THE EFFECTS OF ANTISERA UPON CLEAVAGE AND EARLY DEVELOPMENT OF FERTILIZER RANA PIPTEN S EGGS Thesis for the Degree of M . S . MICHIGAN STATE UNIVERSITY BARBARA JEANNE FREY 1967 IHESSS LIBRARY Michigan State l.111ixr<:11:it}r ‘9' BINDING CY . noun a SflNS’ 7i TEEEJEEEEELJEE ABSTRACT THE EFFECTS OF ANTISERA UPON CLEAVAGE AND EARLY DEVELOPMENT OF FERTILIZED RANA PIPIENS EGGS By Barbara Jeanne Frey A series of experiments were conducted in which Rana pipiens eggs were treated externally and also injected with antisera against various Rana pipiens tissues. Effects on cleavage and subsequent development by each serum were noted and compared with appropriate controls. The results, though inconsistent, did show some cases of total inhibition of cleavage in eggs treated with anti-ovary serum. It is postulated that this was due to an interaction of antibodies with complementary molecular configurations on the surface of the egg, which interfered with the normal function performed by these molecules in cleavage. THE EFFECTS OF ANTISERA UPON CLEAVAGE AND EARLY DEVELOPMENT OF FERTILIZED RANA PIPIENS EGGS By Barbara Jeanne Frey A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Zoology 1967 ACKNOWLEDGMENTS I wish to express my sincere appreciation to Dr. John R. Shaver and Dr. Stephanie H. Barch for their sug- gestions and assistance during the course of this work. I would like to acknowledge and thank the members of my guidance committee, Drs. V. Mallmann and C. Thorton, for their direction in the classroom. A note of thanks is also due to Ronald Pfohl for his generous help and advice on numerous occasions. This study was conducted during the tenure of a National Defense Education Act Fellowship granted to me by the Department of Zoology. ii TABLE OF CONTENTS ACKNOWLEDGMENT LIST OF TABLES I. INTRODUCTION II. MATERIALS AND METHODS A. Production and Fractionation of Antisera B. General Procedure of Jelly Removal and External Treatment of Ferti- lized Rana pipiens Eggs C. General Procedure f0r the Injection of Fertilized Rana pipiens Eggs with Antisera . . . . . . . . III. RESULTS IV. DISCUSSION A. External Treatments B. Injection of Eggs V. SUMMARY LITERATURE CITED . iii Page ii iv 11 ll 12 14 16 33 33 39 39 41 TABLE II. III. IV. VI. VII. VIII. IX. LIST OF TABLES Immediate treatments External treatments of eggs with sera diluted 1:2 with 1/6 f.s. Holt. for 30 minutes . . . . . . . . . . . . External treatments of eggs with sera diluted 1: 1 with 1/6 f. s. Holt. for 20 minutes . . . . . . . . . . . External treatments of eggs with sera diluted 1:1 with 1/6 f.s. Holt. for 30 minutes . . . . . . . . . . . . . . . External treatments of eggs with sera diluted 1: l with 1/10 f. s. Holt. for 30 minutes . . . . . . . . External treatments of eggs with sera. diluted 2:1 with 1/10 f. s. Holt. for 15 minutes . . . . . . . . . . . . External treatments of eggs with sera diluted 2:1 with 1/10 f. s. Holt. for 30 minutes . . . . . . . . . . . . . . External treatments of eggs with sera diluted 1:1 with 1/10 f. s. Holt. for 45 minutes . . . . . . . . . . . . Delayed treatments External treatments of eggs with sera diluted 1:1 with 1/10 f.s. Holt. 4 hours after fertilization for 30 minutes External treatments of eggs with sera diluted 1:1 with 1/10 f.s. Holt. 2 hours after fertilization for 30 minutes iv Page 22 22 23 24 26 26 26 27 28 TABLE XI. XII. XIII. XIV. Injections Injections of eggs with sera diluted 1:2 with f.s. Holt. 1 hour after fertilization . Injections of eggs with sera diluted 1:8 with f.s. Holt. 1 hour after fertilization Injections of eggs with sera diluted 1:4 with 1/10 Holt. 1 hour after fertilization . Injections of eggs with sera diluted 1:8 with 1/10 f.s. Holt. 45 minutes after fertilization . . . . . . . . . . . Injections of eggs with sera diluted 1:4 with 1/10 f.s. Holt. 45 minutes after fertilization ‘. . . . . . . . Page 30 30 31 32 32 I. INTRODUCTION Numerous investigators have employed immunological techniques to study the macromolecular structure of gametes and the role of antigens in fertilization, cleavage, and de- velopment (Tyler,1963, 1965). Knowledge of interacting, well-defined substances involved in such processes as the mechanism of the penetration of the egg surface by the sperm, initiation of the cortical reaction of the egg, the block to polyspermy, and the acrosomal reaction of the Sperm is still largely obscure (Metz, 1961; Monroy, 1965). The use of antigen-antibody reactions as a method of analysis of molecular structure and function is desirable in that antigen-antibody reactions are both sensitive and Specific. It can be seen that if an antigen or its active site plays a role in fertilization or subsequent development, the antibodies produced against the antigen, when used at sublethal doses, would inhibit its normal function. Thus it would be possible to identify the antigen, localize it, and determine its developmental significance and nature of action (Edds, 1956; Tyler, 1955). Lillie (1913, 1914) first observed that the sea water in which unfertilized sea urchin eggs had been stand- ing ("egg water") agglutinated spermatozoa of the same 1 species and temporarily stimulated their motility. He named the agglutinating substance "fertilizin" and elaborated a theory, which stated that fertilizin was continuously se- creted and diffused out into the jelly coat. This led to a number of investigations of fertilization in invertebrates (mainly sea urchins). Tyler (1959) modified Lillie's theory when he noted that fertilizin constituted essentially the whole jelly coat and surface proper of the egg, but that there was no evidence of continuous secretion of fertilizin from the unfertilized egg. He found fertilizin to be tissue and species-specific and when "anti-agglutinin" (sperm ex- tract) and "agglutinin" (egg water) were mixed, there appeared a precipitation layer and neutralization of activity occurred. Experiments by Motomura (1953) and Hagstrom (1956) indicated that a "cytofertilizin" existed in unfertilized, jellyless sea urchin eggs. This was later shown by the use of fluorescine-labelled antibodies against sea urchin fer- tilizin to be present in the plasma membrane rather than the vitelline membrane (Tyler, Seaton, and Signoret, 1961). The material on the surface of the sperm complementary to fertilizin (Lillie's antifertilizin) has been investigated by many of the same techniques as have been used in the analy- sis of fertilizin (Tyler and O'Melveny, 1941). Presence of a substance in the egg proper identical to or closely related to antifertilizin (Tyler, 1940), led Tyler (1947) to the con- cept of a natural auto-antibody reaction. This concept states that cells are composed of systems of complementary sub- stances capable of interacting in a manner similar to antigens and antibodies, and that the formation of certain large molecular substances of cells proceeds by one of the interacting substances serving as a template for the other similar to one of the roles proposed for general antibody formation. The results of these and other experiments seemed to indicate that, in the sea urchin, the interaction of these complementary substances on the plasma membranes of the gametes in a species-specific manner accounts for the specific adherence of the sperm to egg and incorporation of the sperm by a sort of pinocytotic process (Tyler, 1959). To further study the roles of these interacting substances in fertilization and development in the sea ur- chin, antisera against various cell constituents were used. Treatment of eggs prior to fertilization (Perlmann, 1959) indicated the existence of four different types of macro- molecules in the surface layers of the egg, three of which seemed to have some function in sperm attachment and egg activation. Antisera against extracts of unfertilized eggs and of embryos at various stages of development were tested for effects on fertilization and on early development (Tyler, 1959, 1963; Tyler and Brookbank, 1956). Definite cytotoxic and inhibitory effects were noted on unfertilized and fertilized eggs. In the case of the latter, cleavage was blocked by antisera against purified egg jelly (fertilizin) or whole egg homogenates, but not by antisera containing only antibodies against internal constituents of the egg. Recently,papain-digested, univalent antibodies were tested for effects on fertilization and cleavage (Metz and Thompson, 1967). This non-agglutinating, non—precipitating, 3.55 form of gamma globulin failed to produce the morpholog- ical changes in eggs that result from treatment with multi- valent, "7s" antibodies. These univalent antibodies against egg homogenate also failed to affect the fertilizability of dejellied or demembranated eggs. However,-univa1ent anti- egg jelly gamma globulin did inhibit cleavage of fertilized eggs although to a lesser degree than the regular multivalent antibodies. Thus it was concluded in the sea urchin, Ly; techinus variegatus, the fertilization-inhibiting action of multivalent antibodies depends upon the cross-linking of neighboring antigens, not on the blocking of specific anti- genic sites by complementary antibodies. Whether this is true of the cleavage-inhibiting action remains to be as- certained. Similar work has been done on anuran eggs suggest- ing that analogous substances and phenomena interact in fertilization and development in this group (Shaver, 1966). Glaser (cited in Shaver and Barch, 1960) reported agglutination of sperm of Rana pipiens by "egg water" of Rana pipiens. Bernstein (1952) and Shaver, Barch, and Shivers (1962) were unable to repeat this. However, Bern- stein did find that the "egg water" of Rana clamitans irreversibly agglutinated Rana clamitans sperm. This was true of only jellied eggs. It was noted that the jelly of this species was highly soluble compared to that of Rana pipiens. In several species of Anura, coelomic eggs or eggs dejellied chemically or mechanically are generally unferti- lizable. (Bataillon, 1919; Kambara, 1953; Tchou and Wang, 1956; Katagiri, 1965, 1966). Kambara (1953), working with Bufo vulgaris formosus, found that eggs devoid of jelly be- came fertilizable if covered with gelatin or agar. Thus, he postulated a thigmotactic role for the jelly in fertilization. Katagiri (1966), however, investigating Bufo bufo formosus, could not repeat these results with agar, gelatin, or egg albumin, but found a high percentage of dejellied eggs were fertilized when they were inseminated in the pre- sence of either dialyzed jelly material, pronase-digested jelly material, or polyvinylpyrrolidone (PVP). In addition, Shivers (cited in Shaver, 1966) has demonstrated that jellyless body cavity eggs could be ferti- lized by spermatozoa which have had prior contact with jellied uterine eggs. Other work by Katagiri (1965) seemed to indicate that in addition to the presence of jelly (layers J1 and J2 or layer J4) the egg must be cytoplasmically mature to be fertilized. Therefore he suggested that the role of the oviduct may be to establish this cytoplasmic maturity. In other investigations, Nace g£_gl. (1960) found evidence of an antigen (A) in follicle cells, immature oocytes, oviducal epithelium, and oviducal eggs. Believed to be synthesized in the follicle cells and oviduct, this antigen was thought to be either a regulator of meiosis and mitosis, a sperm acceptor, or a factor which "matures" the eggs. Click and Shaver (1963) further demonstrated that Rana pipiens eggs from the middle and lower oviducal regions were more easily fertilized than those from upper segments. Shaver (1966) treated spermatozoa of Rana pipiens with jelly coat materials of different origins and these spermatozoa were used to inseminate jellied uterine eggs. A significant decrease in fertilizing capacity of the sperm was produced by jelly material from oviducts of Rana pipiens, especially by extracts from the lower portion of the oviduct. It was noted that it is from this region that the outer layer of jelly would originate. Shaver and Barch (1960) pretreated both spermatozoa and eggs of Rana pipiens with antisera against the jelly coat material. When the treated gametes were used in crosses with untreated gametes, a significant decrease in numbers of eggs fertilized resulted. Other experiments (Barch, personal communication) in which antisera against other Rana pipiens tissues were used to treat eggs also have produced some in- hibition. The percentages of inhibition of fertilization caused by these antibodies (against ovary, heart, and kidney of R. pipiens, R. clamitans, R. catesbiana, Axolotl, B. mari- nus, B. americanus, and against the egg jelly of the Arbacia punculata) roughly correspond to the number of antigenic com- ponents common to R. pipiens eggs and the tissues of the other species. Very recently, Shaver and Barch (1966) varied the time of insemination in relation to the time of treatment of eggs with various antisera. The results show that the antibodies probably reach the egg surface proper fairly rap~ idly. Therefore, sperm which have traversed the jelly layers may not be able to interact with egg surface sites due to prior blockage of these by the treatment with antibodies. Whether these results would support the existence of a "fer- tilizin—antifertilizin” system in Anuran gametes analogous to that of sea urchin eggs is uncertain. Since the inhibiting action of the antisera could be due to a cross-linked lattice among the jelly molecules act- ing as a mechanical barrier to sperm, Shivers and Metz (1962) used papain—digested, univalent, non-precipitating anti-jelly serum. This also produced a significant inhibition of ferti- lization, but no precipitation of jelly layers occurred. Thus, the authors felt that a direct blocking by the anti- bodies of receptor sites in the jelly which were important in fertilization had occurred. Different antigenic components have been shown to be present at different oviducal levels (lower and upper) by Barch and Shaver (1963). It was also noted that mature uterine eggs of Rana pipiens treated with antisera against the lower segment of the oviduct fertilized in significantly lower numbers than eggs treated with antibodies against the upper segment. Corresponding to oviducal differences are the anti- genic differences in the three jelly layer of mature uterine eggs, investigated extensively by Shivers. Shivers (1961) found that four species of Rana contained both species- specific and common components in their jelly layers. Eggs of Rana pipiens were pretreated with homologous and heterolo- gous antibodies and fertilizability was decreased by antisera both against the species-specific components of Rana pipiens egg jelly and against the common components of heterologous egg jelly. Using fluorescine-conjugated antibodies against homo- logous and heterologous egg jellies to stain the jellies of Rana pipiens eggs, Shivers (1962) found that the species- specific components were localized in the outer two jelly layers and the common component in the inner jelly layer. Investigating various genera and species of amphibians, Shivers (1965) also found that in no case where cross fer- tilization produced viable embryos were jelly antigens shared by the two species lacking. All of these results have led Shaver (1966) to hypo- thesize that the egg jelly antigens are responsible for a series of reactions which involve the complexing of both species-specific and common combining sites on the sperm surface and jelly layers, followed by reactions of comple- mentary sites on the egg and sperm surfaces. “Information about these hypothetical sites on the egg surface is sketchy and inconclusive and it is about these that this present investigation concerns itself. Shaver, Barch, and Shivers (1962), using the double diffusion technique, found a tissue specificity of Rana pipiens egg jelly components similar to that described for the fertilizin of the jelly coat of sea urchin eggs. Some cross reactions did occur between jelly and ovarian material which seemed to indicate the presence in1fluajelly of material secreted by the egg or by oviducal cells. Using antisera against mature ovaries and embryonic stages of Rana pipiens to treat coelomic eggs, an antigen. on the surface of mature oocytes and fertilized eggs (anti- gen F) was found (Lavin, 1963; Nace and Lavin, 1963). Anti- sera against antigen F inhibited activation and cleavage. The role played by antigen F in activation and cleavage was believed to be consistent with the role suggested for mucosubstances in the egg. Although Shivers (1965) showed by the use of fluo- rescine-conjugated antibodies, that egg jelly antigens 10 existed only in the jelly layers, Poirier (1964) found a significant inhibition of fertilization in coelomic eggs of Rana pipiens which had been treated with anti-egg jelly sera, transferred through the oviducts of ovarectomized females, and then inseminated. Cerny (1963) also found a lower number of cleavages in Rana pipiens eggs which had been fertilized, dejellied with potassium cyanide, and then treated with anti-jelly serum. Thus is seemed that by treating dejellied Rana pi- piens eggs, at different times after fertilization, with antisera against various Rana pipiens tissues, further know- ledge of the antigenic components of the frog egg would be gained. The results of exposing eggs to antisera both in culture and by injection will be presented. II. MATERIALS AND METHODS A. Production and Fractionation of Antisera Rana pipiens egg-jelly, heart, and ovary antigens were prepared in the following manner: Jelly was manually removed from mature uterine eggs, lyophilized, and homo- genized (5 mg./m1.) in one-tenth full strength Holtfreter's solution. Heart and ovary were each homogenized (one gram in ten ml.) in one-tenth full strength Holtfreter's solu- tion. Each homogenate was then separately added to equal parts of complete Freunds adjuvant and emulsified. Next, 1.5 m1. of these emulsions were injected half on each side into the subscapular region of rabbits. An equal dose, with incomplete Freund's adjuvant, was injected one week later. In about 3-4 weeks, if antibodies could be detected, bleedings were begun and continued every other week. Anti- gen injections were also continued every two weeks. In all cases, serum was collected from the rabbits, before antigen injections, to be used as control serum. The control serum and the antisera which had been demonstrated, by the Ouchterlony double-diffusion technique (Ouchterlony, 1949; Shaver, 1961), to have antibodies to homologous antigens were each pooled and then fractionated 11 12 as follows: First the serum was filtered and an equal amount of ammonium sulfate was added to cause a precipita- tion. This was placed in the refrigerator overnight. Then it was centrifuged at 2 degrees C. for twenty minutes at a speed of 10,000 rpm. The supernatant (albumin fraction) was poured off and the residue (globulin fraction) was rinsed with 50% ammonium sulfate to get rid of the trapped albumins and centrifuged as before. Again, the supernatant was dis- carded. The residue was dissolved in one-tenth full strength .Holtfreter's solution (one-half the amount of the original \nalume of serum filtered). This dissolved globulin was then (Lialyzed against amphibian saline (0.65% NaCl) for 48 hours, rwnnoved, and frozen for later use. B. General Procedure for Jelly Removal and External Treatment of Fertilized Rana pipiens Eggs Ovulation in female Rana pipiens was induced by pitniitary gland injection (Rugh, 1934). The number of pitui- taries injected depended on the season. Twenty-four to forflxy-eight hours after injection, eggs from l-4 frogs were sturipped onto glass slides and fertilized with sperm suspen- sior1 obtained by the maceration of two testes of Rana pipiens per' 20 ml. of one-tenth full strength Holtfreter's solution. Afte]? 6-8 minutes, excess sperm suspension was decanted and each; slide of eggs was placed in a finger bowl half-full of aeratned water. There the jelly was hydrated for fifteen minutes. One slide of fertilized eggs was left in the l3 aerated water with the jelly intact as a control on the cleavage percentages of that particular batch of eggs. The rest of the eggs were put into 100 ml. of 0.1N cysteine HC1 (M. W. 175. 64) which had been buffered to a pH of about 7.5 with 1N sodium hydroxide. The eggs were vigorously swirled in this solution for about twenty minutes. The cys- teine HCl solution was poured off the eggs and 50 m1. of fresh solution was added and swirling was continued. An- other 50 ml. was added and by this time most of the eggs were visibly dejellied. This could be seen by the way they lay in the beaker touching each other with no jelly coats separating them. At this point they were washed six times with one-sixth or one-tenth full strength Holtfreter's solu- tion and placed into the varidus treatment solutions for different lengths of time (15, 20, 30, and 45 minutes). These treatments began 50-55 minutes, 2 hours, and 4 hours after fertilization. The following treatment solutions were used at various times during the course of this work: full strength Holtfreter's solution; one-sixth full strength Holtfreter's solution; one-tenth full strength Holtfreter's solution; 7.7% Ringers; aerated water; control sera and antisera diluted 1:1, 1:2, 2:1, and 3:1 with one-sixth or one-tenth full strength Holtfreter's solution. In the cases where the eggs were not put into the treatment solutions for 2 or 4 hours after fertilization, the dejellied eggs were kept in one-tenth full strength Holtfreter's solution until 15 included: one-tenth full strength Holtfreter's solution; control serum; heart, ovary, and egg jelly antisera. All the sera were diluted 1:2, 1:4, and 1:8 with one-tenth full strength Holtfreter's solution. A fertilized, non-injected, control slide of eggs was included in each experiment. All eggs were cultured in finger bowls of aerated water at room temperature. Cleavage, gastrula, and tailbud counts were made. III. RESULTS The results of the experiments involving the exter- nal treatment of the dejellied, fertilized frog eggs are presented in Tables I-lX. Each trial in experiments 1-7 in Tables I-VII involved eggs from just one frog (except Exp. 4-H). Cleavage and gastrula counts were made about 4-6 hours and 24—34 hours, respectively, after fertilization. These correspond approximately to Shumway (1940) stages 5 and 11. Although counts were not recorded past gastrula, embryos were allowed to develop and were observed for as long as ten days. More died as the days progressed, but some did appear normal at the end of this period. There are some figures in the data indicating that the number of eggs which successfully reached gastrulation was larger than the number which was recorded as having cleaved. This does not represent a "recovery" but rather a few eggs which were slower to divide at first but had ”caught up" by the gastrula stages.. Eggs which were not counted as developing showed a variety of abnormalities. Often there was cytoplasmic mott- ling or streaking, especially along the cleavage furrows. In other cases the eggs had burst due either to a cytotoxic effect or from being extremely fragile without the jelly l6 17 coat. Many eggs were misshapen and/or contained irregular folds. Some had patches of smooth undivided cytoplasm amidst areas of normally divided calls. Occasionally there were eggs which had partially pushed out of the vitelline mem- brane to form a dumb-bell shaped embryo. Many showed diffuse yolk plugs and/or arrested yolk plug closure. Comparing the percentages, there is a disturbing variation among frogs. This could be due to differences in egg sensitivity or differences among the antisera used. How- ever, the differences among antisera were thought to have been minimized by pooling them. The only antisera that seemed to have effects significantly different from the others were those against ovary. The most striking results appear in the five trials (Experiment 2-A and B; Experiment 4-A, B, and C) where there was no development at all after treatment with anti-ovary antisera. Every egg was smooth without a single cleavage furrow. Since development was satisfactory to excellent after the other treatments, the eggs can be considered normal in these trials. These five trials were done with the same dilutions of sera and with similar times of treatment (20 and 30 minutes). These results could not be repeated although the same methods were used. There were, however, other trials in which noticeably lower percentagesof eggs treated with anti-ovary serum reached the gastrula stage. This is shown by the results in EXperiment 3-A and B. Although the 18 percentages reaching gastrulation were low after treatment with anti-ovary serum in Experiment 4-F and Experiment 6-B, there were also low percentages for eggs treated with anti- heart and anti-jelly sera in both these experiments. The differences were usually greater in the gastrula stages. Sometimes a much lower percentage of eggs reached gastrulation in the jelly controls, or after treatment with v“ l/10 full strength Holtfreter's solution or with the con- trol sera. This appeared only occasionally and did not seem to be due to anything other than differences among eggs from different females. Tables VIII and IX represent experiments in which the eggs were treated with antisera after development had proceeded for 2 or 4 hours. The eggs which were treated at the end of four hours were counted for cleavage after four hours of development, then treated, and counted again an hour after treatment. Of course those which were treated after two hours could not be counted before treatment be- cause it was too early to note cleavage. In each of these trials a regular set of treatments were run in which the eggs were treated immediately as in all the other work. Thus, this served as an overall control for the eggs which were treated after 2 or 4 hours of development. No noticeable differences occurred consistently between the immediate or delayed treatments or between the different types of anti- sera in each experiment. There were some incidences of lower 19 percentages of development in anti-ovary serum. However, these were accompanied by an equally poor development in anti-heart and anti-jelly sera (Exp. 8-B; Exp. 9-C). Appar- ently the time at which the eggs were treated was not a decisive factor here. Eggs from more than one frog were used in each of the trials just mentioned (Experiments 8 and 9). It was hoped that this would minimize differences in egg sensitivi- ties. The eggs used in this phase of the work were cultured in aerated water after treatment rather than Holtfreter's solution, as were those recorded in Tables I-VII. This pro- cedure seemed to be more favorable for survival. Often it was difficult to keep the fractionated sera fully dissolved in the Holtfreter's solution. During the course of the time treatments, a precipitate (probably of denatured protein) would form on the bottom of the dish and around the eggs. This usually only happened with the con- trol and anti-heart sera. Perhaps this meant that active anti-bodies were not effectively reaching the egg surface, but since the results did not show consistent differences in the development of the eggs in these antisera, the sig- nificance of the precipitate on the experiments is uncertain. Unfractionated serum did not precipitate in this fashion, so one experiment was done with unfractionated sera. However, it was not repeated since the unfractionated sera seemed very toxic to the eggs and many more eggs stopped develOping earlier. 20 Tables X—XIV represent the results obtained from injecting antisera into fertilized, jellied Rana pipiens eggs 45 minutes and one hour after fertilization. There was no doubt that the eggs were indeed punctured, since a hole and/or exovate was noticeable. These punctures did not seem to be the reason that development did not proceed normally because many healed as they developed normally. ' Abnormalities were not as varied here as in the ex- ternal treatments. Most of them showed cytoplasmic streak- ing, diffuse yolk plugs in gastrulation, or irregularly shaped embryos in the tailbud stage (often due to edema). |~' Comparison of the percentages here shows even less consistency than the previously mentioned results. There was a general increase in death rate from the four-cell stage to the tailbud stage. This was even true of the non- injected controls and almost all of the eggs injected with 1/10 Holtfreter's solution and control serum. Thus, rather than a specific effect by one or more antisera, there ap- peared to be a general collapse due to the condition of the eggs. The injections were all done in April and May, so perhaps the eggs were not as good as they were earlier in the year. It can only be definitely stated that the percentage of eggs which reached cleavage was good in almost every ex— periment and that of embryos which reached the tailbud stage was low. Thus, the only place where differences could be 21 noticed was in the percentage that reached the gastrula stage. However, the number of embryos reaching the gastrula stage was low regardless of the type of serum injected, al- though injection with anti-ovary serum produced the smallest number of gastrulae. A dilution of 1:1 was not used since the 1:4 dilu- tion seemed sufficiently strong to give an effect and the more concentrated antisera were extremely hard to draw into the pipette. There was also a problem with the fractionated sera precipitating and clogging the needle, so unfractionated sera were tried but proved to be more toxic, as they had in the external treatments. As before, many of these eggs did develop into more advanced normal embryos although no exact counts were made beyond the tailbud stage. 22 TABLE I Experiment 1 Sera diluted 1:2 with 1/6 f.s. Holt.-30 minutes Solution Total Cleaved Percent Gastrula Percent A. J. Cont.* 41 32 78.0 32 78.0 1/6 Holt. 57 55 96.5 55 96.5 Control 57 55 96.5 55 96.5 Heart 52 49 94.2 50 96.2 Ovary 55 54 98.2 55 100.0 Jelly 53 51 96.2 53 100.0 B. J. Cont. 64 62 96.9 62 96.9 1/6 Holt. 73 70 95.9 71 97.3 Control 84 82 97.6 82 97.6 Heart 64 64 100.0 64 100.0 Ovary 81 78 96.3 78 96.3 Jelly 72 68 94.4 68 94.4 TABLE II Experiment 2 Sera diluted 1:1 with 1/6 f.s. Holt.-20 minutes Solution Total Cleaved Percent Gastrula Percent A. J. Cont. 70 70 100.0 70 100.0 1/6 Holt. 88 86 97.7 86 97.7 Control 41 41 100.0 38 92.7 Heart 119 116 97.5 117 98.3 Ovary 70 O 0 0 0 Jelly 58 56 96.6 56 96.6 B. J. Cont. 85 82 96.5 82 96.5 1/6 Holt. 94 85 90.4 85 90.4 Control 79 71 89.9 72 91.1 Heart 64 63 98.4 63 98.4 Ovary 85 0 0 0 0 Jelly 96 94 97.9 90 93.8 *Eggs with jelly, cultured in aerated water after fertiliza- tion--no treatment. 23 TABLE III Experiment 3 Sera diluted 1:1 with 1/6 f.s. Holt.-30 minutes 5 Solution Total Cleaved Percent Gastrula Percent A. J. Cont. 62 59 95.2 59 95.2 1/6 Holt. 92 90 97.8 90 97.8 Control 104 102 98.1 102 98.1 Heart 95 94 98.9 94 98.9 Ovary 73 69 94.5 42 57.5 Jelly 77 72 93.5 65 84.4 B. J. Cont. 41 32 78.0 32 78.0 1/6 Holt. 57 55 96.5 55 96.5 Control 51 49 96.1 37 72.5 Heart 43 42 97.7 39 90.7 Ovary 39 38 97.4 22 56.4 Jelly 52 51 98.1 38 73.1 C. J. Cont. 64 62 96.9 62 96.9 1/6 Holt. 73 70 95.9 71 97.3 Control 93 92 98.9 91 97.8 Heart 57 54 94.7 53 93.0 Ovary 81 78 96.3 78 96 3 Jelly 84 81 96.4 79 94.0 24 TABLE IV Experiment 4 Sera diluted 1:1 with 1/10 f.s. Holt.-30 minutes Solution Total Cleaved Percent Gastrula Percent A. J. Cont. 56 55 98.2 56 100.0 1/10 Holt. 43 43 100.0 43 100.0 Control 54 48 88.9 52 96.3 Heart 55 54 98.2 54 98.2 Ovary 65 0 0 0 0 Jelly 56 52 92.9 43 76.8 B. J. Cont. 59 48 81.4 48 81.4 1/10 Holt. 36 27 75.0 28 77.8 Control 41 31 75.6 33 80.5 Heart 34 26 76.5 27 79.4 Ovary 45 0 0 0 0 Jelly 50 37 74.0 30 60.0 C. J. Cont. 90 88 97.8 84 93.3 1/10 Holt. 55 50 90.9 49 89.1 Control 62 51 82.3 51 82.3 Heart 91 83 91.2 83 91.2 Ovary 72 0 0 0 0 Jelly 85 69 81.2 69 81.2 D. J. Cont. 88 84 95.5 85 96.6 1/10 Holt. 74 72 97.3 79 94.6 Control 90 87 96.7 87 96.7 Heart 76 72 94.7 72 94.7 Ovary 63 57 90.5 57 90.5 Jelly 72 69 95.8 70 97.2 25 TABLE IV, Experiment 4--Continued Solution Total Cleaved Percent Gastrula Percent E. J. Cont. 164 146 89.0 146 89.0 1/10 Holt. 29 25 86.2 23 79.3 Control 45 36 80.0 40 88.9 Heart 46 41 89.1 43 93.5 Ovary 45 42 93.3 42 93.3 Jelly 25 25 100.0 25 100.0 F. J. Cont. 144 112 77.8 112 78.5 Aerat. H20 53 46 86.8 45 84.9 1/10 Holt. 49 42 85.7 42 85.7 Control 25 22 88.0 21 84.0 Heart 38 24 63.2 23 60.5 Ovary 71 51 71.8 48 67.6 Jelly 43 27 62.8 24 55.8 G. J. Cont. 39 37 94.9 33 84.6 Aerat. H20 71 63 88.7 56 78.9 1/10 Holt. 59 59 100.0 59 100.0 Control 57 50 87.7 48 84.2 Heart 55 53 96.4 49 89.1 Ovary 70 66 94.3 63 90.0 Jelly 85 83 97.6 78 91.8 **H. J. Cont. 98 86 87.8 83 84.7 1/10 Holt. 90 79 87.8 78 86.7 Control 76 72 94.7 72 94.7 Heart 82 73 89.0 73 89.0 Ovary 90 82 91.1 72 80.0 Jelly 50 48 96.0 33 66.0 **Eggs used were from four different frogs. 26 TABLE V Experiment 5 Sera diluted 2:1 with 1/10 f.s. Holt.-15 minutes Solution Total Cleaved Percent Gastrula Percent A. J. Cont. 164 146 89.0 146 89.0 1/10 Holt. l9 17 89.5 16 84.2 Control 22 21 95.5 20 90.9 Heart 27 25 92.6 25 92.6 Ovary 40 37 92.5 39 97.5 Jelly 31 28 90.3 28 90.3 TABLE VI Experiment 6 Sera diluted 2:1 with 1/10 f.s. Holt.-30 minutes Solution Total Cleaved Percent Gastrula Percent A. J. Cont. 164 146 89.0 146 89.0 1/10 Holt. 19 17 89.5 16 84.2 Control 27 24 88.9 25 92.6 Heart 18 18 100.0 18 100.0 Ovary 24 18 75.0 19 79.2 Jelly 34 29 85.3 29 85.3 B. J. Cont. 144 112 77.8 113 78.5 Aerat. H20 53 46 86.8 45 84.9 1/10 Holt. 49 42 85.7 42 85.7 Control 62 46 74.2 46 74.2 Heart 69 42 60.9 24 34.8 Ovary 66 34 51.5 23 34.8 Jelly 38 21 55.3 12 31.6 TABLE VII Experiment 7 Sera diluted 1:1 with 1/10 f.s. Holt.-45 minutes Solution Total Cleaved Percent Gastrula Percent A. J. Cont. 164 146 89.0 146 89.0 1/10 Holt. 27 21 77.8 21 77.8 Control 47 44 93.6 43 91.4 Heart 31 28 90.3 28 90.3 OVary 36 29 80.6 29 80.6 Jelly 38 36 94.7 37 97.4 27 TABLE VIII Experiment 8 Sera Diluted 1:1 with 1/10 f.s. Holt.-30 minutes HA. Immediate treatment Solution Total Cleaved Percent Gastrula Percent J. Cont. 101 94 93.1 94 94.1 1/10 Holt. 52 50 96.2 50 96.2 Control 79 72 91.1 73 92.4 Heart 86 80 93.0 80 93.0 Ovary 68 66 97.1 66 97.1 Jelly 74 69 93.2 64 86.5 Delayed treatment after 4 hours 6 Hrs. 4 Hrs. after Fert. after Fert. (1 Hr. Solution Tot. (Before Treat.) % after Treat.) % Gast. % 1/10 Holt. 57 54 94.7 54 94.7 54 94.7 Control 65 64 98.5 64 94.7 64 98.5 Heart 73 70 95.9 70 95.9 70 95.9 Ovary 7O 64 91.4 63 90.0 63 90.0 Jelly 73 68 93.2 68 93.2 68 93.2 HB. Immediate Tfeatment Solution Total Cleaved Percent Gastrula Percent J. Cont. 89 87 97.8 87 97.8— 1/10 Holt. 45 45 100.0 45 100.0 Control 39 28 71.8 32 83.1 Heart 44 25 56.8 29 65.9 Ovary 64 35 54.7 33 51.6 Jelly 51 46 90.2 47 92.2 Delayed treatment after 4 hours 6 Hrs. 4 Hrs. after Fert. after Fert. (1 Hrs. Solution Tot. (Before Treat.) % after Treat.) % Gast. % 1/10 Holt. 48 37 77.1 37 77.1 36 75.0 Control 46 40 87.0 40 87.0 15 32.6 Heart 47 38 80.9 38 80.9 4 8.51 Ovary 36 30 83.3 30 83.3 5 13.9 Jelly 47 37 78.7 37 78.7 22 46.8 **Eggs used were from three different frogs. Experiment 9 28 TABLE IX Sera diluted 1:1 with 1/10 f.s. Holt.-3O minutes Solution Total Cleaved Percent Gastrula Percent *A. Immediate treatment J. Cont. 92 62 67.4 62 67.4 1/10 Holt. 40 24 60.0 27 67.5 Control 46 26 56.5 26 67.5 Heart 55 33 60.0 34 61.8 Ovary 47 29 61.7 28 59.6 Jelly 56 38 67.9 43 76.8 Delayed treatment after 2 hours 1/10 Holt. 56 43 76.8 43 76.8 Control 54 37 68.5 40 74.1 Heart 56 40 71.4 40 71.4 Ovary 52 26 50.0 11 21.2 Jelly 60 42 70.0 37 88.1 **B. Immediate treatment J. Cont. 56 51 91.1 52 92.9 1/10 Holt. 36 27 75.0 25 69.4 Control 41 33 80.5 33 80.5 Heart 34 17 50.0 20 58.8 Ovary 45 32 71.1 30 66.7 Jelly 58 45 77.6 45 77.6 Delayed treatment after 2 hours 1/10 Holt. 38 17 44.7 7 18.4 Control 48 24 50.0 32 66.7 Heart 44 34 77.3 27 61.4 Ovary 44 32 72.7 29 65.9 Jelly 37 23 62.2 21 57.8 *Eggs used were from three different frogs. **Eggs used were from two different frogs. 29 TABLE IX, Experiment 9, Continued Solution Total Cleaved Percent Gastrula Percent *C. Immediate treatment J. Cont. 112 100 89.3 100 89.3 1/10 Holt. 61 57 93.4 56 91.8 Control 70 65 92.9 65 92.9 Heart 52 39 75.0 39 75.0 Ovary 93 70 75.3 72 77.4 Jelly 57 35 61.4 32 56.1 Delayed treatment after 2 hours 1/10 Holt. 48 41 85.4 40 83.3 Control 46 43 93.5 43 93.5 Heart 61 48 78.7 41 67.2 Ovary 77 53 68.8 32 41.6 Jelly 47 28 59.6 22 46.8 *ngs used were from three different frogs. **Eggs used were from two different frogs. 30 TABLE X Experiment 10 Sera diluted 1:2 with 1/10 f.s. Holt.-Inj. 1 hour after fert. Solution Total Cleaved Percent Gast. Percent Tailbud Percent . No Inj. 56 38 67.9 25 54.6 19 33.9 1/10 Holt. 41 18 43.9 18 43.9 12~ 29.3 Control 24 13 54.16 7 29.2 0 0 Heart 27 14 51.9 0 0 0 0 Ovary 38 27 71.0 23 60.5 18 47.4 Jelly 44 31 70.5 19 43.2 9 20.5 . No Inj. 49 48 98.0 41 83.7 38 77.6 1/10 Holt. 32 29 90.6 29 90.6 28 87.5 Control 30 15 50.0 8 26.7 5 16.7 Heart 26 11 42.3 1 3.8 3 11.5 Ovary 25 7 28.0 3 12.0 2 8.0 Jelly 26 10 38.5 4 15.4 6 23.1 TABLE XI Experiment 11 Sera diluted 1:8 with l/10 f.s. Holt.-Inj. 1 hour after fert. Solution Total Cleaved Percent Gast. Percent Tailbud Percent . No Inj. 53 47 88.7 47 88.7 45 84.9 1/10 Holt. 45 45 100.0 39 86.7 26 57.8 Control 38 34 89.5 23 60.5 9 23.7 Heart 32 30 93.8 28 87.5 23 71.9 Ovary 45 39 86.7 34 75.6 20 44.4 Jelly 48 45 93.8 40 83 3 29 60.4 . No Inj. 47 43 91.5 42 89.4 42 89.4 1/10 Holt. 61 56 91.8 17 27 9 3 4.9 Control 46 35 76.1 13 28.3 5 10.9 Heart 34 29 95.3 14 41.2 2 5.9 Ovary 35 32 91.4 1 2 9 l 2.9 Jelly 45 41 91.1 22 48.9 11 24.4 31 TABLE XII Experiment 12 Sera diluted 1:4 with 1/10 f.s. Holt.-Inj. 1 hour after fert. Solution Total Cleaved Percent Gast. Percent Tailbud Percent . No Inj. 44 36 81.8 34 77.3 22 50.0 1/10 Holt. 36 19 52.8 14 38.9 3 8.3 Control 27 13 48.1 11 40.7 4 14.8 Heart 53 36 69.2 9 17.3 2 3.9 Ovary 23 12 52.2 3 13.0 0 0 Jelly 46 31 67.4 19 41.3 5 10.9 . No Inj. 68 63 92.6 57 85.3 48 70.6 1/10 Holt. 40 34 85.0 8 20.0 2 5.0 Control 42 31 73.8 6 14.3 1 2.4 Heart 44 40 90.9 13 29.5 8 18.2 Ovary 38 33 86.8 14 36.8 0 0 Jelly 38 36 94.7 11 28.9 1 2.6 . No Inj. 52 39 75.0 36 69.2 26 50.0 1/10 Holt. 39 29 74.4 4 10.3 0 0 Control 33 23 69.7 5 15.2 1 3.0 Heart 55 46 83.6 6 10.9 0 0 Ovary 40 33 82.5 0 0 0 0 Jelly 30 27 90.0 8 26.7 1 33.3 . No Inj. 64 38 59.4 25 39.1 5 7.8 1/10 Holt. 46 37 80.4 18 39.1 2 4.34 Control 37 26 70.3 11 29.7 3 8.1 Heart 49 40 81.6 13 26.5 0 0 Ovary 34 29 85.3 6 17.6 0 0 Jelly 33 15 45.5 7 21.2 0 0 . No Inj. 45 44 97.8 43 95.6 41 91.1 1/10 Holt. 31 25 80.6 21 67.7 16 51.6 Control 38 33 86.8 27 71.1 14 36.8 Heart 45 45 100.0 31 68.9 16 35.6 Jelly 34 31 91.2 21 61.8 12 35.3 32 TABLE XIII Experiment 13 Sera diluted 1:8 with 1/10 f.s. Holt.1nj. 45 min. after fert. Solution Total Cleaved Percent Gast. Percent Tailbud Percent . No Inj. 35 26 74.3 8 22.9 7 20.0 1/10 Holt. 44 27 61.4 1 2.3 0 0 Control 22 17 77.3 1 4.5 0 0 Heart 32 22 68.8 4 12.5 1 3.1 Ovary 38 29 76.3 0 0 O 0 Jelly 18 10 55.6 0 0 0 0 TABLE XIV Experiment 14 Sera diluted 1:4 with 1/10 f.s. Holt.-Inj. 45 min. after fert. Solution Total Cleaved Percent Gast. Percent Tailbud Percent . No Inj. 3O 26 86.7 16 53.3 4 13.3 1/10 Holt. 25 16 64.0 7 28.0 0 0 Control 30 25 83.3 7 23.3 2 6.7 Heart 29 18 62.1 5 17.2 2 6.9 Ovary 26 20 76.9 3 11.5 0 0 Jelly 19 14 73.7 8 42.1 0 0 . No Inj. 59 49 83.1 46 78.0 42 71.2 1/10 Holt. 33 28 84.8 12 36.4 1 3.0 Control 35 27 77.1 10 28.6 0 0 Heart 29 23 79.3 10 34.5 S 17.2 Ovary 35 30 85.7 1 2.9 0 0 Jelly 46 37 80.4 18 39.1 S 10.9 . No Inj. 55 50 90.9 54 98.2 49 89.1 1/10 Holt. 38 33 86.8 20 52.6 14 36.8 Control 35 32 91.4 26 74.3 24 68.6 Heart 38 36 94.7 29 76.3 25 65.8 Ovary 30 23 76.7 5 16.7 4 13.3 Jelly 29 26 89.7 17 58.6 14 48.3 IV. DISCUSSION A. External Treatments The inconsistent and varied effects of the different ,.— antisera and even of the controls are indeed difficult to interpret as a significant blockage by specific antisera. Instead they must be explained in terms of differences in sensitivity of eggs from different female frogs and differ- ences among antisera. The eggs do vary in their reactions to any kind of manipulations at different times of the year. The health of the frog, the size of the eggs, and the amount and condition of the jelly can all have significant influ- ences on the response of the eggs to treatments. The anti- sera produced by one rabbit can also vary in titer of anti- bodies in relation to the general condition of the animal. Considering the number of animals used and the number of bleedings performed, it is reasonable to suppose that the titer of antibodies would differ, even though they were pooled. I In addition to these inherent variations in the materials used, the process of dejellying the eggs could have had a general deleterious effect on the outcome of the experiments. Eggs with their jelly coats removed are ob- viously more sensitive to any manipulations. Although 33 34 great care was used in the dejellying process, poor develop- ment of the dejellied eggs, treated with both control serum and Holtfreter's solution, in a number of trials, is proof that some protection is afforded by the jelly in manipula- tions of this sort. Eggs without jelly coats are also more sensitive to slight variations in osmolarity. Thus, it can be seen that any cytotoxicity of antisera in general is more likely to have an effect on dejellied eggs. The results obtained in five trials with anti-ovary sera were very interesting and are similar to some other preliminary work done in this laboratory. Barch (personal communication) has injected antisera (diluted 1:1 with one- tenth full strength Holtfreter's solution) against Rana pipiens egg jelly, ovary, heart, and control serum into the body cavity of female Rana pipiens. This was done every other day for four weeks, if possible (most frogs died earlier). The effects on the eggs produced by these females were noted. The frogs injected with anti-ovary serum ovu- lated fewer eggs when artificially stimulated with pituitary glands and when they did ovulate, the percentages of eggs which cleaved were lower. Although this work is incomplete, the general trend seemed to be consistent with the effects of anti-ovary sera on fertilized dejellied eggs. The fact that the inhibition of cleavage in the latter could not be repeated is probably due to differences in sensitivities of the eggs as discussed previously. 35 The reasons that antisera against frog ovary can inhibit cleavage in this way can only be tentatively inter- preted on the basis of the antigenic components of the egg surface and the ovarian tissues. Agar diffusion studies (Shaver, Barch, and Shivers, 1962) have shown that no tissue of Rana pipiens except the oviduct, where the jelly is se- creted, has been found to have components identical to those “ in egg jelly. This is not true of ovary tissue, however, where many antigens common to other frog tissues, including sperm extracts, have been identified. It has also been shown by Barch (personal communication) that there is an ovary (egg)-specific, rather than a species-specific, anti- gen apparently active in fertilization. This supports the presence of an "E" antigen on the surface of the egg as hypothesized by Shaver (1966). Since ovary is mostly egg material, antisera against frog ovary could be expected to contain antibodies which are complementary to antigens in or on the surface of the frog egg. A mucosubstance in Rana pipiens, presumably having a significant role in activation and cleavage, was identi- fied and named "Antigen F" by Lavin (1963). Fluorescine- tagged antisera localized this antigen on the surface (cortex) of developing oocytes and fertilized eggs. Anti-F antisera inhibited both activation and cleavage of fertilized eggs. It is doubtful that antibodies could effectively pass through the vitelline or fertilization membrane 36 (Flickinger and Nace, 1952; Tyler, 1963). Kemp (1956, 1967) has demonstrated with electron microscopy that, in growing oocytes of Rana pipiens, the folded cortex extends as micro— villi into the substance of the developing vitelline membrane intertwining with the microvilli of follicular cells. How- ever, in the mature oocyte, microvilli are withdrawn and the surface becomes smooth. The cortex is separated from the vitelline membrane by a narrow perivitelline space con- taining small vesicles and flocculent material. After fertilization, the perivitelline space increases as the cortical granules erupt and discharge their contents into this space. The short pseudopodial protrusions which had existed in the space soon retract. Thus, the vitelline mem- brane represents a considerable barrier to any antibody which might react with a cortical antigen. It is possible, however, that these antigenic substances, if present on the tips of the microvilli, could have been deposited on the vitelline membrane before retraction. This would allow in- teraction of antibodies complementary to these so-called surface antigens. Assuming that there are antigens that could be reached by antibodies in the anti-ovary sera, there is still a question as to the nature of the antigen-antibody reaction and how it could inhibit cleavage. It seems reasonable that the interaction of antibodies with surface antigens of the fertilized egg could alter its permeability or other 37 properties so as to upset the metabolic and structural or- ganization for cleavage. The inhibition here appeared to be a primary effect and not secondarily due to cytolysis, since cytolysis in uncleaved eggs occurred only after a period of time. Perhaps the antigens and antibodies form a cross-linked lattice with deeper layers or with neighbor- ing unrelated sites since the antibodies used were multi- valent. However, no precipitation layer appeared on the egg surfaces and no clumping of eggs occurred. Tyler and Brookbank (1956) observed that treatment with antiserum increases the tension at the surface of sea urchin eggs. If this is also true of Rana pipiens, the in- crease in tension,perhaps caused by a lattice of antigens and antibodies, could have an external effect on the forma- tion of the cleavage furrows or could lead to later develOp- mental abnormalities. B. Injection of Eggs The damage to the eggs due to the injection of anti- sera can be attributed to the time of year and the time after fertilization at which they were performed. The eggs in April and May are much more sensitive to any kind of treatments as the poor controls show. In many cases, the general injury caused by a puncture was apparently too much of a shock to the eggs. Perhaps the time at which the eggs were injected in relation to insemination was too critical. Various developmental 38 events could have been irreversibly disrupted such as: spindle formation and chromosome movements, furrowing and pinching off of polar bodies, or general protoplasmic streaming. Any drastic derangements could cause immediate cytolysis or later death during the critical periods of gastrulation and hatching. Additional work in these areas with some modifica- fi tions of techniques might lead to more conclusive results I than were obtained in this study. External treatment of the egg as soon as possible after fertilization, if the time of jelly removal can be shortened, might be more effective. {: Injections at a later time after fertilization might also lead to better development. If inhibition similar to that produced here by anti-ovary sera can be repeated, observa- tion of sections of the eggs would give a more complete picture of the nature and extent of the blockage. In evalu- ating the role of egg and jelly-coat antigens in early de- velopment, it must be remembered that when an antibody combines with a.:macromolecule, it does not necessarily follow that it combines with the region of the molecule re- sponsible for the particular developmental activity in question. V. SUMMARY There is considerable literature concerned with the role of antigens in development as investigated by immunolo- gical methods. On the basis of the previous work done on sea urchin and frog eggs, it was of interest to the author to observe, what effect, if any, antisera against egg jelly and various tissues of the frog had on the cleavage and sub- sequent development of frog eggs. Fertilized, dejellied Rana pipiens eggs were treated externally at different times after fertilization, with dif- ferent concentrations of antisera against various Rana pipiens tissues. The same antisera were also injected into jellied Rana pipiens eggs at 45 minutes and 1 hour after insemina- tion. Cleavage, gastrulation, and tailbud (only of the injected eggs) counts were made and percentages of success- ful development in the various antisera and controls were noted. The differences were too varied to be considered significant, except for five trials in which external treat- ment of eggs with anti-ovary sera totally inhibited cleavage in all the eggs involved. 39 40 The possible reasons for the inconsistency of re- sults were discussed. Due to the inability of the author to reproduce the effect with anti-ovary sera, definite conclusions cannot be drawn. 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H. 1960 Experimental studies on the role of the jelly coat material in fertilization in the frog. Acta. Embryol. et Morphol. Exper., 3: 180-189. . 1966 Time relationships of effects of anti-serum treatment of frog eggs. Exptl. Cell Res., 13: 245- 247. Shaver, J. R., Barch, S. H., and Shivers, C. A. 1962 Time- specificity of frog egg-jelly antigens. J. Exptl. 2001., 151: 95-103. Shivers, C. A. (cited in Shaver, 1966). . 1961 Immunobiological studies of the species speci- ficity of egg jellies of the frog. PhD. Thesis, Michigan State University. 1962 Localization of the inhibitory effect of anti- jelly serum on fertilization in frog eggs by fluorescine- tagged antibodies. Am. 2001., 3: 448. 1965 The relationship of antigenic components in egg-jellies of various amphibian species. Biol. Bull., 128: 328-336. Shivers, C. A. and Metz, C. B. 1962 Inhibition of fertiliza- tion in frog eggs by univalent fragments of rabbit antibody. Proc. Soc. Exptl. Biol. Med., 110: 385-387. 44 Shumway, W. 1940 Stages in the normal development of Rana pipiens. Anat. Rec., 13: 139. Tchou-Su and Wang Yu-lan. 1965 E' tudes experimentales sur le role du mucus des oviducts dans la fecondation chez le crapaud, et la consederation generale sur la penetration spermatique. (In Chinese with French summary). Acta Exper. Biologica Sinics, 3: 75-122. Tyler, A. 1940 Agglutination of sea urchin eggs by means of a substance extracted from the eggs. Proc. Natl. Acad. Sci. U.S., 33: 249-256. . 1947 An auto-antibody concept of cell structure. Growth and Differentiation, (Suppl.), 32: 7-19. 1957 Immunological studies of early development, In: The Beginnin s of Embryonic Development, (A. Tyler,TR. C. von orstel, and C. B. Metz, Eds.), pp. 341-382., Amer. Assoc. Adv. Sci. Wash. D.C. 1959 Some immunological experiments on fertiliza- tion and early development in sea urchins. Exptl. Cell Res. (Suppl.), 1: 183-199. 1963 The manipulations of macromolecules sub- stances during fertilization and early development of animal eggs. Am. 2001., 3: 109-126. . 1965 The biology and chemistry of fertilization. Amer. Naturalist, XCIX: 309-334. Tyler, A. and Brookbank, J. 1956 Antisera that block cell division in developing eggs of sea urchins. Proc. Natl. Acad. Sci. U.S., 13: 304-308. 1956 Inhibition of division and deve10pment of sea urchin eggs by antisera against fertilizin. Proc. Natl. Acad. Sci. U.S., 43: 308-313. Tyler, A. and O'Melveny, K. 1941 The role of antifertilizin in the fertilization of sea urchin eggs. Biol. Bull. 115: 337. Tyler, A., Seaton, A., and Signoret, J. 1961 Further analy- sis of antidevelopmental action of antisera against fertilizins and against other egg extracts of sea urchins. Am. 2001., l: 394. MICHI AN STATE UNIVERSITY LIBRARIES I III IIIIII II III I 2 0 3 1 3056 6859 93 -_._--'_.