DR. JOHN W. SAUNDERS. JR. DEPARTMENT OF BIOLOGY MARQUETTE UNIVERSITY MILWAUKEE 3. WISCONSIN QL_| The Beginnings of K^Z Embryonic Development l^S A symposium organized by the Section on Zoological Sciences of the American Association for the Advancement of Science, co- sponsored by the American Society of Zoologists and the Associa- tion of Southeastern Biologists, and presented at the Atlanta Meet- ing, December 27, 1955 Edited by , Albert Tvler, R. C. von Borstel, and ^^ Charles B. Metz Publication No. 48 of the AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE Washington, D. C, 1957 J © 1957 The American Association for the Advancement of Science Library of Congress Catalog Card Number 57-11246 Printed in the United States of America DR. JOHN W. SAUNDERS. JR. DEPARTMENT OF BIOLOGY MARQUETTE UNIVERSITY MILWAUKEE 3. WISCONSIN PREFACE A symposium on "Formation and Early Development of the Embryo," held December 27, 1955, at the Second Atlanta Meet- ing of the AAAS, served as the basis for the present volume. The symposium emphasized problems of early development and of the initiation of development. However, in selecting this general area for discussion the organizing committee did not intend to imply that the problems are considered to be fundamentally different from those encountered in later development. In fact, many of the contributions presented here indicate the generality of the problems of developmental change at any stage along with the special nature of the particular events studied. It may also be said that an understanding of early, as well as later, development depends largely on knowledge of the manner of formation of the egg, that is, of the processes that endow the oocyte, in contrast to other tissue cells, with the capacity to form a new individual. The first paper deals with one aspect of this subject which is also partly considered in some of the others. The next five papers treat principally with the initiation of development, and the remain- ing seven center primarily about subsequent events as related to nuclear and cytoplasmic factors. As with most symposia the general purpose of this one was to bring together a group of individuals working in related fields so that there would be opportunity for participants and audience to profit directly from exchange of knowledge and ideas. It was also decided that the presentations be made available to others in published form. Since the symposium was planned for a one day session, the number of speakers was correspondingly restricted. However, for the purpose of the present volume it was considered desirable that the coverage of the general field be expanded somewhat. This has been done by the inclusion of articles requested from some additional investigators, in this country and abroad, whose work pertains to the subjects under consideration. iv PREFACE It will be readily recognized that the present volume comprises a selected sample, rather than a more comprehensive coverage of investigations into problems of the initial developmental changes undergone by the egg and early embryo. The investi- gations presented in the various communications cover both de- scriptive and experimental work on the biological and chemical levels. Many of the articles contain results of previously unpub- lished researches as well as general reviews of the particular subject. No special attempt was made to force the contributions into a preconceived plan, or to develop an overall general concept. The interrelationships of the individual topics assured a reason- able amount of unity to the work. In addition, there was exchange of articles among some of the contributors, besides the verbal discussions at the symposium, that provided opportunity for fur- ther integration and for elimination of unnecessary repetition. The instances of overlapping that remain were considered desir- able, especially where there were differences in outlook and interpretation. Although such differences reflect, in part, varia- tions in point of view and judgment of the individual contribu- tors, they serve mainly to emphasize the lack of critical informa- tion concerning the particular problem under discussion. In fact we consider much of the value of a work such as this to reside in the extent to which it brings to the attention of students and investigators these regions of uncertainty and indicates the kinds of problems that are in urgent need of solution, along with the modern methods by which answers may be sought. Apart from their intrinsic interest and the measure of progress that they provide, the specific discoveries and analyses presented in this book serve, then, to exemplify various approaches toward our understanding of the manner in which sperm and egg con- trive to produce a new individual. Albert Tyler CONTRIBUTORS C. R. Austin, Division of Experimental Biology, National Insti- tute for Medical Research, Mill Hill, London, England M. W. H. Bishop, Division of Experimental Biology, National Institute for Medical Research, Mill Hill, London, England R, C. VON BoRSTEL, Biology Division, Oak Ridge National Labo- ratory, Oak Ridge, Tennessee M. C. Chang, The Worcester Foundation for Experimental Biol- ogy, Shrewsbury, and Department of Biology, Boston Uni- versity, Boston, Massachusetts Arthur L. Colwin, Department of Biology, Queens College, Flushing, New York Laura Hunter Colwin, Department of Biology, Queens College, Flushing, New York John R. Gregg, Department of Zoology, Columbia University, New York City, New York H. E. Lehman, Department of Zoology, University of North Carolina, Chapel Hill, North Carolina Charles B. Metz, Oceanographic Institute, The Florida State University, Tallahassee, Florida Alberto Monroy, Istituto di Anatomia Comparata, Universita di Palermo, Palermo, Italy Silvio Ranzi, Istituto di Zoologia dell'Universita, Milan, Italy G. Reverberi, Istituto di Zoologia, Universita di Palermo, Palermo, Italy John R. Shaver, Department of Zoology, Michigan State Univer- sity of Agriculture and Applied Science, East Lansing, Michigan Albert Tyler, Division of Biology, California Institute of Tech- nology, Pasadena, California W. S. Vincent, Department of Anatomy, College of Medicine, State University of New York, Syracuse, New York CONTENTS Some Studies on Differentiation and Development of tlie Oocyte W. S. Vincent 1 Specific Egg and Sperm Substances and Activation of the Egg Charles B. Metz 23 Preliminaries to Fertilization in Mammals C. R. Austin and M. W. H. Bishop 71 Some Aspects of Mammalian Fertilization M. C. Chang 109 Morphology of Fertilization: Acrosome Filament Forma- tion and Sperm Entry Arthur L. Col win and Laura Hunter Colwin 135 Studies of Proteins of Sea Urchin Egg and of Their Changes Following Fertilization Alberto Monroy 169 Nucleocytoplasmic Relations in Early Insect Development R. C. von Borstel 175 Nuclear Transplantation, a Tool for the Study of Nuclear Differentiation H. E. Lehman 201 Morphogenesis and Metabolism of Gastrula-Arrested Em- bryos in the Hybrid Rana pipiens $ x Rana sylva- tica $ John R. Gregg 231 Some Observations on Cytoplasmic Particles in Early Echinoderm Development John R. Shaver 263 Early Determination in Development under Normal and Experimental Conditions Silvio Ranzi 291 vii viii CONTENTS The Role of Some Enzymes in the Development of Ascidians G. Reverberi 319 Immmiological Studies of Early Development Albert Tyler 341 Index 383 SOME STUDIES ON DIFFERENTIATION AND DEVELOPMENT OF THE OOCYTE "" W. S. VINCENT: upstate medical center, STATE UNIVERSITY OF NEW YORK, SYRACUSE, NEW YORK When one considers the development of the oocyte, it becomes apparent that there are two general problems to be studied. One of these is the problem of the origin of the egg cell; the other, that of its later development. The study of the origin of the primary germ cell, from which the oocyte is derived, has provoked wide interest and consider- able controversy. Out of this has arisen the following questions: Is there a separate germinal plasm which gives rise to the pri- mordial germ cells, these in turn migrating to the gonad and there forming definitive gonia; or do some or all of the definitive germ cells arise from somatic cells of the germinal epithelium? In some invertebrates it is readily observed that the functional germ cells are derived from a line of cells set apart veiy early in development, although in the coelenterates and annelids this early setting apart is still questionable ( Berrill and Liu, 1948; Wil- son, 1928). In vertebrates, and especially mammals, the evidence for the origin of the definitive germ cell from a primary cell type is far from being conclusive. Although there is general agreement that a primordial germ cell type is found, there is disagreement as to whether these cells migrate into the gonad and there even- tually dififerentiate into mature oocytes, or whether they degen- * Part of the work reported here was done during the tenure of a Ful- bright Fellowship to Belgium. Some of the work was done at the Marine Biological Laboratory, Woods Hole, during the tenure of a Lalor Founda- tion Fellowship, and in addition, while supported by funds from the Atomic Energy Commission, grant AT (30-1) 1343. 1 2 THE OOCYTE erate completely before any definitive cells are formed. In the latter case the oocyte would arise from the germinal epithelium. Some recent studies on this problem include those of Vincent and Dornfeld (1948), Everett (1945), and Jones (1949). An excellent review of the problem of germ cell origin is given by Nelsen (1953). The other aspect of the general problem of the development of the oocyte is that of its differentiation and growth into the mature egg. For the purposes of this discussion, it is convenient to define three general areas of influence on the developing oo- cyte. Two of these might be called external, namely, the contri- butions to the developing egg cell by the surrounding circulatory fluids and the contributions to the oocyte by surrounding cells. The third area is the effects of the oocyte nucleus on the develop- ment of its own cytoplasm. Considerable information is now available regarding the con- tributions of the circulatory media to the developing egg. Clas- sically, the yolk in avian eggs has been considered to be derived from large molecules carried in the circulation (Romanoff and Romanoff, 1949). Probably the most clear-cut demonstration of the transfer of blood proteins to the egg is that of Telfer ( 1954 ) . He found an albumin-like, antigenic protein present in very high quantities in the blood of female Cscropia during the pupal stage. He was then able to demonstrate conclusively that this protein was transferred to the yolk of developing egg cells. Immunologi- cal similarities between the yolk and adult blood proteins have been detected in vertebrates ( Nace, 1953 ) , and this suggests that in toto transfer of protein ( or at least of specific combining sites ) occurs in these forms as well. In his general review of the devel- opment of immunological properties, Tyler (1955) pointed out that there is evidence that similarities exist between blood pro- teins and both the ground substance and the formed elements of the egg cytoplasm. Considerable importance must be attached therefore to the contributions of the circulatory fluids to the de- velopment of the oocyte. The effects of the surrounding cells on the oocyte are varied. W. S. VINCENT 3 Apparently, they must provide some of the differentiating influ- ences that allow the presumptive oocyte to form an egg, since egg cells are not known to differentiate outside the ovary. A dis- cussion of some of these inducing influences is given by Vincent and Dornfeld (1948). The follicle cells which surround the oo- cyte are usually considered to function in some positive manner during the growth of the oocyte — either by direct transfer and/or fusion of all or part of the follicle cell to the developing egg, or by acting as a transfening system for substances provided by the blood stream. In the cases in which the accessory cells contribute directly to the substance of the oocytes^ the accessory cells are known as nurse cells. At times, the egg cell and its associated nurse cells can be shown to have been the offspring of a single parent cell (see discussion in Wilson). Often the transfer of not only cyto- plasm but also nuclear material is observed in the oocyte-nurse cell relationship. (For recent histochemical studies of such rela- tionships see Schrader and Leuchtenberger, 1952.) It seems rea- sonable to assume that the "excess" amount of deoxyribonucleic acid found in the cytoplasm of oocytes may arise from such nuclear transfer (Zeuthen and Hoff-j0rgenson, 1952; Marshak, 1953). The nature of the cytoplasmic materials that are trans- ferred to the egg is discussed by Schrader and Leuchtenberger. Where direct transfer of large cellular fragments is concerned, it is obvious that there does exist a mechanism for considerable modification of oocyte development by its cellular environment. The existence of transferral mechanisms ascribed to the follicle cells is based primarily on inference. Inasmuch as this cellular layer is interposed between the blood stream and the egg, it has generally been assumed that the follicle cell functions in the transfer of materials to the oocyte cytoplasm. Little experimental evidence is available to confirm or deny the validity of such an assumption. The third area of, influence, that of the effects of the oocyte nucleus on its own cytoplasm, has been widely studied. The nu- clear changes which accompany the growth of the oocyte are so 4 THE OOCYTE striking that they have long been correlated with the cytoplasmic changes which occur. The tremendous enlargement of the nu- cleus, specialized modifications of chromosomal structure, and the formation of very large or very many nucleoli, have been in- terpreted as a reflection of nuclear intervention in cytoplasmic differentiation and growth. The many morphological studies of oocyte development have generally led to the conclusion that the nucleoli and the nucleic acid content of the nucleus play a major role in the oocyte. These studies include those of Montgomery (1898), Brachet (1950, 1955), Painter and Taylor (1942), Pani- jel (1951), and Wittek (1952). My own cytochemical and biochemical studies on the role of nucleoli and nucleic acids in the development of the egg are pri- marily concerned with ribonucleic acid (RNA). RNA has been assumed to be related to the synthetic activity of cells since the pioneering studies of Caspersson and co-workers and, independ- ently, Brachet, in the late nineteen thirties (Brachet, 1950; Cas- persson, 1950). Both of these workers believed that nuclear RNA was somehow involved in the cytoplasmic expression of genetic activity. This concept has attained new importance in a more specific way in that RNA has been repeatedly suggested as an agent which could receive genetic specificity residing in nuclear deoxyribonucleic acid and transfer this specificity to synthetic centers in the cytoplasm (Bounce, 1953; Rich and Watson, 1954; Goldstein and Plaut, 1955; Lockingen and DeBusk, 1955; Gamow and Yeas, 1955). In the experiments reviewed below, the starfish oocyte has been used in the study of nuclear RNA. These oocytes can be obtained readily in all stages of development, and in large quantities. They have a large nucleus and nucleolus, the latter of which can be isolated in considerable quantity. In addition, the nucleolus of the starfish oocyte appears to contain all the RNA of the nucleus (Vincent, 1952). By combining qualitative and quantitative his- tochemistry with direct chemical analyses of isolated nucleoli, it has been possible to study certain aspects of RNA metabolism in the starfish oocyte as it relates to the functional activities of this cell. W. S. VINCENT Growth of the Starfish Oocyte In order to relate some of the chemical changes observed in the nucleolus to oocyte growth, it was necessary to determine the size relationships of the nucleolus, nucleus, and cytoplasm of the starfish oocyte. The relationship between nucleus and cyto- plasm in the starfish oocyte follows a rigid pattern during growth. 125 r Fig. 1. Diameter of Asterias ruhens nuclei (large dots) and nucleoli (small dots) plotted against oocyte diameter. Ordinate: diameter of nucleo- lus or nucleus; abscissa: oocyte diameter in microns. Since I have been unable to determine an exact time scale for the growth of the oocyte, changes in size of the nucleus and nucleo- lus are plotted against oocyte diameter ( Fig. 1 ) . The relationship shown here seems to be a general one for oocytes, as similar gi'owth curves are found in amphibians (Gall, 1955) and in rats (Vincent, unpublished data). The volume of nucleus and cyto- plasm increases proportionately until the oocyte reaches about one-half its mature size (Fig. 2). At this time the rate of cyto- Z 10 THE OOCYTE OOCYTE DIAMETER (micro) 50 100 CONCENTRATION TOTAL AMOUNT OF RNA IN ^^'♦-2-— \ OF RNA IN NUCLEOLUS SOLUBLE RNA IN NUCLEOLUS h DIAMETER OF NUCLEOLUS INCREASE OF CYTOPLASMIC VOLUME OVER NUCLEAR VOLUME OOCYTE DIAMETER (micro) Fig. 2. n'owth of A comparison of some of the changes in nucleolar RNA and the the oocyte. (Data from Figs. 1 and 3.) plasmic contribution to cell size increases markedly. This is indi- cated by the break in the slope of the curve for the nucleus in Fig. 1. The diameters of the nucleolus and the oocyte increase proportionately until the latter attains about one-half its mature size, when all growth ceases (curve for the nucleolus in Fig. 1). RNA Content of Individual Nucleoli Some measurements on the RNA content and concentration of individual nucleoli are pertinent in this regard. The ultraviolet absorption of nucleoli isolated in distilled water was compared W. S. VINCENT 7 with the absorption of nucleoU which had been fixed in formahn prior to measurement. The RNA was then removed with hot per- chloric acid and the absorption of nucleoh was remeasured. The latter value was taken as the nonspecific absorption and was sub- tracted from the total absorption of the nucleolus; the difference was considered to be due to the RNA present. The results are shown in Fig. 3 in which the upper curve indicates the amount of RNA in formalin fixed nucleoli and the lower curve, the values 150 100 50 RNA CONTENT of INDIVIDUAL NUCLEOLI TOTAL ISO 100 50 05 10 15 20 " DIAMETER of NUCLEOLUS (Micro) Fig. 3. RNA content of individual Asterias ruhens nucleoli fixed in formalin (dotted and shaded area) and nucleoli isolated in distilled water (dotted area only). Measurements were made with a microphotometer after Lison (1950) employing Beck reflecting objective and condenser N.A. 0.65, lOX quartz ocular. Light source: 2537 A. line of low pressure mercury lamp. obtained from nucleoli isolated in distilled water. The difference between the two curves (shaded in the figure) is of interest. First, it indicates that there is less RNA in isolated nucleoli than in fixed nucleoli of the same size. Second, the pattern of difference in RNA content is proportional to the total amount present until the diameter of the nucleolus reaches about 12 microns. Beyond this, the difiierence between the two remains a constant value. A possible interpretation of this change will be discussed below. The dijfferences in RNA content found in this experiment comple- ment the radiophosphorus studies, suggesting again that starfish THE OOCYTE 3 c o U in X o o .^^ CD O ^ QJ M 'o bO D G *^H 5 X 5J lO ^ Tf *~ 1 — 1 O H-, ■t-T 00 50 ■fe? c < _o M-l (S o G G a C3 tG Si a •^ 15) J-t a O '^ 3 ^ t/5 'a *> rt n QJ 5b Cfl p O o O G 'g tn o :^ o "^ 43 CO ew d G -^ ■Td bb OJ T3 fe G ft W. S. VINCENT 9 nucleoli possess a RNA fraction which is readily soluble in distilled water. A summary of some of the data on nucleolar RNA is given in Fig. 2. Here the data are plotted against oocyte diameter and related to the changes in cytoplasmic volume which are shown on the lower part of the figure. In general, one finds that the oocyte enters a new phase of activity at about 100 microns, with the onset of rapid cytoplasmic growth. This can be attributed to the synthesis of yolk proteins. This new phase of activity is re- flected also in a drop in cytoplasmic RNA concentration not shown here, but readily apparent on slides stained with basic dyes. Such a cytoplasmic picture is typical of oocytes (Vincent and Dornfeld, 1948; Brachet, 1950; Panijel, 1951; Dalcq and Van Egmond, 1953 ) . The nucleolar changes which precede cyto- plasmic synthesis are of considerable interest ( Fig. 2 ) . In general one finds a shifting from curvilinear to linear relationships. RNA concentration drops very rapidly during early growth of the oo- cyte, but with the onset of the production of constant amounts of soluble RNA, maintains a constant concentration, although the nucleolus continues to grow. The nucleolus stops growing shortly after the onset of yolk deposition. RNA Metabolism in Starfish Oocytes By studying isolated nucleoli it has been possible to obtain some information about the metabolism of RNA in the oocyte nucleus. The advantages of working with pure preparations of isolated cell organelles are well known, and therefore a technique was developed whereby nucleoli could be isolated in quantity from starfish oocytes (Vincent, 1952). Distilled water was used as an isolation medium. Baltus (1954) reported that a modified technique in which sugar solutions of high density were used gave greater biochemical integrity than the distilled water me- dium. The technique was tedious and the yield low, however. The results and interpretations reported below are based prima- rily on studies carried out on nucleoli isolated in a Ca-sucrose medium. This process was developed when it became apparent that RNA was lost from the nucleoli into the distilled water iso- 10 THE OOCYTE lation medium. This isolation procedure was modified from the technique of Hogeboom et at. ( 1952 ) for the isolation of mam- malian cell fractions. I am indebted to Dr. Alfred Marshak for his suggestion to use this medium in the isolation of oocyte nu- cleoli. The nature of these preparations is indicated in Fig. 4. Chemical Froperties of Nucleoli. Some of the gross chemical properties of the starfish nucleolus are given in Table I. Table I. Chemical Properties of Isolated Nucleoli Constituent, % Enzymatic acti^dty RNA 2.2-4.6° Guanase 6 5" Adenosine deaminase h 3.15-5.6^ Nucleoside phosphorylase + + ' Adenylic acid (5) Trace" ** DPN synthetase + + " DNA Undetectable"*'^ Dipeptidase " Phosphorus 1.1" DPN reductase a 0.6-1.3^ Alkaline phosphatase " Nitrogen 16-20" Acid phosphatase ??" Calcium Absent"* Ribonuclease . d Dry matter 40-90' " Vincent, 1952. * Baltus, 1954. " Present communication. <* Vincent, 1955a. ' Vincent and Huxley, 1954. RNA Content of Nucleoli. The RNA concentration of nucleoli from two species of starfish, Asterias forhesii and A. ruhens, ap- pears to be about the same, averaging about 4% in each case. Baltus ( 1954 ) found about 5 % RNA in sucrose isolated nucleoli of A. ruhens where the author had found an average of 3.6% in the same species when isolated in distilled water. As shown be- low, no significant differences were found in the concentration of RNA in A. forhesii nucleoli isolated in either distilled water or the modified sucrose medium presented above. Changes of con- centration and total content of RNA of individual A. ruhens nu- cleoli during growth of the oocyte are presented in Figs. 2 and 3. The base content of the RNA of nucleoli and cytoplasmic gran- ules from A. ruhens is shown in Fig. 5 (data recalculated from Vincent, 1952). Significant differences are found in both guanine W. S. VINCENT 11 and uracil content between nucleolus and cytoplasm. Of interest here is the fact that the purine-pyrimidine ratio of the cytoplas- mic RNA is 1:1, while the nucleolar RNA varies considerably from this ratio. The cytoplasmic RNA of animals generally seems to follow the pattern found in the starfish of high guanine and cytosine values, and a purine-pyrimidine relationship of unity. Nuclear RNA's, on the other hand, depart from this relationship (Elson and Chargaff, 1954). Radioisotope Studies on Nuclear RNA. Numerous reports of 40 r 30 20 14C 30 20 -10 GUANINE ADENINE CYTOSINE URACIL Fig. 5. Moles of nitrogenaus base per 100 moles of phosphorus in RNA isolated from Asterias ruhens nucleoli (dotted) and cytoplasmic granules (shaded). (Recalculated from Vincent, 1952.) rapid synthesis of RNA by nuclei have appeared in the last few years (see Smellie, 1955, for review). I have carried out a series of studies on the metabolism of RNA in the starfish oocyte in which radiophosphorus was used as the tracer element. The ova- ries of the starfish are removed, the gonoducts are tied off and then placed in filtered sea water containing inorganic P^^ at ap- propriate levels. The usual dosage has been about 0.25 microcurie of P^" per milliliter of sea water. After exposure for appropriate intervals, the ovaries are removed, washed thoroughly in filtered sea water, and the nucleoli and other fractions are isolated. Some 12 THE OOCYTE preliminary reports of these experiments have appeared ( Vincent, 1954, 1955a,b). When ovaries of A. forbesii were exposed to P^~ at the dose indicated above for periods up to 8 hours at 23° C. and the nu- cleoh were isolated by the distilled water technique, the uptake of radiophosphorus was linear with time (see Fig. 6). As well, P^" was incorporated into nucleolar RNA at about the same, or at a lower, relative rate than it was incorporated into the cyto- plasmic RNA. This relation changes little during the time inter- vals studied (Fig. 7A). The radioautographic studies of Taylor (1953) and Ficq (1953) appeared just after the experiments in Figs. 6 and 7 were com- pleted. These workers found that the nucleolus incorporated radio- isotope into RNA up to 100 times more rapidly than the cytoplasm. In the light of these reports, the experiment described above was repeated in conjunction with a radioautographic control as fol- lows. Ovaries were exposed for 6 hours to sea water containing 0.25 microcurie of P^~ per milliliter. At the end of this time the ovaries were washed thoroughly, a small piece of tissue was fixed in formalin and processed for radioautography, and the nucleoli were isolated from the remainder of the tissue by the distilled water technique. The relative specific activity of the RNA ex- tracted from the isolated nucleoli and cytoplasm was found to be near unity; this is in agreement with the previous experiments. However, the radioautographs prepared from the fixed tissue in- dicated a much more rapid uptake of P^^ by nucleolar RNA, in agreement with the results of the other workers. This experiment indicated that considerable radioactivity incorporated into RNA was lost during isolation. Further demonstration of loss of radio- activity from the nucleoli during isolation was obtained by smear- ing the homogenates of the oocytes on nuclear track plates (Ko- dak NTB-1) at various steps of the isolation procedure. The nucleoli present in the fresh homogenates gave intense blacken- ing of the plate, whereas only a few dozen reduced silver grains were found under the isolated nucleoli. These results indicated a considerable loss of radioactivity from nucleoli during isolation in the distilled water medium. W. S. VINCENT 13 The sucrose procedure mentioned above was then developed in an attempt to maintain a higher integrity in the isolated nucle- oli. The results of the experiments after isolation in sucrose media are compared with the results obtained in pure water isolation in Figs. 6 and 7. The results obtained from these studies compare favorably with those obtained by Ficq ( 1953, 1955 ) in radioau- tographic studies of starfish nucleoli. Ficq, using a value of 10% RNA for cytoplasm, calculated a relative specific activity of nu- cleolar RNA of 100 X that of the cytoplasm. This value, taken a. z SUCROSE ISOLAIION H2O ISOLATION 4 Hours Fig. 6. Specific activity of nucleolar phosphorus after isolation in dis- tilled water and in Ca-sucrose solutions. from Vincent (1952), was for the mixed mitochondrial-micro- some granules of the oocyte cytoplasm. Actual values found for RNA concentration of acetone-dried starfish cytoplasm average 1.7% (range l.l%-2.5%). Ficq's value of lOOX should thus be reduced by a factor of at least 5, which brings the values found by radioautography into reasonable agreement with those found by direct analyses of isolated nucleoli. These data are in agreement with the findings of other workers that nuclear RNA appears to accumulate radiophosphorus more rapidly than cytoplasmic RNA. If one examines the distribution 14 THE OOCYTE of recovered isotope within the nucleokis and cytoplasm a more striking picture appears. As shown in Fig. 7B, at 2 hours, 75% of the total radiophosphorus present in the nucleolus is in the RNA fraction. A more extensive analysis of the time distribution of radiophosphorus in nucleoli is in progress, but the very high proportion of radiophosphorus present in the RNA fraction at the J5 < 5 A 0 SUCROSE isolationkI^ HjO ISOLATION 2 4 Hours 80 60 40 20 Bo NUCLEOLAR RNA CYTOPLASMIC RNA 3..^-'' 4 Hours Fig. 7. A. Comparison of relative specific activities of nucleolar and cytoplasmic RNA when nucleoli were isolated in Ca-sucrose (solid line) and in distilled water (dashed line). B. Per cent of total counts recovered from nucleoli and from cytoplasm which was found in the RNA fraction. short time interval strongly suggests that RNA synthesis occurs in, or immediately adjacent to, the nucleolus. Another fact which has emerged from these studies on isolated nucleoli is that the RNA concentration (3.6% for H-O, 3.97c for sucrose, range identical) is not significantly different in the nu- cleoli isolated by the two different techniques, although very sig- nificant differences in radioactivity of RNA in the two different preparations are apparent, W. S. VINCENT 15 The possibility exists that the higher isotope content of nucle- olar RNA is due to contamination by a small fraction of highly active material other than RNA. Although such a possibility has not been totally eliminated, the procedure used for extraction of RNA (Vincent, 1952) has been highly satisfactory in giving very good separation of this compound. Ultraviolet absorption meas- urements and dii'ect phosphorus determination have been made in every case. The two types of preparations are handled in iden- tical fashion, but agreement between the two has been found to be within the limits of experimental error. This procedure in- cludes thorough washing in cold O.IIV HCl and 60 minutes reflux- ing in boiling alcohol-ether prior to extraction of RNA in hot iV acid. On the basis of these considerations it seems likely that the increased activity present in sucrose nucleoli must be present in a small fraction of the RNA. This fraction is apparently lost in the distilled water media. Structural and Soluble RNA. The data given here are inter- preted as follows. The nucleolus of the starfish oocyte contains at least two types of RNA. One type is bound to the nucleolar struc- ture and has been characterized thus: it is most of the RNA pres- ent in the nucleolus; it is resistant to removal by water solutions; its base ratio differs from the cytoplasmic RNA in guanine and uracil content; it is metabolically relatively inactive. This is called structural RNA. The other RNA is characterized primarily from inference. It appears to be soluble in distilled water; to be pro- duced in amounts related to cytoplasmic synthetic activities; and to be synthesized at a rapid rate, in that it contains nearly all the label in isotope experiments. Its base composition is as yet un- known. This RNA fraction has been designated soluble RNA. ( See Vincent, 1955a, for a more extensive discussion of nucleolar RNA.) Functions of Nuclear RNA I should like to suggest some possible functions for these RNA fractions. The nucleolus contains the highest concentration of dry matter (predominantly protein) of any cell structure (Vincent, 1955a ) . A suggested role for the structural RNA is that it precip- 16 THE OOCYTE itates nuclear proteins, and aids in bringing about this very dehy- drated state. The soluble RNA, produced at a rapid rate and be- ing quite soluble, is apparently passed to the cytoplasm. Its ap- pearance and amount suggest that it is intimately associated with protein synthesis. These qualities are those required of a material which could transfer genetic specificity to the cytoplasm. The data given here will not distinguish between the two al- ternatives, (a) RNA is synthesized within the nucleolus, and ( b ) RNA is synthesized elsewhere in the nucleus and specifically accumulated by the nucleolus. The paper of Taylor et al. (1955) offers a possible answer to this problem, at least in the material which they used (Drosophila). They found that with high reso- lution radioautographs P^- appeared to be localized initially (1 hour after feeding of the isotope) immediately adjacent to the nucleolus, possibly associated with the chromosomes. This sug- gests that the RNA was initially synthesized adjacent to or on the surface of the nucleolus. Within two hours, the label appeared to be evenly distributed in the nucleolus. They found no appar- ent localization of activity about a single region, as would be ex- pected if the nucleolus organizer region were the sole site of synthesis of nucleolar materials. (For a discussion of the origin of nucleolar materials, see Vincent, 1955a. ) The work of Goldstein and Plaut ( 1955 ) also supports the con- tention that RNA is actually synthesized in the nucleus. Their results appear to demonstrate the passage of the label on RNA from the nucleus of the amoeba to the cytoplasm, but no reverse passage appears to occur. Such data seem to be best interpreted by concluding that RNA is synthesized in the nucleus and passes to the cytoplasm. On the other hand, cytoplasmic RNA does not appear to move in the reverse direction. A further question arises in considering the role of the nucle- olus in RNA relationships between nucleus and cytoplasm. Is the nucleolus at all necessary in a system of specificity transfer such as has been suggested? If specificity residing in DNA is trans- ferred to RNA structure by a template mechanism of synthesis (Bounce, 1953; Lockingen and DeBusk, 1955), the RNA product could then diffuse diiectly to the cytoplasm and there impart to W. S. VINCENT 17 the synthesis of new material genetic guidance. As it has been shown that the synthesis of both RNA and specific protein will occur in the absence of the nucleus, although at a diminished rate (Brachet, 1955), one can question the necessity of the nu- clear contribution to synthetic activities. The intimate correlation of nucleolar activities with cytoplasmic synthesis however (see above, also Vincent, 1955a) necessitates the presumption of some sort of relationship between nuclear, nucleolar, and cytoplasmic activities. The finding of Baltus that nucleoli possess high con- centrations (although less than 10% of the cellular activity) of nucleoside phosphorylase and DPN synthetase certainly suggests that the nucleolus can be involved in nucleotide synthesis. The autoradiographic results mentioned above, and the data reported in this paper strongly suggest that RNA synthesis itself occurs in the nucleolus. The data on the RNA of nucleolus and cytoplasm raise another problem in that the base content of the two regions differs widely. Such a difference is found in the starfish (in Fig. 3). This is not in agreement with the hypothesis that nuclear or nucleolar syn- thesis is the sole source of cytoplasmic RNA. The experiments presented above, however, strongly indicate the presence of at least two metabolically quite different RNA fractions in the nu- cleolus. Further characterization of these fractions may help to resolve this apparent disagreement. We are then faced with the conclusion that the chromosomes, the nucleolus, and the cytoplasm are all able to carry out de novo synthesis of RNA. Accepting the contention that specificity is conferred on RNA from a DNA template and that this RNA passes to the cytoplasm, we know that this specificity is main- tained for some time in the cytoplasm (Hammerling, 1953). Whether or not the cytoplasm is able to reduplicate specific RNA molecules (i.e., the plasmagene hypothesis of Spiegelman and Kamen, 1946), has not yet been adequately determined. Enucle- ation experiments suggest that if the cytoplasm is able to perform this function, reduplication is not able to keep up with destruc- tion, and therefore the system eventually runs down. We have mentioned two possibilities for RNA synthesis in the 18 THE OOCYTE nucleus: one, by specificity being imparted from the gene itself (i.e., DNA structure) to RNA structure through a template mechanism, the other by nucleolar enzymes. If one considers a simple case, that of a cell synthesizing a single specific protein molecule at a rapid rate, the question of template yield becomes significant. That is, can a single genetic locus or "master tem- plate" ( such as would limit the rate of production of any end prod- uct ) produce secondary templates rapidly enough to keep up with inactivation which seems to occur during cytoplasmic synthesis? If not, we can suggest the following as a plausible role for the nucleolus which is at least not contradictory to any evidence presently available and seems to offer possibilities for experimen- tal analysis. The nucleolus might be considered to be analogous to a dupli- cating machine with the gene providing the stencil, in that the nucleolus could be assumed to have a specialized mechanism for the rapid synthesis of many new RNA molecules from the specific structure provided by a genetic locus. In this way, identical spe- cific molecules of RNA in large numbers could be manufactured for transfer to the cytoplasm. This general pattern of chromosomal, nucleolar, and cytoplas- mic relationships is essentially that advanced by Caspersson and co-workers in 1940 (Caspersson, 1950, pp. 101 ff.) except that Caspersson's group did not have the advantage of the recent dis- coveries regarding the porosity of the nuclear membrane and the actual transfer of RNA to the cytoplasm. Caspersson's group therefore tended to emphasize the participation of basic proteins in the role which is ascribed to RNA in the preceding paragraph. Summary In this review of some of the many aspects of the origin and development of the oocyte, I have considered in detail only one of the problems which were mentioned in the introduction. In the study of the role of the nucleus in oocyte development re- viewed above, evidence has been presented that indicates that the RNA of the nucleus is intimately involved in the transfer of genetic specificity to the cytoplasm. The nuclear RNA of the W. S. VINCENT 19 starfish oocyte is shown to be metaboHcally heterogeneous m that a small fraction of the RNA possesses different solubility characteristics and is metabolically very active; the rest of the RNA is relatively inert according to these criteria. The correla- tion between chromosomal, nucleolar, and cytoplasmic events, particularly with respect to RNA, has led to a hypothesis of the role of RNA and the nucleolus in the transfer of genetically de- termined specificity to the cytoplasm. The RNA is considered to be a carrier of structural specificity imparted by genetic loci, and the nucleolus is suggested as a site of multiplication of these spe- cific RNA molecules which are then transferred to the cytoplasm. Acknowledgment Gratitude is expressed for the help of Dr. Jean Brachet and mem- bers of his staff in providing laboratory space and for many stimulat- ing conversations at the Laboratory of Experimental Morphology, University of Brussels, Belgium. The assistance of Dr. Maurice Errera is also acknowledged in making the measurements for Fig. 3. REFERENCES Baltus, E. 1954. Observations sur le role biochimique du nucleole. Biochim. et Biophys. Acta, 15, 263-67. Berrill, N. J., and C. K. Liu. 1948. Germplasm, Weismann, and Hy- drozoa. Quart. Rev. Biol, 23, 124-49. Brachet, J. 1950. Chemical Embryology. Interscience Publishers, New York-London. Brachet, J. 1955. The biological role of the pentose nucleic acids. In E. Chargaff and J. N. Davidson (eds.) The Nucleic Acids, Vol. II, pp. 475-519. Academic Press, New York. Caspersson, T. O. 1950. Cell Growth ami Cell Function. W. W. Norton and Co., New York. Dalcq, A., and M. Van Egmond. 1953. Effects de la centrifugation sur I'oocyte de trois mammiferes. Arch. Biol. (Liege), 64, 311-97. Dounce, A. L. 1953. Duplicating mechanism for peptide chain and nucleic acid synthesis. Enzijmologia, 15, 251-58. 20 THE OOCYTE Elson, D., and E. Chargaff. 1954. Regularities in the composition of pentose nucleic acids. Nature, 173, 1037-38. Everett, N. B. 1945. The present status of the germ cell problem in vertebrates. Biol. Revs., Cambridge Phil. Soc., 20, 45-55. Ficq, A. 1953. Incorporation in vitro de glycocolle-l-14C dans les oocytes d'Asteries. Experientia, 9, 377-79. Ficq, A. 1955. Etude autoradiographique du metabolisme de I'oocyte d'Asterias rubens au cours de las croissance. Arch. Biol. (Liege), 66, 509-24. FHckinger, R. A., and G. W. Nace. 1952. An investigation of proteins during the development of the amphibian embryo. Exptl. Cell Re- search, 3, 393-405. Gall, J. 1955, Problems of structure and function in the amphibian oocyte nucleus. Symposia Soc. Exptl. Biol., 9, 358-70. Gamow, G., and M. Yeas. 1955. Statistical correlation of protein and ribonucleic acid composition. Proc. Natl. Acad. Sci., 41, 1011-1019. Goldstein, L., and W. Plant. 1955. Direct evidence for nuclear syn- thesis of cytoplasmic ribose nucleic acid. Proc. Natl. Acad. Sci. U. S., 41, 874-80. Hammerling, J. 1953. Nucleo-cytoplasmic relationships in the develop- ment of Acetabularia. Intern. Rev. Cijtol., 2, 475-98. Hogeboom, G. H., W. C. Schneider, and M. S. Striebach. 1952. Cyto- chemical studies. V. On the isolation and biochemical properties of liver cell nuclei. /. Biol. Chem., 196, 111-20. Jones, R. M. 1949. The use of vital staining in the study of the origin of germ cells in the female rat. /. MorphoL, 84, 293-333. Lison, L. 1950. Etude et realisation d'un photometre a I'usage histo- logique. Acta Anat., 10, 333-47. Lockingen, L. S., and A. G. DeBusk. 1955. A model for intracellular transfer of DNA (gene) specificity. Proc. Natl. Acad. Sci. U. S., 41, 925-34. Marshak, A., and C. Marshak. 1953. Desoxyribonucleic acid in Arba- cia eggs. Exptl. Cell Research, 5, 288-300. Montgomery, T. H. 1898. Comparative cytological studies, with espe- cial reference to the morphology of the nucleolus. /. MorphoL, 15, 265-582. Nace, G. W. 1953. Serological studies of the blood of the developing chick embryo. /. Exptl. Zool, 122, 423-28. Needham, J. 1942. Biochemistry ami Morphogenesis. University Press, Cambridge, England. W. S. VINCENT 21 Nelsen, O. E. 1953. Comparative Embryology of the Vertebrates. Blakiston Co., New York. Painter, T. S., and A. N. Taylor. 1942. Nucleic acid storage in the toads egg. Proc. Natl. Acad. Sci. U. S., 28, 311-16. Panijel, J. 1951. Metabolisme des nucleoproteines dans la gametogenese et la fecondation. Hermann and Cie., Paris. Rich, A., and J. D. Watson. 1954. Some relations between DNA and RNA. Proc. Natl. Acad. Sci. U. S., 40, 759-64. Romanoff, A. L., and A. J. Romanoff. 1949. The Avian Egg. John Wiley and Sons, New York. Schrader, F., and C. Leuchtenberger. 1952. The origin of certain nu- tritive substances in the eggs of Hemiptera. Exptl. Cell Research, 3, 136-46. Smellie, R. M. S. 1955. The metabolism of nucleic acids. In E. Chargaff and J. N. Davidson (eds.), The Nucleic Acids. Vol. II. pp. 393-434. Academic Press, New York. Spiegelman, S., and M. D. Kamen. 1946. Genes and nucleoproteins in the synthesis of enzymes. Science, 104, 581-84. Taylor, J. H. 1953. Intracellular localization of labelled nucleic acid determined by autoradiographs. Science, 118, 555-57. Taylor, J. H., R. D. McMaster, and M. F. Calya. 1955. Autoradio- graphic study of incorporation of P^^ into ribonucleic acid at the intracellular level. Exptl. Cell Research, 9, 460-73. Telfer, W. H. 1954. Immunological studies of insect metamorphosis. II. The role of a sex-limited blood protein in egg formation by the Cecropia silkworm. /. Gen. Physiol, 37, 539-58. Tyler, A. 1955. Ontogeny of immunological properties. In B. H. Willier, P. A. Weiss, and V. Hamburger (eds.). Analysis of Devel- opment. W. B. Saunders Company, Philadelphia. Vincent, W. S. 1952. The isolation and chemical properties of the nu- cleoli of starfish oocytes. Proc. Natl. Acad. Sci. U. S., 38, 139-45. Vincent, W. S. 1954. P^- incorporation in starfish oocyte nucleoli. Biol. Bull, 107, 326. Vincent, W. S. 1955a. Structure and chemistry of nucleoli. Intern. Rev. Cytol, 4, 269-98. Vincent, W. S. 1955b. Phosphate metabolism of starfish oocyte nucle- oli. Biol Bull, 109, '353. Vincent, W. S., and E. J. Dornfeld. 1948. Localization and role of nu- cleic acids in the developing rat ovary. Am. J. Anat., 83, 437-70. 22 THE OOCYTE Vincent, W. S., and A. H. Huxley. 1954. The dry matter content of starfish oocyte nucleoli. Biol. Bull, 107, 290-91. Wilson, E. B. 1928. The Cell in Development and Heredity. Macmil- lan. New York. Wittek, M. 1952. La vitellogenese chez les Amphibiens. Arch. Biol, 63, 133-98. Zeuthen, E., and E. Hoff-J0rgenson, 1952. Evidence for cytoplasmic desoxyribosides in the frog's eggs. Nature, 169, 245-46. SPECIFIC EGG AND SPERM SUBSTANCES AND ACTIVATION OF THE EGG CHARLES B. METZ*: oceanographic institute, FLORIDA STATE UNIVERSITY, TALLAHASSEE At fertilization the egg undergoes a complex series of morpho- logical, physiological, biochemical and, in many cases, morpho- genetic changes that ultimately lead to the differentiation of the new individual. These initial changes constitute the activation of the egg. Under favorable conditions they proceed in an orderly sequence, and the precision of this sequence suggests that the activation changes are interrelated and that they all follow from a few or even a single reaction between the egg and the sperm. Many consider this problem of the activation initiating mecha- nism the central problem of fertilization, but it should be borne in mind that fertilization involves other problems as well. Thus fertilization is characterized by a high order of specificity. This reaches its ultimate expression in those hermaphroditic organ- isms that exhibit self sterility. Finally the sperm must approach the egg, penetrate any extraneous egg membranes, attach to and penetrate the egg surface. These problems of fertilization may be attacked experimen- tally in several ways and of these the one most likely to provide solutions would appear at present to be the study of the role of specific egg and sperm substances in fertilization. The literature of fertilization recounts many attempts to extract a "fertilizing substance" from sperm or to activate eggs with dead spenn. How- ever, none of these efforts appears to have stood the test of criti- cal examination in spite of initial claims of success. Even the most ** These studies have been supported in part by grants from the National Institutes of Health, the American Cancer Society, and the National Sci- ence Foundation. 23 24 EGG AND SPERM SUBSTANCES recent of these (Felix, 1955) may meet a similar fate. Neverthe- less, it should be recalled that paramecia killed by various agents including formalin will specifically activate paramecia of oppo- site mating type (Metz, 1947, 1954a). Therefore this direct ap- proach to the problem may still hold promise even for the more orthodox material. Although these efforts with metazoa appear to have failed in their primary objective, they and related studies have shown that a number of interesting substances may be obtained from both eggs and sperm. Certain of these agents clearly perform a neces- sary role in fertilization and the nature of some others suggests that they too may eventually be assigned a definite if not essen- tial function in fertilization. Serious study of such agents began with Frank R. Lillie's in- vestigation of the sperm isoagglutinin, fertilizin, which he obtained from the eggs of the sea urchin Arbacia and the annelid Nereis. Lillie's (1912, 1913b, 1914, 1919) studies produced the only comprehensive, though now outmoded, theory of fertiliza- tion, namely the Fertilizin Theory. Publication of the Fertilizin Theory stimulated a very considerable series of studies during the following decade. Interest then lagged until the late nineteen thirties when it was revived largely by Hartmann and his associ- ates in Germany and by Tyler in America. This interest has stead- ily increased but with it has come much conflicting data and many divergent statements, owing in part at least to differences in experimental material. Unfortunately, several of these areas of disagreement have not been resolved satisfactorily in the litera- ture. These will receive particular attention here. Some of the well-established aspects of the subject will be treated in less de- taij for they have been discussed in several reviews (Bielig and von Medem, 1949; Tyler, 1948a, 1949, 1955; Runnstrom, 1949a,b, 1952; Rothschild, 1951a,b). Classification of Sex Substances Fertilization presupposes a ripening of the parent animals and an appropriate release of the gametes. These processes have been found to be controlled in many instances by environmental con- C. B. METZ 25 ditions, by sex substances, hormones, etc., or a combination of these factors. These interesting prehminaries to fertihzation are beyond the scope of this review. Only those substances derived from and directly affecting the gametes themselves will be con- sidered here. The more spectacular and more readily investigated sex sub- stances of this sort appear in the natural fluids containing the gametes. Theii' presence in such fluids indicates that they are available under natural conditions and their action upon gam- etes, especially those of the opposite sex, constitutes strong evi- dence that they function in fertilization. In the demonstrable absence of sex substances in natural fluids, the interaction of gametes at fertilization may again be attributed to more or less specific sex substances, but in this case the agents must be insolu- ble in the fluid medium, firmly bound to the gametes or both. The sex substances of Paramecium are exclusively of this type (Metz, 1954a), but comparable agents have not been examined extensively in other organisms. Upon proper analysis such insolu- ble or bound sex substances may be found to occur widely, and an analysis of their role in fertilization may contribute much to our understanding of the physiology of fertilization. Aside from the agents that appear in the media surrounding the gametes, agents of various sorts may be obtained by more or less elaborate extraction of gametes. A number of such agents have definite action upon gametes of the same or opposite sex, but the question of their role in fertilization is not so readily set- tled for the agents may not be available under natural physiolog- ical conditions. Indeed they may be artificial products of the extraction procedures. The basic proteins (protamines, histones) obtained by acid extraction of sperm are notable recent examples of this. Agents from Eggs The simplest preparation from eggs is the supernatant water in which eggs have been suspended. Such egg water may be ex- pected to be charged with soluble and readily diffusible agents from the eggs. The most thoroughly studied of these agents is 26 EGG AND SPERM SUBSTANCES the glycoprotein fertilizin, which specifically agglutinates sperm. However, egg water has been reported to have a variety of other effects on sperm as well. In some instances these effects have also been attributed to fertilizin, but the possibility of action by other agents has not always been eliminated. The effects of egg water on sperm will be considered briefly before discussing fertilizin and the antifertilizin from eggs. Effects of Egg Water on Sperm Chemotaxis and the Approach of the Sperm. When eggs are inseminated, the sperm are frequently observed to accumulate in the vicinity of the egg. This effect is particularly striking in spe- cies whose eggs have a gelatinous coat, and it has led several investigators (Lillie, 1913b; Hartmann, Kuhn, Schartau, and Wallenfels, 1939; Vasseur and Hagstrom, 1946) to the view that diffusible agents (including echinochrome ) from the eggs have chemotactic action upon the sperm. Action of this kind appears to be well established in certain mosses and ferns (see Roths- child, 1951a, 1952, for recent account), but this effect has yet to be demonstrated convincingly in metazoa. Sperm can be shown to accumulate in capillary tubes containing egg water or in drops of egg water injected into sperm suspensions. However, as several investigators (Morgan, 1927; Rothschild, 1951a,b, 1952; Tyler, 1948a, 1955) have emphasized, the possibility of a trap action on the sperai has not been eliminated in these ex- periments. Such trap action could result from agglutination of the sperm or a combination of factors affecting sperm motility (see Rothschild, 1951b, for detailed discussion). In the absence of independent evidence it must be concluded that chemotaxis of metazoan sperm has not been demonstrated and that the sperm probably approach the egg by random motion. Accumulation of sperm about eggs is explained by an adhesion ( trapping ) of sperm in the jelly or at the egg surface following chance contact. Effect of Egg Water on Sperm Motility ami Respiration. Sper- matozoa of the sea urchin become intensely active in the vicinity of eggs. This observation suggests that some product diffusing from the egg activates the sperm. Lillie (1913b) confirmed this C. B. METZ 27 view by demonstrating that specific egg water increases the motil- ity of Arhacia sperm. This sperm activating action of egg water has been confirmed in a number of forms inckiding Megathura (Tyler, 1940a) and other molluscs (von Medem, 1945), the annelid Nereis (Lillie, 1913b), and several sea urchins. However, it does not appear to be a universal property of egg water for Metz (1945) found no sperm activating action of starfish egg water in spite of Loeb's ( 1915 ) claims to the contrary. The nature of the sperm activating agent in sea urchin egg water has been the subject of some dispute. Loeb (1915) be- lieved that this agent differed from fertilizin since egg water pre- pared from fertilizin-free eggs still activated sperm and because calcium ion was required for the agglutination but not the activat- ing action of egg water. In studies that aroused much interest at one time Hartmann, Kuhn, Schartau, and Wallenfels ( 1939 ) reported that crystalline preparations of the substituted naphthaquinone, echinochrome, in sea water solution activated the sperm of Arhacia. Echinochrome occurs in the fonn of granules in eggs of the genus Arhacia. Tyler (1939a) and Cornman (1941), however, were unable to confirm this action of echinoclii^ome on sperm of Strongylocentrotiis ptir- puratus and Arhacia punctulata respectively. Both of these inves- tigators used crystalline preparations. Cornman ( 1941 ) suggested that Hartmann's results may have resulted from failure to control the pH of the solutions. Bielig and Dohrn (1950) found no sperm activating action of echinochrome in buffered systems. It would appear, then, that there is no confirmatory evidence to sup- port the view that echinochrome activates sperm. Volatile or dialyzable sperm activating agents have been re- ported from egg waters by several investigators (Clowes and Bacliman, 1921; Cornman, 1941; Vasseur and Hagstrom, 1946). Tyler and Fox (1939, 1940), however, found that sperm activat- ing action remained with fertilizin through precipitation and dialysis. Tyler (1955) concluded that the activating action of egg water is associated with the fertilizin but that the fertilizin may be split to yield volatile or dialyzable fractions with sperm activating properties. 28 EGG AND SPERM SUBSTANCES In view of its stimulatory action on sperm motility it might be expected that egg water would also increase sperm respiration. This is indeed the case in the sea urchins Echinus escidentiis (Gray, 1928; Carter, 1930, 1931; Vasseur, 1949b), Strongtjlocen- trotus droehachiensis ( Vasseur, 1949b ) , and the gastropod Mega- thura (Tyler, 1948a; Krauss, 1950a). However, in some other forms, notably Arbacia punctulata (Hayashi, 1946), S. purptira- ttis (Tyler, 1948a; Spikes, 1949a), Echinus (Psanimechinus) 7niliaris (Gray, 1928; Carter, 1930, 1931), Lytechinus variegatus (Greenberg, unpublished), and L. pictus (Spikes, 1949a), egg water fails to increase sperm respiration. Indeed egg water treat- ment may actually reduce the rate of oxygen uptake of the sperm of some sea urchins (e.g., Lytechinus, Spikes, 1949a; Arbacia, Hayashi, 1946). Or the egg water may simply delay the usual rapid fall in respiratory rate characteristic of sea water suspen- sions of sperni (e.g., P. miliaris. Gray, 1928; Carter, 1931). Finally starfish egg water has no effect upon either sperm motility or respiration (Metz, 1945; Metz and Birky, unpublished). The agent in egg water which affects the respiration of sperm is usually assumed to be fertilizin. This view is supported by ex- periments (Spikes, 1949a) showing that the agglutinating action and respiratory effects ( in this case a lowering of oxygen uptake ) of sea urchin egg water are destroyed simultaneously by ultravi- olet irradiation. On the other hand, heating at 126° C. destroys the sperm agglutinating action but actually enhances the respira- tion increasing action of E. esculentus egg water. Egg water autoclaved in acid (pH 0.5) failed to increase the oxygen uptake of the sperm (Vasseur, 1949b; see also Biehg and Dohrn, 1950). The discovery (Tyler and Rothschild, 1951; Tyler, 1953; Roths- child and Tyler, 1954) that metal binding or chelating agents increase the motility of sperm may explain the motility increasing action of egg water. As suggested by Tyler and Metz ( 1955), sea urchin fertilizins may possess metal binding action. Low molecular weight substances in egg water and split products of fertilizin may also be found to have this effect. Finally, appreciation of the possibility of this mode of action should materially aid in a further identification of the active agent or agents in egg water. C. B. METZ 29 The situation with respect to increase in respiration appears to be somewhat more compHcated. In some forms metal binding agents fail to increase the oxygen uptake but do increase anaero- bic metabolism of sperm (e.g., Lytechinus pictus, Tyler and Rothschild, 1951 ) . In the starfish, however, similar agents clearly increase oxygen uptake of sperm (e.g., Asterias forbesii, Metz and Birky, 1955). Irrespective of whether the agents increase aerobic or anaerobic energy yielding systems, egg water and metal binding agents should have similar action on sperm respira- tion if their mechanisms of action are the same. In some forms such parallel action has been reported, but in others conflicting data prevent a clear decision. It is evident from the available data (Table I) that the problem requires further investigation. Effect of Egg Water on Sperm Morphology. Studies (re- viewed by Colwin and Colwin, this volume, Dan, 1956, and Metz, 1956) have shown that the sperm acrosome of a variety of forms (24 species in 4 phyla) undergoes a striking change under cer- tain conditions. The change, known as the acrosome reaction, in- volves the conversion of the normally compact acrosome into a filament. This discovery has contributed substantially to our understand- ing of the morphology of fertilization. Specifically it has provided a satisfactory explanation for the filaments that extend between the egg surface and the sperm in the early stages of fertilization of some forms (see Colwin and Colwin, this volume; Dan, 1954a, 1956). The acrosome reaction has been reported to result under a variety of conditions. These include contact of the sperm with a surface such as glass or the egg, exposure to alkaline sea water, or low temperature and treatment with excess calcium or egg water (Dan, 1952, 1954a,b; Dan and Wada, 1955; Wada, Collier, and Dan, 1956; A. L. Colwin and L. H. Colwin, 1955; L. H. Col- win and A. L. Colwin, 1955a,b; Metz and Morrill, 1955). In fact the reaction sometimes occurs in a high proportion of sperm after dilution with sea water (Rothschild and Tyler, 1955). Of these conditions the treatment with egg water is of special in- terest, for it suggests the possibility that the acrosome reaction 30 EGG AND SPERM SUBSTANCES Table I. Effect of Egg Water and Metal Binding Agents on Oxygen Uptake of Sea Urchin Sperm Species Reaction Reference Echinus esculentus -j- Egg water Psammechinus miliaris Lytechinus variegatus Strong ylocentrotus purpuratus + B (thyroxine, desiodo- thyroxine, diiodotyrosine, tyrosine? tryptophane?) — B (versene) — Egg water (maintains but does not increase QO2) + Egg water — B (thyroxine maintains but does not increase QO2) + B (albumen, trypsin, amino acids) Lytechinus pictus — Egg water — B (amino acids) — Egg water — B (alanine) — Egg water Arbacia punctulala — Egg water — B (seminal plasma) Gray, 1928; Carter. 1930, 1931; Vasseur, 1949b Carter, 1931 Rothschild and Tyler, 1954 Gray, 1928; Carter, 1930, 1931 Vasseur, 1949b; Vasseur, Wicklund and Runnstrom, 1950 Carter, 1931 Wicklund, 1954c; Vasseur, Wicklund and Runnstrom, 1950 Spikes, 1949a Tyler and Rothschild, 1951 Greenberg, unpublished Greenberg, unpublished Tyler, 1948a; Spikes, 1949a Hayashi, 1946 Hayashi, 1946 + increase in oxygen uptake. — no substantial increase. B agents which probably bind metals. can be a specific response to an agent from eggs of the species. So far no specificity studies have been carried out with respect to this action of egg water. Furthermore, no substantial attempt has yet been made to determine the active agent in egg water. Its identity with or separation from the sperm isoagglutinin, ferti- hzin, is a problem of special interest. The only data available indicate that egg water can agglutinate sea urchin sperm under conditions (absence of calcium) which do not permit formation C. B. METZ 31 of filaments (Dan, 1954b). On the other hand, a high incidence of filament formation is associated with a strong agglutinating action of egg water in Asterias and Nereis (Metz and Morrill, 1955 ) . In fact in Asterias the two effects are related to the extent that egg water fails to have either action in the absence of a metal binding agent. Egg water treatment also results in a "loosening" and displace- ment of the echinoderm sperm midpiece (Dan, 1954a; Popa, 1927; Rothschild and Tyler, 1955; Tyler, 1952). It is suggested (Tyler, 1952) that this effect may be related to the normal sep- aration of these parts that occurs during fertilization. Fertilizin Agglutination of Sperm The sperm agglutinating action of egg water was reported by several early investigators (see Morgan, 1927, for review) but it was not until Lillie's ( 1913a,b, 1919 ) classical studies on Arbacia and Nereis that a possible relation between this effect and the fertilization of the egg was fully appreciated. Lillie termed the agglutinating agent of egg water jeHilizin. Subse- quently others have employed a variety of terms for the same agent.* Lillie (1913b) showed that fertilizin agglutination of sperm was characterized by a high order of specificity, and this has been confinned on a wide scale (e.g., Metz, 1945; Tyler, 1949; Vasseur, 1951). Lillie (1913b) also found that the agglutinin was used up or absorbed in the agglutination reaction (see also Tyler, 1941; Metz, 1945; Monroy et al., 1954), and he first em- ployed an analogy with antigen-antibody reactions to explain the agglutination of sperm. More recently this analogy has been de- veloped further, particularly by Tyler and his students ( see espe- cially Tyler, 1941, 1948a, 1955; Spikes, 1949b; Metz, 1945). Mechanism of Agglutination. In keeping with modern im- munological theory agglutination is considered to result from a chemical reaction between two substances, antifertilizin on the sperm surface and the fertilizin dissolved in egg water. The reac- * Fertilizin (Lillie, 1913a,b; Tyler, 1948a) - Gynogamone II (Hartmann et al., 1940) = jelly coat substance (Runnstrom, 1949a). 32 EGG AND SPERM SUBSTANCES tion between fertilizin and antifertilizin is further considered to result from interaction of specific reactive groups or sites of fer- tilizin with other reactive groups on the sperm surface (anti- fertilizin). The reactive groups of fertilizin and antifertilizin are endowed with a high order of configurational complementariness and the specificity of fertilizin agglutination resides in the specific "lock and key" nature of this complementary relationship. Finally, Fig. 1. (a) Agglutination of sperm by interaction of multivalent ferti- lizin with antifertilizin of sperm surface, (b) Multivalent fertilizin split into univalent fragments, (c) Failure of sperm to agglutinate with multivalent fertilizin following saturation with univalent fertilizin. (From Rothschild, 1951b.) the agglutinating fertilizin molecule is considered to be multiva- lent with respect to its specific reactive groups. Agglutination results when several sperms are linked together by fertilizin molecules. These relations are illustrated schematically in Fig. 1. In most groups of organisms where it has been reported, fer- tilizin agglutination presents an orthodox antigen-antibody type reaction. In the echinoderms, however, certain interesting excep- tional features have been reported. These include spontaneous C. B. METZ 33 reversal of agglutination in the echinoids and the necessity of an "adjuvant" or second factor in asteroids. Spontaneous Reversal of Agglutination. Lillie (1913b) ob- served that upon mixing Arbacia sperm and egg water the sperm clumped together forming microscopically visible agglutinates, but within a few minutes these clumps broke down, releasing the individual sperms. Similar spontaneous reversal of agglutination has been reported in most of the echinoids so far examined. Lillie ( 1913b) showed that following such reversal of agglutination the sperm could not be reagglutinated by further addition of egg water. Evidently then, the sperm surface is altered in the course of agglutination and reversal. Tyler ( 1941 ) considered several possible explanations for this spontaneous reversal and failure of reagglutination and he concluded that the fertilizin molecule was probably split by action of the sperm into fragments each possessing only a single reactive group. Such univalent fragments should remain in combination with and saturate the antifertilizin groups of the sperm surface without causing agglutination. Fur- thermore, the univalent fragments should protect or block the sperm antifertilizin from reaction with the normal, multivalent, agglutinating fertilizin (see Fig. 1 for schematic illustration). In support of this view Tyler (1941) and Metz (1942a, 1954c) have shown that a number of agents (heating, proteolytic enzymes, x-radiation or ultraviolet radiation, H2O2 oxidation) will convert fertilizin to the univalent form. Such fertilizin will not agglutinate sperni but it will combine with sperm and render it unagglutina- ble by untreated fertilizin. As a final parallel with antigen-anti- body systems Tyler (1945) showed that rabbit antibody could also be converted to a nonagglutinating, univalent form. Fertilizin Agglutination of Starfish Sperm. Some early inves- tigators (Glaser, 1914; Woodward, 1918) reported direct agglu- tination of starfish sperm by specific egg water, but others (Just, 1930; Loeb, 1914; Tyler, 1941; Metz, 1945, 1954b; Metz and Donovan, 1950; Dan^ 1954a) have been unable to confirm this result in the same and other species. Conditions in the starfish were clarified by the discovery (Metz, 1944, 1945) that strong agglutination results when sperm are mixed with specific egg 34 EGG AND SPERM SUBSTANCES Plate I. Agglutination of Luidia clathrata sperm by versene-egg water. 1, Sperm in sea water. 2, Sperm in 0.0025M versene, pH 8.1. 3, Sperm in egg water. 4, Sperm in versene (0.0025M, pH 8.1) -egg water. 5, Spei-m in versene-egg water. Agglutination appears to be strictly head to head. (Phase contrast optics, 1-4, approximately 100 X; 5, approximately 430 X.) C. B. METZ 35 water and an "adjuvant" (Plate I). This fertilizin-adjuvant ag- glutination has been reported in ten species of starfish (Metz, 1945, 1954b; Metz and Donovan, 1950; Dan, 1954a). In this reaction the fertilizin is species specific; the adjuvant is not spe- cific. The first adjuvants found were poorly defined materials ( animal sera, hen's egg white ) , but subsequent study ( Metz and Donovan, 1950; Metz, 1954b) revealed that alkali, amino acids, peptides, SH containing substances, versene, and other metal binding or chelating agents were effective adjuvants. Since metal binding is a common property of these diverse agents and since the adjuvant action is inhibited by addition of metal cations (Metz, 1954b), it appears that the essential function of the ad- juvant is a removal of metal ions. This view explains certain earlier observations. Among these is the report ( Metz, 1945 ) that dena- tmation by heating or ultraviolet radiation increases the adjuvant action of egg white. This enhanced action probably results from exposure of additional SH groups (Mirsky and Anson, 1934). The adjuvant apparently acts upon the sperm, not upon the fertilizin in the fertilizin-adjuvant agglutination reaction. This follows from the fact that sperm, after removal from the adjuvant, can agglutinate on addition of fertilizin. In one experiment (Metz, 1945) spenn were washed free of adjuvant (hen's egg white ) with sea water. In another ( unpublished ) the alkali used as the adjuvant was neutralized with acid. This experiment is summarized in Table II. In both of these experiments the sperm reverted rather rapidly to the nonagglutinable condition. This is explained by the addition of metal ions ( sea water ) in the first experiment. The action of alkali in the second experiment is as- sumed to involve binding metal ions as insoluble or un-ionized hydroxides. Upon addition of acid these hydroxides would be neutralized and thus free the metal cations. Presumably the sperm would remain agglutinable if the adjuvant (and bound met- als) were washed away with salt solutions lacking the offending metal ions. These experiments show that the adjuvants act primarily upon the sperm. Accordingly the fertilizin must be multivalent. This view is supported to the extent that starfish fertilizin can be con- 36 EGG AND SPERM SUBSTANCES Table II. Effect of pH on Fertilizin Agglutination of Patiria Sperm" Sample A Sample C pH7.8 Portion of pH 8.8 (initial pH of Sample B sample readjusted suspension) pH raised to 8.8 to pH 7.3 After After After After After After 10 min. 40 min. 10 min. 40 min. 10 min. 40 min. Fertilizin Patiria — — + + + + + + + ± Astropeden - - - + + + - — Lytechinus — — — + + + — — S. purpuratus — — — + + + — — Sea water — — — + — — Adjuvant (hen's egg white) phis Patiria fertihzin + + + + " Of three samples from a Patiria miniata sperm suspension A served as a control (pH 7.8), B was raised to pH 8.8 with O.lA'' NaOH, and C was raised to pH 8.8 and then lowered to pH 7.3. Ten and 40 minutes after final pH adjustments the suspensions were tested for agglutination with homologous and heterologous fertilizins. Sample A failed to agglutinate. Sample B aggluti- nated specifically at 10 minutes, but gave nonspecific reactions at 40 minutes. Sample C agglutinated weakly, but specifically at 10 minutes, very weakly, if at all, at 40 minutes. verted to the nonagglutinating, univalent form by ultraviolet radiation (Metz, 1945). The experiments cited, then, show that the metal ions affect the ability of starfish sperm to agglutinate with multivalent fertilizin. The metals probably combine with and block the reactive sites of most of the sperm surface antifertilizin. Such action implies that strong anionic groups, SH groups, amino acid, or other groups with a strong affinity for metal ions are essential constit- uents of the "active sites" of starfish antifertilizin, and that these groups must be free to combine with appropriate groups of fer- tilizin. Evidence for such blocking action was obtained in a series of absorption experiments. These showed that untreated starfish sperm can bind fertilizin to a limited extent but that this fertilizin C. B. METZ 37 binding capacity is increased by a factor of at least 128 in the presence of an adjuvant (Metz, 1945). Role of Fertilizin in Fertilization The highly specific and dramatic sperm agglutinating action of fertilizin suggests that this agent may play an important if not essential role in fertiHzation. The effect of egg water on sperm motility, respiration, and the sperm acrosome ( see effects of egg water) may result from action of fertilizin. These effects may be aids to or essential for fertilization. Evidence concerning a more direct role of fertilizin in fertiliza- tion and especially a direct role in the activation of the egg has centered about the following considerations : (a) the universality of occurrence of fertilizin among metazoa, (b) the source of fertilizin, (c) the effect of removal and addition of fertilizin on the fertilizability of the egg, and ( d ) the effect of fertilizin on the fertilizing capacity of sperm. The evidence with regard to these points has been reviewed in detail by Tyler (1941, 1948a, 1955). Therefore it will suffice here to summarize this information and emphasize the most recent work. Occurrence of Fei'tilizin. If fertilizin has a fundamental role in fertilization, it might be expected to occur widely if not uni- versally. Lillie (1919) and Just (1930) believed this to be the true, although they demonstrated agglutination of sperm by egg water in only a limited number of forms. Subsequently, others have added to the Hst (Tyler, 1948a; Bielig and von Medem, 1949) so that positive cases are now known in four of the major phyla (molluscs, annelids, echinoderms, chordates). However, the fact remains that the number of species included is small. Nevertheless, the list is being extended. This extension involves both the discovery of orthodox egg water agglutination of sperm as new species are examined and the development of special methods for detecting fertilizin in egg waters that ordinarily fail to agglutinate homologous sperm. Two such methods have been employed successfully. The first is based on the premise that fer- 38 EGG AND SPERM SUBSTANCES tilizin should combine with sperm even though it fails to aggluti- nate. Tyler (1941) detected such combination in Urechis caupo and Patiria niiniata by the inhibiting action of fertilizin on the fertilizing power of the sperm. Mactra (Spisula) solidissima fer- tilizin was first detected by the same method (Metz and Dono- van, 1949). The second method involves the conversion of the normally nonagglutinating system to an agglutinating one by the addition of an adjuvant. The presence of fertilizin in Patiria egg water, originally demonstrated by the first method, was subse- quently confirmed by this second method. Later studies employ- ing the adjuvant technique have demonstrated agglutinating fertilizins in egg waters of nine other asteroids (see section on starfish agglutination). Finally calcium appears to be necessary for agglutination in some (Loeb, 1915; Vasseur, 1949a) but not all (Dan, 1954b) species. Fortification of the system with an excess of this ion may enhance agglutination (Vasseur, 1949a) or even induce agglutination in nonagglutinating systems. In a number of forms (see effects on sperm morphology, above) egg water has been shown to induce the sperm acrosome reaction. Although it is premature to conclude that fertilizin is the re- sponsible agent in this effect, it is possible that such may prove to be the case and that the acrosome reaction can be developed into a sensitive test for fertilizin. As suggested by the above account, failure to obtain agglutina- tion may result from several causes. In some cases this may be due to a high calcium requirement. In the starfish, metal ions prevent agglutination by blocking most of the antifertilizin gi'oups of the sperm. In some species fertilizin may exist mainly or entirely in the univalent form (Tyler, 1941, 1948a). Finally, fer- tilizin or fertilizin-like material may occur in an insoluble form, firmly bound to or built into the egg surface. The mating type substances of Paramecium are of this nature ( Metz, 1954a ) . Like- wise sea urchin eggs appear to retain a bound layer of fertilizin at their surfaces after the outer gelatinous coat has been removed by acid treatment or enzymatic digestion (Tyler, 1941; Tyler and Metz, 1955). As Tyler (1948a) suggests, the normally jellyless C. B. METZ 39 eggs of many forms may prove to be comparable to jelly-free sea urchin eggs with respect to fertilizin. It should be evident from the foregoing discussion that ferti- lizin may exist in several fomis and that any one of a variety of methods may be required to demonstrate it. Clearly then, the pos- sibility that fertilizin, in one form or another, occurs widely or universally among eggs cannot yet be rejected. Source of Fertilizin. Lillie (1914) believed that fertilizin was produced continuously by Arbacia eggs. Loeb (1914, 1915), how- ever, found that after dissolving the gelatinous coat in acidified sea water no fertilizin could be obtained from sea urchin eggs. This has been confirmed repeatedly (Tyler and Fox, 1939, 1940; Tyler, 1940a; Hartmann, Schartau, and Wallenfels, 1940; see also Runnstrom, 1949a; Tyler, 1948a). In fact fertilizin appears to be the sole component of the jelly for Tyler ( 1949 ) and Runnstrom, Tiselius, and Vasseur ( 1942 ) found that solutions of the coat are homogeneous upon electrophoretic or ultacentrifugal analysis. Although identification of fertilizin with the jelly layer of sea urchin eggs is well established, the cellular origin of the jelly is disputed. For example, on the basis of histological studies Jenkin- son (1911) believed that the jelly is layed down by the follicle cells of the ovary, whereas Lindahl (1932) concluded that the egg itself produces the jelly. Later Vasseur (1951) obtained fer- tilizin from the ovaries of spent sea urchins. Since these ovaries lacked eggs, Vasseur concluded that the fertilizin was produced by ovarian tissue, not by eggs. However, fertilizin from eggs may have remained in the ovary after the eggs were shed. In this connection it is of interest that Mellita quinquiesper- forata egg suspensions obtained by the KCl injection method frequently contain jellies which do not enclose eggs. Indeed, over 90% of jellies from some females are empty (Metz, unpub- lished ) . These empty jellies may have been laid down independ- ently by ovarian tissue in the absence of eggs, or the eggs may have escaped from the jellies. The former possibility seems more likely since the suspensions do not contain proportionate numbers of naked eggs. Nevertheless, in the writer's opinion definite con- 40 EGG AND SPERM SUBSTANCES elusions concerning the cellular origin of the echinoid egg jelly must await further study. Perhaps an investigation of the cellular origin of the echinochrome bodies in sanddollar egg jellies might be rewarding in this regard. Although the jelly surrounding the sea urchin egg is clearly the main source of fertilizin, studies indicate that fertilizin or fer- tilizin like material may also be obtained from less superficial regions of the egg. Thus Motomura ( 1950a ) reported extraction of a sperm agglutinin from unfertilized, jellyless S. piilcherrimus eggs by prolonged standing in sea water, by boiling, and by shak- ing jellyless, fertilized eggs to break the fertilization membranes. On the basis of these experiments Motomura concluded that the sea urchin egg produces a second sperm agglutinin, cytofertilizin. Following certain objections to these experiments (Byers, 1951) Motomura (1953a,b) extended his studies. He found that the surrounding sea water becomes charged with sperm agglutinin upon hatching of blastulae from jellyless eggs. Furthermore, jellyless eggs treated with NaSCN to remove the vitelline mem- brane charge the sea water with agglutinin during the first ten minutes after fertilization. Finally Temnopleurus hardeivickii eggs normally fail to produce a satisfactory agglutinating egg water, but after removal of the jelly and membranes fertilized eggs, blastulae or gastrulae of this species charge the sea water with sperm agglutinin. The exact source of Motomura's cytofer- tilizin has yet to be established, but the hyaline layer material of the egg is one likely possibility ( Tyler, Monroy, and Metz, 1956 ) . This view is supported by cytochemical studies at least to the extent that the egg jelly, the hyaline layer material and the cor- tical granules give positive tests for acid polysaccharides ( Monne and Slautterback, 1950; Monne and Harde, 1951). According to Motomura's experiments the egg jelly fertilizin and cytofertilizin have different pH optima for extraction. This suggests that they may be chemically different, although it may only reflect differences in bonding of the same agent. In any event a comparison of the biological properties of the two agents should be made. Information on the specificity of cytofertilizin agglutination and the effect of cytofertilizin on sperm that have C. B. METZ 41 reversed after agglutination with normal fertilizin will be awaited with particular interest. The source of fertilizin has been examined seriously in only two forms other than the sea urchin, namely the gastropod, Mega- thura, and the annelid, Nereis. Megathum (Tyler, 1940a) is similar to the sea urchin. The unfertilized egg is suiTOunded by a gelatinous coat. When this coat is removed, no further fertilizin can be obtained from the egg. Conditions in Nereis are more complex. The unfertilized egg has no substantial gelatinous coat (Lillie, 1911). However, upon activation of the egg a thick jelly is extruded. The mechanism of this release of jelly has been investigated most recently by Cos- tello (1949). He has shown that the jelly arises by a transforma- tion of certain granules released from the egg cortex. The egg water of unfertilized Nereis eggs contains fertilizin (Lillie, 1913b). Therefore release of some fertilizin does not depend upon visible extrusion of the jelly. However, solutions of the jelly, obtained by treating eggs with alkaline sea water (method of Novikojff, 1939; Costello, 1945) give higher spemi agglutina- tion titers than normal egg water controls (Tyler, 1948b). This suggests that the jelly material of the Nereis egg may be the fertilizin. To account for the fertilizin in normal egg water Tyler suggests that some of the jelly material may diffuse from the normal, unfertilized egg. In any event these experiments are evidence that the fertilizin of Nereis is produced by the egg itself, not by follicle cells of the ovary. Effect of Removal and Addition of Fertilizin on Fertilizability of the Egg. If fertilizin is essential for fertilization, fertilizin-free eggs should fail to fertilize and fertilizability should be restored upon addition of fertilizin. This test for the essentiality of fer- tilizin has been attempted by a number of workers (see Tyler, 1948a for thorough review of this work). Fertilizin cannot be demonstrated in washings from acid-treated, jellyless eggs. Never- theless such eggs can be fertilized. Clearly then, fertilizin is not essential for fertilization or the extraction procedure fails to re- move all the fertilizin. In support of the latter view Frank ( 1939 ) and Tyler ( 1941 ) have shown that jellyless eggs agglutinate when 42 EGG AND SPERM SUBSTANCES treated with antifertilizin from sperm. This result indicates that some fertihzin remains bound to the egg surface. Similar results have been obtained following treatment of Arbacia eggs with ciystalline trypsin and chymotrypsin (Tyler and Metz, 1955). The digestion removes the egg jellies, but these eggs fertilize when inseminated. Interpretation of the ac- tion of the enzymes is complicated by the fact that the treated eggs fail to elevate fertilization membranes upon fertilization. This suggests that the enzymes digest not only the jelly but also the vitelline membrane of the egg and any fertilizin bound to it, including that remaining after acid sea water treatment. Even though the vitelline membrane may be removed and in spite of the fact that such eggs show a greater receptivity to foreign sperm (Hultin, 1948a,b; Bohus Jensen, 1953a,b; Tyler and Metz, 1955), they still agglutinate on addition of antifertilizin (Tyler and Metz, 1955). If it is assumed that antifertilizin suffers no loss in specificity upon extraction from the sperm, it must be concluded that some fertilizin, possible cytofertilizin, resists the action of trypsin for some time. Evidently, then, an unequivocal test of the fertilizability of the fertilizin-free egg has yet to be devised. Nevertheless, removal of the jelly by acid or enzymes lowers the fertilizability of eggs to the extent that more sperm are required to obtain a given per- centage of fertilized eggs. Furthermore, the fertilizability and antifertilizin agglutinability decrease in parallel fashion upon prolonged treatment with trypsin (Tyler and Metz, 1955). Fi- nally, antifertilizin treatment greatly reduces the fertilizability of both control and jellyless eggs (Tyler, 1941; Tyler and Metz, 1955 ) . These experiments, then, show quite clearly that fertilizin in the form of the jelly layer aids fertilization. Furthermore, they strongly suggest that fertilizin at the egg surface is essential for fertilization. If fertilizin is an aid to or essential for fertilization, addition of more fertilizin to eggs, especially jellyless eggs, might be expected to improve fertilization. However, as Tyler ( 1941 ) points out, no way has yet been found to cause added fertilizin to assume its normal relationship ( a jelly layer ) to the egg. Furthermore, Mon- C. B. METZ 43 roy et al. ( 1954 ) have shown that f ertihzm in solution and in the gel form have somewhat different chemical as well as physical properties. Finally, fertilizin in concentrated solution can act upon and saturate the sperm while the latter is still distant from the egg (see below). In view of these considerations it is not sur- prising that addition of fertilizin fails to increase the fertilizability of eggs, even jellyless eggs (Tyler, 1941). Effect of Fertilizin on the Fertilizing Capacity of Sperm. In the sea urchin, sperm agglutination spontaneously reverses. If the sperm is initially treated with an excess of fertilizin, the "re- versed" sperm fail to reagglutinate on further addition of ferti- lizin. This indicates that the surface antifertilizin of the spemi is bound or blocked by univalent fertilizin ( Fig. 1 ) . Such saturation of the surface might be expected to have a profound eflPect upon the fertilizing capacity of the sperm. However, it should be noted at the outset that tests of the fertilizability of reversed sperm are critical only if the sperm surface antifertilizin is completely blocked or unless an elaborate quantitative measurement of sperm fertilizability is performed. With this factor controlled Tyler ( 1941 ) found a marked reduction in fertilizing capacity of reversed S. purpuratus sperm. Similarly, reduction in fertilizing power has been reported in some other sea urchins (e.g., Dan, 1954b). On the other hand, a number of investigators have ob- tained no significant reduction in fertilizing capacity of fertilizin- treated sperm of other species (e.g., Fuchs, 1914, 1915; Hagstrom, 1956; Runnstrom, 1949a). Most of these reports failed to take into account the degree of saturation of the sperm with fertilizin. Finally, in studies of several fornis (Metz, 1942b; Metz and Donovan, 1949; Tyler and Metz, 1955), species differences were found. In Mactra (Spistila) solidissima, and S. purpuratus, fer- tilizin treatment markedly reduced the fertilizing capacity of the sperm, but it failed to do so in Arhacia punctulata* and Mellita quinquiespeiiorata. However, these species differences appear to be relative, not absolute, for Tyler and Metz ( 1955 ) did find a reduction in fertilizing capacity when the reversed sperm of * Lillie (1913b) reported reduction of fertilizing capacity of Arhacia sperm after fertilizin treatment. 44 EGG AND SPERM SUBSTANCES Arbacia and Mellita were tested for their ability to fertilize jelly- less ( trypsin-treated ) eggs. Evidently jellyless eggs provide a more sensitive system for testing the effect of fertilizin on sperm, and use of this system may resolve the conflicting data of the past. The experimental results reviewed in this section, then, show that fertilizin at the egg surface facilitates fertilization whereas the same agent in solution, by virtue of its combination with sperm, acts as a barrier to fertilization. The apparent inconsist- ency of these observations is resolved (Tyler, 1941, 1948a, 1955; Tyler and Metz, 1955) in terms of an essential role of fertilizin in linking the sperm to the egg surface. According to this concept, fertilizin molecules attached to or built into the egg surface com- bine with antifertilizin groups of the sperm to effect the linkage. Thus if the free fertilizin groups at the egg surface are reduced in number by acid extraction, enzymatic digestion or blockage with antifertilizin, the fertilizability of the egg is reduced. Like- wise, blocking the antifertilizin groups of the sperm with univa- lent fertilizin (e.g., "reversed" sperm) or univalent antisera (Tyler, 1946a) reduces the number of such groups available for combination with the egg surface fertilizin and correspondingly lowers the fertilizing capacity of the sperm. It might be expected that the sperm surface would be satu- rated by fertilizin during its passage through the egg jelly and before the sperm reaches the egg surface fertilizin. In considering this possibility Tyler (1948a) suggests that the specific reactive groups of fertilizin in the gel form may be bound by cross link- ages. Such linked groups would not be freely available to com- bine with the sperm. Differences in the metachromatic staining properties of fertilizin in the gel form and in sea water solution ( Monroy et at, 1954 ) are in keeping with this view. Chemistry of Fertilizin The chemistry of fertilizin has been reviewed on several occa- sions (Tyler, 1948a, 1949, 1955; Runnstrom, 1949a, 1952; Vasseur, 1952a ) . Therefore the present treatment will be confined largely to recent work. C. B. METZ 45 Chemical studies have been restricted mainly to the fertilizins from sea urchins. Sea urchin fertilizin (egg jelly or jelly coat substance) can be prepared in rather pure form by brief extrac- tion of washed eggs in acid solution ( see Tyler 1949, for method ) . Such preparations give a single component on ultracentrifugal and electrophoretic examination. These purified fertilizins have been employed in the analytical studies. They are found to be glycoproteins which, on hydrolysis, yield a number of amino acids, one or a few monosaccharides and considerable sulfate. The amino acid composition of several species has been examined. In S. purpurattis chromatographic analyses (Tyler, 1948c, 1949, 1955 ) have revealed fifteen amino acid spots identified as glycine, alanine, serine, threonine, valine, leucine, isoleucine, aspartic, glutamic, arginine, lysine, phenylalanine, tyrosine, tryptophane, and proline. In Echinus esculent us, Paracentrotus lividus, and Strongylocentrotus droebachiensis nine (lysine, arginine, his- tidine, valine, isoleucine, threonine, phenylalanine, tyrosine, and proline) were demonstrated by microbiological assay (Vasseur, 1948, 1952a). Aspartic and glutamic acids were also found in these fertilizins by chromatography (Vasseur, 1948). Thus a considerable number of amino acids are present in sea urchin fertilizin. The proportions of the amino acids showed little varia- tion among the above three species studied by Vasseur. However, Echinocardium cordatum fertilizin was low in lysine and valine. The monosaccharides found in hydrolyzates of several species are listed in Table III. In contradistinction to the amino acids, the number of different monosaccharides in any species is small. The galactose of E. esculentus occurs mainly in the "unnatural" or L-form (Vasseur, 1950). Sulfate has been found to the extent of about 25% in most fertilizins (e.g., S. droebachiensis, Vasseur, 1947; S. purpuratus, Tyler, 1949). Brissopsis hjrifera fertilizin, on the other hand, is low in sulfate (about 4%). This, correlated with the fact that B. hjri- fera fertilizin does not agglutinate sperm (Vasseur, 1952a; Vas- seur and Carlsen, 1948'; Vasseur and Hagstrom, 1946), suggests that sulfate gi^oups are essential for helping endow the molecule with agglutinative properties (see below). 46 EGG AND SPERM SUBSTANCES Table III. Monosaccharides Identified in Hydrolyzatcs of Sea Urchin Fertilizins Monosaccharide Galac- Ghi- Fruc- Species Fucose tose cose tose Reference Echinocardium cor datum X Vasseur, 1952a Strong ylocentrotus droebachiensis X X Vasseur, 1952a StrongyJoccntrotus purpuratus X X Tyler, 1955 Arbacia lixula X X Minganti, cited by Monroy et al, 1954 Paracentrotus lividus X X Vasseur, 1952a Echinus esculentus X Vasseur, 1952a Echinorachnius parnia X Bishop & Metz, 1952 So far fertilizin has not been separated into polysaccharide and protein moieties, at least under mild conditions. Upon oxidation of Echinocardium fertilizin with periodate, Vassem* ( 1952b ) obtained a precipitate. Analysis of the precipitate and supernatant revealed nitrogen in both fractions. Vasseur concluded that the nitrogen in the latter was not fertilizin nitrogen. The carbohydrate was largely confined to the supernatant. Since both the super- natant and precipitate fractions were nondialyzable, Vasseur concluded that he had achieved a separation into protein and carbohydrate components. However, it appears that the evidence for separate protein and carbohydrate moieties in the fertilizin molecule is incomplete. The sulfate is probably esterified to the carbohydrate. Only half of the fucose of Echinocardium cordatum fertilizin is readily attacked by periodate. From this Vasseur (1952b) suggests that the carbohydrate portion consists of a branching fucose sulfate chain. Each fucose sulfate of the "backbone" is linked "laterally" to a single fucose sulfate side group. Only the latter have a-glycol groups free to react with the periodate. Tyler (1948c, 1949) offered another explanation for the failure to readily obtain a sep- aration into protein and polysaccharide components, namely, that fertilizin may not be a conjugated protein in the usual sense, C. B. METZ 47 but that the sugars and amino acids are interhnked. In any event such interhnking is probably not a simple alternation of amino acids and sugars since fertilizin contains peptide linkages as in- dicated by a positive biuret reaction and inactivation by proteo- lytic enzymes (Tyler and Fox, 1940). As might be expected from their- high sulfate content fertilizins are strongly acidic proteins. Even near pH 2 they migrate rapidly to the anode during electrophoresis (Runnstrom, Tiselius, and Vasseur, 1942; Tyler, 1949; Tyler, Burbank, and Tyler, 1954b). Values for the molecular weight of fertilizin have been cal- culated from ultracentrifugation data. Earlier values [e.g., 82,000 for S, purpuratiis fertilizin (Tyler, 1949)] were based on the assumption of a spherical molecule. However, the fertilizin molecule is clearly not spherical for the substance forms gels, precipitates as fibrils (Monroy, 1955), exhibits flow birefringence, and gives sedimentation values that vary with concentration. Accordingly, revised estimates of the molecular weight and axial ratios have been made from more recent sedimentation and dif- fusion data. These give for Arbacia pimctulata fertilizin a molec- ular weight of 300,000 and an axial ratio of 28:1 (Tyler, Burbank, and Tyler, 1954a; Tyler, 1956 ) . When corrected for water of hy- dration, this axial ratio becomes 20:1 (Tyler, 1956). Apparently no detailed study of the chemistry of univalent sea urchin fertilizin has yet been made. Univalent fertilizin, like the multivalent agent, is nondialyzable (Tyler, 1941) and gives the mucin-clot reaction, indicating that the univalent fragments are relatively large stable subunits of fertilizin (Krauss, 1949). Pro- duction of the univalent agent by proteolytic enzymes indicates that peptide bonds may form one type of linkage between such units. Clear understanding of the chemistry of the "reactive groups" of fertilizin has yet to be obtained. Studies of Arbacia punctulata fertilizin with a series of "protein group reagents" ( Metz, 1954c; Runnstrom, 1935 ) showed that free S — S, SH, phenolic and amino groups are not essential constituents of the receptor groups of fertilizin. Strong oxidizing agents (H2O2) destroy the sperm ag- glutinating action by conversion to the univalent form. They fail 48 EGG AND SPERM SUBSTANCES to inactivate the receptors. Reducing agents do not destroy the aggkitinating activity. Periodate oxidation destroys not only the aggkitinating action (Immers and Vasseur, 1949) but also the receptors as shown by inhibition tests ( Metz, 1954c ) . This action suggests that adjacent hydroxyl groups of carbohydrate may be essential, but the action of periodate is rather drastic, at least on long exposure ( Vasseur, 1952b) and may involve configurational changes in the molecule which are largely responsible for the inactivation. Coupling with diazo salts also inactivates the receptor groups (Metz, 1954c), but diazo compounds react with such a variety of groups that fmther study will be required to understand the inactivating ac- tion of this agent. Several investigators (e.g., Vasseur, 1948; Monroy et at, 1954) have suggested that sulfate groups are directly involved in the combination of fertilizin with sperm antifertilizin. Several lines of evidence support this view. Fertilizins low in sulfate {Bris- sopsis lyrifem) fail to agglutinate sperm (Vasseur, 1948; Vasseur and Carlsen, 1948 ) . Sulfate groups are hydrolyzed from fertilizin at pH's that inactivate fertilizin (Vasseur, 1952a), but such inac- tive fertilizin apparently has not been tested for univalence, and blocking the basic groups of the sperm surface prevents agglu- tination (Metz and Donovan, 1951) by fertilizin.* Finally, fer- tilizin is adsorbed to positively charged resins (Monroy et al., 1954). It is also adsorbed to calcium carbonate, aluminum oxide, charcoal, and kaolin ( Tyler and Fox, 1940 ) . A final observation that requires clarification concerns the role of calcium in agglutination. In some forms this ion aids or is re- quired for agglutination (Loeb, 1915; Vasseur, 1949a,b), but in some others it is not essential (Dan, 1954b). * These experiments have been cited by Monroy et al. (1954) in support of the above view. They involved the use of sperm killed with Bouin's fixa- tive. Such sperm will agglutinate with homologous (Arbacia) fertilizin but not with fertilizins from other species. This was originally interpreted to mean that the fixation did not alter the sperm surface antifertilizin. How- ever, subsequent study (Metz, unpublished) revealed that Arbacia fertilizin will agglutinate Bouin's fixative-killed sperm of many species quite nonspe- cifically. C. B. METZ 49 Although sea urchin fertiHzins have been examined most thor- oughly, some information on the chemistry of a few other ferti- lizins and egg jellies is available. The invertebrates include Megathtira and Nereis. Megathura fertilizin (Tyler and Fox, 1940), like sea urchin fertilizin, is nondialyzable, precipitates with protein precipitants, and gives color tests for proteins. Sim- ilarly the nitrogen content (4 to 5%) of both is low. Megathura fertilizin is more stable to heat, acid, and proteolytic enzymes than sea urchin fertilizins. Possibly this is related to the fact that the Megathura sperm agglutination does not reverse spontane- ously (Tyler, 1940a). Tests for univalent Megathura fertilizin following inactivation have not been reported. Nereis egg jelly has been examined by Ferry (cited by Costello, 1949) and Tyler ( 1938b ) . According to the former, the material contains less than 1% nitrogen, at least 75% carbohydrate, part or all of which may be uronic acid, and considerable bound inorganic material. Tyler found that the jelly contained about 5% nitrogen and at least 18% reducing sugar. The fertilizins and egg jellies of some verte- brates have been examined chemically. The forms include the trout (Hartmann et ah, 1947) and amphibian (Minganti, 1955). Antifertilizin from Eggs Lillie (1914) observed that egg water from cytolyzed sea urchin eggs failed to agglutinate sperm strongly. From this Lillie concluded that an agent, antifertilizin, was released from within the egg upon cytolysis and that the agent combined with and neutralized the fertilizin. Later the agent was obtained in active form by freeze-thawing jellyless eggs (Tyler, 1940b). Solutions of the agent form precipitates with and inactivate the sperm ag- glutinating action of fertilizin. They also precipitate egg jellies and agglutinate eggs. The antifertilizin is not highly specific, for Tyler (1948a) ob- tained cross reactions between related echinoids. Extracts of Arhacia lixula and Paracentrotus lividus eggs give an unexpected specificity pattern when tested in the presence of electrolytes (Runnstrom and Monroy, 1950). Arhacia extract was relatively specific whereas Paracentrotus extract was strongly active only 50 EGG AND SPERM SUBSTANCES on Arhacia eggs. In sucrose solution, however, these and other cross tests revealed no specificity (Hagstrom and Hagstrom, 1955). Egg antifertilizin specificity has not been tested in forms other than echinoids. The agent is evidently a protein since it is insoluble in protein precipitants and is inactivated by proteolytic enzymes (Tyler, 1940b, 1948a). According to Tyler (1948a) and Runnstrom, Wicklund, and Low ( 1954 ) egg antifertilizin is quite heat labile. The latter workers find that Arhacia lixula egg antiferti- lizin is inactivated at 57° C in 45 mmutes (Hagstrom and Hag- strom, 1955, report that the same antifertilizin is not inactive after 30 minutes at 100° C). The antifertilizin activity is associated with pigment containing gi'anules (Monroy and Runnstrom, 1952; Wicklund, 1954a) of the egg cytoplasm. These granules failed to sediment at 25,000g in 45 minutes, but did form a precipitate in 45 minutes at 155,- OOOg. Ultracentrifugal and electrophoretic analysis revealed two components. In the ultracentrifuge only one of these was pig- mented. The component with antifertilizin activity has not been identified. Since treatment with ribonuclease appears to enhance the antifertilizin activity of the granular fraction (Runnstrom, Hagstrom, and Low, 1955), it is likely that the active agent is bound to RNA granules. Egg antifertilizin may have a role in fertilization but as yet this remains to be demonstrated. The agent can be extracted from both fertilized and unfertilized eggs (Monroy and Runnstrom, 1952). It seems unlikely, then, that much, if any, of the antiferti- lizin is bound during or participates in the activation changes of the egg. Antifertilizin-treated eggs do not fertilize readily (Tyler, 1940b, 1948a), as might be expected, since the agent precipitates the jelly of the egg in the form of a membrane. As Tyler ( 1940b ) has shown, the membrane acts as a mechanical barrier to the sperm; likewise fertilizability of jellyless eggs is inhibited. This effect has been cited in the case of sperm antifertilizin in support of an essential role of fertilizin in fertilization (Tyler and Metz, 1955). The argument applies equally well for egg antifertilizin. C. B. METZ 51 Apparently, in dilute solution, egg antifertilizin can aid fertiliza- tion to the extent that the "fertilization rate" of both normal and jellyless eggs is increased (Hagstrom and Hagstrom, 1955). This action was most pronounced when fertilization was performed in the solution. This and the fact that the action was duplicated by albumen suggests that the effect may have been upon the sperm. It is probably due to a metal binding action of the egg extract. Antifertilizin evidently facilitates the parthenogenetic action of hypertonic sea water (Runnstrom, Wicklund, and Low, 1954). In several recent reports (Motomura, 1950b; Monroy and Runnstrom, 1950, 1952; Wicklund, 1954b; Runnstrom, Wicklund, and Low, 1954) agents from eggs which thicken, toughen, or facilitate elevation of the fertilization membrane have been de- scribed, and a possible relation of the active agent or agents to egg antifertilizin has been suggested. Evidently the membrane elevating factor differs from antifertilizin since the two effects are associated with separate fractions in the ultracentrifuge and have different heat labilities (Runnstrom, Wicklund, and Low, 1954). With regard to this effect it should be noted that metal binding agents act on sperm to facilitate membrane elevation (Tyler, 1953). The membrane toughening action appears to be associated with pigment granules of the egg ( Monroy and Runnstrom, 1952; Wicklund, 1954a). An agent with similar action is extractable from fertilized eggs with weak (pH 4.5) acid (Motomura, 1950b). The membrane toughening and egg jelly precipitating effects have not been separated. These may be different manifes- tations of antifertilizin action. Upon treatment of eggs with iodosobenzoic acid ( Monroy and Runnstrom, 1952 ) or anisotonic sea water ( Runnstrom and Mon- roy, 1950; Wicklund, 1954a) varying degrees of cytolysis of fertilized eggs are induced. Such cytolysis is associated with a release of pigment from the egg, a thickening of the fertilization membrane and sometimes a partial precipitation of the egg jelly outside the fertilization membrane. These observations in- dicate a release of the agent(s) responsible for membrane tough- ening and jelly precipitation. They also suggest (Motomura, 52 EGG AND SPERM SUBSTANCES 1950b; Riinnstrom, Wicklund, and Low, 1954) the possibility of release of the responsible agent (s) at fertilization under normal conditions. However, the situation is complicated by cytofertilizin which, according to Motomura ( 1950a, 1953a,b ) is also released by the egg at fertilization. Antifertilizin and cytofertilizin might be expected to interact with mutual neutralization. The two agents would not be expected to exist together in active form. Even if they were released separately they should interact in the perivi- telline space, and only the agent in excess should be detectable. In view of this apparent inconsistency, the release of antifertilizin and cytofertilizin must be re-examined with a strict accounting of both agents. Agents from Sperm Several agents have been obtained from sperm which may function in fertilization. These include the antifertilizin from sperm, egg membrane lysins, and preparations which affect the physiological activity of sperm. Before considering these it should be emphasized again that agents in sperm extracts which act upon gametes may be products of the method of preparation. Such agents may not exist in an active form and function in fer- tilization under physiological conditions. Antifertilizin from Sperm Agglutination of sperm by fertilizin implies the existence of a specific receptor on the sperm surface (Fig. 1). The agent, anti- fertilizin,* should have biological properties similar to those of the antifertilizin from eggs. It should occur in appreciable amounts on the sperm surface. In solution it should neutralize fertilizin, precipitate egg jellies and agglutinate eggs. Prepara- tions with such properties have been obtained from sea urchin sperm by heating (Frank, 1939), freeze thawing (Tyler, 1939b), extraction with weak acid ( Tyler and O'Melveny, 1941 ) , or by * Sperm antifertilizin (Tyler and O'Melveny, 1941) = Sperm receptor (Lillie, 1913a, 1914) = Androgamone II (Hartmann et al, 1940) = Jelly precipitating factor from sperm, abbreviated JePpF (S) (Hultin et al., 1952). C. B. METZ 53 centrifuging sperm (Southwick, 1939; see also Tyler, 1948a, for further details). The active agent in these preparations is con- sidered by most investigators to be antifertilizin, the sperm sur- face receptor substance that combines with fertilizin at aggluti- nation. According to the analysis of Tyler and of Runnstrom, Tiselius, and Vasseur (see Tyler, 1948a, for review), the active agent gives the usual protein tests, is relatively heat stable, contains about 16% nitrogen, and can be prepared in electro- phoretically homogeneous form. It is inactivated by proteolytic enzymes. Ultracentrifugation, electrophoretic mobility, and iso- electric precipitation studies indicate a molecule with an acid isoelectric point and a molecular weight under 10,000. Unfortunately, this agent has been confused, as a result of ex- periments of Hultin ( 1947b, 1949 ) , with the basic proteins ( pro- tamines, histones) from sperm. Solutions of basic proteins are readily prepared by acidifying a sperm suspension to pH 1 or lower, separating the insoluble nucleic acid from the dissolved basic protein by centrifugation and neutralizing the basic protein containing supernate. Basic proteins characteristically combine with a wide variety of substances and cells, precipitating the former and agglutinating the latter. It is not suprising, therefore, that sea urchin sperm basic protein agglutinates both the sperm and the eggs of the homologous species (Metz, 1942b, 1949; Hultin, 1947a ) as well as a variety of unrelated forms ( see Metz, Foley, and Donovan, 1949, concerning specificity). Because of the agglutinating action on sea urchin eggs and certain other considerations, Hultin ( 1947b ) concluded that the egg agglu- tinins prepared by freeze thawing, heating, or mild acid extrac- tion of sperm are also basic protein, not the specific receptor substance (antifertilizin) of the sperm surface. However, the latter preparations agglutinate only eggs, whereas the basic proteins agglutinate both sperm and eggs. Furthermore, the egg agglutinating action of antifertilizin is destroyed by the basic protein extraction procedure (pH 1 or lower), and mixture of the two extracts results in mutual neutralization (Metz, 1949). In view of these observations and the acid isoelectric point of antifertilizin (Tyler, 1949; Runnstrom, Tiselius, and Vasseur, 54 EGG AND SPERM SUBSTANCES 1942), it is evident that the egg agglutinating action is not due to basic protein. Hultin ( 1949 ) also ascribed the egg agglutinating action of antifertilizin preparations to the nucleoprotein some- times present in such solutions. However, the main activity of antifertilizin solutions cannot be attributed to nucleoprotein ( Metz and Tyler, independent unpublished studies ) because the nucleic acid content is not related to the biological activity of antifertilizin preparations. Thus one extract prepared by freeze- thawing Arhacia sperm (egg agglutinin titer 16) gave positive reactions with Schiff's reagent, both before and after hydrolysis, no color with diphenylamine (Dische test), and no absorption maximum at 260 m/n when diluted to 1/10. Two other prepara- tions gave absorption peaks at 260 m/x when diluted beyond the range of biological activity. Clearly, then, the egg agglutinating action of antifertilizin is not dependent upon nucleoprotein. In other studies Motomura ( 1954, 1955 ) has prepared acid extracts of acetone-precipitated echinoid sperm. The extracts agglutinated eggs and sperm and precipitated with hen's egg white. It seems most probable that Motomura's agent is also basic protein. The writer has repeated Motomura's procedure and ob- tained an agent which appears to be identical with the basic protein extracted from sperm by less elaborate methods. Evidently, two agglutinins may be obtained from sperm. One of these is a basic protein(s) of histone type (Hamer, 1955). It agglutinates both eggs and sperm. The other is an acidic protein containing very little if any nucleic acid. It agglutinates only eggs. Although the egg agglutinating agent is not a nucleoprotein or basic protein, the question may be raised whether it actually is the spenii receptor substance, antifertilizin. Information on this point might be obtained by consideration of the agent's source and specificity. Extraction of the egg agglutinin with weak acid (pH 3) results in swelling and partial dissolution of the sperm head surface between the acrosome and midpiece (Tyler, 1949). This indicates that the agent may have been removed from this region of the sperm surface. The specificity has been examined C. B. METZ 55 by several workers (Frank, 1939; Tyler, 1940b, 1948a; Hartmann et al, 1940; Hultin, 1947b, 1949; Rminstrom et al, 1942, 1944) all of whom have reported cross reactions. Some of these reac- tions were between distantly related forms clearly beyond the bomids of fertilizin agglutination specificity. However, as Tyler (1948a) pointed out, cross agglutination of eggs by sperm ex- tracts may result from action of natural heteroagglutinins, not from antifertilizin cross reactions. Natural heteroagglutinins for sperm, eggs, and red cells occur widely in invertebrate bloods and seminal fluids (Tyler and Metz, 1945; Tyler, 1946b; Metz, 1949). Another possibility is that the specificity of antifertilizin is altered upon extraction from the sperm. Such loss of specificity could result from alteration of the specific receptor groups or from exposure of other less specific groups that are normally bound. Evidently, the question of identity of the sperm surface antifertilizin with the egg agglutinating agent cannot be settled by the available specificity data. A thorough test of antifertilizin specificity, taking the above factors into consideration, should be performed. Role in Fertilization. If fertilizin is essential for fertilization by virtue of a specific interaction with the sperm, then antiferti- lizin must also be essential. However, the role of fertilizin in fertilization is uncertain. Therefore, independent evidence for the role of antifertilizin has been sought. Thus blocking the sperm surface antifertilizin with fertilizin (Tyler, 1941; Tyler and Metz, 1955) or with univalent antibodies prepared from rabbits im- munized with purified antifertilizin (Tyler, 1946a) reduces the fertilizing capacity of sperm. Likewise partial removal of anti- fertilizin from the sperm surface renders these cells less effective in fertilization (Tyler and O'Melveny, 1941). Finally, treatment of normal or jellyless eggs with antifertilizin solution (Tyler and Metz, 1955) lowers fertilizability of eggs. This action on normal eggs is evidently due to the precipitation of the egg jelly in the form of a membrane, impermeable to the sperm (Tyler and O'Melveny, 1941 ) . Sperm penetration of normal egg membranes is considered below. 56 EGG AND SPERM SUBSTANCES These experiments indicate that antifertihzin at the sperm sur- face is an aid to fertiUzation and render hkely the possibihty that the agent is essential for fertiHzation. Sperm Paralyzing Agents Several workers (e.g., Southwick, 1939; Hartmann, Schartau, and Wallenfels, 1940; Vasseur and Hagstrom, 1946; Runnstrom, Lindvall, and Tiselius, 1944; Rothschild, 1948) have found that seminal fluid or sperm extracts, mainly from sea urchins, inhibit sperm motility. Hartmann et al. ( 1940 ) attributed the action to an agent, androgamone I. Other investigators working on other forms have failed to find comparable action. In certain cases the paralyzing effect is evidently due to O2 lack. Only in the case of the salmon is it probable that the agent is normally functional (Rothschild, 1951b). Tyler (1948a), Runnstrom (1949a), and Rothschild (1951b) have reviewed these studies in detail. Egg Membrane and Surface Ltjsins from Sperm In many forms the egg is surrounded by extraneous jelly lay- ers, membranes, cell layers, or a combination of these. Such mem- branes are mechanical barriers to the fertilizing sperm. These and the egg surface itself must be penetrated for the sperm to enter the egg cytoplasm. Such penetration is evidently achieved, in some forms at least, through the action of egg membrane lysins carried by the sperm. Indeed, of the various "sex substances" that have been obtained from gametes only these membrane lysins have well-understood action in fertilization. The most thoroughly studied of these agents are the membrane lysins from the sperm of molluscs and hyaluronidase in mammals. Since the role of hya- luronidase in mammalian fertilization is discussed elsewhere (Chang, this volume), it will be omitted here. Among molluscs the limpet, Megathura crenulata and the mussel, Mytilus edulis, have been examined in most detail. Freshly shed Megathura eggs are surrounded by a jelly layer and a rather thick membrane. The latter is closely applied to the egg but gradually rises as the egg stands in sea water. Upon heavy insemination, the membrane disappears. The responsible C. B. METZ 57 agent is weakly bound to the sperm surface since, on standing, sperm suspensions gradually charge the sea water with mem- brane lysin (Tyler, 1939b; Krauss, 1950a). The lysin can be ex- tracted with alkali (Krauss, 1950a) or by freeze thawing (Tyler, 1939b). The lytic agent and the Megathura antifertilizin also present in frozen-thawed extracts are distinct since the two agents have different heat stabilities [the lytic agent is rapidly destroyed at 60° C; antifertilizin remains active for some time at 100° C (Tyler, 1939b; Krauss, 1950a)], and they can be extracted sep- arately [the lysin with alkali; antifertilizin with acid (Krauss, 1950a)]. The Megathura egg membrane lysin is evidently a labile protein (Tyler, 1939b; Krauss, 1950a). Release of the lysin by alkali is associated with dissolution of the sperm acrosome ( Tyler, 1949). Krauss (1950a) suggests that the lysin may act by reduc- ing disulfide linkages since cysteine and other sulfhydryl reagents dissolved the membrane. However, he was unable to demonstrate free SH groups in the native extract or block the action of the lysin with iodoacetate. Unfertilized Mytilus edulis eggs are surrounded by a thin, tight vitelline membrane. This membrane is lysed by frozen-thawed or acid extracts of Mytilus sperm (Berg, 1949, 1950). Further- more, if eggs are permitted to cleave in these extracts, the result- ing blastomeres are very loosely joined. The intercellular cement is evidently attacked by the extract. The two effects may result from separate agents, for the membrane lysin is more readily de- stroyed by heat than the cement lysin. Furthermore, only the latter action is inhibited in concentrated sperm extracts. Both agents appear to be proteins (Berg, 1950). Lytic agents com- parable to those from Megathura and Mytilus sperm have been demonstrated in sperm extracts of several other molluscs (Tyler, 1939b; von Medem, 1942, 1945) and the amphibian, Discoglossus pictus (Hibbard, 1928). Concentrated sperm suspensions have similar action on the egg membrane of the annelid Pomatoceros triquester (Monroy, 1948). In fact, it is reasonable to assume some such agent for species with eggs that are surrounded by heavy membranes, even when the lysin is not found in sperm ex- tracts [e.g., Cerebratulus (Metz, unpublished)]. 58 EGG AND SPERM SUBSTANCES These lytic agents not only permit penetration of the egg mem- branes by the sperm, but they may also contribute a specificity factor in fertilization. Thus the lysins and eggs of Haliotus and Megathura do not cross react (Tyler, 1939b). Mtjtilus calif ornia- nus extracts dissolved both the egg membrane and intercellular cement of M. edtdis but cross reaction did not occur in the re- verse combination. Extracts from neither species acted on eggs of more distantly related forms. Likewise, sperm extracts from such foreign species did not act on Mytilus eggs (Berg, 1950). In other molluscs the lytic action is also largely specific (von Medem, 1942, 1945 ) . Krauss ( 1950b ) , however, reported an in- teresting heterologous reaction, namely lysis of the Megathura membrane by a heat-labile agent from sea urchin sperm. Although the cases described above are well established, the situation in the sea urchin has been disputed. In sea urchins a special lytic agent may not be required for penetration of the sperm through the jelly. In most species the jelly is not highly viscous and is readily soluble in weak acid. The COi> produced by the penetrating sperm may be sufficient to dissolve a path to the egg surface. Claims that the sea urchin egg jelly is dissolved by depolymerases from the sperm have not been substantiated (Krauss, 1950c; Monroy and Tosi, 1952; Monroy et al, 1954). Aside from action on the egg jelly, extracts have been prepared from sperm which act upon the egg surface. Runnstrom, Lindvall, and Tiselius (1944) found that the methanol extracts of sea ur- chin sperm caused eggs to shrink without wrinkling in hyper- tonic sea water. This and other tests led to the view that the methanol extracts partially lysed or liquefied the egg surface. This egg surface lysin (androgamone III) was found to be heat stable and dialyzable. These and other properties, including du- plication of the action by detergents, led to the view that the agent is a low-molecular-weight, surface-active agent of fatty acid na- ture (Runnstrom, Tiselius, and Lindvall, 1945; Runnstrom and Lindvall, 1946; Runnstrom, 1947, 1949a). Sperm supernatants have the same action as methanol extracts. Therefore the agent may be available and function under physiological conditions. C. B. METZ 59 The agent inhibits the sperm agghitinating action of fertihzin (presumably in the absence of antifertiHzin ) and fertiHzation. The latter action is reversible to the extent that eggs washed free of the extract will fertilize although development is abnormal (Runnstrom, 1947). Runnstrom and co-workers suggest a num- ber of possible functions for the agent in fertilization. However, judgment as to the function of the agent should be withheld pending further study. Summary It is evident from the account given here that fertilization and especially the activation of the egg cannot as yet be described completely in terms of the interaction of known specific egg and sperm substances. However, it is equally clear that agents are known which have specific action upon gametes of the opposite sex. Of these the egg membrane lysins from sperm perfomi a def- inite, and essential, though secondary, function in fertilization, namely, removal of mechanical barriers between the approaching sperm and the egg surface. Agents from eggs would also appear to facilitate fertilization by physiological action on sperm. In- crease in sperm motility is one of the more obvious of these effects. Fertilizin and antifertilizin on the surfaces of the respective gam- etes may well be the receptor agents by which sperm and eggs make initial union. Furthermore, the presence of these substances on the gametes and not on other tissues can account for the tissue specificity of fertilization. Likewise, the species specificity of fer- tilization may be attributed largely, if not entirely, to these agents, since action of fertilizin on sperm parallels roughly the species specificity of fertilization. Positive evidence for a direct and es- sential role of known substances in the primary activation reac- tions is wanting. On the other hand, the possibility of such action, especially for fertilizin and antifertilizin is not excluded by the available evidence. 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The effect of some agents upon the reaction of Echinocardium spermatozoa toward egg water. Ar- kiv Zool, 38 A, No. 10, 1-9. Runnstrom, J., S. Lindvall, and A. Tiselius. 1944. Gamones of the sperm of the sea urchin and salmon. Nature, 153, 285-86. Runnstrom, J., and A. Monroy. 1950. The effect of egg extract on the surface of the sea urchin eggs and spermatozoa. Arkiv Kemi, 2, 405- 16. Runnstrom, J., A. Tiselius, and A. Lindvall. 1945. The action of andro- gamone III on the sea urchin egg. Arkiv Zool, 36A, No. 22, 1-25. Runnstrom, J., A. Tiselius, and E. Vasseur. 1942. Zur kenntnis der Gamonwirkungen bei Psammechinus miliaris und Echinocardium cordatum. Arkiv Kemi, 15A, No. 16, 1-18. Runnstrom, J., E. Wicklund, and H. Low. 1954. The fertilization and development of the sea urchin egg, Arbacia lixula, under the influ- ence of fractions of egg homogenate. Exptl. Cell Research, 6, 459- 78. Southwick, W. E. 1939. Activity-preventing and egg-sea-water neu- tralizing substances from spermatozoa of Echinometra subangularis. Biol Bull, 77, 147-56. C. B. METZ 67 Spikes, J. D. 1949a. Metabolism of sea urchin sperm. Am. Naturalist, 83, 285-301. Spikes, J. D. 1949b. The prezone phenomenon in sperm agglutination. Biol. Bull, 97, 95-99. Tyler, A. 19.39a. Crystalline echinochrome and spinochrome: Their failure to stimulate the respiration of eggs and of sperm of Stron- gylocentrotus. Proc. Natl. Acad. Sci. U. S., 25, 523-28. Tyler, A. 1939b. Extraction of an egg membrane lysin from sperm of the giant keyhole limpet {Megathura crenulata). Proc. Natl. Acad. Sci. U. S., 25, 317-23. Tyler, A. 1940a. Sperm agglutination in the keyhole limpet, Megathura crenulata. Biol. Bull, 78, 159-78. Tyler, A. 1940b. Agglutination of sea-urchin eggs by means of a sub- stance extracted from the eggs. Proc. Natl. Acad. Sci. U. S., 26, 249- 56. Tyler, A. 1941. The role of fertilizin in the fertilization of eggs of the sea-urchin and other animals. Biol. Bull, 81, 190-204. Tyler, A. 1945. Conversion of agglutinins and precipitins into "uni- valent" (non-agglutinating or non-precipitating) antibodies by pho- todynamic irradiation of rabbit-antisera vs. pneumococci, sheep-red- cells and sea-urchin sperm. /. Immunol, 51, 157-72. Tyler, A. 1946a. Loss of fertilizing power of sea-urchin and Urechis sperm treated with "univalent" antibodies vs. antifertilizin. Proc. Soc. Exptl Biol Med., 62, 197-99. Tyler, A. 1946b. Natural heteroagglutinins in the body-fluids and seminal fluids of various invertebrates. Biol. Bull, 90, 213-19. Tyler, A. 1948a. Fertilization and immunity. Physiol Revs., 28, 180- 219. Tyler, A. 1948b. Fertilizin of Nereis limbata. Biol Bull, 95(2), 271 ( abstract ) . Tyler, A. 1948c. On the chemistry of the fertilizin of the sea-urchin Strongylocentrotus purpuratus. Anat. Record, 101, 658-59 (ab- stract). Tyler, A. 1949. 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BISHOP: NATIONAL INSTITUTE FOR MEDICAL RESEARCH, MILL HILL, LONDON Since von Baer described the mammalian primary oocyte and Barry observed spermatozoa within the rabbit egg, work on fer- tihzation in mammals has been directed chiefly toward the eluci- dation of cytological problems. In recent years, however, increas- ingly more attention has been paid to the events occm'ring between coitus and the arrival of the spermatozoon in the egg cytoplasm. As a result, it is becoming evident that the prelimi- naries to fertilization are of a highly complex nature and further that, in large measure, they determine the successful union of the gametes and the normal outcome of syngamy. Among many invertebrates and lower vertebrates the gametes are released into the surrounding aqueous medium, which con- stitutes their environment of fertilization. As the spermatozoa approach the eggs they experience the influence of chemical substances associated with the eggs, and on their part the sperma- tozoa arouse reactions in the eggs during the course of penetra- tion, so that interaction between egg and spermatozoon becomes a feature of the environment of fertilization. With the occurrence of ovulation and ejaculation the mamma- lian gametes are projected into their environment of fertilization, which thus consists of the accessory secretions of the male and the entire genital tract of the female. From the moment of their release into the female' tract, the spermatozoa come under the manifold influences of this environment, all of which may affect the incidence or course of fertilization. A contribution is also made by the egg to the environment of the spermatozoon, al- 71 72 PRELIMINARIES TO FERTILIZATION though it may not be as generous as that of the externally ferti- lized egg. Since the mammalian egg has yet to pass through the genital tract it is possible that certain substances reaching the lumen of the tract in the liquor folliculi or in the secretions of the mucosa correspond to those actually carried by externally fertilized eggs. The numerous interactions in which the gametes participate with the environment of fertilization and with each other, to- gether with the mechanisms that permit the spermatozoon to reach and enter the egg, may thus be held to be the preliminaries to fertilization. It is the purpose of this review to describe the current trends of research on these events and processes, in the hope of revealing some possibilities for advance in knowledge in the near future. Dilution and Activation of Spermatozoa At ejaculation the spermatozoa are mingled with fluids from the accessory glands, principally the seminal vesicles and the prostate, and this fluid forms the bulk of the seminal plasma. The degree of dilution of spermatozoa varies considerably between species: in the semen of the ram the spermatozoa constitute about 25 per cent of the total volume, whereas in the semen of the boar this proportion is only about 1 per cent. The total num- ber of spermatozoa, the volume of the ejaculate, and the compo- sition of the accessory fluids also vary widely. Despite the fact that much is known about the nature of the seminal plasma (Mann, 1954), its precise role is far from clear. Certainly it func- tions as a vehicle for the spermatozoa, but it by no means pro- vides an ideal medium for their survival. Moreover, the larger the proportion of seminal plasma in the ejaculate the more unfa- vorable it is as a medium (Gunn, 1936). Spermatozoa can be stored in vitro in artificial diluents that are quite unlike seminal plasma much more successfully than in the natural diluent itself. The fertility of spermatozoa, also, is not dependent upon their admixture with the accessory secretions (Ivanov, 1926; Ham- mond, 1930; Walton, 1930 ) . When epididymal spermatozoa were C. R. AUSTIN AND M. W. H. BISHOP 73 used for inseminating cows, the resulting incidence of concep- tion was approximately the same as that which had previously been obtained with ejaculated spermatozoa from the same bulls (Lardy and Ghosh, 1952). The fact that the spermatozoa in the epididymis and vas deferens are almost motionless but become vigorously active when they make contact with the accessory secretions, much as sea urchin spermatozoa do when shed into sea water, suggests that activation of spermatozoa is the only important function of seminal plasma, other than the carriage of the spemiatozoa from male to female tracts. The mechanism of activation is little understood, but it is evi- dently not of a specific nature. It may be due in part, at least, to the dilution of an inhibitory substance (Bishop and Salisbury, 1955a,b) similar in its action to that of potassium in trout and salmon semen (Rothschild, 1951a), or of zinc in echinoderm semen (Fujii, Utida, and Mizuno, 1955). Lardy and his associ- ates, on the other hand, claim that the respiration and aerobic glycolysis of bovine spermatozoa are stimulated at ejaculation by a specific activator, believed to be a sulfite or sulfhydryl com- pound, that has the effect of reducing the metabolic efficiency of these cells as measured by the Meyerhof oxidation quotient (Lardy, Ghosh, and Plant, 1949; Lardy, 1953). Gunn (1936) noted activation of epididymal spermatozoa upon dilution and aeration. Bishop and Matthews (1952) report that quiescence of spermatozoa in the vas deferens is associated with very low oxy- gen tensions and a deficiency of glycolyzable substrate, but not with narcotic accumulations of lactic acid or carbon dioxide. They conclude that oxygen is the principal activating factor, an observation in agreement with Rothschild's (1948) findings on the activation of sea urchin spermatozoa. Electrolytes are also known to stimulate spermatozoon motility ( Milovanov, 1934a,b ) , and they probably play an important part in the activation phe- nomenon. Both in mammalian- and echinoderm spermatozoa increased dilution leads to increased physical and metabolic activity, but to decreased survival of the spermatozoa (Gray, 1928; Salisbury, 74 PRELIMINARIES TO FERTILIZATION Beck, Cupps, and Elliott, 1943; Chang, 1946a,b; Emmens and Swyer, 1948; Cheng, Casida, and Barrett, 1949; Rothschild, 1951b; Willett, 1953; Bishop, 1954). Excessive dilution in vitro is invariably harmful, probably because of the irreparable decay of enzyme systems in the actively metabolizing cells resulting from the stimulating effect of dilution, and because of loss of substances from the cell surface (Emmens and Swyer, 1948). In particular, it is known that electrolytes remove phospholipid ma- terial from cell membranes ( Milovanov, 1934a,b; Lovelock, 1954, 1955; Dallam and Thomas, 1952). The efficiency of removal varies with the electrolyte, according to the lyotropic series of ions (Anderson, 1945). It seems probable that the functions of the osmotically active organic constituents of the seminal plasma, such as fructose, lactic acid, citric acid, amino acids, urea, and inositol, include maintenance of a suitable osmotic environment with a low electrolyte to nonelectrolyte ratio. Kampschmidt, Mayer, and Herman (1953) and Kok (1953) have found that the survival of spermatozoa in vitro is prolonged when the electro- lyte to nonelectrolyte ratio of the diluents is reduced by the ad- dition of metabolizable sugars, and Roy and Bishop ( 1954 ) have obtained similar results by replacing electrolytes with glycine. Although other explanations can be offered, it is likely that the beneficial effect of the sugars and glycine was due to reduction of the stimulating and destructive action of the electrolytes. With sea urchin spermatozoa, on the other hand, glycine provides pro- tection chiefly by chelating heavy metals (Tyler and Rothschild, 1951; Tyler, 1953). Substances such as lecithin, albumin, mucin, and egg yolk also help to protect the spermatozoon membranes from the deleterious effects of dilution: constituents of the semi- nal plasma and of the female genital secretions presumably have a similar function. It would seem, therefore, that the genital secretions, both of male and female but chiefly the former, have the function of ac- tivating the spermatozoa in a controlled manner, the stimulating property being appropriately balanced by the property that miti- gates the deleterious effect of electrolytes. C. R. AUSTIN AND M. W. H. BISHOP 75 Transport and Distribution of Spermatozoa Motility is an almost miiversal characteristic of male gametes throughout the animal kingdom and it undoubtedly facilitates the meeting of egg and spermatozoon. However, the available evidence, particularly that relating to rate of transport of sperma- tozoa in the female tract, indicates that during their passage from the site of deposition to the site of fertilization the sperma- tozoa play a passive role in most species and transport depends little upon their active swimming. The importance of muscular activity of the female tract in the transport of spermatozoa has been appreciated for a long time. Heape (1898) imputed this significance to the activity of the dog uterus which he found to be increased by stimulation of the external genitalia. Westman (1926) observed augmented con- tractions of the Fallopian tubes of female rabbits when in the presence of the male, and Krehbiel and Carstens (1939) found that fluids placed in the rabbit vagina were carried throughout the uterus when the vulva was artificially stimulated. Increased uterine activity in the rabbit following mating was noted by Reynolds (1930), and Van Demark and Hays (1951, 1952, 1953) have shown that strong uterine contractions in the cow are in- duced by various mating stimuli, including the sight of the male, although coitus was the most effective. Hays and Van Demark (1952) claim that rapid spermatozoon transport occurs in the perfused, excised cow uterus, provided that oxytocin is present in the perfusate. It has been shown in the mare (Millar, 1952) that the orgasm of coitus produces a negative pressure within the uterus which would have the effect of rapidly drawing the semen into the uterine lumen. Both the act of ejaculation and the physical and chemical properties of the seminal plasma may also assist transport. In the rodents, the sow, and the mare, the spermatozoa are not de- posited in the vagina but passed directly through the cervix. Gelation of the semen of the boar and stallion presumably plays an important role in retaining the large ejaculate in the uterus. 76 PRELIMINARIES TO FERTILIZATION A similar function may reasonably be imputed to the vaginal plugs formed in many rodents, some bats and insectivores, and the chimpanzee. In the opossum a similar plug is apparently formed by coagulation of female secretions by the seminal fluid. The human seminal gel evidently has a different significance for it soon liquefies under the action of a proteolytic enzyme present in the semen (Huggins and Neal, 1942; Huggins and Vail, 1943). Seminal gels may also have the function of stimulating the female tract by mechanical action, and seminal plasma as a whole by viitue of its physiologically active constituents, such as choline and adrenaline (see Mann, 1954). It has been claimed, notably by Russian workers, that spermatozoa penetrate into the wall of the female tract and exert a stimulating influence upon its func- tions (see Kushner, 1954), For many species the recorded time interval between coitus and the arrival of spermatozoa in the ampulla of the Fallopian tube is surprisingly short: intervals of 15 minutes or less have been noted in the sheep (Starke, 1949), cow (Van Demark and Moeller, 1951), mouse (Lewis and Wright, 1935), rat (Blandau and Money, 1944), and bitch (Whitney, cited by Evans, 1933). By contrast, spermatozoon transport in the rabbit seems to take about 3 or 4 hours (Heape, 1905; Parker, 1931; Florey and Wal- ton, 1932; Braden, 1953; Adams, 1956), although it may occasion- ally take only one hour (Chang, 1952a). It is possible that the slower rate in the rabbit may be associated with the absence of copious uterine fluid; Warren (1938), however, found that re- moval of the uterine fluid from the rat uterus delayed transport by only a few minutes. It is possible that rabbit spermatozoa depend, much more than those of the other species mentioned, upon their own motility to reach their goal. The rabbit, it would seem, does not require rapid spermatozoon transport because it exhibits copulatory ovulation. The biological advantage of rapid transport to animals showing spontaneous ovulation is presum- ably that it increases the period during which coitus can result in normal fertilization — it would be especially important when coitus occurs close to or soon after ovulation. Experimentally delayed coitus or insemination often results in a large increase C. R. AUSTIN AND M. W. H. BISHOP 77 in the incidence of abnormal fertilization in rats ( Blandau, 1952 ) and rabbits (Chang, 1952b) and of polyspermic fertilization in rats and rabbits ( Austin and Braden, 1953a,b ) and mice ( Braden and Austin, 1954b), an increase in the incidence of abnormal pregnancy, and reduction in the number of normal young born in the guinea pig ( Blandau and Young, 1939 ) , rat ( Soderwall and Blandau, 1941; Blandau and Jordan, 1941), rabbit (Hammond, 1934), sheep (Quinlan, Mare, and Roux, 1932), sow (Lewis, 1911), and cow (Trimberger, 1948). Both mechanical and physical attributes of the female genital tract also reduce greatly the numbers of spermatozoa passing to the site of fertilization (Braden, 1953). The chief agents in this process of reduction are the cei^vix (in species having intravagi- nal ejaculation), utero-tubal junction, and tubal isthmus by vir- tue of their narrow lumen, and the uterus, by virtue of its large internal surface area (rabbit) or voluminous fluid content (ro- dents especially ) . Leonard and Perlman ( 1949 ) found that, in the rat, only vigorously motile spermatozoa could pass the utero- tubal junction. Furthermore, not only were inert particles and immotile spermatozoa unable to pass the junction, but active spermatozoa of other species (bull, mouse, and guinea pig) were similarly unable to enter the Fallopian tube, even when mixed with active rat spermatozoa. By contrast, in the cow, dead sper- matozoa were transported as rapidly as living ones (Van Demark and Moeller, 1951 ) , and rat spermatozoa were found to be capa- ble of passing into the Fallopian tube of the ewe (Phillips and Andrews, 1937). Consistently, the pressure required to force flu- ids from the uterus to the tubes is very high in the rat (Alden, 1943) and low in the sheep and cow (Andersen, 1928). Ander- sen's paper contains descriptions of the morphology of the utero- tubal junction in 25 mammals belonging to 10 different orders, which indicate that the restrictive power of the region must differ widely among these species. She noted, too, that fluid could not be forced from the uterus to the tube in the cat and rabbit, and that passage was difficult in the sow at the time when eggs are in the tubes. The comparative freedom of passage through the utero-tubal junction of the cow is illustrated by Rowson's (1955) 78 PRELIMINARIES TO FERTILIZATION observations on the passage of radio-opaque fluids. It may also be relevant that hybrids seem to be more common in ungulates than they are among rodents (Gray, 1954). These data suggest that the exclusion of foreign spermatozoa is a mechanical func- tion of the utero-tubal junction, but reactions of an immunologi- cal nature may also be involved. Within the environment of fertilization there is an ecological niche of special significance which is more clearly defined in mammals than in species having external fertilization. This is the "site of fertilization" and it is characterized not merely by ana- tomical position but by the fact that here the conditions are j^re- sumably optimal for ensuring the penetration of the spermato- zoon into the egg — they may not necessarily be the best for maintaining the viability of the gametes or of the zygote. In most mammals the site of fertilization is the ampulla of the Fallopian tube; in some species spermatozoon penetration may occur in the peri-ovarian sac or bursa which can thus be regarded as an ex- tension of the Fallopian tube, physiologically as well as anatomi- cally (see Braden and Austin, 1954a). A notable exception to the general rule is provided by the tenrecs, in which penetration ap- parently occurs while the egg is still in the ovarian follicle ( Blunt- schli, 1938; Strauss, 1938, 1950). That fertilization within the follicle may occasionally take place in other species also is shown by the fact that ovarian pregnancies have long been known in man (Mahfouz, 1949). The number of spermatozoa that have been observed at the site of fertilization is surprisingly small, especially when compared with the many millions deposited at coitus: rat, mean number of 43 spermatozoa per Fallopian tube (Austin, 1948b), mean of 12 spermatozoa (Blandau and Odor, 1949); mouse, mean of 17 spermatozoa (Braden and Austin, 1954a); rabbit, less than 1000 spermatozoa (Austin, 1948b; Chang, 1951b), mean of 500 spermatozoa (Braden, 1953); sheep, mean of 340 spermatozoa (Braden and Austin, 1954a). In view of the known influence of ovarian function upon the spontaneous muscular activity of the uterus (Reynolds, 1949; Wislocki and Guttmacher, 1924; Cupps and Asdell, 1944; Evans and Miller, 1936) and on the nature of secretions of the repro- C. R. AUSTIN AND M. W. H. BISHOP 79 ductive tract, notably the cervical mucus ( Pommerenke and Viergiver, 1946; Glover and Scott Blair, 1953) it would be unex- pected to find that spermatozoon transport occurred with equal rapidity at all phases of the estrous cycle. However, Green and Winters (1935), Schott and Phillips (1941), and Starke (1949) all consider that the speed of transport in the sheep is independ- ent of ovarian activity. Rowson (1955), on the other hand, ex- presses doubt that this is so in the cow, and Warbritton, McKen- zie, Berliner, and Andrews (1937) suggest that conditions for spermatozoon transport in the ewe are optimal 10 to 12 hours after the beginning of estrus. In the rat the passage of spermato- zoa occurs more readily late in estrus (Braden and Austin, 1954a ) . During pseudopregnancy in the rabbit, conditions do not favor the fertilization of eggs (Wislocki and Snyder, 1931, 1933; Mm-phree, Warwick, Casida, and McShan, 1947; Boyarsky, Bay- lies, Casida, and Meyer, 1947) and fewer spermatozoa reach the uterus and tubes than during estrus (Austin, 1949). Unfavorable conditions obtaining in the tract at other times than estrus may constitute a defense against the possibility of fertilization in preg- nancy, an occurrence that could lead to superfetation. That su- perfetation can arise has been shown experimentally in the rat (Canivenc, Doruville, and Mayer, 1953). From what has been said it appears that the mechanical and physical attributes of the environment of fertilization tend to exert opposing effects upon the spermatozoa. The specialized move- ments of the tract, accompanied in certain instances by pressure gradients, have the effect of quickly and efficiently transporting spermatozoa to the site of fertilization. On the other hand, attri- butes such as the narrow lumen of certain regions, the accumu- lations of fluid, and the large mucosal surface areas exert a dilut- ing influence and greatly diminish the number and density of spermatozoa approaching the site of fertilization. Maintenance of Spermatozoa in the Female Tract Immediately following ejaculation, the spermatozoa, aheady suspended in the male accessory secretions, are further diluted by admixture with the secretions of the female tract. The propor- 80 PRELIMINARIES TO FERTILIZATION tionate contribution made by the seminal plasma to this environ- ment varies widely in different species. The environment afforded by the female tract is presumably ideal for the preparation of the spermatozoa for the task of reaching and entering the eggs. This implies in most animals the existence of conditions favoring vig- orous activity. The female environment is not necessarily the best for the survival of the spermatozoa, and it is well known that the life of the spermatozoon is much shorter in the female tract than in the male (see Hartman, 1939). It would indeed be of little advantage for the spermatozoa long to outlive the eggs. Never- theless, the circumstances may occasionally favor longevity. Rel- atively favorable conditions may exist in the cervical canal, and it has been suggested that this region may act as a reservoir of spermatozoa from which they are steadily released into the uterus (Quinlan, Mare, and Roux, 1932, 1933; Starke, 1949), a situation somewhat analogous to that of the "sperm nests" found in the uterus of the domestic fowl ( Van Drimmelen, 1949, 1951 ) . How- ever, the most remarkable instances of spermatozoon survival are to be found among the bats, wherein the spermatozoa may retain their fertility in the female tract for as long as five months (Re- denz, 1929; Wimsatt, 1942, 1944). During this period they remain embedded in high concentrations in the thick uterine mucus, motionless but capable of exhibiting active motility upon dilu- tion. Clearly, conditions within the bat uterus are highly special- ized, for not only is the capacity of the spermatozoa for motility retained but also the capacity for fertilization and for initiating the development of normal individuals. Spermatozoa in the geni- tal tracts of most other mammals soon enter a state of senescence when these capacities are lost. The losses occur at different times — the ability to form a viable embryo is lost before the ability to fertilize eggs, and fertilizing capacity is lost before motility ( Van Drimmelen and Oettle, 1949). In the fowl malformed, nonviable embryos resulted when the eggs were fertilized with aged sper- matozoa (Crew, 1926; Nalbandov and Card, 1943; Dharmarajan, 1950). There is evidence of similar changes in mammalian sper- matozoa. Young (1931) found that the incidence of nonviable embryos in the guinea pig could be increased from 3.6 to 20 per C. R. AUSTIN AND M. W. H. BISHOP 81 cent by retaining spermatozoa in the epididymis for 20 to 25 days. The observations of SaHsbury, Bratton, and Foote (1952) suggest that the incidence of embryonic mortahty in cattle in- creases with the age of the spermatozoa. It is now well known that mammalian spermatozoa in vitro, unlike sea urchin spermatozoa, metabolize nutrients in the sur- rounding medium to maintain their physiological functions. Un- der anaerobic conditions they maintain their motility by the gly- colytic breakdown of fructose (present in the seminal plasma of most species), or glucose if available (for example, in the secre- tions of the female tract ) ; in the presence of molecular oxygen a number of extracellular substrates can be metabolized. Even with- out exogenous oxidizable material mammalian spermatozoa can respire actively and maintain their motility for an appreciable length of time. Lardy and Phillips (1941, 1945) have attributed this to oxidation of intracellular phospholipids, and Rothschild and Cleland (1952) have found evidence that sea urchin sper- matozoa obtain energy for motility by a similar mechanism. Mammalian spermatozoa are known to contain the enzymes in- volved in the Embden-Meyerhof glycolytic system and the Krebs cycle as well as a complete cytochrome system (see Mann, 1954; Tyler, 1955); these systems are evidently located within the spermatozoon tail since headless spermatozoa can swim ac- tively (Redenz, 1924, 1925, 1926; Moench and Holt, 1929; Han- cock and Rollinson, 1949; Bishop, 1955). Within the female genital tract spermatozoa probably obtain energy for motility through both the glycolytic and respiratory systems, but while the importance of the former is widely appre- ciated that of the latter has received little recognition. Campbell ( 1932 ) has shown that the oxygen tension of the rabbit uterus is comparable to that in many other tissues of the body (20-45 mm. Hg), and in view of the known affinity of the cytochrome system for oxygen this should be ample for aerobic metabolism. Oxida- tive metabolism is ten to fourteen times more productive of en- ergy than glycolytic metabolism (Fruton and Simmonds, 1953), and there is evidence that the motility of mammalian spermatozoa is more closely related to oxygen uptake than to fructolysis 82 PRELIMINARIES TO FERTILIZATION (Bishop, Campbell, Hancock, and Walton, 1954; Bishop and Hancock, 1955; Bishop, 1955). The observation that fluoride in- hibits both fructolysis and motility under aerobic conditions (Lardy and Phillips, 1943) does not invalidate the value of re- spiratory metabolism for motility, because of the nonspecific nature of fluoride inhibition. It is unlikely that the aerobic inhibi- tion of spermatozoon motility by hydrogen peroxide formation, observed under certain conditions in vitro ( McLeod, 1946; Tosic and Walton, 1950) will occur within the female tract, owing to the presence of catalase in the mucosa. The importance of glycolytic metabolism is likely to be greatest when the spermatozoa exist together in dense suspension and con- sequently reduce the local oxygen tension. This will occur at the site of deposition of the semen, and it is of course precisely at this point that the spermatozoa are most under the influence of the seminal plasma. The semen of the dog and cat, however, contains no fructose (Mann, 1954). As already noted, high dilution of spermatozoa occurs in the female genital tract. A well-known result of dilution in vitro is spontaneous head agglutination — it is shown notably by rat, mouse, and guinea pig speiTnatozoa upon suspension in physio- logical saline, but occurs less readily with bull or ram spermato- zoa. This suggests the presence of surface charges on the heads of the spermatozoon and indeed Milovanov and Selivanova ( 1932 ) have reported that unagglutinated spermatozoa do carry a negative charge and migrate to the anode in an electric field. When the charge is neutralized by electrolytes in the medium, head agglutination occurs and the spermatozoa are not moved by the current. Schroder (1940) went further, claiming that she had separated by electrophoresis the spermatozoa bearing the X chromsome, which migrated to the anode, from those bearing the Y chromsome, which migrated to the cathode. This work still awaits confirmation. The surface charge carried by spennatozoa is held to be associated with the lipoid capsule (Milovanov, 1934a,b), which, as mentioned earlier, is readily removed by electrolytes. In the semen or within the female tract, however, head agglutination is not a feature of spermatozoa, presumably C. R. AUSTIN AND M. W. H. BISHOP 83 owing in part to the protective action of nonelectrolytes. There is evidence, too, of a specific protective agent. Lindahl and his associates claim to have isolated a highly potent antagglutin from the semen of several mammals and to have demonstrated its presence also in the secretions of the vagina, cervix, and Fal- lopian tubes and in the ovarian follicle (Lindahl and Kihlstrom, 1954a,b; Lindahl and Nilsson, 1954; Furuhjehn, Nilsson, Lindahl, and Ingleman-Sundberg, 1954; Lindahl and Edkmd, 1955). The antagglutin appears to be a mucoprotein; it is nondialyzable and can be reversibly oxidized. It is evidently produced in the prostate gland and becomes attached, while in the reduced form, to the spermatozoa thus preventing head agglutination, in which func- tion it exhibits no species specificity. On oxidation, the ant- agglutin is inactivated and is detached from the spermatozoa, whereupon head agglutination takes place. The active agent is apparently stabilized in semen by the presence of reducing sub- stances such as ergothionine and ascorbic acid. Such an antag- glutin would have an important function in the seminal plasma and throughout the female genital tract in maintaining the sper- matozoa in a suitable state for reaching and penetrating the eggs. A protective action of vaginal mucus in the rabbit against the specific tail agglutination induced by appropriate antisera has been demonstrated by Smith ( 1949a ) . Maintenance in the female tract therefore involves both sup- ply of metabolites to spermatozoa and protection against the ad- verse effects of the inevitable high dilution that spermatozoa undergo. Capacitation An important function of the female genital tract is the part it plays in the process of capacitation, whereby the spermatozoon gains the ability to pass through the zona pellucida and thus to enter the egg ( see Austin, 1955b ) . That spermatozoa are required to spend a period of a few hours in the female tract before they are able to penetrate eggs was shown by introducing suspensions of spermatozoa into the periovarian sac of the rat or into the Fal- lopian tube of the rabbit shortly after ovulation (Austin, 1951; 84 PRELIMINARIES TO FERTILIZATION Chang, 1951a, 1955a,b). In the rat penetration did not occur until four or more hours later. In the rabbit few or no eggs were fertilized, although control tests, in which spermatozoa were in- troduced before ovulation, yielded a high incidence of fertiliza- tion. The failure of fertilization in the rabbit was explained on the grounds that the eggs became impenetrable, through the deposition of the mucoprotein layer, before the spermatozoa had completed capacitation. Spermatozoa obtained from the uterus of an inseminated rabbit showed the ability to penetrate eggs after a shorter interval of time. Noyes ( 1953 ) found, by insemina- tion into the uterus, that rat uterine spermatozoa penetrated eggs after a shorter time than did epididymal spermatozoa. It ap- peared, therefore, that, while capacitation could take place entirely in the tubes, it could also occur, to some extent at least, in the uterus. The possibility that the effects observed had been due in part to the operative procedures employed was ruled out by the demonstration of a delay between the arrival of the sper- matozoa at the site of fertilization and the penetration of the eggs in intact rats and rabbits when coitus occurred after ovula- tion (Austin, 1952; Austin and Braden, 1954a). It is uncertain what capacitation involves, but it seems to take at least two hours in the rat and four hours in the rabbit, Capacitation is not simply the separation of the spermatozoa from the male accessory secre- tions, for epididymal spermatozoa must also spend a period within the female tract. It is not associated with any obvious change in the structure of the spermatozoa. The most likely explanation seems to be that capacitation involves the activation of an enzyme system; this may possibly be effected by interaction with some- thing originating in the follicular secretions. The Chances of Fertilization It is common experience that, when eggs are recovered from animals such as rats and mice early in the day following coitus, few or no spermatozoa can be seen in the cumulus mass or in the free surrounding fluid, and yet all the eggs are found to have been penetrated. The circumstances certainly suggest the exist- ence of chemotaxis between egg and spermatozoon. Chemotaxis C. R. AUSTIN AND M. W. H. BISHOP 85 seems to occur in ferns, mosses, and seaweeds (Rothschild, 1951a), but so far no unequivocal evidence of attraction between animal gametes has been brought forward; this was pointed out many years ago by BuUer ( 1903 ) and it is apparently still true today (Tyler, 1955). Certain other features may favor the chances of fertilization: the large cumulus mass about many mammalian eggs may help by providing a larger target for the spermatozoa, and the radially arranged cumulus cells, particularly in the near vicinity of the egg, may aid by orientating the spermatozoon toward the egg (Austin and Braden, 1952). The chances of fertilization are, however, chiefly related to the concentration of spermatozoa about the eggs, and this will de- pend both on the total number of spermatozoa present and on the space through which these are distributed, that is, on the size of the site of fertilization. Many more spermatozoa were found at the site of fertilization in the sheep and rabbit than in the rat and mouse (as noted earlier), but the site of fertilization in the for- mer two animals is much larger than in the latter two. The egg itself, the ultimate target for the spermatozoon, also differs in size in these species: sheep and rabbit eggs have approximately four times the surface area of rat and mouse eggs. When all these variables are taken into account, the collision frequency between eggs and spermatozoa is found to be of the same order in all four species (Braden and Austin, 1954a). These considerations relate particularly to the chances of penetration of individual eggs; data on the chances of penetration of the eggs as a group, in any one animal or Fallopian tube, have been reported by Chang ( 1946b,c ) . He found that, when superovulation was induced in rabbits by hormone administration, a higher proportion of eggs was fertilized than in untreated rabbits inseminated with the same numbers of spermatozoa. Evidently either the increased numbers of eggs or the effect of hormone treatment in some way improved the chances of successful fertilization and more than compensated for the need for the penetration of an abnormally large number of eggs. Estrogen administration in mice has also been found to increase the incidence of fertilization, presumably by stimulating uterine and tubal movements and thus increasing 86 PRELIMINARIES TO FERTILIZATION the number of spermatozoa available for fertilization ( Austin and Bruce, 1956). In rats a highly significant association was shown to exist between the number of spermatozoa at the site of fer- tilization and the incidence of extra spermatozoa within eggs (Braden and Austin, 1954a). The data indicate, therefore, that the collision frequency between eggs and spermatozoa is condi- tioned by the mechanical and physical attributes of the female tract in such a way as normally to provide all eggs with good chances of fertilization without too great a risk of penetration by excessive numbers of spermatozoa. However, the eggs themselves also have mechanisms for pro- tection against penetration by extra spermatozoa, namely the zona reaction and the vitelline block to polyspermy, and these devices will be discussed later. For the present it is sufficient to note that in general the entry of extra spermatozoa into the eggs of marsupial and placental mammals must be considered abnor- mal although probably not disadvantageous to the embryo, pro- vided they remain within the perivitelline space. Kushner ( 1954), indeed, reports that Russian workers have observed improved fertility in livestock following insemination with the mixed se- men of several sires. The effect was believed to be due in part to the entry into the egg of several spermatozoa originating from more than one male. The rabbit egg can certainly tolerate the presence of numerous spermatozoa in the perivitelline space ( 20, Van Beneden, 1875; up to 50, Hensen, 1876; a mean of 37, Mori- card and Bossu, 1949; a mean of 17, Chang, 1951b; a mean of 72, Braden, Austin, and David, 1954; up to 20, Adams, 1955). Even in the rabbit egg, however, there is a limit to the invasion: at about 6 hours after ovulation a layer of mucoprotein begins to accumulate on the surface of the zona ( Braden, 1952, 1953 ) , and this layer is impenetrable to spermatozoa (Pincus, 1930; Ham- mond, 1934). Occasionally, more than one spermatozoon enters the vitellus of the mammalian eggs and more than one male pro- nucleus is formed. This constitutes the state of polyspermy which is accepted as being pathological in eutherian mammals, although it is apparently normal in several other species, notably among the insects, elasmobranchs, amphibians, reptiles, birds, and pos- C. R. AUSTIN AND M. W. H. BISHOP 87 sibly monotremes. In mammals it almost certainly gives rise to polyploidy in the embryo (Austin and Braden, 1953b), and poly- ploid embryos seldom go to term (Beatty, 1951). Mechanisms of Spermatozoon Penetration Passage of spermatozoa through the cumulus oophorus is pre- sumably eflFected with the aid of the hyaluronidase that they are known to carry (McClean and Rowlands, 1942; Fekete and Duran-Reynals, 1943; Swyer, 1947a,b). Complete breakdown of the cumulus mass is not a necessary prehminary to spermatozoon penetration and normally does not occur until some time after penetration. The function of hyaluronidase in fertilization, there- fore, appears to be merely that of enabling the individual sperma- tozoa to make paths for themselves through the matrix of the cumulus (Lewis and Wright, 1935; Leonard, Perlman, and Kurzrok, 1947; Austin, 1948a; Austin and Smiles, 1948; Blandau and Odor, 1949; Bowman, 1951 ) . Subsequent denudation of the egg by hyaluronidase, however, may be important in allowing the eggs to start their journey through the tubes and in peniiit- ting a ready gaseous exchange and elimination of metabolites by removing the numerous actively metabolizing follicle cells. Fertilization in rabbits has been prevented by treating the se- men used for insemination with hyaluronidase inhibitors or by injecting the inhibitors into the vagina before insemination ( Pin- cus, Pirie, and Chang, 1948; Parkes, 1953; Chang and Pincus, 1953; Parkes, Rogers, and Spensley, 1954). The results, however, are inconclusive as all the hyaluronidase carried by the spermato- zoa may not have been inhibited and to some extent the failure of fertilization may have been due to the inhibition of other en- zymes, including those involved in the metabolism of the sperma- tozoa. The next step, the passage through the zona pellucida, is even less well understood. In vitro, spermatozoa readily become at- tached to the surface of the zona, particularly of rabbit eggs, and this ability may be important in vivo in ensuring against escape of the spermatozoon and in maintaining close application of the spermatozoon head to pei*mit the action of a possible lytic agent 88 PRELIMINARIES TO FERTILIZATION upon the zona. Adherence of the spermatozoon to the zona pro- vides a parallel in these respects to the trapping of invertebrate spermatozoa in the jelly coat by the fertilizin-antifertilizin reac- tion (Tyler, 1948). It has indeed been shown that there is evi- dence for this reaction in mammals: spermatozoa of the rabbit, mouse, and bull were found to undergo agglutination in vitro in the presence of homologous eggs but showed little effect in the presence of eggs of the other species (Tyler, 1954; Bishop and Tyler, 1956). These authors believe that a fertilizin analogous to that of sea urchin eggs exists in the zona pellucida and that this reacts with antifertilizin on the spermatozoon head so as to bring about both the attachment of the spermatozoon to the zona and its passage through the membrane. On the other hand, ad- herence of the spermatozoon to the zona may well depend upon the same mechanism as head agglutination. If this is so, the ant- agglutin described by Lindahl and his associates would oppose spermatozoon penetration and there would need to be something present about the egg or at the site of fertilization with the prop- erty of counteracting the antagglutin. Following attachment to the zona pellucida, the fertilizing spermatozoon passes through this membrane leaving a small slit that can be recognized many hours after penetration in the eggs of rats (Austin, 1951) and some other rodents. Passage through the zona is evidently rapid, at least in rats, mice, and hamsters, for fertilizing spermatozoa are rarely seen with their heads still within the thickness of the zona ( Austin and Braden, 1956 ) . The possession by the spermatozoon of an agent capable of exerting a lytic action upon the substance of the zona is a reasonable sup- position, but so far no direct evidence of such an agent has been advanced. As already noted, spermatozoa require to spend a pe- riod in the female genital tract in order to undergo capacitation, which may therefore involve either the addition to the sperma- tozoon of a lytic agent produced in the female tract or the acti- vation in the tract of a lytic agent carried by the spermatozoon in an inactive form. A priori, the latter alternative seems prefer- able (Austin, 1951). In varying forms, mammalian spermatozoa possess a structure C. R. AUSTIN AND M. W. H. BISHOP 89 on or near the leading surface of the head with which the sup- posed zona lysin could be associated (Bowen, 1924; Green, 1940), for example, the "vesicle" on the head of the rat spermatozoon (Austin and Sapsford, 1952). It is of interest, too, that a hereditary deformity of the "acrosome" in bull spermatozoa has been found to be associated with sterility ( Hancock, 1949, 1953 ) . The extrac- tion of a lytic agent from the anterior end of the head of Disco- glossus ( toad ) spermatozoa has been reported ( Parat, 1933 ) and similar observations have been made with Megathura (keyhole limpet) spermatozoa (Tyler, 1949) and Mytilus (mussel) sper- matozoa (Wada, Collier, and Dan, 1956). After passing through the zona pellucida the fertilizing sper- matozoon projects into the perivitelline space and because this is small the spermatozoon head soon makes contact with the vitellus or cytoplasmic part of the egg. Contact appears to evoke a reac- tion in the vitellus so that attachment is formed between the spermatozoon head and the surface of the vitellus (Austin and Braden, 1956). The final phase of spermatozoon penetration, the entry into the egg cytoplasm, takes place in a manner suggesting that it is chieHy a function of the vitellus (Austin, 1951). The spermatozoon is absorbed much as a food particle is absorbed by an amoeba, or, as Loeb (1917) pointed out, in a manner re- sembling phagocytosis. Compared with penetration through the zona, entry into the vitellus is comparatively slow, the sperma- tozoon head being attached to the vitelline surface but essentially within the perivitelline space for about half an hour in the rat, mouse, and hamster (Austin and Braden, 1954a; Austin, 1956c). Thereafter, part of the spermatozoon midpiece or tail may still be seen in the perivitelline space or protruding through the hole in the zona at times up to several hours after the entry of the head. Attachment of the spermatozoon head to the vitelline surface appears to be an essential preliminary to entry into the vitellus. Supplementary spermatozoa remain free within the perivitelline space. Probably anything altering the normal state of either sper- matozoon or vitellus may prevent attachment from taking place. It has been reported that unfertilized rabbit eggs with a sperma- tozoon still in the perivitelline space, well after the normal time 90 PRELIMINARIES TO FERTILIZATION of penetration, were seen much more commonly when the semen used for insemination had been treated with a nonspecific hyalu- ronidase inhibitor (Parkes, Rogers, and Spensley, 1954). In mice subjected to hyperthermia, eggs with spermatozoa only in the perivitelline space were chiefly those showing early induced par- thenogenesis (Austin and Braden, 1956). Evidently, artificial ac- tivation of mouse eggs is apt to prevent attachment of spermato- zoon to vitellus. In brief, the passage of the spermatozoon through the cumulus and zona pellucida is presumed to be made possible by enzymes that it carries, whereas entry into the cytoplasm of the egg ap- pears largely to be an active function of the vitellus. Attachment of the spermatozoon to the zona and then to the vitellus must evidently precede penetration of these membranes. Immediate Reactions of the Egg to Spermatozoon Penetration In most mammals — a notable exception being the rabbit — the zona pellucida undergoes a change after the entry of the first spermatozoon, with the result that further spermatozoa tend to be excluded (Braden, Austin, and David, 1954; earlier observa- tions on the incidence of supplementary spermatozoa in mamma- lian eggs are reviewed in this paper ) . This is known as the "zona reaction," and the rate at which it occurs varies between species so that a few extra spermatozoa are often seen in the eggs of the rat, mouse, guinea pig, cat, and ferret, but rarely if ever in those of the dog, sheep, and hamster. In the rat the reaction takes be- tween 10 minutes and 2 hours to reach completion. Smithberg (1953) reported that, in mice, the zona pellucida is removed by proteolytic enzymes more rapidly from unfertilized than from fertilized eggs. This supports the idea that the zona reaction renders the substance of the zona refractory to the action of the supposed zona lysin. The change appears to be induced by an agent released from the vitellus following attachment of a sper- matozoon to its surface (Austin and Braden, 1956). The release of the agent is evidently propagated through the cortex of the egg from the point of attachment of the spermatozoon head, and C. R. AUSTIN AND M. W. H. BISHOP 91 consequently the zona reaction commences at or near the site of penetration and progressively involves the rest of the zona. In the hamster the vitellus of the impenetrated egg displays a large number of cortical granules that disappear when the head of the fertilizing spermatozoon makes contact with the surface (Austin, 1956b). Light scatter by the vitelline surface also dimin- ishes after spermatozoon contact. There is good evidence that a vitelline block to polyspermy does not operate in hamster eggs (Austin, 1956c) so that the cortical changes seen are very likely associated with the zona reaction which is well developed in hamster eggs. In certain respects the responses shown by mammalian eggs are similar to processes involved in membrane elevation in echi- noderm eggs. The fertilization membrane is considered to be the vitelline membrane modified by the products of a change propa- gated through the cortex from the point of spermatozoon contact. The numerous cortical granules, which "explode" when the sper- matozoon enters the egg, evidently play a part in the modification of the vitelline membrane (Moser, 1939; Motomura, 1941; Runn- strom, Monne, and Wicklund, 1944, 1946; Runnstrom and Wick- lund, 1950; Endo, 1952). Changes in the light-scattering prop- erty of the cortex have also been described, although there is disagreement on the direction of the change and on its possible association with the disappearance of cortical granules or the for- mation of the block to polyspermy ( Moser, 1939; Rothschild and Swann, 1949). The zona reaction and the vitelline block to polyspermy are complementary in function. In some species, such as rats and mice, both are operative. In others, such as the hamster, sheep, and dog, protection appears to be vested exclusively in the zona reaction. In others again, such as the rabbit and perhaps the mole and the pocket gopher, the zona reaction is slow or absent and the block to polyspermy seems to be unusually well developed (Braden, Austin, and David, 1954). The block to polyspermy evidently involves a change in the vitelline surface such that attachment will no longer be formed with the head of a contacting spermatozoon (Austin and Braden, 92 PRELIMINARIES TO FERTILIZATION 1956). The rate of its development appears to decrease as the egg ages after ovulation (Austin and Braden, 1953a,b; Braden and Austin, 1954c), and this effect is promoted by local heat (Austin and Braden, 1954b) and by hyperthermia (Austin, 1955a, 1956a; Austin and Braden, 1956). Both aging and heat have long been know^n to increase the incidence of polyspermy in invertebrate eggs (Wilson, 1928). It has not yet been found pos- sible to express quantitatively the normal rate of development of the block in mammals, but estimates have been derived for sea urchin eggs. There is, it seems, a fast partial block covering the egg in less than two seconds and a slower complete component taking about one minute (Rothschild and Swann, 1952; Roths- child, 1954). An early reaction to the penetrating spermatozoon shown by certain invertebrate eggs consists in the formation of a fertiliza- tion cone. Many years ago Asterias (starfish) eggs were observed to develop a filament that moved out to make contact with the nearest spennatozoon and appeared to assist its entry into the egg (Fol, 1877, 1879; see also Colwin and Colwin, this volume). Since then other forms of fertilization cones have been described in different species; in some animals the projection persists after spermatozoon entry, and in others it is withdrawn before this event ( Chambers, 1933 ) . Recent observations on rat, mouse, and hamster eggs show that an analogous elevation develops in the mammalian egg as the spermatozoon head is absorbed through the vitelline surface; it persists for a short time thereafter (Aus- tin and Braden, 1956). It may be seen, therefore, that two of the earliest reactions shown by the mammalian egg to spermatozoon penetration tend to prevent the entry of other spermatozoa. Both reactions are ap- parently evoked by contact of the spermatozoon head with the vitelline surface, but their relative importance varies between species in a complementary manner. Conclusions The evolution of internal fertilization brought with it numerous advantages arising from the complete avoidance of the external C. R. AUSTIN AND M. W. H. BISHOP 93 environment and its vagaries. With internal fertilization there is a much greater likelihood of success, which is achieved through the deposition of the gametes into a highly specialized internal environment. The success of fertilization in this environment, as with the outcome of many other biological situations, is deter- mined by the results of interaction between opposing functions: between those responsible for activating the spermatozoa and those responsible for protection against overactivation, between those favoring rapid transport of spermatozoa and those hinder- ing transport, between those promoting spermatozoon penetra- tion of egg membranes and those preventing excessive penetra- tion. The most important variables appear to be the number of spermatozoa reaching the site of fertilization and their time of arrival. The many integrated processes and reactions that consti- tute the environment of fertilization in mammals have the func- tion of ensuring the meeting of the gametes in the most appro- priate numerical relations and at the optimal phase of their life cycle. In no two species, however, is the mechanism quite the same, so that the preliminaries to fertilization among different species exhibit wide variations in the degree of development of the constituent complementary processes. There may also be disadvantages to internal fertilization which stem from the fact that biological systems are often highly spe- cific within the individual, but they tend to differ between indi- viduals. In particular the immunological processes behave in this way. Spermatozoa are known to show a variety of antigenic prop- erties ( Smith, 1949a,b ) , including the possession of blood group antigens appropriate to the male producing them (Docton, Fer- guson, Lazear, and Ely, 1952 ) . Owing to antigenic diversity be- tween individuals, spermatozoa within the female tract may, to varying degree, be regarded as foreign bodies. According to the "self-not-self" concept, it is a fundamental property of adult organisms that foreign material can be recognized as such and rejected (Burnet and Fenner, 1949; Burnet, 1954). A mechanism of this nature may underlie the failure of heterologous spermato- zoa in many abortive attempts at hybridization. It is possible, also, that antigenic incompatibility between the spermatozoa and 94 PRELIMINARIES TO FERTILIZATION the female genital tract or between spermatozoon and egg, within the same species, may prejudice the chances of fertilization. REFERENCES Adams, C. E. 1955. The frequency of occurrence of supernumerary spermatozoa in rabbit ova. Proc. Soc. Study Fertility, 7, 130-38. Adams, C. E. 1956. Rate of sperm transport in the female reproductive tract of the rabbit. /. Endocrinol, 13, xxi-xxii (proc). Alden, R. A. 1943. The utero-tubal junction in the albino rat. Anat. Record, 85, 290-91. Andersen, D. 1928. Comparative anatomy of the tubo-uterine junc- tion. 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Spontaneous peristalsis of the excised whole uterus and Fallopian tubes of the sow with reference to the ovulation cycle. Bidl Johns Hopkins Hosp., 35, 246-52. Wislocki, G. B., and F. F. Snyder. 1931. On the experimental produc- tion of superfoetation. Bull Johns Hopkins Hosp., 49, 103-5. Wislocki, G. B., and F. F. Snyder. 1933. The experimental acceleration of the rate of transport of ova through the Fallopian tube. Bull. Johns Hopkins Hosp., 52, 379-86. Young, W. G. 1931. A study of the functions of the epididymis. III. Functional changes undergone by spermatozoa during their passage through the epididymis and vas deferens in the guinea-pig. /. Exptl Biol, 8, 151-62. SOME ASPECTS OF MAMMALIAN FERTILIZATION M. C. CHANG: WORCESTER FOUNDATION FOR EXPERIMENTAL BIOLOGY, SHREWSBURY, MASSACHUSETTS, AND DEPARTMENT OF BIOLOGY, BOSTON UNIVERSITY Brief and concise historical accounts of the study of fertiUza- tion have been written by LilHe ( 1923 ) and Austin ( 1953 ) . The Study of fertihzation is a classical subject but lower organisms, especially marine species, were used by early investigators and by most active workers at the present time. In the lower forms, fertilization takes place externally, thus modern physiological, immunological, biochemical, and biophysical techniques can be employed experimentally without much difficulty. In the higher animals, fertilization occurs internally, i.e., in the Fallopian tubes. This makes the experimental analysis of the basic mechanisms of mammalian fertilization very difficult. Moreover, it seems that in the higher animals, owing perhaps to their fertilization in vivo, the process of fertilization is predominantly controlled by internal factors. During the past fifty years, a great deal has been learned about the preparation for fertilization in the mammalian species, such as the growth, maturation, release, and transportation of gametes before fertilization and the morphology and physiology of zygotes after fertilization, but very little is known about the process of fertilization, per se, or the fertilization reaction in mam- mals and the basic mechanisms involved. This is due mainly to the fact that there is no simple procedure for fertilizing mamma- lian eggs in vitro. I shall review here certain facts and some prob- lems involved in the study of mammalian fertilization. The pre- liminaries to fertilization are reviewed by Austin and Bishop in this volume. 109 110 MAMMALIAN FERTILIZATION Fertilization of Mammalian Eggs in vitro The literature on the fertiHzation of mammalian eggs in vitro has been reviewed in detail by Chang and Pincus (1951), Smith (1951), and Austin (1951c). The general procedures employed by various workers were to obtain eggs from the follicles or Fal- lopian tubes and to mix them with sperm collected from the epi- didymis or ejaculate in a physiological solution for certain lengths of time. Then the eggs were either cultured in various ways or transferred into recipient animals. From their reviews it seems that until 1951, except, perhaps, for certain experiments of Pincus (1930), there were scarcely any reports that can be credited as definitive, repeatable experiments demonstrating the successful fertilization of mammalian eggs in vitro although such attempts have been made since Schenk (1878). Reported successes were ascribed to artificial activation or to paithenogenetic cleavage of eggs and other accidental artifacts. Because of the frequency of degenerative fragmentation and of parthenogenetic development and the possibility of gynogenetic development, Smith (1951) suggested that the only certain proof of in vitro fertilization would be to observe and photograph the spermatozoon penetrating the vitellus and the subsequent formation and fusion of male and female pronuclei. Because of technical and optical difficulties, it would be too strict a requirement to ascertain the moment of sperm entry. However, the presence of sperm in the perivitelline space and the formation of a second polar body and a male pro- nucleus would be a good indication of fertilization. Smith (1951) reported that in the presence of Fallopian tube mucosa a spennatozoon penetrated into the ooplasm of 11 of 35 rabbit eggs under a coverslip sealed preparation. Evidence for such penetration was the presence of sperm head within the vitellus, the formation of a male or female pronucleus, or the segmenta- tion of one egg after culture for a longer time. She admitted that sperm penetration was not observed owing to the density of the surrounding corona radiata, and therefore the development might possibly have been parthenogenetic or gynogenetic. It is quite true that when recently shed rabbit eggs are examined under a M. C. CHANG 111 compound microscope a spermatozoon on the top of the zona pellucida can be taken as being in the vitellus and that the pres- ence of a nucleus in the ooplasm may not be a true male pronu- cleus. Venge (1953) reported his fertilization of rabbit eggs in a glass tube under partially anaerobic conditions. Although two litters of young were produced when the treated eggs were trans- ferred into recipient rabbits, he concluded that the development was due to chance and not to controlled processes. Very recently, Shettles ( 1955 ) reported the development of a human egg into a morula after the treatment of an ovarian egg with sperm and tubal mucosa in vitro. He neither excluded the possibility of arti- ficial activation of the egg in his procedme nor mentioned the probability of parthenogenetic cleavage in his paper. Moricard (1950) reported the penetration of a spermatozoon into the ooplasm of 5 out of 21 rabbit eggs (24% ) in vitro under relatively anaerobic conditions obtained by enclosing the eggs in a piece of Fallopian tube placed under petroleum jelly. He stated that the second polar body was not formed and that segmenta- tion was not observed during 7/2 hours culture. Since the for- mation of the second polar body occurs very soon after the penetration of the spermatozoon into the viteUus, and since the transformation of the sperm head into the male pronucleus is also very fast (Pincus and Enzmann, 1932; Chang, 1951a), it is rather peculiar that Moricard's photomicrograph (1950) shows an intact sperm head and no second polar body after 7% hours in culture. Later Moricard (1954) stated that about 30% of rabbit tubal eggs are fertilizable in the utero-tubal secretion con- taining spermatozoa. Since 24% of the eggs can be penetrated in vitro under anaerobic conditions by sperm obtained from the vagina (?), it is not clear whether the 30% of fertilized eggs were due to anaerobic conditions or due to the employment of uterine sperm. After the demonstration that ejaculated sperm require a cer- tain length of time in the female tract to develop their fertilizing capacity (Chang, 1951b; Austin, 1951c), Dauzier et al. (1954) reported that fertilization of rabbit eggs in vitro can be achieved by using sperm recovered from the genital tract of rabbits 12 112 MAMMALIAN FERTILIZATION hours after mating. After the pubHcations by Chang (1951b), Moricard (1954), and Dauzier et at (1954), attempts were made to fertihze rabbit eggs in vitro by uterine or tubal sperm under anaerobic or aerobic conditions, and in the presence or absence of the Fallopian tube. Unfortunately no fertilization was observed (Chang, unpublished). Dr. C. R. Austin told the writer on De- cember 1, 1955, that he was unable to fertilize eggs in vitro with uterine or tubal sperm. It seems that there are still other factors involved beside the "capacitation" of spermatozoa in the female tract. From the above account, it seems that up till now we still do not have a repeatable procedure to fertilize mammalian eggs in vitro. This is not necessarily due to the requirement of an anaer- obic condition because the Fallopian tubes are very vascular ( Plate 1,1). Neither can it be due to temperature change or eggs being rendered unfertilizable once removed from the tube, be- cause freshly shed rabbit eggs can be stored at 10° or 0° C. for 1 day without losing their fertilizability when transferred into the tubes of mated animals (Chang, 1953). Although the physi- cal and chemical environment of the Fallopian tube is important for fertilization, it is not the only place that fertilization can take place because freshly shed rabbit eggs can be fertilized when transferred into the uterus of mated rabbits, but degeneration occurs quickly ( Chang, 1955c ) . It seems that the motility of the Fallopian tube and unknown enzyme systems present in the tube which interact with sperm and eggs may play a role in mamma- lian fertilization. Lillie (1923) stated that "the conditions to be fulfilled in fer- tilization involve, not only penetration of the spermatozoon, or some part of it, into the egg but also reaction between the two." He therefore speaks of "a fertilization reaction when the behav- ior of both partners indicates that the process is proceeding nor- mally." In this respect, the fertilization reaction in the mammalian species may involve a third partner, that is, the Fallopian tube. Parkes ( 1951 ) mentioned that "the biochemical changes involved in penetration, activation and syngamy are almost completely un- known in mammals, and are likely to remain so until fertilization M. C. CHANG 113 Plate I. 1, A portion of a rabbit Fallopian tube showing the blood vessels. 2, The first polar body (A) of an unfertilized rabbit egg and the first polar body (A) and the second polar body (B) of a fertilized rabbit egg. 3, A section of a rat ovaiy, showing the cleavage of a follicular egg and the thick granulosa cells of the follicle. 4, A tripolar spindle of a rabbit blastocyst. 114 MAMMALIAN FERTILIZATION in vitro can be accomplished as a routine experimental proce- dure." Thus in dealing with mammalian fertilization, the fertili- zation reaction as elucidated in the marine species by Hartmann (1949), Runnstrom (1952), and Tyler (1955) is completely ob- scure. Activation and Parthenogenetic Development of Mammalian Eggs In the eggs of mammals, as in those of marine species, the im- mediate consequences of sperm penetration include the develop- ment of a block to prevent polyspermy, the shrinkage of the vitellus, and the resumption of the second maturation division leading to the formation of the second polar body. These proc- esses indicate the activation of the egg from a passive state to an active one. However, activation of eggs even to parthenogenetic development can be achieved by artificial means. Discussions of parthenogenesis include the treatises of Rostand ( 1950 ) and Beatty (1957) and brief reviews of the literature by Tyler (1941), Thibault (1949), Chang and Pincus (1951), Smith (1951), and Austin and Braden (1954b). The elevation of the fertilization membrane in the egg of lower organisms after sperm penetration is one of the earliest criteria of activation, but a fertilization membrane has not been described in mammalian eggs except, perhaps, for one possible instance in the hamster egg (Venable, 1946). Since the mammalian egg at the time of fertilization is enclosed not only with a thick zona pellucida but also with the corona radiata and cumulus oophorus, the observation of any change of the vitelline membrane in a liv- ing egg is extremely difficult. The shrinkage of the vitellus after activation either by sperm penetration or by artificial means is not a reliable criterion. The tonicity of suspending medium and the method as well as the time interval of preparation of eggs for examination are possible causes of shrinkage. In the vitellus of the rat egg, a 14% reduc- tion in volume occurred in the fertilized tubal eggs ( Gilchrist and Pincus, 1932). This is confirmed by Austin and Braden (1954b), who reported also that shrinkage of the vitellus was not consist- M. C. CHANG 115 ently observed after artificial activation by cold shock treatment. In rabbit eggs, sliiinkage of the vitellus after sperm penetration (Pincus and Enzmann, 1932) or after artificial activation (Thi- bault, 1949 ) has also been reported. The formation of the second polar body may be a good criterion of activation by spemi, but one must be sure it is the second polar body, not the cleavage of the first polar body or a fragment of the ooplasm. In the rabbit egg, it is fairly easy to distinguish between the first and second polar body. The cliromosomes of the first one are a group of rods and dots spread in the whole cytoplasm whereas those of the second one are clumped together in a light circular zone (Plate I, 2). The fragment of ooplasm usually has no chromatin. In eggs of many species of marine invertebrates, it is practically only the eggs that fail to extrude polar bodies upon artificial acti- vation that are able to cleave and develop (Tyler, 1941). In the rat egg, the formation of the second polar body is regularly ob- served after artificial activation by cold treatment (Thibault, 1949 ) . This is also confirmed by Austin and Braden ( 1954a ) based on their observation that the first polar body persisted only in 1.3% of untreated eggs. In the rabbit egg, the second polar body is rarely, if ever, observed after cold treatment in vivo or in vitro (Thibault, 1949; Chang, 1954). According to Thibault (1949) eggs were rendered unferti- lizable following artificial activation. In the rat egg, Austin and Braden (1954b) reported that sperm can readily penetrate the eggs that have emitted the second polar body after artificial ac- tivation, and they came to the conclusion that "the development of the block to polyspermy, the shrinkage of the vitellus, and the emission of the second polar body are seen as independent proc- esses, capable of being evoked separately. Only the block to polyspermy appears to be a specific response to sperm penetra- tion." Gynogenetic development comparable to that of frog eggs treated with x-irradiated spermatozoa (Hertwig, 1913; Rugh, 1939) was not obseryed in rabbit eggs (Amoroso and Parkes, 1947). Cleavage of eggs in the ovary of guinea pigs to blastocyst stage 116 MAMMALIAN FERTILIZATION (Courrier, 1923; Bacsich and Wyburn, 1945) and in the ovary of human to the four-cell stage (Krafka, 1939) has been reported. It is probably induced by some abnormal growth or degenerative changes as shown by the thickening of the granulosa layer of the follicles ( Plate 1,3). The cleavage of unfertilized tubal eggs has been reported in the mouse (Charlton, 1917), the rat (Mann, 1924; Austin, 1949), and the ferret (Chang, 1950a). In the rabbit egg, it has been observed on rare occasions a long time after ovu- lation (Chang, 1950a). It may be a manifestation of abnormal cytoplasmic or nuclear activity under certain conditions before degeneration. An ovarian rabbit egg cultivated in vitro by Champy ( 1923 ) cleaved into 8 regular blastomeres in the absence of sperm or any other obvious stimulation. Pincus ( 1930 ) re- ported 63% of unfertilized rabbit eggs divided in a regular way and were indistinguishable from the behavior of fertilized eggs. As in the case of artificial activation of eggs in the lower organ- isms, hyper- or hypotonic solutions, butyric acid, or heat were successfully employed to activate rabbit eggs (Pincus and Enz- mann, 1936; Pincus, 1939). Although anesthesia of the females can also induce the activation of rat eggs, probably owing to the lowering of body temperature ( Thibault, 1949 ) or to the produc- tion of cellular anoxia ( Austin and Braden, 1954b ) , it was found that application of cold either in vivo or in vitro is most effective (Pincus and Shapiro, 1940; Thibault, 1949; Austin and Braden, 1954b; Chang, 1954). The cleavage of eggs to a few blastomeres is quite common after artificial activation. Cleavage up to morula and blastocyst is rare. One 12-celled egg and two blastocysts were obtained by Pincus (1939) when unfertilized eggs were treated with hyper- tonic solution and then transferred into the Fallopian tubes. One collapsed blastocyst was photographed (Pincus and Shapiro, 1940), and one morula and three blastocysts (Thibault, 1949) were found in the rabbit when the Fallopian tubes were previ- ously cooled. However, Chang (1954) has shown that by storage of unfertilized rabbit eggs at 10° C. for one day, 18.6% of 145 eggs developed into blastocysts when transferred into the Fal- M. C. CHANG 117 lopian tubes of recipient rabbits, but no implantation was ob- served after the transfer of 230 treated eggs. According to Chang ( 1954 ) , the transformation of the whole group of chiomosomes in the second maturation spindle into a "pronucleus" and the re- sumption of mitotic division were also observed. The chromo- somes of these blastocysts were determined to be diploid due to the failure of abstriction of the second polar body. Although only a small number of artificially activated eggs de- veloped into blastocysts, three definitely parthenogenetic young rabbits were obtained by Pincus ( 1939 ) when 615 eggs were subjected to various treatments and then transferred to recipient does and "one egg in some 200 developed into a living partheno- genetic rabbit" when freshly ovulated eggs were treated in situ with ice water for 2 to 20 minutes (Pincus and Shapiro, 1940). From the above account, it is quite clear that the activation or parthenogenetic cleavage of mammalian eggs without the pene- tration of sperm is a widely spread phenomenon in vivo as well as in vitro. Since most of the unfertilized eggs do not cleave without the penetration of a spermatozoon, it must be assumed that the cleavage of the unfertilized egg is only induced under certain conditions. This is true also in the artificial activation when eggs were subjected to an adverse environment for a short time. In an abnormal environment, certain systems may be in- jured or enhanced. As F. R. Lillie (1911) stated, "the nature of the inhibition that causes the need of fertilization is a most funda- mental problem." In this respect, the artificial activation of eggs may be a release of the inhibition under certain circumstances. In his review of the general features of artificial partheno- genesis, Tyler ( 1955 ) stated that "in general the percentage of normal development is quite low even when a particular treat- ment initiates development in all of the eggs in a manner indis- tinguishable from that induced by sperm." This is attributable by him to various factors, such as irregularities in distribution of chromosomes, haploidy, lack of a proper division mechanism and, in some instances, to failure to establish a plane of bilateral symmetry. This does not explain why the activated eggs may 118 MAMMALIAN FERTILIZATION divide, develop, and differentiate to an early stage and fail later. Probably the contribution of the male element is more important for more advanced development, differentiation, and adaptation. Sperm Penetration The sperm penetration of the rabbit egg has been observed by Pincus ( 1930 ) , who was under the impression that there was a slight bulging of the ooplasm at the point of sperm entry. The penetration of the rat egg was studied recently by means of phase- contrast microscopy. According to Austin (1951a), the penetra- tion of the rat egg is a very rapid process, taking no more than a few minutes at most for the head to pass through the zona pel- lucida. When the spermatozoon passes through the zona of the rat egg it leaves a slit or potential hole. The entry of the sperma- tozoon into the ooplasm has been observed on several occasions. The spermatozoon may pass straight through the zona into the ooplasm or it may remain for a variable period in the perivitelline space. The head sinks into the vitellus and, the rest of the sper- matozoon, being motionless, is gradually taken in. According to Austin (1951a) the penetration into the vitellus appears to be a function of the vitellus itself, and there must be some property of the head that results in its being absorbed into the vitellus. In a study of the spemi penetration of the rat egg, Blandau and Odor ( 1952 ) did not mention the slit in the zona pellucida, but they stated that the forward progression of the fertilizing s )er- matozoon into the ooplasm is discontinuous and is dependent upon a particular type of undulating movement of the tail which forces the head forward a distance of 10 to 20 microns at a time. They have also observed accessory spermatozoa penetrating into the ooplasm in which the fertilizing spermatozoon has already assumed its final position. The forward movement of accessory sperm, however, is much more continuous and uninterrupted and on one occasion, an accessory spermatozoon was seen to enter and leave the ooplasm a number of times without visible sisjns of decreased activity. But in no instance was the retention of the M. C. CHANG 119 accessory spermatozoon within the ooplasm observed. This is an extraordinary phenomenon, if these ova are not degenerating, and it needs to be confirmed. The modified fertihzation cone described by Pincus in the rabbit egg has not been observed in the rat egg, and the small bit of clear cytoplasm enclosing the fertilizing sperm head in cer- tain rat eggs is considered not a fertilization cone but a result of shrinkage of the vitellus (Blandau and Odor, 1952). Austin and Blandau and Odor agree that several spermatozoa penetrate into the zona pellucida of the rat eggs, 23 in one of Austin's eggs, but they are in disagreement on the manner of sperm entry into the ooplasm. According to the writer's experi- ence with fixed rabbit eggs, the fertilizing spermatozoon is on the very edge of the ooplasm before it changes its shape. If this is true in the rat egg, it is hard to distinguish whether the sperm is absorbed by the vitellus or pushed into the vitellus; maybe both play a part especially considering the viscous nature of the ooplasm. One point of interest is that Austin and Blandau and Odor have observed the penetration of spermatozoa into the vitellus of rat eggs in vitro, but as yet no one has observed penetration through the zona pellucida. Since spermatozoa were never seen entering more than halfway through the zona pellucida during a study of fertilization of rabbit eggs in vitro (Chang, unpub- lished ) , it seems that the penetration of spermatozoa through the zona pellucida is more dependent upon the participation of the Fallopian tube than is the penetration of ooplasm. The problem of polyspermy has been reviewed by Rothschild ( 1954 ) . In many species of animals there is a rapid block at the egg surface after the entry of the fertilizing spermatozoon. In others (birds, salamanders, etc.) many sperm may enter but there is an inhibition of supernumeraiy sperm nuclei to prevent their uniting with the female pronucleus. After a review of the literature on polyspermy in mammalian eggs, Austin and Braden (1953) report that when fertilization is delayed after ovulation, polyspermy occurs in the rabbit and rat eggs. Poly- 120 MAMMALIAN FERTILIZATION spermy induced after hyperthermia in rats has also been reported (Austin, 1955). Several pronuclei and sperm tails were shown in rat eggs, but only male pronuclei were shown in rabbit eggs ( Austin and Braden, 1953 ) . Whether this is due to a division of the female pronucleus as the egg is fertilized at late stages in the case of rabbit egg was not mentioned. Since the male pronuclei in the rat egg "all take part in the formation of first cleavage spindle," the second inhibition mechanism mentioned by Roth- schild is not applicable in the rat egg. As for the tripolar spindle observed in the rat egg (Austin and Braden, 1953, Fig. 9), it is difficult to claim definitely it is a product of polyspenny because a tripolar spindle among normal metaphase plates in a rabbit blas- tocyst has been observed (Plate I, 4). In mammalian eggs, there is probably another mechanism to prevent polyspermy, i.e., the reaction of the zona pellucida to sperm penetration as reported by Braden et al. (1954). Accord- ing to their observation, the penetrability of the zona pellucida by sperm is not influenced by the entry of the first spermatozoon into the rabbit egg, thus 62 sperms were found in the perivitelline space of an egg. The number decreases after the entry of the first sperm in the rat and mouse eggs, for the number of eggs that contained more than one spermatozoon was of a lower fre- quency than expected. The zona pellucida of sheep and dog eggs precludes further penetration of sperm after the entry of the first one because no extra sperms were detected in the perivitelline space of fertilized eggs. Evidence of a change in the zona pellucida has been reported by Smithberg ( 1953 ) , who observed that the zona pellucida of unfertilized mouse eggs was invariably removed by several proteolytic enzymes in less time than that of fertilized eggs. Braden et al. ( 1954) also reviewed the published reports on the number of spermatozoa within the eggs of various mammals. It seems that the zona reaction to the sperm penetration in the eggs of various species falls into the three categories previously mentioned. The zona reaction to sperm penetration is one of the important observations made in this field in recent years. Further study to elucidate this mechanism would be of great interest. M. C. CHANG 121 Pronuclear Behavior during Fertilization Cytological studies of fertilization in the mouse ( Sobotta, 1895; Lams and Doorme, 1908; Gresson, 1941), in the rat (Tafani, 1889; Sobotta and Burckhard, 1911), in the guinea pig (Rubaschkin, 1905; Lams, 1913), in the bat (Van der Stricht, 1910), in the rabbit (Rein, 1883; Pincus, 1939), and in the ferret (Mainland, 1930) are well known. The cytochemistry of mammalian eggs has been investigated recently by Dalcq (1954) and by Ishida (1954). In the rabbit egg, the second polar body is formed about 45 minutes after sperm penetration and is succeeded by the forma- tion of both pronuclei ( Pincus and Enzmann, 1932 ) . The pronu- clear behavior and the correlation between male and female elements at fertilization in the living rat egg were studied by means of phase-contrast microscopy by Austin (1951b,c). He de- scribed the change of the sperm head within 10 minutes after penetration into the vitellus, the occurrence of the second matura- tion division before any alteration of the sperm head, the move- ment and the rotation of the second maturation spindle at telo- phase, the change of the sperm head and that of the female chromosomes into male and female pronucleus, and the three stages of growth and coalescence of the nucleoli of both pronuclei. In supporting the theory of protein synthesis put forward by Caspersson ( 1947 ) , he suggested that the substance produced by the nucleolus associated chromatin, after appropriate modifica- tion by gene action and storage in the nucleoli, passes into the cytoplasm during the terminal reduction of the pronuclei. As the whole process cannot be observed in vitro under the same preparation, Austin (1951a) admitted that "it has been nec- essary to depend upon eggs recovered from rats killed at suc- cessively later times" in order to build up a picture of pronuclear growth. To what extent these processes, especially the coales- cence and division of nucleoli, are due to a degenerative process is difficult to estimate. In the rabbit, eggs recovered at various times after ovulation were stained with vital dyes and 122 MAMMALIAN FERTILIZATION studied under a phase microscope. No obvious coalescence of nucleoli was noted (Chang, unpublished), but a change of the appearance of the cortex at the time of pronucleus formation was observed (Chang, 1955b). Mark ( 1881 ) observed that the pronuclei of Limax eggs come together but do not fuse to form a first cleavage nucleus and stated that "the first cleavage nucleus does not have a morphologi- cal existence." In the rabbit egg, Pincus (1939) stated that "the stage intermediate between pronucleus juxtaposition and spindle formation is rare and must therefore be gone through speedily." In the rabbit eggs recovered at various times after mating the membrane of two pronuclei is clearly seen especially with acetic alcohol fixation but no nuclear membrane was observed before the formation of the cleavage spindle (Chang, unpublished). In Austin's beautiful photomicrographs (1951a), the close contact of pronuclei and the metaphase and anaphase chromosomes are well shown but a clear region without nuclear membrane inter- preted as the prophase chromosomes is not convincing. Since the maturation of pronuclei takes about 10 hours while meta- phase, anaphase, telophase, and first cleavage take about 1 to 2 hours in the rabbit egg ( Chang, 1955b ) , it seems that the growth or maturation of pronuclei corresponds to the prophase of an ordinary cell division. A separate prophase stage after the con- jugation of two pronuclei is still uncertain. Thus the point raised by Mark still requires clarification in mammals. Since Boveri (1888) it has been known that in eggs of most species of animals an important organ of cell division, the cen- trosome, is lacking or inactive, and it is generally introduced by the spermatozoon for the initiation of cell division. There is little understanding of the centrosome in mammalian fertilization al- though the acrosome of the spermatozoon in the rat egg has been discussed (Blandau and Odor, 1950; Austin, 1951a). However, if the centrosome contributed by the sperm is the organ for cleavage, parthenogenetic division may be explained, as Tyler ( 1941 ) has done for the invertebrates and lower vertebrates. M. C. CHANG 123 Hyaluronidase and Antihyaluronidase in Mammalian Fertilization Hyaluronidase in relation to mammalian fertilization has been reviewed by Chang (1950b) and Swyer (1951). According to Mann (1954), the term hyaknonidase, in its widest sense, desig- nates the mucolytic enzyme, or rather a gi'oup of enzymes which bring about the depolymerization and hydrolysis of hyaluronic acid. The mammalian testes and sperm are the richest animaj sources of hyaluronidase. It is known that the hyaluronidase con- tent in the semen is dependent on the concentration of sperm, Swyer (1951) found that the hyaluronidase contents per 100 million spermatozoa per milliliter of semen of rabbit, bull, man, and boar were approximately 20, 7, 0.4, and 0.2 units, respec- tively, but only a very small amount of hyaluronidase, iiTespective of the concentration of spermatozoa, could be detected in dog semen. The recently ovulated rat or rabbit eggs are surrounded by a viscous cumulus oophoris. Yamane (1935), Pincus and Enzmann (1935) brought to light the fact that sperm suspensions or ex- tracts of rabbit sperm could disperse the follicular cells of the cumulus. McClean and Rowlands (1942) discovered that hy- aluronidase obtained from testes, spermatozoa, snake venom, or bacteria can act as a cumulus-dispersing factor by liquefying the viscous gel which cements the follicular cells around freshly ovulated rat eggs. Since the follicular cells surrounding the eggs foiTii an obvious barrier for the penetration of the spermatozoon into the egg, the role of hyaluronidase in mammalian fertilization was very much stressed about ten years ago. Thus, addition of hyaluronidase to rabbit sperm suspension was claimed to increase the fertilizing capacity (Rowlands, 1944), and the application of hyaluronidase for the treatment of human sterility was advocated (Kurzrok et al, 1946; Kurzrok, 1950). It is known that a high concentration of hyaluronidase (Mc- Clean and Rowlands, 1942) or a very high concentration of spermatozoa, 20,000 per cubic millimeter (Pincus and Enzmann, 1936), is needed for the dispersal of follicular cells. This is not in 124 MAMMALIAN FERTILIZATION accordance with mammalian fertilization in the Fallopian tube where only a few spermatozoa are present, about 100 in the rat (Blandau and Odor, 1949), about 1000 to 5000 in the rabbit (Austin, 1948b; Chang, 1951c). It also has been shown that the dispersal of follicular cells is not a prerequisite of sperm penetra- tion because fertilized eggs of the mouse ( Lewis and Wright, 1935), of the rat (Austin, 1948a), and of the rabbit (Chang, 1951a) are still surrounded by follicular cells. The apparent in- crease of fertilizing capacity of rabbit sperm by addition of semi- nal hyaluronidase reported by Rowlands ( 1944 ) was due not to hyaluronidase per se but to the beneficial effect of seminal plasma (Chang, 1947b). This observation, however, does not necessarily exclude the possibility that hyaluronidase present in the individual sperma- tozoon will facilitate its passage through the viscous cumulus oophorus, the cements of corona radiata cells, and the mucopro- tein (Braden, 1952) of the zona pellucida. This postulation, how- ever, cannot reconcile the fact that dog sperm which contains no hyaluronidase is still able to fertilize the dog egg which is not devoid of corona radiata and zona pellucida. It has been suggested that the egg membrane lysin of Tyler ( 1939 ) present in the spermatozoa of marine species, which helps the sperm to penetrate the jelly coat, is a mucopolysaccharase similar, but not identical, with hyaluronidase. This suggestion, according to Mann (1954), is in need of experimental support. Thus the physiological role of hyaluronidase is far from clear. It may be related to the spermiogenetic function as suggested by Mann, or it may function to prevent the spermatozoa from stick- ing together at the end of spermatogenesis in the testes. It may be associated with the activity of the Fallopian tubes which denu- date ( dispersal of corona radiata ) the rabbit eggs ( Swyer, 1947 ) , because in the absence of sperm the denudation of rabbit eggs takes longer than in the presence of sperm ( Pincus, 1930; Chang, 1951a). A hyaluronidase inhibitor, nitrated hyaluronic acid, was re- ported to effect the fertilization of rabbit eggs (Pincus et ah, 1948). Another hyaluronidase inhibitor, phosphoiylated hesperi- M. C. CHANG 125 din, was advocated as an oral antifertility agent (Martin and Beiler, 1952; Sieve, 1952), but when phosphorylated hesperidin was fed to rats or deposited in the Fallopian tubes of rabbits at the time of fertilization, no antifertilization effects were observed ( Chang and Pincus, 1953 ) . Prevention of fertilization by another hyaluronidase inhibitor, trigestistic acid, was examined by Parkes ( 1953 ) , who concluded that although this compound has no effect orally, when added to the sperm-suspension for insemina- tion, "the spermatozoon is incapacitated by it in some way, or other than, or additional to the neutralization of hyaluronidase." According to Parkes (1955), after the treatment of sperm with hyaluronidase inhibitor, there is evidence that spermatozoa are lodged in the perivitelline space of an egg in which no sperma- tozoon has penetrated the vitellus. If this is a fact and not an accidental observation, the inhibition of sperm hyaluronidase may result in the inability of sperm to penetrate vitellus. How- ever, it is not known whether the vitelline membrane contains hyaluronic acid. Agglutination of Spermatozoa and Fertilization "Egg water" is enriched with some substances derived from sea urchin eggs and capable of inducing the activity and agglu- tination of homologous spermatozoa. This sperm-agglutinating agent was called "fertilizin" by Lillie (1923). Agglutination of horse or rabbit sperm occurs at lower (3-6.3) or at higher (8.3) pH, and also occurs in blood serum, in the secretions of the vagina or uterus, or in body fluid (Kato, 1938). It occurs in tissue ex- tracts (Chang, 1947a) and in saline containing Congo red or Chicago blue (Chang, unpublished). According to Parkes et al. ( 1954 ) sperm heads coated with a negatively charged substance are agglutinated in the presence of positively charged substances. It seems therefore that the agglutination of mammalian sperma- tozoa is not a specific reaction but a very general one. It follows then that any condition that induces agglutination may or may not at the same time impair the fertilizing capacity of mammalian spermatozoa. As for the sperm agglutination in relation to ferti- lization reaction of marine species as elaborated by Tyler ( 1948 ) 126 MAMMALIAN FERTILIZATION in immunological tenns, it is a different subject matter. It has been claimed that agglutinated sperm are unable to fertilize (Anderson, 1945; Lindahl and Kihlstrom, 1954), but contradic- tory evidences are available. For instance, intensively aggluti- nated sperm were recovered from the rabbit vagina, but young rabbits were obtained (Kato, 1938). Agglutinated rabbit sperm is invariably recovered from the uterus, and yet fertilization oc- curs after the deposition of uterine sperm into the Fallopian tube (Chang, 1955a). Lindahl and Kihlstrom ( 1952 ) reported that mammalian sper- matozoa agglutinate when semen is stored for some time un- diluted or diluted with saline. They interpreted this to be due to a spontaneous inactivation of "sperm antagglutin" produced in the prostate gland. Lindahl and Nilsson (1954) further reported the finding of a "female sperm antagglutin" present in the fol- licular fluid and in the Fallopian tube of estrous rabbit. Since the mammalian egg is suspended in the follicular fluid, if there is a "fertilizin" diffused from eggs to activate and to agglutinate sperm, as in the case of the sea urchin, follicular fluid should in- duce the activity of sperm as reported by Kurzrok et al. ( 1953 ) rather than induce the disappearance of agglutination as reported by Lindahl and Nilsson ( 1954 ) . This important finding of "ant- agglutin" in the male and female tract of mammals may have other implications for mammalian fertilization which need fur- ther elucidation. Conclusions The understanding of physiological changes at sperm penetra- tion, activation, and syngamy is very limited. This is mainly due to the fact that there is no simple and repeatable procedure to fertilize mammalian eggs in vitro. The reaction substances present in sperm and eggs at fertilization as elucidated in the fertilization of marine species may not be transferred to mammalian fertiliza- tion at present. The Fallopian tube may play a very important role in mam- malian fertilization, especially at the time of sperm penetration through the intercellular spaces of the corona radiata and the M. C. CHANG 127 zona pellucida, but our knowledge of the physiology of the Fallo- pian tube, the corona radiata, the zona pellucida, and the vitel- line membrane is very elementary. The activation or cleavage of eggs in vivo and in vitro without the stimulation of sperm is fairly common, but its mechanism is not known. Although parthenogenetic development to the blas- tocyst stage or to newborn can be achieved by subnormal tem- perature treatment of eggs, there may be still more effective methods to be discovered. The physiological difference between artificial activation and fertilization should be further inves- tigated. Sperm penetration of the vitellus, the formation of male and female pronuclei, and the maturation and conjugation of pronu- clei have been observed, but their physiological properties and their interactions are completely unknown. Even the morphology of conjugated pronuclei and their subsequent division are far from clear. The fertilizability of eggs and its physical basis, the blockage of polyspermy and its chemical nature, and the possibility of the artificial induction of polyspermy and its subsequent events are all obscure. In view of the possibility of a complex of enzymic reactions at fertilization, the role of sperm hyaluronidase at fertilization has been erroneously interpreted. At present, we have certain knowl- edge about what hyaluronidase or hyaluronidase inhibitor may not do, but we do not know the actual function of sperm hyalu- ronidase. It seems that agglutination of mammalian spermatozoa is not a specific reaction to eggs but rather a general reaction. The ag- glutination of sperm in relation to the fertilizing capacity of sperm is uncertain and its relation to the fertilization reaction of mammalian eggs is far from understood. REFERENCES Amoroso, E. C, and A. S. Parkes. 1947. Effects on embryonic develop- ment of x-irradiation of rabbit spermatozoa in vitro. Proc. Roy. Soc. (London), B134, 57-78. 128 MAMMALIAN FERTILIZATION Anderson, J. 1945. The Semen of Animals and Its Use for Artificial Insemination. Imperial Bureau Animal Breeding and Genetics, Edin- burgh. Austin, C. R. 1948a. 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L'oocyte a la fin du stade d'accroissement, au stade de la maturation, au stade de la fe- condation et au debut de la segregation. Mem. acad. roy. med. Bel- gique, 2, 1-176. Venable, T. H. 1946. Pre-implantation stages in golden hamster {Cri- cetus auratus). Anat. Record, 94, 105-19. Venge, O. 1953. Experiments on fertilization of rabbit ova in vitro with subsequent transfer to alien does. In Mammalian Germ Cells, G. E. W. Wolstenholme, editor. Churchill Ltd., London. 134 MAMMALIAN FERTILIZATION Yamane, J. 1935. Kausal-analytische Studien iiber die Befruchtung des Kanincheneies. I. Die Dispersion der Follikelzellen und die Ablo- SLing der Zellen der Corona radiata des Eies diirch Spermatozoen. Cytologia (Tokyo), 6, 233-55. MORPHOLOGY OF FERTILIZATION: ACROSOME FILAMENT FORMATION AND SPERM ENTRY ARTHUR L. COLWIN and LAURA HUNTER COLWIN: QUEENS COLLEGE, FLUSHING, NEW YORK Some Aspects of Sperm Morphology The older studies of spermatozoa by Retzius ( 1904, 1905, 1910) and others revealed that there are wide morphological variations among the spermatozoa of different species. Contem- porary investigations of sperm morphology are in part directed toward a detailed analysis of structure by means of electron mi- croscopy as in the studies of Dan ( 1952, 1954 ) and of Burgos and Fawcett (1955, 1956), and in part toward a cytochemical characterization as in the studies of Clermont, Glegg, and Le- blond (1955). Phase contrast microscopy is facilitating observa- tion of the living spemiatozoon. The present paper will deal chiefly with the acrosomal region, especially with reference to certain changes occurring in this and other regions at fertilization or under other conditions some of which may obtain at fertiliza- tion. The acrosomal region can be seen as a structural entity in sper- matozoa of many species. Often it projects beyond the nucleus in an apical or near apical position. Some investigators have used the terms "acrosome" and "perforatorium" synonymously to in- dicate the entire acrosomal region. Others have used these same terais to distinguish two specific structures within the acrosomal region. It is to be expected that the confusing terminology which exists in the literature concerning the acrosomal region will soon be clarified (vide Burgos and Fawcett, 1955, 1956). Dan and Wada (1955) have described three differentiated 135 136 MORPHOLOGY OF FERTILIZATION 7 8 9 10 I? 12 13 Figs. 1-13. Spermatozoa of bivalve mollusc species to show morphologi- cal changes resulting from the acrosome reaction. (From Dan and Wada, Biol. Bull., 1955.) 1, Mijtiliis edulis spermatozoon in sea water. 2, Mytiltis spermatozoon after egg water treatment. 3, Mijtilus spermatozoon showing partial reaction. 4, Petricola japonica spermatozoon before reaction. 5, Re- acted Petricola spermatozoon in vicinity of egg. 6, Partially reacted Petricola spermatozoon. 7, Mactra sulcataria spermatozoon. 8, M. sulcataria super- numerary spermatozoon. 9, Mactra veneriformis spermatozoon. 10, M. veneriformis spermatozoon after egg water treatment. 11, Spondylus cruen- tus spermatozoon. 12, Spondylus spermatozoon reacted in egg water (side view). 13, Reacted Spondylus spermatozoon as found affixed to under side of coverglass. A. L. COLWIN AND L. H. COLWIN 137 parts of the "acrosome" in spermatozoa of several species of bi- valve molluscs (Figs. 1, 4, 7, 11): a distal section containing what these authors believe to be an egg-membrane lysin, an axial structure possibly related to the precursor of a filament, and a basal structure which may be concerned with the extrusion of the filament. The axial structure described in these acrosomal re- gions would seem to resemble the "stereocil" reported in the spermatozoa of some prosobranch molluscs and in Lumbricus (Grasse and Tuzet, 1933; Tuzet, 1950). Fig. 14. Profiles of heads of sea urchin spermatozoa. (From Afzelius, Z. Zellforsch., 1955.) a-b, Strongijlocentrotus droebachiensis; c, Psammechi- nus miliaris; d, Echinus esculentus; e, Echinocardhim cordatum. Afzelius ( 1955 ) has made electron microscope studies of thin sections of spermatozoa of four species of sea urchin. They reveal that a rather dense acrosomal particle lies in the tip of the acro- somal region. Beneath this particle the nucleus shows a depres- sion or "cave" filled with material less osmiophilic than that of the particle (Figs. 14—15). In one species, Echinocardium corda- tum, the acrosomal region is elongated in a manner unusual for sea urchins (Figs. 14e and 15B). Nevertheless, the acrosomal particle occupies the apical position. Beneath the particle a long stalk is apparently filled with material like that of the "cave" in 138 MORPHOLOGY OF FERTILIZATION the nucleus. In all four species there is some evidence that the material filling the "cave" is oriented as fibers. These structures of the acrosomal region, as well as all other parts of the sperma- Fig. 15. Electron micrographs of thin sections of sea urchin spermato- zoa. (From AfzeUus, Z. Zellforsch., 1955.) Longitudinal sections, A, through head of Strongijlocentrotus droebachiensis and B, through two acrosomal regions of Echinocardium cordatum. tozoon, are completely enclosed within a cell membrane. Afzelius suggests a comparison between the acrosomal particle he ob- served and the particle observed by Dan ( 1952 ) in living sper- matozoa of other species of sea urchin. A. L. COLWIN AND L. H. COLVVIN 139 Response of Spermatozoon to Certain Stimuli Although there have been numerous studies on the morpho- logical changes which the individual egg undergoes during fertili- zation or parthenogenetic activation, there have been few inves- tigations of the also important moiphological changes which the individual spermatozoon undergoes at the critical time when it becomes associated with the egg, or under conditions which may be supposed to obtain at such a time. Yet theories of fertiliza- tion must be in accord with the morphological features observed during sperm-egg association and sperm penetration into the egg. Most of the early studies were based chiefly on fixed material. A few are of particular interest. Kupelweiser ( 1909 ) and Meves (1915) studied fixed material of the mollusc Mytilus edulis; both noted the complete disappearance of the "perfora- torium" by the time the spermatozoon had entered the egg, and Meves further noted that the perforatorium had disappeared even from spermatozoa in process of entering the egg. Lillie (1912b) pointed out that in Nereis the perforatorium of the fixed sperma- tozoon is a rather straight filament, but he called attention to the fact that in the living speniiatozoon the perforatorium is shaped more like "the spike of a helmet" and is shoHer. Popa (1927) studied Arbacia spermatozoa suspended in egg water with the expectation that any changes observed would resemble those preliminary to fertilization. He reported that the treatment caused the appearance of a small granule of sticky substance which he thought was eliminated through the pointed apex of the spermatozoon. Other changes in spenn morphology also were noted. While Popa's observations are difficult to assess, his experiments were at least an attempt at observation of the individual living spermatozoon. Dan (1952, 1954; Dan and Wada, 1955) first demonstrated clearly that on suitable stimulation the spermatozoa of a number of species can undergo rather consistent profound morphological changes. These changes aff^ect the acrosomal region and, to some extent, the middle piece and flagellum. This reaction was termed 140 MORPHOLOGY OF FERTILIZATION the "acrosome reaction" and spermatozoa which showed the changed appearance were called "reacted" spennatozoa. In most species, egg water and/or the presence of unfertilized eggs were effective stimuli. Although these conditions might be supposed to approach those encountered at fertilization, it was found also that the changes could be evoked in other ways, such as by treatment with alkaline sea water, or by "contact" with solid surfaces (e.g., glass slides, collodion membranes). It was not demonstrated whether the causal agent was contact per se or some other as yet undetermined factors associated with the han- dling of the preparations. Dan's studies included two species of sea urchin {Pseudocentrotus depressus and Strongylocentrotus pulcherrimus) , three species of starfish {Asterina pectinifera, Asterias amurensis, and Astropecten scoparius), and twelve spe- cies of bivalve mollusc. Sea Urchins. In studies of living spermatozoa by phase con- trast microscopy and of whole formalin-fixed spermatozoa by electron microscopy, Dan (1952) found that suspension in egg water would induce the acrosome reaction. The reaction was in- terpreted as a breakdown of the acrosomal membrane so that the contained acrosomal material was exposed. Part of the ex- posed material was dispersed rapidly. There remained a central core or fiber which Dan ( 1954 ) termed the "acrosome filament" (Fig. 16; cf. with the untreated spermatozoon, Fig. 18). The length of the filament was about one micron, short as compared with the length of the sperm head. A similar reaction occurred when sea urchin sperm suspensions were placed in alkaline sea water of pH 9.2. Frequently the reacted spermatozoon (Fig. 17) showed the middle piece displaced to a lateral position while the flagellum appeared to emerge from the head at an angle instead of lying in the long axis of the body. Reacted spermatozoa with acrosome filaments were found also in untreated control suspen- sions. Dan interpreted these to be the result of "contact" stim- ulation with a solid surface. Rothschild and Tyler ( 1955 ) studied sea water sperm suspen- sions from two other species of sea urchin. In Echinocardium cordatum they found two forms of the spermatozoon and con- A. L. COLWIN AND L. H. COLWIN 141 Figs. 16-18. Electron micrographs of spermatozoa of the sea urchin, Strongylocentrotus pulcherrimus. (From Dan, Biol. Bull., 1952.) 16, after agglutination with egg' water. 17, after treatment with alkaline sea water. 18, in sea water (control). Figs. 19-20. Electron micrographs of spermatozoa of Echinocardium cordatum. (From Rothschild and Tyler, Exptl. Cell Research, 1955.) 142 MORPHOLOGY OF FERTILIZATION sidered the possibility that either form might be the precursor of the other. In one type of spennatozoon (Fig. 19), fomid in both hving and fixed preparations, the acrosomal region appeared as a short stalk terminating in an apical knob. This is the type of spermatozoon illustrated by Vasseur ( 1947 ) in darkfield studies and, apparently, by Afzelius ( 1955 ) in electron microscope studies of thin sections (Figs. 14e and 15B). The other type of sperma- tozoon (Fig. 20), found only in fixed preparations, has a pro- foundly different appearance. Extending from the apex of the sperm head is a slender filament which is much longer than the acrosomal region of the first type. There is no terminal knob, the middle piece lies in a postero-lateral position, and the flagellum and acrosome filament lie nearly at right angles to each other. All these characteristics are typical of the reacted sea urchin spenna- tozoon as shown by Dan. It seems probable that Fig. 20 represents a reacted spermatozoon. Tyler's (1952) electron microscope stud- ies of fertilizin-treated sea urchin spermatozoa would seem to support this view. After reversal of agglutination, these sper- matozoa too showed the middle piece postero-laterally displaced from its normal location at the base of the head. Since Rothschild and Tyler found spermatozoa with long acrosomal filaments only in -fixed preparations, the possibility exists that the fixative itself or some feature of the process en route to fixation may have stimulated the acrosomal regions to produce these filaments ( vide supra, living i;.s fixed spermatozoa of Nereis; for a more detailed discussion see Colwin and Colwin, 1956 ) . Staiiishes. A more striking example of the acrosome reaction with formation of an acrosome filament was found in several starfish species by Dan ( 1954 ) . When sperm suspensions were made in sea water plus egg albumin ( Metz, 1945 ) to which was then added homologous egg water, irreversible agglutination occurred. Both phase contrast (Fig. 21) and electron microscope studies (Fig. 23) showed that within such preparations there were spermatozoa with long slender acrosome filaments of con- siderable rigidity. The filament extended from the apex of the acrosomal region for about 25 microns. In the reacted sperma- tozoon (Fig. 22A) the middle piece was more nearly spherical A. L. COLWIN AND L. H. COLWIN 143 Fig. 21. Reacted spermatozoa of Asterina pectinifera showing acrosome filaments after suspension in egg water. (From Dan, Biol. Bull., 1954.) a, small cluster of agglutinated spermatozoa; b, single reacted spermatozoon. than in the unreacted spermatozoon (Fig. 22B), and the base of the flagelknn and the acrosome filament lay nearly at right angles to each other. Molluscs. In some of the species of bivalve mollusc studied by Dan and Wada ( 1955 ) , the acrosome reaction was elicited by the species egg water and/or by "contact" with glass surfaces. However, in all the species studied, the spermatozoa produced acrosome filaments when placed in the presence of unfertilized Fig. 22. Head ends of spermatozoa of starfish. (After Dan, Biol. Bull., 1954.) A, reacted, showing the acrosome filament; B, unreacted. 144 MORPHOLOGY OF FERTILIZATION A. L. COLWIN AND L. H. COLWIN 145 eggs of the same species (Figs. 1-13 and cf. Figs. 1 and 2 with Figs. 24 and 25). In all species the reaction resulted in the com- plete disappearance of the original acrosome and the production of a slender rigid filament. The length of the filament varied with the species, being in each case about three times the length of the sperm head. The longest filament measured about 15 microns. In the reacted spermatozoa the middle piece spherules appear to have become less tightly affixed to the base of the sperm head. In some species the reacted condition was observed also in "supernumerary" spermatozoa, that is, in spermatozoa which were associated with the egg (Figs. 5 and 8) but did not sub- sequently enter it. In some species spermatozoa were found in which the acrosome had undergone only a "partial reaction" (Figs. 3 and 6), supposedly because of subnormal condition of the spermatozoa or suboptimal stimulation. In these spermatozoa the filament was always about the length of the original unreacted acrosome. Rothschild and Tyler ( 1955 ) have reported that fixed sperma- tozoa of the chiton Lepidochitorui cinerea, seen with the electron microscope, have an "acrosomal filament" or "anterior process" about twice as long as the rest of the head, whereas in living spermatozoa, seen with the light microscope, "the proximal por- tion of the process is the only part visible." Until a comparison is made between fixed and living spermatozoa under one kind of microscope, a different interpretation cannot be excluded, namely, that the two different relative lengths of the process might repre- sent reacted and unreacted conditions in fixed and living sperma- tozoa, respectively. Some factor in the initial phase of fixation might have stimulated the acrosome reaction. Dan's important observation that spermatozoa undergo an acrosome reaction in response to various stimuli has been con- Fig. 23. Electron micrograph of a reacted (egg water treated) sperma- tozoon of Asterias amtirensis; black arrow points to acrosome filament. (From Dan, Biol Bull, 1954.) Figs. 24-25. Electron micrographs of formalin-fixed spermatozoa of Mytilus edtilis. (From Dan and Wada, Biol Bull, 1955.) 24, in sea water (control). 25, reacted (egg water treated); black arrow points to acrosome filament. 146 MORPHOLOGY OF FERTILIZATION finned in living material in other species of starfish and in the holothurians, Holothiiria atra and Thijone briareus (Colwin and Colwin, 1955a,c, 1956). Quantitative data for the acrosome reaction have been obtained in Thy one. Table I. Acrosome Reaction of Spermatozoa of Thjjone briareus as Obseived in Living Preparations. Production of Acrosome Filaments Evoked by Adding Sea Water Sperm Suspensions to Egg Water or to Sea Water Made Alkaline by Addition of O.liV NH4OH. (From Colwin and Colwin, 1956.) Number Per cent Number Per cent spermatozoa with spermatozoa with E.xperiment No. counted filaments counted filaments Egg water Sea water control ECSN-l" 100 21 100 2 EG,SN-2 52 21 >100 0 EG,SG-3 100 18 100 3 EG,SN-4 104 12.6 100 1 EG,SG-5 73 6.8 100 0 EG,SG-6(a) 111 1.8 sweep'' 0 EG,SG-7(a) 100 0 sweep <1 Alkaline sea water Sea water control EG,SG-6(b) sweep >90^ sweep 0 EG,SG-7(b) sweep >99'= sweep <1 EG,SG-7(c) 97 92'' sweep <1 EG,SG-8 sweep >90'' sweep 0 Note. Experiments bearing the same number were made simultaneously from the same sperm suspension and have the same control. °E eggs; S sperm; N naturalh' shed; G obtained b^' removal from gonad. ^ Sweep Extensive examination of area of coverslip by back and forth move- ment with a mechanical stage. ' pH of alkaline sea water to which sperm suspension was added was slight) v higher than 9.2-9.4 ■^ pH of alkaline sea water to which sperm suspension was added was 9.2-9.4. Thyone briareus. In this species acrosome filaments were formed when sperm suspensions were treated either with egg water or with alkaline (ammoniated) sea water (Table I). Within the concentrations used, the latter was found to be the A. L. COLWIN AND L. H. COLWIN 147 more effective stimulant. Thus as shown in experiment 7 of Table I, with samples from the same original sperm suspension egg water was ineffective whereas alkaline sea water caused at least 92% of the spermatozoa to produce acrosome filaments. Since untreated (control) sperm suspensions sometimes show a very low percentage of spermatozoa with acrosome filaments, it Oj 9r 0 L. zo MO A Fig. 26. Reacted spermatozoa showing acrosome filament, from photo- graphs of living specimens. (From Colwin and Colwin, Biol. Bull., 1956.) a— c, Thyone biiareus; d-e, Asterias forbesii. a, in egg water (35 microns); b, at egg surface (48 microns); c, in alkaline sea water (75 microns) (po- sition of distal part of flagellum modified for reasons of space); d, in insem- inated culture but directed away from egg (15 microns); e, at egg surface (22 microns). would seem possible that other factors, as yet unknown, may also evoke the acrosome reaction. The changes which the spermatozoa undergo when suitably stimulated are seen readily in living preparations. Figure 28 shows an unreacted spermatozoon for comparison with others which have reacted to treatment with alkaline sea water (Figs. 26c, 29 and 30) or with egg water (Figs. 26a and 31). Reacted spermatozoa may be found also in preparations of inseminated eggs. The sperm head is connected with the egg surface by a 148 MORPHOLOGY OF FERTILIZATION long acrosome filament (Figs. 26b and 32). Typically, the reacted spermatozoa show the middle piece rounded in outline, while the filament and the base of the flagellum lie nearly at right angles to each other. This appearance resembles that of reacted starfish spermatozoa (Dan, 1954). Fig. 27. Reacted spermatozoa; bending and curving of acrosome fila- ment, from sketches and photographs of hving specimens. (From Colwin and Colwin, Biol. Bull., 1956.) a-h, Thxjone briareus; i-1, Asterias forbesii; m, A. vulgaris (presumed distal part of acrosome filament dotted), a and k-m, at egg surface; others, unassociated with egg. b, in egg water; c— h, in alkaline sea water (d, same specimen as c, after separating from fixed point); i-j, successive views of one specimen, in inseminated culture but not associated with an egg. A striking feature of the acrosome filament of Thyone is its great length. Whereas the longest acrosome filament previously reported attained a maximum length of 22 to 28 microns ( in star- fishes, Dan, 1954), the filament of Thyone occasionally is as long as 75 microns (Fig. 29), and in one case it measured 90 microns. For comparison, the length of the flagellum is usually A. L. COLWIN AND L. H. COLWIN 149 about 60 microns, and the depth of the jelly hull, through which the acrosome filament must penetrate in order to reach the egg surface, is approximately 55 microns. The spermatozoa of Thyone do not swim through this jelly hull. Other Species. In Holothuria atra (Colwin and Colwin, 1955a), acrosome filaments were found on spermatozoa sus- pended in egg water (with egg albumin as an adjuvant) and on spermatozoa associated with the egg surface (Fig. 38). In starfishes (Colwin and Colwin, 1955a,d, 1956) the acrosome fila- ment has been found on spermatozoa in association with the egg in Asterias amurensis (Fig. 35), A. vulgaris (Fig. 27m) and A. forbesii (Figs. 27k,l and 33 and 34). In all four species sper- matozoa with acrosome filaments were found also in the surround- ing medium but not directly associated with any egg. There is also evidence of acrosome filament formation in spermatozoa of the annelid Sabellaiia vulgaris (Colwin and Colwin, 1955b). Metz and Morrill (1955) have demonstrated that fertilizin treatment of spermatozoa of Asterias forbesii and the annelid Nereis liinbata results in the production of acrosome filaments. In Nereis the acrosome membrane breaks down and a rod-like filament is exposed (or arises); the overall length of the sperm head remains unchanged. This material was fixed in formalin and examined with the electron microscope. From the foregoing evidence it is quite clear that in many spe- cies, representing several phyla, the spermatozoa undergo rather profound changes when suitably stimulated. The most striking change is the production of the more or less straight, relatively rigid acrosome filament. In some species the reaction has been elicited by egg water, in some by alkaline sea water or by surface "contact," but in others it has been seen thus far only when the spermatozoa are in the vicinity of, or in contact with, unfertilized species eggs (some molluscs). As is well known, a number of stimuli other than the normal one (that is, association with the opposite gamete) may cause parthenogenetic activation of the egg. It is not surprising, then, that the spermatozoon too may be activated by other than normal stimuli. It is suggested that the term "acrosome filament" be reserved 150 MORPHOLOGY OF FERTILIZATION A. L. COLWIN AND L. H. COLWIN 151 expressly for the filamentous structure or projection which occurs in the acrosomal region when the spermatozoon undergoes the acrosome reaction and that the term be avoided when referring to the unreacted acrosomal region whatever its length may be. Some Physical Properties of the Acrosome Filament Shape. The threadlike acrosome filament is generally rather straight although this is not invariably the case. The filament may curve gently, or sharply, or even bend ( cf . various examples in Figs. 26-35). Although it appears to have considerable rigidity, it is nevertheless somewhat flexible. This was shown by Dan for certain starfishes (Fig. 21), and it has been found so for other starfishes and also for Thijone (Fig. 27). In some cases the acro- some filament was observed to break. Occasionally spermatozoa were seen in which it appeared that the head had reacted but the acrosome filament was much shorter than usual, or simply not present. Possibly breakage may account for these exceptional specimens. Length. Dan and Wada (1955) have indicated that there does not seem to be a simple taxonomic relationship between species and length of acrosome filament. However, it is evident that a positive correlation exists between the length of the fila- ment and the depth of the barrier through which the sperma- tozoon must pass in order to meet the egg proper. For example, in a number of sea urchins the jelly hull offers little or no barrier and the spermatozoon readily swims through it to the egg; in these the acrosome filament is very short. In contrast, the egg jellies of various starfishes and holothurians ofl^er effective bar- Figs. 28-35. Unretouched photographs of living preparations. (From Colwin and Colwin, Biol Bull, 1956.) Black arrow points to acrosome fila- ment, white arrow to flagellum. The scale is in microns and applies only to Figs. 28-34. 28-31, spermatozoa of Thijone hriareus: 28, unreacted sper- matozoon; 29-30, long acrosome fikiments on reacted spermatozoa in alka- line sea water; 31, egg water treated spermatozoon with acrosome filament; 32, reacted spermatozoon with acrosome filament in contact with egg surface ( no cone formed ) . 33-34, reacted spermatozoa of Asterias forbesii showing acrosome filament in contact with the egg surface (no cone formed). 35, reacted spermatozoon of Asterias amurensis showing the acrosome filament in contact with the egg surface (no cone formed). 152 MORPHOLOGY OF FERTILIZATION riers through which their respective spermatozoa do not swim. In these species the acrosome filament is quite long. In the ex- treme case of Thyone the depth of the barrier approximates 55 microns and the length of the filament is sometimes even greater than this. It has been suggested for some species (Colwin and Colwin, 1954b; Dan, 1954) that the egg reacts only after the filament of the spermatozoon makes physical contact with the true egg surface or with the protoplasm immediately beneath 0 ~ "oTi li nv ^ V V ^ °oo °oooo_ooo OOoV)OOOo«>0 Oo o '>oO°0 0 oo 00 o 0-0 ^O oo CO® 0°o' o Wo o " a O ** o o Fig. 36. Diagrammatic representation of successive stages of sperm en- try as seen in the living egg of Holothiiria atra. (From Colwin and Colwin, /. Morphol, 1955a.) A. L. COLWIN AND L. H. COLWIN 153 the surface. Subsequently, this suggestion has received strong support from observations on sperm entry in several other species (Colwin and Colwin, 1955a, 1956). From the above considera- tions it seems likely that the acrosome filament of a species would have to be at least as long as the depth of the barrier which sep- arates the approaching sperm head from the reactive egg surface in that species. Within a given preparation (e.g., in Thyorie) spermatozoa may be found with acrosome filaments of different lengths. Some filaments are shorter than the depth of the barrier around the species egg. It has been observed frequently that the first spermatozoon, or even the first several spermatozoa, to reach a ripe egg may not succeed in fertilizing it. Inadequate length of the filament might account for such failure. Sperm-Egg Association and Sperm Entry The acrosome filament now may be examined in relation to the egg during sperm entry. The following descriptions are based exclusively on living material. Holothuria atra. The exceptional clarity of the entrance cone in this species made it possible to observe many details readily (Colwin and Colwin, 1955a). The eggs were removed from the gonads. Most were in the germinal vesicle stage when used. Al- though all were moderately polyspermic and failed to cleave, they were considered (op. cit.) to be close to physiological ma- turity and to reflect the essentials of the normal entry pattern of the species. The egg is surrounded by a jelly hull ( Fig. 37 ) through which the spermatozoon does not swim. The chief events of sperm entry are shown diagrammatically in Fig. 36, and representative stages are shown in photographs in Figs. 39 to 44. At the earliest obser- vation an incipient cone was already present, and the acrosome filament extended to the cone through the jelly hull. The main points to be noted are as follows. ( 1 ) The cone rises around the acrosome filament. Often one projection of the cone creeps up the filament in advance of the main body of the cone (Figs. 39- 40). For a time at least, the apex of the cone moves outward while the spermatozoon moves inward. Indeed, the cone may 154 MORPHOLOGY OF FERTILIZATION A. L. COLWIN AND L. H. COLWIN 155 continue to enlarge even after the sperm head and its acrosome filament are well within the egg proper. The cones are always broad. (2) The acrosome filament enters the egg intact as the most anterior part of the sperm head and remains essentially un- altered in diameter and length until at least well into the egg. With less favorable material one might erroneously have con- cluded that the filament connected the sperm head with the apex of the cone and that the filament contracted during sperm entry. (3) At no time was there any basis for confusion between the straight rodlike acrosome filament and the numerous coarse radial striations which occur in the jelly hull (cf. Fig. 37 with Figs. 38-39). Thyone briareus. The process of sperm entry and cone forma- tion in this holothurian is similar to that of H. atra in many re- spects, but it differs in certain significant details (Colwin and Colwin, 1956). The observations were made on eggs removed from the gonads and also on naturally shed noniial eggs. Eggs from both sources were polyspermic in coverslip preparations. Control cultures of artificially obtained eggs did not cleave, but the control cultures of the naturally shed eggs gave 98-99% cleavage and more than 90% active larvae. The chief differences between the entry phenomena in eggs from the two sources were that in the artificially obtained eggs sperm entry was slower and the cone rose higher around the acrosome filament than in the naturally shed eggs. The egg is surrounded by a jelly hull of about 55 microns in thickness. Through this jelly run radial striations which are Figs. 37-45. Unretouched photographs of living preparations showing sperm entry in Holothuria atra. (From Colwin and Colwin, /. Morphol. 1955a.) Fig. 37. Portion of uninseminated egg showing jelly hull with filamen- tous radial projections from the egg surface. Fig. 38. A reacted spermatozoon with the acrosome filament in contact with the egg surface (no cone formed); spermatozoon did not enter egg. Figs. 39—44. Successive stages of sperm entry into the egg (taken from several eggs); note the entry of the intact acrosome filament into the egg proper as the most anterior part of the sperm head. Fig. 45. Compressed cone with exudate emerging from ruptured re- gion; spermatozoon in cone appeared unaffected. 156 MORPHOLOGY OF FERTILIZATION readily distinguishable from the acrosome filament ( Fig. 49 ) . The exceedingly long and tenuous acrosome filament spans the jelly and reaches the egg surface. Presumably in response to a stim- ulus from this filament, the egg forms a hyaline fertilization cone. Figure 46 shows a scheme of the sperm entry process, and Fig. Fig. 46. a-f, scheme of sperm entry process in Thijone briareus. (From Colwin and Colwin, Biol. Bull., 1956.) Part shown in dotted line represents minimum additional length of acrosome filament presumed but not actually seen to enter egg. g, reacted spermatozoon from alkaline sea water suspen- sion for comparison of length of acrosome filament, a, presumed very early stage, based on spermatozoon which failed subsequently to enter; b, low broad incipient cone rises as acrosome filament proceeds into egg proper; c-f, successive stages of sperm entry; acrosome filament enters egg proper as most anterior part of spermatozoon (middle piece, left outside in this case, frequently enters egg). In subsequent stages, entire flagellum passes into egg proper. 47 illustrates successive stages in one egg. Figure 48a-e and Figs. 49 to 51 show stages in several other living eggs. The main points to be noted are as follows. ( 1 ) The cone rises around the acrosome filament to varying heights and sunounds it like a sleeve. In some eggs (Fig. 46) the cone rises around the fila- A. L. COLWIN AND L. H. COLWIN 157 ment for only a short distance. In others (Figs. 47, 49, 50) one projection from a rather broad based cone rises around the fila- ment as a fairly narrow process. In still other eggs (Figs. 48a and e, and 51) a very slender projection creeps up the acrosome fil- ament for some distance and surrounds it in a sleeve of proto- Fig. 47. Successive stages of sperm entry in Tinjone hriareus, from sketches of a living specimen. (From Colwin and Colwin, Biol. Bull., 1956.) A broad cone with filose projections elevated one moderately slender sleeve of protoplasm around the inmoving acrosome filament. Acrosome filament seen in outer part of cone: solid Line. Presumed further course of filament: dotted line. plasm so narrow as to make this portion of the cane itself seem like a filament. ( 2 ) The acrosome filament can sometimes be seen within the hyaline cone (Figs. 48a-e and 49-51). Failure at other times to see the filament within the cone was attributed to optical conditions since often the even much thicker flagellum could not be seen within the cone. (3) While areas beneath the egg surface were vety difficult to observe, nevertheless in a few 158 MORPHOLOGY OF FERTILIZATION cases the acrosome filament was seen to extend into the egg proper for some distance (Figs. 46-48). In a few other cases the filament could not be identified as such, but a long narrow dis- turbance in the arrangement of the yolk granules extended to a depth approximating that to which the filament could be pre- sumed to have entered, as judged by the position of the head. Fig. 48. Variations in sperm entry, from sketches of living specimens. (From Colwin and Colwin, Biol. Bull, 1956.) a-f, Thijone briareiis; g-h, Aster ias forbesii. Acrosome filaments shown only to depth actually seen, a-b and c-d, successive views of two specimens, respectively; e, very slen- der cone embracing acrosome filament; f, acrosome filament within egg proper; g-h, from two specimens of A. forbesii, successive stages which preceded stages closely resembling that shown in f. The overall picture of sperm entry in Thyone, then, agrees with the main observations in H. atra. From the very earliest stage of cone formation the acrosome filament is already in contact with the egg surface and becomes encased by the elevating cone. As part of the sperm head, the acrosome filament enters into the protoplasm of the egg proper. The entire filament enters in H. A. L. COLWIN AND L. H. COLWIN 159 afro and there is at least partial entiy in T. hriareus. There is collateral evidence for this picture of sperm entry in the follow- ing species as well. Astericis. In A. amurensis (Colwin and Colwin, 1955a) the acrosome filament was seen within the cone. In A. forbesii (Col- win and Colwin, 1956) the filament was sometimes seen to be within the cone and to extend a short distance into the egg proper (Figs. 48g-h). Rarely it was observed to be well within the egg proper, as in Thy one (cf. Fig. 48f ). Saccoglossus kowalevskii. In this enteropneust, too, similar events appear to occur during sperm-egg association and sperm entry (Colwin and Colwin, 1954b). The egg is not surrounded by a jelly hull. Instead there are two membranes, an outer and an inner one, Membranes I and II, respectively (Colwin and Col- win, 1954a). From the earliest observation spermatozoon and egg are connected by a filament. As the cone develops it surrounds a portion of the filament. In some cases an advance projection from the broader base of the cone creeps up the filament. This was observed both in polyspermic and in normal eggs (Figs. 52- 54). After Dan's original (1952) publication on the acrosome reac- tion it was "conjectured that in the egg of S. kowalevskii a similar acrosomal breakdown, occurring when the spermatozoon reaches the outside of Membrane I, might discharge a threadlike projec- tion tluough the outer and the vitelline [II] membranes and that this structure or substance, on reaching the true egg surface, might elicit the reaction of the fertilization cone" (Colwin and Colwin, 1954b). The observations on Holothiiria atra and Thijone hriareus, as well as the collateral evidence from other species, tend to support this view that cone formation is a response by the egg to stimulation by the acrosome fialment. This view was expressed also by Dan ( 1954 ) . A Reinterpretation of Earlier Descriptions of Sperm Entry in Echinoderms Fol ( 1878-79 ) noted that in Asterias glacialis a slender tapering cone arose from the egg surface and grew toward the sperma- 160 MORPHOLOGY OF FERTILIZATION A. L. COLWIN AND L. H. COLWIN 161 tozoon even though part of the jelly hull appeared physically to separate the spermatozoon from the egg. He concluded that the egg must send an "attraction cone" to meet the spermatozoon. However, before reaching this conclusion Fol speculated that some connecting filament might arise from the sperm head and extend to the egg surface. He discarded this view because he could not see such a filament. It is now clear that the hypothesis rejected by Fol for A. glacialis is satisfied in Holothuria atra and Thyone briareiis by the demonstration that the spermatozoon does make contact with the egg surface by means of the acrosome filament and that the cone subsequently rises around the fila- ment. It is therefore suggested that what Fol ( 1877 ) described as a fine thread of protoplasm growing from the cone to establish communication with the spermatozoon was, in fact, a projection of the cone growing up around the acrosome filament which had already established the initial communication. Chambers (1923, 1930), working with Asterias (forbesii?), adopted essentially the view of Fol. According to Chambers, a long straight "insemination filament" grew out from the cone to make contact with the sperm head and then contracted, dragging the sperm head through the jelly toward the cone. In fresh mature eggs, however, no cone was seen which was not already con- nected with the sperm head by means of a filament. It is now suggested that the filament described by Chambers in the fresh eggs was the acrosome filament and that the insemination fila- ment which appeared to grow out to meet the sperm head in other cases was, in fact, a very slender projection of the cone Figs. 49-54. 49-51, unretouched photographs of sperm entry as seen in living specimens of Thyone hriareiis. (From Colwin and Colwin, Biol. Bull., 1956.) (Figures have had nonrelevant pieces spliced into upper portions.) Scale of Figs. 28-34 applies. Cones of slender type. Note acrosome filament within cone. 49—50, successive views of one specimen. 52-54, vmretouched photographs of sperm entry in living material of Saccoglossus kowalevskii. (From Colwin and Colwin, /. MorpJiol. 1954b.) 52, a polyspermia egg showing a filament extending from sperm head to and into a very early cone (the heads of two nonentering spermatozoa lie outside the egg membrane). 53, a normal egg with a filament between sperm head and early cone; a narrow projection of the .cone extends up the filament. 54, a normal egg with filament from sperm head apparently extending into hyaline portion of cone. 162 MORPHOLOGY OF FERTILIZATION rising around a more delicate but already established acrosome filament, as occurs in Thyone. In two of Chambers' (1930) cases there is actually evidence that an acrosome filament remains in- tact within the cone. In one case (his Fig. 10) the insemination filament appeared to shorten progressively until the sperm head finally reached the apex of the cone. But the head was rejected and came to lie somewhat removed from the egg surface. At that time a filament of some length extended from the tip of the sperm head. In the second case (his Fig. 19) the egg had been denuded of its jelly hull. No insemination filament was seen. However, the sperm head which entered the cone was later ex- pelled, and after expulsion a filament extended from the tip of the head ( cf . the acrosome filament within the cone of Asterias in Fig.48g-h). Horstadius described long narrow cones in the egg of the star- fish Astropecten aranciacus ( 1939a) and of the holothurian Holo- thuria poll (1939b). These cones were said to traverse the jelly hull and "take possession of" the spermatozoon. His sketch of the cone in this holothurian ( as well as in the starfish ) gives the impression that a thin filament connects the sperm head to the cone. It is now suggested that in both these cases what is por- trayed is a slender cone which has risen around an acrosome fila- ment already present as in Thyone (cf. Figs. 48a,e and Figs. 49- 51). Reinterpreted thus the descriptions by Fol, Chambers, and Horstadius fall into harmony with the general picture of sperm- egg association and sperm entry as they occur in Holothuria atra and Thyone hriareus. The acrosome filament arises from the sperm head and projects through the jelly to the egg surface; the egg is stimulated and responds by cone formation. Depending on the species and perhaps too on the degree of maturity of the egg, the cone may vary from broad to very narrow, rise quickly or slowly and to various heights, and may or may not form advance projections. Whatever these variations may be, the cone surrounds the already established acrosome filament like a sleeve, and the filament (in some cases at least ) enters the egg proper as an essentially intact structm'e. A. L. COLWIN AND L. H. COLWIN 163 Mechanism of Sperm Entry into Egg Little is known of the mechanism by which the movement of the spermatozoon into the egg is effected once the acrosome fila- ment has made its initial association with the egg. It is clear that the mechanism is not direct contraction of the acrosome filament per se, nor recession of the external part of the cone. Lillie's (1912a and 1912b) description of sperm entry in Nereis offers some indication of what the mechanism may be. As mentioned previously, Lillie noted that the "perforatorium" had the form of "the spike of a helmet" in living spermatozoa but that in fixed spermatozoa, whether free or attached to an egg, the perfora- torium was a straight filament-like structure. Metz and Morrill (1955) have found that a comparable change in the acrosomal region was induced by fertilizin treatment of Nereis spermatozoa. It seems reasonable to consider, then, that Lillie's account of the behavior of the spermatozoon and its perforatorium during sperm entry is in reality an account of the behavior of the reacted sper- matozoon and its acrosome filament during sperm entry. In Lil- lie's description based on fixed material, the "fertilization cone" becomes converted into a "specialized cell organ" identifiable by its staining reaction. The sperm head becomes "anchored" in this organ by the tip of its perforatorium (i.e., acrosome filament). This complex, consisting of sperm head and cone, moves slowly from the egg surface and penetrates more deeply into the egg. About 64 minutes after insemination the sperm head becomes separated from this cell organ. The further history of this "cone" is unknown. Lillie's centrifuge experiments with inseminated eggs of Nereis indicate that once the sperm head is associated with the sub- stance of the cone the relationship is a firm one. There is some evidence for such a finn relationship between the spermatozoon and the substance of the cone in HolotJmria atra; in a few cases it was found that if enough pressure were applied to the cover- slip to cause the cone to rupture and form an exudate, the sper- matozoon within the cone was not expelled ( Fig. 45 ) . Moreover, in similar preparations in Thyone hriareus, the spermatozoon 164 MORPHOLOGY OF FERTILIZATION even continued to progress inward. If, as Lillie described it, the "fertilization cone" is a cell organ which moves a considerable distance into the egg, then one might speculate that the mecha- nism which effects the passage of the spemi head through the surface of the egg is the same one which effects the continued movement of the spemi head once it has entered the egg. Lillie (1912b) considered the fertilization cone of Nereis to be quite different from the fertilization cone of echinoderms since in the latter "its significance has been, apparently, merely tem- porary and local, a reaction of the ovum to the spermatozoon with no definable function of its own." In the light of present knowledge this view certainly merits reconsideration. There is no doubt that the formation of an outwardly projecting fertilization cone is a normal phenomenon at least in the eggs of some species. In these the cone is not simply a result of pressure or other con- ditions introduced by placing eggs on a slide and covering them with a coverslip. In Saccoglossus kowalevskii, for example, the relatively large fertilization cone may be seen with the dissecting microscope in eggs inseminated and left in ample water in open Syracuse or stendor dishes. Such eggs develop normally to larval stages (Colwin and Colwin, 1953). Moreover, in at least three echinoderms, T. hriareus, H. atra, and A. forbesii, the acrosome filament which initially may be seen within the outwardly project- ing part of the cone subsequently moves into the egg proper. Further study of these species may reveal a mechanism similar to the one described for Nereis. The great difference in time re- quired for spemi penetration in Nereis as compared with other species may not reflect any fundamental difference in mecha- nism. Summary 1. From the evidence reviewed it is clear that in certain echino- derms, molluscs, annelids, and probably an enteropneust, sper- matozoa undergo a profound reaction when suitably stimulated. The most striking feature of the reaction is the production of a fairly straight, relatively rigid filament, the acrosome filament of Dan. Egg water, alkaline sea water, and the presence of unferti- A. L. COLWIN AND L. H. COLWIN 165 lized eggs elicit this reaction in spermatozoa of one or more of the species studied. In some species the reaction is also elicited by "contact" with a surface and, presumably, other factors as yet unknown. Both quantitative and descriptive data are presented for the acrosome reaction as observed in living sperma- tozoa of Thyone briareus. 2. The length of the acrosome filament varies from species to species, ranging from about 1 micron in some sea urchins to 75 microns or more in Thyone in which it sometimes exceeds the length of the flagellum. It is suggested that in any species the length of the filament of a successful spermatozoon can never be less than the depth of the barrier which effectively separates the sperm head from the reactive egg protoplasm. The jelly hull acts as a barrier in the starfishes and holothurians studied but not in the sea urchins. 3. At the very earliest stage of sperm-egg association observed in Holothuria atra and Thyone, an acrosome filament is already present and extends through the jelly hull to an incipient cone. The cone rises around the acrosome filament to varying heights, surrounding all or part of the filament in a sleeve of protoplasm. In some cases the sleeve is so narrow as to make this portion of the cone itself seem like a filament. The acrosome filament enters the egg intact as the most anterior part of the spenn head and remains essentially unaltered in diameter and length until well into the egg proper, at least in H. atra and probably also in Thyone. Partial evidence suggests essentially the same course of events in several starfishes and an enteropneust. 4. Some of the earlier descriptions of sperm entry in echino- derms are reinterpreted as follows. The filament seen between sperm head and cone (Fol, Chambers) is not of egg origin but is the acrosome filament of the spermatozoon; the narrow cone which appears to rise from the egg to meet the spermatozoon (Horstadius) is simply rising around the acrosome filament which has aheady established contact with the egg proper. 5. Little is known of the mechanism which eftects the move- ment of the spermatozoon into the egg, but this mechanism is not direct contraction of the acrosome filament nor recession of the external part of the cone. 166 MORPHOLOGY OF FERTILIZATION REFERENCES Afzelius, B. A. 1955. The fine structure of the sea urchin spermatozoa as revealed by the electron microscope. Z. Zellforsch., 42, 134-48. Bm-gos, M. H., and D. W. Fawcett. 1955. Studies on the fine structure of the mammalian testis. I. Differentiation of the spermatids in the cat (Fehs domestica). /. Biophys. Biochem. Cyt., I, 287-300. Burgos, M. H., and D. W. Fawcett. 1956. An electron microscope study of spermatid differentiation in the toad, Btifo arenarum Hen- sel. /, Biophys. Biochem. Cyt., 2, 223-40. Chambers, R. 1923. The mechanism of sperm entrance into the star- fish egg. /. Gen. Physiol, 5, 821-29. Chambers, R. 1930. The manner of sperm entry in the starfish egg. Biol Bull, 58, 344-69. Clermont, Y., R. E. Glegg, and C. P. Leblond. 1955. Presence of carbo- hydrates in the acrosome of the guinea pig spermatozoon. Exptl Cell Research, 8, 453-58. Colwin, A. L., and L. H. Colwin. 1953. The normal embryology of Saccoglossus kowalevskii ( Enteropneusta ) . /. Morphol, 92, 401-54. Colwin, A. L., and L. H. Colwin. 1955a. Sperm entry and the acrosome filament (Holothuria atra and Asterias amurensis). }. Morphol, 97, 543-68. Colwin, A. L., and L. H. Colwin. 1955b. Concerning the spermato- zoon and fertilization in the egg of Sabellaria vulgaris. Biol Bull, 109, 357. Colwin, L. H., and A. L. Colwin. 1954a. Fertilization changes in the membranes and cortical granular layer of the egg of Saccoglossus kowalevskii (Enteropneusta). /. Morphol, 95, 1-46. Colwin, L. H., and A. L. Colwin. 1954b. Sperm penetration and the fertilization cone in the egg of Saccoglossus kowalevskii (Enterop- neusta). /. Morphol, 95, 351-72. Colwin, L. H., and A. L. Colwin. 1955c. The spermatozoon and sperm entry in the egg of the holothurian, Thy one briar ens. Biol Bull, 109, 357. Colwin, L. H., and A. L. Colwin. 1955d. Some factors related to sperm entry in two species of Asterias. Biol Bull, 109, 357. Colwin, L. H., and A. L. Colwin. 1956. The acrosome filament and A. L. COLWIN AND L. H. COLWIN 167 sperm entry in Thtjone hriareus (Holothuria) and Asterias. Biol. Bull, 110, 243-55. Dan, J. C. 1952. Studies on the acrosome. I. Reaction to egg-water and other stimuli. Biol Bull, 103, 54-66. Dan, J. C. 1954. Studies on the acrosome. II. Acrosome reaction in starfish spermatozoa. Biol Bull, 107, 203-18. Dan, J. C, and S. K. Wada. 1955. Studies on the acrosome. IV. The acrosome reaction in some bivalve spermatozoa. Biol Bull, 109, 40- 55. Fol, H. 1877. Sur le commencement de I'henogenie chez divers ani- maux. Arch, zool exptl. et gen., 6, 145-69. Fol, H. 1878-1879. Recherches sur la fecondation et le commencement de I'henogenie chez divers animaux. Mem. soc. phys. et hist. not. Geneve, 26, 89^97. Grasse, P. P., and O. Tuzet. 1933. Sur la structure du spermatozoide des metazoaires. Compt. rend. .'ioc. biol, 113, 44-46. Horstadius, S. 1939a. Uber die Entwicklung von Astropecten arancia- cus L. Puhl staz. zool Napoli, 17, 221-312. Horstadius, S. 1939b. tjber die larve von Holothuria poU delle Chiaje. Arkiv Zool, 31A, No. 14. Kupelweiser, H. 1909. Entwicklungserregung bei Seeigeleiern durch Molluscasperma. Arch. Entwicklungsmech. 27, 434—62. LilHe, F. R. 1912a. Studies of fertilization in Nereis. I. The cortical changes in the egg. II. Partial fertiHzation. /. Morphol, 22, 361-93. Lillie, F. R. 1912b. Studies of fertilization in Nereis. III. The morphol- ogy of the normal fertilization of Nereis. IV. The fertilizing powers of portions of the spermatozoon. /. Exptl Zool, 12, 413-78. Metz, C. B. 1945. The agglutination of Starfish sperm by fertilizin. Biol Bull, 89, 84-94. Metz, C. B., and J. B. Morrill. 1955. Formation of acrosome filaments in response to treatment of sperm with fertilizin in Asterias and Nereis. Biol Bull, 109, 349. Meves, F. 1915. Ueber den Befruchtungsvorgang bei der Meismuschel {Mytilus edulis L.). Arch, mikroskop. Anat., 87, Abt. 2, 47-62. Popa, G. 1927. The distribution of substances in the spermatozoon (Arbacia and Nereis). Biol Bull, 52, 238-57. Retzius, G. 1904. Biologische Untersuchungen. N. F. 11. G. Fischer, Jena. Retzius, G. 1905. Biologische Untersuchungen. N. F. 12. G. Fischer, Jena. 168 MORPHOLOGY OF FERTILIZATION Retzius, G. 1910. Biologische Untersuchungen. N. F. 15. G. Fischer, Jena. Rothschild, Lord, and A. Tyler. 1955. Acrosomal filaments in sperma- tozoa. Exptl. Cell Research, Supp. 3, 304-11. Tuzet, O. 1950. Le spermatozoide dans la serie animale. Rev. suisse zool, 57, 433-51. Tyler, A. 1952. Further investigations on fertilizins of eggs of sea- urchins. Anat. Record, 113, 525. Vasseur, E. 1947. The spermatozoon of the sea urchin Echinocardium cordatum (Pennant). Arkiv Zool., 40B, No. 3. STUDIES OF PROTEINS OF SEA URCHIN EGG AND OF THEIR CHANGES FOLLOWING FERTILIZATION ALBERTO MONROY: laboratory of comparative ANATOMY, THE UNIVERSITY OF PALERMO, ITALY The activation of the egg consists fundamentally in the release of the metabolic reactions which are responsible for the initia- tion of morphogenesis. To learn how this is accomplished is the aim of the investigations of the physiological and biochemical basis of morphogenesis. It therefore appears important to investigate the kind of changes that take place in the various systems of the egg upon fertilization since such analysis may help a great deal in under- standing the basic mechanisms of the activation. The study of the changes occurring in the proteins of the egg is obviously one of the most interesting to pursue since it is at the protein level that differentiation takes place and therefore it is hoped that it may eventually help to uncover the basic events of differentiation. This study, although still at an early stage, indicates that im- mediately following fertilization a process of rearrangement of the whole protein pattern of the egg is started. On the other hand, different lines of investigation give consistent evidence that protein synthesis is started much later in development ( Perl- mann and Gustafson, 1948; Perlmann, 1953; Hobermann, Metz, and Graff, 1952; Hultin, 1953). The starting point for the study of the changes of the egg proteins following fertilization was the observation of Mirsky (1936) that in the sea urchin eggs a conspicuous decrease of solubility of a protein fraction takes place during the first ten minutes after fertilization, Mirsky showed, in fact, a 12% de- 169 170 PROTEINS OF SEA URCHIN EGG crease of the proteins soluble in IM KCl, and he was also able to trace this fraction in the extracts of unfertilized eggs as the one that is precipitated by 50% saturation with ammonium sul- fate. Later the present writer and his co-workers undertook a sys- tematic investigation of the changes taking place in the proteins of the egg upon fertilization. The results of these investigations are summarized here. Water extracts of sea urchin eggs were submitted to electro- phoretic analysis, and it was shown that a few minutes after fertilization a small new component appears. However, this com- ponent is no longer detectable thirty minutes after fertilization. The extracts were then fractionated with ammonium sulfate, and the fractions were studied electrophoretically. The following facts were established: (a) a decrease in solubility of one com- ponent immediately after fertilization and (b) a splitting of a component present in the unfertilized eggs, thus giving rise to a new component (Monroy, 1950). Further changes of solubility were also shown to take place, but on the whole these were somewhat irregular and hence difficult to interpret ( Monroy and Monroy-Oddo, 1951). In the first investigations of Mirsky and in those of the pr-esent writer, lyophilized eggs were used. This introduces the possibility of artifacts. Hence in subsequent work only fresh material has been used, and special precautions have also been taken to avoid secondary alterations during preparation (Giardina and Monroy, 1955; Ceas, Impellizzeri, and Monroy, 1955). In the latter in- vestigations attention was focused on a fraction which is precipi- tated by 50% saturation with ammonium sulfate. Three com- ponents are indicated in this fraction by the electrophoretic analysis. Upon treatment with trypsin the fraction prepared from unfertilized eggs is split into live components, whereas the one prepared from fertilized eggs is split into four components (Fig. 1) (D'Amelio, 1955). Comparable results were obtained when the action of trypsin on these proteins was followed by chemical determinations (Fig. 2) (Giardina and Monroy, 1955). Proteins from unfertilized and fertilized eggs also behave differently in A. MONROY 171 respect to heat denaturation. To distinguish between denatured and nondenatured proteins the sohibihty in an acid buffer of high molarity was used. This buffer has been shown to induce precipitation of denatured lactoglobuRn and serum albumin, whereas when they are in the native condition these proteins stay in solution ( Christensen, 1952 ) . In the case of the sea urchin egg extracts, a large proportion of the extracted proteins is precipi- tated by the buffer without any previous denaturing treatment, and the amount of precipitate is greater in the case of the un- fertilized eggs. Although this result does not allow one to draw any conclusion as to the condition that makes such proteins sus- ceptible to the salt buffer, it is nevertheless indicative of a differ- Fig. 1. Electrophoretic patterns after trypsin digestion of the fraction precipitated at 50% saturation of ammonium sulfate from an extract of un- fertilized (solid line) and fertilized (dotted line) eggs. (Redrawn from an experiment of D'Amelio.) ence between the proteins of the unfertilized and of the newly fertilized eggs. A difference between the two is also shown by mere inspection of the precipitate which is flocculent and rapidly settling in the former, whereas in the latter it is finely dispersed, takes some time to appear, and settles only very slowly. The total amount of precipitable proteins upon heating between 50° and 60° C. followed by addition of the buffer is significantly greater in the extracts of unfertilized than in those of fertilized eggs. The same result has been obtained with the fraction pre- cipitated by 50% saturation with ammonium sulfate (Giardina and Monroy, 1955 ) . Two alternative explanations were presented to account for these results, i.e., either a change occurred in the molecular configuration of some proteins or a splitting of a large complex present in the unfertilized egg took place. It was shown 172 PROTEINS OF SEA URCHIN EGG that the intrinsic viscosity of the protein fraction isolated by precipitation by 50% saturation with ammonium sulfate does not vary upon fertilization, thus indicating that no appreciable changes of shape and; or hydration of these protein molecules occurs at fertilization. However, the increase of viscosity induced by urea was considerably greater in the fraction extracted from fertilized than in the one from unfertilized eggs, and a greater §200 / 2 / o c / "o" / CD O A I 1 / c O / 1 ID i / /• 5 . 10 15 20 25 30 Time o[ incubahon in minutes Fig. 2. Release of nonprotein N from the fraction precipitated at 50% saturation of ammonium sulfate from an extract of unfertilized (^) and fertilized (•) eggs of Arbacia lixiila during trypsin digestion. (Calculated from tlje data of Giardina and Monroy.) amount of phenolic groups was exposed in the former than in the latter as a result of this treatment (Ceas, Impellizzeri, and Mon- roy, 1955). These experiments give evidence that, although no changes in the molecular configuration of the proteins of our fraction after fertilization has been demonstrated thus far, these molecules seem to undergo some sort of internal reaiTangement. The ex- A. MONROY 173 perimeiits with urea suggest that in these molecules H-bonds may be more exposed after fertilization. The new configuration appears to render them less susceptible to the attack of trypsin and less easily denatured by heat. A question of considerable importance is whether as a part of this process, end groups, viz., peptides or amino acids, are split off. Some years ago Orstrom (1941) gave evidence of a slight but statistically significant increase of the nonprotein N during the first ten minutes following fertilization. A reinvestigation of this problem is now in progress in om* laboratory. A positive answer to it would make one consider the interesting analogy between activation of the egg at the level of the protein molecules and the activation of some enzymes (e.g., pepsin) in which it is known that the transition from the pro- to the active enzyme is accom- panied by the splitting off of a terminal peptide. One could then speak of an activation of the egg proteins upon fertilization. It is interesting to recall here that Hultin ( 1950 ) found a considerable acceleration of the turnover of the egg proteins after fertilization. On the basis of some model experiments, a working hypothesis has been presented concerning the nature of the cortical reaction in the activation of the sea urchin egg (Maggio and Monroy, 1955; Monroy, 1956). It has been suggested that the key reac- tion of the activation of the egg is the release, followed by the inactivation, of a lysophosphatide-like substance in the cortex of the egg as a result of the reaction with the fertilizing sperm or of any activating treatment. It has also been shown (Tyler, Mon- roy, Kao, and Grundfest, 1956) that coincident with fertilization a change of the membrane potential of the egg occurs. However, how this gioup of early phenomena can possibly be linked to the changes in the proteins of the egg is at present obscure. REFERENCES Ceas, M. P., M. A. Impellizzeri, and A. Monroy. 1955. The action of urea on some proteins of the unfertilized and fertilized sea urchin egg. Exptl. Cell Research, 9, 366-69. Christensen, L. K. 1952. Denaturation and enzymatic hydrolysis of 174 PROTEINS OF SEA URCHIN EGG lactoglobulin. Compt. rend. trav. lab. Carlsberg, Ser. chim., 28, 37- 169. D'Amelio, V. 1955. Trypsin sensitivity of some proteins of the sea urchin egg before and after fertilization. An electrophoretic analy- sis. Experientia, 11, 443. Giardina, G., and A. Monroy. 1955. Changes in the proteins of the sea urchin egg at fertilization. Exptl. Cell Research, 8, 466-73. Hobermann, H. D., C. B. Metz, and J. Graff." 1952. Uptake of deute- rium into proteins of fertilized and unfertilized Arbacia eggs sus- pended in heavy water. /. Gen. Physiol, 35, 639-43. Hultin, T. 1950. The protein metabolism of sea urchin eggs during early development studied by means of N^ '^-labeled ammonia. Exptl. Cell Research, 1, 599-602. Hultin, T. 1953. Metabolism and determination. Proc. Symposium on the Biochemical and Stiaictural Basis of Morphogenesis in Arch, neerl. zool, 10 (Suppl.), 76-91. Maggio, R., and A. Monroy. 1955. Some experiments pertaining to the chemical mechanisms of the cortical reaction in fertilization of sea urchin egg. Exptl. Cell Research, 8, 240-44. Mirsky, A. 1936. Protein coagulation as a result of fertilization. Sci- ence, 84, 333-34. Monroy, A. 1950. A preliminary electrophoretic analysis of proteins and protein fractions in sea urchin eggs and their changes on ferti- lization. Exptl. Cell Research, 1, 92-104. Monroy, A., and A. Monroy-Oddo. 1951. Solubility changes of proteins in sea urchin eggs upon fertilization. /. Gen. Physiol., 35, 245-53. Monroy, A. 1956. Some experiments concerning the chemical mecha- nisms of the activation of the sea urchin egg. Exptl. Cell Research, 10, 320-23. Orstrom, A. 1941. tjber die Stickstoffraktionen im Ei von Paracentro- tus lividus vor und nach der Entwicklungserregung und liber ihre Bedeutung fUr den osmotischen Druck und Stolfwechsel. Arkiv Kemi, Mineral. Geol., 15A, No. 1. Perlmann, P., and T. Gustafson. 1948. Antigens in the egg and early developmental stages of the sea urchin. Experientia, 4, 481-83. Perlmann, P. 1953. Soluble antigens in sea urchin gametes and devel- opmental stages. Exptl. Cell Research, 5, 394-99. Tyler, A., A. Monroy, C. Y. Kao, and H. Grundfest. 1956. Membrane potential and resistance of the starfish egg before and after fertili- zation. Biol. Bull, 111, 153-75. NUCLEOCYTOPLASMIC RELATIONS IN EARLY INSECT DEVELOPMENT* R. C. VON BORSTEL: biology division, oak ridge NATIONAL LABORATORY, OAK RIDGE, TENNESSEE Experimental study of interaction between the nucleus and cytoplasm in development had its origin in the analyses of Boveri (1907) on sea urchins, which showed that alteration of the chromosome complement disrupted development. The role of the cytoplasm has been heavily emphasized by such studies as the classical research of Conklin on ascidian embryos (1931). Fank- hauser (1952, 1955) has comprehensively reviewed nucleocyto- plasmic relations in development of animals other than insects. Since certain aspects of development are revealed with greatest clarity in the insect embryo, and since the genetic background is so well known in certain species, it would seem that information of general interest would be derived from an experimental study of insect development. The work described here is confined for the most part to the parasitic wasp Habrobmcon; the results are used wherever possible to illustrate general principles of develop- ment. The embryology of the young system is presented prima- rily from cytogenetic and cytochemical points of view. Some of the conclusions drawn from the work described depend on a study of gynandromorphs and genetic mosaics, and most of the remainder rely on interpretations of the action of radiation. In insects as in other animals, the fust mitotic divisions are rapid and result in many nuclei within many cells, which consti- tute a pool utilized during tissue formation. As in all animals, much depends on the particular code within the genome at the outset of embryogeny, by which a wild-type Drosophila can be mutated into a mottled peach, a Mormoniella into an oyster, or ' Work performed under USAEC contract No. W-7405-eng-26. 175 176 NUCLEOCYTOPLASMIC RELATIONS a Habrobracon into a black dahlia. Superficially, it would appear that the interaction of cells and cellular components that occurs in embryogenesis has its basis in this coded information con- tained in the genetic nucleus. The simplest model would be an evolving system in which the yolk acts as a nutrient medium and the cytoplasm as a transport system that funnels nourishment to the nucleus and utilizes information from it. Inasmuch as de- velopment of such systems as legs, eyes, wings, antennae, the thorax, and abdomen are under control of the genetic nucleus, by the doctrine of obvious hypothesis, the entire differentiation system has its basis in the genetic nucleus as well. That this model is too simple, however, is apparent upon consideration of determinate development in which egg regions are predestined prior to the existence of nuclei in these regions. The egg cyto- plasm guides the totipotent nuclei into specified but not com- pletely fixed channels of development. The final fixation is a nuclear function. The cytoplasm in determinate eggs therefore must be a repository for a different order of informational organ- ization from that of the nucleus. Since regulative and mosaic development are essentially alike (Penners, 1924; Tyler, 1930), a general model for illustrating development should include a directive action of fixed extent in the cytoplasm. We are interested in the early development of the egg and embryo because the potential exists here for the differentiated, fully developed organism. Before an egg can be activated, its development must be inhibited; prior to embryonic differentia- tion, the potentialities must exist. By analyzing several compo- nents of the predifferentiation system, perhaps the entire em- bryonic organism will be more clearly understood. As the provi- sional model, the young embryo will be considered as a substrate pool for an itinerant nucleus with reproduction as its sole activity until diverted by a determined cytoplasm. General Considerations The experimental animal most often referred to is Habrobracon juglandis (Ashmead), a small Braconid wasp that parasitizes the larva of the Mediterranean flour moth Ephestia kiihniella Zeller. R. C. VON BORSTEL 177 The females of Habrobracon are diploid and the males are hap- loid — unfertilized eggs become haploid males, females originate from fertilized eggs. When eggs are fertilized by sperm contain- ing the same sex alleles as the egg pronuclei, diploid males result (P. W. Whiting, 1943b). This sex determination mechanism appears to occur also in the honey bee, Apis mellifera L. ( Mack- ensen, 1951, 1955). The morphology of meiosis and early embryogeny of Habro- bracon has been studied in detail by Torvik-Greb ( 1935 ) , Hen- schen ( 1928 ) , and Speicher ( 1936 ) . The description given in this review is, for the most part, summarized from the analysis of Speicher. Oogenesis in most insects differs from this form only in details (Wilson, 1928; Sonnenblick, 1950); the description here is restricted to Habrobracon. The female has four ovarioles, each with a uterine sac where mature eggs are stored prior to oviposi- tion (Fig. 1).* One ovariole with attached uterine sac is shown in Fig. 2. The egg arises from a nest of 32 oogonia, 31 of which become nurse cells. (Henschen, 1928, reported a nest of 16 oogonia, but as is apparent from the number of heavily staining nuclei in Fig. 3, more than 15 nurse cells normally exist.) Dur- ing oocyte growth the nurse cells gi^ow too. While the oocyte becomes larger the oocyte nucleus proceeds through the long first meiotic prophase, and the nurse cell nuclei go through several cycles of polysomatic enlargement; the nuclei in nurse cells next to the oocyte are somewhat larger than the nurse cell nuclei farthest from the oocyte. Just prior to entrance of the egg into the uterine sac from the ovariole, the nuclei of the nurse cells disintegrate and are injected into or are engulfed by the egg (Fig. 4). It has been suggested (Painter, 1940; Zeuthen, 1951) that this broken down deoxyribonucleic acid is incorporated into nuclei during the ensuing rapid mitoses. The point might be raised that some of the genetic structure could be passed intact from the deoxyribonucleic acid of the mother to the nuclei of the embryo. This would be equivalent to transformation ( Hotch- kiss, 1955) and has not been observed. Most of the deoxyribo- ** The photomicrographs (Figs. 1-4) were made by Henry H. Jones, of the Carnegie Institution of Washington, Cold Spring Harbor, N. Y. 178 NUCLEOCYTOPLASMIC RELATIONS Fig. 1. The four ovarioles from a Habrobracon female immediately after removal from abdomen. X 10 Fig. 2. Ovariole from a well-fed Habrobracon female showing stages in development of eggs. Mature eggs in uterine sac at right. x40 Fig. 3. Posterior end of developing egg with nurse cells attached. Nu- clear stain employed. X250 Fig. 4. Disintegrating nurse cell nuclei being engulfed into developing egg; smaller stained bodies are follicle cell nuclei on egg periphery. X250 R. C. VON BORSTEL 179 nucleic acid from the nurse cells has broken down into units soluble in hot acid. This implies that it is in the form of mono- nucleotides or oligonucleotides of low order. Since most of the deoxyribonucleic acid is probably in small units, this could ac- count for failure to find transformation, which would be a pre- dictable consequence of incorporation of large blocks of deoxyri- bonucleic acid from nurse cells. Also, except under unusual circumstances, nuclear mutation reflected in the phenotype can be only of an order that is not reparable by cytoplasmic deoxyri- bonucleic acid. After nurse cell incorporation, the mature egg FUSION NUCLEUS AND PRONUCLEUS CLEAVAGE X PERIPHERAL MIGRATION YOLK PHAGOCYTE FORMATION Fig. 5. Diagrammatic representation of nuclear activities during meiosis, nuclear cleavage, and peripheral migration in the Habrohracon egg. now resides in the uterine sac, and the oocyte nucleus proceeds through diakinesis and metaphase. Meiosis is blocked at late metaphase or early anaphase of the first meiotic division, and the egg is ready to be oviposited. The egg is 600 microns long and 150 microns across at its widest place. It is concave-convex in shape and the anterior end of the embryo is the widest. The egg is positioned at least par- tially in accordance with the orientation law of Hallez (1886), that is, with the anterior end of the future embryo pointing to- ward the anterior end of the mother while residing in the uterine 180 NUCLEOCYTOPLASMIC RELATIONS sac. This seems more a fortuitous circumstance than an actual requirement, however, since eggs will occasionally emerge an- terior end first; several have been closely observed and all de- veloped normally. Much of the cytoplasm is distributed in a layer 5 to 10 microns thick around the egg periphery, and the rest is threaded through the yolk that constitutes the major portion of the egg. The nucleus of the unlaid egg lies in the egg interior at the anterior end (see Fig. 5). When oviposition takes place, the egg is tightly squeezed through the ovipositor and emerges with the nucleus now lying against the convex (ventral) surface at the anterior end. It has been suggested that the act of squeez- ing displaces the nucleus and activates the egg into resumption of normal development. The egg obviously is not activated by sperm since fertilization is not necessary for normal development. The mechanical interpretation of egg activation is merely a re- cording of observed events in sequence. It would seem that until eggs can be removed from uterine sacs and artificially activated the activation process will not be fully understood. After oviposition the first meiotic division begins and the chromosomes go through a brief interphase, which is quickly followed by the second division. The divisions leave the four meiotic nuclei in a linear quartet perpendicular to the egg sur- face, all within the egg. The outer polar nucleus quickly disinte- grates, the two middle polar nuclei fuse and form an abortive metaphase plate, and the inner nucleus becomes the functional pronucleus. At 30°, meiosis is completed in thirty minutes after oviposition. The pronucleus migrates into the egg interior, and nuclear cleavages quickly begin. If the egg is fertilized, the sperm nu- cleus comes to lie beside the egg nucleus and forms a double metaphase plate, a characteristic not observed in later cleavages. Nuclear cleavage is a rapid, orderly, and at first completely syn- chronous process. During later cleavages, nuclei in the anterior end divide somewhat sooner than nuclei in the posterior end. This paitial asynchrony is not an artifact, since it is observed whether fixation is done at 50°, 2°, or —83°, or whether either end is punctured before fixation. At the tenth cleavage, when R. C. VON BORSTEL 181 approximately 1024 nuclei are present, the first signs of blastula- tion appear. Some cleavage nuclei remain in the egg interior while the great majority migrate to the periphery. The nuclei in the interior stain more intensely and, as cells, are known as yolk phagocytes or vitellophags (see Sonnenblick, 1950, for review). The nuclei at the periphery undergo several more synchronous divisions, cell membrane formation takes place, and tissue dif- ferentiation begins. Several questions can be asked about relations between the nucleus and cytoplasm. Our work with Habrohracon and studies done in other laboratories on other insects have provided answers for some of these questions. 1. The four meiotic nuclei are apparently all genetically equipotent. Since all four remain inside the egg, why does only one take part in further development? 2. Is there any condition by which accessory sperm can take an active part in development? 3. Can the young embryonic system be regarded as a pool in which mitosis freely occurs (the "pure culture of mitoses" of Zeuthen, 1951) until blastulation, or do critical periods exist dur- ing the cleavage stages? 4. What component of the cytoplasm is the agent of deter- minate development? 5. Is there any general method by which one can distinguish between action of the nucleus and the cytoplasm in bringing about an embryonic event? Genetic Control of the Meiotic Block Genetic Mosaics. During a routine testing following an ir- radiation experiment, a curious mutant was discovered. When this mutant stock was inbred, a relatively high incidence of mo- saicism resulted. These are mosaics in the genetic rather than the embryological sense. In this stock, a virgin female heterozygous for a semidominant body color gene (designated as "lemon lethal"), which is a pupal lethal when homozygous, will occa- sionally produce haploid male offspring that are made up of nuclei carrying the normal allele and nuclei carrying the mutant 182 NUCLEOCYTOPLASMIC RELATIONS allele. Sometimes half the animal bilaterally is lemon lethal, and the other half wild type, each region being autonomous. At other times, certain portions of the body or head are lemon lethal with the rest wild type; or the inverse occurs where only a small patch of wild-type tissue is present on the adult animal. The lethal effect of the gene is nonautonomous, and a male will eclose if only a small patch of wild-type tissue is present. The hereditary background necessary for production of the mosaics is not fully understood at present; not all females of the lemon lethal stock produce mosaics, but when one female is a mosaic producer she MOSAIC HAPLOID EMBRYO TWO PRONUCLEI FROM BINUCLEATE OOCYTE (POSTULATED) TWO PRONUCLEI FROM UNINUCLEATE OOCYTE (ACTUAL) Fig. 6. Representation of normal meiosis, meiosis in a binucleate oocyte, and meiosis in which two meiotic products become pronuclei. tends to produce others. Gynandromorphs from virgins are rare, but they also occur. In any event, the occurrence of a haploid mosaic male means that the egg from which it originated was binucleate. In which sense is it binucleate — two oocyte nuclei undergoing meiosis side by side, or two meiotic products from one oocyte nucleus becoming cleavage nuclei (Fig. 6)? In a cytological investiga- tion of oocytes from females that produced mosaic males, no more than one nucleus was observed in any developing oocyte; however, in several cases, eggs were observed which were not blocked at the first meiotic metaphase. Meiosis was continuing R. C. VON BORSTEL 183 past metaphase I in eggs still in the uterine sacs, a condition never observed in females from other stocks. Meiosis in these cases was observed to be continuing in the interior of the anterior portion of the egg, w^here the blocked metaphase nucleus usually lies. The phenomenon of uninhibited meiosis would seem to be as rare an occurrence as mosaic production. Since in our study only those females that show uninhibited meiosis also produce mo- saics, and vice versa, it seems justifiable to correlate continuation of meiosis in the egg interior with mosaic production and to con- clude that the faulty meiotic block is in some way responsible for induction of mosaics. In agreement with this conclusion, P. W. Whiting (1932) found a wasp that was mosaic for reciprocal crossover types, indicating two pronuclei from the products of a single meiosis. It is conceivable that, if meiosis does not occur at the egg surface, cleavage nuclei of two meiotic nuclei or more (P. W. Whiting, 1934) might survive destruction. Furthermore, this indicates that in normal eggs some agent present in the corti- cal cytoplasm must be responsible for disintegration of the most peripheral polar nucleus and inhibition of further division of the fusion nucleus. The chromosome set that will become the pro- nucleus pushes farther into the interior at each meiotic division and escapes destruction. The simplest explanation then is that, in mosaics, two or more meiotic nuclei must have escaped de- struction by some agent in the egg cortex. That the egg cytoplasm can be incompatible to foreign nuclei has been shown by the interesting cytological analysis of Tchou-Su (1931). Working with amphibian eggs, he has shown that foreign sperm can be destroyed by cytoplasm in a number of different ways before fusion of the egg pronucleus and the foreign sperm nucleus can take place. Nanney (1953), by cen- trifuging Tetmhijmena undergoing conjugation, was able to dem- onstrate that the position of the meiotic products in the cytoplasm determined the later behavior of the nuclei. The cytoplasm in this case certainly has a critical role in directing nuclear events. The most famous mosaics are the Eugster gynandromorph honey bees, which were discovered in the middle of the nine- teenth century and were puzzled over for the next seventy years. 184 NUCLEOCYTOPLASMIC RELATIONS Boveri (1915) studied them and concluded that the male parts were inherited from the mother and the female parts from both parents. He suggested that the sperm had not united with the egg pronucleus but with one of the cleavage nuclei ( Fig. 7 ) . The dis- covery by P. W. Whiting (1924) of a haploid mosaic male, mo- saic in traits inherited from a heterozygous mother, prompted the hypothesis that two products of meiosis had survived and the eggs were binucleate. In 1932, P. W. Whiting discovered a Habrobracon gynandromorph in which the female part came from the sperm and one set of the heterozygous mother's markers FERTILIZATION OFA CLEAVAGE NUCLEUS (BOVERI) FERTILIZATION OF ONE OF TWO PRONUCLEI (WHITING) ZYGOTE FORMATION AND ACCESSORY SPERM NUCLEUS (WHITING AND ROTHENBUHLER) Fig. 7. Diagrammatic representation of three possible mechanisms for gynandromorph fonnation in Hymenoptera. whereas the male portion came from the other set of the mother's markers. This gave strong support to the hypothesis that gynan- dromorphs resulted from fertilization of one of the meiotic products of a binucleate egg (Fig. 7). This is an acceptable al- ternative to Boveri's hypothesis of fertilization of the cleavage nuclei, and, in explaining the origin of this Habrobracon gynan- dromorph, Boveri's hypothesis fails. Meiotic Block. The meiotic block in Habrobracon must be under genetic control, since lack of control is inherited in the lemon lethal stock. Other than this, little is known. Is the block intrinsic? Is there some agent in the egg cytoplasm which inhibits R. C. VON BORSTEL 185 meiosis or does there exist some extrinsic factor produced by the female such as that described by Humphries (1955) in frogs? Humphries' discovery that a low frequency of the frog eggs pre- vented from going down a tied-off oviduct would pass through meiosis uninhibited suggests that a substance secreted by the oviduct causes the block. The alternative hypothesis is still pos- sible although it is less likely that inhibition is intrinsic and does occur; something in the coelom then activates the frog eggs. The possibility that the inhibiting mechanism in the case of the Habrobracon egg is built into the egg is more attractive than extrinsic control, but no evidence that can help in solving this problem is yet available. Karyokinesis In the consideration of the stage of development between one nucleus prepared for cleavage and several thousand nuclei, three items will be examined briefly: (1) prevention of cleavage of ac- cessory sperm, (2) mitosis in broken eggs and egg exudates, and (3) feulgen-negative nuclei in irradiated eggs. In embryogeny proper, of utmost interest is the problem of nondivision of accessory sperm. Rothschild (1954) has briefly considered prevention of polyandrous syngamy (Type II Inhibi- tion of Polyspermy) in marine eggs and vertebrates, but he had very little to say about insects. The accessory sperm in insects do not cleave independently of the zygote nucleus. That sperm nuclei are capable of cleavage, however, has been demonstrated in Habrobracon by A. R. Whiting (1948), who obtained andro- genetic male offspring from fertilization of heavily iiradiated eggs. In these embryos, the egg pronucleus was held back by chromosome bridges and was unable to fuse with the sperm nucleus; the sperm nucleus cleaved normally. Polyspermy is unusual but not rare in Habrobracon. It occurs in approximately 1 % of the eggs ( Speicher, 1936 ) . Gynandromorphs are much less frequent than would be expected if all nuclei in dispermic eggs normally developed. P. W. Whiting (1943a) discovered a strain of Habrobracon that normally produced gynandromorphs with androgenetic parts; Rothenbuhler et al. (1952) discovered a simi- 186 NUCLEOCYTOPLASMIC RELATIONS lar mutant in the honey bee Apis mellifera. The implications are obvious. An accessory sperm nucleus cannot, in general, undergo cleavage as readily as a zygote nucleus, but some genetic differ- ence in Whiting's and Rothenbuhler's stocks allows both the accessory sperm nucleus and zygote nucleus to enter cleavage ( Fig. 7 ) . Rothenbuhler ( 1955 ) has found that the genetic factor for allowing sperm nuclei to be equipotent with zygote nuclei in their ability to enter cleavage has as its basis a modified form of cytoplasmic inheritance. Oddly enough, the influence can be built up in other stocks after several generations of matings by males from the gynandromorph-producing strain (Rothenbuhler, 1955; Rothenbuhler and Gowen, 1955). This is indeed very in- teresting and, as an ideal nucleocytoplasmic interaction, no better example can be found. Normally, an influence must exist in the egg cytoplasm which prevents excess sperm nuclei from dividing as soon as a zygote nucleus forms; this indicates that a substance diffuses with extreme rapidity from zygote nucleus to cytoplasm to sperm. It appears that the mutant honey bee lacks the ability to produce the diffusible agent, Fankhauser (1925) demon- strated the existence of a similar condition in amphibian eggs by a series of elegant experiments in which he separated l^y constric- tion an accessory sperm nucleus from the activated zygote nucleus. Several experiments have been carried out by the author in which organization of the Habrobracon egg has been radically disrupted by breaking the egg and forcing the egg nucleus into the exudate. Of utmost interest is that mitosis under such condi- tions can occur at all. A typical experiment begins when timed eggs have just finished meiosis. Each egg was torn from one end to the other and quickly covered with mineral oil to prevent evaporation. After being incubated for 1 hour longer, the eggs were fixed and stained. In most cases, the nuclei were in the exudate and freely dividing when fixed. Extended experiments have been performed on eggs that were broken and in which cleavage was allowed to continue within the broken membranes (Table I). When newly oviposited eggs were broken widely at their posterior ends and covered with mineral oil to prevent R. C. VON BORSTEL 187 dehydration, 47 of 63 (74.6%) of the eggs were capable of further development. Thirty-three of 47 (70.2%) of the eggs examined proceeded as far as the sixth or seventh cleavage stage. Nineteen of 36 (52.8% ) of the eggs reach the blastula stage. Of those that develop at all but still die before blastulation, most die Table I. Development in Habrobracon Eggs Broken at Completion of Meiosis (30 minutes after oviposition at 30°) Time when development interrupted, hr. 1 1.5 2 3 4 1-cell 2-oell Stage of control 2nd 4th 6th layered layered cleavage ( ,'leavage cleavage blastula blastula Total broken embryos 9 7 11 15 21 Total of embryos stopped at differ- ent nuclear cleav- age stages 1st 1 3 2 6 4 4th — — 0 1 1 6th — — — 1 3 7th — — — 0 1 Broken eggs showing normal develop- ment 8 4 9 7 12 Percentage normal de- velopment 88.9 57.2 81.8 467 57.2 between the fourth and eighth cleavage stages. It would appear from these limited data that the stages around the sixth cleavage are partially critical for nuclei in rapid division. Another characteristic of the sixth or seventh cleavage stage that indicates a critical period of embryogeny is illustrated by a specific action of radiation or nitrogen mustard on eggs or sperm manifested at that time (von Borstel, 1952, 1953b, 1955). When metaphase I eggs are irradiated with 10,000 r, a dose below that which is injurious to cytoplasm as indicated by in- duced androgenesis (A. R. Whiting, 1955), nearly all the em- bryos die at the same stage, the sixth or seventh cleavage. The 188 NUCLEOCYTOPLASMIC RELATIONS conditions of death are unusual. After irradiation of the females, the eggs are oviposited, and meiosis proceeds at the usual rate. Mitosis slowly takes place through two to four cleavages and then nuclear division is blocked for several hours. At approxi- mately the seventh or eighth hour after oviposition, feulgen-neg- ative nuclei are seen here and there in the egg interior. When the total number of nuclei reaches approximately 64 or 128 per egg — the sixth or seventh cleavage — division is blocked completely. The nuclei immediately begin to enlarge, occasionally reaching a size 200 times their normal volume. The nuclei contain pro- tein, but deoxyribonucleic acid, as shown by the feulgen reaction and other methods, is lacking ( von Borstel, 1953a, 1955 ) . This answers the third question, which refers to critical periods during the cleavage stages. When the egg is placed under stress from radiation or breaking, some condition present at the sixth cleavage appears to be some sort of barrier to further develop- ment. Furthermore, it is of interest to note that, in those embryos that get past this cleavage stage (for radiation effects, lower doses are used), development will proceed to the blastula stage at least. Critical periods during cleavage occur normally in Sciara (Du Bois, 1933; Metz, 1938) where the "limited" chromosomes are eliminated at the fifth or sixth cleavage and the sex chromo- somes are eliminated at the seventh or eighth cleavage. That crit- ical periods exist during rapid cleavage is apparent; the mecha- nisms involved are unknown. It is clear from study of the broken Habrohracon eggs that the organization of non-nuclear elements is vital; it is apparent from the radiation experiment that the nucleus is also involved. It seems possible that some basic interac- tion between the nucleus and cytoplasm or some switch in nuclear and cytoplasmic function occurs at this stage of develop- ment in Habrohracon. The Nucleus and Cytoplasm in Determinate Development Depending on the insect, the age of the embryo, and the type of experiment, early insect development has been shown to be completely regulative, completely determined, or somewhere in between. Seidel and his school (see Seidel et at, 1940, for review) R. C. VON BORSTEL 189 laid the groundwork for experimental study of insects; their methods and interpretations have been used on examples from a variety of insect orders. Differentiation in insects has been con- sidered by Bodenstein ( 1955 ) in a short but comprehensive re- view, and no attempt wdll be made here to reevaluate the tre- mendous amount of work that has been done in the field of insect morphogenesis. Suffice it to say that in general the Diptera are considered as being completely determined embryologically and Hymenoptera partially so. Of insects that have been studied ge- netically, Drosophila embryos have been fairly well analyzed by the methods available (Geigy, 1932; Howland and Child, 1935; Rowland and Sonnenblick, 1936; Howland, 1941); Habrobracon embryos have not. The available experimental data on Habro- bracon embryos indicate that development is largely predeter- mined. The few eggs that have been tied in half ha\'e not hatched, and ultraviolet-irradiated eggs ( with the nucleus shielded ) raised to imago have had distorted tergites which were not the result of gene mutations. Bodenstein thoroughly considers the characteris- tics of insect eggs with determinative development. These have the various structures of the future embryo localized in their pe- ripheral cytoplasm. He points out that experiments on determinate eggs show quite conclusively that peripheral cytoplasm is a dif- ferentiated continuum in which localized differentials exert spe- cific influences on totipotent cleavage nuclei, leading them toward special assignments. "One has to assume that the cyto- plasmic regions possess theii^ specific qualities only when in normal topographic relationship to the cortical cytoplasmic layers as a whole. Their influence must be regarded as of a general di- rective nature in that they set up differentials in cleavage nuclei, thereby creating a different pattern within the framework of the blastoderm, which forms the basis of the ensuing de\'elopmental events." That is to say, if any organ-forming region of cytoplasm on the egg surface is injured, no amount of totipotent nuclei com- ing into that region can repair or redirect the cytoplasm. The information for development is locked in the peripheral cyto- plasm, and invading nuclei are guided in their destiny by this cytoplasm. The work of Brauer and Taylor ( 1936 ) is of especial 190 NUCLEOCYTOPLASMIC RELATIONS interest since they were able to demonstrate progressive differen- tiation in the cortical cytoplasm before nuclei had migrated into these regions. Of specific interest are the components of the cytoplasm that contain the information necessary for direction of further devel- UV EXPOSURE (sec) 60 90 (20 150 250 500 750 tOOO (250 1500 UV DOSE (ergs/mm^) Fig. 8. Dose-hatchability curves for Habrobracon eggs irradiated on their convex (nuclear) surfaces (O, ultraviolet; •, ultraviolet plus photo- reactivating light) or concave (non-nuclear) surfaces (A, ultraviolet; A, ultraviolet plus photoreactivating light). From von Borstel and Wolff, 1955. Proc. Natl. Acad. Sci. U. S., by permission of the University of Chicago Press. opment. We assume ultraviolet radiation can destroy the ability for progressive differentiation of regions on the periphery of de- terminate eggs. The Habrobracon egg is unique inasmuch as ultraviolet irradia- tion of the convex side of the egg kills the egg by damage to the nucleus, whereas irradiation of any other place on the egg surface R. C. VON BORSTEL 191 kills the egg by damage to non-nuclear elements. The sm'vival curve is exponential for nuclear killing and sigmoid with respect to cytoplasmic killing ( Fig. 8 ) . Nuclear killing was discussed pre- viously; damage to the cytoplasm, resulting in death, has certain characteristics that make it highly instructive. ( 1 ) Eggs killed by irradiation of cytoplasm die late in development — hatching time is longer in many of those that do hatch, and of those that die, most are characterized by having herniated gut or fragile endo- derm or ectoderm tissues (Amy and von Borstel, 1955). (2) Sur- viving embryos at high doses to cytoplasm have as adults irregu- lar tergites as at least one nonlethal developmental abnormality. (For a similar effect in Drosophila following x-irradiation, see Ulrich, 1951, 1953.) These are gross defects indicating interfer- ence with normal determination and synthesis. (3) The survival curve is steeply sigmoid (von Borstel and Moser, 1956; Ulrich, 1955a,b,c). (4) The action spectrum for cytoplasmic killing indicates that the energy is absorbed by nucleic acid or nucleo- protein (Amy and von Borstel, 1955). (See Loofbourow, 1948, for assumptions and limitations in action spectrum analysis. ) While this work was being done on Habrohracon, an independ- ent study of ultraviolet radiation effects on Drosophila eggs was carried out at Yale by Goldman and Setlow (1956). The research can be considered as mutually confirmatory in most respects, since Goldman and Setlow also observed gut herniation and an action spectrum which they interpreted as indicating energy ab- sorption in nucleic acid when the egg is irradiated. It has been calculated (see Bachem and Reed, 1931) that in normal tissues ca. 80% of the energy of ultraviolet radiation is expended in the first 30 microns. Most of the cytoplasm in Habrohracon eggs is located in a 5- to 10-micron peripheral layer; therefore, it appears reasonable to assume that the cytoplasm comprises the substance sensitive to ultraviolet radiation. Cyto- chemical methods show that peripheral cytoplasm is rich in nucleic acid. This is consistent with action spectrum analysis. The steeply sigmoid shape of the dose-action curve for cyto- plasm suggests a multihit survival curve of high order (von Borstel and Moser, .1956). The shape of the curve appears to be 192 NUCLEOCYTOPLASMIC RELATIONS consistent with the hypothesis that the egg cytoplasm is a multi- unit system, with one hit able to inactivate a unit and not all units having to be hit in order to inactivate the system. This is an ex- tension of Atwood and Norman's theory ( 1949 ) of multihit sur- vival curves (see also Kimball, 1953). It is noteworthy that a great deal of the cytoplasmic pentose nucleic acid appears to be located in particles on the periphery. It should be pointed out that a possible alternative explanation to that of unit inactivation by ul- traviolet radiation is that of production of toxic substances. Wil- bur, Bernheim, and their collaborators (see Bernheim et at, 1952; Fisher and Wilbur, 1954 ) have described fatty acids oxidized by ultraviolet radiation as being such substances. It would be inter- esting to see if irradiated fatty acids can kill eggs and yield re- sults identical to that of direct irradiation of the eggs. However, since the action spectrum for killing indicates that nucleic acid or nucleoprotein absorbs the energy, it appears more profitable at present to explore the consequences of inactivation of cytoplas- mic particles in the egg. The developmental events that follow irradiation of the cyto- plasm suggest that normal synthesis is hampered. This, if true, is of extreme importance. Overly fragile or badly constructed tis- sues and disoriented tergites in surviving adults suggest a disloca- tion of synthesis of structural proteins which may be the conse- quence of inactivation of cytoplasmic pentose nucleic acid. The work of Geigy ( 1932 ) on Drosophila indicated that fewer defects in adults resulted when the egg was irradiated early than when irradiated during differentiation processes. The tergite of the adult is far removed both in time and space from the egg; since the stages between them have not been worked out in Hahro- bracon, any conclusions at present must be considered as tenta- tive. When eggs were irradiated at shorter wavelengths, around 2300 A or less, it was found (Amy and von Borstel, unpublished) that eggs killed by irradiating the cytoplasm have unusual char- acteristics. They die at a much earlier stage of development and the embryos have very little recognizable form. At these wave- lengths, the proteins usually absorb more of the incident energy R. C. VON BORSTEL 193 than nucleic acids. Indeed, Goldman and Setlow (1956) have found a similar circumstance in Drosophila eggs and they explain the effect as being caused by protein denaturation. This phe- nomenon deserves careful analysis because it may reveal a great deal about information content of cytoplasmic elements in de- terminate eggs. Immediately apparent is the possibility that cyto- plasmic protein is used in the determination of egg regions for the first steps in differentiation and the nucleic acid utilized for direction of later development. Alternatively, this phenomenon may be an important clue in deciding between production of toxic substances and inactivation of cytoplasmic particles as a cause of death in these eggs. Photoreactivation Now, is there any general method by which one can distinguish between action of the nucleus and action of the cytoplasm in bringing about an embryonic event? It has already been pointed out that one can distinguish be- tween eggs killed by ultraviolet radiation when the nucleus and cytoplasm are separately exposed. In 1949, Kelner and Dulbecco, working on fungi and viruses respectively, independently dis- covered the phenomenon of photoreactivation. This is the coun- teraction of ultraviolet radiation damage (ca. 2600 A) by radia- tion of a longer wavelength (ca. 3600 A). Photoreactivation is possible after ultraviolet irradiation of a wide variety of living material from protozoa to vertebrates, from viruses and bacteria to the cells of higher plants (see Dulbecco for review, 1955). After ultraviolet irradiation of the nucleus or cytoplasm of the Habrohracon egg, eggs were subjected to reactivating light ( von Borstel and Wolff, 1954, 1955 ) . The results were striking. Injured nuclei can be repaired by light of a longer wavelength, but in- jured cytoplasm cannot (Fig. 8). In both the photorecoverable nucleus and the nonphotorecoverable cytoplasm the energy is absorbed into nucleic acid or nucleoprotein ( Amy and von Borstel, 1955). A similar situation has been reported in Paramecium, where ciliary immobilization from ultraviolet radiation is adjudged to 194 NUCLEOCYTOPLASMIC RELATIONS be a non-nuclear eflFect and the action of the radiation is non- photorecoverable (Brandt and Giese, 1956). The action spec- trum for cihary immobihzation indicates that the ultraviolet radi- ation inducing this effect is absorbed by protein. Blum, Cook, and Loos ( 1954 ) have found four nonphotorecoverable effects in ul- traviolet-irradiated sea urchin eggs. They have suggested that these effects are non-nuclear rather than being indirect effects following irradiation of the egg nucleus. We postulate that this effect of ultraviolet radiation is a general one: Only injury to the nucleus from ultraviolet radiation can he repaired by reactivating light; the cytoplasm is a nonphotore- coverable system. If such a reaction system is truly a general one, it would seem that the system could be profitably employed to follow the interplay of the nucleus and cytoplasm during em- bryonic development. Miss Skreb, working on enucleate amoebae in Brachet's laboratory, has results which indicate that hastening of death by ultraviolet radiation can be delayed by photoreac- tivating light (M. Errera, personal communication). Whether her system or ours is the exceptional one still remains to be deter- mined. Conclusion and Summary Several questions have been asked that are concerned with relations between the nucleus and cytoplasm. 1. The four meiotic nuclei are apparently all genetically equipotent. Since all four remain inside the egg, why does only one take part in further development? By a cytological analysis of oocytes from females that produced a high frequency of hap- loid mosaic adults, it appears that meiosis occurring at the egg periphery leaves three of the four meiotic nuclei susceptible to destruction by some agent in the egg cortex. 2. Is there any condition by which accessory sperm can take an active part in development? In certain genetic strains of Habrobracon and the honey bee, occurrence of partial andro- genesis indicates that normally a zygote nucleus reacts with the cytoplasm in such a way that the cytoplasm can block further development of accessory sperm. The reaction system is incom- R. C. VON BORSTEL 195 plete in certain genetic strains of the wasp Habrobracon and the honey bee. 3. Can the young embryonic system be regarded as a pool in which mitosis freely occurs until blastulation, or do critical periods exist during the cleavage stages? By observing develop- ment taking place in punctured eggs and by irradiating and dam- aging the nuclei of otherwise normal eggs, it was found that the sixth or seventh cleavage is such a critical period in Habrobracon. 4. What component of the cytoplasm is the agent of determi- nate development? By studying the consequences of ultraviolet irradiation of the egg cytoplasm, it appears that nucleic acid or nucleoprotein, perhaps in a particulate system, contains the in- formation necessary for determinate development in the insect egg. 5. Is there any general method by which one can distinguish between action of the nucleus and action of the cytoplasm in bringing about an embryonic event? We suggest that photoreac- tivation of ultraviolet radiation damage may be such a method. When Habrobracon egg cytoplasm is irradiated, dose-reduction by photoreactivation does not occur, but photoreactivation can occur after irradiation of the nucleus. REFERENCES Amy, R. L., and R. 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Zool, 57, 347-407. Ulrich, H. 1951. Sensitive periods and egg-regions in production of the modification "abnormal abdomen" by x-raying eggs of D. mel- anogaster. Dros. Inf. Serv., 25, 131. Ulrich, H. 1953. Induction of "abnormal abdomen" by partial x-raying of Drosophila eggs. Dros. Inf. Serv., 27, 116-17. Ulrich, H. 1955a. Die Bedeutung von Kern und Plasma bei der Abto- tung des Drosopliila-Eies durch Rontgenstrahlen. Naturwissen- schaften, 42, 468. Ulrich, H. 1955b. Comparative studies on the lethal action of x-rays on nucleus and cytoplasm of Drosophila eggs before cleavage. Dros. Inf. Serv., 29, 170-71. Ulrich, H. 1955c. Ein Vergleich der Rontgenstrahlenwirkung auf Kern und Plasma des Drosophila-Eies. Biol. Zentr., 74, 498-515. Whiting, A. R. 1948. Incidence and origin of androgenetic males in x-rayed Habrobracon eggs. Biol. Bull., 95, 354-60. Whiting, A. R. 1955. Androgenesis as evidence for the nature of x-ray- induced injury. Radiation Research, 2, 71-78. Whiting, P. W. 1924. Some anomalies in Habrobracon and tlieir bear- ing on maturation, fertilization, and cleavage. ( Abstr. ) Anat. Rec- ord, 29, 146. Whiting, P. W. 1932. Modification of traits in mosaics from binucleate eggs of Habrobracon. Biol. Bull, 63, 296-309. Whiting, P. W. 1934. Egg-trinuclearity in Habrobracon. Biol. Bull, 46, 145-51. Whiting, P. W. 1943a. Androgenesis in the parasitic wasp Habrobra- con. J. Heredity, 34, 355-66. Whiting, P. W. 1943b. Multiple alleles in complementary sex determi- nation of Habrobracon. Genetics, 28, 365-82. Wilson, E. B. 1928. The Cell in Development and Heredity. The Mac- millan Company, New York. Zeuthen, E. 1951. Segmentation, nuclear growth and cytoplasmic stor- age in eggs of echinoderms and amphibia. Pubbl. staz. zool. Napoli, 23 (Suppl.), 47-69. NUCLEAR TRANSPLANTATION, A TOOL FOR THE STUDY OF NUCLEAR DIFFERENTIATION H. E. LEHMAN: department of zoology, UNIVERSITY OF NORTH CAROLINA, CHAPEL HILL, NORTH CAROLINA The problems of embryonic determination and differentiation are concerned primarily with the mechanisms whereby a limited part of the total capacity of the genome is selected for phenotypic expression in different somatic cells of the developing organism. A substantial body of information derived from nmnerous studies on isolated blastomeres, induction systems, physiological gradi- ents, etc. indicates that the selective mechanism in determination is extranuclear in origin and is dependent primarily upon local cytoplasmic differences. This view is in agreement with the con- ventional opinion which accepts the concept of "constancy of chromosome number" of Hertwig (1916) and assumes that nuclei are quantitatively and qualitatively equivalent throughout the somatic cells of the body. The theory of Weismann ( 1885, 1892 ) which postulated fractional allocation of nuclear materials to different blastomeres as the primary mechanism for somatic dif- ferentiation has generally been abandoned since the equivalence of blastomere nuclei has been repeatedly demonstrated on repre- sentative eggs of most major phyla. Normal development can still take place after the cleavage nuclei are shifted to various "un- natural" positions by compression, centrifugation, or constriction of the egg (see reviews by Wilson, 1925; Morgan, 1927; Spe- mann, 1938). A host of regeneration studies on vertebrates, in- vertebrates, and plants have supplied evidence which, although inconclusive, has been interpreted as an indication that nuclei of 201 202 NUCLEAR TRANSPLANTATION differentiated cells also retain a large measure of their totipotency (see reviews by Schotte, 1939; Child, 1941; Needham, 1952). In the light of this pressure of evidence it is not surprising that the question of nuclear differentiation has received little attention in most experimental studies of development. It should be realized that, although the prevailing consensus accepts the concept of nuclear uniformity in somatic tissues, the possibility of nuclear differentiation has never been conclusively eliminated with regard to the cells of late embryonic or adult tis- sues. During recent years the need for reconsideration of this question has been expressed with increasing frequency (e.g., Huskins, 1947, 1952; Huskins and Steinitz, 1948a,b; Schultz, 1952; Stern, 1955). In this connection it is noteworthy that the embryonic evidence for nuclear uniformity is primarily based upon studies of nuclei in early cleavage stages only, the most striking demonstration being that supplied by the constriction experiments on eggs of Triton in which total development re- sulted in fragments that received only one of the 2-, 4-, or 8-cell stage blastomere nuclei (Spemann, 1914, 1928; Fankhauser, 1925, 1930). When single nuclei from 16- and 32-cell stages en- tered non-nucleated egg fragments, partial development of the fragment resulted. This failure to complete development has gen- erally been attributed to irreversible cytoplasmic changes that resulted from prolonged isolation from a nucleus. Although this may be true, these observations also admit the seldom considered alternative, namely, loss of nuclear totipotency which is nuclear specialization. These and related experiments (see review by Spemann, 1938) have demonstrated conclusively the equivalence of early cleavage nuclei, but they throw no light on the nuclear character of older cells. Predictions from these data that all somatic nuclei remain equivalent during development are particularly insecure in view of the extensive body of information which indicates that for the most part the cytoplasm appears to be almost completely autono- mous during cleavage and blastulation and very few nucleus- dependent functions have been demonstrated during this early phase of development. For example, enucleated eggs and egg H. E. LEHMAN 203 fragments of echinoderms and amphibians have the capacity to cleave and form "blastulae," but none has given any evidence of gastrulating (see reviews by Fankhauser, 1955; J. A. Moore, 1955 ) . The rate and pattern of cleavage until the onset of gastrula- tion in hybrids, and particularly in hybrid andromerogons, pro- ceeds strictly according to the genetic character of the egg cytoplasm and gives no indication of sperm nucleus influence until gastrula and postgastrula stages (see reviews by Needham, 1950; Fankhauser, 1955; J. A. Moore, 1955). Physiological and biochemical studies indicate that, although there is a continuous rise in the metabolic rate during cleavage, the most conspicuous accelerations in respiration and specific syntheses follow the onset of gastrulation (see reviews by Barth and Jaeger, 1947; Brachet, 1947, 1952; Gregg, 1948; Ebert, 1954; Fankhauser, 1954). These observations suggest that, if nuclei become differentiated during development, such changes should be sought, not in early devel- opment when the nuclear influence is slight but, rather, in gas- trula and older tissues when nuclear function can be clearly demonstrated. In examining the possibility of nuclear differentiation, the first question which must be answered relates to whether or not there is sufficient evidence to indicate that cytoplasmic differentiation is accompanied by tissue-specific nuclear change. Until this is settled, it is premature to speculate on secondary questions relat- ing to mechanics of nuclear differentiation, or labile versus ir- reversible determination, or functions of differentiated nuclei in histogenesis and tissue syntheses. Present information does not allow one to generalize in answering the primary question; however, certain evidence unequivocally shows that at least some differentiated somatic cells possess nuclei which vary in a tissue- specific manner from other nuclei in the organism. Evidence for Nuclear Differentiation in Somatic Tissues The nuclei of differentiated tissues possess distinctive varia- tions in size, shape, density, and composition which are histologi- cally diagnostic for specific tissues. Many of these differences show a constant relation to functional activity of the cells con- 204 NUCLEAR TRANSPLANTATION taining them; for example, large nuclei and nucleoli are generally indicative of rapid cytoplasmic synthesis associated with growth or secretion. In addition, numerous cytochemical assays of nu- clear composition show that nuclei of different tissues of the same organism vary both quantitatively and qualitatively (within the sensitivity range of the tests employed) in content and synthesis of special enzymes, proteins, protein precursors, nucleic acids, glycogen, lipoprotein, etc. (see reviews by Boivin, Vendreley, and Vendreley, 1948; Mirsky and Ris, 1949; Brachet, 1950, 1952, 1955; Caspersson, 1950; Caspersson and Schultz, 1951; Novikoff, 1952; Stern et al, 1952; B. C. Moore, 1952; Pollister, 1952, 1954; Fankhauser, 1954). In addition to evidences of chemical variation, unequal chro- mosome composition has been reported in the cells of a number of plants and animals. Of these, the earliest and still one of the most striking examples is found in the work of Boveri and others (see review by Tyler, 1955) on chromatin diminution. This occurs during the early cleavage divisions of the somatic cells of Ascaris, whereas the primordial germ cell retains the full chromatin com- plement. Somewhat comparable differences are found in the somatic and germ cells of certain insects, e.g., the fungus fly, Sciara (Metz, 1938; Berry, 1941), and gall midges, Cecidomidae (White, 1946, 1947, 1948). Of greater interest for present pur- poses, however, are evidences of nuclear differences in histologi- cally distinct somatic tissues. Comprehensive reviews of the oc- currence of normal polyploidy in specific tissues of otherwise diploid animals and plants are given by Berger (1941), Geitler (1941), Huskins (1947, 1952), Schultz (1952), and Fankhauser ( 1954 ) . Polyploid nuclei have been reported in vascular bundles and epidermal structures of plants, in nematode muscle cells, in ganglion cells of snails, and in various arthropod tissues. Similar cases of somatic polyploidy have been reported in the kidney, liver, and epidermis of amphibia, as well as in liver, vascular, and tumor tissues in mammals. Tissue-specific haploidity is claimed for the tail mesenchyme of frog larvae (Green, 1953) and for the mesenchyme-forming micromeres of the sea urchin in which H. E. LEHMAN 205 reduction from diploid to haploid is described as occurring be- tween the 16- and 32-cell stage (Lindahl, 1953; however, con- tested by Makino and Alfert, 1954); the remaining tissues in these embryos retain the normal 2-N count. Without question the most striking examples of tissue-specific chromosomal variation are to be found in insects where many tissues possess specific multiples of the basic diploid number characteristic for the species. The most detailed study is provided by the work of Geitler ( 1934, 1941 ) on chromosome numbers in various tissues of the "pond skater," Gerris lateralis. This species is particularly favorable for study owing to the presence of dis- tinctive heterochromatic regions on the sex chromosomes, which permit them to be identified and to ascertain ploidy during inter- phase. By this means Geitler found the chromosome number in salivary gland nuclei to be 2048-ploid; Malpighian tubules were from 32- to 64-ploids; and mid-gut epithelium and testicular septa were octaploid. He further demonstrated that the nuclei achieved their various polyploid states by endomitosis during the development of germinal diploid cells, the individual chromo- somes undergoing recognizable pro-, meta-, and anaphase changes within the intact nuclear membrane. Somewhat comparable re- sults have been reported for the grasshopper, Romulea (Mickey, 1945); the mosquito, Culex (Berger, 1938, 1941; Grell, 1946a,b); the fungus fly, Sciara (White, 1946, 1947, 1948); and Drosophila (Painter and Reindorp, 1939). The number of such cases prob- ably could be extended considerably if it were certain that multi- strand (polytene) chromosomes represent a preliminary step in the establishment of endopolyploidy. Berger ( 1939, 1941 ) and Grell (1946a,b), for example, have found that the diploid cells of the mosquito intestine increase in size but do not divide dur- ing larval development. During this period the chromosomes duplicate themselves and form bundles of closely associated chro- monemata (equivalent to 8-, 12-, 32-, or 64-ploid), which later separate into individual chromosomes when division is resumed at metamorphosis. If each strand in a polytene chromosome in- deed represents internal chromosomal duplication, variations in 206 NUCLEAR TRANSPLANTATION polyteny in different cells might also be taken as evidence of quantitative multiplication of whole sets of the basic genetic units in the nuclei containing them. The constant relation of definite polytene and polyploid num- bers to certain insect tissues strongly suggests that they are re- lated to differentiation or special functions in these cells. White (1945, pp. 33-34) expresses the opinion of a number of cytolo- gists in attributing developmental significance to these nuclear tissue-specific relationships when he suggests that "The whole process of histological differentiation in insects seems to be inti- mately bound up with this phenomenon of endopolyploidy, each organ and each tissue having its own characteristic degree of ploidy, some being entirely composed of one type of cell while others are mosaics of cells with different multiples of the funda- mental diploid number. To what extent endopolyploidy occurs outside the Insecta is not known at present, but there are indica- tions that it is fairly widespread in many groups of animals." For a critical evaluation of this possibility, one is also referred to Huskins (1947). In addition to the presence of quantitative differences in the number of complete sets of chromosomes in different somatic cells, there is also evidence for tissue-specific variation in the structure of individual chromosomes. Kosswig and Shengiin (1947) and Beermann (1952) working with Chirouomiis larvae have made detailed cytological studies of similar regions on homologous chromosomes obtained from salivary glands, Malpighian tubules, mid-gut, and rectum. They found major tissue-specific differences in polyteny, in overall size of comparable regions and, most note- worthy, differences in the thickness and number of homologous discs at specific loci on the chromosomes. Moreover, these varia- tions showed modification with developmental age with some dis- tinctive structural features appearing or disappearing at various times during differentiation. These observations are contrary to the less extensive, but somewhat comparable, study of Berger (1940) on salivary and mid-gut chromosomes of Sciara in which similar regions of homologous chromosomes from the two tissues were described as having similar structure. The work of Kosswig H. E. LEHMAN 207 and Shengiin and of Beermann indicates, in Chironomus larvae at least, that in addition to other more easily recognized nuclear variations there are also clear-cut tissue-specific differences in the fine structure of chromosomes. These authors consider the possibility that these differences in structure and staining proper- ties of comparable bands on homologous chromosomes may be evidence of activity or inactivity of different specific genes in the respective tissues. A similar suggestion has been made in connec- tion with the possible functional significance of the "lamp brush" chromosomal structure of amphibian oocyte nuclei; the localized enlargement of certain loci on the chromosomes may indicate multiplication of specific regions of the chromosome associated with special syntheses in the developing germ cell (see Duryee, 1950; Gall, 1952). A final category of cytological evidence for nuclear differentia- tion is associated with variability in heterochromatic material demonstrable in interphase nuclei at a time when the nucleus is physiologically most active (Berrill and Huskins, 1936; Huskins and Steinitz, 1948a ) . In a number of instances interphase hetero- chromatin can be traced to specific chromosomes during mitosis and has been useful in determining ploidy in interphase nuclei (Geitler, 1934, 1941; Huskins and Steinitz, 1948b). Heterochro- matin is considered to be for the most part genetically inert (as, for example, in the X and Y chromosomes of Drosophila and in the eliminated chromatin in somatic cells of Ascaris and Sciara); nevertheless, duplications, deletions, and translocations of hetero- chromatin are known to produce sterility and a variety of "posi- tion effects." There has been much speculation concerning the possibility that variations in heterochromatic content might be evidence of differential gene action as was suggested by Muller and Gershenson (1935; see also Schultz, 1939, 1947, 1952; Brachet, 1947; Huskins, 1947; Lewis, 1950; Hannah, 1951; Cas- persson, 1950; Caspersson and Schultz, 1951; Gall, 1952). As an example of one of the present trends of thought, Schultz ( 1952, p. 38) suggested that only the specific genes required for function in a particular type of cell are in an active state; the others are in the "heterochromatic" state and could be dispensed with with- 208 NUCLEAR TRANSPLANTATION out endangering the life of the cell. He points out that this hy- pothesis resembles that of Weismann but differs "in that the ex- periments which discredited the Weismannian point of view (nuclear differentiation at the cleavages) are not relevant to it. It is closer to the hypothesis of changes in the centers of ac- tivity of the genes during differentiation of the tissues " which was earlier suggested by Morgan (1934) as a possible mechanism whereby determined cytoplasm selectively affects genes which in turn maintain the differentiation of the cytoplasm in a reciprocal interaction between these two basic cell components. Until addi- tional information is available concerning the dynamic properties of heterochromatin in interphase nuclei, it is not possible to assay its significance in cell function and differentiation. The essential fact from the foregoing summary of visible dif- ferences in somatic cell nuclei is that there are now numerous "exceptions" to the concept of quantitative and qualitative nu- clear uniformity in somatic tissues. As Huskins (1947) has in- timated, additional studies, unbiased by the preconception of nuclear uniformity, would undoubtedly add materially to the growing body of data that points toward nuclear specialization as a general, rather than an exceptional, aspect of histogenesis. In collecting these data, however, the fact still remains that the cytological evidence of nuclear differentiation in cells of embryos most suitable for experimental study are meager. It is hoped that additional cytogenetic and cytochemical studies will concentrate attention on species favorable for developmental analysis so that the varied techniques from these disciplines can jointly be brought to bear on this morphogenetic problem. One relatively new embryological method, namely, nuclear transplantation, has recently been applied to the question of nuclear differentiation. With more extensive application, it should constitute an impor- tant supplement to the cytological and biochemical information now available on this subject. Technique and Results of Nuclear Transplantation The simplest and most direct manner in which information could be obtained concerning irreversible nuclear determination H. E. LEHMAN 209 during development would be by transplantation of a nucleus from a determined cell into one that was labile or totipotent prior to removal of its own nucleus, as, for example, in an enucleated mature egg. Then, as Briggs and King (1952, p. 356) suggest, "the nature of the ensuing development should re\'eal the char- acter of the transplanted nucleus — complete differentiation would indicate that irreversible nuclear differentiation had not occurred, while limited differentiation would indicate that it had." Accord- ingly, when the methods are finally perfected, nuclear transplan- tation gives promise of being one of the most significant technical advances that has been made in recent years for the study of causal relationships between nucleus and cytoplasm and may well rank in importance with hybridization and merogony as an em- bryological tool for protoplasmic recombination. Nuclear trans- plantation has the unique advantage, heretofore impossible, of permitting one to unite a nucleus and cytoplasm of radically dif- ferent age and prospective fate in a manner comparable to that practiced in the standard transplantation of embryonic tissue fragments. The first demonstration that nuclear transplantation could be successfully carried out was reported by Comandon and deFon- brune (1939) working with Amoeba sphaeronucleus. A blunt microdissection needle was used to force the nucleus of an intact animal through the cell membrane into an enucleated fragment lying adjacent to the donor animal. The fragment with the trans- planted nucleus, although not regaining its total former metabolic capacity, nevertheless gave evidence of "rejuvenation" after the transfer. Torch and Danielh (1950) and DanielH (1952, 1955) extended the study with homo- and heterospecific nuclear trans- fers between Amoeba proteus and A. discoides. Mass, single clone cultures were very readily obtained from homospecific transfers but were only obtained in about 1% of the nucleocytoplasmic hybrids. Species differences in the parent clones involved nuclear size, body form, mode of locomotion, and specific antigenic char- acters. The nuclear size in hybrids was intemiediate between that of the parent clones, indicating that the cytoplasm exerts some influence in determining this character. The general shape and 210 NUCLEAR TRANSPLANTATION action of the hybrids was at first intermediate between that char- acteristic of the parent strains; however, after a few divisions the nucleocytoplasmic hybrids assumed and maintained for over 400 cell generations the characteristics of the host cytoplasm, thereby showing that morphological form and locomotion are almost en- tirely independent of the nucleus. When hybrid antigenic charac- ters were tested they were found to be predominantly of the type characteristic for the species contributing the nucleus. Danielli (1955) suggested that these data may be interpreted as evidence that the nucleus is the controlling agent in synthesis of specific macromolecules, but that the cytoplasm is relatively autonomous with regard to supramolecular organization of these molecules within the cell. If this interpretation be valid, one is faced with a most interesting problem concerning the material basis for this cytoplasmic control of growth products which are apparently largely nucleus-dependent in origin. As illuminating as these studies are, protozoan material is nevertheless inherently incapable of throwing much light on the question of tissue differentiation. For this question metazoan material must be used. No successful nuclear transfers have been reported for other invertebrates although Danielli (1955) stated that preliminary attempts have been made on sea urchin and ascidian eggs. All remaining studies reported to date have dealt with amphibian eggs and, of these, by far the most extensive and successful experiments have been carried out by Briggs and King working with the frog, Rana pipiens (Briggs and King, 1952, 1953, 1955; King and Briggs, 1953, 1954a,b, 1955). In brief, then- transplantation technique involved activation of eggs with a clean glass needle; this is sufficient to cause the egg to rotate and initiate the second maturation division. A few abortive furrows may appear, but no true cleavage or blastulation takes place (Briggs, Green, and King, 1951). Guyer (1907) showed that, in the absence of tissue fluid contaminants, pricking alone is not sufficient to stimulate parthenogenetic development in the frog egg. The egg nucleus was removed by the method of Porter ( 1939 ) in which a needle is inserted under the egg nucleus and drawn upward, causing an exovate of egg cytoplasm which, H. E. LEHMAN 211 when properly executed, contains the egg nucleus. In the hands of Briggs and King, enucleation by this means was approximately 99% successful. Diploid donor cells were isolated from normal blastulae, gastrulae, neurulae, and tail-bud embryos by microdis- section, or better, by the trypsin-versene method for dissociating embryonic cells ( King and Briggs, 1955 ) . Single cells were drawn into fine micropipets in such a manner as to cause the cell mem- brane to rupture without diluting the cell contents with the op- erating medium. With the aid of an Emerson micromanipulator, the whole cell was then injected into an activated and enucleated host egg. No attempt was made to exclude the donor cytoplasm which, however, constituted only a very minute fraction (1:20,- 000 to 1:600,000) of the total egg volume. It is significant in con- nection with the results reported that differentiated cytoplasm lacking nucleus never stimulated any development when injected into enucleated host eggs ( Briggs and King, 1953 ) . The transplantation technique was first tested to determine whether or not the various manipulations damaged either the egg cytoplasm or the transplanted nucleus. Initial experiments (Briggs and King, 1952) employed only undetermined prospec- tive ectodermal cells of normal diploid blastulae. Approximately one-third of the cases gastrulated and, of these, nearly half formed embryos which were quite normal in appearance and capable of developing into swimming larvae, and the remainder underwent somewhat abnormal postgastrula development. Determinations of ploidy in these embryos, based on nuclear size and nucleolar number, revealed that all were either diploid or polyploid (hap- loids would generally be expected if these cases represented failures in enucleation of the egg followed by parthenogenetic de- velopment; however, spontaneous regulation to diploidy can occur in parthenogenetic frog eggs as Parmenter, 1952, and others have shown ) . Although the percentage of successful cases was somewhat lower, essentially similar results were obtained when undetermined prospective neuro-epidermal cells of early gastrulae were used as a source of donor nuclei ( Briggs and King, 1953; King and Briggs, 1954a, 1955). It thus seems certain that these diploid nuclei from blastulae and early gastrulae were not 212 NUCLEAR TRANSPLANTATION irreversibly determined at the time of transplantation since they were capable of replacing the zygote nucleus and of participating in total nomial development. With the foregoing demonstration of the feasibility of this op- erative technique, Briggs and King have proceeded to improve the method and to test the possibility of nuclear differentiation in determined embryonic cells by using gastrulae, neurulae, and tail-bud embryos as sources of donor nuclei. In general it can be said that the older the donor nucleus, the lower is the percent- age of successful cases which show cleavage, gastrulation, and postgastrula development. As yet, no cases involving neurula or tail-bud nuclear transplants have been reported which gastru- lated normally, although a small number have undergone normal cleavage (King and Briggs, 1954a). This may be due to technical difficulties encountered in handling the smaller cells. It is also possible that this developmental failure may be associated with a restriction in the capacity of older nuclei to participate in total development. The strongest evidence in support of the latter pos- sibility is supplied by transplantation of nuclei from determined cells of late gastrulae (Briggs and King, 1953; King and Briggs, 1954a, and, particularly, 1955). The results from transplanting nuclei from recently induced neural ectoderm from late gastrulae into enucleated eggs showed that cases reaching the blastula stage could develop into entirely normal tadpoles (King and Briggs, 1954a). Although the donor nucleus came from a cell determined to form neural tissue, it is apparent that if any tissue-specific nuclear differentiation existed at the time of transfer, it was completely reversible. Briggs and King ( 1955 ) pointed out that these results should be viewed in context with the observations of Grobstein ( 1952 ) and Grobstein and Zwilling ( 1953 ) who have found that, although large masses of explanted mouse or chick neural plate will form nervous tissue, small fragments fail to differentiate. This indicates that the neural plate may be irreversibly determined as a whole while its com- ponent cells remain labile and incapable of self-differentiation. If the same situation were to obtain in Amphibia, the possibility H. E. LEHMAN 213 exists that recently determined neural cells of the frog may possess nuclei in a labile state of differentiation. When late gastrula "chorda-mesoderm" nuclei were injected into enucleated eggs, a few cases developed into entirely normal larvae, one of which was carried to metamorphosis (King and Briggs, 1954a). However, in a great majority of the cases, devel- opment was arrested by the late neurula stage ( King and Briggs, 1955). Histological sections of the abnormal postneurulae re- vealed that brain, spinal cord, notochord, gut, pronephric tubules, heart, blood vessels, somites, and sense organs developed (King and Briggs, 1954a ) . Neural differentiation was generally reduced or entirely absent. It was suggested that the donor "chorda-meso- derm" nucleus, although capable in some instances of giving rise to total nomial development, might be partially deficient in its control of the ectodermal response to neural induction (King and Briggs, 1955). However, on the basis of the best cases ob- tained, it would appear that these nuclei, at most, are determined in an entirely labile manner at the late gastrula stage. The most decisive evidence of nuclear specialization during development was obtained when determined mid-gut endoderm nuclei of late gastrulae were injected into enucleated eggs (King and Briggs, 1955). In 40% of the attempts, complete blastulae were obtained. All failed to develop normally in gastrula and postgastrula stages. The most advanced experimental embryos exhibited a consistent combination of anomalies not observed in other embryos. These included epidermal disorganization in- volving localized deficiencies and unequal thickness, poor neural development with cytological evidence of nuclear degeneration, well-developed notochord, differentiated somites of abnormal form, and a gut "developed as well as the general condition of the embryo allows" (King and Briggs, 1955, p. 324). Endodermal cytoplasm alone did not produce this result when injected into normally fertilized eggs. On the basis of these data, King and Briggs seemed justified in revising their earlier and more con- servative interpretations (King and Briggs, 1954a; Briggs and King, 1955) and in concluding that "it now appears definite that 214 NUCLEAR TRANSPLANTATION nuclei undergo certain changes during differentiation . . . ex- periments not reported here reveal that at later developmental stages there is a loss of the capacity of these nuclei to enter into cleavage of egg cytoplasm. All together, this suggests a progres- sive specialization of nuclear function during cell differentiation" (King and Briggs, 1955, p. 324). The writer is aware of only one attempt to repeat the studies of Briggs and King on eggs of R. pipiens. This consists of an unpub- lished study by Dr. C. L. Markert who has generously given permission to have his data summarized here. The enucleation and transplantation technique was essentially similar to that used by Briggs and King and the results outlined in a written com- munication are of sufficient interest and corroborative value to be quoted in full: "In the initial series of experiments [performed in collaboration with Arthur Freedman] a total of 256 nuclei were transplanted. Of the eggs receiving these transplanted nuclei, 117 showed no signs of development; 34 underwent abortive cleavage; 40 showed extensive cleavage; 32 developed to blastula; 20 gastrulated (gen- erally abnormal); 9 became well developed neurula, and 4 de- veloped into larvae. One of the larvae was tetraploid. I cite these data as illustrative of the discouragingly low level of success that we achieved. Later I resumed this work with a higher percentage of success but the general picture remained essentially un- changed. A variety of cell types was used as a source of nuclei in these experiments. Nearly all of the successful transplants in- volved nuclei from cells of blastula or early gastrula embryos. One of the neurulae — an abnormal one — developed after trans- plantation of a nucleus from an endoderm cell taken from the floor of the archenteron in the late neurula stage of development. This was the most advanced stage of development induced by nuclei taken from post-gastrulae. However, cleavage was induced occasionally even with nuclei from larvae old enough to show muscular responses" (C. L. Markert, unpulDlished experiments). These results are in agreement with those of Briggs and King which indicate that normal development is less frequently ob- tained as the age of the donor nucleus increases. The concluding H. E. LEHMAN 215 statement of Markert is of particular interest in giving promise of success in transplanting nuclei from histologically differentiated cells with further refinement of the operative technique. In evaluating the results of late gastrular nuclear transplants, it can be said with certainty that irreversible nuclear specializa- tion does not take place at a uniform rate throughout the embryo. Apparently none has occurred by this time in prospective ecto- derm derivatives; chorda-mesoderm nuclei may illustrate an inter- mediate condition, whereas endoderm nuclei are clearly special- ized. The data from "mid-gut embryos" probably should be inter- preted as evidence of quantitative, rather than qualitative, modifi- cation in nuclear function since, in the most advanced cases at least, all tissue types were represented to a greater or lesser degree. Even ectoderm, although poorly developed, showed a slight capacity for neural development. An alternative interpretation admits the possibility of tissue-specific (i.e., qualitative) nuclear difl^erentia- tion. For example, current evidence does not peiTnit one to de- cide whether the limited neural capacity in "mid-gut embryos" stemmed from the transplanted nucleus (as is assumed above), or is the expression of a residual effect of the egg nucleus dating from the period of oogenesis. The latter possibility should not be overlooked, since the well-known studies of Hammerling ( 1934, 1953) on regeneration in Acetabiilaria, and of Hadorn (1936) on andromerogonic Triton hybrids [T. palmatus ( 9 ) x T. crista- tiis S ] have shown that species-specific characters of nuclear origin can be maintained in the cytoplasm of enucleated cells and in the presence of a foreign nucleus for extended periods of time (several months in Acetabiilaria; until postmetamorphosis in Triton hybrid merogon tissues when transplanted to diploid hosts). This second possibility gains some support from the cyto- logical condition of the epidermal nuclei in "mid-gut embryos" which showed abnormal degenerati\ e structure that suggests an incompatibility between irreversibly determined cytoplasm and nuclei. This question, along with those relating to the influence of donor cytoplasm injected with the transplanted nuclei, and the possible effects of nuclear damage in operative manipulation (in particular the trypsin-versene method of isolating donor cells) 216 NUCLEAR TRANSPLANTATION cannot be answered in the light of present knowledge. It will be particularly interesting in these connections to learn of the results being obtained with improved techniques for transplantation of postgastrula nuclei which King and Briggs ( 1955 ) indicate are in progress. To complete the picture, it would also be desirable to learn the developmental capacities of blastula and young gastrula endoderm nuclei to discover when nuclear specialization in these cells is first demonstrable. Blastula and gastrula nuclei from R. pipiens 9 xR. catesheiana $ diploid hybrids and from R. pipiens ( 9 ) x R. catesheiana $ andromerogonic haploid hybrids have also been transplanted into enucleated R. pipiens eggs (Briggs and King, 1952; King and Briggs, 1953). These experiments were designed to analyze the role of the nucleus in the well-known lethality which occurs dur- ing gastrulation in R. pipiens 9 x R. catesheiana S crosses (see review by J. A. Moore, 1955). Since this subject is aside the major line of interest here and is treated in a separate section of this volume (see J. R. Gregg), it will suffice to say that potentially lethal hybrid nuclei from undetermined blastula and gastrula cells could be transferred into enucleated eggs and would par- ticipate in normal cleavage and blastulation, but they would not permit complete gastrulation. This was true provided that the donor nucleus had not advanced to the stage when irreversible degenerative changes take place which anticipate lethality in the donor hybrids. The hybrid donor nuclei therefore gave evi- dence of being able to survive long after they would have died had they remained in the donor embryos. These results also show that lathality in this cross is not due to autonomous nuclear change, but it is the result of failure on the part of R. catesheiana nuclear material and R. pipiens cytoplasm to cooperate in postgas- trula development. Related to this study is a brief report (Briggs and King, 1955) of transplanting nuclei from blastulae of the anuran, Triturus pyrrhogaster, into enucleated R. pipiens eggs. Partial cleavage and blastulae were obtained in the best cases. Cytological study showed that the pyrrhogaster cell probably supplied a centrosome which was capable of quite normal func- tion as a cleavage center in pipiens cytoplasm. The foreign nu- H. E. LEHMAN 217 cleus, although incapable of maintaining its integrity during interphase and mitosis, was nevertheless able to increase in chro- matin volume, presumably by utilization of nonspecific protein precursors in the pipiens cytoplasm. The remaining studies on nuclear transplantation that have been reported were carried out on eggs of the salamander, Tri- turus (Triton) pahnatus (Waddington and Pantelouris, 1953; Lehman, 1955). These attempts have been entirely unsuccessful so far as obtaining postblastula is concerned, and no information relative to progressive nuclear differentiation has been obtained from these experiments. Waddington and Pantelouris injected nuclei from diploid blastula, gastrula, and neurula donor cells into non-nucleate halves of normally fertilized eggs. Cleavages were obtained with all types of donor cells. When blastula and gastrula donor cells were used, some of the cases developed into abnormal arrested blastula with a reduced blastocoele filled with loose cells. It was not clear from these results whether the arrest in development at the blastula stage was occasioned by nuclear specialization, or by operative damage, or by lack of totipotency on the part of the host egg fragment. In view of these uncertainties, an attempt was made to repeat these experiments on whole eggs of T. pahnatus ( Lehman, 1955 ) . Unfortunately, Triton eggs cannot be parthenogenetically ac- tivated by pricking, nor was it possible to stimulate development by the injection of blastula nuclei alone. This difficulty was partly circumvented by using Hugh's ( 1939 ) technique of x-iiTadiating sperm at 50,000 r to inactivate the chromatin without destroying the fertilizing capacity of the sperm. Although haploid embryos were consistently obtained from normal eggs fertilized by such sperm, the technique cannot be considered entirely satisfactory since minute disorganized cliromatin fragments, probably of sperm origin, were frequently observed in smear preparations. These fragments, if of sperm origin, had undoubtedly undergone some multiplication during development even though they were not intimately associated with the division figure. The possibility of some developmental effect of these degenerating "sperm chromatin fragments" cannot be ignored. After fertilization with 218 NUCLEAR TRANSPLANTATION irradiated sperm, host eggs were pricked and the egg nucleus was sucked out in the manner described by Curry (1931, 1936); in my experience, this enucleation technique was successful in only about 50% of the attempts (as determined by the incidence of haploids developing from "enucleated" eggs fertilized by nor- mal sperm). Normal diploid blastula ectoderm and endoderm cells were used for injection and they gave similar results. About 65% of the injected eggs cleaved and, of these, about one-third developed into blastulae which externally appeared to be quite normal; however, internally they were invariably atypical and at best had a reduced blastocoele. Most of the cases were sacrificed at the blastula stage for cytological study but, of those permitted to continue development, none progressed beyond the earliest crescent blastopore stage. Since half of these embryos probably retained the egg nucleus after unsuccessful attempts at enuclea- tion and should develop as typical hybrids, these results suggest that the Triton cytoplasm is less able to recover from the injury of injection than is the cytoplasm of R. pipiens. The interpretation of evidence from nuclear transplantation in Triton (Waddington and Pantelouris, 1953; Lehman, 1955) is extremely problematical since no development was obtained in these embryos which cannot be duplicated in totally enucleate eggs of this species ( Fankhauser, 1929, 1934 ) . The injected nuclei from undetermined or determined cells from blastula to neurula stages are probably capable of at least limited participation in cleavage and blastulation, but there is no direct evidence that these Triton nuclei can take part in postblastula development. Whether this is due to operative damage or nuclear specialization is not known. Similar types of arrested blastulae have been de- scribed in various salamander hybrids (e.g., Baltzer et al., 1939; Baltzer and Schonmann, 1951), and in certain instances the indi- vidual tissues have been shown to be completely viable when transplanted to normal diploid hosts. The application of this method to the arrested "injection blastulae" of Triton is the most promising approach available at present for assaying the develop- mental capacities of transplanted nuclei of different age and de- H. E. LEHMAN 219 velopmental history. Although the eggs of Triton appear to be relatively unfavorable for nuclear transplantations, it is hoped that techniques for activation, enucleation, and injection can be improved so that decisive evidence can be obtained concern- ing nuclear differentiation in this classic form for embryonic study. Other species of Triturus (Triton) may be more amenable to nuclear transplantation than T. palmatus, and the search for such a species is particularly desirable in view of the favorable cytological properties of this material. The cytological analysis of "nuclear transplant embryos" is deserving of more thorough study. Nothing is known concerning the actual behavior of the injected nuclei during the initial cleav- age stages, and this subject has considerable intrinsic interest entirely apart from the question of determination. Secondly, it is apparent that the major evidence of successful egg enucleation followed by injection of a diploid donor cell is based on the pres- ence of diploid nuclei in tissues of the experimental embryo; haploidity is indicative of failure in enucleation of the egg. In the studies by Briggs and King, ploidy in "nuclear transplant" embryos that developed to late neurulae and tadpole stages was determined by nuclear size and, wherever possible, by nucleolar number (for method, see Briggs, 1947). Of 67 cases reported (Briggs and King, 1952; King and Briggs, 1954a), only one em- bryo gave evidence of failure in enucleation; this was a haploid- diploid mosaic which had been injected with a young gastrula cell. It probably indicates that both the egg and injected nucleus cooperated in development. The remaining 66 cases represent successful enucleation and transplantation; 34 were diploid and 32 were polyploid (probably tetraploid). Briggs and King (1952) suggested that the high incidence of polyploidy may result from doubling and division of donor chromosomes before the egg cyto- plasm is capable of cleaving. This is probably true, but it does not relieve the necessity of considering the possible develop- mental significance of polyploidy in the experimental animals. This is particularly important when one examines the data given below and finds that the incidence of polyploid individuals in- 220 NUCLEAR TRANSPLANTATION creases with the age and differentiation of the donor nuclei and therefore parallels the evidence interpreted as an indication of nuclear differentiation. Ploidy in "Ahiclear Origin of Donor Cells Transplant" Embryos Diploid Polyploid Blastula ectoderm 21 9 Young gastrula ectoderm 10 7 Late gastrula neural ectoderm and chorda-mesoderm 3 16 (Briggs & King, 1952) (King & Briggs, 1954a) (King & Briggs, 1954a) Note: No data on ploidy were included with the most recent and convincing evidence of nuclear differentiation in late gastrula mid-gut cells reported by Briggs and King (1955). It is well known that, so long as complete sets of chromosomes are duplicated, a moderate increase in ploidy is compatible with normal development (see review by Fankhauser, 1955). The results of Briggs and King have shown that polyploidy in "nuclear transplant" embryos bear this out in that some of their best and most long-lived cases were polyploids. However, when chromo- some duplication involves only part of the chromosome comple- ment (aneuploidy) viability is greatly impaired and differentia- tion is usually abnormal (see reviews by Fankhauser, 1945, 1952, 1955). The latter fact should be considered along with the evi- dence from chromosome counts on "nuclear transplant embryos" of Triton which revealed a high incidence of hyperdiploids and diploid-hyperdiploid mosaics (Lehman, 1955). This alone could be a significant factor contributing to the generally poor develop- ment that was obtained with Triton eggs. The technique for deter- mining ploidy used by Briggs and King ( namely, nuclear size and nucleolar number) cannot yield precise information concerning degrees of aneuploidy. Considerable caution should be exercised in deducing positive evidence for nuclear differentiation from arrested or atypical "nuclear transplant embryos" unless there is a cytological basis for demonstrating euploidy and/or a consistent syndrome in the anomaly obtained with a given type of detennined donor cell nucleus. The latter condition has been satisfactorily met in the H. E. LEHMAN 221 case of mid-gut nuclei from late gastrulae of R. pipiens (King and Briggs, 1955), and this example stands as the only positive evidence of nuclear differentiation yet obtained from nuclear injection experiments. As such, it represents a real advance in the understanding of this problem. However, even this example does not express its nuclear specialization in cytoplasmic responses that are limited to the tissue-specific determination of the in- jected mid-gut donor cells. It is therefore apparent that nuclear and cytoplasmic determinations do not proceed at the same pace since it is well known from the work of Holtfreter ( 1938a,b ) that as early as the late blastula stage the endoderm consists of an almost rigid mosaic of organ-specific irreversibly determined cells. The nucleus of a determined cell may well remain in a labile state of differentiation and/or retain a wider range of histogenic potentialities than the cytoplasm surrounding it. An answer to questions concerning the possibility of tissue-specific nuclear specialization and labile versus irreversible nuclear determination must await additional information from transplantations of more advanced nuclei, preferably from cells showing histological or biochemical differentiation as well as detemiination. It is prob- ably not until quantitative and qualitative differences in the macromolecular composition of the embryonic tissues is distinct that one should expect to find nuclei restricted to tissue-specific functions. Conclusion Evidence from biochemical, cytological, and nuclear transplan- tation studies has been reviewed which indicates that in certain instances at least tissue-specific differences can be demonstrated in the nuclei of histologically determined cells within embryos and adult organisms. These examples stand at present as excep- tions to the generally held concept of quantitative and qualitative nuclear uniformity in somatic cells, a concept which to a large measure is based on evidence obtained from the demonstration of nuclear totipotency in early cleavage cells and regenerating tissue. Nuclear transplantation studies suggest that embryonic nuclei are totipotent until gastrulation and may retain the capacity 222 NUCLEAR TRANSPLANTATION for participating in total development even after the cells contain- ing them are histologically determined. However, with advancing age and progressive differentiation there is an indication that embryonic nuclei become restricted in their developmental ca- pacities. These data would indicate that nuclear determination and differentiation, if they do indeed regularly occur during histogenesis, do not take place concurrently with the initial de- termination of the cell as a whole. One is led to suspect that determination is gradually imposed on the nucleus by its sur- rounding cytoplasm. There is no information available at present to clarify the question of labile versus iireversible nuclear differ- entiation; nor is it possible in the light of current knowledge to decide whether or not cytoplasmic differentiation accompanied by nuclear differentiation is a general or an exceptional aspect of histogenesis. In concluding this summary of evidences for nuclear differen- tiation it is safe to say that even the most guarded and conserva- tive evaluation must admit the possibility of progressive nuclear differentiation in development. Stern ( 1955 ) has pointed out that this hypothesis is not required to account for local developmental differences, but the possibiHty is compatible with the general array of facts provided by genetics, physiology, and embryology. Additional examples of restriction in nuclear function correlated with cytoplasmic differentiation are needed from older tissues and other species before it will be possible to say with any degree of certainty that nuclear differentiation does indeed characterize histogenesis generally. If this primary question is answered af- firmatively by new data, it will be appropriate to consider sec- ondary questions beyond the scope of this paper relative to the manner in which detemiined cytoplasm "induces" nuclear change, as well as the nature of the reciprocal influence of the nucleus in maintaining cytoplasmic function (for theories already ad- vanced relating to these questions, see reviews on: auto-anti- bodies, Tyler, 1946; Weiss, 1950, 1955; Ebert, 1954; Woerdeman, 1955; plusmagenes, Mather, 1948; Schultz, 1950, Sonneborn, 1951; Stern, 1955; enzyme adaptation, Monod, 1947; Spiegehnan, 1948; Stanier, 1954). With regard to these latter questions one of H. E. 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The spermatogenesis of Taxomyia taxi. }. Morphol., 80, 1-24. White, M. J. D. 1948. The cytology of the Cecidomyidae (Diptera). IV. The salivary-gland chromosomes of several species. /. Morphol., 82, 53-80. Wilson, E. B. 1925. The Cell in Development and Heredity. The Mac- millan Company, New York. Woerdeman, M. W. 1955. Immunobiological approach to some prob- lems of induction and differentiation. Biological Specificity and Growth (12th Growth Symposium). Princeton Univ. Press, Prince- ton, N. J. Pages 33-54. MORPHOGENESIS AND METABOLISM OF GASTRULA-ARRESTED EMBRYOS IN THE HYBRID Rana pipiens ? % Rana sylvatica ^ JOHN R. GREGG: zoology department, COLUMBIA UNIVERSITY, NEW YORK The purpose of the account following is to provide a fairly critical and detailed summary of some recent descriptive and experimental work on embryos belonging to the hybrid Rana pipiens 5 x Rana sylvatica S . Embryos of this type become gas- trula-arrested; that is to say, when they are of gastrula age the morphogenetic movements exhibited by their tissues are not correlated in the normal (R. pipiens) pattern, and to casual ob- servation they appear to live out their lives in a state no more developmentally advanced than that of very young R. pipiens gastrula. The fact that such embryos are hybrids, each developing from a zygote formed by the union of an ovum from a member of R. pipiens with a spermatozoon from a member of R. sylvatica, is not yet of much explanatory value in accounting for their defec- tions as developmental systems. Embryos in the two parental species are morphologically similar to a very marked degree. They are of about the same size, they are telolecithal to about the same moderate degree, they cleave totally and in the same pattern, their cells are believed to contain the same number of chromo- somes ( N = 13 ) , and, in general, the sequences of developmental transformations they exhibit are alike to such an extent that Shumway's (1940) normal table for R. pipiens and that of Pol- 231 232 GASTRULA-ARRESTED EMBRYOS Stage Number Age -Hours at Ib'C 0 UNFERTILIZED Stage Number Age -Hours at I6X 7.5 32-CELL Stage Number Age- Hours AT ISX 13 50 NEURAL PLATP 6 14- 62 GRAY CRESCENT MID-CLEAVAGE NEURAL FOLDS 3.5 4.5 5.1 TWO- CELL FOUR- CELL 9 21 10 II 6.5 EIGHT- CELL SIXTEEN-CELL LATE CLEAVAGE 26 34 DORSAL LIP MID-OASTRULA 42 LATE GASTRULA 61 ROTATION 12 64 NEURAL TUBE TAIL BUD Fig. 1. Normal stages in the development of embryos in the species Rana pipiens. (From Shumway, 1940.) J. R. GREGG 233 Stage Numbeir 6 Age: in Hours at 18° Centigrade 96 Length in Millimeters 4 MUSCULAR RESPONSE. 9 16 HEART BEAT 20 140 gill circulation MATCHING 21 I6E MOUTH OPEN CORNE.A TRANSPARENT 22 192 6 TAIL FIN CIRCULATION Fig. 1. {Continued.) 234 GASTRULA-ARRESTED EMBRYOS ST NQ 18* EXTERNAL FORM ST NQ AGE HRS 18* EXTERNAL FORM ST^ NO HRS 18* EXTERNAL FORM 0 13 36 2-5 3+ 4-5 5f 12 16 10 19 24 12 28 14 40 45 50 17 58 Fig. 2. Normal stages in the development of embryos in the species Rana sylvatica. (From PoUister and Moore, 1937.) T. R. GREGG 235 ST. NO. AGE HRS 18° EXTERNAL FORM 18 65 MUSCULAR MOVEMENT 19 73 HEART BEAT 20 90 GILL CIRCULATION SWIMMING- HATCHING . Fig. 2. (Continued.) 236 GASTRULA-ARRESTED EMBRYOS lister and Moore (1937) for R. sylvatica are usable interchange- ably (Figs. 1 and 2). Of course, they are not alike in all respects. For example, their blastopores are shaped differently, and as larvae they are lordotic to different degrees. Under precisely sim- ilar environmental conditions, furthermore, they may undergo corresponding changes at different rates. A R. pipiens zygote, for instance, will develop into an early gastrula in about 26 hours at 18° C, whereas a R. sylvatica zygote will do the same thing in only 19 hours. Also, the temperature ranges within which they develop normally are not the same (Moore, 1939): R. pipiens embryos tolerate any temperature between 6° C and 28° C; R. sylvatica embryos any temperature between 2° C and 24° C. But, generally speaking, our knowledge of the phenotypes of members of the parental species does not suggest explicit hypoth- eses about their genotypes which are presently useful in explain- ing why hybrids develop improperly. The basis of present ideas about these hybrids is Moore's care- ful descriptive study ( 1946 ) together with the experimental work stimulated by it. The latter includes Moore's own analyses of the capacities of hybrid tissues to induce secondary embryonic struc- tures in normal embryos ( 1948 ) and to respond to normal em- bryonic inductors (1947), but the subjects of these particular papers fall outside our scope. In addition, Moore (1955) has published a general review of recent work on anuran hybridiza- tion, in one section of which the interested reader will find briefly summarized most of the findings that will now be presented in greater detail. Morphogenesis of Hybrid Embryos General The following statements are intended to characterize the chief, easily observable characteristics of developing hybrids.* 1. Hybrid embryos cleave and blastulate in the same pattern as * By "hybrid" or "hybrid embryo" we shall mean any embryo whose fe- male parent is a member of R. pipiens and whose male parent is a member of R. sylvatica. J. R. GREGG 237 and synchronously with control embryos.* Small differences be- tween advanced blastulae, however, might be difficult to detect. 2. Hybrid embryos do not begin to gastrulate until controls are in Shumway Stage 10+-11~. We shall express this by saying that hybrids begin to gastrulate only when they are in stage H10+-H11 .f At 18° C this represents a developmental delay of about 3 to 4 hours; but, in appearance, hybrids at this stage are exactly like controls in Stage 10. 3. Once formed, the blastopore of a hybrid embryo does not enlarge appreciably. (Some rare exceptions to this rule are de- scribed in Moore's (1946) paper.) This should not be read as stating that all gastrular movements cease at this stage, for this is not true, as we shall see. 4. When hybrids are in Stage H17 they develop functional external cilia, which rotate them slowly within their tighter-than- normal vitelline membranes. Control embryos develop cilia when they are in Stage 15. 5. By the time they are in Stage H20-H21, hybrid embryos have swollen until their diameters are some 20% greater than their diameters at Stage HIO, and their animal hemispheres have become curiously pitted. Possible mechanisms of swelling were discussed by Moore and also by Gregg ( 1948 ) , but none of their suggestions (all of which postulated hypemormal intrablas- tocoelic hydrostatic pressure ) has yet been put to test. 6. When hybrid embryos are in Stage H20-H21, their blas- tocoel roofs usually rupture, possibly as the result of intolerable internal pressure, and they shrink to their original sizes, approxi- mately. 7. Most hybrids die by progressive cytolysis when they are in Stage H21 (about 160 hours after fertilization, at 18° C). It * We shall use "control" or "control embryo" in accordance with the rule that re is a control for y provided that the following conditions are satisfied: (i) x is a member of R. pipiens; (ii) y is a hybrid; (iii) x and y have the same female parent, and have developed under similar environ- mental conditions for the same length of time. f In general, we shall say that hybrids are in Stage H^ if their controls are in Shumway Stage s. 238 GASTRULA-ARRESTED EMBRYOS should be noted that not all gastrula-arrested anuran hybrids survive this long. Embryos belonging to the hybrid Ranu sylva- tica 2 X Rana pipiens S , for example, cytolyze relatively soon after the onset of gastrulation, when their controls are in Stage 13. Therefore, hybrids of the sort with which we are concerned in this report are exceptionally favorable experimental material. a n o O N a. M M hJ t-5 M E-i >J g CO fe K B O a w^ 0 Wo 80 120 160 200 HOURS DEVELOPMENT 18®C Fig. 3. Development of embryos in the hybrid Rana pipiens 9 x Rana stjlvatica $. (Reconstructed from data of Moore, 1946.) Figure 3 is a schematic representation of the developmental events just outlined. Morphogenetic Movements Unfortunately, we are not in possession of an adequate gen- eral theory of amphibian gastrulation in terms of which we can explain hybrid developmental patterns. Nor, for that matter, do we have precise ways even of describing various tissue move- ments that occur when embryos gastrulate. Nevertheless, gas- trulating amphibians all exhibit movements belonging to some roughly identifiable types. The classification following is useful for our present purpose. A more exhaustive one is given by Nel- son (1953). J. R. GREGG 239 Dorsal Lip Fonnation. The first clearly visible external sign that an embryo is beginning to gastrulate is the appearance of a small pigmented streak to one side of the vegetal hemisphere. If median sagittal sections are made of an embryo in this stage, it is easy to see that the pigmented streak consists of the outermost constricted ends of cells attached to the surface coat and bulging at their free ends inward and upward toward the blastocoel. Vogt (1929) has some exceptionally clear figures of such cells, which we shall call "flask cells." The pigmented streak thus con- stituted indents in the surface of the embryo, the cells just dorsal FLASK-CELL FORMATION ARCHENTERON FORMATION EPIBOLY Fig. 4. Morphogenetic movements in amphibian gastrulation. to it forming the early dorsal lip. Figure 4 contains a highly schematized representation of this process. Since each hybrid embryo finally, if belatedly, develops a normal-looking early dorsal lip, it is not surprising that Moore found no irregularities of histological structure when he examined median sagittal sections of Stage H10+ hybrid embryos. Flask cells were present in normal fashion, attached at their outer ends to the indented and pigmented area adjacent to the forming dorsal lip. It is unlikely, therefore, that incapacity to execute the correct preliminaries to early dorsal lip formation is instrumental in bringing hybrid gastrulae to a developmental impasse. Ex- periments with explant systems reinforce this view. Holtfreter 240 GASTRULA-ARRESTED EMBRYOS ( 1944 ) showed that a bit of nomial head endoderm ( rider ) suit- ably mounted upon the surface of a yolk endoderm substratum ( base ) would in due course embed itself in the latter; and he was able to demonstrate that cells of the rider transformed into flask cells as they infiltrated the cells of the base. Applying this method to hybrids, Gregg and Klein (1955) found that presumptive head endoderm riders from hybrid embryos embed in yolk en- doderm bases with as much dispatch as those from normal em- bryos. Their systems were not studied histologically, but as far as 180»' 90» 0» ■ / PIPIENS HXBRIO 20 "to 60 80 HOURS DEVELOPMENT l8»C 100 Fig. 5. The angle of invagination in developing Rana pipiens and hybrid embryos. (Reconstructed from data of Moore, 1946.) their evidence goes, it sustains the belief that hybrid dorsal lip endoderm is capable of normal morphogenetic behavior. Archenteron Formation and Enlargement. Continued invagi- nation of the pigmented streak just vegetal to the earliest dorsal lip soon results in the formation of a cavity, the archenteron, which in normal amphibian embryos enlarges continuously while gastrulation proceeds (Fig. 4). Moore has devised a method for obtaining a measure of the rate and extent of archenteron forma- tion. It consists in obtaining the value of the angle subtended by two lines in the median sagittal plane, both originating at a point on the center of the blastocoel floor, and tangent respectively J. R. GREGG 241 to the innermost tip of the archenteron and the edge of the dorsal lip (Fig. 5). It is easily seen that elongation of the archenteron will be accompanied by increases in the value of this angle, which Moore calls the angle of invagination. Figure 5 shows that the value of the invagination-angle of an R. pipiens embryo increases sharply throughout the period during which the embryo is gastrulating; for a hybrid, this value in- creases almost as rapidly at first, but thereafter remains nearly constant. Thus, in hybrid embryos, formation of the archenteron 180» " ca il+o» ' 20 M3 60 80 HOURS DEVELOPMENT iSfC 100 Fig. 6. The angle of epiboly in developing Rana pipiens and hybrid embryos. (Reconstructed from data of Moore, 1946.) gets under way, but is brought to a halt when it is only one- quarter complete. Epiboly. As an amphibian embryo gastrulates, its stock of internal tissues is increased by draughts upon that of its external endoderm and mesoderm. The inward transfer is made possible in several ways, but at least partly by epiboly, a thoroughgoing meridional extension of presumptive ectoderm and mesoderm (Fig. 4). Mesodermal tissues are turned inward over the blas- toporal lip; ectodermal ones will eventually constitute the entire external surface. 242 GASTRULA-ARRESTED EMBRYOS Moore has attempted to provide us with a measure of epiboly. He exploited the fact that the dorsal lip of an embryo stands in a determinate geometrical relationship to the plane of its blastocoel floor. This relationship can be expressed by citing the value of an angle which Moore calls the angle of epiholy: it is the angle sub- tended by two lines, both in the median sagittal plane and both originating from the center of the blastocoel floor, one being perpendicular to the plane of the blastocoel floor and the other tangent to the edge of the dorsal lip (Fig. 6). The value of this angle for R. pipiens embryos just commencing to gastrulate is about 106°, and by the time they are in middle Stage 12 its value has increased to 176° at a steady rate. For hybrid embryos the initial value is 138°; with time, the values climb more slowly than normal, and at a decreasing rate, but they finally reach a level of about 168°. The problem now is to interpret those results. Moore believes that the rate and amount of change in this angle are measures of the rate and amount of epiboly. If this interpretation is correct, then Fig. 6 yields the following information: (a) epiboly occurs in hybrid embryos at a rate somewhat slower than normal but to almost the same final extent, and (b) the appearance of a dorsal lip in hybrid embryos is preceded by a considerable amount of epiboly. In connection with (b), it should be remem- bered that the dorsal lips of hybrid embryos do not appear until the latter are in Stage H10+-H11~. However, it may be that Moore's interpretation is correct only by chance, for although it is true that the progress of epiboly will increase the value of the epiboly angle, it is by no means true that an increase in the value of this angle can always be construed as indicating that epiboly has progressed. Any change, whether or not it is accompanied by epiboly, that results in tilting the blastocoel floor away from the dorsal lip will entail an increase in the value of the epiboly angle. One such change that needs to be considered is the stream- ing anteriorly along the archenteron floor of cells originally from the vegetal pole area, for this normally occurring movement might be expected to alter the plane of the blastocoel floor in such a way as to cause an increase in the value of the angle of J. R. GREGG 243 epiboly. Should this actually happen, this value would be a measure not of epiboly alone but of a complex of morphogenetic movements having more than one component. It may not happen in hybrid embryos, for Moore says that "the tip of the archen- teron maintains essentially the same position relative to the blas- tocoel floor throughout. . . ." the length of time required for controls to gastrulate; but it may happen in normal gastrulae. But Moore's interpretation of change in the value of the angle of epiboly is supported by considerable evidence. First, on his interpretation, the final value of this angle for normal embryos is that which would have been predicted from the knowledge that, when they have finished gastrulating, the descendants of their animal pole cells lie about 180° from their blastopores. Sec- ond, one would expect epiboly to wrinkle the animal hemispheres of hybrid embryos ( since little slack is taken up by invagination ) , and this is exactly what happens. Third, there is evidence from experiments with explant systems that hybrid ectoderm is capa- ble of normal epibolic spreading. Holtfreter ( 1944 ) showed that a piece of ventral ectoderm from an amphibian gastrula, when suitably mounted on an endodermal substratum, will spread in all directions over the latter. Gregg and Klein (1955) applied this technique to hybrid embryos and were able to show that hybrid ventral ectoderm will spread normally upon an endodermal base. All this is consistent with Moore's interpretation and is just what would be expected from his results. From our discussion, it is evident that appropriate ectodermal tissues of hybrid gastrulae are capable of epiboly and, in all like- lihood, exhibit it in situ. Thus we must search elsewhere for fac- tors contributory to hybrid juvenile delinquency. Convergent Extension. As they stream toward the blastopore, the presumptive notochordal cells of gastrulating amphibians converge sharply toward the median sagittal plane, and after in- vaginating they migrate cephalad parallel to or convergent to this plane. At the close of gastrulation, therefore, the notochordal cells form a narrow elongated rod of tissue lying centered in the median sagittal plane (Fig. 4). By studying models, Holtfreter (1944) showed that normal 244 GASTRULA-ARRESTED EMBRYOS amphibian presumptive notochord is self-differentiating with re- spect to the morphogenetic movement of convergent extension, for notochordal explants from a young gastrula will elongate in the proper axis within a reasonable time, and so will similar ex- plants mounted suitably upon an endodermal substrate. On the other hand, hybrid presumptive notochord explants l)ehave abnormally ( Gregg and Klein, 1955 ) ; instead of elongat- ing, they merely round up into balls of tissue and remain that way as long as they are cultured. Now, if autonomous convergent extension of notochordal tissue is a necessary concomitant of nor- mal gastrulation, its failure to occur constitutes a sufiBcient con- dition for the nonoccurrence of normal gastrulation. Thus, in its failure, we have a partial explanation for the peculiar course of post-blastula hybrid development. But we also have a generation of further puzzles. It will be recalled that the initial value of the epiboly angle for hybrid embryos (138°) is much greater than that for R. pipiens embryos (106°). Moore, as we have seen, re- gards this as evidence that considerable epiboly precedes dorsal lip formation in hybrid embryos. But that epiboly is one compo- nent (extension) of convergent extension. If the latter is not au- tonomous in hybrid embryos, then the question arises, how is the initial pre-invagination epiboly accomplished? At present, there is no clear-cut answer. It is evident, from Moore's description, that a hybrid invagi- nates enough to move its presumptive head endoderm in under- neath its presumptive chordamesoderm. When this relationship is mimicked in vitro by mounting a bit of hybrid chordameso- derm on an endodermal substratum, the chordamesoderm not only fails to elongate normally, it also spreads upon the substra- tum, in all directions, like ectoderm ( Gregg and Klein, 1955 ) . If this sort of thing happens in situ, then it is easy to see why the notochordal convergence necessary for normal gastrulation does not occur, and why invagination is brought to a sudden halt. Thus, by considering the behavior of explant systems in rela- tion to known features of hybrid development, we can construct a fragmentary explanation in morphological terms for some of the peculiarities of that development. We turn now to consider some J. R. GREGG 245 attempts to analyze hybrid morphogenesis from a physiological or biochemical point of view. Metabolis7n of Hybrid Embryos Respiration Oxygen Uptake. Barth (1946) has made a special study to compare the respiratory rates of hybrid and control embryos. His results are summarized in Fig. 7. The rate at which R. pipiens STAOSS 1.0 5 10 19 14 16 17 18 19 2( / .8 •/ .6 y /^NORMAL A X' ^ .2 »-*-^ ^ ^^^ "~ HYBRID 15 30 46 60 HOURS 76 90 106 120 Fig. 7. Respiration (cu mm Oj/hour embryo ) of developing B.ana pipiens and hybrid embryos. (From Barth, 1946.) embryos utilize environmental oxygen increases with time, roughly in exponential fashion. Hybrid embryos at first respire oxygen at the same rate as R. pipiens control embryos, but when they are late blastulae their respiratory rate becomes constant and remains so until they are in Stage H18, when a small, abrupt increase in respiratory rate (not clearly associated with any mor- phological change, external cilia, for example, begin to function at about Stage 17, according to Moore) initiates a very slow ex- 246 GASTRULA-ARRESTED EMBRYOS ponential rise (possibly associated with the onset of cytolysis) that continues to Stage H20, beyond which respiratory rates have not been measured. It is easy to see that, from late Stage H9 on, hybrid embryos are characterized by increasingly subnormal re- spiratory rates. Respiratory depression seems to be a feature of all parts of hybrid gastrulae. Sze (1953) has compared the respiration of four different regions of the animal hemisphere of hybrid em- bryos in early Stage Hll with that of corresponding regions of control embryos. From the graph of his results (Fig. 8), it can ^ ^ 2 W-D Regions Fig. 8. Respiration of parts of Rami pipiens and hyl^rid gastrulae. Hol- low circles, Rana pipiens; solid circles, hybrid. (Adapted from Sze, 1953.) be seen that the rate of respiration of hybrid tissues from em- bryos in the stage mentioned is everywhere about half that of the corresponding R. pipiens tissues. The difference is a little greater than might be expected if Earth's results on whole embryos are taken as standard, but, pending evidence to the contrary, Sze's results must be interpreted as warranting the supposition that there is no tissue-localized block to respiration in hybrid em- bryos. Respiratory Quotient. In the work cited above, Earth also J. R. GREGG 247 compared the respiiatory quotients (R.Q. = CO2 produced per O2 consumed) of hybrid and R. pipiens embryos (Fig. 9). The respiratory quotients of hybrid embryos in all stages between Stage HI and Stage H12 are not significantly different from those of R. pipiens control embryos. Beginning at Stage H12, however, the respiratory quotients of hybrid embryos become increasingly larger than those of controls. Aging hybrid embryos are thus characterized by hypernormal CO2 production, relative to their oxygen consumption. We have no knowledge at present about the R.Q.'s associated with various parts of hybrid gastrulae. Barth notes that his results are consistent with the view that up to Stage 0.95 '0.85 0.75 + PIPIENS 2"+ lf8 HOURS DEVELOPMENT l8»C 72 Fig. 9. Respiratory quotients of developing Rami pipiens and hybrid embryos, (Reconstructed from data of Barth, 1946.) H12, hybrid embryos are oxidizing substrates of the same kind as control embryos, and that not until thereafter do they oxidize substrates of different kinds. Therefore, to the extent that R.Q. measurements warrant such statements, we are deprived of sup- port for the hypothesis that hybrids fail to gastrulate because they do not oxidize substrates providing the normal types of nec- essary metabolic intermediates. But because of technical difficul- ties involved in the determination of respiratory quotients, espe- cially on pre-gastrula embryos, Barth urges that the absolute values of the R.Q.'s obtained should not be construed as evidence for any hypothesis stating what substrates embryos are oxidizing. 248 GASTRULA-ARRESTED EMBRYOS Evidence relevant to this last point is meager, indeed; but there is at least some. Utilization of Endogenous Carbohydrate In 1948, Gregg made a comparative study of carbohydrate uti- lization by developing hybrid and R. pipiens embryos. Normal €—(^-^'^-€)C9-€>-d^C»—OC ■•^"O'lP I I I I I I I I III I I I I 7 e 10 12 14 17 18 19 DEVELOPMENTAL STAGE 20 21 Fig. 10. Endogenous carbohydrate of developing Rana pipiens em- bryos. Upper curve, total carbohydrate; middle curve, glycogen; lower curve, free carbohydrate. (From Gregg, 1948.) embryos (Fig. 10) are characterized by the fact that they begin to oxidize their carbohydrate store not later than the onset of gastrulation and continue thereafter to do so to such an extent that, by the time they are hatched larvae, they have little more J. R. GREGG 249 than half of it left; while hybrid embryos (Fig. 11) seem to uti- lize their carbohydrates at a slow, almost constant rate through- out their development from Stage HI to Stage H21. The fact that embryos of both types use their glycogen and their total carbo- 110 100 90- >- 80- CQ 1KB 70 (O 60 2 0 50 q: o z 1 40 30 20 10- €8- 08 HYBRIDS ■o^- -oan- .«eoo- • ec-Cte- I I I I I I I I III I I I I I 7 8 10 12 14 17 18 19 20 DEVELOPMENTAL STAGE OF CONTROLS 21 Fig. 11. Endogenous carbohydrate of developing hybrid embryos. Up- per curve, total carbohydrate; middle curve, glycogen; lower curve, free carbohydrate. (From Gregg, 1948.) hydrate at similar rates suggests two things: (a) that glycogen is the only stored carbohydrate oxidized in any great amount throughout embryonic development, and (b) that there is no conversion of glycogen into any other stored carbohydrate — a 250 GASTRULA-ARRESTED EMBRYOS conclusion supported also by the absence from both types of em- bryo of significant amounts of free carbohydrate at any stage in embryogenesis. Glycogen, indeed, seems to be the chief energy source drawn upon by young frog embryos, for we know (Barth and Barth, 1954) that relatively little protein or fat is burned by normal embryos until late in development. Protein and fat me- tabolism of hybrid embryos has not yet been studied. It is not known whether the parts of hybrid gastrulae utilize glycogen at different rates. The work of Jaeger (1945) suggests that they do not, for she found that dorsal tissues of Stage HIO and Stage H12 hybrid embryos did not differ in their glycogen contents, and neither did analogous ventral tissues. It seems prob- able that there is a general depression of glycolysis common to all hybrid cells, corresponding to the general lowering of the respir- atory rate discovered by Sze. Lactic Acid Production In his paper on the respii-atory metabolism of hybrid embryos, Barth (1946) also reported some measurements of their aerobic and anaerobic lactic acid production. Under strictly aerobic con- ditions, neither R. pipiens nor hybrid embryos produce appre- ciable amounts of lactic acid; although embryos allowed to sit 60 • o 20 " 25 50 75 HOURS DEVELOPMENT l8»C Fig. 12. Anaerobic lactic acid production of Rana pipiens and hybrid embryos. (Reconstructed from data of Barth, 1946.) J. R. GREGG 251 close together in a dish of aerated culture medium may excrete considerable amounts of lactic acid, possibly because crowding limits the rate at which environmental oxygen is supplied to them. In nitrogen, both sorts of embryos accumulate large amounts of lactic acid, hybrids to a lesser extent than R. pipiens controls (Fig. 12). Thus, under anaerobic conditions, hybrid embryos produce subnormal amounts of lactic acid. Localization of Metabolic Blocks in Hybrids From the foregoing sections, it is clear that hybrid gastrulae exhibit subnormal oxygen uptakes, subnormal endogenous carbo- hydrate utilization, and subnoniial anaerobic lactic acid produc- tion. In discussing these results we shall make the following assumptions about the intermediary carbohydrate metabolism of hybrid gastrulae: 1. Glycogen, in the presence of ATP (adenosine triphosphate) and inorganic phosphate, is enzymatically oxidized to pyruvic acid, with the production of hydrogen ions and ATP. 2. Under anaerobic conditions, pyruvic acid is enzymatically reduced by hydrogen ions and is converted to lactic acid, 3, Under aerobic conditions, pyruvic acid is enzymatically oxi- dized to carbon dioxide, with the production of ATP and hydro- gen ions. 4, Under aerobic conditions, hydrogen ions enzymatically re- duce respiratory oxygen, with the formation of water. The exact mechanisms by which these events occur will not be critical for the present discussion, but there is no reason to sup- pose that they are unorthodox. Cohen (1954) has recently pre- sented convincing evidence that glycolysis in R. pipiens embryos is of the usual Embden-Meyerhof type, which, in the absence of oxygen, terminates in lactic acid. Hydrogen transport and the oxidative degradation of pyruvate have not been systematically studied in R. pipiens or other amphibian embryos, but there is a comparatively large scattered literature whose review is beyond the scope of this contribution but which is consistent with the hypothesis that hydiogen transport in amphibian embryos is car- ried out in the classic Warburg-Keilin manner and that pyruvate 252 GASTRULA-ARRESTED EMBRYOS is oxidized by a system of intermediate steps of the type usually called a Kiebs cycle. Let us imagine, then, that events of the types diagrammed in Fig. 13 occur both in R. pipiens and in hybrid embryos. Further- more, for simplicity, let us make the possibly counterfactual as- sumption that glycogen is the sole endogenous energy source of pipiens and hybrid gastrulae, and on this basis let us try to local- ize reactions whose subnormal rates may account for the bio- chemical deficiencies just reviewed. From Fig. 13 it will be seen that there are four major places, 1, 2, 3, and 4, at which partial blocks may or may not occur. If S; is a blocked reaction at place i and Si is a nonblocked reaction at place i, then we shall have 2^ = 16 possible situations that may be the state of affairs in hybrid gastrulae : (1) bi bo bg hi (9) Si S2 S3 S4 (2) Si S2 S3 S4 (10) Si S2 S3 S4 (3) Si S2 S3 S4 (11) Si S2 S3 S4 (4) Si S2 S3 S4 (12) Si S2 S3 S4 (5) Si S2 S3 S4 (13) Si S2 S3 S4 (6) Si S2 S3 S4 (14) Si S2 S3 S4 (7) Si S2 S3 S4 (15) Si S2 S3 S4 (8) Si S2 S3 S4 (IG) Si S2 S3 S4 Of these possibilities, only five are clearly disconfirmed by the available data: (4) and (8), because if they were true, then the anaerobic lactic acid production of hybrid gastrulae ought to be, but is not, rate-normal; (12), because if it were true, hybrid an- aerobic lactic acid production ought to be, but is not, rate-normal, whereas aerobic lactic acid production ought to proceed at an ab- normally high rate, but does not; (14), because if it were true, then the oxygen consumption of hybrid gastrulae ought to be, but is not, normal in rate; and (16), because differences between hy- brid and normal embryos in respect to O2 uptake, lactate produc- tion and glycogen utilization should not be, but are, observed. Several other possibilities— (1), (2), (3), (5), (7), (9), (10), and (11) — are neither confirmed nor disconfirmed by existing J. R. GREGG 253 data. To evaluate these cases we shall need additional informa- tion. In particular we shall have to know which of the blocked reactions, if any, become rate-limiting for the entire system, and the degrees to which oxygen and pyruvic acid can compete suc- cessfully for hydrogen ions under each of the postulated condi- tions. Consider case (1), for example, where there is imagined a partial block at all four sites. Under these conditions we should GLYCOGEN ATP + PYRUVIC CO, + ATP Fig. 13. Carbohydrate metabolism schema. expect to find, and do find, that hybrids respire and utilize glyco- gen at a subnormal rate and produce lactic acid anaewbically at a subnormal rate. But we cannot predict how hybrid and R. pipiens gastrulae will compare in respect to aerobic lactic acid produc- tion, for this would depend, among other things, upon knowing (as we do not) which of Si, S2, S3, and S4 are rate-limiting so that we would know which intermediates accumulate, and how well under these restrictions pyruvate and oxygen compete for hydro- gen ions. Therefore, the fact that we actually do find the hybrids aerobically producing lactic acid at a normal, negligible rate is 254 GASTRULA-ARRESTED EMBRYOS neither for nor against this postulate. Analogous considerations can be advanced for the other indeterminate cases. The best we can say is that all these cases remain consistent with the data now on hand. The remaining cases — (6), (13), and (15) — are supported in- discriminately by information that we now have. That is to say, if any of these is the state of affairs in hybrid embryos, then we should expect the hybrids to exhibit subnormal rates of oxygen uptake, glycogen utilization, and anaerobic lactate production, and to produce little or no aerobic lactic acid, and this, in fact, is just what they do. In summary, there are eleven mutually exclusive general pos- sibilities for blocks in hybrid carbohydrate metabolism which must be investigated further before we can decide which of them, if any, actually occur. And this leaves entirely out of considera- tion all other sorts of biochemical deficiencies not directly in- volved with the schema just discussed, not to mention the details of the system that schema represents. Phosphorus Metabolism Hybrid embryos, clearly, are somewhat deficient energy pro- ducers. For this reason, they ought perhaps to find it difficult to maintain their stores of esterified phosphorus, especially when put under stress by being made to develop under conditions where energy production is even further curtailed. In 1947, Barth and Jaeger made some measurements designed to answer the question whether hybrids are as well able as R. pipiens embryos to meet the stresses imposed by anaerobiosis. Their results (Table I ) are summarized below : 1. Aerobic hybrid embryos have been shown not to exhibit a probably significant difference from R. pipiens controls in respect of inorganic phosphorus (I) or ATP-ADP phosphorus (E?). 2. Under anaerobiosis: (a) R. pipiens gastrulae exhibit a prob- ably significant elevation of I, but no significant change in E- is indicated; while (h) hybrid gastrulae exhibit a significant in- crease in I and also a probably significant decrease in Et. The J. R. GREGG 255 Table I. Phosphorus Metabolism of R. pijnens and Hybrid Embryos Aerobic Anaerobic Diff. P X ± s X ± s X ± s Total R. pipiens 43.0 ± 9.4 48.8 ± 4.7 5.8 ± 7.7 >0.05 Hybrid 38.7 ± 6.7 46.1 ± 6.3 7.4 ± 2.0 <0.01 Diff. 4.3 ± 1.7 2.7 ± 1.8 P <0.05 >0.05 I R. pipiens 19.3 ± 2.4 29.9 ± 4.4 10.6 ± 4.8 <0.05 Hybrid 17.3 ± 1.9 30.2 ± 1.0 12.9 ± 0.9 <0.01 Diff. 2.0 dh 3.4 0.3 ± 3.2 2.3 ± 5.6 P >0.05 >0.05 >0.05 E7 R. pipiens 23.7 ±8.3 18.9 ± 2.8 4.8 ± 7.4 >0.05 Hybrid 21.4 ±4.9 15.9 ± 5.3 5.5 ± 2.3 <0.05 Diff. 2.3 ± 4.2 3.0 ± 4.4 P >0.05 >0.05 Source: Reconstructed from Barth and Jaeger, 1947, Table 7, together with original data kindly supplied by these authors. Embryos were allowed to develop 10-22 hr. in aerated or nitrogenatcd me- dium, beginning at Stage 10, after which phosphorus fractions weie measured. Total This is essentially the total inorganic phosphorus in a trichloracetic acid extract after 7 min. hydrolysis in INHCl at 100° C. I This is essentially the total inorganic phosphorus in a trichloracetic acid extract before acid hydrolysis. E7 = Total — I, and is interpreted to be mostly inorganic phosphorus liber- ated by acid hydrolysis from ATP and ADP. Values are in micrograms per 100 embryos. s = standard deviation = ' "^ \n - 1 Probabilities of differences determined by t-tost for small samples, where _ X — m.o s/Vn Throughout, n = 4. hybrid increase in I is shown not to be significantly different from that of R. pipiens embryos, but is in excess of that which might be expected to occur as the result of the liberation of I from breakdown of E7. Thus, if inorganic phosphorus is a reliable indicator, anaerobic 256 GASTRULA-ARRESTED EMBRYOS hybrids do not expend esterified phosphorus more than R. pipiens controls. Since they do not expend enough E? anaerobically to account for the increase in I, it appears that they draw upon some as yet unidentified store of esterified phosphorus for the excess I. Whether R. pipiens embryos do also remains to be established. The evidence thus warrants the conclusion that hybrids meet relatively short anaerobic demands for energy about as well as R. pipiens controls, whatever the actual mechanism, but whether 3^ ^'^ (jjg. P/pg, dry wt.) X 10 hf w. D 1 2 3 4 V \jy Control 27 27 21 21 26 24 Hybrid 26 16 14 22 23 25 Fig. 14. Liberation of inorganic phosphate by acid breis of Rana pipiens and hybrid gastrula tissues. (Reconstructed from data of Mezger- Freed, 1953.) they preserve their entire store of phosphate bond energy as well as controls is open to further inquiry. Additional information about the phosphorus metabolism of hybrid embryos has been obtained by Mezger-Freed ( 1953 ) . This author measured the inorganic phosphorus liberated by acid breis of embryos in various stages of development and also by acid breis of explanted gastrula parts. In this respect, she discov- ered little that would distinguish hybrids from controls, except that breis of dorsal tissues from the animal hemispheres of hybrid gastrulae liberate much less inorganic phosphate than those of corresponding parts of controls ( Fig. 14 ) . She regards her results as evidence for the existence of a phosphatase catalyzing the lib- J. R. GREGG 257 eration of inorganic phosphorus from phosphoprotein, but other- wise their significance in relation to hybrid developmental failure is not clear, Deoxyribosenucleic Acid Synthesis It is plausible to suppose that hybrid embryos may fail to de- velop properly because they cannot synthesize substances neces- IZine in- Hours /OO HJQ 50. 1.0 ControLs:h.r>iv. a O c^ ■ 8 9 10 II IH i5 16 n 18 De-Yizlcypme'ntal Staae, of Controls ( Sfiumumij) Fig. 15. Deoxyribosenucleic acid in developing Rana pipiens and hy- brid embryos. (From Gregg and L0vtrup, 1955.) sary for continued normal morphogenesis. Unfortunately, we have practically no information about the synthetic capacities of hybrid embryos of the type under consideration. The sole pub- lished investigation of this question is that of Gregg and L0vtrup (1955), who claim to have shown that deoxyribosenucleic acid (DNA) is synthesized by hybrids at a normal rate until Stage 258 GASTRULA-ARRESTED EMBRYOS H14, after which no further synthesis occurs (Fig. 15). There- fore, according to them, the faikire of hybrids to gastrulate is not a consequence of subnormal synthesis of DNA. But it should be noted that the analytical method employed by Gregg and L0v- trup did not distinguish between free deoxyribosides and those incorporated in nucleic acids. For this reason, it may be best to regard their data as showing only that the total deoxyriboside content of hybrid embryos is not lower than that of controls until after Stage H14. Whether hybrid DNA synthesis is normal still remains to be demonstrated. In this connection, A. B. C. Moore is in possession of microspectrophotometric data (unpublished) which she regards as showing that as long as hybrid embryos are alive their nuclei each contains a normal amount of DNA. Summary The general picture of hybrid development emerging from the work we have reviewed may now be summarized and commented upon briefly. 1. At all stages precedent to Stage HIO, hybrid embryos exhibit no developmental abnormalities. 2. Hybrid embryos are tardy in forming a dorsal lip, but finally do so in the normal manner at late Stage HIO or early Stage Hll. Epiboly seems to begin at the normal time and progresses to al- most the normal final extent, but at a subnormal rate. Archenteron formation gets underway at a slightly lower than normal speed, but is brought to a standstill while it is only one-quarter com- plete. Chordamesodermal convergent extension does not occur in hybrids. Instead, the chordamesoderm spreads on the substratum provided it by invaginated head endoderm, thus effectively put- ting a stop to further mesodermal invagination. 3. Hybrid embryos, still looking like early gastrulae, cytolyze when they are in Stage H21-H22. 4. On the biochemical side, post Stage HIO hybrid embryos are characterized by subnormal rates of oxygen uptake, endogenous carbohydrate utilization, and anaerobic lactic acid production. At all stages up to Stage H12, however, their respiratory quotients are normal; only thereafter are they increasingly hypernormal. It J. R. GREGG 259 is not known whether, under the stress of anaerobiosis, hybrid gastrulae are less able than jR. pipiens controls to maintain their total stores of esterified phosphorus, but they do not appear to expend more phosphate bond energy under anaerobiosis than do R. pipiens controls. Their only known tissue-localized metabolic difference from normal embryos is the deficient rate at which acid breis of their dorsal tissues will liberate inorganic phosphorus. They probably synthesize deoxyribosenucleic acid at the normal rate until they are in Stage H14, after which there is a fixation of the steady-state level at which this compound is maintained. Unfortunately, it is difficult at the present time to relate the biochemical features of hybrid embryos to their developmental peculiarities. It is tempting to suppose that the lowered rates at which they liberate energy from glycolytic processes is causally connected with their delay in beginning to gastrulate, with the slowness with which epiboly proceeds, or even with the com- plete morphogenetic failme of mesoderm which so effectively disorganizes gastrular movements. For it is plausible, at least, to suppose that morphogenetic processes — tissue movements, cell- shape changes, and the rest — are endergonic processes, and that when an unimpaired supply of energy is not forthcoming such processes must necessarily slow down or fail altogether. But it is also possible that the morphogenetic peculiarities of hybrid em- bryos and their metabolic ones are jointly caused. We do not know what alterations of fine structure may occur in a hybrid as a re- sult of its unusual parentage. It is easy to imagine, however, that they are of such a kind that neither morphogenetic changes nor metabolic processes can occur normally, even in the presence of a nomial energetic coupling between the latter and the former. Clarification of these questions must await further thought and investigation. REFERENCES Earth, L. G. 1946. Studies on the metabolism of development. /. Expth Zool, 103, 463-86. 260 GASTRULA-ARRESTED EMBRYOS Barth, L. G., and L. J. Barth, 1954. The energetics of development. Columbia University Press, New York. Barth, L. G., and L. Jaeger. 1947. Phosphorylation in the frog's egg. Physiol. Zool, 20, 133-46. Cohen, A. I. 1954. Studies on glycolysis during the early development of the Rana pipiens embryo. Physiol. Zool, 27, 128-41. Gregg, J. R. 1948. Carbohydrate metabolism of normal and of hybrid amphibian embryos. /. Exptl. Zool., 109, 119-34. Gregg. J. R., and D. Klein. 1955. Morphogenetic movements of normal and gastrula-arrested hybrid amphibian tissues. Biol. Bull., 109, 265- 70. Gregg, J. R., and S. L0vtrup. 1955. Synthesis of desoxyribonucleic acid in lethal amphibian hybrids. Biol. Bull, 108, 29-34. Holtfreter, J. 1944. A study of the mechanics of gastrulation. Part II. /. Exptl Zool, 95, 171-212. Jaeger, L. 1945. Glycogen utilization by the amphibian gastrula in re- lation to invagination and induction. /. Cellular Comp. Physiol, 25, 97-120. Mezger-Freed, L. 1953. Phosphoprotein phosphatase activity in nor- mal, haploid and hybrid amphibian development. /. Cellular Comp. Physiol, 41, 493-518. Moore, J. A. 1939. Temperature tolerance and rates of development in the eggs of amphibia. Ecology, 20, 459-78. Moore, J. A. 1946. Studies in the development of frog hybrids. I. Em- bryonic development in the cross Rana pipiens 9 x Rana sylvatica $ . J. Exptl Zool, 101, 173-220. Moore, J. A. 1947. Studies in the development of frog hybrids. II. Competence of the gastrula ectoderm of Rana pipiens 9 x Rana sylvatica $ hybrids. /. Exptl. Zool, 105, 349-70. Moore, J. A. 1948. Studies in the development of frog hybrids. III. In- ductive ability of the dorsal lip region of Rana pipiens 9 x Rana sylvatica s hybrids. /. Exptl. Zool, 108, 127-54. Moore, J. A. 1955. Abnormal combinations of nuclear and cytoplasmic systems in frogs and toads. Advances in Genetics, 7, 139-82. Nelsen, O. E. 1953. Comparative embryology of the vertebrates. The Blakiston Company, New York. PolHster, A. W., and J. A. Moore, 1937. Tables for the normal develop- ment of Rana sylvatica. Anat. Record, 68, 489-96. Shumway, W. 1940. Normal stages in the development of Rana pipiens. Anat. Record, 78, 139-49. J. R. GREGG 261 Sze, L. C. 1953. Respiration of the parts of the hybrid gastrula Rana pipiens x Rana sylvatica. Science, 117, 479-80. Vogt, W. 1929. Gestaltungs analyse am Amphibienkeim mit orthcher Vitalfarbimg. II. Teil. Gastrulation und Mesodermbildung bei Uro- delen und Anuren. Wilhelm Roux Arch. Entwickliingsmech. Organ. 120, 384-706. SOME OBSERVATIONS ON CYTOPLASMIC PARTICLES IN EARLY ECHINODERM DEVELOPMENT JOHN R. SHAVER:* kerckhoff laboratories of biology, CALIFORNIA INSTITUTE OF TECHNOLOGY, PASADENA, CALIFORNIA An embryonic system could be defined within the framework of our present concepts of cellular metabolism as one which, in addition to the demands of growth and maintenance, has to meet the unique exigencies of differentiation (cf. recent discussions of Steinbach and Moog, 1955, and Boell, 1955 ) . Our understanding of the ways in which differentiated cells of metazoan organisms carry on such vital functions as energy transfer and protein syn- thesis has been enormously extended in recent years by studies on the structure and function of various kinds of cytoplasmic formed elements. It does not seem surprising, therefore, that numerous suggestions have come from embryologists concerning the role of these subcellular entities in embryonic differentiation (for recent reviews see Brachet, 1950, 1952; Gustafson, 1954). But the information about the interrelationships of cytoplasmic particles and other cell constituents in the life of the cell is still largely descriptive, especially as they concern the intact cell (cf. Porter, 1955 ) . To assign a dominant, if not a unique, place to one or another of the granule populations of protoplasm in the series of transformations leading to specific cell types necessitates an identification of visible structures with causality which still seems very far from being able to be made. * Special Research Fellow, National Cancer Institute, United States Pub- lic Health Service. Present address: Department of Zoology, Michigan State University, East Lansing, Michigan. 263 264 EARLY ECHINODERM DEVELOPMENT The purpose of this brief review is to attempt an examination of cmTent information concerning patterns and gradients of cyto- plasmic particles in echinodemi embryos and to assess some of the recent hypotheses concerning their fmictions in early differ- entiation. Cytoplasmic Particles and Initial Stages of Development As Tyler remarks ( 1955 ) : "An understanding of the factors that endow egg and sperm with the ability to unite and produce a new individual may be expected to depend largely on knowl- edge of the manner of formation of the gametes." An extensive literature exists describing the results of various cyto- and histo- chemical tests performed on cells in various stages of gametogen- esis (for a review of earlier literature, see Brachet, 1950). Al- though there is information on the origin of ribonucleoproteins in the gonial cells of several invertebrate species (Arvy, 1950; Nigon and Delavault, 1952; Faure-Fremiet et ah, 1950; Fautrez- Firlefyn, 1951; Panijel, 1951) and a great deal of discussion about the possible participation of these substances in yolk formation (cf. Wittek, 1952; Urbani, 1955), there seems to be no evidence that ribonucleic acid, demonstrable by the usual cytochemical tests, is associated for the most part with sedimentable fractions of oocytes or unfertilized eggs. Brachet and Chantrenne (1942) have reported about 20% to 40% of the ribonucleic acid to be associated with granules in amphibian egg homogenates, but the granule-free fragments of sea urchin eggs obtained by centrifu- gation were reported to be rich in ribonucleic acid (Harvey and Lavin, 1944). It has long been known, however, that gonial cells and mature gametes contain cytoplasmic elements identical with those found in adult cells (Wilson, 1928). Recent observations with phase contrast and electron microscopy confirm the existence of "typi- cal" mitochondria and/or Golgi bodies in the protoplasm of un- fertilized sea urchin eggs (Shaver, 1955), of germ cells of Helix aspersa (Beams and Tahmisian, 1953), and of frog eggs (Nath and Malhotra, 1954 ) . The flow of these particulate elements from nurse cells into the maturing egg has been described (Wilson, J. R. SHAVER 265 1928; Agaiawal, 1949), but the distribution of various granular constituents along axes, or into "organ-fonning" areas, of eggs is, as has been pointed out on numerous occasions (Wilson, 1928; Watterson, 1955), a result, rather than a cause, of the polarity of the egg. Summarizing the earlier literature on the subject of cytoplasmic inclusions of egg cells, Wilson (1928) states: "It is important to bear in mind the rapid increase of chondriosomes (and of Golgi bodies?) that takes place during the growth of the egg. Only a part of them are used up in the production of other formed elements. The greater number persist, possibly to play some role in the fertilization of the egg, in any case to be handed on to the embryonic cells by cleavage. Perhaps we catch here a glimpse of a mechanism concerned not merely with yolk forma- tion but with the general processes of determination, localization and heredity." But it is precisely the significance for mechanisms of determi- nation and localization of the cytoplasmic inclusions of egg cells (not to mention the still problematical nature of inclusions trans- ported into some egg cells by the sperm) that still eludes us. It has been the universal result of centrifuging egg cells of both mosaic and regulatory types, that the disarrangement of visible cell inclusions, either slightly or quite drastically, does not appear markedly to alter the development of the embryo. The extreme case of this type of experiment is the well-known one of Harvey (1946), whereby fragments of Arbacia eggs, presumably devoid of any granular inclusions, developed into normal embryos after fertilization. Two points may be mentioned in connection with this experi- ment : 1. Harvey states (1946) that mitochondria displaced from the most centripetal egg fragment are replaced during development by de novo formation of these particles, since the pluteus larva developing from it can be seen to have mitochondria. Harvey, however, described in the clear quarter-egg and the "white" halves, a fine line of granules, which sometimes included small fibers, which were thought to be microsomes (see Fig. 1). The identification of mitochondria in the various fragments of centri- s_ !- dj a Q) "T- '^ n ffi .2 o^ • ^ r\ ^~^ »— ) L_ ■—I o ^-^ 05 O i-H O — G 1 < O Z CJ ^ 1 <-) J/| : C-3|— /4- j ■- cr CD < cr o z o X o o UF 5 10 15 20 25 30 35—50 60 70 80 HOURS Fig. 4. Strongijlocentrotiis purpuratus, cultured at 17° C. Numbers of mitochondria per embryo at different times in early development. 25 20 • 15 T • . 10 T level, with a statistical treatment the details of which will be published elsewhere. Several points may be briefly noted in connection with these results : 1. In both species studied, the number of mitochondria re- mains constant during the first twelve or fifteen hours of devel- opment ( temperature of culturing and rates of development were different for each species). This result indicates that mitochon- dria do not multiply during cleavage, but are apportioned among the dividing cells. If equal distribution of the particles is assumed, J. R. SHAVER 283 the number per cell would be approximately halved at each divi- sion, but the total number of mitochondria per embryo would remain the same. The obvious alternative to this interpretation is that mitochondria are destroyed and produced at about the same rates during this period as Agrell ( 1955 ) suggested, but at pres- ent there seems to be no way of deciding between the two possi- bilities. 2. In the advanced blastula stage, just prior to the beginning of migration of the primary mesenchyme cells ( 16 hours for L. pic- tiis, 20 hours for S. piirpiirattts), there is a sharp increase in mito- chondrial number. This increase in number is consistent with the suggestion (Gustafson and Lenicque, 1955) that mitochondria do not increase markedly during cleavage, but there is no evi- dence from this study that mitochondria result from an aggrega- tion of smaller precursors. 3. The level of mitochondria reached at the mesenchyme blas- tula stage is maintained for some hours. At the time when gut differentiation is progressing (about 28 hours in L. pictiis, 33 hours in S. purpuratus) there is a sharp decrease in mitochondria, reaching a level below that of the uncleaved egg. 4. In S. purpuratus, where later developmental stages were studied, mitochondria in pluteus stages have regained the level of the uncleaved egg. The figures presented afford only a rough estimate of mitochon- drial numbers during sea urchin development. The method used, however, appears to be more reliable for quantitative purposes than techniques which depend on the identification of particles in intact cells. If it is assumed that the estimations approximate reality, a few questions raised by such fluctuations of mitochon- drial populations in sea urchin development may be noted: 1. If the numbers of mitochondria do, in fact, remain approxi- mately constant during the cleavage period, what does this mean in relation to the energy-requiring processes occurring at this time? Are we dealing with a situation in which the uncleaved egg has stored enough material, not only to provide for the syntheses which go on during cell division but also to provide for the energy 284 EARLY ECHINODERM DEVELOPMENT demands of the system? Or, as has been suggested, are mitochon- dria destroyed and produced at a rate corresponding to a plas- matic rhythm (Agrell, 1955)? 2. Does the apparent increase in mitochondria in the mesen- chyme blastula reflect new demands of the embryonic system in preparation for, or coincident with, the beginning of primary dif- ferentiation? This question is loaded with imponderables, since we as yet seem to be quite unaware of any specific distinctions that can be made among the demands of the embryonic cell for growth, maintenance, or transformation. To ask whether or not the increase in mitochondria reflects the requirements of the em- bryo at this time for new protein types seems merely to be rephrasing the question. 3. What is the meaning of the decrease in mitochondrial num- ber, shortly after the peak reached at the beginning of gastrula- tion? It has been reported by Gustafson and Lenicque (1952) and Shaver ( 1955 ) that, in intact stained embryos, after gut dif- ferentiation has progressed to some extent, there is a diminution in mitochondrial number in the cells of the body wall and a con- centration of particles in the cells of gut structures and of ciliated bands. An obvious guess as to the nature of the decrease in parti- cles at this time is that synthetic processes or physiological activ- ity, or both, have become restricted to a smaller area of the em- bryo, producing an overall diminution in mitochondria. 4. In addition to questions raised by changes in total numbers of mitochondria, the problem arises of changes in classes of mito- chondria during early development. Recent studies by Paigen (1954) and Novikoff et al. (1953), and others, of mitochondrial heterogeneity, indicate that in adult mammalian cells the chemi- cal structure (and function?) of mitochondria varies with their size. Considering the range of dimensions of the particles counted in this study, it is possible that transitions occur from one size class to another which would be revealed only by differential counts. Although no quantitative data are available, the impres- sion was gained by Shaver (1955) that the larger spherical or oval forms seen in the uncleaved egg and earlier stages were ex- J. R. SHAVER 285 ceeded in numbers by smaller rod-shaped forms in the gastrula and later stages. Caspari (1955) suggested that genetically controlled differ- ences exist in mitochondria of the cells of the same tissues in different strains of mice. From his study there was also some evi- dence of tissue differences in mitochondria in the same animal. The idea is advanced that the composition of particle types in a particular kind of cell is an expression of its developmental and physiological activities. It would be interesting to test this hy- pothesis by studying mitochondrial heterogeneity in the cells of developing embryos, but echinoderm material evidently would not be appropriate, owing to the lack of genetic information a1)0ut this group of animals. Summary A brief review is given of observations and experiments dealing with cytoplasmic particles in development. Some hypotheses con- cerning their role in differentiation are discussed. The results of some current work by the author on changes in number and dis- tribution of mitochondria during development of the sea urchin are presented. Marked changes in number and, possibly, kinds of mitochondria are evident during development. Especially notice- able is an increase in numbers of the particles in mesenchyme blastulae and gastrulae, followed by a sharp decrease in immedi- ately subsequent stages. A gradual return to the earlier level of mitochondrial density was obsei"ved in one of the two species studied. The author has failed to find, in the material studied by him, that mitochondria are distributed along a gradient in mesen- chyme blastulae and gastrulae, as has been reported in another sea urchin species. This result, therefore, does not support hy- potheses of differentiation based on such a distribution pattern of these particles. Technical details are given concerning the iden- tification of mitochondria in intact sea urchin embryos, in living and fixed condition, as well as in homogenates. 286 EARLY ECHINODERM DEVELOPMENT REFERENCES Agaravval, S. C. 1949. The phenomenon of infiltration of Golgi bodies and mitochondria from the egg membranes into the egg. U. Allaha- bad Zool. Sect., pp. 1-7. Agrell, I. 1954. A mitotic gradient in the sea-urchin embryo during gastrulation. Arkiv Zool, 6, 213-17. Agrell, I. 1955. A mitotic rhythm in the appearance of mitochondria during the early cleavages of the sea-urchin egg. Exptl. Cell Re- search, 8, 232-34. Arvy, L. 1950. Donnees histologiques sur I'ovogenese chez Dentaliiim entale Deshayes. Arch. Biol, 61, 187-96. Beams, H. W., and T. Tahmisian. 1953. Phase contrast and electron microscope studies on Golgi bodies and mitochondria of germ cells of Helix aspersa. Cytologia, 18, 157-66. Boell, E. J. 1955. Energy exchange and enzyme development during embryogenesis. In Analysis of Devielopment, B. H. Willier, P. A. Weiss, and V. Hamburger, editors. W. B. Saunders Company, Phil- adelphia, Pa. Pages 520-55. Brachet, J. 1950. Chemical Embryology. Interscience Publishers, New York-London. Brachet, J. 1952. Le role des acides nucleiques dans la vie de la cellule et de Fembryon. Actualites biochimiques. No. 16. Editions Desoer. Liege. Brachet, J., and H. Chantrenne. 1942. Nucleoproteides libres et com- bines sous forme des granules chez I'oeuf d'Amphibiens. Acta Biol Belgica, 2, 451-53. Caspari, E. 1955. The role of genes and cytoplasmic particles in differ- entiation. Ann. N. Y. Acad. Sci., 60, 1026-37. 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EARLY DETERMINATION IN DEVELOPMENT UNDER NORMAL AND EXPERIMENTAL CONDITIONS SILVIO RANZI: zoological department, THE UNIVERSITY OF MILAN, ITALY Development Alterations and How They Are Obtained Working on sea urchin embryos, Runnstrom (1954) and his group showed that there are certain substances such as LiCl that inhibit organ development at the animal half of the embryo by Fig. 1938.) 1. Vegetalized sea urchin embryos. (From Lindahl and Ohman, inducing a developmental increase in the organs formed at the vegetal half (vegetalized larvae) (Fig. 1). On the other hand, other substances such as iodosobenzoate and NaSCN inhibit the organs formed at the vegetal half and favor the development of rudiments which appear at the animal half (animalized larvae) ( Fig. 2 ) . The morphology of the two above mentioned larvae was analyzed by Runnstrom ( 1928 ) , Lindahl ( 1936 ) , and Gustafson 291 292 EARLY DETERMINATION IN DEVELOPMENT (1954): vegetalized embryos show a smaller ciliar tuft at the animal pole than the controls; these embryos develop into larvae in which the ectoderm is reduced whereas the entoderm is hyper- developed and in certain instances it protrudes ( exogastrulae ) . The animalized embryos develop with a very large ciliar tuft; in these embryos entoderm and mesenchyme are reduced. Vertebrate cyclopic embryos, obtained by Stockard (1907) in Fundulus and by Cotronei ( 1922 ) in amphibians through the ac- tion of MgCl2 and LiCl, may be considered as originated from the same causes which induce vegetalized larvae in sea urchins. Adelmann (1934) and Lehmann (1937) showed that LiCl in- duces an inhibition of prechordal plate and of the notochord in Fig. 2. Animalized sea urchin embryos. (From Lindahl, 1936; Tamini, 1941; Backstrom, 1953.) amphibians (Fig. 3). Therefore the cyclopy originates from a disturbance in the induction process, and the hypodevelopment of the notochord and of the prechordal plate corresponds to the sea urchin vegetalization. Later Backstrom ( 1953 ) showed a re- duction in organ development and in the animal half of the Xenopus embryo ( therefore small embryo ) . So far we are unable to state whether this phenomenon can be observed in other am- phibians. An increase of notochord in the amphibians is induced by NaSCN (Ranzi et al, 1946) (Fig. 4). Statistical research carried out by Corti at Milan on Rana esculenta embryos in the tailbud stage showed the following average number of notochordal cell S. RANZI 293 nuclei: 1107 ± 24 m the control embryos; 1233 ± 28 in NaSCN embryos; difference in the means is 126; standard error of differ- ence is ±37, i.e., a significant difference. Furthermore SCN~ in- duces chordal differentiation from cells of other organ rudiments (Corti, 1950; Badinez et at, 1954). These authors obtained larvae which, beside the normal notochord, had more or less extended Fig. 3. Cross sections of Triton embryos: A, control embryo; B, C, Li- treated embryos; c, notochord; mi, somites. (From Lehmann, 1937.) sections of notochord, lateral or dorsal to the brain. Generally, animalizing substances induce notochord formation from cells of other presumptive rudiments; this is also shown by Lallier ( 1955). He showed that sodium thiomalate, an animalizing agent in the sea urchin, induces the formation of notochord in the ventral explants of young amphibian gastrulae in which, therefore, the 294 EARLY DETERMINATION IN DEVELOPMENT Olf.. Fig. 4. Cross sections of amphibian tadpoles: A, control tadpole; B, NaSCN-treated tadpole. Dorsal view of brain and sense organs reconstruc- tion: Ai, normal tadpole; B,, NaSCN-treated tadpole; Co, notochord; Cr, lens; Di, diencephalon; E, epiphysis; Ga, ganglion; G.c, notochord sheath; L.O., optic lobes; Mi, myelencephalon; Oc, optic cup: Olf., olfactory pit; Ot., otocyst; P., first cross section passing through the pronephros; Te., telencephalon. Short dotted line indicates ependymal cavities. Long dotted line indicates notochord. (From Ranzi, 1954.) S. RANZI 295 presumptive rudiment of the notochord is not present. The same phenomena, although with small notochord sections (Fig. 5), were observed by Leone working with urea, when it was recog- nized that proteins are denatured by animalizing substances. On the other hand, notochordal sections appear in the whole embryo treated with urea (Jenkinson, 1906; Fautrez, 1951; Leone, 1953). Leone recognized in these embryos the same developmental al- terations which are induced by SCN" and I~. The alterations shown by older embryos with larger notochords are described by Ranzi et al. ( 1946 ) . These alterations ( Fig. 4 ) are : an increase in hindbrain dimensions, the formation of ganglia from the roof of Fig. 5. Section through ventral explantate of Rana esculenta gastrula treated with urea: c, notochord; 1, lentoid. (From Leone, 1952.) the hindbrain, the epiphysis duplication, the hyperdevelopment of lateral line organs, the formation of epithelial vesicles from the epidermis, the abnormal proliferation of epidermis. Rana escu- lenta embryos treated with iodosobenzoate show the same kind of alterations (unpublished research). Whether the alterations in the nervous system are induced by the enlargement of the notochord or by an excess of active evocat- ing substances is a problem which seems to be solved by the presence of nervous rudiments in axolotl ventral explants treated with NaSCN (Ranzi and Tamini, 1941). In this instance the notochordal rudiment is absent and evocating substances must 296 EARLY DETERMINATION IN DEVELOPMENT originate from materials other than the normal material. The paper by Holtf reter ( 1944 ) , showing that action of several chemi- cal and physical agents bring about the same results, supports the interpretation of the liberation of evocating substances from ab- normal materials. Fig. 6. Petromyzon larvae, head reconstruction: A, control; B, Li+- treated larva; a habenular ganglia; b, first branchial pouch; c, notochord; e, epiphysis; f, pharynx; 1, otocyst; m, midbrain; n, olfactory organ; p, plica rhombo-mesencephalica; pr, forebrain; r, hindbrain; x, protuberance of chiasma; Vi, first trigeminal ganglion; V,, second trigeminal ganglion; VII, facialis; IX, glossopharyngeus; X, vagus. (From Ranzi and JaneseUi, 1941.) The Petromyzon embryos seem to react to Li+ and SCN" just as the amphibians. LiCl-treated embryos are described by Ranzi and JaneseUi (1941) (Fig. 6). NaSCN-treated embryos showed large notochord and hind-brain enlargements ( Ranzi, 1945 ) . This field is open to further research. The lithium chloride-treated embryos of an ascidian {Ciona S. RANZI 297 intestinalis) may develop into exogastrulae (Ranzi and Ferreri, 1945) (Fig. 7). In some of these larvae the notochordal cells do not differentiate. In some other larvae notochordal cells may ap- pear with little difference from entodermal cells. It is concluded, therefore, that there is an entodermization of notochordal cells induced by LiCl. NaSCN-treated embryos show a larger neural plate. In Ascidia malaca embryos, treated with LiCl, Nieuwkoop Fig. 7. Exogastrula from Li + -treated embryo of Ciona: ec, ectoderm; en, entoderm; mi, myoblasts. (1953) found only exogastrulae of the second type.* I did not have the opportunity to study the material further, because I deemed it suflBcient to find certain malformations in ascidians corresponding to those of vertebrates. * Nieuwkoop (1953) interprets the organization of exogastrulae without notochord by admitting that intrinsic differentiation tendencies, extrinsic realization factors, and tissue affinities are required for embryonic differ- entiation, and that realization factors are blocked by LiCl. Perhaps Nieuw- koop's realization factors represent the demoUtion of preexisting protein structures inhibited by L1C1, as discussed below. 298 EARLY DETERMINATION IN DEVELOPMENT Vegetalizing and animalizing agents act on molluscs. Working with LiCl and other salts, Ranzi ( 1928 ) obtained a series of mon- sters of Loligo vulgaris (cephalopod) ranging from convergent eyes, to cyclopy, cyclopy with a small eye and anophthalmia (Fig. 8). SCN~-treated embryos developed a large stomodaeum (stomodaeum rudiment is between eye rudiments) (Ranzi, 1944). Studying embryos with duplicitas cruciata, Ranzi (1931) reached the conclusion that in cephalopod embryos it is most Fig. 8. Li+ effects on Lolifio embryos: A, control; B, convergent eyes; C, cyclopy; D, anophthalmy. (From Ranzi, 1928.) likely that an evocator is acting (entoderm?). I think that pos- sibly in cephalopods a rudiment hyperdeveloped by SCN~ and reduced by LiCl, as occurs for amphibian notochord, is involved. However, I have never carried out direct research to study this point nor have other authors investigated this problem. Thus today we cannot state positively whether an organizer exists in cephalopod embryos. Experimental embryology of cephalopods must be reconsidered. A great deal of material is available and may be handled quite easily. S. RANZI 299 Raven (1952) carried out extensive research on Limnaea stag- nalis ( gasteropod ) . LiCl-treated embryos develop as exogas- trulae ( Fig. 9 ) in which, as in those of ascidians, no entodermiza- tion occurs, but the animal pole organs are reduced just as the ciliar tuft of the sea urchin. Monsters with convergent eyes, cyclopy, and anophthalmia have been described. Arendsen de Wolff-Exalto ( 1947 ) did not notice any change in determination due to NaSCN action. A comparison between the alterations noted in the two mol- luscs has not been made. However, an outstanding fact is the Fig. 9. Topographical relationship in Limnaea exogastrulae: A, nor- mally developed organ rudiments; B,C, reduced organ rudiments at the animal pole; b.ec, large-celled ectodeiTn; en., entoderm; m., marginal zone; s. ect. small-celled ectoderm; st, stomodaeum. (From Raven, 1952.) identity of the series of cyclopic monsters induced by LiCl in both. It is difficult to carry out a comparati\'e analysis based on egg organization. LiCl inhibits rudiment development at the animal half in sea urchins and Limnaea. This does not occur in amphib- ians and in Ciona in which LiCl affects the notochord that is a rudiment on the equatorial level of the egg. As a result, an inter- pretation on the basis of comparative morphology does not seem possible. Differences in Proteins from Embryos Showing Different Malformations Research carried out in our laboratory many years ago showed that ions inducing colloid precipitation also induce vegetalization in sea urchins (Tamini, 1943b) and cyclopic monsters in Am- phibia (Tamini, 1943a). Conversely "swelling" ions induce sea 300 EARLY DETERMINATION IN DEVELOPMENT urchin animalization and the enlargement of the amphibian noto- chord (Ranzi, 1942). By arranging the various ions in the order of their activity, it appears that they follow the lyotropic series of Hofmeister. The greater the activity in inducing cyclopy (or vege- talization) the greater the activity in precipitating the colloids. On the other hand, ions with more swelling activity are more ac- tive in inducing notochord enlargements (or animalization) (Ranzi, 1943). This conclusion brings forth the supposition of a direct action of substances that change embryonic determination on protoplasmic ultrastructures. An attempt to examine this point directly was made by Abruz- zese Sgarlata (1947). Unfertilized eggs of Arhacia were treated either with NaSCN or with LiCl and then centrifuged. In NaSCN- treated eggs stratification was faster than in controls; in LiCl- treated eggs stratification was slower than in controls. This re- search was confirmed and extended to fertilized eggs by Lallier (1955). In order to study the differences between normal, vegetalized and animalized frog embryos, we (Ranzi and Citterio, 1955a,b) have studied the possibilities of extracting the proteins from lyophilized embryos and of precipitating them by using the "salting out" method of Derrien et at ( 1952 ) . With this method we started to study the changes in proteins during normal em- bryonic development. At first, during fertifization, and later, dming the cleavage stages, the amount of protein extracted de- creases. Furthermore, extracted proteins precipitate at a lower concentration of ammonium sulfate. Myosin and actin antigen'- appear during gastrulation. After this stage a gradual process of protein differentiation occurs. New proteins with different char- acteristics of precipitation appear. Plasma and red cell antigens appear during neurulation. On the basis of these data the following experiment is possible. Frog embryos are treated with LiCl or NaSCN at the sensitive stage and then, after a thorough washing in fresh water, are freed from jelly and are lyophilized. The fractions precipitating at dif- ferent concentrations of ammonium sulfate are determined quan- titatively (Fig. 10). In the young gastrulae, after a six-hour treat- S. RANZI 301 20 10 0 10 20 30 40 50 60 70 80 90 C Fig. 10. Graphs of salting out plotted following Derrien et al. for frog embryos at stage lOM left for 6 hours in Holtfreter solution (control), or NaSCN, or LiCl. (From Ranzi and Citterio, 1955.) ment with LiCl, a higher amount of fractions precipitating at a lower concentration of ammonium sulfate (30 to 50% saturation) appears. In the embryos treated with NaSCN, a greater amount of proteins precipitate at a higher concentration of ammonium sulfate (55 to 60% saturation). Embryos after a one-day treatment with NaSCN develop with large notochords and have a far greater quantity of proteins pre- cipitating at higher concentrations of ammonium sulfate (Ranzi and Citterio, 1955a) (Fig. 11). Contr 10 20 30 40 50 60 70 80 90 C Fig. 11. Graphs of salting out like those of Fig. 10 showing NaSCN action. Controls at stage 12. (From Ranzi and Citterio, 1955.) 302 EARLY DETERMINATION IN DEVELOPMENT It is necessary to discuss the nature of different conditions of protein precipitation of embryos treated with LiCl or NaSCN. There may be a difference in physicochemical properties of some proteins or there may be a synthesis of some new proteins. In order to check this point we extracted, following Lawrence et al. (1944), euglobulin a + b* from frog eggs. By adding 1:1 IM KSCN or IM LiCl and storing for one night in a cold room, we plotted the salting out graph. Li+ induces precipitation at a lower concentration of ammonium sulfate; SCN~ induces precipitation at a higher concentration of ammonium sulfate (Fig. 12). That is to say that the fibrillar proteins treated in vivo and in vitro with NaSCN or LiCl show the same transformations. Therefore we conclude that the difference in the salting out graph of em- bryos treated with NaSCN or LiCl originates most likely from the physicochemical conditions of their proteins and not from the synthesis of new proteins. Moreover, it is possible to show that animalizing agents induce a decrease in viscosity of solutions of protein particles which under viscosimetric analysis appear fibrillar. The vegetalizing agents induce instead an increase in viscosity of the same protein solutions (Fig. 13). These changes in viscosity are related to the shape of the particles in solution because they appear only in solutions containing fibrillar proteins and not in solutions of glob- ular proteins (Citterio and Ranzi, 1947). Moreover, both ani- malizing and vegetalizing agents induce an increase in viscosity of the globular protein solutions. The observation of the different actions of animalizing and vegetalizing substances on jSbrillar * It is possible to extract euglobulin a + b from lyophilized eggs with IM KCl. After delipidation with ether, if the solution is diluted to 0.3M, euglobulin a + b precipitate and can be dissolved in IM KCl. This protein does not show flow birefringence but appears highly anisodiametric in the viscosimeter. Its axial ratio, taken with the viscosimeter, seems to increase with dilution; moreover, starting from 0.50 reduced viscosity figures one may reach values exceeding 1.00 (that is, an axial ratio of over 35.1). After Lawrence et al. (1944), I termed this protein fibrillar folded by assuming that its globular-shaped particles may unfold and are unfolded along the walls of the viscosimeter while they appear folded in the axis of the channel where flow birefringence is read. For discussion on other pos- sibilities see Ranzi (1955). S. RANZI 303 AC 0--.'- . 1 1 1 1 i ?-'*-? 10 20 30 40 50 60 70 80 90 C Fig. 12. Salting out graphs plotted as in Fig. 11 for euglobulin a + b diluted 1:1 with IM KCl (control) or with IM KCl + IM LiCl (1:1) or with IM KSCN. As in Fig. 10, LiCl-treated proteins precipitate at lower ammonium sulfate concentrations, KSCN-treated proteins precipitate at higher ammonium sulfate concentrations. (From Ranzi, 1955.) 0.45-1 0.40 0.35-1 0.15 0.10- 0.05 0.20- /' actom. SCN -°^i Li / 0.10 0.05 euglob. a + b °SCN ""I 1 — 0 0.005 0.01 0.05 0.1 0.2 0.5 mol Fig. 13. Specific viscosity of rabbit actomyosin (actom.) solutions and of euglobulin a + b. On the abscissa is plotted the final molar concentration of the added salts (LiCl or KSCN). Ionic strength is I = 1.0 in all samples. LiCl induces an increase in viscosity, KSCN induces a decrease in viscosity. The sketches of the embryos (normal, vegetalized, and animalized) are located according to the active concentration in modifying the development. (From Ranzi, 1955.) 304 EARLY DETERMINATION IN DEVELOPMENT proteins has seemed important since the early stages of this research (Arosio et ah, 1946). The embryonic differentiation is a cytoplasmic phenomenon and some protein fractions which are anisodiametric to viscosimetric analysis and which show in some instances flow birefringence can be extracted from the cytoplasm (Cigada et al., 1954). These fractions can be purified. Therefore, when animalizing and vegetalizing agents induce then- opposite effects on the development, those proteins which may be extracted in a fibrillar shape from cytoplasm are involved. Proteins Are Denatured by Animalizing Agents Viscosimetric data of the action of animalizing agents on glob- ular protein solutions (increasing viscosity) and on fibrillar protein solutions ( decreasing viscosity ) suggest protein denatura- tion. This denaturation by breaking some bonds increases the volume of globular particles and induces a reduction in the anisotropy of the fibrillar particles. This phenomenon was ob- served with the electron microscope on actomyosin treated with SCN~ and I~ (Ranzi, 1947). The research carried out with the Signer apparatus of flow birefringence shows that actomyosin and myosin particles decrease in length after treatment with animahz- ing substances (Rocca, unpublished research). Also the study of chemically detectable free groups favors the conclusion that there is a denaturation process. It is possible to show a greater amount of OH groups and of phenolic groups in actomyosin treated with animalizing substances (Arosio and Bossi, 1954). The same occurs in denaturation processes induced by urea or heat. The ATPase activity of myosin is considerably reduced by the action of animalizing substances. The precipitin test was used to study the action of SCN~ and I~ on actomyosin solutions. SCN~ and I~ induce changes in the antigens and the immunological properties of the changed an- tigens are similar in both cases. The changes indicate a denatura- tion process ( Arosio, 1953 ) . That a denaturation process is the basis of the action of ani- malizing substances is shown ( as mentioned before ) by the fact S. RANZI 305 that Leone ( 1952, 1953 ) found an enlargement of the notochord m amphibians to be induced by urea, a well-known denaturing agent. The formation of notochord and neural rudiments in ex- planted ventral pieces of young gastrulae can be induced by urea. Action of Vegetalizing Agents Vegetalizing substances do not change considerably the afore- mentioned properties of proteins; the flow birefringence is not altered; the intensity of OH and phenolic group reaction is not increased (Arosio and Bossi, 1954). The action of vegetalizing substances becomes evident from another type of experiment. We take into consideration the action of a strong denaturing agent such as urea on proteins treated with animalizing or vegetalizing substances. Two samples are pre- pared of a solution of euglobulin a + b from sea urchin eggs (Fig. 14). A certain quantity of an animalizing or vegetalizing substance sufficient to induce an alteration in the embryonic de- velopment is added to one sample. For example, by using LiCl as a vegetalizing agent, 86 parts of IM KCl plus 14 of IM LiCl are added to 100 parts of the first sample ( tube 2 ) . To the other sample the same amount of IM KCl is added (tube 1). After overnight storage in a cold room we assume that the sample treated with LiCl is vegetalized. The proteins of the second sam- ple are assumed to be proteins of the normal embryo. From both samples we prepare two samples of 5 ml each. Then 0.6 ml of IM KCl are added to one sample (tubes 3,5) and 0.6 ml of 30% irrea is added to the other sample (tubes 4,6). The viscosity read- ings are taken after four hours immersion in the viscosimeter bath. The treatment with vegetalizing substances, as mentioned before, induces an increase in viscosity. Now it is possible to show another fact. The viscosity decreases in the sample with urea and the rate of decrease in proteins treated with vegetaliz- ing agents is lower than in the controls. On the other hand, the rate of decrease in viscosity of proteins treated with animalizing agents is much higher than in the controls (Ranzi and Citterio, 1954). These data (in Fig. 14) show the greater resistance of 306 EARLY DETERMINATION IN DEVELOPMENT 10 CC 1M KCI 8 6cc 1M KCt* 14 cc 1M LiCI • — 10 CC eugiobulin A*b — » ® ® I overnight' in cold room | normal prot-ein vegetatized prol-ein 0.30 0 20 0 10 0 15 0 10 0 05 Q> ♦KCI urea* 5cc © ♦KCI urpa*t=: ♦-5 cc @ © 4 hours of mahurahon (D ® (D (D ^.^/'^.PQ^-'''' < ^sp(d/^sp(D^=°^^^ (D ® coo 0 15 6"'° "?« 0 10 (D 0 05 (D SCN ® l?i£^(.0 59) > 5*£^(=0 50j 5iP:S)(=0 623 >^1£^(.0 4 8) ^ipO) ^4P® ^iP® ^iP® Fig. 14. S. RANZI 307 proteins treated with vegetalizing substances and the lower resistance of proteins treated with animahzing substances. The denaturation induced by animahzing substances leads to an additional denaturation induced by urea. The action of vege- talizing substances is, however, a stabilizing action that opposes the demolition by urea. In the experiment just described all vegetalizing substances exert the same action of protecting from denaturation. All ani- mahzing agents denature and predispose to denaturation (Table I)- Differences in Resistance to Demolition of Proteins from Embryos Showing Different Malformations It is obvious that if the above phenomena are important in the changes of embryonic determination, something must he ob- servable in the embryo itself after treatment with vegetalizing or animahzing substances. To examine this point we carried out the following experiment. Fig. 14. Scheme of the experiments. Into tubes (1) and (2), 10 ml of euglobulin a + b sokition was poured. To tube (1), 10 ml of IM KCl and to tube (2), 10 ml of fluid (1.4 ml IM LiCl + 8.6 ml IM KCl) were added and then stored one night in cold room; 5 ml of mixture (1) were taken and put into tube (3), together with 0.6 ml of IM KCl; 5 ml of mixture (1) plus 0.6 ml 30% urea represent the sample in tube (4). The same procedures were followed for the mixture of tube (2) (vegetalized pro- teins) and samples (5) and (6) were obtained. After 4 hours of matura- tion, the viscosity was read by Ostwald viscosimeter at 14° C. Values of readings are plotted on the column diagram under their respective tubes. The lower column diagrams represent values obtained using iodosobenzoate (left) and thiocyanate (right). In the iodosobenzoate experiment, tube (1) contained the proteins dissolved in Weber and Edsall fluid plus 10 ml of Weber and Edsall fluid; tube (2) contained the proteins plus 10 ml of O.OOIM iodosobenzoic acid dissolved in Weber and Edsall fluid (the last retained a constant pH in spite of the addition of iodosobenzoic acid ) . In the thiocyanate experiment, the mixture of tube ( 1 ) was the same as the Li experiments, while 1 ml of IM KSCN and 9 ml of IM KCl were added to tube (2). It is evident that, with Li, the decrease in viscosity of tube (6) mixture compared with tube (5) was less marked than the controls. With iodosobenzoate and KSCN, the contrary is true. [Decrease of viscosity of tube (6) in comparison with (5) is stronger than the decrease of (4) as compared with (3).] (From Ranzi and Citterio, 1954.) 308 EARLY DETERMINATION IN DEVELOPMENT Table I. Action of Different Agents on Embryonic Development and on Proteins in Solution Sea urchin Amphibian Protein Large Ani- Vege- noto- Cyclopic De- Agent malizes talizes chord monsters natures Stabilizes KCl + + LiCl + + + MgCl2 + + + NaCl + + + NaT + + + NaNs + + NaSCN + + + Na2S04 + + + + + Chymotrypsin + + Citrate + + + + Colchicine + + + Ethanol + + + Ficin + + Glucose + + Glyceraldehyde + + lodosobenzoate + + + Lactate ( + ) + Leucine (+) + Lisine (+) + Maleinate + + Methylene blue + + + Paranitrophenol + + + Pyocyanine + + + Pyruvate + + + + + Tartrate + + + + + Thiomalate + (+) + Thionine + + Thiourea + + Trypan blue + + Trypsin + + Urea + + + Valine (+) + High pH (+) + Note: The crosses in parentheses show for sea urchins action on animal halves, for the amphibians the notochordal formation in ventral explants (from Ranzi, 1955). S. RANZI 309 Three groups of frog embryos at the stage of late blastula, (stage 9) were allowed to develop in fresh water or in 0.05M NaSCN or O.OTAf LiCl. At the stage of young gastrula (stage 10), after 6 hours treatment, the embryos were carefully washed, liberated from jelly, washed again in Holtfreter fluid, and then lyophilized. The powder was extracted overnight in IM KCl. Euglobulin a + b was taken in a solution of IM KCl and the three solutions, (a) euglobulin of control embryos, (b) euglobulin of vegetalized embryos, and (c) euglobulin of animalized embryos, 100 80- 60 ■■•= 4 0- 20- t-pypsin papain Fig. 15. The diagram shows the decrease per cent of viscosity induced by urea (15% final concentration), trypsin (0.5% final concentration pH 7.9), and papain in the euglobulin a + b extracted from embryos: i, con- trols; ii, developed in NaSCN solution; iii, developed in LiCl solution. (The embryos used were taken from the same cultures as those used for the graphs Fig. 10.) (From Ranzi, 1955.) were diluted in such a way as to reach the same optical density at 275 millimicrons in the Beckman spectrophotometer. With each solution the following samples were prepared: (a) euglobulin a -h b diluted 1:1 with the solvent (IMKCl); (b) euglobulin a + b diluted 1:1 with 30% urea; (c) euglobulin a + b diluted 1:1 with trypsin (Merck) dissolved in IM KCl kept at pH 7.9 with Weber and Edsall fluid; (d) euglobulin a + b diluted 1:1 with papain in IM NaCl. After four hours incubation in the vis- cosimeter bath, the viscosity readings were taken (Fig. 15), the 310 EARLY DETERMINATION IN DEVELOPMENT viscosity of proteins diluted in IM KCl being 100. The values show that the proteins from embryos treated with LiCl are much more resistant to the action of urea and to proteolytic enzymes such as trypsin and papain than are control proteins; the proteins of embryos treated with NaSCN are less resistant to urea and proteolytic enzymes. Another experiment was performed with these materials. The 1.0 0.5 A NdSCN Control Fig. 16. The optical densities (at 275 millimicrons) of the solutions of Fig. 15 are assumed to be 100. After the readings, the proteins were precip- itated with trichloracetic acid. The optical density after precipitation is plotted on the ordinates. (From Ranzi, 1955.) solutions used for the viscosimetric test mentioned above were precipitated by diluting 1:1 with 10% trichloracetic acid. The optical density of the supernatant fluid was read at 275 milli- microns in the Beckman spectrophotometer (Fig. 16). The data show that, in the protein solutions of embryos treated with NaSCN, a greater quantity of material remains in solution than in the controls. In the solutions of embryos treated with LiCl a S. RANZI 311 lower quantity of material than in the controls remains in solu- tion. Also with this experiment it can be shown that LiCl, by act- ing on the embryos, inhibits demolition while NaSCN makes demolition easier. Predisposition and Inhibition to Demolition of Protoplasmic Structures The action of animalizing agents which predisposes to demoli- tion and of vegetalizing agents which inhibits demolition has been shown also on some cell structures. Orlandi (1953) showed that Nal, NaSCN, urea, iodosobenzoate, and high pH, disinte- grate the isolated chromosomes, whereas LiCl preserves them. L. Cigada (1954) showed that Nal, NaSCN, methylene blue, and high pH disintegrate the isolated yolk granules whereas LiCl and thiourea preserve them. Brioschi ( 1955 ) working on glyc- erinated muscle fibers showed that while in the presence of vegetalizing substances they regularly contract because of ATP, this property is lost or reduced when animalizing agents are present. The demolishing action induced by animalizing agents and the resistance to demolition induced by vegetalizing agents are there- fore phenomena which concern not only embryonic proteins but also structures in the cell. The Nature of Denaturation Induced by Animalizing Agents Another problem is the nature of the denaturation induced by animalizing agents on these proteins. In the past I have put forward the hypothesis that smaller units could be liberated by the action of animalizing agents. Viscosimetric readings taken this year give an intrinsic viscosity of 0.16 for these subunits (that is, an axial ratio slightly above 11, by admitting a prolate ellipsoid ) . From all the above data it seems today that the predisposition to the demolition phenomenon (of which the formation of these subunits represents only one part of the process) is the aspect of greatest importance of embryonic determination. 312 EARLY DETERMINATION IN DEVELOPMENT Above-Mentioned Conclusions and Data from Experimental Embryology The action of animalizing substances is shown as a demolition of preexisting proteic structures and the action of vegetalizing substances as a resistance to demolition. Does this agree with the findings of experimental embryology? All the authors who, in their studies on vertebrates, speak of developmental inhibition induced by LiCl have encountered the resistance to demolition of proteic ultrastructmes at a microscopic level. The delay in synthesis due to Li+ is in agreement with the smaller increase of many enzymes in the LiCl-treated embryos studied by the Stockholm group (Gustafson, 1954), by Lallier (1955) and by others. On the other hand, the animalization seems in fact to corre- spond to a demolition of preexisting proteic structures. Proteo- lytic enzymes ( chymotrypsin, ficin, trypsin) are animalizing agents (Horstadius, 1949, 1953; Moore, 1952). The observation made by Lindahl et al. (1951) in this field is highly significant: that from a population of more easily animalizable eggs it is possible to extract in 0.6M KI a greater amount of N; that is, they contain a lower ratio of insoluble N. Animalization at times may be induced under the same condi- tions of protein denaturation. Horstadius (1949) found that animalization occurs more easily at a low temperature, Jacobsen et al. ( 1948 ) found that denaturation, induced by urea, is easier to obtain at lower temperatures. Animalization induced by Na- SCN occurs more easily in calcium-free sea water; Ca++ prevents many proteins from undergoing denaturation ( Gorini, 1950 ) . From a comparative viewpoint, the regions overdeveloped by the action of animalizing agents and inhibited by vegetalizing agents show a higher oxydoreduction potential. [See the research by Child ( 1936a,b, 1943), by Ranzi (1939), and by Horstadius ( 1955) ]. I think that this may be interpreted as an indication of a much more active protein breakdown. It is possible to follow the formation of the ciliar tuft and of the mitochondria in the cells of the animal pole of the sea urchin embryo (Gustafson, 1954; S. RANZI 313 Shaver, 1955) which shows a higher oxydoreduction potential. On the other hand, the analyses carried out directly demonstrate the protein breakdown. In the frog blastoporal dorsal lip, that is, the notochordal rudiment at the time at which it is sensitive to animalizing and vegetalizing agents, Deuchar ( 1955 ) found a higher quantity of free amino acids than in other areas of the embryo. The findings of Kavanau (1954) also seem important. He found that the highest quantity of free amino acids in sea urchin embryos occurs exactly during the stage which Biickstrom and Gustafson ( 1953 ) found more sensitive to the action of LiCl. Conclusion The above indicates that animalization and vegetalization orig- inate from the enlargement or inhibition of the areas in which a process of protein demolition, necessary for new protein synthesis, is acting. The following hypothesis may be formulated: It is pos- sible to represent the unfertilized sea urchin egg with much more reactive protoplasm at the animal pole. Fertilization, by activating certain enzymatic systems according to the conception of Runn- strom ( 1949 ) , activates a higher metabolism in the animal pole. If we bear in mind the fact evidenced by the Stockholm School that several metabolites are animalizing substances, the activation of enzymes and the following formation of metabolites, occurring at the animal pole, seem to lead to the development of the ani- mal pole, while the vegetative pole is stabilized because of failure to form these metabolites. Addendum A new train of research was performed in this field by the Milan group during the last year (Ranzi et al., 1957). The salting out diagrams of proteins extracted from the sea urchin, Arbacia lixula L., were studied. The animalized embryos show proteic fractions precipitating at higher ammonium sulfate concentration than those of the controls, whereas the vegetalized embryos show some fractions precipitating at lower ammonium sulfate concentration. Proteins from embryos animalized by iodosobenzoic acid treatment show salting out diagrams identical to those of the NaSCN animalized embryos. 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Experientia, 9, 351-55. Stockard, C. R. 1907. The artificial production of a single median Cyclo- pean eye in the fish embryo by means of sea water solution of mag- nesium chloride. Arch. EnUoicklungsmechan. Organ., 23, 249-58. Tamini, E. 1941. Ricerche sulla animalizzazione nello sviluppo dei ricci di mare. Monit. zool. ital., 52, 81-92. Tamini, E. 1943a. Ricerche sulle cause che determinano la ciclopia degli Anfibi. Wilhelm Roux' Arch. Entwickliingsmech. Organ., 142, 455-89. Tamini, E. 1943b. Ricerche sulla vegetatilizzazione nello sviluppo dei ricci di mare. Rend. ist. lombardo sci., 76, 363-92. THE ROLE OF SOME ENZYMES IN THE DEVELOPMENT OF ASCIDIANS G. REVERBERI: zoological institute, UNIVERSITY, PALERMO, ITALY The cytologists of the past century, closing their research and their discussion on mitochondria, could not suspect that a new brilliant mitochondrial era would be opened in the next century by the work of the biochemists. In any scientific movement it is always difficult to fix exactly the starting point; but perhaps we are not much mistaken in ascribing the beginning of the new golden age of mitochondria to the fundamental discovery of Green et al. ( 1948 ) , by which it was established that the com- plete oxidation of pyruvate and the connected oxidative phos- phorylation are carried out by a group of enzymes and coenzymes that are situated in the particulate elements of the cytoplasm. In the years that followed other important discoveries were made, particularly in the past five years, and enzymology became in a short time one of the most prosperous branches of biology. Morphologists, physiologists, and even the embryologists could not remain indifferent to these new discoveries; they immediately felt that a reconsideration of some of the old, dismissed problems would be very profitable. The morphologists were much helped in such reconsideration by the electron microscope which disclosed, in the mitochondria, unsuspected features. In fact, the electron microscope revealed a mitochondrial structure with transversal bands or "cristae mito- chondriales," as they were named. The problem arose as to their meaning, and of course that problem led to many other problems. The physiologists, for their part, were particularly affected by the new discoveries, as they shed new light on the problems of 319 320 DEVELOPMENT OF ASCIDIANS muscular contraction, cellular secretion, and synthesis of biologi- cal substances. The embryologists, always in search of that "something" re- sponsible for cellular and organic differentiation, felt immediately that they would be greatly helped by the new biochemical re- search: the factors responsible for differentiation and induction must have a chemical basis! Particularly interesting to them was the fact that the mitochondria contain dozens or hundreds of enzymes. These are not distributed at random but, on the con- trary, are assembled in a fixed pattern, that is, locally disposed, spatially connected, and in determined quantities. It is a well-established fact in genetics that the chromosomes also possess a preordered and constant pattern, and there are valid cases in which results have sufficiently demonstrated that genes ai-e enzymes, or at least produce enzymes. Another con- sideration associates in the mind of an embryologist the mitochon- dria and the chromosomes: the fact that, as in the case of the chromosomes, the mitochondria can be separated, with respect to their form and functions, into many different classes. Generally it is assumed that every mitochondrion possesses the power of operating the Krebs cycle, the transfer of electrons to molecular oxygen and the oxidative phosphorylation; but other functions are typical of certain classes of mitochondria. There have been described (Lenicque, 1953) two kinds of mitochondria in the muscles of amphibians, one devoted to the lipidic metabolism, the other implicated in the energetics of contraction. In the liver, too, there are known to exist two kinds of mitochondria which provide two different functions, the synthesis of citrulline and the detoxication of aromatic substances. The brain mitochondria are not able to catalyze the reactions of fat metabolism, and the maximum of differentiation and specialization is represented by the mitochondria of the secretory cells, which perform the most diverse functions. Very often one speaks of "ectodermic" or "en- todermic" mitochondria to indicate theii- principal role. The former would carry out the synthesis of some proteins, and the latter the synthesis of pigments and other substances. In the G. REVERBERI 321 plants, the chloroplasts are mitochondria that have acquired new functions. It is obvious that all these different functions are based on a peculiar enzymatic constitution, from whence possibly derive the different forms of the mitochondria. Electron microscope research shows that the "cristae mitochondriales" are more numerous in the mitochondria that have a high enzymatic content. One of the principal tasks in the future will be to describe where and how the different groups of enzymes are disposed in a mitochondrion. Probably it will be very useful in the future to compare the localization of the enzymes in the mitochondria and the localiza- tion of the genes in the chiomosomes. If the mitochondria are ultimately responsible for the form, the length, and other charac- teristics of a cell, it would be useful to establish what sort of dependence exists between the mitochondria and the genes. Of course it is not intended here to compare the importance of chromosomes with that of mitochondria. It would be sufficient to compare only the ways of their reproduction. Mitochondria grow in length, divide transversely, and are spread at random in the originating cells, because they do not possess that very elabo- rate mechanism which is peculiar to the chromosomes, that is, the "achromatic spindle." Apparently the mitochondria do not possess the very complicated chromosomal structure! Mitochondria of the Ascidian Egg The assumption that the mitochondria are distributed at ran- dom during the mitotic process must be corrected if the cell which divides is a fertilized egg. We shall see, on the contrary, that in some kinds of eggs there is a sort of mechanism, which is rather accurate and which segregates the mitochondria only in certain cells. Probably that segregation is not only quantitative but also qualitative. The importance of this fact is very evident from the embryo- logical point of view, as every cell, while possessing a complete set of genes, does not always have at its disposal the enzymes by which, in consequence of the action of the genes, are initiated 322 DEVELOPMENT OF ASCIDIANS k I Fig. 1. Phalliisia eggs and embryos at different stages of development, after staining of the unfertilized egg with Janus green. See the text (Rever- beri, 1956a). G. REVERBERI 323 those chemical chain reactions that bring about the processes of differentiation. One of the best examples of differential segregation of the mitochondria in certain cells of the developing embryo is offered by the ascidian egg (Reverberi, 1956a). Janus green staining reveals in this egg the presence of a large quantity of mitochon- dria. In the unfertilized egg they are spread' everywhere in the cytoplasm, except at the animal pole, where there is the nuclear sap of the collapsed nuclear vesicle. Between the two zones runs a circular, sinuous line of large granules of an intense green (Fig. 1, a). Upon centrifuging the unfertilized gi^een egg, the colored mitochondria gather between the animal hyaline cap and the large mass of deutoplasm and pigments. The local segregation of the mitochondria starts at fertilization. The uniformly colored egg in a few minutes becomes clear — only the vegetal pole, to which migrates the mass of green-colored mitochondria, becomes green (Fig. 1, b, c, and d). With a new migration the mitochondrial mass reaches the dorsal part of the egg, where normally the yellow crescent is found (Fig. 1, e). The first three segmentations of the egg segregate the mitochondria in the two posterior vegetal blastomeres (Fig. 1, / and g). These blastomeres are the representatives of the mesodermic organs. With the next egg divisions the mitochondria are segre- gated only in the cells of the muscular line (Fig. 1, h, i, j, and k). The tadpoles originating from eggs treated with the Janus green at the beginning of development have green colored tails; more exactly, the green coloration is localized in the muscular cells of the tail (Fig. 1,1). The very impressive movements of migration and segregation of the mitochondria just described and the apparent existence of an accurate mechanism controlling these movements raise the question of the meaning of such processes. It is necessary, however, to remove at once the idea, possibly raised by the above described processes, that the other blas- tomeres of the developing embryo are deprived of mitochondria. First of all, in the ascidian egg there are probably at least two kinds of mitochondria. Meves (1913), Duesberg (1926), Conklin 324 DEVELOPMENT OF ASCIDIANS (1931), Ries (1939), and Tung et al. (1941) described granules which are not stained by the Janus green. They are osmophihc and are brought by centrifugation to the centripetal pole. They are also rich in yellow pigment from which derives the typical "yellow crescent" described by Conklin. Such granules are sup- posed to be mitochondria because they behave like them when treated with ordinary lipidic solvents or with osmic acid. On the other hand, we have described rodlike mitochondria, which take the coloration with the Janus green and which gather, by cen- trifugation, above the equator of the egg, between the hyaline cap and the pigmented zone. Secondly, we have also noticed, especially at the time of their segmentation, that the non-muscle- forming blastomeres of the embryo possess, although in small number, typical mitochondria. Enzymes of the Ascidian Mitochondria The present knowledge on the quality, quantity, and localiza- tion of the enzymes in the mitochondria is generally very poor, because only a few histochemical techniques are at our disposal. Ries (1937) was the first to indicate the presence and localiza- tion in the cells of the developing ascidian egg of "indophenoloxi- dase," "benzidinperoxidase," and "oxidoreductase." These en- zymes were segregated in the cells of the muscle line. Reverberi and Pitotti ( 1939 ) confirmed these points and showed that the pattern of the enzymatic distribution is very different in the un- fertilized and in the fertilized egg. Ries supposed that these enzymes are not localized in the mito- chondria but in the plasm. He attributed a paramount importance to them in relation to the differentiation of the musculature, and he stated that only when the plasm containing these enzymes is displaced by centrifugation, larvae arise which are very defi- cient in musculature. The displacement of the mitochondria is, according to Ries, of no consequence to morphogenesis. Our recent research has shown that the indophenoloxidase of Ries is inhibited by sodium azide; thus, it must be identified as a cytochrome oxidase; also it is localized not in the plasm but in the mitochondria. In fact, the mitochondrial zone of the centrifuged G. REVERBERI 325 egg gives a very intensive reaction to the Nadi reagent. This reaction is negative if the egg was treated before by sodium azide. It also seems likely that the benzidinperoxidase and the oxido- reductase are localized in the mitochondria. Another enzyme which is also supposed to be localized in the mitochondria is the "succinodehydrogenase" which has been studied in the ascidian egg by Mancuso ( 1952). Mancuso showed that this enzyme presents the same pattern of distribution and segregation that is typical for the cytochiome oxidase. We pro- pose to check all these data on cell-free mitochondria. We plan to investigate also the locahzation of these enzymes in the mito- chondrion and their relative quantity. At the moment we can only say, judging from the intensity and rapidity of the Nadi reaction, that the quantity of cytochrome oxidase is very high. Inhibition of Mitochondrial Enzymes in the Ascidian Egg It is well known that the activity of an enzyme can be specifi- cally inhibited by the use of appropriate chemical substances. In some conditions the inhibition can be reversed. Much research has been done by the biochemists on enzyme inhibition; but the methods and the conclusions deduced from their studies cannot be used without reservation for the mitochondria inside the cells. In fact, strong chemical solutions could not be used in the devel- oping eggs without seriously impairing development. Also specific substances acting only on one enzyme and not on other cellular components are scarcely known. In other words, great care must be employed when using inhibitors on living and developing eggs. A specific inhibitor of cytochrome oxidase (CO.) is sodium azide. At appropriate concentrations in vitro it inhibits 80% of the enzyme activity. The sodium azide is not absolutely specific; in fact, it influences also the phosphorylating system. In the ascidian egg the inhibition of the CO. can be easily and at any moment checked by the Nadi reaction, which is negative or re- tarded. Solutions of sodium azide at O.IM inhibit the enzyme in fifteen minutes, but at that concentration the fertilized egg does not develop, polocytes are not emitted, and the egg does not 326 DEVELOPMENT OF ASCIDIANS divide. The same concentration used on the eggs at 2-, 4-, and 8-cell stages produces a similar result; that is, the eggs are blocked. Solutions at O.OIM inhibit the enzyme partially; at this concentration development is also impossible. Only at O.OOIM or 2 X 10~^ and 3 X 10~'^ M is some development possible. Stronger solutions (0.2 or O.IM) can be used only with the un- fertilized egg without any consequent damage to development. After treatment, even prolonged for 12 hours, it can be fertilized in normal sea water and start development. Under such condi- tions the CO. is inhibited, as shown by the Nadi, and the inhibi- tion lasts longer ( Reverberi, 1956b ) . Sodium malonate and selenite are considered good inhibitors of succinodehydrogenase. The inhibition can be checked histo- chemically by tetrazolium salts. In our experiments with ascidian eggs solutions 5 X 10- to 1 X 10"^ M and 6 X 10"^ to 1 X 10"^ M, respectively for malonate and for selenite, are effective. At these concentrations, however, the inhibition of the enzyme is only partial, as revealed by the positive histochemical reaction. Malonate and selenite do not inhibit the activity of the CO. Development of the Ascidian Egg with Blocked Enzymes The central problem of our research was to establish whether the ascidian egg with one or more blocked enzymes develops into a normal larva or into a larva with specific and constant ab- normalities. Eggs at different stages of development were sub- mitted to the action of sodium azide, malonate, or selenite, or sodium azide plus malonate ( or selenite ) . Treatment ivith Sodium Azide. UnfeHilized eggs can be treated at length and at high concentrations (0.1 to O.OIM). In such solutions the CO. is completely inhibited, fertilization and development are possible in normal sea water or in diluted azide solutions, and the inhibition of the enzyme persists. The segmen- tations of the eggs are normal and synchronous with the controls. Tadpoles develop which have a normal "head" but an abnormal tail which is distorted and physiologically impotent. The tadpoles move poorly and verv irregularly, and they are easily exhausted (Fig. 2^7). G. REVERBERI 327 Fig. 2. Phalhisia tadpoles, a, controls; b, treatment with O.lAf sodium azide for 3 hours before fertilization, and development in O.OOlAf azide; c, treatment as in b, interrupted at neurula stage. Fertilized eggs can develop only in dilute solutions (from 2 X 10~^ M); the CO. is only partially inhibited. The developed tad- poles possess normal "head" with brain, sensorial spots, and palps, but very abnormal tails (Fig. 3). The same results are also ob- tained when the treatment was interrupted before the differentia- tion of the musculature, that is, at the neurula stage (Fig. 2c). Treatment with Malonate. Development is affected only at concentrations about 5 X 10~- M or stronger. In Fig. 4 are pre- f ^ ^^J f: k • f Fig. 3. Phallusia tadpoles, a, controls; b, development in 2 X 10 ~^ M sodium azide; c, development in 3 X 10~^ M azide. 328 DEVELOPMENT OF ASCIDIANS Fig. 4. Phalliisia tadpoles, a, development in O.IM sodium malonate; h, development in 6 X 10~2 2Vf malonate. sented some larvae which developed after the treatment. They have almost normal "heads," but the tails are very abnormal — atrophic, distorted, and poorly functional, with the chordal cells partially fused and randomly disposed, apparently as a conse- quence of the disturbed differentiation of the muscle-cells. The above results were obtained also when the treatment was inter- rupted at neurula stage. Treatment with Selenite. Eggs at the 2-cell stage reared in selenite solutions at 1 X 10~^ to 4 X 10"^ M can develop into larvae (Fig. 5a). The larvae from a 1 X 10~^ M solution cannot, however, get out of the membranes, their tails are poorly devel- Fig. 5. Phallusia tadpoles, a, development in 1 X 10-^ M sodium sele- nite; b, development in 6 X lO^^ M selenite, until the neurula stage. G. REVERBERI 329 Oped, without any movement or contraction. The larvae seem to be dead. That they are Uving is however shown by the fact that they enter metamorphosis. The immobihty of the larvae is not due to paralysis, because they do not reacquire motility if they are immersed in normal sea water and are allowed to remain in it for a length of time. Normal larvae, if placed in 1 X 10~^ M selenite solution, do not lose their active movement even after a long period. /? Fig. 6. Phalhisia tadpoles, a, controls; b, development in 1 X 10~^ M sodium azide. c, development in 5 X 10~- M sodium malonate; d, develop- ment in 1 X 10~2 M azide plus 5 X 10^- M malonate. Eggs in weaker solutions (6X10~'*M) develop into mem- brane-free larvae, which, however, have distorted tails and are incapable of any movement. Their "heads" are normal. Their con- dition does not improve in normal sea water. If the treatment is interrupted at the neurula stage (solutions 6 X 10~^ or 4 X 10~^ M) before any muscular differentiation be- gins, larvae arise which also have abnormal tails and are deprived of movement ( Fig. 5b ) . They do not recover if immersed in nor- mal sea water. 330 DEVELOPMENT OF ASCIDIANS Treatment with Sodium Azide Plus Malonate or Plus Selenite. These experiments by which two kinds of mitochondrial enzymes were simultaneously inactivated (CO. and succinodehydroge- nase) were designed to ascertain whether the larvae produced presented more marked caudal abnormalities. The results ob- tained show that, while azide or malonate alone (respectively azide or selenite) do not produce at low concentrations any ab- normality in the larvae, they produce marked tail abnormalities if used together ( Fig. 6 ) . Topographical Distribution of Mitochondria In "Mosaic" Eggs. The above described differential segrega- tion of the mitochondria in particular cells of the developing em- bryo is not an exceptional case. In mosaic eggs other interesting cases have been described. The most notable is that of the Tubi- fex egg. It must be recalled that first Lehmann ( 1941 ) and later Carrano and Palazzo ( 1955 ) demonstrated the presence in this egg, at the opposite poles, of a peculiar plasm rich in indophenol oxidase or cytochrome oxidase, the "polplasma" (Fig. 7). During development the polplasma is differentially distributed in the 2d and 4d cells. Other research has shown that the polplasma is par- ticularly rich in mitochondria, which are differentially segregated, with it, in the 2d and 4d cells. A more accurate analysis (Leh- mann, 1950 ) , confirmed by the use of electron and phase contrast microscope (Lehmann and Wahli, 1954), has shown that larger particles or mitochondria are distributed into the 4d cells, whereas the smaller ones or microsomes, and only few mitochondria, are segregated into the 2d cells. Other equally clear examples of mitochondrial segregation in certain cells of the developing egg are not known, but we have many indications that lead us to believe that such a process is more frequent in "mosaic" eggs than one would suppose. It has been stated (Attardo, 1955a) that the Nadi reaction is positive only in the animal pole of the uncleaved egg of Bithtjnia. In the following stages of development the Nadi reaction is posi- tive only in the quartets of micromeres which will give rise to the ectodemi (Fig. 8). As in this case the Nadi reaction is pre- G. REVERBERI 331 -AB CD-f- iG- ^°"/<^ \\lllc lA j/j//S Sv'^*^ id^r^- "*^2d iD JiD k I Fig. 7. Nadi reaction (dotted) in developing eggs of Tubifex. a, c, e, g, i, from the animal pole; b, d, f, h, j, from the vegetal pole; k, blastula from the animal pole; /, hatched worm (Carrano and Palazzo, 1955). vented by the azide treatment, one can affirm that the zones of the egg which are Nadi positive possess cytoclirome oxidase. If this enzyme is locahzed in the mitochondria, these would ulti- mately be segregated only in the ectodermic blastomeres. Similar results have also been obtained by Mancuso ( 1955a ) in Physa. Mitochondria segregated in the animal pole of the egg have been actually observed by Ries and Gersch (1936) in Aplysia. How- ever, these authors were not able to follow their destiny in the following development. They supposed, probably founded on 332 DEVELOPMENT OF ASCIDIANS some observations, that they were "qiiantitativ imgleich verteilt" in the blastomeres. Other indications of a differential distribution of mitochondria in some blastomeres of a developing embryo are found in the eggs of Nereis, Eucharis, and probably of Myzostoma (Reverberi and Pitotti, 1940; Pitotti, 1947). In these eggs the Nadi reaction showed definite localization of a certain substance. Confirmation that such a substance is cytochrome oxidase, as we suppose, is however lacking. We hope that coloration of the eggs with Janus d e f Fig. 8. Nadi reaction (dotted) in developing egg of Bithiinia (Attardo, 1955a). green will give more information. It is important to note for the moment that, as in the ascidians and in Tiibifex, the Nadi reac- tion is positive only in those blastomeres that will give rise to the larval organs of movement. In the "Regulative" Eggs. In the sea urchin egg the topo- graphical distribution of the mitochondria has been studied by Gustafson and Lenicque (1952; 1955). In the unfertilized egg and in the first stages of development there was a small quantity of mitochondria. Their number suddenly increased at the begin- G. REVERBERI 333 ning of the migration of the mesenchyme cells into the blas- tocoelic cavity. Counts of mitochondria in the early gastrula show that they are more abundant at the animal pole; their number decreases toward the vegetal pole along a gradient. There has been doubt, however, about such mitochrondrial distribution (Shaver, 1955; see also Shaver, this volume). In the amphibian eggs and in other regulative eggs no topo- graphical segregation of mitochondria in particular cells of the developing embryo seems to have been described. Of some in- terest is the work of Shaver ( 1953 ) concerning the differentiation or maturation of mitochondria. In fact, while the mitochondria of the young development stages are not able to stimulate a parthenogenetic development, this property is possessed by the mitochondria from late blastula or gastrula. Inactivation of Enzymes and Morphogenesis In the "Mosaic" Eggs. We have reported above some results which follow the blocking of the cytochrome oxidase or the suc- cinodehydrogenase in ascidian development. Do we have other examples which can confirm such results in the "mosaic" eggs? This can be answered in the affirmative. First we want to recall some results obtained by Raven and Spronk (1952) in the Limnaea egg. The topographical distribu- tion of the alkaline phosphatase in the developing embryo of Limnaea has been described by Minganti (1950) who, by his- tochemical methods, showed that it is localized at early stages of development in the stomodaeum, protonephridium, and shell gland. Beryllium is considered a strong inhibitor of the alkaline phosphatase. Raven and Spronk exposed the eggs of Limnaea to beryllium salts. The embryos which developed from these treated eggs showed very marked abnormalities in the stomodaeum, protonephridium, and shell gland. Similar results have been ob- tained by some of my collaborators in this laboratory. The research of Lehmann (1941) and Carrano and Palazzo (1955) on the localization of the cytochrome oxidase in the de- veloping eggs of Tiihifex has been mentioned above. The CO. is abundant in the mitochondria, particularly in the 4d cell, that 334 DEVELOPMENT OF ASCIDIANS Fig. 9. Hatched Ttibifex worms, a, control; h, c, after treatment with 1 X 10-2 ]Vf sodium azide (Palazzo, 1955). is, the mesodermic cell. Palazzo ( 1955 ) partially blocked the CO. with sodium azide in the first stages of development. She obtained worms with relevant deficiencies in the muscular system and in the circulatory system, both of which are mesodermic derivates (Fig. 9). In the eggs of Bithynia (Attardo, 1955a) and Physa (Mancuso, 1955a), however, the CO. appears localized in the ectodermic blastomeres. Now the blocking of the enzyme by sodium azide (Figs. 10 and 11) produces animals which are abnormal for the ectodermic derivates, i.e., the brain, the eyes, and the tentacles (Attardo, 1955b; Mancuso, 1955b). Another example of relations between the enzymes and mor- phogenesis is offered by the Physa egg. Mancuso ( 1955a ) showed Fig. 10. Larvae of Bithynia. a, control; b, c, after treatment with 7 X 10~2 ]Vf sodium azide for 4 hours, at the earliest stages of development (Attardo, 1955b). G. REVERBERI 335 that the embryos of this mollusc possess in the mantle and in the shell gland a KCN-sensitive oxidase, probably a M-Nadi oxidase. The treatment of the eggs with KCN, which blocks the enzyme, gives rise to embryos with defective or rudimentary mantles and shells (Fig. 12) (Mancuso, 1955c). However, we can add more evidence to the above results in the ascidian egg. This evidence comes from experiments on the d e f Fig. 11. Larvae of Phijsa (schematic representation of the heads). a, control; b to /, after treatment with 0.5% sodium azide for 4 hours (Man- cuso, 1955b). development in anaerobiosis (De Vincentiis, unpublished). In a partially oxygen-deprived atmosphere, that is, in conditions in which the cytochrome oxidase is poorly active, larvae develop which again are abnormal for the tail but normal for the "head," as in the case of the azide-treated larvae. Finally, to remove any objection to the specificity of this result and of the ones reported above, we shall mention some data obtained by Farinella-Ferruzza ( 1955 ) in ascidian eggs after 336 DEVELOPMENT OF ASCIDIANS treatment with a lithium salt. The larvae which developed after such treatment had normal tails, but were without brains, sen- sorial organs, and palps. In the ''Regulative" Eggs. The effects of the enzymatic block- ing on organogenesis have been particularly studied in the sea urchin egg. Many papers on this subject have been published by Rulon (1949, 1950, 1951, 1952, 1955), who organized a well- planned and systematic research. Rulon dedicated most of his attention to the blocking of cytochrome oxidase, succinodehy- Fig. 12. Larvae of Physa. a, control; b to /, after treatment with 0.005% potassium cyanide (Mancuso, 1955c), drogenase, peroxidase, phosphorylases, and enzymes with sulf- hydrylic radicals. He used, respectively, sodium azide, sodium malonate, or maleic acid, sodium selenite, thiourea, glucose, and zinc chloride. He obtained rather generalized abnormalities in the larvae, which also can be produced with other treatments and in that sense are not specific. For example, malonate does not produce the abnormalities that are produced by sodium azide. Both glucose and azide inhibit the phosphorylations, but their effects on development are very different. The results obtained by Rulon are certainly suggestive. However, one would like to know something about the local distribution of these enzymes in the G. REVERBERI 337 egg and on this point histochemical research is still largely want- ing. Other research on the effects of the blocking of the a-keto- glutarase or the aconitase by p-pyruvate or fluoroacetate in the sea urchin egg has been recently published by Montgomery and Bamberger ( 1955 ) . The inhibitors used do not produce any effect on the first stages of development. The embryos arrest at the blastula or early gastrula stages and do not differentiate — the effects of the inhibitors seem to be of rather general character. In the regulative eggs extensive research has been conducted on the amphibians. The eggs have been treated with many en- zymatic inhibitors, for example, KCN, sodium azide, and anaero- biosis, but as far as we know no localized, specific effects have been observed. The blocking of cholinesterase, which is largely present in the nervous system, was not followed by any abnor- mality in that system (Boell, 1946). Conclusions It seems, from the above reported data, that one can affirm with reasonable probability that some enzymes, at least, play a role in morphogenesis. This conclusion emerges more clearly for the mosaic eggs than for the regulative ones. In the latter, as has been mentioned, the inactivation of the enzymes induces abnoiTnalities of general rather than of specific character, as in mosaic eggs. The question arises concerning the mechanism by which the enzymes influence morphogenesis. I suppose that no one can pretend at the present stage of the biological sciences to give a plausible answer to that question. However, on the basis of the data presented above, we would like to offer the following inter- pretation. As we know, every morphogenetic character is under the control of the genes. Where then does the morphogenetic effect of the mitochondrial enzymes enter the picture? As Brachet (1952, 1954) pointed out in several of his papers, the nucleus is the site where nucleotides, either "nucleic acids or coenzymes" (Brachet, 1954, p. 96) are synthesized. It is quite possible that some of these coenzymes activate some mitochondrial enzymes. 338 DEVELOPMENT OF ASCIDIANS Thus the long chain of chemical reactions leading to differentia- tion and organogenesis would be initiated. REFERENCES Attardo, C. 1955a. Localizzazione della citocromo-ossidasi e del lipidi nell'uovo di Bitlujnia codiella. Ricerca sci., 25, 2797-2800. Attardo, C. 1955b. Effetti dell'azide sodico sulle nova di Bithynia codiella. Atti accad. Noz. Lincei, Rend., Ser. 8, 19, 83-84. Boell, E. J. 1946. The effect of di-isopropyl fluorophosphate on the development of behavior and cholinesterase in Ambhjstoma piinc- tatum. Anot. Record (Suppl.), 96, 4-5. Brachet, J. 1952. Le role du noyau cellulaire dans les oxydations et les phosphorylations. Biochim. et Biophys. Acta, 4, 221. Brachet, J. 1954. Nuclear control of enzymatic activities. Colston Pa- pers, 7, 91-102. Carrano, F., and F. Palazzo. 1955. Localizzazione precoce di alcuni enzimi nello sviluppo dell'uovo di Tubifex rivulorum. Riv. biol. (Perugia), 47, 193-202. Conklin, E. G. 1931. The development of centrifuged eggs of ascidians. /. Exptl. Zool, 60, 1-20. Duesberg, J. 1926. Etude cytologique des oeufs centrifuges de Ciona intestinalis. Arch. biol. (Liege), 36, 489. Farinella-Ferruzza, N. 1955. Lo sviluppo embrionale delle Ascidie dopo trattamento con LiCl. Pubbl. staz. zool. Napoli, 26, 42-54. Green, D. E., W. F. Loomis, and V. H. Auerbach. 1948. Studies on the cyclop horase system. I. The complete oxidation of pyruvic acid to carbon dioxide and water. /. Biol. Chem., 172, 389-403. Gustafson, T., and P. Lenicque. 1952. Studies on mitochondria in the developing sea-urchin egg. Exptl. Cell Research, 3, 251-74. Gustafson, T., and P. Lenicque. 1955. Studies on mitochondria in early cleavage stages of the sea urchin egg. Exptl. Cell Research, 8, 114- 17. Lehmann, F. E. 1941. Die Lagerung der Polplasmen des Tubifexeies in ihrer Abhangigkeit von der Eirinde. Natiirwis.senscliaften, 29, 101. Lehmann, F. E. 1950. Elektronenmikroskopische Untersuchungen an den Polplasmen von Tubifex und den Mikromeren von Paracen- trotus. Arch. Klaus-Stiftg., 25, 611-14. G. REVERBERI 339 Lehmann, F. E., and H. R. Wahli. 1954. Histochemische und elek- tronenmikroskopische Unterschiede im Cytoplasma den beiden Somatoblasten des Tubifexkeimes. Z. Zellforsch. u. mikroskop. Anaf., 39, 618-29. Lenicque, P. 1953. Etude sur revolution du chondriome au cours de la genese du tissu musculaire et de sa degenerescence provoquee par la colchicine. Arkiv ZooL, 5, 289-96. Mancuso, V. 1952. Ricerche istochimiche nell'uovo di Ascidie. II. Distribuzione delle ossidasi, perossidasi e della succinodeidrogenasi. Rend. ist. super, sanita, 15, 265-69. Mancuso, V. 1955a. Sostanze Nadi-positive nello sviluppo di Physa rivularis. Ricerca scL, 25, 2843-44, Mancuso, V. 1955b. L'azione dell'azide sodico sullo sviluppo del- I'uovo di Physa rivularis Ph. Riv. hiol. {Perugia), 47, 203-7. Mancuso, V. 1955c. Azione del cianuro di potassio sullo sviluppo di Physa rivularis. Rend, accad. naz. Lincei, ser. 8, 19, 71-73. Meves, Fr. 1913. tJber des Verhaltenes plastosomatischen Bestand- teiles des Spermiums bei der Befruchtung des Eies von PhaUusia mamillata. Arch, mikroskop. Anat., 82, 215. Minganti, A. 1950. Acidi nucleici e fosfatasi nello sviluppo della Limnaea. Riv. hiol. {Perugia), 42, 295. Montgomery, C. M., and J. W. Bamberger. 1955. Action of parapy- ruvate on early development of Strongylocentrotus purpuratus. Sci- ence, 122, 967-68. Palazzo, F. 1955. Effetti dell'azide sodico suU'uovo di Tubifex rivu- lorum. Ricerca sci., 25, 2873-76. Pitotti, M. 1947. La distribuzione delle ossidasi e perossidasi nelle uova di Myzostoma, Beroe e Nereis. Pubbl. staz. zool. Napoli, 21, 93-100. Raven, Chr. P., and N. Spronk. 1952. The action of beryllium on the development of Limnaea stagnalis. Koninkl. Ned. Akad. Weten- schap., 55, 541-53. Reverberi, G. 1956a. Experientia, in press. Reverberi, G. 1956b. Pubbl. staz. zool. Napoli, in press. Reverberi, G., and M. Pitotti. 1939. Differenziazioni fisiologiche del- I'uovo delle Ascidie. Commens. Pont. Acad. Sci., 3, 469-88. Reverberi, G., and M. Pitotti. 1940. Ricerche sulla distribuzione delle ossidasi e perossidasi lungo il "cell-lineage" di uova a mosaico. Pubbl. staz. zool. Napoli, 18, 250-63. Ries, E. 1937. Die Verteilung von Vitamin C, Glutathion, Benzidin- 340 DEVELOPMENT OF ASCIDIANS Peroxydase, Phenolase und Leukomethylenblau-Oxydoreducase wahrend der friihen Embryonalentwicklung verschiedener wirbel- loser Tiere. Pubbl. staz. zool. Napoli, 16, 363^01. Ries, E. 1939. Versuche iiber die Bedeutung des Substanzmosaiks fiir die embryonale Gewebedifferenzierung bei Ascidien. Arch. Exptl. Zellforsch., 23, 95-121. Ries, E., and M. Gersch. 1936. Die ZelldifFerenzierung und Zellspe- zialisierung wahrend der Embryonalentwicklung von Aplysia li- macina L. Zugleich ein Beitrag zu Problemen der vitalen Farbung. Puhhl. staz. zool. Napoli, 15, 223-27. Rulon, O. 1949. The modification of developmental patterns in the sand dollar with maleic acid. Phijsiol. Zool, 22, 247-61. Rulon, O. 1950. The modification of developmental patterns in the sand dollar with sodium azide. Physiol. Zool, 23, 236-47. Rulon, O. 1951. The modification of developmental patterns in the sand dollar by malonic acid. Physiol. Zool, 24, 85-92. Rulon, O. 1952. The modification of developmental patterns in the sand dollar by sodium selenite. Phijsiol Zool, 25, 333-46. Rulon, O. 1955. Developmental modifications in the sand dollar caused by zinc chloride and prevented by glutathione. Biol. Bull, 109, 316-27. Shaver, J. R. 1953. Studies on the initiation of cleavage in the frog egg. /. Exptl Zool, 122, 169-92. Shaver, J. R. 1955. The distribution of mitochondria in sea urchin eggs. Experientia, 11, 351. Tung, T. C., S. H. Ku, and F. Y. F. Tung. 1941. The development of the Ascidian egg centrifuged before fertilization. Biol. Bull, 80, 153-69. IMMUNOLOGICAL STUDIES OF EARLY DEVELOPMENT ALBERT TYLER: kerckhoff laboratories of biology, CALIFORNIA INSTITUTE OF TECHNOLOGY, PASADENA, CALIFORNIA* During recent years there has been mcreasmg interest in the apphcation of immunological methods and concepts to the anal- ysis of problems of the formation and early development of the embryo. The subject was reviewed a short while ago by the au- thor (Tyler, 1955b). In this paper some of the newer develop- ments in this field are presented along with pertinent background of earlier work. The material to be considered concerns the origin of adult antigens and of antibody-forming capacity, effects of antibodies on development, and a concept of natural auto-anti- bodies. A brief account is also included of some current experi- ments of the author on inhibition of cleavage by antisera. Immunologically produced antibodies possess the capacity to react selectively with the specific substances (antigens) em- ployed to induce their formation and with substances possessing certain related stmctural features. The classical experiments of Landsteiner (1917-1946) showed that determinants of antigenic specificity may be represented by relatively small chemical groups on a large complex molecule rather than by the structure of the molecule as a whole. This is illustrated in experiments in which a small molecular substance of known structure, such as arsanilic acid, is conjugated with proteins, such as those of horse serum. When this antigen is injected into a rabbit the antiserum that is * Preparation of this article and original investigations of the author reported herein were supported by a research grant (No. C-2302) from the National Cancer Institute, of the National Institutes of Health, Public Health Service. 341 342 IMMUNOLOGICAL STUDIES produced reacts not only with the original antigen but also with other proteins, such as those of chicken serum, that have been coupled with arsanilic acid. The antiserum reacts also with ordi- nary horse serum, with arsanilic acid alone (no visible precipi- tate ) and with "haptens" structurally similar to the arsanilic acid, but not with chicken serum; nor does an antiserum against horse serum react with chicken serum. From the many experiments of this type with which the immunological literature abounds it is now clear that relatively small parts of a large complex molecule can act as determinants of the specificity of antigens and anti- bodies. Immunochemical research has not, as yet, provided very much information as to the size, number, and diversity of structure of the determinant groups of various native proteins and other large molecular substances of interest to biologists. Landsteiner (1942) showed that products of the hydrolysis of silk protein with molecular weight of about 600 possessed such activity. Another example of investigations in this direction that may be mentioned is the current work of Kabat and Leskowitz ( 1955; cf. Watkins and Morgan, 1955) showing that certain simple sugars possessed the ability to react specifically with antibodies against human blood group substance. They suggest that the determinant struc- tures of the blood group mucopolysaccharidic antigens are prob- ably no larger than tri- to penta- or hexasaccharides. One of the aims of embryologists in applying immunological methods is the determination of the time of appearance of specific constituents of the adult organs. However, this approach is beset with various difficulties, which have not apparently been appre- ciated by all the workers in the field. The prime difficulty is in knowing what is being detected by the antiserum that is used as the test reagent. As indicated above antibodies can detect cer- tain specific structures of a large molecule. A particular protein may possess a number of such structures and they may be of several sorts, each of which may induce the formation of distinct antibodies in the serum, as is illustrated in the recent work of Francis et al. (1955) with "multi-haptenic" antigens. If some of the determinant groups are common to different proteins of an A. TYLER 343 organism, as illustrated in Fig. 1, or if there are similarities in structure, cross reactions are to be expected. Cross-reacting an- tisera are also obtained if the preparation of immunizing antigen contains other active substances as impurities. However, it is not easy to decide in different cases to what extent cross reactions are due to impurities in the original immunizing antigen and to what extent these are due to similarities in determinant groups. Even with highly purified proteins used as antigens, such as the proteins antl-A anti-X antl-B antl-X anti-A antl-Y antl-A-antl-X (antl-AX?) antl-B- anti-X (antl-BX?) antl-A-antl-Y (anti-AY?) Fig. 1. Illustration of the assumption that a particular protein may possess two (or more) kinds of determinant groups, one of which (X) may be present also on other proteins of the same species of organism and the other of which (A) may be present on the same type of protein in a different species. The types of antibodies that may be expected upon im- munization are listed below each antigen. See text for discussion in rela- tion to cross reactions. of chicken egg-white studied by Cohn, Wetter, and Deutsch (1949), cross-reacting antibodies are obtained. Absorption techniques eliminate to some extent certain of these difficulties. However, it is perhaps important to emphasize that in the use of an antiserum as a test reagent, one does not detect a specific protein or other large molecular substance but rather specific determinant groups. These do not necessarily characterize one particular ( chemically defined ) protein, and it is also possible that during embiyonic development they may not remain con- tinuously with the same protein. The term antigen is, therefore, 344 IMMUNOLOGICAL STUDIES used in this discussion primarily with reference to the determi- nant groups. If extracts of various tissues and organs of the adult contained solely or primarily antigens characteristic of the tissue or organ, problems of detection of these antigens in the developing embryo could be rather directly approached. Examples of such organ- specific preparations can be found in the immunological litera- ture, but they are not as numerous or clear-cut as might be de- sired. The classical example is that of lens protein, first described many years ago by Uhlenhuth ( 1903 ) . Antisera against saline- soluble proteins of the lens of the vertebrate eye do not cross- react with other proteins of the same organism. On the other hand, they are largely lacking in species specificity since they cross-react quite generally with lens extracts from various verte- brates from fish to man. Alcoholic extracts of vertebrate brain have also been shown to possess such organ-specific antigenicity although in this case there are cross reactions with testis (see Witebsky, 1929; Lewis, 1933). Another example is thyroglobulin, recently studied by Witebsky et at. (1955). References to other work on this subject may be found in various texts such as those of Landsteiner (1947), Loeb (1945), and Raffel (1953). In most cases the antibodies against various organ or tissue extracts show rather broad cross reactivity. Selective absorption procedures must therefore generally be used to demonstrate distinct anti- genic constituents in various organs, but this suffers from tech- nical limitations when most of the antibodies in an antiserum against a particular preparation are directed against antigens common to various tissues and organs. Relation of Embryonic to Adult Antigens At one time it was thought that eggs and developing embryos possessed no antigens similar to those of the adult, or even that their proteins were of such "general" nature as to lack antigenic properties. The work of many recent investigators has abundantly demonstrated in many species of animals that the eggs and devel- oping embryos possess antigenically active materials, that many of the antigens are similar to certain adult antigens, and that A. TYLER 345 changes occur in extractable and cellular antigens during devel- opment. For reference to investigations in this field during the past decade may be cited the work on frogs by Cooper (1946, 1948, 1950), Ten Gate and Van Doorenmaalen (1950), Flick- inger and Nace (1952), and Spar (1953); on salamanders by Woerdeman (1950, 1953a,b) and Clayton (1953); on birds by Schechtman (1947, 1948, 1952, 1955), Nace and Schechtman (1948), Briles, McGibbon, and Irwin (1948; cf. Ii-win, 1949, 1951), Schechtman and Nace (1950), Ten Cate and Van Dooren- maalen (1950), Schechtman and Hoffman ( 1952 ) , Ebert (1950, 1951, 1952, 1953, 1954, 1955), Miller (1953), and Nace (1953); on mammals by Maculla (1948), Yeas (1949), Chernoff (1953), and Goodman and Campbell (1953); on sea urchins by Perlmann and Gustafson (1948), Perlmann (1953), and Harding, Harding, and Perlmann ( 1954 ) ; and on silkworms by Telfer and Williams ( 1953 ) and Telfer ( 1954 ) . Most of this work has been recently reviewed (see Ebert, 1955; Nace, 1955; Tyler, 1955b) and the present discussion will be limited to a brief consideration of cer- tain special features. One must recognize in the first place that technically it is not possible at present to prove the complete absence of an antigen; that failure to detect an antigen does not prove its absence. Thus Ten Cate and Van Doorenmaalen ( 1950 ) detected lens antigen at earlier stages of development than had Burke et al. (1944). They attribute this to the use of more sensitive procedures. This suggests that the antigen might be detected at still earlier stages of development if further technical sensitivity were achieved. The concentration of lens antigen evidently decreases the earlier the embryonic stage tested, but there is no clear justification for the assumption that it extrapolates to zero. The same considera- tions apply to the interpretation of the various experiments cited above that deal with other organ-specific antigens, such as those of brain, spleen, kidney, and heart. In general, then, the question of preformation in embryonic development is raised again, but in a manner that does not permit easy resolution. Many antigens, such as those of vertebrate red cells that characterize blood groups and blood types, are known to be gene determined. It is 346 IMMUNOLOGICAL STUDIES reasonable to assume that the specific structure of any antigen is represented by some special feature of a corresponding gene. For example, this feature can be considered a structure complemen- tary to that of the antigen (see p. 365). In this sense all antigens would be regarded as preformed in the fertilized egg. At the same time, the actual appearance of the antigen would be epigenetic, dependent upon the time and extent of action of the particular gene. It is also possible that all antigens are present, but some in undetectable amounts, along with their determinant genes (or other self-reproducing cytoplasmic body ) at the start of develop- ment. In either case the processes involved in the determination and differentiation of the various tissues and organs would be as- sumed to entail concomitant activity of specific genes, determin- ing also the formation of characteristic antigens of the tissue or organ. Despite the inability to prove complete absence of an antigen one may, nevertheless, raise the question of whether or not spe- cific "organ" antigens can be detected before the time of visible differentiation. The evidence in the above-cited investigations, although rather incomplete, would answer this in the affirmative. For example, specific antigens of lens, brain, spleen, and heart have been detected at stages well in advance of the differentia- tion of the particular organ. It has also been reported (Woerde- man, 1953a,b, 1955) that lens antigen can be induced to appear in extracts of presumptive lens ectoderm by incubating in the presence of extracts of optic vesicle. A large fraction of the antigens of adult tissue are detectable in the uncleaved egg and even in the oocyte. This appears to be especially true for antigens of adult serum or serum-like antigens of adult organs as Cooper ( 1946 ) showed in the first work to give real impetus to the current investigations on soluble antigens. The extent to which egg and adult serum have antigens in common is illustrated in the work of Schechtman and Hoffman (1952) and of Nace ( 1953 ) . In this work antisera that were produced against whole chicken serum or the separate albumin and globulin frac- tions were found to cross-react quite strongly with egg yolk. Ab- sorption with yolk greatly reduced the reactivity of these antisera A. TYLER 347 with the serum fractions. For example, an antiserum against whole serum which gave a titer of 0.0004, in terms of milligrams of protein in the highest reacting dilution of the antigen, gave a titer of 0.029, a seventy-fold decrease, after absorption with yolk (see Nace, 1953, Table 1, p. 430). With the other antisera against the various separated serum proteins (albumin, a,i8-globulin, y-globulin) there was similar loss in reactivity with original anti- gen upon absorption with yolk or with heterologous serum pro- teins. As noted by these authors contamination of the yolk, preparations with serum can be ruled out as an explanation of the results. They suggest (Nace, 1953, p. 440) that the yolk pro- teins may be a direct transudate from the maternal serum, or that there is a mechanism for resynthesis of antigens very similar to serum from degradation products of serum that pass into the yolk. However, since these experiments and those of others show that some reactivity for serrmi constituents remains after absorp- tion with yolk, it is evident that neither explanation is satisfactory unless it is further assumed that there is a selectivity as to which serum proteins transudate or as to which antigens are resynthe- sized. The same sort of difficulty arises in connection with the presence of serum-like antigens in other tissues of the adult and the developing embryo. An alternative explanation would be based on the suggestion, presented at the beginning of this paper, that many of the chem- ically different proteins, or other large molecular substances, of an organism may each have identical or similar antigenic deter- minant groups along with others that are characteristic of the particular substance. On this basis the reaction of yolk proteins with an antiserum against serum proteins would be attributed to the similarity or identity of one or more of the determinant groups. The great reduction in reactivity of the antiserum with serum proteins after absoiption with yolk indicates that most of the antibodies are directed against these common antigenic struc- tures. The remaining activity after such absorption is due to anti- bodies directed against determinants characteristic of the partic- ular serum proteins. However, it should be emphasized again that even such absorbed" sera do not necessarily detect the particular 348 IMMUNOLOGICAL STUDIES protein against which they were originally produced but rather certain determinant groups. Thus statements as to the time of detection of various proteins during development must be quali- fied to the effect that the substance is not necessarily chemically the same as that in the adult, since the antisera identify only cer- tain parts of the molecule. The cited experiments on the serum proteins of the chick show that the determinant groups of serum albumin are present in sufficient quantity to be detected, by the absorbed antisera employed, at about the fifth day of incubation; those of a,/3-globulin at about the sixth day, and those of -/-globu- lin at the ninth to twelfth day. The only other detailed immuno-embryological experiments with highly purified proteins that have been so far reported are those of Ebert (1953, 1955) on myosin and actin. By the use of antisera made specific for cardiac myosin by absorption with skel- etal myosin he has detected determinant groups of cardiac myo- sin in chick blastoderms in the mid-primitive streak stage, but not earlier. Tests with various portions of the blastoderm at vari- ous stages indicated that the determinant groups are detected throughout the blastoderm (perhaps confined to the epiblast) at the mid-streak stage, but become restricted in the head process and head fold stages to two lateral areas corresponding roughly to the location of potential heart-forming areas (Rawles, 1943). The determinant groups of cardiac actin ( or at least one antigenic component thereof ) were first detected in the head-process stage, with localization similar to that of the myosin. There is, then, evidence that at least the determinant groups of certain proteins ( if not the proteins themselves ) characteristic of adult tissues or organs appear in detectable amounts prior to difl^erentiation of the particular tissue or organ, and that their distribution may coincide with the corresponding "organ-form- ing" areas of the early embryo. This suggests possible causal con- nection with processes of determination and diff^erentiation of various organs and tissues in the embryo, since the general fea- tures of the results obtained with lens, heart, serum proteins, etc., can be assumed to be representative of what may be found for other tissues and organs. A. TYLER 349 In addition to the studies with purified proteins there have been a number (e.g., FHckinger and Nace, 1952; Spar, 1953; Clayton, 1953 ) in which antisera produced against sahne extracts of early embryos have been used, after appropriate absorptions, to detect changes in antigenic composition during early devel- opment. Different stages of development and even different em- bryonic germ layers have thus been shown to provide extracts in which a different assortment of antigens is detectable, new anti- gens becoming detectable at certain stages of development and some apparently disappearing. In these, as well as in the experi- ments with antisera against highly purified adult substances tested against saline extracts of early embryo, there is the uncer- tainty as to whether or not a particular antigenic structure re- mains associated with a saline-soluble constituent, and as to whether or not it is available for reaction in specific absorption procedures. In studies with cellular antigens, such as those of the red blood cells (e.g., Briles et al, 1948; Miller, 1953; Yeas, 1949; cf. Irwin, 1949, 1951; Levine, 1948), the analogous question is to what extent the change in surface antigen that is detected during development represents a change in location rather than new synthesis. For the immediate future the main utility of studies of the type discussed above would appear to be that they illustrate how im- munological procedures may be employed to demonstrate specific chemical changes in an embryonic tissue prior to its actual dif- ferentiation. Further studies of this type, it may be hoped, will shed some light on the question of whether or not this approach will provide pertinent chemical information of some primary change occuiTing in embryonic tissues during the otherwise in- visible process of "determination." Antibody Formation Many early investigators have demonstrated that the early embryo, the fetus, and even the newborn animal lack the capac- ity to produce antibodies ( detectable .in the serum ) in response to the injection of a foreign antigen (see reviews by Needham, 1942; Beveridge and Burnet, 1946; Burnet and Fenner, 1949; 350 IMMUNOLOGICAL STUDIES Topley and Wilson, 1946). Even natural antibodies, such as the isoagglutinins of the human blood groups do not appear in the serum until some time after birth ( see Wiener, 1943 ) . Antibodies that are found in the newborn can be attributed to transfer from the mother through the placenta, in the case of manmials, or to the egg in the case of birds. The discovery by Levine et al. ( 1939, 1941 ) of isoimmunization of the mother by fetal cells demon- strated at the same time, dramatically, the transfer of maternal antibodies to the fetus. Experiments on immunization of the fetus in mammals would be complicated by uncertainties as to whether or not the antigen reached the maternal circulation and antibod- ies produced there passed back into the fetus. However, even the newborn mammal evidently lacks antibody-forming capacity as shown, for example, in experiments by Freund ( 1930 ) on rabbits. In chickens various experiments (cited by Tyler, 1955b) have shown no antibody formation before the fifteenth day of incuba- tion and very weak if any activity at the time of hatching (cf. Wolfe and Dilks, 1948). Correlated with this apparent lack of antibody-forming capac- ity is the ability of foreign tissue grafts to establish themselves in the embryo whereas they consistently fail in the adult. Lack of antibody-forming capacity has also been assumed to account for the ready growth of various viruses on membranes and tissues of chick and mammalian embiyos. It has been previously suggested (Tyler, 1955b) that the vari- ous experiments need not be interpreted as indicating inability of cells of the developing embryo to manufacture antibodies. Quite possibly the embryonic cells do produce antibodies, in re- sponse to foreign antigens, without releasing them into the fluids that are customarily tested. Since the embryo, fetus, and new- born are in a process of rapid gi'owth it is quite conceivable that such antibodies as they might produce would remain a part of the structural proteins of the cell. Thus they would not appear in serum, or even be available for tissue-incompatibility reac- tions. This is consistent with experiments on cells grown in tissue culture where, for example, it has been shown that rat and mouse tissues exhibit no incompatibility in mixed cultures (Harris, A. TYLER 351 1943), even when one of the animals has been previously immu- nized against tissues of the other (Grobstein and Youngner, 1949). As far as phagocytic ability is concerned, tests have shown that this is present in the cells of all three germ layers of early ( 2-day ) chick embryos and becomes progressively delimited to the famil- iar sites of the mature organism (Heine, 1936; Steinmiiller, 1937). Not only do homoplastic and heteroplastic grafts succeed well in embryos but the foreign cells may also persist well into the adult, as illustrated in the experiments of Willier and Rawles (1940, 1944) on neural crest tissue. The same sort of persistence of foreign cells is evident in the obsei-vations of Owen ( 1945 ) on erythrocyte mosaicism of cattle twins, which can be attributed to a transfer of erythropoietic tissue from one twin to the other through the conjoined placentas, a phenomenon also recently re- ported to occur in human twins (Dunsford et al., 1953). Such persistence implies that the adult has become incapable of form- ing antibodies, or a destiiictive amount of antibody, against the foreign antigen introduced in the embryonic stage. The recent remarkable experiments of Billingham, Brent, and Medawar (1953, 1955a,b, 1956) have now demonstrated such failure to react immunologically against specific antigens on the part of adult animals that had been injected with the anti- gen during fetal life. In particular they showed that adult mice of CBA strain that had been injected in utero with tissue (chopped-up adult testis, kidney, and spleen) of A strain can tolerate A strain skin grafts, whereas without such injection the typical incompatibility reaction would ensue. In chickens they accomplished the same thing by transfusing blood between em- bryos of different strains. They termed this phenomenon "actively acquired tolerance" to skin grafts, but it is quite clear from the earlier analyses of the incompatibility reaction that this relates to specific antibody-forming ability. They showed also that the adult lacks only ability to respond to the specific antigens that it re- ceived in fetal life, its ability to form antibodies against other antigens being unimpaired. Furthermore, if lymph nodes from a normal animal are transplanted to a tolerant animal, the latter 352 IMMUNOLOGICAL STUDIES can now react to a tolerated skin graft. Evidently the host's own antibody-forming cells do not respond to the antigen received in fetal life, but antibody-forming cells received from another animal (same strain) can react to antigens in the graft or else- where in the body. This work has now been extended to a number of other sys- tems. In rats it has been reported (Woodruff and Simpson, 1955) that the tolerance to skin grafts can be induced by injection with cells of the prospective donor at the day of birth. In experiments with tumor transplants, mouse Crocker sarcoma S 180 has been found to grow to large size, but not indefinitely, when implanted into rats that had been injected during fetal life with mouse tissue (BoUag, 1955). An ascites tumor of mice {6CSHED lymphosar- coma) has been gotten to grow in strains of mice in which it would ordinarily regress by injection of the 16- to 17-day fetuses with donor strain blood or tumor cells ( Koprowski, 1955 ) . In the latter experiments the tumor cells undergo a change in antigenic specificity upon growth on the tolerant, foreign strain, mouse. A great many earlier experiments particularly by Snell, Kaliss, and their co-workers (see Snell, 1952; Kaliss, 1955) have demon- strated a "conditioning the host" to tumor homografts by pre- injection with various tissue (normal or tumor) homogenates or extracts. This appears to be related to the acquired tolerance phe- nomenon, although the effect is produced by injection of adult animals immediately prior to tumor grafting and can be produced also by injection of antisera (prepared in mice or rabbits) to donor strain mouse tissues. Acquired tolerance is also indicated in experiments in which chick embryos have been injected with killed Salmonella piillorem and the hatched chickens later found to show marked decrease in ability to form antibodies against this antigen ( Buxton, 1954 ) . Similarly the antibody response of cattle to Trichomotms foetus ( freeze-dried or acetone-dried) has been inhibited (Kerr and Robertson, 1954) by injection of this antigen into newborn calves. Specific inhibition of antibody-forming capacity has also been demonstrated by use of simpler protein antigens, such as bovine serum albumin and human serum albumin injected into rabbits A. TYLER 353 during the first days after birth (Hanan and Oyama, 1954; Dixon and Maurer, 1955; Cinader and Dubert, 1955). The unresponsive- ness persists long after the antigen becomes undetectable in the serum, although possibly persistent in the tissues, and does not affect the ability of the animal to respond to an unrelated antigen. Bilhngham et al. ( 1955a,b, 1956 ) suggest that the immunologi- cal paralysis induced by large doses of pneumococcal polysac- charide ( Felton, 1949 ) might be related to the "actively acquired tolerance." However, this seems to involve neutralization of anti- body, as it is formed, by the pneumococcal polysaccharide antigen fixed in the tissues (cf. Dixon and Maurer, 1955), rather than a "central derangement of the antibody-forming capacity" as Bil- lingham et al. describe their results. One possibility that has been suggested (Tyler, 1955b) to account for such a central derange- ment is that the host cells concerned in antibody formation un- dergo a "type-transfonnation" of the sort exhibited by microor- ganisms in response to specific DNA. The experiments with antigens such as serum albumin would rule out a direct action of specific nucleic acid, in the sense of genetic alteration of anti- body-forming cells by DNA. Alternatively one might assume (as suggested on p. 350) that the embryo or newborn manufactures antibodies against foreign antigens but retains them as an integral part of the cell. As such these fixed antibodies might serve as templates (see "The Concept of Natural Auto-Antibodies," be- low) for the formation of antigenic structures of the type origi- nally introduced. If cells of the adult organism have thus acquired a particular antigenic specificity they would not be expected to respond to the same antigen. Other possible interpretations have been discussed by the cited investigators in this field. Since it is still uncertain as to whether or not all the reported experiments represent the same kind of acquired tolerance and since the field is being actively explored, one may expect that current research will soon provide some of the answers. In connection with the above-cited work it is of interest to re- call some early experiments of Murphy (1914a,b). He showed that tumor tissue of a rat could grow readily in the avian embryo, but when the foreign implants were made at the time of hatch- 354 IMMUNOLOGICAL STUDIES ing, they were quickly destroyed. In tissue culture in chicken plasma, rat sarcoma cells grew well in the presence of adult chicken connective tissue, kidney, or liver. However, pieces of adult chicken spleen caused practically total inhibition of growth of the rat sarcoma cells. Chicken bone maiTOw caused a definite retardation but not so marked an inhibition as that brought about by spleen. When the combinations were made in vivo by implan- tation on the outer membrane of the seven-day chick embryo the results were similar, the grafted adult chicken spleen causing a rapid regi"ession of the rat sarcoma tissue. Murphy also demon- strated that pretreatment of adult rats with sublethal total body x-irradiation rendered the animals tolerant to implantation of mouse sarcoma. Recently Lindsley et al. ( 1955 ) have shown that a functional implant of erythropoietic cells can be established in x-irradiated rats by injection of homologous bone marrow carry- ing an immunogenetic marker, and Makinodan (1956) has found that when lethally irradiated mice are injected with rat bone mar- row, being thus protected from the 30-day irradiation death, the mouse red cells can be completely replaced by those of the rat. Effects of Antibodies on Development It is well known that antibodies produced against various kinds of cells and tissues can have a destiaictive action on the homolo- gous material. Reviews of such cytotoxic activity may be found in various immunological texts (e.g., Raffel, 1953). Of more spe- cial interest would be examples of antibody action that were not necessarily lethal and led to specific alterations in development. There have been reports of stimulating efi^ects of antisera, but these have largely lacked substantiation. One of the most noted of these is the reticulo-endothelial immune serum of Bogomolets ( 1943 ) , which has been described as a veritable cure-all. While cytotoxic or inhibiting in high concentration, the serum has been claimed to be highly stimulating to cellular growth and activity in low doses. Experiments by Pomerat (1945, 1946, 1949) and others have failed to confirm the claims. A relative stimulation of growth of homologous organs was reported by Weiss ( 1947 ) in experiments in which antisera against cytolyzed adult liver or A. TYLER 355 kidney were injected into hen's eggs at 2/2 to 8 days of incubation. However, Weiss ( 1953b, 1955 ) more recently considered this to be due to hemorrhage caused by vascular damage rather than to specific stimulation of growth by the antisera. Apart from the above-mentioned work, experiments on the effects of antisera on development deal with inhibitory effects. Much of the current work in this field has been summarized by Nace (1955). In most cases the antisera have been prepared against rather complex extracts, and the various inhibitory eflFects are not readily interpretable in specific terms. Perhaps the best examples of specific action of antisera on embryonic tissues stem from the discovery that destruction of fetal blood cells in human cases of hemolytic disease of the newborn was due to the action of an antibody produced in the mother in response to isoimmuni- zation with fetal antigen (Levine and Stetson, 1939; Levine et al., 1941). This discovery has led to the identification of a large num- ber of human blood cell antigens ( see Levine, 1954 ) in addition to the now well-known Rh factor ( Landsteiner and Wiener, 1940 ) and its variants that were concerned in the original observations. The action of the antisera is evidently specifically on the fetal red cells, other clinical and pathological features of the disease be- ing referable to blood cell destruction ( Levine et al., 1941 ) . This is, then, a selective action of an antiserum on a tissue of the de- veloping organism and one in which much is known, at least genetically, about the particular antigens involved. Unfortunately embryological studies in this field have not kept pace with the genetic, ethnological, and technical advances. This is understand- able by virtue of the relative inaccessibility of the material. The Rh factor has been demonstrated in a 5-cm fetus ( Stratton, 1943; cf. Levine, 1948). However, the effects of isoimmunization ap- pear to be exerted on the almost fully developed fetus or on the newborn infant. There is, of course, uncertainty as to just when the antibodies are produced and reach the fetus in effective con- centration. Experiments with lower mammals in which similar isoimmunization has been reported (see Coombs, 1949; Kellner and Hedal, 1953) may be expected to provide more information of embryological significance. 356 IMMUNOLOGICAL STUDIES An example of a specific effect with an antiserum against a highly purified antigen is contained in the work of Ebert ( 1955 ) . Antisera against cardiac actin when administered in ovo to chick embryos at 48 to 60 hours blocked further development of the heart and resulted in death of the embryo. With appropriately diluted antisera against saline extracts of adult chicken heart, brain, and spleen Ebert ( 1950 ) had earlier reported specific in- hibitory effects on the development of the homologous organ. The more concentrated antisera gave nonspecific lethal and growth- inhibitory effects even after absorption with heterologous organ extracts. Johnson and Leone (1955) report that anti-actomyosin inhibits morphogenesis of the heart in chick embryos, but the de- gree of specificity is not clear since there is also general inhibi- tion of development. Antisera against lens protein have also been reported to produce specific lens damage in 6- to 8-day chick em- bryos ( Burke et ah, , 1944 ) . Experiments with antisera against saline extracts of amphibian embryos by Flickinger and Nace ( 1952 ) and by Clayton ( 1953 ) have given some indication of stage-specific inhibitory action, but the authors consider further tests necessary to establish the selectivity of the action. Nettle- ship (1953) reported that antisera against chicken whole embryo brei, when injected into the incubating egg, blocked development at the corresponding stage. For an un absorbed antiserum of this type to produce a highly specific effect does seem surprising, and one wonders whether or not this might be due to fortuitous varia- tion in the antibody content of the various antisera. The experiments cited point to possible uses of antisera in mod- ifying development in specific ways. As yet, however, they have been too limited in scope to provide information that can be used for further analysis of problems of induction and determination in early development. Inhibition of Cleavage in Sea Urchins by Specific Antisera The author has for many years prepared antisera against vari- ous constituents of the spemi, eggs, developing embryos, and adult tissues of sea urchins as part of a program of investigation A. TYLER 357 of problems of fertilization and early development (of. Tyler, 1955a,b). One aspect of this work, which has now been reported (Tyler and Brookbank, 1955; 1956a,b), will be presented here. Antisera prepared against extracts of developing sea urchin eggs were found to block cleavage of the eggs. At first there ap- peared to be a stage-specific effect, antisera against eggs in early cleavage blocking early, those against later cleavage blocking later. However, this was found to be illusory, the time of cleavage block being dependent upon the strength of antiserum employed. Antisera against eggs in early stages would block at any specific later stage if appropriately diluted, and anti-late stage could block early if sufficiently strong. Immunological literature abounds with experiments showing inhibition of cell division by action of antisera against cells of many kinds of organisms. In the field of cancer research this is an especially active line of investigation, the efforts being di- rected toward the production of antisera that act specifically on the tumor cells and not on normal tissue cells. Various types of material ranging from whole tissue homogenates to washed par- ticulate or viral suspensions have been used as antigens, and in- hibitory effects of the antisera have been described in in vitro and in vivo experiments. This work cannot be reviewed here, but for purpose of reference to some of the investigations in this field may be cited experiments and reviews by Woglom ( 1929 ) , Lums- den (1937), Phelps (1937), Sigurdsson (1942), Spencer (1942), Kidd (1946), Green (1946), Dulaney and Amesen (1949), Law and Malmgren (1951), Barrett (1952), Werder et al. (1952), Hauschka (1952), Nungester and Fisher (1954), Imagawa et al. (1954a,b), and Mountain (1955). A new feature of the present work is that the inhibition of cell division can be obtained with an antiserum produced against a chemically rather well-defined constituent, namely the substance of the gelatinous coat of the egg, known as fertilizin. Tests of antisera produced against the various sea urchin ma- terials showed that the fertilizin antisera were particularly effec- tive in blocking cell division. A summary is given in Table I of the number of rabbits that produced cleavage-blocking antisera 358 IMMUNOLOGICAL STUDIES Table I. Production of Antisera That Block Cleavage of Eggs of Strongylocentrotus purpuratus and Lytechinus pictus (data revised 3/14/57) No. producing antisera that No. of block cleavage of: S. purpuratus L. pictus Immunizing antigen rabbits eggs eggs None 65 1 1 S. purpuratus fertilizin 21 21 21 univalent fertilizin 3 3 2 feitilizin-treated sperm 3 1 1 whole sperm 5 0 0 antifertilizin from sperm 6 1 1 nucleoprotein from sperm 1 0 0 antifertilizin from eggs 6 3 3 extract of fertilized eggs 1 — 1 extract of hatched blastulae 1 1 1 extract of gastrulae 2 2 2 extract of prism larvae 2 2 2 extract of pluteus larvae 2 2 2 blood 3 0 0 extract of epidermis 2 0 0 L. pichis fertilizin 6 4 4 fertilizin-treated sperm 2 2 2 whole sperm 2 0 0 antifertilizin from sperm 6 0 0 antifertilizin from eggs 1 1 1 extract of fertilized eggs 1 — 1 homogenate of 16-cell stage 1 1 1 extract of hatched blastulae 2 2 2 extract of gastrulae 1 1 1 extract of prism larvae 1 1 1 extract of epidermis 2 0 0 S. franciscanus fertilizin 2 2 2 whole sperm 1 0 0 A. punctulata fertilizin 2 0 0 antifertilizin from sperm 1 0 0 antifertilizin from eggs 2 0 0 blood 1 0 0 A. TYLER 359 in response to immunization with various materials of four spe- cies of sea urchins. It may be noted that the fertihzin antisera are consistently effective in this respect and that they are cross reac- tive among three of the species used (S. purpuratus, L. picfus, and S. franciscanus) , but possibly not with the fourth (A. punc- tulata ) . In control sera, cleavage and development generally pro- ceeded normally to swimming late blastula or early gastrula stage. Details of the results with the various antisera cannot be given here. It may be noted, however, that the listing in Table I refers to antisera that blocked cleavage effectively within the space of one division after treatment. Some of the antisera gave retarda- tion of cleavage and cessation of development at a late cleavage stage. The majority of the nonblocking antisera permitted devel- opment to proceed apace with the controls. The absorption with whole sperm, of blocking antisera pro- duced against fertilizin or extracts of fertilized eggs, does not remove the cleavage-blocking effect (Table II). However, an antiserum against sperm extract (antifertilizin) that had retard- ing action lost this property upon absorption with whole spenn. Table II. Absorption of Cleavage-Blocking Antisera Antiserum vs. Absorbed with Cleavage block S. purpuratus fertilizin — + fertilizin S. purpuratus fertilizin — fertilizin S. purpuratus sperm 4- fertilizin L. pictus sperm + L. pictus fertilizin — + fertilizin L. pictus sperm + unfertilized eggs — + unfertilized eggs L. pictus sperm + unfertilized eggs L. pictus fertilized eggs — fertilized eggs — + fertilized eggs L. pictus sperm + 16-cell stage — + 16-cell stage L. pictus 16-cell stage — 16-cell stage S. purpuratus sperm +_ antifertilizin — Partial antifertilizin L. pictus sperm — 360 IMMUNOLOGICAL STUDIES In the performance of the tests of inhibition of cleavage the gelatinous coat ( fertilizin ) and fertilization membrane of the egg are generally first removed before addition of the antiserum. The antisera also act, but usually much more slowly, on eggs that have not been so denuded. It appears, then, that the surface of the denuded egg possesses antigenic groups similar to those of the fertilizin molecule. Further discussion and evidence for the fer- tilizin-like nature of the surface of the denuded egg is given in the report by Tyler, Monroy, and Metz ( 1956; see also Metz, this volume ) on the refertilizability of denuded fertilized eggs. When hatched blastulae or gastrulae are placed in antisera against fertilizin they become immediately immobilized, their further development is inhibited, and they presently cytolyze. Evidently surface antigens of the uncleaved egg persist until these stages in sufficient amount to produce cytotoxic effects upon reaction with the antisera. As noted above (p. 345), there are reports (Perlmann and Gustafson, 1948; Perlmann, 1953; Harding et al., 1954) also of new antigens being detected at the gastrula stage of sea urchins, but it is not known whether or not these are surface antigens. Nuclear as well as cytoplasmic division is blocked by antisera against fertilizin. Division is not blocked at any particular stage of mitosis, and the amount of mitotic progress made depends on the strength of the antiserum. In the most effective antisera (vs. fertilizin) this amounts to about 15 minutes, or about one-fourth to one-sixth of the first division time for L. pictiis and S. piirpura- tus respectively. Treatment as short as 15 to 30 minutes in strong antiserum can block cell division iireversibly. With shorter ex- posures the eggs can resume development. Cleavage block is ef- fected without other visible signs of damage being at first evi- dent, but after several hours exposure to the antiserum cytolytic changes are observed ( Fig. 2 ) . Complement is evidently not nec- essary for the action since heating the antisera at 56° C for one hour does not destroy the cleavage-blocking activity. A marked temporary rise in respiratory rate occurs in eggs treated with blocking antisera. Determinations of sodium content have been made on the supposition that the treatment may have permitted A. TYLER 361 Fig. 2. Inhibition of cleavage of demembranated, fertilized eggs of the sea urchin Lytechimts pictus by treatment with a rabbit antiserum against fertilizin of Strongylocentrotus purpuratus. Figures 2a to f are of eggs in antiserum. Figures 2a' to f are of eggs in control serum. The eggs were placed in the antiserum and control serum at 10 min after insemination and photographs taken at following times (at 17° C) after fertilization: a,a', 2 hr; b,b', 2^2 hr; c,c', 3 hr; d,d', 4^2 hr; e,e', 6 hr; f,f', 24 hr. Magnifica- tion, lOOx. 362 IMMUNOLOGICAL STUDIES entry of this ioii which is nomially in very low concentration in- side the cell. However, no significant increase was found. Deter- minations were also made of the relative tension at the surface of untreated and treated eggs, by the method of separating the egg into light and heavy halves by centrifugal force. An increase, estimated as approximately 40%, was found in the treated eggs. Of special interest in this work is the fact that highly effective antisera are produced by immunization with fertilizin, since much is known concerning the chemical and biological properties of this substance (cf. Tyler, 1948, 1949, 1955a,b, 1956b; Runn- strom, 1949, Vasseur, 1948, 1952). The fertilizins of sea urchins are obtainable in a form that is electrophoretically and ultracen- trifugally homogeneous, and whose purity is further evidenced by the effective removal of nitrogen-containing substance upon absorption with homologous sperm. Molecular weight determi- nation give a value of about 280,000 for the fertilizin of Arhacia punctulata, and fertilizins of other species are in the same range as judged from then- sedimentation constants ( Tyler, 1956b ) . The molecule is highly elongate (axial ratio of about 20:1, calculated as a prolate ellipsoid with 0.4 gram of water of hydration per gram), which is consistent with its gel-forming properties. Chem- ically, sea urchin fertilizins belong to that class of sugar- and amino acid-containing substances termed glycoproteins or muco- polysaccharides. In several species of sea m'chins that have been examined there are two kinds of sugars and 14 kinds of amino acids in the molecule, and the content of the two kinds of residues is approximately the same. Knowledge of the composition of the fertilizins should prove useful in investigation of the determinant groups that are involved in the production of the cleavage-block- ing antisera. Experiments on the biological properties of fertilizin have led to the view (cf. Tyler, 1955a; Tyler and Metz, 1955) that it repre- sents the specific receptor substance for the union of egg and sperm in fertilization and is largely responsible for the tissue and species specificity of the process. When unfertilized eggs are treated with antisera against fertilizin, they lose their fertilizabil- ity. This, however, does not necessarily mean that the antibodies A. TYLER 363 are directed against the particular molecular sites that are con- cerned in the fertilization reaction. The presence of antibody on the surface, due to reaction with determinants at neighboring sites, could suffice to prevent effective interaction between the fertilizin of the egg surface and antifertilizin of the sperm. The fact that, in sea urchins, a cell division-blocking antiseiimi can be produced by immunization with a surface constituent of the cell suggests that this may be possible with various types of cells of other animals. Some recent experiments of Billingham and Sparrow ( 1955 ) may be interpreted as pointing in this direction. These investigators injected saline washings of dissociated epi- dermal cells from one rabbit intradermally into another. When skin grafts were subsequently attempted from the former to the latter, the accelerated incompatibility reaction, typical of an im- munized animal, was exhibited. It seems likely that the washings contained prinjarily, or solely, surface constituents of the cells, and that these served as the effective antigens. In the cited ex- periments no in vitro tests were made of j)Ossible cytotoxic action of the serum of the immunized animal on donor cells, but earlier experiments by Billingham and Sparrow ( 1954 ) showed that treatment of the dissociated cells with the seiTun of an animal that had been immunized by attempted skin graft can completely or partially prevent the cells from giving rise to epithelium upon grafting. They hesitate to term this cytotoxic action since no cyto- lytic effects were observed on the serum-treated cells. However, there was evidently a specific effect of the antibodies that ren- dered the cells susceptible to early disintegration upon trans- plantation. It seems reasonable to expect, then, that antigens effective in engendering the production of specific cytopathogenic sera may be prepared from various kinds of tissues, normal and neoplastic, by extraction of surface constituents. Such extracts, obtained by mild extraction methods, would contain a less complex mixture of substances than obtained by the usual homogenization proce- dures that are employed. Possibly in some cases preparations con- taining a single chemical constituent could be readily obtained, as is the case with sea urchin eggs. The current trend, particularly 364 IMMUNOLOGICAL STUDIES in the field of cancer research, is toward the use of various sedi- mentable particles obtained from cell homogenates as immunizing antigens for the production of cytotoxic antisera. One would expect, however, that the likelihood of obtaining tissue-specific antisera would be greater if simpler antigenic preparations were employed. In any case the present experiments warrant an ex- ploration of the effectiveness of mild procedures, tending to extract surface constituents, for the preparation of immunizing antigens for use in the study of problems of malignant growth as well as in problems of normal development. The Concept of Natural Auto-Antibodies During the earlier investigations by the author on the location of fertilizin in the egg, an antifertilizin was extracted from below the surface of the egg ( Tyler, 1940 ) , after the fertilizin had been found to be the material of the gelatinous coat of the egg. This antifertilizin behaved like antifertilizin derived from sperm, in neutralizing fertilizin, agglutinating a suspension of eggs, form- ing a precipitation membrane on the surface of the gelatinous coat, etc. Since the interaction of fertilizin and antifertilizin is considered analogous to that of antigen and antibody, this find- ing indicated that two substances capable of interacting in that manner could be extracted from a single cell. Various reports on auto-antibodies in the immunological literature could be similarly interpreted as indicating the existence of mutually complemen- tary substances in the same cell or tissue. It was therefore pro- posed that this situation is a general feature of living cells and that it might provide a basis for interpretation and further inves- tigation of specific immunological problems and of problems of growth and differentiation (Tyler, 1942, 1947, 1955b). A some- what similar concept of mutually interacting complementary sub- stances being involved in processes of growth and differentiation has been developed by Weiss (1947, 1949, 1950, 1955). The term auto-antibody concept was used to lend emphasis to the idea that each of the various macromolecular substances of which cells are constructed bears the same sort of relation to an- other of these substances as do antigen and antibody, and they A. TYLER 365 are formed by processes analogous to antibody fomiation. In fact, the formation of immune antibodies is considered a special varia- tion of a process that occirrs normally without the intervention of a foreign antigen (cf. Tyler, 1947, 1948, 1955b). For immune antibody formation it is now quite generally assumed, in accord with the views proposed by Breinl and Haurowitz ( 1930 ) , Alex- ander (1932), Mudd (1932), and Pauling (1940), that foreign antigen becomes incorporated in the site of synthesis of serum globulin, so that, as molecules of the latter are formed, they will bear regional surface configurations complementary to specific structures on the antigen that serves as a template. If one con- siders the situation in the absence of foreign antigen, one may conclude that normal globulin is complementary in structure to whatever specific substances comprise the normal site of syn- thesis. The auto-antibody concept assumes in addition that this mode of origin is general for the formation of all macromolecular constituents of cells. Pauling and Delbriick ( 1940, cf. Pauling, 1955) and Emerson (1945) have shown how this process may be involved also in gene duplication. Examples of the extraction of antigen-antibody-like systems of mutually complementary substances from cells have been previ- ously presented (Tyler, 1940, 1947, 1955b), but these are not as yet very numerous. Experimentally there are difficulties that may well depend upon mutual neutralization during extraction proce- dures, particularly where these involve destruction of the cells, and on lack of suitable testing methods in the event the sub- stances are of the nonprecipitating ( "univalent" ) type ( cf . Tyler, 1945, Tyler, Fiset, and Coombs, 1954 ) . There are also indications that natural auto-antibodies may have the ability of immune anti- bodies to act as protective agents, as illustrated by experiments on the neutralization of the venom of the Gila monster by sei"um of the same animal ( Tyler, 1946, 1956a ) . One of the suggested applications of the auto-antibody concept was to problems of the specific adhesion of cells in the formation of tissues. For these, useful experimental material was evident in the experiments on the reconstitution of sponges from dissociated cells (Wilson, 1907, 1932) and the interactions of dissociated 366 IMMUNOLOGICAL STUDIES cells of amphibian embryos ( Holtfreter, 1943, 1948). Experi- ments along this line have since been performed by Spiegel ( 1954a,b, 1955) on sponges and frog embryos. By use of antisera he has been able to interfere with the reaggregation process in such a way as to indicate a role of specific surface antigens. A striking recent example of specific interaction of cells is given in the experiments by Weiss and Andres (1952) who showed that presumptive melanoblasts of dissociated embryonic chick cell suspensions when injected into the blood stream of early ^ Fig. 3. Diagram illustrating the aggregation of vegetative amoebae (A) of Dictyostelium into a pseudoplasmodium (B) and transformation of the latter into the sorocarp. (From Gregg et ah, 1954.) chick embryos localized in the proper regions characteristic of the cell type and of the donor strain. Other examples may be found in experiments on type-specific reaggregation and differentiation of dissociated embryonic cells in mixed cultures, as in the experi- ments of Trinkaus and Groves ( 1955 ) and of Moscona ( 1956 ) on mixed mesonephric and limb-bud cells of the chick embryo, and of Townes and Holtfreter ( 1955 ) on mixed aggregates of am- phibian gastrula and neurula cells. Another kind of material, the slime mold Dictyostelium, has been investigated recently by J. H. Gregg (1956). In this organ- ism individual amoebae aggregate to form a cell mass which un- dergoes further morphogenetic change ( Fig. 3 ) . With three spe- A. TYLER 367 t> z > ^ \^ _n 1 _n ^ ^ [V a z a S 1.; yv [v: ^ + + a + + + K] + + + + HnaaiDOSia 'q + + a + + + H + + + + HnaandHtid 'a 53 S 00 to 'o ti ^ ° "S ^5 --^ 0) ^ >-i <1^ b a; bC-C a ?! -^ CD -J^ O p CD 2 o &*" 0^ --^ •^ bX) c c a 3 o >> C O (0 W bC . C CD o S ^ o to -^ ^ r-i !2 "o "'' -rl i " g 5 Hi '-' 2 o •c: CO 2. " rn O 1-1 3 ^ c 5 S p^ 2i >- W 0) O ^ ^ ■:;:; bc T3 O 05 ^H 1—1 ^ bC bO OJ g S ° « S rt o &; 0) CO ;-- f-4 l-H •2 S >-l ^ — ' bC bC • C o 3 -G S C 368 IMMUNOLOGICAL STUDIES cies of slime molds Gregg showed that new surface antigens ( as illustrated in Fig. 4) are detected as the time of aggregation is approached. Again one cannot conclude that the antigenic struc- tures that are detected by the antibodies are necessarily the same as those actually concerned in the cell adhesion. However, the antisera have revealed a specific change in cell surface structure correlating with the aggregation phenomenon. It can be expected that this work will lead to extraction and characterization of the specific substances involved. Since the previous discussion (Tyler, 1947) of the relation of the auto-antibody concept to problems of embryonic differentia- tion there have been a few pertinent, if not critical, experiments on the subject. Mention was made on p. 348 of work showing the detection of specific adult tissue antigens at stages prior to visible differentiation. Their time of origin (or increase to detect- able amounts) correlates in some cases with the time that the embryonic tissue becomes "determined." In one particular case, lens protein, experimental induction of the antigen has been re- ported (Woerdeman, 1953a,b, 1955) through action of optic vesicle extract on extracts of competent ectoderm. More particularly the question may be raised as to whether or not processes of induction and detennination can be influenced in specific fashion by substances that can also be described as natural auto-anti]:)odics, extractable from the same organism. There is, as yet, no direct experimental evidence concerning this, nor, for that matter, concerning possible specific action of im- mune antibodies on these processes. As noted above the work with immune antisera has, so far, related to cytotoxic or generally lethal effects in which action on specific de\'elopmental processes are not immediately evident. Changes in cell type would be one of the effects one might seek to obtain. Such changes have been frequently described in experiments with microorganisms gi^own in immune sera (cf. Dubos, 1946), although it is often uncertain to what extent this is due to selection of variants not inhibited by the antibodies. Selection is not involved in the experiments on Parameciinn by Sonneborn (1948, 1950; cf. Beale, 1952) who showed the induction of changes in antigenicity by means of A. TYLER 369 homologous antisera. The transformations also occur m response to certain nonspecific environmental (temperature, pH, etc.) changes. It would be of interest to know whether or not they could be produced by specific cell extracts. In experiments on frog embryos Rose (1952, 1955) reported specific inhibition (mostly temporary) of the differentiation of blood, brain, and heart by culturing the embryos in the presence of the homologous tissue or extracts thereof. On the other hand, somewhat similar experiments with chick embr\os by Ebert (1955) showed no inhibition of the de\elopment of the spleen in response either to transplantation of spleen from the hatched chick or to injection of homogenates of adult spleen. Some time ago several workers (Murphy, 1916; Danchakoff, 1916, 1918; Wil- lier, 1924) reported that chorioallantoic grafts of adult chicken spleen caused an enlargement of the spleen of the host embryo. Ebert (1951, 1952, 1954) investigated this effect in considerable detail and employed also ingenious experiments witli radioac- tively labeled material. He has shown that the effect is class specific and quantitatively tissue specific. The effect first appears with spleens taken from embryos of about 14 days incubation and increases with age of the donor. Altliough certain splenic antigens are first detected at 14 days of incubation Ebert does not consider his experiments to demonstrate that any one of these is the effec- tive agent. With S''-labeled spleens and kidne\'s he has shown a specific transfer of radioactivity from the graft to the homologous organ, which was evidently not due to transfer and localization of cells, as might have been suspected from the experiments of Weiss and Andres (1952). From tlie quantities transferred and other considerations, Ebert (1955) concluded that the results favor a "building block" rather than a "template" or catalytic mechanism and that the material transferred is of the nature of whole, specific splenic, protein molecules or large fragments thereof. The experiments pertain, then, more particularly to giowth rather than differentiation. Grobstein (1955) has recently reviewed his experiments on specific inductive effects between embryonic mammalian tissues separated by filters whose porosity excludes massive cell contacts. 370 IMMUNOLOGICAL STUDIES He suggested that intercellular matrices may play an important part in the inductions, and (Grobstein, 1955, p. 252) that one way in which the matrices might interact is by "molecular com- plementariness, as has been suggested by Weiss (1947) and Tyler (1947) for cell surfaces." He emphasized, however, that there are many possible interpretations of the mechanism of the inductive effects. Again the lack of direct information as to the nature of the specific substances involved precludes further spec- ulation with respect to the auto-antibody concept. In general, while there are suggestions that interactions of the type envisioned in the auto-antibody concept may have a causal connection with processes of induction and determination, direct evidence of this is still lacking. Summary This paper is mainly a review of recent immuno-embryological studies of various investigators, dealing with the detection in the embryo of specific antigens of adult tissues, changes in anti- genicity during development, development of antibody-forming capacity, and eflPects of antibodies on development. These topics are discussed in relation to an auto-antibody concept developed by the author. It contains also results of certain current ex- periments, by the author and his co-workers, in which it has been found that cell division in the early development of eggs of sea urchins can be blocked by antisera prepared against fertilizin. These experiments represent the first, to the author's knowledge, in which a block to cell division has been obtained with antisera prepared against a chemically well-defined antigen. REFERENCES Alexander, J. 1932. Some intracellular aspects on life and disease. Protoplasma, 14, 296-306. Barrett, M. K. 1952. 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INDEX Abruzzese Sgarlata, S., 300 Absorption, 343, 344 Accessory cells, see Nurse cells Acetabiilaria, 215 Acrosome cave, 137 and egg membrane lysin, 57 filament, 135 insemination filament, 161 in mammalian fertilization, 89, 122 particle, 137 physical properties, 151 reaction, 24, 29, 140 Actin antigens, 300 detection in chick blastoderms, 348 efl^ects of antisera against, 356 Actively acquired tolerance, 351 Actomyosin, 304 effects of antisera against, 356 Adams, C. E., 76, 86 Adelmann, H. B., 292 Adenosine triphosphate, 251 Afzelius, B. A., 137, 138, 142 Agarawal, S. C, 265 Agglutination calcium as aid to, 48 of eggs, 49 by fertiHzin, 24, 28, 31 of mammalian sperm, 82, 88, 125 mechanism of, 25, 31, 53 reversal of, 33 of sperm, 33 Agrell, I., 275, 280, 283, 284 Alden, R. A., 77 Alexander, J., 365 Alfert, M., 205 Altman's fluid, 274 Amoeba, 89, 194 passage of RNA in, 16 Amoeba discoides, 209 Amoeba proteus, 209 Amoeba sphaeronucletis, 209 Amoroso, E. C, 115 Amphibians, 86; see also Frogs cyclopic embryos in, 292 dissociated cells of embryos, 366 eggs of, effect of enzyme-inhibi- tion on, 337 embryos of, effects of antisera on, 356 oocyte growth curves of, 5 Amy, R. L., 191-193 Anaerobiosis development in, 335 Andersen, D., 77 Anderson, J., 74, 126 Andres, G.^ 366, 369 Andrews, F., 77, 79 Animal halves dye reduction of, 279 respiration of, 278 Animal pole metabolism at, 313 Animal-vegetal axis differences in particles along, 276 gradient, 272, 279 Animalization, 272, 300 agents, 298, 302, 307, 311, 312 larvae, 291 Anophthalmia, 298 Anson, M. L., 35 Antagglutin for sperm, 83, 88, 126 Antibodies, 341 cytotoxic activity of, 354 in developing chick, 350 developmental effects of, 345 by embryo, fetus, and newborn, 349 formation of, 365 inhibitory effects of, 355 natural, 350 stimulating effects of, 354 univalent, 55 Antifertility agents, 124, 125 383 384 INDEX Antifertilizin, 270 from eggs, 49 extraction of, 364 from sperm, 31, 36, 44, 52, 88 Antigen-antibody reactions, 31 Antigens, 341 bird, 345 determinants of specificity, 341 determination in relation to, 346 embryonic and adult, 344 frog, 345 of hybrids, 210 of mammals, 345 multi-haptenic antigens, 342 organ-specific, 345 of red blood cells, 300, 349 salamander, 345 sea urchins, 345 serum-like, in eggs and embryo, 346, 347 silkworm, 345 of spermatozoa, 93 Antisera cross-reacting, 343 against saline extracts of embryos, 349 against serum albumin, 347 against serum globulin, 347 Apis mellifera, 111; see also Honey bee Aphjsia eggs mitochondria in, 331 Arbacia, 139; see also Sea urchin entries Arbacia eggs fragments of, 265, 272 mitochondria in, 268, 272 NaSCN or LiCl treatment of, 300 Arbacia lixula, 46, 49, 50, 172 fertilization of eggs of, 269 Arbacia piinctulata, 27, 28, 30, 43, 47 fertilizin of, 359, 362 Archenteron formation, 240 Arendsen de Wolff -Exalto, E., 299 Arnesen, K., 357 Arosio, R., 304, 305 Arsanilic acid, 342 Arvy, L., 264 Ascaris, 204, 207 Ascidia malaca LiCl treatment of embr\'os, 297 Ascidian egg centrifugation of, 323 development with blocked en- zymes, 326 effect of anaerobiosis on, 335 mitochondria of, 321 Ascidians enzymes in development of, 319 Asdell, S. A., 78 Asterias, 92, 162; see also Starfish entries Asterias amiirensis, 140, 145, 149, 151, 159 Asterias forbesii, 10, 29, 31, 147, 151, 158, 161, 164 Asterias gJacialis, 159 Asterias pectinifera, 140, 143 Asterias ruhens, 10 Asterias vulgaris, 148 Astropecten, 36 Astropecten aranciacus, 162 Astropecten scoparius, 140 Attardo, C, 330, 334 Attraction cone, 161; see also En- trance cone Atwood, K. C, 192 Austin, C. R., 77-79, 83-92, 109- 112, 114-116, 118-122, 124 Auto-antibodies, 364 determination and, 368 Azide, 325 Bachem, A., 191 Bachman, E., 27 Biickstrom, S., 292, 313 Bacsich, P., 116 Badinez, O. S., 293 Baltus, E., 9, 10, 17 Baltzer, F., 218 Bamberger J. W., 337 Barrett, G. R., 74 Barrett, M. K., 357 Barth, L. C, 203, 245-247, 250 Bat, 76, 80 121 INDEX 385 Baylies, L. E., 79 Beale, G. H., 368 Beams, H. W., 264 Beatty, R. A., 87, 114 Beck, G. H., 74 Beermann, W., 206, 207 Beiler, J. M., 125 Benzidinperoxidase, 325 Berg, W. E., 57, 58 Berger, C. A., 204-206 Berliner, V., 79 Bernheim, F., 192 Berrill, N. J., 1, 207 Berry, R. O., 204 Beveridge, W. I. B., 349 Bielig, H. J., 24, 27, 37, 49 Billingham, R. E., 351, 353, 363 Birds, 86 derivation of yolk in eggs of, 2 Birky, C. W., Jr., 28, 29 Bishop, D. W., 46 Bishop, M. W. H., 73-74, 81-82, 88 Bithijnia eggs cytochrome oxidase in, 334 Nadi reaction in, 330 Blandau, R. J., 76-78, 87, 118-119, 122, 124 Blastomeres mitochondria in, 323 Blastula effects of antisera on, 360 mitochondria in, 283 Blood inhibition of differentiation of, 369 types, 345 Blood groups, 345 antigens in spermatozoa, 93 substances, 342 time of appearance of isoagglutin- ins, 350 Blum, H. F., 194 Bluntschh, H., 78 Bodenstein, D., 189 Body wall mitochondrial number in, 284 Boell, E. J., 263, 278, 337 Bogomolets, A. A., 354 , Bohus Jensen, A., 42 Boivin, A., 204 Bollag, W., 352 von Borstel, R. C., 187, 188, 191- 193 Bossi, J., 305 Bossu, J., 86 Boveri, T., 122, 175, 184, 204 Bowen, R. H., 89 Bowman, R. H., 87 Boyarsky, L. H., 79 Brachet, J., 4, 9, 17, 203 204, 207, 263, 264, 271, 273, 279, 280, 337 Braden, A. W. H., 76-79, 84-92, 114-116, 119, 120, 124 Brain effects of antisera against, 356 extracts of, 344 inhibition of differentiation of, 369 Brandt, C. L., 194 Bratton, R. W., 81 Brauer, A., 189 Breinl, F., 365 Brent, L., 351 Briggs, R., 209-214, 216, 219-221, 223 Briles, W. E., 345, 349 Brioschi, G., 311 Brissopsis lyrifera, 45, 48 Brookbank, J. W., 357 Bruce, H. M., 86 Bull, 73, 82, 89, 123; see also Cow Buller, A. H. R., 85 Burbank, A., 47 Burckhard, G., 121 Burgos, M. H., 135 Burke, v., 345, 356 Burnet, F. M., 93, 349 Buxton, A., 352 Byers, H. L., 40 Campbell, D. H., 345 Campbell, J. A., 81, 82 Canivenc, R., 79 Capacitation, 83, 84, 88, 112 Card, L. E., 80 Carrano, F., 330, 333 386 INDEX Carstens, H. P., 75 Carter, G. S., 28, 30 Casida, L. E., 74, 79 Caspari, E., 285 Caspersson, T., 4, 18, 121, 204, 207 Cat, 77, 90 Ceas, M. P., 170, 172 Cecidomidae, 204 Cecropia, 2 Cell division; see also Cleavage inhibition of by antisera, 356 Cells accessory, see Nurse cells adhesion of, 365 ciliated, particles in, 276 dissociated, 365 epidermal, 363 erythropoietic, implants in x-ir- radiated rats, 354 flask, 239 mesonephric, 366 phagocytic ability of embryonic, 351 ' secretory, mitochondria of, 319 Centrifugation disarrangement of particles by, 292 effect on egg cells, 265 Centrosome in mammalian fertiliza- tion, 122 Cephalopod embryos, 298 Cerebratiilus, 57 Chambers, R., 93, 161, 162, 165 Champy, C. 116 Chang, M. C, 76-78, 84-87, 110- 112, 114-117, 119, 123-126 Chantrenne, H., 264 Chargaff, E., 11 Charlton, H. H., 116 Chelation of metals, see Metal bind- ing Chemotaxis of sperm, 26, 84, 85 Cheng, P. L., 74 Chernoff, A. I., 345 Chicken dissociated cells of embryos, 366 eflFects of antisera on embryos, 356 egg-white, 343 serum, 346 Child, C. M., 189, 202, 271, 279, 312 Chimpanzee, 76 Chironomtis, 206 Cholinesterase, 337 Chondriosomes, 265 Christiansen, L. K., 171 Chromatin diminution, 204 Chromosomes constancy of number, 201 disintegration and preservation, 311 lampbrush, 207 limited, 188 in parthenogenesis, 117 in polar bodies, 115 separation of sperm-bearing, 82 somatic reduction of, 204 Cigada, L., 304, 311 Cinader, B., 353 Ciona intestinalis LiCl treatment of embryos, 296, 299 Citterio, P. 300, 301, 302, 305 Clayton, R. M., 345, 349, 356 Cleavage, 185 effects of granular fractions on, 269 of egg fragments, 202 inhibition of, by antisera, 356 mitochondria during, 280, 282 protoplasmic clotting and, 270 Cleland, K. W., 81 Clermont, Y., 135 Clowes, G. H. A., 27 Cohen, A. I., 251 Cohn, M., 343 Collier, J. R., 29, 89 Colwin, A. L, 29, 92, 146, 148-159, 164 Colwin, L. H., 29, 92, 146, 148-159, 164 Comandon, J., 209 Complement cleavage-block and, 360 INDEX 387 Complementary substances extraction of, 365 Conklin, E. G., 175, 323, 324 Convergent extension, 243 Cook, J. S., 194 Coombs, R. R. A., 355, 365 Cooper, R. S., 345, 346 Cornman, I., 27 Cortex, 268 changes in mammalian egg, 91 cytoplasm, 269 granules, 91, 268 release of calcium from, 269 Corti, C, 293 Costello, D. P., 41, 49 Cotronei, C, 292 Courrier, R., 116 Cow, 73, 75-77, 79, 82, 89; see also Bull Crew, F. A. E., 80 Cristae mitochondriales, 319, 321 Culex, 205 Cumulus oophorus, 87, 123 Cupps, P. F., 74, 78 Curry, H. A., 218 Cyclopy, 292, 298, 308 Cytochrome oxidase, 325 blocking of, 336 Cytofertilizin, 40, 52 C^ytoplasm in determinate development, 188 incompatibility of, 183 inadiation of, 189 maintenance of specificity in, 17 nucleus relationship, 175 particles, 263, 319 synthesis, 7, 17 Dalcq, A., 8, 121 Dallam, R. D., 74 D'Amelio, V., 170, 171 Dan, J. C, 29, 31, 33, 35, 38, 43, 48, 89, 135, 136, 139-143, 145, 148, 151, 152, 159, 164 DanchakofiF, V., 369 Danielli, J. F., 209, 210 Dauzier, L., Ill, 112 David, H. A., 86, 90, 91 " DeBusk, A.G., 4, 16 Delavault, R., 264 Delbriick, M., 365 Denaturation, 304, 311, 312 Deoxyribonucleic acid (DNA), 177, 179, 188, 257, 353; see also Nucleic acid Derrien, Y., 300 Determinant groups, 342, 343, 347 Deuchar, E. M., 313 Deutsch, H. F., 343 Development alterations in, 291 complementary substances in, 364 critical periods of, 195 determinate (mosaic), 189 determination in, 265, 291, 349 model of, 176 Dharmarajan, M., 80 Dictyostelitim effect of antisera against, 366 Differentiation without cleavage, 278 complementary substances in, 364 cytoplasmic particles in, 270 enzymes and, 278 of oocyte, 1 primary, 277, 284 relation to genes and antigens, 346 Dilks, E., 350 Discoglossiis pictiis, 57, 89 Dixon, F. J., 353 DNA, see Deoxijrihoniicleic acid Docton, F. L., 93 Dog, 75, 76, 90, 91, 120, 123 hyaluronidase in semen of, 123 Dohrn, P., 27, 28 Donovan, J. E., 33, 35, 38, 43, 48, 53 Doorme, J., 121 Dornfeld, E. J., 2, 3, 9 Dorsal lip formation of, 239 Double-gradient theory, 271 Dounce, A. L., 4, 16 Drosophila, 175, 189, 191, 205, 207 Drouville, C, 79 Dubert, J. M., 353 388 INDEX DuBois, A. M., 188 Dubos, R. J., 368 Duesberg, J. 323 Dulaney, A. D., 357 Dulbecco, R., 193 Dunsford, I., 351 Duplicitas cruciata, 298 Duran-Reynolds, F., 87 Duryee, W. R., 207 Dye reduction, 279 Ebert, J. D., 203, 222, 345, 348, 356, 369 EcJiinarachniiis parma, 46 Echinocardium cordatitm, 45, 46, 137, 138, 140, 141 Echinochrome, 26, 27 Echinoderm cytoplasmic particles in develop- ment, 263 Echinus esculentus, 30, 45, 46, 137 Echinus miliaris, 28, 30 Edlund, B., 83 Eggs activation of, 23, 59, 89, 114 blood proteins and cytoplasm of, 2 collision frequency of, with sperm, 85,86 cross-reactions with yolk of, 346; see also Yolk granule-free fragments, 267 human, 111, 115 hyalin layer of, 40 lysins of membrane, 56, 88, 124 mammaUan, 110-114, 121 membranes, 56-59, 88-90, 124 mitochondria in fragments, 268 penetration by sperm, 56, 82, 87, 118, 124 reaction of, to mammalian sper- matozoa, 89 ribonucleic acid in fragments, 264 unfertilized, 363 Egg water, 24, 125 agglutination of sperm and, 31 chemotaxis of sperm and, 26 motility of sperm and, 26 respiration of sperm and, 26, 28 Eichenberger, M., 280 Elasmobranchs, 86 Elliott, I., 74 Elson, D., 11 Ely, P., 93 Emerson, S., 365 Emmens, C. W., 74 Endo, Y., 91, 269 Entodermization, 297 Entrance cone, 153, 155-159, 161- 162, 165 Enucleation experiments, 17 Enzmann, E. U., Ill, 115, 116, 121, 123 Enzymes blocked, development with, 326 effect of, on development, 278, 319 genes and, 320 morphogenesis and, 333 proteolytic, 76, 310, 312 Ephestia kiihniella, 176 Epiboly, 241 angle of, 242 Epigenesis, 346 Ernster, L., 273, 280 Errera, M., 19, 194 Erythrocyte mosaicism, 351 Estrous cycle sperm transport and, 79 Eu char is mitochondria in eggs, 332 Euglobin, 305, 309 extraction of, 302 Evans, E. I., 76, 78 Everett, N. B., 2 Exogastrulae, 292, 297 Explants, 239 Fallopian tube role in mammalian fertilization, 110, 126, 127 spermatozoa in, 76, 124 Fankhauser, G., 175, 186, 202-204, 218, 220 Farinella-Ferruzza, N., 335 INDEX 389 Fatty acids effects of ultraviolet radiation on, 192 Faure-Fremiet, E., 264 Fautrez, J., 295 Fautrez-Firlefyn, N., 264 Favvcett, D. W., 135 Fekete, E., 87 Felix, K., 24 Felton, L. D., 353 Female tract, see Genital tract Fenner, F., 93, 349 Ferguson, L. C, 93 Ferns, 26, 84, 85 Ferreri, G., 297 Ferret, 116, 121 Fertilization acrosomes in, 89, 122 chances of, 84 effects of granular fractions on, 269 history, 109 of mammalian egg in vitro, 1 10 site in mammals, 78, 85, 93 specificity, 58 Fertilization cone, 92, 119, 164; see also Entrance cone Fertilization membrane, 51 cortical granules and, 269 Fertilizin, 52, 55, 59, 88, 125, 149 amino acids in, 45 antisera against, 357, 362 axial ratio, 47 biological properties, 362 carbohydrate in, 45 chemistry of, 45-49, 362 effect of, on fertilization, 37-44 electrophoresis of, 45, 47 irradiation of, 33, 36 location, 362 molecular weight, 47 reactive groups, 47 source, 39 sperm agglutination and, 31—37 sperm morphology and, 29-30 sperm motility and, 26, 27, 56, 59 sperm respiration and, 26, 28, 29 sulfate in, 45-47 surface of denuded egg and, 360 theory, 24 ultracentrifugation of, 45, 47 univalent, 32, 43, 47 Ficq, A., 12, 13 Fiset, M. L., 365 Fisher, H., 357 Fisher, W. D., 192 Flickinger, R. A., 345, 348, 356 Florey, H., 76 Flow birefringence, 304, 305 Fol, H., 92, 159, 161, 165 Foley, M. T., 53 Follicle cells of, 3 fertilization in ovarian, 78 de Fonbrune, P., 209 Foote, R. H., 81 Fowl, 80: see also Chicken Fox, S. W., 27, 39, 47-49 Francis, G. E., 342 Frank, J. A., 41, 52, 55 Freedman, A., 214 Freund, J., 350 Frogs; see also Amphibians amino acids in blastoporal dorsal lip, 313 eggs, 264, 270, 302 embrvos, vegetalized, 300, 209, 369 Fruton, J. S., 81, 277 Fuchs, H. M., 43 Fujii, T., 73 Fundulus, 292 Furuhjelm, M., 83 Gall, J., 5, 207 Gamow, G., 4 Gastrulae effects of antisera on, 360 mitochondria in, 276 Geigv, R., 189, 192 Geitler, L., 204, 205, 207 Genes, 346 enzymes and, 320 determination in relation to, 346 duplication, 365 transformation, 177, 179 390 INDEX Genetic mosaics, see Mosaics Genital tract capacitation, 83, 88, 112 muscular activity, 75, 85, 86 spermatozoa transport in, 75-79, 85,86 Germ cell, 1 Gerris lateralis, 205 Gersch, M., 331 Gershenson, S. M., 207 Ghosh, D., 73 Giardina, G., 31, 46, 48, 54, 170 Giese, A. G., 194 Gila monster venom of, 365 Gilchrist, F., 114 Glaser, O., 33 Glegg, Y., 135 Glover, F. A., 79 Goldman, A. S., 191, 193 Goldstein, L., 4, 16 Golgi bodies, 264 Goodman, M., 345 Gopher, 91 Gorini, L., 312 Gowen, J. W., 186 Gradients, 270 Graff, J., 169 Grasse, P. P., 137 Grav, J., 28, 30 Graz, A. P., 73, 78 Green, E. U., 204, 210 Green, R. G., 357 Green, W. W., 79, 89 Greenberg, M., 28, 30 Gregg, J. H., 366-368 Gregg, J. R., 203, 216, 237, 240, 243, 244, 248, 257, 258 Grell, M., 205 Gresson, R. A. R., 121 Grobstein, G., 212, 351, 370 Ground substance, 268 Groves, P. W., 366 Growth, see Development Grundfest, H., 173 Guillermond, A., 280 Guinea pig, 77, 80, 82, 90, 115, 121 Gunn, R. M. G., 72, 73 Gustafson, T., 169, 263, 271, 272, 274-281, 283 284, 291, 312, 313, 332, 345, 360 Gut herniation, 191 Guttmacher, A. F., 78 Guyer, M. F., 210 Gynandromorph, 184 Hahrohracon juglandis, 175-193 Hadorn, E., 215 Hagstrom, B., 26, 27, 43, 45, 50, 51, 56 Haliotus, 58 Hallez, P., 179 Hamburger, V., 278 Hamer, D., 54 Hammerling, J., 17, 215 Hammond, J., 72, 77, 86 Hamster, 88-92; see also Mouse; Rats Hanan, R. Q., 353 Hancock, J. L., 81, 82, 89 Hannah, A., 207 Hapten, 342 Harde, S., 40 Harding, G. V., 345, 360 Harding, D., 345, 360 Harman, J. W., 267, 282 Harris, M., 350 Hartman, C. G., 80 Hartmann, M., 24, 26, 27, 31, 39, 49 52,55,56, 114 Harvey, E. B., 264, 265, 267, 268, 272, 277 Harvey, E. N., 268 Hasselberg, I., 278 Haurowitz, F., 365 Hauschka, T. S., 357 Hayashi, T., 28, 30 Hays, R. L., 75 Heape, W., 75, 76 Heart effects of antisera against, 356 inhibition of differentiation of, 369 Hedal, E. F., 355 Heilbrunn, L. V., 269, 270 Heine, F., 351 Hillier, R., 268 INDEX 391 Helix aspersa, 264 Henschen, W., 177 Hensen, V., 86 Herman, H. A., 74 Hertwig, G., 115 Hertvvig, O., 201 Hesperidin, phosphorylated, 124, 125 Heteroagglutinins, 55 Heterochromatin, 207 Hibbard, H., 57 Histones, 25, 53, Hobermann, H. D., 169 Hoff-J0rgenson, E., 3 Hoffman, H., 345, 346 Hofmeister series, 300 Hogeboom, G. H., 10, 270, 274 Holothuria atria, 146, 152-155, 159- 165 Holothuria poli, 162 Holt, H., 81 Holter, H., 278 Holtfreter, J., 221, 239, 243, 277, 296, 366 Homogenization, 281 Honey bee, 186, 194, 195 of Eugster, 183 Horse, 75, 125 Horstadius, S., 162, 165, 271, 278, 279, 312 Hotchkiss, R. D., 177 Hovvland, R. B., 189 Huggins, C, 76 Hultin, T., 42, 52-55, 169, 173 Humphries, A. A., 185 Huskins, C. L., 202, 204, 206-208 Hyaloplasm, 274 Hyaluronidase, 56, 87 inhibitors of, 87, 90, 123 in semen, 123 Imagawa, D. T., 357 Immers, J., 48 Impellizzeri, M. A., 170, 172 Incompatibility reaction, 351 Indophenol oxidase, 324, 330 Induction effects in mammalian tissues, 369 intercellular matrices and, 370 natural auto-antibodies and, 368 Ingleman-Sunberg, A., 83 Insectivores, 76 Insects, 86 Insemination filament, see Acrosome, filament Invagination angle of, 241 lodosobenzoate treatment of embryos with, 295 Irwin, M. R., 345, 349 Ishida, K., 121 Isoimmunization fetal cells and, 350, 355 Ivanov, E., 72 Jacobsen, C. F., 312 Jaeger, L., 203, 250 Janeselli, L., 296 Janus green, 275, 323 Jeener, R., 280 Jenkinson, J. W., 39, 295 Johnson, I. S., 356 Jones, R. M., 2 Jordon, E. S., 77 Just, E. E., 33, 37 Kabat, E. A., 342 KaUss, N., 352 Kamen, M. D., 17 Kampschmidt, R. F., 74 Kao, C. Y., 173 Karyokinesis, see Cleavage Kato, K., 125, 126 Kavanau, J. L., 313 Kelner, A., 193 Kellner, A., 355 Kerr, W. R., 352 Kidd, J. G., 357 Kihlstrom, J. E., 83, 126 Kimball, A. W., 192 King, T., 209-214, 216, 219-221, 223 Klein, D., 240, 243, 244 Kok, J. C. N.. 74 Koprowski, H., 352 Kosswig, C., 206 392 INDEX Krafka, J., 116 Krauss, M., 28, 47, 57, 58 Krehbiel, R. H., 75 Kriszat, G., 52 Kuff, E., 270 Kuhn, R., 26, 27, 49 Kupelweiser, H., 139 Kurzrok, R., 87, 116 Kushner, K. F., 76, 86 LalHer, R., 293, 300, 312 Lams, H., 121 Landsteiner, K., 341, 342, 344, 355 Lansing, A., 268 Lardy, H. A., 73, 81, 82 Lavin, G. I., 264 Law, L, W., 357 Lazeai", E. J., 93 Leblond, C. P., 135 Lehman, H. E., 217, 218, 220 Lehmann, F. F., 292, 330, 333 Lenicque, P., 271, 272, 274-276, 278-281, 283, 284, 320, 332 Lens antigen, 345 effect of antisera against, 356 extract, lack of species specificity, 344 protein, 344, 368 Leonard, S. L., 77, 87 Leone, C. A., 356 Leone, V., 295, 305 Lepidochitona cinerea, 145 Leskowitz, S., 342 Leuchtenberger, C., 3 Levi, G., 280 Levine, P., 349, 350, 355 Lewis, E. B., 207 Lewis, L. L., 76 Lewis, J. H., 344 Lewis, W. H., 87, 124 Lillie, F. R., 24, 26, 27, 31, 33, 37, 39, 41, 43, 49, 52, 109, 112, 117, 139, 163, 164 Limax, 122 Limiiaea alkaline phosphatase in egg, 333 LiCl treatment of egg, 299 Lindahl, P. E., 39, 83, 88, 205, 271, 278, 291, 312 Lindberg, O., 273, 280 Lindsley, D. L., 354 Lindvall, S., 52, 55, 56, 58 Lithium chloride effects of, 291 Liu, C. K., 1 Lockingen, L. S., 4, 16 Loeb, J., 27, 33, 38, 39, 89 Loeb, L., 344 Loligo vulgaris, 298 Loofbourow, J. R., 191 Loos, G. M., 194 Lorch, I. J., 209 Lovelock, J. E., 74 L0vtrop, S., 257, 258 Low, H., 50-52 Liimbricus, 137 Lumsden, T., 357 Lundblad, G., 52 Lymph nodes transplantation of, 351 Lysins, 56-59, 88-90, 124 Lytechinus pictiis, 28-30, 36 antisera against fertilizin of, 359, 360 mitochondria in, 274, 283 Lytechinus variegatus, 28, 30 McClean, D., 87, 123 McGibbon, W. H., 345 McKenzie, F. F., 79 McLeod, J., 82 McShan, W. H., 79 Mackenson, O., 177 Mactra (Spisiila) solidissima, 38, 43 Mactra sulcataria, 136 Mactra veneriformis, 136 Maculla, E. S., 345 Maggio, R., 31, 46, 48, 54, 173 Mahfouz, N. P., 78 Mainland, D., 121 Makino, S., 205 Makinodan, T., 354 Malhotra, S. K., 264 Malmgren, R. A., 357 INDEX 393 Malonate, 326 Mancuso, V, 325, 331, 334, 335 Mann, T., 72, 76, 81, 82 Mare, G. S., 77, 80 Mark, E. L., 122 Markert, C. L., 214, 215 Marshak, A., 3 Marshak, C, 3 Martin, G. J., 125 Mather, K., 222 Matthews, H., 73 Maurer, P. H., 353 Mayer, D. T., 74 Mayer, G., 79 Medawar, P. B., 351 von Medem, 24, 27, 37, 49, 57, 58 Megathura crenulata, 27, 28, 41, 49, 56, 57, 89 Meiosis, see Oogenesis Meiotic block, 184 Melanoblasts localization of, 366 Mellita quinqiiiesperforata, 39, 43 Metabolism carbohydrate utilization, 248 Embden-Meverhof glycolysis, 81, 251 of hybrid embryos, 245 Krebs cycle, 81, 252 lactic acid production, 250 of phosphorus, 254 Warburg-Keilin hydrogen trans- port, 251 Metal binding, 28, 35, 51, 74 Metz, C. B., 24, 25, 27-29, 31, 33, 35-40, 42-44, 46-48, 50, 53- 55, 57, 142, 149, 163, 169, 357 Metz, C. W., 188, 204 Meves, F., 139, 280, 323 Meyer, R. K., 79 Mezger-Freed, L., 256 Mickey, G. H., 205 Micromeres ribonucleic acid in, 273 Microsomes, 265 distribution of, 330 ribonucleic acid and, 273 Millar, R., 75 Miller, F. W., 78 Miller, W. J., 345, 349 Miloyanov, V. K., 73, 74, 82 Minganti, A., 46, 49, 333 Mirsky, A., 35, 169, 170, 204 Mitochondria, 264, 319 ascidian, enzymes of, 323 de novo formation, 265 differential segregation, 323 dimensions, 267, 274 distribution, 268, 330 ectodermic, 320 electron micrographs of, 275 entodermic, 320 enzymes, inhibition of, 325 fibrillar structure and, 279 gradients, 272 identification, 282 kinds, 285, 320 origin, 280 preservation, 281 quantitative aspects, 279 reproduction, 321 secretory granules and, 279 staining' of, 267, 273 structure, 319 time of function, 277 Mitosis, see Cleavage Mizuno, T., 73 Moeller, A. N., 76, 77 Moench, G. L., 81 Money, W. L., 76 Monne, L., 40, 91 Monod, J., 222 Monotremes, 87 Monroy, A., 31, 40, 42, 44, 46-51, 57, 58, 170-173, 360 Monroy-Oddo, A., 170 Montgomery, C. M., 337 Montgomery, T. H., 4 Moog, F., 263 Moore, A. B. C., 204, 258 Moore, A. R., 312 Moore, J. A., 203, 216, 236-245 Morgan, T. H., 26, 31, 201, 208 Morgan, W. T. J., 342 Moricard, R., 86, 111, 112 Mormoniella, 175 394 INDEX Morphogenesis enzymes and, 333 Morrill, J. B., 29, 31, 149, 163 Mosaics eggs, 265, 330 genetic, 181, 194 Moscona, A., 366 Moser, F., 91, 268 Moser, H., 191 Moss sperm, 26, 84, 85 Motomma, I., 40, 51, 52, 54, 91, 268 Mountain, I. M., 357 Mouse, 76, 82, 84, 88, 116, 120, 124; see also Hamster; Rats Mudd, S., 365 Muller, H. J., 207 Murphree, R. L., 79 Murphy, J. B., 353, 369 Muscle fibers, 311 Myosin, 304 detection in chick blastoderms, 348 Myosin antigen, 300 Mytilus, 89 Mi/tilus californumus, 58 Mytilus edulis, 56, 58, 136, 139, 145 Myzostoma mitochondria in eggs, 332 Nace, G. W., 2 345-348, 355, 356 Nadi reaction, 325, 330 Nalbandov, A. V., 80 Nanney, D. L., 183 NaSCN effects of, 291 explants treated with, 295 Nath, v., 264 Neal, W., 76 Needham, A. E., 202 Needham, J., 203, 349 Nelsen, O. E., 2, 238 Nereis, 139, 142, 163 mitochondria in eggs, 332 Nereis limhata, 24, 27, 31, 41, 49, 149 Nettleship, A., 356 Nieuwkoop, P. D., 297 Nigon, v., 264 Nile blue sulfate, 274 Nilsson, A., 83, 126 Norman, A., 192 Notochord action of different agents on, 308 NaSCN effect on, 292, 301 Novikoff, A. B., 41, 204, 264 Noyes, R. W., 84 Nucleic acid, 191, 193, 195; see also Deoxyribose nucleic acid; Ribo- nucleic acid Nucleolus, 5 chemical properties, 10 duplicating machine and, 18 isolation of, 6 and mammalian pronuclei, 121 organizer region, 16 RNA in, 6-15 ultraviolet absorption of, 7 Nucleoprotein, 191, 193, 195 Nucleoside phosphorylase, 17 Nucleus and cytoplasm, 175-195, 216 in determinate development, 188- 193 differentiation, 203-208 feulgen-negative, 188 irradiation of, 189-194, 217 synthesis in absence of, 17 transplantation, 208-221 Nungester, W. J., 357 Nurse cells, 3, 264 incorporation into eggs, 178 Odor, D. L., 78, 87, 118, 119, 122, 124 Oettle, A. G., 80 O'Melveny, K., 52, 55 Oocyte DNA in, 3 effect of surrounding cells on, 2 growth, 5 nuclear changes, 3 RNA in, 4, 8 role of nucleoli, in, 4 size relationships, 5 starfish, 5 INDEX 395 Oogenesis Habrobracon, 177 Opossum, 76 Orlandi, A., 311 Orstrom, A., 173 Osmium tetroxide, 274 Owen, R. D., 351 Oxidoreductase, 325 Oxytocin, 75 Oyama, J., 353 P^-, see Radiophosphonts Paigen, K., 284 Painter, T. S., 4, 177, 205 Palade, G., 268, 276 Palazzo, F., 330, 333, 334 Pangens, 270 Panijel, J., 4, 8, 264 Pantelouris, E. M., 217, 218 Paracentrotiis lividus, 45, 46, 49 Paralysis, immunological pneumococcal polysaccharide and, 353 Paramecium, 193, 368 Parat, M., 89 Parker, G. H., 76 Parkes, A. S., 87, 90, 112, 115, 125 Parmenter, C. L., 211 Parthenogenesis, mammalian, 90, 110, 114, 127 polar bodies and, 115 Patiria miniata, 36, 38 Pauling, L. 365 Penners, A., 176 Pentose nucleic acid, see Nucleic acid Perforatorium, 135, 139, 163 Perlman, P. L., 77 Perlmann, P., 169, 345, 360 Peroxidase blocking of, 336 Petiicola japonica, 136 Petromijzon, 296 Phelps, H. J., 357 PhilHps, R. W., 77, 79, 81, 82 Phosphatase inhibition by beryllium, 333 Phosphorylases blocking of, 336 Photoreactivation, 193-195 Physa eggs, 331, 334, 335 Pig, 75, 77 Pincus, G., 86, 87, 110, 111, 114- 119, 121-125 Pirie, N. W., 87 Pitotti, M., 324, 332 Placenta transfer of antibodies through, 350 Plant sperm chemotaxis of, 26, 84 Plasmagenes, 270, 280 Plasmatic rhythm, 281, 284 Plaut, W., 4, 16 Plaut, G. W. E., 73 Pluteus mitochondria in, 276, 283 Polarity, 265 PolHster, A. W., 204, 236, 273 Polplasma, 330 Polyploidy, 177, 204, 219, 220 Polyspermy, 77, 185 block to, 85, 91, 114, 120 Pomatocews triquester, 57 Pomerat, C. M., 354 Pommerenke, W. T., 79 Popa, G., 31, 139 Porter, K. R., 263 Prechordal plate, 292 Preformation, 345 Prism larvae, 276 Pronuclei in mammalian fertilization, 121 Protamines, 25, 53 Proteins, 192 action of different agents on, 305 basic, 25, 53 changes following fertilization, 169 demolition, 307, 310, 313 denaturation, 304, 307 fibrillar, 304 globular, 302 in malformed embryos, 299, 307 396 INDEX salting out method for, 300 synthesis, 273, 277, 313 turnover, 173 viscosity changes, 305 Psamniechinus miliaris, 28, 30, 137 Pseiidocentrotus depressus, 140 Pseudopregnancy, 79 Quinlan, J., 77, 78 Rabbit, 75, 81, 84, 89, 110, 115, 118, 123 Radioautography, 12 Radiophosphorus (P-^-), 11 localization, 16 Raffel, S., 344, 354 Rana metabolic blocks in hybrids, 251 respiration in hybrids, 245 Rana catesbeiana, 216 Rana esculenta notochordal cells in, 292 treatment with iodosobenzoate, 295 Rana pipiens, 209, 219, 231, Shumway stages, 232 Rana sylvatica, 231 Pollister and Moore stages, 234 Ranzi, S., 291, 293-298, 300-310, 312,313 Rats, 75, 76, 82, 84, 88, 115, 118 oocyte, growth curves of^ 5 Raven, C. P., 299, 333 Rawles, M. E., 348, 351 Reaggregation, 366 Redenz, E., 80, 81 Reed, C. I., 191 Regulative eggs, 265 enzyme-inactivation in, 336 mitochondria in, 332 Rein, G., 121 Reindorp, E. C, 205 Reptiles, 86 Retzius, G., 135 Reverberi, G., 319, 322-324, 326, 332 Reynolds, S. R. M., 78 Rh factor, 355 Ribonucleic acid (RNA), 264; see also Nucleic acid base content of, 10 in egg fragments, 264 functions of, 15 metabolism, 8 in micromeres, 273 and microsomes, 273 in nucleoli, 6 oocyte diameter and, 7 P^- incorporation into, 11 and protein synthesis, 273 radioisotope studies on, 11 in sea urchin embryos, 273 solubility of 7, 15 synthesis of, 16 ultraviolet absorption of, 7 Ribonucleoproteins, 264 Rich, A., 4 Ries, E., 324, 331 Ris, H., 204 RNA, see Ribomicleic acid Robertson, M, 352 Rodents, see Hamster; Mouse; Rats Rogers, H. J., 87, 90 RolHnson, D. H. L., 81 Rotnalea, 205 Romanoff, A. L., 2 Romanoff, A. J., 2 Romeis, B., 280 Rose, S. M., 369 Rostand, J., 114 Rothenbuhler, W. C., 185, 186 Rothschild, Lord, 24, 26, 28-32, 56, 73, 74, 81, 85, 91, 92, 119, 120, 140-142, 145, 185 Roux, L. L., 77, 80 Rowlands, I. W., 87, 123, 124 Rowson, L. E., 77, 79 Roy, A., 74 Rubaschkin, W., 121 Rugh, R., 115, 217 Rulon, O., 336 Runnstrom, J., 24, 30, 31, 39, 43, 44, 47, 49-53, 55, 56, 58, 59, 91, 114, 269-271, 291, 313, 362 INDEX 397 Sabellaria vulgaris, 149 Saccoglossus kowalevskii, 159, 161, 164 Salisbury, G. W., 73, 81 Salmon, 56, 73 Salmonella piiUorem, 352 Sapsford, C. S., 89 Schartau, O., 26, 27, 31, 39, 52, 55, 56 Schechtman, A. M., 345, 346 Schenk, S. L., 110 Schneider, W. C, 270, 274 Schonmann, H., 218 Schott, R. G., 79 Schotte, O. E., 202 Schrader, F., 3 Schroder, V., 82 Schultz, J., 202, 204, 207, 222 Sciara, 188, 204 Scott Blair, G. W., 79 Sea urchin, 140, see also individual species cytoplasmic particles in eggs, 270 effects of enzyme-blocking on eggs, 336 enzymes in development of, 278 euglobin from eggs, 305 fluoroacetate effect on eggs, 337 Golgi bodies in eggs, 264 gradients in eggs, 270 inhibition of cleavage in, 356 LiCl effect on embryos, 291 mitochondria at different stages of development, 264, 281, 332 p-pyruvate effect on eggs, 337 Seaweeds, 85 Seidel, F., 188 Selenite, 326 Selivanova, O. A., 82 Semen, 72-76, 80, 82, 83; see also Sperm Seminal plasma, see Semen Sera, see also Antisera cytopathogenic, 363 reticulo-endothelial immune, 354 Serum albumin, 347, 352 Serum globulin, 347 Setlow, R. B., 191-193 Sex determination, 177 Sex substances, classification of, 24- 25 Shapiro, H., 116, 117 Shaver, J. R., 263, 264, 266-268, 270, 273-276, 281, 284, 313, 333 Sheep, 76-79, 82, 90, 91, 120 Shelton, E., 281 Shengun, A., 206, 207 Shettles, L. B., Ill Shumway, W., 231 Siekevitz, P., 278 Sieve, B. F., 125 Sigurdsson, B., 357 Silk protein, 342 Simmonds, S., 81 Simpson, L. O., 352 Skreb, Miss, 194 Slautterback, D. B., 40 Slime mold effect of antisera against, 366 Smellie, R. M. S., 11 Smiles, J., 87 Smith, A. U., 83, 93, 110, 114 Smithberg, M., 90, 120 Snell, G. D., 352 Snyder, F. F., 79 Sobotta, J., 121 Soderwall, A. L., 77 Sodium thiomalate, 293 Sonneborn, T. M., 222, 368 Sonnenblick, B. P., 177, 181, 189 Southwick, W. E., 55, 56 Spar, I., 345, 348 Sparrow, E. M., 363 Specificity genetic, transfer of, 16 structural, RNA as carrier of, 19 Speicher, B. R., 177, 185 Spemann, H., 201, 202 Spencer, R. R., 357 Spensley, P. C, 87, 90 Sperm, see also Semen antagglutin for, 83, 88, 126 mammalian, fertilizing capacity, 79, 111 midpiece, 31 398 INDEX penetration of egg by, 56, 82, 87, 118, 124 Spermatozoa, 135-165; see also Ac- rosonies; Agglutination accessory, 194 activation of, 72, 80, 93 age and fertilizing capacity, 80 antigens of, 93 attachment to egg, 89 entry into egg, 153-159, 163 in female tract, 75-88, 111 metabolism of, 81, 82 reacted, 140, 142, 163 separation of chromosome-bearing, 82 storage of, 72, 74 Spiegel, M., 366 Spiegelman, S., 17, 222 Spikes, J. D., 28, 30, 31 Spleen effects of antisera against, 356 induced enlargement of, 369 S35-labeled, 369 Spondtjliis crventiis, 136 Sponges, 366 Spronk, N., 333 Stanier, R. Y., 222 Starfish, 33, 142; see also individual species cytoplasm, RNA concentration of, 13 oocyte, 5 RNA metabolism in oocyte, 8 Starke, N. C, 76, 79, 80 Steinbach, H. B., 263 Steinitz, L. M., 202, 207 Steinmtiller, O., 351 Stereocil, 137 Stern, C, 202, 222 Stern, H., 204 Stetson, R. E., 355 Stockard, C. R., 292 Stratton, F., 355 Sti-auss, F., 78 Strongylocentrotus droebachiensis, 45, 46, 137, 138 Strongylocentrotus franciscanus antisera against fertilizin of, 359 Strongylocentrotus pulcherrimus, 40, 140, 141 Strongylocentrotus purjmratus antisera against fertilizin of, 24, 30, 36, 43, 46, 49, 359, 360 mitochondria in, 274, 283 Succinodehydrogenase, 325 blocking of, 336 inhibitors of, 326 Superfetation, 79 Survival curve, see Ultraviolet radia- tion Swann, M. M., 91, 92 Swver, G. I. M., 74, 123-124 Sze, L. C, 246, 250 Tafani, A., 121 Tahmisian, T., 264 Tamini, E., 295, 299 Taylor, A. C, 189 Tavlor, A. N., 4 Tavlor, J. H., 12, 16 Tchou-Su, M., 183 Telfer, W. H., 2, 345 Temnoplcurus hardeicickii, 40 Ten Gate, G., 345 Tenrecs, 78 Tergites, 189, 191 Testis cross reactions with, 344 Tetrahymena, 183 Thibault, G., 114-116 Thomas, L. E., 74 Thijone, 151, 162 Thyone hriareus, 146, 155, 161, 163 Thyroglobulin, 344 Tiselius, A., 39, 47, 53, 55, 56, 58 Tissue culture, 350 Tissue grafts, 350, 351 Topley, W. W. G., 350 Torvik-Greb, M., 177 Tosi, L., 58 Tosic, J., 82 Totipotency, 202 Trichomonas foetus, 352 Trimberger, G. W., 77 Trinkaus, J. P., 366 Triton, 202 INDEX 399 Triton cristatus, 215 Triton palmatus, 215, 217 Tritnnis palmatus, 218 Triturits pyrrhogaster, 216 Trout, 73 Tnbifex eggs cytochrome oxidase in, 333 mitochondria in, 330 Nadi reaction in, 332 Tumor transplants induced tolerance to, 352 of rat in chick embryo, 353 Tung, T. C, 324 Tuzet, O., 137 Tyler, A., 2, 24, 26-31, 33, 37-58, 74, 81, 85, 88, 89, 114, 115, 117, 122, 124, 125, 140-142, 145, 173, 176, 204, 222, 264, 269, 341, 345, 350, 353, 357, 360, 362, 364, 365, 368, 370 Tyler, J. S., 47 Uhlenhuth, P., 344 Ulrich, H., 191 Ultraviolet radiation, 190-195 Unfertilized eggs, 363 Univalent substances, 365 Urbani, E., 264 Urea, effect on notochord, 295 resistance of proteins to, 310 Urechis caupo, 38 Utero-tubal junction, 77, 78 Utida, S., 73 Vaginal plugs, 76 Vail, V. C, 76 Van Beneden, E., 86 Van Demark, N. L., 75-77 Van der Stricht, O., 121 Van Doorenmaalen, W. J., 345 Van Drimmelen, G. C, 80 Van Egmond, M., 8 Vasseur, E., 26-28, 30, 31, 38, 39, 44-48, 52, 53, 55, 56, 142, 362 Vegetal halves, 278, 279 Vegetalized larvae, 291 ' Vegetalizing agents, 298, 302, 304, 305, 307, 311 Vegetal pole, 313 Venable, T. H., 114 Vendreley, C, 204 Vendreley, R., 204 Venge, O., Ill Venom, 365 Versene, 35 Vier giver, E., 79 Vincent, W. S., 1, 4, 5, 9-11, 13, 15- 17 Viruses, 350 Vogt, W., 239 Wada, S. K., 29, 89, 135, 136, 139, 143, 145, 151 Waddington, C. H., 217 WahU, H. R., 330 Wallenfels, K., 27, 31, 39, 52, 55, 56 Walton, A., 72, 76, 82 Warbritton, V., 79 Warren, M. R., 76 Warwick, E. J., 79 Watkins, W. M., 342 Watson, J. D., 4 Watterson, R. L., 265, 268 Weismann, A., 201, 208 Weiss, P., 222, 354, 355, 364, 366, 369, 370 Werder, A. A., 357 Westman, A. A., 75 Wetter, L. R., 343 White, M. J. D., 204-206 Whiting, A. R., 185, 187 Whiting, P. W., 177, 183-185 Whitney, 76, see Evans (1933), p. 98 Wicklund, E., 30, 50, 51, 52, 91 Wiener, A., 350, 355 Wilbur, K. M., 192 Willett, E. L., 74 Willier, B. H., 351, 369 Wilson, E. B., 1, 92, 177, 201, 264, 265, 271, 277, 280 Wilson, G. S., 350 Wilson, H. v., 365 Wimsatt, W. A., 80 400 INDEX Winters, L. M., 79 Wislocki, G. B., 78, 79 Witebsky, E., 344 Wittek, M., 4, 264 Woerdeman, M. W., 222, 345, 368 Woglom, W. H., 357 Wolfe, H. R., 350 Wolff, S., 193 Woodruff, M. F. A., 352 Woodward, A. E., 33 Wright, E. S., 76, 87, 124 Wyburn, G. M., 116 Xenopus reduction of animal half in em- bryo, 292 X-radiation, 217, 354 Yamane, J., 123 Yeas, M. K. W., 4, 342, 349 Yellow crescent, 323 Yolk absorption of antisera with, 346- 347 disintegration and preservation of granules, 311 formation of, 264 Young, W. G., 77, 80 Youngner, J. S., 351 Zeuthen, E., 3, 177, 181 Zollinger, H., 280 Zona pellucida, 82, 87-92, 119, 124 Zwilling, E., 212