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The effects of superovulation with pregnant mare serum gonadotrophin in uteri, vaginae and serum steroid… Fang, Paul Maximilian 1988

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The E f f e c t s of Superovulation with Pregnant Mare Serum Gonadotrophin i n U t e r i , Vaginae and Serum Steroid Levels of Immature Rats. By Paul Maximilian Fang B.Sc, The University of B r i t i s h Columbia, 1984 B.M.L.Sc, The University of B r i t i s h Columbia, 1986 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTERS OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Human Reproductive Biology Programme) We accept t h i s thesis as conforming to the required standard The University of B r i t i s h Columbia A p r i l 1988 ®Paul Maximilian Fang, 1988 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 lby the head 3ST~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. P a u l M. Fang Department of O b s t e t r i c s & Gynecology The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 n a t o J u l y 7, 19 8 8 DE-6(3/81) Abstract Superovulatory treatment with exogenous gonadotrophins adversely a f f e c t s the uterus through the disruption of the d e l i c a t e balance of ovarian stero i d (estrogens, progestins, androgens) secretion rates. To examine the uterine e f f e c t s of t h i s treatment, 189 animals were given 4, 20 or 40 IU pregnant mare serum gonadotrophin (PMSG) at 28 days of age and s a c r i f i c e d every 24 h u n t i l day 10 (D10) post i n j e c t i o n . To study the long term uterine e f f e c t s , 12 rats were treated with 4 or 40 IU PMSG and k i l l e d on D30. The morphological and h i s t o l o g i c a l changes of control (4 IU) u t e r i mimicked those of the adult on a comparable time course from D2 to D5. Administration of superovulatory doses (2 0, 4 0 IU) of PMSG produced stromal hypertrophy by D2 and fo c a l p a p i l l a r y hyperplasia of the luminal e p i t h e l i a by D3. I t i s suggested that previous exposure to high l e v e l s of estrogen and androgens, secondary to superovulation, are possible causes for t h i s pathology. Levels of 17B-estradiol following 2 0 and 40 IU PMSG treatment were s i g n i f i c a n t l y (p<0.005, p<0.05) elevated above those of controls from DI to earl y D3 and at D2, respectively. Androgen l e v e l s of both groups (20 IU, 40 IU) s i g n i f i c a n t l y (p<0.05, p<0.005) increased from baseline at DI to maxima by D2 and D3, respec t i v e l y . In the 20 IU PMSG group, the hyperplasia gradually regressed a f t e r D3 and was absent by D10. The hyperplasia i n the 40 IU PMSG group, however, had di f f u s e d by D 6 . I t i s suspected that preceding elevated l e v e l s of estrogen may be responsible for t h i s progressive change. At D4, l e v e l s of 17B-estradiol reached a maximum, which was s i g n i f i c a n t l y (p<0.001) greater than those of controls and 20 IU PMSG treated r a t s . Between D6 and D10, the hyperplasia p a r t i a l l y regressed. Examination of u t e r i from D30 revealed no evidence of pathology. In addition to these s t r u c t u r a l e f f e c t s , superovulation induced secretion of a mucinous substance i n both 2 0 IU and 40 IU PMSG groups at D5-D6 and D6-D7, respectively. These r e s u l t s suggest that abnormal changes i n the uterine histology and metabolism may r e s u l t following administration of superovulatory doses of PMSG. Although these dose-dependent alterations appear r e v e r s i b l e , they may i n t e r f e r e with preparations associated with implantation and thus require further investigation. i i i Table of Contents Page # Abstract i i Table of Contents i v L i s t of Figures v i Abbreviations x Acknowledgements x i Introduction 1 I Objective 1 II Human In V i t r o F e r t i l i z a t i o n 1 and Embryo Transfer III Ovulation and Pregnancy i n the 3 PMSG Treated Immature Rat PMSG and Ovulation 4 PMSG and Early Pregnancy 7 IV PMSG Induced Uterine E f f e c t s 10 Materials and Methods 16 I Animals and Materials 16 II Methods 17 Design 17 Histology and Histochemistry 18 Steroid Radioimmunoassay 19 III Assessment of Uterine Tissue 20 Design 20 Degenerate A c t i v i t y 21 Secretory A c t i v i t y 21 iv Table of Contents Page # Synthetic A c t i v i t y ° 22 E p i t h e l i a l P r o l i f e r a t i o n and 22 Hyperplasia IV Assessment of Vaginal Tissue 23 V S t a t i s t i c a l Analysis 24 Results 25 I Endometrium 25 II Vagina 46 III Steroids 57 Discussion 60 Summary 72 References 74 v L i s t of Figures Page # 1. Uterus, 48h +4IU PMSG (40x, H&E) 31 2 . Endometrium, 48h +4IU PMSG (400x, H&E) 31 3 . Uterus, Adult Proestrus (40x, H&E) 31 4 . Uterus, Adult Estrus (40X, H&E) 31 5. Uterus, 72h +4IU PMSG (40x, H&E) 32 6. Endometrium, 72h +4IU PMSG (400X, H&E) 32 7 . Endometrium, 72h +4IU PAS/D) PMSG (400x, AB 2.5/ 32 8 . Uterus, 9 6h +4IU PMSG (40x, H&E) 33 9 . Endometrium, 9 6h +4IU PMSG (400x, H&E) 33 10. Uterus, Adult Metestrus (4Ox , H&E) 33 11. Uterus, Adult Diestrus (40X, H&E) 33 12 . Uterus, 12Oh +4IU PMSG (40X, H&E) 34 13 . Endometrium, 120h +4IU PMSG (400x, H&E) 34 14 . Uterus, 216h +4IU PMSG (40X, H&E) 34 15. Endometrium, 216h + 4IU PMSG (400X, H&E) 34 16. Uterus, 24Oh +4IU PMSG (40X, H&E) 35 17. Endometrium, 240h +4IU PMSG (400X, H&E) 35 18. Uterus, 48h ^  -20IU PMSG (40X, H&E) 35 19. Endometrium, 48h +20IU PMSG (400x, H&E) 35 20. Uterus, 72h +20IU PMSG (40X, H&E) 36 21. Endometrium, 72h +20IU PMSG (400x, H&E) 36 22 . Uterus, 96h +2 0IU PMSG (40X, H&E) 36 v i L i s t of Figures Page # 23 . Endometrium, 96h +20IU PMSG (400X, H&E) 36 24. Uterus, 144h +2 0IU PMSG (40X , H&E) 37 25. Endometrium, 144h +2 0IU PMSG (400x, H&E) 37 26. Endometrium, 2 ..5/PAS/D) 144h +20IU PMSG (400x, AB 37 27 . Uterus 192h +2 0IU PMSG (40X, H&E) 38 28 . Endometrium, 192h +20IU PMSG (4 00x, H&E) 38 29. Endometrium, 216h +20IU PMSG (400x, H&E) 38 30. Uterus, 240h +2 0IU PMSG (40x , H&E) 38 31. Uterus, 48h +4 0IU PMSG (40X, H&E) 39 32 . Endometrium, 4 8h +4 0IU : PMSG (400X, H&E) 39 33 . Uterus, 72h +4 0IU PMSG (40x, H&E) 39 34 . Uterus, 12Oh +4 0IU PMSG (40X , H&E) 40 35. Endometrium, 120h +40IU PMSG (400x, H&E) 40 36. Uterus, 144h +40IU PMSG (40x , H&E) 40 37. Endometrium, 144h +40IU PMSG (400x, H&E) 40 38 . Endometrium, AB 2.5/PAS/D) 144h +40IU PMSG (400x, 41 39 . Uterus, 168h +40IU PMSG (40x , H&E) 41 40. Uterus, 192h +4 0IU PMSG (40x , H&E) 41 41. Uterus, 24Oh +4 0IU PMSG (40x , H&E) 42 42. Endometrium, 240h +40IU PMSG (400x, H&E) 42 v i i L i s t of Figures Page # 43. Perimeter (mm) of the lumen at d i f f e r e n t 43 periods a f t e r i n j e c t i o n of 4, 20 and 40 IU PMSG. 44. M i t o t i c index, expressed i n percent, at 44 d i f f e r e n t periods a f t e r i n j e c t i o n of 4, 20 and 40 IU PMSG. 45. Neutrophil counts of the endometrium at 45 d i f f e r e n t periods a f t e r i n j e c t i o n of 4, 20 and 40 IU PMSG. 46. Vagina, 48h +4IU PMSG (40x, H&E) 49 47. Vagina, 48h +4IU PMSG (40x, AB 2.5/ 49 PAS/D) 48. Vagina, Adult Proestrus (40x, H&E) 49 49. Vagina, Adult Estrus (40x, H&E) 49 50. Vagina, 72h +4IU PMSG (40x, H&E) 50 51. Vagina, 96h +4IU PMSG (40x, H&E) 50 52. Vagina, Adult Metestrus (40x, H&E) 50 53. Vagina, Adult Diestrus (40x, H&E) 50 54. Vagina, 120h +4IU PMSG (40x, H&E) 51 55. Vagina, 120h +4IU PMSG (40x, AB 2.5/ 51 PAS/D) 56. Vagina, 240h +4IU PMSG (40x, H&E) 51 57. Vagina, 240h +4IU PMSG (40x, AB 2.5/ 51 PAS/D) 58. Vagina, 48h +20IU PMSG (40x, H&E) 52 59. Vagina, 72h +20IU PMSG (40x, H&E) 52 v i i i L i s t of Figures Page # 60. Vagina, 96h +20IU ] PMSG (40X, H&E) 52 61. Vagina, 120h +20IU PMSG (40x, H&E) 53 62 . Vagina, PAS/D) 120h + 20IU PMSG (40X, AB 2 .5/ 53 63 . Vagina, 144h + 20IU PMSG (40x, H&E) 53 64 . Vagina, PAS/D) 144h +20IU PMSG (40x, AB 2 .5/ 53 65. Vagina, 216h +20IU PMSG (40x, H&E) 54 66. Vagina, PAS/D) 216h +20IU PMSG (40x, AB 2 .5/ 54 67. Vagina, 120h + 40IU PMSG (40x, H&E) 54 68 . Vagina, PAS/D) 120h + 40IU PMSG (40x, AB 2 -.5/ 54 69. Vagina, 168h + 40IU PMSG (40x, H&E) 55 70. Vagina, PAS/D) 168h +40IU PMSG (40x, AB 2 .5/ 55 71. Vagina, 240h + 40IU PMSG (40x, H&E) 55 72 . Vagina, PAS/D) 240h +40IU PMSG (40x, AB 2 .5/ 55 73. Schematic summary of uterine and vaginal 56 morphological and h i s t o l o g i c a l changes following treatment with 4, 20 and 40 IU PMSG. 74. Changes i n serum l e v e l s of 17B-estradiol, 59 progesterone and androgens a f t e r treatment with 4, 20 and 40 IU PMSG. ix Abbreviations AB Al c i a n Blue 8Gx CI Color Index D Diastase DE Diestrus DEG Degenerate E Estrus ET Embryo Transfer FSH F o l l i c l e Stimulating Hormone HCG Human Chorionic Gonadotrophin H&E Hematoxylin and Eosin IU International Units IVF In V i t r o F e r t i l i z a t i o n LH L u t e i n i z i n g Hormone LHRH Lut e i n i z i n g Hormone Releasing Hormone LPE Late Proestrus ME Metestrus MUC Mucification PAS Periodic Acid S c h i f f PE Proestrus PMSG Pregnant Mare Serum Gonadotrophin SEC Secretory X Acknowledgements I would l i k e extend my deepest gratitude to everyone who made t h i s project possible. Valuable advice on experimental design and interpretation was provided by Dr. Y. S. Moon, Dr. J . E. Dimmick, Dr. G. P. Taylor, Dr. Y. W. Yun, Dr. P. Cagle, Dr. E. Jones, Dr. P. E. Reid, Dr. B. Ho Yuen, Dr. J . T. Emerman, Dr. H. Pritchard and Dr. N. Auersperg. S k i l l f u l t echnical assistance was offered by Ms. Natalie Mari, Mrs. Barbara Barkoczy, Mr. John Leung, Ms. Susan B l a i r and a l l the technologists i n the d i v i s i o n of Anatomical Pathology, Department of Pathology, Children's Hospital. I would also l i k e to thank Mrs. Carol Ford, Mrs. Anne S i n c l a i r , Dan, Kate, Cathy and Jennifer at the the Research Centre for t h e i r s p e c i a l help, patience and generosity. x i Introduction I Objective The impairment of uterine structure and function following superovulatory treatment with exogenous gonadotrophins i s poorly understood. Superovulation i s a widely accepted technique i n synchronizing and inducing ovulation, or increasing the number of ovulations. As these e f f e c t s are achieved through f o l l i c u l a r hyperstimulation, i t i s not s u r p r i s i n g that the d e l i c a t e balance of ovarian s t e r o i d (estrogen, progesterone, androgens) secretion rates i s disrupted. The e f f e c t s of t h i s disruption on s e n s i t i v e t i s s u e s , such as the uterus, have not been thoroughly studied. Thus, the purpose of the present study was to gain knowledge through the examination of the e f f e c t s of superovulatory induction, using pregnant mare serum gonadotrophin (PMSG), on endometrial morphology and histology. The changes i n serum st e r o i d l e v e l s and vaginal histology were also examined to aid i n the interpretation of these endometrial e f f e c t s . II Human In V i t r o F e r t i l i z a t i o n and Embryo Transfer In v i t r o f e r t i l i z a t i o n (IVF) and embryo transfer (ET) provide a means of esta b l i s h i n g pregnancies, a l b e i t with modest success, under a wide range of adverse conditions such as tubal i n f e r t i l i t y , endometriosis, terato-oligospermia, immunological and idiopathic i n f e r t i l i t y (Edwards 1984). Despite achieving mean rates i n f e r t i l i z a t i o n of 90% and i n 1 embryo replacement of 80%, few c l i n i c s have obtained pregnancy rates up to 15-20% per embryo transfer (Edwards 1984, Blankstein 1986). The e t i o l o g i e s responsible for t h i s high wastage rate following embryo transfer are uncertain and poorly understood. Chromosomal abnormalities i n embryos, induced by . superovulation or a r i s i n g from growth i n v i t r o , may be responsible for retarded embryo development and death during early pregnancy (Steptoe et a l . 1980, Frydman 1982). However, because of the few opportunities available to karyotype p r i o r to t r a n s f e r and the d i f f i c u l t y i n obtaining embryos i n the process of being expelled or resorbed, i t i s uncertain how contributory chromosomal imbalances or other occult defects may be. Disorders i n the l u t e a l phase or the uterus, which would i n t e r f e r e with and preclude implantation, are other p o t e n t i a l l y important causes of early abortion (Edwards 1984). Successes i n cases, involving transfer of cryopreserved embryos to a subsequent natural or l i g h t l y stimulated cycle, have been reported to be double those of fresh transfers (Blankstein 1986, Testart 1987), i n d i c a t i n g the presence of a transient deleterious e f f e c t upon uterine r e c e p t i v i t y . Elevated estrogen l e v e l s , secondary to superovulation, may be responsible for t h i s deleterious e f f e c t by prolonging the 2 the uterus. In a rare study, biopsies of endometria from unsuccessful IVF/ET cycles were found to display an advancement of 1-3 days (Jones 1984), suggesting that the desynchronization of the timing of optimum uterine r e c e p t i v i t y with embryo age and development may have led to implantation f a i l u r e or early abortion. More d i r e c t evidence w i l l be required on the deleterious e f f e c t s of superovulation before the r o l e of the uterus i n early embryonic death can be properly assessed. Thus, there are several d e f i c i e n c i e s i n the present understanding of the mechanisms operative following superovulation i n human IVF and ET cycles and t h i s indicates that the study of an appropriate animal model has the po t e n t i a l to extend our knowledge. I l l Ovulation and Pregnancy i n the PMSG Treated Immature Rat The PMSG treated immature rat i s well suited f o r the study of superovulation and has been widely employed. PMSG, possessing both FSH ( f o l l i c u l a r stimulating hormone) and LH ( l u t e i n i z i n g hormone) l i k e a c t i v i t i e s , i s a glycoprotein (Papkoff 1974). The FSH:LH a c t i v i t y r a t i o , dependent upon the PMSG preparation, the assay and the time of i s o l a t i o n during pregnancy, has been estimated between 0.87-1.92 (Stewart et a l . 1976). Like other p i t u i t a r y glycoprotein hormones, PMSG consists of a hormone s p e c i f i c subunit (PMSG-B), s i m i l a r i n amino acid composition to LH-B, and a hormonally non-specific subunit (PMSG-a) (Papkoff et a l . 1978). The high s i a l i c acid 3 content (45%) accounts for the long h a l f l i f e (60 h) and the slow clearance rate of PMSG may be responsible f o r the potency that can be achieved i n a single dose (Austin 1950, Nuti et a l . 1975, Papkoff 1981). Immature r a t s treated with low doses of PMSG (4-8 IU) mimic the phy s i o l o g i c a l events of adult c y c l i n g r a t s : s t e r o i d and gonadotrophin hormone secretion rates, uterine weight, and vaginal smear changes during ovulation and pregnancy as adults (Wilson et a l . 1974, Nuti et a l . 1975, Parker et a l . 1976, Taya et a l . 1981). However, immature rats, when superovulated with moderate (16-24 IU) to high doses of PMSG (30-50 IU), w i l l y i e l d greater numbers of re t r i e v a b l e oocytes compared with adults (Cole 1937, Austin 1950, Zarrow and Quinn 1963, Ying et a l . 1969). In summary, due to i t s convenience, superovulatory response and reproductive performance, the PMSG treated immature rat i s an ide a l model for superovulatory research. PMSG and Ovulation Ovulation, beginning 60 h post administration of low or control doses of PMSG (4-8 IU), y i e l d s 8-12 oocytes over a 12 h period, s i m i l a r to adults (Wilson et a l . 1974, Walton et a l . 1983, Walton and Armstrong 1983, Evans and Armstrong 1984, Yun et a l . 1987). Moderate (16-24 IU) to high (30-50 IU) superovulatory doses, however, i n i t i a t e ovulations within 24 h of administration of PMSG. The ovulation period i s longer (50 4 h) as well. In addition, the ovulation rate following superovulation undergoes s t r i k i n g changes, i n contrast with the steady increase observed i n controls. I n i t i a l l y , the ovulation rate slowly r i s e s to a plateau by 36 h: 8 ova (20 IU) , 18 ova (40 IU). The length of t h i s plateau may vary with dose: 12 h (20 IU), 24 h (40 IU), but invariably a sharp increase i n ovulations w i l l follow: 52 ova/12 h (20 IU), 32 ova/12 h (4 0 IU) (Yun et a l . 1987). Thus, increased stimulation with higher doses of PMSG w i l l not merely r e s u l t i n a proportionate and equal elevation of the ovulation response, but a more complex and anomalous pattern. Estrogen and progesterone secretion patterns i n controls are equivalent to those during the adult estrous cycle, i f one uses vaginal smears an indicator of the stage of the cycle: 48 h post PMSG-proestrus; 72 h-estrus; 96 h-metestrus/diestrus; 120 h-diestrus etc. (Wilson et a l . 1974, Nuti et a l . 1975, Parker et a l . 1976, M i l l e r and Armstrong 1981a). Progesterone l e v e l s , following moderate superovulatory induction (20 IU PMSG), do not d i f f e r greatly from those i n controls; however, 17B-estradiol l e v e l s may increase up to three f o l d of controls by 60 h (Yun et a l . 1987). Severe disruption of the maternal endocrine environment i s more evident with higher PMSG doses. In 40 IU PMSG treated rats, 17B-estradiol l e v e l s may increase two-fold those of controls at 48-60 h and again at 96 h, with progesterone l e v e l s r i s i n g up to four times as much as control l e v e l s by 144 h (M i l l e r and Armstrong 1981b, 5 Walton and Armstrong 1981, Yun et a l . 1987). PMSG has been hypothesized to act both d i r e c t l y and i n d i r e c t l y i n inducing ovulation (Yun et a l . 1987). Superovulatory doses, v i a the FSH-moiety of PMSG, may d i r e c t l y i n i t i a t e the maturation of preovulatory f o l l i c l e s possessing the greatest degree of receptor d i f f e r e n t i a t i o n (Moore et a l . 1980) . I f the hypothalamic-pituitary-ovarian axis i s int a c t , one would expect s i g n i f i c a n t elevations of LH, progesterone and estrogen at the time of ovulation (Short 1984, Pasqualini et a l . 1985). The absence of these elevations i n the f i r s t set of ovulations, 12-36 h following 20-40 IU PMSG, suggests that PMSG i n i t i a l l y acts d i r e c t l y on the ovary. Endogenous and exogenous LH, i n combination, may be important i n f a c i l i t a t i n g the larger second set of ovulations 48-72 h a f t e r superovulatory induction. Longitudinal studies of the ef f e c t s of superovulation have i d e n t i f i e d a LH surge 48-57 h following PMSG treatment (Zarrow and Quinn 1963, Wu and Meyer 1966). In addition, studies using various manipulations (central nervous system depressants, hypophysectomy) indicate t h i s surge i s c r i t i c a l to the completion of ovulation (Strauss and Meyer 1962, Sorrentino et a l . 1972). Basal secretions of estrogens and progesterone, produced from the f i r s t set of ovulations, may s y n e r g i s t i c a l l y stimulate t h i s LH surge through the reduction of threshold l e v e l s at hypophyseal and hypothalamic centres (Kawakami and Sawyer 1959, Ying and Meyer 1969, Hagino 6 and Goldzieher 1970). Low doses of PMSG (4-8 IU) probably also stimulate ovulation by t h i s i n d i r e c t mechanism. Time course studies of gonadotrophin and ste r o i d secretion rates indicate that low doses, although inadequate i n d i r e c t l y stimulating ovulation, are able to i n i t i a t e endogenous LH release i n the same time frame as superovulatory doses through f o l l i c u l a r maturation and steroidogenesis (Wilson et a l . 1974, Parker et a l . 1976). PMSG and Early Pregnancy Ear l y pregnancy i n 4 IU PMSG treated immature rats proceeds along a s i m i l a r time course as normal adults with n e g l i g i b l e embryo losses (Mi l l e r and Armstrong 1981a, Yun et a l . 1984) . By day 5 of pregnancy, a l l zygotes (7-8/rat) have implanted i n the uterus i n 85-100% of a l l rats mated ( M i l l e r and Armstrong 1981a, Walton and Armstrong 1981, 1982) . In addition, these implants are carried to term with no s i g n i f i c a n t f e t a l wastage or loss ( M i l l e r and Armstrong 1981a, Walton and Armstrong 1982). Moderate superovulatory (16-2 0 IU) induction, however, may cause s i g n i f i c a n t pre-implantation losses of embryos and delay implantation of the surviving embryos by as much as two to s i x days. Yun et a l . (1984), studying the e f f e c t s of 20 IU PMSG, found no evidence of implantation on day 5 of pregnancy, but was able to re t r i e v e 5-6 embryos/rat from the oviduct and 1-2 degenerate appearing morulae/rat from the uterus. I t was not u n t i l day 7 of pregnancy that he found any implantation 7 s i t e s (5-7 s i t e s / r a t ) . M i l l e r and Armstrong (1981a), using 16 IU PMSG, estimated, by day 20 of pregnancy f e t a l weights ^ , the delay to tbe as great as 3-6 d i n as many as 38% of a l l mated r a t s . Following high dose induction, except f o r the occasional degenerate zygote (2-3%) recovered from the oviduct (Walton and Armstrong 1981, Yun et a l . 1984), l i t t l e development beyond f e r t i l i z a t i o n has been observed, despite a f e r t i l i z a t i o n rate of 50-60% of ova with no signs of degeneration ( M i l l e r and Armstrong 1981b, Walton and Armstrong 1982, Walton et a l . 1983, Yun et a l . 1984). Oocyte defects may int e r f e r e with normal embryo development and perhaps be associated with the pre-implantation losses of embryos. While very few degenerate (ooplasmic contraction, nuclear fragmentation, partheno-genesis) ova r e s u l t from 4 IU PMSG induction - 0-1.5% ( M i l l e r and Armstrong 1981a, Walton et a l . 1983, Yun et a l . 1987), y i e l d s a f t e r superovulation increase dose dependently: 2 0 IU-17%; 40 IU-40% (Yun et a l . 1987). Superovulation may induce release of degenerate oocytes 1 F e t a l development, following delayed implantation, proceeds at a f i x e d rate; therefore a low end of term (day 2 0 of pregnancy) weight i s a r e f l e c t i o n of the extent of the delay and not of the growth retardation (Hooverman et a l . 1970). 8 from abnormal f o l l i c l e s (Fleming 1982) or perhaps rescue large a n t r a l f o l l i c l e s destined for a t r e s i a (Sherman et a l . 1982). Severe i n t r a f o l l i c u l a r degeneration has been observed as early as 48 h a f t e r 20 or 40 IU PMSG administration (Yun et a l . 1987) . Increased incidence of polyploidy and other chromosomal abnormalities may also be important (Fujimoto 1974, Mauldin and Fraser 1977). These oocyte defects alone, however, cannot account for a l l pre-implantation losses of embryos. M i l l e r and Armstrong (1982) ovariectomized 40 IU PMSG treated rats 74-76 h post i n j e c t i o n (after ovulation) and achieved comparable f e t a l recoveries and implantation s i t e weights as controls on day 10 of pregnancy. In addition, Walton and Armstrong (1983) observed equal f e t a l recovery rates between controls and 40 IU PMSG treated rats following oocyte r e t r i e v a l 60-69 h post i n j e c t i o n and embryo tr a n s f e r into LHRH (LH releasing hormone) synchronized mated adults. These r e s u l t s suggest that the oocytes are capable of normal development, but may be prevented from doing so i n a h o s t i l e hormonal environment. The a f f e c t s of such a hormonal environment may be d i r e c t or mediated i n d i r e c t l y v i a the oviduct. Accelerated embryo transport has been found to be responsible for expulsion i n estrogen treated rats and superovulated rabbits (Greenwald 1961, Bennett 1970, Ortiz et a l . 1979, Tsutsumi et a l . 1981). Moreover, i t has been shown i n rabbits and mice that high 9 estrogen l e v e l s induce oviductal secretion of a low MW substance that i n h i b i t s development of the embryo to the morula (Stone and Hammer 1977, Cline et a l . 1977). IV PMSG Induced Uterine E f f e c t s Recent evidence suggests that superovulatory induction with PMSG adversely a f f e c t s uterine structure and function i n immature rats.' PMSG appears to induce abnormal endometrial and myometrial changes, which may be responsible f o r the increased uterine s i z e also observed a f t e r PMSG treatment. Working with mated and non-mated 4 0 IU PMSG treated rats, M i l l e r et a l . (1981b) found that wet uterine weight increased over 2 00% of control (4 IU PMSG) weights between day 1 2 and 2 of pregnancy (or 72-96 h post injection) and remained elevated u n t i l day 5 of pregnancy. Moreover, as early as day 2 of pregnancy they began observing desquamated c e l l u l a r debris i n uterine lavages from both mated and non-mated superovulated r a t s , which advanced progressively such that by day 3 they were recovering s i g n i f i c a n t amounts of desquamated hyperplastic e p i t h e l i a . In contrast, control uterine lavages were completely negative. In addition to e p i t h e l i a l 2 To achieve mating, male caging i s conducted 60-74 h post PMSG i n j e c t i o n (on the night of proestrus). Mating success i s assessed by a sperm p o s i t i v e vaginal smear the morning of the next day, referred to here as day 1 of pregnancy. 10 p r o l i f e r a t i o n , the increased uterine weight following superovulatory doses of PMSG may be due to abnormal myometrial changes. Rennels (1951), using 30 IU PMSG/20 IU HCG, also observed a marked increase i n uterine weight and proposed t h i s was due to a s i g n i f i c a n t hypertrophy of the inner muscularis of the myometrium, beginning 48 h af t e r treatment. I t i s important to note that, i n addition to s t r u c t u r a l findings, no differences have been observed i n 17B-estradiol and progesterone hormone p r o f i l e s between mated and non-mated 4 0 IU treated rats ( M i l l e r and Armstrong 1981b, Walton and Armstrong 1981). Mating i s an important consideration as i t has been well established that repeated tapping of cervix, as might occur during copulation, may i n i t i a t e pseudopregnancy i n the adult (Long and Evans 1922, DeFeo 1963, Yoshinaga et a l . 1970, Pepe et a l . 1974, Redmond et a l . 1986) and the 4 IU PMSG treated r a t (Nuti et a l . 1975). Pseudopregnancy has been characterized to be a condition mimicking the f i r s t 12 days of pregnancy with regard to a l l hormone p r o f i l e s , vaginal and uterine histology and the onset of uterine r e c e p t i v i t y . Thus, s i m i l a r i t y i n findings between mated and non-mated rats suggests that the underlying changes as a r e s u l t of mating are probably not important and that non-mated rats are acceptable f o r at l e a s t the study of uterine e f f e c t s . Studies involving embryo transfers or decidual reaction inductions on day 5 of pregnancy of PMSG treated immature rats have provided d i r e c t evidence that PMSG impairs uterine 11 function to the point where the endometrium i s no longer able to implant an embryo. Implantation i s the r e s u l t of the 0 embryo and endometrium simultaneously reaching s p e c i f i c stages i n t h e i r development by the time they come into contact with each other (Noyes et a l . 1960). In 4 IU PMSG treated pregnant rats the implantation of nearly a l l of the oocytes ovulated (80-90%) i n the endometrium successfully occurs on day 5 of pregnancy ( M i l l e r and Armstrong 1981a, Walton and Armstrong 1981, 1982, Yun et a l . 1984). Several authors have reported comparable implantation rates following synchronous embryo trans f e r s between 4 IU PMSG treated pregnant rats on day 5 of pregnancy, i n d i c a t i n g normality of the embryo and the endometrium ( M i l l e r and Armstrong 1981b, Walton et a l . 1982). In contrast, i t has been found that s i m i l a r l y conducted tran s f e r s from 4 IU PMSG treated donors to 4 0 IU PMSG treated r e c i p i e n t s r e s u l t i n no implantations (Mi l l e r and Armstrong 1981b). In rodents the c a p a b i l i t y of the endometrium to implant an embryo can be assessed by the decidual response e l i c i t e d by a traumatic stimulus on the anti-mesometrial side (Allen 1931, Khriebel 1937, DeFeo 1963, Shelesnyak et a l . 1963, Yoshinaga et a l . 1971). In embryo transfer studies ( M i l l e r and Armstrong 1981b), while trauma induced deciduomata were observed at the embryo transfer point i n 4 IU PMSG treated rats, none were observed i n 40 IU PMSG treated rats ( M i l l e r and Armstrong 1981b). Similar d i f f i c u l t y i n inducing 12 a normal decidual response has also been reported f o r pseudopregnant immature rats treated with 50 IU PMSG ( G u i l l e t et a l . 1964). These functional study r e s u l t s together with the s t r u c t u r a l findings suggests that high doses of PMSG induce abnormal changes, some manifested s t r u c t u r a l l y , which prevent the uterus from reaching a receptive state. Hormonal studies of implantation imply that elevated estrogen l e v e l s i n rats treated with high doses of PMSG, during the pre-implantation period, may detrimentally a f f e c t uterine s e n s i t i v i t y . In adult rats, three hormone events have been i d e n t i f i e d as c r u c i a l i n c o n t r o l l i n g the onset, duration and magnitude of uterine s e n s i t i v i t y : (1) estrogen surge during the proestrus; (2) slowly r i s i n g l e v e l s of progesterone between ovulation and implantation; (3) pre-nidatory estrogen surge the day before implantation (day 5 of pregnancy)(Yochim et a l . 1963, DeFeo 1967, Psychoyos 1973, Aitken 1978, Finn 1980). These workers also found that the disturbance of t h i s pattern with exogenous ovarian steroids, e s p e c i a l l y estrogens, could reduce or abolish uterine s e n s i t i v i t y . Walton et a l . (1982), administering PMSG antisera at 58 h post 40 IU PMSG i n j e c t i o n , abolished uterine and ovarian weight increases and reduced the 17B-estradiol surge at 96 h, while not influencing progesterone l e v e l s . Even though 17B-estradiol l e v e l s were s t i l l higher than those of controls, the treatment with a n t i -sera s t i l l allowed successful induction of dec i d u a l i z a t i o n i n 47% of a l l mated rats (versus 0% for rats not treated). 13 As the primary t a r g e t t i s s u e o f PMSG i s t h e ovary (Papkoff 1981), s t e r o i d hormones o t h e r than e s t r o g e n , produced d u r i n g f o l l i c u l a r maturation and l u t e i n i z a t i o n , may be a s s o c i a t e d w i t h the abnormal e f f e c t s i n the u t e r u s . In the 40 IU PMSG t r e a t e d r a t , i n c r e a s e d l e v e l s of pr o g e s t e r o n e compared w i t h c o n t r o l s have been i d e n t i f i e d j u s t p r i o r t o i m p l a n t a t i o n (Walton and Armstrong 1981, M i l l e r and Armstrong 1981b). These e l e v a t e d l e v e l s , however, may not be r e s p o n s i b l e f o r a b o l i s h i n g u t e r i n e s e n s i t i v i t y . D e c i d u a l s t u d i e s o f the e f f e c t s o f exogenously administered s t e r o i d s i n pseudopregnant a d u l t r a t s r e v e a l e d t h a t i n c r e a s e d progesterone w i t h i n c r e a s e d o r decreased e s t r o g e n shortened the p e r i o d o f u t e r i n e s e n s i t i v i t y , w h i l e i n c r e a s e d progesterone alone had the r e v e r s e e f f e c t , but i n a l l cases t h e r e was no a f f e c t upon the onset o r magnitude of the d e c i d u a l response (Yochim e t a l . 1963, DeFeo 1967). E l e v a t e d l e v e l s o f androgens observed 48-60 h p o s t 40 and 20 IU PMSG a d m i n i s t r a t i o n (Yun e t a l . 1987) may be important by themselves or i n r e l a t i o n s h i p w i t h e s t r o g e n s . I t i s w e l l e s t a b l i s h e d t h a t h i g h doses o f exogenously a d m i n i s t e r e d aromatizable ( t e s t o s t e r o n e ) o r non-ar o m a t i z a b l e (5a-dihydrotestosterone) androgens can i n d i r e c t l y o r d i r e c t l y , r e s p e c t i v e l y , induce u t e r i n e hypertrophy (Brooksby 1938, Salmon 1938, Korenchevsky e t a l . 1936, McDonald and Doughty 1974, Armstrong e t a l . 1976). Furthermore, a r o m a t i z a b l e androgens, through p e r i p h e r a l and 14 o v a r i a n c o n v e r s i o n t o e s t r o g e n , may p o t e n t i a t e o r exaggerate t h e a f f e c t s o f e x i s t i n g h i g h serum l e v e l s o f e s t r o g e n s . 15 Materials and Methods I Animals and Materials Female Sprague Dawley rats (Charles River Canada Inc., St. Constant, Que.) were obtained at 25 d and at 2 mo of age. A l l animals were housed f o r 3 days under controlled conditions of temperature (20°C) and illumination (12L.12D) p r i o r to eithe r PMSG treatment or determination of vaginal cytology. PMSG (Equinex, Ayerst, Montreal) was administered i n a 4, 2 0 or 4 0 IU dose v i a a subcutaneous dorsal i n j e c t i o n i n 0.2 ml 0.9% NaCl between 0930 and 1130 h. Standard rat chow and water were available ad libitum throughout the treatment period.' • Hematoxylin (C.I.75290), eosin (C.I.45380), diastase (D) (mixed a and B amylase), periodic acid and s c h i f f reagent (PAS) were purchased from BDH Chemicals Ltd. and a l c i a n blue 8GX (AB) (C.I.74240) from Sigma Chemical Co. (St. Louis, MO.). Antisera for 17B-estradiol, progesterone and androgen radioimmunoassays were provided courtesy of Dr. D.T. Armstrong, University of Western Ontario. The respective c r o s s - r e a c t i v i t i e s of the antisera are shown as follows: 17B-Estradiol Antibody 17B-estradiol 96.4% estrone 2.9 e s t r i o l 0.5 other steroids <0.2 Progesterone Antibody progesterone 44.1% 5B-pregnane-3,20-dione 35.5 16 5a-pregnane-3,20-dione 15.7% 3a-hydroxy-5B-pregnane-20-one 2.0 20B-hydroxy-4-pregnene-3-one 1.3 17a-hydroxyprogesterone 1.2 other steroids <0.2 Androgen Antibody 5a-dihydrotestosterone 75.0% 5a-androstane-3a, 17B d i o l 13.5 5a-androstane-3B, 17B d i o l 10.9 19-hydroxytestosterone 4.7 other steroids <0.1 II Methods Design Three separate studies were conducted to determine the e f f e c t s of superovulatory doses of PMSG on the histology of the uterus and the vagina. The short term e f f e c t s of 4, 20 and 4 0 IU PMSG were studied i n immature rats from 48 to 240 h post i n j e c t i o n . In t h i s study 162 animals were divided into 6 animals per treatment dose and s a c r i f i c e d at 9 time i n t e r v a l s , 24 h apart. In addition, the hormonal status of 3 rats per dose was examined at 0, 24 and 60 h. The long term e f f e c t s of 4 and 40 IU PMSG were assessed i n 12 immature rats 3 0 days following administration. In addition, c y c l i n g adult rat s were examined i n order to provide a reference f o r induced c y c l i c a l changes i n immature rats. Twelve adult rat s were followed by vaginal smears for one f u l l cycle and s a c r i f i c e d at the appropriate time to achieve 3 representative animals per proestrus, estrus, metestrus and diestrus phases. The order of s a c r i f i c e was randomized with respect to treatment order. At s a c r i f i c e , the animals were weighed, 17 a n a e s t h e t i z e d u s i n g d i e t h y l e t h e r vapour and exsanguinated v i a t h e t r u n k v e i n . The b l o o d was allowed t o c l o t f o r 4 hours a t room temperature p r i o r t o c e n t r i f u g a t i o n f o r s e r a c o l l e c t i o n . The s e r a was s t o r e d a t -20°C u n t i l s t e r o i d a n a l y s i s . The r e p r o d u c t i v e t r a c t s were d i s s e c t e d whole and a s m a l l crimp was made at the a t the u t e r o - t u b a l j u n c t i o n of the l e f t cornua f o r i d e n t i f i c a t i o n purposes. In a d d i t i o n , t h e v a g i n a was removed and w i t h a l l o t h e r t i s s u e was p l a c e d immediately i n Bouin's f i x a t i v e ( s a t u r a t e d p i c r i c a c i d - 7 5 % , 37% b u f f e r e d formalin-20%, g l a c i a l a c e t i c a c i d - 5 % ) . A f t e r 4 hours, a l l t i s s u e were p l a c e d i n running tap water t o remove excess f i x a t i v e and were s t o r e d i n 10% b u f f e r e d n e u t r a l f o r m a l i n u n t i l h i s t o l o g i c a l p r o c e s s i n g . C l e a r i n g and waxing of a l l t i s s u e was c a r r i e d out u s i n g double l e n g t h i n c u b a t i o n times ( b r a i n v s . r o u t i n e c y c l e ) . H i s t o l o g y and H i s t o c h e m i s t r y During f i x a t i o n , mesenteric t i s s u e was removed and each u t e r i n e horn was d i s s e c t e d from the p e r i o v a r i u m and p a r t i t i o n e d i n t o the f o l l o w i n g segments b e f o r e placement i n t o c a s s e t t e s denoting l e f t and r i g h t s i d e s . The f i r s t 2 mm of u t e r u s (proximal t o the oviduct) were d i s c a r d e d , the next 3 mm were used f o r t r a v e r s e s e c t i o n s , the next 9 mm f o r s a g g i t a l s e c t i o n s , the next 5 mm again f o r t r a v e r s e s e c t i o n s and the r e s t was d i s c a r d e d . The vagina was f i x e d whole f o r s a g g i t a l s e c t i o n s . 18 From each segment of t i s s u e , 4 s e r i a l sections (5 um) were taken for routine H&E staining for t o t a l s of 24 uterine sections and 4 vaginal sections per rat. Vaginal smears, instead of tissue, were taken from h a l f of a l l rats which were treated with 4 and 40 IU PMSG and s a c r i f i c e d between 48 and 192 h. In addition, histochemistry (diastase/alcian blue pH 1.0, 2.5/ PAS (D/AB/PAS)) was performed on 16 s e r i a l sections (5um) from the f i r s t 3 mm uterine segment (right side) and the vagina of each r a t . The s e r i a l sections were divided into four groups and each group was treated i n one of the following ways: AB 2.5/PAS; D/AB 2.5/PAS; AB 2.5; AB 1.0. The regimen was adapted from that used by the Dept. of Anatomical Pathology, Children's Hospital: 1. dewaxing and hydration 5. 1% a l c i a n blue 8GX (pH 1.0 2. 2% diastase - 30 min., 2.5) - 30 min. 30°C 6. 1% periodic acid - 20 min. 3 water - 2 min. 7. water - 5 min. 4. 1% or 3% g l a c i a l acetic 8. s c h i f f reagent - 3 0 min. acid (pH 1.0 or 2.5) - 9. warm water - 5 min. 3 0 min. 10. dehydrate and mount Liver and aorta, respectively, were used as controls fo r the D/PAS and AB staining techniques. S c h i f f reagent controls were performed on a batch basis and the feulgen reaction was used i f pyknosis was present. Steroid Radioimmunoassay Aliquots of 0.5-1.0 mL sera were extracted twice with 4.0 or 4.5 mL d i e t h y l ether. The pooled extracts were evaporated at 3 5°C under nitrogen gas before being reconstituted i n 1.0 mL absolute ethanol. Duplicate 100 uL 19 a l i q u o t s o f the e x t r a c t s were assayed. Approximately 10,000 cpm o f t r a c e r (H 3) was added t o each tube. The b i n d i n g ^ e f f i c i e n c y o f a n t i b o d i e s of the s t e r o i d hormones (17B-e s t r a d i o l , progesterone, androgens) was 40-60% and non-s p e c i f i c b i n d i n g was l e s s than 5%. Q u a l i t y c o n t r o l of the assay was c a r r i e d out u s i n g water blanks and r e f e r e n c e s e r a and r e c o v e r i e s were estimated by a d d i t i o n of t r a c e r t o r e f e r e n c e s e r a a l i q u o t s . I l l Assessment of U t e r i n e T i s s u e  Design T r a v e r s e and l o n g i t u d i n a l h i s t o l o g i c a l s e c t i o n s were a s s e s s e d w i t h r e g a r d t o degenerate, s e c r e t o r y , s y n t h e t i c and p r o l i f e r a t i v e a c t i v i t y as p r e v i o u s l y d e s c r i b e d f o r normal and abnormal r a t and human u t e r i (Long and Evans 1922, Burch e t a l . 1932, Noyes 1950, A r i a s - S t e l l a 1955). The q u a l i t a t i v e o r q u a n t i t a t i v e data were averaged or summated and r e p o r t e d on a p e r r a t b a s i s . S i g n i f i c a n t d i f f e r e n c e s between cornua, o r mesometrial and a n t i - m e s o m e t r i a l e p i t h e l i a , o r l u m i n a l and g l a n d u l a r e p i t h e l i a were noted by e x c e p t i o n . Two independent observers (Drs. E. Jones, P. Cagle) were employed i n s i n g l e b l i n d e v a l u a t i o n s of 10% of a l l s l i d e s t o c o n f i r m i n d i v i d u a l o b s e r v a t i o n s and comparisons o f d i f f e r e n t treatments and times. R e p r e s e n t a t i v e t i s s u e o f each dose and time group were photomicrographed u s i n g the Nikon O p t i p h o t System. 20 Degenerate A c t i v i t y Scoring of a c t i v i t y was determined subjec t i v e l y as follows: 1-no vacuolation; 2-some vacuolation (<50% a l l c e l l s ) ; 3-extensive vacuolation (>50%) ; 4-extensive vacuolation and pyknosis; 5-extensive vacuolation, pyknosis and karyorrhexis. Tissue from adult proestrus and diestrus periods were used as controls for scores "5" and "1", respectively. Characterization of the vacuolated contents was c a r r i e d out by histochemistry. Fluctuations i n leukocytic invasion were used as an i n d i r e c t i n d i c a t o r of degeneration of the epithelium (Long and Evans 1922). Secretory A c t i v i t y Assessment was based upon the presence of intraluminal material, a p i c a l border appearance and cytoplasmic histochemistry. With the techniques applied, not a l l intraluminal material could be v i s u a l i z e d i . e . , during proestrus. Under these conditions i n t e r p r e t a t i o n was based upon observations made during dissection, the degree of uterine distension and vaginal cytology/histology. Histochemistry was used to dis t i n g u i s h the presence of mucin from f o r t u i t o u s l y sectioned c e l l s , which may also give the e p i t h e l i a l surface a shredded appearance. Sections were recut from u t e r i demonstrating secretory material and were restained i n a single batch, to allow for comparisons i n the absence of i n t e r - s t a i n v a riations. 21 Synthetic A c t i v i t y A c t i v i t y was evaluated through time comparisons of cytoplasmic ba s o p h i l l i a / g r a n u l a r i t y and nuclear appearance/polarity of tissue within one treatment group. Nuclear appearance was assessed on the basis of opacity, s i z e and nucleolar s i z e . P o l a r i t y was scored s u b j e c t i v e l y as follows: 1 - a l l n u c l e i resting against basement membrane; 2-<50%; 3-50%; 4->50% and <100%; 5-100%. Tissue obtained from adult diestrus and proestrus phases were used as controls f o r scores "1" and "5", respectively. E p i t h e l i a l P r o l i f e r a t i o n and Hyperplasia Abnormal e p i t h e l i a l p r o l i f e r a t i o n was distinguished from normal growth by both phase and degree. Observed growth outside of proestrus and estrus, both associated with ovulation, was considered the major c r i t e r i o n f o r "out of phase" growth. P r o l i f e r a t i o n , under adult c y c l i n g conditions, occurs during late diestrus/early proestrus p r i o r to the marked d i l a t a t i o n of the uterus with f l u i d i n mid proestrus. Increased growth also occurs during mid-estrus, subsequent to extensive degeneration i n l a t e proestrus/early estrus (Long and Evans 1922). Stromal invasion was considered the major c r i t e r i o n f o r e p i t h e l i a l hyperplasia (Arias-Stella 1955, Meissner et a l . 1957, Dallenbach-Hellweg et a l . 1985). Hyperplasia of the luminal e p i t h e l i a was d i f f e r e n t i a t e d from glandular e p i t h e l i a through the examination of s e r i a l sections and c y t o l o g i c a l 22 comparison of the e p i t h e l i a of i n t e r e s t with intra-stromal glandular e p i t h e l i a . Progressive changes toward a moderate to extensive confluent p a p i l l a r y pattern with stromal r a r e f i c a t i o n were considered evidence of stromal invasion by luminal e p i t h e l i a . Increased e p i t h e l i a l growth was assessed by morphometric measurement of the lumen perimeter (Bioquant II, version 7.2, R&M Biometrics) and mitotic counts. M i t o t i c counts were indexed i n terms of percent e p i t h e l i a l c e l l s . IV Assessment of Vaginal Tissue The e f f e c t s of PMSG treatment were examined i n the histology of the exfoliated c e l l s and in t a c t epithelium and the histochemistry of mucified c e l l s . The exfoliated compartment was examined for the types ( e p i t h e l i a l , granulocytic) and forms (columnar, cuboidal, cornified) of dehiscent c e l l s , the manner of association of these c e l l s (single, clump, sheet) and the r e l a t i v e amount of the d i f f e r e n t c e l l types. The epithelium superior to the germ layer was also examined for the d i f f e r e n t c e l l types and forms as well as the number of layers of each type. The extent of mucification was characterized i n both int a c t and e x f o l i a t e d compartments by histochemistry. Vaginal tissue of histochemical i n t e r e s t was re-stained i n a single batch to allow f o r comparisons i n the absence of i n t e r - s t a i n v a r i a t i o n s . Two independent observers (Drs. Y.W. Yun, P. Cagle) were u t i l i z e d i n v a l i d a t i n g i n d i v i d u a l assessments of 23 t i s s u e and c y t o l o g i c a l preparations. V S t a t i s t i c a l Analysis The s i g n i f i c a n c e of treatment means differences was tested by analysis of variance (completely randomized) and Duncan's multiple range test (1957). Individual interactions were tested by Student's T t e s t and si g n i f i c a n c e was defined as p<0.05. S t a t i s t i c a l analysis was only performed on objective data (counts, RIA and morphometric measurements). 2 4 Results I Endometrium Representative photomicrographs of the u t e r i and endometria from 4, 20, 4 0 IU PMSG treated and adult rats are i l l u s t r a t e d i n Figs. 1-42. The time course of PMSG induced changes i n the perimeter of the lumen, e p i t h e l i a l m i t o t i c and endometrial neutrophil counts are presented respectively i n Figs. 43, 44 and 45. i n addition, these results are summarized i n Fig . 73. The uterine morphological and h i s t o l o g i c a l patterns observed shortly a f t e r 4 IU PMSG treatment were s t r i k i n g l y s i m i l a r to those of adult r a t s . At 48 h, the majority (66%) of control u t e r i (Fig. 1) resembled those of proestrus adults (Fig. 3); the rest appeared delayed i n development (Fig. 14). In the majority of u t e r i , the lumen, f i l l e d with transparent f l u i d , was greatly distended, which was re f l e c t e d i n the large luminal perimeter (7.28 + 0.7 mm). The columnar luminal epithelium (Fig. 2) displayed basophilia, enlarged n u c l e i , with d i s t i n c t n u c l e o l i , but few mitotic figures. The glandular e p i t h e l i a was r e l a t i v e l y inactive and did not appear to follow the changes of the luminal epithelium. By 72 h, the uterine f l u i d had disappeared and the perimeter of the lumen had s i g n i f i c a n t l y (p<0.001) decreased by 56% (Fig. 5). The endometrium, compressed by the collapse of the uterus, was folded and f l a c c i d . This appearance was s i m i l a r to that observed i n the adult estrus state (Fig. 4). The 25 t a l l columnar epithelium was pseudostratified and, i n addition, v i s i b l y degenerate (Fig. 6). An abundant amount of nuclear (feulgen +) debris was observed free i n the c e l l and i n the numerous i n t e r - and i n t r a c e l l u l a r vacuoles observed i n the epithelium. These vacuoles often contained a mucinous (AB 1.0+,2.5+,PAS+,D~) material, which formed a glycocalyx on the luminal surface (Fig. 7). During t h i s episode of degeneration, neutrophil i n f i l t r a t i o n was extensive, e s p e c i a l l y of the stroma. Between 48 and 72 h, the neutrophil count s i g n i f i c a n t l y (p<0.025) increased 58% to 185 + 27/section. From 72 to 120 h, there was an o v e r a l l trend, towards quiescence. The tortuous folds of the endometrium at 72 h had mostly disappeared by 96 h (Fig. 8). Further collapse of the lumen and regressive changes of the endometrium had resulted i n a s l i t - l i k e lumen by 120 h (Fig. 12). These morphological changes were analogous to those observed i n the adult metestrus (Fig. 10) and diestrus (Fig. 11) states, respectively. E p i t h e l i a l degenerative a c t i v i t y (vacuolation, pyknosis) had greatly subsided by 96 h (Fig. 9) and was absent by 120 h (Fig. 13). Concomitantly, the neutrophil count s i g n i f i c a n t l y (p<0.005) decreased from the peak at 72 h to baseline by 120 h. Regenerative a c t i v i t y (mitosis), however, peaked at 96 h (8.56 + 1.5%) before s i g n i f i c a n t l y (p<0.001) f a l l i n g to basal l e v e l s by 144 h. From 120 to 192 h, the epithelium was low columnar and 26 exhibited condensed oval nuclei, both consistent with a state of quiescence. c The uterine response a f t e r 192 h varied greatly. Of a l l the u t e r i , 33% were s t i l l quiescent. Many u t e r i (50%), however, displayed features of early proestrus (Figs. 14 & 16) . The e p i t h e l i a i n these u t e r i presented opaque n u c l e i , d i s t i n c t n u c l e o l i , and crowding of the e p i t h e l i a (Figs. 15 & 17) . Further progressive changes of the endometrium towards maximal d i l a t i o n were evident by 240 h. Compared with those at 192 h, the mitotic index and the luminal perimeter were s i g n i f i c a n t l y (p<0.025) higher at 240 h. At 240 h, 33% of a l l u t e r i were greatly distended, as observed at 48 h (Fig. 1). The changes i n u t e r i treated with 20 IU PMSG followed a s i m i l a r course to the response under the 4 IU PMSG regimen. However, superimposed, over these changes were abnormal p r o l i f e r a t i o n and secretion. At 48 h, only 20% of a l l u t e r i were s i m i l a r to the t y p i c a l proestrus morphology (Fig. 3). The r e s t of the u t e r i presented stromal hypertrophy and moderate i n f o l d i n g of the endometrium (Fig. 18). Very l i t t l e glandular e p i t h e l i a was present, even i n the invaginations of these f o l d s . The predominant luminal epithelium was t a l l columnar and exhibited enlarged nuclei and basophilia (Fig. 19). By 72 h, there was increased i n f o l d i n g and i n addition, the appearance of focal p a p i l l a r y hyperplasia (Fig. 20). Invariably, t h i s hyperplasia occurred at the anti-mesometrial end and was attended by stromal r a r e f i c a t i o n . Glandular 27 e p i t h e l i a did not appear to be involved i n t h i s abnormal p r o l i f e r a t i o n . E p i t h e l i a l degeneration was prominent i n a l l areas except hyperplastic f o c i (Fig. 21). Vacuolation and secretion of a mucinous (AB 1.0+,2.5+,PAS+,D~) material was more frequent than pyknosis. Neutrophil i n f i l t r a t i o n was higher than 48 h l e v e l s , but s i g n i f i c a n t l y (p<0.025) lower than those at 72 h under the 4 IU PMSG regimen. The mitotic index increased s i g n i f i c a n t l y (p<0.001) from 72 h to a maximum at 120 h (3.35 + 0.6%), before returning to baseline by 168 h. After 72 h and at lea s t u n t i l 240 h, the e p i t h e l i a l hyperplasia, stromal hypertrophy and the s i z e of the lumen gradually diminished (Figs. 22-29). The branched hyperplastic pattern at 72 h had disappeared by 9 6 h, through the resorption of smaller p a p i l l a r y buds. By 144 h, the large r p a p i l l a r y extensions as well as e x i s t i n g folds of the endometrium had markedly recessed (Fig. 24). By 192 h, only the folds remained (Fig. 27), which were mostly resorbed by 240 h (Fig. 30). E p i t h e l i a l a c t i v i t y , however, did not begin to recess u n t i l a f t e r 144 h. Between 96-144 h, the columnar epithelium was b a s o p h i l l i c and the nuclei showed d i f f u s e chromatin and d i s t i n c t n u c l e o l i (Figs. 23 & 25). Secretion of a mucinous (AB 1.0+,2.5+,PAS+,D~) and feulgen - substance increased from 120 to 144 h (Fig. 26). The glandular e p i t h e l i a appeared secretory as well. This material appeared as small specks i n the cytoplasm. After 144 h, e p i t h e l i a l 28 a c t i v i t y subsided. By 192 h, the nuclei had condensed and become t i g h t l y packed together (Fig. 28), and within 24 h the basophilia had disappeared (Fig. 29). The a l t e r a t i o n s i n the endometrium r e s u l t i n g from 40 IU PMSG treatment were sim i l a r , but magnified when compared with those i n 2 0 IU PMSG treated r a t s . At 48 h, the e x i s t i n g i n f o l d i n g had created deep invaginations into the endometrium (Fig. 31). The predominant luminal e p i t h e l i a was columnar and displayed enlarged nuclei and basophilia (Fig. 32). By 72 h, p a p i l l a r y hyperplasia appeared f o c a l l y at the anti-mesometrial end (Fig. 33). This was accompanied by the secretion of a mucinous (AB 1.0+,2.5+,PAS+,D~) material. However, unlike the 4 and 2 0 IU PMSG responses, there was no evidence of e p i t h e l i a l degeneration or increased neutrophil i n f i l t r a t i o n . The hyperplasia rapi d l y progressed and by 12 0 h, had spread towards the mesometrial end (Fig. 34). Interestingly, very few mitoses were observed (Fig. 35). By 144 h, the luminal e p i t h e l i a had deeply and m u l t i c e n t r i c a l l y invaded the stroma, forming many f l a c c i d projections of endometria with numerous p a p i l l a r y extensions (Fig. 36). The siza b l e luminal perimeter (7.06 + 0.9 mm) r e f l e c t e d t h i s increase i n the surface area of the endometrium and was s i g n i f i c a n t l y (p<0.005) greater than both the 20 IU PMSG and control responses. The cuboidal epithelium was well d i f f e r e n t i a t e d and displayed enlarged, rounded nuclei (Fig. 37). In addition, the luminal surface was coated heavily with a mucinous (AB 1.0 ,2.5 ,PAS ,D~) material, which was also observed as small specks i n the cytoplasm (Fig. 38)« Glandular e p i t h e l i a was not involved i n t h i s abnormal p r o l i f e r a t i o n , but appeared secretory, s i m i l a r to that of luminal e p i t h e l i a . A f t e r 144 h, a l l a c t i v i t i e s recessed, but quiescence was not attained by 240 h. Many of the p a p i l l a r y extensions observed between 120 and 144 h had regressed by 168 h (Fig. 39). Secretory a c t i v i t y had decreased by 168 h and was absent by 19 2 h. Further reductions i n luminal perimeter and s i z e and stromal hypertrophy were apparent by 192 h (Fig. 40). At 240 h, the e p i t h e l i a was low columnar and appeared inactive (Fig. 42). F u l l regression of the hyperplasia, as observed a f t e r 2 0 IU PMSG treatment, was not achieved by 240 h (Fig 41). U t e r i recovered 40 d a f t e r treatment, however, showed t y p i c a l estrous cycle features and no evidence of hyperplasia or any other h i s t o l o g i c a l abnormalities. 30 F i g . l U t e r u s , 48h +4IU PMSG (40x, H&E) The lumen i s mark e d l y d i l a t e d w i t h f l u i d and appears v a c a n t , e q u i v a l e n t t o t h a t i n a d u l t p r o e s t r u s ( F i g . 3 ) . F i g . 3 U t e r u s , A d u l t P r o e s t r u s (40x, H&E) Fi g . 2 Endometrium, 48h +4IU PMSG (400x, H&E) The b a s o p h i l i a o f t h e c y t o p l a s m and t h e prominence o f t h e n u c l e i and n u c l e o l i a r e c o n s i s t e n t w i t h an a c t i v e e p i t h e l i u m . F i g . 4 U t e r u s , A d u l t E s t r u s (40x, H&E) 31 Fig.5 Uterus, 72h +4IU PMSG (40x, H&E) Collapse of the lumen has led to the compres-sion of the endometrium into f l a c c i d folds, s i m i l a r to the estrus state (Fig.4). Note the tis s u e central i n the lumen. Fig.6 Endometrium, 72h +4IU PMSG (400x, H&E) Vacuolation, pyknosis and karyorhexis of the epithelium are extensive. The compression of the epithelium has led to a t a l l columnar and pseudostratified appearance. Fig.7 Endometrium, 72h +4IU PMSG ( 4 0 0 X , AB 1.0/PAS/D) Vacuolated mucinous (AB 1.0+, 2.5 +,PAS +,D~) material accumulated during degener-ation forms a glycocalyx and i s deposited i n the lumen. 32 F i g . 8 U t e r u s , 96h +4IU PMSG (4Ox, H&E) Through s t r o m a l r e o r g a n i z a t i o n and degener-a t i o n o f t h e e p i t h e l i u m , t h e m a j o r i t y o f t h e f o l d s have been smoothed o u t . Compare w i t h F i g . 1 0 ( a d u l t m e t e s t r u s ) F i g . 9 Endometrium, 96h +4IU PMSG (400x, H&E) E p i t h e l i a l d e g e n e r a t i o n has s u b s i d e d and r e g e n e r a t i v e a c t i v i t y ( m i t o s i s ) i s maximal. F i g . 1 0 U t e r u s , A d u l t M e t e s t r u s F i g . 1 1 U t e r u s , A d u l t D i e s t r u s (40x, H&E) (40x, H&E) 33 F i g . 1 2 U t e r u s , 120h +4IU PMSG (4Ox, H&E) The lumen has become s l i t - l i k e , as d u r i n g d i e s t r u s ( F i g . 1 1 ) , t h r o u g h s t r o m a l r e g r e s s i o n and l o s s o f edema. Note t h e i n c r e a s e d s t r o m a l c e l l u l a r d e n s i t y next t o t h e lumen. Fig.14 U t e r u s , 216h +4IU PMSG (40x, H&E) Enlargement o f t h e lumen i s a t t e n d e d by i n c r e a s e d e p i t h e l i a l s u r f a c e a r e a and s t r o m a l r e o r g a n i z a t i o n . S m a l l i n v a g i n a t i o n s form as a r e s u l t . F i g . 1 3 Endometrium, 120h +4IU PMSG (400x, H&E) R e p a i r o f t h e e p i t h e l i u m i s c o m p l e t e . The e p i t h e l i u m i s low columnar and appears i n a c t i v e . Note t h e condensed n u c l e i , i n d i s t i n c t n u c l e o l i and e o s i n o p h i l i a . F i g . 1 5 Endometrium, 216h +4IU PMSG (400x, H&E) I n c r e a s e d p r o l i f e r a t i o n has r e s u l t e d i n l a t e r a l c o m p r e s s i o n and e p i t h e l i a l c r o w d i n g . The e p i t h e l i u m remains low, but p r e s e n t s a c t i v e n u c l e i . 34 F i g . 1 6 U t e r u s , 240h +4IU PMSG (4Ox, H&E) A c c u m u l a t i o n o f f l u i d i n t h e lumen i s a p p a r e n t . However, maximal d i s t e n s i o n and c o m p l e t i o n o f e n d o m e t r i a l changes have n ot been a t t a i n e d . F i g . 1 8 U t e r u s , 48h +20IU PMSG (40x, H&E) The stroma i s edematous and h y p e r t r o p h i e d , and as a r e s u l t appears f o l d e d . F i g . 1 7 Endometrium, 240h +4IU PMSG (400x, H&E) E x t e n s i v e p r o l i f e r a t i o n accompanies t h e a c c u m u l a t i o n o f f l u i d and d i l a t i o n o f t h e lumen. Note t h e marked c r o w d i n g o f t h e e p i t h e l i u m . F i g . 1 9 Endometrium, 48h +20IU PMSG (400x, H&E) The predominant l u m i n a l e p i t h e l i a i s t a l l columnar and appears v e r y a c t i v e . Note t h e d i f f u s e c h r o m a t i n and d i s t i n c t n u c l e o l i . 35 F i g . 2 0 U t e r u s , 72h +20IU PMSG (40x, H&E) P a p i l l a r y h y p e r -p l a s i a has appeared f o c a l l y a t t h e a n t i - m e s o m e t r i a l end. Note t h a t t h e e p i t h e l i a has i n v a d e d t h e stroma a l m o s t as f a r as the myometrium. Fi g . 2 2 U t e r u s , 96h +20IU PMSG (40x, H&E) Through s t r o m a l r e s o r p t i o n and e p i t h e l i a l d e g e n e r a t i o n , b r a n c h i n g o f t h e e p i t h e l i u m has been reduc e d , r e s u l t i n g i n s l e n d e r s t a l k -p r o j e c t i o n s . F i g . 2 1 Endometrium, 72h +20IU PMSG (400x, H&E) V a c u o l a t i o n and o t h e r d e g e n e r a t i v e changes a r e o n l y s l i g h t i n com p a r i s o n w i t h t h e 4 IU PMSG re s p o n s e ( F i g . 6 ) . The n u c l e i and n u c l e o l i a r e s t i l l e n l a r g e d . F i g . 2 3 Endometrium, 96h +20IU PMSG (400x, H&E) The e p i t h e l i u m appears h e a l t h y and q u i e s c e n t . The n u c l e i a r e o r d e r e d and m o d e r a t e l y condensed. 36 F i g . 2 4 U t e r u s , 144h +20IU PMSG (40x, H&E) The s l e n d e r s t a l k s by t h i s t i m e have been re d u c e d t o s m a l l buds. Note t h e i n c r e a s e d c e l l u l a r d e n s i t y o f t h e stroma i n t h e s e a r e a s . F i g . 2 5 Endometrium, 144h +20IU PMSG (400x, H&E) The e p i t h e l i u m has become d e p o l a r -i z e d and appears s e c r e t o r y . F i g . 2 6 Endometrium, 144h +20IU PMSG (400x, AB 1.0/PAS/D) The mucinous (AB 1.0 +,2.5 +,PAS +, D~) m a t e r i a l , a p p e a r i n g as s m a l l s p e c k s i n t h e c y t o p l a s m , forms a t h i n r i m on t h e l u m i n a l s u r f a c e . 37 F i g . 2 7 U t e r u s 192h +20IU PMSG (40x, H&E) The p a p i l l a r y h y p e r p l a s i a as w e l l t h e f o l d s o f e n d o m e t r i a have r e g r e s s e d . P r o g r e s s i v e changes towards t h e f o r m a t i o n o f a s l i t - l i k e lumen a r e ap p a r e n t . Fig.28 Endometrium, 192h +20IU PMSG (400x, H&E) The l u m i n a l e p i t h e l i u m d i s p l a y s compact and o r g a n i z e d n u c l e i . Note t h e s l i g h t l y d i f f e r e n t a p p e a r i n g , but q u i e s c e n t , g l a n d u l a r t i s s u e . F i g . 2 9 Endometrium, 216h +20IU PMSG (400x, H&E) The e p i t h e l i a i s c o m p l e t e l y i n a c t i v e . The c y t o p l a s m i s c l e a r and p r e s e n t s a s t r o n g e o s i n o -p h i l l i c appearance. F i g . 3 0 U t e r u s , 24Oh +2 0IU PMSG (4Ox, H&E) The lumen i s s t e l l a t e - l i k e and t h e u t e r u s appears s i m i l a r t o t h e m e t e s t r u s ( F i g . 1 0 ) and 96h +4IU PMSG m o r p h o l o g i e s ( F i g . 8 ) . 38 F i g . 3 1 U t e r u s , 48h +40IU PMSG (40x, H&E) The e x i s t i n g i n f o l d i n g has c r e a t e d deep i n v a g i n a t i o n s i n t h e endometrium. Compare w i t h t h e 2 0 IU PMSG re s p o n s e a t 4 8h (F i g . 1 8 ) . Fi g . 3 3 U t e r u s , 72h +40IU PMSG (40x, H&E) S e c r e t o r y p a p i l l a r y h y p e r p l a s i a f o c a l l y appears a t t h e a n t i - m e s o m e t r i a l end, s i m i l a r t o t h a t o b s e r v e d a t 144h +20IU PMSG ( F i g . 2 4 ) . Fig.32 Endometrium, 48h +40IU PMSG (400x, H&E) The predominant l u m i n a l e p i t h e l i a i s t a l l columnar and d i s p l a y s b a s o p h i l i a , e n l a r g e d n u c l e i and d i s t i n c t n u c l e o l i . 39 F i g . 3 4 U t e r u s , 120h +40IU PMSG (40x, H&E) The h y p e r p l a s i a has begun t o s p r e a d c i r c u m -f e r e n t i a l l y t owards t h e meso-m e t r i a l end and t h e s e c r e t o r y a c t i v i t y appears t o have s u b s i d e d t e m p o r a r i l y . F i g . 3 5 Endometrium, 120h +40IU PMSG (400x, H&E) The e p i t h e l i u m appears a c t i v e ; t h e n u c l e i a r e l a r g e and t h e n u c l e o l i a r e d i s t i n c t . M i t o t i c f i g u r e s a r e r a r e . F i g . 3 6 U t e r u s , 144h +40IU PMSG (40x, H&E) The l u m i n a l e p i t h e l i a has d e e p l y and m u l t i c e n t r i c a l l y i n v a d e d t h e stroma, f o r m i n g many f l a c c i d p r o j e c t i o n s w i t h p a p i l l a r y e x t e n s i o n s . F i g.37 Endometrium, 144h +40IU PMSG (400x, H&E) S e c r e t o r y a c t i v i t y was more i n t e n s e t h a n t h a t o b s e r v e d a f t e r 20 IU PMSG ( F i g . 2 5 ) . Note a l s o t h e extreme t h i n n e s s o f t h e stroma i n some p l a c e s . 4 0 F i g . 3 8 Endometrium, 144h +40IU PMSG ( 4 0 0 X , AB 1.0/PAS/D) The s e c r e t i o n o f a s i m i l a r mucinous (AB1.0 +,2.5 +,PAS +, D ) s u b s t a n c e t o t h a t o b s e r v e d w i t h 20 IU PMSG (Fig . 2 6 ) accompanies t h e h y p e r p l a s i a . F i g . 3 9 U t e r u s , 168h +40IU PMSG (4Ox, H&E) Many o f t h e p a p i l l a r y p r o j e c t i o n s had r e g r e s s e d , i n c r e a s i n g t h e t h i c k n e s s o f stroma s e p a r a t i n g t h e myometrium and lumen. S e c r e t o r y a c t i v i t y i s s t i l l p r e s e n t , b u t i s r e d u c e d . F i g . 4 0 U t e r u s , 192h +40IU PMSG (4Ox, H&E) S t r o m a l h y p e r t r o p h y and lumen s i z e have been re d u c e d . F u r t h e r r e g r e s s i o n o f t h e h y p e r p l a s i a by t h i s t i m e i s a p p a r e n t and t h e lumen i s r e l a t i v e l y c l e a r . 41 F i g . 4 1 U t e r u s , 240h +40IU PMSG (40x, H&E) Changes toward f u l l r e g r e s s i o n c o n t i n u e . The s i z e o f t h e u t e r u s and t h i c k n e s s o f th e endometrium a r e s i m i l a r t o t h o s e a t 96 and 120h +4IU PMSG. Note t h e i n c r e a s e d s t r o m a l c e l l u l a r i t y , e s p e c i a l l y n e x t t o t h e lumen. Fi g . 4 2 Endometrium, 240h +40IU PMSG (400x, H&E) The e p i t h -e l i u m appears l e s s a c t i v e . The n u c l e i a r e s m a l l e r and t h e c y t o p l a s m appears e o s i n o p h i l l i c . 42 1.0 J 48 72 96 120 144 ?68 192 216 240 Hours Post PMSG Injection F i g . 4 3 P e r i m e t e r (mm) o f t h e u t e r i n e lumen a t d i f f e r e n t p e r i o d s a f t e r i n j e c t i o n o f 4, 2 0 and 40 IU PMSG. V a l u e s r e p r e s e n t means + SEM, N=6. 43 0 48 72 96~ 120 144 ]~68 192 216 240 Hours Post PMSG Injection F i g . 4 4 E p i t h e l i a l m i t o t i c i n d e x , e x p r e s s e d i n p e r c e n t , a t d i f f e r e n t p e r i o d s a f t e r i n j e c t i o n o f 4, 20 and 40 IU PMSG. V a l u e s r e p r e s e n t means + SEM, N=6. 44 0 48 72 96 120 144 168 192 216 240 Hours Post PMSG Injection F i g . 4 5 N e u t r o p h i l c o u n t s o f t h e endometrium a t d i f f e r e n t p e r i o d s a f t e r i n j e c t i o n o f 4, 20 and 40 IU PMSG. V a l u e s r e p r e s e n t means + SEM, N=6. 45 I I V a g i n a R e p r e s e n t a t i v e p h o t o m i c r o g r a p h s o f t h e v a g i n a e from 4, 20 and 40 IU PMSG t r e a t e d and a d u l t r a t s a r e p r e s e n t e d i n F i g s . 46-74. A summary o f t h e major v a g i n a l changes i s p r o v i d e d i n F i g . 73. The s u c c e s s i o n , d i f f e r e n t i a t i o n and desquamation o f c e l l t y p e s proceeded s t e p w i s e o v e r t h e e n t i r e e p i t h e l i u m . A t 48 h a f t e r 4 IU PMSG, t h e v a g i n a l h i s t o l o g y and c y t o l o g y ( F i g . 46) e x h i b i t e d f e a t u r e s t y p i c a l o f p r o e s t r u s i n t h e a d u l t r a t ( F i g . 4 8 ) . M u c i f i e d (AB 1.0 +,2.5 +,PAS +,D~) e p i t h e l i a l c e l l s p r e d o m i n a t e d t h e e x f o l i a t e d compartment i n most (66%) o f t h e c o n t r o l s ( F i g . 4 7 ) . S t a i n i n g was s l i g h t l y d a r k e r a t AB pH 2.5 t h a n a t AB pH 1.0. I n some r a t s ( 3 3 % ) , however, desquamation had n o t begun. N o n - n u c l e a t e d c o r n i f i e d c e l l s , f o r m i n g e i t h e r t h e most s u p e r f i c i a l o r n e x t s u c c e s s i v e l a y e r , were l o o s e l y o r g a n i z e d i n t o s h e e t s . The r e s t o f t h e e p i t h e l i u m c o n s i s t e d o f squamous p r e - c o r n i f i e d n u c l e a t e d and b a s a l c e l l s . A t 72 h, e x f o l i a t e d s h e e t s o f c o r n i f i e d c e l l s f i l l e d t h e lumen and t h e m u c i f i e d e p i t h e l i a l c e l l s had d i s a p p e a r e d ( F i g . 5 0 ) , s i m i l a r t o t h a t o b s e r v e d d u r i n g e s t r u s ( F i g . 4 9 ) . The e p i t h e l i u m c o n s i s t e d o f a 1-2 s u p e r f i c i a l l a y e r s o f p r e - c o r n i f i e d c e l l s , 3-5 l a y e r s o f c u b o i d a l c e l l s and t h e g e r m i n a t i v u m . N e u t r o p h i l i n f i l t r a t i o n was e x t e n s i v e b u t a b s e n t from t h e lumen. By 96 h, t h e p r e c o r n i f i e d and most s u p e r f i c i a l c u b o i d a l l a y e r s w i t h a s s o c i a t e d n e u t r o p h i l s had become d e h i s c e n t ( F i g . 5 1 ) . T h i s heterogenous p a t t e r n was a l s o 46 o b s e r v e d d u r i n g t h e a d u l t m e t e s t r u s s t a t e ( F i g . 5 2 ) . From 120 t o 192 h, t h e r e was moderate p r o l i f e r a t i o n o f m u c i n o g e n i c c e l l s and c o n c o m i t a n t h y p e r t r o p h y as t h e s e c e l l s d i f f e r e n t i a t e d ( F i g s . 54-55). T h i s appearance was c o m p a r a t i v e l y e q u i v a l e n t w i t h d i e s t r u s i n t h e a d u l t ( F i g . 5 6 ) . A f t e r 192 h, many v a g i n a e (66%) were found t o d i s p l a y f e a t u r e s o f e a r l y ( F i g s . 56-57) o r m i d - p r o e s t r u s ( F i g . 4 8 ) . The v a g i n a l development and d i f f e r e n t i a t i o n s h o r t l y f o l l o w i n g 20 IU PMSG t r e a t m e n t t e m p o r a l l y p a r a l l e l e d t h o s e o f c o n t r o l s ( F i g s . 58-60). A f t e r 96 h, however, t h e e p i t h e l i u m r a p i d l y and p r o g r e s s i v e l y m u c i f i e d . M u c i f i c a t i o n (AB 1.0 +,2.5 +,PAS +,D~) o f t h e most s u p e r f i c i a l e p i t h e l i a l l a y e r s was e v i d e n t by 120 h ( F i g s . 61-62). I n most c a s e s s t a i n i n g was e q u i v a l e n t o r s l i g h t l y d a r k e r w i t h AB pH 2.5 t h a n w i t h pH 1.0. W i t h i n 24 h, t h e l o w e r l a y e r s o f t h e granulosum had begun t o m u c i f y and s e v e r a l s m a l l c y s t s appeared, c o n t a i n i n g s i m i l a r s t a i n i n g mucinous m a t e r i a l ( F i g s . 63-64). I t was not c l e a r whether t h e s e c y s t s were i n t r a c e l l u l a r v a c u o l e s o r i n t r a - e p i t h e l i a l c y s t s . J u d g i n g by t h e s i z e o f t h e s e c y s t s a t 216 h ( F i g . 6 5 ) , however, i t i s u n l i k e l y t h e y were i n t r a -c e l l u l a r . I n a d d i t i o n , as t h e r e was l i t t l e n u c l e a r d e b r i s o r p y k n o s i s , t h e mode o f s e c r e t i o n by s u r r o u n d i n g c e l l s was p r o b a b l y n o n - h o l o c r i n e . The lumen c o n t a i n e d c o p i o u s q u a n t i t i e s o f mucinous (AB 1.0 +,2.5 +, PAS +,D~) m a t e r i a l and numerous c u b o i d a l c e l l s . N e u t r o p h i l i n f i l t r a t i o n was l i g h t 47 and s p o r a d i c compared w i t h t h e response d u r i n g e s t r u s ( F i g . 5 0 ) . W i t h t i m e , t h e number and s i z e o f t h e i n t r a - e p i t h e l i a l c y s t s i n c r e a s e d and by 216 h, t h e e n t i r e e p i t h e l i u m had become c y s t i c ( F i g s . 65-66). The v a g i n a l development f o l l o w i n g 40 IU PMSG was c o m p a r a t i v e l y a c c e l e r a t e d w i t h t h e 2 0 IU PMSG r e s p o n s e . By 9 6 h, t h e m a j o r i t y o f a n i m a l s (83%) had a l r e a d y a c h i e v e d a m e t e s t r u s - l i k e s t a t e . P r o g r e s s i v e m u c i f i c a t i o n (AB 1.0 +, 2.5 +,PAS +,D~) o f t h e whole e p i t h e l i u m , i n v o l v i n g c y s t f o r m a t i o n , was o b s e r v e d a t 120 h ( F i g s . 67-68). The i n t e n s i t y o f s t a i n i n g a t AB pH 1.0 appeared s l i g h t l y l i g h t e r t h a n t h a t a t AB pH 2.5. By 168 h, t h e whole e p i t h e l i u m had become c y s t i c ( F i g s . 69-70). The appearance o f mucinous s e c r e t o r y m a t e r i a l and n e u t r o p h i l i n f i l t r a t i o n was s p o r a d i c . F u r t h e r changes p r i m a r i l y i n v o l v e d i n c r e a s e s i n c y s t s i z e t h r o u g h t h e r u p t u r e o f a d j a c e n t w a l l s ( F i g s . 71-72). 48 F i g . 4 6 V a g i n a , 48h +4IU PMSG (40x, H&E) E p i t h e l i a l c e l l s have e x f o l i a t e d l e a v i n g b e h i n d s e v e r a l l a y e r s o f c o r n i f i e d c e l l s . P r e c o r n i f i e d and c u b o i d a l c e l l s form t h e granulosum. (see Fig.48) Fig.47 V a g i n a , 48h +4IU PMSG (40x, AB 2.5/PAS/D) The d e h i s c e n t e p i t h e l i a l c e l l s c o n t a i n mucin (AB 1.0 +,2.5 +, PAS +,D~). V e r y l i t t l e o f t h i s m a t e r i a l , however, i s s e c r e t e d d i r e c t l y i n t o t h e lumen. 49 F i g . 5 0 V a g i n a , 72h +4IU PMSG (4Ox, H&E) The m u c i f i e d e p i t h e l i a l c e l l s have been r e p l a c e d by e x f o l i a t e d s h e e t s o f c o r n i f i e d c e l l s . N e u t r o p h i l i n f i l t r a t i o n i s c o n f i n e d t o t h e e p i t h e l i u m , (see Fig.49) F i g . 5 1 V a g i n a , 96h +4IU PMSG (40x, H&E) The p r e c o r n i f i e d and most s u p e r f i c i a l c u b o i d a l l a y e r s , c o n t a i n i n g n e u t r o p h i l s , have been e x f o l i a t e d , s i m i l a r t o t h a t seen d u r i n g a d u l t m e t e s t r u s . 50 F i g . 5 4 V a g i n a , 120h +4IU PMSG (40x, H&E) The i n c r e a s e d e p i t h e l i a l h e i g h t i s due t o some p r o l i f e r a t i o n , b u t m a i n l y c e l l u l a r h y p e r t r o p h y , e s p e c i a l l y o f t h e most s u p e r f i c i a l l a y e r s . F i g . 5 6 V a g i n a , 240h +4IU PMSG (40x, H&E) Concomitant w i t h t h e o n s e t o f o v u l a t i o n ( p r o e s t r u s ) , m u c i f i c a t i o n can be o b s e r v e d f a r as t h e p r e c o r n i f i e d l a y e r , seen h e r e as a d a r k i s h band. F i g . 5 5 V a g i n a , 120h +4IU PMSG (40x, AB 2.5/PAS/D) The most s u p e r f i c i a l l a y e r s have become m u c i f i e d (AB 1 . 0 + , 2 . 5 + , PAS +,D~), i n d i c a t i n g f u l l d i f f e r e n t i a t i o n o f t h e s e c e l l s . Compare w i t h F i g . 5 3 . F i g . 5 7 V a g i n a , 240h +4IU PMSG (40x, AB 2.5/PAS/D) Compare t h e d e p t h o f m u c i f i c a t i o n w i t h t h e l o c a t i o n o f t h e p r e c o r n i f i e d band. 51 F i g . 5 8 V a g i n a , 48h +20IU PMSG (40x, H&E) The p a t t e r n r e s e m b l e s t h a t a t 48h +4IU PMSG (F i g . 4 6 ) and a d u l t p r o e s t r u s ( F i g . 4 8 ) . F i g . 6 0 V a g i n a , 9 6h +2 0IU PMSG (40x, H&E) E x f o l i a t e d s u p e r f i c i a l p r e c o r n i f i e d and c u b o i d a l c e l l s and n e u t r o p h i l s occupy t h e lumen. Compare w i t h 96h +4IU PMSG (F i g . 5 1 ) and a d u l t m e t e s t r u s ( F i g . 5 2 ) . Fig . 5 9 V a g i n a , 72h +20IU PMSG (40x, H&E) The corneum as w e l l as some p r e c o r n i f i e d c e l l s have desquamated, s i m i l a r t o t h a t o b s e r v e d a t 72h +4IU PMSG (Fig.50) and a d u l t e s t r u s ( F i g . 4 9 ) . 52 F i g . 6 1 V a g i n a , 120h +20IU PMSG (40x, H&E) H i s t o l o g i c a l l y , i t appears t h a t a l l b u t t h e b a s a l l a y e r has m u c i f i e d . However, o n l y t h e most s u p e r f i c i a l l a y e r s show e v i d e n c e o f t h i s . F i g . 6 2 V a g i n a , 120h +20IU PMSG (40x, AB 2.5/PAS/D) F i g . 6 3 V a g i n a , 144h +20IU PMSG (40x, H&E) The l o w e r l a y e r s o f t h e granulosum had begun t o m u c i f y . I n a d d i t i o n s m a l l s p a c e s , f i l l e d w i t h mucinous m a t e r i a l , had appeared. Fig.64 V a g i n a , 144h +20IU PMSG (40x, AB 2.5/PAS/D) I n a d d i t i o n t o t h e mucinous m a t e r i a l , numerous e p i t h e l i a l c e l l s and some n e u t r o p h i l s had accumulated i n t h e lumen. F i g . 6 5 V a g i n a , 216h +2 0IU PMSG (40x, H&E) The s m a l l spaces had c o a l e s c e d f o r m i n g l a r g e c y s t s . The c e l l s making up t h e w a l l s appeared d i s t e n d e d , b u t h e a l t h y . There was n e g l i g i b l e n u c l e a r d e b r i s . F i g . 6 6 V a g i n a , 216h +20IU PMSG (40x, AB 2.5/PAS/D) F i g . 6 7 V a g i n a , 120h +40IU PMSG (40x, H&E) A l t h o u g h not f u l l y d i f f e r e n t i a t e d , a l l l a y e r s s u p e r i o r t o t h e g e r m i n a t i v u m appear m u c i n o g e n i c . Pi •.•*r Fig.68 V a g i n a , 12Oh +4 0IU PMSG (4OX, AB 2.5/PAS/D) M u c i f i c a t i o n (AB 1.0 +,2.5 +, PAS +,D ) was s p o r a d i c t h r o u g h o u t t h e e p i t h e l i u m , i n d i c a t i n g i n c o m p l e t e d i f f e r e n t i a t i o n . 5 4 F i g . 6 9 V a g i n a , 168h +40IU PMSG (40x, H&E) The e n t i r e e p i t h e l i u m had become c y s t i c , compare w i t h 144 and 216h +20 IU PMSG ( F i g s . 6 3 - 6 6 ) . F i g . 7 1 V a g i n a , 240h +40IU PMSG (40x, H&E) I n t r a - e p i t h e l i a l s e c r e t i o n o f t h e mucinous m a t e r i a l had r u p t u r e d a d j a c e n t w a l l s , r e s u l t i n g i n e n l a r g e d c y s t s . F i g . 7 0 V a g i n a , 168h +40IU PMSG (40x, AB 2.5/PAS/D) I n a d d i t i o n t o desquamated e p i t h e l i a and n e u t r o p h i l s , mucinous s e c r e t o r y m a t e r i a l was p r e s e n t . Fig.72 V a g i n a , 240h +40IU PMSG (40x, AB 2.5/PAS/D) 55 40 IU PMSG Uterine Morpho logy stromal focal hyper papillary ( trophy hyperplasia Epithelial Activity sec Vagina L P E E ME muc 20 IU PMSG | stromal focal • hyper papillary trophy hyperplasia Uterine Morpho logy Epithelial Activity Vagina P E 4 IU PMSG Uterine Morpho logy Epithelial Activity Vagina dilated PE 48 deg sec ME diffuse papillary hyperplasia sec small spaces cystic I sec sec muc small spaces flaccid stellate folds deg proliferation sec & repair 72 ME 96 DE muc 120 partial regression quiescence I I + , - • • large cysts I I full regression i quiescence cystic ' distension proliferation keratogenic & mucinogenic 144 168 differentiation 192 216 240 h F i g . 7 3 S c h e m a t i c summary o f u t e r i n e and v a g i n a l m o r p h o l o g i c a l and h i s t o l o g i c a l changes f o l l o w i n g t r e a t m e n t w i t h 4, 20 "and 40 IU PMSG. A b b r e v i a t i o n s : s e c = s e c r e t o r y ; deg=degenerate; rnuc= m u c i f i c a t i o n ; P E = p r o e s t r u s ; L P E = l a t e p r o e s t r u s ; E = e s t r u s ; ME= m e t e s t r u s ; D E = d i e s t r u s . 56 I l l S t e r o i d s The t e m p o r a l p a t t e r n s o f serum l e v e l s o f 1 7 B - e s t r a d i o l , p r o g e s t e r o n e , and androgens a r e shown i n F i g . 74. Ma j o r a l t e r a t i o n s i n serum s t e r o i d l e v e l s o c c u r r e d s h o r t l y a f t e r 4 IU PMSG t r e a t m e n t . A f t e r 24 h, 1 7 B - e s t r a d i o l l e v e l s s i g n i f i c a n t l y (p<0.001) i n c r e a s e d t o a maximum (421 + 33 pg/ml) a t 48 h, b e f o r e f a l l i n g t o b a s a l l e v e l s by 72 h. P r o g e s t e r o n e l e v e l s s i g n i f i c a n t l y (p<0.01) i n c r e a s e d from 48 t o 60 h (109 + 17 ng/ml) and a l s o r e t u r n e d t o b a s a l l e v e l s by 72 h. E l e v a t i o n s i n 1 7 B - e s t r a d i o l and androgens were o b s e r v e d between 192 and 240 h, but t h e s e were n o t s i g n i f i c a n t . Many s t r i k i n g changes r e s u l t e d from t r e a t m e n t w i t h 20 IU PMSG t r e a t m e n t . By 24 h, 1 7 B - e s t r a d i o l l e v e l s had a l r e a d y 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) and w i t h i n 24 h had peaked (663 + 59 pg/ml). T h i s peak was comparable t o t h a t i n 40 IU PMSG t r e a t e d r a t s , b u t s i g n i f i c a n t l y (p<0.001) h i g h e r t h a n i n c o n t r o l s . Between 48 and 72 h, 1 7 B - e s t r a d i o l l e v e l s s h a r p l y f e l l back t o b a s e l i n e . Androgen l e v e l s r o s e (p<0.05) t h r e e f o l d from 24 t o 48 h (7.59 + 2 . 1 ng/ml) and t h e n d e c r e a s e d s t e p w i s e . They s i g n i f i c a n t l y (p<0.05) d e c l i n e d t o i n t e r m e d i a r y l e v e l s by 96, b u t were s t i l l s i g n i f i c a n t l y (p<0.01) h i g h e r t h a n t h o s e o f c o n t r o l s . T h e r e a f t e r , androgen l e v e l s s l o w l y descended and r e a c h e d b a s e l i n e by 168 h. P r o g e s t e r o n e l e v e l s f o l l o w e d t h o s e o f c o n t r o l s u n t i l a t l e a s t 72 h. By 96 h, however, t h e s e l e v e l s had i n c r e a s e d s i g n i f i c a n t l y (p<0.025) and were g r e a t l y (p<0.001) e l e v a t e d 57 above t h o s e o f c o n t r o l s . A maximum (2 61 + 27 pg/ml) was g r a d u a l l y r e a c h e d by 168 h and t h e n l e v e l s f e l l t o wards b a s a l l e v e l s . S t e r o i d l e v e l s r e s u l t i n g from t h e 40 IU PMSG regimen f l u c t u a t e d g r e a t l y . A f t e r d r a m a t i c a l l y (p<0.001) i n c r e a s i n g between 0 and 48 h (538 + 36 pg/ml), 1 7 B - e s t r a d i o l l e v e l s s i g n i f i c a n t l y (p<0.001) d e c r e a s e d by 68%. The peak a t 48 h was s i g n i f i c a n t l y (p<0.025) g r e a t e r t h a n c o n t r o l l e v e l s . U n l i k e t h e r e s p o n s e s o f l o w e r doses, 1 7 B - e s t r a d i o l l e v e l s s h a r p l y r o s e (p<0.001) a g a i n a f t e r 72 h t o a maximum a t 96 h (537 + 43 pg / m l ) , b e f o r e f a l l i n g t o b a s e l i n e by 120 h. A s i g n i f i c a n t (p<0.005) r i s e i n androgens from 24 h t o a peak (7.92 + 1.3 ng/ml) a t 72 h preceded t h e l a s t i n c r e a s e i n 17B-e s t r a d i o l l e v e l s . Analogous t o t h e 2 0 IU r e s p o n s e , androgen l e v e l s d e c l i n e d i n a s t e p w i s e manner. They f i r s t f e l l (p<0.05) between 72 and 96 h t o p l a t e a u l e v e l s comparable t o t h a t i n 2 0 IU PMSG t r e a t e d r a t s . These l e v e l s t h e n g r a d u a l l y d i m i n i s h e d , w h i l e f o l l o w i n g t h e d e c l i n e o f 2 0 IU PMSG l e v e l s . P r o g e s t e r o n e l e v e l s p a r a l l e l e d t h o s e o f t h e 2 0 IU PMSG re s p o n s e u n t i l 168 h. I t was n o t u n t i l 120 h d i d t h e s e l e v e l s become s i g n i f i c a n t l y (p<0.01) g r e a t e r t h a n t h o s e o f c o n t r o l s . A f t e r 168 h, p r o g e s t e r o n e l e v e l s c o n t i n u e d t o r i s e and r e a c h e d 53 9 + 67 ng/ml by 240 h. 58 6 24 48 72 96 120 144 168 192 216 240 Hours Post PMSG Injection F i g . 7 4 Changes i n serum l e v e l s o f 1 7 B - e s t r a d i o l , p r o g e s t e r o n e and androgens a f t e r t r e a t m e n t w i t h 4, 2 0 and 4 0 IU PMSG. A t 0, 24 and 60 h, 3 r a t s were used; 6 r a t s w e r e used a t a l l o t h e r t i m e s . V a l u e s r e p r e s e n t means + SEM. 59 D i s c u s s i o n The p r e s e n t s t u d y c o n f i r m s t h a t a low dose (4 IU) o f PMSG can i n d u c e immature r a t s t o mimic t h e p h y s i o l o g i c a l e v e n t s o f t h e a d u l t e s t r u s c y c l e . The dynamics o f 17B-e s t r a d i o l and p r o g e s t e r o n e l e v e l s between 24 and 12 0 h p o s t PMSG t r e a t m e n t a r e i n f a i r agreement w i t h t h o s e o b s e r v e d by o t h e r w o r k e r s u s i n g low doses (4-8 IU) o f PMSG ( W i l s o n e t a l . 1974, N u t i e t a l . 1975, P a r k e r e t a l . 1976, M i l l e r and A r m s t r o n g 1981b) and w i t h p e r i - o v u l a t o r y changes r e p o r t e d f o r t h e a d u l t c y c l i n g r a t (Yoshinaga e t a l . 1969, P a s q u a l i n i e t a l . 1985). I n v i e w o f t h e s i m i l a r i t y between hormonal p a t t e r n s o f c o n t r o l s and a d u l t s , t h e c o r r e s p o n d e n c e o f u t e r i n e h i s t o l o g i c a l changes was n o t s u r p r i s i n g . I t has l o n g been r e c o g n i z e d t h a t e n d o m e t r i a l h i s t o l o g i c a l changes d u r i n g t h e e s t r u s c y c l e a r e c o n t r o l l e d by c o n c o m i t a n t f l u c t u a t i o n s o f e s t r o g e n and p r o g e s t e r o n e s e c r e t i o n r a t e s . D u r i n g p r o e s t r u s , t h e u t e r u s becomes m a x i m a l l y d i l a t e d by t h e a c c u m u l a t i o n and r e t e n t i o n o f f l u i d , w h i c h a i d s i n t h e t r a n s p o r t and c a p a c i t a t i o n o f sperm (Warren 193 6, L e o n a r d 1950, K i r t o n e t a l . 1965). I t i s w e l l e s t a b l i s h e d t h a t t h e a c c u m u l a t i o n o f f l u i d as w e l l as t h e i n c r e a s e d l u m i n a l e p i t h e l i a l p r o l i f e r a t i o n t h a t accompanies and a l l o w s f o r l u m i n a l d i s t e n s i o n i s a s s o c i a t e d w i t h t h e e l e v a t i o n o f e s t r o g e n l e v e l s 24 h p r i o r t o maximum d i l a t i o n (Schwarz 1964, Marcus 1974, Kennedy e t a l . 1975, F i n n 1980). D u r i n g e s t r u s , d e c l i n i n g e s t r o g e n l e v e l s and r i s i n g p r o g e s t e r o n e l e v e l s 60 s y n e r g i s t i c a l l y r e l a x t h e c o n s t r i c t e d c e r v i x and a l l o w f o r t h e d r a i n a g e o f u t e r i n e f l u i d t h r o u g h t h e v a g i n a (Kennedy e t a l . 1975). The u t e r u s i n v o l u t e s c o m p r e s s i n g t h e endometrium i n t o f l a c c i d f o l d s . C o n c o m i t a n t l y , a l l s t e r o i d l e v e l s r e t u r n t o b a s e l i n e and t h i s w i t h d r a w a l i n d u c e s s t r o m a l h y p o t r o p h y and e p i t h e l i a l d e g e n e r a t i o n , c h a r a c t e r i z e d by f e u l g e n + n u c l e a r d e b r i s , c y t o p l a s m i c d e p o s i t i o n o f mucins, n e u t r o p h i l i n f i l t r a t i o n and r e p a r a t i v e changes (Long and Evans 1922, A l l e n 1931, Marcus 1974, F i n n e t a l . 1975). S u p e r o v u l a t o r y t r e a t m e n t w i t h PMSG d o s e - d e p e n d e n t l y r e t a r d e d d e g e n e r a t i v e a c t i v i t y . A n i m a l s t r e a t e d w i t h 2 0 IU PMSG d i s p l a y e d t h e c l a s s i c e f f e c t s o f hormonal w i t h d r a w a l a t 72 h, a l b e i t subdued i n comparison w i t h c o n t r o l s and a d u l t s . A l t h o u g h 1 7 B - e s t r a d i o l and p r o g e s t e r o n e l e v e l s had f a l l e n t o b a s e l i n e by 72 h, androgen l e v e l s remained e l e v a t e d and may have p r o v i d e d p a r t i a l hormonal s u p p o r t . E x o g e n o u s l y a d m i n i s t e r e d non- and a r o m a t i z a b l e androgens may i n d u c e u t e r i n e e p i t h e l i a l h y p e r t r o p h y t h r o u g h p r o t e i n and c a r b o h y d r a t e s y n t h e s i s a s s o c i a t e d w i t h growth (Armstrong e t a l . 1976, P a s q u a l i n i e t a l . 1985). D e s p i t e a s h a r p f a l l i n 1 7 B - e s t r a d i o l l e v e l s a f t e r 60 h i n 40 IU PMSG t r e a t e d r a t s , l e v e l s a t 72 h were s t i l l h i g h e r t h a n t h o s e o f c o n t r o l s . These e s t r o g e n s i n c o m b i n a t i o n w i t h e l e v a t e d androgens c o u l d have p r o v i d e d s u f f i c i e n t e p i t h e l i a l s u p p o r t t o a c c o u n t f o r t h e absence o f d e g e n e r a t i o n , n e u t r o p h i l i n f i l t r a t i o n and 61 r e p a i r . W h i l e i n t e r f e r i n g w i t h changes n o r m a l l y a s s o c i a t e d w i t h o v u l a t i o n , i n c r e a s e d l e v e l s o f s t e r o i d s ( 1 7 B - e s t r a d i o l , androgens, p r o g e s t e r o n e ) may have c o n c u r r e n t l y produced e n d o m e t r i a l a t y p i a . H i g h l e v e l s o f 1 7 B - e s t r a d i o l and androgens t o g e t h e r w i t h h y p e r t r o p h i c development o f t h e e n d o m e t r i a l stroma f o l l o w e d e i t h e r 20 o r 40 IU PMSG t r e a t m e n t . As t h e r e was l i t t l e d i f f e r e n c e i n t h e s t e r o i d l e v e l s and t h e e x t e n t o f h y p e r t r o p h y , i t seems l i k e l y t h a t common mechanisms were o p e r a t i v e . I t i s w e l l e s t a b l i s h e d t h a t endogenous and exogenous e s t r o g e n s i n i t i a l l y h y p e r t r o p h y t h e s troma t h r o u g h t h e development o f hyperemia and s t r o m a l edema (Lauson e t a l . 1939, Schwarz 1964, Galand e t a l . 1971, M a r t i n e t a l . 1973). Time c o u r s e s t u d i e s o f o v a r i e c t o m i z e d r a t s g i v e n a s i n g l e dose o f 1 7 B - e s t r a d i o l have r e v e a l e d t h a t edema r a p i d l y d e v e l o p s t o a dose-dependent maximum between 6-10 h, b u t r e g r e s s e s w i t h i n 20 h, even a f t e r t h e use o f p r o l o n g e d t r e a t m e n t (Astwood 1938, C a r r o l l 1945, Homburger e t a l . 1955, Schwarz 1964, F i n n and P o r t e r 1975). F l u i d i s c o n c u r r e n t l y t r a n s f e r r e d from t h e stroma i n t o t h e lumen r e s u l t i n g i n d i s t e n s i o n ( F i n n e t a l . 1975). I n v i e w o f t h e s e e x p e r i m e n t s , t h e p r e s e n c e o f h y p e r t r o p h y a t 4 8 h i n r e l a t i o n t o e l e v a t e d 1 7 B - e s t r a d i o l l e v e l s p r i o r t o 24 h, s u g g e s t s t h e a f f e c t o f e s t r o g e n s may have been secondary. Exogenous a r o m a t i z a b l e and, t o a l e s s e r e x t e n t , n o n - a r o m a t i z a b l e androgens a r e c a p a b l e o f i n d u c i n g s u s t a i n e d h y p e r t r o p h y up t o 62 72 h a f t e r t r e a t m e n t (Rennels 1951, Ar m s t r o n g e t a l . 1976). Androgens may a c t d i r e c t l y upon t h e u t e r u s t h r o u g h t h e i r own o r , i n t h e case o f e l e v a t e d p e r i p h e r a l l e v e l s , e s t r o g e n r e c e p t o r s (Giannopoulos 1973, R o c h e f o r t e t a l . 1974, G a r c i a e t a l . 1977, L o b l e t a l . 1977). As t h e e f f e c t s o f androgens and e s t r o g e n s were n o t d e l i n e a t e d , i t i s p o s s i b l e t h a t t h e s e s t e r o i d s may have s y n e r g i s t i c a l l y i n d u c e d and m a i n t a i n e d h y p e r t r o p h y o f t h e stroma f o r an exten d e d p e r i o d o f t i m e . The most i n t e r e s t i n g f e a t u r e o f t h i s s t u d y was t h e p r o d u c t i o n o f f o c a l and d i f f u s e l u m i n a l p a p i l l a r y h y p e r p l a s i a w i t h s u p e r o v u l a t o r y doses o f PMSG. The development o f h y p e r p l a s i a appeared t o f o l l o w t h e o n s e t o f s t r o m a l h y p e r t r o p h y i n b o t h 20 and 40 IU t r e a t m e n t g r o u p s . I t i s c o n c e i v a b l e t h a t p r o l o n g e d and e l e v a t e d l e v e l s o f 17B-e s t r a d i o l from 24 h t o 48 h a f t e r 2 0 o r 40 IU PMSG t r e a t m e n t i n i t i a t e d t h i s s e q u e n t i a l p a t t e r n . S i m i l a r p a t t e r n s have been o b s e r v e d i n o v a r i e c t o m i z e d r o d e n t s r e p e a t e d l y t r e a t e d w i t h e s t r o g e n s ( C a r r o l 1945, Greenwald e t a l . 1959, F i n n e t a l . 1973, M a r t i n e t a l . 1973). W h i l e p r o l o n g e d t r e a t m e n t i n c r e a s e d u t e r i n e w e i g h t i n i t i a l l y (6-10 h) t h r o u g h s t r o m a l h y p e r t r o p h y , subsequent i n c r e a s e s (24-48 h) r e l i e d upon e p i t h e l i a l p r o l i f e r a t i o n and growth. E p i t h e l i a l h y p e r p l a s i a , however, was i n i t i a l l y f o c a l and i n v a r i a b l y appeared a t t h e a n t i m e s o m e t r i a l end. T h i s f o c a l appearance may have been a r e s u l t o f d i s s i m i l a r hormonal s e n s i t i v i t i e s o f t h e a n t i - and 63 m e s o m e t r i a l r e g i o n s . A u t o r a d i o g r a p h i c s t u d i e s o f o v a r i e c t o m i z e d mice t r e a t e d w i t h a s i n g l e dose o f 17B-e s t r a d i o l showed e q u a l l a b e l i n g r a t e s up t o 16 h between b o t h r e g i o n s , however, a f t e r t h i s t i m e w h i l e m e s o m e t r i a l r a t e s d e c l i n e d , t h o s e o f t h e a n t i m e s o m e t r i a l r e g i o n remained e l e v a t e d up t o a t l e a s t 36 h ( M a r t i n e t a l . 1973). P r o l o n g e d and h i g h l e v e l s o f e s t r o g e n s a s s o c i a t e d w i t h s u p e r o v u l a t o r y t r e a t m e n t may have e x a g g e r a t e d e x i s t e n t e l e v a t e d p r o l i f e r a t i o n r a t e s i n t h e a n t i m e s o m e t r i a l r e g i o n r e s u l t i n g i n f o c a l h y p e r p l a s i a . D i f f e r e n t i a l s e n s i t i v i t y was a l s o o b s e r v e d between l u m i n a l and g l a n d u l a r e p i t h e l i a d u r i n g t h e development o f h y p e r p l a s i a . I t i s w e l l r e c o g n i z e d t h a t t h e a f f e c t s o f s t e r o i d hormones on t h e endometrium a r e dependent upon t h e s e n s i t i v i t y and p r o p o r t i o n o f g l a n d u l a r and l u m i n a l e p i t h e l i a l t i s s u e (Burch e t a l . 1932, W olfe e t a l . 1933, C l e v e l a n d e t a l . 1941, A r i a s - S t e l l a 1954, 1955, M e i s s n e r e t a l . 1957, Buxton e t a l . 1961, F i n n e t a l . 1975, Gondos 1976). I n r a t s and mice, t h e r e i s l i t t l e g l a n d u l a r t i s s u e . Moreover, t h e l u m i n a l e p i t h e l i u m i s f a r more s e n s i t i v e t o hormonal i n f l u e n c e t h a n e i t h e r g l a n d u l a r o r s t r o m a l t i s s u e ( M a r t i n e t a l . 1973, Marcus 1974, F i n n e t a l . 1975, F i n n 1980). These f e a t u r e s might a c c o u n t f o r t h e absence o f s i m i l a r changes i n g l a n d u l a r t i s s u e t o t h a t o f t h e l u m i n a l e p i t h e l i u m w h i l e t h e r e was i n c r e a s e d p r o l i f e r a t i o n . I n a d d i t i o n , t h e d i f f e r e n t i a l s e n s i t i v i t i e s o f t h e l u m i n a l e p i t h e l i u m and t h e stroma may e x p l a i n t h e p a p i l l a r y 64 appearance o f t h e h y p e r p l a s t i c endometrium. The e p i t h e l i u m , g r o w i n g f a s t e r t h a n t h e stroma, i s f o r c e d t o f o l d t o a l l o w f o r an i n c r e a s e i n s u r f a c e a r e a w i t h i n a r e s t r i c t e d volume. Had t h e r e been s y n c h r o n i z e d growth between t h e stroma and e p i t h e l i u m , s t r o m a l r a r e f i c a t i o n would have been a b s e n t and t h e e p i t h e l i u m l e s s c o n v o l u t e d , as o b s e r v e d d u r i n g t h e o n s e t o f p r o e s t r u s i n c o n t r o l s from 216-240 h. From 96 t o 144 h p o s t 40 IU PMSG t r e a t m e n t , t h e r e was i n c r e a s e d h y p e r t r o p h y o f t h e stroma and p r o g r e s s i v e h y p e r p l a s i a . W h i l e i t i s d i f f i c u l t t o p r e c i s e l y i d e n t i f y t h e hormonal e v e n t ( s ) r e s p o n s i b l e , t h e absence o f t h e s e p r o g r e s s i o n s i n 2 0 IU PMSG t r e a t e d r a t s p r o v i d e s a b a s i s f o r co m p a r i s o n . The major d i f f e r e n c e between t h e s t e r o i d a l p a t t e r n s o f t h e 2 0 and 4 0 IU PMSG t r e a t m e n t groups was t h e marked e l e v a t i o n o f 1 7 B - e s t r a d i o l l e v e l s from 72 t o 96 h p o s t 40 IU PMSG. Walton e t a l . (1982), a d m i n i s t e r i n g PMSG a n t i s e r a a t 58 h p o s t 40 IU PMSG i n j e c t i o n , s i m u l t a n e o u s l y r e d u c e d t h e 1 7 B - e s t r a d i o l surge a t 96 h and a b o l i s h e d t h e i n c r e a s e i n u t e r i n e w e i g h t u s u a l l y o b s e r v e d w i t h s u p e r o v u l a t o r y t r e a t m e n t . As p r e v i o u s l y m entioned, h i g h doses o f e s t r o g e n s may in d u c e e n d o m e t r i a l h y p e r t r o p h y and h y p e r p l a s i a . I t i s p o s s i b l e t h a t e a r l i e r e l e v a t i o n s o f e s t r o g e n l e v e l s (24-48 h) may have p r i m e d o r s e n s i t i z e d t h e endometrium t o respond o v e r t l y t o a second s t i m u l u s . The changes i n d u c e d by t h i s second s t i m u l u s would t h e n be 65 superimposed o v e r t h o s e e l i c i t e d by t h e f i r s t s t i m u l u s . M a r t i n e t a l . (1973), u s i n g a s i n g l e i n j e c t i o n o f 17B-e s t r a d i o l , found t h a t u t e r i n e w e i g h t and volume, s t r o m a l edema and e p i t h e l i a l p r o l i f e r a t i o n r a t e s d e c l i n e d a f t e r t h e y r e a c h e d a maximum between 6-24 h. However, i f a second i n j e c t i o n o f t h e same dose (100 ng) was g i v e n 24 h l a t e r , t h e magnitude o f a l l parameters i m m e d i a t e l y i n c r e a s e d t o new and h i g h e r maxima w i t h i n 16-24 h. These r e s u l t s o f t h e use o f s i n g l e and doub l e i n j e c t i o n o f e s t r o g e n s appear t o p a r a l l e l o ur o b s e r v a t i o n s o f 20 and 4 0 IU PMSG t r e a t e d r a t s , r e s p e c t i v e l y . We o b s e r v e d time-dependent i n c r e a s e s i n t h e l u m i n a l p e r i m e t e r and equated t h e s e i n c r e a s e s w i t h p r o l i f e r a t i o n . W i t h t h e e x c e p t i o n o f c o n t r o l s , m i t o t i c r a t e s d i d n o t c o r r e l a t e w e l l w i t h changes i n l u m i n a l p e r i m e t e r . D e s p i t e t h e f o c a l appearance o f h y p e r p l a s i a a t 72 h, t h e m i t o t i c i n d i c e s f o r b o t h 20 and 40 IU PMSG t r e a t m e n t groups a c t u a l l y d e c r e a s e d from 48 t o 72 h. A d d i t i o n a l l y , t h e development o f d i f f u s e h y p e r p l a s i a i n 40 IU PMSG t r e a t e d r a t s was n o t accompanied by i n c r e a s e d m i t o t i c r a t e s . I t i s p o s s i b l e t h a t m i t o s e s were r a p i d , h i g h l y s y n c h r o n i z e d and o c c u r r e d between s a c r i f i c e s . I t i s w e l l e s t a b l i s h e d t h a t e s t r o g e n s i n c r e a s e p r o l i f e r a t i o n r a t e t h r o u g h t h e s h o r t e n i n g o f G-^ and S phases o f t h e c e l l c y c l e ( E p i f a n o v a 1966, L e r o y e t a l . 1969, M a r t i n e t a l . 1973, F i n n e t a l . 1975). I n t h e r e s t i n g s t a t e , t h e m a j o r i t y o f c e l l s can be found i n e i t h e r G-^ o r G Q phases. 66 S t i m u l a t i o n w i t h e s t r o g e n s promotes s y n c h r o n i z e d e n t r y i n t o S and m i t o s i s phases. Exogenous e s t r o g e n s t i m u l a t i o n i n o v a r i e c t o m i z e d r o d e n t s have i n d u c e d c e l l c y c l e l e n g t h s l e s s t h a n 15 h w i t h a m i t o t i c r a t e o f 1 h i n u t e r i n e l u m i n a l e p i t h e l i a l c e l l s ( T a c h i e t a l . 1972, M a r t i n e t a l . 1973, F i n n e t a l . 1975). I t s h o u l d a l s o be noted t h a t a l t h o u g h t i s s u e were i m m e d i a t e l y p l a c e d i n f i x a t i v e , m i t o s i s may have n o t have been i n s t a n t l y a r r e s t e d . B u l l o u g h (1950), u s i n g B o u i n ' s f i x a t i v e , o b s e r v e d p r o g r e s s i v e r e d u c t i o n s i n t h e number o f c e l l s a t each phase o f m i t o s i s up t o 3 h, i n d i c a t i n g t h a t c e s s a t i o n o f a c t i v i t y was dependent upon f i x a t i v e p e n e t r a t i o n and n o t on t h e t i m e o f s a c r i f i c e . Thus, i t would have been p o s s i b l e f o r p r o l i f e r a t i v e changes i n i t i a t e d soon a f t e r a s a c r i f i c e t o f i n i s h p r i o r t o t h e c o m p l e t i o n o f f i x a t i o n o f t h e n e x t s a c r i f i c e . The appearance o f i n t r a - l u m i n a l s e c r e t o r y m a t e r i a l (120-168 h) w i t h d i f f u s e h y p e r p l a s i a i n 40 IU PMSG t r e a t e d r a t s may have been c o i n c i d e n t a l and not r e l a t e d t o t h e p r e s e n c e o f h y p e r p l a s i a . D e s p i t e c o n c u r r e n t e n d o m e t r i a l r e g r e s s i o n i n t h e 20 IU PMSG group, s i m i l a r s t a i n i n g m a t e r i a l was p r e s e n t (96-120 h) i n a l l l u m i n a . H i s t o c h e m i s t r y o f t h i s m a t e r i a l s u g g e s t e d t h e p r e s e n c e o f sul p h o m u c i n s . Under t h e i n f l u e n c e o f e s t r o g e n s , sulphomucins may be produce d and s e c r e t e d by t h e j e n d o m e t r i u m and c e r t a i n segments ( i s t h m u s , a mpulla) o f t h e o v i d u c t o f t h e r a t , mouse and r a b b i t ( L e B l o n d 67 1950, Deane 1952, Greenwald 1969, Hamner 1973, F i n n e t a l . 1975). P h a r m a c o l o g i c a l doses o f e s t r o g e n i n d u c e e n d o m e t r i a l s t r o m a l p r o d u c t i o n o f a c i d mucins, t h a t a r e s u b s e q u e n t l y t r a n s u d a t e d t h r o u g h t h e e p i t h e l i u m i n t o t h e lumen ( Z a c h a r i a e 1958, Heap e t a l . 1962). U t e r i n e e p i t h e l i a l mucin p r o d u c t i o n can be i n i t i a t e d by p r o g e s t e r o n e p r o v i d i n g adequate p r i m i n g w i t h e s t r o g e n has o c c u r r e d . Taken t o g e t h e r , t h e p r e s e n c e o f i n t r a - l u m i n a l s e c r e t o r y m a t e r i a l (96-120 h) may r e p r e s e n t a l a t e n t e f f e c t o f e a r l i e r e l e v a t e d 1 7 B - e s t r a d i o l l e v e l s and an immediate e f f e c t o f r i s i n g p r o g e s t e r o n e l e v e l s , r e s u l t i n g i n combined mucin p r o d u c t i o n and s e c r e t i o n o f t h e stroma and t h e e p i t h e l i u m . There i s a l s o t h e p o s s i b i l i t y t h a t some o r most o f t h e s e c r e t o r y m a t e r i a l o r i g i n a t e d from t h e o v i d u c t . I t i s w e l l r e c o g n i z e d t h a t a c i d mucins, e s p e c i a l l y t h o s e s u l p h a t e d , c o n s t i t u t e a major component o f o v i d u c t a l f l u i d (Hamner 1973, Greenwald 1969). I n a d d i t i o n , t h e p r o d u c t i o n and s e c r e t i o n o f t h e s e mucins i n t h e r a t o v i d u c t a r e e s t r o g e n dependent and o c c u r m a i n l y i n t h e isthmus r e g i o n . Perhaps o v e r t o v i d u c t a l s t i m u l a t i o n by e l e v a t e d e s t r o g e n s a t 9 6 h i n 4 0 IU PMSG t r e a t e d r a t s p r o d u c e d c o p i o u s q u a n t i t i e s o f mucinous m a t e r i a l . The i n t e n s i f i c a t i o n o f a l c i a n o p h i l l i a by 144 h i n t h e s e r a t s may have r e s u l t e d from t h e l e a k a g e o f e x c e s s mucins i n t o t h e u t e r i n e lumen. A l t h o u g h t h e v a g i n a l changes i n d u c e d by 4, 20 o r 40 IU PMSG t r e a t m e n t were analogous t o t h o s e a s s o c i a t e d w i t h o v u l a t i o n i n t h e a d u l t , i t i s u n c e r t a i n whether s i m i l a r 68 mechanisms were p r e s e n t . I t i s w e l l e s t a b l i s h e d t h a t i n t h e a d u l t t h e b a s a l i s i s composed o f a homogenous p o p u l a t i o n o f g e r m - l i n e c e l l s t h a t u n i f o r m l y p a r t i c i p a t e i n k e r a t o g e n i c o r m u c i n o g e n i c d i f f e r e n t i a t i o n when s t i m u l a t e d w i t h e s t r o g e n o r p r o g e s t e r o n e , r e s p e c t i v e l y (Peckham e t a l . 1962, L a d i n s k y e t a l . 1965, B a r k e r e t a l . 1966, P a r a k k a l 1974). Moreover, i t i s t h i s u n i f o r m p a r t i c i p a t i o n t h a t a c c o u n t s f o r t h e w a v e - l i k e p r o g r e s s i o n o f c e l l s t owards t h e lumen. I n our s t u d y , d i f f e r e n t i a t i o n was i n i t i a t e d w i t h i n 48 h o f PMSG t r e a t m e n t and i n v o l v e d two e p i s o d e s o f m u c i f i c a t i o n s e p a r a t e d by a s h o r t - l i v e d k e r a t i n i z a t i o n . I t i s i n t r i g u i n g t h a t t h i s complex sequence o f e v e n t s was n o t p r e c e d e d by s t e r o i d a l p a t t e r n s g e n e r a l l y a s s o c i a t e d w i t h o v u l a t i o n . A u t o r a d i o g r a p h i c s t u d i e s f o l l o w i n g t h e p r o g r e s s i o n o f d i f f e r e n t i a t i o n i n t h e u n t r e a t e d i n t a c t o r s t e r o i d ( e s t r o g e n , p r o g e s t e r o n e ) t r e a t e d o v a r i e c t o m i z e d a d u l t r a t have shown a d e l a y o f l e a s t 48 h between t h e o n s e t o r i n j e c t i o n o f s t e r o i d and t h e o c c u r r e n c e o f i t s e f f e c t s ( S e l y e 1936, B e r t a l a n f l y e t a l . 1963, B a r k e r e t a l . 1966, L e r o y e t a l . 1969). T h r a s h e r e t a l . (1966), however, o b s e r v e d e l e v a t e d l a b e l l i n g i n d i c e s d u r i n g p r o e s t r u s t h a t d r a m a t i c a l l y f e l l by d i e s t r u s , i n d i c a t i n g a good c o r r e l a t i o n between hormonal r e l e a s e from t h e o v a r y and e f f e c t . Our r e s u l t s p r o v i d e c o m p e l l i n g e v i d e n c e t h a t a t l e a s t i n t h e PMSG t r e a t e d immature r a t , t h e v a g i n a l r e s p o n s e t o hormonal s t i m u l a t i o n i s immediate and 69 p r o c e e d s i n a s i m i l a r manner t o t h a t o f a d u l t s . T h i s h y p o t h e s i s i s f u r t h e r s u p p o r t e d by e v i d e n c e o f k e r a t i n i z a t i o n i n c o n t r o l s d u r i n g t h e second o n s e t o f p r o e s t r u s (216-240 h) i n t h e absence o f p r e c e d i n g s t e r o i d f l u c t u a t i o n s . I t i s n o t p o s s i b l e t o p r o v i d e an e x p l a n a t i o n f o r t h e i n d u c t i o n o f m u c i f i c a t i o n t h a t w i l l u n i f y a l l p o t e n t i a l c a u s a t i v e f a c t o r s . M u c i f i c a t i o n has been p r e v i o u s l y d e s c r i b e d i n t h e 10 and 15 d o l d r a t f o l l o w i n g s u p e r o v u l a t o r y t r e a t m e n t w i t h PMSG (Rennels 1951, P a r l a n t i e t a l . 1980). I n o v a r i e c t o m i z e d r a t s , R e n n e l s (1951) a c h i e v e d comparable r e s u l t s w i t h exogenous androgens and pr o p o s e d androgen s t i m u l a t i o n may have been an u n d e r l y i n g a c t i o n o f PMSG. Kennedy e t a l . (1976) demonstrated i n a d r e n a l e c t o m i z e d and o v a r i e c t o m i z e d r a t s t h a t non- and a r o m a t i z a b l e androgens a r e c a p a b l e o f i n d u c i n g m u c i f i c a t i o n . H i g h l e v e l s o f p r o l a c t i n may have a l s o been a c a u s a t i v e f a c t o r . R e n n e l s e t a l . (1977) showed t h a t a s u p e r o v u l a t o r y dose (50 IU) o f PMSG i n c r e a s e d p r o l a c t i n l e v e l s t h r o u g h t h e a c t i o n o f e s t r o g e n s o r a r o m a t i z a b l e androgens on t h e p i t u i t a r y . Moreover, Kennedy e t a l . (1972,1973) r e v e a l e d t h a t p r o l a c t i n s t i m u l a t e d v a g i n a l m u c i f i c a t i o n v i a an e x t r a - o v a r i a n a c t i o n , i n a d d i t i o n t o a l u t e o t r o p h i c - p r o g e s t e r o n e a c t i o n . T h i s e x t r a - o v a r i a n a c t i o n , however, depended upon t h e s y n e r g i s t i c a c t i o n s o f e s t r o g e n and p r o g e s t e r o n e . I n our s t u d y e l e v a t i o n o f p r o g e s t e r o n e l e v e l s appeared t o c o i n c i d e w i t h i n c r e a s e d m u c i f i c a t i o n . I t 70 i s w e l l e s t a b l i s h e d t h a t p r o g e s t e r o n e i n d u c e s m u c i n o g e n i c d i f f e r e n t i a t i o n ( S e l y e 1936, B e r t a l a n f l y e t a l . 1963, B a r k e r e t a l . 1966). E l e v a t e d p r o g e s t e r o n e l e v e l s from 168-240 h p o s t 40 IU PMSG may e x p l a i n t h e e x c e s s i v e m u c i f i c a t i o n and f o r m a t i o n o f e n l a r g e d c y s t s n o t o b s e r v e d i n 20 IU PMSG t r e a t e d r a t s . These r e s u l t s suggest t h a t s u p e r o v u l a t o r y doses o f PMSG i n d u c e p a t h o l o g i c a l s t r u c t u r a l and m e t a b o l i c a l t e r a t i o n s i n t h e u t e r u s and v a g i n a . A l t h o u g h r e v e r s i b l e , many o f t h e s e e f f e c t s p e r s i s t up t o one week a f t e r t r e a t m e n t . W h i l e i t i s not p o s s i b l e t o comment on u t e r i n e r e c e p t i v i t y , i t seems p l a u s i b l e t h a t t h e e f f e c t s i n d u c e d by s u p e r o v u l a t i o n may i n t e r f e r e w i t h p r e - i m p l a n t a t i o n a l p r e p a r a t i o n s and t h e r e f o r e r e q u i r e a t t e n t i o n i n r e f e r e n c e t o i m p l a n t a t i o n . 71 Summary The e f f e c t s o f s u p e r o v u l a t o r y t r e a t m e n t , w i t h r e g a r d t o t h e r a t u t e r u s and v a g i n a , were s t u d i e d by h i s t o l o g i c a l and h i s t o c h e m i c a l t e c h n i q u e s . I n a d d i t i o n , serum s t e r o i d l e v e l s ( 1 7 B - e s t r a d i o l , p r o g e s t e r o n e , androgens) were m o n i t o r e d . A q u a l i t a t i v e and morphometric s t u d y o f t h e t e m p o r a l e f f e c t s o f PMSG has been made. To s t u d y s h o r t t e r m e f f e c t s , u t e r i and v a g i n a e were o b t a i n e d from immature Sprague Dawley r a t s a t 24 h i n t e r v a l s between do and dlO f o l l o w i n g 4, 20 and 40 IU PMSG t r e a t m e n t . E x a m i n a t i o n o f t h e l o n g t e r m e f f e c t s o f PMSG was c a r r i e d out a t d30 p o s t 4 and 40 IU PMSG. A d u l t c y c l i n g r a t s were used t o p r o v i d e h i s t o l o g i c a l r e f e r e n c e s o f t h e p r o e s t r u s , e s t r u s , m e t e s t r u s and d i e s t r u s p hases. U s i n g r o u t i n e h i s t o l o g i c a l t e c h n i q u e , t h e u t e r i n e morphology and t h e c e l l s o f t h e e n d o m e t r i a l stroma and e p i t h e l i u m were s t u d i e d ( l u m i n a l morphometry, s t r o m a l n e u t r o p h i l c o u n t s , e p i t h e l i a l m i t o t i c i n d e x ) . I n a d d i t i o n , d i f f e r e n t i a t i o n o f t h e v a g i n a l e p i t h e l i u m was examined. W h i l e t r e a t m e n t w i t h 4 IU PMSG i n d u c e d changes i n u t e r i and v a g i n a e t h a t mimicked p h y s i o l o g i c a l e v e n t s o f t h e a d u l t e s t r u s c y c l e , s u p e r o v u l a t o r y doses (20, 40 IU PMSG) produced s t r o m a l h y p e r t r o p h y and e p i t h e l i a l h y p e r p l a s i a . F o l l o w i n g 20 IU PMSG t r e a t m e n t , e p i t h e l i a l h y p e r p l a s i a d e v e l o p e d f o c a l l y b e f o r e r e g r e s s i n g c o m p l e t e l y by dl O . However, u t e r i from 40 IU PMSG t r e a t e d ? r a t s d e v e l o p e d more d i f f u s e and i n v a s i v e p a t h o l o g y t h a t was s t i l l e v i d e n t by dlO but absent by d3 0. The r e l a t i o n s h i p o f 72 t h e s e abnormal p a t t e r n s and c o n c o m i t a n t f l u c t u a t i o n s o f serum 1 7 B - e s t r a d i o l and androgen l e v e l s was d i s c u s s e d . H i s t o c h e m i c a l s t u d i e s f o r t h e d e t e c t i o n o f c a r b o h y d r a t e s , e s p e c i a l l y mucins, were u n d e r t a k e n u s i n g t h e d i a s t a s e / a l c i a n b l u e pH 1.0 and 2 . 5 / p e r i o d i c a c i d s c h i f f t e c h n i q u e . I n s u p e r o v u l a t e d r a t s (20, 4 0 IU PMSG), dose-dependent q u a n t i t i e s o f s u l p h a t e d mucins were o b s e r v e d w i t h i n t h e u t e r i n e lumen and v a g i n a l i n t r a - e p i t h e l i a l c y s t s . The e t i o l o g y o f t h i s m u c i f i c a t i o n was d i s c u s s e d i n r e l a t i o n t o 1 7 B - e s t r a d i o l , p r o g e s t e r o n e , androgens and o t h e r hormones, w h i c h l e v e l s may have been g r e a t l y e l e v a t e d by s u p e r o v u l a t o r y t r e a t m e n t . I t was c o n c l u d e d t h a t s u p e r o v u l a t o r y t r e a t m e n t w i t h PMSG p a t h o l o g i c a l l y e f f e c t s t h e u t e r i n e endometrium and v a g i n a l e p i t h e l i u m . Moreover, s u p e r o v u l a t i o n i n d u c e s s t r i k i n g changes i n serum s t e r o i d i e v e l s . These s t e r o i d s may a c t a l o n e , t o g e t h e r o r i n c o m b i n a t i o n w i t h o t h e r hormones i n p r o d u c i n g t h e s e e f f e c t s . 0 73 R e f e r e n c e s A i t k e n , R . J . (1978). The Hormonal C o n t r o l o f I m p l a n t a t i o n . I n " M a t e r n a l R e c o g n i t i o n o f Pregnancy" ( E x c e r p t a M e d i c a , Amsterdam) C i b a F o u n d a t i o n Symposium 64., 53-63. A l l e n , W.M. (1931). I . C y c l i c a l A l t e r a t i o n s o f t h e Endometrium o f t h e Rat D u r i n g t h e Normal C y c l e , Pseudopregnancy and Pregnancy. I I . P r o d u c t i o n o f Deciduomata D u r i n g Pregnancy. Anat. 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