metribolone and Hypospadias

Androgen receptors were partially purified by affinity chromatography of cytosols prepared from cultured genital fibroblasts of normal individuals and patients with androgen receptor abnormalities. Partially purified receptors were covalently labelled with [3H]R1881 (triated methyltrienolone) by ultraviolet photoactivation. Gel electrophoresis of cytosols from normal individuals showed a single radioactive peak, Mr approximately 97 K. Cytosols from patients with decreased nuclear transfer showed a similar peak, Mr approximately 97 K; cytosols from patients with partial androgen insensitivity (PAIS) or receptor positive complete androgen insensitivity (CAIS, R+) showed a peak Mr approximately 30 K-43 K.

Topics: Affinity Labels; Cells, Cultured; Cytosol; Estrenes; Fibroblasts; Humans; Hypospadias; Metribolone; Receptors, Androgen; Reference Values; Skin; Testosterone Congeners

The cause of hypospadias in the majority of patients is unknown. We examined the hypothesis that hypospadias might be explained by androgen receptor abnormalities in the atretic spongiosal tissue commonly known as chordee. We studied 10 patients with relatively severe hypospadias but with a predominantly male phenotype and no readily ascertained explanation for the defect, including no evidence of an abnormality in testosterone biosynthesis. Eight subjects had midshaft hypospadias and 2 had a penoscrotal meatus. All 10 patients had severe chordee. Serum concentrations of testosterone, and luteinizing and follicle-stimulating hormones were measured before human chorionic gonadotropin stimulation and a serum testosterone level was determined 24 hours after the last dose of a 5-day human chorionic gonadotropin stimulation (3,000 units per M.2 per day). Androgen receptor content and binding affinity were assayed in fibroblasts cultured from preputial skin and chordee tissue of patients and foreskin from normal male neonates. With the endogenous ligand dihydrotestosterone the mean number (maximum binding capacity) of androgen receptors was 1,013 fmol. per mg. deoxyribonucleic acid in preputial skin and 833 fmol. per mg. deoxyribonucleic acid in chordee tissue of patients, and 627 fmol. per mg. deoxyribonucleic acid in the foreskin of controls. With the nonmetabolizable, synthetic androgen methyltrienolone (R1881) the mean maximum binding capacity was 1,004, 722 and 758 fmol. per mg. deoxyribonucleic acid, respectively. Dihydrotestosterone receptor affinity (dissociation constant) was similar in preputial skin (0.20 nM.) and chordee tissue (0.21 nM.) from patients, and foreskin (0.29 nM.) from controls. Androgen receptor binding affinity of R1881 also was similar (0.30, 0.24 and 0.21 nM., respectively). Furthermore, the 5 alpha-reductase activity of preputial skin and chordee tissue of patients with hypospadias was similar to that of foreskin from normal neonates. In conclusion, isolated hypospadias in these subjects was not associated with androgen insensitivity of the spongiosal tissues on the basis of either decreased androgen receptor binding affinity, receptor number or conversion of testosterone to dihydrotestosterone.

Topics: 3-Oxo-5-alpha-Steroid 4-Dehydrogenase; Child, Preschool; Dihydrotestosterone; Estrenes; Humans; Hypospadias; Infant; Male; Metribolone; Penis; Receptors, Androgen; Reference Values; Skin

Androgens stimulate development and growth of the external male genitalia. Since hypospadias represents the most common congenital abnormality in the male newborn and the mechanism of action in this disorder is still unclear, androgen binding was assessed in cultured fibroblasts from biopsies from genital skin of 10 patients with idiopathic hypospadias. For comparison, binding was determined in corresponding samples from 8 males with normal penile development and from 9 patients with known androgen resistance syndromes (testicular feminization, Reifenstein syndrome, pseudovaginal perineoscrotal hypospadias). Finally, binding was measured in 10 samples of nongenital skin. Maximum specific binding (Bmax) in idiopathic hypospadias varied from 3.2 to 15.5 (median 6.6) fmol.mg protein-1. Bmax in samples of persons with normal genital development was between 12.2 and 17.9 fmol.mg protein-1 (median 13.2). Bmax in samples of patients with known androgen resistance syndromes was exactly in the range reported previously in the literature. It is evident that Bmax in samples of patients with idiopathic hypospadias differs significantly (P less than 0.01), (Mann Whitney U-test) from those with normal genital development. Thus it seems reasonable to conclude that in some patients with idiopathic hypospadias the genital defect is caused by receptor deficiency.

Topics: Adolescent; Adult; Cells, Cultured; Child; Child, Preschool; Chromatography, Thin Layer; Estrenes; Fibroblasts; Humans; Hypospadias; Infant; Male; Metribolone; Receptors, Androgen

Normal genital skin fibroblast (GSF) monolayers incubated with serum-free medium containing 3 nM [3H]-5 alpha-dihydrotestosterone (DHT) at 37 degrees C for 20 h have about 35% more specific DHT-binding than replicates incubated in serum-free medium with [3H]-DHT for only 1 h to saturate basal specific androgen-receptor activity. If, after 19 h, spent medium is replaced by fresh medium with 3 nM [3H]-DHT for 1 h, specific DHT binding is 85% more than basal. The acquisition of increased binding is temperature dependent (37 greater than 27 degrees C) and cycloheximide (2 microM) suppressible. The increased binding activity is considered to represent an augmentation of androgen receptor concentration because it has the same equilibrium dissociation constant (KD approximately 0.5 nM), rate constant of dissociation (k-1 approximately 6 x 10(-3) min-1) and ligand specificity as basal androgen-receptor activity, and because basal DHT-binding activity is stable in cells preincubated in androgen-free or serum-free medium alone for up to 72 h before assay. Prolonged incubation with methyltrienolone (R1881), a nonmetabolizable synthetic androgen, causes a greater, more persistent increment of androgen receptor activity than does equimolar DHT. The fibroblasts from two subjects with receptor-positive, partial androgen resistance lose their basal receptor activity during prolonged incubation with DHT, but augment it normally with R1881. This suggests that defective DHT metabolism is somehow involved in the pathogenesis of their androgen resistance.

Topics: Cycloheximide; Dihydrotestosterone; Drug Resistance; Estrenes; Fibroblasts; Humans; Hypospadias; Infant; Infant, Newborn; Karyotyping; Kinetics; Male; Metribolone; Receptors, Androgen; Receptors, Steroid; Skin; Temperature; Testosterone Congeners