Clinical and endocrinological characterization of two subjects with Reifenstein syndrome associated with qualitative abnormalities of the androgen receptor

The androgen receptor in fibroblasts cultured from a biopsy of scrotal skin from 1 subject with Reifenstein syndrome has been found to be normal in amount and to bind dihydrotestosterone with normal affinity but to be qualitatively abnormal as evident by thermolability and instability upon ultracentrifugation. The family study of this subject and endocrine studies document androgen resistance in the index patient and his affected uncle. These findings provide evidence for X-linkage of this disorder, and suggest that the mutations that give rise to this phenotype are probably allelic to the mutations of the androgen receptor that cause testicular feminization.     

Androgen binding in cultured human fibroblasts from patients with idiopathic hypospadias

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.     

The physicochemical properties of the cytoplasmic androgen receptor in the kidneys of normal, carrier female (tfm/+) and androgen-insensitive (tfm/y) mice.

The physicochemical properties of the androgen-receptor complex in mouse kidney were determined. The sedimentation coefficient, 7.9S and Stokes radius of 82A, are compatible with an asymmetric protein [frictional ratio f/fo 1.98; axial ratio, assuming a prolate ellipsoid, 20] having a molecular weight of 270,000 daltons. The kidney receptor is a relatively acidic protein of esoelectric point (pI) 4.8 and readily precipitable with protamine sulfate or ammonium sulfate. Studies with protein-specific reagents suggest that both cysteine and tryptophan residues may be necessary for maintaining the functional configuration associated with androgen binding. The kidney receptor can promote the association of testosterone with purified DNA. These properties of the androgen receptor in mouse kidney are remarkably similar to those of male accessory sexual tissue. The receptor detected in carrier female mice (tfm/+ heterozygous for the gene for testicular feminization (tfm), has the same physical properties as that of normal mice. However, due to a decrease in receptor concentration, binding activity is only 69% that of normal. In cytosol from androgen-insensitive mice (tfm/+), a specific androgen receptor cannot be demonstrated by 5 different techniques.     

Steroid hormone toxicity in human fibroblasts does not correlate with high affinity receptor content.

Human diploid skin fibroblasts derived from normal individuals and those with the testicular feminization syndrome (TFM) have been shown to be killed to the same degree by dihydrotestosterone in spite of the absence of high affinity cellular androgen receptors in the TFM fibroblasts. Furthermore, several different normal fibroblast strains from various anatomical sites all showed similar amounts of androgen-induced cytotoxicity even though their respective receptor contents differed by as much as ten-fold. These results suggest that steroid-induced cytotoxicity in human fibroblasts is not correlated with receptor content, unlike murine lymphoid cells in which the receptor content has been shown to be closely related to their ability to survive hormone exposure.     

Male pseudohermaphroditism: diagnosis in cell culture.

Testicular feminization is a classic form of complete male pseudohermaphroditism. The individuals have a normal XY karyotype but unambiguously female external genitalia. They have congenital complete insensitivity to androgen due to an X-linked mutation. In four patients (from tow families with several affected members) with the typical phenotype of testicular feminization, a severe deficit of specific androgen-binding activity was detected in cultured fibroblasts from labium majus skin. Measurement of this activity in genital skin fibroblasts improves the differential diagnosis in patients with complete or imcomplete male pseudohermaphroditism before puberty.     

Androgen receptor in human skin fibroblasts. Characterization of a specific 17beta-hydroxy-5alpha-androstan-3-one-protein complex in cell sonicates and nuclei.

Cultured human skin fibroblasts were shown to contain an androgen binding activity (receptor) which was heat-labile and destroyed by trypsin. Specific binding was seen after incubations of these cells with 1,2-3-H-testosterone, 1,2-3-H17beta-hydroxy-5alpha-androstan-3-one (dihydrotestosterone, DHT) and 1,2-3-H-5alpha-androstane-3alpha, 17beta-diol. This receptor had a high affinity (Kd=0,2-1.6 nM) and a high degree of specificity for DHT. It was measured as a 3-H-DHT-protein complex by gel filtration chromatography using a method which distinguishes specific from nonspecific binding. Receptor activity was distributed about equally between nuclear and extranuclear components at all times studied and was present in both compartments when cell incubations were carried out at 4 degrees and 37 degrees. Saturation analysis indicated that there were 1250-18,600 binding sites per whole cell. By sucrose gradient centrifugation the receptor had a sedimentation coefficient (S20,w) of about 4. Cells grown for 8 days without serum in the medium maintained the same levels of 3-H-DHT binding. Within 15 hours puromycin (20 mug/ml) in serum-free medium caused a 40-60 percent decrease in binding for the same cell lines. Although the highest levels of 3-H-DHT binding were observed in fibroblasts from newborn foreskin, appreciable cytosol and nuclear binding were seen in cells from forearm, neck and abdominal skin. Receptor activity was stable during prolonged culture. Fibroblasts from several skin sites from patients with the androgen insensitivity syndrome (testicular feminization) had no detectable specific DHT binding. In this study it was demonstrated that skin fibroblasts can rapidly convert testosterone to its active form, DHT, bind DHT to a specific receptor protein and transport this complex to their nuclei. Therefore this may prove to be a convenient system for studying androgen action in vitro.     

Binding of sex steroid binding protein to plasma membranes of human testis

The existence of a specific binding site for sex steroid binding protein (SBP or SHBG) was detected on plasma membranes prepared from the testis of a patient affected by a variant form of testicular feminization. A binding technique using [¹²⁵I]SBP as a tracer allowed us to identify a single set of binding sites, characterized by a K_d of 1.917 × 10⁻¹¹ M. The maximum number of binding sites was 5.2 fmol/mg membrane protein. Membranes were also prepared from a sample of genital skin from the same patient, but no binding for [¹²⁵I]SBP was detectable. The evidence of the SBP membrane receptor in the testis of a patient affected by Morris syndrome extends our knowledge about the tissue distribution of the SBP receptor and suggests the possible implication of SBP and its recognition system in a disorder related to peripheral androgen insensitivity.     

Point mutation in the steroid-binding domain of the androgen receptor gene in a family with complete androgen insensitivity syndrome (CAIS)

An exonic single nucleotide substitution in the human androgen receptor gene (hAR) could be detected in an Italian family with two children affected by complete androgen insensitivity syndrome (CAIS), also called testicular feminization. This mutation leads to a guanine to adenine transition in exon 5, changing the sense of the codon from methionine (ATG) to valine (GTG). As this mutation abolishes a NcoI restriction site, a rapid test for the mutation can be performed by digestion of the polymerase chain reaction products with this enzyme. Previous results of indirect gene diagnosis in this family could be confirmed by this method.     

Molecular defects of the androgen receptor

Defects of the androgen receptor cause a wide spectrum of abnormalities of phenotypic male development, ranging from individuals with mild defects of virilization to those with complete female phenotypes. In parallel with this phenotypic spectrum, a large number of different mutations have been identified that alter the synthesis or functional activity of the receptor protein. In many instances, the genetic mutations identified lead to an absence of the intact, full-length receptor protein. Such defects (splicing defects, termination codons, partial or complete gene deletions) invariably result in the phenotype of complete androgen insensitivity (complete testicular feminization). By contrast, single amino acid substitutions in the androgen receptor protein can result in the entire phenotypic spectrum of androgen resistant phenotypes and provide far more information on the functional organization of the receptor protein. Amino acid substitutions in different segments of the AR open-reading frame disturb AR function by distinct mechanisms. Substitutions in the DNA binding domain of the receptor appear to comprise a relatively homogeneous group. These substitutions impair the capacity of the receptor to bind to specific DNA sequence elements and to modulate the function of responsive genes. Amino acid substitutions in the hormone-binding domain of the receptor have a more varied effect on receptor function. In some instances, the resulting defect is obvious and causes an inability of the receptor to bind hormone. In other instances, the effect is subtler, and may result in the production of a receptor protein that displays qualitative abnormalities of hormone binding or from which hormone dissociates more rapidly. Often it is not possible to correlate the type of binding defect with the phenotype that is observed. Instead, it is necessary to measure the capacity of the receptor that is synthesized in functional assays in order to discern any type of correlation with phenotype. Finally, two types of androgen receptor mutation do not fit such a categorization. The first of these—the glutamine repeat expansion that is observed in spinal and bulbar muscular atrophy—leads to a reduction of receptor function that can be measured in heterologous cells or in fibroblasts established from such patients. The expression of ARs containing such expanded repeats in men is associated with a degeneration of motor neurons in the spinal cords of affected patients. Likewise, the alterations of androgen receptor structure that have been detected in advanced forms of prostate cancer also behave as gain-of-function mutations. In this latter type of mutation, the exquisite specificity of the normal androgen receptor is relaxed and the mutant receptors can be activated by a variety of steroidal and non-steroidal ligands.     

Maternal meiosis II nondisjunction in a case of 47,XXY testicular feminization

Summary An 11-year-old patient with incomplete testicular feminization and a 47,XXY karyotype is described. The patient had female external genitalia, clitoromegaly, and some features of Klinefelter's syndrome, including speech delay and delayed intellectual development. DNA analysis using X chromosomal DNA sequences suggests that the supernumerary X chromosome in the patient resulted from maternal nondisjunction during meiosis II. The M II error thereby provides the basis for homozygosity of a mutation in the androgen receptor locus.     

Cultured human skin fibroblasts: a model for the study of androgen action

Human skin may be considered as a target organ for androgens, as are male sex accessory organs, since all events involved in testosterone action have been observed in this tissue. As a corollary, the mechanism of androgen action can be studiedin vitro in cultured skin fibroblasts. The advantages of this system are that studies can be performed with intact human cells under carefully controlled conditions, differentiated genetic and biochemical characteristics of the cells are faithfully preserved and the biological material is renewable from a single biopsy specimen. The metabolism of androgens, in particular the 5α-reduction of testosterone to the active metabolite, dihydrotestosterone, the intracellular binding of androgen to its specific receptor protein and its subsequent translocation to the nucleus have been studied in skin fibroblasts. The intracellular androgen receptor content of genital skin fibroblasts is higher than that from nongenital skin sites. In addition, the androgen receptor has been characterized as a specific macromolecule with properties of high affinity and low capacity similar to that of other steroid hormone receptors. The pathophysiology of three genetic mutations which alter normal male sexual development and differentiation has been identified in the human skin fibroblast system. In 5α-reductase deficiency, an autosomal recessive disorder in which dihydrotestosterone formation is impaired, virilization of the Wolffian ducts is normal but the external genitalia and urogenital sinus derivatives are female in character. At least two types of X-linked disorders of the androgen receptor exist such that the actions of both testosterone and dihydrotestosterone are impaired and developmental abnormalities may involve both Wolffian derivatives and the external genitalia as well. These two forms of androgen insensitivity result from either the absence of androgen receptor binding activity (receptor(−)form) or apparently normal androgen receptor binding with absence of an appropriate biological response (receptor (+) form). In addition, studies with human skin fibroblasts may also be of value in defining the cellular mechanisms underlying the broad spectrum of partial defects in virilization. In summary, we have correlated our studies of the molecular mechanism of androgen action in human genital skin fibroblasts with those of other investigators as these studies contribute to our understanding of male sexual development and differentiation.     

Effect of androgen insensitivity on diabetogenesis in db/db male mice with testicular feminization (Tfm)

In C3H mice, a major component of susceptibility to the diabetogenic action of an obesity mutation (diabetes, db) is male gender associated. We tested whether increased male susceptibility was an androgen receptor mediated process. C3H.SW/Lt-derived db/db males were rendered androgen-receptor function-deficient by introducing the testicular feminization (Tfm) mutation of the X-linked androgen receptor gene. The db/db Tfm/Y males (phenotypically female in appearance) developed severe diabetes indistinguishable from that observed in standard db/db X + Y males. Castration of standard C3H.SW/Lt-db/db males (producing mutants with normal androgen receptors but reduced serum testosterone) also failed to block the gender-enhanced susceptibility. In contrast, female db/db littermates exhibited a milder hyperglycemia, and were more resistant to pancreatic beta cell necrosis and islet atrophy than any of the groups of db/db males. Although these data indicated that male-enhanced sensitivity to diabetogenic stress was independent of circulating androgens, the possibility that the gender dimorphism is predicated upon tissue ratios of active estrogens to androgens in glucose-producing tissues such as liver is discussed.     

Testicular responsiveness to a single hCG dose in patients with testicular feminization

The suggestion that androgens may regulate testosterone (T) production in rat Leydig cells by a receptor-mediated feed-back mechanism, led us to investigate whether in vivo the absence of testicular androgen receptors, as it occurs in testicular feminization (TF), may modify the characteristic testicular response observed in men and prepubertal children after a single dose of hCG. Subjects consist of: 1) six normal men, 2) two adult patients with the complete form of androgen insensitivity syndrome (TF), 3) 12 normal prepubertal boys, 4) one prepubertal boy with the same form of TF. Each subject received i.m. a single dose of hCG 3500 IU/m2 b.s. and blood samples were collected basally and 2, 4, 24, 48, 72 and 96 hours after the hormonal stimulus. Serum levels of T, 17 alpha hydroxyprogesterone (17OHP) and 17 beta estradiol (E2) were measured at each collection time. In normal men a significant increase in T (M +/- SE) was observed at 4 h (758.6 +/- 135 ng/dl, P less than 0.05) and a more significant increase at 48 h (1082 +/- 60.3 ng/dl, P less than 0.001). E2 and 17OHP peaked significantly at 24 h (81.5 +/- 9.6 pg/ml and 460.7 +/- 90.9 ng/dl respectively). This response pattern is characteristic of the testicular desensitization which occurs in normal man after a single hCG dose. The same response pattern has been observed in the two TF adult patients suggesting that human testicular desensitization in vivo does not depend on androgen receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Feminization of rat hepatic P-450 expression by cisplatin. Evidence for perturbations in the hormonal regulation of steroid-metabolizing enzymes

The biochemical basis for the complex effects of the anti-cancer drug cisplatin on hepatic cytochrome P-450 activity was studied in adult male rat liver using P-450 form-specific steroid hydroxylase assays and antibody probes. Cisplatin treatment of adult male rats resulted in a marked and prolonged feminization of the pattern of P-450 enzymes expressed in hepatic tissue. The adult male-specific cytochrome P-450 forms designated P-450 2c (P-450 gene IIC11), P-450 2a (gene IIIA2), and P-450 RLM2 were decreased by 70-90% after 7-14 days, with parallel decreases in their respectively associated microsomal steroid hydroxylase activities. Concomitantly, hepatic levels of the female-predominant enzymes P-450 3 (gene IIA1) and P-450j (gene IIE1) were elevated approximately 2-4-fold. The female-specific microsomal enzyme androstenedione 5 alpha-reductase was induced approximately 20-fold by cisplatin; however, no elevation of the female-specific P-450 2d was detected. The underlying hormonal basis for these effects of cisplatin was then examined. Serum testosterone levels were found to be depleted by cisplatin in a time- and dose-dependent manner which correlated with the observed changes in these hepatic enzymes. Furthermore, castration of adult rats altered the profile of these enzymes in a manner which resembled that observed with cisplatin treatment, suggesting that androgen depletion was the primary cause for the observed feminization of hepatic enzyme expression. Consistent with this possibility, the synthetic androgen methyltrienolone effectively blocked the changes in hepatic enzyme expression induced by cisplatin. Moreover, hepatic enzyme feminization was significantly reversed by chorionic gonadotropin, which fully restored serum testosterone levels in the cisplatin-treated rat. Luteinizing hormone-releasing hormone challenge experiments demonstrated that the responsiveness of the pituitary to this hypothalamic regulator of testicular androgen production was unimpaired by cisplatin treatment, indicating that hypothalamic production or secretion of luteinizing hormone-releasing hormone may be deficient in the cisplatin-treated animals. These studies establish that the effects of cisplatin on hepatic P-450 enzyme expression result from its interruption of the hypothalamic-pituitary stimulation of testicular androgen production and that this, in turn, leads to a depletion of circulating androgens required for maintenance of normal P-450 enzyme expression in adult male rats.     

A 63-kDa protein with androgen-binding activity is not from the androgen receptor.

We have found a new protein in the heart of rat and mice that can be selectively and covalently labelled with the synthetic androgen analog mibolerone. Binding is specific as it can be displaced by excess radioinert ligand. The protein is prominently expressed in liver, kidney, and heart, but not in skeletal muscle. It is water soluble and found in the cytosol. Under denaturing conditions it has a molecular weight of 63,000 and appears on two-dimensional gels with an isoelectric point of 6.3. The protein's affinity for androgen is lower than that of the androgen receptor and it is about 100-fold more abundant than the receptor in the heart. Expression of the protein is not induced by androgen. The presence of this protein in testicular feminization (tfm) mice with a genetical defect of the androgen receptor rules out that it is the androgen receptor or a portion thereof. The biological role of this protein is not yet known.