Androgen receptor in rat liver: Hormonal and developmental regulation of the cytoplasmic receptor and its correlation with the androgen-dependent synthesis of α2u-globulin

213

Biochimica et Biophysica Acta, 3 5 4 ( 1 9 7 4 ) 2 1 3 - - 2 3 2 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s

BBA 27441

ANDROGEN RECEPTOR IN RAT LIVER: HORMONAL AND DEVELOPMENTAL REGULATION OF THE CYTOPLASMIC RECEPTOR AND ITS CORRELATION WITH THE ANDROGEN-DEPENDENT SYNTHESIS OF a2 u-GLOBULIN

A R U N K. ROY*, B R U C E S. MILIN and D O N N A

M. M c M I N N

Department of Biological Sciences, Oakland University, Rochester, Mich. 48063 (U.S.A.) (Received December 17th, 1973)

Summary The cytoplasmic receptor for 5a-dihydrotestosterone has been identified in the rat liver and partially characterized. The receptor is a protein w i t h a sedimentation coefficient of 3.5 S and binds both androgens (5a-dihydrotestosterone and testosterone) and estradiol-17fl with high affinity. At saturating concentration, for every mole of estradiol there seem to be three moles of 5a
* To w h o m all correspondence should be addremed. Abbreviation: PCMB, parachloromercuribenzoate;

214 Introduction Reports from various laboratories have shown that one of the earliest steps in the complete process of the steroidal control of gene action is the binding of the hormone with specific protein receptor present in the soluble cytoplasmic (cytosol) fraction of the target cell [1--9]. This specific interaction between the steroid hormone and its receptor is generally believed to bring about sufficient alteration of the receptor conformation to signal a further chain of events leading to the expression or repression of certain hormone sensitive genomes [10--12]. Although in recent years a considerable body of information on the receptor--steroid interaction in various types of target tissues with different steroid hormones has been accumulated, only a few reports have shown a high degree of correlation between the receptor binding data and the output of specific hormone sensitive protein, and to our knowledge no such studies on androgen action has yet been reported [13,14]. Previous studies of Roy and associates [15,16] have shown that the hepatic tissue of sexually mature rats responds to androgens with the induction of a low molecular weight protein (mol. wt. 26 400) called a 2 u -globulin. ~ 2 u-Globulin which appears as the principal urinary protein in adult male rats has been purified and partially characterized [15,17,18]. The hepatic parenchymal cells have been established as the site of a 2 ~'synthesis [ 19,20 ]. The androgen-dependent synthesis of ~2u-globulin also requires the synergistic influence of various growth and developmental hormones such as pituitary growth hormone, glucocorticoids, thyroid hormones and insulin [21--24]. Investigations concerning the mechanism of the androgenic induction of ~2u-globulin led us to the discovery of a specific androgen receptor in the rat liver cytosol which was found to be absent in the androgen-insensitive pseudohermaphrodite male rats [25]. The discovery of the androgen receptor as well as the androgen
215 the stocks of the Gerontological Research Center of the National Institute of Child Health and Human Development, Baltimore, Md. Bilateral ovariectomy in the female animals was performed through a midventral incision under ether anesthesia, and the animals were allowed 7 days of post-operative rest before any further experimental treatment. Castration of the male rats was also performed under ether anesthesia via the abdominal route, and both testes as well as most of the epididymis were removed. All animals were housed in an airconditioned animal room with regulated 12 h of light and 12 h of darkness and were maintained on Purina rat chow and tap water ad libitum. The procedures for the collection and preservation of urine as well as immunoassay of a2 uglobulin have been described in earlier publications [23,26]. 5~-Dihydrotestosterone (5a-androstan-17~-ol-3-one), estradiol-17~ (A 1,3,5 ( i o )-estratrien-3,17~-diol), testosterone(4-androsten-17~-ol-3-one)androstandiol (5~-androstan-3/~, 17~-diol) and corticosterone (44 -Pregnen-11~, 21-diol-3, 20 dione) were obtained from Sigma Chemical Co. (St. Louis, Mo.). Tritium labeled 5a-[1,2 -3 H2 ]-dihydrotestosterone was obtained in benzeneethanol solution from New England Nuclear Co. (Boston, Mass.) with a specific activity of 44 Ci/mM. Tritium labeled [2,4,6,7-3H4]estradiol,17~ was obtained from Amersham Searle (Arlington Heights, Ill.) in benzene--ethanol solution with a specific activity of 100 Ci/mM. Pronase (A grade) with an activity of 120000 P.U.K./g was obtained from Calbiochem. (Los Angeles, Calif.). Clostridiurn Perfringens neuraminidase (1.1 units/mg using N-acetylneuraminic acid--lactose assay), purified bovine pancreatic DNAase, type VII lipase (from Clostridium cylindracea) and dithiothreitol were obtained from Sigma Chemical Co. (St. Louis, Mo.). Purified RNAase (Spec. act. 2500 units/ mg), ovalbumin, human 7-globulin and methyl testosterone (4-androsten-17amethyl-17~-ol-3-one) were products of Schwartz-Mann (Orangeburg, N.Y.). All of these chemicals were used without any further purifications.

(2) Preparation of the liver cytosol and assay for the androgen receptor activity The procedure for processing the liver a n d the preparation of the 133 000 × g cell supernatant (cytosol) has been described in detail in an earlier publication [25]. The two most important features of the procedure involve (i) maintenance of the operating temperature close to 0°C throughout the experiment, and (ii) homogenization of the liver tissue in the 10 mM Tris--HCl (pH 7.4)--15 mM disodium EDTA--10 mM dithiothreitol buffer at a tissue to buffer ratio of I g:l ml. For binding the steroid ligand to the cytosol receptor, aliquots of 0.396 ml of the cytosol preparation were incubated at 0°C for 40 rain with 0.004 ml of the steroid solution (benzene-ethanol; 9:1, v/v). The above ratio of the steroid solvent to cytosol (1:100) was maintained in all of the in vitro binding experiments. Unless otherwise specified the assay system contained 40 nM labeled steroid. Three different types of incubation tubes, i.e. untreated glass tubes, albumin-coated glass tubes and gelatin-coated glass tubes, were tested for their wall adherence of the labeled steroid (5~-dihydrotestosterone and estradiol). Although all of these three types of tubes were found to bind steroids on their surface, the gelatin-coated tubes showed the least wall adherence and gave the most consistent receptor binding activity with the labeled steroids. Therefore gelatin-coated glass tubes were used for all of

216 the in vitro receptor binding experiments. The following procedure was used to coat the incubation tubes with gelatin. A 0.1% suspension of gelatin was brought to solution by boiling for 1 min, and the incubation tubes were filled with hot gelatin solution. The tubes were allowed to soak in the gelatin solution for 30 min at 60 ° C. After the period of soaking, the gelatin solution was drained off and the tubes were dried at 200°C for 2 h. The receptor--steroid complex was separated on a sucrose density gradient. An 0.1 ml of the cytosol--steroid incubation mixture was layered on a 5--15% sucrose gradient in cellulose nitrate tubes and centrifuged at 47000 rev./min (216000 × g) for 22 h in a Beckman 50.1 Swinging Bucket Rotor. The sucrose gradient was prepared in the same buffer (Tris--HCl(pH 7.4)--disodium EDTA--dithiothreitol) which was used for the preparation of the cytosol. After centrifugation the density gradient was manually fractionated and fractions of 10 drops (about 0.21 ml) were collected in scintillation vials and counted with 5.0 ml of Bray's fluid [28] in a Packard liquid scintillation spectrometer. Quantitative determination of the receptor binding of the steroid ligand was made, according to Hansson et al. [29], by computing the percentage of the total radioactivity bound in the 3.5-S receptor peak after correction for the extrapolated background. The protein concentration of the cytosol was assayed by the Lowry procedure [30]. Results

(1) Sedimentation profile o f the hepatic androgen receptor and receptor stability at various concentrations o f cytosol proteins, KCI, sulfhydryl-protecting reagent and H ÷ Sucrose density gradient analysis of the mature rat liver cytosol incubated at 0°C for 40 min with 5a-[1,2-3H2 ]dihydrotestosterone resulted in the displacement of the radioactivity with a macromolecular fraction having a sedimentation coefficient of approx. 3.5 S (Fig. 1). The above sedimentation coefficient of the 5a-dihydrotestosterone binding peak was calculated according to Martin and Ames [31] with ovalbumin (3.55 S) and human 7-globulin (6.9 S) as standards. The distinctive differences between the hepatic cytosol androgen receptor and the non-specific steroid binding component of the rat serum have already been reported [25]. A relatively high concentration of the cytosol protein (47.7 + 5.5 mg/ml), achieved by homogenizing 1 g of liver with 1 ml of the Tris--HC1 (pH 7.4)--disodium EDTA--dithiothreitol buffer, was found to be required for the stability of the cytosol 5a-dihydrotestosterone receptor. Dilution of the above cytosol preparation with equal volumes of Tris-HC1 (pH 7.4)--disodium EDTA--dithiothreitol buffer resulted in complete inactivation of the receptor (Fig. 1). Presence of sulfhydryl group-protecting reagents such as ~-mercaptoethanol or dithiothreitol augmented the 5a-dihydrotestosterone binding ability of the receptor. Incubation of the cytosol preparation with 2 mM parachloromercuribenzoate (PCMB) at 0°C for 60 min was found to inactivate the receptor activity. In different cytosol preparations, ranges between 6 and 10 mM dithiothreitol added both in the incubation media as well as in the gradients, resulted in maximum 5a-dihydrotestosterone binding to the cytosol receptor. Dithiothreitol concentrations above 10 mM were

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Fig. I . I n f l u e n c e o f c y t o s o l c o n c e n t r a t i o n o n t h e 5 a - [ l , 2 - 3 H 2 ] d i h y d r o t e s t o s t a r o n e and [2,4,6,7-3H4] e s t r a d i o l b i n d i n g b y t h e 3 . 5 - S a n d r o g e n r e c e p t o r . T h e liver c y t o s o l w a s p r e p a r e d a c c o r d i n g t o t h e p r o cedure described under Materials and Methods, and aliquots were diluted with equal volumes of Trls--HCI ( p H 7 . 4 ) - - d i s o d i u m E D T A - - d l t h i o t h r e i t o l b u f f e r p r i o r t o its i n c u b a t i o n w i t h t h e l a b e l e d s t e r o i d s ( 4 0 nM). T h e k e y s f o r t h e s y m b o l s r e p r e s e n t i n g t h e s u c r o s e d e n s i t y g r a d i e n t p a t t e r n s are as f o l l o w s : • •, 5c~-[1,2-3H2]dihydrotestostarone binding with undiluted c y t o s o l (,55.6 m g p r o t e i n / m l ) ; • . • . • , 5a-[l,2-3H2[dihydrotestosterone binding with diluted cytosol (27.8 mg protein/ml); o o, [ 2 , 4 , 6,7-3H4]estradiol binding with undiluted cytosol; s..,~e [2,4,6,7.3H4]estradiol binding with diluted cytosol. Fig. 2. C y t o s o l a n d r o g e n r e c e p t o r a c t i v i t y a t i n c r e a s i n g i o n i c s t r e n g t h . S u c r o s e d e n s i t y g r a d i e n t p a t t e r n o f t h e liver e y t o s o l i n c u b a t e d w i t h 5 a - [ 1 , 2 - 3 H 2 ] d i h y d s o t e s t o s t e r o n e in Trls--HCI (pH 7.4)--dlsodium E D T A - - d i t h i o t h r e i t o l b u f f e r ( n o K C l ) (e "-), in Tris--HC1 ( p H 7 . 4 ) - - d l s o d i u m E D T A - - d i t h i o t h r e i t o l b u f f e r c o n t a i n i n g 0 . 4 M K C l (o o) a n d i n t h e s a m e b u f f e r o o n t a l n i n g 0 . 8 M K C l ( o ~ . . o ) . T h e g r a d i e n t s a l s o c o n t a i n e d t h e s a m e salt c o n c e n t r a t i o n s .

found to inhibit 5a~lihydrotestosterone binding. Therefore, 10 mM dithiothreitol was routinely added to all buffer media used for the receptor assay. The binding of 5a-[1,2 -3 H2 ]dihydrotestosterone to the cytosol receptor appeared to be independent of the salt concentration in the medium. Increasing concentrations of KC1 (both in the incubation medium as well as in the gradient) up to 0.8 M concentration changed neither the sedimentation coefficient nor the 5~-dihydrotestosterone binding ability of the receptor (Fig. 2). Assay of the 5a
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pH Fig. 3. I n f l u e n c e o f p H o n t h e a n d r o g e n r e c e p t o r a c t i v i t y o f r a t liver c y t o s o l . T h e c y t o s o l p r e p a r a t i o n s as well as t h e s u c r o s e d e n s i t y g r a d i e n t s w e r e m a d e in t h e s a m e b u f f e r s o l u t i o n s . C o n d i t i o n s such as t h e t e m p e r a t u r e a n d p e r i o d of i n c u b a t i o n ( 0 ° C , 4 0 r a i n ) , c o n c e n t r a t i o n s o f d i t h i o t h r e i t o l ( 1 0 m M ) a n d E D T A ( 1 5 raM) w e r e t h e s a m e as t h o s e o f t h e s t a n d a r d assay s y s t e m d e s c r i b e d u n d e r Materials a n d M e t h o d s . T h e f o l l o w i n g b u f f e r s y s t e m s w e r e u s e d f o r v a r i o u s p H levels: p H 4.5, 0.1 M a c e t a t e ; p H 6.5, 0.1 M p h o s p h a t e ; p H 7.5 a n d 8.5, 0.1 M Tris-HC1; p H 9.5 a n d 1 0 . 3 , 0.2 M g l y c i n e - - N a O H . D H T , 5 a - d i h y d r o t e s t o s t e r o n e .

(2) Effects of hydrolytic enzymes and temperature variations in the receptor activity In order to establish the nature of the 5a-dihydrotestosterone binding macromolecule, the effects of various hydrolytic enzymes on the receptor activity were tested. Results presented in Table I shows t h a t treatments with TABLE I E F F E C T S O F C E R T A I N H Y D R O L Y T I C E N Z Y M E S ON T H E 5 a - [ 1 , 2 - 3 H 2 ] D I H Y D R O T E S T O S T E R O N E B I N D I N G BY T H E H E P A T I C A N D R O G E N R E C E P T O R A l i q u o t s o f t h e c y t o s o l p r e p a r a t i o n w e r e p r e i n c u b a t e d w i t h t h e e n z y m e s at 0 ° C f o r 6 0 m i n p r i o r to t h e i r use in 5 ( ~ - [ 1 , 2 - 3 H 2 ] d i h y d r o t e s t o s t e r o n e b i n d i n g assay. T h e r e c e p t o r a c t i v i t y w a s d e t e r m i n e d b y c o m p u t a t i o n of t h e c o u n t s w i t h i n t h e 3.5 S p e a k as d e s c r i b e d u n d e r Materials a n d M e t h o d s . All o f e n z y m e s w e r e u s e d at c o n c e n t r a t i o n s of 1 m g p e r m l o f c y t o s o l e x c e p t n e u r a m i n i d a s e t h e c o n c e n t r a t i o n o f w h i c h w a s 2 0 0 ~/g p e r m l o f c y t o s o l . Enzymes

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100 0 99 96 82 89

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Fig. 4. T h e r m a l i n s t a b i l i t y o f t h e c y t o s o l a n d r o g e n r e c e p t o r o f r a t liver. T h e liver c y t o s o l p r e p a r a t i o n s w e r e p r e i n c u b a t e d f o r 5 m i n a t v a r i o u s t e m p e r a t u r e s f r o m 0 t o 5 0 ° C and the 5 a - [ 1 , 2 - 3 H 2 ] d i h y d r o testosterone binding abilities at 0°C of these preincubated cytosol samples were assayed on sucrose d e n s i t y g r a d i e n t s . T h e r e c e p t o r a c t i v i t i e s a r e e x p r e s s e d as p e r c e n t a g e o f t h e c o n t r o l ( p r e i n c u b a t e d a t 0 ° C ) . Fig. 5. I n s t a b i l i t y o f t h e c y t o s o l a n d r o g e n r e c e p t o r o f r a t liver a t 3 8 ° C . T h e r a t liver c y t o s o l w a s p r e i n c u b a t e d a t S 8 ° C f o r v a r i o u s t i m e p e r i o d s p r i o r t o its u s e f o r 5 a - [ 1 , 2 -3 H 2 ] d i h y d r o t e s t o s t e r o n e b i n d i n g a t 0 ° C a n d s u b s e q u e n t s u c r o s e d e n s i t y g]radient s e p a r a t i o n . T h e v a r i o u s p r e i n c u b a t i o n t i m e s a r e e x p r e s s e d with the following symbols: • -', 0 m i n ; c o, I 0 r a i n ; • . . . . . . e , 2 0 m i n ; ~ ~, 30 min; o ...... o, 40 min.

DNAase, RNAase, lipase and neuraminidase at 0°C for 60 min did not significantly decrease the 5a-dihydrotestosterone binding abilityof the cytosol receptor. On the other hand, treatment with pronase at 0°C for 60 rain completely destroyed the 5a-dihydrotestosterone binding property of the 3.5-S cytosol macromolecule. The observed 18% reduction of the receptor activity with lipase may be due to its protease contamination, since the lipase preparation used for the experiment was only "substantially free of protease activity". Exposure of the liver cytosol for 5 rain at various temperatures prior to its incubation with the 5a-[1,2-SH2 ]dihydrotestosterone (at 0°C for 40 rain) showed a sharp drop in 5a
220 T A B L E II C O M P E T I T I O N B E T W E E N V A R I O U S S T E R O I D S F O R R E C E P T O R B I N D I N G IN V I T R O T h e c y t o s o l p r e p a r a t i o n f r o m a d u l t m a l e r a t w a s i n c u b a t e d w i t h 5c~-[1.2-3H2] d i h y d r o t e s t o s t e r o n e ( 4 0 0 nM) a n d 3 0 - f o l d excess o f n o n - r a d i o a c t i v e c o m p e t i n g s t e r o i d s (12 pM). T h e d i s p l a c e m e n t of t h e radioa c t i v i t y f r o m t h e 3.5-S b i n d i n g p e a k w a s u s e d as t h e c o m p e t i t i v e e f f e c t i v e n e s s o f t h e u n l a b e l e d steroids. Competing steroid

R e m a i n i n g c o u n t s in t h e 3.5-S p e a k (%)

None 5~-Dihydr otestost erone Testosterone Androstandiol 17c~-Methyl t e s t o s t e r o n e Corticosterone

100 7 47 91 137 109

(3) Ligand specificity of the cytosol androgen receptor and estradiol binding to the receptor protein Ligand specificity of the receptor protein was tested in competition experiments with 5a-[1,2 -3 H: ] dihydrotestosterone (400 nM) and 30-fold excess of the non-radioactive competing steroid (12/~M). In order to achieve the 400 nM concentration of the 5a-[1,2 -3 H: ] dihydrotestosterone in the incubation media, the specific radioactivity of the stock solution of 5a-[1,2 -3 H2 ] dihydrotestosterone was reduced from 44 Ci/mmole to 2.2 Ci/mmole by the addition of non-radioactive 5a-dihydrotestosterone. For these experiments the liver cytosol was incubated in the presence of both 5a-[1,2-3H2 ]dihydrotestosterone and the competing steroids at 0°C for 40 rain, and the ability of the various steroids to displace the radioactivity from the 3.5-S peak were used as the indices of competition. The results of these experiments presented in Table II and Fig. 6 show that of all the steroids examined only non-radioactive 5a-dihydrotestosterone, testosterone and estradiol showed a high degree of effectiveness in displacing the labeled 5a-dihydrotestosterone from the receptor peak. However, unlike the 5a-[1,2 -3 H2 ] dihydrotestosterone binding, [2,4,6,7 -.3H4 ] estradiol binding to the receptor peak could only be effectively displaced with unlabeled estradiol. Non-radioactive 5a-dihydrotestosterone was found to be only weaMy effective in displacing the labeled estradiol from the receptor protein (Fig. 6b). Labeled 5a-dihydrotestosterone and estradiol binding to the 3.5-S cytosol protein showed a differential sensitivity to dilution. Inactivation of only the 5a-dihydrotestosterone binding activity, leaving the estradiol binding property of the 3.5-S receptor, could be achieved by diluting the cytosol with equal volumes of the homogenizing buffer (Fig. 1). The effects of in vivo administration of high levels of both 5a-dihydrotestosterone and estradiol (receptor binding steroids) and corticosterone (a non-binding steroid) on the androgen receptor activity in vitro was also examined. Mature male rats (300--350 g body weight) were separately injected with 5 mg of the above steroids through intraperitoneal route, and the animals were sacrificed 40 min later for the preparation of the liver cytosol. The cytosol samples were then assayed for the 5a-dihydrotestosterone receptor

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Fig. 6. (a) InhFoition of receptor u p t a k e of 5¢x-[1,2-3H2]dihydrotestosterone b y unlabeled 5a-dihydrotestosterone and estradiol in vitro. The cytosol preparations were incubated simultaneously w i t h 400 nM 5(~-[1.2-3H2]dihydrotestosterone and 12 , M unlabeled steroids and t h e n subjected to sucrose density gradient analysis. Key for the symbols: • •. 4 0 0 n M 5o~-[1.2-3H2]dihydrotestosterone alone; • ...... a 400 nM labeled + 12DM unlabeled 5a-dihydrotestosterone; o ...... o, 400 nM ~ - [ l , 2 - 3 H 2 ] d i h y d r o testosterone + 12 DM estradiol. (b) Inhibition of receptor u p t a k e of [2,4,6,7-3H4]estradiol by unlabeled estradiol and ~ - d i h y d r o t e s t o s t e r o n e . The cytosol preparations were incubated simultaneously with 4 0 0 nM [2,4,6,7-3I-I4]est~diol and 12 DM unlabeled steroids and t h e n subjected to sucrose density gradient analysis. Key for t h e symbols: _'2 ~, 400 nM [2,4,6,7-3H4]es~adlol alone; A...A 400 nM labeled + 12 , M unlabeled estradiol; o...o, 400 nM [2,4,6,7-3H4] est~diol + 12 , M 5a-dlhydrotestosterone.

activity through the usual procedure, as described under Materials and Methods. The results presented in Fig. 7 revealed that both 5a-dihydrotestosterone and estradiol, which showed ,strong affinity for the receptor in vitro, were also able to suppress in vitro binding of 5a-[1,2 -3 H2 ]dihydrotestosterone when administered in vivo; and corticosterone, which did not compete with 5a-dihydrotestosterone for the receptor binding in vitro, also did not affect the receptor activity in vivo.

(4) Receptor affinity and binding kinetics with 5a-dihydrotestosterone and estradiol Binding of both 5a-[1,2- 3 H2 ] dihydrotestosterone and [2,4,6,7 -3 H4 ]estradiol to the cytosol receptor was investigated by following the hormone binding at increasing ligand concentrations. Quantitations of the binding activity in the 3.5-S peak region were done according to the procedure described under Materials and Methods. The results of these experiments were plotted both as simple concentration kinetics of bound vs total steroid, as well as according to Scatchard method [33]. The binding kinetics with 5a
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Fig. 7. E f f e c t of in vivo t r e a t m e n t w i t h 5 ~ - d i h y d r o t e s t o s t e r o n e , e s t r a d i o l a n d c o r t i c o s t e r o n e o n t h e 5 ~ - [ 1 , 2 - 3 H 2 ] d i h y d r o t e s t o s t e r o n e b i n d i n g ability o f t h e h e p a t i c c y t o s o l in v i t r o . 3 0 0 - - 3 5 0 g m a l e r a t s (:> 1 0 0 d a y s o f age) w e r e i n j e c t e d i n t t a p e r i t o n e a l y w i t h 5 m g of t h e s t e r o i d h o r m o n e s p r i o r t o t h e i r sacrifices f o r t h e a n d r o g e n r e c e p t o r assay. • • , c o r t i c o s t e r o n e t r e a t e d ; e . • .e, 5 ( ~ - d i h y d r o t e s t o s t e r one treated; o o, e s t r a d i o l t r e a t e d . Fig. 8. S c a t c h a r d analysis o f 5 a - d i h y d r o t e s t o s t e r o n e ( D H T ) b i n d i n g t o t h e c y t o s o l a n d r o g e n r e c e p t o r . Specific b i n d i n g o f 5 a - [ 1 , 2 : 3 H 2 ] d i h y d r o t e s t o s t e r o n e t o t h e 3.5-S c y t o s o l p r o t e i n w a s d e t e r m i n e d b y s u c r o s e d e n s i t y g r a d i e n t analysis. T h e i n s e t s h o w s a s i m p l e c o n c e n t r a t i o n p l o t of t h e s a m e b i n d i n g d a t a .

tion constants (Kd) of these t w o binding isotherms as estimated from the Scatchard plot were found to be 4.5 • 10 -s M (Kdl) and 2.0 • 10 -6 M (KdIi) , respectively. Estradiol binding to the receptor, on the other hand, gave evidence for only a single binding isotherm b o t h in the concentration plot as well as in the Scatchard plot (Fig. 9). The dissociation constant for the estradiol binding, as calculated from the Scatchard plot, was found to be 3 . 5 . 1 0 -7 M. Extrapolation of Scatchard plots (Figs 8--9) showed that for every mole of estradiol binding there appear to be three moles of 5~-dihydrotestosterone binding within the same a m o u n t of cytosol protein.

(5) Development of the receptor activity in maturing male rats and the effect o f castration on the process Immature male rats do n o t synthesize ~2u-globulin, and, coincident with the gonadal maturation, this androgen-dependent protein normally starts appearing at the time of puberty, around 6 weeks of age [26]. Sucrose density gradient analysis of the hepatic androgen receptor activity in the normal male rats of various ages, starting from 25 days, shows a minimal level of the 3.5-S receptor peak in the 25-day-old rats and a gradual rise of the receptor activity

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Fig. 1 0 . D e v e l o p m e n t o f t h e c y t o s o l a n d r o g e n r e c e p t o r a c t i v i t y i n t h e m a t u r i n g m a l e r a t liver. R e c e p t o z a c t i v i t y in n o r m a l m a l e r a t s o f p r e p u b e r t a l , p u b e r t a l a n d e a r l y p o s t p u b e r t a l ages w e r e a s s a y e d o n s u c r o s e density gradients. The ages of the animals were represented with different symbols: A...A0 25 days; • •, 30 days; 0-..o, 37 days; ¢ -=, 4 8 d a y s ; o . . . o 55days; o o, 6 5 d a y s o f age. T h e i n s e t s h o w s t h e levels o f d a f f y u r i n a r y (~2u o u t p u t 6 f t h e s e Rnlmals i m m e d i a t e l y p r i o r t o t h e i r s a c r i f i c e fox the receptor assay. Fig. 1 1 . E f f e c t o f p r e p u b e r t a l c a s t r a t i o n o n t h e d e v e l o p m e n t o f t h e a n d r o g e n r e c e p t o r a c t i v i t y i n t h e r a t liver c y t o s o l o f m a l e r a t s . T h e a n i m a l s w e r e c a s t r a t e d a t 2 1 d a y s o f age, a n d t h e i r c y t o s o l a n d r o g e n r e c e p t o r a c t i v i t y w a s a s s a y e d a t 3 4 (o o), 5 6 ( A . . . A ) a n d 1 0 8 (o o) d a y s o f age. T h e s u c r o s e d e n s i t y g r a d i e n t p a t t e r n o f t h e s h a m - o p e r a t e d a n i m a l (a -') s a c r i f i c e d a t 1 0 6 d a y s o f a g e serves as a control. None of these castrated animals showed any ~2u-globulin in their urine prior to their sacrifice, w h i l e t h e c o n t r o l a n i m a l s e c r e t e d 2 8 . 2 / ~ g o f t h e p r o t e i n f o r a p e r i o d o f 2 4 h b e f o r e sacrifice.

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Fig. 12. E f f e c t of c a s t r a t i o n o n t h e h e p a t i c a n d r o g e n r e c e p t o r a c t i v i t y in m a t u r e m a l e rats. 3 0 0 - - 3 5 0 g m a l e rats ( ~ , 1 0 0 d a y s o f age) w e r e c a s t r a t e d and their h e p a t i c c y t o s u l a n d r o g e n r e c e p t o r activities w e r e a s s a y e d at v a r i o u s p e r i o d s a f t e r t h e o p e r a t i o n . T h e s u c r o s e d e n s i t y gradient p a t t e r n s o f t h e e x p e r i m e n t a l a n i m a l s at various t i m e p e r i o d s a f t e r c a s t r a t i o n axe e x p r e s s e d w i t h the f o l l o w i n g s y m b o l s ; • •, 0 day c o n t r o l ; o. - .o, 5 d a y s ; • • , 15 d a y s ; D. • . o 18 d a y s . T h e inset s h o w s t h e level o f the 2 4 - h urinary o u t p u t o f a 2 u - g l o b u l i n in t h e s e a n i m a l s i m m e d i a t e l y p r i o r t o t h e i r use for t h e r e c e p t o r assay. Fig. 13. I n d u c t i o n o f h e p a t i c a n d r o g e n r e c e p t o r a c t i v i t y in t h e o v a x i e c t o m i z e d f e m a l e rats t r e a t e d w i t h 5 ~ - d i h y d r o t e s t o s t e r o n e ( 0 . 5 12g/g b o d y w t p e r d a y , s u b c u t a n e o u s i n j e c t i o n s ) . S u c r o s e d e n s i t y gradient p a t t e r n s o f t h e 5 a - [ 1 , 2 - 3 H 2 ] d i h y d r o t e s t o s t e r o n e b i n d i n g t o t h e h e p a t i c c y t o s o l b y the a n i m a l s t r e a t e d w i t h 5 ~ - d i h y d r o t e s t o s t e r o n e f o r v a r i o u s p e r i o d s axe e x p r e s s e d w i t h d i f f e r e n t s y m b o l s : A. • . A 0 d a y control; • A 2 d a y s ; o.-.o, 4 d a y s ; • -', 6 d a y s ; o-.-o, 8 d a y s ; o o, I 0 days. T h e inset s h o w s t h e 2 4 - h urinary o u t p u t o f C~2u-globulin o f t h e s e a n i m a l s i m m e d i a t e l y p r i o r t o their sacrifice for t h e r e c e p t o r assay.

with age. Under the conditions of receptor assay (40 nM 5~-[1,2 -3 H~ ] dihydrotestosterone, approx. 50 mg/ml protein), 3.5-S 5a-dihydrotestosterone binding activity reached a plateau at 48 days of age (Fig. 10). Prior to their sacrifice for the receptor assay, these animals were placed in the metabolism cages and their 24-h urinary output of a2u-globulin was analyzed. The results presented in the inset of Fig. 10 reveal that, although the animals of 40 days of age and younger show a low level of receptor activity, they do not produce any ~2uglobulin. The process of the normal development of the receptor activity in the maturing male rats was found to be prevented by castration at 21 days of age. These prepubertal castrated male rats did not show the normal rise of receptor activity with puberty, and only trace amount of the receptor could be detected even at 108 days of age (Fig. 11). None of these prepubertal castrated male rats used for the receptor assay were found to produce a2u-globulin. The role of androgens in the maintenance of the receptor activity is also shown by the gradual decay of the cytosol androgen receptor activity in the mature male rats after castration {Fig. 12). For these experiments the levels of hepatic

225

cytosol receptor activity were assayed in the adult male rats (300--350 g body weight,> 100 days of age) at different time periods after castration. The results show that after an initial rise in the receptor activity the level gradually declined, and finally only a trace amount of the receptor could be detected at 18 days after castration. The apparent rise in the receptor activity at 5 days after castration may reflect a rise in the level of "free" or "stripped" receptor due to the decline of the endogenous androgens rather than to a rise in the total receptor concentration. The analysis of the 24-h urinary output of aauglobulin in these animals prior to their receptor assay reveals a gradual drop in the urinary ~ u output in these castrated males (Fig. 12, inset).

(6) Androgenic induction of the receptor activity in female rats and androgen insensitivity in prepubertal animals Normal female rats of all ages do not produce ~2u-globulin and show neither 5a-dihydrotestosterone or estradiol binding to the 3.5-S peak in the sucrose gradient. However, the mature female rats could be induced to synthesize the protein after surgical removal of the ovaries and subsequent androgen treatment [16]. Assay of the androgen receptor activity in the hepatic cytosol of the mature female rats (> 100 days of age) after castration and androgen treatment with 5~-dihydrotestosterone at a dose of 0.5 pg/g body weight per day, showed a gradual rise in the cytosol androgen receptor activity of the hepatic tissue which is followed by subsequent induction of ~au-globulin (Fig. 13). Since the prepubertal animals of both sexes do not produce ~2u-globulin even under the influence of high doses of androgens, a developmental block in the androgen responsiveness in the immature animals have been suggested [26]. Unlike the mature animals, treatment of the 21-dayold male rats daffy for 10 days with even 10 #g of 5~-dihydrotestosterone per g body weight (20 times the optimal dose for ~2u induction) did not induce any significant amount of receptor activity as compared to the control animal treated with the vehicle alone (Fig. 14). These results confirm the earlier speculation that androgen insensitivity in prepubertal animals is due to the lack of the receptor induction in the prepubertal age [26].

(7) Estradiol-mediated suppression of the androgen receptor activity and a2usynthesis Estradiol acts as a potent inhibitor of a2u synthesis even at a daily dose of 0.01/~g/g body weight. Estradiol was also found to suppress 5a-dihydrotestosterone binding to the cytosol androgen receptor (Fig. 6). Treatment of the mature male rats (300--350 g body weight, > 100 days of age) with 0.5 #g of estradiol/ g body weight per day for 8 days completely inhibits ~2u synthesis and also brings about a total loss of cytosol androgen receptor activity (Fig. 15). The estradiol treatment also brings about a period of temporary androgen insensitivity in these animals when treatment with androgenic steroids does not induce a~u synthesis. The length of this lag period is dependent on the dose of estradiol. With an estradiol dose of 0.5 #g/g body weight per day for 8 days, the lag period was found to last 4--6 weeks. Results concerning the dose dependency of the estrogenic inhibition of a2u synthesis and the nature of the estrogen-mediated temporary androgen insensitivity are not presented in this paper and will be published elsewhere.

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Fig. 14. N o n - i n d u c t i o n o f t h e c y t o s o l a n d r o g e n r e c e p t o r in t h e liver tissue o f t h e 5 ~ - d i h y d r o t e s t o s t e r o n e t r e a t e d ( 1 0 # g / g b o d y w t p e r d a y f o r 10 d a y s ) p r e p u b e r t a l ( 2 1 - - 3 1 d a y s of age) m a l e rats. T h e sucrose density gradient patterns of the cytosol 5 ~ - [ 1 , 2 - 3 H 2 ] d i h y d r o t e s t o s t e r o n e binding by the 5~-dihydrot e s t o s t e r o n e - t r e a t e d a n i m a l is r e p r e s e n t e d b y t h e solid circles; t h e o p e n circles r e p r e s e n t t h e p a t t e r n o f t h e c o n t r o l a n i m a l i n j e c t e d w i t h t h e v e h i c l e a l o n e . T h e a r r o w s h o w s t h e p o s i t i o n of t h e 3.5-S r e g i o n o f t h e g r a d i e n t . N o n e o f t h e s e a n i m a l s s h o w e d a n y CZ2u-globulin in t h e i r u r i n e b e f o r e t h e i r sacrifice a t 31 d a y s of age. Fig. 15. E s t r a d i o l - m e d i a t e d s u p p r e s s i o n of t h e c y t o s o l a n d r o g e n r e c e p t o r a c t : v l t y in t h e a d u l t m a l e r a t liver. A d u l t m a l e r a t s ( 3 0 0 - - 3 5 0 g b o d y w t , > 1 0 0 d a y s o f age) w e r e t r e a t e d w i t h e s t r a d i o l , 17fl (0.5 p g / g b o d y w t p e r d a y , s u b c u t a n e o u s i n j e c t i o n ) f o r v a r i o u s p e r i o d s of t i m e u p t o S d a y s , a n d t h e i r c y t o s o l a n d r o g e n r e c e p t o r a c t i v i t i e s w e r e a s s a y e d o n s u c r o s e d e n s i t y g r a d i e n t s b o t h w i t h i n t h e a b o v e p e r i o d s of t r e a t m e n t as well as a f t e r t h e w i t h d r a w a l o f e s t r a d i o l t r e a t m e n t . T i m e p e r i o d s in all o f t h e a n i m a l s are c o u n t e d f r o m d a y of t h e s t a r t of t h e e s t r a d i o l t r e a t m e n t . T h e s u c r o s e d e n s i t y g r a d i e n t p a t t e r n s of t h e d i f f e r e n t e x p e r i m e n t a l a n i m a l s , w i t h t i m e p e r i o d s b e g i n n i n g e s t r a d i o l t r e a t m e n t , are e x p r e s s e d b y t h e following symbols: • e, 4 d a y s ( d a i l y e s t r a d i o l t r e a t m e n t ) ; o. • . o 8 d a y s (daily e s t r a d i o l t r e a t m e n t ) ; A . . . A , 14 d a y s (first 8 d a y s of e s t r a d i o l t r e a t m e n t f o l l o w e d b y 6 d a y s o f r e s t ) ; A A, 20 d a y s (first 8 d a y s of e s t r a d i o l t r e a t m e n t f o l l o w e d b y 12 d a y s o f rest). T h e inset s h o w s t h e daily u r i n a r y o u t p u t o f a 2 u - g l o b u l i n in t h e s e a n i m a l s i m m e d i a t e l y p r i o r t o t h e i r sacrifice. T h e p e r i o d of e s t r a d i o l t r e a t m e n t is m a r k e d wJtli a r r o w s ( E S T ) ,

(8) Absence o f the receptor activity and androgen insensitivity in senescen~ male rats In the normal male rats, the urinary output of a2u-globulin starts appearing after about 40 days of age, reaches a peak level (approx. 0.1 mg/g body wt) between 60 and 75 days, and the high level is maintained up to 6 0 0 - - 7 5 0 days of age. Beyond 750 days of age, the animals gradually reach the state of senility which is associated with a rapid decline and complete loss of a2u synthesizing ability in these animals. Examination of the androgen receptor activity in the livers of the non-a2u-producing senile male rats showed virtual absence or, at best, only trace amounts of cytosol androgen receptor activity in the non-a2uproducing senescent male rat liver. In order to reduce the low affinity and

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Fig. 16. L a c k o f a n d r o g e n r e c e p t o r a c t i v i t y and its n o n - i n d u c t i o n in t h e s e n e s c e n t m a l e rat liver. S u c r o s e density gradient p a t t e r n of the liver cytosol incubated wit h 5 a - [ 1 , 2 - 3 H 2 ] d i h y d r o t e s t o s t e r o n e (20 nM); • • , 793-day-old male; o. • .o, 793-day-old male injected for 25 days prior to sacrifice w i t h 1 mg of 5a-dihydrotestosterone per day (subcutaneously); ¢ -', 1 5 6 - d a y - o l d c o n t r o l male. 24-h u r i n a r y outp u t s o f a 2 u - g l o b u l i n w e r e a s s a y e d p r i o r t o t h e i r sacrifice a n d b o t h the u n t r e a t e d as well as t h e 5 a - d i h y d r o t e s t o s t e r o n e - t r e a t e d s e n e s c e n t m a l e s did n o t p r o d u c e a n y a 2 u - g l o b u l i n w h e r e a s t h e c o n t r o l m a l e p r o d u c e d 33.2/~g of a2u-globulin.

non-specific binding of 5a-dihydrotestosterone in the senile rat liver cytosol, the concentration of the 5a-[1,2 -3 H2 ] dihydrotestosterone in the assay system was reduced from 40 to 20 nM. Even after the above modification the cytosol preparations from the non-asu-producing senile male rats did not reveal any receptor peak (Fig. 16). Androgen treatment to these senescent males at a dose of 0.5 #g 5~-dihydrotestosterone/g body weight per day for 20 days neither reinitiated any synthesis of a2u-globulin nor raised the cytosol receptor concentrations in the liver tissues of these animals. The above results indicate that the loss of ~2u synthesis in the senile male rats is primarily due to the fall in the androgen receptor activity in their liver cells, and the androgen insensitivity in these animals is a result of non-inducibility of the receptor activity by androgens. Discussion In spite of early reports of the androgen-dependent appearance of-certain hepatic proteins in the rat [16,34], the mechanism of androgen action on the liver has until recently escaped critical examination. Except limited investigation of the estrogen receptors in the avian and amphibian liver [35,36] most of the studies concerning the sex hormone receptor and their role in macromolecular synthesis have so far been limited primarily to the reproductive

228 tissues, and the hepatic tissue has generally been considered as "non-target" for sex hormones. Contrary to such a concept the results presented in this article show that the rat liver is an important target for sex steroids and could serve as a useful model for studies of androgen action. The published reports regarding the sedimentation coefficients of the cytoplasmic androgen receptors in the different tissues vary from 3.0 S to 10.0 S [5,6,29,37--39]. The sedimentation coefficients for the androgen receptor activity of the hepatic cytosol were found to reside in a 3.5-S protein, and the results do n o t give any indication for heavier receptor species. The inability of 0.8 M KC1 to alter the sedimentation behavior and the 5a-dihydrotestosterone binding property of the receptor suggests that the receptor is a tightly packed protein. It may be of interest to note that in working with the estradiol receptor in the rat uterus, Stancel et al. [40] recently concluded that the cytosol receptor activity in vivo is only associated with 3.8-S to 4.8-S protein and that the heavier forms reported by earlier workers may represent artifactual aggregates. Although some of the differences between the steroid binding c o m p o n e n t of rat serum (albumin) and the cytosol androgen receptor of the liver tissue has been presented in an earlier publication [ 2 5 ] , the salient points may also be presented here: the serum binder and the cytosol androgen receptor have detectable differences in the sedimentation coefficients (3.5 S vs 4.2 S), and the serum peak may be due to 5a-dihydrotestosterone binding by albumin; unlike the cytosol receptor, the serum binder cannot be saturated with 1 0 p M 5~-dihydrotestosterone; immature male, female and pseudohermaphrodite male rats which do n o t reveal any cytosol androgen receptor activity show normal serum binding of 5a-[1,2 -3 H2 ] dihydrotestosterone; unlike the cytosol receptor, the serum binding of 5a-[1,2 -3 H2 ] dihydrotestosterone does n o t show any dilution inactivation. It should also be mentioned that a2u-globulin, whose physiological role is still uncertain, does not bind any steroidal c o m p o u n d (Roy, A.K., unpublished). The t w o distinct types of 5a-dihydrotestosterone binding with different affinities for the 3.5-S receptor peak deserve attention. Multiple equilibria in the binding of estradiol to the ovine endometrial cytoplasm [41] and the vault shaped Scatchard plot with uterine estradiol receptor of the calf [42] have been interpreted as indications of cooperative binding interaction between the c o m p o n e n t binding sites of the receptor protein. Extrapolation of the two 5a-dihydrotestosterone binding c o m p o n e n t s in the Scatchard plot of the hepatic androgen receptor activity indicates that for every type-I binding (Kai = 4.5 • 10 -8 M) there seem to be a b o u t 30 type-II bindings (Kaxi = 2.0 • 10 -6 M). The large molar difference between these two types of 5a-dihydrotestosterone binding c o m p o n e n t s may preclude the possibility of two types of binding sites on a single 3.5-S protein. In the absence of more definitive data we are presently inclined to think that the second t y p e of binding (Kay1) may be the result of partial alteration of the receptor molecule due to preparative procedures. Liao and his associates have reported the existence of t w o different 5~-dihydrotestosterone binding proteins (Complex I and Complex II, also called aprotein and ~3protein) in the cytoplasmic fraction of rat ventral prostate and have suggested the precursor--product relationship between these complexes [37,43]. Pending further experimentation, no com-

229 parison between the two types of 5a-dihydrotestosterone binding observed with the liver cytosol and those of a and ~ proteins of Fang and Liao [37] could be drawn. The higher dissociation constants for the hepatic androgen receptor as compared to prostate and seminal vesicle suggest possible tissue differences in the nature of androgen uptake. Since the experimental data for the calculation of the dissociation constants are obtained from binding studies at 0°C on sucrose density gradients, a non-equilibrium situation, we realize that the observed affinities are expected to be lower than the physiological values. Results of our preliminary experiments with the cytosol 5a-dihydrotestosterone binding in the liver extract showed a Kd of 2.4 • 10 -7 M [25], which was based on smaller numbers of experimental points and may have reflected an intermediate value between the two types of 5a-dihydrotestosterone binding. Unlike 5a-dihydrotestosterone binding, the binding of estradiol to cytosol androgen receptor of the liver tissue showed only one binding isotherm. The Scatchard plot showed a straight line and yielded a dissociation constant (K d ) of 3 . 7 . 1 0 -7 M. The same sedimentation coefficients of the estradiol and 5a-dihydrotestosterone binding peak, cross competition between these two steroids for receptor binding, simultaneous absence of estradiol and 5adihydrotestosterone binding peak in immature male as well as in female rats of all ages, and its simultaneous appearance at the time of maturation in the male and with androgenization in the spayed female, all of these data present a strong argument for a single receptor protein containing both estradiol and 5a-dihydrotestosterone binding sites. None of the above results, however, could completely rule out the possibility for separate and distinct 5a-dihydrotestosterone binding proteins within the 3.5-S peak. Extrapolation of the Scatchard plots for estradiol and 5a-dihydrotestosterone binding show that for the same amount of cytosol protein there are 3 moles of 5a-dihydrotestosterone for every mole of estradiol bound at saturation. This observation, along with the differential sensitivity of the estradiol and 5a-dihydrotestosterone binding to cytosol dilution (Fig. 1), suggests that estradiol and 5a-dihydrotestosterone may occupy two distinct and specific types of binding sites on the receptor molecule. Moreover, the structural differences between estradiol and 5a-dihydrotestosterone make them unlikely candidates for the same active site interaction with high degree of specificity. All of the above arguments lead us to postulate that the binding of estradiol to its specific binding site may allosterically prevent the binding of 5a-dihydrotestosterone on the androgen specific binding sites, and, on the other hand, the binding of 5a-dihydrotestosterone only weakly interferes with estradiol binding. In other words, out of the four possible binding sites on the protein, there are three receptor subunits for 5a-dihydrotestosterone binding and one regulatory subunit for the estradiol binding. All of the results obtained so far in our laboratory are consistent with such a hypothesis. It is of interest to note that both epididymal and prostatic cytosol androgen receptors have also been found to bind estradiol [29,44]. Thus it is possible that the estrogenic regulation of the androgen receptor activity may represent a general physiological modulatory process. Unequivocal verification of the above hypothesis largely hinges on our success in receptor purification. Studies on the physiological role of the cytosol androgen receptor activity

230 on the androgen action in the liver tissue have established a direct correlation between the receptor activity and the urinary output of ~2u-globulin. Immature and senescent males as well as females and pseudohermaphrodite male rats which did not produce ~2u-globulin also lacked the cytosol androgen receptor activities in their liver extracts. Examination of the maturing male and androgen-treated female rat liver cytosol showed that the induction of the receptor activity preceded the induction of ~2u-globulin. The androgen insensitivity of the immature male rats with regard to ~2u induction was also found to be due to the inability of the androgens to induce its own receptor activity in the liver tissues of these animals. A rising shoulder of 5~-dihydrotestosterone binding in the 3.5-S region of the gradient in the non-~2~-producing maturing male rats of 25--37 days of age could be seen in Fig. 10. After about 6 weeks of age this shoulder gave rise to distinct receptor peak with subsequent induction of ~2u-globulin. However, prepubertal castration (at 21 days of age) prevented the maturation of the shoulder into a distinct peak, and these animals failed to produce any ~2~-globulin (Fig. 11). Castration of the adult male rats was also found to cause gradual decrease of the cytosol androgen acceptor activity in the liver tissue as well as of urinary ~2~ output. All of these results support the conclusion that the androgen receptor concentration in the hepatic cytosol is under the inductive control of its own ligand. Constitutive appearance of a small amount of receptor associated with sexual maturation, as indicated by the rising shoulder in the 3.5-S region of the gradient (Fig. 10), may bring only enough androgen into the cell to suffice for the purpose of receptor induction. This may explain the shift in androgen sensitivity around the time of puberty. Other alternative possibilities, such as increased androgen permeability or inactivation of certain inhibitors of androgen action at the time of sexual maturation, may also account for the development of androgen sensitivity around puberty. Although the androgen-dependent induction of the androgen receptor has already been reported in the hair follicles [38] and in the prostate gland [45], a recent report by Sullivan and Strott [39] has indicated a rise in the androgen receptor activity per mg prostatic DNA after castration, and they have proposed the androgen-independent mechanism of receptor regulation in the target tissue [39]. Although the results presented in Fig. 10 show an apparent rise in the cytosol 5~-[1,2 -3 H2 ] dihydrotestosterone binding activity at 5 days after castration, we are inclined to think this rise may be due to an increase in the level of only "stripped" receptor; there may have been an actual drop in the "total" receptor ("stripped" receptor + "charged" receptor) concentration under these situations. The estrogenic inhibition of ~2 u synthesis is clearly shown to be mediated through the androgen receptor activity. Following estrogenization of adult male rats there is a gradual decrease and final loss of both the hepatic cytosol receptor activity as well as of ~2 ~ synthesis within 8 days of treatment (Fig. 15). The loss of androgen receptivity after estrogenization is associated with a relatively long period (4--6 weeks with 0.5 pg estradiol/g body wt) of androgen insensitivity in these animals. The estradiol binding activity is also found to be absent within this refractory period. These results indicate that estradiol not only inhibits the uptake of 5~-dihydrotestosterone by the androgen receptor, as shown by the in vitro competition experiments, but it may also inhibit

231 receptor synthesis through its interference with the receptor induction mechanism. It may be of interest to note that the regulation of progesterone receptor concentration in the guinea pig uterus has recently been discovered to be under antagonistic influence of estrogen and progesterone [46]. The lack of the androgen receptor activity in senile male rats correlates well with their inability to synthesize a2u-globulin. Moreover, the absolute androgen insensitivity in these animals is clearly shown to be due to the noninduction of the receptor activity with androgens. A decreased uptake of both progesterone and estradiol by the aging rabbit uteri [47] and aging rat prostate [48] have also been reported. Receptor inactivation associated with senility may therefore represent one of the primary reasons for the gradual loss of reproductive capacity and hormone responsiveness in the aged animals. Although the situation in the senile males is reminiscent of the immature males, it seems unlikely that these two androgen-insensitive conditions share the same mechanism. Whereas the androgen insensitivity in the immature animals may be due to the absence of a critical level of certain developmental signals, in senile rats it may involve an age-dependent formation of less active as well as inactive receptor proteins as indicated by the discovery of certain enzyme alterations in the aging animals [49--51]. The above defect may be complicated by the loss of androgen-dependent regulatory process for receptor induction in these animals. Based on these results, it seems reasonable to conclude that there are at least three, and may be four, important mechanisms that are involved in the control of androgen action in the hepatic cells, all of which seem to act through receptor regulation. These include: (i) certain developmental signals controlling androgen sensitivity via receptor synthesis; (ii) receptor induction by the androgens; (iii) suppression of receptor activity and receptor synthesis by estradiol; and (iv) gradual loss of receptor induction associated with senescence. The observed correlation between the receptor activity and the androgendependent synthesis of a2u-globulin greatly strengthens the validity of the above conclusions. Acknowledgments The authors thank Drs S.C. Brooks, R.E. Buller, B.W. O'Malley and V.N. Reddy for their criticism of the manuscript. Acknowledgment is made to the Gerontology Research Center, National Institutes of Child Health and Human Development, for use of facilities provided under its Guest Scientist Program and we specially thank Dr N.W. Shock for his generous co-operation. The investigation was supported by the United States Public Health Service, N.I.H. research grant AM-14744. References 1 Jensen, E.V. and Jacobson, H.J. (1962) Recent Prog. Ho rmone Res. 18, 387---414 2 Talwax, G.P., Segal, S,J., Evans, A. and Davidson, O.W, (1964) Proc. Natl. Acad. Sci. U.S. 52, 1059--1066 3 Tort, D. and Gorski, J. (1966) Proc. Natl. Acad. Sci. U.S. 55, 1574--1581 4 Korenman, S.G. and Rao, B.R. (1968) Proc. Natl. Acad. Sci. U.S. 61, 1 0 2 8 - - 1 0 3 3 5 Bruchovsky, N. and Wilson, J.D. (1968) J. Biol. C h e m . 243, 2 0 1 2 ~ 2 0 2 1

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