nodes of the chromatin-reticulum.''^ The relations between these two forms of nucleoli are far from certain, and the variations in staining reaction shown by true nucleoli render it not improbable that intermediate forms exist which may represent an actual transition from one to the other.-*^ In many of the Protozoa, as described beyond, the ''nucleolus" is shown by its behaviour during mitosis to be comparable with an attraction-sphere or centro- some ('Muicleolo-centrosome," Keuten); and even in higher forms there are some cells in which the centrosome is intranuclear (Fig. 148). There is good reason to believe that the chromatin-nucleoli are merely more condensed portions of the chromatin-network. since during cell-division they have the same history as the remaining portion of the chromatin-substance.* The nature of the true nucleoli is still imperfectly known. By some observers, including Flemming, O. and R. Hertwig, and Carnoy, they have been regarded as store- houses of material (para-nuclein, plastin) which contributes to the ^ Montgomery, '98, 2. 2 Flemining first called attention to the chemical difference between the true nucleoli and the chromatic reticulum ('82, pp. 138, 163) in animal-cells, and Zacharias soon afterward studied more closely the difference of staining reaction in plant-cells, showing that the former are especially coloured by alkaline carmine solutions, the latter by acid solutions. Other studies by Carnoy, Zacharias, Ogata, Rosen, Schwarz, Heidenhain, and many others show that the medullary substance (pyrenin) of true nuclei is coloured by acid tar-colours and other plasma stains, while the chromatin has a special affinity for basic dyes. Cf. p. 337. 3 For very full review of the literature of the nucleoli see Montgomery ( '98, 2). 4 Cf. p. 67. THE NUCLEUS 35 formation of chromosomes during division, and hence may play an active role in the nuclear activity. Strasburger ( '95) likewise be- lieves them to contain a store of active material which, however, has no direct relation to the chromosomes but consists of " kinoplasm " Fig. 14. — Special forms of nuclei. A. Permanent spireme-nucleus, salivary gland of Chirowmus larva. Chromatin in a single thread, composed of chromatin-discs (chromomeres), terminating at each end m a true nucleolus or plasmosonie. [Balkianl] B. Permanent spireme-nuclei. intestinal epithelium of dipterous larva Ptychcptfra. [\ AN Gehuchten.] C. The same, side view. D. Polymorphic ring-nucleus, giant-cell of bone-marrow of the rabbit; c. a group of ccntro- somes or centrioles. [Heidenhain.] E. Branching nucleus, spinning gland of butterfly-larva {Picris) . [KORSCHELT.J (p. 322), from which arises the achromatic part of the division- figure (p. 82). On the other hand, Hacker ( '95, '99) ^^^^^ o^^er observers regard the nucleolar material as a passive by-product of the chromatin-activity destined to be absorbed by the active sub- 36 GENERAL SKETCH OF THE CELL Stances. This is supported by the fact that in some forms of mitosis the nucleokis is at the time of division actually cast out of the nucleus into the cytoplasm, where it degenerates without further apparent function. This seems to constitute decisive evidence in support of Hacker's view as api)lied to certain cases ; but without further evidence it must remain doubtful whether it applies to all.' d. The groiiiid-siihstancc, nuclear sap, or kavyolympJi, a clear sub- stance occupying the interspaces of the network and left unstained by most of the dyes that colour the chromatin, the linin, or the plas- mosomes. By most observers the ground-substance is regarded as a liquid filling a more or less completely continuous space traversed by the nuclear network. By Biitschli, however, and some of his fol- lowers the nucleus is regarded as an alveolar structure, the walls of which represent the "netw^ork," while the ground-substance corre- sponds to the alveolar material. Nearly related with this is the view of Reinke ( 94) that the ground-substance consists of large pale frranules of 'Manthanin " or ** oedematin." The configuration of the chromatic network varies greatly in dif- ferent cases. It is sometimes of a very loose and open character, as in many epithelial cells (Fig. i); sometimes extremely coarse and irregular, as in leucocytes (Fig. 49); sometimes so compact as to appear nearly or quite homogeneous, as in the nuclei of spermatozoa and in many Protozoa. In some cases the chromatin does not form a network, but appears in the form of a thread closely similar to the spireme-stage of dividing nuclei (r/. p. 65). The most striking case of this kind occurs in the salivary glands of dipterous \?.x\'^ {^Chiron 0- viHs\ where, as described by Balbiani, the chromatin has the form of a single convoluted thread, composed of transverse discs and termi- nating at each end in a large nucleolus (Fig. 14, A). Somewhat simi- lar nuclei (Fig. 14, B) occur in various epithelial cells of other insects (Van Gehuchten, Gilson), and also in the young ovarian eggs of cer- tain animals (r/. p. 273). In certain gland-cells of the marine isopod Anilocra it is arranged in regular rosettes (Vom Rath). Rabl, fol- lowed bv Van Gehuchten, Heidenhain, and others, has endeavoured to show that the nuclear network shows a distinct polarity, the nucleus having a *' pole " toward which the principal chromatin- threads converge, and near which the centrosome lies.- In many nuclei, however, no trace of such polarity can be discerned. The network may undergo great changes both in physical con- figuration and in staining capacity at different periods in the life of the same cell, and the actual amount of chromatin fluctuates, sometimes to an enormous extent. Embryonic cells are in general 1 Cf. pp. 126-130. 2 cf. the polarity of the cell, p. 55. THE NUCLEUS 17 characterized by the large size of the nucleus; and Zacharias has shown in the case of plants that the nuclei of meristem and other embryonic tissues are not only relatively large, but contain a larger percentage of chromatin than in later stages. The relation of these changes to the physiological activity of the nucleus is still imperfectly understood.^ 2. Finer Stnictiire of tJie Nucleus A considerable number of observers have raised the question whether the nuclear structures may not be regarded as aggregates of more elementary morphological bodies, though there is still no general agreement regarding their nature and relationships. The most definite evidence in this direction relates to the chromatic network. In the stages preparatory to division this network resolves itself into a definite number of rod-shaped bodies known as chroniosoines (Fig. 21), which split lengthwise as the cell divides. These bodies arise as aggregations of minute rounded bodies or microsomes to which various names have been g\vQx\{cJiroi)io- mcres, Fol ; ids, Weismann). They are as a rule most clearly visible and most regularly arranged during cell- division, when the chromatin is ar- ranged in a thread {spireme), or in separate cJironiosoines (Figs. 8, D, 53, B)\ but in many cases they are dis- tinctly visible in the reticulum of the scatteredchromatin-graniiles. [GRUBKK.] "resting" nucleus ,(Fig. 54). It is, however, an open question whether the chromatin-granules of the reticulum are individually identical with those forming the chromo- somes or the spireme-thread. The larger masses of the reticu- Fig. 15. — An infusorian. Trachelo- cerca, with diffused nucleus consisting of 1 Both chromatin-granules and nucleoli have been seen in a considerable number of living cells (Fig. 9). Favourable objects for this purpose are according to Korschelt ('96) the silk- glands of caterpillars, where the whole nucleus may be seen to be filled with fine granules ("microsomes"), among which are scattered many larger granules (" macrosomes "). 'I he later studies of Meves ('97, i) make it probable that the latter are true nucleoli and the for- ?r chromatin-granules. Korschelt, however, regards the "macrosomes" as composed of romatin and the "microsomes" as representing the so-called "achromatic sulistance." me ch 38 GENERAL SKETCH OE THE CELL lum undoubtedly represent aggregations of such granules, but whether the latter completely fuse or remain always distinct is unknown. Even the chromosomes at certain stages appear perfectly homoge- neous, and the same is sometimes true of the entire nucleus, as in the spermatozoon. It is nevertheless possible that the chromatin-gran- ules have a persistent identity and are to be regarded as morpho- logical units of which the chromatin is built up.^ Heidenhain ('93, 94), whose views have been accepted by Rcinke, Waldeyer, and others, has shown that the "achromatic" nuclear net- work is likewise composed of granules, which he distinguishes as /tint/ianiu- or of the chromatic network. Like the latter, the oxychromatin-granules are suspended in a non-staining clear substance, for which he reserves the term //;////. Both forms of granules occur in the chromatic network, while the achromatic network contains only oxychromatin. Thev are sharply differentiated by dyes, the basichromatin being coloured by the basic tar-colours (methyl-green, saffranin, etc.) and other true "nuclear stains"; while the oxychromatin-granules, like many cytoplasmic structures, and like the substance of true nucleoli (pyrenin), are coloured by acid tar-colours (rubin, eosin, etc.) and other "plasma stains." This distinction, as will appear in Chapter VI I., is possibly one of great physiological significance. Still other forms of granules have been distinguished in the nucleus by Reinke ('94) and Schloter ('94). Of these the most important are the " oedematin-granules," which according to the first of these authors form the principal mass of the ground-substance or " nuclear sap " of Hertwig and other authors. These granules are identified by both observers with the " cyanophilous granules," which Altmann regarded as the essential elements of the nucleus. It is at present impossible to give a consistent interpretation of the morphological value and physiological relations of these various forms of granules. The most that can be said is that the basichromatin-granules are probably normal structures ; that they play a principal role in the life of the nucleus ; that the oxychromatin-granules are nearly related to them ; and that not improbably the one form may be transformed into the other in the manner suggested in Chapter VII. The nuclear membrane is not yet thoroughly understood, and much discussion has been devoted to the question of its origin and structure. The most probable view is that long since advocated by Klein i^yd)) and Van Beneden ('83) that the membrane arises as a condensation of the general protoplasmic substance, and is part of the same structure as the linin-network and the cytoplasmic mesh- w^ork. Like these, it is in some cases "achromatic," but in other cases 1 Cf. Chapter VI. THE NUCLEUS 39 it shows the same staining reactions as chromatin, or may be double, consisting of an outer achromatic and an inner chromatic layer. Ac- cording to Reinke, it consists of oxychromatin-granules like those of the linin-network. Interesting questions are raised by a comparison of these facts with the conditions observed in some of the lowest organisms, such as the flagellates and lower rhizopods among animals and the A D «, • / B H O F % '%) 7 E or distrib Fig i6.- Forms of Cyanophyce^, Bacteria, and Flagellates showing the so-called scattered distributed nuclei. [.4-C BuTSCHLi; Z?-/^ Schewiakoff; G-y. Calkin^.] A. Oscillaria. B. Chromafmrn, C. Bacterium lineola. D. Achrowatiuw. E. The same m division. /; Fission of the granules. G. 7l'//-JXiin:nnia«>uiiftuiniff B i\u w,u iviV^*i»»«»»»«»>»«»»?«»jv'!*»«*»<' ::.«.-V".»..*"'„^ti. ■ y^ D Fig. 17. — Ciliated cells, showing cytoplasmic fibriilcTe terminating in a zone of peripheral microsomes to which the cilia are attached. [Engelmann.] A. From intestinal epithelium of Anodonta. B. From gill of Anodonta. CD. Intestinal epi- thelium of Cyclas. tion with which many existing accounts of cell-structure are received. The evidence is nevertheless overwhelmingly strong, as I believe, that not only the fibrillar and alveolar formations, but also the micro- somes observed in cell-structures, are in part normal structures. This evidence is derived partly from a study of the living cell, partly from the resfular and characteristic arrangement of the thread-work and 44 GENERAL SKETCH OF THE CELL microsomes in certain cases. In many Protozoa, for example, a fine alveolar structure may be seen in the living jirotoplasm ; and Flem- ming as well as manv later observers has clearly seen fibrillar struc- tures in the living cells of cartilage, epithelium connective-tissue, and some other animal cells ( Fig. 9). Mikosch, also, has recently described ^;v7;///A7/' threads in living plant-cells. Almost equally conclusive is the beautifully regular arrangement of the fibrillct' in ciliated cells ( Fig. 17, Fngelmann), in muscle-fibres and ncrve-hbres, and especially in the mitotic figure of dividing cells B C D Fig. 18. — Cells of the pancreas in Amphibia. [Mathfavs.] A-C. Nectiiriis ; D. Rami. A and H represent two stages of the " loaded " cell, showing zymogen-granules in the-peripheral and fibrillar structures in tlie basal part of the cell. C shows cells after discharge of the granule-material and invasion of the entire cell by fibrillie. In D por- tions of the fibrillar material are coiled to form the mitosome (" paranucleus " or " Nebenkern "}. (Figs. 2 1, 31), where they are likewise more or less clearly visible in life. A very convincing case is afforded by the pancreas-cells of Nccturiis, which Mathews has carefully studied in my laboratory. Here the thread-work consists of long, conspicuous, defmite fibrillae, some of which may under certain conditions be wound up more or less closely in a spiral mass to form the so-called Nebcjikern. In all these cases it is impossible to regard the thread-work as an accidental coagulation-product. In the case of echinoderm eggs, I have made ('99) a critical comparison of the living structure, as seen under powers THE CYTOPLASM 45 of a thousand diameters and upwards, with the same ohject stained in thin sections after fixation by picro-acetic, subHmate-acetic, and *•/.*••■; ■ ,.'-•,■ «I^- ^*^;s^SsyiiJ^i^/(^j^J^ Fig. 19. — Section through a nephridia! cell of the leech, Clepstite (drawn by Arnold Graf from one of his own preparations). The centre of the cell is occupied by a large vacuole, filled with a watery licinid. The cyto- plasm forms a very regular and distinct reticulum with scattered microsomes which become very large in the peripheral zone. The larger pale bodies, lying in the ground-substance, are cvcretory granules {i.e. metaplasm). The nucleus, at the right, is surrounded by a thick chromatic mem- brane, is traversed by a very distinct linin-network, contains numerous scattered chromatin- granules, and a single large nucleolus within which is a vacuole. Above are two isolated nuclei showing nucleoli and chromatin-granules suspended in the linin-threads. Other reagents. The comparison leaves no doubt that the normal structures are in this case very perfectly preserved, thoui^h the sec- tions give at first sight an appearance somewhat different from that 46 GEXERAL SKE7CII OF THE CELL of the living object, owing to differences of staining capacity. In these eggs the microsomes, thickly scattered through the alveolar walls, stain deeply (Figs. ii. i J ), while the alveolar spheres hardly stain at all. When, therefore, the stained sections are cleared in balsam, the contours of the alveolar spheres almost disappear, and the eye is caught by the walls, which give at first sight quite the appear- ance of a granular reticulum, as it has been in fact described by many observers. Careful study of the sections shows, however, that t lie form and arraui^cjuiut of all the elements is almost idcntieally t/ie same as in life. This result shows that careful treatment by reagents in some cases at least gives a very faithful picture of the normal structure ; and while it should never be forgotten that in sections we are viewing coagulated material, much of which is liquid or semi-liquid in life, w^ should not adopt too pessimistic a view of the results based on fixed material, as I think some of the experimenters referred to above have done. Wherever possible, the structures observed in sections should be compared with those in the living material. When this is imprac- ticable we must rely on indirect evidence; but this is in many cases hardly less convincing than the direct. It is a very interesting and important question whether living protoplasm that appears to the eye to be homogeneous does not really possess a structure that is invisible, owing to the extreme tenuity of the fibrillar or alveolar walls (as was long since suggested by Heitz- mann and Biitschli),^ or to uniformity of refractive index in the structural elements. It is highly prc^bable that such is often the case; indeed, Butschli has shown that such *' homogeneous " protoplasm in Protozoa may show a typical alveolar structure after fixation and staining. This explanation will not, however, apply to the young echinoderm eggs (already referred to at p. 28), where the genesis of the alveolar structure may be follow'ed step by step in the li\ing cell. The protoplasm here appears at first almost like glass, showing at most a sparse and fine granulation ; but after fixing and staining it appears as a mass of fine, closely crowded granules. This may indi- cate the existence of an extremely fine alveolar structure in life; but on the whole I believe that these granules are for the most part coagu- lation-products, since they cannot be demonstrated by staining ijitra vitam, and they very closely resemble the coagulation-granules found in structureless proteids like egg-albumin after treatment by the same reagents. In common with many other investigators, therefore, I believe that protoplasm may in fact be homogeneous dowji to the present limits of microscopical vision. One of the must beautiful forms of cyto-reticulum with which I 1 Cf Butschli, '92, 2, p. 169. THE CYTOPLASM A7 am acquainted has been described by Bolsius and Graf in the ncnh ridial cells of leeches as shown in Fig. 19 (from a preparation by Dr. Arnold Graf). The meshwork is here of great di.stinctness and regularity, and scattered microsomes are found along its threads. It Fig. 20. — Spinal ganglion-cell of the frog. [Lknhossek.] The nucleus contains a single intensely chromatic nucleolus, and a paler linin-network with rounded chromatin-granules. The cytoplasmic fibrillae are faintly shown passing out into the nerve-process below. (They are figured as far more distinct by Flemming.) Tlie dark cyto- plasmic masses are the deeply staining " chromophiiic granules" (.N'issi) of unknown function. (The centrosome, which lies near the centre of the cell, is shown in Fig. 8, C.) At the left, two connective tissue-cells. appears with equal clearness, though in a somewhat different form, in many eggs, where the meshes are rounded and often contain food- matters or deutoplasm in the inter-spaces (Figs. 59, 60). In cartilage- cells and connective tissue-cells, where the threads can be plainly seen 48 GENERAL SKETCH OF THE CELL in life, the network is loose and open, and appears to consist of more or less completely separate threads (F'ig. 9). In the cells of colum- nar epithelium, the threads in the peripheral part of the cell often assume a more or less parallel course, passing outwards from the central region, and giving the outer zone of the cell a striated appear- ance. This is very conspicuously shown in ciliated epithelium, the fibrill:^ corresponding in number with the cilia as if continuous with their bases (Fig. 17).^ In nerve-fibres the threads form closely set parallel fibrillce which may be traced into the body of the nerve-cell; here, according to most authors, they break up into a network in which are suspended numerous deeply staining masses, the "chromo- philic granules" of Nissl (Fig. 20).- In the contractile tissues the threads are in most cases very conspicuous and have a parallel course. This is clearly shown in smooth muscle-fibres and also, as Ballowite has shown, in the tails of spermatozoa. This arrangement is most striking in striped muscle-fibres where the fibrillae are extremely well marked. According to Retzius, Carnoy, Van Gehuchten, and others, the meshes have here a rectangular form, the principal fibrillar having a longitudinal course and being connected at regular intervals by transverse threads ; but the structure of the muscle-fibre is probably far more complicated than this account would lead one to suppo.se, and opinion is still divided as to whether the contractile substance is represented by the reticulum proper or by the ground-substance. Nowhere, perhaps, is a fibrillar structure shown with such beauty as in dividing cells, where (Figs. 21, 31) the fibrillae group themselves in two radiating systems or asters, which are in some manner the immediate agents of cell-division. Similar radiating systems of fibres occur in amoeboid cells, such as leucocytes (Fig. 49) and pigment- cells (Fig. 50), where they probably form a contractile system by means of which the movements of the cell are performed. The views of Biitschli and his followers, which have been touched on at p. 25, differ considerably from the foregoing, the fibrillae being regarded as the optical sections of thin plates or lamelte which form the walls of closed chambers filled by a more liquid substance. Butschli, followed by Rcinke, Eismond, Erlanger, and others, inter- prets in the same sense the astral systems of dividing cells which are regarded as a radial configuration of the lamellae about a central point (Fig. 10, B). Strong evidence against this view is, I believe, 1 The structure of the ciliated cell, as described by Engelmann, may be beautifully demon- strated in the funnel-cells of the nephridia and sperm-ducts of the earthworm. 2 The remarkable researches of Apathy ('97) on the nerve-cells of leeches have revealed the existence within the nerve-cell of networks far more complex and definite than was formerly supposed, and showing definite relations to incoming and outgoing fibrillae within the substance of the nerve-fibres. THE CYTOPLASM 49 afforded by the appearance of the spindle and asters in cross-section. In the early stages of the ^g^ of Nereis, for example, the astral rays are coarse anastomosing fibres that stain intensely and are therefore very favourable for observation (Fig. 60). That they are actual fibres is, I think, proved by sagittal sections of the asters in which the rays are cut at various angles. The cut ends of the branching rays appear in the clearest manner, not as plates but as distinct dots, from which in obHque sections the ray may be traced inwards toward the centro- sphere. Druner, too, figures the spindle in cross-section as consisting of rounded dots, like the end of a bundle of wires, thou^-h these are connected by cross-branches (Fig. 28, F). Again, the crossin^r of Centrospherc con- taining the cen- trosome. Aster. Spindle. Chromosomes forming the equatorial plate. Fig. 21. — Diagram of the dividing cell, showing the mitotic figure and its relation to the cyto- plasmic meshwork. the rays proceeding from the asters (Fig. 128), and their beha^-iour in certain phases of cell-division, is difficult to explain under any other than the fibrillar theory. We must admit, however, that the meshwork varies greatly in differ- ent cells and even in different physiological phases of the same cell ; and that it is impossible at present to bring it under any rule of uni- versal application. It is possible, nay probable, that in one and the same cell a portion of the meshwork may form a true alveolar structure such as is described by Biitschli, while other portions may. at the same time, be differentiated into actual fibres. If this be true the fibrillar or alveolar structure is a matter of secondary moment, and the essential features of protoplasmic organization must be sought in a more subtle underlying structure.^ 1 See Chapter VI. E 50 GENERAL SKETCH OF THE CELL Space would not sufifice for a comparative account of the endless modifications shown by the cytoplasmic substance in different forms of cells. Many of these arise through special differentiations of the active substance, the character of the structure thus being some- times so highly modified, as in the striated muscle-fibre, that it is difficult to trace its exact relation to the more usual forms. More commonly the cytoplasm is modified through the formation of passive or metaplasmic substances which often completely transform the original appearance of the cell. The most frequent of such modifi- cations arise through the deposit of liquid drops and *' granules " (many of the latter, however, being no doubt liquid in life). When the liquid drops arc of watery nature the cavities in which they lie are known as vacuoles^ which are especially characteristic of the pro- toplasm of plant-cells and of Protozoa. These may enlarge or run together to form extensive cavities in the cell, the protoplasm becom- ing reduced to a peripheral layer, or to strands and networks travers- ing the spaces ; while in some forms of unicellular glands the spaces may form branching canals traversing the protoplasm. The vacuolization or meshlike appearance arising through the formation of larger vacuoles or the deposit of other metaplasmic material is not to be confounded with the primary protoplasmic struc- ture. When, however, smaller vacuoles or metaplasmic granules are evenly distributed through the protoplasm, a " pseudo-alveolar " struc- ture (Reinke) arises that can often hardly be distinguished from the " true " alveolar structure of Blitschli.^ Comparative study shows that all gradations exist between the "false " and the "true " alveolar structures and that no logical ground of distinction between the two exists.^ We thus reach ground for the conclusion that the coarser secondary alveolar or reticular formations are to be regarded as only an exaggeration of the primary structure, and that the alveolar mate- rial of *Biitschli's structure belongs in the same general category with the passive or metaplasmic substance.^ E. The Centrosome The centrosome^ is usually an extremely minute body, or more commonly a pair of bodies, staining intensely with haematoxylin and ^ In the latter the alveolar spheres are, according to Biitschli, not more than one or two microns in diameter. ^ This has been demonstrated in the cells of plants by Craio ('96), and more recently by the writer ('99), in the case of echinoderm and other eggs. ^ QC p. 29. * The centrosome was apparently first seen and described by Flemming in 1875, ^" ^^^ egg of the fresh-water mussel Anodonta, and independently discovered by Van Beneden, in THE CENTROSOME 5 I some other reagents, and surrounded by a cytoplasmic radiating aster or by a rounded mass known as the attr'actio}i-spJicrc (Figs. 8, 49, etc.). As a rule it lies in the cytoplasm, not far from the nucleus, and usually opposite an indentation or bay in the latter ; but in a few cases it lies inside the nucleus (Fig. 148). In epithelia the centro- somes (usually double) lie as a rule near the free end of the cell (Fig. 21)} There is still much confusion regarding the relation of the centro- some to the surrounding structures, and this has involved a corre- sponding ambiguity in the terminology. We w^ill therefore only consider it briefly at this point, deferring a more critical account to Chapter VI. In its simplest form it is a single minute granule, which may, however, become double or triple (leucocytes, connective tissue- cells, some epithelial cells) or even multiple, as in certain giant-cells (Fig. 14, D\ and as also occurs in some forms of cell-division (Fig. 52). In some cases (Figs. 8, C, 120, 148) the ** centrosome " is a larger body containing one or more central granules or '* centrioles " (Boveri); but it is probable that in some of these cases the central granule is itself the true centrosome, and the surrounding body is part of the attraction-sphere. During the formation of the spermatozoon the centrosome undergoes some remarkable morphological changes (p. 171), and is closely involved in the formation of the contractile structures of the tail. The nature and functions of the centrosome have formed the sub- ject of some of the most persistent and searching investigations of recent cytology. Van Beneden, followed by Boveri and many later workers, regarded the centrosome as a distinct and persistent cell- organ, which Hke the nucleus was handed on by division from one cell-generation to another. Physiologically it was regarded as being the especial organ of cell-division, and in this sense as the *' dy- namic centre" of the cell. In Boveri's beautiful development of this the following year, in dycyemids. The name is due to Boveri ('88, 2, p. 68). Van Beneden's and Boveri's independent identification of centrosome in Ascaris as a permanent cell-organ ('87) was quicklv supported by numerous observations on other animals and on plants. In rapid succession the centrosome and attraction-sphere were found to be present m pig- ment-cells of fishes (Solger, '89, '90), in the spermatocytes of Amphil)ia (Hermann. Vto), hi the leucocytes, endothelial cells, connective tissue-cells, and lung-epithelium of salamanders (Flemming, '91), in various plant-cells (Guignard, '91), in the one-celled diatoms (Butschli. '91), in the giant-cells and other cells of bone-marrow (Heidenhain, Van Ban.heke, \ an der Stricht, '91), in the flagellate Noctiluca (Ishikawa, '91), in the cells of marine alg:v (Stras- burger, '92), in cartilage-cells (Van der Stricht, '92), in cells of cancerous growths j epitheli- oma, Lustig and Galeotti, '92), in the young germ-cells as already described, in gland-cells (Vom Rath, '95), in nerve-cells (Lenhossek, '95), in smooth muscle-hbres (Lenhossek, 99), and in embryonic cells of manv kinds (Heidenhain, '97)- •'^'any others have conhrmed and extended this list. Guignard's identification of the centrosomes in higher plants i. open to grave doubt (r/ p. 82). Q- P- 57- 52 GEXERAL SKETCH OF THE CELL view it was regarded further as the especial fertiHzing element in the spermatozoon, which, when introduced into the fgg, endowed the latter with the power of division and development. Van Beneden's and Boveri's hypothesis, highly attractive on account of its simplicity and lucidity, is supported by many facts, and undoubtedly contains an element of truth ; yet recent researches have cast grave doubt upon its generality, and necessitate a suspension of judgment upon the entire matter. Many of the most competent recent workers on the cytologv of higher plants have been unable to find centrosomes, whether in the resting-cells, in the apparatus of cell-division, or dur- ing the process of fertilization, notwithstanding the fact that undoubted centrosomes occur in some of the lower plants. Among zoologists, too, an increasing number of recent investigators, armed with the best technique, have maintained the total disappearance of the cen- trosome at the close of cell-division or during the process of fertili- zation, agreeing that in such cases the centrosome is subsequently formed dc novo. Experimental researches, also, have given strong ground for the conclusion that cells placed under abnormal chemical conditions may form new centrosomes (p. 306). If these strongly supported results be well founded. Van Beneden's hypothesis must be abandoned in favour of the view that the centrosome is but a sub- ordinate part of the general apparatus of mitosis, and one which may be entirelv dispensed with. Thus regarded, the centrosome would lose somewhat of the significance first attributed to it, though still remaining a highly interesting object for further research.^ F. Other Organs The cell-substance is often differentiated into other more or less definite structures, sometimes of a transitory character, sometimes showing a constancy and morphological persistency comj^arable with that of the nucleus and centrosome. From a general point of view the most interesting of these are the bodies known as /A? jr//V/jr ox proto- plasts{Y\g. 6), which, like the nucleus and centrosome, are capable of growth and division, and may thus be handed on from cell to cell. The most important of these are the cJirouiatopJiores or cJironioplastids, which are especially characteristic of plants, though they occur in some animals as well. These are definite bodies, varying greatly in form and size, which possess the power of growth and division, and have in some cases been traced back to minute colourless plastids or ^ Cf. pp. Ill, 304. Eisen ('97) asserts that in the blood of a salamander, Bairachoseps, the attraction-sphere (•' archosome ") containing the centrosomes may separate from the remainder of the cell (nucleated red corpuscles) to form an independent form of blood- corpuscle or " plasmocyte," which leads an active life in the blood. OTHER ORGANS 53 leucoplastids in the embryonic cells. By enlargement and differen- tiation these give rise to the starch-builders (amyloplastids), to the chlorophyll-bodies (chloroplastids), and to other ])igment-bodies (chromoplastids), all of which may retain the power of division. The embryonic leucoplastids are also believed to multiply by division and to arise by the division of plastids in the parental organism ; but it remains an open question whether this is their only mode of origin, and the same is true of the more highly differentiated forms of plas- tids to which they may give rise. The contractile or pulsating vacuoles that occur in most Protozoa and in the swarm-spores of many Algae are also known in some cases to multiply by division ; and the same is true, according to the researches of De Vries,. Went, and others, of the non-pulsating vacu- oles of plant-cells. These vacuoles have been shown to have, in many cases, distinct walls, and they are regarded by De Vries as a special form of plastid ("tonoplasts ") analogous to the chromatophores and other plastids. It is, however, probable that this view is only appli- cable to certain forms of vacuoles. The plastids possess in some cases a high degree of morphological independence, and may even live for a time after removal from the remaining cell-substance, as in the case of the "yellow cells" of Radiolaria. This has led to the view, advocated by Brandt and others, that the chlorophyll-bodies found in the cells of many Protozoa and a few Metazoa {Hydra, Spongilla, some planarians) are in reality dis- tinct Alg^ living symbiotically in the cell. This view is probably correct in some cases, e.g. in the Radiolaria ; but it may be doubted whether it is of general appUcation. In the plants the plastids are. almost certainly to be regarded as differentiations of the protoplasmic substance. The existence of cell-organs which have the power of independent assimilation, growth, and division is a fact of great theoretical interest in its bearing on the general problem of cell-organization ; for it is one of the main reasons that have led De Vries, Wiesner, and many others to regard the entire cell as made up of elementary self-propa- gating units. G. The Cell-membrane The structure and origin of the cell-wall or membrane form a subject somewhat apart from our general purpose, since the wall belongs to the passive or metaplasmic products of protoplasm rather than to the living cell itself. We shall therefore treat it very briefly. Broadly speaking, animal cells are in general characterized by the slight development and relative unimportance of the cell-walls, while 54 GEXERAL SKETCH OE THE CELL the reverse is the case in plants, where the cell-walls play a very important role. In the latter the wall sometimes attains a great thickness, usually displays a distinct stratification, and often has a complex sculj)ture. Such massive walls very rarely occur in the case of animal tissues, though the intercellular matrix of cartilage and bone is to a certain extent analogous to them, and the thick and often highly sculptured envelopes of some kinds of eggs and of various Protozoa may be placed in the same category. It is open to question whether any cells are entirely devoid of an enclosing envelope; for even in such ** naked" cells as leucocytes, rhizopods, or membraneless eggs, the boundary of the cell is usually formed by a more resistant layer of protoplasm or " pellicle "(Biitschli) which may be so marked as to simulate a true membrane, as is the case, for example, in the red blood-corpuscles (Ranvier, Waldeyer, etc.). Such pellicles probably differ from true membranes only in degree ; but it is still an open question both in animals and in plants, how far true membranes arise by direct transformation of the periph- eral protoplasmic layer (the " Hautschicht " of botanists), and how far as a secretion-product of the protoplasm. In the case of animal cells, Leydig long since proposed ^ to distinguish between " cuticular " membranes, formed as secretions and usually occurring only on the free surfaces (as in epithelia), from *' true membranes " arising by direct transformation of the peripheral protoplasm. Later researches, including those of Leydig himself, have thrown so much doubt on this distinction that most later writers have used the term cuticular in a purely topographical sense to denote membranes formed only on one (the free) side of the ccll,^ leaving open the question of origin. The formation and growth of the cell-wall have been far more thor- oughly studied in plants than in animals, yet even here opinion is still divided. Most recent researches tend to sustain the early view of Nageli that the cell-wall is in general a secretion-product, though there are some cases in which a direct transformation of protoplasm into membrane-stuff seems to occur. ^ In the division of plant-cells the daughter-cells are in almost all cases cut apart by a cell-plate which arises in the protoplasm of the mother-cell as a transverse series of thickenings of the spindle-fibres in the equatorial region (Fig. 34). This fact, long" regarded by Strasburger and others as a proof of the direct origin of the membrane from the protoplasmic substance, is shown by Strasburger's latest work ('98) to be open to a quite different interpretation, the actual wall being formed by a splitting of the cell-plate into two layers between which the wall appears as a secretion-product. Almost all observers further are ao:reed that the formation of new membranes on naked masses of 1 Cf. '85, p. 12. "^ C/.O. Hertwig, '93. » Cf. Strasburger, '98. POLARITY OF THE CELL 55 protoplasm produced by plasmolysis are likewise secretion-products, and that the secondary thickening of plant-membranes is produced in the same way. These facts, together with the scanty available zoological data, indicate that the formation of membranes by secre- tion is the more usual and typical process. ^ The chemical composition of the membrane or intercellular sub- stance varies extremely. In plants the membrane consists of a basis of cellidose, a carbohydrate having the formula CgHjoOg ; but this sub- stance is very frequently impregnated with other substances, such as silica, Hgnin, and a great variety of others. In animals the inter- cellular substances show a still greater diversity. Many of them are nitrogenous bodies, such as keratin, chitin, elastin, gelatin, and the like ; but inorganic deposits, such as silica and carbonate of lime, are common. H. Polarity of the Cell In a large number of cases the cell exhibits a definite polarity, its parts being symmetrically grouped with reference to an ideal organic axis passing from pole to pole. No definite criterion for the identi- fication of the cell-axis has, however, yet been determined ; for the general conception of cell-polarity has been developed in two differ- ent directions, one of which starts from purely morphological con- siderations, the other from physiological, and a parallelism between them has not thus far been fully made out. On the one hand. Van Beneden ('83) conceived cell-polarity as a primary morphological attribute of the cell, the organic axis being identified as a line drawn through the centre of the nucleus and the centrosome (Fig. 22, A). With this view Rabl's theory ('85) of nuclear polarity harmonizes, for the chromosome-loops converge toward the centrosome, and the nuclear axis coincides with the cell- axis. Moreover, it identifies the polarity of the Qg^. which is so important a factor in development, with that of the tissue-cells; tor the egg-centrosome almost invariably appears at or near one pole of the ovum. Heidenhain ('94, '95) has recently developed this conception of polarity in a very elaborate manner, maintaining that all the struc- tures of the cell have a definite relation to the primary axis, and that this relation is determined by conditions of tension in the astral rays 1 Strasburger ('97, 3, '98) believes membrane-formation in general to be especially con- nected with the activity of the "kinoplasm," or tilar plasm of which he considers the " Haut- schicht," as well as the spindle-fibres, to be largely composed. In support ol this may be mentioned, besides the mode of formation of the partition-walls in the division of plant- cells, Harper's ('97) very interesting observations on the formation of the ascospores m Erysiphe (Fig. IZ), where the spore-membrane appears to arise directly from the astral rays. 56 GENERAL SKETCH OE THE CELL focussed at the centrosome. On this basis he endeavours to explain the position and movements of the nucleus, the succession of division- planes, and many related phenomena.^ Hatschek {^^^) and Rabl ('89, 92), on the other hand, have ad- vanced a quite different hypothesis based on physiological considera- tions. By "cell-polarity" these authors mean, not a predetermined morphological arrangement of parts in the cell, but a polar differen- tiation of the cell-substance arising secondarily through adaptation of the cell to its environment in the tissues, and having no necessary relation to the polarity of Van Beneden (Fig. 22, B, C). This is , \ r.. . ^ • • • • . • • • • •• • • • 9 • • . • • • 1 A Van Beneden. B C Rabl, Hatschek. Fig. 22. — Diagrams of cell-polarity. A. Morphological polarity of Van Beneden. Axis passing through nucleus and centrosome. Chromatin-threads converging toward the centrosome. B.C. Physiological polarity of Rabl and Hatschek, Zj' in a gland-cell, C'in a ciliated cell. typically shown in epithelium, which, as Kolliker and Haeckel long since pointed out, is to be regarded, both ontogenetically and phy- logenetically, as the most primitive form of tissue. The free and basal ends of the cells here differ widely in relation to the food- supply, and show a corresponding structural differentiation. In such cells the nucleus usually lies nearer the basal end, toward the source of food, while the differentiated products of cell-activity are formed either at the free end (cuticular structures, cilia, pigment, zymogen- granules), or at the basal end (muscle-fibres, nerve-fibres). In the non-epithelial tissues the polarity may be lost, though traces of it are often shown as a survival of the epithelial arrangement of the embryonic stages. 1 Cf. p. 105. POLARITY OF THE CELL 57 But, although this conception of polarity has an entirely different point of departure from Van Beneden's, it leads, in some cases at least, to the same result ; for the cell-axis, as thus determined, may coincide with the morphological axis as determined by the position of the centrosome. This is the case, for example, with both the spermatozoon and the ovum ; for the morphological axis in both is also the physiological axis about which the cytoplasmic differentia- tions are grouped. Recent researches have further shown that the same is the case in many forms of epithelia, where the centrosomes lie in the outer end of the cell, often very near the surface.^ (Fig- '2-1) A B WM) km ■ te^J-i '.'.i C • D Fig. 23. — Centrosomes in epithelial and other cells. [A, D, ZiMMERMANN ; E, Heidenhain and COHN; F, Heidenhain.] A. From gastric glands of man ; dead cell at the left. B. Uterine epithelium, man. C. From human duodenum ; goblet-cell, with centrosome in the middle. D. Corneal epithelium of monkev. E. Epithelial cells from mesoblast-somites, embryo duck. F. Red blood-corpuscles from the duck- embryo. The centrosomes are double in nearly all cases. and the recent observations of Henneguy ('98) and Lenhossek (98,1) give reason to believe that the ** basal bodies" to which the ciHa of ciliated epithelium are attached may be the centrosomes.- These facts are of very high significance; for the position of the centro- some, and hence the direction of the axis, is here obviously related to the cell-environment, and it is difficult to avoid the conclusion that the latter must be the determining condition to which the intracellular relations conform. When applied to the germ-cells, this conclusion becomes of high interest ; for the polarity of the Qgg is one of the 1 Zimmermann, '98; Heidenhain and Cohn, '97. 2 cf. p. 356. 58 GEXERAL SKETCH OF THE CELL primary conditions of development, and we have here, as I beUeve, a clue to its determination.^ I. Till: Cell in Relation to the Multicellular Body In analyzing the structure and functions of the individual cell we are accustomed, as a matter of convenience, to regard it as an inde- pendent elementary organism or organic unit. Actually, however, it is such an organism only in the case of the unicelkilar j)lants and animals and the germ-cells of the multicellular forms. When we consider the tissue-cells of the latter, we must take a somewhat dif- ferent view. As far as structure and origin are concerned the tissue- cell is unquestionably of the same morphological value as the one-celled plant or animal ; and i)i tliis sense the multicellular body is equivalent to a colony or aggregate of one-celled forms. Physi- ologically, however, the tissue-cell can only in a limited sense be regarded as an independent unit ; for its autonomy is merged in a greater or less degree into the general life of the organism. From this point of view the tissue-cell must in fact be treated as merely a localized area of activity, provided it is true with the complete apparatus of cell-life, and even capable of independent action within certain limits, yet nevertheless a part and not a whole. There is at present no biological question of greater moment than the means by which the individual cell-activities are coordinated, and the organic unity of the body maintained ; for upon this question hangs not only the problem of the transmission of acquired charac- ters, and the nature of development, but our conception of life itself. Schwann, the father of the cell-theory, very clearly perceived this ; and after an admirably lucid discussion of the facts known to him (*39), drew the conclusion that the life of the organism is essentially a composite ; that each cell has its independent life ; and that " the whole organism subsists only by means of the reciprocal action of the single elementary parts." ^ This conclusion, afterward elaborated by Virchow and Haeckel to the theory of the ** cell-state," took a very strong hold on the minds of biological investigators, and is even now widely accepted. It is, however, becoming more and more clearly apparent that this conception expresses only a part of the truth, and that Schwann went too far in denying the influence of the totality of the organism upon the local activities of the cells. It would of course be absurd to maintain that the whole can consist of more than the sum of its parts. Yet, as far as growth and development are con- ^ Cf. pp. 384, 424. We should remember that the germ-cells are themselves epithelial products. 2 Untersuchungen, Trans., p. 181. THE CELL LN RELATION TO THE MULTICELLULAR BODY 59 cerned, it has now been clearly demonstrated that only in a Hmited sense can the cells be regarded as cooperating units. Thcv are rather local centres of a formative power pervading the growing mass as a whole,^ and the physiological autonomy of the individual cell falls into the background. It is true that the cells may acquire a high degree of physiological independence in the later stages of embryological development. The facts to be discussed in the eighth and ninth chapters will, however, show strong reason for the conclu- sion that this is a secondary result of development, through which the cells become, as it were, emancipated in a greater or less degree from the general control. Broadly viewed, therefore, the life of the multicellular organism is to be conceived as a whole ; and the appar- ently composite character which it may exhibit is owing to a second- ary distribution of its energies among local centres of action. ^ In this light the structural relations of tissue-cells become a ques- tion of great interest ; for we have here to seek the means by which the individual cell comes into relation with the totality of the organ- ism, and by which the general equihbrium of the body is maintained. It must be confessed that the results of microscopical research have not thus far given a very certain answer to this question. Though the tissue-cells are often apparently separated from one another by a non-living intercellular substance, which may appear in the form of solid walls, it is by no means certain that their organic continuity is thus actually severed. Many cases are known in which division of the nucleus is not followed by division of the cell-body, so that multi- nuclear cells or syncytia are thus formed, consisting of a continuous mass of protoplasm through which the nuclei are scattered. Heitz- mann long since contended ( '73), though on insufficient evidence, that division is incomplete in nearly all forms of tissue, and that even when cell-walls are formed they are traversed by strands of protoplasm by means of which the cell-bodies remain in organic continuity. The whole body was thus conceived by him as a syncytium, the cells being no more than nodal points in a general reticulum, and the body forming a continuous protoplasmic mass. This interesting view, long received with scepticism, has been to a considerable extent sustained by later researches, and though it still x^m-d^x^-^siibjiidice, has been definitely accepted in its entirety by some recent workers. The existence of protoplasmic cell-bridges between the sieve-tubes of plants has long been known ; and Tangl's dis- covery, in 1879, of similar connections between the endosperm-cells was followed by the demonstration by Gardiner, Kienitz-Gerloff, A. Meyer, and many others, that in nearly all plant-tissues the cell-walls 1 (7: Chapters VIII.. IX. 2 for a fuller discussion see pp. 388 and 413. 60 GENERAL SKETCH OF THE CELL are traversed by delicate intercellular bridges. Similar bridges have been conclusively demonstrated by Ranvier, J^izzozero, Rctzius, Flem- ming, Pfitzner, and many later observers in nearly all forms of epithe- lium ( Fig. I ) ; and they are asserted to occur in the smooth muscle-fibres, in cartilage-cells and connective tissue-cells, and in some nerve- cells. Dendy ('88), Paladino ( '90), and Retzius ('89) have endeav- oured to show, further, that the follicle-cells of the ovary are connected by protoplasmic bridges not only with one another, but also with tJic oviiDi ; and similar protoplasmic bridges between germ-cells and somatic cells have been also demonstrated in a number of plants, e.g. by Goroschankin ( '83) and Ikeno ( '98) in the cycads and by A. Meyer ('96) in ]\)hox. On the strength of these observations some recent writers have not hesitated to accept the probability of Heitz- mann's original conception, A. Meyer, for example, expressing the opinion that both the plant and the animal individual are continuous masses of protoplasm, in which the cytoplasmic substance forms a morphological unit, whether in the form of a single cell, a multi- nucleated cell, or a system of cells. ^ Captivating as this hypothesis is, its full acceptance at present would certainly be premature ; and as far as adult animal tissues are concerned, it still remains unde- termined how far the cells are in direct protoplasmic continuity. It is obvious that no such continuity exists in the case of the corpuscles of blood and lymph and the wandering leucocytes and pigment-cells. In case of the nervous system, which from an a priori point of view would seem to be above all others that in which protoplasmic con- tinuity is to be expected, its occurrence and significance are still a subject of debate. When, however, we turn to the embryonic stages we find strong reason for the belief that a material continuity between cells here exists. This is certainly the case in the early stages of many arthropods, where the whole embryo is at first an unmistakable syncytium ; and Adam Sedgwick has endeavoured to show that in Pcripatns and even in the vertebrates the entire embryonic body, up to a late stage, is a continuous syncytium. I have pointed out ( '93) that even in a total cleavage, such as that of AnipJiioxus or the echi- noderms, the results of experiment on the early stages of cleavage are difficult to explain, save under the assumption that there must be a structural continuity from cell to cell that is broken by mechan- ical displacement of the blastomeres. This conclusion is supported by the recent work of Hammar ( '96, '97), whose observations on sea-urchin eggs I can in the main confirm. Among the most interesting observations in this direction are those of Mrs. Andrews ('97),^ who asserts that during the cleavage 1 '96, p. 212. Cf. also the views of Hanstein, Strasburger. Russow, and others there cited. ^ Cf. also E. A. Andrews, '98, I, '98, 2. THE CELL IN RELATION TO THE MULTICELLULAR BODY 6 1 of the echinoderm-egg the blastomeres ** spin " dehcate protoplasmic filaments, by which direct protoplasmic continuity is established between them subsequent to each division. These observations, if correct, are of high importance ; for if protoplasmic connections may be broken and re-formed at will, as it were, the adverse evidence of the blood-corpuscles and wandering cells loses much of its weight. Meyer ('96) adduces evidence that in Volvox the cell-bridges are formed anew after division ; and Flemming has also shown that when leucocytes creep about among epithehal cells they rupture the protoplasmic bridges, which are then formed anew behind them.^ We are still almost wholly ignorant of the precise physiological meaning of the cell-bridges ; but the facts indicate that they are not merely channels of nutrition, as some authors have maintained, but paths of subtler physiological impulse. Beside the facts determined by the isolation of blastomeres, referred to above, may be placed Townsend's recent remarkable experiments on plants, described at page 346. If correct, these experiments give clear evidence of the transference of physiological influences from cell to cell by means of protoplasmic bridges, showing that the nucleus of one cell may thus control the membrane-forming activity in an enucleated fragment of another cell. The field of research opened up by these and related researches seems one of the most promising in view; but until it has been more fully explored, judgment should be reserved regarding the whole question of the occurrence, origin, and physio- logical meaning of the protoplasmic cell-bridges. LITERATURE. I •'- Altmann, R.--Die Elementarorganismen und ihre Beziehungen zu den Zellen, 2d ed. Leipzig, 1894. TAnnee Biologique. — /^^7;v>, 1895-96. (Full Reviews and Literature-lists.) Bohm and Davidoff . — Lehrbuch der Histologic des Menschen. Wiesbaden, 1895. Boveri, Th.— (See Lists IV., V.) Biitschli, 0. — Untersuchungen liber mikroskopische Schaume und das Protoplasma. Zt'/^z^s/;^ (Engelmann), 1892. Id. — Untersuchungen liber Struktur. Leipzig, 1898. Carnoy, J. B. — La Biologie Cellulaire. /./Wvr. 1884. Engelmann, T. W. — Zur Anatomic und Pliysiologie der Flimmerzellen: Arch. ges. Phvs., XXIII. 1880. , .^ , Erlanger, R. v. Neuere Ansichten liber die Struktur des Protoplasmas : ZooL CentralbUUl-^j9' 1896. Fischer, A. Fixierung, Farbung und Bau des Protoplasmas. Jemu 1899. Flemming, W. Zellsubstanz, Kern und Zellteikmg. Leipzig, 1882. Id. ZeW^: MerkelundBonHet^sErgebnisse,\.-\'n. 1891-97- (Admirable reviews and literature-lists.) 1 '95, pp. lo-i I ; '97, p. 261 . ' See also Introductory list, p. 14. 62 GENERAL SKETCH OF THE CELL Heidenhain, M. — Uber Kern und Protoplasma : FestscJir. z. ^o-Jii/ir. Doctorjub. 7'Oh 7'. KolUkcr. Leipzig^ i^93- Klein, E. — Observations on the Structure of Cells and Nuclei : (J/^d^'i- Journ. Mic. .SV/.. Will. 1878. Kolliker, A. — Handbucii der Gewebelehre, 6th ed. Leipzig, 1889. Leydig, Fr. — Zelle und Gewebe. Boiui. 1885. Schafer, E. A. — General Anatomy or Histology; in Quaifi's Anatof/ty, I., 2, loth ed. London, 1891. Schiefferdecker & Kossel. — Die Gewebe des Menschlichen Korpers. Braunschweig, I Sc) I . Schwarz, Fr. — Die morphologische und chemische Zusammcnsetzung des Proto- plashias. Jyres/au, 1887. Strasburger, E. — Zcllbildung und Zellteilung. 3d ed. 1880. Id. — Das Botanische Practicum. 3d ed. Jena. 1897. Strasburger, Noll, Schenck, and Schimper. — Lehrbuch der Botanik, 3d ed. Jenay 1897. Strieker, S. — Handbuch der Lehre von den Geweben. Leipzig, 1871. Thoma, R. — Text-book of General Pathology and Pathological Anatomy: trans, by Alex. Bruce. London, 1896. Van Beneden, E. — (See Lists II.. IV.) De Vries, H. — Intracellulare Pangenesis, /ena, 1889. Waldeyer, W. — Die neueren Ansichten liber den Bau und das Wesen der Zelle : Deu'.sch. Med. IVoc/ienschr., Oct., Nov., 1895. Wiesner, J. — Die Elementarstruktur u. das Wachstum der lebenden Substanz : U'ien, Holder. 1892. Wilson, E. B. — The Structure of Protoplasm: Journ. Morph., XV. Suppl. ; also Wood's U oil Biol. Lectures, 1899. Zimmermann, A. — Beitrage zur Morphologic und Physiologic der Pflanzenzelle. Tubingen, 1893. Id. — Die Morphologic und Physiologic des Pflanzlichen Zellkernes. Jena, 1896. CHAPTER II CELL-DIVISION " Wo eine Zelle entsteht, da muss eine Zelle vorausgegangen sein, ebenso wie das Thier nur aus deni Thiere, die Pflanze nur aus der Pflanze entstehen kann. Auf diese Weise ist wenngleich es einzelne Punkte im Korper gibt, wo der strenge Nachweis noch nicht gelie- fert ist, doch das Princip gesichert, dass in der ganzen Reihe alles Lebendigen, dies mogen nun ganze Pflanzen oder thierische Organismen oder integrirende Theile derselben sein, ein ewiges Gesetz der coiithiuir lichen EntzvicklunghQ.%i&\i\.r ViKCHow.^ The law of genetic cellular continuity, first clearly stated by Vir- chow in the above words, has now become one of the primary data of biology, and the advance of research is ever adding weight to the conclusion that the cell has no other mode of origin than by division of a preexisting cell. In the multicellular organism all the tissue- cells arise by continued division from the original germ-cell, and this in its turn arises by the division of a cell preexisting in the parent-body. By cell-division, accordingly, the hereditary substance is split off from the parent-body ; and by cell-division, again, this substance is handed on by the fertilized egg-cell or oosperm to every part of the body arising from it.^ Cell-division is, therefore, one of the central facts of development and inheritance. The first two decades after Schleiden and Schwann ('40-'6o) were occupied with researches, on the part both of botanists and of zool- ogists, which finally demonstrated the universality of this process and showed the authors of the cell-theory to have been in error in asserting the independent origin of cells out of a formative blastema.'"^ The mechanism of cell-division was not precisely investigated until long afterward, but the researches of Remak ('41), Kolliker (44), and others showed that an essential part of the process is a division of both the nucleus and the cell-body. In 1855 {I.e., pp. 174, 175), and again in 1858, Remak gave as the' general result of his researches the following synopsis or scheme of cell-division. Cell-division, he asserted, proceeds from the centre toward the periphery. It begins with the division of the nucleolus, is continued by simple constriction and division of the nucleus, and is completed by division of the cell- 1 Cellularpathologie, p. 25, 1858. 2 Cf. Introduction, p. 10. 3 For a full historical account of this period, see Remak, Untersttchungen iiher die Ent- wickhmg derWirbelthiere, 1855, PP- ^ 64-1 80. Also Tyson on the Cell-doctrine and Sachs's Geschichte der Botajiik. 63 64 CELL-DIVISION body and membrane (Fig. 24). For many years this account was accepted, and no essential advance beyond Rcmak's scheme was made for nearly twenty years. A number of isolated observations were, however, from time to time made, even at a very early period, which seemed to show that cell-division was by no means so simple an operation as Remak believed. In some cases the nucleus seemed to disappear entirely before cell-division (the germinal vesicle of the ovum, according to Reichert, Von Ikier, Robin, etc.); in others to become lobed or star-shaped, as described by Virchow and by Remak himself (Fig. 24,/). It was not until 1873 that the way was opened for a better understanding of the matter. \\\ this year the discoveries of Anton Schneider, quickly followed by others in the same direction by Biitschli, Fol, Strasburger, Van Beneden, Flemming, and Hertwig, showed cell-division to be a far more elaborate process than had been supposed, and to involve a com- plicated transformation of the nucleus to which Schleicher ('78) afterward gave the name of karyokincsis. It soon ap- peared, however, that this mode of division was not of universal occurrence ; and that cell-divi- sion is of two widely different types, which Van Beneden {'j^^ distinguished as fnii^Di 01 ta t io n , T?;„ . T^- ♦ ^- • • f n^-,^ .oUc ;^ Corresponding nearly to the Fig. 24. — Direct division of blood-cells in J^ ^ J ^ the cmbrvo chick, illustrating Remak's scheme. simple proCCSS described by [Remak.] Remak, and division, involving a-e. Successive stasres of division; f. cell ,, t i. j dividing by mitosis. ' -^ the morc Complicated process of karyokincsis. Three years later Flemming ('79) proposed to substitute for these the terms direct and iJidircct division, which are still used. Still later ('82) the same author suggested the terms mitosis (indirect or karyokinetic division) and auiitosis (direct or akinetic division), which have rapidly made their w^ay into general use, though the earlier terms are often em- ployed. ' Modern research has demonstrated the fact that amitosis or direct division, regarded by Remak and his immediate followers as of uni- versal occurrence, is in reality a rare and exceptional process ; and there is reason to believe, furthermore, that it is especially char- acteristic of highly specialized cells incapable of long-continued multiplication or such as are in the early stages of degeneration, for instance, in glandular epithelia and in the cells of transitory embry- onic envelopes, where it is of frequent occurrence. Whether this OUTLINE OF INDIRECT DIVISION 65 view be well founded or not, it is certain that in all the hifjjher and in many of the lower forms of Ufe, indirect division or mitosis is the typical mode of cell-division. It is by mitotic division that the germ- cells arise and are prepared for their union during the process of maturation, and by the same process the oosperm segments and gives rise to the tissue-cells. It occurs not only in the highest forms of plants and animals, but also in such simple forms as the rhizopods, flagellates, and diatoms. We may, therefore, justly regard it as the most general expression of the "eternal law of continuous develop- ment" on which Virchow insisted. A. Outline of Indirect Division or Mitosis (Karyokixesis) In the present state of knowledge it is somewhat difficult to give a connected general account of mitosis, owing to the uncertainty that hangs over the nature and functions of the centrosome. For the pur- pose of the following preliminary outline, we shall take as a type mitosis in which a distinct and persistent centrosome is present, as has been most clearly determined in the maturation and cleavage of various animal eggs, and in the division of the testis-cells. In such cases the process involves three parallel series of changes, which affect the nucleus, the centrosome, and the cytoplasm of the cell-body respectively. For descriptive purposes it may conveniently be divided into a series of successive stages or phases, which, however, graduate into one another and are separated by no well-defined limits. These are: (i) The Pi'opJiases, or preparatory changes; (2) the Mctaphasc, which involves the most essential step in the division of the nucleus ; (3) the Anaphases, in which the nuclear material is distributed ; (4) the Telophases, in which the entire cell divides and the daughter-cells are formed. I. Prophases. — {a) The Nticletis. As the cell prepares for division, the most conspicuous fact is a transformation of the nuclear substance, involving both physical and chemical changes. The chromatin-sub- stance rapidly increases in staining-power, loses its net-like arrange- ment, and finally gives rise to a definite number of separate intensely staining bodies, usually rod-shaped, known as cliroiuosoines. As a rule this process, exemplified by the dividing cells of the salamander-epi- dermis (Fig. i) or those of plant-meristem (Fig. 2), takes place as fol- lows. The chromatin resolves itself little by little into a more or less convoluted thread, known as the 5/r/;/(Knauel)or spireme, and its sub- stance stains far more intensely than that of the reticulum (Fig. 25). The spireme-thread is at first fine and closely convoluted, forming the close spireme." Later the thread thickens and shortens and the n 66 CELL-DIVISION convolution becomes more open ('* open spireme"). In some cases there is but a single continuous thread ; in others, the thread is from E Fig. 25. — Diagrams showing the prophases of mitosis. A. Resting cell with reticular nucleus and true nucleolus; at c the attraction-sphere containing two centrosomes. B. Early prophase ; the chromatin forming a continuous spireme, nucleolus still present : abo%-e, the amphiaster {a). C. D. Two different types of later prophases. C. Disappear- ance of the primary spindle, divergence of the centrosomes to opposite poles of the nucleus (exam- ples, some plant-cells, cleavage-stages of many eggs). D. Persistence of the primary spindle (to form in some cases the " central spindle"), fading of the nuclear membrane, ingrowth of the astral rays, segmentation of the spireme-thread to form the chromosomes (examples, epidermal cells of salamander, formation of the polar bodies). E. Later prophase of type C\ fading of the nuclear membrane at the poles, formation of a new spindle inside the nucleus; precocious splitting of the chromosomes (the latter not characteristic of this tvpe alone). E. The mitotic figure established; e.f. the equatorial plate of chromosomes. {€/. Figs. 21, 27, 32, etc.) OUTLINE OF INDIRECT DIVISION 6/ its first appearance divided into a number of separate pieces or seg- ments, forming a segmented spireme. In either case it ultimately breaks transversely to form the chromosomes, which in most cases have the form of rods, straight or curved, though they are sometimes spher- ical or ovoidal, and in certain cases may be joined together in the form of rings. The staining-povver of the chromatin is now at a maxi- mum. As a rule "the nuclear membrane meanwhile fades away and finally disappears, though there are some cases in which it persists more or less completely through all the phases of division. The chromosomes now lie naked in the cell, and the ground-substance of the nucleus becomes continuous with the surrounding cytoplasm (Fig. 25, A£,/^V The remarkable fact has now been established with high probability that eveiy species of plant or animal has a fixed and characteristic niDH- ber of cJiromosomes, zvhicJi regularly recurs in the division of all of its cells ; and in all forms arising by sexual reproduction the number is even. Thus, in some of the sharks the number is 36 ; in certain gas- teropods it is 32 ; in the mouse, the salamander, the trout, the lily, 24 ; in the worm Sagitta, 18 ; in the ox, guinea-pig, and in man - the num- ber is said to be 16, and the same number is characteristic of the onion. In the grasshopper it is 12; in the hepatic Pallavicinia and some of the nematodes, 8 ; and in Ascaris, another thread-worm, 4 or 2. In the crustacean Artemia it is 168.^ Under certain conditions, it is true, the number of chromosomes may be less than the normal in a given species; but these variations are only apparent exceptions (p. %'j). The even number of chromosomes is a most interesting fact, which, as will appear hereafter (p. 205 ), is due to the derivation of one-half the number from each of the parents. The nucleoli differ in their behaviour in different cases. Net-knots, or chromatin-nucleoli, contribute to the formation of the chromosomes; and in cases such as Spirogyra {ViQ.\m\Q.x, "^6, and Moll, '93 ) or .-^r//- nosphcerium (R. Hertwig, '99), where the whole of the chromatin is at one period concentrated into a single mass, the whole chromatic figure thus appears to arise from a "nucleolus." True nucleoli or plasmo- somes sooner or later disappear ; and the greater number of observers agree that they do not take part in the chromosome-formation. In a considerable number of forms {e.g. during the formation of the j^olar 1 The spireme-formation is by no means an invariable occurrence in mit»)sis. In a consid- erable number of cases the chromatin-network resolves itself ilirectly into tlie chromosomes, the chromatic substance becoming concentrated in separate masses which never form a con- tinuous thread. Such cases are connected by various gradations with the " segmented spi- reme." 2 Flemmino believes the number in man to be considerably greater than 16. ^ For a more complete list see p. 206. 6 8 CELL-Di I vs/oy bodies in various eggs) the nucleolus is cast out into the cytoplasm as the spindle forms, to persist as a " metanucleus " for some time before its final disappearance (Fig. 104). More commonly the nucleolus fades away /;/ s/t//, sometimes breaking into fragments meanwhile, while the chromosomes and spindle are forming. The fate of the material is in this case only conjectural. An interesting view is that of Strasburger ('95, '97), who suggests that the true nucleoli are to be regarded as storehouses of " kinoplasmic " material, which is either directly used in the formation of the spindle, or, upon being cast out of the nucleus, adds to the cytnj-)lasmic store of " kinoplasm " avail- able for future mitosis. {d) The AinpJiiastcr. Meanwhile, more or less nearly parallel with these changes in the chromatin, a complicated structure known as the avipJiiastcr {Yo\, '77) makes its appearance in the position formerly occupied by the nucleus (Fig. 2^,B-F). This structure consists of a fibrous spindle-shaped bodv, the spindle, at either pole of which is a star or aster formed of rays or astral fibres radiating into the sur- rounding cytoplasm, the whole strongly suggesting the arrangement of iron filings in the field of a horseshoe magnet. The centre of each aster is occupied by a minute body, known as the centrosoine (Boveri, 'ZZ\ which may be surrounded by a spherical mass known as the ^rw/rt^j/Z/^/r (Strasburger, '93). As the amphiaster forms, the chro- mosomes group themselves in a plane passing through the equator of the spindle, and thus form what is known as the equatorial plate. The amphiaster arises under the influence of the centrosome of the resting cell, which divides into two similar halves, an aster being developed around each while a spindle stretches between them (Figs. 25, 27). In most cases this process begins outside the nucleus, but the subsequent phenomena vary considerably in different forms. In some forms (tissue-cells of the salamander) the amj^hiaster at first lies tangentially outside the nucleus, and as the nuclear membrane fades away, some of the astral rays grow into the nucleus from the side, become attached to the chromosomes, and finally pull them into posi- tion around the equator of the spindle, which is here called the ecu- tral spiudle (Figs. 25, D, F ; 27). In other cases the original spindle disappears, and the two asters pass to o]:>])osite poles of the nucleus (some plant mitoses and in many animal-cells). A spindle is now formed from ravs that grow into the nucleus from each aster, the nuclear membrane fading away at the poles, though in some cases it may be pushed in by the spindle-fibres for some distance before its disappearance (Figs. 25, 32). In this case there is apparently ho central spindle. In a few exceptional cases, finally, the amphiaster may arise inside the nucleus (p. 304). The entire structure, resulting from the foregoing changes, is OUTLINE OF I.XDIRECT DIVISION 69 known as the karyokiiictic or mitotic figure. It may be described as consisting of two distinct parts; namely, i, the chromatic figure, formed by the deeply staining chromosomes ; and, 2, the achromatic figure, consisting of the spindle and asters which, in general, stain but slightly. The fibrous substance of the achromatic figure is gener- Fig. 26. — Diagrams of the later phases of mitosis. G. Metaphase; splitting of the chromosomes {e.p.^. ». The cast-off nucleolus. //. Ana- phase ; the daughter-chromosomes diverging, between them the interzonal-fibres (/./.). or central spindle; centrosomes already doubled in anticipation of the ensuing -division. /. Late anaphase or telophase, showing division of the cell-body, mid-body at the equator of the spindle and bcgm- ning reconstruction of the daughter-nuclei. J. Division completed. ally known as archoplasm (Boveri, '88), but this term is not applied to the centrosome within the aster. 2. Metaphase. — TYiO, prophases of mitosis are, on the whole, pre- paratory in character. The metaphase, which follows, forms the initial phase of actual division. Each chromosome splits lengthwise into two exactly similar halves, which afterward diverge to opposite poles of the spindle, and here each group of daughter-chromosomes 70 CELL-DIVISIOX finally gives rise to a daiii^^hter-nucleus (Fig. 26). In some cases the splitting of the chromosomes cannot be seen until they have grouped themselves in the equatt)rial plane of the spindle ; and it is only in this case that the term " metaphase " can be applied to the mitotic figure as a whole. In a large number of cases, however, the splitting may take place at an earlier period in the spireme-stage, or even, in a few cases, in the reticulum of the mother-nucleus (Figs. 54. 55). Such variations do not, however, affect the essential fact that the clnvDiatic nctivork is converted into a thread^ which, ivhether continuous or discontinuous, splits tJiroughojit its entire lengtJi into two exactlj equivalent halves. The splitting of the chromosomes, discovered by Flemming in 1880, is the most significant and funda- mental operation' of cell-division; for by it, as Roux first pointed out {^^}i\ the entire substance of the chromatic network is precisely halved, and the daughter-nuclei receive precisely equivalent portions oj chro- matin from the mother-nucleus. It is very important to observe that the nuclear division always shows this exact quality, whether division of the cell-body be equal or unequal. The minute polar body, for example (p. 238), receives exactly the same amount of chromatin as the Q^^g, though the latter is of gigantic size as compared with the former. On the other hand, the size of the asters varies with that of the daughter-cells (Figs. 58, 175), though not in strict ratio. The fact is one of great significance for the general theory of mitosis, as will appear beyond. 3. Anaphases. — After splitting of the chromosomes, the daughter- chromosomes, arranged in two corresponding groups,^ diverge to oppo- site poles of the spindle, where they become closely crowded in a mass near the centre of the aster. As they diverge, the two groups of daughter-chromosomes are connected by a bundle of achromatic fibres, stretching across the interval between them, and known as the interzonal fibres or connecting fibres? In some cases these differ in a marked degree from the other spindle-fibres ; and they are believed by many observers to have an entirely different origin and function. A view now widely held is that of Hermann, who regards these fibres as belonging to a central spindle, surrounded by a peripheral layer of mantle-fibres to which the chromosomes are attached, and only exposed to view as the chromosomes separate.^ Almost invariably in the division of plant-cells and often in that of animal cells these 1 It was this fact that led Flemming to employ the word mitosis (fiiros, a thread). 2 This stage is termed by Flemming the dyasier, a term which should, however, be aban- doned in order to avoid confusion with the earlier word amphiafter. The latter convenient and appropriate term clearly has priority. 3 Verlnndutigsfasern of German authors ; filaments reunissants of Van Beneden. * Cf. p. 105. OUTLINE OF INDIRECT DIVISION 71 fibres show during this period a series of deeply staining thickenings in the equatorial plane forming the cell-plate or mid-body. In plant- mitoses this is a very conspicuous structure ( Fig. 34). In animal cells the mid-body is usually less developed and sometimes rudimentary, being represented by only a few granules or even a single one (Fig. 29). Its later history is described below. 4. Telophases. — In the final phases of mitosis, the entire cell divides in two in a plane passing through the equator of the spindle, each of the daughter-cells receiving a group of chromosomes, half of the spindle, and one of the asters with its centrosome. Meanwhile, a daughter-nucleus is reconstructed in each cell from the group of chromosomes it contains. The nature of this process- differs greatly in different kinds of cells. Sometimes, as in the epithelial cells of Amphibia, especially studied by Flemming and Rabl, and in many plant-cells, the daughter-chromosomes become thickened, contorted, and closely crowded to form a daugJiter-sph'enie , closely similar to that of the mother-nucleus (Fig. 29); this becomes surrounded by a mem- brane, the threads give forth branches, and -thus produce a reticular nucleus. A somewhat similar set of changes takes place in the seg- menting eggs of Ascaris (Van Beneden, Boveri). In other cases, as in many segmenting ova, each chromosome gives rise to a hollow vesicle, after which the vesicles fuse together to produce a single nucleus (Fig. 52). When first formed, the daughter-nuclei are of equal size. If, however, division of the cell-body has been unequal, the nuclei become, in the end, correspondingly unequal — a fact which, as Conklin and others have pointed out, proves that the size of the nucleus is controlled by that of the cytoplasmic mass in which it lies. The fate of the achromatic structures varies considerably, and has been accurately determined in only a few cases. As a rule, the spindle-fibres disappear more or less completely, but a portion of their substance sometimes persists in a modified form i^e.g. the Nebenkern, p. 163). In dividing plant-cells, the cell-plate finally extends across the entire cell and splits into two layers, between which appears the membrane by which the daughter-cells are cut apart.i A nearly similar process occurs in a few animal cells,- but as a rule the cell-plate is here greatly reduced and forms no mem- brane, the cell dividing by constriction through the equatorial plane. Even in this case, however, the division-plane is often indicated before division takes place by a peculiar modification of the cyto- plasm in the equatorial plane outside the spindle (Fig. 30)- This region is sometimes called the cytoplasmic plate, in contradistinction to the spindle-plate, or mid-body proper. In the proi)hases and meta- 1 Cf. Strasburger, '98. 2 Cf. Hoffmann, '98. 7^ CELL-DIVISION phases the astral rays often cross one another in the equatorial region outside the spindle. During the anaphases, however, this crossing disappears, the rays from the two asters now meeting at an angle along the cytoplasmic plate (Fig. 31). Constriction and division of the cell then occur.^ The aster may in some cases entirely disappear, together with the centrosome (as occurs in the mature dgg). In a large number of cases, however, the centrosome persists, lying either outside or more rarely inside the nucleus and dividing into two at a very early period. This'division is clearly a precocious preparation for the ensuing divi- sion of the daughter-cell, and it is a remarkable fact that it occurs as a rule during the early anaphase, before the mother-cell itself has divided. There are apparently, however, some cases in which the centrosome remains undivided during the resting stage and only divides as the process of mitosis begins. Like the centrosome, the aster or its central portion may persist in a more or less modified form throughout the resting state of the cell, forming a structure generally known as the attraction-sphere. This body often shows a true astral structure with radiating fibres (Figs. 8, 49); but it is sometimes reduced to a regular spherical mass which may represent only a portion of the original aster (Fig. 7). B. Origin of the Mitotic Figure The nature and source of the material from which the mitotic figure arises form a problem that has been almost continuously under discussion since the first discovery of mitosis, and is even now but partially solved. The discussion relates, however, almost solely to the achromatic figure (centrosome, spindle, and asters) ; for every one is agreed that the chromatic figure (chromosomes) is directly derived from the chromatin-network, as described above, so that there is no breach in the continuity of the chromatin from one cell-generation to another. With the achromatic figure the case is widely different. The material of the spindle and asters must be derived from the nucleus, from the cytoplasm, or from both ; and most of the earlier research was devoted to an endeavour to decide between these possibilities. The earhest observers {'71-7S) supposed the achro- matic figure to disappear entirely at the close of cell-division, and most of them (Butschli, Strasburger, Van Beneden, '75) believed it to be re-formed at each succeeding division out of the nuclear substance. The entire mitotic figure was thus conceived as a metamorphosed nucleus. Later researches ('75- 85) gave contradic- i See p. 318. Cf. Kostanecki, '97, and Hoffmann, '98. ORIGIN OF THE MITOTIC FIGURE 73 tory and apparently irreconcilable results. Fol ('79) derived the spindle from the nuclear material, the asters from the cytoplasm. Strasburger ('80) asserted that the entire achromatic figure arose A c %-!# »^ D Fig. 27. — The prophases of mitosis (heterotypical form) in primary spermatocytes of Salama)idra. [Meves.] A. Early segmented spireme ; two centrosomes outside the nucleus in the remains of the attraction-sphere. B. Longitudinal splitting of the spireme, appearance of the astral rays, disin- tegration of the sphere. C. Early amphiastcr and central spindle. D. Chromosomes in the form of rings, nuclear membrane disappeared, amphiaster enlarging, mantle-fibres developing. from the cytoplasm, and to that view, in a modified form, he still adheres. Flemming ('82), on the whole, inclined to the opinion that the achromatic figure arose inside the nucleus, yet expressed the y^ CELL-DIVISION opinion that the question of nuclear or cytoplasmic origin was one of minor importance. A long series of later researches on both plants and animals has fully sustained this opinion, showing that the origin of the achromatic figure does in fact differ in different cases. Thus in Infusoria the entire mitotic figure is of intranuclear origin (there are, however, no asters); in echinoderm eggs the spindle is of nuclear, the asters of cytoplasmic, origin ; in the testis-cells and some tissue- cells of the salamander, a complete amphiaster is first formed in the cytoplasm, but to this are afterward added elements probably derived from the linin-network; while in higher plants there is some reason to believe that the entire achromatic figure may be of cytoplasmic origin. Such differences need not surprise us when we reflect that the achromatic part of the nucleus (linin-network, etc.) is probably of the same general nature as the cytoplasm. ^ Many observers have maintained that the material of the astral rays and spindle-fibres is directly derived from the substance of the protoplasmic meshwork, whether nuclear, cytoplasmic, or both ; but its precise origin has long been a subject of debate. This question, critically considered in Chapter VI., will be here only briefly sketched. By Klein {;7%\ Van Beneden ('83), Carnoy ('84, '85), and a large num- ber of later observers, the achromatic fibres, both of spindles and of asters, are regarded as identical with those of a preexisting reticulum which have merely assumed a radiating arrangement about the cen- trosome. The amphiaster has, therefore, no independent existence, but is merely an image, as it were, somewhat like the bipolar figure arising when iron filings are strewn in the field of a horseshoe magnet. Boveri, on the other hand, who has a small but increasing following, maintains that the amphiastral fibres are not identical with those of the preexisting meshwork, but a new formation which, as it were, "crystallizes anew " out of the general protoplasmic substance. The amphiaster is therefore a new and independent structure, arising in, or indirectly from, the preexisting material, but not by a direct mor- phological transformation of that material. This view, which has been advocated by Druner ('94), Braus ('95), Meves ('97, 4, '98), and with which my own later observations ('99) also agree, is more fully discussed at page 318. In 1887 an important forward step was taken through the inde- pendent discovery by Van Beneden and Boveri that in the ^gg of Ascaris the centrosome does not disappear at the close of mitosis, but remains as a distinct cell-organ lying beside the nucleus in the cyto- 1 In the case of echinoderm eggs, I have found reason ('95, 2) for the conclusion that the spindle- fibres are derived not merely from the linin-substance, but also from the chromatm. Despite some adverse criticism, I have found no reason to change my opinion on this point. The possible significance of such a derivation is indicated elsewhere (p. 302). ORIGIN OF THE MITOTIC FIGURE 75 plasm. These investigators agreed that the amphiaster is formed under the influence of the centrosome, which by its division creates two new "centres of attraction" about which the astral systems arise, and which form the foci of the entire dividing system. In them are centred the fibrillae of the astral system, toward them the daughter- F Fig. 28. - Metaphase and anaphases of mitosis in cells (spermatocytes) of the salamander. [Druner.] E Metaphase. Tlie continuous central spindle-fibres pass from pole to pole of the spmdle Outside them the thm layer of contractile mantle-fibres attached to the d.vKlcd ^^^'-:^'^^''^^'^} which only two are shown! Centrosomes and asters. F. Transverse section ^ -- ^ \ ^^ "^ !?^ J figure showing the ring of chromosomes surrounding the central spmdle. the cut ^^'^^/^^ ' rL'''!"^/ a;peanng as ''dots. G. Anaphase; divergence of the ^»-^g'^^--f-°"--"\^.^- ^j^J^^^ "S„f^,^^^ tral spin5leas the interzonal fibres; contractile fibres ^vn^^^'^-\^or..^-' \ '^Xj^^^^^^^ shown. H. Later anaphase (dyaster of Flemming) ; the central spmdle uly exposed to Mev. mantle-fibres attached to the chromosomes. Immediately aftenvard the cell d.vdes (see P .g. ^) . chromosomes proceed, and within their respective spheres of influ- ence are formed the resulting daughter-cells. Both Van Beneden and Boveri fully recognized the importance of then" discovery. "We are justified," said Van Beneden, ^ in regarding the attraction-sphere with its central corpuscle as forming a permanent organ, not only of the early blastomeres, but of all cells, and as constituting a cell-organ equal 76 CELL-DIVISION in rank to the nucleus itself ; and we may conclude that every central corpuscle is derived from a preexisting corpuscle, every attraction- sphere from a preexisting sphere, and that division of the sphere precedes that of the cell-nucleus." ^ Boveri expressed himself in similar terms regarding the centrosome in the same year {"^y, 2, p. 153), and the same general result was reached by Vejdovsky nearlv at the same time,- though it was less clearly formulated than bv either Boveri or Van Beneden. All these observers agreed, therefore, that the achromatic figure arose outside the nucleus, in the cytoplasm ; that the primary impulse to cell-division was given, not by the nucleus, but by the centrosome, and that a new cell-organ had been discovered whose special office Fig. 29. — Final phases (telephases) of mitosis in salamander cells. [Flemming.] /. Epithelial cell from the lung; chromosomes at the poles of the spindle, the cell-body divid- ing; granules of the "mid-body" or '/Auischcnkorpcr ■M the equator of the disappearing spindle. y. Connective tissue-ceil (lung) immediately after division; daughter-nuclei reforming, the cen- trosome just outside of each ; mid-body a single granule in the middle of the remains of the spindle. • was to preside over cell-division. **The centrosome is an indepen- dent permanent cell-organ, which, exactly like the chromatic elements, is transmitted by division to the daughter-cells. 77ic cciitrosojue rep- resents the dynamic cejitre of cell T "^ That the centrosome does in many cases, especially in embryonic cells, behave in the manner stated by Van Beneden and Boveri seems at present to admit of no doubt ; and it has been shown to occur in 1 '87, p. 279. 2 '88, pp. 151, etc. 3 Boveri, '87, 2, p. 153. ORIGIN OF THE MITOTIC FIGURE yy many kinds of adult tissue-cells during their resting state ; for example in pigment-cells, leucocytes, connective tissue-cells, epithelial and endothelial cells, in certain gland-cells and nerve-cells, in the cells of some plant-tissues, and in some of the unicellular plants and ani- mals, such as the diatoms and flagellates and rhizopods. On the other hand. Van Beneden's conception of the attraction-sphere has proved untenable ; for this structure has been clearly shown in some cases to disintegrate and disappear at the close or the beginning of mitosis^ (Fig- 27). Whether the centrosome theory can be maintained is still in doubt ; but evidence against it has of late rapidly accumulated. In the first place, it has been shown that the primary impulse to cell-division cannot be given by fission of the centrosome, for there are several accurately determined cases in which the chromatin-elements divide independently of the centrosome, and it is now generally agreed that the division of chromatin and centrosome are two parallel events, the nexus between which still remains undetermined.^ Secondly, an increasing number of observers assert the total disap- pearance of the centrosome at the close of mitosis ; while some very convincing observations have been made favouring the view that cen- trosomes may be formed de novo without connection with preexisting ones (pp. 213, 305). Thirdly, a large number of recent observers (including Strasburger and many of his pupils) of mitosis in the flowering plants and pteridophytes agree that in these forms no centrosome exists at any stage of mitosis, the centre of the aster being occupied by a vague reticular mass, and the entire achromatic figure arising by the gradual grouping of fibrous cytoplasmic elements (kinoplasm or filar plasm) about the nuclear elements.^ If we can assume the cor- rectness of these observations, the centrosome-theory must be greatly modified, and the origin of the amphiaster becomes a far more com- plex problem than it appeared under the hypothesis of Van Ik-neden and Boveri. That such is indeed the case is indicated by nothing more strongly than by Boveri's own remarkable recent experiments on cell-division (referred to at page 108). C. Details of Mitosis Comparative study has shown that almost every detail of the pro- cesses described above is subject to variation in different forms of cells. Before considering some of these modifications it may be well to pomt out what we are at present justified in regarding as its essential 1 Cf. p. 323. 2 cf. p. 108. « Cf. p. 82. 78 CELL-DIVISION features. These are : (i) The formation of the chromatic and achro- matic figures; (2) the longitudinal splitting of the chromosomes or spireme-thread ; (3) the transportal of the chromatin-halves to the respective daughter-cells. Each of these three events is endlessly varied in detail ; yet the essential phenomena are everywhere the same, with one important exception relating to the division of the chromo- somes that occurs in the maturation of certain eggs and spermatozoa.^ It maybe stated further that the study of mitosis in some of the lower forms (Protozoa) gives reason to believe that the asters are of second- ary importance as compared with the spindle, and that the formation of spireme and chromosomes is but tributary to the division of the smaller chromatin-masses of which they are made up. I. J'aricties of the Mitotic Figure (a) TJic Achromatic Figure. The phenomena involved in the his- tory of the achromatic figure are in general most clearly displayed in embryonic or rapidly dividing cells, especially in egg-cells (Figs. 31, 60), where the asters attain an enormous development, and the centrosomes are especially distinct. In adult tissue-cells the asters are relatively small and difficult of demonstration, the spindle large and distinct ; and this is particularly striking in the cells of higher plants where the asters are but imperfectly developed. Plant-mitoses are characterized by the prominence of the cell-plate (Fig. 34), which is rudimentary or often wanting in animals, a fact correlated no doubt with the greater development of the cell-membrane in plants. With this again is correlated the fact that division of the cell-body in animal cells generally takes place by constriction in the equatorial plane of the spindle ; while in plant-cells the cell is usually cut in two by a cell-wall developed in the substance of the protoplasm and derived in large part from the cell-plate. In animal cells we may distinguish two general types in the forma- tion of the amphiaster, which are, however, connected by interme- diate gradations. In the first of these, typically illustrated by the division of epithelial and testis-cells in the salamander (Flemming, Hermann, Drijner, Meves), a complete amphiaster is first formed in the cytoplasm outside the nucleus, while the nuclear membrane is still intact. As the latter fades away and the chromosomes appear, some of the astral rays grow into the nuclear space and become attached to the chromosomes, which finally arrange themselves in a ring about the original spindle (Figs. 27, 28). In the completed amphiaster, therefore, we may distinguish the original central spi7idle (Hermann, '91) from the surrounding mantle-fibres, the latter being 1 Cf. Chapter V. DETAILS OF MITOSIS 79 attached to the chromosomes, and being, according to Hermann, the principal agents by which the daughter-chromosomes are dragged apart. The mantle-fibres thus form two hollow cones or half-spin- dles, separated at their bases by the chromosomes and completely surrounding the continuous fibres of the central spindle, which come into view as the ''interzonal fibres" during the anaphases (Fig. 28). There is still considerable uncertainty regarding the origin and relation of these two sets of fibres. It is now generally agreed with Van Beneden that the mantle-fibres are essentially a part of the asters, i.e. are simply those astral rays that come into connection with the chromosomes — wholly cytoplasmic in ori- gin (Herma.nn, Driiner, MacFarland), or in part cytoplasmic, in part dif- ferentiated from the linin- network (Flemming, Meves). Driiner ('95), Braus ('95) (salamander), and MacFarland (yPleicro- phyllidia, '97) believe the central spindle to arise secondarily through the union of two opposing groups of astral rays in the area between the centrosomes. On the other hand, Hermann ('91), Flemming ('91), Heidenhain ('94), Kos- tanecki ('97), Van der Stricht ('98), and others believe the central spindle to exist from the first in the form of fibres stretching between the diverging centrosomes ; and Heidenhain believes them to be developed from a special substance, forming a ''primary centrodesmus," which persists in the resting cell, and in which the centrosomes are embedded.^ MacFarland's observa- tions on gasteropod-eggs ('97) indicate that even nearly related torms may differ in the origin of the central spindle, since in Plcurophyllidia it is of secondary origin, as described above, while in Diaiilula it is a primary structure developed from what he describes as the " centro- some," but which, as shown at page 314, is probably to be regarded as ir/p. 315- Fig. 30. — Mid-body in embryornc cells oiUinax. [HOFF- MANN.] Earlier stage above, showing thickenings along the line of cleavage. Later stage, below, showing spindle-plate and cytoplasmic plate. 8o CELL-DIVISIOy an attraction-sphere surrounding; the centrosomes, and is perhaps comparable to Heidenhain's " centrodesmus." In the second type, ilhistrated in the cleavaf]^e of echinoderm, annehd, niolhiscan, and some other egjrs, a central s])indle may be formed, — sometimes already during the anaphases of the preceding mitosis (Figs. 99, 155), — but afterward disappears, the asters moving Fig. 31. — The middle phases ot mitosis in the first cleavage of the Ascaris-^gg. [BOVERI.] y-f. Closing prophase, the equatorial plate forming, B. Metaphase; equatorial plate estab- lished and the chromosomes split; b. the equatorial plate, viewed en face, showing the four chro- mosomes, C. Early anaphase; divergence of the daughter-chromosomes (polar body at one side), D. Later anaphase; p.b. second polar body, (For preceding stages see Fig. 90; for later stages Fig. 145.) to opposite poles of the nucleus. Between these two poles a new spindle is then formed in the nuclear area, while astral rays grow out into the cytoplasm. There is strong evidence that in this case the entire spindle may arise inside the nucleus, i.e. from the sub- stance of the linin-network, as occurs, for example, in the eggs of echinoderms (Fig, 25, E), and in the testis-cells of arthropods. In other cases, however, a part at least of the spindle is of cytoplasmic DETAILS OF MITOSIS 8l origin, since the ends of the spindle begin to form before dissokition of the nuclear membrane, and the latter is pushed inwards in folds by the ingrowing fibres (Figs. 25, C, 99).! In some cases, however, it seems certain that the nuclear membrane fades away before com- pletion of the spindle (first maturation-division of TJialasscma, CJice^- toptcriis), and it is probable that the middle region of the spindle is here formed from the Hnin-network. In most, if not all, mitoses of the second type the chromosomes do not form a ring about the equator of the spindle, but extend in a flat plate completely through u-'---«- / / %l \ N. D Fig. 32. — Mitosis in Sfypocaulon. [SWINGLE.] A. Early prophase witli single aster and centrosonie. B. Initial formation of intraniK car spindle. C. Divergence of the daughter-centrosomes. D. Early anaphase ; nuclear nieniiji..ne still intact. its substance. Here, therefore, it is impossible to speak of a " cen- tral spindle." It is nevertheless probable that the spindle-fibres are of two kinds, viz. continuous fibres, which form the interzonal fibres seen during the anaphases, and half-spindle fibres, extending only from the poles to the chromosomes. It is possible that these two kinds of fibres, though having the same origin, respectively corre- 1 Cf. Platner ('86) on Avion and Lepidoptera, Watase ('91) on Loligo, Braus ('95) on Triton, and Griffin ('96, '99) on Thalassema. Erlanger ('97, 5) endeavours to show that in the mitosis of embryonic cells in the cephalopods (^Scpia'), where the inpushing of the mem- brane was previously shown by Watase, the entire spindle arises from the nucleus. G 82 CELL-DIVISION spond in function to those of the central spindle and to the mantle- fibres. It seems probable that the difference between the two types of spindle-formation may be due to, or is correlated with, the fact that the nuclear transformation takes place relatively earlier in the "first type. When the nucleus lags behind the spindle-formation the centrosomes take up their position prematurely, as it w^ere, the cen- tral spindle disappearing to make w^ay for the nucleus. It is in the mitosis of plant-cells that the most remarkable type of achromatic figure has been observed. In some of the lower forms (Alga?) mitosis has been clearly shown to conform nearly to the process observed in animal cells, the amphiaster being provided with very large asters and distinct centrosomes, and its genesis corre- sponding broadly with the second type described above (Figs. 32, 33), though with some interesting modifications of detail.^ Swingle ('97) describes in Stytopocauloii a process closely similar to that seen in many animal cells, the minute but very distinct centrosomes being surrounded by quite typical cytoplasmic asters, passing to opposite poles of the nucleus, and a spindle then developing between them out of the achromatic nuclear substance (Fig. 32). In the flowering plants and pteridophytes, on the other hand, mitosis seems to be of a quite different type, apparently taking place in tJie entire absence of centrosomes. Guignard ('91, i, '92, 2) clearly described and figured typical centrosomes and attraction-spheres both in the ordinary mitosis (Fig. 34) and in the fertilization of the higher plants, giving an account of their behaviour nearly agreeing with the views then prevaiUng among zoologists. Although these accounts have been supported by some other workers,^ and have recently been in part reiterated by Guignard himself ('98, i), they have not been sustained by some of the best and most careful later observers, who describe a mode of spindle-formation differing radically from that seen in thal- lophytes and in animals generally.-^ According to these observations, begun by Farmer and Belajcff, and strongly sustained by the care- ful studies of Osterhout, Mottier, Nemec, and others, the achromatic figure is almost wholly of cytoplasmic origin, arising from a fibrillar material (" kinoplasm " or " filar plasm," of Strasburger), which at the beginning of mitosis forms a net-like mass surrounding the nucleus, from which fibrilloe radiate out into the cytoplasm. As the nuclear membrane fades, these fibrillae, continually increasing, invade the nuclear area, gather themselves into bundles, converging to a number 1 See especially Swingle ('97) on Sphacelariacea, Strasburger ('97) on Fucus, Mottier ('98) on Dictyota ; cf. also Harper ('97) on Erysiphe and Peziza. 2 Cf. Schaffner ('98), Fulmer ('98). ^ See Osterhout ('97) on Eqidsettim, Mottier ('97, I, '97, 2) on Lilium, Lawson ('98) on Cobcca, Nemec ('99) on Allium, Debski ('97, '99) on Chara ; also Belajeff ('94) and Farmer ('95). DETAILS OF MITOSIS 83 B of centres (without centrosomes), and thus give rise to an irregular multipolar figure (Figs. 36, 133). This figure finally resolves itself into a definite bipolar spindle which is devoid of centrosomes, and in the earlier stages also of asters, though in the later phases some- what irregular asters are formed. On the basis of these observations Mottier^ proposes to distinguish provisionally two well-defined types of mitosis in plants which he designates as the ''thallophyte " and the "cormophyte" types. The latter seems wholly irreconcilable with the process observed in animal-cells ; for the whole course of spindle- formation seems diametrically opposed in the two cases, and should the cormophyte-type be established it would, to say the least, greatly restrict the application of the centrosome-theory of Van Beneden and Boveri. Only future re- search can definitely de- termine the question. There can be no doubt that the descriptions of Guignard and his follow- ers do not rest upon pure imagination ; for it is easy to observe at the spindle- poles in some prepara- tions {e.g. sections of root- tips of AlliuDi, Liliuin, etc.) deeply staining- bodies such as these authors describe. These ''centrosomes" seem, however, to be of quite inconstant occurrence ; and the careful studies of Osterhout, Mottier, and Nemec seem to give good ground for the conclusion that they have no such significance as the centrosomes of lower plants or of animals. It should nevertheless be borne in mind that true centrosomes (*' blepharoplasts ") have been demonstrated in the spermatogenic divisions of some of the vascular cryptogams, and that analogous bodies occur in the corresponding divisions of the cycads (p. 175). We should therefore still hold open the possibility that centrosomes may occur in the vegetative mitoses of the higher plants, their apparent absence being possibly due to lack of staining- capacity or similar conditions rendering their demonstration difificult.- ^ ■■■. - Fig. 33. — Mitosis in ascus-nuclei of a fungus, Erysiphe. [Harper.] . A. Resting nucleus with disc-shaped centrosome [c^. B. Early prophase with aster. C. Later prophase; amphi- aster; intranuclear spindle forming. D. Spindle estab- lished. E. Daughter-nucleus after division ; spore-mem- brane developing from astral rays. 1 ' 97, 2, p. 183. . 2 Mention may here be made of the barrel-shaped truncated spindles described m some of the plants. In Basidiobolus, Fairchild ('97) finds spindles of this type, having no asters 84 CELL-DIVISIOX A no less remarkable mode of spindle-formation, which is in a cer- tain way intermediate between the cormophyte-type and the usual animal type is described by Mead ('97, '98, i) in the first maturation- division of CJuctoptcnis. Here the completed am]:)hiaster is of quite typical form, and the centrosomes persist for the followin^^ mitosis; yet Mead is convinced that the amphiaster is synthetically formed by the union of two separate asters and centrosomes (Fig. 150) which B Fig- 34- — Division of pollen-mother-cells in the lily as described by Gl'IGNARD. A. An.ii)hase of the first division, showing the twelve daughter-chromosomes on eacli side, the interzonal fibres stretching between them, and the centrosomes, already double, at the spindle- poles. D. Later stage, showing the cell-plate at the equator of the spindle and the daughter- spiremes (dispireme-stage of Flemming). C. Division completed; double centrosomes in the resting cell. D. Ensuing division in progress; the upper cell at the close of the proj^hases, the chromosomes and centrosomes still undivided; lower cell in the late anaphase, cell-plate not yet formed. have no genetic connection, arising independently dc novo in the cytoplasm.^ Improbable as such a conclusion may seem on a priori grounds, it is supported by very strong evidence,^ and, taken together and nearly parallel fibres, each of which terminates in a deeply staining granule. Nearly similar spindles have been described by Strasburger ('So) in Spirogyra, and in the embryo- sac o{ Monoiropa. It is not impossible that such spindles may represent a type intermediate between the "cormoptyte " and " thallophyte " types of Mottier. 1 C/: p. 306. 2 I have had the privilege of examining some of Mead's beautiful preparations. DETAILS OF MITOSIS 85 with the facts described in plants, it indicates that the forces involved in sjDindle-formation are far more complex than Van Ik'neden's and Boveri's hypothesis would lead one to suppose.^ The centrosome and centrosphere appear to present great varia- tions that have not yet been thoroughly cleared up and will be more critically discussed beyond. ^ They are known to underg(j extensive changes in the cycle of cell-division and to vary greatly in different forms (Fig. 152). In some cases the aster contains at its centre nothing more than a minute deeply staining granule, which doubtless A ^^g- 36- — Division of spore-mother-cells in Eqiiisetum, sliowing spindle-formation. [OsTERHOfT.] A. Early prophase, " kinoplasmic " fibrillae in the cytoplasm. B. Multipolar fibrillar figure invad- ing the nuclear area, after disappearance of the nuclear membrane. C. Multipolar spindle. D. Quadripolar spindle which finally condenses into a bipolar one. represents the centrosome alone. In other cases the granule is sur- rounded by a larger body, which in turn lies within the centrosphere or attraction-sphere. In still other cases the centre of the aster is occupied by a large reticular mass, within which no smaller body can be distinguished {e.g. in pigment-cells); this mass is sometimes called the centrosome, sometimes the centrosphere. Sometimes, again, the spindle-fibres are not focus.sed at a single point, and the spindle ^ See p. 276 for the peculiar spindles, devoid of asters, observed during the maturation of the egg in certain forms. Cf. also Morgan's experiments on the artificial production of asters and centrosomes, p. 307. - See p. 304. 35 CELL-DIVISIOX appears truncated at the ends, its fibres terminating in a transverse row of granules (maturation-spindles of Ascaris, and some plant-cells). It is not entirely certain, however, that such spindles observed in ]:)reparations represent the normal structure during life. b. Tlic CJinwiatic Fii^mr. — The variations ot the chromatic figure must for the most part be considered in the more special parts of this work. There seems to be no doubt that a single continuous spireme-thread may be formed {rf. p. 113). ^^'^it it is equally certain that the thread may appear from the beginning in a number of distinct segments, i.e. as a segmented spireme, and there are some cases in which no distinct spireme can be seen, the reticulum resolving itself directly into the chromosomes. The chromosomes, when fully formed, vary greatly in appear- ance. In many of the tissues of adult plants and animals they are rod-shaped and are often bent in the middle like a V (Figs. 28, 131 ). Thev often have this form, too, in embryonic cells, as in the segmentation-stages of the Qgg in Ascaris (Fig. 31) and other forms. The rods may, however, be short and straight (seg- menting eggs of echinoderms, etc), and may be reduced to spheres, as in the maturation-stages of the germ-cells. In the equatorial plate the V-shaped chromosomes are placed with the apex of the V turned toward the spindle (Fig. 28), while the straight rods are placed with one end toward the spindle. In either case the daughter- chromosomes first begin to move apart at the point nearest the spindle, the separation proceeding thence toward the free portion. The V-shaped chromosomes, opening apart from the apex, thus give rise in the early anaphase to <>-shaped figures; while rod-shaped chromosomes often produce A- ^'I'ld i-shaped figures (the stem of the 1 being double). The latter, opening farther apart, form straight rods twice the length of the original chromosome (since each consists of two daughter-chromosomes joined at one end). This rod finally breaks across the middle, thus giving the deceptive appearance of a transverse instead of a longitudinal division (Fig. 52). The <>- shaped figures referred to above are nearly related to those that occur in the so-called Jictcrotypical mitosis. Under this ^name Flem- ming ('87) first described a peculiar modification of the division of the chromosomes that has since been shown to be of very great impor- tance in the early history of the germ-cells, though it is not confined to them. In this form the chromosomes split at an early period, but the halves remain united by their ends. Each double chromosome then opens out to form a closed ring (Fig. 37), which by its mode of origin is shown to represent two daughter-chromosomes, each forming half of the ring, united by their ends. The ring finally breaks in two to form two U-shaped chromosomes which diverge to opposite poles DETAILS OF MITOSIS s? of the spindle as usual. As will be shown in Chapter V.,the divisions by which the germ-cells are matured are in many cases of this type ; but the primary rings here in many cases represent not two but four chromosomes, into which they afterward break up. Fig- 37- ~ Heterotypical mitosis in spermatocytes of the salamander. [Flemminc] A. Prophase, chromosomes in the form of scattered rings, each of which represents lui. daughter-chromosomes joined end to end. B. The rings ranged about the equator of the spindle and dividing ; the swellings indicate the ends of the chromosomes. C. The same viewed from the spindle-pole. D.. Diagram (Hermann) showing the central spindle, asters, and centrosomes,and the contractile mantle-fibres attached to the rings (one of the latter dividing). 2. Bivalent and Plurivalent Chroniosomes The last paragraph leads to the consideration of certain varia- tions in the number of the chromosomes. Boveri discovered that the species Ascaris mcgaloccpJiala comprises two varieties which differ in no visible respect save in the number of chromosomes, the germ-nuclei of one form (" variety bivalens " of Hertwig) having two chromosomes, g3 CELL-DIVISIOX while in the other form (" variety iinivalens ") there is but one. Braiier discovered a similar fact in the phyllopod Artcmia, the number of somatic chromosomes being i68 in some individuals, in others only 84 (p. 281). It will appear hereafter that in some cases the primordial germ- cells show only half the usual number of chromosomes, and in Cyclops the same is true, according to Hacker, of all the cells of the early cleavage-stages. In all cases where the number of chromosomes is apparently reduced ("pseudo-reduction" of Riickert) it is highly probable that each chromatin-rod represents not one but two or more chromosomes united together, and Hacker has accordingly proposed the terms bivalent and plnrivaloit for such chromatin-rods.^ • The truth of this view, which originated with Vom Rath, is, I think, conclusively shown by the case of Artcniia described at page 281, and by many facts in the maturation of the germ-cells hereafter considered. In Ascaris we may regard the chromosomes of Hertwig's "variety univalens " as really bivalent or double, i.e. equivalent to two such chromosomes as appear in " variety bivalens." These latter, however, are probably in their turn plurivalent, i.e. represent a number of units of a lower order united together; for, as described at page 148, each of these normally breaks up in the somatic cells into a large number of shorter chromosomes closely similar to those of the related species Ascaris liunbricoides, where the normal number is 24. Hacker has called attention to the striking fact that plurivalent mitosis is very often of the heterotypical form, as is very common in the maturation-mitoses of many animals (Chapter V.), and often occurs in the early cleavages of Ascaris ; but it is doubtful whether this is a universal rule. 3. Mitosis in the Unicellular Plants and Animals The process of mitosis in the one-celled plants and animals has a peculiar interest, for it is here that we must look for indications of its historical origin. But although traces of mitotic division were seen in the Infusoria by Balbiani ('58-61), Stein ('59), and others long before it was known in the higher forms, it has only recently received adequate attention and is still imperfectly understood. Mitotic division has now been observed in many of the main divi- sions of Protozoa and unicellular plants ; but in the present state of 1 The words bivalent and uui-'alcni have been used in precisely the opposite sense by Hertwig in the case of Ascaris. the former term being applied to that variety having two chromosomes in the germ-cells, the latter to the variety with one. These terms certainly have priority, but were applied only to a specific case. Hacker's use of the words, which is strictly in accordance with their etymology, is too valuable for general descriptive purposes to be rejected. DETAILS OF MITOSIS 89 the subject it must be left an open question whether it occurs in all. In some of the gregarines and Heliozoa, the process is of nearly or quite the same type as in the Metazoa. From such mitoses, how- ever, various gradations may be traced toward a much simpler pro- cess, such as occurs in Aniceba and the lower flagellates ; and it is not improbable that we have here representatives of more primitive con- ditions. Among the more interesting of these modifications may be mentioned : — I. Even in forms that nearly approach the mitosis of higher types B D Fig. 38. — Mitotic division in Infusoria, [k. Hkr rwiG.] A-C. jSIacronucleus of Spirochona, showing pole-plates. D-H. Successive stagres in the division of the micronucleus of Paravicecium. D. The earliest stage, showing reticulum. G. Fol- lowing stage (" sickle-form ") with nucleolus. E. Chromosomes and pole-plates. /•: Late ana- phase. H. Final phase. the nuclear membrane may persist more or less completely through every stage {Noctiliica, EuglypJia, ActinospJicerium). 2. Asters maybe present (Heliozoa, gregarines) or wanting (In- fusoria, Radiolaria). 3. In one series of forms the centrosome or sphere is represented by a persistent intranuclear body (nucleolo-centrosome) of consider- able size, which divides to form a kind of central spindle { Huo^/ina Aniocbuy Infusoria.''). 4. In a second series the centrosome or sphere is a persistent 90 CELL-DIVISION cxtranuclear body, as in most Metazoa {Heliozoa, Noctilucay Para- moeba ). 5. In a few forms havin<; a scattered nucleus the chromatin-gran- ules are only collected about the apparently j)ersistent sphere or centrosome at the time of its division, and afterward scatter throuirh the cell, leaving the sphere lying in the general cell-substance ( Tctrainitiis). 6. The arrangement of the chromatin-granules to form chromo- somes appears to be of a secondary importance as compared with A BCD Fig- 39- — Mitosis in the rhizopod, Euglyplia. [SCHEWIAKOFF.] In this form the body is surrounded by a firm shell which prevents direct constriction of the cell-body. The latter therefore divides by a process of budding from the opening of the shell (the initial phase shown at .-/) ; the nucleus meanwhile divides, and one of the daughter-nuclei aftenvard wanders out into the bud. A. Early prophase; nucleus near lower end containing a nucleolus and numerous chromo- somes. D. lu]u.-itorial plate and spindle formed insirle the nucleus; pole-bodies or pole-plates (i.e. attraction-spheres or centrosomes) at the spindle-poles. C. Metaphase. D. Late ana- phase, spindle dividing; after division of the spindle the outer nucleus wanders out into the bud. higher forms, and the essential feature in nuclear division a])pears to be the fission of the individual crranules. We may first consider especially the achromatic figure. The basis of our knowledge in this field was laid by Richard Hertwig through his studies on an infusorian, SpirocJioiia { ^yy), and a rhizo})od, Actiiio- spJicenum{'?i/\). In both these forms a typical spindle and equatorial plate are formed inside tJie nuclear membrane by a direct transfor- mation of the nuclear substance. In SpirocJiona (Fig. 38, A-C) a DETAILS OF MITOSIS 91 hemispherical ''end-plate" or *' pole-plate " is situated at either pole of the spindle, and Hertwig's observations indicated, though they did not prove, that these plates arose by the division of a large "nucleolus." Nearly similar pole-plates were somewhat described by Schewiakoff ('88) in Eiiglypha (Fig. 39), and it seems clear that they are the analogues of the centrosomes or attraction-spheres in higher forms. In Euglcna, as shown by Keuten, the pole-plates, or their analoo-ues, certainly arise by division of a distinct and persistent intra- nuclear body ("nucleolus" or " nucleolo-centrosome ") which elon- Fig. 40. — Mitosis in the flagellate, Eugleua. [KEUTEN.] A Preparing for division ; the nucleus contains a " nucleolus " or nucleolo-centrosome sur- rounded Ty a g-up of chromosomes. B. Division of the ;• nucleolus to form an mtranuclear spindle. C. Later stage. D. The nuclear division completed. gates to form a kind of central spindle around which the chromatin elements are grouped (Fig. 40); and Schaudinn ("95) described a similar process in Am(^ba. Richard Hertwig's latest work_ on Infusoria ('95) indicates that a similar process occurs m the micro- nuclei of Paramcccinm, which at first contain a large " nucleolus and afterward a conspicuous pole-plate at either end ot the spindle (Fio- :;8 D-H\ TJie origin of the pole-plates was not, however, po^tively determined. A corresponding dividing body is foupd m Ccratiinn (Lauterborn, '95), and as in the Infusoria the entire nucleus transforms itself into a fibrillar spindle-like body. 92 CELL-DIVISION Still simpler conditions are found in some of the flagellates.^ In Chilonionas the sphere may still be regarded as intranuclear, since it lies in the middle of an irregular mass of chromatin-granules, though the latter are apparently not enclosed by a membrane. Nuclear division is here accomplished by fission of the sphere and the aggre- gation of the chromatin-granules around the two products. In Tctramitus, finally (Fig. i6), the nucleus is represented by chromatin- granules that are scattered irregularly through the cell and only at the time of division collect about the dividing sphere. t^ CvOl, , <^&< « r Vc. *-*'<■* B C —A ♦I''- , O ^i:^^' --^ F Fig. 41. — Mitosis in the Heliozoa. [SCHAUDINN.] A. Sp/ifrrastruiN : vegetative cell showing nucleus, "central gmnule" (centrosonic), and axial rays. li-G. Acanthocystis. B-D. Prophases of mitosis. E. Budding to form swarm-spores. F. Swarm-spores, devoid of centrosomes. G. Swarm-spores preparing for division ; intranuclear origin of centrosome. In a second series of forms, represented by Noctiliica (Ishikawa, '94, 98), (Calkins, '98, 2), raranurba (Schaudinn, '96, i), Actinophrys and Acanthoi-ystis (Schaudinn, '96, 2), and the diatoms (Lauterborn, '96), the sphere lies outside the nucleus in the cytoplasm and the mitosis is closely similar to that observed in most Metazoa. This is most striking in the Heliozoa, where the centrosome persists through the vegetative condition of the cell as the *• central granule," to which the axial filaments of the pseudopodia converge. Schaudinn ('96, 2) shows that by the division of this body a typical extranuclear amphi- aster and central spindle are formed (Fig. 41), while the chromatin ^ Calkins, '98, I, '98, 2. DETAILS OF MITOSIS 93 passes through a spireme-stage, breaks into very short rod-shaped chromosomes which spHt lengthwise and arrange themselves in the equator of the spindle, while the nuclear membrane fades away. Noctiluca (Fig. 42), as shown by Ishikawa and Calkins, agrees with this in the main points; but the nuclear membrane does not at any period wholly disappear, and a distinct centrosome is found at the centre of the sphere. The latter body, which is very large, gives ••;.-.r-.vA-r.:.r->; ., -■«■"'.•.>•.. TS\:/.;;.\';::... ■. . .. ■■; ^*!?Xi; ^^V}r;:-\:.-:, D Fig. 42. — Mitosis in Noctiluca. [Calkins.] A. Prophase; division of the sphere to form the central spindle; chromosomes convertjing to the nuclear pole. B. Late anapliase. in horizontal section, showing centrosomes; the crntral spindle has sunk into the nucleus; nuclear membrane still intact except at the polos. C. \-A\\y anaphase; mantle-fibres connected with the diverging chromosomes. D. Kinal anaphase (which is also the initial prophase of the succeeding division of spore-forming mitosis) ; doubling of cen- trosome and splitting of chromosomes. rise by a division to a fibrillated central spindle, about which the nucleus wraps itself while mantle-fibres are developed from the sphere-substance and become attached to the chromosomes, the nu- clear membrane fading away along the surface of contact with the central spindle (Calkins). Broadly speaking, the facts are similar in 94 CELL-DIVISION the diatoms {Surirclla, t. Lauterborn), where the central spindle, arisinf( by a peculiar ]:)rocess from an extranuclear centrosome, (sphere ?) sinks into the nucleus in a manner strongly suggesting that observed in Xoctiluca. In the interesting form raiamaini, as described by Schaudinn ('96, I ), the sphere (*' Nebenkorper "), which is nearly as large as the nucleus, divides to form a central spindle, about the equator of which the chromatin-elements become arranged in a ring (Fig. 43); but no centrosome has yet been demonstrated in the sj^here. J\\rai)uvba appears to differ from Rugloia mainly in the fact that at the close of dixision the sphere is in the former left outside the daughter-nucleus and in the latter enclosed within it.^ The connecting link is perfectly given by Tctramitus, where no morphological nucleus is formed, and the sphere lies in the general cell-substance (p. 92); and we could have no clearer demonstration that the extra- or intranuclear position of sphere or centrosome is of quite secondary importance. As regards the formation of the spheres (pole-plates) ActinospJiceriuin (Figs. 44, 45) seems to show a simpler condition than any of the above forms, since no permanent sphere exists, and Brauer ('94) and R. riertwig ('98) agree that the pole-plates are formed by a gradual accumulation of the achromatic substance of the nucleus at opposite poles. A distinct centrosome (centriole t) in the interior of the sphere has thus far only been observed in a few forms {Noctiluca, ActitiospJice- riu7ii), and neither its origin nor its relation to the sphere has yet been sufficiently cleared up. Both Ishikawa('94) and Calkins ('98, 2) somewhat doubtfully concluded that in Xoctiluca the centrosomes arise within the nucleus, migrating thence out into the extranuclear sphere. With this agree R. Hertwig's latest studies on Actiiiosphce- riiiDi ('98), the spindle-poles being first formed from the pole-plates (themselves of nuclear origin), and the centrosomes then passing into them from the nucleus. HertwMg reaches the further remarkable conclusion that the centrosomes arise as portions of the cJiromatin- iictivork extruded at the nuclear poles (Fig. 45), first forming a spongy irregular mass, but afterward condensing into a deeply staining pair of granules which pass to the respective poles of the spindle. It is a remarkable fact that these centrosomes are only found in the two maturation-divisions, and are absent from the ordi- nary vegetative mitoses where the spindle-poles arc formed by two cytoplasmic masses derived, as Hertwig believes, from the intra- nuclear plates. Schaudinn ('96, 3) likewise describes and clearly figures an intranuclear origin of the centrosome in buds of AcantJio- cystis (Fig. 41), which are derived by direct division of the mother- 1 Cf. Calkins, '98, i, p. 388. DETAILS OF MITOSIS 95 nucleus with no trace of a centrosome. In this same form, as described above, the ordinary vegetative mitoses are quite of the metazoan type, with a persistent extranuclcar centrosome. The history of the chromatin in the mitosis of unicellular forms shows some interesting modifications. In a considerable number of forms a more or less clearly marked spireme-stage precedes the forma- tion of chromosomes (diatoms, Infusoria, dinoflagellates, Iuio/yp/i(i)\ in others, long chromosomes are formed without a distinct spireme- stage (N'octihtca). It has been clearly demonstrated that in some cases these chromosomes split lengthwise, as in Metazoa {A^octilnca, V «c^;> X, -O Fig. 43. — Mitosis in Parafnccba. [SCJIAUDINN.] At the left, amoeboid phase, showing nucleus and " Nebenkorper." At the right, four stages of division in the swarm-spores. diatoms, Actinophrys, probably in EuglypJia) ; but in some cases they are stated to divide transversely in the middle (Infusoria according to Hertwig, Ccratiuin according to Lauterborn). These chromosomes appear always to arise, as in Metazoa, through the linear arrangement of chromatin-granules {^Noctihica, Actinospluvriuni, Euglciia\ wliich themselves in many cases arise by the preliminary fragmentation of one or more large chromatin-masses {^c.g. in Noctiluca or ActiuosplicB- rijun). In other forms no such linear aggregates are formed, and direct fission of the chromatin-granules appears to take place without the formation of bodies morphologically comparable with the chromo- somes of such forms as Noctiluca. This is apparently the case in Tetrajnitus, and Achromatium, other forms having a distributed 96 CELL-DIVISION nucleus,! ^nd in such forms as CJiilomouas and Tmchclomonas, where the <^ranules are permanently aggret^ated about a central body. Too little is known of the facts to justify a very positive statement; but on the whole they point toward the conclusion that in the simplest qqOs, o «.«' .'."• ,12 o 'ooo» ,"oOoo .oo'">% ».."""%„ ^ % \ "imd I ^'oirv^Baio^' oOQOooo Fig. 44. — Mitosis in the rhizoped Actinospluvrium. [Braukr.] A. Nucleus and surrounding structures in the early prophase; above and below the reticular nucleus lie the semilunar " pole-plates." and outside these the cytoplasmic masses in which the asters afterward develop. B. Later stage of the nucleus. D. Mitotic figure in the metaphase, showing equatorial plate, intra-nuclear spindle, and pole-plates {p.p.). C. Equatorial plate, viewed ^////r*", consisting of double chromatin-granules. E. Early anaphase. E.G. Later ana- phases. H. F^inal anaphase. /. Telophase; daughter-nucleus forming, chromatin in loop-shaped threads; outside the nuclear membrane the centrosome, already divided, and the aster. J. 1-ater stage; the daughter-nucleus established; divergence of the centrosomes. Beyond this point the centrosomes have not been followed. types of mitosis no true chromosome-formation occurs, thus sustaininp^ Brauer's conclusion that the es.sential fact in the history of the chro- matin in mitosis is the fission of the individual granules.^ 1 The fission of the individual granules is carefully described and figured by Schewiakoff in Achromatimn. ■ '-^ For speculations on the historical origin of the centrosome, etc., see p. 315. DETAILS OF MITOSIS 97 4. Pathological Mitoses Under certain circumstances the delicate mechanism of cell-division may become deranged, and so give rise to various forms of patho- logical mitoses. Such a miscarriage may be artificially produced, as Hertwig, Galeotti, and others have shown, by treating the dividing cells with poisons and other chemical substances (quinine, chloral, nicotine, potassic iodide, etc.). Pathological mitoses may, however, x: \ B ^^^F C D Fig. 45. — Mitosis in Actinosphcerium. [R. Hertwig.] A. Encysted form, with resting nucleus; chromatin aggregated into large nucleolus-like body. B. prophase of division of the encysted form, showing chromosome-like bodies formed of granules, and spindle without centrosomes. C. Earlier prophase of the first maturation division, showing extrusion of chromatic substance to form the centrosome. D. Later stage, showing centrosome and aster. occur without discoverable external cause ; and it is a very interesting fact, as Klebs, Hansemann, and Galeotti have especially pointed out, that they are of frequent occurrence in abnormal growths such as cancers and tumours. The abnormal forms of mitoses are arranged by Hansemann in two general groups, as follows: (i) asymmetrical mitoses, in which the chromosomes are unequally distributed to the daughter-cells, and (2) multipolar mitoses, in which the number of centrosomes is more than H 98 CELL-DIVISIOX two, and more than one spindle is formed. Under the first group are inckided not only the cases of unequal distribution of the daughter- chromosomes, but also those in which chromosomes fail to be drawn into the equatorial })late and hence are lost in the cytoplasm. Klebs first pointed out the occurrence of asymmetrical mitoses in carcinoma-cells, where they have been carefully studied by Hanse- mann and Galeotti. The inequality is here often extremely marked, so that one of the daughter-cells may receive more than twice as much chromatin as the other (Fig. 46). Hansemann, whose conclu- E F Fig. 46. — Pathological mitoses in human cancer-cells. [Galeotti.] A. Asymmetrical mitosis with unequal centrosomes. B. Later stage, showing unequal distri- bution of the chromosomes. C. Quadripolar mitosis. D. Tripolar mitosis. E. Later stage. F. Trinucleate cell resulting. sions are accepted by Galeotti, believes that this asymmetry of mito- sis gives an explanation of the familiar fact that in cancer-cells many of the nuclei are especially rich in chromatin (hyperchromatic cells), while others are abnormally poor (hypochromatic cells). Lustig and Gale(^tti ('93) showed that the unequal distribution of chromatin is correlated with and probably caused by a corresponding inequality in the centrosomes which causes an asymmetrical development of the amphiaster. A very interesting discovery made by Galeotti ('93) is that asymmetrical mitoses, exactly like those seen in carcinoma, may be artificially produced in the epithelial cells of salamanders (Fig. 47) by treatment with dilute solutions of various drugs (antipyrin, cocaine, quinine). DETAILS OF MITOSIS 99 Normal multipolar mitoses, though rare, sometimes occur, as in the division of the pollen-mother-cells and the endosperm-cells of flower- ing plants (Strasburger) ; but such mitotic figures arise through the union of two or more bipolar amphiasters in a syncytium and are due to a rapid succession of the nuclear divisions unaccompanied by fission of the cell-substance. These are not to be confounded with pathological mitoses arising by premature or abnormal division of the centrosome. If one centrosome divide, while the other does not, triasters are produced, from which may arise three cells or a tri- nucleated cell. If both centrosomes divide, tetrasters or polyasters are formed. Here again the same result has been artificially attained by chemical stimulus {cf. Schottlander, '^S). Multipolar mitoses are ^ B Fig. 47. — Pathological mitoses in epidermal cells of salamander caused by poisons. [Galeotti.] A. Asymmetrical mitosis after treatment with 0.05 % antipyrin solution. B. Tripolar mitosis after treatment with 0.5% potassic iodide solution. also common in regenerating tissues after irritative stimulus (Strobe); but it is uncertain whether such mitoses lead to the formation of normal tissue.^ The frequency of abnormal mitoses in pathological growths is a most suggestive fact, but it is still wholly undetermined whether the abnormal mode of cell-division is the cause of the disea.se or the reverse. The latter seems the more probable alternative, since nor- mal mitosis is certainly the rule in abnormal growths ; and Galeotti's experiments suggest that the pathological mitoses in such growths may be caused by the presence of deleterious chemical products in the diseased tissue, and perhaps point the way to their medical treatment. 1 The remarkable polyasters formed in polyspermia fertilization of the egg are de- scribed at page 198. lOO CELL-DIVISIOX D. The Mfxhamsm of Mitosis We now pass to a consideration of the forces at work iii mitotic division, which leads us into one of the most debatable helds of cytological inquiry. I. Function of the Aviphi- astcr CZ- in. z. v-ac a.c All observers agree that the amphiaster is in some manner an expression of the forces by which cell- division is caused, and many accept, in one form or another, the first view clearly stated by Fol,^ that the asters represent in some manner centres of attractive forces focussed in the centrosome or dv- namic centre of the cell. Regarding the nature of these forces, there is, how- ever, so wide a divergence of opinion as to compel the admission that we have thus far accomplished little more than to clear the ground for a ])recise in- vestigation of the subject ; Fig. 48. — Slightly schematic figures of dividing eggs ^ . ' . o{ Ascaris, illustrating Van Bencdcn's theory of mitosis, and the mccnanism Ot mi- [Van Beneden and juLiN.] tosis Still lies before us as A. Early anaphase; each chromosome has divided qj-j^. ^f ^\^^^ moSt fascinating into two. B. Later anaphase during divergence of the ^ . daughter-chromosomes, a.c. Antipodal cone of astral problems 01 CytOlOgy. rays; f.s. cortical zone of the attraction-sphere ; /. inter- (^(ji\ 'J'Jw TllCOiy of Fl- zonal fibres stretching between the daughter-chromo- u,.;i] ^,. r ...f,- , -tUi t^, HTUo somes; m.z. medullary zone of the attraction-sphere; ^^ '^^^^' Coutl Octlllty . —^\x<. p.c. principal cone, forming one-half of the contractile vicVV that haS taken the spindle (the action of these fibres is reenforced by that of cf-rnno'est hold On rCCCnt tlie antipodal cone) ; s.e.c. subequatorial circle, to which t> ^ _ the astral rays are attached. research IS the hypothesis of fibrillar contractility. First suggested by Klein in 1878, this hypothesis was independ- ently put forward by Van Beneden in 1883, and fully outlined 1 ' 73. P- 473- THE MECHANISM OF MITOSIS 10 1 by him four years later in the following words: *' In our opinion all the internal movements that accompany cell-division have their immediate cause in the contractility of the protoplasmic fibrilla:^ and their arrangement in a kind of radial muscular system, composed of antagonizing groups" {i.e. the asters with their rays). " In this sys- tem the central corpuscle (centrosome) plays the part of an organ of insertion. It is the first of all the various organs of the cells to divide, and its division leads to the grouping of the contractile elements in two systems, each having its own centre. The presence of these two systems brings about cell-division, and actively determines the paths of the secondary chromatic asters" {i.e. the daughter-groups of chro- mosomes) '' in opposite directions. An important part of the phe- nomena of (karyo-) kinesis has its efficient cause, not in the nucleus, but in the protoplasmic body of the cell." ^ This beautiful hypothesis was based on very convincing evidence derived from the study of the Ascaris eofo:, and it was here that Van Beneden first demonstrated the fact, already suspected by Flemming, that the daughter-chromosomes move apart to the poles of the spindle and give rise to the two respective daughter-nuclei.^ Van Beneden's general hypothesis was accepted in the following year by Boveri ('88, 2), who contributed many important additional facts in its support, though neither his observations nor those of later investigators have sustained Van Beneden's account of the grouping of the astral rays. Boveri showed in the clearest manner that, during the fertilization of Ascaris, the astral rays become attached to the chromosomes of the germ-nuclei ; that each comes into connection with rays from both the asters ; that the chromosomes, at first irregu- larly scattered in the ^gg, are drawn into a position of equilibrium in the equator of the spindle by the shortening of these rays (Figs. 90. 147); and that tJie rays thicke^i as tJiey sJiorten. He showed that as the chromosome splits, each half is connected only with rays (spindle- fibres) from the aster on its own side ; and he followed, step by step. the shortening and thickening of these rays as the daughter-chromo- somes diverge. In all these operations the behaviour of the rays is 1 '87, p. 280. 2 '83, p. 544. Van Beneden describes the astral rays, both in Ascaris and in tunicates, as differentiated into several groups. One set, forming the "jirincipal cone." are attached to the chromosomes and form one-half of the spindle, and, l)y the contractions of these lilires, the chromosomes are passively dragged apart. An opposite group, forming the " antipodal cone," extend from the centrosome to the cell-periphery, the base of the cone forming the "polar circle." These rays, opposing the action of the principal cones, not only hold the centrosomes in place, but, by their contractions, drag them apart, and thus cause an actual divergence of the centres. The remaining astral rays are attached to the cell-periphery and are limited by a subequatorial circle (Fig. 48). Later observations indicate, however, that this arrangement of the astral rays is not of general occurrence, and that the rays often do not reach the periphery, but lose themselves in the general reticulum. I02 CELL-DIVISIOy precisely like that of muscle-fibres ; and it is difficult to study Bovcri's beautiful figures and clear descriptions without sharing his conviction that "of the contractility of the tibrilke there can be no doubt." ^ Very convincing evidence in the same direction is afforded by pigment-cells and leucocytes or wandering cells, in both of which there is a very large permanent aster (attraction-sphere) even in the resting cell. The structure of the aster in the leucocyte, where it was first discovered by Flemming in 1891, has been studied very carefully by Heidenhain in the salamander. The astral rays here extend throughout nearly the whole cell (Fig. 49), and are believed _. _ B Fig. 49. — Leucocytes or wandering cells of the salamander. [Heidenhain.] A. Cell with a single nucleus containing a very coarse network of chromatin and two nucleoli (plasmosomes) ; s. permanent aster, its centre occupied by a double centrosome surrounded by an attraction-sphere. i9. Similar cell, with double nucleus; the smaller dark masses in the latter are oxychromatin-granules (linin), the larger masses are basichromatin (chromatin proper). by Heidenhain to represent the contractile elements by means of which the cell changes its form and creeps about. A similar con- clusion was reached by Solger ('91) and Zimmermann ('93, 2) in the case of pigment-cells (chromatophores) in fishes. These cells have, in an extraordinary degree, the power of changing their form and of actively creeping about. Solger and Zimmermann have shown that the pigment-cell contains an enormous aster, whose rays extend in every direction through the pigment-mass, and it is almost impos- sible to doubt that the aster is a contractile apparatus, like a radial muscular system, by means of which the active changes of form are produced (Fig. 50). This interpretation of the aster receives addi- tional support through Schaudinn's ('96, 3) highly interesting dis- 1 '88, 2, p. 99. THE MECHANISM OF MITOSIS 10% covery that the ''central granule " of the Heliozoa is to be identified with the centrosome and plays the same role in mitosis (Fig. 41). In these animals the axial filaments of the radiating pseudopodia con- verge to the central granule during the vegetative state of the cell, thus forming a permanent aster which Schaudinn's observations prove to be directly comparable to that of a leucocyte or of a mitotic figure. There is in this case no doubt of the contractility of the rays, and a B .' W/:i/^ -;-^ - Fig. 50. — Pigment-cells and asters from the epidermis of fishes. [ZiMMERMAN'N.] A. Entire pigment-cell, from Bleimius. The central clear space is the central mass of the aster from which radiate the pigment-granules; two nuclei below. D. Nucleus (//) and aster after ex- traction of the pigment, showing reticulated central mass. C. Two nuclei and aster with rod- shaped central mass, from Sargus. strong, if indirect, argument is thus given in favour of contractility in other forms of asters.^ The contraction-hypothesis is beautifully illustrated by means of a simple and easily constructed model, devised by Heidenhain ('94, '96), which closely simulates some of the phenom- ena of mitosis. In its simplest form the model consists of a circle, marked on a flat surface, to the periphery of which are attached at equal 1 For an interesting discussion and develoj ment of the contraction-hypothesis see Watase, '94. 104 CELL-DIVISION Fig. 51. — Heidenhain's model of mitosis (mainly from Heidenhain). A. Dotted lines show position of the rays upon sever- ing connection between the small rings. B. Position upon insertion of " nuclcu=." C. D. Models with fiexiblc hinged hoops, showing division. intervals a series of rub- ber bands (astral rays). At the other ends these bands are attached to a pair of small rings (cen- trosomes) fastened to- gether. In the position of equilibrium, when the rays are stretched at equal tension, the rays form a symmetrical aster with the centrosomes at the centre of the circle (Fig. 51, A). If the con- nection between the cen- trosomes be severed, they are immediately dragged apart to a new position of equilibrium with the rays grouped in two asters, as in the actual cell (dotted lines in Fig. 51, A). If a round pasteboard box of suitable size (nucleus) be inserted between two of the ravs, it assumes an eccentric position, the cell-axis being formed by a line passing through its centre and that of the \)?i\x of small rings {cf. the epithelial cell, p. 57), and upon division of the aster it takes up a position between the two asters. In a second form of the models the circle is formed of two half rings of flexible steel, joined by hinges ; the diver- gence of the small rings is here accompanied by an elongation and partial constriction of the model THE MECHANISM OF MITOSIS IO5 in the equatorial plane ; and if, finally, the hinge-connection be re- moved, each half of the ring closes to form a complete ring.^ Heidenhain has fully worked out a theory of mitosis based upon the analogy of these pretty models. The astral rays of the cell (*' organic radii") are assumed to be in like manner of equal length and in a state of equal tonic contraction or tension, the centrosome forming the common insertion-point of the rays, and equilibrium of the system being maintained by turgor of the cell. Upon disappear- ance of the nuclear membrane and division of this insertion-point, the tension of the rays causes divergence of the centrosomes and forma- tion of the spindle between them, and by further contraction of the rays both the divergence of the daughter-chromosomes and the division of the cell-body are caused. A new condition of equilibrium is thus established in each daughter-cell until again disturbed by division of the centrosome.2 In some cases (leucocytes) the organic radii are visible at all periods. More commonly they are lost to view by breaking up into the cell-reticulum, without, however, losing their essential relations. No one who witnesses the operation of Heidenhain's models can fail to be impressed with its striking simulation of actual cell-division. Closer study of the facts shows, however, that the contraction-hypothe- sis must be considerably restricted, as has been done by the successive modifications of Hermann ('91), Drijner('95), and others. Hermann, to whom the identiiication of the central spindle is due, pointed out that there is no evidence of contractility in the central spindle-fibres, which elongate instead of shorten during mitosis ; and he concluded that these fibres are non-contractile supporting elements, which form a basis on which the movements of the chromosomes take place. The ina7itle-fibres are the only contractile elements in the spindle, and it is by them that the chromosomes are brought into position about the central spindle and the daughter-chromosomes are dragged apart.^ Driiner ('95) still further restricts the hypothesis, maintaining that the progressive divergence of the spindle-poles is caused not by contrac- tion of the astral rays (''polar fibres"), as assumed by Heidenhain (following Van Beneden and Boveri), but by an active growth or elongation of the central spindle, which goes on throughout the whole period from the earliest prophases until the close of the ana- phases. This view is supported by the fact that the central spindle- 1 In a modification of the apparatus devised by Rhumbler ('97), the same effect is pro- duced without the hinges. 2 (7: p. 57. p^or critique of this hypothesis, see Fick ('97), Rhumbler ('96, '97), and Meves ('97, 4). 3 Belajeff ('94) and Strasburger ('95) have accepted a similar view as applied to mitosis in plant-cells. I06 CELL-DIVISIOX fibres are always contorted during the metaphases, as if pushing against a resistance ; and it harmonizes with the facts observed in the mitoses of infusorian nuclei, where no asters are present. This view has been accepted, with slight modifications, by Flemming, Boveri, Meves, Kostanecki, and also by Heidenhain. A nearly decisive argument in its favour is given by such cases as the polar bodies, or the mitosis of salamander spermatocytes as described by Meves ('96, '97, 3), where the spindle-poles are pushed out to the periphery of the cell, the polar astral rays meanwhile nearly or quite disappearing (Fig. 130). This not only strongly indicates the push of the central spindle, but also shows that the assumption of a pull by the polar rays is superfluous. But beyond this both Driincr and Meves have brought arguments against contractility in the other astral rays, endeavouring to show^ that these, like the spindle-fibres, are actively elongating elements, and that (Meves, '97, 3) the actual grouping of the rays during the anaphases is such as to suggest that even the division of the cell-body may be thus caused. A pushing function of the astral rays is also indicated by infolding of the nuclear membrane caused by the development of the aster as described by Platner, Watase, Braus, Griffin, and others.^ The contraction-hypothe- sis is thus restricted by Driiner and Meves to the mantle-fibres alone, though many others, among them Flemming and Kostanecki, still accept the contractility of the astral rays. {b) OtJicr Facts and TJicorics. — Even in the restricted form indi- cated above the contraction-hypothesis encounters serious difficulties, one of which is the fact urged by me in an earlier paper ('95), and subsequently by Richard Hertwig ('98), that in the eggs of echino- derms and many other dividing cells the daughter-chromosome plates, extending through the whole substance of the spindle, wander to the extreme ends of the spindle — a process which demands a contraction of the fibres almost to the vanishing point, while in point of fact not even a shortening and thickening of the fibres can be seen (Fig. 52). Moreover, in these cases, no distinction can be seen between central spindle-fibres and mantle- fibres, and we can only save the contraction-hypothesis by the improbable assumption that fibres indistinguishably mingled, and having the same mode of origin, structure, and staining-reaction, have exactly opposite functions. The inadequacy of the general theory is sufficiently apparent from the fact that in amitosis cells many 1 QC p. 68. It should be pointed out that the originator of the pushing theory was Watase ('93), who ingeniously developed an hypothesis exactly the opposite of Van Bene- den's, assuming both astral rays and spindle-fibres to be actively elongating fibres, dove-tailing in the spindle-region, and pushing the chromosomes apart. This hypothesis is, I believe, in- consistent with the phenomena observed in multiple asters and elsewhere, yet it probably contains a nucleus of truth that forms the basis of Driiner's conception of the central spindle. THE MECHANISM OF MITOSIS 107 divide without any amphiaster whatever. In Infusoria mitosis seems to occur in the entire absence of asters, althou^^h the cells divide by constriction, and the analogy with Heidenhain's model entirely fails. ^vX V ■^> x \ / / -/ ^ o c oo /V'-'^ /'^ X "lr\:\\ Z)' IW Mil''". '' '^ 1 1 \^^- / \ E Fig. 52. — The later stages of mitosis in the G.gg of the sea-urchin Toxopneustcs {.4-D, X 1000; £-F,X5oo). A. Metaphase; daughter-chronaosomes drawing apart but still united at one end. B. Daugh- ter-chromosomes separating. C. Late anaphase ; daughter-chromosomes lying near the spindle- poles. D. Final anaphase; daughter chromosomes converted into vesicles. E. Immediately after division, the asters undivided ; the spindle has disappeared. F. Resting 2-cell stage, the asters divided into tvvo in anticipation of the next division. In Figs. A and /? the centrosome consists of a mass of intensely staining granules, which in Cand D elongates at right angles to the spindle-axis. In /-'the centrosome appears as a single or double granule, which in later stages gives rise to a pluricorpuscular centrum like that in .-/. The connection between D and F is not definitely determined. In Eiiglypha, according to Schewiakoff (Fig. 39), division of the cell- body appears to take place quite independently of the mitotic figure. Again, a considerable number of cases are now known in which dur- ing the fertilization of the ^gg a large amphiaster is formed, with 1 08 CELL-DI J 'I SI ON astral rays sometimes extending throughout almost the entire Qgg^ only to disappear or become greatly reduced without the occurrence of division, the ensuing cleavage being effected by a new am})hiaster or by the recrudescence of the old.^ For these and other reasons we must admit the probability that contractility of the astral fibrillx', if it exists, is but the expression or consequence of a more deeply lying phenomena of more general significance. The subtlety of the prob- lem is strikingly shown by Boveri's remarkable observations on abnormal sea-urchin eggs ('96), which show (i) that the periodic division of the centrosome and formation of the amphiaster may take place independently of the nucleus; (2) that the spindle, as well as the asters, is concerned in division of the cell-body ; and (3) that an amphiaster without chromosomes is unable to effect normal division of the cell-body. The first and third of these facts are shown by eggs in which during the first cleavage all of the chromatin passes to one pole of the spindle, so that one of the resulting halves of the tgg receives no nucleus, but only a centrosome and aster. In this half perfect amphiasters are formed simultaneously with each cleavage in the other half, yit no division of the protoplasmic mass occurs? The second fact is shown in polyspermic eggs, in which multipolar astral svstems are formed by union of the several sperm-asters (Figs. 53, loi ). In such eggs cleavages only occur between asters that are joined by a spindle. Normal cleavage of the cell-body thus requires the complete apparatus of mitosis, and even though the fibres be contractile they cannot fully operate in the absence of chromatin. We may now turn to theories based on the hypothesis, first sug- gested by Fol in 1873, that the astral foci {i.e. centrosomes) represent dynamic centres of attractive or other forces. It should be noted that this hy])othesis involves two distinct questions, one relating to the origin of the amphiaster, the other to its mode of action ; and we have seen that some of the foremost advocates of the contraction-hypothesis, including Van Beneden and Boveri, have held the centrosomes to be attractive centres. Apart from the movements of the chromosomes, the most obvious indication that the centrosomes are dynamic centres is the extraordinary resemblance of the amphiaster to the lines of force in a magnetic field as shown by the arrangement of iron-filings about the poles of a horseshoe magnet — a resemblance pointed out by Fol himself, and urged by many later writers,'^ especially Ziegler ('95) ir/:p. 213. ' This result is opposed to Boveri's earlier work on Ascaris (p. 355), and is modified by Ziegler ('98), who observed in a single case that an irregular cleavage occurred in the enucleated half after two or three divisions of the centrosome. On the other hand, it is sup- ported by Morgan's convincing experiments on the eggs oi Arbacia (p. 308). 2 Cf. the interesting photographic figures of Ziegler ('95). A still closer simulacrum of the amphiaster is produced by fine crystals of sulphate of quinine (a semiconductor) sus- THE MECHANISM OF MITOSIS 109 and Gallardo ('96, '97). It is impossible to regard this analogy as exact ; first, because it is inconsistent with the occurrence of tripolar astral figures ; second, as Meves has recently urged ^ the course of the astral fibres does not really coincide with the lines of force, the most important deviation being the crossing of the rays opposite the equa- torial region of the spindle, which is impossible in the magnetic or electric field. We must, however, remember that the amphiastcr is formed in a viscid medium, that it may perform various movements, and that its fibres probably possess the power of active growth. The C B D F Fig- 53- — Division of dispermic eggs in sea-urchin eggs, schematic. [Boveri.] A. C. E. Eggs before division, showing various connections of the asters. B. D. F. Result- ing division in the three respective cases, showing cleavage only between centres connected by a spindle. physical or chemical effect of the centres, through which the amphias- tcr primarily arises, may thus be variously disturbed (^r modified in later stages, and the crossing of the rays is therefore not necessarily fatal to the assumption of dynamic centres. Biitschli ('92, '98) has, moreover, recently shown that a close sijuii/acntin of the amphiastcr, showing a distinct crossing of the rays, may be produced in an arti- ficial alveolar structure (coagulated gelatine) by tractive forces ccn- pended in spirits of turpentine (a poor conductor) between two electric poles. This experi- ment, devised by Faraday, has recently been applied by Gallardo ('96, '97) to an analysis of the mitotic figure. ^ '96, p. 371. I lo CELL-DIVISION tring in two adjacent points. This result is obtained by warming and then cooling a film of thick gelatine-solution, filled with air-bubbles, and then coagulating the mass in chromic acid. Such a film shows a fine alveolar structure, which assumes a radial arrangement about the air-bubbles, owing to the traction exerted on the surrounding structure by shrinkage of the bubbles on cooling. The amphiastral simjilacra are produced about two adjacent bubbles, — a " spindle " being formed between them, and the " astral rays " sometimes showing a crossing like that seen in the actual amphiaster (Biitschli is himself unable to explain fully how the crossing arises). The protoplasmic asters are maintained by Biitschli to be, in like manner, no more than a radial configuration of the alveolar cell-substance caused by centripetal diffusion-currents toward the astral centres.^ The most interesting part of this view is the assumption that these currents are caused by specific chemical cJianges taking place in the centrosome which causes an absorption of liquid from the surrounding region. ** The astral bodies are structures which, under certain circumstances, function in a measure as centres from which emanate chemical actions upon pro- toplasm and nucleus ; and the astral phenomena which appear about the centrosomes are only a result incidental to this action of the central bodies upon the plasma." ^ Through centripetal currents thus caused arise the asters, and they may even account, in a measure, for the move- ments of the chromosomes.^ This latter part of BiitschU's conception is, I believe, quite inadequate ; but the hypothesis of definite chemical activity in the centrosome is a highly important one, which is sustained by the staining-reactions of the centrosome and by its definite morpho- logical changes during the cycle of cell-division. More or less similar chemical hypotheses have been suggested by several other writers.^ Of these perhaps the most interesting is Strasburger's suggestion,^ that the movements of the chromosomes may be of a chemotactic character, which I suspect may prove to have been one of the most fruitful contributions to the subject. Beside this may be placed Carnoy's still earlier hypothesis ('85), that the asters are formed under the influence of specific ferments emanating from the poles of the nucleus. Mathews ('99, 2) has recently pointed out that there is a considerable analogy between the formation of the astral rays and that of fibrin-fibrils under the infiuence of fibrin-fer- ment, adding the suggestion that the centrosome may actually contain 1 Carnoy ('S5) and Plainer ('86) had previously held a similar view, suggesting that not only the spindle-formation, but also the movements of the chromosomes, might be explained as the result of protoplasmic currents. 2 '92. I, p. 538. 8 '92, 2, p. 160; '92,3, p. 10. * Cf. the first edition of this work, p. 77, also Ziegler ('95). ®'93» 2. THE MECHANISM OF MITOSIS I i j fibrin-ferment. Attention may be called here to the fact, now definitely determined by experiment,^ that cell-division may be incited by chemi- cal stimulus. In most of the cases thus far experimentally examined the divisions so caused are pathological in character, but in others they are quite normal, as shown in Loeb's remarkable results on the production of parthenogenesis in sea-urchin eggs by chemical stimulus as described at pages 215 and 308. While these experiments by no means show that division is itself merely a chemical process, thev strongly suggest that it cannot be adequately analyzed without reckon- ing with the chemical changes involved m it. Resume. A review of the foregoing facts and theories shows how far we still are from any real understanding of the process involved either in the origin or in the mode of action of the mitotic figure. The evidence seems well-nigh demonstrative, in case of the mantle-fibres and the astral rays, that Van Beneden's hypothesis contains an element of truth, but we must now recognize that it was formulated in too simple a form for the solution of so complex a problem. No satisfac- tory hypothesis can, I believe, be reached that does not reckon with the chemical changes occurring at the spindle-poles and in the nucleus ; and these changes are probably concerned not only with the origin of the amphiaster, but also with the movements of the chromosomes. In cases where the centrosome persists from cell to cell we may perhaps regard it as the vehicle of specific substances (ferments ?) which become active at the onset of mitosis, and run through a definite cycle of changes, to initiate a like cycle in the following generation ; and it is quite conceivable that such substances may persist at the nuclear poles, or may be re-formed there as an after-effect, even though the formed centrosome disappears.^ In this consideration we may find a clue to the strange fact — should it indeed prove to be a fact — that the cen- trosome may divide, yet afterward disappear without discoverable connection with the centrosomes of the succeeding mitosis, as several recent observers have maintained. ^ When all is said, we must admit that the mechanism of mitosis in every phase still awaits adequate physiological analysis. The suggestive experiments of Biitschli and Heidenhain lead us to hope that a partial solution of the problem may be reached along the lines of physical and chemical experiment. At present we can only admit that none of the conclusions thus far reached, whether by observation or by experiment, are more than the first 7iazz'e attempts to analyze a group of most complex phenomena of which we have little real understanding. 1 See pp. 306, 308. 2 QT p. 215. SQT p. 213. I 12 CELL-DIVISION 2. Division of tJie CJiroviosovics In de\'eloping his theory of fibrillar contractility, Van Benedcn expressed the view — only, however, as a possibility — that the splitting of the chromosomes might be passively caused by the con- tractions of the two sets of opposing s{)indle-fibres to which each is attached.^ Later observations have demonstrated that this sugges- tion cannot be sustained ; for in many cases the chromatin-thread splits before division of the centrosome and the formation of the achromatic figure — sometimes during the spireme-stage, or even in the reticulum, while the nuclear membrane is still intact. Boveri showed this to be the case in Ascaris, and a similar fact has been observed by many observers since, both in plants and in animals. Fig. 54. — Nuclei in the spireme-stage. A. From the endosperm of the lily, showing true nucleoli. [FLEMMING.] B. Spermatocyte of salamander. Segmented double spireme-thread composed of chromo- meres and completely split. Two centrosomes and central spindle at s. [Hkrmann.] C. Spireme-thread completely split, with six nucleoli. Endosperm of Fritillaria. [FLEM- MING.] The splitting of the chromosomes is therefore, in Bovcri's words, " an independent vital manifestation, an act of reproduction on the part of tJic chromosomes'''^ All of the recent researches in this field point to the conclusion that this act of division must be -referred to the fission of the chromatin-granules or chromomeres of which the chromatin-thread is built. These granules were first clearly described by Balbiani ('76) in the chromatin-network of epithelial cells in the insect- ovary, and he found that the spireme-thread arose by the linear arrangement of these granules in a single row like a chain of bacte- ria."^ Six years later Pfitzner ('82) added the interesting discovery 1 '87, p. 279. 2 '88, p. 113. 8 See '81, p. 638. THE MECHANISM OF MITOSIS 113 that during the mitosis of various tissue-cells of the salamander, the granules of the spireme-thread divide by fission and thus determine the longitudinal splitting of the entire cJironiosoine. This discovery was confirmed by Flemming in the following year ('82, p. 219), and a simi- lar result has been reached by many other observers (Fig. 54). The division of the chromatin-granules may take place at a very early period. Flemming observed as long ago as 1881 that the chromatin- B ^^\ ^\m Fig- 55- — Formation of chromosomes and early splitting of the chromatin-granules in sperma- togonia oi As car is megalocephala, var. bivalens. [BraUER.] A. Ver}^ early prophase; granules of the nuclear recticulum already divided. B. Spireme; the continuous chromatin-thread split throughout. C. Later spireme. A Shortening of the thread. E. Spireme-thread divided into two parts. F. Spireme-thread segmented into four si)lit chromosomes. thread might spHt in the spireme-stage (epithelial cells of the sala- mander), and this has since been shown to occur in many other cases ; for instance, by Guignard in the mother-cells of the pollen in the lily ('91). Brauer's recent work on the spermatogenesis of Ascaris shows that the fission of the chromatin-granules here takes place even before the spireme-stage, when the chromatin is still in the form of a reticulum, and long before the division of the centrosome (Fig. 55). He therefore concludes : " With Boveri I regard the splitting as an 114 CELL-DIVISIOX independent reproductive act of the chromatin. The reconstruction of the nucleus, and in particular the breaking up of the chromosomes after division into small granules and their uniform distribution through the nuclear cavity, is, in the first place, for the purpose of allowing a uniform growth to take place ; and in the second place, after the granules have grown to their normal size, to admit of tJicir precisely equal gnautitative and qualitative division. I hold that all the succeeding phenomena, such as the grouping of the granules in threads, their union to form larger granules, the division of the thread into segments and finally into chromosomes, are of secondary importance ; all these are only for the purpose of bringing about in the simplest and most certain manner the transmission of the daugh- ter-granules (Spalthalften) to the daughter-cells."^ " In my opinion the chromosomes are not independent individuals, but only groups of numberless minute chromatin-granules, which alone have the value of individuals." ^ These obser\'ations certainly lend strong support to the view that the chromatin is to be regarded as a morphological aggregate — as a congeries or colony of self-propagating elementary organisms capable of assimilation, growth, and division. They prove, more- over, that mitosis involves two distinct though closely related factors, one of which is the fission of the chromatic nuclear substance, while the other is the distribution of that substance to the daughter-cells. In the first of these it is the chromatin that takes the active part ; in the second it would seem that the main role is played by the amphiastcr. E. Direct or Amitotic Division I. General Sketch We turn now to the rarer and simpler mode of division known as amitosis ; but as Flemming has well said, it is a somewhat trying task to give an account of a subject of which the final outcome is so unsatisfactory as this ; for in spite of extensive investigation, we still have no very definite conclusion in regard either to the mechan- ism of amitosis or its biological meaning. Amitosis, or direct division, differs in two essential respects from mitosis. First, the nucleus remains in the resting state (reticulum), and there is no formation of a spireme or of chromosomes. Second, division occurs without the formation of an amphiaster; hence the centrosome is not con- cerned with the nuclear division, which takes place by a simple constriction. The nuclear substance, accordingly, undergoes a divi- 1 '93, pp. 203, 204. 2 /.^.^ p. 205. DIRECT OR AMITOTIC DIVISIOX 115 sion of its total mass, but not of its individual elements or chromatin- granules (Fig. 56). Before the discovery of mitosis, nuclear division was generally assumed to take place in accordance with Remak's scheme (p. 63). The rapid extension of our knowledge of mitotic division between the years 1875 and 1885 showed, however, that such a mode of division was, to say the least, of rare occurrence, and led to doubts as to whether it ever actually took place as a normal process. As soon, however, as attention was especially directed to the subject, many cases of amitotic division were accurately determined, though Fig. 56,— Group of cells with amitotically dividing nuclei ; ovarian follicular epithelium of the cockroach. [WHEELER.] very few of them conformed precisely to Remak's scheme. One such case is that described by Carnoy in the follicle-cells of the Qgg in the mole-cricket, where division begins in the fission of the nucleolus, followed by that of the nucleus. Similar cases have been since described, by Hoyer ('90) in the intestinal epithelium of the nematode Rhabdonema, by Korschelt in the intestine of the annelid Ophryotrocha, and in a few other cases. In many cases, how- ever, no preliminary fission of the nucleolus occurs; and Remak's scheme must, therefore, be regarded as one of the rarest forms of cell-division (!). 2. Ccntrosome and Attraction-sphere in Amitosis The behaviour of the centrosome in amitosis forms an interesting question on account of its bearing on the mechanics of cell-division. Fk^iiming obser\-ed ('91 ) that the nucleus of leucocytes might in some cases divide directly without 1 1 6 CELL-Di visioy the formation of an amphiaster, the attraction-sphere remaining undivided mean- while. Heidenhain showed in the following year, however, that in some cases leucocytes containing two nuclei (doubtless formed by amitotic division) might also contain two asters connected by a si)indle. Both Heidenhain and Flemming drew from this the conclusion that direct division of the uulU'hs is in this case inde- pendent of the centrosome, but that the latter might be concerned in the division of the cell-body, though no such process was observed. A little later, however, Meves jniblished remarkable observations that seem to indicate a functional activity of the attraction-sphere during amitotic nuclear division in the "spermatogonia"' of the salamander.! Krause and Flemming observed that in the autumn many of these cells show peculiarly lobed and irregular nuclei (the "polymorphic nuclei " of Bellonci). These were, and still are by some writers, regarded as degenerating nuclei. Meves, however, asserts — and the accuracy of his observations is in the main vouched for by Flemming — that in the ensuing spring these nuclei become uniformly rounded, and may then divide amitotically. In the autumn the attraction- sphere is represented by a diffused and irregular granular mass, which more or less completelv surrounds the nucleus. In the spring, as the nuclei become rounded, the granular substance draws together to form a definite rounded sphere, in which a distinct centrosome may sometimes be made out. Division takes place in the following extraordinary manner: The nucleus assumes a dumb-bell shape, while the attraction-si)here becomes drawn out into a band which surrounds the central part of the nucleus, and finally forms a closed ring, encircling the nucleus. After this the nucleus divides into two, while the ring-shaped attraction-sphere (-archo- plasm") is again condensed into a sphere. The appearances suggest that the ring- shaped sphere actually compresses the nucleus and cuts it through. In a later paper (94) Meves shows that the diffused "archoplasm" of the autumn-stage arises by the breaking down of a definite spherical attraction-sphere, which is re-formed again in the spring in the manner described, and in this condition the cells mav divide cither uiitotically or amitotically. He adds the interesting observa- tion, since confirmed by Rawitz ('94), that in the spermatocytes of the salamander the attraction-spheres of adjoining cells are often connected by intercellular bridges, but the meaning of this has not yet been determined. It is certain that the remarkable transformation of the sphere into a ring during amitosis is not of universal, or even of general, occurrence, as shown by the later studies of \'om Rath (95, 3). In leucocytes, for example, the sphere persists in its typical form, and contains a centrosome, during every stage of the division: but it is an interesting fact that during all these stages the sphere lies on the concave side of the nucleus in the bay which finally cuts through the entire nucleus. Again, in the liver-cells of the isopod Porcellio, the nucleus divides, not by constriction, as in the leucocyte, but by the appearance of a nuclear plate, in the formation of which the attraction sphere is apparently not concerned.'-^ The relations of the centro- some and archoplasm in amitosis are, therefore, still in doubt; but, on the whole, the evidence goes to show that they take no essential part in the process. 3 . Biologica I Sign ifica nee of A m itos is A survey of the known cases of amitosis brings out the following siirnificant' facts. It is of extreme rarity, if indeed it ever occurs in embryonic cells or such as are in the course of rapid and contniued 1 '91, p. 62S. 2 Such a mode of amitotic division was first described by Saliatier in the Crustacea ('89), and a similar mode has been observed by Carnoy and Van der Stricht. DIRECT OR AMITOTIC DIVISION 117 multiplication. It is frequent in pathological growths and in cells such as those of the vertebrate decidua, of the embryonic envelopes of insects, or the yolk-nuclei (periblast, etc.), zvJiicJi arc on the luay toivard degeneration. In many cases, moreover, direct nuclear divi- sion is not followed by fission of the cell-body, so that multinuclear cells and polymorphic nuclei are thus often formed. These and many similar facts led Flemming in 1891 to express the opinion that so far as the higher plants and animals are concerned amitosis is "a process which does not lead to a new production and multiplication of cells, but wherever it occurs represents either a degeneration or an aberration, or perhaps in many cases (as in the formation of multi- nucleated cells by fragmentation) is tributary to metabolism through the increase of nuclear surface." ^ In this direction Flemming sought an explanation of the fact that leucocytes may divide either mitotically or amitotically (/. Peremeschko, Lowit, Arnold, Flemming). In the normal lymph-glands, where new leucocytes are continually regenerated, mitosis is the prevalent mode. Elsewhere (wandering- cells) both processes occur. '* Like the cells of other tissues the leucocytes find their normal physiological origin (Neubildung) in mitosis ; only those so produced have the power to live on and repro- duce their kind through the same process."^ Those that divide ami- totically are on the road to ruin. Amitosis in the higher forms is thus conceived as a purely secondary process, not a survival of a primitive process of direct division from the Protozoa, as Strasburger ('82) and Waldeyer i^^'^) had conceived it. This hypothesis has been carried still further by Ziegler and \o\Vi Rath ('91). In a paper on the origin of the blood in fishes, Ziegler i^'^y) showed that the periblast-nuclei in the ^gg of fishes divide ami- totically, and he was thus led like Flemming to the view that amitosis is connected with a high specialization of the cell and may be a fore- runner of degeneration. In a second paper ('91), published shortly after Flemming's, he points out the fact that amitotically dividing nuclei are usually of large size and that the cells are in many cases distinguished by a specially intense secretory or assimilative activity. Thus, Riige ('90) showed that the absorption of degenerate eggs in the Amphibia is effected by means of leucocytes which creej) into the egg-substance. The nuclei of these cells become enlarged, divide ami- totically, and then frequently degenerate. Other observers ( Korschelt, Carnoy) have noted the large size and amitotic division of the nuclei in the ovarian follicle-cells and nutritive cells surrounding the ovum in insects and Crustacea. Chun found in the entodermic cells of the radial canals of siphonophores huge cells filled with nests of nuclei amitotically produced, and suggested ('90) that the multiplication of 1 '91, 2, p. 291. 1 1 8 CELL-DI VI SI OX nuclei was for the purpose of increasing the nuclear surface as an aid to metabolic interchanges between nucleus and cytoplasm. Amitotic division leading to the formation of multinuclear cells is especially com- mon in gland-cells. Thus, Klein has described such divisions in the mucous skin-glands of Amphibia, and more recently Vom Rath has carefully described it in the huge gland-cells (probably salivary) of the isopod Aiiilocm ('95). Many other cases are known. Dogiel ('90) has ob.served exceedingly significant facts in this field that place the relations between mitosis and amitosis in a clear light. It is a well- known fact that in stratified epithelium new cells are continually formed in the deeper layers to replace those cast off from the super- ficial layers. Dogiel finds in the lining of the bladder of the mouse that the nuclei of the superficial cells, which secrete the mucus cover- ing the surface, regularly divide amitotically, giving rise to huge mul- tinuclear cells, which finally degenerate and are cast off. The new cells that take their place are formed in the deeper layers by mitosis alone. Especially significant, again, is the case of the ciliate Infu- soria, which possess two kinds of nuclei in the same cell, a macro- nucleus and a micronucleus. The former is known to be intimately concerned with the processes of metabolism {cf. p. 342). During con- jugation the macronucleus degenerates and disappears and a new one is formed from the micronucleus or one of its descendants. The macro- nucleus is therefore essentially metabolic, the micronucleus genera- tive in function. In view of this contrast it is a significant fact that while both nuclei divide during the ordinary process of fission the mitotic phenomena are as a rule less clearly marked in the macronu- cleus than in the micronucleus, and in some cases the former appears to divide directly while the latter always goes through a process of mitosis. These conclusions received a very important support in the work of Vom Rath on amitosis in the testis ('93). On the basis of a compara- tive study of amitosis in the testis-cells of vertebrates, mollusks, and arthropods he concludes that amitosis never occurs in the sperm-pro- ducing cells (spermatogonia, etc.), but only in the supporting cells ( Randzellen, Stutzzellen). The former multiply through mitosis alone. The two kinds of cells have, it is true, a common origin in cells which divide mitotically. When, however, they have once become differen- tiated, they remain absolutely distinct ; amitosis never takes place in the series vvhich finally results in the formation of spermatozoa, and the amitotically dividing "supporting-cells" sooner or later perish. Vom Rath thus reached the remarkable conclusion that " when once a cell has undergone amitotic division it has received its death- warrant ; it may indeed continue for a time to divide by amitosis, but inevitably perishes in the end." ^ ^ '91. p. Zl^- SUMMARY AND CONCLUSION Iig There is, however, strong evidence that this conckision is too extreme. Meves ('94) has given good reason for the conckision that in the salamander the nuclei of the sperm-producing cells (spermato- gonia) may divide by amitosis yet afterward undergo normal mitotic division, and Preusse ('95) has reached a similar result in the case of insect-ovaries. Perhaps the most convincing evidence in this direc- tion is afforded by Pfeffer's ('99) recent experiments on Spirogyra. If this plant be placed in water containing 0.5 to 1.0% of ether, active growth and division continue, but only by amitosis. If, however, the same individuals be replaced in water, viitotic division is resmiied and entirely normal growth continues. This seems to show conclusively that amitosis, in lower forms of Hfe at least, does not necessarily mean the approach of degeneration, but is a result of special conditions. Nevertheless, there can be no doubt that Flemming's hypothesis in a general way represents the truth, and that in the vast majority of cases amitosis is a secondary process which does not fall in the generative series of cell-divisions. F. Summary and Conclusion All cells arise by division from preexisting cells, cell-body from cell-body, nucleus from nucleus, plastids (when these bodies are pres- ent) from plastids, and in some cases centrosomes from centrosomes. The law of genetic continuity thus applies not merely to the cell con- sidered as a whole, but also to some of its structural constituents. In mitosis, the usual and typical mode of division, the nucleus under- goes a complicated transformation, and, together with some of the cytoplasmic material, gives rise to the mitotic figure. Of this, the most characteristic features are the chromatic figure, consisting of chromosomes derived from the chromatin, and the achromatic figure, derived from the cytoplasm, the nucleus, or from both, and consisting of a spindle, at each pole of which, as a rule, is a centrosome and aster. There is, however, strong evidence that both these latter struc- tures may in some cases be wanting, and the spindle is therefore prob- ably to be regarded as the most essential element. The chromosomes, always of the same number in a given species (with only apparent exceptions), arise by the transformation of the chromatin-reticulum into a thread which breaks into segments and splits lengthwise throughout its whole extent. The two halves arc thereupon transported in opposite directions along the spindle to its respective poles and there enter into the formation of the two corresponding daughter-nuclei. The spireme-thread, and hence the chromosome, arises from a single series of chromatin-granules or chromomeres which, by their fission, cause the splitting of the thread. 1 20 CELL-DIVISIOX Every individual chromatin-j^ranule therefore contributes its quota to each of the daughter-nuclei, but it is uncertain whether they are persistent bodies or only temporary structures like the chromosomes themselves. The spindle may arise from the achromatic substance of the nucleus, from the cytoplasmic substance, or from both. When cen- trosomes are present it is they, as a rule, that lead the way in divi- sion. About the daughter-centrosomes as foci are formed the asters and between them stretches the spindle, forming an auipJiiastcr which is the most highly developed form of the achromatic figure. When centrosomes are absent, as now appears to be the case in the higher plants, the spindle is formed from fibrous protoplasmic ele- ments that gradually group themselves into a spindle. The mechanism of mitosis is imperfectly understood. Experi- mental studies give ground for the conclusion that the changes undergone by the chromatic and the achromatic figures respectively are parallel but in a measure independent processes, which are how- ever so correlated that both must cooperate for complete cell-division. Thus there is strong evidence that the fission of the chromatin-gran- ules, and the splitting of the thread, is not caused by division of the centrosome or the formation of the spindle, but only accompanies it as a parallel phenomenon. The divergence of the daughter-chromo- somes, on the other hand, is in some manner determined by the spindle-fibres. There are cogent reasons for the view that some of these fibres are contractile elements which, like muscle-fibres, drae: the daughter-chromosomes asunder ; while other s])indle-fibres act as supporting and guiding elements, and probably by their elongation push the spindle-poles apart. The adequacy of this explanation is, however, doubtful, and it is not improbable that the centrosome or spindlc-poles are centres of chemical or other physiological activities that play an essential part in the process and are correlated with those taking place in the chromatin. The functions of the astral rays are likewise still involved in doubt, the rays being regarded by some investigators as contractile elements like muscle-fibres, by others as rigid supporting fibres, or even as actively pushing elements like those of the central spindle. It is generally believed further that they play a definite part in division of the cell-body — a conclusion sup- ported by the fact that the size of the aster is directly related to that of the resulting cell. On the other hand division of the cell-body may apparently occur in the absence of asters (as in amitosis, or among the Infusoria). These facts show that mitosis is due to the coordinate play of an extremely complex system of forces which are as yet scarcely com- prehended. Its general significance is, however, obvious. The effect LITER A TURE I o i of mitosis is to produce a meristic division, as opposed to a viere mass- division, of the chromatin of the mother-cell, and its equal distribution to the nuclei of the daughter-cells. To this result all the operations of mitosis are tributary; and it is a significant fact that this process is characteristic of all embryonic and actively growing cells, while mass-division, as shown in amitosis, is equally characteristic of highly specialized or degenerating cells in which development is approaching its end. LITERATURE. II i Auerbach, L. — Organologische Studien. Breslau, 1874. Van Beneden, E. — Recherches sur la maturation de I'oeuf. la fecondation et la division cellulaire : Arch, de Biol.., IV. 1883. Van Beneden and Neyt. — Nouvelles recherches sur la fecondation et la division mitosque chez TAscaride megalocephale : Bull. Acad. roy. de Belgigue, III. 14, No. 8. 1887. Boveri, Th. — Zellenstudien: I. Jena. Ze2tschr.,XX\. 1887; II .//;/>/. XX II . 1888; III. Ibid. XXIV. 1890. Driiner, L. — Studien liber den Mechanismus der Zelltheilung. Jena. Zeitschr.^ XXIX., II. 1894. Erlanger, R. von. — Die neuesten Ansichten liber die Zelltheilung und ihre Mechanik : Zo'dl. Centra lb.. III. 2. 1896. Id. — Uber die Befruchtung und erste Teilung des Ascariseies : Arcli. mik. Anat., XLIX. 1897. Flamming, W., '92. — Entwicklung und Stand der Kenntnisse liber Amitose : Merkel und Bofinefs Ergebnisse, II. 1892. Id. — Zelle. (See Introductory list. Also general list.) Fol, H. — (See List IV.) Heidenhain, M, — Cytomechanische Studien: Arch. f. Entiuickmcch., I. 4. 1895. Id. — Neue Erlauterungen zum Spannungsgesetz der centrirten Svsteme : Morph. Arb., VII. 1897. Hermann, F. — Beitrag zur Lehre von der Entstehung der karyokinetischen Spindel : Arch. 7mk. Anat.,XXXY\l. 1891. Hertwig, R. — Uber Centrosoma und Centralspindel : Sit:;. -Berg. (Jes. Morph. und Phys. MiincJioi. 1^95' Heft I. Kostanecki and Siedlecki. — Uber das Verhalten der Centrosomen zum Protoplasma : Arch. niik. Anaf., XL\'1I1. 1896. Mark, E. L. — (See List IV.) Meves, Fr. — Zellteilung: Merkel und Bontiefs Ergebnisse, W. 1897. Reinke, F. — Zellstudien : I. Arch. mik. Afiat., XLIII. 1894 : II. /bid. WAV. 1S94. Strasburger, E. — Karyokinetische Probleme : Jahrb.f. l\ 'iss. Jiotan.. XX\'I II. i S95. Strasburger, Osterhout, Mottier, and Others. —Cytologische Studien aus dem Bonner Institut: JaJirb. luiss. Bot.. XXX. 1897. Waldeyer. W. — iJber Karyokinese und ihre Beziehungen zu den Befruchtungsvor- gangen: Arch. 7tiik. Anal, XXXII. 1888. QJ.M.S.. XXX. 1889-90. 1 See also Literature, IV., p. 231. CHAPTER III THE GERM-CELLS " Not all the propjeny of the primary impregnated germ-cells are required for the forma- tion of the liudy in all animals; certain of the derivative germ-cells may remain unchanged and become included in that body which has been composed of their metamorphosed and diversely combined or confluent brethren; so included, any derivative germ-cell may com- mence and repeat the same processes of growth by imbibition and of propagation by spon- taneous fission as those to which itself owed its origin; followed by metamorjihoses and combinations of the germ-masses so produced, which concur to the development of another individual." RlCJiAKU UWEN.i " Es theilt sich demgemass das befruchtete Ei in das Zellenmaterial des Individuums und in die Zellen fiir die Erhaltung der Art." M. NUbSBAUM.'^ The germ from which every living form arises is a single cell, de- rived by the division of a parent-cell of the preceding generation. In the unicellular plants and animals this fact appears in its simplest form as the fission of the entire parent-body to form two new and separate individuals like itself. In all the multicellular types the cells of the body sooner or later become differentiated into two groups, which as a matter of practical convenience may be sharply distin- guished from one another. These are, to use Wcismann's terms : (i) the somatic cells, which are differentiated into various tissues by which the functions of individual life are performed and which col- lectively form the ** body," and (2) \hQ (^ej'm-cells, which are of minor significance for the individual life and are destined to give rise to new individuals by detachment from the body. It must, however, be borne in mind that the distinction between germ-cells and somatic cells is not absolute, as some naturalists have maintained, but only relative. The cells of both groups have a common origin in the parent germ-cell ; both arise through mitotic cell-division during the cleavage of the ovum or in the later stages of development ; both have essentially the same structure and both may have the same power of development, for there are many cases in which a small fragment of the body consisting of only a few somatic cells, perhaps only of one, may give rise by regeneration to a complete body. The dis- tinction between somatic and germ-cells is an expression of the 1 Parthenogenesis, p. 3, 1849. 2 Arch. Mik. Anat., XVIIL, p. 112, 1880. 122 THE GERM-CELLS 123 physiological division of labour; and while it is no doubt the most fundamental and important differentiation in the multicellular body, it is nevertheless to be regarded as differing only in degree, not in kind, from the distinctions between the various kinds of somatic cells. In the lowest multicellular forms, such as Volvox (Fig. 57), the differentiation appears in a very clear form. Here the body consists of a hollow sphere, the walls of which consist of two kinds of cells. The very numerous smaller cells are devoted to the functions of nutri- :?t^Vi \/,-i -^-'n-^ ., ,-■- \ \ ^ Fig. 57. — Volvox, showing the small ciliated somatic cells and eight large germ-cells (drawn from life by J. H. Emerton). tion and locomotion, and sooner or later die. A number, usually eight, of larger cells are set aside as germ-cells, each of which by progressive fission may form a new individual like the parent. In this case the germ-cells are simply scattered about among the somatic cells, and no special sexual organs exist. In all the higher types the germ-cells are more or less definitely aggregated in groups, supported and nour- ished by somatic cells specially set apart for that purpose and forming distinct sexual organs, the ovaj^es and spcrmarics or their equivalents. Within these organs the germ-cells are carried, protected, and nour- ished ; and here they undergo various differentiations to prepare them for their future functions. In the earlier stages of embryological development the progenitors of the germ-cells are exactly alike in the two sexes and are indistin- 124 THE GERM-CELLS guishable from the surrounding somatic cells. As development pro- ceeds, they are first differentiated from the somatic cells and then diverge very widely in the two sexes, undergoing remarkable trans- formations of structure to fit them for their specific functions. The structural difference thus brought about between the germ-cells is, however, only the result of physiological division of labour. The female germ-cell, or ovum, supplies most of the material for the body of the embryo and stores the food by which it is nourished. It is therefore very large, contains a great amount of cytoplasm more or less laden with food-matter (j7^/X' or dciitoplasui), and in many cases becomes surrounded by membranes or other envelopes for the pro- tection of the developing embryo. On the whole, therefore, the early life of the ovum is devoted to the accumulation of cytoplasm and the storage of potential energy, and its nutritive processes are largely constructive or anabolic. On the other hand, the male germ-cell or spermatozoon contributes to the mass of the embryo only a very small amount of substance, comprising as a rule only a single nucleus and a very small quantity of cytoplasm. It is thus relieved from the drudgery of making and storing food and providing protection for the embryo, and is provided with only sufficient cytoplasm to form a locomotor apparatus, usually in the form of one or more cilia, by which it seeks the ovum. It is therefore very small, performs active movements, and its metabolism is characterized by the predominance of the destructive or katabolic processes by which the energy neces- sary for these movements is set free.^ When finally matured, there- fore, the ovum and spermatozoon have no external resemblance ; and while Schwann recognized, though somewhat doubtfully, the fact that the ovum is a cell, it was not until many years afterward that the spermatozoon was proved to be of the same nature. A. The Ovum The animal (i^^^^ (Figs. 5CS-59) is a huge spheroidal cell, sometimes naked, but more commonly surrounded by one or more membranes which may be perforated by a minute opening, the inicropylc, through which the spermatozoon enters (Fig. 63). It contains an enormous nucleus known as the germinal vesicle, within which is a very con- spicuous nucleolus known to the earlier observers as the germinal spot. In many eggs the latter is single, but in other forms many 1 The metaliolic contrast between the germ-cells has been fully discussed in a most sug- gestive manner by Geddes and Thompson in their work on the Evolution of Sex ; and these authors regard this contrast as but a particular manifestation of a metabolic contrast charac- teristic of the sexes in general. THE OVUM 125 nucleoli are present, and they are sometimes of more than one kind as in tissue-cells.i In many forms no centrosome or attraction-sphere is found in the ^gg until the initial stages in the formation of the polar bodies, though Mertens ('93) describes a centrosome and attrac- tion-sphere in the young ovarian eggs of a number of vertebrates (Fig. 79), while Platner ('89) and Stauffacher ('93) find what they believe to be centrosomes in much later stages of AulostoDiuvi and Cyclas, lying outside the nuclear membrane. Beside these cases should be placed those described by Balbiani, Munson, Nemec, and others in which a body closely resembling an attraction-sphere is identified as a "yolk-nucleus" or "vitelline body," as described at page 158. In none of these cases is the identification of this body wholly satisfactory, nor is it known to have any connection with the polar mitoses. Most observers find no centrosome until the prophases of the first polar mitosis. Its origin is still problematical, some observers believing it to arise de novo in the cytoplasm (Mead), others concluding that it is of nuclear origin (Mathews, Van der Stricht, Riickert), still others that it persists in the cytoplasm hidden among the granules. In any case it is again lost to view after forma- tion of the polar bodies, to be replaced by the cleavage-centrosomes which arise in connection with the spermatozoon (p. 187). The egg-cytoplasm almost always contains a certain amount of nutritive matto^, the yoik or deiUoplasin, in the form of liquid drops, solid spheres or other bodies suspended in the meshwork and varying greatly in different cases in respect to amount, distribution, form, and chemical composition. I. TJie Nucleus The nucleus or germinal vesicle occupies at first a central or nearly central position, though it shows in some cases a distinct eccentricity even in its earliest stages. As the growth of the agg proceeds, the eccentricity often becomes more marked, and the nucleus may thus come to lie very near the periphery. In some cases, however, the peripheral movement of the germinal vesicle occurs only a very short time before the final stages of maturation, which may coincide with the time of fertilization. Its form is typically that of a spherical sac, surrounded by a very distinct membrane (Fig. 58); but during the growth of the Q.gg it may become irregular or even amceboid (Fig. j'j), and, as Korschelt has shown in the case of insect-eggs, may move through the cytoplasm toward the source of food. Its structure is 1 Hacker ('95, p. 249) has called attention to the fact that the nucleolus is as a rule single in small eggs containing relatively little deutoplasm (crulenterates, echinodernis, many annelids, and some copepods), while it is multiple in large eggs heavily laden with deutoplasm (lower vertebrates, insects, many Crustacea). 126 THE GERM-CELLS on the whole that of a typical cell-nucleus, but is subject to very great variation, not only in different animals, but also in different stages of ovarian growth. Sometimes, as in the echinoderm ovum, the chro- matin forms a beautiful and regular reticulum consisting of numer- ous chromatin-granules suspended in a network of linin (Fig. 58). In other cases, no true reticular stage exists, the nucleus containing throughout the whole period of its growth the separate daughter-chro- mosomes of the preceding division (copepods, selachians, Amphibia),^ 6- Fig. 58. — Ovarian egg of the sea-urchin, Toxopneustes (x 750). g.v. Nucleus or germinal vesicle, containing an irregular discontinuous network of chromatin; g.i. nucleolus or germinal spot, intensely stained with haematoxylin. The naked cell-body con- sists of a very regular alveolar meshwork, scattered through which are numerous minute granules or microsomes. {Cf. Figs. 11, 12.) Below, at s, is an entire spermatozoon shown at the same enlargement (both middle-piece and fiagellum are slightly exaggerated in size). and these chromosomes may undergo the most extraordinary changes of form, bulk, and staining-reaction during the growth of the Q^gg^ It is a very interesting and important fact that during the growth and maturation of the ovum a large part of the chromatin of the germinal vesicle may be lost, either by passing out bodily into the cytoplasm, by conversion into supernumerary or accessory nucleoli which finally degenerate, or by being cast out and degenerating at the time the polar bodies are formed (Figs. 97, 128). The nucleolus of the egg-cell is, as elsewhere, a variable quantity and is still imperfectly understood. It often attains an enormous development, forming the " Keimfleck " or "germinal spot" of the ^ p- 273. 2 p. 338. THE OVUM 1^7 early observers. There are some cases {e.g. echinoderm eggs) in which it is always a single large spherical body (Fig. 58), and this condition appears to be characteristic of the very young ovarian e^'-f^s of most animals. As a rule, however, the number of nucleoli ^in- creases with the growth of the ovum, until, in such forms as Amphibia and reptiles, they may be numbered by hundreds. In a large number of cases the nucleoli are of two quite distinct types, which Flemming has distinguished as the *' principal nucleolus " Fig. 59. — Ovum of the cat, within the ovary, directly reproduced from a photograph of a preparation by Dahlgren. [Enlarged 235 diameters.] The ovum lies in the Graafian follicle within the discus proligerus, the latter forming the immediate follicular investment {corona radiata) of the Qg^. Within the torona is the clear zona pellucida or egg-membrane. {Cf. Fig. 92.) {Hajiptmicleolus) and ''accessory nucleoli" {Ncbennuclcoli). These differ widely in staining-reaction ; but it does not yet clearly appear whether they definitely correspond to the plasmosomes and karyo- somes of tissue-cells (p. 34). The principal nucleolus, which alone is present in such eggs as those of echinoderms, often stains deeply with chromatin-stains, yet differs more or less widely from the chromatin-network,! and in some cases at least it does not contribute 1 Cf. List, '96, Montgomery, '98, 2, and Obst., '99. 128 THE GERM-CELLS to the formation of chromosomes. It cannot therefore be directly com- pared to the net-knots or karyosomcs of tissue-cells. This nucleolus is often vacuolated and sometimes assumes the form of a hollow vesicle. It is rarely double or multiple. The accessory nucleoli, on the other hand, are in general coloured by plasma-stains, thus resembling the plasmosomes of tissue-cells ; they arc often multiple, and as a rule they arise secondarily during the growth of the (t^g (Fig. 6i). The accessory nucleoli often have no connection with the principal ; but in some mollusks and annelids an accessory and a principal nucleolus are closely united to form a single compound body (Figs. 60, 61). The numerous nucleoli of the amphibian or reptilian ^gg appear to be of the "accessory" type. The singular inconstancy of the nucleolus is evidenced by the fact that even closely related species may differ in this regard. Thus, in Cyclops brcviconiis, according to Hacker, the very young ovum contains a single intensely chromatic nucleolus ; at a later period a number of paler accessory nucleoli appear ; and still later the principal nucleolus disappears, leaving only the accessory ones. In C. strenuus, on the other hand, there is throughout but a single nucleolus. The physiological meaning of the nucleoli is still involved in doubt. Many cases are, however, certainly known in which the nucleolus plays no part in the later development of the nucleus, being cast out or degenerating /;/ situ at the time the polar bodies are formed. It is, for example, cast out bodily in the medusa Alquorca (Hacker) and in various annelids and echinodcrms, afterward lying for some time as a '' metanucleus " in the egg-cytoplasm before degenerating. In these cases the chromosomes are formed in the germinal vesicle inde- pendently of the nucleoli (Fig. 125), which degenerate in j//;/ when the membrane of the germinal vesicle disappears. In such cases it seems quite certain that the nucleoli do not contribute to the forma- tion of the chromosomes, and that their substance represents passive material which is of no further direct use. Hence we can hardly doubt the conclusion of Hacker, that the nucleoli of the germ-cells are, in some cases at least, accumulations of by-products of the nuclear action, derived from the chromatin cither by direct transformation of its substance, or as chemical cleavage-products or secretions. It will be shown in Chapter V. that in some cases a large part of the chromatic reticulum is cast out, and degenerates at the time the polar bodies are formed. The immense growth of the chromatin during the ovarian development is probably correlated in some way with the intense constructive activity of the cytoplasm (p. 339); and when this latter process has ceased a large part of the chromatin-substance, having fulfilled its functions, is cast aside. It seems not improbable that the nucleoli are tributary to the same general process, perhaps THE OVUM 129 Fig. 60. — Eggs of the annelid ^V^^rm, before and after fertilization, X 400 (for int.^rn..^di.itt' stages see Fig. 95). A. Before fertilization. The large germinal vesicle occupies a nearly central position. It con- tains a network of chromatin in which are seen five small darker bodies; these are the quadruple chromosome-groups, or tetrads, in process of formation (not all of them are shown) ; those alone persist in later stages, the principal mass of the network being lost; g.s. double germinal spot. consisting of a chromatic and an achromatic sphere. This egg is heavily laden with yolk, in the form of clear deutoplasm-spheres {d) and fat-drops (/). uniformly distributed through the cyto- plasm. The peripheral layer of cytoplasm (peri-vitelline layer) is free from deutoplasni. Outside this the membrane. B. The egg some time after fertilization and about to divide. The deuto- plasm is now concentrated in the lower hemisphere, and the peri-vitelline layer has disappeared. Above are the two polar bodies {p.b.). Below them lies the mitotic figure, the chromosomes dividing. 130 THE GERM-CELLS serving as storehouses of material formed incidentally to the general nuclear activity, but not of further direct use. Carnoy and Le Brun ('97, '99) reach, however, the conclusion that in the germinal vesicle of Amphibia the chromosomes are derived not from the chromatin-network, but solely from the nucleoli. The apparent contradiction of this result with that of other observers is, ■-. \*. V ¥: •V* ''"'^.^^^ ''-'''''V. '' \ IS •-»r !!?^" .^4. " *-f •J* ■'•% « e. J- Fig. 61. — Germinal vesicles of growing ovarian eggs ut the iaiiiLininiincli, L^f/io {A-D), and the spider, Epeira {E-F). [Ohst.] A. Youngest stage with single (principal) nucleolus. B. Older egg, showing accessory nucle- olus attached to the principal. C. The two nucleoli separated. D. Much older stage, showing the two nucleoli united. E. (ierminal vesicle of Epeira, showing one accessory nucleolus at- tached to the principal, and one free. /•"■. Later stage ; several accessory nucleoli attached to the principal. perhaps, only a verbal one; for the ''nucleoli" are here evidently chromatin-masses, and the disappearance of the chromatic network is comparable with what occurs at a later period in the annelid Qgg (Figs. 97, 128). 2. T/ic Cytoplasm The egg-cytoplasm varies greatly in appearance with the varia- tions of the deutoplasm. In such eggs as those of the echinoderm THE OVUM 131 (Fig. 58), which have Httle or no deutoplasm, the cytoplasm forms a regular meshwork, which is in this case an undoubted alveolar struc- ture, the structure of which has already been described at p. 28. In eggs containing yolk the deutoplasm-spheres or granules are laid down in the spaces of the meshwork and appear to correspond to the alveolar spheres of the echinoderm ^^^g (p. 50). If they are of large size the cytoplasm assumes a *' pseudo-alveolar" structure (Fig. 60), much as in plant-cells laden with reserve starch; but reasons have already been given (p. 50) for regarding this as only a modification of the "primary" alveolar structure of Butschli. There is good reason to believe, however, that the egg-cytoplasm may in some cases form a true reticular structure with the yolk-granules lying in its interstices, as many observers have described. In many cases a pe- ripheral layer of the ovum, known as the cortical or peri-vitelline layer, is free from deutoplasm-spheres, though it is continuous with the protoplasmic meshwork in which the latter lie (Fig. 60). Upon fertilization, or sometimes before, this layer may disappear by a peripheral movement of the yolk, as appears to be the case in Nereis. In other cases the peri-vitelline substance rapidly flows toward the point at which the spermatozoon enters, where a proto- plasmic germinal disc is then formed; for example, in many fish-eggs. The character of the yolk varies so widely that it can here be con- sidered only in very general terms. The deutoplasm-bodies are com- monly spherical, but often show a more or less distinctly rhomb(^idal or crystalloid form as in Amphibia and some fishes, and in such cases they may sometimes be split up into parallel lamellae known as yolk- plates. Their chemical composition varies widely, judging by the staining-reactions; but we have very little definite knowledge on this subject, and have to rely mainly on the results of analysis of the total yolk, which in the hen's ^gg is thus shown to consist largely of pro- teids, nucleo-albumins, and a variety of related substances which are often associated with fatty substances and small quantities of car- bohydrates (glucose, etc.). In some cases the deutoplasm-spheres stain intensely with nuclear dyes, such as hsematoxylin ; e.i:^. in many worms and mollusks; in other cases they show a greater affinity for plasma-stains, as in many fishes and Amphibia and annelids ( Fig. 60). Often associated with the proper deutoplasm-spheres are drops of oil, either scattered through the yolk (Fig. 60) or united to form a .jingle large drop, as in many pelagic fish-eggs. The deutoplasm is as a rule heavier than the protoplasm ; and in such cases, if the yolk is accumulated in one hemisphere, the ^gg assumes a constant position with respect to gravity, the egg-axis standing vertically with the animal pole turned upward, as in the frog, the bird, and many other cases. There are, however, many 132 THE GERM-CELLS cases in which the o.^^ may lie in any position. When fat-drops are present thev usually He in the vegetative hemisphere, and since they are lighter than the other constituents they usually cause the egg to lie with the animal pole turned downwards, as is the case with some annelids ( .AV/'^/.v) and many pelagic fish-eggs. pb — cn Fig. 62. — Schematic figure of a median longitudinal section of the egg of a fly ^.Musca), showing axes of the bilateral egg and the membranes. [From KORSCHEI.T and Hkidkr, after Hf:nkin(; and Blochmann.] e.n. The germ-nuclei uniting; VI. micropyle; p.b. the polar bodies. The fiat side of the egg is the dorsal, the convex side the ventral, and the micropyle is at the anterior end. The deutoplasm (small circles) lies in the centre surrounded by a periph- eral or peri-vitelline layer of proto- plasm. The outer heavy line is the chorion, the inner lighter line the vitelline membrane, both being per- forated by the micropyle, from which exudes a mass of jelly-like substance. 3. The Egi^-ciivclopcs The egg-envelopes fall under three catetcories. These are : — {(I) The ritclli}iL vnnibrauc, secreted bv the ovum itself. {b) The chorion, formed outside the ovum bv the activity of the maternal follicle-cells. {c) Accessory envelopes, secreted by the walls of the oviduct or other maternal structures after the ovum has left the ovary. Only the first of these properly be- lonirs to the ovum, the second and third being purely maternal products. There are some e^^i^s, such as those of certain coelenterates {e.g: Rcnilla\ that are naked throughout their whole develop- ment. In many others, of which the sea-urchin is a type, the fresh-laid ^<^^ is naked but forms a vitelline membrane almost instantaneously after the sperma- tozocin touches it.^ In other forms (in- sects, birds) the vitelline membrane may be present before fertilization, and in such cases the Q.^^g is often surrounded by a chorion as well. The latter is usually very thick and firm and may have a shell-like consistency, its surface sometimes showing various peculiar markings, prominences, or sculptured patterns characteristic of the species (insects).^ 1 That the vitelline membrane does not preexist seems to be estal)lished by the fact that egg-frai^ments likewise surround themselves vlth a membrane when fertilized. [Hertwig.] - In some cases, according to Wheeler, the insect-egg has only a chorion, the vitelline membrane being absent. THE OVUM 133 The accessory envelopes are too varied to be more than touched upon here. They inchide not only the products of the oviduct or uterus, such as the albumin, shell-membrane, and shell of birds and reptiles, the gelatinous mass investing amphibian ova, the capsules of molluscan ova and the like, but also nutritive fluids and capsules secreted by the external surface of the body, as in leeches and earth- worms. When the ^gg is surrounded by a membrane before fertiUzation it is often perforated by one or more openings known as micropylcs, through which the spermatozoa make their entrance (Figs. 62, 63). Where there is but one micropyle, ^ it is usually situated very near the upper or anterior pole (fishes, many insects), but it may be at the opposite pole (some insects and mollusks), or even on the side (insects). In many insects there is a group of half a dozen or more pig. 63.- Upper pole of the egg of Ar^o- micropyles near the upper pole of nanta. [Ussow.] the ^gZ', and perhaps correlated The egg is surrounded by a very thick with this is the fact that several "-.embrane. perforated at m by the funnel- shaped micropyle; below the latter lies the spermatozoa enter the ^gg, though egg-nucleus in 'the peri-vitelline layer of pro- only one is concerned with the topiasm ; M the polar bodies, actual process of fertiUzation. The plant-ovum, w^hich is usually know^n as the oospJicrc ( Figs. 64, 107), shows the same general features as that of animals, being a relatively large, quiescent, rounded cell containing a large nucleus. It never, however, attains the dimensions or the complexity of struc- ture shown in many animal eggs, since it always remains attached to the maternal structures, by which it is provided with food and invested wdth protective envelopes. It is therefore naked, as a rule, and is not heavily laden with reserve food-matters such as the deutoplasm of animal ova. A vitelline membrane is, however, often formed soon after fertilization, as in echinoderms. The most interesting feature of the plant-ovum is the fact that it often contains phistids (leuco- plasts or chromatophores) which, by their division, give rise to tho.se of the embryonic cells. These sometimes have the ionii ot typical chromatophores containing pyrenoids, as in Volvox and many other Algse (Fig. 64). In the higher forms (archegoniate plants), according to the researches of Schmitz and Schimper, the ^gg contains numer- ous minute colourless 'Meucoplasts," which afterward develop into green chromatophores or into the starch-building amyloplasts. This is a point of great theoretical interest ; for the researches of Schmitz, Schimper, and others have rendered it highly probable that these 134 THE GERM-CELLS plastids are persistent niorpholo^c^ncal bodies that arise only by the division of preexisting bodies of the same kind, and hence may be traced continuously from one generation to another through the A C -' Fig. 64. — Germ-cells of Volvox. [OVERTON.] A. Ovum (oosphere) containing a large central nucleus and a peripheral layer of chromato- phores ; /. pyrenoid. B. Spermatozoid ; c.v. contractile vacuoles ; e. " eye-spot " (chromoplastid) ; /. pyrenoid. C. Spermatozoid stained to show the nucleus («). germ-cells. In the lower plants (Algae) they may occur in both germ- cells ; in the higher forms they are found in the female alone, and in such cases the plastids of the embryonic body are of purely maternal origin. B. The Spermatozoon Although spermatozoa were among the first of animal cells ob- served by the microscope, their real nature was not determined for more than two hundred years after their discovery. Our modern knowledge of the subject may be dated from the year 1841, when Kolliker proved that they were not parasitic animalcules, as the early observers supposed, but the products of cells preexisting in the parent body. Kolliker, however, did not identify them as cells, but believed them to be of purely nuclear origin. We owe to Schweigger- Seidel and La Valette St. George the ]:)roof, simultaneously brought forward by these authors in 1865,^ that the spermatozoon is a com- plete cell, consisting of nucleus and cytoplasm, and hence of the same morphological nature as the ovum. It is of extraordinary minute- ness, being in many cases less than t;ooVoo' ^^^ bulk of the ovum.^ 1 Arc/i. Mik. Atiat., I. '65. 2 In the sea-urchin, Toxopiieus/es, I estimate its bulk as being between 4Tnnro7 ^^^ 5 ^ q^q ^ ,) the volume of the ovum. The inequality is in many cases very much greater. THE SPERMATOZOON 135 Its precise study is therefore difficult, and it is not surprisin^^ that our knowledge of its structure and origin is still far from complete. — Apical body or acrosome. Nucleus. End-knob. Middle-piece. Envelope of the tail. .Axial filament. I. Flagellate Sperjrtatozoa In its more usual form the animal spermatozoon resembles a minute, elongated tadpole, which swims very actively about by the vibrations of a long, slender tail morpho- logically comparable with a single cilium or flagellum. Such a spermatozoon con- sists typically of four parts, as shown in Fig. 65 : — 1. The nucleus, which forms the main portion of the **head," and consists of a very dense and usually homogeneous mass of chromatin staining with great intensity with the so-called "nuclear dyes" {e.g. haematoxylin or the basic tar-colours such as methyl-green). It is surrounded by a very thin cytoplasmic envelope. 2. An apical body, or acrosome, lying at the front end of the head, sometimes very minute, sometimes almost as large as the nucleus, and in some cases terminating in a sharp spur by means of which the spermatozoon bores its way into the ovum. 3. The middle-piece, or connecting piece, a larger cytoplasmic body lying behind the head and giving attachment to the tail, from which it is not always dis- tinctly marked off. This body shows the same staining-reactions as the acrosome, having an especial affinity for " plasma- stains " (acid fuchsin, etc.). At its front end it is in some forms (mammals) sepa- rated from the nucleus by a short clear region, the neck. Like the acrosome, the middle-piece is in some cases derived from an " archoplasmic " mass, representing an attraction-sphere {Lumbricus) or a portion of the Nebenkern (insects), and it contains, or according to some authors actually arises from, the centrosome (salamander, mammals, insects, etc.). 4. The tail, or flagellum, in part, at least, a cytoplasmic product developed in connection with the centrosome and " archoplasm " End-piece. Fig. 65. — Diagram of the flagellate spermatozoon. 136 THE GERM-CELLS (attraction-sphere or "Nebenkern") of the mother-cell. Tt consists of a fibrillatcd axial filauioit surrounded by a cytoplasmic envelope, and in certain cases (Amjihibia) bears on one side a fin-like undulat- ing membrane (Fig. 66). Toward the tip of the flagellum the enve- lope suddenlv disappears or becomes very thin, leaving a short cnd-piccc which by some authors is considered to consist of the naked axial filament. The a.xial filament may be traced through the middle-piece up to the head, at the base of which it usually termi- n f Fig. 66. — Spermatozoa of fishes and Amphibia, [Ballowitz.] A. Sturgeon. //. Pike. ('. D. Leuciscus. E. Triton (anterior part). F. Triton (posterior part of flagelkim). G. Raja (anterior part), a. apical body; e. end-piece; /. flagellum ; k. end- knob; w. middle-piece ; //.nucleus; j. apical spur, nates in a minute body, single or double, known as the cnd-kiwb. Recent research has proved that the axial filament grows out from the spermatid-centrosome, the latter in some cases persisting as the end-knob (insects, mollusks, mammals), in other cases apparently enlarging to form the main body of the middle-piece (salamander). The tail-envelopes, on the other hand, arise either from the *'archo- plasm " of the Nebenkern (insects) together with a small amount of unmodified cytoplasm, or from the latter alone (salamander, rat). THE SPERMATOZOON I 37 From a physiological point of view we may arrange the parts of the spermatozoon under two categories as follows : I. The essential structures which play a direct part in fertilization. These are : — {a) The uucleus, which contains the chromatin. (b) The middle-piece, which either contains a formed centrosome or pair of centrosomes (end-knob), or is itself a meta- morphosed centrosome. This is probably to be regarded as the fertilizing element par excellence, since there is reason to believe that when introduced into the Q.gg it gives the stimulus to division. 2. The accessory structures, which play no direct part in fertilization, viz. : — {a) The apex or spur, by which the spermatozoon attaches itself to the ^gg or bores its way into it, and which also serves for the attachment of the spermatozoon to the nurse-cells or supporting cells of the testis. {b) The tail, a locomotor organ which carries the nucleus and centrosome, and, as it were, deposits them in the (•. end-knob ; w. mid- a membrane and Spirally dlffe'^rem'par'tsr''"' ^'" ^' ^' ^' '""''"''"^ ^^ ^'^^ V.^\^l^^ about cach Other; a THE SPERM A TO ZOOM 141 somewhat similar structure occurs in the toad. In some beetles there is a fin-membrane attached to a stiff axial "supporting fibre "( F"io-. 6-], A). The membrane itself is here composed of four parallel fibres, which differ entirely from the supporting fibre in staining-capacity and in the fact that each of them may be further resolved into a large number of more elementary fibrillae. Fig. 69. — Unusual forms of spermatozoa. A. B. C. Living amoeboid spermatozoa of the crustacean Polyp hem us. [Z.xcH.VKl D. E. Spermatozoa of crab, Droniia. P. Oi Et/insa, G. oi Maja. //. of hi.uhus. I. Spermatozoon of lobster, Homarus. [HERRlCK.] J. Spermatozoon of crab, Porcellana. [GROBBEN.] [(■•UfillBKN."! Many interesting details have necessarily been passed over in the foregoing account. One of these is the occurrence, in some mammals, birds. Amphibia (frog). and mollusks, of two kinds of spermatozoa in the same animal. In the birds and Amphibia the spermatozoa are of two sizes, but of the same form, the larger i)eing known as -giant spermatozoa^' (Fig. 67, G, II). In the gasteropod Paluiima the two kinds differ entirelv in structure, the smaller form being of the usual type and not unlike those of biVds. while the larger, or "vermiform," spermatozoa have a worm-like shape and bear a tuft of cilia at one end, somewhat like the spermatozoids of plants (Fig. 67, J, K). In this case only the smaller .spermatozoa are functional (von Brunn). 142 THE GERM-CELLS No less remarkable is the conjugation of spermatozoa in pairs (Fig. 68, //). which takes place in the I'as dtferens in the opossum (Selenka) and in some insects (Hallowitz. Auerbach). IJallowitz's researches ('95) on the double spermatozoa of beetles {Dyiisciiia:) prove that the union is not primary, but is the result of an actual conjugation of previously separate spermatozoa. Not merely two, but three or more spermatozoa may thus unite to form a •• spermatozeugma,"' which swims like a single spermatozoon. Whether the spermatozoa of such a group separate before fertilization is unknown; but Hallowitz has found the groups, after copulation, in the female receptaculum. and he believes that they may enter the egg in this form. The physiological meaning of the process is unknown. 2. OtJicr Fonus of Spermatozoa The principal deviations from the flagellate type of spermatozoon occur among the arthropods and nematodes (Fig. 69). In many of these forms the spermatozoa have no flagellum, and in some cases they are actively amoeboid ; for example, in the daphnid PolypJicnuis ( Fig. 69, A, By C) as described by Ley dig and Zacharias. More commonly they are motionless like the ovum. In the chilognathous myriapods the spermatozoon has sometimes the form of a bi-convex lens {Poly- (hs}fii(s), sometimes the form of a hat or helmet having a double brim {Jiihis). In the latter c^se the nucleus is a solid disc at the base of the hat. In many decapod Crustacea the spermatozoon consists of a cylindrical or conical body from one end of which radiate a num- ber of stiff spine-like processes. The nucleus lies near the base. In none of these cases has the centrosome been identified. ^A.i !! MiB* :r^\M '!&• /,;.vsy \ Spermatozoids of Chara. [Bela- 3. Paternal Genn-cells of Plants In most of the flowering plants the male germ-cells are represented by two "generative nuclei," lying at the tip of the ]:)ollen tube (Fig. 106). On the other hand, in the cycads (Figs. 87, 108) and in a large number of the lower plants (pterido- Fig. 70. JEFF.] y^. Mother-cells with reticular nuclei. /A Later phvtCS, Muscineae, and many stage, with spermatozoids forming C Mature sper- ^th^j-s), the male gCrm-CCll is a matozoid (the elongate nucleus black). '' ^ *^ minute actively swimming cell, known as the spennatozoiel, which is closely analogous to the sper- matozoon. The spermatozoids are in general less highly differenti- ated than spermatozoa, and often show a distinct resemblance to the THE SPERMATOZOON 143 asexual swarmers or zoospores so common in the lower plants (Fi^^s. 70, 71). They differ in two respects from animal spermatozoa : first in possessing not one but tw^o or several flagella ; second, in the fact that these are attached as a rule not to the end of the cell, but on the side. In the lower forms plastids are present in the form of chromatophores, one of which may be dif- ferentiated into a red *' eye-spot," as in Volvox and Fiicus (Figs. 57, 71, A), and they may even contain contractile vacuoles '^^ ( Volvox ) ; but both these structures are wanting in the higher forms. These con- sist only of a nucleus with a very small amount of cytoplasm, and have typically a spiral form. In Chai^a, where their structure and development have recently been carefully studied by Belajeff, the sperma- tozoids have an elon- gated spiral form with two long flagella at- tached near the pointed end, which is directed forward in swimming (Fig. 70). The main body of the spermatozoid is oc- cupied by a dense, apparently homoge- neous nucleus sur- rounded by a very delicate layer of cytoplasm. Behind the nucleus lies a granular mass of cytoplasm, forming one end of the cell, while in front is a slender cytoplasmic tip to which the flagella are attached. Nearly similar spermatozoids occur in the liverworts and mosses. In the ferns and other pteridophytes a somewhat different type occurs Fig. 71.— Spermatozoids of plants. [./. B, C, E, after Guignard; a F, after Strasburgek.] A. Of an alga {Fucus) ; a red chromatophore at the right of the nucleus. B. Liverwort {Pcliia). C. Moss (>///./<'«//w). D. Mars ilia. E. Fern. {Augtoptcris). F. Fern, Fhfi^opterts (the nucleus dark). {Cf. Figs. 87. 88.) 144 THE GERM-CELLS (Figs. 71, %?)). Here the spermatozoid is twisted into a conical spiral and bears numerous cilia attached along the upper turns of the spire. The nucleus occupies the lower turns, and attached to them is a large spheroidal cyto])lasmic mass, which is cast off when the spermatozoid is set free or at the time it enters the archegonium. This, according to Strasburger, proba])ly corresi:)onds to the basal cytoplasmic mass of Cliara. The upper portion of the spire to which the ciHa are attached is composed of cytoplasm alone, as in CJiara. Ciliated spermatozoids, nearly similar in tyj^e to those of the higher cryptogams, have recently been discovered in the cycads by Hirase {Gingko), Ikeno (Cj'cas), and Webber (2'^?;;//^?). They are here hemispherical or pear-shaped bodies of relatively huge size (in Zamia upward of 250 /x in length), with a large nucleus filling most of the cell and a spiral band of cilia making from two to six turns about the smaller end (Figs. 8j, 108). As will be shown farther on (p. 173), the ** anterior " cytoplasmic region of the spermatozoid, to which the cilia are attached, is probably the analogue of the middle-piece of the animal spermatozoon ; and the work of Belajeff, Strasburger, Ikeno, Hirase, Webber, and Shaw gives good ground for the conclusion that it has an essentially simi- lar mode of origin, though w-e are still unable to say exactly how far the comparison can be carried. The *' posterior " region of the sper- matozoid appears to correspond, broadly speaking, to the acrosome. C. Origin of the Germ-cells Both ova and sj^ermatozoa take their origin from cells known as primordial germ-cells, which become clearly distinguishable from the somatic cells at an early period of development, and are at ftrst exactly alike in the two sexes. W^hat determines their subsequent sexual differentiation is unknown save in a few special cases. From such data as we possess, there is very strong reason to believe that, with a few exceptions, the primordial germ-cells are sexually indifferent, i.e. neither male nor female, and that their transformation into ova or spermatozoa is not due to an inherent predisposition, but is a reac- tion to external stimulus. Most of the observations thus far made indicate that this stimulus is given by the character of the food, and that the determination of sex is therefore in the last analysis a prob- lem of nutrition. Thus Mrs. Treat ('73) found that if caterpillars were starved before entering the chrysalis state they gave rise to a preponderance of male imagoes, w^hile conversely those of the same brood that were highly fed produced an excess of females. Yung ('81) reached the same result in the case of Amphibia, highly fed tadpoles producing a great excess of females (in some cases as high as 92%) and underfed ones an excess of males. The same result, again, is ORIGIN OF THE GERM-CEILS 145 given by the interesting experiments of Nussbaum ('97) on the rotifer Hydatina, which is an especially favourable case since sex is here de- termined at a very early period, before tJic egg is laid, the eggs being of two sizes, of which the smaller give rise only to males, and the larger only to females. The earlier experiments of Maupas ('91 ) on this form seemed to show conclusively that the decisive factor was temperature acting on the parent organism, since in a high tempera- ture an excess of females produced male eggs, and in a low tempera- ture the reverse result ensued. Nussbaum shows, however, that this is not a direct effect of temperature, but an indirect one due to the greater birth-rate and the greater activity of the animals under a higher temperature, which result in a speedier exhaustion of food. Direct experiment shows that, under equal temperature-conditions, well-fed females produce a preponderance of female offspring, and vice versa, precisely as in the Lepidoptera and Amphibia. These cases show that sex may be determined by conditions of nutrition either affecting the embryo itself (Lepidoptera, Amphibia) long after the ^^^^^ is laid, or by similar conditions affecting the parent-organism and through it the ovarian ^'g%. Nutrition is, however, not the only determining cause of sex, as is shown by the long-known case of the honey-bee. Here sex is deter- mined by fertilization, the males arising only from unfertilized eggs by parthenogenesis, while the fertilized eggs give rise exclusively to females, which develop into fertile forms (queens) or sterile forms (workers), according to the nature of the food. This is a very excep- tional case, yet here too it is the more highly fed larvae that produce fertile females. It is interesting to compare with this case that of the plant-lice or aphides. In these forms the summer broods, living under favourable conditions of nutrition, produce only females the eggs of which develop parthenogenetically. In the autunni, under less favourable conditions, males as well as females are produced ; and that this is due to the external conditions and not to a fixed cyclical change of the organism is proved by the fact that in the favourable environment of a greenhouse the production of females alone may continue for years. ^ We are not yet able to state whether there is any one causal ele- ment common to all known cases of sex-determination. The observa- tions cited above, as well as a multitude of others that cannot here be reviewed, render it certain, however, that sex as such is not inherited. What is inherited is the capacity to develop into either male or female, the actual result being determined by the combined effect of conditions external to the primordial germ-cell. 1 See Geddes, Sex, in Encyclopcedia Britannua ; Geddes and Thompson, T/ie Evolution of Sex, 1889; Brooks, T/ie Law of Heredity, 1 883; \yatase ('92), The Phenomena of Sex-differentiation. 146 THE GERM-CELLS In the greater number of cases the primordial germ-cells arise in a germinal epithelium which, in the cctlenterates (Fig. 72), may be a part of either the ectoderm or entoderm, and. in the higher types, is a modified region of the peritoneal epithelium lining the body-cavity. In such cases the primordial germ-cells may be scarcely distinguish- able at first from the somatic cells of the epithelium. But in other cases the germ-cells may be traced much farther back in the develop- ment, and they or their progenitors may sometimes be identified in the gastrula or blastula stage, or even in the early cleavage-stages. Thus in the worm Sagitta, Hertwig has traced the germ-cells back to ec ^ B Fig. 72. — Origin of the germ-cells in a hydro-medusa, Cladonevia. [WeismanN.] A. Young stage ; section through wall of manubrium of the medusa ; ova developing in the ectoderm (ec). B. Later stage, showing older ova {o) and "nutritive cells" (ri). The ova contain small nuclei probably derived from engulfed nutritive cells. two primordial germ-cells lying at the apex of the archenteron. In some of the insects they appear still earlier as the products of a large " pole-cell " lying at one end of the segmenting ovum, which divides into two and finally gives rise to two symmetrical groups of germ- cells. Hacker has recently- traced very carefully the origin of the primordial germ-cells in Cyclops from a "stem-cell " (Fig. 74) clearly distinguishable from surrounding cells in the early blastula stage, not only by its size, but also by its large nuclei rich in chromatin, and by its peculiar mode of mitosis, as described beyond. The most beautiful and remarkable known case of early differenti- ation of the germ-cells is that of Ascaris, where Boveri was able to trace them back continuously thiough all the cleavage-stages to the ORIGIN OF THE GERM-CELIS 147 two-cell stage ! Moreover, from the outset the progenitor of the germ- cells differs from the somatic cells not 07ily in the greater size and rich- ness of chromatin of its nuclei, but also in its mode of mitosis; for in all those blastomeres destined to produce somatic cells a portion of ^^g- 73- — Origin of the primordial germ-cells and casting out of chromatin in the somatic cells of Ascaris. [BoVERl.] A. Two-cell stage dividing; s. stem-cell, from which arise the germ-cells. />'. Tlie same from the side, later in the second cleavage, showing the two types of mitosis and the casting out of chromatin {c) in the somatic cell, C. Resulting 4-cell stage; the eliminated onromatin at c. D. The third cleavage, repeating the foregoing process in the two upper cells. the chromatin is cast out into the cytoplasm, where it degenerates, and only in the germ-cells is the sum-total of the chromatin retained. In Ascaris megalocephala univalens the process is as follows (Fig. yi): Each of the first two cells receives two elongated chromosomes. As 148 THE GERM-CELLS the ovum prepares for the second cleavage, the two chromosomes reappear in each, but differ in their behaviour ( F'ig. 73, A, B). In one of them, which is destined to produce only somatic cells, the thickened ends of each chromosome are cast off into the cytoplasm and degen- erate. Only the thinner central part is retained and distributed to the daughter-cells, breaking uj) meanwhile into a large number of segments which split lengthwise in the usual manner. In the other cell, which may be called the stem-all (Fig. J I, s), all the chromatin is preserved and the chromosomes do not segment into smaller pieces. The results are plainly apparent in the four-cell stage, the two somatic nuclei, which contain the reduced amount of chro- matin, being small and pale, while those of the two stem-cells are far larger and richer in chromatin (Fig. 73, C). At the ensuing division (Fig. 73, D) the numerous minute segments reappear in the two somatic cells, divide, and are distributed like ordinary chromosomes ; and the same is true of all their descendants thenceforward. The other two cells (containing the large nuclei) exactly repeat the history of the two-cell stage, the two long chromosomes reappearing in each of them, becoming segmented and casting off their ends in one, but remaining intact in the other, which gives rise to two cells with large nuclei as before. This process is repeated five times (Boveri) or si.\ (Zur Strassen), after which the chromatin- elimination ceases, and the two stem-cells or primordial germ-cells thenceforward give rise only to other germ-cells and the entire chromatin is preserved. Through this remarkable process it comes to pass that in this animal only tJic ij^crni-cclls receive the s/n/i- total of the cgg-cJiromatin Jianded dozen from the parent. All of the somatic cells contain only a portion of the original gcrm-substanee. " The original nuclear constitution of the fertilized (t^^ is transmitted, as if by a law of primogeniture, only to one daughter-cell, and by this again to one, and so on ; while in the other daughter-cells the chromatin in part degenerates, in ])art is transformed, so that all of the descendants of these side-branches receive small reduced nuclei." ^ It would be difficult to overestimate the imi)ortance of this dis- covery ; for although it stands at present an almost isolated case, yet it gives us, as I believe, the key to a true theory of differentiation development,^ and may in the end prove the means of explaining many phenomena that are now among the unsolved riddles of the cell. Hacker ('95) has shown that the nuclear changes in the stem- cells and primordial eggs of Cyclops show some analogy to those of Ascaris, though no casting out of chromatin occurs. The nuclei are very large and rich in chromatin as compared with the somatic cells, and the number of chromosomes, though not precisely determined, 1 Boveri, '91, p. 437- 2 qt p. 426. ORIGIN OF THE GERM-CELLS 149 is less than in the somatic cells (Fig. 74). Vom Rath, workin in the same direction, believes that in the salamander also the number of chromosomes in the early progenitors of the germ-cells is one-half that characteristic of the somatic cells. ^ In both these cases, the chromosomes are doubtless bivalent, representing two Fig. 74. — Primordial germ-cells in Cyclops. [Hackkk.] A. Young embryo, showing stem-cell {st). D. The stem-cell lias ciiviiled into two. giMut; rise to the primordial germ-cell {g). C. Later stage, in section; tlie iirimordial germ-cell has migrated into the interior and divided into two ; two groups of chromosomes in each. chromosomes joined together. In Ascaris, in Hke manner, each of the two chromosomes of the stem-cell or primordial germ-cells is probably plurivalent, and represents a combination of several units of a lower order which separate during the segmenLution ol the thread when the somatic mitosis occurs. 1 Cf. p. 256, Chapter V. 150 THE GERM-CELLS D. Growth and Differentiation of the Germ-cells I. The Oi'it))i {a) GnnvtJi and Xutritiou. — Aside from the transformations of the nucleus, which are considered elsewhere, the story of the ova- rian history of the (tg^^ is largely a record of the changes involved in nutrition and the storage of material. As the primordial germ-cells enlarge to form the mother-cells of the eggs, they almost invariably become intimately associated with neighbouring cells which not only support and protect them, but also serve as a means for the elabora- tion of food for the growing egg-cell. One of the simplest arrange- ments is that occurring in coelenterates, where the ^^^'g lies loose either in one of the general layers or in a mass of germinal tissue, and may crawl actively about among the surrounding cells like an Avia:ba. In such cases (hydroids) the (t'g'g may actually feed upon the surrounding cells, taking them bodily into its substance or fusing with them^ and assimilating their substance with its own. In such cases ( Tubiilaria, Hydra) the nuclei of the food-cells long persist in the egg-cytoplasm, forming the so-called '' pseudo-cells," but finally degenerate and are absorbed by the ^ZZ- ^^ would here seem as if a struggle for existence took place among the young ovarian cells, the victorious individuals persisting as the eggs ; and this view is probably applicable also to the more usual case where the ^gg is only indirectly nourished by its brethren. In most cases, as ovarian development proceeds, a definite associa- tion is established between the Q,gg and the surrounding cells. In one of the most frequent arrangements the ovarian cells form a regular layer ox follicle about the ovum (Figs. 59, 79), and there is very strong reason to believe that the follicle-cells are immediately concerned with the conveyance of nutriment to the o\'um. A num- ber of observers have maintained that the follicle-cells may actually migrate into the interior of the egg, and this seems to be definitely established in the case of the tunicates and mollusks (Fig. 75).^ Such cases are, however, extremely rare; and, as a rule, the material elaborated by the nutritive cells is passed into the o^gg either in solu- tion or in the form of granular or j^rotoplasmic substance.'"^ An interesting case of this kind occurs in the cycads, where, according to Ikeno ('9. Later stage, showing inward migration and absorption of fol- licle-cells. 152 THE GERM-CELLS ^g^ lies in the egg-tube just below a very large nurse-cell, which, when fully developed, has an enormous branching nucleus as shown in Fig. 163. In these two cases, again, the nurse-cell is character- ized by the extraordinary development of its nucleus — a fact which points to an intimate relation between the nucleus and the metabolic activity of the cell.^ In all these cases it is doubtful whether the nurse-cells are sister- cells of the egg which have sacrificed their own development for the sake of their companions, or whether they have had a distinct origin from a verv early period. That the former alternative is possible is shown bv the fact that such a sacrifice occurs in some animals after the eggs have been laid. Thus in the earthworm. Liimbricns tcrrcs- Fig. 76. — Egg and nurse-cell in the annelid, Ophryotrocka, [KORSCHELT.] A. Young stage, the nurse-cell («) larger than the egg {o). B. Growth of the ovum. C. Late stage, the nurse-cell degenerating. tris, several eggs are laid, but only one develops into an embryo, and the latter devours the undeveloped eggs. A similar process occurs in the marine gasteropods, where the eggs thus sacrificed may undergo certain stages of development before their dissolution. - ib) Diffcnntiation of tJic Cytoplasm and Deposit of Dcntoplasm. — In the very young ovum the cytoplasm is small in amount and free from deutoplasm. As the egg enlarges, the cytoplasm increases enormously, a process which involves both the growth of the pro- toplasm and the formation of passive deutoplasm-bodies suspended in the protoplasmic network. During the growth-period a peculiar body known as the yolk-nucIcus appears in the cytoplasm of many ova, and this is probably concerned in some manner with the growth 1 See p. 338. 2 See McMurrich, '96. GROWTH AND DIFFERENTIATION OF THE GERM-CELLS 153 of the cytoplasm and the formation of the yolk. Both its qxW\\\ and its physiological role are, however, still involved in doubt. The deutoplasm first appears, while the eggs are still very small, in the form of granules which seem to have at first no con.stant posi- tion with reference to the egg-nucleus, even in the same species. Thus Jordan ('93) states that in the newt {Dicniyctylus) the yolk may be first formed at one side of the ^g^ and afterward spread to other parts, or it may appear in more or less irregular separate patches which finally form an irregular ring about the nucleus, which at this period has an approximately central position. In some Amphibia Fig- 77- — Ovarian eggs of insects. [Korschklt.J A, Egg of the butterfly, Vanessa, surrounded by its follicle; above, thiee nurse-cells {>i.c.) wiiii branching nuclei; g.v. germinal vesicle. B. Egg of water-beetle, Dyliscus, living; the egg (o.v.) lies between two groups of nutritive cells ; the germinal vesicle sends amoeboid processes into the dark mass of food-granules. the deutoplasm appears near the periphery and advances inward toward the nucleus. More commonly it first appears in a zone surrounding the nucleus (Fig. yS, C, D) and advances thence toward the periphery (trout, Henneguy ; cephalopods, Ussow). In still others {e.g. in myriapods, Balbiani) it appears in irregular jxitches scattered quite irregularly through the ovum (Fig. 78, A). In Braiicliipus the yolk is laid down at the centre of the ^'^^g, while the nucleus lies at the extreme periphery (Brauer). These variations show in general no definite relation to the ultimate arrangement — a fact which proves that the eccentricity of the nucleus and the polarity of the 154 THE GERM-CELLS ^^g cannot be explained as the result of a simple mechanical dis- placement of the germinal vesicle by the yolk, as some authors have maintained. The primary ori^nn of the deutoplasm-grains is a question that involves the whole thcorv of cell-action and the relation of nucleus A B Fig. 78. — Young ovarian eggs, showing yolk-nuclei and deposit of deutoplasm. A. Myriapod {Gcophilits) with single "yolk-nucleus" (perhaps an attraction-sphere) and scat- tered deutoplasm. [Bai.BIAM.] D. The same with several yolk-nuclei, and " attraction-sphere," s. [Balbiam.] C. Fish {Scorpccna), with deutoplasm forming a ring al)out the nucleus, and an irregular mass of "eliminated chromatin" (? yolk-nucleus). [\'an Hammkkk.] D. Ovarian egg of young duck (three months) surrounded by a follicle, and containing a " yolk- nucleus," j.;/. [Mertens.] and cytoplasm in metabolism. The evidence seems perfectly clear that in many cases the deutoplasm arises in situ in the cytoplasm like the zymogen-granules in gland-cells. But there is now also a very considerable body of evidence indicating that a part of the egg-cytoplasm is directly or indirectly derived from the nucleus through the agency of the yolk-nucleus or otherwise ; and the GROWTH AND DIFFERENTIATION OF THE GERM-CELLS 155 subject can best be considered after an account of that bodv. It may be mentioned here, however, that a large number of observers have maintained a giving off of nuclear substance to the cytoplasm, in the form of actual buds from the nucleus (Blochmann, Scharff! Balbiani, etc.) as separate chromatin-rods or portions of the chromatin network (Fol, Blochmann, Van Bambeke, Erlanger, Mertens. Calkins, Nemec, etc.) or as nucleolar substance (Leydig, Balbiani, Will, Lev- dig, Henneguy), but nearly all of these cases demand reexamination. — S C Fig. 79. — Young ovarian eggs of birds and mammals. [Mertens,] A. Egg of young magpie (eight days), surrounded by the follicle and containing germinal vesicle and " attraction-sphere." B. Primordial egg (oogonium) of new-born cat, dividing. C. Kgg of new-born cat containing " attraction-sphere " {s) and centrosome. D. Of young thrusli sur- rounded by follicle and containing besides the nucleus an attraction-sphere and centrosome (s). and a yolk-nucleus (;'.«.). E. Of young chick containing nucleus, attraction-sphere, and f.itty deutoplasm-spheres (black). F. Egg of new-born child, surrounded by follicle ami containmg nucleus and attraction-sphere. {c) Yolk-7i?(c!e?(s.—T\\Q term yolk-nucleus or vitelline body ( Dottcr- kern, corps vitcllin) has been applied to various bodies or masses that appear in the cytoplasm of the growing ovarian ^^'^^•^ and it must be said that the word has at present no well-defined mean- ing. As originally described by von Wittich ('45) in the eggs of spiders, and later by Balbiani ('93) in those of certain myriapods. the yolk-nucleus has the form of a single well-defined spheroidal 156 THE GERM-CELLS mass which appears at a very early period and persists throughout the later ovarian history. In other forms there are several so-called ** yolk-nuclei," sometimes of fairly definite form as described in the Amphibia by Jordan ('93) and in some of the myriapods by Balbiani ('93). In some forms the numerous "yolk-nuclei" are irre,e;ular, ill- defined granular masses scattered through the cytoplasm, as described by Stuhlman {^%6) in the eggs of insects. In still others the "yolk- nucleus" or "vitelline body" closely simulates an attraction-sphere, being surrounded by distinct astral radiations and enclosing one or more central granules like centrosomes ( 6"r/'////.v, Balbiani, '93, and Li)uuliis, Munson, '98). Balbiani is thus led to regard the \'olk- nucleus in general as being a metamorphosed attraction-sphere. Miss Foot ('96) has brought forward evidence to show that the polar rings, observed in the eggs of certain leeches and earthworms, are also to be regarded as "yolk-nuclei" (Fig. 102). Henneguy ('93, '96) finally compares the yolk-nucleus to the macronucleus of the Infusoria (!). In the present state of the subject it is quite impossible to reconcile the discordant accounts that have been given regarding the structure, origin, and fate of the "yolk-nuclei", and from the facts thus far determined we can only conclude that the various forms of " yolk- nuclei " have little more in common than the name. It is, in the first place, doubtful whether the " yolk-nuclei " simulating an attrac- tion-sphere have anything in common with the other forms ; and Mertens ('93), Munson ('98), have shown that the young ovarian ova of various birds and mammals (including man) and of Liinuliis contain one or more "yolk-nuclei" in addition to the " attraction- sphere "(" vitelline body" of Munson). In the second place there seem to be two well-defined modes of origin of the yolk-nucleus. In one type, illustrated by Jordan's observations on the newt ('93), the " yolk-nuclei " arise separately /;/ situ in the cytoplasm without direct relation to the nucleus. The same is true of the small peripheral " yolk-nuclei " of Limulus (Munson). In a second and more frequent type the "yolk-nucleus" first appears very near to or in contact with the nucleus, suggesting that the latter is directly concerned in its formation. The latter is the case, for example, in the eggs of Cyma- too^astcr (Hubbard, '94) .S"j7/<,'-;/c?'/'// /as- (Henneguy, '96), the earthworm (Calkins, '95, Foot, '96), PolyzoiiiuDi and other myriapods (Nemec, '97, Van Bambeke, '98), Limulus (Munson, '98), Cypris (Woltereck, '98), and ]\[olguIa (Crampton, '99). In nearly all of these forms the yolk-nucleus first appears in the form of a cap closely applied to one side of the nucleus (Figs. 80, 81), sometimes so closely united to the latter that it is difficult to trace a boundary between them. At a later period the yolk-nucleus moves away from the nucleus and in GROWTH AND DIFFERENTIATION OF THE GERM-CELLS 157 most, if not in all, cases breaks up into smaller and smaller fra^^ments which contribute, directly or indirectly, to the cytoplasmic f^rowth. In all these cases the history of the yolk-nucleus is such as to indi- cate the participation of the nucleus in its formation. Calkins (95) endeavours to show that the yolk-nucleus in Luvibricus is directly derived from the nucleus by a casting out of a portion of the chro- r^\ r. p o ^^ H Fig. 80.— Yo'k-niicleus in earthworm, spider, and ascidian. [././?, Cai.KINS ; C-E, Va.n Bambeke; F-L Cka.mpton.] A. Early ovarian egg of Z,?Yr;«(5r/V//^. B. Later stage; fragmentation of yolk-nucleus. C. Ova- rian ^gg of PholcHS. D. Later stage; disintegration of yolk-nucleus. E. Remains of the yolk- nucleus scattered through the cytoplasm. F. Early stage of yolk-nucleus in Mo/^^uhi. G-I. Dis- integration of the yolk-nucleus and enlargement of the products to form deutoplasm-spheres. matin-reticulum — a result agreeing in principle with earlier obser- vations on other eggs by Balbiani, Henneguy, Leydig, Will, and other observers. This conclusion rests partly on the apparent direct continuity of yolk-nucleus and chromatin, partly on the staining- reactions. Thus when treated with the Biondi-Khrlich mixture (basic methyl-green, acid red fuchsin), the yolk-nucleus at first stains green like the chromatin, while the cytoplasm is red, and this is the case 158 THE GERM-CELLS even after the yolk-nucleus has quite separated from the nuclear membrane. Later, however, as the yolk-nucleus breaks up, it changes its staining power, and stains red like the cytoplasm. The later observations of Miss Foot ( '96) give ground to doubt the conclusion that the yolk-nucleus is here actually metamorphosed chromatin, for by the combined action of lithium carmine and Lyons blue its substance is sharply differentiated from the chromatin. Still later studies by Crampton (99) on Molgula demonstrate that in this case the volk-nucleus is not directly derived from chromatin, but they nevertheless indicate clearly the formation of the yolk-nucleus by or under the immediate influence of the nucleus — a conclusion also reached on less satisfactory evidence by Hubbard, Van Bambeke, Woltereck, and Nemec. The general morphological history of the yolk-nucleus is here closely similar to that of Lumbricus (Fig. 80), except that no direct continuity between it and the nuclear substance was observed. Stained with methyl-green-fuchsin the yolk-nucleus and major part of the nuclear substance stain red, while the scattered nuclear chromatin-granules and the cytoplasm stain green. Millon's test, combined with digestion-experiments and the foregoing staining- reactions, proves that the yolk-nucleus and the red staining nuclear substance consist of albuminous substance and differ widely from the general cytoplasm, which probably consists largely of nucleo- albumins {cf. p. 331). These reactions give strong ground for the conclusion that the substance of the yolk-nucleus, which progressively accumulates just outside the egg-nucleus, is formed through the direct activity of the latter, perhaps arising within the nucleus and passing out into the cytoplasm. It is possible, further, that 'even the scattered " yolk-nuclei " that seem to be of purely cytoplasmic origin may arise in a similar manner, either, as Crampton suggests, through the early formation and breaking up of a single yolk-nucleus, or in some less obvious way. Interesting questions are suggested by those " yolk-nuclei," such as occur in GcopJiilus and Limulus, that so closely simulate an attraction-sphere. Munson's observations show that this body ("vitelline body") first appears in the very young ova as a crescent applied to the nucleus precisely as in Molo;iila or Lumbricus, but containing one or more central granules (Fig. 81). In later stages it becomes spherical, moves away from the nucleus, and assumes the form of a typical radial attraction-sphere with concentric microsome- circles and astral rays. It is hardly possible to doubt that this body in Limulus is of the same general nature as the yolk-nucleus of Lumbricus, Molgula, Cypris, Cymatogaster, or PJiolcus ; and if it be a true attraction-sphere in the one case we must probably so regard it in all. This identification is, however, by no means complete; GROWTH AND DIFFERENTIATION OF THE GERM-CELLS 159 and even Munson's careful studies do not seem definitely to establish its connection with the attraction-sphere or centrosomc of the last oogonium-division. That a body simulating an attraction-sj)here and containing a central granule may arise dc ?iovo in the cytoplasm is shown by Lenhossek's observations on the spermatids of the rat (p. 170); and the central granule is in this case certainly not a centrosome, since the true centrosomcs are found in another part of the cell. It is quite possible that the "vitelline body" of Liviiihis may have a similar origin. Nemec ('97) finds in Polyzoniuvi in the earliest stages a single body applied to the nucleus and later two bodies, one of which enlarges to form a cap-shaped yolk- D Fig. 81. — Forms of yolk-nuclei in Limidus and Polyzonium. [./-C, MrNSON; D-F, Np:mkc.] A. Very young ovarian eggs oi Llmulus ; at the left "vitelline body" (t-) in the form of a cap on the nucleus; at the right older egg showing astral formation. B. Older stage of the same; "vitelline body" in the form of an attraction-sphere with central granule. C. Peripheral "yolk- nuclei" {y.n.) in Liviulus. D. Very early ovarian egg of a myriapod. Polyzonium, with yolk- nucleus. E. O der egg with yolk-nucleus and astral body ^a). F. Still later stage, beginning disintegration of the yolk-nucleus. nucleus Hke those described above, while the other assumes the structure of a radiating attraction-sphere containing a central granule (centrosome .''), and his observations suggest that the two bodies in question may have a common origin (Fig. 81). In none of these cases do the astral radiations, surrounding this body, seem to have any connection with cell-division, and it is probable that a careful comparison of their physiological significance here, in leucocytes, and in mitotic division, may give us a better under*^tand- ing of the general significance of astral formations in protoplasm. The fate and physiological significance of the yolk-nucleus arc still to a considerable extent involved in doubt. In many cases i1 l6o THE GERM-CELLS breaks up into smaller and smaller ^i^raniiles {Lunibricus, JlToIgida, Pliolciis, some myriapods, Antcdou), which scatter throu^^h the cyto- plasm and are believed by many observers ( Halbiani, Mertens, \Vill, Calkins, Crampton, Nemec), following the earlier views of Allen Thomson, to become directly converted into deutoplasm-spheres (Fig. 80). Other observers (Van Hambeke, Foot, Stiihlman, and others) adopt the original view of Siebold, that the fragments of the yolk-nucleus are absorbed or converted into protoplasmic elements and thus only indirectly contribute to the yolk. In still other cases {^c.g. the "vitelline body" of Lii)iiilus) the yolk-nucleus does not fragment, but seems to serve as a centre about which new deutoplasmic material is formed. A review of the general subject shows that we are justified only in the somewhat vague conclusion that the yolk-nucleus is probably involved in some manner in the general cytoplasmic growth ; and that the facts strongly suggest, though they hardly yet prove, that at least some forms of yolk-nuclei are products of the nuclear activity and form a connecting link between that activity and the constructive processes of the cyto- plasm. That the yolk-nuclei have no very definite morphological value, and that they are not necessary to growth, seems to be shown by Henneguy's observation, that in the eggs of vertebrates it is in some forms invariably present, in others only rarely, and in still others is quite wanting ('96, p. 162). If this be the case, we must conclude that the yolk-nucleus consists of material that contributes to the constructive process, but is not necessarily localized in a definite body. As to its exact role we are, as Henneguy has said, reduced to mere hypotheses.^ The facts indicate that this material is a prod- uct of the nuclear activity, and that it may in some cases contribute directly to formed elements of the cytoplasm. It is probable, how- ever, that beyond this the yolk-nucleus may su])ply materials, perhaps ferments, that play a more subtle part in the constructive process, and of whose physiological significance we are quite ignorant. The whole subject seems a most interesting and important one for further study of the actions of the cell in constructive metabolism, and it is to be hoped that further research will place the facts in a clearer light. 2. Origin of tJic Spermatozoon (a) General. — The relation of the various parts of the sperma- tozoon to the structures of the spermatid is one of the most interesting questions in cytology, since it is here that we must look for a basis of interpretation of the part played by the sperma- 1 '96, p. 170. GROWTH AND DIFFERENTIATION OF THE GERM-CELLS i6l tozoon in fertilization. Obviously the most important of the questions, thus suggested, is the source of the sperm-nucleus and centrosome, though the relation of the other parts to the spermatid- cytoplasm involves some interesting problems. Owing to the extreme minuteness of the spermatozoon, the changes involved in the differentiation of its various parts have always been, and in some respects still remain, among the most vexed of cytological questions. The earlier observations of Kolliker, Schweigger-Seidel, and La Valette St. George, already mentioned, established the fact that the spermatozoon is a cell; but it required a long series of subsequent researches by many observers, foremost among them La Valette St. George himself, to make known the general course of spermatogenesis. This is, briefly, as follows : From the primordial germ-cells arise cells known as spennatogonia} which at a certain period pause in their divisions and undergo a con- siderable growth. Each spermatogonium is thus converted into a spermatocyte, which by two rapidly succeeding divisions gives rise to four spermatozoa, as follows.^ The primary spermatocyte first divides to form two daughter-cells known as spermatocytes of the second order or sperm-mother-cells. Each of these divides again — as a rule, without pausing, and without the reconstruction of the daughter-nuclei — to form two speimatids or sperm-cells. Each of the four spermatids is then directly transformed into a single sperma- tozoon, its nucleus becoming very small and compact, its cytoplasm giving rise to the tail and to certain other structures. The number of chromosomes entering into the nucleus of each spermatid and spermatozoon is always one-half that characteristic of the tissue-cells, and this reduction in number is in most, if not in all, cases effected during the two divisions of the primary spermatocyte. The reduction of the chromosomes, which is the most interesting and significant feature of the process, will be considered in the following chapter, and we are here only concerned with the transformation of the sper- matid into the spermatozoon. All observers are now agreed that the nucleus of the spermatid is directly transformed into that of the spermatozoon, the chromatin becoming extremely compact and losing, as a rule, all trace of its reticular structure. It is further certain that in some cases at least the spermatid-centrosome passes into, or gives rise to, a part of the middle-piece, and that from it the axial filament grows out into the tail. The remaining structures arise, as a rule, from the cytoplasm, and both the acrosome and the envelope of the axial filament otten show a direct relation to the remains of the achromatic figure ( " ar- 1 The terminology, now almost universally adopted, is due to La \'alette St. George. Cj. Fig. 1 1 8. 2 See Fig. 119. M 1 62 THE GERM-CELLS choplasm " or " kinoplasm ") which is found in the spermatid in the form of a sphere (sometimes an attraction-sphere) or *' Nebenkern " or both. Apart from the nuclear history, these facts have been definitely determined in only a few cases, and much confusion still exists in the accounts of different observers. Thus a number of investigators (r.^. Platner, I'^ield, l^enda, Julin, Prenant, Xiessinor) have asserted that the centrosome passes into the acrosome, instead of M N Fig. 82. — Formation of the spermatozoon in an insect, Anasa. [Paulmier.] A. Telophase of secondary spermatocyte-division, showing extra chromosome (small dyad of Fig. 127) below. B. Reconstitution of the nuclei. C. Spermatid witli Nebenkern (A^) and acrosome ia). D. Nebenkern double, with centrosome between the two halves. E. F. G. Elon- gation of the spermatid, outgrowth of axial filament, migration of acrosome, H. Giant spermatid (double size) with two centrosomes and axial filaments. /. Giant spermatid (quadruple size) with four centrosomes and axial filaments. the middle-piece — a result which .stands in contradiction with the fact that durinc; fertilization in a lar^^^e number of accurately known cases the centrosome arises from or in immediate relation to the middle- piece (Amphibia, echinoderms, tunicates, annelids, mollusks, insects, etc.; see p. 212). The clearest and most positive evidence on this question, afforded by recent observations on the spermatogenesis of insects, annelids, mollusks. Amphibia, and mammals, leaves, however, little doubt that the former result was an error and that, as the facts GROWTH AND DIFFERENTIATION OF THE GERM-CELLS 163 of fertilization would lead us to expect, the centrosome of the sper- matid passes into the middle-piece. Accounts vary considerably regarding the origin of the acrosome, which according to most authors is of cytoplasmic origin, while a few describe it as arising inside or from the anterior part of the nucleus. (/7) Composition of the Spermatid. --1:\\q confusion that has arisen in this difficult subject is owing to the fact that the spermatid may contain, besides the nucleus and centrosome, no less than three addi- tional bodies, which were endlessly confused in the earlier studies on the subject. These are the Nebejikern} the attraction-sphere or idiozome (Meves), and the c/uvmatoid lYebenkorper {WQwd^). The Nebenkern (Fig. 82), first described by Biitschli C/ijin the spermatids of butterflies, was afterward shown by La Valette ('86), Platner (^S6, '89), and many later investigators to arise wholly or in part from the remains of the spindle of the second spermatocyte division. Its origin is thus related to that of an attraction-sphere (which it often closely simulates), since the latter likewise arises from the achromatic figure. To the remains of the spindle, however, may be added granular elements, probably forming reserve-material C'centro-deutoplasm of Erlanger), that are scattered through the cyto- plasm or aggregated about the equator of the spindle (Fig. 126). Thus the Nebenkern may have a double origin, though its basis is formed by the spindle-remains. The Nebenkern sometimes takes a definite part in the formation of the tail-envelopes and of the acro- some (insects), but in many cases it seems to be wholly wanting. - The idiozome is in some cases an undoubted attraction-sphere derived from the aster of the last division and at first containing the centro- some, e.g. in the earthworm as shown by Calkins ('95) and Er- langer ('96, 4), in the salamander and guinea-pig, Meves ('96, '99), and in Helix according to Korff ('99), though in later stages the centrosomes usually pass out of the body of the idiozome. In some cases, however (in the rat, according to Lenhossek, '99), the idiozome seems to arise independently through condensation of the cytoplasmic substance into a sphere having no relation to the centrosomes. In some cases the idiozomes of adjoining cells remain for a time con- nected by bridges of material (Fig. 7) representing the remains of the spindle, and hence corresponding to a Nebenkern {e.g. salaman- der, Meves, '96), and the distinction between Nebenkern and idio- zome here fades away. The idiozome is usually concerned in the formation of the acrosome (Amphibia, mammals), but sometimes seems 1 The English equivalent of this should be paranucleus, but the latter word has already been used in various other senses, and it seems preferable to retain Piiilschli's original (ier- man word. ^ For critical discussion, see Erlanger, '97, I. 164 THE GERM-CELLS to degenerate without contributing directly to the sperm-formation {Helix). The chromatoid Nebenkorper, finally, is a small rounded body, staining with plasma-stains, which appear always to degenerate without taking direct part in the formation of the spermatozoon. It is possibly an extruded nucleolus ( Lenhossek ). but its origin and meaning are not definitely known. (r) Traiisforniation of the Spermatid into tJie Spennatozobii. — In the works of earlier authors it is often impossible to distinguish Pig, 3^. — Formation of the spermatozoon from the spermatid in the salamander. [HER- MANN.] A. Young spermatid, showing the nucleus above, and below the colourless sphere, the ring, and the chromatic sphere. D. Later stage, showing the chromatic sphere and ring at the base of the nucleus. C. D. E. F. Later stages, showing the transformation of the chromatic sphere into the middle-piece (?«). which of the various achromatic elements mentioned above have been under observation. We may therefore confine ourselves mainly to the latest works, in which these distinctions are clearly recognized. Owing to their great size, the spermatozoa of Amphibia have been the subject of most careful study; yet a clearer view of the subject GROWTH AND DIFFERENTIATION OF THE GERM-CELLS 165 may, perhaps, be obtained by taking the spermatogenesis of annelids and insects as a basis of comparison. In the insects (butterflies), Biitschli showed, in 1871, that the tail is formed by an elon^Mtion of the cell-body, into which extends the elongated Nebenkern, now divided into two longitudinal halves (Fig. 82). Platner ('89), confirm- ing this observation, further showed that the Nebenkern (in Pyi^icra) consisted of two parts, stating that one ("large mitosome") gives rise to the investment of the axial filament, the other ("small mitosome ") to the middle-piece ; while a third still smaller body, described as a " centrosome," passes to the apex. The later works of HenkingCgi) and Wilcox ('95, '96) render it nearly certain that Platner confu.sed the acrosome with the centrosome, the first-named observer finding in Pyrrhocoris and the second in Calopteniis that Platner's "centrosome" is derived from the Nebenkern, while Wilcox traced the centrosome directly into the middle-piece. Paulmier, finally, has shown in Auasa that the axial filament grows out from the centrosome,^ proving that such is the case by the highly interesting observation that in giant spermatozoa, arising by the non-division of the primary or secondary spermatocytes, either two or four centrosomes are present, each of which gives rise to a single axial filament, though only one Nebenkern is present (Fig. 82). (The bearing of this important fact on the centrosome-question is indicated elsewhere.) These observations, made on three widely different orders of insects, seem to leave no doubt that in insects the centrosome lies in the middle-piece {i.e. at the base of the nucleus), while both the acrosome and the inner tail- envelopes are derived from the Nebenkern. The outer envelope of the tail is derived from unmodified cytoplasm. In the earthworm the phenomena are slightly different, the middle- piece arising from an idiozome or attraction-sphere (Calkins, 95), in which lies the centrosome (Erlanger, '96), while the Nebenkern seems to have no part in the formation of either acrosome or tail-envelopes.- We turn now to the Amphibia, elasmobranchs, and mammals, in which the same general result has been attained, though there is still some divergence of opinion regarding the exact history of the centro- some. Working on the basis laid by Flemming ('87, ^'^^X Hermann ('89) traced the middle-piece in the salamander to a " Nebenkorper," which he believed to be not a Nebenkern but an attraction-sphere. 1 Moore ('95) seems to have been the first actually to describe the outgrowth of the axial filament from the centrosome, in the elasmobranchs. It has since been described by Meves ('97, 2) and Hermann ('97) in the salamander, by Lenhossek ('97), Meves ('98, '99), and Bardeleben ('97) in the rat, guinea-pig, and man; by Godlewski ('97) and Korff ('99) in Helix, and by several others. 2 Calkins's preparations, which I have carefully examined, seem to leave no doubt that the middle-piece arises from a true attraction-sphere derived from the spindle-poles; but Erlanger believes that the granular " centrodeutoplasm " also contributes to the sphere. 1 66 THE GERM-CELLS consisting of three parts, lying side by side in the cytoplasm (Fig. 83). These are {a)2i colourless sphere, shown by Meves's later researches to be probably an attraction-sphere ; (/^) a minute, intensely staining cor- puscle, and ((•) a small, deeply staining ring. The concurrent results of Hermann ('89, '92, '97), IkMula ('93), and Meves ('96, '97, 2) have shown that the small corpuscle (r) is one of the ccutrosoDies of tlie spcnnatiti, and all these observers agree that it passes into or gives Fig. 84. — Formation of the spermatozoon in Amphibia. \^A-E. Salaviandra, MEVES; F-K. Aiiiphiiinta, McGkkijok,] A. Spermatid with peripheral pair of centrosomes lying outside the sphere, and axial filament. B. Centrosomes near the nucleus, outer one ring-shaped. C. Inner centrosome inside the nucleus, enlarging to form middle-piece. D. Portion of much older spermatid, showing divergence of two halves of the ring (/-). E. Portion of mature spermatozoon, showing upper half of ring at r, and the axial filament proceeding from it. F. Spermatid of ^;;///////wa, showing sphere-bridges and ring-shaped mid-bodies. G. Later stage; outer centrosome ring-shaped, inner one double; sphere {s) converted into the acrosome. //. Migration of the centrosomes. /. Middle-piece at base of nucleus, y. The inner centrosome forms the end-knob within the middle-piece, which is now inside the nucleus. K. Enlargement of middle-piece, end-knob within it; elongation of the ring. GROWTH AND DIFFERENTIATION OF THE GERM-CELLS i^y rise to the middle-piece. According to Meves, who has most thor- oughly studied the entire formation of the spermatozoon, the history of these parts is as follows : In the young spermatids the two centro- somes lie quite at the periphery of the cell (Fig. 84),^ and from the outer one growls out the axial filament. The two ccntrosomes, leav- ing the idiozome by which they are first surrounded, now pass inwards toward the nucleus, the outer one meanwhile becoming trans- formed into the ring mentioned above, while the axial filament passes throusfh it to become attached to the inner centrosome. The latter pushes into the base of the nucleus and enlarges enormously to form a cylindrical body constituting the main body of the middle-piece. The ring meanwhile divides into two parts, the anterior of which gives rise to a small, deeply staining body at the posterior end of the middle-piece identical with the "end-knob." The other half of the ring w^anders out along the tail, finally lying at the limit between the main part of the latter and the end-piece. The envelope of the axial filament, here confined to that side opposite the marginal fin (i.e. the ** ventral " side of Czermak), is formed by an outgrowth of the general cytoplasm along the axial filament. The fin and marginal filament are beheved by Meves, as I understand him, to be formed from the axial filament ('97, 2, p. 127).'^ The acrosome, finally, is formed from the idiozome which wanders around the nucleus to its anterior pole. McGregor's results on Ampliiinmi (99) agree in their broader features with those of Meves, but differ on two points, one of which is of great importance. The acrosome here arises from only a part of the sphere (idiozome), while a second smaller part passes to the base of the nucleus and forms the main part of the middle-piece. The inner centrosome passes into the middle-piece to persist as the end- knob from which the axial filament passes out into the tail (Fig. 84). The history of the sphere thus recalls the phenomena seen in the Xe- benkern of the insect-spermatid ; though the posterior moiety does not contribute to the tail-envelope, while the history of the inner centro.s' (^y»u>o^ram,„e (//-A", BeLAJF.FF), and Equisetum (L-N, .-/. Primary spermatogonium (two generations before the primary spermatocytes) in diyision showing centrosomes. B. Primary spermatocyte with pair of " blepharoplastoids " (centrosomes).' C. i^pindle of primary spermatocyte (f^rst maturation-division). D. Four of the eight secondary spermatocytes w,th blepharoplast. Z^-^ Prophase of second maturation-division. H Pair of spermatids (Gymno^^ram,„e) with blepharoplasts. /-J. Formation of the ciliated bandVrom the Dlepnaroplast A. Nearly ripe spermatozoid, showing ciliated band {b), nucleus, and "cyto- plasmic vesicle (the latter is ultimately cast off). E M. Spermatids of Equisetum. N Ripe spermatozoid from above, showing spiral ciliated band. O. Ripe spermatozoid of MarsiUa with very long spiral ciliated band. ". wm STAINING-REACriONS OF THE GERM-NUCLEI 175 Webber and Ikeno have produced apparently stron^r evidence ^ that they arise separately and de novo in the cytoplasm. After the ensu- ing division (by which the two spermatids are formed) the astral rays disappear, and the blepharoplast gives rise by a peculiar process to a long, spiral, deeply staining band, from which the ciha grow forth. The later studies of Shaw ('98, i) and Helajeff ('99) on the blepharo- plasts in Onoclea and Maisilia leave no doubt that these bodies are to be identified with centrosomes. In Marsilia Shaw first found the blepharoplasts lying at the poles of the spindle during the anaphase of the first maturation-division and very closely resembling centro- somes. Each blepharoplast, at first single, divides into two during the late telophase, and during the prophases of the second division the halves diverge to opposite poles of the nucleus and lie at the respec- tive spindle-poles. This account is confirmed by Belajeff, who shows further that during the prophases astral rays surround the blepharo- plasts, and a central spindle is formed between them (Fig. 88). Belajeff also finds centrosomes in all of the earher spermatogenic divisions. The blepharoplasts are thus proved to be, in one case at least, dividing organs w^hich in every way correspond to the centro- somes of the animal spermatocytes; and the justice of Belajeff's comparison is demonstrated. Shaw believed that the primary blepha- roplast, which by division gives rise to those of the two spermatids, arose de novo. He made, however, the significant observation that in Marsilia *' blepharoplastoids," exactly like the blepharoplasts, ap- pear at the spindle-poles of the preceding (antepenultimate) division, and that each of these divides into two in the late telophase. These are said to disappear, without relation to the blepharoplasts which at a slightly later period are found at the spindle-poles of the first matu- ration division ; but in view of the demonstrated continuitv of the blepharoplasts during the second division we may well hesitate to accept this result, as well as Webber's conclusion regarding the independent and separate origin of the blepharoplasts in Zauiia. In any case the facts give the strongest ground for the conclusion that the formation of the spermatozoids agrees in its essential features with that of the spermatozoa, and for the expectation that the history of the achromatic structures in fertilization will yet be found to shnw an essential agreement in plants and animals. E. Staining-reactions of the Germ-nuclei It was pointed out by Ryder in 1883 that in the oyster the germ- nuclei stain differently in the two sexes; for if the hermaphrodite 1 Dr. Webber has kindly given me an opportunity to look through his beautiful prepa- rations. 176 THE GERM-CELLS gland of this animal be treated with a mixture of saffranin and methyl- green, the egg-nuclei are coloured red, the s]:)erm-nuclei bluish green. A similar difference was afterward observed by Auerbach ('91) in the case of many vertebrate germ-cells, where the egg-nucleus was shown to have a special affinity for various red and yellow dyes (eosin, fuchsin, aurantia, carmine), while the sperm-nuclei were esj)e- cially stained with blue and green dyes (methyl-green, aniline-blue, hematoxylin). He was thus led to regard the chromatin of the ^'^^^^ as especially " erythrophilous," and that of the s])erm as " cyanophi- lous." That the distinction as regards colour is of no value has been shown bv Zacharias, Heidenhain, and others ; for staining-agents can- not be logically classed according to colour, but according to their chemical composition ; and a red dye, such as saffranin, may in a given cell show the same affinity for the chromatin as a green or blue dye of different chemical nature, such as methyl-green or hoema- toxylin. Thus Field has shown that the sperm-nucleus of Astcrias may be stained green (methyl-green), blue (haematoxylin, gentian violet), red (saffranin), or yellow (iodine), and it is here a manifest absurdity to speak of '' cyanophilous " chromatin ((/. p. 335). It is certainly a very interesting fact that a difference of staining-reaction exists between the two sexes, as indicating a corresponding difference of chemical composition in the chromatin ; but even this has been shown to be of a transitory character, for the staining-reactions of the germ-nuclei vary at different periods and are exactly alike at the time of their union in fertilization. Thus Hermann has shown that when the spermatids and immature spermatozoa of the salamander are treated with saffranin (red) and gentian violet (blue),^ the chromatic network is stained blue, the nucleoli and the middle-piece red ; while in the mature spermatozoon the reverse effect is produced, the nuclei being clear red, the middle-piece blue. A similar change of staining- capacity occurs in the mammals. The great changes in the staining- capacity of the egg-nucleus at different periods of its history are de- scribed at pages 338-340. Again, Watase has observed in the newt that the germ-nuclei, which stain differently throughout the whole period of their maturation, and even during the earlier phases of fertilization, become more and more alike in the later phases, and at the time of their union show identical staining-reactions. ^ A very similar series of facts has been observed in the germ-nuclei of plants by Strasburger (p. 220). These and many other facts of like import demonstrate that the chemical differences between the germ-nuclei are not of a fundamental but only of a secondary character. They are doubtless connected with the very different character of the meta- bolic processes that occur in the history of the two germ-cells ; and 1 By Flemming's triple method. ^ '92, p. 492. LITERATURE 1 77 the difference of the staining-reaction is probably due to the fact that the sperm-chromatin contains a higher percentage of nucleinic acid, while the egg-chromatin is a nuclein containing a much higher percentage of albumin. LITERATURE. IIP Ballowitz, E. — Untersuchungen tiller die Struktur der Spermatozoen: i. {birds) Arch. )nik. Aiiat., XXXII. 1888; 2. {insects) Zeitschr. iviss. ZooL, L. 1890: 3. {fishes, amphibia., reptiles) Arch. niik. Anat., XXXVI. 1890: 4. {tnani- vtals) Zeit. wiss. Zool.. LII. 1891. Belajeff, W. — Uber die Centrosomen in den spermatogenen Zellen : Ber. d. deuisch. bot. Ges., XVII., 6. 1899. Boveri, Th. — Uber Differenzierung der Zellkerne wahrend der Furchung des Eies von Ascaris meg.: Anat. Anz. 1887. Id. — Die Entwicklung von Ascaris megalocephala mit besonderer Rucksicht auf die Kernverhaltnisse : Festschr. fur C. v. Knpffer. Jena, 1899. Brunn, M. von. — Beitrage zur Kenntniss der Samenkorper und ihrer Entwickelung bei Vogeln und Saugethieren : Arch. mik. Anat., XXXIII. 1889. Hacker, V. — Die Eibildung bei Cyclops und Camptocanthus : Zool. Jahrb.. \. 1892. (See also List V.) Hermann, F. — Urogenitalsystem : Struktur und Histiogenese der Spermatozoen : Merkel nnd Bonnefs Ergebnisse, II. 1892. Ikeno, S. — Untersuchungen Uber die Entwickelung der Geschechtsorgane, t'/t:., bei Cycas : Jahrb. wiss. Bot.., XXXI L, 4. 1898. Kolliker, A. — Beitrage zur Kenntniss der Geschlechtsverhaltnisse und der Samen- fliissigkeit wirbelloser Tiere. Berlin. 1841. Leydig, Fr. — Beitrage zur Kenntniss des thierischen Eies im unbefruchteten Zu- stande : Zool. Jahrb., III. 1889. Moves, F. — Uber die Entwicklung der mannlichen Gescheclitszellen von Salaman- dra : Arch. mik. A?iat.j XLVIII. 1896. Id. — t'ber Struktur und Histogenese der Samenfaden des Meerschweinchens : Arch. mik. Anat., LIV. 1899. Schweigger-Seidel, F. — Uber die Samenkorperchen und ihre Entwicklun-^ : Arch. 7)iik. Anat., I. 1865. Strasburger, E. — Histologische Beitrage; Heft IV.: Das \'erlialten des Pollens und die Befruchtungsvorgange bei den Gymnospermen, Schwarmsporen, prianz- liche Spermatozoiden und das Wesen der Befruchtung. Fischer, Jena, 1892. Thomson, Allen. — Article '' Ovum,'' in Todd's Cyclopedia of Anatomy and Physi- ology. 1859. Van Beneden, E. — Recherches sur la composition et la signification de I'd-'uf : .Mem. cour. de VAcad. roy. de Belgique. 1870. Waldeyer, W. — Eierstock und Ei. Leipzig, 1870. Id. — Bau und Entwickeluns der Samenfaden : I'erh. d. Anat. Gcs. Leipzig, 18S7. o 1 See also Literature, V., p. 287. N CHAPTER IV FERTILIZATION OF THE OVUM " It is conceivable, and indeed probable, that every part of the adult contains molecules derived both from the male and from the female parent; and that, regardcil as a mass of molecules, the entire organism may be compared to a web of which the warp is derived from the female and the woof from the male." IIUXLEY.i In mitototic cell-division we have become acquainted with the means by which, in all higher forms at least, not only the continuity of life, but also the maintenance of the species, is effected ; for through this beautiful mechanism the cell hands on to its descendants an exact dupli- cate of the idioplasm by which its own organization is determined. As far as we can see from an a priori point of view, there is no reason why, barring accident, cell-division should not follow cell-division in endless succession in the stream of life. It is possible, indeed prob- able, that such may be the fact in some of the lower and simpler forms of life where no form of sexual reproduction is known to occur. In the vast majority of living forms, however, the series of cell-divisions tends to run in cycles in each of which the energy of division finally comes to an end and is only restored by an admixture of living mat- ter derived from another eel I. This operation, known as fertiliza- tion or fecundation, is the essence of sexual reproduction ; and in it we behold a process by which on the one hand the energy of division is restored, and by which on the other hand two independent lines of descent are blended into one. Why this dual process should take place we are as yet unable to say, nor do we know which of its two elements is to be regarded as the primary and essential one. Harvey and many other of the early embryologists regarded fer- tilization as a stimulus, given by the spermatozoon, through which the ovum was " animated " and thus rendered capable of development. In its modern form this conception appears in the " dynamic " theories of Herbert Spencer, Biitschli, Hertwig, and others, which assume that protoplasm tends gradually to pass into a state of increasingly sta- ble equilibrium in which its activity diminishes, and that fertilization restores it to a labile state, and hence to one of activity, through mix- ture with protoplasm that has been subjected to different conditions. BiitschU ('76) pointed out that the life-cycle of the metazoon is com- 1 Evolution, in Science and Culture, p. 296, from Enc. Brit., 1878. 178 FERTILIZATION OF THE OVUM 179 parable to that of a protozoan race, a long series of cell-divisions being in each case followed by a mixture of protoplasms through conjuga- tion ; and he assumed that, in both cases, conjugation results in reju- venescence through which the energy of growth and division is restored and a new cycle inaugurated. The same view has been advocated by Minot, Engelman, Hensen, and many others. Mau- pas i^'^Z, '89), in his celebrated researches in the conjugation of Infu- soria, attempted to test this conclusion by following out continuously the hfe-history of various species through the entire cycle of their exist- ence. Though not yet adequately confirmed, and indeed opposed in some particulars by more recent work,^ these researches have yielded very strong evidence that in these unicellular animals, even under normal conditions, the processes of growth and division sooner or later come to an end, undergoing a process of natural ** senescence," which can only be counteracted by conjugation. That fertilization in higher plants and animals does in fact incite division and growth is a matter of undisputed observation. We know, however, that in parthe- nogenesis the ^^% may develop without fertilization, and we do not know whether the tendency to " senescence " and the need for fer- tilization are primary attributes of living matter. The foregoing views maybe classed together as the rejuvenescence theory. Parallel to that theory, and not necessarily opposed to or confirmatory of it, is the view that fertilization is in some way con- cerned with the process of variation. Long since suggested by Tre- viranus and more lately developed by Brooks ^ and Weismann '^ is the hypothesis that fertilization is a source of variation — a conclusion sug- gested by the experience of practical breeders of plants and animals. Weismann brings forward strong arguments against the rejuvenescence- theory, and regards the need for fertilization as a secondaiy acquisi- tion, the mixture of protoplasms to which it leads producing variations — or rather insuring their "mingling and persistent renewal"-' — which form the material on which selection operates. On the other hand, a considerable number of writers, including Darwin. Spencer, O. Hertwig, Hatschek, and others, believe that although crossing may lead to variability within certain limits, its effect in the long run tends to neutralize indefinite variability and thus to hold the species true to the type. It is remarkable that we should still remain uncertain as to the physi- ological meaning of a process so general and one that has been the subject of such prolonged research. Both the foregoing general views are in harmony with the results of Darwin's work on variation and with the experience of practical breeders, which have shown that 1 Cf. Joukowskv, '99. ' Amphimixis, 1S91. 2 The Law of Heredity, 1883. * '99. P- 326. i8o FERTIUZATIOX OF THE OVUM crossing produces both greater vigour and greater variability. In view of all the facts, however, we are constrained to the admission that the essential nature of sexual reproduction must remain undetermined until the subject shall have been far more thoroughly investigated, espe- cially in the unicellular forms, where the key to the ultimate problem is undoubtedly to be sought. A. Preliminary General Sketch Among the unicellular plants and animals, fertilization is effected bv means of conjugation, a process in which two individuals either fuse together permanently or unite temporarily and effect an exchange A "^ ■• "0 c • Fig. 89. — Fertilization of the egg of the snail, P/iysa. [Kostanecki and Wierzejski.] A. The eniire spermatozoon lies in the egg, its nucleus at the right, flagellum at the left, while the minute sperm-amphiaster occupies the position of the middle-piece. The first polar body has been formed, the second is forming. B. The enlarged sperm-nucleus and sperm-amphiaster lie near the centre; second polar body forming and the first dividing. The egg-centrosomes and asters afterward disappear, their place being taken by those of the spermatozoon. of nuclear matter, after which they separate. /;/ (7// the higher forms fertilization consists in the permanent fusion of tivo germ-cells, one of paternal and ofie of maternal origin. We may first consider the fer- tilization of the animal Qgg, which appears to take place in essentially the same manner throughout the animal kingdom, and to be closely paralleled by the corresponding process in plants. PRELIMINARY GENERAL SKETCH l8i Leeuwenhoek, whose pupil Hamm discovered the spermatozoa (1677), put forth the conjecture that the spermatozoon must pene- trate into the egg; and the classical experiments of Spallanzani on the frog's egg (1786) proved that the fertihzing element must be the spermatozoa and not the liquid in which they swim. The penetration of the ovum was, however, not actually seen until 1854, when Newport observed it in the case of the frog's Qg^\ and it was described by Pringsheim a year later in one of the lower plants, Gldigoniuui. The first adequate description of the process was given by Hermann Fol, in 1879,^ though many earlier observers, from the time of Martin Barry ('43) onward, had seen the spermatozoon inside the egg-enve- lopes, or asserted its entrance into the ^^g. In many cases the entire spermatozoon enters the (^^^^^ (mollusks, insects, nematodes, some annehds, Petromyson, axolotl, etc.), and in such cases the long flageilum may sometimes be seen coiled within the ^gg (Fig. 89). Only the nucleus and middle-piece, however, are concerned in the actual fertilization ; and there are some cases (echinoderms) in which the tail is left outside the Qgg. At or near the time of fertilization, the Qgg successively segments off at the upjxT pole two minute cells, known as t\\Q polar bodies {¥\gs. 89, 90, 116) or directive corpuscles, which degenerate and take no part in the subse- quent development. This phenomenon takes place, as a rule, imme- diately after entrance of the spermatozoon. It may, however, occur before the spermatozoon enters, and it forms no part of the process of fertilization proper. It is merely the final act in the process of maturation, by which the ^gg is prepared for fertilization, and we may defer its consideration to the following chapter. I. The Genn-miclei in Fertilization The modern era in the study of fertilization may be said to begin with Oscar Hertwig s discovery, in 1875, of the fate of the sperma- tozoon within the ^gg. Earlier observers had, it is true, paved the way by showing that, at the time of fertilization, the Q.gg contains two nuclei that fuse together or become closely associated before development begins. (Warneck, Biitschli, Auerbach, Wan IkMieden. Strasburger. ) Hertwig discovered, in the iigg of the sea-urchin {Toxopnenstes lividius\ that one of these nuclei belofii^s to the a^g, while the other is derived from the spermatozoon. This result was speedily confirmed in a number of other animals, and has since been extended to every species that has been carefully investigated. The researches of Strasburger, De Bary, Schmitz, Guignard, and others have shown that the same is true of plants. /// every known case an 1 See Pllenogenie, pp. 124 ff., for a full historical account. 1 82 FERTILIZATION OF THE OVUM essoitial pJuiiovioiou of fcrtilizatioi is tJic union of a spciin-uucItHSy of paternal origin, ivith an cgg-nuclcns, of maternal origin, to form tJie primary niielens of the emluyo. TJiis nucleus, knozi'n as tJie cleavage- or segmentation-nucleus, gives rise by divisioii to all t/w nuclei of the body, and hence ever]' nucleus of the child )nay contain nuclear substance y' derived from both parents. And thus Hcrtwi<^ was led to the conclu- sion ('84), independently reached at the same time by Strasburger, Kolliker, and Weismann, that the nucleus is the most essential cle- ment concerned in hereditary transmission. This conclusion received a strong suj^port in the year 1883, through the s]:)lendid discoveries of Van Heneden on the fertilization of the thread-worm, Ascaris megalocephala, the '. C. Immediately after entrance, showing entrance-cone. />». Rotation of the sperm-head, formation of the sperm-aster about the middle- piece. £. Casting off of middle-piece; centrosome at focus of the ravs (r/C Fig. 12). The changes figured occupy about eight minutes. F. G. Approach of the germ-nuclei ; growth of the aster. 1 C/. p. 170. 1 88 FERTILIZATIOX OF THE OVUM nomena in the sea-urchin Toxop)icustcs (Fig. 94). As described at page 197, the tail is in this case left outside, and only the head and middle-piece enter the egg. Within a few minutes after its entrance, and while still very near the periphery, the lance-shaped sperm-head, carrying the niiddle-})iece at its base, rotates through nearly or cpiite 180°, so that the pointed end is directed outward and the middle- piece is turned inward ( P^ig. 94, A-F)} During or shortly after the rotation appears a minute aster centring in or very near the middle- piece. As it enlarges, the middle-piece itself is thrown to one side (Fig. 12), where it soon degenerates, while in the centre of the aster a minute intensely staining centrosome may be seen, l^oth sperm- nucleus and aster now rapidly advance toward the centre of the egg, the aster leading the way and its rays extending far out into the cytoplasm and finally traversing nearly an entire hemisphere. The central mass of the aster comes in contact with the egg-nucleus, divides into two, and the daughter-asters pass to opposite poles of the egg-nucleus, while the sperm-nucleus flattens against the latter and assumes the form of a biconvex lens (Fig. 95). The nuclei now fuse to form the cleavage-nucleus. Shortly afterward the nuclear membrane fades away, a spindle is developed between the asters, and a group of chromosomes arises from the cleavage-nucleus. These are 36 or 38 in number ; and although their relation to the paternal and maternal chromatin cannot in this case be accurately traced, owing to the apparent fusion of the nuclei, there can be no doubt on general grounds that one-half have been derived from each germ- nucleus. The egg then divides into two, four, etc., by ordinary mitosis (Figs. 4, 52). In the type of fertilization just described, the polar bodies are formed long before the entrance of the spermatozoon and the germ- nuclei conjugate immediately upon entrance of the spermatozoon, fusing to form a true cleavage-nucleus. In a second and more frequent type {Ascaris, Fig. 90; PJiysa, Fig. 89; Nereis, Fig. 97; Cyclops, Fig. 98) the sperm-nucleus penetrates for a certain distance, often to the centre of the egg, and then pauses while the polar bodies are formed. It then conjugates with the re-formed egg- nucleus. In this case the sperm-aster always divides to form an amphiaster before conjugation of the nuclei, while in the first case the aster may be still undivided at the time of union. This difference is doubtless due merely to a difference in the time elapsing between entrance of the spermatozoon and conjugation of the nuclei, the amphiaster having, in the second case, time to ^ The first, as far as I know, to observe the rotation of the sperm-head was Flemming in the echinoderm-egg ('8i, pp. 17-19). It has since been clearly observed in several other cases, and is probably a phenomenon of very general occurrence. PRELIMINARY GENERAL SKETCH 189 form during extrusion of the polar bodies. The two types just described (Fig. 96) are connected by various gradations. Thus, in the lamprey, the frog, the rabbit, and in An.pln.xus one polar body is expelled before, and one after, the entrance of the sper- matozoon ; in the annelid Othryotrocha, entrance takes place when the first polar spindle is in the stage of the equatorial plate ; / T<,4»'us,e,, X icoo. (For later stages see t .g.^S^.)^ ^^^^^^^^^^ ^^ ^^^ sperm-nucleus against the A. Union of the nuclei ; extension egg-nucleus ; division of the aster. of the aster. B. Flattening 190 FERTILIZATION OF THE OVUM while in CJuctopterus and Pi en's the first polar spindle has advanced into the anaphase.^ It is an interesting and signiftcant fact that the aster or amphiaster always leads the way in the march toward the egg-nucleus ; and in many cases it may be far in advance of the sperm-nucleus.- Boveri ('87, I) has observed in sea-urchins that the sperm-nucleus may indeed be left entirely behind, the aster alone conjugating with the egg- Fig. 96. — Diagrams of two principal types of fertilization. /. Polar bodies formed after the entrance of the spermatozoa (annelids, mollusks, flat-worms). //. Polar bodies formed before entrance (echinoderms). A. Sperm-nucleus and centrosome at c^ ; first polar body forming at 9 . B. Polar bodies formed ; approach of the nuclei. C. Union of the nuclei. D. Approach of the nuclei. E. Union of the nuclei. F. Cleavage-nucleus. nucleus and causing division of the egg ivitJiout union of tJic gcnn- nuclci, though the sperm-nucleus afterward conjugates with one of the nuclei of the two-cell stage. This process, known as " partial fer- tilization," is undoubtedly to be regarded as abnormal. It affords, however, a beautiful illustration of the view that it is the centro- some alone that incites division of the egg, and is tJierefore the fer- tilizing element proper (Boveri, '87, 2). The foregoing facts lead us to a consideration of Boveri's theory of fertilization, which has for several years formed a central point of discussion. The ground for this theory had been prepared by Oscar ^ QC p. 181. * Cf. Kostanecki and Wicrzejski, '96. PRELIMINARY GENERAL SKETCH 191 Hertwig and Fol. The latter ('73) early reached the conclusion that the asters represented " centres of attraction " lying outside and independent of the nucleus. Oscar Hertwig showed, in 1^75, that Fig 97. — Fertilization of the egg of Nereis, from sections. \ a 400.) A. Soon after the entrance of the spermatozoon, showing the minute '>:^'''^:';^^^^J^:^'^l germinal vesicle disappearing, and the first polar mitotic figure forming ^ '^^^.^^'^ 'P*)^'' '^/'^ fent deutoplasm-spheres (slightly swollen by the reagents), the firm circles o Wrop^ J^,\^^^Z nucleus (J) advancing, a minute amphiaster in front of it; first polar '"'^ .^/.f;^ . "''^'^j ^'^I^ ' polar concentration of the protoplasm. C. Later stage; second polai '^;^'^, '*':" "^^.^.^jllve polar bodies formed; conjugation of the germ-nuclei; the egg-ccn.rosomes and asters have disappeared, leaving only the sperm-amphiaster {cf. tig. 60). in the sea-urchin egg, the amphiaster arises by the division of a single aster that first appears near the sperm-nucleus and accom,ianies it in its progress toward the egg-nucleus. A similar observation was soon afterward made by Fol ('79) i" the eggs of Asl.nas and Sagiita, and in the latter case he determined the fact that the astral 192 FERTILIZATIOX OF THE OVUM rays do not centre in the nucleus, as Hertwig described, but at a point in advance of it — a fact afterward confirmed by Hertwig himself and by l^overi {^'^'S), i ). Hertwig and Fol afterward found that in cases of polyspermy, when several spermatozoa enter the . Apjiroach of the egg-nucleus and sperm-nucleus, the latter accompanied by the sperm-amphiaster. C. Union of the nuclei. D. Later stage of last. E. Prophase of cleavage-spindle. /'. Anaphasr of the same; centrosome divided. G. H. I. Successive stages in the nuclear reconstitution and lormation of the daughter-amphiasters for the second cleavage. J. Two-cell stage. in the trout, in Chcetoptcrus, and in PJiysa. In Ascaris division of the centrosome first occurs at a somewhat later period ( Figs. 90, 176). If now the centrosomes were indeed permanent cell-organs, wc should thus reach the following result: During cleavage the cytoplasm of the blastomeres is derived from that of the egg, the centrosomes from I()5 FERTILIZATION OF THE OVUM the sptnnatozodn, ivJiilc the nuelei {chromatin) are equally derived from both germ-cells. There is very strong reason to accept the first part of this con- clusion (applying to nucleus and cytoplasm), but the question of the centrosomes remains an open one. The array of evidence given above, derived from the study of so many diverse groups, seems to place Boveri's lucid and enticing hypothesis upon a strong foundation. Two essential points still remain, however, to be determined : first, whether the facts observed in Ascaris, l^xhinoderms, PJiysa, Thalas- sema, and the like, are typical of all forms of fertilization ; and, second, whether, if so, the primary cleavage-centrosome is actually imported into the ^gg by the spermatozoon or is only formed under its influence out of the egg-substance. Both these questions have been raised by recent inves^tigators, apparently on good evidence, and some of this evidence is directly opposed to both of the principal assumptions of Boveri's theory. Thus, Wheeler ('97) has found that in Mycostoma both centrosomes are derived from the egg ; Carnoy and Le Brun ('97) maintain that in Ascaris one centrosome is derived from each of the germ-nuclei; in some moUusks, according to MacFarland ('97) and Lillie ('97), both egg-cehtrosomes and sperm-centrosomcs dis- appear, to be replaced by two centrosomes of unknown origin ; while recent botanical workers are unable to find any centrosomes in fertili- zation. These and other divergent results will be critically considered bevond (p. 208) in connection with a more detailed examination of the general subject. It may be pointed out here, however, that recent researches on spermatogenesis (p. 170) render it nearly certain that the centrosome of the sperm-aster cannot be the unmodified cen- trosome of the spermatid, since the latter, in some cases, enlarges to form a " middle-piece " or analogous structure that is far larger than the sperm-centrosome. W. UxMON OF THE GeRM-CELLS It does not lie within the scope of this work to consider the innumerable modes by which the germ-cells are brought together, further than to recall the fact that their union may take place inside the body of the mother or outside, and that in the latter case both eggs and spermatozoa are as a rule discharged into the water, where fertilization and development take place. The spermatozoa may live for a long period, either before or after their discharge, without losing their fertilizing power, and their movements may continue throughout this period. In many cases they are motionless when first discharged, and only begin their characteristic swimming move- ments after coming in contact with the water. There is clear evi- UNION OF THE GERM-CELLS 197 dence of a definite attraction between the germ-cells, which is in some cases so marked (for example in the polyp Roiilla) that when spermatozoa and ova are mixed in a small vessel, each ovum becomes in a few moments surrounded by a dense fringe of sperma- tozoa attached to its periphery by their heads and by their move- ments actually causing the ovum to move about. The nature of the attraction is not positively known, but Pfeffer's researches on the spermatozoids of plants leave little doubt that it is of a chemical nature, since he found the spermatozoids of ferns and of Sclni^iuc/ia to be as actively attracted by solutions of malic acid or malates (con- tained in capillary tubes) as by the substance extruded from the H J — \ '^- ■\stne 202 FERTILIZATIOX OF TJIK OVUM tozoon the germinal vesicle moves toward the periphery, its membrane fades away, and a radially directed mitotic figure appears, bv means of which the first polar body is formed (Fig. 97). Meanwhile the protoplasm flows toward the iipj)er })oIe, the peri-vitelline zone disap- pears, and the eneden), are often applied to the germ-nuclei before their union. These should, I think, be rejected in favour of Hertwig's terms egg-nucleus and sperm-nucleus, on two grounds: (i) The germ-nuclei are true nuclei in every sense, differing from the somatic nuclei only in the reduced number of chromosomes. As the latter character has recently been shown to be true also of the somatic nuclei in the sexual generation of plants (p. 275), it cannot be made the ground for a special designation of the germ-nuclei. (2) The germ-nuclei are not male and female in any proper sense (p- 243). UNION OF THE GERM-CELLS 203 the egg at any point, the sperm-nucleus first moves rapidly inward along an entrance-path that shows no constant relation to the position of the egg-nucleus and is approximately but never exactly radial, i.e. toward a point near the centre of the ^gg. After penetrating a Fig. 103. — Diagrams showing the paths of the germ-nuclei in four different eggs of the sea- urchin, Toxopneustes. From camera drawings of the transparent living eggs. In all the figures the original position of the egg-nucleus (reticulated) is shown at 9 ; the point at which the spermatozoon enters at E (entrance-cone). Arrows indicate the paths traversed by the nuclei. At tlie meeting-point (yl/) the egg-nucleus is dotted. The cleavage-nucleus in its final position is ruled in parallel lines, and through it is drawn the axis of the resulting cleavage- figure. The axis of the &gg is indicated by an arrow, the point of which is turned away from the micromere-pole. Plane of first cleavage, passing near the entrance-point, shown by the curved dotted line. certain distance its direction changes slightly to that of the copula- tion-path, which, again, is directed not precisely toward the egg- nucleus, but toward a meeting-point where it comes in contact with the egg-nucleus. The latter does not begin to move until the 'fc>iD 204 FERTILIZATION OF THE OVUM entrance-path of the sperm-nucleus changes to the copulation-path. It then begins to move slowly in a somewhat curved path toward the meeting-point, often showing slight amoeboid changes of form as it forces its way through the cytoplasm. From the meeting-point the apposed nuclei move slowly toward the point of final fusion, which in this case is near, but never precisely at, the centre of the (t^^^. These facts indicate that the paths of the germ-nuclei are deter- mined bv at least two different factors, one of which is an attraction or other dvnamical relation between the nuclei and the cytoplasm, the other an attraction between the nuclei. The former determines the entrance-path of the sperm-nucleus, while both factors probably operate in the determination of the copulation-path along which it travels to meet the egg-nucleus. The real nature of neither factor is known. Hertwig first called attention to the fact — which is easy to observe in the living sea-urchin egg — that the egg-nucleus does not begin to move until the sperm- nucleus has penetrated some distance into the egg and the sperm-aster has attained a considerable size; and Conklin (94) has suggested that the nuclei are passively drawn together by the formation, attachment, and contraction of the astral rays. While this view has some fiicts in its favour, it is, 1 believe, untenable, for many reasons, among which may be mentioned the fact that neither tlie actual paths of the pro-nuclei nor the arrangement of the rays support the hypothesis ; nor does it account for the conjugation of nuclei when no astral rays are developed (as in Protozoa or in plants). I have often observed in cases of dispermy in the sea-urchin, that both sperm-nuclei move at an equal pace toward the egg-nucleus ; but if one of them meets the egg-nucleus first, the movement of the other is immediately retarded, and only conjugates with the egg-nucleus, if at all, after a considerable interval ; and in polv.spermy the egg-nucleus rarely conjugates with more than two sperm-nuclei. Probably, therefore, the nuclei are drawn together by an actual attraction which is neutralized bv union, and their movements are not improbably of a chemotactic char- acter. Conklin (99) has recently suggested that the nuclei are drawn together by the agency of protoplasmic currents in the egg-substance. 3. Union of tJic Gcrm-nuclci. The CJiromosouics The earlier observers of fertilization, such as Auerbach, Stras- burger, and Hertwig, described the germ-nuclei as undergoing a com- plete fusion to form the first embryonic nucleus, termed by Hertwig the cleavage- or segmentation-nucleus. As early as 1881, however, Mark clearly showed that in the slug Li max this is not the case, the two nuclei merely becoming apposed without actual fusion. Two years later appeared Van Beneden's epoch-making work on Ascaris, in which it was shown not only that the nuclei do not fuse, but that they give rise to two independent groups of chromosomes which separately enter the equatorial plate and whose descendants pass separately into the daughter-nuclei. Later observations have given the strono-est reason to believe that, as far as the chromatin is con- UNION OF THE GERM-CELLS 205 cerned, a true fusion of the nuclei never takes place during fertili- zation, and that the paternal and maternal chromatin may remain separate and distinct in the later stages of development — possibly throughout life (p. 299). In this regard two general classes may be distinguished. In one, exemplified by some echinoderms, by Aviplti- oxus, Phallnsia, and some other animals, the two nuclei meet each other when in the reticular form, and apparently fuse in such a manner that the chromatin of the resulting nucleus shows no visible distinc- tion between the paternal and maternal moieties. In the other class, which includes most accurately known cases, and is typically repre- sented by Ascaris (Fig. 90) and other nematodes, by Cyclops (Fig. 98), and by Pterotrachea (Fig. 93), the two nuclei do not fuse, but only place themselves side by side, and in this position give rise each to its own group of chromosomes. On general grounds we may confi- dently maintain that the distinction between the two classes is only apparent, and probably is due to corresponding differences in the rate of development of the nuclei, or in the time that elapses before their union. ^ If this time be very short, as in echinoderms, the nuclei unite before the chromosomes are formed. If it be more prolonged, as in Ascaris, the chromosome-formation takes place before unicm. With a few exceptions, which are of such a character as not to militate against the rule, tJie number of cJiromosomcs arisitij^ f'om tJic germ-nuclei is always the same in botJi, and is one-half the number characteristic of the tissue-cells of the species. By their union, there- fore, the germ-nitclei give rise to an equatorial^ plate eonfanung the typical niLuiber of chromosomes. This remarkable discovery was first made by Van Beneden in the case of Ascaris, where the number of chromosomes derived from each sex is either one or two. It has since been extended to a very large number of animals and plants, a partial list of which follows. 1 Indeed, Boveri has found that in Ascaris both modes occur, though the fusion of the germ-nuclei is exceptional. (^Cf. p. 296.) 206 FERTILIZATIOX OF THE OVUM A Partial List showing the Number of Chromosomes Char- acteristic OF THE Germ-nuclei and Somatic Nuclei in Various Plants and Animals ^ Germ- Somatic V* -, /-» Nuclei. Nuclei. Name. Group. Authority. I 2 Ascaris me^alocephala. van univalens. Nematodes. Van Beneden, Boveri. 2 4 Id., var. bivalens. • » «% >» Ophi votrocha. Annelids. Korschelt. >» p St\ieop.sis. Tunicates. Julin. 4 Coronilla. Nematodes. Carnoy. » Pallavicinia. Hepatica.'. Farmer. >» Anthoceras. • 4 Davis. 6 I '* Spiroptera. Nematodes. Carnoy. » Prosthecerasus. Polyclades. Klinckostrom, Francotte. >» Nais. Phanerogams. Guignard. w Spirogyra. Conjugatae. Strasburger. ?> [.." Grvllotalpa. Insects. Vom Rath. ;> Caloptenus. ?» Wilcox. D.] /Equorea. Hydromedusae. Hacker. 7 H Pentatoma. Insects. Montgomery. 8 i6 Filaroides. Nematodes. Carnoy. » ~ Prosthiostomum. Polyclades. Francotte. » f" n Leptoplana. M ?> » [••1 Cycloporus. ?? *» » Hydropliilus. Insects. Vom Rath. ?> Phallusia. Tunicates. Hill. >» Li max. Gasteropods. Vom Rath. » r ~ Rat. Mammals. Moore. » p 0.\. guinea-pig. man. • ^ Bardeleben. ?> Ceratozamia. Cvads. Overton, Guignard. » Pinus. Coniferae. Dixon. »> Scilla. TriticLim. Angiosperms. Overton. ?) Allium. » Strasburger, Guignard. » Podoijhyllum. *« Mottier. 9 i8 Echinus. Ecliinoderms. Boveri. ?> Thysanozoon. Polyclades. Van der Stricht. » Sagitta. Chx'tognaths. Boveri. » Chittopterus. Annelids. Mead. » Ascidia. Tunicates. Boveri. lO 20 Lasius. Insects. Henking. II [22] Allolobophora. Annelids. Foot. 12 24 iMyzostoma. » Wheeler. ^ This table is compiled from papers both on fertilization and maturation, brackets are inferred. Numbers in UNION OF THE GEKM-CEU.S 207 Germ- Somatic TVT Nuclei. Nuclei. Name. Group. Authority. 12 24 Thalassema. Annelids. Griffin. II (12) 22 (24) Cyclops strenuus. Copepods. Riickert. 12 24 brevicomis. ?? Hacker. 5? J? Helix. Gasteropods. PlatnerA'cjm Rath. I-) V Branchipus. Crustacea. IJrauer. ?> V Pyrrhocoris. Insects. Henkin;:. J> yy Salmo. Teleosts. liohm. )7 7? Salamandra. Ampliibia. F^lemminjr. I") ?> Rana. «^ Vom Rath. V ?j Mouse. Mammals. Sobotta. •>■> >? Osmunda. Ferns. Strasburs^er. V ?j Lilium. Angiosperms. Strasbur;^er, Gui<(nard. 7> ?? Helleborus. ?7 Strasburger. ?> ?7 Leucojum, Pa^onia, Aconitum. 7? Overton. M 28 Tiara. Hydromedusas. Boveri. ,1? V Pieris. Insects. Henkinsr. i6 32 Cerebratulus, Alicrura. Nemertines. Coe. ?? 77 Pterotrachea, Carinaria, Phyllirhoe. Gastropods. Boveri. )) [,;] Diaptomus, Heterocope. Copepods. Riickert. ?? -"J Anomalocera, Euchaeta. 77 Vom Rath. • « .77J Lumbricus. Annelids. Calkins. i8 36 Torpedo, Pristiurus. Elasmobranchs. Riickert. [18(19)] 36(38) Toxopneustes. Echinoderms. Wilson. 30 [60] Crepidula. Gasteropods. ConkHn. 84 168 Artemia. Crustacea. Brauer. The above data are drawn from sources so diverse and show so remarkable a uniformity as to establish the general law with a very high degree of probability. The few known exceptions are almost certainly apparent only and are due to the occurrence of plurivalent chromosomes. This is certainly the case with Asarris {cf. j). ^j). It is probably the case with the gasteropod Ariou, where, as described by Platner, the egg-nucleus gives rise to numerous chromosomes, the sperm-nucleus to two only ; the latter are, however, plurivalent, for Garnault showed that they break up into smaller chromatin-bodies, and that the germ-nuclei are exactly alike at the time of union. W'c may here briefly refer to remarkable recent observations by Riickert and others, which seem to show that not only the paternal and mater- nal chromatin, but also the chromosomes, may retain their individu- ality throughout development.^ Van Keneden, the pioneer observer 1 '89, pp. 10, 2,2>' 208 FERTILIZATIOX OF THE OVUM in this direction, was unable to follow the paternal and maternal chromatin beyond the first cleavage-nucleus, though he surmised that they remained distinct in later stages as well ; but Rabl and Hoveri broujjht forward evidence that the chromosomes did not lose their identity, even in the resting nucleus. Riickert ('95, 3) and Hacker ('95, I ) have recently shown that in Cyclops \.\\ughout the whole body of the cell, but at the time of fusion of the germ-nuclei they degenerate completely. The peripheral portions of their fibres, however, may be followed, as stated above of PUuro- phyllidia, Prosthecerccus, etc., where the sperm-asters degenerate soon after their forma- tion, so that for a considerable period the tg^ is without trace of aster-llbres. Vet in all of those cases where the sperm-asters disappear and their centrosomes become lost among the other granules of the cell, we are justified in believing that the sperm-centrosomes nevertheless retain their identity, and later reappear in the cleavage-asters " ('98, p. 455). 214 FERTII.IZATroy OF THE OVUM wuuld thus seem to be of the same nature as the disappearance of the sperm-centrosomes, and both Hoveri's theory of fertilization and the f>-eneral hypothesis (^f the ]K"rmanence of the centrosomes would receive a serious bhnv. The investii^ators to whom these observations are due have ranged themselves in two gnnips in the interpretation of the phenomena. On the one hand. Lillie and Child do not hesitate to maintain that the centrosomes actually go out of existence as such, to be re-formed like the asters out of the egg-substance ; and that such a new forma- tion of centrosomes is possible seems to be conclusively shown by the experiments of Morgan and Loeb described at pages 2 1 5 and 307. On the other hand, Sobotta, .MacF'arland, Kostanecki, and Coe, relying partly on the analogy of other forms, partly on the occasional pres- ence of the centrosomes during the critical stage, urge that the dis- appearance of the sperm-centrosomes is only apparent, and is due to the disappearance of the asters, which renders difficult or impossible the identification of the centrosomes among the other protoplasmic granules of the c,^^. These authors accordingly still uphold Boveri's theory. It is difficult to sift the evidence at present, for it has now become very im])ortant to reexamine, in the light of these facts, those cases in which the absolute continuity of the centrosome has been main- tained - for example, in Ascaiis, ChcBtopterus, and TJialassema — in order to determine whether there may not be here also a brief critical period in which the centrosomes disappear. There are, however, some facts which tend to sustain the conclusion that even though the sperm-centrosomes disappear from view, there is some kind of genetic continuity between them and the cleavage-centrosomes. First, both Kostanecki and Wierzejski (96) and Coe ('98) have found that there is some variation in eggs apparently equally well preserved, a few individuals showing the sperm-centrosomes at the poles of the united nuclei at the same period when they are invisible in other individuals. Second, both these observers, Coe most clearly, have shown that the egg-centrosomes di.sappear considerably earlier than the sperm-cen- trosomes, and Coe has traced the sperm-centrosomes continuously to the exact points {tJic poles of tJie united nuclei) at zvhicJi the cleavage- centrosomes afterzvartl appear {V\g. 155). This important observation leads to the suspicion that the apparent disappearance of the centro- somes may be due to a loss of staining-capacity at the critical period, or that even though the formed centrosome disappears its substance reappears in its successor. Here again we come to the view sug- gested at page 1 1 1, that the centrosome may be regarded as the vehicle of a specific chemical substance which is transported to the nuclear poles by its division, and may there persist even though the body of the FERTILIZATION IN PLANTS 215 centrosome be no longer visible. On such a basis we may perhaps find a reconciliation between these observations and Boveri's theory, and may even bring the fertilization of plants into relation with it (p. 221). Even in case of the nucleus, universally recognized as a permanent cell-organ, it is not the whole structure that persists as such during division, but only the chromatin-substance — in some cases only a small fraction of that substance. The law of genetic continuity therefore would not fail in case of the centrosome, though only a portion of its substance were handed on by division ; and even if we take the most extreme negative position, assuming that the sperm-centrosome is wholly formed anew under the stimulus of the spermatozoon, we should still not escape the causal nexus between it and the centrosome of the spermatid. Boveri himself has suggested ^ that the ^gg may be incited to development by a specific chemical substance carried by the sperma- tozoon, and the same view has been more recently urged by IMead,- while Loeb's recent remarkable experiments on sea-urchins ('99) show that the ^^g may in this case {Arbacia) undergo complete parthe- nogenetic development as the result of artificial chemical stimulus.-"^ Assuming such a substance to exist, by what part of the spermato- zoon is it carried .-* It is possible that the vehicle may be the nucleus, which forms the main bulk of that which enters the Q.gg ; and this view seems to be supported by what is at present known of fertiliza- tion in the plants (p. 221). Yet when we regard the facts of fertili- zation in animals, taken in connection with the mode of formation of the spermatozoon, we find it difficult to avoid the conclusion that the substance by which the stimulus to development is normally given is originally derived from the spermatid-centrosome, is conveyed into the Qgg by the middle-piece, and is localized in the sperm-centro- somes which are conveyed to the nuclear poles during the am phi- aster-formation. Accepting such a view, we could gain an intelligible view of the genetic relation between spermatid-centrosome, middle- piece, sperm-centrosome, and cleavage-centrosomes, without commit- ting ourselves to the morphological hypothesis of the persistence of the centrosome as an individualized cell-organ. Such a conclusion, I believe, would retain the substance of Boveri's theory while leaving room for the abandonment of the too simple morphological form in which it was originally cast. D. Fertilization in Plants The investigation of fertilization in the plants has always lagged somewhat behind that of the animals, and even at the i)resent time 1 '91, p. 431. 2 '98, 2, p. 217. 8 (7: p. III. 2l6 FERTIUZATIOX OF THE OVUM uur knowledge of it is rather incomplete. It is, however, sufficient to show that the essential fact is everywhere a union of two germ- j^^jclci — a process agreeing fundamentally with that observed in animals. On the other hand, almost nothing is known regarding the centrosome and the archoplasmic or kinoplasmic structures; and most recent observations point to the conclusion that in the lowering plants and pteridophytes no centrosomes are concerned in fertilization. Manv earl v observers from the time of Pringsheim ('55) onward described a conjugation of cells in the lower plants, but the union of germ-Huclci, as far as I can find, was first clearly made out in the Howering plants by Strasburger in xZ'j'j-'j'^, and carefully described by him in 1884. Schmitz observed a union of the nuclei of the B Fig. 105. — I-'ertilization in Pilularia. [Cami'HELL.] A. /?. Early stages in tlie formation of the spermatozoid. C. The mature spermatozoid ; the nucleus lies above in the spiral turns; below is a cytoplasmic mass containing starch-grains {cf. the spermatozoJds of ferns and of Marsilia, Fig. 71). D. Archegonium during fertilization. In the centre the ovum containing the apposed germ-nuclei (d", 9 ). conjugating cells of Spirogyra in 1879, and made similar observations on other algx in 1S84, Among other forms in which the same phenomenon has been described may be mentioned Gidigoniuin (Klebahn, '92), Vauchcria (Oltmanns, '95), Cystopus (Wager, '96), Splurrothcca and /:";;;'.v////r (Harper, '96), /v/^/m' ( Farmer and Williams, '96, Strasburger, '97), Inisidioboliis (Fairchild, '97), Pilularia (Fig. 105, Campbell, '88), Onoclca (Shaw, '98, 2), Zamia (Webber, '97, 2), and Lilitim (Guignard, '91, Mottier, '97), Ginkgo (Hirase, '97).^ In all of these forms and many others fertilization is effected by the union of a single paternal and a single maternal uninucleated cell, such as occurs throughout the animal kingdom. There are, however, some apparently well-determined exceptions to this rule occurring in the "compound" multinucleate oospheres of some of the lower ^ For unicellular forms see pp. 228, 280. FERTILIZATION IN PLANTS 217 plants. In Albugo bliti (one of the Peronosporece), for example, as shown by the recent work of Stevens ('99), the mature ovum contains about a hundred nuclei, and is fertilized by a multinucleate proto- plasmic mass derived from the anthcridium, each nucleus of the latter conjugating with one of the egg-nuclei. But although the conjugat- ing bodies are here multinucleate, the germ-nuclei conjugate two and two (as is also the case in the multinucleate cysts of ActinospJuEriuDi, p. 279); and the case therefore forms no real exception to the general rule that one paternal nucleus unites with one maternal. C D Fig. 106. — Formation of the ovum and penetration of the pollen-tube in flowering plants. [Strasburger.] A. Embryo-sac of Monotropa, showing the division that follows the two maturation-divisions and produces the upper and lower " tetrads." B. The same, ready for fertihzation. sliowing ovum {0), synergidae {s), upper and lower polar cells (/), and antipodal cells ( Division of the cleavage-nu- cleus. — Cleavage-nucleus. Exchange and fusion of the germ-nuclei. Germ-nuclei. Formation of the polar bodies. Union of the gametes. Fig. 109. — Diagram showing the history of the micronuclei during the conjugation of Para- moecium. [Modified from Maupas.] ♦ -V and F represent the opposed macro- and micronuclei in the two respective gametes; circles represent degenerating nuclei ; Vjlack dots, persisting nuclei. of the species runs in cycles, a long period of multiplication by coil- division being succeeded by an *' epidemic of conjugation." which inaugurates a new cycle, and is obviously comparable in its physio- logical aspect with the period of sexual maturity in the Metazoa. If conjugation does not occur, the race rapidly degenerates and dies out ; and Maupas beheves himself justified in the conclusion that conju- 2^. FERTILIZATIOX OF THE OVUM o-ation counteracts the tendency to senile degeneration and causes rejuvenescence, as maintained bv Hiitschli and Minot.^ In StvloHVi/iia pustulata. which Miiupas followed continuously from the end of February until Juh", the first conjugation occurred on April 29th, after 128 bi-parti- tii.ns; and the epidemic reached its iieight three weeks later, after 175 bi-partitions. 'I'he descendants of individuals prevented from conjugation died out through '-senile iKueneracy." after 316 bii)artitions. Similar facts were observed in many other forms. The degeneracy is manifested by a very marked reduction in size, a partial atrophy of the cilia, and especially by a more or less complete ihxradation of the nuiUar apparatus. I n Stylonycliia pustulata and Onyiliodromus ^i^randis this process especially atTects the micronucleus, which atrophies, and finally disappears, though the animals still actively swim, and for a time divide. Later, the macronucleus becomes irregular, and sometimes breaks up into smaller bodies. In other cases, the degeneration first aflects the macronucleus, which may lose its chromatin, undergo fatty degeneration, and may finally disappear altogether {StylonycJiia tnvtilus), af'ter which the micronucleus soon degenerates more or less completely, and the race dies. It is a very significant fact that toward the end of the cycle, as the nuclei degenerate, the animals become incapable of taking food and of growth ; and it is probable, as Maupas points out. that the degeneration of the cytoplasmic organs is due to disturbances in nutrition caused by the degeneration of the nucleus. The more essential phenomena occurring during conjugation are as follows. The Infusoria possess two kinds of nuclei, a large macnmuclcHS and one or more small micronnclei. During conjuga- tion the macronucleus degenerates and disappears, and the micronu- cleus alone is concerned in the essential part of the process. The latter divides several times, one of the products, the gcrm-imclcus, conjugating with a corresponding germ-nucleus from the other indi- vidual, while the others degenerate as "corpuscules de rebut." The dual nucleus thus formed, which corresponds with the cleavage- nucleus of the ovum, then gives rise by division to both macronuclei and micronuclei of the offspring of the conjugating animals (Fig. 109). These facts may be illustrated by the conjugation of Paramaxium caiidatiim, which possesses a single macronucleus and micronucleus, and in which conjugation is temporary and fertilization mutual. The two animals become united by their ventral sides and the macronu- cleus of each begins to degenerate, while the micronucleus divides twice to form four spindle-shaped bodies (Fig. i 10, A, />). Three of these degenerate, forming- the "corpuscules de rebut," which play no further part. The fourth divides into two, one of which, the •' female pronucleus," remains in the body, while the other, or "male pronucleus," passes into the other animal and fuses with the female pronucleus (Fig. 1 10, C-H ). Each animal now contains a cleavage- nucleus equally derived from both the conjugating animals, and the latter soon separate. The cleavage-nucleus in each divides three 1 C/p. 179. // Fig. no. — Conjugation of Paramascium caudatum. [.-/-C, after R. Hertwic. ; D-K, after Maupas.] (The macronuclei dotted in all the figures.) A. Micronuclei preparing for their first division. R. Second division. C. Third division; three polar bodies or " corpuscules de rebut," and one dividing germ-nucleus in each animal. D. Exchange of the germ-nuclei. E. The same, enlarged. F. Fusion of the germ-nuclei. G. The same, enlarged. H. Cleavage-nucleus, {c) preparing for the first division. /. The cleavage- nucleus has divided twice, y. After three divisions of the cleavage-nucleus; macronucleus breaking up. K. Four of the nuclei enlarging to form new macronuclei. The first fission soon 226 FERTILIZATIOX OF THE OVUM times successively, and of the eight resulting bodies four become macronuclei and four micronuclei (Fig. no, H-K). By two suc- ceeding fissions the four macronuclei are then distributed, one to each of the four resulting individuals. In some other species the micro- nuclei are equally distributed in like manner, but in J\ caudatnni the j)rocess is more complicated, since three of them degenerate, and the fourth divides twice to produce four new micronuclei. In cither case at the close of the process each of the conjugating individuals B Fig. III. — Conjugation of Vorticellids. [MaL'TAS.] A. Attachment of the small frec-swimining microgamete to the large fixed macrogamete ; micronucleus dividing in each (Carc/iesiuin). B. Microgamete containing eight micronuclei; ; • imete four ( I'orticella). C. All but one of the micronuclei have degenerated as polar , . t>r"corpuscules de rebut." D. liach of the micronuclei of the last stage has divided into . to form the germ-nuclei ; two of these, one from each gamete, have conjugated to form the cleavage-nucleus seen at the left ; the other two, at the right, are degenerating. has given rise to four descendants, each containing a macronucleus and micronucleus derived from the cleavage-nucleus. From this time forward fission follows fission in the usual manner, both nuclei divid- ing at each fission, until, after many generations, conjugation recurs. Fssentially similar facts have been observed by Richard Hertwig and Maupas in a large number of forms. In cases of permanent conjugation, as in l\)rticclla, where a smaller microgamete unites with a larger ^nacrogamcte, the process is essentially the same, though the details are still more complex. Here the germ-nucleus derived from each gamete is in the macrogamete one-fourth and in the microgamete CONJUGATION IN UNICELLULAR FORMS 227 one-eighth of the original micronucleus (Fig. i 1 1 ). Each germ- nucleus divides into two, as usual, but one of the products of each degenerates, and the two remaining pronuclei conjugate to form a cleavage-nucleus. The facts just described show a very close parallel to those observed in the maturation and fertilization of the ^g^^. In both cases there is a union of two similar nuclei to form a cleavage-nucleus or its equivalent, equally derived from both gametes, and this is the pro- genitor of all the nuclei of the daughter-cells arising by subsequent divisions. In both cases, moreover (if we confine the comparison to the ^gg), the original nucleus does not conjugate with its fellow until it has by division produced a number of other nuclei all but one of which degenerate. Maupas does not hesitate to compare ^ Be Fig. 112, — Conjugation oi Noctiluca. [Ishikawa.] A. Union of the gametes, apposition of the nuclei. D. Complete fusion of the gametes. Above and below the apposed nuclei are the centrosomes. C. Cleavage-spindle, consisting of two separate halves. these degenerating nuclei or "corpuscules de rebut" with the polar bodies (p. 181), and it is a remarkable coincidence that their number, like that of the polar bodies, is often three, though this is not always the case. A remarkable peculiarity in the conjugation of the Infusoria is the fact that tJie germ-nuclei ujiite ivJien in tJic form of spimiies or mitotic figures. These spindles consist of achromatic fibres, or *'archoplasm," and chromosomes, but no asters or undoubted cen- trosomes have been thus far seen in them. During union the spindles join side by side (Fig. lio, G\ and this gives good reason to believe that the chromatin of the two gametes is equally distrib- uted to the daughter-nuclei as in Metazoa. In the conjugation of some other Protozoa the nuclei unite while in the resting state; but very little is known of the process save in the cystoflagellate Xocti- luca, which has been studied with some care by Cienkowsky and Ishikawa (Fig. 112). Here the conjugating animals completely fuse, but the nuclei are merely apposed and give rise each to one-half of 228 FERriUZATION OF THE OVUM the mitotic ^^wxo.. At either pole of the spindle is a centrosome, the orif(in of which remains undetermined. It is an interestini; fact that in Xoctilnca, in the gregarines, and probably in some other Protozoa, conjugation is followed by a very rapid multiplication of the nucleus followed, by a corresponding divi- sion of the cell-body to form "spores," which remain for a time closely aggregated before their liberation. The resemblance of this D Fig. 113. — Conjugation oi Spirogyta, [OVERTON.] A. Union of the conjugating cells {S. communis). B. The typical, though not invariable, mode of fusion in S. Weber i ; the chromatophore of the "female" cell breaks in the middle, while that of the " male " cell passes into the interval. C. The resulting zygospore filled with pyrenoids. before union of the nuclei. D. Zygospore after fusion of the nuclei and formation of the membrane. process to the fertilization and subsequent cleavage of the ovum is particularly striking. The conjugation of unicellular plants shows some interesting features. Here the conjugating cells completely fuse to form a "zygospore" (Figs. 113, 140), which as a rule becomes surrounded by a thick membrane, and, unlike the animal conjugate, may long remain in a quiescent state before division. Not only do the nuclei SUMMARY AXD COXCLUSION 220 unite, but in many cases the plastids also (chromatophores). In Spirogyra some interesting variations in this regard have been ob- served. In some species De Bary has observed that the long band- shaped chromatophores unite end to end so that in the zvgote the paternal and maternal chromatophores lie at opposite ends. In 5. Weberi, on the other hand, Overton has found that the single maternal chromatophore breaks in two in the middle and the paternal chromatophore is interpolated between the two halves, so as to lie in the middle of the zygote (Fig. 113). It follows from this, as De Vries has pointed out, that the origin of the chromatophores in the daughter-cells differs in the two species, for in the former case one receives a maternal, the other a paternal, chromatophore, while in the latter, the chromatophore of each daughter-cell is equally derived from those of the two gametes. The final result is, however, the same; for, in both cases, the chromatophore of the zygote divides in the middle at each ensuing division. In the first case, therefore, the maternal chromatophore passes into one, the paternal into the other, of the daughter-cells. In the second case the same result is effected by two succeeding divisions, the two middle-cells of the four- celled band receiving paternal, the two end-cells maternal, chro- matophores. In the case of a Spirogyra filament having a single chromatophore it is therefore "wholly immaterial whether the indi- vidual cells receive the chlorophyll-band from the father or the mother" (De Vriesy F. Summary and Conxlusion All forms of fertilization involve a conjugation of cells bv a process that is the exact converse of cell-division. In the lowest forms, such as the unicellular algae, the conjugating cells are, in a morphological sense, precisely equivalent, and conjugation takes place between corresponding elements, nucleus uniting with nucleus, cell-body with cell-body, and even, in some cases, plastid with plastid. Whether this is true of the centrosomes is not known, but in the Infusoria there is a conjugation of the achromatic spindles which certainly points to a union of the centrosomes or their equivalents. As we rise in the scale, the conjugating cells diverge more and more, until in the higher plants and animals they differ widely not only in form and size, but also in their internal structure, and to such an extent that they are no longer equivalent either morphologically (^r physiologically. Both in animals and in plants the paternal gerni- 1 De Vries's conclusion is, however, not entirely certain; for it is impossible to deter- mine, save by analogy, whether the chromatophores maintain their indivitluality in the zygote. 3^0 FERTJLIZATIOX OF THE OVUM cell loses most of its cytoplasm, the main bulk of which, and hence the main body of the embryo, is now supi)lied by the egg; and in the higher plants, the egg alone retains the plastids which are thus supplied by the mother alone. On the other hand, the paternal germ-cell is the carrier of something which incites the (t^z to develoi)ment, and thus constitutes the fertilizing element in the narrower sense. There is strong ground for the conclusion that in the animal spermatozocin this element is, if not an actual centro- some, a body or a substance directly derived from a centrosome of the parent body and contained in the middle-piece. Boveri's theory, according'- to which fertilization consists essentially of the replace- ment of a missing or degenerating egg-centrosomc by the importation of a sperm-centrosome, was stated in too simple and mechanical a form ; for the facts of spermatogenesis show conclusively that the sjiermatid-centrosome is not simply handed on unmodified by the spermatozoon to the Qgg. and the theory wholly breaks down in the case of the higher ])lants. l^ut although the theory probably cannot be sustained in its morphological form, it may still contain a large element of truth when recast in physiological terms. Like mitosis, fertilization is perhaps at bottom a chemical process, the stimulus to development being given by a specific chemical substance carried in some cases by an individualized centrosome or one of its morphological products, in other cases by less definitely formed material. In the case of animals, we cannot ignore the historical continuity shown in the origin of the spermatid-centrosomes, the formation of the middle-piece, and the origin of the sperm-centro- somcs and sperm-amphiaster in the (tgg, even though we do not yet know whether the sperm-centrosome is as such imported into the egg. And this chain of phenomena suggests that even in the higher plants, where no centrosomes seem to occur, the fertilizing substance, even if brought into the ^^^^ in an unformed state, may still be genetically related to the mitotic apparatus of the preceding division.^ Through the differentiation between the paternal and germ-cells in the higher forms indicated above, their original morphological ecjuivalence is lost and only the nuclei remain of exactly the same value. This is shown by their history in fertilization, each giving rise to the same number of chromosomes exactly similar in form, size, and staining-reactions, equally distributed by cleavage to the daughter-cells, and probably to all the cells of the body. Wc tJius find the essential fact of fertilization and sexual reproduction to be a union of equivalent nuclei ; and to tJiis all ot/ier processes are tributary. As regards the most highly differentiated type of fertilization and 1 Cf. Strasburger's view, ]). 221. LITERATURE 231 development we reach therefore the following conception . From the mother comes in the main the cytoplasm of the embryonic body which is the principal substratum of growth and differentiation. From both parents comes the hereditary basis or chromatin by which these pro- cesses are controlled and from which they receive the specific stamp of the race. From the father comes the stimulus inducing the organiza- tion of the machinery of mitotic division by which the ^^^^ splits up into the elements of the tissues, and by which each of the.se elements receives its quota of the common heritage of chromatin. Huxley hit the mark two score years ago when in the words that head this chap- ter he compared the organism to a web of which the warp is derived from the female and the woof from the male. Our principal advance upon this view is the knowledge that this web is probably to be sought in the chromatic substance of the nuclei; and perhaps we shall not push the figure too far if we compare the amphiaster to the loom on which the fabric is woven. LITERATURE. IV 1 Van Beneden, E. — Recherches sur la maturation de roeuf, la fecondation et la divi- sion cellulaire : Arch. Biol., IV. 1883. Van Beneden and Neyt. — Nouvelles recherches sur la fecondation et la division mitosique chez TAscaride megalocephale : Bull. Acad. roy. de Belgique. III. 14. No. 8. 1887. Boveri, Th. — Uber den Anteil des Spermatozoon an der Teilung des Eies : Sitz.- Ber. d. Ges.f. Morph. u. PJiys. in Miincheiu B. III., Heft 3. 1887. Id. — Zellenstudien, II. 1888. j Id. — Befruchtung: Merkel intd Bonnefs Ergeb7tisse,l. 1891. Id. — Uber das Verhalten der Centrosomen bei der Befruchtung des Seeigeleies. etc. : Verhandl. Phys. Med. Ges. Wnrsburg, XXIX. 1895. Biitschli, 0. — Studien Uber die ersten Entwicklungsvorgange der Eizelle, it. s. zc. : Abh. Senckenb. Ges., X. 1876. Coe, W. R., 99. The Maturation and Fertilization of the Egg of Cerebratulus : /^ool. Jahrb., XII. Fick, R. — tjber die Reifung and Befruchtuns; des Axolotleies : Zcitsclir. W'iss.Zodl., LVI. 4. 1893. Griffin, B. B. — Studies on the Maturation, Fertilization, and Cleavage ot Thalassema and Zirphaea: Journ. Morph., XV. 1899. Guignard, L. — Nouvelles etudes sur la fecondation: Ann. d. Sciences nat. Bot., XIV. 1 89 1. Hartog, M. M. — Some Problems of Reproduction, etc. : Quart. Journ. Mu. ^t/.. XXXIII. 1891. Hertwig, 0. — Beitrage zur Kenntniss der Bildung, Befruchtuiii; unci Teilunij iles tierischen Eies, I. : MorpJi. JaJirb., I. 1875. Hertwig, R. — Uber die Konjugation der Infusorien: Abh. d. bayr. Akad. d. ll'iss., II. CI. XVII. 1888-89. Id. — ijber Befruchtung und Konjugation : I'erh. deutsch. Zo'dl. Ges. Berlin, 1892. 1 See also Literature, V.. p. 2S7. 2}2 FERTILIZATION OF THE OVUM Kostanecki, K. v., and Wierzejski, A. — Uber das \'erhalten der sogen. achromati- schen Substanzcn iiu bctruchteten Ei : Arch. mik. A//a/.,XL\'\l. 2. 1896. Mark, E. L. — Maturation. Fecundation, and Segmentation of Umax ca/Jipcstris : Hull. Mm. Com p. Zool. Harvard Collci^e. L'ainhridi:^t\ Mass., \'l. 1881. Maupas. — Lc rejeunissement karyoganiique chez les Cili(5s : An/i. d. Zo'oL, 2"^« sorio. \11. 18S9. Mead, A. D. The Origin and Heliaviour of the Centrosomes of tlie Annelid Egg: Journ. Morph., X I \'. 2 . 1 898. Ruckert, J. — Uber das Selbstiindigbleiben der vaterlichen und miittcrlichen Kern- substanz wiihrend der ersten Entwickiung des befruchteten Cyclops-Eies : Arch. mik. .htat.. \L\'. 3. 1895. Strasburger, E. — Neue L'ntersuchungen iibcr den Hefruchtungsvorgang bei den I'h.inerogamen, als Crundlage t'iir eine Theorie der Zeugung. Jena, 1884. Id. — L'ber Kern- und Zellteilung im Tflanzenreich, nebst einem Anhang liber Bcfnichtung. Jena, 18S8. (See Literature II.) Vejdovsky, F. — EntwickeUmgsgeschichtliche Untersuchungen, Heft i. Reifung, liefruchtung und Furchung des Rhynchehnis-Eies. Prag, 1888. Waldeyer, W. — Befruchlung und \'ererbung : / 'crh. Ges. deutsch. iVaturf. u. Aerzte^ \k\\. 1897. Wilson, Edm. B. — Atlas of Fertilization and Karyokinesis. A\^d' York, 1895. Zoja, R. — Stato Attuale degli Studi sulla Fecondazione : Boll. Scientif. di Pavia-, XVlil.. XIX. 1896-97. CHAPTER V OOGENESIS AND SPERMATOGENESIS. REDUCTION OF THE CHROMOSOMES " Es konimt also in der Generationenreihe der Keimzelle irgendwo zu einer Reduktion der ursprunglich vorhandenen Chromosomenzahl auf die Halfte, und diese Zr?///^';;. reduk- tion ist demnach nicht etwa nur ein theoretisches Postulat, sondern eine Thatsache." BOVKKI.I Van Beneden's epoch-making discovery that the nuclei of the con- jugating germ-cells contain each one-half the number of chromosomes characteristic of the body-cells has now been extended to so many plants and animals that it may probably be regarded as a universal law of development. The process by which the reduction in number is effected, forms the most essential part of the phenomena of matura- tion by which the germ-cells are prepared for their union. No phe- nomena of cell-life possess a higher theoretical interest than these. For, on the one hand, nowhere in the history of the cell do we find so unmistakable and striking an adaptation of means to ends or one of so marked a prophetic character, since maturation looks not to the present but to the future of the germ-cells. On the other hand, the chromatin-reduction suggests questions relating to the morphological constitution of nucleus and chromatin, which have an important bearing on all theories of the ultimate structure of living matter and now stand in the foreground of scientific discussion among the most debatable and interesting of biological problems. Two fundamentally different views have been held of the manner in which the reduction is effected. The earlier and simpler view, which was suggested by Van Beneden and adopted in the earlier works of Weismann, Boveri, and others, assumed an actual degenera- tion or casting out of half of the chromosomes during the growth of the germ-cells — a simple and easily intelligible process. Later researches conclusively showed, however, that this view cannot be sustained, and that reduction is effected by a rearrauiienient and redis- tribution of the 7iuciear siibstance without loss of an\' of its essential constituents. It is true that a lars^e amount of chromatin is lost dur- ing the growth of the Qggr- It is nevertheless certain that this loss is not directly connected with the process of reduction ; for, as Hertwig 1 Zellenstiidien, IH., p. 62. '^ Cf. Figs. 97, 116. 234 REDUCTION OF THE CHROMOSOMES and others have shown, no such loss occurs during spermatogenesis, and even in the oogenesis the evid^ence is clear that an explanation must be sought in another direction. The attempts to find such an explanation have led to some of the most interesting researches of modern cytology ; and though only partially successful, they have raised manv new questions which ])romise to give in the end a deeper insight into some of the fundamental questions of cell-morphology. Kor this reason they deserve careful consideration, despite the fact that taken as a whole the subject still remains an unsolved riddle in the face of which we can only return again and again to Boveri's remark that whatever be its theoretical interpretation the numerical reduction of the chromosomes is itself not a theory but a fact. ABC Fig. 114. — Kormation of the polar bodies before entrance of the spermatozoon, as seen in the living ovarian egg of the sea-urchin, Toxopneustes (X 365). A. Preliminary change of form in the germinal vesicle. B. The first polar body formed, the second forming, C. The ripe egg, ready for fertilization, after formation of the two polar bodies (/. ^. 1. 2) ; c. the egg-nucleus. In this animal the first polar body fails to divide. For its division see Fig. 89. A. General Outline The general phenomena of maturation fall under two heads : viz. Oflgcficsis, which includes the formation and maturation of the ovum, and spcrmatoi:;cn€sis, comprising the corresponding phenomena in case of the spermatozoon. Recent research has shown that maturation conforms to the same type in both sexes, which show as close a paral- lel in this regard as in the later history of the germ-nuclei. Stated in the most general terms, this parallel is as follows : ^ In both sexes the final reduction in the number of chromosomes is effected in the course of the last two cell-divisions, or Diatiiration-divisiojis, by which the definitive germ-cells arise, each of the four cells thus formed having but half the usual number of chromosomes. In the female but one ^ The parallel was first clearly pointed out by Plainer in 1 889, and was brilliantly demon- strated by Oscar Hertwig in the following year. GENERAL OUTLINE 235 of the four cells forms the " ovum " proper, while the other three, known as the polar bodies, are minute, rudimentary, and incapable of development (Figs. 89, 97, 114). In the male, on the other hand, all four of the cells become functional spermatozoa. This difference between the two sexes is probably due to the physiological division of labour between the germ-cells, the spermatozoa being motile and very small, while the ^gg contains a large amount of protoplasm and yolk, out of which the main mass of the embryonic body is formed. In the male, therefore, all of the four cells may become functional ; in the female the functions of development have become restricted to but one Primordial germ-cell. Oogonia. Primary oocyte or ovarian egg. Secondary oocytes (egg and first polar body). Mature egg and three polar bodies. ■ Division-period (the number of divi- sions is much greater). Growth-period. . Maturation-period. Fig. 115. — Diagram showing the genesis of the egg. [After BOVERI.] of the four, while the others have become rudimentary (r/. p. 124). The polar bodies are therefore not only rudimentary cells (Giard, '76), but may further be regarded as abortive eggs — a view first put forward by Mark in 1881, and ultimately adopted by nearly all investigators.^ The evidence is steadily accumulating that reduction is accomplished by two maturation-divisions throughout the animal kingdom, even in the unicellular forms ; though in certain Infusoria an additional divi- sion occurs, while in some other Protozoa only one maturation-division has thus far been made out. Among plants, also, two maturation- 1 A beautiful confirmation of this view is given by Francottes's ('97) observations on a turbellarian, Prosthecerceus. The first polar body is here often abnormally larj^c, all grada- tions having been observed from the normal size up to cells nearly as large as the egg itself. Such polar bodies are occasionally fertilized ^nA develop into small gastrulas, first forming a single polar body like the second polar body of the egg. Here, therefore, two of the four cells are exceptionally capable of development. It may be added that Fol long ago observed the penetration of the small polar bodies by spermatozoa in the echinoderms; and this has been more recently observed by Kostanecki in mollusks. 236 REDUCTIOy OF THE CHROMOSOMES divisions occur in all the hi^i^her forms (Muscineoe, pteridophytes, and phanerogams), and in some, at least, of the lower ones. Here, how- ever, the phenomena are complicated by the fact that the two divi- sions do not as a rule give rise directly to the four sexual germ-cells, but to four asexual spores which undergo additional divisions before the detinitive germ-cells are produced. In the flowering plants there are onlv a few such divisions, which give rise to structures within the pollen-tube or embryo-sac. In the archegoniate cryptogams, on the other hand, each spore gives rise, by repeated divisions, to a " sexual generation" (prothallium, etc.) that intervenes between the process of reduction and that of fertilization. The following account deals primarily with reduction in animals, the plants being afterward con- sidered. I . Reduction ill tlic Female. Fonnation of the Polar Bodies As described in Chapter III., the Q.gg arises by the division of cells descended from the primordial egg-cells of the maternal organism, and these may be differentiated from the somatic cells at a very early period, sometimes even in the cleavage-stages. As development pro- ceeds, each primordial cell gives rise, by division of the usual mitotic type, to a number of descendants known as oogonia (Fig. 115), which are the immediate predecessors of the ovarian (tgg. At a certain period these cease to divide. Each of them then grows to form an ovarian egg, its nucleus enlarging to form the germinal vesicle, its cytoplasm becoming more or less laden with food-matters (yolk or deutoj)lasm), while egg-membranes may be formed around it. The ovum may now be termed the oocyte (Boveri) or ovarian (igg. In this condition the egg-cell remains until near the time of fertili- zation, when the process of maturation proper — i.e. the formation of the polar bodies — takes place. In some cases, e.o;. in the sea-urchin, the polar bodies are formed before fertilization, while the (igg is still in the ovary. More commonly, as in annelids, gasteropods, nema- todes, they are not formed until after the spermatozoon has made its entrance ; while in a few cases one polar body may be formed before fertilization and one afterward, as in the lamprey-eel, the frog, and Ainphioxiis.^ In all these cases the essential phenomena are the same. Two minute cells are formed, one after the other, near the upper or animal pole of the ovum (Figs. 97, 116); and in many cases the first of these divides into two as the second is formed (Fig. 89). A group of four cells thus arises, namely, the mature Q,gg, w^hich gives rise to the embryo, and three small cells or polar bodies which take no part in the further development, are discarded, and soon die 1 Cf. p. 189. GENERAL OUTLINE 2^7 without further change. The egg-nucleus is now ready for union with the sperm-nucleus. f :y.: D — -p.v ^^^1. '-?^/i^ E H Fig. ii6. — Diagrams showing the essential facts in the maturation of the egg. The somatic number of chromosomes is supposed to be four. A. Initial phase; two tetrads have been formed in the germinal vesicle. />'. The two tctr.ids have been drawn up about the spindle to form the equatorial plate of the first polar mitotic figure. C. The mitotic figure has rotated into position, leaving the remains of the germinal vesicle at g.v. D. P'ormation of the first polar body ; each tetrad divides into two dyads. E. First polar body formed; two dyads in it and in the egg. F. lYeparation for the second division. G. Second polar body forming and the first dividing; each dyad divides into two single chromosomes. H. Final result; three polar bodies and the egg-nucleus (9). each con- taining two single chromosomes (half the somatic number) ; c. the egg-centrosome which now degenerates and is lost. 23S K EDUCTION OF THE CHROMOSOMES A Study of the nucleus during; these changes brings out the follow- ing facts. During the multijilication of the oogonia the number of chromosomes is the same as that occurring in the division of the somatic cells, and the same number enters into the formation of the chromatic reticulum of the germinal vesicle. During the formation of the polar bodies this number becomes reduced to one-half, the nucleus of each polar body and the egg-nucleus receiving the reduced number. In some manner, therefore, the formation of the polar bodies is connected with the process by which the reduction is ef- fected. The precise nature of this process is, however, a matter which has been certainly determined in only a few cases. We need not here consider the history of opinion on this subject further than to point out that the early observers, such as Purkinje, \'on Haer, Bischoff, had no real understanding of the process and believed the germinal vesicle to disappear at the time of fertilization. To Hiitschli ('76), Hertwig, and Giard ('76, ^TT) we owe the discovery that the formation of the polar bodies is through mitotic division, the chromosomes of the equatorial plate being derived from the chro- matin of the germinal vesicle.^ In the formation of the first polar body the group of chromosomes splits into two daughter-groups, and this process is immediately repeated in the formation of the second icithout ati intcrccniiig reticular rcstiiig stage. The egg-nucleus therefore receives, like each of the polar bodies, one-fourth of the mass of chromatin derived from the germinal vesicle. But alth(jugh the formation of the polar bodies was thus shown to be a process of true cell-division, the history of the chromosomes was found to differ in some very important particulars from that of the tissue-cells. The essential facts, which were first carefully studied in Ascaris by Van Beneden ('83, '87), and especially by Boveri ('87, i), are in a typical case as follows (Figs. 116, 117): As the &gg prepares for the formation of the first polar body, the chromatin of the ger- minal vesicle groups itself in a number of masses, each of which splits up into a group of four bodies united by linin-threads to form a "quadruple group" or tetrad (Vierergruppe). T/ie number of tetrads is ahuays oie-half the usual nundier of chromosomes. Thus in Ascaris {megalocephalay bivalens) \.\\c.gQrm\v\-a.\ vesicle gives rise to two tetrads, the normal number of chromosomes in the earlier divisions being four ; in the mole-cricket there are six tetrads, the somatic number of chromosomes being twelve ; in Cyclops the respectiv^e numbers are twelve and twenty-four (one of the most frequent cases); while in Artemia there are eighty-four tetrads and one hundred and sixty- ' The early accounts asserting the disappearance of the germinal vesicle were based on the fact that in many cases only a small fraction of the chromatic network gives rise to chromosomes, the remainder disintegrating and being scattered through the yolk. GENERAL OUTLIXE eight somatic chromosomes — the h,Vh» . , counted. As the first polar body foSs LT'^V'"^ '^' ^<^^"-tely to form two double groups, or 4X 2e "th' ^'1^ ^^''^ '^ ,'^^'-d remains in the Q™"'°f°'"'--» i" earlier divisions I^>1 C rb 'T 'k""'' '"<' «^™>'-' -slej. ^'Krrr":'r"''™"'- Z*- F'™ spindle fonninf Jir.-':L^:s;rd!io-'7"¥iyj^^ 3.n.le chro„oso,„es, co„,p,e,in, .h/;eL^'iot''^,^:r'frr-s4s'?etl^r9o.)"^ ''^^''^'' """ '"° 240 HE DUCT! ox OF THE CHROMOSOMES while the other passes into the polar body. Both the (i^g and the first polar body therefore receive each a number of dyads equal to one-half the usual number of chromosomes. The e^*; now proceeds at once to the formation of the second polar body without previous reconstruction of the nucleus. Tlach dyad is halved to form two single chromosomes, one of which, again, remains in the egg while its sister pas.ses into the polar body. Both the n F / / \ ® \ I I © l\ ® \ :)^ AT \ / X" II L Fig. 120. — Reduction in the spermatogenesis of Ascaris mcgalocephala,v7iX. bivalens. [Brauer.] 1 .^l-G. Successive stages in tlie division of the primary spermatocyte. The original reticulum undergoes a ver)' early division of the chromatin-granules which then form a doubly split spireme- tir'ad.Zf. This shortens (6), and breaks in two to form the two tetrads (Z) in profile, .fi" viewed endwise). F. G.H. Urst division to form two secondary spermatocytes, each receiving two dyads. /. Secondary sp divides once longitudinally, thegranulesof each constituent of the tetrad fuse to form i^ ■ -J ^ a. homogeneous sphere. giving the identical pairs or dyads — — £^, and once transversely, giving the tetrads — — -' ab cd '' '^ ^ " Inspection of Fig. 121, I, shows that through the second or transverse division, each member of the tetrad receives only half the number of ids contained in the original segment. This number, four, is the same as that assumed for a single chromosome ; and, since each of the two tetrads contributes one chromosome to the germ-cell, the latter receives 248 REDUCTJOX OF THE CHROMOSOMES but half the usual number both of chromosomes and of ids. This mode of tetrad-formation has been most clearly demonstrated in insects and copepods. and an equivalent ])rocess occurs also in mollusks, annelids, turbellarians, and some other animals, as described beyond. In the second type, illustrated especially by Ascaris, the tetrad is apparently formed by t:^>o longitudinal divisions of each primary chromatin-rod, and no reducing division occurs. If, therefore, we adopt the same terminology as before, we have first ab and cd. then it _ 12/, and hnall V — !— - — — , bv two longitudinal divisions. In ab id ' ab ! ab cd ^ cd this case, according to Brauer's careful studies, each chromatin-granule (** id") divides at each longitudinal division of the primary rod. The four chromosomes of the tetrad are therefore exactly equivalent, being derived from the same region of the spireme-thread, and containing the undiminished number of " ids " (Fig. 121, II). The contradiction may be stated in a different way. In the first type of tetrad formation, the number both of granules and of chro- mosomes is first doubled {i.e. in the assumed case, through the forma- tion of two tetrads, each consisting of four chromosomes, or eight in all), and then reduced to half that number by the two successive matu- ration-divisions. In the second type, on the other hand, the number of chromosomes is likewise doubled, but that of the granules is quad- rupled, so that, although in both types the two maturation-divisions reduce the number of elirouibsonies to one-half, only in the first type do they reduce the number of granules or **ids," as Weismann's hvpothesis demands. We must therefore distinguish sharply between the reduction of the chromosomes and that of the *'ids." The former is primarilv effected b\' the segmentation of the jDrimary s])ireme- thread, or the resolution of the nuclear reticulum, into one-half the usual number of segments (?>. the "pseudo-reduction" of Riickert); and here the real secret of the reduction of the chromosomes lies. The reduction of the "ids," if they have any real existence, is a distinct, and as yet unsolved, question. 2. Detailed Evidence We may now consider some of the phenomena in detail, though the limits of this work will only allow the consideration of a few typical cases. {a) Tetrad for)nation ivith one Longitudinal and one Trajisverse Division. — In many of the cases of this type the tetrads arise from ring-shaped bodies which are analogous to the ring-shaped chromo- somes occurring in heterotypical mitosis (p. %6). First observed by Henking('9i) in /^j';r//^rf7;7i-, tetrad-origin of this type has since been found in other insects by Vom Rath, Toyama, Paulmier, and others, ORIGIX OF THE TETRADS 249 in copepods by Riickert, Hacker, and Vom Rath, in pteridophytcs by Calkins and Osterhout, in the onion, Alliiiui, by Ishikawa, and in various other forms where their history has been less clearly made out. The genesis of the ring was first determined by \k)w\ Rath in the mole cr'ickQt {Gryllotalpa, '92), and has been thoroughly elucidated by the later work of Riickert ('94), Hacker ('95, i), and Paulmier ('99). All these observers have reached the same conclusion; namely, that the ring arises by the longitudinal splitting of a primary chromatin-rod, the two halves remaining united by their ends, and opening out to form a ring. The ring-formation is, in fact, a form of A D E F Fig. 122. — Origin of the tetrads by ring-formation in the spermatogenesis of the mole-cricket Gryllotalpa. [\'(m RATH.] A. Primary spermatocyte, containing six double rods, each of which represents two chromo- somes united' end to end and longitudinally split except at the free ends. B. C. Opening out of the double rods to form rings. D. Concentration of the rings. E. The rings broken up into tetrads. F. First division-figure established. heterotypical mitosis (p. S6). The breaking of the ring into four parts involves, first, the separation of these two halves (corresponding with the original longitudinal split), and second, the tniusrnsc division of each half, the latter being the reducing division of W'cismann. The number of primary rods, from which the rings arise, is one-half the somatic number. Hence each of them is conceived by Vom Rath, Hacker, and Riickert as bivalent or double ; i.e. as representing two chromosomes united end to end. This appears with the greatest clearness in the spermatogenesis of G)-yUotalpa (Fig. 122). Here 2;0 REDVCTIOiY OF THE CHROMOSOMES the spireme-thread splits lengthwise before its segmentation into rods. It then divides transversely to form six double rods (half the usual number of chromosomes), which open out to form six closed rings. These become small and thick, break each into four parts, and thus !©^y* Fig. 125. — Germinal vesicles of various eggs, showing chromosomes, tetrads, and nucleoli. A. .\ copepod {Heterocope) showing eight of the sixteen ring-shaped tetrads and the nucleo- lus. [krcKKki.] /?. I^ter stage of the same, condensation and segmentation of the rings. [RUCKERT.] C. " Cyclops strenuus," illustrating Hacker's account of the tetrad-formation from elongate double rods; a group of " accessory nucleoli." [Hackkr.] D. Germinal vesicle of an annelid {^Op/nyotrocha) -^how-xng nucleolus and four chromosomes. [KORSCHELT.] SO that the transverse division lies in the equatorial plane, and are halved during the formation of the second polar body. The division is accordingly a "reducing division," which leaves eleven single chro- mosomes in the egg. Paulmier's work on Auasa and other Hemip- tera ('99) gives the same result as the above in regard to the origin of the tetrads (Figs. 126, 127). The process is, however, slightly complicated by the fact that no continuous spireme-thread is formed, while the rings are often bent or twisted and never open out to a ORIGIN OF THE TETRADS 253 circular form. They finally condense into true tetrads which are successively divided into dyads and monads by the two divisions; but it is an interesting fact that the order of division occurring in the copepods appears here to be reversed, the first division being the transverse and the second the longitudinal one — a result agreeinc^ with Henking's earlier conclusion in the case of Pyrrochoris. '^Oster'- hout ('97) and Calkins ('97) independently discovered tetrads in the vascular cryptogams (Equisetum, Ptcris), and the last-named observer finds that in Ptej'is they may arise either from rings, as in (iryllotalpa or Heterocope, or from double rods as in Cyclops, the halves in the latter case being either parallel or forming a cross. This longitu- dinal spHt, occurring in the spireme, is followed by a trans\x'rse division by which the tetrad is formed. Tetrads having an essentially similar mode of origin are also described by Atkinson ('99) in Ari- scEma, and tetrad-formation is nearly approached in Allium according to Ishikawa ('99).^ These cases are considered at page 263. Resume. In all the foregoing cases the tetrads arise from a spi- reme which splits lengthwise, segments into one-half the somatic number of rods (each longitudinally divided) and each of the latter divides transversely to form the tetrad. When the ends of the daughter-chromosomes resulting from the longitudinal split remain united (as in insects) ring-forms result, and the earlier phases of tetrad- formation are thus identical with those of heterotypical mitosis. When the split is complete, so that the ends remain free, double rods result; while, if the daughter-chromosomes remain temporarily united at the middle or at the end, X-, Y-, and V-shaped figures may arise. In all these forms tetrad-formation is completed by the com- plete separation of the daughter-rods, the transverse division of each in the middle, and the condensation of the four resulting bodies into a quadruple mass. As will be shown in Section C (p. 258) the transverse division is in many forms delayed until after sepa- ration of the longitudinal halves. In such cases no actual tetrads are formed, though the result is the same. {I)) Second Type. Tetrad-formation ivith two Longitudinal Divi- sions. — The only accurately known case of this type is Ascaris, the object in which tetrads were first discovered by Van Beneden in 1883. Carnoy ('S6, 2) reached the conclusion that the tetrads in some other nematodes {Ophiostomum, Ascaris clavata, .i. lumbricoidcs) arose by a double longitudinal splitting of the primary chromatin-rods. 1 Vom Rath ('93, '59) has endeavoured to show that a process involving the formation of true tetrads occurs in the salamander and the frog, but the later and more accurate studies of Meves ('96) seem to leave little doubt that this was an error, and that the tetrads observed in these forms are not of normal occurrence, as Flemming ('87) had earlier concluded. Cf. p. 259. 234 REDUCTION OF THE CHROMOSOMES In the first of his classical cell-studies Boveri ('87, i) reached the same result through a careful study of Ascaris mcgaloccphala, showing that each tetrad appears in the germinal vesicle in the form of four j)arallel rods, each consisting of a row of chromatin-granules (Fig. 1 17, A-C ). He believed these rods to arise by the double longitudinal splitting of a single primary chromatin-rod, each cleavage being a Fig. 126. — Tetrad-formation in an insect, Anasa. [PauLMIER.] A. Resting spermatogonium with single plasmosome and two chromatin-nucleoli. B. Equa- torial plate of dividing spermatogonium ; twenty large and two small chromosomes, 6'. Final spcrmatogonium-division. D-I. Prophases of first maturation-division. D. E. Synapsis, with single chromatin-nucleolus. /'. Segmented split spireme. G. H. Formation of the tetrad-rings. //. /. Concentration of the rings to form tetrads. preparation for one of the polar bodies. In his opinion, therefore, the formation of the polar bodies differs from ordinary mitosis only in the fact that the chromosomes split very early, and not once, but twice, in preparation for two rapidly succeeding divisions without an intervening resting period. He supported this view by further obser- vations in 1890 on the polar bodies of Sagitta and several gastero- pods, in which he again determined, as he believed, that the tetrads ORIGIN OF THE TETRADS -'55 arose by double longitudinal splitting. An essentially similar view of the tetrads was taken by Hertwig in 1890, in the spermatogenesis of Ascaris, though he could not support this conclusion by very con- vincing evidence. In 1893, finally, Brauer made a most thorough and apparently exhaustive study of their origin in the spermatogene- sis of Ascaris, which seemed to leave no doubt of the correctness of Boveri's result. Every step in the origin of the tetrads from the retic- ulum of the resting spermatocytes was traced with the most pains- taking care. In the early prophases of the first division the nuclear reticulum breaks up more or less completely into granules, which ^^'m'> Fig. 127. — Maturation-divisions in an insect, Afiasa. [Paui.MIER.] A. Primary spermatocyte in metaphase. B. Equatorial plate, showing ten large tetrads and one small one; "odd chromosome" at o. C. Separation of the dyads. D. Telophase, which is also a prophase of the second division. E. Secondary spermatocyte ; division of the dyads ; small dyad shown undivided. F. Final anaphase ; small dyad near the lower chromosome-group. (The figures are numbered from left to right. For later states, see ¥\g. 82.) become in part aggregated in a mass at one side of the nucleus ("synapsis," p. 276), from which delicate threads e.xtend througii the remaining nuclear space (Fig. 120, A). Even at this period the granules of the threads are divided into four parts. As the process proceeds the chromatin resolves itself into a single spircme-thread, consisting of four parallel rows of granules, which break in two to form the two tetrads (var. bivalcns\ or is directly converted into a single tetrad (var. luiivalcns) (Fig. 120). From these observations Brauer concludes that each tetrad arises from a rod, doubly split lengthwise by a process initiated at a very early period through the 256 K EDUCTION OF THE CHROMOSOMES double fission of the chromatin-f^ranulcs. U this be correct, there can be no reduction in Weismann's sense ; tor the four products of each primary chromati^-^^■ranule are equally distributed among the four daughter-cells. A similar conclusion, based on much more incomi)lete evidence, was reached by Hrauer (92) in tlic phyllopod Branchipus. Hrauer's evidently conscientious figures very strongly sustain his conclusion, which, reinforced by the earlier work of Ilertwig and Boveri, has until now seemed to rest upon an unassailable basis. The recent work of Sabaschnikoff ('97) nevertheless raises the possi- bilitv of a different interpretation. l^rauer himself justly urges that the essence of the process lies in the double fission of the chromatin- granules to which the formation of chromosomes is secondary.^ Kvervthing, therefore, turns on the manner in which the quadruple granules arise ; and Sabaschnikoff's work gives some ground for the view that they may arise, not by a double fission, but in some other way. Accordin«r to this author there is a period (in the oiigcnesis) at which the nuclear threads wholly di.sappear, the entire chromatin Ijeing broken up into granules. From this state the granules emerge in quadruple form to arrange themselves in the doubly split spireme exactly as Brauer describes : and a few observations are given (regarding the size and arrangement of the granules) which suggest the possibility that the quadruple granules may arise by the conjugation either of four separate granules or of two pairs of double granules. Since there is ground for the view that tetrads may ari.se by the conjugation of chromosomes (see following section), there is no a priori objection to such a conclusion. Could it be sustained, the maturation- divisions Q>{ Ascaris would in fact involve a true reduction in Weismann s sense; for despite the fact that the chromosomes are only longitudinally divided, the four longitudinal constituents of each tetrad would not be equivalent with respect to the granules, and it is the reduction of the latter ('• ids'") that forms the essence of Weis- mann's hypothesis (p. 245). Another consideration, suggested to mc by Professor T. 11. Morgan, opens still another possibility, which .seems well worthy of test by further research. As already stated (p. 88), the long chromosomes oi Ascaris are plurivalent, since in all but the germ-cells each breaks up into a much larger number of smaller chromosomes (Fig. 73. p. U^)- If- therefore, the latter correspond to the chromosomes of other forms in which tetrads occur (i-.;'-. Cyclops or Arteniia), the .so-called " tetrad " of Ascaris is a compound body : and the true process of reduction mu.st be .sought in the origin of the smaller elements of which it is com- posed, which are, perhaps, directly comparable with Sabaschnikoff's "granules." Until the questions thus opened have been further studied, the case iox Ascaris must remain open : and it is perhaps worth suggesting that a new point of view may here be found for further study al.so of reduction in the vertebrates. - 1 Cf, p. 113. - Bodies closely resembling tetrads are sometimes formed in mitosis, where no reduction should occur. Thus, R. Hertwig ('95) has observed tetrads in the first cleavage-spindle of echinuderm-eggs after treatment with dilute poisons (p. 306). Klinckowstrom figures them in the .f.v^;/7 )■ These do not, however, con- « • ' • • • •• . B yi '• . • • ^ • ••• .•./•. c •• • • ^ • •••- • ,• •••• ' • • • . • • •^: ••• • •• • ^i • •• • • - •••• •• • • •••• • • • • . • • • . •• Fig. 129— Maturation and fertilization in an annelid (armed gephyrean) Thalassema. [(IklKIIN.j A. .\ few moments after entrance of the spermatozoon, showing accessory asters; tetrads forming. />'. T^-irly prophase of first polar mitosis with centrosomes. C. In-pushing of nuclear w;i!l. D. Central spindle established; elimination of nucleolus and nuclear reticulum. E. Slightly : ,t.r stage viewed from above. t\ First polar spindle established, cross-shaped tetrads, crossing of astral rays; sperm-head at j. dense into tetrads, but break apart during the first division at the points corresponding with the ends of the united halves. The first division is therefore an equation-division. As the V-shaped halves sep- arate they again split lengthwise ( Fig. 131), each of the secondary sper- matocytes receiving twelve double V's or dyads. In the telophases and ensuing resting stage, however, all traces of this splitting are lost, the nuclei partially returning to the resting stage, but retaining traces of a spireme-like arrangement (Fig. 131). In the second division twelve double V's reappear, showing a longitudinal division which Flemming and Meves believe to be directly related to that REDUCTION WITHOUT TETRAD-FORMATION 261 seen during the foregoing anaphases. There is therefore no evi- dence of a transverse division. McGregor ('99) describes a nearly sniiilar process in Amphiuma, where the longitudinal division of the A X \ !0 c 4"<** + r D ' I > -/^■^ \ ■'f^?^-^ ^Mil^ i».- y i> ■■ nf ! Z' J V>v^ J»s^n«* J ■^ G // y y Fig. 130. — Maturation in the lamellibranch Zirp/uca and in I/taiasst'/nii. [GRIFFIN.] A-E, Zirp/icea; F-/, Thalassema. A. Unfertilized ^gg, ring-shaped and cross-shaped chromosomes. D. Prophase of first polar mitosis. C. First polar spindle ; double crosses. D. Slightly later stage. E. The double crosses have broken apart (equation-division). G. Ensuing stage; daughter-V's broken apart at ihe apex. H. Telophase of first, early prophase of second, division ; limbs of tlie V's separate but closely opposed. F. Later prophase of second division. /. Second polar spindle in mctaphase. daughter-V's is seen with the greatest clearness throughout the anaphases. The weak point in both the foregoing cases is the fact that all traces of the second longitudinal division are lost during the ensuing 262 REDUCTIOX OF THE CHROMOSOMES resting period ; and I do not think that even the observations of Flemming (97), who has pubHshed the fullest evidence in the case, completely establish the occurrence of a subsequent longitudinal divi- Fig. 131. — (Compare Fig. 27). Maturation-divisions in Salanjatidra. [E from FlemMING, the others from Meves.] A. First division in mctaphase, showing heterotype rings. B. Anaphase; longitudinal split- ting of the daughter-loops. C. Telophase. D. Ensuing pause. E. Early prophase of second division with longitudinally divided segmented spireme. F. Later prophase. G. Metaphase of second division. sion of the chromosomes in the second mitosis. In DesmognatJins, however, where the resting stage is less complete, Kingsbury ('99) finds the longitudinal split in the persistent chromosomes of the REDUCTION WITHOUT TETRAD-FORMATION 263 pause following the first division; and he believes this to be the same division as that seen during the anaphase. Carnoy and Le Hrun ('99) reach the same result in the formation of the polar bodies in Triton, though their general account of the heterotypical mitosis differs very considerably from that of other authors, the rings being stated to arise by a double instead of a single longitudinal split These observers describe the rings of the early anaphase as having almost exactly the same double cross-form as those in Thahisscma ox Ztrphcea (Griffin, '99), but believe them to arise in a manner nearly in accordance with Strasburger's abandoned view of 1895,1 and with Guignard's ('98, 2) and Gregoire's ('99) latest results on the flowering plants, the ring being stated to arise by a double longitudinal split""- ting, as explained at page 265. In the elasmobranch Scy Ilium Moore ('95) finds twelve (the re- duced number) ring-shaped chromosomes at the first division. These closely resemble tetrads ; but a resting stage follows, and the second division is likewise stated to be of the heterotypical form, l^oth divi- sions are stated to be equation-divisions — a conclusion well sup- ported in case of the first, but so far from clear in the .second that a careful reexamination of the matter is highly desirable. In mammals the first division is of the heterotypical form (Her- mann, '89, Lenhossek, '98), though the rings are much smaller than ni the salamander, recalling those seen in arthropods. No true tetrads are, however, formed, and the two divisions are separated by a resting period. The character of the second division is undeter- mined, though Lenhossek believes it to be heterotypical, like the first. (b) Plants. — It is in the flowering plants, where reduction likewise occurs, as a rule, without true tetrad-formation, that the contradiction of results reaches its cHmax ; and it must be said that until further research clears up the present confusion no definite result can be stated. The earlier work of Strasburger and Guignard indicated that no reducing division occurred, the numerical reduction being directly effected by a segmentation of the spireme-thread into half the somatic number of chromosomes. Thus these observers found in the male that the chromosomes suddenly appeared in the reduced number (twelve in the lily, eight in the onion) at the first division of the pollen-mother-cell, and in the female at the first division of the mother-cell of the embryo-sac. The subsequent ])henomena differ in a very interesting way from those in animals, owing to the fact that the two maturation-divisions are followed in the female by one and in the male by two or more additional divisions, in both of which the reduced number of chromosomes persists. In the male the two maturation-divisions give rise to four pollen-grains, in the female to 1 Cf. p. 269. 264 KEDUC710X OF THE CHROMOSOMES the four primary cells of the embryo-sac (Fig. 132); and these two divisions undoubtedly correspond to the two maturation-divisions in animals. In the female, as in the animals, only one of the four resulting cells gives rise to the (i<^^, the other three corresponding to the polar bodies in the animal (t'^^^j:,, though they here continue to divide, and thus form a rudimentary prothalHum.^ The first-men- \\ ■ 7 A ^c- C ' iV^:- m^^ ... IV ^ "4. ^ - • ■ . -V b-,.--'/ F E Fig. 132. — General view of the maturation-divisions in flowering plants. [MOITIER.] A-C, in the male; D-F, in the female. A. The two secondary spermatocytes (pollen-mother- cells) just after the first division (Liliurn). B. Final anaphase of second division {Podophyllum^. C. Resulting telophase, which by division of the cytoplasmic mass produces four pollen-grains. D. Embryo-sac after completion of the first nuclear division {Liliuni). E. The same after the second division. F. The upper four cells resultin.; from the third division {c/. Fig. io6) : o, ovum; /, upper polar cell ; s, synergida;. (For further details, see Figs. 133, 134.) 1 Of these three cells one divides to form the "synergidre," the other two divide to form thriee "antipodal cells" (which like the synergida:; finally degenerate) and a "lower polar cell." The latter sooner or later conjugates with the " upper polar cell " (the sister-cell of the egg) to form the " secondary embryo-sac-nucleus," by the division of which the endo- sperm-cells arise. Of the whole group of eight cells thus arising only the egg contributes REDUCTION WITHOUT TETRAD-FORMATION 265 tioned cell, however, does not directly become the e^^g, but divides once, one of the products being the ^gg and the other the '' upper polar cell" (Fig. 132, F), which contributes to the endosperm-forma- tion (see footnote, and compare page 218). In the male the two maturation-divisions are in the angiosperms followed by two others, one of which separates a "vegetative" trom a "generative" cell, while the second divides the generative nucleus into two definite germ-nuclei. In the gymnosperms more than two such additional divisions take place. In these later divisions, both in the male and in the female (with the exception noted in the footnote below), the reduced number persists, and the principal interest centres in the first two or maturation-divisions. Strasburger and Guignard found in Lilium that while both these divisions differed in many respects from the mitosis of ordinary vegetative cells, neither involved a transverse or reducing division, the chromosomes under- going a longitudinal splitting for each of the maturation-divisions. Further investigations by Farmer ('93), Belajeff ('94), Dixon (96), Sargant ('96, '97), and others, showed that the first division is often of the heterotypical form, the daughter-chromosomes in the late-meta- phase having the form of two V's united by their bases (<>). Despite the complication of these figures, due to torsion and other modifications, their resemblance to the ring-shaped bodies observed in the first maturation-division of so many animals is unmistakable, as was first clearly pointed out by Farmer and Moore ('95). Botanists have differed, and still differ, widely in their interpreta- tion both of the origin and subsequent history of these bodies upon which the question of reduction turns. According to Strasburger's ('95) first account their origin has nothing in common with that of the tetrad-rings, since they were described as arising by a double \o\\- gitudinal splitting of a primary rod, the halves then separating first from one end along one of the division-planes, and then from the other end along the other plane, meanwhile opening out to form a ring such as is shown in Fig. 133. (This process, somewhat difficult to understand from a description, will be understood from the dia- gram. Fig. 135, E~I^ The four elements of the ring are then distrib- uted without further division by the two ensuing maturation-divisions ; and the process, except for the peculiar opening out of the ring, is to the morphological formation of the embryo. It is a highly interesting fact that the nuni- ber of chromosomes shown in the division of the lower of the two nuclei {i.e. the mother- nucleus of the antipodal cells and lower polar-cell) formed at the first division of the embryo-sac-nucleus is inconstant, varying in the lily from 12, 16, 20, to 24 (Cluignard, '91, l), in which respect they contrast with the descendants (egg, synergidx) of the upper nucleus, which always show the reduced number (Mottier, '97, i), i.e. in l.ilium twelve. This exception only emphasizes the rule of the constancy of the chromosome-number in general; for these cells are destined to speedy degeneration. 266 REDUCTIOX OF THE CHROMOSOMES essentially in agreement with the facts described in Ascaris, and involves no reduction-division. Essentially the same result is reached by Guignard (98) in his latest paper on Naias, and by Gregoire ('99) in the Liliacere. Strasburger twice shifted ground in rapid succession. First (97, 2), with M()tticr('97, i ). he somewhat doubtfully adopted a view agreeing , AA'V^S'IA^. \ r?v^ \ X- / A B E /TV ^ Fig. 133. — The first maturation-division in flowering plants. [/>", STRASBURGER and MOT- TlKk , the others from MorilER.] . /. Mother-cell of the embrvo-sac in Lilium ; early prophase of first division : chromatin- threads already longitudinally divided. B. Slightly later stage (split spireme) in the nucleus of the poUen-mo'ther-cell. C. h slightly later prophase (pollen-mother-cell. Podophyllum) with twisted split spireme. D. Earlier prophase (ZL/V/ww, female) ; split twisted chromosomes. E. Equatorial plate {Lilium, male). F.. First maturation-spindle {Fritillaria, male). G. Diver- gence of the daughter-chromosomes {Lilium, male). essentially with the interpretation of Vom Rath, Riickert, etc. (p. 247). The primary rods split once, and bend into a V, the branches of which often come close together, and may be twisted on themselves, thus giving the appearance of the second longitudinal split described in Strasburger's paper of 1895. The two halves of the split U then separate, opening out from the apex, to form the -figure. In the REDUCTION WITHOUT TETRAD-FORMATION 267 second division the limbs of the daughter-V's again come close together, remaining, however, united at one end, where they were believed finally to break apart during the second division. The latter was, therefore, regarded as a true reduction-division, the apparent longitudinal split being merely the plane along which the halves of the V come into contact (Fig. 134, C, D). The two accounts just given represent two extremes, the first agreeing essentially with Ascaris, the second with the copepods or insects. When we compare them with others, we encounter a truly bewildering confusion. Strasburger and Mottier ('97) themselves soon abandoned their acceptance of the reducing division, returning to the conclusion that in both sexes {Liliuin, Podopliylhnn) both divi- sions involve a longitudinal splitting of the chromosomes (Figs. 133, 134). In the first division the longitudinally split spireme segments into twelve double rods, which bend at the middle to form double V's, with closely approximated halves. Becoming attached to the spindle by the apex, the limbs of each separate to form a o -figure. At telophase the daughter-V's shorten, thicken, and join together to form a daughter-spireme consisting of a single contorted thread. This splits lengthwise tJirougJioiit its whole extent^ and then segments into double chromosomes, the halves of which separate at the second division (Fig. 135, L-M). The latter, therefore, like the first, involves no reducing division. This result agrees in substance with the slightly earlier work of Dixon ('96) and of Miss Sargant (96, '97), whose account of the origin of the -figure of the first division differs, however, in some interesting details. It is also in harmony with the general results of Farmer and Moore ('95), of Gregoire ('99), and of Guignard ('98), who, however, describes the first division nearly in accordance with Strasburger's account of 1895, as stated above. On the other hand, Ishikawa (pollen-mother-cells of Allium, 'gy) and especially Belajeff (pollen-mother-cells of Iris, 98) conclude that the second division is a true transverse or reducing division. ^ Ishikawa described the first division as being nearly similar to the ring-forma- tion in copepods, the four elements of the ring being often so condensed as -nearly to resemble an actual tetrad. In the early ana- phases the daughter-V's break at the apex ; and, although in the later anaphases the limbs reunite, Ishikawa is inclined to regard the trans- verse division as being a preparation for the second mitosis. Bela- jeff's earlier work ('94) on Liliuin gave an indecisive result, though one on the whole favourable to a reducing division. In his latest paper, however ('98, i), Belajeff takes more positive ground, stating that after the examination of a large number of forms he has found 1 Schaffner ('97, 2) reaches exactly the reverse result in Lilium philaJelphicum, i.e. the first division is transverse, the second longitudinal. 268 REDUCTIOX OF THE CHROMOSOMES in the pollen-mother-cells of Iris a nuich more favourable object of investi<;ation than Liliu))i, I'^i til/aria, and the other forms on which most of the work thus far has been done, and one in which the sec- ond division takes place with "admirable clearness"; he also gives interesting^ additional details ot the first division in this and other forms. In the first division the spireme splits lengthwise, and then breaks into chromosomes, which assume the shape of a V, Y, or X (Fig. 135, X-Q). The two limbs of these bodies do not, as might be A ■>-'^^ V B C G \^ Fig;. 134. — Tlie seconrl maturation-division in flowering plants. [B. SXRASBURGER and Mullii.K; the others from Moi iiKR.] A. Nucleus of secondary spermatocyte {Podophyllum). D. Prophase of second division {Ltliutn, male) with longitudinally divided chromatin-threads. E. Corresponding stage in the female. E. Metaphase of second division {Podophyllnm, male). G. Initial anaphase {Lilmvi, female). CD. illustrate Mottier's earlier conclusions. ('. Second division {Lilium, male), with chromosomes bent together so as to simulate a sjilit. A Slightly later stage [Eritillaria, male), showing stage supposed to result from breaking apart of the limbs of the U at point of flexure. supposed, represent sister-chromosomes (resulting from the longitu- dinal division of the spireme) attached by one end or at the middle, since each X, Y, or V is double, consisting of two similar superim- posed halves. Belajeff, therefore, regards these figures as longitu- dinally divided bivalent chromosomes, having the value of tetrads, each limb being a longitudinally split single chromosome. The double V's, Y's, and X's take up a position with the apex (or one end of the X) attached to the spindle, and the longitudinal division in the equatorial plane. The halves then progressively diverge from the REDUCTION WITHOUT TETRAD-FORMATION 269 point of attachment, thus giving rise to -shaped, ^>- -shaped, or XX -shaped figures, all of which in the end assume the -shape. This part of the process is in the main similar to that described by Strasburger and Mottier, and the daughter-V's diverge in the same way as these authors describe. The second division, however, differs radically from their account, since no splitting of the spireme-thread occurs. The chromosomes reappear in the V-, Y-, and X-forms, but are ufidivided, and only half as thick as in the first division. Passing to the equator of the spindle, the V- and Y-forms break apart at the apex, while the X-forms separate into the two branches of the X, the daughter-chromosomes having the form of rods slightly bent at the outer end to form a J-figure (Fig. 135, R-T). This division is, accordingly, a transverse or reducing one, which " corresponds com- pletely to the reduction-division in the animal organism" ('98,2, p. 33.) Atkinson ('99) reaches the same general result in Tnlliuni, stating very positively that no longitudinal division occurs in the second mitosis, and believing that the daughter-V's of the first (hete- rotypical) mitosis retain their individuality throughout the ensuing pause, and break apart at the apex (reducing division) in the second mitosis. This observer finds further that in Arisc^ma the heterotvpi- cal rings of the first mitosis condense into true tetrads, by one longi- tudinal and one transverse division, but believes that in this case it is th.Qjirst division that effects the reduction, as in the insects. Such confusion in the results of the most competent observers of reduction in the flowering plants is itself a sufficient commentary on the very great difficulty and uncertainty of the subject; and it would be obviously premature to draw any positive conclusions until further research shall have cleared up the matter.^ 1 Strasburger's new book, entitled Uber Redtiktionsiheiliing, Spindelbildung, Cfutroso- men nnd Cilienhildner im PJlanzenreich (Jena, 1900), is received while this work is in press, too late for analysis in the text. In this treatise the author gives an exhaustive review of the entire subject, contributing also many new and important observations on Liliuni, Iris, Podophylliwi, Tradescantia, Allium, larix, and several other forms. The general result of these renewed researches leads Strasburger to return, in tlie main, to his conclu- sions of 1895, ^^'ith which agree, as stated above, the results of (luignard and Cregoire; and. in a careful critique of Belajeff's work, he shows how the results of this observer may be reconciled with his own. The essence of Strasburger's interpretation is as follows. In the prophases of the first division the chromosomes first undergo a longitudinal division, shorten to f9rm double rods, and then again split lengthwise in a plane at right angles to the lirst. The following stages vary even in the same species {Uliiini) \ and here lies the explanation of much of the divergence between the accounts of different observers, (i) In the typical case, the chromosomes are placed radially, with one end next the spindle; ami, during the metaphase, they open apart along the first division-jilane, from the spindle outwards, to form h- -shaped figures. These figures meanwhile open apart from the free end inwards along the second division-plane. Thus arise the characteristic o -shaped figures, the daughter-V\s having separated along the first (equatorial) division-plane, while the two hmbs ut each V have resulted, not through bending, but from a secoml (axial) split (I'ig. 135, A-//'). The ^ R S T Fig. 135. — Diagrams illustrating different accounts of reduction in the flowering plants. A-D. Vegetative mitoses (heterotypical form) in Picea. [BelajEFF.] E-I. Illustrate Strasburger's earlier account (95) and the later one of Guignard, of the first maturation-division. E. Doubly split rod. F. Metaphase, in profile. G. The same en face, showing the heterotype ring. H. I. Opening out and breaking apart of the ring. J-M. Later account of Strasburger and Mottier {cf. Figs. 133, 134). J. Longitudinally split, V-shaped chromosome of first division. K. Opening out of the ring. L. Prophase of second division, showing longitudinally split segmented spireme. M. Initial anaphase of second division. N-Q. First division. [BELAJEFF.] A'. Longitudinally split chromosomes, viewed in the equa- torial plane. O. The same viewed in the axis of the spindle. P. Separation of the daughter- chromosomes. Q. Anaphase, all the chromosomes assuming the V-form. /?- 7: Second division in /Wj. [Belajeff.] R. Equatorial plate, limbs of X's and V's break- ing apart (reducing division). 6'. Slightly later stage, with daughter-chromosomes still united at one end. T. Anaphase. 270 PECULIARITIES OF REDUCTION IN THE INSECTS 2'Jl Resume. In reduction without tetrad-formation the spireme seg- ments into half the somatic number of chromosomes, which split lengthwise and open out to form rings for the first (heterotypical) mitosis. According to one set of observers, including Flernming, Meves, McGregor, Kingsbury, Moore, Klinckowstrom, Van der Stricht', Francotte, Grifhn, Belajeff, Farmer, Dixon, Strasburger, Sarganti Mottier, Ishikawa, and Atkinson, the ring arises by a single longi- tudinal division. According to another group, including Carnoy, Le Brun, Guignard, and Gregoire, the ring arises through a double longitudinal division, one representing the axial and the other the equatorial plane of the <> -figure. The second group of observers regard both maturation-divisions as longitudinal. Among the first group, Flemming, Meves, McGregor, Kingsbury, Moore, Farmer, Dixon, Strasburger, Sargant, and Mottier likewise believe both divi- sions to be longitudinal, the daughter-V's or their products again splitting lengthwise for the second division ; while Klinckowstrom, Van der Stricht, Francotte, Griffin, Belajeff, Ishikawa, and Atkinson beHeve one of them to be transverse, the daughter-V's breaking apart at the apex, and thus giving the reducing division of Weismann.^ D. Some Peculiarities of Reduction in the Insects We may here briefly consider some interesting observations which show that in some cases the nuclear substance may be unequally distributed to the germ-nuclei. Henking (''90) discovered that in the second spermatocyte-division of Pyrrocho- r is one of the *' chromosomes ^' passes undivided into one of the daughter-cells (spermatids) which receives twelve chromatin-elements while its sister receives but eleven. (The number of chromosomes in the spermatogonia, and of rings in the first spermatocyte-division is twenty-four). This anomalous process is conrirmed with interesting additional details by Paulmier ('99) in Anasa, and obviously rt-lated phenomena are described by Montgomery ('99, i ) in Pentatonia., and by McClung ("99) in XipJiidin7n. breaking apart of the V's at the apex, as described by Belajeff, is, therefore, not a transverse division, but merely the completion of the second longitudinal division. (2) In a second and exceptional type, the chromosomes are placed tnngcniially to the spindle, and the halves separate from the middle, again producing <> -shaped tigures. These, however, arc not of the same nature as those arising in the first case, since they are formed by a bending out of each daughter-chromosome at the middle to form the V, and not by the secoml longi- tudinal split. The effect of the latter is in this case to render each daughter-V in itself double, precisely as in the salamander. The difference between the two types results merely from the difference of position of the chromosome with respect to the spindle, and the final result is the same in both, i.e. two longitudinal divisions and no reducing one. This highly important work brings very strong evidence against the occurrence of trans- verse or reducing divisions in the higher plants, and seems to explain satisfactorily most of the differences of interpretation given by other observers. It will be interesting:: to see whether a similar interpretation is possible in the case of mollusks, anneliiis. and arthropods, where the early stages, in many cases, so strikingly resemble those occurring in the plants. ^ Cf. footnote on page 269. 2-2 REDUCriOX Of THE CI/EOMOSOMES In Pcntatoma the number of chromosomes in the spermatocyte is fourteen. During the final anaphases of the hist division, one of the fourteen daughter-chronio- somes\ssumcs a ilitTerent stainin.i;-cai)acity from the others, and becomes a " chro- matin-nucleohis " which fra.iimcnts into several smaller bodies during the ensuing rcsting-stage. During each of the succeeding spermatocyte-divisions appear seven chronrosomes and a single small chromatin-nucleolus, and both of these kinds t)f bodies are halved at each division, so that each spermatid receives seven chromo- somes and a single chromatin-nucleolus.' In Xipliidittin a body called by McClung the -accessory chromosome," and believed by him to correspond to the "chromatin- nucleolus '* of VVz/A/Av//./, appears in the early prophases of the last spermatogoniuni- division while the remaining chromatin still forms a reticulum. In the equatorial plate this lies outside the ring of chromosomes, but divides like the latter. The same bodv appears in the ensuing resting-stage, and during both of the spermatocyte- divisions.' In these it lies, as before, outside the chromosome-ring, and ditiers markedly from the other chromosomes, but divides like the latter, each of the halves passing into one of the spermatids, where it appears to form an important part of the sperm-nucleus. Despite the peculiarities described above, tlie chromatin, as a whole, seems to be equally distrii)uted in both Pcntatoma and Xiphidimn. In Anasa, however, Paul- niier's studies (98, '99), made in my laboratory, give a result agreeing with that of Henking. and suggest some very interesting further questions. The spermatogonia- nuclei contain two nucleolus-like bodies, and give rise to twenty-two chromosomes, of which two are smaller than the others (Fig. 126). In the first spermatocyte-divi- sion appear eleven tetrads. Ten of these arise from rings like those of uryllotalpa. etc. The eleventh, which is much smaller than the others, seems to arise from a single nucleolus-like body of the spermatocyte-nucleus. and by a process diftering coirsiderably from the others. All of these bodies are halved to form dyads at the first division. In the second spermatocyte-division (Fig. 127) the larger dyads divide to form single chromosomes in the usual manner. The stnall dyad, Jwwevcr, fails to divide, passitii^ over bodily into one of the spermatids. In tliis case, there- fore, half of the spermatids receive ten single chromosomes, while the remainder receive in addition a small dyad. A comparison of the foregoing results indicates that the small tetrad (dyad) corre- sponds to the e.xtra chromosome observed by Henking in Pyrrochoris, and perhaps also to the "accessory chromosome"' oi Xiphidiuni. Whether it corresponds to the '•chromatin-nucleolus'' oi Pentatonia is not yet clear. The most remarkable of these strange phenomena is the formation of the small tetrad, which seems to be a non-essential element, since it does not contribute to all the spermatozoa. Paulmier is inclined to ascribe to it a vestigial significance, regarding it as a "degenerating" chromosome which has lost its functional value, though still undergoing in some measure its original morphological transformation : in this connection it should be pointed out that the spermatocyte-nucleolus, from which it seems to be derived, is represented in the spermatogonia by two such nucleoli, just as the single small tetrad is represented by two small chromosomes in the spermatogonia-mitoses. The real meaning of the phenomenon is, however, wholly conjectural. E. The Early History of the Germ-nuclei There are many peculiarities in the early history of the germ- nuclei, both in plants and animals, that have a special interest in con- ^ On this latter point Montgomery's observations do not seem quite decisive. EARLY HISTORY OF THE GERM-NUCUil 273 nection with the reduction-problem ; and some of these have raised some remarkable questions regarding the origin of reduction. A large number of observers are now agreed that during the growth- period preceding the maturation-division (p. 216), in both sexes, the nucleus of the mother-cell (spermatogonium, oogonium), both in plants and in animals, passes through some of the changes prepara- tory to reduction at a very early period. Thus, in the (ty:^^ the pri- mary chromatin-rods are often present in the very young ovarian eggs, and from their first appearance are already split longitudinally.' Hacker ('92, 2) made the interesting discovery that in some of the copepods {CantJiocamptiis, Cyclops) these double rods could be traced OZ'."- Fig. 136. — Longitudinal section through the ovary of the copepod Canthocamptus. [Hacker,] og. The youngest germ-cells or oogonia (dividing at og.'^) ; a. upper part of the growth-zone; oc. oocyte, or growing ovarian egg; ov. fully formed egg, with double ciiromatin-rods. back continuously to a double spireme-thread, following immediately upon the division of the last generation of oogonia, and that at no period is a true retictibnn formed in tJie gerniiual vesicle (Fig. 136). In the following year Ruckert('93, 2) made a precisely similar discm-- ery in the case of selachians. After division of the last generation of oosconia the daufrhtcr-chromosomes do not give rise to a reticu- lum, but spHt lengthwise, and persist in this condition throughout the entire growth-period of the ^^g. Riickert therefore concluded that the germinal vesicle of the selachians is to be regarded as a " daughter-spireme of the oogonium {Vr-ei) grown to enormous dimensions, the chromosomes of which are doubled and arranged in 1 Hacker, Vom Rath, Riickert, in copepods; Ruckert in selachians; Rom and Fick in Amphibia; HoU in the chick; Ruckert in the rabbit. T -V4 REDUCTIOX OF THE CHROMOSOMES pairs." ^ In this case their number seems to be at first the somatic number (thirty-six), which is afterward halved by conjugation of the elements tw(^ and two ( Riickert ), as in /.//w/t/V//^- (Calkins). It is, however, certain that in many cases (insects, copepods) the double rods first appear in the reduced number, and the observations of Vom Rath ('93) and Hacker ('95, 3 ) K*^'^ some reason to believe that the reduced number mav in some forms be present in the earlier proi^eni- tors of the germ-cells, the former author having found but half the normal number in some of the embryonic cells of the salamander, while Hacker ('95, 3) finds that in Cyclops brcviconiis the reduced number of chromosomes (twelve) appears in the primordial germ-cells which arc differentiated in the blastula-stage (Fig. 74). He adds the inter- esting discovery that in this form the somatic nuclei of the cleavage- staL^es show the same number, and hence concludes that all the chromosomes of these stages are bivalent. As development proceeds, the germ-cells retain this character, while the somatic cells acquire the usual number (twenty-four) — a process which, if the conception of bivalent chromosomes be valid, must consist in the division of each bivalent rod into its two elements. We have here a wholly new light on the historical origin of reduction ; for the pseudo-reduction of the germ-nuclei seems to be in this case a persistence of the embryonic condition, and we may therefore hope for a future explanation of the process by which it has in other cases been deferred until the penul- timate cell-generation, as is certainly the fact in Ascaris? This leads to the consideration of some very interesting recent dis- coveries regarding the relation of reduction to the alternation of gen- erations in the higher plants. As already stated (p. 263), Strasburger, Guignard, and other observers have found that in the angiosperms the two maturation-divisions are in both sexes followed by one or more divisions in which the reduced number persists. The cells thus formed are generally recognized as belonging to the ve.stiges of the sexual generation (prothallium) of the higher cryptogams, the pollen- grains (or their analogues in the female) corresponding to the asexual spores of the archegoniatc cryptogams. We should, therefore, expect to find reduction in the latter forms occurring in the two correspond- ing divisions, by which the "tetrad " of spores is formed (as was first pointed out by Hartog, '91). Botanists were thus led to the surmise, first expressed by Overton in 1892, that the reduced number would be found to occur in the ]-)rothallium-cclls derived from those spores. 1 '92, 2, p. 51. - It may be recalled that in Ascai'is Boveri proved that the ]irimordial germ-cells have the full number of chromosomes, and Hertwig clearly showed that this number is retained up to the last division of the spermatogonia. Ishikawa ('97) finds that in Alliiivi the reduced number (eight) appears in the mitosis of the " UrpoUenzellen " preceding the pollen-mother-cells. This is, however, contradicted by Mottier ('97, 2). EARLY HISTORY OF THE GERM-NUCLEI 2/5 This surmise quickly became a certainty. Overton himself dis- covered ('93 ) that the cells of the endosperm in the i^ymnosperm Ccratozamia divide with the reduced number, namely eight ; and Dixon observed the same fact in Pinus at the same time. In the following year Strasburger brought the matter to a definite conclusion in the case of a fern {Osmitnda), showing that a// the cells of the prothallinni, from the original spore-mother-cell ouzuanls to the for- mation of the germ-cells, have one-half the number of chromosomes found in the asexual generation, namely twelve instead of twentv- four ; in other words, the reduction takes place in the formation of the spore from which the sexual generation arises, many cell-genera- tions before the germ-cells are formed, indeed before the formation of the body from which these cells arise. Similar facts were determined by Farmer in Pallavicinia, one of the Hepaticae, where all of the nuclei of the asexual generation (sporogonium) show eight chromo- somes during division, those of the sexual generation (thallus) four. It now^ seems highly probable that this will be found a general rule. The striking point in these, as in Hacker's observations, is that the numerical reduction takes place so long before the fertilization for which it is the obvious preparation. Speculating on the meaning of this remarkable fact, Strasburger advances the hypothesis that the reduced number is the ancestral number inherited from the ancestral type. The normal, i.e. somatic, number arose through conjugation by which the chromosomes of two germ-cells were brought together. Strasburger does not hesitate to apply the same conception to ani- mals, and suggests that the four cells arising by the division of the oogonium {o-gg plus three polar bodies) represent the remains of a separate generation, now a mere remnant included in the body in somewhat the same manner that the rudimentary prothallium of angi- osperms is included in the embryo-sac. This may seem a highly improbable conclusion, but it must not be forgotten that so able a zoologist as Whitman expressed a nearly related thought, as long ago as 1878 : '' I interpret the formation of polar globules as a relic of the primitive mode of asexual reproduction.'^ ^ Strasburger's \iew is exactly the reverse of this in identifying the polar bodies as the remains of a sexual generation; and as Hacker has pointed out ('98, p. 102), it is difficult to reconcile with the fact that true reduction appears to occur already in the unicellular organisms (p. ijy). The hypothesis is nevertheless highly suggestive and one which suggests a quite new point of view for the study not only of maturation but also of the whole problem of sexuality. We may now return to the consideration of some details. In a considerable number of forms, though not in all, the early prophase is 1 '78, p. 262. 2/6 REDUCTION OF THE CHROMOSOMES characterized, especially in the male, by a more or less complete concentration of the chromatin-substance at one side of the nucleus. This sta<;e, to which Moore has given the name synapsis {Y\g. 120, A\ sometimes occurs when the spireme thread is already split {Ascaris, Lilium), sometimes before the division is visible (insects). In cither case till- cluv}natin-sig)iu)its ijfii?-i^c from the synapsis stage longitndi- nallv divided and in the redneed nnniber, a fact which gives ground for the conclusion that the synapsis is in some way concerned with the rearrangement of the chromatin-substance involved in the numer- ical reduction. During the synapsis the nucleolus remains cjuite distinct from the chromatin, and in many cases it afterward persists beside the tetrads, in the formation of which it takes no j^art, to be cast out into the cytoplasm (Fig. 124) or to degenerate /// situ during the hrst maturation-division. A suggestive phenomena, described by several observers,^ is the casting out of a large part of the nuclear reticulum of the germinal ifc»-«y *•' A io-;yji.iJ-r.- B C Fig. 137. — Types of maturation-spindles in the female. ./. I'irst polar spindle with tetrads, in Hetetocope. [HaCKER]. B. Second polar spindle in Triton. [Caknoy and LeBkun.] C. First polar spindle of ^jrjr/^. [FiJRST.] vesicle at the time the polar bodies are formed (Figs. 97, 128). In these cases {Asterias, PolyeJio^nis, Tlialassenia, Nereis) only a small fraction of the chromatin-substance is preserved to form the chromo- somes, the remainder degenerating in the cytoplasm.- As a final point we must briefly consider the varying accounts of the achromatic maturation-figures in the female already briefly referred to at page 85. In many forms {^e.g. in turbellarians, nemertines, anne- lids, mollusks, echinoderms) the polar am]:)hiasters are of quite ty])ical form, with large asters and distinct centrosomes nearly similar to those of the cleavage-figures. In others, however (nematodes, arthropods, tunicates, vertebrates), the polar spindles differ markedly from those of the cleavage-figures, being described by many authors as entirely devoid of asters and even in some cases of centrosomes (Fig. 137). 1 Cf. Mathews (Wilson and Mathews, '95), Gardiner ('98), Griffin ('99). 2 Cf. the enormous reduction of the chromatin-substance in the elasmobranch egg, p. 338. REDUCTION IN UNICELLULAR lOA'J/S 277 There can be no doubt that these polar spindles differ from the usual type, and that they approach those recently described in the mitosis of the higher plants, but it is doubtful whether the apparent absence of asters and centrosomes is normal. In Ascaris, the first polar spindle arising by a direct transformation of the germinal vesicle (Fig. i ij) has a barrel-shape, with no trace of asters. At the poles of the spindle, however, are one or two deeply staining granules (Fig. 137), w^hich have been identified as centrosomes by Hacker (94) and Erlanger ('97, 4), but by Fijrst ('98) are regarded as central granules, the whole spindle being conceived as an enlarged centrosome.* For the reasons stated at page 314, I believe the former to be the correct interpretation. 2 Spindles without centrosomes have been described in the eggs of tunicates (Julin, Hill, Crampton), in Ajup/iioxits CSohoiin), in some species of copepods (Hacker), and in some vertebrates {Dic- myctyhis, Jordan ; mouse, Sobotta). In Aviphioxus (Sobotta) and Triton (Carnoy and LeBrun) complete asters are not formed, but fibrillas apparently corresponding to astral rays and converging to the spindle-poles are found outside the limits of the spindle (Fig. 137). In the guinea-pig, according to Montgomery ('98), centrosomes and asters are present in the first polar spindle, but absent in the second. The evidence is on the whole rather strong that the achromatic figure in these cases approaches in form that seen in the higher plants ; but it is an open question whether the appearances described may not be a result of imperfect fixation. F. Reduction in Unicellular Forms Although the one-celled and other lower forms have not yet been sufficiently investigated, we have already good ground for the conclu- sion that a process analogous to the reduction of higher types regularly recurs in them. In the conjugation of Infusoria, as already described (p. 223), the original nucleus divides several times before union, and only one of the resulting nuclei becomes the conjugating germ-nucleus, while the others perish, Hke the polar bodies. The numerical corre- spondence between the rejected nuclei or " corpuscules de rebut " has already been pointed out (p. 227). Hertwig could not count the chro- mosomes with absolute certainty, yet he states ('89) that in Piircivuv- cinm caudatum, during the final division, the number of spindle-fibres and of the corresponding chromatic elements is but 4-6, while in the 1 QC p. 312. 2 Sala ('94) and Fiirst have shown that occasionally the polar spindles of Ascaris are provided with large typical asters, and thus resemble those of annelids or mollusks. Sala believed this to be an effect of lowered temperature, but Fiirst 's observations are unfavour- able to this conclusion. 2/8 REDUCTIOX OF THE CHROMOSOMES earlier divisions the number is aj^proxiniately double this (8-9). This observation makes it nearly certain that a numerical reduction of chromosomes occurs in the Protozoa in a manner similar to that of the higher forms ; but the reduction here aj)])ears to be deferred until J \ ^\^J v.^/ o>' A C Fig. 138. — Conjugation and formation of the polar bodies in Actinophrys. [SCHAUDINN.] A. Union of the gametes; first polar spindle. /?, Fusion of the cell-bodies; a single polar body near the periphery of each. C. Fusion of the nuclei. the final division. In the gregarines Wolters ('91) has observed the formation of an actual polar body as a small cell segmented off from each of the two conjugating animals soon after their union ; but the number of chromo- somes was not deter- mined. Schaudinn ('96, 2) has observed a like process in Acti- iwpJuys, each of the gametes segmentmg A C B D off a single polar body, after which the germ- nuclei fuse (Fig. 138). It is ])ossible, as R. Hertwig ('98) points out, that in both these forms a second ])olar body may have been overlooked, owing per- haps to its rapid dis- Fig. 139. — Formation of polar bodies and conjugation in HltCgration. \\\. ActlllO- AcunosphcBrtum. [R. hertwig.] spha^riuvi, accordiug to A. Two gametes ("secondary cysts"), resulting from the p tt • /' o \ fU division of a "primary cyst"; second maturation-spindle in rierLWlg ^ 9*^ A '-^^ each; first polar bodv shown in the right gamete, at/. B. Both nuclcUS of Cach gamete polarbodies(/»i;>2) formed in the right gamete the second |- -^ ^ • • -^ one formmir m the left gamete. C. Subsequent fusion of the ^ gametes; nuclei uniting, two polar bodies (probably the second. SUCCeSSion tO form tWO the first having been absorbed) at/. D. The young AcfinospAcF- num escaping from the cyst-wall; the cleavage-nucleus has the first having been absorbed) at/. D. The yonng Actinosphce- t-)q]ot- bodies (^nnrlei^ divided. which degenerate, after REDUCTION IN UNICELLULAR FORMS 279 Which the germ-nuclei unite (Fig. 139). Whether a reduction in the number of chromosomes occurs in these cases was not determined • B f D H Fig. 140. — Conjugation of Closteriutti. [Klehahn,] dkt^' f'^^'n ^J^^' ""'^"' ^°"' chromatophores. B. Chromatophores reduced to two. nuclei distinct, a Fusion of the nuclei. D. First cleavage of tiie zygote. /.-. Resulting 2-cell stage /-. becond cleavage. G. Resulting stage, each cell bi-nucleate. //. Separation of tl,e cells- one 01 the nuclei in each enlarging to form the permanent nucleus, the other (probably reprd senting a polar body) degenerating. ' 1 Achnosphceriufu forms one of the most extreme known cases of in-iireeding; for the gametes are sister-cells which immediately reunite after forming the polar bodies. The general facts are as follows : The mother animal, containing verv numerous nuclei, l>ecomes encysted, and a very large number of the nuclei degenerate. TJie bodv then segments into 280 REDUCTIOX OF THE CHROMOSOMES Adclea (one of the Coccidire) is a \cry interesting case, for accord- ing to Sicdlecki ('99) polar bodies or their analogues are formed in both sexes. The gametes are here of very unequal size. Upon their union the smaller male cell divides twice to form apparently equiva- lent spermatozoids, of which, however, only one enters the ovum, while three degenerate as polar bodies. These two divisions are of different tvpe ; the first resembles true mitosis, while the second is of sim]-)ler character and is belie\ed bv Siedlecki to effect a reduction in the number of chromosomes. In the meantime the nucleus of the macro- gamete moves to the surface and there expels a portion of its chro- matin, after which union of the nuclei takes place. Interesting facts have been observed in unicellular plants which indicate that the reduction may here occur either before (diatoms) or after (desmids) fusion of the conjugating nuclei. In the ioxxwitx {Rliopalodina) Klebahn ('96) finds that each nucleus divides twice, as in many Infusoria, giving rise to two large and two small nuclei. Each of the conjugates then divides, each daughter-cell receiving one large and one small nucleus. The four resulting individuals then conjugate, two and two, the large nuclei fusing while the small (polar bodies) degenerate. The com- ])arison of this case with that of the Infusoria is highly interesting. In the desmids on the other hand (^'/^^•/mV/;;/ and Cosmariuin, Fig. 140), according to Klebahn ('92), the nuclei first unite to form a cleavage- nucleus, after which the zygote divides into two. Each of the new nuclei now divides, one of the products persisting as the perma- nent nucleus, while the other degenerates and disappears. Chmie- lewski asserts that a similar process occurs in Spirogyra. Although the numerical relations of the chromosomes have not been determined in these cases, it appears probable that the elimination of a nucleus in each cell is a process of reduction occurring after fertilization. G. M.\TUR.\TinN OF Parthexogenetic Eggs The maturation of eggs that develop without fertilization is a sub- ject of special interest, partly because of its bearing on the general theory of fertilization, partly because it is here, as I believe, that one of the strongest supports is found for the hypothesis of the individ- uality of chromosomes. In an early article by Minot {'yj) on the a number (Tive lo twelve) of " jirimary cysts." each containing one of the remaining nuclei. Kach primary cyst divides by mitosis to form two gametes ("secondary cysts "), whicii, after forming the polar bodies, reunite, their nuclei fusing to form a single one. The resulting cell soon creeps out of the cyst-wall and assumes the active life, its nucleus meanwhile mul- tiplying to produce the multinuclear condition characteristic of the adult animal. What is here the physiological motive for the formation of the polar bodies, and how shall it be explained under the Weismann hypothesis? MATURATION OF PARTHENOGEXETIC EGGS 28 I theoretical meaning of maturation, the suggestion is made that parthenogenesis may be due to failure on the part of the Kt^^y:^ to form the polar bodies, the egg-nucleus thus remaining hermaphrodite, and hence capable of development without fertiHzation. This sug- gestion forms the germ of all later theories of parthenogenesis. lial- four ('80) suggested that the function of forming polar cells has been acquired by the ovum for the express purpose of preventing parthe- nogenesis, and a nearly similar view was afterward maintained by Van Beneden.i These authors assumed accordingly that in par- thenogenetic eggs no polar bodies are formed. Weismann ('86) soon discovered, however, that the parthenogenetic eggs of Polyphemus (one of the Daphnidae) produce a single polar body. This observa- tion was quickly followed by the still more significant discovery by Blochmann i^^^) that in Aphis the partJienogcnetic eggs produce a single polar body, zvhile the fertilized eggs produce tzvo. Weismann was al^le to determine the same fact in ostracodes and Rotifera, and was thus led to the view^ which later researches have entirely confirmed, that it is the second polar body that is of special significance in partheno- genesis. Blochmann observed that in insects the polar bodies were not actually thrown out of the ^gg, but remained embedded in its substance near the periphery. At the same time Boveri {'Sy, i) dis- covered that in Ascaris the second polar body might in exceptional cases remain in the egg and there give rise to a resting-nucleus indis- tinguishable from the egg-nucleus or sperm-nucleus. He was thus led to the interesting suggestion that parthenogenesis might be due to the retention of the second polar body in the Qgg and its union with the egg-nucleus. " The second polar body would thus, in a certain sense, assume the role of the spermatozoon, and it might not without reason be said : ^^ PartJienogenesis is the result of fertilizaiiou by the second polar body'' ^ This conclusion received a brilliant confirmation through the obser- vations of Brauer ('93) on the parthenogenetic egg of Arteviia, though it appeared that Boveri arrived at only a part of the truth. Blochmann ('88-89) had found that in the parthenogenetic eggs of the honey-bee tzvo polar bodies are formed, and Platner discov- ered the same fact in the butterfly Liparis ('89) — a fact which seemed to contradict Boveri's hypothesis. Brauer's beautiful re- searches resolved the contradiction by showing that there are txvo types oi partJienogenesis \M\i\Q\\ may occur in the same animal. In the one case Boveri's conception is exactly realized, while the other is easily brought into relation with it. {a) In both modes typical tetrads are formed in the germ-nucleus to the number of eighty-four. In the first and more frequent case i 'ZZ, p. 622. 2 Essay VI., p. 359- ' l-c-, P- 73- 282 REDUCTION OF THE CHROMOSOMES {Y'v^. 141 ) but one polar body is formed, which removes eighty-four dyads, leaving eighty-four in the ^g^2,. There may be an abortive attempt to form a second polar spindle, but no division results, and the eighty-four dyads give rise to a reticular cleavage-nucleus. From uo-^-^^%0 (yo, B V-3-" T ■^/m . ' li. Fig. 141. — First type of maturation in the parthenogenetic egg of Artemia. [BraUER.] A, The first polar spindle; the equatorial plate contains 84 tetrads. D. C. Formation of the first polar body ; 84 dyads remain in the egg, and these give rise to the egg-nucleus, shown in D. F. Appearance of the egg-centrosome and aster. E. G. Division of the aster and formation of the cleavage-figure ; the equatorial plate consists of 84 apparently single but in reality bivalent chromosomes. this arise eighty-four thread-like chromosomes, and t/ic same juimbcr appears in later cleavage-stages. {b) It is the second and rarer mode that realizes Boveri's concep- tion (Fig. 142). Both polar bodies are formed, the first removing eighty-four dyads and leaving the same number in the (tgg. In the formation of the second, the eighty-four dyads are halved to form MATURATION OF PARTIIENOGENETIC EGGS 28 two daughter-groups, each containing eighty-four single chromosomes. Both these groups reviain in the egg, and eaeh gives rise to a singli i^eticular ujccleiis, as described by Boveri in Ascaris. These tzvo niiclei place themselves side by side in the cleavage-figure, and give rise each to eighty-four cJironiosomes, precisely like two germ-nuclei in ordinary fertilization. The one hundred and sixty-eight chromosomes split A B D E Fig. 142. — Second type of maturation in the parthenogenetic egg of Arh-mia. [BraL'ER.] A. Formation of second polar body. D. Return of the second polar nucleus ( />. b?) into the egg; development of the egg-amphiaster. C. Union of the egg-nucleus (?) with the second polar nucleus {p. b?-). D. Cleavage-nucleus and amphiaster. /:". First .l.Mvage-figure wiili equatorial plate containing 168 chromosomes in two groups of 84 each. lengthwise, and are distributed in the usual manner, and reappear in the same number in later stages. In other words, the second polar body here plays the part of a sperm-nucleus precisely as maintained by Boveri. In all individuals arising from eggs of the first type, therefore, the somatic number of chromosomes is eighty-four; in all those arising from eggs of the second type, it is one hundred and sixty-eight. This '284 REDUCTION OF THE CHROMOSOMES difference is clearly due to the fact that in the latter case the chromo- somes are single or univalent, while in the former they are bivalent (actually arising from dyads or double chromosomes). The remark- able feature, on which too much emphasis cannot be laid, is that the numerical difference should persist despite the fact that the mass, and, as far as we can see, the quahty, of the chromatin is the same in both cases. In this fact we must recognize a strong support, not only of Hacker's and Vom Rath's conception of bivalent chromosomes, but also of the more general hypothesis of the individuality of chromo- somes (Chapter VI.). I . Accessory Cells of the Testis It is necessary to touch here on the nature of the so-called " Sertoli-cells," or sup- porting cells of the testis in mammals, partly because of the theoretical ^ significance attached to them by Minot. partly because of their relations to the question of amito- sis in the testis. In the seminiferous tubules of the mammalian testis, the parent- cells of the spermatozoa develop from the periphery inwards toward the lumen, where the spermatozoa are finally formed and set free. At the periphery is a layer of cells next the basement-membrane, having flat, oval nuclei. Within this, the cells are arranged in columns alternating more or less regularly with long, clear cells, con- tainin'g large nuclei. The latter are the Sertoli-cells, or supporting cells :^ they extend nearly^throligh from the basement-membrane to the lumen, and to their inner ends the young sp'ermatozoa are attached by their heads, and there complete their growth. The' spermatozoa are developed from cells which lie in columns between the Sertoh- cells. and which undoubtedly represent spermatogonia, spermatocytes, and sperma- tids, though their precise relationship is, to some extent, in doubt. The innermost of these cells, next the lumen, are spermatids, which, after their formation, are found attached to the Sertoli-cells, and are there converted into spermatozoa without further division. The deeper cells from which they arise are spermatocytes, and the sper- matogonia lie deeper still, being probably represented by the large, rounded cells. Two entirely diiferent interpretations of the Sertoli-cells were advanced as long ago as 1 87 1, and both views still have their adherents. Von Ebner ("71) at first regarded the'Sertoli-cell as the parent-cell of the group of spermatozoa attached to it, and the same view was afterward especially advocated by Biondi ("85) and by Minot (■92). the latter of whom regarded the nucleus of the Sertoli-cell as the physiological analogue of the polar bodies, i.e. as containing the female nuclear substance (92. p. 77). According to the opposing view, first suggested by Merkel ('70- the Sertoli- cell is not the parent-cell, but a nurse-cell, the spermatozoa developing from the columns of rounded cells, and becoming secondarily attached to the Sertoli-cell, which serves merely as a support and a means of conveying nourishment to the growing spermatozoa. This view was advocated by Brown ('85), and especially by Benda ("87). In the following year ("88), von Ebner himself abandoned his early hypothesis and strongly advocated Benda s views, adding the very significant result that/^;/r spermatids arise from each spermatocyte, precisely as was afterward shown to be the case in Ascaris, etc. The very careful and thorough work of Benda and von Ebner. confirmed by that of Lenhossek (98. 2). leaves no doubt that mamma- lian spermatogenesis conforms, in its main outlines, with that of Ascafis. the sala- mander, and other forms, and that Biondi's account is untenable. Minot's theoretical interpretation of the Sertoli-cell. as the physiological equivalent of the polar bodies, therefore collapses. SUMMARY AND CONCLUSION 285 2. Ajuitosis in the Early Sex-cells Whether the progenitors of the germ-cells ever divide amitotically is a question of high theoretical interest. Numerous observers have described amitotic division in testis-cells. and a few also in those of the ovary. The recent observations of Meves ('91), Vom Rath ('93), and others leave no doubt whatever that such divisions occur in the testis of many animals. Vom Rath maintains, after an extended inves- tigation, that all cells so dividing do not belong in the cycle of develo]jmcnt of the germ-cells ('93, p. 164) : that amitosis occurs only in the supporting (;r nutritive cells (Sertoli-cells, etc.), or in such as are destined to degenerate, like the •• residual bodies'' of Van Beneden. Meves has, however, produced strong evidence ("94) that in the salamander the spermatogonia may, in the autumn, divide by amitosi.s, and in the ensuing spring may again resume the process of mitotic division, and give rise to functional spermatozoa. On the strength of these observations Flemming (93) him- self now admits the possibility that amitosis may form part of a normal cycle of devel- opment.^ H. Summary and Conclusion The one fact of maturation that stands out with perfect clearness and certainty amid all the controversies surrounding it is a rcdiiction of the number of chromosoiiies in tJie tdtiniate gerni-cells to one-lialf tJic nnniber cJiaracteristic of the somatic cells. It is equally clear that this reduction is a preparation of the germ-cells for their subsequent union, and a means by which the number of chromosomes is held constant in the species. With a few exceptions the first indication of the numerical reduction appears through the segmentation of the spireme- thread, or the resolution of the nuclear reticulum, into a number of masses one-Jialf that of the somatic cJiromosomes. In nearly all higher animals this process first takes place two cell-generations before the formation of the definitive germ-cells, and the process of reduction is completed by two rapidly succeeding "maturation-divisions," giving rise to four cells, all of which become functional in the male, while in the female only one becomes the ^gg, while the other three — the polar bodies or their analogues — are cast aside. During these two divisions each of the original chromatin-masses gives rise to four chromosomes, of which each of the four daughter-cells receives one ; hence, each of the latter receives one-half the somatic number of chromosomes. In the higher plants, however, the two maturation- divisions are followed by a number of others, in which the reduced number of chromosomes persists, a process most strikingly shown in the pteridophytes, where a separate sexual generation (prothallium) thus arises, all the cells of which show the reduced number. Two general types of maturation may be distinguished according to the manner in which the primary chromatin-masses 'divide. In one, 1 For more recent literature on this subject see Meves, Zelltheilung, in Mcrkel and Bon- net's Ergebnisse, VIIL, 1 898. 2S6 REDUCTION OF THE CHROMOSOMES typically represented by Ascaris and the arthropods, each of these masses divides into four to form a tetrad, thus preparing at once for two rapidly succeeding divisions, which are not separated by a recon- struction of the daughter-nuclei during an intervening resting period. In the other, examples of which are given by the flowering plants and the spermatogenesis of the Amphibia, no true tetrads are formed, the primary chromatin-masses dividing separately for each of the matura- tion-divisions, which are separated by a period in which the nuclei regress toward the resting state, though often not completely return- ing to the reticular condition. These two types differ, however, only in degree, and with few exceptions they agree in the fact that during the prophases of the first division the chromatin-bodies assume the form of rings, the mitosis thus being of the heterotypical form, and each ring having the prospective value of four chromosomes. Thus far the phenomena present no difficulty, and they give us a clear view of the process by which the numerical reduction of the chromosomes is effected. The confusion of the subject arises, on the one hand, from its complication with theories regarding the individu- ality of the chromosomes and the functions of chromatin in inheri- tance, on the other through conflicting results of observation on the mode of tetrad-formation and the character of the maturation-divisions. Regarding the latter question nearly all observers are now agreed that one of these divisions, usually the first, is a longitudinal or equation- division, essentially like that occurring in ordinary mitosis. The main question turns upon the other division, which has been shown in some cases to be transverse and not longitudinal, and thus separates what were originally different regions of the spireme-thread or nuclear substance. The evidence in favour of such a division seems at present well-nigh demonstrative in the case of insects and copepods, and hardly less convincing in the turbellarians, annelids, and mollusks. On the other hand, both divisions are regarded as longitudinal by most of those who have investigated the phenomena in Ascaris and in the vertebrates, and by some of the most competent investigators of the flowering plants. The evidence as it stands is so evenly balanced that the subject is hardly yet ripe for discussion. The principle for which Weismann contended in his theory of reducing div^ision has received strong support in fact; yet should it be finally estabUshed that numerical reduction may be effected either with or without transverse division, as now seems probable, not only will that theory have to be aban- doned or wholly remodelled, but we shall have to seek a new basis for the interpretation of mitosis in general. Weismann's theory is no doubt of a highly artificial character ; but this should not close our eyes to the great interest of the problem that it attempted to solve. LITERATURE 287 The existing contradiction of results has led to the opinion, expressed by a number of recent writers, that the difference between longitudinal or transverse division is of minor importance, and that the entire question of reduction is a barren one. This opinion fails to reckon with the facts on which rests the hypothesis of the individuality of chromosomes (Chap. VI.); but these facts cannot be left out of account. We must find a common basis of interpretation for them and for the phenomena of reduction ; yet how shall we reconcile them with reduction by longitudinal division only } I cannot, there- fore, share the opinion that we are dealing with a barren problem. The peculiarities of the maturation-mitoses are obviously correlated in some way with the numerical reduction, and the fact that they differ in so many ways from the characters of ordinary mitosis gives ground to hope that their exhaustive study will throw further light not only on the reduction-problem itself but also on mitosis in general and on still wider problems relating to the individuality of the chromo- somes and the morphological organization of the nucleus. It is indeed very probable that Weismann's theory is but a rude attempt to attack the problem, and one that may prove to have been futile. The prob- lem itself cannot be ignored, nor can it be dissociated from the series of kindred problems of which it forms a part. LITERATURE. V 1 Van Beneden, E. — Recherches sur la maturation de Toeuf, la fdcondation et la division cellulaire : Arch. Biol.. IV. 1883. Boveri. Th. — Zellenstudien, I., III. Jena, 1887-90. See also " Befruchtung" (List IV.). Brauer, A. — Ziir Kenntniss der Spermatogenese von Ascaris viegaloccphala : An/i. mik. Anat., XLII. 1893. Id. — Zur Kenntniss der Reifung der parthenogenetlsch sich entwickelnden Lies von Artemui Salina : Arch. mik. A?iat., XLIII. 1894. Guignard, L. — Le developpement du pollen et la reduction chromatique dans Ic Naias: Arch. Anat. Mic. II. 1899. (Full literature on reduction in plants.) Griffin, B. B. — See Literature, IV. Hacker, v. — Die Vorstadien der Eireifung (General Review): Arch. mik. A/uii., XLV. 2. 1895. Id. — Uber weitere Ubereinstimmungen zwischen den Fertpflan/.ungsvorgangen der Thiere und Pflanzen : Bio/. Centrally.. X\' II. 1897. Id. — Uber vorbereitende Theilungsvorgange bei Thieren und Prianzcn : \ erh. dentsch. Zool. Ges., VIII. 1898. Id. — Die Reifungserscheinungen : Merkel nnd Bonnet's Ergebnisse. \' 1 1 1 . i S9S . Hertwig, 0.— Vergleich der Ei- und Samenbildung bei Nematoden. Eine Crund- lage fiir cellulaire Streitfragen : Arch. mik. Anat.. XXWI. 1890. Mark, E. L. — (See List IV.) Peter, K. — Die Bedeutung der Nahrzcllen im Hoden : Arch. mik. A>i<7f.. LI 11. ibgb 1 See also Literature, IV., p. 231. 288 REDUCTION OF THE CHROMOSOMES Plainer, G.— Uberdie Bedeutung der Richtungskorperchen : Biol. Centralb.,\\\\. Vom Rath, 0. — Zur Kenntniss der Spermatogenese von Gryllotaipa vulgaris : Arch. inik. Anat.. XL. 1892. ,,..-, c^ a ^- -f Id. — Neue Beitrage zur Frage der Chromatinreduktion in der Samen- und Eireife : Arch. mik. Anat., XLVI. 1895. , , . -r^ • 1 , Riickert, J.— Die Chromatinreduktion der Chromosomenzahl nn Entwicklungsgang der Oro-anismen: Ergebn. d. Anat. u. Entwick.. III. 1893 (1894). Strasburgert E. — Uber periodische Reduktion der Chromosomenzalil im Entwick- luno-'igang der Organismen : Biol. Centralb., XIV. 1894. Id. — Redtktionstheilung. Spindelbildung, etc. : Jena, Fischer, 1900. CHAPTER VI SOME PROBLEMS OF CELL-ORGANIZATION " Wir miissen deshalb den lebenden Zellen, abgesehen von der Molecularstructur der organischen Verbindungen, welche sie enthalt, noch eine andere und in anderer Wcise coni- plicirte Structur zuschieiben, und diese es ist, welche wir mit dem Namen Organization bezeichnen." Brl'cke.^ " Was diese Zelle eigentlich ist, dariiber existieren sehr verschiedene Ansichten." IIackkl.^ The remarkable history of the chromatic substance in the matura- tion of the germ-cells forces upon our attention the problem of the ultimate morphological organization of the nucleus, and this in its turn involves our whole conception of protoplasm and the cell. The grosser and more obvious organization is revealed to us by the micro- scope as a differentiation of its substance into nucleus, cytoplasm, and the like. But, as Strasburger has well said, it would indeed be a strange accident if the highest powers of our present microscopes had laid bare the ultimate organization of the cell. Briicke insisted more than thirty years ago that protoplasm must possess a far more com- plicated morphological organization than is revealed to us in the visible structure of the cell, repeating, though without accepting, an earlier suggestion of Henle's('4i) that the cell might be composed of more elementary vital units ranking between the molecule and the cell. Many biological thinkers since Briicke's time have in one form or other accepted this conception, which indeed lies at the root of nearly all recent attempts to analyze exhaustively the phenomena of cell-life. Without attempting to follow out the history of opinion in detail or to give any extended review of the various theories,^ it may be pointed out that this conception was based both on theoretical a priori grounds and on the observed facts of cell-structure. On the former basis it was developed by Herbert Spencer'' in his theory of '' physiological units " by which he endeavoured to explain the phe- nomena of regeneration, development, and heredity ; while Xageli ('84) developed on the same general lines his theory of micclUc which 1 Elementarorganisjnen, 1861, p. 386. 2 Anthropogenie, 189 1, p. 104. 3 For an exhaustive review see Yves Delage, La structure du protoplasma et Us theories sui Vheredite. Paris, 1895. * Principles of Biology, 1864. U 2S9 290 SOME PROBLEMS OF CELL-ORGANIZATION has been so widely accepted by botanists. In the meantime Darwin ^ introduced a new element into the speculative edifice in his celebrated hypothesis of pangenesis, where for the first time appear the two assumptions of specific differences in the ultra-microscopic corpuscles C'gemmules ") and the power of self-propagation by division. Dar- win did not, however, definitely maintain that protoplasm was actually built of such bodies. The latter hypothesis was added by De Vries ('89), who remodelled the theory of pangenesis on this assumption, thus laying the basis for the theories of development which reached their climax in the writings of Hertwig and Weismann. The views of Spencer and Darwin were based on purely theoretical grounds derived from the general phenomena of growth and inheri- tance. ^ Those of Nageh, De Vries, Wiesner, Altmann, and others were more directly based on the results of microscopical investigation. The view was first suggested by Henle ('41), and at a later period developed by Bechamp and Estor, by Maggi and especially by Alt- mann, that the protoplasmic granules might be actually organic units or bioblasts, capable of assimilation, growth, and division, and hence to be regarded as elementary units of structure standing between the cell and the ultimate molecules of living matter. By Altmann, espe- cially, this view was pushed to an extreme limit, which lay far beyond anything justified by the known facts; and the theory of genetic con- tinuity expressed by Redi in the aphorism ^^ oiniie viviini ex vivo,'' reduced by Virchow to '^ omnis cellula e celhda,'" finally appears in the writings of Altmann as ^^ omne gramdiini e graiuilo'' /^ Altmann's premature generalization rested upon a very insecure foundation and was received with just scepticism. Except in the case of plastids, the division of the cytoplasmic granules was and still remains a pure assumption, and furthermore many of Altmann's ** granules" (zymogen-granules of gland-cells, etc.) are undoubtedly metaplasmic bodies.'^ Yet the beautiful discoveries of Schimper ('85) and others on the origin of plastids in plant-cells give evidence that these cells do in fact contain large numbers of bodies, other than the nuclei, that possess the power of growth and division. The division of the chlorophyll-bodies, observed long ago by Mohl, was shown by Schmitz and Schimper to be their usual if not their only mode of ori- gin ; and Schimper was able to trace them back to minute colourless plastids, scarcely larger than '* microsomes," that are present in large numbers in the protoplasm of the embryonic cells and of the Qgg, and give rise not only to chlorophyll-bodies but also to the amyloplasts or starch-formers and the chromoplasts or pigment-bodies. While it still remains doubtful whether the plastids arise solely by division or also 1 Variation of Atiimals and Plants, 1868. * Cf. Introduction, p. 12. ^ Die Elemeiitarorganismen, Leipsic, 1894, p. 155. ^ Cf. Lazarus, '98. THE NATURE OE CELL-ORGANS 201 by new formation (as now seems to be the case with the centrosome), the foregoing observations on the plastids give a substantial basis for the hypothesis that protoplasm may be built of minute dividing bodies which form its ultimate structural basis. It was these facts, taken in connection with the phenomena of particulate inheritance and varia- tion (Galton), that led De Vries and his followers to the fundamental assumption of '' pangens," ''plasomes," " biophores," and the like as final protoplasmic units ; ^ but these were conceived not as the visible granules, plastids, etc., but as much smaller bodies, lying far bevond the limits of present microscopical vision, through the growth or aggregation of which the visible structures arise. This assumption has been harshly criticised; yet when we recall that in one form or another it has been accepted by such men as Spencer, Darwin, Beale, Hackel, Michael Foster, Nageli, De Vries, Wiesner, Roux, Weis- mann, Oscar Hertwig, Verworn, and Whitman, and on evidence drawn from sources so diverse, we must admit that despite its highly specula- tive character it is not to be lightly rejected. In the present chapter we may inquire how far the known facts of cell-structure speak for or against this hypothesis, incidentally considering a number of detailed questions of cell-morphology which have not hitherto found a place. A. The Nature of Cell-organs The cell is, in Briicke's words, an elementary organism, which may by itself perform all the characteristic operations of Hfe, as is the case with the unicellular organisms, and in a sense also with the germ-cells. Even when the cell is but a constituent unit of a hifrher sfrade of organization, as in multicellular forms, it is no less truly an organism, and in a measure leads an independent life, even though its functions be restricted and subordinated to the common life. It is true that the earlier conception of the multicellular body as a colony of one-celled forms cannot be accepted without certain reservations.- Neverthe- less, all the facts at our command indicate that the tissue-cell possesses the same morphological organization as the egg-cell, or the protozoan, and the same fundamental physiological properties as well. Like these the tissue-cell has its differentiated structural parts or organs, and we have now to inquire how these cell-organs are to be conceived. 1 The following list includes only some of the various names that have been «;ivcn to these hypothetical units by modern writers : Physiological iDiiis (Spencer) ; geinmuU's (Darwin); pangens (De Vries); plasomes (Wiesner); miccllie (Nageli); ,plastiiiules (Hackel and Elssberg) ; inotagmata (Engelmann); hiophores (Weismann); bioblasfs (Beale); so7nacitles {Yo?X.(tx)\ idioblasts (Hertwig); icUosomes (Whitman); biogens (Ver- worn); microzymas (Bechamp and Estor) ; gemnuc (Haacke). These names are not strictly synonymous, nor do all of the writers cited assume the power of division in the units. - Cf. p. 58. 292 SOME PROBLEMS OF CELL-ORGANIZATION The visible organs of the cell fall under two categories, according as they are merely temporary structures, formed anew in each successive cell-generation out of the common structural basis, or permanent struc- tures whose identity is never lost, since they are directly handed on by division from cell to cell.^ To the former category belong, in general, such structures as cilia, pseudopodia, and the like ; to the latter, the nucleus, perhaps also the centrosomes, and the plastids of plant-cells. A peculiar interest attaches to the permanent cell-organs. Closely interrelated as these organs are, they nevertheless have a remarkable degree of morphological independence. They assimilate food, grow, divide, and perform their own characteristic actions like coexistent but independent organisms, of a lower grade than the cell, living together in colonial or symbiotic association. So striking is this morphological and physiological autonomy in the case of the green plastids or chro- matophores that neither botanists nor zoologists are as yet able to dis- tinguish with absolute certainty between those that form an integral part of the cell, as in the higher green plants, and those that are actually independent organisms living symbiotically within it, as is probably the case with the yellow cells of Radiolaria. Even so acute an investigator as Watase ('93, i) has seriously propounded the view that the nucleus itself — or rather the chromosome — should be regarded as a distinct organism living in symbiotic association with the cytoplasm, but having had, in an historical, sense, a different origin. This rather fantastic view has not found much favour, and even were it true w^ould teach us nothing of the origin of the power of division, which must for the present be taken as an elementary process forming one of the primary data of biology. Yet we may still inquire whether the power of division shown by such protoplasmic masses as plastids, chromosomes, centrosomes, nucleoli, and nuclei may not have its root in a like power residing in ultimate protoplasmic units of which they are made up. Could we accept such a view, we might much more easily meet some puzzling cytological difficulties. For under this assumption the difference between transient and permanent cell- organs would become only one of degree, depending on the degree of cohesion between their structural components ; and we could thus con- ceive, for example, how such a body as a centrosome might form, per- sist by division for a number of generations, and finally disintegrate. In connection with this it may be pointed out that even such a typical permanent organ as the nucleus does not persist as siicJi during the ordinary form of division ; for it loses its boundary and many of its other structural characters, becoming resolved into a group of sepa- rate chromosomes. What persists is here not the structural unit, but the characteristic substance which forms its essential constituent, and 1 Cf. footnote, p. 30. STRUCTURAL BASIS OF THE CELL 293 a large part even of this substance may be lost in the process. The term ''persistent organ" is therefore used in rather a figurative sense, and if too literally understood may easily mislead us. With the foregoing considerations in mind let us turn to the actual structural relation of the cell-orgfans. B. Structural Basis of the Cell In Chapter I. some of the reasons have been given for the conclu- sion that none of the obvious structural features of protoplasm (fibrillce, alveoli, granules, and the like) can be regarded as necessary or uni- versal ; and we may now inquire whether there is any evidence that such structures may have such a common structural basis as De \'ries's theory assumes. I shall here take as a point of departure my observa- tions on the structure of protoplasm in echinoderm-eggs,' already briefly reviewed at page 28. The beautiful alveolar structure of these eggs is entirely of secondary origin, and all the visible structural elements arise during the growth of the eggs by the deposit and subsequent enlargement of minute spherical bodies, all apparently liquid drops, in a homogeneous or finely granular basis which is itself a liquid. Some of these spheres enlarge to form the alveolar spheres, whiie the homogeneous basis or continuous substance remains as the interalve- olar material. Others remain much smaller to constitute the " micro- somes " scattered through the interalveolar walls ; and these bodies, like the alveolar spheres, are perfectly visible in life, as well as in section ; they are therefore not coagulation-products or artifacts. From these three elements arise all the other structures observed in these eggs, deutoplasm-spheres {Ophiiira) and pigment-bodies {Arbncia) being formed by further enlargement and chemical alteration of the alveolar spheres, while astral rays and spindle-fibres are differentiated out of the inter-alveolar material and microsomes.^ These various elements show a continuous gradation in size from the largest deuto- plasm-spheres down to the smallest visible granules, the latter being the source of all the larger elements, and in their turn emerging into view from the " homogeneous " basis. Clearly, then, none of these bodies can be regarded as the ultimate structural units ; for the latter, if they exist, must lie in a region at present inaccessible to the micro- scope. This fact, however, no more disproves their existence than it does that of molecules and atoms. It only shows the futiHty of such attempts as those of Altmann and his predecessors to identify " gran- ules " or ''microsomes " as final morphological units, and compels us to turn to indirect instead of direct evidence. It may, however, again be pointed out that it would be quite irrational to conclude that the small- 1 Cf. Wilson, '99. 294 SOME PROBLEMS OF CELL-ORGAXIZATION est visible granules first come into existence when they first come within view of the microscope. The ** homogeneous " substance must itself contain or consist of granules still smaller. The real question is not whether such ultra-microscopical bodies exist, but whether they are permanent organize dho^i^?. possessing besides the power of growth also the power of division. This question can be only indirectly ap- proached ; and we shall find it convenient to do so by beginning at the opposite end of the series, through a reconsideration of the phenomena of nuclear division. C. Morphological Composition of the Nucleus I. TJie Chromatin (a) HypotJiesis of the Individuality of the CJiromosomes. — It may now be taken as a well-estabhshed fact that the nucleus is never formed de novo, but always arises by the division of a preexisting nucleus. In the typical mode of division by mitosis the chromatic substance is resolved into a group of chromosomes, always the same in form and number in a given species of cell, and having the power of assimilation, growth, and division, as if they were morphological individuals of a lower order than the nucleus. That they are such individuals or units has been maintained as a definite hypothesis, es- pecially by Rabl and Boveri. As a result of careful study of mitosis in epithelial cells of the salamander, Rabl ('85) concluded that the cJironiosomes do not lose their individuality at the close of division, but persist in the chromatic reticnhim of the resting nnclens. The reticu- lum arises through a transformation of the chromosomes, which give off anastomosing branches, and thus give rise to the appearance of a network. Their loss of identity is, however, only apparent. They come into view again at the ensuing division, at the beginning of which "the chromatic substance flows back, through predetermined paths, into the primary chromosome-bodies " (Kernfaden), which re- appear in the ensuing spireme-stage in nearly or quite the same posi- tion they occupied before. Even in the resting nucleus, Rabl believed that he could discover traces of the chromosomes in the configuration of the network, and he described the nucleus as showing a distinct polarity having a ''pole" corresponding with the point toward which the apices of the chromosomes converge {i.e. toward the centrosome), and an '' antipole" (Gegenpol) at the opposite point {i.e. toward the equator of the spindle) (Fig. 22). Rabl's hypothesis was precisely formulated and ardently advocated by Boveri in 1887 and 1888, and again in 1891, on the ground of his own studies and those of Van Beneden on the early stages of Ascaris. The hypothesis was supported MORPHOLOGICAL COMPOSITION OF THE NUCLEUS 295 by extremely strong evidence, derived especially from a study of ab- normal variations in the early development of Ascaris, the force of which has, I think, been underestimated by the critics of the hypothesis. Some of this evidence may here be briefly reviewed. In some cases, through a miscarriage of the mitotic mechanism, one or both of the chromosomes destined for the second polar body are accidentally left Fig. 143. — Evidence of the individuality of the chromosomes. Abnormalities in the fertiliza- tion of Ascaris. [BOVERI.J A. The two chromosomes of the egg-nucleus, accidentally separated, have given rise each to a reticular nucleus (?, ?) ; the sperm-nucleus below (-T). B. Later stage of the same, a single chromosome in each egg-nucleus, two in the sperm-nucleus. C. An egg in which the second polar body has been retained; p.b'^ the two chromosomes arising from it; 9 the egg-chromo- somes ; d" the sperm-chromosomes. D. Resulting equatorial plate with six chromosomes. in the ^gg. These chromosomes give rise in the Q.gz to a reticular nu- cleus, indistinguishable from the egg-nucleus. At a later period this nucleus gives rise to the same number of chromosomes as those that entered into its formation, i.e. either one or two. These are drawn into the equatorial plate along with those derived from the germ- nuclei, and mitosis proceeds as usual, the number of chromosomes being, however, abnormally increased from four to five or six (Fig. 143, 296 SOME PROBLEMS OF CELL-ORGANIZATION C, D). Again, the two chromosomes left in the ^gg after removal of the second polar body may accidentally become separated. In this case each chromosome gives rise to a reticular nucleus of half the usual size, and from each of these a single chromosome is afterward formed (Fig. 143, A, B). Finally, it sometimes happens that the two germ-nuclei completely fuse, while in the reticular state, as is normally the case in sea-urchins and some other animals (p. 188). From the cleavage-nucleus thus formed arise four chromosomes. The same general result is given by the observations of Zur Strassen ('98) on the history of giant embryos in Ascaris. These embryos arise by the fusion, either before or after the fertilization, of previ- ously separate eggs, and have been shown to be capable of develop- ment up to a late stage. Not only in the first but also in some, at least, of the later mitoses, such eggs show an increased number of chromosomes proportional to the number of nuclei that have united. Thus in monospermic double eggs (variety bi- valeiis) the number is six instead of four ; in dispermic double eggs the number is increased to eight (Fig. 144). These remarkable observations show that whatever be the imniber of eJironio- sonies entering into the forniation of a reticular nnclens, the same nnniber after- ward issnes from it — a result which de- monstrates that the number of chromo- somes is not due merely to the chemical composition of the chromatin-substance, but to a morphological organization of Fig. 144. — Giant-embryo of y4j^ar/>, the nuclcus. A bcautiful Confirmation of this conclusion was afterward made by Boveri ('93, '95, i) and IMorgan (95, 4), in the case of echinoderms, by rear- ing larvae from enucleated egg-fragments, fertilized by a single sper- matozoon (p. 194). All the nuclei of such larvae contain but half the typical number of chromosomes, — i.e. in Echinns nine instead of eighteen, — since all are descended from one germ-nucleus instead of two ! Equally striking is the remarkable fact, described at page 275, that all of the cells in the sexual generation (oophore) of the higher cryptogams show half the number of chromosomes characteristic of the sporophyte, the explanation being that while reduction occurs at the time of spore-formation, the spores develop without fertilization, the reduced chromosome-number persisting until fertilization occurs var. bivaleiis, arising from a double- fertilized double egg, showing eight chromosomes {7.ur Strasseti). MORPHOLOGICAL COMPOSITION OF THE NUCLEUS 297 long afterward. Attention may be again called to the surprising case of Arteniia, described at page 281, which gives a strong argument in favour of the hypothesis. In addition to the foregoing evidence, Van Beneden and Boveri were able to demonstrate in Ascaris that in the formation of the spireme the chromosomes reappear in the same position as those which entered into the formation of the reticulum, precisely as Rabl Fig. 145. — Evidence of the individuality of the chromosomes in the egg of Ascaris. [Boveri.] E. Anaphase of the first cleavage. F. Two-cell stage with lobed nuclei, the lobes formed by the ends of the chromosomes. G. Early prophase of the ensuing division ; chromosomes re-form- ing, centrosomes dividing. H. Later prophase, the chromosomes lying with their ends in the same position as before ; centrosomes divided. maintained. As the long chromosomes diverge, their free ends arc always turned toward the middle plane (Fig. 31), and upon the re- construction of the daughter-nuclei these ends give rise to corresjiond- ing lobes of the nucleus, as in Fig. 145, which persist throughout the resting state. At the succeeding division the chromosomes reappear exactly in the same position, their ends lyiuj^ in the nuclear lobes as before {¥ig. 145, G, H). On the strength of these facts Boveri con- cluded that the chromosomes must be regarded as " individuals " or '' elementary organisms," that have an independent existence in the 298 SOME PROBLEMS OF CELL-ORGANIZATION- cell. During the reconstruction of the nucleus they send forth pseu- dopodia which anastomose to form a network in which their identity is lost to view. As the cell prepares for division, however, the chro- mosomes contract, withdraw their processes, and return to their "resting state," in which fission takes place. Applying this con- clusion to the fertilization of the Qgg, Boveri expressed his belief that Fig. 146.— Independence of paternal and maternal chromatin in the segmenting eggs of Cyclops. [A-C, from RucKERT; D, from Hacker.] A. First cleavage-figure in C. strenuus ; complete independence of paternal and maternal chromosomes. B. Resulting two-cell stage with double nuclei. C. Second cleavage; chromosomes still in double groups. D. Blastomeres with double nuclei from the eight-cell stage of C brevicornis. " we may identify every chromatic element arising from a resting nucleus with a definite element that entered into the formation of that nucleus, from which the remarkable conclusion follows that in all cells derived in the regular course of division from the fertilized egg, one-half of the chromosojnes are of strictly pater^ial origin, the other half of maternal T ^ i'9i, p. 410. MORPHOLOGICAL COMPOSITION OF THE NUCLEUS 2Qg Boveri's hypothesis has been criticised by many writers, especially by Hertwig, Guignard, and Brauer, and I myself have urged some objections to it. Recently, however, it has received a support so strong as to amount almost to a demonstration, through the remark- able observations of Ruckert, Hacker, Herla, and Zoja on the independence of the paternal and maternal chromosomes. These observations, already referred to at page 208, may be more fully re- view^ed at this point. Hacker ('92, 2) first showed that in Cyclops stre7iiiiis, as in Ascaris and other forms, the germ-nuclei do not fuse but give rise to two separate groups of chromosomes that lie side by side near the equator of the cleavage-spindle. In the two-cell stage (of Cyclops tciiiiicornis) each nucleus consists of two distinct thou^'-h closely united halves, which Hacker believed to be the derivatives of the two respective germ-nuclei. The truth of this surmise was demon- strated three years later by Ruckert ('95, 3) in a species of Cyclops, likewise identified as C. strcimiis (Fig. 146). The number of chromo- somes in each germ-nucleus is here twelve. Ruckert was able to trace the paternal and maternal groups of daughter-chromosomes not only into the respective halves of the daughter-nuclei of the two-cell stage, but into later cleavage -stages. From the bilobed nuclei of the two-cell stage arise, in each cell, a double spireme and a double group of chromosomes, from which are formed bilobed or double nuclei in the four-cell stage. This process is repeated at the ne.xt cleavage, and the double character of the nuclei was in nian\- cases distinctly recognizable at a late stage when the germ-layers were being formed. Finally Victor Herla's ('93) and Zoja's ('95, 2) remarkable obser- vations on Ascaris showed that in Ascaris not only the chromatin of the germ-nuclei, but also the paternal and maternal cliroviosonics, remain perfectly distinct as far as the twelve-cell stage — certainly a brilliant confirmation of Boveri's conclusion. Just how far the dis- tinction is maintained is still uncertain, but Hacker's and Riickert's observations give some ground to believe that it may ])ersist through- out the entire life of the embryo. Both these observers have shown that the chromosomes of the germinal vesicle appear in ti^'o (lisfinct groups, and Ruckert suggests that these may represent the paternal and maternal elements that have remained distinct throughout the entire cycle of development, even down to the formation of the (t^^'g ! Leaving aside all doubtful cases (such as the above suggestion of Riickert's), the well-determined facts form an irresistible proof of the general hypothesis ; and it is one with which every general analysis of the cell has to reckon. I beheve, however, that the hypothesis has received an unfortunate name ; for, except in a few special cases,^ (f/p. ■16- 30O SOME PROBLEMS OF CELL-ORGANIZATION almost no direct evidence exists to show that the chromosomes persist as *' individuals " in the chromatin-reticulum of the resting cell. The facts indicate, on the contrary, that in the vast majority of cases the identity of the chromosomes is wholly lost in the resting nucleus, and the attempts to identify them through the polarity or other morpho- logical features of the nuclear network have on the whole been futile. It is therefore an abuse of language to speak of a persistent ''individ- B C Fig. 147. — Hybrid fertilization of the egg oi As car is megalocephala, var. bivalens, by the sper- matozoon of van z^«/^'a/^«i'. [Herla.] A. The germ-nuclei shortly before union. B. The cleavage-figure forming; the sperm-nucleus has given rise to one chromosome (J'), the egg-nucleus to two (9). C. Two-cell stage dividing, showing the three chromosomes in each cell. D. Twelve-cell stage, with the three distinct chro- mosomes still shown in the primordial germ-cell or stem-cell. uality " of chromosomes. But this verbal difficulty should not blind us to the extraordinary interest and significance of the facts. It is difficult to suppose that the tendency of the chromatin to resolve itself into a particular number of chromosomes is directly due to its chemical or molecular structure, or is analogous to crystallization ; for in the chromatin of the same species, or even in that of the same ^^%^ this tendency varies, not with chemical, but with purely morphological MORPHOLOGICAL COMPOSFnON OF TLIE NUCLEUS ^o\ conditions, i.e. with the number of chromosomes that enter the nucleus. Neither can we assume that it is due merely to the total mass of the chromatin in each case ; for this varies in different nuclei of the same species, or even in the nucleus of the same cell at dif- ferent periods (as in the egg-cell), yet the same number of chromo- somes is characteristic of all. Indeed, we seek in vain for an analogy to these phenomena and can only admit our entire inability to explain them. No phenomena in the history of the cell more clearly indicate the existence of a morphological organization which, though resting upon, is not to be confounded with, the chemical and molecular structure that underlies it ; and this remains true even though we are wholly ignorant what that organization is. {b^ Coviposition of the CJiromosonies. — We owe to Roux ^ the first clear formulation of the view that the chromosomes, or the chr(jmatin- thread, consist of successive regions or elements that are qualitatively different (p. 244). This hypothesis, which has been accepted by Weismann, Strasburger, and a number of others, lends a peculiar interest to the morphological composition of the chromatic substance. The facts are now well estabhshed (i ) that in a large number of cases the chromatin-thread consists of a series of granules (chromomeres) embedded in and held together by the linin-substance, (2) that the splitting of the chromosomes is caused by the division of these more elementary bodies, (3) that the chromatin-grains may divide at a time when the spireme is only just beginning to emerge from the reticulum of the resting nucleus. These facts point unmistakably to the conclu- sion that these granules are perhaps^ to be regarded as independent morphological elements of a lower grade than the chromosomes. That they are not artifacts or coagulation-products is proved by their uniform size and regular arrangement in the thread, especially when the thread is split. A decisive test of their morphological nature is, however, even more difficult than in the case of the chromosomes ; for the chromatin-grains often become apparently fused together so that the chromatin-thread appears perfectly homogeneous, and whether they lose their individuality in this close union is undetermined. Observations on their number are still very scanty, but they point to some very interesting conclusions. In Boveri's figures of the egg- maturation of Ascaris each element of the tetrad consists of six chro- matin-discs arranged in a linear series (Van Beneden's figures of the same object show at most five) which finally fuse to form an appar- ently homogeneous body. In the chromosomes of the germ-nuclei the number is at least double this (Van Beneden). Their number has been more carefully followed out in the spermatogenesis of the same animal (variety bivalens) by Brauer. At the time the chromatin-grains 1 Bedeututtg der Kerntheilungsfigtiren, 1SS3, p. 15. 302 SOME PROBLEMS OF CELL-ORGAXIZATION- divide, in the reticulum of the spermatocyte-nucleus, they are very numerous. His figures of the spireme-thread show at first nearly forty granules in linear series (Fig. 120, />). Just before the breaking of the thread into two the number is reduced to ten or twelve (Fig. 120, C). Just after the division to form the two tetrads the number is four or five (Fig. 120, Z>), which finally fuse into a homogeneous body.^ It is certain, therefore, that the number of chromomeres is not con- stant in a given species, but it is a significant fact that in Ascaris the final number, before fusion, appears to be nearly the same (four to six) both in the oogenesis and the spermatogenesis. The facts re- garding bivalent and plurivalent chromosomes (p. '^j) at once sug- gest themselves, and one cannot avoid the thought that the smallest chromatin-grains may successively group themselves in larger and larsfer combinations of which the final term is the chromosome. Whether these combinations are to be regarded as ** individuals " is a question which can only lead to a barren play of words. The fact that cannot be escaped is that the history of the chromatin-substance reveals to us, not a homogeneous substance, but a definite morpho- logical organization in which, as through an inverted telescope, we behold a series of more and more elementary groups, the last visible term of which is the smallest chromatin-granule, or nuclear microsome, beyond which our present optical appliances do not allow us to see. Are these the ultimate dividing units, as Brauer suggests (p. 113)? Here again we may well recall Strasburger's warning, and hesitate to identify the end of the series with the Hmits reached by our best lenses. Somewhere, however, the series must end in final chromatic units which cannot be further subdivided without the decomposition of chromatin into simpler chemical substances ; and these units must be capable of assimilation, growth, and division without loss of their specific character. It is in these ultimate units that we must seek the ** qualities," if they exist, postulated in Roux's hypothesis; but the existence of such qualitative differences is a physiological assumption that in no manner prejudices our conclusion regarding the ultimate vioi'pJiological composition of the chromatin. D. Chromatin, Lixin, and Cytoplasm What, now, is the relation of the chromatin-grains to the linin-net- work and the cytoplasm } Van Beneden long ago maintained ^ that 1 Eisen ('99) finds that the chromosomes of the spermatogonia of Batrachoseps always consist of six "chromomeres," each of which consists of three smaller granules or " chromi- oles." The latter persist as the chromatin-granules of the resting nucleus; and it is through their successive aggregation that the chromumeres and chromosomes are formed. 2 '83, pp. 580, 583. CHROMATIN, LININ, AND CYTOPLASM 303 the achromatic network, the nuclear membrane, and the cell-mesh- work have essentially the same structure, all consisting of microsomes united by connective substance, and being only " parts of one and the same structure." But, more than this, he asserted that the chromatic and acJiroinatic 7nicrosomcs might be transformed into 07ie another, and were therefore of essentially the same morphological nature. " They pass successively, in the course of the nuclear evolution, through a chromatic or an achromatic stage, according as they imbibe or *^nve off the chromophilous substance." ^ Both these conclusions are borne out by recent researches. Heidenhain ('93, '94), confirmed by Reinke and Schloter, finds that the nuclear network contains granules of two kinds differing in their staining-capacity. The first are the basi-chro- matin granules, which stain with the true nuclear dyes (basic tar-col- ours, etc.), and are identical with the '' chromatin-granules " of other authors. The second are the oxychromatin-granules of the linin-net- work, which stain with the plasma-stains (acid colours, etc.), and are closely similar to those of the cytoreticulum. These two forms gradu- ate into one ajiother, and are conjectured to be diffeirnt phases of the same elements. This conception is furthermore supported by many observations on the behaviour of the nuclear network as a whole. The chromatic substance is known to undergo very great changes in staining-capacity at different periods in the life of the nucleus (p. ll^\ and is known to vary greatly in bulk. In certain cases a very large amount of the original chromatic network is cast out of the nucleus at the time of the division, and is converted into cytoplasm. And, finally, in studying mitosis in sea-urchin eggs I found reason to con- clude ('95, 2) that a considerable part of the linin-network, from which the spindle-fibres are formed, is actually derived from the chromatin. From the time of the earlier writings of Frommann ('65, '67), Arnold {^^j\ Heitzmann ('73), and Klein {^J^\ down to the present, an increasing number of observers have held that the nuclear reticu- lum is to be conceived as a modification of the same structural basis as that which forms the cytoplasm. The latest researches indicate, indeed, that true chromatin (nuclein) is confined to the nucleus.- I^ut the whole weight of the evidence now goes to show that the linin- network is of the same nature as the cell-mesh work, and that the achromatic nuclear membrane is formed as a condensation of the same substance. Many investigators, among whom may be named From- mann, Leydig, Klein, Van Beneden, Carnoy, and Reinke, have de- scribed the fibres of both the intra- and extra-nuclear network as terminating in the nuclear membrane ; and the membrane itself is described by these and other observers as being itself reticular in structure, and by some (Van Beneden) as consisting of closely crowded 1 I.e. p. 583. 2 Cf. Hammarsten ('95). 304 SOME PROBLEMS OF CELL-ORGAXIZATION microsomes arranged in a network. The clearest evidence is, however, afforded by the origin of the spindle-fibres in mitotic division ; for it is now well established that these may be formed either inside or out- side the nucleus, and at the close of mitosis the central portion of the spindle appears always to give rise to a portion of the cytoplasm lying between the daughter-nuclei. In such a case as that of the sea-urchin (see above) we have, therefore, evidence of a direct trans- formation of chromatin into linin-substance, of the latter into spindle- fibres, and, finally, of these into cytoplasm. When all these facts are placed in connection, we find it difficult to escape the conclusion that no definite line can be drawn between the cytoplasmic granules at one extreme and the chromatin-granules at the other. And inasmuch as the latter are certainly capable of growth and division, we cannot deny the possibility that the former may themselves have, or arise from elements having like powers. But while we may take this as a fair working hypothesis, we should clearly recognize that the base of well-determined fact on which it rests is approached by a circuitous route ; that in case of most of the cytoplasmic granules there is not the slightest evidence that they multiply by division ; and that even though some of them may have such powers, we cannot regard them as the ultimate structural units, for the latter must be bodies far more minute. E. The Centrosome From our present point of view the centrosome possesses a peculiar interest as a cell-organ which may be scarcely larger than a cytomi- crosome, yet possesses specific physiological properties, assimilates, grows, divides, and may persist from cell to cell without loss of identity. Nearly all observers of the centrosome have found it lying in the cytoplasm, outside the nucleus; but apart from the Protozoa (p. 94) there is at least one well-established case in which it lies within the nucleus, namely, that of Ascaris, where Brauer made the interesting discovery that in one variety {iinivalens) the centrosome lies inside the nnclens^ in the other variety {bivalens) outside — a fact which proves that its position is non-essential ((/. Figs. 120 and 148). An intra-nuclear origin of the centrosome has also been asserted by Julin ('93) in the primary spermatocytes of Styleopsis, by Riickert ('94) in the eggs of Cyclops, Mathews ('95) in those of Asterias, Car- noy and Le Brun ('97, 2) in Ascaris, Van der Stricht ('98) in the eggs of Thysanozodn, by R. Hertwig ('98) in ActinospJicerinm, Calkins ('98, I ) in Noctiluca, and Schaudinn ('96, 3) in spore-producing buds of Acanthocystis, though in the last-named form the centrosome of the vegetative forms is extra-nuclear (p. 92). 1 THE CEXTROSOME 305 As already stated, ^ it is still undetermined whether a true centro- some may ever arise de novo, but the evidence in favour of such a possibility has of late rapidly increased. Carnoy ('86) long since showed that the ^g^ of Ascaris, during the formation of the polar bodies, sometimes showed numerous accessory asters scattered through the cytoplasm. Reinke ('94) described somewhat similar asters in peritoneal cells of the salamander, distinguishing among them three orders of magnitude, the largest containing distinct centrosomes or "primary centres," while the smaller contained "secondary" and "tertiary" centres, the last named being single H D E a: F ^ Fig. 148. — Mitosis witli intra-nuclear centrosome, in the spermatocytes of Ast\iris tttfx<^h- cephala, var. univalens. [Brauer.] A. Nucleus containing a quadruple group or tetrad of chromosomes (/). nucleolus («). and centrosome {c). B. C. Division of the centrosome, D. E.F. G. Formation of the mitotic tiijure. centrosomes escaping from the nucleus in G. microsomes at the nodes of the cytoreticulum. By successive aggre- gations of the tertiary and secondary centres arise true centrosomes as new formations. Watase ('94-95) ^^^o finds in the q^^ of Mturo- bdella, besides the normal aster containing an undoubted centrosome. numerous smaller asters graduating downwards to such "tertiary asters" as Reinke describes with a microsome at the centre of each, and on this basis concludes that the true centrosome differs from a microsome only in degree and may arise dc novo. iMottier ('97. 2) finds in pollen-mother-cells numerous minute " cyto-asters " having no direct relation to the spindle-formation (Fig. 133). Again Juel 1 r/pp. 52. 214. 3o6 SOME PROBLEMS OF CELL-ORGANIZATION ('97) finds that an isolated chromosome, accidentally separated from the equatorial plate (pollen-mother-cells of Hcmei'ocallis), may give rise to a small vesicular nucleus which may subsequently divide by mitosis, though it is quite out of relation to the spindle-poles of the preceding mitosis (Fig. 149). Strong evidence of the same character as the last is given by the facts in the heliozoon AauitJiocystis, as shown by Schaudinn ('96, 3), the ordinary vegetative cells containing a persistent extra-nuclear centrosome, while in the bud-formation of the swarm-spores a centrosome is formed dc novo, ivitJiojit relation to that of the mother-cell, inside the nucleus of the bud (Fig. 41). The strongest case in favour of the independent origin of centro- somes is, however, given by the observations of Mead on CJiatopteriis ('98) and the remarkable experiments of R. Hertwig ('95, '96) and A C Fig. 149. — Abnormal mitosis in pollen-mother-cells of Hemerocallis, showing formation of small nucleus from one or two stray chromosomes and its subsequent division. [JUEL.] Morgan ('96, i ; '99, i)on the eggs of echinoderms and other animals. When eggs of CJicEtopterns are taken from the body-cavity and placed in sea-water, a multitude of small asters appear in the cytoplasm, two of which are believed to persist as those of the polar spindle, while the others degenerate (Fig. 150). Mead is therefore convinced that the polar centrosomes arise in this case separately and de novo} R. Hertwig showed that when unfertilized eggs of sea-urchins { Strongylocentrotus , EcJiiniis) are kept for some time in sea-water or treated with dilute solutions of strychnine the nuclei undergo some of 1 A number of other authors {^e.g. Griffin, Thalassema, Coe, Cerebratuhis) have likewise found the first polar asters widely separated at their first appearance. On the other hand, Mathews ('95), whose preparations I have seen, finds the polar centrosomes in Asto-ias close together, and Francotte ('97, '98) has demonstrated that in Cydopoi-iis and Prosthece- ra:iis\\v^v arise by the division of a single primary centrosome. The same is stated by Gar- diner ('98) to be the case in Polychccrus. It should be noted, further, that Mead could find no undoubted centrosomes save in the " primary " or definitive polar asters. THE CENTROSOME 30; the changes of mitosis, the chromatin-nctwork giving rise to a group of chromosomes and a spindle, or more frequently a fan-shaped half-spindle, arising from the achromatic substance. In some cases not only a complete spindle appeared but also asters at the poles, though no centrosomes were observed (Fig. 151). Morgan's experi- ments along the same lines were mainly performed upon the sea- urchin Arbacia, but included also the eggs of Astcrias, Sipunculus, and Cerebratnlus (Figs. 150, 151). In these eggs numerous asters may arise in the cytoplasm, if they are allowed to lie some time in sea- v=i-il!.:: /::••.. 9!f^yU -MSB ■:;::. ^ :'i^M--' W- '"■■■ ,j->;_^^- -.^ .— • -: ^- •. ' -.r V-"; *■;. \ ■«* • ; ■ ■ ^^^ - mimm c ■:^^. '■:-->J: ':.-:-:'vk'^^ c#i^ ^ B ••.::-^:: "^^kWM-o c Fig. 150. — Formation de novo (?) of centrosomes. [A, B, MEAD; C, MORGAN.] A. Unfertilized egg of ChcBtopterus with " secondary asters " developed a few minutes after the egg is placed in sea-water. D. Slightly later stage with two definitive polar asters and centrosomes. C. Large "sun" (transformed polar aster) containing numerous small "secondary asters " .in <1 centrosomes, from unfertilized egg of Cerebratnlus after 22 hours in 1.5 '•; sodium chlorult- solution. water or treated by weak solutions of sodium or magnesium chloride. These asters often contain deeply staining, central granules indistin- guishable from the centrosomes of the normal asters ; and, what is ot high interest, such of them as lie near the nucleus take part in the irregular nuclear division that ensues, forming centres toward which the chromosomes pass. These divisions continue for some time, the chromosomes being irregularly distributed through the it^^^, and giving rise to nuclei of various sizes apparently dependent upon the number of chromosomes each receives. After a variable number of such 3o8 SOME PROBLEMS OF CELL-ORGANIZATION divisions the asters disappear, yet the irregular nuclear divisions con- tinue, nuclear spindles with distinct centrosomes being formed at each division, but apparently without relation to the older asters, and they wmi:^^ Fig. 151. — Formation of centrosomes and asters in unfertilized echinoderm-eggs. [A, B, Morgan ; C-E, R. Hertwig.] A. Arbacia, after 4V2 hours in 1.5 % solution of sodium chloride, then 5 hours in sea-water; scattered chromosomes and asters. B. Asters formed after 6^2 hours in XaCl. C-E. Echinus after treatment with 0.5 % strychnine-solution, showing various forms of astral formations (fan- shaped aster, half spindle, and complete mitotic figure). are believed by Morgan to arise de novo from the Qgg substance.^ In the meantime irregular cleavage of the Qgg occurs, though no embryo is produced.'^ Loeb, however, in the remarkable experiments ^ '99, p- 479. - Morgan makes the important observation, which harmonizes with that of Boveri, reported at page 108, that the divisions occur with respect to the nujuber and position of the nuclei, not of the asters, concluding that the former must therefore play an essential rdle as centres of division, and that the activity of the asters is in itself not sufficient to account for division of the cytoplasm. I THE CENTROSOME 309 referred to at page 215, finds that after treatment with magnesium chloride unfertilized sea-urchin eggs {Arbacia) may give rise to perfect Phiteus larvae — a result which if well founded seems to place the new formation of true centrosomes beyond question. Taken together, these researches give strong ground for the con- clusion that true {i.e. physiological) centrosomes may arise de novo from either the cytoplasmic or the nuclear substance and may play the usual role (whatever that may be) in mitosis. If this conclusion be sustained by future research, we shall no longer be able to accept Van Beneden's and Boveri's conception of the centrosome as a per- sistent organ in the same sense as the nucleus ; but on the other hand we shall have gained important ground for further inquiry into the nature and source of that power of division which is so characteristic of living things and upon which the law of genetic continuity rests. Morphology of the Centrosome. — In its simplest form (Fig. 152, A) the centrosome appears under the highest powers as nothing more than a single granule of extraordinary minuteness which stains intensely with iron-haematoxylin, and can scarcely be distinguished from the cyto-microsomes except for the fact that it lies at the focus of the astral rays. In this form it always appears at the centre of the very young sperm-asters during fertilization (Figs. 97, 99), in the earlv phases of ordinary mitosis (Figs. 27, 32), and in some cases also in the resting cell, for example, in leucocytes and connective tissue corpuscles (Figs. 8, 49), where, however, it is often triple or quadruple. In the course of division the centrosome often increases in size and assumes a more complex form, becoming also surrounded by various structures involved in the aster-formation. The relation of these structures to the centrosome itself has not yet been fully cleared up and there is still much divergence of opinion regarding the cycle of changes through which the centrosome passes. It is, therefore, not yet possi- ble to give a very consistent account of the centrosome, still less to frame a satisfactory morphological definition of it. It is convenient to take up as a starting-point Boveri's ('88) account of the centrosomes in the Qgg of Asearis, supplemented by Brauer's ('93) description of those in the spermatocytes of the same animal. During the early prophases of the first cleavage Boveri found the centrosome as a minute granule which steadily enlarges as the spin- dle forms, until shortly before the metaphase it becomes a rather large, well-defined sphere in the centre of which a minute central gratni I c or ceiitriole appears (Fig. 152, B, C). From this time onward the cen- trosome decreases in size until in the daughter-cells it is again reduced to a small granule which divides into two and goes through a similar cycle during the second cleavage and so on. The centrosome is at all stages surrounded by a clear zone ("Heller Hot") in which 310 SOME PROBLEMS OF CELL-ORGANIZATION the astral rays are thinner and stain less deeply than farther out. Brauer's account is substantially the same, though no definite "Heller Hof " was found, and the astral rays were traced directly in to the boundary of the centrosome. He added, however, two impor- tant observations, viz. (i) that the central granule is visible at every period ; and (2) division of tJie centrosome is preceded by division of tJie centi'al granule {Y\g. 148) — an observation recently extended by Boveri to the division of the egg-centrosome.^ Van Beneden and Neyt {^Zj\ on the other hand, gave a quite different account of the t ig. 152. — Diagrams illustrating various accounts of centrosome and aster, A. Centrosome, a simple granule at the centre of the aster; ex. sperm-aster in various animals. B. "Centrosome," a sphere enclosing a central granule or centriole; ex. Brauer's account of spermatocytes oi Ascaris. C Like the last, but " centrosome " surrounded by a "Heller Hof"; ex. Boveri's account of the centrosome of the Ascaris egg. D. Central granule surrounded by a radial sphere ("centrosome") bounded by a microsome-circle, and lying in a "Heller Hof"; ex. polar spindles of Thysatiozodn, Van der Stricht. E. Central granule (" centrosome ") sur- rounded by medullary and cortical radial zones, each bounded by a microsome-circle ; ex. polar spindle of Unio, Lillie, F. Van Beneden's representation of aster of the Ascaris egg; like the last, but the " corpuscule central " consisting of a group of granules. G. " Centrosome," a group of granules surrounded by a "Heller Hof"; ex. the echinoderm-egg. //. "Centrosome" (central granule) surrounded by a vague larger body lying in a reticulated centrosphere ; ex. Thalasseyna. [Griffin.] Structures at the centre of the aster. The " corpuscule central " (usually assumed by later writers to be the centrosome), described as a "mass of granules," is surrounded by two well-defined astral zones, formed as modifications of the inner part of the aster, and constitut- ing the "attraction-sphere." These are an inner "medullary zone," and an outer " cortical zone," each bounded by a very distinct layer of microsomes (Fig. 152, F). 1 Reported by Fiirst, '98, p. in. THE CENTROSOME 311 The discrepancy between these results on the part of the two pioneer investigators of the centrosome has led to great confusion in the terminology of the subject, which has not yet been fully cleared away. Many of the observers who followed Boveri (Flem- ming, Hermann, Van der Stricht, Heidenhain, etc.) found the centro- some, in various cells, as a much smaller body than he had described, often as a single or double minute granule, staining intensely with iron-haematoxylin. Heidenhain ('93, '94) and Druner ('94, '95) found further that the asters in leucocytes and other forms often show several concentric circles of microsomes, and that the sphere bounded by the innermost circle often stains more deeply than the outer por- tions and may appear nearly or quite homogeneous (Fig. 156). To this sphere, with its contained central granule or granules Heidenhain applies the term microcentrum ('94, p. 463), while Kostanecki and Siedlecki suggest the term microsphere ('96, p. 217). Still later Kostanecki and Siedlecki ('97) found that even in Ascaris, as in other forms, sufficient extraction of the colour (iron-haematoxylin) reduces the centrosome to a minute granule to which the astral rays converge, and which is presumably identical with Boveri's ''central granule." Heidenhain ('93, '94) found that in leucocytes the central granule is often double, triple, or even quadruple, while in giant-cells of certain kinds there are numerous deeply staining granules (Fig. 14). He therefore proposed to restrict the term centrosome to the individ- ual granules, whatever be their number, applying the term microccn- triim to the entire group ('94, p. 463). With these facts in mind we can gain a clear view of the manner in which both the confusion of terminology and the contradiction of results has arisen. Brauer ('93) found in Ascaris (see above) that division of the central grmuile precedes division of the ^^ centrosome ,'' and therefore suggested that only the former is equivalent to Wan Beneden's '' corpuscule central," while the body called "centrosome" by Boveri is really the medullary astral zone, the " Heller Hof " being the cortical zone. This is substantially the same conclusion reached by Heidenhain, Rawitz, Lenhossek, Kostanecki and Siedlecki, Krlan- ger, Van der Stricht, Lillie, and several others. The confusion of the subject is owing, on the one hand, to the fact that those who have accepted this conclusion continue to use the word centrosome in two quite different senses, on the other hand to the fact that the conclusion is itself repudiated by Boveri ('95), MacFarland (97), and Fiirst ('98). As regards the terminology we find that most recent writers agree with Heidenhain, Kostanecki and Siedlecki, in restricting the word centrosome to the minute, deeply staining granules, whether one or more, at the centre of the aster. On the other hand, Brauer, Fran- 312 SOME PROBLEMS OF CELL-ORGANIZATION cotte, Van der Stricht, Meves, and others apply the term to the central granule or granules plus the surrounding sphere (" centrosome " of Boveri), which they regard as equivalent to the medullary zone of Van Beneden, the " corpuscule central" of the last-named author being identified with the central granule or "centriole" of Boveri, though the latter structure is considerably smaller than the former as described by Van Beneden. The matter of fact turns largely on the question whether the astral rays traverse the larger sphere to the central granule. That such is the case in Ascaris is positively asserted by Kostanecki and Siedlecki, ('97) and as positively denied by Fiirst ('98) with whose observations Fig. 153. — Structure of the centiosome in the polar asters of a gasteropod, Dinulula. [MaC- Farland.] A. Mitotic figure, formation of first polar body. B. Inner aster at final anaphase; central granule double within the " centrosome." C. Elongation of old " centrosome " to form second polar spindle. those of MacFarland ('97) on gasteropod-eggs agree. On the other hand, in the turbellarians the observations of Francotte ('97, '98) and Van der Stricht ('98, i) seem to leave no doubt that the larger sphere ("centrosome"), here very sharply defined and staining deeply in iron-haematoxylin, is traversed by well-defined astral rays converging to the central corpuscle, and both these observers agree further that hotJi the corpuscle and tJie sphere divide to persist as the ^^ eentrosonies " of tJie daughter-cells — a result in conformity with Van Beneden's con- clusion in the case of Ascaris. Lillie's valuable observations on the polar asters of Unio ('98) afford, I believe, conclusive evidence as to the nature of the sphere. In the THE CENTROSOME earlier stages the aster has cxarM, .u ^'^ w ^ ^ 6^ Fig- 154- - Centrosome and aster in entosphere iound^TK'"' ^^^'^^Ph^-'eJ ^ones. the polar mitoses of C/n/o. [Lillie.] granule (centrosome) surrounded bv medullarv ^. Late anaphase of second polar mitosis raS oftt'oid'rS:,::--"^ ---.• ^ormatt' rays pass (Fig- ic-? /7 p- a dense and deep?y"staininf s!,!!!' '^^l '"""' '^^"'''' '^°"^'^ting of structure and is b^LderbTa i's"' ' f ''■^' '"^ ^>-'^''"' '''-"'^•^^^ anaphase) the central grat,fe di "deTT^to f' '" ^^^ ^''"'^'"-■^ "^^^^ ^o-r or .ore granuies,^of whicJl^^ Co^^ or;rl;^r:cti"l; 314 SOME PROBLEMS OF CELL-ORGANIZATION persist. The inner sphere is now bounded by a definite membrane, and its radiate structure becomes obscure, the astral rays extending only to the boundary of the sphere, though a few rays persist within it (Fig. 154, B). It is clear from this that the inner sphere and central granule pass through phases that bridge the gap between Van Beneden's and Boveri's descriptions. LilHe's observations fully sustain the conclusion that the cmtral gmniUe {^' ceiitriole'' of Boveri) corresponds to the '' corpiiscide cejitraV' of Van Beneden, and the inner spJiere {medullary zone) to BoverVs '' cejitrosomey A comparison of the polar aster of rm'o with that of TJiysanozoon, as described by Van der Stricht ('98), leaves hardly room for doubt that the cortical zone represents Boveri's " Heller Hof " ; for in both forms the rays of the cortical zone are much thinner and Hghter than the more peripheral portions, thus giving a clear zone, which in Uiiio is bounded by only a fairly definite microsome-circle and in TJiysanozoon by none. Lastly, we must recognize the justice of the view urged by Kos- tanecki. Griffin, Mead, Lillie, Coe, and others, that the term centro- some should be applied to the central granule and not to the sphere surrounding it (medullary zone), despite the fact that historically the word was first applied by Boveri to the latter structure. For in both Diauhila (MacFarland) and Unio (Lillie) the second polar spindle arises from the substance of the inner sphere, while the central granule, becoming double, gives rise to the centrosomes at its poles. By following Boveri's terminology, therefore, MacFarland is driven to the strange conclusion that the second polar spindle is nothing other than an enormously enlarged '* centrosome " — a result little short of a reductio ad absnrdnm when we consider that in Ascaris the polar spindle arises by a direct transformation of the germinal vesicle (p. 277). The obvious interpretation is that the central granule is the only structure that should be called a centrosome, the surround- ing sphere being a part of the aster, or rather of the attraction-sphere. Thus regarded, the origin of the spindle in Dianlula presents nothing anomalous and a similar interpretation may be placed on the polar spindles of Ascaris as described by Fiirst ('98).^ 1 In echinoderms the concurrent' results of Reinke ('95), Boveri ('95), myself ('96-'97), show that the " centrosome " is a well-defined sphere containing a large group (ten to twenty) of irregularly scattered, deeply staining granules. I have shown in this case that in the early prophases there is but one such granule, which then becomes double and finally multiple, forming a pluricorpuscular centrum (Fig. 52) not unlike that described by Heidenhain in giant-cells. Kostanecki, who asserts that the centrosome of echinoderms is a single granule ('96, i, '96, 2, p. 248), has not sufficiently studied the later phases of mitosis. Cf. also Erlanger ('98). The centrosomes described in nerve cells by Lenhossek ('95) are apparently of somewhat similar type. Until the facts are more fully known the exact nature of these "centrosomes" remains an open question. Lillie's observations on Unio show that here, too (first polar spindle), the centrosome divides to form a considerable number of THE CEiVTROSOME ^ic The genesis of the concentric spheres surrounding the centrosome will be considered in the following section. We may here only emphasize the remarkable fact that the centres of the dividino- system are bodies which are in many cases so small as to lie almost at the limits of microscopical vision, and which in the absence of the surrounding structures could not be distinguished from other proto- plasmic granules. Full weight should be given to this fact in every estimate of the centrosome theory, and it is no less interestin"- in its bearing upon the corpuscular theory of protoplasm. Watase ('93, '94) made the very interesting suggestion that t/ie cen- trosome is itself notJiing other tJian a microsome of the same morj^ho- logical nature as those of the astral rays and the general meshwork, differing from them only in size and in its peculiar powers.^ Despite the vagueness of the word "microsome," which has no well-defined meaning, Watase's suggestion is full of interest, indicating as it does that the centrosome is morphologically comparable to other elemen- tary bodies existing in the cytoplasmic sti-ucture, and which, minute though they are, may have specific chemical and physiological prop- erties. An interesting hypothesis regarding the historical origin of centrosome is that of Rutschli ("91) and R. Hertwig ("92), who suggest that it may be a derivative of a body comparable with the micro-nucleus of Infusoria, which has lost its chromatin but retained the power of division ; and the last-named author has suggested further that the so-called " archoplasmic loops'' discovered by Platner in puhnonates mav be remnants of the chromatic elements. A similar view has been advocated bv Heidenhain (''93, '94) and Lauterborn ('96). Heidenhain regards central spindle and centrosomes as forming essentially a unit ("-microc^entrum ") homologous with the micro-nucleus of the Infusoria, the centrodesmus (p. 79) representing a part of the original achromatic elements. The metazoan nucleus is compared to the proto- zoan macro-nucleus. The improbability of a direct derivation of the Metazoa from Infusoria, urged by Boveri ('95) and Hertwig ("96), has led Lauterborn ("96) to the view that the metazoan centrosome and nucleus are respectively derivatives of two equivalent nuclei, such as Schaudinn ("95) describes in Ainivba hinucUdta, the ='Nebenkorper" of Paraifia'ba (cf. p. 94), being regarded as an intermediate step, and the micro-nucleus of Infusoria a side-branch. R. Hertwig ('96), on the other hand, regards the metazoon centrosome as a derivative of an intra-nuciear botlv such as the '' nucleolo-centrosome " of Euglena (p. 91), which has itself arisen through a condensation of the general achromatic substance. With this view Calkins (98), on the whole, agrees; but he regards it as probable that the •• nucleoIo-ccntrDsome " granules of which one or two remain as the persistent centrosome. while others are eonverted into microsomes or other cytoplasmic structures. It is probable that somethinj; similar occurs in the echinoderms. 1 The microsome is conceived, if I understand Watase rightly, not as a permanenl mor- phological body, but as a temporary varicosity of the thread, which may lose its i.lentity in the thread and reappear when the thread contracts. The centrosome is in like manner not a permanent organ like the nucleus, but a temporary body formed at the focus of the astral rays. Once formed, however, it may long persist even after disappearance o\ the aster, and serve as a centre of formation for a new aster. 3i6 SOME PROBLEMS OF CELL-ORGANIZATION of Eitgleiia and Ainaba and the sphere of N'octiluca and Paramooba are to be com- pared with the attraction-sphere, while the centrosome may have had a different origin. It appears to me that none of these views rests upon a very substantial basis, and they must be taken rather as suggestions for further work than as w^ell-grounded conclusions. F. The Archoplasmic Structures I. Hypothesis of Fibrillar Persistence The asters and attraction-spheres have a special interest for the study of cell-organs ; for they are structures that may divide and persist from cell to cell or may lose their identity and re-form in suc- cessive cell-generations, and we may here trace with the greatest clearness the origin of a cell-organ by differentiation out of the struc- tural basis. Two sharply opposing views of these structures have been held, represented among the earlier observers on the one hand by Boveri, on the other by Blitschli, Klein, Van Beneden, and Carnoy. The latter observers held that the astral rays and spindle-fibres, and hence the attraction-sphere, arise through a morphological rearrange- ment of the preexisting protoplasmic meshwork, under the influence of the centrosome. This view, which may be traced back to the early work of Fol ('73) and Auerbach ('74), was first clearly formulated by Biitschli ('76), who regarded the aster as the optical expression of a peculiar physico-chemical alteration of the protoplasm primarily caused by diffusion-currents converging to the central area of the aster.^ An essentially similar view is maintained in Biitschli's recent great work on protoplasm,^ the astral " rays " being regarded as nothing more than the meshes of an alveolar structure arranged radially about the centrosomes (Fig. 10, B). The fibrous appearance of the astral rays is an optical illusion, for they are not fibres, but i^at lamellae forming the walls of elongated closed chambers. This view has recently been urged, especially by Erlanger ('97, 4, etc.), who sees in all forms of asters and spindles nothing more than a modified alveolar structure. The same general conception of the aster is adopted by most of those who accept the fibrillar or reticular theory of protoplasm, the astral rays and spindle-fibres being regarded as actual fibres forming part of the general network. One of the first to frame such a con- ception was Klein i^']'^\ who regarded the aster as due to "a radial arrangement of what corresponds to the cell-substance," the latter 1 For a veiy careful review of the early views on this subject, see Mark, Liinax, i88i. 2 '92, 2, pp. 158-169. THE ARCHOPLASMIC STRUCTURES 317 being described as having a fibrillar character.^ The same view is advocated by Van Beneden in 1883. With Klein, Heitznian, and Frommann he accepted the view that the intra-nuclear and extra- nuclear networks were organically connected, and maintained that the spindle-fibres arose from both.^ *'The star-like rays of the asters are nothing but local differentiations of the protoplasmic network.-^ . •• . In my opinion the appearance of the attraction-spheres, the polar corpuscle (centrosome), and the rays extending from it, includ- ing the achromatic fibrils of the spindle, are the result of the appear- ance in the egg-protoplasm of two centres of attraction comparable to two magnetic poles. This appearance leads to a regular arrange- ment of the reticulated protoplasmic fibrils and of the achromatic nuclear substance with relation to the centres, in the same way that a magnet produces the stellate arrangement of iron filings."* This view is further developed in Van Beneden's second paper, pubUshed jointly with Neyt {'^7). " The spindle is nothing but a differentiated portion of the asters." ^ The aster is a ** radial structure of the cell-protoplasm, whence results the image designated by the name of aster." '^ The operations of cell-division are carried out through the " contractility of the fibrillae of the cell-protoplasm and their arrangement in a kind of radial muscular system composed of antagonizing groups." *" An essentially similar view of the achromatic figure has been advocated by many later workers. Numerous observers, such as Rabl, Flemming, Carnoy, Watase, Wilson, Reinke, etc., have ob- served that the astral fibres branch out peripherally into the general meshwork and become perfectly continuous with its meshes, and tracing the development of the aster, step by step, have concluded that the rays arise by a direct progressive modification of the pre- existing structure. The most extreme development of this view is contained in the works of Heidenhain ('93, '94), Buhler (95), Kosta- necki and Siedlecki ('97), which are, however, only a development of the ideas suggested by Rabl in a brief paper published several years before. Rabl ('89, 2) suggested that neither spindle-fibres nor astral rays really lose their identity in the resting cell, being only modified in form to constitute the mitome or filar substance (meshwork), but still beins: centred in the centrosome. Fission of the centrosome is followed by that of the latent spindle-fibres (forming the linin- network); hence each chromosome is connected by pairs of daughter- 1 It is interesting to note that in the same place Klein anticipated the theory of fibrillar contractility, both the nuclear and the cytoplasmic reticulmii being regarded as contractile (/.r., p. 417). 2 'S3, p. 592. * '53, p. 550. 6 /.^.. p. 275. 3 '83, p. 576. 5 '87, p. 263. ' /.^., p. 2S0. 3l8 SOME PROBLEMS OF CELL-ORGANIZATION fibres with the respective centrosomes. Heidenhain, adopting the first of these assumptions, builds upon it an elaborate theory of cell- polarity and cell-division already considered in part at pages 103-105. Sometimes the astral rays ('* organic radii ") retain their radial arrange- ment throughout the life of the cell (leucocytes, Fig. 49) ; more com- monly they are disguised and lost to view in the cytoplasmic meshwork. All, however, are equal in length and in tension — assumptions based on the one hand on the occurrence of concentric circles of microsomes in the aster, on the other hand on the analogy of the artificial model described at page 104. Biihler ('95) and Kostanecki and Siedlecki ('97) likewise unreservedly accept the view that besides the centro- some the entire system of '' organic radii," including astral rays, mantle-fibres, and central spindle-fibres, persists in the resting cell in modified form, and is centred in the centrosome. Kostanecki finally ('97) takes the last step, logically necessitated by the foregoing con- clusion, and apparently supported also by the crossing of the astral rays opposite the equator of the spindle and the relations of their peripheral ends, concluding that the monocentric astral system is con- verted into the dicentric system (amphiaster) by longitudinal fiSsion of the rays} Thus the entire mitome of the mother-cell divides into equal halves for daughter-cells ; and since the radii consist of micro- somes, each of these must likewise divide into two.^ Could this tempting hypothesis be established, Roux's interpretation of nuclear division (p. 224) could be extended also to the cytoplasm ; and the aster- and amphiaster-formation, with the spireme-forma- tion, might be conceived as a device for the meristic division of the entire cell-substance — a result which would place upon a substantial basis the general corpuscular theory of protoplasm. Unfortunately, however, the hypothesis rests upon a very insecure foundation : first, because it is based solely upon the fibrillar theory of protoplasm ; second, because of the very incomplete direct evidence of such a splitting of the rays ; third, because there is very strong evidence that in many cases the old astral rays wholly disappear, to be replaced by new ones.^ We may best consider this adverse evidence in connec- tion with a general account of the opposing archoplasm-hypothesis. 2. The Archoplasm Hypothesis Entirely opposed to the foregoing conception are the views of Boveri and his followers, the starting point of which is given by 1 '97, p. 680. 2 This view had been definitely stated also by O. Schultze in 1890. ^ There is, however, no doubt that the aster as a whole does, in some cases, divide into two — for instance, in the echinoderm-egg. Fig. 95. THE ARCHOPLASMIC STRUCTURES 3IQ Boveri's celebrated archoplasm-hypothesis. Boveri has from the first maintained that the amphiastral fibres are quite distinct from the Gen- eral cell-meshwork. In his earlier papers he maintained ('88, 2) that the attraction-sphere of the resting cell is composed of a distinct sub- stance, '' arcJioplasm,'' consisting of granules or microsomes aggre- gated about the centrosome as the result of an attractive force exerted by the latter. From the material of the attraction-sphere arises the entire achromatic figure, including both the spindle-fibres and the astral rays, and these have nothing to do with the general reticulum of the cell. They grow out from the attraction-sphere into the reticu- lum as the roots of a plant grow into the soil, and at the close of mitosis are again withdrawn into the central mass, breaking up into granules meanwhile, so that each daughter-cell receives one-half of the entire archoplasmic material of the parent-cell. Boveri was further incUned to believe that the individual granules or archoplas- mic microsomes were " independent structures, not the nodal points of a general network," and that the archoplasmic rays arose by the arrange- ment of these granules in rows without loss of their identity.^ In a later paper on the sea-urchin this view underwent a considerable modification through the admission that the archoplasm may not ])re- exist as formed material, but that the rays and fibres may be a new formation, crystallizing, as it were, out of the protoplasm about the centrosome as a centre, but having no organic relation with the gen- eral reticulum ; though Boveri still held open the possibilitv that the archoplasm might preexist in the form of a specific homogeneous sub- stance distributed through the cell, though not ordinarily demonstra- ble by reagents.^ In this form the archoplasm-theory approaches very nearly that of Strasburger, described below. There are three orders of facts that tell in favour of Boveri's modi- fied theory : first, the existence of persistent archoplasm-masses or attraction-spheres from which the amphiasters arise ; second, the origin of amphiasters in alveolar protoplasm ; and, third, the increas- ing number of accounts asserting the replacement of the old asters by others of quite new formation. In at least one case, namely, that of Noctiluca, the entire achromatic figure is formed from a permanent attraction-sphere lying outside the nucleus and perfectly distinct fn^n the general cell-meshwork.^ Other cases of this kind are very rare, and in most cases the attraction-sphere sooner or later disintegrates,^ but in the formation of the spermatozoa we have many examples of archoplasmic masses (Nebenkern, attraction-sphere, idiozome), which apparently consist of a specific substance having a special relation to the achromatic figure. 1 '88, 2, p. 80. 3 Ishikawa, '94, '98, Calkins. '98, 2. 2 '95, 2, p. 40. ^ Cf. p. 323. 120 SOME PROBLEMS OF CEIJ.-OK^_ j^^ ThysanOZOOIl (Van der Stricht) only a single ring of micro- somes exists, and this lies at the boundary between the medullary and cortical zones (Fig. 152, D), the latter differing from the outer region only in the greater delicacy of the rays and their lack of staining-capacity, thus producing a '* Heller Hof." In other cases, no " microsome-circles " exist; but even here a clear zone often surrounds the centrosome {e.g. in Physa, t. Kostanecki and Wierzejski), like that seen in the cortical zone of TJiysanozoon. There are some observations indicating that the entosphere (medul- lary zone) may be directly derived from the centrosome (central granule). This is the conclusion reached by Lillie in the case of Unio referred to above, where, during the prophases of the second polar spindle, the central granule enlarges and breaks up into a group of granules from which the new entosphere is formed. Van der Stricht ('98) reaches a similar conclusion in case of the first polar spindle of TJiysanozoon. We may perhaps give the same interpretation to the large pluricorpuscular centrum of echinoderms (p. 314). This obser- vation may be used in support of the probability that the astral rays rays showing indications of nine concentric circles of microsomes. The area within the second circle probably represents the " attrac- tion-sphere " of Van Beneden. SUMMARY AND CONCLUSION- 327 may be actually derived from the centrosome (p. 321) ; but Lillie finds in some cases that in the same mitosis the entosphere is formed by a different process, arising by a differentiation of the cytoplasm around the central granule. The former case, therefore, may be interpreted to mean simply that the centrosome may give rise to other cytoplasmic elements (as has already been shown in the formation of the sperma- tozoon, p. 172), the material of which may then contribute either directly or indirectly to the building of the aster ; and the facts do not come into collision with the view that the astral rays are in f^en- eral formed from the cytoplasmic substance. G. Summary and Conxlusion A minute analysis of the various parts of the cell leads to the con- clusion that all cell-organs, whether temporary or ** permanent," are local differentiations of a common structural basis. Temporary organs, such as cilia or pseudopodia, are formed out of this basis, persist lor a time, and finally merge their identity in the common basis again. Per- manent organs, such as the nucleus or plastids, are constant areas in the same basis, which never are formed de novo, but arise by the divi- sion of preexisting areas of the same kind. These two extremes are, however, connected by various intermediate gradations, examples of which are the contractile vacuoles of Protozoa, which belong to the category of temporary organs, yet in many cases are handed on from one cell to another by fission, and the attraction-spheres and asters, which may either persist from cell to cell or disappear and re-form about the centrosome. There is now considerable evidence that the centrosome itself may in some cases have the character of a perma- nent organ, in others may disappear and re-form like the asters. The facts point toward the conclusion, which has been especially urged by De Vries and Wiesner, that the power of division, not only of the cell-organs, but also of the cell as a whole, may have its root in a like power on the part of more elementary masses or units of which the structural basis is itself built, tJie degree of permaiieuee in the eell- organs depending on the degree of cohesion manifested by these elemen- tary bodies. If such bodies exist, they must, however, in their primary form, lie beyond the present Hmits of the microscope, the visible struc- tures arising by their enlargement or aggregation. The cell, therefore, cannot be regarded as a colony of ''granules " or other gross morpho- logical elements. The phenomena of cell-division show, however, that the dividing substance tends to differentiate itself into several orders of visible morphological aggregates, as is most clearly shown in the nuclear substance. Here the highest term is the plurivalent chromo- some, the lowest the smallest visible dividing basichromatin-grains, 328 SOME PROBLEMS OF CELL-ORGANIZATION while the intermediate terms are formed by the successive aggrega- tion of these to form the chromatin-granules of which the dividing chromosomes consist. Whether any or all of these bodies are *' indi- viduals " is a question of words. The facts point, however, to the conclusion that at the bottom of the series there must be masses that cannot be further split up without loss of their characteristic proper- ties, and which form the elementary morphological units of the nucleus. In case of the cytoplasm the evidence is far less satisfactory. Could Rabl's theory of fibrillar persistence, as developed by Heiden- hain and Kostanecki, be established, we should indeed have almost a demonstration of panmeristic division in the cytoplasm. At present, however, the facts do not admit the acceptance of that theory, and the division of the visible cytoplasmic granules must remain a quite open question. Yet w^e should remember that the dividing plastids of plant-cells are often very minute, and that in the centrosome we have a body, no larger in many cases than a ** microsome," which is positively known to be in some cases a persistent morphological ele- ment, having the power of growth, division, and persistence in the daughter-cells. Probably these powers of the centrosome would never have been discovered were it not that its staining-capacity ren- ders it conspicuous and its position at the focus of the astral rays isolates it for observation. When we consider the analogy between the centrosome and the basichromatin-grains, when we recall the evidence that the latter graduate into the oxychromatin-granules, and these in turn into the cytomicrosomes, we must admit that Brlicke's cautious suggestion that the whole cell might be a congeries of self- propagating units of a lower order is sufficiently supported by fact to constitute a legitimate working hypothesis. LITERATURE. VI i Van Beneden. E. — (See List IV.) Van Beneden and Julin. — La segmentation chez les Ascidiens et ses rapports avec Torganisation de la larve : Arch. Biol.. V. 1884. Boveri. Th. — Zellenstudien. (See List IV.) Briicke, C. — Die Elementarorganismen : lVie?ier Silz.-Ber., XIAV. 1861. Biitschli, 0. — Protoplasma. (See List I.) Delage. Yves. — La structure du protoplasma, et les theories sur Theredite. Parts, 1895. .. Hacker. V. — Uber den heutigen Stand der Centrosomenfrage : Vcrh. d. deutsch. Zo'dl. Ges. 1894. Heidenhain, M. — (See List I.) Herla. V. — Etude des variations de la mitose chez Pascaride megalocephale : Arch. Biol.yAW. 1893. 1 See also Literature, I., II., IV., V. LITERATURE 329 Morgan, T. H. — The Action of Salt-solutions on the Fertilized and Unfertilized Eggs of Arbacia and Other Animals. Arch. Entiu., VIII. 3. 1898. Kostanecki, K. — Ueber die Bedeutung der Polstrahung wahrend der Mitose. An/i. inik. Anat., XLIX. 1897. Nussbaum, M. — Uber die Teilbarkeit der lebendigen Materie : Arc/i. mik Inat XXVI. 1886. Prenant, A. — Sur le protoplasma superieure (archiplasme, kinoplasme. ergastro- plasme) : Journ. Anat. et Phys., XXIV.-V. 1898-99. (Full Literature-lists.) Rabl, C. — iJber Zellteilung: iJ/(9r///./rt;/^r(^., X. 1885. Anat. AnzeigerAX . 1889. Ruckert, J. — (See List IV.) De Vries, H. — Intracellulare Pangenesis: Jena.. 1889. Watase, S. — Homology of the Centrosome : Journ. Morph., VIII. 2. 1893. Id. — On the Nature of Cell-organization : Woods H oil Biol. Lectures. 1893. Wiesner, J. — Die Elementarstruktur und das Wachstum der lebenden Substanz : Wien, 1892. Wilson. Edm. B. — Archoplasm, Centrosome, and Chromatin in the Sea-urchin Egg: Journ. Morph., Vol. XI. 1895. \ CHAPTER VII SOME ASPECTS OF CELL-CHEMISTRY AND CELL-PHYSIOLOGY " Les phenomfenes fonctionnels ou de depense vitale auraient done leur siege dans le proto- flasme cellulaire. " Le noyau est un appareil de synthese organique, V instrument de la production, le gertne de la cellule" Claude Bernard.i A. Chemical Relations of Nucleus and Cytoplasm It is no part of the purpose of this work to give even a sketch of general cell-chemistry. I shall only attempt to consider certain ques- tions that bear directly upon the functional relations of nucleus and cytoplasm and are of especial interest in relation to the process of nutrition and through it to the problems of development. It has often been pointed out that we know little or nothing of the chemical conditions existing in living protoplasm, since every attempt to examine them by precise methods necessarily kills the protoplasm. We must, therefore, in the main rest content with inferences based upon the chemical behaviour of dead cells. But even here investigation is be- set with difficulties, since it is in most cases impossible to isolate the various parts of the cell for accurate chemical analysis, and we are obliged to rely largely on the less precise method of observing with the microscope the visible effects of dyes and other reagents. This difficulty is increased by the fact that both cytoplasm and karyoplasm are not simple chemical compounds, but mixtures of many complex substances ; and both, moreover, undergo periodic changes of a com- plicated character which differ very widely in different kinds of cells. Our knowledge is, therefore, still fragmentary, and we have as yet scarcely passed the threshold of a subject which belongs largely to the cytology of the future. It has been shown in the foregoing chapter that all the parts of the cell arise as local differentiations of a general protoplasmic basis. Despite the difficulties of chemical analysis referred to above, it has been determined with certainty that some at least of these organs are the seat of specific chemical change ; just as is the case in the various organs and tissues of the organism at large. Thus, the nucleus is 1 Le(;o7ts sur les phenoitiejies de la vie, I., 1878, p. 198. 330 CHEMICAL RELATIONS OF NUCLEUS AND CYTOPLASM 331 characterized by the presence of nuclein (chromatin) which has been proved by chemical analysis to differ widely from the cytoplasmic substances,! while the various forms of plastids are centres for the formation of chlorophyll, starch, or pigment. These facts give -round for the conclusion that the morphological differentiation of cell-or<'-ans IS m general accompanied by underlying chemical specializadons which are themselves the expression of differences of metabolic ac- tivity ; and these relations, imperfectly comprehended as they are are of fundamental importance to the student of development. I. The Proteids and their Allies The most important chemical compounds found in the cell are the group oi protein substances, and there is every reason to believe that these form the principal basis of living protoplasm in all of its forms. These substances are complex compounds of carbon, hydrogen, nitro- gen, and oxygen, often containing a small percentage of sul^Dhur, and in some cases also phosphorus and iron. They form a very extensive group of which the different members differ considerably in physical and chemical properties, though all have certain common traits and are closely related. They are variously classified even by the latest writers. By many authors (for example Halliburton, '93') the word ''proteids " is used in a broad sense as synonymous with albuminous substajtces, including under them the various forms of albumin (eq:<;- albumin, cell-albumin, muscle-albumin, vegetable-albumins), ,<,V^>/;////// (fibrinogin vitellin, etc.), and the /^//^//^j- (diffusible hydrated proteids). Another series of nearly related substances are the albuminoids (reckoned by some chemists among the ''proteids"), examples of which are gelatin, mucin, and, according to some authors also, nuclein, and the 7iucleo-albuniins. Some of the best authorities how- ever, among them Kossel and Hammarsten, follow the usage of Hoppe-Seyler in restricting the woxd. proteid \.o substances of greater complexity than the albumins and globulins. Examples of these are the nuclein s and nucleo-proteids, which are comj^ounds of nu- cleinic acid with albumin, histon, or protamin. The nucleo-proteids, found only in the nucleus, are not to be confounded with the nucleo- ^ It has long been known that a form of " nuclein " may also he obtained from the nucleo- albmiiins of the cytoplasm, e.g. from the yolk of hens' eggs (vitellin). Sucii nucleins tliffcr, however, from those of nuclear origin in not yielding as cleavage-jiroducts the nuclein bases (adenin, xanthin, etc.). The term " paranuclein " (Kossel) or " pseudo-nuclcin " (Ham- marsten) has therefore been suggested for this substance. True nucleins containing a large percentage of albumin are distinguished as ntuleo-protcids. They may be split into albumin (or albumin radicals) and nucleinic acid, the latter yielding as cleavage-products the nuclein bases. Pseudo-nucleins containing a large percentage of albumin are designated as nuclro- albtimins, which, in like manner split into albumin and paranucleinic or pseudo-nucleinic acid, which yields no nuclein bases. (See Hammarsten, '94.) 332 CELL-CHEMISTRY AND CELL-PHYSIOLOGY albumins, which are compounds of pseudo-nucleinic acid with albumin and yield no nuclein-bases (xanthin, hypoxanthin, adenin, guanin) as decomposition products. The distribution of these substances through the cell varies greatly not only in different cells, but at different periods in the life of the same cell. The cardinal fact always, however, remains, that tJiere is a definite and constant contrast between nucleus and cytoplasm. The latter always contains large quantities of nucleo-albumins, certain globulins, and sometimes small quantities of albumins and peptones ; the former contains, in addition to these, nuclein and nucleo-proteids, which form its main bulk, and its most constant and characteristic feature. It is the remarkable substance, nuclein, — which is almost certainly identical with chromatin, — that chiefly claims our attention here on account of the physiological role of the nucleus. 2. The Nuclein Series Nuclein was first isolated and named by Miescher, in 1 871, by subjecting cells to artificial gastric digestion. The cytoplasm is thus digested, leaving only the nuclei ; and in some cases, for instance pus- cells and spermatozoa, it is possible by this method to procure large quantities of nuclear substance for accurate quantitative analysis. The results of analysis show it to be a complex albuminoid substance, rich in phosphorus, for which Miescher gave the chemical formula C29H49N9P3022- The earlier analysis of this substance gave some- what discordant results, as appears in the following table of per- centage-compositions : ^ — These differences led to the opinion, first expressed by Hoppe- Seyler, and confirmed by later investigations, that there are several varieties of nuclein which form a group having certain characters in common. Altmann ('89) opened the way to an understanding of the matter by showing that ** nuclein " may be split up into two sub- stances ; namely, ( i ) an organic acid rich in phosphorus, to which he ^From Halliburton, '91, p. 203. [The oxygen-percentage is omitted in this table.] CHEMICAL RELATIONS OF NUCLEUS AND CYTOPLASM 333 gave the name nucleinic acid, and (2) a form of albumin. Moreover, the nuclein may be synthetically formed by the re-combination of these two substances. Pure nucleinic acid, for which Miescher (96) afterward gave the formula C^oHg^Nj^P^O.^^.^ contains no sulphur, a high percentage of phosphorus (above 9%), and no albumin. l^y adding it to a solution of albumin a precipitate is formed which contains sulphur, a lower percentage of phosphorus, and has the chemical characters of " nuclein." This indicates that the discord- ant results in the analyses of nuclein, referred to above, were probably due to varying proportions of the two constituents ; and Altmann suggested that the "nuclein " of spermatozoa, which contains no sulphur and a maximum of phosphorus, might be uncombined nucleinic acid itself. Kossel accordingly drew the conclusion, based on his own work as well as that of Liebermann, Altmann, Malfatti. and others, that "what the histologists designate as clironiatin con- sists essentially of combinations of nucleinic acid with more or less albumin, and in some cases may even be free nucleinic acid. The less the percentage of albumin in these compounds, the nearer do their properties approach those of pure nucleinic acid, and we may assume that the percentage of albumin in the chromatin of the same nucleus may vary according to physiological conditions." ^ In the same year Halliburton, following in part Hoppe-Seyler, stated the same view as follows. The so-called " nucleins " form a series lead- ing downward from nucleinic acid thus : — (i) Those containing no albumin and a maximum (9-10%) of phos- phorus (pure nucleinic acid). Nuclei of spermatozoa. (2) Those containing little albumin and rich in phosphorus. Chro- matin of ordinary nuclei. (3) Those with a greater proportion of albumin — a series of sub- stances in which may probably be included /jvr;//;/ (nucleoli) and plastin (Hnin). These graduate into (4) Those containing a minimum (0.5 to 1%) of phosphorus — the nucleo-albumins, which occur both in the nucleus and in the cytoplasm (vitellin, caseinogen, etc.). Finally, we reach the globuUns and albumins, especially character- istic of the cell-substance, and containing no nucleinic acid. " We thus pass by a gradual transition (from the nucleo-albumins) to the other proteid constituents of the cell, the cell-globulins, which contain no phosphorus whatever, and to the products of cell-activity, such as the proteids of serum and of egg-white, which are also principally 1 Derived from analysis of the salmon-sperm. ^ '93» P- ^S^- 334 CELL-CHEMISTRY AND CELL-PHYSIOLOGY phosphorus-free." ^ Further, " in the processes of vital activity there are changing relations between the phosphorized constituents of the nucleus, just as in all metabohc processes there is a continual inter- change, some constituents being elaborated, others breaking down into simpler products." This latter conclusion has been well estab- Ushed; the others, as stated by Halliburton, require some modification, on the one hand, through the results of later analyses of chromatin, on the other, because of the failure to distinguish between the nucleo- proteids and the nucleo-albumins. First, it has been shown by Miescher ('96), Kossel ('96), and Mathews ('97, 2) that the chromatin of the sperm-nuclei (in fish and sea-urchins) is not pure nucleinic acid, as Altmann conjectured, but a salt of that acid, with histon, protamin, or a related substance. Thus, in the spermatozoa of the salmon, Miescher's analyses give 60.56% of nucleinic acid and 35.56% of protamin (CigH28N902). In the herring the chromatin is a compound of nucleinic acid (over 63%) and a form of protamin called by Kossel " clupein " (CgoH-^Nj^Og). In the '$>Q,?,-m:z\v\rv Arbacia Mathews finds the chromatin to be a compound of nucleinic acid and '' arbacin," a histon-hke body. Kossel finds also that chromatin (nuclein) derived from the thymus gland, and from leucocytes, is largely a histon salt of nucleinic acid, the proportion of the latter being, however, much less than in the sperm-chromatin, while albumin is also present. In these cases, therefore, the greater part of the nucleinic acid is com- bined not with albumin but with a histon or protamin radical. Second, the nucleo-albumins of the cytoplasm are in no sense transitional be- tween the nucleins and the albumins, since they contain no true nucleinic acid, but only pseudo-nucleinic acid.'-^ The fact nevertheless remains that the nucleins and nucleo-proteids, though confined to the nucleus, form a series descending from such highly phosphorized bodies as the sperm-chromatin toward bodies such as the albumins, which are especially characteristic of the cytoplasm ; and that they vary in composition with varying physiological conditions. The way is thus opened for a more precise investigation of the physiological role of nucleus and cytoplasm in metabolism. 3. Staining-reaction of the Niiclcin Series In bringing these facts into relation with the staining-reactions of the cell, it is necessary briefly to consider the nature of staining- reactions in general, and especially to warn the reader that in the whole field of " micro-chemistry " we are still on such uncertain ground that all general conclusions must be taken with reserve. First, it is still uncertain how far staining-reactions depend upon chemical reaction and how far upon merely physical properties of CHEMICAL RELATIONS OF NUCLEUS AND CYTOPLASM y^ the bodies stained. The prevalent view that staining-reactiuns are due to a chemical combination of the dye with the elements of the cell has been attacked by Gierke ('85), Rawitz ('97), and Fischer ('97> '99 )» al^ of whom have endeavoured to show that these reactions are of no value as a chemical test, being only a result of surface- attraction and absorption due to purely physical qualities of the bodies stained. On the other hand, a long series of experiments, beginning with Miescher's discovery ('74) that isolated nucleinic acid forms green insoluble salts with methyl-green, and continued by Lilienfeld, Heidenhain, Paul Mayer, and others, gives strong reason to believe that beyond the physical imbibition of colour a true chemical union takes place, which, with due precautions, gives us at least a rough test of the chemical conditions existing in the cell.^ Second, similarity of staining-reactioii is by no means always indica- tive of chemical similarity, as is shown, for example, by the fact that in cartilage both nuclei and inter-cellular matrix are intensely stained by methyl-green, though chemically they differ very widely. Third, colour in itself gives no evidence of chemical nature ; for the nucleus and other elements of the same cell may be stained red, green, or blue, according to the dye employed, and to class them as " erythrophilous," " cyanophilous," and the like, is therefore absurd. Fourth, tJie character of the staining-reaction is influenced and in some cases determined by the fixation or other prelimiiiary treat me fit, a principle made use of practically in the operations of mordaunting, but one which may give very misleading results unless carefullv con- trolled. Thus Rawitz ('95) shows that certain colours which ordinarily stain especially the nucleus (saffranin, gentian-violet), can be made to stain only the cytoplasm through preliminary treatment of object with solutions of tannin, followed by tartar-emetic. In like manner Mathews ('98) shows that many of the *' nuclear" tar-colours (saffra- nin, methyl-green, etc.) stain or do not stain the cytoplasm, according as the material has been previously treated with alkaline or with acid solutions. The results with which we now have to deal are based mainly upon experiments with tar-colours (*' aniline dyes"). Ehrlich ('79) long since characterized these dyes as ** acid " or "basic," according as the colouring matter plays the part of an acid or a base in the com- pound employed, showing further that, other things equal, the basic dyes (methyl-green, saffranin, etc.) are especially "nuclear stains" and the acid (rubin, eosin, orange, etc.) "plasma stains." Malfatti ('91), and especially Lilienfeld ('92, '93), following out Miescher's earlier work ('74), found that albumin stains preeminently in the acid stains, nucleinic acid only in the basic ; and, further, that artifi- 1 Cf. Mayer, '91, '92, '97; Lilienfeld, '93; Mathews, '98. 336 CELL-CHEMISTRY AND CELL-PHYSIOLOGY cial nucleins, prepared by combining egg-albumin with nucleinic acid in various proportions, show a varying affinity for basic and acid dyes according as the nucleinic acid is more or less completely saturated with albumin. Lilienf eld's starting-point was given by the results of Kossel's researches on the relations of the nuclein group, which are expressed as follows : ^ — Nucleo-proteid (i% of P or less), by peptic digestion splits into Peptone Nuclein (3-4% P)^ by treatment with acid splits into I ' ^ Albumin Nucleinic acid (9- 1 0% P) , heated with mineral acids splits into « . Phosphoric acid Nuclein bases (A ca?-bohj/drale.) (adenin. guanin. etc.). Now, according to Kossel and Lilienfeld, the principal nucleo- proteid in the nucleus of leucocytes is nucleo-Jiiston, containing about 3% of phosphorus, which may be split into a form of nuclein playing the part of an acid, and an albuminoid base, the Jiiston of Kossel ; the nuclein may in turn be split into albumin and nucleinic acid. These four substances — albumin, nucleo-histon, nuclein, nucleinic acid — thus form a series in which the proportion of phosphorus, which is a measure of the nucleinic acid, successively increases from zero to 9-10%. If the members of this series be treated with the same mixture of red acid fuchsin and basic methyl-green, the result is as follows. Albumin (egg-albumin) is stained red, nucleo-histon greenish blue, nuclein bluish green, nucleinic acid intense green. "We see, therefore, that the principle that determines the staining of the nuclear substances is always the nucleinic acid. All the nuclear sub- stances, from those richest in albumin to those poorest in it, or con- taining none, assume the tone of the nuclear {i.e. basic) stain, but the combined albumin modifies the green more or less toward blue." ^ Lilienfeld explains the fact that chromatin in the cell-nucleus seldom appears pure green on the assumption, supported by many facts, that the proportions of nucleinic acid and albumin vary with different physiological conditions, and he suggests further that the intense staining-power of the chromosomes during mitosis is probably due to the fact that they contain a maximum of nucleinic acid. Very interesting is a comparison of the foregoing staining-reactions with those given by a mixture of a red basic dye (saffranin) and a green acid one (" light green "). With this combination an effect is given which reverses that of the Biondi-EhrUch mixture ; i.e. the nuclein 1 From Lilienfeld, after Kossel ('92, p. 129). ^ I.e., p. 394. CHEMICAL RELATIONS OF NUCLEUS AND CYTOPLASM 337 is coloured red, the albumin green, which is a beautiful demon- stration of the fact that staining-reagents cannot be logically classified according to colour, but only according to their chemical nature, and gives additional ground for the view that staining-reactions of this type are the result of a chemical rather than a merely physical combination. These results must be taken with some reserve for the following'- reasons : Mathews ('98) has shown that methyl-green and other basic dyes will energetically stain albumose, coagulated egg-albumin, and the cell-cytoplasm in or after treatment by alkaline fluids; while con- versely the acid dyes do not stain, or only slightly stain, these sub- stances under the same conditions. This probably does not affect the validity of Heidenhain's results,^ since he worked with acid solu- tions. What is more to the point is the fact that hyaline cartilage and mucin, though containing no nucleinic acid, stain intenselv with basic dyes. Mathews probably gives the clue to this reaction, in the suggestion that it is here probably due to the presence of other acids (in the case of cartilage a salt of chondroitin-sulphuric acid, according to Schmiedeberg); from which Mathews concludes that the basic dyes will, in acid or neutral solutions, stain any element of the tissues that contains an organic acid in a salt combination with a strong base.^ Accepting this conclusion, we must therefore recognize that, as far as the cytoplasm is concerned, the basic or ** nuclear " stains are in no sense a test for nuclein, but only for salts of organic acids in general. In case of the nucleus, however, we know from direct analysis that we are dealing with varying combinations of nucleinic acid, and hence, with the precautions indicated above, may draw provisional conditions from the staining-reactions. Thus regarded, the changes of staining-reaction in the chromatin are of high interest. Heidenhain ('93, '94), in his beautiful studies on leucocytes, has correlated some of the foregoing results with the staining-reactions of the cell as follows. Leucocytes stained with the Biondi-Ehrlich mixture of acid fuchsin and methyl-green sl-.ow the following reactions. Cytoplasm, centrosome, attraction-sj)here, astral rays, and spindle-fibres are stained pure red. The nuclear sub- stance shows a very sharp differentiation. The chromatic network and the chromosomes of the mitotic figure are green. The linin- substance and the true nucleoli or plasmosomes aj)pear red, like the cytoplasm. The Hnin-network of leucocytes is stated by Heidenhain to consist of two elements, namely, of red granules or microsomes suspended in a colourless network. The latter alone is called ** linin " by Heidenhain. To the red granules is applied the term *' ox-ychro- matin," while the green substance of the ordinary chromatic network, 1 See below. - '98. PP- 451-452. 338 CELL-CHEMISTRY AND CELL-PHYSIOLOGY forming the " chromatin " of Flemming, is called *' basichromatin." ^ Morphologically, the granules of both kinds are exactly alike,- and in many cases the oxychromatin-granules are found not only in the " achromatic " nuclear network, but also intermingled with the basi- chromatin-granules of the chromatic network. Collating these results with those of the physiological chemists, Heidenhain concludes that basichromatin is a substance rich in phosphorus (/.^. nucleinic acid), oxychromatin a substance poor in phosphorus, and that, further, '* basichromatin and oxychromatin are by no means to be regarded as permanent unchangeable bodies but may change their colour- reactions by combining with or giving off phosphorus." In other words, ** the affinity of the chromatophilous microsomes of the nuclear network for basic and acid aniline dyes is regulated by certain physio- logical conditions of the nucleus or of the cell."'^ This conclusion, which is entirely in harmony with the statements of Kossel and Halliburton quoted above, opens up the most interest- ing questions regarding the periodic changes in the nucleus. The staining-power of chromatin is at a maximum when in the preparatory stages of mitosis (spireme-thread, chromosomes). During the ensuing growth of the nucleus it always diminishes, suggesting that a com- bination with albumin has taken place. This is illustrated in a very striking way by the history of the egg-nucleus or germinal vesicle, which exhibits the nuclear changes on a large scale. It has long been known that the chromatin of this nucleus undergoes great changes during the growth of the ^gg, and several observers have maintained its entire disappearance at one period. Riickert first carefully traced out the history of the chromatin in detail in the eggs of sharks, and his general results have since been confirmed by Born in the eggs of Triton. In the shark Pristiiirns, Riickert ('92, i) finds that the chromosomes, which persist throughout the entire growth-period of the ^gg, undergo the following changes (Fig. 157): At a very early stage they are small, and stain intensely with nuclear dyes. During the growth of the ^gg they undergo a great increase in size, and progressively lose tJieir staining-capacity . At the same time their surface is enormously increased by the development of long threads which grow out in every direction from the central axis (Fig. 157, A). As the ^gg approaches its full size, the chromosomes rapidly diminish in size, the radiating threads disappear, and the stain- ing-capacity increases (Fig. 157,^). They are finally again reduced to minute, intensely staining bodies which enter into the equatorial plate of the first polar, mitotic figure (Fig. 157, C). How great the change of volume is may be seen from the following figures. At the beginning the chromosomes measure, at most, 12 yi (about o-qVo^ ^^O ^'^ length and ^'94, P- 543- ^^•^•. P- 547- ^^•^•' P- 548- CHEMICAL RELATIONS OF NUCLEUS AND CYTOPLASM 339 1 /z in diameter. At the lieight of their development they are ahnost eight times their original length and twenty times their original diameter. In the final period they are but 2 /x in length and i // in di- ameter. These measurements show a change of volume so enormous, even after making due allowance for the loose structure of the large chromosomes, that it cannot be accounted for by mere swelling or shrinkage. The chromosomes evidently absorb a large amount of Fig. 157. _ Chromosomes of the germinal vesicle in the shark Pnstiurus, at different periods, drawn to the same scale. [RiJCKERT.] A. At the period of maximal size and minimal staining-capacity {tgg 3 mm. in diameter) B. Later period (egg 13 mm. in diameter). C. At the close of ovarian life, of nunmial size and maximal staining-power. matter, combine with it to form a substance of diminished stammg- capacity, and finally give off matter, leaving an intensely stammg substance behind. As Riickert points out, the great mcrease ot sur- face in the chromosomes is adapted to facilitate an exchange of mate- rial between the chromatin and the surrounding substance ; and he concludes that the coincidence between the growth ot the chromo- somes and that of the ^gg points to an intimate connection between the nuclear activity and the formative energy of the cytoplasm. 340 CELL-CHEMISTRY AND CELL-PHYSIOLOGY If these facts are considered in the Hght of the known staining- reaction of the nuclein series, we must admit that the following con- clusions are something more than mere possibilities. We may infer that the original chromosomes contain a high percentage of nucleinic acid ; that their growth and loss of staining-power is due to a combi- nation with a large amount of albuminous substance to form a lower member of the nuclein series, probably a nucleo-proteid ; that their final diminution in size and resumption of staining-power is caused by a giving up of the albumin constituent, restoring the nuclein to its original state as a preparation for di\'ision. The growth and diminished staining-capacity of the chromatin occurs during a period of intense constructive activity in the cytoplasm ; its diminu- tion in bulk and resumption of staining-capacity coincides with the cessation of this activity. This result is in harmony with the obser- vations of Schwarz and Zacharias on growing plant-cells, the per- centage of nuclein in the nuclei of embryonic cells (meristem) being at first relatively large and diminishing as the cells increase in size. It agrees further with the fact that of all forms of nuclei those of the spermatozoa, in which growth is suspended, are richest in nucleinic acid, and in this respect stand at the opposite extreme from the nuclei of the rapidly growing egg-cell. Accurately determined facts in this direction are still too scanty to admit of a safe generalization. They are, however, enough to indi- cate the probability that chromatin passes through a certain cycle in the life of the cell, the percentage of albumin or of albumin-radicals increasing during the vegetative activity of the nucleus, decreasing in its reproductive phase. In other words, a combination of albumin with nuclein or nucleinic acid is an accompaniment of constructive metabolism. As the cell prepares for division, the combination is dissolved and the nuclein-radicle or nucleinic acid is handed on by division to the daughter-cells. A tempting hypothesis, suggested by Mathews on the basis of Kossel's work, is that nuclein, or one of its constituent molecular groups, may in a chemical sense be regarded as the formative centre of the cell which is directly involved in the process by which food-matters are built up into the cell-substance. Could this be established, we should have not only a clear light on the changes of staining-reactions during the cycle of cell-life, but also a clue to the nuclear " control " of the cell through the process of synthetic metabolism. This hypothesis fits well with the conclusions of other physiological chemists that the nucleus is especially con- cerned in synthetic metabolism. Kossel concludes that the formation of new organic matter is dependent on the nucleus,^ and that nuclein in some manner plays a leading role in this process ; and he makes 1 Schiefferdecker and Kossel, Gewebelehre, p. 57. PHYSIOLOGICAL RELATLONS OF NUCLEUS AND CYTOPLASM 341 some interesting suggestions regarding the synthesis of complex organic matters in the living cell with nuclein as a starting-point. Chittenden, too, in a review of recent chemico-physiological dis- coveries regarding the cell, concludes : " The cell-nucleus may be looked upon as in some manner standing in close relation to those processes which have to do with the formation of organic substances. Whatever other functions it may possess, it evidently, through the inherent qualities of the bodies entering into its composition, has a controlling power over the metaboUc processes in the cell, modifying and regulating the nutritional changes " ('94). These conclusions, in their turn, are in harmony with the hypothesis advanced twenty years ago by Claude Bernard ('78), who maintained that the cytoplasm is the seat of destructive metabolism, the nucleus the organ of constructive metabolism and organic synthesis, and insisted that the role of the nucleus in nutrition gives the key to its significance as the organ of development, regeneration, and inheri- tance.^ B. Physiological Relations of Nucleus and Cytoplasm How nearly the foregoing facts bear on the problem of the mor- phological formative power of the cell is obvious ; and they have in a measure anticipated certain conclusions regarding the role of nucleus and cytoplasm, which we may now examine from a somewhat differ- ent point of view. Briicke long ago drew a clear distinction between the chemical and molecular composition of organic substances, on the one hand, and, on the other hand, their definite grouping in the cell b\' which arises organization in a morphological sense. Claude Bernard, in like man- ner, distinguished between eheniical synthesis, through which organic matters are formed, and morphological synthesis, by which the)' are built into a specifically organized fabric ; but he insisted that these two processes are but different phases or degrees of the same phe- nomenon, and that both are expressions of the nuclear activity. We have now to consider some of the evidence that the power of mor- phological, as well as of chemical, synthesis centres in the nucleus, and that this is therefore to be regarded as the especial organ of inheritance. This evidence is mainly derived from the comparison of nucleated and non-nucleated masses of protoplasm ; from the form, 1 " II semble done que la cellule qui a perdu son noyau soit sterilisee au point de vue de la generation, c'est a dire de la synthese morphologique, et qu'elle le soit aussi au point de vue de la synthese chimique, car elle cesse de produire des principes immediats, et ne peut guere qu'oxyder et detruire ceux qui s'y etaient accumules par une elaliDiation anterieure du noyau. II semble done que le noyau soit \& gernie de nutrition de la cellule : il attire autour de lui et elabore les materiaux nutritifs " ('78, p. 523). 342 CELL-CHEMISTRY AND CELL-PHYSIOLOGY position, and movements of the nucleus in actively growing or metab- olizing cells ; and from the history of the nucleus in mitotic cell- division, in fertilization, and in maturation. I. Experiments on Unicellnlar Organisms Brandt i^yj) long since observed that enucleated fragments of Acti- nosphcerium soon die, while nucleated fragments heal their wounds and continue to live. The first decisive comparison be- tween nucleated and non-nu- cleated masses of protoplasm was, however, made by Moritz Nussbaum in 1884 in the case of an infusorian, OxytricJia. If one of these animals be cut into two pieces, the sub- sequent behaviour of the two fragments depends on the presence or absence of the nucleus or a nuclear frag- ment. The nucleated frag- ments quickly heal the wound, regenerate the missing por- tions, and thus produce a perfect animal. On the other hand, enucleated fragments, consisting of cytoplasm only, quickly perish. Nussbaum therefore drew the conclusion that the nucleus is indispens- able for the formative energy of the cell. The experiment was soon after repeated by Gruber('85)in the case of Stentor^ another infusorian, and with the same result (Fig. 159). Fragments possess- ing a large fragment of the nucleus completely regenerated within twenty-four hours. If the nuclear fragment were smaller, the re- generation proceeded more slowly. If no nuclear substance were present, no regeneration took place, though the wound closed and the fragment lived for a considerable time. The only exception — but it is a very significant one — was the case of individuals in which the process of normal fission had begun ; in these a non-nucleated fragment in which the formation of a new peristome had already been initiated healed the wound and completed the formation of the peri- Fig. 158. — Stylonychia, and enucleated irag- ments. [Verworn.] At the left an entire animal, showing planes of section. The middle piece, containing two nuclei, regenerates a perfect animal. The enucleated pieces, shown at the right, swim about for a time, but finally perish. PHYSIOLOGICAL RELATIONS OF NUCLEUS AND CYTOPLASM 343 stome. Lillie ('96) has recently found that Steiitor may by shaking be broken into fragments of all sizes, and that nucleated fragments as small as 2V the volume of the entire animal are still capable of complete regeneration. All non-nucleated fragments perish. These studies of Nussbaum and Gruber formed a prelude to more extended investigations in the same direction by Gruber, Balbiani, Hofer, and especially Verworn Verworn {''^^) proved that in /V/r- stomclla, one of the Foraminifera, nucleated fragments are able to B C Fig. 159. — Regeneration in the unicellular animal Stentor. [From GRUBER after BALBl.\Nr.] A. Animal divided into three pieces, each containing a fragment of the nucleus. B. The three fragments shortly afterward. C. The three fragments after twenty-four hours, each regenerated to a perfect animal. repair the shell, while non-nucleated fragments lack this power. Balbiani ('89) showed that although non-nucleated fragments of Infusoria had no power of regeneration, they might nevertheless continue to live and swim actively about for many days after the operation, the contractile vacuole pulsating as usual. Hofer ('89), experimenting on Auuvba, found that non-nucleated fragments might live as long as fourteen days after the operation (Fig. 160). Their movements continued, but were somewhat modified, and little by little ceased, but the pulsations of the contractile vacuole were but slightly affected; they lost more or less completely the capacity to 344 CELL-CHEMISTRY AND CELL-PHYSIOLOGY digest food, and the power of adhering to the substratum. Nearly at the same time Verworn ('89) pubUshed the results of an extended comparative investigation of various Protozoa that placed the whole matter in a very clear light. His experiments, while fully confirming the accounts of his predecessors in regard to regeneration, added many extremely important and significant results. Non-nucleated fragments both of Infusoria {e.g. Lachrymaria) and rhizopods {Poly^ / .> i •■•' »1" "' ' ''•*•'* ^' ' !.■■■'::•':•!■'''-■::■/ c N.T'X V."..\ % SC? ^^\ \>.;.\ "."N ■ >i''. v^ " /:■:> — v''v>^vV •.••/■':N..-'.'"^...v>it / \. ;..i:; Fig. 160. — Nucleated and non-nucleated fragments of A?na;ba. [HOFER.] A. B. An Amceba divided into nucleated and non-nucleated halves, five minutes after the opera- tion. C. D. The two halves after eight davs, each containing a contractile vacuole. stomcUa, TJialassicolla) not only live for a considerable period, but perform perfectly normal and characteristic movements, show the same susceptibility to stimulus, and have the same power of ingulf- ing food, as the nucleated fragments. TJicy lack, hoivever, the power of digestion and secretion. Ingested food-matters may be slightly altered, but are never completely digested. The non-nucleated frag- ments are unable to secrete the material for a new shell {Polysto- PHYSIOLOGICAL RELATIONS OF NUCLEUS AND CYTOPLASM 345 mella) or the slime by which the animals adhere to the substratum {^Amoeba, Difflugia, Polystomclla). Beside these results should be placed the well-known fact that dissevered nerve-fibres in the higher animals are only regenerated from that end which remains in con- nection with the nerve-cell, while the remaining portion invariably degenerates. A u C D Fig. i6i. — Formation of membranes by protoplasmic fragments of plasmolyzed cells. [Town- send.] A. Plasmolvzed cell, leaf-hair of Cucitrbita, showing protoplasmic balls connected by strands. B. Calyx-hair of Gaillardia ; nucleated fragment with membrane, non-nucleated one naked. C. Root-hair of Marchantia ; all the fragments, connected by protoplasmic strands, have formed membranes. D. Leaf-hair of Cijcurbita ; non-nucleated fragment, with membrane, connected with nucleated fragment of adjoining cell. These beautiful observations prove that destructive metabolism, as manifested by coordinated forms of protoplasmic contractility, may go on for some time undisturbed in a mass of cytoplasm deprived of a nucleus. On the other hand, the building up of new chemical or morphological products by the cytoplasm is only initiated in the presence of a nucleus and soon ceases in its absence. These facts form a complete demonstration that the nucleus plays an essential 346 CELL-CHEMISTRY AND CELL-PHYSIOLOGY part not only in the operations of synthetic metaboUsm or chemical synthesis, but also in the viorpJiological determinatioji of these opera- tions, i.e. the morphological synthesis of Bernard — a point of capital importance for the theory of inheritance, as will appear beyond. Convincing experiments of the same character and leading to the same result have been made on the cells of plants. Francis Darwin (^Ty) observed more than twenty years ago that movements actively continued in protoplasmic filaments, extruded from the leaf-hairs of Dipsaciis, that were completely severed from the body of the cell. Conversely, Klebs ('79) soon afterward showed that naked proto- plasmic fragments of Vaucheria and other algae were incapable of forming a new cellulose membrane if devoid of a nucleus ; and he afterward showed i^^y^ that the same is true of Zygnema and CEdo- gonium. By plasmolysis the cells of these forms may be broken up into fragments, both nucleated and non-nucleated. The former sur- round themselves with a new wall, grow, and develop into complete plants ; the latter, while able to form starch by means of the chloro- phyll they contain, are incapable of utilizing it, and are devoid of the power of forming a new membrane, and of growth and regeneration. A beautiful confirmation of this is given by Townsend ('97), who finds in the case of root-hairs and pollen-tubes, that when the protoplasm is thus broken up, a membrane may be formed by both nucleated and non-nucleated fragments, by the latter however only ivJien they remaiji cofinected with the nucleated masses by protoplasmic strands, however fine. If these strands be broken, the membrane-forming power is lost. Of very great interest is the further observation (made on leaf- hairs in Qicnrbita) that the influence of the nucleus may thus extend from cell to cell, an enucleated fragment of one cell having the power to form a membrane if connected by intercellular bridges with a nucleated fragment of an adjoining cell (Fig. 161). 2. Position and Movements of the Nuclens Many observers have approached the same problem from a dif- ferent direction by considering the position, movements, and changes of form in the nucleus with regard to the formative activities in the cytoplasm. To review these researches in full would be impossible, and we must be content to consider only the well-known researches of Haberlandt i^'J'j) and Korschelt ('89), both of whom have given extensive reviews of the entire subject in this regard. Haberlandt's studies related to the position of the nucleus in plant-cells with especial regard to the growth of the cellulose membrane. He deter- mined the very significant fact that local growth of the cell-wall is always preceded by a movement of the nucleus to the point of growth. Thus, in the formation of epidermal cells, the nucleus lies at first near PHYSIOLOGICAL RELATLONS OF NUCLEUS AND CYTOPLASM 347 the centre, but as the outer wall thickens, the nucleus moves toward it, and remains closely applied to it throughout its growth, after which the nucleus often moves into another part of the cell (Fig. 162, A, B). That this is not due simply to a movement of the nucleus toward the air and light is beautifully shown in the coats of certain seeds, where the nucleus moves, not to the outer, but to the inner wall of the cell and here the thickening takes place (Fig. 162, C). The same position \ k \ \ A A B C Fig. 162. — Position of the nuclei in growing plant-cells. [Haherlandt.] A. Young epidermal cell oi Luzula with central nucleus, before thickening of the membrane. B. Three epidermal cells of Monstera, during the thickening of the outer wall. C. Cell from the seed-coat of Scopulina, during the thickening of the inner wall. D. E. Position of the nuclei dur- ing the formation of branches in the root-hairs of the pea. of the nucleus is shown in the thickening of the walls of the guard- cells of stomata, in the formation of the peristome of mosses, and in many other cases. In the formation of root-hairs in the pea. the pri- mary outgrowth always takes place from the immediate neighbourhood of the nucleus, which is carried outward and remains near the tip of the growing hair (Fig. 162, D, E). The same is true of the rhizoids of fern-prothallia and liverworts. In the hairs of aerial plants this 348 CELL-CHEMISTRY AND CELL-PHYSIOLOGY rule is reversed, the nucleus lying near the base of the hair ; but this apparent exception proves the rule, for both Hunter and Haberlandt show that in this case growth of the hair is not apical, but proceeds from the base ! V^ery interesting is Haberlandt's observation that in the regeneration of fragments of VaiicJieria the growing region, where a new membrane is formed, contains no chlorophyll, but numerous nuclei. The general result, based on the study of a large number of cases, is, in Haberlandt's words, that **the nucleus is in most cases placed in the neighbourhood, more or less immediate, of the points at which growth is most active and continues longest." This fact points to the conclusion that '' its function is especially connected with the developmental processes of the cell," ^ and that "in the growth of the cell, more especially in the growth of the cell-wall, the nucleus plays a definite part." Korschelt's work deals especially with the correlation between form and position of the nucleus and the nutrition of the cell, and since it bears more directly on chemical than on morphological synthesis, may be only briefly reviewed at this point. His general conclusion is that there is a definite correlation, on the one hand, between the position of the nucleus and the source of food-supply, on the other hand, between the size of the nucleus and the extent of its surface and the elabora- tion of material by the cell. In support of the latter conclusion many cases are brought forward of secreting cells in which the nucleus is of enormous size and has a complex branching form. Such nuclei occur, for example, in the silk-glands of various lepidopterous larvae (Meckel, Zaddach, etc.), which are characterized by an intense secretory activity concentrated into a very short period. Here the nucleus forms a labyrinthine network (Fig. 14, E), by which its surface is brought to a maximum, pointing to an active exchange of material between nucleus and cytoplasm. The same type of nucleus occurs in the Malpighian tubules of insects (Leydig, R. Hertwig), in the spinning-glands of amphipods (Mayer), and especially in the nutritive cells of the insect ovary already referred to at page 151. Here the developing ovum is accompanied and surrounded by cells, w^hich there is good reason to believe are concerned with the elaboration of food for the egg-cell. In the earwig Forficiila each ^gg is accompanied by a single large nutritive cell (Fig. 163), which has a very large nucleus rich in chro- matin (Korschelt). This cell increases in size as the ovum grows, and its nucleus assumes the complex branching form shown in the figure. In the butterfly Vanessa there is a group of such cells at one pole of the ^g,g, from which the latter is believed to draw its nutriment (Fig. jf). A very interesting case is that of the annelid OpJuyotrocJia, referred to at page 151. Here, as described by Korschelt, the ^gg floats 1 I.e., p. 99. PHYSIOLOGICAL RELATIONS OF NUCLEUS AND CYTOPLASM 349 in the perivisceral fluid, accompanied by a nurse-cell having a very large chromatic nucleus, while that of the ^gg is smaller and poorer in chromatin. Astheecro: completes its growth, the nurse-cell dwindles away and finally perishes (Fig. jG). In all these cases it is scarcely possible to doubt that the ^gg is in a measure relieved of the task of elaborating cyto- plasmic products by the nurse-cell, and that the great development of the nucleus in the latter is correlated with this function. Regarding the posi- tion and movements of the nucleus, Korschelt reviews many facts pointing toward the same conclusion. Per- haps the most sugges- tive of these relate to the nucleus of the e^s: during its ovarian his- tory. In many of the insects, as in both the cases referred to above, the egg-nucleus at first occupies a central posi- g n Fig. 163. — Upper portion of the ovary in the earw ig /l?/-- Jiciila, showing eggs and nurse-cells. [KoRSCHELT.] Below, a portion of the nearly ripe egg {e), showing deuto- plasm-spheres and germinal vesicle {g.v.). Above it lies the nurse-cell («) with its enormous branching nucleus. Two suc- cessively younger stages of egg and nurse are shown above. tion, but as the Qgg be- gins to grow, it moves to the periphery on the side turned toward the nutritive cells. The same is true in the ovarian eggs of some other animals, good examples of which are afforded by various coelenterates, ^.^. in medusae (Claus, Hertwig) and actinians ( Korschelt, Hertwig), where the germinal vesicle is always near the point of attachment of the ^gg. Most suggestive of all is the case of the water-beetle Dytis- ciis, in which Korschelt was able to observe the movements and changes of form in the living object. The eggs here lie in a single series alter- nating with chambers of nutritive cells. The latter contain granules which are believed by Korschelt to pass into the ^gg, perhaps bodily, perhaps by dissolving and entering in a liquid form. At all events, 350 CELL-CHEMISTRY AND CELL-PHYSIOLOGY the tg^ contains accumulations of similar granules, which extend inward in dense masses from the nutritive cells to the germinal vesi- cle, which they may more or less completely surround. The latter meanwhile becomes amoeboid, sending out long pseudopodia, which are always directed toward the principal mass of granules (Fig. jj). The granules could not be traced into the nucleus, but the latter grows rapidly during these changes, proving that matter must be absorbed by it, probably in a liquid form.^ Among other facts pointing in the same direction may be mentioned Miss Huie's ('97) observations on the gland-cells of Drosera, and those of Mathews ('99) on the changes of the pancreas-cell in Nect7irus. Stimulus of the gland-cells in the leaf of Drosera causes a rapid exhaustion and change of staining-capacity in the cytoplasm. During the ensuing repose the cytoplasm is rebuilt out of material laid down immediately around the nucleus, and agreeing closely in appearance and staining-reaction with the achromatic nuclear constituents. The chromatin increases in bulk during a period preceding the constructive phase, but decreases (while the nucleolar material increases) as the cytoplasm is restored. In the pancreas-cell, as has long been known, the "loaded" cell (before secretion) is filled with metaplasmic zymo- gen-granules, which disappear during secretion, the cell meanwhile becoming filled with protoplasmic fibrils (Fig. 18). During the ensu- ing period of "rest" the zymogen-granules are re-formed at the expense of the fibrillar material, which is finally found only at the base of the cell near the nucleus. Upon discharge of the secretion (granule-material) the fibrillse again advance from the nucleus toward the periphery. Mathews shows that many if not all of them may be traced at one end actually into the nuclear wall, and concludes that they are directly formed by the nucleus. Beside the foregoing facts may be placed the strong evidence reviewed at pages 156-158, indicating the formation of the yolk-nu- cleus, and indirectly of the yolk-material, by the nucleus. All of these and a lars^e number of other observations in the same direction lead to the conclusion that the cell-nucleus plays an active part in nutrition, and that it is especially active during the constructive phases. On the whole, therefore, the behaviour of the nucleus in this regard is in har- mony with the result reached by experiment on the one-celled forms, though it gives in itself a far less certain and convincing result. ^ 1 Mention may conveniently here be made of Richard Hertwig's interesting observation that in starved individuals of Actinospluvriwu the chromatin condenses into a single mass, while in richly fed animals it is divided into fine granules scattered through the nucleus ('98, p. 8). - Loeb ('98, '99) makes the interesting suggestion that the nucleus is especially con- cerned in the oxydative processes of the cell, and that this is the key to its 7-dle in the syn- thetic process. It has been shown that oxydations in the living tissues are probably PHYSIOLOGICAL RELATIONS OF NUCLEUS AND CYTOPLASM -^ ti We now turn to evidence which, though less direct than the above, is scarcely less convincing. This evidence, which has been exhaus- tively discussed by Hertwig, Weismann, and Strasburger, is drawn from the history of the nucleus in mitosis, fertilization, and matura- tion. It calls for only a brief review here, since the facts have been fully described in earlier chapters. 3. TJie Nucleus in Mitosis To Wilhelm Roux i^^i) we owe the first clear recognition of the fact that the transformation of the chromatic substance during mitotic division is manifestly designed to effect a precise division of all its parts, — i.e. a panmeristic division as opposed to a mere mass-division, — and their definite distribution to the daughter-cells. "The essential operation of nuclear division is the division of the mother-granules " {i.e. the individual chromatin-grains) ; "all the other phenomena are for the purpose of transporting the daughter-granules derived from the division of a mother-granule, one to the centre of one of the daughter-cells, the other. to the centre of the other." In this respect the nucleus stands in marked contrast to the cytoplasm, which under- goes on the whole a mass-division, although certain of its elements, such as the plastids and the centrosome, may separately divide, like the elements of the nucleus. From this fact Roux argued, first, that different regions of the nuclear substance must represent different qualities, and second, that the apparatus of mitosis is designed to distribute these qualities, according to a definite law, to the daughter- cells. The particular form in which Roux and Weismann developed this conception has now been generally rejected, and in any form it has some serious difficulties in its way. We cannot assume a precise localization of chromatin-elements in all parts of the nucleus ; for on the one hand a large part of the chromatin may degenerate or be cast out (as in the maturation of the ^ZZ), and on the other hand in the Protozoa a small fragment of the nucleus is able to regenerate the whole. Nevertheless, the essential fact remains, as Hertwig, Kolliker, Strasburger, De Vries, and many others have insisted, that in mitotic cell-division the chromatin of the mother-cell is distributed with the most scrupulous equality to the nuclei of the daughter-cells, and that in this regard there is a most remarkable contrast between nucleus and cytoplasm. This holds true with such wonderful constancy dependent upon certain substances (oxydation ferments) that in some manner, not vet clearly understood, facilitate the process; and the work of Spitzer ('97) has shown that these substances (obtained from tissue-extracts) belong to the group of nucleo-proteids, which are characteristic nuclear substances. The view thus suggested opens a further way toward more exact inquiry into the nuclear functions, though it is not to be supposed that the nucleus is the sole oxydative centre of the cell, as is obvious from the prolonged activity of non-nucleaied protoplasmic masses. 352 CELL-CHEMISTRY AND CELL-PHYSIOLOGY throughout the series of living forms, from the lowest to the highest, that it must have a deep significance. And while we are not yet in a position to grasp its full meaning, this contrast points unmistakably to the conclusion that the most essential material handed on by the mother-cell to its progeny is the chromatin, and that this substance therefore has a special significance in inheritance. 4. The Ahicleus in Fertilization The foregoing argument receives an overwhelming reenforcement from the facts of fertilization. Although the ovum supplies nearly all the cytoplasm for the embry- onic body, and the spermatozoon at most only a trace, the latter is nevertheless as potent in its effect on the offspring as the former. On the other hand, the nuclei con- tributed by the germ-cells, though apparently different, become in the end exactly equivalent in every visible respect — in structure, in staining-reactions, and in the num- ber and form of the chromosomes to which each gives rise. But furthermore the substance of the two germ-nuclei is distributed with absolute equality, certainly to the first two cells of the embrvo, and probably to all later-formed cells. The latter conclusion, which long remained a mere surmise, has been rendered nearly a certainty by the remarkable observations of Ruckert, Zoja, and Hacker, de- scribed in Chapters IV. and VI. We must therefore accept the high probability of the conclusion that the specific character of the cell is in the last analysis determined by that of the nucleus, that is by the chromatin, and that in the equal distribution of paternal and ma- egg-fragment of Sphcer echinus granular is, fertil- tcmal chromatin tO all the CClls of ized with spermatozoon of Echinus microtuber- ^j^^ offsprinP" We find the physio- culatus, ana showing purely paternal characters. . ,1 • r ■, r \ B. Normal Pluteus of Echinus microtuberculatus. loglCal explanation of the fact that Fig. 164. — Normal and dwarf larvae of the sea-urchin. [BOVERI.] A. Dwarf Pluteus arising from an enucleated PHYSIOLOGICAL RELATIONS OF NUCLEUS AND CYTOPLASM 353 every part of the latter may show the characteristics of cither or both parents. Boveri ('89, '95, i) has attempted to test this conckision by a most ingenious and beautiful experiment ; and although his conclusions do not rest on absolutely certain ground, they at least open the way to a decisive test. The Hertwig brothers showed that the eggs of sea- urchins might be broken into pieces by shaking, and that spermatozoa would enter the enucleated fragments and cause them to segment. Boveri proved that such a fragment, if fertilized by a spermatozoon, would even give rise to a dwarf larva, indistinguishable from the nor- mal in general appearance except in size. The nuclei of such larvae are considerably smaller than those of the normal larvae, and were shown by Morgan ('95, 4) to contain only half the nuDibcr of cJiromo- somes, thus demonstrating their origin from a single sperm-nucleus. Now, by fertilizing enucleated egg-fragments of one species ( SphcB- recJiiniis granulans) with the spermatozoa of another {Echinus niicro- tuberculatus), Boveri obtained in a few instances dwarf Plutei show- ing except in size the pure paternal cJiaracters {i.e. those of Echinus, Fig. 164). From this he concluded that the maternal cytoplasm has no determining effect on the offspring, but supplies only the material in which the sperm-nucleus operates. Inheritance is, therefore, ef- fected by the nucleus alone. The later studies of Seehger ('94), Morgan ('95, 4), and Drisch ('98, 3) showed that this result is not entirely conclusive, since hybrid larvae arising by the fertilization of an entire ovum of one species bv a spermatozoon of the other show a very considerable range of varia- tion ; and while most such hybrids are intermediate in character between the two species, some individuals may nearly approximate to the characters of the father or the mother. Despite this fact Boveri ('95, i) has strongly defended his conclusion, though admitting that only further research can definitely decide the question. It is to be hoped that this highly ingenious experiment may be repeated on other forms which may afford a decisive result. 5. The Nucleus in Maturation Scarcely less convincing, finally, is the contrast between nucleus and cytoplasm in the maturation of the germ-cells. It is scarcely an exaggeration to say that the whole process of maturation, in its broadest sense, renders the cytoplasm of the germ-cells as unlike, the nuclei as like, as possible. The latter undergo a scries of com- plicated changes which result in a perfect equivalence between them at the time of their union, and, more remotely, a perfect equality of distribution to the embryonic cells. The cytoplasm, on the other 2 A 354 CELL-CHEMISTRY AND CELL-PHYSIOLOGY hand, undergoes a special differentiation in each to effect a second- ary division of labour between the germ-cells. When this is corre- lated with the fact that the germ-cells, on the whole, have an equal effect on the specific character of the embryo, we are again forced to the conclusion that this effect must primarily be sought in the nucleus, and that the cytoplasm is in a sense only its agent. C. The Centrosome Existing views regarding the functions of the centrosome may con- veniently be arranged in two general groups, the first including those which regard this structure as a relatively passive body, the second those which assume it to be an active organ. To the first belongs the hypothesis of Heidenhain ('94), accepted by Kostanecki ('97, i) and some others, that the centrosome serves essentiall}^ as an insertion- point for the astral rays (''organic radii"), and plays a relatively passive part in the phenomena of mitosis, the active functions being mainly performed by the surrounding structures. To the same category belongs the view of Miss Foot that the formation of the centrosome is, as it were, incidental to that of the aster — ''the expression, rather than the cause, of cell-activity " ('97, p. 810). To the second group belong the views of Van Beneden, Boveri, Biitschli, Carnoy, and others who regard the centrosome as playing a more active role in the life of the cell. Both of the former authors have assumed the centrosomes to be active centres by the action of which the astral systems are organized ; and they are thus led to the conclu- sion that the centrosome is essentially an organ for cell-division and fertilization (Boveri), and in this sense is the "dynamic centre" of the cell.^ To Carnoy and Biitschli is due the interesting suggestion ^ that the centrosomes are to be regarded further as centres of cJiemical action to which their remarkable effect on the cytoplasm is due. That the centrosome is an active centre, rather than a passive body or one created by the aster-formation, is strongly indicated by its behaviour both in mitosis and in fertilization. Griffin ('96, '99) points out that at the close of division in TJialassema the daughter-centro- somes migrate away from the old astral centre and incite about themselves in a different region the new astral systems for the ensuing mitosis (Figs. 99, 155); and similar conditions are described by Coe in Cerebratnlns ('98). In fertilization the aster-formation can- not be regarded as a general action of the cytoplasm, but as a local one due to a local stimulus given by something in the spermatozoon ; for in polyspermy a sperm-aster is formed for every spermatozoon (p. 198). This stimulus is given by something in the middle- 1 Cf. pp. 76, 192. 2 Qr p_ jjQ^ THE CENTROSOME 355 piece (p. 212), which is itself genetically related to the centrosome of the last cell-generation (p. 170). These facts seem explicable only under the assumption that in these cases the centrosome, or a sub- stance which it carries, gives an active stimulus to the cytoplasm which incites the aster-formation about itself, and in the words of Grifhn '' disengages the forces at work in mitosis " ('96, p. 174). For these reasons I incline to the view that in the artificial aster-formation described by Morgan ^ the centrosomes there observed should not be regarded as the creations of the asters, but rather as local deposits of material which incite the aster-formation around them. That the centrosomes or astral centres are centres of division (whether active or passive) is beautifully shown by Boveri's interesting observations on ''partial fertilization" referred to at page 194. Again, Boveri has observed that the segmenting ovum of Ascaris sometimes contains a supernumerary centrosome that does not enter Fig. 165. — Kggs oi Ascaris with supernumerary centrosome. [buVERl.] A. First cleavage-spindle above, isolated centrosome below. B. Result of the ensuing division. into connection with the chromosomes, but lies alone in the cytoplasm (Fig. 165). Such a centrosome forms an independent centre of divi- sion, the cell dividing into three parts, two of which are normal blastomeres, while the third contains only the centrosome and attrac- tion-sphere. The fate of such eggs was not determined, but they form a complete demonstration that it is in this case the centrosome and not the nucleus that determines the centres of division in the cell-body. Scarcely less conclusive is the case of dispermic eggs in sea-urchins. In such eggs both sperm-nuclei conjugate with the egg- nucleus, and both sperm-centrosomes divide (Fig. 166). The cleavage-nucleus, therefore, arises by the union of three nuclei and four centrosomes. Such eggs divide at the first cleavage into four equal blastomeres, each of which receives one of the centrosomes. 1 Cf. p. 307. 356 CELL-CIIEMJSTRY AXD CELL-PHYSIOLOGY The latter must, therefore, be the centres of division ; ^ though it must not be forgotten that, in some cases at any rate, normal division requires the presence of nuclear matter (p. io8). The centrosome must, however, be something more than a mere division-centre ; for, on the one hand, in leucocytes and pigment-cells the astral system formed about it is devoted, as there is good reason to believe, not to cell-division, but to movements of the cell-body as a whole; and, on the other hand, as we have seen (pp. 165, 172), it is concerned in the formation of the flagella of the spermatozoa and spermatozoids, and probably also in that of cilia in epithelial cells. Strasburger ('97) was thus led to the conclusion that the centrosome is essentially a mass of kinoplasm, i.e. the active motor plasm, ^ and a nearly similar view has been adopted by several recent zoologists. Fig. 166. — Cleavage of dispermic egg of Toxopneustes. A. One sperm-nucleus has united with the egg-nucleus, shown at a. b. ; the other Hes above. Both sperm-asters have divided to form amphiasters (a. b. and c. d.). B. The cleavage-nucleus, formed by union of the three germ-nuclei, is surrounded by the four asters. C. Result of the first cleavage, the four blastomeres lettered to correspond with the four asters. Hennecfuv concludes that the centrosomes are " motor centres of the kinoplasm " both for external and for internal manifestations.'^ Lenhossek regards them as " motors " for the control of ciliary action as well as for that of the spermatozoon,"* and perhaps also for that of muscle-fibrillDe.^ Zimmerman concludes that *' the microcentrum is the motor centre of the cell, that is, the * kinocentrum ' opposed to the nucleus as the * chemocentrum.'" ^ Regarding their control of ciliary action, he makes the same suggestion as that of Henneguy and Lenhossek cited above. He adds the further very interesting sug- gestions that the centrosomes may be concerned with the pseudopodial movements in the epithelial cells of the intestine, and that they may 1 This phenomenon was first observed by Ilertwig, and afterward by Driesch. I have repeatedly observed the internal changes in the living eggs of Toxopneustes. 2 cf. p. 221. ^ '98. p- 107- ^ '98. p- 697- 3 '98, p. 495. 5 ' 99. P- 342. THE CENTROSOME 357 also be concerned in the protoplasmic contraction of gland-cells by which the excretion is expelled. [This is based on the fact that the centrosomes are found in the free (pseudopodia-forming) ends of the epitheUal cells, and on the position of the centrosomes in goblet- cells (Fig. 23) and in those of the lachrymal gland.] Peter ('99) has attempted to test these conclusions experimentally by cuttino- or tear- ing off cilia from the cell-body (gut-epitheUum of Aiiodonta) and also by isolating the tails of spermatozoa. In groups consisting of only a few cilia, separated from the nucleus, the movements actively con- tinue, while those that are separated from the basal bodies cease to beat. Spermatozoon tails separated from the head also continue to Fig. 167. — Centrosomes and cilia in spermatocytes of a butterfly. [Henneguy.] move, but only if they remain connected with the middle-piece. Peter, therefore, supports the above conclusions of Henneguy and Lenhossek. On the other hand, Meves ('99) finds that movements of the undulating membrane in the tails of salamander-spermatozoa continue if the middle-piece be entirely removed; while a number of earher observers^ have observed in flagellates that a flagellum separated from the body may actively continue its movements for a considerable time. Further research is therefore required to test these suggestions. The intimate connection of the centrosomes with the formation, on the one hand, of the astral rays, on the other of contractile organs, such 1 See Klebs, '83, Biitschli, '85, Fischer, '94, 2. 358 CELL-CHEMISTRY AND CELL-PHYSIOLOGY as cilia, flagella, and pseudopodia,^ the centrosomes in ciliated cells and spermatozoa, and in the swarm-spores of Noctiluca, is, however, a most striking fact, and is one of the strongest indirect arguments in favour of the general theory of fibrillar contractility in mitosis. D. Summary and Conxlusion The facts reviewed in the foregoing pages converge to the conclu- sion that the differentiation of the cell-substance into nucleus and cytoplasm is the expression of a fundamental physiological division of labour in the cell. Experiments upon unicellular forms demonstrate that, in the entire absence of a nucleus, protoplasm is able for a considerable time to liberate energy and to manifest coordinated activities dependent on destructive metabolism. There is here sub- stantial ground for the view that the cytoplasm is the principal seat of these activities in the normal cell. On the other hand, there is strong cumulative evidence that the nucleus is intimately concerned in the constructive or synthetic processes, whether chemical or morphological. That the nucleus has such a significance in synthetic metaboHsm is proved by the fact that digestion and absorption of food and growth soon cease with its removal from the cytoplasm, while destruc- tive metabolism may long continue as manifested by the phenomena of irritability and contractiHty. It is indicated by the position and movements of the nucleus in relation to the food-supply and to the formation of specific cytoplasmic products. It harmonizes with the fact, now universally admitted, that active exchanges of material go on between nucleus and cytoplasm. The periodic changes of staining-capacity undergone by the chromatin during the cycle of cell- life, taken in connection with the researches of physiological chemists on the chemical composition and staining-reactions of the nuclein series, indicate that the phosphorus-rich substance known as nucleinic acid plays a leading part in the constructive process. During the vegetative phases of the cell this substance is combined with a large amount of the albumin radicles histon, protamin, and related sub- stances, and probably in part with albumin itself, to form nuclein. During the mitotic or reproductive processes this combination appears to be dissolved, the albuminous elements being in large part split off, leaving the substance of the chromosomes with a high percentage of nucleinic acid, as is shown by direct analysis of the sperm-nucleus and is indicated by the staining-reactions of the chromosomes. There is, therefore, considerable ground for the hypothesis that in a chemi- cal sense this substance is the most essential nuclear element handed ^ ^f' PP- 92) 1 02, on the central granule of the Heliozoa. LITERATURE 3^q on from cell to cell, whether by cell-division or by fertilization ; and that it may be a primary factor in the constructive processes of the nucleus and through these be indirectly concerned with those of the cytoplasm. The role of the nucleus in constructive metabolism is intimately related with its role in morphological synthesis, and thus in inheri- tance ; for the recurrence of similar morphological characters must in the last analysis be due to the recurrence of corresponding forms of metabohc action of which they are the outward expression. That the nucleus is in fact a primary factor in morphological as well as chemi- cal synthesis is demonstrated by experiments on unicellular plants and animals, which prove that the power of regenerating lost parts disap- pears with its removal, though the enucleated fragment may continue to live and move for a considerable period. That the nuclear sub- stance, and especially the chromatin, is a leading factor in inheritance is powerfully supported by the facts of maturation, fertilization, and cell-division. In maturation the germ-nuclei are by an elaborate process prepared for the subsequent union of equivalent chromatic elements from the two sexes. By fertilization these elements are brought together, and by mitotic division distributed with exact equal- ity to the embryonic cells. The result, which is especially striking in the case of hybrid-fertilization, proves that the spermatozoon is as potent in inheritance as the ovum, though the latter contributes an amount of cytoplasm which is but an infinitesimal fraction of that supplied by the ovum. It remains to be seen whether the chromatin can actually be re- garded as the idioplasm or physical basis of inheritance, as maintained by Hertwig and Strasburger. Verworn has justly urged that the nucleus cannot be regarded as the sole vehicle of inheritance, since the cooperation of both nucleus and cytoplasm is essential to com- plete cell-life; and, as will be shown in Chapter IX., the cytoplasmic organization plays an important role in shaping the course of devel- opment. Considered in all their bearings, however, the facts seem to accord best with the hypothesis that the cytoplasmic organization is itself determined, in the last analysis, by the nucleus ; ^ and the principle for which Hertwig and Strasburger contended is thus sus- tained. LITERATURE. VII Bernard, Claude. — Lepons sur les Phenomenes de la \'ie : ist ed. 1878: 2d ed. 1885. Paris. Chittenden, R. H. — Some Recent Chemico-phvsiological Discoveries regarding tlie Cell : Am. Nat.. XXVIIL, Feb., 1894. 1 C/p. 431- 36o CELL-CHEMISTRY AND CELL-PHYSIOLOGY Fischer, A. — See Literature I. Gruber. A. — Mikroskopische VMvisekton : Ber. d. Naturf. Ges. Freiburg, \\\., 1893. Haberlandt, G. — tjber die Beziehungen zwischen Funktion und Lage des Zellkerns. Fi seller, 1887. Id. — Physiologische Pflanzenatomie. Leipzig, 1896. Halliburton, W. D. — A Text-book of Cliemical Physiology and Pathology. London^ 1891. Id. — The Chemical Physiology of the Cell (^Gouldstonian Lectures): B?'it. Med. Joitrn. 1893. Hammarsten, 0. — Lehrbuch der physiologische Chemie. 3d ed. Wiesbaden, 1895. Hertwig, 0. and R. — Uber den Befruchtungs- und Teilungsvorgang des tierischen Eies unter dem Einfluss ausserer Agentien. Jena, 1887. Kolliker, A. — Das Karyoplasma und die \'ererbung, eine Kritik der Weismann'schen Theorie von der Kontinuitat des Keimplasmas : Zeitschr. iviss. ZooL, XLIV. 1886. Korschelt, E. — Beitrage sur Morphologic und Physiologic des Zellkernes : Zool. JaJirb. Atiat. it. Oniog., IV. 1889. Kossel, A. — Uber die chemische Zusammensetzung der Zelle : Arch. Anat. u. P/iys. 1891. Id. — Uber die basischen Stoffe des Zellkernes: ZeiL P/iys. C/ieni., XXII., 1896. Lilienfeld, L. — Uber die Wahlverwandtschaft der Zellelemente zu Farbstoffen : Arch. Anat. n. Phys. 1893. Malfatti, H. — Beitrage zur Kenntniss der Nucleine : Zeitschr. Phys. Cheju., XVI. 1891. Mathews, A. P. — The Metabolism of the Pancreas Cell : Journ. Morph., XV. SuppL 1899. Miescher, F. — Physiologisch-chemische Untersuchungen liber die Lachsmilch : Arch. Exp. Path. u'. Pharm., XXXVII., 1896. Prenant, A. — See Literature VI. Riickert, J. — Zur Entwicklungsgeschichte des Ovarialeies bei Selachiern : An. Anz., VII. 1892. Sachs, J. — Vorlesungen liber Pflanzen-physiologie. Leipzig, 1882. Id. — Stofif und Form der Pflanzen-organe : Gesanimelte Abhandlnngen, II. 1893. Strasburger. — See footnote, p. 269. Verworn, M. — Die Physiologische Bedeutung des Zellkerns : Arch, fur die Ges. Phys.,X\A. 1892. Id. — Allgemeine Physiologic. Jena, 1895. Whitman, C. 0. — The Seat of Formative and Regenerative Energy : Journ. Morph., II. 1888. Zacharias, E. — Uber des Verhalten des Zellkerns in wachsenden Zellen : Flora, 81. 1895. CHAPTER VIII CELL-DIVISION AND DEVELOPMENT " Wir konnen demnach endlich den Satz aufstellen, dass samnitliche im cntwickelten Zustande vorhandenen Zellen oder Aequivalente von Zellen durch eine f(jrtschreitende Gliederung der Eizelle in morphologisch ahnliche Elemente entstehen, und dass die in einer embryonischen Organ-Anlage enthaltenden Zellen, so gering auch ihre Zahl scin mag, dennoch die ausschliessliche ungegliederte Anlage fiir sammtliche Formbestandtheile der spateren Organe enthalten," Remak.^ Since the early work of Kolliker and Remak it has been recog- nized that the cleavage or segmentation of the ovum, with which the development of all higher animals begins, is nothing other than a rapid series of mitotic cell-divisions by which the Qgg splits up into the elements of the tissues. This process is merely a contin- uation of that by which the germ-cell arose in the parental body. A long pause, however, intervenes during the latter period of its ovarian life, during which no divisions take place. Throughout this period the egg leads, on the whole, a somewhat passive existence, devoting itself especially to the storage of potential energy to be used during the intense activity that is to come. Its power of division remains dormant until the period of full maturity approaches. The entrance of the spermatozoon arouses in the egg a new phase of activity. Its power of division, which may have lain dormant for months or years, is suddenly raised to the highest pitch of intensity, and in a very short time it gives rise by division to a myriad of de- scendants which are ultimately differentiated into the elements of the tissues. The divisions of the egg during cleavage are exactly comparable with those of tissue-cells, and all of the essential phenomena of mitosis are of the same general character in both. But for two reasons the cleavage of the egg possesses a higher interest than any other case of cell-division. First, the egg-cell gives rise by divi- sion not only to cells like itself, as is the case with most tissue-cells, but also to many other kinds of cells. The operation of cleavage is therefore immediately connected with the process of differentiation, which is the most fundamental phenomenon in development. Second, definite relations may often be traced between the planes of division and the structural axes of the adult body, and these relations are 1 Untersuchungen, 1855, p. 140. 361 362 CELL-DIVISION AND DEVELOPMENT sometimes so clearly marked and appear so early that with the very first cleavage the position in which the embryo will finally appear in the ^g% may be exactly predicted. Such *' promorphological " rela- tions of the segmenting ^^^ possess a very high interest in their bearing on the theory of germinal localization and on account of the light which they throw on the conditions of the formative process. The present chapter is in the main a prelude to that which follows, its purpose being to sketch some of the external features of early development regarded as particular expressions of the gen- eral rules of cell-division. For this purpose we may consider the cleavage of the ovum under two heads, namely : — - 1. TJie Geometrical Relations of Cleavage-forms, with reference to the general rules of cell-division. 2. TJie Promoi'p ho logical Relations of the blastomeres and cleav- age-planes to the parts of the adult body to which they give rise. A. Geometrical Relations of Cleavage-forms The geometrical relations of the cleavage-planes and the relative size and position of the cells vary endlessly in detail, being modified by innumerable mechanical and other conditions, such as the amount and distribution of the inert yolk or deutoplasm, the shape of the ovum as a whole, and the like. Yet all the forms of cleavage can be referred to a single type which has been moulded this way or that by special conditions, and which is itself an expression of two general rules of cell-division, first formulated by Sachs in the case of plant- cells. These are : — 1 . The cell typically tends to divide into eqnal pai'ts. 2. Each new plane of division tends to intersect the pi'cce ding plane at a right angle. In the simplest and least modified forms the direction of the cleavage-planes, and hence the general configuration of the cell- system, depends on the general form of the dividing mass ; for, as Sachs has shown, the cleavage-planes tend to be either vertical to the surface {anticlines) or parallel to it {periclines). Ideal schemes of division may thus be constructed for various geometrical figures. In a flat circular disc, for example, the anticlinal planes pass through the radii; the periclines are circles concentric with the periphery. If the disc be elongated to form an ellipse, the periclines also become ellipses, while the anticlines are converted into hyperbolas confocal with the periclines. If it have the form of a parabola, the periclines and anticlines form two systems of confocal parabolas intersecting at right angles. All these schemes are mutatis mutandis, directly con- vertible into the corresponding solid forms in three dimensions. GEOMETRICAL RELATIONS OF CLEAVAGE-FORMS l^l Sachs has shown in the most beautiful manner that all the above ideal types are closely approximated in nature, and Rauber has applied the same principle to the cleavage of animal-cells. The discoid or spheroid form is more or less nearly realized in the thalloid growths of Fig. i68. — Geometrical relations of cleavage-planes in growing plant-tissues. [From f^AClls. after various authors.] A. Flat ellipsoidal germ-disc of Melobesia (Rosanoff) ; nearly typical relation of eiiiptic periclines and hyperbolic anticlines. B. C. Apical view of terminal knob on epidermal liair of Pinguicola. B. shows the ellipsoid type, C. the circular (spherical type), somewhat modified (only anticlines present). D. Growing point of Salvhiia (Pringsheim), typical ellipsoid type; the single pericline is, however, incomplete. E. Growing point of Azolla (Strasburger) ; circular or spheroidal type transitional to ellipsoidal. F. Root-cap of E(]uisrtinn (Xageli and Leitgeb) ; modified circular type. G. Cross-section of leaf-vein, Trichowanes (Prantl) ; ellipsoidal type with incomplete periclines. H. Embryo oi Alisma ; typical elliyisoid type, pericline incomplete only at lower side. /. Growing point of bud of the pine {AHes) ; typical paraboloid type, both anti- clines and periclines having the form of parabolas (Sachs). various lower plants, in the embryos of flowering plants, and else- where (Fig. 1 68). The paraboloid form is according to Sachs charac- teristic of the growing points of many higher plants ; and here, too, the actual form is remarkably similar to the ideal scheme (Fig. i68, /). 364 CELL-DIVISION AND DEVELOPMENT For our purpose the most important form is the sphere, which is the typical shape of the egg-cell ; and all forms of cleavage may be related to the typical division of a sphere in accordance with Sachs's rules. The ideal form of cleavage would here be a succession of rectangular cleavages in the three dimensions of space, the anticlines passing through the centre so as to split the ^gg in the initial stages successively into halves, quadrants, and octants, the periclines being parallel to the surface so as to separate the inner ends of these cells from the outer. No case is known in which this order is accurately followed throughout, and the periclinal cleavages are of compara- tively rare occurrence, being found as a regular feature of the early cleavage only in those cases where the primary germ-layers are sepa- rated by delamination. The simplest and clearest form of egg- cleavage occurs in eggs like those of echinoderms, which are of spherical form, and in which the deutoplasm is small in amount and equally distributed through its substance. Such a cleavage is beauti- fully displayed in the ^gg of the holothurian Synapta, as shown in the diagrams, Fig. 169, constructed from Selenka's drawings. The first cleavage is vertical, or meridional, passing through the egg-axis and dividing the ^gg into equal halves. The second, which is also meridional, cuts the first plane at right angles and divides the ^gg into quadrants. The third is horizontal, or equatorial , dividing the ^gg into equal octants. The order of division is thus far exactly that demanded by Sachs's rule and agrees precisely with the cleavage of various kinds of spherical plant-cells. The later cleavages depart from the ideal type in the absence of periclinal divisions, the embryo becoming hollow, and its walls consisting of a single layer of cells in which anticHnal cleavages occur in regular rectangular succession. The fourth cleavage is again meridional, giving two tiers of eight cells each ; the fifth is horizontal, dividing each tier into an upper and a lower layer. The regular alternation is continued up to the ninth division (giving 512 cells), when the divisions pause while the gastrulation begins. In later stages the regularity is lost. Hertwig s Development of Sachs's Rules. — Beside Sachs's rules may be placed two others formulated by Oscar Hertwig in 1884, which bear directly on the facts just outHned and which lie behind Sachs's principle of the rectangular intersection of successive division- planes. These are : — 1 . The nucleus tends to take np a position at the centre of its sphere of influence, i.e. of the protoplasmic mass in zvhich it lies. 2. The axis of the 7nitotic figtires typically lies in the longest axis of the protoplasmic mass, and division therefore tends to cut this axis at a right a^tgle. The second rule explains the normal succession of the division- GEOMETRICAL RELATIONS OF CLEAVAGE-FORMS 365 planes according to Sachs's second rule. The first division of a homo- geneous spherical ^^^^, for example, is followed by a second division at right angles to it, since each hemisphere is twice as Ion- in the plane of division as in any plane vertical to it. The mitotic fi-ure of the second division lies therefore parallel to the first plane which forms the base of the hemisphere, and the ensuing division is vertical to It. The same applies to the third division, since each quadrant is as long as the entire ^gg while at most only half its diameter. Divi- sion is therefore transverse to the long axis and vertical to the first two planes. Taken together the rules of Sachs and Hertwig, applied to the ^gg, give us a kind of ideal type or model, well illustrated by the Fig. 169. — Cleavage of the ovum in the \\o\o\h\xx\^Xi Synapta (slightly schematized) f After Selenka.] '■ A-E. Successive cleavages to the 32-cell stage. F. Blastula of 128 cells. cleavage of Synapta, described above, to which all the forms of cleav- age may conveniently be referred as a basis of comparison. Numer- ous exceptions to all four of these rules are, however, known, and they are of little value save as a starting-point for a closer study of the facts. Cleavage of such schematic regularity as that of Synapta is extremely rare, both the form and the order of division being end- lessly varied and in extreme cases showing scarcely a discoverable connection with the ''type." We may conveniently consider these modifications under the following three heads : — 366 CELL-DIVISION AND DEVELOPMENT 1. Variation in the I'JiytJini of division. 2. Displacement of the cells {iticlnding variations in the direction of cleavage). 3. Unequal division of the cells. Nothing is more common than a departure from the regular rhythm of division. The variations are sometimes quite irregular, sometimes follow a definite rule, as, for instance, in the annelid N'eiris (Fig. 171), where the typical succession in the number of cells is with great constancy 2, 4, 8, 16, 20, 23, 29, 32, 37, 38, 41, 42, after which the order is more or less variable. The factors that determine such variations in the rhythm of division are very little understood. Bal- four, one of the first to consider the subject, sought an explanation in the varying distribution of metaplasmic substances, maintaining ('75, '80) that the rapidity of division in any part of the ovum is in general inversely proportional to the amount of deutoplasm that it contains. The entire inadequacy of this view has been demonstrated by a long series of precise studies on cell-lineage, which show that while the large deutoplasm-bearing cells often do divide more slowly than the smaller protoplasmic ones the reverse is often the case, while remarkable differences in the rhythm of division are often observed in cells which do not perceptibly differ in metaplasmic content.^ All the evidence indicates that the rhythm of division is at bottom deter- mined by factors of a very complex character which cannot be disentangled from those which control growth in general. LilHe ('95, '99) points out the very interesting fact, determined through an analysis of the cell-lineage of mollusks and annelids, that the rate of cleavage shows a direct relation to the period at which the prod- ucts become functional. Thus in Unio the more rapid cleavage of a certain large cell (" d. 2"), formed at the fourth cleavage, is obviously correlated with the early formation of the shell-gland to which it gives rise, while the relatively slow rate of division in the first ectomere- quartet is correlated with reduction of the prae-trochal region. The prospective character shown here w^ill be found to apply also to other characters of cleavage, as described beyond. When we turn to the factors that determine the direction of cleav- age or the displacement of cells subsequent to division, we find, as in the case of the division-rhythm, obvious mechanical factors com- bined with others far more complex. The arrangement of tissue-cells usually tends toward that of least resistance or greatest economy of space ; and in this regard they have been shown to conform, broadly speaking, with the behaviour of elastic spheres, such as soap-bubbles when massed together and free to move. Such bodies, as Plateau 1 Cf. Wilson, '92, Kofoid, '94, Lillie, '95, Zur Strassen, '95, Ziegler, '95, and especially Jennings, '97. GEOMEmiCAL RELATIONS OF CLEAVAGE-FORMS 36; and Lamarle have shown, assume a pol^^hedral form and tend toward such an arrangement that the area of surfacccontact /v '-. // a nun^uucn^. Spheres in a mass thus tend o aTsume The £^ Z mterlockm. polyhedrons so arranged that three pCsLterc in a hne, whde four Imes and six planes meet at a point If a ran .cd in a smgle layer on an extended surface, they a'sume the "."'0' A C D Fig. 170. — Cleavage ol Polygordius, from life. fhpl ^°7-^^" ^*^g^- f'«'" above. B. Corresponding view of eight-cell stage. C Side v.ew of the same (contrast Fig. 169, C) . D. Si.xteen-cell stage from the s^de. hexagonal prisms, three planes meeting along a line as before. I^oth these forms are commonly shown in the arrangement of the cells of plant and animal tissues; and Berthold {'^^6) and Errcra {'^6, '%j) carefully analyzing the phenomena, have endeavoured to show that not only the form and relative position of cells, but also the direction of cell-division, is, partially at least, thus determined. It is through displacements of the cells of this type that many of 368 CELL-DIVISION AND DEVELOPMENT the most frequent modifications of cleavage arise. Sometimes, as in Synapta, the alternation of the cells is effected through displacement of the blastomeres after their formation. More commonly it arises during the division of the cells, and may even be predetermined by the position of the mitotic figures before the slightest external sign of division. Thus arises that form of cleavage known as the spiral, oblique, or alternating type, where the blastomeres interlock during their formation and lie in the position of least resistance from the beginning. This form of cleavage, especially characteristic of many worms and mollusks, is typically shown by the ^^^ of Polygordius (Fig. 170). The four-celled stage is nearly like that of Syiiapta, though even here the cells slightly interlock. The third division is, however, oblique, the four upper cells being virtually rotated to the right (with the hands of a watch) so as to alternate with the four lower ones. The fourth cleavage is likewise oblique, but at right angles to the third, so that all of the cells interlock as shown in Fig. 170, D. This alternation regularly recurs for a considerable period. In many worms and mollusks the obliquity of cleavage appears still earlier, at the second cleavage, the four cells being so arranged that two of them meet along a ''cross-furrow" at the lower pole of the ^gg, while the other two meet at the upper pole along a similar, though often shorter, cross-furrow at right angles to the lower {e.g. in Nereis, Fig. 171). It is a curious fact that the direction of the dis- placement is quite constant, the first or upper quartet in the eight- cell stage being rotated to the right, or with the hands of a watch, the second quartet to the left, the third to the right, and so on. Crampton ('94) has discovered the remarkable fact that in P/iysa, a gasteropod having a reversed or sinistral shell, the whole order of displacement is likewise reversed, and the same has recently been shown by Holmes ('99) to be true of Aiicylus. The spiral or alternating type of cleavage beautifully illustrates Sachs's second rule as affected by modifying conditions ; for, as may be seen by an inspection of Figs. 170, 171, each division-plane is approximately at right angles to the preceding and succeeding (whence the " alternation . of the spirals" described by students of cell-lineage), while they are so directed that each cell as it is formed is placed at once in the position of least resistance in the mass, i.e. in the position of minimal surface-contact. It is impossible to resist the conclusion that one of the factors by which the position of the cells (and hence the direction of cell-division) is determined is a purely mechanical one, identical with that which determines the arrangement of soap-bubbles and the like. Very little acquaintance with the facts of development is however GEOMETRICAL RELATIOXS OF CLEAVAGE-EORMS 369 required to show that this purely mechanical factor, though doubtless real, must be subordinate to some other. This is strikingly shown, for example, in the development of annelids and mollusks, where the spiral cleavage, strictly maintained during the earlier stages, finally gives way more or less completely to a bilateral type of division in which the rule of minimal surface-contact is often violated. We see here a tendency operating directly against, and finally overcoming, D E F Fig. 171. — Cleavage oi Nereis. An example of a spiral cleavage, unequal from tlie beginning and of a marked determinate character. ^.Two-cell stage (the circles are oil-drops). B. Four-cell stage ; the second cleavage-plane passes through the future median plane. C. The same from the right side. D. Eight-cell stage. E. Sixteen cells; from the cells marked / arises the prototroch or larval ciliated belt, from X the ventral nerve-cord and other structures, from D the mcsoblast-bands, the germ-cells, and a part of the alimentary canal. F. Twenty-nine-cell stage, from the right side ; /. girdle of prototrochal cells which give rise to the ciliated belt. the mechanical factor which predominates in the earlier stages ; and in some cases, e.g. in the ^g% of Clavclina (Fig. 177) and other tuni- cates, this tendency predominates from the beginning. In both these cases this *' tendency " is obviously related to the growth-jirocess to which the future bilateral embryo will owe its form ; ^ and every attempt to explain the position of the cells and the direction of cleav- age must reckon with the morphogenic process taken as a whole. The blastomere is not merely a cell dividing under the stress of rude ^ Cf. Wilson ('92, p. 444). 2 B 370 CELL-DIVISION AND DEVELOPMENT mechanical conditions ; it is beyond this " a builder which lays one stone here, another there, each of which is placed with reference to future development."^ The third class of modifications, due to unequal division of the cells, not only leads to the most extreme types of cleavage but also to its Fig. 172. — The eight-cell stage of four different animals showing gradations in the inequality of the third cleavage. A. The leech Clepsine (Whitman). B. The oh^Xo'^o^ Rhynchelmis (Vejdovsky). C. The lamellibranch Unto (Lillie). D. Amphioxus. most difficult problems. Unequal divisions appear sooner or later in all forms of cleavage, the perfect equality so long maintained in Synapta being a rare phenomenon. The period at which the in- equality first appears varies greatly in different forms. In Polygordius (Fig. 170) the first marked inequality appears at the fifth cleavage; 1 Lillie, '95, p. 46. GEOMETRICAL RELATIONS OF CLEAVAGE-FORMS 371 in sea-urchins it appears at the fourth (Fig. 3); in AmpJiioxus at the third (Fig. 172); in the tunicate Clavclina at the second ( Fig. 177); in Nereis at the first division (Figs. 60, 171). The extent of the in- equality varies in like manner. Taking the third cleavage as a type, we may trace every transition from an equal division (echinoderms, Polygordiiis), through forms in which it is but slightly marked {Avi- phioxns, frog), those in which it is conspicuous {Nereis, Lymniea, poly- clades, Petroinyzon, etc.), to forms such as Clepsine, where the cells of the upper quartet are so minute as to appear Hke mere buds from the four large lower cells (Fig. 172). At the extreme of the series we reach the partial or meroblastic cleavage, such as occurs in the ceph- alopods, in many fishes, and in birds and reptiles. Here the lower hemisphere of the ^gg does not divide at all, or only at a late period, segmentation being confined to a disc-hke region or blastoderm at one pole of the ^gg (Fig. 173). Very interesting is the case of the teloblasts or pole-cells character- istic of the development of many annelids and mollusks and found in some arthropods. These remarkable cells are large blastomeres, set aside early in the development, which bud forth smaller cells in reg- ular succession at a fixed point, thus giving rise to long cords of cells (Fig. 175). The teloblasts are especially characteristic of apical growth, such as occurs in the elongation of the body in annelids, and they are closely analogous to the apical cells situated at the growing point in many plants, such as the ferns and stoneworts. Still more suggestive is the formation of rudinientaiy cells, arising as minute buds from the larger blastomeres, and, in some cases, appar- ently taking no part in the formation of the embryo (Fig. 174).^ We are as far removed from an explanation of unequal division as from that of the rhythm and direction of division. Inequality of divi- sion, like difference of rhythm, is often correlated with inequalities in the distribution of metaplasmic substances — a fact generalized by Balfour in the statement ('80) that the size of the cells formed in cleavage varies inversely to the relative amount of protoplasm in the region of the ^gg from which they arise. Thus, in all telolecithal ova, where the deutoplasm is mainly stored in the lower or vegetative hemisphere, as in many worms, mollusks, and vertebrates, the cells of the upper or protoplasmic hemisphere are smaller than those of the lower, and may be distinguished as micrGineres from the larger viacro- meres of the lower hemisphere. The size-ratio between micromeres and macromeres is on the whole directly proportional to the ratio between protoplasm and deutoplasm. Partial or discoidal cleavage occurs when the mass of deutoplasm is so great as entirely to prevent cleavage in the lower hemisphere. This has been beautifully con- 1 See Wilson, '98, '99, 2. :>/ CELL-DIVISION AND DEVELOPMENT firmed by O. Hertwig ('98), who, by placing frogs' eggs in a centrifu- gal machine, has caused them to undergo a meroblastic cleavage through the artificial accumulation of yolk at the lower ^^'ole, due to the centrifugal force. While doubtless containing an element of truth, this explanation is, however, no more adequate than Balfour's rule regarding the relation between deutoplasm and rhythm (p. 366); for innumerable cases are known in which no correlation can be made out between the distribu- tion of inert substance and the inequality of division. This is the case, for example, with the teloblasts mentioned above, which contain no deutoplasm, yet regularly divide unequally. It seems to be inap- K ^^ r B Fig. 173. — Partial or meroblastic cleavage in the squid LoUgo. [Watase.] plicable to the inequalities of the first two divisions in annelids and gasteropods. It is conspicuously inadequate in the history of indi- vidual blastomeres, where the history of division has been accurately determined. In Nereis, for example, a large cell known as the first somatoblast, formed at the fourth cleavage (X, Fig. 171, E\ under- goes an invariable order of division, three unequal divisions being fol- lowed by an equal one, then by three other unequal divisions, and again by an equal. This cell contains little or no deutoplasm and undergoes no perceptible changes of substance. The collapse of the rule is most complete in case of the rudi- mentary cells referred to above. In some of the annelids, e.g. in Aricia, where they were first observed,^ these cells are derived from the very large primary mesoblast-cell, which first divides into equal halves. Each of these then buds forth a cell so small as to be no larger than a polar body, and then immediately proceeds to give rise 1 Cf. Wilson, '92, '98. GEOMETRICAL KELATIONS OF CLEAVAGE-IORMS 171 to the mesoblast-bands by continued divisions, always in the same plane at right angles to that in which the rudimentary cells are formed (Fig. 174). The cause of the definite succession of equal and unequal divisions is here wholly unexplained. No less difficult is the extreme inequality of division involved in the formation of the polar bodies. We cannot explain this through the fact that deutoplasm is collected in the lower hemisphere ; for, on the one hand, the succeed- ing divisions (first cleavages) are often equal, while, on the other hand, the inequality is no less pronounced in eggs having equally A B Fig, 174. — Rudimentary blastomeres in the embryo of an aiuK-liu. Aruta. A. From lower pole ; rudimentary cells at^. lanatiancotte on the polar l)odies of Turhellaria, p. 235. ^ For a good review and critique, see Jennings. '97. 376 CELL-DIVISION AXD DEVELOPMEXT the distribution of metaplasmic materials is sufficient to explain the position of the spindle, whether with reference to the direction or the inequality of the cleavage. As regards the direction of the spindle, Berthold i^'^^) long since clearly pointed out that prismatic or cylindrical vegetable cells, for instance, those of the cambium, often divide lengthwise ; and numer- ous contradictions of Hertwig's '' law " have since been observed by students of cell-lineage with such accuracy that all attempts to explain them away have failed.^ In some of these cases the position of the spindle is not that of least but of greatest resistance,"^ the spindle ac- A B Fig. 176. — Segmenting eggs of ^j-car/j. [KOSTANECKI and SlEDLECKl,] A. Early prophase of second division, showing double centrosomes. B. Second cleavage in progress; upper blastomere dividing parallel to long axis of the cell. tually pushing away the adjoining cell to make way for itself. Simi- lar difficulties, some of which have been already considered (p. 372), stand in the way of the attempt to explain the eccentricity of the spindle in unequal division. All these considerations drive us to the view that the simpler mechanical factors, such as pressure, form, and the like, are subordinate to far more subtle and complex operations involved in the general development of the organism, a conclusion strikingly illustrated by the phenomena of teloblastic division (p. 371), where the constant succession of unequal divisions, always in the 1 Cf. Watase ('91), Mead ('94, '97, 2), Heidenhain ('95), Wheeler ('95), Castle ('96), Jennings ('97). 2 See especially the case observed by Mead ('94, '97, 2), in the egg oi Amphiij-ite. GEOMETRICAL RELATIONS OF CLEAVAGE-FORMS 377 same plane, is correlated with a deeply lying law of growth affecting the entire formation of the body. We cannot comprcJiend the forms of cleavage witJiont reference to the end-resnlt ; and thus these phe- nomena acquire a certain teleological character so happily expressed by Lillie (p. 370). This has been clearly recognized in various ways by a number of recent writers. Roux ('94), while seeking to explain many of the operations of mitosis on a mechanical basis, holds that the position of the spindle is partly determined by "immanent" nuclear tendencies. Braem ('94) recognizes that the position of the spindle is determined not merely as that of least resistance for the mitotic figure, but also for that of the resulting products. I pointed out ('92) that the bilateral form of cleavage in annelids must be regarded as a "forerunner" of the adult bilaterality. Jennings ('97) concludes that the form and direction of cleavage are related to the later morphogenetic processes ; and many similar expressions occur in the works of recent students of cell-lineage.^ The clearest and best expression of this view is, however, given bv Lillie ('95, '99), who not only correlates the direction and rate of cleavage, but also the size-relations of the cleavage-cells with the arrangement of the adult parts, pointing out that in general the size, as well as the position, of the blastomeres is directly correlated with that of the parts to which they give rise, and showing that on this basis " one can thus go over every detail of the cleavage, and know- ing the fate of the cells, can explain all the irregularities and pecuH- arities exhibited."^ Of the justice of this conclusion I think any one must be thoroughly convinced who carefully examines the recent literature of cell-lineage. It gives no real explanation of the phenom- ena, and is hardly more than a restatement of fact. Neither does it in any way lessen the importance of studying fully the mechanical conditions of cell-division. It does, however, show how inadequate have been most of the attempts thus far to formulate the " laws " of cell-division, and how^ superficially the subject has been considered by some of those who have sought for such "laws." We now pass naturally to the second or promorphological aspect of cleavage, to a study of which we are driven by the foregoing con- siderations. 1 Conklin ('99) believes that many of the pecuHarities of cleavage may he explained by the assumption of protoplasmic currents which " carry the centrosomes where they will, and control the direction of division and the relative size and quality of the daughter-cells,"' I.e., p. 90. He suggests that such currents are of a chemotropic character, but recognizes that their causation and direction remain unexplained. - cf. ('95), p. 39. 378 CELL-DIVISION AND DEVELOPMENT B. Promorphological Relations of Cleavage The cleavage of the ovum has thus far been considered in the main as a problem of cell-division. We have now to regard it in an even more interesting and suggestive aspect ; namely, in its morpho- logical relations to the body to which it gives rise. From what has been said above it is evident that cleavage is not merely a process by which the ^^g simply splits up into indifferent cells which, to use the phrase of Pfliiger, have no more definite relation to the structure of the adult body than have snowflakes to the avalanche to which they contribute. 1 It is a remarkable fact that in a very large number of cases a precise relation exists between the cleavage-products and the adult parts to which they give rise ; and this relation may often be traced back to the beginning of development, so that from the first division onward we are able to predict the exact future of every indi- vidual cell. In this regard the cleavage of the ovum often goes for- ward with a wonderful clocklike precision, giving the impression of a strictly ordered series in which every division plays a definite role and has a fixed relation to all that precedes and follows it. But more than this, the apparent predetermination of the embryo may often be traced still farther back to the regions of the undivided and even unfertilized ovum. The Qgg, therefore, may exhibit a distinct promorphology ; and the morphological aspect of cleavage must be considered in relation to the promorphology of the ovum of which it is an expression. I . PromorpJiology of the Ovum {a) Polarity and the Egg-axis. — It was long ago recognized by von Baer ('34) that the unsegmented ^gg of the frog has a definite egg-axis connecting two differentiated poles, and that the position of the embryo is definitely related to it. The great embryo] ogist pointed out, further, that the early cleavage-planes also are definitely related to it, the first two passing through it in two meridians inter- secting each other at a right angle, while the third is transverse to it, and is hence equatorial.^ Remak afterward recognized the fact ^ that the larger cells of the lower hemisphere represent, broadly speaking, the ''vegetative layer" of von Baer, i.e. the inner germ-layer or ento- blast, from which the ahmentary organs arise ; while the smaller cells 1 ('83), p. 64. 2 The third plane is in this case not precisely at the equator, but considerably above it, forming a " parallel " cleavage. 3 '55, p. 130. Among others who early laid stress on the importance of the egg-polarity maybe mentioned Auerbach ('74), Hatschek ('77), Whitman ('78), and Van Beneden (^"^l). PROMORPHOLOGICAL RELATIONS OF CLEAVAGE 379 of the upper hemisphere represent the 'f animal layer," outer germ- layer or ectoblast from which arise the epidermis, the nervous s)^tcm, and the sense-organs. This fact, afterward confirmed in a very large number of animals, led to the designation of the two poles as aniimil and vegetative, formative and nutritive, or protoplasmic and dcuto- plasmic, the latter terms referring to the fact that the nutritive deuto- plasm is mainly stored in the lower hemisphere, and that development is therefore more active in the upper. The polarity of the ovum is accentuated by other correlated phenomena. In every case where an egg-axis can be determined by the accumulation of deutoplasm in the lower hemisphere the egg-nucleus sooner or later lies eccentri- cally in the upper hemisphere, and the polar bodies are formed at the upper pole. Even in cases where the deutoplasm is equally distrib- uted or is wanting — if there really be such cases — an egg-axis is still determined by the eccentricity of the nucleus and the corre- sponding point at which the polar bodies are formed. In vastly the greater number of cases the polarity of the ovum has a definite promorphological significance ; for the egg-axis shows a definite and constant relation to the axes of the adult body. It is a very general rule that the upper or ectodermic pole, as marked by the position of the polar bodies, lies in the median plane at a point which is afterward found to lie at or near the anterior end. Through- out the annelids and mollusks, for example, the upper pole is the point at which the cerebral ganglia are afterward formed ; and these organs lie in the adult on the dorsal side near the anterior extremity. This relation holds true for many of the Bilateralia, though the primitive relation is often disguised by asymmetrical growth in the later stages, such as occur in echinoderms. There is, however, some reason to believe that it is not a universal rule. The recent observa- tions of Castle ('96), which are in accordance with the earlier work of Seeliger, show that in the tunicate Ciona the usual relation is reversed, the polar bodies being formed at the vegetative {i.e. deutoplasmic or entodermic) pole, which afterward becomes the dorsal side of the larva. My own observations ('95) on the echinoderm-egg indicate that here the primitive egg-axis has an entirely inconstant and casual relation to the gastrula-axis. It may, however, still be possi]:)lc to show that these exceptions are only apparent, and the principle in- volved is too important to be accepted without further jDroof. {b) Axial Relations of the Primary Cleavage-planes. — Since the egg-axis is definitely related to the embryonic axes, and since the first two cleavage-planes pass through it, we may naturally look for a definite relation between these planes and the embryonic axes ; and if such a relation exists, then the first two or four bkistomeres must likewise have a definite prospective value in the development. Such 38o CELL-DIVISION AND DEVELOPMENT relations have, in fact, been accurately determined in a large number of cases. The first to call attention to such a relation seems to have been Newport ('54), who discovered the remarkable fact that tJie first cleavage-plane in the frog s egg coincides with tJie niediaji plane of the adult body ; that, in other words, one of the first two blastomeres gives rise to the left side of the body, the other to the right. This discovery, though long overlooked and, indeed, forgotten, was con- firmed more than thirty years later by Pfliiger and Roux {'^7). It Fig. 177. — Bilateral cleavage of the tunicate egg. A. Four-celled stage of Clavellna, viewed from the ventral side. B. Sixteen-cell stage (VAN Beneden and JULIN). C. Cross-section through the gastrula stage (Castle) ; a. anterior; p. posterior end ; /. left, /-. right side. [Orientation according to Castle.] was placed beyond all question by a remarkable experiment by Roux ('88), who succeeded in killing one of the blastomeres by puncture with a heated needle, whereupon the uninjured cell gave rise to a half-body as if the embryo had been bisected down the middle line (Fig. 182). A similar result has been reached in a number of other animals by following out the cell-lineage ; e.g. by Van Beneden and Julin ('84) PROMORPHOLOGICAL RELATIOXS OF CLEAVAGE 3S1 in the ^g^ of the tunicate Claveliiia (Fig. 177), and by W^atase ('91) in the eggs of cephalopods (Fig. 178). In both these cases all the early stages of cleavage show a beautiful bilateral symmetry, and not only can the right and left halves of the segmenting o^^^^ be di.'^tin- guished with the greatest clearness, but also the anterior and jjoste- rior regions, and the dorsal and ventral aspects. These discoveries seemed, at first, to justify the hope that a fundamental law of develop- ment had been discovered, and Van Beneden was thus led, as early as 1883, to express the view that the development of all bilateral animals would probably be found to agree with the frog and ascidian in respect to the relations of the first cleavage. This cleavage was soon proved to have been premature. In one series of forms, not the first but the second cleavage-plane was found / — ' V Fig. 178. — Bilateral cleavage of the squid's egg. [\V.\T.\SE.] A. Eight-cell stage. B. The fifth cleavage in progress. The first cleavage {a-p) coincides with the future median plane; the second {l-r) is transverse. to coincide with the future long axis (7\^^;r2>, and some other annelids ; Crepidula, UiJibrclla, and other gasteropods). In another series (A forms neither of the first cleavages passes through the median plane, but both form an angle of about 45° to it {Clcpsinc and other leeches ; RJiynchelniis and other annelids ; Planorbis, Xassa, L 'nio, and other mollusks ; Discococlis and other platodes). In a few cases the first cleavage departs entirely from the rule, and is equatorial, as in Ascans and some other nematodes. The whole subject was finally thrown into apparent confusion, first by the discovery of Clapp ('91 ), Jordan, and Eycleshymer ('94) that in some cases there seems to be no con- stant relation whatever between the early cleavage-planes and the adult axes, even in the same species (teleosts, urodeles); and even in 382 CELL-DIVISION AND DEVELOPMENT the frog Hertwig showed that the relation described by Newport and Roux is not invariable. Driesch finally demonstrated that the direc- tion of the early cleavage-planes might be artificially modified by pressure without perceptibly affecting the end-result {cf. p. 375). These facts prove that the promorphology of the early cleavage- forms can have no fundamental significance. Nevertheless, they are of the highest interest and importance ; for the fact that the forma- tive forces by which development is determined may or may not coincide with those controlling the cleavage, gives us some hope of a 1} V Fig. 179. — Outline of unsegmented squid's egg, to show bilaterality. [Watase.] A. From right side. B. From posterior aspect. a-p. antero-posterior axis ; d-v. dorso-ventral axis ; /. left side ; r. right side. disentangling the complicated factors of development through a com- parative study of the different forms. (^) OtJier Promo7f ho logical CJiaracters of the Ovnm. — Besides the polarity of the ovum, which is the most constant and clearly marked of its promorphological features, we are often able to discover other characters that more or less clearly foreshadow the later develop- ment. One of the most interesting and clearly marked of these is the bilateral symmetry of the ovum in bilateral animals, which is sometimes so clearly marked that the exact position of the embryo may be predicted in the unfertilized ^gg, sometimes even before it is laid. This is the case, for example, in the cephalopod ^gg, as shown by Watase (Fig. 179). Here the form of the new-laid ^gg, before cleavage begins, distinctly foreshadows that of the embryonic body, and forms as it were a mould in which the whole development is cast. Its general shape is that of a hen's ^gg sHghtly flattened on one side, PROMORPIWLOGICAL RELATIOXS OF CLEAVAGE 3S the narrow end, according to Watase, representing the dorsal aspect the broad end the ventral aspect, the flattened side the posterior region, and the more convex side the anterior region. A// the early cleavage-furroivs are bilaterally arranged ivith respect to the plane of a -1^ Fig. i8o. — Eggs of the insect Corixa. [Metschnikoff.] A. Early stage before formation of the embryo, from one side. />'. The same viewed in the plane of symmetry. C. The embryo in its final position. a. anterior end; p. posterior; /. left side, r. right; f. ventral, d. dorsal aspect. (These letters refer to theyf«a/ position of the embryo, which is nearly diametrically opposite to that in which it first develops) ; m. micropyle ; near/ is the pedicle by which the egg is attached. symmetry in the nndivided egg ; and the same is true of the later development of all the bilateral parts. Scarcely less striking is the case of the insect ^^^. as has been pointed out especially by Hallez, Blochmann, and Wheeler (F'igs. 62, 180). In a large number of cases the ^^g is elongated and 384 CELL-DIVISION AND DEVELOPMENT bilaterally symmetrical, and, according to Blochmann and Wheeler, may even show a bilateral distribution of the yolk corresponding with the bilaterality of the ovum. Hallez asserts as the results of a study of the cockroach {Pcriplancta\ the water-beetle {Hydrophilus), and the locust {Locnsta) that '' the egg-cell possesses the same orien- tation as the maternal organism that produces it ; it has a cephalic pole and a caudal pole, a right side and a left, a dorsal aspect and a ventral ; and these different aspects of the egg-cell coincide with the corresponding aspects of the embryo." ^ Wheeler ('93), after ex- amining some thirty different species of insects, reached the same result, and concluded that even when the ^gg approaches the spherical form the symmetry still exists, though obscured. More- over, according to Hallez ('86) and later writers, the Qgg always lies in the same position in the oviduct, its cephalic end being turned forwards toward the upper end of the oviduct, and hence toward the head-end of the mother.^ 2. Meaning of the PromorpJiology of the Ovum The interpretation of the promorphology of the ovum cannot be adequately treated apart from the general discussion of development given in the following chapter; nevertheless it may briefly be considered at this point. Two widely different interpretations of the facts have been given. On the one hand, it has been sug- gested by Flemming and Van Beneden,^ and urged especially by Whitman,* that the cytoplasm of the ovum possesses a definite primordial organization which exists from the beginning of its exist- ence even though invisible, and is revealed to observation thiough polar differentiation, bilateral symmetry, and other obvious characters in the unsegmented Qgg. On the other hand, it has been maintained by Pfliiger, Mark, Oscar Hertwig, Driesch, Watase, and the writer that all the promorphological features of the ovum are of secondary origin; that the egg-cytoplasm is at the beginning isotropous — i.e. indifferent or homaxial — and gradually acquires its promorphological features during its preembryonic history. Thus the ^gg of a bilateral animal is at the beginning not actually, but only potentially, bilateral. Bilaterality once established, however, it forms as it were the mould in which the cleavage and other operations of development are cast, I believe that the evidence at our command weighs heavily on the side of the second view, and that the first hypothesis fails to 1 See Wheeler, '93, p. 67. 2 The micropyle usually lies at or near the anterior end, but may be at the posterior. It is a very important fact that the position of the polar bodies varies, being sometimes at the anterior end, sometimes on the side, either dorsal or lateral (Heider, Blochmann). 3- See p. 298. * Cf. pp. 299, 300. PROMORPHOLOGICAL RELATIONS OF CLEAVAGE 385 take sufficient account of the fact that development docs not nec- essarily begin with fertilization or cleavage, but may begin at a far earlier period during ovarian life. As far as the visible promorpho- logical features of the ovum are concerned, this conclusion is beyond question. The only question that has any meaning is whether these visible characters are merely the expression of a more subtle pre- Fig. 181. — Variations in the axial relations of the eggs of Cyclops. From sections of the eggs as they lie in the oviduct. [HaCKER.] A. Group of eggs showing variations in relative position of the polar spindles and the sperm- nucleus (the latter black) ; in a the sperm-nucleus is opposite to the polar spindle, in b, near it or at the side. B. Group showing variations in the axis of first cleavage with reference to the polar bodies (the latter black) ; a, b, and ^ show three different positions. existing invisible organization of the same kind. I do not believe that this question can be answered in the affirmative save by the trite and, from this point of view, barren statement that every effect must have its preexisting cause. That the ^gg possesses no fixed and predetermined cytoplasmic localization with reference to the adult parts, has, I think, been demonstrated through the remarkable 2C 386 CELL-DIVISION AND DEVELOPMENT experiments of Driesch, Roux, and Boveri, which show that a frag- ment of the ^^g may give rise to a complete larva (p. 353). There is strong evidence, moreover, that the egg-axis is not primordial but is established at a particular period ; and even after its establishment it may be entirely altered by new conditions. This is proved, for example, by the case of the frog's egg, in which, as Pfluger ('84), Born ('85), and Schultze ('94) have shown, the cytoplasmic materials may be entirely rearranged under the influence of gravity, and a new axis established. In sea-urchins, my own observations ('95) render it probable that the egg-axis is not finally established until after fertilization. These and other facts, to be more fully considered in the following chapter, give strong ground for the conclusion that the promorphological features of the ^gg are as truly a result of development as the characters coming into view at later stages. They are gradually established during the preembryonic stages, and the ^gg, when ready for fertilization, has already accomplished part of its task by laying the basis for what is to come. Mark, who was one of the first to examine this subject carefully, concluded that the ovum is at first an indifferent or homaxial cell {i.e. isotropic), which afterward acquires polarity and other promor- phological features.^ The same view was very precisely formulated by Watase in 1891, in the following statement, which I believe to express accurately the truth : " It appears to me admissible to say at present that the ovum, w^hich may start out without any definite axis at first, may acquire it later, and at the moment ready for its cleavage the distribution of its protoplasmic substances may be such as to exhibit a perfect symmetry, and the furrows of cleavage may have a certain definite relation to the inherent arrangement of the protoplasmic substances which constitute the ovum. Hence, in a certain case, the plane of the first cleavage-furrow may coincide with the plane of the median axis of the embryo, and the sundering of the protoplasmic material may take place into right and left, accord- ing to the preexisting organization of the ^gg at the time of cleav- age ; and in another case the first cleavage may roughly correspond to the differentiation of the ectoderm and the entoderm, also accord- ing to the preorganized constitution of the protoplasmic materials of the ovum. " It does not appear strange, therefore, that we may detect a cer- tain structural differentiation in the unsegmented ovum, with all the axes foreshadowed in it, and the axial symmetry of the embryonic organism identical with that of the adult." ^ This passage contains, I believe, the gist of the whole matter, as far as the promorphological relations of the ovum and of cleavage- 1 '81, p. 512. 2 'gi^ p_ 2S0. PROMORPIIOLOGICAL RELATIONS OF CLEAVAGE 387 forms are concerned, though Watase does not enter into the question as to how the arrangement of protoplasmic materials is effected. In considering this question, we must hold fast to the fundamental fact that the ^%g is a cell, like other cells, and that from an a priori point of view there is every reason to believe that the cytoplasmic differ- entiations that it undergoes must arise in essentially the same way as in other cells. We know that such differentiations, whether in form or in internal structure, show a definite relation to the environment of the cell — to its fellows, to the source of food, and the like. We know further, as Korschelt especially has pointed out, that the egg- axis, as expressed by the eecentricity of the gennitial vesicle, often sJiozvs a definite relation to the ovanaji tissues, the germinal vesicle lying near the point of attachment or of food-supply. Mark made the pregnant suggestion, in 1881, that the primary polarity of the egg might be determined by ''the topographical relation of the egg {\\\\k:\\ still in an indifferent state) to the remaining cells of the maternal tis- sue f'om ivhich it is differentiated,'' 2i\iA added that this relation might operate through the nutrition of the ovum. " It would certainly be interesting to know if that phase of polar differentiation which is manifest in the position of the nutritive substance and of the germi- nal vesicle bears a constant relation to the free surface of the epithe- lium from which the egg takes its origin. If, in cases where the egg is directly developed from epithelial cells, this relationship were demonstrable, it would be fair to infer the existence of correspond- ing, though obscured, relations in those cases where (as, for example, in mammals) the origin of the ovum is less directly traceable to an epithelial surface." ^ The polarity of the egg would therefore be comparable to the polarity of epithelial or gland-cells, where, as pointed out at page 57, the nucleus usually lies toward the base of the cell, near the source of food, while the centrosomes, and often also characteristic cytoplasmic products, such as zymogen granules and other secretions, appear in the outer portion. ^ The exact conditions under which the ovarian egg develops are still too little known to allow of a positive conclusion regarding Mark's suggestion. More- over, the force of Korschelt's observation is weakened by the fact that in many eggs of the extreme telolecithal type, where the j^olarity is very marked, the germinal vesicle occupies a central or sub-central position during the period of yolk-formation and only moves toward the periphery near the time of maturation. Indeed, in mollusks, annelids, and many other cases, the germinal vesicle remains in a central position, surrounded by yolk on all sides, until the spermatozoon enters. Only then does the egg-nucleus move i'8i,p. 515. 2 Hatschek has suggested the same CDmparisun (^Zoologie, p. 1 12). 388 CELL-DIVISION AND DEVELOPMENT to the periphery, the deutoplasm become massed at one pole, and the polarity of the ^g^ come into view (7V^;r/j, Figs. 60 and 97).^ In such cases the axis of the ^gg may perhaps be predetermined by the position of the centrosome, and we have still to seek the causes by which the position is established in the ovarian history of the ^gg. These considerations show that this problem is a complex one, involv- ing, as it does, the whole question of cell-polarity ; and I know of no more promising field of investigation than the ovarian history of the ovum with reference to this question. That Mark's view is cor- rect in principle is indicated by a great array of general evidence considered in the following chapter, where its bearing on the general theory of development is more fully dealt with. C. Cell-division and Growth The general relations between cell-division and growth, which have already been briefly considered at page 58 and in the course of this chapter, may now be more critically examined, together with some account of the causes that incite or inhibit division. It has been shown above that every precise inquiry into the rate form, or direc- tion of cell-division, inevitably merges into the larger problem of the general determination of growth. We may conveniently approach this subject by considering hrst the energy of division and the limita- tion of growth. All animals and plants have a limit of growth, which is, how- ever, much more definite in some forms than in others, and differs in different tissues. During the individual development the energy of cell-division is most intense in the early stages (cleavage) and diminishes more and more as the Umit of growth is approached. When the Hmit is attained a more or less definite equilibrium is estab- lished, some of the cells ceasing to divide and perhaps losing this power altogether (nerve-cells), others dividing only under special con- ditions (connective tissue-cells, gland-cells, muscle-cells), while others continue to divide throughout life, and thus replace the worn-out cells of the same tissue (Malpighian layer of the epidermis, etc.). The limit of size at which this state of equilibrium is attained is an heredi- tary character, which in many cases shows an obvious relation to the environment, and has therefore probably been determined and is maintained by natural selection. From the cytological point of view the limit of body-size appears to be correlated with the total number of cells formed rather than with their individual size. This relation has been carefully studied by Conklin ('96) in the case of the gastero- 1 The immature egg of Nereis shows, however, a distinct polarity in the arrangement of the fat-drops, which form a ring in the equatorial regions. CELL-DIVISION AND GROW Til 389 pod Crepidula, an animal which varies greatly in size in the mature condition, the dwarfs having in some cases not more than .,\- the vol- ume of the giants. The eggs are, however, of the same size in all, and their number is proportional to the size of the adult. The same is true of the tissue-cells. Measurements of cells from the epidermis, the kidney, the liver, the alimentary epithelium, and other tissues show that they are on the whole as large in the dwarfs as in the giants. The body-size therefore depends on the total number of cells rather than on their size individually considered, and the same appears to be the case in plants.^ A result which, broadly speaking, agrees with the foregoing, is given through the interesting experimental studies of Morgan ('95, i, '96), supplemented by those of Driesch ('98), in which the number of cells in normal larvae of echinoderms, ascidians, and Aniphioxus is compared with those in dwarf larvae of the same species developed from egg-fragments (Morgan) and isolated blastomeres (Driesch). Unless otherwise specified, the follow^ing data are cited from Driesch. The normal blastula of SpJiccrccJiijius possesses about 500 cells (Morgan), of which from 75 to 90 invaginate to form the archenteron (Driesch). In half-gastrulas the number varies from 35 to 45, occa- sionally reaching 50. In the same species, the normal number of mesenchyme-cells is 54 to 60, in the half-larvae 25 to 30. In Echinus the corresponding numbers are 30 ± and 13 to 15. In the ascidian larvae — a particularly favourable object — there are 29 to 35 (excep- tionally as high as 40) chorda-cells ; in the half-larvae, 1 3 to 17. While these comparisons are not mathematically precise, owing to the diffi- culty of selecting exactly equivalent stages, they nevertheless show that, on the whole, the size of the organ, as of the entire organism, is directly proportional to the number and not to the size of the cells, just as in the mature individuals of Crepidula. The available data are, however, too scanty to justify any very positive conclusions, and it is probable that further experiment will disclose factors at present unknown. It would be highly interesting to determine whether such dwarf embryos could in the end restore the normal number of cells, and, hence, the normal size of the body. In all the cases thus far determined the dwarf gastrulas give rise to larvae {Plutci, etc.) corre- spondingly dwarfed ; but their later history has not yet been suffi- ciently followed out. The gradual diminution of the energy of division during develop- ment by no means proceeds at a uniform pace in all of the cells, and, during the cleavage, the individual blastomeres are often found to exhibit entirely different rhythms of division, periods of active division being succeeded by long pauses, and sometimes by an entire cessa- 1 See Amelung ('93) and Strasburger ('93). 390 CELL-DIVISION AND DEVELOPMENT tion of division even at a very early period. In the echinoderms, for example, it is well established that division suddenly pauses, or changes its rhythm, just before the gastrulation (in Synapta at the 512-cell stage, according to Selenka), and the same is said to be the case in Aniphioxus (Hatschek, Lwoff). In Nereis, one of the blastomeres on each side of the body in the forty-two-cell stage suddenly ceases to divide, migrates into the interior of the body, and is converted into a unicellular glandular organ. ^ In the same animal, the four lower cells (macromeres) of the eight-cell stage divide in nearly regular succes- sion up to the thirty-eight-cell stage, when a long pause takes place, and when the divisions are resumed they are of a character totally different from those of the earlier period. The cells of the ciliated belt or prototroch in this and other annelids likewise cease to divide at a certain period, their number remaining fixed thereafter.^ Again, the number of cells produced for the foundation of particular struc- tures is often definitely fixed, even when their number is afterward increased by division. In annelids and gasteropods, for example, the entire ectoblast arises from twelve micromeres segmented off in three successive quartets of micromeres from the blastomeres of the four- cell stage. Perhaps the most interesting numerical relations of this kind are those recently discovered in the division of teloblasts, where the number of divisions is directly correlated with the number of seg- ments or somites. It is well known that this is the case in certain plants {C/iaracece), where the alternating nodes and intern odes of the stem are derived from corresponding single cells successively segmented off from the apical cell. Vejdovsky s observations on the annelid Dendrobcena give strong ground to believe that the number of meta- merically repeated parts of this animal, and probably of other anne- hds, corresponds in Uke manner with that of the number of cells segmented off from the teloblasts. The most remarkable and accu- rately determined case of this kind is that of the isopod Crustacea, where the number of somites is limited and perfectly constant. In the embryos of these animals there are two groups of teloblasts near the hinder end of the embryo, viz. an inner group of mesoblasts, from which arise the mesoblast-bands, and an outer group of ectoblasts, from which arise the neural plates and the ventral ectoblast. McMur- rich ('95) has recently demonstrated that the mesoblasts always divide exactly sixteen times, the ectoblasts thirty-two (or thirty-three) times, before relinquishing their teleoblastic mode of division and breaking up into smaller cells. Now the sixteen groups of cells thus formed give rise to the sixteen respective somites of the post-naupliar region of the embryo {i.e. from the second maxilla backward). In other 1 This organ, doubtfully identified by me as the head-kidney, is probably a mucus-gland (Mead). 2 cf. Fig. 171. CELL-DIVISION AND GROWTH 301 words, each single division of the mesoblasts and each double division of the ectoblasts splits off the material for a single somite ' The number of these divisions, and hence of the corresponding somites is a fixed inheritance of the species. The causes that determine the rhythm of division, and thus finally estabhsh the adult equilibrium, are but vaguely comprehended The ultimate causes must of course lie in the inherited constitution of the organism, and are referable in the last analysis to the structure of the germ-cells. Every division must, however, be the response of the cell to a particular set of conditions or stimuh ; and it is through the investigation of these stimuh that we may hope to penetrate f'arther into the nature of development. The immediate, specific causes of cell-division are still imperfectly known. In the adult, cells may be stimulated to divide by the utmost variety of agencies — by chemical stimulus, as in the formation of galls, or in hyperplasia induced by the injection of foreign substances into the blood; by mechanical pressure, as in the formation of calluses; by injury, as in the heahng of wounds and in the regeneration of lost parts ; and by a multitude of more complex physiological and pathological conditions, — by any agency, in short, that disturbs the normal equilibrium of the body. In all these cases, however, it is difficult to determine the immediate stimulus to division ; for a long chain of causes and effects may intervene between the primary disturbance and the ultimate reaction of the dividing cells. Thus there is reason to believe that the for- mation of a callus is not directly caused by pressure or friction, but through the determination of an increased blood-supply to the part affected and a heightened nutrition of the cells. Cell-division is here probably incited by local chemical changes ; and the opinion is gain- ing ground that the immediate causes of division, whatever their antecedents, are to be sought in this direction. That such is the case is indicated by nothing more clearly than the recent experiments on the Qgg by R. Hertwig, Mead, Morgan, and Loeb already referred to in part at pages 1 1 1 and 215. The egg-cell is, in most cases, stimu- lated to divide by the entrance of the spermatozoon, but in partheno- genesis exactly the same result is produced by an apparently quite different cause. The experiments in question give, however, ground for the conclusion that the common element in the two cases is a chemical stimulus. In the eggs of ChcEtopterus under normal condi- tions the first polar mitosis pauses at the anaphase until the entrance of the spermatozoon, when the mitotic activity is resumed and both polar bodies are formed. Mead ('98) shows, however, that the same effect may be produced without fertilization by placing the eggs for a few minutes in a weak solution of potassium chloride. In like manner R. Hertwig ('96) and Morgan ('99) show that unfertilized 392 CELL-DIVISION AND DEVELOPMENT echinoderm-eggs may be stimulated to division by treatment with weak solution of strychnine, sodium-chloride, and other reagents, the result being here more striking than in the case of CJicEtopterns, since the entire mitotic system is formed anew under the chemical stimulus. The cUmax of these experiments is reached in Loeb's artificial pro- duction of parthenogenesis in sea-urchin eggs by treatment with dilute magnesium chloride. Beside these interesting results may be placed the remarkable facts of gall-formation in plants, which seem to leave no doubt that extremely complex and characteristic abnormal growths may result from specific chemical stimuli, and many pathologists have held that tumours and other pathological growths in the animal body may be incited through disturbances of circulation or other causes resulting in abnormal local chemical conditions.^ But while we have gained some light on the immediate causes of division, we have still to inquire how those causes are set in opera- tion and are coordinated toward a typical end; and w^e are thus brought again to the general problem of growth. A very interesting suggestion is the resistance-theory of Thiersch and Boll, according to which each tissue continues to grow up to the limit afforded by the resistance of neighbouring tissues or organs. The removal or lessening of this resistance through injury or disease causes a resump- tion of growth and division, leading either to the regeneration of the lost parts or to the formation of abnormal growths. Thus the removal of a salamander's limb would seem to remove a barrier to the proliferation and growth of the remaining cells. These processes are therefore resumed, and continue until the normal barrier is re- established by the regeneration. To speak of such a "barrier" or ** resistance " is, however, to use a highly figurative phrase which is not to be construed in a rude mechanical sense. There is no doubt that hypertrophy, atrophy, or displacement of particular parts often leads to compensatory changes in the neighbouring parts ; but it is equally certain that such changes are not a direct mechanical effect of the disturbance, but a highly complex physiological response to it. How complex the problem is, is shown by the fact that even closely related animals may differ widely in this respect. Thus Fraisse has shown that the salamander may completely regenerate an amputated Hmb, while the frog only heals the wound without further regenera- tion.2 Again, in the case of coelenterates, Loeb and Bickford have shown that the tubularian hydroids are able to regenerate the ten- tacles at both ends of a segment of the stem, while the polyp Cerian- thiis can regenerate them only at the distal end of a section (Fig. 194;. 1 QC p. 97. For a good discussion of this subject, see E. Ziegler, '89. 2 In salamanders regeneration only takes place when the bone is cut across, and does not occur if the limb be exarticulated and removed at the joint. CELL-DIVISION AND GROWTH 393 In the latter case, therefore, the body possesses an inherent polarity which cannot be overturned by external conditions. A very curious case is that of the earthworm, which has long been known to possess a high regenerative capacity. If the posterior region of the worm be cut off, a new tail is usually regenerated. If the same operation be performed far forward in the anterior region, a new head is often formed at the front end of the posterior piece. If, however, the sec- tion be in the middle region the posterior piece sometimes regenerates a head, but more usually a tail, as was long since shown by Spallanzani and recently by Morgan ('99). Why such a blunder should be com- mitted remains for the present quite unexplained. It remains to inquire more critically into the nature of the correla- tion between growth and cell-division. In the growing tissues the direction of the division-planes in the individual cells evidently stands in a definite relation with the axes of growth in the body, as is espe- cially clear in the case of rapidly elongating structures (apical buds, teloblasts, and the like), where the division-planes are predominantly transverse to the axis of elongation. Which of these is the primary factor, the direction of general growth or the direction of the division- planes } This question is a difficult one to answer, for the two phe- nomena are often too closely related to be disentangled. As far as the plants are concerned, however, it has been conclusively shown by Hofmeister, De Bary, and Sachs that tJie groivth of tJic mass is tlic primary factor ; for the characteristic mode of growth is often shown by the growing mass before it splits up into cells, and the form of cell-division adapts itself to that of the mass : *' Die Pflanze bildet Zellen, nicht die Zelle bildet Pflanzen " (De Bary). Much of the recent work in normal and experimental embryology, as well as that on regeneration, indicates that the same is true in prin- ciple of animal growth. Among recent writers who have urged this view should be mentioned Rauber, Hertwig, Adam Sedgwick, and especially Whitman, whose fine essay on the Inadequacy of ilic Cell- theory of Development ('93) marks a distinct advance in our point of view. Still more recently this view has been almost demonstrated through some remarkable experiments on regeneration, which show that definitely formed material, in some cases even the adult tissues, may be directly moulded i?ito new structures. Driesch has shown ('95, 2, '99) that if gastrulas of SpJicerechinus be bisected through the equator so that each half contains both ectoderm and entoderm, the wounds heal, each half forming a typical gastrula, in which the ente- ron differentiates itself into the three typical regions (fore, middle, and hind gut) correctly proportioned, though the whole structure is but half the normal size. Here, therefore, the formative process is in the main independent of cell-division or increase in size. Miss Bickford 394 CELL-DIVISION AND DEVELOPMENT ('94) found that in the regeneration of decapitated hydranths of tubu- larians the new hydranth is primarily formed, not by new cell-formation and growth from the cut end, but by direct transformation of the distal portion of the stem.^ Morgan's remarkable observations on Planarin, finally, show that here also, when the animal is cut into pieces, com- plete animals are produced from these pieces, but only in small degree through the formation of new tissue, and mainly by direct remould- ing of the old material into a new body having the correct propor- tions of the species. As Driesch has well said, it is as if a plan or mould of the new little worm were first prepared and then the old material were poured into it.^ Facts of this kind, of which a considerable store has been accumu- lated, give strong ground for the view that cell-formation is subordi- nate to growth, or rather to the general formative process of which growth is an expression ; and they furnish a powerful argument against Schwann's conception of the organism as a cell-composite (p. 58). That conception is, however, not to be rejected /;/ toto, but contains a large element of truth; for there are many cases in which cells pos- sess so high a degree of independence that profound modifications may occur in special regions through injury or disease, without affect- ing the general equilibrium of the body. The most striking proof of this lies in the fact that grafts or transplanted structures may perfectly retain their specific character, though transferred to a different region of the body, or even to another species. Nevertheless the facts of regeneration prove that even in the adult the formative processes in special parts are in many cases definitely correlated with the organi- zation of the entire mass ; and there is reason to conclude that such a correlation is a survival, in the adult, of a condition characteristic of the embryonic stages, and that the independence of special parts in the adult is a secondary result of development. The study of cell- division thus brings us finally to a general consideration of develop- ment which forms the subject of the following chapter. LITERATURE. VIII Berthold, G. — Studien liber Protoplasma-mechanik. Leipzig, 1886. Boll, Fr. — Das Princip des Wachsthums. Berlin. 1876. Bourne, G. C. — A Criticism of the Cell-theory; being an answer to Mr. Sedgwick's article on the Inadequacy of the Cellular Theory of Development : Quart. Jcmni. Mic.Sci.,XXXN\l\.\. 1895. 1 Driesch suggests for such a process the term reparation in contradistinction to true regeneration. 2 '99, p. 55. It is mainly on these considerations that Driesch ('99) has built his recent theory of vitalism (^cf. p. 417), the nature of the formative power being regarded as a problem sui generis, and one which the "machine-theory of life " is powerless to solve. Cf. also the views of Whitman, p. 416. LITER A TURE 395 Castle, W. E.— The early Embryology of Ciona. Bull. Mus. Conip. ZooL, XXVII. 1896. Conklin, E. G. — The Embryology of Crepidula : Journ. Morpli., XIII. 1 897. Driesch, H. — (See Literature. IX.) Errera, L. — Zellformen unci Seifenblaseii : Tivj^cbl. dcr 60 Vcrsatiuiilniij^ licutsilwr Nafnrforsc/ia'- imd Aerzte zii Wiesbaden. 1887. Hertwig, 0. — Das Problem der Befruchtung und der Isotropic des Eies, eine Theo- rie der Vererbung. Jena^ 1884. Hofmeister. — ^Die Lehre von der Pflanzenzelle. Leipzig., 1867. Jennings, H. S. — The Early Development of Asplanchna : BulL Miis. Coup. Zool., XXX. I. Ca;/ibridge, 1896. Kofoid, C. A. — On the Early Development of Limax : Bidl. Miis. Conip. Zool., XXVII. 1895. Lillie, F. R. — The Embryology of the Unionidae : Journ. Morph.., X. 1895. Id. — Adaptation in Cleavage : Wood'' s Hall Biol. Lectures. 1899. McMurrich, J. P. — Embryology of the Isopod Crustacea: Journ. Morpli., XI. i. 1895. Mark, E. L. —Limax. (See list IV.) Morgan, T. H. — (See Literature, IX.) Rauber, A. — Neue Grundlegungen zur Kenntniss der Zelle : Morph. Ja/irb., \'I1I. 1883. Rhumbler, L. — Allgemeine Zellmechanik : Merkel u. Bonnet., Ergeb., WW. 1898. Sachs, J. — Pflanzenphysiologie. (See list VII.) Sedgwick, H. — On the Inadequacy of the Cellular Theory of Development, etc.: Quart. Journ . Mic . Sci. , XXXVII. i. 1894. Strasburger, E. — Uber die Wirkungssphare der Kerne und die Zellgrcisse : Histo- logische Beitrdge, V. 1893. Zur Strassen, 0. — Embryonalentwickelung der Ascaris : Arch. Ento/n.. III. 1896. Watase, S. — Studies on Cephalopods ; I., Cleavage of the Ovum : Journ. Morph., IV. 3. 1891. Whitman, C. 0. — The Inadequacy of the Cell-theory of Development : Wood's Moll Biol. Lectures. 1893. Wilson, Edm. B. — The Cell-lineage oi Nereis : Journ. Morph., VI. 3. 1892. Id. — Amphioxus and the Mosaic Theory of Development : Journ. Morph., \'I1I. 3. 1893- Id. — Considerations on Cell-lineage and Ancestral Reminiscence: Ann. X. ). Acad., XI. 1898; also Wood's H oil Biol. Lectures, 1899. CHAPTER IX THEORIES OF INHERITANXE AND DEVELOPMENT " It is certain that the germ is not merely a body in which life is dormant or potential, but that it is itself simply a detached portion of the substance of a preexisting living body." HUXLEY.I " Inheritance must be looked at as merely a form of growth." Darwin.2 " Ich mochte daher wohl den Versuch wagen, durch eine Darstellung des Beobachteten Sie zu einer tiefern Einsicht m die Zeugungs- und Entwickelungsgeschichte der organischen Korper zu fiihren und zu zeigen, wie dieselben weder vorgebildet sind, noch auch, wie man sich crewohnlich denkt, aus ungeformter Masse in einem bestimmten Momente plotzlich ausschiessen." Von Baer.3 Every discussion of inheritance and development must take as its point of departure the fact that the germ is a single cell similar in its essential nature to any one of the tissue-cells of which the body is composed. That a cell can carry with it the sum total of the heritage of the species, that it can in the course of a few days or weeks give rise to a mollusk or a man, is the greatest marvel of biological science. In attempting to analyze the problems that it involves, we must from the outset hold fast to the fact, on which Huxley insisted, that the wonderful formative energy of the germ is not impressed upon it from without, but is inherent in the &gg as a heritage from the paren- tal life of which it was originally a part. The development of the embryo is nothing new. It involves no breach of continuity, and is but a continuation of the vital processes going on in the parental . body. What gives development its marvellous character is the rapid- ity with which it proceeds and the diversity of the results attained in a span so brief. But when we have grasped this cardinal fact, we have but focussed our instruments for a study of the real problem. How do the adult characteristics lie latent in the germ-cell ; and how do they become patent as development proceeds ? This is the final question that looms in the background of every investigation of the cell. In approaching it we may well make a frank confession of ignorance ; for in spite of all that the microscope has revealed, we have not yet penetrated the mystery, and inheritance and development still remain in their fun- 1 Evolution, Science and Culture, p. 291. 2 Variation of Anitnals and Plants, II., p. 398. 3 Entwick. der Thieve, II., 1837, p. 8. 396 THE THEORY OF GERMINAL LOCALIZATION 397 damental aspects as great a riddle as they were to the Greeks. What we have gained is a tolerably precise acquaintance with the external aspects of development. The gross errors of the early preformation- ists have been dispelled. ^ We know that the germ-cell contains no predelineated embryo ; that development is manifested, on the one hand, by the cleavage of the ^gg, on the other hand, by a process of differentiation, through which the products of cleavage gradually assume diverse forms and functions, and so accomplish a physiological division of labour. We can clearly recognize the fact that these pro- cesses fall in the same category as those that take place in the tissue- cells ; for the cleavage of the ovum is a form of mitotic cell-division, while, as many eminent naturalists have perceived, differentiation is nearly related to growth and has its root in the phenomena of nutri- tion and metabolism. The real problem of development is the orderly sequence and congelation of these pJic7iomena toward a typical result. We cannot escape the conclusion that this is the outcome of the organization of the germ-cells ; but the nature of that which, for lack of a better term, we call ''organization," is and doubtless long will remain almost wholly in the dark. In the following discussion, which is necessarily compressed within narrow limits, we shall disregard the earlier baseless speculations, such as those of the seventeenth and eighteenth centuries, which attempted a merely formal solution of the problem, confining our- selves to more recent discussions that have grown directlv out of modern research. An introduction to the general subject may be given by a preliminary examination of two central hypotheses about which most recent discussions have revolved. These are, first, the theory of Germinal Localization'^ of Wilhelm His ('74), and, second, the Idioplasm Hypothesis of Nageli ('84). The relation between these two conceptions, close as it is, is not at first sight very apparent ; and for the purpose of a preliminary sketch they may best be con- sidered separately. A. The Theory of Germinal Localization Although the naive early theory of preformation and evolution was long since abandoned, yet we find an after-image of it in the theory of germinal localization which in one form or another has been advo- cated by some of the foremost students of development. It is main- tained that, although the embryo is not \)X^formed in the germ, it must nevertheless be Y>^Qdetermined in the sense that the >). B. Twin gastrulas from a" single egg. C. Double cleavage resulting from the partial separation, by shaking, of the blastomeres of the two-cell stage. D.E.F. Double gastrulas arising from such forms as the last. still forming part of a complete embryo (** partial development"), but in other cases developing directly into a complete dwarf embryo, as if it were an ^g% of diminished size. In 1891 Driesch w^is ab^e to follow out the development of isolated blastomeres of sea-nrchin 410 INHERITANCE AND DEVELOPMENT eggs separated by shaking to pieces the two-cell and four-cell stages. Blastomeres thus isolated segment as if still forming part of an entire larva, and give rise to a half- (or quarter-) blastula (Fig. 183). The opening soon closes, however, to form a small complete blastula, and the resulting gastrula and Pluteus larva is a perfectly formed dwarf of only half (or quarter) the normal size. Incompletely separated blastomeres give rise to double embryos Uke the Siamese twins. Shortly afterward the writer obtained similar results in the case of AnipJiioxHS^ but here tJie isolated blastoinere behaves from the begin- 7iing like a complete ovum of half the usual sice, and gives rise to a complete blastula, gastrula, and larva. Complete embryos have also been obtained from a single blastomere in the teleost Fuiidulus (Morgan, '95, 2), in Triton (Herlitzka, '95), and in a number of hydromedusae (Zoja, '95, Bunting, '99); and nearly complete em- bryos in the tunicates Ascidiella (Chabry, "^'j\ PJiallusia (Driesch, '94), and Molgula (Crampton, '98).^ Perhaps the most striking of these cases is that of the hydroid Clytia, in which Zoja was able to obtain perfect embryos, not only from the blastomeres of the two- cell and four-cell stages, but from eight-cell and even from sixteen- cell stages, the dwarfs in the last case being but one-sixteenth the normal size. These experiments render highly improbable the hypothesis of qualitative division in its strict form, for they demonstrate that the earlier cleavages, at least, do not in these cases sunder fundamentally different materials, either nuclear or cytoplasmic, but only split the ^&g up into a number of parts, each of which is capable of producing an entire body of diminished size, and hence m.ust contain all of the material essential to complete development. Both Roux and Weis- mann endeavour to meet this adverse evidence with the assumption of a " reserve idioplasm," containing all of the elements of the germ- plasm which is in these cases distributed equally to all the cells in addition to the specific chromatin conveyed to them by qualitative division. This subsidiary hypothesis renders the principal one {i.e. that of qualitative division) superfluous, and brings us back to the same problems that arise when the assumption of qualitative division is discarded. The theory of qualitative nuclear division has been practically dis- proved in another way by Driesch, through the pressure-experiments already mentioned at page 375. Following the earlier experiments of Pfluger ('84) and Roux ('85) on the frog's Q,g^, Driesch subjected segmenting eggs of the sea-urchin to pressure, and thus obtained flat plates of cells in which the arrangement of the nuclei differed totally 1 The "partial" development in the earlier stages of some of these forms is considered at page 419. CRITIQUE OF THE ROUX-WEISMANN THEORY 411 from the normal (Fig. 186); yet such eggs when released from press- ure continue to segment, zvitJwiit rearrangeme^it of the nuclei, and give rise to perfectly normal larvae. I have repeated these experi- ments not only with sea-urchin eggs, but also with those of an annelid i^Nereis\ which yield a very convincing result, since in this case the histological differentiation of the cells appears very early. In the normal development of this animal the archenteron arises from four large cells or macromeres (entomeres), which remain after the suc- cessive formation of three quartets of micromeres (ectomeres) and the parent-cell of the mesoblast. After the primary differentiation of the germ-layers the four entomeres do not divide again until a very late period (free-swimming trochophore), and their substance always retains a characteristic appearance, differing from that of the other Fig. 186. — Modification of cleavage in sea-urchin eggs by pressure. A. Normal eight-cell stage of Toxopneustes. B. Eight-cell stage of £'c/^///«j segmentino- under pressure. Both forms produce normal Plutei. blastomeres in its pale non-granular character and in the presence of large oil-drops. If unsegmented eggs be subjected to pressure, as in Driesch's echinoderm experiments, they segment in a flat plate, all of the cleavages being vertical. In this way are formed eight-celled plates in which all of the cells contain oil-drops (Fig. 187, D). If they are now released from the pressure, each of the cells divides in a plane approximately horizontal, a smaller granular micromere being formed above, leaving below a larger clear macromere in which the oil-drops remain. The sixteen-cell stage, therefore, consists of eight deutoplasm-laden macromeres and eight protoplasmic micromeres (instead of four macromeres and twelve micromeres, as in the usual development). These embryos developed into free-swimming trocho- phores containing eight instead of four macromeres, which have the typical clear protoplasm containing oil-drops. In this case there can 412 INHERITANCE AND DEVEIOPMENT be no doubt whatever that four of the entoblastic nuclei were nor- mally destined for the first quartet of micromeres (Fig. 187, B\ from which arise the apical gangha and the prototroch. Under the condi- tions of the experiment, however, they have given rise to the nuclei of cells which differ in no wise from the other entoderm-cells. Even Fig. 187. — Modifications of cleavage by pressure in Nereis. A. B. Normal four- and eight-cell stages. C. Normal trochophore larva resulting, with four entoderm-cells. D. Eight-cell stage arising from an egg flattened by pressure ; such eggs give rise 10 trochophores with eight instead of four entoderm-cells. Numerals designate the successive cleavages. in a highly differentiated type of cleavage, therefore, the nuclei of the segmenting Qgg are not specifically different, as the Roux-Weismann hypothesis demands, but contain the same materials even in the cells that undergo the most diverse subsequent fate. But there is, further- more, very strong reason for believing that this may be true in later NATURE AND CAUSES OF DIFFERENTIATION 413 stages as well, as Koiliker insisted in opposition to Weismann as early as 1886, and as has been urged by many subsequent writers. The strongest evidence in this direction is afforded by the facts of regeneration; and many cases are known — for instance, among the hydroids and the plants — in which even a small fragment of the body is able to reproduce the whole. It is true that the power of regeneration is always limited to a greater or less extent according to the species. But there is no evidence whatever that such hmita- tion arises through specification of the nuclei by quaHtative division, and, as will appear beyond, its explanation is probably to be sought in a very different direction. F. On the Nature and Causes of Differentiation We have now cleared the ground for a restatement of the problem of development and an examination of the views opposed to the Roux-Weismann theory. After discarding the hypothesis of quaH- tative division the problem confronts us in the following form. If chromatin be the idioplasm in which inheres the sum total of heredi- tary forces, and if it be equally distributed at every cell-division, how can its mode of action so vary in different cells as to cause diversity of structure, i.e. dijferentiatio7i ? It is perfectly certain that differen- tiation is an actual progressive transformation of the egg-substance involving both physical and chemical changes, occurring in a definite order, and showing a definite distribution in the regions of the Qgg. These changes are sooner or later accompanied by the cleavage of the Qgg into cells whose boundaries may sharply mark the areas of differentiation. What gives these cells their specific char- acter.? Why, in the four-cell stage of an annelid Qgg, should the four cells contribute equally to the formation of the ahmentary canal and the cephaUc nervous system, while only one of them (the left- hand posterior) gives rise to' the nervous system of the trunk-region and to the muscles, connective tissues, and the germ-cells.' (Figs. 171, 188, B.) There cannot be a fixed relation between the various regions of the &gg which these blastomeres represent and the adult pa'I-ts arising from them; for in some eggs these relations maybe artificially changed. A portion of the Qgg which under normal con- ditions would give rise to only a fragment of the body will, if split off from the rest, give rise to an entire body of diminished size. W hat then determines the history of such a portion.? What influence moulds it now into an entire body, now into a part of a body .? De Vries, in his remarkable essay on Intracellular l\iuo;cmsis ('89), endeavoured to cut this Gordian knot by assuming that the character of each cell is determined by pangens that migrate from 414 INHERITANCE AND DEVEIOPMENT the nucleus into the cytoplasm, and, there becoming active, set up specific changes and determine the character of the cell, this way or that, according to their nature. But what influence guides the migrations of the pangens, and so correlates the operations of devel- opment? Both Driesch and Oscar Hertwig have attempted to f — Fig. i88. — Diagrams illustrating the value of the quartets in a polyclade {Leptoplana), a lamel- libranch {Ufiio), and a gasteropod {Crepiduld). A. Leptoplana, showing mesoblast-formation in the second quartet. B. Crepidula, showing source of ectomesoblast (from a~, b-, c'^) and en- tomesoblast (from quadrant Z)). C. 6'>/2c, 'ectomesoblast formed only from u^. In all the figures the successive quartets are numbered with Arabic figures ; ectoblast unshaded, mesoblest dotted, entoblast vertically lined. answer this question, though the first-named author does not commit himself to the pangen-hypothesis. These writers have maintained that the particular mode of development in a given region or blasto- mere of the tgg is a result of its relation to the remainder of tJie 7nass^ i.e. a product of what may be called the intra-embryonic environ- NATURE AND CAUSES OF DIFEERENTIATION 415 ment. Hertwig insisted that the organism develops as a whole as the result of a physiological interaction of equivalent blastomeres the transformation of each being due not to an inherent specific power of self-differentiation, as Roux's mosaic-theory assumed but to the action upon it of the whole system of which it is a part ''According to my conception," said Hertwig, - each of the first two blastomeres contains the formative and differentiating forces not simply for the production of a half-body, but for the entire organism ; the left blastomere develops into the left half of the bodv only because it is placed in relation to a right blastomere." 1 Again, in a later paper : " The ^gg is a specifically organized elementary organism that develops epigenetically by breaking up into cells and their subsequent differentiation. Since every elementary part {i.e. cell) arises through the division of the germ, or fertilized ^gg, it contains also the ger'm of the w^hole, but during the process of development it becomes ever more precisely differentiated and determined by the formation of cytoplasmic products according to its position with reference to the entire organism (blastula, gastrula, etc.)." 2 An essentially similar view was advocated by the writer ('93, '94) nearly at the same time, and the same general conception was ex- pressed with great clearness and precision by Driesch shortlv after Hertwig: "The fragments {i.e. cells) produced by cleavage are com- pletely equivalent or indifferent." "The blastomeres of the sea- urchin are to be regarded as forming a uniform material, and they may be thrown about, like balls in a pile, without in the least degree impairing thereby the normal power of development." '"^ '' The rehi- tive positio7i of a blastomere iji tJie zi'/io/e determines in general what develops from it ; if its position be e hanged, it gives rise to something different; in other words, its prospective valne is a function of its position. "^ Jn/this last aphorism the whole problem of development is brought to a focus. It is clearly not a solution of the j^roblem, but (Mily a highly suggestive restatement of it; for everything turns upon how the relation of the part to the whole is conceived. W^-y little con- sideration is required to show that this relation cannot be a merelv geometrical or rudely mechanical one, for in the eggs of different 1 '92, I, p. 481. ^ '93» P- 793- It should be pointed out that Roux himself in several papers expressl\ recognizes the fact that development cannot be regarded as a ]iure mosaic-work, and that besides the power of self-differentiation postulated by his hypothesis we must assume a "correlative differentiation" or differentiating interaction of parts in the embryo. Cf. Koux, '92, '93. I- 3 Studien IV., p. 25. * Studien IV., p. 39. Cf. His, " Es muss die Wachsthumserregbarkeit dcs pjcs cine Function des Raumes sein." ('74, p. 153.) 41 6 INHERITANCE AND DEVELOPMENT animals blastomeres may almost exactly correspond in origin and relative position, yet differ widely in their relation to the resulting embryo. Thus we find that the cleavage of polyclades, annelids, and gasteropods (Fig. i88) shows a really wonderful agreement in form, yet the individual cells differ markedly in prospective value. In all of these forms three quartets of micromeres are successively formed according to exactly the same remarkable law of the alternation of the spirals ; ^ and, in all, the posterior cell of a fourth quartet lies at the hinder end of the embryo in precisely the same geometrical relation to the remainder of the embryo ; yet in the gasteropods and annelids this cell gives rise to the mesoblast-bands and their products, in the polyclade to a part of the archenteron, while important differences also exist in the value of the other quartets. The relation of the part to the whole is therefore of a highly subtle character, the pro- spective value of a blastomere depending not merely upon its geomet- rical position, but upon its relation to the whole complex inherited organization of which it forms a part. The apparently simple con- clusion stated in Driesch's clever aphorism thus leads to further prob- lems of the highest complexity. It should be here pointed out that Driesch does not accept Hertwig's theory of the interaction of blasto- meres as such, but, hke Whitman, Morgan, and others, has brought forward effective arguments against that too simple and mechanical conception. That theory is, in fact, merely Schwann's cell-composite theory of the organism applied to the developing embryo, and the general arguments against that theory find some of their strongest \ support in the facts of growth and development.^ Th]s has been ' forcibly urged by Whitman ('93), who almost simultaneously with the statements of Driesch and Hertwig, cited above, expressed the con- viction that the morphogenic process cannot be conceived as merely the sum total or resultant of the individual cell-activities, but operates as a unit without respect to cell-boundaries, precisely as De Bary con- cludes in the case of growing plant-tissues (p. 393), and the nature of that process is due to the organization of the ^gg as a whole. ^ While recognizing fully the great value of the results attained during the past few years in the field of experimental and specula- tive embryology, we are constrained to admit that as far as the essence of the problem is concerned we have not gone very far beyond the conclusions stated above ; for beyond the fact that the inherited organization is involved in that of the germ-cells we remain quite ignorant of its essential nature. This has been recognized by no one more clearly than by Driesch himself, to whose critical researches we owe so much in this field. At the cUmax of a recent elaborate analysis, the high interest of which is somewhat obscured by 1 Cf. p. 368. ^ Cf. pp. 388-394. NATURE AND CAUSES OE DIFEERENTIATION 417 its too abstruse form, Driesch can only reiterate his former aphorism,^ finally taking refuge in an avowed theory of vitalism which assumes the localization of morphogenic phenomena to be determined bv "a wholly unknown principle of correlation,"^ and forms a problem sni generis^^ This conclusion recognizes the fact that the fundamental problem of development remains wholly unsolved, thus confirming from a new point of view a conclusion which it is only fair to point out has been reached by many others. - But while the fundamental nature of the morphogenic process thus remains unknown, we have learned some very interesting facts regard- ing the conditions under which it ta'kes place, and which show that Driesch's aphorism loses its meaning unless carefully qualified. The experiments referred to at pages 353, 410, show that up to a certain stage of development the blastomeres of the early echinoderm, AnipJii- oxiis or medusa-embryo, are "totipotent" (Roux), or "equipotential " (Driesch), i.e. capable of producing any or all parts of the body. Even in these cases, however, we cannot accept the early conclusion of Pfliiger i^'^Z), applied by him to the frog's ^gg, and afterward accepted by Hertwig, that the material of the ^gg,, or of the blasto- meres into which it splits up, is absolutely " isotropic," i.e. consists of quite uniform indifferent material, devoid of preestablished axes. Whitman and Morgan, and Driesch himself, showed that this cannot be the case in the echinoderm Qgg\ for the ovum possesses a polarity predetermined before cleavage begins, as proved by the fact that at the fourth cleavage a group of small cells or micromeres always arises at a certain point, which may be precisely located before cleavage by reference to the eccentricity of the first cleavage-nucleus,'* and which, as Morgan showed,^ is indicated before the third, and sometimes before the second cleavage, by a migration of pigment away from the micromere-pole. These observers are thus led to the assumption of a primary polarity of the egg-protoplasm, to which Driesch, in the course of further analysis of the phenomena, is compelled to add the assumption of a secondary polarity at right angles to the first.'^ The.se polarities, inherent not only in the entire ^gg, but also in each of the blastomeres into which it divides, form the primary conditions under which the bilaterally symmetrical organism develops by epigenesis. To this extent, therefore, the material of the blastomeres, though ''totipotent," shows a certain predetermination with respect to the adult body. 1 '99, pp. 86-87. 2 This phrase is cited by Driesch from an earher work (^'92, p. 59O) as ^ivm- a cncct though " unanalytical " statement of his view. It may be questioned whether many readers will regard as an improvement the '' analytical " form it assumes in his last work. 3 I.e., p. 90. * 'Cf. Fig. 103. ^ '94, I'. 142. 6 See Driesch, '93, pp. 229, 241 ; '96, and '99, p. 44- 2 E 4i8 ISIIERITAXCE AXD DEVELOPMEXT ~\Ve now proceed to the consideration of experiments which show- that in some animal egK'^ ^^»ch ]-)redetermination may go much farther, so that the development does, in fact, show many of the features of a mosaic-work, as maintained ]:>y Roux. The best-determined of these cases is that of the ctenoph()re-e«;K. ^^^ shown by the work of Chun, Fig. 189. — Partial larvcne of the ctenophore Beroc. [Dkiesch and Morgan.] A. Half sixteen-cell stage, from an isolated blastomere. li. Resulting larva, with four rows of swimming-plates and three gastric pouches. C. One-fourth sixtecn-cell stage, from an isolated blastomere. D. Resulting larva, with two rows of plates and two gastric pouches. E. Defective larva, with six rows of plates and three gastric pouches, from a nucleated fragment of an unseg- mented egg. F. Similar larva with five rows of plates, from above. Driesch, and Morgan ('95), and Fischel ('98). These observers have demonstrated that isolated blastomeres of the two-, four-, or eight-cell stage undergo a cleavage which, through the earliest stages, is exactly like that which it would have undergone if forming part of a com- NATURE AND CAUSES OF DIFEERENTIATION 419 plete embryo, and gives rise to a defective larva, having only four two, or one row of swimming-plates (Fig. 189); and Fischel's obser' vations give strong reason to believe that each of the eight micromeres of the sixteen-cell stage is definitely specified for the formation of one of the rows of plates. In like manner Crampton (96) found that in case of the marine gasteropod Ilyanassa isolated blastomeres of two- cell or four-cell stages segmented exactly as if forming part of an entire embryo, and gave rise to fmgmaits of a larva, not to complete dwarfs, as in the echinoderm (Fig. 190). Further, in embryos from which the ''yolk-lobe" (a region of that macromere from which the primary mesoblast normally arises) had been removed, no mesoblast- bands were formed. Most interesting of all, Driesch and Morgan discovered that if a part of the cytoplasm of an imscgjnaitcd cte^'no- phore-egg were removed, the remainder gave rise to an incomplete larva, showing definite defects (Fig. 189, E, F). In none of these cases is the embryo able to complete itself, though it should be remarked that neither in the ctenophore nor in the snail is the partial embryo identical with a fragment of a whole embryo, since the micromeres finally enclose the macromeres, leaving no sur- face of fracture. This extreme is, however, connected by a series of forms with such cases as those of Amphioxns or the medusa, where the fragment develops nearly or quite as if it were a whole. In the tunicates the researches of Chabry i^^y), Driesch ('94), and Crampton ('97) show that an isolated blastomere of the two-cell stage undergoes a typical half-cleavage (Crampton), but finally gives rise to a nearly perfect tadpole larva lacking only one of the asymmetrically placed sense-organs (Driesch). Next in the series may be placed the frog, where, as Roux, Endres, and Walter have shown, a blastomere of the two-cell stage may give rise to a typical half-morula, half-gastrula, and half-embryo 1 (Fig. 182), yet finally produces a perfect larva. A further stage is given by the echinoderm-egg, which, as Driesch showed, undergoes a half-cleavage and produces a half-blastula, which, however, closes to form a whole before the gastrula-stage (Fig. 183). Perfectly formed though dwarf larvae result. Finally, we reach Auiphi- oxus and the hydromasae in which a perfect ** whole develoj)mcnt " usually takes place from the beginning, though it is a verv interest- ing fact that the isolated blastomeres of Aiiiphioxus sometimes show, in the early stages of cleavage, peculiarities of development that recall their behaviour when forming part of an entire embryo.- We see throughout this series an effort, as it were, on the part of the isolated blastomere to assume the mode of development character- istic of a complete ^^^, but one that is striving against conditions that ^ This is not invariably the case, as described beyond. 2 Cf. Wilson, '93, pp. 590, 608. 420 INHERITANCE AND DEVEIOPMENT tend to confine its operations to the role it would have played if still forming part of an entire developing ^g^. In Amphioxits or Clytia this tendency is successful almost from the beginning. In other forms the Umiting conditions are only overcome at a later period, while in the ctenophore or snail they seem to afford an insurmount- Fig. 190. — Partial development of isolated blastomeres of the gasteropod ^gg, Ilyanassa. [Crampton.] A. Normal eight-cell stage. B. Normal sixteen-cell stage. C. Half eight-cell stage, from isolated blastomere of the two-cell stage. D. Half twelve-cell stage succeeding. E. Two stages in the cleavage of an isolated blastomere of the four-cell stage ; above a one-fourth eight-cell stage, below a one-fourth sixteen-cell stage. able barrier to complete development. What determines the limita- tions of development in these various cases ? They cannot be due to nuclear specification ; for in the ctenophore the fragment of an iniseg- mented ^%^, containing the normal egg-nucleus, gives rise to a defec- tive larva ; and my experiments on Nereis show that even in a highly NATURE AND CAUSES OF DIFFERENTIATION 421 determinate cleavage, essentially like that of the snail, the nuclei may be shifted about by pressure without altering the end-result. Neither can they lie in the form of the dividing mass as some authors have assumed ; for in Crampton's experiments the half or quarter blasto- mere does not retain the form of a half or quarter sphere, but rounds A B Fig. 191. — Double embryos of frog developed from eggs inverted when in the two-cell stage. [O. SCHULTZE.] A. Twins with heads turned in opposite directions. B. Twins united back to back. C. Twins united by their ventral sides. D. Double-headed tadpole. off to a spheroid like the ^gg. But if the limiting conditions lie neither in the nucleus nor in the form of the mass, we must seek them in the cytoplasm ; and if we find here factors by which the tendency of the part to develop into a whole may be, as it were, hemmed in, we shall reach a proximate explanation of the mosaic-Hke character of cleavage shown in the forms under consideration, and the mosaic 422 INHERITANCE AND DEVEIOPMENT theory of cytoplasmic localization will find a substantial if somewhat restricted basis. That we are here approaching the true explanation is indicated by certain very remarkable and interesting experiments on the frog's ^gg, which prove that each of the first two blastomeres may give rise either to a half-embryo or to a whole embryo of half size, according to cir- cumstances, and which indicate, furthermore, that these circumstances lie in a measure in the arrangement of the cytoplasmic materials. This most important result, which we owe especially to Morgan, ^ was reached in the following manner. Born had shown, in 1885, that if frogs' eggs be fastened in an abnormal position, — e.g. upside down, or on the side, — a rearrangement of the egg-material takes place, the heavier deutoplasm sinking toward the lower side, while the nucleus and protoplasm rise. A new axis is tints established in the egg, which has the same relation to the body-axes as in the ordinary develop- ment (though the pigment retains its original arrangement). This proves that in eggs of this character (telolecithal) the distribution of deutoplasm, or conversely of protoplasm, is one of the primary forma- tive conditions of the cytoplasm ; and the significant fact is that by artificially changing this distribution the axis of the ejnbryo is shifted. Oscar Schultze( '94) discovered that if the Qgg be turned upside down when in the two-cell stage, a whole embryo (or half of a double embryo) may arise from each blastomere instead of a half-embryo as in the normal development, and that the axes of these embryos show no constant relation to one another (Fig. 191). Morgan (95, 3) added the important discovery that either a half-embryo or a whole half-sized dwarf might be formed, according to the position of the blas- tomere. If, after destruction of one blastomere, the other be allowed to remain in its normal position, a half-embryo always results,^ pre- cisely as described by Roux. If, on the other hand, the blastomere be inverted, it may give rise either to a half-embryo ^ or to a whole dwarf.* Morgan therefore concluded that the production of whole embryos by the inverted blastomeres was, in part at least, due to a rearrangement or rotation of the egg-materials under the influence of gravity, the blastomere thus returning, as it were, to a state of equilib- rium like that of an entire ovum. This beautiful experiment gives most conclusive evidence that each of the two blastomeres contains all the materials, nuclear and cyto- plasmic, necessary for the formation of a whole body ; and that these materials may be used to build a whole body or half-body, according to the grouping that they assume. After the first cleavage takes 1 Ajiat. Anz., X. 19, 1895. ^ Three cases. * Eleven cases observed. * Nine cases observed. NATURE AND CAUSES OF DIFFERENTIATION 423 place, each blastomere is set, as it were, for a half-development, but not so firmly that a rearrangement is excluded. I have reached a nearly related result in the case of both Aniphi- oxus and the echinoderms. In AmpJiioxus the isolated blastomere usually segments like an entire ovum of diminished size. This is, however, not invariable, for a certain number of such bla.stomeres show a more or less marked tendency to divide as if still forming part of an entire embryo. The sea-urchin Toxopnenstcs reverses this rule, for the isolated blastomere of the two-cell stage usually shows a per- fectly typical half-cleavage, as described by Driesch, but in rare cases it may segment like an enth'e ovum of half-size (Fig. 1.83, />>)and give rise to an entire blastula. We may interpret this to mean that in Aniphioxus the differentiation of the cytoplasmic substance is at first very shght, or readily alterable, so that the isolated blastomere, as a rule, reverts at once to the condition of the entire ovum. In the sea- urchin, the initial differentiations are more extensive or more firmlv established, so that only exceptionally can they be altered. In the snail and ctenophore we have the opposite extreme to Avipliioxiis, the cytoplasmic conditions having been so firmly established that they can- not be readjusted, and the development must, from the outset, proceed within the limits thus set up. Through this conclusion we reconcile, as I believe, the theories of cytoplasmic localization and mosaic development with the hypothesis of cytoplasmic totipotence. Primarily the egg-cytoplasm is totipotent in the sense that its various regions stand in no fixed relation with the parts to which they respectively give rise, and the substance of each of the blastomeres into which it splits up contains all of the materials necessary to the formation of a complete body. Secondaril)-, how- ever, development may assume more or less of a mosaic-like character through differentiations of the cytoplasmic substance involving local chemical and physical changes, deposits of metaplasmic material, and doubtless many other unknown subtler processes. Both the ex- tent and the rate of such differentiations seem to vary in different cases ; and here probably lies the explanation of the fact that the isolated blastomeres of different eggs vary so widely in their mode of development. When the initial differentiation is of small extent or is of such a kind as to be readily modified, cleavage is imictcrmi- nate in character and may easily be remodelled (as in AmpJiioxus). When they are more extensive or more rigid, cleavage assumes a mosaic-like or determinate character,^ and qualitative division, in a certain sense, becomes a fact. Conklin's ('99) interesting observa- tions on the highly determinate cleavage of gasteropods {Crepitiit/a) 1 The convenient terms iuJeterminatc and determinate cleavage were suggested by Conklin ('98). 424 INHERITANCE AND DEVELOPMENT show that here the substance of the attraction-spheres is unequally distributed, in a quite definite way, among the cleavage-cells, each sphere of a daughter-cell being carried over bodily into one of the granddaughter-cells (Fig. 192). We have here a substantial basis for the conclusion that in cleavage of this type qualitative division of the cytoplasm may occur. It is important not to lose sight of the fact that development and differentiation do not in any proper sense first begin with the cleavage of the ovum, but long before this, during its ovarian history. ^ The primary differentiations thus established in the cytoplasm form the immediate conditions to which the later development must conform ; and the difference between Aiuphioxiis on the one hand, and the Fig. 192. — Two successive stages in the third cleavage of the egg of Crepidula, seen from the upper pole. [CONKLIN.] In both figures the old spheres (dotted) lie at the upper pole of the embryo, and at the third cleavage they pass into the four respective cells of the first quartet of micromeres. The centro- somes are seen in the new^ spheres. snail or ctenophore on the other, simply means, I think, that the initial differentiation is less extensive or less firmly established in the one than in the other. The origin of the cytoplasmic differentiations existing at the be- ginning of cleavage has already been considered (p. 386). If the conclusions there reached be placed beside the above, we reach the following conception. The primary determining cause of develop- ment lies in the nucleus, which operates by setting up a continuous series of specific metabolic changes in the cytoplasm. This process begins during ovarian growth, establishing the external form of the ^^g, its primary polarity, and the distribution of substances within it. The cytoplasmic differentiations thus set up form as it were a frame- 1 See Wilson ('96), Driesch ('98, i). THE NUCLEUS IN LATER DEVELOPMENT 425 work within which the subsequent operations take place in a course which is more or less firmly fixed in different cases. If the cyto- plasmic conditions be artificially altered by isolation or other dis- turbance of the blastomeres, a readjustment may take place and development may be correspondingly altered. Whether such a read- justment is possible depends on secondary factors — the extent of the primary differentiations, the physical consistency of the e^^g- substance, the susceptibility of the protoplasm to injury, and doubtless a multitude of others. The same doubtless applies to the later stages of development ; and we must here seek for some of the factors by which the power of regeneration in the adult is determined and lim- ited. It is, however, not improbable, as pointed out below, that in the later stages differentiation may occur in the nuclear as well as in the cytoplasmic substance. G. The Nucleus in Later Development The foregoing conception, as far as it goes, gives at least an in- telligible view of the more general features of early development and in a measure harmonizes the apparently conflicting results of experi- ment on various forms. But there are a very large number of facts relating especially to the later stages of differentiation, which it seems to leave unexplained, and which indicate that the nucleus as well as the cytoplasm may undergo progressive changes of its sub- stance. It has been assumed by most critics of the Roux-Weismann theory that all of the nuclei of the body contain the same idioplasm, and that each therefore, in Hertwig's words, contains the germ of the whole. It is, however, doubtful whether this assumption is well founded. The power of a single cell to produce the entire body is in general limited to the earliest stages of cleavage, rapidly diminishes, and as a rule soon disappears entirely. When once the germ-layers have been definitely separated, they lose entirely the power to regener- ate one another save in a few exceptional cases. In asexual repro- duction, in the regeneration of lost parts, in the formation of morbid growths, each tissue is in general able to reproduce only a tissue of its own or a nearly related kind. Transplanted or transposed groups of cells (grafts and the like) retain more or less completely their autonomy and vary only within certain well-defined limits, despite their change of environment. All of these statements are, it is true, subject to exception ; yet the facts afford an overwhelming demonstration that differentiated cells possess a specific character, that their power of development and adaptability to changed conditions becomes in a greater or less degree limited with the progress of development. As indicated above, this progressive specification of the tissue-cells 426 INHERITANCE AND DEVELOPMENT is no doubt due in part to differentiation of the cytoplasm. There is, however, reason to suspect that, beyond this, differentiation may sootier or later involve a specification of the nuclear substance. When we reflect on the general role of the nucleus in metabolism and its signifi- cance as the especial seat of the formative power, we may well hesi- tate to deny that this part of Roux's conception may be better founded than his critics have admitted. Nageli insisted that the idioplasm must undergo a progressive transformation during development, and many subsequent writers, including such acute thinkers as Boveri and Nussbaum, and many pathologists, have recognized the necessity for such an assumption. Boveri's remarkable observations on the nuclei of the primordial germ-cells in Ascaris demonstrate the truth of this view in a particular case ; for here all of the somatic nuclei lose a portion of their chromatin, and only the progenitors of the germ-neclei retain the entire ancestral heritage. Boveri himself has in a measure pointed out the significance of his discovery, insisting that the specific develop- ment of the tissue-cells is conditioned by specific changes in the chromatin that they receive,^ though he is careful not to commit him- self to any definite theory. It hardly seems possible to doubt that in Ascaris the limitation of the somatic cells in respect to the power of development arises through a loss of particular portions of the chromatin. One cannot avoid the thought that further and more specific limitations in the various forms of somatic cells may arise through an analogous process, and that we have here a key to the origin of nuclear specification zvithout recourse to the theory of qualita- tive division. We do not need to assume that the unused chromatin is cast out bodily ; for it m.ay degenerate and dissolve, or may be transformed into linin-substance or into nucleoli. This suggestion is made only as a tentative hypothesis, but the phenomena of mitosis seem well worthy of consideration from this point of view. Its appUcation to the facts of development becomes clearer when we consider the nature of the nuclear ''control" of the cell, i.e. the action of the nucleus upon the cytoplasm. Strasburger, following in a measure the lines laid down by Nageli, regards the action as essentially dynamic, i.e. as a propagation of molecular movements from nucleus to cytoplasm in a manner which might be compared to the transmission of a nervous impulse. When, however, we consider the role of the nucleus in synthetic metaboHsm, and the relation between this process and that of morphological synthesis, we must regard the question in another light ; and opinion has of late strongly tended to the conclusion that nuclear "control" can only be explained as the result of active exchanges of material between nucleus and cytoplasm. De Vries, followed by Hertwig, 1 ' 9^, P- 433- THE NUCLEUS IN LATER DEVELOPMENT 427 assumes a migration of pangens from nucleus to cytoplasm, the character of the cell being determined by the nature of the mi^a'at- ing pangens, and these being, as it were, selected by circumstances (position of the cell, etc.). But, as already pointed out, the pangen- hypothesis should be held quite distinct from the purely physiologi- cal aspect of the question, and may be temporarily set aside ; for specific nuclear substances may pass from the nucleus into the cytoplasm in an unorganized form. Sachs, followed by Loeb, has advanced the hypothesis that the development of particular organs is determined by specific " formative substances " which incite cor- responding forms of metabolic activity, growth, and differentiation. It is but a step from this to the very interesting suggestion of Driesch that the nucleus is a storehouse of ferments which pass out into the cytoplasm and there set up specific activities. Under the influence of these ferments the cytoplasmic organization is deter- mined at every step of the development, and new conditions are established for the ensuing change. This view is put forward only tentatively as a "fiction" or working hypothesis; but it is certainly full of suggestion. Could we establish the fact that the number of ferments or formative substances in the nucleus diminishes with the progress of differentiation, we should have a comparatively simple and intelligible explanation of the specification of nuclei and the limitation of development. The power of regeneration might then be conceived, somewhat as in the Roux-Weismann theorv, as due to a retention of idioplasm or germ-plasm — i.e. chromatin — in a less highly modified condition, and the differences between the various tissues in this regard, or between related organisms, would find a natural explanation. Development may thus be conceived as a progressive transforma- tion of the egg-substance primarily incited by the nucleus, first mani- festing itself by specific changes in the cytoplasm, but sooner or later involving in some measure the nuclear substance itself. This process, which one is tempted to compare to a complicated and progressive form of crystallization, begins with the youngest ovarian Q,^g and pro- ceeds continuously until the cycle of individual life has run its course. Cell-division is an accompaniment but not a direct cause of differen- tiation. The cell is no more than a particular area of the germinal substance comprising a certain quantity of cytoplasm and a mass of idioplasm in its nucleus. Its character is primarily a manifestation of the general formative energy acting at a particular point under given conditions. When once such a circumscribed area has been established, it may, however, emancipate itself in a greater or less degree from the remainder of the mass, and acquire a specific char- acter so fixed as to be incapable of further change save within the limits imposed by its acquired character. 428 INHERITANCE AND DEVELOPMENT H. The External Conditions of Development We have thus far considered only the internal conditions of devel- opment which are progressively created by the germ-cell itself. We must now briefly glance at the external conditions afforded by the environment of the embryo. That development is conditioned by the external environment is obvious. But we have only recently come to realize how intimate the rela- tion is; and it has been especially the service of Loeb, Herbst, and Driesch to show how essential a part is played by the environment in the development of specific organic forms. The hmits of this work will not admit of any adequate review of the vast array of known facts in this field, for which the reader is re- ferred to the works especially of Herbst. I shall only consider one or two cases which may serve to bring out the general principle that they involve. Every liv- ing organism at every stage oFlt? exist^ ence reacts to its environment by physio- logical and morphological changes. The developing embryo, like the adult, is a moving equilibrium — a product of the response of the inherited organization to the external stimuli working upon it. If these stimuli be altered, development is aftered.. This is beautifully shown by the experiments of Herbst and others on the larvae of sea-urchins. [Herbst.] development of sea-urchins. Pouchet A. Normal Piuteus {strongyiocen- ^"^ Chabry showcd that if the cmbryos trotiis). B. Larva {sphcerechinus) at of thcsc auimals bc made to,develop in the same stage as the foresroinsr, devel- ^^„ ,,r^4-^^ ^ <- • * t li. ^i ^^ . • ^ , . • • r 1. sea-water contammg; no hme-salts, the oped m sea-water contammg a slight ^ & ^ ^ oi.i±».o, i.±iv. excess of potassium chloride. larva fails to dcvclop not ouly its calca- reous skeleton, but also its ciHated arms, and a larva thus results that resembles in some particulars an entirely different specific form ; namely, the Toriiai'ia larva of Balanoglossus. This result is not due simply to the lack of necessary material ; for Herbst showed that the same result is attained if a shght excess of potassium chloride be added to sea-water containing the normal amount of Hme (Fig. 193). In the latter case the specific metabolism of the protoplasm is altered by a particular chemical stimulus, and a new form results. Fig. 193. — Normal and modified THE EXTERNAL CONDITIONS OE DEVELOPMENT 429 The changes thus caused by shght chemical alterations in the water may be still more profound. lierbst (92) observed, for example, that when the water contains a very small percentage of lithium chloride, the blastula of sea-urchins fails to invaginate to form a typical gastrula, but evaginatcs to form an hour-glass-shai)ed Fig. 194. — Regeneration in ccelenterates {A, /?. from f.OF.R ; C, D, from BlCKFORP). A. Polyp (Or/a»/'//«j), producing new tentacles from the aboral side of a lateral wound. B. Hydroid ( 7>//7«/a^-/'c?), generating a head at each end of a fragment of the stem susr • ' 1 in water. C. D. Similar generation of heads at both ends, of short pieces of the stem, in E larva, one half of which represents the archenteron. the other halt the ectoblast. Moreover, a much larger number of the blastula-cells undergo the differentiation into entoblast than in the nc^-mal de- velopment, the ectoblast sometimes becoming greatl\- reduced and occasionally disappearing altogether, so that the entire blastula is 430 INHERITANCE AND DEVEIOPMENT differentiated into cells having the histological character of the nor- mal entoblast ! One of the most fundamental of embryonic differen- tiations is thus shown to be intimately conditioned by the chemical environment. The observations of botanists on the production of roots and other structures as the result of local stimuli are famiUar to all. Loeb's interesting experiments on hydroids give a similar result ('91). It has long been known that Tubularia, like many other hydroids, has the power to regenerate its '' head " — i.e. hypostome, mouth, and ten- tacles — after decapitation. Loeb proved that in this case the power to form a new head is conditioned by the environment. For if a Tiibiilaria stem be cut off at both ends and inserted in the sand upside down, i.e. with the oral end buried, a new head is regen- erated at the free (formerly aboral) end. Moreover, if such a piece be suspended in the water by its middle point, a new head is produced at each e7id (Fig. 194); while if both ends be buried in the sand, neither end regenerates. This proves in the clearest manner that in this case the power to form a definite compUcated structure is called forth by the stimulus of the external environment. " These cases must suffice for our purpose. They prove incontesta- bly that normal development is in a greater or less degree the response of tJie developing organism to normal conditions ; and they show that we cannot hope to solve the problems of development without reckon- ing with these conditions. But neither can we regard specific forms of development as directly caused by the external conditions ; for the ^gg of a fish and that of a polyp develop, side by side, in the same drop of water, under identical conditions, each into its predestined form. Every step of development is a physiological reaction, involv- ing a long and complex chain of cause and effect between the stimu- lus and the response. The character of the response is determined, not by the stimulus, but by the inJierited orga7iization. While, there- fore, the study of the external conditions is essential to the analysis of embryological phenomena, it serves only to reveal the mode of action of the germ and gives but a dim insight into its ultimate nature. L Development, Inheritance, and Metabolism In bringing the foregoing discussion into more direct relation with the general theory of cell-action, we may recall that the cell-nucleus appears to us in two apparently different roles. On the one hand, it is a primary factor in morphological synthesis and hence in inheri- tance, on the other hand an organ of metabolism especially concerned with the constructive process. These two functions we may with DEVELOPMENT, INHERITANCE, AND METABOLISM 431 Claude Bernard regard as but different phases of one process. The building of a definite cell-product, such as a muscle-fibre, a nerve- process, a cilium, a pigment-granule, a zymogen-granulc, is in the last analysis the result of a specific form of metabolic activity, as we may conclude from the fact that such products have not only a definite physical and morphological character, but also a definite chemical character. In its physiological aspect, therefore, inheritance is the recurrence, in successive generations, of like forms of metabolism ; and this is effected through the transmission from generation to gen- eration of a specific substance or idioplasm which we have seen reason to identify with chromatin. The validity of this conception is not affected by the form in which we conceive the morphological nature of the idioplasm — whether as simply a mixture of chemical substances, as a microcosm of invisible germs or pangens, as assumed by De Vries, Weismann, and Hertwig, as a storehouse of specific fer- ments as Driesch suggests, or as a complex molecular substance grouped in micellae as in NageU's hypothesis. It is true, as Verworn insists, that the cytoplasm is essential to inheritance ; for without a specifi- cally organized cytoplasm the nucleus is unable to set up specific forms of synthesis. This objection, which has already been con- sidered from different points of view, by both De Vries and Driesch, disappears as soon as we regard the egg-cytoplasm as itself a product of the 7mclcar activity ; and it is just here that the general role of the nucleus in metabolism is of such vital importance to the theory of inheritance. If the nucleus be the formative centre of the cell, if nutritive substances be elaborated by or under the influence of the nucleus while they are built into the living fabric, then the specific character of the cytoplasm is determined by that of the nucleus, and the contradiction vanishes. In accepting this view we admit that the cytoplasm of the egg is, in a measure, the substratum of inheritance, but it is so only by virtue of its relation to the nucleus, which is, so to speak, the ultimate court of appeal. The nucleus cannot operate without a cytoplasmic field in which its peculiar powers may come into play; but this field is created and moulded by itself. J. Preformation and Epigenesis. The Unknown Factor in Development We have now arrived at the farthest outposts of cell-research, and here we find ourselves confronted with the same unsolved problems before which the investigators of evolution have made a halt. For we must now inquire what is the guiding principle of embryological development that correlates its complex phenomena and directs them 432 INHERITANCE AND DEVEIOPMENT to a definite end. However we conceive the special mechanism of development, we cannot escape the conclusion that the power behind it is involved in the structure of the germ-plasm inherited from fore- going generations. What is the nature of this structure and how has it been acquired .'' To the first of these questions we have as yet no certain answer. The second question is merely the general problem of evolution stated from the standpoint of the cell-theory. The first question raises once more the old puzzle of preformation or epigenesis. The pangen-hypothesis of De Vries and Weismann recognizes the fact that development is epigenetic in its external features ; but like Darwin's hypothesis of pangenesis, it is at bottom a theory of preformation, and Weismann expresses the conviction that an epigenetic development is an impossibility.^ He thus ex- plicitly adopts the view, long since suggested by Huxley, that "the process which in its superficial aspect is epigenesis appears in es- sence to be evolution in the modified sense adopted in Bonnet's later writings ; and development is merely the expansion of a potential organism or 'original preformation' according to fixed laws."^ Hert- wig ('92, 2), while accepting the pangen-hypothesis, endeavours to take a middle ground between preformation and epigenesis, by assuming that the pangens (idioblasts) represent only ccll-cJiaracters, the traits of the multicellular body arising epigenetically by permu- tations and combinations of these characters. This conception cer- tainly tends to simpHfy our ideas of development in its outward features, but it does not explain why cells of different characters should be combined in a definite manner, and hence does not reach the ultimate problem of inheritance. What hes beyond our reach at present, as Driesch has very ably urged, is to explain the orderly rhythm of development — the co- ordinating power that guides development to its predestined end. We are logically compelled to refer this power to the inherent organization of the germ, but we neither know nor can we even conceive what that organization is. The theory of Roux and Weis- mann demands for the orderly distribution of the elements of the germ-plasm a prearranged system of forces of absolutely incon- ceivable complexity. Hertwig's and De Yries's theory, though ap- parently simpler, makes no ' less a demand; for how are we to conceive the power which guides the countless hosts of migrating pangens throughout all the long and complex events of development.^ The same difficulty confronts us under anv theory we can frame. If with Herbert Spencer we assume the germ-plasm to be an aggrega- tion of like units, molecular or supra-molecular, endowed with prede- termined polarities which lead to their grouping in specific forms, ^ Germ-plasm, p. 14. 2 Evolution, Science, and Culture, p. 296. PREFORMATION AND EPIGENESIS 433 we but throw the problem one stage farther back, and, as Weismann himself has pomted out,i substitute for one difficulty another of exactly the same kind. The truth is that an explanation of development is at present beyond our reach. The controversy between preformation and epigenesis has now arrived at a stage where it has little meanin£( apart from the general problem of physical causality. What we know is that a specific kind of living substance, derived from the parent, tends to run through a specific cycle of changes during which It transforms itself into a body like that of which it formed \ part • and we are able to study with greater or less precision the mechanism' by which that transformation is effected and the conditions under which it takes place. But despite all our theories we no more know how the organization of the germ-cell involves the properties of the adult body than we know how the properties of hydrogen and oxygen involve those of water. So long as the chemist and physicist ""are unable to solve so simple a problem of physical causality as this, the embryologist may well be content to reserve his judgment on a problem a hundred-fold more complex. The second question, regarding the historical origin of the idio- plasm, brings us to the side of the evolutionists. The idioplasm of every species has been derived, as we must believe, by the modifica- tion of a preexisting idioplasm through variation, and the survival of the fittest. Whether these variations first arise in the idioplasm of the germ-cells, as Weismann maintains, or whether they may arise in the body-cells and then be reflected back upon the idioplasm, is a question to which the study of the cell has thus far given no certain answer. Whatever position we take on this question, the same difficulty is encountered; namely, the origin of that coordi- nated fitness, that power of active adjustment between internal and external relations, which, as so many eminent biological thinkers have insisted, overshadows every manifestation of life. The nature and origin of this power is the fundamental problem of biology. When, after removing the lens of the eye in the larval salamander, we see it restored in perfect and typical form by regeneration from the posterior layer of the iris,^ we behold an adaptive response to changed conditions of which the organism can have had no antece- dent experience either ontogenetic or phylogenetic, and one of so marvellous a character that we are made to realize, as by a flash of light, how far we still are from a solution of this problem. It may be true, as Schwann himself urged, that the adaptive power of living beings differs in degree only, not in kind, from that of unor- 1 Gerjuinal Selection, January, 1896, p. 284. 2 See Wolff, '95, and Muller,''96. 2F 434 INHERITANCE AND DEVEIOPMENT ganized bodies. It is true that we may trace in organic nature long and finely graduated series leading upward from the lower to the higher forms, and we must believe that the wonderful adaptive mani- festations of the more complex forms have been derived from simpler conditions through the progressive operation of natural causes. But when all these admissions are made, and when the conserving action of natural selection is in the fullest degree recognized, we can- not close our eyes to two facts : first, that we are utterly ignorant of the manner in which the idioplasm of the germ-cell can so respond to the influence of the environment as to call forth an adaptive variation ; and second, that the study of the cell has on the whole seemed to widen rather than to narrow the enormous gap that sepa- rates even the lowest forms of Hfe from, the inorganic world. r- \ am well aware that to many such a conclusion may appear reac- ! tionary or even to involve a renunciation of what has been regarded as the ultimate aim of biology. In reply to such a criticism I can ■ only express my conviction that the magnitude of the problem of development, whether ontogenetic or phylogenetic, has been under- estimated ; and that the progress of science is retarded rather than advanced by a premature attack upon its ultimate problems. Yet the splendid achievements of cell-research in the past twenty years stand as the promise of its possibilities for the future, and we need set no Umit to its advance. To Schleiden and Schwann the present standpoint of the cell-theory might well have seemed unattainable. We cannot foretell its future triumphs, nor can we doubt that the way has already been opened to better understanding of inheritance \ and development. LITERATURE. IX Barfurth, D. — Regeneration und Involution: Merkel u. Bonnet^ Ergeb., I.-VIIL 1891-99. Boveri, Th. — Ein geschlechtlich erzeugter Organismus ohne miitterliche Eigen- schaften: Sitz.-Ber. d. Ges.f. Morph. und Phys. in Milnchen, V. 1889. See also Arch. /. Entw. 1 895 . Brooks, W. K. — The Law of Heredity. Baltimore, 1883. Id. — The Foundations of Zoology. N^eiu York, 1899. Davenport, C. B. — Experimental Morphology : L, 11. New York, 1897, 1899. Driesch, H. — Analytische Theorie der organischen Entwicklung. Leipzig, 1894. Id.— Die Localisation morphogenetischer Vorgange : Arch. Entw., WW. i. 1899. Id. — Resultate und Probleme der Entwickelungs-physiologie der Tiere : Merkel u. Bonnet, Ergeb., VIII., 1898. (Full literature.) Herbst, C— iJber die Bedeutung der Reizphysiologie fiir die kausale Auffassung von Vorgangen in der tierischen Ontogenese : Biol. Centralb., XIV., XV. 1894-95. Eertwig, 0. — Altere und neuere Entwicklungs-theorien. Berlin. 1892. LITERATURE 435 Hertwig, 0. — Urmund unci Spina Bifida: Arch. niik. Anat., XXXIX. 1892. Id. — tJber den Werth der Ersten Furchungszellen fiir die Organbildung des Em- bryo: Arch. mik. Anat.yXhW. 1893. Id. — Zeit und Streitfragen der Biologic. I. Berlin, 1894. II. Jena, 1897. Id. — Die Zelle und die Gewebe, II. Jena, 1898. His, W. — Unsere Korperform und das physiologische Problem ihrer Entstehung. Leipzig, 1874. Loeb, J. — Untersuchungen zur physiologischen Morphologie : I. Heteromorphosis. Wurzburg, 1891. II. Organbildung und Wachsthum. Wiirsburg. 1892. Id. — Some Facts and Principles of Physiological Morphology: Wood's El oil BioL Lectures. 1893. Morgan, T. H. — Experimental Studies of the Regeneration of Phanaria iMaculata : Arch. Entiv., VII. 2. 3. 1898. Id. — The Development of the Frog's Egg. New York., 1897. Nageli, C. — Mechanisch-ph3-siologische Theorie der Abstammungslehre. AEiin- chen u. Leipzig, 1884. Roux, W. — tJber die Bedeutung der Kernteilungsfiguren. Leipzig, 1883. Id. — Uber das klinstliche Hervorbringen halber Embryonen durch Zerstorung einer der beiden ersten Furchungskugeln, etc. : VirchoTifs Archiv, 114. 1888. Id. — Fiir unsere Programme und seine Verwirklichung : Arch. Entiu., V. 2. 1897. (See also Gesammelte Abhandlungen liber Entwicklungsmechanik der Organ- ismen, 1895.) Sachs, J. — Stofif und Form der Pflanzenorgane : Ges. Abhandlitngen, II. 1893. Weismann, A. — Essays upon Heredity. First Series. OxJ'onl, 1891. Id. — Essays upon Heredity. Second Series. Oxford, 1892. Id. — Aussere Einfliisse als Entwicklungsreize. Jena, 1894. Id. — The Germ-plasm, A^ew York. 1893. Whitman, C. 0. — Evolution and Epigenesis : Wood's H oil Biol. Lectures. 1894. Wilson, Edm. B. — On Cleavage and Mosaic-work: Arch, fur Entiuicklungsni., III. I. 1896. See also Literature, VIII.. p. 394.) I GLOSSARY [Obsolete terms are enclosed in brackets. The name and date refer to the first use of the word; subsequent changes of meaning are indicated in the definition.] Achro'matin (see Chromatin), the non-staining substance of the nucleus, as opposed to chromatin ; comprising the ground-substance and the linin-network. (Flemming, 1879.) A'crosome ( aKpov. apex, o-co/xa, body), the apical body situated at the anterior end of head of spermatozoon. (Lenhossek, 1897.) [Akaryo'ta] (see Karyota), non-nucleated cells. (Flemming, 1882.) Ale'cithal (d-priv. ; AcKt^os, the yolk of an egg), having little or no yolk (applied to eggs). (Balfour, 1880.) Alloplasma'tic (aAAos, ditferent). Applied to active substances formed by dit^er- entiation from the protoplasm proper, e.g. the substance of cilia, of nerve-hbrillar, and of muscle-fibrillas. Alloplasmatic organs are opposed to •* protoplasmatic."' which arise only by division of preexisting bodies of the same kind. (A. Meyer. 1896.) Aniito'sis (see Mitosis), direct or amitotic nuclear division ; mass-division of the nuclear substance without the formation of chromosomes and amphiaster. (Flemming, 1882.) Am'phiaster {afxcj^L, on both sides ; dar-^p, a star), the achromatic figure formed in mitotic cell-division, consisting of two asters connected by a spindle. ( FoL. 1877.) Amphipy 'renin (see Pyrenin), the substance of the nuclear membrane. (Schwarz, 1887.) Amy'loplasts (a/xyXov, starch: TrAao-ros, TrXdaaeLv. form), the colourless starch- forming plastids of plant-cells. (Errera, 1882.) An'aphase (am, back or again), the later period of mitosis during the divergence of the daughter-chromosomes. (Strasburger. 18S4.) Aniso'tropy (see Isotropy), having a predetermined axis or axes (as applied to the egg). (Pfluger, 1883.) Antherozo'id. the same as Sperniatozoid. Anti'podal cone, the cone of astral rays opposite to the spindle-fibres. (\'an' Bexedex. 1883.) Archiam'phiaster (apxi- = first, + amphiaster), the amphiaster by which the tirst or second polar body is formed. (Whit.al-vx, 1878.) Ar'choplasma or Archoplasm (dpx(^^V' a ruler) (sometimes written <7rc////>/6po^, bearing), the ultimate supra-molecular vital units. Equivalent to the pangens of De Vries, the plasomes of Wiesner, etc. (Weismann, 1893.) Bi'oplasm (^to9, 7rXa?/xa). The active "living, forming germinal material,'' as opposed to ''formed material." Nearly equivalent to protoplasm in the wider sense. (Beale, 1870.) Bi'oplast. equivalent to cell. (Beale, 1870.) Bi'valent. applied to chromatin-rods representing two chromosomes joined end to end. (Hacker, 1892.) Ble'pharoplast (/:^A£c/)apt9, eye-lash or cilium). The centrosome-like bodies in plant-spermatids in connection with which the ciha of the spermatozoids are formed. (Webber. 1897.) Cell-plate (see Mid-body), the equatorial thickening of the spindle-fibres from which the partition-wall arises during the division of plant-cells. (Strasbur- ger, 1875.) Cell-sap. the more liquid ground-substance of the nucleus. [Kolliker, 1865; more precisely defined by R. Hertwig. 1876.] Central spindle, the primary spindle by which the centrosomes are connected, as opposed to the contractile mantle-fibres surrounding it. (Hermann, 1891.) Cen'triole. a term applied by Boveri to a minute body or bodies (" Central-korn ") within the centrosome. In some cases not to be distinguished from the centro- some. (Boveri, 1895.) Centrodes'mus (KeVx/oov, centre; Ses^o?, a band), the primary connection between the centrosomes, formed by a substance from which arises the central spindle. (Heidenhain, 1894.) Centrodeu'toplasm, the granular material of the testis-cells which may contribute to the formation of the Nebenkern or to that of the idiozome. (Erlanger, 1897-) Centrole'cithal (KcVrpov. centre : Acki^os, yolk), that type of ovum m which the deutoplasm is mainly accumulated in the centre. (Balfour, 1880.) Cen'troplasm (Kevrpov. centre; irXdo-fxa), the protoplasm forming the attraction- sphere or central region of the aster ; the substance of the centrosphere. (Er- langer, 1895.) GLOSSARY AT^Q Cen'trosome (Kevrpov, centre ; o-w/xa, body), a body found at the centre of the aster or attraction-sphere, regarded by some observers as the active centre of cell- division and in this sense as the dynamic centre of the cell. Under its influence arise the asters and spindle (amphiaster) of the mitotic fi-nire (Boveim 1888.) Cen'trosphere. used in this work as equivalent to the " astrosphere " of Stras- burger; the central mass of the aster from which the rays proceed and within which Hes the centrosome. The attraction-sphere. [Stkasburger, 1892: applied by him to the " astrosphere " and centrosome taken together.] ' Chloroplas'tids (xAwpoV green; TrAao-ro?, form), the green plastids or chlorophyll- bodies of plant and animal cells. (Schimper, 1883.) Chromatin (xpw/xa, colour), the deeply staining substance of the nuclear network and of the chromosomes, consisting of nuclein. (Fle.m.minc;. 1879.) Chro'matophore (xp^/xa, colour; -cf>6po^, bearing), a general term ai)plied to the coloured plastids of plant and animal cells, including chloroplastids and chromo- plastids. (SCHAARSCHMIDT, 1880; SCHMITZ. 1882.) Chro'matoplasm (xpoifxa. colour; irkacrixa, anything formed or moulded), the sub- stance of the chromoplastids and other plastids. (Strasrurger, 1882.) Chro'miole, the smallest chromatin-granules which by their aggregation form the larger chromomeres of which the chromosomes are composed^ (Eisex, 1899.) Chro'momere (xpoj/xa, colour; /xepos, a part), one of the chromatin-granules of which the chromosomes are made up. Identified by Weis.manx as the "id." See Chromiole. (Fol, 1891.) Chromoplas'tids (xpoj/xa, colour ; TrAao-ro's, form), the coloured plastids or pigment- bodies other than the chloroplasts, in plant-cells. (Schimper. 1883.) Chro'moplasts, net-knots or chromatin-nucleoli : also used by some authors as equivalent to Chromoplastid. (Eisen, 1899.) Chro'mosomes (xpa>/xa, colour: o-w/xa. body), the deeply staining bodies into which the chromatic nuclear network resolves itself during mitotic cell-division. ( Wal- deyer, 1888.) Cleavage-nucleus, the nucleus of the fertilized Qgg, resulting from the union of egg-nucleus and sperm-nucleus. (O. Hertwig, 1875.) Cortical zone, the outer zone of the centrosphere. (Van Benedex, 1887.) Cyano'philous (kwi/os, blue; ^lAetv, to love), having an especial affinity for lilue or green dyes. (Auerbach.) Cy'taster {KvToLy€tv, to love), having an especial affinity for red dyes. (AuERBACH.) ' * Ga'mete (ya/xexT/, wife ; ya/xerrys, husband), one of two conjugating cells. Usually applied to the unicellular forms. Gem'mule (see Pangen), one of the ultimate supra-molecular germs of the cell assumed by Darwin. (Darwix, 1868.) [Ge'noblasts] (yeVos, sex ; fSXaaro^, germ), a term applied by Minot to the mature germ-cells. The female genoblast (egg or '• thelyblast ") unites with the male (spermatozoon or "arsenoblast") to form an hermaphrodite or indifferent cell. (Minot, 1877.) Germinal spot, the nucleolus of the germinal vesicle. (Wagner, 1836.) Germinal vesicle, the nucleus of the egg before formation of the polar bodies. (PuRKiNjE. 1825.) Germ-plasm, the same as idioplasm. (Weis.mann.) Heterokine'sis (erepo';, different), qualitative nuclear division ; a hypothetical mode of mitosis assumed to separate chromatins of different quality ; opposed to homookinesis or equation-division. (Weismann. 1892.) Heterole'cithal (^'Tepo'. C, XIV. — Bohm, A.. "88. Uber Reifung und Befruchtung des Eies von Petromyzon Planeri : A. m. A., XXXII. —Id.. "91. Die Befruchtung des Forelleneies : Sit'z.-Iicr. d. Ges. f. Morph. u. Phys. Munchen, VII. — Boll. Fr.. '76. Das Princip des Wachsthums : Berlin. — Bonnet, C. 1762. Considerations sur les Corps organi.ses : Amsterdam. — Born. G., '85. Uber den Einfluss der Schwere auf das Froschei : ./. ///. .-/., XXIV. — Id., '94. Die Structur des Keimblaschens im Ovarialei von Triton taeniatus : A. ;;/. A., XLIII. — Bourne. G. C, '95. A Criticism of the Cell-theory ; being an Answer to Mr. Sedgwick's Article on the Inadequacy of the Cellular Theory of Development : <2. /•. XXXVIII.. i.— Boveri. Th.. Se. Uber die Bedeutung der Richtungskorper: Sitz.-Ber. Ges. Morpli. u. P/iys. M'lhic/ien, II. — Id., '87, 1. Zellenstudien, Heft I. ; /. Z., XXI. —Id.. '87. 2. C'ber die Befruch- tung der Eier von ^hYvrr/i- ///r<,7?^^v///r7 A/ ; Sdz.-Per. Ges. MorpJi. Phys. M'tinchen, III. — Id., "87, 2. t'ber den Anteil des Spermatozoon an der Teilung des Eies: Sitz.-Ber. Ges. Morph. Phys. Miinchen, III., 3. — Id., "87, 3. C'ber Dif^erenzierung der Zellkerne wahrend der Furchung des Eies von Ascaris meg.: -/. ./., 1887. — Id., '88, 1. Uber partielle Befruchtung : Sitz.-Ber. Ges. Morph. Phys. .Mian hen. I \'., 2. — Id.. '88. 2. Zellenstudien. II.: /. Z.. XXII.— Id.. "89. Ein geschlechtlich erzeugter Organismus ohne mlitterliche Eigenechaften : Sitz.-Ber. Ges. Morph. Phys. Munchen, V. Trans, in.-^w. Nat., March, "93. —Id.. "90. Zellen.studien. Heft III. : /. Z., XXIV. — Id.. '91. Befruchtung : Merkel und Bonnet's Ergebnisse, I. — Id., 452 GENERAL LITERATURE-LIST •95. 1. Uber die Befmchtungs- und Entwickelungsfaihigkeit kernloser Seeigel-Eier, etc. : A. Entwiu. II.. 3. — Id., "95, 2. tJber das Verhalten der Centrosomen bei der Befruchtung des Seeigeleies, nebst allgemeinen Bemerkungen liber Centrosomen und Verwandtes: Verh. d. Fhysikal.-nied. Gesellschaft zu IVih'zbiirg, N. F., XXIX., I. — Id.. "96. Zur Physiologie der Kern- und Zellteilung: Sitzb. Phys.- Med. Ges. W'urzburg. — Braem. F.. "93. Das Prinzip der organbildenden Keim- bezirke und die entwicklungsmechanischen Studien von H. Driesch : B. C XIII., 4.5. Brandt, H.. 11. L'ber Actinosphaerium Eichhornii : Dissertation. Halle, 1877. Brass. A., "83-4. Die Organisation der thierischen Zelle : Halle.— Brauer, A.. "92. Das Ei von Branchipus Grubii von der Bildung bis zur Ablage : Abh. preuss. Akad. IViss., 92. — Id., *93, 1. Zur Kenntniss der Reifung des par- thenogenetisch sich entwickelnden Eies von Artemia Salina : A. ni. A., XLIII. — Id., "93, 2. Zur Kenntniss der Spermatogenese von Ascaris megalocephala : A. in. A.., XLII. — Id., "94. Uber die Encystierung von Actinosph^erium Eich- hornii: Z. iv. Z., LVIII., 2. — Braus. "95. Tber Zellteilung und Wachstum des Tritoneies: /. Z.. XXIX. — Brooks. W. K.. "83. The Law of Heredity: Balti- more. Brown. H. H., "85. On Spermatogenesis in the Rat : Q. /., XXV. — Brown. Robert. "33. Observations on the Organs and Mode of Fecundation in Orchides and Asclepiadeae : Trans. Linn. Sac. 1833. Brucke, C, "61. Die Ele- mentarorganismen : Wiener Sitzbcr., XLIV., i86r . Brnnn. M. von, '89. Beitrage zur Kenntniss der Samenkorper und ihrer Entwickelung bei Vogeln und Sauge- thieren: A. in. A.. XXXIII. -De Bruyne, C, '95. La sphere attractive dans les cellules fixes du tissu conjonctif: Bull. Acad. Sc. de Belgique, XXX.— Burger. O., '91. Uber Attractionsspharen in den Zellkorpern einer Leibesflussigkeit : A. A.y VI. Id. ."92. Was sind die Attractionsspharen und ihre Centralkorper? A. A.j 1892. Butschli. O.. "73. Beitrage zur Kenntniss der freilebenden Nematoden : Nova acta ac ad. Car. Leopold, XXX VI. — Id., "75. Vorlaufige Mitteilungen uber Untersuchungen betrelTend die ersten Entwickelungsvorgange im befruchteten Ei von Nematoden und Schnecken : Z. w. Z., XXV. — Id., '76. Studien liber die ersten Entwickelungsvorgange der Eizelle, die Zellteilung und die Konjugation der Infusorien: Ab/i. des Senckenb. Naturforscher-Ges..X. — Id., '85. Organisations- verhaltnisse der Sog. Cilioflagellaten und der Noctiluca : J/. /.. X. — Id., '90. fber den Bau der Bakterien. etc.: Leipzig. — ldi.,'^\. Uber die sogenannten Centralkorper der Zellen und ihre Bedeutung : Verh. N'aturhist. Med. Ver. Heidel- berg 1891. — Id., '92. 1. t^ber die klinstliche Nachahmung der Karyokinetischen Fi'^-uren: Ibid., N. F., V. — Id., '92. 2. Untersuchungen liber mikroskopische Schaume und das Protoplasma (full review of literature on protoplasmic structure) : Leipzig {Engelniann). — 1A.. '94. Vorlaufige Berichte liber fortgesetzte Unter- suchungen an Gerinnungsschaumen, etc. : Verh. Natnrhist. Ver. Heidelberg^ V. — Id.. '96. Weitere Ausflihrungen liber den Bau der Cyanophyceen und Bakterien : Leipzig. —Id.; '98. Untersuchungen liber Strukturen : Leipzig {Engelniann). CALKINS. G. N.. '95, 1. Observations on the Yolk-nucleus in the Eggs of Lumbricus: Trans. N.Y. Acad. Sci.. June. 1895. — Id., '95, 2. The Spermato- genesis of Lumbricus : /. M., XL, 2. —Id., '97. Chromatin-reduction and Tetrad- formation in Pteridophytes : Bull. Torrey Bot. Club. XXI\\— Id.. '98, 1. The Phylogenetic Significance of Certain Protozoan Nuclei: Ann. N. V. Acad. Sci.. XL, 16. —Id.. "98, 2. Mitosis in Noctiluca : Ginn & Co., Boston, also/. J/., XV., 3. — Calberla. E., '78. Der Befruchtungsvorgang beim Ei von Petromyzon Planeri : Z.w.Z., XXX. — CampbeU. D. H., "88-9. On the Development of Pilularia globulifera : Ann. Bot.. II. — Carnoy, J. B., '84. La biologic cellulaire : Lierre. —■ Id., '85. La cytodierese des Arthropodes : La Cellule. I. — Id.. "86. La cytodie'- rese de l"oeuf: La Cellule, III.— Id., '86. La vesicule germinative et les globules GENERAL LITERATURE-LIST 453 polaires chez qiielqiies Nematodes: La CcUnlc, III. — Id.. "86. La segmentation de IVieuf chez les Nematodes: La Cellule, III., i. — Canioy and Le Brun. "97. 1. '98. "99. La ve'sicule genninative et les globules polaires clic/. 1l-s lialracicns : La Cellule. XII, XIV, XVI. — Id.. "97, 2. La fecondation chez TAscaris megalo- cephala: La Cellule. XIII. — Castle. W. E., "96. Tiic Early Kmbrvology of Ciona intestinalis : Bull. Mus. Coiitp. Zo'dl.y XXVIL, 7. — Chabiy. L.. "87. Conlrihu- tions a Tembryologie normale et pathologique des ascidies simples: Paris. 1887. — Child. C. M.. "97. The Maturation and Fertilization of the Egg of Arenicola : Trans. iV. V. Acad. Sci., X\'I. — Chittenden, R. H.. "94. Some Recent Chemico- physiological Discussions regarding the Cell: .liii.Nat.. X.W'IIl., P'eb., 1894. — Chun. C, "90. Uber die Bedeutung der direkten Zelltheilung : .Sitzb. .Silir. Phvsik.- Okoii. Ges. I\dnigsberg, 1890. — Id., "92. 1. Die Di.ssogonie der Kippenquallen : Festschr. f. Leuckart, Leipsio;^ 1892. — Id., "92. 2. (In Rou.\, "92, p. 55) : \'erli. d. Anat. Ges..V\.., 1892. — Clapp. C. M.. "91. Some Points in the Development of the Toad-Fish :/. J/., V. — Clarke. J. Jack.son. "95. Observations on various Sporozoa: Q.J., XXXVIL, 3. — Coe, W. R.. "99. The Maturation and Fertiliza- tion of the Egg of Cerebratulus : Zo'dl. Jalirb., XII. — Cohn. Ferd.. "51. Xachtrage zur Naturgeschichte des Protococcus pluvialis : Nova Acta. XXII. — Conklin, E. G.. '94. The Fertilization of the Ovum: Biol. Led.. Marine Biol. Lab.. Wood's IIolL Boston., 1894. — Id., "96. Cell-size and Body-size: Kept, of Am. Morp/i. Soc. Science, III., Jan. 10, 1896. — Id., '97, 1. Nuclei and Cytoplasm in the Intestinal Cells of Land Isopods : An/. Nat.. Jan. — Id., '97, 2. The Embryology of Crepidula : /. J/., XIII., I. — Id., "98. Cleavage and Differentiation: Wood's' LI oil Biol. Lec- tures. — Id.. '99. Protoplasmic Movement as a Factor in Differentiation: Wood's LL oil Biol. Lectures. — Crampton, H. E., '94. Reversal of Cleavage in a Sinistral Gasteropod: Ann. N. V. Acad. Sci., March. 1894. —Id., "97. The Ascidian Halt- Embryo: Lbid., June 19. — Id.. '99. The Ovarian History of the Egg of .Molgula: /. J/., XV., Suppl. — Crampton and Wilson, "96. E.xperimental Studies on Gasteropod Development (H. E. Crampton). Appendi.v on Cleavage and Mosaic- Work (E. B. Wilson) : A. Entwni.. III., i. — Czermak. N.. "99. C'ber die Desin- tegration und die Reintegration des Kernkorperchens, etc.: A. A.. X\'.. 22. DARWIN, F., '77. On the Protrusion of Protoplasmic Filaments, e/c. : Q.J. XVII. — Davis, B. M., '99. The Spore-mother-cell of Anthoceros : Bot. Gas., XXVIIL, 2. — Debski, B., '97. Beobachtungen liber Kerntheilung bei Chara : /. w. B., XXX.— Id., "98. Weitere Beobachtungen an Chara: Ibid., XXXII.. 4. — Delage, Yves, "95. La Structure du Protoplasma et les Theories sur rh<5reditc< et les grands Problemes de la Biologic Generale : Paris, 1895. — Id.. "98. Embry- ons sans noyau maternel : C. P., CXXVIL, 15.— Id.. "99. La fecondation m<^ro- gonique et ses resultats : C. A'., Oct. 23. — Demoor, J., "95. Contribution h Tetude de la physiologic de la cellule (inde'pendance fonctionelle du protoplasme et du noyau) : A. B., XIIL — Dendy. A., "88. Studies on the Comparative Anatomy of Sponges: Q.J., Dec, 1888. — Dixon, H. H., "94. Fertilization of P/nus : Ann. Bot., VIII. — Id.. '96. On the Chromosomes of Lilium longlitli)rum : Proc. P. Ir. Ac, III. — Doflein, F. J., "97, 1. Die Eibildung bei Tubularia : Z. w. Z., LXIL, I. — Id., '97, 2. Karyokinesis des Spermakerus : ./. ///. ./.. L. 2. — Dogiel. A. S.. '90. Zur Frage liber' das Epithel der Harnblase : A. ni. A., XXW. — Driesch. H.. "92, 1. Entwickelungsmechanisches : ./../.. VII., 18. — Id. Enlwicklungs- mechanische Studien, L.^IL, 1892. Z. ic. Z.. LIIL: III. -VI.. 1893, Ibid., LV. ; VII.-X., 1893 : JLdt. Zool. St. Neapel, XL, 2. —Id.. "94. Analytische Theorie der organischen Entwicklung : Leipcii^. —Id., '95. 1. \'on der Entwickelung einzelner Ascidienblastomeren : A. Entwm.. L, 3- — ^^i.. ^S- 2. Zur Analysis der Potenzen embryonaler Organzellen : Ibid.y II. — Id., "98,1. I'bcr den Organisation des 454 GENERAL LITERATURE-LIST Eies : Etitwm., IV. — Id.. ""SS, 2. Von der Beendigung morphogener Elemen- tarprocesse: Arch. Entwfn.. VI. —- Id., '98. 3. Ueber rein-miitterliche Charaktere an Bastardlarven von Echiniden : Ibid., \'II., i. — Id., "99. Die Localisation mor- phogenetischer Vorgange : Ibid., VIII., i. — Driesch and Morgan, "95, 2. Zur Analysis der ersten Entwickelungsstadien des Ctenophoreneies : Ibid., II., 2. — Druner, L.. "94. Zur Morphologie der Centralspindel : /. Z., XXVIII. (XXI.). — Id., "95. Studien iiber den Mechanismus der Zelltheilung : Ibid., XXIX., 2. — Dii- sing, C, "84. Die Regulierung des Geschlechtsverhaltnisses : Jena, 1884. VON EBNER, V., '71. Untersuchungen iiber den Ban der Samencanailchen und die Entwicklung der Spermatozoiden bei den Saugethieren und beim Menschen : Inst. PJiys. It. Hist. Graz., 187 1 {Leipzig). — Id., '88. Zur Spermatogenese bei den Saugethieren: A. ni. A., XXXI. — Ehrlich. P., '79. Uber die specifischen Granulationen des Blutes : A. A. P. {FJiys.), 1879, P- 573- — Eisen, G., "97. Plasmocytes : Froc. CaL Acad. Sci., I., i. — Id., "99. The Chromoplasts and the Chromioles : B. C. XIX.. 4. — Eismond, J.. '95. Einige Beitrage zur Kenntniss der Attraktionsspharen und der Centrosomen : A.A.,X. — Endres and Walter, '95. Anstichversuche an Eiern von Rana fusca: A. Entwni., II., i. — Engelniann, T. "W., "80. Zur Anatomic und Physiologic der Flimmerzellen : Arch. ges. Phys., XXIII. — von Erlanger, R., '96. 1. — Die neuesten Ansichten iiber die Zelltheilung und ihre iMcchanik : ZooL Centralb., III.. 2. — Id., '96, 2. Zur Befruchtung des Ascariseies nebst Bemerkungen iiber die Struktur des Protoplasmas und des Centro- somas: Z. A.., XIX. — Id.. "96. 3. Neuere Ansichten iiber die Struktur des Proto- plasmas : ZooL Centralb., IIL, 8,9. — Id., '96. 4. Zur Kenntniss des feineren Baues des Regenwurmhodens. etc. : A. in. A.. XLVII. — Id.. "96. 5. Die \'ersoni- sche Zelle : ZooL Centralb., III.. 3. — Id., "96. 6. Die Entwicklung der mannlichen Geschechtszellen : Ibid.. III.. 12. — Id., "97, 1. Uber Spindelreste und den echten Nebenkern, etc.: ZooL Centralb., IV., i. — Id., '97, 2. Uber die sogenannte Sphare in den mannlichen Geschlechtszellen : Ibid.,\\'.,^. — Id.. '97. 3. Uber die Chromatinreduktion in der Entwicklung der mannlichen Geschlechtszellen : Ibid., IV., 8. — Id.. '97. 4. Beitrage zur Kenntniss des Protoplasmas, etc. A. ni. A.., XLIX. — Id., '97,5. Uber die Spindelbildung in den Zellen der Cephalopoden Keimscheibe : B. C, XVII., 20. — Id., '98. Uber die Befruchtung. etc.. des Seeigeleies : B. C XVIII.. i. — Errera. '86. Eine fundamentale Gleichgewichtsbe- dingung organischen Zellen: Ber. Dentsch. Bot. Ges., 1886. — Id.. '87. Zellformen und Seifenblasen : Tagebl. der 60 Versaminlnng deutscher Naturforscher imd Aerzte Z2i Wiesbaden., 1887. FAIRCHILD. D. G.. '97. t'ber Kerntheilung und Befruchtung bei Basidio- bolus: JaJirb. luiss. Bot., XXX. — Farmer, J, B., '93. On nuclear division of the pollen-mother-cell of Lilium Martagon : Ann. Bot. VII.. 27. — Id., '94. Studies in Hepaticae: Ibid.., VIII., 29. — Id., '95, 1. Uber Kernteilung in Lilium-Antheren, besonders in Bezug auf die Centrosomenfrage : Flora. 1895, p. 57. — Id., 95, 2. On Spore-formation and Nuclear Division in the Hepaticse : Ann. Bot., IX. — Farmer and Moore, '95. On the essential similarities existing between the heterotype nuclear divisions in animals and plants : A. A., XL, 3. — Farmer and "Williams, '96. On Fertilization, etc.. in Fucus : Ann. Bot., X. — Fick. R.. '93. Uber die Reifung und Befruchtung des Axolotleies : Z. lu. Z., LVI.. 4. — Id.. '97. Bemer- kungen zu M. Heidenhain's Spannungsgesetz : Arch. Anat. n. Phys. (Anat.). — Fiedler, C. "91. Entwickelungsmechanische Studien an Echinodermeneiern : Festschr. N^'ds^eli n. Kolliker, Zurich. 1891. — Field. G. "W.. '95. On the Mor- phology and Physiology of the Echinoderm Spermatozoon: /. J/.. XI. — Fischer, A., '94, 1. Zur Kritik der Fixierungsmethoden der Granula : A. A., IX., 22. — GENERAL LITERATURE-LIST 455 Id., 94, 2.— t'ber die Geisseln einiger Flagellaten : /. w. //. XW'II. — Id., '95. Neue Beitrage zur Kritik der Fixiemngsmethoden : - /. ^/., X. — Id.. '9?! Untersuchungen liber den Bau der Cyanopliyceen und Baktcrien : Jena, Fischer. — Id., "99. Fixierung, Farbung und Bau de.s Protopia.sma.s : //^/^/. — Flemming. 'W., '75. Studien in der Entwicklungsge.schichle der Xajaden : Sitzb. d. k. k. Akad. Wiss. Wien, LXXI., 3. — Id., '79, 1. Beitrage zur Kenntni.s.s der Zelle und Hire Lebenserscheinungen, I. : A. m. A., XVI. —Id., '79, 2. C'ber das Vcrlialten des Kerns bei der Zelltheilung, etc.: Virchow's Arch., LXXVII.— Id.. 80. lieitrage zur Kenntniss der Zelle und ihrer Lebenserscheinungen. II. : ./. ///. A., XIX. —Id.. '81. Beitrage zur Kenntniss der Zelle und ihrer Lebenserscheinungen. 111. : IhuL, XX. — Id., '82. Zellsubstanz, Kern und Zellteilung : Lcipzii^, 1882. — Id.. "87. Neue Beitrage zur Kenntniss der Zelle: .;. ///. ^., XXIX. — Id., '88. Wciicre Beobachtungen uber die Entwickelung der Sperniatosomen Ijei Salamandra maculosa • Ibid., XXXI. — Id., '91-97. Zelle, I.-VI. : Er^i^cbn. Anat. u. Entioicklum^s^esch. {Merkel and Bonnet^, 1891-97. — Id., -91. 1. Attraktionsspharen u. Centralklirper in Gevvebs- u. Wanderzellen : A. A. — Id., '91, 2. Neue Beitrage zur Kenntniss der Zelle, II. Teil: A. m. A., XXXVII.— Id., '95, 1. C'ber die Struktur der Spinai- ganglienzellen : VerhandL der anat. Gesellschaft in Basel, i-j Ajjril, 1895, P- '9- — Id., '95, 2. Zur Mechanik der Zelltheilung: A. in. A., XLVI. — Id., *97. 2. Ucbcr den Bau der Bindegewebszellen, etc.: Zeit. iSV^/.. XXXIV. —Floderus, M.. "96. Uber die Bildung der Follikelhlillen bei den Ascidien : Z. lu. Z., LXI.. 2. — Fol. H.. '73. Die erste Entwickelung des Geryonideies : /. Z., VII. —Id., 75. Etudes sur le de'veloppement des Mollusques. — Id., '77. Sur le commencement de Thcnogenie chez divers animaux : Arch. Sci. Nat. et Phys. Genh>e, LVIII. See also Arch. 'zod/. Exp., VI. — Id., '79. Recherches sur'la fecondation et la commencement de rhV/. zur patlK^loi:;. Anat. 11. z. Alli^. i\ithol., XW ., 2, Jena, Fischer, 1893. — Gallardo. Angel, "96. La Carioquinesis : ./////. Soc. Cientif. Argentina, XLII. — Id.. '97. Significado Dinamico de las Figuras Cariocineticas : Ibid., XLIV. — Gardiner. E. G.. "98. The (irowth of the Ovum, etc., in Polychoerus : /. M., XV., r. — Gardiner. W.. "83. Continuity of Proto- plasm in Vegetable Cells: Phil. Trans., CLX.XIW - Garnault. 88. Q%. Sur les phenomenes de la fecondation chez Helix aspera et .Arion empiricorum : Zool. Anz., XL, XII. — Geddes and Thomp.5on. The Evolution of Sex: London, 1899. — — Gegenbaur. C, "54. Beitriige zur naheren Kenntniss der Sch\vimmpolyj)en : Z. w. Z., V. — Van Gehucliten. A., "90. Recherches histologiques sur Tapparei! digestif de la larva de la Ptychoptera contaminata : La Cellule, VL — Giard, A.. "77. 456 GENERAL LITERATURE-LIST Sur la signification morphologique des globules polaires : Revue scientifique, XX. — Id., '90. Sur les globules polaires et les homologues de ces elements chez les infu- soires cilies : BuUeiin scieiitifique de la France et de la Belgiqiie, XXII. — God- lewsky, E., *97. 1, Uber niehrfache bipolar Mitose bei der Spermatogenese von Helix: Ans. Akad. W^iss. Krakaii. — Id., "97, 2. Weitere Untersuchungen liber die Umwandlung der Spermatiden. etc. : Anz. Akad. IViss. K7-akau.. Nov., '97. — Goroschanktin, J., "83. Zur Kenntniss der Corpuscula bei den Gymnosper- men: Bot. Zeit.. LXI. — Graf. A., "97. The Individuality of the Cell: N. Y. State Hosp. Bidl.^ April. — Grdgoire. V.. "99. Les cineses polliniques dans les Liliacees : Bot. Ccntb..XX.. i; La Cellule, XVI., 2. — Griffin, B. B., ^96. The History of the Achromatic Structures in the Maturation and Fertilization of TJialassoiia : Traits. N. Y. Acad. Sci. — Id.. "99. Studies on the Maturation. Fertilization, and Cleavajre of Thalassema and Zirphaea : /. M.. XV. — Gierke. H.. '85. Farberei zu mikro- skopischenZwecken : Zeit. IViss. Mik.. II. — Grobben. C, '78. Beitrage zur Kennt- niss der mannlichen Geschlechtsorgane der Dekapoden : Arb. Zool. Inst. Wien. I. — Gruber. A.. '84. Uber Kern und Kerntheilung bei den Protozoen : Z. iv. Z., XL. — Id.. '85. Uber klinstliche Teilung bei Infusorien : B. C, I\\, 23; V., 5. — Id., '86. Beitrage zur Kenntniss der Physiologie und Biologic der Protozoen : Ber. Naturf. Ges. Freiburg, I. — Id., '93. Mikroscopische Vivisektion : Ber. d. N'aturf. Ges. su Freiburg, WW., i. — Id., "97, Weitere Beobachtungen an vielkernigen Infusorien : Ber. Naturf. Ges. Freiburg, III. — Guignard, L., '89. Developpement et constitution des Antherozoides : Rev. gen. Bot., I. — Id., "91, 1. Nouvelles etudes sur la fecondation : Ann. d. Sciences Nat. Bot., XIV. — Id.. "91, 2. Sur Texistence des " spheres attractives "" dans les cellules vegetales : C.R., 9 Mars. — Id., '98, 1. Les centres cinetiques chez les vegetaux : Ann. Sci. N'at. Bot., (VIII.) V. ; also, Bot. Gaz., XXV. — Id., '98, 2. Le developpement du pollen et la reduction chromatique dans le Nais niajor : Arch. Anat. Mik., II., 4. — Id., '99. Sur les antherozoides et la double copulation sexuelle chez les vegetaux angiospermes : C. R., CXXV^III., 14. HABERLANDT, G.. '87. Uber die Beziehungen zwischen Funktion und Lage des Zellkerns : Fischer, 1887. — Hackel. E.. "66. Generelle Morphologic. — Id., '91. Anthropogenic, 4th ed., Leipzig, 1891. — Hacker. "V., "92. 1. Die Fur- chung des Eies von ^quorea Forskalea : A. ni. A., XL. — Id., "92, 2. Die Eibil- dung bei Cyclops und Canthocamptus : Zool. Jahrb., V. — Id., '92. 3. Die heterotypische Kerntheilung im Cyclus der generativen Zellen : Ber. naturf. Ges. Freibitrg,V\. — Id., '93. Das Keimblaschen, seine Elemente und Lageverander- ungen : A. in. A., XLI. — Id.. "94. Uber den heutigen Stand der Centrosomen- frage : Verhandl. d. deutschen Zool. Ges.. 1894. p. 11. — Id.. "95. 1. Die Vorstadien der Eireifung : A. in. A., XLV., 2. — Id., "95. 2. Zur frage nach dem Vorkommen der Schein-Reduktion bei den Pflanzen : Ibid., XL\'I. Also Ann. Bot., IX. — Id.. '95. 3. Uber die Selbstandigkeit der vaterlichen und mlitterlichen Kernsbe- standtheile wahrend der Embryonalentwicklung von Cyclops: A. in. A.. XLVI., 4. — Id., '97, 1. Die Keimbahn\'on Cyclops : A. in. A., XLIX. — Id., '97, 2. Uber weitere Ubereinstimmungen zwischen den Fortpflanzungsvorgangen der Thiere und Pflanzen : B. C XVII. — Id., "98. t^ber vorbereitende Theilungsvorgange bei Thieren und Pflanzen: Verh. d. Zool. Ges., VIII. — Id., '99. Praxis und Theorie der Zellen und Befruchtungslehre : fena, Fischer. — Hallez. P.. "86. Sur la loi de I'orientation de I'embryon chez les insectes : C. R.. 103, 1886. — Hallibur- ton. W. D.. "91. A Text-book of Chemical Physiology and Pathology: London. — Id., "93. The Chemical Physiology of the Cell: {Gouldstonian Lectures) Brit. Med. fount. — Hammar, J. A., '96. Uber einen primaren Zusammenhang zwi- schen den Furchungszellen des Seeigeleies : A. in. A., XLVIL, i. — Id., '97. Uber eine allgemein vorkommende primare Protoplasmaverbindung zwischen den Bias- GENERAL LITER A TV RE- LI ST 457 tomeren: A. m. A., XLIX. — Hammarsten. O.. "94. Zur Kcnntniss der Nucleo- proteiden : Zcif. P/iys. Clicm., XIX. —Id.. "95. Lehrhuch der phy.siolo^i.schen Chemie, 36 Au.sgabe : Wiesbaden^ 1895. — Hansemann. D.. "91. Karyokinese und Cellularpathologie : Bcrl. Klin. ll'oc/ioiscJnift^ No. 42. — Id.. "93. Spe/iricitat. Altmismu.s und die Anaplasie der Zellen : Berlin, 1893. — Hansteiu. J., "80. Das Protoplasma als Trager der pflanzlichen und thierischen Lcljensverrichtungen. Hcidclbeyg. — Harper, R. A.. "96. Uber das \'erhalten der Kerne W\ der Fruchtentwickelung einiger A.scomyceten : Jahrb. luiss. Hot.. XXIX. — Id.. 97. Kernteiluug und freie Zellbildung im Ascu.s : Ibid.. XXX. — Hardy, W. B.. "99. On the Structure of Cell-protoplasm: Joitr. /'//vs.. XX I \'.. 2. Harvey. Wm.. 1651. Exercitationes de Generatione Animalium : Loudon. Trans, in Sydcn/iani Sac. X.. 1847. — Hartog, M. M., "91. Some Problems of Reproduc- tion, etc. : (2-/-' XXXIII. — Id., "96. The Cytology of Saprolegnia : ./////. /lot.. IX. — Id.. '98. Nuclear Reduction and the Function of Chromatin: Xat.Sci., XII. — Hatschek. B.. *87. Uber die Bedeutung der geschlechtlichen Forti)rian- zung: Prager Med. Woc/iensc/irift. XL\T. — Id.. '88. Lehrbuch der Zooloj^ie. — Heath. H.. "99. The Development of Ischnochiton : y. Z., lAX . — Id.. "95. Tber Kerntheilung, Eireifung und Befruchtung bei (9/'///"jv//7^t7/rf puerilis: Ibid., LX. — Id.. '96. Ktrn- structuren und Zellmembranen in den Spinndriisen der Rau]jen : .1. ///. .1.. XlA'Il. — Id.. '97. t'ber den Bau der Kerne in den Spinndrii.sen der Raupen : Ibid., XLIX. — Kossel, A.. '91. t^ber die chemische Zusammensetzung der /elie : .Irch. .Inat. u. Phys. — Id.. "93. C'ber die Nucleinsaure : Ibid., 1893. — ^^ ■ ^^ ''^tT f^'»* basischen Stofife des Zellkernes : Zeit. Phys. Cheiii., XXII. — von Kostanecki. K . '91. f^ber Centralspindelkorperchen bei karyokinetischer Zellteilung : .\nat. Hefte, 1892, dat. 91. — Id., "96. (ber die Gestalt der Centro.somen im befmchtcten See- igelei : Ibid.. \TI., 2. — Id.. "97. 1. ('ber die Bedeutung der Polstrahlung, etc.: A. ni. A.. LXIX. — Id., '98. Die Befruchtung des Eies von .llyc/'sti'/na : Ibid., LI. — Kostanecki and Siedlecki. "96. t'bcr das \'erhalton der Centrosomen zum Protoplasma: Ibid., XLIX. — Kostanecki and "Wierzejski. "96. I'ber das Verhalten der sos^enannten achromatischen Substanzen im befruchleten Ei : Ibid.. XLII., 2. — Kiihne, "W.. "64. Lhitersuchungen liber das Protoplasma und die Con- 460 GENERAL LITERATURE-LIST tractilitat. — Kupffer, C, '75. tJber Differenzierimg des Protoplasma an den Zellen thieiischer Gewebe : Schr. nafiir. Ver. Schles.-Hoht., I., 3. — Id., "90. Die Entwicklung von Petromyzon Planeri : A. in. A., XXXV. — Id., '96. Uber Ener- giden und paraplastische Bildungen : Rcktoratrede., Miinchoi, 1896. LAMEERE, A., "90. Recherches sur la reduction karyogamique : Briixelks. — Lauterborn. R., '93. Uber Bau und Kerntheilung der Diatomeen : Vcrh. d. N'aturh. Med. Ve)-. in Heidelberg. 1893. —Id., '95. Protozoenstudien, Kern- und Zellteilung von Ceratium hirundinella O. F. M. : Z. w. Z., XLIX. — Id., '96. — La "Valette St. George, '65. Uber die Genese der Samenkcirper : A. ni. A.. I. — Id., "67. Uber die Genese der Samenkorper, II. (Terminology): Ibid., III. — Id., '76. Die Spermatogenese bei den Amphibien : Ibid.., XII. — Id., '78. Die Spermatogenese bei den Saugethieren und dem Menschen : Ibid.., XV. — Id., ■85-"87. Spermatologische Beitrage. I.-V. : Ibid., XXV., XXVII., XXVIII., and XXX. — Lankester, E. Ray, "77. Notes on Embryology and Classification: London. — Lavdovsky. M., "94. Von der Entstehung der cbromatischen und achromatischen Substanzen in den tierischen und pflanzlichen Zellen : Merkel und Bonnefs Anat. Hefte, IV., 13. — Lawson, A. A., "98. Some Observations on the Development of the Karyokinetic Spindle, etc. : Proc. Cal. Acad. Sci., I., 5.— Lazarus, A., '98. Die An^emie: IVien. — Lee, A. Bolles, '96. Sur le Nebenkern, etc., chez Helix: La Cellule, XI. — Id., '97. Les cineses spermatogenetiques chez Helix: Ibid., XIII. —von Lenhossek, M., '95. Centrosom und Sphare in den Spinalganglien des Frosches: A. ni. A., XLVI.— Id., '98, 1. Uber Flimmerzellen : Ver h. An. Ges., XII. — Id., '98, 2. Untersuchungen liber Spermatogenesis : A. m. A.,, LI. — Id., '99. Das Mikrocentrum der glatten Muskelzellen : A. A., XVI., 13, 14. — Leydig. Fr., '54. Lehrbuch der Histologic des Menschen und der Thiere : Frank- fitrt.—16...'Q5. Zelle und Gewebe, i?^;/;/. —Id-, '89. Beitrage zur Kenntniss des thierischen Eies im unbefruchteten Zustande : SpengeVs Jahrb. Anat. Ont., III. — Lilienfeld, L., '92, '93. Uber die Verwandtschaft der Zellelemente zu gewissen Farbstoffen: Verh. Phys. Ges., Berlin, 1892-93. — Id., '93. Uber die Wahlver- wandtschaft der Zellelemente zu Farbstoffen: A. A. P., 1893.— Lillie, F. R., '95. The Embryology of the Unionidae : /. M., X.— Id., '96. On the Smallest Parts of Stentor capable of Regeneration: /. M., XII., i.— Id., "97. On the Origin of the Centres of the First Cleavage-spindle in Unio : Science, V. — Id., '98. Centro- some and Sphere in the Egg of Unio : Zool. Bull., I., 6. — Id., '99. Adaptation in Cleavage : Wood^s Holl Biol. Led. — List, Th., '96. Beitrage zur Chemie der Zelle und Gewebe, I. : Mitth. Zool. St. Neap., XII., 3. — Loeb, J., '91-92. Untersuch- ungen zur physiologischen Morphologic. I. Heteromorphosis : Wiirzburg, 1891. II. Organbildung und Wachsthum : Ibid., 1892. — Id., '92. Experiments on Cleav- age : /. il/., VII. — Id., '93. Some Facts and Principles of Physiological Mor- phology: Wood's Holl Biol. Lectures, 1893. — Id., '94. Uber die Grenzen der Theilbarkeit der Eisubstanz : A. ges. P., LIX., 6, 7.— Id., '95. Uber Kernthei- lung ohne Zelltheilung : Arch. Entwni., II. — Id., '99, 1. Warum ist die Regenera- tion kernloser Protoplasmastlicken unmoglich, etc.: Ibid., VIII.. 4. — Id., '99, 2. On the Nature of the Process of Fertilization and the Artificial Production of Nor- mal Larv£e, etc. : Am. Journ. Phys., III., 3. — Lowit, M., '91. Uber amitotische Kerntheilung: B. CXI. — Lukjanow, '91. Grundzuge einer allgemeinen Patho- logic der Zelle: Leipzig.— liW&ti^ and Galeotti, "93. Cytologische Studien uber pathologische menschliche Gewebe : Beitr. Path. Anat., XIV. MACALLUM, A. B., '91. Contribution to the Morphology and Physiology of the Cell: Trans. Canad. Inst., I., 2. — McClung, C. E., '99. A Peculiar Nuclear Element in the Male Reproductive Cells of Insects : ZooL Bull., II., 4- — MacFar- GENERAL TITERATURE-LIST 46 I land, F. M., '97. Gellulare Studien an Molluskeneiern : Zo'ol. Jahrh. Anat., X. — McGregor, J. H., '99. The Spermatogenesis of .Amphiiima : J. J/., XV'.. Suppl. — McMvirrich. J. P., '86. A Contribution to the Enibryoh>gy of the Prosobranch Gasteropods : Studies Biol. Lab. Johns Hopkins Unii'.. III. — Id., "95. Embry- ology of the Isopod Crustacea: /. J/., XL, i. — Id.. "96. The Volk-Lobe and the Centrosome of Fulgur : A. A.. XII., 23. — Id., "97. The Epithchum of the .Midgut of the Terrestri-1 Isopods : /. JA, XIV., i. — Maggi, L., "78. I plastitluli nei ciliati ed i plastiduli liberamente viventi : Atti. Soc. Ital. Sc. Nat. Milano, X.Xi. (also later papers). — Malfatti, H., '91. Beitrage zur Kenntniss der Nucleine: Zeit.\Phys. Chein.., XVI. — Mark, E. L., "81. Maturation, P'ecundation. and Seg- mentation of Limax campestris : Bull. Mus. Conip. /.ool. Harvard Collct^cW. — Mathews. A. P., "97, 1. Internal Secretions considered in Relation to \'ariation and Development: Science., V., 122. — Id.. "97. 2. Zur Chemie der Six-rmatozoen : Zeit. Phys. CJieui., XXIII., 4, 5. — Id., "98. A Contribution to the Chemistry of Cytological Staining: Am. Journ. P/iys., I.. 4. — Id.. "99, 1. The Origin of Fibri- nogen: Ibid., III.— Id., "99.2. The Metabolism of the Pancreas Cell: /. .1/., XV., Suppl. — Maiipas, M., "88. Recherches experimentales sur la multiplication des Infusoires cilies : Arch. Zool. Exp., 2me serie, VI. — Id.. "89. Le rejeunisse- ment karyogamique chez les Cilies: Ibid., 2me serie, VII. — Id., "91. Sur h- deter- minisme de la sexualite chez THydatina senta : C. /?., Paris. — Mayer. P., "91, Uber das Farben mit Carmin, Cochenille und Hamatein-Thonerde: Mitth. Zool. St. Neapol., X., 3. — Id., "97. Beruht die Farbung der Zellkerne auf einem chem- ischen Vorgang oder nicht?: A. A., XIII., 12. — Mead. A. D.. "95. Some Obser- vations on Maturation and Fecundation in Chaetopteruspergamentaceus Cuv. : /. .)/., X., I. —Id., "97, 1. The Origin of the Egg-centrosomes : Ibid., .Xll. — Id.. "97. 2. The early Development of marine Annelids: Ibid., \. — Id.. "98. 1. Tlic ()r;^in and Behaviour of the Centrosomes in the Annelid Egg : Ibid., Xl\'.. 2. — Id., 98. 2. The Rate of Cell-division and the Function of the Centrosome : Wood's I/oll Bwl. Lectures. — Merkel. F., "71. Die Stiitzzellen des menschlichen Hodens : Mi'illers Arch. — Mertens, H., "93. Recherches sur la signihcation du corps vitcllin de Balbiani dans lovule des Mammiferes et des Oiseaux : A. />'.. XIll. — Metschui- koff, E., "66. Embryologische Studien an Insecten : Z.Il.Z., XVL — Meves, F.. "91. Uber amitotische Kernteilung in den Spermatogonien des Salamanders, und das Verhalten der Attraktionsspharen bei derselben : ./. ./., 1891, No. 22.— Id., '94. Uber eine Metamorphose der Attraktionssphiire in den Spermatogonien von Salamandra maculosa: A. ui. A., XLIV.— Id., '95. t'ber die Zelh-n des Sesambeines der Achillessehne des Frosches {Rana teniporaria) und iiber ihre Cen- tralkorper: //;/V/., XLV. — Id., "96. Uber die Entwicklung der mannlichen (ie- schlechtszellen von Salamandra: Ibid., XLVIIL— Id., '97. 1. Zur Struktur der Kerne in den Spinndriisen der Raupen : Ibid., XlA'lIl. — Id.. '97. 2. Uber Struktur und Histiogenese der Samenfiiden von Salamandra : Ibid., L. - Id . "97. 3. Uber den Vorgang der Zelleinschnlirung : Arch. Pntu'/n., V., 2.— Id.. 97. 4. Zelltlieilung: Jlerkel u. Bonnet, Er^., VI.— Id.. '97. 5. Cher Cc-ntralkiirpt-r in mannlichen Geschlechtszellen von Schmetterlingen : .-/. ./.. Xl\' . i. — Id.. '98. t'ber das Verhalten der Centralkorper bei der Histogenese der Samenfaden vom Men.sch und Ratte : I'erh. An. c;.'^.. XIV. — Id., '99. Cber Struktur und Histo- genesis der Samenfaden des Meerschweinschens : .-/. ///. .-/.. LI\'.- Meyer, A., '96. Die Plasmaverbindungen. etc.: Bat. Zeit., ir, 12.— Meyer. O.. 95. Celhilar- Untersuchungen an Nematodeneiern : /. Z., XXiX. (XXII. ). - Michaelis. L.. -97. Die Befruchtung des Tritoneies : ./. w. -/•. XL\'I1I. — Miescher.^ F.. ^96. Physiologisch-chemische Untersuchungen iiber die Lachsmilch : .Irch. Exp. Path. u.'Pharni.. XXXVII. — Mikosch, "94. C'ber Struktur im ptianzlichen Proto- plasma: Verhandl. d. Ges. deutscher Xaturf. und . /rr/c-. 1 894 : Abteil f. Pjlanzcn- 462 GENERAL LITERATURE-LIST physiologie ii. P/lansenanaiojm'e. — Minot, C. S., '77. Recent Investigations of Embryologists : Froc. Bost. Soc. Nat. Hist.., XIX. — Id., '79. Growth as a Function of Cells : Ibid., XX. — Id., *82. Theorie der Genoblasten : B. C, II., 12. See also Am. Nat., February, 1880, and Froc. Bost. Soc. A'at. Hist., XIX., 1877. — ^^-^ "91- Senescence and Rejuvenation : Joiini. /V/jj'., XII., 2. — Id. ,"92. Human Embryol- ogy: New York.~^o\\ Mohl Hugo, '46. Uber die Saftbevvegung im Innern der Zellen : Bot. Zeitinig. — Moll. J. W., "93. Observations on Karyokinesis in Spiro- gyra : Verh. Kon. Akad., Amsterdam, No. 9. — Montgomery, Th. H.. "98, 1. The Spermatogenesis of Pentatoma, etc. : Zo'dl. Jahrb. — Id., '98, 2. Comparative Cytological Studies, witli Especial Reference to the Morphology of the Nucleolus : J. M., XV., 2. — Moore, J. E. S., '93. Mammalian Spermatogenesis: A. A., VIII. — Id., "95. On the Structural Changes in the Reproductive Cells during the Spermatogenesis of Elasmobranchs : Q. /., XXXVIII. — Morgan, T. H., "93. Experimental Studies on Echinoderm Eggs: A. A., IX., 5, 6. — Id., '95, 1. Studies of the " Partial "' Larvae of Sphaerechinus : A. Entivm., II., i. — Id., '95, 2. Experimental Studies on Teleost-eggs : A. A., X., 19. — Id., '95, 3. Half-embryos and Whole-embrvos from one of the first two Blastomeres of the Frost's Esfor ; Ibid., X., 19. — Id., '95. 4. The Fertilization of non-nucleated P^ragments of Echinoderm-eggs : Arch. Entiviu., II., 2. — Id., '95, 5. The Formation of the Fish- embryo : /. M., X., 2. — Id., "96, 1. On the Production of artificial archoplasmic Centres: Kept, of the Am. Morph. Soc, Science, III., January 10, 1896. — Id., '96, 2. The Number of Cells in Larvae from Isolated Blastomeres of Amphioxus : Arch. Entwm., III., 2. — Id.. "96. 3. The Production of Artificial Astrosphccres : Arch. Entwm., III. — Id., "98. 1. Experimental Studies of the Regeneration of Planaria maculata : Ibid., VII., 2. 3. — Id.. "98, 2. Regeneration and Liability to Injury: Zo'dl. Bull., I., 6. — Id., '99, 1. The Action of Salt-solutions on the Unfertilized and Fertilized Eggs oi Arbacia and other Animals: Arch. Efitii'm., VIII., 3. — Id., '99. 2. A Confirmation of Spallanzani's Discovery, etc.: A. A., XV. 21. — Mottier, D. M., "97, 1. Uber das Verhalten der Kerne bei der Entwick- lung des Embryosacs, etc. : Jahrb. wiss. Bot., XXXI. — Id., '97, 2. Beitraige zur Kenntniss der Kerntheilung in den Pollenmutterzellen. (?/t\ ; Ibid., XXX. — Id., '98. Das Centrosoma bei Dictyota : Ber. D. Bot. Ges., XVI., 5. — Miiller, E., "96. tJber die Regeneration der Augenlinse nach Exstirpation derselben bei Triton : A. m. A., XLVIL. i. — Munson, J. P., '98. The Ovarian Egg of Limulus, etc. : J. M., XV., 2. —Murray, J. A., "98. Contributions to a Knowledge of the Neben- kern in the Spermatogenesis of Pulmonata: Zo'ol. Jahrb., XL, 14. NADSON, G., '95. Uber den Bau des Cyanophyceen-Protoplastes : Script. Botan. Horti. Petropol., W . — Nageli, C, "84. Mechanisch-physiologische Theorie der Abstammungslehre : M'unchen u. Leipzig, 1884. — Nageli und Schwendener, '67. Das Mikroskop. (See later editions.) Leipzig. — Nawaschin. "99. Neue Beobachtungen liber Befruchtung bei Fritillaria und Lilium : Bot. Centb., LXXVIL, 2.— Nemec, B., "97. Uber die Stmktur der Diplopodeneier, A. A., XIII., 10, II. — Id., '99. Uber die karyokinetische Kerntheilung in den WUr- zelspitzen von Allium : J. w. B., XXVIII, 2. — Newport. G. On the Impregnation of the Ovum in the Amphibia: Fhil. Trans., 1851, 1853, 1854. — Norman. "W. 'W., '96. Segmentation of the Nucleus without Segmentation of the Protoplasm : Arch. Entwm., III. — Nussbaum, M.. "80. Zur Differenzierung des Geschlechts im Tier- reich : A. m. A., XVIIL — Id., '84, 1. Uber Spontane und Kunstliche Theilung von Infusorien : Verh. d. naturh. Ver. preus.. Rheinland, 1884. — Id., "84,2. Uber die Versinderungen der Geschlechtsproducte bis zur Eifurchung : A. m. A., XXIII. — Id., "85. Uber die Teilbarkeit der lebendigen Materie, I. : A. ?n. A., XXVI. — GENERAL LITERATURE-UST 463 Id., ^4. Die mit der Entwickeluno- fortschreitende DifTcrenziemng der Zellen : Sitz.-Ber. d. niedcrrhein. Gescllschaft f. Natiir- u. Hcill:undt\ Honn, 5 Nov., 1894; also B. C, XVI., 2, 1896. —Id.. 97. Die Entstehung des Geschlechts bei Hyda- tina: A. 7n. A., XLIX. OBST. P.. "99. Untersuchungen liber das Verhalten der Nucleolen, etc. : Z. w. Z. LXVI., 2. — Ogata. "83. Die Veranderungen der Fancreas/.ellen bei der Secre- tion: A. A. P. — Oppel. A.. 92. Die Befruchtung des Reptilieneies : A. w. A. XXXIX.— Osterhout. W. J. V.. "97. f'ber Entstehung der karyoithe Bedeutung des Zellkerns : Pfl'i'iger's Arch. f. d. ges. Physiol., II.— Id. ,"95. Allge- meine Physiologie : /.?;/^. — Virchow, R., "SS. Cellular-Pathologie : Arch. I\ith. Anat. Phys., VHI., i.— Id., "58. Die Cellularpathologie in ihrer Begriinduncj auf physiologische und pathologische Gewebelehre : Berlin, 1858. — De Vries. H.. '89. Intracellulare Pangenesis : Jena. "WAGER, H., "96. On the StiTicture and Reproduction of Cvstopus. .-/;///. />V)/., X. — Waldeyer, W.. '70. Eierstock und Ei : Leipzij^.—l^.'. 87. Bau und Ent- wickelung der Samenfaden : Verh. A71. Ges. Leipzig, 1887. — Id.. "88. ('ber Karvo- kinese und ihre Beziehungen zu den Befruchtungsvorgangen : ./. m. .7., X.XXII. [Trans, in Q. J.~\ — Id., "95. Die neueren Ansichten liber den Bau und das Wesen der Zelle : Deutsch. Med. Wochenschr., No. 43, flf., Oct. ff., 1895. — Warneck. N, A., '50. Uber die Bildung und Entwickelung des Embryos bei Gasteropoden : Bidl. Soc. Imp. N'at. Moscou, XXIII., i. — "Watase. S.. "91. Studies on Cephaio- pods; I., Cleavage of the Ovum: /. M., IV., 3. — Id.. 92. On the Phenomena of Sex-differentiation: Ibid.^ VL, 2, 1892. — Id.. "93. 1. On the Nature of Cell- organization: Wood's Hall Biol. Lectures, 1893. — Id.. '93. 2. Homology of the Centrosome : J. M., VIII., 2. — Id., "94. Origin of the Centrosome : Biological Lec- tures, Wood'' s LI all, 1894. "Webber, H. J, "97. 1. Peculiar Structures occurring in the Pollen-tube of Zamia: Bot. Gazette, XXII I., 6. — Id.. "97. 2. The Develop- ment of the Antherozoids of Zamia: Ibid., XX1\'.. i. — Id.. "97. 3. Notes on the Fecundation of Zamia and the Pollen-tube Apparatus of liingko: Ibid., XXIV'., 4. — Weismann, A., "83. Uber Vererbung: Jena. — Id.. "85. Die Kontinuit.iit des Keimplasmas als Grundlage einer Theorie der \'ererbung : Jena. — Id.. '86. 1. Richtungskorper bei parthenogenetischen Eiern : Zool. Anz., No. 233. Id.. 86. 2. Die Bedeutung der sexuellen Fortpflanzung fiir die Sclcktionstheorie : Jeita. — Id., '87. i'^ber die Zahl der Richtungskorper und uber ihre Beileutung fiir die Vererbung : /i?;/ (^7. — Id.. "91. 1. Essays upon Heredity. First Series: O.vford. — Id.. "91, 2. Amphimixis, Oder die \'ermischung der Inilividuen : A*//,/. Fischer.— Id., '92. Essays upon Heredity. Second Series: O.x/ord, 1S92. — Id.. "93. The Germ-plasm: A^e^u y^ork. — 16... "94. .Vu.s.sere Einfiii.sse als Entwicklungsreize : Jena. — Id.. "99. Regeneration: Xat. Sci., XIV .. 6. [See al.so A. A.. 1899.] Wheeler. W. M.. "89. The Embryology of />la/ta Gcrmauica and Doryphora decemlineata: J. M., 111. — Id.. "93. .A. Contribution to Insect-embryology: Ibid., VIII., I. — Id., "95. The Behaviour of the Centrosomes in the Fertilized Egg of Myzostoma glabruni: Ibid., X. — Id.. "96. The Sexual Phases of .Myzostoma : / I 468 GENERAL LITERATUKE-LIST Mitth. Zool. St. Neapel. Xll., 2. — Id., "97, The Maturation, Fecundation, and early Cleavage in Myzostoma : Arc/i. Biol., XV. -r. Whitman. C. O., '78. The Embryology of Clepsine: (2- J-^ X\TII. — Id., '87. The Kinetic Phenomena of the Egg during Maturation and Fecundation : /. J/., I., 2. — Id.. "88. The Seat of Formative and Regenerative Energy: Ibid., II. —Id.. "93. The Inadequacy of the Cell-theory of Development : Wood's H oil Biol. Lectures, 1893. — Id., "94. Evolu- tion and Epigenesis : Lbid., 1894. — Wiesner. J.. "92. Die Elementarstruktur und das Wachstum der lebenden Substanz : ll'ieu. — Wilcox. E. "V.. "95. Spermato- genesis of Caloptenus and Cicada : Bull, of tJie ALuseuui of Co)np. Zool., Liarvard College,\' o\.XXX\\.,'^Q. i. — Id., "96. Further Studies on the Spermatogenesis of Caloptenus : Bull. ALus. Comp. Zool., XXIX. — "Will, L.. "86. Die Entstehung des Eies von Colymbetes : Z. w. Z., X LI 1 1. —"Wilson, Edm. B.. "92. The Cell- lin»eage of Nereis : f. M., VI., 3. — Id.. "93. Amphioxus and the Mosaic Theory of Development : Ll?id., \TII., 3. — Id., "94. The Mosaic Theory of Development: Wood's Noll Biol. Led., 1894. — Id.. "95. 1. Atlas of Fertilization and Karyo- kinesis : Ne7i> York, Maonillau. — Id., '95, 2. Archoplasm, Centrosome, and Chromatin in the Sea-urchin Egg: /. J/., XI. — Id., "96. On Cleavage and Mosaic-work. [Appendix to Crampton and Wilson, "96.] : A. Eiitwni., III., i. — Id., "97. Centrosome and Middle-piece in the Fertilization of the Egg. Science-, Vol. v., No. 114. — Id.. "98. Considerations on Cell-lineage and ancestral Remi- niscence: Ann. JV. Y. Acad. Sci., XI. See also JVood's Moll Biol. Lectures, '99. — Id.. "99. On protoplasmic Structure in the Eggs of Echinoderms and some other Animals :/. J/., XV. Suppl. — "Wilson and Mathews. "95. Maturation, Fertilization, and Polarity in the Echinoderm Egg: /. J/., X., i. — "Wolff, Caspar Friedrich, 1759. Theoria Generationis. — "Wolff, Gustav, "94. Bemerkungen zum Darwinismus mit einem experimentellen Beitrag zur Physiologic der Entwick- lung: B. C, XIV., 17. — Id., '95. Die Regeneration der Urodelenlinse : Arc/i. Entu)}n., I., 3. — "Wolters. M., "91. Die Conjugation und Sporenbildung bei Gregarinen: A. m. A., XXXVII. — Woltereck, R., "98. Zur Bildung und Ent- wicklung des Ostrakoden-Eies : Z. w. Z., LXIV. ■yUNG. E.. "81. De Tinfluence de la nature des aliments sur la sexualite : C. R., XCIII ; also Arc/i. Exp. Zool., 2d, I., 1883. ZACH ARIAS. O., "85. Uber die amoboiden Bewegungen der Spermatozoen von Polyphemus pediculus : Z. w. Z., XLI. — Zacharias, E.. "93. 1. t'ber die chemische Beschaffenheit von Cytoplasma und Zellkern : Ber. deutsch. Bot. Ges., II., 5. — Id.. "93. 2. Uber Chrimatophihe : Lbid., 1893. — Id.. "95. Uber das Verhalten des Zellkerns in wachsenden Zellen : Flora, %i, 1895. — Id.. "94. t^ber Beziehungen des Zellenwachstums zur Beschaffenheit des Zellkerns : Berichte der deutschen botan. Gesellschaft, XII., 5. — Id.. "98. t'ber Nachweis und Vorkommen von Nuclein : Ber. d. Bot. Ges., XVI., 7. — Ziegler. E., "88. Die neuesten Arbeiten liber Vererbung und Abstammungslehre und ihre Bedeutung fur die Pathologic : Beitr. zur path. Anat., IV. — Id., "89. Uber die Ursachen der pathologischen Gewebsneubildungen : /;//. Beitr. zur. iviss. Med. Festschrift, R. Virchow, II. — Id.. "92. Lehrbuch der allgemeinen pathologischen Anatomic und Pathogenese, 7th ed., fena. — Ziegler. H. E.. "87. Die Entstehung des Blutes bei Knochenfischen- embryonen : A. in. A. — Id., '91. Die biologische Bedeutung der amitotischen Kerntheilung im Tierreich : B. C, XI. — Id., "94. Uber das Verhalten der Kerne im Dotter der meroblastischen Wirbelthiere : Ber. N'aturf. Ges. Freiburg, 1894. — Id., '95. Untersuchungen liber die Zelltheilung : I'erhandl. d. deutsch. Zool. Ges., 1895. — ^^-j ^^- Einige Betrachtungen zur Entwicklungsgeschichte der Echino- dermen : Verh. d. Zool. Ges. — Id., "98. Experimentelle Studien uber die Zellthei- GENERAL UrERATUKE-LIST 469 lung, I., II.: Arch. Entwm., VI., 2. — Ziegler and vom Rath. Die amitotische Kerntheilung bei den Arthropoden : />'. C".. XI. - Zimmermann, A.. 93. 1. Hei- trage zur Morphologic und Phy.siologie der Frian/.en/.elle : Tubiui^nt. — 1^ . 94 Sammelreferate aus dem Gesammtgebiete der Zellenlehre : Hot. Lnith. lialufte, 1894. Zimmermann. K. W.. 93. 2. Studien iiber i'igmentzellen, etc. : ./. //;../.] XLI.— Id.. "98. Beitrage zur Kenntni.s.s einiger Drusen und Kpithelzellcn : .-/. m. A..LU. — Zoja. R.. "95.1. Sullo .sviluppo dei bla.stomeri isolati dalle uova di alcunemeduse:^. ^;//w///.,I.,4; II., i : II., IV. -Id. 95. 2. .Sulla independenza della cromatina paterna e materna nel nucleo delle cellule embrionali : .-/. .-/.. XI., 10. Id.. '97. Stato attuale degli Studii sulla Fecondazione : />W/. .SV/. di Pavta, XVIII., XIX.— Zur Strassen. O.. -98. C'ber die Rie-senbildung bei Ascaris- Eiern : Arch. E?itwm., \'II., 4. INDEX OF AUTHORS Albrecht, nuclei, 32. Altmann, granule-theory, 25, 27, 290 ; nu- clein, 332. Amici, pollen-tube, 218. Andrews, spinning activities, 61. Apathy, nerve-cells, 48. Aristotle, epigenesis, 8. Arnold, fibrillar theory of protoplasm, 23; leucocytes, 117; nucleus and cytoplasm, 303. Atkinson, reduction, 269. Auerbach, 6; double spermatozoa, 142; staining-reactions, 176; fertilization, i8i. Von Baer, cleavage, lO; cell-division, 64; egg-axis, 378; development, 396. Balbiani, scattered nuclei, 40; spireme- nuclei, 36; mitosis in Infusoria, 88; chro- matin-granules, 112; yolk-nucleus, 155- 156; regeneration in Infusoria, 343. Balfour, polar bodies, 243; rate of division, 366; unequal division, 371. Ballowitz, structure of spermatozoa, 139, 140; double spermatozoa, 142. Van Bambeke, deutoplasm and yolk-nucleus, 156-160; elimination of chromatin, 155. Barry, fertilization, 181. De Bary, protoplasm, 4, 5, 20; conjugation, 181; cell-division and growth, 393. Beale, cell-organization, 291. Bechamp and Estor, microsome-theory, 290, 291. Belajeff, spermatozoids, 172-175; reduction in plants, 267. Benda, spermatogenesis, 163; Sertoli-cells, 284. Van Beneden, cell-theory, i, 6, 7; proto- plasm, 23; nuclear membrane, ^S; cen- trosome and attraction-sphere, 51, 74, 77, 310, 323; cell-polarity, 55; cell-division, 64, 74; origin of mitotic figure, 74-77! theory of mitosis, 100; division of chromo- somes, 112; fertilization oi Ascoris, 7, 1 82; continuity of centrosomes, 75; germ- nuclei, 205; centroNonie in fertilization, 208; theory of sex, 243; j)arlhcnogt:niMs, 281; nucleus and cyt<^plasni, 303; nuckar microsomes, 302 ; promorphology of cleav- age, 381 ; germinal localization, 399. Van Beneden and Julin, first cleavage-plane, 380. Bergmann, cleavage, lO; cell, 17. Bernard, Clauile, nucleus and cytoplasm. ^41 ; organic synthesis, 431. Berthold, protoplasm, 42 ; cell-division, 376- Bickford, regeneration in ctelenierates, 392, 429. Biondi, Sertoli-cells, 284. Biondi-Ehrlich, staining-tluid, 157. Bischoff, cell, 17. Bizzozero, cell-bridges, 60. Blanc, fertilization of trout, 210. Blochmann, insect-egg, 132; budding of nu- cleus, 155; polar bodies, 281 ; bilaterality of ovum, 3S3. Bohm, fertilization in fishes, 192. Bolsius, ne[)hridial cells, 47. Bonnet, theory of development. S, 432. Born, chromosomes in 7>/A)//-cgg, 338; gravitation-experiments, 386. Boveri, centrosome. named. 51 : a i>ermancnl organ, 51, 74; in fertilization, 192, Jii. 215, 230; structure, 309: functions, archoplasm, 69, 318; irigin of inuouc figure. 74, 77, 319: varieties of Asmrif, 87; theory of mitosis. lOi. loS: division of chromosomes, 1 1 2 ; origin of gcrm-ccUs. 147; fertilization of Asraris, 182; of rurotroihen, 1 84; of Echinus, 102: the- ory of fertilization, KK3, 211; of partheno- genesis. 281 ; partial fertilization, I90, I94; retiuction, 233; maturation in Ascitris^ 23S; tetrads, 238; centriole, 309: attrac- tion-sphere, 324; egg-fragments, 353. Braeni, cell-division, 377. Brandt, symbiosis, 53; regeneration in Tro- tozoa, 342. 47 472 INDEX OF AUTHORS Brauer, bivalent chromosomes, 82; mitosis in rhizopod, 96; fission of chromatin- granules, 113; deutoplasm, 153; fertiliza- tion in Bra7ichipus, 192; parthenogenesis in Artemia, 281 ; spermatogenesis in Asca- ris, 255; intra-nuclear centrosome, 304. Braus, 81. Brogniard, pollen-tube, 218. Brooks, heredity, 12; variation, 179. Brown, Robert, cell-nucleus, 18; pollen- tube, 218. Briicke, cell-organization, 289. Von Brunn, spermatozoon, 141. Biihler, astral systems, 318. Biitschli, 6; protoplasm, 25, 36, 50; diffused nuclei, 40; artifacts, 42; asters, 48, 316; cell-membrane, 54; mitosis, 109, no; centrosome in diatoms, 51; rejuvenes- cence, 178; polar bodies, 238. Calberla, micropyle, 200. Calkins, nuclei of flagellates, 40; mitosis in A'octiluca, 92; yolk-nucleus, 157; origin of middle-piece, 165; reduction, 253, 257. Campbell, fertilization in plants, 216. Carnoy, nucleus, 40; muscle-fibre, 48; cen- trosome, no; amitosis, 115, 117; germ- nuclei, 184; asters, 305, 317. Carnoy and Le Brun, nucleoli, 130; fertiliza- tion, 211; reduction, 263. Castle, egg-axis, 379; fertilization, 193. Chittenden, organic synthesis, 341. Chmielewski, reduction in Spirogyj'a, 280. Chun, amitosis, 117; partial development of ctenophores, 418. Clapp, first cleavage-plane, 381. Coe, fertilization, 194, 213; centrosome, 321. Cohn, cell, 17, Conklm, size of nuclei, 71 ; union of germ- nuclei, 204; centrosome in fertilization, 210; centrosome and sphere, 323; un- equal division, 373; protoplasmic cur- rents, 377; cell-size and body-size, 388; types of cleavage, 423. Corda, pollen-tube, 218. Crampton, yolk-nucleus, 158; reversal -of cleavage, 368; experiments on snail, 419, 421 ; on tunicates, 419. Crato, protoplasm, 50. Darwin, evolution, 2, 5; inheritance, 12, 396; variation, ii; pangenesis, 12, 290; gem- mules, 290. Darwin, F., protoplasmic fragments, 346. Dendy, cell-bridges, 60. Dogiel, amitosis, 118. Driesch, dispermy, 198; fertilization of egg- fragments, 200, 353; pressure-experiments, 375,410; regeneration, 393; isolated blas- tomeres, 409; theory of development, 394, 415; experiments on ctenophores, 418; ferment-theory, 427. Driiner, spindle-fibres, 79; central spindle, 105; aster, 321, 326. Von Ebner, Sertoli-cells, 284. Ehrlich, tar-colours, 335. Eismond, structure of aster, 48. Elssberg, plastidules, 291. Endres, experiments on frog's egg, 399, 419. Engelmann, ciliated cells, 44; rejuvenes- cence, 179, Von Erlanger, asters, 48, 316; spindle, 81; elimination of chromatin, 155; Xebenkern, 163,165; fertilization, 194, 212, 213; cen- troplasm, 324. Eycleshymer, first cleavage-plane, 381. Farmer, reduction in plants, 275. Fick, fertilization of axolotl, 192, 212. F'ield, staining-reactions, 176. Fischel, ctenophores, 419. Fischer, nucleus, 40; artifacts, 42; staining- reactions, 335. Flemming, protoplasm, 25, 27 ; chromatin, 2,3 ', centrosome, 51 ; cell-bridges, 60, 61 ; cell- division, 64, 70; splitting of chromosomes, 70; mitotic figure, 79; heterotypical mito- sis, 86; leucocytes, 102; theory of mitosis, 106; division of chromatin, 1 13; amitosis, 117,285; nucleoli, 127; rotation of sperm- head, 188; spermatogenesis, 259-262; astral rays, 317; germinal localization, 399- Floderus, follicle-cells, 150. Fol, I, 6, 64; amphiaster, 68; theory of mi- tosis, 108; sperm-centrosome, 191 ; poly- spermy, 192; attraction-cone, 198; vitel- line membrane, 199; asters, 316. Foot, yolk-nucleus and polar rings, 156, 202; fertilization in earthworm, 187; entrance- funnel, 201; fertilization-centrosome, 212. Foster, cell-organization, somacules, 291. Francotte, polar bodies, 235 ; centrosome, 306; sphere, 312, 325. Frommann, protoplasm, 23; nucleus and cytoplasm, 303. Gaieotti, pathological mitoses, 97. Gallardo, mitosis, 109. INDEX OF AUrilOKS 473 Galton, inheritance, 9. Gardiner, cell-bridges, 59; chroniatin-elimi- nation, 276; sphere, 325, Garnault, fertilization in Ai'ion, 207. Geddes and Thompson, theory of sex, 124. Van Gehuchten, spireme-nuclei, 36; nuclear polarity, 36; muscle-tibre, 48. Giard, polar bodies, 235, 238. Gierke, staining-reactions, 335. Gilson, spireme-nuclei, 36. Godlewski, spermatogenesis, 168. Graf, nephridial cells, 47. Gregoire, reduction, 267. Griffin, fertilization, centrosomes in Thalas- sevia, 193, 194, 213; reduction, 259; structure of centrosome, 314; aster-forma- tion, 321. Grobben, spermatozoa, 141. Gruber, diffused nuclei, 40; regeneration in Stentor, 342. Guignard, mitosis in plants, 82; fertilization in plants, 218, 221; reduction, 263, 267. Haberlandt, position of nuclei, 346. Hackel, inheritance, 7; epithelium, 56; cell- state, 58. Hacker, polar spindles, 276; bivalent chro- mosomes, 88; nucleolus, 125, 128; primor- dial germ-cells, 148; germ-jnuclei, 208, 299; reduction in copepods, 249. Hallez, promorphology of ovum, 384. Halliburton, proteids, 331 ; nuclein, I'i^'}^. Hamm, discovery of spermatozoon, 9, 181. Hammar, cell-bridges, 60. Hammarsten, proteids, 331. Hansemann, pathological mitoses, 97. Hanstein, metaplasm, 19. Hardy, artifacts, 42. Harper, mitosis, 82. Hartsoeker, spermatozoon, 9. Harvey, inheritance, 7; epigenesis, 8. Hatschek, cell-polarity, 56; fertilization, 179. Heidenhain, nucleus, 36; basichromatin and oxychromatin, 38, 337; cell-polarity, 55; position of centrosome, 57; leucocytes, 102; theory of mitosis, 105; amitosis, 116; staining-reactions, 337; nuclear micro- somes, 303; microcentrum, 311; asters, 311, 317; origin of centrosome, 315; po- sition of spindle, 377. Heider, insect-egg, 132. Heitzmann, cell-bridges, 59; nucleus and cytoplasm, 303. Henking, fertilization, 187; insect-egg, 96; spermatogenesis, 165, 248, 253, 27 lo llenle, granules, 289. Henneguy, dcutoplasm, 153; yolk-nucleus, 1 60; centrosome, 356. Hensen, rejuvenescence, 179. Herbst, development and environment, 428. Herla, independence of chromosomes, 208, 299. Hermann, central spindle, 78, 105; division of chromatin, 112; spermatozoon, 165, 166; staining-reactions, 176. Hertwig, O., i, 7, 9; bivalent chromosomes, 88; pathological mitoses, 97; rejuvenes- cence, 178; fertilization, iSi; middle- piece, 187; polyspermy, 199; paths of germ-nuclei, 204; maturati(.n, 241 ; polar bodies, 238; inheritance, 1 82; laws ..f cell-division, 364; theory of development, 415- Hertwig, O. and R., 197; cgg-fragmenls, 199; polyspermy, 199. Hertwig, K., mitosis in Protozoa, <>o; gern)- cells in Sagi/ta, 146; amphiaslers in un- fertilized eggs, 306; conjugation, 222; reduction in Infusoria, 277; in .Utnto- sp/urriui/i, 278; origin of centrosome, 315; cell-division, 391. Hill, fertilization, 187, 193. Hirase, spermatozoids, 144; ferlili/alion, 218. His, germinal localization, 398. Hofer, regeneration in A/na/>a, 343, Hoffman, micropyle, 200. Hofmeister, cell-divismn and growtii, 393. Holmes, cleavage, 368. Hooke, R., cell, 17. Hoyer, amitosis, 115. Huie, Drosern, 350. Huxley, protoplasm, 5; germ, 7, 396; fer- tilization, 1 78, 231 ; evolution and epi- genesis, 432. Ikeno, cell-bridges, 150: blepharoplasts. 17;; fertilization, 221. Ishikawa, Noctilucu, mitosis, 92; conjuga- tion, 227; reduction, 267; tiagcllum. 171. Jennings, cleavage, 377. Jorilan, deutoplasm and yolk-iuKKu>, 153, 156; first cleavage-jdane. 3S1. Tulin, fertilization in Sfy/i<^/>sis, 192. Keuten, mitosis in Eugirna, 91. Klebahn, conjugation and reduction in des- mills and diatoms, 2S0. 474 IXDEX OF AUTHORS Klebs, pathological mitosis, 97, 98; cell- membrane, 346. Klein, nuclear membrane, 38; theory of mitosis, 100; amitosis, 118; nucleus and cytoplasm, 303; asters, 316. Klinckowstrom, fertilization, 213; reduction, 259- Von KolHker, i, 6, 9, 10, 27; epithelium, 56; cell-division, 63; spermatozoon, 9, 134; inheritance, 182; development, 413. Korff, spermatogenesis, 163, 168, 173. Korschelt, nucleus, 37; amitosis, 115; move- ments and position of nuclei, 125, 349, 387; nurse-cells, 151 ; fertilization, 193; tetrads in Ophryotrocha, 258; physiology of nucleus, 348; polarity of egg, 387. Kossel, chromatin, 336; nuclein, 334; or- ganic synthesis, 340. Kostanecki, fertilization, 193; astral rays, 318. Kostanecki and Wierzejski, fertilization of Physa, 193, 210, 212; continuity of cen- trosomes, 211. Kupffer, energids, 30; cytoplasm, 41. Lamarck, inheritance, 12. Lamarle, minimal contact-areas, 361. Lankester, germinal localization, 398. Lauterborn, mitosis in diatoms, 95; origin of centrosome, 315. Leeuwenhoek, spermatozoon, 8; fertiliza- tion, 181. Von Lenhossek, nerve-cell, 21, 47; sperma- togenesis, 169, 315; centrosome, 314, 356. Leydig, cell, 19; protoplasm, 20; cell-mem- brane, 54; spermatozoa, 142; elimination of chromatin, 159. Lilienfeld, staining-reactions of nucleins, 336. Lillie, fertihzation, 196, 213; centrosome and aster, 312, 326, 327; regeneration in Stentor, 343 ; cleavage, 360, 369, 377. Loeb, chemical fertilization, 215, 392; re- generation in coelenterates, 392; theory of development, 427; environment and development, 430. Lustig and Galeotti, pathological mitoses, 98; centrosome, 51. Maggi, granules, 290. Malfatti, staining-reactions of nucleins, 335. Mark, germ-nuclei, 204; polar bodies, 235; polarity of ovum, 387, Mathews, pancreas-cell, 44; aster-formation, iio; fertilization of echinoderms, 192,212; origin of centrosome, 125; nucleic acid, 334; staining-reactions, 337. Maupas, sex in Rotifers, 145; rejuvenes- cence, 179; conjugation of Infusoria, 223. Mayer, staining, 335. McClung, spermatogenesis, 271. MacFarland, spindle, 79; fertilization, 213, 214; centrosome and sphere, 312, 314, 321. ]\IcGregor, spermatogenesis, 167; reduction, 261. McMurrich, gasteropod development, 152; metamerism in isopods, 390. Mead, fertilization of Chcetopterns, 192, 194, 215; sperm-centrosome, 215; centrosomes de novo, 212, 306; cell-division, 391. Merkel, Sertoli-cells, 284. Mertens, yolk-nucleus and attraction-sphere, 156, 159. Metschnikoff, insect-egg, 383. Meves, amitosis, 119, 285; spermatogenesis, 167, 169; reduction, 260; cilia, 357. Meyer, energids, 30; cell-bridges, 60. Miescher, nuclein, 332. Mikosch, protoplasm, 44. Minot, rejuvenescence, 179; cyclical divi- sion, 222; theory of sex, 243; Sertoli- cells, 284; parthenogen sis, 280. Von Mohl, cell-division, 9; protoplasm, 17. Montgomery, nucleolus, 34; spermatogene- sis, 257, 271. Moore, spermatozoon, 167, 171 ; reduction, 263. Morgan, centrosomes, 307; fertilization of egg-fragments, 353; cell-division, 391; effect of fertilization, 201 ; numerical rela- tions of cells, 389; regeneration, 393, 394; isolated blastomeres, 410; polarity, 417; experiments on ctenophores, 418; on frog's egg, 422. Mottier, mitosis, 83; fertilization, 221; re- duction, 266; asters, 305. Munson, yolk-nucleus, 156. Nageli, development, I ; cell-organization, micelloe, 289, 291; polioplasm, 41 ; idio- plasm-theory, 401. Nawaschin, fertilization, 218. Nemec, mitosis, 82; yolk-nucleus, 159. Newport, fertilization, 181 ; first cleavage- plane, 380. Nissl, chromophilic granules, 48. Nussbaum, germ- cells, 122; sex, 145; re- generation in Infusoria, 342; nucleus, 426. Obst, nucleoH, 130; follicle-cells, 151. Osterhout, spindle, 82; tetrads, 253. INDEX OF AUTHORS 475 Overton, germ-cells of Volvox, 134; conju- gation of Spirogyra, 229; reduction, 274, 275- Owen, germ-cells, 122. Paladino, cell-bridges, 60. Paulmier, spermatozoon, 165; reduction, 252, 271. Peremeschko, leucocytes, 117. Peter, cilia, 357. Pfeffer, hyaloplasm, 41; amitosis, 1 19; chemotaxis of germ-cells, 197. Pfitzner, cell-bridges, 60; chromatin-gran- ules, 112. Pfluger, position of spindle, 375; first cleav- age-plane, 3S0; gravitation-experiments, 386; isotropy, 378. Plateau, minimal contact-areas, 366. Platner, mitosis, iio; egg-centrosome, 125; formation of spermatozoon, 163; fertiliza- tion of ^rz^«, 207; maturation, 241. Pouchet and Chabry, development and en- vironment, 428. Prenant, spermatozoon, 162; archoplasm, 322. Preusse, amitosis, 1 19. Prevost and Dumas, cleavage, lO. Pringsheim, Hautschicht, 41 ; fertilization, 181. Purkinje, protoplasm, 17. Rabl, nuclear polarity, 36; cell-polarity, 56; centrosome in fertilization, 210; individu- ality of chromosomes, 294; astral systems, 317- Ranvier, blood-corpuscles, 54. Vom Rath, bivalent chromosomes, 88; ami- tosis, 118, 225; early germ-cells, 149; reduction, 249. Rauber, cell-division and growth, 393. Rawitz, amitosis, 116; staining-reactions, 335- Redi, genetic continuity, 290. Reichert, cleavage, 10, 64. Reinke, pseudo-alveolar structure, 50; nu- cleus, 38, 303; oedematin, 36; asters, 305; nucleus and cytoplasm, 303. Remak, cleavage, i, 10, 361; cell-division, 64; egg-axis, 378. Retzius, muscle-fibre, 48; cell-bridges, 60; end-piece, 140. Rhumbler, 105. Robin, germinal vesicle, 64. Rosen, staining-reactions, 220. Roux, 245, 301, 351 ; meaning of mitosis, 244, 301, 351, 405; position of spindle, 377; first clcavagc-plane, 3S0; frog-experi- ments, nnjsaic thct^ry, 39<;; theory hrri-. 51- Spallanzani, spermatozoa, 9; regeneration, 393- 476 INDEX OF AUTHORS Spencer, physiological units, 289; develop- ment, <32. Stauffacher, egg-centrosome, 125. Stevens, fertilization, 217. Strasburger, i, 7; cytoplasm, 20; Korner- plasma, 41 ; centrosphere, 68, 356, 324; membranes, 55; origin of amphiaster, 82; multipolar mitoses, 99; theory of mitosis, 105, no; spermatozoids, 173; kinoplasm, 27, 82, 322; staining-reactions of germ- nuclei, 220; fertilization in plants, 216, 219, 221; reduction, 265, 269; theory of maturation, 275; organization, 289; in- heritance, 7, 182, 351; action of nucleus, 426. Zur Strassen, giant-embryos, 296; germ- cells, 148. Van der Stricht, spindle, 79; amitosis, 116; fertilization, 210; reduction, 259; centro- some and sphere, 312, 325. Strobe, multipolar mitoses, 99. Stuhlmann, yolk-nucleus, 156. Suzuki, spermatogenesis, 168. Swingle, mitosis, 82. Tangl, cell-bridges, 59. Thiersch and Boll, theory of growth, 392. Townsend, cell-bridges, 61, 346. Treat, sex, 145. Treviranus, variation, 179. Unna, protoplasm, 27. Ussow, micropyle, 133; deutoplasm, 153. Vejdovsky, centrosome, 76; fertilization in Rhynchelmis, 192, 194; metamerism in annelids, 390. Verworn, cell-physiology, 6; regeneration in Protozoa, 344; inheritance, 359, 431. Virchow, I; cell-division, lO, 63; proto- plasm, 25; cell-state, 58. De Vries, organization, pangens, 291, 327, 406; tonoplasts, 53; plastids, 229; chro- matin, 43 1; development, 404. Waldeyer, nucleus, 38; cytoplasm, 41 ; cell- membrane, 54. Walter, frog-experiments, 419. Watase, theory of mitosis, 106; staining- reactions of germ-nuclei, 176; nucleus and cytoplasm, 292; asters, 305; theory of centrosome, 315; astral rays, 321; cleav- age of squid, 381 ; promorphology of ovum, 383, 386. Webber, spermatozoids, 144, 173; fertiliza- tion, 221. Weismann, inheritance, 12; cell-organiza- tion, biophores, 291; somatic and germ cells, 122; amphimixis, 179; maturation, 243-246; constitution of the germ-plasm, 245; parthenogenesis, 281; theory of de- velopment, 404, 407, 432. Went, vacuoles, 53. Wheeler, amitosis, 115; insect-egg, 132; egg of Myzostotna, 151; fertilization in Myzostoma, 208; bilaterality of ovum, 383. Whitman, on Harvey, 7; polar rings, 202; cell-division and growth, 393; polarity, 384; theory of development, 400, 416. Wiesner, cell-organization, 290, 291. Wilcox, sperm-centrosome, 165; reduction, 257- Will, chromatin-elimination, 135. Wilson, protoplasm, 27, 44; mitosis, 106; fertilization in sea-urchin, 187, 212; paths of germ-nuclei, 202; origin of linin, 303; astral rays, 28; centrosphere and centro- some, 314; dispermy, 355; rudimentary cells, 372; pressure-experiments, 41 1; experiments on Amphioxus, 410; theory of development, 415. Von Wittich, yolk-nucleus, 155. Wolff, C. F., epigenesis, 8. Wolff, G., regeneration of lens, 433. Wolters, polar bodies in gregarines, 278. Yung, sex, 144. Zacharias, E., nucleoli, 34; ofmeristem, 37; staining-reactions, 176; nuclein in grow- ing-cells, 340. Zacharias, O., amoeboid spermatozoa, 142. Ziegler, artificial mitotic figure, 108; amito- sis, 117; sphere, 324. Zimmerman, pigment-cells, 102; centrosome, 356. Zoja, independence of chromosomes, 299; isolated blastomeres, 410. INDEX OF SUBJECTS Acanthocystis, 94, 304, 306. Achromatic figure (see Amphiaster), 69; varieties of, 78; nature, 316. AcJiromatium, 39. Actinophrys, 92, 278. Actinosphceriiiin, mitosis, 90, 94; reduction, 278; regeneration, 342. ^quorea, metanucleus, 128. Albugo, 217. Albumin, 331. Allium, 83, 253, 267. Allolobophora, teloblasts, 374. Alveoli, 25, Amitosis, 114; biological significance, 116; in sex-cells, 285. Amoeba, 5; mitosis, 91; experiments on, 343- Amphiaster, 68; asymmetry of, 70, 373; origin, 72, 74, 316; in amitosis, 116; in fertilization, 187, 213; nature, 316; posi- tion, 375. Amphibia, spermatozoa, I40; sex, 145, Atnphioxus, fertilization, 210; polar body, 236, 277; cleavage, 370; dwarf larvK, 389, 410; double embryos, 410. Amphipyrenin, 41. Amphiiitna, 167, 261. Amyloplasts, 53; in plant-ovum, 133, Anaphases, 70; in sea-urchin egg, 106. A>iasa, sperm-formation, 165, 271 ; reduc- tion, 272. Ancylus, 368. Anilocra, gland-cells, nuclei, 36; amitosis, 116. Anodonia, ciliated cells, 43, 357. Antipodal cone, loi. Aphis, 281. Arbacia, 192, 215, 307. Archoplasm, 69; in developing spermatozoa, 171; nature of, 318. Archosome, 52. Argonauta, micropyle, 133. Aricia, rudimentary cells, 372. Arion, spindle, 81 ; germ-nuclei, 207. Ariscema, 269. Ajtetnia, chromosomes, 89; parthcnogcnclic maturation, 281. Artifacts, in protoplasm, 42. Ascaris, chromosomes, 87, 301 ; n»it..sis. So, loi; primordial germ-cells, 146; fcrtili/a- tion, 182, 211; polyspermy, 199; polar bodies, 238; spermatogenesis, 241, 253; individuality of chromosomes. 295; in- tranuclear centrosome, 304; centrosome, 31 1; attraction-sphere, 323; supernumer- ary centrosome, 355. Aster, 68; asymmetry, 70; structure and functions, loi; in amitosis, 116; in fertili- zation, 187, 213; nature of, 316; hncr structure, 326; relative size, 70, 373. Asterias, spermatozoa, 176; sperm-aster, 187; fertilization, 192, 210. Astrocentre, 324. Astrosphere, 324. Attraction-cone, 198. Attraction-sphere, 51. 72; in amitosis, 1 15; of the ovum, 125; of the spermatid, 163; in resting cells, 323; nature of, ^2y Axial filament, 136; origin of, 165. Axis, of the cell, 55; of the nucleus. ^6. 204: of the ovum, 378, 386. Axolotl, fertilization, 192. Bacteria, nuclei, 31, 39. Basichromatin, 38; staining-reaclions, 33S. Bioblast, 290. Biogen, 291. Biophore, 245, 291. Birds, blood-cells, 57; spermatozoa, 13S; young ova, 155. Blastomeres, displacement of, 366; indi- vidual history, 378; prospective value, 415; rhythm of division, 366, 389; «Ic- velopment of single, 409, 418; in normal development. 423. BUunitis, pigment-cells, loj. Blepharoplastoids, 175. HIepharoplasts, 173, 221. Branchipus, yolk, 153; sperm-aster, 192; reduction, 256. 477 478 INDEX OF SUBJECTS Calavus, tetrads, 250. Caloptenus, 165, 257. Cambium, 376. Cancer-cells, mitosis, 98. Canthocamptus, reduction, 251 ; ovarian eggs, -/J- Cell, in general, 4; origin, 9; name, 17; general sketch, 19; polarity of, 55; as a structural unit, 58; structural basis, 23, 293; physiology and chemistry, 330; size and numerical relations, 389; in inheri- tance, 9, 430; differentiation of, 413, 426; independence of, 427. Cell-bridges, 59. Cell-division (see Mitosis, Amitosis), general significance, lo, 63; general account, 65; types, 64; Remak's scheme, 63; indirect, 65; direct, 114; cyclical character, 178, 223; equal and reducing or qualitative, 405 ; relation to development, 388, 405, 410, 427; Sachs's laws, 362; rhythm, 366, 389; unequal, 370; of teloblasts, 371; energy of, 388; relation to metamerism, 390; causes, 391; relation to growth, 388; and differentiation, 427. Cell-membrane, 53. Cell-organization, 289. Cell-organs, 52; nature of, 291; temporary and permanent, 292. Cell-plate, 71. Cell-state, 58. Cell-theory, general sketch, 1-14. Central spindle, 70, 78. Centrodesmus, 79, 315. Centrodeutoplasm, 163, 324. Centroplasm, 324. Centrosome, 22; general sketch, 50, 304; position, 55; in mitosis, 74; a permanent organ, 74; dynamic centre, 76; historical origin, 315; functions, loi, 354; in ami- tosis, 115; of the ovum, 125; of the spermatozoon, 137, 1 65-1 70; in fertiliza- tion, 190, 208; degeneration of, 186, 213; continuity, 74, 77, 194, 214, 321 ; nature, 304; intra-nuclear, 304; supernumerary, 355- Centrosphere, 68, 85; nature of, 324. Cet'aHuin, 91. Ceratozamia, reduction, 275. Cerebratiilus, 1 93, 194, 213, 306, 307, 32 1, 325. Cerianthus, regeneration in, 392. ChcEtopterus, spindle, 81, 84; fertilization, 192; sperm-centrosome, 213; centrosomes de novo, 306; cell-division, 391. Chara, spermatozoids, 143. Chilomonas, 32, 40, 192. Chironomus, spireme-nuclei, 36. Chorion, 132. Chromatic figure, 69; origin, 72; varieties, 86; in fertilization, 181, 204. Chromatin, -^y, i" meristem, 37; in mitosis, 65, 86; in cancer-cells. 98; of the egg- nucleus, 126; elimination of, in cleavage, 147,426; in oogenesis, 233, 276; staining- reactions, 334-340; morphological organi- zation, 37, 245, 294; chemical nature, 332, 404; relations to linin, 302; physiological changes, 338; as the idioplasm, 352; in development, 405, 425, 431. Chromatin-granules, 37; in mitosis, 112; in reduction, 248; general significance, 301- 304; relations to linin, 302, Chromatophore, 53; in the ovum, 133; in fertilization, 229. Chromiole, 302. Chromomere (see Chromatin-granule), 37, 301. Chromoplast, 53. Chromosomes, 67, 70, 86, 112; number of, 67, 206; bivalent and plurivalent, 87; division, 112; of the primordial germ- cell, 148; in fertilization, 182, 204; inde- pendence in fertilization, 204; reduction, 238, 243, 248; in early germ-nuclei, 273; conjugation of, 257; in parthenogenesis, 281; individuality of, 294; composition of, 301; chemistry, 334, 336; history in ger- minal vesicle, 338; in dwarf larvae, 296. Ciliated cells, 44, 57. Ciotia, egg-axis, 379. Clavelina, cleavage, 369, 381. Cleavage, in general, 10 ; geometrical rela- tions, 362; Sachs's rules, 362; Hertwig's rules, 364; modifications of, 366; spiral, 368; reversal of, 368; unequal, 370; under pressure, 375,411 ; promorphology of, 378; bilateral, 381; rhythm, 366, 388; mosaic theory, 399, 423; half cleavage, 410. Cleavage-nucleus, 204. Cleavage-planes, 362; axial relations, 378. Clepsine, nephridial cell, 45; polar rings, 202 ; cleavage, 370. Closteriiim, conjugation and reduction, 280. Cockroach, amitosis, 115; orientation of egg, 384. Coelenterates, germ-cells, 146; regeneration, 392, 393» 430- Conjugation, in unicellular animals, 222; unicellular plants, 228, 280; physiological meaning, 178, 223. INDEX OF SUBJECTS 479 Contractility, theory of mitosis, loo; inade- quacy, 1 06. Copepods, reduction, 251. Corixa^ ovum, 383, Corpuscule central, 310, 314. Crepichda, fertilization, 210 ; dwarfs and giants, 389; cleavage, 323, 423. Cross-furrow, 368. Crustacea, spermatozoa, 142. Ctenophores, experiments on eggs, 41 S. Cucurbita, 346. Cuticular, 54. Cyanophyceae, nucleus, 31, 39. Cycads, spermatozoids, 144, 173; fertiliza- tion, 218, 221. Cyclops, 0x^,12"^', primordial germ-cells, 148; fertilization, 188; reduction, 25 1; attrac- tion-sphere, 325; axial relations, 385. Cytoplasm, 21, 41, 293, 303; of the ovum, 130; of the spermatozoon, 134; morpho- logical relations to nucleus, 302; to archo- plasm, 316, 319; chemical relations to nucleus, 333-341 ; physiological relations to nucleus, 341 ; in inheritance, 352-354, 359 ; in development, 398, 42 1 ; origin, 431. Cytosome, 322. Dendrobczna, metamerism, 390. Determinants, 245. Deutoplasm, 131 ; deposit, 153; effect on cleavage, 366, 371 ; rearrangement by gravity, 422. Development, 1-12; and cell-division, 388; mosaic theory, 399,421; theory of Nageli, 402; Roux-Weismann theory, 404; of single blastomeres, 399, 409, 418; of egg- fragments, 296, 353, 419; De Vries's the- ory, 413; Hert wig's theory, 415, 432; Driesch's theory, 394, 415; partial, 409, 419; half and whole, 419; nature of, 413; external conditions, 428; and metabolism, 430; unknown factor, 431 ; rhythm, 432; adaptive character, 433, Diaptomus, 250. Diatoms, mitosis, 92; centrosome, 51. Dinnlula, 79, 314. Diemyctyliis, yolk, 153; yolk-nuclei, 156. Differentiation, 361 ; theory of De Vries, 404; of Weismann, 405; nature and causes, 413; of the nuclear substance, 425; and cell-division, 427. Dipsacus, 346. Dispermy, 355. Double embryos, 410, 422. Drosera, 350. Dwarfs, formation of, 353. 4,0, 422: size of cells, 389. Dyads (Zwciergruppen;, 239, 241 ; in par- thenogenesis, 284. Dyaster, 70. Dycyemids, centrosome, 51. Dytiscus, ovarian eggs, 153. :}4q. Earthworm, ova, 152; spermatozoon. 165; yolk-nucleus, 154; polar rings, 156. 202; spermatogenesis, 257; tclublasts, 374. Echinoderms, protoplasm, 28. 44, 29 1; sper- matozoa, 137; fertilization, 188. 212; polyspermy, 194, 198; dwarf larva-, 353, 410; half cleavage, 410: eggs under press- ure, 41 1 ; modified larva.*, 428. Echinus, fertilization, 210; centrosome, 314; dwarf larv.-e. 353; number of cells, 389. Ectosphere, 324. Egg-axis, 378; promorphological signifi- cance, 379; determination, 386; alteration of, 422. Egg- fragments, fertilization. 194; develop- ment, 352. Elasmobranchs, spermatozoon, 140, 167, 169; germinal vesicle, 245, 273; reduction, 257. Embryo-sac, 218, 263. Enchylema, 23. End-knob, 136. Endoplasm, 41. End-piece, 140. End-plate, 91. Energid, 19, 30. Entosphere, 324. Envelopes, of the egg, 132. Epigenesis, 8, 432. Equatorial plate, 68. Ei/uist'ium, mit«)sis, 85. Ergastoplasm, 322. Erysiphe, mitosis, '8>2. Eiiclucta, tetrads, 2^0. EugUna, mitosis, 91, 31;;. Euglypha, mitosis, 89, 95. Evolution (preformation), S, 399, 452. Evolution, theory of, 2, 8. Exoplasm, 41. Fertilization, general aspect. 9: physiologi- cal meaning, iSo; general sketch, iSo; Ascaris, 182; mouse, 185; sea-urchin, 188; Nereis, 188; Cychps, '188: 7'haiassema, Cfuctoptertts, 193. 195; pathological, 198; partial, kk). 194; of J/ico^/c^w./. 196, 20S; in plants, 215; egg-fragments, 194; Ho- veri's theory, 192, 211. 48o INDEX OF SUBJECTS Fishes, pigment-cells, 102 ; periblast-nuclei, 117; spermatozoa, 137; young ova, 116; single blastomeres, 410. Flagellates, diffused nuclei, 39. Follicle, of the egg, 150. Forficula, nurse-cells, 151. Fragmentation, 64. Fritillaria, spireme, 112; fertilization, 219. Frog, tetrads, 259; egg-axis, 378; first cleav- age-plane, 380; Roux's puncture experi- ment, 399; post-generation, 409; pressure- experiments, 410; effect of gravity on the egg, 422; development of single blasto- meres, 399, 408, 422; double embryos, 422. Fiicus, 143, 217, 221. Ganglion-cell, 48; centrosome in, 51, 314. Gemmae, 291. Gemmules, 12, 291. Genoblasts, 243. Geophilus, deutoplasm, 154, 158; yolk-nu- cleus, 156. Germ, 7, 396. Germ-cells, general, 8, 9; detailed account, 122; of plants, 133, 142; origin, 144; growth and differentiation, 150; union, 196; results of union, 200 ; maturation, 233; early history of nuclei, 272. Germinal localization, theory of, 397. Germinal spot, 124. Germinal vesicle, 124, 125; early history, 273; movements) 349; position, 387. Germ-nuclei, of the ovum, 125; of the spermatozoon, 135; of plants, 216; stain- ing-reactions, 175; in fertilization, 182, 188; equivalence, 182, 205; paths, 202; movements, 204; union, 204; indepen- dence, 204, 299; in Infusoria, 224; early history, 272. Giant-cells, 31; microcentrum, 314. Gingko, 173. Globulin, 331, 333. Granules (see Microsomes), of Altmann, 290; nuclear, 37, 303; chromophilic, 23, 48; in general, 289. Gravity, effect on the egg, 131, 422. Gregarines, mitosis, 89; polar body, 278. Ground-substance, of protoplasm, 23; of nucleus, 36. Growth, and cell-division, 58, 388. Gryllotalpa, reduction, 249. Guinea-pig, spermatogenesis, 1 70; matura- tion, 277. Heliozoa, 92, 103. Helix, 163, 168, 259. Ilenierocaliis, 306. Heterocope, tetrads, 250. Heterokinesis, 406. Histon, 334, 336. Homoeokinesis, 406. Hydrophiliis, orientation of &gg, 384. Id, in reduction, 245; in inheritance, 406. Idant, 245. Idioblast, 291. Idioplasm, theory of, 401; as chromatin, 403; action of, 406, 414, 431, 432. Idiosome, 291. Idiozome, 163, 165, 324. Ilyanassa, partial development, 419. Infusoria, nuclei, 31, 224; mitosis, 90; con- jugation, 223; reduction, 277. Inheritance, of acquired characters, 12, 433 ; Weismann's theory, 12; through the nucleus, 351-354; and metabolism, 430. Inotagmata, 291. Insect-eggs, 132, 386. Interzonal fibres, 70. Iris, 267. Isopods, metamerism, 390. Isotropy, of the egg, 384, 417. Karyokinesis (see Mitosis), 64. Karyokinetic figure (see Mitotic Figure), 69. Karyolymph, 36. Karyoplasm, 21. Karyosome, 34. Kinoplasm (archoplasm), 54, 77, 82, 173, 322. Lanthanin, 38. Lepidoptera, sex, 144. Leucocytes, structure, 102; division, 117; centrosome, 309; attraction-sphere, 326. Leucoplasts, of plant-ovum, 133. Z/7/?/;«, mitosis, 83; spireme, 112; fertiliza- tion, 219; reduction, 265-271. Umax, germ-nuclei, 204. liniulus, 158. Linin, 32; relations to cytoreticulum and chromatin, 302. liparis, 281. locusta, orientation of egg, 384. loligo, spindle, 81 ; cleavage, 381. lumbricus, yolk-nucleus, 157; reduction, 257- INDEX or SUBJECTS 481 Macrobdella, 305. Macrogamete, 226. Macromeres, 371. Mammals, spermatozoa, 139, 169; young ova, 155. Mantle-fibres, 78, 105. Mar sill a, 175. Maturation (see Reduction), 234; theoreti- cal significance, 243; of parthenogenetic eggs, 280; nucleus in, 353. Medusze, dwarf embryos, 410. ISIeristem, nuclei of, 340. Metamerism, 390. Metanucleus, 128. Metaphase, 69. Metaplasm, 19. Micellae, 291. Microcentrum, 31 1, 315, 324. Microgamete, 226. Micrumeres, 371. Micropyle, 124, 133. Microsomes, 23; of the egg-cytoplasm, 131 ; nature of, 2S9, 290, 293; of the astral sys- tems, 318, 326; of the nucleus, 301, 303; relation to centrosome, 315; staining- reactions, 337. Microsphere, 324. Microzyma, 291. Mid-body, 71, 78. Middle-piece, 135, 139; origin, 161, 165- 170; in fertilization, 187, 212, Mitosis, 64; general outline, 65; modifica- tions of, 77; heterotypical, 86; in unicellu- lar forms, 87; pathological, 88; multipolar, 97; mechanism of, 100; physiological sig- nificance, 351; Roux-Weismann concep- tion of, 245, 406. Mitosome, 165. Mitotic figure (see Mitosis, Spindle), 69; origin, 72; varieties, 78. Molgida, 158. Mouse, fertilization, 185, 193. Aftisca, ovum, 142. Myriapods, spermatozoa, 142; yolk-nucleus, 156. Myzostoma, fertilization, 196, 208. Naias, 266. Nebenkern, pancreas-cells, 44; of spermatid, 163, 165. Nebenkorper, 164, 165. Nectiiriis, pancreas-cells, 44. Nematodes, germ-nuclei, 184. Nereis, asters, 49; perivitelline layer, 131 ; ovum, 129; deutoplasm, 131 ; fertilization. 191 ; attraction-sj^here and centr<». .lue, 325; cleavage. 366, 36*); prcssurf-t-Mn ri- ments on, 411. Nerve-cell, 48. Net-knot, 34. i\W//7//<'(/, mitosis, 93; flagclluni, 171 ; con- jugation, 227; sphere, 319. Nuclear stains, 335. Nuclein, H, 332; staining-reactions, 334; physiological signilicancc, 340. Nuclein-bascs, 331. Nucleinic acid, T,}f, 332-334; stainmg-rcac- tions, 334 ; ])hysiological signiticance, 340. Nucleo-ali)umin, 331, 334. Nucleo-proteid, 331, 334. Nucleolus, }^T^\ in mitosis, 67; of the ovum, 126; physiological meaning, 128, Nucleoplasm, 21. Nucleus, general structure and functions, 31 ; finer structure, 37; polarity, 36, 294; chemistry, 41; in mitosis, 65; of the ovum, 123; of the spermatozoon, 135, 137; relation to cytoplasm, 302; morpht)|ogital composition, 294; in organic synthesis, 340, 430 ; physiolog)', 341; position and movements, 346; in fertilization, 181,352; in maturation, 353; in later tievelopmcnt, 425; in metabolism and inheritance, 430: in inheritance and development. 341, 35S, 405. 425, 431 ; control of the cell, 426. Nurse-cells, 15 1. CEdigoniiim, fertilization, 181; membrane, 346. Onoclea, 175. Oocyte, 236, Oogenesis, 234, 230. Oogonium, 236. Otisphere, 133. Op/iryotrochn, amitosis, 1 15; nursc-cclls, 151; fertilization, iS«), 193; tetrads, 25S. ((possum, siK-rmalozoa, 142. Organization, 289, 2<)l; i>f the nu- 1- 1. ^.^, 301; of the egg, 397, 433- Origin of species. 3. Ch/fiun,if plants. 133; origin and growth, 150; fertilization. 178; effects of spermatozoon upon. 201 ; maturation, 236; parthenogenetic, 280; promorphology, 378; bilaterality, 382. 2 I 482 INDEX OF SUBJECTS Oxychromatin, 38, 303; staining-reactions, 337- Oxydation-ferments, 351. Oxytricha, 342. Oyster, germ-nuclei, staining-reactions, 175. Pallavicuiia, reduction, 275. Pahidina, dimorphic spermatozoa, 141. Pangenesis, 12, 290, 431. Pangens, 291. Parachromatin, 41. Paralinin, 41, Parainceba, mitosis, 94, 315. /'«raw^<"?«;;/, mitosis, 91; conjugation, 224; reduction, 277. Paranucleus, 163. Parthenogenesis, theories of, 281; polar bodies in, 280. Pellicle, 54. Fentatoma^ 271. Felrotnyzon, fertilization, 192, 212. Phallusia, fertilization, 193, 212. Fhysa, fertilization, 193, 210, 212; reversed cleavage, 368. Physiological units, 289. Fieris, spinning-gland, 37. Pigment-cells, 102. Filnlaria, fertilization, 216. Fimis, reduction, 275. Flana7'ia, regeneration, 394. Plant-cells, plastids, 52; membranes, 54; mitosis, 82; cleavage-planes, 363. Plasma-stains, 335. Plasmocyte, 52. Plasmosome, 34. riasome, 291. Plastids, 52; of the ovum, 133; of the sper- matozoid, 143; conjugation of, 229. Plastidule, 291. Plastin, 41, 331. Plen7'ophyllidia, 78, 94. Podophyllum, 267. Polar bodies, 181 ; nature and mode of for- mation, 235-240; division, 236; in par- thenogenesis, 281. Polar rings, 156, 202, Polarity, of the nucleus, 36; of the cell, 55; of the ovum, 378; determination of, 382. Pole-plates, 91. Pollen-grains, formation, 263-265. Pollen-tube, 218. Polyclades, cleavage, 416. Poly cheer us, 276, 325. Polygordius, cleavage, 368. Polyspermy, 198; prevention of, 199. Polystomella, regeneration, 344. Polyzonium, 159. Porcellio, amitosis, 116. Predelineation, 398. Preformation (see Evolution). Pressure, experiments, 375, 410. Principal cone, loi. Pristiurus, 338. Promorphology (see Cleavage, Ovum). Pronuclei, 202. Prophase, 65. Prostheceraus, 213, 235, 256, 259, 306. Prosthiostovnim, 212. Protamin, 334. Proteids, 331. Prothallium, 264; chromosomes in, 275. Protoplasm, 4, 5, 17, 19; structure, 23,42, 293; chemistry, 331. Protoplast (see Plastid). Pseudo-alveolar structure, 50. Pseudo-reduction, 248. Pteris, 253. Pterotrachea, germ-nuclei, 186, 205. Ptychoptera, spireme-nuclei, 35. Pygara, 165. Pyrenin, 41. Pyrenoid, 133. Pyrrhoco7-is, 165, 248. Quadrille of centres, 210. Rat, spermatogenesis, 170. Reduction, general outline, 234; parallel between the two sexes, 241 ; theoretical significance, 243; detailed account, 246; in plants, 263; Strasburger's theory of, 275; in unicellular forms, 277; by conju- gation, 257; modes contrasted, 247. Regeneration, Weismann's theory, 406; in frog-embryo, 409; nature of, 425, 427; in coelenterates, 430; of lens, 433. Rejuvenescence, 179, 224. Keiiilla, ovum, 132, Rhabdoiejua, amitosis, 1 15. KhyncJielinis, fertilization, 192, 193, 212; cleavage, 370. Rotifers, sex, 145. Sagitta, number of chromosomes, 184; pri- mordial germ-cells, 146; germ-nuclei, 184; spermaster, 191. Salamander, epidermis, 3; spermatogonia, 20; mitosis in, 71, 78; pathological mito- sis, 98; leucocytes, 102; spermatozoa, 140; maturation, 259, IXDEX OF SCB/ECTS 483 Sargns, pigment-cells, 103. ScyUimii, 263. Segmentation (see Cleavage). Sehrginelia, spermatozoicls, 197. Senescence, 179. Sepia, spindle, 81. Sertoli-cells, 284. Sex, 9; determination of, 144; Minot's the- ory of, 243. Siphonophures, amitosis, 117. Soma, 13. Somacule, 291. Somatic cells, 122; number of chromosomes. 2-> T 33- Spermary, 123, Spermatid, 161, 163; development into sper- matozoon, 164. Spermatocyte, 161, 241. Spermatogenesis (see Reduction), 234; gen- eral outline, parallel with oogenesis, 241. Spermatogonium, 161, 241. Spermatozeugma, 142. Spermatozoid, structure and origin, 142, 172; in fertilization, 217, 221. Spermatozoon, discovery, 9; structure, 134; essential parts, 135; giant, 141 ; double, 142; unusual forms, 142; of plants, 142; formation, 1 60; in fertilization, 181, 192; entrance into ovum, 197. Sperm-centrosome, 135, 164-171; in fertili- zation, 192, 211-215, 221. Sperm-nucleus, 135; origin, 164-171; in fertilization, 182, 190; rotation, 188; path in the egg, 202; in inheritance, 353; chemistry, 334. Sphcir echinus, fertilization, 193, 210; num- ber of cells, 389; hybrids, 353; regenera- tion, 393. Spindle (see Amphiaster, Central Spindle) , 68 ; origin, 72, 79, 82; in IVotozoa,9o; conjuga- tion of, 227; nature of, 316; position, 375. Spireme, 65. Spirochona, mitosis, 90. Spirogyra, nucleolus, 67; amitosis, 1 19; conjugation, 229; reduction, 2S0. Spongioplasm, 25. Spontaneous generation, 7. Stem-cells, 148. Stentor, regeneration, 342. Siylonychia, senescence, 224. Stypocanlon, mitosis, 82. Siirirelia, 94. Syml)iosis, 53, 292. Syiiapta, cleavage, 364. Syncytium, 59. Teloblasts, 371, 390. Telophase, 71. Tetrads (Vicrcrgruppen), 238: origin, 246; in Ascaris, 241, 253; in arthropods, 248; ring-shaped, 248; in amphibia, 259; ori- gin by conjugation, 257; formulas for, 247- Tetramitus, 40, 92. Thalassema, spindle, 81 ; fcrtili/ati.m, 1^3, 194, 213; reduction, 259, 263; cenlro- some, 321 ; attraction-sphere, 325. Thalassicolla, experiments on, 344, Thysanozpon, 212, 259, 326. Tonoplast, 53. ToxopncHstes, cleavage, lO; mitosis, 107; ovum, 126; spermatozoon, 134; fertiliza- tion, 188; paths of germ-nuclei, 202; polar bodies, 114; double cleavage, 355. Trachelocerca, diffused nuclei, 40. Trilliiiin, 269, Tritou, 140, 212, 263, 277. Trophoplasm, 322, 401. Tiibiilaria, regeneration, 430. Tunicates, egg-axis, 379; cleavage, 381. Unicellular organisms, 5; mitosis, 88; con- jugation, 222; reduction, 277; experi- ments on, 342. Unio, centrosome and aster, 314; cleavage. 381. Urostyla, 40. Vacuole, 50, 53. J'linessa, ovarian egg, 153. Variations, ii; origin of, 433. Wutchcria, meml)rane, 348. \'italism, 394, 417. \'itelline membrane, 132; of egg-fragments 132; formation of, 198; function, I99. J'o/vox, germ-cells, 133. I'ordiellit, conjugation, 226. Xiphidiutn, 271. Vellow cells (of Kadiolaria), 53. Yolk (see Deulojilasm), 152. Volk-nucleus, 155. VulU-plates, 131. ZitrniiJ, 173, 221. ZirpJhca, 259, 263. Zwischenkorper (mid-body), 71. Zygnema, membrane, 346. Zygospore, 228. Columbia University Biological Series. EDITKI) r.V HENRY FAIRFIELD OSBORN, Da Contu PfofesHor cf Zai'i/nr/i/ in ('nh(i,itil,i f'iticer«itu A M > EDMUND B. WILSON, ProjetiHvr [»lication of tlie theory of Evolution. Thus the tirst course outlined the de- velopment of the Descent theory; the second, the a]>]»licati(>n of this theory to the problem of the ancestry of the Vertebrates, largely based upon embryological data; the third, the applica- tion of the Descent theory to the interpretation of the structure and phylogeny of the Fishes or lowest Vertebrates, cliietly based upon comparative anatomy ; the fourth, u])on the problems of individual development and Inheritance, chiefly based upon the structure and functions of the cell. Since their original delivery the lectures have been carofully rewritten and illustrated so as to adapt them to the use of (\d- lege and University students and of general readers. The vol- umes as at present arranged for include: I. From the Greeks to Darwin. By Henry Fairfield OSBORX. II. Aiiipliioxus and the Ancestry of tlie Vertebrates. By Arthur Wili.f.y. III. Fishes, Livinir and F'ossil. By Bashfokp Dkan. lY. The Cell in Development and Iiiiierilance. Uy Edmi^xd B. Wilson. T. The Foundations of Zooloirv. Bv Wimiam Kinn Brooks. THE MACMILLAN COMPANY. 66 FIFTH AVENUE, NEW YORK. I. FROM THE GREEKS TO DARWIN. THE DEVELOPMENT OF THE EVOLUTION IDEA. BY HENRY FAIRFIELD OSBORN, Sc.D., Princeton. Da Costa Professor of Zoology in Columbia University. 8vo. Cloth. $2.00, net. This opening volume, " From the Greeks to Darwin/' is an outline of the development from the earliest times of the idea of the origin of life by evolution. It brings together in a continu- ous treatment the progress of this idea from the Greek philoso- plier Thales (640 B.C.) to Darwin and Wallace. It is based partly upon critical studies of the original authorities, partly upon the studies of Zeller, Perrier, Quatrefages, Martin, and other writers less known to English readers. This history differs from the outlines which have been pre- viously published, in attempting to establish a complete conti- nuity of thought in the growth of the various elements in the Evolution idea, and especially in the more critical and exact study of the pre-Darwinian w^riters, such as Buffon, Goethe, Erasmus Darwin, Treviranus, Lamarck, and St. Hilaire, about whose actual share in the establishment of the Evolution theory vague ideas are still current. TABLE OF CONTENTS. I. The Anticipation^ and Interpretation of Nature. II. Among the Greeks. III. The Theologians and Natural Philosophers.. IV. The Evolutionists of the Eighteenth Century. V. From Lamarck to St. Hilaire. VI. The First Half-century and Darwin. In the opening chapter the elements and environment of the Evolution idea are discussed, and in the second chapter the re- markable parallelism between the growth of this idea in Greece and in modern times is pointed out. In the succeeding chap- ters the various periods of European thought on the subject are covered, concluding with the first half of the present century, especially with the development of the Evolution idea in the mind of Darwin. II. AMPHIOXUS AND THE ANCESTRY OF THE VERTEBRATES. BY ARTHUR WILLEY, B.Sc. LOND., Tutor in Biology, Columbia Fnirersit;/ : Balfour Student of th* Unirernity of Cambridge. 8vo. Cloth. $2.50, net. The purpose of this vohime is to consider tlie proV)h^ni of tlie ancestry of the Vertebrates from the stand i)oint of the anat- omy and development of Ampliioxns and other members of the group Protochordata. The work opens with an Introchiction, in which is given a brief historical sketch of the sj)ecii hit ions of the celebrated anatomists and embryologists, from Ktionnc Geoffroy St. Hilaire down to our own day, upon this problem. The remainder of the first and the whole of tlie second cliai»ter is devoted to a detailed account of the anatomy of Ampiiioxus as compared with that of higher Vertebrates. The third chapter deals with the embrvonic and larval development of Am}>hioxu8, while the fourth deals more briefly with the anatomy, embryoloiry, and relationships of the Ascidians; then the other allied forms, Balanoglossus, Cephalodiscus, are described. The work concludes with a series of di. the Prorlem or Vkktkhkati: Descent. III. FISHES, LIVING AND FOSSIL. ^.Y IXTRODUCrORY STUDY. BY BASHFORD DEAN, Ph.D., Columbia, Instructor in Biology, Columbia University. 8vo. Cloth. $2.50, net. This work has been prepared to meet the needs of the oren- eral student for a concise knowledge of the Fishes. -It contains a review of the four larger groups of the strictly fishlike fornis, Sharks, Chimaeroids, Teleostomes, and the Dipnoans, and adds to this a chapter on the Lampreys. It presents in figures the prominent members, living and fossil, of each group; illustrates characteristic structures; adds notes upon the important phases of development, and formulates the views of investigators as to relationships and descent. The recent contributions to the knowledge of extinct Fishes are taken into special account in the treatment of the entire subject, and restorations have been attempted, as of Dinichthys, Ctenodus, and Cladoselache. The writer has also indicated diagrammatically, as far a? generally accepted, the genetid' relationships of fossil and living forms. The aim of the book has been mainlv to furnish the student with a well-marked ground-plan of Ichthyology, to enable him to better understand special works, such as those of Smith Wood- ward and Giinther. The work is fullv illustrated, mainlv from the writer's original pen-drawings. TABLE OF CONTENTS. CHAPTER I. Fishes. Their Esseniial Characters. Siiarks, Chimaeroids, Teleo- stomes, aud Lung-tishes. Their Appearance iu Time and their Distribution. II. The Lampreys. Their Position with Reference to Fishes. Bdel- lostoma, jVIyxine, Petromyzon, Palaeospondylus. IIL The Shakk Group. Anatomical Cluiracters. Its E.xtiuct 3Iembers, AcaLthodiau, Cladoselachid, Xeuacauthid, Cestraciouts. IV. Chimaer