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The effect of estrogenic alfalfa consumption in cyclic ewes : the plasma gonadotropin profile throughout… Hettle, J. Alan L. 1980

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c. THE EFFECT OF ESTROGENIC ALFALFA CONSUMPTION IN CYCLIC EWES: THE PLASMA GONADOTROPIN PROFILE THROUGHOUT THE ESTROUS CYCLE by J . ALAN L. HETTLE Sc. (Agr.), The U n i v e r s i t y of B r i t i s h Columbia, 197 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Department of Animal Science We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA September 1980 © J . Alan L. H e t t l e , 1980 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree that p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . J . Alan L. H e t t l e Department of Animal Science The U n i v e r s i t y of B r i t i s h Columbia 2357 Main M a l l Vancouver, B r i t i s h Columbia, Canada V6T 2A2 Date: OCT- 0 7 , l%0 ABSTRACT Phyto-estrogens are known to cause r e p r o d u c t i v e f a i l u r e s i n the ewe by i n t e r a c t i n g with u t e r i n e estrogen r e c e p t o r s . Further s t u d i e s have demonstrated anomalous ovarian s t e r o i d l e v e l s which i m p l i c a t e the hypothalamus and p i t u i t a r y . E strogen r e c e p t o r s which b i n d phyto-estrogens have been r e c e n t l y demonstrated i n ovine hypothalamic and p i t u i t a r y t i s s u e s . I t would then seem l o g i c a l to assume that estrogen i n t e r a c t i o n s i n these t i s s u e s w i l l be a l t e r e d and r e s u l t i n abnormal gonadotropic s y n t h e s i s or s e c r e t i o n . T h i s p r o j e c t employed v a g i n a l c y t o l o l o g i c a l s t u d i e s and ovine L u t e i n i z i n g Hormone radioimmunoassays to c h a r a c t e r i z e the plasma LH p r o f i l e s and estrous c y c l e s i n ewes f e d orchard grass hay or p h y t o - e s t r o g e n i c a l f a l f a . R e s u l t s i n d i c a t e t h a t the consumption of p h y t o - e s t r o g e n i c a c t i v i t y w i l l decrease the p u l s a t i l e nature of LH se c r e -t i o n and de l a y the LH peak f u r t h e r i n t o the e s t r u s p e r i o d when compared with ewes f e d a r a t i o n devoid of phyto-e s t r o g e n i c a c t i v i t y . V a g i n a l c y t o l o g i c a l s t u d i e s a l s o demonstrate p h y t o - e s t r o g e n i c a c t i v i t y through e l e v a t e d karyopyknotic index v a l u e s . i i TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS i i i LIST OF TABLES v i LIST OF FIGURES AND ILLUSTRATIONS v i i ACKNOWLEDGEMENT x i INTRODUCTION 1 REVIEW OF LITERATURE 4 1. ESTROGEN AND ESTROGEN RECEPTORS 4 2. ANT I ESTROGENS 13 3. PHYTO-ESTROGENS 21 4. THE HYPOTHALAMUS IN FEMALE REPRODUCTION 31 a. A n a t o m i c a l O r g a n i z a t i o n 31 b. N e u r o t r a n s m i t t e r s and the Hypothalamus 35 c. E x t r a h y p o t h a l a m i c S t r u c t u r e s i n Female R e p r o d u c t i o n 37 d. G o n a d o t r o p i n - R e l e a s i n g Hormone 40 i i i Page 5. THE PITUITARY IN FEMALE REPRODUCTION 43 6. GONADOTROPINS 4 5 a. G e n e r a l C o n s i d e r a t i o n s 45 b. S y n t h e s i s and S e c r e t i o n 46 c. R e c e p t o r s and A c t i v a t i o n 51 d. The O v a r i a n Response 55 7. THE HYPOTHALMIC-HYPOPHYSEAL-OVARIAN AXIS ... 62 a. The Ovine E s t r o u s C y c l e '. 6 2 b. The R o l e o f O v a r i a n S t e r o i d s 6 4 7 ? c. V a g i n a l C y t o l o g y d u r i n g the E s t r o u s C y c l e .. 8. A POTENTIAL ROLE OF PHYTO-ESTROGENS-IN REPRODUCTION 7 6 EXPERIMENTAL PROCEDURE 7 9 I . INTRODUCTION 7 9 I I . MATERIALS 82 I I I . METHODS 8 3 A. E s t r u s S y n c h r o n i z a t i o n 83 B. E x f o l i a t i v e V a g i n a l C y t o l o g y 86 C. Development o f the RIA 87 D. P r o t e i n I o d i n a t i o n 90 i v Page E. Anti-Rabbit Gamma G l o b u l i n T i t e r 9 0 F. Prepa r a t i o n of Gonadotropin Free Plasma .... 9 1 EXPERIMENTAL RESULTS I. PROCEDURAL DEVELOPMENT 9 5 A. P r o t e i n I o d i n a t i o n 9 5 B. Anti-RGG T i t e r Assay 9 7 C. Pr e p a r a t i o n of Gonadotropin Free Plasma .... 9 7 I I . TREATMENT EFFECTS A. Estrus Synchronization 1 0 5 B. E x f o l i a t i v e V a g i n a l Cytology 1 0 6 C. LH Plasma P r o f i l e s 1 1 2 DISCUSSION 1 1 6 BIBLIOGRAPHY • 1 2 1 APPENDIX 1 5 7 I. SPECIFICITY OF # 1 5 ANTI-OLH SERUM 1 5 7 I I . PROTEIN IODINATION 1 5 9 I I I . TNBS ASSAY 1 6 6 IV. ESTRUS SYNCHRONIZATION: BACKGROUND INFORMATION . 1 6 7 v LIST OF TABLES Table Page I Temporal sequence o f events i n the u t e r o t r o p h i c response t o E2~173 8 I I C l a s s i f i c a t i o n o f e s t r o g e n a g o n i s t s and a n t a g o n i s t s 22 I I I P h y t o - e s t r o g e n i c c o n t e n t o f the e x p e r i -m e n t a l r a t i o n s 81 v i Page LIST OF FIGURES AND ILLUSTRATIONS 1. A E s t r o g e n i n t e r a c t i o n w i t h the u t e r i n e t a r g e t c e l l 9a B H y p o t h e t i c a l mechanism f o r gene a c t i o n by s t e r o i d hormones 9b 2. S t r u c t u r a l c o n f i g u r a t i o n s o f n a t u r a l and s y n t h e t i c e s t r o g e n s 12 3. A n t i e s t r o g e n and e s t r o g e n t a r g e t c e l l i n t e r a c t i o n s I 5 4 . S t r u c t u r a l f o r m u l a o f s e v e r a l n o n - s t e r o i d a l a n t i e s t r o g e n s 1 7 5. S t r u c t u r a l f o r m u l a of the p h y t o - e s t r o g e n s 26 6 . P h y t o - e s t r o g e n m e t a b o l i t e s 27 7. A V e n t r a l s u r f a c e o f the sheep b r a i n 33 B Median s e c t i o n o f the sheep b r a i n 33 8. M i d s a g i t a l s e c t i o n o f the r a t d i e n c e p h a l o n 34 / v i i Page 9 . A Mechanism of a c t i o n proposed f o r GnRH .... 49a B Estrogen and GnRH i n t e r a c t i o n i n the pro-posed 2 p o o l hypothesis f o r gonadotropin . 49b 10. P r o s t a g l a n d i n b i o s y n t h e s i s and i n t e r -mediates 50 11. A The mo b i l e - r e c e p t o r hypothesis 55 B The g l y c o p r o t e i n - r e c e p t o r - a d e n y l a t e c y c l a s e i n t e r a c t i o n 55 12. S t e r o i d b i o s y n t h e s i s i n human o v a r i a n t i s s u e 59 13. Hormonal changes d u r i n g the estrous c y c l e of the ewe 63a 14. The v a g i n a l mucosa 71 15. The endocrine r e p r o d u c t i v e system i n females 73 16. E l u t i o n p a t t e r n f o r 1 2 5 I - o L H and 1 2 5 I - o F S H 96 17. A n t i - r a b b i t gamma g l o b u l i n t i t e r a n a l y s i s 98 v i i i Sucrose d e n s i t y c e n t r i f u g a t i o n : e l u t i o n 125 p r o f i l e of r e c e p t o r b i n d i n g to I-oLH 12 5 I-oLH b i n d i n g versus q u a n t i t y of r e c e p t o r used to absorb oLH per m i l l i -l i t r e o f plasma oLH standard curves i n plasma before and a f t e r r e c e p t o r a b s o r p t i o n L o g i t t r a n s f o r m a t i o n of oLH standard curves i n plasma before and a f t e r r e c e p t o r a b s o r p t i o n '. C o n t r o l and experimental karyopyknotic index values V a g i n a l smears obtained d u r i n g the e s t r u s p e r i o d V a g i n a l smears c h a r a c t e r i s t i c of the d i e s t r u s p e r i o d Plasma LH p r o f i l e i n ewes fed orchard grass hay ( c o n t r o l r a t i o n ) i x Plasma LH p r o f i l e i n ewes f e d a l f a l f a cubes ( p h y t o - e s t r o g e n i c ) ACKNOWLEDGEMENT The author wishes to express h i s g r a t i t u d e to those people who c o n t r i b u t e d to t h i s t h e s i s and i n p a r t i c u l a r : Dean W.D. K i t t s f o r h i s encouragement and support throughtout t h i s study. Dr. D.N. Ward, Department Head, Bi o c h e m i s t r y , The M.D. Anderson H o s p i t a l and Tumour I n s t i t u t e , Houston, f o r h i s c o n t r i b u t i o n of p u r i f i e d oLH. Dr. G.D. Niswender, p r o f e s s o r , Department of Phys i o l o g y and B i o p h y s i c s , Colorado State U n i v e r s i t y , F o r t C o l l i n s , f o r h i s c o n t r i b u t i o n of anti-oLH serum and t e c h n i c a l a d v i c e . Dr. L.E. R e i c h e r t , J r . , Department of Bio c h e m i s t r y , Emory U n i v e r s i t y , School of Medicine, A t l a n t a , f o r h i s con-t r i b u t i o n of p u r i f i e d oFSH. K l a r a Shekhtmann and Roger Bragg f o r t h e i r t e c h n i c a l and p r a c t i c a l e x p e r t i s e . xi INTRODUCTION A normal and completely i n t e g r a t e d hypotha-lamo-adenohypophysis-gonadal a x i s i s c r u c i a l to the occurrence o f a r e g u l a r f e r t i l e e s t r o u s c y c l e . The hypothalamus can c o n t r o l the adenohypophysis when i t processes n e u r a l sensory inputs i n t o r e l e a s i n g hormone(s) which are c a r r i e d to the a n t e r i o r p i t u i t a r y through a p o r t a l v a s c u l a r system. Extrahypothalamic areas which i n c l u d e the p i n e a l and l i m b i c s t r u c t u r e s , a l s o i n f l u e n c e the i n c i d e n c e and refinement of these n e u r a l impulses. The R e l e a s i n g Hormone(s) s t i m u l a t e the adeno-hypophysis to r e l e a s e gonadotropins (hence the name Gona-d o t r o p i n R e l e a s i n g Hormone GnRH) i n t o the gene r a l c i r c u l a -t i o n to induce o v a r i a n f o l l i c u l a r development with sub-sequent s t e r o i d b i o s y n t h e s i s , o v u l a t i o n and corpus luteum formation. The o v a r i a n s t e r o i d s , i n p a r t i c u l a r the e s t r o -gens and progestagens, then exert p o s i t i v e and negative feedback e f f e c t s on the hypothalamus, higher c e n t r e s w i t h i the CNS and the a n t e r i o r p i t u i t a r y . Through these s t r u c -t u r e s o v a r i a n s t e r o i d s can modify the s y n t h e s i s and se c r e -t i o n of gonadotropin. 2. The i n t e r p l a y between GnRH, the gonadotropins, i . e . , F o l l i c l e S t i m u l a t i n g Hormone (FSH) and L u t e i n i z i n g Hormone (LH), the estrogens and progesterone i s a c u t e l y important. Therefore any intake of exogenous hormone which i s of s u f f i c i e n t d u r a t i o n and magnitude w i l l d i s r u p t c o n t r o l of the o v a r i a n c y c l e . Such i s the case when animals i n g e s t " e s t r o g e n i c " p a s t u r e s c o n t a i n i n g p r i n c i p a l l y coumestrol and g e n i s t e i n . These phyto-estrogens are capable of a c t i n g as endogenous estrogen a g o n i s t s and a n t a g o n i s t s depending upon the animal's p r e v a i l i n g estrogen blood p r o f i l e . T h i s i s p o s s i b l e because p l a n t estrogens approximate the s t r u c t u r e of E s t r a d i o l -173 (E2~17B) and c o n t a i n the c r i t i c a l f u n c t i o n a l groups. Hence they may b i n d and r e a c t with c y t o s o l r e c e p t o r s i n E2~176 t a r g e t t i s s u e s . Consumption of these compounds i s a s s o c i a t e d w i t h permanent or temporary l o s s i n f e r t i l i t y which has been a t t r i b u t e d p r i n c i p a l l y to impaired sperm and ovum t r a n s p o r t . However, abnormal f o l l i c u l o g e n e s i s and s t e r o i d -ogenesis have been demonstrated i n these animals conse-quently the hypothalamus and p i t u i t a r y have been i m p l i c a t e d . The f o l l o w i n g t e x t w i l l d i s c u s s the estrous c y c l e i n depth with the s p e c i f i c i n t e n t i o n of i d e n t i f y i n g the p o s s i b l e s i t e s where phyto-estrogens may a f f e c t the 3. hypothalamo-adenohypophysis-gonadal axis. Special con-siderations w i l l be developed for the hypothalamus and p i t u i t a r y as i t i s hypothesized that gonadotropin output by the adenohypophysis w i l l be modified by phyto-estrogen consumption. Experimental results w i l l be u t i l i z e d in an attempt to demonstrate this area of concern. 4. REVIEW OF LITERATURE 1. ESTROGENS AND ESTROGEN RECEPTORS N a t u r a l l y o c c u r r i n g estrogens are t y p i c a l l y 18-carbon s t e r o i d s which contain-OH and/or ketone f u n c t i o n a l groups at carbon atoms 3 and 17. Estr o n e , e s t r a d i o l -176 C p h y s i o l o g i c a l responses are a t t r i b u t e d to E2~17B) and e s t r i o l predominate, however, e s t r i o l appears only when s y n t h e s i z e d by a f u n c t i o n a l p l a c e n t a . E s t r a d i o l - 1 7 g i s the major estrogen s e c r e t e d by the theca i n t e r n a o f the ovary ( M a r t i n , 1976). However, recent and c o n c l u s i v e r e s u l t s i n v i t r o ( Dorrington et a l . , 1975; Moon et a l . , 1975; Moon et a l . , 1978) i n d i c a t e t h a t the t h e c a l c e l l s s y n t h e s i z e o v a r i a n androgen which i s then aromatized to e s t r a d i o l - 1 7 6 by the granulosa c e l l s . T h i s w i l l be d i s c u s s e d i n g r e a t e r depth i n the f o l l o w i n g s e c t i o n s . Estrone and e s t r a d i o l - 1 7 B are b i o l o g i c a l l y i n t e r -c o n v e r t i b l e but p r e v a i l i n g c o n d i t i o n s i n the c i r c u l a t o r y system favor formation of estrone hence i t i s one of the major estrogens i n bloo d plasma and along with E2-I73 appears conjugated e i t h e r to s u l f a t e or glucuronate. The f r e e and conjugated 5. estrogens are t r a n s p o r t e d to t h e i r t a r g e t t i s s u e s by b i n d i n g w i t h h i g h a f f i n i t y to sex s t e r o i d - b i n d i n g g l o b u l i n (SSBG) more commonly known as sex s t e r o i d b i n d i n g plasma p r o t e i n (SBP) , ( M a r t i n , 1976; Mercier-Bodard et a l . , 1977; Funder, 1978). T r a n s p o r t a t i o n a l s o occurs when sex s t e r o i d s b i n d to plasma albumins which have a low a f f i n i t y f o r the s t e r o i d s but demonstrate many r e a c t i v e s i t e s which r e s u l t s i n a high c a p a c i t y to b i n d and c a r r y the c i r c u l a t i n g s t e r o i d s . Imperative to the concept of t a r g e t t i s s u e , c e l l s must have the a b i l i t y to r e c o g n i z e and d i s t i n g u i s h between the c i r c u l a t i n g s t e r o i d hormones. The r e c o g n i t i o n mechanisms are achieved because t a r g e t c e l l s c o n t a i n r e c e -p t o r s that have the p r o p e r t i e s which f a c i l i t a t e the a b i l i t y to b i n d s p e c i f i c hormones and as a consequence i n i t i a t e a response through the c e l l ' s molecular apparatus. By i n j e c t i n g t r i t i a t e d e s t r a d i o l - 1 7 6 i n t o immature female r a t s and determining the d i s t r i b u t i o n of r a d i o a c -t i v i t y Jensen ejt al. (1960, 1962) demonstrated that c e l l s are capable of b i n d i n g and c o n c e n t r a t i n g s p e c i f i c s t e r o i d s . The accepted model f o r sex s t e r o i d - r e c e p t o r i n t e r a c t i o n s i s the u t e r u s . Through t h i s model the mech-anism of s t e r o i d a c t i o n has been demonstrated (Gorski et a l . , 1968; Giannopoulos e t al. , 1971; G o r s k i e_t a l . , 1976; O'Malley et a l . , 1976). 6. Upon e n t e r i n g the u t e r i n e c e l l E2~17$ r a p i d l y a s s o c i a t e s w i t h an 8S b i n d i n g p r o t e i n i n the c y t o s o l . T h i s hormone-receptor complex undergoes a c o n f o r m a t i o n a l change and w i t h i n 1 hour appears w i t h i n the n u c l e a r compart-ment (Stumpf, 1969; Giannopoulos et a l . , 1971; O'Malley et a l . , 1976). The estrogen now appears bound to a 4-5S component (Gorski et a l . , 1968) and a s s o c i a t e s w i t h s p e c i -f i c n o n h i s t i n e p r o t e i n s of the chromatin e f f e c t i n g t r a n s -c r i p t i o n a l changes, mRNA formation and subsequent c e l l u l a r m e t a b o l i c responses (Means et a l . , 1972; C o r s k i et a l . , 1968; O'Malley and Schrader, 1976; G o r s k i and Gannon, 1976; G o r s k i et a l . , 1977). Changes w i t h i n the t a r g e t c e l l induced by E^-17g i n c l u d e i n c r e a s e d water uptake and r e t e n t i o n , i n c r e a s e d n i t r o g e n r e t e n t i o n , i n c r e a s e d RNA polymerase a c t i v i t y and b i o s y n t h e s i s of 'induced p r o t e i n ' , an e a r l y i n d i c a t i o n of estrogen a c t i o n (Katzenellenbogen et_ ja l . , 1975a) . For a macromolecule to be c o n s i d e r e d a r e c e p t o r i t must meet s e v e r a l s t r i n g e n t c r i t e r i a (Munck, 1976) : ( i ) The b i n d i n g molecule must be present i n the t a r g e t c e l l s , ( i i ) S p e c i f i c i t y of b i n d i n g ; the b i n d i n g p r o t e i n must have the a b i l i t y to form complexes with molecules of complementary (and 7. o b l i g a t o r y ) s t e r e o c h e m i c a l c o n f i g u r a t i o n s and be able to exclude i n a p p r o p r i a t e s t r u c t u r e s . ( i i i ) High a f f i n i t y ; the a b i l i t y and s t r e n g t h w i t h which a r e c e p t o r w i l l i d e n t i f y and b i n d a s p e c i f i c molecule. T h i s p r i n c i p l e a l s o c o u n t e r a c t s the low c o n c e n t r a t i o n s at which hormones are normally present, ( i v ) The molecule must demonstrate a hi g h a f f i n i t y ( a s s o c i a t i o n ) constant (K^) to i t s s p e c i f i c s t e r o i d hormone, (v) The b i n d i n g p r o t e i n must e x h i b i t f i n i t e or s a t u r a b l e b i n d i n g . N o n - s p e c i f i c b i n d i n g r e p r e s e n t s low a f f i n i t y , non-saturable b i n d i n g . A s s o c i a t i o n of a s t e r o i d to i t s r e s p e c t i v e t a r g e t c e l l r e c e p t o r i n v o l v e s a combination of s e v e r a l p h y s i c a l p r o p e r t i e s . The p r i n c i p a l i n t e r a c t i o n s are hydrophobic and hydrogen bonding. I t i s these molecular f o r c e s that determine a f f i n i t y and s p e c i f i c i t y between s t e r o i d - r e c e p t o r molecules. S t r i c t s t e r e o c h e m i c a l c o n f i g u r a t i o n s are inh e r e n t to the concept of s p e c i f i c i t y and a f f i n i t y w i t h the r e s u l t s t e r o i d s t r u c t u r a l conformation i s c r i t i c a l to proper 8. Table 1 Temporal sequence of metabolic events i n u t e r i of o v a r i e c t o m i z e d r a t s a f t e r i n v i v o a d m i n i s t r a -t i o n of estrogen. TIME METABOLIC RESPONSE 36 sec. 72 sec. 360 sec. 1 hour 5 hours 1-020 hours 30 hours 9.55 s t e r o i d - r e c e p t o r complex T r a n s l o c a t i o n of s t e r o i d - r e c e p t o r complex to n u c l e a r compartment Induced p r o t e i n (I.P.) s y n t h e s i s and nucleoplasmic polymerase II N u c l e o l a r RNA polymerase I (I.P. s y n t h e s i s ) , glucose metabolism P r o t e i n s y n t h e s i s , water i n h i b i t i o n H i s t o n e , DNA, net RNA s y n t h e s i s M i t o s i s From G o r s k i et a l . (1969) and K i t t s (1976). 9a. F I G U R E 1A Uterine Cell Estrogen Figure 1A: Hypothetical model for estrogen interation with the uterine cell (Gorski et al. 1968). FIGURE 1B 9b. Nuclear hormone-receptor complex n o n Histonesopr Acceptor Site E ^ Dissociation mRNA E RNA Polymerase Figure 1B: Hypothetical mechanism of gene action by the steroid hormones (O'Malley and Schrader. 1976) 10. s p e c i f i c b i n d i n g and the subsequent c e l l u l a r response (King and Mainwaring, 1974; Munck, 1976). I t appears that the g r e a t e s t b i n d i n g a f f i n i t y -i s o b t ained i f the n a t u r a l s t e r o i d r i n g c o n f i g u r a t i o n has an unsaturated r i n g A, a p h e n o l i c hydroxyl group on C-3, a l c o h o l i c h y d r o x y l group on C-17 (Hahnel et_ a l . , 1973) and s u b s t i t u t i o n s w i t h i n r i n g D (Korenman, 1969). Maximum b i n d i n g a f f i n i t y between r e c e p t o r and s t e r o i d i s achieved w i t h e s t r a d i o l - 1 7 6 , the most important f e a t u r e i s the p h e n o l i c h y d r o x y l group on C-3. I f t h i s group i s m i s s i n g or s u b s t i t u t e d the a f f i n i t y towards the r e c e p t o r i s l a r g e l y or e n t i r e l y a b o l i s h e d . F u r t h e r to t h i s , r i n g A must be a benzene r i n g w i t h the r e s u l t the hydroxyl group must be p h e n o l i c and not a l c o h o l i c . The second oxygen f u n c t i o n on r i n g D i s important. I t s s t a t e of o x i d a t i o n and p o s i t i o n w i l l i n f l u e n c e the b i n d i n g a c t i v i t y . Maximum b i n d i n g occurs when the oxygen f u n c t i o n i s an a l c o h o l i c h ydroxyl group on C-17 i n the 3 - c o n f i g u r a t i o n (Korenman, 1969; Hahnel e_t a l . , 1973). A d d i t i o n of f u r t h e r oxygen f u n c t i o n s to r i n g D, a d d i t i o n a l s u b s t i t u e n t s on r i n g A and/or u n s a t u r a t i o n o f r i n g B w i l l decrease the a f f i n i t y f o r r e c e p t o r and prove i n h i b i t o r y to b i n d i n g , s p e c i f i c a l l y - O H groups on C - l l or -methyl s u b s t i t u t i o n s on C-2 and C-16. 11. The two n o n - s t e r o i d a l estrogens, h e x e s t r o l and d i e t h y l s t i l b e s t r o l (DES), had b i n d i n g a f f i n i t i e s comparable to e s t r a d i o l - 1 7 6 with b i n d i n g c o n s i d e r e d to occur through the two p h e n o l i c hydroxyl groups (Hahnel et a l . 1973). However, Korenman (1969) u s i n g a r a b b i t c y t o s o l as opposed to human c y t o s o l (Hahnel et a l . , 1973) found a f f i n i t i e s f o r DES and 1 7 6 - e t h y n y l - e s t r a d i o l - 1 7 3 c o n s i d e r a b l y stronger than E2-17.B. Bindin g i s c o n s i d e r e d to i n v o l v e attachment of small molecules ( s t e r o i d s - l i g a n d s ) to r e c e p t o r s at two p o i n t s i n d u c i n g a c o n f o r m a t i o n a l change i n the r e c e p t o r . The f l e x i b l e a c t i v e s i t e of the r e c e p t o r may c o n s i s t of two p a r t s , an a t t r a c t i v e centre which determines the s p e c i -f i c i t y and o r i e n t a t i o n of the l i g a n d , and a second centre l e s s s p e c i f i c than the f i r s t . O b l i g a t o r y to t h i s concept, i t has been demons-t r a t e d t h at there i s a n e c e s s i t y f o r the l i g a n d to have two attachment p o i n t s . The optimum requirements f o r t h i s two p o i n t i n t e r a c t i o n e x i s t w i t h e s t r a d i o l - 1 7 8 where the p h e n o l i c -OH group on C-3 and the C-17B -OH group are a c t i v e b i n d i n g c e n t r e s (Hahnel et a l . , 1973). I n i t i a l l y there i s attachment between the C-3 p h e n o l i c hydroxy group and the primary b i n d i n g c e n t r e . T h i s induces a c o n f o r m a t i o n a l change w i t h i n the r e c e p t o r FIGURE 2 Diethylstilbestrol Figure 2*. Structural configurations of natural synthetic estrogens. 13. which f a c i l i t a t e s the second attachment of the C-17B -OH f u n c t i o n and the second l e s s s p e c i f i c b i n d i n g c e n t r e . For t h i s process to occur, an a d d i t i o n a l requirement must be i n c l u d e d . The d i s t a n c e between the two -OH groups o on the e s t r a d i o l - 1 7 6 molecule i s approximately 10.6 A (Hahnel et a l . , 1973) and i s a p r e r e q u i s i t e f o r optimal b i n d i n g . C o n s i d e r i n g the two n o n - s t e r o i d a l estrogens, h e x e s t r o l i s a f l e x i b l e molecule and i t s two -OH f u n c t i o n a l groups can adapt to t h i s d i s t a n c e . DES i s a more r i g i d molecule due to the carbon-carbon double bond and can o t h e r e f o r e only achieve a d i s t a n c e of 12.0 A between func-t i o n a l groups. 2. ANTIESTROGENS Sankaran and Prasad (1972) and Geynet et a l . (1972) d e f i n e a n t i e s t r o g e n s as compounds which antagonize or i n t e r f e r e with any a c t i o n s of estrogen, p r i n c i p a l l y e s t r a d i o l - 1 7 6. True a n t i e s t r o g e n s demonstrate two proper-t i e s : f i r s t , they e x h i b i t c o m p e t i t i o n with e s t r a d i o l -176 f o r cy t o p l a s m i c r e c e p t o r s i t e s w i t h i n t a r g e t t i s s u e s (Geynet e_t al. , 1972; Rochefort ejt a_l. , 1972 ; Sankaran and Prasad, 1972; Jordan e_t a_l. , 1978); secondly, 14. a n t i e s t r o g e n s e x h i b i t v a r y i n g degrees of e s t r o g e n i c i t y s p e c i f i e d with r e f e r e n c e to t a r g e t t i s s u e , route of adminis-t r a t i o n , dose and time sequence of a c t i o n (Sankaran and Prasad, 1972). Terenius and L j u n g k v i s t (1972) demonstrated a n t i e s t r o g e n e s t r o g e n i c i t y ( i . e . , u t e r o t r o p h i c e f f e c t ) with p o s i t i v e A l l e n Doisy and mouse u t e r i n e growth t e s t s . Through t h e i r b i p h a s i c a c t i o n , a n t i e s t r o g e n s may a l s o be c o n s i d e r e d a g o n i s t i c to a l e s s e r degree than E2~17B as w e l l as a n t a g o n i s t i c to the u t e r o t r o p h i c e f f e c t s of e s t r a d i o l - 1 7 3 • Depending upon the p h y s i o l o g i c a l s t a t e and endogenous estrogen content of the animal, a n t i e s t r o -gens at low c o n c e n t r a t i o n by themselves are able to cause v a r y i n g e s t r o g e n i c e f f e c t s . However, as a c r i t i c a l concen-t r a t i o n i s reached they become an t a g o n i s t s and i n h i b i t the u t e r o t r o p h i c response whether by themselves or i n d i r e c t c o m p e t i t i o n with estrogens (Geynet et a l . , 1972; Ruh and Ruh, 1974; Capony and Rochefort, 1975). The exact s i t e s or mechanism of a c t i o n of the a n t i e s t r o g e n s have not been e l u c i d a t e d , however based on work r e p o r t e d , i t i s l o g i c a l to assume that the s i t e s w i l l p a r a l l e l the u t e r o t r o p h i c responses of e s t r a d i o l -17B (Geynet et a l . , 1972; Rochefort et a l . , 1972). These s e r i e s are demonstrated i n Figure 3 as a f i g u r e of steps: 15. FIGURE 3 Target cell Nucleus 1. Competition with estrogen to enter c e l l 2. I n h i b i t i v e b i n d i n g to c y t o s o l r e c e p t o r 3. Abnormal t r a n s l o c a t i o n of r e c e p t o r - s t e r o i d complex i n t o the nucleus 4. I n h i b i t i o n of t r a n s f o r m a t i o n i n t o the n u c l e a r r e c e p t o r complex 5. A t y p i c a l i n t e r a c t i o n s with the n u c l e a r a c c e p t o r 6. Incomplete or abnormal r e c e p t o r r e c y c l i n g or r e s y n t h e s i s , RNA s y n t h e s i s and subsequent induced p r o t e i n s y n t h e s i s . F i g u r e 3 Proposed s i t e s of a n t i e s t r o g e n a c t i o n (Rochefort et al*. 1972) 16. Step 1. Competition to enter the c e l l . 2. I n h i b i t i v e b i n d i n g to c y t o s o l r e c e p t o r . 3. Abnormal t r a n s l o c a t i o n of r e c e p t o r complex to the nucleus. 4. I n h i b i t i o n of t r a n s f o r m a t i o n to n u c l e a r -r e c e p t o r . 5. A t y p i c a l i n t e r a c t i o n s with n u c l e a r acceptor. 6. Incomplete or abnormal RNA s y n t h e s i s and subsequent induced p r o t e i n s y n t h e s i s . The major n o n - s t e r o i d a n t i e s t r o g e n s used to determine the extent and s i t e s of i n t e r f e r e n c e are: chlomi-phene ( c i s and t r a n s ) , nitromophene c i t r a t e (CI-628), n a f o x i d i n e (U-11,100A), tamoxifen (ICI-46,474), ethamoxy-t r i p h e t o l (MER 25) and to a small extent d i m e t h y l s t i l b e s t r o l (see F i g u r e 4 ) . Two types of a n t i e s t r o g e n s can be d i s t i n g u i s h e d based upon t h e i r b i n d i n g a f f i n i t i e s and a b i l i t y to e f f e c t a u t e r o t r o p h i c response (Korenman, 1969; Rochefort e_t a l . , 1972) . (1) Compounds which i n h i b i t the uptake of 176 i n u t e r i and/or b i n d i n g to i t s r e c e p t o r . The r e l a t i v e a f f i n i t y o f these a n t i e s t r o g e n s f o r r e c e p t o r i s much higher than t h e i r r e l a t i v e u t e r o t r o p h i c response. 17. Figure 4 : Structural formula of several non-steroidal antiestrogens, ) 18. FIGURE 4 (cont'd) GH^ Dimethystilbestrd ( D M 5 ) 19. Consequently, the b i o l o g i c a l a c t i o n i s blocked i n at l e a s t one of the above s t e p s . Examples of t h i s type of a n t i -estrogen are: n a f o x i d i n e , tamoxifen and clomiphene. (2) A n t i e s t r o g e n s of t h i s type e x h i b i t no a f f i n i t y f o r the estrogen c y t o s o l r e c e p t o r which suggest a d i f f e r e n t mechanism of a c t i o n . Examples are progesterone and t e s t o -sterone . Important to t h i s d i s c u s s i o n are the a n t i e s t r o g e n s d e s c r i b e d i n type ( 1 ) . These compounds can be f u r t h e r c a t e g o r i z e d (see Table 2) based on t h e i r proposed mechanism of a c t i o n and time sequence (Terenius and L j u n g k v i s t , 1972; Ruh and Ruh, 1974; Jordan et a l . , 1978): Type 1 ( a ) : Long A c t i n g A n t i e s t r o g e n s The t r i p h e n y l e t h y l e n e estrogen d e r i v a t i v e s n a f o x i -d i n e, tamoxifen, clomiphene and CI 628 have been u t i l i z e d e x t e n s i v e l y to e l u c i d a t e the mechanism of a c t i o n . These compounds occur i n t h i s category due to t h e i r e f f e c t of i n c r e a s i n g the n u c l e a r r e t e n t i o n time of the s t e r o i d -r e c e p t o r complex. Tamoxifen has been shown to maintain a c o n s i s t e n t l y e l e v a t e d c o n c e n t r a t i o n of estrogen r e c e p t o r w i t h i n the nucleus up to 48 hours, whereas the i n i t i a l r i s e i n n u c l e a r accumulation produced by estrogen r e t u r n s to c o n t r o l l e v e l s w i t h i n 24 hours. r 20. Another p r o p e r t y of these compounds i s t h e i r prolonged plasma b i o l o g i c a l h a l f - l i f e (t^) . In the r a t , E2~176 e x h i b i t s a b i o l o g i c a l h a l f - l i f e o f a few hours while tamoxifen i s maintained f o r s e v e r a l days (Jordan et a l . , 1978). C a r r o l l and Cox (1972) have r e p o r t e d the t j f o r E2~176 i n the ewe as 18-22 minutes. Even though estrogen r e c e p t o r i s r e s y n t h e s i z e d and r e c y c l e d the e l e v a t e d l e v e l s of a n t i e s t r o g e n , due to t h e i r prolonged b i o l o g i c a l h a l f - l i f e , w i l l c o n t i n u a l l y b i n d and t r a n s l o c a t e the recep-t o r to the n u c l e a r compartment (Baudendistel et a_l. , 1978) . The t a r g e t t i s s u e then becomes de p l e t e d i n c y t o s o l r e c e p t o r and p r o g r e s s i v e l y r e f r a c t o r y to E2~170 s t i m u l a t i o n . Type 1 (b): Short A c t i n g A n t i e s t r o g e n s or 'Impeded' Estrogens. Compounds such as e s t r i o l and DMS are termed short a c t i n g or 'impeded' estrogens, even though they have r e l a t i v e l y high a f f i n i t y f o r r e c e p t o r s , because they are r a p i d l y l o s t from the r e c e p t o r . Consequently, there i s sub-optimal a c t i v a t i o n of the r e c e p t o r and an a t y p i c a l n u c l e a r r e a c t i o n . The o v e r a l l e f f e c t i s an incomplete and d i m i n i s h e d e s t r o g e n i c response. Not only do the a g o n i s t i c and a n t a g o n i s t i c proper-t i e s o f a n t i e s t r o g e n s depend upon the p h y s i o l o g i c a l s t a t e of the animal due p r i n c i p a l l y to f l u c t u a t i o n s i n c y t o s o l r e c e p t o r (Geynet et _al. , 1972) , they a l s o appear to be 21. a s s o c i a t e d w i t h s p e c i f i c t i s s u e s ( C i d l o w s k i and Muldoon, 1976) . U t i l i z i n g a n t e r i o r p i t u i t a r y , hypothalamus and u t e r i n e r a t t i s s u e s C i d l o w s k i and Muldoon (1976) were able to demonstrate that CI-628 caused ex t e n s i v e d e p l e t i o n of c y t o p l a s m i c r e c e p t o r s by t r a n s l o c a t i o n to the nucleus i n a n t e r i o r p i t u i t a r y and u t e r i n e t i s s u e s but not i n the hypothalamus. R e s u l t s a l s o i n d i c a t e a complete l a c k of re c e p t o r replenishment. A p p l i c a t i o n of MER 25 was f o l l o w e d by a moderate d e p l e t i o n and a short s i g n i f i c a n t phase of replenishment i n c y t o s o l r e c e p t o r . As with CI 628, MER 25 was e f f e c t i v e i n the p i t u i t a r y and uterus but not i n the hypothalamus. DMS demonstrated e f f e c t s intermediate to CI 628 and MER 25 but was a potent inducer of r e c e p t o r replenishment p a r t i c u l a r l y i n the hypothalamus. 3. PHYTO-ESTROGENS Bennetts e_t a l . (1946) were the f i r s t to recog-n i z e that i n f e r t i l i t y i n sheep of western A u s t r a l i a was caused by subterranan c l o v e r consumption. The substances i m p l i c a t e d were of two p r i n c i p a l c a t e g o r i e s ; tlie i s o f l a v o n e s ( g e n i s t e i n , formononetin, etc.) and coumestans (coumestrol, e t c . ) . These compounds as w e l l as b i o c h a n i n A, d i a d z e i n , Table 2 C l a s s i f i c a t i o n of estrogen a g o n i s t s and a n t a g o n i s t s (a) CLASS EXAMPLES NUCLEAR RETENTION PHARMACOLOGIC CHARACTERISTICS UTEROTROPHIC PROPERTIES 1. Short acting 2. Long acting e s t r i o l DMS 16-oxo-estradiol estradiol DES Short (1-4 hr.) Intermediate (6-24 hr.) B. triphenylethy- Long acting, greater lene derivatives than 24-48 hr. e.g. Nafoxidine Tamoxifen antagonistic when injected, agonistic when implanted Agonistic Agonistic - one injection Antagonistic -multiple injec-tions early responses early and late responses early and late responses Early responses: Late responses: water imbibition, hypermia, amino acid and nucleotide uptake, activation of RNA polymerase I and II, stimulation of induced protein, cellular hypertrophy and hyperplasia, sustained RNA polymerase I and II activity. (a) Table reproduced from Mathieson (1979). 23. b e n z o f u r a n o c o u i n a r i n and p s o r a l i d i n were subsequently-i s o l a t e d and c h a r a c t e r i z e d (Bradbury and White, 1951; Bradbury and White, 1954; Moule et a l . , 1963; B i c k o f f et a l . , 1969; L i v i n g s t o n , 1978). The i s o f l a v o n e s (genestein) and coumestans (coumes-t r o l ) have demonstrated t h e i r a b i l i t y to d i s p l a c e and compete wi t h e s t r a d i o l - 1 7 6 f o r estrogen r e c e p t o r s i n t a r g e t t i s s u e s (Shutt, 1967; Foman and Pope, 1966; Folman and Pope, 1969; Lindner, 1976). The phyto-estrogen r e c e p t o r complex i s then able to t r a n s l o c a t e i n t o the nucleus i n d u c i n g an e s t r o g e n i c response (Noteboom and G o r s k i , 1963; Ostrovsky and K i t t s , 1963; Foman and Pope, 1969). The response i s t y p i f i e d by water i m b i b i t i o n , i n c r e a s e s i n RNA, p r o t e i n , p h o s p h o l i p i d and a c i d s o l u b l e u r i d i n e t r i p h o s p h a t e (UTP) i n the uterus of the o v a r i e c t o m i z e d r a t (Noetboom and G o r s k i , 1963; K i t t s , 1976; K i t t s et a l . , 1980). Since the d i s c o v e r y by Bennetts e_t a l . (1946) more than 50 p l a n t s p e c i e s have been shown to c o n t a i n e s t r o g e n i c substances (Bradbury and White, 1954: K i t t s et a l . , 1959; B i e l e y and K i t t s , 1964). A c c o r d i n g to Lindner (review: 1976) the most important pasture and forage crops shown to c o n t a i n phyto-estrogen i n c l u d e : T r i f o l i u m subter- aneum L. ( c l o v e r c u l t i v a r s ; Dwalganup, Mt. Baker, Yarloop, M a r r a r ) , T. pratense (red c l o v e r ) , T. f r a g i f e r u m (strawberry 24. c l o v e r ) , Medicago s a t i v a (Lucerne, a l f a l f a ) and Soya  h i s p i d a (soya beans). V a r i a t i o n i n coumestrol and g e n i s t e i n content and i n c i d e n c e has been a t t r i b u t e d to p l a n t v a r i e t y , c u t t i n g stage of growth, disease and f u n g a l i n f e c t i o n (Moule et a l . , 1963; Hanson et a l . , 1965; B i c k o f f e t > a l . , 1969; K i t t s e_t a_l. , 1969; Cox and Braden, 1974; L i v i n g s t o n , 1978). When dis e a s e and f u n g a l i n f e c t i o n are c o n s i d e r e d , i t i s u n c l e a r whether the p l a n t estrogens are metabolic substances s y n t h e s i z e d i n response to the i n v a s i o n or produced by the i n f e c t i v e agents. Lindner (1976) demons-t r a t e d t h a t coumestrol content i n c r e a s e s i n a l f a l f a i n f e c t e d with the f u n g a l agent Pseudopezia medicagensis and zearalenone formation occurs i n corn d u r i n g storage by the m i c r o b i a l a t t a c k o f Fusarium spp. As s t a t e d p r e v i o u s l y coumestrol and g e n i s t e i n are capable o f competing wi t h e s t r a d i o l - 1 7 3 f o r r e c e p t o r s i t e s i n t a r g e t t i s s u e and i n d u c i n g a u t e r o t r o p h i c response. Whether the response i s a g o n i s t i c (weakly e s t r o g e n i c ) or a n t a g o n i s t i c w i l l depend upon the dose i n g e s t e d and maintained w i t h i n the blood stream. Folman and Pope (1966) demonstrated that coumestrol, g e n i s t e i n , DMS and n o r e t h i s t e r o n e acetate are a g o n i s t i c at low doses and a n t a g o n i s t i c at higher l e v e l s . 25. The s t e r e o c h e m i c a l c o n f i g u r a t i o n of the non-s t e r o i d a l phyto-estrogen molecule enables competition f o r e s t r a d i o l c y t o p l a s m i c r e c e p t o r s . T h i s confirms the importance of the f u n c t i o n a l p h e n o l i c hydroxyl groups as b i n d i n g centre f o r a s s o c i a t i o n with the c y t o s o l r e c e p t o r (Noeboom and G o r s k i , 1963; Shemish e_t al.. , 1972). Coumestrol has a g r e a t e r a f f i n i t y f o r u t e r i n e r e c e p t o r s than do the i s o f l a v o n e s but l e s s than e s t r a d i o l - 1 7 6 . Shutt and Cox (1972) estimate the r e l a t i v e p o t e n c i e s of E^-176: Coumestrol: G e n i s t e i n to be 1:20:111 r e s p e c t i v e l y while Shemish et_ a l . (1972) estimate the r e l a t i v e p o t e n c i e s to be 1: 70 :175. O r i g i n a l s t u d i e s (Bennetts et a l . , 1946) a t t r i -buted i n f e r t i l i t y to coumestrol and g e n i s t e i n , however, metabolic s t u d i e s have demonstrated pathways i m p l i c a t i n g not only coumestrol but equol as the p r i n c i p a l i n f e r t i l i t y agents (see Figure 6). Through a c t i o n of the rumen micro-f l o r a b i o c h a n i n A undergoes O-demethylation to g e n i s t e i n which i s f u r t h e r metabolized to i n e r t p - e t h y l p h e n o l (Braden et a l . , 1966; Shutt et a l . , 1970; Cox and Braden, 1974). Formononetin i s metabolized through O-demethylation to d a i d z e i n which i s f u r t h e r transformed to equol and 0-desmethylangloensin (Cox and Braden, 1974; Lindner, 1976), again through rumen ferme n t a t i o n processes (Shutt, 1976). FIGURE 5 5 B Coumestans: Figure 5 : Structural formula of principle phyto-estrogen 27. FIGURE 6A: | 2 p-Ethyl phenol C H 2 FIGURE 6B O-Desmethyl angdensin Equol FIGURE 6: Structural formula of phyto-estrogen metabolites. 28. I t has been demonstrated that i n g e s t i n g e s t r o g e n i c pasture can be a s s o c i a t e d with such gross p h y s i o l o g i c a l changes as d y s t o c i a , u t e r i n e p r o l a p s e , udder development and m i l k s e c r e t i o n in barren ewes (Noteboom and G o r s k i , 1963), lamb m o r t a l i t y , r e t a i n e d mummified f e t u s , m e t r i t i s , s t e r i l i t y and i n f e r t i l i t y (Moule et a_l. , 1963) . P a t h o l o g i c a l l y the u t e r i n e e p i t h e l i u m , stroma and myometrial l a y e r s undergo hypertrophy and p r o l i f e r a t i o n due to i n c r e a s e d water i m b i b i t i o n and glycogen s y n t h e s i s (Ostrovsky and K i t t s , 1963). A s s o c i a t e d w i t h t h i s abnormal u t e r i n e metabolism i s c y s t i c g l a n d u l a r h y p e r p l a s i a o f the endometrium wi t h u t e r i n e and c e r v i c a l l e s i o n s (Moule et a l . , 1963). T h i s r e s u l t s i n i n c r e a s e d f l u i d i t y of c e r v i c a l and u t e r i n e mucus l e a d i n g to impaired ova and sperm m o b i l i t y w i t h subsequent decreases i n f e r t i l i z a t i o n and c o n c e p t i o n r a t e s (Moule et. al. , 1963; L i g h f o o t and Wroth, 1974a; L i g h t f o o t et al., 1974b; Cox and Braden, 1974; L i v i n g s t o n , 1978). Under such abnormal hormonal c o n d i t i o n s Ostrovsky and K i t t s (1963), Moule et al. (1963), L i g h t f o o t and Wroth (1974), K e l l y et a l . (1976), and L i v i n g s t o n (1978) doubted that t h i s u t e r i n e e p i t h e l i u m c o u l d respond to progesterone priming which would t h e r e f o r e r e s u l t i n u n s u c c e s s f u l i m p l a n t a t i o n , another source of i n f e r t i l i t y . 29. L i g h t f o o t and Wroth (1974) and K e l l y e t a l . (1976) have demonstrated t h a t ewes on coumestan r i c h f e e d e x h i b i t premature r e g r e s s i o n o f the corpus luteum, i n f a c t 581 o f the ewes d i d not have a r e c e n t l y d eveloped corpus luteum. T h i s e l u c i d a t e s two major f e a t u r e s o f a c u t e l y a f f e c t e d ewes; (1) r e g r e s s i o n o f the corpus luteum removes the p r o g e s t e r o n e r e q u i r e d f o r s u c c e s s f u l i m p l a n t a t i o n and (2) a complete absence o f a corpus luteum i n d i c a t e s an a n o v u l a t o r y e s t r u s . ( F i r t h et_ aj.. , 1977). Premature r e g r e s s i o n o f the corpus luteum and a n o v u l a t o r y e s t r u s are two i n d i c a t i o n s o f an a l t e r e d hypo-t h a l a m i c - p i t u i t a r y - o v a r i a n r e l a t i o n s h i p i n which the hypo-thalamus has been s p e c i f i c a l l y i m p l i c a t e d . Noteboom and G o r s k i (1963) demonstrated an i n c r e a s e i n p i t u i t a r y w e i g h t s of a f f e c t e d ewes w i t h subsequent h y p e r a c t i v i t y ( K e l l y e t a l . , 1976). I n c o n t r a s t , Cox and Braden (1974) found p i t u i t a r i e s t o be h i s t o l o g i c a l l y normal i n c l o v e r d i s e a s e d ewes. Any d i s c r e p a n c i e s c o u l d be a r e s u l t o f i n g e s t i n g d i f f e r e n t l e v e l s o f p h y t o - e s t r o g e n . W r i g h t (1963) f e d c o u m e s t r o l to mice w h i c h d i d not suppress g o n a d o t r o p i n b i o s y n t h e s i s but d i d i n h i b i t i t s r e l e a s e . The p l a n t e s t r o g e n s may a c t d i r e c t l y upon the p i t u i t a r y t o p r e v e n t g o n a d o t r o p i n r e l e a s e ( W r i g h t , 19630. However, i t i s more p r o b a b l e t h a t the hypothalamus and i t s s e c r e t i o n o f GnRH i s a f f e c t e d 30 . (Hernshaw et a_l. , 1972; F i n d l a y e_t a l . , 1973; Lindner, 1976, Adams, 1976, Adams e_t a l . , 1979). Permanent i n f e r t i -l i t y amongst ewes i s c h a r a c t e r i s t i c of c l o v e r disease with the hypothalamus and higher nerve ce n t r e s r e s p o n s i b l e . These neuroendocrine c e n t r e s which c o n t r o l the estrous c y c l e become suppressed and permanently d e s e n s i t i z e d to the p o s i t i v e s t i m u l a t o r y e f f e c t of endogenous estrogen (Lindner, 1976; L i v i n g s t o n , 1978; Adams, 1978; Adams et. a l . , 1979). As a consequence of a l t e r e d hypothalamo-hypophyseal f u n c t i o n one would expect abnormal f o l l i c u l o g e n e s i s . K e l l y et a l . (1976) found f o l l i c u l a r a b n o r m a l i t i e s i n d i c a t i v e o f s t r o n g e s t r o g e n i c s t i m u l a t i o n whereas Adams (1976) d i s c o v e r e d s m a l l e r o v a r i e s i n c l o v e r a f f e c t e d ewes i n d i c a t i n g o v a r i a n d e s e n s i t i z a t i o n to c i r c u l a t i n g gonadotropins or decreased l e v e l s of the same gonadotropins. Adams (1979) found ewes f e d subterranean c l o v e r showed e l e v a t e d o v u l a t i o n r a t e s but no d i f f e r e n c e i n p r i m o r d i a l f o l l i c l e s . T h i s i n d i c a t e s an a l t e r e d p a t t e r n of f o l l i c u l a r development r a t h e r than an i n c r e a s e i n the r a t e of i n i t i a t i o n of growth. A f u r t h e r r e s u l t of a t y p i c a l f o l l i c u l o g e n e s i s should be anomalous o v a r i a n s t e r o i d o g e n e s i s which i s i n f a c t the case (Obst and Seamark, 1975; Newsome and K i t t s , 1977). 4. THE HYPOTHALAMUS IN FEMALE REPRODUCTION a) Anatomical O r g a n i z a t i o n The hypothalamus p r o v i d e s the major l i n k between endocrine and n e u r a l systems that i n t e g r a t e the e x t e r n a l and i n t e r n a l environment. I t i s b i l a t e r a l l y symmetrical w i t h d i f f u s e boundaries and forms the w a l l s and f l o o r of the lower r e g i o n o f the t h i r d v e n t r i c l e . The upper boundary i s formed by the hypothalamic s u l c u s , a s t r u c t u r e which d e f i n e s the boundary between the hypothalamus and thalamus. L a t e r a l boundaries are determined by the c e r e b r a l g a n g l i o n , subthalamus and o p t i c t r a c t s . The hypothalamic area a l s o i n c l u d e s the o p t i c chiasma, tuber cinereum, infundibulum and mammillary bodies. The tuber cinereum i s a b u l g i n g p a r t of the f l o o r of the t h i r d v e n t r i c l e which extends downward towards the i n f u n d i -bulum. The expanded upper infundibulum and lower p a r t of the tuber cinereum are r i c h l y s u p p l i e d with blood v e s s e l s and course through to the a n t e r i o r p i t u i t a r y d e f i n i n g the Median Eminence (ME) and hypophyseal p o r t a l system ( M a r t i n , 1976). W i t h i n the hypothalamus i s an e x t e n s i v e nerve f i b r e system which extends throughout other p a r t s of the c e n t r a l nervous system. T h i s neuronal network, which 32. i s r e s p o n s i b l e f o r s y n t h e s i s and s e c r e t i o n of r e l e a s i n g hormones r e g u l a t i n g a n t e r i o r - p i t u i t a r y f u n c t i o n , i s r e f e r r e d to as the P e r i v i c e l l u l a r Neurosecretory System ( S e t a l o , 1977). The v a r i o u s neuron c e l l bodies ( p e r i k a r y a ) aggregate i n s p e c i f i c areas of the hypothalamus termed n u c l e i ( a l s o i s l a n d s ) . S p e c i f i c n u c l e i are i n t e g r a l components of the P e r i v i c e l l u l a r Neurosecretory system and are found i n the medical b a s a l hypothalamus a l s o known as the hypophy-s i o t r o p i c area. The n u c l e i i m p l i c a t e d are the su p r a c h i a s -m a t i c - p r e o p t i c , tuberal-premammillary r e g i o n , ventromedial p e r i v e n t r i c u l a r and arcuate ( S e t a l o , 1977; Dyer, 1977; Brawer and Van Houton, 1977). The f i b r e s from the medial b a s a l hypothalamus form two d i s t i n c t t r a c t s , ( i ) the P r e o p t i c I n f u n d i b u l a r t r a c t and, ( i i ) the T u b e r o i n f u n d i b u l a r t r a c t . These t r a c t s are the endocrine neurones which terminate i n the Median Eminence on the c a p i l l a r i e s of the hypophyseal p o r t a l system. The major c o n c e n t r a t i o n of Gonadotropin R e l e a s i n g Hormone (GnRH) p e r i k a r y a can be found i n the Arcuate Nucleus (Brawer and Van Houten, 1977; K r u l i c h ejt al.. , 1977 ; Zimmerman, 1976; Zimmerman, 1977), the f i b e r s o f which c o n s t i t u t e a major p o r t i o n of the T u b e r i n f u n d i b u l a r t r a c t . There a l s o appears to be a s l i g h t but s i g n i f i c a n t c o n c e n t r a t i o n of GnRH neurons i n the mediobasal zone o f the p r e o p t i c FIGURE 7A Infundibulum FIQJRF 7R C e r e b r a l Infundibulum Figure 7: Ventral (A) and Median (B) surfaces of the s brain (May, 1964). MM Medial Mamillary Nuc. PM Premamillary Nuc. LAHY Pars Distalis of Adenohypophysis LPHY Neural Lobe of Hypophysis SO Supraoptic Nuc. DM Dorsmedial Nuc. VM Ventromedial Nuc. PV Paraventricular Nuc. ARC Arcuate Nucleus NAH Anterior Hypothalamic Area SCH Suprachiasmatic Nuc. Figure 8 : Mid sagital section of the rat diencephalon (Everett, 1978). 35. area ( K r u l i c h et a l . , 1977; Brawer and Van Houton, 1977). It i s thought that the f i b e r s from t h i s r e g i o n a l s o con-t r i b u t e to the T u b e r o i n f u n d i b u l a r t r a c t p r i n c i p a l l y v i a the arcuate n u c l e u s . The GnRH endocrine neurons of the medial b a s a l hypothalamus making up the T u b e r o i n f u n d i b u l a r t r a c t a l s o e x h i b i t branched f i b r e s that extend to other hypothalamic and extrahypothalamic r e g i o n s (Dyer, 1977). There are a l s o p e p t i d e r e l e a s e r e g u l a t i n g interneurones which i n t e g r a t e the v a r i o u s impulses a r i s i n g from the numerous hypothalamic r e g i o n s . T h i s i n d i c a t e s an i n t e g r a t e d c o n t r o l mechanism between other b r a i n c e n t r e s , the CNS, and the hypothalamus (Dyer, 1977; Kordon et a l . , 1976). b) N e u r o t r a n s m i t t e r s and the Hypothalamus Monoamines are c o n s i d e r e d to act as neurotrans-m i t t e r s i n inputs to the n e u r o s e c r e t o r y c e l l s of the hypotha-lamus and i n some cases may even act d i r e c t l y on the a n t e r i o r p i t u i t a r y . The monoamines c o n s i s t of two p r i n c i p a l groups: (1) the catecholamines; dopamine (DA), a d r e n a l i n (A), n o r a d r e n a l i n (NA) and (2) the indolamines; 5-hydroxy-tryptophan (5-HT), melatonin and histamine ( K a l r a , 1977). 36. Dopamine, NA, A and 5-HT pathways o r i g i n a t e i n the lower b r a i n stem ascending to innervate the thalamus, s u b c o r t i c a l and c o r t i c a l l i m b i c systems, b a s a l g a n g l i a and neocortex. Within the hypothalamus there are a number of i n t r a h y p o t h a l a m i c DA systems. The most prominent i s the t u b e r o i n f u n d i b u l a r t r a c t DA system which i n n e r v a t e s the l a t e r a l and medial p a l i s a d e zones o f the e x t e r n a l median eminence (Fuxe et al.. , 1977) . With the involvement o f the t u b e r o i n f u n d i b u l a r t r a c t system i t has been demons-t r a t e d t h a t neurons c o n t a i n i n g DA are conc e n t r a t e d w i t h i n the arcuate and a n t e r i o r p e r i v e n t r i c u l a r n u c l e i (Fuxe et a l . , 1976). C e l l bodies g i v i n g r i s e to the NA neuronal system are l o c a l i z e d i n the pons and medulla oblongata. Axons form the v e n t r a l NA bundle i n the mesencephalon and course through the hypothalamic r e g i o n to terminate i n the medial p a l i s a d e zone and subependymal l a y e r of the median eminence (Fuxe et_ a l . , 1976 ; Fuxe et. a l . , 1977). S t i m u l a t i o n of the DA system i n h i b i t s the r e l e a s e of GnRH from the median eminence while NA w i l l f a c i l i t a t e s e c r e t i o n . The i n t e g r a t i o n o f these two systems y i e l d s the o v e r a l l GnRH response and may be a s i t e of estrogen feedback c o n t r o l . C o n c l u s i o n s drawn from indolamines are u n c l e a r , however, i t i s conceded that 5-HT has a dual 37. e f f e c t o f i n h i b i t i n g b a s a l GnRH s e c r e t i o n and c o n t r o l l i n g the rythmic GnRH s e c r e t i o n p a t t e r n . M e l a t o n i n has been shown to i n h i b i t o v u l a t i o n while histamine w i l l f a c i l i t a t e i t (Brownstein, 1977 ; Fuxe et. a l . , 1976 ; Fuxe e_t a l . , 1977; K a l r a , 1977; Kordon, 1978; McCanne, 1977; Sawyer, 1977) . c) Extrahypothalamic S t r u c t u r e s i n Female Reproduction Higher n e u r a l c e n t r e s are c o n s i d e r e d to i n f l u e n c e the hypothalamus because e x t e r n a l s t i m u l i are known to e f f e c t o v u l a t i o n and sexual behavior. R e a d i l y d i s c e r n i b l e examples of t h i s are: p s y c h o l o g i c a l amenorrhea i n women and induced o v u l a t o r s such as the r a b b i t . The l i m b i c system i s thought to i n t e g r a t e and c o n t r o l c i r c a d i a n , s t r e s s , autonomic nervous system and e x t e r n a l sensory s t i m u l u s responses which i n f l u e n c e the r e p r o d u c t i v e s t a t e o f the animal. S t r u c t u r e s w i t h i n the l i m b i c system are the medial p a r t o f the mesencephalic r e t i c u l a r f ormation (midbrain l i m b i c system), the septum, c i n g u l a t e and p y r i f o r m c o r t e x , hippocampus and amygdala (Mar t i n , 1977; Hutchinson, 1978). The amygdala, hippocampus and a number of mesen-c e p h a l i c n u c l e i a l l have anatomical access to the GnRH d e l i v e r y system. The i n f l u e n c e on t h i s d e l i v e r y system 38. i s achieved through connections w i t h the suprachiasmatic-p r e o p t i c areas (Brawer and Van Houten, 1977; E l l e n d o r f , 1978). More e x t e n s i v e r e s e a r c h has been conducted on the l i m b i c system and i t has been demonstrated that sensory inputs are not a v a i l a b l e to the l i m b i c s t r u c t u r e s w i t h the e x c e p t i o n of the r e t i n o h y p o t h a l a m i c connection to the s u p r a c h i a s m a t i c nucleus (Wilber et a l . , 1976) . Hutchinson (1978) has i l l u s t r a t e d complementary a f f e r e n t and e f f e r e n t n e u r a l pathways between the l i m b i c s t r u c t u r e s and hypotha-lamus i n d i c a t i n g a two-way i n t e r a c t i o n . Sensory inputs are a l s o r e l a y e d to the l i m b i c system through the r e t i c u l a r f o rmation of the thalamus and b r a i n stem (Wilber jet a_l. , 1976) . Hypothalamic i n p u t s from the hippocampus a r i s e from a amassive e f f e r e n t pathway, the f o r n i x ( E v e r e t t , 1978). The f o r n i x i s the major c o n t r i b u t i o n to the mammilary body and a l s o d i r e c t s e f f e r e n t f i b e r s i n t o the arcuate and the ventromedial n u c l e i of the hypothalamus. Some of these f i b e r s a l s o terminate on t u b e r o i n f u n d i b u l a r neurons (Lammers and Lohman, 1974). The amygdala i s perhaps the most s t u d i e d s t r u c -ture of l i m b i c system. P r o j e c t i o n s from the c o r t i c o m e d i a l n u c l e i of the amygdala extend through the s t r i a t e r m i n a l i s to the septum and e x t e r n a l border of the ventromedial and medial p r e o p t i c n u c l e i of the hypothalamus. From the basomedial amygdala, p r o j e c t i o n s of the amygdalofugal pathway pass to the l a t e r a l hypothalamus". The f u n c t i o n of t h i s l a s t pathway i s however u n c l e a r ( E l l e n d o r f , 1976). The amygdala i s capable of e x e r t i n g both a f a c i l i t a -t o r y and i n h i b i t o r y i n f l u e n c e upon the r e l e a s e of gonado-t r o p i n (through the s e c r e t i o n of GnRH). F a c i l i t a t o r y impulses are t r a n s m i t t e d through the s t r i a t e r m i n a l i s and mesencephalo-amygdaloid system while i n h i b i t o r y p u l s e s are implemented through the mesencephalo-hippocampal system. The two systems s t a t e d above may be i n t e g r a t e d to form an o v e r a l l i n h i b i t o r y pathway however the exact i n h i b i t o r y pathway i s u n c l e a r ( T a l e i s n i l e and Beltramino, 1975). The p i n e a l a r i s e s from n e u r o p i t h e l i u m and i s a true endocrine gland because i t s y n t h e s i z e s hormonal f a c t o r s i n s p e c i f i c c e l l s , the p i n e a l o c y t e s , and not i n neurons as does the neuroendocrine hypothalamus (Kappers, 1976). The p i n e a l i n sheep i s both i n d i r e c t l y and d i r e c t l y r e s p o n s i v e to l i g h t (Hutchinson, 1978) which in turn syn-c h r o n i z e s r e p r o d u c t i v e s t a t e s with season. The p i n e a l then a c t s as an i n t e g r a t o r and i n t e r m e d i a r y between seasonal p h o t o p e r i o d i c changes and the neuroendocrine r e p r o d u c t i v e a x i s ( R e i t e r , 1976). 40. Though the mechanism i s unre s o l v e d the main f u n c t i o n i s one of an a n t i g o n a d o t r o p i c nature suppressing gonadotropin a c t i v i t y , again through GnRH s e c r e t i o n . Some s e c r e t o r y products are however r e l e a s e d i n t o the systemic blood system. The s e c r e t o r y products are a n t i g o n a d o t r o p i c and are composed of p o l y p e p t i d e s and indolamines, p r i n c i p a l l y melatonin ( R e i t e r , 1976). d) Gonadotropin R e l e a s i n g Hormone(s) Gonadotropin R e l e a s i n g Hormone a b b r e v i a t e d as GnRH ( a l s o known as LRF, LH-RH, FSH/LH-RH and Gonadoliberin) i s the centre of c o n t r o v e r s y . The q u e s t i o n i s c o n s t a n t l y r a i s e d as to the exact nature of GnRH. Is there j u s t one GnRH r e s p o n s i b l e f o r the r e l e a s e of both gonadotropins (FSH and LH) or are there two r e l e a s i n g hormones? Evidence i n d i c a t e s the e x i s t e n c e of one r e l e a s i n g hormone a c t i n g upon one gonadotroph to evoke a d i f f e r e n t i a l r e l e a s e of e i t h e r LH or FSH. T h i s d i f f e r e n t i a l c o n t r o l of the p i t u i t a r y by the hypothamus i s i n tu r n m o d i f i e d by the p r e v a i l i n g r e p r o d u c t i v e s t a t e and consequently c i r c u l a t i n g o v a r i a n s t e r o i d l e v e l s . These p r e v a i l i n g c o n d i t i o n s w i l l modify the gonadotroph's responsiveness and s e c r e t o r y mechanisms (Meites, 1969; F l e i s c h e r and 41. G u i l l e m i n , 1976; Fink , 1976; Vale et al., 1977; L i n c o l n , 1979; Wise et a l . , 1979). GnRH i s a decapeptide f i r s t i s o l a t e d and charac-t e r i z e d from p r o c i n e hypothalamic e x t r a c t s ( S c h a l l y e_t a l . , 1971; Matsuo e_t al. , 1971). As s t a t e d p r e v i o u s l y , GnRH i s s y n t h e s i z e d w i t h i n the p e r i k a r y a o f the s u p r a c h i -a s m a t i c - p r e o p t i c area and arcuate nucleus of the medial b a s a l hypothalamus. From these n u c l e i the r e l e a s i n g hormone i s d e p o s i t e d and l o c a l i z e d w i t h i n the median eminence to be subsequently r e l e a s e d i n t o the primary c a p i l l a r y plexus of the hypophyseal p o r t a l system CMcCann, 1977). T h i s n e u r o v a s c u l a r pathway forms the c r i t i c a l l i n k from nerve c e l l s o f the hypothalamus to the endr o c r i n e c e l l s o f the a n t e r i o r p i t u i t a r y . D a n i e l and P r i c h a r d (1975) determined that s p e c i f i c r e g i o n s w i t h i n the pars d i s t a l i s of the a n t e r i o r p i t u i t a r y r e c e i v e b l o o d from c e r t a i n areas of the median eminence and neurohypophysis. They a l s o demonstrated that each r e g i o n of the pars d i s t a l i s i s completely c i r c u m s c r i b e d . These p o i n t s may be important because v a r i o u s types of c e l l s of the pars d i s t a l i s tend to be grouped i n p a r t i c u l a r r e g i o n s of the lobe ( D a n i e l and P r i c a r d , 1975; Mathieson, 1979) . 42 . Once i n t o the hypophyseal p o r t a l system the GnRH t r a v e l s down the p i t u i t a r y s t a l k and i n t o s i n u s o i d s w i t h i n the a n t e r i o r p i t u i t a r y . Here the GnRH r e a c t s with s u r f a c e r e c e p t o r s of the gonadotrophs to a c t i v a t e the adenylate c y c l a s e system with subsequent i n c r e a s e s i n cAMP and p r o t e i n kinase a c t i v i t y ( J u s t i z , 1971; McCann, 1977; Conn et a l . , 1979; L a b r i e et. a l . , 1979; L a b r i e et a l . , 1979). As a r e s u l t of t h i s a c t i o n membrane p r o t e i n s are dephosphorylated a l t e r i n g the membrane p e r m e a b i l i t y and e n a b l i n g e x t r u s i o n of the s e c r e t o r y granules c o n t a i n i n g gonadotropin (McCann, 1977) . Wagner et a l . (1979) demons-t r a t e d the e x i s t e n c e of a s i n g l e c l a s s of high a f f i n i t y r e c e p t o r s i t e s (D 2 . 33± . S l x l O ^ M - 1 ) . A complete d i s c u s s i o n of p e p t i d e hormone-membrane r e c e p t o r r e a c t i o n s w i l l f o l l o w i n l a t e r s e c t i o n s . I t should be noted that a second pathway e x i s t s w i t h i n gonadotrophs i n v o l v i n g p r o s t a g l a n d i n which may be r e s p o n s i v e to GnRH. The PG pathway i s thought to be independent of cAMP a c t i v a t i o n (Conn e_t a_l. , 1979; N a i r et a l . , 1979). GnRH a l s o e x e r t s a s e l f - p r i m i n g e f f e c t upon the p i t u i t a r y which magnifies the adenohypophysis response to GnRH. T h i s p r o p e r t y of GnRH i s f u r t h e r enhanced by the a c t i o n of estrogen (Castro-Vazquez and McCann, 1975; McCann, 1977 ; P i c k e r i n g and Fink, 1977 ; L a b r i e e_t a l . , 43. 1978). However, c o n t i n u a l s t i m u l a t i o n by GnRH u l t i m a t e l y leads to d e s e n s i t i z a t i o n of the p i t u i t a r y towards GnRH (Sandow et a l . , 1978). The GnRH i s a l s o l o c a l i z e d i n extra-hypophysio-t r o p i c areas and t h e r e f o r e may have other p h y s i o l o g i c a l a c t i o n s w i t h i n the CNS and b r a i n f u n c t i o n (Moss, 1979). GnRH has been found i n such regions as the p i n e a l , midbrain, c e r e b r a l and c e r e b e l l a r c o r t i c e s and b r a i n stem. A c t i n g through these s t r u c t u r e s GnRH has been demonstrated to a f f e c t mating behavior p a r t i c u l a r l y l o r d o s i s (Moss and McCann, 1973; P f a f f , 1973; Moss, 1979). 5. THE PITUITARY IN FEMALE REPRODUCTION The hypophysis develops i n the r e g i o n d o r s a l to the notochord, a r i s i n g from two ectodermal p r i m o r d i a : thesaccus i n f u n d i b u l a r i s , a v e n t r a l e v a g i n a t i o n from the diencephalon; and Rathke's pocket, a d o r s a l outgrowth of the b u c c a l c a v i t y ( E v e r e t t , 1978). The n e u r a l lobe forms the neurohypophysis ( p o s t e r i o r lobe) while Rathke's pocket forms the adenohypophysis ( a n t e r i o r l o b e ) . The l a t t e r i s c e n t r a l to t h i s d i s c u s s i o n and w i l l be the only h y p o p h y s i a l lobe c o n s i d e r e d . The terms a n t e r i o r and 44. p o s t e r i o r lobes are m i s l e a d i n g because they do not apply to a l l s p e c i e s and w i l l not be used i n subsequent d i s c u s s i o n s . A n a t o m i c a l l y , three zones can be d i s t i n g u i s h e d w i t h i n the adenhypophysis. The pars d i s t a l i s c o n s i s t s of g l a n d u l a r c e l l s arranged i n i r r e g u l a r columns which are i n t i m a t e l y r e l a t e d to a network of s i n u s o i d a l c a p i l -l a r i e s . These c a p i l l a r i e s are the e x t r e m i t i e s of the hypophyseal p o r t a l venules which o r i g i n a t e from the primary plexus (Rennels and Herbert, 1979). The g l a n d u l a r c e l l s are composed of chromophobes, a c i d o p h i l s (somatotrophs, l a c t o t r o p h s ) , and b a s o p h i l s ( t h y r o t r o p h s , gonadotroph). The gonadtrophs are r e s p o n s i -ble for the s y n t h e s i s and s e c r e t i o n of f o l l i c l e s t i m u l a t i n g hormone (FSH) and l u t e i n i z i n g hormone (LH) i n response to GnRH and s t e r o i d feedback s t i m u l a t i o n . S e v e r a l workers have i d e n t i f i e d two d i s t i n c t gonadotroph c e l l types and termed them f o l l i c u l o t r o p h s (which produce FSH) and Luteotrophs (which produce LH), (Holmes and B a l l , 1974; S c h u l s t e r et a l . , 1976; Purandare et a l . , 1978). M a r t i n (1976) and Franchimont (1977) however maintain the presence of one type of gonado-t r o p h which can be i n f l u e n c e d by e x t r a c e l l u l a r c o n d i t i o n s . Evidence f o r t h i s i s based on m i c r o s c o p i c immuno-histochemical techniques which demonstrate FSH and LH w i t h i n the same c e l l s (Nakane, 1975). T h i s dilemma remains unresolved, 45. however, Dacheux and Dubois (1978) implementing e l e c t r o n m i c r o s c o p i c immunocytochemical s t u d i e s have i d e n t i f i e d and c h a r a c t e r i z e d L H - s e c r e t i n g c e l l s i n the ovine p i t u i t a r y . Both LH and i t s subunits (LHa and LH3) were l o c a l i z e d on the s e c r e t o r y granules and on the small granules near the G o l g i complex. The authors a l s o suggest t h a t these techniques can be used to i s o l a t e FSH and as a consequence to determine whether LH and FSH occur i n one or two d i f f e r -ent c e l l types. 6. GONADOTROPINS a) General C o n s i d e r a t i o n s Ovine gonadotropins have been i s o l a t e d and charac-t e r i z e d (Papkoff and Ekblad, 1970) while human gonadotropins have been e x t e n s i v e l y reviewed by Butt (1976). The gonadotropins are dimer g l y c o p r o t e i n s which c o n t a i n up to t h i r t y per cent carbohydrate. P i t u i t a r y FSH has a molecular weight of approximately 35,000, i s r i c h i n glutamic a c i d and c y s t e i n e and 10-30 per cent of the molecule appears as an a l p h a - h e l i c a l s t r u c t u r e s . LH has a molecular weight of approximately 33,000 and 46. i s r i c h i n p r o l i n e which p r o h i b i t s the formation of the a l p h a - h e l i c a l c o n f i g u r a t i o n (Butt, 1976). FSH c o n t a i n s more carbohydrate and s i a l i c a c i d than LH. The hexose amines which occur are p r i n c i p a l l y 2-acetamido-2-deoxy-D-glucose (N-acetylglucosamine) i n FSH, N-acetylglucosamine and N-acetylglucosamine ( r a t i o 6:1 r e s p e c t i v e l y ) i n LH. The hexose amines appear to p l a y a r o l e i n r e c e p t o r b i n d i n g (Butt, 1976) and w i l l be d i s c u s s e d l a t e r . The FSH and LH i n d i v i d u a l subunits (alpha and beta) have no i n t r i n s i c b i o l o g i c a l a c t i v i t i e s ; the dimer i s r e q u i r e d . Hybrid molecules may be obtai n e d , however, the beta subunit determines the nature and c h a r a c t e r i s t i c s of the h y b r i d . ( R e i c h e r t , 1968; R e i c h e r t , 1974; Butt, 1976). b) Gonadotropin Sy n t h e s i s and S e c r e t i o n The gonadotrophs are c o n t r o l l e d through the , a c t i o n of GnRH re a c h i n g the adenohypophysis v i a the hypo-ph y s e a l p o r t a l system. T h i s pathway i s a l s o i n f l u e n c e d by the GnRH i t s e l f and p r e v a i l i n g gonadal s t e r o i d p a t t e r n s ; these t o p i c s w i l l be c o n s i d e r e d i n l a t e r s e c t i o n s . 47. Using phosphodiesterase i n h i b i t o r s , J u t i s z (1971) demonstrated cAMP to be a mediator of GnRH as i t i s able to r e l e a s e FSH and LH from r a t p i t u i t a r i e s i n v i t r o i n the presence of C a + ^ i o n s . By us i n g actinomycin D (RNA s y n t h e s i s i n h i b i t o r ) and cycloheximide ( i n h i b i t s p r o t e i n b i o s y n t h e s i s ) i n v i t r o the r e l e a s i n g a c t i v i t y o f GnRH was only p a r t i a l l y impaired but over a longer p e r i o d FSH r e l e a s e d i d begin to d e c l i n e . T h i s evidence y i e l d e d a two compartment model f o r p i t u i t a r y c e l l gonadotropin s y n t h e s i s and r e l e a s e ( J u t i s z , 1971; Yen, 1977). The model d e p i c t e d i n v o l v e s a r e a d i l y r e l e a s a b l e p o o l o f FSH and a second compartment r e s p o n s i b l e f o r FSH s y n t h e s i s and stor a g e . Evidence i n d i c a t e s the r e l e a s a b l e p o o l to be under d i r e c t i n f l u e n c e of the Gn RH. The syn-t h e s i s compartment appears to be c o n t r o l l e d by i n t r a c e l l u -l a r messengers and feedback mechanisms which may be an i n d i r e c t r e s u l t o f GnRH a c t i o n J u t i s z , 1971). An example of an i n t r a c e l l u l a r messenger c o u l d be the p r o s t a g l a n d i n d e s c r i b e d by Na i r et. a_l. (1979) which i s independent of the adenylate cyclase-cAMP system. T h i s i n t r a c e l l u l a r PG b i o s y n t h e s i s appears to be under the i n f l u e n c e of GnRH as MDA, which i s a d i r e c t m e t a b o l i c product of p r o s t a g l a n d i n endoperoxide, i n c r e a s e s i n p i t u i t a r i e s s t i m u l a t e d with GnRH over c o n t r o l s . P r o s t a g l a n d i n may a l s o be responsible^ 1 48. f o r LH r e l e a s e from gonadotrophs (Nair e_t a^l. , 1979) . In c o n t r a s t to t h i s concept L a b r i e et a l . (1978) suggest p r o s t a g l a n d i n s are not i n v o l v e d i n GnRH a c t i o n at the l e v e l of the a n t e r i o r p i t u i t a r y but s t i m u l a t e LH release at the hypothalamic l e v e l through i n c r e a s e d GnRH s e c r e t i o n . The feedback mechanisms modulating the two p o o l s of gonadotropin may i n v o l v e FSH and LH themselves ( J u t i s z , 1971), the s e l f - p r i m i n g e f f e c t of GnRH (Castro-Vazquez and McCann, 1971; McCann, 1977; P i c k e r i n g and Fink, 1977; Yen, 1977; F o s t e r , 1978; Hoff et a l . , 1979) and o v a r i a n estrogen (Yen, 1977). Through the e a r l y m i d - f o l l i c u l a r phase the second p o o l o f s y n t h e s i z e d and s t o r e d LH p a r a l l e l s the e s t r a d i o l - 1 7 6 p a t t e r n and i s p r e f e r e n t i a l l y augmented. Enlargement of the r e l e a s a b l e p o o l i s not apparent u n t i l the l a t e f o l l i c u l a r phase which r e p r e s e n t s a s h i f t i n g o f LH between the two compartments. Estrogen appears to promote a c c e l e r a t i o n of gonadotropin s y n t h e s i s w i t h i n the second p o o l and t r a n s f e r to the f i r s t p o o l but appears to impede the GnRH mediated LH r e l e a s e . During the mid-l u t e a l phase progesterone and estrogen l e v e l s appear to maintain a l a r g e second p o o l while the f i r s t p o o l i s much smaller. This environment is also associated with extremely low GnRH r e l e a s e (Yen, 1977). Hoff et. a l . (1979) a l s o demonstrated 4 9 a . FIGURE 9A GnRH Secretion Plasma membrane Adenylate cyclase 'ATP cAMP+ PR 5'-AMP Inactive kinase Active kinase O Secretory granules Rough endoplasmic reticulum Figure 9A: Proposed mechanism of action for GnRH (McCann, 1977 and Labrie et al. 1978). FIGURE 9B 49b. GnRH (•) POOL 2 Synthesis and Storage (+) > Activat ion LH Figure 9B: Interrelationship between estrogen and GnRH in the proposed 2 pool hypothesis for gonadotropin (Jutisz,1971 and Yen, 1977) 5 0 FIGURE 10 H OH Hydroxyocid O C 0 0 H Arachidonic acid Hydroperoxide HO' H H OH MDA Figure 10: Prostaglandin biosynthesis ( Nair etal. 1979). 51. that l a r g e elevations of GnRH favo r gonadotropin r e l e a s e whereas small increments promote pr i m i n g . T h i s i s most evident i n the presence of high e s t r a d i o l l e v e l s . c. Gonadotropin Receptors and A c t i v a t i o n P o l y p e p t i d e hormone r e c e p t o r s obey the s t r i n g e n t c r i t e r i a d e s c r i b e d f o r s t e r o i d r e c e p t o r s ; however, hormone s p e c i f i c i t y r e s i d e s i n the s t r u c t u r e of plasma membrane r e c e p t o r s , the enzyme p r o f i l e o f the t a r g e t c e l l and the a b i l i t y to change membrane p e r m e a b i l i t y . T h i s i s i n c o n t r a s t to i n t r a c e l l u l a r s t e r o i d r e c e p t o r s which u l t i m a t e l y a f f e c t gene e x p r e s s i o n (Blake, 1978). The gonads are capable of b i n d i n g 400-500 times more gonadotropin than necessary to evoke maximum s t e r o i d o -g e n e s i s . Only 10-15 per cent of the t o t a l s i t e s need be s a t u r a t e d to achieve the maximum response (Saxena and Rathnam, 1976). Consequently two orders of b i n d i n g s i t e s have been d e s c r i b e d (Saxena and Rathnam, 1976): ( i ) h i g h a f f i n i t y - l o w c a p a c i t y r e c e p t o r s , ( i i ) low a f f i n i t y - h i g h c a p a c i t y , r e p r e s e n t i n g n o n - s p e c i f i c b i n d i n g . In c o n t r a s t , C a t t and Dufau (1977) have indeed demonstrated "spare r e c e p t o r s " and occupation o f any 1 percent o f the 52 . t o t a l l e y d i g c e l l membrane r e c e p t o r s w i l l produce complete a c t i v a t i o n o f LH induced s t e r o i d o g e n e s i s . T h i s i n d i c a t e s e q u i v a l e n t s a t u r a t i o n b i n d i n g and a f f i n i t y between a l l LH r e c e p t o r s . E r i c k s o n e_t a_l. (1979) have determined the LH-Granulosa c e l l r e c e p t o r d i s s o c i a t i o n constant -11 (Kp) to be 1.4 x 10 M. U t i l i z i n g human FSH and semini-ferous tubule homogenates, B h a l l a and R e i c h e r t (1974) have demonstrated the Kp f o r FSH-receptor i n t e r a c t i o n s to be 6.7 x 1 0 " 1 0 M. For optimal hormone-receptor i n t e r a c t i o n s and maximum c e l l response s e v e r a l c r i t i c a l f a c t o r s must be c o n s i d e r e d . Carbohydrate m o i e t i e s are e s s e n t i a l f o r recep-t o r i n t e g r i t y and when FSH i s c o n s i d e r e d , s i a l i c a c i d and mannose r e s i d u e s are r e q u i r e d f o r optimal b i n d i n g (Saxena and Rathnam, 1976; C a t t and Dufau, 1977). Phospho-l i p i d s , p r i n c i p a l l y phosphatidylethanolamine, p h o s p h a t i d y l -s e r i n e and l e c i t h i n are v i t a l requirements f o r f u n c t i o n a l membrane r e c e p t o r s (Saxena and Rathnam, 1976) and d i s u l f i d e groups w i t h i n the LH r e c e p t o r appear to be i m p l i c a t e d f o r the b i o l o g i c a l l y a c t i v e conformation (Catt and Dufau, 1977). The p o l y p e p t i d e hormone-receptor i n t e r a c t i o n i n v o l v e s hydrophobic (consequently hydrogen bonding) and e l e c t r o s t a t i c f o r c e s which are f a c i l i t a t e d by s p e c i f i c 53. amino a c i d r e s i d u e s w i t h i n the hormone and r e c e p t o r s i t e (Catt and Dufau, 1977; Blake, 1978). R e s u l t s obtained with i n s u l i n and glucagon i n d i c a t e that peptide hormones and r e c e p t o r p r o t e i n s are capable of hydrogen bonding w i t h i n themselves exposing hydrophobic r e g i o n s . The comple-mentary hydrophobic r e g i o n s between the hormone and rec e p t o r then a s s o c i a t e i n what i s termed the " z i p p e r concept" (Blake, 1978). B i n d i n g occurs by the i n t e r a c t i o n of a s i n g l e amino a c i d r e g i o n w i t h i n the hormone wit h the appro-p r i a t e s u b s i t e on the r e c e p t o r . The hormone r e o r g a n i z e s i t s conformation with the r e s u l t other s p e c i f i c amino a c i d r e s i d u e s i n t e r a c t i n s u c c e s s i o n w i t h a p p r o p r i a t e r e c e p t o r s u b s i t e s u n t i l the hormone and r e c e p t o r are o p t i m a l l y a l i g n e d (Blake, 1978). I t i s accepted that cAMP i s the secondary messenger w i t h i n FSH and LH t a r g e t c e l l s (Saxena and Rathnam, 1976; Catt and Dufau, 1977; Hay and Moor, 1978; Blake, 1978; Richards e_t al.. , 1979), however, the exact mechanism of cAMP a c t i v a t i o n has only been r e c e n t l y d e s c r i b e d . The o r i g i n a l view that hormone r e c e p t o r s were s t r u c t u r a l l y coupled to adenylate c y c l a s e (EC 4.6.1.1) and served as r e g u l a t o r y s u b u n i t s d u r i n g the enzymatic c o n v e r s i o n o f ATP cAMP has si n c e been r e p l a c e d by other models. T h i s i s a consequence of demonstrations that s e v e r a l hormones can i n f l u e n c e d i s p e r s e d f a t c e l l s through 54. the same adenylate c y c l a s e system and yet s t i l l b i n d to separate s p e c i f i c r e c e p t o r s (Cuatrecasas e_t a l . , 1975) . A model i s a l s o r e q u i r e d which i s more c o n s i s t e n t with the f l u i d nature of plasma membranes. The model proposed i s "The Mobile Receptor Hypothesis" which i s based upon the assumption that r e c e p t o r and enzyme molecules are normally d i s c r e t e and separate s t r u c t u r e s (Cuatrecasas, 1974; Cuatrecasas et a l . , 1975; C a t t and Dufau, 1977; Blake, 1978). Occupancy of the r e c e p t o r s i t e by hormone i n c r e a s e s the a f f i n i t y of the r e c e p t o r f o r other membrane s t r u c t u r e s such as adenylate c y c l a s e (Cuatrecasas, 1974; Blake, 1978). Although the r e c e p t o r s i t e s appear to be p h y s i c a l l y d i s t i n c t from adenylate c y c l a s e (Catt and Dufau, 1977; L e v i t z k i and Helmreich, 1979) they o b v i o u s l y possess i n t i m a t e func-t i o n a l c o n n e c t i o n s . The adenylate c y c l a s e system c o n s i s t s of three components the hormone r e c e p t o r , the GTP r e g u l a t o r y u n i t (GTP B i n d i n g P r o t e i n ) and the adenylate c y c l a s e c a t a l y t i c u n i t (Catt and Dufau, 1977; L e v i t z k i and Helmreich, 1979). A c t i v a t i o n of the membrane bound enzyme r e q u i r e s simultaneous b i n d i n g of hormonal a g o n i s t and guanyl n u c l e o t i d e to t h e i r r e s p e c t i v e s i t e s . W i t h i n the GTP u n i t i s the enzyme GTPase which promotes GTP -> GDP + P i . When the hormone r e a c t s 55. FIGURE 11A Outside mom m o m Inside ATP cAMP FIGURE 11B i r ADENYLATE CYCLASE Catalytic GTP-ase c AMP GTP GDP ATP Figure 11: The hormone mobile receptor adenylate cyclase interaction ( BJake^978: Levitzki and Helmreich, 1979). 56. w i t h the r e c e p t o r i t f a c i l i t a t e s and maintains an exchange of GDP wit h GTP which i s a v i t a l requirement i n adenylate c y c l a s e a c t i v a t i o n ( L e v i t z k i and Helmreich, 1979) . Once a c t i v a t e d adenylate c y c l a s e , through i t s c a t a l y t i c s i t e , r e a c t s with ATP c h e l a t e d to magnesium to produce cAMP. The cAMP then induces the observed t a r g e t c e l l response by a c t i v a t i n g p r o t e i n kinase enzymes e i t h e r alone or i n combination w i t h other i n t r a c e l l u l a r messengers and e f f e c t s membrane p e r m e a b i l i t y depending upon t a r g e t c e l l f u n c t i o n . d) The Ovarian Response to Gonadotropins Ovarian f o l l i c u l a r development i n v o l v e s s t e r o i d and gonadotropin r e g u l a t i o n of granulosa-theca c e l l p r o l i -f e r a t i o n , d i f f e r e n t i a t i o n , morphology and f u n c t i o n . These s p e c i f i c changes i n response appear to be r e l a t e d , i n p a r t , to hormone s p e c i f i c r e g u l a t i o n of hormone r e c e p t o r s . Mammalian o v a r i e s are c o n s i d e r e d to c o n t a i n a n o n - p r o l i -f e r a t i n g p o o l of germ c e l l s which are e v e n t u a l l y surrounded by a l a y e r of c e l l s and through the process of maturation form three c e l l types; granulosa c e l l s , basement membrane, t h e c a l l a y e r s e x t e r n a l to the basement membrane and stromal connective t i s s u e (Saxena and Rathnam, 1976; Richards et a l . , 1978). 57. F o l l i c l e growth i s presumed to begin as a random i n i t i a t i n g event which, once s t a r t e d , proceeds u n t i l ovula-t i o n or a t r e s i a o ccurs. The process i n v o l v e s the formation of a p o o l o f committed growing f o l l i c l e s which subsequently-become l a r g e p r e - a n t r a l f o l l i c l e s . I f t h i s stage o f matura-t i o n and p r e - o v u l a t o r y surge o f gonadotropin are achieved f u r t h e r f o l l i c u l o g e n e s i s w i l l occur simultaneously and culminate with o v u l a t i o n d u r i n g the next r e p r o d u c t i v e c y c l e . A t r e s i a occurs when the gonadotropin surge and p r e - a n t r a l stage are not synchronized (Richards et a_l. , 1978) . F o l l i c u l o g e n e s i s and consequently s t e r o i d o g e n e s i s are b e l i e v e d to i n o v l v e a "Two C e l l Theory". During f o l l i -c l e maturation to the p r e - a n t r a l stage FSH i s bound s o l e l y by the granulosa c e l l s and LH by the t h e c a l c e l l s (Saxena and Rathnam, 1976; Lindner et a l . , 1977; Richards et a l . , 1979) . S y n t h e s i s of o v a r i a n estrogen r e q u i r e s an intimate i n t e r a c t i o n between thecal c e l l s , the so l e source o f androgen, and granulosa c e l l s which are necessary f o r a r o m a t i z a t i o n (D o r r i n g t o n et a_l. , 1975 ; Moon et _al. , 1975 , 1978; Lindner et a l . , 1977; Hay and Moor, 1978; Richards et a l . , 1978). LH f a c i l i t a t e s the c o n v e r s i o n o f c h o l e s t e r o l to pregnenolone with the ensuing formation of androgens, p r i n c i p a l l y dehydroepiandrosterone (DHA), androstenedione 58. and t e s t o s t e r o n e . This conversion and pathway occurs v i a the A^-38-hydroxysteroid pathway w i t h i n the theca c e l l s . The granulosa c e l l s are also capable of androstene-4 dione and testosterone b i o s y n t h e s i s through the A -3-k e t o s t e r o i d pathway w i t h such intermediates as progesterone and 17-hydroxyprogesterone (Fotherby, 1975; Gower and Fotherby, 1975; Gower, 1975a; Marsh et a l . , 1976). FSH then s t i m u l a t e s aromatization of androstenedione and t e s t o -sterone w i t h i n the granulosa c e l l s to produce p r i n c i p a l l y e s t r a d i o l - 1 7 3 (Moon et. a_l. , 1975; Dorrington et. al. , 1975 ; Richards ejt a_l. , 1978) . Gower and Fotherby (1975) contra-d i c t t h i s concept by maintaining that theca c e l l s are r e s p o n s i b l e f o r aromatization and estrogen production during the f i r s t h a l f of the menstrual c y c l e . As e s t r a d i o l - 1 7 3 i s synthesized and secreted i t i n t u r n exerts a p o s i t i v e feedback upon the ovary to increase the f o l l i c u l a r response to gonadotropins. As the f o l l i c l e matures FSH increases i t s own number of recep-t o r s while E2"17B increases the responsiveness of the FSH adenylate cyclase system to FSH. With the approach of the Graafian F o l l i c l e stage FSH and e s t r a d i o l work s y n e r g i s t i c a l l y to induce granulosa c e l l d i f f e r e n t i a t i o n and promote increases i n the number of granulosa c e l l LH receptors (Saxena and Rathnam, 1976; Richards et a l . , 59. FIGURE 12 A c e t y - C o A 1 C h o l e s t e r o l 1 7 - H y d r o x y p r o g e 3 t e r o n e HO Deh ydroepiandrosterone . Pathway Testosterone A Pathway H O ^ , OH Estrone Estradi ol Figure 12: Pathways of steroid biosynthesis in human ovarian tissue (Marsh fit al. 1976) 60. 1976, 1978, 1979; E r i c k s o n et a l . , 1979). T h i s d i s c u s s i o n demonstrates three methods of hormone s p e c i f i c r e g u l a t i o n of hormone r e c e p t o r s which occur with the mammalian ovary (Richards et a l . , 1976); ( i ) A u t o r e g u l a t i o n : hormone a f f e c t s only i t s own r e c e p t o r s , ( i i ) C oordinated r e g u l a t i o n : s t e r o i d - p r o t e i n hormone i n t e r a c t i o n , ( i i i ) H e t e r o r e g u l a t i o n : one hormone a f f e c t s the content of r e c e p t o r f o r an e n t i r e l y d i f f e r e n t hormone. With the appearance of LH r e c e p t o r s on granulosa c e l l s LH b i n d i n g a l t e r s the s t e r o i d o g e n i c f u n c t i o n and morphology of theca and granulosa c e l l s i n i t i a t i n g o v u l a t i o n and spontaneous l u t e i n i z a t i o n (Saxena and Rathnam, 1976; Hay and Moor, 1978). With the onset of LH s t i m u l a t i o n there i s a p r e l i m i n a r y i n c r e a s e i n estrogen s y n t h e s i s which i s then r a p i d l y terminated. T h i s i s f o l l o w e d by a s e r i e s of s e q u e n t i a l but t r a n s i e n t peaks of t e s t o s t e r o n e , 33-17ct-dihydroxypregn-5-en-20-one and pregnenolone a l l of which r e f l e c t a p r o g r e s s i v e l o s s of s t e r o i d o g e n i c a c t i v i t y i n the t h e c a l component. As l u t e i n i z a t i o n of the granulosa 61. c e l l s o c c u r s , the c e l l s e x h i b i t enhanced s y n t h e t i c a c t i v i t y p r o d u c i n g m a i n l y p r o g e s t e r o n e w i t h some 17-hydroxyproges-t e r o n e , 20a-hydroxypregn-4-3n-3-one and 2 0 a - d i h y d r o p r o g e s t e r o n e (Gower and F o t h e r b y , 1975; Hay and Moor, 1978). LH induced s t e r o i d o g e n e s i s i n the human corpus luteum i n v o l v e s the A ^ pathway where LH f a c i l i t a t e s the s y n t h e s i s o f c h o l e s t e r o l from a c e t a t e and the subsequent 2 0 a - h y d r o x y l a t i o n s t e p whereby c h o l e s t e r o l i s c o n v e r t e d t o pregnenolone (Gower, 1975b; Moon e t a l . , 1975; Marsh e t a l . , 1976). LH and p r o l a c t i n c o n s t i t u t e the o v i n e l u t e o t r o p h i c complex (Denamur e t a l . , 1973) and b o t h are r e q u i r e d f o r the e s t a b l i s h m e n t and maintenance o f the o v i n e corpus luteum (Denamur e_t a l . , 1973) . A s s o c i a t e d w i t h LH-induced g r a n u l o s a c e l l l u t e i n i z a t i o n i s a decrease i n the number of r e c e p t o r s f o r LH and FSH w i t h subsequent i n c r e a s e s i n p r o l a c t i n r e c e p t o r . P r o l a c t i n i n t u r n r a i s e s the number o f l u t e a l c e l l r e c e p t o r s f o r LH. P r o l a c t i n and e s t r a d i o l are a l s o r e q u i r e d t o s t i m u l a t e and m a i n t a i n the s t e r o i d o -g e n e s i s w i t h i n the corpus l u t e u m " i n d u c e d by the LH surge (Denamur e_t al . . , 1973; C r o s i g n a n i e t al. , 1976; M a r t i n , 1976; R i c h a r d s e t a l . , 1978; P o i n d e x t e r e t a l . , 1979). The c o n c e n t r a t i o n o f c i r c u l a t i n g p r o l a c t i n , i f s u f f i c i e n t l y e l e v a t e d w i l l i n c o n t r a s t , i n h i b i t human o v a r i a n l u t e a l f o r m a t i o n and f u n c t i o n i n response to LH ( R o l l a n d e t a l . , 1976; F r i e s e n and S h i u , 1977). 62 . In a d d i t i o n to a c t i v a t i o n o f the adenylate c y c l a s e -cAMP system LH and p r o l a c t i n appear to i n c o r p o r a t e p r o s t a g l a n -dins as i n t r a c e l l u l a r messengers i n the l u t e i n i z a t i o n and s t e r o i d o g e n i c processes d u r i n g the ovine e s t r o u s c y c l e . There i s c o n f l i c t i n g evidence that p r o s t a g l a n d i n s , p r i n c i -p a l l y PGE2, may act as an o b l i g a t o r y intermediate between the p r o t e i n hormone-adenylate c y c l a s e system and/or mediators i n other i n t r a c e l l u l a r processes (Kuehl et a l . , 1973, 1976; Weiss et a l . , 1976; Marsh and LeMaire, 1976; Marsh et a l . , 1976; Lindner et a l . , 1977; Hay and Moor, 1978; Behrman, 1979). Gower (1975b) again o f f e r s a c o n t r a d i c t o r y d i s c u s s i o n by s t a t i n g t h at the p r o s t a g l a n d i n - a d e n y l a t e c y c l a s e r e l a t i o n s h i p holds in v i t r o but i s not applicable i n v i v o as there can be a demonstrable r e d u c t i o n of plasma progesterone and 20a-hydroxyprogesterone l e v e l s . Gower (1975b) does not s p e c i f y the type of p r o s t a g l a n d i n , the r e p r o d u c t i v e s t a t e s of the v a r i o u s s p e c i e s or i n f a c t the s p e c i e s i n q u e s t i o n , a l l o f which are c r i t i c a l to h i s c o n c l u s i o n s . 7. THE HYPOTHALAMIC-HYPOPHYSEAL-OVARIAN AXIS a) The Ovine E s t r o u s Cycle The mean es t r o u s c y c l e l e n g t h of the ewe i s determined to" be 16.5-17.5 days ( F r a s e r , 1971; T e r r i l l , 63. 1974; Robertson, 1977) and can be p a r t i t i o n e d i n t o four d i s t i n c t phases depending upon the stage of f o l l i c u l a r m aturation ( T e r r i l l , 1971; McDonald, 1975). Pr o e s t r u s l a s t s approximately two days and i s c h a r a c t e r i z e d by f o l l i -c u l a r growth with i n c r e a s i n g estrogen p r o d u c t i o n . E s t r u s r e p r e s e n t s the p e r i o d of sexual r e c e p t i v i t y which l a s t s 26-40 hours wi t h o v u l a t i o n u s u a l l y o c c u r r i n g 24-27 hours a f t e r the onset of e s t r u s . The l u t e a l phase commences with metestrus, a p e r i o d which l a s t s two days c h a r a c t e r i z e d by the formation of a corpus luteum and progesterone s e c r e -t i o n . D i e s t r u s f o l l o w s r e p r e s e n t i n g the p e r i o d of a func-t i o n a l corpus luteum with l a r g e amounts of progesterone s e c r e t i o n . D i e s t r u s l a s t s approximately 12 days. The endocrine events a s s o c i a t e d with these four phases are e s s e n t i a l f o r c o n t r o l of the o v a r i a n c y c l e . Serum c o n c e n t r a t i o n s of e s t r a d i o l begin to r i s e 12-14 hours p r i o r t o , and peak with , the onset of e s t r u s ( B a i r d et a l . , 1976; Pant et al.. , 1977; Scaramuzzi and Land, 1978). C l o s e l y c o r r e l a t e d with t h i s s e c r e t i o n of o v a r i a n e s t r a d i o l i s androstenedione which indicates a common pathway or site of synthesis ( B a i r d e_t a l . , 1976) . Serum LH remains low d u r i n g the l u t e a l phase (Pant ejt a_l. , 1977), rises to peak l e v e l s 0-16 hours a f t e r the onset of e s t r u s and then r a p i d l y d e c l i n e s to low c o n c e n t r a t i o n s d u r i n g the 63a. FIGURE 13 Figure 13; Schematic reproduction of the hormonal changes during the ovine estrous cycle ( Robert son ,197 7). 64. ensuing l u t e a l phase (Cunningham e_t al. , 1975; Pant et a l . , 1977 ; B a i r d et a l . , 1978). FSH can e x h i b i t two peaks, the f i r s t c o i n c i d e s with the LH surge, the second occurs 24 hours l a t e r (Cunningham e_t al., 1975; Pant et. a l . , 1977). Progesterone l e v e l s vary i n a c y c l i c manner, the h i g h e s t values are a t t a i n e d d u r i n g the m i d - l u t e a l phase and remain e l e v a t e d u n t i l 35 hours p r i o r to the onset of e s t r u s . By t h i s time the u t e r i n e l u t e o l y t i c f a c t o r PGF2 a (Goding, 1974) has f a c i l i t a t e d r e g r e s s i o n of the corpus luteum r e s u l t i n g i n a r a p i d s i g n i f i c a n t decrease i n serum progesterone l e v e l s which remain low u n t i l days 2-4 of the f o l l o w i n g c y c l e ( B a i r d et al., 1976; Pant et a l . , 1977) . b) The Role of Ovarian S t e r o i d s It i s apparent from the p r e v i o u s d i s c u s s i o n concerned w i t h the ovine e s t r o u s c y c l e that there e x i s t d i s t i n c t r e l a t i o n s h i p s between c i r c u l a t i n g hormone l e v e l s and the i n t e g r a t i o n of v a r i o u s endocrine events, a l l of which c o l l e c t i v e l y p r o v i d e accurate c o n t r o l of the repro-d u c t i v e c y c l e . 65. The major s t e r o i d s s e c r e t e d by the ovary, as s t a t e d p r e v i o u s l y , are: progesterone, _ 20oc-dihydroproges-terone, e s t r o n e , 17a-dihydroprogesterone, androstenedione and e s t r a d i o l - 1 7 3 (Scaramuzzi et al., 1974; B a i r d , 1976, 1977). The s t e r o i d s r e l e v a n t to t h i s d i s c u s s i o n are p r i n c i -p a l l y progesterone, androstenedione and e s t r a d i o l - 1 7 B . E s t r o g e n i c e f f e c t s can be g e n i t a l or n o n - g e n i t a l i n nature (Hebert, 1977). In the ewe g e n i t a l e f f e c t s are c h a r a c t e r i z e d by s t i m u l a t i o n i n growth and f u n c t i o n of the o v a r i e s , f a l l o p i a n tubes, u t e r u s , c e r v i x , vagina, e x t e r n a l g e n i t a l i a and mammary glands. T h i s i s achieved through i n c r e a s e d r e c e p t o r , RNA and p r o t e i n metabolism ( M i l l e r , 1976; M i l l e r et a l . , 1977). The n o n - g e n i t a l e f f e c t s of estrogens are summarized by: the development and maintenance of secondary sex c h a r a c t e r i s t i c s , a n a b o l i c e f f e c t s , and c r i t i c a l to the f o l l o w i n g d i s c u s s i o n , feed-back e f f e c t s on c y c l i c gonadotropin s e c r e t i o n and a c t i o n w i t h i n the ovary. The dual and r e c i p r o c a l a c t i o n between gonadal s t e r o i d s and gonadotropin r e l e a s e i l l u s t r a t e s the presence of i n t e g r a t e d c o n t r o l mechanisms. C a s t r a t i o n r e s u l t s i n p i t u i t a r y hypertrophy and a s s o c i a t e d i n c r e a s e s i n gonado-t r o p i n s e c r e t i o n (Dorner, 1976; Hutchinson and Sharp, 1977). A d m i n i s t r a t i o n of estrogen to these c a s t r a t e s 66. r e s u l t s i n a subsequent r e d u c t i o n i n gonadotropin secre-t i o n (Lisk et a l . , 1972). Pant et a l . (1978) and Rawlings et a l . (1978) a c t i v e l y immunized ewes ag a i n s t E2-173. As a consequence the ewes e x h i b i t e d complete absence of estrous b e h a v i o r , the o v a r i e s c o n t a i n e d lar g e G r a a f i a n f o l l i c l e s and plasma LH l e v e l s p a r a l l e l e d i n c r e a s e s i n serum antibody t i t e r . I n j e c t i o n with h i g h doses of s t i l -b e s t r o l d i p r o p i o n a t e reduced the LH c o n c e n t r a t i o n s . The concept presented thus f a r r e p r e s e n t s an o v e r a l l negative feedback f o r estrogen on gonadotropin s e c r e t i o n which maintains gonadotropin l e v e l s w i t h i n p h y s i o -l o g i c a l l i m i t s (Dorner, 1976). Estrogen a l s o e x e r t s a p o s i t i v e feedback upon FSH and LH r e l e a s e . The most noticeable i s the LH surge which induces o v u l a t i o n and l u t e a l forma-t i o n (Hutchinson, 1978). What then, are the primary r o l e s f o r gonadal s t e r o i d s i n the c o n t r o l o f o v u l a t i o n ? During e a r l y p r o e s t r u s the corpus luteum r e g r e s s e s , removing the progesterone b l o c k which has i n h i b i t e d s u f f i c -i e n t gonadotropin r e l e a s e f o r maximal f o l l i c u l a r growth and o v u l a t i o n (Robertson, 1977). With removal of proges-terone, i n t r i n s i c hypothalamic and o v a r i a n rythyms f a c i l i t a t e i n i t i a l r e l e a s e of FSH and e s t r a d i o l respec-t i v e l y . The o v a r i a n s t e r o i d s , i n t h i s i n s t a n c e ££-173, are capable of i n t e r a c t i n g with neuroendocrine t i s s u e 67. (Stumpf, 1970, 1971a,b; Stumpf et a l . , 1975; Stumpf and Sar, 1976; C h a l l i s et a l . , 1976; McEwen, 1976; Kato, 1977a,b; Stumpf and Sar, 1977) to modify the s y n t h e s i s and s e c r e t i o n of GnRH neurons. The p o s i t i v e feedback e f f e c t of estrogen appears to be f a c i l i t a t e d w i t h i n the p r e o p t i c - s u p r a c h i a s m a t i c area (DePaolo and Barraclough, 1979; Fink, 1979). The i n h i b i t o r y a c t i o n of estrogen upon GnRH r e l e a s e occurs i n p a r t w i t h i n the arcuate nucleus of the medial b a s a l hypothalamus (McCann, 1977) and the t u b e r o i n f u n d i b u l a r t r a c t dopamine neurons i n the l a t e r a l p a l i s a d e zone of the median eminence. S t i m u l a t i o n of these dopamine neurons wit h a s s o c i a t e d d e s e n s i t i z a t i o n of norepinephrine neurons r e s u l t s i n reduced GnRH s e c r e t i o n (Fuxe e_t al.. , 1976, 1977). T h i s estrogen mediated r e l e a s e of GnRH f a c i l i -t a t e s GnRH r e l e a s e from the median eminence i n t o the hypophy-s e a l p o r t a l system to s t i m u l a t e the adenohypophysis gonado-tro p h s , i n i t i a l l y and through i t s s e l f - p r i m i n g a f f e c t , to s e c r e t e FSH. E s t r a d i o l - 1 7 B i s a l s o capable o f i n c r e a s i n g the p i t u i t a r y response to GnRH (Fink et a l . , 1977; L a b r i e et a l . i 1977; F o s t e r , 1978; L a b r i e , 1978; Fink , 1979). T h i s promotes f o l l i c u l a r maturation with a s s o c i a t e d i n c r e a s e s i n E ?-173, androstenedione and progesterone. 68. Together FSH and E2~17B induce the formation of granulosa c e l l LH r e c e p t o r s d u r i n g l a t e p r o e s t r u s and e a r l y e s t r u s i n p r e p a r a t i o n f o r o v u l a t i o n (Richards, 1976, 1978, 1979). Estrogen peaks with the onset of e s t r u s and t r i g g e r s the p r e o v u l a t o r y surge of LH i n d u c i n g o v u l a t i o n ( B a i r d and Scaramuzzi, 1976; Franchimont et_ al.. , 1976 ; Yen, 1976; Karsch et a l . , 1977; Fink, 1979). A s s o c i a t e d w i t h the p r e - o v u l a t o r y surge of gonadotropin i s a pr o g r e s s -i v e l y i n c r e a s i n g GnRH turnover. J u s t i z et al. (1973) r e p o r t e d that the hig h e s t l e v e l of GnRH immunoreactivity occurs d u r i n g the p r e o v u l a t o r y surge of LH and FSH and i s not d e t e c t a b l e o u t s i d e the e s t r u s p e r i o d . Gonadotropin-r e l e a s i n g hormone e x h i b i t s p u l s a t i l e r e l e a s e p a t t e r n s ( C r i g h t o n et_ al.. , 1973) with d e t e c t a b l e peaks i n c r e a s i n g i n frequency and magnitude immediately p r i o r to and durin g the p r e - o v u l a t o r y LH surge ( C r i g h t o n et_ a l . , 1974; F o s t e r et a l . , 1976; F o s t e r , 1978). In a d d i t i o n to the p r e - o v u l a t o r y surge i n gonado-t r o p i n , estrogen i s a l s o capable of f a c i l i t a t i n g p r o l a c t i n r e l e a s e from p i t u i t a r y l a c t o t r o p h s (Meites, 1969; Fuxe et a l . , 1977; Wuttke, 1977 ; F e r l a n d et a l . , 1979; Padmanabhan and Convey, 1979; Shupnik e_t a l . , 1979; V i c i a n e_t a l . , 1979). T h i s aspect of estrogen metabolism i s important as p r o l a c t i n and LH form the l u t e o t r o p h i c complex i n the ewe (Denamur, 1973). Dopamine i s a p r o l a c t i n i n h i b i t i n g f a c t o r . Estrogen i s anti-dopaminergic w i t h i n the medial p a l i s a d e zone of the median eminence and can i n d i r e c t l y f a c i l i t a t e p r o l a c t i n r e l e a s e from l a c t o t r o p h s ( F e r l a n d et a l . , , 1979). P r o l a c t i n mediates i t s own s e c r e t i o n v i a a short loop feedback mechanism i n v o l v i n g dopamine turnover i n the medial p a l i s a d e zone. P r o l a c t i n a l s o i n c r e a s e s dopamine turnover i n the l a t e r a l p a l i s a d e zone which i n d i r e c t l y i n h i b i t s GnRH r e l e a s e from the median eminence (Fuxe et a l . , 1976, 1977; Wuttke, 1977; Rudd et a l . , 1979). Under the i n f l u e n c e o f LH and p r o l a c t i n a f u n c t i o n a l corpus luteum i s formed which s e c r e t e s proges-terone d u r i n g d i e s t r u s . Progesterone now appears to be the primary c o n t r o l l e r of t o n i c gonadotropin s e c r e t i o n i n the ewe (Karsch e_t a_l. , 1977). Progesterone a l t e r s the frequency of p u l s a t i l e GnRH r e l e a s e p r o h i b i t i n g LH r e l e a s e a l l o w i n g p i t u i t a r y LH r e s e r v e s to b u i l d up ( P e l l e t i e r and Thiomoner, 1975). Progesterone p r i m i n g i s a l s o necessary f o r the p o s i t i v e estrogen feedback on gonadotropin r e l e a s e ( B a i r d and Scaramuzzi, 1976; Fink et a l . , 1977; Karsch et a l . , 1977 , 1979) . In the ewe Robertson (1977) d e s c r i b e s an i n t r i n s i c f o l l i c u l a r and o v u l a t o r y c y c l e of 4-5 days i n absence of a f u n c t i o n a l corpus luteum and a 4-5 day f o l l i c u l a r c y c l e without 70. o v u l a t i o n i n the presence of a corpus luteum. The f o l l i c u l a r c y c l e c o n s i s t s of 3-4 d i s c r e t e waves of g r a a f i a n f o l l i c u -l a r development f o l l o w e d by a t r e s i a during the normal estrous c y c l e . O v u l a t i o n i n a l l but one f o l l i c l e i s suppressed by progesterone s e c r e t i o n from a f u n c t i o n a l corpus luteum (Karsch et. a l . , 1977 ; Robertson, 1977). The wave of f o l l i c l e development and subsequent a t r e s i a i s a s s o c i a t e d w i t h s l i g h t e l e v a t i o n s i n E2~17B and LH (due to the p o s i t i v e estrogen e f f e c t ) ; however a f u l l p r e o v u l a t o r y surge i s i n h i b i t e d due to the i n c r e a s i n g progesterone s e c r e t i o n ( B a i r d and Scaramuzzi, 1976; Robertson, 1977). The other major o v a r i a n s t e r o i d , androstenedione, p a r a l l e l s the s e c r e t i o n of estrogen and as an androgen may be i m p l i c a t e d i n o v a r i a n f o l l i c u l a r a t r e s i a (Lindner et a l . , 1977; Hay and Moor, 1978). The u l t i m a t e f a t e of f o l l i c l e s may depend on the balance between androgen and estrogen at c r i t i c a l stages of f o l l i c u l a r development. An imbalance i n favour of androgen may a f f e c t the o v a r i a n response to gonadotropin or gonadotropin s e c r e t i o n . In the ewe B a i r d (1976) n e u t r a l i z e d the b i o l o g i c a l a c t i v i t y of androstenedione by a c t i v e immunization which r e s u l t e d i n i n c r e a s e d LH s e c r e t i o n . 71. FIGURE 14 Non Hypothalamic C N S Hypothalamus Area Area Catecholamines + P 1 F GnRH Pituitary LH FSH Prolactin LH Prolactin _L_ FSH Ovary Eg ±-Progesterone t Figure 14 : Schematogram of the endocrine reproductive systems in females . 72. c) E x f o l i a t i v e V a g i n a l Cytology during the E s t r o u s  Cycle Throughout the estrous c y c l e u t e r i n e and v a g i n a l e p i t h e l i u m undergo c y c l i c changes which p a r a l l e l the o v a r i a n s t e r o i d p r o f i l e p r e v a i l i n g i n the blood c i r c u l a t i o n (Papanicolaou et al. , 1948; Sanger et. _al. , 1958; Wachtel, 1969; McDonald, 1975; Sorensen, 1979). The v a g i n a l w a l l c o n s i s t s of a f i b r o u s coat, muscular coat and i n t e r n a l mucosa l i n i n g covered with s t r a t i f i e d squamous e p i t h e l i u m . W i t h i n t h i s e p i t h e l i u m of a mature female 4 d i s t i n c t zones can be r e c o g n i z e d (Figure 15). The p r o l i f e r a t i o n and dominance of any one zone w i l l depend upon the gonadal s t e r o i d hormone p r o f i l e (Wachtel, 1969). The b a s a l c e l l s l i e adjacent to the basement membrane and separate the o v e r l y i n g e p i t h e l i u m from the u n d e r l y i n g stroma. These c e l l s are c u b o i d a l i n shape and form a s i n g l e l a y e r f i r m l y a t t a c h e d to the basement membrane. As a r e s u l t b a s a l c e l l s do not e x f o l i a t e and are absent from v a g i n a l smears. Basal c e l l s are the u n d i f f e r e n t i a t e d c e l l s from which r e g e n e r a t i o n of e p i t h e -lium i s maintained. S u p e r f i c i a l to the b a s a l l a y e r are the Parabasal c e l l s . T h i s zone c o n s i s t s of s e v e r a l rows of p o l y h e d r a l c e l l s with comparatively l a r g e n u c l e i and glycogen processes FIGURE 15 73. x — | - H # i # i « l # l # l « l # l » l » l # ] J A: Basal layer B: Parabasal layer C: Intermediate layer D: Superficial layer i Figure 15: The vaginal mucosa (Wachtel 1969). 74. extending from one c e l l to another. These c e l l s may-e x f o l i a t e , l o s i n g the i n t e r c e l l u l a r b r i d g e s and appear i n smears as round or o v a l shaped c e l l s . The intermediate l a y e r i s composed of s e v e r a l rows of s l i g h t l y l a r g e r f l a t t e r c e l l s whcih a l s o possess i n t e r c e l l u l a r b r i d g e s . T h e i r n u c l e i are v e s i c u l a r and appear s m a l l e r i n r e l a t i o n to c e l l s i z e than those of pa r a b a s a l c e l l s . When e x f o l i a t e d these c e l l s appear l a r g e r and l e s s round than p a r a b a s a l c e l l s . The s u p e r f i c i a l zone c o n s i s t s o f s e v e r a l l a y e r s of l a r g e f l a t c e l l s with p y k n o t i c n u c l e i . In v a g i n a l smears the c e l l s are large and p o l y h e d r a l w i t h a c l e a r t r a n s p a r e n t cytoplasma and p y k n o t i c nucleus. The nucleus s t a i n s u n i f o r m l y dark while the cytoplasm s t a i n s p i n k ( e o s i n o p h i l i c ) . These f o u r zones which make up the v a g i n a l e p i t h e -lium are very s e n s i t i v e to s t i m u l a t i o n by the sex hormones to which they respond. The response c h a r a c t e r i s t i c a l l y i n v o l v e s a l t e r a t i o n of e p i t h e l i u m height (number of c e l l l a y e r s ) and t h i c k n e s s (number of c e l l rows per l a y e r ) . During the f o l l i c u l a r phase estrogen has a pro-l i f e r a t i v e and maturing a f f e c t on the v a g i n a l e p i t h e l i u m which promotes growth and d i f f e r e n t i a t i o n and r e s u l t s 75. in a well developed s u p e r f i c i a l zone. The l e v e l of estrogen and duration of i t s stimulation is depicted by the eventual p r o l i f e r a t i o n and maturation of the e p i t h e l i a l c e l l s . Estrogen also has a clearing effect on c e r v i c a l mucus which becomes transparent, consequently vaginal smears obtained with a high l e v e l of estrogenic stimulation have a clear background. During the l u t e a l phase progesterone has a regressive influence on the mature estrogen primed epithelium. Consequently with the onset of a functional corpus luteum the s u p e r f i c i a l layer i s sloughed off. Once this occurs progesterone w i l l maintain the epithelium within the parabasal and intermediate squamous zones. The c e l l s often appear oval i n shape with thickened c e l l borders, clear blue staining cytoplasm and an oval eccen-t r i c vessicular nucleus. These c e l l s are termed "navicular c e l l s " as they often appear boat shaped. C e l l s of this type have a tendancy to crowd together, f o l d and c u r l t h e i r edges forming the boat shape. Parabasal and inter-mediate c e l l s take up the cyanophilic stain therefore the o v e r a l l staining reaction produces bluish-green smears. Under the influence of progesterone c e r v i c a l mucus i s opaque, exhibits l i t t l e e l a s t i c i t y and contains many leukocytes. 76. 8. A POTENTIAL ROLE FOR PHYTO-ESTROGENS IN FEMALE REPRODUCTION Hauger e_t a_l. (1977) s t a t e the presence or absence of v a r i o u s hormones which may act s y n e r g i s t i c a l l y with, or a n t a g o n i s t i c a l l y t o , estrogen must be c o n s i d e r e d i n i n t e r p r e t i n g the o v e r a l l sequence of events. With t h i s statement i n mind i t becomes obvious that phyto-estrogens w i l l a f f e c t the r e p r o d u c t i v e p h y s i o l o g i c a l responses w i t h i n the animal i n q u e s t i o n . The p o s s i b l e s i t e s f o r coumestan and i s o f l a v o n e a c t i o n p a r a l l e l those f o r endogenous estrogen. Negative and p o s i t i v e feedback a c t i o n at the hypothalamic and p i t u i -t a r y l e v e l s to i n h i b i t or s t i m u l a t e GnRH r e l e a s e becomes an important c o n s i d e r a t i o n . Phyto-estrogens are capable of b i n d i n g to and a l t e r i n g hypothalamic and p i t u i t a r y e s t r a d i o l c y t o s o l (Tang and Adams, 1978; Mathieson, 1980). The p o t e n t i a l of p l a n t estrogens to modify p r o l a c t i n s e c r e -t i o n may a l s o p l a y a r o l e as e l e v a t e d p r o l a c t i n l e v e l s are commonly a s s o c i a t e d w i t h o v a r i a n d y s f u n c t i o n (Besser, 1976; C r o s i g n a n i et al., 1976; R o l l a n d et a l . , 1976). Working w i t h females d u r i n g the puerperium, C r o s i g n a n i et a l . (1976) and Besser (1976) have demonstrated a r e f r a c -t o r i n e s s of gonadotrophs to GnRH, delayed gonadotroph 77. recovery and o v a r i a n i n s e n s i t i v i t y to gonadotropin s t i m u l a -t i o n r e s u l t i n g i n hypogonadism and reduced s t e r o i d o g e n e s i s . Phyto-estrogen a c t i v i t y may a l s o demonstrate i t s e l f w i t h i n the ovary. I t may compound the estrogen-FSH e f f e c t i n d u c i n g g r e a t e r gonadotropin r e c e p t o r formation and increases the o v a r i a n response to c i r c u l a t i n g gonado-t r o p i n . The opposite a l s o holds true i f p l a n t estrogens prove a n t a g o n i s t i c to the a c t i o n o f endogenous estrogen at the o v a r i a n l e v e l . Groom and G r i f f i t h s (1976) u t i l i z e d the a n t i e s t r o g e n tamoxifen i n pre-menopausal women to demonstrate l i t t l e d i f f e r e n c e i n LH, FSH and progesterone s e c r e t i o n ; however, a two to e i g h t - f o l d i n c r e a s e i n e s t r a d i o l l e v e l and a s i g n i f i c a n t decrease i n p r o l a c t i n was found. The a n t i e s t r o g e n CI 628 a l s o demonstrates the a b i l i t y to i n h i b i t the indu6tion of p r o g e s t i n r e c e p t o r s by e s t r a d i o l i n the preoptic-hypothalamus area and p i t u i t a r y (Roy et a l . , 1979). This c a p a b i l i t y of a n t i e s t r o g e n i s of importance a l s o as i t i s now known t h a t progesterone appears to be the primary o r g a n i z e r of the ovine estrous c y c l e (Karsch et a l . , 1977, 1979). The a b i l i t y of p l a n t ' estrogens to modify t h i s aspect of the c y c l e c o u l d a l s o a f f e c t sexual behavior and hormonal sequences during the ensuing o v u l a t o r y c y c l e . The i n t e n t of the r e s e a r c h 78. r e p o r t e d here i s t o determine the e f f e c t , i f any, o f e s t r o -g e n i c a l f a l f a consumption upon the endogenous g o n a d o t r o p i n p r o f i l e i n the c y c l i n g ewe and to deduce the p o s s i b l e pathways i n v o l v e d . 79. EXPERIMENTAL PROCEDURE I. INTRODUCTION The o b j e c t i v e s of t h i s study are to determine the effect of e s t r o g e n i c a l f a l f a consumption on reproduc-t i v e performance i n the ewe. The parameters observed were the response to e s t r u s s y n c h r o n i z a t i o n , e x f o l i a t i v e v a g i n a l c y t o l o g y and blood plasma gonadotropin l e v e l s . To accomplish these o b j e c t i v e s , ten maiden Dorset Horn ewes e x h i b i t i n g normal estrous c y c l e s , as determined through a c t i o n s of a vasectomized marker ram, were randomly assigned i n t o experimental groups. F i v e ewes i n Group I each r e c e i v e d a r a t i o n of Orchard Grass Hay (Dactylus  glomerata; 1.0 kg/day) wi t h a Whole B a r l e y : Beef Concentrate Supplement ( B u c k e r f i e l d s ; 0.30 kg/day). The remaining f i v e ewes i n group II each r e c e i v e d a r a t i o n of Creston A l f a l f a Cubes (Medicago s a t i v a ; 1.2 kg/day), produced and purchased from Kootney Dehydrators, Creston, B.C. Supplement was i n c l u d e d with the Group I r a t i o n i n order to balance p r o t e i n and energy content of the orchard grass hay with the a l f a l f a cubes. P l a n t , b a r l e y and beef concentrate e x t r a c t s were obtained by a m o d i f i e d method of F r a n c i s and 80. M i l l i n g t o n (1965) as d e s c r i b e d by Newsome and K i t t s (1977). The e x t r a c t s were then assayed f o r phyto-estrogen content u s i n g the Competitive B i n d i n g Assay technique of Korenman (1968) and Shutt (1969). Orchard grass hay, b a r l e y and concentrate were shown to c o n t a i n lower amounts of phyto-e s t r o g e n i c a c t i v i t y than a l f a l f a cubes (Table 3). As a consequence group I animals, which r e c e i v e d the orchard grass hay - supplement, formed experimental c o n t r o l s and the group II animals r e c e i v e d an experimental t e s t r a t i o n of a l f a l f a cubes, high i n p h y t o - e s t r o g e n i c a c t i v i t y . Animals were synchronized f o r e s t r u s u s i n g proges-terone impregnated i n t r a v a g i n a l p e s s a r i e s then p l a c e d on t h e i r r e s p e c t i v e r a t i o n s one week p r i o r to the f i r s t scheduled b l e e d i n g . V a g i n a l c y t o l o g i c a l s t u d i e s were conducted i n c o n j u n c t i o n with the use of a vasectomized marker ram to c h a r a c t e r i z e the stage of the estrous c y c l e and determine the onset of b e h a v i o r a l e s t r u s . J u g u l a r venous blood was withdrawn i n t o h e p a r i n i z e d s y r i n g e s , c e n t r i f u g e d and a l i q u o t s of the plasma f r o z e n u n t i l assay. During metestrus, d i e s t r u s and ea r l y - m i d p r o e s t r u s b l o o d samples were obtained on a l t e r n a t e days duri n g l a t e p r o e s t r u s and e s t r u s b l o o d was withdrawn at e i g h t hour i n t e r v a l s . The o b j e c t of t h i s sampling schedule was to pr o v i d e an accurate r e p r e s e n t a t i o n of the ovine gonadotropin p r o f i l e throughout the estrous c y c l e . T a b l e 3 . E s t r o g e n i c c o n t e n t o f e x p e r i m e n t a l r a t i o n s ( i ) S T A R T OF E X P E R I M E N T C O M P L E T I O N O F E X P E R I M E N T G R O U P ~ ~ ~ — A OG B B C A O G B B C 1 " 1 6 . 9 1 2 . 9 0 . 0 1 3 . 8 ± 2 . 1 - 2 2 ± 1 . 5 0 . 0 1 7 . 9 ± 2 . 5 I I . 1 1 8 1 1 2 . 3 - 1 2 1 1 2 6 . 9 -( i ) R e s u l t s e x p r e s s e d a s ' p p m l S . E . M . ' G e n i s t e i n e q u i v a l e n t s ( i i ) A = A l f a l f a c u b e s , OG = O r c h a r d g r a s s , B = W h o l e b a r l e y B C = B e e f c o n c e n t r a t e 82. I I . MATERIALS A. I n t r a v a g i n a l P e s s a r i e s Polyurethane foam (0.036 g/cc) cut i n t o the shape of small plugs 3.5 cm (dia) x 2.54 cm; s y n t h e t i c thread 46 cm i n le n g t h ; d i s t i l l e d e t h a n o l ; progesterone (pregn-4-3n3-3,20-dione,sigma). B. E x f o l i a t i v e V a g i n a l Cytology EA-65 (Papanicalaou S t a i n , F i s h e r ) ; hematoxylin; OG-6 s t a i n ; s y r i n g e with adapter f o r a t e f l o n p i p e t t e ; speculum. C. Ovine L u t e i n i z i n g Hormone (oLH) RIA Anti-oLH serum (#15; G.D. Niswender); a n t i -r a b b i t gamma g l o b u l i n (Anti-RGG, ICN B i o c h e m i c a l s ) ; normal r a b b i t serum; oLH (DNW-9-109-5, 2.43 x NIH-LH-S18, D.N. Ward) . Assay B u f f e r systems; phosphate b u f f e r e d s a l i n e (PBS) c o n t a i n i n g 0.14 M NaCl and 0.01 M sodium phosphate, pH 7.0; PBS made 0.1% g e l a t i n , pH 7.0; PBS-EDTA, .05 M EDTA i n PBS, pH 7.0. 83. D. P r o t e i n R a d i o i o d i n a t i o n Chloramine-T (N-chloro-p-toluene-sulfonomide sodium; Matheson Coleman and B e l l ) ; Hamilton micro-syringe ( P i e r c e ) ; iodine-125 (1 mCi, Amersham); sephadex G-100 (Pharmacia); sodium m e t a b i s u l p h i t e (Na2S20g, F i s h e r ) . B u f f e r systems; 0.05 M phosphate, pH 7.5; 0.05 M phosphate made 0.2% g e l a t i n , pH 7.5; 0.2 M phosphate, pH 7.5. E. P r e p a r a t i o n of Gonadotropin Free Plasma Beckman model L5-65 u l t r a c e n t r i f u g e w i t h T-65 f i x e d angle r o t o r (Bekman); 2-mercaptoethanol (Eastman). Assay b u f f e r system; 0.10 M t r i s - H C l , pH 7.4 c o n t a i n i n g 5.0 mM MgC^, 0.10 M sucrose. Homogenization b u f f e r system; same as above w i t h the a d d i t i o n of 1.0 mM 2-mercaptoethanol. I I I . METHODS A. Progesterone Impregnated I n t r a v a g i n a l P e s s a r i e s For the purposes of an e f f i c i e n t b l e e d i n g schedule and a b i l i t y to c o r r e l a t e the gonadotropin p r o f i l e with 84. the v a r i o u s stages of the estrous c y c l e , i t was imperative that a l l experimental animals be synchronized f o r e s t r u s . T h i s requirement can be e a s i l y achieved with the use of i n t r a v a g i n a l sponges and a s u i t a b l e progestagen. P e s s a r i e s were prepared a c c o r d i n g to the method of Wishart (1967) and Robinson et a l . (1967). Two 46 cm lengths of s y n t h e t i c thread were passed through the polyurethane sponges 1.5 cm apart and knotted twice, once immediately on the s u r f a c e of the pessary and again a p p r o x i -mately 2.5 cm from the lower s u r f a c e opposite the o r i g i n a l knot. T h i s l e f t approximately 30 cm of double s t r a n d which p r o t r u d e s from the v u l v a and p r o v i d e s a means to remove the pessary from the animal. A stock s o l u t i o n of progesterone i n e t h a n o l e q u i v a l e n t to 800 mg/15 ml was prepared and s t o r e d under r e f r i g e r a t i o n u n t i l use. The c o n s t r u c t e d p e s s a r i e s were suspended from a h o r i z o n t a l support and the progesterone s o l u t i o n a p p l i e d i n two 7.5 ml a l i q u o t s a l l o w i n g the sponge to a i r dry completely between a p p l i c a t i o n s . S u f f i c i e n t volume of c a r r i e r s o l v e n t should be used to ensure good d i s p e r s i o n of progestagen throughout the sponge (Haresign, 1978). However, the p i p e t t e method of Robinson £t a l . (1967) proved inadequate f o r t h i s volume and amount of progesterone. As an a l t e r n a t i v e the progesterone was 85. i n j e c t e d s lowly throughout the sponge u t i l i z i n g a s yringe and then p e r m i t t e d to a i r dry. I n s e r t i o n was achieved by f i r s t p o s i t i o n i n g the pessary i n the t i p of the speculum and p l a c i n g i t i n the vagina such that the pessary can be d e p o s i t e d as f a r as p o s s i b l e i n t o the a n t e r i o r vagina adjacent to the c e r v i x . S e v e r a l r e s e a r c h e r s have encountered problems with t h i s l e v e l of progesterone. Moore and H o i s t (1967) found that e s t r u s and o v u l a t i o n were not c o n t r o l l e d as there appears to be a l i m i t to the amount of progesterone that can be absorbed through the vagina. Moore and Robinson (1967) a l s o demonstrated that progesterone i n excess of 500 mg causes the sponge to lose much of i t s e l a s t i c and s o f t q u a l i t i e s producing an i r r i t a n t e f f e c t . Upon pessary withdrawal the author d i d observe blood r e s i d u e s throughout many of the sponges; however, no d e l e t e r i o u s e f f e c t s were observed. P e s s a r i e s were withdrawn a f t e r e ighteen days at which time a l l ewes were immediately run with a vasec-tomized marker ram to determine the onset of b e h a v i o r a l e s t r u s . 86. B. E x f o l i a t i v e V a g i n a l Cytology Throughout the o v a r i a n c y c l e karyopyknotic index (KI) v a l u e s p r o v i d e u s e f u l and r e l i a b l e i n f o r m a t i o n on endogenous estrogen e f f e c t s . In t h i s experiment, KI values w i l l be used as c r i t e r i a to assess a p h y t o - e s t r o g e n i c e f f e c t and perhaps by i n f e r e n c e an abnormal or m o d i f i e d corpus luteum and a s s o c i a t e d progesterone s e c r e t i o n . V a g i n a l smears were obtained by i n s e r t i n g a t e f l o n p i p e t t e through a speculum and a s p i r a t i n g the c e r -v i c a l r e g i o n of the vagina. Smears taken i n t h i s f a s h i o n were obtained on a l t e r n a t e days throughout the course of the experiment and s t a i n e d a c c o r d i n g to Papanicalaou (1954) and W a c h t e l l (1969). The smears were examined m i c r o s c o p i c a l l y to determine the KI v a l u e s . A c c o r d i n g to W a c h t e l l (1969) the KI value e s t a b l i s h e s the percentage of 200 c e l l s counted with p y k n o t i c n u c l e i , o m i t t i n g p a r a b a s a l c e l l s from the , count. The KI value v a r i e s from day to day showing a continuous r i s e throughout the f o l l i c u l a r phase r e a c h i n g a peak at o v u l a t i o n . A f t e r o v u l a t i o n progesterone i s s e c r e t e d and the KI f a l l s s h a r p l y . 87. C. Development o f the oLH RIA To measure g o n a d o t r o p i n q u a n t i t a t i v e l y i n ovine b l o o d plasma a s e n s i t i v e and r e l i a b l e oLH RIA i s r e q u i r e d . The components of such a RIA w i l l i n c l u d e : p u r i f i e d oLH f o r use as s t a n d a r d and l a b e l l e d t r a c e r , o v i n e plasma c l e a r e d o f g o n a d o t r o p i n which s e r v e s as a v e h i c l e t o c a r r y oLH s t a n d a r d s i n the development of a s t a n d a r d c u r v e , an i m m u n o - p r e c i p i t a t i o n r e a c t i o n and f i n a l l y an a n t i -oLH s e r a o f h i g h s p e c i f i c i t y , t i t e r and a f f i n i t y . The oLH used i n t h i s RIA was p r e p a r e d and s u p p l i e d by Dr. D.N. Ward. The p u r i f i e d oLH (DNW-9-109-5) has been assayed by Dr. Ward a c c o r d i n g t o the p r o c e d u r e o f Moyle and Ramachandran (1973) which i n v o l v e s t e s t o s t e r o n e p r o d u c t i o n by i s o l a t e d L e y d i g c e l l s . P o t e n c y e s t i m a t e s were (Ward, p e r s o n a l communication, 1979): 2.15xNIH-LH-S18 (951 CL 1.33-3.81; A = 0.16) 2.72xNIH-LH-S18 (95% CL 1.72-4.54; A = 0.15) Mean potency e s t i m a t e 2.43xNIH-LH-S18 A n t i - o L H s e r a #15 was p r o v i d e d by Dr. G.D. Niswender; i t s p r e p a r a t i o n and c h a r a c t e r i z a t i o n have been d e s c r i b e d 88. elsewhere (Niswender e_t al.. , 1968, 1969). According to the i n s t r u c t i o n of Dr. G.D. Niswender (personal communi-c a t i o n , 1979) the #15 anti-oLH sera was used i n the RIA at a f i n a l d i l u t i o n of 1:40,000. The RIA d e s c r i b e d below i s a m o d i f i c a t i o n of the method o u t l i n e d by Niswender et a l . (1969). Disposable c u l t u r e tubes (12x75 mm) were used f o r the RIA. V a r y i n g amounts of sample, 200 y l i n t h i s i n s t a n c e , or standard i n 200 y l c l e a r e d plasma were p l a c e d i n each tube and PBS-0.1°s g e l a t i n was added to b r i n g the volume to 500 y l . The l y o p h i l i z e d a l i q u o t of #15 anti-oLH serum was r e c o n s t i t u t e d with 10 ml d i s t i l l e d water which then e q u a l l e d a 1:400 d i l u t i o n of antibody i n 0.05 M EDTA-PBS. This was f u r t h e r d i l u t e d to 1:40,000 us i n g 1:400 normal r a b b i t serum which had a l s o been d i l u t e d i n EDTA-PBS. The presence of EDTA i n the r e a c t i o n mixture w i l l perform two f u n c t i o n s ; as a c h e l a t i n g agent i t w i l l minimize d i f f e r e n c e s i n comple-ment and w i l l a l s o i n c r e a s e the t o t a l b u f f e r i n g c a p a c i t y of the incubate. Immuno-precipitation i s dependent to some extent on c o n c e n t r a t i o n of a n t i g e n ( e q u i v a l e n t to the f i r s t antibody with i t s bound hormone). Since t h i s c o n c e n t r a t i o n i s extremely low, non-immune g l o b u l i n of the same s p e c i e s as the f i r s t antibody ( r a b b i t ) must be added to ensure optimal immunoprecipitation (Midgley e_t a l . , 1969). T h i s may be done at d i f f e r e n t times. Some 89. i n v e s t i g a t o r s have added c a r r i e r g l o b u l i n w i t h the second antibody, others add i t as part of the d i l u e n t f o r the sample (Midgley et. a_l. , 1969); however, i n t h i s instance the non-immune r a b b i t g l o b u l i n i s added as pa r t of the d i l u e n t f o r the f i r s t antibody (Niswender e_t a_l. , 1969) . Two hundred m i c r o l i t e r s of the 1:40,000 a n t i -ovine LH serum were added to each assay tube, the contents 1 2 R mixed and incubated at 4°C f o r 24 hours. I-oLH, 50,000 cpm/100 y l , was then added to each tube. The LH-anti-LH complexes were solu b l e at the concentrations employed here the r e f o r e an immunoprecipitation step was r e q u i r e d . This step u t i l i z e d g o a t - a n t i - r a b b i t gamma g l o b u l i n ( a n t i -RGG) at a d i l u t i o n which o p t i m a l l y p r e c i p i t a t e d the RGG. 200 y l of the anti-RGG stock s o l u t i o n was added to each tube and a f t e r 72 hours of f u r t h e r incubation 2.0 ml of c o l d PBS was added to d i l u t e the unbound hormone radioac-t i v i t y . This a d d i t i o n of c o l d PBS negates any wash step r e q u i r e d (Midgley e_t al. , 1969) . The antibody bound hormone was separated from the free hormone by c e n t r i f u g a t i o n i n the c o l d (0-2°C) at 1000 g f o r 30 minutes. The supernatant was poured o f f , the rim of the tube b l o t t e d and the p r e c i p i t a t e counted. Three c r i t i c a l areas i n t h i s oLH RIA, s p e c i f i c a l l y p r o t e i n i o d i n a t i o n , anti-RGG t i t e r and the use of gonado-t r o p i n free plasma w i l l be discussed separately below. 90. D. P r o t e i n I o d i n a t i o n The main o b j e c t i v e i n p r o t e i n i o d i n a t i o n i s to a c h i e v e a h i g h s p e c i f i c a c t i v i t y w i t h m i n i m a l l o s s i n i m m u n o l o g i c a l a c t i v i t y . When b i o l o g i c a l a c t i v i t y i s c o n s i d e r e d the c o n d i t i o n s o f i o d i n a t i o n become even more r i g o r o u s (Sonada and S c h l a m o w i t z , 1970; L e i d e n b e r g e r and R e i c h e r t , 1972; R e i c h e r t and B h a l l a , 1974). The i o d i n a t i o n method employed f o r oLH and oFSH i s a m o d i f i c a t i o n o f the ch l o r a m i n e - T p r o c e d u r e d e s c r i b e d by Greenwood e t aJL. (1963) f o r human growth hormone and i s o u t l i n e d i n the appendix. FSH was i o d i n a t e d a c c o r d i n g to M c N e i l l y and Hagen (1974), LH a c c o r d i n g t o Niswender et a l . (1969) . Both p i t u i t a r y p r e p a r a t i o n s i o d i n a t e d i n t h i s f a s h i o n and s u b s e q u e n t l y p u r i f i e d by g e l f i l t r a t i o n t h r o u g h sephadex G-100 were shown t o be s u i t a b l e f o r use i n the a p p r o p r i a t e RIA w i t h o u t d e c r e a s i n g assay s p e c i f i c i t y o r s e n s i t i v i t y (Niswender et. a l . , 1969; M c N e i l l y and Hagen, 1974). E. Anti-RGG T i t e r A n a l y s i s As s t a t e d p r e v i o u s l y , the oLH-anti-oLH complexes are s o l u b l e i n the RIA system employed. C o n s e q u e n t l y , a method must be d e v i s e d which w i l l q u a n t i t a t e these 91. complexes. S e v e r a l p r o c e d u r e s f o r t h i s purpose are des-c r i b e d (Ransom, 1976); however, the system recommended by Niswender et _ a l . (1969) f o r use i n t h i s p a r t i c u l a r oLH RIA employs an i m m u n o p r e c i p i t a t i o n s t e p i n v o l v i n g anti-RGG. What remains t o be d e t e r m i n e d i s the o p t i m a l anti-RGG d i l u t i o n i n the RIA which w i l l maximize the immuno-p r e c i p i t a t i o n r e a c t i o n . 1 2 5 I-oLH (50,000 cpm/100 y l ) was added to a s e r i e s o f assay tubes c o n t a i n i n g 500 y l PBS-0.11 g e l a t i n . T h i s was f o l l o w e d w i t h a 200 y l a l i q u o t o f the #15 a n t i -oLH serum (1:40,000) and a 24 hour i n c u b a t i o n p e r i o d at 4°C. 200 y l a l i q u o t s from s e r i a l anti.-RGG d i l u t i o n s were then added and the assay tubes a g a i n i n c u b a t e d 72 hours at 4°C. S e r i a l d i l u t i o n s ranged from s t o c k t o 1:1000 i n 0.0 5 M EDTA-PBS, pH 7.0. F. P r e p a r a t i o n o f G o n a d o t r o p i n Free Plasma To o b t a i n a v a l i d s t a n d a r d curve f o r use i n the RIA d e s c r i b e d i t i s n e c e s s a r y t o u t i l i z e o v i n e plasma, as a v e h i c l e to c a r r y s t a n d a r d s , which has been c l e a r e d of endogenous g o n a d o t r o p i n . The use o f c l e a r e d plasma w i l l ensure t h a t endogenous g o n a d o t r o p i n i n the c a r r i e r plasma does not compound the e f f e c t o f added oLH s t a n d a r d s . 92. The use of plasma from hypophysectomized ewes has been demonstrated CMcNeilly et a_l. , 1976); however, the source and a v a i l a b i l i t y of t h i s plasma proved an incon-venience. Rapid and r e a d i l y a c c e s s i b l e methods i n c o r p o r a t e t i s s u e membrane r e c e p t o r p r e p a r a t i o n s s p e c i f i c f o r gonado-t r o p i n and when incubated with plasma w i l l bind and subse-q u e n t l y remove any endogenous gonadotropin. The method d e s c r i b e d here i s a m o d i f i c a t i o n of the procedure o u t l i n e d by Haour and Saxena (1974) which i n c o r p o r a t e s d i f f e r e n t i a l and sucrose d e n s i t y c e n t r i f u g a t i o n to p u r i f y gonadotropin r e c e p t o r from bovine corpus luteum. The p u r i f i e d membrane r e c e p t o r p r e p a r a t i o n was subsequently used a c c o r d i n g to Saxena et a_l. (1974) ; Saito. and Saxena (1976) . Male r a t s of body weight 350-400 grams were s a c r i f i c e d by CC^ a s p h y x i a t i o n and the t e s t e s removed. The r a t t e s t e s homogenate was prepared as d e s c r i b e d by Leidenberger and R e i c h e r t (1972) , R e i c h e r t e_t _al. (1973) . The t u n i c a albuginea was removed, the t e s t e s weighed and homogenized b r i e f l y on i c e i n assay b u f f e r (1 g t e s t e s / 2 ml b u f f e r ) . The r e s u l t a n t homogenate was f i l t e r e d through a double l a y e r of cheese c l o t h to remove the semeniferous tubule f r a c t i o n and the t u r b i d , p i n k f i l t r a t e obtained i s the RTH. 93. The RTH was subsequently re-homogenized and c e n t r i f u g e d 500xg f o r 20 minutes at 2-4°C. The supernatant was again r e - c e n t r i f u g e d 15,000xg f o r 60 minutes at 4°C, the supernatant d i s c a r d e d , the p e l l e t resuspended i n 2.0 ml assay b u f f e r and s u b j e c t e d to sucrose d e n s i t y c e n t r i -f u g a t i o n . Of t h i s p e l l e t suspension 0.2 ml was l a y e r e d onto the s u r f a c e of a d i s c o n t i n u o u s sucrose g r a d i e n t which covered a d e n s i t y range of 0.4-1.6 M sucrose i n assay b u f f e r . U l t r a c e n t r i f u g a t i o n proceeded at 55,000xg f o r 2.0 hours at 2-4°C. At the t e r m i n a t i o n of t h i s c e n t r i f u g a -t i o n 0.75 ml f r a c t i o n s were drawn o f f the bottom of the p o l y a l l o m e r centrifuge tube, each f r a c t i o n d i l u t e d x5 with Tris-HCL B u f f e r and c e n t r i f u g e d 3000 rpm f o r 15 minutes at 4-8°C. The p e l l e t was resuspended i n the o r i g i n a l volume of Tris-HCL and the wash step repeated three times. A 200 y l a l i q u o t of each f r a c t i o n was subsequently incubated with I-oLH (20,000 cpm/100 y l ) and 200 y l T r i s - H C l c o n t a i n i n g 0.1% BSA at 37°C f o r 45 minutes. The pooled membrane r e c e p t o r p r e p a r a t i o n was analyzed f o r p r o t e i n content a c c o r d i n g to the t r i n i t r o -b e n z e n e s u l f o n i c method of Snyder and s o b o c i n s k i (1975, see appendix). A l i q u o t s e q u i v a l e n t to 0.0, 0.5, 1.0, 1.5, 2.5, 5.0, 10.0 and 50.0 mg p r o t e i n were c e n t r i f u g e d 94. and the p e l l e t incubated with 1.0 ml ovine plasma as o u t l i n e d by Haour and Saxena (1974), S a i t o and Saxena (1976). A f t e r a 30-minute i n c u b a t i o n p e r i o d at 37°C the plasma-receptor mixture was c e n t r i f u g e d f o r 15 minutes at 2500 rpm and 4°C. The plasma was withdrawn and assayed f o r LH content with the oLH RIA of Niswender et_ a l . (1969) p r e v i o u s l y d e s c r i b e d . 95. EXPERIMENTAL RESULTS AND DISCUSSION 1. PROCEDURAL DEVELOPMENT A. P r o t e i n I o d i n a t i o n (Refer to Appendix II) Upon completion of the i o d i n a t i o n r e a c t i o n the next o b j e c t i v e i s p u r i f i c a t i o n of the l a b e l l e d hormone. Th i s i s accomplished with f i l t r a t i o n through sephadex-G100 which separates the i o d i n a t i o n mixture i n t o l a b e l l e d hormone, damaged p r o t e i n and f r e e i o d i n e f r a c t i o n s . A t y p i c a l e l u t i o n p a t t e r n f o r i o d i n a t e d gonadotropin i s demonstrated i n Fig u r e 16. For each hormone two peaks are r e a d i l y d i s c e r n i -125 b l e . The f i r s t r e p r e s e n t s I-gonadotropin while the 125 second i n d i c a t e s f r e e I. The system implemented i n t h i s study i n v o l v e d a 1x10 cm sephadex column wi t h a flow r a t e o f 40 ml/hr. which c h a r a c t e r i z e d oFSH and oLH with e l u t i o n volumes (Ve/Vo) of 1.53 and 1.68 r e s p e c t i v e l y . These valu e s d i f f e r from R e i c h e r t e_t a l . (1968) , who i n t h e i r study employed a system which c o n s i s t e d of a 2x90 cm column, 8.0 ml/hr. flow r a t e and produced e l u t i o n volumes f o r hFSH and hLH of 1.65 and 1.78 r e s p e c t i v e l y . 96. FIGURE 16 240 Volume of eluate (milliliters) Figure 16: Elution profiles of 125-oLH (o-o) and 125 I-oFSH (#*). 97. B. Anti-RGG T i t e r A n a l y s i s The anti-RGG d i l u t i o n which w i l l y i e l d the maximum 12 5 I-oLH-anti-oLH bound dpm re p r e s e n t s the optimal immuno-p r e c i p i t a t i o n r e a c t i o n i n the oLH RIA p r e v i o u s l y d e s c r i b e d . The t i t e r a n a l y s i s curve i s presented i n Figure 17 and from i t one can determine the anti-RGG d i l u t i o n f o r the 125 immunoprecipitin step. Maximum I-oLH dpm bound and p r e c i p i t a t e d i s achieved when 200 y l stock (undiluted) anti-RGG are added to each RIA tube. C. P r e p a r a t i o n of Gonadotropin Free Plasma The f i r s t step i n t h i s procedure employs d i s -continuous sucrose c e n t r i f u g a t i o n to p u r i f y LH r e c e p t o r s from the r a t t e s t e s homogenate. F r a c t i o n s were drawn from the bottom of the c e n t r i f u g e v i a l i n volumes which f a c i l i t a t e d c o l l e c t i o n of an i n t e r f a c e i n t o each f r a c t i o n . T h i s approach was used s i n c e c e l l u l a r m a t e r i a l concentrates at the i n t e r f a c e between each sucrose l a y e r i n d i s c o n t i n u -ous sucrose g r a d i e n t c e n t r i f u g a t i o n . F i g u r e 18 d e p i c t s the sucrose d e n s i t y p r o f i l e 125 of these p u r i f i e d f r a c t i o n s f o r b i n d i n g I-oLH. F r a c t i o n s 125 6-8 i n c l u s i v e demonstrated maximum I-oLH bound and were used i n a l l subsequent steps o f t h i s procedure. 9 8 . Figure 17; Titer analysis to d e t e r m i n e the optimal anti-RGG dilution for use in the oLH RIA. 99. FIGURE 18 C\J O X CL C o cD o 1 I ID CAJ -Fraction number Top 01 gradi ent 0.4 M S u c r o s e B o t t o m oi gradient 1.6 M s u c r o s e Figure 18: The sucrose gradient elution profile for 125-f-oLH, 100. A l i q u o t s c o n t a i n i n g i n c r e a s i n g q u a n t i t y o f p u r i -f i e d membrane r e c e p t o r were th e n i n c u b a t e d w i t h 1.0 ml o f ram plasma t o determine t h a t amount o f r e c e p t o r which e f f e c t i v e l y b i n d s a l l d e t e c t a b l e endogenous LH (see F i g u r e 125 19). Maximum I-oLH bound t o i m m u n o p r e c i p i t a t e d a n t i -body, w h i c h r e p r e s e n t s minimum c o m p e t i t i o n w i t h endogenous LH, i s a c h i e v e d w i t h a p p r o x i m a t e l y 10 mg p r o t e i n r e c e p t o r p r e p a r a t i o n . A d d i t i o n a l a l i q u o t s o f ram plasma were i n c u -b a t e d w i t h p u r i f i e d membrane r e c e p t o r at a c o n c e n t r a t i o n o f 10 mg p r o t e i n / 1 . 0 ml plasma. The r e s u l t a n t s t a n d a r d c u r v e s w h i c h compare normal and c l e a r e d ram plasma appear i n F i g u r e 20. In e v a l u a t i n g s t a n d a r d c u r v e s r e g r e s s i o n a n a l y s i s a c c o r d i n g t o the method of B l i s s (1952; 1967) r e q u i r e s the use o f the term lambda ( A ) . Lambda i s d e f i n e d as S /b hence A a p p r o a c h i n g z e r o w i l l i n d i c a t e e i t h e r a y • x d e c r e a s i n g s t a n d a r d e r r o r o f the e s t i m a t e (S„ ) , an i n c r e a s e y • x i n the s l o p e (b) or a c o m b i n a t i o n o f the two. A p r o v i d e s a mechanism f o r comparisons between assays by e v a l u a t i n g the parameters o f p r e c i s i o n and s e n s i t i v i t y i n a g i v e n system. R e g r e s s i o n a n a l y s i s performed i n t h i s manner ( B l i s s , 1972, 1967) y i e l d s A v a l u e s f o r normal and c l e a r e d ram plasma of 0.1168 and 0.1072 r e s p e c t i v e l y . 101. Figure 19? 125-l-oLH bound to r e c e p t o r plotted against the quantity of receptor used to adsorboLH per ml of plasma. 1 0 2 . Performing the L o g i t t r a n s f o r m a t i o n (Feldman and Rodbard, 1 9 7 1 ) where L o g i t B/B Q = l n [ ( B / B 0 ) / ( l - B / B 0 ) ] w i l l produce the curves i l l u s t r a t e d i n Figure 2 1 . P a r a l l e l l i n e a n a l y s i s on the transformed data demonstrate equal s l o p e s f o r each r e g r e s s i o n l i n e ( F Q 0 5 ( 1 ) 1 4 ^ " ^ ' P r o D a " b i l i t y = 0 . 9 7 1 ) . Further t e s t s f o r e q u a l i t y of e l e v a t i o n show that the r e g r e s s i o n l i n e s are d e r i v e d from separate and d i s t i n c t data sets ( F Q Q 5 ( 1 ) 1 s ^ - ^ - U p r o b a b i l i t y = 0 . 0 1 4 ) . 103 FIGURE 20 0 I — , r-O 20 40 oLH (ng/ml) Figure 20: The compari son between normal (o) and gonadotropin cleared (• ) plasma. Figure 21 : Equality of slope between normal (o) and c l e a r e d (•) plasma. 105. 2. TREATMENT EFFECTS A. E s t r u s S y n c h r o n i z a t i o n I f one accepts the argument that i n t r i n s i c mech-anisms are r e s p o n s i b l e f o r the i n i t i a l s e c r e t i o n of FSH and estrogen to i n i t i a t e the o v a r i a n c y c l e then p r e v a i l i n g e n d o c r i n o l o g i c a l c o n d i t i o n s upon pessary removal should have a c r i t i c a l e f f e c t upon the ensuing r e t u r n to e s t r u s w i t h o v u l a t i o n . One f a c t o r which c o u l d c o n t r i b u t e to the o v e r a l l e n d o c r i n o l o g i c a l p a t t e r n i s the occurrence o f c i r c u l a t i n g phyto-estrogens and/or m e t a b o l i t e s from e s t r o g e n i c forages consumed. In both the c o n t r o l and experimental groups a l l ewes g e n e r a l l y e x h i b i t e d b e h a v i o r a l ' e s t r u s 2-4 days a f t e r sponge withdrawal. T h i s r e s u l t i s i n c l o s e agreement with l i t e r a t u r e v a l u e s (Robinson e_t a_l. , 1967) . Ewes which consumed e s t r o g e n i c a l f a l f a d e v i a t e d somewhat from the c o n t r o l ewes. When compared to the c o n t r o l ewes, these ewes demonstrated an onset of b e h a v i o r a l e s t r u s which o c c u r r e d over a l e s s s t r i n g e n t time p e r i o d . Of the 5 ewes fed a l f a l f a one e x h i b i t e d b e h a v i o r a l e s t r u s 8 hours a f t e r sponge withdrawal while another f a i l e d to show b e h a v i o r a l e s t r u s through the 5-day p e r i o d a f t e r pessary removal. 106. B. E x f o l i a t i v e V a g i n a l Cytology The c h a r a c t e r i s t i c c e l l p a t t e r n s d e s c r i b e d i n the human female (Papanicolaou, 1954) have been a p p l i e d to the e s t r o u s c y c l e of the ewe (Sanger et a l . , 1958 a,b). T h i s study again attempted to c h a r a c t e r i z e the estrous c y c l e of the ewe with the methodology of Papanicalaou (1954) and Sanger et a l . (1958) but with the a d d i t i o n of KI v a l u e s and t h e i r i n t e r p r e t a t i o n as o u t l i n e d by Wachtel (1969). KI v a l u e s were obtained from 4 ewes which con-sumed orc h a r d grass hay and 4 which consumed a l f a l f a cubes (Figure 22). In each case a l l ewes e x h i b i t e d an i n i t i a l i n c r e a s e and p l a t e a u i n the KI v a l u e s w i t h i n a p e r i o d 3-10 days p r i o r to e s t r u s . T h i s r e g i o n i s f o l l o w e d with a p e r i o d of r a p i d i n c r e a s e i n the KI to peak l e v e l s which corresponded to e s t r u s . The KI v a l u e s subsequently drop to low l e v e l s with o v u l a t i o n and formation of a f u n c t i o n a l corpus luteum. Upon c l o s e r examination one can see that KI values from ewes which consumed the a l f a l f a used i n t h i s study demonstrate a slower r a t e of descent 0-3 days post o v u l a t i o n . T h i s decreased r a t e of descent becomes more 107. FIGURE 22 -12 -8 -4 0 +4 +8 Days from e s t r u s Figure 22: The karyopyknotic index obtained from ewes which consumed (A) orchard grass hay and (B) alfalfa. 108. pronounced d u r i n g the p e r i o d 3-5 days from e s t r u s . T h i s r e s u l t would seem to i n d i c a t e that g r e a t e r e s t r o g e n i c s t i m u l a t i o n has o c c u r r e d i n ewes f e d e s t r o g e n i c a l f a l f a and the e f f e c t i s maintained through a p e r i o d of low endo-genous estrogen l e v e l s . Such a p e r i o d i s encountered between o v u l a t i o n and the establishment of a f u n c t i o n a l corpus luteum 0-6 days a f t e r e s t r u s (Robertson, 1977). Sanger ejt a l . (1958b) , Folman and Pope (1966) and Newsome and K i t t s (1980) have shown phyto-estrogens to demonstrate e s t r o g e n i c a c t i v i t y when endogenous estrogen l e v e l s are minimal. A c r i t i c a l f e a t u r e of the normal o v a r i a n c y c l e i s the abrupt change from the p r o l i f e r a t i v e to s e c r e t o r y phase which i n d i c a t e s o v u l a t i o n and subsequent formation of a f u n c t i o n a l corpus luteum. This t r a n s i t i o n leads to the c h a r a c t e r i s t i c c e l l p a t t e r n s d e s c r i b e d by Papanicolaou (1954), Sanger et a l . (1958) and Wachtel (1969). These c e l l p a t t e r n s were observed d u r i n g t h i s study and are d e s c r i b e d below. Smears c o l l e c t e d d u r i n g the e s t r u s p e r i o d are t y p i c a l l y c l e a r and c o n t a i n i n d i v i d u a l d i s c r e t e squamous c e l l s with p y k n o t i c n u c l e i (Figure 23). The cytoplasm of these c e l l s i s t r a n s p a r e n t with a s l i g h t p a l e blue hue. V a g i n a l mucus observed d u r i n g t h i s p e r i o d appears t h i n , copious and t r a n s p a r e n t . 109. FIGURE 23 Figure 23: Photograph of exfoliated cells characteristic of the estrus period ( x 4 0 0 ) . 110. With the onset of metestrus there i s a t r a n s i -t i o n from tr a n s p a r e n t p y k n o t i c c e l l s to k a r a t i n i z e d eosino-p h i l i c squamous c e l l s . These c e l l s are l a r g e f l a t squames which appear f o l d e d and o f t e n appear i n dense c e l l masses. The c e l l s observed here are the type ( i i ) c o r n i f i e d eosino-p h i l i c squames d e s c r i b e d by Sanger et _al. (1958) . As metestrus progresses the v a g i n a l m u c u s - c e l l u l a r m a t e r i a l c o l l e c t e d f o r the smear becomes t h i c k e r and opaque-white i n c o l o u r . The c e l l s are now c y a n o p h i l i c and i n d i c a t e the onset of d i e s t r u s . D i e s t r u s i s a s s o c i a t e d w i t h the s e c r e t o r y phase of the e s t r o u s c y c l e consequently the c e l l p a t t e r n i s i n d i c a t i v e o f progesterone s e c r e t i o n . The smears are c h a r a c t e r i s t i c a l l y heavy and composed of c y a n o p h i l i c i n t e r -mediate squamous c e l l s w i t h t h i c k e n e d c e l l borders and f o l d e d edges. The cytoplasm l a c k s transparency and the c e l l s form t h i c k dense masses (Figure 24). Many neutro-p h i l s are a l s o d i s c e r n i b l e d u r i n g t h i s p e r i o d . The o v a r i a n c y c l e i s completed with p r o e s t r u s , a p e r i o d which i n d i c a t e s a c e s s a t i o n of the s e c r e t o r y phase and s t a r t of the p r o l i f e r a t i v e phase. Smears taken d u r i n g p r o e s t r u s are c h a r a c t e r i z e d by d e c r e a s i n g c e l l numbers and t h i n c l e a r e r mucus, a r e s u l t of i n c r e a s i n g estrogen s e c r e t i o n . 111. FIGURE 24 Figure 24: Photograph of exfoliated cells characteristic of the diestrus period ( x 4 0 0 ) . 112. C. LH Plasma P r o f i l e s In t h i s study peak LH l e v e l s were determined to be w i t h i n the range 23.5-46.5 ng/ml and 50.4-83.2 ng/ml f o r ewes f e d orchard grass hay or a l f a l f a cube respec-t i v e l y ( F i g u r e s 25 and 26). L u t e i n i z i n g Hormone l e v e l s were undetectable through the s e c r e t o r y phase which i n d i -cates plasma LH l e v e l s l e s s than 0.5 ng/ml, the lower l i m i t f o r s e n s i t i v i t y i n the RIA employed. These values are i n c l o s e agreement to the oLH l e v e l s p r e v i o u s l y demonstrated. The oLH c o n c e n t r a t i o n s during the cycle have been reported as; 2-3 ng/ml (Geschwind and Dewey, 1968), l e s s than .5-2 ng/ml (Niswender et_ a l . , 1968), 2.9±.9 ng/ml (Goding et a l . , 1969), .5-2.2 ng/ml (Niswender e_t a_l. , 1969) and 2.6 ng/ml (Pant et_ a l . , 1977). Peak l e v e l s o f oLH have been r e p o r t e d as: 80 ng/ml (Geschwind and Dewey, 1968), 25-73 ng/ml with a mean of 47 ng/ml (Niswender et a l . , 1968) , mean of 26 ng/ml (Niswender e_t a l . , 1969) and 75.3±7.4 ng/ml (Pant et a l . , 1977). D i s c r e p a n c i e s between r e s u l t s i n t h i s study and those c i t e d from the l i t e r a t u r e can be a t t r i b u t e d p r i m a r i l y to samples which are obtained from d i f f e r e n t r e g i o n s of the LH peak. For t h i s reason mean peak LH l e v e l s were not o b t a i n e d f o r comparison between treatments. 113. FIGURE 25 i 1 > 1 ' -10 "8 "6 -4 -2 ] 0 Days from estrous Figure 25: Plasma LH levels in ewes fed orchard grass hay with supplement. ( a r r o w indicates onset of e s t r u s ) . 114. FIGURE 26 c 01 e CL 12\ 54 36-18 Ewe n° 244 2 4 5 246 72 54-36 181 0 J Ewe n° 247 24 8 symbol o o symbol o — o 10 -8 -6 -4 -2 Days from e s t r u s 0 t *2 Figure 26: Plasma LH levels in ewes fed alfalfa cubes (arrow indicates onset of e s t r u s ) . al time f r o m onset of est r u s i o LH .peak is significantly longer than the control group (F C a i , > F,o5(i>iy8 ). 115. The r e s u l t s i l l u s t r a t e d i n Fi g u r e s 25 and 26 i n d i c a t e t hat plasma LH l e v e l s peak with the onset of e s t r u s i n ewes fed orchard grass hay, however, ewes fed e s t r o g e n i c a l f a l f a demonstrated plasma LH peaks which occu r r e d 0-24 hours i n t o the e s t r u s p e r i o d . Pre- and post-LH peak l e v e l s were a l s o e l e v a t e d and f l u c t u a t i n g i n ewes f e d orc h a r d grass hay when compared to correspond-ing p e r i o d s i n ewes fed a l f a l f a . One must be c r i t i c a l o f t h i s comparison as i t appears the LH peak i s out of s y n c h r o n i z a t i o n with b e h a v i o r a l e s t r u s i n ewes which consumed a l f a l f a . As a consequence the b l e e d i n g schedule may have i n f a c t missed the p u l s a t i l e r e l e a s e o f LH p r i o r to the LH peak i n these animals. Niswender et a l . (1968), Goding et a l . (1969) and Pant e_t ail. (1977) have r e p o r t e d that the LH peak occurs w i t h i n a 0-16 hour p e r i o d from the onset of e s t r u s and g e n e r a l l y has a 12-hour d u r a t i o n . 116. GENERAL DISCUSSION AND CONCLUDING REMARKS The work of Tang and Adams (1978), Rodgers (1979) and Mathieson (1979; 1980) has demonstrated on ££-173 r e c e p t o r - c o u m e s t r o l or g e n i s t e i n i n t e r a c t i o n i n both the hypothalamus and p i t u i t a r y . These r e s u l t s i m p l i c a t e the hypothalamo-hypophyseal a x i s as a p o s s i b l e s i t e where phyto-estrogens can exert an e f f e c t upon c o n t r o l of the o v a r i a n c y c l e and consequently upon f e r t i l i t y i n tempor-ary and permanent i n f e r t i l e ' c l o v e r i n f e c t e d ' ewes. Rodgers e_t a l . (1980) found permanent c l o v e r -i n f e r t i l e ewes to demonstrate s i g n i f i c a n t l y higher plasma LH l e v e l s than f e r t i l e ewes du r i n g anestrus and breeding seasons. C l o v e r f e r t i l e ewes e x h i b i t e d LH s e c r e t i o n i n t e r -mediate to that of c o n t r o l and c l o v e r - i n f e r t i l e ewes. T h i s r e l a t i o n s h i p p r e v a i l e d even a f t e r the ewes had been removed from e s t r o g e n i c pasture f o r s e v e r a l years (Rodgers et a l . , 1980) . F u r t h e r r e s u l t s r e p o r t e d by Rodgers e_t a l . (1980) i n d i c a t e no s i g n i f i c a n t d i f f e r e n c e i n plasma FSH l e v e l s between f e r t i l e and i n f e r t i l e ewes during anestrus or the breeding season. R e s u l t s from t h i s study were obtained from ewes fed a l f a l f a of low-moderate p h y t o - e s t r o g e n i c a c t i v i t y (Table 3) which d i d not f a c i l i t a t e the occurrence of 117. i n f e r t i l i t y . Mathieson (1979), however, demonstrated that a p h y t o - e s t r o g e n i c content of t h i s l e v e l had the c a p a b i l i t y o f competing w i t h E2~17B at the t i s s u e r e c e p t o r l e v e l . The r e s u l t s here do i n f a c t demonstrate a b e r r a t i o n s i n the e s t r o u s c y c l e and a s s o c i a t e d LH s e c r e t o r y p a t t e r n between c o n t r o l and experimental groups. Mean peak plasma LH l e v e l s o f ewes f e d a l f a l f a appear e l e v a t e d when compared wi t h c o n t r o l ewes which consumed orchard grass hay (66.4±16.8 ng/ml versus 40.1± 5.55 ng/ml r e s p e c t i v e l y ) . T h i s would agree w i t h Rodgers et a l . (198) however t h i s d i f f e r e n c e between mean peak LH l e v e l s may be a t t r i b u t e d to sampling d i f f e r e n t r e g i o ns of the plasma LH peak. Rodgers e_t a l . (1980) a t t r i b u t e the e l e v a t i o n i n LH l e v e l s to e s t r o g e n i c c l o v e r i n t e r f e r -ence w i t h the negative feedback of E2~17B upon LH s e c r e t i o n . Perhaps of g r e a t e r importance i s the demonstra-t i o n of a delayed LH peak ( F i g u r e s 25 and 26). T h i s i n d i -c a t e s a tendency toward abnormal LH s y n t h e s i s and/or s e c r e -t i o n . Using the two p o o l hypothesis f o r LH s y n t h e s i s and r e l e a s e (Figure 9B) d e s c r i b e d by J u s t i z (1971) and Yen (1977), the r e s u l t s may become c l e a r e r . During p e r i o d s of low E2~17B l e v e l s coumestrol, g e n i s t e i n and t h e i r m e t a b o l i t e s may a c t u a l l y augment the a c t i o n of E 9-17B i n i t s i n d u c t i o n of LH s y n t h e s i s i n p o o l 118. 2 and t r a n s f e r to p o o l 1. As endogenous B^-HQ l e v e l s i n c r e a s e through the f o l l i c u l a r phase of the e s t r o u s c y c l e the phyto-estrogens may g r a d u a l l y become a n t a g o n i s t i c to the a c t i o n of E2-178 which co u l d r e s u l t i n d e c r e a s i n g LH p r o d u c t i o n and t r a n s f e r i n t o the r e l e a s i b l e p o o l . As the f o l l i c u l a r phase progresses the p l a n t estrogens may a l s o impede the a c t i o n of GnRH which i s augmented and f a c i l i t a t e d by E2~17B at both the hypothalamic and p i t u i t a r y l e v e l . The e v e n t u a l outcome i s a d e t e c t a b l e LH peak which occurs l a t e r i n the e s t r u s p e r i o d . The d i s c u s s i o n presented above c o u l d a l s o account f o r the apparent absence of, or reduced, f l u c t u a t i o n s i n plasma LH l e v e l s p r i o r to the LH surge and afterwards i n ewes which consume a l f a l f a . T h i s aspect would i n d i c a t e an " a l l or none" response r e q u i r e d to overcome the a n t a g o n i s t i c e f f e c t s o f phyto-estrogens. Rodgers et_ al. (1980) a l s o d e s c r i b e a p e r i o d where LH s e c r e t o r y p a t t e r n s are not w e l l d e f i n e d during the breeding season of c l o v e r i n f e r t i l e ewes . I f the apparent i n c r e a s e i n LH of ' c l o v e r ' i n f e c t e d ewes i s accepted, the LH a u t o r e g u l a t i o n c y c l e i n the r a t as o u t l i n e d by B a r r a c l o u g h et a l . (1979) would a l s o e x p l a i n the reduced f l u c t u a t i o n s i n LH l e v e l s a f t e r the LH peak. The mechanism i m p l i c a t e d i n v o l v e s an LH n e g a t i v e feedback 119. loop from the p i t u i t a r y to the catechalamine pathways c o n t r o l l i n g the MPOA-hypothalamic a c t i v i t y and GnRH r e l e a s e . B arraclough et a l . (1979) and Turgeon (1979) used the r a t to demonstrate a c r i t i c a l aspect of the E2-17B-LH i n t e r r e l a t i o n s h i p . They have shown that a r a p i d drop i n E2"17$ l e v e l s d u r i n g l a t e p r o e s t r u s i n f l u e n c e s the s e c r e t o r y p a t t e r n and magnitude of the LH surge. I f t h i s decrease i n p e r i p h e r a l E2~176 i s prevented the magni-tude of the LH peak i s decreased. I t was a n t i c i p a t e d i n t h i s study that the phytoestrogen c o n c e n t r a t i o n i n the ewe would be s u f f i c i e n t to mask any r a p i d decrease i n endogenous E2~17B r e s u l t i n g i n a r e l a t i o n s h i p s i m i l a r to that d e s c r i b e d by Turgeon (1979). R e s u l t s from Rodgers et a l . (1980) i n d i c a t e t h i s i s not the case and while the r e s u l t s of t h i s study support t h i s view the l e v e l s of phyto-estrogen i n the a l f a l f a r a t i o n consumed were not o f s u f f i c i e n t magnitude to induce a response which would prove or disprove t h i s concept. The KI values demonstrate an e s t r o g e n i c e f f e c t of phytoestrogens once o v u l a t i o n has o c c u r r e d and endogenous E2-173 l e v e l s are low. The KI v a l u e s of the experimental group f a l l to the base l e v e l s o f c o n t r o l ewes 6-7 days from the morning of e s t r u s which a l s o i n d i c a t e s normal l u t e a l formation and f u n c t i o n . 0 120. In summary, permanent i n f e r t i l i t y can be charac-t e r i z e d by impaired ova and sperm t r a n s p o r t and e l e v a t e d LH l e v e l s which are an i n d i c a t i o n of hormonal imbalances which have d e s e n s i t i z e d the hypothalamus to e s t r o g e n i c c o n t r o l mechanisms (Hearnshaw e_t a l . , 1972 ; F i n d l a y et a l . , 1973; Rodgers et a l . , 1980). Temporary i n f e r t i l i t y i n v o l v e s s u b t l e a b e r r a t i o n s w i t h i n the h y p o t h a l a m o - p i t u i t a r y a x i s which may prove cumulative i n nature and r e s u l t i n v a r i a b l e f e r t i l i t y problems. Using a l f a l f a w ith a modest phyto-estrogen c o n c e n t r a t i o n r e s u l t s from t h i s study demonstrate apparent e l e v a t e d plasma LH l e v e l s which c o i n c i d e w i t h c l o v e r -f e r t i l e ewes d e s c r i b e d by Rodgers e_t a l . (1980) . R e s u l t s p r e s e n t e d i n t h i s study a l s o i n d i c a t e phyto-estrogen consump-t i o n w i l l p r o l o n g the e s t r u s p e r i o d by d e l a y i n g the LH surge f u r t h e r i n t o e s t r u s . T h i s c h a r a c t e r i s t i c i n d i c a t e s that normal e s t r u s , p r i m a r i l y between onset and o v u l a t i o n , and the LH surge are not synchronized. T h i s may u l t i m a t e l y i n c r e a s e the i n c i d e n c e of prolonged e s t r u s , abnormal e s t r u s , e s t r u s without o v u l a t i o n , a t y p i c a l f o l l i c u l o g e n e s i s , impaired l u t e a l f u n c t i o n and consequently anomalous hormonal p r o f i l e s which f u r t h e r d i s t u r b the d e l i c a t e c o n t r o l mechanisms w i t h i n the hypothalamo-hypophyseal-ovarian a x i s . 120a. A CRITIQUE Through the course of t h i s study c o n s i d e r a b l e e f f o r t was r e q u i r e d to overcome two important aspects of the RIA employed. The areas of concern were development of the double antibody-immunoprecipitation step and development o f LH-free plasma as a c a r r i e r f o r the LH standards. With r e f e r e n c e to the immunoprecipitation step the q u a l i t y ; a v a i l a b i l i t y and c o s t of commercially prepared immunoglobulins n e c e s s i t a t e s development of a procedure to p r o v i d e p r e c i p i -t a t i n g antibody. To conduct f u r t h e r s t u d i e s of t h i s nature s e v e r a l c o n d i t i o n s should be m o d i f i e d . I n d i v i d u a l animals should serve as t h e i r own c o n t r o l s . T h i s can be accomplished with the animals maintained on t h e i r c o n t r o l d i e t and changed to the experimental r a t i o n . The b l e e d i n g schedule should be r i g o r o u s , a 15-minute i n t e r v a l between sampling would be a p p r o p r i a t e . 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G r i f f i n (1959), "Chroma-tography of L u t e i n i z i n g Hormone from Sheep P i t u i t a r y Glands", B i o c h i m i c a and b i o p h y s i c a Acta 32: 305-314. 155. Ward, D.N., M. Adams-Mayne, N. Ray, D.E. Balke, T. Coffey and M. Showalter (1967), "Comparative S t u d i e s of L u t e i n i z i n g Hormone from Beef, Pork and Sheep P i t u i -t a r i e s " , General and Comparative Endocrinology 8: 44-53. Weiss, T . J . , R.F. Seamark, J.E.A. Mcintosh and R.M. Moor (1976), " C y c l i c AMP i n Sheep Ovarian F o l l i c l e s : S i t e of P r o d u c t i o n and Response to Gonadotropins", J o u r n a l  of Reproduction and F e r t i l i t y 46: 347-353. Wiber, J.F., E. Montoya, N.P., P l o t i n i k o f f , W.F. White, R. Gendrich, L. Renaud and J.B. M a r t i n (1976), "Gonado-t r o p i n - R e l e a s i n g Hormone and T h y r o t r o p i n - R e l e a s i n g Hormone: D i s t r i b u t i o n and E f f e c t s i n the C e n t r a l Nervous System", Recent Progress i n Hormone Research, 32: 117-153. Wise, P.M. (1975), "The Estrogen C y t o s o l Receptor of Female Ovine P i t u i t a r y " , Molecular and C e l l u l a r Endocrinology 3: 385-395. Wise, P.M., N. Ranee, G.D. Barr and C A . Barraclough (1979), "Furt h e r Evidence that L u t e i n i z i n g Hormone-Releasing Hormone i s a l s o F o l l i c l e - S t i m u l a t i n g Hormone-Releasing Hormone", Endocrinology 104: 940-947. Wishart, D.F. (1967), " S y n c h r o n i z a t i o n of Oestrus i n Sheep: The Use of P e s s a r i e s " , The V e t e r i n a r y Record 81: 276-287 . Wong, M.S.F. and R.I. Cox (1971), "Evidence f o r P h y t -oestrogens as Sulpho-Conjugates i n Sheep Plasma", Proceedings, 4th A s i a and Oceania Congress of  End o c r i n o l o g y , No. 53. Wuttke, W. (1977), " R e g u l a t i o n of P r o l a c t i n S e c r e t i n " , pp. 287-292, i n En d o c r i n o l o g y , V o l . I I , V.H.T. James, Ed., Excerpta Medica, Amsterdam. 156. Yen, S.S.C. (1976), "The Adenohypohysis: F u n c t i o n a l Behavior of the Gonadotrophs as Target C e l l s " , pp. 453-469 i n S u b c e l l u l a r Mechanisms i n Reproductive  Neuroendocrinology, F. N a f t o l i n , K.J. Ryan, and I. Davies, Eds., E l s e v i e r / N o r t h H o l l a n d , New York. Yen, S.S.C. (1977), " R e g u l a t i o n of the H y p o t h a l a m i c - P i t u i -t a r y - O v a r i a n A x i s i n Women", J o u r n a l of Reproduction  and F e r t i l i t y 51: 181-191. Zimmerman, E.A. (1976), " L o c a l i z a t i o n of Neurosecretory Peptides i n Neuroendocrine T i s s u e s " , pp. 81-108, i n S u b c e l l u l a r Mechanisms i n Reproductive Neuroendo- c r i n o l o g y , F. N a f t o l i n , K.J. Ryan and I . J . Davies, Eds., E l s e v i e r , New York. Zimmerman, E.A. (1977), " L o c a l i z a t i o n of Hormone S e c r e t i n g Pathways i n the B r a i n by Immunohistochemistry and L i g h Microscopy: A Review", F e d e r a t i o n Proceedings 36: 1964-1967. 157. APPENDIX I SPECIFICITY OF #15 ANTI-oLH SERUMa By u s i n g ovine p i t u i t a r y p r e p a r a t i o n s c o n t a i n i n g v a r i o u s l e v e l s of TSH, FSH, LH, p r o l a c t i n and growth hor-mone, Niswender et. a l . (1969) demonstrated that the r a d i o -immunoassay estimates of LH potency were c o n s i s t e n t with bioass a y estimates [ o v a r i a n a s c o r b i c a c i d d e p l e t i o n t e s t s (OAAD)]. In a l l p r e p a r a t i o n s t e s t e d the i n d i c e s of d i s c r i m i n a t i o n (OAAD LH/RIA LH) approached u n i t y , t h i s suggests that FSH, TSH, growth hormone and p r o l a c t i n do not a f f e c t the e s t i m a t i o n of LH potency by the radioimmuno-assay employed i n t h i s study (see Appendix Table 1). As presented i n Niswender et. al. (1969) . Appendix Table 1 L u t e i n i z ing hormone content of ovine p i t u i t a r y p r e p a r a t i o n s PREPARATION TSH USP U/mg FSH U/mg LH OAAD U/mg LH RIA U/mg OAAD LH RIA LH LER-962 .054 .021 .82 .86 .97 (NIH-LH-S12) LER-1035-3 1.25 - .37 .43 .86 LER-980-1 .03 - .90 1.21 .75 LER-866-3 - 26.0 .003 .005 .60 NIH-P-S8 .0005 .008 .0003 .0003 1.00 NIH-GH-S8 .012 .11 .034 .027 1.26 a From Niswender et a l . (1969). 159. APPENDIX II PROTEIN IODINATION 125 1 mCi Na-( I ) - i o d i d e 25 y l of 0.2 M phosphate b u f f e r - 5.0 yg oLH/25 y l 0.05 M phosphate b u f f e r 40 yg chloramine-T/25 y l 0.05 M phosphate b u f f e r A g i t a t e 2.0 min ( f i n g e r tap) - 240 yg sodium m e t a b i s u l f i t e 30 yl/0.05 M phosphate b u f f e r 1 yg potassium iodide/100 y l 0.05 M phosphate b u f f e r made 16% sucrose T h i s e n t i r e r e a c t i o n mixture was l a y e r e d onto a 1 cc x 10 cc column of sephadex-G 100 e q u i l i b r a t e d i n 0.05 M phosphate b u f f e r and p r e - s a t u r a t e d with 1.0 ml of 50 mg BSA/ml 0.05 M phosphate b u f f e r f o l l o w e d by a 20 ml wash. E l u t i o n was conducted with 0.05 M phosphate b u f f e r . One ml f r a c t i o n s were c o l l e c t e d i n t o 1.0 ml of 0.05 M phosphate c o n t a i n i n g 0.2% g e l a t i n . A l i q u o t s of each f r a c t i o n 160. were counted and the  ±id°I-protein peak d i l u t e d to 50,000 cpm/100 y l PBS c o n t a i n i n g 0.1% g e l a t i n . P r o t e i n i o d i n a t i o n r e q u i r e s i o n i z a t i o n of i o d i n e ( I 2 ) to higher o x i d a t i v e s t a t e s of 0 or +1 to produce a c t i v e ^ O I * molecules which r e a c t with p r o t e i n r e s i d u e s . Reactions with p r o t e i n are g e n e r a l l y i r r e v e r s i b l e i n v o l v i n g i n t e r a c t i o n s between ^ 0 1 * and the t y r o s i n e r e s i d u e s , some h i s t i d i n e r e s i d u e s and s u l f h y d r y l groups of the p r o t e i n . Reactions with -SH groups occur much more r e a d i l y but do not r e s u l t i n s t a b l e bonding of i o d i n e . Consequently i n any l a b e l l i n g experiment the f i r s t i o d i n e consumed, e q u i v a l e n t to -SH content, must be co n s i d e r e d l o s t as d e p i c t e d by the f o l l o w i n g equation (Hughes, 1957): H 2OI ++ R-SH + RSI + H + + H 20 RSI + R-SH + RS-SR + H + 1 (Unusable iodid e ) As a consequence the b a s i s of a l l tagging experiments w i t h i o d i n e i n v o l v e s u b s t i t u t i o n i n t o the t y r o s i n e r e s i d u e s as d e p i c t e d below (Hughes, 1957; 1966). 161. R £ ) OH - ^O0' + H+ I H 2OI + + \_)0~ - *O0~ + H + + H 20 H 2OI + + K(]30" + KZ^ °" + H+ + H 2 ° Chloramine-T i s an o x i d i z i n g agent which f a c i l i -t a t e s the co n v e r s i o n of i o d i d e (I ) to i o d i n e ( I 2 ) a n d subsequently to H 2OI +, presumably by the f o l l o w i n g o v e r a l l e quation (Hughes, 1966); CH 3 Q s 0 2NHCL + 21" •* C H 3 £ ) S 0 2 N H + + CL" + I 2 Chloramine-T i s a l s o capable of r e g e n e r a t i n g i o d i n e from the i o d i d e produced when i o d i n e d i s a s s o c i a t e s i n s o l u t i o n a c c o r d i n g to the f o l l o w i n g equations (Hughes, 1957; 1966): I 2 + H 20 •* H 2OI + + I' 1 2 + OH" •+ IOH + I HOI •+ H + + 01 301 •+ I 0 3 + 21 162. As i t i s an o x i d i z i n g agent, the use of chloramine-T does c r e a t e c e r t a i n disadvantages. Changes w i t h i n the p r o t e i n w i l l occur s i n c e some of the i o d i n e formed may c y c l e between the reagent and p r o t e i n a l t e r n a t e l y decreas-ing the reagent and o x i d i z i n g the p r o t e i n . The p r o t e i n molecule i s a l s o s u s c e p t i b l e to a t t a c k d i r e c t l y by c h l o r -amine-T . As a consequence, the o x i d a t i v e nature of c h l o r -amine-T w i l l impose s t r i c t c o n d i t i o n s on the a c t u a l i o d i n a -t i o n r e a c t i o n . T h i s aspect i s p a r t i c u l a r l y evident when LH and FSH are to be i o d i n a t e d f o r use as a l a b e l l e d l i g a n d i n t i s s u e r e c e p t o r assays where b i o l o g i c a l ( r a t h e r than immunological a c t i v i t y ) i s c r i t i c a l f o r optimal com-p e t i t i v e b i n d i n g to r e c e p t o r . The requirements i n v o l v e the p r o t e i n : Chloramine-T r a t i o , r e a c t i o n temperature and r e a c t i o n time p r i o r to a d d i t i o n of sodium m e t a b i s u l f i t e (Leidenberger and R e i c h e r t , 1972; R e i c h e r t and B h a l l a , 125 1974). Excess p r o t e i n o x i d a t i o n and I s u b s t i t u t i o n may r e s u l t i n g r e a t e r s p e c i f i c a c t i v i t y o f l a b e l l e d p r o t e i n however % n o n s p e c i f i c b i n d i n g i n c r e a s e s , % s p e c i f i c b i n d i n g decreases and % b i o l o g i c a l a c t i v i t y r e t a i n e d a f t e r i o d i n a -t i o n d r a s t i c a l l y decreases ( R e i c h e r t and B h a l l a , 1974). Sonada and Schlamowitz (1970) conducted d e t a i l e d s t u d i e s of parameters which e f f e c t t r a c e i o d i n a t i o n of immunoglobulin G with iodine-125 and chloramine-T. C o n d i t i o n s 163. of oxidant, i o d i d e and p r o t e i n c o n c e n t r a t i o n as w e l l as pH, i o n i c s t r e n g t h , temperature, exposure time and p r o t e i n p u r i t y a l l a f f e c t the o v e r a l l i o d i n a t i o n and subsequent p r o t e i n i n t e g r i t y . Sonada and Schlamowitz (1970) determined a pH 7.0-7.5 and 2°C temperature optimal f o r f ormation of a c t i v e i o d i n e l ^ O I * and i t s i n c o r p o r a t i o n i n p r o t e i n . Oddly enough, Leidenberger and R e i c h e r t (1972) as w e l l as R e i c h e r t and B h a l l a (1974) u t i l i z e d i o d i n a t i o n procedures i n c o r p o r a t i n g pH 7.5 and 2-3°C temperature to ma i n t a i n the b i o l o g i c a l a c t i v i t y of the p r o t e i n i n q u e s t i o n . S e v e r a l a l t e r n a t i v e procedures are a v a i l a b l e f o r p r o t e i n i o d i n a t i o n ; McFarlane's hydrogen peroxide or monochloride methods (1956). The B o l t o n and Hunter 12 5 reagent which u t i l i z e s an I - l a b e l l e d a c y l a t i n g agent (1973) and the enzymatic r a d i o i o d i n a t i o n of gonadotropins by a system composed of l a c t o p e r o x i d a s e and hydrogen peroxide as d e s c r i b e d by M i y a c h i e t a_l. (1972) and P i n t o et a l . (1977). These methods p r o v i d e a m i l d e r i o d i n a t i o n r e a c t i o n which does note p r e d i s p o s e the p r o t e i n molecule to damage by o x i d a t i o n . The chloramine-T method i n c o n t r a s t , i s r a p i d , combines h i g h e f f i c i e n c y w i t h s i m p l i c i t y and consequently i s the most widely used. 164. In the event of p r o t e i n damage proper measures must be taken to ensure a pure homogenous i o d i n a t e d hormone p r e p a r a t i o n i s used i n assays. P u r i f i c a t i o n of the i o d i -nated p r o t e i n , which a l s o separates f r e e unbound i o d i n e -125, can be achieved through s e v e r a l methods. A common procedure i n v o l v e s ge f i l t r a t i o n through sephadex-GlOO (Re i c h e r t and B h a l l a , 1974; M c N e i l l y et a l . , 1976), through B i o - G e l P60 columns (Niswender et al., 1969) or c e l l u l o s e Whatman CF 11 i o n exchange r e s i n s ( M c N e i l l y et a l . , 1976). Assessment of p r o t e i n damage, which would i n d i c a t e f u r t h e r p u r i f i c a t i o n or r e i o d i n a t i o n of f r e s h p r o t e i n m a t e r i a l , can be achieved through s e v e r a l methods. Hunter (1969) d e s c r i b e s three such procedures; the f i r s t i n v o l v e s the s e p a r a t i o n p a t t e r n of l a b e l l e d hormone, damaged l a b e l l e d hormone and f r e e iodine-125 on Whatman 3MC chromatography paper, a m o d i f i c a t i o n consists of u s i n g a small column of c e l l u l o s e (Whatman CF 11) to absorb i n t a c t i o d i n a t e d gonadotropin ( s p e c i f i c a l l y LH) and so separate i t from 125 125 damaged I-LH, I - i o d i d e and other r e a c t a n t s . T h i r d and f i n a l l y the use of p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s 125 to determine the homogeneity of I-FSH. A c c o r d i n g to Sonada and Scholamowitz (1970) i o d i n a t e d p r o t e i n s vary widely i n t h e i r a b i l i t y to r e t a i n n a t i v e s t a t e and f u n c t i o n i n v i v o and in v i t r o depending 165. on the number of atoms of x^Di i n t r o d u c e d , s i d e e f f e c t s due to r e a c t i n g with oxidant and r a d i a t i o n damage by 12 5 I. The i o d i n a t i o n method implemented f o r oLH and oFSH are m o d i f i c a t i o n of the chloramine-T procedure des-c r i b e d by Greenwood et al. (1963) f o r human growth hormone. FSH was i o d i n a t e d a c c o r d i n g to M c N e i l l y and Hagen (1974), LH a c c o r d i n g to Niswender et al. (1969). Both p i t u i t a r y p r e p a r a t i o n s i o d i n a t e d i n t h i s f a s h i o n and subsequently p u r i f i e d by g e l f i l t r a t i o n through sephadex-GlOO were shown to be s u i t a b l e f o r use i n a p p r o p r i a t e radioimmuno-assays without d e c r e a s i n g s p e c i f i c i t y or s e n s i t i v i t y . 166. APPENDIX I I I TRINITROBENZENESULFONIC ACID (TNBS) METHOD FOR DETERMINING AMINES 3 M a t e r i a l s B u f f e r Reagent Procedure Twenty-five mg of TNBS was d i s s o l v e d i n 10 ml of 0.10 M sodium t e t r a b o r a t e b u f f e r , pH 9.3. Twenty m i c r o l i t r e s of t h i s reagent was then added to a known a l i q u o t of unknown i n 200 y l of b u f f e r . The v i a l s were mixed ( f i n g e r tapped), h e l d at room temperature f o r 45 minutes and the analyzed f o r c o l o u r development ( A » r n ) • 0.10 M sodium t e t r a b o r a t e (Na^O-, • 10H 20) , pH 9.3 TNBS [ 2 , 4 , 6 - t r i n i t r o b e n z e n e s u l f o n i c a c i d , ( N 0 2 ) 3 C 6 H 2 S 0 3 H ] a From Snyder and S o b o c i n s k i (1975). 167. APPENDIX IV ESTRUS SYNCHRONIZATION: BACKGROUND INFORMATION The c l a s s i c a l approach f o r e s t r u s s y n c h r o n i z a t i o n simulates p r o l o n g a t i o n of the l u t e a l phase through the use o f a s u i t a b l e progestagen (progesterone or one of i t s analogues). The use of p r o g e s t a t i o n a l agents has r e s u l t e d from three c r i t i c a l r e s u l t s ; Dutt and Casida (1948) found that d a i l y progesterone i n j e c t i o n s given to c y c l i c ewes f o r 14 days i n h i b i t e d both e s t r u s and o v u l a t i o n , removal of the progestagen r e s u l t e d i n a r e t u r n to e s t r u s approximately 3 days l a t e r . Dutt (1952) and Robinson (1952) next a d m i n i s t e r e d progesterone f o l l o w e d by a PMSG i n j e c t i o n to s e a s o n a l l y anestrus ewes which induced c o i n c i d e n t e s t r u s and o v u l a t i o n . F i n a l l y , i t was demons-t r a t e d that progesterone may be taken up from the vagina (Shelton and Moore, 1967; Shelton and Robinson, 1967a). Development of a technique which i n v o l v e d a progestagen c r e a t e d f o u r s p e c i f i c problems: type of progestagen, route of a d m i n i s t r a t i o n , dosage and endocrino-l o g i c a l ( p h y s i o l o g i c a l ) response. Progesterone and s e v e r a l analogues have been ev a l u a t e d on t h e i r a b i l i t y to i n h i b i t e s t r u s , and most important t h e i r a b i l i t y to r e l e a s e t h i s 168. s u p p r e s s i v e e f f e c t upon p r o g e s t i n removal to permit e s t r u s c o i n c i d e n t with o v u l a t i o n (Shelton and Robinson, 1967b). Progesterone, Provera (17a-acetoxy-6a-methylpregn-4-ene-3,20-dione;MAP) and SC-9880 (17a-acetoxy-9a-Fluoro-lla-hydroxypregn-4-ene-3,20-dione;Fluorogestone Acetate; cronolone) have proven the most e f f e c t i v e (Robinson and Moore, 1967; Robinson et_ a l . , 1967; Haresign, 1978). SC-9880 appears i n d i s t i n g u i s h a b l e from progesterone i n a l l c r i t e r i a with a b i o l o g i c a l a c t i v i t y 20-25 times as potent. T h i s i n c o n j u n c t i o n with e a r l i e r e s t r u s f o l l o w i n g SC-9880 versus MAP has proven SC-9880 the p r o g e s t a t i o n a l agent of choice and i s t h e , a c t i v e i n g r e d i e n t i n the commer-c i a l l y prepared synchro-mate p e s s a r i e s (Robinson and Moore, 1967; Robinson e_t a l . , 1967; Shelton and Moore, 1967) . O r a l , i n t r a m u s c u l a r i n j e c t i o n , implants and i n t r a v a g i n a l p e s s a r i e s are the primary routes of adminis-t r a t i o n (Gordon, 1975), however, i n d i v i d u a l animals are not guaranteed an adequate d a i l y intake of p r o g e s t i n when given o r a l l y and i n j e c t i o n r e q u i r e s d a i l y manipula-t i o n of the animals. As a consequence i n t r a v a g i n a l sponges have become the method of choice when s y n c h r o n i z i n g ewes (Robinson, 1965, Robinson and Moore, 1967; Robinson and Smith, 1967; Wishart, 1967). 169. Dose and method of progestagen a d m i n i s t r a t i o n are c r i t i c a l f a c t o r s when r e l e a s e of e s t r u s , o v u l a t i o n and ensuing f e r t i l i t y are c o n s i d e r e d (Haresign, 1978). Low doses of SC-9880 (10 mg/sponge) r e s u l t e d i n poor e s t r u s s y n c h r o n i z a t i o n when p e s s a r i e s are removed. T h i s i s r e l a t e d to the p a t t e r n of p r o g e s t i n a b s o r p t i o n , most i s absorbed dur i n g the f i r s t few days a f t e r i n s e r t i o n , consequently, the d a i l y r a t e of a b s o r p t i o n i n the f i n a l days i s i n s u f f i c i e n t to e x e r t a f u l l n egative feedback. With low dose p e s s a r i e s d u r i n g the f i n a l days of i n s e r t i o n there may a l s o be a sub-optimal amount of s t e r o i d r e q u i r e d f o r maximum e x p r e s s i o n of subsequent e s t r u s and f e r t i l i t y (Morgan et _al. , 1967; Haresign, 1978). Morgan et a l . (1967) s t a t e on the average s i x t e e n per cent of s t e r o i d present at any time w i l l be absorbed over a twenty-four hour p e r i o d . I n c r e a s i n g the l e v e l of s y n t h e t i c p r o g e s t i n over a d e f i n e d l i m i t (40 mg SC-9880) soon has an o v e r a l l d e l e t e r i o u s e f f e c t (Shelton and Moore, 1967). Release to e s t r u s with o v u l a t i o n i s impaired as i s sperm s u r v i v a l and t r a n s p o r t , a l l of which decrease ewe f e r t i l i t y . These e f f e c t s are a t t r i b u t e d to c i r c u l a t i n g endocrine a b n o r m a l i t i e s which r e s u l t p r i m a r i l y i n LH r e l e a s e s i g n i f i c a n t l y e a r l i e r than onset of e s t r u s and i n c r e a s e d c e r v i c a l mucus (Rexroad 170 . and B a r l o , 1977; Haresign, 1978). The optimal and most e f f i c i e n t dose of SC-9880 appears to be w i t h i n the range 20-40 mg/pessary and i n s e r t e d f i f t e e n to eigh t e e n days (Robinson e_t al., 1967; Robinson and Moore, 1967; Robinson and Smith, 1967 a ) . Haresign (1978) found conception r a t e and p r o l i f e r a c y i n c r e a s e s by e i g h t per cent and twelve per cent r e s p e c t i v e l y as the SC-9880 dose i n c r e a s e s from 30 mg up to 40 mg. PMSG treatment (750 I.U. i n 5.0 ml s a l i n e , subcutaneously) zero-twenty-four hours a f t e r pessary removal has demonstrated the a b i l i t y to i n c r e a s e the in c i d e n c e o f e s t r u s , e s t r u s with o v u l a t i o n , c o nception and p r o l i f e r a c y (Robinson and Smith, 1967; Gordon, 1975; Haresign, 1978) . With a PMSG i n j e c t i o n , i t appears a lower l e v e l o f SC-9880; 10-20 mg/pessary, can be used without adverse a f f e c t s (Moore and H o i s t , 1967; Robinson and Smith 1967 b; C h r i s t e n s o n , 1976). The p r e v a i l i n g e n d o c r i n o l o g i c a l c o n d i t i o n s upon pessary removal have a c r i t i c a l e f f e c t upon the ensuing r e t u r n to e s t r u s , o v u l a t i o n with e s t r u s and conception r a t e as s t a t e d p r e v i o u s l y . Time t i l l onset of e s t r u s a f t e r SC-9880 pessary withdrawal occurs i n a does dependent manner. The g r e a t e s t i n c i d e n c e of e s t r u s a s s o c i a t e d 171. with ten, twenty and f o r t y mg SC-9880 oc c u r r e d twenty-n i n e , t h i r t y - f o u r - f o r t y - e i g h t and f o r t y - e i g h t - f i f t y -three hours r e s p e c t i v e l y a f t e r pessary removal (Robinson and Smith, 1967 a ) . Non-returns to s e r v i c e a l s o i n c r e a s e d from 18.4% to 41.7% as the dose of SC-9880 i n c r e a s e d from 20 mg to 40 mg r e s p e c t i v e l y (Robinson and Smith, 1967 a ) . Wishart (1967) demonstrated 95% of a l l ewes t r e a t e d with SC-9880 impregnated p e s s a r i e s (20 mg, 30 mg, 40 mg & PMSG) demonstrated e s t r u s w i t h i n the f i r s t 5 days, Robinson et a l . (1967) found 85% of a l l SC-9880 t r e a t e d ewes (20-40 mg) e x h i b i t e d e s t r u s between days 2 and 4 a f t e r pessary removal while Shelton and Robinson (1967) found i n t r a m u s c u l a r i n j e c t i o n s of progesterone ~ and SC-9880 induced ewes to show e s t r u s 1-4 days a f t e r the l a s t i n j e c t i o n . 

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