Androgen Binding Components Research Articles

Studies on the scrotal testis in unilateral experimental cryptorchidism in rat and guinea pig.

The effects of experimental unilateral cryptorchidism on the scrotal testis regarding weight, morphology and secretion of tubular fluid and the sertoli-cell specific androgen binding protein (ABP) were studied. In the intact guinea pig testis and epididymis an androgen binding component similar to rat ABP was found. In juvenile and adult rats cryptorchid for 17 and 21 days, respectively, and in guinea pigs cryptorchid for 11 weeks, the scrotal testis seemed unaffected regarding all parameters studied. With reference to previous findings of lowered fertility in unilateral cryptorchidism in man the possible mechanisms by which unilateral cryptorchidism may influence the scrotal testis are discussed.

Androgen and oestrogen binding in cytosols of human ovarian tumours.

The specific retention of androgens and oestrogens by cytoplasmic components of human ovarian tumours was investigated. High-affinity, low-capacity binding of androgens was observed in 88% of tumour specimens and oestrogen binding in 32%. Retention of oestrogens did not occur in the absence of androgen binding. The androgen-binding component, of sedimentation coefficient 7.5-8.5S, showed specificity for 5 alpha-dihydrotestosterone and 17 beta-hydroxy-17 alpha-methyl-estra-4,9,11-trien-3-one (R1881). In some instances, competition for R1881-binding sites indicated the presence of progesterone receptor-like binding. The data presented suggest strongly the existence of androgen and oestrogen receptors in some ovarian tumours and may be relevant to histopathological classification and therapeutic rationales.

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.

Nuclear binding of androgens and acid phosphatase activity in prostatic tumors of Nb rats

A transplantable prostatic adenocarcinoma derived from the dorsal lobe of the prostate gland of an Nb rat was analyzed for the concentration of nuclear androgen-binding sites and the presence of acid phosphatase activity. When extracts of nuclei from normal prostatic tissue were labelled with [1,2- 3H]dihydrotestosterone in the absence and presence of competitor, two types of specific binding were observed: type 1 was characterized by an association constant ( K a) of 6 · 10 7 M −1 and involved a molecule that was excluded from Sephadex G-200; type 2 was characterized by a K a of 3 · 10 8 M −1 and depended on a binding component that was retained by Sephadex G-200. Nuclei from androgen-stimulated tumors contained reduced concentrations of both androgen-binding components, whereas nuclei from autonomous tumors had only a trace amount og type 1 sites and were entirely devoid of type 2 sites. In all tumors the acid phosphatase activity per mg of protein was markedly elevated. Relative to normal, the activity of this enzyme was 140% and 350% higher in androgen-stimulated and autonomous tumors, respectively. These findings indicate that prostatic tumors are characterized by a decrease in nuclear androgen-binding, and an increase in specific activity of acid phosphatase, and also that such changes are more pronounced in autonomous than in androgen-stimulated tumors.

Androgen and progesterone binding components in cytosol prepared from cultures enriched in Sertoli cells from immature rat testes.

Androgen and progesterone binding components in cytosol prepared from cultures enriched in Sertoli cells from immature rat testes.

Evidence for an androgen-binding component in the cytosol of the human term placenta

The present communication describes a specific, high-affinity (Kd = 1.9 ± 0.21 × 10−9M; n = 3) binding component for dihydrotestosterone present in the high-speed, hormone-stripped cytosol fraction of the human term placenta. In order to delineate the nature of this substance, the synthetic steroids R1881 (progesterone and androgen receptor specific) and R5020 (progesterone receptor specific) were employed. Minimal binding of H3-R5020 was observed; however, H3-R1881 was found to bind with a Kd = 0.6 ± 0.18 × 10−9M (n = 3). The relative displacing ability of R5020 for H3-R1881 was only 1.3%, substantiating the presence of an androgen binder and negating the presence of a significant level of a progesterone-binding component. Evidence that the binder is of placental origin and does not result from blood contamination includes no significant binding of H3-R1881 to pregnancy serum, binder thermolability at 45° C, and precipitation of the binder by ammonium sulfate at 30% saturation. Speculations as to the receptor or receptor-like nature of the binder are presented.

Specific binding of [ 3H]-methyltrienolone to both progestin and androgen binding components in human benign prostatic hypertrophy (BPH)

As previously reported. [ 3H]-R1881, a stable tracer having high affinity for both androgenic and progestin binding components, binds to BPH cytosol with a predominant progestin-like specificity. However, when incubation was performed in the presence of a large excess of triamcinolone acetonide, a typical androgen binding specificity was demonstrated. This is explained by masking of the progestin binding component by triamcinolone acetonide, a steroid which has high affinity for the progestin receptor and no affinity for the androgen binding component. The present data clearly show that [ 3H]-R1881, in the presence of triamcinolone acetonide, can be efficiently used for determination of androgen binding in BPH, a tissue which contains both androgen and progestin binding components. A similar approach could be adopted for other tissues containing these two receptors.

Androgen Binding Proteins and Androgen Levels in the Human Testis and Epididymis

The human testis and epididymis contain two distinct androgen binding components, one with physicochemical properties identical to the androgen receptor found in other target tissues and another, androgen binding protein (ABP), which shares the same binding characteristics as human testosterone binding globulin (TeBG). Simultaneous assays of ABP and corticosteroid binding globulin (CBG, an exclusive blood protein) in the same testicular extracts suggest that androgen binding to the second component could be readily explained by contamination with blood TeBG rather than by the existence of a testis specific ABP. The levels of dihydrotestosterone (DHT) (the major androgen binding to this protein) in the human epididymis are much lower than those reported for rats and rabbits. Furthermore, there is no concentration gradient in DHT or testosterone along the human epididymis.

Characterization of unoccupied (R) and occupied (RA) androgen binding components of the hyperplastic human prostate

Saturation protocols were developed for measurement of unoccupied (R) and steroid-occupied (RA) androgen binding components of human hyperplastic prostate. The concentration of unoccupied cytoplasmic binding sites (2 hr incubation at 2°C) for the synthetic androgen R1881 (17β-hydroxy-17α-methyl-estra-4,9,11-trien-3-one) and the synthetic progestin R5020 (17α,21-dimethyl-19-norpregna-4,9-diene-3,20-dione) respectively was 10.7 ± 1.4 and 14.3 ± 3.2 fmoles per mg cytosol protein and the apparent steroid affinity respectively was 9.6 ± 0.8 and 1.6 ± 0.4 × 10 8 M −1. Steroid specificity of the unoccupied cytoplasmic R1881 and R5020 binding sites was similar. When R1881 and R5020 were employed as probes of total, R plus RA, cytoplasmic binding components (20–24 hr incubation at 15°C) saturable binding of R5020 was not detectable. Total cytoplasmic R1881 binding site concentration and apparent affinity for R1881 were 51.7 ± 3.3 fmoles per mg cytosol protein and 2.7 ± 0.6 × 10 7 M −1. R5020 was a poor inhibitor of R1881 binding to total cytoplasmic R1881 binding components. Incubation (4 hr at 37°C) of human prostate mince with 5α-DHT (17β-hydroxy-5α-androstan-3-one) demonstrated a single class of KCI extractable nuclear binding component(s) which sedimented at 4–5S on linear sucrose gradients containing 0.6 M KCI. Binding site concentration and apparent affinity for 5α-DHT were 15 fmoles per 100 μg DNA and 1.0 × 10 8 M −1. The crude nuclear pellet obtained from human prostate homogenates contained binding components which were detectable by incubation of the clarified nuclear KCI extract with R1881 at 15°C for 20–24 hr. Binding site concentration and apparent affinity for R1881 were 29 ± 8 fmoles per 100 μg DNA and 3.1 ± 0.4 × 10 8 M −1. Steroid specificity of the nuclear 5α-DHT and R1881 binding sites was comparable. Unoccupied KCI-extractable R1881 binding sites were not detected subsequent to 2 hr incubation at 2°C. The data were consistent with identification of the nuclear binding components and the R1881 cytoplasmic binding component measured subsequent to incubation at 15°C as human prostate androgen receptors.

Androgen receptor in human prostatic cancer: our experience with methyltrienolone

Use of glycerol and the protease inhibitor phenylmethylsulfonyl fluoride resulted in a fourfold increase in the yield of androgen receptor from human neonatal foreskin and a tenfold increase in stability of this receptor. The general physical properties of the androgen-binding component isolated from the cytosol of human neonatal foreskin were found to be consistent with those previously reported for androgen receptors. The synthetic androgen methyltrienolone (R1881) was bound with high affinity (a mean dissociation constant of 0.51 nmol/L) and low capacity (5.6 fmol/mg of protein); maximum binding required 4 hours. Affinity of the receptor was highest for R1881 followed by dihydrotestosterone, testosterone, and progesterone. 17β-Estradiol showed little or no competition. The receptor exhibited a change of sedimentation coefficient from 8S to 4S upon treatment with 0.1 mol/L potassium chloride. Isoelectric focusing demonstrated the existence of two molecular species with apparent isoelectric points of 7.2 and 5.7.

Characteristics of androgen binding in rat adrenal tissue using [su3]H]methyltrienolone (R1881) as tracer

A high level of binding of [ 3H]methyltrienolone (R1881 = 17β-hydroxy-17α-methyl-estra-4, 9, 11-trien-3-one) was found in cytosol prepared from adrenals of castrated male rats. Binding of [ 3H]R1881 was of high affinity (K D = 6.2 nM) and highly specific for androgens. The [ 3H]R1881 complex migrates at 7–9S on sucrose gradients in low ionic strenght buffer and at 4–5S in buffer containing 0.4M KC1. All binding studies have been performed in parallel with rat ventral prostate and adrenal cytosol. The present data suggest the presence of an androgen binding component in rat adrenal tissue.

5alpha-Dihydrotestosterone (DHT) receptors in rat brain and pituitary cell nuclei.

Gonadectomized-adrenalectomized (GX-ADX) adult male rats were injected iv with 2-4 mug/kg [1,2-3H]DHT and sacrificed 2 h later. Whole tissue homogenates and purified cell nuclear fractions were prepared from various brain regions and the pituitary and analyzed for radioactivity. Cell nuclei from pituitary and most limbic-hypothalamic regions (but not cerebral cortex) concentrated radioactivity (per unit protein) over whole tissue levels. The highest levels of nuclear-associated radioactivity were present in pituitary, hypothalamic and septal tissues. Analysis of radioactivity by double isotope dilution, chromatography, and recrystallization revealed that unmetabolized DHT represented 96 and 95% of the nuclear-associated radioactivity in pituitary and pooled limbic-hypothalamic structures, respectively. Simultaneously administered 100-fold molar excesses of unlabeled 5betaDHT, progesterone or corticosterone did not reduce nuclear-retained [3H]DHT, while unlabeled 5alphaDHT competed strongly. The anti-androgen, cyproterone acetate, competed at doses capable of of blocking androgen-mediated neuroendocrine effects. Levels of nuclear-retained [3H]DHT were negligible in intact males, but rose markedly 24 h after castration-adrenalectomy, remaining stable for 2 weeks post-operatively. Density gradient centrifugation of pituitary or brain cell nuclear salt extracts (0.4M KCl) revealed the [3H]DHT bound to a macromolecule sedimenting at 3--4S. The characteristics of DHT brain and pituitary cell nuclear binding are compared with the respective characteristics of neural cytosol and ventral prostate nuclear androgen binding components. These preliminary data suggest the existence of the functional, steroid-specific, stereospecific cell nuclear androgen receptor in the adult rat brain and pituitary.

Identification of limited capacity androgen binding components in nuclear and cytoplasmic fractions of canine prostate.

Nuclear and cytoplasmic androgen receptors were identified in canine prostate preparations. Saturation analysis employing 5α-dihydrotestosterone and tissue from intact dogs demonstrated that crude nuclear pellets contained 85 femtomoles of KCI-extractable and 106 femtomoles of ethanol-extractable receptor sites per 100 μg DNA. The apparent 5α-dihydrotestosterone association constant, 3-3.5 × 108 M−1, and steroid binding specificity of both receptor fractions were identical. 5α-Dihydrotestosterone, 19-nortestosterone, 17β-testosterone, and 5α-androstane-3λ,17β-diol were effective inhibitors of the nuclear binding of radiolabeled 5α-dihydrotestosterone, whereas 5α-androstane-3α,17α-diol, δ4-androstene-3,17-dione, progesterone, estradiol, and cortisol were not. The sedimentation constant of the KCl-extractable nuclear receptor on linear sucrose gradients was 4-5S. Cytoplasmic saturation analysis, employing tissue obtained from dogs 72 hr post-orchiectomy, demonstrated that 5α-dihydrotestosterone and the synt...

Androgen binding in rat uterus cytosol. Study of the specificity

During in vivo infusion of [ 3H]-testosterone, the rat uterus accumulates the radioactivity in the form of testosterone (T) and the prostate in the form of 5α-dihydrotestosterone (DHT), as shown by a DHT/T ratio of 18.1 for the radioactivity extracted from the prostate and 0.014 for the uterus. In vitro the binding of T in uterus cytosol appears to be stronger than the binding of DHT, as opposite to the prostate. Competition studies, involving a large number of steroids do not reveal major differences in specificity of the T-binding component in the uterus and the T- or DHT-binding component in the prostate which have also a similar concentration (66.5 vs 43.3 fmol/mg protein) and affinity for T (k D 1.1 vs 1.2nM). Evidence is presented that the apparently weaker binding of DHT in uterus cytosol is not due to the androgen-binding component itself, but to indirect factors, such as non-specific binding and metabolic conversion of the steroid. Indeed, uterus cytosol contains a 3α-hydroxysteroid dehydrogenase with high activity even at low temperature. The high affinity androgen binding components of uterus and prostate cytosol have thus almost the same specificity with intrinsically preferential binding of DHT. and do not explain the difference in androgen retention observed in vivo.

Androgen binding proteins in the anterior pituitary, hypothalamus, preoptic area and brain cortex of the rat.

The cytosol fractions of the anterior pituitary, hypothalamus, preoptic area and brain cortex of castrated male rats have been found to possess specific androgen binding proteins. The physicochemical characteristics of these binding proteins appear to be very similar. Thus, they were excluded by Sephadex G-100 gel and had a sedimentation coefficient of 6-7S by sucrose gradient centrifugation. The protein nature of the androgen binding components was supported by the fact that protease, but not DNase and RNase eliminated the binding of androgens. In addition, the elimination of the binding by 1 mM p-chloro-mereuriphenylsulfonate (PCMPS) and by heat treatment at 45 C for 30 min indicate that free sulfhydryl groups are necessary for androgen binding and that the proteins are thermolabile. Testosterone, 5alpha-dihydrotestosterone and the antiandrogen Cyproterone acetate were found to possess a much higher affinity than 17beta-estradiol and cortisol for the binding components. Dissociation studies revealed that [3H]testosterone is not easily displaced by unlabeled androgens. In the anterior pituitary, hypothalamus and preoptic area testosterone accounted for the major part of the radioactive material in the total tissue homogenates and also for the greater part of the bound radioactivity 15 min after in vivo administration of [3H]testosterone. [3H]17beta-estradiol accounted for less than 3% of the bound radioactivity under these conditions. If binding of a steroid sex hormone by specific proteins is a prerequisite for the hormonal action, the present study indicates the potential for a direct effect of androgens on the target cells of the anterior pituitary and of the central nervous system

Androgen binding proteins in different testis compartments

Two different high-affinity androgen binding proteins have been demonstrated in the rat testis. The smaller protein (ABP, MW 90,000) is produced in the testis, and transported with the testicular fluid into the epididymis. ABP is found in tubules but not in interstitial tissue or in testicular lymph. Destruction of the spermiogenic cells by physical or chemical agents does not decrease the concentration of ABP in the testis, indicating Sertoli cells to be the site of production. ABP disappears from the testis following hypophysectomy, but reappears following FSH treatment. Other pituitary hormones have no effect. Another androgen binding component, different from ABP, has also been recovered from rat testis tubules. This protein shows characteristics different from ABP but identical to cytoplasmic receptors of epididymis and prostate, indicating the presence of androgen target cells in the testis. Large doses of testosterone or dihydrotestosterone are known to maintain spermatogenesis in the hypophysectomized rat. We postulate that a major action of FSH on spermatogenesis may be mediated by ABP. ABP may facilitate androgen transport into the seminiferous tubules and provide the germ cells with the milieu of androgenic hormones which is required for normal spermatogenesis.

Further Studies on the Accumulation and Binding of Androgen in Rat Epididymis

Two epididymal androgen binding components were separated by polyacrylamide gel electrophoresis and examined in relation to nuclear uptake and binding of androgen after injection of l,2,6,7,3H-testosterone (3H-T) in vivo. The slower moving binding component (CR) was similar to the cytoplasmic receptor in the ventral prostate and was shown to remain in the epididymis after castration or hypophysectomy. The faster moving component, identified as the intraluminal androgen binding protein (ABP), disappeared from the epididymis within 8 days after castration or 10 days after hypophysectomy. The accumulation of androgen in nuclei remained active after the disappearance of ABP, indicating that binding to ABP is not involved in this process. Binding to CR, on the other hand, appeared to be essential for nuclear accumulation. Cyproterone acetate inhibited androgen binding to CR and decreased the nuclear accumulation of androgen without affecting binding to ABP. When rats were castrated for various time intervals (...

Binding of Testosterone to Uterine Components of the Immature Rat

Abstract The mechanism of the uterotrophic and antiuterotrophic action of testosterone in the rat is unknown. Since testosterone does not compete with estradiol-17β for receptor sites, the possible direct interaction of testosterone with uterine components in the rat was investigated. Following the administration of [3H]testosterone to immature female rats, the hormone is selectively taken up by uterine nuclei and is retained for 15 to 30 min. Thereafter, the hormone is gradually released and is virtually eliminated from the uterine nuclei over a period of 2 hours. Very low levels of radioactivity are accumulated in nuclei of diaphragm and other tissues. The simultaneous administration of non-labeled testosterone reduces the nuclear uptake of [3H]testosterone in the uterus by about 80% while it has no effect on the uptake of the labeled hormone by diaphragm nuclei. In the cytosol as well as the nuclear extract of the uterus, [3H]testosterone is bound to macromolecules which are excluded from Sephadex G-50. The nuclear [3H]testosterone macromolecule complex is also excluded from Sephadex G-200. No association of [3H]testosterone to macromolecules in cytosol or nuclei of diaphragm was observed. Uptake and binding of [3H]testosterone to nuclear components were also demonstrated in vitro following incubation of uteri at 37° in Eagle's Hela culture medium. Binding to nuclear components is highly specific for testosterone and the nuclear binding sites are saturated with low concentrations of the hormone. 5α-Dihydrotestosterone is much less effective than testosterone in competing with [3H]testosterone for the nuclear binding sites. Estradiol-17β reduces the nuclear accumulation of [3H]testosterone by only 20 to 30% while progesterone, cortisol, and cyproterone acetate at the concentrations used have no effect. Studies using cell-free systems indicate that binding of testosterone to cytoplasmic components is a prerequisite step for the transfer of the hormone to the nucleus. In vitro and in vivo studies show that the immature rat uterus lacks the capacity to convert testosterone to 5α-dihydrotestosterone or estrogens, to any significant extent. These results demonstrate that the immature rat uterus contains cytoplasmic and nuclear binding components (receptors) with high affinity and specificity for testosterone. Thus the uterotrophic and antiuterotrophic action of testosterone appears to be a direct action of the hormone by a mechanism distinct from that of estradiol-17β. The data also demonstrate that the uterine androgen-binding components are different from those found in the rat prostate with regard to their relative affinity for testosterone and 5α-dihydrotestosterone. Therefore the intracellular active form of androgen may vary from tissue to tissue. Whereas 5α-dihydrotestosterone appears to be the major active androgen in the rat prostate, in other tissues such as the uterus the predominant active androgen may be testosterone itself.

Demonstration of androgen-binding components in rat epididymis cytosol and comparison with binding components in prostate and other tissues.

Rat epididymis cytosol, labeled by incubation with dihydrotestosterone-l,2-3H at 0 C or by injection of testosterone-l,2-3H sc, contained 2 androgen-binding components, one of which demonstrated a high affinity for dihydrotestosterone. The mean constant of dissociation (Kd) of the high affinity component was 7.0 × 109M as determined by methods of dextrancoated charcoal adsorption, sucrose gradient ultracentrifugation and Sephadex gel equilibrium. When epididymis cytosol labeled with dihydrotestosterone-l,2-3H, in amounts which are bound largely by the high affinity binding component, was fractionated by sucrose gradient ultracentrifugation, the radioactivity migrated in a single peak with sedimentation coefficient of 4S in comparison with the 3.5S and 8S peaks of prostate cytosol. Acrylamide gel electrophoresis of similarly labeled epididymis cytosols revealed 2 peaks of radioactivity, while prostate contained only a single peak corresponding to the slower moving epididymis peak. Rat serum and cytosols of...