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Estrogen and phyto-estrogen binding in ewe pituitary, hypothalamus, and other structures Mathieson, Ronna Arlene 1979

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ESTROGEN AND PHYTO-ESTROGEN BINDING IN EWE PITUITARY, HYPOTHALAMUS, AND OTHER STRUCTURES by RONNA ARLENE MATHIESON Bachelor of Science i n Ag r i c u l t u r e The University of B r i t i s h Columbia, 1968 A Thesis Submitted i n P a r t i a l F u l f i l l m e n t 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 thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA APRIL 1979 © Ronna Arlene Mathieson, 1979. In presenting th i s thes is in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make i t f ree l y ava i lab le for reference and study. I further agree that permission for extensive copying of th i s thesis for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t i on of th is thes is fo r f inanc ia l gain sha l l not be allowed without my written permission. n . , ANIMAL SCIENCE Department or The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 aPT?TT. /Q t - I Q 7 Q i i ABSTRACT Phyto-estrogens are known to bind to estrogen receptors of the uterus and they c a n i n i t i a t e the early events of estrogen stimulation i n c l u d i n g water imbibition and synthesis of induced protein as well as l a t e events such as uterine growth. There i s i n d i r e c t evidence that these compounds a f f e c t the functioning of the hypothalamus and p i t u i t a r y with respect to the release of gonadotropins. The purpose of t h i s study was to determine i f the phyto-estrogen compounds, genistein and coumestrol, could i n t e r a c t with the estrogen receptor molecules i n the cytoplasm of p i t u i t a r y and hypothalamus t i s s u e from ewes. Estrogen binding c h a r a c t e r i s t i c s were also examined. Cytosol prepara-tions from the various experimental, tissues were incubated f i f t e e n minutes 3 at t h i r t y degrees C. with H-estradiol; separation of bound from free l a b e l was c a r r i e d out on Sephadex LH-20 columns. Estrogen binding parameters were 3 determined by double r e c i p r o c a l p l o t s . Competitions with H-estradiol i n the presence of either coumestrol or genistein were c a r r i e d out i n a s i m i l a r manner. Apparent i n h i b i t i o n constants (K^ .) were determined from Dixon p l o t s . The.apparent d i s s o c i a t i o n constants (K^) for e s t r a d i o l i n ewe p i t u i t a r y c ytosol was determined to be 0.26^0.12 nM. The apparent f o r coumestrol i n the ewe p i t u i t a r y cytosol was determined to be 59-61 nM and the apparent ILj. f o r genistein was determined to be 130-210 nM. These compounds were shown to displace e s t r a d i o l from receptors i n ewe hypothalamus and amygdala cytosols. Results of preliminary binding experiments with ewe p i n e a l and uterus cytosols are also presented. These r e s u l t s suggest that phyto-estrogens can i n t e r f e r e with normal estrogen feedback mechanisms with respect to gonadotropin release i n the ewe. Signature of Supervisor i i i TABLE OF CONTENTS PAGE ABSTRACT i i TABLE OF CONTENTS i i i LIST OF TABLES v LIST OF FIGURES AND ILLUSTRATIONS v i ACKNOWLEDGMENT v i i i INTRODUCTION 1 REVIEW OF LITERATURE 2 A. THE PITUITARY IN FEMALE REPRODUCTION 2 B. THE HYPOTHALAMUS IN FEMALE REPRODUCTION 4 C. THE PITUITARY-HYPOTHALAMUS RELATIONSHIP 6 D. EXTRAHYPOTHALAMIC STRUCTURES IN REPRODUCTION 7 E. THE HYPOTHALAMIC-HYPOPHYSIAL-0VARIAN RELATIONSHIP 11 F. ESTROGEN RECEPTORS 18 G. GENISTEIN AND COUMESTROL 33 MATERIALS AND METHODS 41 A. MATERIALS 41 B. ESTROGEN PROTEIN BINDING ASSAY 46 C. TREATMENT OF DATA 56 RESULTS 60 A. HYPOTHALAMUS 60 B. PITUITARY 67 C. AMYGDALA 78 D. PINEAL 82 E. UTERUS 82 DISCUSSION 88 i v TABLE OF CONTENTS (continued) BIBLIOGRAPHY APPENDIX I. Appendix Figure 1, Adapter System I I . Lowry assay I I I . TNBS assay IV. Appendix Figure 2, Steroid Estrogens V. Appendix Figure 3, D i e t h y l s t i l b e s t r o l VI. Anti-Estrogen Compounds VII. P h y s i o l o g i c a l Example V LIST OF TABLES PAGE I. Proposed C l a s s i f i c a t i o n System, Estrogen Agonists and 30 Antagonists. I I . Comparison of E s t r a d i o l and Phyto-Estrogen Binding i n various tissues from ewes, as determined i n t h i s study. 92 v i LIST OF FIGURES AND ILLUSTRATIONS PAGE 1. P o t e n t i a l Mechanisms of Actions of Antiestrogens 29 2. Str u c t u r a l Formulae of Phyto-estrogens 35 3. Ventral Surface of the Sheep Brain 43 4. M i d s a g i t t a l View of the Sheep Brain 44, 45 5. 3 H - E s t r a d i o l Binding, P i t u i t a r y , Column e l u t i o n 49 6. E l u t i o n of 3 H - E s t r a d i o l , 6 cm. LH-20 column 51 7. E l u t i o n of 3 H - E s t r a d i o l , 12 cm. LH-20 column 52 8. E l u t i o n of " H-Estradiol, Amygdala, Lowry absorbances 54 9. Preparation 1, Hypothalamus, 3 H - E s t r a d i o l , nM 61 10. Preparation 1, Hypothalamus, Percent H-Estradiol 61 11. Preparation 3 2, Hypothalamus, H-Estradiol 63 12. Preparation 2, Hypothalamus, Percent, 3 H - E s t r a d i o l 64 13. Preparation 3 2, Hypothalamus, H-Estradiol 64 14. Preparation 2, Hypothalamus, Double Reciprocal 65 15. Preparation 3F., Hypothalamus, H-Estradiol 65 16. Preparation 1, P i t u i t a r y , Saturation Curve 68 17. Preparation 1, P i t u i t a r y , Double Reciprocal 69 18. Preparation 2, P i t u i t a r y , Saturation Curve 70 19. Preparation 2, P i t u i t a r y , Double Reciprocal 70 20. Preparation 3, P i t u i t a r y , Saturation Curve 72 21. Preparation 3, P i t u i t a r y , Double Reciprocal 72 22. Preparation 1, P i t u i t a r y , Dixon P l o t , Coumestrol 74 23. Preparation 2, P i t u i t a r y , Dixon P l o t , Coumestrol 75 24. Preparation 2, P i t u i t a r y , I n h i b i t i o n , Genistein 76 25. Preparation 2, P i t u i t a r y , Dixon P l o t , Genistein 77 v i i LIST OF FIGURES AND ILLUSTRATIONS (continued) PAGE 26. Preparation 3, P i t u i t a r y , I n h i b i t i o n Genistein 79 27. Preparation 3, P i t u i t a r y , Dixon P l o t , Genistein 80 28. Amygdala, 3 H - E s t r a d i o l Binding 81 29. P i n e a l , 3 H - E s t r a d i o l Binding 83 30. Pineal, Dixon P l o t , Genistein 83 31. Uterus, E l u t i o n of 3 H - E s t r a d i o l 85 32. Uterus, Saturation Curve 86 33. Uterus, Double Reciprocal Plot 87 v i i i ACKNOWLEDGEMENT Grateful appreciation i s extended to those people who as s i s t e d t h i s study and i n p a r t i c u l a r to Dean W.D. K i t t s , f o r h i s continued encouragement and support during the course of t h i s work, Frances Newsome, for our many useful discussions and for her valuable advice, Dr. Chen and Joe at Richmond Packers, Ltd., for assistance i n c o l l e c t -ing t i s s u e samples, My husband, Geoffrey Webb and daughters Dawn Amber and Fern Marisa, my h e a r t f e l t gratitude.for t h e i r patience and confidence. -1-INTRODUCTION The female reproductive system with i t s many hormonal i n t e r r e l a t i o n -ships has been the subject of study for many years. The e l u c i d a t i o n of the molecular action of hormones has led to intensive research on the complex int e r p l a y and metabolic e f f e c t s brought about by these endogenous messengers. A series of d e l i c a t e checks and balances operate to coordinate the reproduct-ive system and ^modulate hormonal e f f e c t s . The hormonal r e l a t i o n s h i p between ovary and anterior p i t u i t a r y has long been recognized (Moore and P r i c e , 1932). Studies of many workers have demonstrated a further complexity of the gonadal-p i t u i t a r y r e l a t i o n s h i p i n that feedback mechanisms are mediated through the brain , i n p a r t i c u l a r , the hypothalamic area which processes impulses from sensory centres. The hypothalamus exerts co n t r o l over the adenohypophysis by means of r e l e a s i n g hormones with the e s s e n t i a l l i n k being a p o r t a l vascular system. Therefore the hypothalamus provides a major l i n k between the endo-crine system and neural s t i m u l i from the external and i n t e r n a l environments and has been termed a neuro-endocrine transducer. An i n t r i c a t e system r e l y i n g on chemical compounds to convey feedback messages i s vulnerable to interference by compounds which mimic the e f f e c t s of endogenous hormones. Such might be the case of compounds termed phyto-estrogens which are found i n plants and which can cause estrogen e f f e c t s when ingested by animals. This i s possible because they contain c e r t a i n chemical groupings s i m i l a r to s t e r o i d estrogens which are recognized by i n t r a c e l l u l a r estrogen receptors. If these compounds bind to the estrogen receptors of p i t u i t a r y and hypothalamus they could act either as estrogen antagonists against endogenous estrogen or as estrogens when endogenous estrogen l e v e l s are low. It was the purpose of t h i s study to determine i f these compounds do bind to estrogen receptors i n ewe hypothalamus and p i t u i t a r y . This problem -2-has been approached using competitive protein binding techniques and standard methods of binding analysis. Other tissues were examined f o r comparative purposes and as a check on the assay system and analysis used. REVIEW OF LITERATURE A. THE PITUITARY IN FEMALE REPRODUCTION The p i t u i t a r y gland i s extremely complex both i n structure and function. Through i t s hormones, the p i t u i t a r y has d i r e c t e f f e c t s on growth, metabolism, conservation of water, reproduction and l a c t a t i o n as w e l l as an i n d i r e c t i n -fluence on body metabolism through hormone e f f e c t s on the adrenal cortex and thyroid (Holmes and B a l l , 1974). Considerable confusion has a r i s e n regarding nomenclature for the various subdivisions of the p i t u i t a r y due to the great species v a r i a t i o n involved. The terms a n t e r i o r and p o s t e r i o r p i t u i t a r y , commonly used i n the past, are not s a t i s f a c t o r y f or a l l species and tend to be misleading. A generally accepted d i v i s i o n i s on the basis of embryonic o r i g i n of the various t i s s u e s composing the p i t u i t a r y gland. The neurohypophysis which forms the neural component of the gland i s derived from neural ectoderm and consists of the pars nervosa or infundibular process, and the infundibular stem and median eminence which together form the hypophyseal s t a l k . The glandular portion of the p i t u i t a r y i s the adenohypophysis which a r i s e s from buccal ectoderm. I t i s composed of the pars d i s t a l i s , the pars t u b e r a l i s , and the pars intermedia, which i s present only i n some species. The pars d i s t a l i s forms the major portion of the adenohypophysis and constitutes eighty percent or more of the t o t a l p i t u i t a r y gland weight (Martin, 1976). This area has no d i r e c t a r t e r i a l blood supply and receives blood v i a a p o r t a l -3-system which a r i s e s from c a p i l l a r i e s i n the median eminence of the neural component of the p i t u i t a r y (Daniel and Prichard, 1975; Page and Bergland, 1977). The pars d i s t a l i s secretes a number of hormones, two of which are d i r e c t l y involved i n the reproductive process. F o l l i c l e stimulating hormone (FSH) and l u t e i n i z i n g hormone (LH) together form the gonadotropins, so named for t h e i r stimulating e f f e c t on the gonads. The gonadotropic hormones con t r o l the maturation and release of ova d i r e c t l y and i n d i r e c t l y c o n t r o l the devel-opment of secondary sex c h a r a c t e r i s t i c s , reproductive cycles, and f e r t i l i t y , by stimulating the secretion of s t e r o i d sex hormones. There are a number of d i s t i n c t c e l l types i n the pars d i s t a l i s and attempts have been made to r e l a t e p a r t i c u l a r c e l l types to c e r t a i n hormones. While t h i s has to some extent been possi b l e , controversy e x i s t s regarding gonadotrophs, the c e l l s which produce the hormones LH and FSH. Based on h i s t o l o g i c a l s t a i n i n g c h a r a c t e r i s t i c s , the c e l l s designated 6-(delta) baso-p h i l s have been shown to be gonadotrophs. Some workers have i d e n t i f i e d two separate and d i s t i n c t c e l l types wi t h i n t h i s d i v i s i o n and have termed them f o l l i c u l o t r o p h s (FSH producing) and luteotrophs (LH producing) (Schulster, et a l , 1976; Holmes and B a l l , 1974; Costoff, 1973). Others maintain there i s j u s t one type of gonadotroph which can be influenced by e x t r a c e l l u l a r conditions (Franchimont, 1977; Martin, 1976). Evidence f o r t h i s view i s based i n part on electron microscopic-immunochemical techniques which show FSH and LH to be present i n the same c e l l (Nakane, 1975). As yet the question of whether the d i s t i n c t subgroups of gonadotrophs are d i f f e r e n t c e l l types, or f u n c t i o n a l variants of the same c e l l type, remains unresolved (Farquhar, et a l , 1975; Costoff, 1977). A recent study on the ovine p i t u i t a r y with antibodies to i n t a c t LH, and to each of the subunits, «• and 3, of LH has i d e n t i f i e d the LH producing c e l l s , which are located near c a p i l l a r i e s . The - 4 -authors did not l o c a l i z e FSH however, they do suggest t h e i r technique for future study to determine i f FSH i s also located i n the LH producing c e l l s of the sheep p i t u i t a r y (Dacheux and Dubois, 1978). In the context of t h i s discussion the term p i t u i t a r y i s used to mean the pars d i s t a l i s part of the adenohypophysis and to i n d i c a t e that portion of the p i t u i t a r y gland which i s p r i m a r i l y concerned with reproduction. B. THE HYPOTHALAMUS IN FEMALE REPRODUCTION The hypothalamus i s that area of the b r a i n which by d e f i n i t i o n l i e s under the thalamus, however i t s l i m i t s are not c l e a r l y defined (Daniel and Prichard, 1975). I t i s generally accepted to encompass the area from the optic chiasma to the mammillary body and includes the tuber cinereum and by some workers, the median eminence and infundibulum. I t forms the walls and f l o o r of the lower part of the t h i r d v e n t r i c l e of the b r a i n and i t s upper border i s the hypothalamic sulcus. The l a t e r a l boundaries of the hypothalamus are l e s s well defined, and are taken as the optic t r a c t s , cerebral gangalia, and other structures (Martin, et a l , 1977; Daniel and Prichard, 1975). The neurosecretory or hormone producing c e l l s of the hypothalamus, are true neurons i n that they develop action p o t e n t i a l s , have synaptic v e s i c l e s and are dependent on n e u r o g l i a l elements. There i s an extensive nerve f i b r e system i n the hypothalamus for rapid communication within the hypothalamus and between the hypothalamus and other parts of the c e n t r a l nervous system. Neuron c e l l bodies aggregate i n bundles termed n u c l e i , and various n u c l e i have been assigned rol e s i n hypothalamic function. This may be an inaccurate designation as the functions of a given region often extends beyond the ana-tomic margins of the n u c l e i (Martin, et a l , 1977). The anterior-preoptic hypothalamus appears to regulate the a c t i v i t y of other areas of the hypothalamus. The suprachiasmatic preoptic region or -5-medial preoptic area seems to be involved with c i r c a d i a n and c y c l i c reproduc-t i v e : rhythms, inc l u d i n g the induction of ovulation. The e f f e c t s on ovulation are mediated through the basal arcuate area of the hypothalamus. The medial preoptic area receives neural inputs from extrahypothalamic structures of the limbic and midbrain system. Fibres from the anterior-preoptic hypothalamus form the preoptico-tuberal t r a c t which terminates i n the area of the arcuate nucleus of the ventromedial (medial basal) hypothalamus. These f i b r e s must be i n t a c t i n order for the preovulatory surge of LH to occur. The arcuate nucleus which forms the very basal (tuberal) hypothalamus appears to be i n -volved i n the tonic secretion of gonadotropins. The medial basal hypothalamus i s considered to be the l o c a t i o n of r e l e a s i n g and i n h i b i t i n g - f a c t o r producing neurons (Wiittke, 1976). The arcuate nucleus and adjacent area have c l u s t e r s of dopaminergic f i b r e projections into the median eminence. These form the tuberoinfundibular (tuberohypophysial) nerve t r a c t s which terminate i n the external layer of the median eminence at the c a p i l l a r i e s which form the hypo-phy s i a l p o r t a l blood vessels. There i s strong evidence for synaptic linkage between these nerve t r a c t s and afferents a r i s i n g i n the preoptic area. Axonal terminals from noradrenergic and possibly serotonergic f i b r e s from mesencep-h a l i c structures are also found i n the median eminence (Wiittke, 1976). These monoaminergic f i b r e s may modulate a c t i v i t y of r e l e a s i n g hormone neurons but t h e i r r o l e i s unclear. Fibres from the f o r n i x go to the arcuate nucleus and anterior l a t e r a l area as w e l l as to the mammillary body v i a the hippocampo-hypothalamic t r a c t s . The medial mammillary nucleus of the posterior (caudal, mammillary) hypothalamus i s an area of many efferent and afferent pathways and i s involved among other things i n the mediation of behaviour r e l a t e d to sexual a c t i v i t y , however, i t has no known influence on endocrine function (Martin, et a l , 1977). -6-Although the hypothalamus i s involved i n coordinating many functions i n the animal, f o r the purpose of t h i s discussion only those factors d i r e c t l y involved i n reproduction w i l l be discussed. C. THE PITUITARY-HYPOTHALAMUS RELATIONSHIP Much evidence has accumulated suggesting a r e l a t i o n s h i p between the hypo-thalamus and adenohypophysis and ultimately gonadal function (Harris, 1972; Anderson and Haymaker, 1974; Daniel and Prichard, 1975; Dorner, 1976; Fink, 1976; Martin, et a l , 1977; Donovan, 1978a). Observations that the p i t u i t a r y was not autonomous i n maintaining c y c l i c gonadal function, coupled with obser-vations of the e f f e c t of external factors on reproduction, led to the i m p l i c a -t i o n that the p i t u i t a r y was under co n t r o l of the c e n t r a l nervous system. The nature of t h i s c o n t r o l was postulated many years before i t was elucidated. The neurohumoral concept, also c a l l e d the p o r t a l vessel chemotransmitter hypo-thesis postulated that substances (neurohumoral agents) from the hypothalamus passed by a neural and vascular pathway to c o n t r o l the release of anterior p i t u i t a r y hormones. This concept i s now well established and a factor respon-s i b l e f or the release of LH and FSH from the adenohypophysis has been i s o l a t e d , characterized as a s i n g l e decapeptide, and synthesized (Schally, et a l , 1971; Matsuo, et a l , 1971; Guillemin, 1974). As yet, i t appears there i s only one factor responsible for the release of both gonadotropins and i t i s termed va r i o u s l y LH-RH, LH/FSH-RH, or GnRH (gonadotropin r e l e a s i n g hormone) ( R e i c h l i n , et a l , 1976; Convey, 1973). The neurovascular l i n k as outlined above forms the c r i t i c a l pathway from the nerve c e l l s of the hypothalamus to the glandular c e l l s of the pars d i s t a l i s . Daniel and Prichard (1975), i n v e s t i g a t i n g the vascular l i n k , have determined that s p e c i f i c regions of the pars d i s t a l i s receive blood from p a r t i c u l a r areas i n the neurohypophysis (median eminence region) and further that, "The area -7-i n the pars d i s t a l i s that i s fed by any i n d i v i d u a l p o r t a l v e s s e l i s s t r i c t l y circumscribed. These observations are of s p e c i a l i n t e r e s t i n r e l a t i o n to the fa c t that the various types of c e l l s i n 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 regions of the lobe". The lack of evidence for a separate r e l e a s i n g hormone for FSH implies that the separation of FSH and LH release by the adenohypophysis must be a t t r i b u t e d to differences i n the p i t u i t a r y mechanisms of LH and FSH secretion (Fink, 1976; Vale, et a l , 1977). As previously mentioned, there i s evidence for only one type of gonadotroph, and therefore the existence of only one r e l e a s i n g hormone would appear reasonable.. This aspect w i l l be discussed i n a l a t e r section. GnRIi also seems to have b i o l o g i c a l actions within the c e n t r a l nervous system and may a f f e c t sexual behaviour (Moss and McCann, 1973; P f a f f , 1973a; Moss and McCann, 1975; Donovan, 1978b). GnRH i s found i n the p i n e a l , midbrain, and cerebral and c e r e b e l l a r c o r t i c e s and brain stem, as w e l l as i n the hypo-thalamus and p i t u i t a r y . It has an e f f e c t on the e l e c t r i c a l a c t i v i t y of CNS neurons and i s considered a neurotransmitter agent by Wilber, et a l , (1976), who state; "(hypothalamic hormones)...may also subserve c e n t r a l nervous system function i n the r o l e of synaptic modulators". D. EXTRAHYPOTHALAMIC STRUCTURES IN REPRODUCTION Observations of the e f f e c t that external factors have on ovulation and other aspects of reproduction suggested the influence of higher neural centres on hypothalamus function. This evidence includes the p h y s i c a l stimulation of the g e n i t a l area leading to ovulation i n animals that are induced ovulators. Psychological stress can lead to amenorrhea i n women. The presence of v i s u a l o l f a c t o r y , and auditory s t i m u l i can induce or postpone ovulation (Do'rher, 1976). Light plays an important r o l e i n i n i t i a t i n g reproductive cycles and there i s -8-evidence that the pin e a l gland may be involved (Lisk, et a l , 1972). I. The Limbic System The limbic system i s anatomically defined to include the medial part of the mesencephalic r e t i c u l a r formation (midbrain limbic system), the hypo-thalamus, the hippocampus, the septum, the amygdala, and the cingulate and pyriform cortex (Martin, et a l , 1977). The main connecting pathway i s the medial forebrain bundle, a multi-neuronal, multi-synaptic system. There are no d i r e c t sensory inputs into the limbic system with the exception of the retinohypothalamic connection to the suprachiasmatic nucleus. Sensory inputs are probably relayed through the r e t i c u l a r formation of the brain stem and thalamus (Wilber, et a l , 1977). The amygdala sends projections from i t s corticomedial n u c l e i through the s t r i a terminalis to the septum, medial pre-optic hypothalamus, and the external border of the ventromedial n u c l e i of the hypothalamus. From the basomedial amydala, the projections of the v e n t r a l amygdalofugal pathway pass to the l a t e r a l hypothalamus. However the termin-ation and function of t h i s pathway i s unresolved (Ellendorf, 1976; Martin, et a l , 1977). Stimulation of the amygdala evokes an excitatory response i n neurons of the ventromedial n u c l e i (Wilber, et a l , 1976). The f o r n i x forms the efferent p r o j e c t i o n of the hippocampus. It has a major d i s t r i b u t i o n to the mammillary body, and also d i r e c t inputs into the arcuate and ventromedial n u c l e i of the hypothalamus. Some of these seem to terminate d i r e c t l y on the tuberoinfundibular neurons (Lammers and Lohamn, 1974). There i s evidence that afferent impulses to the medial basal hypo-thalamus are required to t r i g g e r the preovulatory discharge of gonadotropins (Tal e i s n i k and Beltramino, 1975). The amygdala area has been the most studied and there i s the i n d i c a t i o n of both a f a c i l i t a t o r y and an i n h i b i t o r y influence on gonadotropin secretion depending on the area stimulated. The f a c i l i t a t o r y -9-impulses are transmitted v i a the s t r i a t e r m i n a l i s . The i n h i b i t o r y pathway i s unknown (Tale i s n i k and Beltramino, 1975). Observations suggest two ant-agonistic systems; the mesencephalo-hippocampal system which has an i n h i b i t o r y e f f e c t on gonadotropin secretion, and the mesencephalo-amydaloid system which has a f a c i l i t a t o r y e f f e c t on gonadotropin secretion ( T a l e i s n i k and Beltramino, 1975). . The limbic-midbrain regions appear to be involved i n the onset of puberty. E l e c t r i c a l stimulation of these areas can a l t e r p i t u i t a r y hormone secretion (Ellendorf, 1976). The hypothalamic and extrahypothalamic pathways are assumed to mediate the c i r c a d i a n rhythms of hormone secretion, stress induced a l t e r a t i o n s i n hormone secretion, i n t e g r a t i o n of neuroendocrine a c t i v i t y with autonomic nervous system responses, and neuroendocrine e f f e c t s triggered by o l f a c t o r y and per i p h e r a l sensory responses (Martin, et a l , 1977). I I . The P i n e a l Gland The pineal'gland.is a true endocrine gland of neural o r i g i n (from neuro-epithelium) but i s not a neuroendocrine organ, as i s the hypothalamus, because synthesis of i t s secretory products takes place i n organ s p e c i f i c c e l l s , the pinealocytes, and not i n neurons (Kappers, et a l , 1974; Kappers, 1976). The function of the p i n e a l has not been f u l l y elucidated but as Reiter (1976) states: " I f any f u n c t i o n a l p o s i t i o n of the p i n e a l gland seems well established, i t i s probably that of the co n t r o l of seasonal reproductive events i n some mammals". It i s evident that l i g h t acts through the p i n e a l gland to synchron-iz e reproduction with the seasons. The mechanisms involved are not f u l l y c l e a r . External l i g h t i n g generates impulses which pass through the accessory o p t i c t r a c t s v i a sympathetic nerve f i b r e s to the p i n e a l (Kappers, 1976; Wurtman and Moskowitz, 1977). The response to l i g h t and dark changes i s r e f l e c t e d i n a p a r a l l e l rhythm i n enzyme a c t i v i t y concerned with indolamine biosynthesis. -10-In some manner, the p i n e a l acts as an intermediary between seasonal photo-p e r i o d i c changes, and the neuroendocrine-reproductive axis (Reiter, 1976). The main function of the p i n e a l appears to be antigonadotropic i n that i t suppresses reproductive a c t i v i t y . P ineal gland a c t i v i t y i s i n v e r s e l y r e l a t e d to the f u n c t i o n a l status of the gonads i n seasonal breeders. The secretory products of the p i n e a l are of two types. The indolamines, e s p e c i a l l y melatonin, appear to have antigonadotropic a c t i v i t y . However, the p i n e a l also appears to secrete polypeptides with antigonadotropic a c t i v i t y . There two products may i n t e r a c t to produce o v e r a l l e f f e c t s (Reiter, 1976). It appears that i n the sheep, melatonin i s secreted into the systemic blood stream (Rollag, e_t a l , 1978). The p i n e a l e f f e c t seems to be on the hypothalamus to influence the secretory a c t i v i t y of the p i t u i t a r y possibly-by>• a f f e c t i n g synthesis and/ or release of GnRH. Reiter (1972) speculates: " i t i s i n t e r e s t i n g that hypo-thalamic i n h i b i t o r y factors...have not been demonstrated for FSH and LH although the existence of r e l e a s i n g . f a c t o r s (or factor) for these hormones are ( i s ) c e r t a i n . Perhaps the p i n e a l provides the important neuroendocrine means for i n h i b i t i n g these reproductively e s s e n t i a l hormones". However, Schally, et a l , (1973a), comments that although both r e l e a s i n g and i n h i b i t i n g f a c t ors are known for growth hormone, melanocyte stimulating hormone and p r o l a c t i n , these hormones do not stimulate t h e i r target c e l l s to produce products which could act as auto-regulatory or "feed-back" agents. Adrenocorticotropic hormone (ACTH), thyroid stimulating hormone (TSH), LH and FSH, for which i n h i b i t i n g f a c t ors have not been demonstrated, a l l stimulate t h e i r target c e l l s to produce hormones which, c a r r i e d i n the blood stream, can exert a feedback e f f e c t on the production and/or release of e i t h e r the hypothalamic r e l e a s i n g hormones or the p i t u i t a r y hormones or both. -11-E. THE HYPOTHALAMIC-HYPOPHYSIAL OVARIAN RELATIONSHIP The r e c i p r o c a l r e l a t i o n s h i p between the p i t u i t a r y and gonads has long been recognized and the mechanism of negative feedback of sex steroids on the release of gonadotropins was formulated i n 1932 (Moore and P r i c e , 1932; reviews, Hutchinson and Sharp, 1977; Dorner, 1976). This closed negative feedback loop, or push-pull system i s based on observations that removal of the p i t u i t a r y r e s u l t s i n cessation of ovarian function. Removal of ovaries leads to increased gonadotropin synthesis and secretion, and to p i t u i t a r y hypertrophy. The i n j e c t i o n of estrogen to castrates leads to decreased gona-dotropin release (Lisk, et a l , 1972). The gonadotropins FSH and LH, separately, and together, bring about mat-uration of the ovarian f o l l i c l e , ovulation, and formation of the corpus luteum. In addition, these hormones stimulate steroidogenesis i n the ovaries. It i s d i f f i c u l t to assign s p e c i f i c r o l e s to these hormones as they appear together i n vivo and, i n p h y s i o l o g i c a l conditions, they probably act synergis-t i c a l l y i n many instances. Generally, FSH i s assigned the r o l e of transforming ovarian f o l l i c l e s i nto Graafian (preovulatory) f o l l i c l e s and, with LH, stimu-l a t i n g estrogen synthesis by the theca c e l l s . Both hormones may have a r o l e i n ovulation although LH appears to predominate. Postovulation both FSH and LH i n i t i a t e l u t e i n i z a t i o n and formation of the corpus luteum, the function of which i s maintained by LH. LH also stimulates progesterone secretion (Sherwood and McShan, 1977). The s t e r o i d hormones, estrogens and progestagens, are secreted into the systemic blood stream and plasma l e v e l s of these two hormones show alternate c y c l i c patterns correlated with the various stages of the estrous cycle. The progestagen produced by the corpus luteum of the ewe i s almost exclus i v e l y progresterone although there may be small quantities of 20 <s -12-dihydroprogesterone and 17 « dihydroxyprogesterone (Baird, 1977). Estrogen i s a general term to encompass numerous compounds which bring about a l l or some estrogenic e f f e c t s (vide i n f r a ) . The endogenous estrogens of the ewe, produced by the theca c e l l s i n the -.ovary--, are the steroids e s t r a d i o l 17 3 and, to a much l e s s e r extent, estrone (Baird, 1977). The ovine ovary also secretes large amounts of androstenedione, peaks being correlated with e s t r a d i o l secretion (Scaramuzzi, et a l , 1974). I t appears that androstenedione may play a r o l e i n the con t r o l of basal LH secretion, possibly through peripheral conversion to estrone (Scaramuzzi and Martensz, 1975). Estrogens produce diverse e f f e c t s which can be divided into g e n i t a l and non-genital categories (Briggs and Brotherton, 1970). The g e n i t a l e f f e c t s of estrogen include the stimulation of growth and function of the uterus, ovaries, cervix, f a l l o p i a n tubes, vagina, external g e n i t a l i a and mammary gland through stimulation of mitosis and protein synthesis. The non-genital e f f e c t s of estrogens include the development and mainten-ance of secondary sex c h a r a c t e r i s t i c s , anabolic e f f e c t s such as increased n i t r o -gen retention, sodium retention, and calcium and phosphorus deposition. Of major importance to t h i s discussion are estrogen feedback e f f e c t s on c y c l i c gonadotropin secretion, and e f f e c t s on the c e n t r a l nervous system i n provoking behavioural estrus (Heap aW Illingworth, 1977; Briggs and Brotherton, 1970; Herbert, 1977). Although the r e c i p r o c a l action of gonadal steroids on gonadotropin release i s w e ll established, the s i t e / s i t e s at which the feedback occurs i s / a r e s t i l l unclear. The bulk of evidence points to a dual system i n which gonadal s t e r o i d s act d i r e c t l y both on the p i t u i t a r y and i n the brain. Evidence that estrogen exerts a negative feedback e f f e c t on, i . e . i n h i b i t s , gonadotropin secretion i s based i n part on c a s t r a t i o n studies i n which removal of ovaries r e s u l t s i n -13-hypersecretion of gonadotropins and hypertrophy of the p i t u i t a r y . Dorner (1976) postulates that gonadotropin function i s kept within physiological, l i m i t s by negative feedback of estrogen. Implanting estrogen into the median eminence of i n t a c t animals leads to ovarian atrophy. It i s considered that a tonic secretion of gonadotropins i s maintained by estrogen action at the medial basal hypothalamus (Ganong, 1977). The s i t u a t i o n regarding the negative feedback of estrogen during parts of the estrous cycle i n the i n t a c t animal i s l e s s c l e a r and i s brought into question by P e l l e t i e r and Thimonier (1975) and Hauger, et a l , (1977) on the basis that estrogen l e v e l changes p a r a l l e l LH changes f o r portions of the cycle. It i s evident that the presence or absence of various 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 to, estrogen must be con-sidered i n i n t e r p r e t i n g the o v e r a l l sequence of p h y s i o l o g i c a l events. During the early f o l l i c u l a r phase, a tonic secretion of GnRH, regulated by the basomedial (tuberoinfundibular) region of the hypothalamus, occurs which stimulates the adenohypophysis to release gonadotropins. FSH predominates and f o l l i c u l a r maturation i s induced (Dorner, 1976; Ladosky and Wandscheer, 1975). At t h i s time the hypothalamic content of LHRH, and the p i t u i t a r y LH content, are lowest (Stelmasiak and Cumming, 1977). In the l a t e .fol'11'c-ular, preovulatory phase, the p o s i t i v e feedback e f f e c t of estrogen on the preovulatory LH surge and on behavioural estrus, i s c l e a r l y established. Scaramuzzi (1976) passively immunized ewes against endogeneous e s t r a d i o l -17 3 and noted the following e f f e c t s . Almost one-third of the treated ewes did not ovulate although they showed normal estrous behaviour. Their ovaries contained large a t r e s i c f o l l i c l e s with marked hypertrophy of the theca interna, and a non-existent granulosa layer. Of those ewes that did ovulate, about one--14-h a l f also showed the same f o l l i c u l a r abnormalities. Treated ewes also had many new corpora lutea i n d i c a t i n g an increased number of developing f o l l i c l e s . These observations c o r r e l a t e with evidence that estrogen i s involved i n a negative feedback loop on p i t u i t a r y gonadotropin secretion. Interference with t h i s feedback res u l t e d i n hypersecretion of FSH and LH, with a r e s u l t i n g hyper-secretion of e s t r a d i o l as indicated by the hypertrophy of thecal c e l l s . Also of i n t e r e s t i n t h i s study was the separation of estrogen e f f e c t s on ovulation from those on estrous behaviour, i n d i c a t i n g that the threshold f or estrogen to induce estrus i s lower than that involved i n negative feedback on gonado-tro p i n release, and also lower than that required to induce the preovulatory LH surge, a p o s i t i v e estrogen feedback e f f e c t . When ewes were a c t i v e l y immunized against e s t r a d i o l -17 3 there was complete absence of estrous behaviour (Cox and Wilson, 1976; Rawlings, et a l , 1978; Pant, et a l , 1978). Ewes a c t i v e l y immunized against estrogen had large Graafian f o l l i c l e s , some of which were haemorrhagic (Rawlings, et a l , 1978). Plasma LH l e v e l s appeared to p a r a l l e l the increase i n serum antibody t i t r e (Rawlings, et a l , 1978) and elevated LH l e v e l s were lowered by a high dose of s t i l b o e s t r o l dipropionate (Pant, et a l , 1978). FSH l e v e l s showed only minor o s c i l l a t i o n s and d i f f e r e n t i a l e f f e c t s to immunization (Pant, et a l , 1978). The corpus luteum i s associated with the absence of ovulation and estrus. Formation of the corpus luteum r e s u l t s i n increased progesterone secretion which suppresses the c y c l i c LH secretion v i a negative feedback (Hendricks and Mayer, 1977). P r o l a c t i n may also be required f or corpus luteum secretory a c t i v i t y (Robertson, 1977). Progesterone may also regulate tonic LH secretion by a l t e r i n g the frequency of p u l s a t i l e release (Yuthasatrakol, et a l , 1977). The negative feedback of progesterone on tonic LH release allows p i t u i t a r y LH stock to b u i l d up ( P e l l e t i e r and Thiomonier, 1975). -15-At l u t e a l regression, progesterone l e v e l s f a l l and thereby expose the p i t u i t a r y to r i s i n g estrogen l e v e l s rendering the stored LH releasable ( S t e l -masiak and Cumming, 1977). In the l a t e f o l l i c u l a r phase, the f i n a l maturation of f o l l i c l e s takes place and the increasing estrogen secretion exerts a p o s i t i v e feedback on the female d i f f e r e n t i a t e d , amygdalar-hypothalamus system (Dorner, 1976). The preovulatory surge of gonadotropins evoked by r i s i n g estrogen l e v e l s requires an i n t a c t hypothalamohypophysial un i t . I t appears that the p o s i t i v e feedback e f f e c t of estrogens on the preovulatory LH peak i s f a c i l i -tated by preceding progesterone l e v e l s . This may be due to induction of receptor synthesis which w i l l be discussed i n a l a t e r section. Stelmasiak and Cumming (1977) postulate that high prog esterone l e v e l s f a c i l i t a t e a "priming e f f e c t mechanism" on the p i t u i t a r y , which, by preventing LH secretion by estrogen waves during the l u t e a l phase, allow the b u i l d up of p i t u i t a r y LH stores. It appears that progesterone as w e l l as estrogen i s responsible for the LH peak and that steroids most l i k e l y i n t e r a c t with neurotransmitter-regulating mechanisms at extra- and intra-hypothalamic l e v e l s (Wuttke, 1976). The preovulatory surge of gonadotropins could be a r e s u l t of estrogen action d i r e c t l y on the brain to cause changes i n the discharge of r e l e a s i n g hormones or d i r e c t l y on the p i t u i t a r y gonadotrophs to a l t e r gonadotropin release e i t h e r by a l t e r i n g secretory a c t i v i t y or by causing changes i n sen-s i t i v i t y to hypothalamic r e l e a s i n g hormones (Finn and Booth, 1977). The hypothalamo-hypophysial p o r t a l system presents t e c h n i c a l d i f f i c u l t i e s f o r sampling to determine the pattern of GnRH release. Sampling of ewe jugular blood to determine the GnRH-like immunoreactivity i n plasma, J u t i s z , et a l , (1973) found the highest l e v e l (6ng/ml) during the preovulatory surge of LH and FSH. These workers did not detect GnRH ( i . e . le s s than 0.5 ng/ml) i n plasma from outside the estrous period. Crighton, et a l , (1973) sampling -16-at estrus on a more frequent basis, found that GnRH showed a p u l s a t i l e release pattern with peak i n t e r v a l s of 1.5 to 6 hours. Foster, et a l , (1974, 1976) sampling ewes v i a jugular cannulae every two hours for twenty days i n the breeding season detected GnRH peaks at various times of the cycle. Peaks increase i n frequency near the end of the cycle with peaks detected before, during and a f t e r the preovulatory LH peak. A f t e r estrus, and at day ten of the cycle, GnRH peaks were unassociated with LH release. The h a l f l i f e of GnRH i s estimated at f i v e minutes (Crighton, et a l , 1973) however i t may be even shorter (Foster, 1974). Although a sing l e i n j e c t i o n of GnRH r e s u l t s i n an LH peak, the amplitude and duration of t h i s peak i s much less than that which occurs during the cycle. M u l t i p l e i n j e c t i o n s to imitate the episodic release of GnRH, resulted i n an LH peak approaching that of a normal cycle, implying that i t i s the frequency of GnRH stimulation that brings.about•the preovulatory LH surge (Crighton, et a l , 1974; Foster, et a l , 1976). There i s now considerable evidence that gonadal steroids a l t e r the response of the p i t u i t a r y to GnRH (Geschwind, 1972; Aiyer and Fink, 1974; Reeves, et  a l , 1970; Schally, et a l , 1973b). Exogenous estrogen or GnRH treatment shows greater e f f e c t on LH release at the onset of behavioural estrus than at any other time i n the cycle (Reeves, et a l , 1970). However the p i t u i t a r y response to GnRH may be due to the plasma estradiol/progesterone r a t i o rather than a response to estrogen alone ( P e l l e t i e r and Thimonier, 1975). The mechanisms by which estrogen exerts i t s e f f e c t s on gonadotropins may involve . protein synthesis but they are poorly understood (Mahesh and McPherson, 1977). E s t r o -gen appears to have some stimulating e f f e c t on the binding capacity of GnRH receptors (Park, et a l , 1976). A negative e f f e c t of estrogen on p i t u i t a r y response to GnRH was not demonstrated by physiologic doses however pharma-c o l o g i c a l i n f u s i o n completely i n h i b i t e d p i t u i t a r y LH/FSH response to GnRH. -17-(Eshkol, et a l , 1975; Gual, et a l , 1975). Estrogen e f f e c t s on extrahypothalamic and intrahypothalamic s i t e s have also been demonstrated. E l e c t r i c a l stimulation of the medial preoptic area leads to gonadotropin release however the magnitude of the response i s re l a t e d to the stage of the estrous cycle, being greatest around estrus (Wuttke, 1976). T a l e i s n i k and Beltramino (1975) state: "...ovarian steroids not only may a l t e r the thresholds of e x c i t a b i l i t y of extrahypothalamic structures but also may determine antagonistic e f f e c t s on the hippocampus and amygdala". Estrogens have been shown to influence enzyme a c t i v i t y i n the p i n e a l , and progesterone i s antagonistic to the e f f e c t of estrogen. High doses of estrogen i n h i b i t hydroxyindole-O-methyl transferase (HIOMT), a key enzyme i n melatonin synthesis, and low doses of estrogen stimulate HIOMT a c t i v i t y . HIOMT a c t i v i t y i s two f o l d higher at diestrus than at estrus i n mature c y c l i n g rats (Preslock, 1977). ' L i t t l e i s known about the feedback mechanisms involved (Kappers, 1976). A summary of the various i n t e r r e l a t i o n s h i p s discussed i s i l l u s t r a t e d below, adapted from Dorner, 1976. The p o s i t i v e and negative feedback loops are not shown. -18-ENVIRONMENT signals SENSE ORGANS neural EXTRAHYPOTHALAMIC BRAIN STRUCTURES A)cortex, B)limbic system, C)pineal D)medial preoptic area, E)mesencephalon neural (neurotransmitter) HYPOTHALAMUS K GnRH ADENOHYPOPHYSIS Gonadotropins (FSH and LH) GONADS sex hormones SECONDARY SEX ORGANS neural-F. ESTROGEN RECEPTORS It became evident i n studies of sex hormones that target c e l l s must possess a mechanism f o r recognizing and d i s t i n g u i s h i n g the various s t e r o i d hormones. The search f o r the recognition system, termed "receptors" was car r i e d out on many fronts; greatly a s s i s t e d by the technology of radi o a c t i v e l a b e l l i n g of steroids to high s p e c i f i c a c t i v i t y enabling i d e n t i f i c a t i o n of hormones i n very low p h y s i o l o g i c a l amounts. I n i t i a l studies involved i n j e c t i n g r a d i o l a b e l l e d hormone, i n vivo, and then l o c a t i n g the hormone d i s t r i b u t i o n , the target organs being those showing s e l e c t i v e retention (Jensen, et a l , 1972). Further examination of d i s t r i b u t i o n was c a r r i e d out eit h e r i n s i t u , -19-by autoradiography, or by su b c e l l u l a r analysis using density gradient c e n t r i -fugation. It i s now w e l l established that s t e r o i d hormones e l i c i t target organ responses by i n t e r a c t i n g with cytoplasmic receptors, which are macromolecular proteins, to form a receptor-hormone complex. The complex undergoes a trans-formation process and t r a v e l s to the nucleus where i t i n t e r a c t s with the chrom-a t i n and brings about t r a n s c r i p t i o n a l changes and the formation of mRNA (reviews: Gorski and Gannon, 1976; Baulieu, et a l , 1975). The model system f o r estrogen action i s the uterus. Many of the biosyn-t h e t i c changes induced by estrogen acting on t h i s system have been elucidated and i n p a r t i c u l a r the synthesis of a s p e c i f i c protein, "induced p r o t e i n " which i s an early event i n estrogen action (Katzenellenbogen and Gorski, 1975). In order f o r a c e l l u l a r component to be designated a receptor, i t should exhibit c e r t a i n c h a r a c t e r i s t i c s among which are: (Clark, et a l , 1977; Baulieu, et a l , 1975) 1. f i n i t e or saturable binding capacity. 2. high a f f i n i t y within p h y s i o l o g i c a l ranges, as c i r c u l a t i n g l e v e l s -9 -10 of estrogen are usually 10 -10 M. This implies a high a f f i n i t y constant (K^) of the receptor f o r estrogen. 3. s p e c i f i c i t y for p a r t i c u l a r s t e r o i d hormones or clas s of hormones. 4. ti s s u e s p e c i f i c i t y i n that target organs are the only ones stimu-l a t e d . 5. c o r r e l a t i o n of macromolecular binding with a b i o l o g i c a l response. Autoradiographic studies have l o c a l i z e d estrogen i n the p i t u i t a r y and brain although there are d i f f e r e n t i n t e n s i t i e s i n d i s t r i b u t i o n throughout target tissues (Stumpf, 1970; Stumpf, 1971a; Stumpf, 1971b). -20-I. P i t u i t a r y L o c a l i z a t i o n 3 Stumpf (1971a) found the H-estradiol concentrated not only i n basophils, but also i n acidophils and chromophobes of the pars d i s t a l i s . No l o c a l i z a t i o n occurred i n the pars intermedia and very l i t t l e i n the neurohypophysis. A combined autoradiographic-immunohistochemical technique by Keefer, et a l , (1975) showed that most but not a l l gonadotrophs, as i d e n t i f i e d with anti-HCG, showed nuclear retention of e s t r a d i o l . I I . Central Nervous System Retention Stumpf(1970, 1971b) summarized the major areas of estrogen accumulation i n neurons. They are concentrated i n the preoptic area of the hypothalamus, the basal tuberal region of the hypothalamus, and i n the c e n t r a l - p o s t e r i o r amygdala. Stumpf and Sar (1976) also include the o l f a c t o r y lobe and tubercle and smaller portions of the septum and hippocampus. These areas correspond to those reported by P f a f f (1973b). Stumpf and Sar (1976) suggest that differences i n apparent uptake by c e l l s i n d i f f e r e n t tissues r e f l e c t differences i n t i s s u e response which they formulate as the " p r i n c i p l e of d i f f e r e n t i a l hormone uptake and response threshold". Under p h y s i o l o g i c a l conditions c e r t a i n target t i s s u e s may be stimulated while others are not (also Rosner, et a l , 1972). They suggest such a d i f f e r e n t i a l uptake and response may be an important factor i n regulating endocrine feedback systems and behavioural responses. Estrogen receptors have been most extensively studied i n the uterus, how-ever s i m i l a r proteins have been i d e n t i f i e d i n the p i t u i t a r y , hypothalamus and other tissues (Liao, 1975). Kato (1977) has recently reviewed the character-i s t i c s of s t e r o i d hormone receptors i n the brai n , hypothalamus and p i t u i t a r y . I I I . Estrogen Receptors of the P i t u i t a r y Several workers have demonstrated that the anterior p i t u i t a r y shows a -21-p r e f e r e n t i a l uptake of estrogen both i n vivo (King, et a l , 1965; Kato and V i l l e e , 1967a) and i n v i t r o (Kato, 1970b; E i s e n f e l d , 1970). Uptake k i n e t i c s were s i m i l a r to the uterus (King, et a l , 1965). The s p e c i f i c i t y of the e s t r o -3 gen binding was demonstrated by displacement of H-estradiol by unlabelled e s t r a d i o l , estrone, e s t r i o l , a non-steroid e s t r o g e n - d i e t h y l s t i l b e s t r o l , and a non-steroid estrogen antagonist-clomiphene (Eisenfeld and Axelrod, 1966; E i s e n f e l d and Axelrod, 1967; Kato and V i l l e e , 1967b; Kato, et a l , 1968; Kato, 1970b; Kahwanago, et a l , 1970). Labelled e s t r a d i o l was found associated with the nuclear f r a c t i o n , both i n vivo (King, et a l , 1965) and i n v i t r o ( L e a v i t t , et a l , 1969; Friend and L e a v i t t , 1972; Anderson, et a l , 1973). Characterization of the estrogen receptors of the anterior p i t u i t a r y by sedimentation gradients, binding s p e c i f i c i t y , and determination of a s s o c i a t i o n (K^) and d i s s o c i a t i o n (KJJ) constants, ind i c a t e that the p i t u i t a r y receptors show very s i m i l a r char-a c t e r i s t i c s to the uterine receptors (Notides, 1970; L e a v i t t , et a l , 1973). IV. Estrogen Receptors of the Hypothalamus The p i t u i t a r y has been shown to contain ten times the number of binding s i t e s of the hypothalamus (Clark, et a l , 1972; Ginsburg, et a l , 1975a; Muldoon, 1977). This i s r e f l e c t e d i n the amount of l a b e l l e d e s t r a d i o l p r e f e r e n t i a l l y taken up by the hypothalamus (Eisenfeld and Axelrod, 1966; Kato and V i l l e e , 1967a; Ei s e n f e l d and Axelrod, 1967; E i s e n f e l d , 1970; Kahwanago, et a l , 1970). Hypothalamic estrogen receptors show s i m i l a r s p e c i f i c i t y c h a r a c t e r i s t i c s to p i t u i t a r y receptors (Kato and ViUee, 1967b; Kato, et a l , 1968). Labelled e s t r a d i o l i s found associated with the nucleus component i n the hypothalamus (Clark, et a l , 1972; Anderson, et a l , 1973; Zigmond and McEwen, 1970). Estrogen receptors occur predominately i n the preoptic-anterior hypothalamus and i n the median eminence region (Kato and V i l l e e , 1967a; Kato, 1973; Ginsburg, et  a l , 1975b). The molecular c h a r a c t e r i s t i c s of the hypothalamus receptors as -22-determined by g e l f i l t r a t i o n , density gradient c e n t r i f u g a t i o n , displacement studies, etc., are s i m i l a r to uterus and p i t u i t a r y receptors ( E i s e n f e l d , 1970; Kahwanago, et a l , 1970). V. Estrogen Receptors of Extrahypothalamic Brain Structures It has been more d i f f i c u l t to demonstrate macromolecular receptors i n other parts of the brain. Although Rosner, et a l , (1972) did not i s o l a t e a s p e c i f i c receptor from the cerebral cortex, they found that estrogen s i g n i f -3 i e a n t l y increased H-cytidine incorporation into RNA as w e l l as increased protein synthesis. Ginsburg, et a l , (1975b) determined that the amygdala contains one hundred times fewer binding s i t e s than the p i t u i t a r y . Few studies on estrogen binding i n the brain have included the pinea l gland. Rosner, et a l (1972) found the uptake of e s t r a d i o l by the p i n e a l to be s i g n i f i c a n t l y higher on a per milligram wet tiss u e basis than that of the uterus i n ovariectomized r a t s . Marks, et a l (1972) determined that the binding 3 of H-estradiol by the macromolecular f r a c t i o n of the pinea l c y t o s o l approached that bound by the anterior p i t u i t a r y , and was about four times that bound by II the hypothalamus on a "disintegration-per-minute per milligram protein basis (DPM/mg. pr o t e i n ) . VI. Binding Sites and the Estrous Cycle 3 The uptake of H-estradiol by p i t u i t a r y and hypothalamus v a r i e s with the stage of the estrous cycle (Kato, 1970a). I t has been shown that the number of high a f f i n i t y estrogen binding s i t e s f l uctuates with the cycle (Ginsburg, et a l , 1975a). A c o r r e l a t i o n between estrogen receptor content and gonadotropin c y c l i c i t y was shown by Parker, et a l , (1976) who found that the depletion of cytosol receptors was followed by the gonadotropin surge. The number of estrogen binding s i t e s i s re l a t e d to the p i t u i t a r y response to GnRH (Greeley, et a l , 1975a,b). At proestrus the cyt o s o l receptor content of the rat an t e r i o r p i t u i t a r y -23-decreases f o r t y percent, and then i s replenished during l a t e proestrus and estrus (Greeley, et a l , 1975b). The p i t u i t a r y response to exogenous GnRH during proestrus p a r a l l e l s the changes i n the content of estrogen receptor i n the cytoplasm and nucleus (Sen and Menon, 1978). VII. P i t u i t a r y and Brain Receptors and the "Steroid Model" The p i t u i t a r y and b r a i n estrogen receptors f i t the s t e r o i d model of the uterus with respect to depletion and replenishment pattern (Cidlowski and Muldoon, 1974; Ginsburg, et a l , 1975a). Although the appearance of "induced pro t e i n " has not been demonstrated there are some in d i c a t i o n s that protein synthesis i s required for estrogen e f f e c t s i n these tissues (Schneider and McCann, 1970). The r e s t o r a t i o n of l o r d o s i s behaviour i n r a t s requires twenty-four hours thereby implying that neurons are acting as s t e r o i d target c e l l s , that i s , responding to estrogen signals by biochemical events l i k e protein synthesis (Beyer, 1976). VIII. Receptor U n i v e r s a l i t y vs. Tissue S p e c i f i c i t y Although the estrogen receptors of the p i t u i t a r y and brain show many c h a r a c t e r i s t i c s s i m i l a r to uterine receptors there may be t i s s u e s p e c i f i c c h a r a c t e r i s t i c s as yet undetected. Ginsburg, et a l , (1975a) found that the d i s s o c i a t i o n constants (K Q) f o r p i t u i t a r y tissues were c o n s i s t e n t l y lower than those obtained from hypothalamus and uterus t i s s u e . The rates of reaction to reach equilibrium were also d i f f e r e n t . It took uterus and hypothalamus less than f i v e minutes (30°C) while p i t u i t a r y , cortex, and amygdala took more than ten minutes (30°C) (Ginsburg, et a l , 1975b). Further evidence that p i t u i -tary receptors may be d i f f e r e n t i s presented by Ginsburg, et a l , (1976b) who estimated the a f f i n i t y constants for eleven compounds using cytosols from f i v e d i f f e r e n t t i s s u e s . The ranking orders were the same, however there were differences between the tissues i n the absolute values. There was a tendency -24-toward higher a f f i n i t y i n the p i t u i t a r y , s i g n i f i c a n t l y so f o r seven of the eleven compounds. I t i s speculated that i n the p i t u i t a r y there i s an a d d i t i o n a l estrogen binding moiety of even higher a f f i n i t y of such low quantity as to be undetected by Scatchard a n a l y s i s . As yet, whether estrogen receptors are iden-t i c a l throughout the reproductive system remains unresolved. Evidence both i n favour of, and against, t i s s u e s p e c i f i c i t y of estrogen receptors i s presented by Kato (1977). IX. S t r u c t u r a l Requirements f or Binding There are c e r t a i n chemical c h a r a c t e r i s t i c s of estrogenic steroids which are recognized by estrogen receptors i n target c e l l s . The binding of a hormone to the receptor to form an activated (transformed) receptor hormone complex, which then translocates to the nucleus, involves various types of chemical i n t e r a c t i o n s between the protein receptor and s t e r o i d hormone. Hydrophobic in t e r a c t i o n s , hydrogen bonding, and s t e r i c f a c t o rs a l l play a part i n high a f f i n i t y , s p e c i f i c binding (King and Mainwaring, 1974). An estrogenic s t e r o i d i s mainly non-polar i n nature and therefore hydrophobic i n t e r a c t i o n s are import-ant i n binding, the s p e c i f i c i t y being determined by the s p a c i a l arrangements of polar substituents (Liao, et a l , 1973; King and Mainwaring, 1974). In order f o r a compound to exhibit estrogenic a c t i v i t y the presence of at l e a s t one aromatic r i n g i s considered c r i t i c a l (Frieden, 1976). The binding a f f i n i t y of a s t e r o i d i s strongly dependent on the presence of a phenolic hydroxyl, and on substituents on r i n g D (Korenman, 1969). The highest a f f i n i t y occurs f o r a s t e r o i d with a phenolic hydroxyl group on C 3 and an a l c o h o l i c hydroxyl at C 17 i n a g-configuration (Hahnel, et a l , 1973). A 17 3 hydroxyl on r i n g D i s common to both androgen and estrogen steroids (Busetta, et a l , 1977). The presence of an oxygen function on r i n g D i s important i n binding, and i t s state of oxidation and p o s i t i o n influences binding a f f i n i t y (Hahnel, -25-et a l , 1973). Binding a f f i n i t y i s reduced i f the C 17 hydroxyl i s changed from 3 to « configuration or i f the 17 3 hydroxyl i s oxidized to a ketone, as i n estrone (King and Mainwaring, 1974). Binding a f f i n i t y may be enhanced by c e r t a i n D r i n g s u b s t i t u t i o ns such as i n e t h i n y l e s t r a d i o l and estrone acetate (Korenman, 1969). The receptor binds the D r i n g loose enough to permit c e r t a i n molecular f l e x i b i l i t y (Hospital, et a l , 1975) , and may i n fa c t form two hydrogen bonds with the 17 3 hydroxyl (Busetta, et a l , 1977). The d i f f e r -ence between estrogenic and androgenic steroids occurs i n the A r i n g , which i s f u l l y saturated or contains a C 4 - C 5 double bond i n androgens, and i s aromatic i n estrogens (Busetta, et a l , 1975). The free phenolic hydroxyl on r i n g A i s e s s e n t i a l and i t s p o s i t i o n i s of c r i t i c a l importance for estrogen a c t i v i t y (Hahnel, et a l , 1973; King and Mainwaring, 1974). Hormone s p e c i f i c i t y i s assigned to the A r i n g ; however Busetta, et a l (1977) speculate that i t i s only the oxygen substituent at C 3 and not the aromatic nature of the r i n g that i s important. Removal of the C 3 hydroxyl blocks binding (King and Main-waring, 1974) while C 3 methylation r e s u l t s i n a reduction i n binding a f f i n i t y (Korenman, 1969; Geynet, et a l , 1972; King and Mainwaring, 1974) and a c e t y l a -t i o n of the C 3 hydroxyl retains a c t i v i t y (Korenman, 1969). Substitutions on C 2 are i n h i b i t o r y (Korenman, 1969). Addition of a l k y l groups or oxygen functions to the s t e r o i d leads to le s s a c t i v e compounds (Frieden, 1976; Korenman, 1969). . i A C 11 3 hydroxyl or C 16 hydroxyl diminishes binding (King and Main-waring, 1974; Korenman, 1969). A d d i t i o n a l unsaturation on the estrogen s t e r o i d nucleus i n h i b i t s binding (Korenman, 1969; Hahnel, et a l , 1973). C 7 meth-oxylation (Geynet, et a l , 1972) or the presence.or absence of an angular methyl group on C 13 (Hahnel, et a l , 1973) have l i t t l e influence on binding. In general, a d d i t i o n a l oxygen functions on r i n g D, a d d i t i o n a l s u b s t i t u t i o n s on r i n g A, and unsaturation of r i n g B, decrease a f f i n i t y (Hahnel, et a l , 1973). -26-In order to bind to the estrogen receptor a s t e r o i d must possess two p o t e n t i a l o hydrogen bonding groups about 15 A apart at e i t h e r end of the molecule (King and Mainwaring,. 1974). Hahnel, et a l , (1973) consider that the s t e r o i d attaches to the binding s i t e f i r s t through a hydrogen bond at the C 3 hydroxyl which may induce a s t e r e o s p e c i f i c conformational change i n receptor to permit the C 17 3 hydroxyl attachment through a strong hydrogen bond at a l e s s s p e c i f i c s i t e . Busetta, et a l (1977) however, consider the f i r s t attachment to be v i a the D r i n g with estrogenic or androgenic properties r e l a t e d to the o r i e n t a t i o n o of the A r i n g substituents, a d i f f e r e n c e of approximately 3 A. They state that a compound with a D type r i n g and with the same thickness of a s t e r o i d "would have the corresponding a c t i v i t y of an estrogen or androgen depending on the l o c a t i o n of the terminal atom of the A r i n g " . Korenman (1969) i n studying various compounds found that, with some exceptions, the r e l a t i v e binding a f f i n i t y of a compound to the uterine receptor p a r a l l e l s the uterotro-phic properties of that compound. X. Non Steroid Estrogens: Antiestrogen Properties Non s t e r o i d compounds possessing some of the c h a r a c t e r i s t i c s mentioned above also exhibit varying degrees of e s t r o g e n i c i t y (Geynet, et a l , 1972). A potent non s t e r o i d estrogen, d i e t h y l s t i l b e s t r o l (DES), binds to the uterine estrogen receptor with equal or greater a f f i n i t y than e s t r a d i o l (Hospital, et  a l , 1972). A number of classes of compounds which can bind to the same, or part of the same, receptor binding s i t e as does e s t r a d i o l 17 B can i n t e r f e r e with or compete with e s t r a d i o l f or binding s i t e s when both are present. The various classes of non s t e r o i d compounds with these properties have been reviewed by Geynet, et a l , (1972), Frieden (1976) and others (Lunan and Klopper, 1975; Baulieu, 1976). Many of these compounds can also exhibit agonist, that i s , estrogenic c h a r a c t e r i s t i c s , often.to a l e s s e r extent than e s t r a d i o l . -27-Because these compounds can i n t e r f e r e with normal estrogen action i n v i v o , they f a l l into the broad catagory termed antiestrogens. Antiestrogenic sub-stances have been defined as compounds which i n t e r f e r e with any actions of an endogenous estrogen l i k e e s t r a d i o l 17 3 or estrone (Sankaran and Prasad, 1972). These agents can be subdivided further into two catagories: a) those which have no a f f i n i t y for estrogen receptors, that i s , do not compete with e s t r a d i o l f o r binding s i t e s , and i n h i b i t estrogen a c t i o n through means other than the estrogen receptor mechanism; and b) those that i n t e r f e r e with estrogen action d i r e c t l y through the receptor mechanism by binding to the cytoplasmic receptor thereby i n h i b i t i n g the uptake of e s t r a d i o l (Rochefort, et a l , 1972). Examples of the former group, (a), are androgens, progestagens and enzyme i n h i b i t o r s . Examples of the l a t t e r group, (b), are numerous, and i n many cases these com-pounds mimic the b i o l o g i c a l actions of e s t r a d i o l and are competitive i n h i b i t o r s of estrogen binding i n target t i s s u e s (Sankaran and Prasad, 1972). I t i s the l a t t e r type of compound that can bind to the estrogen receptors which i s of i n t e r e s t to t h i s discussion. It i s becoming in c r e a s i n g l y evident that i t i s not the i n i t i a l competition for estrogen binding s i t e s i n target t i s s u e alone, but the subsequent chain of events which occurs, that determines the a n t i - e s t r o g e n i c i t y of a compound (Clark, et a l , 1974; Cidlowski and Kuldoon, 1976; Baulieu, 1976). In the target c e l l there are several p o t e n t i a l mechanisms of a c t i o n or points of interference by antiestrogens. These are outlined i n Figure 1, (Jordan, et a l , 1978a). Competitive antagonism, 1, could occur for the cytoplasmic receptor. Such competition would be a function of the r e l a t i v e a f f i n i t i e s and concentrations of estrogen and competitor. This p o s s i b i l i t y i s considered a s i m p l i s t i c explan-at i o n of antiestrogen a c t i v i t y (Clark, et a l , 1974; Cidlowski and Muldoon, 1976; Jordan, et a l , 1978a). -28-Antagonists could i n h i b i t the transformation, 2, to the activated receptor hormone complex. This mechanism has yet to be demonstrated. It i s possible that the tr a n s l o c a t i o n to the nucleus, 3, of the activated complex could be i n h i b i t e d , however compounds with anti-estrogenic a c t i v i t y have been shown to induce t r a n s l o c a t i o n (Rochefort, et a l , 1972). It appears that there i s not ne c e s s a r i l y a common mechanism for a l l classes of antiestrogens and attempts have been made to c l a s s i f y groups of antiestrogenic compounds on the basis of length of nuclear retention (Clark, et a l , 1977b) (see Table I ) . a. long acting antiestrogens The most studied group of antiestrogenic compounds i s the triphyenylethylene derivatives which includes clomiphene, tamoxifen, CI 628, and nafoxidine (see Appendix). These compounds have been termed "long a c t i n g " antiestrogens i n that they cause abnormally long nuclear retention of the receptor complex (Clark, et a l , 1977b). I t was postulated that t h i s retention could i n t e r f e r e with cytoplasm estrogen receptor resynthesis, 4, and/or replenishment (Clark, et a l , 1974). However, these compounds also have long plasma h a l f l i v e s , e.g. tamoxifen tj i s several days, and thus even i f resynthesis of cytoplasmic receptors does occur, as indicated by Koseki, et a l , (1977), l i t t l e accumula-t i o n of receptors i n the cytoplasm would r e s u l t as there would be immediate tra n s l o c a t i o n to the nucleus (Jordan, et a l , 1977b). Thus the ti s s u e becomes re f r a c t o r y to subsequent estrogen stimulation. Koseki, et a l , (1977), suggest that the i n a b i l i t y of the c e l l to process or remove the receptor/antiestrogen complex from the nucleus may somehow i n t e r f e r e with continuous uterine estrogen stimulation. b. short acting antiestrogens Of d i r e c t i n t e r e s t to t h i s discussion are the compounds which have been FIGURE 1: POTENTIAL MECHANISMS OF ACTION OF ANTIESTROGENS IN THE TARGET TISSUE CELL 3 CYTOPLASM NUCLEUS estrogen receptor + -> \ estrogen \ \ \ \ transformed receptor complex receptor resynthesis (24 hours) ^ -1. competitive antagonism 2. i n h i b i t i o n of transformation 3. i n h i b i t i o n of tr a n s l o c a t i o n 4. i n h i b i t i o n of receptor resynthesis a: as from Jordan, et a l , 1978a. TABLE I: PROPOSED CLASSIFICATION SYSTEM, ESTROGEN AGONISTS AND ANTAGONISTS3 CLASS EXAMPLES NUCLEAR RETENTION PHARMACOLOGIC CHARACTERISTICS UTEROTROPHIC PROPERTIES 1. short acting e s t r i o l DMS 16 oxo-estradiol short (1-4 hr) p a r t i a l agonist/ antagonist when i n j ec t ed. Agoni s t when implanted. early responses long acting A. e s t r a d i o l DES intermediate (6-24 hr) Agonist early and l a t e responses B. t r i p h e n y l -ethylene d e r i v a t i v e s eg. Nafoxidine CI 628 Tamoxifen long acting greater than 24-48 hrs. Agonist - one in j ection. Antagonist -multiple i n j ections. early and l a t e responses Early responses: water imbibition, hyperemia, amino acid and nucleotide uptake, a c t i v a t i o n of RNA poly-merase I and I I , stimulation of induced protein. Late responses: c e l l u l a r hypertrophy and hyperplasia, sustained RNA polymerase II and II a c t i v i t y C l a s s i f i c a t i o n based on events that occur a f t e r a single i n j e c t i o n of the compound. a: as given i n Clark, et a l , 1977b. -31-termed "impeded", based on uterotrophic e f f e c t s (Terenius and Ljungkvist, 1972) or "short acting", based on s u b c e l l u l a r retention (Clark, et a l , 1977b). These compounds bind to the cytoplasmic receptor and translocate to the nucleus, however t h e i r nuclear retention i s of short duration, and although early events of estrogen stimulation are induced, l a t e r events are not, thereby demonstrat-ing the need for sustained estrogen stimulation for f u l l estrogenic e f f e c t (Lan and Katzenellenbogen, 1976). If these compounds are administered repeat-edly, thereby sustaining plasma concentration for longer periods they can bring about f u l l estrogen stimulation (Baulieu, 1976). Examples of compounds i n t h i s category are e s t r i o l (Lan and Katzenellenbogen, 1976) and d i m e t h y l s t i l -b e s t r o l (DMS) (Capony and Rochefort, 1977). Capony and Rochefort, (1977), 3 using H-DMS determined d i r e c t l y that the receptor-DMS a s s o c i a t i o n rate was slower and d i s s o c i a t i o n rate f a s t e r than f o r the e s t r a d i o l - r e c e p t o r complex. DMS translocated to the nucleus, however the nuclear retention was short. Katzenellenbogen, et a l , (1978), compared the actions of d i e t h y l s t i l b e s t r o l (DES) and DMS. This study showed that both DES and DMS r a p i d l y translocated receptor to the nucleus but nuclear receptor l e v e l s r a p i d l y returned to control l e v e l by 6 hours with DMS while they remained elevated with DES. Methyl-ati o n of DMS prolonged i t s e f f e c t and the methyl ether become much more l i k e DES. c. differences i n target t i s s u e responses As indicated, the estrogenic or antiestrogenic properties of compounds have been characterized i n the uterine system; depending on physiologic s t a t e , compounds can act e i t h e r as agonists or as antagonists to e s t r a d i o l . Few studies of antiestrogen action i n other target tissues have been reported; however, there are i n d i c a t i o n s that there may be t i s s u e differences i n response to these compounds. Cidlowski and Muldoon, (1976), studying DMS, CI 628 and -32-MER 25, (a t r i a r y l a l k a n e estrogen antagonist with no agonist properties) i n anterior p i t u i t a r y , hypothalamus and uterus found the same order of binding i n h i b i t i o n f o r a l l tissues but the degree of i n h i b i t i o n v a r i e s with t i s s u e , p a r t i c u l a r l y with the long acting compounds. Luine and McEwen (1977) studied the e f f e c t of CI 628 on various estrogen-affected enzyme systems i n the uterus, brain and p i t u i t a r y . As an agonist CI 628 equalled e s t r a d i o l i n a l t e r i n g enzyme a c t i v i t y i n the brain, ( i . e . increased acetyl-CoA-:choline-0-acetyl transferase, E.C.23.3.1.6., CAT; and decreased monoamine:02 oxidoreductase, E.C.I.4.3.4, MAO), however was less e f f e c t i v e (approximately one-half that of e s t r a d i o l ) on increasing G6PDH (D-glucose-6-phosphate:NADPH+ oxidoreductase, E.C.1.1.1.49) a c t i v i t y i n the uterus. CI 628 showed even les s e f f e c t i n the p i t u i t a r y , i n that, while e s t r a d i o l doubled G6PDH a c t i v i t y , there was no s i g n i f -icant change with CI 628. As an antagonist CI 628 did decrease nuclear estrogen binding i n a l l three t i s s u e s , but the estrogen binding recovery times were d i f f e r e n t . The p i t u i t a r y and uterus were slower to recover than the hypothal-amus, preoptic and amygdala areas. Antagonist properties were d i f f e r e n t i n each t i s s u e . In the uterus and p i t u i t a r y CI 628 attenuated the e f f e c t of e s t r a d i o l on G6PDH a c t i v i t y whereas i n the brai n , CI 628 did not block estrogen changes to CAT or MAO. The authors concluded that CI 628 i s a more potent agonist and l e s s e r antagonist i n the brain than i n the p i t u i t a r y or uterus. Etgen and Whalen (1978) in d i c a t e that DMS induces normal l o r d o s i s behaviour i n rats at concentrations which are weakly estrogenic on uterine growth. DMS also i n h i b i t s compensatory ovarian hypertrophy at the same concentration. The e f f e c t s of these compounds on receptor replenishment and nuclear binding i n the brain and p i t u i t a r y are not yet c l e a r and i t i s not known i f they act i n the same manner as i n the uterus. -33-G. GENISTEIN AND COUMESTROL Two compounds which appear to act i n a s i m i l a r manner to e s t r i o l and DMS are genistein and coumestrol (Newsome and K i t t s , 1979). They are representatives of the classes of compounds known as isoflavones and coumestans, r e s p e c t i v e l y . These compounds are of p a r t i c u l a r i n t e r e s t as they occur i n plants but can produce estrogenic e f f e c t s i n animals, (review: Farnsworth, et a l , 1975), and have been termed phyto-estrogens. I. Binding Properties Genistein and coumestrol have been shown to bind to the uterine estrogen receptors of sheep (Shutt and Cox, 1972) and pregnant rabbit s (Shemesh, et a l , 1972). These compounds can induce estrogen-like a c t i v i t i e s i n the uterus including water imbibition and incorporation of l a b e l l e d precursors into protein, l i p i d and RNA (Notebloom and Gorski, 1963). They also bring about the synthesis of "induced p r o t e i n " i n the rat uterus, an early event c h a r a c t e r i s t i c of estrogen stimulation (Somjen and Kaye, 1976). Recently these compounds have been shown to bind to estrogen receptors i n human breast cancer c e l l s and they can enhance tumor c e l l p r o l i f e r a t i o n (Martin, et a l , 1978). The binding a b i l i t y of coumestrol coupled with i t s fluorescent c h a r a c t e r i s t i c s has made t h i s com-, pound a t o o l i n the study of binding properties of c a l f uterine estrogen recept-ors (Lee, et a l , 1977). Coumestrol shows greater a f f i n i t y f o r the uterine receptor than isoflavones although s t i l l l e s s than e s t r a d i o l . The reported values f o r coumestrol range from twenty times (Shutt and Cox, 1972) to seventy times (Shemesh, et a l , 1972) and genistein at 111 (Shutt and Cox, 1972) to 175 times (Shemesh, et a l , 1972) le s s e f f e c t i v e than e s t r a d i o l i n competitive studies. The ranking of various coumestan and isoflavone compounds on a r e l a t i v e binding a b i l i t y basis confirms the importance of phenolic groups to binding a f f i n i t y (Shutt and Cox, 1972, -34-Shemesh, et a l , 1972). The i n v i t r o binding a f f i n i t i e s of coumestrol and genistein are greater and closer together than was expected from t h e i r i n vivo uterotrophic potencies (Shutt and Cox, 1972). On a uterotrophic basis coumestrol i s 160 times and genistein 2500 times le s s e f f e c t i v e than e s t r a d i o l i n rats (Perel and Lindner, 1970). These compounds have been shown to i n h i b i t the uptake and utero-vagino-trophic response to e s t r a d i o l (Folman and Pope, 1966, 1969; Shutt, 1967). II . Presence i n Plants Plants containing phyto-estrogens have been implicated i n i n f e r t i l i t y problems i n grazing animals, i n p a r t i c u l a r sheep (reviews: B i c k o f f , 1968a; Braden and McDonald, 1970; Cox and Braden, 1974; Shutt, 1976; Livingston, 1978). The isoflavones are the major phyto-estrogens i n clover and the coumestans predominate i n the medics (Braden and McDonald, 1970). a. isoflavones The isoflavones genistein.and formononetin were i s o l a t e d from subterranean clover ( T r i f o l i u m subterraneum) following research into the causative agents of severe reproductive disorders i n sheep grazing subterranean clover i n A u s t r a l i a (Bradbury and White, 1951). The syndrome was characterized by symptoms of hyperestrogenic stimulation.therefore estrogen-like compounds i n the sub-terranean clover.were suspected. The permanent i n f e r t i l i t y which resulted from long term grazing of estrogenic pastures became known as "clover disease". Other estrogenic isoflavones subsequently i s o l a t e d from subterreanean clover and red clover ( T r i f o l i u m pratense) as well as other plants include biochanin A, diadzein and pratensein. S t r u c t u r a l formulae are shown i n Figure 2a. For many years confusion existed regarding species d i f f e r e n t responses to phyto-estrogen compounds depending on route of administration (review: Morley, et a l , 1968). Af t e r extensive studies on the metabolism of isoflavones i n the -35 a: ISOFLAVONES R, R2 GENISTEIN H OH BIOCHANIN A CH 3 OH DAIDZEIN H H FORMONONETIN CH 3 H (-CH3 in 4-methoxycoumestroD-^OH b: COUMESTANS 0 coumestrol Others: P s o r a l i d i n , Lucernol, T r i f o l i o l , S a t i v o l , Medicagol, etc., (Bickoff, 1968). Figure 2: St r u c t u r a l Formulae of Phyto-estrogens -36-sheep i t was found that biochanin A and genistein are degraded i n the rumen to e s t r o g e n i c a l l y i n a c t i v e phenols, such as p-ethylphenol (Braden, et a l , 1967). Formononetin, however, i s demethylated to diadzein and then metabolized to equol (7,4'-dihydroxyisoflavan) and, to a much lesser degree, O-desmethyl-angolensin, compounds which are e s t r o g e n i c a l l y a c t i v e (Shutt and Braden, 1968). Equol i s considered the agent responsible for "clover disease" i n sheep grazing pasture with high formononetin content (Shutt and Braden, 1968; Shutt, et a l , 1970). b. coumestans Reports that ladino clover ( T r i f o l i u m repens) and a l f a l f a (Medicago sativa) also contained estrogenic substances (Engle, et a l , 1957; Coop and Clark, 1960) led to the discovery of coumestrol, a representative of a new cl a s s of compounds, the coumestans (Bickoff, et a l , 1957). Other coumestans include t r i f o l i o l , 4'-0-methoxycoumestrol, medicagol, l u c e r n o l , s a t i v o l , 3-methyoxy-coumestrol and 11,12 dimethoxy-7-hydroxycoumestan (Bickoff, et a l , 1969). The major ones are coumestrol and 4'-O-methylcoumestrol. S t r u c t u r a l formulae are shown i n Figure 2b. L i t t l e i s known about the metabolism of coumestrol; however 4'-0-methyl-coumestrol i s believed to be demethylated to coumestrol i n the rumen. (Shutt, et a l , 1969). Absorption of coumestrol from the rumen takes place and l i t t l e or no i n a c t i v a t i o n occurs as r e f l e c t e d i n unchanged plasma l e v e l s of conjugated coumestrol (Kelly and Lindsay, 1978). c. blood l e v e l s i n animals Although the phyto-estrogens and t h e i r metabolites show r e l a t i v e l y low a f f i n i t y f o r estrogen receptors, t h e i r concentration i n the blood stream can be several orders of magnitude greater than endogenous estrogen l e v e l s (Shutt, et a l , 1967; Shutt, et a l , 1970; Lindner, 1967). The greatest proportion of -37-blood phyto-estrogens are i n a conjugated form as glucosiduronates which are considered i n a c t i v e (Shutt, et a l , 1967). A smaller proportion appears as sulpho-conjugates (Wong and Cox, 1971) and le s s as " f r e e " or unconjugated: these forms being considered b i o l o g i c a l l y active (Shutt, 1976). The plasma l e v e l s of " f r e e " and sulpho-conjugated phytoestrogen can reach the nanogram per m i l l i l i t e r range (Wong and Cox, 1971; Shutt, et a l , 1967; Braden, et a l , 1971; K e l l y and Lindsay, 1978) whereas endogenous estrogen l e v e l s are i n the picogram per m i l l i l i t e r range, d. i n f e r t i l i t y problems Although severe clover disease i s now rare due to changes i n management practi s e s (Lightfoot, 1974) s u b c l i n i c a l f e r t i l i t y problems rel a t e d to clover disease s t i l l e x i s t (Adams, 1977b). Ewes, termed " c l o v e r - a f f e c t e d " , show a lowered f e r t i l i t y rate believed r e l a t e d to the impairment of f e r t i l i z a t i o n due to decreased sperm m o t i l i t y i n the increased f l u i d i t y of c e r v i c a l mucus (Cox and Braden, 1974). Attempts have been made to r e l a t e changes i n c e r v i c a l mucus to s u b f e r t i l i t y and to u t i l i z e i t as a diagnostic t o o l , however the v a r i a b l e nature and observation that h i s t o l o g i c changes i n the cervix are not ne c e s s a r i l y associated with the production of abnormal mucus, have so f a r l i m i t e d t h i s technique (Adams, 1977a, b). Another form of reproductive problem i s termed "temporary i n f e r t i l i t y " which occurs i n ewes grazing estrogenic pasture p r i o r to and during the mating season (Morley, Axelson and Bennett, 1964). Although having a marginal e f f e c t d i f f i c u l t to detect without.close study, t h i s type of i n f e r t i l i t y could never-theless be important economically (Coop and Clark, 1960; Braden and McDonald, 1970; Scales, et a l , 1977). Of a temporary nature • (Engle, et a l , 1957) t h i s i n f e r t i l i t y i s character-ized by a reduction i n the number of ewes showing estrus (Lightfoot and Wroth, -38-1974; Coop and Clark, 1960; Clark, 1965) and delayed f i r s t detectable estrus (Engle, et a l , 1957). There i s a lowered f e r t i l i z a t i o n rate (Lightfoot and Wroth, 1974; Engle, et a l , 1957) and delayed f i r s t conception (Engle, et a l , 1957; Coop and Clark, 1960; Clark 1965). While impaired sperm transport may be a cause, i t i s also possible that there i s a reduction i n ovulation rate (Lightfoot and Wroth, 1974; Wroth and Lightfoot, 1976; Scales, et a l , 1977). This i s also indicated by a reduced number of ewes twinning (Coop and Clark, 1960; Scales, et a l , 1977). Although most studies into the e f f e c t s of phyto-estrogens have concentrated on changes i n the reproductive t r a c t there are i n d i c a t i o n s that these compounds can i n t e r f e r e with the normal hormone balance between ovaries and hypothalamus/ hypophysis. I I I . Indirect Evidence for E f f e c t s on Hypothalamo-Hypophysis Area Observations of estrus without ovulation i n f l o c k s grazing estrogenic pasture by F i r t h , et a l , (1977) suggest that anovulatory estrus may be a s i g -n i f i c a n t contributing factor to the low f e r t i l i t y of c l o v e r - a f f e c t e d ewes. K e l l y , et a l , (1976) noted that some ewes disp l a y i n g estrus had no recent corpus luteum when laparotomized. Ewes on phyto-estrogens showed f o l l i c u l a r abnormal-i t i e s such as numerous f o l l i c l e s , d e f i c i e n t antrum formation and signs of early a t r e s i a (Adams, 1977c; K e l l y , et a l , 1976). Adams (1976) indicated that there was a f a i l u r e of ovarian compensatory hypertrophy i n clover-affected ewes implying an a l t e r a t i o n i n hormone balance of the hypothalamus-pituitary-ovarian axis. K e l l y , et a l , (1976) noted that very high l e v e l s of coumestans i n h i b i t e d the expression of estrus i n ewes and suggested t h i s was due to a lack of endo-genous estrogen. Newsome and K i t t s (1977) reported that ewes consuming forage containing phyto-estrogens had plasma estrogen l e v e l s lower and more uniform than controls suggesting an e f f e c t on gonadotropin secretion. K e l l y , et a l , -39-(1976) reported that h i s t o l o g i c a l examination of the p i t u i t a r y of clover affected ewes showed basophils with r e l a t i v e l y enlarged vacuolated n u c l e i and degranu-lat e d cytoplasm. Adams (1977c) also noted degranulation of the 6-(delta) basophils and c e l l s hyperactive i n appearance. Hearnshaw, et a l , (1972) however, found that there were no consistent changes i n the p i t u i t a r i e s of clover-diseased ewes confirming the observations of Gardiner and Nairn (1969). Examination of the hypothalamus revealed groups of shrunken hyperchromatic neurons (Adams, 1977c; Gardiner and Nairn, 1969) p o s s i b l y associated with the development of permanent i n f e r t i l i t y i n ewes. Hearnshaw, et a l , (1971) noted that the plasma LH concentration of ovariectomized ewes r i s e s a f t e r ingesting estrogenic subter-ranean clover s i m i l a r to the LH response to s t i l b e s t r o l i n j e c t i o n . Hearnshaw, et a l , (1972) reported that the estrogen evoked LH release response i n o v a r i -ectomized ewes i s i n h i b i t e d i n ewes with clover disease. Extending these observations, Findlay, et a l , (1973) infused clover-diseased ovariectomized ewes with GnRH and did provoke an LH surge thus demonstrating that the p i t u i t a r y was capable of r e l e a s i n g LH and therefore the f a i l u r e of e s t r a d i o l to provoke the LH surge was due to interference with the c a p a b i l i t y of the hypothalamus to respond to estrogen. There have been few reports on the e f f e c t s of i s o l a t e d phyto-estrogen compounds on the hypothalamus or p i t u i t a r y . L e a v i t t and Wright (1965) attempted to determine the r o l e of a plant estrogen on the feedback mechanism of the p i t u i t a r y of mice and reported that coumestrol simulated the e f f e c t s of e s t r a d i o l i n the i n h i b i t i o n of gonadotropin release determined by h i s t o l o g i c a l examination. Leavitt (1965) determined that the coumestrol increased the number of gonado-trophs i n the p i t u i t a r y of mice before i t had uterotrophic e f f e c t s . Coumestrol also appears to produce pe r s i s t e n t anovulatory estrus i n adult female r a t s when injected as neonates (Leavitt and Meismer, 1968). -40-The foregoing observations imply that phyto-estrogens do act on the p i t -u i t a r y and hypothalamus as well as the reproductive t r a c t . In order f o r phyto-estrogens to act as estrogens or antiestrogens i n the p i t u i t a r y and hypothala-mus they must f i r s t i n t e r a c t with estrogen receptors. EXPERIMENTAL OBJECTIVES This study was undertaken to determine the i n t e r a c t i o n of the phyto-estrogen compounds, coumestrol and genistein, with the cytoplasmic estrogen receptors of p i t u i t a r y and hypothalamus t i s s u e from sheep. Information regard-ing the nature of the i n t e r a c t i o n was sought. I n i t i a l l y , the e s t r a d i o l binding c h a r a c t e r i s t i c s of target t i s s u e s were investigated. -41-MATERIALS AND METHODS A. MATERIALS I. Buffer composition Estrogen binding assay buffer: 0.01 M sodium phosphate with 0.25 M sucrose, pH 7.3, containing 0.02% azide. Homogenization buffer: the same as above with the a d d i t i o n a l concentration of 0.1 M 2-mercaptoethanol. I I . Chemicals. Sources are indicated a f t e r the compounds. Blue Dextran-Pharmacia Fine Chemicals. Potassium Ferricyanide - Fisher Chemicals. E s t r a d i o l 17 3 (1,3,5,(10) oestratrien-3, 17 3-diol) -Sigma Chemical Co.. D i e t h y l s t i l b e s t r o l (DES) (<s ^'-diethyl-4, 4'-stilbenediol) -Matheson, Coleman and B e l l , Manufacturing Chemists. Coumestrol (7', 6-dihydroxycoumarino (3', 4'-3, 2) coumarone) -Eastman Chemicals. Genistein (4', 5, 7-trihydroxyisoflavone) - ICN-K&K Laborator-i e s , Inc.. 3 H-estradiol 17 3 (2, 4, 6, 7, (n)- JH) e s t r a d i o l - Amersham Corp. S p e c i f i c a c t i v i t y of batches ranged from 89 Ci/mmol to 104 Ci/mmol. Stock solutions of estrogen compounds were made by d i s s o l v i n g the compound i n f r e s h l y d i s t i l l e d ethanol. Stock solutions were stored i n a freezer and were d i l u t e d with buffer for use. Labelled e s t r a d i o l (250 uCi) i n benzene/ -42-ethanol was dried under nitrogen, dissolved i n buffer and stored at 4°C. I I I . Tissue Samples a. Source A l l tissues were obtained from mature ewes of undetermined age slaughter-ed at Richmond Packers, Ltd., Richmond, B.C.. In a l l , r e s u l t s are given for several groups of ewes sampled over the period of these experiments. A t o t a l of f i f t y - o n e ewes were sampled. b. Tissue sampling The brain was exposed immediately post-slaughter either by a) d r i l l i n g a hole through the p a r i e t a l and f r o n t a l bones with a 2.5 inch hole saw driven by a commercial duty, 1/2 inch capacity Black and Decker e l e c t r i c d r i l l , or b) by chopping the s k u l l with a large cleaver. The l a t t e r method proved to be quicker although damage to brain t i s s u e was more extensive. The average time from slaughter to having the t i s s u e i n i c e was f i v e minutes. The cerebral hemispheres were l i f t e d up and back (dorsocaudally) and the o l f a c t o r y bulbs and optic t r a c t s severed to expose the v e n t r a l surface of the brain. (Figure 3). Hypothalamic t i s s u e was removed from an area approximately 5 mm r o s t r a l to the optic chiasma, caudally to the mammillary body and l a t e r a l l y bounded by the hypothalamic f i s s u r e s , to a depth of approximately 5 mm. Average weight of t i s s u e sample was 0.6 gms. This area encompasses the main centres of the hypothalamus known to contain e s t r a d i o l receptors as shown by auto-radiographic studies (Stumpf, 1970, 1971a,b, 1972; et a l , 1975). (Figure 4a,b). The brain was e n t i r e l y removed from the s k u l l . The diaphragma p e l l a e was cut and the e n t i r e p i t u i t a r y was l i s t e d from the s e l l a t u r c i c a . Average weight of the p i t u i t a r y gland was 0.8 gms. The p i t u i t a r y was used without -43-FIGURE 3 Figure 3: View of the Ventral Surface of the Sheep Brain, (May, 1970). M A S S A  I N T E R M E D I A C F R F R R A L H E M I S P H E R E S P I N E A L C E R E B E L L U M O L F A C T O R Y B U L B C H I A S M A \ H Y P O P H Y S 1 S M A M M I L L A R Y  B O D Y 33 m > i -P-I Figure 4a: M i d s a g i t t a l View of the Sheep Brain (Ranson and Clark, 1959). AC MI Massa Intermedia AC Anterior Commissure MB Mammillary Body OC Optic Chiasma I P Infundibular Process P I Pars Intermedia PD Pars D i s t i l a s 5mm PD Figure 4b: M i d s a g i t t a l View of the Sheep Brain, section through hypothalamus and p i t u i t a r y . (Daniel and Prichard, 1975). -46-separation into component parts due to t e c h n i c a l d i f f i c u l t i e s , however no estrogen binding has been detected i n the posterior portion (Ginsburg, et a l , 1975). Other tissues obtained were pine a l glands, uterus, and amygdala. A l l tissues were placed i n i c e - c o l d homogenization buffer and kept on i c e for transport and subsequent procedures, c. Cytosol preparation Pooled samples for each tis s u e were b l o t t e d with cheesecloth and weigh-ed. Homogenization with approximately three volumes of homogenization buffer was done by hand i n a glass-Teflon t i s s u e homogenizer immersed i n i c e . Uterus and p i t u i t a r y t i s s u e required s c i s s o r chopping p r i o r to homogenization. The homogenates were centrifuged at 4°C f o r 60 minutes at 100,000 x g i n a °ave. Beckman Model L5-65 Ul t r a c e n t r i f u g e . The supernatants (cytosols) for each t i s s u e were pooled and d i s t r i b u t e d i n 0.5-0.7 ml portions to glass ampoules, frozen i n dry i c e (-78°C), flame sealed, then transferred to l i q u i d nitrogen (-195.8°C). The ampoules were stored i n l i q u i d nitrogen i n a cryostat u n t i l use. For assays, the ampoules were opened and cytosol allowed to thaw at room temperature, then kept i n i c e for immediate use. B. ESTROGEN PROTEIN BINDING ASSAY I. Introduction The method used i n t h i s study i s an adaptation of that of Ginsburg, et a l , (1974). I t involves incubation of cytosol with t r i t i a t e d e s t r a d i o l with or without unlabelled estrogen. Separation of free from bound e s t r a d i o l i s accomplished by passing the incubate through a Sephadex LH-20 column at 4°C with a t h i r t y minute d i s s o c i a t i o n time on the column to permit d i f f e r -e n t i a t i o n of high a f f i n i t y binding from low a f f i n i t y , non-specific binding. -47-The c e l l u l a r components which bind e s t r a d i o l with high a f f i n i t y and mediate the action of estrogen are termed receptors. This i n t e r a c t i o n i s termed s p e c i f i c binding and i t i s a saturable r e a c t i o n , that i s , the number of spec-i f i c binding s i t e s i s f i n i t e . In addition to s p e c i f i c , high a f f i n i t y binding s i t e s , high capacity binding s i t e s of lower a f f i n i t y may be present i n c y t o s o l preparations. The binding of estrogen to these low a f f i n i t y s i t e s i s termed non-specific binding and the presence of non-specific binding must be taken into account i n the i n t e r p r e t a t i o n of binding data. (Mester, et a l , 1970). Cochet, et a l , (1976), have determined that the non-specific binding components f a l l i n a range of 40,000 to 100,000 molecular weight whereas the s p e c i f i c binding occurs with proteins of approximately 240,000 molecular weight. Both types of s i t e s would elute i n the same f r a c t i o n s from Sephadex LH-20. Ginsburg, et a l , (1974), has developed a cold d i s s o c i a t i o n step to overcome t h i s d i f f i c u l t y . C h i l l i n g the incubate to around 0°C slows the d i s s o c i a t i o n of bound hormone from the high a f f i n i t y s i t e s : however the non-s p e c i f i c s i t e s , that i s , those with lower a f f i n i t y , release bound hormone at greater than an order of magnitude f a s t e r than the s p e c i f i c , high a f f i n i t y sites. (Mester, et a l , 1970). The released estrogen i s retained by the LH-20 so that at the end of the cold d i s s o c i a t i o n period, only the high a f f i n i t y , s p e c i f i c s i t e s , r e t a i n l a b e l l e d e s t r a d i o l which i s then eluted from the column and counted. There are numerous methods a v a i l a b l e to correct f o r non-specific binding. P a r a l l e l incubations can be c a r r i e d out with unlabelled estrogen i n considerable excess. The unlabelled estrogen displaces l a b e l l e d estrogen from the high a f f i n i t y , l i m i t e d capacity, s p e c i f i c s i t e s while l a b e l l e d estrogen i s not displaced from the non-saturable, high capacity, non-specific s i t e s . Therefore bound l a b e l i n tubes with excess unlabelled estrogen may be a t t r i b u t e d to non-specific binding and s p e c i f i c binding determined by d i f f e r --48-ence from the t o t a l binding. This necessitates using twice as much c y t o s o l . As cytosol i n t h i s study was i n l i m i t e d supply, a c o n t r o l experiment was c a r r i e d out to determine i f the cold d i s s o c i a t i o n period was s u f f i c i e n t to minimize the presence of non-specific binding i n the column eluate. Incuba-t i o n with excess DES resulted i n n e g l i g i b l e binding of l a b e l l e d e s t r a d i o l , as given i n Figure 5. This indicates that minimal non-specific binding i n the p i t u i t a r y c ytosol remains a f t e r the cold d i s s o c i a t i o n procedure. Column eluates were counted by l i q u i d s c i n t i l l a t i o n . I I . Columns Glass tubing of approximately 0.5 mm i n t e r n a l diameter was formed into columns 20-25 cm. long with one end drawn to a small diameter. Teflon tubing was attached to the drawn ends and sealed with metal paper clamps, Double C l i p s . Glass wool plugs were used to r e t a i n the g e l . a. g e l Sephadex LH-20, Pharmacia Fine Chemicals, was swollen i n cold assay buffer at l e a s t twelve hours before use. Sephadex LH-20 i s a hydroxypropylated form of Sephadex G-25 and as w e l l as separating on the basis on molecular s i z e , (exclusion l i m i t 5000 molecular weight), exhibits hydrophobic and hydrophilic properties due to an increased r a t i o of carbon to hydroxyl groups, (Pharmacia information booklet). The l i p o p h i l i c character of Sephadex LH-20 permits the p r e f e r e n t i a l retention of s t e r o i d s , including e s t r a d i o l , i n aqueous sol u t i o n . This property enabled the use of small columns to minimize d i l u t i o n of the bound f r a c t i o n of e s t r a d i o l while r e t a i n i n g the unbound l a b e l l e d e s t r a d i o l . The a f f i n i t y of the g e l f o r s t e r o i d s also f a c i l i t a t e d the d i f f e r e n t i a l d i s s o c -i a t i o n of low a f f i n i t y binding, (Ginsburg, et a l , 1974). b. c h a r a c t e r i z a t i o n of columns The method of Ginsburg, et a l , (1974), u t i l i z e s small glass columns FIGURE 5 -49-I i ' « 5 F R A C T I O N N U M B E R Figure 5: 3 H - E s t r a d i o l Binding of P i t u i t a r y Cytosol, Column e l u t i o n , without and with excess DES, (duplicates) to show presence of s p e c i f i c e s t r a d i o l binding a f t e r t h i r t y minute cold d i s s o c i a t i o n period on LH-20 column. - 0 — O — O - 3 9 m > • H-Estradiol at 0.17 nM. - A — A — A - 3 H-Estradiol at 0.22 nM and DES at 6.5 nM. -50-( i . d . 0.45 mm), with Sephadex LH-20 to a bed height of 6 cm. Blue Dextran and potassium f e r r i c y a n i d e , located v i s u a l l y , were used to determine the ex-cluded, (void), volume and included volume, r e s p e c t i v e l y , of the column. I n i t i a l studies using Blue Dextran and potassium f e r r i c y a n i d e indicated that there was the p o s s i b i l i t y of free e s t r a d i o l e l u t i n g from the column i n the Blue Dextran region where the large c y t o s o l proteins, including estrogen receptors, were expected. Puca, et a l , (1971), used Sephadex G-25 to separate bound e s t r a d i o l from free at 4°C and found the bound f r a c t i o n to emerge i n the macromolecular peak. The e l u t i o n of r a d i o a c t i v i t y , 3 H - e s t r a d i o l , from a 6 cm column i n the absence of c y t o s o l when 200 u l of a mixture containing 3 100 u l Blue Dextran, 100 u l potassium f e r r i c y a n i d e and 50 u l of H-estradiol was layered on top i s given i n Figure 6. To avoid t h i s possible overlap of bound and free r a d i o a c t i v i t y a l l separations i n t h i s study u t i l i z e d gel heights of 12 cm. Ginsburg, et a l , (1974), state that the r a d i o a c t i v i t y i n eluates a t t r i b u t a b l e to the high a f f i n i t y complexes i s independent of column length. E l u t i o n p r o f i l e s of cyto-s o l incubates from 12 cm columns are given i n Figure 7. TNBS assay absorb-ances, vide i n f r a , are p l o t t e d as well to show the s p e c i f i c i t y of the radio-active counts i n r e l a t i o n to the protein, peptide, and amine absorbances. Each column was used once, the gel removed, the column decontaminated and repacked with fresh LH-20 at l e a s t four hours before reuse. In a comparison of various assay methods, Jungblut, et a l , (1972), determined that Sephadex chromatography and agar electrophoresis both stood out over several other separation methods as the most s e n s i t i v e . c. incubation procedure Cytosol, 200 u l , was added to tubes containing t r i t i a t e d e s t r a d i o l and buffer alone, or with unlabelled estrogenic compound, coumestrol, genistein -51-FIGURE 6 2 0 0 4 CL o S 150-o u 100-50-0-0 potassium ferr icyanide h H blue dextran 5 0 100 150 DROP NUMBER Figure 6: E l u t i o n of H-Estradiol from 6 cm Sephadex LH-20 column i n absence of cy t o s o l . (cCPM are quench corrected counts per minute). -52-FIGURE 7 FRACTION NUMBER Figure 7: E l u t i o n of H-Estradiol Bound by P i t u i t a r y Cytosol from 12 cm Sephadex LH-20 column, with TNBS absorb-ances shown. (cCPM are quench corrected counts per minute). -53-or d i e t h y l s t i l b e s t r o l (DES), i n a volume of 50 u l . Therefore t o t a l incubation volume was 250 u l . Incubation for f i f t e e n minutes was c a r r i e d out i n a 30°C water bath. Two 100 u l a l i q u o t s , duplicates, were each layered on LH-20 columns and run i n with 100 u l of cold assay b u f f e r . Columns were maintained at 4°C i n i c e water baths. After the incubation mix was run into the g e l bed, column flow was stopped for t h i r t y minutes to allow d i s s o c i a t i o n of the low a f f i n i t y , non-specific binding, (Ginsburg, et a l , 1974). A 25 u l aliquot of the incubation mix was transferred to a s c i n t i l l a t i o n v i a l f o r a count of t o t a l r a d i o a c t i v i t y present in the incubate. Columns were eluted i n t o glass m i n i - s c i n t i l l a t i o n v i a l s , 7 ml volume, V i a l e t t e , Amersham Corp., mounted i n t e s t tube racks of a LKB f r a c t i o n c o l -l e c t o r f i t t e d with drop counting head. Two columns, duplicates were run at the same time using two f r a c t i o n c o l l e c t o r s . d. l o c a t i o n of protein i n column eluates In order to determine accurately the p o s i t i o n of c y t o s o l protein i n the column eluates, the c o l l e c t e d f r a c t i o n s were subjected to a protein detection procedure. To avoid loss of radioactive counts through trans f e r to other containers a l l reagents were added to the m i n i - s c i n t i l l a t i o n v i a l s containing f r a c t i o n s from the columns. Absorbances were read i n a Spectronic 20 spectrophotometer-colorimeter, Bausch and Lomb, using an adapter designed to hold the m i n i - s c i n t i l l a t i o n v i a l i n the l i g h t path while activating, the "gate", Appendix Figure A - l . I n i t i a l l y the method of Lowry, et a l , (1951) was used to v i s u a l l y locate the protein peak, Appendix 2. As shown i n Figure 8, from an i n i t i a l experi-ment, the peak of protein absorbance correlated w e l l with the peak of r a d i o -a c t i v i t y . This method presented d i f f i c u l t i e s due to the large volume of aqueous reagent involved. The assayed samples did not mix w e l l i n the s c i n t i l -- 54 -FIGURE 8 o-F 1 1 1 1— O 5 10 15 20 FRACTION NUMBER Figure 8: E l u t i o n of H-Estradiol Bound by Amygdala Cytosol, with Lowry absorbances shown. - 55 -l a t i o n f l u i d , PCS, Amersham Corp., and there was frequent, non-uniform phase separation i n the s c i n t i l l a t i o n counter. An a d d i t i o n a l d i f f i c u l t y was numerous spurious counts r e s u l t i n g from chemiluminescence i f samples did not stand f o r some time before adding s c i n t i l l a t i o n f l u i d . Chemiluminescence can r e s u l t from oxidation of unsaturated compounds by molecular oxygen and t h i s can be catalyzed by bases such as are present i n the Lowry reagents, (Wang, et a l , 1975, pg. 269). For these reasons an alternate method was sought that would be more se n s i t i v e , thereby requiring l e s s reagent to be added to the samples but with an e a s i l y detected colour reaction f or v i s u a l l o c a t i o n of protein. The 2,4,6-trinitrobenzenesulfonic acid (TNBS) method of Snyder and Sobocinski, (1975), f o r the determination of amines, gives a yellow coloured product with small amounts of reagent and i s considered by the authors to be twice as s e n s i t i v e as the Lowry method, Appendix 3. This method i s p a r t i c -u l a r l y s u i t a b l e f o r l o c a t i n g protein peaks as i t requires the addition of only one reagent to the sample v i a l . As a q u a l i t a t i v e , not qua n t i t a t i v e , assessment of eluates was required, reagents were mixed and added i n minimum amounts, usually 75 u l per v i a l . As shown i n Figure 7, the f i r s t peak absorb-ance correlated with peak r a d i o a c t i v i t y . For routine use the p o s i t i o n of protein peaks was v i s u a l l y determined. Assayed samples mixed r e a d i l y i n 6 ml s c i n t i l l a t i o n f l u i d , B i o f l u o r , New England Nuclear, and there was no chemi-luminescence observed. e. measurement of r a d i o a c t i v i t y The m i n i - s c i n t i l l a t i o n v i a l s containing column eluates and s c i n t i l l a t i o n f l u i d were placed i n glass V i a l e t t e adapters, Amersham Corp., for counting i n a Nuclear Chicago Isocap 300 l i q u i d s c i n t i l l a t i o n spectrometer. Counting e f f i c i e n c y f o r t r i t i u m was approximately f o r t y percent with a background of -56-20 cpm. Quenching was monitored by the instrument external standard r a t i o . Background corrected counts per minute were standardized using a t r i t i u m quench serie s and expressed i n terms of corrected counts per minute, cCPM. Bound cCPM were obtained by summing the counts i n those v i a l s containing the macromolecular protein peak eluates and unboundj "f r e e " , cCPM were obtained as the d i f f e r e n c e between the t o t a l cCPM i n the incubate and the bound portion, f. multi-channel e l u t i o n system The l a t t e r portion of t h i s study involved a modified assay system. Incubations were c a r r i e d out as above except that f i v e incubates were run simultaneously, i n duplicate, using a ten column e l u t i o n system. Ten glass columns, Econo-columns, Biorad Laboratories, with Luer t i p s , packed with Sephadex LH-20, were connected to 18 gauge, 1% inch needles, Yale, B-D, set into a p l e x i g l a s s tank containing i c e and water. Columns were eluted using a Desaga Multi-channel P e r i s t a l t i c pump, Brinkman, and a modified Gilson Micro Fractionator, Model FG-100K, with a p l e x i g l a s s bar containing ten needles as drop tubes which permitted the simultaneous e l u t i o n of the ten columns into ten f r a c t i o n s each, (adapted from Webb, 1978). Subsequent procedures were as outlined above. C. TREATMENT OF DATA I. Introduction Apparent d i s s o c i a t i o n constants, K^, were determined by double r e c i p r o c a l a n a l y s i s , (Lineweaver and Burk, 1934). Apparent i n h i b i t i o n constants, K , were determined by Dixon p l o t s , (Dixon, 1953). The slopes and intercepts of l i n e s were determined by simple l i n e a r regression using the method of l e a s t squares. Intercepts, K^ ., f o r Dixon pl o t s were determined g r a p h i c a l l y and a l g e b r a i c a l l y from the equations of the two i n t e r s e c t i n g l i n e s . -57-I I . Methods The a p p l i c a t i o n of enzyme k i n e t i c s p l o t t i n g methods to s t e r o i d hormone-receptor i n t e r a c t i o n s has been outlined by Rodbard, (1973), i n a review of the analysis of bimolecular reactions. Some of these p l o t s were used i n t h i s study to gain i n s i g h t into the i n t e r a c t i o n of e s t r a d i o l 17 3 and phytoestrogen, genistein or coumestrol, with estrogen receptors i n c y t o s o l prepared from sheep p i t u i t a r y and hypothalamus and to determine the competitive nature of these i n t e r a c t i o n s . The actual hormone-receptor i n t e r a c t i o n i s possibly more complex than the assumed simple bimolecular reaction on which these p l o t s are based. Sanborn, et a l , (1971), have noted the presence of at l e a s t two interdependent binding s i t e s with s i m i l a r a f f i n i t i e s and the presence of p o s i t i v e cooperat-i v i t y at low e s t r a d i o l concentrations. However, Rodbard, (1973), states: " u n t i l both the hormone(s) and the receptor(s) are a v a i l a b l e i n homogeneous form i t w i l l be nearly impossible to delineate the i n t r i c a s i e s of the reaction mechanisms, and obtain r e a l i s t i c mathematical and physical-chemical models". In order to evaluate competitive i n t e r a c t i o n s , an estimate of the a f f i n i t y of s p e c i f i c receptors f o r e s t r a d i o l 17 3 i s required as competition between two molecules f o r the same binding s i t e on a receptor i s a function of both the concentrations of the molecules and the a f f i n i t y of the binding s i t e f o r each of the competing species. a. the determination of apparent d i s s o c i a t i o n constants, K^. The determination of an apparent K^, d i s s o c i a t i o n constant, value f o r the estrogen receptors was c a r r i e d out for each cytosol preparation using some of the p l o t t i n g methods described by Rodbard, (1973). The nomenclature equivalents to enzyme k i n e t i c s parameters are given below: -58-Enzyme k i n e t i c s Binding reactions substrate II f r e e " , unbound hormone enzyme receptor v e l o c i t y bound hormone Vmax t o t a l concentration of binding s i t e s Km , apparent d i s s o c i a t i o n constant i . s a turation analysis The equivalent of the Michaelis-Menten p l o t of enzyme k i n e t i c s f o r binding analysis i s the saturation binding curve. The d i s s o c i a t i o n constant i s defined as that concentration of ligand at which the binding s i t e s are one ha l f saturated. This i s analogous to Km of enzyme k i n e t i c s where a substrate concentration equal to Km y i e l d s a react-i o n / v e l o c i t y equal to one ha l f the maximal or saturation v e l o c i t y . Both the d i s s o c i a t i o n constant, K^, and i t s r e c i p r o c a l , the asso c i a t i o n constant K^, are measures of the a f f i n i t y of a binding s i t e f o r a p a r t i c u l a r ligand mole-cule. The rate at which the binding reaction approaches saturation with increasing ligand (hormone) concentration i s r e l a t e d to the a f f i n i t y of the receptor f o r the ligand. The l e v e l of binding at saturation i s a measure of the number of binding s i t e s present. i i . double r e c i p r o c a l analysis In a simple binding system obeying Michaelis-Menten k i n e t i c s the double r e c i p r o c a l p l o t , (Lineweaver-Burk, 1934) may be used to determine apparent binding a f f i n i t i e s , ( E d s a l l and Wyman, 1958 , pp. 620-622). In the double r e c i p r o c a l plot the apparent a f f i n i t y constant K^, i s obtained from the absolute value.of i n t e r s e c t i o n on the x axis of the extrapolated l i n e j o i n i n g the data points. The apparent d i s s o c i a t i o n constant K^, i s the r e c i p r o c a l of t h i s value. -59-The determination of values permits the s e l e c t i o n of appropriate experimental conditions for competitive studies. Incubations with genistein or coumestrol were c a r r i e d out with l a b e l l e d e s t r a d i o l concentrations approx-imately equal to Kp or to s i x times i n order to determine the i n t e r s e c t i o n point on Dixon p l o t s , vide i n f r a , from.which the apparent d i s s o c i a t i o n constants, or i n h i b i t i o n constants, values, f o r the phytoestrogens were obtained, (Dixon, 1953). The Kj., i n h i b i t i o n constant, i s the d i s s o c i a t i o n constant of the i n h i b -i t o r - r e c e p t o r complex, and thus i s a measure of the a f f i n i t y of the receptor for the i n h i b i t i n g compound. K or i n h i b i t i o n constants for genistein and coumestrol were obtained by the use of Dixon p l o t s , a graphical procedure i n which the r e c i p r o c a l of the concentration of bound ligand (hormone) i n the presence of i n h i b i t o r i s p l o t t e d versus the i n h i b i t o r (phyto-estrogen) con-centration. Dixon pl o t s have been used to determine K^ . values for a number of estrogens and anti-estrogens at the uterine receptor by Geynet, et a l , (1972). This p l o t also distinguishes between competitive and non-competitive i n h i b i t i o n . Binding data i s obtained for a range of i n h i b i t o r concentrations at two ligand concentrations and graphed i n the manner described. In the case of non-competitive i n h i b i t i o n the i n t e r s e c t i o n point of the two l i n e s thus obtained occurs on the x axis at a value equal to minus K^ .. For the competitive case the i n t e r s e c t i o n occurs above the x axis but s t i l l at an x value equal to minus K , (Webb, 1963). -60-RESULTS A number of experiments to determine estrogen and/or phyto-estrogen binding properties were c a r r i e d out on ewe hypothalamus, p i t u i t a r y , amygdala, pinea l and uterus cytosol preparations. The binding assay involves incubation of cytosol prepared from the test t i s s u e with a range of concentrations of t r i t i a t e d e s t r a d i o l to determine the estrogen binding parameters. Phyto-estrogen binding assays were c a r r i e d out with a range of competitor concen-t r a t i o n s i n the presence of a constant amount of t r i t i a t e d e s t r a d i o l . Results of e s t r a d i o l and phyto-estrogen binding experiments f o r ewe hypothalamus, p i t u i t a r y , amygdala, pine a l and uterus are presented below. A. HYPOTHALAMUS Estrogen binding experiments were c a r r i e d out on four d i f f e r e n t cytosol preparations of ewe hypothalamus t i s s u e . Experiments using DES, coumestrol and genistein were included to determine the i n h i b i t o r y a c t i v i t y of these compounds with respect to e s t r a d i o l i n binding to the estrogen receptor pro-teins i n hypothalamus c y t o s o l . The r e s u l t s f o r separate studies of each hypothalamus cytosol preparation are presented below. I. Preparation 1, Hypothalamus, September, 1976. 3 The amount of H-estradiol binding i n t h i s hypothalamus preparation was low, the maximum binding observed being 0.2 percent of the t o t a l e s t r a d i o l present, without a t h i r t y minute d i s s o c i a t i o n time to minimize non-specific binding, Figure 9. DES added at a concentration greater than one hundred 3 times that of the H-estradiol present decreased by approximately twenty 3 percent the binding of H-estradiol i n t h i s preparation. Genistein at 4.12 3 uM decreased H-estradiol binding by the same amount, as shown i n Figure 10. 3 These observations i n d i c a t e that of the t o t a l H-estradiol bound approximately FIGURE 9 0 . 0 2 0 4 0.0154 0.010 0 . 0 0 5 Figure 9: Preparation 1, Hypothalamus. E2: 3 H - e s t r a d i o l binding (nM) at concentration =7.5 nM (6 experiments); DES: % - e s t r a d i o l binding (nM) at 7.5, nM i n presence of DES=84.8 nM (2 experiments); Gen: % - e s t r a d i o l binding (nM) at 7.5 nM i n presence of Genistein=4.12 nM (2 exper iments). FIGURE 10 100 Figure 10: Preparation 1, Hypothalamus. Percent 3 H - e s t r a d i o l binding i n presence of DES or Genistein, at concentrations i n Figure 9. 100% binding 3H-estradiol=0.0168 nM. -62-20 percent was due to s p e c i f i c binding and t h i s s p e c i f i c a l l y bound H-estradiol 3 represented 0.04% of the t o t a l H-estradiol present i n the incubation. This amount of s p e c i f i c e s t r a d i o l binding of approximately 0.0034 nM was near the minimal detectable binding l e v e l and thus e s t r a d i o l binding parameters could not be determined for t h i s hypothalamus cytosol preparation. Large excesses 3 of DES and genistein i n h i b i t e d t h i s s p e c i f i c binding of H-estradiol. I I . Preparation 2. Hypothalamus. December. 1976, Preliminary binding experiments, omitting the 30 minute d i s s o c i a t i o n 3 time to minimize non-specific binding, yielded low l e v e l s of t o t a l H-binding 3 i n t h i s hypothalamus preparation. The maximum H-estradiol bound represented 3 3 le s s than 0.6 percent of the H-estradiol present at incubation. The H-e s t r a d i o l binding data without and with a t h i r t y minute d i s s o c i a t i o n time are shown i n Figure 11. 3 Competition assays with genistein and coumestrol reduced the H-estradiol binding approximately 20 percent as shown i n Figure 12. An attempt to deter-mine the s p e c i f i c e s t r a d i o l binding c h a r a c t e r i s t i c s of t h i s c y t o s o l prepar-ation by using p a r a l l e l incubations without and with greater than one hundred times molar excess of DES i s shown i n Figure 13. Curve C represents the difference between the t o t a l binding, curve A, and the binding i n the presence of excess DES, curve B, and i s considered to be the amount bound to the e s t r o -3 gen receptor (Ginsburg, et a l , 1974). Maximal s p e c i f i c H-estradiol binding was approximately 0.005 nM, again too low to accurately assess e s t r a d i o l binding parameters (Clark and Peck, 1977). The data curve C, Figure 13, i s shown i n double r e c i p r o c a l form i n Figure 14. This cytosol preparation 3 showed low l e v e l s of s p e c i f i c H-estradiol binding s i m i l a r to preparation 1 and i n s u f f i c i e n t data was obtained to present complete e s t r a d i o l binding 3 information. The s p e c i f i c H-estradiol binding was i n h i b i t e d by high l e v e l s 2 _ i 0.16-1 O Q 0.14-1 | 0.12-1 in L U 0.10 ^~ 0.08\ a Z0.06] §0.04 0.02 0 i ON I 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 TOTAL 3H-ESTRADIOL (nM) Figure 1 1 : Preparation 2 , Hypothalamus, 3 • H-estradiol binding l e v e l s obtained 3 A H-estradiol binding l e v e l s obtained to minimize nonspecific binding. H-Estradiol Binding, nM. without cold d i s s o c i a t i o n time, with t h i r t y minute d i s s o c i a t i o n time H- cu H* OQ rt OQ 3 3 H i-i i i ro H-OQ ro •—s Cu rt M 3' ro M CU o ro W rt I—1 rt 3 C/ >,r u> rt co o rt O CO CO 3" ro O &• CO Co .. s—' rt O 3 rt rt fu .. ro i-i ^ O 3 i-i i i H H- TJ 3 CD cu O ro cu fu Co 3 i i o Cu cT 3 CO p. 9 hi 3* (D ro d n o rt H- H- 3 i i H- CO cn ro • O 3 ro i-i O O co ro CT ro ^—\ 3 O 1—1 M ** T3 H- 3 i-h cu ti- rt fu rt o O OJrj ll 3 i-i H- W i-ti i i ro i-i ffi CU OQ cu O 3 O ro cu CO 1 rt O rt i-i 3 i-i rt o rt ro H- • o H- T3 3 O H-ro I-h O CO o O i-h O OQ i i p. ro o n rt rt 3 3 ro ro 3 3 H- rt> Co i i LO CO 3 cn fu rt i-h X M cu to H' ro rt ro H- o - 3 11 CD cn 3 n ro CT H- g rt O ON CO t ; - O ffi o r  ro 1 Efi cr CO 3 H- a. p 3 O 0 a H- O • i-h o M 3 3 I 1 H» C/3 OQ 3 OQ —' BOUND 3H-E< fu c r o X Q =t %3H-ESTRADI0L BOUND a On n O O u •—i t i i i i • o o y/////////////mm nm i mi XXXXXX^XX^XX^X RvXXXXXXXXXXXXXvX^I -ESTRADIOL (nM) £ 1 * Z[ S38n9U - 1 7 9 -H- Co OQ rt C i-S p. fa 00 CT* c q i-i < fD fD (BOUND ^H-ESTRADIOL, nM)-1 _. ro w ^ m 0 ) o o o o o o o o o o o o -si o o CO o o > I-i 1—1 • • • H- Ui rt fD -P-3 •• s—' n • • Cu H-H- 13 o CO H 3 hd i-i H- w O CN i-i O i-i hh 1 rt - fD O fD Hi fD Co X) Co 13 ro cn 3 Co H Co I-i rt S « i-i fD i-i Co • Co 13 Co a CO rt i-i rt o Cu 1 H" fD H-fD H- fD O CO O O cn 3 fD 3 M rt 3 > i-i Co rt K> 1 cr P3 Co w O Cu rt (3 H- H- a 3 O « O V! Cu M s 13 CO •d O 13 SI cr o O rt fD H- O rt l-h 3-O rt C 3- Co H* 3" 3 Co O h-1 hh Cu M C Co H* o fo i-i S O w B <! c n w C ro. CO CD o o CO o « 3 c I Cr fD •iJ 1—1 CO H" fD rt OQ i-i (3 P H Cu fD H> O h-' h-1 Co BOUND 3H-ESTRADI0L (nM| 5t CD - g 9 --66-of DES, genistein and coumestrol. I I I . Preparation 3, Hypothalamus, May 1877, Fresh A binding assay was conducted using hypothalamus, cy t o s o l the day of preparation without l i q u i d nitrogen freezing. Maximal binding was 0.7 percent of the t o t a l e s t r a d i o l present i n the incubation. Coumestrol at 69 nM reduced 3 H-estradiol binding to 63 percent of the c o n t r o l . IV. Preparation 3F, Hypothalamus, May 1977, Frozen i n l i q u i d nitrogen A d d i t i o n a l experiments were conducted with the above preparation a f t e r 3 storage i n l i q u i d nitrogen. No binding was detected at a t o t a l H-estradiol concentration l e s s than 0.5 nM e s t r a d i o l . Maximal binding was l e s s than 0.5 percent of the t o t a l e s t r a d i o l present. Attempts to determine the amount of s p e c i f i c binding with excess DES gave e r r a t i c r e s u l t s . Competition with coumestrol was inconclusive. Results are presented i n Figure 15. V. Preparation 4, Hypothalamus, May 1978 E s t r a d i o l binding i n t h i s preparation was l e s s than 0.5 percent of the t o t a l present. No further experiments were conducted. 3 The amount of H-estradiol bound by these four hypothalamus cy t o s o l 3 preparations ranged from 0.2 to 0.7 percent of the t o t a l H-estradiol present i n the incubations. It was concluded that t h i s low l e v e l of t o t a l detectable 3 H-estradiol binding coupled with the even lower s p e c i f i c binding component did not permit the r e l i a b l e determination of e s t r a d i o l binding parameters. Likewise, r e l i a b l e determination of i n h i b i t i o n parameters of coumestrol and 3 genistein was not possible at these low l e v e l s of H-estradiol binding due to the d i f f i c u l t y i n detecting degrees of i n h i b i t i o n . Coumestrol at a concentra-t i o n approximately equal to the apparent determined for p i t u i t a r y c ytosol (vide i n f r a ) i n h i b i t e d e x t r a d i o l binding i n hypothalamus cy t o s o l preparations to approximately 60 percent while genistein at a concentration greater than a hundred times apparent K as determined i n p i t u i t a r y c ytosol (vide i n f r a ) - 67 -completely suppressed s p e c i f i c e s t r a d i o l binding in hypothalamus c y t o s o l , preparations 1 and 2.. These r e s u l t s suggest that the competitive e f f i c i e n c y of both coumestrol and genistein in hypothalamus cytosol i s probably compar-able to that determined f o r the p i t u i t a r y (vide i n f r a ) . B. PITUITARY The close hormonal r e l a t i o n s h i p between the p i t u i t a r y and the hypothal-amus coupled with i t s known response to estrogen suggested t h i s t i s s u e as a suitable experimental material to study the binding of estrogen and phyto-estrogens. Experiments on three p i t u i t a r y c ytosol preparations were c a r r i e d out both to determine e s t r a d i o l binding c h a r a c t e r i s t i c s and to determine the competitive action of coumestrol and genistein. Results are presented f i r s t for estrogen binding parameters followed by phyto-estrogen binding character-i s t i c s . I. Determination of Apparent K p Values for P i t u i t a r y Estrogen Receptor The d i s s o c i a t i o n constant, K^, i s a measure of the binding a f f i n i t y between a hormone and i t s receptor and i s a c h a r a c t e r i s t i c of a p a r t i c u l a r hormone receptor p a i r . Apparent determinations for the r e c e p t o r - e s t r a d i o l 3 i n t e r a c t i o n were obtained from double r e c i p r o c a l analysis of the H-estradiol binding data for each p i t u i t a r y c ytosol preparation and are presented below. a. Preparation 1, P i t u i t a r y , May 1977 The saturation binding curve for t h i s preparation i s shown i n Figure 16. The double r e c i p r o c a l analysis of t h i s data i s shown in Figure 17. The apparent for e s t r a d i o l which i s the r e c i p r o c a l of the X intercept was determined to be 0.39 nM and r e c i p r o c a l maximal binding, (B ) \ as deter-r max mined by the Y intercept 2as 3.1 nM \ equivalent to 0.32 nM. b. Preparation 2, P i t u i t a r y , September 1977 Saturation analysis for t h i s preparation i s shown i n Figure 18. Double r e c i p r o c a l analysis i s shown in Figure 19. The apparent for e s t r a d i o l of t h i s preparation as above was determined to be 0.26 nM and r e c i p r o c a l maximal Figure 16: Preparation 1,. P i t u i t a r y , Saturation Curve," (points are means of d u p l i c a t e s ) . Data presented i n Double Reciprocal form i n Figure 17. -69-FIGURE 17 (FREE 3 H - E S T R A D I O L , nM) Figure 17: Preparation 1, P i t u i t a r y , Double Reciprocal p l o t , (r=0.95, n=14), Apparent 1^=0.39 nM. -70-FIGURE 18 0 0.5 1.0 1.5 2.0 2.5 F R E E . 3 H - E S T R A D ! O L (nM) Figure 18:. Preparation 2, P i t u i t a r y , Saturation Curve, (points are means of du p l i c a t e s ) . Data presented i n Double Reciprocal form in-;.Figure 19. _ FIGURE 19 i I 2 20-0 10 2 0 3 0 4 0 (FREE 3 H - E S T R A D I 0 L , nM)"1 Figure 19: Preparation 2, P i t u i t a r y , Double Reciprocal p l o t . (r=0.94, n=19), Apparent Kg=0.26 nM. - 71 -binding, (B ) \ was 1.19 nM \ equivalent to 0.84 nM, therefore the amount max 3 of H-estradiol binding in t h i s c ytosol preparation was approximately 2% times greater than preparation 1. The r e c i p r o c a l B ^ ^ values were used f o r the Dixon p l o t s of phyto-estrogen- compounds to determine K^ . values (vide i n f r a ) 3 i n the cases of i n h i b i t i o n data being a v a i l a b l e f o r only one H-estradiol concentration. c. Preparation 3, P i t u i t a r y , May 1978 Saturation analysis for t h i s preparation i s shown i n Figure 20. The 3 amount of maximal H-estradiol i n t h i s preparation was s i m i l a r to preparation 1. Double r e c i p r o c a l analysis i n Figure 21 determined the apparent K n for e s t r a d i o l to be 0.14 nM. 3 The amount of H-estradiol binding present i n p i t u i t a r y c ytosol pre-parations 1 and 3 were s i m i l a r , however preparation 2 showed double the amount of binding capacity. In a l l three cases the amount of s p e c i f i c e s t r a d i o l binding was s u f f i c i e n t to assess e s t r a d i o l binding parameters and thus binding i n h i b i t i o n c h a r a c t e r i s t i c s of phyto-estrogens. The apparent values for e s t r a d i o l determined f o r these three p i t u i t a r y c y t o s o l preparations when considered together i n d i c a t e an apparent K D of 0.26 * 0.12 nM, a high a f f i n i t y of p i t u i t a r y estrogen receptor f o r e s t r a d i o l . Following the above determinations, studies to assess the competitive e f f i c i e n c y of phyto-estrogen compounds were c a r r i e d out. I I . Determination of Apparent K-r Values for Phyto-estrogens The K p i n h i b i t o r constant, determined for a compound from competitive studies with hormone present, equals the of the hormone receptor f o r the i n h i b i t i n g compound i n the case of competitive i n h i b i t i o n and thus can be used to evaluate the a f f i n i t y of a receptor f o r various compounds which may show d i f f e r e n t degrees of binding a b i l i t y . Apparent K^ . values f o r two phyto-estrogen compounds were determined from Dixon plot analysis f o r coumestrol and genistein with d i f f e r e n t p i t u i t a r y FIGURE 20 Figure 20: Preparation 3, P i t u i t a r y , Saturation Curve, (points are means of d u p l i c a t e s ) . Data presented i n Double Reciprocal form i n Figure 21. FIGURE 21 Figure 21: Preparation 3, P i t u i t a r y , Double Reciprocal p l o t . (r=0.74, n=16), Apparent K^O.14 nM. -73-cytosol preparations and r e s u l t s are presented separately f o r each compound. a. Coumestrol Apparent K^ . determinations were made on two p i t u i t a r y cytosol prepara-tions . i . Preparation 1, P i t u i t a r y , May 1977 3 Dixon analysis for coumestrol i n t h i s preparation as determined by H-e s t r a d i o l binding with various coumestrol concentrations i s shown i n Figure 3 22. As analysis at only one H-estradiol concentration (0.5 nM) was completed, the apparent K^ . was determined at the point of i n t e r s e c t i o n of the experiment-a l l i n e with a l i n e drawn at r e c i p r o c a l maximal binding, (B ) \ as deter-max mined from double r e c i p r o c a l analysis f o r t h i s c ytosol preparation (vide supra). The apparent K^ . for coumestrol was determined to be 61 nM. i i . Preparation 2, P i t u i t a r y , September 1977 Dixon analysis f o r coumestrol on t h i s cytosol preparation i s shown i n 3 Figure 23. Analysis at only one concentration of H-estradiol was a v a i l a b l e (2.8 nM). The apparent was determined at the point of i n t e r s e c t i o n with the r e c i p r o c a l maximal binding, (B ) \ l i n e as determined from double r ° max r e c i p r o c a l analysis of t h i s c ytosol preparation (vide supra). The apparent determined f or coumestrol was 58.6 nM, that i s 59 nM. b. Genistein Genistein apparent K^ . values were determined on two cy t o s o l preparations. I n h i b i t i o n curves to show the p a r a l l e l binding i n h i b i t i o n of genistein at d i f -3 ferent H-estradiol concentrations are also presented. i . Preparation 2, P i t u i t a r y , September 1977 3 The i n h i b i t i o n curves f o r genistein with H-estradiol at 0.46 nM and 2.9 nM are presented i n Figure 24: Dixon a n a l y s i s , shown i n Figure 25, deter-mined the apparent K for genistein i n t h i s preparation to be 130 nM. -74-FIGURE 22 - A p p a r e n t =. "61 nM 5 0 100 150 COUMESTROL (nM) 2 0 0 Figure 22: Preparation 1, P i t u i t a r y , Dixon P l o t , Coumestrol, •^H-Estradiol at 0.5 nM (r=0.8, n=10), Apparent coumestrol determined at (B )~^- from Figure 17. max for Figure 23: Preparation 2, P i t u i t a r y , Dixon P l o t , Coumestrol, 3 H - E s t r a d i o l at 2.8 nM (r=0.86, n=8), Apparent % for coumestrol determined at (B )~1 from Figure 19. max -76-FIGURE 24 •4 4 i i • > i • i i i i o o o o o o o o o o O c r > c o r ^ c r > L 0 ^ r r o c \ i ' — QND09 nOIQVcJlS3-Hc ! N 3 0 d 3 d o • 2 o ' cn o ' 00 . o r- . C . o CD . o If) ISTE o ' ^ L L I o " ro Figure 24: Preparation 2, P i t u i t a r y , I n h i b i t i o n Curves, Genistein with ^H-estradiol at concentrations of 2.9 nM # and 0.46 nM ^ GENISTEIN (nM) Figure 25: Preparation 2, P i t u i t a r y , Dixon P l o t , Genistein, Apparent K for genistein determined to be 130 nM. -'H-estradiol binding i n h i b i t i o n determined with concentration of 3H-estradiol at 0.46 nM A (r=0.97, n=6) 3 H - e s t r a d i o l at 2.9 nM % (r=0.9, n=6) -78-i i . Preparation 3, P i t u i t a r y , May 1978 I n h i b i t i o n curves f o r genistein with e s t r a d i o l at 0.4 and 2 nM are shown i n Figure 26. Dixon pl o t a n a l y s i s , shown i n Figure 27, determined the apparent K^ . f o r genistein i n t h i s preparation to be 210 nM. Results recorded i n Figures 16-21 show the apparent d i s s o c i a t i o n con-stant, f o r e s t r a d i o l i n ewe p i t u i t a r y c y t o s o l as determined f o r three cytosol preparations to be 0.26 - 0.12 nM. Apparent K determinations on two p i t u i t a r y cytosol preparations each for coumestrol and genistein revealed coumestrol (apparent K^=59-61 nM) to be about three times more e f f i c i e n t a competitor than genistein (apparent K^.=130-210 nM) i n ewe p i t u i t a r y c y t o s o l , that i s , the a f f i n i t y of the estrogen receptor f o r coumestrol i s about three times greater than i t s a f f i n i t y f o r genistein. The receptor a f f i n i t y f o r coumestrol i s approximately 230 times l e s s , and for genistein approximately 650 times l e s s , than f o r e s t r a d i o l . In a d d i t i o n to the hypothalamus and p i t u i t a r y , f o r comparative purposes other possible estrogen target tissues were chosen to examine e s t r a d i o l and/ or phyto-estrogen i n t e r a c t i o n with cytosol estrogen receptors. In t h i s supplementary study two extrahypothalamic brain structures, the amygdala and the p i n e a l , were examined. In addition, the ewe uterus, i n p a r t i c u l a r the caruncles of the endometrium, was studied f o r estrogen binding parameters. C. AMYGDALA The amygdala was chosen as a representative of extrahypothalamic b r a i n structures believed involved i n reproduction, p a r t i c u l a r l y onset of puberty 3 and ovulation. An experiment to determine H-estradiol binding and competition with 2.71 uM and 5.42 uM genistein i n amygdala cytosol was conducted. Results are shown i n Figure 28. Although e s t r a d i o l binding was reduced, the concen-tr a t i o n s of both e s t r a d i o l and genistein i n t h i s experiment were high. The -79-FIGURE 26 T r 80 100 i ' r 300 400 500600 GENISTEIN (nM) Figure 26: Preparation 3, P i t u i t a r y , I n h i b i t i o n Curves, Genistein, with 3 H - e s t r a d i o l concentrations of • 2.0 nM and A 0.4 nM - ApparentK=-210n - 2 0 0 -100 100 2 0 0 3 0 0 GENISTEIN (nM) 5 0 0 Figure 27: Preparation 3, P i t u i t a r y , Dixon Plot, Genistein. Apparent Kj_ for genistein determined to be 210 nM. 3 H - e s t r a d i o l binding i n h i b i t i o n determined with concen-t r a t i o n of 3 H - e s t r a d i o l at 0.4 nM 3 H - e s t r a d i o l at 2 nM (r=0.91, n=6) (r=0.8, n=7) 00 o i -81-FIGURE 28 0 22 H-estradiol = 6 nM H-estradiol Genistein ^H-estradiol Genistein 6 nM 2.7 uM 6 uM 5.42 uM 5 0 100 PERCENT BINDING OF "H-ESTRADIOL Figure 28: Amygdala, H-Estradiol Binding alone and i n presence of Genistein. -82-closeness of the reduction, even though the genistein concentration was doubled, indicates that the amount of s p e c i f i c binding i s low, that i s approx-imately 10 percent of t o t a l binding detected and that genistein completely i n h i b i t e d t h i s s p e c i f i c binding. The remaining bound e s t r a d i o l detected i s i n the non-specific f r a c t i o n . In t h i s experiment the genistein dissolved i n ethanol was added to incubation tubes, then dried under, nitrogen. I t i s possible that a l l did not r e d i s s o l v e i n the cytosol. For a l l other competi-tions with other tissues the genistein or coumestrol was added i n ethanol d i r e c t l y to the c y t o s o l to ensure accurate competitor concentration. As t h i s preliminary experiment determined that the amount of e s t r a d i o l binding was low i n amygdala c y t o s o l , t h i s p a r t i c u l a r aspect was not pursued. An e l u t i o n p r o f i l e for•amygdala'binding is- shown i n Figure 8. D. PINEAL The p i n e a l gland has been implicated i n the control of reproductive rhythms and i s possibly influenced by s t e r o i d hormones. An experiment to determine the p o s s i b i l i t y of s p e c i f i c e s t r a d i o l binding i n the p i n e a l was conducted using p a r a l l e l incubations with 100 times molar excess d i e t h y l s t i l -b e s t r o l . In addition, competitions with 300 nM and 500 nM genistein were ca r r i e d out. As shown i n Figure 29, saturation binding was not reached. 3 Excess DES did not detectably decrease H-estradiol binding i n d i c a t i n g that either s p e c i f i c binding was not present or was not detected by t h i s assay system. This observation i s confirmed i n the Dixon pl o t of the genistein competitions as zero slope indicates the absence of detectable competition, Figure 30. E. UTERUS Experiments were c a r r i e d out to study the estrogen binding character-i s t i c s of sheep uterine cytosol prepared from caruncles of endometrium. In -83-FIGURE 29 0.5 -£0.4 < tr 0.3 Ixl I X r o 5 0.2 o m 0.1 • H-Estradiol alone A DES, 100 x excess • Genistein, 300-500 nM fe 8 1.0 2.0 3.0 FREE ^H-ESTRADIOL (nM) 4.0 Figure 29: Pi n e a l , H-Estradiol Binding i n Presence of DES and Genistein. FIGURE 30 :20 o Q < \— t/) x Q Z 3 10 O 0 CD 0 H-Estradiol=0.5 nM H-Estradiol=2.5 nM 100 200 300 GENISTEIN (nM) 400 500 Figure 30: P i n e a l , Dixon P l o t , Genistein -84-order to determine the amount of non-specific binding present i n t h i s uterine 3 cytosol preparation a binding experiment with H-estradiol and p a r a l l e l incu-bations containing 100 times molar excess DES was conducted. Results as shown by column e l u t i o n p r o f i l e i n Figure 31 indi c a t e that the amount of non-s p e c i f i c binding present was les s than ten percent of t o t a l binding. Satura-3 t i o n analysis i s shown i n Figure 32 with a maximal H-estradiol binding greater than 4 nM. Determination of the apparent f o r e s t r a d i o l by double r e c i p r o c a l analysis i s presented i n Figure 33. The apparent f o r e s t r a d i o l i n uterine cytosol was determined to be 0.6 nM. -85-FIGURE 31 NUMBER OF DROPS Figure 31: Uterus, E l u t i o n of Bound H-Estradiol from LH-20 column, (cCBM are quench corrected counts per minute), with 3 % H-Estradiol at 0.46 nM A> as above with excess DES. -86-F1GURF 7>? 9 Figure 32: Uterus, Saturation Curve, H-estradiol binding, nM. (Points are means of d u p l i c a t e s ) . Data shown i n Double Reciprocal f o r m i n Figure 33. -87-FIGURE 33 6 . 0 -0 5 10 15 2 0 , 2 5 (FREE 3 H-ESTRADI0L, nM) Figure 33: Uterus, Double Reciprocal Plot (r=0.93, n=16), Apparent ^=0.6 nM. -88-DISCUSSION 3 Results of H-estradiol binding studies f o r the various tissues are discussed i n terms of estrogen binding parameters. The r e s u l t s of the phyto-estrogen experiments are then discussed with possible p h y s i o l o g i c a l i m p l i c a -tions mentioned. I. E s t r a d i o l Binding Parameters 3 It was determined that the amount of s p e c i f i c H-estradiol binding i n these hypothalamus cytosol preparations was low, maximum detected being 0.003 nM i n preparation 1 and about 0.008 nM i n preparation 2. The non-specific 3 binding component of the t o t a l H-estradiol binding was high (Figure 13). In such a system i n which the quantity of non-specific binding i s great with respect to t o t a l binding the p r o b a b i l i t y of accurate determination of s p e c i f i c binding i s very low (Clark and Peck, 1977). For t h i s reason the determination of s p e c i f i c e s t r a d i o l binding parameters i n ewe hypothalamus cytosol was not pursued. It therefore follows that i n h i b i t i o n studies under these binding conditions "can lead to highly erroneous r e s u l t s " , (Clark and Peck, 1977). Because the main emphasis of t h i s study was to determine estrogen receptor a f f i n i t i e s for phyto-estrogens no attempt was made to quantitate the amount of estrogen binding for the d i f f e r e n t t i s s u e s . However, i t was obvious that cytosol preparations of p i t u i t a r y and uterus contained greater estrogen binding capacity than c y t o s o l of hypothalamus amygdala and p i n e a l and therefore e s t -r a d i o l binding parameters could be more c l o s e l y examined i n these two t i s s u e s . Apparent e s t r a d i o l values i n t h i s study were determined on cy t o s o l pools of p i t u i t a r y t i s s u e from i n t a c t ewes. The reports of other workers are based on r e s u l t s from i n d i v i d u a l ovariectomized ewes. The apparent values for e s t r a d i o l determined i n t h i s study, 0.14-0.39 mean 0.26*0.12 nM, are i n -89-the range reported by other workers. Wise and Payne (1975) reported on f i v e i n d i v i d u a l ewes a mean of 0.103 nM for e s t r a d i o l and 0.14 nM f o r estrone, using density gradient c e n t r i f u g a t i o n . These workers also reported a d i f f e r -ence i n values equivalent to apparent K^ between anoestrus, breeding and ~ e s t r a d i o l treated anoestrus ewes ranging from 0.02 nM to 0.08 nM (Wise, et a l , (1975). It i s possible that the ewes which formed the cyt o s o l pools for t h i s study were i n varying reproductive states and thus i f p i t u i t a r y e s t r a d i o l changes with phase of reproduction then s l i g h t differences i n i n d i v i d u a l ewes may influence the apparent value determined on a cytosol pool. Tang and Adams (1978) reported values which are equivalent to values of 0.02 nM for p i t u i t a r i e s from both control and clover affected ewes using the dextran-charcoal assay. Depending on. the method of determination, the e s t r a d i o l values reported for female rat p i t u i t a r i e s range from 0.07 to 0.32 nM (Ginsburg, et a l , 1974) and Kato (1977) reported the Kp of rat p i t u i t a r i e s to be 1.4 nM. As i s evident from the figures quoted f o r the female r a t , the species most thoroughly studied with regard to hypothalamus and p i t u i t a r y estrogen binding c h a r a c t e r i s t i c s , the determination of apparent K^ values i s subject to some v a r i a t i o n due to method, reproductive state of experimental animals and pos-s i b l y other factors and thus although absolute values appear d i f f e r e n t they f a l l within the same order of magnitude, i n d i c a t i n g the high a f f i n i t y of the receptors f o r e s t r a d i o l . The apparent K^ f o r e s t r a d i o l determined i n t h i s study for ewe uterus, i n p a r t i c u l a r the caruncles of the endometrium, was 0.6 nM and t h i s tends to confirm the value reported by Shutt and Cox (1972) of 0.11 to 0.16 nM for the estrogen-primed, ovariectomized sheep uterus. Values reported f o r other species are also i n the same range. The K^ values reported for c a l f and r a t uterus are 0.2-0.5 nM (Geynet, et a l , 1972) and Senior (1975) reports the ^  of cow -90-uterus to be 0.05-0.25 nM. The values for rat uterus have been reported as 0.18-0.46 nM (Ginsburg, et a l , 1974) 0.14-0.34 nM (Feherty, et a l , 1970), and 1.4 nM (Kato, 1977). Again the high a f f i n i t y of estrogen receptors i n the uterus for e s t r a d i o l was evident. S p e c i f i c e s t r a d i o l binding was detected i n the ewe amygdala, confirming the reports of such binding i n t h i s part of the brain i n the r a t . As with the hypothalamus, the amount of detectable s p e c i f i c e s t r a d i o l binding i n the amygdala cytosol preparation was too low for r e l i a b l e determination of e s t r a d i o l binding parameters and was not pursued. The presence of s p e c i f i c e s t r a d i o l binding receptors i n the p i n e a l gland, as has been previously reported f o r the rat ( C a r d i n a l i , et a l , 1975), was not detected i n the ewe p i n e a l under the conditions of these experiments. As the volume of cytosol prepared was l i m i t e d due to the small s i z e of the p i n e a l gland, i t was not possible to continue experiments on t h i s aspect of estrogen binding. The determination of e s t r a d i o l binding parameters i n the p i t u i t a r y per-mitted the s e l e c t i o n of experimental conditions to assess the a f f i n i t y of estrogen receptors of t h i s t i s s u e f or the phyto-estrogens, genistein and coumestrol. A d d i t i o n a l experiments on other tissues are reported. I I . The Determination of Phyto-estrogen Binding C h a r a c t e r i s t i c s The nature of the i n t e r a c t i o n between e s t r a d i o l and phyto-estrogens with the estrogen receptors of the p i t u i t a r y was determined to be competitive, that i s , these compounds i n t e r a c t with the same binding s i t e as e s t r a d i o l . This conclusion was drawn from Dixon pl o t analysis (Figures 25,27). The i n t e r s e c t i o n of the experimental l i n e s f or coumestrol and genistein at a point above the abscissa to the l e f t of the ordinate i s consistent with comp-e t i t i v e i n h i b i t i o n taking place. This implies that at concentrations high -91-enough, r e l a t i v e to the appropriate a f f i n i t i e s involved, coumestrol or geni-s t e i n can e f f e c t i v e l y compete with e s t r a d i o l for estrogen receptors. Further-more i n the absence of e s t r a d i o l these compounds can bind to the estrogen receptors of the p i t u i t a r y and possibly act i n the manner of e s t r a d i o l . This competition with e s t r a d i o l by the phyto-estrogens coumestrol and genistein has been demonstrated, i n t h i s study to occur with estrogen receptors i n cytosol from ewe hypothalamus, p i t u i t a r y and amygdala. Previously t h i s competition has been reported at uterine receptors of various species and at estrogen receptors from human breast cancer c e l l s (Shemesh, et a l , 1972; Shutt and Cox, 1972; Geynet, et a l , 1972; Martin, et a l , 1978). In order to quantitate the binding of phyto-estrogen compound to p i t -u i t a r y estrogen receptors the determination of i n h i b i t o r constants, K^ . values, by Dixon pl o t s was c a r r i e d out for genistein and coumestrol. In the case of competitive i n h i b i t i o n , as has been herein determined for genistein and coumestrol, the i s equal to the of the i n h i b i t o r and thus i s a quanti-t a t i v e measure of the a f f i n i t y of the receptor for i n h i b i t o r . The apparent K values determined for coumestrol and genistein at the ewe p i t u i t a r y estrogens receptors i n t h i s study are s i m i l a r to those reported by Geynet, et a l , (1972) for the same compounds i n rat and c a l f uterine cytosol. The apparent K^ . f o r genistein determined i n t h i s study i s 130-210 nM, comparable with the f i g u r e quoted by Geynet, et a l , (1972) of 360 nM. These r e s u l t s suggest a s l i g h t l y greater a f f i n i t y f o r genistein i n the ewe p i t u i t a r y c y t o s o l than that of c a l f or rat. uterine cytosol however not enough information i s a v a i l a b l e to consider i f t h i s d i f f e r e n c e i s s i g n i f i c a n t . The apparent f o r coumestrol determined for p i t u i t a r y cytosol as 59^61 nM c l o s e l y approaches the figures of Geynet, et a l , (1972) of 42-50 nM f o r uterine cytosol of c a l f and rat suggesting s i m i l a r a f f i n i t y of p i t u i t a r y and uterine estrogen receptors for coumestrol. -92-The q u a l i t a t i v e studies of phyto-estrogen binding i n hypothalamus and amygdala did not determine competitive c h a r a c t e r i s t i c s or e s t r a d i o l binding i n h i b i t i n g e f f i c i e n c y and therefore the determination of a f f i n i t y of the estrogen receptors of these tissues for the phyto-estrogens was. not p o s s i b l e . The i n d i c a t i o n for.hypothalamus cytosol was that a s i m i l a r a f f i n i t y f o r coumestrol was present as was determined i n the p i t u i t a r y . A comparison of the various binding c h a r a c t e r i s t i c s determined for the tissues of t h i s study i s shown i n Table I I . TABLE I I : Comparison of E s t r a d i o l and Phyto-Estrogen Binding i n various tissues from ewes, as determined i n t h i s study. TISSUE CYTOSOL POOL ESTRADIOL ^ GENISTEIN K^ . COUMESTROL Hypothalamus * n. d. <2710 nM s i m i l a r to p i t u i t a r y P i t u i t a r y 0.26*0.12 nM 130-210 nM 59-61 nM Amygdala n. d. <2710 n. d, P i n e a l S p e c i f i c e s t r a d i o l binding not detected Uterus 0.6 nM n.d. n.d. n.d. means not determined i n t h i s study The i s defined i n p h y s i c a l terms as the product of the concentrations of the i n t e r a c t i n g species, i . e . receptor and estrogen, divided by the concen-t r a t i o n of the bound form, i . e . estrogen-receptor complex at equilibrium. Therefore at an estrogen concentration such that the concentration of receptor i n free and bound form i s equal, then the i s equal to the concentration of unbound ligand. -93-E + R t ER, = (E)(R) , so that when (R) = (ER), then (ER) = (E), i . e . , i s equal to the concentration of estrogen at which the receptors (R) are one-half saturated. As the Kj. of a competitive i n h i b i t o r i s equal to the of receptor for that i n h i b i t o r , then the same conditions apply. Therefore the lower the f o r a compound/receptor p a i r , the greater the a f f i n i t y of the receptor for that compound. Thus a comparison of or Kj. values can give a quantitative assessment of the a f f i n i t y of the estrogen receptor for various compounds. As determined i n t h i s study the p i t u i t a r y shows approximately three times greater a f f i n i t y f o r coumestrol than for genistein, and 230 times le s s a f f i n i t y f o r coumestrol and 650 times le s s af-f i n i t y f o r genistein than for e s t r a d i o l . The s i g n i f i c a n c e of these observa-tions i n r e l a t i o n to p h y s i o l o g i c a l conditions w i l l be discussed presently. Other reports of genistein and coumestrol competitions with e s t r a d i o l at the uterine estrogen receptors have determined r e l a t i v e binding a f f i n i t i e s or the concentration of phyto-estrogen necessary to reduce e s t r a d i o l binding 50 percent (Shemesh, et a l , 1972; Shutt and Cox, 1972). Such determinations are a function of e s t r a d i o l concentration present and thus are a q u a l i t a t i v e measure of i n h i b i t i n g e f f i c i e n c y . The apparent values f o r phyto-estrogens determined i n t h i s study show a s i m i l a r trend i n the r e l a t i v e magnitude of i n h i b i t i o n reported for genistein and coumestrol by Shemesh, et a l , (1972) and Shutt and Cox (1972). The apparent values herein reported are equivalent to 16 ng/ml f o r coumestrol and 35-57 ng/ml f o r genistein. Assuming that the i n t r a c e l l u l a r l e v e l s of these compounds are s i m i l a r to blood l e v e l s , that i s , there i s no -94-s p e c i f i c i n t r a c e l l u l a r concentration of compound, then the reported l e v e l s of unconjugated, that i s , " f r e e " phyto-estrogens i n sheep blood are somewhat below the l e v e l s necessary for h a l f maximal saturation of receptors as c a l c u -lated from the data i n t h i s study. However, i n r e l a t i o n to c i r c u l a t i n g e s t r a d i o l l e v e l s i n the picogram per m i l l i l i t e r range, unconjugated phyto-estrogen l e v e l s can reach up to a thousand f o l d greater and thus can be assumed to exert considerable e f f e c t . P a r t i c u l a r l y as competition between estrogen t and phyto-estrogen for receptor i s a function not only of r e l a t i v e a f f i n i t y ** but also concentration. ** For estrogen and i n h i b i t o r (phyto-estrogen), the f r a c t i o n a l binding ( f) of each to receptor i s represented by f^ = (E) and f = (I) . • . , .. ( E ) + K ^ 1 (I) + K T (Weatphal, 1971) A p h y s i o l o g i c a l example i s presented i n the Appendix. In addition, while the major amount of phyto-estrogen i s i n the form of i n a c t i v e glucosiduronates, a portion of the phyto-estrogen conjugates are i n the form of sulphoconjugates and the l e v e l s i n t h i s form exceed plasma "f r e e " l e v e l s (Wong and Cox, 1971). Sulphate conjugates of estrogen play an important r o l e i n s t e r o i d a c t i v i t y (Brooks, et a l , 1978) and thus sulphoncon-jugates of phyto-estrogens may also be of b i o l o g i c a l s i g n i f i c a n c e , (Wong and Cox, 1971). As determined i n t h i s study, phyto-estrogen can i n t e r a c t with estrogen receptors i n ewe p i t u i t a r y , hypothalamus and amygdala. In the case of severe clover disease i t has been demonstrated that permanent e f f e c t s occur i n the hypothalamus, which no longer i s able to respond to estrogen stimulation -95-although the p i t u i t a r y s t i l l responds to exogenous LH-RH (Hearnshaw, et a l , 1972; Findlay, et a l , 1973). It has been speculated that changes i n the estrogen receptors of the hypothalamus and p i t u i t a r y are responsible for t h i s e f f e c t however no mechanism has been proposed (Tang and Adams, 1978). S l i g h t l y reduced expression of behavioural signs of estrus has also been observed i n clover diseased ewes (Adams, 1978). It i s possible that the permanent changes brought about by phyto-estrogens i n the hypothalamus are a r e s u l t of these compounds i n t e r a c t i n g with estrogen receptors i n these t i s s u e s . Direct evidence that phyto-estrogens i n t e r f e r e with gonadotropin release i s d i f f i c u l t to demonstrate, however numerous reports imply that t h i s i s the case and thus may be an important f a c t o r i n temporary i n f e r t i l i t y . 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Palmer and B.E. Howland (1977), "Release of LH i n Anoestrous and C y c l i c Ewes", Journal of Reproduction and F e r t i l i t y 50:319-321. Appendix Figure 1: M i n i s c i n t i l l a t i o n v i a l adapter for Spectronic 20. -115-APPENDIX 2 Lowry Protein Assay Reagents A 2.0% Na 2C0 3 i n 0.1 N NaOH B 0.5% CuSO^'S^O i n 1% Na + or K + t a r t r a t e made from mixing on part of 1% CuSO^-5H20 with one part of 2% t a r t r a t e . C mix 50 ml. A plus 1 ml B, fresh each day D mix 1 part phenol, F o l i n - C i o c a l t e a u , reagent plus 2 parts water. Method f o r Q u a l i t a t i v e Determination Six hundred u l of reagent C was added to s c i n t i l l a t i o n v i a l s containing column eluates, mixed and allowed to stand at room temperature for ten minutes. Then 60 u l of reagent D was added and mixed immediately. V i a l s were allowed to stand twenty minutes and absorbances read, A,--, i n a Spectronic 20 spec-b U U trophotometer-colorimeter with adapter system as i n Appendix Figure A l . A l t e r -n a t i v e l y , peaks were located v i s u a l l y . Adapted from Lowry, et a l , (1951) as i n Chaykin, (1966). -116-APPENDIX 3 Trinitrobenzenesulfonic Acid (TNBS) (method f o r determining amines Materials Buffer: 0.10 M sodium tetraborate ( N a ^ C ^ • lOH^O) , pH 9.3 Reagent: TNBS (2,4,6-trinitrobenzenesulfonic a c i d , (N0 2) 3C 6H 2S0 3H)) Procedure used, as modified for Q u a l i t a t i v e Determination Twenty f i v e mg. of TNBS was dissolved i n 100 mis. of 0.1 M sodium t e t r a -borate b u f f e r , pH 9.3. Seventy-five m i c r o l i t r e s of t h i s reagent was added to each s c i n t i l l a t i o n v i a l of column eluate. V i a l s stood at room temperature for t h i r t y minutes to permit colour development. Location of pro t e i n peaks was v i s u a l l y determined. Adapted from Snyder and Sobocinski, (1975). -117-APPFND1X FIGURE 2 • STEROID ESTROGENS R, R* ESTRADIOL 17 g OH H ESTRONE =0 H ESTRIOL OH OH APPFNniX FlflllRF 7>: DIETHYLSTILBESTROL -118-APPENDIX II An t i Estrogen Compounds Common Number Common Name MRL 41 ICI 46, 474 CI 628 U 11, 100A MER 25 Clomiphene Tamoxiphen Nitromophene c i t r a t e Nafoxidine Ethamoxytriphetol These compounds are reported i n the l i t e r a t u r e either by name or number. The most common usage has been used i n the text. APPENDIX I I I P h y s i o l o g i c a l Example of Estrogen and Phyto-Estrogen Interactions with Ewe P i t u i t a r y Cytosol Estrogen Receptors Assuming maximal peripheral plasma e s t r a d i o l l e v e l to be 15 pg/ml as reported i n Hauger, et a l (1977) and Rawlings, et a l (1978) and the for e s t r a d i o l to be 0.26 nM, then the f r a c t i o n a l occupancy of binding s i t e s , f , would be E as follows: 15 pg/ml = .055 nM f = (E) = .055 = 0.17 or 17% of the receptors s i t e s (E) + .055 + 0.26 would be occupied by e s t r a d i o l at t h i s concentration. Considering the highest preovulatory e s t r a d i o l l e v e l s reported by Scaramuzzi and Land (1978) or 3.9 pg/ml (equivalent to .014 nM) then, fT7 = (E) = 0.014 = 0.051 or 5% of the receptor s i t e s (E) + 0.014 + 0.26 would be occupied with e s t r a d i o l at t h i s concentration. -119-For the phyto-estrogen, coumestrol, assuming a blood l e v e l of 5 ng/ml (Lindner, 1967) with a = 60 nM, then the f r a c t i o n a l occupancy of binding s i t e s , f-j., would be: (5 ng/ml being equivalent to 19 nM) f = (I) = 19 nM = .24 or 24% of the estrogen receptors (I) + K 19 nM + 60 NM of the ewe p i t u i t a r y would be occupied with coumestrol at t h i s concentration and i t can be assumed that t h i s would exert considerable e f f e c t on the estrogen dependent mechanisms present. 

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