Correlation of hepatic cytosolic androgen binding proteins with androgen induction of hepatic microsomal ethylmorphine N-demethylase in the rat

Previous reports have demonstrated the presence of moderate to high affinity binding for androgens in the cytosol of livers from male rats. This binding was significantly lower in female rats or in immature rats of either sex. The hepatic androgen binding protein, which sedimented at approx. 4 S on sucrose density gradients, has been called a receptor which mediates the actions of androgens in the liver. The experiments in the present study were designed to evaluate the hepatic androgen binding protein for characteristics which have been attributed to receptors in other tissues and to correlate the presence of androgen binding with androgen induction of hepatic drug metabolism. In the current studies, we have shown that cytosol from the livers of male rats bound [3H]dihydrotestosterone [( 3H]DHT) and translocated this steroid ligand to the nucleus in a time and temperature dependent manner. Cytosol prelabeled with [3H]DHT, when passed over a column of denatured DNA cellulose, eluted in three radioactive peaks. Two of these peaks were absent when cytosol from livers of female or hypophysectomized males was used. In addition, the presence of high concentrations of hepatic androgen binding correlated well with the ability of androgen to induce ethylmorphine N-demethylase, a marker of microsomal cytochrome P-450-dependent drug metabolism. Values for both parameters were higher in males than in either females or hypophysectomized males. Testosterone treatment induced both parameters in ovariectomized females and 17 beta-estradiol repressed both in males. However, testosterone treatment failed to induce hepatic androgen binding in hypophysectomized males and immature males, both of which are also unresponsive to androgen induction of drug metabolism. The results suggest that one or more hepatic cytosolic androgen binding proteins possess several characteristics associated with steroid receptors in reproductive tract tissue. Furthermore, this binding may be implicated as a mediator for the androgen induction of at least one component of hepatic drug metabolism.     

Characterization of a [3H]methyltrienolone (R1881) binding protein in rat liver cytosol

The binding of radiolabelled methyltrienolone 17 beta-hydroxy-17 alpha-methyl-estra-4,9,11-trien-3-one (R1881) to adult male rat liver cytosol has been characterized in the presence of Na-molybdate to stabilize steroid-hormone receptors, and triamcinolone acetonide to block progestin receptors. Using sucrose density gradient analysis, male liver cytosol contains a [3H] R1881 macromolecular complex which sediments in the 8-9S region. 8S binding of R1881 to male rat serum, female liver cytosol or cytosol from a tfm rat cannot be demonstrated. Further metabolism of [3H] R1881 following 20h incubation with male rat liver cytosol was excluded: In the 8S region 97% of [3H] R1881 was recovered by thin layer chromatography. Characteristics of this [3H] R1881-8S binding protein include high affinity (Kd = 2.3 +/- 41 nM) and low binding capacity (18.8 +/- 3.3 fmol/mg cytosol protein), precipitability in 0-33% ammonium sulfate, and translocation to isolated nuclei following in vivo R1881 treatment. Whereas, the cytosol R1881-receptor is competed for by dihydrotestosterone, testosterone, and estradiol, [3H] estradiol binding in the 8S region is not competitive with androgens but does compete with diethylstilbestrol. The nuclear androgen binding site has a Kd = 2.8 nM for [3H] R1881, and is androgen specific (testosterone greater than 5 alpha-dihydrotestosterone greater than estradiol greater than progesterone greater than cyproterone acetate greater than diethylstilbestrol greater than dexamethasone greater than triamcinolone). Since a number of liver proteins including the drug and steroid metabolizing enzymes are, in part, influenced by the sex-hormone milieu, the presence of a specific androgen receptor in male rat liver may provide valuable insight into the regulation of these proteins.     

Identification and partial characterization of the cytoplasmic androgen receptor in bovine ovarian capsule

[3H]Dihydrotestosterone (DHT) binding to a specific protein in the cytosol of bovine ovarian capsule was studied in vitro. The specific androgen-binding protein in the cytosol was analyzed by chromatographic and ultracentrifugal techniques. From Scatchard analysis, the dissociation constant was 7.4 nM and the number of binding sites was 58.8 fmol/mg protein. Testosterone and 17 alpha-methyltrienolone (R1881) compete for [3H]DHT binding. In the presence of sodium molybdate and at low salt concentrations, the steroid-protein complex sediments as a 9S form, while in the presence of high salt, it sediments at 3.5S. In the absence of molybdate or in the presence of high salt, the 9S form dissociates in a temperature-dependent manner into smaller units. These properties are consistent with the presence of a typical androgen receptor in the bovine ovarian capsule.     

Interaction of medroxyprogesterone acetate with cytosol androgen receptors in the rat hypothalamus and pituitary

The binding of medroxyprogesterone acetate (MPA) with cytosol androgen receptors from rat pituitary and hypothalamus was studied. The pituitary and hypothalamic cytosol androgen receptors from adult castrated female rats were in vitro labeled using 3H natural (testosterone (T) and 5 alpha-dihydrotestosterone (DHT] and [3H]synthetic (methyltrienolone) androgens as radioligands. The [3H]androgen-receptor complexes sedimented with a coefficient of 8S in linear sucrose gradients. When incubated with an excess of radioinert MPA, specific binding was abolished indicating interaction of MPA with androgen receptors. Furthermore specific [3H]MPA-androgen cytosol receptor complexes could be identified in these neuroendocrine tissues when a post-gradient receptor labeling technique was used in the absence or presence of radioinert MPA, DHT, and triamcinolone acetonide. A study of binding kinetics disclosed that the equilibrium dissociation constant and saturation binding capacity for the MPA binder, were similar to those exhibited by DHT binding to androgen receptors in both studied tissues under identical experimental conditions. The overall results were interpreted as demonstrating that MPA interacts with cytosol steroid receptors other than those of progesterone in the rat hypothalamus and anterior pituitary. The data are consistent with MPA binding to androgen receptors.     

A male specific hepatic estrogen binding protein: characteristics and binding properties

Mammalian liver is a sex-steroid responsive tissue in that androgen and estrogen receptors are present and mediate differential hepatic hormonal effects. Further, we and others have found a sexual dimorphism in the hepatic cytosolic content of estrogen binding proteins. In addition to the estrogen receptor, the male has a high-capacity (12.0-15.0 pmol/mg protein) estrogen binding protein (MEB) which demonstrates a moderate affinity for estradiol (Kd = 31.0-43.2 nM) if estradiol metabolizing enzymes are first precipitated with protamine sulfate. This protein exhibits a unique specificity for steroidal estrogens: 2-methoxyestriol greater than estradiol greater than estriol = 2-methoxyestradiol greater than 2-hydroxyestradiol greater than estrone greater than 2-methoxyestrone greater than estriol 3-glucuronide greater than 2-hydroxyestrone = 3-methoxyestriol greater than androstanediol greater than dihydrotestosterone greater than testosterone. Other androgens such as androstenedione and methyltrienolone, nonsteroidal estrogens such as diethylstilbestrol, and the antiestrogens tamoxifen and 4-hydroxytamoxifen do not compete for [3H]estradiol ([3H]E2) binding. MEB is a relatively small-molecular-weight protein with a Sr of 20.4 A as determined by gel filtration on Sephadex G-100. The kinetics of [3H]E2 association and dissociation at 4 degrees C are very rapid, with t1/2 values of less than 5 s. Sodium molybdate, generally used to stabilize steroid receptors, inhibits MEB-[3H]estradiol binding activity in cytosol in a time- and dose-dependent manner, an effect not observed with partially purified MEB. Magnesium chloride inhibits binding activity of the Sephadex G-100 MEB pool, an effect reversed by EDTA. Other divalent cations also inhibit binding: Mn2+ greater than Mg2+ greater than Ca2+. Furthermore, EDTA complexes of these cations slightly enhance binding relative to EDTA alone: Ca2+ EDTA greater than Mg2+ EDTA greater than Mn2+ EDTA. These results demonstrate that MEB is a unique sex-steroid binding protein, albeit of unknown function, which is distinct from hepatic steroid receptors.     

Purification and immunochemical characterization of the cytoplasmic androgen-binding protein of rat liver

The cytoplasmic androgen-binding (CAB) protein of the male rat liver has been implicated to play a role in the androgen-dependent regulation of alpha 2u-globulin synthesis. The liver of the adult male rat contains about 50 fmol of specific high-affinity androgen-binding activity per milligram of total cytosolic protein. Photoaffinity labeling with [3H]R-1881 followed by SDS-polyacrylamide gel electrophoresis and autoradiography shows that the CAB is a 31-kilodalton protein. By means of DEAE-cellulose chromatography and preparative SDS-polyacrylamide gel electrophoresis, we have purified the CAB protein to electrophoretic homogeneity and have raised polyclonal rabbit antiserum that is monospecific to this protein. In the sucrose density gradient, the antiserum reacted with the androgen-binding component of the male liver cytosol prelabeled with tritiated dihydrotestosterone. Western blot analysis of the liver cytosol showed that the antiserum recognizes only the 31-kDa androgen-binding component. Such immunoblotting also showed that unlike the young adult, the androgen-insensitive states during prepuberty and senescence are associated with a marked reduction in the hepatic concentration of the immunoreactive CAB protein. No immuno-chemical cross-reactivity between CAB and another androgen-binding component of Mr 29K (which is associated with androgen insensitivity during prepuberty and senescence) was observed. The latter finding favors the possibility that 31- and 29-kDa androgen-binding components may have distinct sequence structure.     

Intracellular Ca2+ stimulates the binding to androgen receptors in platelets

In this study, we demonstrated that ADP-induced platelet aggregation activates the binding of testosterone (T) to its receptor. It is well known that binding of ADP to its receptors induced the release of Ca2+ ions from dense bodies into the cytosol of platelets. In this work, we compared the binding of testosterone or dihydrotestosterone to their receptors using cytosol obtained from ADP-treated and non-treated platelets. These experiments were repeated using EGTA (a calcium chelator) or U73122 (a phospholipase C enzymatic activity inhibitor) to the ADP-treated platelets. In addition, we also developed a competition analysis for the androgen receptors (AR) using [3H]DHT, non-radioactive T, DHT or cyproterone acetate from ADP-treated platelets cytosol. The results from this study indicate that the cytosol obtained from non-ADP-treated platelets did not show any binding to [3H]T or [3H]DHT, whereas cytosol from ADP-treated platelets binds to the radio-labeled androgens. Furthermore cytosol from ADP plus U73122-treated platelets did not show binding to [3H]T or [3H]DHT. These data suggest that intracellular Ca2+ ions stimulates the binding of androgens to their receptors in platelets cytosol. The competition analysis shows that T and DHT have high affinities for the androgen receptors with similar IC50 values, whereas cyproterone acetate shows a lower affinity. The results from these data clearly indicate the presence of androgen receptors in platelets.     

Relative binding affinity of novel steroids to androgen receptors in hamster prostate

The in vivo and in vitro antiandrogenic activity of four aromatic esters 10a-10d, one aliphatic ester 10e based on the pregna-4,16-diene-6, 20-dione structure and two aromatic 17c, 17d and two aliphatic valeroyloxy esters 17a, 17b based on the more saturated 4-pregnene-6,20-dione skeleton was examined. The biological activity of steroids 9, 10a-10e and 17a-17d, was determined using prostate glands from gonadectomized adult male golden hamsters. In the in vitro studies, the relative binding affinity of these steroids to cytoplasmic androgen receptor (AR) of hamster prostate was determined from, the corresponding IC50 values obtained from the competitive binding plots. The standards dihydrotestosterone (DHT) and cyproterone (CA) acetate used have displaced [3H]DHT from the AR with an IC50 value of 3.2 and 4.4 nM respectively. All steroidal compounds synthesized in this study showed a binding affinity for the androgen receptor, present in the cytosol from prostate hamster; compounds 10a-10c showed the highest affinities for this receptor. The in vivo experiments showed that all steroidal derivatives were subcutaneously active, since they decreased the weight of the prostate gland in gonadectomized hamsters treated with DHT, and are antagonists for the androgen receptor since they block the DHT-induced prostate weight gain. The derivatives having the more conjugated 4,16-pregnadiene-6, 20-dione system (10a-10c) exhibited a higher antiandrogenic activity than the corresponding steroids (17a-17d) based on the more saturated 4-pregnene-6,20-dione system.     

Modulation of steroid affinity for the androgen receptor by sucrose

The effects of sucrose on androgen binding to its receptor were investigated. Sucrose decreased the rate of thermal inactivation of unoccupied and occupied androgen receptor (AR) and the rates of [3H]5 alpha-dihydrotestosterone [( 3H]DHT) dissociation from both activated and nonactivated AR complexes. Binding of [3H]DHT to AR in vivo, or in intact cells at 37 degrees C, caused reduction of [3H]DHT dissociation from cytosolic and nuclear complexes, as compared to in vitro labeled receptor complexes. Further, exposure of these complexes to sucrose at 0 degrees C caused an additional reduction of dissociation rates. Thus, the decrease of [3H]DHT dissociation induced by sucrose is independent of the reaction that reduces DHT dissociation from activated and transformed AR. Sucrose also reduced the ability of mersalyl acid to inactivate AR complexes. This effect of sucrose was markedly diminished in the presence of 2M urea. Sucrose did not significantly affect the association rate, sedimentation properties, or nuclear binding ability of AR complexes, but it did decrease the equilibrium dissociation constant. Other monosaccharides and disaccharides also stabilized AR. These data suggest that sucrose induces conformational changes in the steroid binding domain of androgen receptor, thereby reducing the rates of inactivation, steroid dissociation, and the accessibility of sulfhydryl groups to mersalyl.     

Steroid metabolism and binding activity in a murine renal tumor cell line

The purpose of this study was to partially characterize the steroid binding activity of murine renal tumor cells in continuous culture. The steroid receptor content of a cloned renal tumor cell line (RAG) and a subline RAG-2 was examined by sucrose gradient analysis, hydroxylapatite and dextran-coated charcoal methods. The RAG cells lacked estrogen- and progestin-binding activity, whereas specific 5 alpha-dihydrotestosterone (DHT) and dexamethasone (Dx) binding activities were detected as 8S peaks on low salt gradients. The specificity of DHT binding was examined by sucrose gradient analysis: DHT, R1881 and ORG2058 all completely inhibited [3H]DHT binding whereas diethylstilbestrol and Dx were ineffective. The androgen receptor content of the RAG cells was approx. 15 fmol/mg cytosol protein by the hydroxylapatite-filter assay, with an estimated Kd for methyltrienolone (R1881) of 5 nM at 0 degrees C. Scatchard analysis of [3H]Dx binding by RAG cytosol showed a Kd of 6 nM for Dx and 44 nM for corticosterone at 0 degrees C. Glucocorticoid receptor levels were estimated to be 182 fmol/mg cytosol protein by dextran-coated charcoal assay. Metabolism of [3H]testosterone and [3H]DHT by RAG cells was examined 1, 4 and 6 h after exposure to labeled hormone. Radioactive DHT was the primary intracellular metabolite recovered after exposure to [3H]testosterone. There was little conversion of DHT to androstanediol.     

Demonstration and partial characterization of cytosol receptors for testosterone.

Androgen uptake was investigated in several peripheral organs after administration of (1,2,6,7 minus -3H)testosterone to castrated male rats. The animals were killed after 30 min, the organs were taken out, and the radioactivity was determined after tissue combustion. A relatively high accumulation of androgen was found in pancreas, adrenals, spleen, thigh muscle, kidneys, and liver in addition to the classical androgen target organs coagulation glands, seminal vesicles, prostate, preputial glands, and harderian glands. In a second serier of experiments, nuclear and cytosol fractions were prepared from prostate, seminal vesicles, coagulation glands, preputial glands, spleen, submaxillary glands, kidneys, and pancreas from castrated male rats give (1,2,6,7 minus -3H)testosterone, and these fractions were then characterized by thin-layer and radio-gas chromatography with respect to their patterns of labeled steroids. Only prostate and seminal vesicles were found to contain significant amounts of nuclear 5alpha-(-3H)dihydrotestosterone. The major nuclear androgen was (-3H)testosterone that was the only detectable labeled steroid in coagulation glands, preputial glands, and spleen and that constituted 70% or more of the nuclear radioactivity in seminal vesicles, submaxillary glands, kidneys, and pancreas. These results indicate that testosterone itself may be the predominant active androgen principle in vivo in most androgen target organs and that conversion to 5alpha-dihydrotestosterone is generally not a prerequisite for androgen activity. Using an ultrasensitive micromodification of isoelectric focusing (cf. M. Katsumata and A. S. Goldman (1974), Biochem. Biophys. Acta 359, 112. It was possible to show that cytosol from kidney; submaxillary gland, thigh muscle, and levator ani muscle and nuclei from kidney and submaxillary gland contained androgen-binding proteins with pI's in the region 4.6-5.1 ("4.6 minus 5.1 Complex"). This complex also formed in vitro after incubation of (1,2,6,7 minus -3H)testosterone with cytosol from kidney and submaxillary gland. (1,2,6,7 minus -3H)Testosterone was bound with high affinity to receptor proteins in cytosol from both kidney, submaxillary gland, and thigh muscle with dissociation constants of 5.0 x 10 minus -12 M (kidney), 3.3 x 10 mi;nus -11 M and 4.1 x 10 minus -10 M (two types of binding sites, submaxillary gland), 2.4 x 10 minus -12 M (thigh muscle) and 1.9 x 10 minus -12 M (levator ani muscle). The number of binding sites was in all cases between 1 and 20 fmol/mg of protein. On the basis of these results the hypothesis is presented that a common class of testosterone receptors is present in most organs and that these receptors can be detected both in vivo and in vitro provided methods sensitive enough are utilized.     

Cortisol-binding components of cytosol receptor in normal and adrenalectomized rat liver.

[3H] cortisol was found to be bound to two distinct components of receptor protein in hepatic cytosol of both normal and adrenalectomized adult male rats. In liver cytosol of normal rats, 78% of total bound [3H] cortisol to receptor was associated with the first receptor component, and about 20% to the second one. However, the first component of cytosol receptor in adrenalectomized rats was found to bind 95% of total [3H] cortisol bound to both receptor components, while only 4.5% of the bound hormone was found associated with the second receptor component. These alterations in binding capacity are discussed in relation to possible regulation of nuclear RNA synthesis by cortisol-receptor complexes.     

Cytoplasmic androgen binding protein of rat liver: molecular characterization after photoaffinity labeling and functional correlation with the age-dependent synthesis of alpha 2u-globulin

The liver of the mature male rat contains a moderate affinity (Kd = 10(-8)M), low-capacity, cytoplasmic androgen binding protein (CAB) whose appearance during puberty and disappearance during senescence correlate with the androgen-dependent synthesis of alpha 2u-globulin. Molecular properties of CAB were examined by photoaffinity labeling with tritiated methyltrienolone (R-1881), a synthetic androgen, and by its localization within the hepatocytes which are competent to produce alpha 2u-globulin. Photoaffinity labeling of the liver cytosol derived from postpubertal male rats, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, showed a predominant androgen binding band corresponding to Mr 31,000. This 31-kilodalton (kDa) binding component was conspicuously absent in the liver of androgen-insensitive prepubertal and senescent male rats and in adult male rats treated with estradiol-17 beta. In addition, unlike the cytoplasmic extract, the nuclear lysate of the male rat hepatocytes did not contain the 31-kDa androgen binder. Disappearance of the 31-kDa androgen binding band from the cytosolic fraction of androgen-insensitive animals was associated with a concomitant appearance of a minor androgen binding component of apparent Mr 29,000. The livers of postpubertal male rats normally contain two subpopulations of hepatocytes, only one of which is highly active (competent) in alpha 2u-globulin synthesis. Separation of these two subpopulations through a fluorescence-activated cell sorter followed by whole cell labeling showed more than a 2-fold higher uptake of R-1881 by the competent cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Androgen binding in peripheral tissues of fetal rhesus macaques: effects of androgen metabolism in liver

In rhesus monkeys sexual differentiation of the brain and reproductive tract (RT) is androgen-dependent. Presumably these effects are mediated through the androgen receptor (AR). The AR has not been characterized in fetal tissues such as liver, kidney, heart, spinal cord and RT in this species. We characterized AR binding using [3H]R1881 as the ligand in cytosols from tissues obtained on days 100-138 of gestation. Scatchard analyses revealed a single, saturable, high affinity AR in liver, kidney, heart, spinal cord and RT. The apparent dissociation constant (Kd) ranged from 0.52 to 0.85 nM with no significant tissue differences. The number of AR (Bmax; fmol/mg protein) differed significantly (P less than 0.01) between tissues (liver greater than RT much greater than kidney greater than or equal to heart greater than or equal to spinal cord). Radioinert testosterone (T) and 5 alpha-dihydrotestosterone (DHT) but not androstenedione, progesterone, estradiol-17 beta, estrone or cortisol in a 50-fold molar excess inhibited [3H]R1881 binding to the AR in spinal cord, heart, kidney and RT. However, in liver only DHT competed significantly (P less than 0.01) for binding. This difference in binding of DHT vs T in the liver was further investigated by incubating liver and kidney cytosols with [3H]DHT and [3H]T at 4 degrees C. We identified the metabolic products by mobility on Sephadex LH-20 columns and reverse isotope dilution. Liver cytosols metabolized [3H]DHT to 5 alpha-androstane- 3 alpha,17 beta-diol (5 alpha-diol) and [3H]T to 5 beta-androstane-3 alpha, 17 beta-diol (5 beta-diol) at 4 degrees C. In contrast, kidney cytosols metabolized [3H]DHT while [3H]T remained unchanged. Further studies indicated that a 50-fold molar excess of 5 alpha-diol inhibited the binding of [3H]R1881 in liver cytosols by about 50% whereas the same molar concentration of 5 beta-diol had no effect. These data demonstrate the presence of AR in peripheral tissues of fetal rhesus monkeys and suggest that androgens through their receptors may affect development of these tissues. Liver cytosols are capable of metabolizing T and DHT at 4 degrees C at conditions similar to those used for measuring cytosolic AR. However, T and DHT are metabolized differently, generating different isomers which have different affinities for hepatic AR.     

Androgen receptors in brain and pituitary of female rats: cyclic changes and comparisons with the male

The in vitro binding of a synthetic androgen, methyltrienolone ([3H]-R1881), to brain and pituitary (PIT) cytosol and nuclear extracts was determined in male and female rats. Purified cytosol was prepared from PIT or hypothalamic-preoptic area-amygdala (HPA) and incubated in the presence of 0.1 to 10 nM [3H]-R1881. Scatchard analysis revealed the presence of a single, saturable, high-affinity binding site in PIT cytosol with a dissociation constant (Kd) of 0.42 X 10(-10) M in females and 0.95 X 10(-10) M in intact males. The Kd of HPA cytosol was much less in castrated males [0.47 +/- 0.05 (SEM) X 10(-10)M, n = 7] and females (0.63 +/- 0.1 X 10(-10) M, n = 4) than in intact males (5.8 +/- 1.1 X 10(-10) M, n = 8). Treatment of castrated males with dihydrotestosterone (DHT) for 24 h (250 micrograms/100 g of body weight) increased the Kd of HPA cytosol only slightly (1.6 X 10(-10) M, mean of two replicates). Scatchard analysis of salt-extracted nuclear androgen receptor (ARn) showed a single, high-affinity binding site with similar Kd values in PIT and HPA of intact and castrated, DHT-treated male rats (PIT Kd = 7.3 X 10(-10) M, 9.3 X 10(-10) M; HPA Kd = 1.5 X 10(-9) M, 1.3 X 10(-9) M, respectively). Competition studies involving a range of several radioinert steroids revealed that the binding of [3H]-R1881 to cytosol (ARc) and nuclear extract was specific for androgen receptor when triamcinolone acetonide (10 microM) was added. The ARc and ARn levels were quantified in PIT, preoptic area (POA), hypothalamus (HT), amygdala, hippocampus, and cortex by single point estimation. Significantly (p less than 0.01) greater amounts of ARc were detected in PIT of ovariectomized females (32.7 +/- 2.9 fmol/mg of protein) than in that of orchidectomized males (22.33 +/- 1.6 fmol/mg of protein). The highest levels in the brain were seen in HT and POA. Pituitary ARc in females varied throughout the estrous cycle. Significantly (p less than 0.01) greater amounts were detected on estrus (45.8 +/- 2.2 fmol/mg of protein) and proestrus (39.0 +/- 1.9 fmol/mg of protein) than on diestrus (29.2 +/- 1.5 fmol/mg of protein). These data confirm the existence of specific receptors for androgen in male and female brain and PIT, and suggest an important role for androgen in the control of PIT hormone secretion in the female.     

Ontogeny of an 8S androgen binding protein in rat liver cytosol

Studies were performed to elucidate the ontogeny of a single class of androgen binding protein in male rat liver cytosol which exhibits characteristics of a ligand specific, high affinity (Kd = 2.3 nM), 8S-receptor capable of nuclear translocation. Detectable levels of receptor first appear at 45 days of age in the male and reach maximum concentration at 65 days. Barely detectable levels are seen in females throughout the duration of study (80 days). Gonadectomy in both sexes (65 days) and androgen treatment of oophorectomized females do not alter the normal development of sexual differentiation of the high affinity androgen receptor. After neonatal castration (2 days) and DES replacement however, receptor sites do not undergo differentiation and adult males exhibit female levels. Conversely, neonatal androgen replacement in 2-day castrates partially restores the level of binding sites to control males values (TP, 71%; DHT, 51%). Neonatal castration without replacement retards but does not fully eliminate sexual differentiation of levels of receptor sites in adult males. Likewise, neonatal androgen treatment in females results in a partial masculinization of binding sites. Following hypophysectomy, levels of receptor sites in females are similar to intact or hypophysectomized males; sexual differences in the adult are abolished. These studies suggest that sexual differentiation of specific liver cytosol androgen binding sites in the adult may be partially programmed at birth by testicular androgen and furthermore, adult sexual dimorphism is maintained through an inhibitory influence of the pituitary in the female.