.\ T. 1......w?.-I.....4 .n. . v“... o.l...o ,Ol. c..d..45.¢..outs l oooofioo‘ 00.0.10. — V-‘ 3.7.1; .Cf. fofo'ol.17.ol|.‘ ‘ y 1 onguvflh Q o “- m. 0 . Q. C II D 'l h. ‘. (I O O ' I O ’ . . v .lvuoOOa. I '1 . anur.%ufll. ¢.30..hI . 2...: ...Z...s...:. ~.'o~.~p~ :0... 'I..-"J.~.~O.-Acx‘l.l .‘OM r”. “ohnnu “4:. . flap. _4' 3 :0. 7,1.)(1... .v. _ 0.9... r _ MiChzvfll time University Thaw»; ABSTRACT SPERM - EGG JELLY INTERACTIONS IN RANA PIPIENS By Chester Ronald Roberts Immunobiological techniques were used to examine the role of egg jelly in fertilization of Rana pipiens. Eggs of R. pipiens were treated prior to insemination with various antisera which block the normal function of the egg jelly and thus inhibit fertilization. Treated ("capac- itated") sperm were then used for fertilization in an effort to by—pass the antisera inhibition. Treatment (capacitation) consisted of mixing sperm with egg water or various oviducal extracts for a period of time prior to insemination. A biochemical analysis of the basic com- ponents of the egg jelly was done to investigate possible chemical variations in different jelly layers. In addition a cytological study was done on the jelly producing organ, the oviduct, using light and transmission electron microscopy in order to investigate any structural differences at various oviducal levels which could be correlated to anti- genic and histochemical differences that have been found by other investigators. By capacitation of the sperm prior to insemination, the inhibition of fertilization due to the various treat— Chester R. Roberts ments with antisera of the eggs was by-passed. No major variations in the ability of the different "capacitating" solutions to affect a higher percentage of fertilization were noted. The factors present in the egg water which could be responsible for capacitation of sperm seems to be stable over a period of time and can be removed by centrifugation in the presence of excess sperm. A cyto- logical study revealed some ultrastructural aspects of the female oviduct during egg jelly production but no evidence was found that could associate fine structural differences with histochemical and antigenic differences in the oviduct. SPERM — EGG JELLY INTERACTIONS IN RANA PIPIENS BY Chester Ronald Roberts A THESIS submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Zoology 1971 To Lisa ACKNOWLEDGMENTS I wish to express my deep appreciation for the help of Dr. J.R. Shaver and Dr. S.H. Barch who made this thesis possible. I would also like to thank Dr. G. Spink and Dr. H. Ozaki for their helpful suggestions as members of my guidance committee. iii TABLE OF CONTENTS DEICATION .00.0.0...0.0.0.0....OOOOOOOOOOOOOOOOOCO ACKNOWLEDGEWIITS OOOOOOOOO0.000000000000IOOOOOOO... TABLE OF COIITENTS 00......0.0.0.0...OOOOOOOOOOOOOOO LIST OF TABLES OOOOOOOOOOOOOOOOOOOOOOOCOOOOOOOOOOCO LIST OF FIGIJRES 0.00.00.00.00.000IOOOOOOOOOOOOOOOO. INTRODUCTION 0.0.0.0...OOOOOOOOOOOOOOOOOOO000...... quTERIAI‘s AND RETHODS OOOOOOOOOOOOOOODOOOOOOOOOO... capaCitation Of sperm 0 O O O O O O O O O O O O O O O O O O O O C O 0 ChemiCfl Analysis 0 O O O O O O O O O O O O O O 0.. O O O O O C O O 0.. O MicrOSCOPy OOOOOCOOOOO0.0.00.00.00.00 0.... 0... RESULTS .OOOOOOOOOOOOOO00.000.00.000.00000000000000 Capacitation of Sperm ........................ Chemical Composition ......................... Microscopy 0.0.0.0....OOOOOOOOOOOOOOOOOOOOOOOO DISCUSSIOIJ OOOOOCOOOOO...0.00.00.00.00...0.0.000... SMIARY COOOOOOOOOOOOOOOO0.0.0.0...OOOOOOOOOOOOOOO. BIBLIOGRAPHY .0.0IOOOOOOOOOOOOOOIOOOO0.000.000.0000 APPENDIX iv Page ii iii iv vi 18 18 25 3O 47 53 55 LIST OF TABLES Table l a. Fertilization values from capacitation . experiment # l ............................. appendix 1 b. Analysis of variance of capacitation . experiment # 1 ............................. appendix 2 a. Fertilization values from capacitation emeriment#2 OOOOOOOOOOOOOOO0.0.0.0.000... appendix 2 b. Analysis of variance of capacitation experiment # 2 ............................. appendix 3 a. Fertilization values from capacitation experiment # 3 oeoeeeoeoooooeeooooeeeeeoocoo appendix 3 b. Analysis of variance of capacitation emeriment#3 .000OOOOOOOIOOOOOOOOOOOOOOOOO appendix 4 a. Fertilization values from capacitation experiment # 4 ............................. appendix 4 b. Analysis of variance from capacitation experiment # 4 ............................. appendix Figure l. 2. 3. 4. 5. 6. 7a. 7b. 7c. 7d. 7e. 8. 9. 10. 11. 12. 13. 14. LIST OF FIGURES Oviducal segments used to obtain extracts for capacitation experiment # 3 ...................... Fertilization values from capacitation emeriment # 1 0.00.00.00.00...00.0000...00000000. Fertilization values from capacitation emeriment # 20.00.000.000...OOOOOOOOOOOOOOOOOOOOO Fertilization values from capacitation experiment # 3 oeooeeeoeeeooeeeeeeoeoeeeoeoocoeeeo Fertilization values from capacitation eXPerimen-t # 4' 00.000.000.000...000.000.000.000... Protein concentration of solutions used to capaCj-tate Sperm OOOOOOOOOOOOOO.IOOOOOOOOOOOOOOOOO Nelson's test .................................... Fiske-Subarow inorganic phosphate determination .. Mejbaum pentose determination .................... Deoxypentose determination ....................... Lowry protein determination ...................... Amino acid content of various oviducal materials from several species of Rana and one species of MO 0.00.00.00.00.00000COOOOOO0.0.0.0000...0.0... Section of an oviduct of an adult female Rana pipiens showing the three cell types ............. Outermost edge of the oviduct .................... Jelly-producing cells packed with secretory prOduCtS OOOOOOOOOOOOOOOOOO0.000.00.000.00...00.o. Mitochondria of a jellyhproducing cell ........... Blood vessel surrounded by jelly producing cells . Secretory duct which connects the jellyhproducing cells With the lumen OOOOOOOOOOOOO.90.000.00.00... vi Page 13 19 21 23 24 26 27 27 27 27 27 29 33 34 35 56 37 38 Figure 15. l6. 17. 18. 19. 20. 21. 22. Boundary between the jelly producing cells and mucus secreting cells ........................... NUcleus of mucus secreting cell ................. Mucus secretory cells bordering the lumen ....... Secretory product or mucus cells being exuded into the lulnen 0f the OViduCt OOOOOOOOOOOOOOOOOO. Section showing all three cell types of the oviduct. Products of both secretory cell types are being exuded into the lumen ................. Ciliated epithelial cell ........................ Ciliated epithelial cell and secretion of mucus cell material oooeoeeeeeeeeeeoeoeeeoeeeeeeeooeeoe Section showing all three cell types of the oviduct bordering the lumen...................... vii Page 39 4o 41 42 43 44 45 46 INTRODUCTION Many animal eggs have some sort of surounding capsule. These capsules have a broad range of morphology from a mucus envelope to distinct layers of cells. In the case of the amphibian, the egg is surrounded by homogeneous thick, jelly-like capsules. Some of the earliest work done on the jelly envelOpes of frogs appeared in 1883 (Giacosa,1883). The jelly was noted to swell enormously when contact was made with water after the eggs left the ovisac, and was assumed to have a protective function to save individual embroys from any sudden, harmful environ- mental change (eg. mechanical, chemical, or thermal shock). Later observations by E. Bataillon pointed toward another function of amphibian.egg jelly besides that of protection. He noted that eggs taken from the body cavity (coelomic eggs) could not be fertilized. Such eggs do not have a jelly envelOpe since during ovulation, the eggs rupture the wall of the follicle and pass into the body cavity before being forced into the ostium of the oviduct by cilia (Noble, 1931 ; Rugh, 1935). As the egg progresses down the oviduct, it becomes surrounded by a mucoid or gelatinous material (the egg jelly) which is secreted by specialized cells of the oviduct. Additional evidence that the jelly enveloPes of amphibians have a significant role in fertilization was 2 furnished by the observation that the eggs that have passed through the oviduct and then mechanically or chemically dejellied still could not be fertilized in any appreciable amount (Shaver, 1960; Kambara, 1953; Tchou-Su, 1956; Hugh, 1935). One of the more important tools used by investigators to study the role of the egg jelly envelopes are immuno- biological techniques. It has been demonstrated that the fertilizability of fighpgpigng eggs, pretreated with anti- sera of rabbits immunized against the jelly coat of the eggs, was decreased (Shaver and Barch, 1960). Whether this inhibitidn is due to mechanical blockage of sperm gig a precipitation lattice network, or to the blockage of specific sperm receptor sites in the jelly is not yet clear. Vork with "univalent" antibodies (papain digested and thus non-precipitating) is inconclusive (Shivers and Metz, 1962; Arakelian, 1971). It is evident, however, that antibodies against the jelly-coat engage complementary sites in the jelly and prevent in some way the initial sperm-jelly interactions prerequisite for fertilization in amphibians. To further investigate the role of antigenic components of the egg jelly, attention has been given to the various jelly layers found around amphibian eggs. Three to six layers have been noted in R. pipiens (Shaver, 1966; Race et al.,l96l; Shivers and James, 1970 A.). four in the toad §g£9_bufo asiaticus (Kambara, 1953), and four in fiyla arboreggjaponica (Katagiri, 1963). The importance of different jelly layers has been demonstrated by noting 3 the fertilizability of eggs removed from various levels of the oviduct. Kambara used this technique with Bufo bufo asiaticu§_in which four distinct layers are visible in spawned eggs. Results showed that the first two layers deposited around the egg are necessary for fertilization to occur. The innermost alone is not sufficient, indicating that the second layer or an alliance of the two innermost layers (called C and D) is a necessary requirement. In a similiar manner, Glick and Shaver (1963) studied the fer— tilizability of eggs from various levels of the oviduct of anpgpgeps. As in Kambara's experiments, an essential con- stituent(s) is put around the egg from the middle and lower levels of the oviduct. The lack of fertilizability of the upper oviducal eggs reflects the absence of these constitu- ents from the other levels. This of course does not mean that the upper oviducal secretions are non-essential. Using the agar-gel diffusion technique (Ouchterlony, 1949), Barch and Shaver (1963) have found antigenic com- ponents unique to the upper or lower levels of the oviduct. In addition there are antigenic components common to all levels. All of these antigens have also been shown to be present in the egg jelly. Localization of these antigenic constituents in the egg jelly was achieved by using fluorescein—conjugated antibodies (Shivers, 1952)- These antigens consist of two types: non— specific antigenic components which are shared between closely related species of 39g; and species-specific anti— genic components which are present in only one species of 4 figgg. The non-specific antigenic components appear to be confined to the inner layers of the egg jelly whereas the species-specific antigenic components appear to be located in the middle and outer layers. The significance of these egg jelly antigens in fer- tilization was tested by noting the effects of antibodies directed against these antigens (Shivers, 1961). It seems that both the species-specific and the non-specific anti- gens are necessary for fertilization. In addition, The presence of an antigen produced by the egg itself has been detected (Race and Davin, 1963) and shown to have a signifi- cant role in fertilization. Examination of the function of the egg jelly in fer- tilization has been accomplished by treatment of frog sperm prior to imsemination with secretions of the female repro- ductive tract, as has been done with sperm of other animal groups (Austin, 1951; Austin and Bishop, 1958; Chang, 1951; Kirton and Hafs, 1965). It has been shown that sperm of mammals and of several invertebrate species require a period of contact or residency within the female reproductive tract before being able to fertilize. During such exposure the sperm becomes "capacitated" and thereby becomes able to initiate fertilization. It is thought that capacitation is a process which in some way produces the acrosome reaction which is important in sperm-egg membrane fusion (Dan, 1956; Colwin and Colwin, 1964).Several studies with invertebrate and mammalian spermatozoa indicate that a release of sperm materials, believed to be lytic enzymes (lysins), from the 5 acrosome is accomplished when contact has been made with some activator substance(s) of the female reproductive tract (Monroy, 1965; Stambough and Buckley, 1969). Recent studies (Shivers and James, 1970 B.; Gusseck and Hedrick, 1971; Welf and Hedrick, 1971) indicate that the sperm of R. pipieng and XenOpus leavis can also be "capacitated" by exposure to jelly coat materials. By such exposure the sperm was able to fertilize mechanically and chemically dejellied Xenopus laevis eggs and the body cavity (coelomic) eggs of R.pipien§. Thus the role of the egg jelly has been experimentally demonstrated through pre- treatment (capacitation) of the sperm with jelly coat materials. Electron micrographic studies of R.pipiens spermatozoa (Poirier, 1970) indicate that acrosomal structures do exist and are, in general, similiar to those of invertebrate and mammalian sperm. However, the classical acrosome reaction has yet to be observed in Raga species with electron microscopy. Although the enzymatic nature of the acrosomal reaction has been demonstrated in mammals (Hartree and Allison, 1970; Stambough and Buckley, 1969; Monroy, 1956), the number and nature of acrosomal substances have not been explicitly defined in amphibian spermatozoa. The tissue responsible for the production of frog egg-jelly material has also been investigated as to pos- sible clues to early sperm-egg interactions. The oviducts are not uniform in their distribution of glands and mucus cells (LeBrun, 1891; Lee, 1965). The gross structure of the oviduct can be divided into three parts: a short straight 6 upper portion around the ostium, a long curled, twisted middle portion, and a lower curved protion near the ovisac and cloaca. Histological studies have revealed three cell types which constitute the oviduct (Lee, 1965): jelly gland (tubular) cells, mucus cells, and ciliated epithelial cells which line the lumen. In addition connective tissue anchors the oviduct to the body wall. The structural differences in the various portions of the oviduct seem to be reflected by the distinct layers of jelly materials seen in the jelly envelopes (Kambara, 1953; Metz and Monroy, 1967; Shivers and James, 1970 A.) In R._pipiens six distinct regions have recently been reported by Shivers and James (1970 A.) using differential histochemical staining techniques on the cells of the oviduct. It was also noted that the jelly was depOSit- ed in six layers instead of the three reported by other workers. Based on an average oviducal length of 39 cm., the regions measured 1, 5, 6, 3, 20, and 3 cm. respectively, in a cranial — caudal direction. Histochemically, all regions except the fifth (20 cm.) showed large amounts of acid mucopolysaccharides with sulfated mucopolysaccharides found in regions 1,5,4, and 6. Neutral mucopolysaccharides were found in regions 2 and 5. These different histochemical properties possibly reflect the distribution of oviducal antigens previously noted (Barch and Shaver, 1963). Recent evidence (Pereda, 1971) on the nature and distribution of sialomucins and proteins also showed differences in the oviduct which could be correlated to the jelly layers. 7 In analyzing the chemical composition of amphibian egg jelly, the most common method used other than histochemical techniques has been acid hydrolysis (Flood et al., 1948). Such hydrolysates yeild chiefly monosaccharides and as- sociated proteins by splitting of glycosidic bends. Amphibian egg jelly consists of two types of compounds: carbohydrates and proteins. The major carbohydrate contents of the egg jelly are glucosamine, galactosamine, fucose, and galactose (Folkes et al., 1950; Minganti, 1955; Hiyama, 1945; Schultz and Becher, 1955; Bray and James, 1949; Schultz and Ditthorn, 1900). Forty to fifty percent of the frog egg jelly consists of these reducing sugars. The protein moiety consists about an equal porportion of the egg jelly. A complete spectrum of the twenty amino acids is present (Metz and Monroy, 1967) but the exact stoichiometric relation— ships between the proteins and the carbohydrates as muco- polysaccharides is not clear. Disulfide bonds have been shown to be important in maintaining the structural integrity of the jelly (Gusseck and Hedrick, 1971). However, variance in the chemical composition of the egg jelly from species to species is apparent (Monroy, 1965) indicating again that species—specific chemical differences may be involved in mechanisms of fertilization. This report further investigates the role of the egg jelly in fertilization of R. pipiens. Immunobiological tech- niques were employed to examine initial sperm reactions, sometimes termed "capacitation" of sperm. The normal role of the egg jelly is blocked by the use of inhibitory antisera. 8 Sperm was then treated with various capacitating solutions in order to overcome this block. The capacitating solutions consisted of egg water and dilute extracts from various levels of the oviduct. Various levels of the oviduct were used in preparing capacitating solutions in order to note if their different antigenic composition could have differ- ent effects on enabling the sperm to fertilize antisera- blocked eggs. The work of Shivers and James (1970 A.) was used as a guideline in obtaining extracts from various oviducal levels. A general biochemical analysis of oviducal materials was done in order to correlate antigenic differ- ences with the basic chemical composition. A cytological study was also performed which perhaps reflects structural differences in the oviduct which could be linked to histo- logical and antigenic differences. MATERIALS AND METHODS Capacitation of Sperm In all experiments R. pipiens purchased from commercial dealers in Wisconsin and Vermont were used. Eggs were obtain- ed by pituitary and hormone induced ovulation (wright and Flathers, 1961). Five mg. of progesterone in corn oil was injected into the dorsal lymph sac plus one adult frog pitu- itary injected into the body cavity for each female. Sperm was obtained by dissection of the testis and maceration in 1:10 Holtfreter's solution. A "normal" sperm suspension con- tained one testis per 10 m1. of solution. The eggs of females used in all the experiments and those eggs used to obtain the egg water were pretested by inseminating sma 1 samples with a normal sperm suspension and noting the success of fertilization. This was done to insure that fertilizable eggs were used. Antisera used in all experiments were obtained by previously employed methods (Barch and Shaver, 1963; Shaver et a1. 1970). Approximately 5 mg. of antigen was mixed with 1 m1. of 1:10 Holtfreter's solution and suspended in 1 m1. of complete Freund's adjuvant. This mixture was then inject- ed into the subscapular muscles of rabbits. After one week a similiar dose with incomplete adjuvant was administered. 10 Every 4 - 6 weeks thereafter booster shots of the antigen — incomplete adjuvant mixture was given. The rabbits were bled from a marginal ear vein. Blood for control sera was taken prior to immunization. All sera were tested for anti- body content on Ouchterlony double diffusion plates (Ouch- terlony, 1949; Shaver, 1961) and dialized against 0.65% Na01 prior to use. In the first experiment sperm was treated (capacitated) by exposure to egg water for various time intervals. Egg water was obtained by stripping all the eggs of a female into 75 - 80 cc of 1:10 Holtfreter's solution, allowing them to sit at room temperature in the Holtfreter's solution for 1 to 1% hrs. and drawing off the fluid with a Pasteur pipette. A protein determination, using an Hitachi Model 101 UV—Vis spectrOphotometer, was run on all egg water used. Eggs were exuded onto glass microscope slides and covered with antijelly serum, control serum, or 1:10 Holt- freter's solution for 2 minutes. The eggs were washed by thorough pipetting with 1:10 Holtfreter' solution. Batches of eggs from each of the three treated groups were then in— seminated with the different sperm-egg water suspensions listed below or with normal sperm suspension. In this first experiment the eggs from 16 different frogs were used. Capacitated sperm were obtained by diluting 1 m1. of a concentrated sperm suspension ( 1 testis per 1 m1. of l: 10 Holtfreter's solution) with 9 ml. of egg water. This mixture was used immediately or allowed to react for 5, 10, 11 or 15 minutes before being applied to treated eggs. All sperm suspensions (capacitated and normal) were allowed to react for 10 minutes with eggs, which were then placed in finger bowls filled with aerated water. After several hours the number of cleaving eggs (2 — 8 cell stage) and the total number of eggs were counted and recorded. The percent of cleavage was calculated and transformed into are sin equivalents for a statistical test (analysis of variance; Snedecor, 1956). Significant dif- ferences were calculated by the Q test (see appendix). In the second experiment capacitated sperm were prepared as described previously except the sperm were exposed to egg water for one time Period (10 minutes) only. Eggs on glass microscope slides were treated as before with 10 different types of antisera from immunized rabbits, non-immune control sera, and 1: 10 Holtfreter's solution. The antisera were directed against 3, pipiens egg jelly, upper oviduct material, middle oviduct material, lower oviduct material, spleen, kidney, ovary, sperm, and 3, clamitggg egg jelly. Batches of eggs from each of the above treated groups were then inseminated with the capaci- tated sperm or with the normal sperm suspension. The eggs from 23 different females were used. After several hours the eggs were counted and a statistical analysis of var— iance was done as in experiment # 1. Sperm capacitation in the third experiment was ac- complished by mixing sperm for 10 minutes with materials exuded from various regions of the oviduct of R. pipiens 12 and with egg water. Oviducts fron non-ovulating adult females were dissected and divided into segments cor- responding to the regions described by Shivers and James (1970 A.) or into thirds ( Figure 1). A whole oviduct was also used to obtain a capacitating solution. All oviducal segments were individually homogenized in 1:10 Holtfreter's solution and stirred for several hours. The extracts were filtered through glass wool and stored in the cold. A protein determination was made on each extract. The extracts were kept in a highly diluted form so as to prevent excess gel formation. Eggs on glass microscope slides were treated as previously mentioned with either antisera against the egg jelly or non—immune control sera. Batches of eggs from these two treated groups were then inseminated with the different solutions of capacitated sperm or with normal sperm suspension. The eggs of 19 different frogs were used. After several hours the eggs were counted and an analysis of variance was done as in experiment 51 (see appendix). In the fourth experiment, four different solutions were tested as to their capacitating ability. The four solutions were a) normal egg water, b) egg water stored 24 hrs. in the cold and allowed to reach room temperature prior to use, 0) egg water spun at 11,500 x g in an Inter- national High Speed Refrigated Centrifuge Model HR-l for 15 minutes, d) the supernatant of a dense sperm - egg water suspension. To obtain the supernatant, 4 testis were 13 ma psmaflhomxo sowpmpflommmo mom mpomupxo sflmppo on new: ohm: whamemmm omens .mvmflzp Opsfi can “.4 omoav .umEMh use mpo>wnm on mafivmooom m zmsomnp H mQOHMom ousfi m: use was smpoommflv mposufi>o .H opsmfim 14 macerated in 10 m1. of fresh egg water and allowed to re— act for 10 minutes. This suspension was then spun at 11,500 x g for 15 minutes and the supernatant saved. A protein de- termination was done on all four solutions. Sperm were mixed with these solutions 10 minutes prior to insemination. Eggs on glass microscope slides were treated as mentioned above either with antisera against the egg jelly or with non-immune control sera. Batches of eggs from these two treated groups were then inseminated with the different sperm - capacitating solution mixtures or with normal sperm suspension. The eggs of 15 different frogs were used. After several hours the success of fertilization was noted and an analysis of variance was done as in experiment # 1 (see appendix). Chemical Analysis Biochemical tests were run on 1yophylized extracts from the whole oviduct of R. pipiens. The extracts were obtained by homogenizing whole oviducts from adult females in 1:10 Holtfreter's solution and stirring for several hours. The extracts were then filtered through glass wool and lyOphylized (freeze-dried). In all of the chemical methods listed below, 2 mg. of 1y0phylized extract was hydrolyzed in 1 N E1480,1 for 1% hrs. at 105 C. The chemical composition was determined by the following methods: reducing sugars by the Nelson test (Nelson, 1944), inorganic phosphate by the modified Fiske - Subbarow method (Fiske and Subbarow, 1925), protein by the Lowry method (Lowry et al., 1951), pentose sugar by the Hejbaum method ( Sdhneider,195® , and deoxypentose by the 15 Dische method (Dische, 1955). The colorimetric determinations were done on a Spectronic 20. An amino acid analysis was performed on 1yophylized extracts prepared in the manner described above from the whole oviduct of R. pipiens, upper & oviduct, middle third oviduct, lower third oviduct, the outer jelly layers of R. pipiens eggs, and the whole oviduct extracts from R; clamitans, R. catesbieana, and Bufo fowleri. In the case of each of the above materials, 6 mg. were hydrolyzed in 2 m1. of 6 N H01 for 24 hrs. at 105 C in sealed glass tubes. The HCl was evaporated and replaced with distilled water so that an approximate protein concentration of 1 mg. per ml. was present. A 100 ul sample was analyzed by an amino acid analyzer using Technicon Chromobeads C. The separated amino acids were detected with a Gilford Micro-Sample Spectrophoto- meter set at 540 mu. Use of the amino acid analyzer was kindly provided by Dr. Derek Lamport, ABC Plant Research Lab, M.S.U. hicrogcgpy For electron microscopy whole oviducts from adult female R. pipiens were fixed at room temperature with 6.25 % glutaraldehyde in Sorensen's phosphate buffer (.2M) at pH 7.2 for 50 minutes. They were then divided into six segments corresponding to the regions described by Shivers and James (1970 A.) and washed thoroughly with Sorensen's phosphate buffer (.2 M). The tissues were post-fixed in 1 % osmium tetroxide in Sorensen's phosphate buffer (.2 M) 16 for 45 minutes and dehydrated through an alcohol series of 25%, 50%, 75%, 95% ETOH for 10 minutes each and 100% BTOH twice for 15 minutes each. The tissues were transferred into two changes of propylene oxide of 30 minutes each and then placed into a 1:1 mixture of propylene oxide and Epon 812 mixture (Luft, 1961). This 1:1 mixture was changed four times over a 48 hr period. The tissues were flat embedded in Epon 812 and left in a dessicator for 48 hrs. to allow for complete penetration. The Epon was polymerized in a 60 oven for 48 hrs. Thin sections, with an interference color of silver to gold, approximately 75 to 100 nm thick,were cut with glass knives on a Porter - Blum MT—2 ultramicrotome. The sections were collected on 150 or 200 mesh uncoated copper ethene grids. The sections were stained for 15 minutes by floating them on a concentrated aqueous uranyl— acetate solution and then for 5 minutes on an aqueous lead citrate solution (Reynolds, 1965). The sections were observed on a Philips 100 B trans— mission electron microscope Operating at 60 KV. Pictures were taken with 35 mm fine grain release positive film. For light microscopy, whole oviducts were fixed in 10% neutral formalin for 1 hour. The oviducts were divided into segments corresponding to the six regions described by Shivers and James (1970 A.) and dehydrated through a series of alcohol changes as described above and embedded in parafin. Sections were cut on an A.0. Spencer No 820 Rotary Microtome at a thickness of 5 u meters. The sections were stained in Mayer's hematoxylin and eosin (Luna, 1968) 17 and observed with an A.O. Spencer light microscope. Pictures were taken with Kodak Pan-X 35 mm negative film. RESULTS Capacitation of Sperm The first sperm capacitation experiment indicated that preinsemination treatment of sperm with egg water enhanced the percentage of cleavage of antijelly sera - treated eggs. The fertilization values from this experiment are shown in figure 2. Except for the eggs inseminated with sperm exposed to egg water for 15 minutes (J-l5') the enhancement in cleavage does not reach the levels shown by the controls. The increase, however, is significantly higher than the anti— jelly sera - treated eggs inseminated with normal sperm. The use of control sera and Holtfreter - treated eggs inseminated with the treated sperm and the untreated sperm demonstrated two points. First the antijelly sera treatment was inhibitory to fertilization and second that the sperm exposure to egg water was not deleterious to normal fertilization. The cap- acitated sperm had the same fertilizing ability as normal sperm when inseminating Holtfreter or control sera - treated eggs. For example, the cleavage of Holtfreter — treated eggs was the same whether the sperm was exposed to egg water for 15 minutes (H-15') or untreated (H-U). These values were not significantly different from those of control sera - treated eggs inseminated with normal sperm (0-0) or sperm exposed to egg water for 15 minutes (0-15'). 18 19 J .OIO camp psosmmmflw >H IpszHmstHm mmSHm> u + .DIO nmsu mmnms: mezmonHanm mmsz> u * .SOanemmsn fiaomm Hman: n D .AmHv mopsch mH no .AOHV mepchE OH .Amv mopssHE m .AOV meSCHE O soy seams mmm 0p Esomm we mammomxm sh cmanHmEooom mm: GOHpmuHomamo spasm .Amv QOHpSHom m.hmpmpmuHor so sAOV when Hopscoo oQSEEHusoc .va pmoo hHHmn map quHmmm mammHugm szB pcmspmoau :CHmeHEomcham hmpmm wmwe msmHme,.m wCH>MoHo mo mowmpcmohmm mo mpsmHmbstm :Hm ohm CH mmSHm> GOHpmNHHHpamm " HW psmsflsmmxm cOHpmpHommmO .m mhsuflm LR Dr! "J :11 U "D :1: mH OH OH OH m m m o mH m J. rd .8 \ .¢.mm 8.5m m.sm {moflw gnon * 9 20 When other types of antisera were used as in experi- ment # 2 (figure 3), it can be noted that in all cases, except that of antispleen sera (Sp-U and Sp-lO'), treat- ment of sperm with egg water succeeded in allowing more eggs to be fertilized. As seen before in experiment # l the exposure to the egg water was not successful in increas- ing the fertilization of the eggs to the level seen with the control sera or Holtfreter - treated eggs ( C-U, H—U, C-lO', H—lO'). In all cases treatment of sperm with egg water increased the success of fertilization of eggs treated with inhibitory antisera to approximately the same degree. The enhancement seen also corresponds to that observed in experiment # l, keeping in mind that the sperm were exposed only for 10 minutes. Antisperm sera were the most inhibitory of all sera used. Treatment with egg water significantly increased the cleavage of the antisperm sera - treated eggs (S—lO') but the percent of cleavage was still far below the control treatments. Antijelly, antilower oviduct, and antimiddle oviduct sera inhibited to approximately the same degree. Anticlamitans jelly sera treatment produced similiar values. Antiovary, antikidney, and antiupper oviduct sera were also inhibitory to about the same degree, which was less than the inhibition mentioned above. Antispleen serum was the only antiserum used (except control serum) which showed no significant inhibition. In experiment # 3 different parts of the oviduct were used for making capacitating solutions, as well as egg water. 21 .DIO sosa unopemch thcmonHcmHm mmsz> n + .DIO cusp aosmHz meCQQHchmHm wmsz> n * .QOszHom ampmz mmm cm :sz mopsuHE OH mom cmxHE Susan H OH .QOHmstmzm Esomm Hmaso: u D .Amv sOHpSHom n.9mpmMMpHom :pHs was .AOV when Honpnoo eQSEEHlso: .A.O.mv pmoo mHHow mmmpHEmHo .m .Ammv soeHam .Amv hmGon .AOV hambo .AHV posvH>o soBOH .sz posvH>o stUHB .Ambv pod©H>o seam: .AmV sammm .Abv deo hHwa was pwsHmmm whomHuns zpflz psmspmmsp COHpmsHEcmmam awugm mmmo mcmHmmm,.m mnH>mmHo mo nmmmpcmoamm mo mpqum>szo ch chm CH mmSHm> GOHpMNHHHpHmh “m % pumaflpsmxs sOHpmpHcmamo ahmgm .m mhsmHh OH D OH D OH D OH D OH D OH D OH D D 6.x .o.m mm mm s m .H .H 2 a a: a: m m a A + L KO 0 + 7 .10 o + ..b O + l. . +3) 8 7+ 1 o( .2. . o 2 o 1.... c) +v 7 +¥ O 2 Z/ 0 2 2 EU 0 l 2 l + . a 3 .. 3 f0 1* +3 +n. .br +1. 0 QU 5 2 0 +8. C) D 1 +3. 0 O 0 IL! 2 O 1.... . 5 5.. 7 b. 1.... o r) t. 0 Al Al IIT O. I“! * i. 91 Q, h“ 1. o L... .o . L... t t t 7 5 «d l 7 r), o o o l l O 6 6 f0 on 0w Om 22 The results of this experiment are shown in figure 4. As seen in the previous experiments, antijelly sera - treated eggs inseminated with normal sperm (J—U) had a significantly lower fertilization rate. Treatment of sperm with the homo— genates of upper, middle, or lower thirds or with whole oviducts as well as with homogenates of the six histochemical- ly different oviducal regions described by Shivers and James (1970 A.), or with normal egg water raised the percentage of cleaving eggs treated with antijelly sera. No significant differences between the various capacitating solutions as to their ability to supress the inhibition was noted. The exception to this is a very small significant difference between the capacitating solution from oviducal region 1 (J—l) and the capacitating solution from the whole oviducal extract (J-W). Again the enhancement in cleavage with the treated sperm does not reach the levels shown by the controls. In the fourth experiment the capacitation activity of the egg water was tested. The results of this experiment are shown in figure 5. 0f the four solution used, only the supernatant (J-S) was no longer able to increase the for— tilizability of the antijelly sera - treated eggs. The abil- ity of the egg water to effect a higher percentage of fer- tilizability can thus be used up or removed. The mere spinning of the egg water in the centrifuge did not sig- nificantly remove the activity of the egg water (J-Spun). The activity of the egg water also seems to be stable over a 24 hr. period of time. As with the normal egg water, the 23 .Dlw can» ponmfln hfipswoflmfi:MHm ohm mosamp HH< .AOHV hops: MMo was .AEV poscfi>o mach: .Aqv posvfl>o csflnp hosed .sz poscfl>o vpflnp mavvfle .Ambv poncfl>o vhflnp swam: .Awlav m ammonnp H m:0fimon awozvfl>o Scum voUSXm mamflsmpme mHHmw on whamOQXo muSCHE OH hp vmnwfiamsooom mm: soapmpwowmmo ahmmm .on when Hospsoo ocsaafllzos so adv mpmoo maamw on» pmsfiwmm whomfipqm spa: pcospwmhp soapMGHSomcflohm moans mmmo msmfimfim,.m qu>MoHo mo mmMMpcmohom mo mammdw>fisvo can ohm :H mmzam> :OHpmNHHthoh um % unmaflpomxo cowpm»Hommmo Epomm .: muswflm sssqszspspmommaammmmaaoaoass U h o h o n o a U a U h o w 0 h o m U h o b u h .h. o, 1* 2/ oo rm 5 Mo. Aw 8 L . . h. . 2/ 9 m «o, s a A 4. 3 a, m o 8 OO 2) 1 7) I/ L: o/ n/ an a 1 l O l...- a/m o 2/ 0/ 5 r) 5 WU, OO 0/ 0 OO 0/ o o o o o O 5 5 2 l 2 1 2 [o ,b ro ,b ro ,o 4. h. 7. OH ON on 3 om or on 0w 00 24 .puw swap pagans meQMOHMHsMHm moSHm> u * .COHmsmmmsm Shown HmEHom u 2 .Amv cOHmsommsm Human I houmB mmo mpscHs OH m mo pcmusqhmmsm map was .Assmmv m K oom.HH pm mmpsts mH mom damn seams mmm .A.pn :Nv mops: mmo wHo .ms :m .AOHV pops: mmo sH mouSCHS OH sow Ehomm muHNHS an vostHm Iaooom mm: coHpMpHommwo Enema .AOV whom Hoppcoo ossnsHlso: so Hwy “moo mHHom on» vmcflmmw whomfipzm anz pumapmopp COHpmsHEmmcHopm houmm mmwo mamHMHml.mtmcH>mmHo mo newspsmohom mo muanm>H5vo GHw ohm sH moSHm> cOHpmNHHHpAom u: % psoBHsomKo sOHpMpHoMQMo Smmmm .m mHSMHh .9: :m .hn sm zzmm csmm O O O O OH :.mm on 0: swomw sfiomw {memo B O 25 increase in percentage of cleavage (J-24 hr.) is the same. In accordance with the other experiments, treatment of sperm with fresh egg water, 24 hr. old egg water, and spun egg water increased the success of fertilization significantly above that of the untreated sperm (J—U) but the level seen in the controls was still not reached. The results of the protein determinations of all egg water and oviducal extracts used are given in figure 6. Such determinations were used as an indicator to note if jelly or egg substances had diffused into the surrounding media. Note that the egg water — sperm supernatant of experiment # 4 had a tremendous increase in protein content but showed little capacitating ability. ghemical Composition The results of Nelson's test, which gives an indication of reducing sugars present in the jelly,is shown in figure 7a. Approximately 25% of the jelly is reducing sugars, a value consistent with other investigators’ work. Inorganic phosphate as determined by the Fiske-Subbarow method (figure 7b) is not present. Results from the Mejbaum method of pentose determination as shown in figure 70 disclosed no pentose sugars. Also the deoxypentose determination revealed no amounts of this sugar present in the jelly (figure 7 d). The Lowry method for protein determination, shown in figure 7e, indicated that approximately 47.5% of the egg jelly is protein. Optical Protein Solution Dansity Cbncentration (mg/m1) Egg water for .165 .30 experiment # l. .245 .44 .400 .70 .160 .27 Average .43 .100 .15 Egg water for .140 .24 experiment # 2. .195 .32 average .24 Egg water for .16 .26 experiment # 3 .14 .24 .14 .24 Extracts used in .21 .36 average .38 experiment # 3 from oviducal regions: 1 .15 .26 2 .15 .26 3 .14 .24 4 .12 .18 5 .13 .22 6 .64 1.14 Whole oviduct .20 .34 upper‘ " .28 .50 middle " .37 .66 lower " .31 .55 Egg water for .16 .26 experiment # 4 .14 .24 .14 .24 .21 .36 average .38 Sperm-egg water .67 1.2 supernatant for .93 1.7 experiment # .84 1.5 .50 .9 average 1.32 24 hr. old egg water .08 .12 for experiment # 4 .165 .30 o.n- values did not .115 .18 change over the 24 hrs. .31 .54 average .28 Figure 6. Protein determinations on solutions used to treat or capacitate sperm. A standarchurve of bovine albumen was used to convert absorbancy to mg. of protein per ml. All solutions were read at 280 mu on a.Hitachi Model 101 UV’Vis Spectrophotometer. 26 Figure 7 a. Nelson's test Sample Cbncentration Optical Density % reducing 4540 mu sugar Standard (galactosel .07 mg/ml .61 Standard. " .10 ” .92 Jelly .10 " . 21 25% Figure 7 b. Flake-Subbarow inorganic phosphate determination Sample Concentration Optical Density % Pi 660 mu Standard .8 umoles/ml .95 Standard .4 umoles/ml .489 Jelly .1 mg/ml 4.046 1.1% Figure 7 c. Mejbaum pentose determination Sample Concentration Optical Density % Pentose 660 mu . sugar Standard 1 u mole ATP .492 JellL .1 mg/ml .020 L 1% Figure 7 d. Deoxypentose determination Sample Concentration Optical Density % Deoxypentose 595 mu 81111811“ Standard 1 u mole deoxyadenosine-- .43 5' -phosphate Jelly .1 rug/ml .01 1.1% Figure 7 e. Lowry protein determination Sample Concentration Optical Density % Protein 660 mu Standard. 50 ug/ml .20 Jelly 100 ug/ml .19 47.5% 27 28 The results of the amino acid analysis on various oviducal extracts of R. pipiens, R. clamitans, R. catesbieana, and Bufo fowleri are shown in figure a. The values of valine, cystine, and methionine are consolidated as one since sugar residue contamination appears at the same time these amino acids come off the column. Therefore it must be kept in mind that the values for these three amino acids also in— F' corporate some sugar contamination in the hydrolyzed sample. The lysine content of R. clamitans oviducal extract was lost due to a mechanical failure of the recording instrument. 1 Those amino acids which absorb at 440 mu and proline were E not analyzed. The amino acid content of the different parts of the R. pipiens oviduct has minor variations as can be noted in the lysine, glutamic acid, and threonine values. The middle and lower oviducal extracts consistently showed almost the same percentages throughout the amino acids tested. In addition some amino acids such as tyrosine, isoleucine, and phenylalanine are present in practically the same amount in the oviduct extracts of R. clamitans, R. catesbieana, and Bufo fowleri as well as R. pipiens. It should be noted,none- theless, that variations are present between the species tested. This is seen, for example, in the serine, glutamic acid, and threonine percentages. However the differences apparent in the amino acid content have no set pattern. 29 W C . R a B Amino ' ‘ ' Acid W U M L O C. t F. Serine 8.0. 9.0 7.5 7.5 7.8 9.7 ‘;7.8 12.4 Glycine 8.1 9.0 8.5 9.1 7.8 9.3 8.3 5.2 Asp. Acid 10.0' 10.3 9.6 9.3 8.1 11.0 8.5 8.7 Val., Cys., 19.5 17.4 19.5 20.5 22.3 16.4 15.5 17.4 & Meth. Glut. Acid 9.3 11.5 9.4 9.1 7.7 12.2 8.0 9.3 Threonine 17.5 11.0 17.5 16.8 21.6 13.8 16.6 18.2 Tyrosine 2.5 2.7 2.6 2.6 2.3 2.8 1.9 2.7 Alanine 4.3 5.5 4.5 4.3 3.7 6.5 8.4 4.5 Histidine 1.2 1.8 1.6 1.5 1.1 1.6 0.7 - 1.0 Leucine 4.3 5.1 4.8 4.8 3.5 6.6 3.2 4.9 Phenylalanine 2.0 2.6 2.2 2.1 1.4 3.0 1.5 2.7 Arginine 4.8 2.9 2.4 2.3 3.1 3.2 1.5 2.7 Isoleucine 4.1 4.1 4.3 4.0 4.2 4.0 2.1 3.7 Lysine 4'5 702 602 602 505 ""‘" 609 509 Figure.8. Amino acid content of 1yophylized homogenate extracts from the whole oviduct of R. pipiens (W), upper % oviduct (U), middle third oviduct (M), lower third oviduct (L), the outer jelly layers of R.ppipiens eggs (0), the whole oviduct of R. clamitans (w R.C-), the whole oviduct of R. catesbieana rant), and Bufo fowleri (B.F.). The numbers represent percent of the protein content. 30 Microscopy Although sections were taken at the various oviducal levels described in the materials and methods, few if any ultrastructural differences were noted which could be cor- related to differences in the jelly secretions. However much information was obtained on the morphology of the oviduct. Sections shown in figures 9 through 22 were taken at various r oviducal levels but are representative of the oviduct as a ~ whole. A major portion of the mature female oviduct was noted to have three basic cell types. First ciliated epithelial cells were present along the folded lining of the lumen. Interspersed with these are mucus - secreting cells which also border the lumen of the oviduct (figure 9). With light microscopy these two cell types were difficult to distinguish due to their common location. They both appear distally to the third cell type commonly called the jelly - secreting or jelly - producing cells. At the time of sacrifice of the females, the oviducts were near the end of glandular growth and so were filled with jelly secretions. The jelly - secret— ing cells constitute the bulk of the oviduct and are arranged as tubular glands (figure 9). Starting with the outermost edge of the oviducts, several layers of muscle and connective tissue are seen which anchor the oviducts to the body wall (figure 10). Immediately adjacent are the jelly - producing gland cells which constitute most of the oviduct. They are packed with 31 secretory material which consists of electron - dense secre- tory granules surrounded by a white mucus type capsule (figures 10, 11, 12). Due to the large amount of secretory material produced by the cells, many of the normal cell constituents such as the nucleus, mitochondria, golgi, etc. are pushed to the periphery of the cell or crowded into a very small area (figure 12) so that, depending on the plane of section, these cells appear to be filled only with the secretory material (figure 11). Throughout the evi- duct there is also evident a rich capillary network (figure 13) which sometimes appears very close to the lumen. Per— meating the glandular portion of the oviduct are many small secretory ducts which seem to connect with the lumen (figure 14). The ducts seem to be lined with micro - villi which perhaps aid the movement of the secretory material toward the lumen during ovulation. ioving inward toward the lumen there is a sharp bound- ary between the jelly - producing cells and the mucus - secreting cells (figure 15). The mucus - secreting cells are also packed with a secretory substance. This substance, however, lacks the granular structure of the jelly - produc- ing cells. The nuclei and other cytoplasmic constituents also seem to be crowded into a small area by the secretory product (figures 16, 17, 18). There is usually a single layer of this type of cell present. The secretory product seems to be exuded directly into the lumen (figure 18). The third cell type, the ciliated epithelial cell, does 32 not appear to have any secretory product. It usually possesses a large and folded nucleus with an abundant amount of ribosomes and mitochondria (figures 19, 20, 21). Numerous cilia are present with anchoring elements ex— tending into the cytoplasm. These cilia display the typical 9-+2 structure. Projecting between the cilia of these cells are micro - villi. All three cell types alternately border on the lumen (figure 22). The secretory product of the jelly producing cells seems to change before exiting into the lumen. The electron dense core seems to lose its white mucus envelope (figure 19). This could be due to the time of fixation. The animal was not induced to ovulate and so the cells shown are in a state of jelly production, not jelly secretion. 33 Figure 9. Section of an oviduct of an adult female Rana pipiens. Three cell types are present: (1) jelly-producing, tubular gland cells, (2) mucus-secreting cells, (3) ciliated epithelial cells. Hematoxylin and eosin 150 X Figure 10. Outermost edge of the oviduct showing muscle (Mu) which anchors the oviduct to the body wall. N--nucleus, S :secretory material, A- artifact 11,000 X 35 Figure 11. Jellybproducing cells packed with secretory products. Five cells are shown. 9,000 X 36 c r r Figure 13. Blood vessel (B) surrounded by jelly-producing cells. Artifacts (A) are holes in the Epon and stain residue. N =nucleus, S =secretory granules. 8,000 X 38 Figure 14. Secretory duct (SD) which connect the jelly- producing cells with the lumen. Microvilli (Mv) are present. m 39 Figure 15. Boundary between the jellyaproducing cells and mucus secreting cells. A blood vessel is also apparent (B). S =secretory product of jelly-producing cell, M: secretory product of the mucus secreting cells, C=ecytcp1asm of mucus cell, A =artifacts. 8,000 X 4o Figure 16 . mu 1 cell's c eus Of mu secretory Product cus secreting cell surr ounded by th e . 30,000 X 41 Figure 17. Mucus secretory cells bordering the lumen (L) of the oviduct. Parts of a ciliated epithelial cell (E) are also present. Ne nucleus, 8: secretory product of jellyeproducing cells, Mu: secretory product of mucus cell, C-=cilia, Mv : microvilli, A: artifacts. 9,000 X 42 Figure 18. Secretory product of mucus cells being exuded into the lumen of the oviduct. C: cilia, Mv= microvilli. 19,000 X \v‘ I t . K . ' 5 . p y ’4‘}, ‘..7‘1‘ 1 L . ' b a» ' ‘ ' . ._.. .. g "v . ‘ , r.‘ a, . '1.“ i ’ J .. “X r" ' Figure 19. Section shownig all three cell types of the oviduct. Products of both secretory cell types are being exuded into the lumen. N: nucleus of ciliated epithelial cell, M: mitochondria, C: cilia, My: microvilli, Srmmao mo m0MMpswoumm mo mps0Hm>H500 can cam 2H mosam> cowuwnaaapamm 0H magma Table Ib. Three way analysis of variance on the effect of egg water on sperm used to inseminate eggs of Rana pipiens treated with antisera against the jelly coat, non- immune control sera, or Holtfreter's solution. df Sum of Sguarex. Mean Square F Animals 15 15145.11 876.2 9.9: Treatment 2 25254.91 12617.5 143.2* Time 4 4564.95' 1141.2 15.0* Interaction 8 8018.35 1002.5 11.4: Error 210 18493.70 88.1 Total 259 69455.02 * values significant to the 1% probability level O n .0 fiw 5.797 .0) . {L :n.. 1.5.2)... 3.". m.00 0.0m 0.00 0.0m m.00 0.m0 0.00 0.Hm H.00 H.00 m.a0 0.00 m m.00 0.m0 0.m0 m.00 0.00 0.0m 0:00 m.H0 0.00 0.00 0.00 0:00 0m 0.00 m.0a 0.00 0.00 0.00 0.00 0.00 H.Hm 0.00 0.00 0-00 0.00 mm m.00 asan 0.m0 0.0m 0.m0 ~.00 0.m0 0.0m 0.H0 0.00 ms00 0.00 Hm m.~0 0.0a 0.00 0.00 ms00 0.0m m.00 H.0m 0.00 0.m0 0:00 m.a0 0m 0.H0 0.00 0.00 0.00 0. 00 0.00 0.00 0.0m 0.00 0.~0 0.H0 0.H0 0H 0.00 H.0m 0.00 0.00 m.00 m.0m 0.00 0.0a 0.00 0.00 0.00 0.00 ms 0.00 H.00 m.00 0.00 0.00 0.00 m.m0 0.0m 0.00 0.00 0.00 0.00 0H 0.m0 H.0H 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 m.m0 0H 0.00 0.0m 0.H0 0.mm 0.00 0.0m 0.00 m.0m H.H0 0.00 0.a0 m.00 0H ~.00 H.0m 0.m0 0.0a 0.00 0.00 0.00 0.0m 0.00 0.00 0.00 0.00 0H 0.m0 0.0 H.00 H.0H 0.00 0:0m 0.a0 0.mH 0.00 0.00 0.00 0.00 0H m.00 0.0a m.00 0.m0 0.00 0.00 0.m0 0.0a 0.00 0.m0 m.0a 0.0m NA 0.00 0. m0 0. 00 0. 00 H.00 0.m0 m.00 0.0m 0.00 0.00 0.00 0.00 as 0.00 0. 00 H.00 0. 00 0.00 0.00. 0.00 0.00 0.00 «.00 0.00 .H.00 0H 0.00 0.0m 0.m0 0.00 0.00 m.00 0.00 0.0a 0.00 0.00 H.00 0.H0 0 0.00 0.00 0.00 0:00 0.m0 0.00 m.00 0.0m 0.H0 m.00 0.00 0.H0 0 0.00 0.0m 0.m0 0.00 0.00 0.00 H.00 0.00 0.00 0.00 0.H0 0.00 0 m.00 0.0m 0.00 0.mm 0.00 0.0m 0.00 0.00 0.00 0.00 0.00 m.00 0 0.00 H.0H 0.00 0.0 0.0m 0.0a m.m0 0.0 0.H0 0.00 m.00 0.00 0 H.00 0.00 0.00 0.0m 0.00 m.00 0.00 m.mm 0.00 0.00 0.00 0.00 0 0.0m 0.0a 0. 00 0.00 m. 00 0.0m 0.m0 0.0a 0.00 0.00 0.m0 0.00 0 m.00 0.0m 0. 00 0.0m x .00 0.00 0.00 m.0H 0.00 m.00 H.00 0.00 m m.00 m.00 0.00 moHN 0.00 0.00 0.00 -m.00 0. 00 0.00 atas 0.m0 H cans 01A 0a-: 0.: 00:00 0-00 oats 0.0 0H 0 are 0aum sum 0 finesse mpswapmoha .scflpsaom M0003 mM0 :0 spa: mopsqwe OH 909 00x08 Sammw H OH .coansmmmsm 890mm Hassos u D .Amv soapsaom 0.9000a90H0m was .on mnmm Hoapsoo .Aomv umoo haaon waspfismao.m .on mhm>o .nmmv somamm .AMV hennax .Amv Eammm .Aqv posvw>o noon .AZV posvfl>o educfla .AmmV uosua>o swam: .Ahv pwoo maaon 0:» pwsflmwu 09000920 £003 0:08pmmap soapmnH50mqficam 000mm wwwo mnmflmflm scam msa>wmao mo momspsmohmm mo mpsoas>flswm can one :0 M05Hm> soapmnwaflpuoh a HH manna '2'.- 0.H0 0.0m 0.00 0.00 m.00 0.00 0.00 0.00 0.0a 0.0 0.m0 H.0H 0.00 0.0m H.00 m.00 0.00 0.0m 0.Hm 0.0a 0.00 0.0m 0.00 H.0m 0.00 0.00 0.00 0.0m w.ma 0.00 0.00 H.00 0.00 0-00 0.00 m.00 0.00 0.0 0.0a 0.0a 0.00 m.0a 0.00 0.0m 0.00 0.00 0.00 m.00 0.00 0.00 0.m0 0.0m 0.00 0.0m 0.00 0.0. 0.00 0.00 .H.0H 0.0m H.00 0.0m 0.00 m.00 0.00 0.00 m.00 m.0m 0.ma 0.00 0.00 m.00 0:m0 0.00 0.00 0.00 0.00 0.00 0.00 0.0 0.00 m.00 0.00 .00 0.00 0.00 0.00 0.00 H.Hm 0.00 m.00 m.0m a.00 0.00 m.m0 0.00 0.m0 0.00 0.00 0.0 0.00 0.0m 0.H0 0.0m m.00 m.00 H.H0 0.00 0.0a 0.00 0.00 0.00 H.m0 m.0a H.00 0.00 0.00 0.00 0.ma 0.00 H.0n 0.0a 0.00 m.00 m.H0 m.00 m.00 0.00 0.00 H.0 m.00 0.00 0.00 0.00 H.H0 0.00 0.00 0.00 0.0a 0.00 0.m0 0.m0 0.00 0.00 0.00 0.00 0.00 0.00 0.ma 0.00 0.m0 m.mm 0.00 0.00 0.00 0.00 0.00 0.00 0.00 m.0H 0.00 0.0m 0.m0 0.00 0.00 0.00 0.00 0.00 H.0H 0.0 0.00 0.m0 0.00 m.00 m.00 0.00 0.m0 0.00 0.00 0.0a m.00 H.0a 0.H0 0.0m 0.00 0.00 0.00 m.00 0.0a 0.ma m.00 0.0m 0.0m 0.0a 0.00 0.00 0.00 0.0a H.Hm 0.00 0.00 H.0m 0.H0 m.00 0.m0 0.m0 0.00 0.0m 0.0m 0.0a 0.00 0.m0 0.00 0.0m 0.00 m.H0 0400 0.0a 0.0m 0.0a 0.00 0.00 0.00 0.0a m.00 m.0m m.mm 0.00 0.0a 0.0 0.00 0.00 0.a0 0.00 0.00 0.00 0.00 0.m0 0.0m 0.0 0Huum slum 00-0 010 00:00 0-00 oats 0-x 0HIm 0:0 mmeE#6®HB macsCflpsoov |>< MN MW Hm Om M¢LRKOL\OOO\ r-lr-ir-lr-lr-{r-ir-i m H H HNMJ‘WKOBOOQS n sesame 0 HH mHnma Table II b. Three way analysis of variance on the effect egg water on sperm used to inseminate eggs of Rana pipiens treated with different antisera, non-immune control sera, and Holtfreter's solution. df Sum of Squares Mean Square F Animals 22 9817.01 446.2 6.8: Treatment 1 85060.09 85060.09 1300.6‘ Sera 10 19997.56 1999.8 50.6* Interaction 10 6514.80 651.5 9.7‘ Error 462 30194.96 65.4 Total 505 -151584.42 ‘ values significant to the 1% probability level 0.00 0.m0 0.00 0.m0 0.00 0.00 0.H0 0.0a 0.00 M 0.00 0.00 0.00 0.H0 m.0m 0.00 0.00 0.Hm H.00 0H 0.00 0.00 m.00 0.00 0.0m 0.00 0.00 0.0m 0.00 0H 0.H0 0.00 H.0m 0.00 H.00 H.00 0.00 0.0m 0.00 0H 0.H0. H.00 0.00 0.00 0.00 0.00 0.00 0.00 0. a 00 0.00 0.00 0.m0 0.00 o.-0 0.00 0.00 0.00 0..0 ma 0.00 0.00 0.00 0.00 0.0a 0.0. 0. 0 0.0m 0.H0 0H 0.00 H.00 0.00 0.00 ..a0 0.00 0.00 0.x: 0.00 0H 0.00 0.00 0.00 0.00 0.0m 0.00 0.H0 0.00 0.00 ms 0.00 0.00 0.H0 0.00 0.00 0.00 0.00 0.00 0.00 HH 0.00 0.00 0.00 0.00 0.00 m.00 0.00 0.0m 0.00 00 0.00 0.00 0.00 0.H0 0.00 o.00 0.00 .00 0.00 0 0.00 0.H0 0.00 0.00 H.00 0.00 m.00 0.00 0.00 0 0.m0 m.00 0.00 m.00 0.00 0.00 0.00 H.0m 0.00 0 0.0m 0.0m 0.0a 0.0m 0.0a 0.00 0.00 0.00 0.00 0 0.mm 0.00 0.0m 0.H0 0.0m 0.00 0.00 0.00 0.00 0 0.00 0.H0 0.00 0.00 0.00 0.00 0.mH 0.0m H.00 0 0.0m 0.00 0.00 H.0m 0.00 0.0m 0.00 0.0a 0.00 0 0.0m m.00 H.00 0.m0 0.00 0.00 0.00 0.0a 0.00 m 0.H0 0.00 0.00 0.H0 0.00 0.00 m.00. 0.00 0.00. H 0:0 0:0 m:0 m:0 H:0 00:0 0H:0 0:0 0:0 % Hosans 00:0800009 .Ea0mn Hmsaos u D .AOHV H0003 mm0 000 .sz pospw>o 0H0:3 .AAV posuw>o 000:0 A030H .sz pozcfi>o 000:0 0H00HE .Ambv pozvfi>o sham» p0mm: .Aw I HV 0 nmsoanp H msdflm0a H0050H>o 800m v0usx0 nasfla0pma haamh 0p 0hsmomx0 0pscfls OH 00 u0nmaam§ooow 003 soapmufiomgmo aa0mm .nov 0900 Hospcoo 0::EEHICOG no va 0000 0HH0h 0gp pmsH0M0 0&00Hpsw Spas ps0epm0ap soaumsfla0wsfl0sm 00000 wmw0 0:0Hmwmiom mcfi>00flo Mo 00mmps0oa0m mo wps0H0>st0 can 000 :0 m0SH0> soapmafiawpa0m 0 HHH magma 0.00 0.00 0.00 0.00 0.00 m.00 0.00 .H.00 0.m0 0.00 0.00 0.00 M 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0:00 0.00 0.00 0.00 0.00 0H 0.00 0.00 0.00 0:H0 0.00 0.00 0:00 0.00 0.00 0.00 0.00 a.a0 0H 0.00 0.00 0.00 0.00 0.00 0.00 0:00 0:00 0.00 0.00 0.00 0.00 0H 0.00 0.00 0.00 0:00 0.00 0.00 0.00 0.00 0.00 0.00 H.m0 0.00 0H 0.00 0.00 0:00 0.00 0:00 0:00 0.00 0-00 0.00 H.00 0.00 0.00 0H 0.00 0.a0 0.00 0.00 0.00 0.H0 0.00 0.H0 0.00 0.00 m.00 0.00 0H 0:m0 0.00 0.00 0:00 0.00 0.00 0.00 0:00 0.00 0.00 0.00 0.00 00 H.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 H.am 0.00 NA 0:00 0.00 0.00 0:00 0.00 0.00 H.00 0:00 0:00 0.00 0.00 0.00 HH 0.00 0:00 0.00 m.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 00 0.00 0.00 H.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 «.00 0.00 0 0.00 0.00 0.00 0.00 0.0m 0.00 0:00 0.00 0.00 0.H0 0.00 0. 0 0 0.00 0.00 0.00 0:00 0.00 0.00 0.00 0.00 0.00 0:00 0:00 0.00 0 0:00 0.00 0.00 0:00 0:00 0:00 0:00 0:00 0.00 0:00 0.00 0.00 0 H:H0 0.00 0.00 0:00 a.H0 0.00 0:0m 0.00 0.00 0:00 0.00 0:00 0 0.00 0.00 H.mm 0.00 0.00 0.00 H.0 0.0m 0.0 0.00 0.0m 0:0m 0 0.00 0.00 0.00 0.00 0.00 0.a0 0.00 0.00 0.00 0.00 m.00 0.00 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0:00 0.00 H.00 0.00 m 0.00 0.00 0M0 0.00 0.a0 0:00 0.00 0.00 04mm 0.0 0.00 0.00 H 3:0 3:0 0:0 0:0 2:0 2:0 00:0 00:0 0:0 0:0 0:0 0:0 0 senses mpc0aus0ae anoanspeoov n HHH manna Table III b. Three way analysis of variance on the effect of oviducal extracts and egg water on sperm 100d to insem- inate eggs of Rana_pipiens treated with antisera against the jelly coat or non-immune control sera. df Sum 0f.§229293 Kean souare F Animals 18 59450.61 1858.57 22.2* Treatment 1 74049.47 74049.47 885.1* bnra 11 5527.89 520.72 3-3‘ Interaction 11 6914.73 628.62 7.5* Error 414 54655.71 33.66 Total 455 152576.46 * Values significant to the 16 probability level amawm H< m wwswwHHnmawos <0H00m H: 000 00: onsw