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The effects of superovulation on ovarian function and embryo development in rats Yun, Young Won 1989

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THE EFFECTS OF SUPEROVULATION ON OVARIAN FUNCTION AND EMBRYO DEVELOPMENT IN RATS By YOUNG WON YUN D.V.M., Seoul National University, 1982 M.Sc., Seoul National University, 1984 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Human Reproductive Biology Programme) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August 10, 1989 <c) Young Won Yun, 1989 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Obste t r ics and Gynaecology The University of British Columbia Vancouver, Canada Date A u g u s t 1 7 , 1 9 8 9 DE-6 (2/88) i i ABSTRACT This study examined primary defects and related mechanisms leading to impaired f e r t i l i t y following superovulation with exogenous gonadotropin. Experiments were designed to address the ovulatory response, oocyte quality (gross morphology and nuclear maturation) and embryo development with reference to ovarian steroid and circulating LH levels in immature rats treated with 4, 20 or 40 IU pregnant mare's serum gonadotropin (PMSG). Superovulatory doses (20 and 40 IU) of PMSG induced the f i r s t ovulation as early as 24 hr and prolonged multiple ovulations with two increases: one before 36 hr and the other after 48 hr, while a low dose (4 IU) of PMSG induced a burst of ovulation between 60 and 72 hr. The biphasic superovulatory response was paralleled with two distinct luteinizing hormone (LH) peaks in circulating blood. Early ovulation could be, in part, due to the exogenous LH activity derived from high doses of • PMSG. The i n i t i a l prolonged elevation of serum LH before 54 hr resulted from actual cross-reaction of the injected PMSG with LH antibody in the assay and was dose-dependent, while a precipitous second elevation between 54 and 60 hr resulted primarily from an endogenous LH surge. Additionally, the observation of a marked ovulation inhibition by using a progesterone antagonist (RU 486) and an estrogen antagonist (tamoxifen) indicated the active participation of progesterone as well as estrogen i n the PMSG-induced ovulation. A dose-dependent increase in the percentage of degenerate oocytes following superovulation was noticed from 36 hr onwards and positively correlated with the timing of increased levels of ovarian and serum ) androgens. Furthermore, superovulated oocytes with even normal appearance displayed substantially d i f f e r e n t stages of nuclear maturation varying from prophase I to metaphase I I , while a majority of control oocytes (4 IU PMSG) was synchronized at metaphase I I . The incidence of mei o t i c a l l y aberrant oocytes i n superovulated rats was cl o s e l y associated with abnormal f o l l i c u l a r steroidogenesis, i . e . a marked a l t e r a t i o n of f o l l i c u l a r contents of progesterone and p a r t i c u l a r l y androgens and/or a consistent disruption of sequential changes i n o v e r a l l r a t i o s of androgens/17B-estradiol, progesterone/17B-estradiol and progesterone/androgens. Administration of an androgen antagonist (flutamide) i n superovulated rats s i g n i f i c a n t l y reduced the proportion of degenerate oocytes and c e l l u l a r degeneration of preimplantation embryos, and improved the developmental potential (embryo cleavage), at least i n part, v i a decreased production of ovarian androgens. These results r e f l e c t a si g n i f i c a n t role of augmented ovarian androgen secretion i n the perturbation of oocyte quality and subsequent embryo development following superovulation. However, the pharmacological effects of flutamide were res t r i c t e d to early stage (Day 2) of pregnancy. Therefore, the actual improvement of the q u a l i t y or development of early embryos by flutamide was ascribed to a substantial reduction of abnormal oocytes before f e r t i l i z a t i o n . The results of an experiment using the RU486 and tamoxifen indicated that estrogen, but not progesterone, influenced oocyte q u a l i t y i n superovulated rats. RU486 treatment did not af f e c t the oocyte morphology, but tamoxifen treatment was associated with a marked increase i n the percentage of degenerate oocytes. iv The results of this research provide direct evidence of atypical ovulations of superovulated oocytes with premature or asynchronous nuclear maturation as a primary defect leading to impaired f e r t i l i t y including abnormal embryo development, and demonstrate a close relationship between meiotically aberrant oocytes and abnormal f o l l i c u l a r steroidogenesis in superovulated rats. Increased levels of ovarian and circulating androgens preceding f e r t i l i z a t i o n were particularly implicated i n the perturbation of oocyte quality and embryo development following superovulation. Finally, the process of multiple ovulations induced by superovulatory doses of PMSG appears to involve the exogenous LH activity of PMSG as well as active participation of progesterone and estrogen. V TABLE OF CONTENTS Page Abstract i i Table of Contents v L i s t of Tables v i i L i s t of Figures v i i i Glossary x Acknowledgments x i i i INTRODUCTION 1 REVIEW OF LITERATURE 4 I. Induction of Superovulation . . . . 4 II. Biological and Biochemical Properties of PMSG 7 III. Endocrine Aspects of Superovulation 9 A. General 9 B. Gonadotropin Secretion and Induced Ovulation . . . . 11 C. Ovarian Steroids and Oocyte Maturation 14 IV. Developmental Capability of Superovulated Oocytes . . . . 17 A. General 17 B. Genetic Condition 20 C . Oocyte Quality . . . : . . . . . . , , „ . . » , „ . . . • . . 21 D.. Maternal Environment 24 MATERIALS AND METHODS . 28 I. Animals 28 II. Experimental Design (Protocols 1-5) 28 v i I I I . Assessment of Ovulation, Oocyte Morphology and Embryo Development 33 IV. Assessment of Oocyte Nuclear Maturation 34 V. Histological Examination 37 VI. Determination of Hormones 37 A. 17B-estradiol, Progesterone and Androgens 37 B. Androgenic Steroids 40 C. LH 44 VII. S t a t i s t i c a l Analysis 45 RESULTS 46 I. Oocyte Morphology, Ovulatory and Steroid Responses (Protocol 1) 46 II. Follicular Steroid Contents and Oocyte Maturation (Protocol 2) 61 III. Serum LH Response (Protocol 3) 68 IV. Effects of Flutamide on Oocyte Morphology and Embryo Development (Protocol 4) 73 V. Effects of RU486 and Tamoxifen on Ovulation and Oocyte Morphology (Protocol 5) 84 DISCUSSION o . . . . . . . . „ • B 92 SUMMARY AND CONCLUSIONS . . . . . . . . . . . . 116 BIBLIOGRAPHY 121 v i i L I S T OF TABLES Table Page I. Radiochemical identification of androstenedione, 5a-dihydrotestosterone and testosterone 43 II. Effects of superovulatory treatment on nuclear maturation of oocytes recovered from oviducts in rats . . 65 III . Oocyte recovery and ovarian weights 72 hr after administration of PMSG to immature rats 72 IV. Effects of flutamide on recovery and normality of oocytes from oviducts of 40 IU PMSG-injected rats . . . . 74 V. Effects of flutamide on recovery and developmental capacity of superovulated ova in preimplantation stage . . 78 VI. Effects of flutamide on normality and cleavage of superovulated ova from both oviducts and uterine horns in preimplantation stage . . 80 VII. Effects of RU486 and tamoxifen on recovery and normality of oocytes from oviducts, and ovarian weight in 4 IU PMSG-injected rats 85 VIII. Effects of RU486 and tamoxifen on recovery and normality of oocytes from oviducts, and ovarian weight in 40 IU PMSG-injected rats 88 v i i i LIST OF FIGURES Figure Page 1. Diagrammatic representation of preovulatory nuclear maturation stages in the rat oocyte 36 2. Separated steroids on TLC s i l i c a gel-coated plate, developed in a solvent system of chloroform: methylene chloride: diethyl ether (40:40:20,v:v:v) 41 3. Percentage of rats ovulated at different time periods after injection of 4, 20 or 40 IU PMSG 47 4. Recovery of oocytes from oviducts per rat at different time periods after injection of 4, 20 or 40 IU PMSG . . . 48 5. Morphology of normal and abnormal oocytes recovered from oviducts after treatments with a control dose (4 IU) and superovulatory doses (20 and 40 IU) of PMSG, respectively 49 6. Percentage of abnormal oocytes recovered from oviducts of superovulated rats at different time periods after injection of 20 or 40 IU PMSG 50 7. Ovarian histology obtained at 24 hr after 20 IU PMSG treatment 52 8. Follicular and oocyte morphology of control ovary obtained at 48 hr after 4 IU PMSG treatment 53 9. Follicular and oocyte morphology of superovulated ovary obtained at 48 hr after 40 IU PMSG treatment . . . . 54 10. Changes in ovarian 17B-estradiol, progesterone and androgens after administration of 4, 20 or 40 IU PMSG to immature rats 55 11. Changes in serum 17B-estradiol, progesterone and androgens after administration of 4, 20 or 40 IU PMSG to immature 12. Correlation between the percentage of abnormal oocytes and content of ovarian androgens from 24 to 60 hr after injection of 20 or 40 IU PMSG to immature rats 59. 13. Correlation between the percentage of abnormal oocytes and concentration of serum androgens from 24 to 60 hr after injection of 20 or 40 IU PMSG to immature rats . . . 60 ix 14. Changes in f o l l i c u l a r contents of 17fl-estradiol, progesterone and androgens after administration of 4 or 20 IU PMSG to immature rats 62 15. Changes in ratios of androgens/176-estradiol, progesterone/ 176-estradiol and progesterone/androgens in f o l l i c u l a r fluid after administration of 4 or 20 IU PMSG to immature rats 63 16. Typical configuration of nuclear maturation of oocytes recovered from oviducts after PMSG 66 17. Changes in serum LH concentrations after administration of 4 or 20 IU PMSG to immature rats 69 18. LH assay of PMSG and serum pools, showing the cross-reactivity of the injected PMSG with LH antibody . . . . . 71 19. Ovarian contents of androgens, 17B-estradiol, and progesterone after treatment with vehicle or flutamide in 40 IU PMSG-injected rats 75 20. Serum levels of androgens, 176-estradiol, and progesterone after treatment with vehicle or flutamide in 40 IU PMSG-injected rats 77 21. Serum levels of androgens, 176-estradiol, and progesterone on Days 2 to 5 of pregnancy after administration of 4 IU PMSG alone and 40 IU PMSG plus vehicle or flutamide to immature rats 81 22. Contents of serum androgenic steroids, androstenedione, testosterone, and 5a-dihydrotestosterone after administration of 4 IU PMSG alone and 20 IU PMSG plus vehicle or flutamide to immature rats 82 23. Serum levels of 176-estradiol, progesterone and androgens 24 hr after treatment with vehicle, RU486 or tamoxifen in 4 IU PMSG-injected rats 87 24. Serum levels of 176-estradiol, progesterone and androgens before or after treatment with vehicle, RU486 or tamoxifen in 40 IU PMSG-injected rats 90 X GLOSSARY B/Bo Antigen binding ratio per maximum binding °C Degrees Celsius 14c A radioactive isotope of carbon Ci Curie (= 3.7xl0 1 0 disintegrations per second) CIN Color index number CL Corpora lutea cm Centimetre cpm Radioactive counts per minute CV Coefficient of variation DPBS Dulbecco's phosphate-buffered saline dpm Radioactive disintegrations per minute e.g. For example (exempli gratia) FSH F o l l i c l e stimulating hormone g Gram Gly Glycine 3H Tritium, a radioactive isotope of hydrogen hCG Human chorionic gonadotropin hMG Human menopausal gonadotropin hr(s) Hour(s) 125j A radioactive isotope of iodine i.d. Inner diameter i.e. That i s (id est) IU International unit IVF In vitro f e r t i l i z a t i o n x i kg Kilogram L:D Light:Dark, e.g. 12L:12D = 12 hours light:12 hours dark LH Luteinizing hormone LHRH LH releasing hormone mCi M i l l i c u r i e mg Milligram min Minute ml M i l l i l i t r e mm Millimetre mmol Millimole n Number ng Nanogram NSB Non-specific binding OAAD Ovarian ascorbic acid depletion pg Picogram pH -Log H+ concentration in a solution Phe Phenylalanine PLSD - Protected least significant difference p.m. Afternoon (post meridiem) PMSG Pregnant mare's serum gonadotropin Pro Proline RIA Radioimmunoassay rpm Revolutions per minute SE Standard error sec Second(s) x i i SEM Standard error of means Ser Serine Sp. act. Specific activity TLC Thin layer chromatography TSH Thyroid stimulating hormone v:v:v Volume ratio vs Versus v l Microlitre ym Micrometre yM Micromolar A Change in two values x i i i ACKNOWLEIX^ENTS I would l i k e to g r a t e f u l l y acknowledge my supervisor, Dr. Y.S. Moon, for providing me the opportunity to pursue t h i s study and for his continuous support, patience and encouragement throughout the time spent i n t h i s laboratory. I am also indebted to Drs. B. Ho Yuen and A.J. Duleba for their invaluable guidance, c r i t i c a l review and professional advice. I also wish to express my appreciation to the members of my research supervisory committee, Drs. P. Hahn and R. Rajamahendran for thei r c areful reading of the thesis and t h e i r interest i n t h i s work. Special thanks are also extended to Mrs. N.M. Duleba, Mr. F.H. Yu and Dr.. R.F. Weick for t h e i r enthusiastic and technical assistance and to Dr. D.T. Armstrong for the generous, g i f t of steroids antisera. Miss M.R. Ungless took much care i n the typing of this manuscript. This study was supported by the grants of Drs. Y.S. Moon and B. Ho Yuen from the Medical Research Council of Canada and the B r i t i s h Columbia Health Care Research Foundation. - 1 -INTRCOUCTION Induction of superovulation with exogenous gonadotropins i s a well-established technique i n the livestock industry for enhanced commercial production of genetically superior individuals and in human IVF and embryo replacement programmes for increased yield of oocytes. This technique has also been ut i l i z e d to f a c i l i t a t e basic studies in reproductive biology including ovulation, f e r t i l i z a t i o n , embryo development and implantation. Despite i t s widespread application, the use of superovulatory doses of gonadotropin has been reported to result i n reduced f e r t i l i t y in large domestic animals (Betteridge and Moore, 1977; Evans and Robinson, 1980; Armstrong and Evans, 1983) and in small laboratory animals (Adams, 1960; Beaumont and Smith, 1975; Miller and Armstrong, 1981a). The impairment i s characterized by the highly variable yield of viable embryos, and the frequent recovery of retarded or abnormal embryos (Betteridge, 1977). Results of numerous studies using immature rats indicate that low doses (4-8 IU) of PMSG induce a synchronized ovulation within 72 hr through e l i c i t i n g an endogenous LH surge between.54 and 57 hr after injection of the gonadotropin (Sorrentino et a l . , 1972; Kostyk et a l . , 1978) and produce normal pregnancy without an increased embryonic or fetal wastage (Nuti et a l . , 1975). The patterns of circulating steroid hormones (17fl-estradiol and progesterone) and LH during the 24 hr preceding 72 hr ovulation (Meyer et a l . , 1971; Wilson et a l . , 1974) and the temporal relationship between LH levels and oocyte maturation (Hillensjo et a l . , 1974) in this regimen are comparable to those observed on the days of proestrus and estrus in cycling adult rats (Barraclough et a l . , 1971; Linkie and Niswender, 1972). Thus, - 2 -PMSG-treated immature rat i s a model well suited for studying the mechanisms and effects of superovulation. In immature rats, a single injection of superovulatory doses (16-40 IU) of PMSG leads to a great reduction in f e r t i l i z a t i o n rates (Walton et a l . , 1983), a substantial loss and degeneration of preimplantation embryos (Miller and Armstrong, 1981b), and a partial or complete failure of implantation (Miller and Armstrong, 1982). This impairment has previously been attributed to the va r i a b i l i t y in timing of ovulation (Evans and Armstrong, 1983; Walton et a l . , 1983), excessive stimulation of the genital tract (Miller and Armstrong, 1981b) and resultant asynchrony between embryonic and uterine development by disturbances in the oviductal and uterine environments (Walton and Armstrong, 1982). However, the major factor(s) and c r i t i c a l time involved in the low f e r t i l i t y following superovulation are not f u l l y understood. Primary defects could occur in the process of oocyte maturation and ovulation prior to f e r t i l i z a t i o n by hyperstimulation of ovarian tissues or acceleration of f o l l i c u l a r development because of the prolonged biochemical action of PMSG reported by Schams et a l . (1978). Superovulatory dose of PMSG in immature rats has been found to induce an unusually precocious and prolonged ovulatory response (Walton et a l . , 1983). It appears that a portion of superovulated oocytes i s derived from abnormal f o l l i c l e s , which would not ovulate at a regular cyclic estrus (Braw and T s a f r i r i , 1980; Fleming, 1982). Administration of superovulatory doses of PMSG to sheep alters the pattern of steroid hormones secreted by the f o l l i c l e s cultured i n  vitro and results in premature activation of oocytes during maturation (Moor et a l . , 1985). In addition, alterations in the normal pattern of steroid - 3 -secretion during f o l l i c u l a r maturation induce intracellular changes in sheep oocytes expressed as gross abnormalities at f e r t i l i z a t i o n (Moor et a l . , 1980b). It has been suggested that f o l l i c u l a r steroid microenvironment i s a key factor determining f o l l i c u l a r growth and oocyte maturation (McNatty and Baird, 1978). This study was done in an attempt to examine the primary defects and possible mechanisms related to i n f e r t i l i t y in immature rats following superovulation with PMSG. Experiments were carried out to investigate: (1) Time course of ovulatory response and oocyte morphology with reference to steroid hormone levels in ovarian tissues and peripheral blood during different time periods of treatments with .4, 20, or 40 IU PMSG; (2) Follicular steroid contents and oocyte maturation following PMSG treatments; (3) Circulating LH response to PMSG treatments; (4) Effects of steroid antagonists (flutamide, RU486 and tamoxifen) on ovulatory response and oocyte morphology and/or developmental potential of preimplantation embryos after treatments with PMSG. - 4 -REVIEW OF LITERATURE I. Induction of Superovulation. More than 60 years ago, the discovery of pituitary and placental gonadotropins led to an attempt to increase the number of mammalian oocytes capable of f e r t i l i z a t i o n and normal development. The phenomenon of superovulation was i n i t i a l l y described by the studies of Smith and Engle (1927) and Engle (1927) in which ovulation could be induced i n adult rats and mice by the daily implantation of anterior pituitary tissues. This procedure produced excessive numbers of oocytes (20-48 oocytes per animal) and implantation sites (19-29) in mice treated with mouse pituitary implants. Cole (1936) further simplified the procedure with a single injection of unfractionated pregnant mare's serum which could induce the ovulation of up to 54 oocytes. Using this simplified procedure, a maximum of 33 embryos and 23 offsprings in one l i t t e r was obtained (Cole, 1940). As regards the optimal age of experimental animals, i t has been found that female rats in the period immediately before sexual maturity were more satisfactory for this purpose than adults (Cole, 1937). Under optimal conditions, 80 or more oocytes could be obtained from prepubertal mice (Gates, 1971), while 30 to 40 oocytes ovulated from mature mice (Fowler and Edwards, 1957; Biggers et a l . , 1971). Two possible reasons may explain the varying sensitivity to exogenous gonadotropins between prepubertal and mature animals; (1) the number of large f o l l i c l e s capable of responding to gonadotropins in mature mice i s lower than that found in the ovaries of immature mice (Jones and Krohn, 1961), and (2) i t i s generally d i f f i c u l t to synchronize the time of administration of exogenous gonadotropins with - 5 -changes of the normal estrous cycle (McLaren and Michie, 1959; Edwards et a l . , 1963). Superovulation technique in sexually immature rodents appears to be more convenient, economical, and less time-consuming than spontaneous ovulation in mature donors. When a large number of pregnant animals are required for experimental purposes, we must either obtain the pregnant females from commercial distributors, or maintain enormous colonies of adult animals for mating. Both are very expensive and the latter necessitates laborious and time-consuming examination of vaginal smears. Thus, immature females in which synchronous ovulation can be induced by a single injection of PMSG may be a suitable model or substitute for adult animals in basic studies of reproduction. It has been suggested that both the amounts of FSH and LH play a central role in ovulation and superovulation (Pincus, 1940; Evans and Simpson, 1940; Rowlands, 1944; Bodemer et a l . , 1959) and FSH appears to limit the a b i l i t y of the f o l l i c l e to respond to LH (Cole, 1936; Pincus, 1940; Willett et a l . , 1952; Payne and Runser, 1958). Hisaw (1947) emphasized the requirement of both adequate amount and optimum period for FSH to act before LH to ensure ovulation induction. Thus, the use of combination of PMSG, primarily a long-acting FSH-like hormone preparation, and hCG has been developed as a means of superovulation. The optimum interval between the injection of PMSG and hCG was reported to be 56 hr in the rat (Rowlands, 1944; Zarrow et a l . , 1958), 40 hr in the mouse .(Gates and Runner, 1957; Fowler and Edwards, 1957) and 54 to 56 hr in the hamster (Bodemer et a l . , 1959). However, in later reports, hCG has been phased out of the two consecutive gonadotropin treatment regimens, since endogenous LH - 6 -s e c r e t i o n a n d / o r exogenous L H - l i k e a c t i v i t y o f h i g h d o s e o f PMSG a r e t h o u g h t t o be s u f f i c i e n t t o e f f e c t s u p e r o v u l a t i o n i n f e m a l e s t r e a t e d w i t h PMSG a l o n e (Whyman e t a l . , 1 9 7 9 ; Newcomb e t a l . , 1 9 7 9 ; M i l l e r and A r m s t r o n g , 1 9 8 1 b ) . A maximum o v u l a t o r y r e s p o n s e i n d u c e d b y PMSG w i t h o r w i t h o u t hCG h a s b e e n f o u n d t o be c o m p a r a b l e i n immatu re r a t s a t t h e a g e s o f 28 t o 30 d a y s , a l t h o u g h t h e s e n s i t i v i t y o f immatu re r a t s t o a s i n g l e i n j e c t i o n o f PMSG was l o w e r t h a n t h a t o f two c o n s e c u t i v e g o n a d o t r o p i n t r e a t m e n t s b e f o r e o r a f t e r t h e s e ages ( Z a r r o w and Q u i n n , 1 9 6 3 ) . T h e r e i s a l s o c o n s i d e r a b l e e v i d e n c e t h a t i n d u c t i o n and s y n c h r o n i z a t i o n o f t h e f i r s t e s t r u s , o v u l a t i o n , m a t i n g and p r e g n a n c y c a n be e f f i c i e n t l y i n i t i a t e d b y t h e i n j e c t i o n o f a s i n g l e d o s e o f PMSG i n immature r a t s ( S t r a u s s a n d M e y e r , 1 9 6 2 ; McCormack and M e y e r , 1 9 6 3 ; Z a r r o w and Q u i n n , 1 9 6 3 ) . The p o t e n t i a l o f f u l l n o r m a l d e v e l o p m e n t o f o o c y t e s o r embryos o b t a i n e d by s u p e r o v u l a t i o n i s o f o b v i o u s i m p o r t a n c e t o b o t h r e s e a r c h e r s a n d s t o c k b r e e d e r s . S u p e r o v u l a t i o n w i t h PMSG h a s b e e n a c h i e v e d i n numerous s p e c i e s . I n t h e r a t ( C o l e , 1 9 3 7 ; S a t o , 1 9 6 2 ) , mouse ( F o w l e r and E d w a r d s , 1 9 5 7 ; Edwards and F o w l e r , 1 9 5 9 ; W i l s o n and Z a r r o w , 1 9 6 2 ) , r a b b i t ( K e n n e l l y and F o o t e , 1 9 6 5 ; S e i t z e t a l . , 1 9 7 0 ) , h a m s t e r ( G r e e n w a l d , 1 9 6 2 ) , cow ( H a f e z and R a j a k o w s k i , 1 9 6 4 ; F o o t e and Onuma, 1 9 7 0 ) , and p i g ( H u n t e r , 1 9 6 6 ; D z i u k , 1 9 6 8 ; L o n g e n e c k e r and D a y , 1 9 6 8 ) , many o o c y t e s a f t e r s u p e r o v u l a t i o n c o u l d f e r t i l i z e and i m p l a n t t o p r o d u c e s u p e r p r e g r i a n c y . The economic b e n e f i t s o f p r o d u c i n g g r e a t e r numbers o f o f f s p r i n g J r o m g e n e t i c a l l y s u p e r i o r a n i m a l s h a s p r o m p t e d t h e l i v e s t o c k i n d u s t r y t o u s e t h i s s u p e r o v u l a t i o n t e c h n i q u e i n a t t e m p t s t o o b t a i n s u p e r p r e g n a n c y o r t o m a x i m i z e t h e r e p r o d u c t i v e p o t e n t i a l o f a g i v e n f e m a l e i n d i v i d u a l ( F o o t e a n d Onuma, 1 9 7 0 ; Saumande a n d C h u p i n , - 7 -1 9 7 7 ; B a k e r and J i l l e l l a , 1 9 7 8 ; B o l a n d e t a l . , 1 9 7 8 ) . The more r e c e n t i m p o r t a n c e i n human r e p r o d u c t i o n , where s u p e r o v u l a t i o n u s i n g hMG e q u i v a l e n t t o PMSG has b e e n u s e d f o r t h e t r e a t m e n t o f o v a r i a n d y s f u n c t i o n (Wyshak , 1 9 7 8 ; Hack and L u n e n f e l d , 1979) and f o r I V F and embryo t r a n s f e r programme ( L o p a t a e t a l . , 1 9 7 8 ; E d w a r d s , 1 9 8 1 ; Moon e t a l . , 1 9 8 5 ) , r a i s e s new c l i n i c a l c o n s i d e r a t i o n s . I I . B i o l o g i c a l a n d B i o c h e m i c a l P r o p e r t i e s o f PMSG. PMSG i s one o f t h e mos t w i d e l y u s e d g o n a d o t r o p i n p r e p r a t i o n t o i n d u c e s u p e r o v u l a t i o n i n v a r i o u s mammalian s p e c i e s . The s i m p l i f i e d p r o c e d u r e a c h i e v e d b y a s i n g l e d o s e o f PMSG and i t s c o m m e r c i a l a v a i l a b i l i t y o f mass p r o d u c t i o n ( P a p k o f f , 1981) have l e d t o i t s w i d e - s c a l e u s e a s a s u p e r o v u l a t o r y a g e n t i n e x p e r i m e n t a l and d o m e s t i c a n i m a l s . PMSG h a s b e e n f o u n d t o be a g l y c o p r o t e i n hormone s e c r e t e d f r o m e n d o m e t r i a l c u p s o f t h e p r e g n a n t mare ( C o l e and G o s s , 1943) and i s p r e s e n t i n h i g h c o n c e n t r a t i o n i n t h e c i r c u l a t i n g b l o o d be tween Days 40 and 130 o f g e s t a t i o n ( C o l e and H a r t , 1 9 3 0 ) . S i n c e t h e i n i t i a l f i n d i n g o f t h e d u a l F S H - l i k e and L H - l i k e b i o l o g i c a l a c t i v i t i e s o f PMSG ( C o l e and H a r t , 1 9 3 0 ; C o l e , 1 9 3 8 ) , i t h a s b e e n f u r t h e r d e m o n s t r a t e d t h a t PMSG i s a s i n g l e m o l e c u l e w h i c h c o n s i s t s o f a h o r m o n a l l y n o n - s p e c i f i c s u b u n i t (PMSG-a) and a h o r m o n e - s p e c i f i c s u b u n i t (PMSG-B) b e i n g r e s p o n s i b l e f o r b o t h F S H - l i k e and L H - l i k e a c t i v i t i e s ( G o s p o d a r o w i c z and P a p k o f f , 1967? Papkoff, . 1974 : P a p k o f f e t a l . , 1 9 7 8 ) . The amino a c i d c o m p o s i t i o n o f PMSG—a b e g i n n i n g w i t h t h e s e q u e n c e N ^ - P h e - P r o ( G l y o r P r o ) . , and t e r m i n a t i n g w i t h i s o l e u c i n e a t c a r b o x y l t e r m i n u s h a s b e e n f o u n d t o be v e r y s i m i l a r t o e i t h e r L H - a , F S H - a , - 8 -o r T S H - a , and t h e c o m p o s i t i o n o f PMSG-fi w i t h t h e s e q u e n c e N r L ^ - S e r - P r o - G l y . . and y e t u n d e t e c t e d C - t e r m i n a l r e s i d u e i s c l o s e r t o L H - f i ( P a p k o f f , 1 9 7 8 ) . When c o m p a r i s o n s have been made be tween PMSG and t h e FSH and L H , much more c a r b o h y d r a t e (41-45%) h a s b e e n d e t e c t e d i n PMSG: 13.0% o f n e u t r a l s u g a r s (mannose , g a l a c t o s e , and f u c o s e ) , 17.6% o f h e x o s a m i n e s ( g l u c o s a m i n e and g a l a c t o s a m i n e ) , and 10.8% o f s i a l i c a c i d ( L a n d e f e l d , e t a l . , 1 9 7 2 ; L a n d e f e l d and M c S h a n , 1 9 7 4 ) . I t h a s b e e n f u r t h e r c h a r a c t e r i z e d t h a t PMSG-a h a s a 18.6% o f c a r b o h y d r a t e c o n t e n t , s i m i l a r t o F S H - a a n d L H - a , and t h a t PMSG-fi h a s a 55.3% o f c a r b o h y d r a t e c o n t e n t w h i c h i s 2 . 2 t o 3 . 0 f o l d h i g h e r t h a n t h a t o f FSH- f i and L H - B ( P a p k o f f , 1 9 7 8 ) . O f i t s t o t a l o f 55.3% c a r b o h y d r a t e , s i a l i c a c i d has been d e m o n s t r a t e d t o be p r e s e n t i n t h e g r e a t e s t amount (21.3%) ( P a p k o f f , 1 9 7 8 ) . I n t h e same s t u d y , i t h a s b e e n s u g g e s t e d t h a t t h e r a t i o o f FSH and LH a c t i v i t y i n a PMSG m o l e c u l e v a r i e s d e p e n d i n g upon c a r b o h y d r a t e c o m p o s i t i o n . A r a d i o r e c e p t o r a s s a y u t i l i z i n g a p r e p a r a t i o n o f r a t t e s t i c u l a r s e m i n i f e r o u s t u b u l e s f o r measurement o f FSH c o n c e n t r a t i o n a n d r a t t e s t i c u l a r i n t e r s t i t i a l t i s s u e s f o r measurement o f LH l e v e l s has shown t h e r a t i o o f F S H : L H i n PMSG r a n g e d f r o m 0 . 8 7 t o 1 .92 d e p e n d i n g upon t h e t i m e o f PMSG i s o l a t i o n d u r i n g p r e g n a n c y and i t s p r e p a r a t i o n ( S t e w a r t e t a l . , 1 9 7 6 ) . Upon r e c o m b i n a t i o n o f PMSG-a a n d PMSG-B i n a 1:1 r a t i o , i t h a s b e e n shown t h a t s i g n i f i c a n t amounts o f b o t h L H (27.4%) and FSH (33.6%) a c t i v i t i e s w e r e r e g e n e r a t e d ( P a p k o f f , 1 9 7 8 ) . The known c a r b o h y d r a t e c o n t e n t o f PMSG, s p e c i f i c a l l y a v e r y h i g h amount o f . s i a l i c a c i d , a c c o u n t s f o r t h e l o n g h a l f - l i f e and s l o w c l e a r a n c e r a t e (Schams e t a l . , 1 9 7 8 ) . - 9 -I t a p p e a r s t h a t t h e b i o c h e m i c a l p r o p e r t i e s o f p r o l o n g e d a c t i o n c o u p l e d w i t h FSH and L H m i x t u r e may b e , i n p a r t , r e s p o n s i b l e f o r t h e p o t e n c y w h e r e b y s u p e r o v u l a t i o n c a n be i n d u c e d b y a s i n g l e d o s e o f PMSG ( Z a r r o w and Q u i n n , 1 9 6 3 ; Whyman e t a l . , 1 9 7 9 ; Newcomb e t a l . , 1 9 7 9 ; M i l l e r a n d A r m s t r o n g , 1 9 8 1 b ) , s i n c e f o l l i c u l o g e n e s i s i n mammals depends o n t h e p r e s e n c e and a c t i v i t i e s o f b o t h FSH and L H (Murphy e t a l . , 1 9 8 4 ) . However , o v a r i a n and s u p e r o v u l a t o r y r e s p o n s e t o a s t a n d a r d d o s e o f PMSG v a r i e s c o n s i d e r a b l y i n f a r m a n i m a l s ( B e l l o w s and S h o r t , 1 9 7 2 ; Newcomb, 1 9 7 6 ; Schams e t a l . , 1978) and has b e e n a t t r i b u t e d t o t h e p o s s i b l e v a r i a t i o n s i n t h e r a t i o o f FSH and LH a c t i v i t y i n d i f f e r e n t e x t r a c t s a n d c o m m e r c i a l l y a v a i l a b l e b a t c h e s o f t h e hormones (Newcomb and R o w s o n , 1 9 7 6 ) . I I I . E n d o c r i n e A s p e c t s o f S u p e r o v u l a t i o n . A. G e n e r a l . The d i s t r i b u t i o n and m e t a b o l i s m o f t h e PMSG i n j e c t e d t o a n i m a l s have n o t y e t b e e n d e s c r i b e d , b u t a l o n g h a l f - l i f e c o u l d be a n t i c i p a t e d f r o m t h e s t u d i e s i n v a r i o u s s p e c i e s ; Endogenous PMSG i n mares h a s a h a l f - l i f e o f a b o u t 6 days a f t e r h y s t e r e c t o m y ( C o l e e t a l . , 1967). I n j e c t e d PMSG has a h a l f - l i f e o f a b o u t 6 d a y s i n h o r s e s and 26 h r i n r a b b i t s ( C a t c h p o l e e t a l . , 1 9 3 5 ) , 26 h r i n r a t s ( P a r l o w and W a r d , 1961) a n d a b o u t 21 h r i n s h e e p ( M c i n t o s h e t a l . , 1 9 7 5 ) . I n s h e e p , M c i n t o s h e t a l . ( 1 9 7 5 ) f o u n d no e v i d e n c e t h a t t h e o v a r i e s . removed PMSG f r o m t h e c i r c u l a t i n g b l o o d ; b u t , i n c a t t l e , l o w d o s e s o f PMSG i n j e c t e d d i r e c t l y i n t o t h e o v a r y a p p e a r t o b i n d t o r e c e p t o r s and p r o d u c e a l o c a l e f f e c t ( B e t t e r i d g e , 1974). The h a l f - l i f e o f d i f f e r e n t p r e p a r a t i o n s o f PMSG c a n be e x p e c t e d t o v a r y a c c o r d i n g t o t h e - 10 -e x t e n t t o w h i c h s i a l i c a c i d i s removed f r o m t h e i n j e c t e d n a t i v e m o l e c u l e ( M c i n t o s h e t a l . , 1 9 7 5 ) . I n c y c l i n g a d u l t h a m s t e r s t r e a t e d w i t h PMSG, G r e e n w a l d (1963) h a s i n v e s t i g a t e d t h e i n t e r a c t i o n o f e x o g e n o u s a n d endogenous g o n a d o t r o p i n s o n f o l l i c u l a r d e v e l o p m e n t u s i n g a n t i - P M S G s e r u m and shown t h a t t h e m a t u r a t i o n o f r e s e r v e f o l l i c l e s depends o n a d e q u a t e c i r c u l a t i n g l e v e l s o f PMSG t h r o u g h o u t t h e e s t r o u s c y c l e . I f PMSG i s r e q u i r e d t h r o u g h o u t t h e e n t i r e c o u r s e o f f o l l c u l a r d e v e l o p m e n t a n d m a i n t e n a n c e i n i n t a c t immature r a t s t r e a t e d w i t h PMSG, p r e m a t u r e i n a c t i v a t i o n o f PMSG may r e s u l t i n f o l l i c u l a r d e g e n e r a t i o n and t h e l o s s o f o v u l a t i n g c a p a b i l i t y (Sasamoto and K e n n a n , 1 9 7 2 ) . I n t h e i r s u b s e q u e n t s t u d y u s i n g a n t i - P M S G s e r u m , i t has b e e n shown t h a t PMSG i n i t i a l l y s t i m u l a t e s f o l l i c u l a r d e v e l o p m e n t d u r i n g t h e f i r s t 36 h r f o l l o w i n g i n j e c t i o n o f t h e g o n a d o t r o p i n , and t h e r e a f t e r t h e endogenous g o n a d o t r o p i n s e l i c i t e d b y PMSG v i a t h e p o s i t i v e f e e d b a c k mechanism o f s t e r o i d hormones p r o d u c e d a r e r e s p o n s i b l e f o r f u r t h e r d e v e l o p m e n t and m a i n t e n a n c e o f t h e f o l l i c l e s i n a n o v u l a t i n g s t a t e (Sasamoto and K e n n a n , 1 9 7 3 ) . The a b i l i t y o f PMSG t o p r o d u c e s u p e r o v u l a t i o n a p p e a r s t o depend n o t o n l y o n i t s p r o l o n g e d b i o l o g i c a l h a l f - l i f e b u t a l s o o n t h e l e v e l s b e i n g i n i t i a l l y h i g h enough t o r e c r u i t more f o l l i c l e s a t c r i t i c a l s t a g e s o f t h e i r d e v e l o p m e n t ( G r e e n w a l d , 1 9 7 3 ) . S y s t e m i c i n j e c t i o n o f PMSG i n c a t t l e i n i t i a l l y e x e r t s a l u t e o t r o p h i c e f f e c t ( H e n r i c k s et a l . , 1 9 7 3 ; H a l l f o r d e t a l . , 1 9 7 5 ; M o o r e , 1975) , w h i c h h a s b e e n r e l a t e d to i t s L H - l i k e a c t i v i t y (Newcomb a n d R e a s o n , 1976) . T h i s p r o p e r t y h a s been p resumed t o b e r e s p o n s i b l e f o r t h e o c c a s i o n a l p r e m a t u r e o v u l a t i o n o f a l a r g e f o l l i c l e p r e s e n t a t t h e t i m e o f PMSG i n j e c t i o n (Newcomb and Rowson , 1976) and p e r s i s t e n c e o f l a r g e n o n l u t e i n i z e d - 11 -o r c y s t i c f o l l i c l e s ( B e t t e r i d g e , 1 9 7 7 ) . I t h a s b e e n s u g g e s t e d t h a t t h e p r e c o c i o u s f o r m a t i o n a n d p r e m a t u r e r e g r e s s i o n o f C L f o l l o w i n g s u p e r o v u l a t i o n w i t h PMSG i n h i b i t f u r t h e r o v u l a t i o n , and r e s u l t i n a b n o r m a l d e v e l o p m e n t o r r e t a r d a t i o n o f embryos i n e a r l y p r e g n a n c y ( B e t t e r i d g e , 1 9 7 7 ; du M e n s i l du B u i s s o n e t a l . , 1 9 7 7 ) . I n e a c h c a s e , t h e r e a r e a l s o s u g g e s t i o n s t h a t s u p e r o v u l a t o r y d o s e s o f PMSG a l t e r t h e o v a r i a n s t e r o i d a l m i l i e u a f t e r o v u l a t i o n and r e s u l t i n p e r t u r b a t i o n o f o v i d u c t a l and u t e r i n e f u n c t i o n f o r embryo s u r v i v a l a n d / o r d e v e l o p m e n t . B o u t e r s e t a l . (1983) have r e p o r t e d t h a t s u p e r o v u l a t o r y t r e a t m e n t w i t h PMSG i n c a t t l e i n d u c e s a s e c o n d wave o f f o l l i c l e s a f t e r o v u l a t i o n , c a u s i n g h i g h c o n c e n t r a t i o n s o f 1 7 B - e s t r a d i o l i n t h e p e r i p h e r a l b l o o d d u r i n g e a r l y e m b r y o n i c d e v e l o p m e n t , w h i c h a p p e a r s t o be due t o t h e l o n g h a l f - l i f e o f PMSG (Schams e t a l . , 1 9 7 8 ) . A l t h o u g h g o n a d o t r o p i n t r e a t m e n t f o r m u l t i p l e o v u l a t i o n s h a s f r e q u e n t l y been r e a s s e s s e d (Ryan e t a l . , 1 9 8 4 ; Evans e t a l . , 1 9 8 4 ) , t h e r e i s l i t t l e i n f o r m a t i o n o n t h e s y n c h r o n y o f o v u l a t i o n f o l l o w i n g t h e s u p e r o v u l a t o r y t r e a t m e n t , d e s p i t e t h e p r o b a b i l i t y t h a t p o o r s y n c h r o n y i s a l i k e l y c a u s e o f f e r t i l i z a t i o n f a i l u r e a n d f u r t h e r d e v e l o p m e n t a l d e f e c t s o f e a r l y e m b r y o s . B . G o n a d o t r o p i n S e c r e t i o n and I n d u c e d O v u l a t i o n . S p i l m a n e t a l . , (1973) r e p o r t e d t h a t PMSG l e a d s t o t h e r e l e a s e o f endogenous LH w i t h i n 24 t o 48 h r a f t e r i n j e c t i o n i n c a t t l e , b u t t h i s was n o t c o n f i r m e d b y o t h e r s ( H e n r i c k s e t a l . , ' 1 9 7 3 ; Saumande a n d P e l l e t i e r , 1975; H a l l f o r d e t a l . , 1975) who d e s c r i b e no LH p e a k s b e f o r e t h e o n e c o i n c i d i n g w i t h e s t r u s . P r e o v u l a t o r y LH s e c r e t i o n h a d no r e l a t i o n s h i p w i t h o v u l a t i o n r a t e s when e x p r e s s e d a s maximum p l a s m a L H l e v e l s ( H e n r i c k s e t a l . , 1 9 7 3 ) , - 12 -b u t a p o s i t i v e r e l a t i o n s h i p was n o t i c e d when e x p r e s s e d a s t h e t o t a l q u a n t i t y o f LH r e l e a s e d (Saumande and P e l l e t i e r , 1 9 7 5 ) . I n immatu re r a t s , t i m e c o u r s e s t u d i e s o f t h e o v u l a t o r y r e s p o n s e e m p l o y i n g d e f i n e d l o w d o s e s ( 4 - 8 IU) o f PMSG d e m o n s t r a t e d a n i n t e g r a t i o n o f s e q u e n t i a l c h a n g e s i n t h e e n d o c r i n e r e s p o n s e i n c l u d i n g g o n a d o t r o p i n s e c r e t i o n a s s o c i a t e d w i t h a s y n c h r o n i z e d p r o c e s s o f o o c y t e m a t u r a t i o n : c r i t i c a l t i m e o f endogenous L H s u r g e a t 5 2 - 5 7 h r ( S o r r e n t i n o e t a l . , 1 9 7 2 ; C o s t o f f e t a l . , 1 9 7 4 ) , m e i o t i c r e s u m p t i o n o f f o l l i c u l a r o o c y t e s 2 t o 3 h r t h e r e a f t e r ( H i l l e n s j o e t a l . , 1974) and o v u l a t i o n a t 6 0 - 7 2 h r a f t e r PMSG ( W a l t o n e t a l . , 1 9 8 3 ) . The endogenous g o n a d o t r o p i n s u r g e i n t h e P M S G - t r e a t e d immature f e m a l e s h a s f o l l o w e d t h e d r a m a t i c i n c r e a s e s i n p r e o v u l a t o r y se rum l e v e l s o f 1 7 f l - e s t r a d i o l and p r o g e s t e r o n e w h i c h a r e s i m i l a r t o t h o s e o b s e r v e d d u r i n g t h e a d u l t p r o e s t r u s ( S h a i k h , 1 9 7 1 ; B u t c h e r e t a l . , 1 9 7 4 ; P a r k e r e t a l . , 1 9 7 6 ) . The p i t u i t a r y i s a c t i v a t e d t o r e l e a s e o v u l a t o r y g o n a d o t r o p i n s o n t h e a f t e r n o o n b e f o r e e s t r u s be tween 2 : 0 0 and 4 : 0 0 p . m . (known a s t h e c r i t i c a l t i m e p e r i o d ) w h i c h c a u s e o v u l a t i o n a b o u t 12 h r s l a t e r i n c y c l i n g a d u l t r a t s ( E v e r e t t e t a l . , 1 9 4 9 ) . T h u s , t h e o v a r y t h r o u g h e s t r o g e n s e c r e t i o n , a p p e a r s t o a c t a s a t r i g g e r f o r t h e p r e o v u l a t o r y g o n a d o t r o p i n s u r g e ( S c h w a r t z , 1 9 6 9 ) . H i l l e n s j o e t a l . , (1974) have r e p o r t e d t h a t t h e t e m p o r a l r e l a t i o n s h i p be tween se rum LH l e v e l s and o o c y t e m a t u r a t i o n i n P M S G - t r e a t e d immature r a t s i s exactly t h e same a s t h a t s e e n i n p r o e s t r o u s a d u l t r a t s . I n g o n a d o t r o p i n ^ - t r e a t e d immatu re r a t s , a l l o v u l a t i o n s i n a p a r t i c u l a r o v a r y have been f o u n d t o be c o m p l e t e d i n l e s s t h a n 1.5 h r s a n d f r e q u e n t l y s e v e r a l f o l l i c l e s a p p e a r t o r u p t u r e a t t h e same t i m e ( B l a n d a u , 1 9 5 5 ) . I n t h e same s t u d y , t h e t i m e r e q u i r e d f o r a f o l l i c l e t o r u p t u r e h a s - 13 -b e e n d e m o n s t r a t e d t o v a r y f r o m 5 t o 760 s e c , d e p e n d i n g l a r g e l y upon t h e p o s i t i o n o f c u m u l u s o o p h o r u s i n . r e l a t i o n t o t h e s t i g m a . Howeve r , i t h a s b e e n shown t h a t h i g h s u p e r o v u l a t o r y d o s e s o f PMSG i n immatu re r a t s i n d u c e p r e c o c i o u s o r m u l t i p l e waves o f o v u l a t i o n f o r an u n u s u a l l y e x t e n d e d p e r i o d , a t l e a s t 48 h r , w h i c h a r e p r e s u m a b l y due t o t h e exogenous L H a c t i v i t y o f PMSG i n i t s e l f (De L a L a s t r a e t a l . , 1 9 7 2 ; M i l l e r and A r m s t r o n g , 1 9 8 1 b ) . D i r e c t a c t i o n o f h i g h d o s e s o f PMSG o n t h e o v a r y h a s b e e n d e m o n s t r a t e d b y t h e s t u d i e s o f R o w l a n d s a n d W i l l i a m s (1943) and W i l l i a m s (1945) i n w h i c h h y p o p h y s e c t o m i z e d immatu re r a t s o v u l a t e d a f t e r a s i n g l e d o s e o r t w o c o n s e c u t i v e h i g h d o s e s o f PMSG u p t o 4 d a y s a f t e r t h e o p e r a t i o n . D e s p i t e c o n s i d e r a b l e a t t e n t i o n t o LH a c t i v i t y o f PMSG and i t s d i r e c t a c t i o n o n t h e o v a r y , no p u b l i s h e d d a t a a r e a v a i l a b l e o n t h e r e l a t i o n s h i p be tween a t y p i c a l . o v u l a t o r y r e s p o n s e and amounts o f e x o g e n o u s o r endogenous L H i n t h e c i r c u l a t i o n o f s u p e r o v u l a t e d r a t s . F u r t h e r m o r e , i t has b e e n s u g g e s t e d t h a t n e u r a l a c t i v a t i o n s y s t e m f o r p i t u i t a r y r e l e a s e o f o v u l a t i n g hormone may be d i f f e r e n t d e p e n d i n g upon t h e d o s e s o f PMSG a d m i n i s t e r e d t o immatu re r a t s ( Y i n g and M e y e r , 1 9 6 9 ) . I n c o n s i d e r i n g t h e d o s e r e s p o n s e t o PMSG, i t m i g h t be r e l e v a n t t h a t , i n r a t s , t h e r e i s a d e p r e s s i o n o f o v u l a t i o n r a t e and a n o v u l a t i o n a t t h e d o s e s h i g h e r t h a n 40 I U a n d a t t h e d o s e s r a n g e d f r o m 12 t o 15 I U , r e s p e c t i v e l y ( W i l l i a m s , 1 9 4 5 ; Y i n g a n d M e y e r , 1969; W i l s o n e t a l . , 1 S 7 4 ) . T h i s s u p p r e s s i o n o r " p a r a d o x i c a l " a n o v u l a t o r y r e s p o n s e h a s b e e n a t t r i b u t e d - t o a d e f i c i e n c y o f p r o g e s t e r o n e w h i c h s y n e r g i z e s w i t h e s t r o g e n . i n t h e r e l e a s e o f p r e o v u l a t o r y FSH a n d L H (McCormack and M e y e r , 1963; C a r l s o n , 1 9 6 8 ) . I n t h e s t u d i e s a b o v e , a d m i n i s t r a t i o n o f exogenous p r o g e s t e r o n e o r L H p r i o r t o t h e c r i t i c a l t i m e p e r i o d o v e r c a m e t h e i n h i b i t o r y o v u l a t o r y - 14 -r e s p o n s e . I t h a s b e e n r e p o r t e d t h a t p r o g e s t e r o n e e x h i b i t s b o t h f a c i l i t a t i n g and i n h i b i t i n g a c t i o n s o n P M S G - i n d u c e d o v u l a t i o n i n immature r a t s , d e p e n d i n g upon t h e t i m i n g and d o s e o f exogenous p r o g e s t e r o n e (McCormack and M e y e r , 1 9 6 3 ; Za r row and H u r l b u r t , 1 9 6 7 ) . The e f f e c t s o f exogenous p r o g e s t e r o n e o n advancement o f t h e o v u l a t i o n t i m e may be e x e r t e d b y s h i f t i n g f o r w a r d t h e c r i t i c a l p e r i o d o f endogenous LH r e l e a s e ( Z e i l m a k e r , 1 9 6 6 ; L i s k , 1 9 6 9 ) . F a c i l i t a t i o n o f L H r e l e a s e and o v u l a t i o n o c c u r s o n l y i f p r o g e s t e r o n e i s p r e s e n t f o r no more t h a n 6 t o 12 h r s ; o v u l a t i o n i s b l o c k e d f o r 24 o r 48 h r s i f p r o g e s t e r o n e i s p r e s e n t f o r a l o n g e r i n t e r v a l ( L i s k , 1 9 6 9 ) . I t h a s a l s o been s u g g e s t e d t h a t a n a d e q u a t e c i r c u l a t i n g l e v e l o f e s t r o g e n i s r e q u i r e d f o r t h e s p o n t a n e o u s r e l e a s e o f LH and f o r t h e f a c i l i t a t i o n o f L H r e l e a s e b y p r o g e s t e r o n e (Docke and D o r n e r , 1 9 6 5 ; G r a y b u r n and B r o w n - G r a n t , 1 9 6 8 ) . C. O v a r i a n S t e r o i d s and O o c y t e M a t u r a t i o n . O o c y t e s r e q u i r e a s p e c i f i c i n t r a f o l l i c u l a r s t e r o i d e n v i r o n m e n t f o r t h e c o m p l e t i o n o f t h e f u l l m a t u r a t i o n p r o c e s s . A l t e r a t i o n s i n t h e s t e r o i d p r o f i l e d u r i n g m a t u r a t i o n , e s p e c i a l l y i n p r e o v u l a t o r y f o l l i c l e s , i n d u c e m o r p h o l o g i c a l c h a n g e s i n t h e o o c y t e w h i c h a r e e x p r e s s e d a s g r o s s a b n o r m a l i t i e s a t f e r t i l i z a t i o n i n c l u d i n g p o l y s p e r m y , a r r e s t e d spe rm c o n d e n s a t i o n and f r a g m e n t e d m a l e p r o n u c l e i (Moor e t a l . , 1 9 8 0 b ) . I t h a s been shown t h a t s u p e r o v u l a t o r y t r e a t m e n t i n c a t t l e r e s u l t s i n a n a b n o r m a l p r o g e s t e r o n e / e s t r o g e n r a t i o i n a m a j o r i t y o f f o l l i c l e s d u r i n g t h e p r e - and p e r i - o v u l a t o r y p e r i o d , a n d p r o d u c e d p r e m a t u r e l y a c t i v a t e d o r m e i o t i c a l l y a r r e s t e d o o c y t e s ( C a l l e s e n e t a l . , 1 9 8 6 ) . I n s h e e p t r e a t e d w i t h s u p e r o v u l a t o r y d o s e s o f PMSG , 33% o f o o c y t e s d i s p l a y e d a p r e m a t u r e - 15 -a c t i v a t i o n c h a r a c t e r i z e d b y a b n o r m a l p a t t e r n o f p r o t e i n s y n t h e s i s a n d w e r e a s s o c i a t e d w i t h h y p e r s e c r e t i o n o f f o l l i c u l a r 1 7 B - e s t r a d i o l (Moor e t a l . , 1 9 8 4 , 1 9 8 5 ) . The v a r i a t i o n i n t h e f o l l i c u l a r s t e r o i d o g e n e s i s o b s e r v e d , d u r i n g and a f t e r t h e p r e o v u l a t o r y LH s u r g e i s b e l i e v e d t o be c a u s e d f i r s t b y t h e h e t e r o g e n e i t y o f p o o l s o f s u p e r o v u l a t e d f o l l i c l e s w h i c h w e r e r e c r u i t e d a t t h e t i m e o f g o n a d o t r o p i n s t i m u l a t i o n , a n d s e c o n d a r i l y b y t h e d i f f e r e n t a b i l i t y o f t h e s e f o l l i c l e s t o d e v e l o p a f t e r b e i n g s t i m u l a t e d (Moor e t a l . , 1 9 8 0 a ) . L i k e t h e a b n o r m a l f o l l i c u l a r s t e r o i d o g e n e s i s i n s u p e r o v u l a t e d c a t t l e , t h e o o c y t e m a t u r a t i o n , e x p r e s s e d b y t h e n u c l e a r and c y t o g e n e t i c a p p e a r a n c e , has been s u b j e c t t o a h i g h d e g r e e o f a s y n c h r o n y w i t h i n and b e t w e e n a n i m a l s a t t h e same t i m e i n t e r v a l s f o l l o w i n g t h e L H p e a k ( C a l l e s e n e t a l . , 1 9 8 6 ) . T h i s a s y n c h r o n y phenomenon h a s been d e s c r i b e d i n s u p e r o v u l a t e d sheep (Moor e t a l . , 1984) a n d c a t t l e (Greve e t a l . , 1 9 8 4 ) , a n d may be a c o n s e q u e n c e o f p r e m a t u r e m e i o t i c a c t i v a t i o n o f o o c y t e s f r o m c e r t a i n f o l l i c l e s ( C a l l e s e n e t a l . , 1 9 8 6 ) . S t u d i e s o n s h e e p o v a r i a n f o l l i c l e s have d e m o n s t r a t e d t h a t LH i n h i b i t s e s t r o g e n and a n d r o g e n s e c r e t i o n f r o m t h e G r a a f i a n f o l l i c l e , w h i l e t h e s e c r e t i o n o f p r o g e s t e r o n e i s e n h a n c e d (Semark e t a l . , 1 9 7 4 ; M o o r , 1 9 7 4 ) . T he se s t u d i e s h a v e s u g g e s t e d t h a t L H h a s a d i s p a r a t e a c t i o n o n t h e s t e r o i d o g e n e s i s o f p r e o v u l a t o r y f o l l i c l e s : s t i m u l a t i o n o f p r o g e s t e r o n e p r o d u c t i o n and i n h i b i t i o n o f e s t r o g e n s e c r e t i o n . A t r a n s i e n t s t i m u l a t i o n o f e s t r o g e n s e c r e t i o n seems , however-; to p r e c e d e t h e i n h i b t i o n ( M c C r a c k e r i ; e t a l . , 1 9 7 1 ) . S i m i l a r e v i d e n c e h a s b e e n o b t a i n e d by c u l t u r i n g p r e o v u l a t o r y G r a a f i a n f o l l i c l e s , i s o l a t e d f r o m t h e P M S G - t r e a t e d immature r a t s p r i o r t o t h e endogenous g o n a d o t r o p i n s u r g e ( H i l l e n s j o e t a l . , 1 9 7 6 ) . T h i s s t u d y h a s - 16 -shown t h a t d u r i n g t h e f i r s t 4 h r s t h e r e i s a n i n c r e a s e i n t h e s e c r e t i o n o f 1 7 B - e s t r a d i o l and a n d r o g e n s ( a n d r o s t e n e d i o n e a n d t e s t o s t e r o n e ) . L a t e r , a f t e r L H t r e a t m e n t , t h e r e i s a s i g n i f i c a n t s h i f t f r o m e s t r o g e n / a n d r o g e n t o p r o g e s t e r o n e . S e v e r a l i n v e s t i g a t o r s have shown t h e e f f e c t s o f e s t r o g e n and p r o g e s t e r o n e o n mammalian o o c y t e m a t u r a t i o n i n v i t r o . 1 7 B - e s t r a d i o l , a t v a r i o u s c o n c e n t r a t i o n s , s i g n i f i c a n t l y r e d u c e d t h e i n c i d e n c e o f m a t u r a t i o n o f c u m u l u s - e n c l o s e d r a b b i t a n d b o v i n e o o c y t e s ( R o b e r t s o n and B a k e r , 1 9 6 9 ) , denuded p o r c i n e o o c y t e s (McGaughey, 1 9 7 7 ) , a n d denuded mouse o o c y t e s ( N e k o l a and S m i t h , 1 9 7 4 ; E p p i g and K o i d e , 1 9 7 8 ) . P r o g e s t e r o n e , a t a h i g h c o n c e n t r a t i o n (100 y M ) , h a s b e e n r e p o r t e d t o enhance t h e m a t u r a t i o n o f denuded b o v i n e and r a b b i t o o c y t e s ( R o b e r t s o n a n d B a k e r , 1 9 6 9 ) , c u m u l u s - e n c l o s e d r a b b i t o o c y t e s (Bae and F o o t e , 1 9 7 5 ) , a n d denuded o o c y t e s f r o m g o n a d o t r o p i n - t r e a t e d p r e p u b e r t a l r h e s u s monkeys ( G o u l d and Graham, 1 9 7 6 ) , w h i l e a t l o w c o n c e n t r a t i o n s ( 1 0 - 3 0 y M ) , i t d e c r e a s e d m a t u r a t i o n f r e q u e n c y o f denuded mouse o o c y t e s ( N e k o l a and S m i t h , 1 9 7 4 ; E p p i g and K o i d e , 1978) and c o r o n a - e n c l o s e d r a b b i t o o c y t e s ( S m i t h e t a l . , 1 9 7 8 ) . A s t u d y o n t h e r o l e o f hormones i n gap j u n c t i o n f u n c t i o n s u g g e s t s t h a t s t e r o i d hormones h a v e a m i n o r b u t s t a t i s t i c a l l y s i g n i f i c a n t e f f e c t o n t h e p a s s a g e o f m o l e c u l e s f r o m c u m u l u s c e l l s t o o o c y t e s (Moor e t a l . , 1980c). However , t h e ma jo r r e g u l a t o r s o f i n t r a c e l l u l a r c o u p l i n g a n d amino a c i d t r a n s p o r t a c r o s s t h e oolemma., name ly FSH and L H , a r e n o t l i k e l y t o be d e p e n d e n t upon s t e r o i d s y n t h e s i s f o r t h e i r a c t i o n o n t h e membrane (Moor a n d S m i t h , 1978; Moor e t a l , 1980c). T h e r e f o r e , i t h a s been s u g g e s t e d t h a t t h e a c t i o n o f s t e r o i d hormones i s n o t p r i m a r i l y o n t h e membranes o f t h e o o c y t e (Moor e t a l , - 17 -1 9 8 0 c ) . L i e b e r m a n e t a l . (1976) have i n d i c a t e d t h a t t h e e a r l y s e q u e n c e o f m a t u r a t i o n c h a n g e s i n t h e n u c l e u s l e a d i n g t o g e r m i n a l v e s i c l e b r e a k d o w n a n d f o r m a t i o n o f t h e f i r s t me taphase p l a t e a r e i n d e p e n d e n t o f s t e r o i d s u p p o r t . I t h a s , h o w e v e r , been shown t h a t t h e a b s e n c e o r a l t e r a t i o n o f s t e r o i d hormones d u r i n g t h e l a t e r s t a g e s o f m a t u r a t i o n i n c r e a s e s t h e i n c i d e n c e o f ch romosomal a b n o r m a l i t y e s p e c i a l l y a t t e l o p h a s e I and me taphase I I (McGaughey, 1 9 7 7 ; Moor e t a l . , 1 9 8 0 b ) . I V . D e v e l o p m e n t a l C a p a b i l i t y o f S u p e r o v u l a t e d O o c y t e s . A . G e n e r a l . D e s p i t e w i d e s p r e a d u s e o f t h e s u p e r o v u l a t i o n t e c h n i q u e i n r e s e a r c h , c o m m e r c i a l and c l i n i c a l f i e l d s , t h e a t t e m p t s a t i n d u c t i o n o f s u p e r p r e g n a n c i e s and e x c e s s i v e l i t t e r s i z e s i n v a r i o u s mammal ian s p e c i e s have been d i s a p p o i n t i n g . E a r l i e r s t u d i e s f o u n d t h e d i s p a r i t y b e t w e e n t h e number o f i m p l a n t a t i o n s i t e s a n d l i t t e r s i z e s ( C o l e , 1 9 3 7 ; E v a n s a n d S i m p s o n , 1940 i n r a t s ; F o w l e r a n d E d w a r d s , 1 9 5 7 ; S a t o , 1963 i n m i c e ; Adams , 1960 i n r a b b i t s ) and be tween o v u l a t i o n and i m p l a n t a t i o n r a t e s ( A u s t i n , 1950 i n r a t s ) . R e s u l t s o f Evans a n d S i m p s o n (1940) showed t h a t a f t e r s u p e r o v u l a t i o n w i t h exogenous g o n a d o t r o p i n s i n immature r a t s , t h e maximum number o f i m p l a n t a t i o n s i t e s was 34 b u t t h e maximum number o f l i v i n g f e t u s e s was s u b s t a n t i a l l y r e d u c e d t o 1 7 . The h i g h w a s t a g e r a t e h a s a l s o b e e n r e p o r t e d f o l l o w i n g embryo t r a n s f e r i n human I V F programme ( E d w a r d s , 1 9 8 4 ; B l a n k s t e i n , 1 9 8 6 ) . D e s p i t e a c h i e v i n g h i g h r a t e s o f f e r t i l i z a t i o n (90%) a n d embryo r e p l a c e m e n t (80%), p r e g n a n c y r a t e a t t e r m was o n l y 15-20% p e r embryo t r a n s f e r . I n t h e s t u d i e s a b o v e , t h e d i s p a r i t y f o l l o w i n g s u p e r o v u l a t i o n was - 18 -mainly ascribed to preimplantation loss of embryos, failure of implantation, or spontaneous abortion in early or mid-pregnancy. Postimplantation resorption or death of embryos in itself may not be a major factor leading to the poor reproductive performance following superovulation. Austin (1950) was the first to focus upon preimplantation developmental failure of superovulated oocytes as a cause for the known disparity between ovulation and implantation rates and the high mating rates with few or no implantation sites in rats. Until Day 2 of pregnancy, the number of embryos flushed out of oviducts and uterine horns was comparable to ovulation counts. However, from Day 3 of pregnancy/ there was a definite decline in the number of recovered embryos associated with an increase in degenerate oocytes and retarded embryo development (Wu and Meyer, 1966; Miller and Armstrong, 1981a; Sherman et al., 1982; Yun and Kwun, 1984). Early loss of preimplantation embryos following superovulation could be accounted for by the observation of vaginal expulsion of ova in both 17B-estradiol-treated rats and superovulated rabbits (Greenwald, 1961; Tsutsumi et al., 1981) and by the suggestion of excessive stimulation of the genital tract arising from hyperstimulated large follicles which persist even after normal ovulation (Miller and Armstrong, 1981b). In contrast, uterine ligation at cervical ends of superovulated rats did not increase the recovery rates in the preimplantation period (Sherman et al., 1984). Thus, expulsion of embryos to the vagina does not appear to be a main course of preimplantation loss following superovulation in rats. There are several lines of evidence on developmental failure of superovulated oocytes. The use of exogenous gonadotropins has been linked - 19 -with detrimental effects such as chromosomal abnormalities, embryonic death and congenital defects (Bradford et al., 1974; Hofsaess and Meadham, 1971; Fujimoto et al., 1974; Takagi and Sasaki, 1976). Previous results of my M.Sc. study and others with an immature rat model have indicated that although normal pregnancy with no significant embryonic or fetal wastage can be obtained after ovulation induction with low doses of PMSG, high superovulatory doses are associated with abnormal embryo development and implantation failure (Nuti et al., 1975; Miller and Armstrong, 1981a; Sherman et al., 1982; Yun and Kwun, 1984). It has been reported that embryonic or fetal death following superovulation most commonly occurs during cleavage (Allen and McLaren, 1971), about the time of implantation and at or shortly after parturition (Edwards and Fowler; 1959; McLaren and Michie, 1959). A number of investigators have ascribed the embryo loss or death after superovulation to abnormalities or failure of the process of fertilization (Walton et al., 1983), genetic deficiencies in superovulated oocytes (Beaumont and Smith, 1975), or to environmental factors; e.g. competition between embryos (Edwards and Fowler, 1959; Edwards et al., 1963; McLaren and Michie, 1959; Allen and McLaren, 1971) or maternal systemic factors such as nutritional stress or insufficiency of progesterone (McLaren and Michie, 1956, 1959). It appears that the detrimental effects and mechanism(s) of superovulation may be multifactoral including the disturbances in oocytes/embryos themselves or in the maternal environment for their survival. - 20 -B . G e n e t i c C o n d i t i o n . G e n e t i c a b n o r m a l i t i e s i n d u c e d i n o o c y t e s / e m b r y o s m i g h t be mos t damag ing t o t h e u s e f u l n e s s o f t h e s u p e r o v u l a t i o n p r o c e d u r e , s i n c e t h e p r o g e n y w o u l d be d e s t i n e d t o d e a t h a t a n y s t a g e o f p r e g n a n c y r e g a r d l e s s o f t h e n o r m a l i t y o f m a t e r n a l e n v i r o n m e n t ( F l e m i n g , 1 9 8 2 ) . R e p o r t s f o r t h e r a b b i t and mouse h a v e i n d i c a t e d a n i n c r e a s e i n g e n e t i c a b n o r m a l i t i e s a f t e r s u p e r o v u l a t o r y t r e a t m e n t s c o m p a r e d t o u n t r e a t e d c o n t r o l s ( F u j i m o t o e t a l . , 1 9 7 4 ; T a k a g i a n d S a s a k i , 1976) a n d s u g g e s t e d t h a t d i g y n i c t r i p l o i d s c o n s t i t u t e a n a p p r e c i a b l e p r o p o r t i o n o f p r e - and p o s t - i m p l a n t a t i o n embryo l o s s e s ( T a k a g i and S a s a k i , 1 9 7 6 ) . I n humans, t h e i n c r e a s e d f r e q u e n c y o f t r i p l o i d y and o t h e r t y p e s o f c h r o m o s o m a l a n o m a l i e s have a l s o b e e n o b s e r v e d i n s p o n t a n e o u s a b o r t u s e s a f t e r s u p e r o v u l a t i o n i n d u c t i o n t h e r a p y b y hMG and hCG (Boue and B o u e , 1973) a n d a t t r i b u t e d t o t h e t r e a t m e n t o f g o n a d o t r o p i n i t s e l f o r n o r m a l f l u c t u a t i o n s o f FSH s e c r e t i o n i n p r e g n a n c y ( M a u d l i n and F r a s e r , 1 9 7 7 ) . On t h e o t h e r h a n d , i n t h e s t u d i e s o f f i r s t - c l e a v a g e mouse embryos a f t e r s u p e r o v u l a t i o n , a l o w l e v e l , s t a t i s t i c a l l y n o t s i g n i f i c a n t , o f ch romosomal a n o m a l i e s h a s been d e m o n s t r a t e d : a n i n c i d e n c e o f 1.8% a n e u p l o i d y a n d 1.5% p o l y p l o i d y (Donahue , 1 9 7 2 ) . I t h a s a l s o b e e n r e p o r t e d t h a t t h e r e w e r e no i n c r e a s e s i n g e n e t i c o r d e v e l o p m e n t a l d e f e c t s i n o f f s p r i n g a f t e r s u p e r o v u l a t i o n i n t h e human (Hack a n d L u n e n f e l d , 1 9 7 9 ) , t h e h a m s t e r ( F l e m i n g a n d Y a n a g i m a c h i , 1980) and t h e r a b b i t ( M a u r e r e t a l . , 1 9 6 8 ) . I n t h e above s t u d i e s , g e n e t i c c o n d i t i o n o f s u p e r o v u l a t e d o o c y t e s / e m b r y o s l e a d i n g t o embryo l o s s o r f e t a l d e a t h i n s u p e r o v u l a t e d a n i m a l s i s s t i l l c o n t r o v e r s i a l , d e p e n d i n g upon d i f f e r e n t s p e c i e s and s t r a i n s , and r e m a i n s t o be c l a r i f i e d . - 21 -C . O o c y t e Q u a l i t y . N o r m a l embryo d e v e l o p m e n t a f t e r f e r t i l i z a t i o n r e q u i r e s t h a t o o c y t e s h a v e f i r s t a t t a i n e d n u c l e a r and c y t o p l a s m i c m a t u r i t y . The o o c y t e s s h o u l d be o v u l a t e d a n d made a v a i l a b l e f o r f e r t i l z a t i o n a t a t i m e c o o r d i n a t e d w i t h t h e a t t a i n m e n t o f t h e i r m a t u r i t y ( F l e m i n g , 1 9 8 2 ) . The same a u t h o r h a s s u g g e s t e d t h a t o o c y t e s r e l e a s e d b e f o r e m a t u r i t y may be d e f i c i e n t i n m a t e r i a l s o r mechanisms needed f o r f e r t i l i z a t i o n and f u r t h e r d e v e l o p m e n t , w h i l e m a t u r e o o c y t e s r e t a i n e d i n f o l l i c l e s may s u f f e r t h e l o s s o r d e g e n e r a t i o n o f t h e s e m a t e r i a l s o r m e c h a n i s m s . I n t h e p i g , t h e p r o p o r t i o n o f p r i m a r y o o c y t e s w h i c h w e r e o v u l a t e d i n t o t h e o v i d u c t i n c r e a s e d w i t h t h e number o f t o t a l o v u l a t i o n s i n d u c e d b y i n c r e a s i n g d o s e s o f PMSG ( H u n t e r , 1 9 7 9 ) . C o n v e r s e l y , i n t h e s u p e r o v u l a t e d r h e s u s monkey ( B a t t a e t a l . , 1 9 7 8 ) , f o l l i c u l a r l y aged o o c y t e s w e r e o b t a i n e d upon a n a l y s i s o f f o l l i c u l a r o o c y t e s a t v a r i o u s t i m e s a f t e r o v u l a t o r y t r e a t m e n t , and o f o v u l a t e d o o c y t e s r e c o v e r e d f r o m o v i d u c t s o r u t e r i . I n t h e p i g , l a r g e d o s e s o f PMSG may h a v e s t i m u l a t e d l e s s ^ m a t u r e f o l l i c l e s t o o v u l a t e , w h e r e a s i n t h e r h e s u s monkey , s t i m u l a t i o n b y PMSG when begun d u r i n g t h e m i d - f o i l i c u l a r p h a s e o f t h e m e n s t r u a l c y c l e may h a v e r e s u l t e d i n e a r l y f o l l i c u l a r d e v e l o p m e n t a n d e n c l o s e d o o c y t e s t o be o v e r s t i m u l a t e d w i t h r e s p e c t t o d o s e o r l e n g t h o f e x p o s u r e t o FSH a c t i v i t y o f PMSG ( F l e m i n g , 1 9 8 2 ) . I t h a s a l s o b e e n s u g g e s t e d t h a t l a t e t r e a t m e n t o f PMSG i n r h e s u s monkeys m i g h t n o t " r e s c u e " d e v e l o p i n g f o l l i c l e s f r o m a t r e s i a and c o u l d l e a d t o t h e o v u l a t i o n o f p o o r q u a l i t y o o c y t e s ( F l e m i n g , 1 9 8 2 ) , I n t r a f o l l i c u l a r a g i n g o f t h e o o c y t e h a s b e e n a s s o c i a t e d w i t h a n i n c r e a s e d i n c i d e n c e o f d e v e l o p m e n t a l a n o m a l i e s w h i c h i n c l u d e a n i n c r e a s e i n p o l y s p e r m y , d e g e n e r a t i o n o f f e r t i l i z e d o o c y t e s , i r r e g u l a r c e l l membranes , - 22 -u n e q u a l c l e a v a g e , l a r g e p o l a r b o d i e s , r e t a r d e d d e v e l o p m e n t a n d a b n o r m a l m o r p h o l o g y o f b l a s t o c y s t s ( B u t c h e r and P a g e , 1 9 8 1 ) . A s t u d y o f e x p e r i m e n t a l d e l a y o f o v u l a t i o n b y b l o c k i n g p r e o v u l a t o r y LH s u r g e w i t h s o d i u m p e n t o b a r b i t a l i n c y c l i n g a d u l t r a t s h a s shown t h a t p r e o v u l a t o r y a g i n g o f o o c y t e s i n t h e f o l l i c l e s r e s u l t s i n a d e c r e a s e i n f e r t i l i z a t i o n r a t e (78% v s 93%) and a n i n c r e a s e i n a b n o r m a l d e v e l o p m e n t a t t h e p r o n u c l e a r , 2 - c e l l and b l a s t o c y s t s t a g e s (29.0% v s 3.6%, 14 .1% v s 4.3% and 53.6% v s 2 0 . 3 % , r e s p e c t i v e l y ) , when c o m p a r e d t o u n t r e a t e d c o n t r o l s (Fugo a n d B u t c h e r , 1 9 6 6 ) . The i n t r a f o l l i c u l a r l y aged o o c y t e s a f t e r a 48 h r d e l a y o f o v u l a t i o n a r e c h a r a c t e r i z e d b y t h e l a c k o f a c o n t i n u o u s l a y e r o f c o r t i c a l g r a n u l e s w h i c h l i n e t h e p l a s m a membrane ( P e l u s o and B u t c h e r , 1 9 7 4 ) . S i n c e t h e c o r t i c a l l a y e r i s i n v o l v e d i n t h e p r o t e c t i v e mechan i sm a g a i n s t p o l y s p e r m y ( A u s t i n , 1 9 5 6 ) , t h e r e d u c e d c o r t i c a l complement o f t h e aged o o c y t e s c o u l d be i n e f f e c t i v e i n b l o c k i n g p o l y s p e r m y , t h u s p r o v i d i n g a p o s s i b l e e x p l a n a t i o n f o r t h e i n c r e a s e d i n c i d e n c e o f t h e d e v e l o p m e n t a l a n o m a l i e s i n t h e aged o o c y t e s ( P e l u s o and B u t c h e r , 1 9 7 4 ) . The same a u t h o r s have a l s o s u g g e s t e d t h a t p r e m a t u r e i n d u c t i o n o f m i t o c h o n d r i a l m o r p h o l o g y o f t h e f o l l i c u l a r l y aged o o c y t e s s h o w i n g e l o n g a t i o n w i t h a s h e l f - l i k e c r i s t a e s t r u c t u r e a s s o c i a t e d w i t h a h i g h m e t a b o l i c s t a t e may r e s u l t i n t h e i n c r e a s e d i n c i d e n c e o f s u b s e q u e n t d e v e l o p m e n t a l a b n o r m a l i t i e s . A n o t h e r i m p o r t a n t s o u r c e o f d e v e l o p m e n t a l d e f e c t s i s a n o m a l o u s f e r t i l i z a t i o n c a u s e d b y a g i n g o f gametes w i t h i n t h e o v i d u c t a f t e r o v u l a t i o n ( J u e t t e n and B a v i s t e r , 1 9 8 3 ) . A number o f s t u d i e s h a v e shown t h a t d e l a y e d m a t i n g a d v e r s e l y a f f e c t s e m b r y o g e n e s i s ( B l a n d a u and Y o u n g , 1939; H u n t e r , 1 9 8 0 ; E d w a r d s , 1 9 8 0 ) . R a p i d d e t e r i o r a t i o n o f t h e a g e d o o c y t e s a f t e r - 23 -o v u l a t i o n i s b e l i e v e d t o a c c o u n t f o r more t h a n two p o l a r b o d i e s , f o r m a t i o n o f a n e x t r a p r o n u c l e u s and d e c r e a s e d a b i l i t y t o g o t h r o u g h e a r l y c l e a v a g e d i v i s i o n s (Chang a n d F e r n a n d e z - C a n o , 1 9 5 8 ; J u e t t e n a n d B a v i s t e r , 1 9 8 3 ) . Chang (1952) i n s e m i n a t e d r a b b i t s a t t h e t i m e o f i n d u c e d o v u l a t i o n and f o u n d t h a t o n l y 51% o f o o c y t e s were f e r t i l i z e d ; a b o u t h a l f t h e f e r t i l i z e d o o c y t e s d e v e l o p e d i n t o b l a s t o c y t s , and 12% o f t h e s e were p l a i n l y a b n o r m a l . E v e n when r a b b i t s w e r e i n s e m i n a t e d 2 h r b e f o r e o v u l a t i o n , 60% o f t h e o o c y t e s w e r e l o s t b e f o r e i m p l a n t a t i o n ; a t a l a t e r s t a g e , t h e same a u t h o r n o t e d t h a t o n l y 35% o f i m p l a n t a t i o n s i t e s had a b n o r m a l f e t u s e s — 6% w e r e s t i l l d e v e l o p i n g b u t were a b n o r m a l , w h i l e 59% w e r e r e s o r b i n g o r had a l r e a d y d i s a p p e a r e d . The t i m e r e l a t i o n s h i p b e t w e e n o o c y t e a g i n g and a n o m a l i e s o f f e r t i l i z a t i o n h a s c o n v i n c i n g l y b e e n d e m o n s t r a t e d b y B l a n d a u (1952) who f o l l o w e d up h i s e a r l i e r s t u d i e s b y a r t i f i c i a l l y i n s e m i n a t i n g a l b i n o r a t s o f t h e W i s t a r s t r a i n a t v a r i o u s t i m e s i n r e l a t i o n t o o v u l a t i o n , and k i l l i n g them s u f f i c i e n t l y e a r l y t o be a b l e t o o b s e r v e t h e c o u r s e o f f e r t i l i z a t i o n a n d e a r l y c l e a v a g e . The r e s u l t s o f a b o v e s t u d y showed t h a t , whe reas o n l y 1.4% o f o o c y t e s w e r e u n d e r g o i n g a b n o r m a l f e r t i l i z a t i o n when i n s e m i n a t i o n was done b e f o r e o v u l a t i o n , t h e r e w e r e p r o g r e s s i v e l y h i g h e r f r e q u e n c i e s w i t h t h e t i m e o f i n s e m i n a t i o n a f t e r o v u l a t i o n , r i s i n g t o 14% a t 6 t o 8 h r a n d 43% a t 9 t o 12 h r . D e g e n e r a t i v e c h a n g e s o f c u m u l u s and c o r o n a r a d i a t a c e l l s a n d o t h e r m o r p h o l o g i c a l c h a n g e s i n c l u d i n g t h e l o s s o f p r o x i m i t y o f c o r t i c a l g r a n u l e s t o t h e c e l l membrane h a v e b e e n n o t e d i n mouse o o c y t e s a g e d f o r 18 t o 24 h r a f t e r o v u l a t i o n ( S z o l l o s i , 1 9 7 1 ) . I n m o s t mammals, o v u l a t i o n i s c l o s e l y s y n c h r o n i z e d w i t h m a t i n g , s o t h e r e i s l i t t l e c h a n c e t h a t , u n d e r n o r m a l c i r c u m s t a n c e s , a g e d o o c y t e s w i l l - 24 -become f e r t i l i z e d ( J u e t t e n and B a v i s t e r , 1 9 8 3 ) . S i m i l a r l y , i n i m m a t u r e r a t s t r e a t e d w i t h l o w d o s e s o f PMSG, n o r m a l p r e g n a n c y w i t h o u t a n y s i g n i f i c a n t e m b r y o n i c and f e t a l w a s t a g e c o u l d b e m a i n t a i n e d ( N u t i e t a l . , 1 9 7 5 ) , s i n c e o v u l a t o r y r e s p o n s e i n c l u d i n g p r e o v u l a t o r y g o n a d o t r o p i n s e c r e t i o n a n d o o c y t e m a t u r a t i o n i s s y n c h r o n i z e d ( S o r r e n t i n o e t a l . , 1 9 7 2 ; C o s t o f f e t a l . , 1 9 7 4 ; H i l l e n s j o e t a l . , 1 9 7 4 ; W a l t o n e t a l . , 1 9 8 3 ) . However , s u p e r o v u l a t o r y d o s e s o f PMSG l e a d s t o a s y n c h r o n o u s i n d u c t i o n o f o v u l a t i o n w i t h a n e x t e n d e d p e r i o d i n immature r a t s (De L a L a s t r a e t a l . , 1 9 7 2 ; W a l t o n e t a l . , 1 9 8 3 ) , i n c y c l i n g a d u l t m i c e ( S t e r n and S c h e u t z , 1 9 7 0 ) , and i n c a t t l e ( B e l l o w s and S h o r t , 1 9 7 2 ) . I f more t h a n one o v u l a t i o n wave i s o c c u r i n g w i t h x s u p e r o v u l a t o r y t r e a t m e n t , a g i n g o f t h e o o c y t e s may c o n t r i b u t e t o a n i n c r e a s e d i n c i d e n c e o f a b n o r m a l embryos ( G r a y and C h r i s m a n , 1 9 7 9 ) . I t a p p e a r s t h a t a p o o l o f s u p e r o v u l a t e d o o c y t e s may i n c l u d e h e t e r o l o g o u s m a t u r i t y . F l e m i n g (1982) h a s s u g g e s t e d t h a t s u p e r o v u l a t o r y t r e a t m e n t may e i t h e r " f o r c e " l e s s - m a t u r e f o l l i c l e s , w h i c h l a c k t h e a b i l i t y t o r e s p o n d f u l l y t o g o n a d o t r o p i c s t i m u l a t i o n , t o o v u l a t e immature o o c y t e s ; o r i f i n i t i a t e d a f t e r t h e d i s t i n c t i o n o f d e v e l o p i n g f o l l i c l e s i n t o o v u l a t o r y and a t r e t i c s u b g r o u p s may i n d u c e o v u l a t i o n o f o v e r - m a t u r e d o r d e g e n e r a t i n g o o c y t e s . D . M a t e r n a l E n v i r o n m e n t . An a l t e r e d endocrine e n v i r o n m e n t , e s p e c i a l l y e l e v a t e d s t e r o i d c o n c e n t r a t i o n s , i s commonly a s s o c i a t e d w i t h s u p e r o v u l a t o r y t r e a t m e n t s i n t h e h a m s t e r ( B a r a n c z u k and G r e e n w a l d , 1 9 7 3 ; G r e e n w a l d , 1 9 7 6 ) , t h e r a b b i t ( F u j i m o t o and T a n a k a , 1 9 7 7 ) , t h e r a t ( M i l l e r and A r m s t r o n g , 1 9 8 1 b ; W a l t o n - 25 -and A r m s t r o n g , 1 9 8 1 , 1 9 8 2 ) , t h e cow (Saumande, 1980) and t h e human ( V a i t u k a i t i s a n d R o s s , 1 9 7 6 ) . E l e v a t e d s t e r o i d l e v e l s a t o v u l a t i o n have b e e n s u g g e s t e d t o e x e r t d e l e t e r i o u s e f f e c t s o n t h e n o r m a l d e v e l o p m e n t o f s u p e r o v u l a t e d o o c y t e s i n c l u d i n g a l t e r a t i o n o f s p e r m c a p a c i t a t i o n i n t h e f e m a l e t r a c t and o v i d u c t a l t r a n s p o r t o f c l e a v e d e m b r y o s , a n d i n t e r f e r e n c e i n t h e f o r m a t i o n and f u n c t i o n o f t h e C L ( G r e e n w a l d , 1 9 6 7 ; C h a n g , 1 9 7 0 ; Humphrey, 1 9 7 5 ) . H i g h c i r c u l a t i n g and o v a r i a n 1 7 f i ^ e s t r a d i o l may be d e t r i m e n t a l t o e a r l y embryo d e v e l o p m e n t , s i n c e 2 - c e l l mouse embryos c u l t u r e d i n o v i d u c t a l f l u i d f r o m e s t r o g e n - d o m i n a t e d d o n o r s w e r e s i g n i f i c a n t l y l e s s a b l e t o d e v e l o p t o m o r u l a e o r b l a s t o c y s t s ( C l i n e e t a l . , 1 9 7 7 ) . S i m i l a r l y , i t h a s b e e n shown t h a t t h e i n f l u e n c e o f 1 7 6 - e s t r a d i o l i n t h e a m p u l l a o f t h e o v a r i e c t o m i z e d r a b b i t i s d e l e t e r i o u s t o n o r m a l embryo d e v e l o p m e n t i n v i v o ( S t o n e a n d Hamner, 1977) and i n v i t r o ( S t o n e e t a l . , 1 9 7 7 ) . G r e e n w a l d (1961) f o u n d t h a t a s i n g l e i n j e c t i o n o f 1 7 f l - e s t r a d i o l c y c l o p e n t y l p r o p i o n a t e o n Day 1 o f p r e g n a n c y i n immature r a t s c a u s e d t h e i n c r e a s e d t u b a l and u t e r i n e m o t i l i t y s o t h a t f e r t i l i z e d o o c y t e s w e r e e x p e l l e d f r o m t h e u t e r u s w i t h i n 48 h r p o s t c o i t u m . T h u s , i t h a s b e e n s u g g e s t e d t h a t t h e r a p i d l o s s o f p r e i m p l a n t a t i o n embryos f r o m t h e o v i d u c t s a n d u t e r i n e h o r n s o f s u p e r o v u l a t e d r a t s may be c a u s e d b y t h e p r e - o r p o s t - o v u l a t o r y e l e v a t i o n o f s e r u m 1 7 f l - e s t r a d i o l l e v e l s o b s e r v e d Days 2 - 5 a f t e r i n j e c t i o n o f a s u p e r o v u l a t o r y d o s e o f PMSG ( M i l l e r a n d A r m s t r o n g , 1 9 8 1 b ) . On t h e o t h e r h a n d , i n t h e same s t u d y , t h e embryos i n s u p e r o v u l a t e d r a t s f r e q u e n t l y r e m a i n e d i n t h e o v i d u c t s u n t i l Days 6 a n d 7 a f t e r t r e a t m e n t , and t h e i r f u r t h e r d e v e l o p m e n t t o m o r u l a e was i m p a i r e d . The d e v e l o p m e n t o f r a t embryos i n v i v o t o t h e b l a s t o c y s t - 26 -s t a g e was r e t a r d e d i n a n i m a l s w h i c h h a v e e x p e r i e n c e d h i g h s e r u m 1 7 f l - e s t r a d i o l l e v e l s f o r a n e x t e n d e d p e r i o d p r i o r t o o v u l a t i o n (Fugo a n d B u t c h e r , 1 9 6 6 ; B u t c h e r and P o p e , 1 9 7 9 ) . I t h a s b e e n shown t h a t t h e o v i d u c t s u n d e r e s t r o g e n dominance s e c r e t e a l o w m o l e c u l a r w e i g h t s u b s t a n c e t o i n h i b i t t h e d e v e l o p m e n t o f embryos t o m o r u l a e ( S t o n e a n d Hamner, 1 9 7 7 ; S t o n e e t a l . , 1 9 7 7 ; C l i n e e t a l . , 1 9 7 7 ) . T h e r e f o r e , t h e r e c o u l d be t w o p o s s i b i l i t e s o f d e t r i m e n t a l e f f e c t s o f h i g h l e v e l s o f c i r c u l a t i n g 1 7 f i - e s t r a d i o l a f t e r s u p e r o v u l a t i o n o n embryo d e v e l o p m e n t i n o v i d u c t s a n d u t e r i . I f t r a n s p o r t i s a c c e l e r a t e d , embryos a r r i v i n g i n t h e u t e r u s p r e m a t u r e l y do n o t d e v e l o p n o r m a l l y and t h u s may d e g e n e r a t e o r a r e p r o m p t l y e x p e l l e d f r o m t h e u t e r u s v i a t h e c e r v i x ( G r e e n w a l d , 1 9 6 1 , 1 9 6 7 ; B e n n e t t , 1 9 7 0 ) . I t h a s a l s o b e e n s u g g e s t e d t h a t h i g h l e v e l s o f s t e r o i d s , p a r t i c u l a r l y e s t r o g e n , t h r o u g h t h e e f f e c t s o n o v i d u c t a l m u s c u l a t u r e a n d s p h i n c t e r , may r e t a i n t h e embryos i n t h e o v i d u c t and p r e v e n t o r d e l a y t h e i r e n t r y i n t o t h e u t e r u s s u c h t h a t t h e s y n c h r o n y be tween embryo and s e n s i t i z e d u t e r u s i s l o s t ( F l e m i n g , 1 9 8 2 ) . Under n o r m a l c i r c u m s t a n c e s , t h e s e c r e t i o n o f s t e r o i d hormones b y o v a r i a n t i s s u e s d u r i n g t h e e s t r o u s c y c l e and e a r l y p r e g n a n c y e n s u r e s t h a t t h e u t e r i n e s e n s i t i v i t y f o r i m p l a n t a t i o n c o i n c i d e s w i t h t h e p r e s e n c e o f a m a t u r e b l a s t o c y s t ( P s y c h o y o s , 1 9 7 3 ) . The same a u t h o r i n d i c a t e d t h a t , i n t h e r a t , b o t h e s t r o g e n a n d p r o g e s t e r o n e a r e e s s e n t i a l f o r i m p l a n t a t i o n . I t h a s b e e n f u r t h e r s u g g e s t e d t h a t t h e o p t i m a l c o n d i t i o n s f o r embryo a t t a c h m e n t a n d i m p l a n t a t i o n depend o n a d e l i c a t e l y b a l a n c e d s y n e r g i s t i c a c t i o n o f e s t r o g e n a n d p r o g e s t e r o n e o n t h e e n d o m e t r i u m ( O o u r r i e r , 1 9 5 0 ) , a n d t h i s b a l a n c e may h a v e b e e n d i s t u r b e d b y t h e d i f f e r e n t r a t i o o f 1 7 f l - e s t r a d i o l t o p r o g e s t e r o n e i n s u p e r o v u l a t e d r a t s ( W a l t o n a n d A r m s t r o n g , 1 9 8 2 ) . A l t h o u g h , DeFeo (1963) - 27 -has shown that high levels of progesterone do not inhibi t uterine sens i t iv i ty , the high levels of preovulatory progesterone in superovulated rats (Walton and Armstrong, 1981) may cause uterine changes that are detrimental to blastocyst implantation by disturbing the optimum rat io of 17B-estradiol to progesterone (Walton et a l . , 1982). In addition to local or systemic endocrine changes, superovulation may alter the endocrine microenvironment through the steroidogenic capabi l i t ies of the more abundant cumulus masses and preimplantation embryos (Heap et a l . , 1979; Saurer, 1979; Sherizly and Kraicer, 1980). - 28 -MATERIALS AND METHODS I. Animals. Immature female Sprague-Dawley rats were purchased from Charles River Canada Inc. (St. Constant, Quebec) at 22 days of age. All animals were kept under temperature- and light-controlled conditions (20-25°C, 12L:12D) in an air-conditioned room and fed standard rat chow and water ad  libitum throughout the treatment period. II. Experimental Design. Protocol 1. The effects of different doses of PMSG on ovulatory response and oocyte quality (gross morphology) were examined with reference to steroid hormone levels in ovarian tissues and peripheral blood. At the age of 28 days, animals received 4 IU (control dose), 20 IU (intermediate superovulation dose), or 40 IU (high superovulation dose) of PMSG between 0830 and 0900 h. PMSG (Equinex, Ayerst, Montreal, Quebec) was subcutaneously administered in 0.4 ml of 0.9% NaCl solution. Following PMSG injection, the rats were killed by cervical dislocation at 6 or 12 hr intervals (n=5-8 rats at each time point). Trunk blood was collected in glass tubes and allowed to stand at room temperature for 1/2-1 hr to clot. The samples were then stored for 12-24 hr at 4°C and centrifuged at 2000 rpm for 10 min to separate serum. At the time of sacrifice, ovaries were dissected free from oviducts, cleaned of bursae, connective tissue and fat, and crushed in 1 ml absolute ethanol with a glass rod. The sera and ovarian homogenate were stored at -20°C - 29 -f o r s u b s e q u e n t a s s a y o f 1 7 B - e s t r a d i o l , p r o g e s t e r o n e a n d a n d r o g e n s u s i n g R I A . Some o v a r i e s o b t a i n e d a t 24 o r 48 h r a f t e r PMSG i n j e c t i o n w e r e p r o c e s s e d f o r h i s t o l o g i c a l e x a m i n a t i o n o f f o l l i c l e s . O o c y t e s w e r e c o l l e c t e d f r o m o v i d u c t s a n d i m m e d i a t e l y s u b j e c t e d t o t h e a s s e s s m e n t o f o v u l a t o r y r e s p o n s e and o o c y t e g r o s s m o r p h o l o g y . P r o t o c o l 2 . To examine t h e f o l l i c u l a r s t e r o i d c o n t e n t s and o o c y t e m a t u r a t i o n f o l l o w i n g PMSG t r e a t m e n t s , a n i m a l s w e r e a l l o t e d t o t h e two g r o u p s r e c e i v i n g 4 I U o r 20 I U o f PMSG a t t h e age o f 28 d a y s , and k i l l e d a t 24 , 4 8 , 60 o r 72 h r a f t e r PMSG (n=5-7 r a t s a t e a c h t i m e p o i n t ) . O v a r i e s o b t a i n e d a t e a c h t i m e p e r i o d w e r e d i s s e c t e d f r e e , c l e a n e d o f b u r s a e , c o n n e c t i v e t i s s u e , and f a t . A h e a t - d r a w n m i c r o p i p e t t e c o n n e c t e d t o 20 cm o f p l a s t i c t u b i n g ( i . d . , 1 mm) was a t t a c h e d t o a 10 m l s y r i n g e . Under a d i s s e c t i n g m i c r o s c o p e ( N i k o n , O p t i p h o t ) , e a c h f o l l i c l e was c a r e f u l l y p u n c t u r e d a n d f o l l i c u l a r f l u i d was c o l l e c t e d b y s u c t i o n . The f o l l i c u l a r f l u i d p o o l e d f r o m a l l v i s i b l e a n t r a l f o l l i c l e s o n e a c h p a i r o f o v a r i e s was e x p e l l e d i n t o a n a r r o w c e n t r i f u g e t u b e . A f t e r c e n t r i f u g a t i o n a t 1000 rpm f o r 5 m i n , t h e v o l u m e o f s u p e r n a t a n t f o l l i c u l a r f l u i d was measu red w i t h a n E p p e n d o r f m e c h a n i c a l p i p e t t e ( 0 . 5 - 1 0 y l ) . The v o l u m e s o f f o l l i c u l a r f l u i d o b t a i n e d f r o m e a c h p a i r o f o v a r i e s r a n g e d f r o m 2 - 1 0 y l ; t h e y were made up t o a t o t a l v o l u m e o f 1 .0 m l w i t h s a l i n e and s t o r e d a t - 2 0 ° C f o r t h e s u b s e q u e n t a s s a y o f 17fl - e s t r a d i o l , p r o g e s t e r o n e , a n d a n d r o g e n s . The o o c y t e s , r e c o v e r e d f r o m o v i d u c t s a t 72 h r (4 I U PMSG) a n d 2 4 , 48 o r 72 h r (20 I U P M S G ) , w e r e c l a s s i f i e d a s d e g e n e r a t e o r n o r m a l i n a p p e a r a n c e . O n l y m o r p h o l o g i c a l l y n o r m a l o o c y t e s w e r e s u b j e c t e d t o t h e s t a i n i n g o f n u c l e u s f o r t h e a s s e s s m e n t o f e a c h s t a g e o f n u c l e a r m a t u r a t i o n . - 30 -P r o t o c o l 3 . To e x a m i n e t h e c i r c u l a t i n g LH r e s p o n s e t o PMSG t r e a t m e n t s , a n i m a l s were d i v i d e d i n t o t h e two g r o u p s r e c e i v i n g 4 I U o r 20 I U o f PMSG (n=7 s a m p l e s a t e a c h t i m e p o i n t ) . One d a y b e f o r e t h e e x p e r i m e n t (Day 27 o f a g e ) , t h e r a t s w e r e i n s t a l l e d w i t h c h r o n i c a l l y i n d w e l l i n g c a t h e t e r s u s i n g t h e method d e s c r i b e d b y Harms a n d O j e d a ( 1 9 7 4 ) . B r i e f l y , t h e r i g h t j u g u l a r v e i n was e x p o s e d a f t e r i n d u c t i o n o f a n e s t h e s i a w i t h s o d i u m p e n t o b a r b i t a l ( S o m n o t a l , S i g m a C h e m i c a l C o . , S t . L o u i s , MO) a t a d o s e o f 35 mgAg o f b o d y w e i g h t and a c u r v e d 22-gauge n e e d l e was u s e d t o p l a c e a c a t h e t e r made o f s i l a s t i c t u b i n g ( D o w - C o r n i n g C o r p . , M i d l a n d , M I ) i n t o t h e v e i n . The c a t h e t e r was s l i d i n t o t h e v e i n and made t o e n t e r o r a p p r o a c h t h e r i g h t a t r i u m . The c a t h e t e r was t h e n a n c h o r e d o n t o t h e p e c t o r a l m u s c l e u n d e r w h i c h t h e j u g u l a r v e i n p a s s e s . The e x p o s e d end o f t h e c a t h e t e r was t h e n p a s s e d u n d e r n e a t h t h e s k i n and e x t e r i o r i z e d a t t h e back o f t h e neck a n d t i e d s h u t . On t h e f o l l o w i n g d a y , t h e j u g u l a r c a t h e t e r was c o n n e c t e d w i t h a f l e x i b l e p i e c e o f h e p a r i n i z e d p o l y e t h y l e n e t u b i n g ( F i s h e r S c i e n t i f i c C o . , N J , P E - 5 0 ) f o r w i t h d r a w a l o f b l o o d s a m p l e s . To p r o v i d e t h e b a s a l l e v e l o f se rum L H , 0 . 5 m l o f w h o l e b l o o d was c o l l e c t e d f r o m i n d i v i d u a l r a t s v i a t h e j u g u l a r c a t h e t e r i m m e d i a t e l y b e f o r e a d m i n i s t r a t i o n o f PMSG. A l l r a t s i n b o t h g r o u p s were b l e d 0 . 5 m l w h o l e b l o o d a t 12 h r and s u b s e q u e n t l y a t 6 h r i n t e r v a l s u n t i l s a c r i f i c e a t 72 h r a f t e r PMSG. To p r e v e n t a c u t e s h o c k f r o m b l o o d l o s s and c l o t f o r m a t i o n i n t h e c a t h e t e r , e a c h b l o o d s a m p l e was f o l l o w e d b y r e p l a c e m e n t o f a n a n e q u a l v o l u m e o f a d i l u t e h e p a r i n - s a l i n e s o l u t i o n (25 I U / m l ) a s d e s c r i b e d e l s e w h e r e ( W a y n f o r t h , 1 9 8 0 ) . Serum s a m p l e s ( a b o u t 200 u l p e r t u b e ) were s e p a r a t e d b y c e n t r i f u g a t i o n and s t o r e d a t - 2 0 ° C u n t i l L H R I A . - 31 -At the time of sacri f ice at 72 hr , ovaries were dissected and weighed as a pa ir . Ovulation was also assessed by counting the oocytes recovered from oviducts. Protocol 4. The effects of an androgen antagonist (flutamide) on oocyte quality (gross morphology) and developmental potential of preimplantation embryos after treatments with PMSG were examined. I n i t i a l l y , animals were divided into the two treatment groups receiving 5 mg flutamide or ethanol-saline vehicle at 30 and 36 hr, and one pre-treatment control group receiving no further treatment after administration of 40 IU PMSG at the age of 28 days. The animals of treatment groups were k i l l ed at 48, 60 or 72 hr after PMSG (n=6 or 7 rats at each time point) and those of pre-treatment control group were k i l l ed at 24 hr (n=6 rats) . Flutamide (a,a,a-trifluoro-2-^nethyl- 4'-nitro-m-lacto-toluidide, Schering Pharmaceuticals, Kenilworth, NJ) was dissolved in ethanol and then diluted with an equal volume of saline as described previously (Peluso et a l . , 1981), and subcutaneously administered in a volume of 0.4 ml. In an extension to t h e . i n i t i a l experiment, the rats subsequently to be mated, were randomly al lotted to the three different groups. The females of one group received 4 IU PMSG alone. The females of the other two groups were given 40 IU PMSG with either flutamide or ethanol-saline vehicle as described previously. At 60 hr after PMSG, the females had their vaginae gently opened with saline-soaked cotton swabs and were caged with mature f e r t i l e Sprague-Dawley males (one male and two females per breeding cage). The females were separated from males and scored for the occurrence of mating at 72 hr (designated as Day 1 of pregnancy). A sperm-positive score - 32 -was n o t e d b y t h e p r e s e n c e o f a c o p u l a t o r y p l u g i n t h e v a g i n a a n d / o r s p e r m a t o z o a i n v a g i n a l s m e a r s . The ma ted f e m a l e s were k i l l e d b e t w e e n 0900-1000 h o n Days 2 , 3 , 4 o r 5 o f p r e g n a n c y (n=5 o r 6 r a t s o n e a c h d a y o f p r e g n a n c y ) . A t t h e t i m e o f s a c r i f i c e i n b o t h t h e e x p e r i m e n t s , o v a r i a n t i s s u e s a n d / o r b l o o d w e r e p r e p a r e d and s t o r e d f o r t h e a s s a y o f 1 7 B - e s t r a d i o l , p r o g e s t e r o n e , and a n d r o g e n s . O o c y t e s a n d embryos were c o l l e c t e d f r o m o v i d u c t s a n d / o r u t e r i n e h o r n s , and s u b j e c t e d t o t h e a s s e s s m e n t o f o v u l a t i o n , g r o s s m o r p h o l o g y o f o o c y t e o r p r e i m p l a n t a t i o n e m b r y o , and d e v e l o p m e n t a l s t a g e . A n o t h e r s e p a r a t e e x p e r i m e n t was p e r f o r m e d t o d e t e r m i n e m a j o r a n d r o g e n i c s t e r o i d s i n s e rum o f PMSG-and f l u t a m i d e - t r e a t e d " r a t s . The r a t s r e c e i v e d 4 ID PMSG a l o n e o r 20 I U PMSG w i t h e i t h e r a c o m b i n a t i o n o f f l u t a m i d e o r e t h a n o l - s a l i n e v e h i c l e , and t h e i r t r u n k b l o o d s a m p l e s w e r e c o l l e c t e d a t 2 4 , 4 8 , o r 60 h r (n=10 s a m p l e s a t e a c h t i m e p o i n t ) . The d o s e and t i m e s c h e d u l e f o r t h e t r e a t m e n t o f f l u t a m i d e was i d e n t i c a l t o t h e p r e c e d i n g s . The s e r a were s e p a r a t e d b y c e n t r i f u g a t i o n and s t o r e d a t - 2 0 ° C u n t i l t h e s u b s e q u e n t s e p a r a t i o n o f a n d r o g e n i c s t e r o i d s u s i n g T L C . The l e v e l s o f e a c h s t e r o i d were d e t e r m i n e d u s i n g R I A . P r o t o c o l 5 . The e f f e c t s o f a p r o g e s t e r o n e a n t a g o n i s t (RU486) a n d a n e s t r o g e n a n t a g o n i s t ( t a m o x i f e n ) o n o v u l a t o r y r e s p o n s e a n d o o c y t e q u a l i t y ( g r o s s m o r p h o l o g y ) a f t e r t r e a t m e n t s w i t h PMSG w e r e e x a m i n e d . I n i t i a l l y , a n i m a l s r e c e i v e d 4 I U PMSG a t t h e age o f 28 d a y s a n d were d i v i d e d i n t o t h r e e g r o u p s r e c e i v i n g t h r e e c o n s e c u t i v e i n j e c t i o n s o f 1 mg R U 4 8 6 , 1 mg t a m o x i f e n o r v e h i c l e a l o n e a t 2 4 , 36 and 48 h r a f t e r PMSG. A l l r a t s w e r e s a c r i f i c e d - 33 -a t 72 h r (n=7 r a t s ) . RU486 (RU38486 , l l f i - ( 4 - d i m e t h y l a n u . n o p h e n y l ) - 1 7 f i -h y d r o x y , - 1 7 a - ( p r o p - l - y n y l ) - e s t r a - 4 , 9 - d i e n - 3 - o n e , R o u s s e l U c l a f , R o m a i n v i l l e , F r a n c e ) and t a m o x i f e n ( I . C . I . 4 6 , 4 7 4 , t r a n s i s o m e r o f l - ( p - B - d i m e t h y l a m i n o e t h o x y p h e n y l ) - 1 , 2 - d i p h e n y l b u t - l - e n e , S i g m a C h e m i c a l C o . , S t . L o u i s , MO; f r e e b a s e ) w e r e s u b c u t a n e o u s l y a d m i n i s t e r e d i n 0 . 4 m l o f sesame o i l s u s p e n s i o n c o n t a i n i n g 5% e t h a n o l a s a v e h i c l e . I n a n e x t e n s i o n t o t h e i n i t i a l e x p e r i m e n t , t h e r a t s w e r e i n j e c t e d w i t h 40 I U PMSG and d i v i d e d i n t o f o u r g r o u p s r e c e i v i n g e i t h e r no f u r t h e r i n j e c t i o n s ( p r e - t r e a t m e n t c o n t r o l ) , 1 mg RU 4 8 6 , 2 . 5 mg t a m o x i f e n , o r v e h i c l e a l o n e f o l l o w i n g t h e i n j e c t i o n s c h e d u l e d e s c r i b e d p r e v i o u s l y . The f e m a l e s w e r e k i l l e d a t 12 h r ( p r e - t r e a t m e n t c o n t r o l ) , 60 o r 72 h r a f t e r PMSG (n=5-7 r a t s a t e a c h t i m e p o i n t ) . A t e a c h t i m e o f s a c r i f i c e , t r u n k b l o o d was c o l l e c t e d a n d s t o r e d a t 4 ° C p r i o r t o t h e s e p a r a t i o n o f s e r u m b y c e n t r i f u g a t i o n f o r t h e a s s a y o f 1 7 B - e s t r a d i o l , p r o g e s t e r o n e , a n d a n d r o g e n s . O v a r i e s w e r e d i s s e c t e d and w e i g h e d a s a p a i r . O o c y t e s w e r e r e c o v e r e d f r o m o v i d u c t s a n d s u b j e c t e d t o t h e a s s e s s m e n t o f o v u l a t i o n and o o c y t e g r o s s m o r p h o l o g y . I I I . Asses smen t o f O v u l a t i o n , O o c y t e M o r p h o l o g y and Embryo D e v e l o p m e n t . O v i d u c t s and u t e r i n e h o r n s were s e p a r a t e d a t t h e u t e r o t u b a l j u n c t i o n . The o v i d u c t s o b t a i n e d f r o m e a c h f e m a l e were p l a c e d i n a few d r o p s o f DPBS i n a 10x35 mm p e t r i d i s h ( F a l c o n P l a s t i c s , L o s A n g e l e s , C A ) . Unde r a s t e r e o d i s s e c t i n g m i c r o s c o p e (10X m a g n i f i c a t i o n , N i k o n S M Z - 1 0 ) , o o c y t e mass was r e a d i l y a c c e s s i b l e i n t h e e n l a r g e d and t r a n s l u c e n t a m p u l l a r e g i o n o f t h e o v i d u c t . T h i s d i s t e n d e d segment was t h e n p u n c t u r e d a n d t h e o o c y t e -• 34 -mass was e x p e l l e d . S u b s e q u e n t l y , i n o r d e r t o f a c i l i t a t e t h e o o c y t e c o u n t i n g , t h e e x t r a c o r o n a l c u m u l u s c e l l s s u r r o u n d i n g t h e o o c y t e s w e r e d i s p e r s e d a f t e r b e i n g e x p o s e d t o a few d r o p s o f DPBS c o n t a i n i n g 0.1% o f h y a l u r o n i d a s e ( o v i n e t y p e II , S igma C h e m i c a l C o . , S t . L o u i s , MO) f o r 5 -10 m i n . S p e c i f i c a l l y , i n p r e g n a n t f e m a l e s o f t h e p r o t o c o l 4 , t h e o v i d u c t was f l u s h e d w i t h 0 . 2 m l DPBS by i n s e r t i n g a b l u n t - e n d e d 3 0 ' g a u g e n e e d l e t h r o u g h t h e i n f u n d i b u l u m , and t h e u t e r i n e h o r n was f l u s h e d w i t h 0 . 5 m l DPBS by i n s e r t i n g a b l u n t - e n d e d 21 gauge n e e d l e t h r o u g h t h e w a l l o f i t s c e r v i c a l e n d . The r e c o v e r e d o o c y t e s o r embryos were c o u n t e d unde r t h e s t e r e o d i s s e c t i n g m i c r o s c o p e (40X m a g n i f i c a t i o n ) and e x a m i n e d w i t h o u t s t a i n i n g u n d e r a p h a s e - c o n t r a s t m i c r o s c o p e (100X m a g n i f i c a t i o n , N i k o n O p t i p h o t ) . The o c c u r r e n c e o f f r a g m e n t a t i o n and o t h e r d e g e n e r a t i v e changes was a s s e s s e d , a s d e s c r i b e d e l s e w h e r e ( E l s d e n e t a l . , 1 9 7 8 ; M i l l e r and A r m s t r o n g , 1 9 8 1 b ) . B r i e f l y , t h o s e e g g s s h o w i n g a n i r r e g u l a r c e l l mass w i t h d e b r i s , a n a rmophous opaque mass o f v i t e l l i n e m a t e r i a l , empty z o n a p e l l u c i d a , a g r e a t d i f f e r e n c e i n s i z e o f b l a s t o m e r e s and a h a z y b l a s t o m e r e o u t l i n e were c l a s s i f i e d a s a b n o r m a l . A m a j o r p r o p o r t i o n o f a b n o r m a l i t i e s was d e r i v e d f r o m f r a g m e n t a t i o n . The d e v e l o p m e n t a l s t a g e s o f p r e i m p l a n t a t i o n embryos w e r e a l s o r e c o r d e d . I V . A s s e s s m e n t o f O o c y t e N u c l e a r M a t u r a t i o n . N o r m a l a p p e a r i n g o o c y t e s w e r e p l a c e d i n t o t h e F a l c o n p e t r i d i s h c o n t a i n i n g h y p o t o n i c s o d i u m c i t r a t e ( 1 . 0 % , A n a l a R , T o r o n t o ) a n d s w o l l e n a t - 35 -room temperature for about 10 min. The oocytes were subsequently transferred onto a grease-free slide with a thin coat of Mayer's albumen (Jones, 1966), a mixture of an equal volume of chicken egg white and glycerol (BDH Chemicals Ltd., Toronto, Ontario), and allowed to dry over a hot plate (45-53°C) to enhance chromosome spreading. Mayer's albumen was used to attain better adhesion of the oocytes onto the slide, thus preventing the loss in the next step of fixation. The dried oocytes were then fixed with acetic alcohol, freshly made up with one part of glacial acetic acid (BDH Chemicals Ltd., Toronto, Ontario) and two parts of absolute ethyl alcohol (BDH Chemicals Ltd., Toronto, Ontario), in a Coplin jar for 45 min and stained with 2% aceto-orcein for 30 min. A stock solution of this stain had been made up in a fumehood by dissolving 2.2 g of orcein (B34063, BDH Chemicals Ltd., Toronto, Ontario) in 45 ml of hot glacial acetic acid with continuous st i r r i n g for 30 min and by adding 55 ml of 0.9% NaCl solution (Tjio and Whang, 1962). A working staining solution was prepared immediately before use by mixing two parts of the stock solution with three parts of 0.9% NaCl solution and by f i l t e r i n g with Millex PF f i l t e r (Millipore Products Division, Bedford, MA). Finally, the stained oocytes were allowed to a ser i a l dehydration through 50%, 60%, 80% and 100% ethanol followed by xylene (Anala R, Toronto, Ontario) for 5 min i n each step, and subjected to a microscopic evaluation of nuclear maturation. Various stages of jaeiosis observed in this experiment were identified according to the c r i t e r i a of Austin (1961, see Figure 1). FIGURE 1 Diagrammatic representation of preovulatory nuclear maturation stages in the rat oocyte (re-drawn from Austin, 1961). a: 'Germinal vesicle 1 stage b: 'Germinal vesicle' break-down (GVBD) stage, migration of germinal vesicle to the periphery of ooplasm, and disappearance of nucleolus and nuclear membrane c: Prophase I, speckled appearance with short segments of chromosomal threads. d: Metaphase I, arrangement of the bivalents at the equator of meiotic spindle. e: Anaphase I, movement of the bivalents to the opposite ends of the spindle. f,g: Telophase I, rotation of the spindle through 90°, and start of polar body separation with elevation of vi t e l l u s near to ••the chromosomes, .• V. h , i : Metaphase I I , emission of the f i r s t polar body with formation of a furrow around the outermost set of chromosomes. \ V. Histological Examination. Ovaries obtained at 24 or 48 hr after 4, 20 or 40 IU PMSG were immediately fixed in Bouin's solution (saturated picric acid - 75%, buffered formalin - 20%, glacial acetic acid - 5%, a l l reagents obtianed from BDH Chemicals Ltd., Toronto, Ontario) for about 4 hr and washed for 2 to 3 hrs in running tap water to remove excess fixative. The ovarian tissue was subsequently dehydrated in sequential concentrations of ethanol (70, 80, 90 and 100%), cleared, and embedded in paraffin wax. Serial sections of the tissue block were then cut 10 ym thick and stained with hematoxylin (CIN 75290, BDH Chemicals Ltd., Toronto, Ontario) and eosin (CIN 45380, BDH Chemicals Ltd., Toronto, Ontario). All sections were examined for evidence of precocious ovulation or follicular atresia, and representative sections were taken for photomicroscopy. Advanced stages of follicular atresia were defined by nuclear pyknosis and/or a loss of homogeneity of the granulosa cells in conjunction with thecal hypertrophy and varying degrees of oocyte degeneration. VI. Determination of Hormones. A. 17fi-estradiol, Progesterone and Androgens. Ovarian supernatants obtained by centrifugation, 0.5-1.0 ml aliquots of sera and follicular fluid samples were extracted twice with •:. 5.0 ml diethyl ether (anhydrous r- 3DH Chemicals Ltd., Toronto, Ontario)by vortexing for 2 min; the extracts were evaporated under nitrogen flow at 35°C and reconstituted in 1.0 ml absolute ethanol. A range of 20-100 yl aliquots of the extracts, i f necessary, after 10 or 100 times of dilution - 38 -w i t h a b s o l u t e e t h a n o l , were d r i e d down a n d a s s a y e d i n d u p l i c a t e f o r 1 7 f i - e s t r a d i o l , p r o g e s t e r o n e and a n d r o g e n s b y s p e c i f i c R I A u s i n g t h e a n t i s e r a k i n d l y d o n a t e d b y D r . D . T . A r m s t r o n g , U n i v e r s i t y o f W e s t e r n O n t a r i o , L o n d o n , O n t a r i o . N o n - r a d i o l a b e l l e d s t e r o i d s , 1 7 B - e s t r a d i o l , p r o g e s t e r o n e a n d t e s t o s t e r o n e p u r c h a s e d f r o m S i g m a C h e m i c a l C o . ( S t . L o u i s , M O ) , and r a d i o l a b e l e d s t e r o i d s [ 2 , 4 , 6 , 7 , 1 6 , 1 7 - 3 H ] 1 7 f i - e s t r a d i o l ( s p . a c t . 140 3 C i / m m o l ) , [ 1 , 2 , 6 , 7 , 1 6 , 1 7 - H ] p r o g e s t e r o n e ( s p . a c t . 112 C i / m m o l ) and 3 [ 2 , 6 , 7 - H ] t e s t o s t e r o n e ( s p . a c t . 80 C i / m m o l ) o b t a i n e d f r o m Amersham C o . ( A r l i n g t o n H e i g h t s , I L ) were u s e d a s s t a n d a r d a n d t r a c e r , r e s p e c t i v e l y . S o l v e n t s o b t a i n e d f o r s t e r o i d R I A w e r e o f a n a l y t i c a l g r a d e and u s e d w i t h o u t f u r t h e r p u r i f i c a t i o n , e x c e p t f o r e t h a n o l , w h i c h was r e d i s t i l l e d . 3 I n t h e a s s a y p r o c e d u r e s , a p p r o x i m a t e l y 1 0 , 0 0 0 cpm o f t r a c e r ( H) was added t o e a c h t u b e . The unbound hormone was removed b y t h e d e x t r a n - c o a t e d c h a r c o a l a d s o r p t i o n m e t h o d , and t h e bound s t e r o i d was c o u n t e d i n a LKB 1217 R a c k b e t a l i q u i d s c i n t i l l a t i o n c o u n t e r . The a s s a y s were l i n e a r a t 25 -300 p g / t u b e . The b i n d i n g e f f i c i e n c y (Bo) o f t h e s t e r o i d a n t i b o d i e s was 40-60% and NSB was l e s s t h a n 5%. The i n t r a - and i n t e r - a s s a y CV f o r t h e s t e r o i d a s s a y s were a s f o l l o w s : 7% a n d 5% f o r 1 7 f i - e s t r a d i o l , 8% and 9% f o r p r o g e s t e r o n e , 5% and 6% f o r a n d r o g e n s , r e s p e c t i v e l y . The r e s p e c t i v e . c r o s s - r e a c t i v i t i e s o f t h e a n t i s e r a w e r e a s f o l l o w s : - 39 -17f i-estradiol Antibody 17f i-estradiol 100.0% estrone 2.9% e s t r i o l 0.5% other major steroids <0.2% Progesterone Antibody progesterone 100.0% 5fi-pregnane-3,20-dione 35.5% 5a-pregnane-3,20-dione 15.7% 3a-hydroxy-5 g-pregnan-20-one 2.0% 206-hydroxy-4-pregnen-3-one 1.3% 17a-hydroxyprogesterone 1.2% other major steroids <0.2% Testosterone Antibody testosterone 100.0% 5a-dihydrotestosterone 75.0% 5ct-androstane-3ct,17B-diol 13.5% 5a-androstane-3B,17B-diol 10.9% 19-hydroxytestosterone 4.7% ' •••••••Qjtiier-wjor steroids <0.1% Based on the above c r o s s - r e a c t i v i t y data, the testosterone antibody was r e l a t i v e l y nonspecific, thus the steroids measured using t h i s antiserum were referred to as androgens rather than testosterone. Hormone le v e l s were - 40 -e x p r e s s e d a s n g / r a t f o r o v a r i a n s t e r o i d s , a n d n g / m l f o r s e rum a n d f o l l i c u l a r s t e r o i d s . B . A n d r o g e n i c S t e r o i d s . The s e p a r a t i o n o f m a j o r a n d r o g e n i c s t e r o i d s ( a n d r o s t e n e d i o n e , t e s t o s t e r o n e , a n d 5 a - d i h y d r o t e s t o s t e r o n e ) f r o m s e r a was u n d e r t a k e n b y T L C . The 1 .0 m l a l i q u o t s o f s e r a w e r e e x t r a c t e d t w i c e w i t h f i v e v o l u m e s o f d i e t h y l e t h e r b y v o r t e x i n g f o r 2 m i n ; t h e e x t r a c t s w e r e e v a p o r a t e d u n d e r o 14 n i t r o g e n f l o w a t 35 C . A p p r o x i m a t e l y 4000 dpm o f [ 4 - C ] 14 a n d r o s t e n e d i o n e ( s p . a c t . 5 9 . 0 m C i / m m o l ) , [ 4 - C ] 5 a - d i h y d r o t e s t o s t e r o n e 14 ( s p . a c t . 5 9 . 0 m C i / m m o l ) , and [ 4 - C ] t e s t o s t e r o n e ( s p . a c t . 5 6 . 9 mCi /mmol ) o b t a i n e d f r o m Aroersham C o . ( A r l i n g t o n H e i g h t s , I L ) were added a s i n t e r n a l s t a n d a r d s t o t h e d r i e d e x t r a c t s a n d r e c o n s t i t u t e d i n 70 p i o f a b s o l u t e e t h a n o l . The e x t r a c t s were a p p l i e d o n t o s i l i c a - c o a t e d T L C p l a t e s (Eas tman Kodak C o . , R o c h e s t e r , N Y ) , and t h e a n d r o g e n i c s t e r o i d s were s e p a r a t e d i n a s y s t e m o f c h l o r o f o r m : m e t h y l e n e c h l o r i d e : d i e t h y l e t h e r ( 4 0 ; 4 0 ; 2 0 , v : v : v ) a t 4 ° C . These s o l v e n t s , o b t a i n e d f r o m BDH C h e m i c a l s L t d . ( T o r o n t o , O n t a r i o ) , w e r e o f a n a l y t i c a l g r a d e and u s e d w i t h o u t f u r t h e r p u r i f i c a t i o n . The i s o l a t e d s t e r o i d s w e r e d e t e c t e d b y e x p o s i n g t h e ch roma tograms t o n o - s c r e e n m e d i c a l x - r a y f i l m (Eas tman Kodak C o . , R o c h e s t e r , NY) f o r 7 d a y s . L a t e r , t h e s t a n d a r d s t e r o i d s w e r e v i s u a l i z e d b y s p r a y i n g t h e ch roma tograms w i t h i^i s u l f u r i c a c i d ( J , T , B a k e r Cfcerpical Q> . f f B h U H p s b u r g , N J . ) ? e t h a n o l ( l ; l , v : v ) ^ : a n d by c h a r r i n g f o r 15 m i n a t 1 2 0 ° C i n F i s h e r 30 G L a b . o v e n ( s e e F i g u r e 2 ) . The s t e r o i d s p o t s o n t h e ch roma tog rams were i n i t i a l l y i d e n t i f i e d b y a l i g n i n g and t r a n s i l l u m i n a t i n g w i t h t h e c o r r e s p o n d i n g 41 FIGURE 2 S e p a r a t e d s t e r o i d s o n T L C s i l i c a g e l - c o a t e d p l a t e , d e v e l o p e d i n a s o l v e n t s y s t e m o f c h l o r o f o r m : m e t h y l e n e c h l o r i d e : d i e t h y l e t h e r ( 4 0 : 4 0 : 2 0 , v : v : v ) . The d e v e l o p e d ch roma tog rams w e r e e x p o s e d t o n o - s c r e e n x - r a y f i l m i n o r d e r t o l o c a l i z e l a b e l l e d a n d r o g e n i c s t e r o i d s . 4i<a PROGESTERONE ANDROSTENEDIONE 5a-DIHYDROTESTOSTERONE 17P-ESTRADIOL TESTOSTERONE ORIGIN a u t o r a d i o g r a m s . F i n a l i d e n t i f i c a t i o n o f e a c h s t e r o i d was made b y r e c r y s t a l l i z a t i o n t o a c o n s t a n t s p e c i f i c a c t i v i t y ( s e e T a b l e I ) , w h i c h was c a r r i e d o u t on n o n c h a r r e d s t e r o i d s e l u t e d f r o m t h e a p p r o p r i a t e z o n e s o f t h e c h r o m a t o g r a m s . The s p o t s were c u t o u t a n d s t o r e d i n e t h e r - r i n s e d c u l t u r e t u b e s . The a n d r o g e n i c s t e r o i d s w e r e e l u t e d f r o m t h e c u t - o u t s p o t s b y u s i n g t w i c e 5 . 0 m l o f d i e t h y l e t h e r . A f t e r e v a p o r a t i o n , t h e f i n a l e x t r a c t s were r e c o n s t i t u t e d i n 1 . 0 m l o f a b s o l u t e e t h a n o l and s t o r e d a t - 2 0 ° C p r i o r t o s t e r o i d R I A . To d e t e r m i n e t h e p r o c e d u r a l l o s s f o r T L C s e p a r a t i o n o f a n d r o g e n i c s t e r o i d s , s t e r o i d f r e e se rum was u s e d . N o r m a l r a t s e r u m o b t a i n e d f r o m immature r a t s w i t h o u t a n y t r e a t m e n t was m i x e d w i t h a c t i v a t e d N o r i t - A c h a r c o a l ( F i s h e r S c i e n t i f i c C o . , N J ) a t a r a t i o o f 2 g i n 10 m l se rum and t h e n i n c u b a t e d f o r 24 h r a t 4 ° C . The r e s u l t i n g s l u r r y was c e n t r i f u g e d f o u r t i m e s a t 4000 rpm f o r 30 m i n . T h i s p r o c e d u r e was r e p o r t e d t o remove o v e r 99% o f s t e r o i d s f r o m se rum w i t h o u t a f f e c t i n g t o t a l p r o t e i n c o n c e n t r a t i o n o r pH ( H o l l a n d e r and Shenkman, 1 9 7 4 ) . S u b s e q u e n t l y , known q u a n t i t i e s o f a l l t h e t h r e e r a d i o l a b e l e d a n d r o g e n i c s t e r o i d s were added t o t h e s t e r o i d - f r e e s e r u m and s e p a r a t e d b y T L C a s d e s c r i b e d p r e v i o u s l y . The p r o c e d u r a l l o s s f o r T L C s e p a r a t i o n was l e s s t h a n 30%; r e c o v e r y r a t e s f o r t h e t h r e e a n d r o g e n i c s t e r o i d s w e r e t h e f o l l o w i n g s : a n d r o s t e n e d i o n e , 81%; 5 a - r d i h y d r o t e s t o s t e r o n e , 71%; t e s t o s t e r o n e , 82%. •<The a n d r o g e n i c s t e r o i d s s e p a r a t e d f r o m se rum s a m p l e s w e r e d e t e r m i n e d b y t h e same s t e r o i d R I A p r o c e d u r e d e s c r i b e d p r e v i o u s l y . I n t h e a s s a y p r o c e d u r e , s i n c e t e s t o s t e r o n e a n t i s e r u m showed a s u b s t a n t i a l l y h i g h c r o s s - r e a c t i v i t y t o 5 a - d i h y d r o t e s t o s t e r o n e , t h e c o n t e n t s o f t h e two - 43 -TABLE I R a d i o c h e m i c a l i d e n t i f i c a t i o n o f a n d r o s t e n e d i o n e , 5 a - d i h y d r o t e s t o s t e r o n e and t e s t o s t e r o n e * Compound C a r r i e r S p e c i f i c a c t i v i t y (dpm/mg) a f t e r r e c r y s t a l l i z a t i o n number (mg) 0 1 2 A n d r o s t e n e d i o n e 1 1 . 7 391 557 549 5 a ^ 3 i h y d r o - 1 2 . 4 495 534 536 t e s t o s t e r o n e T e s t o s t e r o n e 1 1 . 3 362 556 552 * R e c r y s t a l l i z a t i o n s were c a r r i e d o u t f r o m a c e t o n e a n d h e x a n e . - 44 -steroids, testosterone and 5a-dihydrotestosterone were assayed with testosterone antiserum. However, the measurement of androstenedione contents was made with its specific antiserum kindly donated by Dr. D.T. Armstrong. In this assay, non-radiolabelled androstenedione obtained from Sigma Chemical Co. (St. Louis, MO) and radiolabeled steroid [1,2,6,7-^H]-androstenedione (sp. act. 110 Ci/mmol) purchased from Amersham Co. (Arlington Heights, IL) were used as standard and tracer, respectively. Hormone levels were expressed as ng/ml for serum androgenic steroids. C. LH. The serum samples collected at 6 or 12 hr intervals for 72 hr after administration of 4 or 20 IU PMSG were analyzed in duplicate by a standard LH assay procedure with a slight modification of a double-antibody RIA method as described by Murphy and Mahesh (1984). The purified LH antigen (NlH-rLH-I-6), standard (NIH-rLH-RP-2) and the first antibody (NIH-anti-rLH-S-8) were obtained from the National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases (NIADDK) National Hormone and 125 Pituitary Program. The purified LH antigen was iodinated with I by the Chloramine-T method (Bolton, 1977). The second antibody (sheep anti-rabbit gamma globulin) was purchased from Arnell Products Co., Inc. (Cherokee Station, New York, NY). A 25% binding: was obtained at the final dilution of 1:180, 000. The, assay was linear at80--64.0 pg/tube. The volume of the serum aliquots (100 yl) was adjusted to work in the middle range of the standard curve. All samples were measured in a single assay, in which a pool of serum from intact cycling adult rats on the day of diestrus had a - 45 -l e v e l o f 0 . 9 8 ng L H / m l w i t h t h e i n t r a - a s s a y CV o f 6.4%. The minimum d e t e c t a b l e l e v e l o f LH was 40 p g / t u b e , o r 0 . 4 n g / m l i n t h e s a m p l e s . The v a l u e s o f s e rum L H w e r e e x p r e s s e d i n t e r m s o f ng N I H - r L H - R P - 2 / m l . I n t h e a s s a y p r o c e d u r e , PMSG was f o u n d t o c r o s s - r e a c t w i t h t h e L H a n t i s e r u m , a l t h o u g h t h e b i n d i n g was n o t p a r a l l e l t o t h a t o f N I H - L H - R P - 2 ( s e e F i g u r e 1 8 ) . The a d d i t i o n o f 0 . 1 2 5 I U PMSG t o 100 p i s e r u m f r o m t h e i n t a c t r a t s r a i s e d t h e a p p a r e n t l e v e l o f " L H " i n t h e a s s a y b y a b o u t 300 p g N I H - L H - R P 2 e q u i v a l e n t . T h i s was a p p r o x i m a t e l y e q u a l t o t h e i n c r e a s e i n s e rum i m m u n o r e a c t i v e L H t h a t was measu red i n t h e r a t s o f s u p e r o v u l a t i o n g r o u p f o l l o w i n g t h e i n j e c t i o n o f 20 I U PMSG ( s e e F i g u r e 1 7 ) . V I I . S t a t i s t i c a l A n a l y s i s D a t a g e n e r a t e d f r o m t h e e x p e r i m e n t o f P r o t o c o l 1 w e r e e v a l u a t e d b y a c o m p l e t e l y r a n d o m i z e d d e s i g n o f a n a l y s i s o f v a r i a n c e ( S t e e l and T o r r i e , 1 9 6 0 ) . When s i g n i f i c a n t i n t e r a c t i o n s were f o u n d , i n d i v i d u a l F - t e s t s were p e r f o r m e d t o d e t e r m i n e t h e s i g n i f i c a n c e o f d i f f e r e n c e s be tween a n y means o f g r o u p s . D i f f e r e n c e s b e t w e e n means f o r a l l t h e d a t a w e r e c o n s i d e r e d t o be s i g n i f i c a n t a t p < 0 . 0 1 . C o r r e l a t i o n be tween o v a r i a n o r s e r u m s t e r o i d l e v e l s and p e r c e n t a g e s o f a b n o r m a l o o c y t e s was a l s o a s s e s s e d u s i n g t h e S p e a r m a n ' s r a n k c o r r e l a t i o n t e s t . D a t a f r o m t h e e x p e r i m e n t s o f P r o t o c o l s 2 t o 5 w e r e a n a l y z e d e m p l o y i n g S t u d e n t ' s t - t e s t , o r when a p p r o p r i a t e , a n a l y s i s o f v a r i a n c e f o l l o w e d b y F i s h e r ' s PLSD t e s t w i t h t h e a i d o f a c o m p u t e r i z e d p r o g r a m . C o m p a r i s o n s w i t h p<0 .05 w e r e c o n s i d e r e d t o be s i g n i f i c a n t . - 46 -RESULTS I . O o c y t e M o r p h o l o g y , O v u l a t o r y and S t e r o i d R e s p o n s e s ( P r o t o c o l 1 ) . The t i m e c o u r s e o f P M S G - i n d u c e d o v u l a t o r y r e s p o n s e i s p r e s e n t e d i n F i g u r e s 3 and 4 . A l l c o n t r o l r a t s (4 I U PMSG) o v u l a t e d a t 6 0 - 7 2 h r , w h i l e t h e r a t s g i v e n s u p e r o v u l a t o r y d o s e s (20 a n d 40 IU) o f PMSG b e g a n o v u l a t i n g a s e a r l y a s 24 h r a f t e r g o n a d o t r o p i n i n j e c t i o n . B o t h s u p e r o v u l a t o r y t r e a t m e n t s y i e l d e d a c o m p a r a b l e p e r c e n t a g e (28.6%) o f o v u l a t i n g a n i m a l s a t 24 h r a f t e r i n j e c t i o n . T h e r e a f t e r , t h i s e f f e c t was d o s e - d e p e n d e n t ; 20 a n d 40 I U PMSG i n d u c e d o v u l a t i o n i n o v e r 60% o f t h e a n i m a l s a t 48 a n d 36 h r , r e s p e c t i v e l y . T h e r e were two i n c r e a s e s i n t h e number o f o o c y t e s r e c o v e r e d f r o m o v i d u c t s o f t h e s u p e r o v u l a t e d r a t s : a d o s e - d e p e n d e n t i n c r e a s e b e t w e e n 24 and 36 h r and a d o s e - i n d e p e n d e n t i n c r e a s e be tween 48 a n d 72 h r . A f t e r 48 h r , t h e s u p e r o v u l a t e d a n i m a l s showed a s i g n i f i c a n t (p<0 .01) i n c r e a s e i n t h e number o f r e l e a s e d o o c y t e s above t h a t o f 4 I U P M S G - t r e a t e d r a t s . The maximum c o u n t s o f t h e o o c y t e s were a t t a i n e d f o r 20 I U PMSG t r e a t m e n t a t 60 h r ( 5 7 . 6 + 2 . 9 o o c y t e s / r a t ) and f o r 40 I U PMSG a t 72 h r ( 5 0 . 5 + 5 . 0 o c c y t e s / r a t ) . A b n o r m a l o o c y t e s w i t h v a r i o u s t y p e s o f d e g e n e r a t i o n w e r e r e c o v e r e d f r o m 48 and 36 h r onwards i n t h e r a t s g i v e n 20 and 40 I U PMSG, r e s p e c t i v e l y ( F i g . 5 ) . I n c o n t r a s t , no d e g e n e r a t e o o c y t e s were f o u n d i n a n y c o n t r o l r a t s t h r o u g h o u t t h e w h o l e e x a m i n a t i o n p e r i o d . The p e r c e n t a g e o f a b n o r m a l o o c y t e s g e n e r a l l y i n c r e a s e d i n a d o s e - d e p e n d e n t manner i n t h e s u p e r o v u l a t e d r a t s g i v e n b o t h 20 and 40 I U PMSG ( F i g . 6 ) . S h a r p i n c r e a s e s i n t h e p e r c e n t a g e o f a b n o r m a l o o c y t e s o c c u r r e d f r o m 54 h r o n w a r d s f o r 20 I U P M S G - t r e a t e d r a t s a n d a t 36 -48 h r and 54 -60 h r f o r 40 I U P M S G - t r e a t e d r a t s . The maximum d e g e n e r a t i o n o f o o c y t e s was o b s e r v e d i n 20 I U P M S G - t r e a t e d r a t s a t 72 h r FIGURE 3 P e r c e n t a g e o f r a t s o v u l a t e d a t d i f f e r e n t t i m e p e r i o d s a f t e r i n j e c t i o n o f A , 20 o r 40 I U PMSG. Numbers above e a c h b a r r e p r e s e n t t h e number o f r a t s s a c r i f i c e d a t e a c h t i m e ; ^ d e n o t e s 0%. FIGURE 4 R e c o v e r y o f o o c y t e s f r o m o v i d u c t s p e r r a t a t d i f f e r e n t t i m e p e r i o d s a f t e r i n j e c t i o n o f 4 , 20 o r 40 IU PMSG. V a l u e s r e p r e s e n t means + SEM ( n = 5 - 8 ) . 42* J I —I I o o o o •O CN FIGURE 5 M o r p h o l o g y o f n o r m a l a n d a b n o r m a l o o c y t e s r e c o v e r e d f r o m o v i d u c t s a f t e r t r e a t m e n t s w i t h a c o n t r o l d o s e (4 IU) and s u p e r o v u l a t o r y d o s e s (20 and 40 IU) o f PMSG, r e s p e c t i v e l y . a : Norma l a p p e a r i n g o o c y t e w i t h t h e f i r s t p o l a r b o d y ( P B ) a t 72 h r (4 I U P M S G ) , X400. b - d : Abnorma l o o c y t e s w i t h d i f f e r e n t s i z e s o f o o p l a s m i c f r a g m e n t s a t 4 8 - 7 2 h r (20 I U P M S G ) , x 4 0 0 . e : Abnorma l o o c y t e w i t h c o n t r a c t e d o o p l a s m a t 72 h r (40 I U PMSG) , x 4 0 0 . A p a r t o f o o p l a s m i s p u l l e d away f r o m t h e f r a c t u r e d z o n a p e l l u c i d a . f : A b n o r m a l o o c y t e w i t h "empty z o n a " s t r u c t u r e a t 72 h r (40 I U PMSG) , x 4 0 0 . 43Z FIGURE 6 P e r c e n t a g e o f a b n o r m a l o o c y t e s r e c o v e r e d f r o m o v i d u c t s o f s u p e r o v u l a t e d r a t s a t d i f f e r e n t t i m e p e r i o d s a f t e r i n j e c t i o n o f 20 o r 40 IU PMSG. Numbers above e a c h b a r r e p r e s e n t t h e t o t a l number o f o o c y t e s r e c o v e r e d a t e a c h t i m e ; ^ d e n o t e s 0%. - 51 -(30.9%) and i n 40 I U P M S G - t r e a t e d r a t s a t 60 h r ( 6 1 . 0 % ) . The m a j o r i t y o f o o c y t e s u n d e r g o i n g d e g e n e r a t i o n was f r e e o f c u m u l u s m a s s e s . H i s t o l o g i c a l e x a m i n a t i o n o f o v a r i e s f r o m t h e s u p e r o v u l a t e d r a t s showed f r e s h l y r u p t u r e d and l u t e i n i z e d f o l l i c l e s i n d i c a t i n g p r e c o c i o u s o v u l a t i o n a s e a r l y a s 24 h r a f t e r PMSG t r e a t m e n t ( F i g . 7 ) . I n c o n t r a s t , t h e o v a r i e s o f 4 I U P M S G - t r e a t e d r a t s showed no e v i d e n c e o f o v u l a t i o n up t o 48 h r . I n l a r g e f o l l i c l e s o f t h e c o n t r o l r a t s , t h e r e was l i t t l e e v i d e n c e o f f o l l i c u l a r a t r e s i a w i t h o o c y t e d e g e n e r a t i o n a t 24 o r 48 h r a f t e r i n j e c t i o n ( F i g . 8 ) . Howeve r , i n l a r g e f o l l i c l e s o f t h e s u p e r o v u l a t e d r a t s , a p r e d o m i n a n c e o f f o l l i c u l a r a t r e s i a w i t h f r a g m e n t a t i o n o f o o c y t e s was o b s e r v e d a t 4 8 , b u t n o t a t 24 h r , a f t e r t r e a t m e n t ( F i g . 9 ) . O v a r i a n c o n t e n t s o f 1 7 f i - e s t r a d i o l , p r o g e s t e r o n e a n d a n d r o g e n s a r e shown i n F i g u r e 1 0 . A t 0 h r , t h e o v a r i e s c o n t a i n e d 0 . 2 + 0 . 0 ng 1 7 B - e s t r a d i o l / r a t , 1 .9+0 .6 ng p r o g e s t e r o n e / r a t a n d 0 . 5 + 0 . 0 ng a n d r o g e n s / r a t . F o l l o w i n g PMSG i n j e c t i o n , t h e 1 7 B - e s t r a d i o l c o n t e n t o f o v a r i e s f r o m c o n t r o l r a t s (4 I U PMSG) r o s e g r a d u a l l y t o a peak o f 4 . 6 + 0 . 5 n g / r a t a t 48 h r ( p r o e s t r u s ) and t h e n s h a r p l y d e c l i n e d t o l e s s t h a n 0 . 7 n g / r a t a t 60 and 72 h r a f t e r i n j e c t i o n . Compared t o c o n t r o l s , t h e 1 7 B - e s t r a d i o l c o n t e n t o f s u p e r o v u l a t e d o v a r i e s i n c r e a s e d s i g n i f i c a n t l y (p<0 .01) be tween 36 and 60 h r . The peak v a l u e s o f 1 7 B - e s t r a d i o l w e r e 1 8 . 4 + 3 . 2 n g / r a t and 9 . 1 + 0 . 5 n g / r a t f o r 20 I U PMSG ( a t 60 h r ) and f o r 40 I U PMSG ( a t 5 4 . h r ) , r e s p e c t i v e l y . B y 72 h r , o v a r i a n 1 7 B - e s t r a d i o ! c o n t e n t was c o m p a r a b l e i n 4 I U ( 0 . 2 + 0 . 0 n g / r a t ) , 20 I U ( 0 . 6 + 0 . 0 n g / r a t ) a n d 40 I U ( 1 . 3 + 0 . 2 n g / r a t ) P M S G - t r e a t e d r a t s . O v a r i a n p r o g e s t e r o n e c o n t e n t i n 20 I U P M S G - t r e a t e d r a t s i n c r e a s e d s t e a d i l y f r o m 12 h r onwards a n d r e a c h e d a peak 52 FIGURE 7 Ovarian histology obtained at 24 hr after 20 IU PMSG treatment. Note a ruptured f o l l i c l e with abundant luteal c e l l s in the antrum, a) xlOO, b) xlOOO. FIGURE 8 F o l l i c u l a r a n d o o c y t e m o r p h o l o g y o f c o n t r o l o v a r y o b t a i n e d a t 48 h r a f t e r 4 IU PMSG t r e a t m e n t . N o t e a p p e a r a n c e o f h e a l t h y l a r g e f o l l i c l e w i t h a n o r m a l o o c y t e . * a ) x l O O , b) x l O O O . 533 54 FIGURE 9 F o l l i c u l a r and o o c y t e m o r p h o l o g y o f s u p e r o v u l a t e d o v a r y o b t a i n e d a t 48 h r a f t e r 40 I U PMSG t r e a t m e n t . N o t e a t r e t i c l a r g e f o l l i c l e w i t h a f r a g m e n t e d o o c y t e ; t h e r e a r e l o s s o f g r a n u l o s a c e l l c o n t i n u i t y (1) and h y p e r t r o p h y o f t h e c a l a y e r ( 2 ) . a ) x l O O , b) x l O O O . FIGURE 10 Changes i n o v a r i a n 1 7 f i - e s t r a d i o l , p r o g e s t e r o n e a n d a n d r o g e n s a f t e r a d m i n i s t r a t i o n o f 4 , 20 o r 40 I U PMSG t o immature r a t s . V a l u e s r e p r e s e n t means + SEM f o r p a i r e d o v a r i e s ( n = 5 - 8 ) . 55a 20 15 10 5 0 200 150 lOOh 170- ESTRADIOL O 4IU PMSG 9 20IU PMSG A 40IU PMSG PROGESTERONE 0 12 24 36 Time (hour) 48 60 - 56 -a t 60 h r ( 1 3 3 . 0 + 1 8 . 8 n g / r a t ) , w h e r e a s i n t h e r a t s g i v e n 40 I U PMSG, i t showed a b i p h a s i c i n c r e a s e w i t h t h e peak v a l u e s a t 36 h r ( 1 9 6 . 3 + 1 2 . 1 n g / r a t ) and a t 60 h r ( 2 0 5 . 1 + 1 7 . 6 n g / r a t ) . I n 4 I U P M S G - t r e a t e d r a t s , t h e r e was no s i g n i f i c a n t i n c r e a s e i n t h e p r o g e s t e r o n e c o n t e n t d u r i n g t h e f i r s t 54 h r , b u t t h e r e a f t e r i t i n c r e a s e d s h a r p l y t o a peak a t 60 h r ( 1 2 7 . 0 + 9 . 1 n g / r a t ) . B y 72 h r , o v a r i a n p r o g e s t e r o n e c o n t e n t was 7 2 . 5 + 1 7 . 3 n g / r a t f o r 4 I U P M S G - t r e a t e d r a t s . I t w a s , h o w e v e r , s i g n i f i c a n t l y (p<0 .01) h i g h e r i n t h e s u p e r o v u l a t e d r a t s : 1 3 0 . 4 + 2 3 . 4 n g / r a t (20 IU) and 1 6 2 . 2 + 6 . 6 n g / r a t (40 I U ) . O v a r i a n a n d r o g e n c o n t e n t i n t h e c o n t r o l r a t s g i v e n 4 I U PMSG d e m o n s t r a t e d two p e a k s : 5 0 . 9 + 6 . 0 n g / r a t a t 12 h r and 5 2 . 9 + 8 . 0 n g / r a t a t 36 h r a f t e r i n j e c t i o n . I t was s i g n i f i c a n t l y (p<0 .01) h i g h e r be tween 12 and 36 h r , when compared t o t h e s u p e r o v u l a t o r y t r e a t m e n t o f 20 o r 40 I U PMSG. From t h e n o n w a r d s , t h e o v a r i a n a n d r o g e n c o n t e n t f o r 4 I U P M S G - t r e a t e d r a t s s h a r p l y d e c l i n e d t o l e s s t h a n 5 .7 n g / r a t a t 60 o r 72 h r . I n c o n t r a s t , a n d r o g e n c o n t e n t o f t h e s u p e r o v u l a t e d o v a r i e s i n c r e a s e d a n d be tween 48 and 60 h r was s i g n i f i c a n t l y (p<0.01) h i g h e r t h a n t h a t o f c o n t r o l o v a r i e s , w i t h t h e peak v a l u e s o f 5 8 . 6 + 7 . 7 n g / r a t a t 60 h r a f t e r t r e a t m e n t w i t h 20 I U PMSG and 61 .2+5 .1 n g / r a t a t 54 h r a f t e r a d m i n i s t r a t i o n o f 40 I U PMSG. By 72 h r , o v a r i a n a n d r o g e n c o n t e n t was c o m p a r a b l e i n 4 I U ( 2 . 5 + 0 . 8 n g / r a t ) and 20 I U ( 2 . 9 + 0 . 6 n g / r a t ) b u t h i g h e r i n 40 I U ( 1 4 . 3 + 2 . 6 n g / r a t ) P M S G - t r e a t e d r a t s . The c h a n g e s i n t h e 1 7 B - e s t r a d i o l , p r o g e s t e r o n e and a n d r o g e n s o f o v a r i a n t i s s u e s and s e r a f o l l o w e d c o m p a r a b l e p r o f i l e s t h r o u g h o u t t h e w h o l e e x a m i n a t i o n p e r i o d ( F i g s . 10 and 1 1 ) . The peak l e v e l s o f o v a r i a n s t e r o i d s w e r e , h o w e v e r , much h i g h e r t h a n t h o s e o f s e r u m s t e r o i d s : up t o 4 - f o l d f o r 1 7 B - e s t r a d i o l , up t o 2 - f o l d f o r p r o g e s t e r o n e and up t o 7 - f o l d f o r a n d r o g e n s . 57 FIGURE 11 Changes i n s e r u m 1 7 0 - e s t r a d i o l , p r o g e s t e r o n e and a n d r o g e n s a f t e r a m i n i s t r a t i o n o f 4 , 20 o r 40 I U PMSG t o immatu re r a t s . V a l u e s r e p r e s e n t means + SEM ( n = 5 - 8 ) . 1.2 0.8 0.4 0 E 150 C CO 100 0 i Q) 50 Co E D k . 0 0) CO 170- E S T R A D I O L O 41U PMSG 9 201U PMSG A 401U PMSG 15 10 0 P R O G E S T E R O N E A N D R O G E N S 12 24 36 48 Time (hour) - 58 -T h e r e was a s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n b e t w e e n o v a r i a n o r s e r u m a n d r o g e n l e v e l s a n d p e r c e n t a g e s o f a b n o r m a l o o c y t e s d u r i n g t h e i n t e r v a l o f a n d r o g e n i n c r e a s e , i . e . be tween 24 and 60 h r a f t e r i n j e c t i o n o f 20 o r 40 I U PMSG ( F i g s . 12 and 1 3 ) . R e g r e s s i o n a n a l y s i s g i v e s t h e e q u a t i o n s : f o r 20 I U PMSG t r e a t m e n t , y = 0 . 2 3 x ^ 2 . 4 9 ( r = 0 . 8 1 , p < 0 . 0 1 , n=16) and y = 1 . 0 7 x 2 - 2 . 9 2 ( r = 0 . 8 5 , p < 0 . 0 1 , n=16) ; f o r 40 I U PMSG t r e a t m e n t , y = 0 . 7 0 x ^ 1 . 5 8 ( r = 0 . 5 1 , p < 0 . 0 5 , n=25) and y = 4 . 0 5 x 2 - 5 . 0 7 ( r = 0 . 7 0 , p < 0 . 0 1 , n = 2 5 ) , w h e r e y = p e r c e n t a g e o f a b n o r m a l o o c y t e s , x ^ = c o n t e n t o f o v a r i a n a n d r o g e n s , and x 0 = c o n c e n t r a t i o n o f s e rum a n d r o g e n s . FIGURE 12 Correlation between the percentage of abnormal oocytes and content of ovarian androgens from 24 to 60 hr after injection of 20 or 40 IU PMSG to immature rats. 53 a Ovarian Androgens (ng/rat) 60 FIGURE 13 C o r r e l a t i o n be tween t h e p e r c e n t a g e o f a b n o r m a l o o c y t e s and c o n c e n t r a t i o n o f s e r u m a n d r o g e n s f r o m 24 t o 60 h r a f t e r i n j e c t i o n o f 20 o r 40 I U PMSG t o immature r a t s . Serum Androgens (ng/ml) - 61 -II. F o l l i c u l a r S t e r o i d C o n t e n t s and O o c y t e M a t u r a t i o n ( P r o t o c o l 2 ) . The v a l u e s f o r mean c o n t e n t s o f 1 7 B - e s t r a d i o l , p r o g e s t e r o n e a n d a n d r o g e n s i n f o l l i c u l a r f l u i d a r e p r e s e n t e d i n F i g u r e 1 4 . I n c o n t r o l r a t s t r e a t e d w i t h 4 I U PMSG, t h e c o n t e n t o f 1 7 B - e s t r a d i o l was 1 1 6 . 2 + 1 1 . 5 n g / m l a t 24 h r and s i g n i f i c a n t l y (p<0 .05) i n c r e a s e d t o t h e v a l u e o f 2 6 5 . 7 + 6 2 . 8 n g / m l by 48 h r ; no f u r t h e r i n c r e a s e was n o t e d a t 60 h r . In s u p e r o v u l a t e d r a t s t r e a t e d w i t h 20 I U PMSG, t h e c h a n g e s i n 1 7 J 3 - e s t r a d i o l c o n t e n t w e r e c o m p a r a b l e t o c o n t r o l v a l u e s t h r o u g h o u t t h e p e r i o d e x a m i n e d . P r o g e s t e r o n e c o n t e n t i n c o n t r o l r a t s r o s e s t e a d i l y f r o m 24 h r ( 2 6 0 . 6 + 2 1 . 2 n g / m l ) t o r e a c h a maximum b y 60 h r ( 6 2 6 6 . 2 + 7 9 2 . 1 n g / m l ) . A l t h o u g h t h e c h a n g e s i n p r o g e s t e r o n e l e v e l s b e t w e e n c o n t r o l a n d s u p e r o v u l a t e d r a t s f o l l o w e d s i m i l a r p a t t e r n s , t h e r e was a s i g n i f i c a n t (p<0 .05) d i f f e r e n c e i n t h e l e v e l s a t 48 h r : 2076 .6+217 .7 n g / m l i n c o n t r o l and 4274 .4+664 .1 n g / m l i n s u p e r o v u l a t e d r a t s . A g r e a t d i f f e r e n c e be tween c o n t r o l and s u p e r o v u l a t e d r a t s was c h a r a c t e r i z e d b y d i f f e r e n t p a t t e r n s o f and rogens o b s e r v e d a t d i f f e r e n t t i m e s . A n d r o g e n c o n t e n t i n c o n t r o l r a t s was 8 8 2 . 1 + 9 4 . 8 n g / m l a t 24 h r and s h a r p l y d e c l i n e d t o t h e l e v e l o f 3 7 . 1 + 1 4 . 4 n g / m l b y 60 h r . The c o n t e n t i n s u p e r o v u l a t e d r a t s was 1 8 6 . 4 + 3 3 . 0 n g / m l a t 24 h r w h i c h was s i g n f i c a n t l y (p<0 .01) l o w e r t h a n c o n t r o l v a l u e . T h e r e a f t e r , i t s i g n i f i c a n t l y (p<0.05) r o s e t o t h e v a l u e o f 4 7 4 . 6 + 7 5 . 8 n g / m l b y 48 h r and r e m a i n e d a t t h i s l e v e l . These v a l u e s o b t a i n e d a t 48 h r o r 60 h r w e r e s i g n i f i c a n t l y (p<0.05) h i g h e r t h a n c o n t r o l s . As shown i n F i g u r e 1 5 , i n c o n t r o l r a t s , t h e mean r a t i o s o f a n d r o g e n s : 1 7 f i - e s t r a d i o l ( A / E ) , p r o g e s t e r o n e : 1 7 f l - e s t r a d i o l ( P / E ) a n d p r o g e s t e r o n e : a n d r o g e n s ( P / A ) r e f l e c t e d s e q u e n t i a l changes a t v a r i o u s t i m e s . 62 FIGURE 14 Changes i n f o l l i c u l a r c o n t e n t s o f 1 7 f i - e s t r a d i o l , p r o g e s t e r o n e a n d a n d r o g e n s a f t e r a d m i n i s t r a t i o n o f 4 o r 20 I U PMSG t o immatu re r a t s . V a l u e s a r e g i v e n a s means + SE ( n = 5 - 7 ) . The means w i t h no s u p e r s c r i p t s i n common a r e s i g n i f i c a n t l y d i f f e r e n t ( p < 0 . 0 5 ) . 500 400-300 -200-100-620-17B-estradlol • 4 IU PMSG C • 20 IU PMSG be ab m 13, X m abc ab 24 48 60 8000 6000 4000 -2000 -1200 1000 i 800 600-400-200-0 d ab Androgens ab be T VA a 24 48 Time after PMSG (hr) 60 FIGURE 15 Changes i n r a t i o s o f a n d r o g e n s / 1 7 £ - e s t r a d i o l , p r o g e s t e r o n e / 1 7 i 3 - e s t r a d i o l and p r o g e s t e r o n e / a n d r o g e n s i n f o l l i c u l a r f l u i d a f t e r a d m i n i s t r a t i o n o f 4 o r 20 I U PMSG t o immatu re r a t s . V a l u e s a r e g i v e n a s means + S E ( n = 5 - 7 ) . The means w i t h no. s u p e r s c r i p t s i n common a r e s i g n i f i c a n t l y d i f f e r e n t ( p < 0 . 0 5 ) . 12 10 -8-£ 3 a Androgens/17B-estradlol • 4 IU PMSG • 20 IU PMSG 6 -2-0' 24 ab a b a b 7} 48 60 tit o a> CO o 3 o o LL O CB Time after PMSG (hr) - 64 -I n c o n t r a s t , t h e o v e r a l l r a t i o s i n s u p e r o v u l a t e d r a t s w e r e c o n s i s t e n t l y s t e a d y o v e r t h e t i m e p e r i o d o f e x p e r i m e n t . I n c o n t r o l r a t s , t h e A / E r a t i o d e c r e a s e d s h a r p l y , b u t P / E and P / A r a t i o s i n c r e a s e d g r a d u a l l y f r o m 24 h r o n w a r d s . S i g n i f i c a n t c h a n g e s w e r e o b s e r v e d a t 48 h r f o r A / E r a t i o ( p < 0 . 0 1 ) , and a t 60 h r f o r P / E (p<0 .05 ) and P / A r a t i o s ( p < 0 . 0 1 ) . The A / E a n d P / E r a t i o s i n s u p e r o v u l a t e d r a t s were s i g n i f i c a n t l y (p<0 .05) l o w e r and h i g h e r t h a n e a c h r a t i o i n c o n t r o l r a t s a t 24 h r , r e s p e c t i v e l y . T h e r e a f t e r , t h e s e r a t i o s i n s u p e r o v u l a t e d r a t s r e m a i n e d c o n s t a n t w i t h no s u b s t a n t i a l c h a n g e s . The P / A r a t i o i n s u p e r o v u l a t e d r a t s was c o n s i s t e n t l y l o w o v e r t h e w h o l e p e r i o d e x a m i n e d and c o m p a r a b l e t o t h e r a t i o o b s e r v e d a t 24 o r 48 h r i n c o n t r o l r a t s . However , a t 60 h r , i t was s i g n i f i c a n t l y (p<0 .01 ) l o w e r t h a n t h e r a t i o i n c o n t r o l r a t s . The v a r i o u s s t a g e s o f n u c l e a r m a t u r a t i o n o f o o c y t e s r e c o v e r e d f r o m o v i d u c t s a t d i f f e r e n t t i m e s a f t e r PMSG a r e s u m m a r i z e d i n T a b l e I I . M o r p h o l o g i c a l l y d e g e n e r a t e o o c y t e s i n c o n t r o l r a t s ( 1 / 5 7 o o c y t e s a t 72 h r ) and i n s u p e r o v u l a t e d ( 6 / 8 8 o o c y t e s a t 48 h r a n d 2 5 / 1 3 9 o o c y t e s a t 72 h r ) w e r e e x c l u d e d f r o m t h e m e i o t i c e v a l u a t i o n . The p r o p o r t i o n o f o o c y t e s a c t u a l l y a n a l y z a b l e f o r t h e c l a s s i f i c a t i o n o f e a c h s t a g e was 73% i n c o n t r o l r a t s and 67-91% i n s u p e r o v u l a t e d r a t s , s i n c e some o o c y t e s l o s t o r s c a t t e r e d t h e i r chromosomes by o c c a s i o n a l r u p t u r e o f c e l l membrane d u r i n g t h e p r o c e s s o f p r e p a r i n g t h e o o c y t e s . I n c o n t r o l r a t s , o b s e r v a t i o n o f o o c y t e s r e v e a l e d c i r c u l a r i n d e n t a t i o n o r c o n s t r i c t i o n o f o o c y t e membrane and c o m p l e t e a g g r e g a t i o n o f chromosomes i n t o p o l a r b o d y , t y p i c a l o f me taphase I I ( F i g . 1 6 ) . However , i n s u p e r o v u l a t e d r a t s , i t was a c t u a l l y n o t p o s s i b l e t o d i s t i n g u i s h be tween me taphase I and me taphase I I b e c a u s e o f i n c o n s i s t e n t TABLE I I E f f e c t s o f s u p e r o v u l a t o r y " t r e a t m e n t on n u c l e a r m a t u r a t i o n o f o o c y t e s r e c o v e r e d f r o m o v i d u c t s i n r a t s Time a f t e r # o f o o c y t e s # o f o o c y t e s S t a g e s o f M e i o s i s (%) PMSG ( h r ) r e c o v e r e d 9 examined P r o p h a s e I Anaphase I T e l o p h a s e I Metaphase I / I I U n d e t e r m i n e d C o n t r o l b 72 5 7 ( 5 / 5 ) 56 - 1 ( 1 . 8 ) 5 1 ( 9 1 . 0 ) * 4 ( 7 . 1 ) S u p e r -o v u l a t i o n 0 24 1 5 ( 2 / 5 ) 15 1 0 ( 6 6 . 7 ) - - - 5 ( 3 3 . 3 ) 48 8 8 ( 5 / 7 ) 82 1 9 ( 2 3 . 2 ) 1 3 ( 1 5 . 9 ) - 3 9 ( 4 7 . 6 ) 1 1 ( 1 3 . 4 ) 72 1 3 9 ( 6 / 6 ) 114 1 ( 0 . 9 ) 5 1 ( 4 4 . 7 ) 7 ( 6 . 1 ) 4 5 ( 3 9 . 5 ) 1 0 ( 8 . 8 ) a Number o f r a t s e x h i b i t i n g o v u l a t i o n s / t o t a l number o f r a t s k i l l e d a t e a c h t i m e a r e g i v e n i n p a r e n t h e s e s . R e c o v e r e d o o c y t e s w i t h v i s i b l e s i g n s o f d e g e n e r a t i o n were i n c l u d e d i n t o t a l c o u n t i n g . b R a t s were g i v e n 4 I U o f p r e g n a n t mare se rum g o n a d o t r o p i n (PMSG). C R a t s were g i v e n 20 I U PMSG. • O o c y t e s showed c o n s i s t e n t l y t y p i c a l me taphase I I c o n f i g u r a t i o n . - " <;v66 FIGURE 16 T y p i c a l c o n f i g u r a t i o n o f n u c l e a r m a t u r a t i o n o f o o c y t e s r e c o v e r e d f r o m o v i d u c t s a f t e r PMSG. (a) p r o p h a s e I a t 48 h r (20 I U P M S G ) . x 4 0 0 , (b) metaphase I a t 72 h r (20 I U P M S G ) . x 4 0 0 , ( c ) t e l o p h a s e I a t 72 h r (20 I U PMSG) . x l O O O , (d) me taphase I I a t 72 h r (4 I U PMSG) . x400 - 67 -formation of t y p i c a l polar body. Prior to or immediately after the completion of membrane a b s t r i c t i o n , polar bodies i n some portion of superovulated oocytes became rapidly degenerated. In control r a t s , of the 56 oocytes examined at 72 hr f only one oocyte was at anaphase I, but the majority (91.0%) showed metaphase I I configuration. In contrast, superovulated oocytes displayed considerably d i f f e r e n t stages of meiotic maturation over the time period of experiment (Fig. 16). Although, of the 15 oocytes examined at 24 hr, 10 oocytes (66.7%) were consistently at prophase I except for the unanalyzable 5 oocytes, the oocytes superovulated at 48 or 72 hr were not synchronized to any stage of nuclear maturation. The percentages of oocytes at prophase I, anaphase I, telophase I and metaphase I / I I were 23.2% (19/82 oocytes), 15.9% (13/82 oocytes), 0% and 47.6% (39/82 oocytes) at 48 hr, and 0.9% (1/114 oocytes), 44.7% (51/114 oocytes), 6.1% (7/114 oocytes) and 39.5% (45/114 oocytes) at 72 hr, respectively. - 68 -I I I . Se rum L H R e s p o n s e ( P r o t o c o l 3 ) . The t i m e c o u r s e s and p a t t e r n s o f L H r e l e a s e i n t h e b l o o d o f c o n t r o l (4 I U PMSG) a n d s u p e r o v u l a t e d (20 I U PMSG) r a t s a r e i l l u s t r a t e d i n F i g u r e 1 7 . B o t h g r o u p s showed a s i m i l a r t i m e r e l a t i o n s h i p o f b i p h a s i c LH r e s p o n s e t o PMSG. H o w e v e r , a g r e a t d i f f e r e n c e was n o t e d i n t h e m a g n i t u d e o f s e r u m L H l e v e l s be tween t h e two g r o u p s . Mean b a s a l l e v e l s o f se rum LH p r i o r t o a d m i n i s t r a t i o n o f PMSG w e r e 0 . 3 7 t o 0 . 5 0 n g / m l . Serum LH l e v e l s b e g a n t o r i s e s i g n i f i c a n t l y (p<0.01) b e t w e e n 0 h r a n d 12 h r , and r e a c h e d t h e f i r s t p e a k s o f 1 .81+0 .21 n g / m l a t 12 h r a n d 3 . 7 8 + 0 . 1 8 n g / m l a t 18 h r i n c o n t r o l and s u p e r o v u l a t e d r a t s , r e s p e c t i v e l y . T h e r e a f t e r , t h e LH l e v e l s i n b o t h g r o u p s g r a d u a l l y t a p e r e d o f f b y 54 h r . The f i r s t p e a k s were f o l l o w e d b y enormous and s i g n i f i c a n t (p<0 .001) e l e v a t i o n s o f se rum LH w i t h t h e s e c o n d p e a k s w h i c h were s y n c h r o n i z e d a t 60 h r i n b o t h g r o u p s . The peak v a l u e s o f s e c o n d e l e v a t i o n s were 1 4 . 9 8 + 0 . 9 2 n g / m l and 6 .85+1 .29 n g / m l i n c o n t r o l a n d s u p e r o v u l a t e d r a t s , r e s p e c t i v e l y . S u b s e q u e n t l y , t h e LH l e v e l s i n b o t h g r o u p s r a p i d l y f e l l b y 66 h r w i t h no f u r t h e r c h a n g e s a p p a r e n t a t 72 h r . A s compared t o c o n t r o l s , be tween 12 h r a n d 54 h r , se rum L H l e v e l s i n s u p e r o v u l a t e d r a t s w e r e s i g n i f i c a n t l y (p<0 .001) e l e v a t e d . T h i s e l e v a t i o n was c o n s i s t e n t a t e a c h t i m e p o i n t . Howeve r , a t 60 h r , t h e mean p e a k v a l u e of the s e c o n d e l e v a t i o n i n s u p e r o v u l a t e d r a t a was s i g n i f i c a n t l y r e d u c e d b y .5.4% beloi?' t h a t o f c o n t r o l r a t s . S i m i l a r l y - a maximum i n c r e a s e o f mean A L H b e t w e e n t h e t w o p e a k s was much l e s s i n s u p e r o v u l a t e d r a t s t h a n t h a t i n c o n t r o l r a t s . The mean A L H a f t e r s u b t r a c t i o n o f t h e f i r s t peak f r o m t h e s e c o n d peak was 3 . 0 7 n g / m l i n s u p e r o v u l a t e d r a t s a n d 1 3 . 1 7 n g / m l i n c o n t r o l r a t s . FIGURE 17 Changes i n s e rum L H c o n c e n t r a t i o n s a f t e r a d m i n i s t r a t i o n o f 4 o r 20 I U . P M S G t o immature r a t s . S e q u e n t i a l b l o o d s a m p l e s w e r e t a k e n f r o m t h e same a n i m a l u s i n g c h r o n i c c a t h e t e r i z a t i o n i n t o e x t e r n a l j u g u l a r v e i n . V a l u e s a t e a c h p o i n t r e p r e s e n t t h e means + S E ( n = 7 ) . Time after PMSG (hr) - 70 -The f i r s t i n c r e a s e s o f s e r u m LH w e r e f o u n d t o r e s u l t f r o m t h e c r o s s - r e a c t i o n o f t h e i n j e c t e d PMSG w i t h L H a n t i b o d y i n t h e a s s a y p r o c e d u r e . The i n i t i a l i n c r e a s e s were d o s e - d e p e n d e n t w i t h a p r o l o n g e d h a l f - l i f e o f 3 0 - 3 6 h r w h i c h was much l o n g e r t h a n t h a t f o r g e n u i n e L H ( 0 . 5 h r , r e p o r t e d by Bogdanove and G a y , 1 9 6 9 ) . I n a d d i t i o n , t h e r e s u l t o f d i l u t i o n b i n d i n g o f N I H - L H - R P 2 , PMSG a n d se rum p o o l s i n t h e LH a s s a y p r o c e d u r e ( F i g . 18) showed t h a t PMSG a c t u a l l y c r o s s - r e a c t e d w i t h L H a n t i b o d y , a n d t h a t t h e s e r i a l d i l u t i o n c u r v e o f se rum p o o l s o b t a i n e d f r o m s u p e r o v u l a t e d r a t s d u r i n g t h e i n i t i a l i n c r e a s e was n o t p a r a l l e l t o t h a t o f t h e N I H - L H - R P 2 s t a n d a r d b u t was much c l o s e r t o t h a t o f PMSG. On t h e o t h e r h a n d , t h e s e r i a l d i l u t i o n c u r v e o f s e rum p o o l s o b t a i n e d f r o m c o n t r o l r a t s a t 60 h r was p a r a l l e l t o t h e N I H - L H - R P 2 s t a n d a r d c u r v e . A f t e r f i n a l c o l l e c t i o n o f b l o o d s a m p l e s a t 72 h r , t h e o v u l a t o r y r e s p o n s e a n d c h a n g e s o f o v a r i a n w e i g h t i n r e s p o n s e t o t w o d i f f e r e n t d o s e s o f PMSG were e x a m i n e d ( T a b l e I I I ) . I n c o n t r o l r a t s , t h e t o t a l mean number o f o o c y t e s r e c o v e r e d was 7 . 6 + 0 . 5 o o c y t e s p e r r a t , a n d mean w e t w e i g h t o f p a i r e d o v a r i e s was 3 2 . 8 + 1 . 9 mg p e r r a t . On t h e o t h e r h a n d , a s u p e r o v u l a t o r y d o s e o f PMSG was shown s i g n i f i c a n t l y t o i n c r e a s e t h e o v u l a t o r y r e s p o n s e ( 3 6 . 0 + 8 . 5 o o c y t e s p e r r a t , p<0 .05) a n d o v a r i a n we t w e i g h t ( 6 8 . 7 + 8 . 2 mg p e r r a t , p < 0 . 0 1 ) a b o v e t h o s e o b t a i n e d b y a c o n t r o l d o s e o f PMSG. — 7 1 FIGURE 18 LH a s s a y o f PMSG a n d se rum p o o l s , s h o w i n g t h e c r o s s - r e a c t i v i t y o f t h e i n j e c t e d PMSG w i t h LH a n t i b o d y . N I H - L H - R P 2 : L H s t a n d a r d p r e p a r a t i o n . PMSG: N o n - i n j e c t e d a n d d i l u t e d p r e p a r a t i o n . INTACT POOL: Serum p o o l s c o l l e c t e d f r o m t h e d i e s t r o u s a d u l t r a t s w i t h o u t i n j e c t i o n o f PMSG. . POOL C 6 0 : Serum p o o l s c o l l e c t e d f r o m t h e immature r a t s a t 60 h r a f t e r i n j e c t i o n o f a c o n t r o l d o s e (4 IU) o f PMSG. POOL S 1 2 - 2 4 : Serum p o o l s c o l l e c t e d f r o m t h e immatu re r a t s a t 1 2 , 18 a n d 24 h r a f t e r i n j e c t i o n o f a s u p e r o v u l a t o r y d o s e (20 IU) o f PMSG. 1.0 0.8H o CO co 0.6-1 0.4H 0.2H - ® - NIH-LH-RP2 PMSG + INTACT SERUM POOL -<$>- C60 SERUM POOL SI2-24 SERUM POOL °-°-r—T 1 20 40 80 - 72 -TABLE I I I O o c y t e r e c o v e r y and o v a r i a n w e i g h t s 72 h r a f t e r a d m i n i s t r a t i o n o f PMSG t o immatu re r a t s T r e a t m e n t N o . o f a n i m a l s 9 N o . o f o o c y t e s (mean + SE) O v a r i a n w e i g h t (mg) (mean+SE) 4 I U PMSG 7 7 . 6 + 0 . 5 3 2 . 8 + 1 . 9 20 I U PMSG 7 3 6 . 0 + 8 . 5 1 3 6 8 . 7 + 8 . 2 c a A l l i n d i v i d u a l s i n e a c h g r o u p e x h i b i t e d o v u l a t i o n s . k p < 0 . 0 5 a n d c p < 0 . 0 1 , s t a t i s t i c a l l y s i g n i f i c a n t compared t o 4 I U P M S G - t r e a t e d g r o u p . - 73 -IV. Effects of Flutamide on Oocyte Morphology and Embryo Development  (Protocol 4). The ovulatory response in rats superovulated with 40 IU PMSG was unaffected by flutamide treatment (Table IV). Apart from one rat in the vehicle treatment group, which showed l i t t l e evidence of ovarian stimulation at 48 hr, a l l rats in the vehicle or flutamide treatment groups ovulated consistently 48 to 72 hr after PMSG. The total number of oocytes recovered at each time varied considerably between animals in each treatment group (between 15 and 64 oocyes/rat). No significant differences were observed in the mean number of oocytes between vehicle- and flutamide-treated groups throughout the period examined, nor did the mean number of oocytes in each treatment group increase significantly after 48 hr. No degenerate oocytes were found in any pre-treatment control rats which ovulated at 24 hr (8.5+0.5 oocytes/rat). Flutamide treatment in rats superovulated with PMSG significantly reduced the percentage of degenerate oocytes ovulated at 48 hr (p<0.01), 60 hr (p<0.01) and 72 hr (p<0.01), compared to the vehicle-treated rats (Table IV). The maximum decrease in degenerate oocytes was observed at 72 hr (26-fold); one or two degenerate oocytes were occasionally recovered from three of the six flutamide-treated rats. The changes in ovarian eontehts of androgens, 17fi-estradiol and progesterone after treatment with vehicle cr flutamide are shown in Figure 19. By 48 hr, a l l the three ovarian steroid hormones in both groups of treated rats were significantly (p<0.01) elevated above the values of pre-treatment controls obtained at 24 hr. Compared to the vehicle regimen, - 74 -TABLE I V E f f e c t s o f f l u t a m i d e o n r e c o v e r y a n d n o r m a l i t y o f o o c y t e s f r o m o v i d u c t s o f 40 I U P M S G - i n j e c t e d r a t s Time a f t e r PMSG ( h r ) a 48 60 72 V e h i c l e 1 3 N o . o f o o c y t e s 3 1 . 0 + 7 . 0 3 1 . 1 + 1 . 9 3 4 . 5 + 2 . 9 % d e g e n e r a t e o o c y t e s 1 9 . 0 + 5 . 0 5 4 . 7 + 3 . 5 4 1 . 7 + 4 . 2 F l u t a m i d e c N o . o f o o c y t e s 3 5 . 4 + 5 . 6 2 7 . 1 + 2 . 1 3 9 . 2 + 4 . 9 % d e g e n e r a t e o o c y t e s 2 . 1 + 0 . 8 d 1 9 . 3 + 1 . 4 e 1 . 6 + 0 . 7 f a R e s u l t s a r e e x p r e s s e d a s means + SEM f o r t h e number o f o v u l a t e d r a t s ( n = 6 o r 7 ) . D ' c R a t s were t r e a t e d w i t h v e h i c l e o r 5 mg f l u t a m i d e at 30 a n d 36 h r a f t e r i n j e c t i o n o f 40 I U PMSG. d-f p < 0 . 0 1 , compared .to corresponding v e h i c l e t r e a t m e n t g r o u p -FIGURE 19 Ovarian contents of androgens, 17B-estradiol, and progesterone a f t e r treatment with vehicle or flutamide i n 40 IU PMSG-injected r a t s . Values are means _+ SEM for paired ovaries (n=6 or 7). **p<0.01, compared to pre-treatment controls (40 IU PMSG only). a , f ip<0.01 and Yp<0.05, compared to corresponding vehicle treatment group. 40 30 -20 -Androgens • 401 u PMSG only 0 40IU PMSG*veh H 40IU PMSG*Flu 24 48 60 72 20 m O u cn c a tfm «9 > 10 176-estradiol * 24 60 72 300 -200 -100 Progesterone 24 I 4 * 60 72 Time after PMSG (hr) - 76 -f l u t a m i d e t r e a t m e n t was a s s o c i a t e d w i t h a 58% d e c r e a s e i n a n d r o g e n l e v e l ( p < 0 . 0 1 ) , a 64% d e c r e a s e i n 1 7 f i - e s t r a d i o l l e v e l (p<0 .01) a n d a 30% i n c r e a s e i n p r o g e s t e r o n e l e v e l (p<0.05) a t 60 h r . T h e r e were no s i g n i f i c a n t d i f f e r e n c e s i n a l l t h r e e o v a r i a n s t e r o i d hormones be tween v e h i c l e - a n d f l u t a m i d e - t r e a t e d g r o u p s b e f o r e a n d a f t e r 60 h r . W i t h t h e e x c e p t i o n o f 1 7 6 - e s t r a d i o l , c h a n g e s i n se rum s t e r o i d hormones f o l l o w e d c o m p a r a b l e p r o f i l e s t o t h o s e d e t e r m i n e d f o r o v a r i a n t i s s u e s t h r o u g h o u t t h e i n t e r v a l s e x a m i n e d ( F i g s . 19 and 2 0 ) . D a t a f o r t h e r e c o v e r y a n d p r e i m p l a n t a t i o n d e v e l o p m e n t o f s u p e r o v u l a t e d o v a a r e p r e s e n t e d i n T a b l e V . The number o f o v a r e c o v e r e d f r o m s u p e r o v u l a t e d r a t s t r e a t e d w i t h v e h i c l e d i m i n i s h e d r a p i d l y a f t e r Day 2 and r e a c h e d 0 o n Day 5 o f p r e g n a n c y . I n c o n t r a s t , 8 t o 12 embryos w e r e c o n s i s t e n t l y r e c o v e r e d f r o m c o n t r o l a n i m a l s o v e r t h e same t i m e p e r i o d . F l u t a m i d e t r e a t m e n t d i d n o t p r e v e n t t h e e a r l y l o s s o f p r e i m p l a n t a t i o n embryos w h i c h t o o k p l a c e a f t e r Day 2 , a l t h o u g h o n Day 5 embryos w e r e o c c a s i o n a l l y r e c o v e r e d f r o m t h e f l u t a m i d e - t r e a t e d r a t s . T h e r e w e r e no s i g n i f i c a n t d i f f e r e n c e s be tween t h e two g r o u p s t r e a t e d w i t h v e h i c l e o r f l u t a m i d e i n t h e a b s o l u t e number o f o v a r e c o v e r e d f r o m Days 2 t o 5 . Compared t o c o n t r o l r a t s , embryo d e v e l o p m e n t i n b o t h v e h i c l e - a n d f l u t a m i d e - t r e a t e d r a t s was r e t a r d e d c o n s i s t e n t l y f r o m Day 2 , and maximum c l e a v a g e was a r r e s t e d f r o m Day 3 o n w a r d s . Embryos i n t h e f l u t a i a i d e t r e a t m e n t ;group d e v e l o p e d to a soaximum o f 6 - c e l l s . w h i l e t h o s e iv». the v e h i c l e t r e a t m e n t g r o u p d i d n o t d e v e l o p b e y o n d t h e 4 - c e l l s t a g e . The o v a w i t h v i s i b l e s i g n s o f d e g e n e r a t i o n w e r e c o n s i s t e n t l y r e c o v e r e d f r o m b o t h o v i d u c t s and u t e r i o f t h e v e h i c l e - o r f l u t a m i d e - t r e a t e d FIGURE 20 Serum l e v e l s o f a n d r o g e n s , 1 7 f i - e s t r a d i o l , and p r o g e s t e r o n e a f t e r t r e a t m e n t w i t h v e h i c l e o r f l u t a m i d e i n 40 IU P M S G - i n j e c t e d r a t s . V a l u e s a r e means +_ SEM (n=6 o r 7 ) . **p<0 .01 , compared t o p r e - t r e a t m e n t c o n t r o l s (40 IU PMSG o n l y ) . a p < 0 . 0 1 and B ' Y p < 0 . 0 5 , compared t o c o r r e s p o n d i n g v e h i c l e t r e a t m e n t g r o u p . 12 10 8 -6 -A -2 0 7^5 Androgens • 4 0 I U PMSG o n l y * E3 4 0 i u P M 5 G * v e h * U 4 0 I U PMSG+Flu 2 4 4 8 6 0 £ e 8) cn E 3 0.8 -0.6 0 .4 0 .2 0 .0 176-estradiol 2 4 4 8 6 0 2 0 0 4 8 6 0 T i m e a f t e r P M S G (hr) 7 2 - 7 8 -TABLE V Effects of flutamide on recovery and developmental capacity of superovulated ova in preimplantation stage 2 a Day of pregnancy 3^ 4 c 5<3 4 IU PMSG No. of ova recovered oviduct uterus Developmental stage 40 IU PMSG plus vehicle e No. of ova recovered oviduct uterus Developmental stage 40 IU PMSG plus flutamidef No. of ova recovered oviduct uterus Developmental stage 9.8+0. 0 2-cell 22.7+3.8 0.8+0.7 1-to 2-cell 21.0+3.1]9 0.5+0.5 10.2+0.5 0 2-to 6-cell 13.2+3.1 1.7+0.7 1-to 4-cell 16.3+2.5i h 3.7+1.5 10.3+0.3 0 8-cell 1.2+0.5 0.8+0.7 1-to 4-cell 2.3+1.0]i 0 0 9.5+0.7 blastocyst 0 0 1.4+1.2]} 0 1-to 2-cell 1-to 6-cell 2-to 6-cell 4-to 6-cell a _ d Results are. expressed as means + SEM for the number of pregnant rats; (a-c) 6 rats, and (d) 5 rats each group. e?f Rats were treated with vehicle or 5 mg flutamide at 30 and 36 hr af'tev injection of 40 IU PMSG. 9~j No significant difference (p>0.05), compared to corresponding vehicle treatment group. - 79 -rats from Day 2 to 5 (Table VI) . In contrast, no degenerate ova in control rats (4 IU PMSG) were found throughout the examination period. In superovulated animals, compared to the vehicle group, flutamide treatment s ignif icant ly (p<0.01) decreased the percentage of degenerate ova on Day 2. However, after Day 3, this' percentage in the flutamide-treated rats rapidly increased to reach a maximum of 83.4% on Day 5 which was similar to that in the vehicle-treated rats on Day 4. On the other hand, flutamide treatment s ignif icant ly (p<0.01) increased the percentage of cleaved embryos above that from the vehicle-treated rats on Day 2 (Table VI) . However, after Day 3, the percentage of cleaved embryos in the flutamide-treated rats gradually decreased to a low of 16.7% on Day 5, which was comparable to that in the vehicle-treated rats on Day 4. Serum androgens and progesterone from Day 2 to 5 (p<0.01) and serum 17fi-estradiol on Day 2 (p<0.05) in superovulated rats treated with vehicle were s ignif icant ly elevated above the values in the control rats treated with 4 IU PMSG (Fig. 21). Compared to the results of vehicle regimen, flutamide treatment after 40 IU PMSG decreased serum androgen level by 39% on Day 2 (p<0.05). On Day 2, serum 17fi-estradiol in the flutamide-treated rats greatly decreased by 51% below that in the vehicle-treated rats but the difference was not s ta t i s t i ca l l y s ignif icant . Data obtained from a separate experiment show the changes in serum contents of androstenedione, testosterone and 5a--dihydrotestosterone after treatment of a control dose (4 IU) of PMSG alone and a superovulatory dose (20 IU) of PMSG with vehicle or flutamide (Fig . 22). Throughout the examination period, the values of a l l three androgenic steroids in the - 80 -TABLE V I Effects of flutamide on normality and cleavage of superovulated ova from both oviducts and uterine horns i n preimplantation stage. Day o f p r e g n a n c y 2 a 3 b 4 c 5 d V e h i c l e e % d e g e n e r a t e o v a 4 5 . 9 + 6 . 0 3 5 . 7 + 1 2 . 2 7 6 . 3 + 1 7 . 7 % c l e a v e d embryos 1 3 . 2 + 4 . 4 3 7 . 0 + 1 0 . 1 1 7 . 5 + 1 1 . 8 F l u t a m i d e ^ % d e g e n e r a t e o v a 12.6+5.0*3 1 3 . 4 + 5 . 6 4 1 . 7 + 2 5 . 0 8 3 . 4 + 1 6 . 7 % c l e a v e d embryos 7 3 . 6 + 6 . 7 n 7 5 . 6 + 6 . 5 5 8 . 3 + 2 5 . 0 1 6 . 7 + 1 6 . 7 a~d R e s u l t s a r e e x p r e s s e d a s means + SEM f o r t h e number o f p r e g n a n t r a t s i n w h i c h more t h a n one ovum w e r e r e c o v e r e d : ( a , b ) 6 r a t s , ( c ) 4 r a t s , a n d (d) 2 r a t s e a c h g r o u p . e ' f R a t s w e r e t r e a t e d w i t h v e h i c l e o r 5 mg f l u t a m i d e a t 30 and 36 h r a f t e r i n j e c t i o n o f 40 IU PMSG. 9 » n p<o.01 w i t h i n e a c h p a r a m e t e r , c o m p a r e d t o c o r r e s p o n d i n g v e h i c l e t r e a t m e n t g r o u p . FIGURE 21 Serum l e v e l s o f a n d r o g e n s , 1 7 f i - e s t r a d i o l , a n d p r o g e s t e r o n e o n Days 2 t o 5 o f p r e g n a n c y a f t e r a d m i n i s t r a t i o n o f 4 I U PMSG a l o n e and 40 I U PMSG p l u s v e h i c l e o r f l u t a m i d e t o immature r a t s . V a l u e s a r e means + SEM (n=5 o r 6 ) . *p<0.05 a n d * * p < 0 . 0 1 , compared t o c o r r e s p o n d i n g c o n t r o l s (4 I U P M S G ) . a p < 0 . 0 5 , compared t o c o r r e s p o n d i n g v e h i c l e t r e a t m e n t g r o u p . Androgens • 41U PMSG 0 40IUPMSG*Veh 40IU PMSG*F1U * T E "9 •s— O L. O cn E e cn 500 i 400 300 200 100 0 Progesterone 3 4 Day o f P r e g n a n c y T Sail FIGURE 22 C o n t e n t s o f s e rum a n d r o g e n i c s t e r o i d s , a n d r o s t e n e d i o n e , t e s t o s t e r o n e , a n d 5 a - d i h y d r o t e s t o s t e r o n e a f t e r a d m i n i s t r a t i o n o f 4 IU PMSG a l o n e and 20 I U PMSG p l u s v e h i c l e o r f l u t a m i d e t o immatu re r a t s . V a l u e s a r e means + SEM (n=10) . *p<0.05 and * * p < 0 . 0 1 , compared t o p r e - t r e a t m e n t c o n t r o l s (20 I U PMSG o n l y ) . a , e , Y p < 0 . 0 5 , c o m p a r e d t o c o r r e s p o n d i n g v e h i c l e t r e a t m e n t g r o u p a t e a c h t i m e . Androstenedione • 4IUPMSQ • 2011) PMSG only 0 20IUPMSG+Veh 101 20IU PMSG+Flu X i . 4 24 48 60 24 48 60 5 a-d i hy d rotestoste ro ne * . * I liii! 24 48 I m 60 Time after PMSG (hr) - 83 -c o n t r o l r a t s (4 I U PMSG) d e c r e a s e d s t e a d i l y f r o m t h e maximum v a l u e s a t 24 h r ( a n d r o s t e n e d i o n e , 1.53+0.17 n g / m l ; t e s t o s t e r o n e , 1.67+0.31 n g / m l ; 5 a - d i h y d r o t e s t o s t e r o n e , 0.56+0.08 n g / m l ) t o t h e i r r e s p e c t i v e minimum v a l u e s b y 60 h r ( a n d r o s t e n e d i o n e , 0.04+0.03 n g / m l ; t e s t o s t e r o n e 0.08+0.04 n g / m l ; 5 a - d i h y d r o t e s t o s t e r o n e , 0.06+0.02 n g / m l ) . I n c o n t r a s t , t h e p r o f i l e o f a n d r o g e n i c s t e r o i d s i n t h e v e h i c l e - t r e a t e d s u p e r o v u l a t e d r a t s e x h i b i t e d g e n e r a l l y i n c r e a s i n g t r e n d f r o m 24 h r . Compared t o t h e s t e r o i d l e v e l s i n p r e - t r e a t m e n t c o n t r o l r a t s o f s u p e r o v u l a t o r y r e g i m e n a t 24 h r , t e s t o s t e r o n e and 5 a - d i h y d r o t e s t o s t e r o n e c o n t e n t s s i g n i f i c a n t l y (p<0.01) i n c r e a s e d by 48 h r , w h i l e a n d r o s t e n e d i o n e c o n t e n t showed a s i g n i f i c a n t (p<0.05) e l e v a t i o n b y 60 h r . F l u t a m i d e t r e a t m e n t t o t h e s u p e r o v u l a t e d r a t s s i g n i f i c a n t l y (p<0.05) d e c r e a s e d t h e c o n t e n t s o f a r o m a t i z a b l e a n d r o g e n i c s t e r o i d s b e l o w t h o s e o f v e h i c l e r e g i m e n : a n d r o s t e n e d i o n e b y 76% a t 48 h r ; t e s t o s t e r o n e b y 75% a t 48 h r and b y 87% a t 60 h r . The l o w e r l e v e l s o f n o n a r o m a t i z a b l e a n d r o g e n , 5 a - d i h y d r o t e s t o s t e r o n e w e r e o b t a i n e d f r o m t h e r a t s o f v e h i c l e t r e a t m e n t g r o u p , a s compared t o t h o s e o f f l u t a m i d e r e g i m e n , b u t n o t s t a t i s t i c a l l y s i g n i f i c a n t . - 84 -V . E f f e c t s o f RU486 a n d T a m o x i f e n o n O v u l a t i o n and O o c y t e M o r p h o l o g y  ( P r o t o c o l 5 ) . E f f e c t s o f t h e s t e r o i d a n t a g o n i s t s (RU486 and t a m o x i f e n ) o n o v u l a t i o n , o o c y t e n o r m a l i t y and o v a r i a n w e i g h t i n r a t s i n j e c t e d w i t h a c o n t r o l d o s e (4 IU) o f PMSG a r e p r e s e n t e d i n T a b l e V I I . A l l r a t s i n v e h i c l e - t r e a t e d g r o u p o v u l a t e d a r a n g e o f 9 -14 o o c y t e s p e r r a t . The o v u l a t o r y r e s p o n s e t o 4 I U PMSG was a p p a r e n t l y i n h i b i t e d b y t h e t r e a t m e n t w i t h e i t h e r RU486 o r t a m o x i f e n . I n R U 4 8 6 - t r e a t e d g r o u p , a l t h o u g h t h e p r o p o r t i o n o f r a t s o v u l a t i n g seemed t o be s l i g h t l y r e d u c e d b y 1 4 . 3 % , t h e mean number o f o o c y t e s r e c o v e r e d f r o m e a c h r a t was s i g n i f i c a n t l y (p<0 .01 ) r e d u c e d b y 39.3% a s c o m p a r e d t o v e h i c l e - t r e a t e d g r o u p . I n t a m o x i f e n - t r e a t e d g r o u p , b o t h t h e p r o p o r t i o n o f r a t s o v u l a t i n g a n d t h e mean number o f o o c y t e s p e r r a t w e r e s i g n i f i c a n t l y r e d u c e d b y 71.4% and 94.9% ( p < 0 . 0 0 1 ) , r e s p e c t i v e l y , a s compared t o e a c h p a r a m e t e r o b s e r v e d f rom v e h i c l e - t r e a t e d g r o u p o f a n i m a l s . T h u s , t a m o x i f e n seemed t o be more e f f e c t i v e i n t h e i n h i b i t i o n o f o v u l a t o r y r e s p o n s e t h a n RU486 when a d m i n i s t e r e d t o 4 I U P M S G - i n j e c t e d r a t s a t e q u i p o t e n t d o s e s ( t h r e e c o n s e c u t i v e i n j e c t i o n s o f 1 mg p e r a n i m a l ) . M o r e t h a n 95% o f o o c y t e s r e c o v e r e d f r o m t h e r a t s o f v e h i c l e - t r e a t e d g r o u p a p p e a r e d t o be n o r m a l ; o n l y one o r two d e g e n e r a t e o o c y t e s w e r e o c c a s i o n a l l y r e c o v e r e d f r o m t h r e e o f t h e seven v e h i c l e - t r e a t e d r a t s . No s i g n i f i c a n t d i f f e r e n c e was o b s e r v e d i n g r o s s m o r p h o l o g y o f t h e o o c y t e s r e c o v e r e d f r o m t h e two g r o u p s o f r a t s t r e a t e d w i t h v e h i c l e o r RU486 a f t e r 4 I U PMSG. I n c o n t r a s t , t a m o x i f e n t r e a t m e n t s i g n i f i c a n t l y (p<0.001) i n c r e a s e d t h e p r o p o r t i o n o f o o c y t e s e x h i b i t i n g v i s i b l e s i g n s o f d e g e n e r a t i o n a s compared t o t h a t i n v e h i c l e - t r e a t e d g r o u p o f a n i m a l s . - 85 -TABLE V I I E f f e c t s o f RU486 and t a m o x i f e n o n r e c o v e r y and n o r m a l i t y o f o o c y t e s f r o m o v i d u c t s , a n d o v a r i a n w e i g h t i n 4 I U P M S G - i n j e c t e d r a t s a T r e a t m e n t P r o p o r t i o n o f r a t s o v u l a t i n g N o . o f o o c y t e s 0 % d e g e n e r a t e o o c y t e s 0 mg o v a r i a n t i s s u e d V e h i c l e 7 / 7 1 1 . 7 + 0 . 7 4 . 7 + 2 . 3 4 3 . 7 + 3 . 9 RU486 6 /7 7 . 1 + 1 . 3 e 7 . 5 + 4 . 8 5 0 . 3 + 4 . 8 T a m o x i f e n 2 / 7 0 . 6 + 0 . 4 f 7 5 . 0 + 2 5 . 0 9 2 9 . 3 + 2 . 0 n a R a t s were t r e a t e d w i t h v e h i c l e , 1 mg RU486 o r 1 mg t a m o x i f e n a t 2 4 , 36 and 48 h r a f t e r i n j e c t i o n o f 4 I U PMSG. D ~ d R e s u l t s o b t a i n e d 24 h r a f t e r t r e a t m e n t a r e e x p r e s s e d a s means + S E . e p < 0 . 0 1 , f / 9 p < 0 . 0 0 1 , ° p < 0 . 0 5 , c o m p a r e d t o c o r r e s p o n d i n g v e h i c l e -t r e a t e d g r o u p . - 8 6 -O v a r i a n w e i g h t r e s p o n s e t o 4 I U PMSG was n o t a f f e c t e d b y RU486 t r e a t m e n t , s i n c e t h e r e w e r e no s i g n i f i c a n t d i f f e r e n c e s i n t h e mean w e i g h t o f o v a r i a n t i s s u e s b e t w e e n v e h i c l e - and R U 4 8 6 - t r e a t e d g r o u p s o f r a t s . I n c o n t r a s t , t a m o x i f e n t r e a t m e n t s i g n i f i c a n t l y (p<0 .05) r e d u c e d t h e mean o v a r i a n w e i g h t b y 33.0% a s compared t o t h a t i n v e h i c l e - t r e a t e d g r o u p . The l e v e l s o f s e rum 1 7 1 3 - e s t r a d i o l , p r o g e s t e r o n e and a n d r o g e n s a f t e r t h e t r e a t m e n t w i t h v e h i c l e , RU486 o r t a m o x i f e n i n 4 I U P M S G - i n j e c t e d r a t s a r e i l l u s t r a t e d i n F i g u r e 2 3 . T h e r e w e r e no s i g n i f i c a n t d i f f e r e n c e s i n t h e l e v e l s o f a l l t h r e e se rum s t e r o i d hormones b e t w e e n v e h i c l e - and R U 4 8 6 - t r e a t e d g r o u p s o f r a t s . However , compared t o v e h i c l e - t r e a t e d g r o u p , t a m o x i f e n t r e a t m e n t was a s s o c i a t e d w i t h a 200.0% i n c r e a s e i n 1 7 B - e s t r a d i o l l e v e l (p<0 .05) a n d a 40.3% d e c r e a s e i n p r o g e s t e r o n e l e v e l ( p < 0 . 0 5 ) . E f f e c t s o f t h e RU486 , and t a m o x i f e n o n o v u l a t i o n , o o c y t e n o r m a l i t y , a n d o v a r i a n w e i g h t i n r a t s i n j e c t e d w i t h a s u p e r o v u l a t o r y d o s e (40 IU) o f PMSG a r e shown i n T a b l e V I I I . A l t h o u g h no o v u l a t i o n s w e r e o b s e r v e d i n a n y r a t s o f p r e - t r e a t m e n t c o n t r o l g r o u p , a l l r a t s i n t h e v e h i c l e - t r e a t e d g r o u p o v u l a t e d c o n s i s t e n t l y t h r o u g h 60 t o 72 h r a f t e r 40 I U PMSG. The mean number o f o o c y t e s r e c o v e r e d f r o m t h e r a t s o f t h e v e h i c l e - t r e a t e d g r o u p was 2 4 . 4 + 1 . 4 o o c y t e s p e r r a t a t 60 h r and f u r t h e r i n c r e a s e d t o r e a c h 4 0 . 1 + 2 . 5 o o c y t e s p e r r a t a t 72 h r . I t was e v i d e n t t h a t t h e s u p e r o v u l a t o r y r e s p o n s e t o 40 I U PMSG was i n h i b i t e d b y t h e t r e a t m e n t w i t h R U 4 8 6 . • I n R U 4 8 6 - t r e a t e d g r o u p , t h e p r o p o r t i o n o f r a t s e x h i b i t i n g o v u l a t i o n was r e d u c e d b y 42.9% a t 60 h r a n d 11 .1% a t 72 h r a s compared t o v e h i c l e - t r e a t e d g r o u p a t e a c h t i m e . The mean number o f o o c y t e s r e c o v e r e d f r o m e a c h r a t o f t h i s g r o u p was s i g n i f i c a n t l y (p<0 .01 ) r e d u c e d b y 70.1% a t 60 h r a n d 47 .1% a t 72 h r when compared t o t h e FIGURE 23 Serum l e v e l s o f 1 7 0 - e s t r a d i o l , p r o g e s t e r o n e and a n d r o g e n s 24 h r a f t e r t r e a t m e n t w i t h v e h i c l e , RU486 o r t a m o x i f e n i n 4 I U P M S G - i n j e c t e d r a t s . V a l u e s a r e means + S E (n=7) . The means w i t h s u p e r s c r i p t s i n common a r e s i g n i f i c a n t l y d i f f e r e n t ( p < 0 . 0 5 ) . TABLE VIII E f f e c t s o f RU486 and t a m o x i f e n o n r e c o v e r y and n o r m a l i t y o f o o c y t e s f r o m o v i d u c t s , and o v a r i a n w e i g h t i n 40 I U P M S G - i n j e c t e d r a t s ^ T r e a t m e n t Time a f t e r PMSG ( h r ) b 60 72 N o . o o c y t e s d e g e n e r a t e o o c y t e s mg o v a r i a n t i s s u e N o . o o c y t e s % d e g e n e r a t e o o c y t e s mg o v a r i a n t i s s u e V e h i c l e RU486 T a m o x i f e n •24 .4+1 .4 ( 7 / 7 ) C 7 . 3 + 2 . 8 d ( 4 / 7 ) 22 .6+1.2 ( 5 / 5 ) 5 0 . 4 + 9 . 8 4 5 . 1 + 6 . 7 7 3 . 2 + 1 0 . 8 1 4 7 . 7 + 8 . 2 1 4 2 . 9 + 6 . 0 1 2 1 . 1 + 4 . 7 f 4 0 . 1 + 2 . 5 ( 8 / 8 ) c 2 1 . 2 + 4 . 1 e ( 8 / 9 ) 2 4 . 8 + 4 . 1 ? ( 5 / 5 ) 41 .6+5 .1 37 .5+11 .8 8 2 . 6 + 8 . 0 h 1 6 1 . 0 + 3 . 9 1 3 8 . 1 + 1 0 . 1 1 1 8 . 0 + 3 . 9 1 a R a t s w e r e t r e a t e d v d t h . v e h i c l e , 1 mg RU486 o r 2 . 5 mg t a m o x i f e n a t 24 , 36 and 48 h r a f t e r i n j e c t i o n o f 40 I U PMSG. ^ R e s u l t s a r e e x p r e s s e d a s means+ SE. c N u m b e r of r a t s e x h i b i t i n g o v u l a t i o n s / t o t a l number o f r a t s k i l l e d a t e a c h t i m e a r e g i v e n i n p a r e n t h e s e s . d/e ,gp<o.oi, f p < 0 . 0 5 , n ' i p < 0 . 0 0 1 , compared t o c o r r e s p o n d i n g v e h i c l e - t r e a t e d g r o u p . - 89 -v e h i c l e - t r e a t e d g r o u p a t e a c h t i m e . On t h e o t h e r h a n d , t a m o x i f e n was m a r g i n a l l y e f f e c t i v e f o r i n h i b i t i o n o f s u p e r o v u l a t o r y r e s p o n s e , a l t h o u g h i t was a d m i n i s t e r e d t o t h e 40 I U P M S G - i n j e c t e d r a t s a t e v e n h i g h e r d o s e s ( t h r e e c o n s e c u t i v e i n j e c t i o n s o f 2 . 5 mg p e r a n i m a l ) t h a n RU486 . When compared t o t h e v e h i c l e - t r e a t e d g r o u p , a s i g n i f i c a n t d i f f e r e n c e was n o t e d i n t h e mean number o f o o c y t e s o n l y a t 72 h r . I n t h i s g r o u p , a l l r a t s o v u l a t e d c o n s i s t e n t l y t h r o u g h o u t t h e p e r i o d e x a m i n e d . R e l a t i v e l y h i g h p r o p o r t i o n s o f d e g e n e r a t e o o c y t e s w e r e o b s e r v e d i n v e h i c l e - t r e a t e d s u p e r o v u l a t e d r a t s : 50.4+9.8% a t 60 h r a n d 41.6+5.1% a t 72 h r a f t e r 40 I U PMSG. T h e r e w e r e no s i g n i f i c a n t d i f f e r e n c e s i n t h e p e r c e n t a g e s o f d e g e n e r a t e o o c y t e s r e c o v e r e d f r o m t h e r a t s i n v e h i c l e - and R U 4 8 6 - t r e a t e d g r o u p s t h r o u g h o u t t h e p e r i o d e x a m i n e d . I n c o n t r a s t , t a m o x i f e n t r e a t m e n t s i g n i f i c a n t l y (p<0 .001) i n c r e a s e d t h e p r o p o r t i o n o f o o c y t e s e x h i b i t i n g s i g n s o f d e g e n e r a t i o n a t 72 h r a s compared t o t h a t i n v e h i c l e - t r e a t e d g r o u p o f a n i m a l s . The w e i g h t r e s p o n s e o f s u p e r o v u l a t e d o v a r i e s a f t e r 40 I U PMSG was n o t a f f e c t e d by RU 486 t r e a t m e n t b u t m a r k e d l y i n h i b i t e d b y t a m o x i f e n t r e a t m e n t . V i r t u a l l y no s i g n i f i c a n t d i f f e r e n c e s w e r e r e c o r d e d i n t h e mean o v a r i a n w e i g h t b e t w e e n v e h i c l e - and R U 4 8 6 - t r e a t e d g r o u p o f r a t s . I n c o n t r a s t , t a m o x i f e n t r e a t m e n t s i g n i f i c a n t l y r e d u c e d t h e mean o v a r i a n w e i g h t b y 18.0% a t 60 h r (p<0 .05 ) a n d 26.7% a t 72 h r ( p<0 .001 ) a s compared t o t h a t i n v e h i c l e - t r e a t e d g r o u p of a n i m a l s . The c h a n g e s i n s e r u m l e v e l s o f 1 7 f i - e s t r a d i o l , p r o g e s t e r o n e a n d a n d r o g e n s b e f o r e o r a f t e r t h e t r e a t m e n t w i t h v e h i c l e , RU486 o r t a m o x i f e n i n r a t s i n j e c t e d w i t h 40 I U PMSG a r e p r e s e n t e d i n F i g u r e 2 4 . A t 60 h r a f t e r F I G U R E 24 Serum l e v e l s o f 1 7 0 - e s t r a d i o l , p r o g e s t e r o n e a n d a n d r o g e n s b e f o r e o r a f t e r t r e a t m e n t w i t h v e h i c l e , RU486 o r t a m o x i f e n i n 40 I U P M S G - i n j e c t e d r a t s . V a l u e s a r e means + S E ( n = 5 - 7 ) . The means w i t h no s u p e r s c r i p t s i n common a r e s i g n i f i c a n t l y d i f f e r e n t ( p < 0 . 0 5 ) . OH 40IUF • 40IUF Q 40IUF • 40IUF a 17B >MSG only 'MSG+Veh >MSG+RU486 •MSG+Tam -est T l rad d I iol a r r d I a iliij! Progc jste de c Tr iron r ab I e i c tx ibc T T cc I e 12 60 72 Time after PMSG (hr) - 91 -PMSG, t h e l e v e l s o f a l l t h r e e s t e r o i d hormones i n v e h i c l e - t r e a t e d g r o u p o f s u p e r o v u l a t e d r a t s w e r e s i g n i f i c a n t l y (p<0 .05 ) e l e v a t e d a b o v e t h o s e o f p r e - t r e a t m e n t c o n t r o l s o b t a i n e d a t 12 h r . Compared t o t h e v e h i c l e - t r e a t e d g r o u p , t h e r e w e r e no s i g n i f i c a n t c h a n g e s i n a n y o f t h e t h r e e s t e r o i d hormones t h r o u g h o u t t h e p e r i o d e x a m i n e d a f t e r RU486 t r e a t m e n t . Howeve r , t a m o x i f e n t r e a t m e n t r e s u l t e d i n m a r k e d a l t e r a t i o n s i n t h e l e v e l s o f o v e r a l l s t e r o i d h o r m o n e s : 102.7% (p<0 .01) a n d 800.0% (p<0.001) i n c r e a s e s f o r 1 7 B - e s t r a d i o l , 63.9% (p<0 .05 ) a n d 180.6% (p<0 .001) i n c r e a s e s f o r a n d r o g e n s a t 60 h r and 72 h r , r e s p e c t i v e l y , and a 46.2% (p<0.05) d e c r e a s e f o r p r o g e s t e r o n e a t 60 h r . - 92 -D I S C U S S I O N D e s p i t e w i d e s p r e a d a p p l i c a t i o n o f s u p e r o v u l a t i o n t e c h n i q u e s f o r t h e y i e l d o f m u l t i p l e o o c y t e s a n d embryos i n l i v e s t o c k i n d u s t r y a n d human I V F and embryo r e p l a c e m e n t p rog rammes , s u p e r o v u l a t o r y t r e a t m e n t w i t h e x o g e n o u s g o n a d o t r o p i n s f r e q u e n t l y r e s u l t s i n i m p a i r e d f e r t i l i t y s u c h a s r e d u c e d f e r t i l i z a t i o n , a b n o r m a l embryo d e v e l o p m e n t and i m p l a n t a t i o n f a i l u r e i n v a r i o u s mammalian s p e c i e s . T h i s i m p a i r m e n t h a s p r e v i o u s l y b e e n a s s o c i a t e d w i t h v a r i a b i l i t y i n t i m e o f o v u l a t i o n ( E v a n s a n d A r m s t r o n g , 1 9 8 3 ; W a l t o n e t a l . , 1983) and e x c e s s i v e e s t r o g e n i c s t i m u l a t i o n o f r e p r o d u c t i v e t r a c t t o r e s u l t i n a s y n c h r o n y b e t w e e n e m b r y o n i c a n d u t e r i n e e n v i r o n m e n t ( M i l l e r and A r m s t r o n g , 1 9 8 1 b ; W a l t o n a n d A r m s t r o n g , 1 9 8 2 ) . However , m a j o r f a c t o r s and c r i t i c a l t i m e i n v o l v e d i n t h e r e d u c e d f e r t i l i t y f o l l o w i n g s u p e r o v u l a t i o n w e r e n o t f u l l y u n d e r s t o o d . The d e f e c t s c o u l d o c c u r i n t h e p r o c e s s o f t h e o o c y t e m a t u r a t i o n t h r o u g h o v u l a t i o n p r i o r t o f e r t i l i z a t i o n b y h y p e r s t i m u l a t i o n o f o v a r i a n t i s s u e s and a c c e l e r a t i o n o f f o l l i c u l a r d e v e l o p m e n t b e c a u s e o f t h e b i o c h e m i c a l p r o p e r t i e s o f PMSG w i t h i t s p r o l o n g e d a c t i o n and a m i x t u r e o f FSH and L H t o s t i m u l a t e f o l l i c u l a r s t e r o i d s e c r e t i o n ( G o s p o d a r o w i c z and P a p k o f f , 1 9 6 7 ; Schams e t a l . , 1 9 7 8 ) . P r e - o r p e r i - o v u l a t o r y e n d o c r i n e a s p e c t s i n c l u d i n g t h e s e c r e t i o n o f f o l l i c u l a r s t e r o i d s and endogenous g o n a d o t r o p i n s a r e b e l i e v e d t o a f f e c t t h e f i n a l m a t u r a t i o n asid:further f e r t i l e l i f e o f o o c y t e s m'ior t o f e r t i l i z a t i o n . . I n t h e p r e s e n t s t u d y c o n d u c t e d w i t h f i v e e x p e r i m e n t a l , p r o t o c o l s , t h e p r i m a r y d e f e c t s and p o s s i b l e mechan i sms l e a d i n g t o i n f e r t i l i t y f o l l o w i n g s u p e r o v u l a t i o n have b e e n d i s c u s s e d w i t h s p e c i a l a t t e n t i o n t o t h e o v u l a t o r y r e s p o n s e , o o c y t e q u a l i t y ( g r o s s m o r p h o l o g y a n d n u c l e a r m a t u r a t i o n ) , - 93 -developmental potential of preimplantation embryos, and pre- or peri-ovulatory responses of ovarian steroid hormones and circulating LH in the rats superovulated with PMSG. Protocol 1. The results of this experiment show that a single injection of superovulatory doses (20 and 40 IU) of PMSG in rats induced the firs t ovulation as early as 24 hr with two distinct increases in the number of released oocytes: one before 36 hr and the other after 48 hr of treatment. In addition, superovulatory treatments caused a dose-dependent increase in the population of abnormal oocy t e s an& 9 marked alteration of ovarian steroid pattern from 36 hr onwards after gonadotropin administration. Time-course studies of ovulation using various neuropharmacologic central nervous system depressants (Sorrentino et al., 1972; Hillensjo et al., 1974) and hypophysectomy (Strauss and Meyer, 1962; Zarrow and Quinn, 1963) suggested that in immature rats the release of endogenous LH occurs at 53-57 hr following PMSG injection. The present results, however, revealed that a single injection of 20 or 40 IU PMSG caused the first ovulation 24 hr after treatment. Walton et al. (1983) mentioned the occurrence of the f i r s t ovulation 38-46 hr after injection of 40 IU PMSG in immature rats. The variation of the first ovulation induction time may be due to the different model system, i.e. i t may depend upon body weight or age of rats. The occurrence of the first ovulation as.early as 24.hr is evidenced by»-tfe observation of ruptured l u t e i n i z e d folldcles i n the stainad?se<SiGias::of ovaries from rats sacrificed 24 hr after injection of 20 or 40 IU PMSG. These observations indicate that 20 or 40 IU PMSG may have contained sufficient LH-like activity to trigger ovulation as early as 24 hr after injection. - 94 -The p r e c i s e mechan i sm o f P M S G - i n d u c e d o v u l a t i o n i s n o t k n o w n . A c c o r d i n g t o Schams e t a l . ( 1 9 7 8 ) , PMSG h a s a l o n g h a l f - l i f e a n d a s l o w c l e a r a n c e r a t e a s a c o n s e q u e n c e o f i t s v e r y h i g h s i a l i c a c i d c o n t e n t , a s w e l l a s a m i x t u r e o f F S H - and L H - l i k e a c t i v i t y when m e a s u r e d b y b i o a s s a y . The se c h e m i c a l p r o p e r t i e s o f PMSG may be r e s p o n s i b l e f o r a p r e m a t u r e a c t i v a t i o n o f o o c y t e s , e s p e c i a l l y i n t h o s e f o l l i c l e s t h a t show t h e g r e a t e s t d e g r e e o f r e c e p t o r d i f f e r e n t i a t i o n a t t h e t i m e o f g o n a d o t r o p i n a d m i n i s t r a t i o n (Moor e t a l . , 1 9 8 5 ) . I t h a s b e e n s u g g e s t e d t h a t PMSG i n d u c e s o v u l a t i o n t h r o u g h b o t h d i r e c t a n d i n d i r e c t mechan i sms i n immatu re r a t s (McCormack and M e y e r , 1 9 6 3 ) . B e c a u s e o f i t s d o m i n a n t a n d p r o l o n g e d FSH a c t i v i t y , PMSG p r i m a r i l y a c t s a t o v a r i a n f o l l i c u l a r l e v e l t o s t i m u l a t e f o l l i c u l a r d e v e l o p m e n t p r e s u m a b l y b y i n c r e a s i n g t h e r e c r u i t m e n t f r o m n o n - g r o w i n g p o o l o f p r i m o r d i a l f o l l i c l e s , and e n h a n c e s o v a r i a n s t e r o i d hormone p r o d u c t i o n ( C h a t t e r j e e e t a l . , 1 9 7 7 ; D o t t e t a l . , 1 9 7 9 ) . S u b s e q u e n t l y , t h e i n c r e a s e d s t e r o i d s p r o d u c e e i t h e r a p o s i t i v e o r a n e g a t i v e f e e d b a c k o h t h e endogenous LH s u r g e t o t r i g g e r o v u l a t i o n ( Y i n g and M e y e r , 1 9 6 9 ) . I n t h e p r e s e n t e x p e r i m e n t , s u p e r o v u l a t o r y t r e a t m e n t w i t h 20 o r 40 I U PMSG showed two d i f f e r e n t p a t t e r n s : i n c r e a s e s o f o v u l a t i o n b e t w e e n 2 4 - 3 6 h r and be tween 4 8 - 7 2 h r b u t a p l a t e a u b e t w e e n 36-48 h r a f t e r i n j e c t i o n . The number o f o o c y t e s r e c o v e r e d d u r i n g t h e f i r s t i n c r e a s i n g p e r i o d was l e s s t h a n o r s i T r i i i a r t o n o r m a l r e c o v e r y ( u p t o 15 o o c y c e s / r a t ) , b u t above c h a t i n t h e 1 s s i c o r i i l ! - i n c r e a s i n g p e r i o d (up t o 58 o o c y t e s / r a t ) . T h i s f i n d i n g i n d i c a t e s t h a t t h e r e s p o n s e s t o s u p e r o v u l a t o r y d o s e s o f PMSG t a k e p l a c e p o s s i b l y b y d i f f e r e n t mechanisms d e p e n d i n g u p o n t h e t i m e a f t e r t h e t r e a t m e n t w i t h PMSG. O v a r i a n s t e r o i d hormones a r e , i n g e n e r a l , c r u c i a l i n r e g u l a t i n g o v u l a t o r y - 95 -r e s p o n s e . The c i r c u l a t i n g l e v e l o f e s t r o g e n s p r o d u c e d b y PMSG e x e r t s i t s s t i m u l a t o r y e f f e c t o n t h e h y p o t h a l a m u s a n d f u r t h e r , p r o g e s t e r o n e may s y n e r g i z e w i t h e s t r o g e n s t o f a c i l i t a t e g o n a d o t r o p i n r e l e a s e i n i m m a t u r e f e m a l e r a t s ( H a g i n o and G o l d z i e h e r , 1 9 7 0 ) . T h i s f a c i l i t a t i n g a c t i o n o f p r o g e s t e r o n e may be due t o i t s l o w e r i n g t h e t h r e s h o l d o f t h e h y p o t h a l a m u s f o r g o n a d o t r o p i n r e l e a s e (Kawakami a n d S a w y e r , 1 9 5 9 ) . I n t h e p r e s e n t e x p e r i m e n t , t h e f i r s t i n c r e a s e o f o v u l a t i o n w i t h i n 36 h r f o l l o w i n g s u p e r o v u l a t i o n a p p e a r s t o be due t o t h e L H - l i k e m o i e t y o f PMSG i t s e l f . T h i s c o n c e p t may be a c c o u n t e d f o r b y t h e a b s e n c e o f s i g n i f i c a n t e l e v a t i o n o f 1 7 f i - e s t r a d i o l and p r o g e s t e r o n e l e v e l s d u r i n g t h e f i r s t s t a g e . H o w e v e r , t h e s e c o n d i n c r e a s e f r o m 48 h r onwards may b e due t o t h e endogenous L H s u r g e v i a p o s i t i v e f e e d b a c k s t i m u l u s i n i t i a t e d b y c o i n c i d e n t a l e l e v a t i o n o f t h e s t e r o i d s o r due t o b o t h endogenous and e x o g e n o u s L H a c t i v i t i e s . T h i s g o n a d o t r o p i n h a s a b i o l o g i c a l h a l f - l i f e o f 54 -60 h r (Sasamoto e t a l . , 1 9 7 2 ) . T h e r e f o r e , a f t e r t h e f i r s t s t a g e o f i n c r e a s e , l a r g e s u p e r o v u l a t o r y d o s e s o f PMSG may s t i m u l a t e t h e g r o w t h o f o v a r i a n f o l l i c l e s t o o v u l a t e b y i t s own L H - l i k e m o i e t y . I t h a s b e e n s u g g e s t e d t h a t h i g h d o s e o f PMSG i n c r e a s e s o v a r i a n s t e r o i d o g e n i c e f f i c i e n c y , and p r o d u c e s p r e m a t u r e a c t i v a t i o n o f o o c y t e s m o s t p r o b a b l y b y i t s s u f f i c i e n t L H - l i k e a c t i v i t y (Moor e t a l . , 1 9 8 5 ) . T h i s e a r l i e r activation? of' vjocytas may c a u s e abnotaalities because ..of t h e . l i k e l y u n s u i t a b l e follicKJlar; e n S i c o n m e n t f o r t h e i r m a t u r a t i o n and o v e r - r i p e n e s s a t o v u l a t i o n . I n s u p e r o v u l a t e d immatu re r a t s , e x c e s s i v e r i s e s i n b o t h o v a r i a n and se rum s t e r o i d l e v e l s ( m a i n l y 1 7 B - e s t r a d i o l ) w e r e n o t e d p r i o r a n d s u b s e q u e n t t o n o r m a l l y e x p e c t e d t i m e o f o v u l a t i o n ( M i l l e r and A r m s t r o n g , - 96 -1981b; Walton and Armstrong, 1981). Walton and Armstrong (1981) suggested that the superovulatory treatment with 40 IU PMSG, because of i t s prolonged biological ac t iv i ty , s tr ik ingly elevated estrogen levels for longer than normal periods. However, no published data are available on the ovarian androgen response during the extended period of ovulation. Further, the correlation between gonadotropin-induced changes of overal l ovarian steroid responses (estrogens, progesterone and androgens) and oocyte abnormalities following superovulation has not yet been studied. . The results of the present experiment suggest that the alterations in ovarian steroid levels following superovulation may be responsible for the occurrence of oocyte degeneration 36-48 hr after administration of 20 or 40 IU PMSG. From this period onwards, superovulatory treatments with PMSG produce an early r ise or prolonged elevation of 176-estradiol and progesterone above control levels . In addition, two peaks of androgens in control rats occur before this period, whereas in superovulated rats, there was a persistent elevation with similar magnitude to control regimen after this period. The alterations of overal l steroid patterns from 36 hr onwards coincide with the timing of r i s ing percentage of abnormal oocytes. The abnormality of oocytes i s consistent with the observation of a predominance of oocyte degeneration in the large f o l l i c u l a r sections from superovulated rats sacrif iced 48 hr after PMSG injection. A portion of degenerating .(ovocytes previously trapped in large follicles may contribute to ovulating: pool of the abnormal oocytes. This poss ib i l i ty i s supported by the suggestion of Fleming (1982) that superovulatory treatment may induce the ovulation of degenerating oocytes from the abnormal f o l l i c l e s , which would - 97 -n o t o v u l a t e in n o r m a l c y c l i c e s t r o u s s t a g e . A d d i t i o n a l l y , o o c y t e a g i n g due t o e a r l i e r a c t i v a t i o n by t h e LH a c t i v i t y of PMSG may a l s o be a c o n t r i b u t i n g f a c t o r f o r t h e o c c u r r e n c e o f a b n o r m a l o o c y t e s . T h i s may be a c c o u n t e d f o r by t h e p r e s e n t r e s u l t s d e m o n s t r a t i n g t h a t 36 h r a f t e r a d m i n i s t r a t i o n o f 40 I U PMSG, a p o r t i o n o f a b n o r m a l o o c y t e s f r e e o f c u m u l u s masses were o b s e r v e d d e s p i t e no s i g n i f i c a n t e l e v a t i o n o f 17B - e s t r a d i o l and a n d r o g e n l e v e l s a b o v e c o n t r o l . I n t h e p r e s e n t e x p e r i m e n t , no a s s e s s m e n t was made o f f e r t i l i z a b i l i t y and d e v e l o p m e n t a l c a p a c i t y o f " n o r m a l - a p p e a r i n g " s u p e r o v u l a t e d o o c y t e s , b u t s u c h o o c y t e s w o u l d s t i l l l i k e l y have t h e s e p o t e n t i a l s . W a l t o n and A r m s t r o n g (1983) d e m o n s t r a t e d t h a t f r e s h l y o v u l a t e d " n o r m a l - a p p e a r i n g " o o c y t e s f o l l o w i n g s u p e r o v u l a t i o n w i t h 40 I U PMSG have a n e q u a l a b i l i t y t o f e r t i l i z e and d e v e l o p t o f e t a l s t a g e a s do o o c y t e s f r o m 4 IU P M S G - t r e a t e d r a t s . The p a r t i c i p a t i o n o f s t e r o i d s i n t h e m a t u r a t i o n o f mammalian o o c y t e s i n v i t r o h a s p r e v i o u s l y been d o c u m e n t e d . 17fl - e s t r a d i o l , a t a v a r i e t y o f c o n c e n t r a t i o n s , s i g n i f i c a n t l y r e d u c e d t h e i n c i d e n c e o f o o c y t e m a t u r a t i o n i n r a b b i t and c a t t l e ( R o b e r t s o n a n d B a k e r , 1969), p i g (McGaughey , 1977), and mouse ( N e k o l a and S m i t h , 1974; E p p i g a n d K o i d e , 1978). P r o g e s t e r o n e , a t a h i g h c o n c e n t r a t i o n (102 yM), e n h a n c e d t h e o o c y t e m a t u r a t i o n in r a b b i t and c a t t l e ( R o b e r t s o n a n d B a k e r , 1969), a n d gohadot rop ia-treated p r e p u b e r t a l r h e s u s monkey (Gould'and Graham, 1976)? w h i l e a t l w c o n c e n t r a t i o n s (10 a n d 31.8 yM) i t decreased m a t u r a t i o n f r e q u e n c y in mouse ( N e k o l a and Smith, 1974; Eppig a n d K o i d e , 1978) and rabbit ( S m i t h et a l . , 1978). Moor et a l . , (1980b) d e m o n s t r a t e d that a l t e r a t i o n s in the n o r m a l p a t t e r n of s t e r o i d s s e c r e t e d d u r i n g m a t u r a t i o n - 98 -i n d u c e d i n t r a c e l l u l a r c h a n g e s i n s h e e p o o c y t e s e x p r e s s e d a s g r o s s a b n o r m a l i t i e s a t f e r t i l i z a t i o n . I n N e m b u t a l - t r e a t e d r a t s , p r o l o n g e d e x p o s u r e o f t h e i n t r a f o l l i c u l a r o o c y t e s t o 1 7 B - e s t r a d i o l d u r i n g t h e o v u l a t o r y d e l a y i s c a u s a l l y r e l a t e d t o s u b s e q u e n t a b n o r m a l d e v e l o p m e n t and e m b r y o n i c d e a t h ( B u t c h e r and P o p e , 1 9 7 9 ) . The mechan i sm b y w h i c h e s t r o g e n i n d u c e s a b n o r m a l embryos i s unknown b u t r e l a t e d t o a l t e r a t i o n s i n t h e o o c y t e p r i o r t o f e r t i l i z a t i o n ( P e l u s o and B u t c h e r , 1 9 7 4 ) . The r e s u l t s o f p r e s e n t e x p e r i m e n t have r e v e a l e d t h e r i s i n g p e r c e n t a g e o f a b n o r m a l o o c y t e s f o l l o w i n g s u p e r o v u l a t i o n i s d o s e - d e p e n d e n t . H o w e v e r , 1 7 B - e s t r a d i o l l e v e l i n 20 I U P M S G - t r e a t e d r a t s was c o n s i s t e n t l y h i g h e r t h a n t h a t i n 40 I U P M S G - t r e a t e d r a t s . E x p l a n a t i o n o f t h i s phenomenon needs f u r t h e r e x p e r i m e n t a t i o n . T h i s r a i s e s t h e q u e s t i o n o f w h e t h e r t h e p r o l o n g e d e l e v a t i o n o f 1 7 B - e s t r a d i o l i n s u p e r o v u l a t e d r a t s m i g h t be a m a j o r f a c t o r l e a d i n g t o t h e i n c i d e n c e o f a b n o r m a l o o c y t e s f r o m 36 h r a f t e r i n j e c t i o n o f PMSG. I n t h e p r e s e n t e x p e r i m e n t , t h e most i n t e r e s t i n g r e s u l t i s t h e s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n be tween o v a r i a n o r s e r u m a n d r o g e n l e v e l s and p e r c e n t a g e s o f a b n o r m a l o o c y t e s be tween 24 a n d 60 h r f o l l o w i n g s u p e r o v u l a t i o n . P r o t o c o l 2 . The s t e r o i d p a t t e r n s i n f o l l i c u l a r f l u i d o f c o n t r o l (4 I U PMSG) and s u p e r o v u l a t e d r a t s (20 I U PMSG) c l o s e l y r e s e m b l e t h e c h a n g e s i n o v a r i a n t i s s u e s and s e r u m r e p o r t e d p r e v i o u s l y (Yun e t a l . , 1987) i n d i c a t i n g t h a t s u p e r o v u l a t o r y treatment r e s u l t s - i n a b n o r m a l follieala-r s t e r o i d o g e n e s i s , m the . p res^ |v | ; . , ; j i . x r j e r ime .n fcssuperovu la t ion with- „2iL J&JfSSGu. i n immature r a t s s u b s t a n t i a l l y a l t e r e d t h e f o l l i c u l a r c o n t e n t s o f p r o g e s t e r o n e and p a r t i c u l a r l y a n d r o g e n s , and c o n s i s t e n t l y i n t e r f e r e d with s e q u e n t i a l changes i n o v e r a l l r a t i o s o f A / E , P / E a n d P / A . A d d i t i o n a l l y , i t - 99 -p r o d u c e d a t y p i c a l o v u l a t i o n s o f m e i o t i c a l l y p r e m a t u r e o r a s y n c h r o n o u s o o c y t e s . I n immatu re r a t s , t h e d o s e s o f 4 - 8 I U PMSG a r e known t o p r o d u c e a p a t t e r n o f c i r c u l a t i n g s t e r o i d hormones s i m i l a r t o t h a t s e e n i n c y c l i n g a d u l t r a t s , and i n d u c e a n L H s u r g e w h i c h g e n e r a t e s a p h y s i o l o g i c a l number o f o v u l a t i n g f o l l i c l e s ( W i l s o n e t a l , 1 9 7 4 ; P a r k e r e t a l . , 1 9 7 6 ) . Time c o u r s e s t u d i e s o f o v u l a t o r y r e s p o n s e i n r a t s d e m o n s t r a t e d t h e c r i t i c a l t i m e o f L H s u r g e a t 53-57 h r ( S o r r e n t i n o e t a l . , 1 9 7 2 ; H i l l e n s j o e t a l . , 1974) and o v u l a t i o n a t 6 0 - 7 2 h r a f t e r PMSG ( W a l t o n e t a l . , 1 9 8 3 ; Y u n e t a l . , 1 9 8 7 ) . F o l l i c u l a r o o c y t e s o b t a i n e d f r o m P M S G - i n j e c t e d r a t s showed a r e s u m p t i o n o f m e i o s i s , b a s e d o n g e r m i n a l v e s i c l e b r e a k - d o w n , 2 - 3 h r a f t e r L H s u r g e ( H i l l e n s j o e t a l . , 1 9 7 4 ) . T h e r e f o r e , i t i s mos t l i k e l y t h a t i n immature r a t s l o w d o s e s ( 4 - 8 IU) o f PMSG i n d u c e o v u l a t i o n s w i t h i n t e g r a t e d s e q u e n t i a l c h a n g e s i n e n d o c r i n e r e s p o n s e a s w e l l a s w i t h s y n c h r o n i z e d p r o c e s s o f o o c y t e m a t u r a t i o n . T h i s c o n c e p t c o u l d be s u p p o r t e d b y t h e c u r r e n t o b s e r v a t i o n s o f c o n s i s t e n t r e c o v e r y o f metaphase I I o o c y t e s a t o v u l a t i o n and s e q u e n t i a l c h a n g e s i n f o l l i c u l a r s t e r o i d c o n t e n t s i n c o n t r o l r a t s t r e a t e d w i t h 4 I U PMSG. F o l l i c u l a r s t e r o i d p r o f i l e , e x c e p t f o r a n d r o g e n s , i n c o n t r o l r a t s g e n e r a l l y a c c o r d s w i t h t h e r e s u l t s o f s e r u m s t e r o i d s ( P a r k e r e t a l . , 1976). I n t h e p r e s e n t e x p e r i m e n t , t h e f o l l i c u l a r c o n t e n t s o f b o t h 1 7 f l - e s t r a d i o l a n d progesterone f r o m c o n t r o l r a t s w e r e s i g n i f i c a n t l y elevated b y 48 h r a f t e r PM.SGv.^Jfee . e l e v a t i o n s o f these f o l l i c u l a r steroids^ p r i o r to'the-expected c r i t i c a l t i m e a r e b e l i e v e d t o t r i g g e r o r s t i m u l a t e t h e p r e o v u l a t o r y g o n a d o t r o p i n s u r g e a s s u g g e s t e d b y H a g i n o and G o l d z i e h e r (1970). On t h e o t h e r h a n d , t h e c u r r e n t o b s e r v a t i o n o f c h a n g e i n f o l l i c u l a r a n d r o g e n c o n t e n t - 100 -d o e s n o t c o n f i r m t h e p r o f i l e o f c o n s i s t e n t e l e v a t i o n o f t e s t o s t e r o n e a s r e p o r t e d by P a r k e r e t a l ( 1 9 7 6 ) . A g r e a t d i f f e r e n c e i n a n d r o g e n r e s p o n s e t o PMSG may be due t o t h e d i f f e r e n c e i n a n d r o g e n i c s t e r o i d s m e a s u r e d ; i n t h e p r e s e n t s t u d y , t h e s t e r o i d s measured u s i n g t e s t o s t e r o n e a n t i s e r u m w e r e r e f e r r e d t o a s a n d r o g e n s b e c a u s e o f i t s h i g h c r o s s - r e a c t i v i t y w i t h o t h e r a n d r o g e n s . The r o l e o f i n i t i a l s t i m u l a t i o n and s u b s e q u e n t s u p p r e s s i o n o f a n d r o g e n s e c r e t i o n d u r i n g f o l l i c u l a r d e v e l o p m e n t i n c o n t r o l r a t s r e m a i n s t o be c l a r i f i e d . The r e s u l t s o f p r e s e n t e x p e r i m e n t s u g g e s t t h a t a b n o r m a l f o l l i c u l a r s t e r o i d o g e n e s i s f o l l o w i n g s u p e r o v u l a t i o n may b e a s s o c i a t e d w i t h a t y p i c a l o v u l a t i o n s o f o o c y t e s w i t h m e i o t i c a b e r r a t i o n . S u p e r o v u l a t o r y t r e a t m e n t w i t h 20 IU PMSG i n r a t s r e s u l t e d i n s i g n i f i c a n t e l e v a t i o n o f p r o g e s t e r o n e , and i n i t i a l d e c r e a s e and s u b s e q u e n t i n c r e a s e i n a n d r o g e n s a s compared t o c o n t r o l , and c o n s i s t e n t l y d i s r u p t e d t h e s e q u e n t i a l c h a n g e s w i t h p r o g r e s s i v e d e c r e a s e i n A / E r a t i o and p r o g r e s s i v e i n c r e a s e s i n P / E and P / A r a t i o s o b s e r v e d i n c o n t r o l r a t s . L i k e t h e a b n o r m a l s t e r o i d o g e n e s i s , s u p e r o v u l a t e d o o c y t e s were s u b j e c t t o a h i g h d e g r e e o f p r e m a t u r i t y o r a s y n c h r o n o u s n u c l e a r m a t u r a t i o n v a r y i n g f r o m p r o p h a s e I t o metaphase I I . These r e s u l t s i n d i c a t e t h a t o o c y t e a n d f o l l i c l e m a t u r a t i o n i n s u p e r o v u l a t e d r a t s may o c c u r i n d e p e n d e n t l y o f t h e n o r m a l p h y s i o l o g i c a l e v e n t s . I t h a s p r e v i o u s l y b e e n d e m o n s t r a t e d t h a t s u p e r c v u i a t o r y d o s e s (20 and 40 IU) o f PMSG i n ' r a t s p r o d u c e d m u l t i p l e o v u ^ a t . i b p s . v j h i c h accompany t h e f i r s t o v u l a t i o n a s e a r l y a s 24 h r a f t e r t r e a t m e n t s ( Y u n e t a l . , 1 9 8 7 ) . S u c h a t y p i c a l o v u l a t i o n h a s a l s o b e e n o b s e r v e d i n g o n a d o t r o p i n - t r e a t e d m a t u r e m i c e ( S t e r n a n d S c h e u t z , 1 9 7 0 ) , and i n p r o g e s t e r o n e - a n d P M S G - t r e a t e d r a t s ( Z a r r o w and G a l l o , 1 9 6 9 ; G a l l o a n d Z a r r o w , 1 9 7 0 ) . - 101 -The a s y n c h r o n y phenomenon h a s p r e v i o u s l y b e e n r e p o r t e d i n s u p e r o v u l a t e d sheep (Moor e t a l . , 1984) a n d c a t t l e ( G r e v e e t a l . , 1 9 8 4 ) , a n d a s c r i b e d t o p r e m a t u r e m e i o t i c a c t i v a t i o n o f o o c y t e s f r o m c e r t a i n f o l l i c l e s and m e i o t i c a r r e s t a f t e r g e r m i n a l v e s i c l e b r e a k - d o w n a n d p r i o r t o o r a t t h e d i a k i n e s i s s t a g e (Moor e t a l . , 1 9 7 3 ) . Howeve r , i n t h e s e s t u d i e s , t h e o b s e r v a t i o n o f a s y n c h r o n o u s n u c l e a r m a t u r a t i o n f o l l o w i n g s u p e r o v u l a t i o n h a s b e e n made i n i n t r a f o l l i c u l a r o o c y t e s p r i o r t o o v u l a t i o n . I n t h e p r e s e n t e x p e r i m e n t , t h e o v i d u c t a l r e c o v e r y o f s u p e r o v u l a t e d o o c y t e s w i t h v a r i o u s s t a g e s o f n u c l e a r m a t u r a t i o n p r o v i d e s d i r e c t e v i d e n c e o f a t y p i c a l o v u l a t i o n s a f t e r PMSG, and s u p p o r t s t h e p r e v i o u s s u g g e s t i o n t h a t a p o r t i o n o f s u p e r o v u l a t e d o o c y t e s c a n be d e r i v e d f r o m p h y s i o l o g i c a l l y u n f i t f o l l i c l e s , w h i c h w o u l d n o t o v u l a t e a t a r e g u l a r c y c l i c e s t r u s (Braw and T s a f r i r i , 1 9 8 0 ; F l e m i n g , 1 9 8 2 ; Yun e t a l . , 1 9 8 7 ) . The p r e c i s e mechan i sm f o r t h e a t y p i c a l o v u l a t i o n s o f s u p e r o v u l a t e d o o c y t e s w i t h m e i o t i c a b e r r a t i o n i s n o t y e t c l e a r . B y f a r , t h e p r e c o c i o u s o r m u l t i p l e o v u l a t i o n s r e s u l t i n g f r o m s u p e r o v u l a t o r y t r e a t m e n t s h a v e b e e n a t t r i b u t e d t o t h e exogenous L H a c t i v i t y o f h i g h d o s e s o f PMSG (De L a L a s t r a e t a l . , 1 9 7 2 ; M i l l e r and A r m s t r o n g , 1 9 8 1 b ; Y u n e t a l . , 1 9 8 7 ) . D e v e l o p m e n t a l p o t e n t i a l o f s u p e r o v u l a t e d o o c y t e s w i t h v a r i o u s s t a g e s o f n u c l e a r m a t u r a t i o n was n o t a s s e s s e d i n t h e p r e s e n t e x p e r i m e n t . 'However/'"-a portion o f these o o c y t e s i s l i k e l y to be susceptible t o ' h o s t i l e oviidf^al'ejivirpnment p r e s u m a b l y i n f l u e n c e d by altered follicular steroid r e s p o n s e , and may c o n t r i b u t e t o t h e p o o l o f p r o g r e s s i v e i n c r e a s e i n d e g e n e r a t e o o c y t e s o b s e r v e d i n t h e c u r r e n t a n d p r e v i o u s e x p e r i m e n t s ( Y u n e t a l . , 1987) o r t o t h e r e d u c e d f e r t i l i z a t i o n r a t e ( W a l t o n e t a l . , 1 9 8 3 ) . - 102 -In conclusion, the results of present experiment provide direct evidence of atypical ovulations of superovulated oocytes with premature or asynchronous nuclear maturation which may contribute to the pool of abnormal oocytes prior to fertilization in rats after PMSG. Current data also indicate a close association between the incidence of meiotically aberrant oocytes and abnormal follicular steroidogenesis following superovulation with PMSG in rats. Protocol 3. In the present experiment, both a control dose (4 IU) and a superovulatory dose (20 IU) of PMSG elicited two distinct LH peaks in rats: a first slight rise at 0-18 hr and a second sharp rise at 54-60 hr after treatment. This result, in general, agrees with the patterns produced by a different dose (10 IU) of PMSG (Hillensjo et al., 1974). Together, these findings indicate a similar and dose-independent time relationship of PMSG to induce LH surge. However, by analyzing the magnitude of the LH response, a superovulatory dose of PMSG was associated with a significant attenuation of the endogenous LH surge accompanied by prolonged elevations of serum LH, as compared to that corresponding to control regimen.. The present observation of two LH increases coupled with previous findings of two distinct ovulations after superovulatory treatment in rats (De La Lastra et al. , 1972; Yun et al., 1987) suggests that the LH response to PMSG. has two differen t components: a slight and prolonged elevation with the first peak iindi^<g^de?jt^of pituitary secretion and a prec.iptitous second elevation of the pituitary-dependent surge. PMSG is known to possess an exceptionally high sialic acid content and a consequent slow clearance rate as well as a mixture of FSH- and LH-like activity when measured by bioassay - 103 -(Schams et al., 1978). The circulating half l i f e of PMSG (30-36 hr) observed in the present experiment is consistent with the prolonged disappearance rate reported by others (Parlow and Ward, 1961; Sasamoto et al., 1972; Aggarwal and Papkoff, 1981). This estimation is considerably longer than the half l i f e of endogenous gonadotropins which were reported to be 2.5 hr for FSH and 0.5 hr for LH in rats (Bogdanove and Gay, 1969). A study of neutralization with anti-PMSG serum demonstrated the active biological l i f e of PMSG of 54 to 60 hr in mice (Sasamoto et al., 1972) and its circulating inactivation time of 36 hr in rats (Sasamoto and Kennan, 1973). On the basis of these chemical properties of PMSG and a significant cross-reaction between PMSG and rat LH antibody in the current assay system, one can conclude that the elevated serum LH around the time of the first peak is actually an intrinsic component of PMSG. Additionally, timing of the surge between 54 and 60 hr in the present experiment is well in agreement with the critical time of endogenous LH secretion established in numerous time-inhibition studies using various neuropharmacologic central nerve depressants (Sorrentino et al., 1972; Hillensjo et a l . , 1974) and hypophysectomy (Strauss and Meyer, 1962; Zarrow and Quinn, 1963). It has previously been reported that in immature rats a low dose (3 IU) of PMSG initially stimulates a rapid follicular development during the first 36 hr prior to its inactivation, in blood, and then by the next 25 hx endogenous gonadotropin is responsilole for the maintenance of follicles to ensure final ovulation (Sasamoto and Kennan, 1973). In the present experiment, a superovulatory dose of PMSG resulted in the in i t i a l prolonged elevations of serum LH around the first peak and subsequent - 104 -suppression of the second, endogenous, LH surge as compared to control levels of serum LH. Therefore, in superovulated rats , the current observation of increased ovulatory response and ovarian weights as well as the previous findings of precocious ovulation as early as 24 hr (De La Lastra et a l . , 1972; Yun et a l . , 1987) reflect the effectiveness of PMSG-derived in tr ins i c gonadotropin for ovarian hyperstimulation. However, this interpretation does not rule out the involvement of endogenous gonadotropin secretion in the process of f o l l i c u l a r maturation and ovulations even in superovulated rats , since the second elevation of serum LH remains higher than the preceding peak. Several l ines of evidence indicate that superovulatory regimens using exogenous gonadotropins inhib i t the onset of endogenous LH surge and attenuate i t s magnitude in humans (Ferraretti et a l . , 1983; Messinis and Templeton, 1986) and monkeys (Littman and Hodgen, 1984). The results of present experiment are consistent with these findings, and provide further evidence on a nonprimate model. In rats, a superovulatory dose of PMSG signif icantly suppressed the endogenous LH surge without affecting the timing of i t s onset. This suppression has previously been attributed to the action(s) of inhibin- l ike protein and/or nonsteroidal ovarian factor(s) produced from hyperstimulated f o l l i c l e s which mediates d irect ly a negative feedback of LH;secretion (Ferraretti et a l . f 1983) or interferes with estrc»gen-mei6iiat.ed;?positive; feedback (Littman- and Hodgen,,,*19.84). The substance responsible for this effect was further shown to be a factor of ovarian origin with a short c irculat ing h a l f - l i f e , since b i l a t e r a l ovariectomy restored normal p i tu i tary responsiveness within 30 min (Schenken - 105 -and Hodgen , 1 9 8 3 ) . T h e r e f o r e , i t seems l i k e l y t h a t a s i g n i f i c a n t s u p p r e s s i o n o f endogenous L H s u r g e i n s u p e r o v u l a t e d r a t s i s a r e f l e c t i o n o f h y p e r s t i m u l a t i o n o f m u l t i p l e f o l l i c l e s b y a s u p e r o v u l a t o r y d o s e o f PMSG. The f a c t o r ( s ) and i t s r e g u l a t o r y mechan i sm f o r t h e endogenous L H s e c r e t i o n i n s u p e r o v u l a t e d r a t s r e m a i n t o be f u r t h e r c l a r i f i e d . P r o t o c o l 4 . O o c y t e d e g e n e r a t i o n f o l l o w i n g s u p e r o v u l a t o r y t r e a t m e n t s w i t h PMSG i n immature r a t s h a s b e e n a s s o c i a t e d w i t h i n c r e a s e d l e v e l s o f o v a r i a n a n d r o g e n s (Yun e t a l . , 1 9 8 7 ) . I n t h e p r e s e n t e x p e r i m e n t , a d m i n i s t r a t i o n o f an a n d r o g e n a n t a g o n i s t ( f l u t a m i d e ) a f t e r s u p e r o v u l a t o r y d o s e (40 IU) o f PMSG i n immature r a t s s i g n i f i c a n t l y r e d u c e d o o c y t e d e g e n e r a t i o n , w i t h o u t i n f l u e n c i n g t h e o v u l a t o r y r e s p o n s e , and embryo d e g e n e r a t i o n i n p r e i m p l a n t a t i o n s t a g e . A d d i t i o n a l l y , i t i m p r o v e d t h e d e v e l o p m e n t a l p o t e n t i a l o f s u p e r o v u l a t e d o o c y t e s a f t e r f e r t i l i z a t i o n , s i n c e t h e r e was a s i g n i f i c a n t i n c r e a s e i n t h e p r o p o r t i o n o f c l e a v e d e m b r y o s . The a b n o r m a l i t i e s i n e m b r y o n i c m o r p h o l o g y and d e v e l o p m e n t r e s u l t i n g f r o m s u p e r o v u l a t i o n have p r e v i o u s l y b e e n a s c r i b e d t o t h e a b n o r m a l s e c r e t i o n o f o v a r i a n s t e r o i d hormones ( M i l l e r and A r m s t r o n g , 1982) and t h e e l e v a t e d l e v e l s o f c i r c u l a t i n g s t e r o i d s ( B o o t h e t a l . , 1 9 7 5 ) . The e x p o s u r e t o e x c e s s i v e amounts o f e s t r o g e n and p r o g e s t e r o n e i n v i t r o c a u s e d s t r u c t u r a l c h a n g e s i n f e r t i l i z e d embryos t o b l o c k p r o t e i n s y n t h e s i s ( W i l l m e r , 1 9 6 1 ) . I n a d d i t i o n , e x c e s s amounts o f " e s t r o g e n a n d p r o g e s t e r o n e r e s u l t e d in c l e a v a g e a r r e s t in mouse (WMfcten^ : 1957^ K i r k p a t r i c k ? 1971) -andcr^gfert. •}• embryos ( D a n i e l , 1 9 6 4 ) . I n t h e p r e s e n t e x p e r i m e n t , s u p e r o v u l a t i o n s i g n i f i c a n t l y e l e v a t e d t h e c i r c u l a t i n g s t e r o i d hormones d u r i n g t h e p r e i m p l a n t a t i o n p e r i o d : 1 7 f l - e s t r a d i o l o n Day 2 a n d p r o g e s t e r o n e and - 106 -a n d r o g e n s on Days 2 t o 5 o f p r e g n a n c y . F l u t a m i d e t r e a t m e n t i n s u p e r o v u l a t e d r a t s s i g n i f i c a n t l y d e c r e a s e d a n d r o g e n l e v e l , b u t t h i s e f f e c t was l i m i t e d o n Day 2 w i t h o u t a f f e c t i n g t h e l e v e l s o f 1 7 f i - e s t r a d i o l and p r o g e s t e r o n e . A l s o , f l u t a m i d e d i d n o t e f f e c t i v e l y p r e v e n t t h e e a r l y l o s s o f p r e i m p l a n t a t i o n embryos r e s u l t i n g f r o m s u p e r o v u l a t i o n and f a i l e d t o i m p r o v e embryo d e v e l o p m e n t beyond t h e 6 - c e l l s t a g e . F u r t h e r m o r e , i t s e f f e c t s o n p r e i m p l a n t a t i o n embryo d e v e l o p m e n t g r a d u a l l y d e c r e a s e d f r o m Day 3 . The p a r t i a l e f f e c t s o f f l u t a m i d e o n p r e i m p l a n t a t i o n d e v e l o p m e n t may be a c c o u n t e d f o r b y t h e s t e r o i d hormone r e s p o n s e ; i t was i n s u f f i c i e n t t o r e s t o r e t h e p r o l o n g e d e l e v a t i o n o f a n d r o g e n s a n d p r o g e s t e r o n e r e s u l t i n g f r o m s u p e r o v u l a t i o n (40 I U PMSG) t o c o n t r o l l e v e l s (4 I U P M S G ) . T h e r e f o r e , a s i g n i f i c a n t d e c r e a s e i n c e l l u l a r d e g e n e r a t i o n o f p r e i m p l a n t a t i o n embryos and a s i g n i f i c a n t i n c r e a s e i n t h e p r o p o r t i o n o f c l e a v e d embryos a f t e r f l u t a m i d e t r e a t m e n t may be a s c r i b e d t o a s u b s t a n t i a l r e d u c t i o n o f a b n o r m a l o o c y t e s . b e f o r e f e r t i l i z a t i o n . T i m i n g o f o v u l a t i o n a p p e a r s t o be i m p o r t a n t t o a s s e s s t h e o c c u r r e n c e o f o o c y t e d e g e n e r a t i o n e i t h e r w i t h i n t h e f o l l i c l e o r i n t h e o v i d u c t and t h e i m p a c t o f f l u t a m i d e o n t h i s e v e n t . The p r e s e n t d a t a f o r t i m e - d e p e n d e n t o v u l a t o r y r e s p o n s e b y PMSG h a v e shown a c o n s i s t e n t l y s t e a d y s t a t e w i t h an unchanged number o f s u p e r o v u l a t e d o o c y t e s be tween 48 a n d 72 hi'. These r e s u l t s a r e d i f f i c u l t t o r e c o n c i l e w i t h t h e p r e v i o u s ofes&owstiotfs^(Walton e t a l.y 1983; Y u n e t a!*y 1987) i n d i c a t i n g that s u p e r o v u l a t o r y d o s e s o f PMSG i n d u c e m u l t i p l e o v u l a t i o n s w i t h a n i n c r e a s e d number o f o o c y t e s a f t e r 48 h r . I t may be e x p l a i n e d b y b a t c h t o b a t c h v a r i a t i o n i n t h e r e l a t i v e a c t i v i t y o f FSH a n d L H i n c o m m e r c i a l PMSG - 107 -p r e p a r a t i o n s (Murphy e t a l . , 1 9 8 4 ) . H o w e v e r , i n v i e w o f t h e d a t a o f t h i s e x p e r i m e n t , a c t u a l t i m i n g o f o v u l a t i o n i n s u p e r o v u l a t e d r a t s a p p e a r s t o be c o m p l e t e b y 48 h r , whe reby t h e p r e v i o u s l y o v u l a t e d o o c y t e s c o n s t i t u t e a ma jo r p o p u l a t i o n o f d e g e n e r a t e o o c y t e s o b s e r v e d b e t w e e n 48 and 72 h r . A g e d o o c y t e s a f t e r o v u l a t i o n u n d e r g o a p r o g r e s s i v e d e t e r i o r a t i o n w i t h i n t h e o v i d u c t w h i c h r e s u l t s i n a b n o r m a l i t i e s o f f e r t i l i z a t i o n and e a r l y e m b r y o n i c d e v e l o p m e n t ( A u s t i n , 1 9 7 0 ; L o n g o , 1 9 8 0 ) . I t i s , t h e r e f o r e , l i k e l y t h a t f l u t a m i d e i n s u p e r o v u l a t e d r a t s may e n h a n c e t h e v i a b i l i t y and s u r v i v a l o f t h e p r e v i o u s l y o v u l a t e d o o c y t e s w i t h i n t h e o v i d u c t p r e s u m a b l y b y a l t e r i n g s t e r o i d hormone r e s p o n s e w h i c h may i n f l u e n c e t h e o v i d u c t a l e n v i r o n m e n t . However , t h i s i n t e r p r e t a t i o n d o e s n o t d i s c o u n t t h e p o s s i b i l i t y t h a t p r e o v u l a t o r y s t e r o i d l e v e l s c h a n g e d b y f l u t a m i d e may c o n t r o l t h e i n t r a f o l l i c u l a r e n v i r o n m e n t w h i c h i n t u r n i n f l u e n c e s t h e v i a b i l i t y and f e r t i l i z a b i l i t y o f t h e o o c y t e s . I n t h e p r e s e n t e x p e r i m e n t , f l u t a m i d e s u b s t a n t i a l l y r e d u c e d a n d r o g e n s a s w e l l a s 1 7 B - e s t r a d i o l i n b o t h o v a r i a n t i s s u e and s e r u m , and s u b s e q u e n t l y r e s u l t e d i n a s i g n i f i c a n t d e c r e a s e i n t h e p r o p o r t i o n o f d e g e n e r a t e o o c y t e s a n d a m a r k e d d e c r e a s e i n e a r l y embryo d e g e n e r a t i o n . A l s o , i n t h e p r e v i o u s e x p e r i m e n t (Yun e t a l . , 1 9 8 9 ) , a l t e r a t i o n o f f o l l i c u l a r s t e r o i d c o n t e n t s ( p a r t i c u l a r l y a n d r o g e n s ) f o l l o w i n g s u p e r o v u l a t o r y t r e a t m e n t h a s b e e n a s s o c i a t e d w i t h a t y p i c a l o v u l a t i o n s o f m e i o t i c a l l y p r e m a t u r e oi:•••asynchronous o o c y t e s w h i c h n a y c o n t r i b u t e t o t h e p o o l o f a b n o r m a l o t c r j t e s ; p M o r t o f e r t i l i z a t i o n , , M o r i e t a l . (1977) have s u g g e s t e d t h a t a n d r o g e n s a c t i v e l y p a r t i c i p a t e i n t h e o v u l a t o r y p r o c e s s i n r a t s t r e a t e d w i t h a l o w d o s e o f PMSG. By n e u t r a l i z i n g endogenous t e s t o s t e r o n e w i t h i t s a n t i s e r u m o r b y - 1 0 8 -i n h i b i t i n g t h e p r e o v u l a t o r y LH s u r g e w i t h a n o n s t e r o i d a l a n d r o g e n a n t a g o n i s t ( h y d r o x y f l u t a m i d e ) , t h e o v u l a t o r y r e s p o n s e i n d u c e d b y l o w d o s e s o f PMSG was m a r k e d l y r e d u c e d ( M o r i e t a l . , 1 9 7 7 ; O p a v s k y e t a l . , 1 9 8 7 ) . I n c o n t r a s t , i n t h e p r e s e n t e x p e r i m e n t , t h e o v u l a t o r y r e s p o n s e i n d u c e d b y a s u p e r o v u l a t o r y d o s e o f PMSG was n o t a f f e c t e d b y f l u t a m i d e a d m i n i s t r a t i o n . P o s s i b l y , s u f f i c i e n t L H - l i k e a c t i v i t y i n a s u p e r o v u l a t o r y d o s e o f PMSG may o v e r r i d e t h e i n h i b i t o r y e f f e c t o f f l u t a m i d e o n t h e s e c r e t i o n o f p r e o v u l a t o r y L H . I n t e r e s t i n g l y , f l u t a m i d e h a s r e c e n t l y b e e n f o u n d t o e x e r t no e f f e c t o n t h e m o d u l a t i o n o f L H s u r g e i n c y c l i n g a d u l t r a t s ( J o n e s e t a l . , 1 9 8 7 ) . D i f f e r e n c e s i n t h e e f f e c t s o f a n d r o g e n s o n f o l l i c u l a r d e v e l o p m e n t and f u n c t i o n c o u l d m o s t l i k e l y be a s c r i b e d t o t h e d i f f e r e n c e s i n d o s a g e , d u r a t i o n o f a c t i o n , o r t h e p a r t i c u l a r a n d r o g e n i n v o l v e d ( F a r o o k h i , 1 9 8 5 ) . I t h a s b e e n r e p o r t e d t h a t f l u t a m i d e e x e r t s i t s a n t i a n d r o g e n i c e f f e c t s on m a l e s e x o r g a n s b y i n h i b i t i o n o f a n d r o g e n u p t a k e a n d / o r n u c l e a r b i n d i n g o f a n d r o g e n s i n v i v o w i t h o u t o t h e r h o r m o n a l a c t i v i t i e s ( N e r i e t a l . , 1 9 7 2 ; P e e t s e t a l . , 1 9 7 4 ) . Howeve r , i n t h e p r e s e n t e x p e r i m e n t , b o t h o v a r i a n a n d se rum a n d r o g e n l e v e l s i n t h e f l u t a m i d e - t r e a t e d r a t s d e c r e a s e d s i g n i f i c a n t l y a t 60 h r a f t e r PMSG a n d o n Day 2 o f p r e g n a n c y . A d d i t i o n a l l y , a t 60 h r a f t e r PMSG, f l u t a m i d e s i g n i f i c a n t l y d e c r e a s e d 1 7 B - e s t r a d i o l l e v e l . I n a s e p a r a t e e x p e r i m e n t , f l u t a m i d e s i g n i f i c a n t l y d e c r e a s e d a r o m a t i z a b l e a n d r o g e n i c s t e r o i d s , ' a n d r o s t e n e d i o n e - a n d ; t e s t o s t e r o n e b u t no t . a n o n a r o m a t i z a t j l e a n d r o g e n , 5 a - d i h y d r o t e s t o s t e r o n e i n s e r u m s a m p l e s o b t a i n e d f r o m 20 I U P M S G - t r e a t e d r a t s . These r e s u l t s s u g g e s t t h a t t h e d e c r e a s e d 1 7 B - e s t r a d i o l l e v e l b y f l u t a m i d e i s due t o t h e r e d u c t i o n o f a r o m a t i z a b l e a n d r o g e n s . I n c o n t r a s t , e a r l i e r s t u d y r e p o r t e d t h a t f l u t a m i d e f a i l e d t o - 109 -a l t e r t h e o v a r i a n s t e r o i d c o n t e n t i n i m m a t u r e r a t s t r e a t e d w i t h 5 I U PMSG ( P e l u s o e t a l . , 1 9 8 0 ) . The d i f f e r e n c e i n r e s p o n s e t o f l u t a m i d e o n s t e r o i d hormone l e v e l s may be due t o t h e d i f f e r e n t d o s e s o f PMSG e m p l o y e d . S u p e r o v u l a t o r y r e g i m e n may p r o v i d e a d i f f e r e n t r e s p o n s e t o f l u t a m i d e a t t h e o v a r i a n l e v e l b y a u g m e n t i n g a n d r o g e n p r o d u c t i o n . The r e s u l t s o f p r e v i o u s e x p e r i m e n t h a v e shown t h a t s u p e r o v u l a t o r y d o s e s o f PMSG s i g n i f i c a n t l y i n c r e a s e d o v a r i a n a n d r o g e n l e v e l s , w h i l e a c o n t r o l d o s e s i g n i f i c a n t l y d e c r e a s e d t h e l e v e l s a f t e r 36 h r (Yun e t a l . , 1 9 8 7 ) . I t s eems , t h e r e f o r e , t h a t f l u t a m i d e may e x e r t i t s e f f e c t s i n s u p e r o v u l a t e d r a t s b y a n a l t e r a t i o n i n s t e r o i d h o r m o n e s , and c o n c e i v a b l y b y a n i n h i b i t i o n o f a n d r o g e n b i n d i n g i n t h e o v a r y . T h i s c o n c e p t may e x p l a i n t h e d i s c r e p a n c y i n s t e r o i d hormone a l t e r a t i o n s o b s e r v e d o n f l u t a m i d e t r e a t m e n t : no s i g n i f i c a n t d i f f e r e n c e a t 48 h r and s i g n i f i c a n t d i f f e r e n c e a t 60 h r . The p r e c i s e mechan i sm o f f l u t a m i d e a c t i o n a t t h e o v a r i a n l e v e l i n s u p e r o v u l a t e d r a t s r e m a i n s t o be f u r t h e r e l u c i d a t e d . F rom t h e s e o b s e r v a t i o n s , i t c a n b e c o n c l u d e d t h a t 1) f l u t a m i d e g r e a t l y r e d u c e s o o c y t e d e g e n e r a t i o n and c e l l u l a r d e g e n e r a t i o n o f p r e i m p l a n t a t i o n embryos i n s u p e r o v u l a t e d r a t s ; 2) f l u t a m i d e i m p r o v e s t h e d e v e l o p m e n t a l p o t e n t i a l o f s u p e r o v u l a t e d o o c y t e s a s j u d g e d b y a m a r k e d i n c r e a s e i n t h e p r o p o r t i o n o f c l e a v e d e m b y r o s ; 3) f l u t a m i d e a p p e a r s t o a c t b y d e c r e a s i n g f o l l i c u l a r a n d r o g e n p r o d u c t i o n f o l l o w i n g s u p e r o v u l a t i o n , b u t ; 4) t h e e f f e c t s , o f f l u t a m i d e o n embryo d e v e l o p m e n t g r a d u a l l y . d e c r e a s e d i n t h e p r o g r e s s o f e a r l y p r e g n a n c y . The p r e s e n t r e s u l t s s t r o n g l y s u g g e s t t h a t t h e a u g m e n t a t i o n o f o v a r i a n a n d r o g e n s e c r e t i o n p r e c e d i n g f e r t i l i z a t i o n e x e r t s a s i g n i f i c a n t r o l e i n t h e p e r t u r b a t i o n o f o o c y t e q u a l i t y a n d s u b s e q u e n t embryo - 110 -d e v e l o p m e n t a f t e r s u p e r o v u l a t i o n . F u r t h e r r e s e a r c h i s r e q u i r e d t o d e f i n e t h e p a r t i c u l a r mechan i sms i n w h i c h a n d r o g e n s d e l e t e r i o u s l y a f f e c t o o c y t e q u a l i t y and s u b s e q u e n t embryo d e v e l o p m e n t i n s u p e r o v u l a t e d r a t s . P r o t o c o l 5 . S t e r o i d hormones s e c r e t e d f r o m P M S G - s t i m u l a t e d o v a r i a n t i s s u e s i n r a t s a r e t h o u g h t t o c o n t r o l t h e o v u l a t o r y r e s p o n s e and o o c y t e m o r p h o l o g y . E v i d e n c e f o r a r o l e o f a n d r o g e n s i n t h e o v u l a t o r y p r o c e s s h a s b e e n p r o v i d e d b y t h e s t u d i e s i n w h i c h a n a n d r o g e n a n t a g o n i s t ( f l u t a m i d e ) was f o u n d t o i n t e r f e r e w i t h s p o n t a n e o u s L H s u r g e and o v u l a t i o n i n 4 I U P M S G - i n j e c t e d r a t s ( C p a v s k y e t a l . , 1987) and r e d u c e o o c y t e d e g e n e r a t i o n r e s u l t i n g f r o m s u p e r o v u l a t i o n w i t h 40 I U PMSG i n r a t s ( Y u n e t a l . , 1 9 8 8 ) . These s t u d i e s s u g g e s t t h e i m p o r t a n t r o l e s o f a n d r o g e n s i n t h e c o n t r o l o f o v a r i a n f u n c t i o n and o o c y t e q u a l i t y i n P M S G - t r e a t e d r a t s ; h o w e v e r , t h e y do n o t e x c l u d e t h e p o s s i b i l i t y o f i n v o l v e m e n t o f o t h e r s t e r o i d hormones i n t h e p r o c e s s o f P M S G - i n d u c e d o v u l a t i o n a n d m a i n t e n a n c e o f o o c y t e q u a l i t y . I n t h e p r e s e n t e x p e r i m e n t u s i n g t h e r a t s i n j e c t e d w i t h e i t h e r a c o n t r o l d o s e (4 IU) o r a s u p e r o v u l a t o r y d o s e (40 IU) o f PMSG, a d m i n i s t r a t i o n o f a p r o g e s t e r o n e a n t a g o n i s t (RU486) s i g n i f i c a n t l y r e d u c e d o v u l a t o r y r e s p o n s e w i t h o u t a f f e c t i n g o o c y t e m o r p h o l o g y , w h i l e a n e s t r o g e n a n t a g o n i s t ( t a m o x i f e n ) t r e a t m e n t was a s s o c i a t e d w i t h a s i g n i f i c a n t d e c r e a s e i n o v u l a t o r y r e s p o n s e a s w e l l a s a n i n c r e a s e i n t h e p r o p o r t i o n o f d e g e n e r a t e o o c y t e s . The c o m p a r i s o n be tween t h e t r e a t m e n t s o f RU486 and t a m o x i f e n f o r t h e o v u l a t i o n - i n h i b i t i n g e f f e c t s showed t h a t t he . e f f e c t i v e n e s s i n i t s p e r f o r m a n c e i s g r e a t e r f o r t a m o x i f e n i n 4 I U P M S G - i n j e c t e d r a t s a n d f o r RU486 i n 40 I U P M S G - i n j e c t e d r a t s . - I l l -The p r e s e n t o b s e r v a t i o n o f m a r k e d i n h i b i t i o n o f o v u l a t o r y r e s p o n s e a f t e r RU486 and t a m o x i f e n i n P M S G - i n j e c t e d r a t s s u g g e s t s p o t e n t i a l p h y s i o l o g i c a l r o l e s o f p r o g e s t e r o n e and e s t r o g e n i n t h e p r o c e s s o f P M S G - i n d u c e d o v u l a t i o n . E a r l i e r s t u d i e s ( C o s t o f f e t a l . , 1 9 7 4 ; P a r k e r e t a l . , 1976) d e m o n s t r a t e d t h e p a t t e r n s o f p r e o v u l a t o r y i n c r e a s e s i n c i r c u l a t i n g l e v e l s o f t h e s e s t e r o i d hormones a c c o m p a n i e d b y p r e o v u l a t o r y s u r g e s o f FSH and L H i n P M S G - i n j e c t e d r a t s . I t a p p e a r s t h a t t h e r i s i n g l e v e l o f p r e o v u l a t o r y e s t r o g e n s p r o d u c e d b y PMSG e x e r t s i t s s t i m u l a t o r y a c t i o n a t t h e h y p o t h a l a m i c l e v e l p r o m o t i n g t h e r e l e a s e o f LHRH, and a t t h e a n t e r i o r p i t u i t a r y t o i n c r e a s e r e s p o n s i v e n e s s t o LHRH l e a d i n g t o g o n a d o t r o p i n s e c r e t i o n ( L a b r i e e t a l . , 1 9 7 8 ) . A l s o , i n c r e a s e d l e v e l s o f p r e o v u l a t o r y p r o g e s t e r o n e may s y n e r g i z e w i t h e s t r o g e n s t o f a c i l i t a t e g o n a d o t r o p i n s e c r e t i o n b y l o w e r i n g t h e t h r e s h o l d o f t h e h y p o t h a l a m u s t o e s t r o g e n i c s t i m u l u s (Kawakami and S a w y e r , 1959) a n d b y i n c r e a s i n g p i t u i t a r y s e n s i t i v i t y t o LHRH ( M c P h e r s o n and M a h e s h , 1 9 7 9 ) . The p o s i t i v e f e e d b a c k r e l a t i o n s h i p b e t w e e n t h e s t e r o i d hormones a n d g o n a d o t r o p i n s e c r e t i o n c o u l d be f u r t h e r s u p p o r t e d b y t h e s t u d i e s u s i n g a n t i b o d i e s o r a n t a g o n i s t s o f 1 7 f i - e s t r a d i o l and p r o g e s t e r o n e ( F e r i n e t a l . , 1 9 6 9 ; L a b h s e t w a r , 1 9 7 0 ; Rao and Mahesh , 1986) a n d e m p l o y i n g t h e s e e x o g e n o u s s t e r o i d hormones i n o v a r i e c t o m i z e d r a t s ( L e g a n e t a l . , 1 9 7 5 ; M c P h e r s o n e t a l . , 1975). T h e r e f o r e , t h e i n t e g r a l a c t i o n s o f e s t r o g e n s a n d p r o g e s t e r o n e a t hypothsi.lamb^pituitai"y a x i s seem t o be n e c e s s a r y for -'-tbs e x p r e s s i o n o f s p o n t a n e o u s g o n a d o t r o p i n s u r g e e n s u r i n g c o n s e q u e n t o v u l a t i o n . However , i n t h e i n t e r p r e t a t i o n o f t h i s mechan i sm, i t i s i m p o r t a n t t o p o i n t o u t t h e d i f f e r e n c e s i n r e l a t i v e c o n t r i b u t i o n o f e a c h s t e r o i d hormone f o r t h e - 112 -stimulation of ovulatory response between the two groups of rats injected with different doses of PMSG. The results of present experiment in which tamoxifen was administered to 4 IU PMSG-injected rats at equipotent dose and to 40 IU PMSG-injected rats at even 2.5-fold higher dose compared to that of RU486 showed that the effectiveness of tamoxifen to inhibit ovulatory response was greater in 4 IU PMSG-injected rats but less in 40 IU PMSG-injected rats than that of RU486. This finding indicates that the major steroid hormone involved in PMSG-induced ovulation may be different depending upon the dose of this gonadotropin to stimulate ovaries: 17fi-estradiol for a control dose-stimulation and progesterone for a superovulatory dose-stimulation. It is probable that in PMSG-injected rats RU486 exerts its antagonism of the action of progesterone on hypothalamo-pituitary axis rather than on ovary, since i t did not affect a l l other parameters of oocyte quality, tissue weight and steroid hormone secretion at ovarian level consistently in both groups of rats injected with 4 or 40 IU PMSG. This concept concerning the mode of action of RU486 is well in agreement with the study (Rao and Mahesh, 1986) in which RU486 treatment in 8 IU PMSG-injected immature rats and cycling adult rats significantly reduced the preovulatory surge levels of FSH and LH, but i t had no effect on gonadotropin levels in immature rats 7 days after ovariectomy. The mode of^inl^bjUjoj^ £.actipn of tamoxifen on ovulatory response appears to be similar to that of RU486. A number of studies demonstrated that antiestrogenic effect of tamoxifen to inhibit the secretion of preovulatory gonadotropins is mediated principally via the - 113 -h y p o t h a l a m o - p i t u i t a r y a x i s r a t h e r t h a n v i a o v a r y ( W a l p o l e , 1968; L a b h s e t w a r , 1970; B i l l a r d and M c D o n a l d , 1973). H o w e v e r , i n t h e i n t e r p r e t a t i o n f o r t h e w h o l e mechanism o f t a m o x i f e n t o m o d u l a t e o v a r i a n f u n c t i o n , some c a u t i o n i s r e q u i r e d . C u r r e n t o b s e r v a t i o n o f a g r e a t i n c r e a s e i n t h e p r o p o r t i o n o f d e g e n e r a t e o o c y t e s and r e d u c t i o n i n o v a r i a n w e i g h t a s w e l l a s marked c h a n g e s i n se rum s t e r o i d hormones a f t e r t r e a t m e n t o f t a m o x i f e n may i n d i c a t e t h e d i r e c t a c t i o n o f t a m o x i f e n a t o v a r i a n l e v e l . The e f f e c t s o f t a m o x i f e n o n t h e s e p a r a m e t e r s w e r e c o n s i s t e n t i n b o t h g r o u p s o f r a t s i n j e c t e d w i t h 4 o r 40 I U PMSG. I n t h e p r e s e n t e x p e r i m e n t , t a m o x i f e n t r e a t m e n t was a s s o c i a t e d w i t h a s i g n i f i c a n t i n c r e a s e i n s e r u m 1 7 B - e s t r a d i o l a n d / o r s e rum a n d r o g e n s , and a s i g n i f i c a n t d e c r e a s e i n s e r u m p r o g e s t e r o n e i n P M S G - i n j e c t e d r a t s . E v i d e n c e s u g g e s t s t h e d i s p a r a t e a c t i o n o f e s t r o g e n a n t a g o n i s t s i n c l u d i n g t a m o x i f e n , c l o m i p h e n e c i t r a t e and n a f o x i d i n e o n c u l t u r e d r a t g r a n u l o s a c e l l s , whereby t h e s e a n t a g o n i s t s s u b s t a n t i a l l y augmented F S H - s t i m u l a t e d e s t r o g e n p r o d u c t i o n and i n h i b i t e d F S H - s t i m u l a t e d p r o g e s t e r o n e p r o d u c t i o n i n a d o s e - r e l a t e d manner ( W e l s h e t a l . , 1984). T h i s o b s e r v a t i o n h a s b e e n a s c r i b e d t o enhancement o f a r o m a t a s e a c t i v i t y a n d s u p p r e s s i o n o f p r e g n e n o l o n e b i o s y n t h e s i s v i a d i r e c t m e d i a t i o n t h r o u g h g r a n u l o s a c e l l b i n d i n g s i t e s s p e c i f i c f o r e s t r o g e n a n t a g o n i s t s ( S g a r l a t a e t a l . , 1984; W e l s h e t a l . , 1984). E a r l y o r s u s t a i n e d e l e v a t i o n df c i r c u l a t i n g p r e o v u l a t o r y 17.8-estradiol has b e e n f o u n d t o be eatoK^otexic p r e s u m a b l y t h r o u g h a d i r e c t a c t i o n o n t h e p r e o v u l a t o r y r a t o o c y t e s ; t h e e f f e c t c o u l d be p a r t i a l l y r e s t o r e d by a n t i s e r a t o 1 7 B - e s t r a d i o l ( B u t c h e r a n d P o p e , 1979). F u r t h e r m o r e , t h e o v i d u c t s u n d e r t h e i n f l u e n c e o f h i g h l e v e l s o f o v a r i a n a n d - 114 -c i r c u l a t i n g 1 7 f i - e s t r a d i o l h a v e shown t o s e c r e t e a l o w m o l e c u l a r w e i g h t s u b s t a n c e t o i n h i b i t embryo d e v e l o p m e n t i n m i c e ( C l i n e e t a l . , 1977) and r a b b i t ( S t o n e and Hamner, 1977; S t o n e e t a l . , 1977). On t h e o t h e r h a n d , i t h a s been s u g g e s t e d t h a t c l o m i p h e n e c i t r a t e , r e l a t e d t o t a m o x i f e n i n i t s c h e m i c a l s t r u c t u r e , may e x e r t a d i r e c t e s t r o g e n - a n t a g o n i s t i c e f f e c t o n t h e i n t r a f o l l i c u l a r o o c y t e s t o p r o m o t e i t s d e g e n e r a t i o n a n d f u r t h e r t o i n c r e a s e p r e i m p l a n t a t i o n embryo d e g e n e r a t i o n r a t e s a s s o c i a t e d w i t h r e d u c t i o n i n d e v e l o p m e n t a l p o t e n t i a l i n m i c e ( L a u f e r e t a l . , 1983) a n d r a b b i t s ( Y o s h i m u r a e t a l . , 1985). S i m i l a r l y , a n e x p o s u r e o f c l o m i p h e n e c i t r a t e t o c u l t u r e d p r e o v u l a t o r y f o l l i c l e s e x h i b i t e d d o s e - d e p e n d e n t a t r e t i c - l i k e c h a n g e s i n r a t s ( L a u f e r e t a l . , 1982). I t s eems , t h e r e f o r e , l i k e l y t h a t t h e p r o m o t i o n o f o o c y t e d e g e n e r a t i o n b y t a m o x i f e n t r e a t m e n t may b e m e d i a t e d b y a d i r e c t a n t i e s t r o g e n i c a c t i o n o f t a m o x i f e n i n t h e p r e o v u l a t o r y f o l l i c l e s a n d / o r b y i t s e l e v a t i o n o f c i r c u l a t i n g e s t r o g e n s t o p r o d u c e h o s t i l e e n v i r o n m e n t i n t h e o v i d u c t s . Howeve r , i n t h e p r e s e n t e x p e r i m e n t , t h e p r i m a r y f a c t o r ( s ) l e a d i n g t o t a m o x i f e n - a s s o c i a t e d o o c y t e d e g e n e r a t i o n be tween t h e t w o g r o u p s o f r a t s i n j e c t e d w i t h d i f f e r e n t d o s e s o f PMSG c o u l d be d i f f e r e n t d e p e n d i n g upon t i m i n g o f o v u l a t i o n . T i m e - c o u r s e o f o v u l a t o r y r e s p o n s e t o i n c r e a s i n g d o s e s o f PMSG h a s p r e v i o u s l y b e e n w e l l d e s c r i b e d ( W a l t o n e t a l . , 1983: Y u n e t a l . , 1987). T h u s , i n 4 I U P M S G - i n j e c t e d r a t s , t h e p r e s e n t o b s e r v a t i o n o f a n i n c r e a s e d p r o p o r t i o n o f d e g n e r a t e o o c y t e s a f t e r t a m o x i f e n r e f l e c t s p o s t o v u l a t o r y e v e n t a t 72 h r a f t e r PMSG a n d s u g g e s t s t h e i m p a c t o f t a m o x i f e n i n t h e o v i d u c t t h r o u g h i t s e l e v a t i o n o f c i r c u l a t i n g 1 7 B - e s t r a d i o l a s a p r i m a r y f a c t o r . On t h e o t h e r h a n d , i n 40 I U P M S G - i n j e c t e d r a t s , s i n c e o v u l a t i o n s were p e r s i s t e n t b e t w e e n 60 a n d 72 h r a f t e r PMSG, t h e - 115 -t a m o x i f e n - a s s o c i a t e d o o c y t e d e g e n e r a t i o n may be a s c r i b e d t o b o t h f a c t o r s i n c l u d i n g t h e d i r e c t a n t i e s t r o g e n i c a c t i o n o f t a m o x i f e n i n p r e o v u l a t o r y f o l l i c l e s and i t s s e c o n d a r y i m p a c t i n t h e o v i d u c t . A d d i t i o n a l l y , t h e e l e v a t e d l e v e l o f c i r c u l a t i n g a n d r o g e n s b y t a m o x i f e n may be a n o t h e r c o n t r i b u t i n g f a c t o r t o t h e p r o d u c t i o n o f h o s t i l e o v i d u c t a l e n v i r o n m e n t hence p r o m o t i n g o o c y t e d e g e n e r a t i o n . I t h a s p r e v i o u s l y been shown t h a t a g r e a t r e d u c t i o n o f o o c y t e d e g e n e r a t i o n b y a n a n d r o g e n a n t a g o n i s t ( f l u t a m i d e ) i n s u p e r o v u l a t e d r a t s w i t h PMSG h a s b e e n a s s o c i a t e d w i t h s i g n i f i c a n t d e c r e a s e s i n c i r c u l a t i n g a n d r o g e n s a s w e l l a s 1 7 B - e s t r a d i o l (Yun e t a l . , 1 9 8 8 ) . I n summary, b o t h a p r o g e s t e r o n e a n t a g o n i s t (RU486) a n d a n e s t r o g e n a n t a g o n i s t ( t a m o x i f e n ) e x h i b i t e d i n h i b i t o r y a c t i o n o f o v u l a t i o n i n P M S G - i n j e c t e d r a t s i n d i c a t i n g t h e p o t e n t i a l p h y s i o l o g i c a l r o l e s o f p r o g e s t e r o n e and e s t r o g e n f o r a c t i v e p a r t i c i p a t i o n i n P M S G - i n d u c e d o v u l a t o r y p r o c e s s . The r e l a t i v e o v u l a t i o n - i n h i b i t i n g e f f e c t i v e n e s s o f t h e two s t e r o i d a n t a g o n i s t s was d i f f e r e n t b e t w e e n t h e r a t s i n j e c t e d w i t h two d i f f e r e n t d o s e s o f PMSG i n d i c a t i v e o f m a j o r s t e r o i d hormone i n v o l v e d i n P M S G - i n d u c e d o v u l a t i o n : 1 7 B - e s t r a d i o l f o r a c o n t r o l d o s e - s t i m u l a t i o n a n d p r o g e s t e r o n e f o r a s u p e r o v u l a t o r y d o s e - s t i m u l a t i o n . A d d i t i o n a l l y , t h e d e t r i m e n t a l e f f e c t o f t a m o x i f e n o n o o c y t e m o r p h o l o g y h a s b e e n d e m o n s t r a t e d i n P M S G - i n j e c t e d r a t s . I t a p p e a r s t h a t t a m o x i f e n - a s s o c i a t e d o o c y t e d e g e n e r a t i o n may be . m e d i a t e d b y i t s known a n t i e s t r o g e n i c a c t i o n i n p r e o v u l a t o r y f o l l i c l e s , and/or i t s e l e v a t i o n o f c i r c u l a t i n g 1 7 B - - e s t r a d i o ! a n d / o r a n d r o g e n s t o p r o d u c e h o s t i l e o v i d u c t a l e n v i r o n m e n t . - 116 -SUMMARY AND CONCLUSIONS O v u l a t o r y r e s p o n s e , o o c y t e q u a l i t y ( g r o s s m o r p h o l o g y a n d n u c l e a r m a t u r a t i o n ) , and d e v e l o p m e n t a l p o t e n t i a l o f p r e i m p l a n t a t i o n e m b r y o s a f t e r a d m i n i s t r a t i o n o f d i f f e r e n t d o s e s ( 4 , 2 0 , o r 40 IU) o f PMSG t o i m m a t u r e r a t s w e r e s t u d i e d w i t h r e f e r e n c e t o t h e s t e r o i d hormone l e v e l s i n o v a r i a n t i s s u e s , p e r i p h e r a l b l o o d , o r f o l l i c u l a r f l u i d a n d c i r c u l a t i n g L H r e s p o n s e . I n a d d i t i o n , t h e e f f e c t s o f a n a n d r o g e n a n t a g o n i s t ( f l u t a m i d e ) , a p r o g e s t e r o n e a n t a g o n i s t ( R U 4 8 6 ) , a n d a n e s t r o g e n a n t a g o n i s t ( t a m o x i f e n ) o n t h e s e p a r a m e t e r s were a l s o e x a m i n e d . A l o w d o s e (4 IU) o f PMSG i n d u c e d a c o n s i s t e n t o v u l a t i o n o f up t o 15 o o c y t e s p e r r a t w h i c h was s y n c h r o n i z e d a t 6 0 - 7 2 h r . However , s u p e r o v u l a t o r y d o s e s (20 and 40 IU) o f PMSG i n d u c e d p r o l o n g e d m u l t i p l e o v u l a t i o n s w i t h maximum a v e r a g e c o u n t s o f 51 t o 58 o o c y t e s p e r r a t b e t w e e n 60 and 72 h r . S u p e r o v u l a t o r y t r e a t m e n t s r e s u l t e d i n t h e f i r s t o v u l a t i o n a s e a r l y a s 24 h r w i t h two i n c r e a s e s i n t h e number o f o o c y t e s r e c o v e r e d f r o m o v i d u c t s : one b e f o r e 36 h r and t h e o t h e r a f t e r 48 h r . The e a r l y o v u l a t i o n , i n s u p e r o v u l a t e d r a t s c o u l d b e , i n p a r t , a s c r i b e d t o t h e exogenous L H a c t i v i t y o f h i g h d o s e s o f PMSG. The r e s u l t o f s e r u m LH r e s p o n s e t o PMSG showed two d i s t i n c t LH p e a k s i n w h i c h t h e i n i t i a l p r o l o n g e d e l e v a t i o n o f s e r u m LH b e f o r e 54 h r r e s u l t e d f r o m a c t u a l c r o s s - r e a c t i o n o f t h e i n j e c t e d PMSG W i i ; h * L H a n t i b o d y i n t h e a s s a y ar id was d o s e - d e p e n d e n t w h i l e a p r e c i p i t o u s -*>ect)hd;;elevation b e t w e e n .54 a n d 60 h r r e s u l t e d p r i m a r i l y f rorn a p i t u i t a r y - d e p e n d e n t LH s u r g e . A d d i t i o n a l l y , t h e r o l e o f s t e r o i d hormones i n t h e P M S G - i n d u c e d o v u l a t i o n was i m p l i c a t e d i n t h e e x p e r i m e n t s u s i n g t h e s t e r o i d a n t a g o n i s t s . A d m i n i s t r a t i o n o f t h e RU486 o r t a m o x i f e n i n t h e r a t s - 117 -i n j e c t e d w i t h d i f f e r e n t d o s e s (4 o r 40 IU) o f PMSG s i g n i f i c a n t l y i n t e r f e r e d w i t h o v u l a t i o n b e t w e e n 60 a n d 72 h r a f t e r PMSG i n d i c a t i n g a c t i v e p a r t i c i p a t i o n o f b o t h p r o g e s t e r o n e and e s t r o g e n i n t h e g o n a d o t r o p i n - i n d u c e d o v u l a t i o n . The mode o f i n h i b i t o r y a c t i o n s o f t h e s e a n t a g o n i s t s o n o v u l a t o r y r e s p o n s e was d i s c u s s e d . On t h e o t h e r h a n d , t h e t r e a t m e n t w i t h f l u t a m i d e i n s u p e r o v u l a t e d r a t s d i d n o t a f f e c t t h e o v u l a t o r y r e s p o n s e . S u p e r o v u l a t o r y t r e a t m e n t s w i t h PMSG c a u s e d a d o s e - d e p e n d e n t i n c r e a s e i n t h e p r o p o r t i o n o f d e g e n e r a t e o o c y t e s a n d a m a r k e d a l t e r a t i o n o f o v a r i a n a n d s e r u m s t e r o i d l e v e l s f r o m 36 h r o n w a r d s . T h e r e was a s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n be tween t h e p e r c e n t a g e o f d e g e n e r a t e o o c y t e s a n d a n d r o g e n l e v e l s i n b o t h o v a r i a n t i s s u e s a n d s e r u m f r o m 24 t o 60 h r f o l l o w i n g s u p e r o v u l a t i o n . I n t h e e x p e r i m e n t t o e x a m i n e t h e s t e r o i d c o n t e n t s i n f o l l i c u l a r f l u i d and o o c y t e m a t u r a t i o n , t h e r e s u l t s showed a c l o s e a s s o c i a t i o n b e t w e e n t h e i n c i d e n c e o f m e i o t i c a l l y a b e r r e n t o o c y t e s and a b n o r m a l f o l l i c u l a r s t e r o i d o g e n e s i s i n s u p e r o v u l a t e d r a t s . S u p e r o v u l a t e d o o c y t e s d i s p l a y e d s u b s t a n t i a l l y d i f f e r e n t s t a g e s v a r y i n g f r o m p r o p h a s e I t o metaphase I I be tween 24 and 72 h r , w h i l e t h e n u c l e a r m a t u r a t i o n o f a m a j o r i t y o f c o n t r o l o o c y t e s r e c o v e r e d f r o m t h e o v i d u c t s o f 4 I U P M S G - i n j e c t e d r a t s a t 72 h r was s y n c h r o n i z e d a t me taphase I I . The p r o f i l e s o f f o l l i c u l a r s t e r o i d c o n t e n t s i n s u p e r o v u l a t e d r a t s w e r e c h a r a c t e r i z e d b y b o t h a ma rked a l t e r a t i o n of p r o g e s t e r o n e and p a r t i c u l a r l y a n d r o g e n s arid a c o n s i s t e n t d i s r u p t i o n of sequential changes i n o v e r a l l r a t i o s o f A / E , P / E -and P / A , w h i c h r e m a i n e d c o n s t a n t w i t h no s u b s t a n t i a l c h a n g e s a s compared t o c o n t r o l r e g i m e n . The p a t t e r n o f e a c h f o l l i c u l a r s t e r o i d hormone i n b o t h c o n t r o l and s u p e r o v u l a t e d r a t s c l o s e l y r e s e m b l e d t h e c h a n g e i n o v a r i a n - 118 -tissues and sera. It was, therefore, considered that (1) the maturation of follicle and oocyte in superovulated rats might occur independently of the normal physiological events by an altered intrafollicular steroid environment, and (2) a portion of superovulated oocytes with premature or asynchronous nuclear maturation might be susceptible to hostile oviductal environment presumably influenced secondarily by an altered follicular steroid response and contribute to the pool of a progressive increase in degenerate oocytes prior to fertilization. The possibility of atypical ovulations with abnormal oocytes derived from physiologically unfit follicles following superovulation was discussed. To explore further the action of elevated androgens on the perturbation of oocyte quality in superovulated rats, the effects of flutamide in superovulated rats were examined on oocyte gross morphology and embryo development in preimplantation stage. Flutamide treatment after injection of a superovulatory dose of PMSG significantly reduced oocyte degeneration throughout the whole period examined between 48 and 72 hr, and cellular degeneration of preimplantation embryos. Additionally, i t improved the developmental potential of superovulated oocytes after fertilization, since a great increase in the proportion of cleaved embryos was noticed. These effects exerted by flutamide were, in part, attributed to the decreased production of ovarian androgens (particularly aromatizable androgenic steroids, androstenedione and testosterone) which might influence,; the oviductal environment and in turn enhance the viability and survival of the previously ovulated oocytes within the oviduct. Flutamide treatment in superovulated rats significantly decreased ovarian and/or serum levels of - 119 -a n d r o g e n s a t 60 h r and o n Day 2 o f p r e g n a n c y . The i n t e r p r e t a t i o n o f t h e i m p a c t o f f l u t a m i d e o n o o c y t e q u a l i t y i n t h e o v i d u c t was b a s e d o n t h e o b s e r v a t i o n o f a c t u a l t i m i n g o f o v u l a t i o n s h o w i n g a s t e a d y s t a t e w i t h a n unchanged number o f s u p e r o v u l a t e d o o c y t e s be tween 48 a n d 72 h r i n t h i s p a r t i c u l a r e x p e r i m e n t . On t h e o t h e r h a n d , a g r a d u a l d e c r e a s e i n t h e e f f e c t s o f f l u t a m i d e o n embryo deve lopmen t , was o b s e r v e d i n t h e p r o g r e s s o f e a r l y p r e g n a n c y , i . e . f r o m Day 3 o f p r e g n a n c y . A l s o , f l u t a m i d e d i d n o t e f f e c t i v e l y p r e v e n t t h e e a r l y l o s s o f p r e i m p l a n t a t i o n embryos r e s u l t i n g f r o m s u p e r o v u l a t i o n and f a i l e d t o i m p r o v e embryo d e v e l o p m e n t b e y o n d t h e 6 - c e l l s t a g e . The p a r t i a l e f f e c t s o f f l u t a m i d e o n p r e i m p l a n t a t i o n embryo d e v e l o p m e n t c o u l d be a c c o u n t e d f o r b y t h e s t e r o i d r e s p o n s e t o f l u t a m i d e , s i n c e i t was i n s u f f i c i e n t t o r e s t o r e t h e p r o l o n g e d e l e v a t i o n o f c i r c u l a t i n g a n d r o g e n s and p r o g e s t e r o n e r e s u l t i n g f r o m s u p e r o v u l a t i o n t o c o n t r o l l e v e l s . P h a r m a c o l o g i c a l e f f e c t o f f l u t a m i d e t o d e c r e a s e a n d r o g e n l e v e l was l i m i t e d o n Day 2 o f p r e g n a n c y w i t h o u t a f f e c t i n g t h e l e v e l s o f o t h e r s t e r o i d hormones . F i n a l l y , a s i g n i f i c a n t d e c r e a s e i n c e l l u l a r d e g e n e r a t i o n o f p r e i m p l a n t a t i o n embryos a n d a s i g n i f i c a n t i n c r e a s e i n t h e p r o p o r t i o n o f c l e a v e d embryos a f t e r f l u t a m i d e t r e a t m e n t were a s c r i b e d p r i n c i p a l l y t o a s u b s t a n t i a l r e d u c t i o n o f a b n o r m a l o o c y t e s b e f o r e f e r t i l i z a t i o n . The e f f e c t s o f RU486 and t a m o x i f e n o n o o c y t e m o r p h o l o g y w e r e a l s o examined t o u n d e r s t a n d the potential roles o f p r o g e s t e r o n e and e s t r o g e n i n t h e p e r t u r b a t i o n o f superovulated oocyte q u a l i t y . RU486 treatment.after a d m i n i s t r a t i o n o f d i f f e r e n t d o s e s o f PMSG d i d n o t a f f e c t g r o s s m o r p h o l o g y o f o o c y t e s r e c o v e r e d f r o m o v i d u c t s . However , i t was f o u n d t h a t t a m o x i f e n t r e a t m e n t i n b o t h c o n t r o l and s u p e r o v u l a t e d r a t s was a s s o c i a t e d w i t h a - 120 -c o n s i s t e n t i n c r e a s e i n t h e p r o p o r t i o n o f d e g e n e r a t e o o c y t e s . The p r o m o t i o n o f o o c y t e d e g e n e r a t i o n a f t e r t a m o x i f e n t r e a t m e n t was c o n s i d e r e d t o be d u e t o (1) i t s d i r e c t a n t i e s t r o g e n i c a c t i o n i n t h e p r e o v u l a t o r y f o l l i c l e s , s i n c e t h e r e was a s i g n i f i c a n t r e d u c t i o n o f o v a r i a n w e i g h t 12 and 24 h r a f t e r t r e a t m e n t , (2) i t s s e c o n d a r y a c t i o n t o e l e v a t e c i r c u l a t i n g 1 7 6 - e s t r a d i o l a n d / o r a n d r o g e n s w h i c h m i g h t p r o d u c e h o s t i l e o v i d u c t a l e n v i r o n m e n t . T a m o x i f e n t r e a t m e n t s i g n i f i c a n t l y i n c r e a s e d s e r u m l e v e l s o f 1 7 6 - e s t r a d i o l a n d / o r a n d r o g e n s i n b o t h c o n t r o l a n d s u p e r o v u l a t e d r a t s . The r e s u l t s o f t h i s e x p e r i m e n t have i n d i c a t e d , i n p a r t , t h e i n v o l v e m e n t o f e s t r o g e n , b u t n o t p r o g e s t e r o n e , t o i n f l u e n c e t h e o o c y t e q u a l i t y i n s u p e r o v u l a t e d r a t s . C o l l e c t i v e l y , t h e r e s u l t s o f t h i s s t u d y s u g g e s t t h a t t h e i m p a i r e d f e r t i l i t y s e e n a f t e r s u p e r o v u l a t o r y t r e a t m e n t s w i t h PMSG r e s u l t s p r i m a r i l y f r o m a t y p i c a l o v u l a t i o n s o f s u p e r o v u l a t e d o o c y t e s w i t h p r e m a t u r e o r a s y n c h r o n o u s n u c l e a r m a t u r a t i o n a s s o c i a t e d w i t h a b n o r m a l f o l l i c u l a r s t e r o i d o g e n e s i s b y e x c e s s i v e g o n a d o t r o p i c s t i m u l a t i o n . E l e v a t e d l e v e l s o f o v a r i a n and c i r c u l a t i n g and rogens p r i o r t o f e r t i l i z a t i o n were p a r t i c u l a r l y i m p l i c a t e d a s a ma jo r f a c t o r c a u s i n g t h e p e r t u r b a t i o n o f o o c y t e q u a l i t y a n d embryo d e v e l o p m e n t a f t e r s u p e r o v u l a t i o n . F i n a l l y , t h e p r o c e s s o f m u l t i p l e o v u l a t i o n s i n d u c e d b y s u p e r o v u l a t o r y d o s e s o f PMSG a p p e a r e d t o i n v o l v e t h e exogenous L H a c t i v i t y o f PMSG a s w e l l a s a c t i v e p a r t i c i p a t i o n o f p r o g e s t e r o n e and e s t r o g e n . - 121 -BIBLIOGRAPHY Adams, C . E . ( 1 9 6 0 ) . P r e n a t a l m o r t a l i t y i n t h e r a b b i t o r y c t o l a g u s c u n i c u l u s . J . R e p r o d . F e r t . , 1_:36-44. A g g a r w a l , B . B . and P a p k o f f , H . ( 1 9 8 1 ) . S t u d i e s o n t h e d i s a p p e a r a n c e o f e q u i n e c h o r i o n i c g o n a d o t r o p i n f r o m t h e c i r c u l a t i o n i n t h e r a t : t i s s u e u p t a k e and d e g r a d a t i o n . E n d o c r i n o l o g y , 1 0 9 : 1 2 4 2 - 1 2 4 7 . A l l e n , J . and M c L a r e n , A . ( 1 9 7 1 ) . C l e a v a g e r a t e o f mouse e g g s f r o m i n d u c e d and s p o n t a n e o u s o v u l a t i o n . J . R e p r o d . P e r t . , 2 7 : 1 3 7 - 1 4 0 . A r m s t r o n g , D . T . and E v a n s , G . ( 1 9 8 3 ) . F a c t o r s i n f l u e n c i n g s u c c e s s o f embryo t r a n s f e r i n sheep and g o a t s . T h e r i o g e n o l o g y , 1 9 : 3 1 - 4 2 . A u s t i n , C R . ( 1 9 5 0 ) . The f e c u n d i t y o f t h e immature r a t f o l l o w i n g i n d u c e d s u p e r o v u l a t i o n . J . E n d o c r i n o l . , 6^ :293-301 . A u s t i n , C R . ( 1 9 5 6 ) . C o r t i c a l g r a n u l e s i n h a m s t e r e g g s . E x p . C e l l . R e s . , 1 0 : 5 3 3 - 5 4 0 . A u s t i n , C R . ( 1 9 6 1 ) . The mammal ian E g g . O x f o r d : B l a c k w e l l S c i e n t i f i c P u b l i c a t i o n s . A u s t i n , C R . ( 1 9 7 0 ) . A g e i n g a n d r e p r o d u c t i o n : p o s t - o v u l a t o r y d e t e r i o r a t i o n o f t h e e g g . J . R e p r o d . F e r t . ( s u p p l . ) , 1 2 : 3 9 - 5 3 . B a e , I . H . and F o o t e , R . H . ( 1 9 7 5 ) . E f f e c t s o f hormones o n t h e m a t u r a t i o n o f r a b b i t o o c y t e s r e c o v e r e d f r o m f o l l i c l e s o f v a r i o u s s i z e s . J . R e p r o d . F e r t . , 4 2 : 3 5 7 - 3 6 0 . B a k e r , A . A . and J i l l e l l a , D . ( 1 9 7 8 ) . T e c h n i q u e s o f s u r g i c a l and n o n - s u r g i c a l o v a c o l l e c t i o n o f s u p e r o v u l a t e d c o w s . V e t . R e c . , 1 0 3 : 5 5 8 - 5 6 2 . B a r a n c z u k , R . and G r e e n w a l d , G . S . ( 1 9 7 3 ) . P e r i p h e r a l l e v e l s o f e s t r o g e n i n t h e P M S - p r i m e d c y c l i n g h a m s t e r . E n d o c r i n o l o g y , 9 2 : 1 5 4 7 - 1 5 5 0 . B a r r a c l o u g h , C A . , C o l l u , R . , M a s s a , R . a n d M a r t i n i , L . ( 1 9 7 1 ) . T e m p o r a l i n t e r r e l a t i o n s h i p s be tween p l a s m a L H , o v a r i a n s e c r e t i o n r a t e s a n d p e r i p h e r a l p l a s m a p r o g e s t i n c o n c e n t r a t i o n s i n t h e r a t : E f f e c t o f nembutal a n d exogenous g o n a d o t r o p i n s . E n d o c r i n o l o g y , 8 8 : 1 4 3 7 - 1 4 4 7 . B a t t a , S.K., Stark, R . A . a n d B r a c k e t t , B . G . ( 1 9 7 8 ) . O v u l a t i o n i n d u c t i o n b y g o n a d o t r o p i n and p r o s t a g l a n d i n t r e a t m e n t s o f r h e s u s monkeys a n d o b s e r v a t i o n s o f t h e o v a . B i o l . R e p r o d . , 1 8 : 2 6 4 - 2 7 8 . Beaumont , H . M . and S m i t h , A . F . ( 1 9 7 5 ) . E m b r y o n i c m o r t a l i t y d u r i n g t h e p r e -a n d p o s t - i m p l a n t a t i o n p e r i o d s o f p r e g n a n c y i n m a t u r e m i c e a f t e r s u p e r o v u l a t i o n . J . R e p r o d . F e r t . , 4 5 : 4 3 7 - 4 4 8 . - 122 -B e l l o w s , R . A . a n d S h o r t , R . E . (1972). S u p e r o v u l a t i o n and m u l t i p l e b i r t h s i n b e e f c a t t l e . 1 0 t h B i e n n i a l Sympos ium o n A n i m a l R e p r o d u c t i o n . 34  ( s u p p l . 1):67-79. B e n n e t t , J . P . ( 1 9 7 0 ) . The e f f e c t o f d r u g s o n e g g t r a n s p o r t . A d v . B i o s c i . , 4 : 1 6 5 - 1 8 0 . B e t t e r d i g e , K . J . ( 1 9 7 4 ) . U n i l a t e r a l s t i m u l a t i o n o f b o v i n e o v a r i e s b y l o c a l i n j e c t i o n o f p r e g n a n t m a r e ' s s e rum g o n a d o t r o p i n . J . R e p r o d . F e r t . , 3 7 : 1 0 1 - 1 0 4 . B e t t e r i d g e , K . J . ( 1 9 7 7 ) . S u p e r o v u l a t i o n . I n Embryo T r a n s f e r i n Farm A n i m a l s . K . J . B e t t e r i d g e e d . , C a n . D e p t . A g r i c , M o n o g r a p h , 1 6 : 1 - 9 . B e t t e r i d g e , K . J . and M o o r e , N . W . ( 1 9 7 7 ) . T e c h n i q u e s and r e s u l t s i n s h e e p a n d g o a t s . S u p e r o v u l a t i o n . I n Embryo T r a n s f e r i n Farm A n i m a l s . A R e v i e w o f T e c h n i q u e s a n d A p p l i c a t i o n s . K . J . B e t t e r i d g e e d . , C a n . D e p t . A g r i c , Mo n o g ra p h , 1 6 : 3 7 - 3 8 . B i g g e r s , J . D . , W h i t t e n , W . K . and W h i t t i n g h a m , D . G . ( 1 9 7 1 ) . The c u l t u r e o f mouse embryos i n v i t r o . I n M e t h o d s i n Mammal ian E m b r y o l o g y . J . C . D a n i e l , J r . e d . , F reeman , San F r a n c i s c o , C h . 6 , p p . 8 6 - 1 1 6 . B i l l a r d , R . and M c D o n a l d , P . G . ( 1 9 7 3 ) . I n h i b i t i o n o f o v u l a t i o n i n t h e r a t b y i n t r a h y p o t h a l a m i c i m p l a n t s o f a n a n t i e s t r o g e n . J . E n d o c r i n o l . , 5 6 : 5 8 5 - 5 9 0 . B l a n d a u , R . J . ( 1 9 5 2 ) . The f e m a l e f a c t o r i n f e r t i l i t y and i n f e r t i l i t y . I . The e f f e c t s o f d e l a y e d f e r t i l i z a t i o n o n t h e d e v e l o p m e n t o f t h e p r o n u c l e i i n r a t o v a . F e r t i l . S t e r i l . , 2 : 3 4 9 - 3 6 5 . B l a n d a u , R . J . ( 1 9 5 5 ) . O v u l a t i o n i n t h e l i v i n g a l b i n o r a t . F e r t i l . S t e r i l . , 6 : 3 9 1 - 4 0 4 . B l a n d a u , R . J . a n d Y o u n g , W . C . ( 1 9 3 9 ) . The e f f e c t s o f d e l a y e d f e r t i l i z a t i o n o n t h e d e v e l o p m e n t o f t h e g u i n e a p i g ovum. Am. J . A n a t . , 6 4 : 3 0 3 - 3 2 9 . B l a n k s t e i n , J . ( 1 9 8 6 ) . I n v i t r o f e r t i l i z a t i o n and embryo T r a n s f e r . I n O v u l a t i o n I n d u c t i o n and I n V i t r o F e r t i l i z a t i o n . J . B l a n k s t e i n , S . M a s i a c h and B . L u n e n f e l d e d s . , Y e a r Book M e d i c a l P u b l i s h e r s , C h i c a g o , p p . 1 5 5 - 1 9 2 . Bodemer , C . W . , Rumery , R . E . and B l a n d a u , R . J . ( 1 9 5 9 ) . S t u d i e s o n i n d u c e d o v u l t i o n i n t h e i n t a c t immature h a m s t e r . F e r t i l . S t e r i l . , 1 0 : 3 5 0 - 3 6 0 . B o g d a n o v e , E . M . and G a y . V . L . (1969). S t u d i e s o n t h e d i s a p p e a r a n c e o f L H a n d FSH i n t h e r a t : A q u a n t i t a t i v e a p p r o a c h t o a d e n o h y p o p h y s i a l k i n e t i c s . E n d o c r i n o l o g y , 8 4 : 1 1 1 8 - 1 1 3 1 . B o l a n d , M . P . , C r o s b y , T . F . and G o r d o n , I . ( 1 9 7 8 ) . M o r p h o l o g i c a l n o r m a l i t y o f c a t t l e embryos f o l l o w i n g s u p e r o v u l a t i o n u s i n g p r e g n a n t mare se rum g o n a d o t r o p i n . T h e r i o g e n o l o g y , 1 0 : 1 7 5 - 1 8 0 . - 123 -B o l t o n , A . E . ( 1 9 7 7 ) . E x p e r i m e n t a l p r o t o c o l s f o r t h e r a d i o i o d i n a t i o n o f p r o t e i n s and o t h e r compounds . I n R a d i o i o d i n a t i o n T e c h n i q u e s , R e v i e w 1 8 . A . E . B o l t o n e d . , A r l i n g t o n H e i g h t s : A m e r s h a m / S e a r l e C o r p . , p . 4 5 . B o o t h , W . D . , Newcomb, R . , S t r a n g e , H . , R o w s o n , L . E . A . a n d S a c h e r , H . B . ( 1 9 7 5 ) . P l a s m a e s t r o g e n a n d p r o g e s t e r o n e i n r e l a t i o n t o s u p e r o v u l a t i o n a n d egg r e c o v e r y i n t h e c o w . V e t . R e c . , 9 7 : 3 6 6 - 3 6 9 . B o u e , J . G . and B o u e , A . ( 1 9 7 3 ) . I n c r e a s e d f r e q u e n c y o f c h r o m o s o m a l a n o m a l i e s i n a b o r t i o n s a f t e r i n d u c e d o v u l a t i o n . L a n c e t , i _ : 6 7 9 - 6 8 0 . B o u t e r s , R . , M o y a e r t , I . , C o r i j n , M . a n d V a n d e p l a s s c h e , M . ( 1 9 8 3 ) . The u s e o f a PMSG a n t i s e r u m i n s u p e r o v u l a t e d c a t t l e : e n d o c r i n o l o g i c a l c h a n g e s a n d e f f e c t s o n t i m i n g o f o v u l a t i o n . Z u c h t h y g i e n e , 1 8 : 1 7 2 - 1 7 7 . B r a d f o r d , G . E . , T a y l o r , S t . C . S . , Q u i r k e , J . F . and H a r t , R . ( 1 9 7 4 ) . An e g g t r a n s f e r s t u d y o f l i t t e r s i z e , b i r t h w e i g h t and l amb s u r v i v a l . A n i m . P r o d . , 1 8 : 2 4 9 - 2 6 3 . B r a w , R . H . and T s a f r i r i , A . ( 1 9 8 0 ) . E f f e c t o f PMSG o n f o l l i c u l a r a t r e s i a i n t h e immature r a t o v a r y . J . R e p r o d . F e r t . , 5 9 : 2 6 7 - 2 7 2 . B u t c h e r , R . L . , C o l l i n s , W . E . and F u g o , N . W . ( 1 9 7 4 ) . P l a s m a c o n c e n t r a t i o n o f L H , F S H , p r o l a c t i n , p r o g e s t e r o n e a n d e s t r a d i o l - 1 7 B t h r o u g h o u t t h e 4 - d a y e s t r o u s c y c l e o f t h e r a t . E n d o c r i n o l o g y , 9 4 : 1 7 0 4 - 1 7 0 8 . B u t c h e r , R . L . and P a g e , R . D . ( 1 9 8 1 ) . R o l e o f t h e a g i n g o v a r y i n c e s s a t i o n o f r e p r o d u c t i o n . I n D y n a m i c s o f O v a r i a n F u n c t i o n . N . B . S c h w a r t z a n d M . H u n z i c k e r - D u n n e d s . , R a v e n P r e s s , New Y o r k , p p . 2 5 3 - 2 7 1 . B u t c h e r , R . L . and P o p e , R . S . ( 1 9 7 9 ) . R o l e o f e s t r o g e n d u r i n g p r o l o n g e d e s t r o u s c y c l e s o f t h e r a t o n s u b s e q u e n t e m b r y o n i c d e a t h o r d e v e l o p m e n t . B i o l . R e p r o d . , 2 1 : 4 9 1 - 4 9 5 . C a l l e s e n , H . , G r e v e , T . a n d H y t t e l , P . ( 1 9 8 6 ) . P r e o v u l a t o r y e n d o c r i n o l o g y and o o c y t e m a t u r a t i o n i n s u p e r o v u l a t e d c a t t l e . T h e r i o g e n o l o g y , 2 5 ( 1 ) : 7 1 - 8 6 . C a r l s o n , I . H . ( 1 9 6 8 ) . E f f e c t o f r e p r o d u c t i v e s t a t e o n t h e b l o o d and t i s s u e p r o g e s t i n s o f a d u l t and immature f e m a l e r a t s . P h . D . T h e s i s , U n i v e r s i t y o f W i s c o n s i n . C a t c h p o l e , H . R . , C o l e , H . H . and P e a r s o n , P . B . ( 1 9 3 5 ) . S t u d i e s o n t h e r a t e o f d i s a p p e a r a n c e and f a t e o f mare g o n a d o t r o p i c hormone f o l l o w i n g i n t r a v e n o u s i n j e c t i o n . A m . J . P h y s i o l . , 1 1 2 : 2 1 - 2 6 . C h a n g , M . C . ( 1 9 5 2 ) . E f f e c t s o f d e l a y e d f e r t i l i z a t i o n o n s e g m e n t i n g o v a , b l a s t o c y s t s and f e t u s e s i n r a b b i t . F e d . P r o c . F e d . Am. S o c . E x p . 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The i n f l u e n c e o f hormone i n d u c t i o n o f o v u l a t i o n on t h e f e t u s a n d n e w b o r n . I n t h e I n f l u e n c e o f M a t e r n a l Hormones o n t h e F e t u s and Newborn . M . N i t z a n e d . , P e d i a t r . A d o l e s c . E n d o c r i n o l . , V o l . 5 , p p . 1 9 1 - 2 1 2 . H a f e z , E . S . E . and R a j a k o w s k i , E . ( 1 9 6 4 ) . P l a c e n t a l a n d f e t a l d e v e l o p m e n t d u r i n g m u l t i p l e b o v i n e p r e g n a n c y . A n a t o m i c a l and p h y s i o l o g i c a l s t u d i e s . A n a t . R e c , 1 5 0 : 3 0 3 - 3 1 6 . H a g i n d , N . and G o l d z i e h e r , J . W . ( 1 9 7 0 ) . E f f e c t o f t i m i n g a n d q u a n t i t y o f e s t r o g e n o n g o n a d o t r o p i n - i n d u c e d o v u l a t i o n i n immatu re r a t s . E n d o c r i n o l o g y , 8 6 : 2 9 - 3 3 . H a l l f o r d , D . M . , Turman, E . J . , We t t eman , R . P . , P o p e , C E . a n d M e y e r h o e f f e r , D . C ( 1 9 7 5 ) . R e p r o d u c t i v e r e s p o n s e o f t h e b o v i n e t o PMSG. J . A n i m . S c i . , 4 0 : 1 8 7 , A b s t r . H a r m s , P . G . and O j e d a , S . R . ( 1 9 7 4 ) . A r a p i d and s i m p l e p r o c e d u r e f o r c h r o n i c c a n n u l a t i o n o f t h e r a t j u g u l a r v e i n . J . A p p l . P h y s i o l . , 3 6 : 3 9 1 - 3 9 2 . Heap , R . B . , F l i n t , A . P . F . , G a d s b y , J . E . a n d R i c e , C . ( 1 9 7 9 ) . Hormones , t h e e a r l y embryo and t h e u t e r i n e e n v i r o n m e n t . J . R e p r o d . F e r t . , 5 5 ( 1 ) : 2 6 7 - 2 7 5 . H e n r i c k s , D . M . , H i l l , J . R . , J r . , D i c k e y , J . F . a n d Lamond, D . R . ( 1 9 7 3 ) . P l a s m a hormone l e v e l s i n b e e f cows w i t h i n d u c e d m u l t i p l e o v u l a t i o n s . J . R e p r o d . F e r t . , 3 5 : 2 2 5 - 2 3 3 . H i l l e n s j o , T . , B a r n e a , A . , N i l l s o n , L . , H e r l i t z , H . a n d A h r e n , K . ( 1 9 7 4 ) . T e m p o r a l r e l a t i o n s h i p b e t w e e n s e r u m L H l e v e l s a n d o o c y t e m a t u r a t i o n i n p r e p u b e r t a l r a t s i n j e c t e d w i t h p r e g n a n t m a r e ' s s e r u m g o n a d o t r o p i n . E n d o c r i n o l o g y , 9 5 : 1 7 6 2 - 1 7 6 6 . H i l l e n s j o , T . , B a u m i n g e r , S . a n d A h r e n , K . ( 1 9 7 6 ) . E f f e c t o f l u t e i n i z i n g hormone on t h e p a t t e r n o f s t e r o i d p r o d u c t i o n b y p r e o v u l a t o r y f o l l i c l e s o f p r e g n a n t m a r e ' s se rum g o n a d o t r o p i n - i n j e c t e d immature r a t s . E n d o c r i n o l o g y , 9 9 : 9 9 6 - 1 0 0 2 . H i s a w , F . L . ( 1 9 4 7 ) . D e v e l o p m e n t o f t h e g r a a f i a n f o l l i c l e a n d o v u l a t i o n . P h y s i o l . R e v . , 2 7 : 9 5 - 1 1 9 . 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The e f f e c t o f PMSG d o s e o n t h e i n c i d e n c e o f ch romosomal a n o m a l i e s i n mouse embryos f e r t i l i z e d i n v i t r o . J . R e p r o d . F e r t . , 5 0 : 2 7 5 - 2 8 0 . M a u r e r , R . E . , H u n t , W . L . , V a n V l e c k , L . D . a n d F o o t e , R . H . ( 1 9 6 8 ) . D e v e l o p m e n t a l p o t e n t i a l o f s u p e r o v u l a t e d r a b b i t o v a . J . R e p r o d . F e r t . , 1 5 : 1 7 1 - 1 7 5 . McCormack , C . E . and M e y e r , R . K . ( 1 9 6 3 ) . O v u l a t i o n i n d u c e d b y p r o g e s t e r o n e i n immature r a t s p r e t r e a t e d w i t h p r e g n a n t mare se rum g o n a d o t r o p h i n . G e n . Comp. E n d o c r i n o l . , 3 ^ 3 0 0 - 3 0 7 . M c C r a c k e n , J . A . , B a i r d , D . T . and G o d i n g , J . R . ( 1 9 7 1 ) . F a c t o r s a f f e c t i n g t h e s e c r e t i o n o f s t e r o i d s f r o m t r a n s p l a n t e d o v a r y i n s h e e p . R e c e n t P r o g . Horm. 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( 1 9 7 9 ) . Dose r e l a t e d e f f e c t o f a s i n g l e i n j e c t i o n o f p r o g e s t e r o n e o n g o n a d o t r o p i n s e c r e t i o n and p i t u i t a r y s e n s i t i v i t y t o LHRH i n e s t r o g e n - p r i m e d c a s t r a t e f e m a l e r a t s . B i o l . R e p r o d . , 2 0 : 7 6 3 - 7 7 2 . - 132 -M e s s i n i s , I . E . a n d T e m p l e t o n , A . ( 1 9 8 6 ) . The e f f e c t o f p u l s a t i l e f o l l i c l e s t i m u l a t i n g hormone o n t h e endogenous l u t e i n i z i n g hormone s u r g e i n women. C l i n . E n d o c r i n o l . , 2 5 : 6 3 3 - 6 4 0 . M e y e r , R . K . , K a r a v o l a s , H . J . , K l a u s i n g , M . a n d N o r g a r d , D . W . ( 1 9 7 1 ) . B l o o d p r o g e s t e r o n e and p r e g n e n o l o n e l e v e l s d u r i n g p h e n o b a r b i t a l (PB) b l o c k o f P M S - i n d u c e d o v u l a t i o n i n immatu re r a t s . E d n o c r i n o l o g y , 8 8 : 9 8 3 - 9 9 0 . M i l l e r , B . G . and A r m s t r o n g , D . T . ( 1 9 8 1 a ) . S u p e r o v u l a t o r y d o s e s o f p r e g n a n t mare se rum g o n a d o t r o p i n c a u s e d e l a y e d i m p l a n t a t i o n and i n f e r t i l i t y i n immature r a t s . B i o l . R e p r o d . , 2 5 : 2 5 3 - 2 6 0 . M i l l e r , B . G . a n d A r m s t r o n g , D . T . ( 1 9 8 1 b ) . E f f e c t s o f a s u p e r o v u l a t o r y d o s e o f p r e g n a n t mare se rum g o n a d o t r o p i n o n o v a r i a n f u n c t i o n , s e r u m e s t r a d i o l , and p r o g e s t e r o n e l e v e l s a n d e a r l y embryo d e v e l o p m e n t i n immatu re r a t s . B i o l . R e p r o d . , 2 5 : 2 6 1 - 2 7 1 . M i l l e r , B . G . and A r m s t r o n g , D . T . ( 1 9 8 2 ) . I n f e r t i l i t y i n s u p e r o v u l a t e d immatu re r a t s : r o l e o f o v a r i a n s t e r o i d h y p e r s e c r e t i o n . B i o l . R e p r o d . , 2 6 : 8 6 1 - 8 6 8 . Moon , Y . S . , Ho Y u e n , B . , P r i d e , S . M . , Rowe, T . C . , P o l a n d , B . J . , McComb, P . F . and G o m e l , V . ( 1 9 8 5 ) . A p r e l i m i n a r y r e p o r t i n an i n v i t r o f e r t i l i z a t i o n ( I V F ) programme a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a ( U B C ) . Gamete R e s . , 1 1 : 2 8 9 - 2 9 6 . M o o r , R . M . ( 1 9 7 4 ) . The o v a r i a n f o l l i c l e o f t h e s h e e p : I n h i b i t i o n o f e s t r o g e n s e c r e t i o n b y l u t e i n i z i n g hormone . J . E n d o c r i n o l . , 6 1 : 4 5 5 - 4 6 3 . M o o r , R . M . , C a h i l l , L . P . and S t e w a r t , F . ( 1 9 8 0 a ) . O v a r i a n s t i m u l a t i o n o r egg p r o d u c t i o n a s a l i m i t i n g f a c t o r o f egg t r a n s f e r . I X t h I n t . C o n g r . A n i m . R e p r o d . and A . I . , M a d r i d , p p . 4 3 - 5 4 . M o o r , R . M . , Hay , M . F . , M c i n t o s h , J . E . A . a n d C a l d w e l l , B . V . ( 1 9 7 3 ) . E f f e c t o f g o n a d o t r o p i n s on t h e p r o d u c t i o n o f s t e r o i d s b y s h e e p o v a r i a n f o l l i c l e s c u l t u r e d i n v i t r o . J . E n d o c r i n o l . , 5 8 : 5 9 9 - 6 1 1 . M o o r , R . M . , K r u i p , T h . A . M . and G r e e n , D . ( 1 9 8 4 ) . I n t r a o v a r i a n c o n t r o l o f f o l l i c u l o g e n e s i s : l i m i t s t o s u p e r o v u l a t i o n ? T h e r i o g e n o l o g y , 2 1 : 1 0 3 - 1 1 6 . M o o r , R.,M., O s b o r n , J . C . and C r o s b y * . I ' l M . ( 1 9 8 5 ) . G o n a d o t r o p h i n - i n d u c e t f : a b n o r m a l i t i e s i n s h e e p o o c y t e s a f t e r s u p e r o v u l a t i o n . J . R e p r o d . F e r t . , 7 4 : 1 6 7 - 1 7 2 . M o o r , R . M . , P o l g e , C . a n d W i l l a d s e n , S . M . ( 1 9 8 0 b ) . E f f e c t o f f o l l i c u l a r s t e r o i d s o n t h e m a t u r a t i o n and f e r t i l i z a t i o n o f mammal ian o o c y t e s . J . 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E v i d e n c e f o r a n d r o g e n p a r t i c i p a t i o n i n i n d u c e d o v u l a t i o n i n immatu re r a t s . E n d o c r i n o l o g y , 1 0 1 : 6 2 3 - 6 2 6 . M u r p h y , L . L . a n d M a h e s h , V . B . ( 1 9 8 4 ) . S e l e c t i v e r e l e a s e o f l u t e i n i z i n g hormone b y 3 a - h y d r o x y - 5 a - p r e g n a n - 2 0 - o n e i n immatu re o v a r i e c t o m i z e d e s t r o g e n - p r i m e d r a t s . B i o l . R e p r o d . , 3 0 : 7 9 5 - 8 0 3 . M u r p h y , B . D . , M a p l e t o f t , R . J . , Manns , J . and Humphrey, W . D . ( 1 9 8 4 ) . V a r i a b i l i t y i n g o n a d o t r o p h i n p r e p a r a t i o n s a s a f a c t o r i n t h e s u p e r o v u l a t o r y r e s p o n s e . T h e r i o g e n o l o g y , 2 1 ( 1 ) : 1 1 7 : 1 2 5 . N e k o l a , M . V . and S m i t h , D . M . ( 1 9 7 4 ) . S t e r o i d i n h i b i t i o n o f o o c y t e m a t u r a t i o n i n v i t r o . 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I n t e r f e r e n c e w i t h p r e o v u l a t o r y l u t e i n i z i n g hormone s u r g e and b l o c k a d e o f o v u l a t i o n i n immature p r e g n a n t m a r e ' s s e r u m g o n a d o t r o p i n - p r i m e d r a t s w i t h t h e a n t i - a n d r o g e n i c d r u g , h y d r o x y f l u t a m i d e . B i o l . R e p r o d . , 3 6 : 6 3 6 - 6 4 2 . P a p k o f f , H . ( 1 9 7 4 ) . C h e m i c a l a n d b i o l o g i c a l p r o p e r t i e s o f t h e s u b - u n i t s o f p r e g n a n t mare se rum g o n a d o t r o p h i n . B i o c h e m . B i o p h y s . R e s . Comm., 5 8 : 3 9 7 - 4 0 4 . - 134 -P a p k o f f , H . ( 1 9 7 8 ) . I n t r o d u c t i o n o f r e l a t i o n s h i p o f PMSG t o t h e p i t u i t a r y g o n a d o t r o p h i n s . I n C o n t r o l o f R e p r o d u c t i o n i n t h e Cow. J . M . S r e e n a n e d . , The Hague , M a r t i n u s N i j h o f f , p p . 7 4 - 8 6 . P a p k o f f , H . ( 1 9 8 1 ) . V a r i a t i o n i n t h e p r o p e r t i e s o f e q u i n e g o n a d o t r o p h i n . T h e r i o g e n o l o g y , 1 5 : 1 - 1 6 . P a p k o f f , H . , B e w l e y , T . A . a n d Ramachandran , J . ( 1 9 7 8 ) . P h y s i c o c h e m i c a l a n d b i o l o g i c a l c h a r a c t e r i z a t i o n o f p r e g n a n t mare s e r u m g o n a d o t r o p h i n and i t s s u b u n i t s . B i o c h e m . B i o p h y s . A c t a . , 5 3 2 ( 1 ) : 3 7 3 - 3 7 7 . P a r k e r , C . R . , J r . , C o s t o f f , A . , M u l d o o n , T . G . and M a h e s h , V . B . ( 1 9 7 6 ) . A c t i o n s o f p r e g n a n t mare s e r u m g o n a d o t r o p i n i n t h e immature f e m a l e r a t : C o r r e l a t i v e c h a n g e s i n b l o o d s t e r o i d s , g o n a d o t r o p i n s , and c y t o p l a s m i c e s t r a d i o l r e c e p t o r s o f t h e a n t e r i o r p i t u i t a r y a n d h y p o t h a l a m u s . E n d o c r i n o l o g y , 9 8 : 1 2 9 - 1 3 8 . P a r l o w , A . F . and W a r d , D . N . ( 1 9 6 1 ) . R a t e o f d i s a p p e a r a n c e o f L H , PMS and. HCG f r o m p l a s m a . I n Human P i t u i t a r y G o n a d o t r o p h i n s . A . A l b e r t a n d C h a r l e s C . Thomas e d s . , S p r i n g f i e l d , I l l i n o i s , p p . 2 0 4 - 2 0 9 . P a y n e , R . W . and R u n s e r , R . H . ( 1 9 5 8 ) . The i n f l u e n c e o f e s t r o g e n and a n d r o g e n o n t h e o v a r i a n r e s p o n s e o f h y p o p h y s e c t o m i z e d immatu re r a t s t o g o n a d o t r o p i n s . E n d o c r i n o l o g y , 6 2 : 3 1 3 - 3 2 1 . P e e t s , E . A . , H e n s o n , M . F . a n d N e r i , R . O . ( 1 9 7 4 ) . On t h e mechan i sm o f t h e a n t i a n d r o g e n i c a c t i o n o f f l u t a m i d e ( a - a - a - t r i f l u o r o - 2 - m e t h y l - 4 ' - n i t r o - m -p r o p i o n o t o l u i d i d e ) i n t h e r a t . E n d o c r i n o l o g y , 9 4 : 5 3 2 - 5 4 0 . P e l u s o , J . 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P r e g n a n t mare se rum g o n a d o t r o p i n : r a t i o o f f o l l i c l e - s t i m u l a t i n g hormone and l u t e i n i z i n g hormone a c t i v i t i e s measured b y r a d i o r e c e p t o r a s s a y . J . E n d o c r i n o l . , 7 1 ( 3 ) : 4 7 1 - 4 8 2 . S t o n e , S . L . and Hamner, C E . ( 1 9 7 7 ) . Hormona l and r e g i o n a l i n f l u e n c e s o f t h e o v i d u c t on t h e d e v e l o p m e n t o f r a b b i t e m b r y o s . B i o l . R e p r o d . , 1 6 : 6 3 6 - 6 4 6 . S t o n e , S . L . , R i c h a r d s o n , L . L . , Hamner , C E . and O l i p h a n t , G . ( 1 9 7 7 ) . P a r t i a l c h a r a c t e r i z a t i o n o f h o r m o n e - m e d i a t e d i n h i b i t i o n o f embryo d e v e l o p m e n t i n r a b b i t o v i d u c t a l f l u i d . B i o l . R e p r o d . , 1 6 : 6 4 7 - 6 5 3 . S t r a u s s , W . F . and M e y e r , R . K . ( 1 9 6 2 ) . 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Effects of superovulatory doses of pregnant mare serum gonadotropin on oocyte quality and ovulatory and steroid hormone responses in rats. Gamete Res., 16:109-120. Yun, Y.W., Ho Yuen, B. and Moon, Y.S. (1988). Effects of an antiandrogen, flutamide, on oocyte quality and embryo development in rats superovulated with pregnant mare's serum gonadotropin. B i o l . Reprod., 39:279-286. Yun, Y.W. and Kwun, C.K. (1984). Studies on the possible cause of implantation failure in immature rats given high doses of pregnant mare serum gonadotropin. Seoul Univ. J. Vet. Sci., 9(2):139-155. Yun, Y.W., Yu, F.H., Ho Yuen, B. and Moon, Y.S. (1989). Effects of superovulatory dose of pregnant mare serum gonadotropin on f o l l i c u l a r steroid contents and oocyte maturation in rats. Gamete Res., 23:289-298. Zarrow, M.X., Caldwell, A . L . , Hafez, E.S.E. and Pincus, G. (1958). Superovulation in the immature rat as a possible assay for LH and HCG. Endocrinology, 63:748-758. Zarrow, M.X. and.Gallo, R.V. (1969). Action of progesterone on PMS-induced ovulation in the immature rat. Endocrinology, 84:1274-1276. Zarrow, M.X. and Hurlburt, E.C. (1967). Inhibition and f a c i l i t a t i o n of PMS-induced ovulation in the immature rat following treatment with progesterone. Endocrinology, 80(4):735-740. Zarrow, M.X. and Quinn, D.L. (1963). Superovulation in the immature rat following treatment with PMS alone and inhibition of PMS-induced ovuation. J, • Endocrinol., 2j6:181-188. Zeilmaker, G.H. (1966). The biphasic effect of progesterone on ovulation in the -'rat.''" •^ta^Endocrinoi." (Kbh.), 51(3): 461-468. PUBLICATIONS 1. Lee, J.N., Han, S.N., Lee, C.U., Kwun, C.K. and Yun, Y.W. (1983): Effects of histamine, 5-HT and bradykinin on implantation and parturition in the rat. Seoul Univ. J, Vet. Sci. 8(8):21-28. 2. Yun, Y.W., and Kwun, C.K. (1984): Studies on the possible cause of implantation failure in immature rats given high doses of PMSG. Seoul Univ. J. Vet. Sci. 9(2):139-155. 3. Yun, Y.W. and Moon, Y.S. (1985): Effectof high doses of PMSG on embryo development and implantation in rats. Soc. Exp. Biol. Med., October 9, Univ. British Columbia, Vancouver, B.C. (abstract). 4. Yun, Y.W., Ho Yuen, B. and Moon, Y.S. (1986): The effects of superovulatory doses of PMSG on oocyte quality, ovulatory and steroid hormone responses in rats. Am. Fertil. Soc/Can Fertil. and Androl. Soc., September 27 to October 2, Toronto, Ontario (abstract). 5. Yun, Y.W., Ho Yuen, B. and Moon, Y.S. (1987): Effects of superovulatory doses of pregnant mare serum gonadotropin on oocyte quality, ovulatory and steroid hormone responses in rats. Gamete Res. 16:109-120. 6. Yun, Y.W., Ho Yuen, B. and Moon, Y.S. (1987): Effects of an antiandrogen, flutamide on oocyte quality and embryonic development after superovulation with PMSG in rats. 20th Annual Meeting of the Society for the Study of Reproduction, July 20-23, Univ. Illinois, Urbana, IL (abstract). 7. Yun, Y.W., Ho Yuen, B. and Moon, Y.S. (1988): Effects of an antiandrogen, flutamide on oocyte quality and embryo development in rats superovulated with pregnant mare serum gonadotropin. Biol. Reprod. 39:279-286. 8. Yun, Y.W., Ho Yuen, B. and Moon, Y.S. (1988): Changes in follicular steroid contents and asynchronous nuclear maturation of oocytes in rats superovulated with PMSG. 21st Annual Meeting of the Society for the Study of Reproduction, August 1-4, Univ. Washington, Seattle, WA (abstract). 9. Moon, Y.S., Duleba, A.J., Mari, N., Yun, Y.W. and Ho Yuen, B. (1988): Effectof clomiphene citrate on PMSG and hCG induced ovulation in hypophysectomized rats. 8th International Congress of Endocrinology, July 17-23, Kyoto, Japan (abstract). 10. Yun, Y.W., Yu, F.H., Ho Yuen, B. and Moon Y.S. (1989): Effects of superovulatory dose of pregnant mare serum gonadotropin of follicular steroid contents and oocyte maturation in rats. Gamete Res. 23:289-298. 11. Moon, Y.S., Duleba, A.J., Yun, Y.W. and Ho Yuen, B. (1989): Effects of clomiphene citrate on ovarian function in hypophysectomized rats. J. Reprod. Fert. 86:753-757. 12. Yun, Y.W., Weick, R.F., Yu, F.H., Ho Yuen, B. and Moon, Y.S. (1989): Serum luteinizing hormone response in pregnant mare serum gonadotropin-treated rats. 45th Annual Meeting of the American Fertility Society, November 11-16, San Fransisco, CA (abstract). 13. Feng, T.B., Yun, Y.W., Ho Yuen, B. and Moon, Y.S. (1989): Inhibitory effects of clomiphene citrate on ovulation induced by pregnant mare serum gonadotropin in immature rats. The Meeting of Canadian Fertility and Andrology Society, November 9-11, Vancouver, B.C. (abstract). 

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