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Comparative effects of naturally occurring, synthetic and plant estrogens on uterine metabolism Kitts, David D. 1976

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THE COMPARATIVE EFFECTS OF NATURALLY OCCURRING, SYNTHETIC AND PLANT ESTROGENS ON UTERINE METABOLISM by D a v i d Dale K i t t s B . S c , U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1974 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n The F a c u l t y o f Graduate S t u d i e s (Department o f A n i m a l S c i e n c e ) We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA September, 1976 © D a v i d Dale K i t t s , 1976 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r ee t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r ag ree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y pu rpo se s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . David D. K i t t s Department o f The U n i v e r s i t y o f B r i t i s h Co l umb i a 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date Jefite/nier 2o . ABSTRACT V a r i o u s e s t r o g e n s b i o l o g i c a l l y e q u i v a l e n t t o 1.0 jig e s t r a d i o l - 1 7 3 were a d m i n i s t e r e d i n t r a p e r i t o n e a l l y t o immature f e m a l e r a t s t o m o n i t o r t h e r e l a t i v e d o s e and t i m e c o u r s e e f f e c t s on w a t e r i m b i b i t i o n . E s t r a d i o l - 1 7 3 , e s t r i o l and d i e t h y s t i l -b e s t r o l (DES) were t h e s t r o n g e s t e s t r o g e n s i n i n d u c i n g t i s s u e edema. P l a n t e s t r o g e n s , g e n i s t e i n and c o u m e s t r o l , i n p u r e c r y s t a l l i n e f o r m were a p p r o x i m a t e l y 10~3 t i m e s as p o t e n t i n s t i m u l a t i n g w a t e r i m b i b i t i o n a s e s t r a d i o l - 1 7 3 . The a d m i n i s t r a t i o n o f e s t r a d i o l - 1 7 3 , g e n i s t e i n and coumes-t r o l was shown t o enhance t h e p e r m e a b i l i t y o f u t e r i n e v a s c u l a t u r e as i n d i c a t e d by t h e d i f f u s i o n o f i n t r a v e n o u s l y i n f u s e d I n d i a i n k . L a r g e d o s e s (5.0 .ug) o f e s t r a d i o l - 1 7 3 were a d m i n i s t e r e d t o immature f e m a l e r a t s t o measure t h e r a t e o f s y n t h e s i s o f RNA and DNA i n t h e u t e r u s . I t was o b s e r v e d t h a t t h e u t e r i n e c e l l c y c l e was s i g n i f i c a n t l y (P ^ 0.025) r e d u c e d a f t e r t h e a d m i n i s t r a t i o n o f e s t r a d i o l - 1 7 3. N e t a c c u m u l a t i o n o f RNA and DNA was shown t o o c c u r a f t e r 12 and 24 h o u r s r e s p e c t i v e l y . 3 The i n c o r p o r a t i o n o f [5,6- H] u r i d i n e i n t o t h e u t e r i n e t i s s u e was s t u d i e d by a d m i n i s t e r i n g n a t u r a l , s y n t h e t i c and p l a n t e s t r o g e n s i n t r a p e r i t o n e a l l y t o immature f e m a l e r a t s . A f t e r a s i x h o u r p e r i o d t h e r e was a d e c r e a s e d u t e r i n e s p e c i f i c a c t i v i t y 3 o f [5,6- H] u r i d i n e i n e s t r o g e n t r e a t e d r a t s when compared t o c o n t r o l a n i m a l s . E s t r a d i o l - 1 7 3 p r o d u c e d t h e g r e a t e s t r e d u c t i o n i n t h e s p e c i f i c a c t i v i t y o f t h e h y d r o l y z e d RNA w h i l e DES, e s t r i o l and e s t r a d i o l - 1 7 o t p r o d u c e d r e l a t i v e l y s m a l l e r r e d u c t i o n s . i The r e d u c t i o n of s p e c i f i c a c t i v i t y s i x hours a f t e r i n v i v o p u l s i n g w i t h e s t r a d i o l - 1 7 3 was probably due to a d i l u t i o n e f f e c t a s s o c i a t e d w i t h changes o c c u r r i n g i n c e l l p e r m e a b i l i t y . G e n i s t e i n 3 and coumestrol a l s o reduced the s p e c i f i c a c t i v i t y of C5,6- H] u r i d i n e s i m i l a r to t h a t observed w i t h e s t r i o l . E x t r a c t s of a l f a l f a hay and soybean meal were analyzed q u a n t i t a t i v e l y and q u a l i t a t i v e l y f o r p l a n t estrogens. S h o r t i n v i v o p u l s i n g of 30 minutes of v a r i o u s estrogens was employed to 3 determine the uptake of C5,6- HH u r i d i n e by the u t e r i n e t i s s u e . E s t r a d i o l - 1 7 3 , p u r i f i e d g e n i s t e i n , a l f a l f a and soybean e x t r a c t s 3 were found to i n c o r p o r a t e L~5,6- Ej u r i d i n e a t g r e a t e r r a t e s than c o n t r o l groups. TABLE OF CONTENTS Page Abstract 1 L i s t of Tables v L i s t of Figures V 1 L i s t of Appendix Figures v i i Acknowledgements v i i i Introduction 1 Literature Review 3 1. Antiestrogens 3 2. E f f e c t of Estrogen on C e l l Metabolism 7 3. Phytoestrogens . 17 Experimental Procedure 2 6 Experiment A : The Comparative E f f e c t s of Estrogens and Phytoestrogens on Water Imbibition and Hyperemia of the Rat Uterus 26 Introduction 26 Materials and Methods 2 8 Results 30 Discussion 31 Conclusions, 33 Experiment B : E f f e c t of Estrogen on Hyperemia of Uterine Tissue 40 Introduction 40 Materials and Methods 40 Results 41 Discussion and Conclusions 41 Experiment C : E f f e c t s of Estradiol-17g on RNA and DNA Synthesis i n Immature Uterine Tissues . ... ... . .45 Introduction 45 i i i Page M a t e r i a l s and Methods 46 R e s u l t s 48 D i s c u s s i o n 50 C o n c l u s i o n s 52 E x p e r i m e n t D : E f f e c t s o^ E s t r o g e n s and P h y t o e s t r o g e n s on t h e I n c o r p o r a t i o n o f H U r i d i n e i n t o RNA by t h e U t e r u s - S i x Hour In. V i v o P u l s i n g . . . : 58 I n t r o d u c t i o n 5 8 M a t e r i a l s and Methods 60 R e s u l t s 62 D i s c u s s i o n 64 C o n c l u s i o n s 66 E x p e r i m e n t E : E f f e c t o f E s t r o g e n s and P h y t o e s t r o g e n s on t h e I n c o r p o r a t i o n o f T r i t i a t e d U r i d i n e I n t o RNA -S h o r t In. V i v o P u l s i n g 70 I n t r o d u c t i o n 70 M a t e r i a l s and Methods 71 R e s u l t s 74 D i s c u s s i o n 75 C o n c l u s i o n s 76 E x p e r i m e n t F : E a r l y E f f e c t s o f E s t r o g e n s and P l a n t E x t r a c t s on t h e I n c o r p o r a t i o n o f T r i t i a t e d U r i d i n e I n t o RNA by U t e r i n e T i s s u e 80 I n t r o d u c t i o n 80 M a t e r i a l s and Methods 80 R e s u l t s 82 D i s c u s s i o n 83 C o n c l u s i o n s 84 G e n e r a l C o n c l u s i o n s 89 B i b l i o g r a p h y 92 Appendix - . 102 i v LIST OF TABLES T a b l e Page 1 E f f e c t o f t ime on w a t e r i m b i b i t i o n by r a t u t e r i n e t i s s u e s f o l l o w i n g a d m i n i s t r a t i o n o f e s t r a d i o l - 1 7 3 (Expt. A-I) . . 37 2 E f f e c t o f dose on water i m b i b i t i o n by r a t u t e r u s t i s s u e s f o l l o w i n g a d m i n i s t r a t i o n o f e s t r a d i o l - 1 7 3 (Expt. A-2) 38 3 E f f e c t s o f s t e r i o d and p l a n t e s t r o g e n s on w a t e r i m b i b i t i o n by r a t t u e r i n e t i s s u e s i x hours f o l l o w i n g a d m i n i s t r a t i o n (Expt. A-3) 39 4 RNA and DNA c o n c e n t r a t i o n s i n homogenates of d i f f e r e n t immature r a t u t e r i n e w e i g h t s (Expt. C) . . 56 5 E f f e c t o f t i m e on RNA and DNA s y n t h e s i s by r a t u t e r i n e t i s s u e (Expt. C ) . . . . . 57 6 The i n v i t r o i n c o r p o r a t i o n o f r a d i o a c t i v i t y i n d i f f e r e n t f r a c t i o n s o f r a t u t e r i , s i x hours f o l -l o w i n g i n v i v o e s t r o g e n a d m i n i s t r a t i o n (Expt. D) . . 68 7 RNA c o n t e n t and s p e c i f i c a c t i v i t y o f RNA e x t r a c t e d from r a t u t e r i s i x hours f o l l o w i n g e s t r o g e n a d m i n i -s t r a t i o n (Expt. D) 69 8 Rf v a l u e s o f s t a n d a r d p h y t o e s t r o g e n s and t h o s e o b t a i n e d from p l a n t e x t r a c t s as measured by T h i n L a y e r Chromatography (Expt. E) 79 9 The in_ v i t r o i n c o r p o r a t i o n o f r a d i o a c t i v i t y i n d i f f e r e n t f r a c t i o n s o f r a t u t e r i , 30 minutes f o l -l o w i n g i n v i v o e s t r o g e n a d m i n i s t r a t i o n (Expt. F) . . 87 10 I n c o r p o r a t i o n o f [5,6 H] u r i d i n e i n t o t h e h y d r o -l y z e d RNA f r a c t i o n o f immature female r a t u t e r i 30 minutes f o l l o w i n g e s t r o g e n a d m i n i s t r a t i o n . . . . 88 v LIST OF FIGURES F i g u r e Page 1 Temporal sequence o f m e t a b o l i c e v e n t s i n u t e r i o f immature or o v a r i e c t o m i z e d r a t s a f t e r i n v i t r o a d m i n i s t r a t i o n o f e s t r o g e n 9 2 S t r u c t u r e and m e t a b o l i s m o f e s t r o g e n i c compounds . . 19 3 Wet u t e r i n e w e i g h t s i x hours f o l l o w i n g e s t r o g e n a d m i n i s t r a t i o n (Expt. A-3) 35 4 E f f e c t o f t i m e on water i m b i b i t i o n i n r a t u t e r i n e t i s s u e s f o l l o w i n g a d m i n i s t r a t i o n o f e s t r a d i o l - 1 7 3 (Expt. A - l ) . . . " . . 36 5 The d i s t r i b u t i o n o f I n d i a i n k i n t h e u t e r i n e v a s -c u l a t u r e o f c o n t r o l and e s t r a d i o l - 1 7 3 t r e a t e d r a t s (Expt. B) x 100 43 6 The d i s t r i b u t i o n o f I n d i a i n k i n t h e u t e r i n e v a s -c u l a t u r e o f c o u m e s t r o l and g e n i s t e i n t r e a t e d r a t s (Expt. B) x 100 44 7 S t a n d a r d c u r v e s f o r RNA and DNA (Expt. C) 5 3 8 RNA and DNA c o n c e n t r a t i o n i n homogenates o f d i f f e r e n t immature r a t u t e r i n e w e i g h t s (Expt. C) 54 9 E f f e c t o f t ime on RNA and DNA s y n t h e s i s i n r a t u t e r i n e t i s s u e f o l l o w i n g e s t r o g e n a d m i n i s t r a t i o n (Expt. C) 55 10 Q u a l i t a t i v e e x a m i n a t i o n o f p h y t o e s t r o g e n c o n t e n t by t h i n l a y e r chromatography 78 11 C o m p e t i t i v e b i n d i n g a s s a y of p h y t o e s t r o g e n s (Expt. E) 86 v i LIST 0F: APPENDIX FIGURES Figure Page A Nucleic Acid Extraction Procedure 103 B Extraction of Phytoestrogens From Plant Materials 104 C Quench Correction Curve for Tritium 105 v i i AC KNOWLEDGEMENTS The a u t h o r w i s h e s t o e x p r e s s h i s g r a t i t u d e t o t h e Department o f A n i m a l S c i e n c e and t h e U n i v e r s i t y o f B r i t i s h Columbia R e s e a r c h Committee f o r f i n a n c i a l s u p p o r t o f t h i s r e s e a r c h . The accommo-d a t i o n p r o v i d e d by Dean W.D. K i t t s , t h e t h e n chairman o f A n i m a l S c i e n c e i s g r a t e f u l l y acknowledged. I am i n d e b t e d t o Dr. C R . K r i s h n a m u r t i f o r h i s u s e f u l sug-g e s t i o n s , w h i c h were v i t a l i n the p r e p a r a t i o n o f t h i s t h e s i s . The a u t h o r would a l s o l i k e t o thank Ms. F r a n c e s Newsome f o r her t e c h n i c a l a s s i s t a n c e and her many u s e f u l s u g g e s t i o n s . S p e c i a l t h a n k s a r e extended t o M i s s E. R o s s i t e r f o r her a s s i s t a n c e and encouragement d u r i n g t h e c o u r s e o f t h e s t u d y . v i i i 1..' INTRODUCTION F o l l o w i n g t h e p u b l i c a t i o n by J a c o b and Monod (1961) o f t h e o p e r o n model f o r r e g u l a t i n g gene e x p r e s s i o n , t h e mechanism o f s t e r o i d hormone a c t i o n h a s become one o f t h e most p o p u l a r a r e a s o f r e s e a r c h i n e n d o c r i n o l o g y and a n i m a l r e g u l a t o r y b i o l o g y . I n t h e l a s t two d e c a d e s an i n c r e a s i n g amount o f e v i d e n c e h a s a c c u m u l a t e d s u g g e s t i n g t h a t v a r i o u s hormones r e g u l a t e g r o w t h , d i f f e r e n t i a t i o n and m e t a b o l i c a c t i v i t y i n t a r -g e t t i s s u e s t h r o u g h t h e i r e f f e c t s on RNA m e t a b o l i s m . To d a t e t h e r e i s an i n c r e d i b l e amount o f l i t e r a t u r e a v a i l a b l e on t h i s s u b j e c t , however no u n i f y i n g c o n c e p t has emerged. The most a p p a r e n t s e q u e n c e o f e v e n t s w o u l d be f o r a hormone t o a c t i v a t e o r r e p r e s s c e r t a i n f u n c t i o n a l l y l i n k e d genes and a l l o w t r a n s -c r i p t i o n o f new s p e c i e s o f mRNA w h i c h w o u l d t h e n c o d e f o r s y n t h e s i s o f s p e c i f i c p r o t e i n s . I n a d d i t i o n t o t h i s , t h e r e a p p e a r s t o be a s e c o n d f u n c t i o n o f most a n a b o l i c hormones, t h e r e l e a s e o f b i o g e n i c amines ( i . e . h i s t a m i n e ) . S zego and D a v i s (1967) r e p o r t e d t h a t t h e e a r l i e s t known e f f e c t s o f e s t r o g e n s were a s s o c i a t e d w i t h t h e u t e r i n e c e l l membrane and h i s t a m i n e r e l e a s e . I n c r e a s e s i n 3 J 5 ' c y c l i c AMP i n t h e u t e r i n e c e l l membrane were o b s e r v e d 15 s e c o n d s a f t e r e x p o s u r e t o e s t r a d i o l -173. I n a d d i t i o n t o t h e n a t u r a l l y o c c u r r i n g e s t r o g e n s , e s t r o -g e n i c a c t i v i t y has b e en r e p o r t e d i n many f o r a g e s and o t h e r p l a n t m a t e r i a l s (Guggolz e t aJL. 1 9 6 1 ; Beck, 1964; A l l i s o n and K i t t s , 1 9 6 4 ) . B e n n e t t e t a l . (1946) f i r s t r e p o r t e d t h e p r e s e n c e o f p h y t o e s t r o g e n s i n s u b t e r r a n e a n c l o v e r ( T r i f o l i u m 2. subterraneum). Since that time plant estrogens have been re-cognized as agents responsible for the i n f e r t i l i t y of grazing stock. Plant constituents which are regarded as estrogenic and are responsible for many reported cases of i n f e r t i l i t y i n animals grazing estrogenic forages and pasture.legumes are the isoflavones, genistein, biochanin A, daidzein, equol and formononetin (Bradbury and White, 1954; Cheng et a l . 1953(b); Cheng et a l . 1954) and coumestrol (Cheng et al_. 1953 (a); Bickoff et a l . 1957). The estrogenic potential present i n various plant sources i s generally much weaker than naturally occurring steroid or synthetic estrogens. However, the large amount of phytoestrogens ingested during grazing may elevate the plasma leve l s of estrogen to a degree which would i n t e r f e r e with reproductive processes. The objectives of t h i s study were to e s t a b l i s h the base l i n e s regarding the time course and dose ef f e c t s of estradiol-17g and i t s various analogues on uterine tissue metabolism and secondly to employ an i n v i t r o bioassay which would elucidate the i n i t i a l metabolic events occurring i n the c e l l a f t e r estro-gen, phytoestrogen or plant extract administration. 3. REVIEW OF LITERATURE 1. Antiestrogens The word "antiestrogen" i s a widely used and grossly mis-leading term used to describe compounds which e x i s t n a t u r a l l y (Cook and K i t t s , 1964; Chow et a l . 1972) or are produced syn-t h e t i c a l l y (Callantine et a_l. 1968; Davidson et aJL. 1968; Humphrey, 1968) and which decrease the response of female reproductive organs to estrogen administered or injested con-comitantly. Estrogenicity of a compound i s usually assessed by gross, h i s t o l o g i c and biochemical changes i n the uterus, cervix and vagina of ovariectomized animals. D i f f i c u l t y i n defining the degree of estrogenicity ari s e as a r e s u l t of d i f -ferent thresholds of the compound required for e l i c i t i n g an estrogenic response. The dose, mode of administration and physiological status of the animal w i l l govern the absorption and retention of the hormone by a p a r t i c u l a r target organ. These variables play s i g n i f i c a n t roles i n determining an estro-genic response and therefore whether or not a compound i s a n t i -estrogenic . Kato et a l . (1968) and E i s i n f e l d (1970) reported that the binding of estrogens to receptors i n the hypothalamus and p i t u i t a r y as well as other target tissues constitutes an impor-tant mechanism regulating reproduction. The mechanism whereby antiestrogens i n h i b i t b i o l o g i c a l responses to endogenous estrogens involves the competition for s p e c i f i c macromolecular components i n the uterus (Wyss et al_. 1968) p i t u i t a r y and hypo-thalamus (Kato and V i l l e e , 1967); Leavitt et a l . 1973). 4. Emmens e t a l . (1960) r e p o r t e d t h a t s t e r o i d s and n o n s t e r o i d s are h i g h l y a c t i v e when administered subcutaneously o r when l o c a l l y i n j e c t e d a g a i n s t estrogens. Nonsteroids which are w e l l known an t a g o n i s t s of estrogens i n c l u d e DES and mesobutesterol w h i l e androgens and p r o g e s t i n s are c o n s i d e r e d c l a s s i c a l s t e r o i d a n t a g o n i s t s . The mode of a c t i o n of a n t i e s t r o g e n s i s b e l i e v e d by many workers (Emmens e t a l . 196 0; Kato e t a l . 1968; Newsome and K i t t s , 1975) to e n t a i l a s u c c e s s f u l c o m p e t i t i o n w i t h the n u c l e a r r e c e p t o r s as w e l l as to c y t o s o l r e c e p t o r s . They prevent the i n i t i a t i o n of some r e l a t i v e l y e a r l y events o c c u r r i n g i n the c e l l which are the r e s u l t o f m o d i f i c a t i o n s i n the t r a n s p o r t of e s t r a d i o l - 1 7 3 through c e l l u l a r and/or n u c l e a r membranes (Emmens e t a l . 1960; Rochefort and Capony, 1972). Rochefort and Capony (1972) r e p o r t e d t h a t the i n h i b i t i o n r e s u l t e d from a n t i e s t r o g e n s competing wi t h e s t r a d i o l - 1 7 3 f o r b i n d i n g s i t e s i n the t a r g e t organ. T h i s would be an example of c o m p e t i t i v e i n h i b i t i o n of the r e c e p t o r s i t e . A n t i e s t r o g e n s may a l s o i n h i b i t the r e c e p t o r s i t e n o n c o m p e t i t i v e l y by ca u s i n g a r e d u c t i o n of the product formed or p r e v e n t i n g uptake and r e t e n t i o n o f the hormone. M o d i f i c a t i o n by a n t i e s t r o g e n s o f the met a b o l i c steps subsequent to the i n i t i a l hormonal stimulus may a l s o be r e s p o n s i b l e i n a l t e r i n g the p h y s i o l o g i c a l a c t i o n o f an estrogen (Rochefort and Capony, 1972). In t h i s case a n t i e s t r o g e n s would have a modi-f y i n g e f f e c t on the secondary response of the estrogen, but no r e a l e f f e c t on the primary a c t i o n o f the hormone. These a n t i e s t r o g e n s which reduce the c o n c e n t r a t i o n of estrogen a t the s i t e o f the t a r g e t t i s s u e s may do so by forming 5. a durable complex of low b i o l o g i c a l a f f i n i t y with estrogenic binding s i t e s (Callantine et a l . 196 8). A l t e r n a t i v e l y they can form a s h o r t - l i v e d complex with the binding s i t e s which are too transient to exert any b i o l o g i c a l e f f e c t such as growth stimu-l a t i o n (Rochefort and Capony, 1972). Nevertheless, the avai-l a b i l i t y of receptor s i t e s for c i r c u l a t i n g estrogens i s reduced. Antiestrogens i n h i b i t the binding of estrogens to t h e i r receptor s i t e s i n varying degrees (Lee, 1974). One of the major factors governing the binding a f f i n i t y of various a n t i -estrogens i s the structure of the i n d i v i d u a l compounds. Huggins 3 and Jenson (1955) showed that maximum competition with H estradiol-17g for receptor s i t e s i n the uterine cytosol depends strongly on the presence of phenolic hydroxyl groups located on the 3' p o s i t i o n . They reported that substituents at the C-16 and C-17 position i n the D-ring may also a f f e c t binding, and v a r i a t i o n from the 17-hydrcxyl group of e s t r a d i o l resulted i n reduced a f f i n i t y . Shutt and Cox (1972) studied the mechanism of phytoestrogen action and reported that within the isoflavone series, the presence of a phenolic hydroxyl group i n both rings A and B was associated with the highest r e l a t i v e binding a f f i n i t y . The presence of an additional 5' hydroxyl group i n genistein increased i t s a f f i n i t y for binding. Upon methylation to biochanin A there was a sharp decrease i n binding a f f i n i t y . I t appears from these reports that to be capable of competing with endogenous estrogens for similar protein subunits an a n t i -estrogen must be s t r u c t u r a l l y s i m i l a r to the naturally occurring estrogens. The degree of hydrophobic bonding i s important i n regard 6. t o t h e b i n d i n g a f f i n i t i e s o f v a r i o u s e s t r o g e n s . D i m e t h y l s t i l -b e s t r o l (DMS) and m e s o - b u t e s t r o l d i f f e r from t h e i r more p o t e n t c o u n t e r p a r t s DES and m e s o - h e x o e s t r o l o n l y by the s i z e o f t h e i r h y d r o p h o b i c h y d r o c a r b o n c h a i n . The dose o r c i r c u l a t o r y l e v e l o f a n t i e s t r o g e n s i n t h e plasma may a l s o have an e f f e c t on t h e b i n d i n g a f f i n i t i e s . C a l l a n t i n e cat a l _ . (1968) r e p o r t e d t h a t a maximum response o f a hormone i s c h a r a c t e r i z e d by s a t u r a t e d r e c e p t o r s i t e s . The degree o f r e sponse i s t h u s p r o p o r t i o n a l t o t h e amount i n j e c t e d . The s t e r o i d - p r o t e i n i n t e r a c t i o n i n t h e c y t o p l a s m i s a random p r o c e s s and i s a n e c e s s a r y s t e p f o r the f o r m a t i o n o f e s t r o g e n p r o t e i n complex. The p r e s e n c e o f an a n t i e s t r o g e n would r e s u l t i n t h e r e d u c t i o n o f e s t r o g e n s a t u r a t i o n . I n t e r f e r e n c e w i t h v a r i o u s p h y s i o l o g i c a l e v e n t s , a s s o c i a t e d w i t h r e p r o d u c t i v e f a i l u r e due t o t h e e f f e c t o f numerous a n t i -e s t r o g e n s , has been documented (Engle e t a l _ . 1957; L e a v i t t and W r i g h t , 1965; Cox and Braden, 1974). Kato e t a l . (1968) r e p o r t e d t h a t clomiphene, an a n t i e s t r o g e n p o s s e s s i n g a n t i -f e r t i l i t y c h a r a c t e r i s t i c s s u p p r e s s e d t h e p i t u i t a r y f u n c t i o n and g o n a d o t r o p h i n r e l e a s e . Davidson e t a l . (1968) r e p o r t e d t h a t d a i l y doses of clomiphene i n h i b i t e d e s t r a d i o l - 1 7 g and t h e s e c r e t i o n o f l u t e n i z i n g hormone. T h i s was found t o have an e f f e c t on s u c c e s s f u l i m p l a n t a t i o n w h i c h r e q u i r e s an optimum c o n c e n t r a t i o n o f e s t r o g e n as w e l l as p r o g e s t e r o n e . S e g a l e t a l . (1958) demonstrated t h a t clomiphene was b l a s t o t o x i c and p r e -v e n t e d i m p l a n t a t i o n by i t s e f f e c t on t h e endometrium as w e l l as by damaging the z y g o t e a t b i o c h e m i c a l l e v e l s . P r a s a d e t a l . (1965) suggested t h a t most a n t i e s t r o g e n s were not z y g o t o x i c , but t h e y p r e v e n t e d i m p l a n t a t i o n by t h e i r e f f e c t s on t h e m a t e r n a l 7. environment. C a r t e r e t a i . (1955) w o r k i n g w i t h p h y t o e s t r o g e n s demon-s t r a t e d t h a t g e n i s t e i n when f e d t o mice a t 0.02% o f d i e t i n d u c e d the premature opening o f the c e r v i x and r e s u l t e d i n fewer l i t t e r s b e i n g b o r n . S i m i l a r s t u d i e s r e g a r d i n g m a t e r n a l environment a l t e r a t i o n s , due t o a n t i e s t r o g e n s were r e p o r t e d by F i n n (1966) and Humphrey (1968). I m p l a n t a t i o n and t h e i n d u c t i o n o f deciduomata i n i n t a c t o r o v a r i e c t o m i z e d mice a r e s t r o n g l y depend-ent on optimum e s t r o g e n and p r o g e s t e r o n e l e v e l s . F i n n (1966) demonstrated t h a t d e c i d u a l i z a t i o n i s even more dose dependent t h a n i m p l a n t a t i o n . 2. E f f e c t o f E s t r o g e n on C e l l M e t a b o l i s m E s t r o g e n i c hormones e x e r t an i n f l u e n c e on t h e m e t a b o l i s m o f many t a r g e t organs by b i n d i n g e f f e c t i v e l y t o i n t r a c e l l u l a r components. There e x i s t s a s t r o n g , r e v e r s i b l e a s s o c i a t i o n between t h e c i r c u l a t o r y hormone and i t s r e c e p t o r s i t e ( T o f t and G o r s k i , 1966; Jenson e t a l . 1969; Means and O'Malley, 1972; O'Malley and S c h r a d i e r , 1976). T h i s a s s o c i a t i o n i s t h e p r i m a r y s t e p i n u t e r o t r o p h i c p r o c e s s e s and i s governed by two main c r i t e r i a : 1. The uptake o f t h e hormone i s not s a t u r a t e d and i s independent o f any h y p e r p h y s i o l o g i c a l l e v e l . 2. The r e t e n t i o n o f the hormone i s s a t u r a t e d and i s dependent on t h e dose whether o r not i t exceeds the p h y s i o l o g i c a l l e v e l . H a m i l t o n (1963) demonstrated t h a t t h e r a t u t e r u s p o s s e s s e s the c a p a c i t y under e s t r o g e n i c s t i m u l u s t o s y n t h e s i z e 8. RNA i n v i v o and i n v i t r o by mechanisms s i m i l a r t o t h o s e p r e -v i o u s l y r e p o r t e d i n t h e r a t l i v e r n u c l e i . The sequence of e v e n t s f o l l o w i n g the a d m i n i s t r a t i o n o f e s t r a d i o l - 1 7 g i s i n i t i a t e d a t t h e c e l l u l a r l e v e l and p r o g r e s s e s t o t h e genome l e v e l r e s u l t i n g i n t h e s t i m u l a t i o n o f the s y n t h e s i s o f RNA, p r o t e i n and e v e n t u a l l y DNA and c e l l u l a r d i v i s i o n ( F i g . 1 ) . Two t h e o r i e s have been proposed by G o r s k i and Raker (1974) t o e x p l a i n the mode o f a c t i o n o f e s t r o g e n s . The "Domino Theory" d e s c r i b e s t h e e a r l y r e s p o n s e s o f e s t r o g e n ( c y t o s o l b i n d i n g and i n d u c e d p r o t e i n - R N A s y t h e s i s ) w h i c h s e t o f f l a t e r c e l l u l a r e v e n t s . The " S u s t a i n e d Output Theory" d e s c r i b e s t h e l a t e r e v e n t s which a r e dependent on t h e c o n t i n u e d p r e s e n c e o f e s t r o g e n i n t h e n u c l e u s and on t h e e a r l y e v e n t s . G o r s k i and Raker (1974) demonstrated t h a t b o t h e s t r a d i o l - 1 7 3 and e s t r i o l have the same e f f e c t on e a r l y r e s p o n s e s (2-4 hours) o f t h e r a t u t e r u s . However, a t 18-24 hours e s t r i o l had l i t t l e o r no e f f e c t , i n c o n t r a s t t o t h e 300% i n c r e a s e o v e r th e c o n t r o l caused by e s t r a d i o l - 1 7 3. These workers suggested t h a t t h e p r e s e n c e o f an e s t r o g e n and the s u s t a i n e d o u t p u t of some c r i t i c a l f a c t o r s seems t o be e s s e n t i a l f o r t h e f u l l p h y s i o l o g i c a l e f f e c t s o f e s t r o g e n i c hormones. The i n i t i a l r esponse o f a hormone i s i t s n o n c o v a l e n t b i n d i n g t o a s e r i e s o f c a r r i e r p r o t e i n s r e s p o n s i b l e f o r t r a n s -p o r t i n g t h e hormone t o t h e genome o f i t s r e s p e c t i v e t a r g e t organ. P r e s e n t i n t h e e u k a r y o t i c organism a r e two t y p e s o f p r o t e i n s w h i c h a r e i m p o r t a n t i n t h e t r a n s p o r t and b i n d i n g o f e s t r a d i o l - 1 7 3. Serum p r o t e i n a l b u m i n i s t h e p r i n c i p a l b i n d i n g p r o t e i n i n v o l v e d i n the t r a n s p o r t o f the s t e r o i d t o i t s t a r g e t o r g a n . The UTERINE CELL METABOLISM 36 72 360 1 5 10-20 30 SEC. SEC. SEC. HOUR HOURS HOURS HOURS F_ig. 1. T e m p o r a l s e q u e n c e o f m e t a b o l i c e v e n t s i n U t e r i o f i m m a t u r e o r o v a r i e c t o m i z e d r a t s a f t e r i n v i v o a d m i n i s t r a t i o n o f e s t r o g e n 10. c i r c u l a t i n g s t e r o i d s a r e a l s o p r e s e n t i n a c o m p l e x i n v o l v i n g a g l y c o p r o t e i n w i t h t h e c a r b o h y d r a t e m o i e t y b e i n g g l u c u r o n i c a c i d . T h i s a s s o c i a t i o n w i t h g l u c u r o n i c a c i d i s r e f e r r e d t o a s t h e c o n j u g a t e d f o r m o r i n a c t i v e f o r m o f t h e hormone and c l e a v a g e i s r e q u i r e d b e f o r e i t c a n e n t e r t h e c e l l ( J e n s o n a n d DeSombre, 1 9 7 3 ) . The s e c o n d b i n d i n g p r o t e i n i n v o l v e s t h e s e l e c t i v e u p -t a k e o f t h e s t e r o i d by t h e t a r g e t o r g a n (Chamness, 1 9 7 2 ) . T a l w a r e t a l . (1968) c h a r a c t e r i z e d t h e n o n c o v a l e n t l i n k a g e b e t w e e n t h e s t e r o i d a n d i t s s p e c i f i c c a r r i e r r e c e p t o r i n t h e t a r g e t o r g a n t o be g r e a t e r i n i t s s p e c i f i c i t y t h a n t h e s e r u m a l b u m i n b i n d i n g . T a r g e t t i s s u e s o f e s t r a d i o l - 1 7 g p o s s e s s r e c e p t o r s i t e s c a p a b l e o f r e c o g n i z i n g and b i n d i n g o n t o t h e hormone. The mech-a n i s m b y w h i c h s t e r o i d s p e n e t r a t e t h e c e l l membrane i s h y p o t h e s i z e d t o be a f a c i l i t a t e d d i f f u s i o n ( K a t z e n e l l e n b o g e n and G o r s k i , 1 9 7 5 ) . A f t e r e n t r y i s made, t h e hormone i s a d s o r b e d b y s p e c i f i c p r o t e i n s p o s s e s s i n g h i g h a f f i n i t i e s . The r e c e p t o r p r o t e i n b i n d s e s t r a d i o l - 1 7 B a n d DES w i t h h i g h e r a f f i n i t y and g r e a t e r s p e c i f i t y t h a n e s t r a d i o l - 1 7 a , p r o g e s t e r o n e a n d t e s t o s t e r o n e ( T e r e n i u s , 1 9 6 9 ) . T h e r e c e p t o r , a t e r m u s e d t o d e s -c r i b e a p a r t i c u l a r p r o t e i n p r e s e n t i n t h e t i s s u e a n d whose i n t e r -a c t i o n w i t h t h e s t e r o i d r e s u l t s i n a hormone i n d u c e d r e s p o n s e , i s a m a c r o m o l e c u l e w i t h a minimum m o l e c u l a r w e i g h t o f 100,000 ( G o r s k i a n d R a k e r , 1 9 7 4 ) . T o f t a n d G o r s k i (1966) u s i n g e x p l i c i t s e d i m e n t a t i o n p r o f i l e s o f l o w s a l t a n d s u c r o s e g r a d i e n t s , r e p o r t e d t h a t t h e s t e r o i d hormone a s s o c i a t e s s p o n t a n e o u s l y w i t h t h e e x t r a c e l l u l a r p r o t e i n t o f o r m a 8S (200,000 d a l t o n s ) r e c e p t o r - s t e r o i d c o m p l e x . H i g h e r s a l t c o n c e n t r a t i o n s a n d t h e u s e o f u r e a r e s u l t e d i n v a r i o u s s l o w e r s e d i m e n t a t i o n s p e c i e s . They s u g g e s t e d t h a t a s m a l l e r s u b u n i t , 4S (45,000 d a l t o n s ) was t h e n a t i v e e s t r o g e n b i n d i n g u n i t . O ' M a l l e y and Means (1974) a l s o r e p o r t e d t h a t t h e 8S c l a s s i c a l c y t o p l a s m i c e s t r a d i o l - 1 7 3 r e c e p t o r was e s t r o g e n s p e c i f i c and was t r a n s f e r r e d t o t h e n u c l e u s where i t s t i m u l a t e d RNA s y n t h e s i s . J e n s o n and DeSombre (1973) c o n f i r m e d t h a t c y t o p l a s m i c b i n d i n g was n e c e s s a r y f o r t h e t r a n s p o r t a t i o n o f t h e s t e r o i d t o t h e n u c l e u s . The p r o t e i n - s t e r o i d complex i s v e r y s e n s i t i v e t o low t e m p e r a t u r e s (Puca e t a l . 1 9 7 1 ) , p r o t e o l y t i c enzymes, h i g h s a l t c o n c e n t r a t i o n s (Chamness, 1972) and s p e c i f i c i n h i b i t o r s s u c h as v a r i o u s s u l p h y d r y l b l o c k i n g r e a g e n t s ( K i n g e t a l . 1 9 7 1 ) . Raynaud e t a l . (1971), S o l o f e t a l . (1972) and J e n s o n e t a l . (1969) e s t a b l i s h e d t h a t t h e e s t r a d i o l - 1 7 3 r e c e p t o r p r o t e i n p l a c e d u n d e r t h e s e c o n d i t i o n s w i l l d i s s o c i a t e and d e g r a d e t o r e s p e c t i v e 4S s u b u n i t s . I f n o t i n t e r f e r e d w i t h , t h e 8S c o m p l ex, p o s s e s s i n g an a f f i n i t y f o r t h e n u c l e u s w i l l e n t e r i t i n t h i s f o r m . Once p r e s e n t i n t h e n u c l e u s , t h e s t e r o i d r e c e p t o r p r o t e i n u n d e r g o e s a change w h i c h s e d i m e n t s t o a 5S complex a f t e r e x t r a c t i o n w i t h a d i l u t e p o t a s s i u m c h l o r i d e s o l u t i o n . W i l l i a m s and G o r s k i (1971) and J e n s o n and DeSombre (1973) d i s c l o s e d t h a t i n l i v i n g c e l l s b i n d i n g s i t e s a r e bound t o t h e n u c l e u s . The 5S n u c l e a r p r o t e i n i s f o u n d i n t h e a c i d p o r t i o n o f t h e c h r o m a t i n and u n l i k e t h e c y t o p l a s m i c r e c e p t o r s i t e , i t d i f f e r s i n i t s s i z e and a f f i n i t y f r o m t h e s t e r o i d . A n d e r s o n e t a l . (1972) have c o n -f i r m e d t h e p r e s e n c e o f a n u c l e a r p r o t e i n w i t h e x p e r i m e n t s i n -v o l v i n g t r i t i a t e d e s t r a d i o l - 1 7 3 . They showed t h a t w i t h i n t h e n u c l e u s , t h e e s t r o g e n i s bound t o t h e n u c l e a r r e c e p t o r p r o t e i n w h i c h i s s m a l l e r i n s i z e a n d g r e a t e r i n i t s a f f i n i t y f o r t h e s t e r o i d t h a n t h e c y t o p l a s m i c r e c e p t o r p r o t e i n . The n u c l e a r and c y t o p l a s m i c r e c e p t o r s h a v e v a r i o u s immun-o l o g i c a l , c h e m i c a l and p h y s i c a l p r o p e r t i e s i n common. The p r o -t e i n m o i e t y i s an e s s e n t i a l p a r t o f t h e b i n d i n g p r o p e r t i e s i n b o t h r e c e p t o r s . A f t e r a p e r i o d f o l l o w i n g t h e a s s o c i a t i o n w i t h t h e c y t o p l a s m i c p r o t e i n , t h e s t e r o i d - p r o t e i n c o m p l e x d i s a p p e a r s f r o m t h e c y t o s o l a n d r e a p p e a r s i n t h e n u c l e i w i t h a s m a l l e r s p e c i f i c i t y b u t g r e a t e r a f f i n i t y . The n u c l e a r p r o t e i n r e c e p t o r s t e r m e d " n e o - r e c e p t o r s " a r e p r e c u r s o r s o f t h e c y t o p l a s m i c r e c e p -t o r s . K i n g e t ajl. (1971) h a v e p r o p o s e d t h a t i t i s t h e r e q u i r e d a b i l i t y o f t h e n u c l e i t o t r a n s f o r m t h e c y t o p l a s m i c r e c e p t o r t o a f o r m w h e r e i t c a n t h e n a t t a c h o n t o t h e c h r o m a t i n . T h i s t r a n s -f o r m a t i o n f r o m c y t o p l a s m i c r e c e p t o r t o a n u c l e a r r e c e p t o r i s a l s o t e m p e r a t u r e d e p e n d e n t a n d s e n s i t i v e t o s u l f h y d r y l b l o c k i n g r e a g e n t s . They a l s o s u g g e s t e d t h a t t h e c h r o m a t i n r e c e p t o r i s p o s s i b l y a h i s t o n e , w h i c h w o u l d e x p l a i n f o r i t s a d d i t i v e s p e c i f i c i t y . T h i s h y p o t h e s i s p r o p o s e s t h a t t h e s t r u c t u r e o f a n a c c e p t o r i s c o m p l i m e n t a r y t o t h e e s t r a d i o l - 1 7 3 r e c e p t o r a n d t h e e f f e c t o f an e s t r o g e n r e s p o n s i v e c e l l w o u l d be d e t e r m i n e d by t h e p o s i t i o n o f t h e l o c u s o f t h e a c c e p t o r on t h e genome. The c o n c e n t r a t i o n o f c y t o p l a s m i c and n u c l e a r r e c e p t o r p r o -t e i n s a p p e a r s t o be a f u n c t i o n o f t h e c i r c u l a t i n g e s t r o g e n l e v e l i n t h e p l a s m a ( S o l o f e t a l . 1972). A f t e r o v a r i e c t o m y t h e a c c e p t o r p r o p e r t y o f t h e n u c l e a r r e c e p t o r i s d e c r e a s e d . K i n g e t a _ l . (1971) d e m o n s t r a t e d t h a t a r e d u c t i o n i n 4S r e c e p t o r : : s i t e s a s w e l l a s a d e c r e a s e i n t h e t o t a l amount o f c y t o p l a s m i c b i n d i n g s i t e s o c c u r r e d a f t e r o v a r i e c t o m y o r h y p o p h y s e c t o m y was performed. T h i s would i n d i c a t e t h a t t h e number o f c y t o p l a s m i c r e c e p t o r s and e s p e c i a l l y t h e 4S component i s c o n t r o l l e d by o v a r i a n and p i t u i t a r y hormones. Ten minutes a f t e r t h e a d m i n i s t r a t i o n o f an e s t r o g e n i c com-pound a p a r t i c u l a r RNA i s s y n t h e s i z e d f o r a s p e c i f i c e s t r o g e n i n d u c e d p r o t e i n (IP) ( G o r s k i e t aJL. 1975). O b s e r v a t i o n s made by N o t i d e s and G o r s k i (1966), Barnea and G o r s k i (1970) and K a t z e n e l l e n b o g e n and G o r s k i (1972) have l e d t o t h e conc e p t t h a t e s t r o g e n s a r e r e s p o n s i b l e f o r t h e i n d u c t i o n o f t h e s y n t h e s i s o f a p a r t i c u l a r u t e r i n e p r o t e i n . T h i s p r o t e i n f r a c t i o n d e t e c t a b l e a f t e r 4 0-60 minutes i s e s s e n t i a l f o r t h e i n c r e a s e i n m e t a b o l i c a c t i v i t i e s o f t h e t i s s u e . P u r i f i c a t i o n o f I P , has shown t h a t i t i s v e r y s i m i l a r i n c h a r a c t e r i s t i c s t o ovalbumin w i t h a b i o -l o g i c a l h a l f l i f e o f n i n e hours. Ruh e t a_l. (1973) demonstrated t h a t t h e s y n t h e s i s o f IP can be r e g u l a t e d o n l y by th o s e compounds whi c h p o s s e s s e s t r o g e n i c p r o p e r t i e s and w h i c h w i l l b i n d t o t h e u t e r i n e e s t r o g e n r e c e p t o r p r o t e i n s . Among s e v e r a l e s t r o g e n i c compounds t e s t e d e s t r a d i o l - 1 7 3, DES, e s t r i o l and e s t r o n e were e f f e c t i v e i n IP s y n t h e s i s i n t h a t o r d e r . P r o g e s t e r o n e and t e s t o s t e r o n e had no e f f e c t . R e c e n t l y G o r s k i e t a l . (1975) r e p o r t e d t h a t t h e l a g p e r i o d o f 40-60 minutes was t h e time n e c e s s a r y f o r mRNA t o be p r o c e s s e d and moved from t h e n u c l e u s t o t h e c y t o p l a s m , where i t a s s o c i a t e s w i t h the polysomes, t h e s i t e o f p r o t e i n s y n t h e s i s . E s t r o g e n i s t h e r e f o r e c o n s i d e r e d t o ind u c e t he s y n t h e s i s o f mRNA whi c h i n t u r n i s e s s e n t i a l f o r s y n t h e s i s o f an in d u c e d p r o t e i n . Though t h e s y n t h e s i s o f RNA as a p r i m a r y e v e n t i n e a r l y a c t i o n o f e s t r o g e n s has been e s t a b l i s h e d , t h e mechanism by whi c h 14. e s t r o g e n i n f l u e n c e s RNA s y n t h e s i s has been s u b j e c t t o q u e s t i o n . I t i s n o t c l e a r w h e t h e r t h e h o r m o n a l s t i m u l a t i o n r e s u l t s f r o m an e f f e c t on t h e c h r o m a t i n t e m p l a t e a c t i v i t y ( B a r k e r and W a rren, 1967), RNA p o l y m e r a s e a c t i v i t y (Maul and H a m i l t o n , 1 9 6 7 ) , t h e t r a n s p o r t o f RNA f r o m t h e n u c l e u s t o t h e c y t o p l a s m ( H a m i l t o n e t a l . .1968), o r a c o m b i n a t i o n o f a l l t h e s e f a c t o r s . DNA d e p e n d e n t RNA p o l y m e r a s e enzymes have been i s o l a t e d f r o m t h e n u c l e o l u s and t h e n u c l e o p l a s m o f e u k a r y o t i c c e l l s . By t h e u s e o f r a d i o -a u t o g r a p h y t h e s e q u e n t i a l s t i m u l a t i o n o f a Mg d e p e n d e n t p o l y -merase by 2.0 h o u r s o c c u r r i n g i n t h e n u c l e o l u s , and a M n + + d e p e n d e n t p o l y m e r a s e by 4.0 h o u r s i n t h e n u c l e o p l a s m was o b s e r v e d i n t h e o v a r i e c t o m i z e d r a t (Maul and H a m i l t o n 1 9 6 7 ) . The s u c c e s s -i v e s t i m u l a t i o n o f t h e s e enzymes and t h e a c c e l e r a t e d s y n t h e s i s o f a l l t y p e s o f RNA i m m e d i a t e l y f o l l o w i n g e s t r o g e n a d m i n i s t r a t i o n p r o v i d e e v i d e n c e t h a t i n c r e a s e d r a t e o f p r o t e i n s y n t h e s i s i s o c c u r r i n g i n t h e u t e r u s . Greenman and Kenny (1964) d e m o n s t r a t e d t h a t o v a r i e c t o m y l o w e r s t h e a c t i v i t y o f t h e u t e r i n e r i b o s o m e s and s u g g e s t e d t h a t e s t r o g e n s a l s o have t h e a b i l i t y t o r e g u l a t e t h e c a p a c i t y o f t h e f u n c t i o n a l r i b o s o m e s t o s y n t h e s i z e p r o t e i n s i n a d d i t i o n t o i n f l u e n c i n g t h e s y n t h e s i s o f r i b o s o m e s . S e v e r a l m e t a b o l i c e v e n t s have been shown t o i n c r e a s e 1-2 h o u r s a f t e r e s t r a d i o l - 1 7 3 a d m i n i s t r a t i o n . G l u c o s e , p h o s p h o l i p i d (Pepe and Yochim, 1971) and t h e a c t i v i t y o f RNA p o l y m e r a s e I (Noteboom and G o r s k i , 1963) a r e among t h e i n i t i a l c h a n g e s n o t i c e d a f t e r a s i n g l e e s t r o g e n i n j e c t i o n . Raynaud et_ a l . (1971) were t h e f i r s t w o r k e r s t o d e m o n s t r a t e an i n v i t r o e f f e c t o f e s t r a d i o l -173 on RNA s y n t h e s i s i n i s o l a t e d u t e r i n e n u c l e i . By i n c u b a t i n g c a l f u t e r i n e c y t o s o l w i t h e s t r a d i o l - 1 7 3 and a d d i n g i s o l a t e d n u c l e i t h e y a s s a y e d f o r RNA p o l y m e r a s e a c t i v i t y and were a b l e t o d e m o n s t r a t e a s u c c e s s f u l i n t e r a c t i o n between t h e s t e r o i d and i t s s p e c i f i c r e c e p t o r s i t e . An i n c r e a s e i n n u c l e a r DNA d e p e n d e n t RNA p o l y m e r a s e a c t i v i t y was n o t e d as w e l l . B a r k e r and W a r r en (1967) r e p o r t e d t h a t a s i n g l e i n t r a v e n o u s i n j e c t i o n o f e s t r a d i o l - 1 7 3 t o o v a r i e c t o m i z e d r a t s r e s u l t e d i n an i n c r e a s e i n t e m p l a t e o f u t e r i n e c h r o m a t i n f o r RNA s y n t h e s i s w h i c h i s t i s s u e s p e c i f i c . The t e m p l a t e c a p a c i t y o f l i v e r and l u n g c h r o m a t i n f r o m t h e same a n i m a l was n o t e l e v a t e d by e s t r o g e n . I n a d d i t i o n t o b e i n g t i s s u e s p e c i f i c , t h e e f f e c t was a l s o s t e r o i d s p e c i f i c . A f t e r i n c u b a t i o n i n v i t r o f o r 12 h o u r s , e s t r a d i o l - 1 7 3 e n h a n c e d t h e a b i l i t y o f u t e r i n e c h r o m a t i n t o s e r v e as a t e m p l a t e f o r DNA d e p e n d e n t RNA p o l y m e r a s e t o a g r e a t e r e x t e n t t h a n e s t r a d i o l - 1 7 3 . Teng and H a m i l t o n (1967) o b s e r v e d t h a t e s t r a d i o l - 1 7 3 was bound t o u t e r i n e c h r o m a t i n i n v i v o as e a r l y as 2 m i n u t e s a f t e r i t s a d m i n i s t r a t i o n w i t h maximal b i n d i n g o c c u r r i n g a f t e r 8 h o u r s . Twenty m i n u t e s a f t e r e s t r o g e n t r e a t -ment H a m i l t o n e t a l . (1968) n o t i c e d t h a t t h e r a t e o f n u c l e a r RNA s y n t h e s i s i n v i v o was as h i g h a s 500-600% o f c o n t r o l . The a c t i v i t y o f RNA p o l y m e r a s e was s i g n i f i c a n t l y e l e v a t e d a f t e r one h o u r a l t h o u g h an i n c r e a s e i n u t e r i n e RNA c o n t e n t was n o t a c h i e v e d u n t i l 8-12 h o u r s ( M e u l l e r e t _ a l . 1958 ).Other r e p o r t s ( B i l l i n g s e t a l . 1969(b) i n d i c a t e t h a t t h e t o t a l RNA and p r o t e i n l e v e l s do n o t r i s e u n t i l 6 and 12 h o u r s r e s p e c t i v e l y . T h e s e s e r i e s o f e v e n t s s u g g e s t t h a t t h e r e e x i s t s a t r a n s f e r o f a p a r t o f n ewly s y n t h e s i z e d RNA f r o m t h e n u c l e u s t o t h e c y t o p l a s m . T h i s i s i n a c c o r d a n c e w i t h t h e i n c r e a s e d a c c u m u l a t i o n o f w h o l e t i s s u e RNA and p r o t e i n between 2 and 24 h o u r s w i t h o u t any d e t e c t a b l e i n c r e a s e i n t h e RNA/DNA and protein/DNA r a t i o s o f t h e n u c l e i . On the b a s i s o f h i s t o l o g i c a l e v i d e n c e , M e u l l e r e t a_l. (1958) demonstrated t h a t i n c r e a s e d n u c l e o l a r s i z e , g r e a t e r abundancy o f endoplasmic r e t i c u l u m and i n c r e a s e d b a s o p h i l i c c h a r a c t e r o f t h e u t e r i n e c e l l c y t o p l a s m were n o t i c e d 24 hours a f t e r e s t r o g e n a d m i n i s t r a t i o n and t h e s e were c o r r e l a t e d w i t h i n c r e a s e d p r o -d u c t i o n and a c c u m u l a t i o n o f RNA and enhanced p r o t e i n s y n t h e s i s . H a m i l t o n (1963) r e p o r t e d t h a t the u t e r u s i n a normal r a t c o n t a i n s t w i c e t h e amount o f RNA and s i x t i m e s t h e q u a n t i t y o f p r o t e i n t h a n i n t h e o v a r i e c t o m i z e d r a t . The u t e r u s o b t a i n e d from a r a t d u r i n g e s t r u s c o n t a i n e d 0.3 t o 6 t i m e s t h e amount o f p r o t e i n t h a n d u r i n g d i e s t r u s . I n a d d i t i o n t o t h e e f f e c t on RNA and p r o t e i n s y n t h e s i s , e s t r o g e n s have a l s o been shown t o s t i m u l a t e many enzymes i n t h e u t e r u s (Pepe and Yochim, 1971; H a l l , 1973). The i n c r e a s e i n the a c t i v i t y o f many enzymes i s r e f l e c t e d i n h i g h i n c o r p o r a t i o n 14 14 of C a c e t a t e and C g l u c o s e i n t o l i p i d s (Noteboom and G o r s k i , 3 3 1963(a) , H u r i d i n e and H - c y t i d i n e i n t o RNA ( M i l l e r and Emmens, 14 1967; B i l l i n g e t al_. 1969 (a)) and C - g l y c i n e i n t o p r o t e i n ( M e u l l e r e t a_l. 1958). I n c r e a s e s i n p h o s p h o l i p i d , g l y c o g e n and l a c t a t e m e t a b o l i s m i n the r a t endometrium have a l s o been a t t r i b u t e d t o e s t r a d i o l - 1 7 3 (Walaas e t a l . 1952). E s t r a d i o l - 1 7 3 and i t s analogues a r e a l s o a s s o c i a t e d w i t h changes i n t h e u t e r i n e membrane and h i s t a m i n e r e l e a s e . Lieberman e t a l . (1963) r e p o r t e d i n c r e a s e d l e v e l s o f 3',5'-c y c l i c AMP i n t h e u t e r i n e membrane 15 seconds a f t e r t h e a d m i n i s -t r a t i o n o f e s t r a d i o l - 1 7 3 . S p a z i a n i e t a l . (1958)demonstrated t h a t the r e l e a s e o f h i s t a m i n e from t h e u t e r u s was r e s p o n s i b l e f o r i m b i b i t i o n o f water w h i c h i s one o f the e a r l y e f f e c t s o f e s t r a d i o l - 1 7 $ on t h e c e l l . Hyperemia o f u t e r i n e t i s s u e s i s d e t e c t a b l e 1-2 hours a f t e r e s t r o g e n a d m i n i s t r a t i o n and f l u i d uptake i n t o t h e u t e r u s r e a c h e s a maximum a f t e r 6 hours (Astwood,. .1938). I n c r e a s e s i n d r y w e i g h t o f u t e r i n e t i s s u e s a f t e r e s t r o g e n a d m i n i s t r a t i o n a r e acco u n t e d f o r by i n c r e a s e i n p r o t e i n c o n t e n t o f t h e u t e r u s f i r s t seen a t a p p r o x i m a t e l y 18 hours ( B i l l i n g e t a l . 1 9 6 9 ( b ) ) . I n c r e a s e s i n t h e r a t e o f DNA (Kaye e t a l . 1972) and h i s t o n e s y n t h e s i s ( W i l l i a m s and G o r s k i , 1971; Anderson e t a l . 1972) b e g i n a t about 18 hours a f t e r e s t r o g e n t r e a t m e n t . T h i s i s f o l l o w e d by c e l l u l a r d i v i s i o n w h i c h b e g i n s i n t h e u t e r u s a t a p p r o x i m a t e l y 24 hours (Kaye e t a l . 1972; Lee, 1974). 3. P h y t o e s t r o g e n s The e f f e c t s o f p h y t o e s t r o g e n s on l i v e s t o c k r e p r o d u c t i o n have been r e c o g n i z e d f o r a l o n g time ( B a r t l e t t e t a l . 1948; O c h i e t a l . 1964; Cayen e t a l . 1965; Cox and Braden, 1974). The p o t e n t i a l economic l o s s may be a s c r i b e d t o t h e i r antagonism t o the b i o c h e m i c a l and p h y s i o l o g i c a l r e s p o n s e s t o e s t r o g e n . I n a d d i t i o n t o e s t r o g e n i c s u b s t a n c e s o t h e r compounds p r e s e n t i n c e r t a i n s p e c i e s o f p l a n t s have a l s o been found t o a f f e c t r e p r o -d u c t i v e p r o c e s s e s . F o r example, compounds e x t r a c t e d from t h e ponderosa p i n e n e e d l e s ( P i n u s ponderosa) a r e b e l i e v e d t o be r e s p o n s i b l e f o r weak c a l v e s a t b i r t h as w e l l as a b o r t i o n i n g r a z i n g a n i m a l s i n the w e s t e r n p a r t o f U.S.A. and Canada (Cook and K i t t s , 1964; Chow e t a l . 1 9 7 2 ; Stevenson e t a l . 1972). 1 8 . The v a r i o u s f a c t o r s i n f l u e n c i n g t h e pre s e n c e o f p h y t o -e s t r o g e n s and t h e i r e f f e c t s on a n i m a l r e p r o d u c t i o n have been s t u d i e d by many w o r k e r s . The f i r s t r e p o r t o f e s t r o g e n i c sub-s t a n c e s i n p l a n t s was made i n 1926 by Dohrn e t a l . Loewe e t a l . a n d F e l l n e r \ who r e p o r t e d e s t r o g e n i c c h a r a c t e r i s t i c s from t h e e x t r a c t s o f o v a r i e s o f the water r o s e and w i l l o w c a t k i n s . Sub-s e q u e n t l y many e s t r o g e n analogues i n f o r a g e s have been i d e n t i f i e d and r e l a t e d t o r e p r o d u c t i v e problems. These i n c l u d e t h e s t e r o l s , c o u m e s t r o l and o t h e r coumestans (benzofuranocoumarins) and t h e i s o f l a v o n e s , f o r m o n o n e t i n , b i o c h a n i n A, d a i d z e i n , g e n i s t e i n and the most r e c e n t l y i s o l a t e d e q u o l . The p r e s e n c e o f e s t r o g e n i c compounds i n f o r a g e s was d i s -c o v e r e d by Be n n e t t e t a l . (194 6 ), who r e p o r t e d on t h e e s t r o g e n i c a c t i v i t y i n s u b t e r r a n e a n c l o v e r ( T r i f o l i u m s u b t e r r a n e u m ) . L a t e r many e s t r o g e n i c compounds have been i d e n t i f i e d from e x t r a c t s o f B r i t i s h and American p a s t u r e p l a n t s , i n p a r t i c u l a r r e d c l o v e r ( T r i f o l i u m p r a t e n s e t ) , (Pope e t a]L. 1953 ; Wong and F l u x , 1 9 6 2 ) . Bradbury and White (1951) i s o l a t e d t h e i s o f l a v o n e g e n i s t e i n from s u b t e r r a n e a n c l o v e r and a l f a l f a , w h i l e Pope e t a l . (1953) i s o -l a t e d a n o t h e r e s t r o g e n i c i s o f l a v o n e , b i o c h a n i n A ( 5 , 7 , d i h y d r o x y H - m e t h o x y - i s o f l a v o n e ) from r e d c l o v e r . G e n i s t e i n has a l s o been i s o l a t e d from soybean o i l meal (Cheng e t a l . 1 9 5 3 ( a ) ) . Pope (1954) r e p o r t e d t h a t g e n i s t e i n was not o n l y p r e s e n t i n soybean meal but a l s o o c c u r r e d t o g e t h e r w i t h b i o c h a n i n A and formono-n e t i n i n b o t h s u b t e r r a n e a n and r e d c l o v e r s . The i s o f l a v o n e , e q u o l , was f i r s t e x t r a c t e d and i s o l a t e d by M a r r i a n and Haselwood (1932) from a p h e n o l i c f r a c t i o n o f e t h e r - s o l u b l e . (•*" c i t e d from Bradbury and Whit e , 1954) F i g . 2. Structure and metabolism of estrogenic compounds 20. e x t r a c t s o f pregnant mare's u r i n e s u g g e s t i n g t h a t t h i s compound may have o r i g i n a t e d from t h e m e t a b o l i s m o f t h e i n g e s t e d f o r a g e . S i n c e t h e n i t has been i s o l a t e d from c a t t l e and f o w l u r i n e samples (Klyne and W r i g h t , 1959; Cayen e t a l . 1965), t h e b i o -l o g i c a l s i g n i f i c a n c e o f w h i c h i s d i s c u s s e d l a t e r . The e s t r o g e n i c a c t i v i t y o f t h e i s o f l a v o n e s was demonstrated by Chang e t al_. (1954) who f e d them t o immature mice and measured t h e e f f e c t on u t e r i n e w e i g h t . R e s u l t s o f p r e l i m i n a r y b i o l o g i c a l t e s t s showed t h a t g e n i s t e i n had an e s t r o g e n i c a c t i v i t y -5 o f a p p r o x i m a t e l y 10 t i m e s t h a t o f e s t r o n e . Formononetm was found t o be i n a c t i v e as a s s e s s e d by t h e A l l e n - D o i s y method w h i c h i s based on t h e e s t i m a t i o n o f the c o r n i f i c a t i o n o f r a t v a g i n a . The m e t a b o l i s m o f v a r i o u s p h y t o e s t r o g e n s by t h e rumen m i c r o b e s has been s t u d i e d by many wo r k e r s . I n a s e r i e s o f e x p e r i m e n t s N i l s s o n (1961 a and b) and (1962) demonstrated t h a t t h e rumen mi c r o o r g a n i s m s d e m e t h y l a t e d t h e c o m p a r a t i v e l y weaker i s o f l a v o n e s , f o r m o n o n e t i n and b i o c h a n i n - A t o y i e l d more p o t e n t e s t r o g e n i c compounds such as d a i d z e i n and g e n i s t e i n ( F i g . 2 ) . Braden e t a l . (1967) found t h a t i n sheep t h e i s o f l a v o n e s , g e n i s t e i n , b i o c h a n i n A, and f o r m o n o n e t i n were a l l e s t r o g e n i c when a d m i n i s t e r e d i n t r a r u m i n a l l y , but had o n l y 5% o f the a c t i v i t y when g i v e n i n t r a m u s c u l a r l y . B i o c h a n i n A i s m e t h y l a t e d a t t h e 4' p o s i t i o n on the C - r i n g and a f t e r m i c r o b i a l m e t a b o l i s m , t h e m e t h y l group i s c l e a v e d and g e n i s t e i n i s produced (Braden e t a l . 1967; Cox and Braden, 1974). Formononetin w h i c h i s a l s o m e t h y l a t e d a t t h e 4' p o s i t i o n i s c o n v e r t e d t o d a i d z e i n a f t e r s i m i l a r a c t i o n o f the m i c r o f l o r a . D a i d z e i n i n t u r n i s m e t a b o l i z e d t o e q u o l , w h i c h i s a v e r y p o t e n t e s t r o g e n analogue and i s b e l i e v e d t o be t h e c h i e f p l a n t c o n s t i t u e n t r e s p o n s i b l e f o r " C l o v e r D i s e a s e " ( L i g h t f o o t and Wroth, 1974). C o u m e s t r o l i s found p r e d o m i n a n t l y i n a l f a l f a . I n the f o w l i t i s degraded a l o n g pathways q u i t e d i f f e r e n t from t h o s e o f g e n i s t e i n , d e s p i t e t h e f a c t t h a t t h e b i o s y n t h e t i c pathway o f c o u m e s t r o l i n t h e p l a n t r e sembles t h a t o f t h e i s o f l a v o n e s (Cayen e t a l . 1965). P h y t o e s t r o g e n s have been r e p o r t e d t o a c t l i k e e s t r a d i o l - 1 7 ^ . a f t e r t h e i r consumption and subsequent m e t a b o l i s m by t h e g r a z i n g a n i m a l (Braden e t a l . 1967; S h u t t £t a l . 1967; L i n d n e r , 1967). Endogenous e s t r o g e n s c i r c u l a t e i n a c o n j u g a t e form w h i c h r e n d e r s them i n a c t i v e . P h y t o e s t r o g e n s , a f t e r t h e i r m e t a b o l i s m and a b s o r p t i o n i n t h e g u t , a r e c o n j u g a t e d w i t h g l u c u r o n i c a c i d i n the l i v e r and c i r c u l a t e i n t h e plasma i n t h e form o f i n a c t i v e g l u c u r o n a t e s . A f t e r t h e i r i n t e r a c t i o n w i t h the r e c e p t o r p r o t e i n s t h e c o v a l e n t c o n j u g a t i o n w i t h g l u c u r o n i c a c i d i s s p l i t by t h e a c t i o n o f g l u c u r o n i d a s e (Cox and Braden, 1974) . The r a t e c o n j u g a t i o n and t h e m e t a b o l i s m w i l l govern t h e e f f e c t o f p h y t o e s t r o g e n s on the r e p r o d u c t i v e performance o f g r a z i n g a n i m a l s . D i f f e r e n c e s have been o b s e r v e d i n t h e m e t a b o l i s m o f p h y t o e s t r o g e n s between sheep and c a t t l e . Braden e t a l . (1971) r e p o r t e d t h a t c i r c u l a t i n g p h y t o e s t r o g e n s were more e f f i c i e n t l y c o n j u g a t e d i n c a t t l e t h a n i n sheep, r e n d e r i n g them l e s s s u s -c e p t i b l e t o the a c t i o n o f p h y t o e s t r o g e n s . - 3 - 5 P h y t o e s t r o g e n s a r e 10 t o 10 t i m e s as e s t r o g e n i c as e s t r a d i o l - 1 7 3 i n t h e l a b o r a t o r y mouse (Bradbury and Whi t e , 1951; C a r t e r e t all955).However, w i t h c o n t i n u o u s g r a z i n g t h e r e l a t i v e l y h i g h i n t a k e o f p h y t o e s t r o g e n s can i n d u c e s i g n i f i c a n t b i o l o g i c a l e f f e c t s ( B a r r e t , 1961). R e s u l t s on t h e degree o f e s t r o g e n i c i t y o f v a r i o u s p l a n t compounds have been c o n t r a d i c t o r y , p r i m a r i l y b e c a u s e o f t h e e r r o r s a s s o c i a t e d w i t h t h e i r low s o l u b i l i t y i n w a t e r . R e l a t i v e e s t r o g e n i c a c t i v i t i e s were i n v e s t i g a t e d by Wong and F l u x (1962) u s i n g t h e s e m i - q u a n t i t a t i v e mouse u t e r i n e w e i g h t a s s a y method d e v e l o p e d by K i t t s e t a l . ( 1959). Shemesh e t a l . (1971) r e p o r t e d t h a t a p p r o x i m a t e l y one u n i t w e i g h t o f e s t r a d i o l - 1 7 3 was e q u i v a l e n t t o 70 u n i t s o f c o u m e s t r o l and 175 u n i t s o f g e n i s t e i n . The e s t r o g e n i c p o t e n c y o f p h y t o e s t r o g e n s i s r e l a t e d t o t h e i r b i n d i n g a f f i n i t y f o r m a c r o m o l e c u l a r components l o c a t e d i n t h e u t e r i n e c y t o s o l . The m e t h y l a t e d i s o f l a v o n e s , b i o c h a n i n A and f o r m o n o n e t i n d i d n o t s i g n i f i c a n t l y i n h i b i t e s t r a d i o l - 1 7 3 b i n d i n g . T h i s s u g g e s t s t h a t t h e f r e e h y d r o x y l g r o u p s m e t h y l a t e d i n b i o c h a n i n A and f o r m o n o n e t i n a r e e s s e n t i a l f o r t h e e f f e c t i v e i n t e r a c t i o n w i t h t h e e s t r o g e n r e c e p t o r . T h e r e have been many r e p o r t s o f p r o f o u n d e f f e c t s on t h e g r o w t h , l a c t a t i o n and r e p r o d u c t i o n o f a n i m a l s g r a z i n g e s t r o g e n i c f o r a g e p l a n t s o r p a s t u r e s ( B a r t l e t t e t a l . 1948; Pope, 1954; S a n g e r and B e l l , 1960; M a r s h a l l , 1 9 7 4 ) . I n many c a s e s i n f e r -t i l i t y i s t e m p o r a r y i n n a t u r e and r e c y c l i n g and c o n c e p t i o n r e t u r n t o n o r m a l f i v e t o s i x weeks a f t e r t h e c o n s u m p t i o n o f t h e e s t r o -g e n i c m a t e r i a l i s s t o p p e d ( L i g h t f o o t and Wroth, 1 9 7 4 ) . Permanent i n f e r t i l i t y ( c l o v e r d i s e a s e ) may r e s u l t when ewes have been g r a z i n g e s t r o g e n i c c l o v e r p a s t u r e s f o r a number o f y e a r s ( L i g h t -f o o t and Wroth, 1974; M a r s h a l l , 1 9 7 4 ) . The m a g n i t u d e o f t h e e f f e c t o f p h y t o e s t r o g e n s depends on many e n v i r o n m e n t a l v a r i a b l e s . S t a g e o f m a t u r i t y and d e f o l i a t i o n ( K i t t s e t a l . 1 9 5 9 ) , t r a c e m i n e r a l d e f i c i e n c y ( R o s s i t e r , 1 9 7 0 ) , r o u t e o f a d m i n i s t r a t i o n ( O s t r o v s k y and K i t t s , 1962), f o l i a r d i s e a s e (Saba e t a i . 197 2) and i n d i v i d u a l p l a n t p a r t s d e t e r m i n e t h e amount o f e s t r o g e n i c m a t e r i a l p r e s e n t i n f o r a g e and p a s t u r e p l a n t s . I n f e r t i l i t y r e s u l t i n g from th e i n g e s t i o n o f e s t r o g e n i c p a s t u r e s may be m a n i f e s t e d i n many forms (Sanger and B e l l , 1960; B a r r e t t , 1961; L i g h t f o o t and Wroth, 1974). The e f f e c t s o f p h y t o e s t r o g e n s on sperm p r o d u c t i o n and v i a b i l i t y i n rams have been shown t o be n e g l i g i b l e (George and T u r n b u l l , 1966). L i g h t f o o t and Wroth (1974) r e p o r t e d a r e d u c t i o n i n t h e o n s e t o f e s t r u s i n ewes g r a z i n g e s t r o g e n i c p a s t u r e s . O v u l a t i o n r a t e s o f ewes on s i m i l a r p a s t u r e s were a l s o r e p o r t e d t o be r e d u c e d , t h e r e b y a f f e c t i n g t h e normal l e n g t h o f t h e e s t r o u s c y c l e . D i f f e r e n c e s on ovum t r a n s -f e r and t h e passage o f t h e egg t h r o u g h t h e F a l l o p i a n tube f o r s u c c e s s f u l f e r t i l i z a t i o n have a l s o been a t t r i b u t e d t o t h e e f f e c t o f p h y t o e s t r o g e n s . The e s t a b l i s h m e n t o f a p o o l o f sperm i n t h e c e r v i x immediate-l y a f t e r mating i s i m p o r t a n t f o r s u c c e s s f u l f e r t i l i z a t i o n i n sheep ( R e s t a l l and Wales, 1966; S m ith, 1970). L i g h t f o o t and Wroth (1974) c o l l e c t e d eggs from ewes f e d e s t r o g e n i c f o r a g e s and found t h a t t h e y had l o w e r numbers o f spermatozoa p r e s e n t on t h e zona p e l l u c i d a , w h i c h reduced t h e p r o b a b i l i t y o f f e r t i l i z a t i o n . No e v i d e n c e has been e s t a b l i s h e d s u g g e s t i n g a b n o r m a l i t i e s i n t h e o v a r y , p i t u i t a r y o r a d r e n a l c o r t e x as a consequence o f g r a z i n g on e s t r o g e n i c p a s t u r e s . Obst and Semark (197 0) r e p o r t e d f l u c t u a t i n g l e v e l s o f plasma p r o g e s t e r o n e due t o f u n c t i o n a l changes i n t h e c o r p u s luteum o f a n i m a l s g r a z i n g v a r i o u s c l o v e r p a s t u r e s . L e a v i t t and W r i g h t (196 5) r e p o r t e d t h a t t h e p r i n c i p a l e f f e c t o f p h y t o e s t r o g e n s was t o i n h i b i t t h e r e l e a s e o f gona-d o t r o p h i c hormones by servomechanism on t h e hypothalamus. A g r e a t e r i n c i d e n c e o f i r r e g u l a r e s t r o u s c y c l e s i n d a i r y cows f e d e s t r o g e n i c f o r a g e s was r e p o r t e d by A l d e r (1965). B a r r e t t (1961) found a c o r r e l a t i o n between f e r t i l i t y of.' t h e cow and s i z e o f m a c r o s c o p i c c y s t s seen a t a u t o p s y . Other changes o b s e r v e d i n c a t t l e g r a z i n g s u b t e r r a n e a n c l o v e r i n c l u d e t h e s i z e o f u t e r u s and udder even i n non-pregnant and n o n l a c t a t i n g cows, and i n c r e a s e d c l i n i c a l i n c i d e n c e o f s w o l l e n v u l v a s , c y s t i c o v a r i e s and hyperemic mucous membranes. P a t h o l o g i c a l con-d i t i o n s c h a r a c t e r i z e d by c y s t i c g l a n d u l a r h y p e r p l a s i a o f t h e endometrium were a l s o n o t i c e d i n ewes. V a r i a b l e degrees o f h y p e r p l a s i a i n t h e stroma c e l l s r e s e m b l i n g l e s i o n s d e s c r i b e d by Novak and R i c h a r d s o n (1941) i n p o s t menopausal women were ob s e r v e d i n t h e e p i t h e l i a l l i n i n g o f the c y s t i c g l a n d s . The e p i t h e l i a l l i n i n g was c h a r a c t e r i z e d as b e i n g f l a t and d e v o i d o f a l l m i t o t i c a c t i v i t y . The i n c i d e n c e o f a b o r t i o n f o l l o w i n g t h e i n g e s t i o n o f p i n e n e e d l e s by c a t t l e i s not e n t i r e l y known. The s u b s t a n c e r e s -s p o n s i b l e f o r a b o r t i o n s i n c a t t l e , sheep o r deer g r a z i n g p i n e n e e d l e s i s a t e i t e r p e n e ( G l y c y r r h e t m i c a c i d ) . Chow e t a l . (1972) r e p o r t e d a f t e r e x a m i n i n g th e u t e r u s o f p r e gnant a n i m a l s , consuming p i n e n e e d l e s t h a t f e t a l development had been a r r e s t e d . The u t e r i were r e p o r t e d t o be hyperemic pr n o d u l a r and t h e f e t u s was i n t h e p r o c e s s o f r e a b s o r p t i o n . A l l e n and K i t t s (1961) and Cook and K i t t s (1964) r e p o r t e d the p r e s e n c e o f an agent i n p i n e n e e d l e s which s u p p r e s s e d growth of t h e u t e r u s o f immature mice and r e s u l t e d i n embryonic m o r t a l i t y . Aqueous e x t r a c t s from y e l l o w p i n e have been r e p o r t e d t o p o s s e s s e s t r o g e n i c c h a r a c t e r -i s t i c s . Attempts have been made to reduce the incidence of repro-ductive disorders i n animals consuming estrogenic plant materials by a l t e r i n g the metabolism of phytoestrogens i n animals and by immunologic methods. The former requires a better under-standing of the chemical changes to these compounds by the action of the rumen microorganisms and subsequent metabolism. Cox et a l . (1972) reported that there i s a covalent attachement of antigenic protein with suitable phytoestrogens r e s u l t i n g i n haptens. Sera obtained from sheep possessing antibodies against various phytoestrogens reacted s p e c i f i c a l l y with plant estrogens. Antibody t i t r e s were reported to p e r s i s t in sheep for about one year, with no e f f e c t on the normal estrous cycle of the animal. E x p e r i m e n t A : The C o m p a r a t i v e E f f e c t s o f E s t r o g e n s and P h y t o e s t r o g e n s on Water I m b i b i t i o n and H y p e r e m i a o f t h e R a t U t e r u s . I n t r o d u c t i o n E s t r o g e n i c hormones e x e r t an i n f l u e n c e on t h e l e v e l o f m e t a b o l i s m i n many t a r g e t t i s s u e s . Most o f t h e s e e f f e c t s a r e p r o b a b l y s e c o n d a r y phenomena r e s u l t i n g f r o m an i n i t i a l s t i m u -l a t i o n o f some key b i o c h e m i c a l p r o c e s s . I t i s a l s o p o s s i b l e t h a t e s t r o g e n s e x e r t more t h a n one p r i m a r y e f f e c t on many c e l l u l a r components i n t h e t a r g e t c e l l . I n p a r t i c u l a r t h e e s t r o g e n i n d u c e d r e l e a s e o f h i s t a m i n e by t h e u t e r u s r e s u l t s i n e s t r o g e n r e c e p t o r i n t e r a c t i o n and genome a c t i v a t i o n and may t h e r e f o r e r e p r e s e n t a s e p a r a t e h o r m o n a l a c t i o n . S t i m u l a t i o n o f t h e u t e r i n e g r o w t h i s a g e n e r a l c h a r a c t e r -i s t i c o f e s t r o g e n i c compounds w h i c h may d i f f e r q u a n t i t a t i v e l y i n t h e i r c a p a c i t y t o promote g r o w t h o f t h e f e m a l e r e p r o d u c t i v e t r a c t . T h e r e a r e a l s o marked s p e c i e s d i f f e r e n c e i n r e s p o n s e t o e s t r o g e n s , some b e i n g more s e n s i t i v e t h a n o t h e r s . S t a n d a r d e s t r o g e n a s s a y t e c h n i q u e s i n l a b o r a t o r y a n i m a l s have been b a s e d on r e l a t i v e l y l a t e r e s p o n s e s o f t h e u t e r i n e and v a g i n a l t i s s u e s . R e s p o n s e s most f r e q u e n t l y s e l e c t e d i n t h e p a s t were t h e v a g i n a l e p i t h e l i a l c o r n i f i c a t i o n and i n c r e a s e d t e t r a z o l i u m r e d u c t i o n r e a c t i o n s ( M a r t i n , 1964) . T h e s e r e s p o n s e s o c c u r c o n s i d e r a b l y l a t e r t h a n c e r t a i n v a s c u l a r and m e t a b o l i c e v e n t s and a r e a l s o n o n s p e c i f i c t o v a r i o u s e s t r o g e n s . The e a r l i e r s i x - h o u r w a t e r i n h i b i t i o n t e s t , d e v e l o p e d by Astwood (1938) measures t h e a c c u m u l a t i o n o f w a t e r i n t h e u t e r u s d u r i n g v e r y i n i t i a l s t a g e s o f e s t r o g e n a d m i n i s t r a t i o n . S p a z i a n i e t a_l. (1958) f i r s t r e p o r t e d t h a t t h e a c c u m u l a t i o n o f w a t e r i n t h e u t e r u s f o l l o w i n g e s t r o g e n a d m i n i s t r a t i o n was s e c -ondary t o hyperemia and t h a t i n c r e a s e d p e r m e a b i l i t y was t h e r e s u l t o f e s t r o g e n i n d u c e d r e l e a s e o f h i s t a m i n e . The per c e n t o f water i n g e n e r a l r e f l e c t s t h e p h y s i o l o g i c a l s t a t e o f t h e u t e r u s (Hinshaw, 1959). F i n n and M a r t i n (1973) s t u d i e d t h e e f f e c t o f e s t r o g e n on c e l l p r o l i f e r a t i o n i n b o t h the g l a n d u l a r and l u m i n a l e p i t h e l i a l c e l l s o f mouse u t e r u s and found t h a t c e l l d i v i s i o n i n b o t h t y p e s was s t i m u l a t e d i n response t o e s t r o g e n . However, t h e i n i t i a l r e s p onse t o each was d i f -f e r e n t . The l u m i n a l e p i t h e l i u m showed maximal m i t o s i s a f t e r 24 hours i n response t o a s i n g l e d a i l y i n j e c t i o n whereas the g l a n d s showed l i t t l e r e sponse u n t i l 72 hours and r e q u i r e d a t l e a s t t h r e e d a i l y i n j e c t i o n s f o r maximal r e s p o n s e . Kaye e t a_l. (1972) r e p o r t e d an i n c r e a s e i n c e l l d i v i s i o n and maximum m i t o t i c a c t i v i t y between 24 and 28 hours a f t e r a s i n g l e dose o f e s t r o g e n was g i v e n . The a c c e l e r a t e d c e l l u l a r d i v i s i o n was more marked i n t h e e p i t h e l i u m t h a n th e stroma o f myometrium. These f i n d i n g s s uggest t h a t t i s s u e growth i s r e s p o n s i b l e f o r t h e n o t a b l e i n c r e a s e i n u t e r i n e d r y w e i g h t s o f e s t r o g e n t r e a t e d a n i m a l s over c o n t r o l groups. The o b j e c t o f the p r e s e n t s e r i e s o f e x p e r i m e n t s was t o d e t e r m i n e t h e mode o f a c t i o n o f v a r i o u s e s t r o g e n s w i t h p a r -t i c u l a r r e f e r e n c e t o t h e e a r l y b i o c h e m i c a l mechanisms i n v o l v e d and t h e r o l e o f p h y t o e s t r o g e n s i f any, i n m o d i f y i n g t h e same. As a p r e l i m i n a r y s t e p i n t h i s d i r e c t i o n E x p e r i m e n t A was under-t a k e n t o t e s t the p o t e ncy o f v a r i o u s e s t r o g e n i c compounds and compare t h e i r t i me c o u r s e e f f e c t s on t h e b a s i s o f water i n h i b i t i o n 28. o f immature r a t u t e r i n e t i s s u e s . M a t e r i a l s and Methods A n i m a l s Immature female r a t s (35-40 g ) , d e r i v e d from W i s t a r s t o c k were pur c h a s e d from UBC Co l o n y and Woodlyn L a b o r a t o r i e s , Guelph, O n t a r i o . They were housed i n a i r c o n d i t i o n e d q u a r t e r s w i t h u n i f o r m exposure t o 12 hours o f l i g h t and 12 hours o f d a r k n e s s . A n i m a l s had f r e e a c c e s s t o p e l l e t e d f o o d ( P u r i n a L a b o r a t o r y Chow) and wa t e r . M a t e r i a l s E s t r a d i o l - 1 7 3 ( e s t r - 1 , 3 , 5 ( 1 0 ) - t r i e n e - 3 ,17c* d i o l ) , E s t r i o l ( e s t r - 1 , 3 , 5 ( 1 0 ) - t r i e n e 3,16 , 1 7 3 - t r i o l ) , E s t r o n e ( e s t r - 1 , 3 , 5 ( l O ) . t r i e n e 3-01-17-one) , . DES (a , a , - d i m e t h y l - s t i l b e n e - 4 - 4 ' - d i o l ) , E s t r a d i o l 17a ( e s t r - 1 , 3 , 5, (10 )-triene - 3-17,);diol) , and T e s t o s t e r o n e (173 Hydroxy-4-androsten - 3-one) were o b t a i n e d from Sigma C h e m i c a l s Co. The f o l l o w i n g p l a n t e s t r o g e n s were donated by Dr. A.B. Beck o f C.S.I.R.O., Western A u s t r a l i a , Dept. o f A g r i c u l t u r e : Formononetin ( 7 - h y d r o x y - 4 ' m e t h o x y i s o f l a v o n e ) , B i o c h a n i n - A (4'methoxy-5-7-hydroxyisoflavone) and C o u m e s t r o l (7-hydroxy-coumarono-coumarins). P l a n t e x t r a c t s were made from a l f a l f a by t h e method o f F r a n c i s and M i l l i n g t o n (1965). A d m i n i s t r a t i o n o f e s t r o g e n s and P l a n t e s t r o g e n s I n Experiment A - I , e i g h t groups o f a n i m a l s (4 a n i m a l s / group) w i t h comparable body w e i g h t s were g i v e n a s i n g l e i n j e c t i o n o f 1.0 mg o f e s t r a d i o l - 1 7 3 i n t r a p e r i t o n e a l l y t o s t u d y t h e time c o u r s e e f f e c t o f e s t r o g e n on water i m b i b i t i o n by r a t u t e r u s t i s s u e s . E x p e r i m e n t s A-2 and A-3 were d e s i g n e d t o s t u d y t h e e f f e c t o f v a r y i n g c o n c e n t r a t i o n s o f e s t r a d i o l - 1 7 3 and t h e r e l a t i v e e f f e c t s o f v a r i o u s e s t r o g e n s on water i m b i b i t i o n . A s t o c k s o l u t i o n o f e s t r a d i o l - 1 7 3 was made by d i s s o l v i n g 5.0 mg i n 2.0 ml o f 95% e t h a n o l . T h i s was broug h t t o a volume of 5.0 ml with 0.9% NaCI to give a concentration of 100 ug/0.1 ml. A 100 u l aliquot of t h i s solution was made up to a volume of 10.0 ml with 0.9% NaCI to give an administration concentration of 1.0 ug/0.1 ml. Solutions of DES, e s t r i o l and testosterone were made i n similar concentrations. Estradiol-17d and estrone were prepared in concentrations of 2.0 ug/0.1 ml and 6.0 ug/0.1 ml respectively. The concentration of phytoestrogens was made to correspond to estradiol-173 i n terms of uterotrophic a c t i v i t y (Perel and Lidner, 1 9 7 0 ) . Coumestrol solutions were made by dis s o l v i n g 3.0 mg of p u r i f i e d coumestrol i n 10.0 ml of propylene g l y c o l giving a concentration of 90.0 ug /0.3 ml. Genistein (8.0 mg) was dissolved i n 3.0 ml of propylene g l y c o l to give a f i n a l concentration of 0.8 mg/0.3 ml. Formononetin (100 mg/ml) and biochanin-A (100 mg/ml) stock solutions were made up i n toluene. An aliquot from both was taken and evaporated to dryness under a stream of nitrogen. The residue was dissolved i n propylene glyc o l to give a concentration of 100 mg/0.1 ml. To test for uterotrophic a c t i v i t y the compounds were injected i n t r a p e r i t o n e a l l y into immature female rats. Control animals were given intraperitoneal i n j e c t i o n s of 0.5% alcohol i n corresponding volumes of 0.9% NaCI or propylene g l y c o l . After appropriate time inter v a l s the animals were s a c r i f i c e d by placing them i n a jar f i l l e d with carbon dioxide. Their u t e r i were excised and stripped of adhering f a t and mesentery. Uterine tissues were blotted dry and weighed on a microprecision torque balance. Tissues were then transferred to an oven and dried to a constant weight at 90°C for 12 hours. The degree of water imbibition was-.'calculated from the per cent moisture. 30. A l l s t a t i s t i c a l a n a l y s i s ' was done - by S t u d e n t ' s t - t e s t . R e s u l t s I n E x p e r i m e n t A - I i t was n o t i c e d t h a t d u r i n g t h e f i r s t few h o u r s a f t e r e s t r a d i o l - 1 7 3 a d m i n i s t r a t i o n , w a t e r i m b i b i t i o n i n -c r e a s e d g r a d u a l l y , r e a c h i n g a maximum o f 4 0.6% o v e r c o n t r o l l e v e l s a f t e r s i x h o u r s ( F i g . 4 ) . T h i s was f o l l o w e d by a s l i g h t d e c r e a s e i n t i s s u e m o i s t u r e c o n t e n t between 12 and 24 h o u r s . Water i m b i b i t i o n was f o u n d t o i n c r e a s e s i g n i f i c a n t l y (P < 0.025) a g a i n a t 24 and 30 h o u r s w h i c h c o r r e s p o n d e d w i t h i n -c r e a s e s i n u t e r i n e d r y w e i g h t s ( T a b l e 1 ) . I n c r e a s e s i n m o i s t u r e c o n t e n t n o t i c e d a t 3 0 h o u r s were, however, n o t as g r e a t as t h o s e r e a c h e d a t s i x h o u r s . E x p e r i m e n t A-2 was p e r f o r m e d t o s t u d y t h e i n c r e a s e i n w a t e r i m b i b i t i o n a t s i x h o u r s i n r e s p o n s e t o d i f f e r e n t d o s e s o f e s t r a d i o l - 1 7 3 . I t was n o t i c e d t h a t i n c r e a s e s i n w e i g h t due t o m o i s t u r e c o n t e n t were s i g n i f i c a n t l y (P< 0.025) g r e a t e r a t l o w e r d o s e s (50-1000 ng) t h a n i n h i g h e r o n e s (5000-50,000 ng) ( T a b l e 2 ) . S i g n i f i c a n t (P< 0.025) i n c r e a s e s i n u t e r i n e d r y w e i g h t s were n o t i c e d w i t h i n c r e a s e d d o s e s o f e s t r o g e n . I n E x p e r i m e n t A -3 e s t r o g e n s were a d m i n i s t e r e d i n v a r i o u s d o s e s and were compared t o t h e e f f e c t o f e s t r a d i o l - 1 7 3 s t a n d a r d s g i v e n i n a d o s e o f l*0.^ig i n t r a p e r i t o n e a l l y . The r e s u l t s ( F i g . 3, T a b l e 3) show t h e r e l a t i v e e f f e c t s o f t h e s t e r o i d e s t r o g e n s were i n t h e o r d e r o f e s t r i o l , e s t r a d i o l - 1 7 3 , DES, e s t r a d i o l - 1 7 a and e s t r o n e . The e f f e c t s o f p l a n t e s t r o g e n s on u t e r i n e w a t e r i m b i b i t i o n were compared t o e s t r a d i o l - 1 7 3 and t e s t o s t e r o n e (1.0 p.g I.P.) 31. s t a n d a r d s . C o u m e s t r o l and g e n i s t e i n d i s p l a y e d s i g n i f i c a n t (P 1 0.025) e f f e c t s o v e r c o n t r o l s i n r e g a r d t o t h e i r degree o f water i m b i b i t i o n , but a t much l a r g e r dosages (Table 3 ) . Formononetin and b i o c h a n i n - A d i d not show any s i g n i f i c a n t (P>0.025) i n c r e a s e i n water i m b i b i t i o n . D i s c u s s i o n R e s u l t s o f Exp e r i m e n t s A - l , 2, 3 i n d i c a t e t h a t t h e degree o f w ater i m b i b i t i o n v a r i e s w i t h v a r i o u s e s t r o g e n s . The a b i l i t y o f an e s t r o g e n t o produce t h i s e f f e c t i s r e l a t i v e l y c o n s t a n t , governed m a i n l y by t h e time and dose l e v e l s c h a r a c t e r i s t i c o f the e s t r o g e n . Among t h e e a r l i e s t known e f f e c t s o f e s t r o g e n s a r e t h o s e a s s o c i a t e d w i t h t h e u t e r i n e membrane and h i s t a m i n e r e l e a s e ( S p a z a n i e t al_. 1958; Szego e t a l . 1967). Experiment 1-A demonstrated t h a t a s i n g l e dose o f e s t r o g e n r e s u l t s i n a c c u m u l a t i o n o f f l u i d i n u t e r i n e t i s s u e s w h i c h w i l l be a t i t s maximum a t s i x hours. The per c e n t m o i s t u r e r e a c h e d a maximum a t t h i s p a r t i c u l a r t i m e . T h i s r e s u l t i s i n a c c o r d -ance w i t h t h e o b s e r v a t i o n o f Hinshaw (1959) who r e p o r t e d t h a t t h e g a i n i n w e i g h t d u r i n g t h e f i r s t s i x hours was m a i n l y due t o the a c c u m u l a t i o n o f f l u i d i n t h e lumen o f t h e u t e r u s . The de c r e a s e i n p e r c e n t m o i s t u r e c o n t e n t o f t h e t i s s u e a f t e r s i x hours i s p r o b a b l y due t o p a r t i a l r e a b s o r p t i o n o f f l u i d p r i o r t o i t s e n t e r i n g i n t o t h e u t e r i n e .growth phase a t 12-14 hours as suggested by Hinshaw (1959). In E xperiment A-2 t h e amount o f an e s t r o g e n a d m i n i s t e r e d over and above 50 ng d i d not produce any f u r t h e r g a i n i n wet u t e r i n e w e i g h t o r per c e n t m o i s t u r e . A c c o r d i n g t o Hinshaw (1959) t h e d e c r e a s e s i n u t e r i n e w e i g h t w h i c h o c c u r s s i x h o u r s f o l l o w i n g a s i n g l e l a r g e d o s e o f e s t r a d i o l - 1 7 3 was n o t due t o s e l f i n h i b i t o r y e f f e c t s b e c a u s e g r e a t e r u t e r i n e w e i g h t s o c c u r r e d t h r e e a n d f o u r h o u r s a f t e r e s t r o g e n a d m i n i s t r a t i o n . I n E x p e r i m e n t A-3 i t was e v i d e n t t h a t w a t e r i m b i b i t i o n was a common r e s p o n s e o f a l l e s t r o g e n s t h o u g h t h e i r a b i l i t y t o p r o d u c e s u c h a n e f f e c t may be a f f e c t e d by t h e t i m e f o l l o w i n g t h e a d m i n i s -t r a t i o n o f e s t r o g e n s a n d t h e d o s e . F r o m t h e d a t a o b t a i n e d i n t h i s e x p e r i m e n t , e s t r a d i o l - 1 7 3 a n d e s t r i o l p o s s e s s e d s i m i l a r a b i l i t i e s i n p r o d u c i n g t h i s e f f e c t . To o b t a i n a 32% i n c r e a s e i n u t e r i n e w e i g h t , e s t r i o l was t h e most e f f e c t i v e , f o l l o w e d b y e s t r a d i o l - 1 7 3 , DES, e s t r a d i o l - 1 7 a , e s t r o n e a n d t e s t o s t e r o n e . T h i s i s i n a g r e e m e n t w i t h t h e f i n d i n g s o f S z e g o a nd R o b e r t s (1953) who r e p o r t e d t h a t i n 24-27 d a y o l d r a t s , t h e r e l a t i v e e f f e c t i v e n e s s o f e s t r o g e n s i n p r o m o t i n g u t e r i n e w a t e r i m b i b i t i o n s i x h o u r s a f t e r a d m i n i s t r a t i o n was i n t h e o r d e r o f e s t r i o l , e s t r a d i o l - 1 7 3 a n d e s t r o n e . Among t h e p h y t o e s t r o g e n s c o u m e s t r o l a n d g e n i s t e i n w e r e c a p a b l e o f b r i n g i n g a b o u t a n e s t r o g e n i c r e s p o n s e . When a d m i n -i s t e r e d i n t r a p e r i t o n e a l l y e i g h t t o t e n t i m e s t h e c o n c e n t r a t i o n o f g e n i s t e i n was r e q u i r e d t o e l i c i t a r e s p o n s e s i m i l a r t o t h a t o f c o u m e s t r o l . The u s e o f u t e r i n e w e i g h t i n c r e a s e s f o r t h e a s s a y o f p h y t o -e s t r o g e n a c t i v i t y h a s y i e l d e d c o n f l i c t i n g r e s u l t s i n t h e p a s t . R e s u l t s o b t a i n e d i n E x p e r i m e n t A-3 a r e n o t i n c o m p l e t e a g r e e -ment w i t h r e p o r t s o f p h y t o e s t r o g e n u t e r o t r o p h i c a c t i v i t y ( B i c k o f f e t a l . 1 9 62, N i l s s o n , 1962; B r a d e n e t a l . 1 9 6 7 ) . The v a r i a b l e s w h i c h m u s t be t a k e n i n t o a c c o u n t when e v a l u a t i n g such d i f f e r e n c e s a r e t h e r a t e o f a b s o r p t i o n , r a t e o f m e t a b o l i s m and method o f a d m i n i s t r a t i o n o f t h e e s t r o g e n i c compounds. T h i s has l e d t o c o n f l i c t i n g r e p o r t s i n t h e l i t e r a t u r e r e n d e r i n g t h e p r o p e r assessment o f u t e r i n e w e i g h t b i o a s s a y s d i f f i c u l t . I n v iew o f t h i s i t was c o n s i d e r e d d e s i r a b l e t o u n d e r t a k e more a c c u r a t e e x p e r i m e n t s w h i c h would d e s c r i b e t h e e a r l y e v e n t s f o l l o w i n g t h e a d m i n i s t r a t i o n o f e s t r o g e n i c compounds. C o n c l u s i o n s These s e r i e s o f e x p e r i m e n t s were d e s i g n e d t o e s t a b l i s h an o p t i m a l time c o u r s e and dose e f f e c t o f e s t r o g e n s f o r use i n subsequent e x p e r i m e n t s . The purpose o f i n c r e a s e d i m b i b i t i o n o f water due t o e s t r o g e n i s not e n t i r e l y known, however i t appears t o be i m p o r t a n t due t o i t s r e l a t i v e l y e a r l y and dynamic e f f e c t . A s i x hour t i m e c o u r s e p r o v e d t o be o p t i m a l t i m e a f t e r a s t a n d a r d 1.0 ug dose o f e s t r a d i o l - 1 7 3 was g i v e n i n t r a p e r i t o n -e a l l y . D u r i n g t h i s t i m e and a t t h i s dose, i t was found t h a t the t i s s u e reached i t s maximum a b i l i t y t o respond. I n Experiment A-3 r e s u l t s o b t a i n e d ( F i g . 3, T a b l e 3) showed v a r i o u s e s t r o g e n s have t h e c a p a c i t y t o i n d u c e water i m b i b i t i o n ; however dose l e v e l s and t i m e a f t e r dose were c h a r a c t e r i s t i c o f p a r t i c u l a r e s t r o g e n s . From t h e s e r e s u l t s i t may be c o n c l u d e d t h a t some e s t r o g e n s a r e more e f f e c t i v e i n enhancing water i m b i b i t i o n by the u t e r u s w h i l e o t h e r s a r e more a c t i v e i n t h e p r o m o t i o n o f u t e r i n e growth. E s t r a d i o l - 1 7 3, t h e most promine n t e s t r o g e n produced endogenously, i s t h e s t r o n g e s t n a t u r a l e s t r o g e n f o r growth and water uptake by the uterus even at low doses. Phytoestrogens also demonstrated some degree of estrogenic a c t i v i t y i n regard to water imbibition, although i t was f e l t that the carrying vehicle and route of administration were important c r i t e r i a i n influencing t h e i r response. WET U T E R I N E W E I G H T ( M G ) CD CD i—I I CD CN] CD I CD •cr C O N T R O L E S T R A D I O L - 1 7 / ^ (1,0 JUG) E S T R A D I O L - 1 7 C ^ ( 2 . 0 JUG) D E S (1.0 JUG) E S T R O N E (6.0 JUG) E S T R I O L (1.0 JUG) T E S T O S T E R O N E (l.OjJG) C O U M E S T R O L (90 JUG) G E N I S T E I N ( 0 , 8 7 T S G ) B I O C H-A (100 JUG) F O R M O N O N E T I N (100 JUG) g. 3. Wet uterine weight six hours following estrogen administration (Expt. A-3) 36. 70 r F i g . 4. E f f e c t of time on water imbibition i n rat uterine tissues following adminis-t r a t i o n of estradiol-173 (Expt. A-I) Table 1 : E f f e c t of time on water imbibition by rat uterine tissues following administration of estradiol-17 3 (Expt. A-I) Animal Characteristics Time (hours) following administration of estradiol-17 3 Number of Animals Body Weight (g) Uterine Wet Weight (mg) Uterine Dry Weight (mg) Moisture Content of Uterus (%) Control (0) 4 37.6 ± 0.60 19.0 ± 0.01 ( a ) 4.0 ± 0.01 ( a ) 79.4 * a ) 2 4 37.6 ± 0.90 22.0 ± 0.01 ( b ) 4.2 ± 0.0.1 ( b ) 80.1 ( a ) 3 4 37.5 ± 1.20 22.0 ± 0.02 ( b ) 4.2 ± 0.01 ( b ) 81.3 ( b ) 6 4 37.5 ± 0.70 32.1 ± 0.02 ( c ) 4.6 ± 0.01 ( c ) 85.2 ( d ) 12 4 37,. 4 ± 0.60 28.1 ± 0.01 ( C ) 5.0 ± 0.01 ( d ) 82.1 ( b ) 14 4 36.7 ± 1.20 28.0 ± 0.02 ( c ) 5.1 ± 0.01 ( d ) 81.0 ( b ) 24 4 37.5 ± 1.70 32.0 ± 0.02 ( d ) 5.5 ± 0.01 ( e ) 82.2 ( b ) 30 4 37.2 ± 0.60 37.0 ± 0.02 ( e ) 5.9 ± 0.02 ( f ) 83.7 ( C ) (a,b,c,d,e,f) Means with d i f f e r e n t subscripts are s i g n i f i c a n t l y d i f f e r e n t (P < 0.025) T a b l e 2: E f f e c t o f d o s e on w a t e r i m b i b i t i o n by r a t u t e r u s t i s s u e s f o l l o w i n g a d m i n i s t r a t i o n o f e s t r a d i o l - 1 7 $ ( E x p t . A-2) A n i m a l C h a r a c t e r i s t i c s Dosage (ng) o f e s t r a d i o l - 1 7 3 i n j e c t e d Number o f A n i m a l s Body W e i g h t (g) U t e r i n e Wet Weig h t (mg) U t e r i n e Dry Weig h t (mg) M o i s t u r e C o n t e n t o f U t e r u s (%) C o n t r o l (0) 4 37.6 ± 1.6 19.0 ± 1.10 ( a ) 4.0 ± 0.10 ( a ) 79.0 ( a ) 50 4 38.6 ± 2.1 32.6 ± 0.80 ( b ) 4.6 ± 0.10 ( b ) 86.0 ( b ) 100 4 41.6 ± 3.6 33.6 ± 0.40 ( c ) 4.8 ± 0.10 ( c ) 85.7 ( b ) 500 4 44.1 ± 2.1 33.1 ± 0.20 ( b ) 4.9 ± 0.05 ( c ) 85.2 ( b ) 1000 4 42.9 ± 1.8 33.8 ± 0.20 ( c ) 5.0 ± 0.10 ( C ) 85.2 ( b ) 5000 4' 43.7 ± 1.2 32.8 ± 0.90 ( b ) 5.2 ± 0.05 ( C ) 84.2 ( C ) 50000 4 42.8 ± 1.1 32.9 ± 1.20 ( b ) 5.1 ± 0.01 ( c ) 84.2 ( C ) (a,b,c) Means w i t h d i f f e r e n t ' s u p e r s c r i p t s a r e s i g n i f i c a n t l y d i f f e r e n t (P < 0.025) T a b l e 3 : E f f e c t s o f s t e r o i d and p l a n t e s t r o g e n s on water i m b i b i t i o n by r a t u t e r i n e t i s s u e s i x hours f o l l o w i n g a d m i n i s t r a t i o n (Expt. A - 3 ) Animal C h a r a c t e r i s t i c s E s t r o g e n s i n j e c t e d and dose Number o f A n i m a l s Body Weight (g) U t e r i n e Wet Weight (mg) U t e r i n e Dry Weight (mg) M o i s t u r e C o n t e n t o f U t e r u s (%) -Conto 1 (0 ug) 4 3 7 . 2 ± 1 . 3 0 2 3 . 1 ± 3 . 1 0 ( a ) • 4 . 6 . ± 0 . 1 0 ( a ) 7 9 . 8 ( a ) E s t r a d i o l - 1 7 3 ( 1 . 0 ug) 4 4 2 . 9 ± 0 . 5 0 3 3 . 8 ± 1 . 0 ( c ) 5 . 0 ± 0 . 4 0 ( b ) 8 5 . 2 ( b ) E s t r a d i o l - 1 7 a ( 2 . 0 ug) 4 4 3 . 1 ± 1 . 3 0 3 0 . 5 ± 0 . 8 ( b ) 4 . 8 ± 0 . 1 0 ( C ) 8 3 . 9 ( c ) DES ( 1 . 0 ug) 4 4 6 . 9 ± 0 . 8 0 . 3 3 . 1 ± 0 . 6 ( C ) 4 . 9 ± 0 . 1 0 ( C ) 8 5 . 0 ( b ) E s t r o n e ( 6 . 0 ug) 4 4 8 . 9 ± 1 . 0 0 3 4 . 1 ± 1 .6 ( c ) 5 . 0 ± 0 . 2 0 ( b ) 8 5 . 1 ( b ) E s t r i o l ( 1 . 0 ug) 4 4 7 . 2 ± 1 . 0 0 3 6 . 9 ± 1 . 2 ( d ) 4 . 8 ± 0 . 1 0 ( C ) 8 5 . 6 ( b ) T e s t o s t e r o n e ( 1 . 0 ug) 4 3 6 . 5 ± 1 . 3 0 2 3 . 6 ± 1.1 ( a ) 4 . 7 ± 0 . 1 0 ( C ) 8 0 . 1 ( a ) C o u m e s t r o l ( 9 0 . 0 ug) 4 4 3 . 9 ± 1 . 5 0 3 3 . 9 ± 1 . 2 ( c ) 4 . 6 ± 0 . 3 0 ( a ) 8 6 . 3 ( b ) G e n i s t e i n ( 0 . 8 mg) 4 4 7 . 1 ± 1 . 9 0 3 2 . 7 ± 1 .5 ( C ) 4 . 7 ± 0 . 6 0 ( C ) 8 5 . 6 ( b ) B i o c h a n i n A ( 1 0 0 ug) 4 4 8 . 2 ± 1 . 3 0 2 4 . 7 ± 1 . 5 ( a ) 4 . 8 ± 0 . 1 0 ( C ) 8 0 . 5 ( a ) Formononetin ( 1 0 0 ug) 4 4 7 . 5 ± 1 . 9 0 2 5 . 3 ± 1 .3 ( a ) 4 . 3 ± 0 . 7 0 ( a ) 8 2 . 8 ( C ) (a,b,c,d) Means with d i f f e r e n t subscripts are s i g n i f i c a n t l y d i f f e r e n t (P <: 0.025) 40. Experiment B : E f f e c t o f E s t r o g e n on Hyperemia o f U t e r i n e T i s s u e I n t r o d u c t i o n I n E x p e r i m e n t B, changes i n the u t e r i n e v a s c u l a t u r e were s t u d i e d i n immature female r a t s by i n t r o d u c i n g I n d i a i n k i n t o t h e v a s c u l a r system s i x hours a f t e r t h e a d m i n i s t r a t i o n o f e s t r a d i o l - 1 7 3 , g e n i s t e i n o r c o u m e s t r o l . M a t e r i a l s and Methods Immature female r a t s ( 2 5 - 3 0 g) were employed. They were m a i n t a i n e d and housed as d e s c r i b e d i n Experiment A. E s t r a d i o l -1 7 3 , g e n i s t e i n and c o u m e s t r o l ( 1 . 0 ug, 8 0 . 0 mg and 9 0 . 0 ug r e s p e c t i v e l y ) were a d m i n i s t e r e d , i n t r a p e r i t o n e a l l y . A n i m a l s were p r e p a r e d f o r p e r f u s i o n by op e n i n g t h e t h o r a c i c w a l l and e x p o s i n g t h e t h o r a c i c a o r t a . The esophagus was s e v e r e d and a l l c o n n e c t i v e t i s s u e was removed. P o l y e t h y l e n e t u b i n g ( 0 . 4 7 " x 0 . 6 7 " ) was t h r e a d e d i n t o a P a s t e u r p i p e t t e w h i c h i n t u r n was f i t t e d t o an a d a p t o r (Leur-Lok T u o h y - ^ . The o t h e r end o f the ad a p t o r was a t t a c h e d t o a 2 6 G ne e d l e w h i c h was i n s e r t e d i n t o t h e a r t e r y . A P o l y s t a l t i c p e r f u s i o n pump ( B u c h l e r I n s t r . ) was employed t o i n f u s e warm 0 . 9 % s a l i n e ( 3 7 ° C , pH 7 . 2 ) a t a r a t e o f 3 . 2 ml min ^  t h r o u g h t h e p o l y e t h y l e n e t u b i n g . The d u r a t i o n o f the p e r f u s i o n was d e t e r m i n e d by the appearance o f the v i s c e r a and u s u a l l y l a s t e d 3 . 5 minutes depending on t h e s i z e o f t h e a r t e r y . C a u t i o n was t a k e n n o t t o m a n i p u l a t e t h e u t e r u s t o o much d u r i n g t h e p e r f u s i o n . Warm, u n d i l u t e d I n d i a i n k ( 1 0 - 2 0 cm ) was i n t r o d u c e d i n t o t h e t u b i n g by means o f a hypodermic s y r i n g e . F o l l o w i n g the p e r f u s i o n w i t h I n d i a i n k , t h e r e p r o d u c t i v e t r a c t was removed w i t h a m i n i m a l amount o f h a n d l i n g and was r i n s e d i n warm 0.9% normal s a l i n e . The u t e r i n e t i s s u e s were e x c i s e d t o form open sigments c l o s e t o t h e e x t r a uterine p l e x u s and p l a c e d on m i c r o s c o p e s l i d e s f o r an e x a m i n a t i o n o f t h e a n t i -m e s o m e t r i a l r e g i o n . U t e r i were f i x e d w i t h 10% f o r m a l i n f o r 24 hours and t h e n d e h y d r a t e d s u c c e s s i v e l y w i t h 50, 80, and 2 X 95% a l c o h o l . F o l l o w i n g f i x a t i o n and d e h y d r a t i o n , t h e t i s s u e s were c l e a r e d w i t h x y l e n e f o r 5 m i n u t e s . The p r e p a r e d s l i d e s were o b s e r v e d under 100X power u s i n g a phase c o n t r a s t m i c r o -scope. R e s u l t s From F i g . 5 and 6 i t may be n o t e d t h a t e s t r a d i o l - 1 7 3 p o s s e s s e d t h e g r e a t e s t a b i l i t y t o i n d u c e hyperemia i n t h e immature female r a t u t e r u s . G e n i s t e i n and c o u m e s t r o l a l s o enhanced u t e r i n e v a s c u l a r p e r m e a b i l i t y when compared t o c o n t r o l t i s s u e s . The degree o f hyperemia i n d u c e d by g e n i s t e i n and c o u m e s t r o l d i d not appear t o be as d i s t i n c t as e s t r a d i o l - 1 7 3 , even though r e l a t i v e l y l a r g e r doses were a d m i n i s t e r e d . D i s c u s s i o n and C o n c l u s i o n s The use o f t h i s t e c h n i q u e has d e f i n i t e drawbacks i n s t u d y -i n g e s t r o g e n i c p o t e ncy o f v a r i o u s compounds. The d i f f i c u l t i e s a r e a s s o c i a t e d w i t h t h e m a n i p u l a t i o n o f u t e r i n e t i s s u e s d u r i n g p r e p a r a t i o n and t h e consequent damage. To m i n i m i z e t h e s e d i s a d v a n t a g e s e v e r y e f f o r t was made t o t r e a t t h e specimens the same way including the i n j e c t i o n of saline at constant rate, temperature and the preparation of sections with the same f i x i n g and clearing reagents. C a p i l l a r i e s i n any given region were found to vary i n diameter though almost a l l specimens were f i l l e d with India ink. Some appeared as exceedingly fine l i n e s under a magnification of 100X. By means of vi s u a l comparison of various treatments with controls, i t was noted that the blood vessels of the uterus r e f l e c t a gradient of response to estrogen concentration. C a p i l l a r i e s around the uterine horns become an e a s i l y recogniz-able feature of vascular architecture of the uterus as seen in the injected specimens. Genistein and coumestrol (0.8 mg and 90. ug, respectively) had some e f f e c t on the vascular permea-b i l i t y of the uterine c e l l . Perel and Lidner (1970) reported si m i l a r responses occurring with hyperemia aft e r the i n j e c t i o n of coumestrol and genistein (80 ;ug, 0.62 mg respectively). These phytoestrogens did not provide that same magnitude of response as did estradiol-17 3 (1.0 ug). 43. CONTROL F i g . 5. The d i s t r i b u t i o n of I n dia ink i n the u t e r i n e vasculature of c o n t r o l and e s t r a d i o l - 1 7 8 t r e a t e d r a t s (Expt. B) x 100. COUMESTROL GENISTEIN F i g . 6. The d i s t r i b u t i o n of India ink i n the uterine vasculature of coumestrol and genistein treated rats (Expt. B) x 100 E x p e r i m e n t C : E f f e c t s o f E s t r a d i o l - 1 7 3 on RNA and-DNA S y n t h e s i s i n Immature U t e r i n e T i s s u e s . I n t r o d u c t i o n The e f f e c t o f e s t r o g e n i c hormones on RNA and DNA s y n t h e s i s i n t h e mammalian u t e r u s has been r e v i e w e d i n d e t a i l by Means and O'Malley (1972). I n o r d e r t o s t u d y t h e e a r l i e s t d e t e c t a b l e r esponse o c c u r r i n g i n the u t e r i n e c e l l a f t e r t h e a d m i n i s t r a t i o n o f e s t r o g e n s , a t t e m p t s have been made t o s t u d y t h e e f f e c t s o f a n t i b i o t i c i n h i b i t o r s o f RNA and DNA s y n t h e s i s . As a r e s u l t o f t h e s e s t u d i e s a c h r o n o l o g i c a l sequence o f b i o c h e m i c a l e v e n t s o c c u r r i n g i n v i v o a f t e r the a d m i n i s t r a t i o n o f e s t r o g e n s has been e s t a b l i s h e d ( G o r s k i and K a t z e l l e n b o g e n , 1975). From s t u d i e s based on the i n c o r p o r a t i o n o f v a r i o u s l a b e l l e d p r e c u r s o r s , t h e r a t e o f RNA and DNA s y n t h e s i s has been d e t e r -mined by many w o r k e r s . G o r s k i and N i c o l e t t e (1963) r e p o r t e d an i n c r e a s e d r a t e i n RNA s y n t h e s i s o c c u r r i n g i n t h e n u c l e u s one hour a f t e r e s t r a d i o l - 1 7 3 a d m i n i s t r a t i o n . A d d i t i o n a l i n f o r m a t i o n r e g a r d i n g t h e e a r l y a c t i o n o f e s t r a d i o l - 1 7 3 on RNA s y n t h e s i s was o b t a i n e d by Noteboom and G o r s k i (1963^b)) who r e p o r t e d an i n c r e a s e d a c t i v i t y i n the RNA polymerase enzyme as e a r l y as one t o f o u r hours a f t e r e s t r o g e n a d m i n i s t r a t i o n . A l t h o u g h RNA polymerase a c t i v i t y i s s i g n i f i c a n t l y e l e v a t e d one hour a f t e r the a d m i n i s -t r a t i o n o f e s t r o g e n an i n c r e a s e i n n e t u t e r i n e RNA c o n t e n t i s not a c h i e v e d u n t i l 8-12 hours ( B i l l i n g e t al.1969(a) ) . The newly s y n t h e s i z e d RNA appears t o r e p r e s e n t a l l t y p e s o f RNA, but i s p r i m a r i l y r i b o s o m a l RNA and may d i f f e r i n c o m p o s i t i o n from t h a t produced i n t h e absence o f e s t r o g e n s as i n d i c a t e d by DNA-RNA h y b r i d i z a t i o n s t u d i e s (Hahn e t a_l. 1'968). A c o n v e r s i o n o f r i b o s o m e s t o p o l y s o m e s o c c u r s due t o t h e e n t r y o f new mes-s e n g e r RNA i n t o t h e c y t o p l a s m , ( M e a n s and O ' M a l l e y , 1 9 7 2 ) . T h e s e p o l y r i b o s o m e s s y n t h e s i z e a q u a n t i t a t i v e l y d i f f e r e n t p o p u -l a t i o n o f p e p t i d e s i n v i t r o w h i c h s h o u l d r e f l e c t t h e e a r l i e r a l t e r a t i o n s i n m e s s e n g e r RNA p r o d u c t i o n . T h i s s e q u e n c e o f e v e n t s i n d i c a t e s t h a t e s t r o g e n e x e r t s i t s p r i m a r y e f f e c t i n t h e n u c l e u s t o promote s e l e c t i v e gene a c t i v a t i o n and s u b s e q u e n t s y n t h e s i s o f new p r o t e i n m o l e c u l e s on c y t o p l a s m i c p o l y s o m e s . E s t r a d i o l - 1 7 3 was a l s o r e p o r t e d t o p r o d u c e an i n c r e a s e i n t h y m i d i n e i n c o r p o r a t i o n i n t o DNA, 16 h o u r s a f t e r e s t r a d i o l - 1 7 3 i n j e c t i o n ( G o r s k i and R a k e r , 1974). I n c r e a s e s i n t h e r a t e o f DNA and h i s t o n e s y n t h e s i s b e g i n a b o u t 18 h o u r s a f t e r e s t r o g e n t r e a t m e n t (Kaye e t a l . 1 9 72). T h e s e e v e n t s o c c u r p r i o r t o c e l l d i v i s i o n w h i c h b e g i n s i n t h e u t e r u s a t a p p r o x i m a t e l y 24 h o u r s a f t e r e s t r a d i o l - 1 7 3 a d m i n i s t r a t i o n ( M e u l l e r e t a i . 1 9 5 8 ) . I n v i e w o f t h e i n c r e a s e s i n u t e r i n e d r y w e i g h t s o b s e r v e d a f t e r t h e a d m i n i s t r a t i o n o f e s t r o g e n i n E x p e r i m e n t A, s t u d i e s were i n i t i a t e d t o d e t e r m i n e t h e e f f e c t s o f e s t r a d i o l - 1 7 g on o v e r a l l n u c l e i c a c i d m e t a b o l i s m i n t h e u t e r i n e c e l l . The p r o c e d u r e f o r RNA and DNA e x t r a c t i o n was s t a n d a r d i z e d . T h i s was f o l l o w e d by q u a n t i t a t i v e d e t e r m i n a t i o n o f t h e e f f e c t s o f e s t r a d i o l - 1 7 3 on RNA and DNA s y n t h e s i s by t h e u t e r i n e t i s s u e s . M a t e r i a l s and Methods A n i m a l s Immature f e m a l e r a t s (40-50 g) were u s e d f o r t h i s s t u d y . They were h o u s e d and m a i n t a i n e d as p r e v i o u s l y d e s c r i b e d u n d e r Experiment A. M a t e r i a l s E s t r a d i o l - 1 7 3 was o b t a i n e d from S i g n a C h e m i c a l s . RNA ( y e a s t , Sigma Chemicals) and s-RNA (A g r a d e , Calbiochem) donated by Dr. B.C. Sung, Dept. o f N e u r o l o g i c a l S c i e n c e s , UBC were used as RNA s t a n d a r d s . C a l f thymus DNA o b t a i n e d from Mann Res e a r c h L a b o r a t o r i e s was used as DNA s t a n d a r d s . S t a n d a r d c u r v e s were o b t a i n e d u s i n g s-RNA and c a l f thymus DNA i n K O H - T r i c h l o r o a c e t i c a c i d (TCA) and HCIO^ r e s p e c t i v e l y . Methods 1. S t a n d a r d i z a t i o n o f RNA and DNA E x t r a c t i o n P r o c e d u r e s I n i t i a l e x p e r i m e n t s were performed t o s t a n d a r d i z e t h e nuc-l e i c a c i d e x t r a c t i o n p r o c e d u r e s . T h i s was a c c o m p l i s h e d by e x t r a c t i n g RNA and DNA from i n c r e a s i n g w e i g h t s o f u t e r i n e t i s s u e -as d e s c r i b e d below. A n i m a l s were s a c r i f i c e d by e x p o s i n g them t o carbon d i o x i d e i n a s e a l e d j a r and u t e r i n e t i s s u e s were p o o l e d i n i c e c o l d 0.9% N a C l . Wet t i s s u e s l i c e s were randomly s e l e c t e d , b l o t t e d and weighed i n q u a n t i t i e s o f 40.0, 50.0 and 100.0 mg r e s p e c t i v e l y . N u c l e i c a c i d c o n t e n t was e x t r a c t e d by t h e methods o f Schmidt-Thannhauser (1945) and C e r i o t t i (1955) w i t h minor m o d i f i c a t i o n s as summarized i n Appendix, F i g . A. Three whole u t e r i were p o o l e d i n 4.0 ml o f i c e c o l d d e i o n i z e d water and homogenized u s i n g a t i s s u e homogenizer (ASCO I n d u s t r i e s ) f o r 5 m i n u t e s . To t h e homogenate 7.0 ml o f i c e c o l d 10% TCA were added. The tubes were a l l o w e d t o s t a n d f o r 10 minutes on i c e , c e n t r i f u g e d , and the p r e c i p i t a t e resuspended and washed w i t h 10 ml o f i c e c o l d 95% e t h a n o l . F o l l o w i n g c e n t r i f u g a t i o n 48. the r e s u l t i n g p r e c i p i t a t e was suspended i n 2.0 ml o f IN KOH f o r 16 hours a t 37°C. A f t e r a d d i t i o n o f 0.4 ml o f 6N HC1, p r o t e i n and DNA were p r e c i p i t a t e d w i t h c o l d 5% TCA. RNA i n t h e s u p e r -n a t a n t f r a c t i o n was e s t i m a t e d by r e a d i n g t h e absorbance a t 260 nm i n an Unicam 800 s p e c t r o p h o t o m e t e r a g a i n s t an a p p r o p r i a t e KOH-TCA b l a n k . The p r e c i p i t a t e o f DNA and p r o t e i n was suspended i n 2.5 ml o f 10% HC10 4 and heated a t 70-80°C f o r 25 m i n u t e s . T h i s m a t e r i a l was c e n t r i f u g e d and t h e DNA i n t h e s u p e r n a t a n t was measured a t 260 nm u s i n g an a p p r o p r i a t e HCIO^ b l a n k . S t a n d a r d c u r v e s w i t h y e a s t RNA o r pure s-RNA and c a l f thymus DNA were used t o d e t e r m i n e the n u c l e i c a c i d c o n t e n t o f t h e e x t r a c t s . A n a l y s i s o f d a t a was done by S t u d e n t ' s t - t e s t . 2. Time Course E f f e c t s o f E s t r a d i o l - 1 7 3 on N u c l e i c A c i d  S y n t h e s i s by the u t e r i n e t i s s u e . A f t e r s t a n d a r d i z i n g t h e e x t r a c t i o n p r o c e d u r e o f RNA and DNA, a time c o u r s e e f f e c t o f e s t r a d i o l - 1 7 3 on RNA and DNA s y n t h e s i s by the u t e r i n e t i s s u e was s t u d i e d . R a t s were d i v i d e d i n t o groups o f t h r e e a n i m a l s and each a n i m a l was g i v e n an i n t r a p e r i t o n e a l i n j e c t i o n o f 5 jug o f e s t r a d i o l - 1 7 g . C o n t r o l a n i m a l s r e c e i v e d i n j e c t i o n s o f e q u a l volumes o f 0.9% NaCI con-t a i n i n g 1% e t h a n o l . They were s a c r i f i c e d a f t e r 0,6,12,24,4 8 and 72 hours by p l a c i n g them i n a j a r o f ca r b o n d i o x i d e . RNA and DNA were', e x t r a c t e d from t h e u t e r i n e t i s s u e s t o d e t e r m i n e t h e t i m e when maximum e f f e c t o f e s t r o g e n on n u c l e i c a c i d s y n -t h e s i s was m a n i f e s t e d . R e s u l t s 1. S t a n d a r d i z a t i o n o f RNA and DNA E x t r a c t i o n P r o c e d u r e s Due t o t h e i m p u r i t i e s i n t h e y e a s t RNA i t was found neces-s a r y t o s t a n d a r d i z e t h i s p r e p a r a t i o n a g a i n s t known s o l u t i o n s o f p u r i f i e d s-RNA. The m o l a r a b s o r p t i o n c o e f f i c i e n t o f s-RNA was d e t e r m i n e d by d i s s o l v i n g i t i n 0.5 mg/ml o f 0.1 M a c e t a t e b u f f e r , pH 5.0. s-RNA i n c o m p l e t e s o l u t i o n had a m o l a r a b s o r p -t i o n c o e f f i c i e n t o f 18 O.D.^^Q X mg s-RNA - 1. A f t e r o b t a i n i n g t h i s v a l u e , i t was p o s s i b l e t o d e t e r m i n e t h e c o n c e n t r a t i o n o f t h e c o r r e s p o n d i n g y e a s t RNA f i l t r a t e . S t a n d a r d c u r v e s o f RNA and DNA a r e g i v e n i n F i g . 7. The a p p r o x i m a t e c o n t e n t o f RNA and DNA p e r u t e r u s o f immature r a t s was 112.0 ± 1 0 p.g and 237 ± 1 1 . 0 y g r e s p e c t i v e l y ( T a b l e 5 ) . The RNA c o n c e n t r a t i o n s i n 40.0, 50.0 and 100.0 mg o f u t e r i n e t i s s u e s were 1 7 6 . 1 ± 2 . 1 , 2 2 3 . 2 ± 1 . 9 , 540.7+2.5 y g r e s p e c t i v e l y ; c o r r e s p o n d i n g v a l u e s f o r DNA were 232+11, 515+9, 1023±152 jag r e s p e c t i v e l y ( F i g . 8, T a b l e 4 ) . 2. Time C o u r s e E f f e c t o f E s t r a d i o l - 1 7 3 on N u c l e i c A c i d  S y n t h e s i s by t h e u t e r i n e t i s s u e When e s t r a d i o l - 1 7 3 was a d m i n i s t e r e d t o immature r a t s p o s -s e s s i n g e q u i v a l e n t body w e i g h t s n e t RNA s y n t h e s i s was s e e n a f t e r 12 h o u r s ( T a b l e 5 ) . Maxi m a l RNA s y n t h e s i s i n t h e immature r a t u t e r u s was n o t i c e d 48 h o u r s f o l l o w i n g a s i n g l e i n j e c t i o n o f e s t r a d i o l - 1 7 3 . The c o n c e n t r a t i o n o f RNA i n t h e u t e r i n e t i s s u e d e c l i n e d s l i g h t l y between 48 and 72 h o u r s , b u t i t n e v e r r e a c h e d i t s c o n t r o l l e v e l ( F i g . 9 ) . The e f f e c t o f e s t r a d i o l - 1 7 ^ on t h e DNA c o n t e n t o f t h e u t e r u s showed a d e l a y e d r e s p o n s e w h i c h l a s t e d l o n g e r . Maximum i n c r e a s e s i n n e t u t e r i n e DNA d i d n o t o c c u r u n t i l 48 h o u r s a f t e r e s t r o g e n a d m i n i s t r a t i o n . S l i g h t d e c r e a s e s i n u t e r i n e DNA c o n -c e n t r a t i o n s were n o t i c e d a f t e r 48 h o u r s . A s t e a d y d e c r e a s e i n t h e RNA/DNA r a t i o n s was noted between 24 and 72 h o u r s , b u t th e d e c r e a s e i n RNA/DNA r a t i o s d i d n o t r e t u r n t o c o n t r o l l e v e l s . A l l d a t a was s t a t i s t i c a l l y a n a l y z e d by s t u d e n t ' s t - t e s t . ' D i s c u s s i o n The l e v e l s o f n u c l e i c a c i d i n the u t e r u s o f immature r a t s a re i n agreement w i t h t h e r e s u l t s o b t a i n e d by G o r s k i and K a t z e l l e n b o g e n (1975). They r e p o r t e d t h a t c o n c e n t r a t i o n s o f RNA when e x p r e s s e d i n terms o f c e l l u l a r RNA were c o m p a r a t i v e l y l o w e r i n t h e u t e r u s o f immature r a t s t h a n i n o t h e r t i s s u e s . The RNA/DNA r a t i o s i n t h e immature r a t u t e r u s was 0.47 w h i c h c o r r e s p o n d s t o t h e r a t i o o f 0.4 r e p o r t e d by G o r s k i and K a t z e l l e n b o g e n (1975). T h i s i s i n c o n t r a s t t o r a t i o s o f 2.0 and 5.0 i n l i v e r and E - c o l i r e s p e c t i v e l y . R e p o r t s by Kaye e t a_l. (1972) have r e v e a l e d t h a t t h e i n -3 c o r p o r a t i o n o f (Me-H) t h y m i d i n e i n t o DNA i s dependent on age and dose. I n r a t s l e s s than 15 days o l d , s i n g l e i n j e c t i o n s o f e s t r a d i o l - 1 7 3 d i d n o t r e s u l t i n an i n c r e a s e i n t h e u t e r i n e w e i g h t , RNA c o n t e n t , o r r a t e o f DNA s y n t h e s i s . Twenty day o l d r a t s , on t h e o t h e r hand, showed s i g n i f i c a n t i n c r e a s e s i n th e r a t e o f RNA and DNA s y n t h e s i s . Amounts o f e s t r a d i o l - 1 7 3 as low as 50 pg t o 20 day o l d r a t s , w e i g h i n g 33 grams were a l s o r e p o r t e d t o i n c r e a s e t h e r a t e o f DNA s y n t h e s i s . W i t h t h i s work i n mind immature female r a t s were employed i n t h i s s t u d y and a r e l a t i v e l y l a r g e dose o f e s t r a d i o l - 1 7 3 (5 ug) was a d m i n i s t e r e d t o e l i c i t a d e f i n i t e r e s p o n s e . R e s u l t s from t h i s e x p e r i m e n t i n d i c a t e t h a t t h e e f f e c t s o f e s t r a d i o l - 1 7 3 on t i s s u e growth a r e mediated by changes i n u t e r i n e RNA and DNA. H a m i l t o n e t a l . (1968) and B i l l i n g e t a l . ( 1 9 6 9 ( b ) ) r e p o r t e d t h a t t h o u g h no i n c r e a s e i n t h e u t e r i n e RNA c o n t e n t as m e asured by c h e m i c a l methods was n o t i c e d o v e r t h e f i r s t s e v e n h o u r s , a s i g n i f i c a n t i n c r e a s e was o b s e r v e d 12 h o u r s a f t e r a d m i n i s t r a t i o n o f e s t r a d i o l - 1 7 3 . D a t a f r o m t h i s e x p e r i -ment i s i n a c c o r d a n c e w i t h t h e s e f i n d i n g s . The i n c r e a s e i n n e t s y n t h e s i s i n t h e u t e r i n e t i s s u e f o l l o w i n g a d m i n i s t r a t i o n o f e s t r a d i o l - 1 7 3 has b e en a t t r i b u t e d t o i n c r e a s e d s y n t h e s i s o f t h e enzyme RNA p o l y m e r a s e . However, on t h e b a s i s o f t h e t i m e l a g n o t i c e d d u r i n g t h e e a r l y s t a g e s o f e s t r o g e n a d m i n i s t r a t i o n and t h e c h a n g e s i n p r e -c u r s o r p o o l s i z e s , G o r s k i and K a t z e l l e n b o g e n (1975) have e x p r e s s e d d o u b t s t h a t RNA s y n t h e s i s was g r e a t l y a f f e c t e d a t t h e s e e a r l y t i m e s . They have s u g g e s t e d t h a t t h e mode o f a c t i o n o f e s t r o g e n s i s more g e n e r a l i n n a t u r e and r e q u i r e s t o be s u b -s t a n t i a t e d by more e x p e r i m e n t a l d a t a . They a s c r i b e d t h e i n c r e a s e d a c t i v i t y o f v a r i o u s n u c l e a r f u n c t i o n s i n r e s p o n s e t o e s t r o g e n was d e p e n d e n t on t h e programming o f t h e c e l l , i . e . t h e p r o d u c t i o n o f s p e c i f i c mRNA and rRNA s p e c i e s . , I n c r e a s e s i n t h e DNA c o n t e n t o f t h e u t e r i n e t i s s u e were n o t i c e d t o o c c u r a t a s l o w e r r a t e t h a n t h e RNA c o n t e n t . T h e s e r e s u l t s a g r e e w i t h t h o s e o f M e u l l e r e t a l . (1958); B i l l i n g e t a l . ( 1 9 6 9 ( b ) ) ; Kaye e t a l . ( 1 9 7 2 ) , who r e p o r t e d t h a t t h o u g h s i g n i f i c a n t c e l l u l a r h y p e r t r o p h y o c c u r r e d a f t e r s i x h o u r s , i n c r e a s e s i n c e l l number c o u l d be n o t i c e d o n l y a f t e r 24 h o u r s f o l l o w i n g e s t r o g e n a d m i n i s t r a t i o n . S i m i l a r l y , E p i f a n o v a (1966) u s i n g t r i t i a t e d t h y m i d i n e and a u t o r a d i o g r a p h i c t e c h n i q u e s , f o u n d i n c r e a s e s i n m i t o t i c i n d e x e s o f mouse u t e r i n e e p i t h e l i u m 42-24 h o u r s f o l l o w i n g e s t r o g e n a d m i n i s t r a t i o n r e s u l t i n g i n 1.5 f o l d s h o r t e n i n g o f t h e c e l l g e n e r a t i o n t i m e . T h i s r e d u c t i o n o f c e l l g e n e r a t i o n t i m e o c c u r r e d a t t h e e x p e n s e o f t h e G-^  and s t a g e s w h i c h r e f e r t o t h e t i m e o f p r e p a r a t i o n f o r DNA s y n t h e s i s and DNA r e p l i c a t i o n r e s p e c t i v e l y . The t i m e gap between t h e u p t a k e o f w a t e r and t h e maximum a c t i v i t y o f t h e p o l y s o m e s , p a r t i c u l a r l y rRNA, t h e p r o t e i n s y n -t h e s i z i n g o r g a n e l l e s o f t h e c e l l s has been a t t r i b u t e d by G o r s k i e t a l . (1975) t o be t h e t i m e r e q u i r e d f o r t h e p r o d u c t i o n o f mRNA needed f o r t h e s y n t h e s i s o f i n d u c e d p r o t e i n ."(I.P.). C o n c l u s i o n s E x p e r i m e n t C was d e s i g n e d t o s t a n d a r d i z e t h e method o f RNA and DNA e x t r a c t i o n f r o m u t e r i n e t i s s u e s and d e t e r m i n e t h e t i m e c o u r s e o f t h e e f f e c t o f e s t r a d i o l - 1 7 3 on t h e s y n t h e s i s o f RNA and DNA. RNA and DNA c o n c e n t r a t i o n s i n t h e immature f e m a l e r a t u t e r u s were 112±10 and 237±21 ug r e s p e c t i v e l y . From t h e r e s u l t s o f E x p e r i m e n t s A, B and C i t may be c o n -c l u d e d t h a t f o l l o w i n g e s t r o g e n a d m i n i s t r a t i o n t o immature r a t s t h e r e i s a d i s t i n c t p a t t e r n o f i n c r e a s e d w a t e r r e t e n t i o n a t s i x h o u r s , i n c r e a s e d RNA s y n t h e s i s a t 12 h o u r s and a c c e l e r a t e d DNA m e t a b o l i s m and c e l l u l a r p r o l i f e r a t i o n a t 24 h o u r s . RNA/DNA r a t i o s w h i c h i n d i c a t e c e l l u l a r p r o l i f e r a t i o n i n c r e a s e d g r a d u a l l y a f t e r 12 h o u r s . Maximum c e l l u l a r p r o l i f e r a t i o n was n o t i c e d 24 h o u r s a f t e r a d m i n i s t r a t i o n o f e s t r a d i o l - 1 7 3 , i n d i c a t i n g a l e v e l l i n g o f f o f RNA p r o d u c t i o n and an i n c r e a s e i n DNA m e t a b o l i s m . RNA AND DNA CONCENTRATION (uG/ML) Fig.. 7. Standard curves for RNA and DNA (Expt. C) 54. F i g . 8. RNA and DNA concentration i n homogenates of d i f f e r e n t immature rat uterine weights (Expt. C) 500 12 24 48 72 T I M E (HOURS) F i g . 9. E f f e c t o f t i m e on RNA and DNA s y n t h e s i s i n r a t u t e r i n e t i s s u e f o l l o w i n g e s t r o g e n a d m i n i s t r a t i o n ( E x p t . C) Table 4 : RNA and DNA concentrations in homogenates of d i f f e r e n t immature rat uterine weights (Expt. Wet weight of Uterus (mg) RNA concentrations (ug) . DNA concentrations (ug). RNA/DNA 40.0 176.1 ± 2.1 232.0 ± 11.0 0.75 50.0 223.3 ± 1.9 515.6 ± 9.03 0.4.3 100.0" 540.7 ± 2.5 1023.6 ± 1.52 0.52 T a b l e 5 : E f f e c t of time on RNA and DNA s y n t h e s i s by r a t u t e r i n e t i s s u e (Expt. C) Animal C h a r c t e r i s t i c s Time (hours) f o l l o w i n g a d m i n i s t r a t i o n of e s t r a d i o l - 1 7 3 Number of Animals Body Weight (g) U t e r i n e Wet Weight (mg) RNA co n c e n t r a t i o n (ug/uterus) DNA c o n c e n t r a t i o n (ug/uterus) RNA/DNA C o n t r o l (0) 9 4 3 + 2.0 25.7 + 3.0 ( a ) 112 ± 10 ( a ) 237 ± 21 ( a ) 0. 47 6 9 45 ± 3.0 31.1 ± 5.0 ( b ) 117 ± 12 ( a ) 232 ± 20 ( a ) 0. 50 12 9 44 ± 2.5 44.5 ± 2.5 ( C ) 200 ± 19 ( b ) 253 + 29 ( a ) 0. 79 24 9 43 ± 3.0 66.1 ± 3.9 ( d ) 357 ± 37 ( C ) 271 ± 25 ( a ) 1.30 48 9 46 ± 1.5 75.3 ± 4 . 8 ( e ) 452 ± 29 ( d ) 402 ± 31 ( c ) 1.12 72 9 ,44 ± 1.8 ,47.2 ± 6.9 ( C ) 302 ± 19 ( c ) 309 ± 28 ( b ) 0.97 (a,b,c,d,e) Means with d i f f e r e n t subscripts are s i g n i f i c a n t l y d i f f e r e n t (P ^  0.025) 58. Experiment D : E f f e c t s o f E s t r o g e n s and P h y t o e s t r o g e n s on t h e I n c o r p o r a t i o n o f 3H U r i d i n e i n t o R N A by t h e U t e r u s - S i x Hour I n V i v o P u l s i n g . I n t r o d u c t i o n A n e t s y n t h e s i s o f R N A by t h e u t e r u s i n r e s p o n s e t o e s t r o g e n a d m i n i s t r a t i o n has been demonstrated i n t h e p r e v i o u s e x p e r i m e n t . Other workers have a l s o shown t h e i n c o r p o r a t i o n o f v a r i o u s p r e -c u r s o r s i n t o d i f f e r e n t t y p e s o f R N A ( H a m i l t o n e t a l . 1968; B i l l i n g e t al_. 1969 (B) ) . However, i s o t o p i c t r a c e r s t u d i e s d e s i g n e d t o m o n i t o r t h e r a t e o f u t e r i n e R N A s y n t h e s i s have y i e l d e d con-f l i c t i n g r e s u l t s . B i l l i n g e t a l . (1969(b)) a l l o w e d l a b e l l e d a denosine t o e q u i l i b r a t e w i t h t h e u t e r i n e a d enosine n u c l e o t i d e p o o l p r i o r t o and f o r a l i m i t e d t i m e a f t e r e s t r a d i o l - 1 7 3 a d m i n i s t r a t i o n , and n o t i c e d t h a t i n c o r p o r a t i o n o f l a b e l l e d a d e nosine i n t o u t e r i n e R N A i n c r e a s e d o n l y s l i g h t l y d u r i n g t h e i n i t i a l phase o f t h e response and was not s u b s t a n t i a l u n t i l 5 hours. On t h e c o n t r a r y , H a m i l t o n e t a l . (1968) have r e p o r t e d 3 t h a t d u r i n g a 10 minute p u l s e i n v i v o , 5- H u r i d i n e i n c o r p o r a t i o n i n t o u t e r i n e n u c l e a r R N A was maximal 20 minutes a f t e r e s t r a d i o l - 1 7 3 a d m i n i s t r a t i o n . The e x t e n t o f i s o t o p e i n c o r p o r a t i o n i n t o R N A i s governed not o n l y by t h e r a t e o f R N A s y n t h e s i s but a l s o by t h e s p e c i f i c a c t i v i t y a s s o c i a t e d w i t h i t s n u c l e o t i d e p r e c u r s o r s a t t h e t i m e o f i n c o r p o r a t i o n . I n r e g a r d t o the u t e r u s , e s t r o g e n t r e a t m e n t has been shown t o i n f l u e n c e t h e s p e c i f i c a c t i v i t i e s o f v a r i o u s R N A p r e c u r s o r s by i n c r e a s i n g t h e v a s c u l a r i t y (Szego, 1967), o r the p e r m e a b i l i t y o f p r e c u r s o r s ( B i l l i n g cat al. 1969(b)) and p o o l s i z e s ( M u e l l e r e t a l . 1958). G o r s k i e t a l . (1975) have drawn a t t e n t i o n t o t h e d i f f i c u l t i e s i n d r a w i n g c o n c l u s i o n s f r o m e x p e r i m e n t s i n v o l v i n g p r e c u r s o r i n c o r p o r a t i o n . Munns and Katzman (1971) e m p l o y i n g an i n v i t r o s y s t e m o f p r e c u r s o r i n c o r -p o r a t i o n p r e c e d e d by an i n v i v o a d m i n i s t r a t i o n o f e s t r a d i o l - 1 7 3 14 r e p o r t e d t h a t L - [ m e t h y l - C] m e t h i o n i n e was a u s e f u l p r e c u r s o r o f m e t h y l a t e d RNA 1s ( i . e . tRNA and rRNA). The a p p a r e n t a d v a n t a g e o f t h i s l a b e l was i t s i n d e p e n d e n c e on f l u c t u a t i n g n u c l e o t i d e p r e c u r s o r p o o l s i z e s . S i m i l a r d i f f e r e n c e s have n o t been e n c o u n t e r e d w i t h t h y m i d i n e l a b e l l e d DNA. Numerous w o r k e r s have employed t r i t i a t e d t h y m i d i n e i n b o t h i n v i v o and i n v i t r o a s s a y s (Kaye e t al_. 1972; C a r t e r e t a l . 1 9 75). Kaye e t a l . (1972) f a v o r e d t h e use o f i n v i t r o s y s t e m s due t o g r e a t e r i n c o r p o r a t i o n and b e t t e r r e c o v e r y r a t e s 3 . . o f H - t h y m i d i n e . R e c e n t l y K a t z e l l e n b o g e n and G o r s k i (1975) c o n c l u d e d f r o m an e x t e n s i v e r e v i e w o f t h e methods employed by v a r i o u s w o r k e r s t h a t no c l e a r c u t e v i d e n c e o f e a r l y i n c r e a s e s i n t h e i n c o r p o r a t i o n o f p r e c u r s o r s i n t o RNA due t o e s t r a d i o l - 1 7 3 t r e a t m e n t e x i s t e d . The p u r p o s e o f t h e f o l l o w i n g e x p e r i m e n t s was t o s t u d y i n v i t r o t h e d e g r e e o f t r i t i a t e d u r i d i n e i n c o r p o r a t i o n i n t o RNA by t h e u t e r u s f o l l o w i n g e s t r o g e n o r p h y t o e s t r o g e n a d m i n i s t r a t i o n . I n v i e w o f d a t a o b t a i n e d i n E x p e r i m e n t s A, B and C r e g a r d i n g t h e e s t r o g e n i c a c t i v i t y o f v a r i o u s e s t r o g e n s , E x p e r i m e n t D was i n i t i a t e d t o s t u d y t h e c o m p a r a t i v e e f f e c t s o f s t e r o i d e s t r o g e n s and p h y t o e s t r o g e n s i n i n d u c i n g t h e i n c o r p o r a t i o n o f t h e i s o t o p i c n u c l e o s i d e , u r i d i n e . 60. M a t e r i a l s and Methods A n i m a l s Immature f e m a l e r a t s (4 0-50 g) were m o s t l y u s e d i n t h i s s t u d y . I n a d d i t i o n t o t h e s e a n i m a l s two g r o u p s o f r a t s (60 g) were a l s o u s e d i n a few e x p e r i m e n t s . They were h o u s e d and m a i n -t a i n e d as d e s c r i b e d i n E x p e r i m e n t A. M a t e r i a l s 3 [5,6- H] u r i d i n e ( s p e c i f i c a c t i v i t y , 44.5 Ci/;m mole ..was o b t a i n e d f r o m Amersham S e a r l e . TC medium 199, c h e m i c a l l y d e f i n e d b i o l o g i c a l medium was o b t a i n e d f r o m D i f c o L a b o r a t o r i e s . I t was s u p p l e m e n t e d w i t h 1.0 mM g l u t a m i n e ( D i f c o ) and 2% b o v i n e a l b u m i n ( S i g m a ) . Y e a s t RNA and c a l f thymus DNA were o b t a i n e d f r o m Sigma c h e m i c a l s and Mann R e s e a r c h L a b . r e s p e c t i v e l y . Methods A d m i n i s t r a t i o n o f E s t r o g e n s i n v i v o Immature f e m a l e r a t s were g i v e n s i n g l e i n j e c t i o n s o f t h e s t e r o i d e s t r o g e n s and p h y t o e s t r o g e n s i n d o s e s m e n t i o n e d i n E x p e r i m e n t A. C o n t r o l a n i m a l s r e c e i v e d s i m i l a r v o l u m e s o f t h e t e s t v e h i c l e s , a l c o h o l - s a l i n e o r p r o p y l e n e g l y c o l . The i n c o r -3 p o r a t i o n o f H - u r i d i n e by r a t u t e r i was d e t e r m i n e d as d e s c r i b e d below. _3 I n c o r p o r a t i o n o f [5,6 H] u r i d i n e by t h e u t e r u s  i n v i t r o A n i m a l s were s a c r i f i c e d s i x h o u r s a f t e r t h e a d m i n i s t r a t i o n o f e s t r o g e n s by p l a c i n g them i n a s e a l e d j a r c o n t a i n i n g c a r b o n d i o x i d e . The u t e r i n e t i s s u e s were removed and t r a n s f e r r e d t o i c e c o l d 0.9% NaCI. T i s s u e s were s t r i p p e d o f a d h e r i n g f a t and c o n n e c t i v e t i s s u e and p l a c e d i n a prewarmed (37°C) f i v e ml s t o p p e r e d b o t t l e c o n t a i n i n g 1.0 ml o f TCM 199 and a p p r o x i m a t e l y 3 1.6 juCi o f H - u r i d i n e . Uterxne t i s s u e s were i n c u b a t e d under an atmosphere o f 95%0 2-5%CC> 2 f o r 1.0 hour i n a water b a t h (37°C) a t a s h a k i n g speed o f one s t r o k e p e r second. A f t e r t h e i n c u b a t i o n t h e media was removed and t h e u t e r i p l a c e d on d r y i c e t o t e r m i n a t e t h e r e a c t i o n . Whole u t e r i n e t i s s u e s were r i n s e d t h r e e t i m e s s u c c e s s i v e l y w i t h i c e c o l d d i s t i l l e d w a t e r t o remove the e x t r a c e l l u l a r r a d i o a c t i v i t y . T h i s f r a c t i o n r e p r e s e n t i n g t i s s u e wash was p o o l e d and an a l i q u o t t a k e n f o r d e t e r m i n i n g the r a d i o a c t i v i t y . D u p l i c a t e a l i q u o t s were added t o 10 ml o f PCS s c i n t i l l a t i o n f l u i d (Amersham S e a r l e ) and counted on an Is o c a p 300 l i q u i d s c i n t i l l a t i o n c o u n t e r ( N u c l e a r C h i c a g o ) . The e f f i c i e n c y o f t h e c o u n t i n g was 38-41% as d e t e r m i n e d by the c h a n n e l r a t i o method. T i s s u e s were b l o t t e d d r y , weighed and s t o r e d a t 20°C u n t i l RNA and DNA e x t r a c t i o n p r o c e d u r e s were c a r r i e d o u t . D e t e r m i n a t i o n o f S p e c i f i c A c t i v i t y o f RNA  i n U t e r i n e T i s s u e s Three u t e r i from each group were p o o l e d and t i s s u e homo-genates were p r e p a r e d as d e s c r i b e d i n Experiment C (Appendix F i g . The homogenates were f i r s t washed w i t h 7.0 ml o f c o l d 10% TCA. The r e s u l t i n g p r e c i p i t a t e was resuspended i n 7.0 ml o f c o l d 5% TCA and t h e two washes p o o l e d . D u p l i c a t e a l i q u o t s o f t h e TCA a c i d s o l u b l e f r a c t i o n were added t o 10 ml o f PCS and counted i n the l i q u i d s c i n t i l l a t i o n c o u n t e r . The e f f i c i e n c y o f c o u n t i n g under t h e s e c o n d i t i o n s was 33.5%. The r a d i o a c t i v i t y i n t h i s f r a c t i o n r e p r e s e n t s t h e e x t e n t o f p r e c u r s o r uptake w h i c h has not y e t been i n c o r p o r a t e d i n t o RNA. A n a y l s i s o f d a t a was done by S t u d e n t ' s t - t e s t . F o l l o w i n g t h e c o l l e c t i o n o f t h e a c i d s o l u b l e f r a c t i o n , t h e p r e c i p i t a t e was r e s u s p e n d e d i n 10 ml o f i c e c o l d 95% e t h a n o l t o remove t h e g r e a t e r p o r t i o n o f t h e f a t s o l u b l e f r a c t i o n . T h i s was a l l o w e d t o s t a n d f o r 10 m i n u t e s t h e n c e n t r i f u g e d a t 3500 RPM on a d e s k c e n t r i f u g e f o r f i v e m i n u t e s . The r e s u l t i n g s u p e r n a t a n t showed no r a d i o a c t i v i t y b e yond b a c k g r o u n d l e v e l s . F o l l o w i n g a l k a l i n e h y d r o l y s i s , t h e s o l u t i o n s were n e u t r a l i z e d w i t h HC1 and p r o t e i n and DNA were p r e c i p i t a t e d w i t h c o l d TCA. Under t h e s e c o n d i t i o n s RNA w i l l be p r e s e n t i n t h e f o r m o f m o n o n u c l e o t i d e s i n t h e s u p e r n a t a n t w h i c h w i l l be r e f e r r e d t o a s t h e h y d r o l y z e d RNA f r a c t i o n . RNA washes were p o o l e d and d u p l i c a t e 50 u l a l i q u o t s were t a k e n f o r l i q u i d s c i n t i l l a t i o n c o u n t i n g . The e f f i c i e n c y o f t r i t i u m c o u n t i n g u n d e r t h e s e c o n -d i t i o n s was 28-33%. The r e m a i n i n g p o r t i o n o f t h e f r a c t i o n was u s e d f o r q u a n t i t a t i v e d e t e r m i n a t i o n by s p e c t r o p h o t o m e t r y . S p e c i f i c a c t i v i t y o f t h e RNA f r a c t i o n s was e x p r e s s e d as DPM p e r ug RNA. DNA e x t r a c t e d i n h o t p e r c h l o r i c a c i d was a l s o q u a n t i t a t e d on t h e s p e c t r o p h o t o m e t e r and a l i q u o t s were t a k e n t o d e t e r m i n e i s o t o p e r a d i o a c t i v i t y . No r a d i o a c t i v i t y s i g n i f i c a n t l y above b a c k g r o u n d was r e c o r d e d i n t h i s f r a c t i o n . The e f f i c i e n c y o f t h e o v e r a l l e x t r a c t i o n p r o c e d u r e was c h e c k e d by a d d i n g t h e r a d i o a c t i v i t y i n i n d i v i d u a l f r a c t i o n s and e x p r e s s i n g i t as a p e r c e n t o f t o t a l r a d i o a c t i v i t y a d m i n i s t e r e d . R e s u l t s The e x t r a c t i o n p r o c e d u r e u s e d i n E x p e r i m e n t D a c c o u n t e d f o r 85-90% r e c o v e r y o f t h e i s o t o p i c a l l y l a b e l l e d u r i d i n e n u c l e o s i d e . I n g e n e r a l t h e mean r a d i o a c t i v i t y i n t h e h y d r o l y z e d RNA f r a c t i o n s was l e s s i n t h e e s t r o g e n t r e a t e d a n i m a l s t h a n i n t h e c o n t r o l s ( T a b l e 6 ) . The i n c o r p o r a t i o n o f t r i t i a t e d u r i d i n e i n t h e RNA f r a c t i o n o b t a i n e d f r o m t h e w h o l e u t e r u s was l o w e r i n t h e e s t r o g e n t r e a t e d r a t s ( T a b l e 7 ) . The o n l y e x c e p t i o n was i n t h e c a s e o f e s t r o n e t r e a t e d a n i m a l s w h e r e t h e r a d i o a c t i v i t y i n t h e h y d r o l y z e d RNA f r a c t i o n was s l i g h t l y h i g h e r t h a n i n t h e c o n t r o l . The u p t a k e o f t h e r a d i o a c t i v i t y by t h e u t e r i o f t e s t o s t e r o n e t r e a t e d r a t s r e s e m b l e d t h a t o f c o n t r o l r a t s . When t h e r a d i o a c t i v i t y i n t h e RNA f r a c t i o n was e x p r e s s e d p e r u n i t o f u t e r i n e wet w e i g h t t h e u t e r i o f e s t r a d i o l - 1 7 g t r e a t e d r a t s h a d s i g n i f i c a n t l y (P< 0.05) l o w e r i n c o r p o r a t i o n o f t h e l a b e l t h a n t h o s e o f c o n t r o l r a t s ( T a b l e 7 ) . I n t h e r a t s t r e a t e d w i t h DES, e s t r i o l and e s t r a d i o l - 1 7 a t h e i n c o r p o r a t i o n o f t h e l a b e l a p p e a r e d t o be l o w e r t h a n i n t h e c o n t r o l and t e s t o s -t e r o n e t r e a t e d o n e s . The s p e c i f i c a c t i v i t y o f RNA i n t h e u t e r i o f r a t s t r e a t e d w i t h e s t r a d i o l - 1 7 g was s i g n i f i c a n t l y ( P < 0 . 0 5 ) l o w e r t h a n i n t h e c o n t r o l ( T a b l e 7 ) . S i m i l a r t r e n d s i n t h e s p e c i f i c a c t i v i t y o f RNA w e r e o b s e r v e d i n DES, e s t r i o l a n d e s t r a d i o l - 1 7 a t r e a t e d r a t s . E s t r o n e t r e a t e d a n i m a l s a p p e a r e d t o show no d i f f e r e n c e f r o m t h e c o n t r o l s . I n p h y t o e s t r o g e n t r e a t e d g r o u p s ( T a b l e 7 ) , c o u m e s t r o l a n d _3 g e n i s t e i n t r e a t e d r a t s i n c o r p o r a t e d [5,6 H] u r i d i n e i n t o t h e h y d r o l y z e d RNA f r a c t i o n p e r u t e r u s a t l e v e l s h i g h e r t h a n t h o s e t r e a t e d w i t h e s t r a d i o l - 1 7 g , e s t r i o l a nd DES, and a t r a t e s s i m i l a r t o c o n t r o l a n d t e s t o s t e r o n e t r e a t e d g r o u p s . U n d e r t h e i n f l u e n c e o f t h e w e a k e r p h y t o e s t r o g e n s , f o r m o n o n e t i n and -3 b i o c h a n i n - A , t h e i n c o r p o r a t i o n r a t e o f [5,6 H] u r i d i n e i n t o t h e h y d r o l y z e d RNA f r a c t i o n was s i m i l a r t o t h a t o b s e r v e d w i t h t h e s t e r o i d e s t r o g e n s . When t h e r a d i o a c t i v i t y was e x p r e s s e d i n t e r m s o f u n i t u t e r i n e w e i g h t , c o u m e s t r o l , g e n i s t e i n , b i o c h a n i n and f o r m o n o n e t i n i n d u c e d a l e s s e r i n c o r p o r a t i o n o f u r i d i n e t h a n t e s t o s t e r o n e a n d c o n t r o l t r e a t e d g r o u p s . The s p e c i f i c a c t i v i t y o f t h e h y d r o l y z e d RNA f r a c t i o n i n p h y t o e s t r o g e n t r e a t e d r a t s was g e n e r a l l y l o w e r t h a n i n t h e c o n -t r o l a n d t e s t o s t e r o n e t r e a t e d a n i m a l s . P a r t i c u l a r l y t h e l o w s p e c i f i c a c t i v i t y o f RNA i n g e n i s t e i n t r e a t e d r a t s a s c o m p a r e d t o t h e c o n t r o l was s t a t i s t i c a l l y s i g n i f i c a n t (P<0..05). G e n i s t e i n a n d c o u m e s t r o l r e s e m b l e d e s t r i o l i n t h e i r a b i l i t y t o s t i m u l a t e i n c o r p o r a t i o n o f t h e i s o t o p i c p r e c u r s o r . B i o c h a n i n A and f o r m o n o n e t i n w e r e a l s o l o w e r t h a n t h e c o n t r o l i n t h e i r a b i l i t y t o p r o m o t e u p t a k e o f t h e l a b e l a n d r e s e m b l e d t h e t r e n d s o b s e r v e d w i t h DES and e s t r a d i o l - 1 7 $ i n t h i s r e s p e c t . D i s c u s s i o n E x p e r i m e n t D was u n d e r t a k e n t o compare t h e a b i l i t y o f d i f -f e r e n t e s t r o g e n s i n e n h a n c i n g t h e i n c o r p o r a t i o n o f an i s o t o p i c p r e c u r s o r by t h e r a t u t e r i n e t i s s u e . H a v i n g e s t a b l i s h e d i n E x p e r i m e n t A t h a t maximum w a t e r i m b i b i t i o n o c c u r r e d s i x h o u r s a f t e r e s t r o g e n a d m i n i s t r a t i o n t h i s t i m e i n t e r v a l was c h o s e n i n t h i s e x p e r i m e n t t o a l l o w f o r m a x i m a l w a t e r i m b i b i t i o n a n d t h e r e -f o r e g r e a t e r p e r m e a b i l i t y o f t h e c e l l membrane t o a n e x o g e n o u s s u p p l y o f t h e l a b e l l e d n u c l e o s i d e . Though one w o u l d h a v e e x p e c t e d an i n c r e a s e i n t h e s p e c i f i c a c t i v i t y o f RNA a f t e r t h e a d m i n i s t r a t i o n o f e s t r o g e n s i t was s u r p r i s i n g t o f i n d t h a t b o t h t h e s t e r o i d a n d p h y t o e s t r o g e n s d i d i n f a c t r e d u c e t h e s p e c i f i c a c t i v i t y a f t e r s i x h o u r s . B e f o r e b e i n g i n c o r p o r a t e d i n t o t h e u t e r i n e RNA, u r i d i n e must p a s s t h r o u g h an u r i d i n e n u c l e o t i d e p o o l , t h e s i z e o f w h i c h i s e x -panded by t h e a c t i o n o f e s t r o g e n s as s u g g e s t e d by M u e l l e r e t a l The r e d u c t i o n i n t h e s p e c i f i c a c t i v i t y o f RNA n o t i c e d s i x h o u r s a f t e r a d m i n i s t r a t i o n o f e s t r o g e n s may be a s c r i b e d t o t h e i n c r e a s e d s i z e o f t h e n u c l e o t i d e p o o l s u f f i c i e n t t o r e d u c e t h e s p e c i f i c a c t i v i t y . T h i s i s p a r t i c u l a r l y n o t i c e a b l e i n t h e c a s o f e s t r a d i o l - 1 7 3 and l e s s so i n DES and e s t r i o l . The a b s e n c e change i n t h e s p e c i f i c a c t i v i t y o f RNA i n e s t r o n e t r e a t e d r a t s o v e r t h e c o n t r o l s u g g e s t s t h a t i t i s a c o m p a r a t i v e l y weak e s t r o g e n and owes i t s b i o l o g i c a l a c t i v i t y s o l e l y t o i t s c o n -v e r s i o n t o e s t r a d i o l - 1 7 3 ( T e r e n i u s , 1 9 6 9 ) . The h i g h e r s p e c i f i a c t i v i t y o f RNA i n r a t s t r e a t e d w i t h e s t r a d i o l - 1 7 a t h a n e s t r a d i o l - 1 7 3 i n d i c a t e s t h e i m p o r t a n c e o f c o n f i g u r a t i o n on h o r -monal e f f e c t s . M i l l e r and Emmens (1967) o b s e r v e d an i n c r e a s e i n t h e i n -c o r p o r a t i o n o f l a b e l l e d u r i d i n e i n t h e mouse f o l l o w i n g e s t r a d i o l - 1 7 3 , e s t r i o l and e s t r o n e t r e a t m e n t . Munns and Katzman (1971) f o u n d t h a t e x p o s u r e t o e s t r o g e n i n v i v o e v e n f o r s h o r t p e r i o d s e n h a n c e d t h e u p t a k e o f t r i t i a t e d u r i d i n e by t h e u t e r u s i n v i t r o . I f t h e d u r a t i o n between t h e a d m i n i s t r a t i o n o f e s t r o g e n s and l a b e l l e d u r i d i n e was t o o l o n g a d e c r e a s e i n t h e u p t a k e o f r a d i o a c t i v i t y by t h e u t e r i n e t i s s u e s was n o t i c e d . To d e t e r m i n e w h e t h e r o r n o t t h i s was t r u e , s h o r t i n v i v o p u l s i n g s t u d i e s were a t t e m p t e d l a t e r on i n E x p e r i m e n t E. Comparable r e s u l t s on t h e e f f e c t o f p h y t o e s t r o g e n s on u r i d i n e u p t a k e by t h e r a t u t e r u s a r e n o t a v a i l a b l e i n t h e l i t e r a t u r e . As already indicated the s o l u b i l i t y of phyto-estrogens i n various solvents should be considered while assessing t h e i r estrogenic potency. In t h i s study d i f f i c u l t y was exper-ienced i n d i s s o l v i n g coumestrol. The low s p e c i f i c a c t i v i t y of RNA i n the u t e r i of rats treated with the phytoestrogens, biochanin A and formononetin i s of interest i n the l i g h t of t h e i r r e l a t i v e l y poor a b i l i t y to increase water imbibition as shown i n Experiment A. In par-t i c u l a r the s p e c i f i c a c t i v i t y of RNA i n rats treated with biochanin A and formononetin was even lower than i n those treated with estradiol-17 3 and DES, two potent estrogens. How-ever, r e l a t i v e l y heavier (60 g) rats were included i n the group treated with biochanin A and formononetin. On the basis of the increased uterine weight and RNA content i t i s possible that these animals may have entered the early phase of the estrous cycle and the c i r c u l a t i n g endogenous estrogens may have supple-mented the action of the phytoestrogens. Conclusions This experiment was i n i t i a t e d to study the r e l a t i v e e f f e c t s of estrogenic steroids and phytoestrogens i n terms of t h e i r a b i l i t y i n v i t r o to stimulate incorporation of l a b e l l e d uridine i n the RNA extracted from rat u t e r i six hours aft e r administering various estrogens i n vivo. Contrary to expectations data ob-tained i n t h i s experiment showed that estrogen treated u t e r i had a lower s p e c i f i c a c t i v i t y of RNA than control tissues. It i s possible that the period of six hours following estrogen administration was too long for the i n v i t r o e f f e c t s to be m a n i f e s t e d . The l o w s p e c i f i c a c t i v i t y may a l s o be due t o t h e i n c r e a s e d s i z e o f t h e u t e r i n e n u c l e o t i d e p o o l . D e c r e a s i n g t r e n d s i n t h e s p e c i f i c a c t i v i t y o f RNA w e r e n o t i c e d i n t h e o r d e r o f DES £ e s t r i o l < e s t r a d i o l - 1 7 a < e s t r o n e . E s t r a d i o l -' s (1.0 ;jg) a n d g e n i s t e i n (0.8 mg) p r o d u c e d a s i g n i f i c a n t (P< 0.05) and (P< 0.1) r e s p e c t i v e l y d e c r e a s i n g e f f e c t o n t h e -3 i n c o r p o r a t i o n o f [5,6 H] u r i d i n e i n t o t h e h y d r o l y z e d RNA f r a c t i o n . H o wever, t h e s p e c i f i c a c t i v i t y o f RNA i n c o u m e s t r o l t r e a t e d r a t s was n o t s i g n i f i c a n t l y l o w e r t h a n c o n t r o l a n i m a l s . B o t h g e n i s t e i n and c o u m e s t r o l r e s e m b l e d e s t r i o l i n t h e i r c a p a c i t y t o p r o m o t e t h e i n c o r p o r a t i o n o f l a b e l l e d u r i d i n e . I n r a t s t r e a t e d w i t h b i o c h a n i n A o r f o r m o n o n e t i n t h e s p e c i f i c a c t i v i t y o f RNA i n t h e u t e r u s was v e r y l o w w h i c h may be due t o t h e p h y s i o l o g i c a l s t a t e o f t h e a n i m a l r a t h e r t h a n t o t h e t r u e e s t r o g e n i c p o t e n t i a l o f t h e s e p h y t o e s t r o g e n s . Table 6 : The i n v i t r o incorporation of r a d i o a c t i v i t y i n d i f f e r e n t fractions of rat uterl~7 s i x hours following i n vivo estrogen administration (Expt. D) Di s t r i b u t i o n of r a d i o a c t i v i t y i n d i f f e r e n t fractions (DPM) Treatment Groups of animals (n) Medium and tissue wash Cold TCA soluble f r a c t i o n Hydrolyzed RNA fr a c t i o n Recovery of administered l a b e l (%) Control 4 2 8 9 3 8 8 6 + 1 0 7 , 9 9 0 20 4 5 3 1 ± 2 6 , 2 0 5 8 9 , 1 4 6 ± 1 4 , 7 8 0 8 8 . 1 ± 4 . 1 Estrone 1 2 8 5 9 4 3 9 2 8 3 6 7 0 9 3 , 7 7 1 8 9 . 5 E s t r i o l 1 2 9 1 7 3 1 7 2 4 5 7 1 2 5 5 , 1 9 9 8 8 . 9 DES 1 2 9 4 6 7 3 0 2 8 4 0 2 6 5 8 , 2 5 9 9 0 . 9 E s t r a d i o l - 1 7 c t 1 2 8 2 9 4 3 8 2 8.0 0 9 8 7 8 , 1 3 8 8 8 . 1 Estradiol-17g 4 2 8 1 0 3 2 1 ± 1 0 0 , 0 1 0 2 8 5 3 1 6 ± 1 8 5 2 0 5 4 , 2 9 9 ± 1 0 , 2 7 0 8 7 . 7 ± 3 . 5 Testosterone 1 2 8 7 2 2 8 1 2 2 0 1 3 3 9 0 , 4 7 7 8 8 . 0 Genistein 3 2 8 4 8 5 0 5 ± 2 1 5 , 4 4 1 1 6 4 5 7 8 ± 1 2 , 1 5 8 7 8 , 5 2 7 + 1 0 , 2 9 0 8 5 . 4 ± 6 . 5 Coumestrol 2 2 8 1 1 2 3 5 ±. 6 4 , 2 4 9 1 8 3 6 3 9 ± 3 5 , 6 6 3 8 7 , 8 7 1 ± 2 , 5 4 3 8 5 . 2 Biochanin A 1 2 9 1 9 0 0 0 1 4 4 2 6 8 6 7 , 4 1 9 8 6 . 5 Formononetin 1 2 9 2 4 5 1 9 1 1697 . 3 4 3 , 3 5 1 . 8 5 . 3 T a b l e 7 : RNA c o n t e n t and s p e c i f i c a c t i v i t y o f RNA e x t r a c t e d from r a t u t e r i s i x hours f o l l o w i n g e s t r o g e n a d m i n i s t r a t i o n (Expt. D) RNA c o n t e n t R a d i o a c t i v i t y i n h y d r o l y z e d RNA Treatment Mean Body Weight (g) ug RNA ug RNA DPM DPM DPM u t e r u s mg u t e r u s u t e r u s mg u t e r i n e w e i g h t ug RNA C o n t r o l 42.0 ± 8.3 134.0 ±7.1 4.3 ± 0.2 29715 ± 4928 933.9 ± 1.5 ( a ) 6.9 ± 1.2 ( a ) E s t r o n e 41.0 134.8 3.9 31257 930. 3 6.9 E s t r i o l 39. 4 133 3.9 18399 541.2 4.0 DES 46. 8 143 3.8 19419 516.5 3.6 E s t r a d i o l - 1 7 g 38.3 ± 5.1 143.2 ± 3.2 4.1 ± 0.4 17659 ± 1583 501.7 ± 4419 ( b ) 3.5 ± 0.3 ( C ) E s t r a d i o l - 1 7 a 49.1 161.0 5.2 26659 851.2 5.2 T e s t o s t e r o n e 35.6 109.0 3.5 30159 985.4 9.0 G e n i s t e i n 50.3 ± 3.7 156.5 ± 4.7 4.2 ± 0.1 26916 ± 1400 725.7 ± 106 ( c ) 4.6 ± 0.7 ( b ) C o u m e s t r o l 50.3 ± 4 . 7 141.5 ± 0.9 3.5 ± 0.3 29306 ± 1498 741.9 ±. 3.7.9 ( C ) 5.2 ± 0.3 ( a ) R i n r h a n i n A 59. 3 196. 8 4.4 . . 22473 510.7 2.5 59 7 176. 5 3.9 144 50 580.3 3.2 ((a,b,c,d) Means w i t h d i f f e r e n t s u b s c r i p t s a r e s i g n i f i c a n t l y d i f f e r e n t (P < 0.05). 70. Experiment E : E f f e c t of Estrogens and Phytoestrogens on the I n c o r p o r a t i o n o f T r i t i a t e d U r i d i n e Into RNA - Short i n v i v o P u l s i n g I n t r o d u c t i o n The very low c o n c e n t r a t i o n of phytoestrogens i n p l a n t m a t e r i a l and t h e i r weak e s t r o g e n i c potency have made i t d i f f i c u l t t o study t h e i r b i o l o g i c a l e f f e c t s . The problem i s f u r t h e r complicated by the d i f f i c u l t i e s a s s o c i a t e d w i t h v a r i o u s e x t r a c t i o n procedures which do not permit q u a n t i t a t i v e r e c o v e r y of the p l a n t estrogens. Consequently the development of s u i t a b l e b i o a s s a y s which c o u l d be employed t o t e s t t h e i r e s t r o g e n i c potency and to pr o v i d e knowledge on t h e i r mode of a c t i o n has been hampered. Recently the i n t r o d u c t i o n of competi-t i v e p r o t e i n b i n d i n g techniques, which are s e n s i t i v e enough t o measure the b i o l o g i c a l a c t i v i t y of ve r y s m a l l q u a n t i t i e s o f phytoestrogens has p a r t l y overcome the l i m i t a t i o n s of b i o a s s a y s . The s e n s i t i v i t y of bio a s s a y procedures has a l s o been g r e a t l y i n c r e a s e d by the development o f techniques i n which the i n c o r -p o r a t i o n of a l a b e l l e d p r e c u r s o r by u t e r i n e c e l l u l a r o r g a n e l l e s i n v i t r o i s determined q u a n t i t a t i v e l y a f t e r the a d m i n i s t r a t i o n of e s t r o g e n i c compounds i n v i v o . The r e s u l t s o f Experiment D have i n d i c a t e d t h a t the i n v i t r o a d m i n i s t r a t i o n of t r i t i a t e d u r i d i n e to the u t e r i n e t i s s u e s i x hours a f t e r i n j e c t i o n o f estrogens i n v i v o has not r e s u l t e d i n l a r g e i n c o r p o r a t i o n of the l a b e l as expected. I t i s p o s s i b l e t h a t the s i x hour d u r a t i o n a f t e r i n v i v o a d m i n i s t r a t i o n of estrogens was too long to observe the e a r l y e f f e c t s produced i n the t i s s u e s . M i l l e r (1964) reported that reduction of tetrazolium s a l t s occurred as early as 2 8 minutes a f t e r i n j e c t i o n of e s t r a d i o l -173 and suggested that t h i s short duration i s c r i t i c a l i n observing the early e f f e c t s of estrogens. Shutt (1967) employed a sim i l a r technique as an index of the b i o l o g i c a l e f f e c t of genistein. Subsequently i s o t o p i c precursors have been widely used with d i f f e r e n t degrees of success i n studying the early action of estrogen. Munns and Katzman (1971) observed that the extent of incorporation of l a b e l l e d precursors was high when the l e v e l was administered 30 minutes a f t e r i n j e c t i o n of estradiol-173. In the l i g h t of these experiments t h i s experi-ment was undertaken to study the very early e f f e c t s occurring i n the uterine c e l l a f t e r a b r i e f exposure to d i f f e r e n t estro-gens using the basic technique of Munns and Katzman (1971). Phytoestrogens were f i r s t extracted from plant materials. The early e f f e c t s which these extracts produced on the uterine tissue were then compared with those caused by estradiol-173. Estrogenic a c t i v i t y of ce r t a i n plant materials has been attributed to the presence of nonsteroidal phytoestrogens capable of competing with estradiol-173 for s p e c i f i c binding s i t e s located i n the uterine cytosol. This study presents the results of q u a l i t a t i v e and quanitative determinations of various phytoestrogens and th e i r a b i l i t y for the uterine binding protein. Materials and Methods Materials F i r s t cuttings of orchard grass hay (Dactylus glomerata) and a l f a l f a hay (Medicago sativa) were obtained from Agassiz Research Station. Soyabean (Glycine max) meal was obtained from Buckerfields. [2,4,6,7 (n) H ] estradiol-173 ( s p e c i f i c a c t i v i t y , 96 Ci/mmole) was obtained from Amersham/Searle. Dextran T-40 was obtained from Pharmacia Chemicals and activated charcoal f rom S igma. Methods Extraction and I d e n t i f i c a t i o n of Phytoestrogens Phytoestrogens were extracted from 2.0 g samples of oven dried ground orchard grass hay, a l f a l f a hay and soyabean meal by treating them successively with absolute alcohol and peroxide-free ether (Francis and M i l l i n g t o n 1965). The f i n a l extract was concentrated to dryness under a stream of nitrogen and absolute alcohol was added. Samples were then stored at -2 0°C u n t i l ready for use. The e f f i c i e n c y of extraction was checked by adding known concentrations of genistein to those samples and by c a l c u l a t i n g the per cent recovery. Qualitative determination of phytoestrogens was done on two d i r e c t i o n a l t h i n layer chromatography using G-25 s i l i c a gel plates (D.C. Fertigplatten-Macheray-Nagel Co.). The plates were activated at 100°C f o r one hour p r i o r to use and 10-20 u l aliquots of plant extracts were placed on each plate. F i f t y m i c r o l i t e r aliquots of formononetin, genistein, coumestrol and biochanin-A dissolved i n alcohol were used as standards. The developing solvent systems used were chloroform-methanol (91:9,V/V) i n the f i r s t d i r e c t i o n and ammonia saturated chloro-form-methanol (91:9T,V/V) i n the second. The plates were allowed to dry at room temperature for approximately 15 minutes before running them i n the second d i r e c t i o n . Developed chroma-tograms were observed under u l t r a v i o l e t l i g h t for the detection of formononetin, daidzein and coumestrol. I d e n t i f i c a t i o n of these compounds was made by comparing t h e i r Rf values with standards and by t h e i r v i s u a l c h a r a c t e r i s t i c s as seen under u l t r a v i o l e t l i g h t . Areas representing the phytoestrogens were c i r c l e d with a pencil and the plates transferred to a fume hood where they were sprayed with a solution of cold 1.0N s u l p h a n i l l i c acid containing 10%Na2CO3 and 4.5% NaN02 for the detection of the nonfluorescent compounds, genistein, biochanin-A and equol. The former two compounds were i d e n t i f i e d by comparing t h e i r Rf values with known standards. Quantitative Determination of Phytoestrogens The competitive protein binding assay of Korenman(1968) was employed to determine the concentration of phytoestrogens quantitatively. Uterine cytosol was obtained from a s ix day pregnant rabbit and homogenized at 4°C i n three volumes of T r i s buffer (pH,4.0 W/V) using a Waring blender (Sorvall). The homo-genate was centrifuged at 7000 g for 15 minutes at 0°C. The super-natant f r a c t i o n was removed and recentrifuged (Beckman, u l t r a -centrifuge L5-65) at 100,000 g for 90 minutes at 0°C. Immedi-ately following u l t r a c e n t r i f u g a t i o n the second supernatant f r a c t i o n containing the cytosol proteins was co l l e c t e d by Pasteur pipette and stored i n l i q u i d nitrogen u n t i l ready for use. Standard curves for competitive protein binding assays were obtained using d i f f e r e n t concentrations of p u r i f i e d genistein. Aliquots of increasing concentration of p u r i f i e d genistein were added to test tubes and dried under a stream of nitrogen. To each sample were added successively 100 LII of T r i s b u f f e r , pH 8.0, 50 u l o f ^ H - e s t r a d i o l - l V ^ and 100 u l o f r a b b i t u t e r i n e c y t o s o l . The tubes were mixed a f t e r t h e a d d i t i o n o f t h e u t e r i n e c y t o s o l , samples were a l l o w e d t o s t a n d a t room temperature f o r 30 m i n u t e s a t t h e end of w h i c h 1.0 ml o f d e x t r a n c o a t e d c h a r c o a l was added t o each tube. The tubes were t h e n mixed, k e p t a t 4°C f o r 15 minutes and c e n t r i f u g e d on a desk c e n t r i g u g e . The s u p e r n a t a n t was c o l l e c t e d w i t h a P a s t e u r p i p e t t e and an a l i q u o t t a k e n f o r t h e d e t e r m i n a t i o n o f r a d i o a c t i v i t y . Based on t h e c o m p e t i t i o n f o r b i n d i n g s i t e s between e s t r a d i o l - 1 7 3 and t h e added g e n i s t e i n t h e r a d i o a c t i v i t y i n t he s u p e r n a t a n t w i l l be d i r e c t l y p r o p o r t i o n a l t o t h e added g e n i s t e i n . P l a n t e x t r a c t s were d r i e d i n a stream o f n i t r o g e n and t h e c o n c e n t r a t i o n o f t o t a l p h y t o e s t r o g e n s was measured by t h e compe-t i t i v e p r o t e i n b i n d i n g assay and t h e v a l u e s e x p r e s s e d i n terms o f e s t r o g e n i c a f f i n i t y o f g e n i s t e i n . R e s u l t s Q u a l i t a t i v e a n a l y s i s o f o r c h a r d g r a s s hay e x t r a c t s on two d i m e n s i o n a l t h i n l a y e r chromatography d i s c l o s e d no i d e n t i f i a b l e s p o t s c o r r e s p o n d i n g t o p h y t o e s t r o g e n s . The Rf v a l u e s o f p u r i -f i e d p h y t o e s t r o g e n s s t a n d a r d s a r e g i v e n i n T a b l e 8. I n t h e a l f a l f a hay e x t r a c t s s p o t s c o r r e s p o n d i n g t o c o u m e s t r o l , formo-n o n e t i n and g e n i s t e i n were o b s e r v e d (Table 8 ) . A l t h o u g h no s t a n d a r d s o f e q u o l were used a weak s p o t c o r r e s p o n d i n g t o t h e Rf v a l u e o f e q u o l was n o t i c e d i n t h e a l f a l f a e x t r a c t . Both g e n i s t e i n and d a i d z e i n were p r e s e n t i n t h e soyabean e x t r a c t ( F i g . 1 0 ) . The s t a n d a r d c u r v e f o r the c o m p e t i t i v e p r o t e i n b i n d i n g assay of p u r i f i e d genistein i s shown i n F i g . 11. When known quantities of genistein was added to orchard grass hay, the extraction procedure gave a recovery of 59% as determined by the competitive protein binding assay. The concentration of phytoestrogens i n the orchard grass hay extract was very low and equivalent to 1.5-2.0 ug genistein units per 2.0 g of the sample. A l f a l f a hay and soyabean meal extracts contained much larger concentrations of phytoestrogens equivalent to 70 and 126 rig respectively of genistein units. Discussion The presence of estrogenic a c t i v i t y i n forages has been reviewed by many researchers working i n t h i s f i e l d (Bickoff et a l . 1957; Kohler and Bickoff, 1961). Beck et a l . (1964) reported the i s o l a t i o n and subsequent i d e n t i f i c a t i o n of various plant estro-gens by thin layer chromatography. The plant constituents currently believed to contribute to estrogenic action are the isoflavongs, genistein, biochanin-A, formononetin^daidzein and equol as well as the coumarin derivative, coumestrol. The i s o l a t i o n and i d e n t i f i c a t i o n of coumestrol, formononetin and genistein i n the a l f a l f a extract i n t h i s study support the findings of Kohler and Bickoff (1961) who reported that coume-s t r o l accounts for the majority of the b i o l o g i c a l a c t i v i t y i n a l f a l f a while the isoflavones are responsible for the remaining a c t i v i t y . The i d e n t i f i c a t i o n of genistein i n soyabean meal extract also agrees with Cheng et a l . (1953(b) who reported high leve l s of genistein i n soyabean meal. Phytoestrogens have since been reported to bind on s p e c i f i c uterine proteins (Shutt and Cox, 1972) and act i n various areas of c e l l metabolism (Noteboom and Gorski, 1963Xa)• It was also evident that s u f f i c i e n t estrogenic a c t i v i t y was present i n various plant extracts strong enough to displace estradiol-173 from uterine binding proteins. Though no e v i -dence for the presence of phytoestrogens i n orchard grass hay could be detected by the TLC method, i t was possible to detect very small quantities of phytoestrogen a c t i v i t y when the sensi-t i v e competitive protein binding assay method was used. I t i s l o g i c a l to expect that following the i n i t i a l binding of the phytoestrogen to uterine cytosol proteins a d d i t i o n a l estrogen mediated responses would occur sequentially l a t e r . To test t h i s hypothesis i t was decided to undertake further studies involving the i n v i t r o incorporation of lab e l nucleoside by the uterine tissue a f t e r a b r i e f i n vivo exposure to estrogens. Conclusions In Experiment E the quantitative and q u a l i t a t i v e determina-t i o n of the phytoestrogen content of various forages was made. Qualitative studies of the a l f a l f a extract revealed the presence of coumestrol, genistein and formononetin. From v i s u a l observations of the chromatograms from a l f a l f a extracts, the isoflavones appeared to be i n a much smaller quantity than coumestrol. In the soyabean extract, genistein and daidzein were i s o l a t e d and although no standards for daidzein were availa b l e , i d e n t i f i c a t i o n could be made from i t s c h a r a c t e r i s t i c fluorescence under u l t r a v i o l e t l i g h t . According to the v i s u a l observations made from the soyabean meal chromatograms, genistein appeared to be i n greater concentration than daidzein. Qu a l i t a t i v e examination of the orchard grass hay extract did not disclose any spot corresponding to phytoestrogens. Quantitatively, estrogenic a c t i v i t y was observed i n the genistein spiked hay and a l f a l f a hay and soyabean meal extracts. A very small amount of estrogenic a c t i v i t y (1.5-2.0 ug) per 2.0g of sample was recorded i n the orchard grass hay extract. A l f a l f a hay and soyabean meal extracts possessed a substantial amount of a c t i v i t y of 70 ug and 126 ug respectively. 78. F O R M O N O N E T I N o Q o E Q U O L G E N I S T E I N C O U M E S T R O L A L F A L F A H A Y ( M E D I C A G O S A T I V A ) G E N I S T E I N D A I D Z E I N o C O U M E S T R O L S O Y B E A N M E A L ( G L Y C I N E M A X ) F i g . 10. Q u a l i t a t i v e e x a m i n a t i o n o f p h y t o e s t r o g e n c o n t e n t by t h i n l a y e r chromatography A = Ch l o r o f o r m - m e t h a n o l (91:9 v/v) B = Ammonia S a t u r a t e d C h l o r o f o r m - m e t h a n o l (91 T a b l e 8 : Rf v a l u e s o f s t a n d a r d p h y t o e s t r o g e n s and t h o s e o b t a i n e d from p l a n t e x t r a c t s as measured by T h i n Layer Chromatography (Expt. E) Sample Used Co u m e s t r o l G e n i s t e i n Formononetin D a i d z e i n Equol B i o c h a n i n A S t a n d a r d 0.41 (0.85) 0.58 (0.91) 0.74 (0.74) - 0.7 6 (0.79) A l f a l f a Hay 0.47 (0.87) 0.56 (0.94) 0.70 (0.82) 0.62 (0.53) -Soybean Meal 0.51 (0.83) 0.63 (0.89) - 0.54 (0.14) - -Orchard Grass Hay - - - - - -- F i g u r e s o u t s i d e b r a c k e t s c o r r e s p o n d t o Rf v a l u e s i n f i r s t s o l v e n t - F i g u r e s w i t h i n b r a c k e t s c o r r e s p o n d t o Rf v a l u e s i n second s o l v e n t 80 . Experiment F : Early E f f e c t s of Estrogens and Plant Extracts on the Incorporation of T r i t i a t e d Uridine Into RNA by Uterine Tissue. Introduction In the l i g h t of.quantitative and q u a l i t a t i v e determination of phytoestrogen a c t i v i t y i n plant extracts made i n Experiment E further e f f o r t s were made to obtain information on the mode of action of estrogenic compounds. Based on the competition for binding s i t e s between estradiol-17 3 and the various phyto-estrogens i t was f e l t that additional experiments designed s p e c i f i c a l l y to study the uptake of a radioactive nucleoside into the RNA f r a c t i o n by uterine tissues would be informative i n defining the mode of action of estrogenic compounds i n various plant extracts. In Experiment D the six hour duration between the i n vivo administration of estrogen and i n v i t r o uptake of lab e l l e d nucleoside by the uterine tissue resulted i n a lower s p e c i f i c a c t i v i t y of RNA. Therefore i t was decided to shorten the duration following estrogen administration to 30 minutes before undertaking i n v i t r o uptake studies. Materials and Methods Animals Immature female rats-(33-43 g) were used i n t h i s study. They were housed and maintained as described i n Experiment A. Materials Plant extracts of orchard grass hay, a l f a l f a hay and soya-bean meal were prepared as described i n Experiment E. Addi-81. t i o n a l materials required for i n v i t r o studies were the same as described i n Experiment D. Methods Administration of Estrogens i n vivo Immature female rats were given single i n j e c t i o n s of estradiol-17 3, genistein, a l f a l f a hay extract and soyabean meal extract i n doses of 1.0 ug, 6 0.0 ug, 14.5 ug and 40.0 }ig respec-t i v e l y . Estradiol-173, genistein and soyabean meal extract administered i n t r a p e r i t o n e a l l y i n 0.2 ml of aqueous 1% ethanol. A l f a l f a hay extract was administered i n t r a p e r i t o n e a l l y i n 0.2 ml of 50% propylene g l y c o l . Control animals received similar volumes of 1% ethanol i n s a l i n e . _3 Incorporation of [5,6 H] Uridine By Uterus In V i t r o Animals were s a c r i f i c e d 30 minutes aft e r the administration of estrogens by placing them i n a sealed j a r containing CG^. The whole u t e r i were removed within one minute, freed of f a t and connective tissue and placed i n i c e cold i s o t o n i c s a l i n e . Two to three groups per treatment containing three whole u t e r i per group were gently blotted and transferred to prewarmed (37°C) 5 ml incubation v i a l s containing 2.5 ml of TCM 199, 10.0 uCi of 3 H-uridine and 1.0 mM glutamine. Uterine tissues were incubated for 1.0 hour at 37°C i n a water bath at a shaking speed of one stroke per second under an atmosphere of 95% 02~5% CG^. After incubation the medium was removed by Pasteur pipette and 0.5 ml of 0.6 N HCIO^ was added to terminate the reaction. Whole uterine tissues were rinsed with ice cold d i s t i l l e d water 82. t o remove e x t r a c e l l u l a r r a d i o a c t i v i t y . T h i s f r a c t i o n r e p r e s e n t i n g t i s s u e wash was p o o l e d and p r o c e s s e d as d e s c r i b e d i n E x p e r i m e n t D. T i s s u e s were b l o t t e d , weighed and s t o r e d a t -20°C u n t i l RNA and DNA e x t r a c t i o n s were c a r r i e d o u t . The r a d i o a c t i v i t y i n the c e l l u l a r f r a c t i o n was d e t e r m i n e d as d e s c r i b e d under E x p e r i m e n t D. Recovery r a t e s of the i s o t o p e were a l s o made t o det e r m i n e t h e e f f i c i e n c y o f t h e e x t r a c t i o n p r o c e d u r e . A l l r e s u l t s were a n a l y s e d s t a t i s t i c a l l y by t h e s t u d e n t s ' t - t e s t . R e s u l t s The p e r c e n t o f t o t a l r a d i o a c t i v i t y w h i c h was r e c o v e r e d i n the d i f f e r e n t f r a c t i o n s amounted t o 79-91% (Table 9 ) . There was a s l i g h t l y g r e a t e r i n c o r p o r a t i o n o f t r i t i a t e d u r i d i n e i n the h y d r o l y z e d RNA f r a c t i o n i n u t e r i o f e s t r a d i o l - 1 7 3 and p h y t o -e s t r o g e n t r e a t e d r a t s t h a n i n c o n t r o l a n i m a l s . T h i s a p p a r e n t i n c r e a s e was however not s i g n i f i c a n t (P < 0.05) when a n a l y z e d by th e s t u d e n t s ' t - t e s t . The r a d i o a c t i v i t y i n the h y d r o l y z e d RNA f r a c t i o n when e x p r e s s e d p e r u n i t o f u t e r i n e wet w e i g h t was s i g n i -f i c a n t l y (P < 0.05) h i g h e r i n t h e u t e r i o f e s t r a d i o l - 1 7 3 ahd pure g e n i s t e i n t r e a t e d r a t s t h a n c o n t r o l s (Table 1 0 ) . A l f a l f a hay and soyabean meal e x t r a c t s a l s o i n c r e a s e d t h e i n c o r p o r a t i o n o f the l a b e l l e d p r e c u r s o r i n t o t h e h y d r o l y z e d RNA f r a c t i o n p e r u n i t o f u t e r i n e wet w e i g h t , however t h i s i n c r e a s e was n o t s i g n i f i c a n t . The s p e c i f i c a c t i v i t y o f RNA i n t h e e s t r a d i o l - 1 7 3 and p h y t o -e s t r o g e n t r e a t e d r a t u t e r i was s i g n i f i c a n t l y (P < 0.05) g r e a t e r t h a n t h e c o n t r o l s (Table 1 0 ) . The a l f a l f a and soyabean e x t r a c t s a l s o s i g n i f i c a n t l y (P < 0.05) i n c r e a s e d t h e s p e c i f i c a c t i v i t y o f RNA i n t h e immature female r a t u t e r i when compared t o c o n t r o l 8 3 . animals (Table 10) . Discussion Having established i n Experiment E that d i f f e r e n t phyto-estrogens were present i n a l f a l f a hay and soyabean, and that they were capable of binding onto s p e c i f i c uterine proteins, Ex-periment F was i n i t i a t e d to determine whether or not they were potent enough to enhance the early incorporation of l a b e l l e d uridine into RNA of uterine tissues. To determine t h i s short i n vivo pulsing of the estrogenic compound i n question was em-ployed. The increased uptake of the la b e l l e d uridine observed i n this experiment i s i n agreement with that reported by Munns and Katzman (19 71). I t i s p a r t i c u l a r l y noteworthy that the estra-diol-173 or phytoestrogen treated r a t u t e r i had a higher s p e c i f i c a c t i v i t y of RNA unlike i n Experiment D where they had a lower s p e c i f i c a c t i v i t y than the controls (Tables 6 and 9). Increases i n [5, 6 H] uridine incorporation into RNA during the early phase of estrogen and phytoestrogen action may be attributed to the increased permeability of the uterine c e l l membrane. Further, during the r e l a t i v e l y long duration a f t e r i n vivo estra-diol-173 administration i n Experiment D, there may be an increased i n f l u x of endogenous supply of nucleosides which would cause a reduction i n the uterine s p e c i f i c a c t i v i t y as suggested by Munns and Katzman (1971). Hence the duration time of 30 minutes chosen for i n vivo pulsing with estradiol-17 3 and various phytoestrogens i n Experiment F appeared to r e s u l t i n higher s p e c i f i c a c t i v i t i e s of RNA. 84. The doses of v a r i o u s e s t r o g e n i c compounds a d m i n i s t e r e d were chosen on the b a s i s o f t h e e s t r o g e n i c a c t i v i t y d e t e r m i n e d by c o m p e t i t i v e p r o t e i n b i n d i n g a s s a y s . D e s p i t e t h e f a c t t h a t t h e s e compounds were a d m i n i s t e r e d i n d i f f e r e n t q u a n t i t i e s t h i s d i d not appear t o i n f l u e n c e t h e s p e c i f i c a c t i v i t y o f t h e i n c o r -p o r a t e d r a d i o a c t i v e p r e c u r s o r . When t h e p h y t o e s t r o g e n a c t i v i t y o f t h e e x t r a c t s was e x p r e s s e d i n terms o f g e n i s t e i n u n i t s , a l f a l f a and soyabean e x t r a c t s (14.5 ug and 40.0 ug r e s p e c t i v e l y ) were s i m i l a r t o e s t r a d i o l - 1 7 6 (1.0 ug) and g e n i s t e i n (60.0 ug) i n t h e i r a b i l i t y t o enhance i n c o r p o r a t i o n o f t r i t i a t e d u r i d i n e . E s t r a d i o l - 1 7 3 has been r e p o r t e d t o i n c r e a s e t h e a c t i v i t y o f DNA dependent RNA polymerase enzyme (Maul and H a m i l t o n 196 7 ) . A l t h o u g h no e x p e r i m e n t s were done t o de t e r m i n e whether o r n o t v a r i o u s p h y t o e s t r o g e n s a r e c a p a b l e o f i n c r e a s i n g polymerase a c t i v i t y , i t seems r e a s o n a b l e t h a t t h e g r e a t e r u r i d i n e i n c o r p o r a -t i o n o b s e r v e d i n t h e h y d r o l y z e d RNA f r a c t i o n may be due t o g r e a t e r a c t i v i t y o f t h i s enzyme. More e x p e r i m e n t a l d a t a i s r e q u i r e d b e f o r e c o n c l u s i o n s can be made r e g a r d i n g p h y t o e s t r o g e n i n d u c e d polymerase a c t i v i t y . Based on t h e e f f e c t s on e a r l y e v e n t s o c c u r r i n g i n c e l l u l a r m e t a b o l i s m due t o a s i n g l e i n j e c t i o n o f p h y t o e s t r o g e n s o b t a i n e d from a s m a l l q u a n t i t y o f p l a n t m a t e r i a l i t i s i n t e r e s t i n g t o s p e c u l a t e t h e magnitude o f e f f e c t s w h i c h c o u l d be produced i n g r a z i n g a n i m a l s i n j e s t i n g l a r g e q u a n t i t i e s o f p h y t o e s t r o g e n s . C o n c l u s i o n s I n E xperiment F t h e o b j e c t i v e was t o examine t h e e s t r o g e n i c p o t e n t i a l of d i f f e r e n t f o r a g e s by e s t a b l i s h i n g a b i o a s s a y u s e f u l 85. i n s t u d y i n g an e s t r o g e n i n d u c e d r e s p o n s e , namely t h e i n c o r p o r a -t i o n o f a s p e c i f i c i s o t o p i c p r e c u r s o r . Exposure o f u t e r i n e t i s s u e t o e s t r o g e n s f o r 30 minutes i n v i v o was s u f f i c i e n t t o s t u d y t h e i n i t i a l e f f e c t s o f e s t r a d i o l - 1 7 3 and p h y t o e s t r o g e n s . S i g n i f i c a n t l y g r e a t e r (P < 0.05) d i f f e r e n c e s were o b t a i n e d between e s t r o g e n t r e a t e d r a t s and c o n t r o l a n i m a l s i n r e g a r d t o i n c o r p o r a t i o n o f t r i t i a t e d u r i d i n e i n t o t h e h y d r o -l y z e d RNA f r a c t i o n o f the u t e r i n e c e l l . I t i s p r o b a b l e t h a t a s h o r t e r p u l s i n g p e r i o d and exposure o f u t e r i n e t i s s u e s t o l a r g e r q u a n t i t i e s (10 u C i ) o f i s o t o p i c p r e c u r s o r t h a n were used i n Experiment D e n a b l e d t h e i n i t i a l e f f e c t s o f e s t r a d i o l - 1 7 3 and p h y t o e s t r o g e n s t o be o b s e r v e d . More e x p e r i m e n t a l d a t a r e g a r d i n g t h e s p e c i f i c mode o f a c t i o n o f p h y t o e s t r o g e n s on u t e r i n e c e l l m e t a b o l i s m ( i . e . i n c r e a s e d polymerase a c t i v i t y ) a r e r e q u i r e d b e f o r e c o n c l u s i o n s r e g a r d i n g p h y t o e s t r o g e n mediated RNA s y n t h e s i s can be made. 11. C o m p e t i t i v e B i n d i n g A s s a y o f P h y t o e s t r o g e n s (Expt. E) I n c r e a s i n g c o n c e n t r a t i o n s o f g e n i s t e i n were added t o 100 u l t r i s b u f f e r ; 50 u l 3H E s t r a d i o l - 1 7 B and 100 r i l p r egnant r a b b i t u t e r i n e c y t o s o l t o d e t e r m i n e t h e G e n i s t e i n S t a n d a r d Curve. S E M I - L O G A R I T H M I C 4 6 6 0 1 0 4 C Y C L E S X 7 0 D I V I S I O N S H»0£ IK U.S . A . K E U F F E L a E S S E R C O . 8 6 a . Ul CD N CO 13 O GENISTEIN ADDED TO ASSAY TUBE (NG) Table 9 : The i n v i t r o incorporation of r a d i o a c t i v i t y i n d i f f e r e n t fractions of rat u t e r i , 30 minutes following i n vivo estrogen administration (Expt. F) D i s t r i b u t i o n of r a d i o a c t i v i t y i n d i f f e r e n t fractions (DPM) Treatment Groups of Animals Medium and tissue wash Cold TCA soluble f r a c t i o n Hydrolyzed RNA fr a c t i o n Recovery of administered lab e l % Control 2 22692616 ± 66,578 2248490 ± 179538 929984 ± 82521 91 ± 2% Estradiol-17 3 2 22158070 ± 321857 2507680 ± 194458 1186117 ± 22805 91 * 1% Genistein 3 18844267 ± 232915 2672025 ± 508445 1033235 ± 17193 79 i 1% A l f a l f a hay extract 2 19836720 ± 105723 2351821 ± 51510 1178381 ± 32684 82 ~ 1% Soyabean meal extract 2 20929731 ± 148367 2484358 ± 313876 1128354 ± 31858 86 i 3% Table 10 : Incorporation of [5,6 H] uridine into the hydrolyzed RNA f r a c t i o n of immature female rat u t e r i 30 minutes following estrogen administration Radioactivity i n hydrolyzed RNA Treatment Groups of Animals (n) DPM DPM mg uterine weight DPM uterus ug RNA Control 2 309,994 ± 27,507 8,732 ± 774 ( a ) 69.3 ± 6.2 ( a ) Estradiol-173 2 395:,372 ± 10,750 11,526 ± 221 ( b ) 92.5 ± 1.4 ( b ) -Genistein 3 329,411 ± 17,477 11,124 ± 802 ( b ) 89.2 ± 6.2 ( b ) A l f a l f a extract 2 392,793 ± 10,894 11,385 ± 315 ( a ) 91.1 ± 2.5 ( b ) Soyabean extract 2 376,118 ± 10,619 11,062 ± 220 ( a ) 87.8 ± 2.5 ( b ) (a,b) Means with d i f f e r e n t subscripts are s i g n i f i c a n t l y d i f f e r e n t (P < 0.05) 89 . GENERAL CONCLU SIONS Estradiol-17 3, the major c i r c u l a t i n g estrogen i n majority of mammalian species was chosen as the standard with which other estrogens were compared. I n i t i a l experiments involving dose levels and sequential time i n t e r v a l s following estradiol-173 administration were designed to esta b l i s h baseline values regarding target organ metabolism. . Six hours following the intraperitoneal administration of a single i n j e c t i o n of estradiol-173 (1.0 ug) maximal tissue edema was observed. The a b i l i t y of other steroid, synthetic and plant estrogens to produce s i m i l a r water imbibition was also studied. E s t r i o l and DES were equivalent to estradiol-173 i n inducing water imbibition. The plant estrogens, genistein and coumestrol were capable of inducing tissue edema however they -3 were only 10 times as potent as estradiol-17 3 i n doing so. Administration of estradiol-17 3, genistein and coumestrol were shown to enhance uterine vascular permeability s i x hours a f t e r t h e i r administration as indicated by India ink perfusion experi-ments . Studies on the metabolism of RNA and DNA were c a r r i e d out by experiments designed to monitor the synthesis of nucleic acids following estradiol-17 3 administration. I t was found that estradiol-17 3 s i g n i f i c a n t l y shortened the uterine c e l l cycle. Net accumulation of RNA and DNA was noticed to occur 12 and 2 4 hours respectively following the administration of estradiol-173. I t was f e l t that these series of data would be useful i n future i s o t o p i c precursor experiments. The f i r s t l a b e l l e d nucleoside experiment employed the data obtained from e a r l i e r 90. experiments involving a six hour (iri vivo) pulsing period of estradiol-17 3 or i t s equivalent followed by a one hour i n v i t r o exposure to a l a b e l l e d precursor. The e s t r o g e n treated groups displayed lower s p e c i f i c a c t i v i t i e s than control groups. With estradiol-17 3 having the lowest s p e c i f i c a c t i v i t y , i t was f e l t that the data actually s i g n i f i e d those compounds which possessed the greatest a f f i n i t y to a l t e r the c e l l membrane and by doing so resulted i n the d i l u t i o n of the tracer compound by similar endogenous precursors. Therefore the lower the s p e c i f i c a c t i v i t y , the greater the d i l u t i o n of the tracer and the stronger the estrogen. Estradiol-17 3 s i g n i f i c a n t l y reduced the s p e c i f i c a c t i v i t y of a la b e l l e d precursor. Plant estrogens, genistein and coumestrol also showed reductions i n the s p e c i f i c a c t i v i t i e s of the l a b e l l e d precursor, and were sim i l a r to the trend noticed with e s t r i o l . The physiological status of the animals was seen to be an important factor as the re s u l t s of bio-chanin-A and formononetin were misleading due to the suspected onset of estrus of these treated animals. The early action of estradiol-17 3, genistein and various plant extracts was studied i n the f i n a l experiment aimed at looking at the comparative a b i l i t y of each to incorporate an isot o p i c precursor. Quantitative and q u a l i t a t i v e determina-tions of estrogenic a c t i v i t y of the plant extracts were made pr i o r to employing a short in_ vivo pulsing method used to prevent the d i l u t i o n of the radioactive tracer. From these studies, estradiol-173, genistein, a l f a l f a hay and soyabean meal extracts contained s u f f i c i e n t estrogenic a c t i v i t y to incorporate uridine 91. into the RNA f r a c t i o n of the uterus. 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The E s t r o g e n i c A c t i v i t y o f Red C l o v e r I s o f l a v o n e s and Some o f T h e i r D e g r e d a t i o n P r o d u c t s . J . E n d o c r i n o l . 24:341:348, 1962. Wyss, R.H., K a r s z n i a , R., H e i n r i c h s , W.L. and Hermann, W.L. I n h i b i t i o n o f U t e r i n e R e c e p t o r B i n d i n g o f E s t r a d i o l - 1 7 3 by a n t i e s t r o g e n s . J . C l i n . E n d o c r i n o l . 28:1824-1828, 1968. 102 . APPENDIX 103. Three. Whole U t e r i n e T i s s u e s - Homogenize i n 2.0 ml i c e c o l d d e m i n e r a l i z e d water f o r approx. 5 minutes - add 7.0 ml i c e c o l d 10% TCA - s t a n d 10 minutes and c e n t r i f u g e S u p e r n a t a n t - TCA, a c i d S o l u b l e F r a c t i o n P e l l e t - add 2.0 ml 1 N KOH - s t a n d f o r 16 h r s . , 37°C - add 0.4 ml 6 N HC1 - add 5% TCA a t 0°C - mix and p l a c e i n i c e f o r 10 minutes - c e n t r i f u g e P e l l e t add 2.5 ml 10% HCIO4 heat t o 70-85°C f o r 25 min, C o o l - s t a n d f o r 10 min. C e n t r i f u g e P e l l e t P r o t e i n - h y d r o l y z e i n 2.0 ml NaOH P e l l e t r e suspend i n 95% ETOH a t 0°C C e n t r i f u g e S u p e r n a t a n t S u p e r n a t a n t H y d r o l y z e d RNA F r a c t i o n - r e a d a t 260 nM Su p e r n a t a n t - DNA F r a c t i o n - r e a d a t 260 nM Appendix F i g . A. N u c l e i c A c i d E x t r a c t i o n P r o c e d u r e 2.0 Oven Dried Sample.of Plant Materials 104. 1. grind, add 3.0 ml 0.01 M HCI stand 20 minutes 2. add 3.0 ml Abs. ethanol Reflux 10 minutes Decant Liquid Phase 1 Residue Reflux 10 ml ETOH Decant Liquid Phase 2 - pool phases 1 & 2 - reduce volume - f i l t e r into extraction tubes Residue - discard F i l t r a t e Residue - add 15 ml D i s t i l l e d Ligroine Ether - discard - s i t 10 min. at 45° angle - suction o f f ether - add 10 ml peroxide free ethyl ether - shake 20 min. Freeze i n Dry Ice Liquid Phase 1 Soli d - c o l l e c t ether and - add 10 ml peroxide evaporate under free ethyl ether N 2 (40°) Refreeze Liquid phase 2 S o l i d phase - add to Liquid phase 1 - discard - evaporate under N 2 add 0.5 ml Absolute Ethanol wait 20 min. and store at 4°C. Appendix Fig. B. Extraction of Phytoestrogens From Plant Materials 70-A p p e n d i x F i g . C. Quench C o r r e c t i o n C u r v e f o r T r i t i u m 

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