Testosterone-receptor Complex Research Articles

Interaction of hepatic chromatin with androgen-receptor complex.

When the chromatin prepared from male rat livers interacted with [3H] testosterone-receptor complex prepared from male rat livers, the binding was much higher than when interacted with free [3H] testosterone, indicating that the formation of testosterone-receptor complex is prerequisite for interaction with chromatin. When the [3H] testosterone-receptor complex was interacted with the chromatin and the bound chromatin proteins were extracted into three fractions based on their extractability or solubility in salt solutions, higher radioactivities were found in the 0.35 M NaCl-extract and 2.0 M NaCl residual fraction. Chromatin was fractionated into 0.35 M NaCl-soluble, 2.0 M NaCl-soluble and insoluble fractions, and interaction of free [3H] testosterone or [3H] testosterone-receptor complex with chromosomal fractions was studied. The binding of the chromosomal fractions with free [3H] testosterone was negligible compared with that of [3H] testosterone-receptor complex. The binding of 0.35 M NaCl-soluble fraction with [3H] testosterone-receptor complex was higher than that of 2.0 M NaCl-soluble fraction. The binding ability of 2.0 M NaCl-insoluble fraction with [3H] testosterone-receptor complex was twice as much as that of the intact chromatin. This observation indicated that salt-soluble fractions of chromatin contributes only partly to the binding of chromatin with [3H] testosterone-receptor complex and that other components in 2.0 M NaCl-insoluble fraction may participate in the binding.

Binding of cytoplasmic testosterone-receptor complex to nucleic from male rat livers.

A crude cytoplasmic androgen receptor preparation of rat livers or its partially purified one by a 30% ammonium sulfate precipitation was prelabeled with [3H] testosterone ([3H]T) and then incubated with nuclei from rat livers at 30 degrees C for 30 min. The formations of the [3H]T-cytosol receptor complex and its nuclear bound complex were determined. The formation of testosterone-cytosol receptor complex was a prerequisite for the nuclear binding of testosterone. Nuclear fraction, when incubated with the prelabeled crude cytosol, bound [3H]T-receptor complex as much as when incubated with the prelabeled partially purified cytosol. The addition of unlabeled crude cytosol to the prelabeled partially purified cytosol increased the nuclear binding of the receptor. The presence of a thermostable, low molecular inhibitor and a thermolabile, macromolecular activator in the crude cytosol preparation was suggested by dialysis, heating and gel chromatography.

Kinetic evidence for a unique testosterone-receptor complex in 5α-reductase sufficient genital skin fibroblasts and the effects of 5α-reductase deficiency on its formation

When 5α-reductase-sufficient genital skin fibroblast (GSF) monolayers are incubated with testosterone (T), they first form androgen (A)-receptor (R) complexes that dissociate at a fast rate [ k(37° C = 0.024 min −1]. As T is converted to 5α-dihydrotestosterone (DHT), this population of T-R complexes is eventually replaced by one that dissociates much more slowly [ k(37° C) = 0.006 min −1], at a rate typical of DHT-R complexes. During the course of T to DHT conversion, one may observe a population of A-R complexes that has a linear (monophasic) intermediate dissociation rate constant [ k(37° C) = 0.012 min −1]; this population cannot simply reflect a mixture of T- and DHT-R complexes. The rate at which the complexes are processed from one dissociative form to the next varies with the incubation temperature and the presence or absence of serum in the medium; it also varies within and among GSF strains under apparently constant conditions. To explain these facts, we propose a model that enables the 5α-reductase enzyme to influence the processive dissociative behaviour of T-R complexes by engaging in some sort of coupling with the AR. The proposal is strengthened by a set of observations in cells with constitutive, mendelian or inhibitor-induced 5α-reductase deficiency that preclude a simple quantitative relation between A-R complex processing and the extent of T to DHT conversion.

The binding of 3H-labelled androgen-receptor complexes to hypothalamic chromatin of neonatal mice: Effect of sex and androgenization

The binding of 3H-labelled androgen-receptor complexes, prepared by (NH 4) 2SO 4 precipitation from the 105,000 g supernatant of hypothalamic cytosol, to hypothalamic chromatin of neonatal mice covalently coupled to cellulose was measured in vitro. Saturation binding was also determined after extraction of histones and the masking of acidic proteins with high molarities of guanidine hydrochloride. This investigation showed the presence of high-affinity, low-capacity acceptor sites for [ 3H]-testosterone-receptor complexes in male hypothalamic chromatin ( K d value = 0.39 × 10 −10 M and binding sites of 41 fmol per mg of DNA). Acceptor activity seems to be associated with the acidic protein fraction of chromatin. No specific acceptor sites of similar nature were found in chromatin taken from the hypothalami of female mice. On the basis of these results, it is suggested that the androgen-unresponsiveness of female mice is related to the absence of acceptors for the androgen-receptor in female mice hypothalami.

Testosterone at High Concentrations Interacts with the Human Androgen Receptor Similarly to Dihydrotestosterone*

Testosterone and dihydrotestosterone are believed to exert their androgenic effects by interacting with a single intracellular receptor protein in androgen target tissues. During fetal life, however, testosterone mediates the virilization of the Wolffian ducts into the epididymis, vas deferens, and seminal vesicles, whereas the urogenital sinus and external genitalia require the in situ conversion of testosterone to dihydrotestosterone to undergo male development. The reason why the signal provided by testosterone needs to be amplified in some androgen target tissues but not in others remains an enigma. To provide insight into the different actions of these androgens we studied their interaction with the human androgen receptor in fibroblasts cultured from the genital skin of a patient with 5 alpha-reductase deficiency. Dihydrotestosterone was formed in negligible amounts in these cells, and in some experiments the residual 5 alpha-reductase activity was further blocked with the 5 alpha-reductase inhibitor finasteride. Saturation analysis in fibroblast monolayers disclosed similar amounts of binding with testosterone and dihydrotestosterone, and the affinity of binding of dihydrotestosterone was, on the average, about 2-fold greater than that of testosterone. [3H]Testosterone also exhibited a 5-fold faster dissociation rate from the receptor than [3H]dihydrotestosterone. In thermolability experiments the [3H]testosterone-receptor complex displayed marked instability at 42 C with 2 nM [3H] testosterone, whereas with 20 nM [3H]testosterone, receptor stability was similar to that seen with [3H]dihydrotestosterone. In up-regulation experiments, 2 nM [3H]testosterone produced a 34% increase in specific androgen receptor binding after 24 h, whereas 20 nM [3H]testosterone produced an average increase of 64%. Our results suggest that the weaker androgenic potency of testosterone compared to that of dihydrotestosterone resides in its weaker interaction with the androgen receptor, most clearly demonstrable as an increase in the dissociation rate of testosterone from the receptor. When present in relatively high concentrations, however, testosterone overcomes this defect by mass action.(ABSTRACT TRUNCATED AT 400 WORDS)

Interaction of testosterone and testosterone receptor complexes with nuclei of skeletal muscle from intact male mice and from mice bearing the testicular feminization (Tfm) mutant gene.

With the nuclear exchange assay and the nuclear retention assay it is shown that the androgen insensitivity of Tfm mice is probably due to a defect of the nuclear acceptor sites for the testosterone receptor complex. Further on the results obtained point strongly to the possibility that hormone free androgen receptor is localized in the nuclei and in the cytoplasma according to the "equilibrium model". A practicable method for separation of unbound steroids from nuclei is described.

A mutation that causes lability of the androgen receptor under conditions that normally promote transformation to the DNA-binding state.

Dihydrotestosterone-receptor complexes formed in human fibroblast cytosol prepared at 0 degrees C in the presence of sodium molybdate can be readily transformed to the DNA-binding state by heating at 25 degrees C. Under these conditions 50-70% of dihydrotestosterone-receptor complexes bind to DNA. We describe here studies of the transformation process in cytosols derived from normal cells and from fibroblasts propagated from subjects with syndromes of androgen resistance. In contrast to the situation with dihydrotestosterone, normal testosterone-receptor complexes are unstable under in vitro transforming conditions. Although equal amounts of hormone-receptor complex are formed at 0 degrees C, only 15% of testosterone-receptor complexes remain stable and acquire DNA-binding capacity after warming. This instability is not reversible upon lowering the temperature and is corrected by low concentrations (0.25 microM) of the protease inhibitor leupeptin. We have also identified two cousins with androgen resistance whose androgen-receptor complexes exhibit similar in vitro transformation lability with both dihydrotestosterone and testosterone. Phenotypic evidence in these subjects indicates that dihydrotestosterone-mediated processes are more completely impaired than are testosterone-mediated events. These findings suggest that dihydrotestosterone may amplify the androgenic signal at its targets not only by its higher affinity for the receptor but also by its more efficient conversion to the DNA-binding state and that such amplification may be less critical in target tissues in which testosterone suffices for androgenic effect. This offers one possible explanation of how a mutation that affects a single receptor protein may differentially impair the actions of two binding ligands of the receptor.

Possible mechanisms of androgen resistance in 5α-reductase deficiency: Implications for the physiological roles of 5α-reductases

Studies of testosterone and 5α-dihydrotestosterone (DHT) binding in 5α-reductase deficient human genital skin fibroblasts demonstrated the presence of a single class of androgen receptor which bound both steroids in cytoplasm and nucleus. The dissociation constant ( K d = 0.44 nmol/1) and dissociation rate of testosterone-receptor complexes were greater than for DHT-receptor complexes ( K d = 0.20 nmol/ l). As judged by competition with testosterone and DHT, progesterone bound to the androgen receptor with a K d of about 7 nmol/l. The affinity of 5α-pregnane-3,20-dione for androgen receptors was about 5-times less than that of progesterone; 3α/β or 20α reduction lowered the affinity even further. It is suggested that one function of the high levels of 5α-reductase in human embryonic external genitalia and urogenital sinus may be to prevent the antagonism of androgen binding to its receptor by progesterone. In support of this concept it has been found that the concentrations of progesterone required to inhibit testosterone or DHT binding to androgen receptors are lower in 5α-reductase deficient fibroblasts than in control cells with high 5α-reductase activity. Studies of the specificity of the 5α-reductase activity in human prostate microsomes have shown that conversion of testosterone into DHT ( K m = 12 nmol/ l) was competitively inhibited by crotonyl CoA ( K i = 125 μmol/l), a model substrate for enoyl CoA reductase of the microsomal fatty acid elongase system. It is speculated that 5α-reduction of testosterone and reduction of enoyl CoA could be properties of a single enzyme.

Nuclear testosterone receptors in the ovine testis

Nuclear [ 3H]testosterone-receptor complexes were demonstrated in hypophysectomized ram testis after in vitro direct labelling. The nuclear binding was maximal after a 45 min incubation of the tissue. The receptors are extractable by 0.4 M KC1 or NaSCN with a 25–30% efficiency. They migrate towards the anodic region during electrophoresis on agar gel. Nuclear androgen receptors were characterized in intact lamb testis by a testosterone exchange assay. After precipitation by protamine sulphate, the receptors were labelled with [ 3 H]testosterone during a 12 h incubation at 4°C. The exchange activity was linear between 0.1 and 0.9 mg of DNA per ml of incubation buffer. The receptors bind testosterone with a limited capacity (40–180 fmoles per mg DNA) and a dissociation constant K d of 2 × 10 −9 M. Their relative affinities for steroids are dihydrotestosterone > testosterone > estradiol > progesterone > 5α-androstanediol > cyproterone acetate > R5020.

Binding of androgens in 5 alpha-reductase-deficient human genital skin fibroblasts: inhibition by progesterone and its metabolites.

Binding of [3H]testosterone and 5alpha-dihydro[3H]testosterone ([3H]DHT) to specific androgen-receptor sites of 5alpha-reductase-deficient human genital skin fribroblasts (five cell-lines) was studied in the intact cultured cells at 37 degrees C. Under the conditions of the experiments, conversion of [3H]testosterone into [3H]DHT was negligible. Both steroids bound to the same set of high-affinity saturable sites in cytoplasmic and nuclear fractions of the cells. Unlabelled testosterone DHT and methyltrienolone competed effectively with the labelled steroids. Progesterone and oestradiol were weaker competitors; cortisol did not compete. The dissociation constant (Kd) for high-affinity complexes with [3H]testosterone (0.44 +/- 0.035 nmol/l) was higher than that for [3H]DHT complexes (0.20 +/- 0.090 nmol/l). Unlabelled DHT was more effective than unlabelled testosterone in competing with either radioactive steroid. Complexes of [3H]DHT and receptor dissociated more slowly than [3H]testosterone-receptor complexes and [3H]DHT bound more extensively to low-affinity non-saturable sites in fibroblasts. As judged by competition with the radioactive androgens, progesterone bound to the androgen receptor with a Kd of about 7 nmol/l. 5alpha-Pregnane-3-20-dione had an approximately fivefold lower affinity than progesterone for androgen receptors; 3alpha/beta- or 20alpha-reduction lowered its affinity further. It is suggested that in 5alpha-reductase deficiency in man progesterone in amniotic fluid and blood could effectively inhibit testosterone binding to androgen receptors in the male embryonic external genitalia. One function of the high levels of 5alpha-reductase activity normally found in embryonic external genitalia and urogenital sinus may be to protect these tissues from the potentially antiandrogenic action of progesterone.

Androgen receptor complex binding in murine skeletal muscle nuclei

Examination of binding of androgen-receptor complexes from murine skeletal muscle cytosol was performed by modified nuclear retention assay and modified nuclear acceptor assay. The experiments showed the binding of androgen-receptor complexes to the nuclear acceptor sites to be a cooperative process. Hill analysis of the data obtained resulted in a Hill coefficient of 3,6. The apparent dissociation constant for binding of cytosolic [ 3H]-testosterone-receptor complexes to nuclei was found to be in the range of K D = 6 − 8 × 10 −11 M. The nuclear matrix was able to bind androgen-receptor complexes in a saturable way, too.

Inhibition of 5 alpha-reductase, receptor binding, and nuclear uptake of androgens in the prostate by a 4-methyl-4-aza-steroid.

17 beta-N,N-Diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one (DMAA) is a potent reversible inhibitor of 5 alpha-reductase. The inhibition by DMAA of the conversion of testosterone to 5 alpha-dihydrotestosterone by rat prostate 5 alpha-reductase is competitive with testosterone, the apparent Ki being 5 nM, and uncompetitive with NADPH, DMAA inhibited both membrane-bound and solubilized 5 alpha-reductase. DMAA has moderate affinity for the prostate cytosol androgen receptor: 3 X 10(-6) M gives 50% inhibition of the binding of 10(-9) M 5 alpha-[3H]dihydrotestosterone to this receptor. This affinity to the androgen receptor is 1,000-, 500-, 120-, and 40-fold lower than that of 5 alpha-dihydrotestosterone, testosterone, spironolactone, and cyproterone acetate, respectively, and 7-fold higher than that of cimetidine. After incubation of [3H]testosterone with minced prostate, more than 90% of the radioactivity extracted from the nuclei co-chromatographed with 5 alpha-dihydrotestosterone and the rest with testosterone. DMAA at low concentrations decreased the ratio of 5 alpha-dihydrotestosterone to testosterone in the nuclei without significantly reducing the total uptake. DMAA at high concentrations also reduced the total radioactivity in the nuclei. This differential effect may reflect a higher affinity of DMAA for 5 alpha-reductase than for the androgen receptor. When 5 alpha-[3H]dihydrotestosterone was used in the tissue incubations, all radioactivity extracted from nuclei co-chromatographed with 5 alpha-dihydrotestosterone, regardless of whether or not DMAA was present. This nuclear uptake of 5 alpha-dihydrotestosterone is inhibited only by high concentrations of DMAA. In a cell-free system, the nuclear uptake of 5 alpha-[3H]dihydrotestosterone prebound to the cytosol receptor was not inhibited by DMAA. These results suggest that DMAA may inhibit nuclear uptake of 5 alpha-dihydrotestosterone by inhibiting the receptor binding. Sucrose gradient centrifugation of the radioactive KCl nuclear extracts prepared from the tissue incubations showed that the nuclear [3H]testosterone-receptor complex has a greater rate of dissociation than does the nuclear 5 alpha-[3H]dihydrotestosterone-receptor complex. [3H]Testosterone prebound to the prostate cytosol receptor also dissociates faster than 5 alpha-[3H]dihydrotestosterone prebound to the cytosol receptor.

Intact testosterone receptor complex does not induce RNA synthesis of Tfm-nuclei in multinucleated urethral muscle fibres of mosaic mice.

The X-linked testicular feminization mutation (Tfm) of the mouse leads to androgen insensitivity of target cells. Through the autosomal sex reversed (Sxr) factor we have converted female heterozygotes into males. Due to X-inactivation, mosaic animals arise which are composed of androgen sensitive wild-type and androgen insensitive Tfm cells. In the androgen dependent striated urethral muscle, Tfm and wild-type cells fuse and form multinucleated muscle fibres. In the muscle fibres the Tfm nuclei are exposed to the intact cytoplasmic testosterone receptor complex coded for by the wild-type nuclei. We ask the question whether under these conditions RNA synthesis can be stimulated in the Tfm nuclei. Castrated mosaic animals were injected with testosterone, and incorporation of 3H-uridine was studied by autoradiography. We found two classes of muscle cell nuclei, those with low grain counts corresponding to the Tfm controls and those with high grain counts corresponding to the stimulated male controls. The results indicate that the Tfm nuclei are not stimulated by the intact testosterone receptor complex.

Nuclei in testicular feminization (Tfm) are not activated by intact testosterone receptor complexes: a morphometric study in striated urethral muscle of mosaic mice.

Sex reversed mice heterozygous for the X-linked Tfm mutation are mosaics with respect to the Tfm locus. In the androgen-dependent striated urethral muscle, nuclei coding for the intact testosterone receptor protein and nuclei coding for the defective Tfm receptor protein are incorporated in the same multinucleate muscle fibres. The intact testosterone receptor complex can thus be expected to enter the Tfm nuclei. Our measurements show that the fibre diameters of the mosaic muscle form a homogeneous population, intermediate in size between induced male and non-inducible Tfm phenotypes. By contrast, the nuclear size shows a bimodal distribution, the subpopulations corresponding to Tfm and wild type nuclei. The results indicate that the Tfm nuclei are not activated by the intact testosterone receptor complex.

Effect of pyridoxal phosphate on the androgen receptor from rat prostate: inhibition of receptor aggregation and receptor binding to nuclei and to DNA-cellulose.

Abstract The 5α-dihydro[ 3 H]-testosterone-receptor complex of rat ventral prostate binds to DNA-cellulose. Binding can be inhibited by incubation of the androgen-receptor complex with pyridoxal phosphate; pyridoxal, pyridoxine, pyridoxamine and pyridoxamine phosphate are not inhibitors. Reduction of pyridoxal phosphate with NaBH 4 or conversion of pyridoxal phosphate to pyridoxal phosphate oxime with NH 2 OH prior to incubation with the androgen-receptor complex causes a loss of inhibition. These results are consistent with the view that inhibition of receptor binding to DNA is due to the formation of a Schiff base between pyridoxal phosphate and an amino group(s) of the receptor. Androgen-receptor complex binds to prostate nuclei. Incubation of the androgen-receptor complex and prostate nuclei with pyridoxal phosphate inhibits the nuclear retention of receptor. If the androgen-receptor complex is bound to nuclei before addition of pyridoxal phosphate, receptor can be removed from nuclei by the addition of pyridoxal phosphate. The prostate androgen-receptor complex has a sedimentation coefficient of 3–4S in sucrose gradients containing a high concentration of salt and 8–9S in sucrose gradients containing a low concentration of salt. Androgen-receptor complex treated only with pyridoxal phosphate shows no change in sedimentation behavior. However, androgen-receptor complex treated first with pyridoxal phosphate and then with NaBH 4 sediments to the same position (3–4S) in sucrose gradients containing low or high concentrations of salt. Covalent modification of androgen-receptor complex with pyridoxal phosphate, therefore, prevents aggregation of receptor in low salt gradients.

Studies on the androgen receptor of the rat seminal vesicle: comparison of the binding characteristics of dihydrotestosterone and testosterone.

The postmitochondrial supernatant (PMS) prepared from homogenized seminal vesicles of castrated rats contains the same number of specific binding sites (105–120 fmol/mg protein) for testosterone and dihydrotestosterone (DHT). The identical rate of inactivation of these binding sites in the absence of ligand and the identical velocity of sedimentation of the bound steroids through sucrose density gradients suggest that they bind to the same androgen receptor. Furthermore, competition studies favor the view that the same binding site of the receptor is occupied by these androgens. The affinity of testosterone to the receptor is lower than that of DHT. Our results indicate that a higher dissociation rate of the testosterone-receptor complex is primarily responsible for this difference in affinities. In contrast with the association reaction which attains an apparent equilibrium within 60 min, the dissociation of these steroid-receptor complexes is a relatively long process with half times of several hours. Moreover, with respect to the androgenicity of a steroid, the rate of dissociation from the receptor may be decisive, since only the results of those competition experiments in which long incubation times (days) have been employed reflect the relative androgenic potency of various steroids. Using a reconstituted in vitro system a PMS dependent and tissue specific uptake of testosterone by vesicular nuclei has been demonstrated. Under identical conditions about half as much testosterone as DHT is translocated into the nuclei. On the basis of this finding a direct role of testosterone-receptor complex in the regulation of nuclear functions is postulated.

Characterization of a cytoplasmic androgen receptor in the male secondary sexual character of green frog (Rana esculenta)

In the thumb pad of male Rana esculenta an androgen receptor has been demonstrated. The cytoplasmic receptor has a proteinaceous nature with a KD for testosterone of 3.12 ± 3.4 S.D. × 10−10 M and a number of sites of 333 ± 133 S.D. fmol/mg of proteins. A Stokes radius of 40 A and a sedimentation coefficient of 3.40 ± 0.5 S.D. were determined. Using these parameters a molecular weight of ~ 57,000 and a f/f = 1.58 were calculated. Optimum binding was found at 0–10°C at pH 7.5. The [H3]-testosterone-receptor complex is focused on a pH 5–7 gradient at pH 5.4. A t12 of 105min and a dissociation rate of 0.00413 min−1 for testosterone were determined. Using the nuclear exchange assay, carried out at 0–4°C. a KD of 2.85 × 10−9 M and a number of sites of 9 pmol/mg DNA were estimated for nuclear receptor.

Demonstration of an androgen receptor in rat pancreas

[1,2,6,7-3H]Testosterone (250 muCi) was administered to castrated male rats; after 30 min a labelled testosterone-receptor protein complex with a pI of 5.1 was recovered from the pancreatic cytosol. A labelled testosterone-receptor complex with an identical pI was also extracted from the nuclear fraction of rat pancreas after incubation of minced pancreatic tissue with 0.1 muM-]1,2,6,7-3H]testosterone for 30 min at 37 degrees C. Studies in vitro showed that [1,2,6,7-3H]testosterone was bound to a receptor protein focusing at a pI of 5.1 and with a Kd of 2 nM and a number of binding sites of 4.7 fmol/mg of protein in castrated male rats. The testosterone-receptor complex sedimented at 3.5 S in high-salt sucrose-density gradients, was excluded from Sephadex G-200 and Ultragel ACA-34, was stable towards treatment with dextran-coated charcoal, was relatively sensitive to heat, and was stable to treatment with deoxyribonuclease and ribonuclease, but was sensitive to treatment which proteinase. It is suggested that the pancreatic androgen receptor, which was also present in castrated female rats, may play a role in sex-steroid regulation of pancreatic function.

Characterization of the androgen receptors in the hypothalamus, preoptic area and brain cortex of the rat

The specific androgen receptors for testosterone (T) (1) and 5α-dihydrotestosterone (DHT) in the cytosol fraction of the hypothalamus, preoptic area and brain cortex of the rat have been characterized using electrophoresis and isoelectric focusing in polyacrylamide gels. After labeling of the cytosol fractions in vivo and in vitro we were able to demonstrate androgen-receptor complexes moving with an electrophoretic mobility (R f) of 0.5 in 3.25% acrylamide gels containing 0.5% agarose and 10% glycerol. Polyacrylamide gel electrophoresis was used as a quantitative assay for androgen receptors in the tissues. The hypothalamus, preoptic area and brain cortex were found to possess a single class of high affinity binding sites for androgens and the dissociation constants (K D) were estimated to be 3.4, 4.3 and 2.6 × 10 −10 M respectively. The binding capacities were 3.7 (hypothalamus), 3.5 (preoptic area) and 1.8 × 10 −15 (brain cortex) moles of high affinity binding sites per mg protein. Like other androgen-receptor complexes, the testosterone-receptor complexes of the hypothalamus, preoptic area and brain cortex were temperature labile, sulfhydryl dependent and revealed a very slow rate of dissociation at 0°C (t 1 2 > 36 hr). The receptors in all the tissues had an isoelectric point of 5.8. The steroid specificity of the cytoplasmic androgen receptors was tested in vitro by the competing efficiency of different unlabeled steroids for ( 3H)-testosterone binding. In the three tissues in investigation the following order of affinity was found: DHT > T > Cyproterone acetate > progesterone > androstenedione > 17β-estradiol. Cortisol did not effect androgen binding significantly. Thus, the physicochemical characteristics of the cytoplasmic androgen receptors of the hypothalamus, preoptic area and brain cortex are very similar, if not identical, to those of the androgen receptors described in the anterior pituitary, ventral prostate, epididymis and testis.

Characterization of the Androgen Receptor in the Anterior Pituitary of the Rat

The specific androgen receptors for testosterone (T) and 5alpha-dihydrotestosterone (DHT) in the cytosol fraction of the anterior pituitary of rats have been further characterized using electrophoresis and isoelectric focusing in polyacrylamide gels. After labeling of the cytosol fraction in vivo and in vitro, we were able to demonstrate androgen-protein complexes moving with an electrophoretic mobility (Rf) of 0.5 in 3.25% acrylamide gels containing 0.5% agarose and 10% glycerol. This method was used for quantitative measurements of pituitary androgen receptors, allowing multiple samples to be run simultaneously with little or no non-specific binding. There was no measurable dissociation of the androgen-receptor complexes during electrophoresis. When radioactive testosterone (1 nM) was added to pituitary cytosol fractions in vitro, there was an increase in the binding up to 4 hours of incubation at 0 C and little or no increase between 4 and 24 hours. All the binding studies therefore were done by incubation overnight at 0 C. When cytosol fractions were incubated with increasing concentrations of radioactive testosterone, a typical saturation curve was found. Scatchard plot analysis showed a binding capacity of 12.0 femtomoles/mg protein and the equilibrium constant of dissociation was estimated to be 3.4 +/- 0.7 (SD) X 10(-10)M. Like other androgen-receptor complexes, the testosterone-receptor complex in the anterior pituitary gland had an extremely slow rate of dissociation at 0 C (t1/2 greater than 4 days). The steroid specificity of the cytoplasmic androgen receptors was tested in vitro by the competing efficiency of different unlabeled steroids for [3H] testosterone binding. T and DHT caused the same inhibition of [3H]T to the receptors. However, since metabolism, of DHT to 5alpha-androstane-3alpha,17beta-diol occurred even at 0 C, the affinity of DHT for the receptor is probably somewhat underestimated. Cyproterone acetate had approximately half the affinity for the receptor compared with T, whereas lower affinities were found for progesterone and 17beta-estradiol. Cortisol did not appear to have any affinity for the receptors. Isoelectric focusing in polyacrylamide gels showed a peak of bound radioactivity with an isoelectric point of 5.8. Thus, the characteristics of the cytoplasmic androgen receptors of the anterior pituitary gland are very similar to those of the androgen receptors described in the ventral prostate, epididymis, and testis.