KiHiffiS i pliii Sir " Marine Biological Laboratory WOODS HOLE. MASSACHUSETTS IN MEMORY OF Edward Gardiner Gardiner 1854-1907 I THE CELL IN DEVELOPMENT AND INHERITANCE Columbia SEnibrrsi'tu Biological Series. EDITED BY HENRY FAIRFIELD OSBORN. I 1. FROM THE GREEKS 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, LIVING 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. COLUMBIA UNIVERSITY BIOLOGICAL SERIES. LV. THE CELL IN Development and Inheritance BY EDMUND B. WILSON, Ph.D. PROFESSOR OF INVERTEBRATE ZOOLOGY, COLUMBIA UNIVERSITY " Natura nusquam magis est tota quam in minimis " PI.INY THE MACMILLAN COMPANY LONDON: MACMILLAN & CO., Ltd. 1896 All rights reserved Copyright, 1896, By the MACMILLAN COMPANY. 3 3// ICorliioolj ^rcss J. S. Ciisliing & Co. - Berwick & Smith Norwood Mass. I'.S.A. 2E0 mg Jricuti THEODOR BOVERI V 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 Zcllc iind Gctvcbe 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 egg, — 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 Hertvvig.^ 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^otis sur la cellule is received, too late for further notice, as this volume is going through the press. - Allen Thomson. > PREFACE ix inv^estigators, 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. 343). 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. VV. Columbia Univeksitv, Xew York, July, 1S96. TABLE OF CONTENTS INTRODUCTION List of Figures Historical Sketch of the Cell-theory; its Relation to the Evolution-theory. Earlier Views of Inheritance and Development. Discovery of the Germ-cells. Cell- division, Cleavage, and Development. Modern Theories of Inheritance. Lamarck, Darwin, and Weismann ........... Literature ..........••••• PAGE XV I 12 CHAPTER I General Sketch of the Cell A. General Morphology of the Cell . B. Structural Basis of Protoplasm C. The Nucleus .... 1. General Structure 2. Finer Structure of the Nucleus 3. Chemistry of the Nucleus . D. The Cytoplasm E. The Centrosome F. Other Cell-organs . G. The Cell-membrane H. Polarity of the Cell I. The Cell in Relation to the Multicellular Body Literature, I. ...... . 14 17 27 28 29 30 37 38 38 41 43 CHAPTER II Cell-Division A. Outline of Indirect Division or Mitosis . B. Origin of the Mitotic Figure .... C. Modifications of Mitosis ..... 1. \^arieties of the Mitotic Figure . 2. Heterotypical Mitosis .... Bivalent and Plurivalent Chromosomes Mitosis in the Unicellular Plants and Animals Patliological Mitoses ..... I). The Mechanism of Mitosis ..... 1. Function of the Amphiaster ()/) Theory of I'ihrillar Cijutractility . {l>) Other Theories .... 2. Division of the Chromosomes xi J- 4- 5- 47 53 57 57 00 61 62 f'7 70 70 70 75 77 Xll TABLE OF CONTENTS E.' Direct or Amitotic Division 1. General Sketch ...... 2. Centrosome and Attraction-sphere in Amitosis 3. Biological Significance of Amitosis F. Summary and Conclusion ..... Literature, II. . . • PAGE 80 80 81 82 85 86 CHAPTER III The Gekm-Cells A. The. Ovum 1. The Nucleus 2. The Cytoplasm . 3. The Egg-envelopes B. The Spermatozoon 1. The Flagellate Spermatozoon 2. Other Forms of Spermatozoa 3. Paternal Germ-cells of Plants C. Origin and Growth of the Germ-cells . D. Growth and Differentiation of the Germ-cells 1. The Ovum ..... («) Growth and Nutrition . (^) Differentiation of the Cytoplasm. () Composition of the Chromosomes Chromatin, Linin, and the Cytoret The Centrosome . The Archoplasmic Structures 1. Asters and Spindle 2. The Attraction-sphere Summary and Conclusion culum the Chromosomes A. B. C. D. E. F. Literature, VI. .... CHAPTER VII Some Aspects of Cell-Chemistry and Cell-Physiology A. Chemical Relations of Nucleus and Cytoplasm 1. The Proteids and their Allies 2. The Nuclein Series .... 3. Staining-reactions of the Nuclein Series B. Physiological Relations of Nucleus and Cytoplasm Experiments on Unicellular Organisms Position and Movements of the Nucleus The Nucleus in Mitosis The Nucleus in Fertilization The Nucleus in Maturation C. The Centrosome . . . ■ D. Summary and Conclusion I. 2. 3- 4- 5- Literature, VII. PAGE 198 199 199 202 205 208 208 209 209 211 212 214 215 221 223 224 229 229 232 236 237 238 239 240 242 248 248 252 256 257 259 259 261 26'? CHAPTER VIII Cell-Division and Development A. Geometrical Relations of Cleavage-forms .... B. Promorphological Relations of Cleavage .... 1. Promorphology of the Ovum . . . • • (a) Polarity and the Egg-axis .... {b) Axial Relations of the Primary Cleavage-planes \c~) Other Promorphological Characters of the Ovum 2. Meaning of the Promorphology of the Ovum . 265 278 278 278 280 282 285 XIV TABLE OF CONTENTS C. The Energy of Division D. Cell-division and Growth Literature, VIII. . 289 293 294 CHAPTER IX Theories of Inhekhance and Development The Theory of Germinal Localization . The Idioplasm Theory .... Union of the Two Theories . The Roux-Weismann Theory of Development Critique of the Roux-Weismann Theory On the Nature and Causes of Differentiation The Nucleus in Later Development The External Conditions of Development Development, Inheritance, and Metabolism Preformation and Epigenesis. The Unknown Factor in Development A. B. C. D. E. F. G. H. I. J- Literature, IX. Glossary . . . . General Literature-List Index ok Authors . Index of Subjects 296 ;oo ;o2 306 3" 321 526 327 33 o 343 359 365 LIST OF FIGURES lo. II. 12. 13- 14. 15- 16. 17- 18. 19- 20. 21. 22. 23- 24. 26. 27. 28. 29. 30. 31- 32. 33- 34- 35- 36. 37- 38. 39- 40. 41- 42- PAGE Epidermis of larval salamander 2 Amceba Proteus 4 Cleavage of the ovum in Toxopneustes 8 Diagram of inheritance 11 Diagram of a cell 14 Spermatogonium of salamander 15 Group of cells, showing cytoplasm, nu- cleus, and centrosome 16 Alveolar or foam-structure of proto- plasm, according to Biitschli 18 Living cells of salamander, showing fibrillar structure 20 Nuclei from the crypts of Lieberkiihn. 24 Special forms of nuclei 25 Diffused nucleus in Trackelocerca. ... 26 Ciliated cells 30 Nephridial cell of Clepsine 32 Nerve-cell of frog 33 Diagram of dividing cell 35 Diagrams of cell-polarity 39 Remak's scheme of cell-division 46 Diagram of the prophases of mitosis. . 48 Diagram of later phases of mitosis. ... 50 Prophases in salamander cells 54 Metaphase and anaphases in salaman- der cells 55 Telophases in salamander cells 56 Middle phases of mitosis in Ascaris- eggs 58 Mitosis in pollen-mother-cells of lily. . 59 Heterotypical mitosis 60 Mitosis in Infusoria 62 Mitosis in Euglypha 63 Mitosis in Euglena 64 Mitosis in Noctiluca 65 Mitosis in Actiiiosphcsrium 66 Pathological mitoses in cancer-cells.. . 68 Pathological mitosis caused by poisons 69 Mechanism of mitosis in Ascaris 71 Leucocytes 72 Pigment-cells 73 Mitosis in the egg of Toxopneustes. ... 76 Nuclei in the spireme-stage 78 Early division of chromatin in Ascaris 79 Amitotic division 81 Volvox 89 Ovum of Toxopneustes gi 43- 44- 45- 46. 47- 48. 49- 50- 51- 52- 53- 54- 55- 56. 57. 58. 59- 60. 61. 62. 63- 64. 65- 66. 67. 68. 69. 70. 71- 72. 73- 74- 75- 76. 77- 79- 80. 81. 82. PAGE Ovum of Nereis 95 Insect-egg 96 Micropyle in Argonauta 97 Germ-cells of / 'olvox 98 Diagram of the flagellate spermatozoon 99 Spermatozoa of fishes and amphibia. . 100 Spermatozoa of birds and other ani- mals 102 Spermatozoa of mammals 104 Unusual forms of spermatozoa 105 Spermatozoids of Ckara 106 Spermatozoids of various plants 107 Germ-cells of Hydractinia 109 Primordial germ-cells of Ascaris, no Primordial germ-cells of Cyclops 112 Egg and nurse-cell in Opkryotrocha . . . 114 Ovarian eggs of insects 115 Young ovarian eggs of various animals 116 Young ovarian eggs of birds and mam- mals iiS Young ovarian eggs of earthworm. . . 120 Formation of the spermatozoon 124 Transformation of the spermatids of the salamander 125 Fertilization of Physa 131 Fertilization of Ascaris 133 Germ-nuclei of nematodes 134 Fertilization of the mouse 136 Fertilization of Pterotrachea 137 Entrance and rotation of sperm-head in Toxopneustes 138 Conjugation of the germ-nuclei in Tox- opneustes 139 Fertilization of Nereis 141 Fertilization of Cyclops 142 Continuity of centrosomes in 'Ihalas- seina 144 Entrance of spermatozoon into the egg 146 Pathological polyspermy 147 Polar rings of Clepsine 150 Paths of the germ-nuclei in Toxo- pneustes 152 Fertilization of Myzostonia 158 Fertilization of Pilularia 160 Fertilization of the lily 161 Diagram of conjugation in Infusori.i. . 164 Conjugation of Paramaccium 166 XVI LIST OF FIGURES 83- 84. 85- 86. 87. 88. 8g. 90. 91- 92. 93- 94- 95- 96. 97- 98. 99. 102. 103. 104. 106. 107. 108. 109. no. PAGE Conjugation of I 'orticella 167 112. Conjugation of .\octiliica 168 j 113. Conjugation of Spi7-ogyra 169 ! Polar bodies in Toxopneustes 174 j 114. Genesis of the egg 175 ' 1 15. Diagram of formation of polar bodies 177 ' 116. Polar bodies in .-{scans 178 i Genesis of the spermatozoon 180 j 117. Diagram of reduction in the male. .. . 181 ! 118. Spermatogenesis of Asca) is 184 ' Tetrads of Gryllotalpa 188 I 119. Tetrads and polar bodies in Cyclops.. 189 Diagrams of tetrad-formation in ar- ! 120. thropods 191 121. Germinal vesicles and tetrads 192 122. Ovary of Caiitkocamptiis 194 123. Possible tetrad-formation in the lily. . 197 124. Conjugation and reduction in Closte- 125. rnim 198 126. First type of parthenogenetic matura- 127. tion in Ariemia 203 128. Second type of parthenogenetic mat- 129. uration in .Irtemia 204 130. Modes of tetrad-formation contrasted 206 131. .Abnormalities in the fertilization of 132. .hcaris 216 133. Individuality of chromosomes in .As- caris 217 134. Independence of chromosomes in fer- tilization of Cyclops 218 135. Hybrid fertilization of .4scaris 220 Mitosis with intra-nuclear centrosome' 136. in .Ascar/s 225 Diagram of different types of centro- , 137. some and centrosphere 233 Structure of the aster in spermatogo- 138. nium of salamander 234 139. History of chromosomes in the germi- 140. nal vesicle of sharks 245 141. Nucleated and enucleated fragments of Stylonychia 249 142. P.AGE Regeneration in Stentor 250 Nucleated and enucleated fragments of Amoeba 25 1 Position of nuclei in plant-cells 253 Ovary of Forficula 255 Normal and dwarf larvae of sea- urchins 258 Supernumerary centrosome in .Ascaris 260 Cleavage of dispermic egg of Toxo- pneustes 261 Geometrical relations of cleavage- planes in plants 266 Cleavage of Synapta 268 Cleavage of Polygordius 269 Cleavage of Nereis 271 Variations in the third cleavage 272 Meroblastic cleavage in the squid. . . . 273 Teloblasts of the earthworm 274 Bilateral cleavage in tunicates 281 Bilateral cleavage in Loligo 282 Eggs of Loligo 283 Eggs and embryos of Corixa 284 Variations in axial relations of Cyclops 286 Half-embryos of the frog 299 Half and whole cleavage in sea-urchins 306 Normal and dwarf gastrulas of Amptii- oxus 307 Dwarf and double embryos of .Amphi- o.vus 308 Cleavage of sea-urchin eggs under pressure 309 Cleavage of yV^rifw-eggs under press- ure 310 Diagrams of cleavage in annelids and polyclades 313 Partial larvae of ctenophores 314 Partial cleavage in Ilyaiiassa 316 Double embryos of frog 318 Normal and modified larvse of sea- urchins 324 Regeneration in coelenterates 325 INTRODUCTION -ooJ^^Oo- "Jedes Thier erscheint ah eine Sunune vitaUr Einheiten, von denen jede den vollen Charakter des Lebens an sich tragt." ViRCHOW.' During the half-century that has elapsed since the enunciation of the cell-theory by Schleiden and Schwann, in 1838-39, it has become ever more clearly apparent that the key to all ultimate biological problems must, in the last analysis, be sought in the cell. It was the cell-theory that first brought the structure of plants and animals under one point of view by revealing their common plan of organization. It was through the cell-theory that Kolliker and Remak opened the way to an understanding of the nature of embryological development, and the law of genetic continuity lying at the basis of inheritance. It was the cell-theory again which, in the hands of Virchow and Max Schultze, inaugurated a new era in the history of physiology and pathology, by showing that all the various functions of the body, in health and in disease, are but the outward expression of cell-activi- ties. And at a still later day it was through the cell-theory that Hert- wig, Fol, Van Beneden, and Strasburger solved the long-standing riddle of the fertilization of the Qgg, and the mechanism of hereditary transmission. No other biological generalization, save only the theory of organic evolution, has brought so many apparently diverse phe- nomena under a common point of view or has accomplished more for the unification of knowledge. The cell-theory must therefore be placed beside the evolution-theory as one of the foundation stones of modern biology. And yet the historian of latter-day biology cannot fail to be struck with the fact that these two great generalizations, nearly related as they are, have been developed along widely different lines of research, and have only within a very recent period met upon a common ground. The theory of evolution originally grew out of the study of natural history, and it took definite shape long before the ultimate structure of living bodies was in any degree comprehended. The evolutionists ^ Cellnlarpathologie , p. 12, 1S5S. B I ^ INTRODUCTION of the Lamarckian period gave little heed to the finer details of internal organization. They were concerned mainly with the more obvious characters of plants and animals — their forms, colours habits, distribution, their anatomy and embryonic development — and witli the systems of classification based upon such characters- and long afterwards it was, in the main, the study of like characters with reference to their historical origin that led Darwin to his splen- a F IWt'-'^^-'^^ \ I "^l^^^^r X b did triumphs. The study of micro.scopical anatomy, on which the cell-theory was based, lay in a different field. It was begun and long carried forward with no thought of its bearing on the oHgin of living forms; and ev;en at the present day the fundamental problems k organization with which the cell-theory deals, are far less accessible o2Trt "T""^\ '^'" '^"■'^^ suggested by the more obvious, cxteinal characters of plants and animals. Only within a few years INTR OD UCTION 3 indeed, has the ground been cleared for that close alliance of the evolutionists and the cytologists which forms so striking a feature of contemporary biology. We may best examine the steps by which this alliance has been effected by an outline of the cell-theory, fol- lowed by a brief statement of its historical connection with the evolu- tion-theory. 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 may 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 anatomy has shown us the nature of the material on which it has operated, demonstrating that the obvious characters of plants and animals are but varying 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- 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 (Fig. i). 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. The microscope shows it to be an aggre- gate composed of innumerable minute bodies, as if it were a colony or congeries of organisms more elementary than itself. These elemen- tary bodies, the cells, are essentially minute masses of living matter ox protoplasm, a substance characterized by Huxley many years ago as the "physical basis of life " and now universally recognized as the immediate substratum of all vital action. Endlessly diversified in the details of their form and structure, cells nevertheless possess a charac- teristic type of organization common to them all ; hence, in a certain sense, they may be regarded as elementary organic units out of which the body is compounded. In the lowest forms of life the entire body consists of but a single cell (Fig. 2). In the higher multi- cellular forms the body consists of a multitude of such cells asso- ciated in 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. ^ From the physiological point of view a like comparison may be drawn. In the one-celled forms all of the • This comparison must he taken with some reservation, as will appear beyond. 4 INTRODUCTION vital functions are performed by a single cell ; in the higher types they are distributed by a physiological division of labour among different groups of cells specially devoted to the performance of specific functions. The cell is therefore not only a unit of structure, but also a unit of function. " It is the cell to which the consideration of every bodily function sooner or later drives us. In the muscle- cell lies the riddle of the heart-beat, or of muscular contraction ; in the gland-cell are 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 slumbering in the ganglion-cell. ... If then physiology is not to rest content with the mere extension of our ■;-.v-..N • -■.;-■:■-■*«- ■• '-.{1 ;-i f ■• c (""n ^i^'To '.V ~ C V Fig. 2. — Amxba Proteus, an animal consisting of a single naked cell, X 280. (From Sedgwick and Wilson's Biology.) n. The nucleus ; w.v. Water-vacuoles ; c.v. Contractile vacuole ; f.v. Food-vacuole. knowledge regarding the more obvious operations of the human body, if it would seek a real explanation of the fundamental phe- nomena of life, it can only attain its end through the study of ccll- pJiysiologyy ^ Great as was the impulse which the cell-theory gave to anatomical and physiological investigation, it 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, if we except the theory of pangenesis, did Darwin attempt at any later period to bring it into any very definite relation to his views. The cell-theory first came in contact with the evolution-theory nearly twenty years ^ Verworn, AUgeiiteine Physiologic, p. 53, 1895. INTRODUCTION later through 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 by 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. The identification of the ccll-nncleus as the vehicle of inheritance, made independently and almost simultaneously in 1884-85 by Oscar Hertwig, Strasburger, Kblliker, and Weismann, must be recognized as the first definite advance '1 towards the internal problems of inheritance through the cell-theory ; and the discussions to which it gave rise, in which Weis- mann has taken the foremost place, must be reckoned as the most interesting and significant of the post-Darwinian period. These discussions have set forth in strong relief the truth that the general problems of evolution and heredity are indissolubly bound up with those of cell-structure and cell-action. This can best be appreciated from an historical point of view. The views of the early embryologists 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 ^zg^ he says, "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 egg 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 approximationto the modern doctrine of germinal continuity about which all theories of heredity are revolving. To the modern student the germ is, m Huxley's words, simplv a detached living portion of the substance of a pre-existing living body^ carrying with it a definite structural organization characteristic of the species. Harvey's view is only superficiallv similar to this ; for, as Huxley pointed out, it was obscured by his belief that the germ might arise "spontaneously," or through 1 It must not be forgotten that Haeckel expressed the same vieu- in 1866— only how- ever, as a speculation, since the data necessary to an inductive conclusion were not obtained until long afterwards. "The internal nucleus provides for the transmission of heredUary characters, the external plasma on the other hand for accommodation or adaptation to the external world" {Gen. Morph., p. 287-9). -De Genera Hone, 1651; Trans., p. 271. 3 Evolution in Biology, 1878; Science and Culture, p. 291. 6 INTRODUCTION the influence of a mysterious " calidniii iuuatiiiu,'' out of not-living matter. Whitman, too, in a recent brilHant essay,i has shown how- far Harvey was from any real grasp of the law of genetic continuity, which is well characterized as the central fact of modern biology. Neither could the great physiologist of the seventeenth century have had the remotest conception of the actual structure of the egg. The cellular structure of living 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 specu- lation. For a hundred years after Harvey's time embryologists sought in vain to penetrate the mysteries enveloping the beginning of the individual life, and despite their failure the controversial writ- ings of this period form one of the most interesting chapters in the history of biology. By the extreme "evolutionists" or " praeforma- tionists" the &gg was believed to contain an embryo fully formed in miniature, as the bud contains the flower or the chrysalis the butter- fly. Development was to them merely the unfolding of that which already existed ; inheritance, the handing down from parent to child of an infinitesimal reproduction of its own body. It was the service of Bonnet to push this conception to its logical consequence, the theory of cviboitcmcnt or encasement, and thus to demonstrate the absurdity of its grosser forms ; for 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 infinitiiui, like an infinite series of boxes, one within another — hence the term " emboitement." Bonnet himself renounced this doctrine in his later writings, and Caspar Frederich Wolff (1759) led the way in a return to the teach- ings of Harvey, showing by precise actual observation that the egg does not at first contain any formed embryo whatever ; that the struct- ure is wholly different from that of the adult ; that development is not a mere process of unfolding, but a progressive process, involving the continual formation, one after another, of new parts, previously non- existent as such. This is somewhat as Harvey, himself following Aristotle, had conceived it — a process of epigoicsis 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) ^I'ld his immediate followers to recognize the fact, conclusively demonstrated by all later researches, that the ej^x is a eel! having the same essential structure as other cells of the '^Evolution and Epigenesis, Wood's HoU Biological Lectures, 1894. INTRODUCTION 7 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 afterwards 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- ment, and therefore regarded by the early observers as parasitic animalcules or infusoria, a view which gave rise to the name spcruia- to.zoa (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- ing 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 Qgg, 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 .-^ Schwann and Schleiden held that cells might arise in two different ways ; viz. either by the division or fission of a pre-exi.sting mother- cell, or by " free cell-formation," new cells arising in the latter case not from pre-existing cells, but by crystallizing, as it were, out of a formative or nutritive substance, termed the " cytoblastema." It was only after many years of painstaking research that " free ccll- 1 The discovery of the spermatozoa is generally accredited to Ludwig Ilamm, a pupil of Leeuwenhoek (1677), though Hartsoeker afterwards claimed the merit of having seen them as early as 1674 (Dr. Allen Thomson"). 8 INTRODUCTION formation " was absolutely proved to be a myth, though many of Schwann's immediate followers threw doubts upon it, and as early as 1855 Virchow positively maintained the universality of cell-divis- ion, contending that every cell is the offspring of a pre-existing parent-cell, and summing up in the since famous aphorism, " omnis A B H C 1 Fig. 3. — Cleavage of the ovum of the sea-urchin Toxopneusles, X 330, from hfe. The suc- cessive divisions up t'o the i6-cell stage {H) occupy about two hours. / is a section of the embryo (blastula) of three hours, consisting of approximately 128 cells surrounding a central cavity or blastocoel. celbila e cellular^ At the present day this conclusion rests upon a foundation so firm that we are 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 1 Arch, filr Path. Ana/., VIII., p. 23, 1855. INTR ODUC TION 9 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 ^g^ into two parts, each of which is a cell, like the Q.gg itself. The two then divide in turn to form four, eight, sixteen, and so on in more or less regular progres- sion (Fig. 3) until step by step the ^fgg has spHt up into the multitude of cells which build the body of the embryo, and finally of the adult. This process, known as the cleavage or segmentation of the egg, was observed long before its meaning was understood. It seems to have been first definitely described in the case of the frog's egg, by Prevost and Dumas (1824), though earlier observers had seen it; but at this time neither the &gg nor its descendants were known to be cells, and its true meaning was first clearly perceived by Bergmann, Kolliker, Reichert, von Baer, and Remak, some twenty years later. The interpretation of cleavage as a process of cell-division was fol- lowed by the demonstration that cell-division does not begin with cleavage, but can be traced back into the foregoing generation ; for the egg-cell, as well as the sperm-cell, arises by the division of a cell pre- existing in the parent-body. It is therefore deidved by direct descent from an egg-cell of the foregoing generation, and so on ad infimtnm. Embryologists thus arrived at the conception so vividly set forth by Virchow in 1858 ^ of an uninterrupted series of cell-divisions extend- ing backw^ard from existing plants and animals to that remote and unknown period when vital organization assumed its present form. Life is a continuous stream. The death of the individual involves no breach of continuity in the series of cell-divisions by which the life of the race flows onwards. The individual body dies, it is true, but the germ-cells live on, carrying with them, as it were, the traditions of the race from which they have sprung, and handing them on to their descendants. These facts clearly define the problems of heredity and variation as they confront the investigator of the present day. All theories of evolution take as fundamental postulates the facts of variation and heredity ; for it is by variation that new characters arise and by heredity that they are perpetuated. Darwin recognized two kinds of variation, both of which, being inherited and maintained through the conserving action of natural selection, might give rise to a permanent transformation of species. The first of these includes congenital or inborn variations ; i.e. such as appear at birth or are developed "spontaneously," without discoverable connection with the activities of the organism itself or the direct effect of the environment upon it. In a second class of variations are placed the so-called acquired char- 1 See tlie quotation from the original edition of the Celliilarpatliologie at the head of Chapter II., p. 45. lO INTRODUCTION acters ; i.e. changes that arise in the course of the individual Ufe as the effect of use and disuse, or of food, cHmate, and the Hke. The inheritance of congenital characters is now universally admitted, but it is otherwise v^'ith acquired characters. The inheritance of the latter, now the most debated question of biology, had been taken for granted by Lamarck a half-century before Darwin ; but he made no attempt to show how such transmission is possible. Darwin, on the other hand, squarely faced the physiological requirements of the prob- lem, recognizing that the transmission of acquired characters can only be possible under the assumption that the germ-cell definitely reacts to all other cells of the body in such wise as to register the changes taking place in them. In his ingenious and carefully elab- orated theory of pangenesis, ^ Darwin framed a provisional physio- logical hypothesis of inheritance in accordance with this assumption, suggesting that the germ-cells are reservoirs of minute germs or gemmules derived from every part of the body ; and on this basis he endeavoured to explain the transmission both of acquired and of con- genital variations, reviewing the facts of variation and inheritance with wonderful skill, and building up a theory which, although it forms the most speculative and hypothetical portion of his writings, must always be reckoned one of his most interesting contributions to science. The theory of pangenesis has been generally abandoned in spite of the ingenious attempt to remodel it made by Brooks in 1883.^ In the same year the whole aspect of the problem was changed, and a new period of discussion inaugurated by Weismann, who put forth a bold challenge of the entire Lamarckian principle.'^ "I do not propose to treat of the whole problem of heredity, but only of a certain aspect of it, — the transmission of acquired characters, which has been hitherto assumed to occur. In taking this course I may say that it was impossible to avoid going back to the foundation of all phenomena of heredity, and to determine the substance with which they must be connected. In my opinion this can only be the sub- stance of the germ-cells ; and this substance transfers its hereditary tendencies from generation to generation, at first unchanged, and always uninfluenced in any corresponding manner, by that which happens during the life of the individual which bears it. If these views be correct, all our ideas upon the transformation of species re- quire thorough modification, for the whole principle of evolution by means of exercise (use and disuse) as professed by Lamarck, and accepted in some cases bv Darwin, entirely collapses " {I.e., p. 69). 1 Variation of .Animals and Plants, Chapter XXVIl. - The Im7u of Heredity, Baltimore, 18S3. 3 Ueber Vcrerhiin:^, 1S83. See Essays jipon Heredity, I., by A. Weismann, C'laremloii Press, Oxford, 1S89. iNrRODUcriox 1 1 It is impossible, he continues, that acquired traits should be trans- mitted, for it is inconceivable that definite changes in the body, or " soma," should so affect the protoplasm of the germ-cells, as to cause corresponding changes to appear in the offspring. How, he asks, can the increased dexterity and power in the hand of a trained piano- player so affect the molecular structure of the germ-cells as to produce a corresponding development in the hand of the child ? It is a physi- ological impossibility. If we turn to the facts, w^e find, Weismann affirms, that not one of the asserted cases of transmission of acquired characters will stand the test of rigid scientific scrutiny. It is a reversal of the true point of view to regard inheritance as taking place from the body of the parent to that of the child. The child inherits from the parent germ-cell, not from the parent-body, and the germ-cell owes its characteristics not to the body which bears it, but to its descent from a pre-existing germ-cell of the same kind. Thus the body is, as it were, an offshoot from the germ-cell (Fig. 4). As " .S Line of succession. (T) Line of inheritance. G Fig. 4. — Diagram illustrating Weismann's theory of inheritance. G. The germ-cell, which by division gives rise to the body or soma (5) and to new germ-cells (f?) which separate from the soma and repeat the process in each successive generation. far as inheritance is concerned, the body is merely the carrier of the germ-cells, which are held in trust for coming generations. Weismann's subsequent theories, built on this foundation, have given rise to the most eagerly contested controversies of the post- Darwinian period, and, whether they are to stand or fall, have played a most important part in the progress of science. For aside from the truth or error of his special theories, it has been Weismann's great service to place the keystone between the work of the evolutionists and that of the cytologists, and thus to bring the cell-theory and the evolution-theory into organic connection. It is from this point of view that the present volume has been written. It has been my endeavour to treat the cell primarily as the organ of inheritance and development ; but, obviously, this aspect of the cell can only be apprehended through a study of the general phenomena of cell-life. The order of treatment, which is a convenient rather than a strictly logical one, is as follows : — The opening chapter is devoted to a general sketch of cell-struct- 1 2 INTR OD UCTION ure, and the second to the phenomena of cell-division. The follow- ing three chapters deal with the germ-cells, — the third with their structure and mode of origin, the fourth with their union in fertiliza- tion, the fifth with the phenomena of maturation by which they are prepared for their union. The sixth chapter contains a critical dis- cussion of cell-organization, completing the morphological analysis of the cell. In the seventh chapter the cell is considered with reference to its more fundamental chemical and physiological properties as a^ prelude to the examination of development which follows. The suc- ceeding chapter approaches the objective point of the book by con- sidering the cleavage of the ovum and the general laws of cell-division of which it is an expression. The ninth chapter, finally, deals with the elementary operations of development considered as cell-functions and with the theories of inheritance and development based upon them. SOxME GENERAL WORKS ON THE CELL-THEORY Bergh, R. S. — Vorlesungen liber die Zelle unci die einfachen Gewebe : Wiesbaden, 1S94. Delage, Yves. — La Structure du Protoplasma et les Theories sur Tlieredite et les grands Problemes de la Biologie Generale : Paris, 1895. Geddes & Thompson. — The Evolution of Sex : Ne-d) York, 1890. Henneguy, L. F. — Lecons sur la cellule : Paris. 1896. Hertwig, 0. — Die Zelle und die Gewebe: Fischer, Jena, 1892. Translation, pub- lished by Macinillaii, Londoti and N'ew York, 1895. Huxley, T. H. — Review of the Cell-theory : British and Foreign Medico-Chirurgical Review, XH. 1853. Minot, C. S. — Human Embryology: New York, 1892. Remak, R. — Laitersuchungen liber die Entwicklung der Wirbelthiere : BerliUy 1850-55. Schleideri, M. J. — Beitrage zur Phytogenesis : Miiller's Archiv, 1838. Translation in Sydenham Soc, XH. London. 1847. Schwann, Th. — Mikroscopische Untersuchungen liber die Uebereinstimmung in der Structur und dem Wachsthum der Thiere und Pflanzen : Berlin. 1839. Translation in Sydenham Soc, XII. London, 1847. Tyson, James. — The Cell-doctrine, 2d ed. Philadelpliia. 1878. Virchow, R. — Die Cellularpathologie in ihrer Begrlindung auf physiologische und pathologische Gewebelehre. Berlin. 1858. Weismann, A. — Essays on Heredity. Translation: First series, Oxford, 1891 ; Second series, Oxford. 1892. Id. — The Germ-plasm. IVew York, 1893. CHAPTER I GENERAL SKETCH OF THE CELL " Wir haben gesehen, dass alle Organismen aus wesentlich gleichen Theilen, namlich aus Zellen zusammengesetzt sind, dass diese Zellen nach wesentlich denselben Gesetzen sich bilden und wachsen, dass also diese Prozesse iiberall auch durch dieselben Krafte hervorge- bracht werden miissen." Schwann. ^ The term " cell " is a biological misnomer ; for whatever the living cell is, it is not, as the word implies, a hollow chamber surrounded by solid walls. The term is merely an historical survival of a word casually employed by the botanists of the seventeenth century to designate the cells of certain plant-tissues which, when viewed in section, give somewhat the appearance of a honeycomb.- The cells of these tissues are, in fact, separated by conspicuous solid walls which were mistaken by Schleiden, unfortunately followed by Schwann in this regard, for their essential part. The living sub- stance contained within the walls, to which Hugo von Mohl gave the n3.Vi\Q protoplasm^ (1846) was at first overlooked or was regarded as a waste-product, a view based upon the fact that in many im- portant plant-tissues such as cork or wood it may wholly disappear, leaving only the lifeless walls. The researches of Bergmann, Kolliker, Bischoff, Cohn, Max Schultze, and many others, showed, however, that some kinds of cells, for example, the corpuscles of the blood, are naked masses of living protoplasm not surrounded bv walls, — a fact which proves that not the wall, but the cell-contents, is the essential part, and must therefore be the seat of life. It was found further that with the possible exception of some of the lowest forms of life, such as the bacteria, the protoplasm invariably contains a definite rounded body, the nucleus,'^ which in turn may contain a still ^ UnteisHchungen, p. 227, 1839. - The word seems to have been first employed by Robert Hooke, in 1665, to designate the minute cavities observed in cork, a tissue which he described as made up of " little boxes or cells distinct from one another" and separated by solid walls. ^ The same word had been used by Purkyne some years before (1840) to designate the formative material of young animal embryos. ■* First described by Robert Brown in 1833. 13 14 GENERAL SKETCH OE THE CELL smaller body, the nucleolus. Thus the cell came to be defined by Max Schultze and Leydig as a mass of protoplasm containing a nucleus, a morphological definition which remains sufficiently satis- factory even at the present day. Nothing could be less appropriate than to call such a body a " cell " ; yet the word has become so firmly established that every effort to replace it by a better has failed, and it probably must be accepted as part of the established nomenclature of science.^ Attraction-sphere enclosing two centrosomes. I Plasmosnme or true nucleolus. Chromatin- network. Nucleus -i Linin-network. Karyosome or net-knot. Plastids lying in the cytoplasm. Vacuole. Lifeless bodies (meta- plasm) suspended in the cytoplasmic reticu- lum. Fig. 5. — Diagram of a cell. Its basis consists of a thread-work (viitome, or teticuluiii) com- posed of minute granules (//uc/oso/?ies) and traversing a transparent ground-substance. A. General Morphology of the Cell The cell is a rounded mass of protoplasm which in its simplest form is approximately spherical. This form is, however, seldom realized save in isolated cells such as the unicellular plants and animals or the egg-cells of the higher forms. In vastly the greater number of cases the typical spherical form is modified by unequal growth and differentiation, by active movements of the cell-substance, or by the mechanical pressure of surrounding structures. The 1 Sachs has proposed the convenient word energid (^Elora, '92, p. 57) to designate the essential living i)art of the cell, i.e. the nucleus with that portion of the active cytoplasm that falls within its sphere of influence, the two forming an organic unit both in a morpho- logical and in a physiological sense. It is to be regretted that this convenient and appro- jiriale term has not come into general use. (See also Elora, '95, p. 405.) GENERAL MORPHOLOGY OE THE CELL 15 protoplasm which forms its living basis is a viscid, translucent, granular substance, often forming a network or sponge-like structure extending through the cell-body and showing various structural modifications in different regions and under different physiological states of the cell. Besides the living protoplasm the cell almost invariably contains various lifeless bodies suspended in the meshes of the network ; examples of these are food-granules, pigment-bodies, drops of oil or water, and excretory matters. These bodies play a purely passive part in the activities of the cell, being either reserve food-matters destined to be absorbed and built up into the living substance, or by-products formed from the protoplasm as waste matters, or in order to play some role subsidiary to the actions of the protoplasm itself. The lifeless inclusions in the protoplasm have been collectively designated as nictaplasiu (Hanstein) in contradis- tinction to the Xw-xw-^ protoplasm ; but this convenient term is not in general use. Among the lifeless products of the protoplasm must be reckoned also the cell-wall or vicDibranc by which the cell-body may be sur- rounded ; but it must be remembered that the cell-wall in many cases arises by a direct transformation of the protoplasmic substance, and that it often retains the power of growth by intussusception like living matter. In all save a few of the lowest and simplest forms, perhaps even in them, the protoplasmic substance is differ- entiated into two very distinct parts, viz., the cell-body, forming the princi- pal mass of the cell, and a smaller body, the iincleiis, which lies in its interior (Fig. 5). Both structurally Fig. 6. — A resting cell {ipermatogo- oiArl /^Ko,-.-,;,^oU,r frU^r.^ 4- ^ 4- 1 riiiim) from the testis of the salamander, and cnemicaliy these two parts show , • .u . ■ 1 . m, .11 J 1 li o oi.wvv showing the typical parts. Above, the large differences of so marked and constant nucleus, with scattered masses of chro- a character that they must be re- "i^^''". .''"i™work and membrane. ■' Around it, the cyloplasuiic tlircad-work. garded as the most important of all Below, the attract'ion-sphere (a) and cen- protoplasmic differentiations. The ^'■«^o'»*= W- [After Rawitz.] nuclear substance is therefore often designated as nucleoplasm or karyoplasm ; that of the cell-body as cytoplasm (Strasburger). Some of the foremost authorities, however, among them Oscar Hertwig, re- ject this terminology and use the word "protoplasm " in its historic sense, applying it solely to the cytoplasm or substance of the cell-body. i6 GENERAL SKETCH OF THE CELL At a first examination the nucleus appears to be a perfectly dis- tinct body suspended in the cytoplasm. Most of the latest researches point, however, to the conclusion that nucleus and cytoplasm are pervaded by a com.mon structural basis, morphologically continuous A B C D P'g- 7- — Various cells showing the typical parts. A. From peritoneal epithelium of the salamander-larva. Two centrosomes at the right. Nucleus showing net-knots. [P'LEMMING.] B. Spermatogonium of frog. Attraction-sphere (aster) containing a single centrosome. Nucleus with a smgle |ilasniosome. [Hermann.] C. Spinal ganglion-cell of frog. Attraction-sphere near the centre, containing a single centro- some with several centrioles. [Lenhossek.] D. Spermatocyte of Proleus. Nucleus in the spireme-stage. Centrosome single ; attraction- sphere containing rod-shaped bodies. [HERMANN.] under certain conditions from one to the other, and that both are to be regarded as specially differentiated areas in that basis. ^ The terms ^ The fact tliat the nucleus may move actively throui^h the cvtoplasm, as occurs iluring the fertilization of the egg and in some other cases, seems to show that the morphological continuity may at times be interrupted. STRUCTURAL BASIS OF PROTOPLASM 1/ "nucleus" and "cell-body" are therefore only topographical expres- sions, and in a measure the same is true of the terms " karyoplasm " and " cytoplasm." The latter, however, acquire a special significance from the fact that there is on the whole a definite chemical contrast be- tween the nuclear substance and that of the cell-body, the former being characterized by the abundance of a substance rich in phos- phorus known as niicli'iH, while the latter contains no true nuclein and is especially rich in proteids and related substances (nucleo-albumins, albumins, globulins, and others), which contain a much lower per- centage of phosphorus. The differentiation of the protoplasmic substance into nucleus and cytoplasm is a fundamental character of the cell, both in a morpho- logical and in a physiological sense ; and, as will appear hereafter, there is reason to believe that it is in a measure the expression of a corresponding localization of the operations of constructive and destructive metabolism which lie at the basis of the individual cell- life. A third element, the centrosovie (Figs. 5-7), present in many if not in all cells, is especially concerned with the process of division and cell-reproduction. Recent research has rendered it probable that in point of morphological persistency the centrosome is comparable with the nucleus ; but this conclusion is not yet definitely established. B. Structural Basis of Protoplasm As ordinarily seen under moderate powers of the microscope proto- plasm shows no definite structural organization. A more precise ex- amination under high powers, especially after treatment with suitable fixing: and staining reagents, reveals the fact that both nucleus and cytoplasm possess a complicated structure. Regarding the pre- cise nature of this structure opinion still differs. According to the view most widely held, one of its essential features is the presence of two constituents, one of which, the ground-substance, cyto- lyiupJi, or cncJiylcma, is more liquid, while the other, the spongio- plasni or reticulum, is of firmer consistency, and forms a sponge-like network or alveolar structure extending everywhere through the more liquid portion. At the present time it seems probable that the more solid portion is the more active and is perhaps to be identified as the living substance proper, the ground-substance being passive ; but the reverse of this view is maintained by Leydig, Schafer, and some others. The most elaborate and painstaking investigation has moreover failed to determine with absolute certainty even the physi- cal configuration of the network. Butschli and a considerable school of followers among both i8 GENERAL SKETCH OE THE CELL zoologists and botanists regard protoplasm as essentially a liquid, or I'ather a mixture of liquids, which forms a foam-like alveolar structure^ like an emulsion, in which the firmer portion forms the walls of sepa- rate chambers, filled with the more liquid substance (Fig. 8). By r A c Fig. 8. — Alveolar or foam-structure of protoplasm, according to Biitschli. [BuTSCHLI.] A. Epidermal cell of the earthworm. B. Aster, attract ion-sphere, and centrosome from sea- urchin egg. C. Intra-capsular protoplasm of a radiolarian {Thalasucolla) with vacuoles. D. Peripheral cytoplasm of sea-urchin egg. E. Artificial emulsion of olive-oil, sodium chloride, and water. special local modifications of this structure all the parts of the cell are formed. Biitschli has shown that artificial emulsions, variously pre- pared, may show under the microscope a marvellously close resem- 1 " Wabenslriik/ur. " STRUCTURAL BASIS OF PRO'J'OPI.ASM IQ blance to actual protoplasm, and that drops of oil-emulsions suspended in water may even exhibit amoeboid changes of form. Opposed to Butschli's conception is the view, first clearly set forth by Frommann and Arnold ('65-'67), and now maintained by such authorities as Flemming, Van Beneden, Strasburger, and perhaps the greater number of contemporary investigators, that the more solid portion consists of coherent threads which extend through the ground- substance, either separately or connected by branches to form a mesh- work like the fibres of a sponge (Figs. 7, 9). In the present state of the subject it is difficult, indeed, impossible, to decide which of these opposing views should be accepted ; for the evidence is very strong that each expresses a part of the truth. It is generally admitted that such an alveolar structure as Biitschli de- scribes is characteristic of many unicellular forms, and occurs in many higher forms where the cell-substance is filled with vacuoles or with solid inclusions such as starch-grains or deutoplasm-spheres. In the latter case the structure has been termed "pseudo-alveolar" (Reinke); but it remains to be seen whether there is any real dis- tinction between this and the true alveolar structure described by Biitschli. On the other hand the evidence of true fibrillar or reticular structure in many tissue-cells, especially during cell-division, is very convincing ; and my own observations have led me to regard this structure as the more typical and characteristic. For descriptive pur- poses I shall accordingly adopt the terms of the fibrillar or reticular hypothesis, designating the more solid portion of protoplasm as the tlircad-ivork or rcticiduvi ("Geriistwerk," "Fadenwerk" of German writers) in contradistinction to the more liquid gnv -d-substance. It should be clearly understood, however, that these terms are used only as a matter of convenience, and are not meant to exclude the possibility that the "fibres" or the "reticulum" may in many cases be open to Biitschli's interpretation. From a theoretical point of view the finer structure of the network is a question of very great interest and importance. The earlier investigators, such as Virchow and Max Schultze, failed to observe the thread-work, and described protoplasm as consisting of a clear homogeneous basis in which were embedded numerous granules. Even at the present time a similar view is held by a few investi- gators, more especially among botanists {e.g., Berthold, Schwarz), who regard the thread-work either as an artificial effect produced by reagents, or, if normal, as an inconstant and hence unimportant feature. The best and most careful recent studies on proto- plasm have, however, yielded very convincing evidence that, what- ever be the precise configuration of the protoplasmic reticulum, it is not only a normal structure, but t)ne of very wide occurrence. 20 GENERAL SKETCH OF THE CELL \ -^i:f-. S^" *^. I / r^( /_ :y7 A . ' ,, ^ ^ i._"^ \ ^., H^lfff^-^- VI 1 C D ^ ;• -\ iogens (Ver- worn) ; inicrozy»ins (Bechamp and Estor) ; getnmct (Haacke). THE XL- CLE us 23 eel], and through this is especially concenicd with the formative proc- esses involved in growth and development. For these and many other reasons, to be discussed hereafter, the nucleus is generally re- garded as a controlling centre of cell-activit)^ and hence a primary factor in growth, development, and the transmission of specific quali- ties from cell to cell, and so from one generation to another. I. General St rue tare The cell-nucleus passes through two widely different phases, one of which is characteristic of cells in their ordinary or vegetative con- dition, 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 conspicuous irregular network (Figs. 5, 7, 10). Its form, though subject to variation, is on the whole singularly constant, and shows no definite relation to that of the cell in which it lies. Typically spherical, it may, in certain cases, assume an irregular or amoeboid form, may break up into a group of more or less completely sepa- rated lobes (polymorphic nuclei), or may be perforated to form an irregular ring (Fig. 11, D). 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. 11, E). Interesting modi- fications of the nucleus occur in the unicellular forms. In the cdiate Infusoria the body contains nuclei of two kinds, viz. a large macronnclcus and one or more smaller inicronuclei. The first of these shows a remarkable diversity of structure in different forms, being often greatly elongated and sometimes showing a moniliform structure like a string of beads. In Ti^acJielocerca and some other Infusoria, according to Gruber ('84), the nucleus is not a single definite body, but is represented by minute granules scattered throughout the cell-substance (Fig. 12); Biitschli describes somewhat similar diffused nuclei in some of the Flagellates, and in the Bacteria. In the ordinary forms of nuclei in their resting state the following structural elements may as a rule be distinguished (Figs. 5,6,7, 10, 1 1): — a. The nuclear membrane, a well-defined delicate wall which gives the nucleus a sharp contour and differentiates it clearly from the surrounding cytoplasm. /;. The nuclear reticulum. This, the most essential part of the nucleus, forms an irregular branching network or reticulum which consists of two very different constituents. The first of these, the 24 GENERAL SKETCH OF THE CELL nuclear substance par excellence, is known as chromatin (Flemmino) on account of its very marked staining capacity wlien treated witli various dyes. In some cases the chromatin forms a nearly continu- ous network, but it often appears in the form of more or less detached rounded granules or irregular bodies. The second constituent is a transparent substance, invisible until after treatment by reagents, known as linin (Schwarz). This substance, which is probably of the same nature as the cyto- plasmic network outside the nucleus, surrounds and supports the chromatin, and thus forms the basis of the nuclear net- work. c. The nucleoli, one or more larger rounded or irregular bodies, suspended in the net- work, and staining intensely with many dyes ; they may be absent. The bodies known by this name are of at least two different kinds. The first of these, the so-called true nucleoli or plasmosovics (Figs. 5, 7, B, 10), are of spherical form, and by treatment with differential stains such as ha;matoxylin and eosin are found to consist typi- T- k'^,'-m°'~T"° ""'''" ^'■°'" ^'^'^ ">'P'^ °^ cally of a central mass stainino- Lieberkuhn in the salamander. [Heidenhain.I in,/ <-! >. i ^ The character of the chromatin-net.ork , ^^'^ Cytoplasm, SUrrOUuded (^a«c/zw;«a//«) is accurately shown. The upper '^Y ^ shell which Staius like nucleus contains three plasmosomes or true chromatin. Those of the Other nucleoli ; the lower, one. A few fine linin-threads r "J. c ui uic OLUer (oxychromathi) are seen in the upper nucleus lOrm, the " Uet-kuotS " (NetZ- running off from the chromatin-masses. The \x\Ol< r^y^yi .v>' .'-v yy-.'V' 5 ^^^--iaa"y..>S'?:'i;fe*vr; '""'"''•*"''' C E Fig. II. — Special forms of nuclei. A. Permanent spireme-nucleus, salivary gland of Chironotn us larva. Chromatin in a single thread, composed of chromatin-discs (chromomeres), terminating at each end in a true nucleolus or plasmosome. [BalbiaN'I.] B. Permanent spireme-nuclei, intestinal epithelium of dipterous larva Ptychoptera. [VAN Gehuchten.] C. The same, side view. D. Polymorphic ring-nucleus, giant-cell of bone-marrow of the rabbit ; <■, a group of centro- somes or centrioles. [Heidenhain.] E. Branching nucleus, spinning-gland of butterfly larva {Pier is). [KORSCHELT.] stance occupying the interspaces of the network and left unstained by many dyes which colour the chromatin intensely. Until recently former are especially coloured by alkaline carmine solutions, the latter by acid solutions. Still later studies by Zacharias, and especially by Heidenhain, show that the medullary r.ubstance (pyrenin) of true nuclei is coloured by acid anilines and other plasma stains, while the chromatin has a special affinity for basic anilines. Cf. p. 242. 26 GENERAL SKETCH OF THE CELL the ground-substance has been regarded as a fluid or semi-fluid, but recent researches by Reinke and others have thrown doubt on this view, as described at p. 28. 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 epithelia.1 cells (Fig. i) ; sometimes extremely coarse and irregular, as in leucocytes (Fig. 10) ; 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 net- work, but appears in the form of a thread closely similar to the spireme- stage of dividing nuclei (cf. p. 47). The most striking case of this kind occurs in the salivary glands of dip- terous larvae {CJiiroiiomus), where, as described by Balbiani, the chromatin has the form of a single convoluted thread, composed of transverse discs and terminating at each end in a large nucleolus (Fig. w, A). Some- what similar nuclei (Fig. 11, B) occur in various glandular cells of other insects (Van Gehuchten, Gilson), and also in the young ovarian eggs of cer- tain animals (cf. p. 193). In certain gland-cells of the marine isopod Aui- locra it is arranged in regular rosettes (Vom Rath). Rabl, followed by Van Gehuchten, Heidenhain, and others, has endeavoured to show that the nuclear network shows a distinct polarity, the nucleus having a "pole" towards 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. Fmbryonic cells are in general Fig. 12. — An infusorian, Trachelo- cerca, with diffused nucleus consi=ting of scattered chromatin-granules. [Gruber.] ^ ( 'f. the polarity of tlie cell, THE NUCLEUS 27 characterized by the large size of tlie 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.^ A description of the nucleus during division is deferred to the fol- lowing chapter. 2. Fi)ur St nut lire of the Nucleus Many recent researches indicate that some at least of the nuclear structures are 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 revolves itself into a definite number of rod-shaped bodies known as chrojuosovies (Fig. i6), which split lengthwise as the cell divides. These bodies arise as aggregations of minute rounded bodies or microsomes to which various names have been given {cJu'oniomeres, Fol ; ids, Weismann). They are as a rule most clearly visible and most regularly arranged during cell-division, when the chromatin is arranged in a thread {spireme), or in separate cJirouiosoines (Figs. 7, D, 38, B) ; but in many cases they are dis- tinctly visible in the reticulum of the "resting" nucleus (Fig. 39). It is, however, an open question whether the chromatin-granules of the reticulum are individually identical with those forming the chromosomes or the spireme-thread. The larger masses of the reticulum undoubtedly represent aggregations of such granules, but whether the latter completely fuse or remain always distinct is unknown. Even the chromosomes may appear perfectly homogene- ous, and the same is sometimes true of the entire nucleus, as in the spermatozoon. The opinion is nevertheless gaining ground that the chromatin-granules have a persistent identity and are to be regarded as morphological units of which the chromatin is built up.- Heidenhain ('93, '94), whose views have been accepted by Reinke, Waldeyer, and others, has shown that the "achromatic" nuclear net- work is likewise composed of granules which he distinguishes as lanthanin- or oxycJironiatin-^x-^xwXo^'s, from the basic/i7'0]natiu-gr:x\M\\ts of the chromatic network. Like the latter, the oxychromatin-granules are suspended in a non-staining clear substance, for whicli he reserves 1 See Chapter VII. -' Cf. Chapter VI. 28 GENERAL SKETCH OF THE CELL the term "linin." Both forms of granules occur in the chromatic network, while the achromatic network contains only oxychromatin. They are sharply differentiated by dyes, the basichromatin being coloured by the basic anilines (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 anilines (rubin, eosin, etc.) and other "plasma stains." This distinction, as will appear in Chapter VII., is probably 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 ('78) and Van Beneden ('83) that the membrane arises as a condensation of the general protoplasmic reticulum, and is part of the same structure as the linin-network and the cyto-reticulum. Like these, it is in some cases "achromatic," but in other cases it shows the same staining reactions as chromatin, or may be double, con- sisting of an outer achromatic and an inner chromatic layer. _ Ac- cording to Reinke, it consists of oxychromatin-granules like those of the linin-network. 3. Chemistry of the Nucleus The chemical nature of the various nuclear elements will be considered in Chapter VII., and a brief statement will here suffice. The following classification of the nuclear substances, proposed by Schwarz in 1887. has been widely accepted, though open to criticism on various grounds. 1. CJiroDiatin. The chromatic substance (basichromatin) of the network and of tliose nucleoli known as net-knots or karyosomes. 2. Li)ii)i. The achromatic network and the spindle-fibres arising from it. THE CYTOPLASM 29 3. Paralinin. The ground-substance. 4. Pyrenin or Parachromatin. The inner mass of trae nucleoH. 5. Amphipyrenin. The substance of the nuclear membrane. CJiroiiiatin is probably identical with nitclein (p. 240). which is a compound of «7^c/^/t rt67V/ (a com ple.x organic acid, rich in phosphorus) and albumin. In certain cases (nuclei of spermatozoa, and probably also the chromosomes at the time of mitosis), chromatin may be composed of nearly pure nucleic acid. The Unin is probably composed of "plastin," a substance similar to nuclein, but containing a lower percentage of phosphorus, and either belonging to the nucleo-proteids or approaching them. It is nearly related with the substance of the cyto-reticulum. Pyrenin consists of a plastin-substance which stains like linin. Amphipyrenin is probably identical with linin. since the nuclear membrane is probably a condensed portion of the general reticulum which forms the boundary between the intra- and extra-nuclear networks. It should be borne in mind, however, that the membrane often has an inner chromatic layer composed of chromatin. D. The Cytoplasm It has long been recognized that in the unicellular forms the cytoplasmic substance is often differentiated into two well-marked zones ; viz. an inner medullary substance or endoplasm in which the nucleus lies, and an outer cortical substance or exoplasm (ectoplasm) from which the more differentiated products of the cytoplasm, such as cilia, trichocysts, and membrane, take their origin. Indications of a similar differentiation are often shown in the tissue-cells of higher plants and animals,^ though it may take the form of a polar differ- entiation of the cell-substance, or may be wholly wanting. Whether the distinction is of fundamental importance remains to be seen ; but it appears to be a general rule that the nucleus is surrounded by protoplasm of relatively slight differentiation, while the more highly differentiated products of cell-activity are laid down in the more peripheral region of the cell, either in the cortical zone or at one end of the cell.^ This fact is full of meaning, not only because it is an expression of the adaptation of the cell to its external environment, but also because of its bearing on the problems of nutrition. ^ For if, as we shall see reason to conclude in Chapter VII., the nucleus be immediately concerned with synthetic metabolism, we should expect to find the immediate and less differentiated products of its action in its neighbourhood, and on the whole the facts bear out this view. 1 This fact was first pointed out in the tissue-cells of animals by Kupffer ('75), and ils importance has since been urged by Waldeyer, Reinke, and others. The cortical layer is by Kupffer termed paraplasm, by Pfeffer hyaloplasm, by Pringsheini the Haiiischiclit. The medullary zone is termed by Kupffer, protoplasm, sensu strictti ; by Strasburger Korner- plastna. bv Nageli polioplasrn. 2 Cf. p'. 38. ' 3 See Kupffer ('90), pp. 473-476- ^o GENERAL SKETCH OF THE CELL The most pressing of all questions regarding the cytoplasmic structure is whether the sponge-like, fibrillar, or alveolar appearance is a normal condition existing during life. There are many cases, especially among plant-cells, in which the most careful examination has thus far failed to reveal the presence of a reticulum, the cyto- plasm appearing, even under the highest powers and after the most \ \ ■■. :as^-; (OinBiitiiMiinnitiaiunuiiiif i;;;;f.'!!;;:;;;.'i,'::i!ti!i:;|i;;i;.'i,' !j;;|ii;;j;;;;;;;;;|;i!i;fi;||;;;i!!f B uw*«i»"»*"'!"*,"'";"""i: C D Fig. 13. — Ciliated cells, showing cytoplasmic fibrill^ terminating in a zone of peripheral microsomes to which the cilia are attached. [Engelmann.] A. From intestinal ii\)\\.\-\fi\\\xm oi A>iodo)ita. B. Yxom. g\\\ o{ Aiwdonta. CD. Intestinal epi- thelium of Cyclas. careful treatment, merely as a finely granular substance. This and the additional fact that the cytoplasm may show active streaming and flowing movements, has led some authors, especially among bota- nists, to regard the reticulum as non-essential and as being, when present, a secondary differentiation of the cytoplasmic substance specially developed for the performance of particular functions. It has been shown, moreover, that structureless protcids, such as egg- THE CYTOPLASM 3 I albumin and other substances, when coagulated by various reagents, often show a structure closely similar to that of protoplasm as ob- serv^cd in microscopical sections. Bijtschli has made careful studies of such coagulation-phenomena which show that coagulated or dried albumin, starch-solutions, gelatin, gum arable, and other substances show a fine aveolar structure scarcely to be distinguished from that which he believes to be the normal and typical structure of pro- toplasm. Fischer ('94, '95) has made still more extensive tests of solutions of albumin, peptone, and related substances, in various degrees of concentration, fixed and stained by a great variety of the reagents ordinarily used for the demonstration of cell-structures. The result was to produce a marvellously close sinudacruui of the appear- ances observed in the cell, reticulated and fibrillar structures being- produced that often consist of rows of granules closely similar in every respect to those described by Altmann and other students of the cell. After impregnating pith with peptone-solution and then hardening, sectioning, and staining, the cells may even contain a central nucleus-like mass suspended in a network of anastomosing threads that extend in every direction outward to the walls, and give a remarkable likeness of a normal cell. These facts show how cautious we must be in judging the appear- ances seen in j^reserved cells, and justify in some measure the hesita- 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 regular and characteristic arrangement of the thread-work and microsomes in certain cases. In many Protozoa, for example, a fine alveolar structure may be seen in the living protoplasm ; and Flem- ming as well as many later observers has clearly seen fibrillar struct- ures in the living cells of cartilage, epithelium connective-tissue, and some other animal cells (Fig. 9). Mikosch, also, has recently described granular threads in living plant-cells. Almost equally conclusive is the beautifully regular arrangement of the fibrillse in ciliated cells (Fig. 13, Engelmann), in muscle-fibres and nerve fibres, and especially in the mitotic figure of dividing-cells (Figs. 16, 24), where they are likewise more or less clearly visible in life. A very convincing case is afforded by the pancreas-cells of Nccturus, which Mathews has carefully studied in my laboratory. Here the thread-work consists of long, conspicuous, definite fibrillae. some of which may under certain conditions be wound up more or less clearly in a spiral mass to form the so-called Ncbenkcni. In all these cases it is impossible to regard the thread- work as an accidental 32 GENERAL SKETCH OF THE CELL coagulation-product. On the whole, therefore, it is probable that careful treatment by reagents gives at least an approximately true picture of the normal thread-work, though we must always allow for the possible occurrence of artificial products. ?^^^iiu •"■•■■■'■.•'• • "••'i ijpfr--;- %ry ■-:-■■:-. *• * :• <- • *z*\ Fig. 14. — Section through a nephridial cell of the leech, Clepsine (drawn bv Arnold Graf from one of his own preparations). The centre of the cell is occupied by a large vacuole, filled with a watery liquid. 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 excretory granules {i.e. metaplasm). The nucleus, at the right, is surrounded by a thick chromatic inem- brane, is traversed by a very distinct linin-network, contains nuinerous scattered chromatin- granules, and a single large nucleolus within which is a vacuole. Above are two isolated nuclei showing nucleoli and chromatin-granules suspended on the linin-threads. One of the most beautiful forms of cyto-reticulum with which I am acquainted has been described by Bofeius and Graf in the ne- THE CYTOPLASM II phridial cells of leeches as shown in Fig. 14 (from a preparation by Dr. Arnold Graf). The reticulum is here of great distinctness and regularity, and scattered microsomes are found along its threads. It appears with equal clearness, though in a somewhat different form, ';S?»«»."tv Fig. 15. — Spinal ganglion-cell of the frog. [VON Lenhossek.] 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 Plemming.) The dark cyto- plasmic masses are the deeply staining " chromophilic granules" (.\issl) of unknown function. (The centrosome, which lies near the centre of the cell, is shown in Fig. 7, 6".) At the left, two connective tissue-cells. in many eggs, where the meshes are rounded and often contain food- matters or deutoplasm in the inter-spaces (Figs. 42, 43). In cartilage- cells and connective tissue-cells, where the threads can be plainly seen in life, the network is loose and open, and appears to consist of more or less completely separate threads (Fig. 9). In the cells of colum- D 34 GENERAL SKETCH OF THE CELL 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 fibrillse corresponding in number with the cilia as if continuous with their bases (Fig. 13).^ In nerve-fibres the threads form closely set parallel fibrillae 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. 15). 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 Ballowitz 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 fibrillae 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 suppose, and opinion is still divided as to whether the contractile substance is represented by the reticulum proper or by the ground-substance. Nowhere, however, is the thread-work shown with such beauty as in dividing-cells, where (Figs. 16, 24) 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. 35) and pigment- cells (Fig. 36), 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. 18, differ considerably from the foregoing, the fibrillae being regarded as the optical sections of thin plates or lamellae which form the walls of closed chambers filled by a more liquid substance. Biitschli, followed by Reinke, Eismond, Erlanger, and others, interprets 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. 8, B). Strong evidence against this view is, I believe, afforded by the appearance of the spindle and asters in cross-section. In the early stages of the egg of Nereis, for example, the astral rays are coarse anastomosing fibres that stain intensely and are therefore very favourable for observation (Fig. 43). 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 ^ The structure of the ciliated cell, as described by Engelmann, may be beautifully demonstrated in the funnel-cells of the nephridia and sperm-ducts of the earthworm. THE CYTOPLASM 35 cut ends of the branching rays appear in the clearest manner, not as plates but as distinct dots, from which in oblique sections the ray may be traced inwards towards the centrosphere. Driiner, too, figures the spindle in cross-section as consisting of rounded dots, like the end of a bundle of wires, though these are connected by cross-branches (Fig. 22, F\ Again, the crossing of the rays pro- ceeding from the asters (Fig. 69), and their behaviour in certain phases of cell-division, is difficult to explain under any other than the fibrillar theory. We must admit, however, that the network varies greatly in Centrosphere con- taining the cen- trosome. Aster. Spindle. Chromosomes forming the equatorial plate. Fig. 16. — Diagram of the dividing cell, showing the mitotic" figure and its relation to the cyto- reticuluni. different cells and even in different physiological phases of the same cell ; and that it is imj^ossible at present to bring it under any rule of universal application. It is possible, nay probable, that in one and the same cell a portion of the network 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 organ- ization must be sought in a more subtle underlying structure.^ ^ See Chapter \'I. 36 GENERAL SKETCH OF THE CELL E. The Centrosome No element of the cell has aroused a wider interest of late than the remarkable body known as the centrosome, which is now gener- ally regarded as the especial organ of cell-division, and in this sense as the dynaviic centre of the cell (Van Beneden, Boveri).^ In its simplest form the centrosome is a body of extreme minuteness, often indeed scarce larger than a microsome, which nevertheless exerts an extraordinary influence on the cytoplasmic network during cell- division and the fertilization of the Qgg. As a rule it lies out- side, though near, the nucleus, in the cyto-reticulum, surrounded by a granular, reticular, or radiating area of the latter known as the attraction-spJiere or centrosphere (Figs. 5, 6, 7).- It may, however, lie within the nuclear membrane in the linin-network (Fig. 107). In some cases the centrosome is a single body which divides into two as the cell prepares for division. More commonly, it becomes double during the later phases of cell-division, in anticipa- tion of the succeeding division, the two centrosomes thus formed lying passively within the attraction-sphere during the ordinary life of the cell. They only become active as the cell prepares for the ensuing division, when they diverge from one another, and each becomes the centre of one of the astral systems referred to at p. 49. Each of the daughter-cells receives one of the centrosomes, which meanwhile again divide into two. The centrosome seems, therefore, to be in some cases a permanent cell-organ, like the nucleus, being handed on by division from one cell to another. There are, however, some cells, e.g. muscle-cells, most gland-cells, and many unicellular organisms, in which no centrosome has thus far been discovered in the resting-cell ; but it is uncertain whether the centrosome is really absent in such cases, for it may be hidden in the nucleus, or too small to be distinguished from other bodies in the cytoplasm. There is, however, good reason to believe that it degenerates and disappears in the mature eggs of many animals, and this may likewise occur in other cells. At present, therefore, we are not able to say whether the centrosome is of equal constancy with the nucleus.^ 1 The centrosome was discovered by Van Beneden in the cells of Dycyemids ('76), and first carefully described by him in the egg of Ascaris seven years later. The name is due to P.overi ('88, 2, p. 68). ' 2 Cf. p. 229. ^ Its nature is more fully discussed at p. 224. OTHER ORGANS 37 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 comparable with that of the nucleus and centrosome. From a general point of view the most interesting of these are the bodies known 3.s p last ids or proto- plasts {¥\g. 5), 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 cJiromatopJiorcs or clironioplasts, 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 never arise spontaneously, so far as known, but always by the division of pre-existing bodies of the same kind. They 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 distinct Algae living symbiotically in the cell. This view is probably correct in some cases, e.g. in the Radiolaria ; but it may well be doubted whether it is of general application. In the plants the chlorophyll- bodies and other chromoplasts are almost certainly to be regarded as differentiations of the cytoplasmic substance. The same is true of the aniyloplasts, which act as centres for the formation of starch. 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 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- propagating units. 38 GENERAL SKETCH OF THE CELL G. The Cell-membrane From a general point of view the cell-membrane or intercellular substance is of relatively minor importance, since it is not of constant occurrence, belongs to the lifeless products of the cell, and hence plays no direct part in the active cell-life. In plant-tissues the mem- brane is almost invariably present and of firm consistency. Animal tissues are in general characterized by the slight development or absence of cell-walls. Many forms of cells, both among unicellular and multicellular forms, are quite naked, for example Aviceba and the leucocytes ; but in most, if not in all, such cases, the outer limit of the cell-body is formed by a more resistant layer of protoplasm — the " pelhcle " of Blitschli — that may be so marked as to simulate a true membrane, for example, in the red blood-corpuscles (Ranvier, Wal- deyer) and in various naked animal eggs. Such a " pellicle " differs from a true cell-membrane only in degree ; and it is now generally agreed that the membranes of plant-cells, and of many animal-cells, arise by a direct physical and chemical transformation of the periph- eral layer of protoplasm. On the other hand, according to Leydig, Waldeyer, and some others, the membrane of certain animal-cells may be formed not by a direct transformation of the protoplasmic substance, but as a secretion poured out by the protoplasm at its surface. Such membranes, characterized as " cuticular," occur mainly or exclusively on the free surfaces of cells (Waldeyer). It remains to be seen, how- ever, how far this distinction can be maintained, and the greatest diversity of opinion still exists regarding the origin of the different forms of cell-membranes in animal-cells. The chemical composition of the membrane or intercellular sub- stance varies extremely. In plants membrane consists of a basis of cellulose, a carbohydrate having the formula Q^Yi^^Or^ ; but this sub- stance is very frequently impregnated with other substances, such as silica, lignin, 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 orga/ac axis passing from pole to pole. No definite criterion for the identi- fication of the cell-axis has, however, yet been determined; for the POLARITY OF THE CELL 39 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 very clearly made out. On the one hand, Van Beneden {^"^l) 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. 17, A). With this view Rabl's theory ('85) of nuclear polarity harmonizes, for the chromosome-loops converge tow- ards the centrosome, and the nuclear axis coincides with the cell-axis. Moreover, it identifies the polarity of the egg, which is so important a factor in development, with that of the tissue-cells; for the egg- r-A t tt A Van Beneden. B C Rabl, Hatschek. Fig. 17. — Diagrams of cell-polarity. A. Morphological polarity of Van Beneden. Axis passing through nucleus and centrosome. Chromatin-threads converging towards the centrosome. B. C. Physiological polarity of Rabl and Hatschek, B in a gland-cell, 6" in a ciliated cell. 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 struct- ures 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 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. In the present state of the subject, Heidenhain's theories must be regarded as somewhat trans- cendental, though they give many suggestions for further investigation. 40 GENERAL SKETCH OF THE CELL Hatschek ('88) 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 jn 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. 17, B, C.) This is typically shown in epithelium, which, as Kolliker and Hackel 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, towards the source of food, while differentiated products of the 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. 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 differentiations are grouped. Moreover, the observations of Heidenhain, Lebrun, and Kostanecki indicate that the same is true in epithelium ; for, accord- ing to these authors, the centrosome is always situated on that side of the nucleus turned towards the free end of the cell. How far this law holds good remains to be seen, and, until the facts have been further investigated, it is impossible to frame a consistent hypothesis of cell-polarity. The facts observed in epithelial cells, are, however, of great significance ; for the position of the centrosome, and hence the direction of the axis, is here obviously related to the cell-environ- ment, and it is difficult to avoid the conclusion that the latter must be the determining condition to which the intracellular relations con- form. When applied to the germ-cells, this conclusion becomes of high interest ; for the polarity of the Q,g^ is one of the primary con- ditions of development, and we have here, as I believe, a clue to its origin.^ ' Cf. pp. 288, 320. THE CELL LN RELATLON TO THE MULTLCELLULAR BODY 4 1 I. The 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 unicellular plants 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 /// this 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 appa- ratus 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 co-ordinated, 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 (1839), 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, afterwards elaborated by Virchow and Hackel 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- cerned, it has now been clearly demonstrated that only in a limited sense can the cells be regarded as co-operating units. They are rather local centres of a formative power pervading the growing ^ Unkrsuckinigen, p. igi. 42 GENERAL SKETCH OF THE CELL 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 becomes 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 equilibrium 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 in a measure sustained by later researches, though it still remains sub judicc. Tangl, Gardiner, and many later observers have shown that the cell-walls of many plant-tissues are traversed by delicate intercel- lular bridges, and similar bridges have been conclusively demon- strated by Bizzozero, Retzius, Flemming, Pfitzner, and many others in the case of animal epithelial cells (Figs, i, 9). The same has been asserted to be the case with the smooth muscle-fibres, with car- tilage-cells and connective-tissue cells, and in a few cases with nerve- cells. Paladino and Retzius ('89) have endeavoured to show, further, that the follicle-cells of the ovary are connected by protoplasmic 1 Cf. Chapters VIII., IX. 2 For a fuller discussion see pp. 293 and 311. THE CELL IX RELATION TO THE MULTICELLULAR BODY 43 bridges not only with one another, but also xcit/i the ovnvi, a conclu- sion which, if estabhshed by further research, will be of the greatest interest. 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 the structure in which protoplasmic continuity is to be expected, the latest researches are rendering it more and more probable that no such continuity exists, and that nerve-impulses are transmitted from cell to cell by contact-action. When, however, we turn to the embryonic stages we find strong reason for the belief that a material continuity between cells must exist. This is certainly the case in the early stages of many arthro- pods, where the whole embryo is at first an unmistakable syncytium ; and Adam Sedgwick has endeavoured to show that in Fcripatits 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 Ainphioxiis or the echinoderms, 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 mechanical displacement of the blastomeres. This conclusion is supported by the recent work of Hammar ('96), whose observations on sea-urchin eggs I can in the main confirm. As the subject now lies, however, the facts do not, I believe, jus- tify any general statement regarding the occurrence, origin, or physi- ological meaning of the protoplasmic continuity of cells ; and a most important field here lies open for future investigation. LITERATURE. P Altmann, R. — Die Elementarorganismen und ihre Beziehungen zu den Zellen, 2d ed. Leipzig, 1894. Van Beneden, E. — (See Lists IL. IV.) Boveri, Th. — (See Lists IV., V.) Butschli, 0. — Untersuchungen iiber mikroskopische Schaume und das Protoplasma. Leipzig (Engelmann), 1892. Engelmann, T. W. — Zur Anatomie und Physiologic der Flimmerzellen : Arch. ges. Pliys..XyA\\. 1880. von Erlanger, R. — Neuere Ansichten liber die Struktur des Protoplasmas : Zool. Cetitralbl., III. 8, 9. 1896. 1 See also Introductory list, p. 12. 44 GENERAL SKETCH OF THE CELL Flemming, W. — Zellsubstanz, Kern unci Zellteilung. Leipzig, 1882. Id. — Zelle: Merkel unci Bo7i)iefs Ergeb)iisse,\.-\N . 1891-94. (Admirable reviews and literature-lists.) Heidenhain, M. — Uber Kern und Protoplasma : FestscJir. z. ^o-jahr. Doctorjiib. 71011 V. KolUker. Leipzig, 1893. Klein, E. — Observations on the Structure of Cells and Nuclei: (2uart. Joiirn. Mic. Sci.,XN\\\. 1878. Kolliker, A. — Handbuch der Gewebelehre, 6th ed. Leipzig, 1889. Leydig, Fr. — Zelle und Gewebe. Bonn, 1885. Schafer, E. A. — General Anatomy or Histology; in (2!iain's Anatomy, I. 2, loth ed. London, 1891. Schiefferdecker & Kossel. — Die Gewebe des Alenschlichen Kbrpers. Braiinschweig, 1891. Schwarz, Fr. — Die morphologische und chemische Zusammensetzung des Proto- plasmas. Breslau, 1887. Strasburger, E. — Zellbildung und Zellteilung. 3d ed. 1880. 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. De Vries, H. — Intracellulare Pangenesis. Jena, 1889. Waldeyer, W. — Die neueren Ansichten liber den Bau und das Wesen der Zelle: DcjiiscJi. j]Led. JJ'oc/iensc/ir., Oct., Nov., 1895. Wiesner, J. — Die Elementarstruktur u. das Wachstum der lebenden Substanz : Wien, Lioldcr. 1892. Zimmerman, A. — Beitrage zur Morphologic und Physiologie der Pflanzenzelle. Tilbitigen, 1893. CHAPTER II CELL-DIVISION " Wo eine Zelle entsteht, da muss eine Zelle vorausgegangen sein, ebenso wie das Thier nur aus dein Thieve, die Pflanze nur aus der Ptianze entstehen kann. Auf diese Wt-ise ist, wenngleich es einzelne Punkte im Korper gibt, wo der strange Nachweis noch nicht gelie- fert ist, doch das Princip gesichert, dass in der ganzen Reihe alles Lebendigen, dies nio"en nun ganze Ptlanzen oder thierische Organismen oder integrirende Theile derselben sein, ein ewiges Gesetz der coniininrlichen Enhvicklung besteht." Virchow.^ 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. The cell has no other mode of origin than by division of a pre-existing cell. In the multicellular organism all the tissue-cells have arisen by continued division from the original germ-cell, and this in its turn arose by the division of a cell pre-existing in the parent-body. By cc/l-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 (1840-60) 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 afterwards, 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 (/.r., 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 1 Celhdarpathologie, p. 25, 1858. 2 Cf. Introtluction, p. 9. •5 For a full historical account of this period, see Remak, Untersuchun^en ilber die Ent- wicklung der Wirhdthiere, 1855, pp. 164-180. ■* Unlersuchiingeti, p. 175. 45 46 CELL-DIVISION with the division of the nucleolus, is continued by simple constriction and division of the nucleus, and is completed by division of the cell- body and membrane (Fig. i8). For many years this account was accepted, and no essential advance beyond Remak'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 sim- ple 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 Baer, Robin, etc.); in others to become lobed or star-shaped, as described by Virchow and by Remak himself (Fig. i8,/). It was not until 1873 that the way was opened for a better understanding of the matter. In 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) afterwards gave the name of Karyokiiiesis. It soon ap- peared, however, that this mode d ' € Fig. 18. — Direct division of blood-cells in the embryo cluck, illustrating Remak's scheme. [Remak.] a-e. Successive stages of division; / Cell of division waS not of UniVCr- dividing by mitosis. g^l occurrcncc ; and that cell- division is of two widely different types, which Van Beneden ('76) distinguished as fragvientatwn, corresponding nearly to the simple process described by Remak, and division, involving the more com- plicated process of karyokinesis. Three years later Flemming ('79) proposed to substitute for these the terms direct and indirect division, which are still used. Still later ('82) the same author suggested the terms mitosis (indirect or karyokinetic division) and aviitosis (direct or akinetic division), which have rapidly made their way into general use, though the earlier terms are often employed. 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, in the cells of transitory em- OUTLINE OF IXDIRECr DIVISIOX OR MITOSIS 47 bryonic envelopes, and in tumours and other pathological forma- tions, where it is of frequent occurrence. Whether this view be well founded or not, it is certain that in all the higher and in many of the lower forms of life, 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 matura- tion, and by mitotic division 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, Flagel- lates, and Diatoms. We may, therefore, justly regard it as the most general expression of the "eternal law of continuous development" on which Virchow insisted. A. Outline of Indirect Division or Mitosis (Karyokinesis) The process of mitosis 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, how- ever, graduate into one another and are separated by no well-defined limits. These are : ( i ) The Prophases, or preparatory changes ; (2) the JMctapJiasc, 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 Nucleus. As the cell prepares for division the most conspicuous fact is a transformation of the nuclear sub- stance, involving both physical and chemical changes. The chroma- tin resolves itself little by little into a more or less convoluted thread, known as the skein (Knauel) or spireme, and its substance stains far more intensely than that of the reticulum (Fig. 19). In some cases there is but a single continuous thread ; in others, the thread is from its first appearance divided into a number of separate pieces or segments forming a segmented spireme. In either case it ulti- mately breaks transversely into a definite number of distinct bodies, known as chromosomes (Waldeyer, '88), which in most cases have the form of rods, straight or curved, though they are sometimes spherical or ovoidal, and in certain cases may be joined together in the 'form of rings. The staining power of the chromatin is now at a maximum. As a rule the nuclear membrane meanwhile fades away and finally disappears. The chromosomes now lie naked in the cell, and the ground-substance of the nucleus becomes continuous with the surrounding cytoplasm (Fig. 19, D, E, F). 48 CELL-DIVISION Every species of plant or animal has a fixed and characteristic num- ber of chromosomes, zvJiich regularly recurs in the division of all of its cells; and in all forms arising by sexual reproduction the number is D Fig. 19. —Diagrams showing the prophases of mitosis. A Resting-cell with reticular nucleus and true nucleolus ; at c the attraction-sphere contain- in'^ two centrosomes. B. Earlv prophase ; the chromatin forming a continuous spireme, nucleolus sti'll present- above, the amphh.ster {a). C. D. 'IVo different types of later prophases; C. Dis- appearance'of the primary spindle, divergence of the centrosomes to opposite poles of the nucleus (examples manv plant-cells, cleavage-stages of many eggs). /.). Persistence of the' primary spindle (to form' in some cases the "cent.al spindle"), fading of the nuclear membrane, ingrowth of the astral ravs segmentation of the spireree-thread to form the chromosomes (examples, epi- d-rmal cells of salamander, formation of the polar bodies). E. Later prophase of tvpe C ; fadmg 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 type alone). I. The mitotic figure established ; e.p. The equatorial plate of chromosomes. (Cf. Figs. 16. 21, 24.) OUTLINE OF INDIRECT DIVISION OR MITOSIS 49 cveji. Thus, in some of the sharks the number is 36; in certain gasteropods it is 32 ; in the mouse, the salamander, the trout, the Hly, 24; in the worm Sagitta, 18; in the ox, guinea-pig, and in man the number 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 Artcmia it is 168.1 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. 61). The even number of chromosomes is a most interestins: fact, which, as will appear hereafter (p. 135), 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, consisting of true chromatin, probably enter into the formation of the spireme-thread. True nucleoli seem to dissolve and disappear, or in some cases are cast out bodily into the cytoplasm, where they degen- erate and have no further function. Whether they ever contribute to the formation of chromosomes is uncertain. {b) The Aviphiastcr. Meanwhile, more or less nearly parallel with these changes in the chromatin, a complicated structure known as the aviphiastcr {Yo\, 'yj) makes its appearance in the position formerly occupied by the nucleus (Fig. 19, B-F). This structure consists of a fibrous spindle-shaped body, the spindle, at either pole of which is a star or asfer 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 ccntrosome (Boveri, '88), which may be surrounded by a spherical mass known as the centrosphcre (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 (Fig. 19, A-D). 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 amphiaster 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 cen- tral spindle (Figs. 19, D, F\ 21). In other cases the original spindle ^ For a mure complete list see p. 154. 50 CELL-DIVISION disappears, and the two asters pass to opposite poles of the nucleus (most plant mitoses and in many animal cells). A spindle is now formed from rays 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 Fig. 20. — Diagrams of the later phases of mitosis. G. Metaphase ; spHtting of the chromosomes {e. p.) ; «. The cast-off nucleolus. H. 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 begm- ning reconstruction of the daughter-nuclei. J. Division completed. disappearance (Fig. 19, C, E). In this case there is apparently no central spindle. In a few exceptional cases, finally, the amphiaster may arise inside the nucleus (p. 225). The entire structure, resulting from the foregoing changes, is known as the karyokinctic or mitotic fiirurc. It may be described as consi-sting of two distinct parts; namely, i, the chromatic figure, formed by the deeply staining chromosomes ; and, 2, the achromatic OUTUNE OF INDIRECT DTVrSION OR MITOSIS 5 1 figure, consisting of the spindle and asters which, in general, stain but slightly. The fibrous substance of the achromatic figure is gener- ally known as arcJioplasni (Boveri, "i^), but this term is not applied to the centrosome within the aster. 2. Metaphasc. — The prophases of mitosis are, on the w^hole, pre- paratory in character. The metapJiasc, which follows, forms the initial phase of actual division. Each chromosome splits lengthwise into two exactly similar halves, which afterwards diverge to opposite poles of the spindle, and here each group of daughter-chromosomes finally gives rise to a daughter-nucleus (Fig. 20). In some cases the splitting of the chromosomes cannot be seen until they have grouped themselves in the equatorial 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. 38, 39). Such variations do not, however, affect the essential fact that the chromatic network is converted into a tJiread^ ivhich, zvhether continuous or discontinuous, splits throughout its entire length into two exactly 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 ('83), the entire substance of the chromatic network is precisely halved, and the daughter-nuclei receive precisely equivalent portions 0/ chro- matin from the mother-nucleus. It is very important to observe that the nuclear division always shows this exact equality, whether division of the cell-body be equal or unequal. The minute polar body, for example (p. 131), receives exactly the same amount of chromatin as the Qgg, 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 (cf. Figs. 43, 71) 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 grou^is of daughter-chromosomes are connected by a bundle of achromatic fibres, stretching across the interval between them, and known as the interzonal fibres QX cotinecting fibres? In some cases, these differ \\\ a 1 It was this fact that led Flemming to employ the woxA "mitosis" (/iiros, a thread). - This stage is termed by Flemming the dyaster, a term which should, however, be aban- doned in order to avoid confusion with the earlier word amphiaster. The latter convenient and appropriate term clearly has priority. ^ Verbinduugsfasern of German authors; filaments reiinissanls of Van Beneden. ,2 CELL-DIVISION marked degree from the other spindle-fibres ; and they are believed by many observers to have an entirely different origm and function. A view now widely held is that of Hermann, who regards these fibres as belono-ing to a central spindle, surrounded by a peripheral layer of manUe-fibres to which the chromosomes are attached, and only exposed to view as the chromosomes separate.^ They are sometimes thickened in the equatorial region to form a body known as the cell- plate or mid-body, which, in the case of plant-cells, takes part in the formation of the membrane by which the daughter-cells are separated. 4 Telophases. — \x^ the final phases of mitosis, the entire cell divides in two in a plane passing through the equator of the spmdle, each of the daughter-cells receiving a group of chromosomes, half of the spindle, and one of the asters with its centrosome. Meanwhile, a dauo-hter-nucleus is reconstructed in each cell from the group of chrom^osomes 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, contort.ed, and closely crowded to form a danghter-spireuie, closely similar to that of the mother-nucleus (Fig. 23); 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 m the seg- mentino- eggs of Ascaris (Van Beneden, Boveri). In other cases, as in man^'y segmenting ova, each chromosome gives rise to a hollow vesicle after which the vesicles fuse together to produce a smgle nucleus (Fig. 37). 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 m which it lies. , .J ui 11 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. In dividing plant- cells the interzonal fibres become thickened at the equator of the spindle and form a transverse plate of granules, known as the cell- plate (Fig. 25), which gives rise to the membrane by which the two daughtertcells are separated. The remainder of the spindle disap- pears. A similar cell-plate occurs in some animal cells; but it is often greatly reduced, and may form only a minute body known as the mid-body (Zwischenkorper), which lies between the two cells after 1 Cf. p. 74- ORIGIN OF THE MITOTIC FIGURE 53 their division (Fig. 23). In other cases, as in the cells of the testis, the remains of the spindle in each cell sometimes gives rise to a more or less definite body known as \.\iQ. paranucleus or Nebenkeru (Fig. 62). The aster may in some cases entirely disappear, together with the centrosome (as occurs in the mature <^g,^). 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, however, some undoubted cases (cf. Figs. 6, 7) 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-spJiere. This body often shows a true astral structure with radiating fibres (Figs. 7, 35); but it is sometimes reduced to a regular spherical mass which may represent only the centrosphere of the original aster (Fig. 6). B. Origin of the Mitotic Figure The chromatic figure (chromosomes) is derived directly from the chromatic network of the resting-nucleus as described above. The derivation of the achromatic figure (spindle and asters) is a far more difficult question, which is still to some extent involved in doubt. By the earlier observers (1873-75) the achromatic figure was supposed to disappear entirely at the close of cell-division, and most of them (Biitschli, Strasburger, Van Beneden, '75) believed it to be reformed at each succeeding division out of the nuclear substance. Later re- searches (1875-85) gave contradictory 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 from the cytoplasm. Flemming ('82) was in doubt, and regarded the question of nuclear or cytoplasmic origin as one of minor importance, yet on the whole inclined to the opinion that the achromatic figure arose inside the nucleus.^ In 1887 a new face was put on the whole question through the independent discovery by Van Beneden and Boveri that the centrosome docs not disappear at the close of mitosis, but remains as a distinct cell-organ lying beside the nucleus in the cytoplasm. These investigators agreed that the amphiaster is formed under the influence of the centrosome, •* Zellsiibstanz, p. 226. 54 CELL-DIVISION which leads the Avay in cell-division by dividing into two similar halves to form the centres of division. "Thus we are justified," said Van Beneden, "in regarding the attraction-sphere with its central •7^^ ■^ Sf-*: \ B A ' 'l \ Z> Fig. 21. — The prophases in cells (spermatogonia and spermatocytes) of the salamander. [DrUner.] A. Spermatogonium in the spireme-stage ; the chromatin-thread lies in the linin-network, still surrounded by the membrane ; above, the two centrosomes, the central spmdle not yet formed. B. Later stage (spermatocyte) ; the nuclear membrane has disappeared, leaving the naked chro- mosomes; above, the amphiaster, with centrosomes and central spindle; astral rays extending towards the chromosomes. D. Following stage ; splitting of the chromosomes, growth of the aster; mantle-fibres and central spindle clearly distinguished. C. The fully formed mitotic figure (metaphase) ; the chromosomes, fully divided, grouped in the equatorial plate. corpuscle as forming a permanent organ, not only of the early blas- tomeres, but of all cells ; that it constitutes a cell-organ equal in rank to the nucleus itself ; that every central corpuscle is derived from a pre-existing corpuscle, every attraction-sphere from the pre-existing OA'/CLY OF THE MITOTIC FIGURE 55 sphere, and that division of the sphere precedes that of the cell- nucleus."^ Boveri expressed himself in similar terms in the same year (^?>7, 2, p. 153), and the same general result was reached by Vejdovsky nearly at the same time,^ though it was less clearly formu- lated than by either Boveri or Van Beneden. Fig. 22. — Metaphase and anaphases of mitosis in cells (spermatocytes) of 'the salamander. [DrUner.] E. Metaphase. The continuous central spindle-fibres pass from pole to pole of the spindle. Outside them the thin layer of contractile mantle-fibres attached to the divided chromosomes, of which only two are shown. Centrosomes and asters. F. Transverse section through the mitotic figure showing the ring of chromosomes surrounding the central spindle, the cut fibres of the latter appearing as dots. G. Anaphase ; divergence of the daughter-chromosomes, exposing the cen- tral spindle as the interzonal fibres; contractile fibres (principal cones of Van Btsneden) clearly shown. H. Later anaphase (dyaster of Flemming) ; the central spindle fully exposed to view; mantle-fibres attached to the chromosomes. Immediately afterwards the cell divides (see Fig. 23). 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 ofifice 1 '87, p. 279. 2 '88, pp. 151, etc. 56 CELL-DIVISION 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-rr/A-. The centrosome rep- resents the dynamic centre of the cell." ^ This view has been widely accepted by later investigators, and the centrosome has been shown to occur in a large number 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 many plant-tissues, and in some of the unicellular Fig. 23. — Final phases (telophases) of mitosis in salamander cells. [FlemmiNG.] /. Epithelial cell from the lung; chromosomes at the poles of the spindle, the cell-body divid- ing; gianules of the "mid-body" or 7.ivischenkdiper v.i the equator of the disappearing spindle. y. Connective-tissue cell (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. plants, and animals, such as the Diatoms and Flagellates. That the centrosome gives the primary impulse to cell-division by its own division has, however, been disproved ; for there are several accu- rately determined cases in which the chromatin-elements divide long before the centrosome, and it is now generally agreed that the division of chromatin and centrosome are two parallel events, the causal relation between which still remains undetermined. (Cf. P- 77 ■) 1 Boveri, '87, 2, p. 153. MODIFICATIONS OF MITOSIS 57 C. Modifications of Mitosis The evidence steadily accumulates that the essential phenomena of mitosis are of the same general type in all forms of cells, both in plants and in animals. Everywhere, with a single important exception (maturation), the chromatin-thread splits lengthwise through- out its whole extent, and everywhere an achromatic spindle is formed that is in some manner an agent in the transportal of the chromatin- halves to the respective daughter-cells. The exception to this general law, which occurs during the preparation of the germ-cells for their development and constitutes one of the most significant of all cyto- logical phenomena, is considered in Chapter V. We have here only to glance at a number of modifications that affect, not the essential character, but only the details of the typical process. I. Varieties of the Mitotic Figiwe All of the mitotic phenomena, and especially those involved in the history of the achromatic figure, are in general most clearly displayed in embryonic cells, and especially in the egg-celP (Fig. 24). In the adult tissue-cells the asters are relatively small, the spindle relatively large and conspicuous. The same is true of plant-cells in general where the very existence of the asters was at first overlooked. Plant-mitoses are characterized by the prominence of the cell-plate (Fig. 25), 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. 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 undergo extensive changes in the cycle of cell-division and to vary greatly in different forms (Fig. 108). In some cases the aster contains at its centre nothing more than a minute deeply staining granule, which doubtless ^ A very remarkable modification of the achromatic figure occurs in the spiral asters, ch.o 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. 65 ; for later stages, Fig. 104.) spindle-fibres are not focussed at a single point, and the spindle appears truncated at the ends, its fibres terminating m a transverse row of granules (maturation-spindles of Ascans, and some plant-cells). It is not entirely certain, however, that such spindles observed m preparations represent the normal structure during life.^ 1 Hacker asserts in a recent paper ('94) that the truncate>-''--»'?5>. Pig- 30- — Mitosis in the Flagellate .V(7t//- A. Nucleus (fi) in the early prophase; outside it the attraction-sphere [s), containing two centrosomes (Ishikawa). B. The mitotic figure; n. the nucleus, containing rod-shaped chromosomes; s. attraction-sphere; s./>. ex- tra-nuclear central spindle. (Drawn by G. N. Calkins from one of his own preparations.) in the higher types, except that the nuclear membrane does not disappear. ^ Regarding the history of the chromatin the most thorough obser- vations have been made by Schewiakoff in EiiglypJia and Brauer in Actuwsphcerinin. In the former case a segmented spireme arises from the resting reticulum, and long, rod-shaped chromosomes are formed, which are stated to split lengthwise as in the usual forms of mitosis. The nuclear membrane persists throughout, and the entire mitotic .AH of the essential features in this process, as described by Ishikawa, have been con- lirmed by Calkins in the Columbia laboratory. F 66 CELL-DIVISION figure, except the minute asters, is formed inside it (Fig. 28). In ActinospJicerimn, on the other hand, there is no true spireme stage, and no rod-shaped chromosomes are at first formed. The reticulum breaks up into a large number of granules which give rise to an equatorial plate, divide by fission, and are distributed to the daughter-nuclei. sO,"." .oo'">\ ^^-in^t. '0<' 00 ■ o O O0O6OO Fig. 31. — Mitosis in the rhizopod ^t/ww/^«r2w;«. [BRAUER.] 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 e/t face, consisting of double chromatin-granules. E. Early anaphase. F. G. Later ana- phases. H. Final anaphase. /. Telophase; daughter-nucleus forming, chromatin in loop-shaped threads ; outside the nuclear membrane the centrosome, already divided, and the aster. J. Later stage ; the daughter-nucleus established ; divergence of the centrosomes. Beyond this point the centrosomes have not been followed. Only in the late anaphase {telophase) do these granules arrange them- selves in threads (Fig. 31,/), and this process is apparently no more than a forerunner of the reticular stage. This case is a very convincing argument in favour of the view that the formation and splitting of chro- mosomes is secondary to the division of the ultimate chromatin-granules. MODIFICATIONS OF MITOSIS 67 (Cf. pp. y?, and 221.) Richard Hertwig's studies on Infusoria and those of Lauterborn on Flagellates indicate that here also no longitu- dinal splitting of the chromatin-threads occurs and that the divi^sion must be referred to the individual chromatin-granules. Ishikawa de- scribes a peculiar longitudinal splitting of chromosomes in Noctihica, but Calkins' studies indicate that the latter observer has probably mis- interpreted certain stages and that the division probably takes place in a somewhat different manner. A typical spireme and chromosome- formation has also been described by Lauterborn in the Diatoms ('93). In none of the foregoing cases does the nuclear membrane dis- appear. In the gregarines, however, the observations of Wolters ('91) and Clarke ('95) indicate that the membrane does not persist, and that a perfectly typical mitotic figure is formed. To sum up : The facts at present known indicate that the unicellu- lar forms exhibit forms of mitosis that are in some respects transi- tional from the typical mitosis of higher forms to a simpler type. The asters may be reduced (Rhizopods) or wanting (Infusoria); the spindle is typically formed inside the nucleus, either by division of an intra-nuclear " nucleolo-centrosome " {Eicglena, Amceba), or possibly by rearrangement of the chromatic substance without a differentiated centrosome (.?micronuclei of Infusoria). In every case the essential fact in the history of the chromatin is a division of the chromatin- granules ; but this may be preceded by their arrangement in threads or chromosomes {Euglypha, Diatoms) or may not {Actinosphcenimi). These facts point toivards the conclusion Jhat centrosome, spindle, and chromosomes are all secondary differentiations of the primitive nuclear staictiire, and indicate that the asters and attraction-spheres may be historically a later acquisition developed in the cytoplasm after the dif- ferentiation of the centrosome. 5. Pathological Mitoses Under certain circumstances the delicate mechanism of cell-division may become deranged, and so give rise to various forms of patholog- ical 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, occur without discoverable external cause ; and it is a very interest- ing 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 68 CELL-DIVISION o-eneral groups, as follows: (i) asyinvictrical 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 two, and more than one spindle is formed. Under the first group are included not only the cases of unequal distribution of the daugh- ter-chromosomes, but also those in which chromosomes fail to be drawn into the equatorial plate 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- Pig, 22. — Pathological mitoses in human cancer-cells. [Galeotti.] A. Asymmetrical mitosis with unequal centrosomes. B. Later stage, showing unequal dis- tribution of the chromosomes. C. Quadripolar mitosis. D. Tripolar mitosis. E. Later stage. F. Tri-nucleate cell resulting. 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. 32). Hansemann, whose conclu- 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 (hyper-chromatic cells), while others are abnormally poor (hypochromatic cells). Lustig and Galeotti ('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 MODIFICATIONS OF MITOSIS 69 that asymmetrical mitoses, exactly like those seen in carcinoma, may be artificially produced in the epithelial cells of salamanders (Fig. 33) by treatment with dilute solutions of various drugs (antipyrin, cocaine, quinine). 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- ^ B Fig. 33. — Pathological mitoses in epidermal cells of salamander caused by poisons. [Galeotti.] .-/. Asymmetrical mitosis after treatment with 0.05% antipyrin solution. D. Tripolar mitosis after treatment with 0.5% potassic iodide solution. 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, '88). Multipolar mitoses are 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 disease or the reverse. The latter seems the more probable alternative, since normal mitosis is certainly the rule in abnormal growths ; and Galeotti's 1 The remarkable polyasters formed in polyspermia fertilization of the egg are de- scribed at p. 147. / -O CELL-DIVISION 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. D. The Mechanism of Mitosis We now pass to a consideration of the forces at work in mitotic division, which leads us into one of the most debatable fields of cytological inquiry. I. Fi/nctioji of the AmpJiiastcr 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 view first clearly stated by Fol,^ that the asters represent in some manner centres of attractive forces focussed in the centrosome or dynamic centre of the cell. Regarding the nature of these forces, there is, however, so wide a divergence of opinion as to compel the admission that we have thus far accom- phshed little more than to clear the ground for a precise investigation of the subject ; and the mechanism of mitosis still lies before us as one of the most fasciniiting problems of cytology. {a) The Theory of Fibrillar Coiitraetility. — The view that has taken the strongest hold on recent research is the hypothesis of fibrillar contractility. First suggested by Klein in 1878, this hypoth- esis was independently put forward by Van Beneden in 1883, and fully outlined 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 fibrillae and their arrangement in a kind of radial muscular system, composed of antagonizing groups" (/.r. the asters with their rays). " In this system 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 chromosomes) " in opposite directions. An important part of the phenomena of (karyo-) .kinesis has its effi- cient cause, not in the nucleus, but in the protoplasmic body of the cell."- This beautiful hypothesis was based on very convincing ' '73- p- 473- ' '87. P- 280. THE MECHANISM OF MITOSIS 71 K-ac cz- a.c evidence derived from the study of the Ascaris Qgg, and it was here that Van Beneden first demonstrated the fact, already sus- pected by Flemming, that the daughter-chromosomes move apart to the poles of the spindle, and give rise to the two re- spective daughter-nuclei.^ Van Beneden describes the astral rays, both in Asca7-is and in tunicates, as differentiated into sev- eral groups (Fig. 34). One set, forming the " principal cone," are attached to the chromosomes and form one-half of the spindle, and, by the contractions of these fibres, the chro- mosomes are passively dragged apart. An oppo- site 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 con- tractions, 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 sub-equatorial circle. Later observations indi- cate, 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.- Van Beneden's general hypothesis was accepted in the following year by Boveri {'88, 2), who contributed many important additional 1 '83, p. 544- m.z. Fig. 34. — Slightly schematic figures of dividing eggs of Ascaris, illustrating Van Beneden's theory of mitosis. [Van Beneden and Julin.] A. Early anaphase; each chcomosome has divided into two. B. Later anaphase during divergence of the daughter-chromosomes, a.c. Antipodal cone of astral rays ; c.z. cortical zone of the attraction-sphere ; i. in- terzonal fibres stretching between the daughter-chromo- somes ; m.z. medullary zone of the attraction-sphere; p.c. principal cone, forming one-half of the contractile spindle (the action of these fibres is reinforced by that of the antipodal cone) ; s.e.c. sub-equatorial circle, to which the astral ravs are attached. 72 CELL-DI VISION 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 irregularly scattered in the Q,g^, are drawn into a position of equilibrium in the equator of the spindle by the shortening of these rays (Figs. 65, 104); and that tJie rays thicken as tJicy sJiortcn. 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, B / Fig. 35. — 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. B. Similar cell, with double nucleus; the smaller dark masses in the latter are oxychromatin-granules (linin), the larger masses are basichromatin (chromatin proper). the shortening and thickening of these rays as the daughter-chromo- somes diverge. In all these operations the behaviour of the rays is precisely like that of muscle-fibres ; and it is difficult to study Boveri's beautiful figures and clear descriptions without sharing his conviction that "of the contractility of the fibrillae 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 1 '88, 2, p. 99. THE MECHANISM OF MITOSIS 71 carefully by Heidenhaiii in the salamander. The astral rays here extend throughout nearly the whole cell (Fig. 35), and are believed 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 Zimmerman ('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 Fig. 36. — Pigment-cells and asters from the epidermis of fishes. [ZIMMERMAN.] .-/. Entire pigment-cell, fi cm Blennius. The central clear space is the central mass of the aster Irom which radiate the pigment-granules; two nuclei below. B. Nucleus {?/) and aster after ex- traction of the pigment, showing reticulated central mass. C. Two nuclei and aster with rod- shaped central mass, fiom Sargiis. actively creeping about. Solger and Zimmerman 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. 36). But although these observations seem to place the theory of fibrillar contractility upon a firm basis, it has since undergone various modifi- 74 CELL-DIVISION cations and limitations, which show that the matter is by no means so simple as it first appeared. The most important of these modifi- cations are due to Hermann ('91) and Driiner ('95), who have relied mainly on the study of mitosis in various cells of the salamander, well known as extremely favourable objects for study. These observers have demonstrated that in this case the spindle-fibres are of two kinds which, apparently, differ both in origin and i;i mode of action. Hermann showed that the primary amphiaster is formed outside the nucleus, without connection with the chromosomes, and that the original spindle persists as a "central spindle" (Figs. 21, 22), which he regards as composed of noii-contmctilc fibres, and merely forming a support on which the movements of the chromosomes take place. The contractile elements are formed by certain of the astral rays which grow into the nucleus, and become attached to the chromo- somes, as Boveri described. By the contraction of these latter fibres the chromosomes are now dragged towards the spindle, and around its equator they are finally grouped to form the equatorial plate. The fully formed spindle consists, therefore, of two elements ; namely, («) the original " central spindle," and {b) a surrounding mantle of con- tractile'^" mantle-fibres " attached to the chromosomes, and originally derived from astral rays. In the anaphase, as Hermann believes, the daughter-chromosomes are dragged apart solely by the contractile mantle- fibres, the central spindle fibres being non-contractile and serving as a support or substratum along zvhich the chromosomes viove. As the chromosomes diverge, the central spindle comes into view as the m- terzonal fibres (Fig. 22, G, H). Strasburger ('95) is now inclined to accept a similar view of mitosis in the cells of plants. Druner ('95) in his beautiful studies on the mechanism of mitosis has advanced a step beyond Hermann, maintaining that the pro- gressive divergence of the spindle-poles is caused 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 anaphases. This view is supported by the fact that the central spindle-fibres are always contorted during the metaphases, as if pushing against a resistance; and, as Richard Hertwig points out ('95), it harmonizes with the facts observed in the mitoses of in- fusorian nuclei. The same view is adopted by Braus and by Reinke. Flemming ('95) is still inclined, however, to the view that the divergence of the centres may be in part caused by the trac- tion of the antipodal fibres, as maintained by Van Beneden and Boveri. Heidenhain, finally, while accepting the contractility-hypothesis, ascribes only a subordinate role to an active physiological contrac- tility of the fibres. The main factor in mitosis is ascribed to elastic THE MECHANISM OF MITOSIS 75 tension of the astral rays which are attached at one end to the cen- trosome, at the other to the cell-periphery. By turgor of the cell the rays are passively stretched, thus causing divergence of the spindle-poles and of the daughter-chromosomes to which the spin- dle-fibres are attached. An active contraction of the fibres is only invoked to explain the closing phases of mitosis. {!)) Other Theories. — Watase's ingenious theory of mitosis ('93) is exactly the opposite of Van Beneden's, assuming that the spindle- fibres are not pulling but pushing agents, the daughter-chromo- somes being forced apart by continually lengthening fibres which grow out from the centres and dovetail in the region of the inter- zonal fibres. Each daughter-chromosome is therefore connected with fibres from the aster, not of its own, but of the opposite side. This view is, I believe, irreconcilable with the movements of chromosomes observed in multiple asters, and also with those that occin- during the fertilization of the Qgg, where the chromo- somes are plainly drawn towards the astral centres and not pushed away from them. Biitschli, Carnoy, Platner, and others have sought an explanation in a totally different direction from any of the foregoing, regarding the formation of the amphiaster as due essentially to streaming or osmotic movements of the fluid constituents of the protoplasm, and the movements of the chromosomes as being in a measure mechan- ically caused by the same agency. Oscar Hertwig adopts a some- what vague dynamical view, regarding the formation of the mitotic figure as due to an interaction between nucleus and cytoplasm, which he compares to that taking place in a magnetic field between a mag- net and a mass of iron filings : " The interaction between nucleus and protoplasm in the cell finds its visible expression in the formation of the polar centres and astral figures ; the result of the interaction is that the nucleus always seeks the middle of its sphere of action."^ He gives, however, no hint of his view regarding the nature of the action or the causes of the chromosomal movements. Ziegler ('95) accepts a somewhat similar view ; and he has shown that surpris- ingly close sininhxcra of the mitotic figure in many of its different phases may be produced by placing bent wires (representing the chromosomes) in the field of a horseshoe magnet strewn with iron filings. My own studies on the eggs of echinoderms ('95, 2) and annelids have convinced me that no adequate hypothesis of the mitotic mech- anism has yet been advanced. In these, as in many other forms, the spindle-fibres show no differentiation into central spindle and peri- ^ Zelle und Geivebe, p. 172. 76 CELL-DIVISION pheral mable-fibres ; and the chromosomes extend entirely through the substance of the spnidle in its equatorial plane. If there be sup- porting, as opposed to contractile, fibres, they must be intermingled with the latter; and both forms must have the same origin. The \^^v^^M■/ '■/ -^*- ^§^;^^i^ M "M. #ifi^-^^^^^ lil' ^o ^- 0^ ^gm^n'm^ c ■"■>•'■ D E pig_ 27. — The later stages of mitosis in the egg of the sea-urchin Toxopiienstes {A-D, X 1000; L-F, X 500). A. Metaphase; daughter-chromosomes drawing apart but still united at one end. B. Daugh- ter-chromosomes separating. C. Late anaphase; daughter-chromosomes lying at the spindle- poles. D. Final anaphase; daughter-chromosomes converted into vesicles. £. Immediately after division, the asters undivided; the spindle has disappeared. K Resting 2-cell stage, the asters divided into two in anticipation of the next division. In Figs. A to D, the centrosphere appears as a large reticulated mass from which the rays pro- ceed. It is probable that a minute centrosome, or pair of centrosomes, lies near the centre ot the centrosphere, but this is not shown. daughter-chromosomes appear to move towards the poles tJinnigh Ihe substance of the spindle, and do not travel along its periphery as described by Hermann and Drtiner in amphibia and by Strasburger ('93, 2) in the plants No shortening or thickening of the rays can THE MECHANISM OF MITOSIS jy be observed, and the chromosomes proceed to the extreme limit of the spindle-poles and appear actually to pass into the interior of the huge reticulated centrosphere. I cannot see how this behaviour of the chromosomes is to be explained as the result solely of a con- traction of fibres stretching between them and the centrosphere. It is certain, moreover, that another factor is at work. Throughout the anaphases, the centrosphere steadily grows until, at the close, it attains an enormous size (Fig. 37), and its substance differs chem- ically from that of the rays, for after double staining with Congo red (an acid aniline) and haematoxylin it becomes bright red while the ra^'s are blue. It seems probable, therefore, that the movements of the chromosomes are affected by definite chemical changes occur- ring in the centrosphere, as Butschli^ and Strasburger^ have main- tained ; and it is possible that the substance of the spindle-fibres may be actually taken up into the centrosphere, and the chromo- somes thus drawn towards it. Strasburger has made the interesting suggestion, which seems well worthy of consideration, that the move- ments of the chromosomes may be of a chemotactic character. In any case, I believe that no satisfactory hypothesis can be framed that does not reckon with the chemical and physical changes going on in the centrosphere, and take into account also the probability of a dynamic action radiating from it into the surrounding struct- ures. Van Beneden's hypothesis is probably, in principle, correct ; but, as Boveri himself admits in his latest paper ('95), it seems cer- tain that other factors are involved besides the contractility of the achromatic fibres, and the mechanism of mitosis still awaits adequate physiological analysis. 2. Division of the CJiromosomes In developing his theory of fibrillar contractility Van Beneden 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 spindle-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. The splitting of the chromosomes is therefore, in Boveri's words, 1 '92, pp. 158, 159. - '93, 2. 3 'S;, p. 279. 7S CELL-DIVISION ''ail independent vital manifestatio)i, an act of reproduction on the part of the cJiroviosoines.'" ^ All of the recent researches m 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 {^'j6) 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 ('72) added the interesting discovery, that during the mitosis of various tissue-cells of the salamander, the granules of the spireme-thread divide by fission and thus determine the Fig. 38. — Nuclei in the spireme-stage. A. From the endosperm of the Hly, 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. [HERMANN.] C. Spireme-thread completely split, with si.\ nucleoli. Endosperm of Fritlllaria. [Flem- ming.] lon^i^itudinal splitting of the entire chromosome. This discovery was confirmed by Flemming in the following year ('82, p. 219), and a sim- ilar result has been reached by many other observers (Fig. 38). The division of the chromatin-granules may take place at a very early period. Flemming observed as long ago as i88r that the chromatin- thread might split 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 Asearis 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 i'88, p. 113. 2 See '81, p. 638. THE MECHANISM OF MITOSIS 79 reticulum, and long before the division of the centrosome (Fig. 39). He therefore concludes : " With Boveri I regard the splitting as an 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 B Fig. 39. — Formation of chromosomes and early splitting of the chromatin-granules in sperma- togonia of Ascaris megalocephala, var. bivalens. [Bkauer.J A. Very early prophase; granules of the nuclear reticulum already divided. D. Spireme; the continuous chromatin-thread split throughout. C. Later spireme. D. Sliortenmg of tlie thread. E. Spireme-thread divided into two parts. F. Spireme-thread segmented into four split chromosomes. allowing a uniform growth to take place ; and in the second place, after the granules have grown to their normal size, to admit of their precisely eqiial quantitative 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 80 CELL-DIVISION the simplest and most certain manner, the transmission of the daugh- ter-granules (Spalthalften) to the daughter-cells."! "In my opmion the^'chromosomes are not independent individuals, but only groups of numberless minute chromatin-granules, which alone have the value of individuals."^ These observations certainly lend strong support to the view that the chromatin is to be regarded as a morphological aggregate — as a cono-eries 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 archoplasm, or in the last analysis, the centrosome. 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 o-ive 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-" sion of its total mass, but not of its individual elements or chromatin- granules (Fig. 40). Before the discovery of mitosis, nuclear division was generally assumed to take place in accordance with Remak's scheme (p. 45)- 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, 1 '93, pp- 203, 204. " ''■'^•' P- -°5- DIRECT OR AMITOTIC DIVISION 8i many cases of amitotic division were accurately determined, though very few of them conformed precisely to Remak's scheme. One such case is that described by Carnoy in the follicle-cells of the egg in the mole-cricket, where division begins in the fission of the nucleolus, followed by that of the nucleus. Similar cases have Fig. 40. — Group of cells with amitotically dividing nuclei; ovarian follicular epithelium of the cockroach. [WHEELER.] been since described, by Hoyer ('90) in the intestinal epithelium of the nematode Rhabdoncma, 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. Centrosome 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. Flemming ob- served (91) that the nucleus of leucocytes might in some cases divide directly without the formation of an amphiaster, the attraction-sphere remaining undivided meanwhile. 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 spindle. Both Heidenhain and Flemming drew from this the conclusion that direct division of the nucleus is in this case independent 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 published remarkable observations that seem to indicate a functional activity of the attraction-sphere during amitotic nuclear division in the "sperma- 82 CELL-DIVISION too-onia" 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 deo-enerating nuclei. Meves, however, asserts — and the accuracy of his obser- vations 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 granu- lar mass, which more or less completely 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-sphere becomes drawn out into a band which surrounds the central part of the nucleus, and finally forms a closed ring, encirclino' the nucleus. After this the nucleus divides into two, while the ring- shaped attraction-sphere (" archoplasm ") 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 "'archo- plasm " of the autumn-stage arises by the breaking down of a definite spherical attraction-sphere, which is reformed again in the spring in the manner described, and in this condition the cells may divide either initotieally or aniitotically. He adds the interesting observation, 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 vom 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. Biological Significance of Amitosis A survey of the known cases of amitosis brings out tlie following significant facts. It is of extreme rarity, if indeed it ever occurs in embryonic cells or such as are in the course of rapid and continued 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.), w/iic/i arc on tlic zvay toivards degeneration. In many cases, moreover, direct nuclear divi- sion is not followed by fission of the cell-body, so that multinuclear 1 '91, p. 628. ■■^ Such a mode of amitotic division was lirst described by Sabatier in the Crustacea ('89), and a similar mode has been observed by Carnoy and Van der Stricht. DIRECT OR AMITOTIC DIVISION 83 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(t. 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 ('88) had conceived it. This hypothesis has been carried still further by Ziegler and vom Rath ('91). In a paper on the origin of the blood in fishes, Ziegler ('87) showed that the periblast-nuclei in the eggs of fishes divide amitotically, 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 forerunner 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 assimi- lative activity. Thus, Riige ('90) showed that the absorption of degenerate eggs in the amphibia is effected by means of» leuco- cytes which creep into the egg-substance. The nuclei of these cells become enlarged, divide amitotically, 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 nrtritive-cells surrounding the ovum in insects and Crusta- cea. 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 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 1 ' 91, 2, p. 291. 84 CELL-DIVISION common 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 Anilocm ('95). Many other cases are known. Dogiel ('90) has observed 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 superficial layers. Dogiel finds in the lining of the bladder of the mouse that the nuclei of the superficial cells, which secrete the mucus covering the surface, regularly divide amitotically, giving rise to huge multinuclear 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 Infusoria, which possess two kinds of nuclei in the same cell, a macronucleus and a micronucleus. The former is known to be intimately concerned with the processes of metabolism (cf. p. 165). During conjugation the macronucleus degenerates and disappears and a new one is formed from the micronucleus or one of its descendants. The macronucleus is therefore essentially metabolic, the micronucleus generative 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 macronucleus than in the micronucleus, and in some cases the former appears to divide directly while the latter always goes through a process of mitosis. In view of all these facts and others of like import Ziegler, like Flemming, concludes that amitosis is of a secondary character, and that when it occurs the series of divisions is approaching an end. This conclusion received a very important support in the work of vom R^th 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- producing 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 differentiated, they remain absolutely distinct; amitosis never takes place in the series which finally results in the formation of spermatozoa, and the amitotically dividing " supporting-cells " sooner or later perish. Vom Rath thus reached the remarkable con- clusion 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. 33 1-) SUMMARY AND CONCLUSION 85 Whether this conckision can be accepted without modification remains to be seen. Flemming himself regards it as too extreme, and is incHned to accept Meves' conclusion that amitosis may occur in the sperm-producing cells of the testis. The same conclusion is reached by Preusse in the case of insect-ovaries. There can be no doubt, however, that Flemming's hypothesis in a general way repre- sents 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 Three distinct elements are involved in the typical mode of cell- division by mitosis ; namely, the centrosome, the chromosome, and the cell-body. Of these, the centrosome may be considered the organ of division par excellence ; for as a rule it leads the way in division, and under its influence, in some unknown manner, is organized the astral system which is the immediate instrument of division. This system appears in the form of two asters, each containing one of the daughter-centrosomes and connected by a spindle to form an aviphi- aster. It arises as a differentiation or morphological rearrangement of the general cell-reticulum, the asters being formed from the extra- nuclear reticulum, the spindle sometimes from the linin-network, sometimes from the cyto-reticulum, sometimes from both. 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 are 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, is formed as a single series of chromatin-granules or chromomeres which, by their fission, cause the splitting of the thread. Every individual chromatin-granulc therefore contributes its quota to each of the daughter-nuclei. The mechanism of mitosis is imperfectly understood. There is good reason to believe that the fission of the chromatin-granules, and hence the splitting of the thread, is not caused by division of the centrosome, but only accompanies it as a parallel phenomenon. The divergence of the daughter-chromosomes, on the other hand, is in some manner determined by the spindle-fibres developed under the influence of the centrosomcs. There arc cogent reasons for the view that some at least of these fibres are contractile elements which, like 86 CELL-DIVISION muscle-fibres, drag the daughter-chromosomes asunder ; while other spindle-fibres act as supporting and guiding elements, and probably by their elongation push the spindle-poles apart. The contractility hypothesis is, however, difficult to apply in certain cases, and is prob- ably an incomplete explanation which awaits further investigation. The functions of the astral rays are involved in even greater doubt, being regarded by some investigators as contractile elements like those of the spindle, by others as rigid supporting fibres like those of the central spindle. In either case one of their functions is prob- ably to hold the kinetic centre in a fixed position while the chromo- somes are pulled apart. Whether they play any part in division of the cell-body is unknown ; but it must be remembered that the size of the aster is directly related to that of the resulting cell (p. 51) — a fact which indicates a very intimate relation between the aster and the dividing cell-body. On the other hand, in amitosis the cell-body may divide in the absence of asters. These facts show that mitosis is due to the co-ordinate play of an extremely complex system of forces which are as yet scarcely com- prehended. Its purpose is, however, as obvious as its physiological explanation is difficult. // is tlic cud of mitosis to divide every part of the e/iroiuatin of the inother-eell equallv betiveen the dajighter-uiielei. All the other operations are tributary to this. We may therefore regard the mitotic figure as essentially an apparatus for the distri- bution of the hereditary substance, and in this sense as the especial instrument of inheritance. LITERATURE. II Auerbach, L. — Organologische Studien. Breslaii, 1874. Van Beneden, E. — Recherches .sur la maturation de Poeuf, la fecondation et la division cellulaire : Arcti. de Biol., IV. 1883. Van Beneden & Neyt. — Nouvelles recherches sur la fecondation et la division mitosique chez I'Ascaride megalocephale : Bull. Acad. roy. de Belgique, 1887. III. 14, No. 8. Boveri, Th. — Zellenstudien: \. Jena. Zedschr., XXI. 1887; U. Il^id. XXII. 1888; III. Ibid. XXIV. 1890. Brauer, A. — Qber die EncN'stinmg von Actinosphaerium Eichhorni : Zcitschr. IViss. Zool., LVIII. 2. 1894. Driiner, L. — Studien Uber den Mechanismus der Zelltheilung. Jena. Zedsckr., XXIX., II. 1894. Erlanger, R. von. — Die neuesten Ansichten iil)er die Zellthcilung und ihre Mechanik : Zool. Centralb., III. 2. 1896. Flemming, W.,'92. — Entwicklung und Stand der Kenntnisse liber Amitose : Merkel und Boruief-i Ergebnisse. II. 1 892 . Id. — Zellc. (See introdnctdiv list. Also general list.) SUMMARY AND CONCLUSION 8/ Fol, H. — (See List IV.) Heidenhain, M. — Cytomechanische Studien : Arch.f. Entwickmech., 1. 4. 1895. Hermann, F. — Beitrag zur Lehre von der Entstehung der karyokinetischen Spindel : Arch. Mik. Aiiai., XXXVII. 1891 . Hertwig, R- — Ober Centrosoma und Centralspindel : Sitz.-Ber. Ges. Morph. nnd Pliys. Miinc/ien, 1895, Heft I. Mark, E. L. — (See List IV.) Reinke, F. — Zellstudien : I. Arch. Mik. Anat., XLIII. 1894; II. Ibid. XLIV. 1894. Strasburger, E. — Karyokinetische Vrohltmc : Jahrb. f. Wiss.Botan. XXVIII. 1895. Waldeyer, W. — IJber Karyokinese und ihre Beziehungen zu den Befruchtungsvor- ^ransen : Arch. Mik. Anat., XXXII. 1888. Q.J.M.S., XXX. 1889-90. CHAPTER III THE GERAI-CELLS " Not all the progeny of the primary impregnated germ-cells are required for the forma- tion of the body 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 inhibition and of propagation by spon- taneous hssion as those to which itself owed its origin; followed by metamorphoses and combinations of the germ-masses so produced, which concur to the development of another individual." Richard Owen.i " Es theilt sich demgemass das befruchtete Ei in das Zellenmaterial des Individuums und in die Zellen fiir die Erhaltung der Art." M. NussBAUM.^ The germ from which every living form arises is a single cell, de- rived by the division of a parent-cell of the precedmg 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 Weismann's terms : ( i ) the somatic cells, which are differentiated into various tissues by which the functions of individual life are performed and which collectively form the "body," and (2) \X-\.. is then found to lie .^^':'B0m:fMmi^ just outside the ger- minal vesicle on the side nearest the egg- periphery where the polar bodies are sub- sequently formed After extrusion of the polar bodies (p. 131) the egg-centrosome as a rule degenerates and disappears. The ;7::;^^v^;•■.v■•i>•'v^'•.^^:; e> " t •". jT-J; '■'■J ■^'^'V■•!^' ''»*V *^\>i.' M egg thus loses the power of division Avhich is afterwards restored during fer- tilization through the introduction of a new centrosome by the spermatozoon. In parthenogenetic eggs, on the other hand, the egg-centrosome persists, and the egg "•-J&.Vf.C\i^''-'""'"' Fig. 42. — Ovarian egg of the sea-urchin Toxopneustes (X750)- g.v. Nucleus or germinal vesicle, containing an irregular dis- continuous network of chromatin ; g-.s. nucleolus or germinal spot, intensely stained with haeniatoxylin. The naked ceii-body consists of a very regular network, the threads of which appear as irregular rows of minute granules or microsomes. Below, at s, is an entire spermatozoon shown at the same enlargement (both middle-piece and flagellum are slightly exaggerated in size). accordingly retains the power of division without fertilization. The disappearance of the egg-centrosome would, therefore, seem to be in some manner a provision to necessitate fertilization and thus to guard against parthenogenesis. The egg-cytoplasm almost always contains a certain amount of nutritive matter, the yolk or denioplasjii, in the form of solid spheres or other bodies suspended in the meshes of the reticulum and vary- ing greatly in different cases in respect to amount, distribution, form, and chemical composition. 1 Hacker ('95, p. 249) has called attention to the fact that the nucleolus is as a rule single in small eggs containing relatively litUe deutoplasm (ccelenterates, echinoderms, many annelids, and some copepods), while it is multiple in large eggs heavily laden with '.«.). E. Of young chick containing nucleus, attraction-sphere and fatty deuto- plasm-spheres (black). F. Egg of new-born child, surrounded by follicle and containing nucleus and attraction-sphere. those of myriapods, having the form of a single well-defined sphe- roidal mass which appears at a very early period and persists through- out the later ovarian history. At the other extreme are "diffused yolk-nuclei " having the form of numerous irregular and ill-defined masses scattered through the cytoplasm, as described by Stuhlmann ('86) in the eggs of insects and more recently by Calkins and Foot in earthworms. An intermediate form is represented in the amphibia I GROWTH AND D/FFEEENTIATION OF THE GERM-CELLS I IQ (Jordan, '93) and niyriapods (Balbiani, '93), where the &gg contains a number of fairly well defined yolk-nuclei. In Lumbricus the "yolk- nucleus" first appears as a single irregular deeply staining body closely applied to the nucleus and afterwards breaks up into numer- ous smaller bodies (Calkins, '95). The most diverse accounts have been given of the structure and origin of these problematical bodies. This is in part owing to the fact, recently pointed out by Mertens, that two entirely different structures have been confounded under the one term. One of these is the attraction-sphere of the young &gg with its centrosome. Such a "yolk-nucleus" has been described by Balbiani in the eggs of the myriapod Geophilus (Fig. 59, B). The other is a body, variously described as arising from the nucleus or in the cytoplasm, which is not improbably concerned in some manner with the constructive metabolism involved in the growth of the egg-cytoplasm and perhaps indirectly concerned with the formation of deutoplasm. It seems clear that the latter form alone should receive the name of yolk- nucleus, if indeed the term is worth retaining. Mertens ('93) has recently described the ova of a number of birds and mammals (including man) as containing a very distinct attrac- tion-sphere containing one or more intensely staining centrosomes (Fig. 60). This has, however, nothing to do with the true yolk- nucleus which may sometimes be seen in the same o-gg, lying beside the attraction-sphere (Fig. 60, D). The latter sooner or later fades away and disappears. The yolk-nucleus, on the other hand, may long persist. This observation probably explains the strange result reached by Balbiani in the case of myriapods {GeopJiilns), where the "yolk- nucleus" is described as arising by a budding of the nucleus, yet is identified with an attraction-sphere ! The " yolk-nucleus " of Balbiani has here the typical appearance of an attraction-sphere, surrounded by rays and containing two or several centrosomes or centrioles. Besides this, however, the Q.gg contains several other bodies which are described as arising by budding off from the nucleus and per- haps represent the true yolk-nuclei (Fig. 59, B). The origin of the yolk-nucleus proper appears to differ in different cases. Jordan's observations on the newt seem to leave no doubt that the bodies described as yolk-nuclei in this animal arise in situ in the cytoplasm ; and a similar origin of the yolk-nucleus has been described by a number of earlier observers. On the other hand, a number of observers have asserted its origin from the nucleus, either by a process of nuclear budding, by a casting out of the nucleolus of separate chromatin-rods, or of portions of the chromatic reticulum. That such a casting-out of nuclear substance occurs during the ova- rian history of some eggs appears to be well established ; but it is I20 THE GERM-CELLS uncertain whether the bodies thus arising have the same physiologi- cal significance as the "yolk-nuclei" of cytoplasmic origin. Calkins ('95, i), working in my laboratory, has brought forward strong evi- dence that the "yolk-nucleus" of Liivibricns is derived from a sub- stance nearly related with chromatin (Fig. 61). The yolk-nucleus Fig. 61. — Young ovarian eggs of the earthworm (Lumbricus) , showing yolk-nucleus. [Calkins.] A. Very early stage ; the irregular yolk-nucleus (>'.«.) closely applied to the germinal vesicle and staining like chromatin. B. Later stage; the yolk-nucleus separating from the germinal vesicle and changing its staining-power. C. Still later stage; the yolk-nucleus broken up into rounded bodies staining like the cytoplasm. here first appears as an irregular granular body lying directly on the nuclear wall, which in some cases appears to be interrupted, as if yolk- nucleus and chromatin were directly in continuity. Later the yolk- nucleus separates from the germinal vesicle and lies beside it in the cytoplasm. It finally breaks up into a considerable number of sec- ondary yolk-nuclei scattered through the Q^,g. The action of differ- ential stains at different periods indicates that the substance of the GROWTH AND DIFFERENTIATION OF THE GERM-CELIS 121 yolk-nucleus is nearly related with chromatin, if not directly derived from it. When treated with the Biondi-Ehrlich 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 even after the yolk-nucleus has quite separated from the nuclear mem- brane. Later, however, as the yolk-nucleus breaks up, it loses its nuclear staining power, and stains red like the cytoplasm. This conclusion is, however, disputed in a later work by Foot ('96), who maintains that the yolk-nucleus in Allolobophora is not of nuclear but of " archoplasmic " origin, though no relation between it and an attraction-sphere is established. ^ She adds the very interesting dis- covery that the " polar rings " (cf. p. i 50) are probably to be identi- fied with the yolk-nucleus, or are at least derived from a similar substance. Calkins's observations taken in connection with those of Balbiani, Van Bambeke, and other earlier workers give, however, strong evi- dence, as I believe, that the "yolk-nucleus" of Lmnbriciis is de- rived, if not from the nucleus, at any rate from a substance nearly related with chromatin, which is afterwards converted into cyto- plasmic substance. It is certain, in this case, that the appearance of the yolk-nucleus is coincident with a rapid growth of cytoplasm ; but we cannot suppose that the latter grows entirely at the expense of the yolk-nucleus. More probably the yolk-nucleus supplies certain materials necessary to constructive metabolism, and it is not impo.s- sible that these may be ferments. We may perhaps interpret in the same manner the elimination of separate nuclear elements {i.e. not forming a definite yolk-nucleus) as described by Van Bambeke, Mertens, v. Erlanger, and many earlier writers. The meaning of the yolk-nuclei of purely cytoplasmic origin is very obscure, and we have at present really no ground for assigning to them any particular function. It can only be said that their appearance coincides in time approximately with the period of great- est constructive activity in the cytoplasm, but there is no evidence of their direct participation in the yolk-formation, and we do not know whether they are active constructive physiological centres, or merely stores of reserve substances or degeneration-products. 1 Miss Foot's use of the term "archoplasm" largely deprives the word of the definite meaning attached to it by Boveri. To identify as "archoplasm" everything stained by Lyons blue is indeed a broad use of the term. 122 THE GERM-CELLS 2. Formation of the Spermatozoon Owino- 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 speinnatogonia} 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 spermatids 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 many cases effected during the two divisions of the primary spermatocyte. In some cases, however {e.g. in the salamander), the reduced number appears during the divi- sion of the spermatogonia and may even appear in the very early germ-cells (cf. p. 194). The reduction of the chromosomes, which is the most interesting and significant feature of the process, will be con- sidered in the following chapter, and we are here only concerned with the transformation of the spermatid 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 generally agreed, further, that the envelope of the tail-substance is derived from the cytoplasm of the spermatid. Beyond this point opinion is still far from unanimous, though it is probable that the other structures — viz. the axial filament, the 1 The terminology, now almost universally adopted, is due to I.a Valette St. George. Cf. Fig. 90. - See Fig. 91. GROWTH AND DIFFERENTIATION OF THE GERM-CELLS 123 middle-piece, and the point — are likewise of cytoplasmic origin ; and it is certain that the middle-piece is in some cases derived from the attraction-sphere of the spermatid, and contains the centrosome. As the spermatid develops into the spermatozoon it assumes an elongate form, the nucleus lying at one end while the cytoplasm is drawn out to form the flagellum at the opposite end. The origin of the axial filament is still in doubt. Many authors (for example, Flemming and Niessing) have described it as growing out from the nucleus; but more recent work by Hermann, Moore, and others, shows that this is probably an error and that the axial filament is derived from the substance of the attraction-sphere. The greatest uncertainty relates to the origin of the middle-piece and the apex. By one set of authors the centrosome is believed to pass into the point of the spermatozoon (Platner, Field, Benda, Pre- nant) ; by another set, into the middle-piece (Hermann, Wilcox, Cal- kins). That the latter is a correct view is absolutely demonstrated by the fact that during fertilization the centrosome in every accu- rately known case is derived from the middle-piece (amphibia, echino- derms, tunicates, earthworm, insects, mollusks, etc.). The observations of Platner and others in support of the other view are, however, too detailed to be rejected on this ground alone, and it is not impossible that the position of the centrosome may vary in different forms. The uncertainty is due to the difficulty of tracing out the fate of the cen- trosome and archoplasmic structures of the spermatid. It is certain that each spermatid receives a centrosome or attraction-sphere from the preceding amphiaster. But besides the centrosome (attraction- sphere) the spermatid may also contain a second "achromatic" body known as the paranucleus (Nebenkern) or initosome, which has un- doubtedly been mistaken for the attraction-sphere in some cases ^ and to this circumstance the existing confusion may be in part due. The concurrent results of La Valette St. George, Platner, and several others have shown that the " Nebenkern " is derived from the re- mains of the spindle-fibres ; but the most divergent accounts of its later history have been given by different investigators. According to Platner's studies on the butterfly Pygcera ('89), it consists of a larger posterior and a smaller anterior body, which he calls respectively the large and small niitosonia (Fig. 62, C). The former gives rise to the investment of the axial filament of the tail, the latter to the middle- piece, while the " centrosome " lies at the anterior end of the nucleus at the " apex" (Fig. 62, D). Field ('95) reaches an essentially similar result in the echinoderm spermatozoon, the single " Nebenkern " forming the middle-piece, while the "centrosome" lies at the tip ^ Compare the confusion between yolk-nucleus and attraction-sphere in the ovum, p. 119. 124 THE GERM-CELLS (Fig. 62, B). Benda describes the " Nebenkern " in the mammals as consisting of two parts, one of which passes backward and takes part in the formation of the tail-envelope, while the other passes forward to form the apex (head-cap or apical knob) and represents the attraction-sphere (archoplasm). A somewhat similar account was given by Platner of the " Nebenkern " of pulmonates. Accord- ing to the more recent work of Moore on elasmobranchs, both middle-piece and apex are derived from the attraction-sphere, the centrosome passing into the former (Fig. 62, A\ The work of Platner and Field appears to have been carefully Jl C Fig. 62. — Formation of the spermatozoon from the spermatid. A. Late stage of spermatid of the shark Scyllium. [Moore.] B. Spermatid of starfish ChcBtaster. [FIELD.] C. Spermatid of butterfly Pygcsra. D. Young spermatozoon of the same. [PLATNER.] a. apical body; a.f. axial filament; c. "centrosome;" e. envelope of tail; m. middle-piece (" small mitosoma" of Platner) ; n. nucleus ; /. paranucleus (" Nebenkern," or " large mitosoma" of Platner). done, yet there is good reason to believe that both these observers are in error, since their results are contradicted by the history of the spermatozoon in fertilization. As regards the insects, Henking's observations on the fertilization of the butterfly Picris leave little doubt that the sperm-centrosome is here derived from the middle- piece; and, moreover, in the grasshopper Caloptenits, Wilcox ('95) has traced the centrosome of the spermatid into the middle-piece. In the case of echinoderms, Boveri, Mathews, and myself, confirmed by several later observers, have independently traced the sperm-centro- some to the middle-piece during fertilization, and have shown that GROWTH AND DIFFERENTIATION OF THE GERM-CELLS 1 25 ■ ' ' f '% i^' »iJ Fol was in error in referring it to the tip. Field's conclusion is there- fore almost certainly erroneous, and he has probably confounded the centrosome with the " Nebenkern " or paranucleus. Diametrically opposed, moreover, to the results of Platner and Field are those of Hermann ('89) and Calkins ('95, 2) on amphibia and earthworms, and both these observers have devoted especial attention to the origin of the middle-piece. The evidence brought forward bv the last- named author, whose preparations I have critically examined, seems perfectly con- clusive that the at- traction-sphere or centrosome passes into the middle-piece. The " Nebenkern," which is rarely pres- ent, appears in this case to take no part in the formation of the fiagellum, but degenerates without further change. In the salamander the origin of the middle- piece has been care- fully studied by Flemming and Her- mann. The latter ('89) has traced the middle-piece back to Fig. 63. — Formation of the spermatozoon from the sper- matid in the salam.mder. [HERMANN.] A. Young spermatid showing the nucleus above, and below the colorless sphere, the ring, and tlie chromatic sphere. B. 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 m, an " accessory body " (Nebenkorper), which he believes to be not a " Nebenkern " (derived from the spindle-fibres), but an attraction-sphere derived from the aster of the preceding division, as in Liunbricus. This body differs greatly from an ordinary attraction-sphere, consisting of the following three parts lying side by side in the cytoplasm (Fig. 63). These are: {a) a colourless sphere, {b) a minute rounded body which stains red with saffranin like the nucleoli or plasmo- 126 THE GERM-CELLS somes of the spermatid-nucleus, ic) a ring-shaped structure staining purple with gentian violet, like the chromatin. The colourless sphere ultimately vanishes, the red rounded body gives rise to the middle- piece, while the ring gives rise to the envelope (fin) of the flagellum. The apex or spur is developed from the nuclear membrane. ^ Her- mann's results on the mouse agree in a general way with those on the salamander; but the apex (head-cap) is here derived from the cytoplasm. A " Nebenkorper" lies in the cytoplasm, consisting of a pale sphere and a smaller deeply staining body. From the latter arises the " end-knob," which Hermann accordingly homolo- gizes with the middle-piece of the salamander spermatozoon, and from it the axial filament appears to grow out into the flagellum. The colourless sphere disappears as in the salamander, and the envelope of the axial filament is derived from .the cytoplasm. M.oore ('95) describes the flagellum of elasmobranchs as growing out from the attraction-sphere (archoplasm) of the spermatid (Fig. 62, A). Sin/nnary. — The foregoing account shows that our positive know- ledge of the formation of the spermatozoon still rests upon a some- what slender basis. But despite the discrepancies in existing accounts, all agree that the spermatozoon arises by a direct meta- morphosis of the spermatid, receiving from it a nucleus and a small amount of cytoplasm containing a centrosome or attraction- sphere. All agree, further, that the middle-piece is of archoplasmic origin, being derived, according to some authors, from a true attrac- tion-sphere (or centrosome); according to others, from a " Neben- kern" formed from the spindle-fibres. The former account of its origin is certainly true in some cases. The latter cannot be accepted without reinvestigation, since it stands in contradiction to what is known of the middle-piece in fertilization, and is possibly due to a confusion between attraction-sphere and " Nebenkern." Similar doubts exist in regard to the origin of the apex, which is variously described as arising from the nuclear membrane, from the general cytoplasm, from the "Nebenkern," and from the centrosome. Most late observers agree, further, that the flagellum is developed in intimate relation with the archoplasmic material (attraction-sphere or " Nebenkern "). This conclusion tallies with that of Strasburger, who regards the flagella of plant-spermatozoids as derived from the " kinoplasm " (archoplasm), and it is of especial interest in view of Van Beneden's hypothesis of the contractility of the archoplasm- fibrilte. It is, however, possible that the axial filament may be derived from the nucleus, in which case it would have an origin comparable with that of the spindle-fibres in many forms of mitosis. ' Flemminp; described the middle-piece as arising inside the nucleus ; liut Hermann's observations leave no doubt that this was an error. STAINING-REACTIONS OF THE GERM-NUCLEI 12/ 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 gland of this animal be treated with a mixture of saffranin and methyl- green, the egg-nuclei are coloured red, the sperm-nuclei bluish-green. A similar difference was afterwards 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, carmin), while the sperm-nuclei were espe- cially stained with blue and green dyes (methyl-green, aniline blue, hsematoxylin). He was thus led to regard the chromatin of the egg as especially " erythrophilous," and that of the sperm as " cyanophi- lous." That the distinction as regards colour is of no value has been shown by Zacharias, Heidenhain, and others; for staining agents cannot 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 haema- toxylin. Thus Field has shown that the sperm-nucleus of Asterias may be stained green (methyl-green), blue (hsematoxylin, gentian violet), red (saffranin), or yellow (iodine), and it is here a manifest absurdity to speak of " cyanophilous " chromatin (cf. p. 243). 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 tha.t when the spermatids and immature spermatozoa of the salamander are treated with saffranin (red) and gentian violet (blue),i 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 described at pp. 245, 246. 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 1 By Flemming's triple method. - '92, p. 492. 128 THE GERM-CELLS similar series of facts has been observed in the germ-nuclei of plants by Strasburger (p. 163). 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 the difference of the staining-reaction is probably due to the fact that the sperm-chromatin consists of pure or nearly pure nucleic acid, while the egg-chromatin is a nuclein containing a much higher per- centage of albumin. LITERATURE. Ill Ballowitz, E. — Untersuchungen Liber die Strulvtur der Spermatozoen : I. {birds) Arch. Mik. Anat. XXXII., 1888; 2. {insects) Zeitschr. Wiss. ZooL, L., 1890; 3. {fishes, ainpJiibia, reptiles) Arch. Mik. Anat., XXXVI., 1890; 4. {niain- inals) Zeit. M'iss. Zoo/., LI I., 1891. Van Beneden, E. — Recherches sur la composition et la signification de Poeuf : Mem. coi/r. de I Acad. ray. de s. de Bclgiqne, 1870. Boveri, Th. — tJber Differenzierung der Zellkerne wahrend der Furchung des Eies von Ascaris meg. : Anat. Ans., 1887. Brunn, M. von. — Beitrage zur Kenntniss der Samenkorper und ihrer Entwickelung bei Vogeln und Saugetliieren : Arch. Mik. Anat., XXXIII., 1889. Hacker, V. — Die Eibiidung bei Cyclops und Camptocanthus : Zool. Jahrb., V., 1892. (See also List V.) Hermann, F. — Urogenitalsystem ; Struktur und Histiogenese der Spermatozoen : Mer/cel und Bonnet^' Ergebnisse, II., 1892. Kblliker, A. — Beitrage zur Kenntniss der Geschlecbtsverhaltnisse und der Samen- fliissigkeit wirbelloser Tiere. Berlin, 1841. Leydig, Fr. — Beitrage zur Kenntniss des thierischen Eies im unbefruchteten Zu- stande. Zool. Jahrb., III. 1889. Schweigger-Seidel, F. — Uber die Samenkorperchen und ihre Entwicklung: Arch. Mik. Anat., I. 1865. Strasburger, E. — Histologische Beitrage; Heft IV: Das Verhalten des Pollens und die Befruchtungsvorgange bei den Gymnospermen, Schwarmsporen, ptlanz- liche Spermatozoiden und das Wesen der Befruchtung. Fischer, Jena, 1892. Thomson, Allen. — Article. "Ovum,'" in Todd's Cyclopedia of Anatomy and Physi- ology. 1859. Waldeyer, W. — Eierstock und Ei. Leipzig, 1870. Id. — Bau und Entwickelung der Samenfaden : Verh. d. Aiiat. Ges. Leipzig, 1887. 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, regarded 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." HuXLEY.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 duplicate 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 probable, 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 gradually comes to an end and is only restored by an admixture of living- matter derived from anotJier cell. This operation, known :is fertilisation ox fecundation, is the essence of sexual repro- duction ; 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. According to the older and more familiar "dynamic " hypothesis, brought forward by Biitschli ('76) and Minot i'n^ '79) and afterwards supported by such investigators as Engel- mann, Hensen, Hertwig, and Maupas, the essential end of sexuality is rejuvenescence, i.e. the restoration of the growth-energy and the inauguration of a new cycle of cell-division. Maupas's celebrated experiments on the conjugation of Infusoria, although not yet ade- 1 Evolution, in Science and Culture, p. 296, from Enc. Bril., 1878. K 129 130 FERTILIZATION OF THE OVUM quately confirmed, 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 conjugation or fertilization actually has such a dynamic effect is disputed by no one. What is not determined is whether this is the primary motive for the process — i.e. whether the need of fertilization is a primary attribute of living matter — or whether it has been secondarily acquired in order to ensure a mixture of germ-plasms derived from different sources. The latter view has been urged with great force by Weismann, who rejects the rejuve- nescence theory in toto and considers the essential end of fertilization to be a mixture of germ-plasms ("Amphimixis") as a means for the production, or rather multiplication, of variations which form the material on which selection operates. On the other hand, Hatschek ('87, i) sees in fertilization exactly the converse function of checking variations and holding the species true to the specific type. The present state of knowledge does not, I believe, allow of a decision between these diverse views, and the admission must be made that the essential nature of sexual reproduction must remain undetermined until the subject shall have been far more thoroughly investigated, especially in the unicellular forms, where the key to the ultimate problem is undoubtedly to be sought. A. General Sketch Among the unicellular plants and animals, fertilization is effected by means of conjugation, a process in which two or more individuals permanently fuse together, or in which two unite temporarily and effect an exchange of nuclear matter, after which they separate. In all the higher forms fertilisation consists in the pennancnt fusion of tzvo genn-cells, one of paternal and one of maternal origin. We may first consider the fertilization of the animal &^g, 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. Leeuwenhoek, whose pupil Hamm discovered the spermatozoa (1677), put forth the conjecture that the spermatozoon must pene- trate into the egg ; but the process was not actually seen until nearly two centuries later (1854), when Newport observed it in the case of the frog's ^^^\ and it was described by Pringsheim a year later in one of the lower plants, GLdigoniiDn. The first adequate description of the process was given by Hermann Fol, in 1879,^ though many ^ See I Ilhtogenie, pp. 124 ff., for a full liistorical account. GENERAL SKETCH I.^I earlier observers, from the time of Martin Barry ('43) onwards, had seen the spermatozoa inside the egg-envelopes, or asserted its entrance into the Q.gg. In many cases the entire spermatozoon enters the Q,gg (mollusks, insects, nematodes, some annelids, Petromyzon, axolotl, etc.), and in such cases the long fiagellum may sometimes be seen coiled within the Qg^ (Fig- 64). 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 egg. At or near .0 •^, 00 o.r,; ^ d» Fig. 64. — Fertilization of the egg of the snail P/iysa. [Kostanfxki and Wierzejski.] A. The entire spermatozoon lies in the egg, its nucleus at the right, fl.igellum 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 afterwards disappear, their place being taken by those of the spermatozoon. the time of fertilization, the egg successively segments off at the upper pole two minute cells, known as the po/ar bodies (Figs. 64, 65, 89) 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 egg is prepared for fertilization, and we may defer its consideration to the following chapter. 132 FERTILIZATIOX OF THE OVUM I. The Gcnu-iiiiclci in Frrti/i. 'nation 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 egg. Earlier observers had, it is true, paved the way by showing that, at the time of fertilization, the Qgg contains Hvo nuclei that fuse together or become closely associated before development begins. (Warneck, Biitschli, Auerbach, Van Beneden, Strasburger.) Hertwig discovered, in the O-gg of the sea-urchin ( Toxopnciistcs lividiis), that one of these nuclei belongs to the egg, ivhile the other is derived from the sperniatozodn. 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. In every hnoivn ease an essential phenomenon of fertilirjation is the union of a sperm-nucleus, of paternal origin, ivitJi an egg-nucleus, of maternal origin, to form the primary nnclens of the embryo. This nucleus, kjiozvn as the cleavage- or segmentation-7iucleus, gives rise by division to all the nuclei of the body, and hence every nnclens of tJie child may contain nuclear substance derived from both parents. And thus Hertwig 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 ele- ment concerned in hereditary transmission. This conclusion received a strong support in the year 1883, through the splendid discoveries of Van Beneden on the fertilization of the thread- worm, Ascaris mcgalocepJiala, the egg of which has since ranked with that of the echinoderm as a classical object for the study of cell- problems. Van Beneden's researches especially elucidated the struct- ure and transformations of the germ-nuclei, and carried the analysis of fertilization far beyond that of Hertwig. In Ascaris, as in all other animals, the sperm-nucleus is extremely minute, so that at first sight a marked inequality between the two sexes appears to exist in this respect. Van Beneden showed not only that the inequality in sjze totally disappears during fertilization, but that the two nuclei undergo a parallel series of structural changes which demonstrate their precise morphological equivalence down to the minutest detail ; and here, again, later researches, foremost among them those of Boveri, Strasburger, and Guignard, have shown that, essentially, the same is true of the germ-cells of other animals and of plants. The facts in Ascaris (variety bivalens) are essentially as follows (Fig. 65) : After the entrance of the spermatozoon, and during the for- mation of the polar bodies, the sperm-nucleus rapidly enlarges and GENERAL SKETCH 133 E ^ Fig. 65. - Fertilization of the egg of Ascaris megalocepkala, var. bivaleus. [BOVERI.] (tor later stages see Fig. 104.) A. The spermatozoon has entered the egg, its nucleus is shown a. c?; ^''^^f^ '"^^I'^^J-^^Z^ lar mass of '■ archoplasm" (attraction-sphere) ; above are the clos.ng phase n the ^ -» "J ° the second polar body (two chromosomes in each nucleus). B. ^^''^-''f'^'i^'J^^^:^^^^^ lar stage ; the attraction-sphere (a) contains the dividmg centrosome. C. '-^'^'^^;'''^'; '^^^^^^l in the ^e m-nuclei; the centrosome divided. D. Each germ-nucleus resolved mto t^vo chromo so nesT attraction-;phere (.) double. E. Mitot.c figure forming for the ^rst cleavage ^he chro- mosomes (.) alreadv split. /••. First cleavage in progress, showmg divergence of the daughter chromosomes towards the spindle-poles (only three chromosomes shown). 134 FERTILIZATION OF THE OVUM finally forms a typical nucleus exactly similar to the egg-nucleus. The chromatin in each nucleus now resolves itself into two long, worm-like chromosomes, which are exactly similar in form, size, and staining reaction in the two nuclei. Next, the nuclear membrane fades away, and the four chromosomes lie naked in the egg-substance. Every trace of sexual difference has now disappeared, and it is impossible to distinguish the paternal from the maternal chromo- somes (Figs. 65, D, E). Meanwhile an amphiaster has been devel- oped which, with the four chromosomes, forms the mitotic figure for the first cleavage of the ovum, the chromatic portion of zuhich has been syjithctically formed by the union of two equal germ-nuclei. The A B Fig. 66. — Germ-nuclei and chromosomes in tlie eggs of nematodes. [Carnoy.] A. Egg of nematode parasitic in Scyllium ; the two germ-nuclei in apposition, each containing four chromosomes; the two polar bodies above. B. Y^gg oi Filar aides ; each germ-nucleus with eight chromosomes ; polar bodies above, deutoplasm-spheres below. later phases follow the usual course of mitosis. Each chromosome splits lengthwise into equal halves, the daughter-chromosomes are transported to the spindle-poles, and here they give rise, in the usual manner, to the nuclei of the two-celled stage. Each of these nuclei, therefore, receives exactly equal amounts of paternal and mater)ial chromatin. These discoveries were confirmed and extended in the case of Ascaris by Boveri and by Van Beneden himself in 1887 and 1888 and in several other nematodes by Carnoy in 1887. Carnoy found the number of chromosomes derived from each sex to be in Coronilla 4, in Ophiostomum 6, and in Filaroides 8. A little later Boveri GENERAL SKETCH 135 ('90) showed that the law of numerical equality of the paternal and maternal chromosomes held good for other groups of animals, being in the sea-urchin Echinus 9, in the worm Sagitta 9, in the medusa Tiara 14, and in the vi\oWvi'i\i PterotracJica 16 from each sex. Similar results were obtained in other animals and in plants, as first shown by Guignard in the lily ('91), where each sex contributes 12 chromosomes. In the onion the number is 8 (Strasburger) ; in the annelid Ophryo- troclia it is only 2 from each sex (Korschelt). In all these cases the iiuuibcr contributed by each is onc-Jialf the number characteristic of the body-cells. The union of two germ-cells thus restores the normal number, and thus we find the explanation of the remarkable fact commented on at p. 48 that tJie ninnber of chromosomes in sexually produced organisms is ahvays evcji} These remarkable facts demonstrate the two germ-nuclei to be in a morphological sense precisely equivalent, and they not only lend very strong support to Hertwig's identification of the nucleus as the bearer of hereditary qualities, but indicate further that these qualities must be carried by the chromosomes ; for their precise equivalence in number, shape, and size is the physical correlative of the fact that the two sexes play, on the whole, equal parts in hereditary transmis- sion. And thus we are finally led to the view that chromatin is the physical basis of inheritance, and that the smallest visible units of structure by which inheritance is effected are to be sought in the chromatin-granules or chromomeres. 2. The Centrosome in Fertilization The origin of the centrosomes and of the amphiaster, by means of which the paternal and maternal chromosomes are distributed and the Q^g^ divides, is still in some measure a matter of dispute. In a large number of cases, however, it is certainly known tljat the egg-cen- trosome disappears before or during fertilization and its place is taken by a nczv ccjitrosonte which is introduced by the spcj'matododn and divides into tivo to form the cleavage-amphiaster. This has been conclusively demonstrated in several forms (various echinoderms, annelids, nematodes, tunicates, mollusks, and vertebrates) and estab- lished with a high degree of probability in many others (insects, Crus- tacea). In every accurately known case, moreover, the centrosome has been traced to the middle-piece of the spermatozoon ; e.g. in sea-urchins (Hertwig, Boveri, Wilson, Mathews, Hill), in the axolotl (Fick), in the tunicate Phallusia (Hill), probably in the earthworm, 1 Cf. p. 154. 136 FERTILIZATION OF THE OVUM Allolobophoj-a (Foot), in the butterfly Picris (Henking), and in the gasteropod Physa (Kostanecki and Wierzejski). The agreement between forms so diverse is very strong evidence that this must be regarded as the typical derivation of the centrosome.^ The facts may be illustrated by a brief description of the phe- Fig. 67. — Maturation and fertilization of tlie egg of the mouse. [SOKOTTA.] A. Tlie ovarian egg still surrounded by the follicle-cells and the membrane {:./., zona pel- lucida) ; the polar spindle formed. B. Egg immediately after entrance of the spermatozoon (sperm-nucleus at ,'^). C. The two germ-nuclei (j', ? ) still unequal; polar bodies above. D. Germ-nuclei approaching, of equal size. E. The chromosomes forming. F. The minute cleavage-spindle in the centre; on either side the paternal and maternal groups of chromosomes. nomena in the sea-urchin Toxopnc/istcs (Fig. 69). As described at p. 146, the tail is in this case left outside, and only the head and middle-piece enter the Qgg. Within a few minutes after its entrance, and while still very near the periphery, the lance-shaped sperm-head, carrying the middlcrpiece at its base, rotates through nearly or quite 1 Cf. p. 156. GENERAL SKETCH m 1 80°, SO that the pointed end is directed outward and the middle- piece is turned inward (Fig. 69 A-F)} During the rotation a minute aster is developed about the middle-piece as a centre, and at the B Fig. 68. — Fertilization of the egg of the gasteropod Pterotracbea. [BOVERI.] A. Tlie egCT-nucleus {E) and sperm-nucleus (5) approaching after formation of the polar bodies; the latter shown above {P.B.)\ each germ-nucleus contains sixteen chromosomes; the sperm-amphiaster fully developed. B. The mitotic figure for the first cleavage nearly established ; the nuclear membranes have disappeared leaving the maternal group of chromosomes above the spindle, the paternal below it. ' 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. 1^.8 FERTILIZATION OF THE OVUM central point a minute intensely staining centrosome may be seen.^ As the sperm-nucleus advances, the aster leads the way, and at the same time rapidly grows, its rays extending far out into the cytoplasm and finally traversing nearly an entire hemisphere of the egg. The central mass of the aster comes in contact with the egg-nucleus, di- vides 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. 70). The nuclei now fuse to form the cleavage-nucleus. Shortly afterwards the nuclear mem- brane fades away, a spindle is developed between the asters, and i m X B C D '-^'f^ Fig. 69. — Entrance and rotation of tlie sperm-head and formation of tlie sperm-aster in the sea-urchin Toxopiiensfes (A.-F., X 1600; G. //., X 800). A. Sperm-head before entrance; «, nucleus; m, middle-piece and part of the flagellum. B. C. Immediately after entrance, showing entrance-cone. D.-F. Rotation of the sperm-head, formation of the sperm-aster about the middle-piece (the minute centrosome not shown). G. H. Approach of the germ-nuclei ; growth of the aster. 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. Throughout these changes no trace of an egg-centrosome is to be discovered. This centrosome, though present in earlier stages, has been lost after the polar bodies were formed by the ovarian ^g^. 1 I was unable to find such a centrosome in Toxopneustes, but the observations of Boveri and Hill prove that it is certainly present in other sea-urchins, and I now lielieve my own account to have been at fault in this respect. GENERAL SKETCH 139 The facts just described are now known to be typical of a large number of cases. We may, however, distinguish two types of ferti- Fig. 70. — Conjugation of the germ-niiclei and division of the sperm-aster in the sea-urchin Toxopneustes, X 1000. (For later stages see Fig. 37.) . /. Union of the nuclei, extension of the aster. D. Flattening of the sperm-nucleus against the egg-nucleus, division of the aster. ' lization according as the polar-bodies are formed before or after the entrance of the spermatozoon. In the first case, well illustrated by the sea-urchin (Fig. 69), the germ-nuclei conjugate immediately after I40 FERTILIZATION OF THE OVUM entrance of the spermatozoon. In the second and more frequent case {Ascaris, Fig. 65 ; Physa, Fig. 64; Nereis, Fig. 71 ; Cyclops, Fig. 72), the sperm-nucleus penetrates for a certain distance, often to the cen- tre of the Q.gg, and then pauses while the polar bodies are formed. It then conjugates with the reformed egg-nucleus. In this case, the sperm-aster always divides to form an amphiaster before conju- gation 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 form during extrusion of the polar bodies. It is an interesting and significant fact that the aster or amphiaster always leads the way in the march towards the egg-nucleus ; and in many cases it may be far in advance of the sperm-nucleus.^ Bovori ('88, i) has observed in sea-urchins that the sperm-nucleus may indeed be left entirely behind, the aster alone conjugating with the egg- nucleus and causing division of the egg without wiion of the gcrni- nuclci, though the sperm-nucleus afterwards 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 demonstration of the fact that // is the centro- soDie a/one that ca?ises division of the egg, and it is therefore the fer- tilising element proper (Boveri, '8"/, 2). We may therefore conclude that the end of fertilization is the union of the germ-nuclei and the equal distribution of their substance, while the active agent in this process is the centrosome. The earliest investigators of fertilization, such as Biitschli and Fol, had no knowledge of the centrosome, and hence no clear idea as to the origin of the asters, but Fol stated in 1873 that the asters repre- sented "centres of attraction" lying outside and independent of the nucleus. Oscar Hertwig showed, in 1875, that in the sea-urchin egg the amphiaster arises by the division of a single aster that first appears near the sperm-nucleus and accompanies it in its progress toward the egg-nucleus. A similar observation was soon afterwards made by Fol ('79) in the eggs of Asterias and Sagitta, and in the latter case he determined the fact that the astral rays do not centre in the nucleus, as Hertwig described, but at a point in advance of it, — a fact afterwards confirmed by Hertwig himself and by Boveri ('88, I ). Hertwig and Fol afterwards found that in cases of poly- spermy, when several spermatozoa enter the 0.^2,, each sperm-nucleus is accompanied by an aster, and Hertwig proved that each of these might give rise to an amphiaster (Fig. 75). ^ Cf. Kostanecki and Wierzejski, '96. GENERAL SKETCH 141 It was Boveri ("87) who first accurately traced the complete history of the centrosome and clearly formulated the facts, proving that in Ascans a single centrosome is brought in by the spermatozoon and that it divides to form two centres about which are developed the two Fig. 71. — Fertilization of the egg of A'^^ifM, from sections. ( X 400.) . /. Soon after the entrance of the spermatozoon, showing the minute sperm-nucleus at Q., the germinal vesicle disappearing, and the first polar mitotic figure forming. The empty spaces repre- sent deutoplasm-spheres (slightly swollen by the reagents), the firm circles oil-chops. B. Sperm- nucleus (d") advancing, a minute amphiaster in front of it; first polar mitotic figure established; polar concentration of the protoplasm. C. Later stage; second polar body forming. D. The polar bodies formed; conjugation of the germ-nuclei; the egg-centrosomes and asters have disappeared, leaving only the sperm-amphiaster (cf. Fig. 64). asters of the cleavage-figure. He was thus led to the following con- clusion, which I believe still accurately expresses the truth: ''The ripe eger possesses all of the organs and qualities necessary for division excepting the centrosome, by zvhich division is initiated. The sperma- 142 FERTILIZATION OF THE OVUM to::odn, on the other hand, is provieled zvitJi a centrosome, bnt lacks the substance in ivJiich this organ of division may exert its activity. Through the union of the tzvo cells in fertilization all of the essential organs necessary for division are brought togetJier ; the egg notv contains a centrosome which by its own division leads the ivay in the embryonic development} Boveri did not actually follow the disappearance of the egg-centrosome, but nearly at the same time this process was carefully described by Vejdovsky in the case of a fresh-water annelid Rhynchelmis. Here, again, very strong evidence was brought for- Fig. 72. — Fertilization of the egg in tlie copepod Cyclops stienuus. [RuCKERT.] A. Sperm-nucleus soon after entrance, the sperm-aster dividing. B. The germ-nuclei ap- proaching; c?,the enlarged sperm-nucleus with a large aster at each pole; $,the egg-nucleus reformed after formation of the second polar body, shown at the right. C. The apposed reticular germ-nuclei, now of equal size; the spindle is immediately afterwards developed between the two eiioimous sperm-asters ; polar body at the left. ward to show that the cleavage-amphiaster arises by the division of a single sperm-aster. Very numerous observations to the same effect have been made by later observers. Bohm could find in Petromyzon ('88) and the trout ('91) no radiations near the egg-nucleus after the formation of the polar-bodies, while a beautiful sperm-aster is devel- oped near the sperm-nucleus and divides to form the amphiaster. Platner ('86) had already made similar observations in the snail Arion, and the same result was soon afterwards reached by Brauer ('92) in the case of Branchipus, and by Julin ('93) in Styleopsis. Pick's careful study of the fertilization of the axolotl ('93) proved in 1 -87, 2, p. 155. GENERAL SKETCH . 1 43 a very convincing manner not only that the amphiaster is a product of the sperm-aster, but also that the latter is developed about the middle-piece as a centre. The same result was indicated by Foot's observations on the earthworm ('94), and it was soon afterwards conclusively demonstrated in echinoderms through the independent and nearly simultaneous researches of myself on the ^g^ of Toxo- pncustes, of Mathews on Aj-bacia, and of Boveri on EcJiimis. Nearly at the same time a careful study was made by Mead ('95) of the annelid Chceoptents, and of the starfish Asterias by Mathews, both observers independently showing that the polar spindle contains dis- tinct centrosomes, which, however, degenerate after the formation of the polar bodies, their place being taken by the sperm-centrosome, which divides to form an amphiaster before union of the nuclei, as in Rhyjichelmis. Exactly the same result has since been reached by Hill ('95) in SpJi(zrcchiniis and the tunicate Phallusia, and by Kos- tanecki and Wierzejski ('96) in PJiysa (Fig. 64) ; and in all of these the centrosome is likewise shown to arise from the middle-piece. The origin of the centrosome from the spermatozoon alone has also been shown by Riickert ('95, 2) in Cyclops (Fig. -72), and is indicated by Sobotta's work ('95) on the fertilization of the mouse (Fig. 67). Such an array of evidence, derived from the study of so many diverse groups, places Boveri's conception of fertilization (p. 141) on a very strong foundation, and justifies the conclusion that the origin of the first cleavage-centrosomes from the spermatozoon alone is a phenomenon of very wide, if not of universal, occurrence. The descendants of these centrosomes may be traced continuously into later cleavage-stages, and there can be little doubt that they are the progenitors of all the centrosomes of the adult body. Boveri and Van Beneden, followed by a number of later observers,^ have followed the daughter-centrosomes through every stage of the first cleavage into the blastomeres of the two-cell stage, where they persist and give rise to the centrosomes of the four-cell stage, and so on in later stages. This is beautifully shown in the Q.gg of Thalassemia (Fig. 73), which has been carefully followed out in my laboratory by Mr. B. B. Griffin. The centrosome is here a minute granule at the focus of the sperm- aster, which divides to form an amphiaster soon after the entrance of the spermatozoon. During the early anaphase of the first cleavage each centrosome divides into two, passes to the outer periphery of the centrosphere, and there forms a minute amphiaster for the second cleavage before the first cleavage takes place (Fig. 73) ! The minute centrosomes of the second cleavage are therefore the direct descendants of the sperm-centrosome ; and there is good reason to believe that the 1 See Mead on Chisopierus, '95, and Kostanecki and Wierzejski on Physa, '96. 144 FERTFLTZATIOX OF THE OVUM continuity is not broken ni later stages. An exactly similar process is described by Kostanecki and Wierzejski in the &^^ of PJiysa. We thus reach the following remarkable conclusion : During cleavage the cytoplasm of tlic blastoinercs is derived from that of the egg, the centrosomcs from the spermatozoon, ivhile the nuclei {cJiromatin) are Fig. .73. — Persistence of the centrosomes from cell to cell, in ihc cleavage of the egg of the gephyiean Thalasscina. [GRIFFIN.] A. Mitotic figure for the first cleavage ; the centrosome already double in each centrosphere (the small black bodies are deutoplasni-spheres). B. Early anaphase ; migration of the centro- somes to the periphery of the centrosphere. C. Middle anaphase (only one-half of the mitotic figure shown) ; daughter-amphiaster already formed. D. Telophase : the egg dividing and nuclei reforming ; the old amphiaster has disappeared, leaving only the daughter-amphiaster in each cell. UNION OF THE GERM-CELLS 1 45 equally derived from both gcnn-cclls ; and certainly it would be hard to find more convincing evidence that the chromatin is the controlling factor in the cell by which its specific character is determined. We now proceed to a more detailed and critical examination of fertilization. B. Union of the Germ-cells It docs 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- dence of a definite attraction between the germ-cells, which is in some cases so marked (for example in the polyp Rcnilld) 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 Pfeffcr'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 Selaginclla to be as actively attracted by solutions of malic acid or malates (con- tained in capillary tubes) as by the substance extruded from the neck of the archegonium. Those of mosses, on the other hand, are indifferent to malic acid, but are attracted by cane-sugar. These experiments indicate that the specific attraction between the germ- cells of the same species is owing to the presence of specific chemical substances in each case. There is clear evidence, furthermore, that the attractive force is not exerted by the egg-nucleus alone, but by the egg-cytoplasm; for, as- the Hertwigs and others have shown, spermatozoa will readily enter egg-fragments entirely devoid of a nucleus. In naked eggs, such as those of some echinoderms, and coelen- terates, the spermatozoon may enter at any point ; but there are some cases in which the point of entrance is predetermined by the presence of special structures through which the spermatozoon 146 FERTILIZATION OF THE OVUM enters (Fig. 74). Thus, the starfish Q^g, according to Fol, pos- sesses before fertilization a pecuhar protoplasmic " attraction-cone " to which the head of the spermatozoon becomes attached, and through which it enters the Qgg. In some of the hydromedusse, on the other hand, the entrance point is marked by a funnel-shaped depression at the egg-periphery (Metschnikoff). When no preformed attraction- cone is present, an " entrance-cone " is sometimes formed by a rush of protoplasm towards the point at which the spermatozoon strikes the ^gg and there forming a conical elevation into which the sperm- head passes. In the sea-urchin (Fig. 74) this structure persists only a short time after the spermatozoon enters, soon assuming a /- m n ■■-■ a lilO . ■ Fig. 74. — Entrance of the spermatozoon into the egg. A.-G. In the sea-urchin Toxppneustes. //. In the medusa il/z/wcowi?. [Metschnmkoff.] /. In the star-fish .-:/.f/tV7aj. [FoL.] A. Spermatozoon of Toxopneustes, X 2000; a, the apical body, ;/, nucleus, m, middle-piece, f, fiagellum. D. Contact with the egg-periphery. C. D. Entrance of the head, formation of the entrance-cone and of the vitelline membrane (y), leaving ihe tail outside. E.F. Later stages. G. Appearance of the sperm-aster {s) about 3-5 minutes after first contact; entrance-cone break- ing up. H. Entrance of the spermatozoon into a preformed depression. /. Approach of the spermatozoon, showing the preformed attraction-cone. ragged flame-shape and breaking up into slender rays. In some cases the Q.gg remains naked, even after fertilization, as appears to be the case in many ccelenterates. More commonly a vitelline mem- brane is quickly formed after contact of the spermatozoon, — e.g. in Amphioxiis, in the echinoderms, and in many plants, — and by means of this the entrance of other spermatozoa is prevented. In eggs surrounded by a membrane before fertilization, the spermato- zoon either bores its way through the membrane at any point, as is probably the case with mammals and amphibia, or may make its entrance through a micropyle. In some forms only one spermatozoon normally enters the ovum, UNION OF THE GERM-CELLS H7 as in cchinoderms, mammals, many annelids, etc., while in others several may enter (insects, elasmobranchs, reptiles, the earthworm, Petromyzoii, etc.). In the former case more than one spermatozoon may accidentally enter (pathological polyspermy), but development is then always abnormal. In such cases each sperm-centrosome gives rise to an amphiaster, and the asters may then unite to form the most complex polyasters, the nodes of which are formed by the Fig- 75- — Pathological polyspermy. A. Polyspermy in the egg oi .{scans ; below, the egg-nucleus ; above, three entire spermatozoa within the egg. [Sala.] D. Polyspermy in sea-urchin egg treated with 0.005 % nicotine-solution ; ten sperm-nuclei shown, three of which have conjugated with the egg-nucleus. C. Later stage of an egg similarly treated, showing polyasters formed by union of the sperm-amphiasters. [O. and R. HERlWic;.] centrosomes (Fig. 75). Such eggs either do not divide at all or undergo an irregular multiple cleavage and soon perish. If, how- ever, only two spermatozoa enter, the egg may dcv^elop for a time. Thus Driesch has determined the interesting fact, which I have con- firmed, that sea-urchin eggs into which two spermatozoa have acci- dentally entered undergo a double cleavage, dividing into four at the first cleavage, and forming eight instead of four micromeres at the 148 FERTILIZATIOX OF THE OVUM fourth cleavage. Such embryos develop as far as the blastula stage, but never form a gastrula.^ In cases where several spermatozoa normally enter the *^'g^^ (physiological polyspermy), only one of the sperm-nuclei normally unites with the egg-nucleus, the supernumerary sperm-nuclei either degenerating, or in rare cases — e.g. in elasmo- branchs and reptiles — living for a time and even dividing to form " merocytes " or accessory nuclei. The fate of the latter is still in doubt ; but they certainly take no part in fertilization. It is an interesting question how the entrance of supernumerary spermatozoa is prevented in normal monospermic fertilization. In the case of echinoderm-eggs Fol advanced the view that this is mechanically effected by means of the vitelline membrane formed instantly after the first spermatozoon touches the Q^^. This is indi- cated by the following facts. Immature eggs, before the formation of the polar bodies, have no power to form a vitelline membrane, and the spermatozoa always enter them in considerable numbers. Polyspermy also takes place, as O. and R. Hertwig's beautiful ex- periments showed ('87), in ripe eggs whose vitality has been dimin- ished by the action of dilute poisons, such as nicotine, strychnine, and morphine, or by subjection to an abnormally high temperature (31° C.) ; and in these cases the vitelline membrane is only slowly formed, so that several spermatozoa have time to enter.^ Similar mechanical explanations have been given in various other cases. Thus Hoffman believes that in teleosts the micropyle is blocked by the polar-bodies after the entrance of the first spermatozoon ; and Calberla suggested {Pctnv/iyrjon) that the same result might be caused by the tail of the entering spermatozoon. It is, however, far from certain whether such rude mechanical explanations are adequate ; and there is considerable reason to believe that the egg- may possess a physiological power of exclusion called forth by the first spermatozoon. Thus Driesch found that spermatozoa did not enter fertilized sea-urchin eggs from which the membranes had been removed by shaking.'^ In some cases no membrane is formed (some coelenterates), in others several spermatozoa are found inside the membrane (nemertines), in others the spermatozoon may penetrate the membrane at any point (mammals), yet monospermy is the rule. ^ For an account of the internal changes, see p. 261. - The Hertwigs attribute this to a diminished irritability on the part of the egg-substance. Normally requiring the stimulus of only a single spermatozoon f(jr the formation of the vitel- line meml:>rane, it here demands the more intense stimulus of two, three, or more before the membrane is formed. Tliat the membrane is not present before fertilization is admitted by Hertwig on the ground stated at p. 97. ^ On the other hand, Morgan states ('95, 5, p. 270) that one or more spermatozoa will enter nucleated or enucleated egg-fragments whether obtained before or after fertilization. UNION OF THE GERM-CELLS 1 49 I. Innncdiatc Results of Union The union of the germ-cells calls forth profound changes in both. (a) The Spermatozoon. — Almost immediately after contact the tail ceases its movements. In some cases the tail is left outside, being carried away on the outer side of the vitelline membrane, and only the head and middle-piece enter the Q.g^ (echinoderms. Fig. 74). In other cases the entire spermatozoon enters (amphibia, earthworm, insects, etc., Fig. 64), but the tail always degenerates within the ovum and takes no part in fertilization. Within the ovum the sperm-nucleus rapidly grows, and both its structure and staining- capacity rapidly change (cf. p. 127). The most important and signifi- cant result, however, is an immediate resumption by the spcjin-nucleus and sperm-eentrosoj/ie of the poiver of division which has hitherto been suspended. This is not due to the union of the germ-nuclei ; for, as the Hertwigs and others have shown, the supernumerary sperm-nuclei in polyspermic eggs may divide freely without copu- lation with the egg-nucleus, and they divide as freely after entering enucleated egg-fragments. The stimulus to division must therefore be given by the egg-cytoplasm. It is a very interesting fact that in some cases the cytoplasm has this effect on the sperm-nucleus only after formatioji of the polar bodies ; for when in sea-urchins the spermatozoa enter immature eggs, as they freely do, they penetrate but a short distance, and no further change occurs. (/;) The Ovum. — The entrance of the spermatozoon produces an extraordinary effect on the Qg%, which extends" to every part of its organization. The rapid formation of the vitelline membrane, already described, proves that the stimulus extends almost instantly through- out the whole ovum.^ At the same time the physical consistency of the cytoplasm may greatly alter, as for instance in echinoderm eggs, where, as Morgan has observed, the cytoplasm assumes immediately after fertilization a peculiar viscid character which it afterwards loses. In many cases the Q.g^ contracts, performs amoeboid move- ments, or shows wave-like changes of form. Again, the egg-cyto- plasm may show active streaming movements, as in the formation of the entrance-cone in echinoderms, or in the flow of peripheral proto- plasm towards the region of entrance to form the germinal disc, as in many pelagic fish-eggs. An interesting phenomenon is the formation, behind the advancing sperm-nucleus, of a peculiar funnel-shaped mass of deeply staining material extending outwards to the periphery. This has been carefully described by Foot ('94) in the earthworm, 1 I have often observed that the formation of the membrane, in 7'oxopitftts/es^ proceeds like a wave from the entrance-point around the periphery, but this is often irregular- I50 FERTILIZATION OF THE OVUM where it is very large and conspicuous, and I have since observed it also in the sea-urchin (Fig. 69). The most profound change in the ovum is, however, the migration of the germinal vesicle to the periphery, and the formation of the polar bodies. In many cases either or both these processes may occur before contact with the spermatozoon (echinoderms, some vertebrates). In others, however, the egg awaits the entrance of the spermatozoon (annelids, gasteropods, etc.), which gives it the necessary stimulus. This is well illustrated by the Q^g of Nereis. In the newly-dis- charged egg the germinal vesicle occupies a central position, the yolk, consisting of deutoplasm- spheres and oil-globules, is uni- formly distributed, and at the periphery of the egg is a zone of clear perivitelline protoplasm (Fig. 43). Soon after entrance of the spermatozoon the germinal vesicle moves towards the periphery, its membrane fades away, and a radi- ally directed mitotic figure appears, by means of which the first polar body is formed (Fig. 71). Mean- while the protoplasm flows towards the upper pole, the perivitelline zone disappears, and the Q,g^ now shows a sharply marked polar differentiation. A remarkable phe- nomenon, described by Whitman in the leech ('78), and later by Foot in the earthworm ('94), is the formation of " polar rings," a process which follows the entrance of the spermatozoon and accompanies the formation of the polar bodies. These are two ring-shaped cytoplasmic masses which form at the periphery of the egg near either pole and advance thence towards the poles, the upper one surrounding the point at which the polar bodies are formed (Fig. 76). Their meaning is unknown, but Foot ('96) has made the interesting discovery that they are probably of the same nature as the yolk-nuclei (p. 121). Fig. 76. — Egg of the leech Clepsiiic, dur- ing fertilization. [Whitman.] I>.b., polar bodies ; p.r., polar rings ; cleav- age-nucleus near the centre. UNION OF THE GERM-CELLS 151 2. Paths of the Gcrui-nnclei {Pro-nuclei) ^ After the entrance of the spermatozoon both germ-nuclei move through the egg-cytoplasm and finally meet one another. The paths traversed by each vary widely in different forms. In general two classes are to be distinguished, according as the polar-bodies are formed before or after entrance of the spermatozoon. In the former case (echinoderms) the germ-nuclei unite at once. In the latter case the sperm-nucleus advances a certain distance into the tgg and then pauses while the germinal vesicle moves towards the periphery, and gives rise to the polar-bodies (Ascaris, annelids, etc.). This significant fact proves that the attractive force between the two nuclei is only exerted after the formation of the polar-bodies, and hence that the entrance-path of the sperm-nucleus is not determined by such at- traction. A second important point, first pointed out by Roux, is that the path of the sperm-nucleus is curved, its " entrance-path " into the Q.gg forming a considerable angle with its "copulation-path " towards the egg-nucleus. These facts are well illustrated in the sea-urchin ^gg (Fig- 77), where the egg-nucleus occupies an eccentric position near the point at which the polar bodies are formed (before fertilization). Entering the Qgg 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. towards a point near the centre of the ^g%. After penetrating a certain distance its direction changes slightly to that of the copulation- path, which, again, is directed not precisely towards the egg-nucleus, but towards a meeting-point where it comes in contact with the egg-nucleus. The latter does not begin to move until the entrance- path of the sperm-nucleus changes to the copulation-path. It then begins to move slowly in a somewhat curved path towards 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 ^gg. These facts indicate that the paths of the germ-nuclei are deter- 1 The terms "female pro-nucleus," "male pro-nucleus" (Van Beneden), are often ap- plied to the germ-nuclei before their union. These should, I think, be rejected in favour of Hertwig's terms egg-niuleiis -icnA sperin-mideus, 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. 196), 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. 183). 152 FERTILIZATION OF THE OVUM mined by at least two different factors, one of which is an attraction or other dynamical relation between the nuclei and the cytoplasm, the other an attraction between the nuclei. The former determines Fig- 77- — Diagrams showing the paths of the germ-nuclei in four different eggs of the sea- urchin Toxopiu'iistes. 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 the meeting-point {M) 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 egg 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. 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. UX/OX OF THE GKKM-CELLS 153 Hertwisi 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 toi^ether by the formation, attachment, and contraction of the astral rays. While this view has some facts in its favour, it is, I believe, untenable, for many reasons, among which mav l^e mentioned the fact that neither the 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 are insignificant as compared with the nuclei (as in plants). I have often observed in cases of dispermy in the sea-urchin, that both sperm-nuclei move at an equal pace towards 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 polyspermy, 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 by union, and their movements are not improbably of a chemotactic character. 3. Union of the Gcnn-nnclci. The Chromosomes t 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 clca-eage- or segmentation-niteleus. As early as 1881, however, Mark clearly showed that in the slug Limax 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 strongest reason to believe that, as far as the chromatin is con- cerned, a true fusion of the nuclei never takes place during fertiliza- tion, and that the paternal and maternal chromatin jnay remain separate and distinct in the later stages of development — possibly throughout life (p. 219). In this regard two general classes may be distinguished. In one, exemplified by some echinoderms, by Amphi- oxns, P/iail/isia, 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 rej^re- sented by Ascaris (Fig. 65) and other nematodes, by Cje/ops {¥\g. 72), and by Pterotrachea (Fig. 6?>), 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- 154 FERTILIZATION OF THE OVUM 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 union. With a few exceptions, which are of such a character as not to militate against the rule, the tunnber of chromosomes arising frovi the gcrui-mtclei is alivays the same in both, and is one-half the number characteristic of the tissne-cells of the species. By their nnion, tJiere- fore, the germ-iuiclci give rise to an equatorial plate co7itaining the typical nnmbcr 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. A Partial List showing the Number of Chromosomes Char- acteristic OF THE Germ-Nuclei and Somatic Nuclei in Various Plants and Animals.^ Germ- Nuclei. Somatic Nuclei. Name. Group. Authority. I 2 Ascaris megalocephala, var. univalens. Nematodes. Van Beneden, Boveri. 2 4 Id., var. bivalens. 77 7? If ?? Ophryotrocha. Annelids. Korschelt. T> ["] Styleopsis. Tunicates. Julin. 4 8 Coronilla. Nematodes. Carnoy. ?9 1? Pallavicinia. HepaticEe. Farmer. 6 12 Spiroptera. Nematodes. Carnoy. v> U Gryllotalpa. Insects. vom Rath. If ?? Caloptenus. 77 Wilcox. GO *? /Equorea. Hydromedusse. Hacker. 8 i6 Filaroides. Nematodes. Carnoy. )) 77 Hydrophilus. Insects. vom Rath. J) j; Phallusia. Tunicates. Hill. ■)■> ?; Li max. Gasteropods. vom Rath. 77 u Rat. Mammals. Moore. f> [.] Ox, guinea-pig, man. 77 Bardeleben. ■>■> 77 Ceratozamia. Cycads. Overton. }} 77 Pinus. Conifera?. Dixon. 1 Indeed, Boveri has found that in Ascaris both modes occur, though the fusion of the germ-nuclei is exceptional. (Cf. p. 216.) 2 The above table is compiled from papers both on fertilization and maturation. Num- bers in brackets are inferred. IWION OF THE GERM-CELLS 155 Germ- Somatic Name. Group. AUTHOKITV. Nl'CLEI. Nuclei. 8 16 Scilla, Triticum. Angiosperms. Overton. ?5 ■>■> Allium. >> Strasburger, Guignard. 9 18 Echinus. Echinoderms. Boveri. 55 ?? Sagitta. Chaitognaths. j> *•! ^9 Ascidia. Tunicates. ?7 II [22] Allolobophora. Annelids. Foot. II (12) 22 (24) Cyclops strenuus. Copepods. Rlickert. 12 24 brevicornis. ■>i Hacker. ■>•! Helix. Gasteropods. Platner, vom Rath. j» If Branchipus. Crustacea. Brauer. j; D,] Pynhocoris. Insects. Henking. )j >> Salmo. Teleosts. Bohm. jj 5« Salamandra. Amphibia. Flemming. ^j ?7 Rana. >7 vom Rath. J7 Mouse. Mammals. Sobotta. 4^ V Osmunda. Ferns. Strasburger. ?? » Lilium. Angiosperms. Strasburger, Guignard. jy J> Helleborus. ?> Strasburger. ?? 7? Leucojum, Paeonia, Aconitum. 1^ Overton. 14 28 Tiara. Hydromedusae. Boveri. 16 32 Pterotrachea, Carinaria, • Phyllirhoe. Gastropods. •\1 „ [..] Diaptomus, Heterocope. Copepods. Rlickert. ^y [•,] Anomalocera, Euchaeta. *>% vom Rath. J? D,] Lumbricus. Annelids. Calkins. 18 36 Torpedo, Pristiurus. Ellasmobranchs. Rlickert. [18(19)] 36(38) Toxopneustes. Echinoderms. Wilson. 84 168 Artemia. Crustacea. Brauer. The above data are drawn from sources so diverse and show so remarkable a uniformity as to estabHsh 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 Ascaris (cf. p. 61). It is probably the case with the gasteropod Avion, where, as described by Plainer, 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-bodics, and that the germ-nuclei are exactly alike at the time of union. ^ We may here briefly refer to remarkable recent observations by Ruckert and others, which seem to show that not only the paternal and mater- 1 '89, pp. 10, 33. 156 FERTILIZATION OF THE OVUM nal chromatin, but also the chromosomes, may retain their individu- ality throughout development.^ Van Beneden, the pioneer observer in this direction, was unable to follow the paternal and maternal chro- matin beyond the first cleavage-nucleus, though he surmised that they I remained distinct in later stages as well ; and Rabl and Boveri {j brought forward evidence that the chromosomes did not lose their identity, even in the resting nucleus. Ruckert ('95, 3) and Hacker ('95, i) have recently shown that in Cyclops, the paternal and mater- nal chromatin-groups not only remain distinctly separated during the anaphase, but give rise to double nuclei in the two-cell stage (Fig. 105). Each half again gives rise to a separate group of chromosomes at the second cleavage, and this is repeated at least as far as the blas- tula stage. Herla and Zoja have shown furthermore that if in A scans the egg of variety diva/ens, having two chromosomes, be fertilized with the spermatozoon of variety nnivalcns having one chromosome, the three chromosomes reappear at each cleavage, at least as far as the twelve-cell stage (Fig. 106); and according to Zoja, the paternal chromosome is distinguishable from the two maternal at each step by its smaller size. We have thus what must be reckoned as more than a possibility, that every cell in the body of the child may receive from each parent not only half of its chromatin substance, but one-half of its chromosomes, as distinct and individual descendants of those of the parents. C. Centrosome and Archoplasm in Fertilization We have now finally to consider more critically the history of the centrosomes in fertilization, already briefly reviewed at p. 135. The account there given considers only the more usual and typical history of the centrosome, viz. the degeneration of the egg-centrosome and the introduction of a new centrosome by the spermatozoon. There is, however, one phenomenon which indicates « /r/t?;-/ the possibility that other modes of fertilization may occur, namely, partJicnogcncsis, in which the Q.gg develops without fertilization. In this case, as Brauer ('93) has clearly shown in Arteviia, the egg-centrosome remaining after the formation of the polar bodies does not degenerate, but divides into two to form the cleavage-amphiaster. The degeneration of the egg-centrosome is therefore not a necessary or invariable phenome- non, and as a matter of fact several accounts have been given of its persistence and active participation in the process of fertilization. These accounts fall under three categories, as follows : — I. Each germ-cell contributes a single centrosome, one of which 1 Cf. p. 219. CENTROSOME AXD ARCHOPLASM LV FERTILIZATION I 5/ forms the centre of each aster of the first mitotic figure (Van Beneden, in Ascaris, '83, "87, p. 270). 2. Each germ-cell contributes two centrosomes (or one which im- mediately divides into two), which conjugate, paternal with maternal, to form those of the cleavage-amphiaster (Fol, in sea-urchins, '91 ; Guignard, in flowering plants, '91 ; Conklin, in gasteropods, '93). 3. The centrosome is derived not from the spermatozoon, but from the egg (Wheeler, in the case of Myzostoma, 95). The first of these accounts, which rested rather on surmise than on adequate observation, may probably be safely rejected, for it con- tradicts the universal law that the centrosome divides into two before cell-division, and is unsupported by later observers (Meyer, Erlanger, etc.)- The second view, as embodied in the statements of Fol, Gui- gnard, and Conklin, demands fuller consideration. All these authors agree that each germ-cell contributes two centrosomes, or one which divides into two during fertilization. The daughter-centrosomes thus formed conjugate two and two in such a manner that each of the centrosomes of the cleavage-spindle is formed by the union of a cen- trosome derived from each germ-cell. It is an interesting and sig- nificant fact that a conjugation of centrosomes was predicted by Rabl ('89) on the a priori ground that if the centrosome is a perma- nent cell-organ, as Boveri and Van Beneden maintain, then a union of germ-cells must involve a union not only of nuclei, but also of centrosomes. Unusual interest was therefore aroused when Fol, in 1891, under the somewhat dramatic title of the "Quadrille of Cen- tres," described precisely such a conjugation of centrosomes as Rabl had predicted. The results of this veteran observer were very posi- tively and specifically set forth, and were of so logical and con- sistent a character as to command instant acceptance on the part of many authorities. Moreover, a precisely similar result was reached through the careful studies, in the same year, of Guignard, on the lily, and of Conklin ('93), on the marine gasteropod Crcpidnla, a confirmation which seemed to place the quadrille on a firm basis. Fol's result was, however, opposed to the earlier conclusions of Boveri and Hertwig, and a careful re-examination of the fertilization of the echinoderm egg, independently made in 1894-5 by Boveri {Echinus), by myself {Toxopneiistes), and Mathews {Arbacia, Astcrias), demon- strated its erroneous character. In the echinoderm, as in so many other cases, the egg-centrosome disappears. The cleavage-amphi- aster arises solely by division of the sperm-aster, and the centrosome of the latter is derived not from the tip of the spermatozoon, as asserted by Fol, but from the middle-piece, as already described. The same result has been since reached by Hill and Erlanger. Various attempts have been made to explain Fol's results as based 158 FERTILIZATION OF THE OVO'M on double-fertilized eggs, on imperfect method, on a misinterpreta- tion of the double centrosomes of the cleavage-spindle, yet they still remain an inexplicable anomaly of scientific literature. Fig. 1^8. — Fertilization of the egg of the parasitic annelid Myzostoma. [WHEELER.] A. Soon after entrance of tlie spermatozoon ; the sperm-nucleus at J ; at ? the germinal vesicle ; at c the double egg-centrosome. B. First polar body forming at ? ; n, the cast-out nucle- olus or germinal spot. C. The polar bodies formed {p.b) ; germ-nuclei of equal size ; at c the persistent egg-centrosomes. D. Approach of the germ-nuclei; the egg-amphiaster formed. In all other known cases this amphiaster is derived from the jr/t'r;«-amphiaster. Serious doubt has also been thrown on Conklin's conclusions by subsequent research. Kostanecki and Wierzejski ('96) have recently made a very thorough study, by means of serial sections, of the fertil- CENTROSOME AND ARCHOPLASM IN FERTILIZATION I 59 ization of the gasteropod P/iysa, and have reached exactly the same result as that obtained in the echinoderms. Here also the egg-centre degenerates, and its place is taken by a centrosome brought in by the spermatozoon and giving rise to a sperm-amphiaster, which per- sists as the cleavage-amphiaster (Fig. 64). A strong presumption is thus created that Conklin was in error ; and if this be the case, the last positive evidence of a conjugation of centrosomes in the animal &gg disappears.^ In view of this result we may well hesitate to accept Guignard's conclusions in the case of flowering plants. The figures of this author show in the clearest manner four centrosomes lying in the neighbourhood of the apposed germ-nuclei (Fig. 80) ; but the conju- gation of these centrosomes was an inference, not an observed fact, and has not been confirmed by any subsequent observer. Until such confirmation is forthcoming we must receive Guignard's results with scepticism.^ The third view, based upon the single case of Myzostoma as described by Wheeler ('95), apparently rests on strong evidence, though its force cannot be exactly estimated until a more detailed account has been published. In this case no sperm-aster can be seen at any period, with which is correlated the fact that no middle- piece can be made out in the spermatozoon. The egg-centrosome, on the other hand, is stated to persist after the formation of the second polar body, to become double at a very early period, and to give rise directly to the cleavage-amphiaster (Fig. 78). I can find no ground in Professor Wheeler's paper to doubt the accuracy of his conclusions. Nevertheless, an isolated case, which stands in contradiction to all that is known of other forms, must rest on irre- fragable evidence in order to command acceptance. Since, more- over, the case involves the whole theory of fertilization based on other animals (cf. p. 141), it must, I think, await further investiga- tion. 1 Richard Ilertwig has, however, recently published a very interesting observation which indicates that we may not yet have fully fathomed the facts in the case of echinoderms. If unfertilized echinoderm-eggs, after formation of the polar-bodies, lie for many hours in water or be treated with dilute poisons (strychnine), they may form a more or less perfectly developed amphiaster, and the nucleus may even make an abortive attempt at division. No centrosomes, however, could be discovered, even by the most approved methods. This remarkable phenomenon is probably of the same nature as the formation of artificial asters observed by Morgan (p. 226), but its meaning is not clear. - Van der Stricht, in a recent paper on Amphioxtts ('95), is inclined to believe that a fusion between the egg-centre and the sperm-centre occurs; but the evidence is very incom- plete, and a comparison with the case of Physa indicates that his conclusion cannot be sustained. The same criticism applies to the earlier work of Blanc ('91, '93) on the trout's i6o FERriLIZAriOiX OF THE OVUM D. Fertilization in Plants The investigation of fertilization in the plants has always lagged somewhat behind that of the animals, and even at the present time our knowledge of it is less complete, especially in regard to the history of the centrosome and the archoplasmic structures. It is, however, sufficient to show that the process is here essentially of the same nature as in animals in so far as it involves a union of two germ-nuclei de- rived from the two respective sexes. Many early observers from the time of Pringsheim ('55) on- ward described a con- jugation of cells in the lower plants, but the union of gcnii- nnclci, as far as I can lind, was first clearly made out in the flow- ering plants by Stras- burger in 1877-8, and carefully described by him in 1884. Schmitz observed a union of the nuclei of the conjugating cells of Spirogyra in 1879, and made similar obser- vations on other algce in 1884. The same has been shown to be true in MuscinccE. and Ptcridophytcs by Strasburger, Cambell, and others (Fig. 79). Up to the present time, however, the only thorough investigation of fertilization has been made in the case of the flowering plants, and our knowledge of the process here is due in the first instance to Strasburger ('84, '88) and Guignard (91), supplemented by the work of Belajeff and Overton. The ovum or oosphere of the flower- ing plant is a large, rounded cell containing a large nucleus and numerous minute colourless plastids from which arise, by division, the plastids of the embryo (chromatophorcs, amyloplasts). The ovum lies in the " embryo-sac," which represents morphologically the female prothallium or sexual generation of the Pteridophyte, and is itself embedded in the ovule within the ovary. The male germ-cell is here non-motile, and is represented by a "generative nucleus," with a Fig. 79. — Fertilization in Pibularia. [Cambell.] A. B. Early stages in the formation of the spermatozoid. B. The mature spermatozoid; the nucleus lies above in the spiral turns; below is a cytoplasmic mass containing starch- grains (cf. the spermatozoids of ferns and oi Marsilia, Fig. 53). D. Archegonium during fertilization. In tlie centre the ovum containing the apposed germ-nuclei (d", ?). FERTILIZATION IX PLANTS i6i small quantity of cytoplasm and two centrosomes (Guignard), lying near the tip of the pollen-tube (Fig. 80, A), which is developed as an outgrowth from the pollen-grain and represents, with the latter, a rudimentary male prothallium or sexual generation. The formation D Fig. 80. —Fertilization of the lily. [Guionaru.] A. The tip of the pollen-tube entering the embryo-sac ; below, the ovum (ocisphere) with its nucleus at ? and two centrosomes; at the tip of the pollen-tube the sperm-nucleus (cf) with two centrosomes near it. B. Union of the germ-nuclei. C. Later stage of the same, showing the asserted fusion of the centrosomes. R. The first cleavage- figure in the metaphase. D. Early anaphase of the same; precocious division of the centrosomes. M 1 62 FERTILIZATION OF THE OVUM of the pollcn-tube, and its growth clown through the tissue of the pistil to the ovule, was observed by Amici ('23), Brogniard ('26), and Robert Brown ('31); and in 1833-34 Corda was able to follow its tip through the micropyle into the ovule. ^ Strasburger ('77-88) first demonstrated the fact that the generative nucleus carried at the tip of the pollen-tube enters the ovum and unites with the egg-nucleus. On the basis of these observations he reached, in 1884, the same conclusion as Hertwig, that the essential phenomena of fertilization is a union of two germ-nuclei, and that the nucleus is the vehicle of hereditary transmission. Strasburger did not, however, observe the centrosome in fertilization. This was accomplished in 1891 by Guignard, who demonstrated in the case of the lily {Liliiim Martagon) that the generative nucleus as it enters the egg is accompanied by a small quantity of cytoplasm and by two centrosomes (Fig. 80). He showed further that the o.^^ also con- tains two centrosomes; and according to his account the conjugation of the nuclei is accompanied by a conjugation of the centrosomes, as already described. Guignard also first cleared up the history of the chromosomes, reaching results closely in accord with those of Van Beneden in the case of Ascaris. The two germ-nuclei do not actually fuse, but remain in contact, side by side, and give rise each to one-half the chromosomes of the equatorial plate, precisely as in animals (Fig. 80). The number of chromosomes from each germ-nucleus is, in the lily, twelve. The later history is identical with that of the animal egg, each chromosome splitting lengthwise, and the halves passing to opposite poles of the spindle. Each daughter-nucleus therefore receives an equal number of chromosomes from the maternal and paternal germ-nuclei.^ As in the case of animals (p. 127), the germ-nuclei of plants show marked differences in structure and staining-reaction before their union, though they ultimately become exactly equivalent. Thus, according to Rosen ('92, p. 443), on treatment by fuchsin-methyl-blue 1 It is interesting to note that the botanists of the eighteentli century engaged in the same fantastic controversy regarding the origin of the embryo as that of the zoologists of the time. Moreland (1703), followed by Etienne Francjois Geoffroy, Needham, and others, placed himself on the side of Leeuwenhoek and the spermatists, maintaining that the pollen supplied the embryo which entered the ovule through the micropyle. (The latter had been described by Grew in 1672.) It is an interesting fact that even Schleiden adopted a similar view. On the other hand, Adanson (1763) and others maintained that the ovule contained the germ which \vas excited to development l)y an aura or vapour emanating from the pollen and entering through the tracheae of the pistil. '^ Guignard's observations on the conjugation of the centrosomes have already been con- sidered at p. 159. They stand at present isolated as the only precise account of the history of the centrosomes in plant-fertilization, and no general conclusions on this subject can therefore at present be drawn. CONJUGATION IN UNICELLULAR FORMS 1 63 the male germ-nucleus of phanerogams is "cyanophilous," the female "erythrophilous," as described by Auerbach in animals. Stras- burger, while confirming this observation in some cases, finds the reaction to be inconstant, though the germ-nuclei usually show marked differences in their staining-capacity. These are ascribed by Strasburger ('92, '94) to differences in the conditions of nutrition ; by Zacharias and Schwarz to corresponding differences in chemical composition, the male nucleus being in general richer in nuclein, and the female nucleus poorer. This distinction disappears during ferti- lization, and Strasburger has observed, in the case of gymnosperms (after treatment with a mixture of fuchsin-iodine-green) that the paternal nucleus, which is at first "cyanophilous," becomes "erythro- philous," like the egg-nucleus before the pollen-tube has reached the &g^^. Within the egg both stain exactly alike. These facts indicate, as Strasburger insists, that the differences between the germ-nuclei of plants are as in animals of a temporary and non-essential character. E. Conjugation in Unicellular Forms The conjugation of unicellular organisms possesses a peculiar inter- est, since it is undoubtedly a prototype of the union of germ-cells in the multicellular forms. Biitschli and Minot long ago maintained that cell-divisions tend to run in cycles, each of which begins and ends with an act of conjugation. In the higher forms the cells pro- duced in each cycle cohere to form the multicellular body ; in the unicellular forms the cells separate as distinct individuals, but those belonging to one cycle are collectively comparable with the multi- cellular body. The validity of this comparison, in a morphological sense, is generally admitted.^ No process of conjugation, it is true, is known to occur in many unicellular and in some multicellular forms, and the cyclical character of cell-division still remains sub jiidicc? It is none the less certain that a key to the fertilization of higher forms must be sought in the conjugation of unicellular organisms. The diflficulties of observation are, however, so great that we are as yet acquainted with only the outlines of the process, and have still no very clear idea of its finer details or its physiological meaning. The phenomena have been most closely followed in the Infusoria by Butschli, Maupas, and Richard Hertwig, though many valuable ob- servations on the conjugation of unicellular plants have been made by De Bary, Schmitz, Klebahn, and Overton. All these observers have reached the same general result as that attained through study of the fertilization of the ii-g^ ; namely, that an essential phenomenon ' Cf. p. 41. 2Cf. p. 129. 164 FERTILIZATION OF THE OVUM of conjugation is a union of the nuclei of the conjugating cells. Among the unicellular plants both the cell-bodies and the nuclei completely fuse. Among animals this may occur ; but in many of the Infusoria union of the cell-bodies is only temporary, and the con- jugation consists of a mutual exchange and fusion of nuclei. It is Second fission. First fission, alter separation. Difierentiation of micro- and macronuclei. Separation of the gametes. > 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. 81. — Dingrnm showin? the history of the mictonuclei during the conjugation of Para- vimciuin. [Modified from MauPAS.] A' and 1" represent the opposed macro- and micronuclei in the two respective gametes; circles represent degenerating nuclei ; black dots, persisting nuclei. impossible within the limits of this work to attempt more than a sketch of the process in a few forms. We may first consider the conjugation of Infusoria. Maupas's beautiful observations have shown that in this group the life-history of the species runs in cycles, a long period of multiplication by cell- division being succeeded by an "epidemic of conjugation," which inauo-urates a new cycle, and is obviously comparable in its physio- CONJUGATIOX IX UNICELLULAR FORMS 1 65 logical aspect with the union of germ-cells in the Metazoa. If conju- gation do not occur, the race rapidly degenerates and dies out ; and Maupas believes himself justified in the conclusion that conjugation counteracts the tendency to senile degeneration and causes rejuve- nescence, as maintained by Biitschli and Minot.^ In Stvloiivchia pustulata, whicli Maupas followed continuously from the end of February until July, the tirst conjugation occurred on April 29th. after 128 bi-parti- tions ; and tlie epidemic reached its height three weeks later, after 175 bi-partitions. The descendants of individuals prevented from conjugation died out through -senile degeneracv," after 316 bi-partitions. 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 degradation of the nuclear apparatus. In Stylonyclna pttstulata and 0)iychodroniiis grandis this process especially affects 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 afiects the macronucleus, which may lose its chromadn, undergo fatty degeneration, and may finally disappear altogether {Stylonychia mytilus), after which the micronucleus soon degenerates more or less completely, and the race dies. It is a very significant fact that towards the end of the cycle, as the nuclei degenerate, the animals become incapable of taking food and of growth ; and it is probable, as ]\Iaupas points out, that the degeneration of the cytoplasmic organs is clue to disturbances in nutrition caused bv the degeneration of the nucleus. -&^ The more essential phenomena occurring during conjugation are as follows. The Infusoria possess two kinds of nuclei, a large viacnmuclcns and one or more small micromtclci. 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 gcrvi-uuclc7is, 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 bv division to both macronuclei and micronuclei of the offspring of the conjugating animals (Fig. 81 ). These facts may be illustrated by the conjugation of Paj-ania'ciiiin caiidatiini, 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. 82, A, B). Three of these degenerate, forming the " corpusculcs 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 1 Cf. p. 129. Fig. 82. — Conjugation oi Paramcsairm caudatum. \A-C, after R. Hertwu;; D-K, after Maupas.] (Tlie macronuclei dotted in all the figures.) 4 Micronuclei preparing for their first division. i?. Second division. C. Third division: three polar bodies or " corpuscules de rebut," and one dividing germ-nucleus m each animal. D. Exchange of the germ-nuclei. E. The same, enlarged. F. Fusion of the germ-nuc ei. G. The same enlarged. H. Cleavage-nucleus (t). preparing for the first division /. The cleavage- nucleus has divided twice. J. After three divisions of the cleavage-nuc eus ; macronucleus breaking up. K. Four of the nuclei enlarging to form new macronuclei. The first fission soon takes place. CONJUGATION IN UNICELLULAR FORMS 167 5 pronucleus (Fig. ^2, 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 times successively, and of the eight resulting bodies four become macronuclei and four micronuclei (Fig. 82, 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 dis- tributed in like man- ner, but in /'. cauda- tuui the process is more complicated, since three of them degenerate, and the fourth divides twice to produce four new micronuclei. In either case at the close of the process each of the conju- gating individuals has given rise to four descendants, each containing a macro- nucleus and micro- nucleus derived from the cleavage-nucleus. From this time for- ward fission follows fission in the usual manner, both nuclei dividing at each fis- sion, until, after many generations, conjuga- Fig. 83. — Conjugation of Vorticellids. [Maupas.] A. Attachment of the small free-swimming microgamete to the large fixed macrogamete ; micronucleus dividing in each {Carchesiuni). B. Microgamete containing eight micronuclei; macrogamete four {Voiiice/hi) . C. All but one of the micro- nuclei have degenerated as polar bodies or " corpuscules de rebut." D. Each of the micronuclei of the last stage has divided into two 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. tion recurs. Essentially similar facts have been observed by Richard Hertwig and Maupas in a large number of forms. In cases of permanent conjugation, as in Vorticella, where a smaller microgamete unites with a larger macroi^aj/iftr, 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 microgam- ete one-eighth of the original micronucleus (Fig. 83). 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. i68 FERTILIZATIOX OF THE OVUM 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 egg) 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 these degenerating nuclei or " corpuscules de rebut" with the polar bodies (p. 175), 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 A B C Fig. 84. — Conjugation of Noctiluca. [ISHIKAWA.] A. Union of the gametes, apposition of the nuclei. B. Complete fusion of the gametes. Above and below the apposed nuclei are the centrosomes. C. Cleavage-spindle, consisting of two separate halves. is the fact that the gcnn-niiclci unite zchen in the form of spindles 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. 82, G ), and this gives good reason to believe that the chromatin of the two gametes is equally dis- tributed 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 Noctiluca, which has been studied with some care by Cienkowsky and Ishikawa (Fig. 84). Here the conjugating animals completely fuse, but the nuclei are merely apposed and give rise each to one- half of the mitotic figure. At either pole of the spindle is a cen- trosome, the origin of which remains undetermined. It is an interesting fact that in Noctiluca, in the Gregarines, and probably in some other Protozoa, conjugation is followed by a very CONJUGATION IN UNICELLULAR FORMS 169 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 process to the fertilization and subsequent cleavage of the ovum is particularly striking. The conjugation of unicellular plants shows some interesting D Fig. 85. — Conjugation of Spirogyra. [OVERTON.] A. Union of the conjugating cells {S. communis). D. The typical, though not invariable, mode of fusion in 5. Weberi ; 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 pryrenoids, before union of the nuclei. D. Zygospore after fusion of the nuclei and formation of the membrane. features. Here the conjugating cells completely fuse to form a "zygospore" (Figs. 85, 99), 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 unite, but in many cases the plastids also (chromatophores). In Spirogyra some interesting variations in this regard have been observed. In some species De Bary has observed that the long band-shaped chro- matophores unite end to end so that in the zygote the paternal and lyO FERTILIZATION OF THE OVUM maternal chromatophores lie at opposite ends. In 5. Wcbcri, on the other hand, Overton has found that the single maternal chromato- phore 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. 85). 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, chromatophores. In the case of a Spirogyra filament having a single chromatophore it is therefore "wholly immaterial whether the individual cells re- ceive the chlorophyll-band from the father or the mother " (De Vries), — a result which, as Wheeler has pointed out, is in a measure analo- gous to that reached in the case of the centrosome of the animal ^gg?- F. Summary and Conclusion All forms of fertilization involve a conjugation of cells by 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 or physiologically. Both in animals and in plants the paternal germ- cell loses most of its cytoplasm, the main bulk of which, and hence the main body of the embryo, is now supplied by the Qgg. But, 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 individuality in the zygote. SUMMARY AXD CONCLUSION I/I more than this, the germ-cells come to differ in their morphological composition ; for in plants the male germ-cell loses its plastids, which are supplied by the mother alone, while in most if not all animals the egg loses its centrosome, which is then supplied by the father. The loss of the centrosome by the egg is, I believe, to be regarded as a provision to guard against parthenogenesis and to ensure amphimixis. The equivalence of the germ-cells is tints finally lost. Only the germ-nuclei retain their primitive morphological equivalence. Hence zve find the essential fact of fertilization and sexual reproduction to be a union of equivalent nuclei; and to this all other processes are tributary. The substance of the germ-nuclei, giving rise to the same number of chromosomes in each, is equally distributed to the daughter-cells and probably to all the cells of the body. As regards the most highly differentiated type of fertilization and development we thus reach 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 processes are controlled and from which they receive the spe- cific stamp of the race. From the father comes the centrosome to organize the machinery of mitotic division by which the egg splits up into the elements of the tissues, and by which each of these 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 woof from the male. What has since been gained is the knowledge that this web is to be sought in the chro- matic substance of the nuclei, and that the centrosome is the weaver at the loom. LITERATURE IV Van Beneden, E. — Recherches sur la maturation de Toeuf, la fe'condation et la division ccllulaire: Arc/i. Biol.,lY. 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. — tjber den Anteil des Spermatozoon an der Teilung des Eies : Sit2.- Bcr. d. Ges.f. Morph. 11. P/iys. in Munchen. B. III.. Heft 3. 1887. Id. — Zellenstudien, II. 1888. Id. — Befmchtung : Merkel wui Bonnefs Ergebnissc. I. 1891 . Id. — Uber das Verhalten der Centrosomen bei der Befruclitung des Seeigeleies, etc.: Ver/miidl. P/iys. Med. Ges. IVnrsbitrg, XXIX. 1895. Fick, R. — ijber die Reifung und Befmchtung des Axolotleies : Zeitschr. U'/ss. Zo'dl., LVI. 4. 1893. 1/2 FERTILIZATION OF THE OVUM Guignard, L. — Nouvelles etudes sur la fecondation : Aim. d. Sciences nat. Bot.y XIV. 1891. Hartog, M. M. — Some Problems of Reproduction, etc.: (luart. Jouni. Mic. Sci., XXXIII. 1 89 1. Hertwig, 0. — Beitrage zur Kenntniss der Bildung, Befruchtung und Teilung des tierischen Eies, I.: Morpli. Jalirb.. I. 1875. Hertwig, R. — Uber die Konjugation der Infusorien: Abh. d. bayr. Akad. d. ll'iss., II. CI. XVII. 1888-89. Id. — Uber Befruchtung und Konjugation : ]'crli. deittsch. Zool. Ges. Berlin, i%c)2. Kostanecki, K. v., and Wierzejski, A. — Uber das Verhalten der sogen. achro- matischen Substanzen im befruchteten Ei {oi P/iysa) : Arch. mik. Anal, XLVII. 2. 1896. Mark, E. L. — Maturation, Fecundation, and Segmentation of Liviax campestris: Bull. Miis. Co/up. Zool. Harvard Collei^e, Cambridge. Mass., VI. 1881. Maupas. — Le rejeunissement karyogamique chez les Cilie's : Arch. d. Zool., 2"'" se'rie. VII. 1889. Riickert, J. — Uber das Selbstandigbleiben der vaterlichen und mlitterlichen Kern- substanz walirend der ersten Entwicklung des befruchteten Cyclops-Eies : Arch. jnik. Anal, XLV. 3. 1895. Strasburger, E. — Neue Untersuchungen liber den Befruchtungsvorgang bei den Phanerogamen. als Grundlage fiir eine Theorie der Zeugung. JeJia, 1884. Id. — Uber Kern- und Zellteilung im Pflanzenreich, nebst einem Anhang iiber Befruchtung. Jena. 1888. Vejdovsky, F. — Entvvickelungsgeschichtliche Untersuchungen. Heft i, Reifung, Befruchtung und Furchung des Rhynchelmis-Eies. Brag. 1888. Wilson, Edm. B. — Atlas of Fertilization and Karyokinesis. New York, 1895. CHAPTER V OOGENESIS AND SPERMATOGENESIS. REDUCTION OF THE CHROMOSOMES " Es komnit also in der Generationenreihe der Keimzelle irgendwo zu eiiier Reduktion der urspriinglich vorhandenen Chromosomenzahl auf die Hiilfte, und diese Za/iUn-reduk- tion ist demnach nicht etwa nur ein theoretisches Postulat, sondern eine Thatsache." BovERl.i Van Beneden's epoch-making discovery that the nuclei of the con- iuo-atinsf srerm-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 uiatui-a- 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 problems relating to the morphological constitution of nucleus and chromatin which have an important bearing on all theories of development, and which now stand in the foreground of scientific discussion among the most debatable and interesting of biological problems. It must be said at the outset that the phenomena of maturation belong to one of the most difficult fields of cytological research, and one in which we are confronted not only by diametrically opposing theoretical views, but also by apparently contradictory results of observation. Two fundamentally different views have been held of the manner in which the reduction is effected. The earlier and simpler view, which was somewhat doubtfully suggested by Boveri i^'&'J, i ), and has been more recently supported by Van Bambeke ('94) and some others, 1 Zelleiistudien, III. p. 62. 173 174 REDUCTION OF THE CHROMOSOMES assumed an actual degeneration or casting out of half the chromo- somes during the growth of the germ-cells — a simple and easily intelligible process. The whole weight of the evidence now goes to show, however, that this view cannot be sustained, and that reduction is effected by a rearrangement and redistribution of tlie nnclear sub- stance without loss of any of its essential constituents. It is true that a large amount of chromatin is lost during the growth of the It is nevertheless certain that this loss is not directly con- po'ir nected with the process of reduction ; for, as Hertwig and others have shown, no such loss occurs during spermatogenesis, and even in the oogenesis the evidence is clear that an explanation must be. sought in another direction. We have advanced a certain distance towards such an explanation and, indeed, apparently have found it Fig. 86. — Formation of the polar bodies before entrance of the spermatozoon, as seen in the living ovarian egg of the sea-urchin Toxopneusfes (X 365). A. Preliminary change of form in the germinal vesicle. D. The first polar body formed, the second forming. C. The ripe egg, ready for fertilization, after formation of the two polar bodies (p.b.,1, 2); ^, the egg-nucleus. In this animal the second polar body fails to divide. For division of the second polar body see Fig. 64. in a few specific cases. Yet when the subject is regarded as a whole, the admission must be made that the time has not yet come for an understanding of the phenomena, and the subject must there- fore be treated in the main from an historical point of view. A. General Outline The general phenomena of maturation fall under two heads ; viz. oogenesis, which includes the formation and maturation of the ovum, and spermatogenesis, comprising the corresponding phenomena in case of the spermatozoon. Recent research has shown that matura- tion conforms to the same type in both sexes, which show as close a parallel in this regard as in the later history of the germ-nuclei. Stated 1 Cf. Figs. 71, 88. \ GENERAL OUTLIAE 175 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 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 of the four cells forms the "ovum" proper, while the other three, known as t\\Q. polar bodies, are minute, rudimentary, and incapable of development (Figs. 64, 71, 86). 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 q^^ contains a large amount of protoplasm and yolk, out of which the 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. 87. — Diagram showing the genesis of the egg. [After Boaeri.] main mass of the embryonic body is formed. In the male, therefore, all of the four cells may become functional ; in the female the func- tions of development have become restricted to but one of the four, while the others have become rudimentary (cf. p. 182). The polar bodies are therefore to be regarded as abortive eggs — a view first put forward by Mark in 1881, and ultimately adopted by nearly all inves- tigators. I. Reduction in tJie Female. Formation of the Polar Bodies As described in Chapter III., the Qgg 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 ^ The parallel was first clearly pointed out by Plainer in 1889, ^f^' was brilliantly demon- strated by Oscar Hcrtwig in the following year. 1/6 KEDUCriOX OF THE CHROMOSOMES 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. 87), which are the immediate predecessors of the ovarian egg. 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 deutoplasm), while egg-membranes may be formed around it. The ovum may now be termed the oocyte (Boveri) or ovarian egg. 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.g. in the sea-urchin, the polar bodies are formed before fertilization while the ^gg 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 afterwards, as in the lamprey-eel, the frog, and Ampliioxus. 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. 71, 86); and in many cases the first of these divides into two as the second is formed (Fig. 64). A group of four cells thus arises, namely, the mature egg, which 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 without further change. The egg-nucleus is now ready for union with the sperm-nucleus. A study of the nucleus during these changes brings out the follow- ing facts. During the multiplication of the oogonia the number of chromosomes is, in some cases at any rate, 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 effected. 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, von Baer, Bischoff, had no real understanding of the process and believed the germinal vesicle to disappear at the time of fertilization. ^ See, however, p. 194. GENERAL OUTLINE \77 To Butschli ('76,) Hertwig, and Giard {'yj) 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- D G Fig. 88. -Diagrams showing the essential facts in the maturation of the egg. The somatic number of chromosomes is supposed to be four. .^.Initial phase ; two tetrads have been formed in the germinal vesicle. B. The two tetrads have been drawn up about the spindle to form the equatorial plate of the first polar mitotic ngure. c. 1 he mUotic figure has rotated into position, leaving the remains of the germinal vesicle at gv D. Formation of the first polar body: each tetrad divides into two dvads. Hi;,!/. polar body formed; two dyads in it and in the egg. F. Preparation for the second division G. Second polar body forming and the first dividing; each dyad divides into two single chromosomes H. Final result; three pol.ar bodies and the egg-nucleus ( 9 ). each con- taining two single chromosomes (half the somatic number); ^. the egg-cemrosome which now degenerates and is lost. 178 REDUCTION OF THE CHROMOSOMES .4:s?>-^ .:. w^^^m^^cm^^-^' F I ^^ K Fig. 89. — Formation of the polar bodies in Ascaris megalocephala, var. bivaleiis. [BOVERI.] A. The egg with the spermatozoon just entering at J" ; the germinal vesicle contains two rod- shaped tetrads (only one clearly shown), the number of chromosomes in earlier divisions having been four. B. The tetrads seen in profile. C. The same in end view. D. First spindle forming (in this case inside the germinal vesicle). E. First polar spindle. F. The tetrads dividing. G. First polar body formed, containing, like the egg, two dyads. H. I. The dyads rotating into position for the second division. J. The dvads dividing. K. Each dyad has divided into two single chromosomes, completing the reduction. (For later stages see Fig. 65.) GENERAL OUTLINE 1 79 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 wit/ioNt ail intervening reticular resting 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 although 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 accurately deter- mined by Boveri in Ascaris ('87, i), are in a typical case as follows (Figs. 88, 89) : As the Qgg prepares for the formation of the first polar body, the chromatin of the germinal vesicle groups itself in a num- ber 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 ahvays one-half the usual number of chromosomes. Thus in Ascaris {megalocephala, bivalens) the germinal vesicle gives rise to two tetrads, the normal number of chromosomes in the earlier divisions being four ; in the salamander and the frog there are twelve tetrads, the somatic number of chro- mosomes being twenty-four (Fleming, vom Rath), etc. As the first polar body forms, each of the tetrads is halved to form two double groups, or dyads, one of which remains in the egg while the other passes into the polar body. Both the &gg and the first polar body therefore receive each a number of dyads equal to one-half the usual number of chromosomes. The Qgg now proceeds at once to the formation of the second polar body without previous reconstruction of the nucleus. Each dyad is halved to form two single chromo- somes, one of which, again, remains in the egg while its sister passes into the polar body. Both the Qgg and the second polar body accord- ingly receive two single chromosomes (one-half the usual number), each of which is one-fourth of an original tetrad group. From the two remaining in the egg a reticular nucleus, much smaller than the original germinal vesicle, is now formed.^ Essentially similar facts have now been determined in a consider- able number of animals, though the form of the tetrads varies greatly, and there are some cases in which no actual tetrad-formation has been observed (apparently in the flowering plants). It is clear from the 1 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. ^ It is nearly certain that the division of the first polar body (which, however, may be omitted) is analogous to that by which the second is formed, i.e. each of the dyads is similarly halved. i8o REDUCTION OF THE CHROMOSOMES foregoing account that the numerical reduction of c\\xovci?L\Jvi\-})iasscs takes place before the polar bodies are actually formed, through the operation of forces which determine the number of tetrads within the germinal vesicle. The numerical reduction is therefore deter- mined in the grandmother-cell of the Q.gg. The actual divisions by which the polar bodies are formed merely distribute the elements of the tetrads. 2. Reduction ill the Male. Spermatogenesis The researches of Platner ('89), Boveri, and especially of Oscar Hertwig ('90, i) have demonstrated that reduction takes place in the Primordial germ-ce Spermatogonia. Division-period (the number of divi sions is much greater). Maturation-period. Growth -period. Primarj- spermatocyte. Secondary spermatocytes. Spermatids. Spermatozoa. Fig. 90. — Diagram showing the genesis of the spermatozoon. [After BOVERI.] male in a manner almost precisely parallel to that occurring in the female. Platner first suggested ('89) that the formation of the polar bodies is directly comparable to the last two divisions of the sperm mother-cells (spermatocytes). In the following year Boveri reached the same result in Ascaris, stating his conclusion that reduction in the male must take place in the "grandmother-cell of the sperma- tozoon, just as in the female it takes place in the grandmother-cell of the Q.^^,'' and that the egg-formation and sperm-formation really agree down to the smallest detail ('90, p. 64). Later in the same year appeared Oscar Hertwig's splendid work on the spermato- genesis of Ascaris, which established this conclusion in the most striking manner. Like the ova, the spermatozoa are descended from primordial germ-cells which by mitotic division give rise to the GENERAL OUTLINE l8l spcruiatogotiia from which the spermatozoa are ultimately formed (Fig. 90). Like the oogonia, the spermatogonia continue for a time to divide with the usual (somatic) number of chromosomes ; i.e. four in Ascaris vicgaloccpliala bivalcns. Ceasing for a time to divide, they Fig. 91. — Diagrams showing the essential facts of reduction in the male. The somatic num- ber ot chromosomes is suppos(;d to be four. A. B. Division of one of the spermatogonia, sliowing the fuH number (four) of chromosomes. C. Primary spermatocyte prt- p.iiing for division ; tlie chromatin forms two tetrads. D, E. F. First division to form two secondary spermatocytes each of which receives two dyads. G. H. Division of the two secondary spermatocytes to form four spermatids. Each of the latter receives two single chromosomes and a centrosome which persists in the middle-piece of the spermatozoon. now enlarge considerably to form s/^n-inaton'fc\s\ each of which is morphologically equivalent to an unripe ovarian ovum, or oocyte. Each spermatocyte finally divides twice in rapid succession, giving rise first to two daughter-spermatocytes and then to four spermatids, each of which is directly converted into a single spermatozoon. The 1 82 KEDUCTIOX OF THE CHROMOSOMES history of the chromatin in these tzvo divisions is exactly parallel to that in tJic formation of the polar bodies (Figs. 91, 92). From the chromatin of the spermatocyte are formed a number of tetrads equal to one-half the usual number of chromosomes. Each tetrad is halved at the first division to form two dyads which pass into the respec- tive daughter-spermatocytes. At the ensuing division, which occurs without the previous formation of a resting reticular nucleus, each dyad is halved to form two single chromosomes which enter the respective spermatids (ultimately spermatozoa). From each sperma- tocyte, therefore, arise four spermatozoa, and each sperm-nucleus receives half the usual number of single chromosomes. The par- allel with the egg-reduction is complete. These facts leave no doubt that the spermatocyte is the morpho- logical equivalent of the oocyte or immature ovarian egg, and that the group of four spermatozoa to which it gives rise is equivalent to the ripe ^gg plus the three polar bodies. Hertwig was thus led to the following beautifully clear and simple conclusion : " The polar bodies are abortive eggs which are formed by a final process of division from the egg-mother-cell (oocyte) in the same manner as the spermatozoa are formed from the sperm-mother-cell (sperma- tocyte). But while in the latter case the products of the division are all used as functional spermatozoa, in the former case one of the products of the egg-mother-cell becomes the ^gg, appropriating to itself the entire mass of the yolk at the cost of the others which persist in rudimentary form as the polar bodies." ^ 3. Theoretical Significa7ice of Maturation Up to this point the facts are clear and intelligible. When, how- ever, we attempt a more searching analysis by considering the origin of the tetrads and the ultimate meaning of reduction, we find our- selves in a labyrinth of conflicting observations and hypotheses from which no exit has as yet been discovered. And we may in this case most readily approach the subject by considering its theoretical aspect at the outset. The process of reduction is very obviously a provision to hold con- stant the number of chromosomes characteristic of the species ; for if it did not occur, the number would be doubled in each succeeding generation through union of the germ-cells. But why should the number be constant .'' In its modern form this problem was first attacked by Weismann in 1885, and again in 1887, though many earlier hypotheses regard- 1 '90, I, p. 126. GEXERAL OUTLINE 1 83 ing the meaning of the polar bodies had been put forward.^ His interpretation was based on a remarkable paper published by Wil- helm Roux in 1883,^ in which are developed certain ideas which afterwards formed the foundation of Weismann's whole theory of in- heritance and development. Roux argued that the facts of mitosis are only explicable under the assumption that chromatin is not a uniform and homogeneous substance, but differs qualitatively in differ- ent regions of the nucleus ; that the collection of the chromatin into a thread and its accurate division into two halves is meaningless unless the chromatin in different regions of the thread represents different qualities which are to be divided and distributed to the daughter- cells according to some definite law. He urged that if the chromatin were qualitatively the same throughout the nucleus, direct division would be as efficacious as indirect, and the complicated apparatus of mitosis would be superfluous. Roux and Weismann, each in his own way, subsequently elaborated this conception to a complete theory of inheritance and development, but at this point we may confine our attention to the views of Weismann. The starting-point of his theory is the hypothesis of De Vries that the chromatin is a congeries or colony of invisible self- propagating vital units or biopJiorcs somewhat like Darwin's " gemmules " (p. 303), each of which has the power of determining the development of a particular quality. Weismann conceives these units as aggregated to form units of a higher order known as " determinants," which in turn are grouped to form ^ Of these we need only consider at this point the very interesting suggestion of Minot ('77), afterwards adopted by Van Beneden ('83), that the ordinary cell is hermaphrodite, and that maturation is for the purpose of producing a unisexual germ-cell by dividing the mother-cell into its sexual constituents, or " genoblasts." Thus, the male element is removed from the egg in the polar bodies, leaving the mature egg a female. In like manner he believed the female element to be cast out during spermatogenesis (in the " Sertoli cells"), thus rendering the spermatozoa male. By the union of the germ-cells in fertiliza- tion the male and female elements are brought together so that the fertilized egg or oosperm is again hermaphrodite or neuter. This ingenious view was independently advocated by A'an Beneden in his great work on Ascaris ('83). A fatal objection to it, on which both Strasburger and Weismann have insisted, lies in the fact that male as well as female quali- ties are transmitted by the egg-cell, while the sperm-cell also transmits female qualities. The germ-cells are therefore non-sexual; they are physiologically as well as morphologi- cally equivalent. The researches of Hertwig, Brauer, and Boveri show, moreover, that in Ascaris, at any rate, all of the four spermatids derived from a spermatocyte become func- tional spermatozoa, and the beautiful parallel between spermatogenesis and oogenesis thus established becomes meaningless under Minot's view. This hypothesis must, therefore, in my opinion, be abandoned. Balfour probably stated the exact truth when he said, "In the formation of the polar cells part of the constituents of the germinal vesicle, which are requisite for its functions as a complete and independent nucleus, is removed to make room for the supply of the necessary parts to it again by the spermatic nucleus" ('80, p. 62). He fell, however, into the same error as Minot and Van Beneden in characterizing the germ-nuclei as " male " and " female." ^ tJber die Bedeutung der Kerntheilungsfiguren. 1 84 REDUCTION OF THE CHROMOSOMES " ids," the latter being idcntiiied with the visible chromomeres or chromatin-granules. The ids finally are associated in linear groups to form the " idants " or chromosomes. Since the biophores differ qualitatively, it follows that the same must be true of the higher units Fig. 92. — Reduction in the sperm.ifogenesis of Ascarh megalocephala, var. bivalens. [Brauer.] ^ A-G. Successive stages in the division of the primary spermatocyte. The original reticuhmi undergoes a very early division of the chromatin-granules which then form a doubly split spireme- tliread, D. This shortens (C), and breaks in two to form the two tetrads {D in profile, E viewed endw ise) . F. G. H. First division to form two secondary spermatocytes, each receiving two dyads. /. Secondary spermatocyte. J. K. The same dividing. L. Two resulting spermatids, each with two single chromosomes and a centrosonie. formed by their aggregation. Hence each chromosome has a dis- tinct and definite character of its own, representing a particular group of hereditary qualities. From this it follows that the number of 1 For division of the spermatogonia see Pig. 39 ; for the corresponding phenomena in var. iiiiii'alens see Fig. 107. GENERAL OUTLINE 1 85 specifically distinct chromosomes is doubled by the union of two germ-cells, a process which if unchecked would quickly lead to an infinite complexity of the chromatin or germ-plasm. The end of maturation, or reduction, is therefore to prevent "the excessive accumulation of different kinds of hereditary tendencies or germ- plasms " 1 through the progressive summation of ancestral chromatins. We now come to the vital point of Weismann's hypothesis of reduction, about w^hich all later researches have revolved. Assuming with Roux that the different qualities or "ancestral germ-plasms" are arranged in a linear manner in the spireme-thread and in the chromosomes derived from it, he ventured the prediction ('87) that two kinds of mitosis would be found to occur. The first of these is characterized by a longitudinal splitting of the thread, as in ordi- nary cell-division, "by means of which all the ancestral germ-plasms are equally distributed in each of the daughter-nuclei after having been divided into halves." This form of division, w^hich he called " equal division " (Aequationstheilung), was then a known fact. The second form, at that time a purely theoretical postulate, he assumed to be of such a character that each daughter-nucleus should receive only half the number of ancestral germ-plasms possessed by the mother-nucleus. This he termed a "reducing division" (Reduk- tionstheilung), and suggested ^ that this might be effected either by a transverse division of the chromosomes, or by the divergence and separation of entire chromosomes without division. By either method the number of " ids " would be reduced; and Weismann argued that such reducing divisions must be involved in the formation of the polar bodies, and in the parallel phenomena of spermatogenesis. The fulfilment of Weismann's prediction is one of the most inter- esting results of recent cytological research. It has been demon- strated, in a manner which I believe is incontrovertible, that the reducing divisions postulated by Weismann actually occur, though not precisely in the manner conceived by him. Unfortunately, how- ever, this demonstration has been made in only a few specific cases, — the complete demonstration, indeed, in but a single group, namely, the copepod Crustacea, —while careful studies by the most accom- plished observers have led to an entirely different result in other cases; namely, in Ascaris and the flowering plants. We are in fact confronted by an apparent contradiction of so absolute a character that no middle ground between the conflicting results can at ]:)resent be discovered. We may best appreciate the nature of this contra- diction by a preliminary consideration of the tetrad groups ; for it is plain that the nature of the maturation-divisions can only be approached through a study of the origin of the tetrads. 1 Essay VI., p. 366. ^ l-c, p. 375- 1 86 REDUCTION OF THE CHROMOSOMES B. Origin of the Tetrads I. General SketcJi It is generally agreed that each tetrad arises by a double division of a single primary chromatin-rod. Nearly all obser\'ers agree further that the number of primary rods at their first appearance in the o-erminal vesicle or in the spermatocyte-nucleus is one-Jialf the usual nuviber of chromosomes, and that this numerical reduction is due to the fact that the spireme-thread segments into one-half the usual num- ber of pieces. The contradiction relates to the manner in which the primary rod divides to form the tetrad. According to one account, mainly based on the study of Ascaris by Boveri, Hertwig, and Brauer, and supported in principle by the observations of Guignard and Strasburger on the flowering plants, each tetrad arises by a double lojigitudinal splitting of the primary chromatin-rod caused by the division of each chromatin-granule into four parts. In this case the four resulting bodies — - i.e. the four chromosomes of the tetrad — must be exactly equivalent, since all are derived from the same region of the spireme-thread and consist of equivalent groups of ids or chromatin-granules { Fig. \02, A). No reducing division can therefore occur in Weismann's sense. There is only a reduction in the number of chromosomes, not a reduction in the number of qualities represented by the chromatin-granules. This may be graphically expressed as follows: — If the original spireme-thread be represented by abed, normal mitosis consists in its segmentation into the four chromosomes a — b — c — d, which split lengthwise to form -. -r* -> -,- In matu- ^ ^ abed ration the thread segments into tn'o portions, ab — cd, each of which then split into four equivalent portions, giving the equivalent tetrads, ab Mb , cd i cd . x x y y ■ v • ^ \ thus, — r\—r and — 7 — -, . or . =^ — , smce it is not known ab\ab cd cd x x y y whether ab really is equal to a + b. The second account, which finds its strongest support in the observations of Ruckert, Hacker, and vom Rath on the maturation of arthropods, asserts that each tetrad arises by one longitudinal and one transverse division of eacJi primaiy chroinatiji-ivd {¥'\g. \02, B). Thus the spireme abed segments as before into two segments ab and cd. These first divide longitudinally to form -- and — and then trans- ab cd versely to form — - and -j-. Each tetrad therefore consists, not of a b c \d ORIGIN OF THE TETRADS 1 8/ four equivalent chromosomes, but of two different pairs ; and the second or transverse division by which a is separated from b, or c from d, is the reducing division demanded by Weismann's hypoth- esis. The observations of Riickert and Hacker prove that the transverse division is accompHshed during the formation of the second polar body. 2. Detailed Evidence We may now consider some of the evidence in detail, though the limits of this work will only allow the consideration of some of the best known cases. We may first examine the case of Asearis, on which the first account is based. In the first of his classical cell-studies Boveri showed that each tetrad appears in the ger- minal vesicle in the form of four parallel rods, each consisting of a row of chromatin-granules (Fig. 89, A-C). He believed these rods to arise by the double longitudinal splitting of a single primary chro- matin-rod, each cleavage being a 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 sup- ported this view by further observations in 1890 on the polar bodies of Sagitta and several gasteropods, in which he again determined, as he believed, that the tetrads arose by double longitudinal splitting. An essentially similar view of the tetrads was taken by Hertwig in 1890, in the spermatogenesis of Asearis, though he could not support this conclusion by very convincing evidence. In 1893, finally, Brauer made a most thorough and apparently exhaustive study of their origin in the spermatogenesis of Asearis, which seemed to leave no doubt of the correctness of Boveri's result. Every step in the origin of the tetrads from the reticulum of the resting spermatocytes was traced with the most painstaking care. The first step observed was a double splitting of the chromatin-threads in the reticulum, caused by a divi- sion of the chromatin-granules into four parts (Fig. 92, A). From the reticulum arises a continuous spireme-thread, which from its first appearance is split into four longitudinal parts, and ultimately breaks in two to form the two tetrads characteristic of the species. These have at first the same rod-like form as those of the germinal vesicle. Later they shorten to form compact groups, each consisting of four spherical chromosomes. Brauer's figures are very convincing, and, if correct, seem to leave no doubt that the tetrads here arise by a double longitudinal splitting of the spireme-thread, initiated even in the reticular stage before a connected thread has been formed. If i88 REDUCTION OF THE CHROMOSOMES this really be so, there can be here no reducing division in Weis- mann's sense. The reduction of chromatin, caused by the ensuing cell-division, is therefore only a quantitative mass-reduction, as Hert- wig and Brauer insist, not a qualitative sundering of different ele- ments, as Weismann's postulate demands.^ The work of Strasburger and Guignard, considered at p. 195, has given in principle the same general result in the flowering plants, though the details of the pro- cess are here considerably modified, and apparently no tetrads are formed. A E F Fig. 93. — Origin of the tetrads by ring-formation in the spermatogenesis of the mole-cricket Gryllotalpa. [voM 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. We now return to the second view, referred to at p. 186, which accords with Weismann's hypothesis, and flatly contradicts the con- clusions drawn from the study of Ascaris. This view is based mainly on the study of arthropods, especially the Crustacea and insects, but has been confirmed by the facts observed in some of the lower verte- brata. In many of these forms the tetrads first appear in the form of closed rings, each of which finally breaks into four parts. First observed by Henking ('91) in the insect PyrrocJwris, they have since been found in other insects by vom Rath and Wilcox, in various cope- 1 In an earlier paper on Braiichipiis ('92) Brauer reached an essentially similar result, which was, however, based on far less convincing evidence. ORIGIN OF THE TETRADS 189 pods by Riickert, Hacker, and vom Rath, in the frog by vom Rath, and in elasmobranchs by Moore. The genesis of the ring was first determined by vom Rath in the mole-cricket {Gryllotalpa, '92), and has been thoroughly elucidated by the later work of Riickert ('94) and Hacker ('95, i). All these observers, excepting Wilcox and °00§ 0\\ • •; ... , . Cl_ OOOO 0 0 1)0 ^O 0 ° ° o ^J^^ D Fig. 94. — Formation of the tetrads and polar bodies in Cyclops, slightly schematic. (The full number of tetrads is not shown.) [RiicKERT.] A. Germinal vesicle containing eight longitudinally split chroniatin-rods (half the somatic number). D. Shortening of the rods; transverse division (to form the tetrads) in progress. C. Position of the tetr.ids in the first polar spindle, tlie longitudinal split horizontal. D. Ana- phase ; longitudinal division of the tetrads. E. The first polar body formed ; second polar spindle with the eight dyads in position for the ensuing division, which will be a transvetse or reducing division. Moore (see p. 201), 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 heterotypi- cal mitosis (p. 60). The breaking of the ring into four parts involves IQO REDUCTION OF THE CHROMOSOMES first the separation of these two halves (corresponding with the origi- nal longitudinal split), and second, the transverse division of each half, the latter being the reducing division of Weismann. 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 chro- mosomes united end to end. This appears with the greatest clear- ness in the spermatogenesis of Gryllotalpa (Fig. 93). Here 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 give rise to six typical tetrads. An essentially similar account of the ring-formation is given by vom Rath in Euclueta and Calaiiiis, and by Riickert in Hcterocope and Diaptouius. That the foregoing interpretation of the rings is correct, is beauti- fully demonstrated by the observations of Hacker, and especially of Riickert, on a number of other copepods {Cyclops, Canthocamptus), in which rings are not formed, since the splitting of the primary chromatin-rods is complete. The origin of the tetrads has here been traced with especial care in Cyclops strciiiiiis, by Riickert ('94), whose observations, confirmed by Hacker, are quite as convincing as those of Brauer on Ascaris, though they lead to a diametrically opposite result. The normal number of chromosomes is here twenty-two. In the germinal vesicle arise eleven threads, which split lengthwise (Fig. 94), and finally shorten to form double rods, manifestly equivalent to the closed rings of Diaptouius. Each of these now segments transversely to form a tetrad group, and the eleven tetrads then place themselves in the equator of the spindle for the first polar body (Fig. 94, C), in such a manner that the longitudinal split is transverse to the axis of the spindle. As the polar body is formed, the longitudinal halves of the tetrad separate, and the formation of the first polar body is thus demonstrated to be an "equal division" in Weismann's sense. The eleven dyads remaining in the eggs now rotate (as in Ascaris), 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 chromo- somes in the egg, and it is a curious fact that this conclusion, which apparently rests on irrefragable evidence, completely confirms Weis- mann's earlier views, published in 1887,^ and contradicts the later interpretation upheld in his book on the germ-plasm. 1 Essay VI. ORIGIN OF THE TETRADS [91 Hacker ('92) has reached exactly similar results in the case of CantJiocamptus and draws the same conclusion. In Cyclops stt'enuiis he finds in the case of first-laid eggs a variation of the process which seems to approach the mode of tetrad formation in some of the lower vertebrates. In such eggs the primary double rods become sharply a Fig. 95. — Diagrams of various modes of tetrad-formation. [Hacker.] a. Common starting-point, a double spireme-thread in the germinal vesicle ; d. common re- sult, the typical tetrads; b. c. intermediate stages: at the left the ring-formation (as in Diaptomus, Gryllotalpa, Heterocope) ; middle series, complete splitting of the rods (as in Cyclops according to Ruckert, and in Cauthocamptns) ; at tlie right by breaking of the V-shaped rods (as in Cyclops sti cnuHs, according to Hacker, and in the salamander, according to vom Rath). bent near the middle to form V-shaped loops (Fig. 96, C\ which finally break transversely near the apex to form the tetrad ^ — a process which clearly gives the same result as before. An exactly similar process seems to occur in the salamander as described by Flemming and ^ Hacker upholds this account ('95, i) in spite of the criticisms of RUckert and vom Rath. 192 REDUCTION OF THE CHROMOSOMES vom Rath. Flemming observed the double V-shaped loops in 1887, and also the tetrads derived from them, but regarded the latter as "anomalies." Vom Rath ('93) subsequently found that the double V's break at the apex, and that the four rods thus formed then draw- together to form four spheres grouped in a tetrad precisely like those of the arthropods. Still later ('95, i) the same observer traced a nearly similar process in the frog ; but in this case the four ele- Fig. 96. — Germinal vesicles of various eg^s, showing chromosomes, tetrads, and nucleoli. A. A copepod (Hcterocope) showing eight of the sixteen ring-shaped tetrads and the nucleo- lus. [RUCKERT.] B. Later stape of tlie same, condensation and segmentation of the rings. [ROCKERT.] C. "Cyclops sdeniiif'," illustrating Hacker's account of the tetrad-formation from elongate double rods; a group of " accessory nucleoli." [Hackf.r] D. (jerminal vesicle of an annelid {Ophrvofi-ocha) showing nucleolus and four chromosomes. [K(_)RSCHF,l/r.] ments appear to remain for a short time united to form a ring before breaking up into separate spheres. To sum up : The researches of Riickert, Hacker, and vom Rath, on insects, Crustacea, and amphibia have all led to the same result. However the tetrad-formation may differ in matter of detail, it is in all these forms the same in principle. Each primary chromatin-rod has the value of a bivalent chromosome ; i.e. two chromosomes joined end to end, ab. By a longitudinal division a ring or double THE EARLY HISTORY OF THE GERM-NUCLEI 193 rod is formed, which represents two equivalent pairs of chromosomes ab ab During the two maturation-divisions the four chromosomes are spHt apart, — nr» and Riickert's observations demonstrate that a\b the first division separates the two equivalent dyads, ab and ab, which by the second division are split apart into the two separate chromo- somes, a and b. Weismann's postulate is accordingly realized in the second division. It is clear from this account that the primary halving of the number of chromatin-rods is not an actual reduction, since each rod represents two chromosomes. Ruckert therefore proposes the convenient term " pseudo-reduction " for this pre- liminary halving.^ The actual reduction is not effected until the dyads are split apart during the second maturation-division. C. The Early History of the Germ-Nuclei We may for the present defer a consideration of accounts of reduc- tion differing from the two already described and pass on to a consideration of the earlier history of the germ-nuclei. A consider- able number of observers are now agreed that the primary chromatin- rods appear at a very early period in the germinal vesicle and are longitudinally split from the first. (Hacker, vom Rath, Ruckert, in copepods ; Ruckert in selachians ; Born and Pick in amphibia ; Holl in the chick; Ruckert in the rabbit.) Hacker ('92, 2) made the interesting discovery that in some of the copepods {Cajithocaviptus, Cyclops^ these double rods could be traced 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 germinal vesicle (Fig. 97). In the following year Rlick- ert ('93, 2) made a precisely similar discovery in the case of selachians. After division of the last generation of oogonia the daughter-chro- mosomes do not give rise to a reticulum, but split lengthwise, and persist in this condition throughout the entire growth-period of the Qg^^. Ruckert therefore concluded that the germinal vesicle of the selachians is to be regarded as a " daughter-spireme of the oogonium {Ur-ei) grown to enormous dimensions, the chromosomes of v/hich are doubled and arranged in pairs." ^ In the following year ('93) vom Rath, following out the earlier work of Flemming, discovered an exactly analogous fact in the spermatogenesis of the salamander. The tetrads were here traced back to double chromatin-rods, indi- vidually identical with the daughter-chromosomes of the preceding 1 '93, 2, p. 541. - '92, 2. p. 51. 194 REDUCTION OF THE CHROMOSOMES spermatogonium-clivision, which spHt lengthwise during the anaphase and pass into the spermatocyte-nucleus without forming a reticulum. Flemming had observed in 1887 that these daughter-chromosomes split in the anaphase, but could not determine their further history. Vom Rath found that each double daughter-chromosome breaks in two at the apex to form a tetrad, which passes into the ensuing spermatocyte without the intervention of a resting stage. ^ It is clear that in such cases the " pseudo-reduction " must take place at an earlier period than the penultimate generation of cells. In the salamander Flemming ('87) found that the " chromosomes " of the spermatogonia appeared in the reduced number (twelve) in at least three cell-generations preceding the penultimate. Vom Rath ('93) Fig. 97. — Longitudinal section through the ovary of the copepod Canthocamptus. [HACKER.] og. The youngest germ-cells or oogonia (dividing at ^^. -) ; a. upper part of the growth- zone ; oc. oocyte, or growing ovarian egg ; ov. fully formed egg, with double chromatin-rods. traced the pseudo-reduction in both sexes back to much earlier stages, not only in the larva;, but even in the embryo (!). This very remark- able discovery showed that the pseudo-reduction miglit appear in the early progenitors of the germ-cells during embryonic life — perhaps even during the cleavage. This conjecture has apparently been substan- tiated by Hacker ("95, 3), who finds that in Cyclops brevicornis the 1 It is certain that these facts do not represent a universal type of maturation, for in Ascaris there is no doubt that a true reticular resting stage occurs in the primary spermato- cytes, and probably also in the germinal vesicle. Hacker found, moreover, that the same species might show differences in this regard; for in Cyclops strenuus the first-laid eggs have no resting stage, the double daughter-chromosome passing directly into the tetrads, while in later broods of eggs a daughter-spireme, composed of long double threads, is formed. The difference is believed by Hacker to be due to the fact that the earlier eggs are quickly laid, while the later broods are long retained in the oviduct. REDUCTION IN THE PI A NTS 195 reduced number of chromosomes (twelve) appears in the primordial germ-cells which are differentiated in the blastula-stage (Fig. 56). He adds the interesting discovery that in this form the somatic nuclei of the cleavage-stages show the same number, and hence concludes that all the chromosomes of these stages are bivalent. As develop- ment 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 penultimate cell-generation, as is certainly the fact m Ascaris} The foregoing facts pave the way to an examination of reduction in the plants, to which we now proceed. D. Reduction in the Plants Guignard's and Strasburger's observations on reduction in the flowering plants gave a result which in substance agrees with that obtained by Boveri and Brauer in the case of Ascaris. These observers could find absolutely no evidence of a transverse or reduc- ing division, and asserted that the reduction in number is directly effected by a segmentation of the spireme-thread into half the usual number of chromosomes; i.e. by a process exactly corresponding With the "pseudo-reduction" of Riickert (see Fig. 25). These observers find that in the male the chromosomes suddenly appear m the reduced number (twelve in the lily, eight in the onion) at the finst division of the pollen-mother-cell, from which arise four pollen-grains. In the female the same process takes place at the f^rst division of the mother-cell of the embryo-sac. Strasburger and Guignard agree that in the subsequent divisions these chromo- somes do not form tetrads, but undergo simple longitudinal split- ting at each successive division. In case of the male there are at least four of these divisions; viz. two divisions to form the four pollen-grains, a third division to form the vegetative and generative cell of the pollen-grain, and finally a fourth division of the generative nucleus in the pollen-tube. In all these mitoses the reduced number of chromosomes appears, and each division is followed by a return of the nucleus to the resting state. In the 'It may be recallc-,1 that in .Ascaris Boveri proved that the primordial 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. 196 REDUCTION OF THE CHROMOSOMES mother-cell of the embryo-sac the number of divisions before fertiliza- tion is three, four, five, or sometimes even more, the reduced number persisting throughout. These facts led to the suspicion, first expressed by Overton in 1892, that the reduced number of chromosomes might be found in the sexual generation of higher cryptogams (which corresponds with the cells derived from the pollen-grain, or from the mother-cell of the embryo-sac). This surmise quickly became a certainty. Overton himself discovered ('93) that the cells of the endosperm in the Gymnosperm Ccmtozaniia divide with the reduced number, namely eight ; and Dixon observed the same fact in Pinits at the same time. In the following year Strasburger brought the matter to a definite conclusion in the case of a fern {Osmnnda), showing that all the crlls of the protJiallimn, from the original spore-motJier-cell omvarcis to the fonnation of the germ-cells, have ouc-half the nmnher of cJiroviosomcs found in the asexual generation, namely twelve instead of twenty-four; in other words, the reduction takes place in the formation of the spore from which the sexual generation arises, scores of cell-generations 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 four chromosomes dur- ing division, those of the sexual generation (thallus) eight. It now seems highly probable that this will be found a general rule. The striking point in these, as in vom Rath's and Hacker's obser- vations, 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 tJie 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 animals, and suggests that the four cells arising by the division of the oogonium (egg 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 pro- thallium of angiosperms 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:'^ Could Strasburger's hypothesis be substantiated, it would place the entire problem, not merely of maturation, but of sexuality itself, in a new light. 1 '78, p. 262. REDUCTIOX IX THE PLANTS 197 Strasburger's hypothesis is, however, open to a very serious a priori objection, as Hacker has pointed out ; for if the account of "reduction" in the plants given by Guignard and Strasburger be correct, it corresponds exactly to the " pseudo-reduction " in animals, and the "chromosomes" of the sexual generation must be bivalent like those of the early germ-cells in animals. The recent observa- tions of Belajeff, Farmer, and especially those of Sargant, give, how- ever, good reason to believe that both Guignard and Strasburger have overlooked some of the most essential phenomena of reduction. These observations have not yet revealed the exact nature of the process, yet they show that the first division of the pollen- mother-cells (in the lily) is of the lictcro typical form ; i.e. that the cJiromosonies have the form of rings. It is impos- sible to avoid the suspicion that these rings may be of the same nature as the ring- shaped tetrads in animals, though apparently they do not actually break up into a tetrad. Until this point has been cleared up by fur- ther investigation the nature of reduction in the plants _.._-... ,., , ,-,, > a '■ . Fig. 98. — Division of the chromosomes (? tetrad- remains an open question. . formanon) in the first division of the pollen-mother- cell of the lily. (a. b. after FARMER and MoORE; c-g. after Sargant.) a. b. Two stages in the ring-formation (hetero- typical mitosis), c-f. Successive stages, in profile view, of the separation of the daughter-chromosomes. i^. The daughter-chromosomes, seen en face, at the shape, and Miss Sargant's moment of separation -, this stage is perhaps 10 be very interesting observations interpreted as a tetrad like those occurring in the -' '^ . salamander. give some reason to believe that the V breaks at the apex precisely as described by Hacker in Cyclops and vom Rath in the salamander (Fig. 98, g). Should this prove to be the case the way would be opened for an interpretation of reduction in the plants agreeing in principle with that of Riick- ert, Hacker, and vom Rath ; and as far as the plants are concerned, the a priori objection to Strasburger's interesting hypothesis might be removed. Belajeff and Farmer showed that as the daughter-chromo- somes diverge after the first division they assume a V- 198 liEDUCTION OF THE CHROMOSOMES E. Reduction in Unicellular Forms A reduction of the number of chromosomes as a preparation for conjugation in the one-celled forms has not yet been certainly deter- mined, but there are many facts that render it highly probable. In B C D E H Fig. 99. — Conjugation of Closteriitm. [Klebahn.] A. Soon after union, four chromatophores. B. Chromatophores leduced to two, nuclei distinct. C. Fusion of the nuclei. D. First cleavage of the zygote. E. Rrsulting 2-cell stage. /•: Second cleavage. G. Resulting stage, each cell bi-nuoleate. H. Separation of the cells; one of the nuclei in each enlarging to form the permanent nucleus, the other (proliably repre- senting a polar body) degenerating. DIVERGENT ACCOUNTS OF REDUCTION 1 99 the conjugation of infusoria, as already described (p. 165), the original nucleus divides several times before union, and only one of the result- ing nuclei becomes the conjugating germ-nucleus, while the others perish, like the polar bodies. The numerical correspondence be- tween the rejected nuclei or "corpuscles de rebut" has already been pointed out (p. 168). Hertwig could not count the chromosomes with absolute certainty, yet he states ('89) that in Pammocciuui ca7idatinn, during the final division, the number of spindle-fibres and of the corresponding chromatic elements is but 4-6, while in the earlier divisions the number is approximately 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 appears to be deferred until the final division. ^ In the gregarinesWolters('9i)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 chromosomes was not determined. In the unicellular plants there are indications of a similar process, but the few facts at our command indicate that the reduction may here take place not before, but after, conjugation of the nuclei. Thus in the dermids Clostcrinm and Costnarii/ni, according to Klebahn (Fig. 99), 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 permanent nucleus, while the other degenerates and disappears. Chmielewski 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. F. Divergent Accounts of Reduction We can only touch on a few of the accounts of reduction which differ from both the modes already considered. Of these the most interesting are observations which indicate the possibility of, I. The Format ioii of Tetrads by Conjugation A considerable number of observers have maintained that reduc- tion may be effected by the union or conjugation of chromosomes that were previously separate. This view agrees in principle with that of Ruckert, Hacker, and vom Rath ; for the bivalent chromo- 1 Cf. Moore on the spermatogenesis of mammals, p. 201. 200 REDUCTION OF THE CHROMOSOMES somes assumed by these authors may be conceived as two conjugated chromosomes. It seems to be confirmed by the observations of Born and Fick on amphibia and those of Riickert on selachians {Pristi- iirus) ; for in all these cases the number of chromatin-masses at the time the first polar body is formed is but half the number observed in younger stages of the germinal vesicle. In Pristinnis there are at first thirty-six double segments in the germinal vesicle. At a later period these give rise to a close spireme, which then becomes more open, and is found to form a double thread segmented into eighteen double segments ; i.e. the reduced number. In this case, therefore, the preliminary pseudo-reduction is almost certainly effected by the union of the original thirty-six double chromosomes, two by two. The most specific accounts of such a mode of origin have, however, been given by Calkins (earthworm) and Wilcox (grasshopper). The latter author asserts ('95) that in Caloptcnus the spireme of the first spermatocyte first segments into the normal number (twelve) of dumb- bell-shaped segments, which then become associated in pairs to form six tetrads. Each of these dumb-bell-shaped bodies is assumed to be a bivalent chromosome, and the tetrad-formation is therefore inter- preted as follows : — ^bcd-l ab-cd-kl a^ 4(, etc. (tetrads). (spireme) (segmented spireme) c\a K^'^ There is, therefore, no longitudinal splitting of the chromosomes. A careful examination of the figures does not convince me of the correctness of this conclusion, which is, moreover, inconsistent with itself on Wilcox's own interpretation. Since each germ-nucleus receives six chromosomes, the somatic number must be 12, and Wilcox has observed this number in the divisions of the sperma- •togonia. The 12 dumb-bell-shaped primary segments must there- fore represent single chromosomes, not bivalent ones, as Wilcox assumes, and his primary tetrad must therefore be not — -^> as he ^ ^ c\d assumes, but either - or (if we assume that the normal number of chromosomes undergoes a preliminary doubling) . • Until this o r ^ ^ b\ 0 contradiction is cleared up Wilcox's results must be received with considerable scepticism. The second case, which is perhaps better founded, is that of the earthworm {Lwnbriciis tejTestris), as described by Calkins ('95, 2); whose work was done under my own direction. Calkins finds, in accordance with all other spermatologists save Wilcox, that the spireme-thread splits longitudinally and then divides transversely into 32 double segments. These then unite, two by two, to form 16 tetrads. The 32 primary double segments therefore represent DIVERGENT ACCOUXTS OF REDUCTION 20I chromosomes of the normal number that have spHt longitudinally, a b , , r 1 r , . a b a x „ , I.e. T» etc., and the lormula tor a tetrad is j- or Such a b a b a X a tetrad, therefore, agrees as to its composition with the formulas of Hacker, vom Rath, and Ruckert, and agrees in mode of origin with the process described by Ruckert in the eggs of Pristiiirus. While these observations are not absolutely conclusive, they nevertheless rest on strong evidence, and they do not stand in actual contradiction of what is known in the copepods and vertebrates. The possibility of such a mode of origin in other forms must, I think, be held open. Under the same category must be placed Korschelt's unique results in the egg-reduction of the annelid OpJiryotrocJia ('95), which are very difficult to reconcile with anything known in other forms. The typical somatic number of chromosomes is here four. The same minibcr of chromosomes appear in the germinal vesicle (Fig. 96, D). They are at first single, then double by a longitudinal split, but after- wards single again by a reunion of the halves. The four chromo- somes group themselves in a single tetrad, two passing into the first polar-body, while two remain in the O-^Z, but meanwhile each of them again splits into two. Of the four chromosomes thus left in the (t^^, two are passed out into the second polar body, while the two remain- ing in the Q'gg give rise to the germ-nucleus. From this it follows that the formation of the first polar body is a reducing division (!) — a result which agrees with the earlier conclusions of Henklng on PyiTochoris, but differs entirely from those of Ruckert, Hacker, and vom Rath. The meaning of this remarkable result cannot here be discussed. A clue to its interpretation is perhaps given by Hacker's interesting observations on the two modes of maturation in Cautho- caniptns, for which the reader is referred to Hacker's paper ('95, i). Moore ('95) has given an account of reduction in the spermatogen- esis of mammals and elasmobranchs which differs widely in many respects from those of all other observers. In both cases there is said to be a resting stage between the two spermatocyte-divisions, and in mammals (rat) the reduced number of chromosomes first appears in the prophase of the last division. In elasmobranchs both spermatocyte-divisions are of the heterotypical form, with ring- shaped chromosomes. On all these points Moore's account contra- dicts those of all other investigators of reduction in the animals, and he is further in contradiction with Ruckert on the number of chromosomes. His general interpretation accords with that of Brauer and Strasburger, reducing divisions being totally denied. The evidence on which this interpretation rests will be found in his original papers. 202 REDUCTION OF THE CHROMOSOMES G. Maturation of Parthenogenetic 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 i^Jj) on the theoretical meaning of maturation the suggestion is made that parthenogenesis may be due to failure on the part of the &g^ to form the polar bodies, the egg-nucleus thus remaining hermaphrodite, and hence capable of development without fertilization. This sug- gestion forms the germ of all later theories of parthenogenesis. Bal- 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 afterwards maintained by Van Beneden.^ These authors assumed accordingly that in par- thenogenetic eggs no polar bodies are formed. Weismann ^'^^) soon discovered, however, that the parthenogenetic eggs of Poly- pJicuins (one of the Daphnidse) produce a single polar-body. This observation was quickly followed by the still more significant dis- covery by Blochmann ('88) that /// ApJiis the partJicnogcjietic eggs produce a single polar body lohile the fertilized eggs produce txvo. Weismann was able 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 parthenogenesis. Blochmann observed that in insects the polar bodies were not actually thrown out of the egg, but remained embedded in its substance near the periphery. At the same time Boveri (^7, i) discovered that in Ascaris the second polar body might in exceptional cases remain in the Q.g^ and there give rise to a resting-nucleus indistinguishable 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 ^^^j:^ 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 : Partheno- genesis is the result of fertilization by the second polar body.'" '^ This conclusion received a brilliant confirmation through the obser- vations of Brauer ('93) on the parthenogenetic ^ €r (yoo^om B :■■'■■ '^'^. E X' -'-'<^2- Fig. 100. — First type of maturation in the parthenogenetic egg of Artemia. [Brauer.] A. The first polar spindle; the equatorial plate contains 84 tetrads. B. 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. 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 (Fig. too) but one polar body is formed, which removes eighty-four dyads, leaving eighty-four in the o.^^. There may be an abortive attempt to form a second polar spindle, but no division results, and 204 REDUCTION OF THE CHROMOSOMES the eighty-four dyads give rise to a reticular cleavage-nucleus. From this arise eighty-four thread-like chromosomes, and the same munber appears in later cleavage-stages. {b) It is the second and rarer mode that realizes Boveri's concep- tion (Fig. loi). Both polar bodies are formed, the first removing eighty-four dyads and leaving the same number in the egg. In the ~^"m D /•: Fig. loi. — Second type of maturation in the parthenogenetic egg of Artetnia. [Brauer.] A. Formation of second polar body. B. Return of the second polar nucleus {p.b:-) into the egg ; devvlopment of the egg-amphiaster. C. Union of the egg-nucleus ( 9 ) with the second polar nucleus {p.b?).^ D. Cleavage-nucleus and amphiaster. E. First cleavage-figure with equatorial plate containing i68 chromosomes in two groups of 84 each. formation of the second, the eighty-four dyads are halved to form two daughter-groups, each containing eighty-four single chromosomes. BotJi these groups remain in the egg, and each gives rise to a sttigle reticular nucleus, as described by Boveri in Ascaris. These tiva nuclei place the VIS elves side by side in the cleavage figure, and give rise each to eighty-four chromosomes, precisely like tivo germ-nuclei in ordinary fertilization. The one hundred and sixty-eight chromosomes split SUMMARY AA'D CONCLUSION 205 lengthwise, and are distributed in the usual manner, and reappear ill the same unuiber in all later stages. In other words, the second polar body here plays the part of a sperm-nucleus, precisely as main- tained 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. It is impossible to doubt that the chromosomes of the first class are bivalent; i.e. represent two chromosomes joined together — for that the dyads have this value is not a theory, but a known fact. It remains to be seen whether these facts apply to other parthenogenetic eggs ; but the single case of Artemia is little short of a demonstration not only of Hacker's and vom Rath's conception of bivalent chromo- somes, but also of the more general hypothesis of the individuality of chromosomes (Chapter VI.). Only on this hypothesis can we explain the persistence of the original number of chromosomes, whether eighty-four or one hundred and sixty-eight, in the later stages. How important a bearing this case has on Strasburger's theory of reduction (p. 196) is obvious. 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 reductioji in the number of chromosoines in the ttltimate genn-cells to one-half the number eJiaracteristic 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. As soon, however, as we attempt to advance beyond this point we enter upon doubtful ground, which becomes more and more uncertain as we proceed. With a few exceptions the reduction in number first appears in the direct progenitors of the germ-cells by a segmentation of the spireme-thread into one-ha^f the ns?tal number of rods. This process is, however, not an actual reduction in the num- ber of cJiromosomcs, but only a preliminary " pseudo-reduction " in the number of chromatin-///c?'i'.s-rjr. In what we may regard as the typical case {e.g. Ascaris) the pseudo-reduction first appears at the penultimate division ; i.e. in the grandmother-cell of the germ-cell (primary oocyte or spermatocyte). It may, how^ever, appear at a very much earlier period, even in the embryonic germ-cells, the reduced number appearing in every succeeding division until the germ-cells are formed. This is the case in the salamander and in Cyclops. It appears in its most striking form in the higher plants, where the re- jo6 REDUCTION OF THE CHROMOSOMES A .t- ( r-H ■■■■1 1' > h-1 ^' ■ < h-H ■'1 r t -< '■■1 ■' tH '"• r 1— 1 "i r h-H "n ,-\ '< r ^ ( 1-1 \ (I— (> (HI cluced number appears in all the cells of the sexual generation (pro- thallium, pollen-tube, embryo-sac), beginning with the mother-cell of the asexual spores from which this generation arises. In every case we must distinguish carefully between the primary pseudo-reduction in the number of chromatin-masses, and the actual reduction in the number of chromosomes ; for the former is in some cases certainly not an actual halving of the number of cJiromosonics, since each of the primary chromatin-rods is proved by its later history to be bivalent, representing two chromosomes united end to end (sal- amander, copepods). In these cases the actual reduction takes place in the course of the last two divisions (formation of the polar bodies and of the spermatids), each bivalent chromatin-rod di- viding transversely into the two chromosomes which it repre- sents, and at the same time (or earlier) splitting lengthwise. Each primary rod thus gives rise to a tetrad consisting of two pairs of chromosomes which, by the two final divisions, are distributed one to each of the four resulting cells. In the copepods the first division sepa- rates the longitudinal halves of the chromosomes and is there- fore an " equal division " (Weis- mann). The second division corresponds with the transverse division of the primary rod, and therefore is the "reducing division " postulated by VVeismann. This result gives a perfectly clear conception of the process of actual reduction and its relation to the preparatory pseudo-reduction that precedes it. It has, however, been absolutely demonstrated in only two groups of animals, viz. the copepods and the vertebrates (amphibia), and a diametrically opposite result has been reached in the case of Ascaris (Boveri, Hertwig, Brauer) and in the plants (Gui- gnard, Strasburger). In Ascaris typical tetrads are formed, but all observers agree that they arise by a double longitudinal splitting of the original chromatin-rod. In the plants no tetrads have been ob- served, but the precise nature of the maturation-divisions is still in doubt. We have thus two diametrically opposing results. In the one Fig. 102. — Diagram contrasting the two modes of tetrad-formation. A. Ascaris-type. Double longitudinal split- ting of the primary rod; no reduction in the number of granules ("ids"). B. Copepod-type. A longitudinal followed by a transverse division of the primary rod; the number of granules halved by the second division. SUMMARY AXD CONCLUSION 20/ case the primary halving in number is a pseudo-reduction, and each tetrad arises by one longitudinal and one transverse division of a bivalent chromosome, representing two different regions of the spireme-thread (Hacker, vom Rath, Riickert, Weismann). In the other case the primary halving appears to be an actual reduction, and if tetrads are formed, they arise (Ascaris) by a double longitudi- nal splitting of the primary rod, and all of its four derivatives repre- sent the same region of the spireme-thread. Since the latter consists primarily of a single series of granules (" ids " of Weismann, or chromomeres), by the fission of which the splitting takes place, the difference between the two views comes to this : that in the second case the four chromosomes of each tetrad must represent identical groups of granules, while in the first case they represent two differ- ent groups (Fig. 102). In the second case the maturation-divisions cannot cause a reduction in the number of different kinds of ids. In the first case the number of ids is reduced to one-half by the second division by which the second polar body is formed, or by which two spermatids arise from the daughter-spermatocyte (Riickert, Hacker, vom Rath). The first view must obviously stand or fall with the conception of the primary chromatin-rods as bivalent chromosomes. That this is a valid conception is in my judgment demonstrated by Brauer's remarkable observations on A^'temia ; for in this case it is impossi- ble to escape the conclusion that the "chromosomes" of those parthenogenetic embryos in which the number is halved are bivalent, — i.e. have the value of two chromosomes united by their ends, — and they lend the strongest support to vom Rath's and Hacker's hypothesis. For if the number of chromosomes be merely the expression of a formative tendency, like the power of crystalliza- tion, inherent in each specific kind of chromatin, why should the chromatin of the same animal differ in the two cases though derived from the same source in both .? Yet if the cleavage-nucleus arises from eighty-four dyads the same number of chromatin-rods appears in all later stages ; whereas if the dyads break each into two separate chromosomes before their union, the number is thenceforward one hundred and sixty-eight. So great is the force of this evidence that I think we must still hesitate to accept the results thus far attained in Ascaris and the plants, and must await further research in this direction. Until the contradiction is cleared up the problem of reduction remains unsolved. 208 REDUCTION OF THE CHROMOSOMES APPENDIX I . Accessory Cells of the Testis It is necessary to touch here on the nature of the so-called " SertoH-cells," or supporting cells of the testis in mammals, partly because of the theoretical signifi- cance attached to them by iMinot, partly because of their relations to the question of amitosis in the testis. In the seminiferous tubules of the mammalian testis, the parent-cells of the spermatozoa develop from the periphery inwards towards the lum.en, 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, containing large nuclei. The latter are the Sertoli-cells, or supporting cells ; they extend nearly through from the basement-membrane to the lumen, and to their inner ends the young spermatozoa are attached by their heads, and there complete their growth. Tlie spermatozoa are developed from cells which lie in columns between the Sertoli-cells, and which undoubtedly represent spermatogonia, spermatocytes, and spermatids, though their precise relationship is, to some extent, in doubt. The innermost of these cells, next the lumen, are sperma- tids, 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 spermatogonia lie deeper still, being probably represented by the large, rounded cells. Two entirely different interpretations of the Sertoli-cells were advanced as long ago as 1871, 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 afterwards especially advocated by Biondi ('85), and is .still maintained by Minot ('92), who regards the nucleus of the Sertoli-cell as the physio- logical 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 (71), 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 four spermatids arise from each spermatocyte, precisely as was afterwards shown to be the case in Ascaris, etc. The very careful and thorough work of Benda and von Ebner leaves no doubt, in my opinion, that mammalian spermato- genesis conforms, in its main outlines, with that of Ascaris, the salamander, and other forms, and that Biondi's views, which Minot unfortunately adopts, are without foundation. If this be the case, Minot's theoretical interpretation of the Sertoli-cell as the physiological equivalent of the polar bodies, of course collapses. Various other attempts have been made to discover in the spermatogenesis a casting out of material which might be compared with the polar bodies, but these attempts have now only an historical interest. Van Beneden and Julin sought such material in the " residual corpuscles " left behind in the division of the sperm-forming cells of Ascaris. Other authors have regarded in tlie same light the '■ Nebenkern " (Waldeyer) and the "residual globules" (Lankester, Brown) thrown off by the developing spermatozoa of mammals. All of these views are, like iMinofs, wide of the mark, and they were advanced before the real parallel between spermato- genesis and ovogenesis had been made known by Platner and Hertwig. APPENDIX 209 2. Atrn'tosis 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 Preusse C95), leave no doubt whatever that such divisions occur in the testis of many animals. Vom Rath, however, maintains, after an extended investigation, that all cells so dividing do not belong in the cycle of development of the germ-cells ("93, p. 164) : that amitosis occurs only in the sup- porting or nutritive cells (Sertoli-cells, etc.), or in such as are destined to degenerate, like the -residual bodies"' of Van Beneden. Aleves has, however, produced strong evidence ("94) that in the salamander the spermatogonia may, in the autumn, divide by amitosis, and in the ensuing spring may again resume the process of mitotic division, and give rise to functional spermatozoa. On the strength of these observa- tions. Flemming ('93) himself now admits the possibility that amitosis may form part ot a normal cycle of development, and Preusse has recently shown that amitosis may continue through several generations in the early ovarian cells of Hemiptera without a sign of degeneration. LITERATURE. V Van Beneden, E. — Recherches sur la maturation de Toeuf, la fecondation et la division cellulaire : Arch. Biol., \W . 1883. Boveri, Th. — Zellenstudien. I., III. Jena, 1887-90. See also " Befruchtung " (List IV.). Brauer, A. — Zur Kenntniss der Spermatogenese von Ascaris megalocepJiala : Arch. jiiik. Aiiat., XLII. 1893. Id. — Zur Kenntniss der Reifung der parthenogenetisch sich entwickelnden Eies von Artcmia Saliiia : Arch. mile. Aitaf., XLIII. 1894. Hacker, V. — Die \'orstadien der Eireifung (General Review) : Arch. inik. Atiat., XLV. 2. 1895. Hertwig, 0. — Vergleich der Ei- und Samenbildung bei Nematoden. Eine Grund- lage fiir cellulaire Streitfragen : Arch. mile. Anal., XXXVI. 1890. Mark, E. L. — (See List IV.) Plainer, G. — tJber die Bedeutung der Richtungskorperchen : Biol. Centralb., VIII. 1889. vom Rath, 0. — Zur Kenntniss der Spermatogenese von Gryllotalpa vulgaris : Arch mik. Anal.. XL. 1892. Id. — Neue Beitrage zur Frage der Chromatinreduction in der Samen- und Eireife : Arch. mik. Aiiat., XLVI. 1895. Ruckert, J. — Die Chromatinreduktion der Chromosomenzahl im Entwicklungsgang der Organismen: Ergebn. d. Anat. u. Ejitwick., III. 1893 (1894). Strasburger, E. — Uber periodische Reduktion der Chromosomenzahl im Entwick- lungsgang der Organismen : Biol. Centralb.,XlW 1894. CHAPTER VI SOME PROBLEMS OF CELL-ORGANIZATION " Wir miissen deshalb den lebenden Zellen, abgesehen von der Alolecularstructur der organischen Verbindungen, welche sie enthalt, noch eine andere und in anderer Weise com- plicirte Structur zuschreiben, und diese es ist, welche wir mit dem Namen Oi-ganization bezeichnen." Brucke.i "Was diese Zelle eigentlich ist, dariiber existieren sehr verschiedene Ansichten." Hackel.2 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 centrosome. 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. Brlicke 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, and suggested the possible existence of vital units ranking between the molecule and the cell. Many biologi- . cal 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. I shall make no attempt to review the a priori arguments that have been urged in favour of this conception,^ but will rather inquire what are the extreme conclusions justified by the known facts of cell- structure. ^ Ele7ncntaro7-ganismen, 1861, p. 386. '^ Aiiihropogenie, 1891, p. 104. 3 For an exhaustive review of the subject see Yves Delage, La Slructiirc dit protoplasina, ct les theories sur Vheredite. Paris, 1895. 210 THE NATURE OE CELL-ORGAXS 211 A. The Nature of Cell-organs The cell is, in Briicke's words, an cloncntary organism, which may by itself perform all the characteristic operations of life, 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 higher grade of organization, as in multicellular forms, it is no less truly an organ- ism, 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.^ Nevertheless, 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. The visible organs of the cell fall under two categories according as they are merely temporary structures, formed anew in each suc- cessive cell-generation out of the common structural basis, or per- manent structures 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, probably also the centrosome, and the plastids of plant-cells. A peculiar interest attaches to the per- manent cell-organs. Closely inter-related as these organs are, they nevertheless have a remarkable degree of morphological indepen- dence. 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 chromatophores that neither botanists nor zoologists are as yet able to distinguish with absolute certainty between those that form an integral part of the cell, as in the higher green plants, and those that are actually inde- pendent organisms living symbiotically within it, as is probably the ca.se with the yellow cells of Radiolaria. Even so acute an investi- gator as Watase ('93, i) has not hesitated to regard the nucleus itself — or rather the chromosome — as a distinct organism living in symbiotic association with the cytoplasm, but having had, in an his- torical sense, a different origin. It is but a short step from this con- 1 Cf. p. 41. 2 12 SOME PROBLEMS OF CELL-ORGANIZATION elusion to the view that the centrosome, too, is such an independent organism and that the cell is a symbiotic association of at least three dissimilar living beings ! Such a conception would, however, as I believe, be in the highest degree misleading, even if with Watase we limit it to the nucleus and the cytoplasm. The facts point rather to the conclusion that all cell-organs arise as differentiated areas in the common structural basis of the cell, and that their morphological character is the outward expression of localized and specific forms of metabolic activity. It is certain that some of the cell-organs are the seat of specific chemical changes. Chromatin (nuclein) is formed only in the nucleus. The various forms of plastids have specific metabolic powers, giving rise to chlorophyll, to pigment, or to starch, according to their nature. The centrosome, as Butschli, Strasburger, and Heidenhain have in- sisted, possesses a specific chemical character to which its remarkable effect on the cytoplasm must be due.^ Even in regions of the cyto- plasm not differentiated into distinct cell-organs the metabolic activities may show specific and constant localization, as shown by the deposit of zymogen-granules, the secretion of membranes, the formation of muscle-fibres, and a multitude of related facts. Physiologically, therefore, no line of demarcation can be drawn between permanent cell-organs, transient cell-organs, and areas of the cell-substance that are physiologically specialized but not yet morphologically differen- tiated into organs. When we turn to the structural relations of cell- organs, we find, I think, reason to accept the same conclusion in a morphological sense. The subject may best be approached by a consideration of the structural basis of the cell and the morphologi- cal relations between nucleus and cytoplasm. B. Structural Basis of the Cell It has been pointed out in Chapter I. that the ultimate structural basis of the cell is still an open question ; for there is no general agreement as to the configuration of the protoplasmic network, and we do not yet know whether the fibrillar or the alveolar structure is the more fundamental. This question is, however, of minor impor- tance as compared with the microsome-problem, which is, I think, the most fundamental question of cell-morphology, and which is equally pressing whatever view we may hold regarding the configuration of the network. Are the granules described as " microsomes " accidental and non- essential bodies, produced, it may be, by the coagulating effects of 1 Cf. p. 77. STRUCTURAL BASIS OF THE CELL 213 the reagents, as Fischer's experiments suggest ? Or are they normal and constant morphological elements that have a definite significance in the life of the cell ? It is certain that the microsomes are not merely nodes of the network, or optical sections of the threads, as the earlier authors maintained ; for the fibrillae may often be seen to consist of regular rows of granules. Van Beneden gave the first clear description of the microsomes in this regard in the following words : " I have often had occasion to note facts that establish the essential identity of the moniliform fibrillas and the homogeneous fibrillae of the protoplasm. In my opinion every fibrilla, though it appear under the microscope as a simple line devoid of varicosities, is formed at the expense of a moniliform fibril composed of micro- somes connected with one another by segments of uniting fibrils." ^ Again, in a later work he says of the fibrils of the astral system in Ascaris : "It is easy to see that the achromatic fibrils are monili- form, that they are formed of microsomes united by inter-fibrils." ^ Similar observations have been made by many later writers. In the eggs of sea-urchins and annelids, which I have carefully studied, there is no doubt that after some reagents, e.g. sublimate-acetic, picro- acetic, chromo-formic, the entire astral system has exactly the struct- ure described by Van Beneden in Ascaris. Although the basal part of the astral ray appears like a continuous fibre, its distal part may be resolved into a single series of microsomes, like a string of beads, which passes insensibly into the cytoreticulum. The latter is composed of irregular rows of distinct granules which stain intensely blue with haematoxylin, while the substance in which they are em- bedded, left unstained by haematoxylin, is colored by red acid aniline dyes, such as Congo red or acid fuchsin. The difficulty is to determine whether this appearance represents the normal structure or is produced by a coagulation and partial dis- organization of the threads through the action of the reagents. A justifiable scepticism exists in regard to this point ; for it is perfectly certain that such coagulation-effects actually occur in the proteids of the cell-substance, and that some of the granules there observed have such an origin. It is very difficult to determine this point in the case of the cyto-microsomes, owing to their extreme minuteness. The question must, therefore, be approached indirectly by way of an examination of the nucleus and its relation to the cytoplasm. Here we find ourselves on more certain ground and are able to make an analysis that in a certain measure justifies the hypothesis that the cyto- microsomes may be true morphological elements having the power of growth and division like the cell-organs formed by their aggregation. 1 ^%Z> P- 576, 577- ■' '87- P- 266. 2 14 SOME PROBLEMS OF CELL-ORGANIZATION I. Xuclcus and Cytoplasm From the time of the earlier writings of Frommann ('65, '6f), Arnold ('67), Heitzmann ('73), and Klein i^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.^ But the whole weight of the evidence now goes to show that the linin-network is of the same nature, both chemically and physically, as the cyto-reticulum, and that the achromatic nuclear membrane is formed as a condensation of the same substance. Many investi- gators, among whom may be named Frommann, Leydig, Klein, Van Beneden, and Reinke, have described the threads 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 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 outside the nucleus, and there is a pretty general agreement among cytologists, with the important exception of Boveri, that both spindle-fibres and astral rays arise by a direct rearrangement of the pre-existing structures.''^ 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; and in the division of the (tg2, in the sea-urchin I have obtained strong evidence that the spindle-fibres are directly resolved into a portion of the general reticulum. These fibres are in this case formed inside the nucleus from the linin-network ; and we have therefore proof positive of a direct genetic continuity be- tween the latter and the cytoplasmic structures. But more than this, I have found reason to conclude that in this case a considerable part of the linin-network is derived from the chromatin, that the entire nuclear reticulum is a continuous structure, and that it is no more than a specially differentiated area of the general cell-network {'95, 2). This conclusion finds, I believe, a very strong support in the studies of Van Beneden, Heidenhain, and Reinke reviewed beyond (p. 223) ; but the bearing of these only becomes plain after considering the morphological differentiations of the nuclear net- work and its transformations during mitosis. ' Cf. Hammarsteii ("95). -The long-standing dispute as to tlie origin of the nuclear menihranc (whether nuclear or cytoplasmic) is therefore of little moment. JfONF/rOLOG/CAL COMPOSITION OF yilE NUCLEUS 215 C. Morphological Composition of the Nucleus I. The CJironiatin (a) Hypothesis of the Individuality of the Chromosomes. — It may now be taken as a well-established fact that the nucleus is never formed dc novo, but always arises by the division of a pre- existing 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, especially by Rabl and Boveri. As a result of a careful study of mitosis in epithelial cells of the salamander, Rabl ('85) concluded that the ehromosomes do not lose their individuality at the close of division, but persist in the chromatic reticulum of the resting nucleus. The reticulum arises through a transformation of the chromosomes, which give off anastomizing 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 reappear in the ensuing spireme-stage in nearly or quite the same position 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 de- scribed the nucleus as showing a distinct polarity having a " pole " corresponding with the point towards which the apices of the chro- mosomes converge {i.e. towards the centrosome), and an " anti- pole " (Gegenpol) at the opposite point {i.e. towards the equator of the spindle) (Fig. 17). 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 by extremely strong evidence, derived especially from a study of abnormal 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 in the egg. These chromosomes give rise in tlie egg to a reticular nucleus, indistinguishable from 2l6 SOME PROBLEMS OF CELL-ORGANIZATION 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, how- ever, abnormally increased from four to five or six (Fig. 103 C,D). Again, the two chromosomes left in the egg after removal of the Fig. 103. — Evidence of the individuality of the chromosomes. .Abnormalities in the fertiliza- tion of Ascaru. [BOVERI.] A. The two chromosomes of the egg-nucleus, accidentally separated, have given rise each to a reticular nucleus (?, ?) ; the sperm-nucleus below (cf). B. Later stage of tlie 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 ; /. b.'^ the two chromosomes arising from it, 9 'h^ egg-chromo- somes, cf the sperm-chromosomes. D. Resulting equatorial plate with six chromosomes. 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 sins^le chromosome is afterwards formed (Fig. 103, A, B). Finally, it sometimes happens that the two germ-nuclei completely fuse while in the reticular state, as is nor- mally the case in sea-urchins and some other animals (p. I53)- From the cleavage-nucleus tlius formed arise four chromosomes. MORPHOLOGICAL COMPOSITION OF THE NUCLEUS 217 These remarkable observations show that whatever be the number of cJirovwsonies entering into the formation of a reticular miclens, the same number afterwards issue from it — -a. result which demonstrates that the number of chromosomes is not clue merely to the chemical composition of the chromatin-substance, but to a morphological organ- ization of the nucleus. A beautiful confirmation of this conclusion was afterwards made by Boveri ('93, '95, i) and Morgan ('95, 4) in the case of echinoderms, by rearing larvae from enucleated egg- Fig. 104. — Evidence of the individuality of the chromosomes in the egg of Ascaris. [BOVEKI.] 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. fragments, fertilized by a single spermatozoon (p. 258). All the nuclei of such larvae contain but half the typical number of chromo- somes,— i.e. nine instead of eighteen, — since all are descended from one germ-nucleus instead of two ! Van Beneden and Boveri were able, furthermore, to demonstrate in Ascaris that in the formation of the spireme the chromosomes reappear in the same position as those which entered into the forma- tion of the reticulum, precisely as Rabl maintained. As the long 2l8 SOME PROBLEMS OE CELL-ORGANIZATION chromosomes diverge, their free ends are always turned towards the middle plane ( Fig. 69), and upon the reconstruction of the daughter- nuclei these ends give rise to corresponding lobes of the nucleus, as in Fig. 104, which persist throughout the resting state. At the suc- ceeding division the chromosomes reappear exactly in the same posi- Fig. 105. • — 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. D. Resulting 2-cell stage with double nuclei. C. Second cleavage ; chromosomes still in double groups. D. Blastomeres with double nuclei from the 8-cell stage of C. breviconiis. tion, tJicir cuds lying in the nuclear lobes as before (Fig. 104, G, H). On the strength of these facts Boveri concluded that the chromosomes must be regarded as "individuals" or "elementary organisms," that have an independent existence in the cell. During the reconstruc- tion of the nucleus they send forth pseudopodia which anastomose to form a network in which their identity is lost to view. As the cell MORPHOI.OCICAL COMPOSiriON OF THE NUCLEUS 219 prepares for division, however, the chromosomes contract, withdraw their processes, and return to their '' resting state," in which iission takes place. Applying this conclusion to the fertilization of the egg, Boveri expressed his belief that "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 remark- able conclusion follows tJiat in all cells derived in the regular course of division from the fertilized egg, one-half of the chroviosonies are of strictly paternal origin, the other Jialf of maternal ^ ^ 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 re- markable observations of Ruckert, Hacker, Herla, and Zoja on the independence of the paternal and maternal chromosomes. These observations, already referred to at p. 1 56, may be more fully reviewed at this point. Hacker ('92, 2) first showed that in Cyclops strenuus, 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 tenuicornis)&2iQh nucleus consists of two distinct though closely united halves, which Hacker believed to be the derivatives of the two respec- tive germ-nuclei. The truth of this surmise was demonstrated three years later by Ruckert (95, 3) in a species of Cyclops, likewise identi- fied as C strenuus {¥\g. 105). The number of chromosomes in each germ-nucleus is here twelve. Ruckert was able to trace the pater- nal 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 arises, 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 next cleavage, and the double character of the nuclei was in many cases distinctly recog- nizable at a late stage when the germ-layers were being formed. Finally Victor Herla' s remarkable observations on Ascaris ('93) showed that in Ascaris not only the chromatin of the germ-nuclei, but also the paternal and maternal chromosomes, remain perfectly distinct as far as the twelve-cell stage — certainly a brilliant confirma- tion of Boveri's conclusion. Just how^ far the distinction is main- tained is still uncertain, but Hacker's and Riickert's observations give some ground to believe that it may persist throughout the entire life of the embryo. Both these observers have shown that 1 '91, p. 410. 220 SOME PROBLEMS OF CELL-ORGANIZATION the chromosomes of the germinal vesicle appear in tzoo distinct 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 egg ! When to these facts is added the evidence afforded by Brauer's beautiful observations on Artcmia, no escape is left from the hypothesis of the individuality of the chromosomes in one form or B C Fig. io6. — Hybrid fertilization of the egg of Ascaris megalocephala, var. bivaUiis, by the sper- matozoon of var. univaleiis. [Herla.] A. The germ-nuclei shortly before union. B. The cleavage-figure forming; the sperm-nucleus has given rise to one chromosome (cf), 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. another, even though we admit that Boveri's statement may have gone somewhat too far. The only question is how to state the facts without introducing obscure conceptions as to what constitutes an " individual." It is almost certain, as pointed out beyond (p. 221), that the chromosomes are not the ultimate units of nuclear structure, for they arise as aggregations of chromatin-grains that have likewise the power of growth and division. The fact remains — and it is one of MORPHOLOGICAL COMPOSITION OF THE XUCLEUS 221 the highest significance^ that these more elementary units group themselves into definite aggregates of a higher order that show a certain degree of persistent individual existence. It may be said that the tendency to assume such a grouping is merely a question of nuclear dynamics, and is due to a "formative force" innate in the chromatin-substance. This is undoubtedly true ; but it is only another form of expression for the facts, though one that avoids the use of the quasi-metaphysical term "individual." Whether a chro- mosome that emerges from the resting nucleus is individually the same as one that entered into it can only be determined when we know whether it consists of the same group of chromatin-granules or other elementary bodies. It must not be forgotten, however, that in the case of the egg the chromosomes may persist without loss of their boundaries from one division to another, since no reticulum is formed (cf. p. 193). {b) Compositio)i of the Cliromosomes. — We owe to Roux^ the first clear formulation of the view that the chromosomes, or the chro- matin-thread, consist of successive regions or elements that are qualitatively different (p. 183). 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 established (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 unmis- takably to the conclusion that these granules are perhaps to be re- garded as independent morphological elements of a lower grade than the chromosomes. That they are not artefacts 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 morpholog- ical nature is, however, even more difficult than in the case of the chro- mosomes ; 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 unde- termined. 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 chromatin-disks arranged in a linear series (Van Beneden's figures of the same object show at most five) which finally fuse to 1 Bedeuliing der Kertitheilungsfiguren^ 1883, p. 15. 222 SOME PROBLEMS OF CELL-ORGANIZATION form an apparently 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 sperma- togenesis of the same animal (variety bivalens) by Brauer. At the time the chromatin-grains divide, in the reticulum of the spermato- cyte-nucleus, they are very numerous. His figures of the spireme- thread show at first nearly forty granules in linear series (Fig. 92, A). Just before the breaking of the thread into two the number is reduced to ten or twelve (Fig. 92, C). Just after the division to form the two tetrads the number is four or five (Fig. 92, D), 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. 61) at once sug- gest themselves, and one cannot avoid the thought that the smallest chromatin-grains may successively group themselves in larger and larger 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 visi- ble 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. 79) .? Here again we may well recall Strasburger's warning, and hesitate to identify the end of the series with the limits 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. These units must be capable of assimilation, growth, and division without loss of their specific character. This I believe is an absolute logical necessity. 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 assump- tion that in no manner prejudices our conclusion regarding the ultimate viorpJiological composition of the chromatin. CHROMATIN, LIN IN, AND THE CYTORETICULUM 223 D. Chromatin, Linin, and the Cytoreticulum What, now, is the relation of the smallest visible chromatin-grains to the linin-network and the cytoreticulum ? Van Beneclen long- ago maintained ^ that the achromatic network, the nuclear mem- brane, and the cytoreticulum 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 tJie chromatic and achromatic microsomes miirht be transformed into one another, and zuc7'-e 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 give off the chromophilous substance.""'^ Both these conclusions are borne out by recent researches. Heidenhain ('93, '94), confirmed by Reinke and Schlo- ter, finds that the nuclear network contains granules of two kinds differing in their staining-capacity. The first are the basi- chromatin granules, which stain with the true nuclear dyes (basic anilines, etc.), and are identical with the " chromatin-granules " of other authors. The second are the oxychromatin-granules of the linin-network, which stain with the plasma-stains (acid anilines, etc.), and are closely similar to those of the cytoreticulum. These tzvo forms graduate into one another, and arc conjectured to be different phases of the same elements. This conception is furthermore sup- ported by many observations on the behaviour of the nuclear net- work 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. 244), and is known to vary greatly in bulk. In certain cases a very large amount of the original chromatic net- work 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 was forced to the conclusion ('95, 2) that a consid- erable part of the linin-network, from which the spindle-fibres are formed, is actually derived from the chromatin. 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 microsomes at one extreme and the chromatin-gran- ules at the other. And inasmuch as the latter are certainly capable of growth and division, we cannot deny the possibility that the former may have like powers. It may well be that our present reagents do not give us a true picture of these elementary units — that "micro- somes " are but a rude semblance of reality. That they arc never- 1 '83, p, 580, 583. ^ Lc, p. 583. 224 SOME PROBLEMS OF CELL-ORGANIZATION theless an expression of the morphological aggregation of the proto- plasmic network out of more elementary units, must, I think, be accepted as a working hypothesis. Whether they are elementary organisms in Altmann's sense, whether they have a persistent mor- phological identity, whether they arise solely by the division of pre- existing microsomes, or may undergo dissolution and reformation, whether, in short, they are the self-propagating elementary bodies postulated by so many eminent naturalists as the essential basis of the cell, — all these are entirely open questions which the cytology of the future has to solve. E. The Centrosome When we turn to the centrosome, we find clear evidence of the existence of a cell-organ which, though scarcely larger than a cyto- microsome, possesses specific physiological powers, assimilates, grows, divides, and may persist from cell to cell, without loss of identity. It is far easier to define the centrosome in physiological than in mor- phological terms. In the former sense Boveri ('95, 2) defines it as a single permanent cell-organ zuhich forms the dynamic centre of the cell and mnltiplics by division to form the centres of the daughter-cells} A centrosome is necessarily present in all cells at the time of mitosis. Whether, however, it persists in the resting state of all cells is un- known. The most careful search has thus far failed to reveal its presence in many tissue-cells, e.g. in muscle-cells and many gland- cells ; but these same cells may, under certain conditions, divide by mitosis, as in regeneration or tumour-formation, and the centrosome may be hidden in the nucleus, or so minute as to escape observation. We must, however, remember that the centrosome often disappears in the mature egg, and the same may be true of some tissue-cells. Van Beneden's and Boveri's independent identification of centrosome in Ascaris as a permanent cell-organ ('Sy) was quickly supported by numerous observations on other animals and on plants. In rapid succession the centrosome and attraction- sphere were found to be present in pigment-cells of fishes (Solger. 'Sg. '90). in the spermatocytes of Amphibia (Hermann, '90), in the leucocytes, endothelial cells, con- nective tissue-cells and lung-epithelium of salamanders (Flemming, '91), in various plant-cells (Guignard, "91). in the one-celled diatoms (Blitschli, '91), in the giant- cells and other cells of bone-marrow (Heidenhain, Van Bambeke, Van der Stricht, '91), in the flagellate Nottiliica (Ishikawa, '91). in the cells of marine algae (Stras- burger, '92), in cartilage-cells (Van der Stricht, "92). in the cells of cancerous growths (epithelioma, Lustig and Galeotti, '92), in the young germ-cells as already described, and finally, in gland-cells (vom Rath, '95), and in nerve-cells (Lenhossek, '95). They have not yet been found in resting muscle-cells. 1 The fact that the centrosome is double in many cells does not conflict with this defini- tion, for the doubling is obviously a precocious preparation for the ensuing division. THE CENTROSOME 225 The earlier observers of the centrosome always found it lying in the cytoplasm, outside the nucleus. Almost simultaneously, in 1893, three investigators indepen- dently discovered it inside the nucleus of the resting cell, — Wasielewsky, in the vouno- ovarian eggs (oogonia) of Ascaris ; Brauer. in the spermatocytes of the same animal; and Karsten. in the cells of a plant. Psilotitm (Humphrey states, however, that Karsten's observations were erroneous). Several later observers have described a similar intra-nuclear origin of the centrosome, and several of these (Zimmermann. Lavdovsky, Knuten) have followed Wasielewsky in locating it in tlie nucleolus. Evidence against this latter view has been brought forward, especially by Humphrey and Brauer. The latter observer found both nucleoli and centrosome as separate bodies within the nucleus. He made further the interesting discovery that in t c ^-^ — // A ♦ B C D E ^^^'^ a Fig. 107. — Mitosis with intra-nuclear centrosome, in the spermatocytes of Ascaris megalo- ccphala, var. univalciis. [BraUKR.] 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 figure, ccntrosomes escaping from the nucleus in G. Ascaris the centrosome lies, in one variety {mtivalcns) inside the nucleus, in the other variety {bivalens') outside — z. fact which proves that its position is non-essential (cf. Figs. 92 and 107). Oscar and Richard Hertwig maintain that the intra-nuclear position of the centrosome is the more primitive, the centrosome having been originally differentiated from a part of the nuclear substance. This view is based in the main on tlie facts of mitosis in the Infusoria, where the whole mitotic tigure appears to arise within the nuclear membrane (cf. p. 62). Whether a true centrosome may ever arise de novo is Hkewise undetermined. The possibiHty of such an origin has been conceded by a number of recent writers, among them Biirger, Watase, Richard Hertwig, Heidenhain, and Reinke. The latter author ('94) would Q 226 SOME FJWBLEMS OF CELL-ORGANIZAriOM distinguish in the cell, besides the " primary centres " or centrosomes, secondary and tertiary centres, the latter being single microsomes formed at the nodes of the network. By the successive aggrega- tions of the latter may arise the secondary and primary centres as new formations. Watase ('94) advocates a somewhat similar view, and states that he has observed numerous gradations between a true aster and such "tertiary asters" as Reinke describes. Further evi- dence in the same direction is afforded by Morgan's remarkable observations on the formation of "artificial asters" in vmfertilized sea-urchin eggs which have lain for some time in sea-water ('96). Such eggs often contain numerous asters, each of which contains a body resembling a centrosome.^ Beside these observations must be placed those of Richard Hertwig, on the formation of an amphiaster in ripe unfertilized sea-urchin eggs (p. 159). All these observations are of high interest in their bearing on the historical origin of the centrosome ; but they do not prove that the centrosome of the nor- mal aster ever arises by free formation. On the whole, the evidence has steadily increased that the centrosome is to be classed among the permanent cell-organs ; but whether it ranks with the nucleus in this regard must be left an open question. The known facts are still too scanty to enable us to state precisely what a centrosome is in a morphological sense, either as regards its actual structure or its relation to other parts of the cell. In its sim- plest form (Fig. 108, 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 appears at the centre of the young sperm-aster in various animals — for example in the sea-urchin (Boveri), iji Chcetoptcnis (Mead), and in Nereis? In almost all cases, however, the centrosome after- wards assumes a more complex structure and becomes surrounded by certain envelopes, the relation of which, on the one hand, to the centrosome and, on the other hand, to the astral rays have not yet been fully cleared up. Boveri, whose observations have been confirmed by Brauer, Hacker, and others, described the centrosome in the cleavage-asters of Asearis as a small sphere containing a minute central granule ; and Brauer's careful studies on the spermatogenesis of the same animal showed 1 I have had the privilege of examining Professor Morgan's preparations, and can contirm his statement that these eggs contain but a single nucleus and hence are not polyspermic. 2 This appearance is not due to the shrinkage of a larger and more complex structure, as some authors have suggested; for in Nereis such a structure — i.e. the centrosphere — is afterwards developed around the centrosome. THE CENTROSOME 22 J that both these structures are persistent and that division of the sphere is preceded by division of the granule (Fig. 107). The central granule is exactly like the simple centrosome of the sperm-aster as described above, but we do not yet know with certainty the genesis of the sphere surrounding it, and hence cannot state whether this is part of the centrosome proper or a part of the centrosphere surrounding it. The former view is adopted by Boveri, who suggests the word " centriole " for the central granule; and, according to his observa- tions on Ascaris and on sea-urchins, the simple centrosome of the original sperm-aster enlarges to form the sphere, while the centriole afterwards appears within it. In the case of T/ialassenia, however, Grififin's observations leave no doubt that the central granule per- sists in its original form from its first appearance in the sperm-aster through every stage of the cleavage-amphiaster, dividing during the early anaphase in each aster and giving rise to the centrosomes of the daughter-asters in which it again appears as a simple granule at the focus of the rays without a trace of surrounding envelopes (Fig. 73). In the cleavage-amphiaster it is surrounded by a some- what vague, rounded mass (apparently representing the entire " cen- trosome " of Boveri and Brauer), which in turn lies in a reticulated centrosphere, from which the rays radiate. Both these structures disappear during the late anaphase, leaving only the central granule. Here, therefore, the true centrosome certainly corresponds to the central granule or centriole ; and all the surrounding structures be- long to the centrosphere. As soon as we look further we find apparent departures from this simple type of centrosome. In leucocytes Heidenhain finds at the centre of the centrosphere not one or two, but always three, and some- times four, granules, which he conceives as centrosomes forming a central group or microcentrum. In the giant-cells of bone-marrow the central group consists of a very large number (a hundred or more) of such granules, each of which is again conceived as a "centrosome " (Fig. \\,D). In the sea-urchin {Echinus) Boveri states that the original simple centrosome of the sperm-aster enlarges greatly to form a relatively large, well-defined sphere in which appear numerous granules (centrioles), which he would compare individually with the elements of Heidenhain's "central group." I have given a somewhat similar account of the facts in Toxopjicn- stcs, describing the centrosphere as a reticulated mass derived from an original granule or centrosome at the focus of the rays,^ and many 1 Professor Boveri informs me tliat I was in error in attributing to him the view that the entire central mass of the aster — i.e. the centrosphere — here represents the centrosome. The large spherical centrosome of Echinus is surrounded by a clear area which he regard? as the centrosphere. 228 SOME PROBLEMS OF CELL-ORGANIZATION other investigators have been unable to find a distinct body to be identified as a centrosome within the centrosphere. As far as the sea-urchins are concerned, there is, I think, good reason to doubt not only my own former concUisions, but also those of Boveri. Both vom Rath ('95, 2) and Hill ('95) find at the centre of the centrosphere in sea-urchins a distinct black granule ("centrosome"), which becomes double in the early anaphase precisely as in Thalasscma. More- over, Grifhn's studies under my direction show that the minute single centrosome of Thalasscvia entirely loses its staining-power after cer- tain reagents and only comes into view after other treatment.^ I am now, therefore, inclined to believe that many if not all of the accounts asserting the absence of a minute central centrosome in the centro- sphere are based on unsuitable methods, and that in most of such cases, if not in all, it is really present. However this may be, it is now certainly known that the centro- some is in some cases a granule so small as to be almost indistin- guishable from the microsomes ; that in this form it is able to organize the surrounding cytoplasm into the astral system ; and that in this form it may be handed on by division from cell to cell. It may well be that in some cases such a centrosome may multiply to form a cen- tral group, as in leucocytes and giant-cells ; that it may enlarge to form a granular or reticular sphere, as Boveri describes ; and that the individual granules within such a sphere do not have the value of centrosomes. Such secondary morphological modifications do not affect the physiological significance of the centrosome as a perma- nent cell-organ, but they have an important bearing on the question of its relation to the other constituents of the cell. The latter cjuestion has not been definitely answered. Biitschli, who has been followed by Erlanger, regards the centrosome as a small differentiated area in the general alveolar structure ; and he describes it in the sea-urchin as actually made up of a number of minute vesicles (Fig. 8, B). Burger ('92) suggested that the entire attraction-sphere and aster arise by a centripetal movement of micro- somes to form a radiating group the centre of which (centrosome) is represented by a condensed mass of the ground-substance. Watase ('93, '94) added the very interesting suggestion that tlic centrosome is itself nothing other than a viicrosonic of the same morphological nature as those of the astral rays and the general thread-work, differ- ing from them only in size and in its peculiar powers.^ Despite the 1 The centrosome disappears after fixation with sublimate-acetic, Init is perfectly shown after pure sublimate or picro-acetic. .See Science, Jan. lo, 1S96. - The microsome is conceived, if I understand Watase rightly, not as a permanent mor- phological body, but as a temporary varicosity of the thread, which may lose its identity 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 THE ARCHOPLASMIC STRUCTURES 229 ambiguity of the word "microsome" Watase's suggestion is full of interest, indicating as it does that the centrosome is morphologically comparable to other elementary bodies existing in the cytoplasmic structure, and which, minute though they are, may have specific chemical and physiological properties. F. The Archoplasmic Structures I. Asters and Spindle The asters and attraction-spheres have a special interest for the study of cell-organs ; for these are structures that may divide and persist from cell to cell or may lose their identity and reform 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 struct- ural basis. Two sharply opposing views of these structures are now held. Boveri ('88, 2), who has been followed in a measure by Stras- burger, maintains that the attraction-sphere of the resting cell is com- posed of a distinct substance, " arc/ioplasni,'' consisting of granules or microsomes aggregated 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 reticulum as the roots of a plant grow into the soil, and at the close of mitosis are again withdrawn into the cen- tral mass, breaking up into granules meanwhile, so that each daugh- ter-cell receives one-half of the entire archoplasmic material of the parent-cell. This material is, however, wholly distinct from that of the general reticulum, not, as many earlier observers have maintained, identical with it. Boveri was further inclined to believe that the individual granules or archoplasmic microsomes were " independent structures, not the nodal points of a general network," and that the archoplasmic rays arose by the arrangement of these granules in rays. Once formed, however, it may long persist even after disappearance of the aster and serve as a centre of formation for a new aster. In the latter case the astral rays are con- ceived as actual derivatives of the centrosome which, as it were, spins them out in the cyto- plasm. "The aster, from this point of view, may he considered as a physiological device for concentrating the cytoplasmic substance in a form which can be spun out again into filaments in the direction which will produce a definite physiological effect" ('94, p. 284). This part of Watase's conce]5tion is, on the whole, I think, opposed to the facts, though it certainly explains the injiushing of the nuclear membrane during the prophases of mitosis. It is im|jossible to believe that the rays of the enormous sperm- aster are developed out of the minute granule at their centre or that they flow back into it at the close of division. The centrosome increases in size during the formation of the aster, decreases during its disappearance, which is the reverse of what the hypothesis demands. Many other argu- ments in the same direction might be urged. 230 SOME PROBLEMS OF CELL-ORGANIZATION rows without loss of their individuality.^ In a later paper on the sea-urchin ('95) this view is somewhat modified by the admission that in this case the archoj^lasm may not pre-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 general reticulum. Strong evidence against the archoplasm-theory has been brought forward by many investigators, and I believe it to be in principle untenable. Nearly all recent workers have accepted in one form or another the early view of Biitschli, Klein, and Van Beneden that the astral rays and spindle-fibres, and hence the attraction-sphere, arise through a morphological rearrangement of the pre-existing protoplas- mic network, under the influence of the centrosome. Although this view may be traced back to the early work of Fol ('73) and Auerbach {'74), it was first clearly formulated by Biitschli ^"jQ), 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 centrosome (Fig. 8, B). The fibrous appearance of the astral rays is an optical delu- sion, for they are not fibres, but flat lamellae forming the walls of elongated closed chambers. This view has more recently been urged by Reinke and Eismond. 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 ('78), who regarded the aster as due to " a radiar arrangement of what corresponds to the cell-substance," the latter being described as having a fibrillar character.^ The same view is advocated by Van Beneden in 1883. With Klein, Heitzman, 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 1 '88, 2, p. 80. ^ I.e., p. 40. •^ For a very careful review of the early views on this subject, see Mark, Umax, 1881. * '92, 2, pp. 158-169. ^ It is interesting to note that in the same place Klein anticipated the theory of fibrillar contractility, both the nuclear and the cytoplasmic reticulum being regarded as contractile {I.e., p. 417). '■' '83. P- 59-- ' '83> P- 576- THE ARCHOPLASMIC STRUCTURES 23 I 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, published jointly with Neyt ('87). ''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, Eismond, Reinke, etc., have ob- served that the astral fibres branch out peripherally into the general reticulum and become perfectly continuous with its meshes. This is very clearly shown in the formation of the sperm-aster about the middle-piece of the spermatozoon. In the sea-urchin {Toxopncnstcs) the formation of the rays from the cytoplasmic reticulum can be fol- lowed step by step, and there can, I think, be no doubt that the astral rays arise by a direct transformation or morphological rearrangement of the pre-existing structure, and that they extend themselves at their outer ends, as the sperm-aster moves through the egg-substance, by progressive differentiation out of this reticulum.^ Once formed, how- ever, the rays may possess a considerable degree of persistence and may actively elongate by growth. Only thus can we explain the pushing in of the nuclear membrane by the ingrowing spindle-fibres during the prophases of mitosis in certain forms (p. 50) and the bending of the rays when two asters collide, as recently described by Kostanecki and Wierzejski ('96). It seems certain, furthermore, that during the rotation of the amphiaster in the formation of the polar bodies (Fig. 71) and in similar cases, the spindle, at lea.st, moves bodily. The substance of the spindle or of the asters may, moreover, persist in the resting cell, after the close of mitosis, as the attraction- sphere or paranucleus (Nebenkern), and in such cases the term " archoplasm " may conveniently be retained for descriptive purposes. To regard the archoplasm as a primary and independent constituent of the cell would, however, as I believe, be an error. 1 '5 3, p. 550. - I.e., p. 263. 3 i_c_^ p. 275. * I.e., p. 280. 5 '95, 2, p. 446. 232 SOME PROBLEMS OF CELL-ORGAXJZATION 2. The Attraction-Sphere The foregoing conception of the asters receives a strong support from the study of the attraction-sphere in resting cells. It is agreed by all observers that this structure is derived from the aster of the dividing cell ; but there is still no general agreement regarding its precise mode of origin from the aster, and the subject is confused by differences in the terminology of different authors. There are some cases in which the entire aster persists throughout the resting cell (leucocytes, connective tissue-cells) and the term " attraction-sphere " has by some authors been applied to the whole structure. As origi- nally used by Van Beneden, however,^ the word was applied (in Ascaris) not to the entire aster but only to its central portion — a spherical mass bounded by a circle of microsomes from which the astral rays proceed. At the close of division the rays fade away in the general network, leaving only the central sphere containing the ccntrosome. Boveri's account of the same object was entirely differ- ent ; for he conceived the attraction-sphere (" archoplasm-sphere ") of the resting cell as representing the entire aster, the rays being withdrawn towards the centrosome and breaking up into a mass of granules. Later workers have proposed different terminologies, which are at present in a state of complete confusion. Fol (91) proposed to call the centrosome the astroccntre, and the spherical mass sur- rounding it (attraction-sphere of Van Beneden) the astrosphere. Strasburger accepted the latter term and proposed the new word " centrosphere " for the astrosphere and the centrosome taken to- gether.2 This terminology has been accepted by most botanists and by some zoologists. A new complication was introduced by Boveri ('95), who applied the word "astrosphere" to the r;////v ^^sVr/- exclusive of the centrosome, in which sense the phrase "astral sphere" had been employed by Mark in 1881. The word "astrosphere" has therefore a double meaning and would better be abandoned in favour of Strasburger's convenient term "centrosphere," which may be understood as equivalent to the "astrosphere" of Fol. As regards the structure of the centrosphere, two well-marked types have been described. In one of these, described by Van Beneden in Ascaris, by Heidenhain in leucocytes, by Drliner and Braus in divid- ing cells of amphibia, the centrosphere has a radiate structure, being traversed by rays which stretch between the centrosome and the peripheral microsome-circle (Figs. 34, 108, G). In the other form, described by Vejdovsky in the eggs of RJiyncIichiiis, by Solger and Zimmermann in pigment-cells, by myself in sea-urchin eggs and in ^'83, p. 548. -'92, p. 5i- THE ARCHOPLASMIC STRUCTURES 233 Nereis, by Riickert in Cyclops, and in a number of other cases, the centrosphere has a non-radiate reticular structure (Figs. 71, 108, E). In some cases no centrosome has been found in this sphere ; but for reasons ah-eady stated (p. 228) I incline to believe that a centrosome is really present. In many, if not in all cases of both types, the sphere consists of an outer and an inner zone, the latter enclosing the centrosome ; but the relation of the inner zone to the centrosome still remains, in a meas- -:^Wm> B -///, / /Ijfr; t F G H Fig. 108. — Diagrams illustrating various descriptions of centrosome and centrosphere. A. Simplest type; only a minute centrosome at the focus of the ra\s (sp'rm-aster in many forms). B. Rays proceeding directly from a centrosome of considerable size within which is a central granule. Example, Brauer's description of the spermatocytes of Ascaris. C. Ra\s pro- ceeding from a clear centrosphere (astrosphere of Strasburger) , enclosing a centro>ome like the last but with no central granule (in flowering plants according to Guignard, Strasburger, and others). D. An e.xtremely minute centrosome lying in the middle of a large reticulated cen- trosphere {e£'. Hill's description of the sperm-aster in sea-urchins and tunicates). £. Like the last, but with a small spherical body surrounding the centrosome (examples, the eggs of Ihalas- scma 2,x\6. Nereis). F. No centrosome as distinguished from tiie rrticulated centrosphere. Ex- amples in the pigment-cells of fishes according to Zimmerman, in the eggs of echinoderms ac- cording to Wilson ; many similar accounts have been given, but all are open to question. G. In Ascaris, according to Van Beneden, outside the centrosome lie tlie cortical and medullary zones of the attraction-sphere. H. The same according to Boveri. The centrosome contains a cen- tral granule or centriole (cf. B.) ; outside this is a clear zone (medullary zone of Van B- neden), and outside this a vaguely defined granular zone, probably corresponding to \"an Beneden's cortical zone. ure, in doubt. Van Beneden described the centrosphere in Ascaris as consisting of an outer cortical and an inner vicdiillary zone, both of which were conceived as only a modification of the inner region of the aster. Boveri's account is somewhat different. The centrosome is described as surrounded by a clear zone (" heller Hof "), — probably corresponding with Van Beneden's "medullary zone," — while the " cortical zone " of the latter author is not recognized as distinct from the aster (or archoplasm-sphere). The centrosome itself contains a 234 SOME PROBLEMS OF CELL-ORGAXIZATION minute central granule or ccntriolc. This discrepancy between Boveri and Van Beneden was cleared up in a measure by Heidenhain's beautiful studies on the asters in leucocytes, and the still more thorough later work of Driiner on the spermatocyte-divisions of the salamander. In leucocytes (Fig. 35) the large persistent aster has at its centre a well-marked radial sphere bounded by a circle of microsomes, as described by Van Beneden, but without division into cortical and medullary zones. The astral rays, however, show indications of other circles of microsomes lying out- side the centrosphere. Driiner found that a whole series of such concentric circles might exist (in the cell shown in Fig. 109 no less than nine), but that the inner- most two are often especially distinct, so as to mark off a cen- trosphere composed of a medul- lary and a cortical zone precisely as described by Van Beneden. These observations show conclu- sively that the centrosphere of the radial type is merely the inner- most portion of the aster, which acquires an apparent boundary through the especial development of a ring of microsomes. And thus Van Beneden's original view is confirmed, that not only the aster as a whole, but also the centro- sphere, is but a modified area of the general cytoplasmic thread-work. Heidenhain points out that there are many cases — for instance, the young sperm-aster — in which there is at first no clearly marked central sphere, and the rays proceed outward directly from the centro- some. The sphere, in such cases, seems to arise secondarily through a modification of the inner ends of the astral rays. Heidenhain there- fore concludes that the centrosome is the only constant element in the sphere, the latter being a secondary formation and not entitled to rank as a persistent cell-organ, though it may in certain cases persist and dividt^ like the centrosome. Vom Rath, who has made a very careful study of the attraction-spheres in a large number of cells among both vertebrata and invertebrata, arrives at a nearly similar view, though he lays greater stress on the differentiation and independence of the sphere. In asters of dividing cells he could find in many cases no Fig. 10^. — SnjcniKitogonium of salaman- der. [DiU'NF.R.] The nucleus lies below. Above is the enormous aster, the centrosome at its centre, its rays showing indications of nine concentric circles of microsomes. The area within the second circle probably represents the " attrac- tion-sphere " of Van Beneden. THE ARCHOPLASMIC STRUCTURES 235 limit between sphere and aster, though in other cases it is distinctly present. In the resting cell, on the other hand, the boundary of the sphere is often very sharply marked, so that the sphere appears as a well-defined spherical body. The origin of such a definite sphere from the aster has not been very definitely determined, but Driiner's obser- vations indicate that it arises in the manner described by Van Bene- den, through the disappearance of the more peripheral portions of the astral rays. It is, in other words, the persistent centrosphere.^ The genesis of the reticular type of centrosphere is not so well determined. In Xcrcis the aster (maturation-asters, sperm-aster) has at first nothing more than a minute centrosome at its centre. This becomes surrounded at a later period by a large reticulated centrosphere, showing no sign of radial arrangement, that appears to arise by a transformation of the inner ends of the astral rays. A nearly similar account is given by Hill in the case of the sperm- aster in Strongyloccutrotus and Phallnsia. In these latter cases the centrosphere shows no differentiation into cortical and medullary zones. In TJialasseina and Nereis, on the other hand, the minute cen- trosome becomes surrounded by a somewhat vague body distinctly different from the reticulum of the outer centrosphere, and this body perhaps represents a "medullary zone." This body, with the centrosome, corresponds very nearly to the " centrosome " of Ascaris with its " centriole " or central granule as described by Boveri and Brauer ; but in Tlialassema Griffin's observations show conclusively that the minute central granule alone is the centrosome, and that the surrounding body does not persist after division. I cannot avoid the suspicion that the body described by Boveri as the " centrosome " in Echiinis may represent this medullary region of the centrosphere, and that he, like myself, may have overlooked the centrosome. Nor does it seem impossible that the " centriole " or central granule of Ascaris (Boveri, Brauer) may likewise represent the true centrosome. These questions can only be cleared up by further investigation. To sum up : The history of the " archoplasmic " structures gives strong ground for the conclusion that attractiou-spheres, asters, and spindle are, like the nucleus, differcntiatioiis of the general cell-netivork, which is, as it zvere, moulded by the centrosome into a specific form. If this be well founded, the word "archoplasm" has no significance save in a topographical or descriptive sense. In this light it is an interesting fact that the aster or attraction-sphere may either persist and divide, like a permanent cell-organ, or may disappear and re-form in successive cell-generations. 1 The same general result is indicated in the case of plants, though the phenomena have here been less carefully examined. 236 SOME PROBLEMS OF CELL- ORGANIZATION G. Summary and Conclusion A minute analysis of the various parts of the cell leads to the conclusion 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 for a time, and finally merge their identity in the common basis again. Permanent organs, such as the nucleus or centrosome, are constant areas in the same basis, which never are formed dc novo, but arise by the division of pre-existing areas of the same kind. These two extremes are, however, connected by various interme- diate 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. The facts point strongly to the conclusion, which has been espe- cially urged by De Vries and Wiesner, that in many if not in all cases the division of cell-organs is in the last analysis brought about by the division of more elementary masses of which they are made up; and furthermore that tJic degree of permanence depends on the degree of coJiesion nianfested by these masses. The clearest evi- dence in this direction is afforded by the chromatic substance of the nucleus, the division of which does not take place as a single mass- division, but through the fission of more elementary discrete bodies of which it consists or into which it is resolved before division. Several orders of such bodies are visible in the dividing nucleus, forming a series of which the highest term is the plurivalent chro- mosome, the lowest the smallest visible dividing basichromatin-grains, while the intermediate terms are formed by the successive aggrega- tion of these to form the chromomeres of which the dividing chromo- somes consist. Whether any or all of these bodies are "individuals " 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 properties, and which form the elementary morphological units of the nucleus. There is reason to beheve that the linin-network is likewise com- posed of minute bodies, the oxychromatin-granules, which are closely similar m appearance to the smallest chromatin-grains, and differ from them only in chemical nature as shown by the difference of staining-power. Whether the oxychromatin-granules have also the power of growth and division is unknown ; but if, as Van Beneden and Heidenhain maintain, the basichromatin- and oxychromatin-gran- SUMMARY AND CONCLUSION 23/ ules be only different modifications of the same element, a presump- tion certainly exists that they have such powers. When we extend this comparison to the cytoplasm, the ground becomes more uncer- tain. It seems well established that the cytoreticulum is of the same nature as the linin-network. If this be admitted, we are led to accept on a priori grounds that some at least of the cytomicrosomes are not artefacts, but morphological bodies comparable with those of the linin and chromatin networks, and like them capable of growth and division. This conclusion is, as yet, no more than a somewhat doubtful inference. In the centrosome, however, we have a body, no larger in many cases than a "microsome," which is positively known to be a persistent morphological element, having the power of growth, division, and persistence in the daughter-cells. Probably these powers of the cen- trosome would never have been discovered were it not that its stain- ing-capacity renders 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 oxy- chromatin-granules, and these in turn into the cytomicrosomes, we must admit that Briicke's cautious suggestion that the whole cell might be a congeries of self-propagating units of a lower order is to-day not entirely without the support of facts. LITERATURE. VI 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 : Wiener Sitz.-Ber., WAN . 1861. Biitschli, 0. — Protoplasma. (See List I.) Hacker, V. — Uber den heutigen Stand der Centiosomenfrage : I'erh. d. deiitsch. Zool. ues. 1894. Heidenhain, M. — (See List I.) Heria, V. — Etude des variations de la mitose chez Tascaride megalocephale : /Ire/i. />VV7/.. XIII. 1S93. Nussbaum, M. — Uber die Teilbarkeit der lebendigen Alaterie: Arc/i. mile. Anat., XXVL 1886. Rabl, C. — Uber Zellteilung : Morph. Jahrh.. X. 1885. Riickert, J. — (See List IV'.) De Vries, H. — Intracellulaie Pangenesis: yena, 1889. Watase, S. — Homology of the Centrosome: Joitrn. Morph., VIII. 2. 1893. Id. — On the Nature of Cell-organization : U'ooi/s Holl Biol. Lectures. 1893. Wiesner, J. — Die Elementarstruktur und das Wachstum der lebenden Substanz : Wien, 1892. Wilson, Edm. B. — .\rchoplasm. Centrosome, and Chromatin in the Sea-urchin Egg: Joitrii. Morph.., \'ol. XI. 1895. CHAPTER VII SOME ASPECTS OF CELL-CHEMISTRY AND CELL-PHYSIOLOGY " Les phenomenes fonctionnels ou de depense vitale anraieut done leur siege dans le protoplasme celhilaire. " Le noyau est un appareil de syntlu'se organiqiu, I' instrument de la production, le gertne de la celllller CLAUDE BERNARD.^ 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 chemi- cal 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 investiga- tion is beset 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 complicated character which differ very wddely in dif- ferent 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 an all-pervading substratum which in the greater number of cases, perhaps in all, has the form of ^ I.e^ons sur les pkenomefies de la 77>, L, 1878, p. 198. 238 CHEMICAL RELATIONS OF NUCLEUS AND CYTOPLASM 239 a sponge-like network. Cell-organs, such as the nucleus, the spindle and asters, the centrosome, are to be regarded as specialized areas in this network, just as the visible organs of the multicellular body are specialized regions in the all-pervading cellular tissue. And pre- cisely as the various organs and tissues are the seat of special chemi- cal activities leading to the formation and characteristic transformation of specific substances, — as for instance haemoglobin is characteristic of the red blood-corpuscles, or chlorophyll of the assimilating tissues of plants, — so in the cell the various morphological regions are areas of specific chemical activities and are characterized by the presence of corresponding substances. The morphological differentiation of cell-organs is therefore in a way the visible expression of underlying chemical specializations ; and these are in the last analysis reducible to differences of metabolic action. I. The Protcids and tJicir Allies The most important chemical compounds found in the cell are the group of 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 sulphur, and in some cases also phosphorus and iron. They form a very exten- sive 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. Halliburton ('93) employs the word "proteids" as synonymous with albuminous substajices, including under them the various forms of albumin (egg-albumin, cell-albumin, muscle-albumin, vegetable-albumins), globulin (fibrinogen, vitellin, etc.), and the pep- tones (diffusible hydrated proteids). This author places in a sepa- rate class of albuminoids another series of nearly related substances (reckoned by some chemists among the " proteids "), examples of which are gelatine, mucin, and especially nuclein, and the nucleo- albumiiis. The three last-named bodies are characterized by the presence of phosphorus, in which respect they show a very definite contrast to the " proteids," many of which, such as egg-albumin, con- tain no phosphorus, and others only a trace. By Hammarsten and some others the word " proteid " is, however, emplovcd in a more restricted sense, being applied to substances such as the nucleins and nucleo-proteids, of greater complexity than the albumins and globulins. The latter, together with the nucleo-albumins, are classed as albuminous bodies (Eiweisskorper).i 1 See Hammersten, '95, p. 16. 240 SOME ASPECTS OF CELL-CHEMISTRY AND CELL-PHYSIOLOGY The distribution of these substances throughout 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 there is a definite and constant contrast between widens 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, unclein and nncleo-firoteids, which as the names indicate, forms its main bulk and its most con- stant 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 Nnclein Series Nuclein was first isolated and named by Miescher in 1871, by subjecting cells to artificial gastric digestion. The cytoplasm is thus digested, leaving only the nuclei; and in some cases, for m- stance pus-cells and spermatozoa, it is possible by this method to procure large quantities of nuclear substance for accurate quanti- tative analysis. The results of analysis show it to be a complex albuminoid substance, rich in phosphorus, for which Miescher gave the chemical formula C.gH^gN.PgO.^^. Later analyses gave some- what discordant results, as appears in the following table of per- centage-compositions : ^ — C H N P PrS-CELLS. (Hoi>pe-Seyler.) 49.58 7.10 15.02 2.28 Spermatozoa of Salmon. (Miescher.) 36.11 5-15 13.09 5-59 Human Brain. (v. Jaksch.) 50.6 7.6 13.18 1.89 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 substances; namely, (i) an organic acid rich in phosphorus, to which he gave the name nucleic acid, and (2) a form of albumin. Moreover, 1 From Halliburton, '91, p. 203. [The oxygen-percentage is omittea in this table.] CHEMICAL KELATIOXS OF NUCLEUS AXD CYTOPLASM 241 the nuclein may be synthetically formed by the re-combination of these two substances. Pure nucleic acid contains no sulphur, a high percentage of phosphorus (above 9 %), and no albumin. By adding it to a solution of albumin a precipitate is formed which contains sulphur, a lo\ver 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 con- tains no sulphur and a maximum of phosphorus (over 9.5 %), might be uncombined nucleic acid itself. Kossel accordingly drew the conclusion, based on his o\\\\ work as well as that of Liebermann, Altmann, Malfatti, and others, that " what the histologists designate as chromatin consists essentially of combinations of nucleic acid with more or less albumin, and in some cases may even be free nucleic acid. The less the percentage of albumin in these compounds, the nearer do their properties approach those of pure nucleic acid, and we may assume that the percentage of albumin in the chromatin of the same nucleus mav vary according to physiological condi- tions." ^ In the same year Halliburton, following in part Hoppe- Seyler, stated the same view as follows. The so-called " nucleins " form a series leading downward from nucleic acid thus : — (i) Those containing no albumin and a maximum (9-10 %) of phos- phorus (pure nucleic 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 pyrenin (nucleoli) 2i\\(\ plastin (linin). These graduate into (4) Those containing a minimum (0.5 to i %) of phosphorus — the nucleo-albumins, which occur both in the nucleus and in the cytoplasm (vitellin, caseinogin, etc.). Finally, we reach the globulins and albumins, especially character- istic of the cell-substance, and containing no nucleic 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 phosphorus-free."- Further, "in the processes of vital activity there are changing relations between the phosphorized constituents of the nucleus, just as in all metabolic processes there is a continual inter- 1 '93, p. 158. 2 '53^ p. 5-4. 242 SOME ASPECTS OF CELL-CHEMISTRY AND CELL-PHYSIOLOGY change, some constituents being elaborated, others breaking down into simpler products." ^ These conclusions established a probability that the chemical differences between chromatin and cytoplasm, striking and constant as they are, are differences of degree only ; and they opened the way to a more precise investigation of the physiological ivle of nucleus and cytoplasm in metabolism. 3. Staining-rcactioiis of tJic Ahiclein-scries We may now bring these facts into relation with the staining- reactions of chromatin and cytoplasm when treated with the aniline dyes. These dyes are divided into two main classes,^ viz. the " basic " anilines and the " acid " anilines, the colouring-matter playing the part of a base in the former and of an acid in the latter. The basic anilines {e.g. methyl-green, Bismarck brown, saffranin) are in general "nuclear stains," having a strong affinity for chromatin, while the acid anilines (acid fuchsin, Congo red, eosin, etc.) are "plasma-stains," colouring more especially the cytoplasmic elements. We owe to Malfatti and Lilienfeld the very interesting discovery that tJic various members of tJie nuclcin series shoiv an affinity for tJie basic dyes in direct proportion to the amount of niie/eie acid {as measured by the amount of phosphorus) they contain. Thus the nuclei of spermatozoa, known to consist of nearly pure nucleic acid, stain most intensely with basic dyes, those of ordinary tissue-cells, which contain less phosphorus, less intensely. Malfatti ('91) tested various members of the nuclein-series, synthetically produced as combinations of egg-albumin and nucleic acid from yeast, with a mixture of red acid fuchsin and basic methyl-green. With this combination free nucleic acid was coloured pure green, nucleins containing less phosphorus became bluish-violet, those with little or no phosphorus pure red. Lilienfeld' s more precise experiments in this direction ('92, '93) led to similar results. His starting-point was given by the results of Kossel's researches on the relations of the nuclein group, which are expressed as follows:^ — ■ 1 It has long been known that a form of " nuclein " may also be obtained from the nucleo-albumins of the cytoplasm, e.g. from the yolk of hens' eggs (vitellin). Such nu- cleins differ, however, from those of nuclear origin in not yielding as cleavage-products the nuclein bases (adenin, xanthin, etc.). The term " paranuclein " (Kossel) or " pseudo-nuclein " (Ilammarsten) has therefore been suggested for this substance. True nucleins containing a large percentage of albumin are distinguished as micleo-proteids. They may be split into ali)umin and nucleic acid, the latter yielding as cleavage-products the nuclein bases. Pseudo- nucleins containing a large percentage of albumin are designated as nucleo-allniimns, which in like manner split into albumin and paranucleic or pseudo-nucleic acid, which yields no nuclein bases. (See Hammarsten, '94.) 2 See Ehrlich, '79. ^ From Lilienfeld, after Kossel, '92, p. 129. CHEMICAL RELATIONS OF NUCLEUS AND CYTOPLASM 243 Nucleo-albi(ijti)i (i % of P or less), by peptic digestion splits into Peptone Nudein (3-4 % P), by treatment with acids splits into Albumin Nncleic acid (9-10 % P). heated with mineral acids splits into Phosphoric acid Nnclein bases (adenin, guanin, etc.). i^A carbohydrate.^ Now, according to Ko.ssel and Lilienfeld, the principal nucleo- albumin (nucleo-proteid) in the nucleus of leucocytes is micleo-Jiiston, containing about 3 % of phosphorus, which may be split into a form of nnclein playing the part of an acid, and an albuminoid base, the histon of Kossel ; the nuclein may in turn be split into albumin and nucleic acid. These four substances — albumin, nucleo-histon, nu- clein, nucleic acid — thus form a series in which the proportion of phosphorus, i.e. of nucleic 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, nucleic acid intense green. " We see, therefore, that the principle that determines the staining of the nuclear substances is always the nucleic 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 towards blue." ^ Lilienfeld explains the fact that chromatin in the cell-nucleus seldom appears pure green on the assumption, supported by many facts, that the proportion of nucleic 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 consist, like the chromatin of spermatozoa, of pure or nearly pure nucleic acid. Very interesting and con- vincing 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-Ehrlich mixture ; i.e. the nuclein is coloured red, the albumin green. This is a beautiful demonstration of the fact that staining-reagents cannot be logically classified according to coloin-, but onlv according to their chemical 1 I.e., p. 394. 244 ^OME ASPECTS OF CELL-CHEMISTRY AND CELL-PHYSIOLOGY nature. Such terms as " erythrophilous," " cyanophilous," and the like have therefore no meaning apart from the chemical compo- sition both of the dye and of the substance stained. ^ The constancy and accuracy of these reactions await further test, and until this has been carried out we should be careful not to place too implicit a trust in the staining-reactions as an indication of chemi- cal nature, especially as they are known to be affected by the pre- ceding mode of fixation. They afford, nevertheless, a rough method for the micro-chemical test of the proportion of nucleic acid present in the nuclear structures, and this in the hands of Heidenhain has led to some suggestive results. Leucocytes stained with the Biondi- Ehrlich mixture of acid fuchsin and methyl-green show the following reactions. Cytoplasm, centrosome, attraction-sphere, astral rays, and spindle-fibres are stained pure red. The nuclear substance shows a verv sharp differentiation. The chromatic network and the chromo- somes of the mitotic figure are green. The linin-substance and the true nucleoli or plasmosomes appear red, like the cytoplasm. The linin-network of leucocytes is stated by Heidenhain to consist of two elements, namely, of red granules or microsomes sus- pended in a colourless network. The latter alone is called " linin " by Heidenhain. To the red granules is applied the term "oxychro- matin," while the green substance of the ordinary chromatic network, 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 basichromatin-granules of the chromatic network. Collating these results with those of the physiological chemists, Heidenhain concludes that basichromatin is a substance rich in phosphorus {i.e. nucleic 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 are regulated by cer- tain physiological 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- iCf. p. 127. 2 '94, p. 543. 3/.^., p. 547. s W^iM^ r^>^ n Fig. 113. — Nucleated and non-nuoleated fragments of Amxba. [HOP'ER.] A. D. An Ammha divided into nucleated and non-nucleated halves, five minutes after the opera- tion. C. D. The two halves after eight days, each containing a contractile vacuole. dctcr»iination of these operations, 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 unicellular plants. Klebs observed as long ago as 1879 that naked protoplasmic fragments of Vanchcria and other algae were incapable of forming a new cellulose membrane if devoid of a nucleus ; and he afterwards showed ('87) 252 SOME ASPECTS OF CELL-CHEMISTRY AND CELL-PHYSIOLOGY that the same is true of Zygnciiia and CEdigoniitvi. By plasmolysis the cells of these forms may be broken up into fragments, both nucleated and non-nucleated. The former surround themselves with a new wall, grow, and develop into complete plants ; the latter, while able to form starch by means of the chlorophyll they contain, are incapable of utilizing it, and are devoid of the power of forming a new membrane, and of growth and regeneration.^ Although Verworn's results confirm and extend the earlier work of Nussbaum and Gruber, he has drawn from them a somewhat different conclusion, based mainly on the fact, determined by him, that a nucleus deprived of cytoplasm is as devoid of the power to regenerate the whole as an enucleated mass of cytoplasm. From this he argues, with perfect justice, that the formative energy cannot properly be ascribed to the nucleus alone, but is rather a co-ordinate activity of both nucleus and cytoplasm. No one will dispute this conclusion; yet in the light of other evidence it is, I think, stated in somewhat misleading terms which obscure the significance of Verworn's own beautiful experiments. It is undoubtedly true that the cell, like any other living organism, acts as a whole, and that the integrity of all of its parts is necessary to its continued existence ; but this no more pre- cludes a specialization and localization of function in the cell than in the higher organism. The experiments certainly do not prove that the nucleus is the sole instrument of organic synthesis, but they no less certainly indicate its especial importance in this process. The sperm-nucleus is unable to develop its latent capacities without be- coming associated with the cytoplasm of an ovum, but its significance as the bearer of the paternal heritage is not thereby lessened one iota. 2. Position and Movements of the Xiieleus 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 impos- sible, and we must be content to consider only the well-known researches of Haberlandt (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 determined the very significant fact that local growth of the cell-wall is always preceded by a movement of the nucleus to the 1 Palla ('90) has disputed this result, maintaining that enucleated masses of protoplasm pressed out from pollen-tubes might surround themselves with membranes and grow out into long tubes. Later observations, however, by Acqua CgO, throw doubt on Palla's conclusion. PHYSIOLOGICAL RELATIONS OF NUCLEUS AND CYTOPLASM 253 point of growth. Thus, in the formation of epidermal cells the nucleus lies at first near the centre, but as the outer wall thickens, the nucleus moves towards it, and remains closely applied to it throughout its growth, after which the nucleus often moves into another part of the cell (Fig. 114, A, B). That this is not due simply to a movement of the nucleus towards the air and light is beautifully shown in the coats of certain seeds, where the nucleus Fig. 114. — Position of the nuclei in growing plant-cells. [tiAKERLAXnT.] A. Young epidermal cell of Lmula with central nucleus, before thickening of the membrane. B. Three epidermal cells of Momtera, 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. moves not to the outer, but to the inner wall of the cell, and here the thickening takes place (Fig. 114, C). The same position 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 primary outgrowth alwavs takes place from the immediate neighbour- hood of the nucleus, which is carried outward and remains near the tip of the growing hair (Fig. 114, D, E). The same is true of the 254 SOME ASPECTS OF CELL-CHEMISTRY AND CELL-PHYSIOLOGY rhizoids of fern-prothallia and liverworts. In the hairs of aerial plants this 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 ! Very interesting is Haberlandt's observation that in the regeneration of fragments of Vauchcria the o-rowincf resrion, 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 morphologi- cal 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 elaboration 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 larvas (Meckel, Zaddach, etc.), which are characterized by an intense secretory activity con- centrated into a very short period. Here the nucleus forms a labyrinthine network (Fig. ii, 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 p. 114. Here the develop- ing ovum is accompanied and surrounded by cells, which 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. 115), which has a very large nucleus rich in chromatin (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 1 I.e., p. 99. PHYSIOLOGICAL RELATIONS OF NUCLEUS AND CYTOPLASM 255 of such cells at one pole of the o.^^ from which the latter is believed to draw its nutriment (Fig. 58). A very interesting case is that of the annelid OpJiryotrocha, referred to at p. 114. Here, as described by Korschelt, the ^g^ floats in the perivisceral fluid, accompanied by a nurse-cell having a very large chromatic nucleus, while that of the egg is smaller and poorer in chromatin. As the ^tgg completes its growth, the nurse- cell dwindles away and finally perishes (Fig. 57). In all these cases it is scarcely possible to doubt that the egg is in a measure relieved of the task of elaborat- ing cytoplasmic products by the nurse-cell, and that the great develop- ment 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 towards the same conclusion. Per- g haps the most sugges- tive of these relate to the nucleus of the Q^'g during its ovarian his- tory. In many of the Fig. 115. — Upper portion of the ovary in the earwig Forjicula, showing eggs and nurse-cells. [KORSCHELT.] Below, a portion of the near!)' ripe egg {e), showing deuto- plasm-spheres and germinal vesicle (£'v). Above it lies the mseCtS, as in both the mnse-cell («) with its enormous branching nucleus. Two cases referred to above successivelyyounger stages of egg and nurse are shown above, the egg-nucleus at first occupies a central jjosition, but as the egg begins to grow, it moves to the periphery on the side turned towards the nutritive cells. The same is true in the ovarian eggs of some other animals, good examples of which are afforded by various coelenterates, e.g. in medusae (Claus, Hertwig) and actinians (Korschelt, Hertwig), where the germinal vesicle is always near the point of attachment of the Qgg. Most suggestive of all is the case of the water-beetle Dytiscns, in which Korschelt was able to observe the movements and changes of form in the living object. The eggs 256 SOME ASPECTS OF CELL-CHEMISTRY AND CELL-PHYSIOLOGY here lie in a single series alternating with chambers of nutritive cells. The latter contain granules which are believed by Korschelt to pass into the egg, perhaps bodily, perhaps by dissolving and entering in a liquid form. At all events, the egg contains accumulations of similar granules, which extend inwards in dense masses from the nutritive cells to the germinal vesicle, which they may more or less completely surround. The latter meanwhile becomes amoeboid, sending out long pseudopodia, which are always directed towards the principal mass of granules (Fig. 58). The granules could not be traced into the nucleus, but the latter grows rapidly during these changes, proving that mat- ter must be absorbed by it, probably in a liquid form.^ All of these and a large 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 its constructive phase. On the whole, therefore, the behaviour of the nucleus in this regard is in harmony with the result reached by experiment on the one-celled forms, though it gives in itself a far less certain and con- vincing result. We now turn to evidence which, though less direct than the experi- mental proof, is scarcely less convincing. This evidence, which has been exhaustively discussed by Hertwig, Weismann, and Strasburger, is drawn from the history of the nucleus in mitosis, fertilization, and maturation. It calls for only a brief review here, since the facts have been fully described in earlier chapters. 3. The Xnclcus in Mitosis To Wilhelm Roux ('83) w^e owe the first clear recognition of the fact that the transformation of the chromatic substance dur- ing mitotic division is manifestly designed to effect a precise di- vision 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 divi- sion 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 undergoes 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 1 Some observers have maintained that the nucleus may take in as well as give off solid matters. This statement rests, however, on a very insecure foundation. PHYSrOLOGICAL KELATIOXS OF NUCLEUS AND CYTOPLASM 257 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 VVeismann developed this conception has now been gener- ally rejected, and in any form it has some serious difficulties In its way. We cannot assume a precise localization of chromatin-ele- ments 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 matu- ration of the Qgg), and on the other hand in the Protozoa a small fragment of the nucleus is able to regenerate the whole. Neverthe- less, the essential fact remains, as Hertwig, Kolliker, Strasburger, De Vries, and many others have insisted, that in mitotic cell-divi?ion the chromatin of the mother-cell is distributed with the most scrupu- lous 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 through- out 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 posi- tion 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 Xitclcits ill Fertilization The foregoing argument receives an overwhelming reinforce- ment from the facts of fertilization. Although the ovum supplies nearly all the cytoplasm for the embryonic body, and the sper- matozoon at most only a trace, the latter is nevertheless as potent m Its effect on the offspring as the former. On the other hand, the nuclei contributed by the two germ-cells, though apparently different, become in the end exactly equivalent in every visible respect — in structure, in staining-reactions, and in the number and form of the chromosomes to which each gives rise. But further- more the substance of the two germ-nuclei is distributed with abso- lute equality, certainly to the first two cells of the embryo, and probably to all later-formed cells. The latter conclusion, which long remained a mere surmise, has been rendered nearly a cer- tainty by the remarkable observations of Riickert, Zoja, and Hacker described in Chapters IV. and VI. The conclusion is irresistible that the specific character of the cell is in the last analvsis deter- mined by that of the nucleus, that is by the chromatin, and that in the equal distribution of paternal and maternal chromatin to all the cells of the offspring we find the physiological explanation of the s 258 SOME ASPECTS OF CELL-CIIEMISTKY AND CELL-PHYSIOLOGY fact that every part of the latter may show the characteristics ot either or both parents. , • i .4. Boveri ('89. '95, i ) has attempted to test this cone usion by a most ingenious and beautiful experiment; and although his cone usions do ^ not rest on absolutely certam ground, they at least open the way to a decisive test. The Hertwig brothers showed that the eggs of sea-urchins might be enucleated by shaking, and that spermatozoa would enter the enucleated fragments and cause them to segment. Boveri proved that such fragments would even give rise to dwarf larva;, indistinguishable from the normal in general appear- ance and differing from the latter only in size and in the very significant fact that their nuclei contain only half the nor- mal number of chromosomes. Now, by fertilizing enucleated egg-fragments of one species {SphcvrccJiinus granulans) with the spermatozoa of another ( Ecliiniis Diicrotiiberculatns), Bo- veri obtained in a few instances dwarf Plutei showing purely paternal characteristics ( h ig. 116). 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, effected by the nucleus alone.i Boveri's result is unfortunately not quite conclusive, _ as has ^een pointed^^^t bv Seeli-er and Morgan, yet his extensive experiments establish, 1 hink a s? ong presumption in its favour. Should they be positively fonfii-med, the' would furnish a practical demonstration of mheritance through the nucleus. Fig. 116. — Normal and dwarf larvte of the sea-urchin. [BOVERI.] A. Dwarf Pluteus arising from an enucleated eg<^-fragment ot Sp/uerechnnis gra>ii„°^''^'''- ' '"" repeateclh observed U,e Internal changes in .be bv.ng eggs of r«../»e„./«. SUMMARY AND CONCLUSION 261 somes of the fibres must be in some manner polarized by an influence emanating from the centrosome, but in the present state of know- ledge it would be useless to speculate on the nature of this influence. One fact, however, should be borne in mind, namely, that the centro- some differs chemically from the substance of the fibres as shown by Its staining-reactions ; and this may form a clue to the further inves- tigation of this most interesting problem. The principal point in connection with our present theme is that the centrosome cannot be regarded as taking any important part in Fig. 118. — Cleavage of dispermic egg of Toxoptieustes. A. One sperm-nucleus has united with the egg-nucleus, shown at <^. ^ ; the other lies above Both sperm-asters have divided to form amphiasters («, b and ., d) . B The cleavage nuc^eu^ formed by unton of the three germ-nuclei, ,s surrounded by the fo^r asters. C. ResJt of the fi.^ cleavage, the four blastomeres lettered to correspond with the four asters. the general metabolism of the cell, nor can it be an organ of inheri- tance ; for on the one hand it is absent or so small as to be indistin- guishable in many actively metabolizing cells, such as those of the pancreas or kidney, or the older ovarian eggs, and, on the other hand in fertilization it may be derived from one sex only. The conclusion regarding inheritance would not be invalidated, even if it could be positively shown that in some cases both germ-cells might contribute a centrosome ; for a single case of its one-sided origin would be con- clusive, and many such are actually known. D. SUMM.-XRV AND CONCLUSION All of the facts reviewed in the foregoing pages converge, I think to the conclusion drawn by Claude Bernard, that the nucleus is the formative centre of the cell in a chemical sense, and through this is the especial seat of the formative energy in a morphological sense. That the nucleus has such a significance in .synthetic metabolism is proved by the fact that digestion and absorption of food, growth, and 262 SOME ASPECTS OF CELL-CHEMISTRY AND CELL-PHYSIOLOGY secretion cease with its removal from the cytoplasm, while destructive metabohsm may long continue as manifested by the phenomena of irritability and contractility. It is indicated by the position and move- ments of the nucleus in relation to the food-supply and to the forma- tion 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 stammg- capacity undergone by the chromatin during the cycle of cell-life, taken in connection with the researches of physiological chemistson the chemical composition and staining-reactions of the nuclem-series indicate that the substance known as nucleic acid plays a leading part in the constructive process. During the vegetative phase of the cell this substance appears to enter into combination with proteid or albuminous substance to form a nuclein. During its mitotic or repro- ductive phase the albumin is split off, leaving the substance o the chromosomes as nearly pure nucleic acid. When this is correlated with the fact that the sperm-nucleus, which brmgs with it the pater- nal heritage, likewise consists of nearly pure nucleic acid, the pos- sibility is opened that this substance may be in a chemical sense not only the formative centre of the nucleus but also a primary factor m the constructive processes of the cytoplasm. The role of the nucleus in constructive metabolism is intimately related with its rdle in morphological synthesis and thus m inheri- tance • for the recurrence of similar morphological characters must in the last analysis be due to the recurrence of correspondmg fornis o metabolic action of which they are the outward expression. That the nucleus is in fact a primary factor in morphological aswe 1 as chemical synthesis is demonstrated by experiments on unicellular plants and animals, which prove that the power of regenerating los parts disappears with its removal, though the enucleated fragment may continue to live and move for a considerable period. _ This fact establishes the presumption that the nucleus is, if not the actual' seat of the formative energy, at least the controlling factor m that energy, and hence the controlling factor in inheritance. This presumption becomes a practical certainty when we turn to the facts of maturation, fertilization, and cell-division. All of these con- vero-e to the conclusion that the chromatin is the most essential ele- ment in development. 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 ele- ments are brought together and by mitotic division distributed with exact equality to the embryonic cells. The result proves that he spermatozoon is as potent in inheritance as the ovum though the latter contributes an amount of cytoplasm which is but an infini- SUMMARY AXD CONCLUSION 263 tesimal fraction of that supplied by the ovum. The centrosome, finally, is excluded from the process of inheritance, since it may be derived from one sex only. LITERATURE. VII Bernard. Claude. — Le(;ons sur les Phenomenes de la Vie: ist ed. 1878; 2d ed. 1885. Finis. Chittenden, R. H. — Some Recent Chemico-physiological Discoveries regardinjj the Cell: Am. Nat., XXVIII., Feb., 1894. Haberlandt. G. — Uber die Beziehungen zwischen Funktion und Lage des Zellkerns. Fisc/wr. 1887. Halliburton, W. D. — A Text-book of Chemical Physiology and Pathology. London, 1 89 1. Id. — The Chemical Physiology of the Cell {Gouldstonian Lectures): Brit. Med. JoilDl. 1893. Hammarsten. 0. — Lehrbuch der physiologische Chemie. 3d ed. Wiesbaden, 1895. Hertwig, 0. & R. — Uber den Befruchtungs- und Teilungsvorgang des tierischen Eies unter dem Eintiuss Jiusserer Agentien. Jena, 1887. Kolliker, A. — Das Karyoplasma und die Vererbung, eine Kritik der Weismann'schen Theorie von der Kontinuitat des Keimplasmas : Zeitschr. iviss. Zo'ol., XLIV 1886. Korschelt, E. — Beitrage sur Morphologie und Physiologic des Zell-kernes : Zo'ol. Jahrb. Anat. 11. Onto_i^.. 1\'. 1889. Kossel, A. — Uber die chemische Zusammensetzung der Zelle : Arc/i. Anat. u. Fhys 1S91. Lilienfeld, L. — Uber die Wahlverwandtschaft der Zellelemente zu Farbstoffen : .bxh. Anat. 11. Fhys. 1893. Malfatti, H. — Beitrage zur Kenntniss der Nucleine : Zeitschr. Fhys. Chevi., XVI 1891. Riickert, J. — Zur Entwicklungsgeschichte des Ovarialeies bei Selachiern : An. Anz. VII. 1892. Sachs, J. — Vorlesungen uber Pflanzen-physiologie. Leipzig, 1882. Id. — Stoff und Form der Pflanzen-organe : Gesammelte Abhandlnngen, II. 1893. Verworn, M. — Die Phvsiologische Bedeutung des Zellkerns : Arch, fiir die Ge<;. Fhys.,XL\. 1892. Id. — Allgemeine Physiologic. Jena, 1895. Zacharias, E. — Uber Chromatophilie : Ber. d. dentsch. Bot. Ges. 1893. Id. — Uber des Verhalten des Zellkerns in wachsenden Zellen : Flora. 81. 1895. Whitman, C. 0. — The Seat of Formative and Regenerative Energv : Journ. Morph.. II. 1888. CHAPTER VIII CELL-DIVISION AND DEVELOPMENT " Wir koiinen demnach endlich den Satz aufstellen, dass siimmtliche im entwickelten Zustande vorhandenen Zellen oder Aequivalente von Zellen durch eine fortschreitende Gliederung der Eizelle in morphologisch ahnliche Elemente entstehen, und dass die in einer embryonischen Organ-Anlage enthaltenden Zellen, so gering auch ihre Zahl sein mag, dennoch die ausschliessliche ungegliederte Anlage filr sammtliche Formbestandtheile der spateren Organe enthalten." Remak.i 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 fnitotic cell-divisions by which the egg 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, bringing with it a new centrosome, 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 descendants 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, 1 Untersucliiingen, 1855, p. 140. 264 GEOMETRICAL RELATIONS OF CLEAVAGE-FORMS 265 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 sometimes so clearly marked and appear so early that with the very first cleavage the position in w^hich the embryo will finally appear in the ^%g may be exactly predicted. Such " promorphological " rela- tions of the segmenting egg 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 laws of cell-division. For this purpose we may consider the cleavage of the ovum under two heads, namely : — ■ 1. TJic Geometrical Relations of Cleavage-fonns, with reference to the general laws of cell-division. 2. T/ie PromorpJiological 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 are variants of a single type which has been moulded this way or that by special conditions, and which is itself an expression of two general laws of cell-division, first formulated by Sachs in the case of plant- cells. These are : 1. TJie cell typically tends to divide into equal parts. 2. EacJi new plane of division tends to intersect the preceding plane at a rig J it 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 266 CELL-DIVISION AND DEVELOPMENT 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 Fig. 119. — Geometrical relations of cleavage-planes in growing plant-tissues. [From Sachs, after various authors.] A. Flat ellipsoidal germ-disc of Melobesia (Rosanoff) : nearly typical relation of elliptic periclines and iiyperbolic anticlines. B. C. Apical view of terminal knob on epidermal iiair of Pinguicola. B. shows the ellipsoid type, C. the circular (spherical type), somewhat modified (only anticlines present). D. Growing point of Salvinia (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 Eqiiisetum (Niigeli and Leitgeb) ; modified circular type. G. Cross-section of leaf-vein, Triclioinanes (Prantl) ; ellipsoidal type with incomplete periclines. H. Embryo of Alismci; typical ellipsoid type, pericline incomplete only at lower side. /. Growing point of bud of the pine {Ad/£s) ; typical paraboloid type, both anti- clines and periclines having the form of parabolas (Sachs). right angles. All these schemes are, mutatis mutandis, directly con- vertible into the corresponding solid forms in three dimensions. Sachs has shown in the most beautiful manner that all the above ideal types are closely approximated in nature, and Rauber has applied GEOMETRICAL RELATIONS OF CLEAVAGE-FORMS 26y 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 various lower plants, in the embryos of flowering plants, and elsewhere (Fig. 119). The paraboloid form is according to Sachs characteristic of the growing points of many higher plants; and here too the actual form is remarkably similar to the ideal scheme (Fig. 119, /). For our purpose the most important form is the sphere, which is the typical shape of the egg-cell; and all forms of cleavage are deriv- atives of the typical division of a sphere in accordance with Sachs's laws. 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 i^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 most typical 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 Qgg of the holothurian Synapta, as shown in the diagrams. Fig. 120, constructed from Selenka's drawings. ^ The first cleavage is vertical, or Jiuridional, passing through the egg-axis and dividing the egg into equal halves. The second, which is also meridional, cuts the first plane at right angles and divides the egg into quadrants. The third is horizontal, or equatorial, dividing the (tgg into equal octants. The order of division is thus far exactly that demanded by Sachs's law 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 wall consisting of a single layer of cells in which anticlinal 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. This simple and regular mode of division forms a type to which nearly all forms of cleavage may be referred ; but the order and form 1 Cf. also Fig. 3. 268 CELL-DIVISION AND DEVELOPMENT of the divisions is endlessly varied by special conditions. These modifications are all referable to the three following causes : — 1. Disturbances in the rhythm of division. 2. Displacement of the cells. 3. Unequal division of the cells. The first of these requires little comment. Nothing is more com- mon than a departure -from the mathematical regularity of division. The variations are sometimes quite irregular, sometimes follow a definite law, as, for instance, in the annelid Nereis {Y\g. 122), where the typical succession in the number of cells is with great constancy D E Fig. 120. — Cleavage of the ovum in the holothurian Synapta (slightly schematized). [After Selenka.] A-E. Successive cleavages to the 32-cell stage. F. Blastula of 128 cells. 2, 4, 8, 16, 20, 23, 29, 32, n, 38, 41, 42, after which the order is more or less variable. The meaning of such variations in particular cases is not very clear. They are certainly due in part to variations in the amount of deutoplasm ; for, as Balfour long since pointed out ('75), the rapidity of division in any part of the ovum is in general inversely proportional to the amount of deutoplasm it contains. E.xceptions to this law are, however, known. The second series of modifications, due to displacements of the cells, are probably due to mutual pressure, however caused, ^ which ^ The pressure is probably due primarily to an attraction between the cells {cyto/ropisin of Koux), but may be increased by the presence of membranes, by tur_<^(ir, or by sjiccial processes of gr. \o. 300 THEORIES OF INHERITANCE AND DEVELOPMENT are expressed with great caution and with a full recognition of the difficulty and complexity of the problem. From his latest essay, in- deed ('94); it is not easy to gather his precise position regarding the theory of cytoplasmic localization. Through all his writings, never- theless, runs the leading idea that the germ is definitely organized before development begins, and that cleavage only reveals an organ- ization that exists from the beginning. "That organization precedes cell-formation and regulates it, rather than the reverse, is a conclu- sion that forces itself upon us from many sides." ^ "The organ- ism exists before cleavage sets in, and persists throughout every stage of cell-multiplication."^ In so far as this view involves the assumption that the organization of the egg-cytoplasm at the be- ginning of cleavage is a primordial character of the egg, Whitman's conception must, I think, be placed on the side of the localization theory ; but his point of view can only be appreciated through a study of his own writings. All of these views, excepting those of Roux, lean more or less distinctly towards the conclusion that the cytoplasm of the egg-cell is from the first mapped out, as it were, into regions which corre- spond with the parts of the future embryonic body. The cleavage of the ovum does not create these regions, but only reveals them to view by marking off their boundaries. Their topographical arrange- ment in the o.^'g does not necessarily coincide with that of the adult parts, but only involves the latter as a necessary consequence — some- what as a picture in the kaleidoscope gives rise to a succeeding pic- ture composed of the same parts in a different arrangement. The germinal localization may, however, in a greater or less degree, fore- shadow the arrangement of adult parts — for instance, in the egg of the tunicate or cephalopod, where the bilateral symmetry and antero- posterior differentiation of the adult is foreshadowed not only in the cleavage stages, but even in the unsegmented t2,g. By another set of writers, such as Roux, De Vries, Hertwig, and Weismann, germinal localization is primarily sought not in the cyto- plasm, but in the nucleus ; but these views can best be considered after a review of the idioplasm hypothesis, to which we now proceed. B. The Idioplasm Theory We owe to Nageli the first systematic attempt to discuss heredity regarded as inherent in a definite physical basis ; ^ but it is hardly necessary to point out his great debt to earlier writers, foremost among them Darwin, Herbert Spencer, and Hackel. It was the 1 '93, p. 115. - /.<■., p. 112. 3 j'/igorie der Abstaviinungslehre, 1884. THE IDIOPLASM THEORY 3OI oreat merit of Nageli's hypothesis to consider inheritance as effected iDy the transmission not of a cell, considered as a whole, but of a par- ticular substance, the idioplasm, contained within a cell, and forming the physical basis of heredity. The idioplasm is to be sharply dis- tinguished from the other constituents of the cell, which play no direct part in inheritance and form a " nutritive plasma " or trophoplasui. Hereditary traits are the outcome of a definite molec- ular organization of the idioplasm. The hen's egg differs from the frog's because it contains a different idioplasm. The species is as completely contained in the one as in the other, and the hen's Q.^g differs from a frog's as widely as a hen from a frog. The idioplasm was conceived as an extremely complex substance consisting of elementary complexes of molecules known as niiccUcB. These are variously grouped to form units of higher orders, which, as development proceeds, determine the development of the adult cells, tissues, and organs. The specific peculiarities of the idioplasm are therefore due to the arrangement of the micellse ; and this, in its turn, is owing to dynamic properties of the micellae themselves. Dur- ing development the idioplasm undergoes a progressive transforma- tion of its substance, not through any material change, but through dynamic alterations of the conditions of tension and movement of the micellae. These changes in the idioplasm cause reactions on the part of surrounding structures leading to definite chemical and plastic changes, i.e. to differentiation and development. Nageli made no attempt to locate the idioplasm precisely or to identify it with any of the known morphological constituents of the cell. It was somewhat vaguely conceived as a network extending through both nucleus and cytoplasm, and from cell to cell through- out the entire organism. Almost immediately after the publication of his theory, however, several of the foremost leaders of biologi- cal investigation were led to locate the idioplasm in the nucleus, and succeeding researches have rendered it more and more highly probable that it is to be identified with chroiiiatiu. The grounds for this conclusion, which have already been stated in Chapter VII., may be here again briefly reviewed. The beautiful experi- ments of Nussbaum, Gruber, and Verworn proved that the regenera- tion of differentiated cytoplasmic structures in the Protozoa can only take place when nuclear matter is present (cf. p. 248). The study of fertilization by Hertwig, Strasburger, and Van Beneden proved that in the sexual reproduction of both plants and animals the nucleus of the germ is equally derived from both sexes, while the cytoplasm is derived almost entirely from the female. The two germ-nuclei, which by their union give rise to that of the germ, were shown by Van Beneden to be of exactly the same morphological nature, since each 302 THEORIES OF INIIERITANCE AND DEVELOPMENT gives rise to chromosomes of the same number, form, and size. Van Beneden and Boveri proved (p. 134) that the paternal and maternal nuclear substances are equally distributed to each of the first two cells, and the more recent work of Hacker, Ruckert, Herla, and Zoja establishes a strong probability that this equal distribution con- tinues in the later divisions. Roux pointed out the telling fact that the entire complicated mechanism of mitosis seems designed to effect the most accurate division of the entire nuclear substance in all of its parts, while fission of the cytoplasmic cell-body is in the main a mass-division, and not a meristic division of the individual parts. Again, the complicated processes of maturation show the significant fact that while the greatest pains is taken to prepare the germ-nuclei for their coming union, by rendering them exactly equivalent, the cytoplasm becomes widely different in the two germ-cells and is devoted to entirely different functions. It was in the main these considerations that led Hertwig, Stras- burger, Kolliker, and Weismann independently and almost simultane- ously to the conclusion that the nucleus contains the physical basis of inheritance, ajid that chromatin, its essential constituent, is the idio- plasm postulated in Nageli s theory. This conclusion is now widely accepted ; and notwithstanding certain facts which at first sight may seem opposed to it, I believe it rests upon a basis so firm that it may be taken as one of the elementary data of heredity. To accept it is, however, to reject the theory of germinal localization in so far as it assumes a pre -localization of the egg-cytoplasm as a fundamental character of the Qg'g. For if the specific character of the organism be determined by an idioplasm contained in the chromatin, then every characteristic of the cytoplasm must in the long run be determined from the same source. A striking illustration of this fact is given by the phenomena of colour-inheritance in plant-hybrids, as De Vries has pointed out. Pigment is developed in the embryonic cytoplasm, which is derived from the mother-cell ; yet in hybrids it may be inherited from the male through the nucleus of the germ-cell. The specific form of cytoplasmic metabolism by which the pigment is formed must therefore be determined by the paternal chromatin in the germ-nucleus, and not by a pre-determination of the egg-cyto- plasm. C. Union of the Two Theories We have now to consider the attempts that have been made to transfer the localization-theory from the cytoplasm to the nucleus, and thus to bring it into harmony with the theory of nuclear idio- plasm. These attempts are especially associated with the names of THE ROUX-IVEISMANN THEORY OF DEVELOPMENT 303 RoLix, Dc Vries, Weismann, and Hertwig; but all of them may be traced back to Darwin's celebrated hypothesis of pangenesis as a prototype. This hypothesis is so well known as to require but a brief review. Its fundamental postulate assumes that the germ-cells contain innumerable ultra-microscopic organized bodies or gcinvuilcs, each of which is the germ of a cell and determines the development of a similar cell during the ontogeny. The germ-cell is, therefore, in Darwin's words, a microcosm formed of a host of inconceivably mi- nute self-propagating organisms, every one of which predetermines the formation of one of the adult cells. De Vries ('89) brought this conception into relation with the theory of nuclear idioplasm by assuming that the gemmules of Darwin, which he czWo.^ pang ens, are contained in the nucleus, migrating thence into the cytoplasm step by step during ontogeny, and thus determining the successive stages of development. The same view was afterwards accepted by Hert- wig and Weismann.^ The theory of germinal localization is thus transferred from the cytoplasm to the nucleus. It is not denied that the egg-cytoplasm may be more or less distinctly differentiated into regions that have a constant relation to the parts of the embryo. This differentiation is, however, conceived, not as a primordial characteristic of the ^2,2,, but as one secondarily determined through the influence of the nucleus. Both De Vries and Weismann assume, in fact, that the entire cyto- plasm is a product of the nucleus, being composed of pangens that migrate out from the latter, and by their active growth and multipli- cation build up the cytoplasmic substance.^ D. The Roux-Weismann Theory of Development We now proceed to an examination of two sharply opposing hy- potheses of development based on the theory of nuclear idioplasm. One of these originated with Roux ('83) and has been elaborated especially by Weismann. The other was clearly outlined by De Vries ('89), and has been developed in various directions by Oscar Hertwig, 1 The neo-pangenesis of De Vries differs from Darwin's hypothesis in one very important respect. Darwin assumed that the gemmules arose in the body, being thrown off as germs by the individual tissue-cells, transported to the germ-cells, and there accumulated as in a reservoir; and he thus endeavoured to explain the transmission of acquired characters. De Vries, on the other hand, denies such a transportal from cell to cell, maintaining that the pangens arise or pre-exist in tlie germ-cell, and those of the tissue-cells are derived from this source in' cell-division. - This conception obviously harmonizes with the role of the nucleus in the synthetic process. In accepting the view that the nuclear control of the cell is effected by an emana- tion of specific substances from the nucleus, we need not, however, necessarily adopt the pangen -hypothesis. 304 THEORIES OF INHERITANCE AND DEVELOPMENT Driesch, and other writers. In discussing them, it should be borne in mind that, although both have been especially developed by the advocates of the pangen-hypothesis, neither necessarily involves that hypothesis in its strict form, i.e. the postulate of discrete self-propa- gating units in the idioplasm. This hypothesis may therefore be laid aside as an open question, and will be considered only in so far as it is necessary to a presentation of the views of individual writers. The Roux-Weismann hypothesis has already been touched on at p. 183. Roux conceived the idioplasm {i.e. the chromatin) not as a .single chemical compound or a homogeneous mass of molecules, but as a highly complex mixture of different substances, representing dijfcrcnt qualities, and having their seat in the individual chromatin- granules. In mitosis these become arranged in a linear series to form the spireme-thread, and hence may be precisely divided by the splitting of the thread. Roux assumes, as a fundamental postulate, that division of the granules may be either quantitative or qualitative. In the first mode the group of qualities represented in the mother- granule is first doubled and then split into equivalent daughter-groups, the daughter-cells therefore receiving the same qualities and remain- ing of the same nature. In "qualitative division," on the other hand, the mother-group of qualities is split into dissimilar groups, which, passing into the respective daughter-nuclei, lead to a correspoudiiig differentiation in the daughter-eells. By qualitative divisions, occur- ring in a fixed and predetermined order, the idioplasm is thus split up during ontogeny into its constituent qualities, which are, as it were, sifted apart and distributed to the various nuclei of the embryo. Every cell-nucleus, therefore, receives a specific form of chroniatiti which determines the nature of the cell at a given period and its later his- tory. Every cell is thus endowed with a power of self determination, which lies in the specific structure of its nucleus, and its course of development is only in a minor degree capable of modification through the relation of the cell to its fellows (" correlative differentiation "). Roux's hypothesis, be it observed, does not commit him to the theory of pangenesis. It was reserved for Weismann to develop the hypothesis of qualitative division in terms of the pangen-hypothesis, and to elaborate it as a complete theory of development. In his first essay ('85), published before De Vries's paper, he went no fur- ther than Roux. "I believe that we must accept the hypothesis that in indirect nuclear division, the formation of non-equivalent halves may take place quite as readily as the formation of equivalent halves, and that the equivalence or non-equivalence of the subsequently pro- duced daughter-cells must depend upon that of the nuclei. Thus, during ontogeny a gradual transformation of the nuclear substance takes place, necessarily imposed upon it, according to certain laws. THE ROUX-WEISMANN THEORY OF DEVELOPMENT 305 by its own nature, and such transformation is accompanied by a gradual change in the character of the cell-bodies." ^ In later writ- ings Weismann advanced far beyond this, building up an elaborate artificial system, which appears in its final form in the remarkable book on the germ-plasm ('92). Accepting De Vries's conception of the pangens, he assumes a definite grouping of these bodies in the germ-plasm or idioplasm (chromatin), somewhat as in Nageli's concep- tion. The pangens or biopJiorcs are conceived to be successively ag- gregated in larger and larger groups ; namely, (i) dcteinniiiants, which are still beyond the limits of microscopical vision ; (2) ids, which are identified with the visible chromatin-granules ; and (3) idants, or chromosomes. The chromatin has, therefore, a highly complex fixed architecture, which is transmitted from generation to generation, and determines the development of the embryo in a definite and specific manner. Mitotic division is conceived as an apparatus which may distribute the elements of the chromatin to the daughter-nuclei either equally or unequally. In the former case {'' lioiiiccokinesis,'" integral or quantitative division), the resulting nuclei remain precisely equiva- lent. In the second case {'' /wtcrokinesis,'' qualitative or dijfcrential division), the daughter-cells receive different groups of chromatin- elements, and hence become differently modified. During ontogeny, through successive qualitative divisions, the elements of the idioplasm or genn-plasni (chromatin) are gradually sifted apart, and distributed in a definite and predetermined manner to the various parts of the body. " Ontogeny depends on a gradual process of disintegration of the id of germ-plasm, which splits into smaller and smaller groups of determinants in the development of each individual. . . . Finally, if we neglect possible complications, only 07ie kind of determinant re- mains in each cell, viz. that which has to control that particular cell or group of cells. ... In this cell it breaks up into its constituent bi- ophores, and gives the cell its inherited specific character."'-^ Devel- opment is, therefore, essentially evolutionary and not epigenetic ; '^ its point of departure is a substance in which all of the adult characters are represented by preformed, prearranged germs; its course is the result of a predetermined harmony in the succession of the qualitative divisions by which the hereditary substance is progressively disinte- grated. In order to account for heredity through successive genera- tions, Weismann is obliged to assume that, by means of quantitative or integral division, a certain part of the original germ-plasm is car- ried on unchanged, and is finally delivered, with its original architecture unaltered, to the germ-nuclei. The power of regeneration is explained, in like manner, as the result of a transmission of unmodified or slightly modified germ-plasm to those parts capable of regeneration. 1 Essay IV., p. 193, 1885. - Gerni-plasin, pp. 76, 77. -^ I.e., p. 15. X 3o6 THEORIES OF INHERITANCE AND DEVELOPMENT E. Critique of the Roux-Weismann Theory From a logical point of view the Roux-Weismann theory is unas- sailable. Its fundamental weakness is its (///cri-Z-metaphysical char- acter, which indeed almost places it outside the sphere of legitimate scientific hypothesis. Not a single visible phenomenon of cell-divi- C D Fig. 132. — Half and whole cleavage in the eggs of sea-urchins. A. Normal i6-cell stage, showing the four micromeres above (from Diiesch, after Selenka). B. Half i6-cell stage developed from one blastomere of the 2-cell stage after killing the other by shaking (Driesch). C. Half blastula resulting, the dead blastomere at the right (Driesch). D. Half-sized i6-cell stage of Toxopneiistes, viewed from the micromere-pole (the eight lower cells not shown). This embryo, developed from an isolated blastomere of the 2-cell stage, segmented like an entire normal ovum. sion gives even a remote suggestion of qualitative division. All the facts, on the contrary, indicate that the division of the chromatin is carried out with the most exact equality. The theory of qualita- tive division was suggested by a totally different order of phenom- ena, and is an explanation constructed ad 'hoc. Roux, it is true, was led to the hypothesis through an examination of mitosis ; but it is CRITIQUE OF THE ROUX-WEISMANN THEORY 307 safe to say that he would never have maintained in the same breath that mitosis is expressly designed for quantitative and also for qual- itative division, had he fixed his attention on the actual phenomena of mitosis alone. The hypothesis is in fact as complete an a priori assumption as any that the history of scholasticism can show, and every fact opposed to it has been met by equally baseless subsidiary hypotheses, which, like their principal, relate to matters beyond the reach of observation. Such an hypothesis cannot be actually overturned by an appeal to fact. When, however, we make such an appeal, the improbability of A B Fig. 133. — Normal and dwarf gastrulas of Amphioxus. A. Normal gastrula. B. Half-sized dwarf, from an isolated blastomere of the 2-cell stage. C. Quarter-sized dwarf, from an isolated blastomere of the 4-cell stage. the hypothesis becomes so great that it loses all semblance of reality. It is rather remarkable that Roux himself led the way in this direc- tion. In the course of his observations on the development of a half- embryo from one of the blastomeres of the two-cell stage he determined the significant fact that the half-embryo afterivards regenerated the missing Iialf, and gave rise to a complete embryo. Essentiall)' the same result was reached by later observers, both in the frog (Endres, Walter, Morgan) and in a number of other animals, with the impor- tant addition that the half-formation is sometimes characteristic of only the earliest stages and may be entirely suppressed. In 1891 Driesch was able to follow out the development of isolated blasto- 3o8 THEORIES OE INHERITANCE AND DEVEIOPMENT meres of sea-urchin 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 quar- ter-) blastula (Fig. 132). The opening soon closes, however, to form a Fig. 134. — Dwarf and double embryos of Amphioxus. A. Isolated blastomere of the 2-cell stage segmenting like an entire egg (cf. Fig, 123, Z)). B. Twin gastrulas from a single egg. C. Double cleavage resulting from the partial separation, by shaking, of the blastomeres of the 2-cell stage. D. E. F. Double gastrulas arising from such forms as the last. 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 gave rise to double embryos like the Siamese twins. Shortly afterwards the writer obtained similar result in the case of AnipJiioxus, but here the isolated blastomere seg- CRITIQUE OF THE ROUX-WEISMANN THEORY 309 ments from the beginning like an entire ovitni of diuiinisJicd size (Figs. 133, 124). The same result has since been reached by Morgan in the teleost fishes, and by Zoja in the medusa. The last-named experi- menter was able to obtain perfect embryos not only from blasto- meres 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 ^^g the normal size ! These experiments gave a fatal blow to the entire Roux-Weismann theory ; for the results showed that the cleavage of the ovum does not in these cases sunder different materials, either nuclear or cytoplasmic, but only splits it up into a number of similar parts, each of which may give rise to an entire body of diminished size. The theory of qualitative nuclear division has been practically Fig. 135. — Modification of cleavage in sea-urchin eggs by pressure. A. Normal 8-cell stage of Toxopneustes. B. Eight-cell stage of Echinus segmenting under pressure. Both forms produce normal Plutei. disproved in another way by Driesch, through the pressure-experi- ments already mentioned at p. 275. Following the earlier experiments of Pfliiger and Roux 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 from the normal (Fig. 134) ; yet such eggs when released from pressure continue to segment, zvitJiont rearrangement of the nuclei, and give rise to per- fectly normal larvae. I have repeated these experiments not only with sea-urchin eggs, but also with those of an annelid {Nereis), which yield a very convincing result, since in this case the histological differentia- tion of the cells appears very early. In the normal development of this animal the archenteron arises from four large cells or macro- meres (entomeres), which remain after the successive formation of three quartets of micromeres (ectomeres) and the parent-cell of the lO THEORIES OF INHERITANCE AND DEVEIOPMENT 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 charac- teristic appearance, differing from that of the other blastomeres in its pale non-granular character and in the presence of large oil-drops. Fig. 136. — Modification of cleavage by pressure in Nereis. A. B. Normal 4- and 8-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 to trocho- phores with eight instead of four entoderm-cells. Numerals designate the successive cleavages. If unsegmented eggs be subjected to pressure, as in Driesch's echino- derm 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. 136, 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 ON THE XATURE AND CAUSES OE DIFFERENTIATION 31I 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 trochophores contain- ing eight instead of four macromeres, which have the typical clear protoplasm containing oil-drops. In this case there can be no doubt whatever that four of the entoblastic nuclei were normally destined for the first quartet of micromeres (Fig. 136, B), from which arise the apical ganglia and the prototroch. Under the conditions of the experiment, however, they have given rise to the nuclei of cells which differ in no wise from the other entoderm-cells. Even in a highly differentiated type of cleavage, therefore, the nuclei of the segmenting ^gg are not specifically different, as the Roux-Weismann hypothesis demands, but contain the same materials even in cells that undergo the most diverse subsequent fate. But there is, furthermore, very strong reason for believing that this may be true in later stages as well, as Kolliker insisted in opposition to Weismann as early as 1886, and as has been urged by many subsequent writers. The strong- est 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 repro- duce 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 limitation arises through specifica- tion of the nuclei by qualitative 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 prob- lem of development, and an examination of the views opposed to the Roux-Weismann theory. After discarding the hypothesis of quali- 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. dijfcrcntiationf 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 egg. These changes are sooner or later accompanied by the cleavage of the egg into cells whose boundaries may sharply mark the 312 THEORIES OF INHERITANCE AND DEVELOPMENT areas of differentiation. What gives these cells their specific char- acter ? Why, in the four-cell stage of an annelid egg, should the four cells contribute equally to the formation of the alimentary canal and the cephalic 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. 122, 137, B). There cannot be a fixed and necessary relation of cause and effect between the various regions of the ^ZZ which these blas- tomeres represent and the adult parts arising from them ; for, as we have seen, these relations may be artificially altered. A portion of the egg which under normal conditions 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. What 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 Pangenesis ('89), endeavoured to cut this Gordian knot by assuming that the character of each cell is determined by pangens that migrate from 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 migration of the pangens, and so correlates the operations of devel- opment .^ Both Driesch and Oscar Hertwig have attempted to 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 egg is a result of its relation to the remainder of the mass, i.e. a product of what may be called the intra-embryonic environ- ment. Both at first assumed not only that the nuclei are equivalent, but also that the cytoplasmic regions of the Qgg are isotropic, i.e. primarily composed of the same materials and equivalent in struct- ure. Hertwig insisted that the organism develops as a whole as the result of a formative power pervading the entire mass ; that differen- tiation is but an expression of this power acting at particular points ; and that the development of each part is, therefore, dependent on that of the whole. ^ " According to my conception," said Hertwig, "each of the first two blastomeres contains the formative and differ- entiating 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 body only because it is placed in relation to a right blasto- mere." ^ Again, in a later paper : — " The egg is a specifically 1 Whitman had strongly urged this view several years before, and a nearly similar concep- tion lay at the bottom of Herbert Spencer's theory of development. Cf. pp. 41, 293. 2 '92, I, p. 481. ox THE NATURE AND CAUSES OF DIFFERENTIATION 313 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 o.^^^ it contains also the germ of the whole,^ but during the process of development it becomes ever more precisely differentiated and determined by the formation of cytoplasmic prod- ucts according to its position with reference to the entire organism (blastula, gastrula, etc)."^ Driesch expressed the same view with great clearness and pre- cision shortly after Hertwig : — " The fragments {i.e. cells) produced by cleavage are completely equivalent or indifferent." "The blasto- A B Fig. 137. — Diagrams contrasting the value of the blastomeres in polyclades and annelids. A. Plan of cleavage in the polyclade egg (constructed from the figures of Lang). B. Corre- sponding plan of the annelid egg. In both cases the ectoblast is unshaded, with the exception of X ; the mesoblast is ruled in vertical lines and the entoblast in horizontal. In both, three succes- sive quartets of micromeres are budded forth from the four primary cells A. B. C. D. In the polyclade the first quartet is ectoblastic, the second and third mesoblastic. In the annelid all three quartets are ectoblastic, while the mesoblast (;!/) arises from the posterior cell of a fourth quartet of which the remaining three are entoblastic. meres 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 develop- ment." ^ "■The relative position of a blastoniere in the zvliole de- termines in general ivJiat develops from it; if its position be changed, it gives rise to something different ; in other words, its prospective value is a function of its position^' ** This conclusion undoubtedly expresses a part of the truth, though, as will presently appear, it is too extreme. The relation of the part 1 That is, in the specifically organized chromatin within the nucleus. 93. P- 793- Stuilien IV. p. 25. ^ Studien IV. p. 39. 314 THEORIES OF INHERITANCE AND DEVELOPMENT to the whole must not, however, be conceived as a merely geometri- cal or mechanical one ; for, in different species of eggs, blastomeres may exactly correspond in origin and relative position, yet have entirely different morphological value. This is strikingly shown by Fig. 138. — Partial larvae of the ctenophore Beroc. [DRiESCii and Morgan.] A. Half i6-cell stage, from an isolated blastomere. B. Resulting larva, with four rows of swim- ming plates and three gastric pouches. C. One-fourth i6-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 unsegmented egg. F. Similar larva with five rows of plates, from above. a comparison of the polyclade egg with that of the annelid or gasteropod (Fig. 137). In both cases three quartets of micromeres are successively budded off from the four cells of the four-cell stage in exactly the same manner. The first quartet in both gives rise to ectoderm. Beyond this point, however, the agreement ceases : ox THE NA TURE AND CA USES OF DIFFERENTIA TION 3 I 5 for the second and third quartets form mesoblast in the polyclade, but ectoblast in the annelid and gasteropod ! In the latter forms the mesoblast lies in a single cell belonging to a fourth quartet of which the other three cells form entoblast. This shows conclusively that the relation of the part to the whole is of an exceedingly subtle character, and that the nature of the individual blastomere depends, not merely upon its geometrical position, but upon its physiological relation to the inJicritcd organization of which it forms a part. Meanwhile, and subsequently, however, facts were determined that threw doubts on the hypothesis of cytoplasmic isotropy and led Driesch to a profound modification of his views, and in a measure rehabilitated the theory of cytoplasmic localization. Whit- man, Morgan, and Driesch himself showed that the cytoplasm of the echinoderm Qgg is not strictly isotropic, as Hertwig assumed; for the ovum possesses a polarity predetermined before cleavage begins, as proved by the fact that a group of small cells or micro- meres always arises at a certain point which may be precisely located before cleavage by reference to the eccentricity of the first cleavage- nucleus.^ Experiments on the eggs of other animals proved that the predetermination of the cytoplasmic regions may be more extensive. In the egg of the ctenophore, for example, Driesch and Morgan (95)> confirming the earlier observations of Chun, proved that an isolated blastomere of the two- or four-cell stage gives rise not to a whole dwarf body, but to a half- or quarter-body, as Roux had observed in the frog2(Fig. 135, A-D). But, more than this, these experimenters made the interesting discovery that if a part of the cytoplasm of an jmscgDicntcd ctenophore-egg were removed, the remainder gave rise to an incomplete larva, sJioiving certain defects li'hich represent the portions removed (Fig. 138, E, F). Again, Crampton 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 X.o fragments of a larva, not to complete dwarfs, as in the echinoderm (Fig. 139). These results demonstrate that the ovum may show a high degree of cytoplasmic localization and that in such cases cleavage may be in fact a mosaic-work, as Roux maintained in case of the frog. But they also show that the localization, and the resulting mosaic-like cleavage, is not determined by specific differences in the nuclei ; for in the ctenophore the fragment of an nnsegmentcd 'i'^i,^^, though con- taining an entire nucleus, gives rise to a defective larva, and in Nereis the nuclei may be shifted about at will without altering the develop- 1 Cf. Fig. 77. - The larva is, however, not a strict partial one, since it makes an abortive attempt to form the normal number of gastric pouches. i6 THEORIES OF INHERITANCE AND DEVELOPMENT ment. And if the germinal localization is not directly determined by the nuclei it must here be determined by a pre-organization of the cytoplasmic substance. How is this result to be reconciled with the experiments on Ampliioxjis and the echinoderms, and with the more o-eneral conclusion that the ultimate determining causes of differentia- Fig. 139. — Partial development of isolated blastomeres of the gasteropod egg, llyanassa. [CRAMI'TON.] A. Normal 8-cell stage. B. Normal i6-cell stage. C. Half 8-cell stage, from isolated blasto- mere of the 2-cell stage. D. Half 12-cell stage succeeding. E. Two stages in the cleavage of an isolated blastomere of the 4-cell stage; above a one-fourth 8-cell stage, below a one-fourth i6-cell stage. tion are to be sought in the nucleus .? The difficulty at once disap- pears when we recall 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 ON THE NATURE AND CAUSES OF DIFFERENTIATION 317 between AinpJiioxus on the one hand, and the snail or ctenophorc 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. We thus arrive at the central point of my own conception of devel- opment, and of Driesch's later views, which were developed in a most able and suggestive though somewhat abstruse manner in his Atialy- tiscJtc Thcoric dcr organiscJicn Entivickliing{^()^), and slightly modified in a later paper published jointly with Morgan, ('95, 2). The gist of Driesch's theory is as follows. All the nuclei are equivalent, and all contain the same idioplasm equally distributed to them by mitotic division. Through the influence of this idioplasm the cytoplasm of the egg, or of the blastomeres derived from it. undergoes specific and progressive changes, each change reacting upon the nucleus and thus inciting a new change. These changes differ in different regions of the Qgg because of pre-existing differences, chemical and physical, in the cytoplasmic structure ; and these form the conditions ("Form- bildungsfaktoren ") under which the idioplasm operates. Some of these conditions are purely mechanical, such as the shape of the ovum, the distribution of deutoplasm, and the like. Others, and probably the more important, are far more subtle, such as the distri- bution of different chemical substances in the cytoplasm, and the unknown polarities of the cytoplasmic molecules. A nearly related conception was developed with admirable clear- ness by Oscar Hertwig ('94) nearly at the same time. Both Driesch and Hertwig thus retreated in a measure towards the theory of germinal localization in the cytoplasm, which both had at first rejected ; but only to a middle ground which lies between the two extremes of the strict predestination theory and the theory of cytoplasmic isotropy. For these writers now maintain that the initial cytoplasmic localization of the formative conditions is of limited extent and determines only the earlier steps of development. With each forward step new conditions (chemical differentiations and the like) are established which form the basis for the ensuing change, and so on in ever-increasing complexity. This view is substantially the same as that which I have myself urged in several earlier works, and I have pointed out how it enables us to reconcile the apparent contradiction between the partial development of isolated blastomeres of such forms as the ctenophorc, on the one hand, with the total development of such forms as Aviphioxus or the echinoderm, on the other. In the latter case we may suppose the cytoplasmic differentiation to be but feebly established at the beginning, and the blastomeres remain for a time in a plastic state, which enables them on isolation to revert to the condition of the original entire ovum. In the former case the initial differentiation is more extensive or more rigidly fixed, so that 3i8 THEORIES OF INHERITANCE AND DEVELOPMENT the development of the blastomcre is from the beginning hemmed in by the cytoplasmic conditions, and its powers are correspondingly limited. In such cases the cleavage may exhibit more or less of a mosaic-like character, and the theory of cytoplasmic localization acquires a real meaning and value. That we are here approaching the true explanation is indicated by Fig. 140. — Double embryos of frog developed from eggs inverted when in the 2-cen stage. [O. SCHULTZE.] A. Twins with heads turned in opposite directions. B. Twins united Ijack to back. C. Twins united by their ventral sides. D. Double-headed tadpole. certain very remarkable and interesting experiments on the frog's Q-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 circumstances, and which indicate, furthermore, that these circum- stances lie in a measure in the arrangement of the cytoplasmic materials. This most important result, which we owe especially to ON THE NATURE AND CAUSES OF DIFFERENTIATION 319 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 towards the lower side, while the nucleus and protoplasm rise. A new axis is thus established in the egg, which has the same relation to the body-axes as in the ordinary development (though the pigment retains its original arrange- ment). This proves that in eggs of this character (telolecithal) the distribution of deutoplasm, or conversely of protoplasm, is one of the primary formative conditions of the cytoplasm; and the significant fact is that by artificially changing this distribution the axis of the embryo is shifted. Oscar Schultze ('94) discovered that if the ^^^ be turned upside down when in the two-cell stage, a whole embryo (or half of a double embryo) might 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. 140). Morgan ('95,3) added the important discovery that either a half- embryo or a w^hole half-sized dwarf might be formed, according to the position of the blastouiere. If, after destruction of one blastomere, the other be allowed to remain in its normal position, a half-embryo always results,^ precisely 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 equilibrium 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 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 both of Aniphi- oxHS and the echinoderms. In Aniphioxns the isolated blastomere usually segments like an entire ovum of diminished size. This is, however, not invariable, for a certain proportion of the blastomeres show a more or less marked tendency to divide as if still forming part of an entire embryo. The sea-urchin Toxopncustes reverses this rule, for the isolated blastomere of the two-cell stage usually shows a perfectly typical half-cleavage, as described by Driesch, but in rare 1 Anat. Aiiz., X. 19, 1895. ^ Three cases. '^ Eleven cases observed. * Nine cases observed. 320 THEORIES OF INHERITANCE AND DEVEIOPMENT cases it may segment like an entire ovum of half-size (Fig. 132, D) and give rise to an entire blastula.^ We may interpret this to mean that in AmphioxiLS the differentiation of the cytoplasmic substance is at first very slight, 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 firmly established, so that only exceptionally can they be altered. In the snail we have the opposite extreme to Amphioxus, the cytoplasmic conditions having been so firmly established that they cannot be altered, 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 isotropy. Primarily the egg-cytoplasm is isotropic in the sense that its various regions stand in no fixed and necessary rela- tion with the parts to which they respectively give rise. Secondarily, however, it may undergo differentiations through which it acquires a definite regional predetermination which becomes ever more firmly established as development advances. This process does not, how- ever, begin at the same time, or proceed at the same rate in all eggs. Hence the eggs of different animals may vary widely in this regard at the time cleavage begins, and hence may differ as widely in their power of response to changed conditions. The origin of the cytoplasmic differentiations existing at the be- ginning of cleavage has already been considered (p. 285). 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 ^gg, its primary polarity, and the distribution of substances within it. The cytoplasmic differentiations thus set up form as it were a frame- work within which the subsequent operations take place, in a more or less fixed course, and which itself becomes ever more complex as development goes forward. If the cytoplasmic conditions be artifi- cially altered by isolation or other disturbance of the blastomeres, a readjustment may take place and development may be correspond- ingly altered. Whether such a readjustment is possible, depends on secondary factors — the extent of the primary differentiations, the physical consistency of the egg-substance, the susceptibility of the protoplasm to injury, and doubtless a multitude of others. 1 I have observed this only twice. In both cases the cleavage up to the sixteen-cell stage was exactly like that of the entire egg except that the micromeres were relatively larger, as shown in the figure. THE NUCLEUS IN LATER DEVELOPMENT 32 1 G. The Nucleus in Later Development Tlie 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 leaves wholly 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. There are, however, a multitude of well-known facts which cannot be explained, even approximately, under this assumption. 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 regenerate one another save in a few exceptional cases. In asexual reproduction, 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. How can we explain this progressive specification of the tissue-cells and how interpret the differences in this regard between related species } To these questions the Roux-Weismann theory gives a definite and intelligible answer; namely, that dijfcrcntiatiou sooner or later involves a specification of the nuclear substance ivhich differs in degree in different cases. W'hen we reflect on the general role of the nucleus in metabolism and its significance as the especial seat of the formative power, we may well hesitate to deny that this i)art of Roux's conception may be better founded than his critics have admitted. Nageli insisted that the idioplasm must undergo a progressive trans- formation during development, and many subsequent writers, including such acute thinkers as Boveri and Nus.sbaum, and many pathologists, have recognized the necessity for such an assumption. Boveri's re- markable observations on the nuclei of the primordial germ-cells in 322 THEORIES OF INHERITANCE AND DEVELOPMENT 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 gcrm-nuclci retain the entire ancestral heritage. Boveri hirnself has in a measure pointed out the significance of his discovery, insisting- that the specific development of the tissue-cells is condi- tioned by specific changes in the chromatin that they receive.i though he is careful not to commit himself to any definite theory. It hardly seems possible to doubt that in Asca7ds 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 ivitJiout recourse to the theory of qualitative division. We do not need to assume that the unused chromatin is cast out bodily ; for it may 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 application 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 metabolism, and the relation between this process and the morphological formative power, 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, assumes a migration of pangens from nucleus to cytoplasm, the character of the cell being determined by the nature of the migrat- mg pangens, and these bemg, 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. ^ '91. P- 433- THE EXTERXAL CONDITIONS ofi DEVELOPMENT 323 It is but a step from this to the very interestini;" 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 theory, as due to a retention of idioplasm or germ-plasm—/.^, 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 egg 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. H. The External Condition.s 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 relation is ; and it has been espe- ]24 THEORIES OF INHERITANCE AND DEVELOPMENT cially 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 limits 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 referred 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 living organism at every stage of its existence reacts to its envn-on- ment by physiological and morpho- logical 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 altered. This is beautifully shown by the experiments of Herbst and others on the development of sea- urchins. Pouchet and Chabry showed that if the embryos of these animals be made to develop in sea-water con- taining no lime-salts, the larva fails to develop not only its calcareous skele- ton, but also its ciliated arms, and a larva thus results that resembles in some particulars an entirely different specific form ; namely, the Tornaria larva of Balanoglossns. This result is not due simply to the lack of neces- sary material; for Herbst showed that the same result is attained if a slight excess of potassium chloride be added to sea-water containing the nor- mal amount of lime (Fig. I40- Ii"^ the latter case the specific metabolism of the protoplasm is altered by a particular chemical stimulus, and a new form results. The changes thus caused by slight chemical alterations m the water may be still more profound. Herbst ('92) observed, for ex- ample, that when the water contains a very small percentage of lithium chloride, the blastula of sea-urchins fails to invagmate to form a typical gastrula, but evaghiatcs to form an hour-glass-shaped larva, one half of which represents the archenteron, the other half the ectoblast. Moreover, a much larger number of the blastula-cells Fig. 141. — Normal and modified larvae of sea-urchins. [HERBST.] A. Normal Pluteus {Strongyloce7itro- tus). B. Larva {Spkcsreckhiits) at the same stage as the foregoing, developed in sea-water containing a slight excess of potassium chloride. THE EXTERNAL CONDITIONS OF DEVELOPMENT 325 undergo the differentiation into entoblast than in the normal de- velopment, the ectoblast sometimes becoming greatly reduced and occasionally disappearing altogether, so that the entire blastula is differentiated into cells having the histological character of the normal entoblast ! One of the most fundamental of embryonic differentia- Fig. 142. — Regeneration in coelenterates (. /. D. from LoEB ; C. D. from BiCKFORD). ./. Polyp {Cerianthus) producing new tentacles from the aboral side of a lateral wound. B. Hydroid ( Ttibularia) generating a head at each end of a fragment of the stem suspended in water. C. D. Similar generation of heads at both ends of short pieces of the stem, in Ttibularia. tions 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 familiar to all. Loeb's interesting experiments on hydroids gave a similar result ('91). It has long been known that Tubularia, like many other hydroids, has 326 THEORIES OF INHERITANCE AND DEVELOPMENT 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 Tiibularia 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 eacJi end (Fig. 142) ; 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 complicated structure is called forth by the stimulus of the external environment. These cases must suffice for our purpose. They prove incontesta- bly that nonnnl developineiit is in a greater or less degree tJie response of tJie developing oiganisni to iiormal 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 direetly eansed by the external conditions ; for the egg 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- ino- 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 inherited organization. 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 idioplasm and gives but a dim insight into its ultimate nature. I. Development, Inheritance, and Metabolism In brinoino- the foreooins; 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 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-granule, 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 ; PREFORMATION AND EPIGENESIS 12 J 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. This remains true however we may conceive the morphological nature of the idioplasm — whether as a microcosm of invisible germs or pangens, as conceived by De Vries, Weismann, and Hertwig, as a storehouse of specific ferments as Driesch suggests, or as complex molecular substance grouped in micellae as in Nageli's hypothesis. It is true, as Vervvorn 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, both by De Vries and Driesch, disappears as soon as we regard the egg-cytoplasm as itself a product of the nuclear 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 &g^ 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. Both are necessary to development ; the nucleus alone suffices for the inheritance of specific possibilities of development. J. Preformation and Epigenesis. The Unknown Factor in Development We have now arrived at the furthest 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 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. 328 THEORIES OF INHERITANCE AND DEVELOPMENT 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 cell-characters, the traits of the multicellular body arising epigenetically by permu- tations and combinations of these characters. This conception cer- tainly tends to simplify 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 lies 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 this 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 Vries'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 any 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, we but throw the problem one stage further back, and, as Weismann himself has pointed out,^ 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 1 Gerin-plas//i , p. 14. 2 Evohition, Science and Culture^ p. 296. ^ Gerniinal Selection, January, 1S96, p. 284. PREFORMATION AND EPIGENESIS 329 epigencsis has now arrived at a stage where it has Httle meaning apart from the general problem of physical causality. What we know is that a specific kind of livdng 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 a 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 properties of the idioplasm involve 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 hundredfold 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 pre-existing 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 on which, as far as I can see, the study of the cell has not thus far thrown a ray of light. Whatever position we take on this question, the same difficulty is encountered; namely, the origin of that co-ordinated 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 w^e 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- 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 1 See Wolff, '95, and MuUer, '96. 2 See Wolff, '94, for an admirably clear and forcible discussion of this case. 330 THEORIES OF INHERITANCE AND DEVELOPMENT 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 play of physical forces upon it 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 life from the inorganic world. I 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 limit 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 repress the hope that step by step the way may yet be opened to an understanding of inheritance and development. LITERATURE. IX Boveri, Th. — Ein geschlechtlich erzeugter Orgaiiismus ohne miitterliche Eigen- schaften: Sitz.-Ber. d. Ges. f. Morph. unci P/iys. hi Miinchen, V. 1889. See iX'i.o ArcJi. f. Eiitiviii . 1895. Brooks, W. K.— The Law of Heredity. Ballimorc, 1883. Driesch, H. — Analytische Theorie der organischen Entwicklung. Leipzig, 1894. Herbst, C — Uber die Bedeutung der Reizphysiologie fur die kausale Auffassung von Vorgangen in der tierischen Ontogenese : BioL Centralb., XIV., XV. 1894-95. Hertwig, 0. — Altere and neuere Entwicklungs-theorieen. Berlin, 1892. Id._Urmund und Spina Bifida: Arch. mik. Anat.. XXXIX. 1892. Id. — iJber den Werth der ersten Fiu-chungszellen fur die Organbildung des Embryo: Arch. mik. Anat., XIA\. 1893. Id. — Zeit und Streitfragen der Biologie. Berlin, 1894. His, W. — Unsere Korperform und das physiologische Problem ihrer Entstehung. Leipzig, 1874. Loeb, J. — Untersuchungen zur physiologischen Morphologic: I. Heteromorphosis. Wiirzburg, \?>()i . II. Organbildung und Wachsthum. IVnrzdurg, iSg2. Id. — Some Facts and Principles of Physiological Morphology: J food's Moll Biol. Lectures, i S93 . Nageli, C. — Mechani.sch-physiologische Theorie der Abstammungslehre. MYin- chen u. Leipzig, 1884. Roux, W. — Uber die Bedeutung der Kernteilungsiiguren. Leipzig. 1883. PREFORMATION AND EPIGENESIS 33! Roux, W. — iJber das klinstliche Hervorbringen lialber Embryonen durch Zerstorung einer der beiden ersten Furchungskugeln, etc. : Vircliow's Archiv, 114. 1888. Sachs, J. — StofFund Form der Pfianzenorgaiie : Ges. Ah/iandlungeji, II. 1893. Weismann, A. — Essays upon Heredity, First Series. Oxford, 1891. Id. — Essays upon Heredity, Second Series. Oxford, 1892. Id. — Aussere Einflusse als Entwicklungsreize. Jena, 1894. Whitman, C. 0. — Evolution and Epigenesis : IVood^s Holl Biol. Lectures. 1894. Wilson, Edm. B. — On Cleavage and Mosaic-work: Arch, fiir Entwicklungsm., III. I. 1896. GLOSSARY rObsolete terms are enclosed in brackets. The name and date refer to the first use of the word ; LUDsoiete ^^^^^^^^^^t changes of meaning are indicated m the definition.] Achro'matin (see Chromatin), the non-staining substance of the nucleus, as opposed to chromatin: comprising the ground-substance and the hnm-netwo,k. (Flemmixg. 1880.) TAkaryo'ta] (see Karyota). non-nucleated cells. (Flemmixg. 1882.) kte cithal (ipriv. : A.'k.^o., the yolk of an egg), having httle or no yolk (applied to eo-o-sV (Balfour. 1880.) . . Amito'sls (see Mitosis), direct or amitotic nuclear division; mass-division ot the nuclear substance without the formation of chromosomes and amphiaster. AtipMalteM^aJx^?' on both sides; aar^p. a star), the achromatic tigure formed in mitotic cell-division, consisting of two asters connected by a spindle. (FOL. Amphipy'renin (see Pyrenin). the substance of the nuclear membrane. Amyloplasts '[Z^Xov, starch: TrXacrro., 7rU/xa, skin), the living protoplasm asserted to fomr a part of the cell-membrane in plants. (Wiesner, 18S6.) Der'matosomes (8e'p/xa, skin ; o-w/xa. body), the plasomes which form the cell-mem- brane. (WiESXER, 1886.) Determinant, a hypothetical unit formed as an aggregation of biophores, determin- ing the development of a single ceil or independently variable group ot cells. (Wels-maxx, 1 891.) [Deuthy'alosome] (8euT(epos), second ; see Hyalosome), the nucleus remaining in the egg after formation of the tirst polar body. (Vax Bexedex. 1883.) Deu'toplasm (SturCepos), .second ; TrAatr/xu, anything formed), yolk, lifeless food- matters deposited in the cytoplasm of the &%% ; opposed to -protoplasm."' (Van Benedex, 1870.) Directive bodies, the polar bodies. (Fr. Ml'LLER, 1848.) Directive sphere, the attraction-sphere. (Guigxard. 1891.) Dispermy, the entrance of two spermatozoa into the egg. 336 GLOSSARY Dispi'reme (see Spireme), that stage of mitosis in wliich eacli daugliter-nucleus has given rise to a spireme. (Flemming, 1882.) Dy 'aster (8m?. two; see Aster. 2). the double group of chromosomes during the anaphases of cell-division. (FlemiMING, 1882.) Egg-nucleus, the nucleus of the egg after formation of the polar bodies and before its union with the sperm-nucleus. Equivalent to the -female pronucleus'' of Van Beneden. (O. Hertwig, 1875.) Enchyle'ma (eV. in; x"'^os, juice), i. The more fluid portion of protoplasm, consisting of "hyaloplasma." (Hanstein, 1882.) 2. The ground-substance (cvtolvmph) of cytoplasm as opposed to the reticulum. (Carnov, 1883.) Biier'gid', the cell-nucleus together with the cytoplasm lying within its sphere of influence. (Sachs, 1892.) Equatorial plate, the group of chromosomes lying at the equator of the spindle during mitosis. (Van Beneden. 1875.) Erythro'philous (ipvOpo^, red; c^tAeu'. to love), having an especial affinity for red dves. (Auerbach.) Ga'mete (ya/xeVr/, wife: yajxh-q^. husband), one of two conjugating cells. Usually applied to the unicellular forms. G-em'mule (see Pangen). one of the ultimate supra-molecular germs of the cell assumed by Darwin. (Darwin. 1868.) [Gre'noblasts] (ycVos, se.x ; /^Aacrros. 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. (ATixoT. 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. (Weismann.) Heterole'cithal (eVepos. different; AeKt^os, yolk), having unequally distributed deutoplasm (includes telolecithal and centrolecithal). (Mark, 1892.) Heterotyp'ical mitosis (crepo?, different; see Mitosis), that mode of mitotic division in which the daughter-chromosomes remain united by their ends to form rings. (Flemming, 1887.) [Holoschi'sis] (0A05. whole ; or_;^i'^eti/. to split), direct nuclear division. Amitosis. (Flemming, 1882.) Homole'cithal (6/xo?. the same, uniform; AeKt^os, yolk), equivalent to alecithal. Having little deutoplasm, equally distributed, or none. (Mark. 1892.) Homceotyp'ical mitosis (o/xoto?. like: see Mitosis), a form of mitosis occurring in the spermatocytes of the salamander, differing from the usual tvpe only in the shortness of the chromosomes and the irregular arrangement of the daughter- chromosomes. (Flemming, 1887.) Hy'aloplasma (ilaAo?, glass; TrXdafxa, anything formed), i. The ground-sub- stance of the cell as distinguished from the granules or microsomes. [Hanstein, 1880] 2. The ground-substance as distinguished from the reticulum or "spon- gioplasm." (Levdig, 1885.) 3. The exoplasm or peripheral protoplasmic zone in plant-cells. (Pfeffer.) Hy'alosomes ( JaAo?. glass ; aw/xa, body), nucleolar-like bodies but slightly stained by eitlier nuclear or plasma stains. (Lukjanow. 1888.) [Hy'groplasma] (vyp6<;, wet; TrAarr/xa. something formed), the more liquid part of protoplasm as opposed to the firmer stereoplasm. (Nageli, 1884.) Id, the hypothetical structural unit resulting from the successive aggregation of biophores and determinants. Identified by Weismann as the chromomere, or chromatin-granule. (Weismann, 1891.) GLOSSARY 337 Idant, the hypothetical unit resulting from tlie successive aggregation of biophores, determinants, and ids. Identified by Weismann as the chromosome. (Weis- MANX, 1 891.) Id'ioblasts (tStos, one's own, /^Aucrro?. germ), the hypothetical ultimate units of the cell; the same as biophores. (O. Hertwig. 1893.) Id'ioplasm (I'Sios, one's own; 7rAao-/Aa, a thing formed), equivalent to the germ- plasm of Weismann. The substance, now generally identified with chromatin. which bv its inherent organization involves the characteristics of the species. The physical basis of inheritance. (Nageli, 18S4. ) Id'iosoine (18105, one's own; awfxa, body), the same as idioblast or plasome. (WaiTMAX. 1893.) Interfilar substance, the ground-substance of protoplasm as opposed to the thread-work. (Flemming, 1882.) Interzonal fibres ('• Filaments reunissants '' of Van Beneden. " Verbindungs- fasern " of Flemming and others). Those spindle-fibres that stretch between the two groups of daughter-chromosomes during the anaphase. Equivalent in some cases to the central spindle. (Mark., iS8r.) Iso'tropy (i,'cros, equal; rpoTrr;, a turning), the absence of predetermined axes (as applied to the egg). (Pfluger, 1883.) [Ka'ryaster] {Kapvov, nut, nucleus ; see Aster. 2). the star-shaped group of chromo- somes in mitosis. Opposed to cytaster. (Flem.mixg, 1882.) Karyenchy'ma (Kapvov, nut. nucleus; ev, in; x^^/x-o's, juice), the "nuclear sap." (Flemmixg, 1882.) Karyokine'sis (Kapvov, nut, nucleus; KtVr;o-ts, change, movement), the same as mitosis. (Schleicher. 1878.) [Karyoly'ma], the "karyolytic" (mitotic) figure. (Auerbach, 1876.) Ka'ryolymph. The nuclear sap. (Hackel. 1891 .) [Karyo'lysis] (Kapvov. nut, nucleus; Avert?, dissolution), the supposed dissolution of the nucleus during cell-division. (Auerbach, 1874.) [Karyoly'tic figure] (see Karyolysis). a term applied by Auerbach to the mitotic figure in living cells. Believed by him to result from the dissolution of the nucleus. (Auerbach. 1874.) Karyomi'crosome (see Microsome), the same as nucleo-microsome. Ka'ryomite. the same as chromosome [? Schiefferdecker]. Karyomi'tome (Kapvov, nut. nucleus; p.LTo}p.a, from /AtVos, a thread), the nuclear as opposed to the cytoplasmic thread-work. (Flemmixg. 1882.) Karyomito'sis (Kapvov, nut. nucleus; see Mitosis), mitosis. (Flemmixg, 1882.) Ka'ryon (Kapvov. nut. nucleus), the cell-nucleus. (Hackel. 1891.) Ka'ryoplasm ( Kaovoi/. nut. nucleus ; TrAao-jua. a thing formed), nucleoplasm. The nuclear as opposed to the cytoplasmic substance. (FLE^^MIXG. 1882.) Ka'ryosome {Kapvov, nut. nucleus; o-w/xa, body), i. Nucleoli of the "net-knot" type, staining with nuclear dyes, as opposed to plasmosomes or true nucleoli. (Ogata, 1883.) 2. The same as chromosome. (Platxek. 1886.) 3. Caryo- some. The cell-nucleus. (Wata.se, 1894. ) [Karyo'ta] {Kapvov, nut. nucleus), nucleated cells. (Fle.mmixg. 1882.) Karyothe'ca (Kapvov, nut. nucleus; 6y]Kr], case. box), the nuclear membrane. (H.ACKKL. 1S91.) Ki'noplasm (klvuv, to move; irXaiTp-'i, a thing formed), equivalent to archoplasm ; opposed ])y Strasburger to the '• trophoplasm " or nutritive plasm. (Stras- burger, 1892.) [Lanthanin] {kavOavw, to conceal), equivalent to oxychromatin. (Heidex- haix, 1892.) z 338 GLOSSARY Leucoplas'tids (Xet-Kos, white; TrAaoros, form), the colourless plastids of plant- cells from which arise the starch-formers (amyloplastids), chloroplastids, and chromoplastids. (Schimper, 1883.) Li'nin (linum, a linen thread), the substance of the "achromatic" nuclear reticulum. (Schw'ARZ. 1887.) Maturation, the final stages in the development of the germ-cells. More spe- cifically, the processes by which the reduction of the number of chromosomes is effected. Metakine'sis (see Metaphase) duera, beyond {i.e. further) ; K^vvrjcn^, movement), the middle stage of mitosis, when the chromosomes are grouped in the equa- torial plate. (Flemming, 1882.) Metanu'cleus, a term applied to the egg-nucleus after its extnision from the germinal vesicle. (Hacker, 1892.) Met'aphase, the middle stage of mitosis during which occurs the splitting of the chromosomes in the equatorial plate. (Strasburger, 1884.) Met'aplasm (/xera. after, beyond; TrAacr/Lia, a thing formed), a term collectively applied to the lifeless inclusions (deutoplasm, starch, etc.) in protoplasm as op- posed to the living substance. (Hanstein, 1880.) Micel'la, one of the ultimate supra-molecular units of the cell. (Nageli, 1884.) Microcen'trum. the dynamic centre of the cell, consisting of one or more centro- somes. (Heidenhain. 1894.) Mi'cropyle (^i/cpds, small; ttvXt], orifice), the aperture in the egg-membrane through which the spermatozoon enters. [First applied by TuRPiN, in 1806, to the opening through which the pollen-tube enters the ovule, t. Robert Brown.] Mi'crosome (/xtKpo?. small ; aw/xa, body), the granules as opposed to the ground- substance of protoplasm. (Hanstein, 1880.) Middle-piece, that portion of the spermatozoon lying behind the nucleus at the Iwse of the flagellum. (Schweigger-Seidel, 1865.) Mid-body (•• Zwischenkbrper"). a body or group of granules, probably comparable with the cell-plate in plants, formed in the equatorial region of the spindle during the anaphases of mitosis. (Flemming, 1890.) Mi'tome (/Atrcofta, from /aitos, a thread), the reticulum or thread-work as opposed to the ground-substance of protoplasm. (Flemming, 1882.) [Mitoschi'sis] du-tro?, thread; cr;>^t'^etv, to split), indirect nuclear division; mito- sis. (Fle.mming, 1882.) Mito'sis (|U,tTo?. a thread), indirect nuclear division typically involving: a. the formation of an am]3hiaster; ^, conversion of the chromatin into a thread (spireme) ; c, segmentation of the thread into chromosomes ; d, splitting of the chromosomes. (Flemming, 1882.) Mi'tosome (ixlto?, a thread ; (rw/xa, body), a body derived from the spindle-fibres of the secondary spermatocytes, giving rise, according to Platner, to the mid- dle-piece and the tail-envelope of the spermatozoon. Equivalent to the Neben- kern of La Valette St. George. (Platner, 1889.) Nebenkern (Paranucleus), a name originally applied by Blitschli (1871) to an extranuclear body in the spermatid; afterwards shown by La Valette St. George and Platner to arise from the spindle-fibres of the secondary spermatocyte. Since applied to many forms of cytoplasmic bodies (yolk-nucleus, etc.) of the most diverse nature. Nuclear plate, i. The equatorial plate. (Strasburger. 1875.) 2. The parti- tion-wall which sometimes divides the nucleus in amitosis. Nucleic acid, a complex organic acid, rich in phosphorus, and an essential constituent of chromatin. GLOSSARY 339 Nuclein, the chemical basis of chromatin ; a compound of nucleic acid and albumin. (MiESCHER, 1874.) Nucleo-albumin, a nuclein having a relatively high percentage of albumin. Distinguished from nucleo-proteids by containing paranucleic acid wliich yields no xanthin-bodies. Nucleochylema (x^Aos, juice), the ground-substance of the nucleus as opposed to that of the cytoplasm. ( Strasburger, 1882.) Nucleohy'aloplasma (see Hyaloplasm), the achromatic substance (linin) in which the chromatin-granules are suspended. (Strasburger, 1882.) Nucleomi'crosomes (see Microsome), the nuclear (chromatin) granules as opposed to those of the cytoplasm. (Strasburger, 1882.) Nu'cleoplasm. i. The reticular substance of the (egg-) nucleus. (Van Benedex, 1875.) -• "^^^^ substance of the nucleus as opposed to that of the cell-body or cytoplasm. (Strasburger, 1882.) Nucleo-pro'teid, a nuclein having a relatively high percentage of albumin. May be split into albumin and true nucleic acid, the latter yielding xanthin-bodies. CEdematin {oihr]ixa, a swelling), the granules or microsomes of the nuclear ground- substance. (Reixke. 1893.) O'ocyte (Ovocyte), (cJdi/. egg; Kt'ro?, hollow (a cell)), the ultimate ovarian egg before formation of the polar bodies. The primary oocyte divides to form the first polar bodv and the secondary oocyte. The latter divides to form the second polar body and the mature egg. (Boveri. 1891.) Oogen'esis, Ovogenesis (wdv, egg; yeVeo-is, origin), the genesis of the egg after its origin by division from the mother-cell. Often used more specifically to denote tlie process of reduction in the female. Oogo'nium, Ovogonium (cJdi'. egg ; yovrj. generation), i. The primordial mother- cell from which arises the egg and its follicle. (Pfluger.) 2. The descendants of the primordial germ-cell which ultimately give rise to the oocytes or ovarian eggs. (Boveri, 1891.) Ookine'sis (cJdi/, egg; KLvrjcn?. movement), the mitotic phenomena of the egg dur- ing maturation and fertilization. (Whitman, 1887.) O'vocentre. the egg-centrosome during fertilization. (FOL. 1891.) Oxychro'matin (ofws, acid ; see Chromatin), that portion of the nuclear substance stained by acid aniline dyes. Equivalent to -linin" in the usual .sense. (Heidenhain, 1894.) Pangenesis (7r5s (Trai/-), all; yeVecrt?, production), the theory of gemmules, ac- cording to which hereditary traits are carried by invisible germs thrown off by the individual cells of the Ixidy. (Darwin. 1868.) Pangeas (ttSs (vrav-), all; -yei'T^?. producing), the hypothetical ultimate supra- molecular units of the idioplasm, and of the cell generally. Equivalent to gemmules, micellae, idioblasts. biophores. etc. (De Vries. 1889.) Panmeri'stic (-rrav. all; /aepo?. part), relating to an ultimate protoplasmic structure consisting of independent units. See Pangen. Parachro matin (see Chromatin), the achromatic nuclear substance (linin of Scliwarz) from which the spindle-fibres arise. (Pfitzner. 1883.) Parali'nin (see Linin), the nuclear ground-substance or nuclear sap. (ScHWARZ, 1887.) Parami'tome (see Mitome). the ground-substance or interfilar substance of pro- toplasm, opposed to mitome. (Fle.m.mixc;. 1892.) Paranu'clein (see Nuclein). the .substance of true nucleoli or plasmosomes. Pyrenin of Schwarz. (O. Hertwtg, 1878.) Applied by Kossel to "nucleins'" derived from the cytoplasm. These are compounds of albumin and paranucleic acid which yields no xanthin-bodies. 340 GLOSSARY Par'aplasni {Trapd, beside; TrAacr/xa, something formed), the less active portion of the cell-substance. Originally applied by Kupffer to the cortical region of the cell (exoplasm), hut now often applied to the ground-substance. (Kupffer, I875-) Per'iplast (TrepL around ; TrAacrros, form), a term somewhat vaguely applied to the attraction-sphere. The term daughter-periplast is applied to the centrosome. (\'FjnovsKV, 1888.) Plas'mosome (TrXdcrfxa, something formed (/.c. protoplasmic) ; crw/xa. body), the true nucleolus, distinguished by its affinity for acid anilines and other •' plasma- stains."' (Ogata, 1883.) Pla'sonie (TrAacr/xa, a thing formed; croj/xa. body), the ultimate supra-molecular vital unit. See Biophore. Pangea. (Wiesner, 1890.) Plas'tid (TrAacTTos, form), i. A cell, whether nucleated or non-nucleated. (Hackel, 1866.) 2. A general term applied to permanent cell-organs (chloroplasts, etc.) other than the nucleus and centrosome. (Schimper. 1883.) Plas'tidule. the ultimate supra-molecular vital unit. (Elssberg, 1874; Hackel, 1876.) Plas'tin, a term of vague meaning applied to a substance related to the nucleo- proteids and nucleo-albumins constituting the linin-netwoik (Zacharias) and the cytoreticulum (Carnoy). (Reinke and Rodewald, 1881.) Pluriva'lent {plus, more ; 7'alere. to be worth), applied to chromatin-rods that have the value of more than one chromosome sensn strictii. (Hacker. 1892.) Polar bodies (Polar globules), two minute cells segmented off from the ovum before union of the germ-nuclei. (Disc, by Carus, 1824; so named by Roiiix. 1862.) Polar corpuscle, the centrosome. (Va\ Bexeden. 1876.) Polar rays (Polradien). a term sometimes applied to all of the astral ravs as opposed to the spindle-fibres, sometimes to the group of astral ravs opposite to the spindle-fibres. Pole-plates (End-plates), the achromatic spheres or masses at the poles of the spindle in the mitosis of Protozoa, probably representing the attraction-spheres. (R. Hertwig. 1S77.) Polyspermy, the entrance into the ovum of more than one spermatozoon. Prochro 'matin (see Chromatin), the substance of true nucleoli, or plasmosomes. Equivalent to paranuclein of O. Hertwig. (Pfitzner, 1883.) Pronuclei, the germ-nuclei during fertilization; /.t'.. the egg-nucleus (female pro- nucleus) after formation of the polar bodies, and the sperm-nucleus (male pro- nucleus) after entrance of the spermatozoon into the egg. ( Vax Bexeden, 1875.) [Prothy'alosome] (see Hyalosome). an area in tlie germinal vesicle {oi Ascan's) by which the germinal spot is surrounded, and which is concerned in formation of the first polar body. (Van Bexeden, 1S83.) Pro'toblast (Trpwro?. first : ^Aaoros, a germ), a naked cell, devoid of a membrane. (KOLLIKER.) Pro'toplasm (TrpoiTos. first ; TrAacr/xa. a thing formed or moulded), i. The living substance of the cell, comprising cytoplasm and karyoplasm. (Purkyxe. 1840; H. VON MoHL. 1846.) 2. The cytoplasm as opposed to the karyoplasm. Pro'toplast (TrpwTo?. first; TrAaord?. formed), i. The protoplasmic body of the cell, including nucleus and cytoplasm, regarded as a unit. Nearly equi\-alent to the energid of Sachs. (Han.steix. 1880.) 2. Used by some authors synony- mous! v with plastid [Pseudochro'matin] (see Chromatin), the same as prochromatin. (Pfitz.ner, 1886.) GLOSSARY 341 Pseudonu'cleiii (see Nuclein). the same as the paranuclein of Kossel. (Ham- MARSTKX. 1894.) Pseudo-reduction, the preliminary halving of the number of chromatin-rods as a prelude to the formation of the tetrads and to the actual reduction in tlie number of chromosomes in maturation. (RuCKERT, 1894.) Pyre'nin {-n-vpi'^v. the stone of a fruit: i.e. relating to the nucleus), the substance of true nucleoli. Equivalent to the paranuclein of Hertwig. (Schwarz, 1887.) Pyre'noid {Trvprjv, the stone of a fruit; like a nucleus), colourless plastids (leuco- plastids ). occurring in the chromatophores of lower plants, forming centres for the formation of starch. (Sch.mitz, 1883.) Reduction, the halving of the number of chromosomes in the germ-nuclei during maturation. Sertoli-cells. the large, digitate, supporting, and nutritive cells of tlie mammalian testis to which the developing spermatozoa are attached. (Equivalent to - sper- matoblast"' as originally used by vox Ebxer, 1871.) Spermatid {cnrepixa, seed), the tinal cells which are converted without further division into spermatozoa ; they arise by division of the secondary spermatocytes or "Samenmiitterzellen." (La Valette St. George. 1886.) Sper'matoblasts {aTrep/xa, seed; /iJAaards. germ), a word of vague meaning, originally applied to the supporting cell or Sertoli-cell. from which a group of spermatozoa was supposed to arise. By various later writers used synonymously with spermatid, (vox Ebxer, 1871.) Sper'matocyst (o-7re'p/i,a, seed ; Kuort?, bladder), originally applied to a group of sperm-producing cells ("spermatocytes "), arising by division from an •• Ursa- menzelle"' or "spermatogonium." (La Valette St. George. 1876.) Spermatocyte ( o-Trepjua, seed ; kv'tos, hollow^ (a cell)), the cells arising from the spermatogonia. The primary spermatocyte arises by growth of one of the last generation of spermatogonia. By its division are formed two secondary sper- matocytes, each of which gives rise to two spermatids (ultimately spermatozoa). (La Valette St. George. 1876.) [Spermatogem'ma] {cnr(.pp.a. seed; gemma, bud), nearly equixalent to spermato- cyst. Differs in the absence of a surrounding membrane. [In mammals. La Valette St. George, 1878.] Spermatogen'esis {(nvipp-a, seed; yeVecns. origin), the phenomena involved in the formation of the spermatozoon. Often used more specifically to denote the process of reduction in the male. Spermatogo'nium (" Ursamenzelle " ) {(Jiripjxa, seed; yovr/. generation), the descendants of the primordial germ-cells in the male. Each ultimate sper- matogonium typically gives rise to four spermatozoa. (La Valette St. George. 1876.) Spermatome'rites (a-n-epixa, seed; /xepo?. a part), the chromatin-granules into which the sperm-nucleus resolves itself after entrance of the spermatozoiin. (In Petroiiiyson. Bohm. 1887.) Sper'matosome ( o-Trep/xu. seed; a^p-a, body), the same as spermatozoon. (La \\vlette St. George, 1878.) Spermatozo'id (see Spermatozoon), the ciliated paternal germ-cell in plants. Tlie word was first used by von Siebold as synonymous with spermatozoon. Spermatozoon {(j-Kkppxu seed; ^woi/, animal), the paternal germ-cell of animals. (Lei:l\vk\hoek. 1677.) Sperm-nucleus, the nucleus of the spermatozoon ; more especially applied to it after entrance into the egg before its union with the egg-nucleus. In this sense equivalent to the -male pronucleus" of Van Beneden. (O. Hertwtg. 1875.) Sper'mocentre. the sperm-centrosome during fertilization. (FoL, 1891.) 342 GLOSSARY Spi'reme ((T7reip>7jaa. a thing wound or coiled ; a skein), the skein or "Knauel" stage of the nucleus in mitosis, during which the chromatin appears in the form of a thread, continuous or segmented. (Flemming. 1882.) Spon'gioplasm {(nroyyiov, a sponge; 7rAacr/x,a, a thing formed), the cytoreticulum. (Levdig, 1885.) Ste'reoplasm {uTepto'i, solid), the more solid part of protoplasm as opposed to the more fluid " hygroplasm." (Nageli, 1884.) Substantia hyalina, the protoplasmic ground-substance or "hyaloplasm." (Levdig. 1885.) Substantia opaca, the protoplasmic reticulum or " spongioplasm." (Leydig, 1885.) Te'loblast (re'Aos, end; /^AacrTo?, a germ), large cells situated at the growing end of the embryo (in annelids, etc.), which bud forth rows of smaller cells. (Whitman, Wilson, 1887.) Telole'cithal (reAos. end ; AeKt^os. yolk), that type of ovum in which the yolk is mainlv accumulated in one hemisphere. (Balfour, 1880.) Telophases. Telokine'sis (reAos, end), the closing phases of mitosis, during which the daughter-nuclei are re-formed. (Heidenhain, 1894.) To'noplasts (two?, tension ; TrAao-ro?, form), plastids from which arise the vacuoles in plant-cells. (De Vries, 18S5.) Trophoplasni (rpocfiy, nourishment ; irXdafxa) . 1 . The nutritive or vegetative substance of the cell, as distinguished from the idioplasm. (Nageli, 1884.) 2. The active substance of the cytoplasm other than the •• kinoplasm " or archoplasm. (Strasburger, 1892.) Tro'phoplasts (rpocfyy]. nourishment; TrAao-ro?, form), a general term, nearly equiv- alent to the "plastids" of Schimper, including '■ anaplasts " (amyloplasts), "autoplasts" (chloroplasts), and chromoplasts. (A. Meyer, 1882-83.) Yolk-nucleus, a word of vague meaning applied to a cytoplasmic body, single or multiple, that appears in the ovarian egg. [Named "Dotterkern" bv Carus, 1850.] Zy'gote or Zy'gospore (^uyoV, a yoke), the cell produced by the fusion of two conjugating cells or gametes in some of the lower plants. GENERAL LITERATURE-LIST The following list includes only the titles of works actually referred to in the text and those immediately related to them. For more complete bibliography the reader is referred to the literature-lists in the special works cited, especially the following. For reviews of the early history of the cell-theory see Remak"s Untersiiduuigen C50-'55). Huxley on the Cell-theory ('53), and Tyson's Cell-doetrine (^8). An exhaustive review of the earlier literature on protoplasm, nucleus, and cell- division will be found in Flemming's Zellsubstanz ('82), and a later review of theories of protoplasmic structure in Butschli's Protoplasnia ('92). The earlier work on mitosis and fertilization is very thoroughly reviewed in Whitman's Clep- sine C78). Fol's Henogenie ('79), and Mark^s Umax ('81). For more recent general literature-lists see especially Hertwig's Zelle iind Gewebe ('93), Yves Delage ("95), Henneguy's Cellule ('96), and the admirable reviews by Flemming, Boveri, Rlickert, Roux, and others in Merkel and Bonnet's Ergebnisse ("91 -'94). The titles are arranged in alphabetical order, according to the system adopted in Minot's Human Embryology. Each author's name is followed by the date of publi- cation (the first two digits being omitted, except in case of works published before the present century), and this by a single number to designate the paper, in case two or more works were published in the same year. For example, Boveri, Th., '87, 2, denotes the second paper published by Boveri in 1S87. In order to economize space, the following abbreviations are used for the journals most frequently referred to : — ABBREVIATIONS A. A. Anatomischer Anzeiger. A. B. Archives de Biologic. A. A. P. Archiv fur Anatomic und Physiologic. A. in. .4. Archiv fiir mikroscopische Anatomic. A. Entm. Archiv fur Entwicklungsmcchanik. B. C. Biologischcs Centralblatt. C. R. Comptes Rendus. /. i\I. journal of Morphology. J. Z. Jenaischc Zeitschrift. M. A. Miiller's Archiv. M.J. Morphologisches Jahrbuch. Q. J. Quarterly Journal of Microscopical Science. • Z. A. Zoologischer Anzeiger. Z. ru. Z. Zeitschrift fiir wissenschaftliche Zoologie. ACQUA, "91. Contribuzione alia conoscenza della cellula vegetale : Malpighia, V. — Altmaii. R., "86. Studicn liber die Zelle. 1. : Leipzig. — Id., '87. Die Genese der Zcllen: Leipzig. — lA., '89. Uber Nucleinsaure : A. A. /'., p. 524. — Id., '90. "94. Die Elementarorganismen und ihre Beziehung zu den Zellen : Leipzig. — Amelung, E.. '93. IMjer mittlere Zellgrcisse : Elora. p. 176. — Arnold, J., '79. 343 344 GENERAL LITERATURE-LIST Uber feinere Struktur der Zellen, etc. : Virclunv^s Arch., 1879. (See earlier papers.) — Aiierbach. L.. '74. Organologische Studien : Ii?-eslaii. — Id.. "91. Uber einen se.xuellen Gegensatz in der Chromatophilie der Keim.substanzen : Sitziingsber. der KonigL preiiss. Akad. d. W/ss. Berlin, XXXV^ VON BAER. C. E., '28, '37. Uber Entwickelung.sgescliiclite der Thiere. Beo- bachtung und Relie.xioii : I. Koiiigsberg, 1828; II. 1837. — Id., '34. Die Metamor- phose des Eies der Batrachier: MYiUer^s Arch. — Balbiani. E. G., "64. Sur la constitution du germe dan.s I'oeuf animal a\ant la fecondation : C. R., LVIII. — Id., '76. Sur les phcnomenes de la division du noyau cellulaire : C. R , XXX., Octo- ber, 1876. — Id., "81. Sur la structure du noyau des cellules salivares chez les larves de Chironomus : Z. .1.. 1881, Nos. 99, 100. — Id., "89. Recherches experimen- tales sur la merotomie des Infusoires cilies : Reciieil ZooL Suisse, Januarv, 1889. — Id., '91, 1. Sur les regenerations successives du peristome chez les Stentors et sur le role du noyau dans ce phenomene : Z. A., 372, 373. — Id., "91, 2. Sur la struc- ture et division du noyau chez les Spirochona gemmipara : Ann. d. Micrographie. Id., '93. Centrosome et Dotterkern : your)i. de Tauat . et de la phvsiol ., XXIX. — — Balfour, F. M.. "80. Comparative Embryology: I. 1S80. — Ballowitz, ■88-"91. Untersuchungen iiber die Struktur der Spermatozoen : i. (birds) A. in. A., XXXII., 1888; 2. (insects) Z. iv. Z., LX., 1890; 3. (fishes, amphibia, reptiles) .-/. ///. A. XXXVI., 1890: 4. (mammals) Z. iv. Z., 1891. — Id.. "89. Fibrillare Struktur und Contractilitat : Arch. ges. Rhys., XLVI. — Id., "91, 2. Die innere Zusammensetz- ung des Spermatozoenkopfes der Saugetiere : Centralb. f. Rhys., V. — Id., "95. Die Doppelspermatozoa der Dytisciden : Z. «'. Z.. XLV., 3. — "Van Bambeke. C, "93. Elimination d'elements nucleaires dans I'oeuf ovarien de Scorp;tna scrofa : A. B., XIII. I. — De Bary. "58. Die Conjugaten. — Id.. "62. Uber den Bau und das Wesen der Zelle : RIora, 1862. — Id.. "64. Die Alycetozoa : 2d Ed., Leip- zig.— Barry, M. Spermatozoa observed within the JMammiferous Ovum: Rhil. Trans., 1843. — Beale, Lionel S.. "61. On the Structure of Simple Tissues of the Human Body: London. — Bechamp and Ester, '82. De la constitution elemen- taire des tissues : Montpellier. — Belajeff, "94, 1. Zur Kenntniss der Karvokinese bei den Pflanzen : Flora, 1894. Erganzungsheft. — Id., "94, 2. Uber Bau und Entwicklung der Spermatozoiden der Pflanzen: Flora, LIV. — Benda. C, "87. Untersuchungen iiber den Bau des funktionirenden Samenkenkalchens einiger Sau- gethiere : A. ni. A. — Id., '93. Zellstrukturen und Zelltheilungen des Salaman- derhodens : I'crh. d. Anal. Ges., 1893. — "Van Beneden, E., '70. Recherches sur la composition et la signification de I'oeuf: Ulem. cour. de TAc. roy. d. S. de Belgique, 1870. — Id., '75. La maturation de Toeuf, la fecondation et les premieres phases du developpement embryonnaire des mammiferes d"apres des recherches faites chez le lapin : Bull. Ac. roy. de Belgique, XI. — Id., "76, 1. Recherches sur les Dicyemides: Bull. Acad. Roy. Belgique, XLI., XLII. — Id., '76, 2. Contribution a I'histoire de la vesicule germinative et du premier noyau embryon- naire : Ibid., XLI.: also Q.J., XVI. — Id., '83. Recherches sur la maturation de I'oeuf, la fecondation et la division cellulaire: A. B., \W . — "Van Beneden and Julin, '84. 1. La segmentation chez les Ascidiens et ses rapports av^c Torgani- sation de la larve : Ibid., V. — Id., "84. 2. La spermatogenese chez I'Ascaride megalocephale : Bull. Acad. Roy. Belgique, 3me ser., VII. — "Van Beneden, E., et Neyt, A.. "87. Nouvelles recherches sur la fecondation et la division mitosique chez TAscaride megaloctfphale : Ibid.. 1887. — Bergh, R. S., "94. Vorlesungen Uber die Zelle und die einfachen Gewebe : Wiesbaden. — Id., "95. Uber die relativen Theilungspotenzen einiger Embryonalzellen : . /. Entin., II., 2. — Bernard, Claude. Legons sur les Phenomenes de la Vie: ist Ed. 1878, 2d Ed. 1885, Raris. — Ber- tliold, G., "86. Studien iiber Protoplasma-mechanik : Leipzig. — Bickford, E. E., GENERAL LITERATURE-LIST 345 '94. Notes on Regeneration and Heteromoqshosis of Tubulaiian Hydroids : /. M., IX., 3. — Biondi, D.. '85. Die Entwicklung der Spermatozoiden : A. m. A., XXV. — Blanc, H.. '93. Etude sur la fccondation de I'neuf de la truite : Ber. Natiir- forsch. Ut's. zii FrL-il>i/>\^\ VIII. — Blochmann, F., '87.2. Liber die Richtungs- korper bei Insekteneiern : M. J.. XII. — Id., "88. ('ber die Richtungskorper bei imbefruclitet .sich entwickelnden Insekteneiern: Verli. natiirh. med. Ver. Heidel- berg, N. F., IV.. 2. — Id.. '89. L'ber die Zahl der Riclitungskorper bei befruchteten und unbefruchteten Bieneneiern : J/. J. — Id., '94. Cber die Kerntheilung bei Euglena : B. C, XIV. — Bohm. A.. '88. Uber Reifung und Befruchtung des Eies von Petromyzon Planeri : A. iii. A., XXXII. — Id.. '91. Die Befruclitung des Forelknieies : Sitz.-Be?'. d. Ges.f. Morph. it. Phys. MYiiiclwn. VII. — Boll, Fr., '76. Das Princip des Wachstliums : Berlin. — Bonnet, C, 1762. Considerations sur les Corps organises: .linsterdain. — Born. G.. "85. ('ber den Einfluss der Schwere auf das Froschei : A. in. A.. XXIV'. — Id.. '94. Die Structur des Keim- blaschens im Ovarialei von Triton taeniatus : A. ;//. A., XLIII. — Bonrne, G. C, '95. A Criticism of tlie Cell-theory ; being an answer to Mr. Sedgwick's Article on the Inadequacy of the Cellular Theory of Development: O. y., XXXVIII., i. — Boveri, Th., "86. Uber die Bedeutung der Richtungskorper: Sitz.-Ber. Ges. Morph. 11. FJiys. Miincheii. II. — Id.. '87.^. Zellenstudien, Heft I. : J. Z., XXL — Id., '87, 2. Uber die Befruchtung der Eier von Ascaris niegalocephala : .Sitz.-Ber. Ges. Morph. Phys. Miuuheii, III. — Id., '87, 2. Uber den Anteil des Spermatozoon an der Teilung des Eies: Sitz.-Ber. Ges. Morph. Phys. MYinchieiu III., 3- — Id., '87. 3. Uber Dififerenzierung der Zellkerne wahrend der Furchung des Eies von Ascaris meg.: A. A., 1887. — Id., '88, 1. Uber partielle Befruchtung: Sitz.-Ber. Ges. Morph. Phys. Miincheiu IV.. 2.— Id., '88, 2. Zellenstudien,'' II. : J. Z., XXII. — Id., "89. Ein geschlechtlich erzeugter Organismus ohne nflitterliche Eigenschaften : Sitz.-Ber. Ges. Morph. Phys. M'lhiehen,^ . Trans, in Am. Nat., March, '93.— Id., '90. Zellenstudien, Heft III.: J.Z., XXIV. —Id., '91. Be- fruchtung: Merkel imd BonnePs Ergebiiissc, I. — Id.. '95, 1. Uber die Befruch- tungs-und Entwickelungsfahigkeit kernloser Seeigel-Eier, etc: A. Entiii., II., 3. — Id., '95, 2. Uber das Verhalten der Centrosomen bei der Befruchtung des Seeige- leies, nebst allgemeinen Bemerkungen uber Centrosomen und Verwandtes : Verh. d. Physikal.-iiied. Gesellschaft zu Wiirzburg, N. F., XXIX., i. — Braem, F., '93. Des Prinzip der organbildenden Keimbezirke und die entwicklungsmechanischen Studien von H. Driesch : B. C, Xlll., 4, 5. — Brandt, H., "77. Uber Actino- sphxrium lOichhornii : Dissertation, Halle, i?,J7 . — Brass. A.. "83-4. Die Organi- sation der thierischen Zelle : Halle. — Brauer. A., '92. Das Ei von Branchipus Grubii von der Bildung bis zur Ablage : Abh. preiiss. A/cad. ll'iss., '92. — Id., '93. 1. Zur Kenntniss der Reifung des parthenogenetisch sich entwickelnden Eies von Artemia Salina : A. m. A., XLIII. — Id.. '93, 2. Zur Kenntniss der Spermato- genese von Ascaris megalocephala : ./. ni. A., XLII. — Id.. '94. Uber die En- cystierung von Actinosphirrium Eichhornii : Z. w. Z., LVllL, 2. — Braus. '95. Uber Zellteilung und Wachstum des Tritoneies: J. Z., XXIX. — Brooks. "W. K., '83. The Law of Heredity: Baltimore. — 'Rxovvw, H. H., "85. On Spermato- genesis in the Rat: Q. J., XXV. — Brown, Robert. "33. Observations on the Organs and Mode of Fecundation in Orchides and AsclepiadecX : Trans. Linn. Sac, 1833. — Brunn. M. von, '89. Beitrjige zur Kenntniss der Samenkiirper und ilircr Entwickelung bei Vogeln und Saugethieren : A. m. A., XXXIII. — Briicke, C. '61. Die Elementarorganismen : Wiener Sitzber., XLIV., 1861. — Burger. O., '91. Uber Attractionsspharen in den Zellkorpern einer Leibes- fliissigkeit : A. A.. VI. — Id.. 92. Was sind die Attractionsspharen und ihre Centralkdrper ? A. .L. 1892. — De Bruyne, C, '95. La sphere attractive dans les cellules fixes du tissu conjonctif : Bull. Acad. Sc. de Belgique, XXX. — 34b GENERAL LITERATURE-LIST Biitschli, O.. "73. Beitrage zur Kenntniss der freilebenden Nematoden : Nova acta acad. Car. Leopold, XXXVI. — Id., '75. Vorlaufige Mitteilungen iiber Unter- suchuiigen betrefifend die ensten Entwickelungsvorgange im befmchteten Ei von Nematoden und Schnecken : Z. w. Z.. XXV. — Id., "76. Studien iiber die ersten Entwickelungsvorgange der Eizelle. die Zellteilung und die Konjugation der Infu- sorien : AbJi. des Senckeiib. A^atiirforscher-Ges., X. — Id., "91. Uber die soge- nannten Centralkorper der Zellen und ilire Bedeutung : I'erJi. Au-iturhist. Med. Ver. Heidelberg, 1891. — Id., '92. 1. Uber die kiinstliche Nachalnnung der Karyoki- netischen Figuren : Ibid., N. F., V. — Id.. "92. 2. Untersuclumgen iiber mikro- skopische Schaume und das Protoplasma (full review of literature on protoplasmic structure): Leipzig {Engeliuanii'). — Id.. "94. Vorlaufige Bericht iiber fortgesetzte Untersuchungen an Gerinnungsschaumen, etc. : l^erh. Nat iir hist. Ver. Heidelberg, V. CALKINS. G. N.. "95. 1. Observations on the Yolk-nucleus in the Eggs of Lumbricus: Trans. A^.V. Acad. Sci.. June. 1895. — Id.. "95. 2. The Spermato- o-enesis of Lumbricus : '7. M.. XL. 2. — Carnoy, J. B.. "94. La biologic cellulaire : Liege. — Id., '85. La cytodierese des Arthropodes : La Cellule A. — Id.. '86. La cytodierese de I'oeuf: La Cellule, III. — Id., '86. La vesicule germinative et les globules polaires chez quelques Nematodes: La Cellule, III. — Id., "86. La seg- mentation de Toeuf chez les Nematodes : La Cellule, III., i. — Calberla. E.. "78. Der BefiT.chtungsvorgang beim Ei von Petromyzon Planeri : Z. %u. Z., XXX . — Campbell, D. H., '88-9. On the Development of Pilularia globulifera : A/ui. Bot., II. — Castle. "W. B.. '96. The Early Embryology of Ciona intestinalis : Bull. Miis. Coinp. Ztfi>7., XXVII. , 7. — Chabry. L.. '87. Contributions a I'embryologie normale et pathologique des ascidies simples: Paris, 1887. — Chittenden. R. H., '94. Some Recent Chemico-physiological Discussions regarding the Cell : Ann. Nat., XXVIII., Feb., 1894.— Chun. C, '90. Uber die Bedeutung der direkten Zelltheilung : Sitzb. Schr. Fhysik.-Okon. Ges. Konigsberg, 1890. — Id., '92, 1. Die Dissogonie der Rippenquallen : Festschr. f. Lei/ckart, Leipzig, 1892. — Id., '92, 2. (In Roux, "92, p. 55): J'er/i. d. Anat. Ges., VI.. 1892. — Clapp, C. M.. '91. Some Points in the Development of the Toad-Fish: J. J/.. V. — Clarke. J. Jackson. '95. Ob- servations on various Sporozoa : Q. J., XXXVII., 3. — Cohn, Ferd., "51. Nach- trage zur Naturgeschichte des Protococcus pluvialis : Nova Acta, XXII. — Conklin, E. G., '94. The Fertilization of the Ovum : Biol. Led., Marine Biol. Lab., IVood's Holl, Boston, 1894. — Id., '96. Cell-size and Body-size: Rept. of Am. MorpJi. Sac, Science All.. ]^-^. 10, 1896. — Crampton, H. E.. '94. Reversal of Cleavage in a Sinistral Gasteropod : Ann. N. V. Acad. Sci., March. 1894. — Crampton and 'Wilson. '96. Experimental Studies on Gasteropod Development (H. E. Cramp- ton). Appendix on Cleavage and Mosaic-Work (E. B. Wilson) : A. Entm., III., i. DELAGE. "Y"VES, "95. La Structure du Protoplasma et les The'ories sur I'here- dite et les grands Problemes de la Biologie Generale : Paris. 1895. — Demoor, J., '95. Contribution a I'etude de la physiologic de la cellule (independance fonction- elle du protoplasme et du noyau) : A. B., XIII. — Dogiel. A. S.. "90. Zur Frage iiber das Epithel der Harnblase : A. m. A., XXXV. — Driesch. H. Entwicklungs- mechanische Studien: I.. II.. 1892. Z. w. Z, LIII.; III.-VI.. 1893, Z*?-/'^/.. LV. ; VII.- X., 1893: Mitt. Zool. St. Neapel, XL, 2.— Id., '94. Analytische Theorie der organischen Entwicklung : Leipzig. — Id.. '95. 1. Von der Entwickelung einzelner Ascidienblastomeren : A. Entni.A-. 3- — Driesch and Morgan. "95. 2. Zur Analysis der ersten Entwickelungs stadien des Ctenophoreneies : Ibid., II., 2. — Driiner, L., "94. Zur Morphologic der Centralspindel : J. Z, XXVIII. (XXI.). — Id., '95. Studien iiber den Mechanismus der Zelltheilung: Ibid., XXIX.. 2. — Diising, C, '84. Die Regulicrung des Geschlechtsverhailtnisses : Jena, 1884. GENERAL LITERATURE-LIST 347 VON EBNER, V.. '71. Untersuchungen uber den Bau der Samencanalchen und die Entwickluag der Spermatozoiden bei den Saiugethieren und beim Menschen : Inst. Phys. u. Hist. Graz., 1871 {Leipzig). — 1di.,QQ. Zur Spermatogenese bei den Saiugethieren: A. m. A., XXXI. — Ehrlich, P.. "79. Uber die specifischen Granulationen des Blutes : A. A. P. {Phys.), 1879, P- 573- — 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. Entin., II., I. — Eiigelmann, T. W., "80. Zur Anatomic und Physiologic der Flimmerzellcn : Arch.ges. Phys., XXIII. — von Erlanger, R.. "96, 1. Die ncuesten Ansichten liber die Zelltheilung und ihre Mechanik : Zool. Centralb., III., 2. — Id.. '96, 2. Zur Befmchtung des Ascariseies nebst Bemerkungen liber die Struktur des Protoplasmas und des Centrosomes : Z. A., XIX. — Id.. "96, 3. Neuere Ansichten liber die Stmktur des Protoplasmas : Zool. Centralb., III.. 8. 9. — Errara, "86. Eine fundamentale Gleichgewichtsbedingung organischen Zellen : Ber. Deutsch. Bot. Ges., 1886. — Id., "87. ZcUformcn und Seifenblasm : Tagebl. der 60 Versammlnng deiitscher Naturforscher und Aerzte zii Wiesbaden, 1887. FARMER, J. B.. "93. On nuclear division of the pollen-mother-ccll of Lilium Martagon: Ann. Bot., VII., 27.— Id., "94. Studies in Hcpatica? : Ibid., VIII., 29. — Id., "95. 1. tJber Kernteilung in Lilium-.Antheren, besonders in Bezug auf die Centrosomcnfrage : Flora, 1895. p. 57. — Farmer and Moore. "95. On the essential similarities e.xisting between the heterotype nuclear divisions in animals and plants : A. A., XL, 3. — Fiok, R., '93. ttber die Reifung und Befruchtung des Axolotleies : Z. w. Z., LVI., 4. — Fiedler. C, "91. Entwickelungsmechanische Studien an Echinodermeneier : Festschr. Xijgeli u. Ko Hiker. Zurich. 1891. — Field, G. "W., "95. On the Morphology and Physiology of the Echinoderm Spermatozoon : /. M.. XI. — Fischer. A., '94. Zur Kritik der Fixierungsmethoden der Granula : A. A., IX., 22. — Id. '95. Neue Beitrage zur Kritik der Fixiemngsmethoden : Ibid.,X. — Flem- ming, W., "79. Beitrage zur Kenntniss der Zelle und ihre Lebenserscheinuno-en. I. : A. >n. A., XVI. — Id., "79. Tber das Verhalten des Kerns bei der Zelltheilung, etc. : Virchow's Arch., LXXVII. — Id., "80. Beitrage zur Kenntniss der Zelle und ihrer Lebenserscheinungen, II. : A. ni. A., XIX. — Id., "81. Beitrage zur Kenntnis der Zelle und ihrer Lebenserscheinungen, III. : Ibid., XX. — Id., '82. Zellsubstanz, Kern und Zellteilung: Leipzig, 1882. — Id., "87. Neue Beitrage zur Kenntnis der Zelle : A. m. A., XXIX. — Id., '88. Weitere Beobachtungen liber die Entwickelung der Spermatosomen bei Salamandra maculosa: Ibid., XXXI. — Id., "91-4. Zelle, I.-IV. : Ergebn. Anat. 11. Enttvickhingsgesch. {Merkel and Bonnet), 1891-94. — Id. '91, 1. Attraktionsspharen u. Centralkorper in Gewebs- u. Wanderzellen : A. A. — Id., "91, 2. Neue Beitrage zur Kenntnis der Zelle, II. Teil : A. in. A., XXXVII. — Id., '95, 1. Uber die Struktur der Spinalganglienzellen : Verhandl. der anat. Gesellschaft in Basel, 17 April, 1895, p. 19. — Id., "95, 2. Zur Mechanik der Zell- theilung: A. m. A., XLVL — Floderus. M., '96. C'ber die Bildung der Follikel- hlillen bei den Ascidien : Z. iu. Z., LXL, 2. — Fol, H., '73. Die erste Entwickelung des Geryonideies : J. Z., VII. — Id., "75. Etudes sur le developpement des Mol- lusques. — Id., "77. Sur le commencement de I'henogenie chez divers animaux : Arch. Sci. i\at. et Phys. Geneve, L\'I1I. See also Arch. Zool. Exp., VI. — Id., '79. Recherches sur la fecondation et la commencement de rhenogenie; Mem. de la Soc. de physique et d'hist. nat., Geneve, XXVI. — Id., '91. Le Quadrille des Centres. Un episode nouveau dans I'histoire de la fecondation : Arch, des sci. phys. et nat., 15 Avril, 1891 ; also, /i. A., 9-10, 1891. — Foot, K., "94. Preliminary Note on the Maturation and Fertilization of Allolobophora : /. M., IX.. 3. '94. — Id., '96. Yolk-nucleus and Polar Rings: Ibid.,X\\.. i. — Frenzel, J.. '93. Die Mitteldarmdrlise des Flusskrebses und die amitotische Zelltheilung: A. m. A.., 348 GENERAL LITERArURE-LIST XLI. — Fromman, C, '65. Uber die Struktur der Bindesubstanzzellen des Rlickenmarks : Centrl. f. iiied. IJ'/ss.. III.. 6. — Id.. "75. Zur Lehre von der Structur der Zellen : J. Z.. IX. (earlier papers cited). — Id., "84. Untersuchungen liber Struktur, Lebenserscheinungen und Reactionen thierischer uiid pflanzlicher Zellen: /. Z., XVII. GALEOTTI, GINO, '93. Uber experimentelle Erzeugung von Unregelmassig- keiten des karyokinetischen Processes: Be/, siir patholog. Anat. u. s. Allg. Pathol., XIV.. 2. Jena, Fischer, 1893. — Gardiner. W.. "83. Continuity of Protoplasm in Vegetable Cells: Phil. Trans., CLXXIV. — Garnault. "88. '89. Sur les pheno- menes de la fecondation chez Helix aspera et Arion empiricorum : Zool. Anz., XI., XII. — Geddes and Thompson. The Evolution of Sex. — Gegenbaiir, C. '54. Beitrage zur naheren Kenntniss der Schwimmpolypen : Z. w. Z., V. — Van Gehnchten. A.. "90. Recherches histologiques sur Tappareil digestif de la larve de la Ptvchoptera contaminata: La Celliile.,V\. — Giard, A., "77. Sur la significa- tion morphologique des globules polaires : Revue scientifiquc, XX. — Id., '90. Sur les globules polaires et les homologues de ces elements chez les infusoires cilies : BuUeti)i scientifique de la France et de la Belgiqiie, XXII. — Grobben, C, '78. Beitrage zur Kenntniss der mannlichen Geschlechtsorgane der Dekapoden : Arb. Zool. Lnst. \]ien,\. — Gruber, A.. "84. Uber Kern und Kerntheilung bei den Protozoen : Z. iu. Z., XL. — Id., 85. Uber klinstliche Teilung bei Infusorien: B. C, IV., 23; v., 5. — Id., "86. Beitrage zur Kenntniss der Physiologic und Biologic der Protozoen: Ber. Natnrf- Ges. F'reibnrg, I. — Id., '93. Mikroscopische Vivisektion : Ber. d. Naturf. Ges. zn Freiburg, VII., i — Guignard, L.. "89. Developpement et constitution des Antherozoides : Rct. gen. Bot., I. — Id.. "91, 1. Nouvelles etudes sur la fecondation : Ann. d. Sciences Nat. Bot., XIV. — Id.. '91. 2. Sur I'existence des "spheres attractives " dans les cellules vegetales : C. R., 9 Mars. HABERLANDT, G.. '87. Uber die Beziehun-it. and Foreign Med.-Chir. Beview, XII. — Id., "78. Evolution in Biology, Enc. Brit., 9th ed., 187S ; Science and Culture, N. Y., 1882. ISHIKA"WA, M., "91. Vorlautige Alitteilungen liber die Konjugations- erscheinungen bei den Noctiluceen : Z. A., No. 353, 1891. — Id., "94. Studies on Reproductive Elements : II.. iVoctiluca miliaris, Sur., its Division and Spore-forma- tion ; Journ. College of Sc. Imp. Univ. Japan, VI. JENSEN. O. S.. "83. Recherches sur la spermatogenese : A. B., IV.— Johnson. H. P.. "92-. Amitosis in the embryonal envelopes of the Scorpion: Bull. Mus. Conip. Zo'dl.. XXII., 3. — Jordan. E. O., "93. The Habits and Development of the Newt: J. J/., VIII., 2. — Jordan and Eycleshymer. "94. On the Cleav- age of Amphibian Ova: J. J/.. IX.. 3, 1894. — ■ Julin, J., "93. 1. Structure et developpement des glandes sexuelles, ovogenese, spermatogenese et fecondation chez Styleopsis grossularia: L)ull. Sc. de France et de Belgique., XXIV. — Id., '93. 2. Le corps vitellin de Balbiani et les elements des Metazoaires qui corre- spondent au Macronucleus des Infusoires cilies : Bull. Sc. de France et de Belgique, XXIV. KEUTEN. J.. "95. Die Kerntheilung von Euglena viridis Ehr: Z. iu. Z., LX. — Kienitz-Gerloff, F., '91. Review and Bibliography of Researches on Proto- plasmic Connection between adjacent Cells: in Bot. Zeitung, XIAX. — Klebahn, '92. Die Befruchtung von Qidigonium : Zeit. iviss. Bot., XXIV. — Id. Die Keimung von Closterium und Cosmarium : JaJirb. f. wiss. Bot.,XXll. — Klebs. G., '84. L'ber die neueren Forschungen betretfs der Protoplasmaverbindungen benach- barter Zellen : Bot. Zeit.. 1884. — Id., '87. Uber den Einfluss des Kerns in der Zelle: B. C, VII. — Klein. E.. "78-9. Observations on the Structure of Cells and Nuclei : Q. 7.. XVIII., XIX. — von Kolliker. A., '41. Beitraigezur Kenntnis der Geschlechtsverhaltnisse und der Samenfllissigkeit wirbelloser Tiere : Berlin. — Id., '44. Entwicklungsgeschichte der Cephalopoden: Zurich. — Id.. "85. Die Bedeutung der Zellkerne flir die Vorgange der Vererbung : Z. w. Z., XLII. — Id., '86. Das Karyoplasma und die Vererbung. eine Kritik der Weismann'schen Theorie von der Kontinuitat des Keimplasmas : Z. w. Z., XLIII. —Id., "89. Hand- buch der Gewebelehre, 6th ed. : Leipzig. — Korsclielt. E.. '89. Beitrage zur Mor- phologic und Physiologie des Zell-Kernes : Zool. Jahrb. Anat. u. Ontog., IV. — Id.. '93. L'ber Ophryotrocha puerilis : Z. w. Z.. LIV. — Id.. "95. t^ber Kern- tlieilung, Eireifung und Befrachtung bei Ophryotrocha puerilis: Z. iv. Z.,'LX. — Kossel, A.. '91. Uber die chemische Zu.sammensetzung der Zelle : Arch. Anat. u. Phys. — Id., '93. Uber die Nucleinsaure : Ibid., 1893. — von Kostanecki. '91. Uber Centralspindelkorperchen bei karyokinetischer Zellteilung : Anat. Hefte. 1892, dat. gi. — Kostanecki and "Wierzejski. '96. Uber das Verhalten der sogenann- ten achromatischen Substanzen im befruciiteten Ei : A. m. A., XLII.. 2. — Kiihne. "W., "64. Untersuchungen liber das Protoplasma und die Contractilitait. — Kupffer, GENERAL LITERATURE-LIST 351 C, '75. t'ber Differenzierung des Protoplasma an den Zellen thierischer Gewebe : Schr. natur. V'er. Schles.-Holst., I., 3. — Id.. "90. Die Entwicklung von Petromy- zon Planeri : A. m. A., XXXV. LAMEERE. A.. "90. Recherches sur la reduction karyogamique : Bnixelles. — Lauteiborn, R., "93. Uber Bau und Kerntlieilung der Diatomeen : Verh. d. Naturh. Med. Ver. in Heidelberg, 1893. — Id.. "95. Protozoenstudien. Kern- und Zellteilung von Ceratium iiirundinella O. F. M. : Z. iv. Z., XLIX. — La Valette St. George. '65. Ueber die Genese der Samenkorper: A. m. A.. I. — Id.. "67. Uber die Genese der .Samenkorper. II., (Terminology) : A. 111. A., III. — Id., '76. — Die Spermatogenese bei den Amphibien : Idid.,Xll. — Id., '78. Die Spermatogenese bei den Saugethieren und dem Menschen : //>id., XV. — Id. Spermatologi.sche Beitrage, I.-V. : A. w. A.. XXV.. XXVII.. XXVIII., and XXX., 1885-87. —"^Lankester.E. Ray. '77. Notes on Embryology and Classification : London. — Lavdovsky, M., 94. Von der Entstehung der chromatischen und achromatischen Substanzen in den tierischen und prianzliclien Zellen: Mcrkel und Bonnet's Anat. Hefte, IV., 13. — von Lenhossek, M., "95. Centrosom und Sphare in den Spinalganglien des Frosches ; A. in. A.. XLVI. — Leydig. Fr. "54. Lehrbuch der Histologic des Menschen und der Thiere : Frankfurt. ^\di.. "85. — Zelle und Gewebe. Bonn. — Id., "89. Beitrage zur Kenntniss des thierischen Eies im unbefruchteten Zustande : Spengel's Jahrb. Anat. Ont., III. — Lilienfeld. L.. '92. "93. tlber die Verwand- schaft der Zellelemente zu gewissen Farbstotien: I'er/i. Fhys. Ges., Berlin, 1892-3. — Id., "93. t'ber die Wahlverwandtschaft der Zellelemente zu Farbstoffen : A. A. /'., 1893. — Lillie. F. R . "95. The Embryology of the Unionid?e: J. Af., X. — Id.. "96. On the Limit of Size in the Regeneration of Stentor: Be/>t. Am. JMorplt. Soc. Science.. III., Jan. 10. 1896. — Loeb. J., "91-2. Untersuchungen zur phvsiologischen iMorphologie. I. Heteromorphosis : U "/irzbnrg, iSgi. II. Organ- bildung und Wachsthum : U'iirzburg, 1892. — Id.. '93. Some Facts and Principles of physiological Morphology : Wood's Holl Biol. Lectures, 1893. — Id.. "94. L'ber die Grenzen der Theilbarkeit der Eisubstanz : A. ges. P., LIX., 6, 7. — Lowit. M.. ■91. rber amitotische Kerntheilung : I). C. XI. — Lukjanow. "91. ("rrundzlige einer allgemeinen Pathologic der Zelle: Leipzig. — Lustig and Galeotti, "93. Cytologische Studien uber pathologische menschliche Gewebe: Beitr. Path. Anat.. XIV. MACALLXJM, A. B., "91. Contribution to the Morphology and Physiology of the Cell : Trans. Canad. Inst.. I., 2. — McMurrich. J. P.. "86. A Contribution to the Embryology of the Prosobranch Gasteropods : Studies Biol. Lab. Johns Hopkins Univ., III. — Id.. "95. Embryology of the Isopod Crustacea: /. J/.. XI.. i. — Maggi, L., '78. I plastiduli nei ciliati ed i plastiduli liberamente viventi : .4tti. Soc. Ital. .Sc. Nat. Milano, XXI. (also later papers). — Malfatti. H.. "91. — Beitrage zur Kenntniss der Nucleine : Zeit. Phys. Cheni.. XVI. — Mark. E. L.. '81. Maturation. Fecundation and Segmentation of Lima.K campestris : Bull. Jfus. Conip. Zo'ol. Har- vard College, VI. — Maupas. M.. "88. Recherches experimentales sur la multipli- cation des Infusoires cilies : Arch. Zool. Pxp., 2me .serie. VI. — Id.. '89. Le rejeunissement karyogamique chez les Cilies: Ibid., 2me .serie, VII. — Id.. "91. Sur le determinisme de la se.xualite chez FHydatina senta: C. R-, Paris. — Mead, A. D.. '95. Some observations on maturation and fecundation in Chaetopterus pergamentaceus Cuv. : J M., X.. i . — Merkel. F.. "71. Die Stlitzzellen des mensch- lichen Hodens : JIuller's Arch. — Meitens, H., "93. Recherches sur la signifi- cation du corps vitellin de Balbiani dans I'ovule des Mammiferes et des Oiseaux : A. B.. XIII. — Met.schnikoff. E.. '66. Embryologische Studien an Insecten: Z.ti'. Z.. XVI. — Meves, F.. '91. C'ber amitotische Kernteilung in den Sperma- 352 G ENERA L L I TERA T I ^RE-LIS T togonien des Sahimanders, und das Verhalten der Attraktionsspharen bei derselben : A. .-/.. 1891. No. 22. — Id., '94. Uber eine Metamorphose der Attraktionssphare in den Spermatogonien von Salamandra maculosa: A. m. A., XLIV. — Id., '95. Uber die Zellen des Sesambeines der Achillessehne des Frosches {Kana tein- poraria) und iiber ihre Centralkorper : Ibid.. XLV. — Msyer. O.. '95. Cellulaire Untersuchungen an Nematodeneiern : J. Z., XXIX. (XXII.)- — Mikosch. "94. Uber Struktur im pflanzlichen Protoplasma : I'erhandl. d. Ges. deiitschcr Xaturf. und Arzte, 1894; Abteil f. Pflansenphysiologie u. Pflaiizenanattviiie. — Minot, C. S., '77. Recent Investigations of Embryologists : Froc. Bost.Soc. Nat. Hist., XIX. — Id.. "79. Growth as a Function of Cells : /Z'/<'/., XX. — Id., '82. Theorie der Genoblasten: B. C, II., 12. See also A//i. Nat., February, 1880.— Id., "87. Theorie der Genoblasten: B. C. II., 12, 1887; also, Pfoc. Post. Soc. Nat. Hist., XIX., 1877. — Id., '91. Senescence and Rejuvenation:/^';/;-;/. /V/i'.r., XII., 2. — Id., '92. Human Embryology: New York. — von Mohl, Hugo, "46. Uber die Saftbewegung im Innern der Zellen : Bot. Zeitung. —lAooxe. J. E. S.. "93. Mam- malian Spermatogenesis: ^. .-i., VIII. — Id., '95. On the Structural Changes in the Reproductive Cells during the Spermatogenesis of Elasmobranchs : Q,. J., XXX\'III. Morgan, T. H.. '93. E.xperimental Studies on Echinoderm Eggs: A. .-/., IX., 5, 6. — Id., '95. 1. Studies of the "Partial"" Larvae of Sphaerechinus : A. Entm.. II., i. — Id., 95. 2. Experimental Studies on Teleost-eggs : A. A., X.. 19. Id.. "95, 3. Half-embryos and Whole-embryos from one of the first two Blastomeres of the Frog"s Egg: Ibid.. X., 19. —Id., "95, 4. The Fertilization of non-nucleated Fragments of Echinoderm-eggs : Arch. Iiiiti/i., II., 2. — Id., "95, 5. The Formation of the Fish-embryo : J. J/.. X.. 2. — Id., "96. 1. On the Production of artiticial archoplasmic Centres: Rept. of the Am. Morph. Soc., Science. III., January 10, 1896. — Id., '96, 2. The Number of Cells in Larvce from Isolated Blastomeres of Amphioxus : Arch. Eutni.. III., 2. — Muller. E., "96. Uber die Re"-eneration der Augenlinse nach Exstirpation derselben bei Triton: A. jji. A., XLVII., I. NAGELI. C, "84. Mechanisch-physiologische Theorie der Abstammungslehre : Miinchen //. Leipzig. 1884. — Nageli und Schwendener, "67. Das Mikroskop. (See later editions.) Leipzig. — Newport, G. On the Impregnation of the Ovum in the Amphibia: Phil. Trans.. 185 1. 1853. 1854. — Nussbaum, M., "80. Zur Dift'erenzierung des Geschlechts im Tierreich : A. in. A.. XVIII. — Id., '8?:, 1. i'ber Spontane und Kiinstliche Theilung von Infusorien: I'erJi. d. naturh. I'er. preiiss. Rhineland. 1884. — Id., '84, 2. ("ber die Veranderungen der Geschlechts- producte bis zur Eifurchung : A. in. A., XXIII. — Id., "85. — Uber die Teilbarkeit der lebendigen Materie. I. : A. in. A., XXVI. — Id., "94. Die mit der Entwickelung fortschreitende Differenzierung der Zellen : Sitz.-Ber. d. niederrhein. Gescllschaft f. Natiir- //. Heiliciinde, Bonn. 5 Nov., 1894; also B. C XVI., 2, 1896. OGATA, "83. Die Veranderungen der Pancreaszellen bei der Secretion : A. A. P. — Oppel. A., '92. Die Befruchtung des Reptilieneies : A. in. A , XXXIX. — Overton, C. E., "88. Uber den Conjugationsvorgang bei Spirogyra : Per. deutsch. Bot. Ges.. VI. — Id., "89. Beitrag zur Kentniss der Gattung Volvox : Pot. Ceiitrb: XXXIX. — Id., "93. Uljer die Reduktion der Chromosomen in den Kernen der PHanzen : Vierteljahrschr. natiirf. Ges. ZYirich, XXXVllI. Also Ann. Pot., VII., 25. PALADINO, G , "90. I ponti intercellulari tra I' novo ovarico e le cellule folli- colari, etc.: A. A., V. — Palla. "90. Beobachtungen liber Zellhautbildung an des Zellkerns beraubten Protoplasten. P''/ora, 1890. — Pfitzner, "W., "82. Uber GENERAL LITERA TURK-LIST 353 den feineren Ban der bei der Zelltheilung auffretenden fadenfdrmigen Differenzier- ungen des Zellkerns : J/. J.. VII. — Id., "83. Beitrage zur Lehre vom Baue des Zellkenis und seinen Theilungseischeinungen : A. 111. A.. XXII. — Pfluger, E.. "83. Uber den Einfluss der Schwerkraft auf die Theilung der Zellen : 1., Arch. i^es. F/iys.. XXXI.: II.. /diW., XXXII.; abstract in BioL Centb., III., 1884. — Id., "84. Uber die Einwirkung der Schwerkraft und anderer Bedingiingen auf die Rich tun o- der Zelltheilung : Arch. o-cs. Fhys.. XXXIW — Id.. '89. Die allgemeinen Lebenser- scheinungen : Bonn. — Platner. G., "86. Tber die Befruchtung von .Ir/o/i empiri- coriim: A. m. A., XXXVII. — Id., "89.1. L'ber die Bedeutung der Richtungs- korperchen : B. C. VIII. — Id.. "89. 2. Beitrage zur Kenntniss der Zelle und ihrer Teilungserscheinungen, I.-VI. : ./. m. A., XXXIU. — Poirault and Raci- borski. "96. l'ber konjugate Kerne und die konjugate Kerntheilung : B. C, XVI., I . — Prenant. "94. Sur le corpuscule central : Bi/IL Soc. Sci., Nancy, 1 894. — Preusse. F., "95. Uber die amitotische Kerntheilung in den Ovarien der Hemi- pteren : Z. w. Z.. LIX.. 2. — Prevost and Dumas. '24. Nouvelle the'orie de la generation: .liiii. Sci. N'at.. \.. II. — Pringsheim. N.. "55. L'ber die Befruchtuno- der Algen: Moiiatsb. Berl. Akad., 1855-6. — Purkyne : JaJirb. f. u. Z., LX.. I. — Raiiber. A.. '83. Neue Grundlegungen zur Kentniss der Zelle: M. y., VIII. — Rawitz. B.. "95. Centrosoma und Attraktionsphiire in der nihenden Zelle des Salamanderhodens : A. iii. A., XLIV.. 4. — Reinke. Fr. '94. Zellstudien. \., A. m. A.. XLIIL; II.. /bid.. XLIV.. 1894. — Id.. "95. Untersuchungen iiber Befruchtung und Furchung des Eies der Echinodermen : Sitz.-Ber. Akad. d. JJ'iss. Berlin. 1895, June 20. — Reiuke and Rodewald. "81. Studien liber das Proto- plasma : Untersuch. aiis d. bot. Inst. Gidii/n^eii. II. — Remak. R.. "41. t'ber Theilung rother Blutzellen beim Embryo: Med. Ver. Zeit., 1841. — Id., "50-5., Untersuchungen liber die Entwicklung der Wirbelthiere : Berlin. 1850-55. — Id., '58. t'ber die Theilung der Blutzellen beim Embryo: MYillers Arch.. 1858. — Retzius, G., "89. Die Intercellularbriicken des Eierstockeies und der Follikelzellen : Verh. Anal. Ges., 1889. — Rhumbler. L.. '93. t'ber Entstehung und Bedeutung der in den Kernen vieler Protozoen und im Keimblaschen von Metazoen vorkom- menden Binnenkorper (Nucleolen) : Z. %u. Z.. LVI. — Rompel. '94. Kentrochona Nebaliae n. g. n. sp.. ein neues Infusor aus der Familie der Spirochoninen. Zugleich ein Beitrag zur Lehre von dei" Kernteilung und dem Centrosoma: Z. ia. Z.. LVIII., 4. — Rosen, 92. t'ber tinctionelle L^nterscheidung verschiedener Kernbestand- theile und der Sexual-kerne : Cohn's Beitr. z. Biol. d. Pflanzen, V. — Id., '94. Neueres Uber die Chromatophilie der Zellkerne : Schles. Ges. vHIerl. Kiilt. 1894. — Roux. "W.. '83, 1. t'ber die Bedeutung der Kernteilungsfiguren : Leipzig. — Id., '83, 2. t^ber die Zeit der Bestimmung der Hauptrichtungen des Froschembryo : Leipzig. — Id . "85. L'ber die Bestimmung der Hauptrichtungen des Froschembryos im Ei, und iiber die erste Theilung des Froscheies : Breslaner iirtzl. Zeitg.. 1885. — Id., '87. Bestimmung der medianebene des f^roschembryo durch die Kopulationsricht- ung des Eikernes und des Spermakernes : A. rn. A..XX\X. — Id., "88. Uber das 2 A 354 GENERAL LITERATURE-LIST klinstliche Hervorbringen halber Embryonen durch Zerstorung einer der beiden ersten Furchungskugehi. etc.: Virchow's Archiv, 1 14. — Id., '90. Die Entwickel- ungsmechanik derOrganismen. ^fVev/, 1890. — Id., '92.1. Entwickelungsmechanik : M'erkel and Bonnet, Erg., II. — Id., "92. 2. Uber das entwickelungsmechanische Vermogen jeder der beiden ersten Fuicliungszellen des Eies : Verh. Anat. Ges., VI. — id., '93, 1. Uber Mosaikarbeit und neuere Entwickelangshypothesen : An. Hefte, Feb., 1893. — Id., '93, 2. Uber die Spezifikation der Furchungzellen, etc.: B. c] XIII., 19-22.— Id., '94, 1. Uber den " Cytotropismus" der Furchungszellen des Gra.sfrosches : Arc/i. Entni., I., i, 2. — Id., '94, 2. Aufgabe der Entvvickel- unc^smechanik. etc: Arch. Entni., I., i. Trans, in Biol. Lectures, n'ood's Noll, 1894. — Rtickert, J., '91. Zur Befruchtung des Selachiereies : A. A., VI. — Id., '92. 1. Zur Entwicklungsgeschichte des Ovarialeies bei Selachiern : A. A., VII.— Id. '92, 2. Uber die Verdoppelung der Chromosomen ini Keimblaschen des Se- lachiereies : Ibid., VIII. — Id., "93, 2. Die Chromatinreduktion der Chromosomen- zahl im Entwicklungsgang der Organismen : Merkel and Bonnet, Erg., III. — Id., '94. Zur Eireifung bei Copepoden : An. Hefte. — ld., '95,1. Zur Kenntniss des Befruchtungsvorganges : Sitsb. Bayer. Akad. ll'iss., XXVI., i. — Id., '95, 2. Zur Befruchtung von C}'c/o/>s strenuiis : A. A., X., 22. — Id., '95, 3. Uber das Selb- standigbleiben der vaterlichen und mliterlichen Kernsubstanz wahrend der ersten Entwicklung des befruchteten Cyclops-Eies : A. ni. A., XLV^, 3. — Ruge, G., '89. Vorgange am Eifollikel der Wirbelthiere : M. J., XV. — Ryder, J. A., '83. — The microscopic Sexual Characteristics of the Oyster, etc : Bull. U. S. Fish. Comin., March 14, 1883. Also, Ann. Mag. Nat. Hist., XII., 1883. SABATIER, A., '90. De la Spermatogenese chez les Locustides ; Comptes Rend., CXI., '90. — Sachs., J., '82. Vorlesungen liber Pflanzen-physiologie ; Leip- zig, Id. Uber die Anordnung der Zellen in jungsten Pflanzentheile : Arb. Bot. Inst. UTerzbnrg, II. — Id., '92. Physiologische Notizen, II., Beitrage zur Zel- lentheorie: Flora, 1892, Heft I. — Id., '93. Stoff und Form der Pflanzen-organe ; Gesammelte Abhandhtng, II., 1893. — Id., '95. Physiologische Notizen, IX., weitere Betrachtungen liber Energiden und Zellen: Flora, LXXXI., 2. — Sala, L., '95. E.xperimentelle Untersuchungen liber die Reifung und Befruchtung der Eier bei Ascaris megalocephala ; A. ni. A., XL. — Sargant. Ethel, '95. Some details of the first nuclear Division in the Pollen-mother-cells of Lilium martagon ; Joitrn. Roy. Mic. Soc, 1895, part 3. — Schafer, E. A.. '91. General Anatomy or Histol- oo-y: in Quain's Anatomy, I., 2, loth ed., London. — Schaudinn. F., '95. Uber die Theiluno- von Antaba binucleata Gmber: Sitz.-Ber. Ges. Naturforscli., Freunde, Berlin, Jahrg. 1895, No. 6. — Id., '96. Uber den Zeugungskreis von Paranuvba Eilliardi: Sitz.-Ber. Ges. Naturforsch., Freunde, Berlin, 1896, Jan. 13. — Schewi- akoff, "W., '88. tJber die karyokinetische Kerntheilung der Euglypha alveolata: M. J., XIII. — Schiefferdecker and Kossel, "91. Die Gewebe des Menschlichen Korpers : Braunsc/nccig. — Schimper, "85. Untersuchungen liber die Chlorophyll- korper, etc. : Zeitsch. loiss. Bot., XVI. — Schleicher, W., '78. Die Knorpelzell- theiluno-. Ein Beitrag zur Lehre der Theilung von Gewebezellen : Ccntr. i/ied. Wiss. Berlin^iSjS. Also A. m. A., XVI, 1879. — Schleiden, M. J., '38. Beitrage zur Phytogenesis : Aliiller's Arc/iiv, \%^%. [Trans, in Sydenham Soc, XII.: London, 1847.] — Schloter, G., '94. Zur Morphologie der Zelle : A. in. A.. XLIV.. 2. — Schmitz, '84. Die Chromatophoren der Algen. — Schneider, A., '73. LTnter- suchungen liber Plathelminthen : Jahrb. d. oberhess. Ges. f. Natur- Heilkunde, XIV., Giesscn. — Schneider, C, "91. Untersuchungen liber die Zelle: Arb. Zool. Inst. Jl'ien, IX.. 2. — Schottlander, J., '88. Uber Kern und Zelltheilungsoor- gilnge in dem Endothel der entziindeten Hornhaut: A. in. A.. XXXI. — Schultze, Max, '61. liber Muskelkdrperchen und das was man eine Zelle zu nennen hat : CENEKAL LITERATURE-IJSr 355 Arch. Anat. P/iys., 1861. — Schultze, O., "87. Untersuchungen iiber die Reifung unci Befmchtung des Amphil)ien-eies : Z. w. Z., XLV. — Id., '94. Die kunstliche Erzeugung von Doppelhildungen bei Frosclilarven, etc.: Arcli. Entiii.^ I., 2. — Schwann. Th., '39. Alikroscopisclie Untersuchungen liber die Uebereinstimmung in der Structur und dem Wachsthum der Tiiiere und Pfianzen : Berlin. [Trans, in Sydenham Soc.,X\\.: London, 1847.] — Schwarz, Fr., '87. Die iMorphologische und chemische Zusammensetzung des Protoplasmas : Breslait. — Schweigger- Seidel. O., "65. Uber die Samenkorperclien und ihre Entwickelung : A. m. A., I. — Sedgwick. A.. "85-8. The Development of the Cape Species of Peripatus, I-VI.: (2- 7- XXV.-XXVIII. — Id., "94. On the Inadequacy of the Cellular Theory of Development, etc.: Q. J., XXXVII., i. — Seeliger. 6., "94. Giebt es geschlechtlicherzeugte Organismen ohne miitterliche Eigenschaften ?: A. Ent., I.. 2. — Selenka, E., '83. Die Keimblatter der Echinodermen : Stitdien uber Entwick., II, Wiesbaden, 1883. — Sertoli, E., '65. Dell' esistenza di particolori cellule ramificate dei canaliculi seminiferi del testicolo umano : // Morgagni. — Sied- lecki, M., '95. Uber die Struktur und Kerntheilungsvorgange bei den Leucocy- ten der Urodelen : Anz. Akad. It'iss., Krakaii, 1895. — Sobotta, J., '95. Die Befruchtung und Furchung des Eies der Maus : A. m. A., XL. — Solger. B., '91. Die radiaren Strukturen der Zellkorper im Zustand der Ruhe und bei der Kerntheilung: Berl. Klin. Vl'ochenschr., XX., 1891. — Spallanzani, 1786. Ex- periences pour servir a I'histoire de la generation des animaux et des plantes : Geiieva. — Strasbnrger, E., '75. Zellbildung und Zelltheilung : ist ed., Je)ia, 1875. — Id., -77. i'ber Befruchtung und Zelltheilung : J. Z., XI. — Id., '80. Zell- bildung und Zellteilung : 3d ed. — Id. ,'82. Uber den Theilungsvorgang der Zell- kerne und das Verhaltniss der Kerntheilung zur Zelltheilung: A. ni. A.. XXI. — Id.. '84. 1. Die Controversen der indirecten Zelltheilung: Ibid., XXIII. — Id., '84. 2. Neue Untersuchungen liber den Befruchtungsvorgang bei den Phaneroga- men. als Grundlage fur eine Theorie der Zeugung: Jena, 1884. ^ Id., '88. Uber Kern- und Zellteilung im Pflanzenreich, nebst einem Anhang liber Befruchtung : Jena. — Id., '89. Vh^x das Wachsthum vegetabilischer Zellhaute : Hist. Bei., II., Jena. — Id., "91. Das Protoplasma und die Reizbarkeit : Rektoratsrede. Bonn, Oct. 18, 1891. Jena, Fisc/ier. — IA.,'92. Histologische Beitrage, Heft IV. : Das Verhalten des Pollens und die Befruchtungsvorgange bei den Gymnospermen, Schuarmsporen, pflanzliche Spermatozoiden und das Wesen der Befruchtung: Fischer, Jena, 1892. — Id., '93. 1. Uber die Wirkungssphare der Kerne und die Zellengrcisse : Hist. Beitr., V. — Id., '93, 2. Zu dem jetzigen Stande der Kern- und Zelltheilungsfragan : A. A.,V\\\., \). 177. — Id., '94. Uber periodische Reduktion der Chromosomenzahl im Entwicklungsgang der Organismen: B. C, XIV. — Id., '95. Kuryokinetische Probleme : Jahrb. f. luiss. Botanik, XXVIII., i . — Van der Stricht, O.. '92. Contribution a I'etude de la sphere attractive: A. B., XII., 4. — Id.. "95. 1. La maturation et la fecondation de I'oeuf dWmphioxus lanceolatus : Bnll. Acad. Roy. Bel^iqitc, XXX., 2. — Id., '95, 2. De Torigine de la figure achro- matique de I'ovule en mitose chez le Thvsanozoon Brocchi : Verhandl. d. anat. I'ersaninil. in Strassburg 1895, p. 223. — Id., "95, 3. Contributions a I'etude de la forme, de la structure et de la division du noyau : Bull. Acad. Roy. Sc. Belgiqne, XXI.X. — Strieker. S., '71. Handbuch der Lehre von den Geweben : Leipzig. — Stuhlmann, Fr., '86. Die Reifung des Arthropodeneies nach Beobachtungen an Insekten. Spinnen, Myrio])oden und Peripatus: Ber. Natiirf. Ges. Freiburg, I. — Swaen and Masquelin. "83. Etude sur la Spermatogenese : A. />., IV. THOMA, R., "96. Text-book of (General Pathology and Pathological Anatomy : Trans, by A. Bruce, London. — Thomson. Allen. Article "Generation" in Todd's Cyclopedia. — Id. .Article ••Ovum" in Todd's Cyclopedia. — Tyson, James. "78. The Cell-doctrine: 2d ed., Philadelphia. 356 GENERAL LITERATURE-LIST USSOW, M., '81. Untersuchungen liber die Entwickelung der Cephalopoden : Arch. Biol.. II. VEJDOVSKY, F., '88. Entwickelungsgeschichtliche Untersuchungen, Heft I. : Reifung, Befruchtungund Furchungdes Rhynchelmis-Eies : Prag, i888. — Verworn, M. '88. Biologische Protisten-studien : Z. -w. Z., XLVI. — Id.. "89. — Psyclio- physiologische Protisten-studien: Jena. — Id., '91. Die physiologisclie Bedeutung des Zellkerns : I'/iiiger's Arch. f. d. ges. Pliysiol.. LI. — Id.. "95. Allgemeine Physiologie : Jena. — Virchow, R.. '55. Cellular-Pathologie : Arcli. Patli. Anat. Pliys.. V'lII., I. — Id., "58. Die Cellularpathologie in ihrer Begrlindung auf phvsio- logische und pathologische Gewebelehre : Berlin. 1858. — De Vries, H., '89. Intracellulare Pangenesis : Jena. WALDEYER. W.. -70. Eierstock und Ei : Leipzig. — Id.. '87. Bau und Entwickelung der Samenfaden : I'erh. d. Anal. Leipzig. 1887. — Id.. "88. Uber Karyokinese und ihre Beziehungen zu den Befruchtungsvorgangen : A. m. A., XXXII. [Trans, in Q.J.^ — Id., '95. Die neueren Ansichten iiber den Bau und das Wesen der Zelle : Deiitscli. Med. Woc/iensc/ir.. No. 43, IT.. Oct. ff., 1895. — Warneck, N. A., '50. Ueber die Bildung und Entwickelung des Embryos bei Gasteropoden : Bull. Soc. Imp. Nat. Moscon. XXIII.. i. — Watase, S.. "91. Studies on Cephalopods ; I.. Cleavage of the Ovum : J. J/.. IV.. 3. — Id., '92. On the Phenomena of Sex-differentiation: Ibid.. VI., 2, 1892. — Id.. '93. 1. On the Nature of Cell-organization: IVood^s Hall Biol. Lectures. 1893. — Id., '93, 2. Homology of the Centrosome: J. M., VIII., 2. — Id.. '94. Origin of the centro- some : Biological Lectures, IVood^s Holl. 1894. — "Weismann. A.. '83. Uber Vererbung : Je/ia. — Id.. "85. Die Kontinuitat des Keimplasmas als Grundlage einer Theorie der Vererbung: Jena. — Id., '86. 1. Richtungskorper bei partheno- genetischen Eiern: Zool. Ans., No. 233. — Id.. "86. 2. Die Bedeutung der se.xuel- len Fortpflanzung fiir die Selektionstheorie : Jena. — Id., '87. Uber die Zahl der Richtungskorper und iiber ihre Bedeutung fiir die Vererbung: Jena. — Id., '91, 1. Essays upon Heredity. First Series : Oxford. — Id., '91, 2. Amphimixis, oder die Vermischung der Individuen : Jena. Fischer. — Id.. "92. Essays upon Hereditv, Second Series : O.x'ford, \%()2. — Id. .'93. The Germ-plasm : iVew y'ork. — Id., "94. Aeussere Einflusse als Entwicklungsreize : Jena. — "Wheeler, "W. M., '89. The Embryology of Blatta Gernianica and Doryphora decenilmeata : J. M.. III. — Id.. '93. A Contribution to Insect-embryology: //;/>/.. VIII. i. — Id.. '95. The Behavior of the Centrosomes in the Fertilized Egg oi Myzostonia glabruin : Ibid.. X. — 'Whitman, C. O.. '78. The Embryology of Clepsine : (J. /., XVIII. — Id.. '87. The Kinetic Phenomena of the Egg during Maturation and Fecundation : J. AL. I.. 2. — Id., 88. The Seat of Formative and Regenerative Energy: /bid.. II. — Id. .'93. The Inadequacv of the Cell-theorv of Development: Wood's Noll Biol. Lectures, 1893. — Id., 94. Evolution and Epigenesis : 7/'/^/., 1894. — Wiesner, J.. '92. Die Elementarstruktur und das Wachstum der lebenden Substanz : IJ'ie//. — "Wilcox, E. v.. '95. Spermatogenesis of Caloptenus and Cicada: Bull, of the Museum of Camp. Zool., Harvard College. Vol. XXVII., Nr. i. — Will, L.. '86. Die Entstehung des Eies von Colymbetes : Z. w. Z., XLIII. — 'Wilsou, Edni. B., '92. The Cell-lineage of Nereis: J. M, VI., 3. — Id.. "93. Amphioxus and the Mosaic Theory of Development : Ibid . VIII . 3. — Id.. "94. The Mosaic Theory of Development: U'ood^s Holl Biol. Led.. 1894. — Id.. '95. 1. Atlas of Fertilization and Karyokinesis : New I'ork. Macmillan. — Id.. '95. 2. Archoplasm, Centro- some, and Chromatin in the Sea-urchin Egg: J. J/.. XI. — Id.. '96. On Cleavage and Mosaic-work: A. Entm.. III., i. — Wilson and Mathe'ws. "95. Maturation, Fertilization, and Polarity in the Echinoderm Egg: /. J/., X., i. — Wolff, Caspar GENERAL LITERATURE-LIST 357 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 : .Irc/i. Entin., I., 3. — Welters, M.. "91. Die Conjugation und Sporenbildung bei Gregarinen : ./. ///. ./.. XXXV'II. ZACH ARIAS. O., '85. Uber die amoboiden Bewegungen der Spermatozoen von I'()l\ plit-mus pediculus : Z. iv. Z., XLI. — Zacharias, E.. "93. 1. L'ber die chemische Beschatfenheit von Cytoplasma und Zellkern : Ber. deutsch. bot. Ges., II., 5. — Id.. "93, 2. Uber Chroniatophilie : Ibid., 1893. — Id., '95. t'ber da.s Verhalten des Zellkerns in \vach.senden Zellen : flora, 'i\. 1895. — Id., "94. I'ber Beziehungen des Zellenwachstums zur Beschaffenheit des Zellkerns : Berichte der deutschen botan. Gesellschaft, XII., 5. — Ziegler. E.. "88. Die neuesten Arbeiten liber Vererbung und Abstammungslehre und ihre Bedeutung fur die Pathologie : Beitr. zur path. Aiiat.. IV. — Id.. "92. Lehrbuch der allgemeinen pathologischen Anatomie und Pathogenese, 7th ed. : Jena . —Ziegier. H. E., "87. Die Entsteh- ung des Blutes bei Knochenfischenembryonen : A. m. .4. — Id.. "91. Die biolo- gische Bedeutung der amitotischen Kernthcilung im Tierreich : B. C. XI. — Id., "94. Uber das Verhalten der Kerne im Dotter der meroblastischen Wirbelthiere : Ber. N'ati/rf. Ges. Freiburg, 1894. — Id.. "95. Untersuchungen Uber die Zelltheilung : Verhandl. d. deutsch. Zool. Ges., 1895. — Ziegler and vom Rath. Die amitotische Kerntheilung bei den Arthropoden : /S'. C. XI. ^Zimmermann. A., '93,1. Bei- trage zur Morphologic und Physiologic der Pflanzenzelle : Tubingen. — Zimmer- mann. K. "W.. '93. 2. Studien iiber Pigmentzellen. etc: A. m. A., XLI. — Zoja, R., "95, 1. Sullo sviluppo dei blastomeri isolati dalle uova di alcune medusc : A. Entvi., I., 4: II.. I : II.. I\'. — Id.. '95. 2. .Sulla independenza della cromatina paterna e materna nel nucleo delle cellule emljrionali: A. A.. XI.. 10. INDEX OF AUTHORS Altmann, granule-theory, 21, 22, 28, 31, 224; nuclein, 240. Amici, pollen-tube, 162. Aristotle, epigenesis, 6. Arnold, fibrillar theory of protoplasm, 19; leucocytes, 83; nucleus and cytoplasm, 214. Auerbach, 5; double spermatozoa, 106; staining-reactions, 127; fertilization, 132. von Baer, cleavage, 9; cell-division, 46; egg-axis, 278; development, 295. Balbiani, spireme-nuclei, 25, 26; mitosis in Infusoria, 62; chromatin-granules, 78; yolk-nucleus, 116-121; regeneration in Infusoria, 249. Balfour, polar bodies, 183; une(iual division, 273- Ballov\itz, structure of spermatozoa, 34, 100- 104; double spermatozoa, 106. Van Bambeke, deutoplasm and yolk-nucleus, 116, 117, 121; elimination of chromatin, 116; reduction, 173. Barry, fertilization, 131. De Bary, conjugation, 163, 169; cell-division and growth, 293. Beale, cell-organization, 21, 22. Bechamp and Estor, microsome-theory, 21; microzymas, 22. Belajeff, spermatozoids, 106, 107; reduction in plants, 197. Bellonci, polymorphic nuclei, 82. Benda, spermatogenesis, 123, 124; Sertoli- cells, 208. Van Beneden, cell-theory, i, 4; protoplasm, 19; nuclear membrane, 28; centrosome and attraction-sphere, 36, 53, 70, 224,230, 232, 233; cell-polarity, 39, 40; cell-divi- sion, 46, 51, 52; origin of mitotic figure, 53, 54; theory of mitosis, 70-75; division of chromosomes, 77; fertilization of As- caris, 134; continuity of centrosomes, 143; germ-nuclei, 153, 154; centrosome in fer- tilization, 157; theory of sex, 183; par- thenogenesis, 202; microsomes, 213; nucleus and cytoplasm, 214; individuality of chromosomes, 217; nuclear microsomes, 223; promorphology of cleavage, 281; germinal localization, 298. Van Beneden and Julin, first cleavage-plane, 280. Bergmann, cleavage, 9; cell, 13. Bernard, Claude, nucleus and cytoplasm, 238, 247; organic synthesis, 248, 261, 326. Berthold, protoplasm, 19. Bickford, regeneration in coelenterates, 293, 325- Biondi, Sertoli-cells, 208. Biondi-Ehrlich, staining-fluid, 121. Bischoff, cell, 13. Bizzozero, cell-bridges, 42. Blanc, fertilization of trout, 159. Blochmann, insect-egg, 96; budding of nucleus, 117; polar bodies, 202; bilater- ality of ovum, 283. Bohm, fertilization in fishes, 142. Bolsius, nephridial cells, 32. Bonnet, theory of development, 6, 328. Born, chromosomes in T)-iton-tgg, 245; gravitation-experiments, 285. Boveri, centrosome, named, 36,49; a jier- manent organ, 56; in fertilization, 124, 135, 140, 141 ; continuity of, 143; defini- tion of, 224; structure, 226, 227; func- tions, 259; archoplasm, 51, I2i, 229; origin of mitotic figure, 53, 55 ; mitosis in Ascaris, >,?>■, varieties of ./jir^? ;-/.?, 61 ; the- ory of mitosis, 71, 72; division of cliromo- somes, 77; origin of germ-cells, iio, ill, 322; fertilization of .hearts, 133, 134; of Pterotrachea, 137; of Echinus, 143, 157; theory of fertilization, 140, 14I; of par- thenogenesis, 202; partial fertilization. 559 36o INDEX OF AUTHORS 140, 259; reduction, 173; maturation in Asca>-is, i-jg, iSj; tetrads, 221; centriole, 227, 235; attraction-sphere, 233; egg- fragments, 258; position of polar bodies, 280. Brandt, symbiosis, 37; regeneration in Pro- tozoa, 248. Brauer, bivalent chromosomes, 61 ; mitosis in rhizopod, 65; fission of chromatin- granules, 78; deutoplasm, 117; fertiliza- tion in i?;-rt«f/«)>;«, 142; parthenogenesis in Ai-iemia, 156, 202-205; spermatogene- sis in Ascaris, 184, 187; tetrads, 222; intra-nuclear centrosome, 225. Braus, mitosis, 74. Brogniard, pollen- tube, 162. Brooks, heredity, 10. Brown, Robert, cell-nucleus, 13; pollen- tube, 162. Briicke, cell-organization, 21, 210, 237, 249. von Brunn, spermatozoon, 102, 105. Buffon, organization, 21. Bunting, germ-cells, 109. Burger, centrosome, 228. Biitschli, 5; protoplasm, 17-19; diffused nuclei, 23; artefacts, 31 ; asters, 34, 230; cell-membrane, 38; mitosis, 46, 53, 75; centrosome in diatoms, 65, 224; rejuve- nescence, 129; cyclical division, 163; polar bodies, 175; nature of centrosome, 228. Calberla, micropyle, 148. Calkins, mitosis in Noctilnca, 65, 67; yolk- nucleus, 117-121; origin of middle-piece, 123, 125; reduction, 200. Campbell, fertilization in plants, 160. Carnoy, muscle-fibre, 34; mitosis, 75; ami- tosis, 81-83; germ-nuclei, 134. Castle, egg-axis, 279 ; bilateral cleavage, 281. Chittenden, organic synthesis, 247. Chmielewski, reduction in Sfirogyra, 199. Chun, amitosis, 83; partial development of ctenophores, 315. Clapp, first cleavage-plane, 282. Clarke, mitosis in gregarines, 67. Cohn, cell, 13. Conklin, size of nuclei, 52; union of germ- nuclei, 153; centrosome in fertilization, 157, 158; unequal division, 275; cell-size and body-size, 289. Corda, pollen-tube, 162. Crampton, reversal of cleavage, 270; experi- ments on snail, 315. Darwin, evolution, 2, 4; inheritance, 7, 295 ; variation, 9; pangenesis, 10, 303; gem- mules, 21, 22. Dogiel, amitosis, 84. Driesch, dispermy, 147; fertilization of egg- fragments, 148; pressure-experiments, 275, 282,309; isolated blastomeres, 308; theory of development, 312, 317, 328; experi- ments on ctenophores, 315; ferment- theory, 327. Driiner, spmdle-fibres, 35 ; central spindle, 74. 76; aster, 234. Diising, sex, 109. von Ebner, Sertoli-cells, 208. Eismond, structure of aster, 34. Elssberg, plastidules, 22. Endres, experiments on frog's egg, 307. Engelmann, inutagmata, 22; ciliated cells, 30, 31, 34; rejuvenescence, 129. von Erlanger, asters, 34; elimination of chromatin, 117, 121 ; fertilization, 157; centrosome, 22S. Eycleshymer, first cleavage-plane, 282. Farmer, reduction in plants, 196, 197. Pick, fertilization of axolotl, 135, 142. Field, formation of spermatozoon, 123-125: staining-reactions, 127. Fischer, artefacts, 31, 213. Flemming, protoplasm, 19, 31 ; chromatin, 24; cell-bridges, 42; cell-division, 46; splitting of chromosomes, 51 ; mitotic fig- ure, 53; heterotypical mitosis, 60; leuco- cytes, 72; theory of mitosis, 74; division of chromatin, 78; amitosis, 80-84, 209; axial filament, 123; middle-piece, 125, 126; rotation of sperm-head, 137; spermato- genesis, 193, 194; astral rays, 231; ger- minal localization, 298. Floderus, follicle-cells, 113. Fol, I, 5,46; amphiaster, 49, 53; theory of mitosis, 70; sperm-centrosome, 125; fer- tilization in echinoderms, 130, 157; poly- spermy, 140; attraction-cone, 146; vitel- line memlirane, 148; asters, 230. Foot, yolk-nucleus and polar rings, 119, 121, 150; archoplasm, 121; fertilization in earthworm, 136, 143; entrance-cone, 149. Foster, cell-organization, somacules, 22. Frommann, protoplasm, 19; nucleus and cytoplasm, 214. Galeotti, pathological mitoses, 67-69. GaltDH, inheritance, 7. Cardiner, cell-bridges, 42. Garnault, fertilization in A?-ioii. 155. INDEX OF AUTHORS 361 Geddes and Thompson, theory of sex, 90. Van Gehuchten, spireme-nuclei, 25; nuclear polarity, 26; muscle -hbre, 34. Giard, polar bodies, 177. Gilson, spireme-nuclei, 26. Graf, nephridial cells, 32. Griffin, fertilization, centrosomes in Thalas- sema. 143. 144; structure of centrosome, 235- Grobben, spermatozoa, 105. Gruber, diffused nuclei, 23, 26; regeneration in Stentor, 248. Guignard, mitosis in plants, 59, 78; sperma- tozoiids, 107; fertilization in plants, 157, 159, 161 ; reduction, 195; centrosome, 224. Haacke, gemmae, 22. Haberlandt, position of nuclei, 252. Hackel, inheritance, 5; cell-organization, 21,22,210; epithelium, 40; cell-state, 41. Hacker, polar spindles of Ascaris, 58; bi- valent chromosomes, 61, 62; nucleolus, 91,93; primordial germ-cells, no, I12; germ-nuclei, 156, 193, 194, 219; reduc- tion in copepods, 189, 191; polar bodies, 280. Hallez, promorphology of ovum, 2S3. Halliburton, proteids, 239; nuclein, 240, 241. Hamm, discovery of spermatozoon, 7, 130. Hammar, cell-bridges, 43. Hamraarsten, proteids, 239. Hansemann, pathological mitoses, 67, 68. Hanstein, metaplasm, 15; microsomes, 21. Hartsoeker, spermatozoon, 7. Harvey, inheritance, 5; epigenesis, 6. Hatschek. cell-polarity, 39. 40; fertilization, 130. Heidenhain, nucleus. 24, 25; basichromatin and oxychromatin, 27, 244; cell-polarity, 39; position of centrosome, 40; leuco- cytes, 72, 73; theory of mitosis, 74; ami- tosis, 81; staining-reactions, 127, 144; nuclear microsomes, 223; microcentrum, 227; asters, 234; position of spindle, 277. HeiHer, insect-egg, 96. Heitzmann, theory of organization, 42; nu- cleus and cytoplasm, 214. Henking. fertilization, 124,136; insect-egg, 96; tetrads, 188; reduction, 201. Henle, granules, 21. Henneguy, deutoplasm, 117. Hansen, rejuvenescence, 129. Herbst. development and environment. 324. Herla, independence of chromosomes, 156, 219. Hermann, spermatogonia, 16; central spin- dle, 52, 74, 76; division of chromatin, 78; spermatozoon, 123-126; staining- reactions, 127; centrosome, 224. Herrick, spermatozoon, 105. Hertwig, O., i, 5, 7, 15, 21 ; idioblasts, 22; cell-division, 46; bivalent chromosomes, 61; pathological mitoses, 67; theory of mitosis, 75; rejuvenescence, 129; fertiliza- tion, 132; middle-piece, 135; polyspermy, 140; paths of germ-nuclei, 153; matura- tion, 175, 180-182; polar bodies, 177; inheritance, 257,302; laws of cell-division, 276; cleavage-planes, 282; theory of de- velopment, 312, 317. 322, 328. Hertwig, O. and R., origin of centrosome, 64; egg-fragments, 145; polyspermy, 148. Hertwig, R., mitosis in Protozoa, 63, 64, 67; central spindle, 74; amphiasters in un- fertilized eggs, 159, 226; conjugation, 167; reduction in Infusoria, 199. Hill, fertilization, 135, 143, 157; centro- sphere, 235. His, germinal localization, 297. Hofer, regeneration in Amceha, 249. Hoffman, micropyle. 148. Hofmeister, cell-division and growth, 293. Hooke, R., cell, 13. Hoyer, amitosis, 81. Humphrey, centrosome, 225. Huxley, protoplasm, 3; germ, 5, 295; fer- tihzation, 129, 171 ; evolution and epi- genesis, 328. Ishikawa, A'octiluca, mitosis, 65, 67; conju- gation, 168. Jordan, deutoplasm and yolk-nucleus, 116, 119; first cleavage-plane, 282. Julin, fertilization in Styleopsis, 142. I Karsten, centrosome, 225. i Keuten, mitosis in Eiigletia^ 64. Klebahn, conjugation and reduction in des- mids, 199. Klebs, pathological mitosis, 67, 68; cell- membrane, 251. Klein, nuclear membrane, 28; theory of mitosis, 70, 230; amitosis, 84; nucleus and cytoplasm, 214; asters, 230. von Kolliker, i, 5, 7, 9, 13; epithelium. 40; cell-division, 45; spermatozoon, 98, 122; inheritance, 257. 302; development, 311. Korschelt. nucleus, 25; amitosis, 81, 83; movements and position of nuclei, 92, 254-256; insect-egg, 96; nurse-cells. 113, 36: INDEX OF AUTHORS 114; ovarian ova, 115; fertilization, 135; tetrads in Ophryotrocha, 201 ; physiology of nucleus, 252, 254-256; polarity of egg, 287. Kossel, chromatin, 241; nuclein, 243; or- ganic synthesis, 247. Kostanecki, position of centrosome, 40. Kostanecki and Wierzejski, fertilization of Physa, 131, 136, 143, 159; continuity of centrosomes, 144; collision of asters, 231. Krause, polymorphic nuclei, 82. Kupffer, cytoplasm, 29. Lamarck, inheritance, 10. Lamarle, minimal contact-areas, 269. Lankester, germinal localization, 297. Lauterborn, mitosis in diatoms, 65, 67. Lebrun, position of centrosome, 40. Leeuwenhoek, spermatozoon, 7 ; fertiliza- tion, 130. von Lenhossek, nerve-cell, 16, 33; centro- some, 224. Leydig, cell, 14; protoplasm, 17; cell-mem- brane, 38; spermatozoa, 106; eUmination of chromatin, 117. Lilienfeld, staining-reactions of nucleins, 242, 243. Lillie, regeneration in Stentor, 249. Loeb, regeneration in coelenterates, 293, 325; theory of development, 322; envi- ronment and development, 324. Lustig and Galeotti, pathological mitoses, 68; centrosome, 224. Maggi, granules, 21. Malfatti, staining-reactions of nucleins, 242. Mark, spiral asters, 57; germ-nuclei, 153; polar bodies, 175; promorphology of ovum, 287. Mathews, pancreas-cell, 31; fertilization of echinoderms, 124, 135, 143, 157; nucleic acid, 247. Maupas, sex in Rotifers, 108; rejuvenes- cence, 129; conjugation of Infusoria, 165, 168. McMurrich, gasteropod development, 115; metamerism in isopods, 291. Mead, fertilization of Chcctoptertis, 143; sperm-centrosome, 226. Merkel, Sertoli-cells, 208. Mertens, yolk-nucleus and attraction-sphere, 116-121. Metschnikoff, insect-egg, 284. Meves, amitosis, 81-85, 209. Miescher, nuclein, 240. Mikosch, protoplasm, 31. Minot, rejuvenescence, 129; cyclical divi- sion, 163; theory of sex, 183; Sertoli-cells, 208; parthenogenesis, 202. von Mohl, protoplasm, 13. Moore, spermatozoon, 123-126; reduction, 189, 201. Morgan, fertilization of egg-fragments, 148; effect of fertilization, 149; numerical rela- tions of cells, 288; isolated blaslomeres, 309; experiments on ctenophores, 315; on frog's egg, 319. Nageli, cell-organization, 21; micellae, 22, 301; polioplasm, 29; idioplasm-theory, 300. Newport, fertilization, 130; first cleavage- plane, 280. Niessing, axial filament, 123. Nissl, chromophilic granules, ^t„ 34. Nussbaum, germ-cells, 88; regeneration in Infusoria, 248; nucleus, 321. Overton, germ-cells of Vohwx, 98; conjuga- tion of Spirogyra, 169, 170; reduction, 196. Owen, germ-cells, 88. Paladino, cell-bridges, 42. Peremeschko, leucocytes, 83. Pfeffer, hyaloplasm, 29; chemotaxis of germ- cells, 145. Pfitzner, cell-bridges, 42; chromatin-gran- ules, 78. Pfliiger, position of spindle, 277; first cleavage-plane, 280 ; gravitation-experi- ments, 285; isotropy, 278. Plateau, minimal contact-areas, 269. Platner, mitosis, 75; formation of spermato- zoon, 123-125; fertilization of .4rion; maturation, 175, 180. Pouchet and Chabry, development and en- vironment, 324. Prenant, spermatozoon, 123. Preusse, amitosis, 85, 209. Prevost and Dumas, cleavage, 9. Pringsheim, Hautschicht, 29; fertilization, 130. Purkyne, protoplasm, 13. Rabl, nuclear polarity, 26; cell-polarity, 39, 40, 52; centrosome in fertilization, 157; individuality of chromosomes, 215. Ranvier, blood-corpuscles, 38. vom Rath, nucleus, 26; bivalent chromo- somes, 61; amitosis, 82-84; early germ- cells, 112; reduction, 189, 192; tetrads. INDEX OF AUTHORS 363 193; centrosome, 224; attraction-sphere, 234- Rauber, cell-division and growth, 293. Rawitz, spermatogonium, 15; amitosis, 82. Redi, genetic continuity, 21. Reichert, cleavage, 9, 46. Reinke, pseudo-alveolar structure, 19; nu- cleus, 26, 27, 223; fedematin, 28; cyto- plasm, 29; asters, 34, 226, 231; central spindle, 74; nucleus anti cytoplasm, 214. Reniak, cleavage, l, 9, 264; cell-division, 45' 46 ; egg-axis, 279. Retzius, muscle-tibre, 34 ; cell-bridges, 42 ; entl-pic'ce, 104. Robin, germinal vesicle, 46. Rosen, staining-reactions, 162. Roux, cell-organization, 21; meaning of mi- tosis, 51, 183, 221, 256; position of spindle, 277; first cleavage-plane, 277, 280; frog- experiments, mosaic theory, 298; theory of development, 303; post-generation, 307- Riickert, pseudo-reduction, 6r, 193; fertili- zation of Cyclops. 142; independence of germ-nuclei, 156, 219; reduction in cope- pods, 189; early history of germ-nuclei, 193, 245; reduction in selachians, 200; history of germinal vesicle, 245. Riige, amitosis, 83. Ryder, staining-reactions, 127. Sabatier, amitosis, 82. Sachs, energid, 14 ; laws of cell-division, 265; cell-division and growth, 293; de- velopment, 322. St. George, La Valette, spermatozoon, 7, 98 ; spermatogenesis (terminology), 122. Sala. polyspermy, 147. Sargant, reduction in plants, 197. Schafer, protoplasm, 17. Scharff, budding of nucleus, 117. Schaudinn, mitosis in Amxha. 64. Schewiakoff, mitosis in Etiglypha, 63-65. Schimper, plastids, 98. Schleicher, karyokinesis, 46. Schleiden, cell-theory, i ; cell-division, 7 ; nature of cells, 13 ; fertilization, 162. Schloter. granules, 28, 223. Schmitz, plastids, 98 ; conjugation, 160. Schneider, discovery of mitosis, 46. Schottlander, multipolar mitosis, 69. Schultze, M., cells, i, 13, 14; protoplasm, 19. Schultze, O., gravitation-experiments, 285 ; double embryos, 318. Schwann, cell-theory, i ; the egg a cell, 6 ; origin of cells, 7; nature of cells, 13 ; or- ganization, 41 ; adaptation, 329. Schwarz, protoplasm, 19; linin, 24; chem- istry of nucleus, 28 ; nuclei of growing cells, 246. Schweigger-Seidel, spermatozoon, 7,98, 122. Sedgwick, cell-bridges, 43. Seeliger, egg-fragments, 258 ; egg-axis, 279. Selenka, double spermatozoa, 106. Sobotta, fertilization of mouse, 136, 143. Solger, pigment-cells, 73 ; attraction-sphere, 224. Spallanzani, spermatozoa, 7. Spencer, physiological units, 21, 22 ; devel- opment, 32S. Strobe, multipolar mitoses, 69. Strasburger, I, 5 ; cytoplasm, 15 ; proto- plasm, 19 ; Kornerplasma, 29; centro- sphere, 49, 232 ; origin of amphiaster, 53 ; multipolar mitoses, 69 ; theory of mitosis, 74, 76, 77 ; amitosis, 83 ; sper- matozoids, 107, 108, 126; kinoplasm, 108, 126; staining-reactions of germ-nuclei, 128; fertilization in plants, 135, 160, 162 ; reduction, 188, 195 ; theory of maturation, 196; organization, 210 ; in- heritance, 257, 302 ; action of nucleus, 322. zur Strassen, primordial germ-cells in Asca- ris. III. Van der Stricht, amitosis, 82 ; attraction- sphere, 224 ; fertilization in Amphioxus^ 159. Stuhlmann, yolk-nucleus, 1 19. Tangl. cell-bridges, 42. Thiersch and Boll, theory of growth, 292. Treat, sex, 109. Ussow, micropyle, 97; deutoplasm. 117. Vejdovsky, centrosome, 55 ; fertilization in Rhynchelmis^ 142 ; metamerism in an- nelids, 291. Verworn, cell-physiology, 4 ; cell-organiza- tion, 21 ; biogens, 22 ; regeneration in Protozoa^ 249 ; nucleus and cytoplasm, 252 ; inheritance, 327. Virchow, i ; cell-division, 8, 9, 21, 45-47 ; protoplasm, 19 ; cell-state, 41. De Vries, organization, 21 ; pangens, 22 ; tonoplasts, 37 ; plastids, 170 ; chromatin, 183 ; panmeristic division, 236 ; pangene- sis, 303; development, 312. Waldeyer. nucleus, 27 ; cytoplasm, 29 ; cell- 364 INDEX OF AUTHORS membrane, 38 ; chromosomes, 47 ; amito- sis, 83. Walter, frog-experiments, 307. Wasielewsky, centrosome, 225. Watase, theory of mitosis, 75 ; staining- reactions of germ-nuclei, 127; nucleus and cytoplasm, 211 ; asters, 226; theory of centrosome, 228 ; astral rays, 231 ; cleav- age of squid, 273, 283 ; promorphology of ovum, 283, 287. Weismann, inheritance, 10, 11, 302; cell- organization, 21; biophores, 22; ids, 27; somatic and germ cells, 88 ; amphimixis, 130; maturation, 1 83-1 85 ; constitution of the germ-plasm, 183, 305; partheno- genesis, 202; theory of development, 303- 305. 328. Went, vacuoles, 37. Wheeler, amitosis, 81 ; insect-egg, 97 ; fertilization in Alyzostonia^ ^57" '59'' plastids, 170; bilateralitv of ovum, 283. Whitman, on Harvey, 6 ; cell-organization, 21 ; idiosome, 22 ; polar rings, 150 ; cell- division and growth, 293 ; theory of de- velopment, 297, 299. Wiesner, cell-organization, 21, 137 ; pla- some, 22 ; panmeristic division, 236. Wilcox, sperm-centrosome, 123, 124; re- duction, 189, 200. W'ill, chromatin-ehmination, 117. Wilson, fertilization in sea-urchin, 135, 136, 143; paths of germ-nuclei, 152; origin of linin, 223 ; astral rays, 231 ; centro- sphere and centrosome, 232-235 ; di- sperniy, 260 ; pressure-experiments, 309 ; first cleavage-plane, 277 ; experiments on Amphioxus, 308, 319; theory of develop- ment, 317. von Wittich, yolk-nucleus, 118. Wolff, C. F., epigenesis, 6. Wolff, G., regeneration of lens, 329. Wolters, mitosis in gregarines, 67 ; polar body in gregarines, 199. Yung, sex, 109. Zacharias, E,, nucleoli, 24, 25 ; of meristem, 27 ; staining-reactions, 127 ; nuclein in growing cells, 246. Zacharias, O., amoeboid spermatozoa, 105. Ziegler, artificial mitotic figure, 75 ; amito- sis, 83, 84, Zimmerman, pigment-cells, 73. Zoja, independence of chromosomes, 156, 219 ; isolated blastomeres, 309. INDEX OF SUBJECTS Achromatic figure (see Amphiaster), 50; varieties of, 57; nature, 229. Actinosphariitm, mitosis, 63, 66; regenera- tion, 248. Adaptation, 329. ALqieort'a, metanucleus, 93. Albumin, 239, 241. Allolobophora^ fertilization, 136; teloblasts, 274. Amphiaster, 49; asymmetry of, 51, 275; origin, 49, 75, 231; in amitosis, 81; in fertilization, 134, 140, 142, 156; nature, 260; position, 275-277. Amitosis, 46, 80; biological significance, 82; in sex-cells, 209. Aiiitvlm, 4; mitosis, 64; experiments on, 249. Amphibia, spermatozoa, 100. Amphimixis. 130, 1 71. Aniphio.xiis^ fertilization, 153, 159; polar body, 176; cleavage, 270, 271; dwarf larvce, 289, 307; double embryos, 308. Amphipyrenin, 29. Amyloplasts, 37; in plant-ovum, 98, 160. Anaphases, 47, 51 ; in sea-urchin egg, 77. Aniioci-a, gland-cells, nuclei, 26; amitosis. 84. Auodonta, ciliated cells, 30. Antipodal cone, 71. Archoplasm, 51; in developing sperma- tozoa, loi, 123, 126, in spermatozoids, 108, 126; and yolk-nucleus, 121 ; nature of, 229-231. Argoiiauta, micropyle, 97. Arioii, germ-nuclei, 155. Artefacts, in protoplasm, 31, 213. Artemia, chromcsomes, 49, 61, 205; par- thenogenetic maturation, 202-205. Ascaris, chromosomes, 49; mitosis, 52, 58, 71, 78; primordial germ-cells, no, 332; fertilization, 132-134, 141 ; polyspermy, 147; polar bodies, 179; spermatogenesis. 180-182, 184; individuality of chromo- somes, 215-218; intranuclear centrosome, 225; attraction-sphere, 233; supernum- erary centrosome, 259. Aster, 34; asymmetry, 51 ; spiral, 57 ; structure and functions, 71; in amitosis, 81; in fertilization, 138, 157; nature of, 229-231; finer structure, 231, 233, 244; relative size, 275. As/erias, spermatozoa, 127; sperm-aster, 140; fertilization, 143, 146. Astrocentre, 232. Astrosphere, 232. Attraction-cone, 146. Attraction-sphere, 36, 53, 54; in amitosis, 81; of the ovum, 119; of the spermatid, 125; in resting cells, 224; nature of, 232-235. Axial filament, 99; origin of, 123. Axis, of the cell, 38; of the nucleus. 26, 215; of the ovum, 40, 278-280, 298, 319. Axolotl, fertilization, 131. Bacteria, nuclei, 23. Basichromatin, 27, 223; staining-reactions, 223, 245. Bioblast, 22. Biogen, 22. Biophore, 22, 183, 305. Birds, blood-cells, 46; spermatozoa, 102; young ova, 119. Blastomeres, displacement of, 270; in- dividual history, 273; prospective value, 280, 313; rhythm of division, 290; de- velopment of single, 298, 307-309, 315, 319; in normal development, 312. Bleuiiius, pigment-cells, 73. Branchipiis, yolk, 117; sperm-aster, 142; reduction, 1 88. Calanus, tetrads, 190. Cancer-cells, mitosis, 68. 366 INDEX OF SUBJECTS Canthocainptus^ reduction, 190 ; ovarian eggs, I93> 194- Cell, in general, 3; origin, 7, 8; name, 13; general sketch, 14; polarity of, 38; as a structural unit, 41 ; structural basis, 16-22, 212; physiology and chemistry, 238; size and numerical relations, 289-292; in in- heritance, 7, 295, 328; differentiation of, 311-315; independence of, 323. Cell-bridges, 42. Cell-division (see Mitosis, Amitosis), general signilicance, 9, 45; general account, 45; types, 46; Remak's scheme, 46; indirect, 47; direct, 80; cyclical character, 129, 164; equal and reducing or qualitative, 185, 304. 305; relation to development, 264; Sachs's laws, 265; rhythm, 268, 289; unequal, 270-276; of teloblasts, 271; energy of, 289; relation to metamerism, 291 ; causes, 292; relation to growth, 293; and differentiation, 312, 323. Cell-membrane, 38. Cell-organs, 37; nature of, 21 1; temporary and permanent, 211, 236. Cell-organization, 21; general discussion, 210-237. Cell-plate, 52. Cell-state, 41. Cell-theory, general sketch, i-io. Central spindle, 49, 52; origin and function, 74- Centrosome, 17; general sketch, 36; posi- tion, 39; in mitosis, 49; a permanent organ, 54, 224; dynamic centre, 56; historical origin, 67; functions, 70, 259; in amitosis, 81; of the ovum, 91 ; of the spermatozoon, 99, loi, 123; in fertiliza- tion, 135, 141, 144, 156-159, 171; degen- eration of, 135, 142, 171, 224; continuity, 143, 227; in parthenogenesis, 156, 203; nature, 224-229; intra-nuclear, 64, 225; effect on cytoplasm, 36, 212; supernum- erary, 260. Centrosphere, 36, 49, 77; nature of, 232. Ceraiozainia, reduction, 196. Cerianthns^ regeneration in, 293. Chcrtopterus, fertilization, 143; sperm-centro- some, 226. Chara, spermatozoids, 106. Chii-onomtis, spireme-nuclei, 26. Chorion, 96. Chromatic figure, 50; origin, 53; varieties, 59; in fertilization, 134. Chromatin, 24; in meristeni, 27; in mitosis, 47; in cancer-cells, 68; of the egg-nucleus, 92; elimination of, in cleavage, iii, in oogenesis, 117, 121 ; staining-reactions, 127, 243, 244; morphological organization, 78, 80, 183-185, 215-222, 304, 305; chemical nature, 28, 241-244; relations to linin, 223; physiological changes, 244- 247; as the idioplasm, 257, 301, 302; in development. 321, 322, 326. Chromatin-granules, 27; in mitosis, 78; in reduction, 206; general significance, 221, 222, 305 ; relations to linin, 223. Chromoplast, 37. Chromatophore, 37, 211 ; in the ovum, 98; in fertilization, 169. Chromomere (see Chromatin-granule), 27, 221. Chromosomes, 27; in mitosis, 47-52; num- ber of, 48, 49, 154, 219; variation of, 59; bivalent and plurivalent, 61, 190, 205- 207; division, 77; of the primordial germ- cell, III; in fertilization, 134, 135, 154; independence in fertilization, 156, 219; reduction, 173; in early germ-nuclei, 193; conjugation of, 199; in parthenogenesis, 203,204; individuality of, 215-221 ; com- position of, 221, 304, 305; chemistry, 243; history in germinal vesicle, 245; in dwarf larv£e, 258. Ciliated cells, 30, 34. Ciona, egg-axis, 280. Clavelina, cleavage, 270, 281. Cleavage, in general, 9; geometrical rela- tions, 265-278; Sachs's laws, 265; modi- fications of, 268; spiral, 270; reversal of, 270; meroblastic, 271 ; under pressure, 275, 309; Hertwig's laws, 276; promor- phology of, 278; bilateral, 280; rhythm, 290 ; mosaic theory, 299; half cleavage, 308; and development, 309-320, 323; partial, 315. Cleavage-nucleus, 153. Cleavage-planes, 267; determination of, 277; axial relations, 280-285, 287. Clepsine, nephridial cell, 32; polar rings, 150; cleavage, 270. Closteriuf7i, conjugation and reduction, 198. Cockroach, amitosis, 81; orientation of egg, 283. Coelenterates, germ-cells, 109; regeneration, 325- Conjugation, in unicellular animals, 163-168; unicellular plants, 169; physiological mean- ing, 129, 165. Contractility, theory of mitosis, 70-74; in- adequacy, 77. Copepods, reduction, 190. Corixa, ovum, 284. INDEX OF SUBJECTS 367 Crepidula^ fertilization, 157; position of spindles, 277; dwarfs and giants, 289. Cross- furrow, 270. Crustacea, spermatozoa, 105, 106. Ctenophores, experiments on eggs, 314. Cyclas, ciliated cells, 30. Cyclops, ova, 93 ; primordial germ-cells, 1 1 2 ; fertilization, 142, 156,218; reduction, 189- 191; attraction-sphere, 233; axial rela- tions, 286. Cytolyniph, 17. Cytoplasm, 15, 29, 213, 236; of the ovum, 97, 115, 170; of the spermatozoon, 107; morphological relations to nucleus, 214; to archuplasm, 230-235; chemical rela- tions to nucleus, 238, 240, 241 ; physio- logical relations to nucleus, 248; in inheritance, 297, 298, 327; in develop- ment, 315-320; origin, 327. Dendrobcsna, metamerism, 291. Determinants, 183, 305. Deutoplasm, 90, 91, 94; deposit, 115; ar- rangement, 117, 279; effect on cleavage, 273; re-arrangement by gravity, 285, 319. Development, i, 6; and cell-division, 264; mosaic theory, 298; theory of Nageli, 301 ; Roux-Weismann theory, 303; of single blastomeres, 298, 307, 315; of egg-frag- ments, 217, 258, 285, 315; Hertwig's theory, 312, 317; Driesch's theory, 313, 317; partial, 315; half and whole, 319; nature of, 320; external conditions, 323; and metabolism, 326; unknown factor, 327; rhythm, 328; adaptive character, 329- Diatoms, mitosis, 67; centrosome, 224. Dieinyctyliis, yolk, 116; yolk-nuclei, 1 19. Differentiation, 264. 296; theory of De Vries, 303; of Weismann, 305; nature and causes, 311-320; of the nuclear substance, 321 ; and cell-division, 323. Dispermy, 147, 260. Double embryos, 308, 319. Dwarfs, formation of, 258, 289, 307-309, 315; size of cells, 289. Dyads (Zweiergrappen), 179, 181, 184, 189; in parthenogenesis, 203-205. Dyaster, 51. Dycyemids, centrosome, 36. Dytisciis, ovarian eggs, 115, 256. Earthworm, ova, 115; spermatozoon, 125; yolk-nucleus, I2i; fertilization, 135; polar rings, 150; spermatogenesis, 200; telo- blasts, 274. Echinoderms, spermatozoa, 123; fertiliza- tion, 143, 157; polyspermy, 147; dwarf larva:, 258, 289; half cleavage, 306; eggs underpressure, 309; modified larvse, 324. Echinus, fertilization, 143,157; centrosome, 235; dwarf larvje, 258; number of cells, 291. Egg-axis, 278; promorphological signifi- cance, 279, 298; determination, 285, 287, 322; alteration of, 319. Egg-centrosome, 91, 119; degeneration of, 91, 138, 141, 142, 171; asserted persist- ence, 157-159. Egg-fragments, fertilization, 97, 145, 148; development, 217, 258, 285, 289, 315. Elasmobranchs, spermatozoon, 100, 124; germinal vesicle, 193, 245; reduction, 200. Embryo-sac, 160. Enchylema, 17. Endoplasni, 29. End-piece, 100, 104. End-plate, 64. Envelopes, of the egg, 96. Epigenesis, 6, 305, 327, 328. Equatorial plate, 49, 58, 66; formation, 74. £?/c/i(2'/'y conjugation, 199 ; formulas for, 186, 193, 200, 201. Tka/asse>?ia, fertilization, 143 ; centrosome, 228 ; attraction-sphere, 235. Thalassicolla, experiments on. 250. Tonoplast, 37. Toxopneustes, cleavage, 8 ; mitosis, 76 ; ovum, 91 ; spermatozoon. 99; fertili- zation, 146 ; paths of germ-nuclei, 152; polar bodies, 174; double cleavage, 260. Trachelocerca, diffused nuclei, 26. .Trophoplasm, 301. Tiibularia, regeneration, 293, 325. Tunicates, egg-axis, 280; cleavage, 281. Unicellular organisms, 3 ; mitosis, 62 ; con- jugation, 163; reduction, 198; experi- ments on, 248-252. Unio, cleavage, 272. Vacuole, 37. Vanessa, ovarian egg, 115. Variations, 9 ; origin of, 329, 330. Vaticheria, membrane, 251, 254. Vitelline membrane, 96 ; of egg- fragments, 97; formation of, 146; function, 148. Volvox, germ-cells, 89, 98. Vorticella, conjugation, 167. Yellow cells (of Radiolaria), 37, 211. Yolk (see Deutoplasm), 90, 94. Yolk-nucleus, 115, 118. Yolk -plates, 94. Zwischenkorper (mid-body), 52. Zygnema, membrane, 252. Zygospore, 169. V I 33/7