THESIS : LIBRARY Michigan Stat: University .54 This is to certify that the thesis entitled XENOGENOUS FERTILIZATION 0F SQUIRREL MONKEY AND GOLDEN HAMSTER OOCYTES presented by Francesco John DeMayo has been accepted towards fulfillment of the requirements for M.S. dggree in Phy51010qy £9 / 70% M w k; ‘/L {fizz ‘ v // é fiEZ/(j/ Major professor Date May 7, 1981 0-7639 Igflflfixus L $64 . ~3--i‘irlll OVERDUE FINES: 25¢ per day per item RETURNING LIBRARY MATERIALS: Place in book return to remove charge from circulation records XENOGENOUS FERTILIZATION OF SQUIRREL MONKEY AND GOLDEN HAMSTER OOCYTES By Francesco John DeMayo A THESIS Submitted to Michigan State University in partial fulfillment of requirements for the degree of MASTER OF SCIENCE Department of Physiology 1981 ABSTRACT XENOGENOUS FERTILIZATION OF SQUIRREL MONKEY AND GOLDEN HAMSTER OOCYTES by Francesco John DeMayo The ability of the rabbit oviduct to support the fertilization of squirrel monkey and hamster ova, namely, xenogenous fertilization, was studied. The following parameters were examined: sperm concentra- tion, day of rabbit pseudopregnancy, time of recovery and number of oocytes deposited into the rabbit oviduct. Hamster ova recovered from superovulated females were placed in the oviduct of day l, 2, 4, 7 or 10 pseudopregnant rabbits and the oviducts were inseminated. Epididymal sperm in concentrations of 106, 107, 108 or 109 sperm/m1 were tested. The ova were then recovered from the rabbit oviduct 28, 29, 30 or 32 hours later. Squirrel monkey ova recovered by laparoscopic follicular aspira- tion from gonadotropin stimulated ovaries were placed in the oviduct of l, 2 or 3 day pseudopregnant rabbits. These oviducts were insemi- nated with 106, 107 or 108 sperm/m1 and recovered 12 to 98 hours after insemination. Fertilization of squirrel monkey and hamster ova were judged by the presence of 2 polar bodies and 2 pronuclei or cleavage. Xenogenous fertilization rates for squirrel monkey and hamster ova were 25/79 (31.6%) and 119/198 (60.1%) respectively with 3/25 Franceso John DeMayo (12.0%) and 38/119 (31.9%) of the fertilized squirrel monkey and hamster ova cleaving, respectively. Day of rabbit pseudopregnancy and sperm concentration had no effect on the fertilization or cleavage rates of hamster and squirrel monkey ova. Cleavage time was observed to be at least 28 hours for hamster ova xenogenously fertilized and at least 31 hours for xenogenously fertilized squirrel monkey ova. These developmental times correspond to normal i§_gizg_developmental rates. The number of hamster ova deposited into the rabbit oviduct had no effect on fertilization. To my mother ii ACKNOWLEDGEMENTS I wish to express my gratitude to Dr. W. R. Dukelow, my advisor, for the opportunities, advice and friendship given to me during this work. I would like to thank the members of my committee, Drs. E. M. Convey, H. Ozaki and T. Adams for their assistance and guidance. Finally, I wish to express my appreciation to Mr. Patricio Hirst, Mrs. Bonnie Cleeves and everyone else at the Endocrine Research Unit for their assistance and making this work enjoyable. iii TABLE OF CONTENTS LIST OF TABLES INTRODUCTION- LITERATURE REVIEW In Vitro Fertilization 'Msnogametic Fusion Blastomere Separation Xenogenous Embryo Culture Xenogenous Fertilization MATERIALS AND METHODS Animal Care Xenogenous Fertilization Procedure Medium Ovum Collection Sperm Collection Rabbit Surgery Embryo Recovery Statistical Analysis RESULTS DISCUSSION SUMMARY AND CONCLUSIONS BIBLIOGRAPHY APPENDICES A. Publications by the Author B. Vita iv Page N -|-\\OO\O\N 17 18 18 18 20 21 21 22 23 29 33 35 A1 B1 Table 10 LIST OF TABLES Page In_vitro fertilization of mammalian ova The viability of embryos produced by blastomere sepa— ration The culture of laboratory animal ova in the rabbit oviduct The culture of domestic animal ova in the rabbit ovi- duct Xenogenous fertilization of mammalian ova The contents of the medium used in the manipulation of squirrel monkey and hamster ova Xenogenous fertilization of hamster and squirrel monkey ova deposited into rabbit oviducts during different stages of pseudopregnancy The effects of sperm concentrations on the xenogenous fertilization of hamster and squirrel monkey ova ------- Xenogenous fertilization and development of hamster and squirrel monkey ova at varying times of recovery after insemination The effects of the number of oocytes deposited in the pseudopregnant rabbit oviduct on xenogenous fertiliza- 12 13 15 19 24 26 27 28 tion of hamster ova INTRODUCTION The study of the fertilization process can benefit both the pre— vention of unwanted human pregnancies and the productivity of domestic animals raised for food consumption. The ability to efficiently gene— rate normal embryos has application to: 1) some types of human infer— tility; 2) embryo transfer in domestic and endangered species; 3) screening of potentially teratologic, mutagenic and toxicologic com— pounds; 4) testing of fertility and infertility drugs and 5) scien- tific research. Presently there are five possible methods for the generation of mammalian embryos other than natural fertilization: l) in vitgg fertilization; 2) monogametic fusion; 3) blastomere separation; 4) parthanogenic activation and 5) xenogenous fertilization. The latter is the fertilization of oocytes with homologous sperm in the oviduct of a heterologous species. The term xenogenous comes from the Greek roots "xeno" (foreign) and "genous" (nuture). Possibly the oviducts of the pseudopregnant rabbit would constitute a suitable environment for normal fertilization and development of hamster and squirrel monkey gametes. This possibility was investigated. LITERATURE REVIEW In Vitro Fertilization The discovery of the need for sperm to be incubated in the female reproductive tract prior to fertilization, i.e. capacitation (Chang, 1951; Austin, 1951) was followed by many reports of in vitrg fertili- zation (Table 1). Live births have resulted from embryo transfer of igLXi££g_ferti1ized ova has been accomplished in mice, rats, rabbits (Brackett, 1979) and man (Steptoe and Edwards, 1978; Lopata g£_§13, 1980). Ig_vitro fertilization of golden hamster GMesocricetus auratus) ova was first accomplished by incubating ova and oviductal components with sperm recovered from the uteri of mated females or the epidydimi of males. Sperm, collected from the uterus, fertilized a higher percentage of the hamster ova (Yanagimachi and Chang, 1963, 1964). This higher fertilization rate can be attributed to the female tract and not male accessory sex gland secretions, since male secretions decreased the fertilizability of epididymal sperm in 31353 (Tsunoda and Chang, 1977). The penetration of ova by sperm was slower in git£9_than in vizg_(Yanagimachi, 1966) and development of hamster ova fertiilized iglzi££g_was halted at the two cell stage (Yanagimachi and Chang, 1964). A scanning electron microscopic examination of the ultrastructure of hamster ova fertilized in_vitro or in_vivo showed TABLE 1 In_Vitro Fertilization of Mammalian Ova Species Investigators Guinea Pig Rabbit Mouse Rat Gerbil Golden Hamster Chinese-Hamster Cat Dog Pig Sheep Cow Squirrel Monkey Baboon Man Yanagimachi, 1970b, 1972 Chang, 1954; Dauzier and Thibault, 1959 Whittingham, 1968; Pavlok, 1968 Toyoda and Chang, 1974 Noske, 1972 Yanagimachi and Chang, 1963, 1964 Pickworth and Chang, 1969 Hamner g£_al,, 1970; Bowen, 1977 Mahi and Yanagimachi, 1976 Harns and Smith, 1970; Iritani g£_§1,, 1975; Iritani at al., 1978 Dauzier and Thibault, 1959; Kraemer, 1966; Biondioli and Wright, 1980 Bregalla gt 31., 1974; Iritani and Niwa, 1977; Bracket gt 31., 1977, 1978, 1980 Cline gt a1., 1972, Johnson gt_§1,, 1972; Gould gnglg, 1973; Kuehl and Dukelow, 1975, 1979 Kraemer 35 a1., 1979 Blandau, 1980 4 that the mode of sperm binding to the vitelline membrane was differ— ent. For example, EE;ZEE£22 the anterior tip of the sperm appears to bind to the vitellus first while in vivg_the post acrosomal collar binds to the vitellus first (Shalgi and Phillips, 1980). Despite these differences there have been no other reports citing differences in the early events of fertilization between in vizg_and EE;XEEEQ systems and the in;vi££g_system for hamster fertilization has been extensively used to study sperm capacitation and other processes in fertilization. In vitrg fertilization of hamster ova can occur in an environment where the osmolality ranges from 232 to 452 mosmolal with maximum fertilization at 292 to 390 mosmolal (Miyamoto and Chang, 1973). '13 zitrg_fertilization of hamster ova can occur in media ranging in pH from 6.7 to 8.7 with optimum fertilization at a pH of 6.8 to 8.2 (Miyamoto §£_§1,, 1944). Sperm concentration is important for in zitrg_fertilization. The optimum sperm concentration for EE.XE££2. fertilization of hamster ova was reported to be 2x107 sperm/ml with declining fertilization rates at greater or lesser sperm concentra— tions (Talbot g£_al,, 1974). Ovum concentration also affects the rate of in zi££9_fertiliza- tion in a defined medium (Niwa at 31., 1980). The importance of ovum concentration on in XEEEE fertilization is not due to the ova them— selves but rather their follicular constituents (Barros, 1968). Ferti- lization rates of hamster ova that were washed to remove all the follicular fluid was decreased (Barros and Austin, 1966, 1967) and hamster ova devoid of the cumulus cells were not fertilized (Gwatkin 5 gt 31., 1972). The contribution of the follicular fluid, cumulus cells and cumulus matrix to fertilization can be mimicked with: mouse or rat oviductal fluid (Barros, 1968); heat inactivated bovine folli— cular fluid (Gwatkin and Anderson, 1969; Yanagimachi, 1969a), mouse or rat follicular fluid (Yanagimachi, 1969b); and heat inactivated ham- ster CMiyamoto and Chang, 1972), rat, guinea pig, rabbit, bull or human sera (Barros and Garavagno, 1970; Yanagimachi, 1970a). There- fore, for 39L3§££g_fertilization of hamster ova to occur, there must be present a nonspecific factor(s) to facilitate sperm capacitation. This factor may be an albumin-like compound, since hamster sperm have been capacitated 32:22E52 in defined media to which bovine serum albumin was added. Albumin, however, did not cause the acrosome reaction in these spermatozoa and other factors may be involved (Bavister, 1969, 1973). Sperm will attach to the zona pellucida of squirrel monkey ova igLvi££g_(Johnson gt 31., 1972). Others have demonstrated that ferti— lization of squirrel monkey will occur ig;vitrg_and these fertilized ova will develop to the 2 cell (Cline gt a1., 1972; Gould 35 31., 1973), 4 cell (Kuehl and Dukelow, 1975) and 8 cell stages (Kuehl and Dukelow, 1979). The time of development of in gitrg fertilized ova of squirrel monkeys have been shown to be similar to that occurring in vixg_in the baboon, rhesus monkey and human embryos, i.e. second polar body extrusion 6-22 hours after insemination; first cleavage 20—40 hours after insemination; second cleavage 46-52 hours after insemi— nation and third cleavage 52—74 hours after insemination (Kuehl and Dukelow, 1979). Monogametic Fusion The production of embryos by fusion of two homologous oocytes is termed monogametic fusion. This has been accomplished in mice by fusing two zona-free oocytes using inactivated sendai vivus to acti— vate the fusion process (Soupart gt_al,, 1977, 1979). Zena-free mouse ova were incubated for 3 minutes with ultraviolet light inactivated sendai vivus. The two ova were then placed together to allow fusion. One hour after fusion occurred it was observed that the cortical granules were extruded from the fused oocytes and meiosis resumed. Five hours after fusion, two second polar bodies were extruded (Sou— part, 1977). When fused mouse ova were cultured for 5.5 days the zygotes developed to the blastocyst stage with 120 to 130 nuclei. The development of the zygote was dependent on the three dimensional integrity of the blastomeres. If this was lost, development ceased at the eight cell stage. When three dimensional integrity was maintained by placing the blastomeres in an empty rabbit zona pellucida, develop- ment proceeded (Soupart 3£_213, 1979). Thus, the fusion of two oocytes restored the diploid number of chromosomes and resulted in the events similar to those which occur after sperm—ovum fusion during the fertilization process. Blastomere Separation Separation of the blastomeres of two cell rat embryos and trans— fer of these blastomeres to pregnant recipients resulted in the development of those embryos to the egg cylinder stage (Nicholas and Mall, 1942). In rabbits (Seidel, 1952) and mice (Tarkowski, 1959) 7 destruction of one of the blastomeres of a two cell embryo and subse- quent transfer of the remaining blastomeres can result in the birth of live offspring. Thus, each blastomere of an early developing zygote possesses the potential for complete development. The separation of the blastomeres of an early developing zygote could be used to in— crease the number of embryos produced by natural fertilization. Blastomere separation and transfer of the resultant monozygotic twins has been accomplished in mice (Moustafa and Hahn, 1978), sheep (Willadsen, 1979, 1980) and cattle (Willadsen g£_a1,, 1981). Early developing embryos are collected and the zona pellucida are enzy- matically or mechanically removed. The blastomeres are then mechani— cally separated with fine needles and placed in empty zona pellucida. In mice the embryos were transferred to recipient females (Moustafa and Hahn, 1978) but in the domestic species the embryos were embedded in a small cylinder of 1% agar in 0.9% NaCl. This agar chip is then encapsulated in a larger 1.2% agar shell. The embryos are then allowed to develop to the late morula or early blastocyst stage in a ligated oviduct of a ewe. The embryos are recovered and transferred to synchronized recipients (Willadsen, 1981). The results of blasto— mere separation and transfer of these species is shown in Table 2. When damage due to micromanipulation was minimized and the embryos were separated into sets of blastomeres of equal cell number, the success of transfer did not differ significantly from normal embryo transfer results (Willadsen, 1980; Willadsen gt_al,, 1981). 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