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A morphological investigation of the effects of pregnant mare serum gonadotrophin on oocyte maturation,… Britton, Ann Patricia 1991

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A MORPHOLOGICAL INVESTIGATION OF THE EFFECTS OF PREGNANT MARE SERUM GONADOTROPHIN ON OOCYTE MATURATION, FERTILIZATION AND EMBRYONIC DEVELOPMENT IN RATS By ANN PATRICIA BRITTON D.V.M., University of Guelph, 1981 M.Sc, University of Guelph, 1986 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Human Reproductive Biology Program) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA January 28, 1991 (c) Ann Patricia Britton, 1991 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 fltis4cif\{/) -f UipraJJ^bzc, The University of British Columbia Vancouver, Canada Date Feb. Af \ c \ ° ) \ DE-6 (2/88) i i ABSTRACT A delicate balance of steroid and gonadotrophic hormones is essential for intrafollicular oocyte maturation and successful f e r t i l i z a t i o n and embryonic development. Previous studies have demonstrated that a superovulatory dose of pregnant mare serum gonadotrophin (PMSG) has excessive gonadotrophic activity and alters intrafol1icular steroid hormone levels. In a series of four experiments, the morphology of oocytes and embryos retrieved from immature rats, treated with either a low or high dose of PMSG, and mature, cycling rats was compared to determine whether a superovu-latory dose of PMSG has an adverse effect on oocyte maturation and subsequent f e r t i l i z a t i o n and embryonic development in immature rats. Morphological criteria for the assessment of intraoviductal oocyte aging were established in the f i r s t experiment. During intraoviductal aging, progressive morphological changes directed by the intrinsic developmental program of the oocyte were observed. Further alterations in morphology were attributed to abnormalities of cytoskeletal function. In the second experiment, no difference in morphology was observed between oocytes retrieved from immature rats treated with either 4 or 40 IU PMSG. When compared with mature rats, changes attributable to cytoskeletal instability were observed in aged oocytes from immature rats treated with both doses of PMSG. This was concluded to be a manifestation of altered intrafollicular oocyte maturation as a result of the administration of exogenous gonadotrophin. In the third and fourth experiments, delayed f e r t i l i z a t i o n and i i i a significant reduction in fe r t i l i z a t i o n rate were observed in superovulated, immature rats. The major cause of fe r t i l i z a t i o n failure was determined to be intraoviductal oocyte aging. A significant increase in abnormal embryos was observed as a result of parthenogenetic activation of the aged oocytes. Abnormal, fert i l i z e d embryos retrieved from the superovulated group were concluded to be the manifestation of delayed f e r t i l i z a t i o n . In conclusion, the major effect of a superovulatory dose of PMSG on oocyte f e r t i l i z a b i l i t y and embryonic development was intraoviductal oocyte aging and delayed f e r t i l i z a t i o n . Changes attributed to altered intrafol1icular maturation were manifested during oocyte aging in immature rats treated with either the low or high dose of PMSG. iv TABLE OF CONTENTS Page Abstract . i i Table of Contents... iv List of Tables.... ix List of Figures x Glossary •• x i i i Acknowledgements . xv INTRODUCTION A. THE MORPHOLOGY OF OOCYTES AND EARLY CLEAVAGE STAGE EMBRYOS 1 1. Intraf ol licular Growth and Maturation 1 2. Fertilization 4 3. Early Cleavage Stage Embryos 6 B. THE ACTION OF PREGNANT MARE SERUM GONADOTROPHIN IN RATS 7 1. Background 7 2. Source and Biological Activity of PMSG....... 8 3. Reproductive Patterns A. Mature Rat 12 B. Immature Rat 15 4. Effects of a Low Dose of PMSG in Immature Rats A. Endocrine Response. 17 B. Ovulatory Response..... 18 C. Failure of PMSG to Initiate Puberty 19 5. Effects of a Superovulatory Dose of PMSG in Immature Rats A. Endocrine Response.... 20 V B. Ovulatory Response 24 C. Mechanism of F o l l i c l e Selection by PMSG... 26 6. Oocyte Maturation in Immature Rats A. Meiotic Competence 29 B. Intrafollicular Oocyte Maturation......... 30 C. Chromosomal Abnormalities 33 7. Effects of a Superovulatory Dose of PMSG on Fecundity A. General 34 B. Fertilization 35 C. Embryonic Development 39 D. Implantation 42 8. Summary 44 C. RATIONALE AND OBJECTIVES 46 MATERIALS AND METHODS 1. Animals 48 2. Experimental Design A. Part 1 - Morphological Investigation of Intraoviductal Aging in the Mature Rat... 49 B. Part 2 - Morphological Investigation of Post-Ovulatory Oocyte Quality in Immature, PMSG-Treated Rats and Mature Rats 49 C. Parts 3 and 4 - Morphological Investigation of the Causes of Fertilization Failure and Abnormal Embryo Development in Immature Rats Superovulated with PMSG 49 3. Oocyte and Embryo Retrieval and Gross Assessment A. Parts 1 and 2 - Morphological Investigation of Intraoviductal Aging in Mature Rats and Post-Ovulatory Oocyte Quality in Immature, PMSG-Treated Rats and Mature Rats 50 vi B. Parts 3 and 4 - Morphological Investigation of the Causes of Fertilization Failure and Abnormal Embryo Development in Immature Rats Superovulated with PMSG 51 4. Light and Electron Microscopy A. Parts 1, 3 and 4 - Morphological Investigation of Intraoviductal Aging in the Mature Rat and the Causes of Fertilization Failure and Abnormal Embryo Development in Immature Rats Superovulated with PMSG. , . 52 B. Part 2 - Morphological Investigation of Post-Ovulatory Quality in Immature, PMSG-Treated Rats and Mature Rats 54 5. Statistical Analysis 55 RESULTS AND DISCUSSION PART 1: A Morphological Investigation of Intraoviductal Aging in the Mature Rat A. RESULTS 5 6 1. Ovulation and Gross Morphology... 56 2. Light Microscopy A. Intact Oocytes 56 B. Fragmented Oocytes 62 3. Electron Microscopy A. Intact Oocytes. . 63 B. Fragmented Oocytes 70 B. DISCUSSION. 73 PART 2: A Morphological Investigation of Tubal Oocytes Retrieved from Immature Rats Treated with Either 4 or 40 IU PMSG and Mature, Spontaneously Ovulating Rats A. RESULTS 86 1. Oocyte Retrieval and Gross Assessment 86 v i i 2. Light Microscopy 91 3. Electron Microscopy . 97 B. DISCUSSION 103 1. Ovulation and Gross Observations 103 2. Light and Electron Microscopy.. 110 PART 3: A Morphological Investigation of the Causes of Fertilization Failure in Superovulated Immature Rats A. RESULTS 12 2 1. Gross Embryo Retrieval and Assessment.... 122 2. Light and Electron Microscopy 124 B. DISCUSSION 128 PART 4: A Morphological Investigation of Fertilization and Embryonic Development in Immature Rats Treated with 4 or 40 IU PMSG and Spontaneously Ovulating Rats A. RESULTS. •••• 137 1. Breeding Rates 137 2. Embryo Retrieval and Gross Assessment . 140 3. Light Microscopic Assessment 147 4. Electron Microscopy 158 B. DISCUSSION 171 1. Breeding and Pregnancy Rates 171 2. Gross Embryo Retrieval 173 3. Parthenogenesis 176 4. Multinucleated Embryos 181 5. Effects of Delayed Fertilization... 183 6. Abnormal Embryonic Development 185 7. Nucleolar Activation and Nucleolar-Like Bodies. 189 v i i i 8. Role of the Cytoskeleton in Embryonic Development 191 9. Summary 192 SUMMARY .' 194 BIBLIOGRAPHY 197 ix LIST OF TABLES Table Page 1. Oocyte retrieval from immature rats treated with either 4 or 40 IU PMSG and mature, spontaneously cycling rats on day one of pregnancy 123 2. Gross assessment and f e r t i l i z a t i o n rate of oocytes retrieved on day one of pregnancy from immature and mature rats 123 3. Light microscopic assessment of unfertilized oocytes retrieved from mature and immature rats on day one of pregnancy 125 4. Gross assessment of oocytes retrieved from immature PMSG treated rats which failed to mate and exhibited a metestrus smear 138 5. Range of embryos retrieved from each group of rats on each day of pregnancy 141 6. Occurrence of sperm in the cytoplasm of normal and abnormal embryos 154 7. Nucleolar activation in normal and abnormal embryos.. 154 X LIST OF FIGURES Fi g u r e Page 1. Hormonal patt e r n s of mature, c y c l i n g r a t s and immature, PMSG-treated r a t s 22 2. O v u l a t i o n r a t e s d u r i n g i n t r a o v i d u c t a l aging i n mature r a t s 57 3. Gross observations d u r i n g i n t r a o v i d u c t a l oocyte aging 57 4. M i c r o s c o p i c changes observed during i n t r a o v i d u c t a l oocyte aging 58 5. P r o g r e s s i o n of changes observed by l i g h t microscopy during i n t r a o v i d u c t a l oocyte aging 60 6. E l e c t r o n micrographs of oocytes r e t r i e v e d through es t r u s to metestrus .. 65 7. E l e c t r o n micrographs of oocytes r e t r i e v e d at metestrus and d i e s t r u s . . . . 67 8. E l e c t r o n micrographs of oocytes r e t r i e v e d at d i e s t r u s and p r o e s t r u s 69 9. E l e c t r o n micrographs of oocytes r e t r i e v e d at p r o e s t r u s 72 10. O v u l a t i o n r a t e s of immature r a t s 87 11. Average number of oocytes r e t r i e v e d . . . 87 12. Percentage of o v u l a t o r y r a t s with ampullary d i s t e n s i o n 88 13. Gross assessment of cumulus from immature r a t s 88 14. Gross assessment of oocytes from immature r a t s 90 15. M i c r o s c o p i c assessment of oocytes r e t r i e v e d from immature r a t s (4 IU) 93 16. M i c r o s c o p i c assessment of oocytes r e t r i e v e d from immature r a t s (40 IU) 93 17. Li g h t microscopy of cumulus-enclosed oocytes 94 18. L i g h t microscopy of cumulus-free oocytes 96 19. E l e c t r o n micrographs of oocytes r e t r i e v e d at various times from immature r a t s 98 x i 20. Electron micrographs of aged oocytes retrieved from superovulated immature rats 100 21. Electron micrographs of fragmented oocytes retrieved from immature rats 102 22. Electron micrographs of unfertilized oocytes retrieved from superovulated immature rats 127 23. Percentage of bred rats from which embryos were retrieved. 139 24. Average number of embryos retrieved 139 25. Gross retrieval of degenerated oocytes 142 26. Pattern of retrieval of degenerated oocytes - mature 143 27. Pattern of retrieval of degenerated oocytes - 4 IU.. 143 28. Pattern of retrieval of degenerated oocytes - 40 IU 143 29. Gross embryo development - mature 145 30. Gross embryo development - 4 IU. 145 31. Gross embryo development - 40 IU 145 32. Gross embryo quality on day 2 of pregnancy 146 33. Gross embryo quality on day 3 of pregnancy 146 34. Microscopic assessment of embryos with expected development 148 35. Light micrographs of embryos retrieved from mature rats on days 1 to 3 of pregnancy 150 36. Light micrographs of embryos retrieved from immature rats 152 37. Light micrographs of embryos and fragmented oocytes retrieved from superovulated rats 156 38. Microscopic assessment of developmental1y advanced embryos 157 39. Microscopic assessment of fragmented oocytes 157 40. Electron micrographs of embryos retrieved from mature rats on days 1 and 2 of pregnancy 159 x i i 41. Electron micrographs of embryos retrieved from mature rats on days 2 and 3 of pregnancy 161 42. Electron micrographs of embryos retrieved from immature rats on days 1 and 2 of pregnancy 164 43. Electron micrographs of embryos retrieved from immature rats on day 3 of pregnancy 165 44. Electron micrographs of abnormal embryos on day one of pregnancy 167 45. Electron micrographs of abnormal embryos on day 2 of pregnancy 169 x i i i GLOSSARY BSA bovine serum albumin BSAD Dulbecco's phosphate buffered saline with 10% BSA added C centigrade CG cortical granules CEO cumulus-enclosed oocyte CFO cumulus-free oocyte DNA deoxyribonucleic acid DPBS Dulbecco's phosphate buffered saline FSH f o l l i c l e stimulating hormone FA f i b r i l l a r array GA 2.5% glutaraldehyde in 0.1 molar Sorenson's phosphate buffer GVBD germinal vesicle breakdown hr hours HCG human chorionic gonadotrophic hormone IU International Units LH luteinizing hormone ml mi 11i1itre mm millimetre MC mitochondria MN multinucleated MVB multivesicular body MVF microvillus free nm nanometre NLB nucleolar-like body NOR nucleolus organizer region OP organelle polarization X I V PB 0.1 molar Sorenson's phosphate b u f f e r PH - l o g H* c o n c e n t r a t i o n i n a s o l u t i o n PMSG pregnant mare serum gonadotrophin rDNA ribosomal d e o x y r i b o n u c l e i c a c i d RNP r i b o n u c l e o p r o t e i n SER smooth endoplasmic r e t i c u l u m urn micrometre VA v e s i c u l a r aggregate XV ACKNOWLEDGEMENTS I would f i r s t like to thank the Medical Research Council of Canada who awarded me a five year fellowship and made this work possible. The Medical Research Council of Canada and the British Columbia Health Care Research Foundation supported the research. I would especially like to thank Dr. Peter Hewitt, Dr. Ron Lewis and Dr. John Robinson of the Animal Health Centre, British Columbia Ministry of Agriculture and Fisheries at Abbotsford, B.C. for kindly providing the electron microscopy suite used for the project. My supervisory committee, Dr. Y.S. Moon (supervisor), Dr. D. Rurak (chairman), Dr. B. Ho Yuen, Dr. R. Rajamahendran and Dr. D. Kalousek, provided valuable advice and guidance. Dr Y.S. Moon deserves special mention for his support throughout the project. I would like to thank Frank Yu for his indispensable assistance with the rats. Natalie Duleba, Dr. Pathma Rajamahendran, Susanne Leung and Dr. Y.W. Yun also provided valuable technical assistance. The staff of the Research Centre animal holding f a c i l i t y and in particular, Carol Ford, were extremely helpful in solving husbandry problems. Dr. Sally Lester kindly provided her photomicroscope for the light micrographs. Most of a l l I would like to acknowledge my family, who tolerated my absence without complaint, took on more than their fair share of the chores and continually provided moral support. This work is dedicated to you, Brad, Eddie, Irma and Marge, for you are the truly important part of my l i f e . 1 INTRODUCTION A. THE MORPHOLOGY OF OOCYTES AND EARLY CLEAVAGE STAGE EMBRYOS 1. Intrafol1icular Growth and Maturation Oocytes in primordial, or non-growing f o l l i c l e s , are spherical and surrounded by a single layer of granulosa cells with which they share multiple foci of c e l l contact (Odor, 1960; Zamboni, 1974). The oocytes are arrested at the dictyate stage of meiosis and contain a nucleus (germinal vesicle) with cytoplasmic organelles aggregated on i t s periphery (Odor, 1960; T s a f r i r i and Kraicer, 1972; Zamboni, 1974). At this stage, large, lamellar Golgi, small round, electron dense mitochondria, smooth endoplasmic reticulum and a few multives-icular bodies, clusters of variably sized vesicles with vesicular or dense homogeneous content, are observed (Odor, 1960; Zamboni, 1970). In addition, a small number of small, round, membrane bound structures with highly electron dense content (cortical granules) are scattered in the cytoplasm (Szollosi, 1967 ). As f o l l i c l e s enter the growing pool, the granulosa cells multiply and a thick glycoprotein capsule (zona pellucida) is la i d down between the surface of the oocyte (oolemma) and the surrounding granulosa c e l l s (Odor, 1960; Zamboni, 1970; Zamboni, 1974). Although the zona pellucida separates the 2 oocyte from the granulosa c e l l s , contact i s maintained via cytoplasmic processes of the granulosa cells which traverse the zona pellucida (Odor, 1960; Zamboni, 1970; Zamboni, 1974; McGaughey, 1984). M i c r o v i l l i appear on the oolemma and extend a short distance into the zona pellucida and organelles move away from the germinal vesicle in scattered aggregates through-out the cytoplasm (Odor, 1960; Zamboni, 1970). As oocytes grow, large lamellar Golgi decrease in size and transform into an increasing number of complex multivesicular bodies scattered on the periphery of the oocyte (Odor, 1960; Zamboni, 1970; Peluso and Butcher, 1974). The multivesicular bodies synthesize cortical granules (Szollosi, 1967; Peluso and Butcher, 1974), which increasingly appear just beneath the oolemma (Sz o l l o s i , 1967). An increased number of stacks of parallel f i b r i l s ( f i b r i l l a r arrays) (Burkholder et al. , 1971) and clusters of short lengths of smooth endoplasmic reticulum (vesicular aggregates) (Vazquez-Nin and Sotelo, 1967; Peluso and Butcher, 1974) appear in the cytoplasm (Sotelo and Porter, 1959; Odor, 1960; Szollosi, 1967; Zamboni, 1970). At the time of antrum formation, the large reticulated, fibril1ogranular nucleolus transforms to a dense, f i b r i l l a r , compact, spherical mass and ribosome synthesis ceases (Antoine et al . , 1987, 1989). With completion of oocyte growth, an even layer of microfilaments is present just beneath the oolemma (Amsterdam et a l . , 1977 ) . 3 The gonadotrophin surge has a profound effect on oocyte morphology prior to ovulation. Granulosa c e l l processes retract from the oolemma, resulting i n loss of c e l l communication and nutritional support for the oocyte (cumulus-oocyte c e l l coup-ling) (Sotelo and Porter, 1959; Odor, 1960; Zamboni, 1974; Crosby and Moor, 1984; McGaughey, 1984). A space between the oolemma and the inner surface of the zona pellucida (perivit-elline space) appears as a result of both the loss of cumulus-oocyte c e l l coupling and retraction of m i c r o v i l l i from the inner layer of the zona pellucida (Odor, 1960). Meiosis resumes and i s associated with a sequence of programmed changes in morphology (Albertini, 1987). Organelles, i n particular the multivesicular bodies (MVB), aggregate around the germinal vesicle (Ezzell and Szego, 1979). The chromosomes condense, the nucleolus disappears and the nuclear membrane breaks down (germinal vesicle breakdown (GVBD)) (Zamboni, 1970; McGaughey, 1984). Spindle microtubules are synthesized, the organelles disperse again in the cytoplasm, the meiotic apparatus is formed and the f i r s t polar body is extruded ( A l b e r t i n i , 1987; McGaughey, 1984). In the rat, the f i r s t polar body is highly unstable and degenerates quickly ( T s a f r i r i and Kraicer, 1972). Following extrusion of the f i r s t polar body, the second meiotic spindle assembles below the oolemma and the oocyte arrests at metaphase of the second meiotic d i v i s i o n (Zamboni, 1970; McGaughey, 1984; A l b e r t i n i , 1987). The presence of the 4 chromosomes induces formation of a protrusion of the overlying c e l l surface with a thick suboolemmal microfilament web and loss of m i c r o v i l l i and c o r t i c a l granules (Chen and Longo, 1984; Van Blerkom and Bell, 1986; Maro et a l . , 1986). Thus, subse-quent to.ovulation, tubal oocytes are separated from the zona pellucida by a perivitel1ine space, have a specialized c e l l surface overlying chromosomes which are arrested at metaphase two, have a layer of cortical granules underlying the remainder of the plasma membrane, and have organelles scattered i n the cytoplasm (Sotelo and Porter, 1959; Odor, 1960; Zamboni, 1970; Peluso and Butcher, 1974). 2. F e r t i l i z a t i o n The zona pellucida is a f i b r i l l a r structure which affords physical protection and containment of. the developing embryo prior to implantation (Dvorak et a l . , 1984). In order to f e r t i l i z e an oocyte, the sperm binds to the outer surface of the zona pellucida and then penetrates to the p e r i v i t e l 1ine space (Dvorak et al . , 1984). The sperm and oocyte plasma membranes fuse at the s i t e of contact between gametes (Szollosi and Ris , 1961; Dvorak et a l . , 1984) and the sperm is incorpor-ated into the oocyte. Gamete fusion triggers massive exocytosis of the suboolemmal c o r t i c a l granules which alters the character of both the oolemma and the zona pellucida, preventing poly-spermy (Dvorak et a l . , 1977 ). Simultaneously, the oocyte 5 resumes meiosis, the spindle rotates and the second polar body is extruded (Maro et al ., 1984; Dvorak et a l . , 1984 ). All of these processes triggered by gamete fusion are mediated by the suboolemmal microfilament web (Maro et a l . , 1984, 1986; Cran, 1987). Following sperm incorporation and extrusion of the second polar body, the chromosomes decondense (Szollosi and Ris, 1961; Dvorak et al . , 1984), dense, f i b r i l l a r nucleoli reappear (Sotelo and Porter, 1959; Schuchner, 1970; S z o l l o s i , 1965; Dvorak et a l . , 1984) and the spindle breaks down (Zamboni and Mastroianni, 1966). Discontinuous segments of smooth endoplasmic reticulum surround individual chromosomes and fuse to form the pronuclear membrane (Zamboni and Mastroianni, 1966; Dvorak et a l . , 1984). The newly reconstituted pronuclei then migrate centrally (Maro et a l . , 1984). Following pronuclear reconstitution, small dense, f i b r i l l a r bodies (Sotelo and Porter, 1959; Schuchner, 1970), lamellar Golgi and linear lengths of smooth endoplasmic reticulum appear in the cytoplasm (Sotelo and Porter, 1959; Schlafke and Enders, 1967). Prior to the f i r s t cleavage division, organelles cluster centrally around the pronuclei (Sotelo and Porter, 1959; Zamboni and Mastroianni, 1966; Dvorak et al . , 1984). The pronuclear membranes become increasingly undulent and break down, the mitotic spindle appears and the condensed chromosomes 6 from both pronuclei aggregate on the spindle equator and divide (Zamboni et a l . , 1972). 3. Early Cleavage Stage Embryos From the 1 to the 4 c e l l stage of early development, organelles are scattered i n clusters throughout the cytoplasm and increased numbers of secondary lysosomes and residual bodies, appear (Dvorak et al . , 1977). The MVB and vesicular aggregates progressively decrease i n both size and number (Schlafke and Enders, 1967; Dvorak et a l . , 1977). At the late 2 c e l l to 4 c e l l stage, f i b r i l l a r arrays commence to decline in the cytoplasm and a reticulated, granulofibri11 ar network appears on the surface of the dense nucleoli, signalling i n i t i a t i o n of transcriptional a c t i v i t y of the embryonic genome (Izquierdo and V i a l , 1962; Schuchner, 1970). In summary, oocytes undergo significant changes i n morphol-ogy during i n t r a f o l l i c u l a r growth and preovulatory maturation. This i s followed by further morphological alterations in association with f e r t i l i z a t i o n and early embryonic development. All of these architectural alterations are accompanied by metabolic changes ( A l b e r t i n i , 1987) and, thus, functional changes are correlated with structural changes during early development (Cech and Sedlackova, 1983). 7 B. THE ACTION OF PREGNANT MARE SERUM GONADOTROPHIN IN RATS 1. Background The r o l e of the p i t u i t a r y and g o n a d o t r o p h i c hormones i n mammalian o v u l a t i o n has been r e c o g n i z e d s i n c e the 1920's when Smith and Engle (1927) r e p o r t e d t h a t d a i l y i m p l a n t s of the a n t e r i o r p i t u i t a r y c o u l d induce o v u l a t i o n i n r a t s . F u r t h e r s t u d i e s l e d to the d i s c o v e r y t h a t a s i n g l e i n j e c t i o n of un-f r a c t i o n a t e d pregnant mare serum indu c e d o v u l a t i o n i n p r e p u b e r t a l r a t s . The number of oo c y t e s shed was d i r e c t l y r e l a t e d to the dose of pregnant mare serum a d m i n i s t e r e d w i t h i n a c r i t i c a l range, above which d e c r e a s i n g o v u l a t o r y e f f i c i e n c y was observed ( C o l e , 1936). A l t h o u g h o v u l a t i o n , m a t i n g and i m p l a n t a t i o n c o u l d be i n d u c e d i n r a t s as young as 2 2 days of age, maximum p r o l i f i c a c y was o b s e r v e d i n r a t s j u s t b e f o r e the normal time of m a t u r i t y ( C o l e , 1940) . When a superovu1 a t o r y dose was a d m i n i s t e r e d to these r a t s , a maximum of 54 o o c y t e s were r e t r i e v e d , up t o 33 i m p l a n t a t i o n s i t e s were counted and the l a r g e s t l i t t e r born was 23 ( C o l e , 1937; Co l e , 1940). As a r e s u l t of the l o s s of o v u l a t o r y o o c y t e s and r e s o r p t i o n of i m p l a n t a t i o n s i t e s , the number o f v i a b l e young born was o n l y i n c r e a s e d i n a small number of r a t s ( C o l e , 1940). The f e c u n d i t y of superovu1 a t e d immature r a t s was sub s e q u e n t l y s t u d i e d i n d e t a i l by A u s t i n (1950). With 20 I n t e r n a t i o n a l U n i t s (IU) p r e g n a n t mare serum g o n a d o t r o p h i n 8 (PMSG) f o l l o w e d 56 hours l a t e r by 20 IU human c h o r i o n i c g o n adotrophin (HCG), an average o f 37 o o c y t e s were o v u l a t e d ( A u s t i n , 1950). However, numerous ad v e r s e e f f e c t s were i d e n t -i f i e d i n c l u d i n g low mating r a t e s , low f e r t i l i z a t i o n r a t e s and low i m p l a n t a t i o n r a t e s ( A u s t i n , 1950). Summation of these e f f e c t s on f e c u n d i t y r e v e a l e d t h a t o n l y 9% of the. r a t s mated, f e r t i l i z e d and underwent embryonic i m p l a n t a t i o n ( A u s t i n , 1950). D e s p i t e the poor r e p r o d u c t i v e performance of immature r a t s g i v e n a s u p e r o v u l a t o r y dose of PMSG, r e p r o d u c t i v e b i o l o g i s t s were drawn to the model due to the l a b o u r and expense i n v o l v e d when us i n g mature r a t s f o r r e p r o d u c t i v e s t u d i e s ( N u t i et a l . , 1975). In 1975, N u t i et a l demonstrated t h a t a s i n g l e dose of 8 IU PMSG g i v e n to 30 day o l d r a t s r e s u l t e d i n a r e p r o d u c t i v e p r o f i l e analogous t o t h a t of s p o n t a n e o u s l y o v u l a t i n g mature r a t s . I t was thus con c l u d e d t h a t t h e immature PMSG t r e a t e d rat c o n s t i t u t e d a s u i t a b l e model of the mature r a t f o r r e p r o d u c t i v e s t u d i e s ( N u t i e t a l . , 1975). 2. Source and B i o l o g i c a l A c t i v i t y of PMSG Pregnant mare serum g o n a d o t r o p h i n was one of the f i r s t com-m e r c i a l l y a v a i l a b l e g o n adotrophins and has been w i d e l y used f o r the i n d u c t i o n of s u p e r o v u l a t i o n i n d o m e s t i c a n i m a l s and f o r r e p r o d u c t i v e s t u d i e s ( A l l e n and S t e w a r t , 1978; P a p k o f f , 1981; Reeves, 1987). Pregnant mare serum g o n a d o t r o p h i n i s a g l y c o p r o t e i n which i s s y n t h e s i z e d and s e c r e t e d by t r o p h o b l a s t c e l l s of the equine e n d o m e t r i a l cups (McDonald, 1977 ; P a p k o f f , 1981). Serum l e v e l s are d e t e c t a b l e i n pregnant mares on day 40, peak from day 60 t o 80 and then d e c l i n e to day 140 of g e s t a t i o n (McDonald, 1977). Pregnant mare serum g o n a d o t r o p h i n c o n s i s t s of a l p h a and beta s u b u n i t s s i m i l a r to the p i t u i t a r y g o n a d o t r o p h i n s , f o l l i c l e s t i m u l a t i n g hormone (FSH) and l u t e i n i z i n g hormone (LH) (Pap-k o f f , 1978; Pap k o f f , 1981). The b e t a s u b u n i t c o n t a i n s the hormone s p e c i f i c a c t i v i t y and the c o m b i n a t i o n of al p h a and be t a s u b u n i t s c o n f e r s r e c e p t o r r e c o g n i t i o n (Reeves, 1987).. The al p h a s u b u n i t has a s i m i l a r c a r b o h y d r a t e c o n t e n t to t h a t of FSH and LH, w h i l e the c a r b o h y d r a t e c o n t e n t of the b e t a s u b u n i t i s 2 to 3 times g r e a t e r ( P a p k o f f , 1978; Aggarwal and P a p k o f f , 1981). The a l p h a s u b u n i t of PMSG i s s i m i l a r to FSH, w h i l e the beta s u b u n i t i s almost i d e n t i c a l to LH i n amino a c i d sequence (Pap-k o f f , 1978). T h i s i s c o n s i s t e n t w i t h s t u d i e s i n the mare, where PMSG was shown to have a p r e d o m i n a n t l y l u t e o t r o p h i c e f f e c t ( C o l e et a l . , 1931; S q u i r e s and G i n t h e r , 1975; A l l e n and Stewart, 1978). However, i n the immature r a t , PMSG has both FSH and LH a c t i v i t y ( C o l e and H a r t , 1930; P a p k o f f , 1974; Pap k o f f , 1978). The low l e v e l of FSH a c t i v i t y i n t h e mare appears to be a f a i l u r e o f . equine FSH r e c e p t o r s to r e c o g n i z e PMSG, w h i l e a f f i n i t y of PMSG f o r both FSH and LH r e c e p t o r s was demonstrated i n t he r a t (Moore and Ward, 1980). PMSG t h e r e f o r e has dual , 10 s p e c i e s s p e c i f i c a c t i v i t y ( C o l e et a l . , 1940; P a p k o f f , 1978; Moore & Ward, 1980 ) . Pregnant mare serum g o n a d o t r o p h i n i s produced by the endom e t r i a l cups as a s i n g l e m o l e c u l e w i t h p r e d o m i n a n t l y l u t e o t r o p h i c a c t i v i t y w h i c h then undergoes m o d i f i c a t i o n of the beta l o o p , endowing i t w i t h FSH a c t i v i t y (Moore and Ward, 1980) . When r e t r i e v e d d i r e c t l y from e n d o m e t r i a l cups, PMSG c o n s i s t e n t l y has l e s s FSH a c t i v i t y than t h a t r e t r i e v e d from serum (Papkoff et a l ., 1978; Papkoff, 1981). T h i s v a r i a b i l i t y was a t t r i b u t e d to the p r e s e n c e of m o l e c u l e s i n d i f f e r e n t stages of b i o s y n t h e s i s ( P a p k o f f , 1978; Papkoff e t a l . , 1 978 ; P a p k o f f , 1981) . Post s e c r e t o r y m o d i f i c a t i o n of a s i n g l e amino a c i d i n the beta loop can a l t e r t h e net charge and c o n f e r the molecule w i t h FSH a c t i v i t y (Ward and Moore, 1978 ). S i m i l a r l y , a 90% r e d u c t i o n i n b i o l o g i c a l a c t i v i t y was a s s o c i a t e d w i t h an a l t e r e d amino a c i d sequence i n the be t a loop o f PMSG r e c o v e r e d from mares wi t h d e c l i n i n g t i t r e s ( P a p k o f f , 1981 ) . There i s marked v a r i a t i o n i n o v u l a t o r y response to exogenous PMSG (Stewart et a l . , 197 6; A l l e n and Stewa r t , 1978; Schams et al . , 1978). T h i s i s p a r t l y a t t r i b u t a b l e to dosage and d i f f e r e n c e s i n response between i n d i v i d u a l animals (Schams et al . , 1978; Newcomb et a l . , 1979). D i f f e r i n g F SH/LH r a t i o s were a l s o found i n d i f f e r e n t commercial batches of PMSG, depending on the time d u r i n g g e s t a t i o n they were i s o l a t e d , and t h i s was c o r r e l a t e d w i t h d i f f e r i n g o v u l a t o r y response i n immature r a t s (Murphy e t a l . , 1984). The number of o v u l a t i o n s was d i r e c t l y r e l a t e d t o t h e l e v e l of FSH a c t i v i t y (Murphy et a l . , 1984). The b i o l o g i c a l a c t i v i t y o f PMSG r e t r i e v e d from equine t r o p h o b l a s t c u l t u r e s c o r r e l a t e d c l o s e l y w i t h the v a r i a b l e c a r b o h y d r a t e c o n t e n t ( P a p k o f f , 1981) and i t was suggested t h a t p o s t - s e c r e t o r y a l t e r a t i o n of the c a r b o h y d r a t e c o m p o s i t i o n may i n c r e a s e FSH a c t i v i t y (Ward and Moore, 1980). S i n c e d e s i a l y l a -t i o n of HCG leads to d e c r e a s e d s t e r o i d o g e n i c a c t i v i t y due to reduced s i g n a l g e n e r a t i o n i n the t a r g e t c e l l (Moyle et a l . , 1975), i t was s u g g e s t e d t h a t the r a t i o of FSH and LH a c t i v i t y may depend on the c a r b o h y d r a t e c o m p o s i t i o n of PMSG (Stewart et a l . , 1976; M c i n t o s h et a l . , 1976). However, d e s i a l y l a t i o n of PMSG leads to a s i m i l a r magnitude of change i n both FSH and LH a c t i v i t y ( P a p k o f f , 1974; Papkoff, 1978; Moore and Ward, 1980). P r o g r e s s i v e d e s i a l y 1 at i o n of go n a d o t r o p h i n s a l s o reduces b i o l o g i c a l a c t i v i t y (Van H a l l et a l . , 1971; Rajaniemi and Vanha-Per t t u 1 a, 1973 ) by s h o r t e n i n g t h e h a l f l i f e of the mol e c u l e ( M c i n t o s h et a l . , 1975). S i a l i c a c i d i n h i b i t s r e c e p t o r mediated h e p a t i c uptake of g o n a d o t r o p h i n s and d e g r a d a t i o n ( M o r e l l et a l . , 1971; Van H a l l et a l . , 1971; Rajaniemi and V a n h a - P e r t t u l a , 1973 ; Schams e t a l . , 1978). A l t h o u g h equine g o n a d o t r o p h i n s have more s i a l i c a c i d than o t h e r s p e c i e s , PMSG i s p a r t i c u l a r l y h i g h w i t h a t o t a l c o n t e n t of 11% (Papkoff, 1978). C o n s e q u e n t l y , PMSG has a l o n g h a l f l i f e , i n excess of 20 hours (Parlow and Ward, 1961; M c i n t o s h et a l . , 1975; Schams et 12 a l . , 1978). T h i s i s s i g n i f i c a n t l y l o n g e r than t h a t of FSH (2 hours) or LH (30 m i n u t e s ) (Bogdanove and Gay, 1969). T h e r e f o r e , the v a r i a b l e FSH and LH a c t i v i t y of PMSG stems from the mul-t i p l e e f f e c t s of c a r b o h y d r a t e c o n t e n t on h a l f l i f e and s i g n a l t r a n s d u c t i o n (Van H a l l e t a l . , 1971; Rajaniemi and Vanha-P e r t t u l a , 1973; Moyle et a l . , 1975; M c i n t o s h et a l . , 1975) and of amino a c i d sequence on net m o l e c u l a r charge and r e c e p t o r r e c o g n i t i o n (Ward and Moore, 1978).. 3. R e p r o d u c t i v e P a t t e r n s i n t h e Mature and Immature Rat A. Mature Rat In the mature r a t , f o l l i c u l a r growth and development l e a d i n g to o v u l a t i o n are r e g u l a t e d by a h i g h l y i n t e g r a t e d sequence of hormonal e v e n t s . C i r c u l a t o r y e s t r adi ol -17(3 ( e s t r a d i o l ) l e v e l s commence t o r i s e on the a f t e r n o o n of d i -e s t r u s , peak on the morning of p r o e s t r u s and then d e c l i n e (Brown-Grant et a l . , 1970; N a f t o l i n et a l . , 1972). The d e c l i n e of e s t r a d i o l i s f o l l o w e d by the g o n a d o t r o p h i n s u r g e , c h a r a c t e r -i z e d by r a p i d l y r i s i n g and d e c l i n i n g l e v e l s of LH and FSH, on the a f t e r n o o n of p r o e s t r u s and a t r a n s i t o r y e l e v a t i o n of progesterone and t e s t o s t e r o n e (Brown - Grant et a l . , 1970; B a r r a c l o u g h et a l . , 1971; N a f t o l i n et a l ., 1972; Gay and Tomac-s, 1974). As a r e s u l t of t h e c i r c a d i a n rhythm, the endogenous gonadotrophin surge o c c u r s at 14:00 to 16:00 of p r o e s t r u s 13 ( E v e r e t t e t a l . , 1949). A second FSH s u r g e o c c u r s i n the e a r l y morning of e s t r u s (Gay and Tomacs, 1974). T h i s p a t t e r n of hormone r e l e a s e l e a d i n g to o v u l a t i o n depends on the response of the p i t u i t a r y t o s e c r e t o r y s t i m u l i . I n c r e a s i n g s e n s i t i v i t y of the p i t u i t a r y to hypothalamic r e l e a s i n g hormones commences on t h e a f t e r n o o n of d i e s t r u s , reaches a maximum on the a f t e r n o o n of p r o e s t r u s and then d e c l i n e s . ( A i y e r et a l . , 1974). T h i s a l t e r a t i o n i n p i t u i t a r y s e n s i t i v i t y i s c r i t i c a l i n d e t e r m i n i n g the magnitude of the go n a d o t r o p h i n s u r g e and i s bot h c a u s a l l y and t e m p o r a l l y a s s o c i a t e d w i t h the e l e v a t i o n of e s t r a d i o l on the a f t e r n o o n of d i e s t r u s and the morning of p r o e s t r u s (Kobayashi et a l . , 1969; Arimura and S c h a l l y , 1971). During the e a r l y phase of the go n a d o t r o p h i n surge, an a m p l i f i c a t i o n e f f e c t i s mediated by pr o g e s t e r o n e and r e l e a s i n g hormones ( A i y e r et a l . , 1974; Rao and Mahesh, 1986) . T h e r e f o r e , the r i s i n g e s t r a d i o l l e v e l s i n c o n c e r t w i t h p r o g e s t e r o n e prime t h e hypothaiamo-hypophyseal a x i s f o r a r a p i d and l a r g e s c a l e r e l e a s e of gonadotrophins which i n d u c e o v u l a t i o n ( E r i c k s o n , 1986). W h i l e e s t r a d i o l and pr o g e s t e r o n e r e g u l a t e t h e surge of p i t u i t a r y g o n a d o t r o p h i n s , androgens and t h e d e c l i n e of i n h i b i n mediate the second phase of FSH r e l e a s e (Gay and Tomacs, 1974; Lee, 1983). The c y c l i c s u r g e s of LH and FSH ensure the m a t u r a t i o n of 10 to 12 f o l l i c l e s every 4 days ( H i r s h f i e l d , 1986a). T h i s appears to be the number of f o l l i c l e s r e q u i r e d f o r s y n t h e s i s of an 14 adequate l e v e l of e s t r a d i o l f o r g o n a d o t r o p h i n r e l e a s e ( H i r s h -f i e l d , 1986a). At e s t r u s , a group of f o l l i c l e s e x i s t s on the ovary which have completed g o n a d o t r o p h i n i n d e p e n d e n t develop-ment and r e q u i r e h i g h l e v e l s of FSH f o r c o n t i n u e d growth, without which they w i l l undergo a t r e s i a ( H i r s h f i e l d and M i d g l e y , 1978a). The second phase o f FSH r e l e a s e f o l l o w i n g the g o n a d o t r o p h i n surge s e l e c t s t h i s c o h o r t of f o l l i c l e s which w i l l o v u l a t e at the next e s t r u s ( H i r s h f i e l d and M i d g l e y , 1978b; Osman, 1985). Once a f o l l i c l e i s thus s t i m u l a t e d , exposure to FSH must be m a i n t a i n e d f o r c o n t i n u e d growth and d i f f e r e n t i a t i o n ( R i c h a r d s et a l . , 1976; Rao et a l . , 1978). FSH a l s o primes the s e l e c t e d f o l l i c l e s to become more s e n s i t i v e t o the low g o n a d o t r o p h i n l e v e l s which p r e v a i l at d i e s t r u s so t h a t preovu-l a t o r y development w i l l c o n t i n u e (Schwart z , 1974; R i c h a r d s and Midgley, 1976; Weischen and D u l l a r t , 1976). E s t r a d i o l , FSH and LH p l a y e s s e n t i a l r o l e s i n t h e growth and d i f f e r e n t i a t i o n of the f o l l i c l e s . E s t r a d i o l and FSH are c r i t i c a l f o r the p r o l i f e r a t i o n and d i f f e r e n t i a t i o n of g r a n u l o s a c e l l s w h i l e t o n i c LH s t i m u l a t e s t h e c a l growth and d i f f e r e n t i -a t i o n (Rao et a l . , 1978 ; R i c h a r d s and Kersey, 1979; R i c h a r d s et "al.., 1980; Bogovich e t a l . , 1981; D a n i e l and Armstrong, 1984). These c e l l s then a c t i n c o n c e r t w i t h t h e i r r e s p e c t i v e s t i m u l a -t o r y hormones as a s t e r o i d o g e n i c u n i t s y n t h e s i z i n g e s t r a d i o l , androgens and s u b s e q u e n t l y p r o g e s t e r o n e , which i n t u r n a c t with 15 gonadotroph!ris to mediate f o l l i c u l a r m a t u r a t i o n (Rao et a l . , 1978;" R i c h a r d s and Kersey,. 197 9). B. Immature Rat F o l l i c u l a r development and growth o c c u r i n an environment dominated by c y c l i c hormonal changes i n t h e mature r a t . In the PMSG r e s p o n s i v e immature r a t , c y c l i c hormonal a c t i v i t y i s absent ( H i r s h f i e l d , 1985 ). Therefore,. knowledge of the f o l l i c u l a r and hormonal dynamics of the p r e p u b e r t a l r a t a r e key to u n d e r s t a n d i n g how PMSG ind u c e s o v u l a t i o n i n these a n i m a l s . Rats e x p e r i e n c e f i r s t e s t r u s and the commencement of c y c l i c r e p r o d u c t i v e a c t i v i t y at a p p r o x i m a t e l y 40 days of age (Dohler and Wuttke, 1975). From 9 to 21 days of age, i n c r e a s i n g c o n c e n t r a t i o n s o f FSH, LH and e s t r a d i o l can be measured i n the c i r c u l a t i o n ( M e i j s - R o e l e f s e t a l . , 1973; D o h l e r and Wuttke, 1975). While FSH and e s t r a d i o l l e v e l s remain c o n s t a n t l y h i g h , LH f l u c t u a t e s d i u r n a l l y w i t h very h i g h peak l e v e l s d u r i n g t h i s p e r i o d (Dohler and Wuttke, 1975). E s t r a d i o l a c t s i n a p o s i t i v e feedback manner on the hypotha1amo-hypophysea 1 a x i s to induce the p u l s a t i l e LH r e l e a s e which matures the hy p o t h a l a m i c c y c l i c c e n t r e f o r f u t u r e a c t i v i t y ( D o h ler and Wuttke, 1975). In con-t r a s t , the e l e v a t e d e s t r a d i o l l e v e l s have a n e g a t i v e feedback e f f e c t on FSH ( M e i j s - R o e l e f s et a l . , 1973). From 21 to 25 days of age, e s t r a d i o l , LH and FSH l e v e l s drop and p r o g e s t e r o n e and p r o l a c t i n l e v e l s g r a d u a l l y s t a r t to i n c r e a s e ( M e i j s - R o e 1 e f s et 16 a l . , 1973; Dohler and Wuttke, 1975). The d e c l i n e o f gonado-t r o p i n s i n c i r c u l a t i o n r e f l e c t s i n c r e a s e d s e n s i t i v i t y of the hypothalamo-hypophyseal a x i s to the n e g a t i v e feedback e f f e c t of e s t r a d i o l ( M e i j s - R o e l e f s e t a t . , 1973) or r i s i n g p r o l a c t i n and pr o g e s t e r o n e (Dohler and Wuttke, 1975). The s e n s i t i v i t y of the hypothalamo-hypophyseal a x i s t o p o s i t i v e s t e r o i d hormone f e e d -back g r a d u a l l y i n c r e a s e s from day 21 to puberty ( D o h l e r and Wuttke, 1975). As p u b e r t y n e a r s , l e v e l s of e s t r a d i o l and androgens r i s e and c y c l i c a c t i v i t y i s i n i t i a t e d (Aguado and Ojeda, 1985). A d m i n i s t r a t i o n of PMSG i n d u c e s an i n c r e a s e i n f o l l i c u l a r diameter i n the immature r a t as e a r l y as 8 days of age (Golden-berg et a l . , 1973). The magnitude of the i n c r e a s e i n f o l l i c u l a r diameter induced by PMSG re a c h e s a maximum a t 21 days of age and a t t h i s time o v u l a t i o n can a l s o be induced (Goldenberg et al . , 1973; H i r s h f i e l d , 1986b). Maximal o v u l a t o r y r e s p o n s i v e n e s s i s o b s erved at 26 days of age (Zarrow and Quinn, 1963). In the absence of g o n a d o t r o p h i n s u r g e s , s i g n i f i c a n t f o l l i c u l a r development occurs i n the immature r a t which p r o g r e s s e s to the e a r l y a n t r a l stage, the c r i t i c a l r e g u l a t o r y p o i n t at which i n the mature r a t , they a r e r e c r u i t e d by the FSH s u r g e f o r o v u l a t i o n at the next e s t r u s ( H i r s h f i e l d , 1986b). T h e r e f o r e , at the time when immature r a t s become maximally r e s p o n s i v e to PMSG, endogenous g o n a d o t r o p h i n l e v e l s are low, t h e r e i s i n c r e a s i n g s e n s i t i v i t y o f t h e hypothalamo-hypophyseal a x i s to 17 s t e r o i d hormone feedback and t h e r e i s a p o p u l a t i o n of f o l l i c l e s which can be r e c r u i t e d by h i g h l e v e l s of FSH. 4. E f f e c t s of a Low Dose of PMSG i n Immature Rats • A. E n d o c r i n e Response Many s t u d i e s have s c r u t i n i z e d the hormonal events t r i g g e r e d by PMSG i n the immature r a t t o de t e r m i n e i f the mechanism of i n d u c t i o n of f o l l i c u l a r growth and o v u l a t i o n i s the same as t h a t observed i n the mature r a t . A d m i n i s t r a t i o n of a low dose (3 to 10 IU) of PMSG to 26 t o 33 day o l d r a t s i n d u c e s e l e v a t i o n of androgens 12 hours p o s t - t r e a t m e n t which p e r s i s t s u n t i l 36 hours then d e c l i n e s over the en s u i n g 36 hour p e r i o d (Yun et a l . , 1987 ). E s t r a d i o l r i s e s p r o g r e s s i v e l y and peaks at 54 hours ( M i l l e r and Armstrong, 1981a; Yun e t a l . , 1987) w h i l e plasma p r o g e s t e r o n e l e v e l s i n c r e a s e a t 54 to 60 hours ( W i l s o n et a l . , 197 4; Yun et al ., 1987). An endogenous g o n a d o t r o p h i n surge i s observed 52 to 54 hours post t r e a t m e n t , concomitant w i t h a d e c l i n e i n p i t u i t a r y LH ( W i l s o n e t a l . , 1974). T h i s induces o v u l a t i o n of 8 to 12 o o c y t e s ( S t r a u s s and Meyer, 1962; Fuxe et a l . , 1972; H i l l e n s j o et a l . , 1974; Kostyk et a l . , 1978; M i l l e r and Armstrong, 1981a; Walton e t a l . , 1983; Yun e t a l . , 1987) ap p r o x i m a t e l y 10 hours a f t e r the end of the c r i t i c a l p e r i o d ( S t r a u s s and Meyer, 1962; H i l l e n s j o et a l . , 1974; Wilson et al . , 1974 ; Sasomoto and Johke, 1975; Ahren et a l . , 1978 ; M i l l e r and Armstrong, 1981a; Yun e t a l . , 1987). 18 T h i s p a t t e r n of s t e r o i d and g o n a d o t r o p h i n hormones i s s i m i l a r to the p r e o v u l a t o r y p a t t e r n of mature r a t s ( W i l s o n et a l . , 1974). As i n the mature r a t , the c i r c a d i a n rhythm i s c e n t r a l t o t h i s p a t t e r n ( S t r a u s s and Meyer, 1962; M i l l e r and Armstrong, 1981a). Neuropharmaco1ogic b l o c k a d e of the endog-enous surge j u s t p r i o r to the c r i t i c a l p e r i o d w i l l d e l a y o v u l a t i o n by 24 hours i n the immature r a t ( S t r a u s s and Meyer, 1962). Delay of the g o n a d o t r o p h i n surge can a l s o be i n d u c e d by exposing immature r a t s t o c o n t i n u o u s l i g h t f o r s e v e r a l days p r i o r to treatment (McCormack and Bennin, 1970). B. O v u l a t o r y Response The o v u l a t o r y e f f e c t of PMSG i s mediated by e s t r a d i o l (Zarrow and D i n i u s , 1971). The i n c r e a s i n g e s t r a d i o l l e v e l s act wi t h p r o g e s t e r o n e to s t i m u l a t e LH r e l e a s e and induce o v u l a t i o n as i n the mature r a t (McCormack and Meyer, 1963; Zarrow and G a l l o , 1969; Hagino and G o l d z i e h e r , 1970; Meyer et a l . , 1971). O v u l a t i o n i n PMSG t r e a t e d immature r a t s i s b l o c k e d by admin-i s t r a t i o n of a n t i - e s t r a d i o 1 ( F e r i n et a l . , 1969) and a s i m i l a r g o n a d o t r o p h i n and o v u l a t o r y response can be a c h i e v e d w i t h a s i n g l e i n j e c t i o n of e s t r a d i o l benzoate (Ying et a l . , 1971). T h e r e f o r e , an adequate number of f o l l i c l e s must be s e l e c t e d f o r growth and development by PMSG so t h a t a s u f f i c i e n t l e v e l of e s t r a d i o l i s p r e s e n t to t r i g g e r an o v u l a t o r y r e l e a s e of LH. 19 A d m i n i s t r a t i o n of PMSG s t i m u l a t e s development of FSH r e s p o n s i v e f o l l i c l e s and s t e r o i d o g e n e s i s i n the immature rat (Sasamoto and Kennan, 1973 ). Due t o the long h a l f l i f e of PMSG, t h e r e i s a p r o l o n g e d g o n a d o t r o p h i n e f f e c t s i m i l a r to the endogenous FSH sur g e and t o n i c LH a c t i v i t y i n the mature r a t , which a r e necessary f o r f i n a l f o l l i c u l a r growth and d i f f e r e n t i -a t i o n ( H i r s h f i e l d , 1985). A d m i n i s t r a t i o n of anti-PMSG w i t h i n 36 hours of treatment w i l l b l o c k o v u l a t i o n , i l l u s t r a t i n g that f o l l i c u l a r development i s dependent on exogenous g o n a d o t r o p h i n a c t i v i t y u n t i l t h i s time (Sasamoto and Kennan, 1973). The g o n a d o t r o p h i n requirement f o r f o l l i c l e maintenance i s 10% of t h a t r e q u i r e d f o r i n i t i a t i o n of growth (Sasamoto et a l . , 1972 ; R i c h a r d s and Kersey, 1979). Thus, a f t e r 36 hours, endogenous g o n a d o t r o p i n s can m a i n t a i n the p r e o v u l a t o r y f o l l i c l e s u n t i l the endogenous g o n a d o t r o p h i n surge i n d u c e s o v u l a t i o n (Sasamoto and Kennan, 1973; P e l u s o et a l . , 1981). C. F a i l u r e of PMSG to I n i t i a t e Puberty A l t h o u g h the hormone and o v u l a t o r y p a t t e r n s a re very s i m i l a r i n the mature and immature r a t , a d m i n i s t r a t i o n of PMSG does not i n i t i a t e r e p e t i t i v e c y c l i c a c t i v i t y i n the immature r a t (Sasamoto and Johke, 1975; Aguado and Ojedo, 1985). It was suggested t h a t the mechanism of o v u l a t i o n i n d u c t i o n i n the immature r a t may d i f f e r from t h a t of mature r a t s (Zarrow and 20 D i n i u s , 1971). C l o s e s c r u t i n y of p i t u i t a r y and serum g o n a d o t r o p h i n l e v e l s i n d i c a t e t h a t d i f f e r e n c e s do e x i s t . In the immature r a t , peak l e v e l s of plasma FSH are s i g n i f i c a n t l y h i g h e r and d e c l i n e e a r l i e r (Sasamoto and Johke, 1975). P i t u -i t a r y FSH drops g r a d u a l l y r a t h e r than p r e c i p i t o u s l y , w i t h slower and p o o r e r r e c o v e r y (Sasamoto and Johke, 1975). The LH surge i s longer w i t h a lower peak (Sasamoto and Johke, 1975) and p i t u i t a r y LH r e l e a s e i s slow ( K l a u s i n g and Meyer, 1968; Sasamoto and Johke, 1975), w i t h more e f f i c i e n t r e c o v e r y (Sasamoto and Johke, 1975). These d i f f e r e n c e s i n d i c a t e t h a t the p i t u i t a r y and hypothalamus of mature and immature r a t s d i f f e r i n s e n s i t i v i t y to r e g u l a t o r y s t i m u l i and t h i s l i k e l y , r e f l e c t s i m m a t u r i t y of the hypothalamo-hypophysea 1 a x i s i n p r e p u b e r t a l r a t s (Sasamoto and Johke, 1975). 5. E f f e c t s of a SuperovuLatory Dose of PMSG i n Immature Rats A. E n d o c r i n e Response S i m i l a r to the low dose, a s u p e r o v u l a t o r y dose (15 to 40 IU) of PMSG s t i m u l a t e s an e l e v a t i o n of e s t r a d i o l , androgens and pr o g e s t e r o n e ( S o r r e n t i n o et a l . , 1972; Wi l s o n et a l . , 1974; M i l l e r and Armstrong, 1981a; Yun . et a l . , 1987). P i t u i t a r y LH r i s e s to a maximum at 42 to 52 hours p o s t - t r e a t m e n t (Zarrow and D i n i u s , 1971; W i l s o n et a l . , 1974). P i t u i t a r y g o n a d o t r o p h i n s d e c l i n e and a g o n a d o t r o p h i n surge i s observed 52 to 57 hours p o s t - t r e a t m e n t ( S o r r e n t i n o et a l . , 1972; Wilson et a l . , 1974). 21 In the s u p e r o v u l a t e d r a t , as i n the p h y s i o l o g i c a l l y s t i m u l a t e d r a t , e s t r a d i o l and p r o g e s t e r o n e a c t v i a p o s i t i v e feedback at the hypothalamo-hypophyseal l e v e l t o t r i g g e r an endogenous gonadotrophin s u r g e (Zarrow and G a l l o , 1969; Hagino and G o l d z i e h e r , 1970; Zarrow and D i n i u s , 1971; W i l s o n et a l . , 1974). A d m i n i s t r a t i o n of h i g h doses of t e s t o s t e r o n e (Klawon et al . , 1971; Fuxe et a l . , 1972), p r o g e s t e r o n e (Zarrow and G a l l o , 1969) or es t r o g e n (Hagino and G o l d z i e h e r , 1970) i n h i b i t the endogenous g o n a d o t r o p h i n s u r g e . S i g n i f i c a n t d i f f e r e n c e s i n hormone p r o f i l e s e x i s t between immature r a t s t r e a t e d w i t h e i t h e r a low or s u p e r o v u l a t o r y dose of PMSG ( F i g u r e 1 ) . In c o n t r a s t to the p h y s i o l o g i c a l l y stimu-l a t e d r a t , the s u p e r o v u l a t e d r a t e x p e r i e n c e s an e l e v a t i o n of androgens c o i n c i d e n t w i t h e s t r a d i o l (Yun et a l . , 1987). The l e v e l s of e s t r a d i o l p r i o r to the endogenous g o n a d o t r o p h i n surge are h i g h e r i n the s u p e r o v u l a t e d r a t ( W i l s o n et a l . , 1974; M i l l e r and Armstrong, 1981a; Yun et a l . , 1987). There i s an i n i t i a l p r o l o n g e d but m i l d e l e v a t i o n of LH from 12 t o a p p r o x i -mately 36 hours p o s t - t r e a t m e n t f o l l o w e d by an a t t e n u a t e d endogenous g o n a d o t r o p h i n s u r g e ( W i l s o n e t a l . , 1974; Yun, 1989). No c o r r e l a t i o n was i d e n t i f i e d between the magnitude of the LH surge and the number of ova shed (Klawon et a l . , 197 2). A g r e a t e r c o r r e l a t i o n was found between the number of ova shed and e s t r a d i o l l e v e l s ( W i l s o n e t a l . , 1974). LOW DOSE OF PMSG 126-1 M2S _ J A r - j Page 2 2 f\ FIGURE 1 The hormonal p a t t e r n s observed i n mature, s p o n t a n e o u s l y o v u l a t i n g r a t s and immature r a t s t r e a t e d w i t h e i t h e r a low or h i g h dose of PMSG are i l l u s t r a t e d . The changes which o c c u r subsequent to treatment w i t h PMSG and d u r i n g the e s t r u s c y c l e of the mature r a t are d e p i c t e d . (Data f o r mature r a t adapted from Brown-Grant et a l . , 1970; B a r r a c l o u g h et a l . , 1971; N a f t o l i n e t a l . , 1972; Gay and Tomacs, 1974) . (Data f o r immature r a t s adapted from W i l s o n et a l . , 1974; Yun, 1989). 23 The marked e l e v a t i o n i n both e s t r a d i o l and androgen l e v e l s was a t t r i b u t e d to the l a r g e number of f o l l i c l e s r e c r u i t e d by the h i g h dose of PMSG and i t s FSH/LH a c t i v i t y ( W i l s o n and Zarrow, 1962 ; M i l l e r and Armstrong, 1981a; Yun et a l . , 1987; L e v e i l l e and Armstrong, 1989). The long h a l f l i f e of PMSG may l e a d to c o n t i n u e d s t i m u l a t i o n of l a r g e u n l u t e i n i z e d f o l l i c l e s and s y n t h e s i s o f e s t r a d i o l ( M i l l e r and Armstrong, 1981a; Armstrong et al ., 1983). A n t i s e r a to PMSG a d m i n i s t e r e d 57 hours p o s t - t r e a t m e n t s i g n i f i c a n t l y reduces e s t r a d i o l l e v e l s (Walton and Armstrong, 1981). Hyperstimu1 at i o n of a l a r g e number of f o l l i c l e s i s l i k e l y a l s o r e s p o n s i b l e f o r s u p p r e s s i o n of the LH sur g e . The f i r s t m i l d e l e v a t i o n of LH i n the sup ero vul a t e d r a t i s l i k e l y exogenous LH a c t i v i t y of PMSG (Yun, 1989). The second, endoge-nous, LH peak i s s u p p r e s s e d without a f f e c t i n g the time of onset (Yun, 1989 ). T h i s phenomenon was a l s o o b s e r v e d i n s u p e r o v u l a ted c a t t l e (Jensen et a l . , 1982) and p r i m a t e s . In pri m a t e s , s u p e r o v u l a t i o n was a s s o c i a t e d w i t h the p r o d u c t i o n o f i n h i b i n or other n o n - s t e r o i d a l o v a r i a n f a c t o r s which e i t h e r d i r e c t l y or i n d i r e c t l y i n h i b i t e d g o n a d o t r o p h i n s e c r e t i o n ( F e r r a r e t t i et a l . , 1983; L i t t m a n and Hodgen, 1984). A d i r e c t dose response r e l a t i o n s h i p between the p r o d u c t i o n of f o l l i c u l a r i n h i b i n and a d m i n i s t r a t i o n of PMSG was observed i n immature r a t s (Lee et a l . , 1981; Lee et a l . , 1982; Davis et a l . , 1986). Although i n h i b i n p r e d o m i n a n t l y i n h i b i t s s e c r e t i o n o f FSH i n the PMSG t r e a t e d immature a l s o d e c r e a s e LH 24 r a t (Lee, 1983) , h i g h doses of i n h i b i n l e v e l s ( F e r r a r e t t i et a l . , 1983 ). w i l l B. O v u l a t o r y Response As demonstrated o r i g i n a l l y by C o l e (1936), the number of o v u l a t i o n s i n d u c e d by PMSG i n immature r a t s i s dose r e l a t e d . While a low dose w i l l i n d u c e a p h y s i o l o g i c number of o v u l a t i o n s (8 to 12), h i g h e r doses induce a superovu1 a t o r y number of o v u l a t i o n s (30 to 60) (Klawon et a l . , 1971; Kostyk et a l . , 1978; M i l l e r and Armstrong, 1981a; Walton et a l . , 1983; Yun et a l . , 1987; L e v e i l l e and Armstrong, 1989). However, t h e r e i s wide v a r i a t i o n i n s u p e r o v u l a t o r y response between i n d i v i d u a l s (Sherman et a l . , 1982; Walton et a l . , 1983; Evans and Armstr-ong, 1984; Sherman et a l . , 1984) and superovu1at ion i s in d u c e d i n some cases w i t h 8 to 10 IU PMSG ( W i l s o n and Zarrow, 1962; Evans and Armstrong, 1984). Maximal o v u l a t o r y response i s a c h i e v e d w i t h doses of PMSG lower than 40 IU (Evans and.Armstrong, 1984; Yun e t a l . , 1987). Above 40 IU PMSG, o v u l a t i o n f a i l u r e and c y s t i c f o l l i c l e s are observed ( W i l s o n and Zarrow, 1962; Ko s t y k et a l . , 1978). Con-ti n u o u s exposure of g r a n u l o s a c e l l s to LH leads t o downregula-t i o n of LH r e c e p t o r s (Schwall and E r i c k s o n , 1983). The pro-longed h a l f l i f e of PMSG and i t s h i g h LH a c t i v i t y may cause a decrease i n LH r e c e p t o r s which r e s u l t s i n poor o v u l a t o r y response. However, the number of LH r e c e p t o r s p r e s e n t on 25 g r a n u l o s a c e l l s at the time o f o v u l a t i o n i s f a r i n excess of t h a t r e q u i r e d f o r maximal o v u l a t o r y response ( D i e r s c h k e e t a l . , 1983). Thus, the f a i l u r e of o v u l a t i o n may i n s t e a d be due to o v e r s t i m u l a t i o n of f o l l i c u l a r growth and premature l u t e i n -i z a t i o n (Wilson and Zarrow, 1962). S u p e r o v u l a t i o n o f immature r a t s i nduces a b i p h a s i c o v u l a t o r y response (De La L a s t r a e t a l . , 1972; Ko s t y k et a l . , 1978; M i l l e r and Armstrong, 1981a; Walton et a l . , 1983; Yun et a l . , 1987) which c o i n c i d e s t e m p o r a l l y w i t h the two peaks of LH a c t i v i t y (Yun, 1989). A s u p e r o v u l a t o r y dose of PMSG s t i m u l a t e s the f i r s t wave of o v u l a t i o n d i r e c t l y as a r e s u l t of i t s i n t r i n s i c LH a c t i v i t y and the second wave i n d i r e c t l y v i a p o s i -t i v e s t e r o i d hormone feedback a t the hypothalamo-hypophyseal a x i s and an endogenous g o n a d o t r o p h i n surge (McCormack and Meyer, 1963; Ying and Meyer, 1969a,b; Y i n g and Meyer, 1973; De La L a s t r a et al ., 197 2; S o r r e n t i n o et a l . , 1972). The f i r s t wave of o v u l a t i o n i n immature r a t s can be re p r o -duced by the a d m i n i s t r a t i o n of HCG a l o n e (Sugawara et a l . , 1969; De La L a s t r a e t a l . , 1972; Neal and Baker, 1973; Kostyk et a l . , 1978). Thus, the f i r s t wave of o v u l a t i o n i n PMSG-t r e a t e d immature r a t s i s a d i r e c t e f f e c t of the exogenous LH a c t i v i t y on p r e e x i s t a n t a n t r a l f o l l i c l e s (Sugawara et a l . , 1969; Kostyk et a l . , 1978; M i l l e r and Armstrong, 1981a). Low doses o f PMSG f a i l t o s t i m u l a t e o v u l a t i o n d i r e c t l y due to a s u b o v u l a t o r y l e v e l o f LH a c t i v i t y (De La L a s t r a e t a l . , 1972). 2 6 However, e a r l y o v u l a t i o n i s o c c a s i o n a l l y seen w i t h the low dose (De La L a s t r a et a l . , 1972; Kostyk et a l . , 1978; M i l l e r and Armstrong, 1981a). C. Mechanism of F o l l i c l e S e l e c t i o n by PMSG Large numbers of p r e a n t r a l and a t r e t i c f o l l i c l e s are pr e s e n t i n immature r a t s due to low c i r c u l a t i n g gonadotrophin l e v e l s ( P e l u s o et a l . , 1977 ; H i r s h f i e l d , 1985 ). PMSG may induce s u p e r o v u l a t i o n by rescue of a t r e t i c f o l l i c l e s ( T h i b a u l t , 1977; Braw and T s a f r i r i , 1980; Sherman et a l . , 1982; Moor et a l . , 1984; Monniaux e t a l . , 1984). The number of a t r e t i c f o l l i c l e s were observed t o d e c r e a s e and the number of p r e o v u l a t o r y f o l l i -c l e s t o i n c r e a s e 48 hours f o l l o w i n g a superovu1 a t o r y dose of PMSG (Braw and T s a f r i r i , 1980). In a more recent study of s u p e r o v u l a t i o n i n immature r a t s , a t r e s i a was a r r e s t e d but not re v e r s e d , w h i l e a marked i n c r e a s e i n t h e number of growing a n t r a l f o l l i c l e s was seen ( H i r s h f i e l d , 1989). It was concluded t h a t PMSG s t i m u l a t e s o v u l a t i o n of r a p i d l y growing, r a t h e r t han a t r e t i c , f o l l i c l e s ( H i r s h f i e l d ,. 1 989 ) . F o l l i c l e s i n the immature r a t d e v e l o p under d i f f e r e n t hormonal c o n d i t i o n s than those i n the mature r a t , s i n c e t h e r e i s a l a c k o f c y c l i c a c t i v i t y , c o r p o r a l u t e a and p r e o v u l a t o r y f o l l i c l e s ( H i r s h f i e l d , 1985; H i r s h f i e l d , 1986a). In c o n t r a s t to the mature r a t , f o l l i c l e s a t the c r i t i c a l s t a g e of development, where they can e i t h e r be s e l e c t e d f o r f u r t h e r growth by FSH or 27 undergo a t r e s i a ( H i r s h f i e l d and M i d g l e y , 1978a,b; H i r s h f i e l d and Schmidt, 1987), s y n t h e s i z e no e s t r a d i o l ( D a n i e l and Armstrong, 1984), have 50% fewer g r a n u l o s a c e l l s and have no c e n t r i p e t a l d i f f e r e n t i a t i o n of mural g r a n u l o s a c e l l s ( H i r s h -f i e l d , 1986a). In the mature r a t , p r e o v u l a t o r y growth proceeds under the i n f l u e n c e of FSH and LH f o r 36 t o 48 hours ( R i c h a r d s and Kersey, 1979; Bogovich et a l . , 1981) f o l l o w i n g which HCG w i l l i n d u c e o v u l a t i o n ( H i r s h f i e l d and Schmidt, 1987). However, i n the immature r a t , these f o l l i c l e s w i l l o v u l a t e w i t h i n 24 hours of HCG a d m i n i s t r a t i o n ( H i r s h f i e l d and Schmidt, 1987). A d m i n i s t r a t i o n of PMSG i n d u c e s a marked i n c r e a s e i n LH r e c e p t o r s (Peluso e t a l . , 1977; Vidyashankar and Moudgal, 1984; H i r s h f i e l d and Schmidt, 1987 ) and 3- (3 - h y d r o x y s t e r o i d d e h y d r o -genase and s i d e c h a i n c l e a v a g e enzyme a c t i v i t y i n g r a n u l o s a c e l l s w i t h i n 48 hours of trea t m e n t ( H i r s h f i e l d and Schmidt, 1987; O r l y , 1989), i n d i c a t i v e of 1 u t e i n i z a t i o n ( Z e l e z n i k et a l . , 1974). These changes cannot be induced by a d m i n i s t r a t i o n of e s t r o g e n alone and r e f l e c t the d i r e c t e f f e c t of gonadotrophin on f o l l i c l e s ( Z e l e z n i k e t a l ., 1974 ). T h e r e f o r e , the g r e a t e r s e n s i t i v i t y of f o l l i c l e s to LH s t i m u l a t i o n i n the immature r at compared w i t h the mature r at enables PMSG to induce e a r l y o v u l a t i o n accompanied by 1 u t e i n i z a t i o n . In the immature r a t , PMSG s t i m u l a t e s p r o l i f e r a t i o n i n a l l s i z e s of f o l l i c l e s ( H i r s h f i e l d , 1985). A d m i n i s t r a t i o n of PMSG switches the s t e r o i d o g e n i c pathway to a r o m a t i z a b l e androgen and 28 e s t r a d i o l p r o d u c t i o n (Aguado and Ojeda, 1985; Johnson and G r i s w o l d , 1984) and i n c r e a s e s the m i t o t i c index of g r a n u l o s a c e l l s ( P e l u s o e t a l . , 1977; V i d y a s h a n k a r and Moudgal, 1984; H i r s h f i e l d , 1985; H i r s h f i e l d and Schmidt, 1987). In response to PMSG, f o l l i c l e s , which are one sta g e of development behind those which can be i n d u c e d to o v u l a t e d i r e c t l y , undergo r a p i d synchronous growth and r e a c h p r e o v u l a t o r y s i z e w i t h i n 48 hours of treatment ( P e l u s o et a l . , 1977; O r l y , 1989). T h i s a c t i o n i s mediated by e s t r a d i o l (Braw and T s a f r i r i , 1980). I n h i b i t i o n of aromatase leads to a marked decrease i n the number of a n t r a l f o l l i c l e s and t h i s i s r e v e r s e d by the a d m i n i s t r a t i o n of est r o g e n ( D i e r s c h k e e t a l . , 1983). The a c t i o n of PMSG i n the immature r a t t h e r e f o r e stems from both dual gonadotrophi c a c t i v i t y and a long h a l f l i f e which l e a d s t o a p r o l o n g e d p e r i o d of s t i m u l a t i o n ( H i r s h f i e l d , 1985). A s u p e r o v u l a t o r y dose of PMSG d i r e c t l y s t i m u l a t e s d i f f e r e n t i -a t i o n and o v u l a t i o n o f FSH r e s p o n s i v e f o l l i c l e s and s t i m u l a t e s r a p i d growth of s m a l l e r f o l l i c l e s than would have been r e c r u i t e d w i t h e i t h e r a lower dose of PMSG or the endogenous FSH surge a t e s t r u s ( D o t t et a l . . , 1979; Braw and T s a f r i r i , 1980; H i r s h f i e l d , 1985; D r i a n c o u r t et a l . , 1987; D r i a n c o u r t , 1987; H i r s h f i e l d , 1989). In these ways, PMSG i n d u c e s super-o v u l a t i o n i n immature r a t s ( H i r s h f i e l d , 1985; H i r s h f i e l d , 1986a; H i r s h f i e l d and Schmidt, 1987; H i r s h f i e l d , 1989). 29 6. Oocyte M a t u r a t i o n i n Immature Rats A. M e i o t i c Competence The o b s e r v a t i o n t h a t f o l l i c l e s o f mature and immature r a t s are q u a l i t a t i v e l y and q u a n t i t a t i v e l y d i f f e r e n t r a i s e s c o ncerns over the m a t u r i t y of the o o c y t e s i n d u c e d to o v u l a t e by PMSG. In  v i t ro s t u d i e s demonstrated t h a t GVBD o c c u r s more s l o w l y i n oocytes r e t r i e v e d from immature r a t s ( K a p l a n et a l . , 1977). However, t h i s may not have a s i g n i f i c a n t e f f e c t on r e p r o d u c t i v e p o t e n t i a l as l i v e o f f s p r i n g were produced f o l l o w i n g in. v i t r o m a t u r a t i o n and f e r t i l i z a t i o n of murine o o c y t e s r e t r i e v e d from p r e a n t r a l f o l l i c l e s ( E p p i g and Sc h r o e d e r , 1989). The development of m e i o t i c competence i s i n f l u e n c e d by the s y n e r g i s t i c a c t i o n of FSH and e s t r a d i o l i n p r e p u b e r t a l l i f e (Bar-Ami and T s a f r i r i , 1981; Bar-Ami e t a l . , 1983; Bar-Ami and T s a f r i r i , 1986). Oocytes from p r e p u b e r t a l r a t s a r e incompetent to resume m e i o s i s i f r e t r i e v e d p r i o r to day 21 t o 25 of age (Bar-Ami and T s a f r i r i , 1981; Bar-Ami e t a l . , 1983; Bar-Ami and T s a f r i r i , 1986) due t o d e f e c t i v e p r o t e i n s y n t h e s i s o r abnormal s p i n d l e assembly (Bar-Ami and T s a f r i r i , 1981). T h e r e f o r e , at the age when immature r a t s become m a x i m a l l y r e s p o n s i v e to PMSG, they have a l s o a c q u i r e d m e i o t i c competence (Zarrow and Quinn, 1963). However, d u r i n g the f i n a l s t a g e s of p r e o v u l a t o r y f o l l i c l e development, f u r t h e r o o c y t e m a t u r a t i o n o c c u r s i n the oocyte, which i s c r u c i a l f o r subsequent f e r t i l i z a t i o n and embryonic development (Moor and Trounson, 1977; Zhang and 30 Armstrong, 1989). A t t e n u a t i o n of p r e o v u l a t o r y m a t u r a t i o n i n the s u p e r o v u l a t e d immature r a t may l e a d to a b n o r m a l i t i e s of f i n a l oocyte m a t u r a t i o n . B. I n t r a f o l l i c u l ar Oocyte M a t u r a t i o n The low dose of PMSG s t i m u l a t e s the most mature of the FSH r e s p o n s i v e f o l l i c l e s t o grow and d i f f e r e n t i a t e over a time course s i m i l a r to t h a t i n the mature r a t ( W i l s o n and Zarrow, 1962). However, the f o l l i c l e s s e l e c t e d f o r growth and d i f f e r e n -t i a t i o n by the low dose w i l l be those which are i n d u c e d to o v u l a t e i n the f i r s t wave of o v u l a t i o n w i t h t h e superovu1 a t o r y dose ( W i l s o n and Zarrow, 1962 ). The s m a l l e r s i z e of the f o l l i c l e s which o v u l a t e i n the f i r s t wave suggest t h a t they do not undergo complete p r e o v u l a t o r y m a t u r a t i o n ( M i l l e r and Armstrong, 1981a). S i m i l a r l y , f o l l i c l e s which o v u l a t e i n the second wave of s u p e r o v u l a t i o n , mature over t h e same time frame as f o l l i c l e s s e l e c t e d by the low dose of PMSG but a r e r e c r u i t e d at an e a r l i e r s t a g e of development ( W i l s o n and Zarrow, 1962). S u p e r o v u l a t o r y doses of PMSG induc e f l u i d i m b i b i t i o n i n a n t r a l f o l l i c l e s ( H i r s h f i e l d , 1989), s u g g e s t i n g t h a t t h e s e f o l l i c l e s may not complete m a t u r a t i o n p r i o r t o o v u l a t i o n , a l t h o u g h they a c h i e v e a normal p r e o v u l a t o r y s i z e . T h e r e f o r e , i n c o n t r a s t to the low dose, a s u p e r o v u l a t o r y dose of PMSG reduces the p e r i o d of p r e o v u l a t o r y f o l l i c u l a r m a t u r a t i o n i n both waves of o v u l a -t i o n . 31 Cumu1 us - o o c y t e c e l l c o u p l i n g and f o l l i c u l a r s e c r e t i o n s are e s s e n t i a l f o r promoting normal c y t o p l a s m i c m a t u r a t i o n of the oocyte ( M a t t i o l i et al . , 1988; Vanderhyden and Armstrong, 1989). D u r i n g the g o n a d o t r o p h i n surge, FSH reduces cumulus-oocyte c e l l c o u p l i n g w h i l e LH i n d u c e s germinal v e s i c l e break-down (Kaplan et al..-, 1977; Moor et a l . , 1981). In the mature r a t , g e r m i n a l v e s i c l e breakdown and p o l a r body f o r m a t i o n commence a p p r o x i m a t e l y 1.5 and 6 hours a f t e r the g o n a d o t r o p h i n surge, r e s p e c t i v e l y ( T s a f r i r i and K r a i c e r , 1972; A y a l o n e t a l . , 1972; Vermeiden and ZeiImaker , 197 4). D u r i n g t h i s p e r i o d , o o c y t e s a c q u i r e zona p e l l u c i d a s e n s i t i v i t y to sperm p e n e t r a -t i o n , male p r o n u c l e a r growth f a c t o r and p r o t e i n s which d i r e c t c l e a v a g e (Moor and Trounson, 1977; T h i b a u l t , 1977). These changes a r e mediated by both gonadotrophins and s t e r o i d hormones (Ayalon et a l . , 1972; H i l l e n s j o , 1976; Moor and Trounson, 1977; T h i b a u l t , 1977; Moor et al . , 1981; Zhang and Armstrong, 1989). Once i n i t i a t e d , an adequate p e r i o d of expo-s u r e and s y n c h r o n i z a t i o n of s t e r o i d and g o n a d o t r o p h i n hormones are r e q u i r e d t o s u c c e s s f u l l y complete m a t u r a t i o n ( T h i b a u l t , 1977; Moor et al . , 1980 ). D i s s o c i a t i o n of t h i s p r o c e s s l e a d s to o v u l a t i o n of d e f e c t i v e o o c y t e s ( T e s a r i k et a l ., 1984). In the immature r a t t r e a t e d w i t h a low dose of PMSG, the LH surge i s f o l l o w e d by the same time sequence of ger m i n a l v e s i c l e breakdown, p o l a r body one f o r m a t i o n and o v u l a t i o n as seen i n the mature r a t ( H i l l e n s j o et a l . , 1974; Ahren et a l . , 32 1978). Oocytes a r e f u l l y grown and c a p a b l e of resuming m e i o s i s at the time o v u l a t i o n can be m a x i m a l l y i n d u c e d by PMSG (Bar-Ami and T s a f r i r i , 1981). S i n c e b o t h the time course and the gon a d o t r o p h i n and s t e r o i d hormone p a t t e r n of p r e o v u l a t o r y f o l l i c u l a r m a t u r a t i o n are s i m i l a r t o those o f . t h e mature r a t , oocytes which mature under the i n f l u e n c e of a low dose of PMSG l i k e l y undergo normal m a t u r a t i o n . Many f u n c t i o n a l s t u d i e s i n d i c a t e t h a t t h e s e oocytes have the same r e p r o d u c t i v e poten-t i a l as those of mature r a t s ( N u t i et a l . , 1975 ; M i l l e r and Armstrong, 1982; Walton and Armstrong, 1983). In the s u p e r o v u l a ted r a t , the s h o r t e n e d phase of f o l l i c u -l a r growth and development p r i o r to o v u l a t i o n s u g g e s t s t h a t o o c y t e s may not e x p e r i e n c e an adequate p e r i o d of m a t u r a t i o n ( M i l l e r and Armstrong, 1981a). Moreover, o v a r i a n l e v e l s of e s t r a d i o l and androgens are low p r i o r to the f i r s t wave of o v u l a t i o n (Yun et a l . , 1987). In c o n t r a s t , f o l l i c u l a r s t e r o i d hormone l e v e l s are e l e v a t e d d u r i n g the second wave of p r e o v u l a -t o r y matu rat i o n ( M i l l e r and Armstrong, 1981a; Yun e t a l . , 1987; Yun e t a l . , 1988). The pr e s e n c e o f s t e r o i d hormones i n proper b a l a n c e i s e s s e n t i a l d u r i n g o o c y t e m a t u r a t i o n f o r normal f e r t i l i z a t i o n and subsequent c l e a v a g e (Moor, 1978; Moor et a l . , 1980; Moor et a l . , 1985). A l t e r a t i o n of s t e r o i d o g e n e s i s d u r i n g m a t u r a t i o n leads to oocyte a b n o r m a l i t i e s which are expressed at f e r t i l i z a t i o n (Moor, 1978; Moor e t a l . , 1980). 33 Oocyte m a t u r a t i o n can occur i n d e p e n d e n t l y of o v u l a t i o n , s i n c e the amount of g o n a d o t r o p h i n r e q u i r e d f o r m a t u r a t i o n i s much lower than t h a t r e q u i r e d f o r o v u l a t i o n (Vermeiden and Ze i l m a k e r , 1974). B r i e f e x p o s u r e t o LH i s s u f f i c i e n t to i n i t i a t e ovum n u c l e a r m a t u r a t i o n ( A y a l o n et a l . , 1972). In the human, an i n c r e a s e i n t o n i c serum LH was a s s o c i a t e d w i t h decreased f e r t i l i z a b i l i t y due t o premature i n t r a f o l 1 i c u 1 a r oocyte m a t u r a t i o n and ag i n g ( S t a n g e r and Y o v i c h , 1985). The LH a c t i v i t y of PMSG a l s o l e a d s to o v u l a t i o n of aged and abnormal oocytes i n sheep (Moor e t a l . , 1985) . In the s u p e r o v u l a t e d immature mouse, no c o r r e l a t i o n c o u l d be made between f o l l i c l e s i z e and oocy t e n u c l e a r m a t u r a t i o n (Neal and Baker, 1973). S i m i l a r l y , i n the r a t , the f o l l i c l e and oocyte undergo i n d e -pendent and asynchronous m a t u r a t i o n and o v u l a t i o n under the i n f l u e n c e of a l t e r e d s t e r o i d hormone l e v e l s i n d u c e d by a sup erovul ato ry dose of PMSG (Yun et a l . , 1989 ). However, t r a n s -f e r of f r e s h l y o v u l a t e d o o c y t e s from s u p e r o v u l a t e d r a t s to normal r e c i p i e n t s l e a d s t o normal embryonic and f e t a l d e v e l -opment (Walton and Armstrong, 1983). T h e r e f o r e , adequate time and exposure t o g o n a d o t r o p h i n s and s t e r o i d hormones f o r oocyte m a t u r a t i o n e v i d e n t l y o c c u r s i n the s u p e r o v u l a t e d r a t . C. Chromosomal A b n o r m a l i t i e s Abnormal m e i o t i c m a t u r a t i o n may l e a d to chromosomal anomalies i n embryos from s u p e r o v u l a t e d immature r a t s (Yun et 34 a l . , 1989) . An i n c r e a s e i n chromosomal anomalies was 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 doses of PMSG i n the mouse, sheep and hamster (Chang, 1977; M a u d l i n and F r a s e r , 1977; Murray et a l . , 198 6; Sengoku and Dukelow, 1988; C a t a l a et a l . , 1988). Although an i n c r e a s e d i n c i d e n c e of a n e u p l o i d y was observed i n oocytes from s u p e r o v u l a t e d immature mice ( C a t a l a e t a l . , 1988), t h i s was not observed i n o t h e r s t u d i e s . In the mouse, e l e v a t e d e s t r a d i o l l e v e l s d e c r e a s e d p o l a r body one f o r m a t i o n in. v i t r o (Eppig and K o i d e , 1978). However, the m a j o r i t y of the chromosomal anomalies were r e l a t e d to i n t r a o v i d u c t a l oocyte aging p r i o r t o f e r t i l i z a t i o n which l e d to e r r o r s of f e r t i l i z -a t i o n such as f a i l u r e to extrude t h e second p o l a r body and polyspermy (Murray e t a l ., 1986; C a t a l a e t a l . , 1988 ). Examin-a t i o n of f e r t i l i z e d o o c y t e s from s u p e r o v u l a t e d immature r a t s r e v e a l e d no i n c r e a s e i n polyspermy (Walton et a l . , 1983 ). In one in. v i t r o f e r t i l i z a t i o n study, a s i g n i f i c a n t i n c r e a s e i n polyspermy was noted w i t h 24 IU PMSG but not w i t h other super-o v u l a t o r y doses (Evans and Armstrong, 1984). To d a t e , karyo-t y p i n g of oocytes from s u p e r o v u l a t e d immature r a t s has not been condue ted . 7. E f f e c t s of a S u p e r o v u l a t o r y Dose of PMSG on F e c u n d i t y A. General S u p e r o v u l a t i o n o f immature r a t s w i t h PMSG i s a s s o c i a t e d w i t h r e t r i e v a l of. i n c r e a s e d numbers of degenerated and abnormal 35 o o c y t e s ( M i l l e r and Armstrong, 1981a; Yun et a l . , 1987). S i n c e fragmented and c l e a v e d oocytes a r e o b s e r v e d i n f o l l i c l e s i n advanced stage s of a t r e s i a , i t was s u g g e s t e d t h a t the i n d u c t i o n of o v u l a t i o n of a t r e t i c f o l l i c l e s may l e a d to the appearance of these o o c y t e s d u r i n g s u p e r o v u l a t i o n (Braw and T s a f r i r i , 1980; Yun et a l . , 1987). However, due to the b i p h a s i c o v u l a t o r y p a t t e r n and the l a g p e r i o d between commencement of o v u l a t i o n and the appearance of abnormal o o c y t e s , i t was s u g g e s t e d t h a t these d e g e n e r a t i v e changes may be due t o i n t r a o v i d u c t a l oocyte aging ( M i l l e r and Armstrong, 1981a; Walton et a l . , 1983 ; Yun et a l . , 1987 ). P e r t u r b a t i o n of i n t r a f o l 1 i c u 1 a r s t e r o i d o g e n e s i s d u r i n g p r e o v u l a t o r y oocyte 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 immature r a t s may l e a d to o v u l a t i o n of abnormal oocytes (Yun e t a l . , 1987; Yun et a l . , 1988 ; Yun et a l ., 1989). The appearance of abnormal oocytes was a s s o c i a t e d t e m p o r a l l y w i t h a s i g n i f i c a n t r i s e i n androgens and e s t r a d i o l (Yun e t a l . , 1987; Yun e t a l . , 1988; Yun, 1989). A d m i n i s t r a t i o n of the a n t i a n d r o g e n , 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 o v a r i a n androgen and e s t r a d i o l l e v e l s and t h i s was a s s o c i a t e d w i t h improved post-ovu1 a t o r y v i a b i l i t y of o o c y t e s (Yun et a l . , 1988). B. F e r t i l i z a t i o n The f e r t i l i z a b l e l i f e of mammalian oocytes ranges from 6 to 15 hours (Yanagimachi and Chang, 1961; Adams and Chang, 1962; 36 Marston and Chang, 1964; Hunter, 1967; J u e t t e n and B a v i s t e r , 1983). With i n c r e a s i n g t i me between o v u l a t i o n and f e r t i l i z a t i o n ( i n t r a o v i d u c t a l oocyte aging) i n mammals, t h e r e i s a p r o g r e s s -i v e d e c l i n e i n normal embryonic development, i n c r e a s e d occur-rence of f e r t i l i z a t i o n e r r o r s , f a i l u r e of sperm head decondens-a t i o n and u l t i m a t e l y , f a i l u r e of sperm p e n e t r a t i o n (Odor and Blandau, 1956; Y a n a g i m a c h i and Chang, 1961; W i t s c h i and Laguens, 1963; Marston and Chang, 1964; Fugo and B u t c h e r , 1966; A u s t i n , 1967; B u t c h e r and Fugo, 1967; Hunter, 1967; Shaver and C a r r , 1967; Chang and Hunt, 1968; V i c k e r s , 1969;' Shaver and C a r r , 1969; A u s t i n , 1970; Meyer and Longo, 1979; J u e t t e n and B a v i s t e r , 1983; G i a n f o r t o n i and Gulyas, 1985). Oocyte a g i n g i s a s s o c i a t e d with l o s s of cumulus 18 to 24 hours (Yanagimachi and Chang, 1961; Longo, 1981; G i a n f o r t o n i and G u l y a s , 1985), and f r a g m e n t a t i o n more t h a n 24 hours (Odor and B l a n d a u , 1956; Marston and Chang, 1964; Hunter, 1967; S z o l l o s i , 1971; Meyer and Longo, 1979; Longo, 1980), a f t e r o v u l a t i o n . In immature s u p e r o v u l a t e d r a t s , o o c y t e s c o l l e c t e d 24 hours p o s t - t r e a t m e n t were cu m u l u s - e n c l o s e d and of normal appearance when the cumulus was removed (Yun et a l . , 1987). By 3 6 to 48 hours, i n c r e a s e d numbers of cumulus-free and fragmented oocytes were r e t r i e v e d , w h i c h d e c l i n e d p r o p o r t i o n a t e l y a t 72 hours p o s t - t r e a t m e n t i n a s s o c i a t i o n w i t h the second wave of o v u l a t i o n ( M i l l e r and Armstrong, 1981a; Walton et a l . , 1983; Yun et a l . , 1987). F e r t i l i z a t i o n r a t e s i n c u m u l u s - f r e e o o c y t e s were s i g -37 n i f i c a n t l y reduced compared w i t h cumulus-enclosed o o c y t e s (Walton and Armstrong, 1983). The poor f e r t i 1 i z a b i 1 i t y of oo c y t e s was thus a t t r i b u t e d t o p o s t - o v u l a t o r y aging ( M i l l e r and Armstrong, 1981a; Walton and Armstrong, 1983; Walton et a l . , 198 3; Yun et a l ., 198 7) . An i n v e r s e dose response r e l a t i o n s h i p i s observed between PMSG and f e r t i l i z a t i o n r a t e i n immature r a t s (Walton and Armstrong, 1983; Walton et a l . , 1983; L e v e i l l e and Armstrong, 1989). The major cause of in. v i v o f e r t i l i z a t i o n f a i l u r e i n immature s u p e r o v u l a t e d r a t s i s asynchrony of o v u l a t i o n and mating. When immature r a t s were t r e a t e d with 4 or 40 IU PMSG and exposed t o males 54 hours p o s t - t r e a t m e n t , mating r a t e s of 67% and 77%, r e s p e c t i v e l y , were observed ( M i l l e r and Armstrong, 1982). When r a t s t r e a t e d w i t h 40 IU PMSG were exposed to males 30 hours p o s t - t r e a t m e n t , o n l y 21% mated ( M i l l e r and Armstrong, 1982). S i m i l a r l y , i n mice, s u p e r o v u l a t i on with PMSG d i d not s t i m u l a t e mating behaviour c o n c u r r e n t w i t h e a r l y o v u l a t i o n s ( S t e r n and Schuetz, 1970). T h i s i s l i k e l y due t o in a d e q u a t e e s t r a d i o l l e v e l s to induce e s t r u s i n t h e m a j o r i t y of super-o v u l a t e d immature r a t s on day 1 ( B o l i n g and Blandau, 1939; Walton et a l . , 1983). S i n c e r a t s mate i n a s s o c i a t i o n w i t h the second wave of o v u l a t i o n , those o o c y t e s r e l e a s e d i n the f i r s t wave w i l l be too aged to undergo f e r t i l i z a t i o n (Walton et a l . , 1983; L e v e i l l e and Armstrong, 1989). A d m i n i s t r a t i o n of a combi-n a t i o n of FSH/HCG w i t h lower LH a c t i v i t y than PMSG r e s u l t e d i n 38 a s i g n i f i c a n t i n 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 , presumably due to abolishment of the f i r s t wave of o v u l a t i o n ( L e v e i l l e and Armstrong, 1989). In t r a n s f e r s t u d i e s , no r e d u c t i o n i n f e r t i l i t y or f e t a l development was found i n cumu1 us-enc1osed o o c y t e s r e t r i e v e d i n a s s o c i a t i o n w i t h e i t h e r the f i r s t or second wave of o v u l a t i o n (Walton and Armstrong, 1983). A s i g n i f i c a n t l y lower f e r t i l i z -a t i o n r a t e was observed i n eggs r e t r i e v e d at 42 hours, which were presumably aged (Walton and Armstrong, 1983). On a p r o p o r t i o n a l b a s i s , aging of o o c y t e s from the f i r s t wave of o v u l a t i o n d i d not c o m p l e t e l y account f o r the d e c r e a s e d f e r t i -l i z a t i o n r a t e observed i n one in. v i t ro s t u d y , prompting the aut h o r s to suggest t h a t abnormal o o c y t e s p r o b a b l y a l s o account f o r a p o r t i o n of the f e r t i l i z a t i o n f a i l u r e (Evans and Arm-s t r o n g , 1984). However, t h e r e i s g r e a t v a r i a b i l i t y i n o v u l a t i o n time between s u p e r o v u l a t e d r a t s , and s i n c e g r o s s morphology i s not a r e l i a b l e i n d i c a t o r of p o s t - o v u l a t o r y age, aged o o c y t e s without any g r o s s a l t e r a t i o n c o u l d account f o r t h i s d i s c r e p a n c y (Walton e t a l . , 1983; Evans and Armstrong, 1984). Delayed f e r t i l i z a t i o n was a s s o c i a t e d w i t h a p r o g r e s s i v e l o s s of p o t e n t i a l f o r normal embryonic development (Yanagimachi and Chang, 1961; Marston and Chang, 1964; Maurer et a l . , 1969; Thompson and Zamboni, 1975; U s u i and Yanagimachi, 1976; G i a n -f o r t o n i and Gulyas, 1985). T h i s was p a r t l y due to l o s s of c y t o -p l a s m i c f a c t o r s c r i t i c a l f o r development and f u n c t i o n o f the 39 male p r o n u c l e u s (Niwa and Chang, 1975; J u e t t e n and B a v i s t e r , 1983; T e s a r i k and Kopecny, 1989). It was noted d u r i n g i n v i v o f e r t i l i z a t i o n t h a t s i g n i f i c a n t l y fewer p e n e t r a t e d ova from the s u p e r o v u l a t e d group d e v e l o p e d p r o n u c l e i (Walton et a l . , 1983). Male p r o n u c l e a r growth f a c t o r i s s y n t h e s i z e d 4 hours a f t e r the LH surge and i s l o s t 10 t o 12 hours p o s t o v u l a t i o n i n the r a t (Niwa and Chang, 1975). T h e r e f o r e , t h i s d i f f e r e n c e c o u l d be due to a g i n g , i n c o m p l e t e oocyte m a t u r a t i o n , o r s i m p l y the presence of a number of f e r t i l i z e d o ocytes w h i c h had y e t to undergo p r o n u c l e u s f o r m a t i o n (Walton e t a l . , 1983). Oocytes from s u p e r o v u l a t e d r a t s f e r t i l i z e d in. v i t ro a l l d e v e l o p e d n o r m a l l y to the 2 c e l l s t age (Evans and Armstrong, 1984) s u g g e s t i n g t h a t i f oocytes from s u p e r o v u l a t e d r a t s a r e f e r t i l i z e d they w i l l undergo f i r s t c l e a v a g e . C. Embryonic Development S u p e r o v u l a t i o n of immature r a t s w i t h PMSG a d v e r s e l y a f f e c t s embryonic development ( C o l e 1936; A u s t i n 1950). S i m i l a r l y , i n mice, an i n v e r s e r e l a t i o n s h i p was n o t e d between dose of PMSG and embryonic development (Edgar et a l . , 1987). Numerous s t u d i e s have i n v e s t i g a t e d the mechanism by which h i g h doses of PMSG i n t e r f e r e w i t h embryonic development i n . the r a t . S i n c e t r a n s f e r s t u d i e s demonstrated t h a t s u c c e s s f u l f e t a l development c o u l d be a c h i e v e d i n f r e s h l y o v u l a t e d o o c y t e s or embryos from s u p e r o v u l a t e d immature r a t s i f they developed i n a normal 40 r e c i p i e n t (Walton and Armstrong, 1983; Walton e t a l . , 1982), a t t e n t i o n was d i r e c t e d toward the maternal environment. E s t r a d i o l and androgen l e v e l s n o r m a l l y r i s e on days 1 to 4 of pregnancy i n the r a t , l i k e l y due to p r e o v u l a t o r y development of f o l l i c l e s r e c r u i t e d by the FSH surge at e s t r u s (Osman, 1986; L e v e i l l e and Armstrong, 1989) or from c o r p o r a l u t e a ( G i b o r i et a l . - , 1978). E s t r a d i o l has b o t h a n i d a t o r y and s t i m u l a t o r y e f f e c t , on embryonic development (Sanyal and Meyer, 1970; Jacobson et a l . , 1970). However, 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 are s i g n i f i c a n t l y h i g h e r than normal, and have a d e l e t e r i o u s e f f ect . In r a t s s u p e r o v u l a t e d w i t h PMSG, e l e v a t e d e s t r a d i o l , androgen and p r o g e s t e r o n e l e v e l s p e r s i s t from day 1 to 4 of pregnancy ( M i l l e r and Armstrong, 1981a; Walton and Armstrong, 1981; Yun e t a l . , 1988; L e v e i l l e and Armstrong, 1989 ). T h i s i s a s s o c i a t e d w i t h l o s s of embryos from the r e p r o d u c t i v e t r a c t , d e c l i n i n g q u a l i t y of embryos, embryonic d e v e l o p m e n t a l r e t a r d a -t i o n and f a i l u r e o f i m p l a n t a t i o n ( M i l l e r and Armstrong, 1981a, b; Walton and Armstrong, 1981; M i l l e r and Armstrong, 1982; Walton and Armstrong, 1982; Walton et a l . , 1982; Sherman et a l . , 1982 ; Yun et a l . , 1988 ; L e v e i l l e and Armstrong, 1989 ). In the r a t , e l e v a t e d e s t r a d i o l l e v e l s and a l t e r a t i o n of the e s t r a d i o l / p r o g e s t e r o n e r a t i o l e a d t o r a p i d l o s s of embryos from the r e p r o d u c t i v e t r a c t (Greenwald, 1961; Greenwald, 1967; Wu et a l . , 1971; O r t i z e t a l . , 1979; Sherman et a l . , 1982). Immature 41 r a t s t r e a t e d w i t h 40 IU PMSG a l s o e x p e r i e n c e r a p i d l o s s of embryos from t h e r e p r o d u c t i v e t r a c t (Walton and Armstrong, 1982; L e v e i l l e and Armstrong, 1989). Ovariectomy or i n h i b i t i o n of s t e r o i d o g e n e s i s d e c r e a s e e s t r a d i o l 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 and i n c r e a s e t h e number of embryos r e t a i n e d ( M i l l e r and Armstrong, 1981b; Walton and Armstrong, 1981; M i l l e r and Armstrong, 1982; Walton et a l . , 1982; Sherman et a l . , 1984). Numerous s t u d i e s i n the mouse (Wu et a l . , 1971; K i r k p a t r i c k , 1971; C l i n e e t a l . , 1977; R o b l e r o et a l . , 1983) and r a b b i t (Greenwald, 1963; D a n i e l , 1964; McGaughey and D a n i e l , 1966; Chang and Harper, 1966; Stone e t al . , 1977 ) have demonstrated t h a t e s t r a d i o l and p r o g e s t e r o n e i n pr o p e r b a l a n c e are r e q u i r e d f o r normal embryonic development. When the l e v e l of e s t r a d i o l i s i n c r e a s e d d u r i n g e a r l y development, r e t a r d e d development and f r a g m e n t a t i o n o c c u r i n d e p e n d e n t l y of im p l a n t a -t i o n f a i l u r e . E l e v a t e d e s t r a d i o l l e v e l s i n the r a t were a s s o c i a t e d w i t h r e t a r d e d embryonic development and the ap-pearance of abnormal embryos ( B u t c h e r and Pope, 1979). T h i s e f f e c t was l i k e l y m ediated i n d i r e c t l y by a l t e r a t i o n of o v i d u c t a l e p i t h e l i a l p r o t e i n s y n t h e s i s and, thus, o v i d u c t a l f l u i d c o m p o s i t i o n ( C l i n e et a l ., 1977; Stone et a l . , 1977 ). P r o g r e s s i v e d e g e n e r a t i o n of embryos o c c u r s i n s u p e r o v u l a t e d immature r a t s from day 1 t o 3 of pregnancy (Sherman et a l . , 1982). By r e d u c i n g LH a c t i v i t y d u r i n g s u p e r o v u l a t i o n , e s t r a d i o l and androgen l e v e l s were s i g n i f i c a n t l y lowered and t h i s was 42 a s s o c i a t e d w i t h a marked i n c r e a s e i n the number of n o r m a l l y d e v e l o p i n g embryos ( L e v e i l l e and Armstrong, 1989). However, abolishment of the f i r s t wave of o v u l a t i o n presumably o c c u r r e d w i t h the r e d u c t i o n i n LH a c t i v i t y and t h i s would a l s o improve embryo development by m i n i m i z i n g the e f f e c t of p o s t - o v u l a t o r y oocyte aging ( L e v e i l l e and Armstrong, 1989). D. I m p l a n t a t i o n High doses of PMSG (40 IU) cause r a p i d l o s s of embryos from the r e p r o d u c t i v e t r a c t and f a i l u r e of i m p l a n t a t i o n (Walton and Armstrong, 1982). Both e s t r a d i o l and p r o g e s t e r o n e a r e r e q u i r e d i n a d e l i c a t e b a l a n c e to i n d u c e n i d a t i o n (Yochim and De Feo, 1963). E s t r a d i o l i s r e q u i r e d to s e n s i t i z e the u t e r u s to the n i d a t o r y e f f e c t of p r o g e s t e r o n e ( K r a i c e r et a l . , 1989). If e s t r a d i o l i s i n c r e a s e d , d e c r e a s e d s e n s i t i v i t y of the endometrium t o the n i d a t o r y s i g n a l r e s u l t s (Yochim and De Feo, 1963). While the a b s o l u t e v a l u e of e s t r a d i o l a f f e c t s s e n s i t i v -i t y of the endometrium to the n i d a t o r y s i g n a l , the d e c i d u a l r e a c t i o n i t s e l f depends on a p r o p e r r a t i o of e s t r a d i o l / p r o g e s t e r o n e (Yochim and De Feo, 1963). E l e v a t e d e s t r a d i o l i n h i b i t s i m p l a n t a t i o n and t h e d e c i d u a l r e a c t i o n i n the r a t (Greenwald, 1961; Butcher and Pope, 1979). Reduction of the e l e v a t e d e s t r a d i o l l e v e l i n s u p e r o v u l a t e d r a t s by o v a r i e c t o m y or a d m i n i s t r a t i o n of anti-PMSG s i g n i f i c a n t l y i n c r e a s e d the number of i m p l a n t e d embryos and t h i s b e n e f i c i a l 43 e f f e c t was r e v e r s e d by a d m i n i s t r a t i o n of e s t r o g e n ( M i l l e r and Armstrong, 1982; Walton et a l . , 1982). I m p l a n t a t i o n f a i l u r e was a l s o a t t r i b u t e d to abnormal b l a s t o c y s t development (Walton and Armstrong, 1982). A l a r g e dose of e s t r a d i o l may i n h i b i t d e c i d u a l i z a t i o n by d i s r u p t i o n of a u t e r i n e r e s p o n s e e l i c i t e d by the b l a s t o c y s t ( K r a i c e r et a l . , 1989). In the immature mouse s u p e r o v u l a t e d w i t h PMSG, reduced numbers of m i c r o v i l l i were observed on b l a s t o c y s t s and i t was suggested t h a t t h i s would i n t e r f e r e w i t h i m p l a n t a t i o n (Champlin et a l . , 1987). However, i n the r a t , i t was shown t h a t b l a s t o c y s t s t r a n s f e r r e d to a normal r e c i p i e n t were capable of normal f e t a l development (Walton et a l . , 1982). Delayed i m p l a n t a t i o n , as e v i d e n c e d by low b i r t h weights, was observed i n immature r a t s t r e a t e d w i t h 16 IU PMSG alone or 40 IU f o l l o w e d by anti-PMSG ( M i l l e r and Armstrong, 1981b; Walton and Armstrong, 1982). I t was concluded t h a t p r e v i o u s exposure t o e l e v a t e d e s t r a d i o l l e v e l s l e d to reduced s e n s i t i v -i t y to the n i d a t o r y s t i m u l u s and a d e l a y i n d e c i d u a l i z a t i o n ( M i l l e r and Armstrong, 1981b). D e s p i t e t h e l a r g e number of o v u l a t i o n s induced by a s u p e r o v u l a t o r y dose of PMSG, l i t t e r s i z e i s not c o r r e s p o n d i n g l y i n c r e a s e d ( A u s t i n , 1950; M i l l e r and Armstrong, 1981b; Sherman et a l . , 1982; Sherman e t a l . , 1984). The number of i m p l a n t a t i o n s i t e s i n s u p e r o v u l a t e d immature r a t s a r e g r e a t e r than the number of l i v e b o r n s u g g e s t i n g t h a t p o s t - i m p l a n t a t i o n l o s s a l s o 44 a c c o u n t s f o r a s i g n i f i c a n t p r o p o r t i o n of the embryonic wastage ob s e r v e d (Sherman et a l . , 1982; Sherman et a l . , 1984 ). T h i s may be due to the wide spectrum of i n d i v i d u a l r e s ponse t o superovu-l a t i o n (Walton et a l . , 1982 ), s i n c e r a t s w i t h h i g h and low p o t e n t i a l f o r l a r g e l i t t e r s were observed (Sherman e t a l . , 1982). A l t e r n a t i v e l y , i t was s u g g e s t e d t h a t the low number of l i v e b o r n was dues to l i m i t e d c a r r y i n g c a p a c i t y of the ute r u s ( M i l l e r and Armstrong, 1981b). However, l i t t e r s i z e was i n c r e a s e d to 30 f e t u s e s by a d m i n i s t r a t i o n of a FSH/HCG m i x t u r e which c o n t a i n e d s i g n i f i c a n t l y lower LH a c t i v i t y than PMSG ( L e v e i l l e and Armstrong, 1989). T h i s was a s s o c i a t e d w i t h lowered e s t r a d i o l and androgens l e v e l s ( L e v e i l l e and Armstrong, 1989) s u g g e s t i n g t h a t the e f f e c t s o f a l t e r e d s t e r o i d o g e n e s i s i n immature r a t s s u p e r o v u l a t e d w i t h PMSG extend i n t o the p o s t -i m p l a n t a t i o n p e r i o d . 8. Summary In summary, s i g n i f i c a n t d i f f e r e n c e s i n p r e v a i l i n g hormone p a t t e r n s and f o l l i c u l a r s t a t u s e x i s t between immature and mature r a t s . The q u a l i t a t i v e and q u a n t i t a t i v e d i f f e r e n c e s between f o l l i c l e s of immature and mature r a t s l e a d to d i f f e r e n t r e sponses to PMSG s t i m u l a t i o n and f a c i l i t a t e the s u p e r o v u l a t o r y response i n immature r a t s . A low dose of PMSG a d m i n i s t e r e d to immature r a t s l e a d s to an e n d o c r i n e and o v u l a t o r y r e s p o n s e s i m i l a r but not i d e n t i c a l 45 to t h a t observed i n the mature r a t at p r o e s t r u s and e s t r u s . The most mature f o l l i c l e s i n the ovary are s t i m u l a t e d t o grow and d i f f e r e n t i a t e over a 48 to 60 hour p e r i o d , l e a d i n g t o o v u l a t i o n i n response to an endogenous g o n a d o t r o p h i n s u r g e . These r a t s w i l l mate and c a r r y a normal pregnancy t o term i l l u s t r a t i n g t h a t the im m a t u r i t y of the hypothalamo-hypophyseal a x i s i n immature r a t s s i g n i f i c a n t l y a f f e c t s o n l y t h e i n i t i a t i o n of c y c l i c r e p r o d u c t i v e a c t i v i t y . High doses of PMSG d i r e c t l y t r i g g e r o v u l a t i o n of the most mature f o l l i c l e s , w i t h an a t t e n u a t e d p e r i o d of growth and d i f f e r e n t i a t i o n , and i n d u c e r a p i d growth and d i f f e r e n t i a t i o n of s m a l l e r f o l l i c l e s which then o v u l a t e i n r e s p o n s e to an endoge-nous g o n a d o t r o p h i n surge. S u p e r o v u l a t o r y doses of PMSG have e x c e s s i v e LH a c t i v i t y , and, wit h the p r o l o n g e d p e r i o d of s t i m u l a t i o n , l e a d t o r a d i c a l l y a l t e r e d s t e r o i d o g e n e s i s and a cascade of p a t h o p h y s i o l o g i c a l e v e n t s : asynchrony of mating and o v u l a t i o n and d y s f u n c t i o n of embryo t r a n s p o r t , embryonic development, i m p l a n t a t i o n and f e t a l development. However, even w i t h t h i s marked a l t e r a t i o n i n f o l l i c u l a r m a t u r a t i o n and hormone p r o f i l e , o o c y t e s and embryos from s u p e r o v u l a t e d r a t s w i l l i m p l a n t and produce l i v e b o r n i f t r a n s f e r r e d to normal animals. A b o l i s h m e n t o f the e 1 e v a t e d A S t e r o i d hormone l e v e l s and the f i r s t wave of o v u l a t i o n d u r i n g s u p e r o v u l a t i o n , by r e d u c i n g both the l e v e l o f LH a c t i v i t y and the p r o l o n g e d p e r i o d of 46 s t i m u l a t i o n , improves f e r t i l i t y and l e a d s to a s i g n i f i c a n t i n c r e a s e i n the number of l i v e b o r n . C. RATIONALE AND OBJECTIVES Oocytes and embryos undergo s p e c i f i c , p r e d e t e r m i n e d changes i n morphology d u r i n g m a t u r a t i o n , f e r t i l i z a t i o n and e a r l y embryonic development, and these a r e c o r r e l a t e d w i t h changes i n metabolism ( A l b e r t i n i , 1987; Moor and G a n d o l f i , 1987). S i n c e f u n c t i o n a l changes a r e r e f l e c t e d by s t r u c t u r a l changes (Cech and Sedlackova, 1983; C h e v i l l e , 1983), a b n o r m a l i t i e s of oocyte m a t u r a t i o n , f e r t i l i z a t i o n and embryonic development i n immature r a t s s u p e r o v u l a t e d w i t h PMSG would be e v i d e n c e d by a l t e r e d c e l l u l a r morphology. A m o r p h o l o g i c a l s t u d y of o o c y t e s and embryos from immature r a t s s u p e r o v u l a t e d w i t h PMSG would thus r e v e a l the n a t u r e of these a b n o r m a l i t i e s and t h e e f f e c t s on embryonic development. The o b j e c t i v e of t h i s i n v e s t i g a t i o n was to more c l e a r l y d e f i n e the adverse e f f e c t s of a s u p e r o v u l a t o r y dose of PMSG on oocyte m a t u r a t i o n , f e r t i l i z a t i o n and embryonic development i n the immature r a t . Four experiments were conducted t o d e t e r m i n e , on a m o r p h o l o g i c a l b a s i s , whether s u p e r o v u l a t i o n of immature r a t s w i t h PMSG le a d s to: 1) i n t r a o v i d u c t a l o o c y t e a g i n g 2) o v u l a t i o n of abnormal o o c y t e s 47 3) f e r t i l i z a t i o n f a i l u r e of abnormal o o c y t e s 4) abnormal or d e l a y e d embryonic development. In Experiment 1, t h e p r o g r e s s i v e m o r p h o l o g i c a l changes which occur d u r i n g i n t r a - o v i d u c t a 1 o o c y t e a g i n g i n the r a t were documented i n or d e r to e s t a b l i s h m o r p h o l o g i c a l c r i t e r i a f o r the subsequent a n a l y s i s of o o c y t e s and embryos. The o b j e c t i v e of Experiment 2 was t o deter m i n e whether s u p e r o v u l a t i o n of immature r a t s w i t h PMSG l e a d s to o v u l a t i o n of immature or abnormal o o c y t e s . The o b j e c t i v e of Experiment 3 was t o de t e r m i n e whether s u p e r o v u l a t e d immature r a t s have a reduced f e r t i l i z a t i o n r a t e and, i f so, to determine whether the r e d u c t i o n i n f e r t i l i z a t i o n was due t o i n t r a o v i d u c t a l a g i n g and/or the pres e n c e of immature or abnormal o o c y t e s . In Experiment 4, t h e o b j e c t i v e was to determine the r e l a t i v e impact of i n t r a o v i d u c t a l oocyte a g i n g , d e l a y e d f e r t i l i z a t i o n and abnormal oo c y t e m a t u r a t i o n on embryonic development. S i n c e f o l 1 i c 1 e s and o o c y t e s from immature r a t s were shown to be q u a l i t a t i v e l y d i f f e r e n t from t h o s e o f mature r a t s ( K a p l a n et a l . , 1977; H i r s h f i e l d , 1986a), o o c y t e s and embryos from immature PMSG-treated r a t s were compared w i t h oocytes and embryos from mature, c y c l i n g r a t s . T h i s would p r o v i d e f u r t h e r i n s i g h t i n t o any a b n o r m a l i t i e s i n d u c e d by treatment of immature r a t s w i t h PMSG. 48 MATERIALS AND METHODS 1. Animals Immature (22 days o l d ) and mature (3-6 months o l d ) female and mature, f e r t i l i t y - p r o v e n , male Sprague-Dawley r a t s were obtained from Charles R i v e r Canada Inc (S t . Constant, Quebec). The r a t s were kept at 20-25° c e n t i g r a d e (C) on a 12 hours l i g h t : 12 hours dark c y c l e and were fed standard r a t chow and water ad l i b i t u m . At 28 days of age, e i t h e r a low (4 IU) or s u p e r o v u l a t o r y (40 IU) dose of PMSG (Equinex, A y e r s t , M o n t r e a l , Quebec), prepared from three separate batches of the commercial gonadotrophin, was adm i n i s t e r e d subcutaneously i n 0.4 m i l l i l i t r e (ml) s a l i n e to the immature r a t s between 8:00 and 9:00. A v a g i n a l smear was taken d a i l y from each mature r a t between 8:00 and 1:000 to e s t a b l i s h the stage of the es t r u s c y c l e . Mature r a t s with three c o n s e c u t i v e f o u r day e s t r u s c y c l e s were s e l e c t e d f o r the study. A l l r a t s were s a c r i f i c e d by ether overdose. F o l l o w i n g s a c r i f i c e , the o v a r i e s , o v i d u c t and uterus were removed from each rat and placed i n Dulbecco's phosphate b u f f e r e d s a l i n e (DPBS;pH 7.3). A f t e r s e p a r a t i n g the o v i d u c t s from the o v a r i e s and uterus with the a i d of a s t e r e o d i s s e c t i n g microscope, the oocytes and embryos were r e t r i e v e d from the o v i d u c t by i n s e r t i n g a b l u n t 30 gauge needle i n t o the infundibulum and f l u s h i n g w i t h 0.3-0.5 ml DPBS c o n t a i n i n g 1% bovine serum albumin (BSA) (Sigma Chemical Co., St. L o u i s , MO) i n t o a 10x35 m i l l i m e t r e (mm) p e t r i d i s h . 49 2. Experimental Design A. Part 1 - Mor p h o l o g i c a l I n v e s t i g a t i o n of I n t r a o v i d u c t a l Aging i n the Mature Rat Mature female r a t s were s a c r i f i c e d at 9:00, 12:00, 18:00 and 24:00 of estrus and at 9:00 of metestrus, d i e s t r u s and p r o e s t r u s . The ampullae were assessed f o r d i s t e n s i o n and the presence of an egg mass. On the morning of p r o e s t r u s , the u t e r i n e horns were separated, a bl u n t 23 gauge needle was i n s e r t e d and 1 ml of DPBS c o n t a i n i n g 1% BSA was f l u s h e d through each horn i n t o a 10x35 mm p e t r i d i s h . B. Part 2 - Mo r p h o l o g i c a l I n v e s t i g a t i o n of Post-Ovulatory Oocyte Q u a l i t y i n Immature, PMSG-Treated Rats and Mature Rats Immature r a t s were s a c r i f i c e d between 8:00 and 1:000 at 0, 24, 48, 72 and 96 hours (hr) post-treatment with e i t h e r 4, or 40, IU PMSG. Mature r a t s were s a c r i f i c e d between 9:00 and 1:000 of e s t r u s . C. Parts 3 and 4 - M o r p h o l o g i c a l I n v e s t i g a t i o n of the Causes of F e r t i l i z a t i o n F a i l u r e and Abnormal Embryo Development i n Immature Rats Superovulated with PMSG At 30 days of age, the vaginae of the immature r a t s were gen t l y opened with a s m a l l , heat p o l i s h e d , s a l i n e - s o a k e d g l a s s probe. Immature and mature females were placed with male r a t s 55-56 hr f o l l o w i n g a d m i n i s t r a t i o n of e i t h e r 4, or 40, IU PMSG or at 15:00 of p r o e s t r u s . The mating r a t i o was two females to one male. 50 Immature females which r e c e i v e d the same1 dose of PMSG were placed together with the same male. The females were removed between 8:00 and 9:00 the f o l l o w i n g morning (day 1 of pregnancy) and checked f o r the presence of a v a g i n a l plug and/or semen i n a vag i n a l smear. Females with evidence of mating were s a c r i f i c e d between 9:00 and 12:00 on day 1 of pregnancy (Part 3) and between 9:00 and 15:00 on days 1, 2, 3 and 4 of pregnancy (Part 4). 3. Oocyte and Embryo R e t r i e v a l and Gross Assessment A. P a r t s 1 and 2 - Morphological I n v e s t i g a t i o n of I n t r a o v i d u c t a l Oocyte Aging i n Mature Rats and P o s t - O v u l a t o r y Oocyte Q u a l i t y i n Immature, PMSG-Treated Rats and Mature Rats The oocytes recovered from each r a t were counted and the gross morphology of the cumulus on each oocyte was assessed with a s t e r e o d i s s e c t i n g microscope at 40x m a g n i f i c a t i o n . The cumulus mass was c o n s i d e r e d to be good i f the c e l l s surrounding the oocyte were dense, and evenly d i s t r i b u t e d and the oocyte was b a r e l y v i s i b l e w i t h i n the mass. If the oocyte was p l a i n l y v i s i b l e w i t h i n a mass c o n s i s t i n g of a reduced number of d i s p e r s e d and clumped c e l l s , i t was c o n s i d e r e d to be f a i r . The absence of a cumulus mass was recorded. Cumulus-enclosed oocytes were l e f t i n t a c t . The q u a l i t y of cumulus-free oocytes was judged to be good i f one large r e t r a c t i l e c e l l , round to s l i g h t l y e l l i p s o i d a l i n shape, f i l l e d most of the space w i t h i n the zona p e l l u c i d a and had a homogeneous, f i n e l y g r a n u l a r content. An oocyte was c o n s i d e r e d fragmented i f there were two or more c e l l fragments, both s m a l l e r than the o r i g i n a l c e l l , 51 with a l a r g e p e r i v i t e l 1 i n e space. If the s i n g l e c e l l was markedly reduced i n s i z e with a corresponding l a r g e p e r i v i t e l 1 i n e space or i f the c e l l had i l l - d e f i n e d c e l l borders with opaque cytoplasm, i t was considered degenerated. B. Parts 3 and 4 - Mor p h o l o g i c a l I n v e s t i g a t i o n of the Causes of F e r t i l i z a t i o n F a i l u r e and Abnormal Embryo Development i n Immature Rats Superovulated with PMSG The number of embryos r e t r i e v e d from each r a t was recorded and embryos were assessed f o r stage of development under a stereo d i s s e c t i n g microscope at 80x m a g n i f i c a t i o n . On day 1 of pregnancy, oocytes were p l a c e d i n DPBS c o n t a i n i n g 2 drops of 0.2% hyalur-onidase (Ovine Type I I , Sigma Chemical Co., St. L o u i s , MO) f o r 5-10 minutes to d i s p e r s e any remaining cumulus mass. The oocytes were then assessed as d e s c r i b e d above f o r Parts 1 and 2. Oocytes were considered to be f e r t i l i z e d i f a second p o l a r body was present i n the p e r i v i t e l 1 i n e space. Embryos were c o n s i d e r e d to be of good q u a l i t y i f the blastomeres occupied most of the space w i t h i n the zona p e l l u c i d a and were of approximately equal s i z e , with well demarcated c e l l borders and r e t r a c t i l e , f i n e l y g ranular cytoplasm. Good embryos at the two and four c e l l stage u s u a l l y had one or two p o l a r bodies present i n the p e r i v i t e l 1 i n e space. Embryos were co n s i d e r e d to be of poor q u a l i t y i f th e r e was v a r i a t i o n i n the s i z e of c e l l s , i f there were one or more opaque, poorly d e f i n e d c e l l s w i t h i n the c e l l mass or i f there was accumulation of c e l l d e b r i s and small fragments i n the p e r i v i t e l 1 i n e space. Embryos were considered 52 fragmented i f they c o n s i s t e d of m u l t i p l e c e l l fragments with marked v a r i a t i o n i n s i z e and shape. U n f e r t i l i z e d oocytes (Part 3) and a l l oocytes and embryos (Part 4) were prepared f o r l i g h t and e l e c t r o n microscopy. 4. L i g h t and E l e c t r o n Microscopy A. Parts 1, 3 and 4 - Morphological I n v e s t i g a t i o n of I n t r a o v i d u c t a l Oocyte Aging i n the Mature Rat and the Causes of F e r t i l i z a t i o n F a i l u r e and Abnormal Embryo Development i n Immature Rats Superovulated with PMSG Immediately f o l l o w i n g r e t r i e v a l and gross assessment, the oocytes and embryos were p l a c e d i n 2.5% gl u t a r a l d e h y d e (Ted Pel l a , Redding, CA) i n 0.1 molar Sorenson's phosphate b u f f e r (GA;pH 7.4) fo r one hour. The oocytes and embryos were washed twice i n 0.1 molar Sorenson's phosphate b u f f e r (PB;pH 7.4) f o l l o w i n g GA f i x a t i o n , t r a n s f e r r e d with a 10 ul heat p o l i s h e d m i c r o p i p e t t e to a depression s l i d e c o n t a i n i n g 10% BSA i n Dulbecco's. phosphate b u f f e r e d s a l i n e (BSAD) and then t r a n s f e r r e d to a d u s t - f r e e Beem capsule (J.B. EM, Do r v a l , Quebec) c o n t a i n i n g one drop of BSAD. The oocytes and embryos were l e f t f o r 30 minutes to s e t t l e on the bottom of the cap s u l e s . Any specimens adherent to the s i d e s of the capsules were g e n t l y d i s l o d g e d with the cl o s e d end of a heat p o l i s h e d m i c r o p i p e t t e . When the specimens were s i t u a t e d on the bottom of the cap s u l e s , they were c e n t r i f u g e d h o r i z o n t a l l y f o r 15 minutes at 1800 g ( r e l a t i v e c e n t r i f u g a l f o r c e ) . F o l l o w i n g c e n t r i f u g a t i o n , 3 drops of GA were added to each capsule which were then c e n t r i f u g e d as d e s c r i b e d f o r an a d d i t i o n a l 60 minutes. The 53 capsules were f i l l e d with GA and r e f r i g e r a t e d at 4° C o v e r n i g h t . The f o l l o w i n g morning, the GA i n each capsule was poured o f f and the top h a l f of the ca p s u l e s removed with a s c a l p e l b l ade. The capsules were then observed at lOx m a g n i f i c a t i o n while the BSAD molds c o n t a i n i n g the oocytes and embryos were removed by running the f i n e end of a heat p o l i s h e d m i c r o p i p e t t e around the top edge and then down one corner on the s i d e of the mold o p p o s i t e to the oocytes. With g e n t l e p r e s s u r e , the molds were l i f t e d out wit h the heat p o l i s h e d m i c r o p i p e t t e and t r a n s f e r r e d to g l a s s v i a l s . The samples were washed twice i n PB, p o s t - f i x e d i n 1% osmium t e t r o x i d e (Marivac, H a l i f a x , Nova S c o t i a ) i n PB, dehydrated i n a graded s e r i e s of acetone ( F i s h e r S c i e n t i f i c , F a i r Lawn, NJ), i n f i l t r a t e d with Spurrs epoxy r e s i n (J.B. EM, Dor v a l , Quebec) and c a r e f u l l y r e a l i g n e d i n Beem capsules (J.B. EM, D o r v a l , Quebec) c o n t a i n i n g 3 drops of r e s i n . The capsules were f i l l e d with r e s i n and polymerized at 60°C f o r 18 hours. S e r i a l s e c t i o n s of oocytes and embryos were cut at 0.5 to 1.0 micrometre (um) on an LKB Ultratome (LKB, Bromma, Sweden), heat f i x e d to a glass s l i d e , s t a i n e d with 1% Methylene Blue (BDH Chemicals, Toronto, Ontario) i n 1% borax (BDH Chemicals, Toronto, Ontario) and examined at 400x m a g n i f i c a t i o n . Thin (60-90 nanometre (nm)) s e c t i o n s were taken at 8 to 10 um i n t e r v a l s a f t e r l o c a t i n g the oocytes i n the r e s i n b l o c k , mounted on u l t r a h i g h t r a n s m i s s i o n copper g r i d s (J.B. EM, D o r v a l , Quebec) and s t a i n e d with u r a n y l acetate (Ted P e l l a , Redding, CA) and lead c i t r a t e (Reynolds, 1963) fo r twenty minutes each. The t h i n s e c t i o n s were examined on a Z e i s s EM10C e l e c t r o n microscope ( C a r l Z e i s s , West Germany). 54 B. Part 2 - Morphological I n v e s t i g a t i o n of Post-Ovulatory Oocyte Q u a l i t y i n Immature, PMSG-Treated Rats and Mature Rats The oocytes were suspended i n a warm 3% s o l u t i o n of agarose (Marine C o l l o i d s Inc., Rockland, MN) on a g l a s s s l i d e f o l l o w i n g glutaraldehyde f i x a t i o n . A f t e r s e t t i n g , plugs c o n t a i n i n g the oocytes were cut with a s c a l p e l blade and f i x e d i n GA. The samples were washed twice i n PB, p o s t - f i x e d i n 1% osmium t e t r o x i d e (Marivac, H a l i f a x , Nova S c o t i a ) i n PB, dehydrated i n a graded s e r i e s of acetone ( F i s h e r S c i e n t i f i c , F a i r Lawn, NJ) and embedded i n Spurrs epoxy r e s i n (J.B. EM, Dor v a l , Quebec). P r i o r to suspen-s i o n i n agarose, cumulus-enclosed, GA f i x e d oocytes from four mature r a t s were p l a c e d i n 0.1% h y a l u r o n i d a s e (Ovine Type I I , Sigma Chemical Co., St. L o u i s , MO) i n DPBS f o r up to 5 minutes to remove the cumulus. A l l samples were then processed as d e s c r i b e d . Oocytes were l o c a t e d i n the r e s i n b l o c k by s e r i a l s e c t i o n i n g and s t a i n i n g . When a specimen was l o c a t e d , s e r i a l s e c t i o n s of the oocyte were cut at 0.5 to 1.0 um and s t a i n e d with 1% Methylene Blue (BDH Chemicals, Toronto, Ontario) i n 1% borax (BDH Chemicals, Toronto, O n t a r i o ) . Every f i f t h s e c t i o n was examined f o r the presence of chromosomes. Four randomly s e l e c t e d t h i n s e c t i o n s were cut at 60-90 nm du r i n g the s e r i a l s e c t i o n i n g and three or four t h i n s e c t i o n s were taken when the chromosomes were found. T h i n s e c t i o n s were s t a i n e d with uranyl a c e t a t e (Ted Pel l a , Redding, CA) and lead c i t r a t e (Reynolds, 1963) f o r twenty minutes each. The oocytes with cumulus removed were compared with cumulus-enclosed oocytes from mature r a t s to determine whether treatment of cumulus-enclosed oocytes with 0.1% h y a l u r o n i d a s e f o l l o w i n g f i x a t i o n 55 i n GA had any e f f e c t on oocyte morphology. Oocytes from immature r a t s i n each of the treatment groups and from mature r a t s were then compared. 5. S t a t i s t i c a l A n a l y s i s Data was analyzed when i n d i c a t e d by Chi square t e s t f o r independence or Student's t t e s t . D i f f e r e n c e s were co n s i d e r e d to be s t a t i s t i c a l l y s i g n i f i c a n t at the p<0.05 confidence i n t e r v a l . 56 PART 1: A MORPHOLOGICAL INVESTIGATION OF INTRAOVIDUCTAL OOCYTE AGING IN THE MATURE RAT A. RESULTS 1. O v u l a t i o n and Gross Morphology The range and average number of oocytes r e t r i e v e d from mature r a t s are presented i n F i g u r e 2. The assessment of ampullary d i s t e n -s i o n and gross cumulus and oocyte morphology are presented i n F i g u r e 3. Cumulus d i s p e r s i o n commenced between 12:00 and 18:00 on the day of e s t r u s and was completed by 9:00 of metestrus. Concomi-tant with complete d i s p e r s a l of the cumulus between 24:00 of e s t r u s and metestrus, ampullary d i s t e n s i o n completely receded and fragmented oocytes appeared. Fragmented oocytes contained two to f i f t e e n v a r i a b l y s i z e d fragments with i r r e g u l a r o u t l i n e but those which c l o s e l y resembled two, three or f o u r c e l l embryos were a l s o observed. The percentage of t o t a l oocytes which were fragmented rose s t e a d i l y from metestrus to p r o e s t r u s and the number of fragments i n many oocytes i n c r e a s e d while fragment s i z e decreased. 2. L i g h t Microscopy A. In t a c t Oocytes The p r o g r e s s i o n of morphological changes which occurred over the o b s e r v a t i o n p e r i o d are summarized i n F i g u r e s 4 and 5. At 9:00 of e s t r u s , i n t a c t oocytes ( F i g u r e 5a) were c h a r a c t e r i z e d by an undulent e e l 1 out 1ine with obvious p e r i v i t e l 1 i n e space (Figure 5a). F i r s t p o l a r bodies were observed i n 6/26 (23%) of the oocytes. Large, multi 1oculated m u l t i v e s i c u l a r bodies were l o c a t e d c e n t r a l l y 57 AVERAGE NUMBER AND RANGE OF OOCYTES RETRIEVED FROM MATURE RATS E9:00 E12:00 E18:00 E24:00 MET SACRIFICE TIME DI PRO (A 120 110-100-90-80 70-60-50 40 30-20-10-0 GROSS OBSERVATIONS DURING INTRAOVIDUCTAL OOCYTE AGING 41 * 45 43 * 39 * E9:00 E12:00 E18:00 E24:00 MET SACRIFICE TIME DI PRO | | GOOD CUMULUS £ 3 F A I R CUMULUS [gj] NO CUMULUS I FRAGMENTED Z'l. 'iZt& Page 57 f{ FIGURE 2 The range and average number of oocytes r e t r i e v e d from each r a t are i l l u s t r a t e d . The number of r a t s examined i s i n d i c a t e d above each s a c r i f i c e time. E = e s t r u s ; MET = metestrus; DI = d i e s t r u s ; PRO = p r o e s t r u s FIGURE 3 Gross assessment of ampullary d i s t e n s i o n , cumulus d i s p e r -s i o n and oocyte morphology are i l l u s t r a t e d . An a s t e r i s k present over s a c r i f i c e time i n d i c a t e s the presence of ampullary d i s t e n s i o n and a v i s i b l e egg mass. At 24:00 of e s t r u s , ampullary d i s t e n s i o n was reduced i n each r a t . The t o t a l number of oocytes r e t r i e v e d are d i s p l a y e d above the bars f o r each group of r a t s . 58 120 CHANGES IN OOCYTE MORPHOLOGY DURING INTRAOVIDUCTAL AGING E9:00 E12:00 El 8:00 E24:00 MET SACRIFICE TIME E=3 IRREG M 0 V T • N L B .> Page 58 fj FIGURE 4 The p r o g r e s s i o n of l i g h t and e l e c t r o n m i c r o s c o p i c changes noted i n i n t a c t oocytes over the o b s e r v a t i o n p e r i o d are i l l u s t r a t e d . IRREG = i r r e g u l a r i t y of the protuberance; CHR MOVT = e i t h e r r o t a t i o n or c e n t r a l m i g r a t i o n of the m e i o t i c apparatus; NLB = the presence of one or more n u c l e o l a r - 1 i k e bodies. The number of oocytes examined i n each group i s i n d i c a t e d above the b a r s . 59 i n the c e l l ( F i g u r e 5a), while s m a l l e r MVB were observed p e r i p h -e r a l l y . V e s i c u l a r aggregates (VA) were l o c a t e d p e r i p h e r a l l y and at l e a s t one VA i n each oocyte c o n t a i n e d one or more s m a l l , round, b a s o p h i l i c f o c i . The chromosomes were t i g h t l y aggregated on the metaphase p l a t e j u s t below the su r f a c e of a v a r i a b l y s i z e d , smooth to undulent, m i c r o v i l l u s - f r e e (MVF) protuberance of the c e l l . The metaphase p l a t e i n l o n g i t u d i n a l s e c t i o n was s i t u a t e d p e r p e n d i c u l a r to the o v e r l y i n g c e l l s u r f a c e and the s p i n d l e f i b r e s extended a s h o r t d i s t a n c e from e i t h e r s i d e of the chromosomes to convergent p o i n t s i n the c y t o s o l (Figure 5a). At 12:00 of e s t r u s , only minor changes were observed i n the cytoplasm. Although m u l t i p l e MVB were s t i l l present, they tended to be s m a l l e r with fewer m u l t i l o c u l a t e d forms. More s i g n i f i c a n t changes were observed i n the m e i o t i c apparatus. The s u r f a c e of the MVF protuberance o v e r l y i n g the chromosomes was i r r e g u l a r i n 2/25 oocytes. In 3/25 oocytes, the metaphase chromosomes were r o t a t e d 45°-90° on the s p i n d l e a x i s . The m e i o t i c apparatus had moved c e n t r a l l y i n 3/25. A l l 6 of these oocytes with r o t a t i o n and c e n t r a l movement of the m e i o t i c apparatus had l o s t the MVF p o r t i o n of the oolemma. The remainder of the oocytes s t i l l maintained a m i c r o v i 1 l u s - f r e e s u r f a c e over metaphase chromosomes with unchanged o r i e n t a t i o n . By 18:00 of e s t r u s , the s u r f a c e of the MVF protuberance was i r r e g u l a r i n 10/26 oocytes. The m e i o t i c apparatus had r o t a t e d i n 6/26 oocytes and moved c e n t r a l l y i n 2/26 oocytes. While both of the oocytes with c e n t r a l m i g r a t i o n of the chromosomes had l o s t the MVF p o r t i o n of the oolemma, 3/6 of the oocytes with chromosome r o t a t i o n Page 60 f\ FIGURE 5 A. Int a c t oocyte r e t r i e v e d at 900 of e s t r u s . Note the wide p e r i v i t e l 1 i n e space (small arrow) with undulent c e l l o u t l i n e . Large m u l t i v e s i c u l a r bodies are present (arrowheads). The m e i o t i c apparatus l i e s i n a m i c r o v i l l u s - f r e e protuberance, j u s t below the oolemma ( l a r g e arrowhead). M a g n i f i c a t i o n = 625x. B. Int a c t oocyte r e t r i e v e d at 2400 of e s t r u s . Note the i r r e g u l a r protuberance o v e r l y i n g the m e i o t i c apparatus (arrow). One n u c l e o l a r - l i k e body i s present (arrowhead). The m e i o t i c apparatus has moved s l i g h t l y c e n t r a l i n the oocyte. M a g n i f i c a t i o n ^ 625x. C. Int a c t oocyte r e t r i e v e d at 2400 of e s t r u s . Note the elonga-t i o n and r o t a t i o n of s p i n d l e inward (arrow). N u c l e o l a r - l i k e bodies are s i t u a t e d along the p e r i p h e r y of the c e l l (arrow-heads). M a g n i f i c a t i o n = 625x. D. Intact oocyte r e t r i e v e d at 2400 of e s t r u s . Note the c e n t r a l l y l o c a t e d m e i o t i c apparatus (arrow). The chromo-somes remain on the s p i n d l e equator. M a g n i f i c a t i o n = 625x. E. Intact oocyte r e t r i e v e d at 2400 of e s t r u s . The s p i n d l e i s elongated (arrowhead). One n u c l e o l a r - l i k e body i s present (arrow). M a g n i f i c a t i o n = 625x. F. Intact oocyte r e t r i e v e d at metestrus. The c e n t r a l chromosomes have s c a t t e r e d i n the cytoplasm (arrow). Inset lower l e f t : I n t a c t oocyte r e t r i e v e d at p r o e s t r u s . Note the wider s c a t t e r of chromosomes i n the cytoplasm (arrowheads). M a g n i f i c a t i o n = 350x. G. Fragmented oocytes r e t r i e v e d at metestrus. Note the m u l t i p l e v a r i a b l y s i z e d and shaped c e l l fragments. N u c l e i c o n t a i n n u c l e o l i (arrows) and n u c l e o l a r - l i k e bodies are present i n the cytoplasm (arrowheads). M a g n i f i c a t i o n = 375x. H. Fragmented oocyte r e t r i e v e d at p r o e s t r u s . Note the i n c r e a s e d number of s m a l l e r fragments. The l a r g e fragment i s m u l t i n u c l e a t e d (arrowheads). One i n t a c t oocyte has s e v e r a l b a s o p h i l i c f o c i i n a l a r g e v e s i c u l a r aggregate (arrow). M a g n i f i c a t i o n = 375x. 61 maintained a MVF protuberance. E l o n g a t i o n of the s p i n d l e was observed i n 3/26 oocytes, one of which a l s o had an i r r e g u l a r s u r f a c e . B a s o p h i l i c f o c i were observed more o f t e n i n the VA. One to two s m a l l , round, densely b a s o p h i l i c , n u c l e o l a r - l i k e bodies (NLB) were observed p e r i p h e r a l l y i n 6/26 oocytes. At 24:00 of e s t r u s , the MVF protuberance was i r r e g u l a r i n 10/33 i n t a c t oocytes ( F i g u r e 5b). The m e i o t i c apparatus was r o t a t e d i n 9/33 (F i g u r e 5c) and chromosomes had migrated c e n t r a l l y i n 8/33 of the oocytes ( F i g u r e 5d). The MVF p o r t i o n of the c e l l s u r f a c e was absent i n a l l oocytes with c e n t r a l l y migrated chromosomes and 5/9 oocytes with r o t a t i o n . The s p i n d l e was elongated i n 9/33 of the oocytes ( F i g u r e 5e), of which 3/9 had an i r r e g u l a r s u r f a c e , 2/9 had c e n t r a l l y migrated chromosomes and 1/9 had chromosome r o t a t i o n ( F i g u r e 5c) . The chromosomes had s c a t t e r e d i n the cytoplasm and the s p i n d l e was no longer v i s i b l e i n one oocyte. N u c l e o l a r - l i k e bodies were observed p e r i p h e r a l l y i n 23/33 oocytes ( F i g u r e 5b,c,e). The number of NLB observed per oocyte v a r i e d from one to two i n most to f i v e to seven i n three oocytes. At metestrus, the chromosomes were c e n t r a l l y l o c a t e d i n 24/28 i n t a c t oocytes w i t h l o s s of the MVF p o r t i o n of the c e l l membrane. Ro t a t i o n of the m e i o t i c apparatus was noted i n 1/28 oocytes and another oocyte had an i r r e g u l a r MVF protuberance. In 4/24 oocytes, the chromosomes were s c a t t e r e d i n the cytoplasm ( F i g u r e 5 f ) . In 1/24 oocytes, a nucleus with a densely b a s o p h i l i c , compact n u c l e o l u s was present i n the c e n t r e of the c e l l . The MVB were in c r e a s e d again i n s i z e and were c e n t r a l l y l o c a t e d . A l l oocytes contained NLB (4-7 per c e l l ) which were o f t e n l o c a t e d adjacent to the VA and sometimes l o c a t e d c e n t r a l l y . Of 5 a d d i t i o n a l oocytes 62 with small fragments i n the p e r i v i t e l 1 i n e space, 3 had a second p o l a r body. At d i e s t r u s , 17/17 i n t a c t oocytes had c e n t r a l , s c a t t e r e d chromosomes which were l e s s b a s o p h i l i c and l a r g e r than those seen aggregated c e n t r a l l y at e a r l i e r times. N u c l e o l a r - l i k e bodies were i n v a r i a b l y present i n the cytoplasm (12 to 13 per o o c y t e ) . The VA contained m u l t i p l e , densely b a s o p h i l i c f o c i which tended to be l a r g e r than those seen at metestrus. At p r o e s t r u s , i n t a c t oocytes were v a c u o l a t e d and contained l a r g e r l e s s b a s o p h i l i c s c a t t e r e d chromosomes ( F i g u r e 5 f ) . B. Fragmented Oocytes Fragmented oocytes had a more b a s o p h i l i c cytoplasm with fewer o r g a n e l l e s , which tended to be c l u s t e r e d m u l t i f o c a l l y i n the cytoplasm. Up to 10 n u c l e i with n u c l e o l i were observed i n each fragmented oocyte with a maximum of 6 n u c l e i i n any one i n d i v i d u a l fragment (Figure 5g) . More than 20 NLB were s c a t t e r e d between fragments i n i n d i v i d u a l oocytes ( F i g u r e 5g). From d i e s t r u s to p r o e s t r u s , oocytes c o n s i s t i n g of more than twenty v a r i a b l y s i z e d but small fragments appeared ( F i g u r e 5h) . Many of the fragments i n these oocytes were v a c u o l a t e d and contained few o r g a n e l l e s . Although they contained a s i m i l a r number of n u c l e i , these were d i s t r i b u t e d between a l a r g e r number of fragments so that the l a r g e s t fragments only contained a maximum of 3 n u c l e i . The fragments were o f t e n so s m a l l , that many contained only a nucleus. There was p r o g r e s s i v e d i l a t i o n and l y s i s of the smal l e r fragments with a concomitant i n c r e a s e i n p e r i v i t e l 1 i n e c e l l d e b r i s . 63 3. E l e c t r o n Microscopy A. I n t a c t Oocytes Oocytes r e t r i e v e d at 9:00 of e s t r u s had a small amount of c e l l d e b r i s c o n s i s t i n g of v a r i a b l y s i z e d v e s i c l e s and f l o c c u l e n t m a t e r i a l i n an obvious p e r i v i t e l 1 i n e space. The s u r f a c e of the oocyte was undulent i n nature with b l u n t c y t o p l a s m i c processes extending a short d i s t a n c e i n t o the p e r i v i t e l 1 i n e space at v a r i a b l e i n t e r v a l s ( F i g u r e 6a,b). F o c a l patches of m i c r o v i l l i were occa-s i o n a l l y observed on the oolemma. Coated p i t s and v e s i c l e s were commonly observed. The c y t o s o l was homogeneous and moderately e l e c t r o n dense with a small number of 15 to 20 nm diameter e l e c t r o n dense granules s c a t t e r e d randomly throughout the cytoplasm. F i b r i l l a r a r r a y s (FA) c o n s i s t e d of p a r a l l e l s t a c k s of l a t t i c e - l i k e s t r u c t u r e s 20 to 40 nm wide and 2.4 to 4 urn long. The FA were d i s t r i b u t e d throughout the cytoplasm with a tendency to be arranged i n l a r g e aggregates devoid of o r g a n e l l e s other than small v e s i c l e s and ribosomes ( F i g u r e 6b). Mitochondria (MC) were round to c y l i n d r i c a l i n shape with h i g h l y e l e c t r o n dense m a t r i x and c o n c e n t r i c to t r a n s v e r s e c r i s t a e . Round MC ranged i n diameter from 400 to 800 nm while c y l i n d r i c a l forms ranged from 300 by 600 nm to 400 by 1600 nm i n s i z e . The MC were u s u a l l y arranged i n c l u s t e r s , o f t e n a s s o c i a t e d with other o r g a n e l l e s . V e s i c u l a r aggregates ranged i n diameter from 2 to 5.6 urn and c o n s i s t e d of an i n t e r l a c i n g network of small tubules with moderately e l e c t r o n dense, homogeneous, f i n e l y g r a n u l a r content. The VA were surrounded by mitochondria and o c c a s i o n a l l y contained one or more e l e c t r o n dense, f i n e l y f i b r i l l a r f o c i 190 to 300 nm i n diameter ( F i g u r e 6b). S i n g l e elements of smooth endoplasmic 64 r e t i c u l u m (SER) were c l o s e l y a s s o c i a t e d with MC, co n t a i n e d f i n e l y g r anular, homogeneous content and were o c c a s i o n a l l y m i l d l y to moderately d i l a t e d . C l u s t e r s of small v e s i c l e s with scant content were observed i n the cytoplasm, u s u a l l y i n a s s o c i a t i o n with o r g a n e l l e s . The MVB c o n s i s t e d of s a c c u l e s and f l a t t e n e d t ubules i n a cy t o s o l matrix of i n c r e a s e d e l e c t r o n d e n s i t y . Numerous v a r i a b l y s i z e d , membrane-bound v e s i c l e s w i t h a mixture of v e s i c u l a r , g ranular, membranous, homogeneously e l e c t r o n dense and f l o c c u l e n t content were s i t u a t e d w i t h i n the MVB background matrix or j u s t p e r i p h e r a l to i t ( F i g u r e 6a). The MVB v a r i e d i n both s i z e and complexity so that small to l a r g e masses were arranged e i t h e r i n sheets or i n a m u l t i 1 o c u l a t e d manner. C o r t i c a l g r a n u l e s (CG) with a p e r i p h e r a l space and c e n t r a l , h i g h l y condensed, f i n e l y f i b r i l l a r content, o c c a s i o n a l l y with a c c o r d i o n - l i k e p e r i o d i c i t y were of t e n observed adjacent to MVB and i n the c o r t i c a l cytoplasm. C o r t i c a l granules were l o c a t e d p r i m a r i l y i n the outer cortex and o f t e n ju s t beneath the oolemma ( F i g u r e 6b). There was marked v a r i a t i o n i n the d i s t r i b u t i o n of suboolemmal CG w i t h i n each oocyte. In some areas, the d e n s i t y was high w h i l e adjacent areas of the oocyte were completely devoid of CG. C o r t i c a l granules were never observed i n the m i c r o v i l l u s - f r e e p o r t i o n of the oocyte (Figure 6a). E x o c y t o s i s of c o r t i c a l granules was o c c a s i o n a l l y observed. Condensed chromosomes were present i n the outer p o r t i o n of a small protuberance of the oocyte with an undulent s u r f a c e f r e e of e i t h e r m i c r o v i l l i or c y t o p l a s m i c processes (Figure 6a). A t h i c k web (150 to 250 nm wide) of m i c r o f i l a m e n t s was present beneath the oolemma i n the protuberance ( F i g u r e 6a). No o r g a n e l l e s other than 65 >A " Page 65 FIGURE 6 A. Oocyte r e t r i e v e d at 900 of e s t r u s . Metaphase chromosomes (small arrowheads) l i e i n the m i c r o v i l l u s f r e e protuberance with a t h i c k , suboolemmal m i c r o f i l a m e n t web (small arrow). The s p i n d l e microtubules are embedded i n a matrix devoid of o r g a n e l l e s ( l a r g e arrow). The m u l t i v e s i c u l a r body has numerous v e s i c l e s with v a r i a b l e content a s s o c i a t e d with saccules and tubules i n a matrix of i n c r e a s e d e l e c t r o n d e n s i t y ( l a r g e arrowhead). M a g n i f i c a t i o n = 5,000x. B. Oocyte r e t r i e v e d at 900 of e s t r u s . There i s a wide p e r i -v i t e l l i n e space with numerous c y t o p l a s m i c processes on the c e l l s u r f a c e ( l a r g e arrow). Suboolemmal c o r t i c a l granules are v i s i b l e (small arrowhead). C l u s t e r s of o r g a n e l l e s are present between f i b r i l l a r a r r a y s ( l a r g e arrowhead). Ves-i c u l a r aggregates with e l e c t r o n dense f o c i are v i s i b l e (small arrows) i n a moderately e l e c t r o n dense and homo-geneous c y t o s o l . M a g n i f i c a t i o n = 3150x. C. Oocyte r e t r i e v e d at 2400 of e s t r u s . Cytoplasmic processes are present on the protuberance ( l a r g e arrow). F i b r i l l a r a r r a y s are d i s r u p t e d and reduced i n l e n g t h (small arrow). Chromosomes ( l a r g e arrowhead) have moved away from the surface of the c e l l i n a s s o c i a t i o n w i t h the s p i n d l e (small arrowhead). M a g n i f i c a t i o n = 2,500x. D. Oocyte r e t r i e v e d at 900 of metestrus. A l a r g e m u l t i v e s i c u -l a r body i s present i n the cytoplasm ( l a r g e arrowhead). F i b r i l l a r arrays are broken up (small arrowhead). There i s moderate d i l a t i o n of smooth endoplasmic r e t i c u l u m (arrow). M a g n i f i c a t i o n = 2,500x. 66 small v e s i c l e s were present i n the c y t o s o l between or immediately around the chromosomes and s p i n d l e m i c r o t u b u l e s . By 12:00 of e s t r u s , l i t t l e change was observed i n the oocytes. The MVB were l e s s o f t e n m u l t i 1 o c u l a t e d and CG were s t i l l observed i n the surrounding cytoplasm. There was more v a r i a t i o n between oocytes i n the presence of SER d i l a t i o n . At 18:00 of e s t r u s , some oocytes were i n d i s t i n g u i s h a b l e from those observed at 9:00 of e s t r u s , w h ile others were s i g n i f i c a n t l y a l t e r e d . The VA were more o f t e n l o c a t e d c e n t r a l l y w i t h i n the oocyte and h i g h l y e l e c t r o n dense f o c i were more commonly observed w i t h i n the substance of the VA. There was v a r i a t i o n between oocytes i n the d e n s i t y of PA. Condensed chromosomes, s t i l l a s s o c i a t e d with s p i n d l e m icrotubules, were observed more c e n t r a l l y i n the cytoplasm with concomitant l o s s of the MVF protuberance and a r e d u c t i o n i n the width of the suboolemmal m i c r o f i l a m e n t web ( F i g u r e 6c). At 24:00 of e s t r u s , the FA tended to c o n t a i n fewer l a t t i c e s which were o f t e n reduced i n length (0.7 to 2.8 um) i n some oocytes. Linear elements of SER were more of t e n observed f r e e i n the cytoplasm without obvious a s s o c i a t i o n with m i t o c h o n d r i a . The CG were o c c a s i o n a l l y enlarged with i n c r e a s e d e l e c t r o n lucency i n the suboolemmal p o s i t i o n and CG e x o c y t o s i s was o c c a s i o n a l l y observed i n many oocytes. At metestrus, more m i c r o v i l l i were observed on the oolemma. The MVB were i n c r e a s e d i n s i z e and o r i e n t e d as l a r g e sheets of sa c c u l e s , tubules and v e s i c l e s which, i n a s s o c i a t i o n with v e s i c u l a r aggregates, formed very l a r g e complexes c e n t r a l l y i n the oocytes (Figure 6d) . Small, h i g h l y e l e c t r o n dense, non-membrane bound, f i b r i l l a r , 500 to 960 nm diameter NLB were observed i n the 67 Page 67$ FIGURE 7 A. Oocyte r e t r i e v e d at metestrus. The c y t o s o l i s e l e c t r o n lucent with l o s s of g r a n u l a r i t y . F i b r i l l a r a r r a y s are reduced i n number. A m u l t i v e s i c u l a r body ( l a r g e arrowhead) and v e s i c u l a r aggregate (small arrowhead) are present. There i s a nascent G o l g i (open arrow) and a p e r i p h e r a l n u c l e o l a r - l i k e body (small arrow). There i s m i l d to moderate smooth endoplasmic r e t i c u l u m d i l a t i o n ( l a r g e arrow). M a g n i f i c a t i o n = 2,500x. B. Fragmented oocyte r e t r i e v e d at d i e s t r u s . Numerous n u c l e o l a r - l i k e bodies are s i t u a t e d on the p e r i p h e r y of v e s i c u l a r aggregates. Segments of smooth endoplasmic r e t i c u l u m abut the s u r f a c e of one n u c l e o l a r - l i k e body (arrow). Small e l e c t r o n dense f o c i are present at the' p e r i p h e r y of the v e s i c u l a r aggregates (arrowheads). Numerous m i c r o v i l l i are present on the s u r f a c e . M a g n i f i c a t i o n = 5,000x. C. Fragmented oocyte r e t r i e v e d at d i e s t r u s . The n u c l e o l a r - l i k e body has numerous small vacuoles. E a r l y t r a n s f o r m a t i o n of G o l g i i s v i s i b l e i n the adjacent cytoplasm (arrowhead). M a g n i f i c a t i o n = 9,450x. D. Fragmented oocyte r e t r i e v e d at d i e s t r u s . The l a r g e n u c l e o l a r - l i k e body and surrounding cytoplasm are enveloped by a tortuous network of smooth endoplasmic r e t i c u l u m . H i g h l y condensed granules are f o c a l l y present on the in n e r s u r f a c e of the double membrane (small arrow). Non-membrane bound NLB are adjacent (arrowheads). A nucleus with obvious nucleoplasm i s present ( l a r g e arrow). M a g n i f i c a t i o n = 9,450x. 68 cytoplasm (Figure 7a). V e s i c u l a r aggregates o f t e n had NLB s i t u a t e d at the periphery and e l e c t r o n dense f o c i up to 720 nm i n diameter w i t h i n the substance ( F i g u r e 7a). Lamellar and c o n c e n t r i c arrange-ments of double membranes were observed adjacent to VA and appeared to be e a r l y d i f f e r e n t i a t i o n of G o l g i ( F i g u r e 7a). The FA were decreased i n number with l a t t i c e s broken up i n t o s h o r t e r (160 nm to 0.5 um) segments i n many oocytes ( F i g u r e 6d) . Elongated SER elements tended to compartmentalize the cytoplasm. At d i e s t r u s , i n t a c t oocytes had an i r r e g u l a r s u r f a c e with v a r i a b l e numbers of c y t o p l a s m i c processes and m i c r o v i l l i . Mitochondria were enlarged (up to 1000 nm i n diameter) with decreased e l e c t r o n d e n s i t y of the matrix and the appearance of e l e c t r o n dense granules and o c c a s i o n a l f o c a l d i l a t i o n of c r i s t a e or compartmental v a c u o l i z a t i o n . Some very l a r g e VA, up to 8 um i n diameter, were present. An i n c r e a s e d number of independent v e s i c l e s , with complex content s i m i l a r to that of MVB, were observed. The MVB were u s u a l l y s m a l l e r with a concomitant r e d u c t i o n i n v e s i c l e content to predominantly g r a n u l a r and f l o c c u l e n t m a t e r i a l and o c c a s i o n a l m i l d d i l a t i o n of c o n s t i t u e n t t u b u l e s . The FA were reduced i n cyt o p l a s m i c d e n s i t y and were o f t e n p o l a r i z e d w i t h i n the c e l l . The chromosomes were c e n t r a l l y l o c a t e d i n a l l oocytes, with or without s p i n d l e remnants a s s o c i a t e d . Elements of SER, MC and v e s i c l e s were i n t e r s p e r s e d with the chromosomes. By proestrus ( F i g u r e 8a), a r e d u c t i o n i n coated p i t s , m i c r o v i l l i and cytoplasmic processes was observed with a decrease i n c y t o s o l e l e c t r o n d e n s i t y , e l e c t r o n dense granules and FA. The MC o f t e n contained markedly e l e c t r o n l u c e n t , f l o c c u l e n t r a t h e r than granular, matrix. The SER were o f t e n d i l a t e d with f l o c c u l e n t r Page 69 f\ FIGURE 8 A. Intact oocyte r e t r i e v e d on the morning of p r o e s t r u s from a mature r a t . The oolemma i s smooth with few c o r t i c a l gran-u l e s ( l a r g e arrowhead). F i b r i l l a r a r r a y s are reduced i n num-ber and fragmented. There i s d i l a t i o n of the smooth endo-plasmic r e t i c u l u m and mitochondria (small arrowheads). The chromosomes are s c a t t e r e d i n the c e l l and p y k n o t i c (arrows). No m u l t i v e s i c u l a r bodies, v e s i c u l a r aggregates or s p i n d l e microtubules are present. M a g n i f i c a t i o n = 2,812x. B. Fragmented oocyte r e t r i e v e d at d i e s t r u s . M u l t i p l e n u c l e i with f i b r i l l a r n u c l e o l i (arrowheads) and numerous m i c r o v i l l i are present. Two p e r i n u c l e a r and one c y t o p l a s m i c n u c l e o l a r -l i k e body are present (arrows). M a g n i f i c a t i o n = 3,543x. 70 content. The MVB were s m a l l e r , predominantly devoid of v e s i c l e s and the saccules and tubules were m i l d l y to moderately d i l a t e d . Cytoplasmic vacuoles c o n t a i n e d scant m a t e r i a l . The o r g a n e l l e s were p o l a r i z e d en masse w i t h i n the cytoplasm. The s c a t t e r e d chromosomes c o n s i s t e d of i r r e g u l a r , clumped chromatin and s p i n d l e remnants were not observed. B. Fragmented Oocytes Fragmented oocytes d i f f e r e d from i n t a c t oocytes i n many resp e c t s . More m i c r o v i l l i were observed on the s u r f a c e of oocyte fragments (Figure 7b, 8b) and there was an i n c r e a s e d amount of c e l l d e b r i s i n the p e r i v i t e l 1 i n e space. There was an i n c r e a s e i n c y t o s o l e l e c t r o n d e n s i t y with a concomitant i n c r e a s e i n g r a n u l a r i t y . Organelles tended to be reduced i n number and s i t u a t e d p e r i p h e r a l l y with loss of the VA. M i t o c h o n d r i a ranged i n s i z e to 1200 nm diameter. F i b r i l l a r array l a t t i c e s were unchanged but i r r e g u l a r l y d i s t r i b u t e d between fragments. The MVB were reduced i n s i z e with d i s s o c i a t i o n of the v e s i c l e s from the s a c c u l e s and t u b u l e s . There was more v a r i a t i o n i n v e s i c l e content and s i z e . Small, round to i r r e g u l a r , n u c l e i were always present i n fragments and u s u a l l y contained 1 to 3 l a r g e , e l e c t r o n dense, f i b r i 1 1 a r n u c l e o l i ( F i g u r e 7d, 8b). N u c l e o l a r - l i k e b o d ies were observed p e r i p h e r a l l y i n fragments of a l l s i z e s . The NLB v a r i e d i n s i z e , were u s u a l l y a s s o c i a t e d with VA and m u l t i p l e , small e l e c t r o n lucent spaces were of t e n observed w i t h i n the f i b r i l l a r substance (Figure 7b,c,d). At d i e s t r u s , small f o c i of condensed chromatin were o f t e n present on the inner n u c l e a r l e a f l e t and on the dense n u c l e o l i . The NLB were l a r g e r , v a r y i n g i n s i z e from 500 to 1200 nm diameter, 71 and SER elements o c c a s i o n a l l y completely e n c l o s e d them ( F i g u r e 7b, 9a). Granular, e l e c t r o n dense m a t e r i a l was a l s o o c c a s i o n a l l y observed on the s u r f a c e of NLB ( F i g u r e 9b). Some NLB were a s s o c i -ated with tortuous lengths of double membranes which enclosed segments of the cytoplasm and NLB, resembling n u c l e i ( F i g u r e 7d). Continuous lengths of SER i n c r e a s i n g l y compartmentalized the cytoplasm, completely e n c i r c l i n g many v e s i c l e s i n the process ( F i g u r e 9a). Cytoplasmic v e s i c l e s were l a r g e r and often contained h i g h l y e l e c t r o n dense, c e n t r a l , amorphous content (Figure 9b). At p r o e s t r u s , s a c c u l e s and tubules of the MVB were f r e q u e n t l y d i l a t e d ( F i g u r e 9b). N u c l e i o f t e n contained condensed chromatin on the s u r f a c e of many n u c l e o l i ( F i g u r e 9c). C l u s t e r s of d i l a t e d mitochondria and SER with scant f l o c c u l e n t content were observed p e r i p h e r a l l y i n many fragments ( F i g u r e 9d) . There was v a c u o l a t i o n and i n c r e a s e d e l e c t r o n lucency of the mito c h o n d r i a and d i l a t i o n of SER i n the oocytes ( F i g u r e 9d). Residual bodies were o c c a s i o n a l l y seen. Very l a r g e NLB, up to 2.7 um i n diameter were observed. Reduced numbers of c o r t i c a l granules and FA were present. Oocytes with more than twenty small fragments contained l a r g e , cytoplasmic vacuoles with v a r i a b l e content. There was o f t e n d i l a t i o n of the nu c l e a r membrane i n the small fragments. 72 / Page 72 FIGURE 9 A. Fragmented oocyte r e t r i e v e d at p r o e s t r u s . Smooth endo-plasmic r e t i c u l u m i s surrounding i n t r a c y t o p l a s m i c v e s i c l e s (small arrowheads), a v e s i c u l a r aggregate ( l a r g e arrowhead) and n u c l e o l a r - l i k e body (arrow). The smooth endoplasmic r e t i c u l u m around the v e s i c u l a r aggregate and n u c l e o l a r - l i k e body has completely i s o l a t e d these s t r u c t u r e s from the surrounding cytoplasm. M a g n i f i c a t i o n = 5,625x. B. Fragmented oocyte r e t r i e v e d at p r o e s t r u s . A l a r g e v e s i c l e i s present i n the cytoplasm ( l a r g e arrow). The m u l t i v e s i c u l a r body i s devoid of v e s i c l e s with d i l a t i o n of tubules (small arrow). E l e c t r o n dense f o c i are present i n the m i t o c h o n d r i a l matrix (small arrowhead). Coarse granules have aggregated around a n u c l e o l a r - l i k e body ( l a r g e arrowhead). M a g n i f i c a t i o n = 7,500x. C. Fragmented oocyte r e t r i e v e d at p r o e s t r u s . Mitochondria c o n t a i n e l e c t r o n dense granules i n the matrix (small arrow). Numerous l a r g e v e s i c l e s are p r e s e n t . Coarse granular chromatin i s c l u s t e r e d on the s u r f a c e of the f i b r i l l a r n u c l e o l u s ( l a r g e arrow). One p e r i p h e r a l n u c l e o l a r - l i k e body i s present (arrow-head). M a g n i f i c a t i o n s 5,000x. D. Fragmented oocyte r e t r i e v e d at p r o e s t r u s . There i s v a r i a t i o n i n the presence of m i c r o v i 1 1 i , c o r t i c a l granules, f i b r i l l a r a r r a y s and cytoplasmic g r a n u l a r i t y between f r a g -ments. N u c l e o l a r - l i k e bodies ( l a r g e arrowheads) and l a r g e v e s i c l e s with v a r i a b l e but scant content (arrow) are present. There i s m i l d to moderate smooth endoplasmic r e t i c u l u m d i l a t i o n and m i t o c h o n d r i a l v a c u o l a t i o n (small arrowhead) i n 2 fragments. There i s m i t o c h o n d r i a l and smooth endoplasmic r e t i c u l u m d i l a t i o n with o r g a n e l l e p o l a r i z a t i o n i n a t h i r d fragment (open arrow). M a g n i f i c a t i o n = 2,500x. 73 B. DISCUSSION The l i g h t and e l e c t r o n microscopic morphology of oocytes r e t r i e v e d at 9:00 of e s t r u s was c o n s i s t e n t with that r e p o r t e d by other workers ( S o t e l o and P o r t e r , 1959; Odor, 1960; Zamboni, 1970; Peluso and Butcher, 1974). However, i n the present study, c o r t i c a l granules were more v a r i a b l y d i s t r i b u t e d beneath the oolemma than d e s c r i b e d by Peluso and Butcher (1974). V a r i a b l e d i s t r i b u t i o n of suboolemmal c o r t i c a l g r a n u l e s was a l s o d e s c r i b e d by S z o l l o s i (1962) and Guraya (1982). The f i r s t p o l a r body of the r a t was observed i n a maximum of 30% of oocytes s h o r t l y a f t e r o v u l a t i o n , and thus was reported to be very u n s t a b l e ( T s a f r i r i and K r a i c e r , 1972). T h i s was supported by the o b s e r v a t i o n of a f i r s t p o l a r body i n l e s s than 25% of r e c e n t l y o v u l a t e d oocytes i n the present study. The morphological changes observed during i n t r a o v i d u c t a l oocyte aging i n the r a t were s i m i l a r to those d e s c r i b e d i n the mouse, rabbit and hamster (Yanagimachi and Chang, 1961; Marston and Chang, 1964; S z o l l o s i , 1971; Longo, 1974a,b; Longo, 1975; Meyer and Longo, 1979; Longo, 1980). In these s p e c i e s , a s e r i e s of coordinated morphological a l t e r a t i o n s analagous to those observed i n f e r t i l i z e d oocytes ( r o t a t i o n and c e n t r a l m i g r a t i o n of the m e i o t i c apparatus, appearance of f i b r i l l a r n u c l e o l i and n u c l e a r r e c o n s t i t u t i o n with c e l l d i v i s i o n ) were observed d u r i n g aging ( S z o l l o s i , 1971; Longo, 1974a,b; Longo, 1975). These changes were reported to occur a f t e r l o s s of oocyte f e r t i 1 i z a b i 1 i t y (Marston and Chang, 1964). The f e r t i l i z a b l e l i f e of the r a t oocyte i s 12 to 14 hr (Niwa and Chang, 1975). Morphological changes of the m e i o t i c apparatus were i n i t i a l l y observed i n a small percentage of oocytes between 12:00 and 18:00 of e s t r u s , corresponding with the end of 7 4 the f e r t i l i z a b l e l i f e of the oocyte. Since oocytes r e t r i e v e d at t h i s time were s t i l l cumulus-enclosed and e x h i b i t e d no gross a b n o r m a l i t i e s , u n f e r t i l i z a b l e oocytes would not be d e t e c t a b l e g r o s s l y . In the present study, dynamic changes i n the m e i o t i c appar-atus, MVB, VA, MC, FA, c e l l number and n u c l e a t i o n were s i m i l a r to the e a r l y developmental processes observed i n f e r t i l i z e d r a t oocytes. Dynamic changes i n MVB, n u c l e a r and n u c l e o l a r r e c o n s t i t u t i o n , c y t o k i n e s i s and t r a n s f o r m a t i o n of MC and SER occur d u r i n g e a r l y development i n the r a t ( S o t e l o and P o r t e r , 1959; S z o l l o s i , 1966; Schuchner, 1970; Dvorak et a l . , 1977; A l b e r t i n i , 1987). Secondary lysosomes and r e s i d u a l bodies i n c r e a s e as cleavage progresses while MVB, VA and FA d e c l i n e (Dvorak et a l ., 1977). Hence, duri n g i n t r a o v i d u c t a l aging i n the r a t , oocytes f o l l o w a p r e d i c t a b l e course of e a r l y developmental processes. T h i s supports the concept that an i n t r i n s i c developmental program i s a c t i v a t e d i n the rat oocyte at the time of o v u l a t i o n ( S h a l g i and K r a i c e r , 1978; S h a l g i et a l . , 1985). This program i s b e l i e v e d to confer the oocyte with the p o t e n t i a l to i n i t i a t e development (Howlett and B o l t o n , 1985) and oocytes which undergo these developmental processes are r e f e r r e d to as a c t i v a t e d (Longo, 1974a). During oocyte growth and maturation, compounds which d i r e c t embryonic development are s y n t h e s i z e d and s t o r e d f o r subsequent u t i l i z a t i o n (Moor a n d G a n d o l f i , 1987; T e s a r i k , 1989). E x p r e s s i o n of these compounds commences a f t e r the second metaphase a r r e s t (Kubiak, 1989) and i n i t i a t e s and d i r e c t s e a r l y embryonic develop-ment (Moor and G a n d o l f i , 1987). The male gamete does not i n i t i a t e , but a c c e l e r a t e s and enhances, the developmental program (Golbus and 75 S t e i n , 1976; Howlett and B o l t o n , 1985; E i c h e n l a u b - R i t t e r et a l ., 1986; Moor and G a n d o l f i , 1987). Thus, the developmental program i s expressed i n u n f e r t i l i z e d oocytes ( E i c h e n l a u b - R i t t e r et a l . , 1986; Paynton et a l . , 1988) and a r t i f i c i a l l y a c t i v a t e d parthenogenotes (Tarkowski, 1970; Van Blerkom and Runner, 1976; Kubiak, 1989), but at a slower r a t e . However, i n the mouse, the n u c l e o l a r o r g a n i z e r r e g i o n (NOR) a c t i v a t e s i n 2 c e l l embryos and aged, u n f e r t i l i z e d oocytes at the same p o s t - o v u l a t o r y time s u g g e s t i n g that some aspects of the developmental program operate on a s t r i c t l y c h r o n o l o g i c a l b a s i s (Hansmann et a l . , 1978). The developmental program leads to s p e c i f i c u l t r a s t r u c t u r a l changes i n the oocyte (Moor and G a n d o l f i , 1987). Dense, f i b r i l l a r , non-membrane bound s t r u c t u r e s appeared i n the p e r i p h e r a l cytoplasm of aged rat oocytes. Due to the frank s i m i l a r i t y w i t h the q u i e s c -ent, f i b r i l l a r n u c l e o l i observed i n the germinal v e s i c l e of pr e o v u l a t o r y rat oocytes ( M e r v e i l l e et a l . , 1985; Antoine et a l . , 1989), these s t r u c t u r e s were c a l l e d NLB. Cytoplasmic, non-membrane bound NLB were observed i n the cytoplasm of p r o n u c l e a r (Schuchner, 1970), pre- and p o s t - i m p l a n t a t i o n r a t embryos ( S z o l l o s i , 1966; Takeuchi, 1980; Takeuchi and Takeuchi, 1982). The NLB appeared to be d i s t i n c t e n t i t i e s from the extruded n u c l e o l i d e s c r i b e d by other i n v e s t i g a t o r s i n 1 and 2 c e l l r a t embryos, which were membrane-bound, p e r i n u c l e a r and disappeared q u i c k l y from the cytoplasm ( S o t e l o and Porter, 1959; Izquierdo and V i a l , 1962; S z o l l o s i , 1965). E x t r a n u c l e o l a r bodies m o r p h o l o g i c a l l y and cytochemical1y i d e n t i c a l to quiescent n u c l e o l i appear i n the germinal v e s i c l e d uring preovulatory maturation i n the r a t (Antoine and Goessens, 76 1990). A f t e r germinal v e s i c l e breakdown, these bodies p e r s i s t near the condensed chromosomes as small masses and then fuse to g i v e r i s e to the f i b r i l l a r n u c l e o l i of p r o n u c l e i (Antoine and Goessens, 1990). This suggests that e x t r a n u c l e o l a r bodies may be u n r e l a t e d to the NLB, s i n c e m u l t i p l e n u c l e i with up to 11 n u c l e o l i were a l s o observed i n aged r a t oocytes. E x t r a n u c l e o l a r bodies were not observed i n a s s o c i a t i o n with condensed chromosomes i n e i t h e r aged or unaged oocytes i n the present study. The NLB observed i n the aging r a t oocytes were s i m i l a r to the NLB reported i n pre- and p o s t - i m p l a n t a t i o n embryos. During i n t r a o v i d u c t a l oocyte aging, the NLB i n c r e a s e d i n s i z e and i n some cases developed vacuoles or accumulated segments of SER on the surface. S i m i l a r l y , i n p o s t - i m p l a n t a t i o n r a t embryos the NLB appeared to grow i n the cytoplasm and were o f t e n vacuolated or surrounded by endoplasmic r e t i c u l u m (Takeuchi and Takeuchi, 1982). This suggests that the appearance of NLB i s d i r e c t e d by the i n t r i n s i c developmental program of the oocyte, independent of f e r t i l i z a t i o n . Cytoplasmic NLB i n p o s t - i m p l a n t a t i o n r a t embryos c o n s i s t of r i b o n u c l e o p r o t e i n (RNP) (Takeuchi and Takeuchi, 1982). They are bel i e v e d to a r i s e from the n u c l e o l u s and represent a method of d i s p e r s i n g RNP, necessary f o r a c t i v e and r a p i d metabolism during ear l y embryogenesis (Takeuchi and Takeuchi, 1982), to the cytoplasm ( S z o l l o s i , 1966). S i m i l a r s t r u c t u r e s were observed i n hypothalamic neurons and adenohypophyseal c e l l s of the r a t (Hindelang-Gertner et al . , 1974). These were observed to v a c u o l a t e as metabolic a c t i v i t y increased and were hypothesized to be preribosomal RNP of n u c l e o l a r o r i g i n (Hindelang-Gertner et a l . , 1974). The o r i g i n of the 77 cytoplasmic NLB i n the aged r a t oocytes was u n c l e a r . E l e c t r o n dense f o c i w i t h i n the VA i n c r e a s e d i n number and s i z e p r i o r to, and c o i n c i d e n t with, the appearance of NLB suggesting that NLB may a r i s e from recondensation of n u c l e o l a r RNP i n v e s i c u l a r aggregates. The accumulation of SER around NLB and the formation of n u c l e a r - l i k e s t r u c t u r e s suggest t h a t NLB are capable of i n d u c i n g nuclear membrane r e c o n s t i t u t i o n and may thus c o n t a i n chromatin. Polypeptides from the n u c l e a r lamina bind to s p e c i f i c s i t e s on chromatin and mediate r e c o n s t i t u t i o n of the n u c l e a r envelope, which o r i g i n a t e s from SER ( A l b e r t s et a l . , 1983). Since VA are part of the SER network (Vazquez-Nin and S o t e l o , 1967; Peluso and Butcher, 1974), they l i k e l y c o n t a i n segments of the germinal v e s i c l e envelope ( A l b e r t s et a l . , 1983). T h i s may e x p l a i n why NLB are observed i n a s s o c i a t i o n with VA. E x t r a n u c l e o l a r bodies which appear i n the germinal v e s i c l e of mouse and human oocytes are the morphological m a n i f e s t a t i o n of a m p l i f i e d ribosomal d e o x y r i b o n u c l e i c a c i d (rDNA) t r a n s c r i p t i o n (Chouinard, 1973; Wolgemuth et a l . , 1979; Hartung et a l . , 1983). E x t r a n u c l e o l a r bodies i n human d i p l o t e n e oocytes were shown to c o n t a i n rDNA (Wolgemuth-Jarashow et a l . , 1977). The unequivocal presence of rDNA was not demonstrated i n NLB by s p e c i f i c NOR-silver s t a i n i n g (Takeuchi and Takeuchi, 1982; Takeuchi and Sonta, 1983). However, the NOR-silver method only l a b e l s a c t i v e l y t r a n s c r i b i n g rDNA (Hartung et a l . , 1983). T h e r e f o r e , i f NLB represent rDNA gene a m p l i f i c a t i o n which oc c u r r e d d u r i n g p r e o v u l a t o r y maturation i n the r a t , n o n - t r a n s c r i b i n g rDNA may p e r s i s t f o l l o w i n g germinal v e s i c l e breakdown. A m p l i f i e d , n o n - t r a n s c r i b i n g rDNA p e r s i s t s i n Xenopus  l a e v i s oocytes through e a r l y cleavage as Feulgen p o s i t i v e bodies i n 78 the cytoplasm (Thomas et a l . , 1977). The f i b r i l l a r n u c l e o l u s of p r e o v u l a t o r y oocytes i s the morphological m a n i f e s t a t i o n of c e s s a t i o n of RNP s y n t h e s i s (T e s a r i k et a l . , 1983; Geuskens and Alexandre, 1984). Thus, the NOR-silver method does not l a b e l these s t r u c t u r e s (Hansmann et a l . , 1978; Takeuchi, 1984; Takeuchi and Takeuchi, 1986; Antoine et al.., 1987). F o l l o w i n g c e s s a t i o n of t r a n s c r i p t i o n a l a c t i v i t y , rDNA withdraws from compacting n u c l e o l i and condenses on the s u r f a c e (Mirre and S t a h l , 1978; M i r r e and S t a h l , 1981; Geuskens and Alexandre, 1984; Takeuchi, 1984; Antoine et a l . , 1987; T e s a r i k et a l . , 1987a). Thus, a m p l i f i e d rDNA on the s u r f a c e of NLB may induce n u c l e a r membrane r e c o n s t i t u t i o n . The accumulation of granules on the s u r f a c e of some NLB during aging i s s u g g e s t i v e of chromatin. Although, rDNA gene a m p l i f i c a t i o n has not been s p e c i f i c a l l y demonstrated i n the r a t , e x t r a n u c l e o l a r bodies and a t r a n s i t o r y i n c r e a s e i n the number of NOR were observed d u r i n g r a t spermatogenesis (Hofgartner et a l . , 1979; Schultz et a1., 1984). This suggests t h a t rDNA gene a m p l i f i -c a t i o n may a l s o occur i n r a t gametes. If NLB do not induce nuclear membrane fo r m a t i o n , accumulation of SER around NLB may precede autophagocytosis. More than 30 hours p o s t - o v u l a t i o n , SER membranes accumulated around o r g a n e l l e s and i n c r e a s e d numbers of r e s i d u a l bodies were observed i n the r a t oocytes. Increased lysosomal a c t i v i t y and autophagocytosis were a l s o reported i n aged mouse oocytes ( S z o l l o s i , 1971; Longo, 1980) and cleavage stage r a t embryos (Dvorak et a l . , 1977). Autophago-c y t o s i s i s an i n t e g r a l p a r t of i n t r a c e l l u l a r turnover of macro-molecular compounds and senescent o r g a n e l l e s ( A r s t i l a et a l . , 1972). Thus, c y t o p l a s m i c NLB may represent a storage form of 79 maternal macromolecules which are u t i l i z e d during embryogenesis v i a autophagocytosis. The zona p e l l u c i d a undergoes physicochemical a l t e r a t i o n s during i n t r a f o l 1 i c u l a r maturation, which render i t permeable to sperm ( T e s a r i k and Kopecny, 1986; T e s a r i k et a l . , 1988a; Cran, 1989). Chemical t r a n s f o r m a t i o n of the zona continues subsequent to o v u l a t i o n and i s b e l i e v e d to be mediated by low l e v e l CG e x o c y t o s i s (Cran and Cheng, 1985; Yang and Yanagimachi, 1989). In the p i g , p r o g r e s s i v e morphologicai t r a n s f o r m a t i o n of the contents of CG from h i g h l y e l e c t r o n dense (dark) to more e l e c t r o n lucent ( l i g h t ) was observed f o l l o w i n g o v u l a t i o n (Cran, 1989). L i g h t CG underwent premature e x o c y t o s i s i n both p o r c i n e (Cran and Cheng, 1985; Cran, 1989) and murine oocytes ( N i c o s i a et a l . , 1977). The o b s e r v a t i o n of l i g h t CG and o c c a s i o n a l CG e x o c y t o s i s i n the present study suggests that low l e v e l CG e x o c y t o s i s a l s o occurs subsequent to o v u l a t i o n i n the rat.• C o r t i c a l granules which were exocytosed were l i k e l y replaced by newly s y n t h e s i z e d CG s i n c e no apparent r e d u c t i o n i n the number of suboolemmal CG was observed u n t i l 48 to 72 hours post-o v u l a t i o n . Increased numbers of cytoplasmic CG were observed subsequent to o v u l a t i o n due to ongoing p o s t - o v u l a t o r y s y n t h e s i s (Odor, 1960; Zamboni, 1970). P r i o r to movement of the m e i o t i c apparatus d u r i n g aging, i r r e g u l a r i t i e s of the o v e r l y i n g MVF oolemma developed i n many oocytes. The contour of the oolemma i s mediated by the a c t i o n and d i s t r i b u t i o n of a c t i n m i c r o f i l a m e n t s , i n concert with microtubules (Lehtonen and Badley, 1980; Chen and Longo, 1984; Cran, 1987; Lehtonen et a l . , 1988). Loss of suboolemmal a c t i n at the s i t e of p o l a r body formation leads to marked c o n v o l u t i o n s of the membrane 80 adjacent to the midbody (Maro et a l . , 1984). D i s r u p t i o n of the continuous m i c r o f i l a m e n t web l e a d i n g to m u l t i f o c a l a c t i n d i s t r i b u -t i o n i n the MVF protuberance causes d i m p l i n g of the oolemma (Maro et a l . , 1984). Thus, d i s r u p t i o n of the suboolemmal m i c r o f i l a m e n t web was l i k e l y r e s p o n s i b l e f o r the appearance of oolemma i r r e g u l a r -i t i e s on the MVF protuberance. Since the suboolemmal m i c r o f i l a m e n t web i s induced by the presence of chromosomes (Chen and Longo, 1984; Van Blerkom and B e l l , 1986; Maro et a l . , 1986), the appear-ance of membrane i r r e g u l a r i t i e s may i n d i c a t e s u b t l e movement of the u n d e r l y i n g m e i o t i c apparatus or a l t e r a t i o n of i t s i n f l u e n c e on m i c r o f i l a m e n t d i s t r i b u t i o n . Displacement of metaphase chromosomes from the equator of an i n t a c t s p i n d l e was observed i n mouse oocytes aged i n v i v o f o r 15 to 30 hours ( E i c h e n l a u b - R i t t e r et a l . , 1986). This was due to molecular changes i n the s p i n d l e s i m i l a r to those observed during anaphase and telophase of mei o s i s two ( E i c h e n l a u b - R i t t e r et a l . , 1986). In the r a t , displacement of chromosomes on an i n t a c t s p i n d l e was not observed. Chromosome d i s p e r s a l was observed 24 to 30 hours p o s t - o v u l a t i o n i n the present study and was a s s o c i a t e d with frank s p i n d l e d i s o r i e n t a t i o n and d e g r a d a t i o n . D i s p e r s a l of chromosomes was a l s o a s s o c i a t e d with s p i n d l e d i s r u p t i o n i n aged mouse oocytes (Longo, 1980). S p i n d l e d i s r u p t i o n and d i s p e r s a l of chromosomes can be reproduced experimental 1y i n murine oocytes with the m i c r o t u b u l e i n h i b i t o r , nocodazole (Maro et a l . , 1986; Van Blerkom and B e l l , 1986). Thus, t h i s change r e p r e s e n t s f u n c t i o n a l l o s s of s p i n d l e m i c r o t u b u l e s i n aged oocytes. The d i f f e r e n c e i n the e a r l y f a t e of chromosomes during oocyte aging i n the v a r i o u s species may be due to d i f f e r e n t e x p r e s s i o n of the developmental program. However, 81 chromosome displacement on the m e i o t i c s p i n d l e was a s s o c i a t e d with aging i n oocytes r e t r i e v e d from immature, PMSG-treated r a t s i n Part 2. Hence, expression of the developmental program i n aging oocytes of the same sp e c i e s may a l s o vary under d i f f e r e n t p h y s i o l o g i c a l c o n d i t i o n s . During oocyte aging i n the r a t , segmented and i n t a c t oocytes were observed. In oocytes which f a i l e d to segment, the chromosomes s c a t t e r e d i n the c e l l and became pyknotic and the c e l l underwent degeneration. This process was a l s o observed i n aged r a b b i t oocytes ( A u s t i n , 1967). The remainder of the oocytes developed n u c l e i and underwent fragmentation or cleavage. S i m i l a r l y , i n the r a b b i t (Longo, 1974b) and the mouse (Longo, 1980), n u c l e i were only observed i n segmented oocytes. The developmental program thus a r r e s t s p r i o r to n u c l e a r r e c o n s t i t u t i o n and c e l l d i v i s i o n i n a p r o p o r t i o n of aged oocytes. Formation of a second p o l a r body was observed 24 to 30 hours p o s t - o v u l a t i o n i n a small number of oocytes i n the present study. Oocytes from mature r a t s were reported to l o s e the c a p a c i t y to form a second p o l a r body on the afternoon of e s t r u s , i n a s s o c i a t i o n with s c a t t e r of the chromosomes (Zeilmaker and Verhamme, 1974). The MVF protuberance and m i c r o f i l a m e n t web are l o s t as the m e i o t i c apparatus migrates c e n t r a l l y i n the mouse (Webb et a l . , 1986) and i n the r a t . Since the m i c r o f i l a m e n t web mediates c o n s t r i c t i o n of the m e i o t i c furrow, l o s s of the m i c r o f i l a m e n t web precludes p o l a r body formation i n aged oocytes (Webb et a l . , 1986). At 24 hours p o s t - o v u l a t i o n , 50% of aged r a t oocytes s t i l l had suboolemmal chromosomes, which induce the formation of a m i c r o f i l a m e n t web and m i c r o v i l l u s - f r e e s u r f a c e on the o v e r l y i n g oolemma (Chen and Longo, 82 1984; Maro et a l . , 1986; Van Blerkom and B e l l , 1986). T h e r e f o r e , a b s t r i c t i o n of a second p o l a r would presumably be p o s s i b l e i n these oocytes. Fragmented oocytes resembled 2, 3 and 4 c e l l embryos i n a percentage of aged oocytes. In the r a b b i t (Meyer and Longo, 1979), mouse (Longo, 1980) and hamster (Longo, 1975), oocytes morphologi-c a l l y i d e n t i c a l to cleavage stage embryos were a t t r i b u t e d to parthenogenetic a c t i v a t i o n . In the r a t , c l e a v e d oocytes were a l s o m u l t i n u c l e a t e d . M u l t i n u c l e a t i o n was a s s o c i a t e d with i n v i t r o aging i n human (Zamboni et a l . , 1972) and r a t (Niwa and Chang, 1975) oocytes. M u l t i n u c l e a t e d c e l l s were commonly observed i n a s s o c i a t i o n with parthenogenetic a c t i v a t i o n i n the mouse (Braden, 1957; S o l t e r et a l . , 1974; Tarkowski, 1971). Therefore, the presence of m u l t i p l e n u c l e i i n an embryo would suggest that i t was an u n f e r t i l i z e d , a c t i v a t e d oocyte. The occurrence of m u l t i n u c l e a t e d embryos was p r e v i o u s l y re p o r t e d ( A u s t i n and Braden, 1954; Thompson and Zamboni, 1975) and was considered to be the most common abnormality of human preim-p l a n t a t i o n embryos (Lopata et al'. , 1983). M u l t i n u c l e a t e d human embryos were hypothesized, to a r i s e from impaired c y t o s k e l e t a l f u n c t i o n i n v o l v i n g s p i n d l e microtubules ( T e s a r i k et a l . , 1987b). Thus, c y t o s k e l e t a l d y s f u n c t i o n may a l s o be a consequence of i n t r a o v i d u c t a l aging of oocytes. In the human, f a i l u r e of the embryo to a c t i v a t e t r a n s c r i p t i o n was a s s o c i a t e d with m u l t i n u c l e a t e d blastomeres and quiescent n u c l e o l i ( T e s a r i k et a l . , 1987b). Parthenogenotes undergo delayed a c t i v a t i o n of t r a n s c r i p t i o n and, thus, the e a r l y stages a l s o have quiescent n u c l e o l i ( S o l t e r et a l . , 1974; Van Blerkom and Runner, 83 1976). Although coarse granules accumulated on the s u r f a c e of quiescent n u c l e o l i i n a percentage of the n u c l e i , d e f i n i t i v e n u c l e o l a r a c t i v a t i o n was not observed during aging i n the r a t . Thus, m u l t i n u c l e a t i o n and f a i l u r e of t r a n s c r i p t i o n are a s s o c i a t e d with numerous abnormal s t a t e s i n oocytes and embryos. Oocytes aged i n the o v i d u c t were u l t r a s t r u c t u r a l 1 y d i s t i n c t from oocytes aged under d i f f e r e n t circumstances i n the r a t . N u c l e o l a r - l i k e bodies were not observed i n oocytes aged during neuropharmacologic blockade of o v u l a t i o n (Peluso and Butcher, 1974) or i n oocytes from aged r a t s (Peluso et a l . , 1980). In these s t u d i e s , the oocytes remained i n the f o l l i c l e p r i o r to a c t i v a t i o n by the LH surge (Peluso and Butcher, 1974; Peluso et a l . , 1980) and t h e r e f o r e underwent changes which were l i k e l y not d i r e c t e d by the p o s t - o v u l a t o r y developmental program. Morphological evidence of i n t r a f o l 1 i c u l a r and c h r o n o l o g i c a l aging i n c l u d e d a r e d u c t i o n i n the number of suboolemmal c o r t i c a l granules (Peluso and Butcher, 1974; Peluso et a l . , 1980). The authors concluded that t h i s was due to reduced s y n t h e s i s or degeneration of CG i n oocytes aged i n the f o l l i c l e (Peluso and Butcher, 1974) or CG e x o c y t o s i s i n oocytes from aged r a t s (Peluso et a l . , 1980). Fragmentation and m u l t i p l e n u c l e i formation were noted i n a t r e t i c oocytes i n the p r e p u b e r t a l rat (Vazquez-Nin and Sotelo, 1967). The unequal s i z e of fragments and v a r i a b l e presence of up to seven n u c l e i i n i n d i v i d u a l fragments was s i m i l a r t o the a c t i v a t e d oocytes observed i n the present study. While p e r i p h e r a l , e l e c t r o n dense s t r u c t u r e s were observed i n the a t r e t i c oocytes, these were suboolemmal, membrane bound and appeared to be CG (Vazquez-Nin and So t e l o , 1967). T h e r e f o r e , the presence of NLB i n fragmented, aged 84 r a t oocytes would d i s t i n g u i s h them from fragmented, a t r e t i c oocytes. Reduced v e s i c l e content was observed i n MVB of oocytes aged i n the f o l l i c l e during neuropharmacology blockade of o v u l a t i o n (Peluso and Butcher, 1974). In r a t oocytes, MVB are dynamic s t r u c t u r e s and morphology i s h i g h l y v a r i a b l e ( S z o l l o s i , 1967), making i t d i f f i c u l t to assess the s i g n i f i c a n c e of a l t e r e d mor-phology. However, MVB s y n t h e s i z e CG ( S z o l l o s i , 1967; Peluso and Butcher, 1974). The r e d u c t i o n i n MVB content d u r i n g i n t r a f o l 1 i c u l a r aging was c o n s i s t e n t with the decreased number of CG (Peluso and Butcher, 1974). In c o n t r a s t , p o s t - o v u l a t o r y r e g r e s s i o n of MVB was not a s s o c i a t e d with any obvious decrease i n CG during i n t r a o v i d u c t -a l aging. M u l t i v e s i c u l a r bodies may a l s o p l a y an important r o l e i n k a r y o k i n e s i s , as they aggregate around the germinal v e s i c l e and p r o n u c l e i p r i o r to n u c l e a r membrane breakdown (Sot e l o and P o r t e r , 1959; A l b e r t i n i , 1987). Therefore, a l t e r a t i o n of MVB morphology during i n t r a o v i d u c t a l oocyte aging i n the r a t l i k e l y r e f l e c t s the m u l t i p l e f u n c t i o n s performed by t h i s o r g a n e l l e i n p r e i m p l a n t a t i o n development. D i s r u p t i o n and r e d u c t i o n of FA with a decrease i n c y t o p l a s m i c g r a n u l a r i t y was observed i n n o n - a c t i v a t e d oocytes i n the present study and i n oocytes r e t r i e v e d from aged r a t s (Peluso- et a l . , 1980). In the rat and mouse, FA are a storage form of ribosomes r e q u i r e d f o r e a r l y embryonic development (Burkholder et a l . , 1971; Dvorak et a l . , 1977). Breakdown of the FA may t h e r e f o r e be the source of the i n c r e a s e d number of ribosomes observed i n fragmented oocytes i n the present study. However, i n oocytes which f a i l to segment, FA may be c a t a b o l i z e d to m a i n t a i n homeostasis. 85 During the l a t t e r stages of i n t r a o v i d u c t a l oocyte aging i n the r a t , changes c o n s i s t e n t with c e l l u l a r d egeneration were observed. M i t o c h o n d r i a l and SER d i l a t i o n , c l u s t e r i n g of o r g a n e l l e s and i n c r e a s e d lucency of the cytoplasm represent d e g e n e r a t i v e processes a s s o c i a t e d with a f a i l u r e to maintain c e l l u l a r homeostasis ( C h e v i l l e , 1983). S i m i l a r changes were observed i n a t r e t i c and c h r o n o l o g i c a l l y aged r a t oocytes (Vazquez-Nin and S o t e l o , 1967; Peluso et a l . , 1980). These are n o n - s p e c i f i c , degenerative processes common to a l l types of c e l l s ( C h e v i l l e , 1983). In c o n c l u s i o n , t h i s study e s t a b l i s h e d morphological c r i t e r i a f o r the p a t h o l o g i c a l a n a l y s i s of oocytes and embryos of the r a t . I n t r a o v i d u c t a l oocyte aging, was accompanied by s p e c i f i c morphol-o g i c a l a l t e r a t i o n s which r e p r e s e n t e d a c t i v a t i o n of the inherent developmental program and, as such, d i f f e r e d from other forms of u l t r a s t r u c t u r a l pathology i n r a t oocytes. S i m i l a r to other s p e c i e s , the morphological changes i n the r a t were not c o n s i s t e n t l y observed i n oocytes u n t i l a f t e r l o s s of f e r t i 1 i z a b i 1 i t y . While the major changes i n oocyte morphology observed d u r i n g i n t r a o v i d u c t a l aging were s i m i l a r to those observed i n other s p e c i e s , the appearance of cytoplasmic NLB was s p e c i f i c to the r a t . The development of i r r e g u l a r i t i e s on the protuberance and the formation of m u l t i p l e n u c l e i during aging were a t t r i b u t e d to c y t o s k e l e t a l d y s f u n c t i o n . 86 PART 2: A MORPHOLOGICAL INVESTIGATION OF TUBAL OOCYTES RETRIEVED FROM IMMATURE RATS TREATED WITH EITHER 4 OR 40 IU PMSG AND MATURE, SPONTANEOUSLY OVULATING RATS A. RESULTS I. Oocyte R e t r i e v a l and Gross Assessment The o v u l a t i o n r a t e s and range and average number of oocytes r e t r i e v e d from the immature r a t s are presented i n F i g u r e s 10 and I I , r e s p e c t i v e l y . O v u l a t i o n commenced at 24 and 48 hours i n the 40 and 4 IU groups, r e s p e c t i v e l y . A s i g n i f i c a n t i n c r e a s e i n number of oocytes r e t r i e v e d was observed i n the s u p e r o v u l a t e d group from 24 to 48 (p<0.025) and 48 to 72 (p<0.005) hours and i n the 4 IU group from 72 to 96 hours (p<0.025) post-treatment. Although there was a s i g n i f i c a n t d i f f e r e n c e i n the number of oocytes r e t r i e v e d between the superovulated and 4 IU groups at 48 (p<0.025), 72 and 96 (p<0.005) hours, no d i f f e r e n c e was noted between the superovulated group at 48 hr and the 4 IU group at 96 hours. The percentages of ov u l a t o r y r a t s which had ampullary d i s t e n s i o n and the percentages of oocytes which were cumulus-enclosed are shown i n F i g u r e s 12 and 13, r e s p e c t i v e l y . In the 4 IU group, the percentage of o v u l a t o r y r a t s with ampullary d i s t e n s i o n d e c l i n e d from 48 to 72 hr. In c o n t r a s t , the percentage of superovu-l a t e d r a t s with ampullary d i s t e n s i o n d e c l i n e d from 24 to 48 hr and in c r e a s e d again at 72 hr. Ampullary d i s t e n s i o n was not observed i n e i t h e r treatment group at 96 hr. The changes i n the r e t r i e v a l of cumulus-enclosed oocytes f o l l o w e d a s i m i l a r p a t t e r n to that of ampullary d i s t e n s i o n i n both treatment groups. The percentage of cumulus-enclosed oocytes r e t r i e v e d from the 4 IU group decreased s i g n i f i c a n t l y (p<0.01) from 48 to 72 hr, whereas i n the superovu-87 RETRIEVAL OF OOCYTES FROM IMMATURE RATS 60 55-50-45- T SACRIFICE TIME 4IU - • - 40 IU z ' '_••*:< Page 87ft FIGURE 10 The percentage of immature r a t s which had ovulated at each s a c r i f i c e time are i l l u s t r a t e d . The t o t a l number of r a t s i n each group are i n d i c a t e d above the s a c r i f i c e time. FIGURE 11 The range and average number of oocytes r e t r i e v e d from each treatment group of immature r a t s at each s a c r i f i c e time are i l l u s t r a t e d . Range i s denoted by the v e r t i c a l l i n e s . Note that the range of oocytes r e t r i e v e d v a r i e s more between r a t s i n the 40 IU group. The number of o v u l a t o r y r a t s i s i n d i c a t e d beside each p o i n t on the graph. 88 PERCENTAGE OF OVULATORY RATS WITH AMPULLARY DISTENSION 120-1 = :  SACRIFICE TIME |~~ | 4IU 40 IU RECOVERY OF CUMULUS-ENCLOSED OOCYTES FROM IMMATURE RATS Q 120-1 § 110-2 100-g 90-^ 80-0 70-& 60-8 ^ 40-$ 30-1 2D" UJ o 10-53 45 187 140 592 | 86 194 a o 0 24 48 72 96 SACRIFICE TIME • 4IU f^40 IU ••• Page 88fl FIGURE 12 The percentage of o v u l a t o r y r a t s with ampullary d i s t e n s i o n i s i l l u s t r a t e d . The number above each bar denotes the number of r a t s which ovulated i n each group. FIGURE 13 The percentages of oocytes which were e n c l o s e d i n a good cumulus are i l l u s t r a t e d . The number above each bar denotes the number of oocytes r e t r i e v e d at each time. 89 l a t e d group cumulus-enclosed oocytes decreased and i n c r e a s e d s i g n i f i c a n t l y (p<0.005) from 24 to 48 and 48 to 72 hr, r e s p e c t i v e -l y . The percentage of t o t a l oocytes r e t r i e v e d which were f r a g -mented i s presented i n F i g u r e 14. The percentage of fragmented oocytes r e t r i e v e d i n c r e a s e d s i g n i f i c a n t l y (p<0.005) from 48 to 72 hr i n the 40 IU group and from 72 to 96 hr i n both treatment groups. Twenty-six mature r a t s were s a c r i f i c e d on the morning of e s t r u s . Oocytes were r e t r i e v e d b i l a t e r a l l y from d i s t e n d e d ampullae i n 19/26 (73%) of the mature r a t s . Of the seven remaining r a t s , two had marked u t e r i n e d i s t e n s i o n , absence of ampullary d i s t e n s i o n and l a r g e o v a r i a n a n t r a l f o l l i c l e s , two had corpora hemorrhagica but.no ampullary d i s t e n s i o n or oocytes i n the o v i d u c t , two had ampullary d i s t e n s i o n and oocytes i n only one oviduct and one had o v i d u c t a l oocytes but no ampullary d i s t e n s i o n . These 7 r a t s were dis c a r d e d from the study. The number of oocytes r e t r i e v e d from 19 mature r a t s ranged from 12 to 17, with an average of 13.5 oocytes per r a t . A l l the oocytes r e t r i e v e d from the mature r a t s were cumulus-enclosed. The cumulus was removed from 59 oocytes r e t r i e v e d from f o u r r a t s . Ten of the 59 oocytes (17%) were degenerated. Of the 10 degenerated oocytes, 8 were r e t r i e v e d from one r a t (8/13 degenerated) while two were r e t r i e v e d from a second r a t (2/17 degenerated). Fragmented oocytes were not recovered from mature r a t s . 90 GROSS ASSESSMENT OF OOCYTES FROM IMMATURE RATS 120-, : SACRIFICE TIME | | 4 IU £7] 40 IU Page 90 FIGURE 14 The gross assessment of oocyte morphology i s shown. Fragmented oocytes f i r s t appeared at 48 hr i n the 40 IU group and 72 hr i n the 4 IU group. The t o t a l number of oocytes r e t r i e v e d i n each group are denoted by the number above the b a r s . 91 2. L i g h t Microscopy Oocytes t r e a t e d with h y a l u r o n i d a s e were m o r p h o l o g i c a l l y i n d i s t i n g u i s h a b l e from oocytes l e f t w i t h i n the cumulus mass at both the l i g h t and e l e c t r o n m i c r o s c o p i c l e v e l s . A t o t a l of 57 cumulus-enclosed oocytes (CEO) r e t r i e v e d from nine mature r a t s were examined by l i g h t microscopy. Of the 57 oocytes, s i x (10.5%) were degenerated. F i v e of the s i x degenerated oocytes were r e t r i e v e d from one r a t . The degenerated oocytes had a d i s c o n t i n u o u s plasma membrane with a marked r e d u c t i o n i n c y t o p l a s m i c b a s o p h i l i a , FA and o r g a n e l l e content. The o r g a n e l l e s were o f t e n c l u s t e r e d together on one s i d e of the cytoplasm or were s c a t t e r e d randomly throughout the cytoplasm. The chromosomes were u s u a l l y s c a t t e r e d c e n t r a l l y i n the c y t o p i asm with v a r i a b l e degrees of decondensation. Some degenerated oocytes were completely l y s e d and c o n s i s t e d of a l a r g e body of c e l l d e b r i s bounded by the zona p e l l u c i d a . The morphology of v i a b l e , cumulus-enclosed oocytes r e t r i e v e d from mature r a t s was f u l l y d e s c r i b e d i n Part 1. Thus, only the s a l i e n t f i n d i n g s w i l l be d e s c r i b e d here. The oocytes were charac-t e r i z e d by an undulent c e l l o u t l i n e with an obvious p e r i v i t e l 1 i n e space. A f i r s t p o l a r body was observed i n 13/51 (25%) of the v i a b l e oocytes and 5/13 (38.5%) of the p o l a r bodies were l y s e d . Large, m u l t i l o c u l a t e d MVB were observed i n 45/59 (76%) of the oocytes. A t o t a l of 133 CEO were examined from immature r a t s (48 and 85 oocytes from the 4 and 40 IU treatment groups, r e s p e c t i v e l y ) . O v e r a l l , 6% of the CEO r e t r i e v e d at 48 and 72 hr i n the 4 IU group and 7% of those r e t r i e v e d at 24 and 72 hr i n the 40 IU group were degenerated. At 48 hr i n the 40 IU group, 9/9 (100%) of the CEO 92 examined were degenerated and 3/9 had chromosomes s c a t t e r e d on an i n t a c t s p i n d l e s i m i l a r to anaphase. The v i a b l e CEO i n both treatment groups c o n t a i n e d a r e c o g n i z a b l e f i r s t p o l a r body i n 18% and 39% of the oocytes i n the 4 and 40 IU groups, r e s p e c t i v e l y . The m i c r o s c o p i c assessment of oocytes from the 4 and 40 IU groups i s summarized i n Figures 15 and 16. No morphological d i f f e r e n c e s were noted at the l i g h t m i c r o s c o p i c l e v e l between CEO from the immature r a t s t r e a t e d with e i t h e r 4 or 40 IU PMSG. Therefore, these oocytes were grouped t o g e t h e r f o r comparison with CEO from the mature r a t s . The CEO from the immature r a t s d i f f e r e d from those r e t r i e v e d from the mature r a t s i n s e v e r a l regards. The p e r i v i t e l 1 i n e space of CEO from the immature r a t s was more narrow and the s u r f a c e contour of the oocytes was smoother with a more v a r i a b l y s i z e d , cytoplasmic protuberance at one pole ( F i g u r e 17a). The oolemmal s u r f a c e of the protuberance was i r r e g u l a r i n 7/34 (20%) oocytes at 72 hr i n the 4 IU group and 3/16 (19%) and 22/52 (42%) at 24 and 72 hr, r e -s p e c t i v e l y , i n the 40 IU group ( F i g u r e 17b). Large, m u l t i 1 o c u l a t e d MVB (Figure 17c) were present i n the cytoplasm of 57/116 (49%) v i a b l e CEO from the immature r a t s , s i g n i f i c a n t l y fewer than observed i n CEO from mature r a t s (p<0.01). Small MVB were commonly observed i n a l l oocytes. Two morphological changes were observed s p e c i f i c a l l y i n CEO r e t r i e v e d at 72 hr. Two to three but o c c a s i o n a l l y up to ei g h t well demarcated, densely b a s o p h i l i c , n u c l e o l a r - l i k e bodies were observed i n the cytoplasm of 9/34 (26.5%) and 2/52 (4%) of oocytes i n the 4 and 40 IU groups, r e s p e c t i v e l y . Unusual o r i e n t a t i o n of the chromosomes was a l s o observed. In 5/34 (15%) and 11/52 (21%) of the 93 120 MICROSCOPIC ASSESSMENT OF OOCYTES FROM IMMATURE RATS (4 IU) 24 48 72 SACRIFICE TIME | | NORMAL AGED MICROSCOPIC ASSESSMENT OF OOCYTES FROM IMMATURE RATS (40 IU) 12Ch SACRIFICE | | NORMAL £ 3 AGED Page 93 ft FIGURE 15 The percentages of oocytes which were assessed to be normal or aged at each s a c r i f i c e time i n the 4 IU group are shown. The numbers above the bars denote the t o t a l number of oocytes examined. FIGURE 16 The percentages of oocytes which were assessed to be normal or aged at each s a c r i f i c e time i n the 40 IU group are shown. The a s t e r i s k above the bar of fragmented oocytes at 24 hr i n d i c a t e s that 2 degenerated, germinal v e s i c l e stage oocytes were r e t r i e v e d . The numbers above the bars denote t o t a l number of oocytes examined. 94 V Page 94 fi FIGURE 17 A. Inta c t oocyte r e t r i e v e d at 72 hr from a r a t t r e a t e d with 40 IU PMSG. Note the smooth c e l l o u t l i n e and small p e r i v i t -e l l i n e space (arrow). The m e i o t i c apparatus i s perpendicu-l a r to the o v e r l y i n g MVF oolemma (arrowhead). M a g n i f i c a t i o n = 625x. B. Intact oocyte r e t r i e v e d at 72 hr from a s u p e r o v u l a t e d r a t . Note the i r r e g u l a r protuberance (arrowhead). The o v e r a l l o u t l i n e of the c e l l i s more undulent. M a g n i f i c a t i o n = 565x. C. Intact oocyte r e t r i e v e d at 72 hr from a r a t t r e a t e d with 40 IU PMSG. A l a r g e m u l t i v e s i c u l a r body i s present i n the centre (arrow). The p e r i v i t e l 1 i n e space i s small and the c e l l contour i s smooth (arrowhead). M a g n i f i c a t i o n = 625x. D. Intact oocyte r e t r i e v e d at 72 hr from a r a t t r e a t e d with 4 IU PMSG. Chromosomes are s c a t t e r e d on an i n t a c t s p i n d l e beneath the oolemma (arrow). M a g n i f i c a t i o n = 565x. E. Int a c t oocyte r e t r i e v e d at 72 hr from a r a t t r e a t e d with 40 IU PMSG. Note the i r r e g u l a r s u r f a c e o v e r l y i n g the me i o t i c apparatus (arrow). The chromosomes have s c a t t e r e d on the s p i n d l e (arrowhead). M a g n i f i c a t i o n = 565x. F. Int a c t oocyte r e t r i e v e d at 72 hr from a r a t t r e a t e d with 4 IU PMSG. Note the markedly i r r e g u l a r protuberance with r o t a t e d m e i o t i c apparatus (arrowhead). The protuberance has developed shoulders (small arrows) s i m i l a r to an ext r u d i n g p o l a r body. A l a r g e v e s i c u l a r aggregate i s adjacent to the me i o t i c apparatus ( l a r g e arrow). M a g n i f i c a t i o n = 565x. 95 CEO i n the 4 and 40 IU groups, r e s p e c t i v e l y , the chromosomes were s c a t t e r e d along the s p i n d l e ( F i g u r e 17d). An i r r e g u l a r protuberance and NLB were observed on 3/5 and 8/11 of the oocytes with s c a t t e r e d chromosomes i n the 4 and 40 IU groups, r e s p e c t i v e l y ( F i g u r e 17e). In 2/32,(6%) and 1/52 (2%) oocytes i n the 4 and 40 IU groups, r e s p e c t i v e l y , the chromosomes were present i n a prominent, markedly i r r e g u l a r protuberance with s h o u l d e r s ( F i g u r e 1 7 f ) . NLB were a l s o present i n the cytoplasm of 2 of these oocytes. In 5/52 (10%) oocytes i n the 40 IU group, the m e i o t i c apparatus was l o c a t e d more c e n t r a l l y i n the cytoplasm with l o s s of the MVF protuberance ( F i g u r e 18a). Cumulus-free oocytes (CFO) r e t r i e v e d from immature r a t s were a l s o examined. Of 65 CFO, 6 (9%) were degenerated, 2 were degener-ated germinal v e s i c l e stage oocytes, 36 (55%) were i n t a c t and 21 (32%) were fragmented. S i m i l a r to CEO, the CFO d i d not d i f f e r m o r p h o l o g i c a l l y between treatments. T h e r e f o r e , these oocytes were pooled i n t o two groups f o r d e s c r i p t i o n : i n t a c t and fragmented. I n t a c t CFO had a more i r r e g u l a r oolemmal su r f a c e than CEO with a wider p e r i v i t e l 1 i n e space and 7/36 (16%) were elongated with a concave f a c e . P o l a r bodies were not observed. Chromosomes were suboolemmal i n 21/36 (58%) i n t a c t oocytes and of these 16/21 (76%) had an i r r e g u l a r protuberance and 3/16 (19%) had chromosomes s c a t t e r e d on the s p i n d l e . Chromosomes were c e n t r a l l y l o c a t e d i n 15/36 (42%) and these were e i t h e r i n metaphase (7), s c a t t e r e d on the s p i n d l e (1) or s c a t t e r e d i n the cytoplasm (7) with no s p i n d l e v i s i b l e . N u c l e o l a r - l i k e bodies were observed i n 33/36 (92%) oocytes. Two of the i n t a c t oocytes w i t h suboolemmal chromosomes had a markedly prominent and i r r e g u l a r protuberance with shoulders 96 ..j. Page 96 ft FIGURE 18 A. Intact oocyte r e t r i e v e d at 72 hr from a r a t t r e a t e d with 4 IU PMSG. Note the i r r e g u l a r s u r f a c e (arrow) above the m e i o t i c apparatus which has r o t a t e d and moved s l i g h t l y c e n t r a l l y with s c a t t e r e d chromosomes (arrowhead). M a g n i f i c a t i o n = 565x. B. Intact oocyte r e t r i e v e d at 72 hr from a superovulated r a t . The protuberance i s i r r e g u l a r (arrowhead). A shoulder (arrow) i s present on one s i d e of the oocyte. M a g n i f i c a t i o n = 565x. C. Oocyte r e t r i e v e d at 72 hr from a superovulated r a t . The m e i o t i c apparatus has been extruded i n t a c t (arrow). Note the re d u c t i o n i n f i b r i l l a r a r r a y s i n the cytoplasm ( l a r g e arrowheads). M a g n i f i c a t i o n = 565x. D. Fragmented oocytes r e t r i e v e d at 72.hr from a super-ovulated r a t . One oocyte has 2 l a r g e c e l l s with c l o s e c e l l a p p o s i t i o n . The other oocyte has v a r i a b l y s i z e d fragments, one of which c o n t a i n s m u l t i p l e n u c l e i (arrow). M a g n i f i c a t i o n = 350x. E. Fragmented oocytes r e t r i e v e d at 96 hr from a super-ovulated r a t . The cytoplasm i s darker than that of i n t a c t oocytes. Fragments are m u l t i n u c l e a t e d (arrow) with organ-e l l e p o l a r i z a t i o n (small arrowheads) and o c c a s i o n a l l y s i s ( l a r g e arrowhead). M a g n i f i c a t i o n = 280x. 97 ( F i g u r e 18b). Both of these oocytes were r e t r i e v e d from the super-ovulated group. Two a d d i t i o n a l oocytes from the superovulated group had extruded the e n t i r e m e i o t i c apparatus i n an apparent p o l a r body (Figure 18c). No chromosomes remained i n the cytoplasm of these two c e l l s . Fragmented oocytes ( F i g u r e 18d,e) c o n s i s t e d of 2 to 12 c e l l fragments of v a r i a b l e s i z e and shape. The p e r i v i t e l 1 i n e space was l a r g e r than that of i n t a c t oocytes and contained more c e l l d e b r i s . Fragmented oocytes contained m u l t i p l e , s m a l l , round to i r r e g u l a r n u c l e i , u s u a l l y with one compact n u c l e o l u s . V a r i a b l y s i z e d NLB were present i n a l l fragmented oocytes. Vacuoles and c l u s t e r e d c y t o p l a s -mic o r g a n e l l e s were commonly observed, while MVB and VA were r a r e l y observed. 3. E l e c t r o n Microscopy The u l t r a s t r u c t u r a l morphology of cumulus enclosed oocytes from mature, spontaneously o v u l a t i n g r a t s was d e s c r i b e d f u l l y i n Part 1. I n t a c t CEO from the immature r a t s d i f f e r e d from those of mature r a t s i n two ways. The contour of CEO from immature r a t s was more smooth and l e s s undulent. W i t h i n the p e r i v i t e l 1 i n e space, membrane loops e i t h e r f r e e or tenuously attached to the oocyte s u r f a c e were observed. The MVB tended to be s m a l l e r with reduced v e s i c u l a r content i n some oocytes ( F i g u r e 19a). A s i m i l a r continuum of morphological changes was observed i n CEO from the immature r a t s i n both treatment groups. Metaphase chromosomes were observed i n a smooth to i r r e g u l a r MVF protuberance and, with movement of the chromosomes away from the oolemma, the MVF protuberance and the suboolemmal m i c r o f i l a m e n t web were l o s t . Page 98R FIGURE 19 A. Intact oocyte r e t r i e v e d 72 hr post-treatment from a r a t t r e a t e d with 4 IU PMSG. The p e r i v i t e l 1 i n e space i s small with cytoplasmic processes on the s u r f a c e of the oocyte. N u c l e o l a r - l i k e bodies are present i n the cytoplasm (arrowheads). M a g n i f i c a t i o n = 2,500x. B. High m a g n i f i c a t i o n of a n u c l e o l a r - l i k e body i n an oocyte r e t r i e v e d 48 hr post-treatment from a superovulated r a t . The s t r u c t u r e i s not bound by a membrane. M a g n i f i c a t i o n = 20,000x. C. Intact oocyte r e t r i e v e d 48 hr post-treatment from a superovulated r a t . Note the m i c r o v i l l i on the s u r f a c e ( l a r g e arrowhead) and the n u c l e o l a r - l i k e bodies (small arrowheads) i n the cytoplasm. M a g n i f i c a t i o n = 3,543x'. D. Degenerated germinal v e s i c l e stage oocyte r e t r i e v e d 24 hr post-treatment from a superovulated r a t . The cytoplasm i s e l e c t r o n lucent and la c k s a g r a n u l a r , homogeneous matrix. F i b r i l l a r arrays are d i s r u p t e d and reduced i n number. There i s moderate smooth endoplasmic r e t i c u l u m d i l a t i o n (arrowheads). M u l t i v e s i c u l a r bodies and v e s i c u l a r aggregates are absent. Two aggregates of chromatin are present on the s u r f a c e of the nucleolus (arrows). M a g n i f i c a t i o n = 2,500x. 99 In some oocytes, chromosomes were s c a t t e r e d on the suboolemmal s p i n d l e , s i m i l a r to anaphase. Cumulus-enclosed oocytes r e t r i e v e d at 24 and 48 hr i n the 40 and 4 IU groups, r e s p e c t i v e l y , had o r g a n e l l e s which were c l u s t e r e d c e n t r a l l y and p e r i p h e r a l l y . Oocytes r e t r i e v e d at 72 hr tended to have more randomly s c a t t e r e d o r g a n e l l e s and t h i s was a s s o c i a t e d with fewer and s m a l l e r FA i n the cytoplasm. E l e c t r o n - d e n s e , f i b r i l l a r , 300 to 600 nm diameter NLB were observed i n the cytoplasm i n both treatment groups at 72 hr (Fi g u r e 19a,b). The degenerated germinal v e s i c l e stage oocytes had a marked r e d u c t i o n i n c y t o s o l g r a n u l a r i t y , VA, MVB and FA. There was d i l a t i o n of MC and SER. The germinal v e s i c l e had numerous prominent n u c l e a r pores and an undulent o u t l i n e . Two f o c i of condensed chromatin were present on the s u r f a c e of the l a r g e quiescent n u c l e o l u s ( F i g u r e 19d). Coincident w i t h cumulus d i s p e r s a l , the contour of the i n t a c t oocytes and the MVF protuberance became more i r r e g u l a r ( F i g u r e 20a). In some cases, there was marked i r r e g u l a r i t y of the protuber-ance (Figure 20b). Electron-dense, homogeneous, f i b r i l l a r n u c l e o l i were a s s o c i a t e d w i t h chromosomes both p r i o r t o, and a f t e r , membrane r e c o n s t i t u t i o n ( F i g u r e 20c). C e n t r a l l y l o c a t e d chromosomes, d i v o r c e d from s p i n d l e m icrotubules, underwent n u c l e a r membrane r e c o n s t i t u t i o n , forming m u l t i p l e , small round n u c l e i ( F i g u r e 20d). The NLB i n c r e a s e d i n s i z e from 300-600 nm to up to 3000 nm diameter (Figure 19c, 20c,d). The l a r g e r NLB o c c a s i o n a l l y c o ntained m u l t i p l e small spaces through which the c y t o s o l was e v i d e n t . Some NLB had attenuated lengths of SER on the s u r f a c e and others were completely enclosed w i t h i n a double membrane. A p e r i p h e r a l aggregate of coarse granules was o c c a s i o n a l l y present on the 100 ~, Page 100 ft FIGURE 20 A. I n t a c t oocyte r e t r i e v e d 72 hr post-treatment from a superovulated r a t . The s u r f a c e o v e r l y i n g the chromosomes i s i r r e g u l a r (arrows). The cytoplasm i s moderately e l e c -tron dense and homogeneously g r a n u l a r . A m u l t i v e s i c u l a r body ( l a r g e arrowhead) and v e s i c u l a r aggregate (small arrowhead) are present. Mitochondria are s c a t t e r e d between c l u s t e r s of f i b r i l l a r a r r a y s . M a g n i f i c a t i o n = 2,500x. B. I n t a c t oocyte r e t r i e v e d 48 hr post-treatment from a superovulated r a t . There i s marked b l e b b i n g of the micro-v i l l u s f r e e s u r f a c e . Chromosomes have s c a t t e r e d ( l a r g e arrows) on the d i s r u p t e d s p i n d l e (small arrow). The sub-oolemmal microfilament web i s i n t a c t . F i b r i l l a r arrays are reduced i n number and l i n e a r segments of smooth endo-plasmic r e t i c u l u m compartmentalize the cytoplasm (small arrowhead's). M a g n i f i c a t i o n = 2,500x. C. I n t a c t oocyte r e t r i e v e d 96 hr post-treatment from a superovulated r a t . The chromosomes ( l a r g e arrowhead) have moved c e n t r a l l y i n the cytoplasm i n a s s o c i a t i o n with a d i s r u p t e d s p i n d l e (small arrowhead). Recondensation of a nu c l e o l u s i s apparent (open arrow). The m u l t i v e s i c u l a r bodies have aggregated i n t o a l a r g e sheet ( l a r g e arrow). N u c l e o l a r -l i k e bodies (small arrows) are present i n the cytoplasm and m i c r o v i l l i can be seen on the c e l l s u r f a c e . M a g n i f i c a t i o n = 2,500x. D. I n t a c t oocyte r e t r i e v e d 48 hr post-treatment from a superovulated r a t . Few m i c r o v i l l i or cyt o p l a s m i c processes are present on the c e l l s u r f a c e . Chromosomes have moved c e n t r a l l y and s c a t t e r e d o f f the s p i n d l e (arrowheads). There i s n u c l e a r membrane r e c o n s t i t u t i o n around i n d i v i d u a l chromo-somes. L i n e a r lengths of smooth endoplasmic r e t i c u l u m extend from VA and mitochondria i n t o the adjacent cytoplasm (small arrows). Few f i b r i l l a r a r r a y s are present i n the cytoplasm. One l a r g e suboolemmal n u c l e o l a r - l i k e body i s present ( l a r g e arrow). M a g n i f i c a t i o n = 2,812x. 101 s u r f a c e of NLB, e i t h e r with or without a membrane. Lamellar G o l g i s t r u c t u r e s appeared and the SER became i n c r e a s i n g l y more elongated, g i v i n g the cytoplasm a segmented appearance ( F i g u r e 20d). Oocyte fragmentation was observed i n a s s o c i a t i o n with n u c l e i formation. The n u c l e i , c o n s i s t i n g of an e l e c t r o n - l u c e n t nucleoplasm alone or with f o c i of condensed chromatin, were o c c a s i o n a l l y c l u s t e r e d together and the c l u s t e r f u r t h e r surrounded by an a d d i t i o n a l double membrane. Many n u c l e i c o ntained a dense, f i b r i l l a r n u c l e o l u s which i n some cases had coarse granules c l u s t e r e d on the n u c l e o l a r s u r f a c e ( F i g u r e 21a). The nuclear membrane was moderately to markedly d i l a t e d i n some n u c l e i ( F igure 21a). Fragmented oocytes had numerous m i c r o v i l l i on the s u r f a c e with a c t i v e m i c r o p i n o c y t o s i s . Suboolemmal c o r t i c a l granules were v a r i a b l y present between fragments w i t h i n the same oocyte ( F i g u r e 21a,c). Fewer MVB were observed, with fewer c o n s t i t u e n t v e s i c l e s and MVB devoid of v e s i c l e s with m i l d d i l a t i o n of the s a c c u l e s and tubules were o f t e n p r e s e n t . As observed i n the i n t a c t oocytes, NLB oft e n had attenuated lengths of SER on the s u r f a c e and others were completely enclosed w i t h i n a double membrane ( F i g u r e 21b). Coarse granules were o c c a s i o n a l l y present on the s u r f a c e of NLB (F i g u r e 21b). Lamellar G o l g i ( F i g u r e 21b) and d i l a t e d m i t o c h o n d r i a and SER were commonly observed. Cytoplasmic p o l a r i z a t i o n of o r g a n e l l e s was o f t e n seen and many smal l fragments c o n s i s t e d s o l e l y of packed FA. A l a r g e amount of c e l l d e b r i s was present i n the p e r i v i t e l 1 i n e space (Figure 21c). 102 Page 102R FIGURE 21 A. Fragmented oocyte r e t r i e v e d 96 hr post-treatment from a r a t t r e a t e d with 4 IU PMSG. C o r t i c a l granules are present (small arrowhead). F i b r i l l a r a r r a y s are v a r i a b l y present i n fragments. N u c l e i are s c a t t e r e d between fragments. There i s m i l d n u c l e a r membrane d i l a t i o n ( l a r g e arrowhead). Condensed chromatin i s forming a rim around a f i b r i l l a r n u c l e o l u s (arrow). M a g n i f i c a t i o n = 5,000x. B. Fragmented oocyte r e t r i e v e d 72 hr post-treatment from a superovulated r a t . Segments of smooth endoplasmic r e t i c u l u m completely enclose the n u c l e o l a r - l i k e body (arrow). One n u c l e o l a r - l i k e body with p e r i p h e r a l coarse granules i s completely enclosed w i t h i n a double membrane ( l a r g e arrow-head). One nucleus with t y p i c a l nucleoplasm and f i b r i l l a r n u c l e o l u s i s present. A nascent G o l g i i s present (small arrowhead). Note the m i c r o v i l l i on the c e l l s u r f a c e . M a g n i f i c a t i o n = 5,000x. C. Fragmented oocyte r e t r i e v e d 96 hr post-treatment from a superovulated r a t . One l a r g e fragment i s i r r e g u l a r i n shape with a remnant of the midbody i n the adjacent p e r i v i t e l 1 i n e space ( l a r g e arrow). There i s marked v a r i a t i o n i n c o r t i c a l granule d i s t r i b u t i o n between fragments (small arrow). M u l t i v e s i c u l a r bodies are s c a t t e r e d among fragments with scant content. F i b r i l l a r a r r a y s are numerous and one n u c l e o l a r - l i k e body i s present adjacent to a nascent g o l g i (small arrowhead). Note the moderately e l e c t r o n dense c y t o s o l with decreased g r a n u l a r i t y . There i s an i n c r e a s e i n p e r i v i t e l 1 i n e space d e b r i s and small c y t o p l a s m i c blebs are being pinched o f f (open arrow). M a g n i f i c a t i o n = 2,500x. 103 B. DISCUSSION 1. O v u l a t i o n and Gross Observations In the present study, o v u l a t i o n was observed p r i o r , and subsequent, to the estimated time of the endogenous gonadotrophin surge i n both the 4 and 40 IU treatment groups. T h i s led to a pro-g r e s s i v e and p r o p o r t i o n a l i n c r e a s e i n the number of aged oocytes r e t r i e v e d at each subsequent s a c r i f i c e time. No d i f f e r e n c e s a t t r i b u t a b l e to a d i r e c t e f f e c t of the s u p e r o v u l a t o r y dose of PMSG were observed. However, morphological d i f f e r e n c e s between both unaged and aged oocytes from mature and immature r a t s were observed. O v u l a t i o n i n immature r a t s g i v e n 4-10 IU PMSG occurs 10 to 12 hours subsequent to the endogenous gonadotrophin surge 52 to 54 hours post-treatment ( S t r a u s s & Meyer, 1962; Zarrow and G a l l o , 1969; Hagino and G o l d z i e h e r , 1970; De La L a s t r a et al . , 1972; H i l l e n s j o et a l . , 1974; Wilson et a l . , 1974; Sasamoto and Johke, 1975; Ahren et a l . , 1978; Kostyk et a l . , 1978; M i l l e r and Arm-stro n g , 1981a; Walton et a l . , 1983; Yun et a l . , 1987; Yun, 1989). T h i s c o r r e l a t e s w i t h the present f i n d i n g that 100% of ra t s i n the 4 IU group had ov u l a t e d at 72 hours. However, e a r l y o v u l a t i o n i n the low dose group was not re p o r t e d by a l l workers. In the present study, an average of 11 oocytes were r e t r i e v e d from 33% of the r a t s s a c r i f i c e d at 48 hours. M i l l e r & Armstrong (1981a) reported e a r l y o v u l a t i o n i n 14% of immature r a t s t r e a t e d with 4 IU PMSG, but no mention was made of oocyte number or exact time of o v u l a t i o n . Kostyk et a l (1978) recovered one oocyte from one of twelve r a t s at both 24 and 48 hours a f t e r a d m i n i s t r a t i o n of 10 IU of PMSG. De La 104 L a s t r a et a l (1972) recovered an average of 0.5 oocytes at 48 hours from 27% of r a t s g i v e n 10 IU PMSG. Ther e f o r e , the present study agrees with the f i n d i n g s of others that e a r l y o v u l a t i o n i s induced i n up to 27% of animals p r i o r to the endogenous gonadotrophin surge (De La L a s t r a et a l . , 1972; Kostyk et a l . , 1978; M i l l e r and Armstrong, 1981a). O v u l a t i o n p r i o r to the endogenous surge i s commonly seen i n immature r a t s given a superovulatory dose of PMSG (Zarrow and G a l l o , 1969; De La L a s t r a et a l . , 1972; Kostyk et a l . , 1978; M i l l e r and Armstrong, 1981a; Walton et a l . , 1983; Yun et a l . , 1987). The f i r s t wave of o v u l a t i o n i s due to the d i r e c t a c t i o n of the LH moiety of PMSG on f o l l i c l e s at the most advanced stages of gonadotrophin independent growth (Wilson and Zarrow, 1962; M i l l e r and Armstrong, 1981a; H i r s h f i e l d , 1989) and can be reproduced by the a d m i n i s t r a t i o n of HCG alone (Sugawara et a l . , 1969; De La L a s t r a et a l . , 1972; Neal and Baker, 1973; Kostyk et a l . , 1978). A d m i n i s t r a t i o n of HCG alone to immature r a t s induces o v u l a t i o n of an average of 7 oocytes i n 25% of animals at 24 hours (Kostyk et a l . , 1978). " In the superovulated group, 86% and 100% of the r a t s o vulated 24 and 48 hours post-treatment, r e s p e c t i v e l y . These f i n d i n g s agree with those of De La L a s t r a et a l (1972) and Walton et a l (1983). However, o v u l a t o r y r a t e s of 20-29% and 22-60% at 24 and 48 hours, r e s p e c t i v e l y , were r e p o r t e d by others (Zarrow and G a l l o , 1969; Kostyk et a l . , 1978; Yun et a l . , 1987). These d i f f e r e n c e s may be due to the s t r a i n of r a t s used, although, no s i g n i f i c a n t d i f f e r -ences i n o v u l a t o r y r a t e s were found to e x i s t between Purdue and Sprague-Dawley r a t s (Zarrow and Quinn, 1963). 105 The e a r l y o v u l a t o r y response i n r a t s g i v e n e i t h e r a low or superovulatory dose of PMSG may r e f l e c t d i f f e r e n t i a l s e n s i t i v i t y of i n d i v i d u a l r a t s to the LH moiety of PMSG. Subpopul a t i o n s of f o l l i c l e s a l s o e x i s t i n the ovary with d i f f e r i n g s e n s i t i v i t y to hormonal s t i m u l a t i o n ( E r i c k s o n et a l . , 1983). Wide v a r i a t i o n i n i n d i v i d u a l response was re p o r t e d i n immature r a t s s u p e r o v u l a t e d with PMSG (Sherman et a l . , 1982; Walton et a l . , 1983; Evans and Armstrong, 1984) and s u p e r o v u l a t i o n i s o c c a s i o n a l l y observed with the low dose (Wilson and Zarrow, 1962; Evans and Armstrong, 1984). However, the g r e a t e r magnitude of the e a r l y o v u l a t o r y response i n both treatment groups compared with those r e p o r t e d p r e v i o u s l y (De La L a s t r a et a l . , 1972; Kostyk et a l . , 1978) may be due to higher FSH/LH a c t i v i t y i n the batch of PMSG used (De La L a s t r a et a l . , 1972; Kostyk et a l . , 1978; Murphy et a l . , 1984; Yun et a l . , 1987). The l e v e l of FSH/LH a c t i v i t y i s d i r e c t l y r e l a t e d to the number of o v u l a t i o n s induced by PMSG (Murphy et a l . , 1984). The FSH/LH a c t i v i t y i n the batch of PMSG used i n t h i s study was l i k e l y high s i n c e an average of 11 oocytes were r e t r i e v e d at 48 hours from 33% of the r a t s t r e a t e d w i t h 4 IU. In most cases, the low dose of PMSG has a subovulatory l e v e l of LH a c t i v i t y (De La L a s t r a et a l . , 1972). However, i n c r e a s i n g doses of HCG alone do not induce o v u l a t i o n of i n c r e a s e d numbers of oocytes i n immature r a t s (Kostyk et a l . , 1978). T h e r e f o r e , i t would appear that the FSH a c t i v i t y of PMSG plays a r o l e i n determining the magnitude of e a r l y o v u l a t i o n at both the high and low dose, as i t does with s u p e r o v u l a t o r y doses i n a s s o c i a t i o n with the second wave of o v u l a t i o n (Murphy et a l . , 1984). The f i n a l o v u l a t o r y p a t t e r n would then be due to the combined e f f e c t s of i n d i v i d u a l s e n s i t i v i t y to g o n a d o t r o p i n s and 106 the l e v e l of exogenous FSH/LH a c t i v i t y . In the present study, 3/14 su p e r o v u l a t e d r a t s s a c r i f i c e d at 72 hours had apparently completed o v u l a t i o n 24 hours e a r l i e r as ampullary d i s t e n s i o n and cumulus-enclosed oocytes were absent, yet 30-48 oocytes were r e t r i e v e d from each r a t . Yun et a l (1988) a l s o recovered a superovulatory number of oocytes from immature r a t s at 48 hours with no f u r t h e r i n c r e a s e i n number at 72 hours. T h i s was a t t r i b u t e d to batch to batch v a r i a b i l i t y i n FSH/LH a c t i v i t y of PMSG and to the wide v a r i a t i o n i n o v u l a t o r y response between i n d i v i d u a l s (Yun et a l . , 1988). Within a c r i t i c a l dosage range, a d i r e c t dose response r e l a t i o n s h i p e x i s t s between PMSG and number of oocytes o v u l a t e d (Wilson and Zarrow, 1962; Kostyk et a l . , 1978). A dose range of 3-10 IU PMSG w i l l induce o v u l a t i o n of 8-18 oocytes, while 15-40 IU w i l l induce o v u l a t i o n of 32-63 oocytes (Wilson and Zarrow, 1962; De La L a s t r a et a l . , 1972; Kostyk et a l . , 1978; Walton et a l . , 1983; Yun et a l . , 1987). These f i n d i n g s are i n agreement with the present study where an average of 10 and 42 oocytes were r e t r i e v e d 72 hours post-treatment from r a t s g i v e n 4 or 40 IU PMSG, r e s p e c t i v e l y . From 24-48 hours post-treatment, 9-20 oocytes were r e t r i e v e d on average from the 40 IU group, which i s a l s o c o n s i s t e n t with the 4-22 oocytes reported i n t h i s time p e r i o d by other workers (Zarrow and G a l l o , 1969; De La L a s t r a et a l . , 1972; Walton et a l . , 1983; Yun et a l . , 1987). This number d i d not d i f f e r s i g n i f i c a n t l y from the number of oocytes r e t r i e v e d at 72 hours from r a t s t r e a t e d with 4 IU PMSG (Zarrow and G a l l o , 1969; De La L a s t r a et a l . , 1972; Walton et a l . , 1983; Yun et a l . , 1987). I t was thus suggested t h a t the oocytes which ovulated i n the f i r s t wave were the same cohort as 107 those which ovulated at 72 hours i n the 4 IU group (Wilson and Zarrow, 1962; Kostyk et a l . , 1978). There was a s i g n i f i c a n t i n c r e a s e i n the number of oocytes r e t r i e v e d i n the 40 IU group from 24 to 48 hours and 48 to 72 hours post-treatment. While there was no s i g n i f i c a n t i n c r e a s e i n the num-ber of oocytes o v u l a t e d from 48 to 72 hours i n the 4 IU group, there was a s i g n i f i c a n t i n c r e a s e from 72 to 96 hours. Since oocytes r e t r i e v e d at 96 hours i n the 4 IU group l a c k e d a cumulus, they were l i k e l y ovulated 24 hours e a r l i e r s u g g e s t i n g t h a t , at 72 hours, the ra t s were s a c r i f i c e d j u s t b e f o r e the end of o v u l a t i o n or before a l l the ovulated oocytes reached the ampullae. There was no s i g n i f i c a n t d i f f e r e n c e i n the number of oocytes r e t r i e v e d from superovulated r a t s at 48 hours and from the 4 IU group at 96 hours, suggesting that the oocytes o v u l a t e d i n the f i r s t wave of s u p e r o v u l a t i o n were those which o v u l a t e d i n response to the endogenous surge induced by the low dose. The s i g n i f i c a n t i n c r e a s e i n number of oocytes r e t r i e v e d from the 40 IU group at 72 and 96 hours compared with the 4 IU group i n d i c a t e s the recruitment of a d d i t i o n a l oocytes f o r the second wave of s u p e r o v u l a t i o n . The recovery of degenerated oocytes was r e p o r t e d to in c r e a s e with time f o l l o w i n g a d m i n i s t r a t i o n of a s u p e r o v u l a t o r y dose of PMSG ( M i l l e r and Armstrong, 1981a; Yun et a l . , 1987). These authors considered a l l abnormal forms to be degenerated oocytes ( M i l l e r and Armstrong, 1981a; Yun et a l . , 1987). In the present study, abnormal oocytes were c l a s s i f i e d as e i t h e r degenerated or fragmented to determine whether the treatment had d i f f e r e n t e f f e c t s on oocyte q u a l i t y . Both v i a b l e and degenerated cumulus-enclosed oocytes were r e t r i e v e d from mature and immature r a t s . The occurrence of degen-108 e r a t e d oocytes d i d not i n c r e a s e w i t h p o s t - o v u l a t o r y time and the high number of degenerated oocytes o c c a s i o n a l l y r e t r i e v e d was apparently a phenomenon of i n d i v i d u a l r a t s r a t h e r than an e f f e c t of treatment with PMSG or i n t r a o v i d u c t a l aging. Sherman et a l (1982) a l s o recovered a d i s p r o p o r t i o n a t e number of degenerated oocytes from a small percentage of mature r a t s . S i m i l a r l y , v i a b l e and degenerated cumulus-enclosed oocytes were r e t r i e v e d from mature mice (Longo, 1980). T h i s p a t t e r n of oocyte degeneration was a l s o observed i n Parts 3 and 4. Fragmented oocytes f i r s t appeared 24 hours a f t e r the com-mencement of o v u l a t i o n . A p r o g r e s s i v e i n c r e a s e was then observed i n both groups over the remainder of the o b s e r v a t i o n p e r i o d . Frag-mented oocytes were not observed i n the f i r s t wave of o v u l a t i o n by others but were r e t r i e v e d w i t h i n c r e a s i n g frequency from 36 to 60 hours a f t e r a d m i n i s t r a t i o n of 40 IU PMSG ( M i l l e r and Armstrong, 1981a; Walton et a l . , 1983; Walton and Armstrong, 1983; Yun et a l . , 1987; Yun et a l . , 1988). At 72 hours, the percentage of abnormal oocytes dropped due to the second wave of o v u l a t i o n (Walton et a l . , 1983; Yun et a l . , 1987). In the mouse, hamster, r a b b i t and r a t , oocyte fragmentation occurs w i t h i n 24 hours of o v u l a t i o n (Marston and Chang, 1964; Hunter, 1967; S z o l l o s i , 1971; Niwa and Chang, 1975; Meyer and Longo, 1979; Longo, 1980). These o b s e r v a t i o n s suggest that fragmentation was an e f f e c t of p o s t - o v u l a t o r y aging r a t h e r than a d i r e c t e f f e c t of the treatment on oocyte q u a l i t y . It was shown i n Part 1, that fragmentation of mature r a t oocytes occurred i n 38% of oocytes w i t h i n 30 hours of o v u l a t i o n . Despite the f a c t that almost h a l f of the oocytes r e t r i e v e d i n the f i r s t wave of s u p e r o v u l a t i o n were present i n the ampullae 24 hours 109 post-treatment, only 9% of oocytes were fragmented at 48 hours. This may be due to the longer and more v a r i a b l e f i r s t wave of o v u l a t i o n (Walton et a l . , 1983) and t h e r e f o r e , more v a r i a b l e post-ovulatory age of oocytes. However, i n the mouse, fragmentation of oocytes occurred with g r e a t e r frequency i n mature than immature females (Marston and Chang, 1964) s u g g e s t i n g that the d i f f e r e n c e may be p a r t l y a t t r i b u t a b l e to maternal age. Yun et a l (1987) observed a higher percentage of abnormal oo-cytes 48 hours post-treatment i n the s u p e r o v u l a t e d group than was observed i n the present study. T h i s i s s u r p r i s i n g , s i n c e a higher p r o p o r t i o n of r a t s o v u l a t e d at 24 hours i n the present study. How-ever, Yun et a l (1987) a l s o examined oocytes f o r parthenogenesis, whereas t h i s was not conducted i n the present study. Thus, the i n c l u s i o n of both parthenogenotes and degenerated oocytes i n the abnormal group of oocytes by Yun et a l (1987) may account f o r the d i f f e r e n c e i n the two s t u d i e s . D i s t e n s i o n of the ampullae with cumulus-enclosed oocytes i s i n d i c a t i v e of recent o v u l a t i o n as demonstrated i n Part 1. The presence of ampullary d i s t e n s i o n and cumulus-enclosed oocytes c o r r e l a t e d c l o s e l y with o v u l a t i o n i n the 4 IU group. At 48. and 72 hours post-treatment, 100% and 72-80% of r a t s , r e s p e c t i v e l y , had ampullary d i s t e n s i o n and cumulus-enclosed oocytes. The decrease i n percentage of r a t s with ampullary d i s t e n s i o n and cumulus-enclosed oocytes at 72 hours (20-28%) corresponds with the 33% o v u l a t i o n rat e at 48 hours and r e f l e c t s the 24 hour aging p e r i o d i n these r a t s . Both cumulus-enclosed and cumulus-free oocytes were a l s o r e t r i e v e d at 72 hours from r a t s t r e a t e d with 4 IU PMSG i n another study ( M i l l e r and Armstrong, 1981a). 110 The p a t t e r n of ampullary d i s t e n s i o n v a r i e d more i n the 40 IU group. At 24 hours, f o u r r a t s had ampullary d i s t e n s i o n and cumulus-enclosed oocytes while two r a t s had no ampullary d i s t e n s i o n . One cumulus-enclosed oocyte was r e t r i e v e d from one r a t which had l i k e l y j u s t commenced o v u l a t i o n . The other r a t had a t o t a l of four oocytes w i t h i n a poor cumulus l i k e l y due e i t h e r to aging (Walton et a l . , 1983) or o v u l a t i o n of oocytes from a t r e t i c or abnormal f o l l i c l e s (Yun et a l . , 1987). At 48 and 96 hours, the decrease i n ampullary d i s t e n s i o n was a s s o c i a t e d with a d e c l i n e i n cumulus-enclosed oocytes due to i n t r a o v i d u c t a l oocyte aging. At 72 hours, the 40 to 50% i n c r e a s e i n r a t s with ampullary d i s t e n s i o n c o i n c i d e d with the second wave of o v u l a t i o n . Three r a t s l a c k e d ampullary d i s t e n s i o n and cumulus and l i k e l y d i d not undergo a second wave of o v u l a t i o n . The lower value of cumulus-enclosed oocytes when compared with ampullary d i s t e n s i o n at 72 hours i s due to the presence of both cumulus-enclosed and cumulus-free oocytes i n the same r a t s . A s i m i l a r temporal r e l a t i o n s h i p between o v u l a t i o n , loss of cumulus and oocyte fragmentation was r e p o r t e d by others ( M i l l e r and Armstrong, 1981a; Walton et a l . , 1983; Yun et a l . , 1987) and t h i s i s c o n s i s t e n t with the b i p h a s i c p a t t e r n of o v u l a t i o n i n superov-u l a t e d r a t s . 2. L i g h t and E l e c t r o n Microscopy M i c r o s c o p i c examination, both at the l i g h t and e l e c t r o n l e v e l s , r e v e a l e d no d i f f e r e n c e s between CEO or CFO r e t r i e v e d from the two treatment groups. Due to the a s s o c i a t i o n of cumulus d i s p e r s a l with i n t r a o v i d u c t a l oocyte aging, oocytes were d i v i d e d i n t o CEO and CFO f o r examination i n an attempt to separate aged from f r e s h l y ovulated o o c y t e s . However, i t was shown i n Part 1 that cumulus d i s p e r s a l i s not a r e l i a b l e i n d i c a t o r of oocyte age, s i n c e the cumulus i s l o s t 18-24 hours a f t e r o v u l a t i o n while morphological changes at the c e l l u l a r l e v e l commence as e a r l y as 12 hours i n some oocytes. I r r e g u l a r s u r f a c e of the MVF protuberance was observed i n 19% and 20% of CEO at 24 and 72 hours i n the 40 and 4 IU groups, r e s p e c t i v e l y . In mature r a t s , t h i s change was an e a r l y s i g n of aging which appeared i n 10 t o 40% of oocytes 7 to 14 hours a f t e r o v u l a t i o n , r e s p e c t i v e l y . In the 4 IU group at 72 hours, the i r r e g u l a r protuberance was l i k e l y due to the presence of oocytes from r a t s which o v u l a t e d e a r l y . In the 40 IU group, t h r e e r a t s at 24 hours each had 11 to 16 oocytes i n the ampullae. T h e r e f o r e , i t i s l i k e l y that o v u l a t i o n commenced 12 hours post-treatment i n the 40 IU group s i n c e i t takes 12 hours f o r the f i r s t wave of o v u l a t i o n to occur (Walton et a l . , 1983) and approximately two hours f o r oocytes to reach the ampullae (Rowlands, 1944). O v u l a t i o n i n the superovulated immature r a t was observed as e a r l y as 12 hours post-treatment by others (Walton et a l . , 1983). The oocytes which ovu-l a t e d f i r s t may then have been present i n the oviduct s u f f i c i e n t l y long'to undergo aging. S i g n i f i c a n t l y more oocytes (42%) had an i r r e g u l a r protuberance at 72 hours i n the 40 IU group. T h i s d i f f e r e n c e may be p a r t l y due to the e a r l i e r s t a r t of o v u l a t i o n i n the superovulated r a t (Walton et a l . , 1983). A l a r g e r percentage of the oocytes may a l s o o v u l a t e i n the e a r l y stage of the second wave, and t h e r e f o r e undergo aging. A l t e r n a t i v e l y , oocytes from s u p e r o v u l a t e d immature r a t s may undergo premature i n t r a f o l 1 i c u l a r a c t i v a t i o n and aging as a r e s u l t of the 112 h i g h LH a c t i v i t y of PMSG as demonstrated i n sheep (Moor et a l . , 1985). N u c l e o l a r - l i k e bodies were noted i n 26.5% of the CEO i n the 4 IU group at 72 hours. In the mature r a t , NLB were observed i n over 20% of oocytes 12 to 14 hours a f t e r o v u l a t i o n . If NLB appear at the same p o s t - o v u l a t o r y time i n oocytes from immature r a t s , the presence of cytoplasmic NLB i n the 4 IU group suggests that o v u l a t i o n commenced e a r l i e r than 64 hours post-treatment. O v u l a t i o n had commenced by 60 hours i n 28% of r a t s i n the 4 IU group i n the experiment of Yun et a l (1987). By 72 hours these oocytes would be aged but s t i l l cumulus-enclosed, and t h i s may account f o r the mi c r o s c o p i c s i g n s of aging i n the low dose group. F u r t h e r , although 33% of the r a t s o v u l a t e d at 48 hours i n the 4 IU group, only 18% of the oocytes r e t r i e v e d at 72 hours were cumulus-free. T h i s i s c o n s i s t e n t with the ob s e r v a t i o n that the cumulus p e r s i s t s longer around oocytes from immature r a t s than mature r a t s ( A u s t i n , 1950). T h e r e f o r e , CEO from e a r l y o v u l a t i o n may account f o r the oocytes with NLB. In c o n t r a s t t o the 4 IU group, only 4% of cumulus-enclosed oocytes i n the 40 IU group had NLB i n the cytoplasm at 72 hours. T h i s i s i n c o n s i s t e n t with the f i n d i n g t h at 42% of the oocytes had other s i g n s of aging. It i s p o s s i b l e that these oocytes may have aged s u f f i c i e n t l y to develop an i r r e g u l a r protuberance but not enough to develop NLB. A l t e r n a t i v e l y , an i r r e g u l a r protuberance on oocytes from immature r a t s s uperovulated with PMSG may r e s u l t from c e l l u l a r d y s f u n c t i o n s other than c h r o n o l o g i c a l aging. F u l l y grown i n t r a f o l l i c u l a r r a t oocytes have an even l a y e r of mi c r o f i l a m e n t s beneath the oolemma (Amsterdam et a l . , 1977; 113 Lehtonen and Badley, 1980). F o c a l i n t e n s i f i c a t i o n of t h i s l a y e r occurs subsequent to resumption of m e i o s i s . The presence of chromosomes below the oolemma induces a t h i c k suboolemmal web of a c t i n m i c r o f i l a m e n t s , l o s s of m i c r o v i l l i and e v a g i n a t i o n of the o v e r l y i n g oolemma (Chen and Longo, 1984; Longo and Chen, 1984; Okada et a l . , 1986; Van Blerkom and B e l l , 1986). The markedly i r r e g u l a r protuberances observed i n aged oocytes from immature r a t s may r e f l e c t e i t h e r an abnormal i n f l u e n c e of suboolemmal chromosomes or abnormal response of a c t i n m i c r o f i l a m e n t s to the u n d e r l y i n g chromosomes. Polar body formation i s mediated by, and depends on the presence of, the t h i c k m i c r o f i l a m e n t web a s s o c i a t e d w i t h suboo-lemmal chromosomes (Webb et a l . , 1986). I n h i b i t i o n of the m i c r o f i l a m e n t web r e s u l t s i n f a i l u r e of both chromosome r o t a t i o n and e x t r u s i o n of the p o l a r body (Webb et a l . , 1984). A l a r g e number of i r r e g u l a r cytoplasmic processes are normally seen adjacent to the s i t e of p o l a r body a b s t r i c t i o n (Odor and Renninger, 1960; Odor, 1960) due to l o s s of suboolemmal a c t i n when the m e i o t i c apparatus r o t a t e s (Maro et a l . , 1984). S i m i l a r l y , i r r e g u l a r suboolemmal a c t i n d i s t r i b u t i o n leads to s u r f a c e i r r e g u l a r i t i e s (Maro et a l . , 1984). P o s t - t r a n s l a t i o n a l m o d i f i c a t i o n and r e o r g a n i z a t i o n of the c y t o s k e l -eton occurs i n the absence of f e r t i l i z a t i o n , but at a slower r a t e ( E i c h e n l a u b - R i t t e r et a l . , 1986; Webb et a l . , 1986; Paynton et a l . , 1988). Therefore, the i r r e g u l a r protuberances observed i n oocytes from the immature r a t s may be due to r e o r g a n i z a t i o n of the c y t o s k e l e t o n with aging. T h i s i s c o n s i s t e n t with the f i n d i n g that preprogrammed developmental processes are under way i n the oocyte p r i o r to f e r t i l i z a t i o n ( S h a l g i et a l . , 1985; E i c h e n l a u b - R i t t e r et 114 a l . , 1986; Moor and G a n d o l f i , 1987) and are r e s p o n s i b l e f o r the morphological changes observed d u r i n g i n t r a o v i d u c t a l oocyte aging (Webb et a l . , 1986) as d i s c u s s e d i n Part 1. In the mouse, d e p o l y m e r i z a t i o n of k i n e t o c h o r e m i c r o t u b u l e s was observed 15 to 30 hours p o s t - o v u l a t i o n ( E i c h e n l a u b - R i t t e r et a l . , 1986). This r e s u l t e d i n displacement of metaphase chromosomes from the equator of the s p i n d l e ( E i c h e n l a u b - R i t t e r et a l . , 1986). T h i s change was s i m i l a r i n nature to the k i n e t o c h o r e m i c r o t u b u l e d i s a s -sembly observed d u r i n g anaphase and t e l o p h a s e subsequent to f e r t i l i z a t i o n , s u g g e s t i n g that c y t o s k e l e t a l t r a n s f o r m a t i o n s occur independently of f e r t i l i z a t i o n ( E i c h e n l a u b - R i t t e r et a l . , 1986). In the hamster, oocytes i n anaphase were observed w i t h i n 12 hr of o v u l a t i o n due to spontaneous a c t i v a t i o n (Yanagimachi and Chang, 1961). E l e c t r i c a l a c t i v a t i o n of r a b b i t , mouse and hamster oocytes a l s o led to asynchronous movement of chromosomes (Gulyas, 1976). Anaphase and telophase oocytes were a l s o observed i n super-ovulated r a t s from 48 to 72 hours post-treatment i n the study of Yun et a l (1989). Yun et a l (1989) observed oocytes i n prophase p r i o r to the recovery of anaphase and telophase forms and concluded that asynchrony of oocyte maturation with o v u l a t i o n had occurred. In the present study, no oocytes i n prophase were observed. Oocytes i n anaphase f i r s t appeared 24 hours a f t e r the commencement of o v u l a t i o n i n both treatment groups and the m a j o r i t y had an i r r e g u l a r protuberance or other morphological a l t e r a t i o n s c o n s i s t -ent with aging. T h i s suggests that, s i m i l a r t o aged and a c t i v a t e d mouse and hamster oocytes (Yanagimachi and Chang, 1961; Gulyas, 1976; E i c h e n l a u b - R i t t e r et a l . , 1986), chromosome displacement on the m e i o t i c s p i n d l e may be a consequence of i n t r a o v i d u c t a l aging 115 and expression of the p o s t - o v u l a t o r y developmental program. U n l i k e s p i n d l e r o t a t i o n and p o l a r body f o r m a t i o n , anaphase and tel o p h a s e are m i c rofilament independent f u n c t i o n s (Maro et a l . , 1984). Displacement of chromosomes from the s p i n d l e equator was not observed i n oocytes from mature r a t s d u r i n g i n t r a o v i d u c t a l aging and a c t i v a t i o n . Thus, s p i n d l e i n s t a b i l i t y and the exaggeratedly i r r e g u l a r protuberance seen i n immature r a t oocytes suggest that there may be e i t h e r a b n o r m a l i t i e s or asynchrony of c y t o s k e l e t a l f u n c t i o n i n oocytes from immature r a t s which are expressed d u r i n g i n t r a o v i d u c t a l aging. The appearance of cleavage furrows at the base of the MVF protuberance and the e x t r u s i o n of the i n t a c t m e i o t i c apparatus i n a small number of aged oocytes f u r t h e r suggests that there are a b n o r m a l i t i e s of c y t o s k e l e t a l f u n c t i o n as r o t a t i o n of the m e i o t i c apparatus p r i o r to p o l a r body f o r m a t i o n can only occur i f one pole and the equator of the s p i n d l e are anchored by the microfilament web (Maro et a l . , 1984). Since the p r o g r e s s i v e changes observed d u r i n g aging a re r e g u l a t e d by the i n t r i n s i c developmental program of oocytes, s i m i l a r a b n o r m a l i t i e s of c y t o s k e l e t a l f u n c t i o n may occur i n f e r t i l i z e d oocytes from PMSG t r e a t e d immature r a t s . A d m i n i s t r a t i o n of PMSG to immature r a t s may have an adverse e f f e c t on s y n t h e s i s , assembly or f u n c t i o n of the oocyte c y t o -s k e l e t o n . A c t i n m i c r o f i l a m e n t s a re capable of r a p i d rearrangement and, i n a s s o c i a t i o n with m i c r o t u b u l e s , mediate changes i n c e l l shape (Lehtonen and Badley, 1980; Chen and Longo, 1984; Cran, 1987; Lehtonen et a l . , 1988). T h i s c e l l system i s h i g h l y l a b i l e ( A l b e r t s et a l . , 1983; Lehtonen et a l . , 1988) and t h e r e f o r e l i k e l y suscep-t i b l e to changes i n r e g u l a t o r y s t i m u l i caused by a l t e r a t i o n of the 116 i n t r a f o l l i c u l a r m i l i e u or cumulus oocyte coupling. Protein synthesis and the appearance of acti n during oocyte maturation depend on cumulus oocyte coupling mediated by gonado-t r o p i n s (Osborn and Moor, 1982; Moor and Gandolfi, 1987; M a t t i o l i et al . , 1988). D i s s o c i a t i o n of thi s coupling leads to reduced synthesis of a c t i n (Moor and Gandolfi, 1987). Therefore, the prolonged and high l e v e l of gonadotrophin a c t i v i t y i n PMSG-treated immature rats may lead to premature cumulus oocyte d i s s o c i a t i o n and reduced actin synthesis which may d e s t a b i l i z e the cytoskeleton of the oocyte. Cumulus-enclosed oocytes from mature and immature rats were morphologically s i m i l a r i n a l l regards except oocyte contour and the prevalence of large MVB. As a result of the gonadotrophin surge during preovulatory f o l l i c u l a r maturation i n the mature rat, a large p e r i v i t e l 1 i n e space appears and the oolemmal surface assumes an undulent outline (Odor, 1960; S z o l l o s i , 1966). These changes were lacking in oocytes retrieved from the immature rats treated with either dose of PMSG. However, enlargement of the pe r i v i t e l 1 i n e space was observed i n CFO suggesting that t h i s change occurs during intraoviductal aging. This observation suggests that oocytes from immature rats stimulated to ovulate with either a low or a high dose of PMSG, and by either exogenous or endogenous gonadotrophin do not complete maturation u n t i l after ovulation. The c o r t i c a l layer of microfilaments provides c e l l s with structural r i g i d i t y (Lehtonen and Badley, 1980) and mediates c o r t i c a l granule release (Cran, 1987). Enlargement of the p e r i -v i t e l l i n e space i n the hamster involves p a r t i a l CG exocytosis (Yang and Yanagimachi, 1989). In the mouse, CG release occurs i n 117 a s s o c i a t i o n with formation of the MVF protuberance ( D u c i b e l l a et a l . , 1988a). S i m i l a r to the present study, no a p p r e c i a b l e decrease i n suboolemmal CG, other than at the MVF protuberance, was observed by e l e c t r o n microscopy i n mature mouse oocytes ( D u c i b e l l a et a l . , 1988b). Increased a b i l i t y to undergo CG e x o c y t o s i s i s observed with time f o l l o w i n g GVBD and i s a s s o c i a t e d with c y t o p l a s m i c maturation ( D u c i b e l l a et a l . , 1990). Thus, the small p e r i v i t e l 1 i n e space of oocytes from immature r a t s may r e f l e c t c y t o p l a s m i c immaturity at the time of o v u l a t i o n . The appearance of a l a r g e r p e r i v i t e l 1 i n e space with p o s t - o v u l a t o r y age i n these oocytes supports the co n t e n t i o n that c y t o p l a s m i c maturation i s completed subsequent to o v u l a t i o n . T herefore, the d i f f e r e n c e i n contour may represent d i f f e r e n c e s i n c y t o s k e l e t a l m a t u r i t y between oocytes from immature, PMSG-treated r a t s and mature r a t s . The c y t o s k e l e t o n p l a y s a c e n t r a l r o l e i n cy t o p l a s m i c organ-i z a t i o n w i t h i n the oocyte (Moor and G a n d o l f i , 1987), mediating s p i n d l e r o t a t i o n , movement of p r o n u c l e i and o r g a n e l l e aggregation and d i s p e r s a l (Maro et a l . , 1984; Maro et a l . , 1986; A l b e r t i n i , 1987). In the r a t , the gonadotrophin surge induces c l u s t e r i n g of MVB around the germinal v e s i c l e ( E z z e l l and Szego, 1979). F o l l o w i n g GVBD, the MVB r e d i s t r i b u t e i n the cytoplasm ( E z z e l l and Szego, 1979) and at the time of o v u l a t i o n aggregate as l a r g e m u l t i l o c -u l a t e d s t r u c t u r e s ( S o t e l o and P o r t e r , 1959; Odor, 1960). S i g n i f i c a n t l y fewer l a r g e , m u l t i l o c u l a t e d MVB were observed i n CEO from the immature r a t s than were observed i n the mature r a t s . T h i s i s c o n s i s t e n t w i t h i n t r a o v i d u c t a l oocyte aging i n the cumulus-enclosed oocytes. However, the p o s s i b i l i t y that the absence of l a r g e m u l t i l o c u l a t e d MVB i n these oocytes may be due to e i t h e r 118 abnormal, or incomplete, oocyte maturation or a l t e r a t i o n of microtubule f u n c t i o n i n oocytes from immature r a t s cannot be r u l e d out s i n c e the absence of MVB was not always a s s o c i a t e d with other e a r l y signs of aging i n the cumulus-enclosed oocytes. As noted i n Part 1, d e c l i n e of l a r g e MVB was not c o n s i s t e n t l y a s s o c i a t e d with aging i n a l l oocytes and t h e r e was v a r i a t i o n i n MVB s i z e and morphology even i n f r e s h l y o v u l a t e d oocytes. Therefore, t h i s s t r u c t u r e appears to be one which undergoes r a p i d and dynamic changes i n the oocyte and, on i t s own, i s not a r e l i a b l e morphol-o g i c a l i n d i c a t o r of the s t a t u s of the oocyte. Great v a r i a t i o n i n the o r g a n e l l e d i s t r i b u t i o n of immature bovine oocytes was a l s o observed but was not a s s o c i a t e d with a d i f f e r e n c e i n maturation c a p a b i l i t y (de Loos et a l . , 1989). Superovul a t i o n of immature r a t s with PMSG may lead to o v u l a t i o n of degenerated oocytes from a t r e t i c f o l l i c l e s (Braw and T s a f r i r i , 1980). Two degenerated, germinal v e s i c l e stage oocytes were r e t r i e v e d from one s u p e r o v u l a t e d r a t at 24 hours, suggesting t h a t these oocytes o r i g i n a t e d from a t r e t i c f o l l i c l e s . Oocytes from a t r e t i c f o l l i c l e s have c y t o p l a s m i c v a c u o l a t i o n with fewer o r g a n e l l e s i n a f l o c c u l e n t c y t o s o l matrix (Vazquez-Nin, 1967; Zamboni et a l . , 1972) s i m i l a r to the ones examined i n the present study. No other evidence of o v u l a t i o n of abnormal f o l l i c l e s was observed at any time d u r i n g the study. However, i n Part 3, abnormal oocytes were a l s o r e t r i e v e d from one superovulated immature r a t . During a t r e s i a , the oocyte may undergo m e i o t i c maturation or remain i n the germinal v e s i c l e stage ( E r i c k s o n , 1986). Oocytes with s i g n s of aging may have a r i s e n from a t r e t i c f o l l i c l e s where m e i o t i c maturation occurred. Oocytes which resume meiosis during a t r e s i a 119 may undergo segmentation resembling cleavage and these segments may c o n t a i n up to 7 n u c l e i or be anucleate (Vazquez-Nin, 1967; Zamboni et a l . , 1972; E r i c k s o n et a l . , 1983) s i m i l a r to the fragmented oocytes r e t r i e v e d at 72 and 96 hours from the immature r a t s . However, the s i g n s of oocyte aging i n the present study c o r r e l a t e d c l o s e l y with the o v u l a t i o n p a t t e r n , ampullary d i s t e n s i o n and cumulus d i s p e r s a l , making i t u n l i k e l y that the i n d u c t i o n of o v u l a t i o n of a t r e t i c f o l l i c l e s would account f o r a s i g n i f i c a n t p r o p o r t i o n of oocytes r e t r i e v e d from s u p e r o v u l a t e d immature r a t s . T h e r e f o r e , o v u l a t i o n of a t r e t i c f o l l i c l e s d u r i n g s u p e r o v u l a t i o n with PMSG appears to be an event i s o l a t e d to a small percentage of r a t s and l i k e l y r e f l e c t s i n d i v i d u a l v a r i a t i o n i n response t o , ra t h e r than a d i r e c t e f f e c t of, PMSG treatment. An i n c r e a s e i n abnormal oocytes was p o s i t i v e l y c o r r e l a t e d with a c o i n c i d e n t r i s e i n s t e r o i d hormone l e v e l s by other workers ( M i l l e r and Armstrong, 1981a; Yun et a l . , 1987). E l e v a t e d e s t r a d i o l l e v e l s are a s s o c i a t e d with i n c r e a s e d fragmentation and degeneration of r a b b i t ( D a n i e l , 1964; McGaughey and D a n i e l , 1966), mouse ( K i r k p a t r i c k , 1971), and r a t (Butcher and Pope, 1979) embryos. T h i s e f f e c t i s mediated by a l t e r a t i o n of o v i d u c t a l e p i t h e l i a l p r o t e i n s y n t h e s i s and hence a l t e r e d o v i d u c t a l f l u i d composition ( C l i n e et a l . , 1977; Stone et a l . , 1977). Treatment of supe r o v u l a t e d immature r a t s with the antiandrogen, f l u t a m i d e , leads to a s i g n i f i c a n t d e c l i n e i n o v a r i a n androgen and e s t r a d i o l l e v e l s and enhanced v i a b i l i t y of oocytes i n the o v i d u c t (Yun et a l . , 1988). T h i s suggests that aging alone may not be r e s p o n s i b l e f o r the d e t e r i o r a -t i o n of oocytes observed f o l l o w i n g s u p e r o v u l a t i o n . In the present study, no morphological evidence of an adverse 120 e f f e c t of s u p e r o v u l a t i o n on i n t r a f o l 1 i c u l a r oocyte maturation was observed. A s u b t l e e f f e c t of the su p e r o v u l a t o r y doses of PMSG on oocyte maturation may have been masked by the v a r i a b i l i t y i n mor-phology observed i n a l l three groups of r a t s . A l t e r n a t i v e l y , changes i n the p r o t e i n s s y n t h e s i z e d as observed by Moor et a l (1985) i n sheep oocytes f o l l o w i n g a d m i n i s t r a t i o n of a superovula-t o r y dose of PMSG may not be a s s o c i a t e d with immediate changes i n morphology. A l t e r e d c e l l f u n c t i o n i s not always accompanied by an obvious and immediate change i n c e l l morphology (Vazquez-Nin et a l . , 1979; C h e v i l l e , 1983). F u n c t i o n a l s t u d i e s would b e t t e r reveal whether adverse e f f e c t s on oocyte maturation occur. However, there i s no doubt t h a t , due to the prolonged, b i p h a s i c p a t t e r n of o v u l a t i o n induced by a su p e r o v u l a t o r y dbse of PMSG, i n t r a o v i d u c t a l oocyte aging i s a s i g n i f i c a n t cause of i n f e r t i l i t y i n the immature r a t . The d i f f e r e n c e s i n oocyte contour and aging observed between the immature and mature r a t s suggests that complete maturation of c o n t r a c t i l e p r o t e i n s , and/or c o o r d i n a t i o n of c y t o s k e l e t a l f u n c t i o n with germinal v e s i c l e breakdown was not achieved p r i o r to o v u l a t i o n i n prepubertal r a t s . T h i s may be an e f f e c t of exogenous gonadotrophin a c t i v i t y e i t h e r due to a d i r e c t e f f e c t on i n t r a f o l 1 -i c u l a r oocyte maturation, mediated by gross d i s t o r t i o n of 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 or premature d i s s o c i a t i o n of cumulus oocyte c o u p l i n g , or recruitment of immature f o l l i c l e s which have i n s u f f i c i e n t time t o undergo maturation p r i o r t o o v u l a t i o n . If t h i s i s the case, i t appears t o have l i t t l e f u n c t i o n a l s i g n i f i c a n c e , s i n c e r e c e n t l y o v u l a t e d oocytes r e t r i e v e d from immature PMSG-t r e a t e d r a t s w i l l produce l i v e b o r n i f permitted t o develop i n a 121 non-superovulated female (Walton et a l . , 1982; Walton and Arm-stro n g , 1983). S i m i l a r l y , haphazard o r i e n t a t i o n of s p i n d l e microtubules was not a s s o c i a t e d with any adverse e f f e c t on re p r o d u c t i v e p o t e n t i a l i n human oocytes matured i n v i t r o (Zamboni et a l . , 1972) . In summary, morphological examination of oocytes r e t r i e v e d from immature r a t s t r e a t e d w i t h e i t h e r 4 or 40 IU PMSG and mature, c y c l i n g r a t s r e v e a l e d that no morphological d i f f e r e n c e s e x i s t between oocytes r e t r i e v e d from immature r a t s t r e a t e d w i t h e i t h e r the low or high dose of PMSG. The observed changes i n morphology i n these two groups were a t t r i b u t a b l e to i n t r a o v i d u c t a l aging and t h i s c o r r e l a t e d with the observed o v u l a t o r y p a t t e r n i n each group. D i f f e r e n c e s a t t r i b u t e d t o a l t e r a t i o n of c y t o s k e l e t a l f u n c t i o n were noted between r e c e n t l y o v u l a t e d and aged oocytes from immature and mature r a t s . This was concluded to be the r e s u l t of an adverse e f f e c t of PMSG treatment on i n t r a f o l 1 i c u l a r oocyte maturation. 122 PART 3: A MORPHOLOGICAL INVESTIGATION OF THE CAUSES OF FERTILIZATION FAILURE IN SUPEROVULATED IMMATURE RATS A. RESULTS 1. Gross Embryo R e t r i e v a l and Assessment The number of r a t s s a c r i f i c e d and both the number and assessment of embryos r e t r i e v e d are presented i n Tables 1 and 2. Degenerated and fragmented oocytes were s u b t r a c t e d from the t o t a l number of oocytes r e t r i e v e d i n each group and f e r t i l i z a t i o n r a t e s were computed on the b a s i s of number of f e r t i l i z e d oocytes per t o t a l number of i n t a c t oocytes. T h i s ensured t h a t the f e r t i l i z a t i o n r a t e s were expressed as a percentage of the t o t a l number of f e r t i l i z a b l e oocytes. T h i s adjustment was based on the assumption that the degenerated and fragmented oocytes were n o n - f e r t i 1 i z a b l e . The f e r t i l i z a t i o n r a t e s of each treatment group of r a t s are presented i n Table 2. In the 40 IU group, two r a t s had no f e r t i l -i z e d oocytes d e s p i t e the presence of sperm i n the v a g i n a l smear. To avoid p o s s i b l e b i a s of the r e s u l t s of the 40 IU treatment group, the f e r t i l i z a t i o n r a t e was computed both w i t h , and without, the oocytes r e t r i e v e d from these two r a t s (Table 2). The f e r t i l i z a t i o n r a tes d i d not d i f f e r s i g n i f i c a n t l y between mature r a t s and immature r a t s t r e a t e d with 4 IU PMSG (91% and 96%, r e s p e c t i v e l y ) . However, the f e r t i l i z a t i o n r a t e of immature r a t s t r e a t e d w i t h 40 IU was s i g n i f i c a n t l y lower (p<0.005), e i t h e r with (41%), or without (59%), i n c l u s i o n of the oocytes from the two r a t s which f a i l e d to f e r t i 1 i z e . 123 TABLE 1. OOCYTE RETRIEVAL FROM IMMATURE RATS TREATED WITH EITHER 4 OR 40 IU PMSG AND MATURE SPONTANEOUSLY CYCLING RATS ON DAY ONE OF PREGNANCY GROUP NUMBER OF RATS TOTAL OOCYTES AVERAGE PER RAT MATURE 10 149 14.9 4 IU 13 130 10 40 IU 10 47 9 47 .9 TABLE 2. GROSS ASSESSMENT AND FERTILIZATION RATE OF OOCYTES RETRIEVED ON DAY ONE OF PREGNANCY FROM IMMATURE AND MATURE RATS GROUP TOTAL FERT UNFERT DEG FRAG RATE % MATURE 149 118 11 20 0 118/ 129 91% 4IU 130 100 4 24 2 100/ 104 96% 40 IU 479 137 202 15 124 137/ 339 41% 40lU a - 137 96 - - 137/ 233 59% a-40 IU group w i t h omission of oocytes r e t r i e v e d from 2 r a t s RATE = f e r t i l i z a t i o n r a t e (number of f e r t i l i z e d oocytes per t o t a l oocytes r e t r i e v e d minus degenerated & fragmented oocytes) % = f e r t i l i z a t i o n r a t e expressed as a percentage of t o t a l i n t a c t oocytes FERT = f e r t i l i z e d ; UNFERT = u n f e r t i l i z e d ; DEG = degenerated; FRAG = fragmented 124 2. L i g h t and E l e c t r o n Microscopy A summary of the l i g h t m i c r o s c o p i c assessment of u n f e r t i l i z e d oocytes i s presented i n T a b l e 3. Oocytes with an i r r e g u l a r MVF protuberance, cytoplasmic NLB or chromosome movement ( r o t a t i o n or c e n t r a l m i g r a t i o n of the m e i o t i c apparatus or chromosome s c a t t e r ) were observed predominantly i n the 40 IU group. Table 3 i l l u s t r a t e s t h a t , i n the 40 IU group, 78/172 (45%) of the u n f e r t i l i z e d oocytes e x h i b i t e d morphological changes c o n s i s t e n t with aging. Two (3%) of the 78 oocytes had sperm i n the p e r i v i t e l l i n e space and a m e i o t i c apparatus which had moved away from the oolemma, w h i l e 7 (9%) had s c a t t e r e d chromosomes on an i n t a c t s p i n d l e i n the absence of sperm p e n e t r a t i o n . F i v e of these 7 oocytes a l s o had other morphological changes c o n s i s t e n t w i t h aging. Of the 94 metaphase oocytes examined, 5 (5%) had sperm i n the p e r i v i t e l 1 i n e space and were l i k e l y undergoing f e r t i l i z a t i o n at the time of r e t r i e v a l . One of these 5 oocytes with sperm i n the p e r i v i t e l 1 i n e space was r e t r i e v e d from 1 of the 2 r a t s w i t h complete f a i l u r e of f e r t i l i z a t i o n . When the u n f e r t i l i z e d oocytes were examined a f t e r removal of the oocytes from the 2 r a t s which f a i l e d to f e r t i l i z e , 55/86 (64%) of the oocytes had mo r p h o l o g i c a l evidence of aging. Of these 55 oocytes, 2 (4%) had sperm i n the p e r i v i t e l l i n e space and 7 (13%) had s c a t t e r e d chromosomes on the m e i o t i c apparatus. Of the 31 metaphase oocytes, 4 (13%) had sperm i n the p e r i v i t e l l i n e space. U l t r a s t r u c t u r a l l y , u n f e r t i l i z e d oocytes from the 40 IU group d i s p l a y e d a continuum of d i s t i n c t changes. However, the oocytes r e t r i e v e d from 1 of the 2 r a t s which f a i l e d to undergo f e r t i l i z -a t i o n d i f f e r e d and w i l l be d e s c r i b e d at the end of t h i s s e c t i o n . The morphology of normal metaphase two oocytes was d e s c r i b e d f u l l y TABLE 3. LIGHT MICROSCOPIC ASSESSMENT OF UNFERTILIZED OOCYTES RETRIEVED FROM MATURE AND IMMATURE RATS ON DAY ONE OF PREGNANCY GROUP TOTAL META-PHASE IRREG SURFACE NLB CHROM MOVT TOTAL ALTERED % MATURE 6 2 0 0 4 4/6 66 4 IU 4 3 0 1 0 1/4 25 40 IU 172 94 29 16 33 78/172 45 40 IU a 86 31 23 11 21 55/86 64* a-40 IU group w i t h omission of u n f e r t i l i z e d oocytes from 2 r a t s • - s i g n i f i c a n t l y h i g h e r than u n c o r r e c t e d 40 IU group (p<0.01) TOTAL ALTERED = number of oocytes w i t h a l t e r e d morphology over t o t a l number of oocytes examined % = percentage of t o t a l oocytes examined which had a l t e r e d morphology IRREG = i r r e g u l a r ; NLB = n u c l e o l a r - l i k e b o d i e s ; CHROM MOVT = chromosome movement 126 i n Part 1. I r r e g u l a r i t y of the protuberance ( F i g u r e 22a), NLB, r o t a t i o n and m i g r a t i o n of the m e i o t i c apparatus and s c a t t e r of the suboolemmal chromosomes on the s p i n d l e were observed at the e l e c t r o n m i c r o s c o p i c l e v e l i n u n f e r t i l i z e d oocytes. These oocytes a l s o had a v a r i a b l e r e d u c t i o n i n f i b r i l l a r a r r a y s , c y t o p l a s m i c compartmentalization by l i n e a r segments of SER, l a m e l l a r G o l g i and r e t e n t i o n of suboolemmal c o r t i c a l granules. Of the 51 u n f e r t i l i z e d oocytes r e t r i e v e d from one r a t t r e a t e d with 40 IU PMSG, 42 oocytes were examined. An i r r e g u l a r MVF protuberance, chromosome movement and cytoplasmic NLB were observed i n 6 (14%), 8 (19%) and 3 (7%) of the oocytes, r e s p e c t i v e l y . The 25 remaining metaphase oocytes d i f f e r e d from the metaphase oocytes p r e v i o u s l y d e s c r i b e d . These oocytes had a more e l e c t r o n ' lucent cytoplasm with reduced g r a n u l a r i t y , fewer MVB and fewer FA which were a l s o reduced i n l a t t i c e l e n g t h (Figure 22b,c). Large non-membrane bound c l u s t e r s of e l e c t r o n dense granules d i s p l a c e d c y t o s o l matrix ( F i g u r e 22d). Spaces devoid of v i s i b l e content were a l s o observed i n the cytoplasm (Figure 22b). Fewer cyt o p l a s m i c processes and m i c r o v i l l i were present on the oolemma, the number of suboolemmal CG was reduced and CG exocytosis was observed ( F i g u r e 22b,c,d). Smooth endoplasmic r e t i c u l u m was m i l d l y t o moderately d i l a t e d . 127 _ P a g e 127$ FIGURE 22 A. U n f e r t i l i z e d oocyte r e t r i e v e d from a superovulated r a t . The s u r f a c e o v e r l y i n g the m e i o t i c apparatus i s i r r e g u l a r (arrow) but f r e e of m i c r o v i l l i and cytoplasmic processes. A n u c l e o l a r - l i k e body i s present i n the v e s i c u l a r aggregate adjacent to the chromosomes (arrowhead). Note the cumulus c e l l s on the s u r f a c e of the zona p e l l u c i d a . M a g n i f i c a t i o n = 2,500x. B. Oocyte from one s u p e r o v u l a t e d r a t which mated but f a i l e d to f e r t i l i z e . C l u s t e r s of e l e c t r o n dense granules are num-erous i n the cytoplasm (arrows). M u l t i v e s i c u l a r bodies are s c a r c e and vacuoles are present i n the cytoplasm (arrowhead). Note the e l e c t r o n l u c e n t cytoplasm. M a g n i f i c a t i o n = 2,500x. C. Oocyte from one superovulated r a t which mated but f a i l e d to f e r t i l i z e . Suboolemmal c o r t i c a l granules are decreased i n number. C o r t i c a l g r a n u l e e x o c y t o s i s (arrow) i s e v i d e n t . Cytoplasmic processes on the oolemma are reduced i n l e n g t h and number. M a g n i f i c a t i o n = 2,500x. D. C o r t i c a l granule e x o c y t o s i s i n an oocyte from one super-ovulated r a t which mated but f a i l e d to f e r t i l i z e (arrow). Note the c l u s t e r of e l e c t r o n dense granules i n the c y t o -plasm (open arrow). M a g n i f i c a t i o n = 8,000x. 128 B. DISCUSSION The s u p e r o v u l a t o r y dose of PMSG s i g n i f i c a n t l y reduced in. v i v o f e r t i l i z a t i o n r a t e s when compared w i t h mature r a t s and immature r a t s t r e a t e d with 4 IU. Th i s d i f f e r e n c e e x i s t e d d e s p i t e the e x c l u s i o n of fragmented and degenerated oocytes from the u n f e r t i l -i z e d group and the e x c l u s i o n of oocytes from two r a t s which showed no evidence of f e r t i l i z a t i o n . Decreased f e r t i l i z a t i o n r a t e s , both i n v i v o and in. v i t r o , were p r e v i o u s l y r e p o r t e d i n immature r a t s s u p e r o v u l a t e d with PMSG ( A u s t i n , 1950; M i l l e r and Armstrong, 1981a; Walton et a l . , 1983; Walton and Armstrong, 1983; Evans and Arm-stro n g , 1984). Walton et a l . (1983) suggested that the d i f f e r e n c e i n f e r t i l i z a t i o n r a t e between the 4 and 40 IU groups was l i k e l y due to the presence of oocytes which were aged, and t h e r e f o r e u n f e r t i l -i z a b l e , but looked normal g r o s s l y . T h i s was s u b s t a n t i a t e d by the present study which demonstrated t h a t 64% of the u n f e r t i l i z e d , i n t a c t oocytes had m i c r o s c o p i c evidence of aging. While f e r t i l i z a t i o n r a t e s i n immature r a t s t r e a t e d with 4 IU PMSG and mature r a t s were comparable ( A u s t i n , 1950; Walton and Arm-strong, 1983; Walton et a l . , 1983), the f e r t i l i z a t i o n r a t e i n supe r o v u l a t e d r a t s i n the present study was lower than that observed by others ( M i l l e r and Armstrong, 1981a; Walton and Armstrong, 1983; Walton et a l . , 1983; Evans and Armstrong, 1984; L e v e i l l e and Armstrong, 1989). Walton et a l . (1983) a l s o excluded degenerated and fragmented oocytes p l u s r a t s with one or no f e r t i l i z e d oocytes i n the r e t r i e v a l . Under these c o n d i t i o n s , the supero v u l a t e d group had an 89% f e r t i l i z a t i o n r a t e , s i g n i f i c a n t l y lower than the c o n t r o l group, yet h i g h e r than the present f i n d i n g . T h i s d i f f e r e n c e i s most l i k e l y due t o the d i f f e r e n c e i n r e t r i e v a l 129 times as Walton et a l . (1983) s a c r i f i c e d at 14:00 to 16:00, whereas i n the present study, the r a t s were s a c r i f i c e d between 9:00 and 12:00. This suggests that f e r t i l i z a t i o n occurs over a prolonged p e r i o d i n the s u p e r o v u l a t e d immature r a t . In the present study, 36% of the u n f e r t i l i z e d oocytes examined were i n metaphase, of which 13% had sperm i n the p e r i v i t e l 1 ine space and were a p p a r e n t l y undergoing f e r t i l i z a t i o n . No sperm were seen i n or around the remaining oocytes s u g g e s t i n g that an inadequate number of sperm to c o n s t i t u t e a normal sperm:oocyte r a t i o were present i n the ampullae at the time of r e t r i e v a l . A u s t i n (1950) demonstrated t h a t the number of sperm at the f e r t i l i z a t i o n s i t e i n superovulated r a t s was s i m i l a r to, or lower than, the mature r a t . In the mature r a t , the r a t i o of ampullary sperm to oocytes i s approximately 1-2:1 ( S h a l g i and K r a i c e r , 1978). While hundreds of sperm reach the isthmus w i t h i n 20 minutes of mating, only three to four sperm ascend the o v i d u c t to the ampulla per hour and t h i s i s l i k e l y the f i r s t major b l o c k to polyspermy ( S h a l g i and K r a i c e r , 1978). In the mature r a t , sperm are present i n the ampulla at the commencement of o v u l a t i o n and continue to ascend the oviduct t h e r e a f t e r ensuring that t h e r e i s p r o g r e s s i v e f e r t i l i z a t i o n over the 3 hr ovu l a t o r y p e r i o d ( S h a l g i and K r a i c e r , 1978). The second wave of o v u l a t i o n i n the superovulated immature rat occurs over a 6 hr p e r i o d (Walton et a l . , 1983) and, as i n the mature r a t , there i s l i k e l y p r o g r e s s i v e f e r t i l i z a t i o n . O v u l a t i o n stops at 6:00 (Walton et a l . , 1983). Therefore, from 9:00 to 12:00 those u n f e r t i l i z e d oocytes would l i k e l y be the most r e c e n t l y ovulated i n the wave and t h e r e f o r e s t i l l f e r t i l i z a b l e . By 14:00 to 130 16:00 these oocytes would be ne a r i n g the end of the f e r t i l i z a b l e p e r i o d (Niwa and Chang, 1975), when an i n c r e a s e d i n c i d e n c e of abnormal f e r t i l i z a t i o n occurs ( A u s t i n , 1967; G i a n f o r t o n i and Gulyas, 1985). T h i s i s c o n s i s t e n t w i t h the s i g n i f i c a n t l y lower percentage of pr o n u c l e a r forms i n the superovulated group found by Walton et al.. (1983), presumably due to f a i l u r e of sperm head decondensation. However, these oocytes may a l s o have been r e c e n t l y f e r t i l i z e d and had not yet undergone p r o n u c l e i formation. Since s u p e r o v u l a t i o n w i t h PMSG i n c r e a s e s the number of ovulatory oocytes but does not concomitantly enhance sperm t r a n s p o r t , the p e r i o d r e q u i r e d f o r f e r t i l i z a t i o n w i l l be prolonged. T h i s would e x p l a i n the lower f e r t i l i z a t i o n r a t e and the l a r g e percentage of unfer-t i l i z e d metaphase oocytes observed i n the present study compared with that observed by Walton et a l . (1983). A second p o l a r body was not observed i n 106 oocytes r e t r i e v e d from 2 superovulated r a t s , d e s p i t e post-breeding v a g i n a l smears p o s i t i v e f o r sperm. A sperm was observed i n the p e r i v i t e l l i n e space of only 1 of the 86 oocytes examined from these 2 r a t s . Superovu-l a t e d immature r a t s which f a i l e d to f e r t i l i z e were reported by other workers and i t was suggested that a superovulatory dose of PMSG i n t e r f e r e s with sperm t r a n s p o r t ( A u s t i n , 1950; M i l l e r and Armstrong, 1981a; Walton et a l . , 1983). T h i s i s l i k e l y due to the e f f e c t of the e l e v a t e d s t e r o i d hormone l e v e l s on the rep r o d u c t i v e t r a c t , which mediates sperm t r a n s p o r t (Hawk, 1975; Blandau, 1978). I n t r a o v i d u c t a l oocyte aging, due to the delay i n mating a s s o c i a t e d with the f i r s t wave of o v u l a t i o n and the delay i n f e r t i l i z a t i o n a s s o c i a t e d w i t h the second, has a major impact on f e r t i l i z a t i o n i n immature superovulated r a t s . Recently ovulated 131 oocytes from e i t h e r wave o f . o v u l a t i o n i n superovulated immature r a t s have an equal chance of f e r t i l i z a t i o n and f e t a l development i f t r a n s f e r r e d to a normal r e c i p i e n t (Walton and Armstrong, 1983; Evans and Armstrong, 1984). However, Evans et a l . (1984) concluded that aging df oocytes from the f i r s t wave of o v u l a t i o n d i d not completely account f o r the d e c l i n e i n f e r t i l i z a b i l i t y (Evans and Armstrong, 1984). In the study of Evans et a l . (1984), there was great v a r i a b i l i t y i n o v u l a t o r y response between r a t s and oocytes were assessed only at the gross l e v e l (Evans and Armstrong, 1984). Th e r e f o r e , i t i s probable t h a t at l e a s t a p o r t i o n of the non-degenerated, u n f e r t i l i z e d oocytes had undergone aging without any obvious gross m a n i f e s t a t i o n . Loss of f e r t i l i z a b i l i t y i s a s s o c i a t e d with a l t e r a t i o n of the cytoplasm, c e l l membrane and zona p e l l u c i d a and occurs before any morphological changes are manifested (Yanagimachi and Chang, 1961; Marston and Chang, 1964; Longo, 1981; G i a n f o r t o n i and Gulyas, 1985). S i m i l a r l y , i n Part 1 i t was concluded that gross morphology i s not a r e l i a b l e i n d i c a t o r of po s t - o v u l a t o r y age and, t h e r e f o r e , f e r t i 1 i z a b i 1 i t y . The zona p e l l u c i d a of metaphase one oocytes i s not pe n e t r a b l e by sperm (Tesarik et a l . , 1988a). The f i r s t p o l a r body of the rat i s extremely unstable and r a p i d l y degenerates f o l l o w i n g emission ( T s a f r i r i and K r a i c e r , 1972; H i l l e n s j o , 1976), making i t d i f f i c u l t to d i f f e r e n t i a t e between metaphase one and metaphase two oocytes. T h e r e f o r e , i t i s p o s s i b l e that a p o r t i o n of the u n f e r t i l i z e d metaphase r a t oocytes may have been i n meiosis one. However, the hi g h l e v e l s of gonadotrophin a c t i v i t y i n PMSG i n i t i a t e premature n u c l e a r a c t i v a t i o n (Moor et a l . , 1980) and a l t e r cumulus-oocyte c o u p l i n g (Moor and G a n d o l f i , 1987) i n preov u l a t o r y oocytes of the 132 sheep. In the r a t , l o s s of cumulus-oocyte c o u p l i n g i s a s s o c i a t e d with resumption of meiosis (Larsen et a l . , 1986). T h e r e f o r e , i t i s u n l i k e l y that a s i g n i f i c a n t p o r t i o n of the u n f e r t i l i z e d oocytes were i n metaphase one. Th i s i s c o n s i s t e n t with the o b s e r v a t i o n that a l l oocytes r e t r i e v e d from superovulated hamsters were i n metaphase two (Roldan. et a l . , 1987). During p r e o v u l a t o r y oocyte maturation, physicochemical changes, mediated by i n t a c t cumulus-oocyte c o u p l i n g , occur i n the zona p e l l u c i d a which render i t pene t r a b l e ( T e s a r i k and Kopecny, 1986; Moor and G a n d o l f i , 1987; T e s a r i k et a l . , 1988a; Cran, 1989). If the i n t e g r i t y of the cumulus-oocyte c o u p l i n g i s d i s r u p t e d during maturation, p e n e t r a b i l i t y of the zona i s l o s t (Moor and G a n d o l f i , 1987). Since g o n a d o t r o p i n s a c t i v e l y mediate cumulus-oocyte co u p l i n g and s i g n a l t r a n s m i s s i o n (Kaplan et a l . , 1977; Moor et a l . , 1981; Moor and G a n d o l f i , 1987), the prolonged p e r i o d of gonadotrophin s t i m u l a t i o n a s s o c i a t e d with PMSG treatment may d i s r u p t c o u p l i n g and i n h i b i t the development of zona p e n e t r a b i l i t y . In the mouse, zona s o l u b i l i t y i s lower i n oocytes from superovulated immature than mature females (Longo, 1981) suggesting that the s u p e r o v l a t o r y process i n t e r f e r e s with zona t r a n s f o r m a t i o n . Increased p o t e n t i a l f o r CG exo c y t o s i s develops w i t h time f o l l o w i n g GVBD and i s a s s o c i a t e d with cytoplasmic m a t u r a t i o n ( D u c i b e l l a et a l . , 1990). Th e r e f o r e , a p o r t i o n of the u n f e r t i l i z e d cohort may have been i n c a p a b l e of sperm p e n e t r a t i o n due to immaturity of the zona p e l l u c i d a and asynchrony of n u c l e a r and cy t o p l a s m i c matura-t i o n . T h i s may be a compounding f a c t o r with sperm t r a n s p o r t i n the delayed f e r t i l i z a t i o n observed i n metaphase oocytes r e t r i e v e d from superovulated r a t s . 133 Spontaneous r e l e a s e of CG i s observed i n human (Rousseau et a l . , 1977), mouse ( N i c o s i a et a l . , 1975; D u c i b e l l a et a l . , 1990) and hamster oocytes (Longo, 1974). A c q u i s i t i o n of zona p e l l u c i d a p e n e t r a b i l i t y to sperm i n v o l v e s low l e v e l CG e x o c y t o s i s from the oocytes, i n a d d i t i o n to the i n f l u e n c e of cumulus c e l l s ( T e s a r i k and Kopecny, 1986; T e s a r i k et al. ' , 1988a). As observed i n Part 1, occa-s i o n a l exocytosis of CG o c c u r r e d i n both unaged and aged oocytes. Thus, i n a percentage of r a t oocytes, the zona may a c q u i r e p e n e t r a b i l i t y subsequent to o v u l a t i o n i n su p e r o v u l a t e d r a t s r e s u l t i n g i n delayed f e r t i l i z a t i o n . In c o n t r a s t , overwhelming dehiscence of CG i s a s s o c i a t e d with l o s s of zona s o l u b i l i t y and l o s s of f e r t i l i z a b i l i t y d u r i n g aging (Yanagimachi and Chang, 1961; Dodson et a l . , 1989). Unequivocal r e d u c t i o n i n suboolemmal CG was noted i n only one r a t i n the present study. However, the marked v a r i a t i o n i n suboolemmal CG d i s t r i b u t i o n made q u a n t i t a t i v e assessment d i f f i c u l t and more s u b t l e r e d u c t i o n s i n the number of CG may have been present but beyond the l i m i t s of q u a l i t a t i v e d e t e c t i o n . Sperm p e n e t r a t i o n of the zona p e l l u c i d a was observed i n 2 of the aged oocytes. P e n e t r a t i o n of aged r a t oocytes was r e p o r t e d by A u s t i n (1950), Niwa and Chang (1975) and Walton et a l . (1983). In Part 4, f e r t i l i z a t i o n of a small percentage of aged and fragmented oocytes was a l s o observed. The low number of p e n e t r a t e d aged oocytes suggests that sperm d i f f e r e n t i a t e between aged and unaged oocytes at the l e v e l of the zona. A l t e r a t i o n of the zona p e l l u c i d a may occur i n the r a t oocyte d u r i n g aging i n the absence of copious CG exocytosis. T h i s may represent an advanced stage of the pr o g r e s s i v e physicochemical t r a n s f o r m a t i o n of the zona mediated by 134 low l e v e l CG e x o c y t o s i s . In the mouse, s t r u c t u r a l a l t e r a t i o n of the zona occurred i n the absence of obvious CG e x o c y t o s i s and t h i s was a s s o c i a t e d with decreased zona p e n e t r a b i l i t y (Longo, 1981). S i g n i f i c a n t l y more aged oocytes were present i n the u n f e r t i l -i z e d group a f t e r removal of oocytes from the two r a t s which f a i l e d to f e r t i l i z e s u g g e s t i n g t h a t , when the e f f e c t of a l t e r e d sperm tr a n s p o r t i s removed, aging i s the major f a c t o r i n v o l v e d i n poor f e r t i l i z a t i o n r a t e . While the m a j o r i t y of u n f e r t i l i z e d oocytes from one of these r a t s were a p p a r e n t l y normal, those of the other r a t were abnormal. T h i s suggests t h a t , i n a subpopulation of r a t s , f a c t o r s other than sperm t r a n s p o r t and delayed f e r t i l i z a t i o n may a f f e c t f e r t i l i z a t i o n r a t e s . A p a u c i t y of suboolemmal CG was noted i n u n f e r t i l i z e d oocytes r e t r i e v e d from one r a t . In the mouse ( G i a n f o r t o n i and Gulyas, 1985; Dodson et a l . , 1989) and hamster (Yanagimachi and Chang, 1961), overwhelming CG e x o c y t o s i s i s a s s o c i a t e d with zona hardening and f a i l u r e of f e r t i l i z a t i o n . Although oocytes with unequivocal signs of aging were observed, many of the oocytes from t h i s r a t had non-s p e c i f i c morphological changes. Overwhelming CG e x o c y t o s i s was not observed during i n t r a o v i d u c t a l aging i n the mature r a t nor was a redu c t i o n i n CG observed i n the aged, u n f e r t i l i z e d oocytes r e t r i e v e d from the other r a t s i n the present study. T h e r e f o r e , i t i s u n l i k e l y that the r e d u c t i o n i n suboolemmal CG i n the oocytes r e t r i e v e d from one r a t was due to i n t r a o v i d u c t a l oocyte aging. A r e d u c t i o n i n suboolemmal CG was observed d u r i n g i n t r a f o l l -i c u l a r oocyte aging induced by neuropharmacology blockade i n the mature r a t (Peluso and Butcher, 1974). Since e s t r a d i o l l e v e l s were elev a t e d during the d e l a y of o v u l a t i o n , i t was concluded that the 135 morphological a l t e r a t i o n s were the r e s u l t of the adverse e f f e c t s of the e s t r a d i o l (Peluso and Butcher, 1974). In the present study, a marked r e d u c t i o n i n cytoplasmic g r a n u l a r i t y and e l e c t r o n d e n s i t y with empty spaces and m u l t i p l e c l u s t e r s of e l e c t r o n dense granules was observed, s i m i l a r to the morphology d e s c r i b e d f o r a t r e t i c r a t oocytes (Vazquez-Nin and S o t e l o , 1967). Thus, these oocytes may have o r i g i n a t e d from a t r e t i c f o l l i c l e s as suggested by Braw and T s a f r i r i (1980). However, these c y t o p l a s m i c changes are non-s p e c i f i c with regard to e t i o l o g y and are t y p i c a l of e a r l y c e l l s w e l l i n g due to a l t e r e d p e r m e a b i l i t y of the plasma membrane ( C h e v i l l e , 1983). This i s a d e g e n e r a t i v e change s i m i l a r to that observed i n the l a t e r stages of i n t r a o v i d u c t a l oocyte aging i n Part 1. Thus, the abnormal, u n f e r t i l i z e d oocytes may have o r i g i n a t e d from e i t h e r a t r e t i c f o l l i c l e s or f o l l i c l e s which underwent a l t e r e d f o l l i c u l a r maturation due to a l t e r e d s t e r o i d hormone l e v e l s . A l t e r n a t i v e l y , these oocytes may have undergone p o s t - o v u l a t o r y degeneration. The marked decrease i n suboolemmal CG i n the u n f e r t i l i z e d , abnormal oocytes may be the r e s u l t of decreased s y n t h e s i s due to the low number of MVB. However, t h i s i s u n l i k e l y s i n c e CG s y n t h e s i s occurs over a prolonged p e r i o d d u r i n g f o l l i c u l a r growth and matura-t i o n as well as subsequent to o v u l a t i o n ( S z o l l o s i , 1967; Zamboni, 1970). Therefore, i t i s more l i k e l y t h a t CG e x o c y t o s i s occurred, p o s s i b l y due to i n s t a b i l i t y of suboolemmal a c t i n m i c r o f i l a m e n t s (Cran, 1987) as d i s c u s s e d i n Part 2. Enlargement of the p e r i v i t e l l -i n e space i s observed i n a s s o c i a t i o n with CG e x o c y t o s i s i n the hamster (Yang and Yanagimachi, 1989). The abnormal oocytes had no i n c r e a s e i n the width of the p e r i v i t e l 1 i n e space suggesting that 136 the l o s s of f l u i d and e l e c t r o l y t e balance may have counteracted any e f f e c t of CG e x o c y t o s i s on oocyte diameter. In t h i s r a t , t h e r e f o r e , p e n e t r a t i o n f a i l u r e may have been due to oocyte degeneration and overwhelming CG e x o c y t o s i s l e a d i n g to zona hardening. However, the p o s s i b i l i t y of f a i l u r e of sperm t r a n s p o r t cannot be r u l e d out. In summary, a s i g n i f i c a n t decrease i n f e r t i l i z a t i o n r a t e was observed i n n a t u r a l l y mated, superovulated r a t s 72 to 75 hr post-treatment. S i x t y - f o u r percent of the u n f e r t i l i z e d oocytes had a l t e r e d morphology c o n s i s t e n t with i n t r a o v i d u c t a l oocyte aging and were l i k e l y u h f e r t i l i z a b l e . The lack of f e r t i l i z a t i o n i n the metaphase oocytes was a t t r i b u t e d to the long p e r i o d r e q u i r e d f o r f e r t i l i z a t i o n i n the s u p e r o v u l a t e d r a t as a r e s u l t of the low r a t i o of ampullary sperm to oo c y t e s . Thus, the s i g n i f i c a n t r e d u c t i o n i n f e r t i l i z a t i o n r a t e i n s u p e r o v u l a t e d immature r a t s was concluded to be the r e s u l t of i n t r a o v i d u c t a l oocyte aging and delayed f e r t i l -i z a t i o n . 137 PART 4: A MORPHOLOGICAL INVESTIGATION OP FERTILIZATION AND EMBRYONIC DEVELOPMENT IN IMMATURE RATS TREATED WITH 4 OR 40 IU PMSG AND SPONTANEOUSLY OVULATING RATS A. RESULTS 1. Breeding Rates Of 170 immature r a t s s e l e c t e d f o r the study, 84 and 86 were t r e a t e d with 4 and 40 IU PMSG, r e s p e c t i v e l y . In the 4 IU group, 49/84 (59%) had a post-breeding smear p o s i t i v e f o r sperm, whereas 70/86 (81%) of the r a t s i n the 40 IU group had p o s i t i v e post-breeding smears. The d i f f e r e n c e i n b r e e d i n g r a t e s was s i g n i f i c a n t at the p<0.005 l e v e l . Of the r a t s which f a i l e d to breed, 25/35 (72%) and 6/16 (35%) i n the 4 and 40 IU groups, r e s p e c t i v e l y , had approximately equal numbers of n e u t r o p h i l s and e p i t h e l i a l c e l l s i n the v a g i n a l smear and were c o n s i d e r e d to be i n metestrus. The number of r a t s with a metestrus smear was s i g n i f i c a n t l y h i g h e r i n the 4 IU group (p<0.05). To f u r t h e r c l a r i f y t h i s f i n d i n g , 9/35 and 6/16 of the r a t s which f a i l e d to breed i n the 4 and 40 IU groups, r e s p e c t i v e l y , were s a c r i f i c e d the morning f o l l o w i n g placement with a male. As i l l u s t r a t e d i n Table 4, these 15 r a t s had reduced ampullary d i s t e n s i o n and a l a r g e number of cumulus-free and fragmented oocytes, s u g g e s t i n g that o v u l a t i o n had occurred 24 hr e a r l i e r . Two of 48 mature r a t s had an e s t r u s smear with no sperm the morning f o l l o w i n g breeding. These r a t s were not f u r t h e r examined. The remaining 46 r a t s (96%) had a v a g i n a l smear p o s i t i v e f o r sperm. The breeding r a t e i n mature r a t s was s i g n i f i c a n t l y higher (p<0.05) than the 40 IU group. 138 TABLE 4. GROSS ASSESSMENT OF OOCYTES RETRIEVED FROM IMMATURE PMSG TREATED RATS WHICH FAILED TO MATE AND EXHIBITED A METESTRUS SMEAR TREATMENT GROSS ASSESSMENT 4 IU 40 IU number of r a t s examined 9 6 number of r a t s o v u l a t e d 7 6 t o t a l embryos r e t r i e v e d 59 133 average per r a t 8.4 22 degenerated oocytes 16 (26%) 6 ' (4.5%) fragmented oocytes 12 (19.5%) 33 (25%) i n t a c t oocytes 18 (29%) 65 (49.5%) 2 c e l l 7 (11.3%) 21 (15.5%) 3 c e l l 6 (9.7%) 7 (5%) 4 c e l l 0 1 (0.5%) ampullary d i s t e n s i o n 1/7 2/6 cumulus mass 1/7 1/6 139 120 PERCENTAGE OF BRED RATS FROM WHICH EMBRYOS WERE RETRIEVED 2 3 DAY OF PREGNANCY | | MATURE (23 4 IU g 3 40 IU 50 40" er 8 30 fe 20 10 AVERAGE NUMBER OF EMBRYOS RETRIEVED 20 * \ 17 19 *. m— 3 * 2 3 DAY OF PREGNANCY 4 IU ••+• 40 IU - * - MATURE Page 139 FIGURE 23 The percentage of bred r a t s from which embryos were r e t r i e v e d i s i l l u s t r a t e d . A p r o g r e s s i v e decrease i n pregnant r a t s was observed from day 2 to 4 i n the 40 IU group. The number of bred r a t s i n each group i s i n d i c a t e d above the bars. FIGURE 2 4 The average number of embryos and oocytes r e t r i e v e d from r a t s i n the 3 treatment groups i s i l l u s t r a t e d . A pro-g r e s s i v e decrease i n the number of embryos r e t r i e v e d was observed i n the 40 IU group over the f i r s t 4 days of pregnancy. The number of r a t s from which embryos were r e t r i e v e d i n the superovulated group i s i n d i c a t e d . 140 2. Embryo R e t r i e v a l and Gross Assessment The percentages of r a t s i n each group from which embryos were r e t r i e v e d on each day of pregnancy are presented i n F i g u r e 23. The range and average number of embryos r e t r i e v e d per r a t are shown i n Table 5 and Figure 24, r e s p e c t i v e l y . Embryos were r e t r i e v e d from a l l r a t s i n the mature and 4 IU groups at each s a c r i f i c e time. In c o n t r a s t , fewer embryos were r e t r i e v e d from a d e c r e a s i n g percentage of superovulated r a t s over the i n i t i a l four day o b s e r v a t i o n p e r i o d . Since extremely low numbers of . embryos were r e t r i e v e d from superovulated r a t s on day 4 of pregnancy, the o b s e r v a t i o n p e r i o d was l i m i t e d to days 1, 2 and 3 of pregnancy. The gross assessment of embryos r e t r i e v e d from a l l 3 treatment groups are presented i n F i g u r e s 25 to 31, i n c l u s i v e . The number of degenerated oocytes r e t r i e v e d from each group at each s a c r i f i c e time as a percentage of the t o t a l number of embryos r e t r i e v e d i s presented i n Figure 25. S i g n i f i c a n t l y fewer degenerated oocytes were r e t r i e v e d from superovulated r a t s on day 1 of pregnancy (p<0.005). Fewer degenerated oocytes were r e t r i e v e d from the mature r a t s on day 2 and day 3 (p<0.025). S i g n i f i c a n t l y fewer degenerated oocytes were r e t r i e v e d from the superovulated group than the 4 IU group on day 3 (p<0.005). F i g u r e s 26, 27 and 28 i l l u s t r a t e that the m a j o r i t y of the degenerated oocytes i n a l l groups were r e t r i e v e d from a d i s p r o p o r t i o n a t e number of r a t s . T herefore, degenerated oocytes were d i s c a r d e d from the study. Figures 29, 30 and 31 i l l u s t r a t e gross embryonic development over the f i r s t 3 days of pregnancy. In the mature and 4 IU groups, f e r t i l i z e d oocytes and 2 c e l l embryos predominated on days 1 and 2 of pregnancy, r e s p e c t i v e l y . By day 3 of pregnancy, 2, 3 and 4 c e l l 141 TABLE 5. RANGE OF EMBRYOS RETRIEVED FROM EACH GROUP OF RATS ON EACH DAY OF PREGNANCY GROUP DAY MATURE 4 IU 40 IU 1 9-19 1-12 4-84 2 11-19 1-13 1-54 3 12-19 7-13 1-71 4 10-17 12-14 1-4 142 120-1 110-100-i 90-LU CC 80-i 70-9 r-LL 60-O UJ 50-o z 40-LU & 30-It 20-10-o-RETRIEVAL OF DEGENERATED OOCYTES 204 135 843 190 152 360 181 352 ^ 1 1 2 DAY OF PREGNANCY | I MATURE [ 2 3 * IU £ ~ ] 40 IU Vr -T?^ . ' Page 142 f) FIGURE 25 The percentage of the t o t a l r e t r i e v a l which, on gross examination, were degenerated oocytes i s i l l u s t r a t e d . No e f f e c t of treatment on the occurrence of degenerated oocytes was observed. The percentage of degenerated oocytes was lower i n the 40 IU group than the 4 IU group on day 1 and 3 of pregnancy. The t o t a l number of oocytes and embryos r e t r i e v e d from each group i s i n d i c a t e d above the bars. 143 PATTERN OF RETRIEVAL OF DEGENERATED OOCYTES - MATURE 120-110-100-90-80-UJ 3 70-60-GC UJ 50-CL 40-30-20-10-o-PATTERN OF RETRIEVAL OF DEGENERATED OOCYTES - 4 IU 14 18 14 1 2 CAY OF PREGNANCY PATTERN OF RETRIEVAL OF DEGENERATED OOCYTES - 40 IU DAY OF PREGNANCY • Page 143 (\ FIGURES 26, 27 and 28 These graphs i l l u s t r a t e that the m a j o r i t y of degenerated oocytes were r e t r i e v e d from a small percentage of r a t s i n each group on a l l days of pregnancy. The percentage of r a t s which had 50% or more degenerated oocytes i s i l l u s t r a t e d by the bar l a b e l l e d r a t s . The bar l a b e l l e d oocytes represents the percentage of the t o t a l degenerated oocytes which were recovered from these r a t s . 144 embryos c o n s t i t u t e d the major p a r t of the r e t r i e v a l . S i g n i f i c a n t l y more 3 c e l l embryos and fewer 4 c e l l embryos were r e t r i e v e d from the 4 IU group on day 3 of pregnancy than the mature group (p<0.025). A small percentage of fragmented and u n f e r t i l i z e d oocytes were r e t r i e v e d on each day of pregnancy. In c o n t r a s t to the mature and 4 IU groups, no one type of embryo predominated on any day of pregnancy i n the 40 IU group. S i g n i f i c a n t l y more u n f e r t i l i z e d , fragmented and developmental l y advanced embryos and s i g n i f i c a n t l y fewer f e r t i l i z e d embryos were r e t r i e v e d from the 40 IU group on day 1 of pregnancy (p<0.005). The l a r g e p r o p o r t i o n of u n f e r t i l i z e d oocytes r e t r i e v e d on day 1 i n the 40 IU group was s i g n i f i c a n t l y reduced on day 2 with a concomitant s i g n i f i c a n t i n c r e a s e i n fragmented forms and 2, 3 and 4 c e l l embryos (p<0.005). On day 2 and 3 of pregnancy, embryos at the expected developmental stage and fragmented forms were r e t r i e v e d i n approximately equal p r o p o r t i o n s . Fewer 2 c e l l embryos on day 2 and 4 c e l l embryos on day 3 were recovered from the 40 IU group than the other two groups (p<0.005). S i g n i f i c a n t l y more 3 c e l l embryos were r e t r i e v e d from the 40 IU group on day 3 (p<0.005). Figure 27 i l l u s t r a t e s that s i g n i f i c a n t l y more fragmented forms (p<0.005) were r e t r i e v e d from the 40 IU group over the o b s e r v a t i o n p e r i o d than the mature and 4 IU groups. However, the i n c r e a s e w i t h i n the 40 IU group was only s i g n i f i c a n t on day 2 (p<0.005). When developmental r a t e s were compared f o l l o w i n g e x c l u s i o n of fragmented oocytes from a l l groups, there was no s i g n i f i c a n t d i f f e r e n c e i n the number of 2 c e l l embryos r e t r i e v e d on day 2 or 3 of pregnancy between groups. S i g n i f i c a n t l y more 3 c e l l embryos and fewer 4 c e l l embryos were observed i n the 40 IU group on day 3 145 GROSS EMBRYO DEVELOPMENT - MATURE 120 110 100 90 80 70 60 SO 40 30 20 10 0 i o I UI 8 UI FRAG CD UNFERT H i 1CEU • 2 CELL E3 3 CELL K] 4CEU 150 180 226 DAY OF PREGNANCY GROSS EMBRYO DEVELOPMENT - 4 IU 112 146 167 1 2 3 DAY OF PREGNANCY GROSS EMBRYO DEVELOPMENT - 40 IU 120 110 100-90-80 70 60 50 40 30 20 10 0 812 344 336 > 1 1 2 3 DAY OF PREGNANCY . ..x Page' 145 R FIGURES 29, 30 and 31 These graphs i l l u s t r a t e gross embryo development from day 1 to 3 of pregnancy i n the three groups. FRAG = fragmented oocyte; UNFERT = u n f e r t i l i z e d i n t a c t oocyte; FERT = f e r t i l i z e d i n t a c t oocyte. The numbers above the bars i n d i c a t e the t o t a l r e t r i e v a l minus degenerated oocytes. 146 120 GROSS EMBRYO QUALITY ON DAY 2 OF PREGNANCY MAT TREATMENT GROUP • 2 CELL 3 CELL ^ 4 CELL GROSS EMBRYO QUALITY ON DAY 3 OF PREGNANCY 120-1 TREATMENT GROUP • 2 CELL f23 3 CELL gxj 4 CELL . . u Page 146 FIGURES 32 and 33 These graphs i l l u s t r a t e the gross q u a l i t y of embryos i n t h three treatment groups. Poorer q u a l i t y of 3 and 4 c e l l embryo i s observed i n the 40 IU group on day 2 and 3 of pregnancy. The number of embryos examined i s i n d i c a t e d above the b a r s . 147 (p<0.005). Within the 40 IU group, the t o t a l percentage of 2, 3 and 4 c e l l embryos d i d not change s i g n i f i c a n t l y from day 2 to 3 of pregnancy. However, the number of 2 c e l l embryos s i g n i f i c a n t l y decreased (p<0.005) and the number of both 3 and 4 c e l l embryos s i g n i f i c a n t l y i n c r e a s e d (p<0.005) from day 2 to 3 of pregnancy. The percentages of embryos judged g r o s s l y to be of good q u a l i t y on days 2 and 3 of pregnancy are shown i n F i g u r e s 32 and 33. In the mature and 4 IU groups, gross embryo q u a l i t y was maintained over the o b s e r v a t i o n p e r i o d . There was no d i f f e r e n c e between groups i n the q u a l i t y of 2 c e l l embryos. The gross q u a l i t y of 3 and 4 c e l l embryos was s i g n i f i c a n t l y lower (p<0.005) on day 3 of pregnancy i n the 40 IU group than i n the mature and 4 IU groups. The gross q u a l i t y of 3 and 4 c e l l embryos was s i g n i f i c a n t l y lower than that of 2 c e l l embryos w i t h i n the 40 IU group on day 2 (p<0.025) and 3 (p<0.005) of pregnancy. 3. Light M i c r o s c o p i c Assessment The l i g h t m i c r o s c o p i c morphology of embryos from the immature r a t s i s summarized i n F i g u r e 34. In the mature group, 31 f e r t i l i z e d oocytes were examined on day 1 of pregnancy. Of these, 2/31 were undergoing f e r t i l i z a t i o n with obvious sperm head f u s i o n to the oocyte (Figure 35a). One oocyte was i n e a r l y t e l ophase with the s p i n d l e s t i l l p a r a l l e l to the s u r f a c e of the c e l l ( F i g u r e 35b). The telophase s p i n d l e i n the other c e l l had r o t a t e d and was i n the process of p i n c h i n g o f f the second p o l a r body ( F i g u r e 35c). The remaining 29 oocytes had a second p o l a r body i n the p e r i v i t e l 1 i n e space, which o c c a s i o n a l l y had f u r t h e r d i v i d e d i n t o two equal p a r t s . Two of the 29 oocytes had a second MVF protuberance on the pole 148 | | MATURE 2 2 4 IU g>3 40 IU Page 148 A FIGURE 34 This graph i l l u s t r a t e s the percentage of normal embryos assessed m i c r o s c o p i c a l l y i n the three treatment groups. A s i g n i f i c a n t d e c l i n e i n embryo q u a l i t y was observed i n the 40 IU group on day 2 and 3 of pregnancy. The number of embryos examined i n each group i s i n d i c a t e d above the bars. 149 o p p o s i t e to the second p o l a r body. E a r l y sperm head decondensa t ion was o c c u r r i n g i n these a d d i t i o n a l MVF p r o t u b e r a n c e s and the sperm t a i l was a l s o v i s i b l e a d j a c e n t to the male c h r o m a t i n . The s u r f a c e c o n t o u r of the f e r t i l i z e d oocytes was undulent to s l i g h t l y i r r e g u l a r , o f t e n w i t h a smal l r a i s e d i n c o r p o r a t i o n cone which conta ined the sperm t a i l . The sperm t a i l was s i t u a t e d p e r i p h e r a l l y i n most o o c y t e s and an extranumerary sperm was o f t e n v i s i b l e i n the p e r i v i t e l l i n e s p a c e . P e r i p h e r a l VA w i t h dense, b a s o p h i l i c f o c i and s m a l l t o l a r g e , o c c a s i o n a l l y m u l t i l o c u l a t e d , MVB were observed. The c y t o s o l b a s o p h i l i a v a r i e d from l i g h t to dark and a sperm t a i l was v i s i b l e i n a l l o o c y t e s . Two p r o n u c l e i were observed i n 27 f e r t i l i z e d o o c y t e s . These were u s u a l l y l o c a t e d p e r i p h e r a l l y e i t h e r near the second p o l a r body or near the sperm t a i l ( F i g u r e 35d) . The p r o n u c l e i ranged from smal l and round w i t h h i g h l y b a s o p h i l i c nuc l eop lasm w i t h up to four s m a l l , p e r i p h e r a l , d e n s e l y b a s o p h i l i c n u c l e o l i to l a r g e and i r r e g u l a r wi th f a i n t l y b a s o p h i l i c cy top lasm w i t h up to s i x l a r g e , c e n t r a l or p e r i p h e r a l n u c l e o l i . One pronuc leus i n most oocytes had one very l a r g e c e n t r a l n u c l e o l u s ( F i g u r e 3 5 d , e ) . One to two smal l dense ly b a s o p h i l i c NLB were observed i n 6/27 (22%) oocytes w i th two p r o n u c l e i ( F i g u r e 35e ) . On day 2 of p r e g n a n c y , 2 c e l l embryos c o n s i s t e d of two i r r e g u l a r , s l i g h t l y e l o n g a t e d c e l l s w i t h a f l a t t e n e d opposing s u r f a c e and a l a r g e i n t e r c e l l u l a r space . One or two p o l a r bodies were v i s i b l e i n the p e r i v i t e l 1 i n e space . The p o l a r body was u s u a l l y s i t u a t e d at the j u n c t i o n of the two b l a s t o m e r e s . In those oocytes which had two p o l a r body remnants , the p o l a r bodies were s i t u a t e d at oppos i t e e x t r e m i t i e s of the i n t e r c e l l u l a r space . The sperm t a i l 150 r - ^ / i ^ P a g e 150 fl FIGURE 35 A. F e r t i l i z e d oocyte r e t r i e v e d from a mature rat on the morning f o l l o w i n g breeding. Note the sperm head being i n c o r p o r a t e d i n t o the oocyte (arrowhead). M a g n i f i c a t i o n = 565x. B. F e r t i l i z e d oocyte r e t r i e v e d from a mature r a t on the morning f o l l o w i n g breeding. The oocyte i s i n telophase with the s p i n d l e p a r a l l e l to the oolemma (arrow). M a g n i f i c a t i o n = 565x. C. F e r t i l i z e d oocyte r e t r i e v e d from a mature r a t on day one of pregnancy. The telophase s p i n d l e has r o t a t e d 90° on i t s a x i s (arrow) and has commenced p o l a r body 2 e x t r u s i o n (arrowhead). M a g n i f i c a t i o n = 565x. D. Zygote r e t r i e v e d from a mature r a t on day one of pregnancy. There i s e a r l y p r o n u c l e i formation. The p r o n u c l e i (arrows) are p e r i p h e r a l i n the c e l l , below the second p o l a r body ( l a r g e arrowhead). The sperm t a i l can be observed i n the adjacent cytoplasm (small arrowhead). M a g n i f i c a t i o n = 625x. E. Zygote r e t r i e v e d from a mature r a t on day one of pregnancy. The p r o n u c l e i are c e n t r a l i n the c e l l (arrows). Note the second p o l a r body with chromosomes forming a nucleus ( l a r g e arrowhead). One p e r i p h e r a l n u c l e o l a r - l i k e body i s present (small arrowhead). M a g n i f i c a t i o n = 625x. F. Two c e l l embryo r e t r i e v e d from a mature r a t on day 3 of pregnancy. Note the large, c e n t r a l i r r e g u l a r n u c l e i with compact n u c l e o l i (arrows) and c l o s e c e l l a p p o s i t i o n (arrowhead). M a g n i f i c a t i o n = 625x. 151 was of t e n observed extending from one blastomere to the other v i a themidbody. P e r i p h e r a l VA tended to be sma l l e r and o f t e n contained b a s o p h i l i c f o c i . Smaller MVB were commonly observed. P e r i p h e r a l and cytoplasmic NLB were p r e s e n t e i t h e r s i n g l y or i n c l u s t e r s . The cytoplasmic NLB were o f t e n c oncentrated around the nucleus. There was i n c r e a s e d p e r i n u c l e a r b a s o p h i l i a and one nucleus was present i n each blastomere. The n u c l e i were l a r g e , i r r e g u l a r and c e n t r a l . The nucleoplasm was p a l e w i t h up to four l a r g e , round n u c l e o l i and numerous s m a l l e r , round, d e n s e l y b a s o p h i l i c f o c i e i t h e r w i t h i n the nucleoplasm or immediately i n a s s o c i a t i o n with the i n n e r n u c l e a r membrane. On day 3 of pregnancy, the blastomeres of the 2 c e l l embryos were more c l o s e l y apposed with a smaller i n t e r c e l l u l a r space (Figure 3 5 f ) . The n u c l e o l i were o c c a s i o n a l l y ring-shaped, contained one or more vacuoles or had developed an i n d i s t i n c t , i r r e g u l a r b a s o p h i l i c border i n 12/15 (80%) of the embryos. One embryo had one normal blastomere and one p a l e , l y s e d blastomere. Another embryo had one c e l l i n m i t o s i s . Four c e l l embryos c o n s i s t e d of four angular, s l i g h t l y i r r e g u l a r c e l l s of equal s i z e which were c l o s e l y apposed to one another with a small i n t e r c e l l u l a r space and f o c a l p o i n t s of c e l l c o n t a c t. The second p o l a r body was only o c c a s i o n a l l y observed. P e r i p h e r a l VA and MVB were r a r e l y seen. Each blastomere contained one l a r g e , c e n t r a l , s l i g h t l y i r r e g u l a r nucleus with v a r i a b l e nucleoplasmic b a s o p h i l i a and 1 to 4 n u c l e o l i . The n u c l e o l i had a compact centre with marked i r r e g u l a r b a s o p h i l i a p e r i p h e r a l l y . N u c l e o l a r - l i k e bodies were commonly observed i n the p e r i p h e r a l and p e r i n u c l e a r cytoplasm. One embryo had three normal blastomeres and 152 Page 152 A FIGURE 36 A. F e r t i l i z e d oocyte r e t r i e v e d from a superovulated r a t on day 1 of pregnancy. Two p r o n u c l e i are present. Note the. i n c o r p o r a t i o n cone with sperm (arrowhead) and the v a r i a t i o n i n cytoplasmic b a s o p h i l i a between oocytes. M a g n i f i c a t i o n = 350x. B. F e r t i l i z e d oocyte r e t r i e v e d from a superovulated r a t on day 1 of pregnancy. Note the marked i r r e g u l a r i t y of the oocyte where the second p o l a r body was extruded (arrow). The c e l l has a more undulent contour i n ge n e r a l . M a g n i f i c a t i o n = 375x. C. Two c e l l embryos r e t r i e v e d from an immature rat t r e a t e d with 4 IU PMSG. Note the c e n t r a l nucleus with n u c l e o l u s . The second p o l a r body (arrow) and a l a r g e c e l l fragment (arrow-head) are present i n the p e r i v i t e l 1 i n e space. M a g n i f i c a t i o n = 565x. D. Two c e l l embryo r e t r i e v e d from a superovulated r a t on day 2 of pregnancy. The sperm t a i l i s v i s i b l e i n the cytoplasm (arrow). A la r g e l y s e d c e l l fragment i s present (arrowhead). M a g n i f i c a t i o n = 565x. E. Two c e l l embryo r e t r i e v e d from a superovulated r at on day 2 of pregnancy. The c e l l s are m u l t i n u c l e a t e d (small arrow-heads) with o r g a n e l l e p o l a r i z a t i o n ( l a r g e arrowheads). Two c e l l fragments are present i n the p e r i v i t e l 1 i n e space. N u c l e o l a r - l i k e bodies are i n d i c a t e d by arrows. M a g n i f i c a t i o n = 625x. F. Three c e l l embryo r e t r i e v e d from a superovulated r at on day 3 of pregnancy. The embryo i s m u l t i n u c l e a t e d with o r g a n e l l e p o l a r i z a t i o n (arrowhead). M a g n i f i c a t i o n = 625x. G. Four c e l l embryo r e t r i e v e d from a r a t t r e a t e d with 4 IU PMSG. Note the c l o s e c e l l a p p o s i t i o n of the four c e l l s and the a c t i v a t e d n u c l e o l i (arrows). M a g n i f i c a t i o n = 375x. H. Four c e l l embryo r e t r i e v e d from a superovulated rat on day 3 of pregnancy. Note the a c t i v a t e d n u c l e o l i (arrows). A supernumerary sperm i n present i n the p e r i v i t e l 1 i n e space (arrowhead). One c e l l has dark cytoplasm with m u l t i p l e small vacuoles. M a g n i f i c a t i o n = 625x. 153 one p a l e , lysed blastomere. The number of embryos i n a l l groups on each day of pregnancy with sperm i n the cytoplasm and n u c l e o l a r a c t i v a t i o n are presented i n Tables 6 and 7, r e s p e c t i v e l y . The occurrence of sperm i n the cytoplasm d i d not d i f f e r s i g n i f i c a n t l y between embryos with normal m i c r o s c o p i c morphology i n a l l groups on each day of pregnancy. F e r t i l i z e d oocytes r e t r i e v e d from the immature r a t s were s i m i l a r to those of the mature r a t s ( F i g u r e 36a). Zygotes from the immature r a t s v a r i e d more i n s u r f a c e contour and 8/135 (6%) of the zygotes from the superovulated r a t s had a markedly i r r e g u l a r s u r f a c e adjacent to the second p o l a r body ( F i g u r e 36b). The percen-tage of oocytes with NLB i n the 4 (9/25 (36%)) and 40 (57/135 (42%)) IU groups was higher than that observed i n the mature r a t s (6/27 ( 2 2 % ) ) , but t h i s was not s t a t i s t i c a l l y s i g n i f i c a n t . Two c e l l embyros r e t r i e v e d on day 2 of pregnancy from immature r a t s t r e a t e d with 4 IU PMSG d i d not d i f f e r m o r p h o l o g i c a l l y from those r e t r i e v e d from the mature r a t s ( F i g u r e 36c). There was a s i g n i f i c a n t and p r o g r e s s i v e i n c r e a s e i n abnormal embryo morphology from day 1 to 3 i n the 40 IU group (p<0.005). On day 2 of preg-nancy, 31/43 (72%) of the 2 c e l l embryos were s i m i l a r to those r e t r i e v e d from the mature and 4 IU groups ( F i g u r e 36d). Twelve (28%) of the 2 c e l l embryos were u n f e r t i l i z e d and had abnormal morphology. Nine (75%) were m u l t i n u c l e a t e d (MN), having up to s i x sm a l l , round to s l i g h t l y i r r e g u l a r n u c l e i c o n t a i n i n g one or two round, densely b a s o p h i l i c n u c l e o l i i n each blastomere. Marked p e r i p h e r a l p o l a r i z a t i o n of o r g a n e l l e s (OP) was observed i n 10 (83%) of the abnormal embryos, and seven of these were a l s o multinuc-l e a t e d ( F i g u r e 36e). When u n f e r t i l i z e d 2 c e l l embryos were excluded 154 TABLE 6. OCCURRENCE OF SPERM IN THE CYTOPLASM OF NORMAL AND ABNORMAL EMBRYOS GROUP DAY 1 DAY 2 DAY 3 MATURE 27/27 12/12 35/41 4 IU 25/25 27/29 28/35 40 IU 135/135 28/31 8/10 4 IU ABNORMAL 0/6 - 0/4 40 IU ABNORMAL 6/29* 2/21* 5/24* * Day 1 - three 2 c e l l embryos (2/3 m u l t i n u c l e a t e d ) - two m u l t i n u c l e a t e d 4 c e l l embryos - one 3 c e l l embryo with n u c l e o l a r a c t i v a t i o n Day 2 - two 2 c e l l embryos (1 m u l t i n u c l e a t e d ) Day 3 - two m u l t i n u c l e a t e d 2 c e l l embyros - two m u l t i n u c l e a t e d 3 c e l l embyros - one m u l t i n u c l e a t e d 4 c e l l embryo TABLE 7. NUCLEOLAR ACTIVATION IN NORMAL AND ABNORMAL EMBRYOS GROUP DAY 1 DAY 2 DAY 3 MATURE 0/27 0/12 12/15 2 c e l l 26/26 4 c e l l 4 IU 0/25 0/29 8/12 2-3 c e l l 23/23 4 c e l l 40 IU 0/135 0/31 7/7 2-3 c e l l 4/4 4 c e l l 4 IU ABNORMAL 0/6 - 0/4 40 IU ABNORMAL 1/29* 0/21 0/24 * - one 3 c e l l embryo 155 from the t o t a l number of 2 c e l l embryos r e t r i e v e d , s i g n i f i c a n t l y fewer (p<0.005) 2 c e l l embryos were observed i n the 40 IU group compared with the other two groups. However, the percentage of f e r t i l i z e d embryos remained s i g n i f i c a n t l y h igher (p<0.005) on day 2 compared with day 1 w i t h i n the 40 IU group. On day 3 of pregnancy, 4/16 (25%) of 2 and 3 c e l l embryos r e t r i e v e d from the 4 IU group were m u l t i n u c l e a t e d . The remaining 12 embryos d i d not d i f f e r from those examined i n the mature group and four were i n m i t o s i s . In the 40 IU group, 6/13 (46%) 2 c e l l embryos were MN and one a l s o had OP. Of the seven u n i n u c l e a t e d 2 c e l l embryos, one had OP. S i x of seven 3 c e l l embryos were MN, of which 3 had p o l a r i z a t i o n of o r g a n e l l e s ( F i g u r e 3 6 f ) . Four c e l l embryos r e t r i e v e d from immature r a t s t r e a t e d with 4 IU PMSG d i d not d i f f e r from those of mature r a t s ( F i g u r e 36g), whereas those recovered from s u p e r o v u l a t e d r a t s d i f f e r e d substan-t i a l l y (Figure 36h) . Ten of 15 (67%) of the 4 c e l l embryos recovered from superovulated r a t s were MN ( F i g u r e 37a), of which one had OP. One of the f i v e u n i n u c l e a t e d 4 c e l l embryos a l s o had OP. Embryos with abnormal m i c r o s c o p i c morphology r e t r i e v e d on both day 2 and 3 of pregnancy had a s i g n i f i c a n t l y lower f e r t i l i z a t i o n r a t e , as evidenced by absence of sperm remnants i n the cytoplasm (p<0.005). A s i g n i f i c a n t i n c r e a s e i n MN embryos was noted on day 3 (p<0.005). The f i n d i n g s of the morphological assessment of developmental-l y advanced embryos are summarized i n F i g u r e 38. Developmentally advanced embryos were more o f t e n m u l t i n u c l e a t e d (p<0.005) and s i g n i f i c a n t l y fewer were f e r t i l i z e d (p<0.005) than embryos at expected stages of development. S i x 2 c e l l embryos were recovered 156 Page 156 A FIGURE 37 A. Four c e l l embryo r e t r i e v e d from a s u p e r o v u l a t e d r a t on day 3 of pregnancy. Three blastomeres are m u l t i n u c l e a t e d . There i s no a c t i v a t i o n of n u c l e o l i . M a g n i f i c a t i o n = 565x. B. Two c e l l embryo r e t r i e v e d from a superovulated r a t on day 1 of pregnancy. There i s good c e l l a p p o s i t i o n and one c e l l i s m u l t i n u c l e a t e d (arrow). M a g n i f i c a t i o n = 625x. C. Four c e l l embryo r e t r i e v e d on day 1 from a superovulated r a t . A sperm head i s present i n one c e l l (arrow). C e l l s are rounded with poor c e l l a p p o s i t i o n . There i s o r g a n e l l e p o l a r i z a t i o n i n one c e l l (arrowhead). M a g n i f i c a t i o n = 565x. D. Fragmented oocyte r e t r i e v e d from a superovulated r a t on day 2 of pregnancy. The c e l l i s d i v i d i n g (arrows), forming a new fragment with m u l t i p l e n u c l e i . M a g n i f i c a t i o n = 625x. E. Fragmented oocyte r e t r i e v e d from a superovulated r a t on day 2 of pregnancy. There i s o r g a n e l l e p o l a r i z a t i o n i n a l l fragments and m u l t i p l e n u c l e i are present i n one fragment (arrowhead). F i b r i l l a r a r r a y s are absent. M a g n i f i c a t i o n = 625x. F. Fragmented oocyte r e t r i e v e d from a superovulated r a t on day 3 of pregnancy. One fragment i s packed with f i b r i l l a r a r rays ( l a r g e arrowhead). O r g a n e l l e s have c l u s t e r e d around n u c l e i (small arrowheads) and i n small groups w i t h i n the cytoplasm. M a g n i f i c a t i o n = 625x. 157 120 MICR0SC0PIC ASSESSMENT OF DEVELOPMENT ALLY ADVANCED EMBRYOS DAY OF PREGNANCY Q M N L ^ F E R T I ^ O P 120-. £! 110-100-§ 90-Q 80-I 70-60-LL U. 50-O 40-i 30-ED o cc 20-B 10-o-MICROSCOPIC ASSESSMENT OF FRAGMENTED OOCYTES 16 20 11 DAY OF PREGNANCY • MN EIFERTC^OP :• j Page 157 R FIGURE 38 This graph i l l u s t r a t e s the occurrence of m u l t i n u c l e a t e d blastomeres (MN), f e r t i l i z a t i o n (FERT) and o r g a n e l l e p o l a r i z a t i o n (OP) i n developmentally advanced embryos i n the 40 IU group. The number of developmentally advanced embryos examined i s i n d i c a t e d above the bars. FIGURE 39 This graph summarizes the m i c r o s c o p i c morphology of fragmented oocytes recovered from the superovulated r a t s . MN = m u l t i n u c l e a t e d ; FERT = f e r t i l i z e d ; OP = o r g a n e l l e p o l a r i z a t i o n . The number of fragmented oocytes examined i s i n d i c a t e d above the b a r s . 158 from the 4 IU group on day 1 of pregnancy, of which three were MN and one had no nucleus i n one c e l l . In the 40 IU group, 24/29 of the 2, 3 and 4 c e l l embryos were m u l t i n u c l e a t e d ( F i g u r e 37b). One of the 4 c e l l embryos with sperm i n the cytoplasm had an uncondensed sperm head ( F i g u r e 37c). The morphology of fragmented oocytes recovered, over the o b s e r v a t i o n p e r i o d i s summarized i n F i g u r e 39. S i m i l a r to the abnormal and developmentally advanced embryos, fragmented oocytes were f r e q u e n t l y MN ( F i g u r e 37d,e) wi t h OP ( F i g u r e 37d,e,f). Fragmented oocytes w i t h s u r f a c e c o n s t r i c t i o n s were o c c a s i o n a l l y observed (Figure 37d). 4. E l e c t r o n Microscopy A d i s t i n c t and dynamic p r o g r e s s i o n of u l t r a s t r u c t u r a l change was.observed i n embryos r e t r i e v e d from mature r a t s . On day 1 of pregnancy, f e r t i l i z e d oocytes had an i r r e g u l a r c e l l s u r f a c e with i n c r e a s e d numbers of m i c r o v i l l i and a c t i v e m i c r o p i n o c y t o s i s and many coated p i t s . There was a s u b s t a n t i a l , but not complete, r e d u c t i o n i n suboolemmal c o r t i c a l g r a n u l e s ( F i g u r e 40a). Sperm was v i s i b l e i n the p e r i v i t e l 1 i n e space, cytoplasm and i n c o r p o r a t i o n cone. Round (375-850 nm diameter) to c y l i n d r i c a l (180 x 700 to 250 x 1500 nm), e l e c t r o n dense mitochondria with t r a n s v e r s e and c o n c e n t r i c c r i s t a e were observed s i n g l y and c l u s t e r e d , e i t h e r alone or with other o r g a n e l l e s , i n the cytoplasm. F i b r i l l a r a r r a y s were densely packed i n the cytoplasm ( F i g u r e 40a). V e s i c u l a r aggregates with e l e c t r o n dense f o c i were o f t e n observed as were sheets of MVB. These o r g a n e l l e s were u s u a l l y s i t u a t e d around the nucleus. Small stacks of l a m e l l a r membranes were observed near VA and were 159 .3;-. Page 159 fl FIGURE 40 A. Zygote r e t r i e v e d from a mature r a t on day 1 of pregnancy. C o r t i c a l granules are markedly reduced i n number, m i c r o v i l l i are present on the oolemmal s u r f a c e (arrow) and small n u c l e o l a r - l i k e bodies are present (arrowheads). N u c l e o l i are i n i n t i m a t e contact with the i n n e r s u r f a c e of the pronuclear membrane and are p r o t r u d i n g i n t o the adjacent cytoplasm. M a g n i f i c a t i o n = 3,150x. B. F e r t i l i z e d oocyte r e t r i e v e d from a mature r a t on day 1 of pregnancy. Sperm head decondensation i s o c c u r r i n g ( l a r g e arrowhead). The sperm t a i l i s present i n the adjacent cytoplasm (arrow). The presence of the male chromosomes has induced an o v e r l y i n g m i c r o v i 1 l u s - f r e e s u r f a c e with a t h i c k sub-oolemmal microfilament web (small arrowhead). The s u r f a c e i s i r r e g u l a r , s i m i l a r to that seen dur i n g i n t r a o v i d u c t a l aging. M a g n i f i c a t i o n = 2,500x. C. Two c e l l embryo r e t r i e v e d on day 2 of pregnancy from a mature r a t . Note the wide i n t e r c e l l u l a r space (arrow) with m i c r o v i l l i oh apposing blastomere s u r f a c e s . The cytoplasm i s moderately e l e c t r o n dense and homogeneously granular with m u l t i p l e f i b r i l l a r arrays and mitochondria. V e s i c u l a r aggregates, c o n t a i n i n g many small n u c l e o l a r - l i k e bodies (arrowheads), and m u l t i v e s i c u l a r bodies are c l u s t e r e d around the nucleus, which contains a l a r g e n u c l e o l u s . M a g n i f i c a t i o n = 2,500x. D. High m a g n i f i c a t i o n of p e r i n u c l e a r n u c l e o l a r - l i k e bodies i n a 2 c e l l embryo r e t r i e v e d f r o m a mature r a t on day 2 of pregnancy. The n u c l e o l a r - l i k e bodies are s t r u c t u r a l l y i d e n t i c a l to the small n u c l e o l i i n the nucleus (arrows). The n u c l e o l a r - l i k e bodies o f t e n have d i s c o n t i n u o u s segments of smooth endoplasmic r e t i c u l u m on the s u r f a c e and are a s s o c i a t e d with v e s i c u l a r aggregates and nascent G o l g i . Note the granular cytoplasm. M a g n i f i c a t i o n = 6,300x. 160 considered to be e a r l y morphological t r a n s f o r m a t i o n of G o l g i . Smooth endoplasmic r e t i c u l u m was c l o s e l y a s s o c i a t e d with mitochondria and was o c c a s i o n a l l y m i l d l y d i l a t e d . N u c l e o l a r - l i k e bodies, 500 to 900 nm diameter, were observed i n the cytoplasm ( F i g u r e 40a) . Two p r o n u c l e i were observed i n most oocytes. These were large and i r r e g u l a r i n shape with v a r i a b l e decondensation of chromatin. Large, h i g h l y e l e c t r o n dense, f i b r i l l a r n u c l e o l i were present e i t h e r b u l g i n g i n t o the cytoplasm on the i n n e r p r o n u c l e a r membrane or f r e e i n the nucleoplasm (Figure 40a). Small f o c i of dense granules were o c c a s i o n a l l y seen on the s u r f a c e of the n u c l e o l i . One oocyte was undergoing sperm head decondensation below a MVF i r r e g u l a r protuberance of the oocyte with a suboolemmal mi c r o f i l a m e n t web ( F i g u r e 40b). On the s i d e of the oocyte opposite to the sperm head decondensation, a second p o l a r body and nascent female pronucleus were observed. The second p o l a r body had a smooth contour with a suboolemmal micr o f i l a m e n t web and was devoid of c o r t i c a l g r a n u l e s . I t contained a mass of condensed or p a r t i a l l y decondensed chromatin, microtub-u l e s , mitochondria and small v e s i c l e s . The c y t o s o l v a r i e d but was u s u a l l y moderately e l e c t r o n dense and g r a n u l a r . Two c e l l embryos had an i r r e g u l a r c e l l o u t l i n e w i t h numerous m i c r o v i l l i , a c t i v e m i c r o p i n o c y t o s i s and few suboolemmal c o r t i c a l granules ( F i g u r e 40c). M i c r o v i l l i were a l s o observed along the wide i n t e r c e l l u l a r space ( F i g u r e 40c). The cytoplasm was more granular than pronuclear embryos. Mitochondria were more e l e c t r o n lucent and l a r g e r (400 to 1200 nm diameter). V e s i c u l a r aggregates often contained NLB and had small transforming G o l g i at the periphery. 161 "Page 161 FIGURE 41 A. Two c e l l embryo r e t r i e v e d from a mature r a t on day 3 of pregnancy. A ring-shaped n u c l e o l u s i s present. The f i b r i l 1ogranular r e t i c u l u m i n the adjacent nucleoplasm i s i n d i c a t i v e of ribosome s y n t h e s i s (arrows). Several small n u c l e o l i (small arrowhead) are v i s i b l e i n the nucleus. A n u c l e o l a r - l i k e body i s present i n the adjacent cytoplasm ( l a r g e arrowhead). M a g n i f i c a t i o n = 2,500x. B. Two c e l l embryo r e t r i e v e d from a mature rat on day 3 of pregnancy. A p e r i p h e r a l f i b r i l l o g r a n u l a r network i s present on the s u r f a c e of the n u c l e o l u s (arrow). Micro-v i l l i are no longer present on apposing blastomere s u r f a c e s . The i n t e r c e l l u l a r space i s diminished with numerous poin t s of contact between the two c e l l s (arrow-heads). M a g n i f i c a t i o n = 2,500x. C. Two c e l l embryo r e t r i e v e d from a mature rat on day 2 of pregnancy. Three n u c l e o l a r - l i k e bodies are present at the pe r i p h e r y of the c e l l i n a s s o c i a t i o n with v e s i c u l a r aggregates. One n u c l e o l a r - l i k e body.and the surrounding cytoplasm are i s o l a t e d by a continuous l e n g t h of smooth endoplasmic r e t i c u l u m (arrow). The sperm t a i l i s present i n the midbody (arrowhead). M a g n i f i c a t i o n = 4,095x. D. Four c e l l embryo r e t r i e v e d from a mature rat on day 3 of pregnancy. Two c e l l s have undergone i r r e v e r s i b l e c e l l s w e l l i n g and degeneration. Note the marked cytoplasmic lucency with f l o c c u l e n t matrix, d i l a t e d mitochondria (arrowhead), n u c l e o l a r - l i k e body (arrow) and f l o c c u l e n t nucleoplasm with n o n - a c t i v a t e d n u c l e o l u s . There i s c l o s e a p p o s i t i o n of the c e l l s . M a g n i f i c a t i o n = 2,500x. 162 The VA and MVB o f t e n formed l a r g e complexes i n the cytoplasm ( F i g u r e 40c). Large numbers of v e s i c l e s i n the MVB c o n t a i n e d h i g h l y e l e c t r o n dense, c e n t r a l , amorphous content. A small number of r e s i d u a l bodies were a l s o observed. L i p i d d r o p l e t s were occa-s i o n a l l y observed i n the cytoplasm. The n u c l e i c o n t a i n e d m u l t i p l e s m a l l , f i b r i l l a r , h i g h l y e l e c t r o n dense n u c l e o l i ( F i g u r e 40d) and the l a r g e n u c l e o l i more o f t e n had coarse granules on the s u r f a c e . P e r i n u c l e a r v e s i c u l a r aggregates with NLB 300 to 830 nm diameter were prominent ( F i g u r e 40c,d). N u c l e o l a r - l i k e bodies were more numerous, o c c u r r i n g s i n g l y or i n c l u s t e r s of up to f i v e , were o c c a s i o n a l l y rimmed by segments of SER and were i n c r e a s e d i n s i z e to 1800 nm diameter ( F i g u r e 41c). One NLB i n one embryo was s i t u a t e d w i t h i n a l a r g e area of c o a r s e l y granular cytoplasm i s o l a t e d by a continuous l e n g t h of SER from the surrounding cytoplasm ( F i g u r e 41c). Remnants of the sperm were observed i n the cytoplasm but the i n c o r p o r a t i o n cone was gone. The sperm t a i l was f r e q u e n t l y observed extending from one blastomere to the other v i a the midbody (Figure 41c). The second p o l a r body o f t e n contained a nucleus w i t h v a r i a b l e chromatin decondensation and compact n u c l e o l u s . One or more NLB were o c c a s i o n a l l y observed i n the p o l a r body cytoplasm. Two c e l l embryos r e t r i e v e d on day 3, had a more narrow i n t e r c e l l u l a r space and more c l o s e l y apposed blastomeres with fewer m i c r o v i l l i on the i n t e r c e l l u l a r s u r f a c e s ( F i g u r e 41b). They contained more e l e c t r o n l u c e n t mitochondria up to 1200 nm i n diameter. R e t i c u l a t e d , f i b r i l l o g r a n u l a r s t r u c t u r e s extended from the surface of the n u c l e o l i i n t o the surrounding nucleoplasm (F i g u r e 41a,b). One embryo had a more e l e c t r o n l u c e n t blastomere, 163 with d i s r u p t e d FA and moderately to markedly d i l a t e d SER. Four c e l l embryos had an i r r e g u l a r e x t e r n a l c e l l o u t l i n e w i t h fewer m i c r o v i l l i than 2 c e l l embryos. The i n t e r c e l l u l a r space was small and the blastomeres were c l o s e l y apposed. M i c r o v i l l i were r a r e l y observed on apposing c e l l s u r f a c e s and m u l t i p l e p o i n t s of c e l l contact were present. Increased numbers of r e s i d u a l bodies were observed i n the cytoplasm with a r e d u c t i o n i n MVB. V e s i c u l a r aggregates were reduced i n both s i z e and number. N u c l e o l a r - l i k e bodies were i n c r e a s e d i n s i z e up to 2200 nm i n diameter. One embryo had two degenerated blastomeres w i t h e l e c t r o n l u c e n t , f l o c c u l e n t c y t o s o l with d i l a t e d SER and MC ( F i g u r e 41d). One of the blastomeres had an i n t a c t nucleus w i t h a quiescent n u c l e o l u s (Figure 41d)'. The n u c l e i of 4 c e l l embryos c o n t a i n e d more c o a r s e l y g r a n u l a r nucleoplasm and pores were more commonly observed i n the n u c l e a r membrane. Coarse d e p o s i t s of e l e c t r o n dense m a t e r i a l were present on the inner s u r f a c e of the nuc l e a r membrane. At l e a s t one l a r g e n u c l e o l u s i n each blastomere had a l a r g e area of r e t i c u l a t e d , f i b r i l l o g r a n u l a r m a t e r i a l on the s u r f a c e . Embryos r e t r i e v e d from the immature r a t s at the expected stage of development f o r the day of r e t r i e v a l were m o r p h o l o g i c a l l y s i m i l a r to embryos r e t r i e v e d from the mature r a t s ( F i g u r e 42a-d). However, embryos from superovulated, immature r a t s d i f f e r e d i n the se v e r a l regards. Four c e l l embryos from superovulated, immature r a t s tended to have fewer m i c r o v i l l i on the c e l l s u r f a c e and more e l e c t r o n lucent c y t o s o l with reduced g r a n u l a r i t y , a r e d u c t i o n i n the d e n s i t y of FA and fewer r e s i d u a l bodies ( F i g u r e 43a,b). 1 64 Page 164 ft FIGURE 42 A. Zygote r e t r i e v e d from a su p e r o v u l a t e d r a t on day 1 of pregnancy. The male pronucleus with s i n g l e l a r g e n u c l e o l u s and adjacent sperm t a i l (arrow) are v i s i b l e . Suboolemmal c o r t i c a l granules are gone and th e r e i s e a r l y d i f f e r e n t i a t i o n of m i c r o v i l l i (arrowhead). M a g n i f i c a t i o n = 2,500x. B. F e r t i l i z e d , 2 c e l l embryo r e t r i e v e d from a superovulated ra t on day 2 of pregnancy. Chromosome decondensation i s evident i n the second p o l a r body (arrow). M i c r o v i l l i are present on the c e l l s u r f a c e . Condensed chromatin has formed a rim around the n u c l e o l u s (arrowhead). M a g n i f i c a t i o n = 2,500x. C. High m a g n i f i c a t i o n of a n u c l e o l a r - l i k e body i n the periphery of a 2 c e l l embryo r e t r i e v e d on day 2 of pregnancy from a r a t t r e a t e d with 4 IU PMSG. Segments of smooth endoplasmic r e t i c u l u m abut the s u r f a c e of the l a r g e n u c l e o l a r -l i k e body (small arrowhead). The n u c l e o l a r - l i k e bodies are as s o c i a t e d with a v e s i c u l a r aggregate ( l a r g e arrowhead) and a nascent G o l g i (arrow). M a g n i f i c a t i o n = 9,450x. D. F e r t i l i z e d , 2 c e l l embryo r e t r i e v e d on day 2 of pregnancy from a rat t r e a t e d with 4 IU PMSG. Numerous m i c r o v i l l i are present on the outer and i n t e r c e l l u l a r s u r f a c e s of the blastomeres. The sperm t a i l i s present i n the c e l l u l a r end of the midbody ( l a r g e arrow). The i n t e r c e l l u l a r space i s wide. A small v e s i c u l a r aggregate (arrowhead) and m u l t i v e s i c u l a r body (open arrow) are present i n the cytoplasm, i n a d d i t i o n to nascent Golg i (small arrow). M a g n i f i c a t i o n = 2,500x. 165 Page 165 ft FIGURE 43 A. F e r t i l i z e d 4 c e l l embryo r e t r i e v e d from a r a t t r e a t e d with 4 IU PMSG on day 3 of pregnancy. The c e l l s are c l o s e l y apposed with few m i c r o v i l l i i n a narrow i n t e r c e l l u l a r space. The n u c l e o l u s i s a c t i v a t e d (arrow). Few m u l t i v e s i c u l a r bodies and v e s i c u l a r aggregates remain and t h e r e i s an i n c r e a s e i n r e s i d u a l bodies (arrowheads). M a g n i f i c a t i o n = 2,500x. B. F e r t i l i z e d 4 c e l l embryo r e t r i e v e d from a superovulated rat on day 3 of pregnancy. The midbody i s s t i l l a t t a c h e d to two c e l l s (open arrow). The blastomeres are c l o s e l y apposed. The cytoplasm i s l e s s homogeneously g r a n u l a r than 4 c e l l embryos from the other two groups. There are fewer r e s i d u a l bodies. F i b r i l l a r a r r a y s are o f t e n c o a l e s c e d i n l a r g e s t a c k s (arrow-head). One n u c l e o l u s i s a c t i v a t e d (arrow). M a g n i f i c a t i o n = 2,500x. C. F e r t i l i z e d 4 c e l l embryo r e t r i e v e d from a superovulated rat on day 3 of pregnancy. Smooth endoplasmic r e t i c u l u m i s moderately d i l a t e d with scant content (arrowhead). There i s o c c a s i o n a l d i l a t i o n ( l a r g e arrow) and compartmental d i l a t i o n (small arrow) of mitochondria. Numerous c l u s t e r s of e l e c t r o n dense granules are v i s i b l e i n the cytoplasm (open arrow). A supernumerary sperm i s present i n the p e r i v i t e l 1 i n e space. M a g n i f i c a t i o n = 2,500x. D. M u l t i n u c l e a t e d 4 c e l l embryo r e t r i e v e d from a super-ovulated r a t on day 3 of pregnancy. There i s marked d i l a t i o n of mitochondria (small arrowhead) and smooth endoplasmic r e t i c u l u m ( s m a l l arrow) with p e r i n u c l e a r c l u s t e r i n g of o r g a n e l l e s . The m u l t i v e s i c u l a r bodies are d e v o i d of v e s i c l e s with moderate d i l a t i o n of tubules and s a c c u l e s ( l a r g e arrowhead). M i c r o v i l l i are absent on the blastomere surface. The f i b r i l l a r a r r a y s have formed l a r g e s t a c k s (open arrow). M a g n i f i c a t i o n = 2,500x. 166 Mitochondria were more e l e c t r o n dense and were o c c a s i o n a l l y v a cuolated (Figure 43c). Although the range of m i t o c h o n d r i a l diameter was the same as t h a t observed i n the other groups, the sma l l e r mitochondria predominated. One 4 c e l l embryo had moderately d i l a t e d SER with reduced c y t o s o l e l e c t r o n d e n s i t y and fewer FA ( F i g u r e 43c). The u l t r a s t r u c t u r a l morphology of developmentally advanced embryos and fragmented oocytes v a r i e d from that of normal embryos i n many regards. On day 1 of pregnancy, 2, 3 and 4 c e l l embryos were f r e q u e n t l y m u l t i n u c l e a t e d . Those embryos with sperm i n the cytoplasm had moderately reduced numbers of suboolemmal CG. One 4 c e l l embryo, i n which sperm head decondensation had f a i l e d to occur, had no apparent r e d u c t i o n i n CG. The presence of suboolemmal CG was v a r i a b l e i n the remainder of the embryos. Some had CG i n one blastomere and not the other, while others were e i t h e r devoid of CG or had a normal complement i n a l l c e l l s . The i n t e r c e l l u l a r space was wide with m i c r o v i l l i i n a l l embryos, except one 4 c e l l embryo which had c l o s e blastomere a p p o s i t i o n . The e l e c t r o n d e n s i t y of the c y t o s o l v a r i e d more between embryos and two of the m u l t i n u c l e a t e d 2 c e l l embryos had e l e c t r o n lucent c y t o s o l with markedly reduced g r a n u l a r i t y and fewer FA. Two of the u n f e r t i l i z e d 2 c e l l embryos had second p o l a r bodies. Mitochondria were more e l e c t r o n l u c e n t i n most embryos and diameter ranged up to 1000 nm. There was v a r i a b l e accumulation of coarse granules on the s u r f a c e of f i b r i l l a r n u c l e o l i i n both m u l t i n u c l e -ated and u n i n u c l e a t e d c e l l s . One f e r t i l i z e d 3 c e l l embryo had a p e r i n u c l e o l a r g r a n u l a r r e t i c u l a t e d s t r u c t u r e and mitochondria up to 1200 nm diameter. The v e s i c l e s of the MVB i n t h i s embryo had 167 Page 167 A FIGURE 44 A. Fragmented, m u l t i n u c l e a t e d oocyte r e t r i e v e d from a super-ov u l a t e d r a t on day 1 of pregnancy. Note the sperm t a i l i n the c o r t i c a l cytoplasm (arrowhead) and the r e t e n t i o n of c o r t i c a l granules (arrow). M a g n i f i c a t i o n = 4,000x. B. F e r t i l i z e d , fragmented oocyte r e t r i e v e d from a super-ovulated r a t on day 1 of pregnancy. A second p o l a r body with chromosomes (arrows) was extruded and c o r t i c a l granule exo-c y t o s i s occurred. M i c r o v i l l i , sperm t a i l ( l a r g e arrowhead) and nascent G o l g i ( s m a l l arrowhead) are present. A c e l l fragment with suboolemmal c o r t i c a l granules i s adjacent to the p o l a r body. M a g n i f i c a t i o n = 2,500x. C . U n f e r t i l i z e d , fragmented, m u l t i n u c l e a t e d oocyte r e t r i e v e d from a superovulated r a t on day 1 of pregnancy. There i s marked v a r i a t i o n i n the presence of c o r t i c a l granules (arrow) between fragments. One n u c l e o l u s has condensed chromatin on the perimeter ( l a r g e arrowhead). Note the wide' space between fragments. M a g n i f i c a t i o n = 2,500x. D. U n f e r t i l i z e d , f o u r c e l l embryo r e t r i e v e d from a super-ovulated r a t on day 1 of pregnancy. Note the c l o s e c e l l a p p o s i t i o n , nascent G o l g i (small arrowhead), moderate smooth endoplasmic r e t i c u l u m d i l a t i o n (small arrow), m u l t i p l e n u c l e i w i t h f i b r i l l a r n u c l e o l i , l a r g e n u c l e o l a r - l i k e body with smooth endoplasmic r e t i c u l u m on the s u r f a c e ( l a r g e arrowhead), suboolemmal c o r t i c a l granules (open arrow), decrease i n f i b r i l l a r a r r a y s and i n c r e a s e d e l e c t r o n lucency of the cytoplasm. M a g n i f i c a t i o n = 2,500x. 168 reduced content. One u n f e r t i l i z e d , m u l t i n u c l e a t e d 4 c e l l embryo had c l o s e c e l l a p p o s i t i o n , reduced content i n v e s i c l e s of the MVB and d i l a t e d SER (Figure 44d). Fragmented oocytes had more v a r i a b l e morphology than abnormal embryos ( F i g u r e 44a-c). Fragmented forms were m u l t i n u c l e a t e d w i t h o r g a n e l l e p o l a r i z a t i o n i n one or more fragments. One fragmented oocyte, which c o n s i s t e d of one l a r g e c e l l and many small p e r i p h e r a l fragments, was f e r t i l i z e d and had a second p o l a r body and a marked r e d u c t i o n i n CG (Figure 44b). There was v a r i a b l e b l e b b i n g ( p i n c h i n g o f f of cytoplasm i n t o the p e r i v i t e l l i n e space) and cytoplasmic v a c u o l a t i o n with v a r i a b l e r e d u c t i o n i n FA and e l e c t r o n d e n s i t y of the c y t o s o l between fragments. M i t o c h o n d r i a ranged i n diameter to 1000 to 1200 nm. The v e s i c l e s of the MVB were o f t e n empty. The presence of c o r t i c a l granules a l s o v a r i e d between fragments ( F i g u r e 44c). On day 2 and 3 of pregnancy, abnormal embryos commonly had o r g a n e l l e p o l a r i z a t i o n ( F i g u r e 43d, 45b). The c y t o s o l was more o f t e n e l e c t r o n lucent with decreased d e n s i t y of FA and there was o f t e n a r e d u c t i o n i n the number of m i c r o v i l l i on the c e l l s u r f a c e ( F i g u r e 45a). M i l d to moderate d i l a t i o n of SER and MVB s a c c u l e s and t u b u l e s , with a r e d u c t i o n i n v e s i c l e s was observed ( F i g u r e 45a,c). M i t o c h o n d r i a ranged i n diameter up to 1000 to 1200 nm i n a l l embryos. D i l a t i o n and v a c u o l a t i o n of mitochondria was observed only i n 4 c e l l embryos (Figure 45c,d). One 2 c e l l embryo had m u l t i p l e double membranes around c e n t r a l l y c l u s t e r e d n u c l e i ( F i g u r e 45b). Most embryos had a wide i n t e r c e l l u l a r space with v a r i a b l e m i c r o v i l l i . One f e r t i l i z e d , m u l t i n u c l e a t e d 2 c e l l embryo had r e t e n t i o n of CG i n one blastomere and a l a r g e amount of c e l l d e b r i s 169 .<• Page 169 FIGURE 45 A. U n f e r t i l i z e d 2 c e l l embryos r e t r i e v e d on day 2 of preg-nancy from a superovulated r a t . There i s c l o s e c e l l appos-i t i o n . F i b r i l l a r a r r a y s are markedly decreased i n the c y t o -plasm. There i s d i l a t i o n of smooth endoplasmic r e t i c u l u m i n one c e l l (arrow). There i s d i l a t i o n of m u l t i v e s i c u l a r body tubules (arrowhead). Two c e l l fragments resemble a d i v i d e d second p o l a r body. A c l u s t e r of one l a r g e and four small n u c l e o l a r - l i k e bodies i s present i n the cytoplasm of one c e l l (open arrow). Note the r e t e n t i o n of c o r t i c a l g r a n u l e s . M a g n i f i c a t i o n = 2,500x. B. M u l t i n u c l e a t e d , u n f e r t i l i z e d 2 c e l l embryo r e t r i e v e d from a superovulated r a t on day 2 of pregnancy. M u l t i p l e double membranes are present on two n u c l e i (arrows). O r g a n e l l e s are c l u s t e r e d p e r i p h e r a l l y and p e r i n u c l e a r l y . F i b r i l l a r a r r a y s are d i s r u p t e d and d i s p e r s e d throughout the cytoplasm. There i s c o r t i c a l granule r e t e n t i o n and m i l d d i l a t i o n of mitochondria (small arrowhead). Note the wide i n t e r c e l l u l a r space. M a g n i f i c a t i o n = 2,500x. C. U n f e r t i l i z e d 4 c e l l embryo r e t r i e v e d from a superovulated rat on day 3 of pregnancy. Note the l a r g e vacuole, d i l a t i o n of m u l t i v e s i c u l a r body tubules ( l a r g e open arrow) with cytoplasmic c o r t i c a l granules (small open arrow), compart-mental m i t o c h o n d r i a l d i l a t i o n (small arrowhead), r e d u c t i o n i n f i b r i l l a r a r r a y s , i n c r e a s e i n p e r i v i t e l 1 i n e space d e b r i s ( l a r g e arrowhead) and l o s s of m i c r o v i l l i . One n u c l e o l u s has condensed chromatin on the s u r f a c e (small arrow). M a g n i f i c a t i o n = 2,500x. D. U n f e r t i l i z e d , m u l t i n u c l e a t e d 4 c e l l embryo r e t r i e v e d from a superovulated r a t on day 2 of pregnancy. There i s c l o s e c e l l a p p o s i t i o n . Some mitochondria are d i l a t e d . One c e l l has more e l e c t r o n lucent cytoplasm with l o s s of g r a n u l a r i t y , decreased number of f i b r i l l a r arrays and d i l a t i o n of smooth endoplasmic r e t i c u l u m (arrow) with compartmentalization of the cytoplasm (open arrow). M a g n i f i c a t i o n = 2,500x. 170 i n the p e r i v i t e l 1 i n e space with a second p o l a r body. On day 3, abnormal embryos had fewer a c t i v a t e d n u c l e o l i (p<0.005). On day 2 and 3 of pregnancy, fragmented forms more o f t e n had d i l a t i o n of the SER and tubules and s a c c u l e s of the MVB. N u c l e o l i had more accumulation of coarse granules on the s u r f a c e and NLB were often s l i g h t l y v a c u o l a t e d . Fewer c o r t i c a l g ranules were observed. On day 3 of pregnancy, vacuolated and d i l a t e d mitochondria were observed. 171 B. DISCUSSION 1. Breeding and Pregnancy Rates S i g n i f i c a n t l y fewer r a t s mated i n the 4 IU group than i n the 40 IU or mature groups. Marked v a r i a t i o n i n the mating r a t e , from 50 to 96%, was observed i n immature r a t s t r e a t e d with a low dose of PMSG by others (Nuti et a l . , 1975; M i l l e r and Armstrong, 1981b; M i l l e r and Armstrong, 1982; Walton et a l . , 1983; L e v e i l l e and Armstrong, 1989). The tendency f o r immature r a t s to mate l e s s c o n s i s t e n t l y when t r e a t e d with 4 IU PMSG was a t t r i b u t e d to lower e s t r a d i o l l e v e l s than superovulated r a t s (Walton et a l . , 1983), premature o v u l a t i o n and f a i l u r e to ovulate ( M i l l e r and Armstrong, 1981b). S i g n i f i c a n t l y more r a t s which f a i l e d t o breed had a metestrus smear i n the 4 IU group. Rats t r e a t e d with 4 IU PMSG e x h i b i t e d an es t r u s smear on day 1 followed by a metestrus smear on day 2 of pregnancy, s i m i l a r to that observed i n the mature r a t ( M i l l e r and Armstrong, 1981b). Hence, the metestrus smear 72 hours post-treatment i n the present study suggests that these r a t s had been i n est r u s the p r e v i o u s day. F u r t h e r , a hig h percentage of oocytes r e t r i e v e d from these r a t s were cumulus-free and fragmented, i n d i c a t i v e of i n t r a o v i d u c t a l aging. T h i s i s c o n s i s t e n t with the obse r v a t i o n i n Part 2 that 33% of r a t s i n the 4 IU group ovulated e a r l y . However, i n Part 2, i t was concluded t h a t e a r l y o v u l a t i o n was the r e s u l t of the d i r e c t a c t i o n of the LH moiety of PMSG. The present o b s e r v a t i o n of a metestrus smear i n immature r a t s which f a i l e d to breed suggests that e a r l y o v u l a t i o n i n the low dose group was a s s o c i a t e d w i t h e l e v a t e d e s t r a d i o l l e v e l s , presumably from f o l l i c l e s s t i m u l a t e d to ovulate and suggests t h a t an endogenous 172 gonadotrophin surge may have o c c u r r e d . The metestrus smear i n s u p e r o v u l a t e d r a t s which f a i l e d to breed a l s o suggests that e s t r u s o c c u r r e d 24 hours e a r l i e r . These animals may represent the small percentage of r a t s which mate i n a s s o c i a t i o n with the f i r s t wave of o v u l a t i o n ( M i l l e r and Armstrong, 1982). In the superovulated r a t , an e s t r u s smear i s u s u a l l y observed to day 3 of pregnancy due to u n l u t e i n i z e d a n t r a l f o l l i c l e s ( M i l l e r and Armstrong, 1981a) and p e r s i s t e n t e s t r a d i o l s y n t h e s i s ( M i l l e r and Armstrong, 1981b). Thus, the metestrus smear observed i n superovulated r a t s suggests t h a t l u t e i n i z a t i o n o c c u r r e d and e s t r a d i o l l e v e l s dropped ( M i l l e r and Armstrong, 1981b). L u t e i n i z a t i o n s u b s t a n t i a l l y reduces e s t r a d i o l p r o d u c t i o n ( Z e l e z n i k et a l . , 1974). T h i s i s c o n s i s t e n t w i t h the o b s e r v a t i o n t h a t super-o v u l a t i o n was completed i n a percentage of immature r a t s p r i o r to the expected time of the endogenous gonadotrophin surge as observed i n Part 2 and by Yun et a l . (1988). A p r o g r e s s i v e decrease i n the number of r a t s from which embryos were r e t r i e v e d and the t o t a l number of embryos r e t r i e v e d per r at was observed from day 2 to 4 of pregnancy i n the 40 10 group. By day 4 of pregnancy, an average of two embryos were r e t r i e v e d from l e s s than 50% of the r a t s . Of these, h a l f were u n f e r t i l i z e d oocytes. Over t h i s time p e r i o d , an i n c r e a s i n g percentage of the embryos were a l s o r e t r i e v e d from the u t e r u s . T h i s p a t t e r n agrees with the o b s e r v a t i o n s of others (Sherman et a l . , 1982; L e v e i l l e and Armstrong, 1989) and was a t t r i b u t e d t o the elevated e s t r a d i o l l e v e l s d u r i n g the f i r s t 3 to 4 days of pregnancy (Walton and Armstrong, 1981; M i l l e r and Armstrong, 1981a; Sherman et a l . , 1982; Yun et a l . , 1988; L e v e i l l e and Armstrong, 1989). 173 High l e v e l s of e s t r a d i o l induce r a p i d t r a n s p o r t of embryos through the r e p r o d u c t i v e t r a c t and v a g i n a l e x p u l s i o n i n the r a t (Greenwald, 1961; Greenwald, 1967; Wu et a l . , 1971; O r t i z et a l . , 1979). A s i g n i f i c a n t improvement i n embryo r e t e n t i o n was achieved i n superovulated r a t s when e s t r a d i o l and androgen l e v e l s were decreased by ovariectomy, anti-PMSG or f l u t a m i d e treatment (Walton and Armstrong, 1981; M i l l e r and Armstrong, 1982; Yun et a l . , 1988). S i m i l a r l y , s u p e r o v u l a t i o n with an FSH/HCG p r e p a r a t i o n with low LH a c t i v i t y r e s u l t e d i n lower androgen and e s t r a d i o l l e v e l s and normal r e t e n t i o n of embyros i n the r e p r o d u c t i v e t r a c t ( L e v e i l l e and Armstrong, 1989). 2. Gross Embryo R e t r i e v a l Degenerated oocytes were r e t r i e v e d from a l l groups during the present study. As observed i n P a r t s 1, 2 and 3, the m a j o r i t y of degenerated oocytes were recovered from a m i n o r i t y of r a t s . T h i s phenomenon was a l s o observed by Sherman et a l . (1982). No i n c r e a s e i n the in c i d e n c e of degenerated oocytes was observed i n the superovulated group over the p e r i o d of study and t h e r e f o r e no c o r r e l a t i o n between degeneration of oocytes and treatment with PMSG was made. The p r o p o r t i o n of degenerated oocytes was i n f a c t lower i n the 40 IU group, s u g g e s t i n g t h a t i n any r a t , there i s a f i n i t e number of degenerated oocytes i n f o l l i c l e s which can be s t i m u l a t e d to o vulate. Therefore, the s i g n i f i c a n t r e d u c t i o n i n degenerated oocytes i n the supe r o v u l a t e d group c o u l d not be considered a b e n e f i c i a l e f f e c t of PMSG treatment. In the present study, u n f e r t i l i z e d and fragmented oocytes accounted f o r approximately 10% of the t o t a l r e t r i e v a l from the 4 174 IU and mature groups on days 1 t o 3 of pregnancy. Poor q u a l i t y embryos on days 2 and 3 of pregnancy c o n s t i t u t e d an a d d i t i o n a l 5 t o 10% of the t o t a l i n these groups. The r e s u l t a n t 15 to 20% t o t a l abnormal embryos i s s i m i l a r to t h a t observed by others (Sherman et a l . , 1982; L e v e i l l e and Armstrong, 1989). However, the present values i n the mature r a t are h i g h e r than the 2 to 11% abnormal embryos observed by Sherman et a l . (1982). T h i s may be due to s t r a i n d i f f e r e n c e s between Sprague-Dawley and Wistar r a t s or may represent the normal range of b i o l o g i c a l v a r i a t i o n . Oocyte fragmentation i s i n d i c a t i v e of i n t r a o v i d u c t a l aging and l o s s of f e r t i l i z a b i l i t y (Marston and Chang, 1964). Since superovu-l a t e d immature r a t s e x h i b i t b i p h a s i c o v u l a t i o n , but mate only i n a s s o c i a t i o n with the second wave, the oocytes which ovulate i n the f i r s t wave are u n f e r t i l i z a b l e at the time of i n s e m i n a t i o n ( M i l l e r and Armstrong, 1982; Walton and Armstrong, 1983). Therefore, the fragmented oocytes observed on day 1 of pregnancy represent oocytes which ovulated e a r l y and f a i l e d t o undergo f e r t i l i z a t i o n . The low percentage of fragmented oocytes i n the 4 IU group, d e s p i t e the o b s e r v a t i o n that 33% of r a t s o v u l a t e d e a r l y i n Part 2, r e f l e c t s e x c l u s i o n of r a t s which f a i l e d to mate from the study. The presence of fragmented oocytes i n the mature r a t suggests that asynchrony of mating and o v u l a t i o n may a l s o occur i n a small percentage of spontaneously o v u l a t i n g females. S i g n i f i c a n t l y fewer f e r t i l i z e d oocytes were r e t r i e v e d from superovulated r a t s on day 1 of pregnancy. W i t h i n the 40 IU group, a s i g n i f i c a n t decrease i n u n f e r t i l i z e d oocytes was a s s o c i a t e d w i t h an i n c r e a s e i n the number of 2 c e l l embryos and fragmented oocytes on day 2 of pregnancy. T h i s suggests that u n f e r t i l i z e d oocytes 175 e i t h e r underwent f e r t i l i z a t i o n or fragmentation subsequent to the time of s a c r i f i c e on day 1. As d i s c u s s e d i n Part 3, over h a l f of the u n f e r t i l i z e d , i n t a c t oocytes r e t r i e v e d from superovulated r a t s were aged. The remainder were apparently normal metaphase oocytes, of which 13% were undergoing f e r t i l i z a t i o n at the time of r e t r i e v a l . The s i g n i f i c a n t i n c r e a s e i n 2 c e l l embryos on day 2 of pregnancy suggests t h a t delayed f e r t i l i z a t i o n o ccurred i n a s i g -n i f i c a n t p o r t i o n of these oocytes. The time to f i r s t cleavage f o l l o w i n g f e r t i l i z a t i o n i n the rat i s approximately 22 hours ( S h a l g i et a l . , 1985). Thus, i f delayed f e r t i l i z a t i o n o c c u r r e d i n the superovulated r a t , one would expect to observe s i g n i f i c a n t l y more f e r t i l i z e d oocytes i n the superovu-l a t e d group on day 2 of pregnancy than the 4 IU or mature groups. However, metabolic events which d i r e c t e a r l y development are set i n motion i n the r a t oocyte when o v u l a t i o n i s t r i g g e r e d ( S h a l g i et a l . , 1985). Aged r a t oocytes cleaved more than two hours e a r l i e r p o s t - f e r t i l i z a t i o n than oocytes which were f e r t i l i z e d s h o r t l y a f t e r o v u l a t i o n (Freeman et a l . , 1970; S h a l g i and K r a i c e r , 1978; S h a l g i et a l . , 1985). Thus, a c t i v a t i o n of the developmental program may allow oocytes which undergo delayed f e r t i l i z a t i o n to c a t c h up with promptly f e r t i l i z e d oocytes ( E i c h e n l a u b - R i t t e r et a l . , 1986). This may represent a s t r a t e g y t o maximize r e p r o d u c t i v e p o t e n t i a l and prevent delayed embryonic development and thus, i s c o n s i s t e n t with the s i g n i f i c a n t r e d u c t i o n i n f e r t i l i z e d oocytes observed i n the present i n v e s t i g a t i o n on day 2 of pregnancy i n the superovulated group. Embryos at advanced developmental stages f o r the day of recovery were r e t r i e v e d on day 1 and 2 of pregnancy. The percen-176 tages of developmental 1 y advanced embryos were higher than those observed by L e v e i l l e and Armstrong (1989) and Sherman et a l . (1982) l i k e l y due to the i n c l u s i o n of poor q u a l i t y embryos i n the present study. Embryos at unexpected stages of development were r e t r i e v e d i n low numbers from the 4 IU group and t h i s was a l s o r e p o r t e d by others (Sherman et a l . , 1982; L e v e i l l e and Armstrong, 1989). The recovery of embryos i n advanced stages of development was r e p o r t e d i n superovulated mice (Beaumont and Smith, 1975). T h i s was i a t t r i b u t e d to the occurrence of cleavage f i v e hours e a r l i e r i n embryos from superovulated mice than spontaneously o v u l a t i n g mice ( A l l e n and McLaren, 1971; Beaumont and Smith, 1975). A s i m i l a r o b s e r v a t i o n was made i n the present study where the s a c r i f i c e time f o r mature r a t s had to be delayed f o r f i v e hours to ensure r e t r i e v a l of embryos at the same stage of development as the immature r a t s . However, no d i f f e r e n c e i n cleavage r a t e was noted between the two dose l e v e l s of PMSG. T h i s f i n d i n g suggests that a c t i v a t i o n of the developmental program i n oocytes from immature PMSG-treated r a t s may commence p r i o r to o v u l a t i o n as observed i n sheep superovulated with PMSG (Moor et a l . , 1985). However, the e a r l i e r cleavage time does not account f o r the degree of develop-mental advancement observed i n the present study. Two, 3 and 4 c e l l embryos were r e t r i e v e d 12 hours a f t e r the commencement of o v u l a -t i o n , a time frame over which cleavage c o u l d not occur ( S h a l g i et a l . , 1985), even i f the developmental r a t e had been advanced by f i v e hours. 3. Parthenogenesis During i n t r a o v i d u c t a l aging i n the mature r a t , oocytes which 177 resembled 2, 3 and 4 c e l l embryos were r e t r i e v e d as d e s c r i b e d i n Part 1. Cleavage of aged oocytes was d e s c r i b e d i n the hamster (Chang and Fernandez-Cano, 1958; Yanagimachi and Chang, 1961; Longo, 1974), mouse (Marston and Chang, 1964; S z o l l o s i , 1971; Longo, 1980) and r a b b i t (Meyer and Longo, 1979) and was a t t r i b u t e d to spontaneous parthenogenetic a c t i v a t i o n ( O ' N e i l l and Kaufman, 1988). Cytoplasmic fragments, some of which are l a r g e enough to mimic a second p o l a r body, a l s o appear i n a s s o c i a t i o n with parthenogenetic c y t o k i n e s i s (Longo, 1974). Parthenogenotes develop at a re t a r d e d r a t e (Chang and Fernandez-Cano, 1958; Tarkowski, 1971; Longo, 1974; S o l t e r et a l . , 1974). The apparent developmental r a t e i n parthenogenotes depends p a r t l y on the d i s t r i b u t i o n of a c t i n and the m e i o t i c s p i n d l e at the time of a c t i v a t i o n (Webb et a l . , 1986). Most of the changes which occur i n the c y t o s k e l e t o n of developing embryos a l s o occur i n aging oocytes, but at a slower r a t e (Paynton et a l . , 1988). Aged oocytes e i t h e r cleave immediately a f t e r a c t i v a t i o n and appear to be one cleavage stage ahead of other parthenogenotes or cl e a v e 24 hours l a t e r (Graham, 1970; Webb et a l . , 1986). Since the f i r s t wave of o v u l a t i o n commences as e a r l y as 12 hours post-treatment (Walton et a l . , 1983), there would be adequate time f o r parthenogenetic a c t i v a t i o n and cleavage of aged oocytes p r i o r to r e t r i e v a l at 72 hours i n the supe r o v u l a t e d group. Thus, embryos at advanced stages of development i n the present study may have been p a r t h e n o g e n e t i c a l l y a c t i v a t e d oocytes from the f i r s t wave of o v u l a t i o n . Parthenogenotes u s u a l l y undergo developmental a r r e s t at the 2 to 4 c e l l stage (Van Blerkom and Runner, 1976). Developmental l y 178 advanced embryos beyond the 4 c e l l stage were not observed i n the present study and by day 3 of pregnancy no developmentally advanced embryos were observed. In the 40 IU group, 2, 3 and 4 c e l l embryos c o n s t i t u t e d the same percentage of non-fragmented oocytes on day 2 and 3 of pregnancy. Since parthenogenotes tend to cleave i r r e g u l a r -l y and fragment (Graham, 1970; S o l t e r et a l . , 1974; Longo, 1974; Webb et a l . , 1986), they would be of poorer gross q u a l i t y than f e r t i l i z e d embryos. In the present study, the gross q u a l i t y of developmentally advanced 3 and 4 c e l l embryos was s i g n i f i c a n t l y lower than embryos at the expected stage of development. The gross q u a l i t y of 3 and 4 c e l l embryos i n the superovulated group on day 3 of pregnancy was s i g n i f i c a n t l y lower than the other two groups. These observations suggest that'parthenogenotes a l s o comprised a percentage of embryos at expected stages of development on day 3 of pregnancy. T h i s c o n c l u s i o n was f u r t h e r supported by the m i c r o s c o p i c f i n d i n g s . Parthenogenesis i s a s s o c i a t e d w i t h r e t e n t i o n of c o r t i c a l granules, f a i l u r e to extrude a p o l a r body and the appearance of m u l t i n u c l e a t e d c e l l s (Braden, 1957; Yanagimachi and Chang, 1961; Marston and Chang, 1964; Tarkowski, 1971; Longo, 1974; S o l t e r et a l . , 1974; Thompson and Zamboni, 1975; Longo, 1975; Meyer and Longo, 1979; O ' N e i l l and Kaufman, 1988). In the present study, developmentally advanced embryos were u s u a l l y m u l t i n u c l e a t e d with o r g a n e l l e p o l a r i z a t i o n and r e t e n t i o n of c o r t i c a l g r a n u l e s . They were r a r e l y f e r t i l i z e d or contained a r e c o g n i z a b l e second p o l a r body. M u l t i n u c l e a t e d 2 and 3 c e l l embryos i n the 4 IU group l i k e l y represent parthenogenetic a c t i v a t i o n of aged oocytes i n r a t s which mated but ovulated e a r l y . 179 A s i g n i f i c a n t percentage of embryos at the expected stage of development on day 2 and 3 i n the 40 IU group were u n f e r t i l i z e d and mul t i n u c l e a t e d with o r g a n e l l e p o l a r i z a t i o n and thus, were l i k e l y parthenogenotes. When the percentage of 2 c e l l embryos r e t r i e v e d from the 40 IU group on day 2 of pregnancy was c o r r e c t e d f o r the presence of parthenogenotes, a s i g n i f i c a n t r e d u c t i o n i n f e r t i l i z e d 2 c e l l embryos was observed. Thus, the s u p e r o v u l a t o r y treatment s i g n i f i c a n t l y reduced the f e r t i l i z a t i o n r a t e . T h i s i s c o n s i s t e n t with the f i n d i n g s of o t h e r s (Walton et a l . , 1983; Evans and Arm-strong, 1984; L e v e i l l e and Armstrong, 1989). The percentage of embryos which were at the 2 c e l l stage was not s i g n i f i c a n t l y d i f f e r e n t between groups on day 3 of pregnancy. However, the percentage of 4 c e l l embryos was lower i n the 40 IU group on day 3, su g g e s t i v e of delayed embryonic development. Since parthenogenotes develop at a slower rate than f e r t i l i z e d eggs and u s u a l l y a r r e s t at the 2 t o 4 c e l l stage (Tarkowski, 1971; Kaufman and Sachs, 1976; Van Blerkom and Runner, 1976), the presence of parthenogenotes i n a s u p e r o v u l a t o r y cohort would g i v e the appear-ance of retarded development on day 3 and 4 of pregnancy. However, d i v i s i o n time between embryos undergoing second cleavage can vary up to 13 hours (Smith and Johnson, 1986). Thus, the s i g n i f i c a n t decrease i n 2 c e l l embryos and the i n c r e a s e i n 3 c e l l embryos w i t h i n the 40 IU group from day 2 to 3 of pregnancy, suggests that c e l l d i v i s i o n was impending i n a s i g n i f i c a n t percentage of embryos. The retarded embryonic development i n the 40 IU group when compared with the other two groups on day 3 of pregnancy was l i k e l y due to the lower f e r t i l i z a t i o n r a t e and the presence of parthen-ogenotes. Therefore, development of f e r t i l i z e d embryos to the 4 180 c e l l stage may not be s i g n i f i c a n t l y d e l a y e d . Due to r a p i d l o s s of embryos from the r e p r o d u c t i v e t r a c t and the r e s u l t a n t r e s t r i c t e d p e r i o d of o b s e r v a t i o n , retarded embryonic development was d i f f i c u l t to a c c u r a t e l y a s s e s s . However, r e t a r d e d embryonic development was not observed p r e v i o u s l y on day 3 of pregnancy i n superovulated immature r a t s ( M i l l e r and Armstrong, 1981a; Sherman et a l . , 1982; L e v e i l l e and Armstrong, 1989). M u l t i n u c l e a t e d oocytes were observed 20 hours p o s t - o v u l a t i o n i n the r a t by Niwa and Chang (1975). M u l t i n u c l e a t e d oocytes were not observed on day 1 of pregnancy i n the p r e s e n t study, l i k e l y due to the e a r l i e r p o s t - o v u l a t o r y s a c r i f i c e time. However, there was no s i g n i f i c a n t i n c r e a s e i n the t o t a l percentage of m u l t i n u c l e a t e d embryos from day 1 to 2 of pregnancy i n the superovulated group, suggesting that the m u l t i n u c l e a t e d embryos on day 2 of pregnancy were the same cohort observed on day 1. The s i g n i f i c a n t i n c r e a s e i n m u l t i n u c l e a t e d 3 and 4 c e l l embryos on day 2 suggests that there was cleavage of 2 c e l l parthenogenotes. M u l t i n u c l e a t e d embryos i n c r e a s e d s i g n i f i c a n t l y from day 2 to 3 of pregnancy i n the 40 IU group. T h i s cannot be accounted f o r by f u r t h e r parthenogenetic a c t i v a t i o n , as no s i g n i f i c a n t decrease i n u n f e r t i l i z e d oocytes was observed over the corresponding p e r i o d . M u l t i n u c l e a t e d embryos on day 3 r a r e l y had a c t i v a t e d n u c l e o l i . Parthenogenotes f a i l to undergo n u c l e o l a r a c t i v a t i o n , or do so at a r e t a r d e d stage of development ( S o l t e r et a l . , 1974), suggesting that f u r t h e r parthenogenotes were becoming m u l t i n u c l e a t e d . However, f e r t i l i z e d m u l t i n u c l e a t e d embryos a l s o f a i l to undergo n u c l e o l a r a c t i v a t i o n ( T e s a r i k et a l . , 1987b). 181 4. M u l t i n u c l e a t e d Embryos F e r t i l i z e d embryos w i t h m u l t i n u c l e a t e d blastomeres i n c r e a s e d from one on day 2 to f i v e on day 3. M u l t i n u c l e a t i o n was r e p o r t e d to be one of the most common a b n o r m a l i t i e s observed i n e a r l y human pre i m p l a n t a t i o n embryos (Lopata et a l . , 1983). T h i s c o n d i t i o n was a t t r i b u t e d to polyspermy as m u l t i n u c l e a t e d embryos were observed to a r i s e from t r i p r o n u c l e a r oocytes f o l l o w i n g f e r t i l i z a t i o n i n v i t r o (Van Blerkom et a l . , 1984). In the present study, t r i p r o n u c l e a r oocytes were not observed. Thus, the f e r t i l i z e d m u l t i n u c l e a t e d embryos l i k e l y represent a b n o r m a l i t i e s other than t r i p l o i d y . M u l t i n u c l e a t e d human embryos were a l s o observed to a r i s e from oocytes apparently f e r t i l i z e d normally i n v i t r o ( T e s a r i k et a l . , 1987b). The m u l t i p l e n u c l e i were a t t r i b u t e d to d e f e c t i v e m i g r a t i o n of chromosomes at anaphase due to impaired t u b u l i n f u n c t i o n as a r e s u l t of hormonal imbalances induced by the s t i m u l a t i o n p r o t o c o l during i n t r a f o l l i c u l a r oocyte maturation ( T e s a r i k et a l . , 1987b). S i m i l a r to m u l t i n u c l e a t e d embryos i n the present study, the mu l t i n u c l e a t e d human embryos d i d not undergo n u c l e o l a r a c t i v a t i o n and were g r o s s l y i n d i s t i n g u i s h a b l e from u n i n u c l e a t e d embryos (Tesa r i k et a l . , 1987b). Thus, i n the present study, m u l t i n u c l e -ated, f e r t i l i z e d embryos may be the morphological m a n i f e s t a t i o n of d e f e c t i v e c y t o s k e l e t a l f u n c t i o n r e s u l t i n g from delayed f e r t i l i z -a t i o n or abnormal i n t r a f o l 1 i c u l a r oocyte maturation as d i s c u s s e d i n Part 2. During n u c l e a r envelope r e c o n s t i t u t i o n , a d i s c o n t i n u o u s double membrane aggregates i n i t i a l l y around i n d i v i d u a l chromosomes, forming karyomeres, which then fuse to form the nucleus (Greenwald, 1961; Zamboni and M a s t r o i a n n i , 1966). M u l t i p l e n u c l e i appear i n 182 d i v i d i n g c e l l s when th e r e i s d y s f u n c t i o n of t u b u l i n p o l y m e r i z a t i o n (Hernandez-Verdun et a l . , 1979). Abnormal movement of chromosomes on the s p i n d l e at anaphase leads to chromosome s c a t t e r i n the cytoplasm, f a i l u r e of karyomere f u s i o n and the formation of m u l t i p l e small n u c l e i (Hernandez-Verdun et a l . , 1979). In Part 2, karyomeres were seen to be s c a t t e r e d i n the cytoplasm i n aged i n t a c t oocytes and i t was suggested that c y t o s k e l e t a l i n s t a b i l i t y was expressed d u r i n g aging i n oocytes from immature r a t s . T h i s i s c o n s i s t e n t with the o b s e r v a t i o n of o r g a n e l l e p o l a r i z a t i o n i n the present study, s i n c e d i s t r i b u t i o n and movement of o r g a n e l l e s i n the cytoplasm i s a l s o mediated by the c y t o s k e l e t o n ( D u c i b e l l a et a l . , 1977; Maro et a l . , 1984). T h e r e f o r e , the c y t o s k e l e t o n appears to be the s t r u c t u r e most s e n s i t i v e to the e f f e c t s of i n t r a o v i d u c t a l oocyte aging and a l t e r e d i n t r a f o l 1 i c u l a r maturation. A u s t i n (1950) r e p o r t e d f e r t i l i z a t i o n of fragmented oocytes i n superovulated r a t s . Niwa and Chang (1975) demonstrated that t h i s was the r e s u l t of delayed f e r t i l i z a t i o n i n the r a t . In the present study, f i v e m u l t i n u c l e a t e d embryos on day 1 had sperm i n the cyt o -plasm. In one 4 c e l l embryo, the sperm head had f a i l e d to decon-dense suggesting that f e r t i l i z a t i o n occurred 12 to 14 hours a f t e r o v u l a t i o n when the a c t i v i t y of male pron u c l e a r growth f a c t o r i s l o s t (Niwa and Chang, 1975). Only one of the remaining 4 embryos had undergone c o r t i c a l g r a n u l e e x o c y t o s i s and extruded a second p o l a r body. F e r t i l i z a t i o n l i k e l y occurred near the end of the f e r t i l i z a b l e l i f e of these oocytes, when i n c r e a s e d e r r o r s of f e r t i -l i z a t i o n occur ( J u e t t e n and B a v i s t e r , 1983). Since suboolemmal a c t i n m i c r o f i l a m e n t s mediate c o r t i c a l granule e x o c y t o s i s (Cran, 1987), r e t e n t i o n of c o r t i c a l granules i n f e r t i l i z e d oocytes 183 suggests that the programmed changes i n the c y t o s k e l e t o n had advanced to the point where c o r t i c a l granule e x o c y t o s i s was precluded d e s p i t e gamete f u s i o n . A c t i n m i c r o f i l a m e n t s do not mediate gamete f u s i o n and sperm i n c o r p o r a t i o n (Maro et a l . , 1984), thus, f e r t i l i z a t i o n could s t i l l o ccur. A l t e r n a t i v e l y , changes to the oolemma may have i n t e r f e r e d w i t h c o r t i c a l granule e x o c y t o s i s . Changes i n the macromolecular composition of the oolemma occur i n aged mouse oocytes (Longo, 1981). 5. E f f e c t s of Delayed F e r t i l i z a t i o n With i n c r e a s i n g post o v u l a t o r y age, oocytes p r o g r e s s i v e l y lose the p o t e n t i a l f o r normal and advanced embryonic development (Yanagimachi and Chang, 1961; Marston and Chang, 1964; Maurer et• a l . , 1969; Thompson and Zamboni, 1975; Usui and Yanagimachi, 1976; G i a n f o r t o n i and Gulyas, 1985). I f f e r t i l i z a t i o n i s delayed beyond a c r i t i c a l p o i n t i n the r a t , abnormal embryonic development i s observed ( S h a l g i and K r a i c e r , 1978). Therefore, decreasing p o t e n t i a l f o r embryonic development may occur i n a percentage of oocytes from the second wave of o v u l a t i o n which, as d i s c u s s e d i n Part 3, undergo delayed f e r t i l i z a t i o n . Polyspermy and digyny were repo r t e d to be i n c r e a s e d i n occurrence due to delayed f e r t i l i z a t i o n i n the r a b b i t (Shaver and Carr, 1967; Chang and Hunt, 1968; Shaver and Carr, 1969) and mouse (Marston and Chang, 1964; V i c k e r s , 1969). Superovulation with PMSG i n mice (Chang, 1977; Maudlin and F r a s e r , 1977) and hamsters (Sengoku and Dukelow, 1988) l e d to an i n c r e a s e i n t r i p l o i d y and t h i s was b e l i e v e d to be the r e s u l t of delayed f e r t i l i z a t i o n (Chang, 1977). An i n c r e a s e d i n c i d e n c e of polyspermy was reported i n a s s o c i -184 a t i o n with delayed f e r t i l i z a t i o n i n the r a t (Odor and Blandau, 1956). Therefore, i n the superovulated r a t , where f e r t i l i z a t i o n of oocytes i s delayed by a low sperm to oocyte r a t i o , an i n c r e a s e d i n c i d e n c e of polyspermy and digyny may occur. A s i g n i f i c a n t i n c r e a s e i n polyspermy was observed at one super o v u l a t o r y dose l e v e l during i n v i t r o f e r t i l i z a t i o n i n the immature r a t (Evans and Armstrong, 1984). In the human, t r i p r o n u c -l e a r oocytes can undergo f i r s t cleavage to 3 c e l l s with abnormal chromosome c o n s t i t u t i o n (Kola et a l . , 1987). Thus, a percentage of the abnormal embryos r e t r i e v e d from superovulated immature r a t s may be t r i p l o i d s . However, n e i t h e r digyny nor polyspermy were observed' i n f e r t i l i z e d embryos at expected stages of development i n the present study. Nor was an inc r e a s e d i n c i d e n c e of polyspermy observed i n the s t u d i e s of Walton et a l . (1983) and Niwa and Chang (1975). S i m i l a r l y , e r r o r s of f e r t i l i z a t i o n were not reported i n sheep sup e r o v u l a t e d with PMSG (Murray et a l . , 1986). Therefore, t r i p l o i d y i s u n l i k e l y to c o n t r i b u t e s i g n i f i c a n t l y to i n f e r t i l i t y i n the superovulated r a t . In the hamster, delayed f e r t i l i z a t i o n i s a s s o c i a t e d with a decrease i n normal pro n u c l e a r and 2 c e l l embryos ( J u e t t e n and B a v i s t e r , 1983). F a c t o r s r e q u i r e d f o r male pro n u c l e a r development appear d u r i n g f i n a l maturation (Niwa and Chang, 1975; T e s a r i k and Kopecny, 1989). In the r a t , t h i s f a c t o r i s l o s t 12 to 14 hours p o s t - o v u l a t i o n (Niwa and Chang, 1975). In the human, aging f o r 22 hours had no e f f e c t on male pronuclear growth f a c t o r ( T e s a r i k and Kopecny, 1989).. However, d e s p i t e normal sperm decondensation, male pronuclear development f a i l e d i n aged murine oocytes (Borsuk and Tarkowski, 1989). 185 A s i g n i f i c a n t r e d u c t i o n i n p r o n u c l e a r formation was observed i n oocytes from superovulated r a t s by Walton et a l . (1983), presumably due e i t h e r to oocyte aging or recent f e r t i l i z a t i o n . In the present study, f a i l u r e of p r o n u c l e a r development was not observed. Marked i r r e g u l a r i t y of s u r f a c e contour at the s i t e of p o l a r body e x t r u s i o n was observed i n a small percentage of pronuclear oocytes i n the s u p e r o v u l a t e d r a t s . T h i s may be the e x p r e s s i o n of c y t o s k e l e t a l i n s t a b i l i t y of aged oocytes as d i s c u s s e d i n Part 2 and i t may be these embryos which subsequently develop m u l t i n u c l e a t e d blastomeres. Thus, s u r f a c e i r r e g u l a r i t y and m u l t i n u c l e a t e d blastomeres i n embryos from superovulated r a t s l i k e l y represent delayed f e r t i l i z a t i o n . 6. Abnormal Embryonic Development A p r o g r e s s i v e i n c r e a s e i n abnormal embryos was observed from day 1 to 3 i n superovulated immature r a t s by Sherman et a l . (1982) and L e v e i l l e and Armstrong (1989). S i m i l a r l y , a s i g n i f i c a n t i n c r e a s e i n poor q u a l i t y embryos was observed from day 2 to 3 of pregnancy i n the present study. The i n c r e a s e i n poor q u a l i t y embryos on day 3 of pregnancy i n the absence of a s i g n i f i c a n t decrease i n u n f e r t i l i z e d oocytes suggests that there was an adverse e f f e c t of treatment on embryo q u a l i t y . While t h i s may be due to the e f f e c t s of delayed f e r t i l i z a t i o n or parthenogenetic a c t i v a t i o n , the e l e v a t e d s t e r o i d hormone l e v e l s i n superovulated r a t s may a l s o have an adverse e f f e c t on embryonic development (Yun et a l . , 1988). E s t r a d i o l l e v e l s are e l e v a t e d p r i o r to the second wave of o v u l a t i o n i n the superovulated immature r a t and f o r three days subsequently ( M i l l e r and Armstrong, 1981a,b; Yun et a l . , 1987, 186 1988) . In the b a r b i t u r a t e - t r e a t e d mature r a t , prolonged exposure of i n t r a f o l 1 i c u l a r oocytes to e s t r a d i o l leads to a s i g n i f i c a n t i n c r e a s e i n degenerated, r e t a r d e d and abnormal embryos (Butcher and Fugo, 1967; Butcher et a l . , 1969; Butcher et a l . , 1974; Butcher and Pope, 1979). However, i t i s u n l i k e l y that exposure of i n t r a f o l l -i c u l a r oocytes to e l e v a t e d l e v e l s of e s t r a d i o l i n the superovulated ra t has a permanent e f f e c t on the p o t e n t i a l f o r embryo development. Numerous s t u d i e s have shown that r e c e n t l y o v u l a t e d oocytes and b l a s t o c y s t s from superovulated immature r a t s are capable of i m p l a n t a t i o n and f e t a l development i f a normal maternal environment i s provided (Walton and Armstrong, 1981; Walton et a l . , 1982; Walton and Armstrong, 1982; M i l l e r and Armstrong, 1982; Walton and Armstrong, 1983; Sherman et a l . , 1984; L e v e i l l e and Armstrong, 1989) . Elev a t e d e s t r a d i o l l e v e l s d u r i n g e a r l y pregnancy cause fragmentation and degeneration of embryos i n the mouse and r a b b i t (McGaughey and D a n i e l , 1966; K i r k p a t r i c k , 1971). T h i s appears to be the r e s u l t of prolonged exposure to estrogen, s i n c e e l e v a t i o n of estrogen l e v e l s f o r 10 to 15 hours on day 1 of pregnancy has no e f f e c t on embryo q u a l i t y ( O r t i z et a l . , 1979). The adverse e f f e c t of estrogen on embryonic development i s mediated by a l t e r a t i o n of o v i d u c t a l f l u i d p r o t e i n ( C l i n e et a l . , 1977; Stone et a l . , 1977). A p r o g r e s s i v e i n c r e a s e i n abnormal and degenerated embryos i s observed i n superovulated immature r a t s b e f o r e e x p u l s i o n from the repr o d u c t i v e t r a c t (Walton and Armstrong, 1981; M i l l e r and Armstrong, 1981a; Sherman et a l . , 1982; Yun et a l . , 1988; L e v e i l l e and Armstrong, 1989). Improvement i n embryo v i a b i l i t y i s noted when androgen and e s t r a d i o l l e v e l s are reduced by ovariectomy, anti-PMSG 187 or a d m i n i s t r a t i o n of the antiandrogen, f l u t a m i d e (Walton and Arm-strong, ,1981 ; Sherman et a l ., 1984; Yun et a l . , 1988). Lowering the LH a c t i v i t y of the s u p e r o v u l a t o r y regime a l s o reduces e s t r a d i o l and androgen l e v e l s and s i g n i f i c a n t l y improves embryo v i a b i l i t y ( L e v e i l l e and Armstrong, 1989). In the present study, d e g e n e r a t i v e changes were most commonly observed i n m u l t i n u c l e a t e d and d e v e l -opmentally advanced embryos. However, an i n c r e a s e d i n c i d e n c e of degenerative changes was noted i n f e r t i l i z e d 4 c e l l embryos on day 3 of pregnancy i n the s u p e r o v u l a t e d group. Thus, the d e c l i n e i n embryo q u a l i t y i n the present study may be p a r t l y a t t r i b u t a b l e to e l e v a t e d s t e r o i d hormone l e v e l s . The m i c r o s c o p i c o b s e r v a t i o n s of f e r t i l i z e d embryos from the 1 to 4 c e l l stage were c o n s i s t e n t w i t h the f i n d i n g s of other workers (S o t e l o and P o r t e r , 1959; I z q u i e r d o and V i a l , 1962; S z o l l o s i and R i s , 1963; S z o l l o s i , 1965; S z o l l o s i , 1966; S c h l a f k e and Enders, 1967; Schuchner, 1970; Dvorak et a l . , 1977). At the 4 c e l l stage, mitochondria are more e l e c t r o n l u c e n t and l a r g e r , MVB and VA are decreased i n s i z e and number and an i n c r e a s e i n autophagocytic vacuoles and r e s i d u a l bodies are n o r m a l l y observed (Dvorak et a l . , 1977). A p r o g r e s s i v e decrease and d i s o r i e n t a t i o n of FA commences at the b l a s t o c y s t stage ( I z q u i e r d o and V i a l , 1962; S c h l a f k e and Enders, 1967). F e r t i l i z e d 4 c e l l embryos r e t r i e v e d from the 40 IU group on day 3 of pregnancy e x h i b i t e d d e g e n e r a t i v e changes not observed i n 4 c e l l embryos from the other two groups. Four c e l l embryos i n the 40 IU group had fewer FA, l e s s g r a n u l a r and more e l e c t r o n l u c e n t cytoplasm with fewer r e s i d u a l b o d i e s . D i l a t i o n of smooth endoplasmic r e t i c u l u m and o r g a n e l l e p o l a r i z a t i o n were observed i n 188 i n d i v i d u a l blastomeres of some embryos. In the mouse, morphological t r a n s f o r m a t i o n of e l e c t r o n dense mitochondria at the 4 c e l l stage i s a s s o c i a t e d with i n c r e a s e d u t i l i z a t i o n of glucose f o r o x i d a t i v e r e s p i r a t i o n , i n c r e a s e d oxygen consumption and i n c r e a s e d m e t a b o l i c a c t i v i t y ( S t e r n et a l . , 1971). Therefore, r e t e n t i o n of e l e c t r o n dense mitochondria i n 4 c e l l embryos from the 40 IU group suggests that metabolic a c t i v i t y was lower i n the superovulated group. C e l l s normally turnover senescent o r g a n e l l e s and macromol-e c u l a r compounds by autophagocytosis ( A r s t i l a et a l . , 1972). Autophagocytosis and r e s i d u a l bodies are observed i n a s s o c i a t i o n with c e l l d i f f e r e n t i a t i o n i n embryos (Longo and Anderson, 1969). Increased autophagocytosis i s a l s o observed i n p a t h o l o g i c a l s t a t e s when there i s i r r e v e r s i b l e damage to o r g a n e l l e membranes ( C h e v i l l e , 1983). The reduced number of r e s i d u a l bodies i n 4 c e l l embryos suggests that blastomere d i f f e r e n t i a t i o n was r e t a r d e d and that i r r e v e r s i b l e o r g a n e l l e damage had not occurred. A premature decrease and d i s o r i e n t a t i o n of FA with decreased cytoplasmic e l e c t r o n d e n s i t y and g r a n u l a r i t y was observed i n 4 c e l l embryos i n the 40 IU group. F i b r i l l a r a r r a y s are considered to be a storage form of the RNP u t i l i z e d d u r i n g embryogenesis i n the mouse (Garcia et a l . , 1979) and the r a t (Burkholder et a l . , 1971). Premature degradation of FA suggests that there was i n c r e a s e d u t i l i z a t i o n i n an attempt t o maintain homeostasis. The i n c r e a s e i n cytoplasmic lucency and l o s s of g r a n u l a r i t y i s f u r t h e r suggestive of p r o t e o l y s i s and a s l o w l y p r o g r e s s i v e i n s u l t ( C h e v i l l e , 1983), as observed during aging i n non-segmented oocytes i n Part 1. D i l a t i o n of SER and v a c u o l a t i o n of mitochondria are a l s o s i g n s of non-speci-189 f i c , r e v e r s i b l e i n j u r y (Trump and A r s t i l a , 1975; C h e v i l l e , 1983). Due to the low number of 4 c e l l embryos r e t r i e v e d on day 3 and the prevalence of m u l t i n u c l e a t e d c e l l s , only f i v e 4 c e l l embryos were examined and thus the low sample number may have introduced b i a s . However, the homogeneity of u l t r a s t r u c t u r a l morphology between 4 c e l l embryos i n the 4 IU and mature r a t groups and the i n c r e a s e d i n c i d e n c e of degenerative changes i n embryos of the 40 IU group suggests that there was an adverse e f f e c t of treatment, e i t h e r mediated by e l e v a t e d s t e r o i d hormones and a l t e r a t i o n of the maternal environment or reduced p o t e n t i a l f o r embryonic development due to delayed f e r t i l i z a t i o n . C e l l death i s an i n t e g r a l p a r t of embryogenesis and t i s s u e d i f f e r e n t i a t i o n but i s not normally observed u n t i l the morula to b l a s t o c y s t stage of embryonic development ( H i n c h c l i f f e , 1981). Blastomere l y s i s with marked d i l a t i o n and v a c u o l a t i o n of o r g a n e l l e s and l o s s of membrane i n t e g r i t y i s i r r e v e r s i b l e and i n d i c a t i v e of c e l l death (Trump and A r s t i l a , 1975). Blastomere l y s i s was o c c a s i o n a l l y observed i n oocytes and embryos from both mature and immature r a t s , suggesting that t h i s change i s n o n - s p e c i f i c and may i n v o l v e i n t r i n s i c d e f i c i e n c i e s of i n d i v i d u a l c e l l s or embryos. 7. N u c l e o l a r A c t i v a t i o n and N u c l e o l a r - L i k e Bodies Although 4 c e l l embryos i n the 40 IU group showed other s i g n s of c e l l degeneration, n u c l e o l a r a c t i v a t i o n was not i n h i b i t e d except i n one embryo with marked m i t o c h o n d r i a l and SER d i l a t i o n , and thus i r r e v e r s i b l e damage. The f i r s t appearance of r e t i c u l a t e d n u c l e o l i i s a s s o c i a t e d with a c t i v a t i o n of the embryonic genome and q u a n t i -t a t i v e changes i n o r g a n e l l e s ( T e s a r i k et a l . , 1988b). N u c l e o l a r 190 pr e c u r s o r s c o n s i s t of a homogeneous mass of den s e l y packed f i b r i l s (Geuskens and Alexandre, 1984; T e s a r i k et a l . , 1986; T e s a r i k et a l . , 1987a). I n f i l t r a t i o n by adjacent NOR chromatin i s followed by t r a n s c r i p t i o n and the appearance of a p e r i p h e r a l r e t i c u l a t e d g r a n u l o f i b r i l l a r network ( T e s a r i k et a l . , 1986; T e s a r i k et a l . , 1987a). In the mouse, immature n u c l e o l i a c q u i r e p e r i p h e r a l granules at the 2 c e l l stage and commence r e t i c u l a t i o n at the 4 c e l l stage (Hillman and Tasca, 1969; C a l a r c o and Brown, 1969). S i m i l a r l y , i n the r a t , a granula r network f i r s t appears on the s u r f a c e of n u c l e o l a r p r e c u r s o r s at the l a t e 2 c e l l stage which in c r e a s e s i n s i z e and becomes r e t i c u l a t e d from the 4 to the 8 c e l l stage ( I z q u i e r d o and V i a l , 1962; S z o l l o s i , 1966). T h i s was confirmed i n the present study. Cytoplasmic NLB were observed i n fragmented, developmental 1 y advanced and 1, 2, 3 and 4 c e l l embryos i n a l l groups. Cytoplasmic NLB were not repo r t e d by Sc h l a f k e and Enders (1967), while s i m i l a r s t r u c t u r e s were c o n s i d e r e d to be s e c t i o n i n g a r t e f a c t by Izquierdo and V i a l (1962). N u c l e o l a r - l i k e bodies were observed i n pronuclear zygotes w i t h i n s i x hours of f e r t i l i z a t i o n by Schuchner (1970) and remnants of n u c l e o l i were observed i n the cytoplasm of pronuclear oocytes by Sotelo and P o r t e r (1959). The presence of p e r i n u c l e a r NLB i n zygotes was a t t r i b u t e d to n u c l e o l a r e x t r u s i o n from p r o n u c l e i ( S z o l l o s i and R i s , 1963; S z o l l o s i , 1965; Schuchner, 1970). These s t r u c t u r e s were e l e c t r o n dense, f i b r i l l a r , bound by a double membrane and were r a p i d l y l o s t from the cytoplasm ( S z o l l o s i and R i s , 1963; S z o l l o s i , 1965; Schuchner, 1970). In the present study, f i b r i l l a r n u c l e o l i were observed on the in n e r aspect of the pron u c l e a r membrane, some of which were 191 p r o t r u d i n g s l i g h t l y , but e x t r u s i o n of n u c l e o l i was not observed. N u c l e o l a r - l i k e bodies were f i r s t observed i n the p e r i p h e r a l cytoplasm i n a s s o c i a t i o n with VA. These were u s u a l l y non-membrane bound, although d i s c o n t i n u o u s segments of SER were observed on the su r f a c e of the l a r g e r NLB. P e r i n u c l e a r NLB i n 2 c e l l embryos were a l s o a s s o c i a t e d w i t h VA and o f t e n surrounded by SER segments. T h i s suggests, as d i s c u s s e d i n Part 1, t h a t VA are the t a r g e t o r g a n e l l e f o r RNP of n u c l e o l a r o r i g i n , whether extruded as f i b r i l l a r n u c l e o l i or as decondensed RNP with subsequent recondensation i n the cytoplasm. Cytoplasmic NLB i n r a t embryos represent t r a n s i e n t accumula-t i o n of RNP of n u c l e o l a r o r i g i n r e q u i r e d f o r e a r l y embryogenesis, which g r a d u a l l y disappear during development ( S z o l l o s i , 1966; Takeuchi, 1980; Takeuchi and Takeuchi, 1982; Takeuchi and Sonta, 1983). S i m i l a r s t r u c t u r e s i n t r o p h o b l a s t c e l l s were observed to be u t i l i z e d during delayed i m p l a n t a t i o n i n the r a t ( S c h l a f k e and Enders, 1963). The presence of NLB i n aged oocytes and normal and abnormal cleavage stage embryos suggests that these s t r u c t u r e s f u l f i l an important requirement of p r e i m p l a n t a t i o n development. 8. Role of the C y t o s k e l e t o n i n Embryonic Development The c y t o s k e l e t o n mediates a l t e r a t i o n and maintenance of c e l l shape ( D u c i b e l l a and Anderson, 1975; Lehtonen and Badley, 1980). Blastomere f l a t t e n i n g d u r i n g compaction i s mediated by s u r f a c e contact (Ziomek and Johnson, 1980) and i s followed by r a p i d changes i n the c y t o s k e l e t o n (Johnson and Maro, 1984). Enhanced blastomere a p p o s i t i o n leads to d i f f e r e n t i a t i o n of j u n c t i o n a l type plasma membrane,: l o s s of m i c r o v i l l i on apposed c e l l surfaces and develop-192 ment of f o c a l submembrane d e n s i t i e s (Johnson et a l . , 1988; T e s a r i k , 1989). In the mouse, t h i s t r a n s f o r m a t i o n does not occur u n t i l the 4 to 8 c e l l stage i n a s s o c i a t i o n with a c t i v a t i o n of the embryonic genome (Calarco and E p s t e i n , 1973; D u c i b e l l a et a l . , 1977; Reeve, 1981). However, i n the human, plasma membrane d i f f e r e n t i a t i o n was observed as e a r l y as the 2 c e l l stage, well i n advance of i n i t i -a t i o n of genome t r a n s c r i p t i o n ( T e s a r i k , 1989). In the present study, j u n c t i o n a l d i f f e r e n t i a t i o n of plasma membrane was observed as e a r l y as the 2 c e l l stage on day 3 of pregnancy. This was a s s o c i a t e d with morphological n u c l e o l a r changes suggestive of genome a c t i v a t i o n . J u n c t i o n a l d i f f e r e n t i a t i o n was a l s o observed i n parthenogenotes where n u c l e o l a r a c t i v a t i o n to the r e t i c u l a t e d stage was not observed. Since a c t i v a t i o n and expre s s i o n of the embryonic genome i n the human embryo occurs subsequent to blastomere plasma membrane r e o r g a n i z a t i o n ( T e s a r i k , 1988; T e s a r i k et a l . , 1988b), i t was suggested that development of j u n c t i o n a l type plasma membrane i s r e g u l a t e d by maternal messages i n the oocyte ( T e s a r i k , 1989), which are a c t i v a t e d by blastomere contact (Ziomek and Johnson, 1980). Aged oocytes undergo c y t o s k e l e t a l a l t e r a t i o n s t y p i c a l of the f e r t i l i z e d oocyte but at a slower r a t e (Paynton et a l . , 1988) and thus can mediate j u n c t i o n a l d i f f e r e n t i -a t i o n t y p i c a l of blastomeres. T h i s a b i l i t y to undergo c y t o c o r t i c a l r e o r g a n i z a t i o n t y p i c a l of e a r l y cleavage may e x p l a i n why parthen-ogenotes are of t e n i n d i s t i n g u i s h a b l e from normal embryos (Yanagi-machi and Chang, 1961). 9. Summary In summary, s u p e r o v u l a t i o n of immature r a t s with PMSG l e d to 193 a s i g n i f i c a n t decrease i n f e r t i l i z a t i o n r a t e and a s i g n i f i c a n t i n c r e a s e i n abnormal embryos. T h i s was p r i m a r i l y a t t r i b u t a b l e to i n t r a o v i d u c t a l oocyte aging. Parthenogenetic a c t i v a t i o n of aged oocytes s i g n i f i c a n t l y i n c r e a s e d the number of abnormal and developmentally advanced embryos i n the superovulated group. Abnor-m a l i t i e s of c y t o s k e l e t a l f u n c t i o n were demonstrated i n a small group of f e r t i l i z e d embryos and a t t r i b u t e d to delayed f e r t i l i z -a t i o n . Thus, i n t r a o v i d u c t a l oocyte aging and delayed f e r t i l i z a t i o n were shown to have a major impact on p r e i m p l a n t a t i o n embryonic development i n superovulated r a t s . 194 SUMMARY A s e r i e s of four experiments were performed to determine, on a morphological b a s i s , whether s u p e r o v u l a t i o n of immature r a t s with PMSG leads to i n t r a o v i d u c t a l aging of oocytes, abnormal i n t r a f o l 1 -i c u l a r oocyte maturation, f e r t i l i z a t i o n f a i l u r e of abnormal oocytes and abnormal embryonic development. I t was demonstrated that during i n t r a o v i d u c t a l aging, oocytes undergo s p e c i f i c morphological changes which were d i r e c t e d by the i n t r i n s i c developmental program of the oocyte. While these changes were s i m i l a r i n nature to those observed i n other s p e c i e s d u r i n g i n t r a o v i d u c t a l oocyte aging, the appearance of n u c l e o l a r - l i k e bodies was unique to the r a t . F u r t h e r changes i n plasma membrane contour and chromosome d i s t r i b u t i o n were a t t r i b u t e d to ab n o r m a l i t i e s of c y t o s k e l e t a l f u n c t i o n . The study of i n t r a o v i d u c t a l oocyte aging e s t a b l i s h e d morphol-o g i c a l c r i t e r i a f o r the subsequent assessment of oocytes and embryos i n the remaining three experiments. I t was determined that no d i f f e r e n c e s i n morphology e x i s t e d between r e c e n t l y o v u l a t e d oocytes from immature r a t s t r e a t e d with e i t h e r 4 or 40 IU PMSG. Aged oocytes were r e t r i e v e d from immature r a t s t r e a t e d with both dose l e v e l s of PMSG and the occurrence of aged oocytes corresponded c l o s e l y with the o v u l a t o r y p a t t e r n s i n each group. Comparison of oocytes from mature and immature r a t s r e v e a l e d d i f f e r e n c e s i n oocytes from the immature r a t s which were a t t r i b u t e d to c y t o s k e l e -t a l d y s f u n c t i o n . These were concluded to be the m a n i f e s t a t i o n of a l t e r e d i n t r a f o l 1 i c u l a r oocyte maturation due to the a d m i n i s t r a t i o n of e i t h e r a low or s u p e r o v u l a t o r y dose of PMSG to immature r a t s . Thus, unequivocal evidence of a s p e c i f i c adverse e f f e c t of a superovulatory dose of PMSG on i n t r a f o l l i c u l a r oocyte maturation 195 was not obtained. A s i g n i f i c a n t r e d u c t i o n i n f e r t i l i z a t i o n r a t e was observed i n superovulated immature r a t s , predominantly due to i n t r a o v i d u c t a l oocyte aging. Approximately o n e - t h i r d of the u n f e r t i l i z e d oocytes were m o r p h o l o g i c a l l y normal, metaphase oocytes and i t was suggested that there may be a prolonged f e r t i l i z a t i o n p e r i o d i n superovulated r a t s . The study of embryonic development s u b s t a n t i a t e d that superovulated immature r a t s had a s i g n i f i c a n t l y reduced f e r t i l i z -a t i o n r a t e and a prolonged p e r i o d of f e r t i l i z a t i o n . The major e f f e c t of a su p e r o v u l a t o r y dose of PMSG on embryonic development was i n t r a o v i d u c t a l oocyte a g i n g . Parthenogenetic a c t i v a t i o n of aged oocytes l e d to the gross impression of develop-mentally advanced embryos on day 1 and 2 of pregnancy and i n c r e a s e d numbers of poorer q u a l i t y embryos on day 3 of pregnancy. Micro-s c o p i c a l l y , parthenogenotes e x h i b i t e d m o r p h o l o g i c a l changes t y p i c a l of oocytes which had undergone i n t r a o v i d u c t a l aging. Small numbers of parthenogenotes were a l s o observed i n the c o n t r o l groups suggesting that parthenogenesis i s not a s p e c i f i c e f f e c t of, but i s enhanced by, s u p e r o v u l a t i o n . Morphological a l t e r a t i o n s t y p i c a l of those a t t r i b u t e d to c y t o s k e l e t a l d y s f u n c t i o n i n aged oocytes were a l s o observed i n embryos i n the s u p e r o v u l a t e d group. Thus, i t was suggested that these abnormal embryos had undergone delayed f e r t i l i z a t i o n , w ith subsequent e x p r e s s i o n of a c q u i r e d d e f e c t s i n c y t o s k e l e t a l f u n c t i o n . In c o n c l u s i o n , s u p e r o v u l a t o r y doses of PMSG exert an adverse e f f e c t on p r e i m p l a n t a t i o n embryonic development v i a a myriad of pathways, a l l of which can be a t t r i b u t e d to the high FSH/LH a c t i v i t y and long h a l f l i f e of the molecule. I n t r a o v i d u c t a l oocyte 196 aging was shown to be the major c o n t r i b u t i n g f a c t o r to the gross o b s e r v a t i o n of poor oocyte q u a l i t y , reduced f e r t i l i z a t i o n r a t e and abnormal embryonic development i n superovulated immature r a t s . I n t r a o v i d u c t a l oocyte aging occurred as a consequence of both the b i p h a s i c o v u l a t o r y p a t t e r n i n superovulated r a t s and delayed f e r t i l i z a t i o n f o l l o w i n g mating. Although a l t e r a t i o n of i n t r a 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 has been demonstrated i n s u p e r o v u l a t e d r a t s , an adverse e f f e c t of PMSG on oocyte maturation has not been u n e q u i v o c a l l y demonstrated. Exogenous gonadotrophin may not a f f e c t i n t r a f o l l i c u l a r m aturation to the extent that there i s s i g n i f i c a n t impairment of oocyte f u n c t i o n and a l t e r a t i o n of c e l l morphology. A l t e r n a t i v e l y , oocytes may be capable of responding and adapting to a l t e r e d metabolic s t a t e s i n order to maintain p r e i m p l a n t a t i o n development. 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