Androgen Binding Capacity and 5α-Reductase Activity in Pubic Skin Fibroblasts from Hirsute Patients*

ABSTRACT In vivo binding of [3H] testosterone2), [3H]5α-dihydrotestosterone and [3H]3α-androstanediol to cytosolic and nuclear fractions of LA/BC and thigh muscles has been studied in functionally hepatectomized castrated rats following a 1 h infusion of the labelled steroid. The identification of metabolites formed from each steroid has also been determined in tissue cytosols. In each experiment, ventral prostate was used as reference target tissue. After [3H] testosterone and [3H]5α-dihydrotestosterone perfusions, cytosolic binding could be demonstrated in a 8–10S peak on sucrose gradient with LA/BC and ventral prostate or in the macromolecular fraction after filtration through Sephadex G-25 with thigh muscles. In both types of muscles, [3H]5α-dihydrotestosterone binding represented only one tenth of [3H] testosterone binding. This behaviour seems to be related to the high rate of 5α-dihydrotestosterone metabolism observed in these tissues; testosterone, on the contrary, was not metabolized. After [3H]3α-androstanediol perfusion, cytosolic [3H] androgen binding in LA/BC and in thigh muscles was almost non-existent. In muscles [3H]3α-androstanediol remained essentially unconverted. In ventral prostate, with every hormone studied level of cytosolic binding was comparable. It was observed that in this tissue [3H] testosterone and [3H]3α-androstanediol were metabolized into [3H]5α-dihydrotestosterone. Androgen binding to 0.4 m KCl extracted nuclear proteins has been demonstrated in ventral prostate as a 3.5–4.5S binding peak on sucrose gradient and this with each steroid perfused. In LA/BC, only [3H] testosterone gave a well defined binding peak. In thigh muscles, levels of nuclear binding were too low to be determined. In summary, these results suggest that rat perineal and skeletal muscles possess cytosolic androgen binding proteins similar to those found in ventral prostate. However, it appears that steroid metabolism is quite different in ventral prostate and muscles with respect to presence of 5α-reductase activity and extent of conversion of 5α-dihydrotestosterone into androstanediols. These differences may explain why, in vivo, muscles bind testosterone instead of 5α-dihydrotestosterone as in ventral prostate.

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Stroma of human benign prostatic hyperplasia: preferential tissue for androgen metabolism and oestrogen binding

Acta Endocrinologica ◽

10.1530/acta.0.0960422 ◽

1981 ◽

Vol 96 (3) ◽

pp. 422-432 ◽

Cited By ~ 91

Author(s):

M. Krieg ◽

G. Klötzl ◽

J. Kaufmann ◽

K. D. Voigt

Keyword(s):

Benign Prostatic Hyperplasia ◽

Binding Sites ◽

Reductase Activity ◽

Enzyme Reaction ◽

Prostatic Hyperplasia ◽

Binding Studies ◽

Normal Prostate ◽

Layer Chromatography ◽

Androgen Binding ◽

Tissue Fraction

Abstract. Because of the well known stromal-epithelial interaction of various urogenital organs, it was of interest to compare quantitatively steroid metabolism and binding in epithelium (E) and stroma (S) of the human benign prostatic hyperplasia (BPH). Testosterone 5α-reductase activity was determined by thin-layer chromatography and androgen as well as oestrogen binding sites by a charcoal adsorption technique after a steroid incubation period of 18 h at 0°C, using methyltrienolone (R1881) and oestradiol-17β as tritiated ligands and unlabelled R1881 and diethylstilboestrol as the respective competitors. The main results were as follows: (1) using biochemical markers (acid phosphatase, hydroxyproline), an on average 17% contamination of E by S and 6% of S by E was found, (2) the molar optimum of NADPH for the enzyme reaction was nearly identical in E and S, ranging between 1 and 0.1 mm, (3) the apparent Michaelis constant (Km) of 5α-reductase was in both fractions identical, the mean being 0.15 (μm, (4) the maximal rate of 5α-reductase activity (pmol 5α-reduced metabolites · mg protein−1 · 1 h−1) was 161 ± 28 (sem; n = 20), 66 ± 4.6 and 148 ± 6.6 in S, E and whole tissue fraction of BPH, respectively. In two normal prostates the means were: 88, 53 and 73, respectively, (5) the androgen binding sites were evenly distributed between the cytosol of E and S, while measurable oestrogen binding sites were found in 42% of the analyzed S but only in 5% of analyzed E. In conclusion: the 2.4 times higher 5·-reductase activity in S compared to E of the BPH is responsible for the about 2 to 2.5 times higher activity in the whole tissue fraction of BPH if compared with the normal prostate. Furthermore, due to our preliminary binding studies, oestrogens might play an important role in the S fraction of BPH.

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Androgen receptor disorder in three brothers with bifid prepenile scrotum and hypospadias

Acta Endocrinologica ◽

10.1530/acta.0.1230271 ◽

1990 ◽

Vol 123 (3) ◽

pp. 271-276 ◽

Cited By ~ 15

Author(s):

Monika Bals-Pratsch ◽

Hans-Udo Schweikert ◽

Eberhard Nieschlag

Keyword(s):

Androgen Receptor ◽

Binding Capacity ◽

Reductase Activity ◽

Serum Concentrations ◽

Equilibrium Dissociation Constant ◽

Normal Range ◽

Undescended Testes ◽

Skin Biopsies ◽

17 Hydroxyprogesterone ◽

Equilibrium Dissociation

Abstract Three brothers with congenital transposition of the penis, scrotal hypospadias, bifid scrotum, and bilateral undescended testes are described. Further signs of incomplete virilization, but no gynecomastia were seen. LH and FSH were elevated, whereas testosterone levels were reduced or in the normal range. Serum concentrations of 17-hydroxyprogesterone, dehydroepiandrosterone, androstenedione, 5α-dihydrotestosterone and estradiol measured in two affected brothers were in the normal range. Fibroblasts from scrotal skin biopsies performed in two patients showed normal 5α-reductase activity (419 and 214 pmol · (mg protein)−1 · h−1; normal >1), whereas androgen receptors had reduced maximal binding capacity (Bmax 4 and 14 fmol · (mg protein)−1; normal ≥ 18) and an increased equilibrium dissociation constant (0.7 and 1.26 nmol/l; normal 0.2±0.08) indicating a quantitative and qualitative androgen receptor defect. These patients represent a further variant of androgen insensitivity.

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Hormone regulation of the rodent Harderian gland: binding properties of the androgen receptor in the male golden hamster

Journal of Endocrinology ◽

10.1677/joe.0.1120003 ◽

1987 ◽

Vol 112 (1) ◽

pp. 3-8 ◽

Cited By ~ 37

Author(s):

F. Vilchis ◽

A. Hernandez ◽

A. E. Perez ◽

G. Perez-Palacios

Keyword(s):

Androgen Receptor ◽

Binding Capacity ◽

Sedimentation Coefficient ◽

Harderian Gland ◽

Hormone Regulation ◽

Apparent Dissociation Constant ◽

Receptor Complexes ◽

Male Golden Hamster ◽

Sucrose Gradient Ultracentrifugation ◽

Androgen Binding

ABSTRACT Studies were conducted in castrated golden hamsters to assess whether sexual dimorphism and sensitivity to sex steroid hormones in the rodent Harderian gland are mediated by an interaction of androgens with specific intracellular receptors. Physical properties, binding kinetics and stereospecificity of the androgen receptor were analysed using [3H]mibolerone as the radioligand. The presence of [3H]mibolerone–androgen receptor complexes with a sedimentation coefficient of 7–8S was demonstrated in Harderian gland cytosol by a linear sucrose gradient ultracentrifugation technique using a vertical rotor. Kinetic analysis revealed an androgen-binding site with an apparent dissociation constant of 0·3±0·07 (s.d.) nmol/l and a saturation binding capacity of 113±15 fmol/mg protein. Displacement studies indicated that unlabelled mibolerone, methyltrienolone, 5α-dihydrotestosterone and testosterone were efficient competitors for the androgen-binding sites, while progesterone, 17β-oestradiol, dexamethasone, dehydroepiandrosterone, ethiocholanolone and 5α-16-androsten-3-one were not. Experiments in long-term castrated animals revealed that the Harderian gland androgen receptor concentration and sedimentation coefficient remained unmodified. The results of these studies were interpreted as demonstrating the presence of a specific high-affinity intracellular androgen receptor in the male hamster Harderian gland. J. Endocr. (1987) 112, 3–8

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

Journal of Endocrinology ◽

10.1677/joe.0.0940415 ◽

1982 ◽

Vol 94 (3) ◽

pp. 415-427 ◽

Cited By ~ 13

Author(s):

M. B. Hodgins

Keyword(s):

Androgen Receptor ◽

Androgen Receptors ◽

Cultured Cells ◽

Reductase Activity ◽

External Genitalia ◽

Skin Fibroblasts ◽

Urogenital Sinus ◽

Receptor Complexes ◽

High Affinity ◽

Receptor Sites

Binding of [3H]testosterone and 5α-dihydro[3H]testosterone ([3H]DHT) to specific androgen-receptor sites of 5α-reductase-deficient human genital skin fibroblasts (five cell-lines) was studied in the intact cultured cells at 37 °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. 5α-Pregnane-3,20-dione had an approximately fivefold lower affinity than progesterone for androgen receptors; 3α/β- or 20α-reduction lowered its affinity further. It is suggested that in 5α-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 5α-reductase activity normally found in embryonic external genitalia and urogenital sinus may be to protect these tissues from the potentially antiandrogenic action of progesterone.

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