@prefix vivo: . @prefix edm: . @prefix ns0: . @prefix dcterms: . @prefix skos: . vivo:departmentOrSchool "Land and Food Systems, Faculty of"@en ; edm:dataProvider "DSpace"@en ; ns0:degreeCampus "UBCV"@en ; dcterms:creator "Kitts, David D."@en ; dcterms:issued "2010-02-11T19:04:45Z"@en, "1976"@en ; vivo:relatedDegree "Master of Science - MSc"@en ; ns0:degreeGrantor "University of British Columbia"@en ; dcterms:description """Various estrogens biologically equivalent to 1.0 μg estradiol-17β were administered intraperitoneally to immature female rats to monitor the relative dose and time course effects on water imbibition. Estradiol-17β, estriol and diethystil-bestrol (DES) were the strongest estrogens in inducing tissue edema. Plant estrogens, genistein and coumestrol, in pure crystalline form were approximately 10⁻³ times as potent in stimulating water imbibition as estradiol-17β. The administration of estradiol-17β, genistein and coumestrol was shown to enhance the permeability of uterine vasculature as indicated by the diffusion of intravenously infused India ink. Large doses (5.0 μg) of estradiol-17β were administered to immature female rats to measure the rate of synthesis of RNA and DNA in the uterus. It was observed that the uterine cell cycle was significantly (P < 0.025) reduced after the administration of estradiol-17β. Net accumulation of RNA and DNA was shown to occur after 12 and 24 hours respectively. The incorporation of [5,6-³H] uridine into the uterine tissue was studied by administering natural, synthetic and plant estrogens intraperitoneally to immature female rats. After a six hour period there was a decreased uterine specific activity 3 of [5,6-³H] uridine in estrogen treated rats when compared to control animals. Estradiol-17β produced the greatest reduction in the specific activity of the hydrolyzed RNA while DES, estriol and estradiol-17α produced relatively smaller reductions. The reduction of specific activity six hours after in vivo pulsing with estradiol-17β was probably due to a dilution effect associated with changes occurring in cell permeability. Genistein and coumestrol also reduced the specific activity of C5,6-³H] uridine similar to that observed with estriol. Extracts of alfalfa hay and soybean meal were analyzed quantitatively and qualitatively for plant estrogens. Short in vivo pulsing of 30 minutes of various estrogens was employed to determine the uptake of [5,6-³H] uridine by the uterine tissue. Estradiol-17β, purified genistein, alfalfa and soybean extracts were found to incorporate [5,6-³H] uridine at greater rates than control groups."""@en ; edm:aggregatedCHO "https://circle.library.ubc.ca/rest/handle/2429/20075?expand=metadata"@en ; skos:note "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 "@en ; edm:hasType "Thesis/Dissertation"@en ; edm:isShownAt "10.14288/1.0093861"@en ; dcterms:language "eng"@en ; ns0:degreeDiscipline "Animal Science"@en ; edm:provider "Vancouver : University of British Columbia Library"@en ; dcterms:publisher "University of British Columbia"@en ; dcterms:rights "For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en ; ns0:scholarLevel "Graduate"@en ; dcterms:title "Comparative effects of naturally occurring, synthetic and plant estrogens on uterine metabolism"@en ; dcterms:type "Text"@en ; ns0:identifierURI "http://hdl.handle.net/2429/20075"@en .