Androgen receptors in ventral prostate glands of zinc deficient rats

Androgen binding has been studied in the prostate cytosol of zinc deficient rats by charcoal assays. Rats were housed individually in plastic cages and maintained on a zinc deficient diet for 3 months. The cytosol fraction of prostate gland was incubated with various concentrations of tritiated methyltrienolone (3H-R1881, a synthetic androgen) alone or in the presence of 500-fold excess of radioinert R1881. Scatchard analysis of the data revealed that the number of androgen binding sites in the cytosol fraction of the zinc deficient rat prostate was 31 +/- 5.2 fmol/mg cytosol protein. This was significantly lower than that (84 +/- 11.5 fmol/mg protein) of the controls. Their dissociation constant (Kd = 1.6 +/- 0.6 nM) on the other hand was not different from that (1.7 +/- 0.7 nM) of control animals. This decrease in the concentration of cytosol receptor sites in the zinc deficient state suggests that this metal is involved in the androgen-binding process in the target cells.     

Androgen receptor in human placental villi

In studies with a synthetic androgen, R 1881, an androgen-binding component was found in the cytosol of human placental villi. Kinetic analysis indicated that the Kd value of this component was 1.4 nM at 0-4 degrees C and that binding of R 1881 amounted to 277 +/- 73 fmol/mg protein. glycerol density gradient ultracentrifugation showed a peak of binding activity in the 8S region in a medium of low ionic strength, but in the 4.5S region in a medium containing 9.5 M KCl. The R 1881-binding component was inactivated by mild heat- or trypsin-treatment, but not by treatment with DNase or RNase. Most of the R 1881-binding activity was sedimented at 20 to 40% saturation of ammonium sulfate. These findings indicate that the R 1881-binding component in human placental cytosol is quite similar in its characteristics to androgen receptors, which are present in various androgen-responsive organs. Testosterone was a more potent competitor of R 1881-binding than DHT or cyproterone acetate. Scatchard plots indicated that the binding site of testosterone was identical with that of R 1881. These findings suggest that the androgen receptor in placental cytosol is specific for testosterone. The Kd value for testosterone was calculated to be 3.2 nM.     

Binding of [3H]methyltrienolone to androgen receptor in rat liver

The synthetic androgen methyltrienolone is superior to testosterone and androstenedione for the measurement of androgen receptor in tissues where the native ligands are metabolized into inactive derivatives. [3H]Methyltrienolone binds with a high affinity to androgen receptor in cytosol prepared from male rat livers, as the Scatchard analysis revealed that the Kd value was 3.3 X 10(-8) M and the number of binding sites was 35.5 fmol/mg protein. Since methyltrienolone also binds glucocorticoid receptor which exists in rat liver, the apparent binding of androgen receptor is faulty when measured in the presence of glucocorticoid receptor. The binding of methyltrienolone to glucocorticoid receptor can be blocked by the presence of a 100-fold molar excess of unlabeled synthetic glucocorticoid, triamcinolone acetonide, without interfering in its binding to androgen receptor, because triamcinolone does not bind to androgen receptor. Triamcinolone-blocked cytosol exhibited that the Kd value was 2.5 X 10(-8) M and the number of binding sites was 26.3 fmol/mg protein, indicating a reduction to 3/4 of that in the untreated cytosol. The profile of glycerol gradient centrifugation indicated that [3H]methyltrienolone-bound receptor migrated in the 8-9 S region in both untreated and triamcinolone-blocked cytosols, but the 8-9 S peak in triamcinolone-blocked cytosol was reduced to about 3/4 of that of untreated cytosol.     

Growth hormone receptors in ovary and liver during gametogenesis in female rainbow trout (Oncorhynchus mykiss).

Changes of growth hormone receptivity in the ovary during the reproductive cycle were studied in rainbow trout (Oncorhynchus mykiss). A method for characterizing growth hormone receptors in crude ovary homogenate was required for this. Binding of radiolabelled recombinant rainbow trout growth hormone (125I-labelled rtGH) to crude ovary preparation was dependent on ovarian tissue concentration. The sites were specific to growth hormone, with no affinity for prolactins and gonadotrophins. Similar high affinities for 125I-labelled rtGH were obtained with crude ovary (4.2 x 10(9) +/- 0.3 mol l-1) and crude liver preparations (4.9 x 10(9) +/- 0.1 mol l-1) at all stages of ovogenesis, and with ovarian membrane preparations (8.2 x 10(9) mol l-1) tested at the beginning of vitellogenesis. Ovarian growth hormone receptor concentration was highest during the early phases of follicular development (endogenous vitellogenesis: 315-310 fmol g-1 ovary) and decreased regularly during oocyte and follicular growth (exogenous vitellogenesis) to reach a minimal value at oocyte maturation (42 fmol g-1 ovary). In postovulated fish, binding was at a similar level (297 fmol g-1 ovary) to that found in endogenous vitellogenesis. Conversely, the absolute binding capacity of the whole ovary was low from immaturity to early exogenous vitellogenesis (0.1-0.6 pmol per pair of gonads), increased slowly during vitellogenesis and more markedly during rapid oocyte growth and at the time of final maturation (10.8 pmol per pair of gonads). In postovulated fish, the absolute binding capacity decreased partially (4.4 pmol per pair of gonads). Mean hepatic growth hormone receptor concentration did not vary with the reproductive stage for most of the cycle (3.0-4.5 pmol g-1 liver) except in endogenous vitellogenesis where significantly higher concentrations were observed (6.7 pmol g-1 liver). Individual ovarian growth hormone receptor concentrations were correlated with hepatic growth hormone receptor concentrations, indicating that they are regulated in a similar way. We conclude that growth hormone receptors are present in the ovary during the entire ovarian cycle in rainbow trout, probably mainly in somatic cells as indicated by the same concentration of binding sites in immature and in postovulated fish. Growth hormone is potentially important during oocyte recruitment in vitellogenesis and initiation of growth and during final follicular maturation.     

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.     

Increase in testicular androgen receptor during sexual maturation in the rat

Androgen receptor concentration was measured by exchange with 3H-dimethylnortestosterone (DMNT) in cytosol and nuclear extracts from testes of rats 15-90 days of age. Dissociation kinetics verified the necessity of an extended incubation (86 h) for maximum exchange at 4 degrees C. Nuclear androgen receptor concentration per mg DNA decreased between 15 and 25 days of age, from 375 to 146 fmol per mg DNA, then increased to 584 fmol per mg DNA at 90 days. Testicular receptor content also increased between 25 and 90 days of age. Cytosol receptor concentration patterns were similar to nuclear androgen receptor patterns. The affinity of the receptor for the ligand did not change with age (mean Kd = 0.88 nM). No significant difference in androgen receptor concentration per cell was detected between cultured peritubular cells from animals 25 and 45 days of age. Androgen receptor concentrations in freshly isolated peritubular cells could not be determined. There also was no difference in receptor concentration per cell in a Leydig cell-enriched fraction from animals between 25 and 45 days of age. Although androgen receptor concentrations per Sertoli cell increased between 15 and 35 days of age, the increase in Leydig cell number over the same period probably accounted for approximately 75% of the increase in receptor per testis between 25 and 45 days of age.     

Characterisation and covalent labeling of the human placental methyltrienolone binding protein

Human placental cytosol contains an androgen binding protein which binds the synthetic androgen methyltrienolone (R 1881) with high affinity (Kd 8.7 nM) and with an average binding capacity of 518 fmol/mg cytosol protein. This study provides further evidence that this protein is distinguishable from classical androgen receptors on the basis of steroid specificity and sulphydryl group sensitivity. Covalent labeling studies have shown this protein, which we have called "the methyltrienolone binding protein", to have a mol. wt of 67,000 daltons.     

Oestrogen receptor of calf mammary gland. Purification by use of sodium bromide and heparin-sepharose.

1. Calf mammary-gland cytosol apparently has a single oestrogen receptor capable of auto- and/or hetero-association of varying complexity. Computation of the dissociation constant for oestradiol-17beta gives Kd = 0.5 nM. The number of binding sites is 40 fmol/mg of cytosol protein. The oestrogen receptor in the presence of NaBr, a chaotropic salt that inhibits the interaction of receptor with other cytosol components, sediments through sucrose density gradients as a single sharp peak at 4S, and it has a Stokes radius of 3.4 nm measured by gel filtration. 2. A large-scale purification procedure of the calf mammary-gland oestrogen receptor based on the inhibition of receptor aggregation by NaBr and interaction with heparin-Sepharose is reported. The receptor is purified more than 1500-fold over that in the 27,000g supernatant of the homogenate, with a 30% yield. In 'low-salt' buffer the purified receptor sediments through sucrose gradients at 4S and the Stokes radius, measured by gel filtration in the presence of heparin, is 3.4 nm. The mol.wt computed from these values is about 60,000, and the frictional ratio is 1.3.     

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.     

Characteristics of cytosol androgen receptor in rat thymus

Male and female rat thymic cytosol contained specific androgen receptor. The apparent dissociation constants (Kd) were 2.4 nM in males and 2.5 nM in females, and the number of binding sites (NBS) were 23.7 fmol/mg protein in males and 34.2 fmol/mg protein in females. Transformation of receptor to the DNA binding state was achieved by heat or KCl treatment of [3H]R1881-receptor complex, and the characteristics of transformed and nontransformed receptors were investigated. The nontransformed androgen-receptor complex eluted at 0.20-0.25 M KCl from DEAE-Sephacel and sedimented at 9.1 S and its molecular weight was 255,000 on agarose gel chromatography, while the transformed receptor complex eluted at 0.03-0.15 M KCl with a broad peak and sedimented at 4.5 S and its molecular weight was 80,000-85,000. The minicolumn binding assay revealed that approximately 57% of the total receptor complexes bound to DNA-cellulose following heat treatment (20 degrees C, 1 h). Castration exerted no effect on the physicochemical properties of cytosol androgen receptor, but it increased the number of binding site to the female level.     

In vitro effects of beta-endorphin on testicular release of androgens in the lizard podarcis sicula raf

The effects of the proopiomelanocortin-derived opioid peptide, beta-endorphin (β-EP), and of the opioid antagonist, naloxone (NAL), on both basal and pituitary-stimulated androgen secretion from superfused quiescent and active testes were assessed in the adult lizard, Podarcis sicula. In the absence of the homologous pituitary, in vitro treatment with β-EP and/or NAL did not affect basal secretion of androgens from quiescent and active testes. Conversely, in the presence of the homologous pituitary, treatment with β-EP brought about a decrease in androgen secretion in active testes, but no effect on quiescent ones Naloxone counteracted the inhibitory effect of β-EP in active testes, and enhanced maximal pituitary-stimulated secretion of androgens in quiescent but not in active testes. The effects produces by β-endorphin and naloxone were reversible. These results suggest that, in this lizard, opioids might be involved in the control of androgen release. The lack of effect of β-EP and naloxone when added directly to the testes seems to suggest that the opioid agonist and antagonist act on androgen release by modulating pituitary gonadotrophin output. © 1996 Wiley-Liss, Inc.     

Evidence for androgen receptors in sexually dimorphic perineal muscles of neonatal male rats. Absence of androgen accumulation by the perineal motoneurons

During development, survival of the sexually dimorphic spinal nucleus of the bulbocavernosus (SNB) and its target perineal muscles, the bulbocavernosus (BC) and the levator ani (LA) is androgen-dependent. To define androgen's site of action in masculinizing SNB system structures, we examined whether or not androgen receptors are present in SNB motoneurons and/or BC/LA muscles of neonatal male rats. Using a receptor binding assay, we have identified androgen-binding factors in the neonatal BC/LA (Bmax = 13.5 fmol/mg protein; Kd = 4.69 nM) for the first time. In contrast, androgen autoradiography provided no evidence that neonatal spinal motoneurons accumulate androgens. These results support the hypothesis that BC/LA muscles are a primary site of androgen action for masculinizing SNB system structures, and that androgen need not interact with SNB motoneurons directly to sexually differentiate them.     

Characteristics of a testosterone-estradiol binding globulin (TEBG) in goldfish serum

Neuroendocrine tissues in teleost fish aromatize androgen to estrogen at extraordinarily high rates. As part of a project in which we are studying the dynamics of sex steroid uptake, metabolism, and receptor binding in goldfish (Carassius auratus) brain and pituitary, we have identified and characterized a sex-steroid-binding component of serum. This protein has been designated a testosterone-estradiol-binding globulin (TEBG) since it bound testosterone (T) and estradiol-17 beta (E2) with high affinity (Kd = 1.9 and 2.1 nM, respectively) whereas other steroids were less effective ligands (5 alpha-dihydrotestosterone greater than progesterone = 11-ketotestosterone greater than estrone = estriol = diethylstilbestrol greater than cortisol). Scatchard analysis, disc gel electrophoresis and sucrose gradient centrifugation all indicate that T and E2 are bound by the same protein. The number of available serum binding sites (Bmax = 10(-7) M) greatly exceeded reported maximal levels of T and E2 in the same species and showed no obvious sex or seasonal differences. However, the steroid-TEBG interaction was unstable, exhibiting very short half-times of association (less than 15 min) and dissociation (less than 10 min). On the basis of comparison of the physicochemical characteristics of TEBG with other intracellular androgen-binding proteins in goldfish brain (androgen receptor, aromatase), we predict that the serum-binding protein would not limit but rather enhance exchange of T and E2 in central tissues.     

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.     

Explanation of the pseudohypoaldosteronism (PHA)-stress syndrome with an artificial aldosterone receptor model

A receptor for aldosterone was studied in the cytosol of rectal mucosa of two sisters (M.A., M.B.) with the clinical manifestations of pseudohypoaldosteronism (PHA). Compared to age matched controls the patients showed a decreased affinity for aldosterone (M.A. Kd1: 0.18 nM, Kd2: 4.55 nM; Nmax1: 0.185 fmol/mg cytosol protein (CP), Nmax2: 3.12 fmol/mg CP, respectively). In an attempt to find an explanation for the phenomenon of stress-induced electrolyte imbalance in PHA patients an experimental set up was designed, using aldosterone antibody material as artificial aldosterone receptor. Specific binding was evaluated in addition with and without a 25-100-fold molar excess of dexamethasone (DEX) in order to overcome the glucocorticoid affinity of the aldosterone receptor, a phenomenon proposed to be the cause for the severe consequences of stress in some patients with PHA. The aldosterone antiserum showed two binding sites, similar to the natural receptor (Kd1: 0.15 nM, Kd2: 1.30 nM; Nmax: 30 fmol/mg CP and 130 fmol/mg CP, respectively). Under the influence of DEX the high affinity binding site (Kd1) was occupied by the glucocorticoidanalogon (Kd: 1.30 nM; Nmax: 125 fmol/mg CP). In conclusion, in stress situations, with increased quantities of glucocorticoid circulating, the high affinity binding site of the aldosterone receptor might be occupied by the glucocorticoids, while the low affinity binding site in PHA patients might not have sufficient binding capacity to maintain the electrolyte balance.     

Assay of primate seminiferous tubule androgen receptors using [3H]mibolerone

The synthetic radiolabelled androgen mibolerone (7 alpha, 17 alpha-dimethyl-19-nortestosterone) was used to characterize androgen receptor binding in the seminiferous tubules from Cynomolgus monkey testis. Mibolerone binding was of high affinity (Kd = 0.6-5.4 nM) and limited capacity (37-50 fmol/mg protein), and was androgen specific. Sucrose density gradient centrifugation using a vertical tube rotor permitted the identification of a 9S molybdate-stabilized receptor under low salt conditions. The receptor bound to DEAE-cellulose. Methyltrienolone, but not mibolerone, also bound to a low affinity high capacity binding site in tubule cytosol, which probably represents glucocorticoid receptor binding, since it could be displaced by excess dexamethasone. However, occupancy of this low-affinity binding site by dexamethasone in an androgen receptor assay with [3H]methyltrienolone lead to a 33% underestimation of receptor binding, which appeared to relate to radioactive decomposition. Mibolerone, as well as methyltrienolone, bound to a progestin-binding protein in seminiferous tubule cytosol. These studies provide methods for the study of seminiferous tubule androgen receptors in subhuman primates and indicate that, due to its greater stability and lack of binding to glucocorticoid receptor, mibolerone is a useful new ligand in the study of androgen receptors in testis and its constituent cells.     

Androgen receptor in rat liver: characterization and separation from a male-specific estrogen-binding protein

Many liver processes are sexually dimorphic. In particular, the microsomal content of specific enzymes and the synthesis of specific proteins are under sex steroid hormone control. Because the liver of male rats is strikingly androgen responsive, we sought evidence for an androgen receptor in this tissue. We detected and characterized both cytosolic and nuclear androgen-binding proteins. Both forms bind [3H]R1881 (methyltrienolone, 17 beta-hydroxy-17 alpha-methyl-4,9,11-estratriene-3-one) with the high affinity, low capacity, and specificity for androgens and antiandrogens characteristic of androgen receptors. No high-affinity binding of [3H]DHT could be detected in unfractionated cytosol because of the rapid metabolism of this ligand; however, binding of a DHT metabolite to the high-capacity male-specific estrogen binder (MEB) of cytosol was observed. Both gel filtration and heparin-Sepharose affinity chromatography separate the cytosolic androgen receptor from MEB. Incubation of cytosol in the absence of sodium molybdate resulted in androgen-binding activity which was retained by DNA-cellulose. Castration of male rats results in a time-dependent loss of both cytosolic and nuclear androgen binding, as well as a loss in MEB activity. Androgen-binding activity is low in livers from female rats, but can be induced by testosterone treatment. An intact pituitary is necessary for maintenance of androgen-binding activity, as hypophysectomy results in complete loss of activity.     

Characterization of estrogen binding proteins in mouse liver cytosol: absence of sexual dimorphism

Estrogen binding proteins in mouse liver cytosol were characterized by separation on Sephadex G-75 columns, by Scatchard plot analysis, and by hormonal competition studies. A high affinity receptor (56-70 fmol/mg cytosolic protein) with a mol. wt greater than 75,000, Kd of 5.7-8.4 X 10(-10) M was identified in male and female C3H liver. A second high capacity low affinity (HCLA) binder (200-300 fmol/mg cytosolic protein) with a mol. wt of about 50,000, Kd of 1.7-7.2 X 10(-8) was also identified. Following partial purification of the estrogen binders by ammonium sulfate precipitation, Scatchard plot analysis revealed selective removal of HCLA. On Sephadex G-75 filtration, the purification also resulted in selective removal of the 17 beta-estradiol binding component with a mol. wt of 50,000. Comparison with rat cytosol separations show that the sexual dimorphism in HCLA binding proteins (5 times higher in male than female rat liver) was absent in the mouse liver. These studies document the presence of a specific high affinity estrogen binding protein in mouse liver and indicate that the sexual dimorphism in HCLA proteins is not a universal feature of all rodent species.     

Androgen binding by skeletal muscle nuclei

A study was made of 3H-19-nortestosterone binding by isolated nuclei and 0.4 M KCl nuclear extract of the rat skeletal muscle. Binding specificity was ascertained by incubation in the presence of various unlabeled steroids. The Kd values were measured for nuclei and 0.4 M KCl nuclear extract (11.6 +/- 2.5 nM and 9.9 +/- 1.6 nM, respectively). The amount of binding sites was 24.1 +/- 1.7 fmol/mg DNA or 13.7 +/- 1.0 fmol/g tissue. Enzymatic treatment with pronase and DNase shows that nuclear androgen receptors are proteins. DNA was noted to have a stabilizing effect. DNase treatment of nuclei during extraction with 0.4 M KCl was shown to significantly increase the amount of specifically bound radioactivity in the extract.