.V_.__-.. ..— a-.--—q%y*q.mmqw THE MORPHOLOGY, msmocv AND FUNCTION OF THE .SEMINAI. VESICLES V IN THE ADULT MALE FROG, - , ‘ f RAMA PIPIENS 4 . I Thesis for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY PAMELA KAY McALLIST ER 1973 L I B R R Y Michigan S Late _ U111}? "v W This is to certify that the thesis entitled The Morphology, Histology and Function of the Seminal Vesicles in the Adult Male Frog, Rana Eigiens. presented by Pamela Kay McAll is ter has been accepted towards fulfillment of the requirements for Ph. D. degree in Zoology é ‘FQWW W my / A Date. I) \ 0'7“. ABSTRACT THE MORPHQLOGY, HISTOLOGY AND FUNCTION OF THE SEMINAL VESICLES IN THE ADULTIMALE 320G, ENE! PTPTEWS By Pamela Kay MmAllister The seminal vesicles in Hana pipisns are multiple and branched evaginations of the Wolffian ducts consisting of a layer of pseudo- stratified columnar epithelium.surrounded by connective tissue. The secretory products of the seminal vesicles were seen to contain neutral mucopolysaccharides whereas acid mucopolysaccharides were absent. Several experiments revealed that materials from the seminal vesicles are able to prolong the fertilizable life of spermatozoa which are stored at refrigerator temperatures. Low levels of fertility were seen if sperm‘which have been incubated with seminal vesicle homogenate were washed off the eggs after short periods of time. If sperm remained on the eggs for longer periods of time, high levels of fertility were seen. High levels of fertilizability were obtained even if spermwwere washed off shortly after insemination if the sperm were exposed to diffusible factors obtained from freshly ovulated eggs before their use in fertilization. Results suggest that the seminal. vesicles may be the source of a factor or factors which may bind to the sperm thereby rendering them incapable of fertilizing and that one role of the egg jellies may be to capacitate the sperm. This may Pamela Kay McAllister be of significance in natural fertilization in that sperm may thereby be maintained in a fertiliaable state during the time they are stored. in the seminal vesicles. THE MDRPHOLOGY, HISTOLOGY AND FUNCTIGN OF THE SEMINAL VESICLES IN THE ADULT MALE FRCG..RANH PTPTENS l . '\ I' 3 By ‘fidji‘l‘ 0‘. Pamela KayAMcAllister A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR 0F PHILGSOPHY Department of Zoology _ 1973 “*9 mms I would like to express my sincere appreciation to my major pro- fessor, Dr. John R. Shaver, for his guidance and constructive criticism during my graduate work and his help in the preparation of this ‘manuacript. I also acknowledge and thank the members of my guidance committee, Drs. w. R. Dukelw. H. Ozaki. and S. Aggarwsl, for their helpful suggestions for this thesis. ii LIST OF TABLES I O O O O Q LIST or was 0 C O O O 0 LIST 0F TEXT FIGURES . . . LIST 01’ APPENDIX TABLES. . 9 OF CONTENTS meDUCTION O O O O I O O O O I O O O The morphology and Histology of Vesicles in Anuran Amphibians Functions of the Seminal Vesicles in Anuran Effects of Hormones on the Semdnal Vesicles AnuranAmphibians............ The Seminal Vesicles in Other Non-Hanalian The Seminal Vesicles in.Hammals . . the Seminal METHODSANDMATENIALS........... “we U.“ D O O 0 Preparation of Sperm Suspensions. Castration of Frogs . . . . . . . Histological Procedures . . . . . RESULTS 0 O O O O O O O O O O O O O O O O O O 0 Q Amphib ians . of Vertebrates O O O C O . ”Orphelogy and Rifltfilafly. e e e e e e e e e e e e e e 0 Effects of Seminal Vesicles on Fertilization. . . . . . DISCUS s ION O O O O C O O O O O O O O O O O O O O O O O O O O O LITEMNRE CITE O O O O O O O O O O I O O O O O O O O O O O 0 APPENDIX C O O O O O O O O O O O O O O O O O O O O I O O O O 0 iii Page iv vii \1 #UN NH \DQNN 11 ll 20 37 43 46 LIST OF TABLES Table Page 1 Effect of castration on "seminal vesicle weights" . . . . 18 2 Cytochemical observations on the secretions of the seminal vesicles and‘Wolffian ducts of the frog. m pipim O O I O O Q 0 O O O O I O O O O O O I O O O O 19 iv LIST OF PLATES Figure I Page 1 The urogenital system of an adult male.Rhna pipiens . . . l3 2 A.representative cross section through the seminal vesicleofanintactfrog................ l7 3 A representative cross section through the seminal vesicle of a castrated frog . . . . . . . . . . . . . . . l7 Figure II III IV VI VII VIII IX XI LIST OF TEXT FIGURES Diagram-atic representation of the urogenital system of an adult male Rana pipiens . . . . . . . . . . . . . Experimental procedure. . . . . . . . . . . . . , . . . Effect of seminal vesicle homogenate on the fertilizing capacity of sperm incubated less than 12 hours. . . . . Effect of seminal vesicle homogenate on the fertilizing capacity of sperm incubated 24 hours. . . . . . . . . . Effect of seminal vesicle homegenate on the fertilizing capacity of sperm incubated 48 hours. . . . . . . . . . Effects of brain, oviduct, and seminal vesicle homogen- ates on the fertilizing capacity of frog sperm. . . . . The effect of brain, oviduct, and seminal vesicle homogenates on the fertilizing capacity of frog sperm. Data plotted by the method of least squares . . . . . . The effect of egg water on the fertilizability of sperm incubated in seminal vesicle homogenate or Holtfreter's 801“: ion “ma 0 O O O O O I C O O O O C O O O C Q O O O The effect of egg water on the fertilizability of frog sperm incubated in seminal vesicle homogenate or in Holtfreter's solution alone. Changes in fertilization with increasing insemination times. . . . . . . . . . . A comparison of the effect of seminal vesicle homogenates from intact and castrated animals on the fertilizing cap‘c ity 0f 1 to: 8p em . O O I O O Q 0 I O O O Q 0 O O O A comparison of the effect of seminal vesicle homogenates from intact and castrated animals on the fertilizing capacity of frog sperm. Changes in fertilization with increasing insemination times . . . . . . . . . . . . . vi Page 15 21 22 23 25 28 29 32 33 35 35‘ Table LIST OF APPENDIX TABLES Sperm incubated for less than 12 hours in Holtfreter's solution and seminal vesicle homogenate. . . . .,. s . . Sperm incubated for 24 hours in Holtfreter's solution and seminal vesicle homogenate . . . . . . . . . . . . . Sperm incubated for 48 hours in Holtfreter's solution and seminal vesicle homogenate . . . . . . . . . . . . . Sperm incubated for 48 hours in Holtfreter's solution, seminal vesicle homogenate, brain homogenate and Widucal hmgaute O O O O O O O O O 0 fl 0 l I O Q 0 O O 0 Basic statistics, regression statistics. and calcula- tion of higher order regression coefficients for seminal vesicle data . . . . . . . . . . . . . . . . . . Effect of egg water on the fertilizability of sperm incubated in seminal vesicle homogenate or Holtfreter's so 1“ t ion 0 O O O O O Q 0 O O O O O O O O O C O O O O O 0 Regression statistics based on data in Appendix Table 5. Effect of sperm treatments on subsequent embryonic development 0 O O O O O I O O O O O O O O O O I I I O O 0 Effect of castration on the ability of seminal vesicle homogenates to alter fertilizing capacity at spam 0 O I O O O O O O O O O O O O O O O O O O O I 0 vii Page 46 47 49 50 51 53 55 56 58 INTRODUCTION The morphology and Histology of the Seminal Vesicles in.Angran.Amphibians Although considerable attention has been directed towards under- standing the conditions necessary for fertilization in anuran amphibians, including the role of the oviductal secretions and egg jellies, little information is available on the role that the male accessory glands, the seminal vesicles, may play. The seminal vesicles are generally described as outgrowths of the vas dbfbrens or Helffisn duct and have been described as the site of sperm storage prior to ejaculation. Seminal vesicles have been described in,a number of anuran species including Discoglossus piatus (Mann, Lutwak-Msnn and Hay, 1963) , Rana ssculsnta, Rona tsmponaria (Aron, 1926), Epic crueifbr and Rana pipiens (Rngh, 1934 and 1939). In histological sections transverse to the long axis the seminal vesicles of D. pictus were seen to consist of a thick epithelial layer supported by a thin layer of connective tissue. The epithelium consists of three types of cells: basal cells, long columnar cells containing PAS-positive granules, and small cells surrounding the lumen. The fluid within the seminal vesicles is hypo- tonic and contains a high content of glycoprotein. The seminal vesicles of.R. esculenta and R. tamporaria are described as enlargements of the distal one-third of the Wblffian duct. Histological sections show them lined with a simple epithelium of mucous cells which invaginate into the connective tissue forming 2 pseudo-glands. During the breeding season the lumina within the seminal vesicles are filled with sperm. At this time the glandular evaginations are larger and more complex. The seminal vesicles of H. aruoifbr and.R. pipiens are described only as enlargements of the posterior portions of the wolffian ducts. ‘ggpctions of the Seminal Vesicles in Annran ggphibians Although the functional significance of the seminal vesicles in anuran amphibians is unknown, several suggestions have been made. In.R. pipisns spermatozoa are stored in the seminal vesicles and are expelled during amplexus. Spermatozoa removed from various portions of the male reproductive tract have been found to be functional in regards to fertilizing capacity (Hugh. 1939). In E. aruoifbr the seminal vesicles are not only a storage place for spermatozoa but also may be copulatory accessories. since in Hyla as in toads each egg is inseminated singly and the seminal vesicles together with the muscular cloaca supply each egg with a small amount of spermatozoa (Hugh, 1941). In addition to their storage function the seminal vesicles may be the source of seminal fluids which serve as a vehicle for the passage of sperm to the outside (Mann, 1964; Mann, Lutwik-Mann and Hay, 1963). Effects of Hormones on the Seminal Vesicles of Anuran Amphibians The endocrine control of wolffian duct development has been best studied in.R. temporaria. The duct is formed independently of the presence or absence of male or female hormones. Later. towards the end of the larval period, frogs treated with androgen display hypertrophy of the wolffian ducts and develop a seminal vesicle. The sensitivity to testosterone follows a decreasing caudo-cephalic gradient (Gallien, 1955). 3 In adult R. pipiens and Bufo merieanus the Wolff ian ducts includ- ing the seminal vesicles hypertrophy following treatment with testosterone propionate (Puckett, 1939). Sperm release from the testes and enlarge- ment of the Wolffian ducts and seminal vesicles has been reported in R. pip‘tsns and H. crucifsr following treatment with pituitary gonado- tropins (Rugh, 1939, 1941). Regression of the Wolffian ducts, including a decrease in diameter of the ducts and a decrease in the height of the epithelium, has been observed in R. ssculsnta following hypophysectomy (Sluter, van 00rdt and Highorst, 1950). Transplantation of testicular tissue into castrated male frogs has been reported to induce development of nuptial pads and seminal vesicles (Parkes, 1960). The Seminal Vesicles in Other Hon-Mammalian Vertebrates Seminal vesicles have also been described in a number of other non-mammalian vertebrates. In teleost fish seminal vesicles have been described as fan-shaped glandular structures lined with typical secre- tory epithelium. They are believed to produce a fluid resembling mammalian prostatic and vesicular secretions, which may have the function of providing nutrients for the spermatozoa or may secrete a substance which maintains the sperm in an imobilized state (Mann, 1964). Histochemical and biochemical studies on the secretions of the seminal vesicles of the catfish, Heteropnsustes, indicate the presence of mucoproteins, mucopolysaccharides, proteoses, phospholipids and native proteins (Hayyar and Sundararaj, 1970). Seminal vesicle weights were found to decrease following treatment with an anti-androgen, I cyproterone acetate (Sundararaj and Nayyar, 1969). Eggs were success- fully fertilized using testis squash with or without seminal vesicles indicating that the seminal vesicles are not essential to 4 fertilization. Thus, while the seminal vesicles are not essential, the seminal fluids probably help in natural fertilization in that they maintain the sperm in an inactive but viable condition (Sundararaj, 1958). Although seminal vesicles have not been described in reptiles, hypertrophy and secretory activity has been demonstrated in a variety of male lizards and snakes to include the preterminal and terminal segments of the urinary tubules, the collecting canal, and the ureter. The femoral glands, epididymides and vase dbfbrantia are all affected by castration. The vas deferens in castrated males of Anolis carolinensis were reported to undergo a 63% reduction in diameter when compared to sexually active males and a 37% reduction when compared to sexually quiescent males. Reduction in the height of the epithelial cells lining the was dbfbrsns was also reported (Parkes, 1960). I Seminal vesicles have also been reported in passerine birds as swollen portions of the vas dbfbrens and are considered to be sites of sperm storage. The seminal vesicles are small during periods of sexual inactivity but enlarge and become highly convoluted during the breeding season (Mann, 1964). During the height of the breeding season the sperm-laden seminal vesicles descend into cloacal protuberances where the temperature is lower than body temperature. Thus the major function of the seminal vesicles in birds as well as in fish may be to maintain the sperm in a viable state until intromission (Parkes, 1960). The Seminal Vesicles in Mammglg The seminal vesicles in mammals, as in non-mammalian vertebrates with the exception of fishes, are glandular developments of the wolffian ducts and are dependent on androgens for the maintenance of the 5 epithelium and secretory activity. In some species the secretions of the seminal vesicle contribute a substantial portion to the whole ejaculate, although the secretory output of the seminal vesicles varies considerably and in some species may contribute relatively little volume of the total ejaculate. Seminal vesicles are totally absent in some species such as the dog and cat. In addition to providing the fluid medium suspending the sperm, the male accessory glands in.mammals may be the source of sperm- coating antigens which may have significance in fertilization. The need for a uterine incubation period for spermatozoa (capacitation) was first demonstrated in 1951 (Austin, 1951; Chang, 1951). It was sub- sequently demonstrated that capacitation could be functionally reversed (decapacitation) by a substance from the seminal plasma referred to as decapacitation factor (Chang, 1957). In one study utilizing immunofluoreacence a single sperm-coating antigen was demonstrated in the human and in the rabbit which was firmly attached to ejaculated sperm. The origin of this antigen was the seminal vesicle (wail and Rodenberg, 1962). Subsequent studies demonstrated the presence of several spermrcoating antigens of accessory gland origin. Ejaculated sperm did not fluoresce when exposed to antibodies against testicular sperm, indicating that at ejaculation the sperm.are coated with seminal plasma antigens which block the sites with which antibodies against testicular sperm would interact (Hunter and Nornss, 1969; Johnson and Hunter, 1972). Two of the spermrcosting antigens were demonstrated to be glycoproteins and to have activity similar to that of decapacitation factor since they were able to block fertilisation when spermwwere incubated with them.(Hunter, 1969). 6 The present investigation was begun for the purpose of elucidating the functional significance of the seminal vesicles in male Rana pipisns since in amphibians, as in mussels, the seminal vesicles may be the source of sperm-coating antigens which may alter the fertilizing capacity of the sperm. For this purpose a series of experiments were performed in which sperm were incubated with a variety of substances, including materials from the seminal vesicles, to determine whether they could influence the fertilizing capacity of the sperm when they were used to fertilize normal jellied eggs. Additionally, seminal vesicles from castrated animals were compared to those from intact animals as to their ability to alter the fertilizing capacity of testicular sperm. The histology and cytochemistry of the seminal vesicles from intact and castrated animals were also compared. METHODS AND MATERIALS Animals Used Adult male and female.Rena pipiens were obtained from a commercial dealer (Bay Biologicals, Ltd., Port Credit, Canada). The animals were kept in hibernation until use (4-6'0.). Breparation of Sperm.Suspensions Male frogs were pithed and the testes and seminal vesicles were removed. In some experiments the brains and male rudimentary oviducts were also removed. The tissues were placed in 10% Holtfreter's solu- tion over ice until all dissections were complete. The testes were crushed with forceps. filtered through glass wool, and the resulting suspension diluted with 102 Holtfreter's solution to give a concentra- tion of about 2000 sperm/mmS. A hemocytometer was used in making the sperm counts. Aliquots of the sperm suspension were then mixed with equal volumes of filtered tissue homogenate or 102 Holtfreter's solution giving a final concentration of 1000 sperm/mmP. The suspensions were stored for varying times in the refrigerator until use in fertilization experiments. Tissue homogenates were prepared by homogenizing in a glass homogenizer with 102 Holtfreter's solution (lgm. tissue/10ml. Holtfreter's). In one set of experiments the sperm suspensions were mixed with an equal volume of egg water prior to use. In these experiments the sperm suspension was initially diluted to a concentration of 4000 sperm/mmé. 8 Aliquots were mixed with a 202 tissue homogenate. Egg water was pre- pared by extruding freshly ovulated eggs into a dish and covering them with 102 Holtfreter's solution. One hour later the Holtfreter's solu- tion.was pipetted off and was mixed with the sperm suspensions which had been removed from the refrigerator and allowed to come to room temperature. This gave a final sperm.concentration of 1000 sperm/mm; as in the other experiments. The sperm concentration was again checked for each suspension just prior to use in fertilization experiments. Eggs were obtained by injecting mature females with progesterone and frog pituitaries (Wright and Plathers, 1961). Eggs were extruded onto microscope slides and inseminated. An average of 25-50 eggs were fertilized from each female for each time period. At varying times after insemination the spermwwere poured off the slides and the slides were immersed in a large volume of aerated tap water. Three to five hours later the percentage of cleaving eggs was determined and used as the criterion for fertilization. For statistical analysis, the percentage of cleaving eggs was transformed by the angular transforma- tion (0 - arc sin vi; ). Castration of Frogs Frogs were castrated under ether anesthesia. An incision was made in the lateral body wall midway between the pectoral and pelvic girdles. The testis on that side was pulled through the incision, the mesorchium was sutured to close the testicular blood vessels and the testis removed. The incision was sutured and the testis on the other side was then removed by the same procedure. The frogs were kept at room temperature until use. 9 Histological Procedures The seminal vesicles were fixed in Smith's modification of Bouin's fixative (Guyer, 1947), dehydrated, cleared, and embedded in paraffin. One seminal vesicle from each animal was sectioned at 10 microns and stained with hematoxylin and eosin for routine histological examina- tion. The other seminal vesicle from each animal was sectioned at 5 microns and stained for carbohydrates and proteins. The periodic acid- Schiff (PAS) procedure for neutral mucopolysaccharides was used both ‘with and without prior treatments to determine more accurately the nature of the PAS-positive compounds. Schiff's reagent was prepared by the method of de Tomasi (Pearse, 1970). PAS-reactive protein was removed by incubation of sections at 37°C. for 1 hour with trypsin (0.12 in 0.1M sodium phosphate buffer at pH 7.4). Control sections were incubated in buffer only. A 1:1 solution of chloroform and methanol was used to remove PAS-positive lipids. Glycogen was removed from some sections by incubating for 1 hour at 37'C. in a 0.12 malt diastase solution (Nutritional Biochemicals Corp.) in 0.02M sodium phosphate buffer at pH 6.0 containing 0.652 NaCl. The specificity of the reaction was determined by acetylation with a 2:3 mixture of acetic anhydride and pyridine for 2 hours at 60°C. and also by placing slides directly into Schiff's reagent without periodic acid oxidation (Pearse, 1970). Acid mucopolysaccharides were determined by staining with alcian blue at pH 2.5 (Barks and Anderson, 1963; Mowry, 1963). The ninhydrineSchiff procedure was used for the detection of protein. The specificity of the reaction was determined by omitting the ninhydrin oxidation step and also by incubating some sections in a 10 solution of 12 acetic acid in acetic anhydride for 1 hour at 60°C (Umpierre, 1971). RESULTS Moghology and His tologz The seminal vesicles in.Rana pipisns are glandular swellings of the posterior portions of the wolffian ducts and are enclosed in a connective tissue sheath along with the posterior portions of the male rudimentary oviducts. The urogenital system of an adult male Rana pipisns is seen in Plate I, Figure l, and is illustrated diagram- matically in Text Figure 1. The sperm after leaving the testes pass through the vase afTbrentia and into the anterior tubules of the kidneys. The sperm then pass into the wolffian ducts and are stored in the seminal vesicles. During amplexus the sperm pass into the cloaca and are emitted over the eggs (Hugh, 1939). In transverse sections the seminal vesicles are seen to consist of multiple and highly branched evaginations of the Welffian duct. Surrounding the lumina of the seminal vesicles and of the wolffian duct is a pseudo-stratified columnar epithelium. A layer of connective tissue surrounds the epithelium (Plate II, Figure 2). Five weeks after castration, seminal vesicles were removed from four castrated and four sham-operated controls. Seminal vesicles together'with the adjacent portions of the.oviducts were removed, weighed, and fixed. 0ne seminal vesicle from each animal was sectioned for routine histological examination and the other was stained for carbohydrates and proteins. Five weeks after castration the height of 11 12 Figure 1. The urogenital system of an adult male Rana pipiens. Key to Abbreviations B - Urinary Bladder Cl - Cloaca FB - Fat Body K - Kidney 0 - Oviduct SV - Seminal Vesicle T - Testis WE - wolffian Duct 13 PLATE I 14 TEXT FIGURE I Diagrammatic representation of the urogenital system of an adult male Rana pipiens. Key to Abbreviations B - Urinary Bladder C — Colon FB - Fat Body K - Kidney O - Oviduct SV - Seminal Vesicle T - Testis ' VB - Vasa Effsrsntia WD - wolffian Duct 15 TEXT FIGURE I 16 Figure 2. A representative cross section through the seminal vesicle of an intact frog. Figure 3. A representative cross section through the seminal vesicle of a castrated frog. Figure 2. Intact Figure 3. Castrated 18 the epithelium of the seminal vesicles and the wolffian duct was reduced (Plate II, Figure 3). The weights of the seminal vesicles and adjacent portions of the oviducts were reduced following castration (Table 1) although the dif- ference was not significant (0.1 MMDOHM HNNH MHZZNUOZON A4023 >0 seconds on ma ea ma 6H d ma A<2qum nuances nounmfia on ma GN mH 9H H on mm ON ma OH H Hp gun—Hm HE ZGHHDnem m.mmaumhaaom mousse: on mm ow ma OH H 6H 8. on co 29 TEXT FIGURE VII The effect of brain, oviduct, and seminal vesicle homogenates on the fertilizing capacity of frog sperm. Data plotted by the method of least squares. -€+-- Holtfreter's Solution -—4|—-- Brain Homogenate r-43-- 0viducal Homogenate ---0- - Seminal Vesicle Homogenate so -- so A 40 w ; ,.-- / N - 30 h 20 w 10 w III 1:) l; 2; 2; '30 time in minutes 30 before use in fertilization. As in the previous experiment, the sperm were incubated with seminal vesicle homogenate or with Holtfreter's solution alone for 48 hours in the refrigerator. The sperm suspensions were divided into two aliquots and egg water added to one aliquot of each suspension. Ten percent Holtfreter's solution was added to the other aliquot of each suspension. The data were analyzed by three—way analysis of variance. The data are plotted from regression lines calculated by the method of least squares. Results show that egg water has no effect on sperm incubated in Holtfreter's solution alone. In both cases (sperm in Holtfreter's solution with and without egg water added) there was no significant increase in fertilization as a function of time and the average fertility does not differ. Egg water did have a significant effect on sperm incubated in seminal vesicle homogenate. As in the previous experiment, fertility was lowwwhen seminal vesicle treated sperm which were not incubated with egg water were allowed short periods of time to interact with the eggs. Fertility increased when increasing time was allowed for interaction. The overall fertility seen with seminal vesicle- treated sperm without egg water did not differ from that seen with Holtfreter's-treated sperm because of the low fertility of the seminal vesicle—treated sperm at the short time periods. If seminal vesicle- treated sperm were treated with egg water prior to insemination fer- tility was high at all times and there was no significant increase in fertility over time. Further, the average fertility seen when seminal vesicle-treated sperm were incubated with egg water was significantly higher than the fertility achieved by sperm in Holtfreter's solution with or without egg water or with sperm in seminal vesicle homogenates 31 without egg water (Text Figure VIII and Text Figure IX based on data in Appendix Table 6 and Appendix Table 7). Since the differences in fertility could have been due to differu ences in activation rather than fertilization embryos were counted in the tailbud stage and the percentage of developing embryos was compared with the percent fertilization. Since many embryos had the external characteristics of the haploid syndrome, embryos were scored as normal or abnormal. Embryos which resulted from fertilization with sperm incubated in Holtfreter's solution alone were frequently retarded in their development as compared to the embryos resulting from fertiliza- tion with sperm incubated in seminal vesicle homogenate. Since these embryos were otherwise normal in appearance they were scored as normals. There were no significant differences either in the percent of embryos which developed or in the percent of the developing embryos which were abnormal in appearance (Appendix Table 8). Thus, the differences seen in previous experiments were due to differences in the fertilizing capacity of the sperm. Since the materials from the seminal vesicle which influence the fertilizing capacity of the sperm.may be dependent on androgens or other factors from.the testes, male frogs were castrated and after five weeks their seminal vesicles were removed, homogenized, and incubated with sperm. Sperm'were incubated in the refrigerator for 48 hours as in the previous experiments. Sperm.incubated with Holtfreter's solution alone and with seminal vesicle homogenate from intact frogs were used as controls. Eggs from only one female were used due to the small amount of seminal vesicle homogenate available from castrated animals. Results show that seminal vesicles from castrated and intact animals do not differ in their ability to maintain the sperm in a 32 TEXT FIGURE VIII The effect of egg water on the fertilizability of sperm incubated in seminal vesicle homogenate or Holtfreter's solution alone. 50 a ‘ 40 w 30 w- e '. 20 W 10 u “ I no egg egg no egg egg water water water water HOLTFRETER'S SEMINAL VESICLE SOLUTION HOMOGENATE 33 TEXT FIGURE IX The effect of egg water on the fertilizability of frog sperm incu- bated in seminal vesicle homogenate or in Holtfreter‘s solution alone. Changes in fertilization with increasing insemination times. —Q—- - sperm in Holtfreter's solution alone + - sperm in Holtfreter's solution, egg water added —-9— - sperm in seminal vesicle homogenate —-O— - sperm in seminal vesicle homogenate, egg water added 60 50 40 30 20 1' 10 +- L l i 10 20 30' time in minutes 34 fertilizable state during 48 hours of sperm storage. Sperm incubated with seminal vesicle homogenate from.both intact and castrated animals fertilized significantly*more eggs than sperm.incubated with Holtfreter's solution alone (Text Figure X and Text Figure XI based on data in Appendix Table 9). 35 TEXT FIGURE X A comparison of the effect of seminal vesicle homogenates from intact and castrated animals on the fertilizing capacity of frog sperm. 40 4*- 30 «b j 6 20 «L- 10 w intact ' fl castrated HOLTFRETER'S SOLUTION SEMINAL VESICLE HOMOGENATE 36 TEXT FIGURE XI A comparison of the effect of seminal vesicle homogenates from intact and castrated animals on the fertilizing capacity of frog sperm. Changes in fertilization with increasing insemination times. -E— - Holtfreter's Solution - Seminal Vesicle Homogenate - Intact - Seminal Vesicle Homogenate - Castrated 50 40 9 30 20 1*- 10 l- of!” / / / L l L g 5 10 15 20 25 30 time in minutes DISCUSSION The seminal vesicles of the male frog, Ranarpipians, were demon- strated to be similar histologically to those described in other species of Rana (Aron, 1926). They consist of multiple evaginations of the wolffian ducts lined by pseudo-stratified columnar epithelium which is surrounded by connective tissue. Histochemical techniques which can identify general classes of carbohydrates were used to demonstrate the nature of the materials which are secreted by the seminal vesicles. Although the techniques employed can demonstrate only general classes of carbohydrates, by use of the appropriate controls differences in the chemical nature of their constituents can be detected. The PAS reaction is used to demonstrate carbohydrates rich in neighboring hydroxyl groups or equivalent amino substitutions (Mowry, 1963). Greater accuracy in identification of the reactive compounds can be obtained with controls such as protein digestion, lipid extraction, and diastase digestion (Barks and Anderson, 1963). Alcian blue at pH 2.5 stains carbohydrates rich in free acidic groups although sulfate groups may also contribute to the staining (Howry, 1963). The complex carbohydrates in the epithelium and lumina of the seminal vesicles appear to be in the _ form of neutral mucopolysaccharides since they are PAS-positive and diastase-resistant and stain.weakly with a protein stain. Acidic mucopolysaccharides are absent. 37 38 Results clearly indicate that materials from.the seminal vesicles can indeed influence the fertilizing capacity of sperm. The fertilizable life of the sperm was prolonged when they were exposed to materials from the seminal vesicles. In fact, sperm which are incubated in seminal vesicle homogenate still retain some fertility after three days at refrigerator temperatures whereas sperm.in Holtfreter's solution alone are totally inviable at this time (data not presented). Interestingly, after two days at refrigerator temperatures, sperm incubated in seminal vesicle homogenate were capable of fertilizing greater numbers of eggs than sperm incubated with brain, oviduct, or Holtfreter's solution alone but only if allowed considerable time (25- 30 minutes) to interact with materials from the eggs or their enveloping jelly coats. That the lag in fertilization was due to an interaction between the sperm.and some material which diffuses-from the egg or its jelly cost was demonstrated since pre-incubation of seminal vesicle- treated spermuwith egg water resulted in elimination of the lag. This interaction can be called capacitation by analogy with the phenomenon in mammals where sperm must interact with materials from the female reproductive tract before gaining fertilizing capacity (Austin, 1951; Chang, 1951). Incubation of sperm with male rudimentary oviduct results in an almost complete loss of fertilizability. This raises the possibility that the materials from the male oviduct may contain the some materials as the female oviduct and in this instance the sperm may have been. capacitated during the two day incubation period and that once capaci- tated the sperm have a short fertilizable life. Most of the sperm incubated with male oviduct were immotile and were probably dead. That the male rudimentary oviduct does contain some materials similar to 39 those found in female oviducts has been demonstrated (Umpierre, 1971). If indeed the loss of fertilizability of sperm incubated in oviduct homogenate was due to premature capacitation, it would be fruitful to utilize male oviducts rather than female ones in an attempt to isolate the capacitating materials since the secretions of the male oviduct are less complex than those from the female (umpierre, 1971). In anuran amphibians it has been established that the egg jellies are essential in fertilization since coelomic eggs or eggs from which the jellies have been removed are not fertilizable (Shaver and Barch, 1960; Shaver, 1966; Katagiri, 1966a and 1966b). Coelomic eggs, however, can be fertilized by sperm which have been exposed to jellied eggs (Shaver, 1966). The nature of the dependency on the jelly coat for fertilization is unknown although experimental evidence indicates that changes in the sperm are involved. The egg jellies are hetero- geneous and may contain more than one factor which is necessary for fertilization. Hydrated anuran eggs are not normally fertilizable but fertility can be restored by treating hydrated eggs with materials which diffuse from them. The "diffusible factor" which is produced by the oviducts and retained by the jelly before hydration would under physiological conditions presumably activate the sperm.hefore they penetrate the jelly coats (Barbieri and Raisman, 1969). The authors also report that de-jellied eggs could be fertilized either in the presence of solubilized jelly or the diffusible factor. Sperm.which have been exposed to diffusible materials from egg jellies are also capable of fertilizing eggs whose jelly costs have been blocked by antibodies. Eggs which have been exposed to antibodies prepared against egg jellies are normally not fertilizable. In this case the alteration of the sperm induced by diffusible materials from 40 uterine eggs has been called capacitation (Shivers and James, 1970; Roberts, 1970). ' In all of these experiments the authors have been primarily interested in elucidating the role of the egg jellies in the fertili- zation process and have employed fertilization of eggs which are abnormal (either lacking in jelly or with jellies blocked with anti- bodies) in an attempt to demonstrate jelly-induced sperm alterations. The authors have also employed sperm obtained from the testes which may not be the same as sperm which are emitted during amplexus, since in normal fertilization sperm must be exposed to materials from the seminal vesicles. The present experiments suggest that the seminal vesicles may be the source of materials which decapacitate the sperm inasmuch as sperm ‘which have had contact with seminal vesicle materials require a long period of interaction with eggs or diffusible materials from the eggs if high levels of fertilization are to be achieved. The sperm may be coated by materials from the seminal vesicles and this coating material may then be removed during exposure of the sperm to diffusible materials from the egg jellies. Alternatively, the materials from the seminal vesicles may simply alter the sperm surface without actually binding to it. The role of the seminal vesicle materials may thus be to bind to or alter the sperm surface such that the sperm is maintained in a fertilizable state during storage of the sperm.in the seminal vesicle. The seminal vesicles may not be the only source of factors which render the sperm in a non-capacitated state (decapacitating factors) since immunological studies in our laboratory have failed to demonstrate any antigenically unique material in the seminal vesicles when compared 41 to other portions of the male reproductive tract. The decapacitating factor(s) may also be present in the testis but insufficient in amount to maintain the fertilizability of a dilute sperm suspension for a prolonged period of time. In all previous experiments in which sperm capacitation in anuran amphibians has been described testicular sperm were utilized. From.these studies it appears that capacitation is a two step phenomenon involving a small diffusible molecule which inter- acts with the sperm before contact with the egg jelly and a non- diffusible molecule with which the sperm interacts as it passes through the jelly (Barbieri and Raisman, 1969; wolf and Hedrick, 197; Katagiri, 1966a and 1966b). In all of these cases capacitation occurred very quickly. In the present experiments capacitation required 25 to 30 minutes. The difference appears to be only a matter of the time required since in this case, as in the previous experiments, capaci- tation could be induced by the same materials (diffusible factors from the egg jellies). This does not imply that in natural fertilization capacitation requires such a long period of time, since under the artificial conditions of these experiments the sperm may have been exposed to a much greater concentration of decapacitating factors than would be the case in natural fertilization. Exposure of sperm in 101 Holtfreter's solution to egg jelly materials prior to use in fertiliza- tion of normal jellied eggs has been reported to reduce the percent of eggs fertilized by about 172 (Shivers and James, 1971). The authors suggest that the decrease in fertility is due to premature capacitation possibly involving an acrosomal reaction. Similarly, in the experie ments reported here, sperm which have been incubated in Holtfreter's solution and then exposed to egg water showed a slight decrease in fertilizing capacity although the difference was not significant. 42 This suggests that sperm from the testis are capacitated by diffusible materials from the eggs and that capacitation in this case occurs rapidly. Thus the difference in capacitation between the experiments described herein and those of other authors may be due simply to the amount of time required and not to differences in the nature of the changes which are occurring in the sperm. Sperm.exposed to the seminal vesicles may have a greater amount of material bound to their surface and thus a longer period of time may be required for its removal. The fact that seminal vesicles from castrated and intact animals do not differ in their ability to alter the fertilizing capacity of the sperm.may reflect the fact that they are not dependent upon cone tinuous stimulation by materials from the testes for their function or may have been due to the fact that insufficient time had elapsed after castration for the level of hormones to decline sufficiently for the loss of function to occur. In conclusion, the present experiments suggest that the role of the seminal vesicles in Rana pipiens may be to maintain the sperm in a fertilizable state during the time sperm are stored in it. Upon release and exposure to materials from the egg jellies the sperm then acquire fertilizing capacity. The seminal vesicles may be the source of materials which cost or alter the sperm.aurface. The role of the egg jellies would then be to remove the coating substance and/or further alter the sperm so that fertilizing capacity is attained. LITERATURE CITED :‘illlllll LITERATURE CITED Aron, M. 1926. Recherches morphologiques et experimentales sur le determinisme des caracteres sexuales males chez les Anoures. Arch. de 3101., Paris, _3_6_:3. Austin, C. R. 1951. Observations on the penetration of the sperm into the mamalian egg. Aust. J. Scient. Res. B., 4:581. Barbierri, F. D. and Raisman, J. S. 1969. Non-genetic factors involved in the fertilization of Bufo arenarwn oocytes. Embryologia, _1_g: 363-372. Barks, T. and Anderson, P. J. 1963. Histochemistry, Theory, Practice and Bibliography. Harper and Row, H.Y., pp. 65-95. Chang, M. C. 1951. Fertilizing capacity of spermatozoa deposited into the fallopian tubes. Nature, Lond., 168:697. . 1957. A detrimental effect of seminal plasma on the fertiliz- ing capacity of sperm. Nature, Lond., £72: 258. Gallien, L. 1955. The action of sex hormones on the development of sex in Amphibia. In: Memoirs of the Society for Endocrinology of Vertebrates. Part I. The Comparative Physiology of Repro- duction and the Effects of Sea: Hormones in Vertebrates. Cambridge University Press, pp. 188-204. Guyer, M. F. 1947. Animal Micrology. University of Chicago Press, Chicago, p. 242. Hunter, A. G. 1969. Differentiation of rabbit sperm antigens from those of seminal plasma. J. Reprod. Fert., 22:413-418. Hunter, A. G. and Homes, H. 0. 1969. Characterization and isolation of a sperm coating antigen from rabbit seminal plasma with capacity to block fertilization. J. Reprod. Fert., 293419-427. Johnson, w. L. and Hunter, A. G. 1972. qunofluorescent evaluation of male rabbit reproductive tract for sites of secretion and absorption of seminal antigens. Biol. of Reprod., _6_:13-22. Katagiri, C. 1966a. Fertilization of dejellied uterine toad eggs in various experimental conditions. meryologia, 9:159-169. . 1966b. The fertilizing capacity of frog sperm in the homolo- gous and heterologous egg-j ellies and polyvinyl-pyrolidone (PVP) . J. Pac. Sci. Hokkaido Univ. (Zool.), _]_._6_:77-84. 43 44 Mann, T. 1964. The Biochemistry of Semen and of the Male Reproductive Tract. Methuen and Co., London. and Lutwak-Mann, C. and Hay, M. F. 1963. A note on the so- called seminal vesicles of the frog, Discoglossus pictus. Acts Embryologia et Morph. Expt1., _6_:21-25. Mowry, R. W. 1963. The special value of methods that color both acidic and vicinyl hydroxyl groups in the histochemical study of mucins. With revised directions for colloidal iron stain, the use of alcian blue 68X and their combination with the periodic acid- Schiff reaction. Ann. N.Y. Acad. Sci., M:402—423. Meyer, 8. K. and Sundararaj, B. I. 1970. Seasonal reproductive activity in the testes and seminal vesicles of the catfish, Heteropneustes fossilis (Bloch). J. Morph., _l_3_0_:207-226. Parkes, A. s. 1960. Marshall '8 Physiology of Reproduction. Vol. I, Part 2. Longmans, Green and Co., Ltd., London. Pearse, A. G. E. 1970. Histochamistry, Theoretical and Applied. 3rd edition. J. and A. Churchill, London. Puckett, w. 0. 1939. Some effects of crystalline sex hormones on the reproductive structures of several anurans. Anat. Rec., _7_§_:127. Roberts, C. R. 1971. Sperm-egg interactions in Rana pipiens. Master's Thesis, Michigan State University. Rugh, R. 1934. Induced ovulation and artificial fertilization in the frog. Biol. .Bull. , 62:22. . 1939. The reproductive processes of the male frog, Rana pipiens. J. Exp. Zool., goal-105. . 1941. Experimental studies on the reproductive physiology of the male spring peeper, Hyla crucifer. Proc. Amer. Philosoph. Shaver, J .R. 1966. Immunological studies of the jelly-coats of anuran eggs. Am. Zoo1., 6:75-87. and‘ Barch, S. H. 1960. Experimental studies on the role of jelly coat material in fertilization in the frog. Acts Embryol. Morphol. Exp., 35180-189. Shivers, C. A. and James, J. M. 1971. Fertilization of antiserum-.- inhibited frog eggs with "capacitated" sperm. Biol. of Reprod. , 53229-235. Sluter, J. W., 0ordt, G. J. van and Mighorst, J. C. A. 1950. A study of the testis tubules, interstitial tissue and sex characters (thump pads and Wolffian ducts) of normal and hypophysectomized frogs (Rana esculenta). Quart. J. Micr. Sci., 19:131. lilII-l‘lf 45 Sundararaj, B. I. 1958. The seminal vesicles and their seasonal changes in the Indian catfish, Esteropnsustes. Copeia, 4:289-297. and Nayyar, S. R. 1969. Effects of estrogen, 811-9055, and cyperterone acetate on the hypersecretory activity in the seminal vesicles of the castrate catfish, Heteropnsustes fossilis (Bloch). J. Exp. Zool., _1_7__2_:399-408. Umpierre, C. C. 1971. Studies on the distribution of mucopolysaccharides in adult Rana pipiens male rudimentary oviducts after hormonal stimulation. Doctoral Thesis, Michigan State University. Weil, A. J. and Rodenberg, J. M. 1962. The seminal vesicles as a source of the spermatozoa-coating antigen of seminal plasma. Proc. Soc. Exp. Biol. Med., 122567-570. Wolf, D. P. and Hedrick, J. L. 1971. A molecular approach to fertiliza- tion. III. Development of a bioassay for sperm capacitation. Develop. Biol., 32:360-376. Wright, P. A. and Flathers, N. R. 1961. Facilitation of pituitary- induced frog ovulation by progesterone in early fall. Proc. Soc. Exp. Biol. Med., _1_0_§:346-347. APPENDIX 46 Appendix Table 1. Sperm incubated for less than 12 hours in Holtfreter's solution and seminal vesicle homogenate Holtfreter's Solution Seminal Vesicle Homogenate Insemination l cleaved I cleaved time total 3 Z Fert. a total # Z Fert. e ~3- 32 1 34 51 -9- 39 1 38 7o 28 O O 23 O O 13 10 20 20 28 38 24 15 23 28 -§3- 88.5 70.18 28 100 90.00 ANOVA Table source of variation df SS MS F Treatments 1 50.9232 50.9232 .2382 Times 1 1698.844 1698.844 7.9478* Interaction 1 .6721 .6721 .0031 Error .8 1710.0004 213.75 Total 11 3460.439? _. _. e a: ' — - LSD c.01 34.57 Holtfreter s 21 X30 23.32 _. _. * t.05 - 22.2 Seminal Vesicle xl-x30 - 24.27 Test for Homogeneity of Variances szmax./s2mdn. 6.4527 e Significant at the 52 level. 47 Appendix Table 2. Sperm incubated for 24 hours in Holtfreter's solu- tion and seminal vesicle homogenate Holtfreter's Solution Seminal Vesicle Homo enate Insemination T cleaved 1 cleaved time total 7 I Fert. a total 7 X Fert. 6 2 7 25 8.0 16.43 -§§ 24.1 29.4 3 , 23 33 9.0 17.56 31 74.2 59.47 0 4 l‘minute 1 5 -J§ 13 8 21 81 -13 34 6 36 03 29 O O 26 O 0 -—§ 29 6 32 96 -—1 29 2 32 71 27 ' ' 24 ' ' 21 20 3:; 39.6 39.00 36 55.6 48.22 11 ‘25 s .21 -57 13.5 21.56 41 63.4 52.77 30 minutes -—9 21 4 27 56 15- 51 9 46 69 28 ' ' 27 ° ° 14 .11 16 24 ANOVA Table source of variation df SS MS F Treatments 1 1724.8322 1724.8322 16.2663* 48 Appendix Table 2 (cont'd.) source of variation df SS MS F rm: 1 1923.5342 1923.5342 18.1402' Interaction 1 6.448 6.448 .0608 Error .29 2120.7322 106.0366 Total 23 5775.5476 ' . v " ." .. LSD t.01 16.9 Holtfreter s 21 X3° l8.9* t.05 - 13.6 Seminal vesicle xl-x30 - 16.9* 2 Test for Homogeneity of Variances s max./s2min. . 12.37 *Significant at the 12 level. 49 Appendix Table 3. Sperm incubated for 48 hours in Holtfreter's solution and seminal vesicle homogenate 7* FT Holtfreter's Solution; Seminal Vesicle Homggenate Insemination cleaved Y cleaved time total 3 1 Fert. 6 total 3 Z Fert. e 3 2 42 7.1 15.34 -55 8.6 16.43 1 minute 3 3 2 20 30 minutes -8- 14 3 22 22 L7- 37 8 37 94 56 ° ' 45 ' ' ANOVA Table source of variation df SS MS F Treatments 1 428.8656 428.8056 15.69* Times 1 394.6645 394.6645 14.44* Interaction 1 254.1385 254.1385 9.36* Error .4 109.2881 27.322 Total 7 1186.8967 ** LSD t.01 - 24.07 Seminal Vesicle x1-x30 - 25.32 t.05 - 14.51 Test for Homogeneity of variances szmax./s2min. - 29.2737 r 1' vvv fl W *Significant at the 52 level. **Significant at the 12 level. Appendix Table 4. 50 Sperm incubated for 48 hours in Holtfreter's solu- tion, seminal vesicle homogenate, brain homogenate and oviducal homogenate Holtfreter's Seminal Vesicle Brain 0viducal Solution* Homogenate Homogenate Homogenate Time ‘ a e O 6 1 min. 39.03110.4 28.031826 20.78;I-_18.63 2.86:7.0 10 min. 23.43:19.l 25.07:l4.9 29.65115.5 9.30:1l.0 15 min. 28.40:l7.3 26.10:il.2 33.60:l0.6 15.50118.8 20 min. 40.47:?2.2 45.12113.7 45.67116.6 13.37120.9 25 min. 28.3817.7 50.671§.l 37.58115.l 20.22:7.9 30 min. 42.8&t§.8 Sl.8818.4 37.141ll.‘ 10.32:12.l ANOVA Table source of variation df SS HS P Treatments 3 15526.8665 5175.6221 26.559l** Times 5 4677.7593 935.5518 4.8008** Interaction 15 4306.126? 287.0751 l.4731*** Error {120 23384.5467 194.8712 Total 143 47895.2994 * Each value is an average of six replicates and represents the average of eggs fertilized for of the arc sin equivalents of the percentages each replicate j; the standard deviation. ** Significant at the 12 level. * * *Significant at the 52 level. Appendix Table 5. 51 Basic statistics, regression statistics. and calcu- lation of higher order regression coefficients for seminal vesicle data sperm incubated for 48 hours in:_ Holtfreter's 'rSeminal Vesicle Brain Oviducalj Solution Homogenate Homogenate Homogenate Basic statistics 22 1215.51 1362.14 1286.36 429.93 222 49836.6699 59957.5872 53686.3662 12645.6748 zrzln 42957.3237 56389.5142 46985.6748 6189.4626 n 36 36 36 36 a 6 6 6 6 znx 606 666 666 666 m:2 13566 13566 13566 13566 zxczr) 21623.65 26373.35 22787.1 8479.5 Regression statistic; 2x2 3365.6 3365.6 3365.6 3365.6 21y 562.565 3443.9934 1133.3734 1242.345 33 gtpt 1916.6415 4849.9204** . 1626.5735 1655.6236 ss lin. 95.7577 3588.8322* 388.6642 466.9957 regress. ss dev. 1826.8838 315,272. 631.9693 588.6279 regress. 38 within 6878.6862 118.9357 6695.2914 5855.8635 by.‘ 6.1762 1.642 6.3429 0.3758 7 33.76 37.84 35.73 11.94 i 16.83 16.83 16.83 16.83 I inter. 36.8991 26.297 29.9661 5.6166 52 Appendix Table 5 (cont'd.) sperm incubated for 48 hours inzl. _# HoltfreterTs' Seminal Vesicle Brain 0viducal Solution Homogenate Homogenate Homogenate Y—' Calculation of higher order regression coefficients for seminal vesicle data. Data were coded for analysis. Regression coefficients only have been decoded. x2 - 5.5084 xzy - 196.3861 88 86v. - 186.4264 2 2 3 regress. (x ) - 57.6751 x y - 585.3866 3 2 * 88 within - 3568.073 (x ) - 545.3462 SS grps - 4849.9204 2 2 inter. - 32.8387 xx - 17.1692 ss lin. - 3931.9776** 3 regress. b ‘ - -4.2469 xx - 49.5461 7' 2 3 ss quad. - 113.6699 b x2 - 6.3857 x x - 174.9045 regress. y. b 3 - -6.6674 xy - 57.411 83 cubic - 624.4465* "‘ regress. a * Significant at the 52 level. **Significant at the 11 level. Appendix Table 6. 53 Effect of egg water on the fertilizebility of sperm incubated in seminal vesicle homogenate or Holtfreter's solution Holtfreter's Seminal Vesicle Holtfreter's Solution + Seminal.Vesicle Homogenate + Solution Egg water Homogenate Egg Water Time 8 8 8 8 l min. 44.28113.9 31.09:l$.1 33.14j§.7 40.48112.0 10 min. 40.68112.9 34.61:18.8 34.71110.5 43.7117.6 15 min. 43.04:§.6 42.97:8.4 35.05:l3.0 48.5514.6 20 min. 42.28:l4.2 39.62:ll.4 51.00:].2 52.0316.3 25 min. 49.79:9.6 37.73116.8 46.24102.5 52.01:;0.0 30 min. 40.68:9.8 49.56314.8 53.8319.3 55.05110.9 ANOVA Table source of variation df SS HS F Treatments ' (3°1‘f““‘ ° 1 688.4939 688.4939 5.3126* or seminal vesicles) Times 5 3150.3374 630.0674 4.86l8** Egg water (present or 1 23.4498 23.4498 0.1809 absent) Times vs 5 701.6723 140.3344 1.0828 Treatments Treatments vs 1 1090.2653 1090.2653 8.4128*** Egg Water 54 Appendix Table 6 (cont'd.) source of variation df SS HS F Times vs 5 629.1357 125.8271 0.9709 Egg Water Times vs Treat- ments vs Egg 5 946 .974 Water Error $3.; 15681 . 0315 Total 143 Bartlett's Test for Homogeneity of Variances 189 . 3948 1 . 4614 129. 5953 x2 - 23.6297 0.5