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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 o f S u p e r o v u l a t i o n w i t h Pregnant Mare Serum Gonadotrophin i n U t e r i , Vaginae and Serum S t e r o i d L e v e l s of Immature Rats. By Paul M a x i m i l i a n Fang B . S c , The U n i v e r s i t y o f B r i t i s h Columbia, 1984 B.M.L.Sc, The U n i v e r s i t y 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 in THE FACULTY OF GRADUATE STUDIES (Human Reproductive  Biology  Programme)  We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard  The U n i v e r s i t y of B r i t i s h April  Columbia  1988  ®Paul M a x i m i l i a n 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 by the head 3ST~my l  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  DE-6(3/81)  July  7,  19 8 8  Abstract S u p e r o v u l a t o r y treatment w i t h exogenous  gonadotrophins  a d v e r s e l y a f f e c t s t h e u t e r u s through t h e d i s r u p t i o n o f t h e d e l i c a t e balance o f o v a r i a n s t e r o i d androgens) s e c r e t i o n r a t e s .  (estrogens, p r o g e s t i n s ,  To examine t h e u t e r i n e  effects  o f t h i s treatment, 189 animals were g i v e n 4, 20 o r 40 IU pregnant mare serum gonadotrophin  (PMSG) a t 28 days o f age  and s a c r i f i c e d every 24 h u n t i l day 10 (D10) p o s t To study t h e l o n g term u t e r i n e e f f e c t s ,  injection.  12 r a t s were t r e a t e d  w i t h 4 o r 40 IU PMSG and k i l l e d on D30. The 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 o f c o n t r o l (4 IU) u t e r i mimicked those o f t h e a d u l t on a comparable time c o u r s e from D2 t o D5.  A d m i n i s t r a t i o n o f s u p e r o v u l a t o r y doses  (2 0, 4 0 IU) o f PMSG produced  stromal hypertrophy by D2 and  f o c a l p a p i l l a r y h y p e r p l a s i a o f t h e l u m i n a l e p i t h e l i a by D3. I t i s suggested t h a t p r e v i o u s exposure t o h i g h l e v e l s o f e s t r o g e n and androgens,  secondary t o s u p e r o v u l a t i o n , a r e  p o s s i b l e causes f o r t h i s pathology.  Levels of 17B-estradiol  f o l l o w i n g 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) e l e v a t e d above those o f c o n t r o l s from DI t o e a r l y D3 and a t D2, r e s p e c t i v e l y . groups  Androgen l e v e l s o f both  (20 IU, 40 IU) s i g n i f i c a n t l y  (p<0.05, p<0.005)  i n c r e a s e d from b a s e l i n e a t DI t o maxima by D2 and D3, respectively.  In t h e 20 IU PMSG group, t h e h y p e r p l a s i a  g r a d u a l l y r e g r e s s e d a f t e r D3 and was  absent by D10. The  hyperplasia D6.  i n t h e 40 IU PMSG group, however, had d i f f u s e d by  I t i s suspected t h a t preceding  elevated  l e v e l s of  e s t r o g e n may be r e s p o n s i b l e  for t h i s progressive  D4,  reached a maximum, which was  l e v e l s of 17B-estradiol  significantly  change.  At  (p<0.001) g r e a t e r than those o f c o n t r o l s and 20 Between D6 and D10, t h e h y p e r p l a s i a  IU PMSG t r e a t e d r a t s . p a r t i a l l y regressed.  Examination o f u t e r i from D30 r e v e a l e d  no evidence o f pathology. e f f e c t s , superovulation  In a d d i t i o n t o these s t r u c t u r a l  induced s e c r e t i o n o f a mucinous  substance i n both 2 0 IU and 40 IU PMSG groups a t D5-D6 and D6-D7, r e s p e c t i v e l y . These r e s u l t s suggest t h a t abnormal changes i n t h e u t e r i n e h i s t o l o g y and metabolism may r e s u l t f o l l o w i n g administration  o f superovulatory  doses o f PMSG.  Although  t h e s e dose-dependent a l t e r a t i o n s appear r e v e r s i b l e , they may i n t e r f e r e with preparations  a s s o c i a t e d w i t h i m p l a n t a t i o n and  thus r e q u i r e f u r t h e r i n v e s t i g a t i o n .  iii  Table o f Contents  Page #  Abstract  i i  T a b l e o f Contents  iv  L i s t of Figures  vi  Abbreviations  x  Acknowledgements  xi  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 and Embryo T r a n s f e r  1  I I I O v u l a t i o n and Pregnancy i n t h e PMSG T r e a t e d Immature Rat  IV  3  PMSG and O v u l a t i o n  4  PMSG and E a r l y Pregnancy  7  PMSG Induced U t e r i n e E f f e c t s  M a t e r i a l s and Methods  10 16  I  Animals and M a t e r i a l s  16  II  Methods  17  Design  17  H i s t o l o g y and H i s t o c h e m i s t r y  18  S t e r o i d Radioimmunoassay  19  I I I Assessment o f U t e r i n e T i s s u e  20  Design  20  Degenerate A c t i v i t y  21  Secretory A c t i v i t y  21  iv  T a b l e o f Contents  Page #  Synthetic A c t i v i t y  °  Epithelial Proliferation Hyperplasia  22 and  22  IV  Assessment of V a g i n a l T i s s u e  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.  U t e r u s , 48h +4IU PMSG (40x, H&E)  31  2.  Endometrium, 48h +4IU PMSG (400x, H&E)  31  3 . U t e r u s , A d u l t P r o e s t r u s (40x, H&E)  31  4.  U t e r u s , A d u l t E s t r u s (40X, H&E)  31  5.  U t e r u s , 72h +4IU PMSG (40x, H&E)  32  6.  Endometrium, 72h +4IU PMSG (400X, H&E)  32  7.  Endometrium, 72h +4IU PMSG (400x, AB 2.5/ PAS/D)  32  8.  U t e r u s , 9 6h +4IU PMSG (40x, H&E)  33  9.  Endometrium, 9 6h +4IU PMSG (400x, H&E)  33  10. U t e r u s , A d u l t Metestrus (4Ox , H&E)  33  11. U t e r u s , A d u l t D i e s t r u s (40X, H&E)  33  12 . U t e r u s , 12Oh +4IU PMSG (40X, H&E)  34  13 . Endometrium, 120h +4IU PMSG (400x, H&E)  34  14 . U t e r u s , 216h +4IU PMSG (40X, H&E)  34  15. Endometrium, 216h + 4IU PMSG (400X, H&E)  34  16. U t e r u s , 24Oh +4IU PMSG (40X, H&E)  35  17. Endometrium, 240h +4IU PMSG ( 4 0 0 X , H&E)  35  18. U t e r u s , 48h ^-20IU PMSG (40X, H&E)  35  19. Endometrium, 48h +20IU PMSG (400x, H&E)  35  20. U t e r u s , 72h +20IU PMSG (40X, H&E)  36  21. Endometrium, 72h +20IU PMSG (400x, H&E)  36  22 . U t e r u s , 96h +2 0IU PMSG (40X, H&E)  36  vi  L i s t of Figures  Page #  23 . Endometrium, 96h +20IU PMSG (400X, H&E)  36  24. U t e r u s , 144h +2 0IU PMSG (40X , H&E)  37  25. Endometrium, 144h +2 0IU PMSG (400x, H&E)  37  26. Endometrium, 144h +20IU PMSG (400x, AB 2 ..5/PAS/D)  37  27 . U t e r u s 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. U t e r u s , 240h +2 0IU PMSG (40x , H&E)  38  31. U t e r u s , 48h +4 0IU PMSG (40X, H&E)  39  32 . Endometrium, 4 8h +4 0IU :PMSG (400X, H&E)  39  33 . U t e r u s , 72h +4 0IU PMSG (40x, H&E)  39  34 . U t e r u s , 12Oh +4 0IU PMSG (40X , H&E)  40  35. Endometrium, 120h +40IU PMSG (400x, H&E)  40  36. U t e r u s , 144h +40IU PMSG (40x , H&E)  40  37. Endometrium, 144h +40IU PMSG (400x, H&E)  40  38 . Endometrium, 144h +40IU PMSG (400x, AB 2.5/PAS/D)  41  39 . U t e r u s , 168h +40IU PMSG (40x , H&E)  41  40. U t e r u s , 192h +4 0IU PMSG (40x , H&E)  41  41. U t e r u s , 24Oh +4 0IU PMSG (40x , H&E)  42  42. Endometrium, 240h +40IU PMSG (400x, H&E)  42  vii  L i s t of Figures  Page #  43. P e r i m e t e r (mm) o f t h 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, 20 and 40 IU PMSG.  43  44. M i t o t i c index, expressed i n percent, 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.  44  45. N e u t r o p h i l counts o f the 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.  45  46. Vagina, 48h +4IU PMSG (40x, H&E)  49  47. Vagina, 48h +4IU PMSG (40x, AB 2.5/ PAS/D)  49  48. Vagina, A d u l t P r o e s t r u s (40x, H&E)  49  49. Vagina, A d u l t E s t r u s (40x, H&E)  49  50. Vagina, 72h +4IU PMSG (40x, H&E)  50  51. Vagina, 96h +4IU PMSG (40x, H&E)  50  52. Vagina, A d u l t Metestrus  50  (40x, H&E)  53. Vagina, A d u l t D i e s t r u s (40x, H&E)  50  54. Vagina, 120h +4IU PMSG (40x, H&E)  51  55. Vagina, 120h +4IU PMSG (40x, AB 2.5/ PAS/D)  51  56. Vagina, 240h +4IU PMSG (40x, H&E)  51  57. Vagina, 240h +4IU PMSG (40x, AB 2.5/ PAS/D)  51  58. Vagina, 48h +20IU PMSG (40x, H&E)  52  59. Vagina, 72h +20IU PMSG (40x, H&E)  52  viii  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, 120h + 20IU PMSG (40X, AB 2 .5/ PAS/D)  53  63 . Vagina, 144h + 20IU PMSG (40x, H&E)  53  64 . Vagina, 144h +20IU PMSG (40x, AB 2 .5/ PAS/D)  53  65. Vagina, 216h +20IU PMSG (40x, H&E)  54  66. Vagina, 216h +20IU PMSG (40x, AB 2 .5/ PAS/D)  54  67. Vagina, 120h + 40IU PMSG (40x, H&E)  54  68 . Vagina, 120h + 40IU PMSG (40x, AB 2 -.5/ PAS/D)  54  69. Vagina, 168h + 40IU PMSG (40x, H&E)  55  70. Vagina, 168h +40IU PMSG (40x, AB 2 .5/ PAS/D)  55  71. Vagina, 240h + 40IU PMSG (40x, H&E)  55  72 . Vagina, 240h +40IU PMSG (40x, AB 2 .5/ PAS/D)  55  73. Schematic 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 treatment w i t h 4, 20 and 40 IU PMSG.  56  74. 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 treatment w i t h 4, 20 and 40 IU PMSG.  59  ix  Abbreviations AB  A l c i a n Blue 8 G x  CI  C o l o r Index  D  Diastase  DE  Diestrus  DEG  Degenerate  E  Estrus  ET  Embryo T r a n s f e r  FSH  F o l l i c l e S t i m u l a t i n g Hormone  HCG  Human C h o r i o n i c  H&E  Hematoxylin and E o s i n  IU  International  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  L u t e i n i z i n g Hormone R e l e a s i n g  LPE  Late  ME  Metestrus  MUC  Mucification  PAS  Periodic Acid  PE  Proestrus  PMSG  Pregnant Mare Serum Gonadotrophin  SEC  Secretory  Gonadotrophin  Units  Hormone  Proestrus  Schiff  X  Acknowledgements I would l i k e extend my deepest g r a t i t u d e t o everyone made t h i s p r o j e c t p o s s i b l e .  who  V a l u a b l e a d v i c e on  experimental d e s i g n and i n t e r p r e t a t i o n was p r o v i d e d by Dr. Y. S. Moon, Dr. J . E. Dimmick, Dr. G. P. T a y l o r , Dr. Y. W.  Yun,  Dr. P. Cagle, Dr. E. Jones, Dr. P. E. R e i d , Dr. B. Ho Yuen, Dr. J . T. Emerman, Dr. H. P r i t c h a r d and Dr. N. Auersperg. S k i l l f u l t e c h n i c a l a s s i s t a n c e was  o f f e r e d by Ms.  Mrs. Barbara Barkoczy, Mr. John Leung, Ms.  N a t a l i e Mari,  Susan B l a i r and a l l  the t e c h n o l o g i s t s i n the d i v i s i o n o f Anatomical Pathology, Department o f Pathology, C h i l d r e n ' s H o s p i t a l .  I would a l s o  l i k e t o thank Mrs. C a r o l Ford, Mrs. Anne S i n c l a i r ,  Dan,  Cathy and J e n n i f e r a t the the Research Centre f o r t h e i r s p e c i a l h e l p , p a t i e n c e and g e n e r o s i t y .  xi  Kate,  Introduction I Objective The  impairment  of u t e r i n e s t r u c t u r e and  function  f o l l o w i n g s u p e r o v u l a t o r y treatment w i t h exogenous gonadotrophins  i s p o o r l y understood.  Superovulation i s a  w i d e l y a c c e p t e d technique i n s y n c h r o n i z i n g and i n d u c i n g o v u l a t i o n , o r i n c r e a s i n g the number of o v u l a t i o n s . e f f e c t s are  As these  achieved through f o l l i c u l a r h y p e r s t i m u l a t i o n , i t  i s not s u r p r i s i n g t h a t the d e l i c a t e balance of o v a r i a n s t e r o i d (estrogen, progesterone, androgens) s e c r e t i o n r a t e s i s disrupted.  The e f f e c t s of t h i s d i s r u p t i o n on  sensitive  t i s s u e s , such as the u t e r u s , have not been thoroughly s t u d i e d . Thus, the purpose  of the p r e s e n t study was  knowledge through the examination  to gain  of the e f f e c t s of  s u p e r o v u l a t o r y i n d u c t i o n , u s i n g pregnant mare serum gonadotrophin  (PMSG), on endometrial morphology and  histology.  The changes i n serum s t e r o i d l e v e l s and v a g i n a l h i s t o l o g y were a l s o examined t o a i d i n the i n t e r p r e t a t i o n of these endometrial  effects.  I I Human In V i t r o F e r t i l i z a t i o n and Embryo T r a n s f e r In v i t r o f e r t i l i z a t i o n  (IVF) and embryo t r a n s f e r  (ET)  p r o v i d e a means of e s t a b l i s h i n g pregnancies, a l b e i t w i t h modest s u c c e s s , under a wide range of adverse c o n d i t i o n s such as t u b a l i n f e r t i l i t y , immunological  endometriosis, terato-oligospermia,  and i d i o p a t h i c i n f e r t i l i t y  (Edwards 1984).  D e s p i t e a c h i e v i n g mean r a t e s 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 o b t a i n e d  r a t e s up t o 15-20% per embryo t r a n s f e r (Edwards  pregnancy  1984,  B l a n k s t e i n 1986). The  e t i o l o g i e s r e s p o n s i b l e f o r t h i s h i g h wastage r a t e  f o l l o w i n g embryo t r a n s f e r are u n c e r t a i n and p o o r l y understood. Chromosomal a b n o r m a l i t i e s i n embryos, induced by . s u p e r o v u l a t i o n or a r i s i n g from growth i n v i t r o , may  be  r e s p o n s i b l e f o r r e t a r d e d embryo development and death d u r i n g e a r l y pregnancy  (Steptoe e t a l . 1980,  Frydman 1982).  However,  because o f the few o p p o r t u n i t i e s a v a i l a b l e t o karyotype p r i o r t o t r a n s f e r and the d i f f i c u l t y i n o b t a i n i n g embryos i n the process  o f b e i n g e x p e l l e d or resorbed,  i t i s uncertain  how  c o n t r i b u t o r y chromosomal imbalances or o t h e r o c c u l t d e f e c t s may  be. D i s o r d e r s i n the l u t e a l phase or the u t e r u s , which would  i n t e r f e r e w i t h and p r e c l u d e p o t e n t i a l l y important Successes i n cases,  i m p l a n t a t i o n , are  causes of e a r l y a b o r t i o n  i n v o l v i n g t r a n s f e r of  other (Edwards 1984).  cryopreserved  embryos t o a subsequent n a t u r a l or l i g h t l y s t i m u l a t e d c y c l e , have been r e p o r t e d t o be double those of f r e s h t r a n s f e r s ( B l a n k s t e i n 1986,  T e s t a r t 1987), i n d i c a t i n g the presence of a  t r a n s i e n t d e l e t e r i o u s e f f e c t upon u t e r i n e r e c e p t i v i t y . Elevated estrogen  l e v e l s , secondary t o s u p e r o v u l a t i o n , may  r e s p o n s i b l e f o r t h i s d e l e t e r i o u s e f f e c t by p r o l o n g i n g  2  the  be  the u t e r u s .  I n a r a r e study, b i o p s i e s o f endometria from  unsuccessful  IVF/ET c y c l e s were found t o d i s p l a y an  advancement o f 1-3 days (Jones 1984), s u g g e s t i n g desynchronization  that the  o f t h e t i m i n g o f optimum u t e r i n e r e c e p t i v i t y  w i t h embryo age and development may have l e d t o i m p l a n t a t i o n f a i l u r e or early abortion.  More d i r e c t evidence w i l l be  r e q u i r e d on t h e d e l e t e r i o u s e f f e c t s o f s u p e r o v u l a t i o n the r o l e o f t h e u t e r u s properly  before  i n e a r l y embryonic death can be  assessed.  Thus, t h e r e a r e s e v e r a l d e f i c i e n c i e s i n t h e p r e s e n t u n d e r s t a n d i n g o f t h e mechanisms o p e r a t i v e superovulation  following  i n human IVF and ET c y c l e s and t h i s  t h a t t h e study o f an a p p r o p r i a t e  indicates  animal model has t h e  p o t e n t i a l t o extend our knowledge. Ill  Ovulation The  and Pregnancy i n t h e PMSG Treated  PMSG t r e a t e d immature r a t i s w e l l s u i t e d f o r t h e  study o f s u p e r o v u l a t i o n possessing  and has been w i d e l y employed.  PMSG,  both FSH ( f o l l i c u l a r s t i m u l a t i n g 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 , (Papkoff  Immature Rat  1974).  PMSG p r e p a r a t i o n ,  i s a glycoprotein  The FSH:LH a c t i v i t y r a t i o , dependent upon t h e t h e assay and t h e time o f i s o l a t i o n  during  pregnancy, has been estimated between 0.87-1.92 (Stewart e t al.  1976).  L i k e other p i t u i t a r y g l y c o p r o t e i n hormones, PMSG  c o n s i s t s o f a hormone s p e c i f i c subunit  (PMSG-B), s i m i l a r i n  amino a c i d composition t o LH-B, and a hormonally n o n - s p e c i f i c subunit  (PMSG-a) (Papkoff  e t a l . 1978). 3  The h i g h s i a l i c  acid  content  (45%)  accounts f o r t h e long h a l f l i f e  (60 h) and t h e  slow c l e a r a n c e r a t e o f PMSG may be r e s p o n s i b l e f o r t h e potency t h a t can be achieved  i n a s i n g l e dose ( A u s t i n 1950,  N u t i e t a l . 1975, Papkoff 1981).  Immature r a t s t r e a t e d w i t h  low doses o f PMSG (4-8 IU) mimic t h e p h y s i o l o g i c a l events o f a d u l t c y c l i n g r a t s : s t e r o i d and gonadotrophin hormone s e c r e t i o n r a t e s , u t e r i n e weight, and v a g i n a l smear changes d u r i n g o v u l a t i o n and pregnancy as a d u l t s (Wilson e t a l . 1974, N u t i e t a l . 1975, Parker e t a l . 1976, Taya e t a l . 1981). However, immature r a t s , when superovulated  w i t h moderate (16-  24 IU) t o h i g h doses o f PMSG (30-50 IU), w i l l y i e l d  greater  numbers o f r e t r i e v a b l e oocytes  (Cole  1937,  compared w i t h a d u l t s  A u s t i n 1950, Zarrow and Quinn 1963, Y i n g e t a l . 1969).  In summary, due t o i t s convenience, s u p e r o v u l a t o r y and  response  r e p r o d u c t i v e performance, t h e PMSG t r e a t e d immature r a t  i s an i d e a l model f o r s u p e r o v u l a t o r y  research.  PMSG and O v u l a t i o n O v u l a t i o n , beginning  60 h post a d m i n i s t r a t i o n o f low o r  c o n t r o l doses o f PMSG (4-8 IU), y i e l d s 8-12 oocytes  over a 12  h p e r i o d , s i m i l a r t o a d u l t s (Wilson e t a l . 1974, Walton e t a l . 1983, et  Walton and Armstrong 1983, Evans and Armstrong 1984, Yun  a l . 1987).  superovulatory  Moderate (16-24 IU) t o h i g h  (30-50 IU)  doses, however, i n i t i a t e o v u l a t i o n s w i t h i n 24 h  o f a d m i n i s t r a t i o n o f PMSG.  The o v u l a t i o n p e r i o d i s l o n g e r (50  4  h) as w e l l .  In addition, the ovulation rate following  s u p e r o v u l a t i o n undergoes s t r i k i n g changes, i n c o n t r a s t w i t h the steady  i n c r e a s e observed i n c o n t r o l s .  I n i t i a l l y , the  o v u l a t i o n r a t e s l o w l y r i s e s t o a p l a t e a u by 36 h: 8 ova (20 IU) , 18 ova (40 IU).  The l e n g t h o f t h i s p l a t e a u may v a r y  with  dose: 12 h (20 IU), 24 h (40 IU), b u t i n v a r i a b l y a sharp i n c r e a s e i n o v u l a t i o n s w i l l f o l l o w : 52 ova/12 h (20 IU), 32 ova/12 h (4 0 IU) (Yun e t a l . 1987).  Thus, i n c r e a s e d  s t i m u l a t i o n w i t h h i g h e r doses o f PMSG w i l l not merely r e s u l t i n a p r o p o r t i o n a t e and equal e l e v a t i o n o f t h e o v u l a t i o n response, b u t a more complex and anomalous p a t t e r n . Estrogen  and progesterone s e c r e t i o n p a t t e r n s i n c o n t r o l s  a r e e q u i v a l e n t t o those d u r i n g t h e a d u l t e s t r o u s c y c l e , i f one uses v a g i n a l smears an i n d i c a t o r o f t h e stage o f t h e c y c l e : 48 h p o s t PMSG-proestrus; 72 h - e s t r u s ; 96 h - m e t e s t r u s / d i e s t r u s ; 120  h-diestrus etc.  Parker levels,  (Wilson e t a l . 1974, N u t i e t a l . 1975,  e t a l . 1976, M i l l e r and Armstrong 1981a). f o l l o w i n g moderate s u p e r o v u l a t o r y  PMSG), do n o t d i f f e r g r e a t l y from those  Progesterone  i n d u c t i o n (20 IU  i n c o n t r o l s ; however,  1 7 B - e s t r a d i o l l e v e l s may i n c r e a s e up t o t h r e e f o l d o f c o n t r o l s by 60 h (Yun e t a l . 1987). maternal endocrine PMSG doses.  Severe d i s r u p t i o n o f t h e  environment i s more e v i d e n t w i t h  higher  In 40 IU PMSG t r e a t e d r a t s , 1 7 B - e s t r a d i o l  levels  may i n c r e a s e t w o - f o l d those o f c o n t r o l s a t 48-60 h and again a t 96 h, w i t h progesterone l e v e l s r i s i n g up t o f o u r times as much as c o n t r o l l e v e l s by 144 h ( M i l l e r and Armstrong 1981b, 5  Walton and Armstrong 1981, Yun e t a l . 1987). PMSG has been hypothesized  t o a c t both d i r e c t l y and  i n d i r e c t l y i n i n d u c i n g o v u l a t i o n (Yun e t a l . 1987). Superovulatory  doses, v i a t h e FSH-moiety o f PMSG, may d i r e c t l y  i n i t i a t e the maturation of preovulatory  follicles  the g r e a t e s t degree o f r e c e p t o r d i f f e r e n t i a t i o n 1980) .  I f the hypothalamic-pituitary-ovarian  possessing  (Moore e t a l .  axis i s intact,  one would expect s i g n i f i c a n t e l e v a t i o n s o f LH, p r o g e s t e r o n e and  estrogen  a t t h e time o f o v u l a t i o n (Short 1984, P a s q u a l i n i  e t a l . 1985).  The absence o f these e l e v a t i o n s i n t h e f i r s t  s e t o f o v u l a t i o n s , 12-36 h f o l l o w i n g 20-40 IU PMSG, suggests t h a t PMSG i n i t i a l l y  a c t s d i r e c t l y on t h e 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 l a r g e r second s e t o f o v u l a t i o n s 48-72 h a f t e r superovulatory  induction.  of s u p e r o v u l a t i o n PMSG treatment  Longitudinal studies of the effects  have i d e n t i f i e d a LH surge 48-57 h f o l l o w i n g  (Zarrow and Quinn 1963, Wu and Meyer 1966).  a d d i t i o n , s t u d i e s u s i n g v a r i o u s manipulations system depressants, critical 1962,  (central  In  nervous  hypophysectomy) i n d i c a t e t h i s surge i s  t o t h e completion o f o v u l a t i o n (Strauss and Meyer  S o r r e n t i n o e t a l . 1972).  Basal s e c r e t i o n s o f estrogens  and p r o g e s t e r o n e , produced from t h e f i r s t may s y n e r g i s t i c a l l y  set of ovulations,  s t i m u l a t e t h i s LH surge through t h e  r e d u c t i o n o f t h r e s h o l d l e v e l s a t hypophyseal and hypothalamic centres  (Kawakami and Sawyer 1959, Ying and Meyer 1969, Hagino  6  and G o l d z i e h e r 1970).  Low doses o f PMSG (4-8 IU) p r o b a b l y  a l s o s t i m u l a t e o v u l a t i o n by t h i s i n d i r e c t mechanism.  Time  c o u r s e s t u d i e s o f gonadotrophin and s t e r o i d s e c r e t i o n  rates  i n d i c a t e t h a t low doses, although inadequate i n d i r e c t l y s t i m u l a t i n g o v u l a t i o n , a r e a b l e t o i n i t i a t e endogenous LH r e l e a s e i n t h e same time frame as s u p e r o v u l a t o r y doses through f o l l i c u l a r m a t u r a t i o n and s t e r o i d o g e n e s i s  (Wilson e t a l .  1974,  P a r k e r e t a l . 1976). PMSG and E a r l y  Pregnancy  E a r l y pregnancy i n 4 IU PMSG t r e a t e d immature r a t s proceeds a l o n g a s i m i l a r time course as normal a d u l t s w i t h n e g l i g i b l e embryo l o s s e s al.  1984) .  ( M i l l e r and Armstrong 1981a, Yun e t  By day 5 o f pregnancy, a l l zygotes (7-8/rat) have  implanted i n t h e u t e r u s i n 85-100% o f a l l r a t s mated and Armstrong  1981a, Walton and Armstrong  (Miller  1981, 1982) . I n  a d d i t i o n , t h e s e implants a r e c a r r i e d t o term w i t h no s i g n i f i c a n t f e t a l wastage o r l o s s Walton and Armstrong Moderate  ( M i l l e r and Armstrong  1981a,  1982).  s u p e r o v u l a t o r y (16-2 0 IU) i n d u c t i o n , however,  may cause s i g n i f i c a n t p r e - i m p l a n t a t i o n l o s s e s o f embryos and d e l a y i m p l a n t a t i o n o f t h e s u r v i v i n g embryos by as much as two t o s i x days.  Yun e t a l . (1984), s t u d y i n g t h e e f f e c t s o f 20 IU  PMSG, found no evidence o f i m p l a n t a t i o n on day 5 o f pregnancy, but was a b l e t o r e t r i e v e 5-6 embryos/rat from t h e o v i d u c t and 1-2 degenerate appearing morulae/rat from t h e u t e r u s .  I t was  not u n t i l day 7 o f pregnancy t h a t he found any i m p l a n t a t i o n 7  sites  (5-7 s i t e s / r a t ) .  M i l l e r and Armstrong (1981a), u s i n g 16  IU PMSG, estimated, by day 20 o f pregnancy f e t a l weights ^, t h e d e l a y t o b e as g r e a t as 3-6 d i n as many as 38% o f a l l t  mated r a t s . F o l l o w i n g h i g h dose i n d u c t i o n , except f o r t h e o c c a s i o n a l degenerate zygote (2-3%) recovered from t h e oviduct  (Walton and Armstrong 1981, Yun e t 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, d e s p i t e a f e r t i l i z a t i o n r a t e o f 50-60% o f ova w i t h no s i g n s of degeneration  ( M i l l e r and Armstrong 1981b, Walton and  Armstrong 1982, Walton e t a l .  1983, Yun e t a l .  1984).  Oocyte d e f e c t s may i n t e r f e r e with normal embryo development and perhaps be a s s o c i a t e d w i t h t h e p r e i m p l a n t a t i o n l o s s e s o f embryos.  While v e r y few degenerate  (ooplasmic c o n t r a c t i o n , n u c l e a r fragmentation, p a r t h e n o g e n e s i s ) ova r e s u l t from 4 IU PMSG i n d u c t i o n - 0-1.5% and Armstrong 1981a, Walton e t a l .  1983, Yun e t a l .  (Miller  1987),  y i e l d s a f t e r s u p e r o v u l a t i o n i n c r e a s e dose dependently: 2 0 IU17%; 40 IU-40% (Yun e t a l .  1987).  S u p e r o v u l a t i o n may induce r e l e a s e o f degenerate oocytes  1  F e t a l development, f o l l o w i n g delayed i m p l a n t a t i o n , proceeds  a t a f i x e d r a t e ; t h e r e f o r e a low end o f term (day 2 0 o f pregnancy) weight i s a r e f l e c t i o n o f t h e e x t e n t o f t h e d e l a y and not o f t h e growth r e t a r d a t i o n  8  (Hooverman e t a l .  1970).  from abnormal f o l l i c l e s  (Fleming 1982) o r perhaps rescue l a r g e  antral f o l l i c l e s destined for atresia Severe  (Sherman e t a l . 1982).  i n t r a f o l l i c u l a r degeneration has been observed  as e a r l y  as 48 h a f t e r 20 o r 40 IU PMSG a d m i n i s t r a t i o n (Yun e t a l . 1987) .  Increased i n c i d e n c e o f p o l y p l o i d y and o t h e r  chromosomal a b n o r m a l i t i e s may a l s o be important 1974,  Mauldin and F r a s e r 1977).  however, cannot account embryos.  (Fujimoto  These oocyte d e f e c t s alone,  for a l l pre-implantation losses of  M i l l e r and Armstrong (1982) o v a r i e c t o m i z e d 40 IU  PMSG t r e a t e d r a t s 74-76 h post i n j e c t i o n  ( a f t e r o v u l a t i o n ) and  a c h i e v e d comparable f e t a l r e c o v e r i e s and i m p l a n t a t i o n s i t e weights  as c o n t r o l s on day 10 o f pregnancy.  Walton and Armstrong  (1983) observed  In a d d i t i o n ,  equal f e t a l  recovery  r a t e s between c o n t r o l s and 40 IU PMSG t r e a t e d r a t s f o l l o w i n g 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 t r a n s f e r i n t o LHRH (LH r e l e a s i n g hormone) s y n c h r o n i z e d mated a d u l t s . These r e s u l t s suggest t h a t the oocytes a r e capable o f 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 o f 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 t h e o v i d u c t .  A c c e l e r a t e d embryo  t r a n s p o r t has been found t o be r e s p o n s i b l e f o r e x p u l s i o n i n e s t r o g e n t r e a t e d r a t s and s u p e r o v u l a t e d r a b b i t s  (Greenwald  1961,  e t a l . 1981).  Bennett  1970, O r t i z e t a l . 1979, Tsutsumi  Moreover, i t has been shown i n r a b b i t s and mice t h a t h i g h  9  e s t r o g e n l e v e l s induce o v i d u c t a l s e c r e t i o n o f a low MW substance t h a t i n h i b i t s development o f t h e embryo t o t h e morula  (Stone and Hammer 1977,  C l i n e e t a l . 1977).  IV PMSG Induced U t e r i n e E f f e c t s Recent evidence suggests t h a t s u p e r o v u l a t o r y w i t h PMSG a d v e r s e l y  induction  a f f e c t s u t e r i n e s t r u c t u r e and f u n c t i o n i n  immature r a t s . ' PMSG appears t o induce abnormal e n d o m e t r i a l and myometrial changes, which may be r e s p o n s i b l e  f o r the  i n c r e a s e d u t e r i n e s i z e a l s o observed a f t e r PMSG treatment. Working w i t h mated and non-mated 4 0 IU PMSG t r e a t e d r a t s , M i l l e r e t a l . (1981b) found t h a t wet u t e r i n e weight over 2 00%  increased  o f c o n t r o l (4 IU PMSG) weights between day 1  o f pregnancy  (or 72-96 h post  u n t i l day 5 o f pregnancy.  i n j e c t i o n ) and remained  2  and 2  elevated  Moreover, as e a r l y as day 2 o f  pregnancy they began o b s e r v i n g  desquamated c e l l u l a r d e b r i s i n  u t e r i n e lavages from both mated and non-mated  superovulated  r a t s , which advanced p r o g r e s s i v e l y such t h a t by day 3 they were r e c o v e r i n g  s i g n i f i c a n t amounts o f desquamated  hyperplastic epithelia. were completely n e g a t i v e .  2  In c o n t r a s t , c o n t r o l u t e r i n e  lavages  In a d d i t i o n t o e p i t h e l i a l  To a c h i e v e mating, male caging  i s conducted 60-74 h p o s t  PMSG i n j e c t i o n (on t h e n i g h t o f p r o e s t r u s ) .  Mating success i s  a s s e s s e d by a sperm p o s i t i v e v a g i n a l smear t h e morning o f the next day,  r e f e r r e d t o here as day 1 o f pregnancy. 10  p r o l i f e r a t i o n , t h e i n c r e a s e d u t e r i n e weight f o l l o w i n g superovulatory changes.  doses o f PMSG may be due t o abnormal myometrial  Rennels (1951), u s i n g 30 IU PMSG/20 IU HCG, a l s o  observed a marked i n c r e a s e i n u t e r i n e weight and proposed t h i s was  due t o a s i g n i f i c a n t hypertrophy o f t h e i n n e r  muscularis  of t h e myometrium, b e g i n n i n g 48 h a f t e r treatment. I t i s important t o note t h a t , i n a d d i t i o n t o s t r u c t u r a l f i n d i n g s , no d i f f e r e n c e s have been observed i n 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 hormone p r o f i l e s between mated and non-mated  4 0 IU t r e a t e d r a t s ( M i l l e r and Armstrong 1981b, Walton and Armstrong 1981). has  Mating i s an important c o n s i d e r a t i o n as i t  been w e l l e s t a b l i s h e d t h a t repeated tapping  o f c e r v i x , as  might occur d u r i n g c o p u l a t i o n , may i n i t i a t e pseudopregnancy i n the a d u l t 1970,  (Long and Evans 1922, DeFeo 1963, Yoshinaga e t a l .  Pepe e t a l . 1974, Redmond e t a l . 1986) and t h e 4 IU PMSG  t r e a t e d r a t (Nuti e t a l . 1975).  Pseudopregnancy has been  c h a r a c t e r i z e d t o be a c o n d i t i o n mimicking t h e f i r s t pregnancy w i t h r e g a r d  12 days o f  t o a l l hormone p r o f i l e s , v a g i n a l and  u t e r i n e h i s t o l o g y and t h e onset o f u t e r i n e r e c e p t i v i t y .  Thus,  s i m i l a r i t y i n f i n d i n g s between mated and non-mated r a t s suggests t h a t t h e u n d e r l y i n g  changes as a r e s u l t o f mating a r e  p r o b a b l y n o t important and t h a t non-mated r a t s a r e a c c e p t a b l e f o r a t l e a s t t h e study o f u t e r i n e Studies inductions  effects.  i n v o l v i n g embryo t r a n s f e r s o r d e c i d u a l  reaction  on day 5 o f pregnancy o f PMSG t r e a t e d immature r a t s  have p r o v i d e d  d i r e c t evidence t h a t PMSG impairs 11  uterine  f u n c t i o n t o t h e p o i n t where t h e endometrium to  implant an embryo.  embryo and endometrium  i s no l o n g e r a b l e  Implantation i s the r e s u l t of the  0  simultaneously reaching s p e c i f i c stages  i n t h e i r development by t h e time they come i n t o c o n t a c t w i t h each o t h e r  (Noyes e t a l . 1960).  I n 4 IU PMSG t r e a t e d pregnant  r a t s t h e i m p l a n t a t i o n o f n e a r l y a l l o f t h e oocytes o v u l a t e d (80-90%)  i n t h e endometrium  pregnancy 1981,  s u c c e s s f u l l y occurs on day 5 o f  ( M i l l e r and Armstrong 1981a, Walton and Armstrong  1982, Yun e t a l . 1984).  S e v e r a l authors have r e p o r t e d  comparable i m p l a n t a t i o n r a t e s f o l l o w i n g synchronous embryo t r a n s f e r s between 4 IU PMSG t r e a t e d pregnant r a t s on day 5 o f pregnancy, i n d i c a t i n g n o r m a l i t y o f the embryo and t h e endometrium In  ( M i l l e r and Armstrong 1981b, Walton e t a l .  1982).  c o n t r a s t , i t has been found t h a t s i m i l a r l y conducted  t r a n s f e r s from 4 IU PMSG t r e a t e d donors t o 4 0 IU PMSG t r e a t e d r e c i p i e n t s r e s u l t i n no i m p l a n t a t i o n s 1981b).  ( M i l l e r and Armstrong  In rodents t h e c a p a b i l i t y o f t h e endometrium t o  implant an embryo can be a s s e s s e d by t h e d e c i d u a l response e l i c i t e d by a t r a u m a t i c s t i m u l u s on the anti-mesometrial  side  ( A l l e n 1931, K h r i e b e l 1937, DeFeo 1963, Shelesnyak e t a l . 1963, Yoshinaga e t a l . 1971).  In embryo t r a n s f e r s t u d i e s  ( M i l l e r and Armstrong 1981b), w h i l e trauma induced deciduomata were observed a t t h e embryo t r a n s f e r p o i n t i n 4 IU PMSG t r e a t e d r a t s , none were observed i n 40 IU PMSG t r e a t e d ( M i l l e r and Armstrong 1981b).  12  rats  Similar d i f f i c u l t y i n inducing  a normal d e c i d u a l response has a l s o been r e p o r t e d f o r pseudopregnant immature r a t s t r e a t e d with 50 IU PMSG e t a l . 1964).  (Guillet  These f u n c t i o n a l study r e s u l t s t o g e t h e r  with  the s t r u c t u r a l f i n d i n g s suggests t h a t h i g h doses o f PMSG induce  abnormal changes, some manifested  prevent  the uterus  s t r u c t u r a l l y , which  from r e a c h i n g a r e c e p t i v e s t a t e .  Hormonal s t u d i e s o f i m p l a n t a t i o n imply t h a t e l e v a t e d estrogen  l e v e l s i n r a t s t r e a t e d with h i g h doses o f PMSG,  d u r i n g t h e p r e - i m p l a n t a t i o n p e r i o d , may d e t r i m e n t a l l y a f f e c t uterine sensitivity.  In a d u l t r a t s , t h r e e 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 t h e onset, and magnitude o f u t e r i n e s e n s i t i v i t y : during the proestrus;  surge  (2) s l o w l y r i s i n g l e v e l s o f p r o g e s t e r o n e  between o v u l a t i o n and i m p l a n t a t i o n ; surge t h e day b e f o r e  (1) e s t r o g e n  duration  implantation  (3) p r e - n i d a t o r y  estrogen  (day 5 o f pregnancy)(Yochim  e t a l . 1963, DeFeo 1967, Psychoyos 1973, A i t k e n 1978, F i n n 1980).  These workers a l s o found t h a t t h e d i s t u r b a n c e  p a t t e r n w i t h exogenous o v a r i a n s t e r o i d s , e s p e c i a l l y c o u l d reduce o r a b o l i s h u t e r i n e s e n s i t i v i t y .  of t h i s  estrogens,  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 , a b o l i s h e d u t e r i n e and o v a r i a n weight i n c r e a s e s and reduced t h e 1 7 B - e s t r a d i o l surge a t 96 h, w h i l e n o t i n f l u e n c i n g progesterone l e v e l s . still  h i g h e r than those o f c o n t r o l s , the treatment w i t h  sera s t i l l 47%  Even though 1 7 B - e s t r a d i o l l e v e l s were  allowed  anti-  successful induction of d e c i d u a l i z a t i o n i n  o f a l l mated r a t s (versus 0% f o r r a t s not t r e a t e d ) . 13  As  t h e primary  (Papkoff during  t a r g e t t i s s u e o f PMSG i s t h e o v a r y  1 9 8 1 ) , s t e r o i d hormones o t h e r t h a n  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 ,  a s s o c i a t e d w i t h t h e abnormal e f f e c t s  c o n t r o l s have been i d e n t i f i e d  (Walton  a n d A r m s t r o n g 1981, M i l l e r  These e l e v a t e d l e v e l s ,  adult  o f exogenously  rats  just prior  estrogen shortened  sensitivity,  while  1981b).  Decidual studies o fthe i n pseudopregnant with  increased  the period of uterine  increased progesterone  alone had t h e  b u t i n a l l c a s e s t h e r e was no a f f e c t  onset  o r magnitude o f t h e d e c i d u a l response  1963,  DeFeo 1 9 6 7 ) .  h post  compared  t o implantation  and Armstrong  administered steroids  or decreased  60  I n t h e 40  of progesterone  revealed that increased progesterone  reverse effect,  may b e  however, may n o t b e r e s p o n s i b l e f o r  abolishing uterine sensitivity. effects  produced  i n the uterus.  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 with  estrogen,  Elevated levels  upon t h e  (Yochim e t a l .  o f androgens observed  48-  40 a n d 20 I U PMSG a d m i n i s t r a t i o n (Yun e t a l . 1987)  may b e i m p o r t a n t  by themselves  or i n relationship  with  estrogens.  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  administered  aromatizable  ( t e s t o s t e r o n e ) o r non-  aromatizable  ( 5 a - d i h y d r o t e s t o s t e r o n e ) androgens c a n i n d i r e c t l y  or d i r e c t l y ,  respectively,  (Brooksby  induce u t e r i n e  hypertrophy  1938, Salmon 1938, K o r e n c h e v s k y e t a l . 1936,  M c D o n a l d a n d D o u g h t y 1974, A r m s t r o n g e t a l . 1 9 7 6 ) . Furthermore,  a r o m a t i z a b l e androgens, through  14  p e r i p h e r a l and  ovarian conversion to estrogen, the a f f e c t s  of  may p o t e n t i a t e o r  e x i s t i n g h i g h serum l e v e l s  15  of  exaggerate  estrogens.  M a t e r i a l s and Methods I Animals and M a t e r i a l s Female Sprague Dawley r a t s  (Charles R i v e r Canada Inc.,  S t . Constant, Que.) were o b t a i n e d a t 25 d and a t 2 mo o f age.  A l l animals were housed  c o n d i t i o n s o f temperature  f o r 3 days under  controlled  (20°C) and i l l u m i n a t i o n  (12L.12D)  p r i o r t o e i t h e r PMSG treatment o r d e t e r m i n a t i o n o f v a g i n a l cytology.  PMSG (Equinex, A y e r s t , Montreal) was a d m i n i s t e r e d  i n a 4, 2 0 o r 4 0 IU dose v i a a subcutaneous d o r s a l i n 0.2 ml 0.9% NaCl between 0930 and 1130 h.  injection  Standard r a t  chow and water were a v a i l a b l e ad l i b i t u m throughout t h e treatment p e r i o d . ' Hematoxylin  • (C.I.75290), e o s i n  (C.I.45380), d i a s t a s e (D)  (mixed a and B amylase), p e r i o d i c a c i d and s c h i f f reagent (PAS) were purchased from BDH Chemicals L t d . and a l c i a n b l u e 8GX  (AB) (C.I.74240) from Sigma Chemical Co. (St. L o u i s , MO.). A n t i s e r a f o r 1 7 B - e s t r a d i o l , progesterone and androgen  radioimmunoassays  were p r o v i d e d c o u r t e s y o f Dr. D.T.  Armstrong, U n i v e r s i t y o f Western O n t a r i o .  The r e s p e c t i v e  c r o s s - r e a c t i v i t i e s o f t h e a n t i s e r a a r e shown as f o l l o w s : 1 7 B - E s t r a d i o l Antibody 17B-estradiol estrone estriol other s t e r o i d s  96.4% 2.9 0.5 <0.2  Progesterone Antibody progesterone 5B-pregnane-3,20-dione 16  44.1% 35.5  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 o t h e r s t e r o i d s <0.2 Androgen  Antibody  5 a - d i h y d r o t e s t o s t e r o n e 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 o t h e r s t e r o i d s <0.1 I I Methods Design Three separate s t u d i e s were conducted  t o determine t h e  e f f e c t s o f s u p e r o v u l a t o r y doses o f PMSG on t h e h i s t o l o g y o f t h e u t e r u s and t h e v a g i n a . and  The s h o r t term e f f e c t s o f 4, 20  4 0 IU PMSG were s t u d i e d i n immature r a t s from 48 t o 240 h  post i n j e c t i o n .  I n t h i s study 162 animals were d i v i d e d i n t o 6  animals p e r treatment 24 h a p a r t .  dose and s a c r i f i c e d a t 9 time  intervals,  I n a d d i t i o n , t h e hormonal s t a t u s o f 3 r a t s p e r  dose was examined a t 0, 24 and 60 h. The l o n g term e f f e c t s o f 4 and 40 IU PMSG were assessed i n 12 immature r a t s 3 0 days following administration.  In a d d i t i o n , c y c l i n g adult r a t s  were examined i n o r d e r t o p r o v i d e a r e f e r e n c e f o r induced c y c l i c a l changes i n immature r a t s .  Twelve a d u l t r a t s were  f o l l o w e d by v a g i n a l smears f o r one f u l l c y c l e and s a c r i f i c e d a t t h e a p p r o p r i a t e time t o achieve 3 r e p r e s e n t a t i v e animals per p r o e s t r u s , e s t r u s , metestrus  and d i e s t r u s phases.  The o r d e r o f s a c r i f i c e was randomized w i t h r e s p e c t t o treatment  order.  A t s a c r i f i c e , t h e animals were weighed, 17  anaesthetized  using  via  vein.  the  trunk  d i e t h y l e t h e r v a p o u r and The  blood  was  allowed  exsanguinated  to c l o t  for 4  h o u r s a t room t e m p e r a t u r e p r i o r t o c e n t r i f u g a t i o n f o r collection. analysis. small  The The  left  the  vagina  immediately 37%  made a t t h e  cornua was  buffered  s t o r e d a t -20°C u n t i l  reproductive  c r i m p was  the  s e r a was  a t the  with  i n Bouin's f i x a t i v e formalin-20%,  a l l t i s s u e were p l a c e d  excess  fixative  formalin until  (brain vs. Histology During  cassettes uterus mm  and  the  saggital  i n 10%  using  acid-75%, After 4  water t o  buffered  remove  neutral  C l e a r i n g and  double length  waxing  incubation  cycle).  m e s e n t e r i c t i s s u e was  r e m o v e d and  f o l l o w i n g segments b e f o r e  left  t o the  and  right  oviduct)  sides.  s e c t i o n s , the  n e x t 5 mm  discarded.  The  sections.  18  The  again vagina  placement first  n e x t 9 mm  into  2 mm  the  of  next  3  for  for traverse was  each  and  were d i s c a r d e d ,  f o r t r a v e r s e s e c t i o n s , the  r e s t was  placed  acetic acid-5%). tap  of  addition,  t i s s u e was  d i s s e c t e d from the p e r i o v a r i u m  denoting  were u s e d  In  a  Histochemistry  i n t o the  (proximal  saggital  and  fixation,  u t e r i n e h o r n was partitioned  junction  (saturated p i c r i c  processing.  c a r r i e d out routine  a l l other  i n running  were s t o r e d  histological  o f a l l t i s s u e was times  utero-tubal  glacial  hours,  and  steroid  t r a c t s were d i s s e c t e d w h o l e and  f o r i d e n t i f i c a t i o n purposes. removed and  sera  sections  f i x e d whole  for  From each segment of t i s s u e , 4 s e r i a l s e c t i o n s were taken f o r r o u t i n e H&E s e c t i o n s and  s t a i n i n g f o r t o t a l s of 24  4 v a g i n a l s e c t i o n s per r a t .  Vaginal  (5  um)  uterine  smears,  i n s t e a d of t i s s u e , were taken from h a l f of a l l r a t s which were t r e a t e d with 4 and and pH  192 1.0,  h.  s i d e ) and  s a c r i f i c e d between  In a d d i t i o n , h i s t o c h e m i s t r y  2.5/  sections  40 IU PMSG and  PAS  (5um)  (D/AB/PAS)) was  from the f i r s t  the  f o l l o w i n g ways: AB  The  regimen was  The  2.5/PAS; D/AB  t r e a t e d i n one  2.5/PAS; AB  2.5;  adapted from t h a t used by the Dept.  1. dewaxing and h y d r a t i o n 2. 2% d i a s t a s e - 30 min., 30°C 3 water - 2 min. 4. 1% or 3% g l a c i a l a c e t i c a c i d (pH 1.0 or 2.5) -  1.0.  of  Hospital:  10.  dehydrate and mount  a o r t a , r e s p e c t i v e l y , were used as  f o r the D/PAS and AB  AB  of  5. 1% a l c i a n blue 8GX (pH 1.0 2.5) - 30 min. 6. 1% p e r i o d i c a c i d - 20 min. 7. water - 5 min. 8. s c h i f f reagent - 3 0 min. 9. warm water - 5 min.  3 0 min.  s t a i n i n g techniques.  used i f pyknosis was  controls  S c h i f f reagent  c o n t r o l s were performed on a batch b a s i s and r e a c t i o n was  (right  s e r i a l s e c t i o n s were  each group was  Anatomical Pathology, C h i l d r e n ' s  L i v e r and  serial  u t e r i n e segment  the v a g i n a of each r a t .  d i v i d e d i n t o f o u r groups and  ( d i a s t a s e / a l c i a n blue  performed on 16  3 mm  48  the  feulgen  present.  S t e r o i d Radioimmunoassay A l i q u o t s of 0.5-1.0 mL 4.0  or 4.5  mL  d i e t h y l ether.  s e r a were e x t r a c t e d t w i c e w i t h The  pooled e x t r a c t s were  evaporated a t 3 5°C under n i t r o g e n gas before r e c o n s t i t u t e d i n 1.0  mL  absolute 19  ethanol.  being  Duplicate  100  uL  a l i q u o t s o f t h e e x t r a c t s were a s s a y e d . cpm o f t r a c e r  Approximately  ( H ) was added t o e a c h t u b e .  The b i n d i n g  3  ^efficiency  o f a n t i b o d i e s o f t h e s t e r o i d hormones (17B-  estradiol,  p r o g e s t e r o n e , androgens)  s p e c i f i c b i n d i n g was a s s a y was and  carried  l e s s t h a n 5%.  out u s i n g water  was  10,000  40-60% a n d n o n -  Quality  control  of the  b l a n k s and r e f e r e n c e s e r a  r e c o v e r i e s were e s t i m a t e d b y a d d i t i o n o f t r a c e r t o  reference sera  aliquots.  Ill  of Uterine Tissue  Assessment Design  T r a v e r s e and l o n g i t u d i n a l  histological  assessed w i t h regard t o degenerate, proliferative abnormal al.  activity  q u a n t i t a t i v e d a t a were a v e r a g e d Significant  Two  f o r normal  and  The q u a l i t a t i v e o r  o r summated and r e p o r t e d on a  d i f f e r e n c e s between c o r n u a , o r  observers  i n single blind  o r l u m i n a l and  to confirm individual  (Drs. E. Jones,  each dose  and t i m e group  Representative tissue of  were p h o t o m i c r o g r a p h e d  20  Cagle)  o b s e r v a t i o n s and c o m p a r i s o n s  t r e a t m e n t s and t i m e s .  O p t i p h o t System.  P.  e v a l u a t i o n s o f 10% o f a l l  different  Nikon  s y n t h e t i c and  were n o t e d b y e x c e p t i o n .  independent  were employed slides  1955).  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 ,  glandular epithelia  were  (Long and E v a n s 1922, B u r c h e t  1932, N o y e s 1950, A r i a s - S t e l l a  mesometrial  secretory,  as p r e v i o u s l y d e s c r i b e d  r a t a n d human u t e r i  per r a t basis.  sections  using the  of  Degenerate A c t i v i t y S c o r i n g o f a c t i v i t y was determined s u b j e c t i v e l y as f o l l o w s : 1-no v a c u o l a t i o n ; 2-some v a c u o l a t i o n cells);  3-extensive v a c u o l a t i o n  v a c u o l a t i o n and p y k n o s i s ; and  karyorrhexis.  (>50%) ; 4-extensive  5-extensive v a c u o l a t i o n ,  T i s s u e from a d u l t p r o e s t r u s  p e r i o d s were used as c o n t r o l s f o r scores respectively.  (<50% a l l  and d i e s t r u s  "5" and "1",  Characterization of the vacuolated  was c a r r i e d out by h i s t o c h e m i s t r y .  pyknosis  contents  Fluctuations i n  l e u k o c y t i c i n v a s i o n were used as an i n d i r e c t i n d i c a t o r o f degeneration  of the epithelium  Secretory  (Long and Evans 1922).  Activity  Assessment was based upon t h e presence o f i n t r a l u m i n a l m a t e r i a l , a p i c a l border appearance and c y t o p l a s m i c histochemistry.  With t h e techniques  applied, not a l l  i n t r a l u m i n a l m a t e r i a l c o u l d be v i s u a l i z e d i . e . , d u r i n g proestrus.  Under these c o n d i t i o n s i n t e r p r e t a t i o n was based  upon o b s e r v a t i o n s  made d u r i n g d i s s e c t i o n , t h e degree o f  u t e r i n e d i s t e n s i o n and v a g i n a l c y t o l o g y / h i s t o l o g y . Histochemistry  was used t o d i s t i n g u i s h t h e presence o f mucin  from f o r t u i t o u s l y s e c t i o n e d c e l l s , which may a l s o g i v e t h e e p i t h e l i a l s u r f a c e a shredded appearance.  S e c t i o n s were  r e c u t from u t e r i demonstrating s e c r e t o r y m a t e r i a l and were r e s t a i n e d i n a s i n g l e batch,  t o allow f o r comparisons i n t h e  absence o f i n t e r - s t a i n v a r i a t i o n s .  21  Synthetic A c t i v i t y A c t i v i t y was  e v a l u a t e d through time comparisons of  c y t o p l a s m i c b a s o p h i l l i a / g r a n u l a r i t y and appearance/polarity Nuclear  of t i s s u e w i t h i n one treatment  appearance was  and n u c l e o l a r s i z e .  nuclear group.  assessed on the b a s i s of o p a c i t y , 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  f o l l o w s : 1 - a l l n u c l e i r e s t i n g a g a i n s t basement membrane; 2<50%;  3-50%; 4->50% and <100%; 5-100%.  Tissue obtained  from  a d u l t d i e s t r u s and p r o e s t r u s phases were used as c o n t r o l s f o r s c o r e s "1" and  "5", r e s p e c t i v e l y .  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 d i s t i n g u i s h e d from normal growth by both phase and degree.  Observed growth  o u t s i d e o f p r o e s t r u s and e s t r u s , both a s s o c i a t e d w i t h o v u l a t i o n , was phase" growth.  c o n s i d e r e d the major c r i t e r i o n f o r "out  of  P r o l i f e r a t i o n , under a d u l t c y c l i n g  c o n d i t i o n s , occurs d u r i n g l a t e d i e s t r u s / e a r l y p r o e s t r u s  prior  t o 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 a l s o occurs d u r i n g  subsequent t o e x t e n s i v e degeneration estrus  in late proestrus/early  (Long and Evans 1922). Stromal  i n v a s i o n 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 h y p e r p l a s i a ( A r i a s - S t e l l a 1955, 1957,  mid-estrus,  Dallenbach-Hellweg  l u m i n a l e p i t h e l i a was  e t a l . 1985).  Meissner  H y p e r p l a s i a o f the  d i f f e r e n t i a t e d from g l a n d u l a r  through the examination  of s e r i a l s e c t i o n s and 22  et a l .  epithelia  cytological  comparison of the e p i t h e l i a of i n t e r e s t with i n t r a - s t r o m a l glandular e p i t h e l i a .  P r o g r e s s i v e changes toward a moderate to  e x t e n s i v e c o n f l u e n t p a p i l l a r y p a t t e r n with r a r e f i c a t i o n were c o n s i d e r e d luminal e p i t h e l i a .  evidence  Increased  stromal  of stromal  i n v a s i o n by  e p i t h e l i a l growth was  assessed  by morphometric measurement of the lumen perimeter I I , v e r s i o n 7.2,  R&M  counts were indexed  Biometrics)  The  and m i t o t i c counts.  i n terms of p e r c e n t  IV Assessment of V a g i n a l  epithelial  cells.  e f f e c t s of PMSG treatment were examined i n the intact epithelium  the h i s t o c h e m i s t r y of m u c i f i e d c e l l s . compartment was  examined f o r the types  g r a n u l o c y t i c ) and cells,  (epithelial,  forms (columnar, c u b o i d a l , c o r n i f i e d ) of the manner of a s s o c i a t i o n of these  d i f f e r e n t c e l l types.  The  cells  the  e p i t h e l i u m s u p e r i o r t o the germ  a l s o examined f o r the d i f f e r e n t c e l l types  forms as w e l l as the number of l a y e r s of each type. e x t e n t of m u c i f i c a t i o n was  histochemical interest  was  The and  V a g i n a l t i s s u e of  r e - s t a i n e d i n a s i n g l e batch  a l l o w f o r comparisons i n the absence o f Two  and  c h a r a c t e r i z e d i n both i n t a c t  e x f o l i a t e d compartments by h i s t o c h e m i s t r y .  variations.  and  The e x f o l i a t e d  ( s i n g l e , clump, sheet) and the r e l a t i v e amount of  l a y e r was  Mitotic  Tissue  h i s t o l o g y of the e x f o l i a t e d c e l l s and  dehiscent  (Bioquant  independent observers  to  inter-stain (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 V Statistical The  preparations.  Analysis  s i g n i f i c a n c e o f treatment means d i f f e r e n c e s was  t e s t e d by a n a l y s i s o f v a r i a n c e  (completely  Duncan's m u l t i p l e range t e s t (1957).  randomized) and  Individual interactions  were t e s t e d by Student's T t e s t and s i g n i f i c a n c e was defined  as p<0.05.  on o b j e c t i v e data  Statistical (counts,  a n a l y s i s was o n l y performed  RIA and morphometric measurements).  24  Results I Endometrium Representative  photomicrographs o f t h e u t e r i and  endometria from 4, 20, 4 0 IU PMSG t r e a t e d and a d u l t r a t s a r e i l l u s t r a t e d i n F i g s . 1-42.  The time course of PMSG induced  changes i n t h e p e r i m e t e r o f t h e lumen, e p i t h e l i a l m i t o t i c and e n d o m e t r i a l n e u t r o p h i l counts a r e presented r e s p e c t i v e l y i n F i g s . 43, 44 and 45.  i n a d d i t i o n , these r e s u l t s a r e  summarized i n F i g . 73. The  u t e r i n e 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  patterns  observed s h o r t l y 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 t o those o f a d u l t r a t s . of c o n t r o l u t e r i (Fig.  At 48 h, t h e m a j o r i t y (66%)  ( F i g . 1) resembled those o f p r o e s t r u s  adults  3 ) ; t h e r e s t appeared delayed i n development ( F i g . 14).  In t h e m a j o r i t y  o f u t e r i , t h e lumen, f i l l e d with  f l u i d , was g r e a t l y distended, large luminal perimeter luminal  epithelium  transparent  which was r e f l e c t e d i n t h e  (7.28 + 0.7 mm).  The columnar  ( F i g . 2) d i s p l a y e d b a s o p h i l i a ,  enlarged  n u c l e i , w i t h d i s t i n c t n u c l e o l i , but few m i t o t i c f i g u r e s . glandular  The  e p i t h e l i a was r e l a t i v e l y i n a c t i v e and d i d not  appear t o f o l l o w t h e changes o f t h e l u m i n a l e p i t h e l i u m .  By  72 h, t h e u t e r i n e f l u i d had disappeared and t h e p e r i m e t e r o f the lumen had s i g n i f i c a n t l y 5).  (p<0.001) decreased by 56% ( F i g .  The endometrium, compressed by t h e c o l l a p s e o f t h e  uterus,  was f o l d e d and f l a c c i d .  T h i s appearance was s i m i l a r  to t h a t observed i n t h e a d u l t e s t r u s s t a t e 25  ( F i g . 4 ) . The  tall  columnar e p i t h e l i u m was  p s e u d o s t r a t i f i e d and, i n  a d d i t i o n , v i s i b l y degenerate nuclear  ( F i g . 6).  ( f e u l g e n ) d e b r i s was +  An abundant amount of  observed f r e e 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 v a c u o l e s observed i n the e p i t h e l i u m .  These v a c u o l e s o f t e n c o n t a i n e d a mucinous  (AB 1.0 ,2.5 ,PAS ,D~) m a t e r i a l , which formed a g l y c o c a l y x on +  +  +  t h e l u m i n a l s u r f a c e ( F i g . 7).  During t h i s episode of  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 was e x t e n s i v e , e s p e c i a l l y of the stroma.  Between 48 and 72 h, the  n e u t r o p h i l count s i g n i f i c a n t l y + 27/section.  (p<0.025) i n c r e a s e d 58% t o  From 72 t o 120 h, t h e r e was  towards quiescence.  an o v e r a l l  185  trend,  The t o r t u o u s f o l d s of the endometrium a t  72 h had mostly disappeared by 96 h ( F i g . 8 ) .  Further  c o l l a p s e of the lumen and r e g r e s s i v e changes of the endometrium had r e s u l t e d i n a s l i t - l i k e lumen by 120 h ( F i g . 12).  These m o r p h o l o g i c a l changes were analogous  observed 11)  i n the a d u l t metestrus  states, respectively.  t o those  ( F i g . 10) and d i e s t r u s ( F i g .  E p i t h e l i a l degenerative a c t i v i t y  ( v a c u o l a t i o n , pyknosis) had g r e a t l y subsided by 96 h ( F i g . 9) and was  absent by 120 h  ( F i g . 13).  n e u t r o p h i l count s i g n i f i c a n t l y  Concomitantly,  (p<0.005) decreased from the  peak a t 72 h t o b a s e l i n e by 120 h.  Regenerative  ( m i t o s i s ) , however, peaked a t 96 h (8.56 + 1.5%) significantly  the  activity before  (p<0.001) f a l l i n g t o b a s a l l e v e l s by 144  From 120 t o 192 h, the e p i t h e l i u m was  26  low columnar and  h.  e x h i b i t e d condensed o v a l n u c l e i , both c o n s i s t e n t w i t h a s t a t e of  quiescence. c  all  The u t e r i n e response the uteri,  a f t e r 192 h v a r i e d g r e a t l y .  33% were s t i l l  quiescent.  Many u t e r i  Of  (50%),  however, d i s p l a y e d f e a t u r e s o f e a r l y p r o e s t r u s ( F i g s . 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 o f t h e e p i t h e l i a 17) .  F u r t h e r p r o g r e s s i v e changes o f t h e endometrium towards  maximal d i l a t i o n were e v i d e n t by 240 h. at  ( F i g s . 15 &  Compared w i t h  those  192 h, t h e m i t o t i c index and t h e l u m i n a l p e r i m e t e r were  significantly  (p<0.025) h i g h e r a t 240 h.  u t e r i were g r e a t l y d i s t e n d e d , as observed  A t 240 h, 33% o f a l l a t 48 h ( F i g .  1).  The changes i n u t e r i t r e a t e d w i t h 20 IU PMSG f o l l o w e d a s i m i l a r course t o t h e response under t h e 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 s e c r e t i o n .  A t 48 h, only 20% o f a l l u t e r i  were s i m i l a r t o t h e t y p i c a l p r o e s t r u s morphology ( F i g . The  3).  r e s t o f t h e u t e r i presented stromal hypertrophy and  moderate i n f o l d i n g o f t h e endometrium ( F i g . 18). Very  little  g l a n d u l a r e p i t h e l i a was present, even i n t h e i n v a g i n a t i o n s o f these f o l d s .  The predominant l u m i n a l e p i t h e l i u m was t a l l  columnar and e x h i b i t e d enlarged n u c l e i and b a s o p h i l i a ( F i g . 19).  By 72 h, t h e r e was i n c r e a s e d i n f o l d i n g and i n a d d i t i o n ,  the appearance o f f o c a l p a p i l l a r y h y p e r p l a s i a ( F i g . 2 0 ) . I n v a r i a b l y , t h i s hyperplasia occurred a t the anti-mesometrial end and was attended by stromal r a r e f i c a t i o n . 27  Glandular  e p i t h e l i a d i d not appear t o be i n v o l v e d i n t h i s abnormal proliferation.  E p i t h e l i a l degeneration  a r e a s except h y p e r p l a s t i c f o c i  was prominent i n a l l  ( F i g . 21). V a c u o l a t i o n and  s e c r e t i o n o f a mucinous (AB 1.0 ,2.5 ,PAS ,D~) m a t e r i a l was +  more f r e q u e n t higher  than pyknosis.  +  +  Neutrophil  i n f i l t r a t i o n was  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  t h a n those a t 72 h under the 4 IU PMSG regimen. index i n c r e a s e d s i g n i f i c a n t l y  The m i t o t i c  (p<0.001) from 72 h t o a  maximum a t 120 h (3.35 + 0.6%), b e f o r e r e t u r n i n g t o b a s e l i n e by  168 h.  A f t e r 72 h and a t l e a s t u n t i l 240 h, t h e  e p i t h e l i a l h y p e r p l a s i a , stromal hypertrophy and t h e s i z e o f the lumen g r a d u a l l y diminished  ( F i g s . 22-29).  h y p e r p l a s t i c p a t t e r n a t 72 h had disappeared the r e s o r p t i o n o f s m a l l e r p a p i l l a r y buds. l a r g e r p a p i l l a r y extensions  The branched by 9 6 h, through  By 144 h, t h e  as w e l l as e x i s t i n g f o l d s o f the  endometrium had markedly recessed  ( F i g . 24).  By 192 h, o n l y  the  f o l d s remained  ( F i g . 27), which were mostly resorbed by  240  h ( F i g . 30). E p i t h e l i a l a c t i v i t y , however, d i d not begin  t o r e c e s s u n t i l a f t e r 144 h.  Between 96-144 h, t h e columnar  e p i t h e l i u m was b a s o p h i l l i c and t h e n u c l e i 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 ( F i g s . 23 & 25).  Secretion  o f a mucinous (AB 1.0 ,2.5 ,PAS ,D~) and f e u l g e n  substance  +  increased  +  +  -  from 120 t o 144 h ( F i g . 26). The g l a n d u l a r  e p i t h e l i a appeared s e c r e t o r y as w e l l . as s m a l l specks i n the cytoplasm.  28  T h i s m a t e r i a l appeared  A f t e r 144 h, e p i t h e l i a l  a c t i v i t y s u b s i d e d . By 192 h, t h e n u c l e i had condensed and become t i g h t l y packed  t o g e t h e r ( F i g . 28), and w i t h i n 24 h t h e  b a s o p h i l i a had disappeared  ( F i g . 29).  The a l t e r a t i o n s i n t h e endometrium r e s u l t i n g from 40 IU PMSG treatment were s i m i l a r , but m a g n i f i e d when compared w i t h those i n 2 0 IU PMSG t r e a t e d r a t s .  A t 48 h, t h e e x i s t i n g  i n f o l d i n g had c r e a t e d deep i n v a g i n a t i o n s i n t o t h e endometrium ( F i g . 31). The predominant l u m i n a l e p i t h e l i a was columnar and d i s p l a y e d e n l a r g e d n u c l e i and b a s o p h i l i a ( F i g . 32).  By 72 h, p a p i l l a r y h y p e r p l a s i a appeared  f o c a l l y at the  a n t i - m e s o m e t r i a l end ( F i g . 33). T h i s was accompanied by t h e s e c r e t i o n o f a mucinous (AB 1.0 ,2.5 ,PAS ,D~) m a t e r i a l . +  +  +  However, u n l i k e t h e 4 and 2 0 IU PMSG responses, t h e r e was no evidence o f e p i t h e l i a l degeneration o r i n c r e a s e d n e u t r o p h i l infiltration.  The h y p e r p l a s i a r a p i d l y p r o g r e s s e d and by 12 0  h, had spread towards t h e mesometrial  end ( F i g . 34).  I n t e r e s t i n g l y , v e r y few mitoses were observed  ( F i g . 35). By  144 h, t h e l u m i n a l 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 t h e stroma, endometria  forming many f l a c c i d p r o j e c t i o n s o f  w i t h numerous p a p i l l a r y e x t e n s i o n s ( F i g . 36). The  s i z a b l e luminal perimeter  (7.06 + 0.9 mm) r e f l e c t e d  this  i n c r e a s e i n t h e s u r f a c e area o f t h e endometrium and was significantly  (p<0.005) g r e a t e r than both t h e 20 IU PMSG and  c o n t r o l responses.  The c u b o i d a l e p i t h e l i u m was w e l l  d i f f e r e n t i a t e d and d i s p l a y e d enlarged, rounded n u c l e i ( F i g . 37).  I n a d d i t i o n , t h e l u m i n a l s u r f a c e was coated h e a v i l y  with a mucinous (AB 1.0 a l s o observed  ,2.5  ,PAS  ,D~)  m a t e r i a l , which  as small specks i n the cytoplasm  G l a n d u l a r e p i t h e l i a was  was  ( F i g . 38)«  not i n v o l v e d i n t h i s abnormal  p r o l i f e r a t i o n , but appeared s e c r e t o r y , s i m i l a r t o t h a t of luminal e p i t h e l i a .  A f t e r 144  quiescence  was  extensions  observed  168  h  and was  perimeter 192  h  not a t t a i n e d by 240 h.  ( F i g . 39). absent  h, a l l a c t i v i t i e s recessed,  between 120  and  144  Many of the  by 19 2 h.  ( F i g . 42).  h y p e r p l a s i a , as observed achieved by 240 treatment, no evidence  h  by 168  h  were apparent by  At 240 h, the e p i t h e l i a was  and appeared i n a c t i v e  by  Further reductions i n luminal  and s i z e and stromal hypertrophy  ( F i g . 40).  papillary  h had r e g r e s s e d  S e c r e t o r y a c t i v i t y had decreased  but  low  columnar  F u l l r e g r e s s i o n of the  a f t e r 2 0 IU PMSG treatment,  ( F i g 41).  U t e r i recovered  was  40 d a f t e r  however, showed t y p i c a l e s t r o u s c y c l e f e a t u r e s of h y p e r p l a s i a or any other  abnormalities.  30  not  histological  and  Fig.l U t e r u s , 4 8 h +4IU PMSG ( 4 0 x , H&E) The l u m e n i s markedly d i l a t e d w i t h f l u i d and a p p e a r s v a c a n t , e q u i v a l e n t to that i n adult proestrus (Fig.3).  F i g . 2 E n d o m e t r i u m , 4 8 h +4IU PMSG ( 4 0 0 x , H&E) T h e b a s o p h i l i a of t h e cytoplasm and t h e p r o m i n e n c e 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 epithelium.  Fig. 3 Uterus, (40x, H&E)  Fig.4 (40x,  Adult  Proestrus  31  Uterus, H&E)  Adult  Estrus  Fig.5 Uterus, 72h +4IU PMSG (40x, H&E) C o l l a p s e o f t h e lumen has l e d t o t h e compress i o n o f t h e endometrium i n t o f l a c c i d f o l d s , s i m i l a r t o the e s t r u s s t a t e ( F i g . 4 ) . Note t h e t i s s u e c e n t r a l i n t h e lumen.  Fig.6 Endometrium, 72h +4IU PMSG (400x, H&E) V a c u o l a t i o n , pyknosis and k a r y o r h e x i s o f the e p i t h e l i u m a r e e x t e n s i v e . The compression o f t h e e p i t h e l i u m has l e d t o a t a l l columnar and p s e u d o s t r a t i f i e d appearance.  Fig.7 Endometrium, 72h +4IU PMSG ( 4 0 0 X , AB 1.0/PAS/D) V a c u o l a t e d mucinous (AB 1.0 , 2.5 ,PAS ,D~) m a t e r i a l accumulated d u r i n g degenera t i o n forms a g l y c o c a l y x and i s d e p o s i t e d i n t h e lumen. +  +  32  +  Fig.8 U t e r u s , 9 6 h +4IU PMSG (4Ox, H&E) T h r o u g h s t r o m a l r e o r g a n i z a t i o n and degeneration of the epithelium,the m a j o r i t y o f t h e f o l d s have b e e n s m o o t h e d o u t . Compare with Fig.10 (adult metestrus)  Fig.9 E n d o m e t r i u m , 9 6 h +4IU PMSG ( 4 0 0 x , 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 regenerative a c t i v i t y (mitosis) i s maximal.  Fig.10 Uterus, A d u l t Metestrus ( 4 0 x , H&E)  Fig.11 Uterus, Adult Diestrus ( 4 0 x , H&E)  33  F i g . 1 2 U t e r u s , 1 2 0 h +4IU PMSG (4Ox, H&E) The l u m e n h a s become s l i t - l i k e , a s d u r i n g d i e s t r u s (Fig.11), through 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. N o t e t h e i n c r e a s e d stromal c e l l u l a r density next t o t h e lumen.  F i g . 1 3 E n d o m e t r i u m , 1 2 0 h +4IU PMSG ( 4 0 0 x , 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 l o w 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 4 U t e r u s , 2 1 6 h +4IU PMSG (40x, H&E) E n l a r g e m e n t o f t h e lumen i s a t t e n d e d b y 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 stromal reorganization. Small i n v a g i n a t i o n s form as a result.  F i g . 1 5 E n d o m e t r i u m , 2 1 6 h +4IU PMSG ( 4 0 0 x , 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 compression 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 presents active 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, m a x i m a l 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 o t been a t t a i n e d .  F i g . 1 7 E n d o m e t r i u m , 240h +4IU PMSG ( 4 0 0 x , 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 epithelium.  F i g . 1 8 U t e r u s , 4 8 h +20IU PMSG ( 4 0 x , H&E) The s t r o m a i s edematous and h y p e r t r o p h i e d , and a s a r e s u l t a p p e a r s folded.  F i g . 1 9 E n d o m e t r i u m , 4 8 h +20IU PMSG ( 4 0 0 x , 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 very 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 nucleoli.  35  F i g . 2 0 U t e r u s , 7 2 h +20IU PMSG ( 4 0 x , 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 the 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 invaded the stroma almost as f a r as t h e myometrium.  F i g . 2 1 E n d o m e t r i u m , 7 2 h +20IU PMSG ( 4 0 0 x , 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 c h a n g e s are o n l y s l i g h t i n comparison w i t h t h e 4 I U PMSG r e s p o n s e ( F i g . 6 ) . The n u c l e i a n d nucleoli are s t i l l enlarged.  F i g . 2 2 U t e r u s , 9 6 h +20IU PMSG ( 4 0 x , H&E) T h r o u g h 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 degeneration, branching o f the e p i t h e l i u m has been reduced, resulting i n slender stalkprojections.  F i g . 2 3 E n d o m e t r i u m , 9 6 h +20IU PMSG ( 4 0 0 x , H&E) T h e 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 ordered and m o d e r a t e l y condensed.  36  F i g . 2 4 U t e r u s , 1 4 4 h +20IU PMSG ( 4 0 x , H&E) T h e s l e n d e r s t a l k s by t h i s t i m e have been reduced t o s m a l l buds. Note the increased c e l l u l a r d e n s i t y o f t h e stroma i n these areas.  F i g . 2 5 E n d o m e t r i u m , 1 4 4 h +20IU PMSG ( 4 0 0 x , H&E) T h e e p i t h e l i u m h a s 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 E n d o m e t r i u m , 1 4 4 h +20IU PMSG ( 4 0 0 x , AB 1.0/PAS/D) The m u c i n o u s (AB 1 . 0 , 2 . 5 , P A S , D~) m a t e r i a l , a p p e a r i n g a s small specks i n t h e cytoplasm, forms a t h i n r i m on t h e luminal surface. +  37  +  +  F i g . 2 7 U t e r u s 1 9 2 h +20IU PMSG ( 4 0 x , 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 of endometria have r e g r e s s e d . P r o g r e s s i v e changes towards the formation o f a s l i t - l i k e lumen a r e a p p a r e n t .  F i g . 2 8 E n d o m e t r i u m , 1 9 2 h +20IU PMSG ( 4 0 0 x , H&E) T h e 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 . N o t e t h e s l i g h t l y d i f f e r e n t appearing, but q u i e s c e n t , g l a n d u l a r tissue.  F i g . 2 9 E n d o m e t r i u m , 2 1 6 h +20IU PMSG ( 4 0 0 x , H&E) T h e 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 presents a strong eosinop h i l l i c appearance.  F i g . 3 0 U t e r u s , 24Oh +2 0 I U PMSG (4Ox, H&E) T h e l u m e n 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 ) a n d 96h +4IU PMSG m o r p h o l o g i e s (Fig.8).  38  F i g . 3 1 U t e r u s , 4 8 h +40IU PMSG ( 4 0 x , H&E) T h e e x i s t i n g i n f o l d i n g h a s c r e a t e d deep invaginations i n the e n d o m e t r i u m . Compare w i t h t h e 2 0 I U PMSG r e s p o n s e a t 4 8h (Fig.18) .  F i g . 3 3 U t e r u s , 7 2 h +40IU PMSG ( 4 0 x , 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 the 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 observed a t 144h +20IU PMSG ( F i g . 2 4 ) .  39  F i g . 3 2 E n d o m e t r i u m , 4 8 h +40IU PMSG ( 4 0 0 x , H&E) T h e 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 basophilia, enlarged nuclei and d i s t i n c t n u c l e o l i .  F i g . 3 4 U t e r u s , 1 2 0 h +40IU PMSG ( 4 0 x , H&E) The h y p e r p l a s i a h a s 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 o w a r d s t h e mesom 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 E n d o m e t r i u m , 120h +40IU PMSG ( 4 0 0 x , H&E) T h e 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 nucleoli are distinct. Mitotic figures are rare.  F i g . 3 6 U t e r u s , 1 4 4 h +40IU PMSG ( 4 0 x , 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 invaded the s t r o m a , f o r m i n g many f l a c c i d projections with papillary extensions.  F i g . 3 7 E n d o m e t r i u m , 1 4 4 h +40IU PMSG ( 4 0 0 x , 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 I U 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 .  40  F i g . 3 8 E n d o m e t r i u m , 1 4 4 h +40IU PMSG ( 4 0 0 X , AB 1.0/PAS/D) The secretion of a similar mucinous (AB1.0 ,2.5 ,PAS , D ) substance t o t h a t observed w i t h 20 I U PMSG ( F i g . 2 6 ) accompanies t h e h y p e r p l a s i a . +  +  +  F i g . 4 0 U t e r u s , 1 9 2 h +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 reduced. Further 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 time i s a p p a r e n t a n d t h e lumen i s relatively clear. 41  F i g . 3 9 U t e r u s , 1 6 8 h +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 regressed, increasing the 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. Secretory a c t i v i t y i s s t i l l present, b u t i s reduced.  F i g . 4 1 U t e r u s , 240h +40IU PMSG (40x, H&E) C h a n g e s t o w a r d 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 of t h e u t e r u s and t h i c k n e s s o f the endometrium a r e s i m i l a r t o t h o s e a t 96 a n d 1 2 0 h +4IU PMSG. N o t e t h e i n c r e a s e d stromal 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.  42  F i g . 4 2 E n d o m e t r i u m , 240h +40IU PMSG ( 4 0 0 x , H&E) The e p i t h e l i u m a p p e a r s 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 cytoplasm appears eosinophillic.  1.0  J  48  72  96  120  144  ?68  192  216  240  Hours Post PMSG Injection Fig.43 P e r i m e t e r (mm) o f t h e u t e r i n e l u m e n 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 a n d 40 I U 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 Fig.44 E p i t h e l i a l m i t o t i c index, expressed i n percent, 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 a n d 40 I U 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 Fig.45 N e u t r o p h i l counts 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 a n d 40 I U PMSG. V a l u e s r e p r e s e n t means + SEM, N=6.  45  II  Vagina Representative  20  and  Figs.  40  I U PMSG t r e a t e d and  46-74.  provided  photomicrographs of the v a g i n a e from  73.  succession,  d i f f e r e n t i a t i o n and  types proceeded stepwise  desquamation of  over the e n t i r e epithelium.  a f t e r 4 I U PMSG, t h e v a g i n a l h i s t o l o g y and exhibited 48).  features t y p i c a l of proestrus  Mucified  (AB  i n the  +  ( F i g . 47).  t h a n a t AB had  not  pH  begun.  1.0.  of  a t AB  the pH  2.5  I n some r a t s ( 3 3 % ) , h o w e v e r , d e s q u a m a t i o n  Non-nucleated c o r n i f i e d c e l l s ,  i n t o sheets.  The  and  c e l l s had  either  l a y e r , were l o o s e l y consisted  basal c e l l s .  e x f o l i a t e d sheets of c o r n i f i e d c e l l s f i l l e d epithelial  forming  r e s t of the e p i t h e l i u m  o f squamous p r e - c o r n i f i e d n u c l e a t e d  mucified  (66%)  s l i g h t l y darker  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 organized  46)  cells  +  S t a i n i n g was  (Fig.  h  adult rat (Fig.  1.0 ,2.5 ,PAS ,D~) e p i t h e l i a l +  cell  A t 48  cytology  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 controls  in  A summary o f t h e m a j o r v a g i n a l c h a n g e s i s  in Fig.  The  adult r a t s are presented  4,  disappeared  the  A t 72  l u m e n and  ( F i g . 50),  the  similar  t o t h a t observed during estrus  ( F i g . 49).  c o n s i s t e d o f a 1-2  l a y e r s of p r e - c o r n i f i e d c e l l s ,  3-5  superficial  l a y e r s o f c u b o i d a l c e l l s and  the  Neutrophil  i n f i l t r a t i o n was  lumen.  96 h, t h e p r e c o r n i f i e d and  By  cuboidal dehiscent  The  h,  germinativum.  extensive but most  absent from  become  T h i s h e t e r o g e n o u s p a t t e r n was 46  the  superficial  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 ( F i g . 51).  epithelium  also  observed during t h e adult metestrus state  ( F i g . 5 2 ) . From 120  t o 192 h , t h e r e was m o d e r a t e 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 concomitant hypertrophy as these differentiated  ( F i g s . 54-55).  cells  T h i s appearance  was  comparatively equivalent with diestrus i n the adult A f t e r 192 h , many v a g i n a e of early  (66%) were found t o d i s p l a y f e a t u r e s  ( F i g s . 56-57) o r m i d - p r o e s t r u s  The  (Fig. 56).  (Fig. 48).  v a g i n a l development and d i f f e r e n t i a t i o n  shortly  f o l l o w i n g 20 I U 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 controls  (Figs.  58-60).  A f t e r 96 h , h o w e v e r , 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 +  +  +  was e v i d e n t b y 120 h ( F i g s . 6 1 - 6 2 ) .  I n most c a s e s  layers  staining  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 g r a n u l o s u m h a d  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, s i m i l a r s t a i n i n g mucinous m a t e r i a l  (Figs.  63-64).  containing I t was n o t  c l e a r w h e t h e r 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 intra-epithelial cysts. 216  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  h ( F i g . 6 5 ) , however, i t i s u n l i k e l y t h e y were  cellular.  I n a d d i t i o n , a s t h e r e was l i t t l e  intra-  nuclear debris or  p y k n o s i s , t h e mode o f s e c r e t i o n b y s u r r o u n d i n g c e l l s was probably non-holocrine. q u a n t i t i e s o f mucinous  The l u m e n c o n t a i n e d c o p i o u s (AB 1 . 0 , 2 . 5 , PAS ,D~) m a t e r i a l a n d  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 50).  compared w i t h t h e r e s p o n s e d u r i n g e s t r u s ( F i g .  With time,  cysts increased cystic  t h e number a n d s i z e o f t h e i n t r a - e p i t h e l i a l a n d b y 216 h , t h e e n t i r e e p i t h e l i u m h a d become  ( F i g s . 65-66). The v a g i n a l d e v e l o p m e n t  comparatively  f o l l o w i n g 40 I U PMSG was  a c c e l e r a t e d w i t h t h e 2 0 I U 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%) h a d a l r e a d y a c h i e v e d a metestrus-like state.  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 +  +  formation,  epithelium,  involving cyst  was o b s e r v e d a t 120 h ( F i g s . 6 7 - 6 8 ) .  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 a p p e a r e d s l i g h t l y l i g h t e r t h a n a t AB pH 2.5. cystic  By 168 h , t h e w h o l e  ( F i g s . 69-70).  e p i t h e l i u m h a d become  The a p p e a r a n c e  o f mucinous  m a t e r i a l a n d 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 . changes  p r i m a r i l y involved increases  rupture  of adjacent walls  secretory Further  i n cyst s i z e through the  ( F i g s . 71-72).  48  that  F i g . 4 6 V a g i n a , 4 8 h +4IU PMSG ( 4 0 x , 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 behind several layers o f cornified cells. Precornified and c u b o i d a l c e l l s f o r m t h e granulosum. (see Fig.48)  49  F i g . 4 7 V a g i n a , 4 8 h +4IU PMSG ( 4 0 x , AB 2.5/PAS/D) T h e dehiscent e p i t h e l i a l c e l l s c o n t a i n m u c i n (AB 1 . 0 , 2 . 5 , PAS ,D~). Very 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. +  +  +  F i g . 5 0 V a g i n a , 7 2 h +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 of 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 confined t o the e p i t h e l i u m , (see Fig.49)  50  F i g . 5 1 V a g i n a , 9 6 h +4IU PMSG (40x, H&E) The p r e c o r n i f i e d and m o s t s u p e r f i c i a l c u b o i d a l layers, containing n e u t r o p h i l s , have been exfoliated, similar t o that seen d u r i n g a d u l t m e t e s t r u s .  F i g . 5 4 V a g i n a , 1 2 0 h +4IU PMSG ( 4 0 x , H&E) T h e 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 hypertrophy, e s p e c i a l l y o f t h e most superficial layers.  F i g . 5 5 V a g i n a , 1 2 0 h +4IU PMSG ( 4 0 x , AB 2.5/PAS/D) T h e m o s t s u p e r f i c i a l l a y e r s h a v e 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 of these c e l l s . Compare w i t h F i g . 5 3 .  F i g . 5 6 V a g i n a , 240h +4IU PMSG ( 4 0 x , H&E) C o n c o m i t a n t w i t h the onset o f o v u l a t i o n (proestrus), 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 a s t h e p r e c o r n i f i e d l a y e r , seen here as a d a r k i s h band.  F i g . 5 7 V a g i n a , 2 4 0 h +4IU PMSG (40x, AB 2.5/PAS/D) Compare the depth o f m u c i f i c a t i o n with the 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 , 4 8 h +20IU PMSG ( 4 0 x , H&E) T h e p a t t e r n r e s e m b l e s t h a t a t 4 8 h +4IU PMSG ( F i g . 4 6 ) a n d a d u l t proestrus (Fig.48).  F i g . 6 0 V a g i n a , 9 6h +2 0 I U PMSG ( 4 0 x , 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 o c c u p y t h e l u m e n . Compare w i t h 96h +4IU PMSG ( F i g . 5 1 ) a n d adult metestrus ( F i g . 52). 52  F i g . 5 9 V a g i n a , 7 2 h +20IU PMSG ( 4 0 x , H&E) The c o r n e u m a s w e l l a s 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 7 2 h +4IU PMSG (Fig.50) and a d u l t e s t r u s (Fig.49).  F i g . 6 1 V a g i n a , 1 2 0 h +20IU PMSG ( 4 0 x , 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 h a s 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 , 1 2 0 h +20IU PMSG ( 4 0 x , AB 2.5/PAS/D)  F i g . 6 3 V a g i n a , 1 4 4 h +20IU PMSG ( 4 0 x , H&E) The l o w e r l a y e r s o f t h e granulosum h a d begun t o mucify. I n a d d i t i o n small spaces, f i l l e d w i t h mucinous m a t e r i a l , had appeared.  F i g . 6 4 V a g i n a , 1 4 4 h +20IU PMSG (40x, AB 2.5/PAS/D) I n a d d i t i o n t o t h emucinous m a t e r i a l , numerous e p i t h e l i a l c e l l s a n d some n e u t r o p h i l s h a d accumulated i n t h e lumen.  F i g . 6 5 V a g i n a , 2 1 6 h +2 0 I U PMSG ( 4 0 x , H&E) The s m a l l s p a c e s 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 m a k i n g u p 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 . T h e r e was n e g l i g i b l e nuclear debris.  F i g . 6 6 V a g i n a , 2 1 6 h +20IU PMSG ( 4 0 x , AB 2.5/PAS/D)  Pi  •.•*r F i g . 6 7 V a g i n a , 1 2 0 h +40IU PMSG ( 4 0 x , H&E) A l t h o u g h n o t f u l l y differentiated, a l l layers s u p e r i o r t o t h e germinativum appear mucinogenic.  54  F i g . 6 8 V a g i n a , 12Oh +4 0 I U 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 throughout t h e e p i t h e l i u m , indicating incomplete differentiation. +  +  +  F i g . 6 9 V a g i n a , 1 6 8 h +40IU PMSG ( 4 0 x , H&E) The e n t i r e e p i t h e l i u m h a d become c y s t i c , c o m p a r e w i t h 144 a n d 2 1 6 h +20 I U PMSG ( F i g s . 6 3 - 6 6 ) .  F i g . 7 0 V a g i n a , 1 6 8 h +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 .  F i g . 7 1 V a g i n a , 240h +40IU PMSG ( 4 0 x , 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 adjacent walls, r e s u l t i n g i n enlarged cysts.  F i g . 7 2 V a g i n a , 2 4 0 h +40IU PMSG ( 4 0 x , AB 2.5/PAS/D)  55  40 IU PMSG Uterine Morpho logy  stromal focal hyper papillary trophy hyperplasia  Epithelial Activity  Vagina  (  LPE  E  stromal hyper trophy  Epithelial Activity  ME  partial regression  sec  sec  20 IU PMSG Uterine Morpho logy  diffuse papillary hyperplasia  muc  |  quiescence  I  •  large cysts  I  I  full regression  focal • papillary hyperplasia  deg sec  I ,-•  +  cystic  small spaces  I  i  sec  sec  muc  small spaces  quiescence  cystic Vagina  PE  ME  '  4 IU PMSG Uterine Morpho logy  dilated  Epithelial Activity  Vagina  flaccid folds  deg sec  PE  48  stellate  distension  proliferation & repair  proliferation  ME  72  96  keratogenic & mucinogenic differentiation  DE muc  120  144  168  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 a n d v a g i n a l m o r p h o l o g i c a l a n d h i s t o l o g i c a l c h a n g e s 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 I U 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 ; d e g = d e g e n e r a t e ; 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= metestrus; DE=diestrus.  56  Ill  Steroids 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 , a n d a n d r o g e n s a r e shown i n F i g . 7 4 . 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 I U PMSG t r e a t m e n t . significantly  shortly after 4  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  (p<0.001) i n c r e a s e d  pg/ml) a t 48 h , b e f o r e  t o a maximum ( 4 2 1 + 33  f a l l i n g t o b a s a l l e v e l s b y 72 h .  Progesterone 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  t o 60 h (109 + 17 ng/ml) a n d a l s o r e t u r n e d 72 h .  Major  to basal  f r o m 48 l e v e l s by  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 b e t w e e n 192 a n d 240 h , b u t t h e s e w e r e n o t significant.  Many s t r i k i n g c h a n g e s r e s u l t e d f r o m  w i t h 20 I U PMSG t r e a t m e n t . already peaked  significantly  By 24 h , 1 7 B - e s t r a d i o l  increased  (663 + 59 p g / m l ) .  sharply three  fell  fold  l e v e l s had  (p<0.005) a n d w i t h i n 24 h h a d  T h i s p e a k was c o m p a r a b l e t o t h a t i n  40 I U 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 than i n c o n t r o l s .  treatment  (p<0.001)  higher  B e t w e e n 48 a n d 72 h , 1 7 B - e s t r a d i o l l e v e l s  back t o b a s e l i n e .  Androgen l e v e l s rose  (p<0.05)  f r o m 24 t o 48 h ( 7 . 5 9 + 2 . 1 ng/ml) a n d t h e n  decreased stepwise. intermediary  They s i g n i f i c a n t l y  l e v e l s by 96, b u t were s t i l l  (p<0.01) h i g h e r  than those of c o n t r o l s .  (p<0.05) d e c l i n e d t o significantly T h e r e a f t e r , androgen  l e v e l s s l o w l y d e s c e n d e d a n d r e a c h e d b a s e l i n e b y 168 h . Progesterone l e v e l s followed those o f c o n t r o l s u n t i l 72 h .  at least  By 96 h , h o w e v e r , t h e s e l e v e l s h a d i n c r e a s e d  significantly  (p<0.025) a n d w e r e g r e a t l y (p<0.001)  57  elevated  above t h o s e o f c o n t r o l s .  A maximum (2 61 + 27 p g / m l ) was  g r a d u a l l y r e a c h e d b y 168 h a n d t h e n l e v e l s levels.  Steroid  fluctuated  levels resulting  greatly.  After  towards  f r o m t h e 40 I U PMSG  dramatically  b e t w e e n 0 a n d 48 h (538 + 36 p g / m l ) , significantly  fell  (p<0.001)  regimen  increasing  17B-estradiol  (p<0.001) d e c r e a s e d b y 6 8 % .  basal  levels  T h e p e a k 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  levels.  U n l i k e t h e responses o f lower doses,  levels  sharply rose  17B-estradiol  (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 p g / 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 b y 120 h . significant  (p<0.005) r i s e i n a n d r o g e n s f r o m 24 h t o a p e a k  (7.92 + 1.3 ng/ml) a t 72 h p r e c e d e d estradiol  A  levels.  levels declined  t h e l a s t i n c r e a s e i n 17B-  Analogous t o t h e 2 0 IU response, androgen  i n a s t e p w i s e manner.  They f i r s t  fell  (p<0.05) b e t w e e n 72 a n d 96 h t o p l a t e a u l e v e l s c o m p a r a b l e t o t h a t i n 2 0 I U PMSG t r e a t e d diminished, while following  rats.  the decline  Progesterone l e v e l s p a r a l l e l e d response u n t i l  168 h .  These l e v e l s t h e n  gradually  o f 2 0 I U PMSG l e v e l s .  t h o s e o f t h e 2 0 I U PMSG  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 controls. and r e a c h e d  After  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  53 9 + 67 n g / m l b y 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 , progesterone and a n d r o g e n s a f t e r t r e a t m e n t w i t h 4, 2 0 and 4 0 I U PMSG. A t 0, 24 a n d 60 h , 3 r a t s w e r e u s e d ; 6 r a t s w e r e u s e d 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  Discussion The  p r e s e n t study c o n f i r m s t h a t a low dose  (4 I U ) o f  PMSG c a n i n d u c e i m m a t u r e r a t s t o m i m i c t h e p h y s i o l o g i c a l events o f t h e adult estrus  cycle.  The d y n a m i c s o f 17B-  e s t r a d i o l a n d p r o g e s t e r o n e l e v e l s b e t w e e n 24 a n d 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 other workers using 1974,  agreement w i t h t h o s e o b s e r v e d by  low doses  (4-8 IU) o f PMSG ( W i l s o n  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) a n d w i t h p e r i - o v u l a t o r y the al.  adult cycling rat 1985).  patterns  changes r e p o r t e d f o r  (Yoshinaga e t a l . 1969, P a s q u a l i n i e t  I n v i e w o f t h e s i m i l a r i t y between hormonal  o f c o n t r o l s and a d u l t s , t h e correspondence o f  u t e r i n e h i s t o l o g i c a l c h a n g e s was n o t s u r p r i s i n g . been r e c o g n i z e d the  estrus  proestrus,  during  c y c l e a r e c o n t r o l l e d by concomitant f l u c t u a t i o n s During  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 b y 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 , transport  I t has long  t h a t endometrial h i s t o l o g i c a l changes  of 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 .  1950,  et al.  a n d c a p a c i t a t i o n o f sperm  K i r t o n e t a l . 1965).  accumulation of f l u i d  which aids  i n the  ( W a r r e n 193 6,  Leonard  I t i s well established that the  as w e l l as t h e i n c r e a s e d  luminal  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 luminal  distension  i sassociated  with the elevation of  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,  M a r c u s 1974, Kennedy e t a l . 1975, F i n n  During  1980).  estrus,  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 the drainage o f u t e r i n e f l u i d through t h e vagina al.  1975).  into flaccid to and  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 folds.  t h e endometrium  Concomitantly, a l l s t e r o i d l e v e l s  b a s e l i n e and t h i s withdrawal induces stromal epithelial  (Kennedy e t  hypotrophy  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, infiltration A l l e n 1931,  and r e p a r a t i v e changes  return  +  neutrophil  (Long a n d E v a n s 1 9 2 2 ,  M a r c u s 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 retarded degenerative a c t i v i t y .  Animals  dose-dependently treated with 20 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 h o r m o n a l w i t h d r a w a l a t 72 h , a l b e i t s u b d u e d i n c o m p a r i s o n Although  w i t h c o n t r o l s and a d u l t s .  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 b y 72 h , a n d r o g e n l e v e l s r e m a i n e d have p r o v i d e d p a r t i a l hormonal support.  e l e v a t e d a n d may Exogenously  a d m i n i s t e r e d n o n - a n d a r o m a t i z a b l e a n d r o g e n s may i n d u c e u t e r i n e e p i t h e l i a l hypertrophy through 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 al.  1976, P a s q u a l i n i e t a l . 1985).  (Armstrong e t  Despite a sharp  fall 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 I U PMSG t r e a t e d l e v e l s a t 72 h w e r e s t i l l  rats,  higher than those o f c o n t r o l s .  These e s t r o g e n s i n combination 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  support t o account 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  repair. 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 ovulation,  increased  associated  levels of steroids (17B-estradiol,  a n d r o g e n s , p r o g e s t e r o n e ) may h a v e c o n c u r r e n t l y endometrial  atypia.  androgens together  produced  High l e v e l s o f 1 7 B - e s t r a d i o l and with hypertrophic  development o f t h e  endometrial  s t r o m a f o l l o w e d e i t h e r 20 o r 40 I U PMSG  treatment.  A s t h e r e was l i t t l e  l e v e l s and t h e extent  difference i n the steroid  o f h y p e r t r o p h y , i t seems l i k e l y  common m e c h a n i s m s w e r e o p e r a t i v e .  that  I t i swell established  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 the  with  hypertrophy  stroma t h r o u g h t h e development o f hyperemia and s t r o m a l  edema ( L a u s o n e t a l . 1 9 3 9 , S c h w a r z 1 9 6 4 , G a l a n d e t a l . 1 9 7 1 , Martin  e t a l . 1973).  rats given  Time c o u r s e s t u d i e s o f  ovariectomized  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  that  edema r a p i d l y d e v e l o p s t o a d o s e - d e p e n d e n t maximum b e t w e e n 610 h , b u t r e g r e s s e s  w i t h i n 20 h , e v e n a f t e r t h e u s e o f  prolonged treatment  (Astwood 1938, C a r r o l l  al.  1945, Homburger e t  1955, Schwarz 1964, F i n n and P o r t e r 1975).  concurrently resulting  Fluid i s  t r a n s f e r r e d from t h e stroma i n t o t h e lumen  i n d i s t e n s i o n (Finn e t a l . 1975).  I n view o f these  experiments, t h e presence o f hypertrophy a t 4 8 h i n r e l a t i o n to elevated  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 h a v e b e e n s e c o n d a r y . aromatizable  and, t o a l e s s e r extent,  androgens a r e capable o f i n d u c i n g  62  Exogenous  non-aromatizable  s u s t a i n e d h y p e r t r o p h y up t o  72 h a f t e r t r e a t m e n t  (Rennels 1951, Armstrong  e t a l . 1976).  A n d r o g e n s may a c t d i r e c t l y u p o n t h e u t e r u s t h r o u g h t h e i r own or,  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  receptors  (Giannopoulos  levels,  estrogen  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 w e r e 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 h a v e s y n e r g i s t i c a l l y i n d u c e d a n d 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 The  f o r an extended  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  luminal papillary hyperplasia  w i t h s u p e r o v u l a t o r y d o s e s o f PMSG. h y p e r p l a s i a appeared hypertrophy  p e r i o d o f time.  The development o f  t o f o l l o w t h e onset o f stromal  i n b o t h 20 a n d 40 I U 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 17Bestradiol  f r o m 24 h t o 48 h a f t e r 2 0 o r 40 I U PMSG  i n i t i a t e d this sequential pattern. been observed  (Carrol  1945, Greenwald e t a l . 1959, F i n n e t  1973, M a r t i n e t a l . 1973).  increased u t e r i n e weight hypertrophy,  p a t t e r n s have  i n ovariectomized rodents repeatedly treated  with estrogens al.  Similar  treatment  subsequent  While prolonged  initially  a n t i m e s o m e t r i a l end.  ( 6 - 1 0 h) t h r o u g h  stromal  i n c r e a s e s (24-48 h) r e l i e d u p o n  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. h o w e v e r , was i n i t i a l l y  treatment  Epithelial hyperplasia,  f o c a l and i n v a r i a b l y  appeared  a tthe  T h i s f o c a l a p p e a r a n c e may h a v e b e e n 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  mesometrial regions. ovariectomized  Autoradiographic  studies  of  mice t r e a t e d w i t h a s i n g l e dose of  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  17B-  t o 16 h b e t w e e n 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 d e c l i n e d , those e l e v a t e d up and  of the antimesometrial  t o a t l e a s t 36 h  high l e v e l s of estrogens  treatment  may  region  rates  remained  (Martin e t a l . 1973). associated with  Prolonged  superovulatory  have exaggerated 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 rates i n the antimesometrial in focal hyperplasia.  Differential  region  resulting  s e n s i t i v i t y was  also  o b s e r v e d b e t w e e n l u m i n a l and  glandular epithelia during  the  development of h y p e r p l a s i a .  I t i s well recognized  the  a f f e c t s o f s t e r o i d hormones on t h e e n d o m e t r i u m a r e u p o n t h e s e n s i t i v i t y and epithelial tissue  ( B u r c h e t a l . 1932,  e t a l . 1941,  al.  B u x t o n e t a l . 1961,  I n r a t s and  dependent  p r o p o r t i o n o f g l a n d u l a r and  Cleveland 1957,  that  Arias-Stella  Wolfe et a l . 1954,  1955,  F i n n e t a l . 1975,  mice, there i s l i t t l e  glandular  luminal 1933,  Meissner  et  Gondos 1 9 7 6 ) .  tissue.  M o r e o v e r , 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  to  hormonal i n f l u e n c e than e i t h e r g l a n d u l a r or stromal  tissue  ( M a r t i n e t a l . 1973,  Finn  1980).  M a r c u s 1974,  F i n n e t a l . 1975,  These f e a t u r e s might account f o r the absence  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 of t h e e p i t h e l i u m w h i l e t h e r e was  increased p r o l i f e r a t i o n .  addition, the 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 of the  e p i t h e l i u m and  t h e s t r o m a may  64  e x p l a i n the  of  luminal In  luminal  papillary  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 ,  growing f a s t e r than 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 Had  i n surface area w i t h i n a r e s t r i c t e d  t h e r e been s y n c h r o n i z e d  epithelium,  stromal  growth between t h e stroma and  r a r e f i c a t i o n would have been absent and  the epithelium l e s s convoluted, of proestrus  as observed during t h e onset  i n c o n t r o l s f r o m 216-240 h .  F r o m 96 t o 144 h p o s t 40 I U PMSG t r e a t m e n t , increased  volume.  t h e r e was  hypertrophy o f t h e stroma and p r o g r e s s i v e  hyperplasia.  While 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 the  hormonal event(s)  r e s p o n s i b l e , t h e absence o f these  progressions  i n 2 0 I U PMSG t r e a t e d r a t s p r o v i d e s  comparison.  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  patterns  a basis f o r  o f t h e 2 0 a n d 4 0 I U PMSG t r e a t m e n t g r o u p s was t h e  m a r k e d e l e v a t i o n o f 1 7 B - e s t r a d i o l l e v e l s f r o m 72 t o 96 h p o s t 40  I U PMSG.  Walton e t a l . (1982),  administering  a n t i s e r a a t 58 h p o s t 40 I U PMSG i n j e c t i o n ,  PMSG  simultaneously  r e d u c e d t h e 1 7 B - e s t r a d i o l s u r g e a t 96 h a n d a b o l i s h e d t h e increase  i n u t e r i n e weight u s u a l l y observed  superovulatory  treatment.  As p r e v i o u s l y m e n t i o n e d ,  d o s e s o f e s t r o g e n s may i n d u c e e n d o m e t r i a l hyperplasia. estrogen  with high  h y p e r t r o p h y and  I t i spossible that e a r l i e r elevations of  l e v e l s (24-48 h) may h a v e 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 . changes induced by t h i s second s t i m u l u s would t h e n be  65  The  superimposed over those e l i c i t e d by t h e f i r s t M a r t i n e t a l . (1973), estradiol,  stimulus.  u s i n g a s i n g l e i n j e c t i o n o f 17B-  found t h a t u t e r i n e weight and volume,  stromal  edema a n d 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 b e t w e e n 6-24 h .  However, i f a s e c o n d  i n j e c t i o n o f t h e same d o s e (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 immediately increased h i g h e r maxima w i t h i n 16-24 h.  t o new a n d  These r e s u l t s o f t h e u s e o f  s i n g l e and double 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 our  observations  o f 20 a n d 4 0 I U PMSG t r e a t e d  rats,  respectively. We o b s e r v e d t i m e - d e p e n d e n t i n c r e a s e s perimeter  and equated t h e s e i n c r e a s e s w i t h  With t h e exception  i n the luminal proliferation.  of controls, mitotic rates d i d not  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 . the  Despite  f o c a l a p p e a r a n c e 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 a n d 40 I U PMSG t r e a t m e n t g r o u p s a c t u a l l y d e c r e a s e d f r o m 48 t o 72 h . diffuse hyperplasia  A d d i t i o n a l l y , t h e development o f  i n 40 I U PMSG t r e a t e d r a t s was n o t  accompanied by increased m i t o t i c r a t e s . 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 sacrifices.  I t i spossible and o c c u r r e d  I t i s well established that estrogens  p r o l i f e r a t i o n r a t e through t h e shortening of t h e c e l l c y c l e  (Epifanova  between increase  o f G-^ a n d S p h a s e s  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).  In the resting state, the  m a j o r i t y o f c e l l s c a n be f o u n d i n e i t h e r G-^ o r G 66  that  Q  phases.  S t i m u l a t i o n w i t h estrogens and  m i t o s i s phases.  ovariectomized  promotes s y n c h r o n i z e d  Exogenous e s t r o g e n  rodents  have induced  entry into S  stimulationi n  cell  cycle lengths  less  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 epithelial  cells  e t a l . 1975).  ( 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  I t should  were i m m e d i a t e l y  placed  a l s o be n o t e d t h a t a l t h o u g h  i n f i x a t i v e , m i t o s i s may h a v e n o t  have been i n s t a n t l y a r r e s t e d . fixative, cells  Bullough  (1950),  observed progressive reductions  using  a t e a c h p h a s e o f m i t o s i s up t o 3 h , i n d i c a t i n g  n o t on t h e t i m e o f s a c r i f i c e .  Bouin's  i n t h e number o f  c e s s a t i o n o f a c t i v i t y was d e p e n d e n t u p o n f i x a t i v e and  tissue  that  penetration  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 the completion the next The  of fixation of  sacrifice. 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 I U PMSG t r e a t e d r a t s may h a v e b e e n c o i n c i d e n t a l a n d n o t r e l a t e d t o t h e presence o f hyperplasia.  Despite  concurrent  endometrial  r e g r e s s i o n i n t h e 20 I U PMSG g r o u p , s i m i l a r s t a i n i n g m a t e r i a l was  present  ( 9 6 - 1 2 0 h) i n a l l l u m i n a .  Histochemistry  m a t e r i a l suggested t h e presence o f sulphomucins. influence o f estrogens,  of this  Under t h e  s u l p h o m u c i n s may b e p r o d u c e d a n d  s e c r e t e d b y t h e e n d o m e t r i u m and c e r t a i n segments j  (isthmus,  a m p u l l a ) o f t h e o v i d u c t o f t h e r a t , mouse a n d r a b b i t  67  (LeBlond  1950, 1975).  Deane 1 9 5 2 , G r e e n w a l d 1969, Pharmacological  Hamner 1973,  Finn eta l .  doses o f estrogen induce  endometrial  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 subsequently transudated through 1958,  t h e e p i t h e l i u m i n t o t h e lumen  Heap e t a l . 1 9 6 2 ) .  (Zachariae  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 progesterone with estrogen has occurred.  p r o v i d i n g adequate p r i m i n g  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 progesterone  levels,  resulting 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 epithelium.  T h e r e 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 m o s t  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 . 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 those c o n s t i t u t e a m a j o r component o f o v i d u c t a l f l u i d Greenwald 1969).  I t i s  sulphated,  (Hamner 1 9 7 3 ,  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 occur mainly  i n t h e isthmus  region.  s t i m u l a t i o n by elevated estrogens  Perhaps o v e r t o v i d u c t a l  a t 9 6 h i n 4 0 I U PMSG  t r e a t e d r a t s produced copious q u a n t i t i e s o f mucinous material. these  T h e 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 b y 144 h i n  r a t s may h a v e r e s u l t e d f r o m t h e l e a k a g e  o f excess  m u c i n s i n t o t h e u t e r i n e lumen. Although PMSG t r e a t m e n t  t h e v a g i n a l c h a n g e s i n d u c e d b y 4, 20 o r 40 I U were analogous t o those 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 68  similar  mechanisms were p r e s e n t .  I t i sw e l l established that i n the  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 germ-line  c e l l s that uniformly p a r t i c i p a t e i n keratogenic or  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 progesterone, al.  r e s p e c t i v e l y (Peckham e t a l . 1 9 6 2 , L a d i n s k y e t  1965, Barker  i s t h i s uniform  e t a l . 1966, P a r a k k a l  1974).  Moreover, i t  p a r t i c i p a t i o n t h a t accounts f o r t h e wave-like  p r o g r e s s i o n o f c e l l s t o w a r d s t h e lumen.  I n our study,  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 and  i n v o l v e d two e p i s o d e s  treatment  o f m u c i f i c a t i o n separated  short-lived keratinization.  I t i s intriguing that  by a this  c o m p l e x s e q u e n c e o f e v e n t s was n o t p r e c e d e d b y s t e r o i d a l patterns generally associated with ovulation. Autoradiographic  studies following the progression of  d i f f e r e n t i a t i o n i n the untreated  intact or steroid  progesterone) t r e a t e d ovariectomized  (estrogen,  a d u l t r a t h a v e shown a  d e l a y o f l e a s t 48 h b e t w e e n 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 occurrence  al.  1963, Barker  e t a l . (1966),  o f i t s e f f e c t s ( S e l y e 1936,  e t a l . 1966, L e r o y  Bertalanflyet  e t a l . 1969).  Thrasher  however, observed e l e v a t e d l a b e l l i n g  during proestrus that dramatically f e l l  indices  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 the ovary  and e f f e c t .  Our r e s u l t s p r o v i d e  from  compelling  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 i m m a t u r e r a t , t h e v a g i n a l response 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 . hypothesis  i s f u r t h e r supported  This  by evidence 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 onset o f proestrus  (216-240 h) i n t h e a b s e n c e o f p r e c e d i n g  steroid  fluctuations. I t i snot possible t o provide induction of mucification that w i l l causative factors.  an e x p l a n a t i o n  unify a l lpotential  M u c i f i c a t i o n has been p r e v i o u s l y  described  i n t h e 10 a n d 15 d o l d r a t f o l l o w i n g  treatment  w i t h PMSG ( R e n n e l s  ovariectomized  for the  r a t s , Rennels  1951,  superovulatory  P a r l a n t i e t a l . 1980).  (1951) a c h i e v e d  In  comparable  r e s u l t s w i t h exogenous androgens and proposed  androgen  s t i m u l a t i o n may h a v e b e e n a n u n d e r l y i n g a c t i o n o f PMSG. K e n n e d y e t a l . (1976) d e m o n s t r a t e d i n a d r e n a l e c t o m i z e d ovariectomized capable  r a t s t h a t non- and a r o m a t i z a b l e  of inducing mucification.  androgens a r e  High l e v e l s o f p r o l a c t i n  may h a v e a l s o b e e n a c a u s a t i v e f a c t o r . showed t h a t a s u p e r o v u l a t o r y  R e n n e l s e t a l . (1977)  d o s e (50 I U ) o f PMSG  p r o l a c t i n l e v e l s through t h e a c t i o n o f estrogens aromatizable  and  a n d r o g e n s on t h e p i t u i t a r y .  increased  or  M o r e o v e r , 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 luteotrophic-progesterone  action.  This extra-ovarian action,  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  progesterone.  I n our study  elevation of  progesterone  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 established that progesterone induces mucinogenic differentiation e t a l . 1966).  ( S e l y e 1936, Elevated  B e r t a l a n f l y e t a l . 1963, B a r k e r  p r o g e s t e r o n e l e v e l s f r o m 168-240 h  p o s t 40 I U PMSG may e x p l a i n t h e e x c e s s i v e formation  o f enlarged  m u c i f i c a t i o n and  c y s t s n o t o b s e r v e d i n 20 I U PMSG  treated rats. 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 induce 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 the uterus  and vagina.  alterationsi n  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 . not  d o s e s o f PMSG  While i ti s  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 induced by s u p e r o v u l a t i o n interfere with pre-implantational require attention i n reference  71  preparations  t o implantation.  may  and t h e r e f o r e  Summary The  e f f e c t s of superovulatory treatment,  with regard 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 b y h i s t o l o g i c a l a n d histochemical techniques.  In addition,  (17B-estradiol, progesterone,  serum s t e r o i d  androgens) were  levels  monitored.  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 h a s b e e n made. u t e r i and vaginae  To s t u d y s h o r t t e r m  effects,  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 b e t w e e n do a n d d l O f o l l o w i n g 4, 20 a n d 40 I U PMSG t r e a t m e n t .  Examination  o f t h e long term  PMSG was c a r r i e d o u t a t d30 p o s t 4 a n d 40 I U PMSG.  effects of Adult  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 the p r o e s t r u s , e s t r u s , metestrus  and d i e s t r u s phases.  Using  r o u t i n e h i s t o l o g i c a l technique, 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 , mitotic index).  In addition, differentiation of thevaginal  e p i t h e l i u m was e x a m i n e d . induced  While treatment  changes i n u t e r i and vaginae  p h y s i o l o g i c a l events doses  t h a t mimicked superovulatory  ( 2 0 , 40 I U PMSG) p r o d u c e d s t r o m a l h y p e r t r o p h y a n d F o l l o w i n g 20 I U PMSG  e p i t h e l i a l h y p e r p l a s i a developed c o m p l e t e l y by dlO. r a t s developed still  w i t h 4 I U PMSG  of the adult estrus cycle,  epithelial hyperplasia.  ?  epithelial  treatment,  f o c a l l y before regressing  However, u t e r i  f r o m 40 I U PMSG t r e a t e d  more d i f f u s e a n d i n v a s i v e p a t h o l o g y  e v i d e n t by dlO b u t absent 72  b y d3 0.  t h a t was  The r e l a t i o n s h i p o f  t h e s e a b n o r m a l p a t t e r n s and c o n c o m i t a n t 1 7 B - e s t r a d i o l a n d a n d r o g e n l e v e l s was  f l u c t u a t i o n s o f serum  discussed.  Histochemical studies f o r the detection of carbohydrates,  e s p e c i a l l y m u c i n s , were u n d e r t a k e n  d i a s t a s e / a l c i a n b l u e pH 1.0 technique.  and 2 . 5 / p e r i o d i c a c i d  In superovulated rats  using the schiff  ( 2 0 , 4 0 I U PMSG), d o s e -  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  within  t h e u t e r i n e lumen a n d 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 , progesterone, w h i c h l e v e l s may  androgens and o t h e r  have been g r e a t l y e l e v a t e d by  hormones,  superovulatory  treatment. 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