Luteinizing hormone pulsatility in patients with major ovarian hyperandrogenism.

Hyperandrogenism and ovulatory dysfunction are common in women with either polycystic ovary (PCOS) or ovarian virilizing tumor. However, contrasting with the numerous studies that have extensively described gonadotropin secretory abnormalities, principally increased LH pulse amplitude and frequency, few studies have concerned gonadotropin secretion in patients with ovarian virilizing tumors; low gonadotropin levels have occasionally been reported, but never extensively studied. The goal of the present study was to further evaluate the pulsatility of LH secretion in women with ovarian virilizing tumor compared with that of PCOS patients. Eighteen women with major hyperandrogenism (plasma testosterone level >1.2 ng/ml) were studied (5 women with ovarian virilizing tumor, 13 women with PCOS, and 10 control women). Mean plasma LH level, LH pulse number and amplitude were dramatically low in patients with ovarian tumors when compared to both PCOS (p<0.001) and controls (p<0.001). In case of major hyperandrogenism, LH pulse pattern differs markedly between women with ovarian virilizing tumor or PCOS, suggesting different mechanisms of hypothalamic or pituitary feedback.

Expression of androgen receptor coactivators in normal and cancer prostate tissues and cultured cell lines.

BACKGROUND: In prostate cancer cell lines, androgen receptor (AR) coactivators modulate the transcriptional activity of AR. However, very little is known about their expression in normal prostate tissue and during progression to cancer. METHODS: AR and coactivators ARA54, ARA55, ARA70, and SRC1 RNA were analyzed by RT-PCR in normal and tumoral tissues of the same prostate, in prostate cell lines, and after hormonal treatments of prostate epithelial cells. RESULTS: AR-coactivators were expressed in normal and tumoral tissues and in cultured prostate cells; only ARA55 expression was decreased in tumoral relative to normal tissue of all seven prostates analyzed. It was not expressed in LNCaP and DU145 cancer cells and low in PNT2 immortalized cells in which all coactivator's expression were down regulated by DHT and up regulated by E2. In addition, coactivator's expression was increased in hyperplastic relative to normal prostate fibroblasts. CONCLUSIONS: ARA55 is both an AR coactivator and a focal adhesion protein (Hic-5). Its role in the progression of prostate carcinoma may therefore involve these two different functions. Its decrease in cancer tissue suggests that it plays a different role than that expected, namely, facilitate cell proliferation and therefore mobility and metastasis.

Alterations of expression and regulation of transforming growth factor beta in human cancer prostate cell lines.

TGF beta can promote and/or suppress prostate tumor growth through multiple and opposing actions. Alterations of its expression, secretion, regulation or of the sensitivity of target cells can lead to a favorable environment for tumor development. To gain a better insight in TGF beta function during cancer progression, we have used different cultured human prostate cells: preneoplastic PNT2 cells, the androgen-dependent LNCaP and the androgen-independent PC3 and DU145 prostate cancer cell lines. We have studied by specific ELISA assays in conditioned media (CM), the secretion of TGF beta 1 and TGF beta 2 in basal conditions and after hormonal treatment (DHT or E2) and the expression of TGF beta 1 mRNA by Northern blot. We have also compared the effect of fibroblast CM on TGF beta secretion by the different cell types. Compared to PNT2 cells, cancer cell lines secrete lower levels of active TGF beta which are not increased in the presence of fibroblast CM. LNCaP cells respond to androgen or estrogen treatment by a 10-fold increase of active TGF beta secretion while PC3 and DU145 are unresponsive. In conclusion, prostate cancer cell lines have lost part of their ability to secrete and activate TGF beta, and to regulate this secretion through stromal-epithelial interactions. Androgen-sensitive cancer cells may compensate this loss by hormonal regulation.

Estrogen and antiestrogen actions on transforming growth factorbeta (TGFbeta) in normal human breast epithelial (HBE) cells.

We have previously shown that estradiol (E2) increases the growth of normal human breast epithelial (HBE) cells and the antiestrogen 4-hydroxytamoxifen (4-OHT) inhibits estrogen-induced proliferation. These effects of estrogens and antiestrogens on proliferation have also been well documented in breast cancer cells. One mechanism for the antiproliferative effects of antiestrogens is the stimulation of TGFbeta in hormone-dependent MCF-7 and T47D cells. The role of this inhibitory growth factor in normal human breast cells has not been well studied. Accordingly, we measured the amounts of total and active TGFbeta1 and TGFbeta2 by specific E(max) immunoassay (EIA) in culture medium from normal breast cells (epithelial and fibroblasts) and from various ER- and ER+ breast cancer cell lines. We established that HBE cells are sensitive to the antiproliferative effect of TGFbetas, and studied the effect of E2 and 4-OHT, alone or in combination, on the secretion and activation of TGFbetas by HBE cells. HBE cells secrete TGFbeta1 and even more TGFbeta2, and are sensitive to these factors. However, in contrast to MCF-7 cells, TGFbeta secretion in normal breast cells is not regulated by E2 and 4-OHT.

Transforming growth factor beta in the human prostate: its role in stromal-epithelial interactions in non-cancerous cell culture.

BACKGROUND: Stromal-epithelial interactions play a critical role in prostate development, but the precise mechanisms are still unknown. Transforming growth factor-beta (TGFbeta) could be a potential mediator of these interactions, but there is as yet no clear demonstration of its role. METHODS: Separate cultures and co-cultures of fibroblasts and epithelial human prostate cells were performed. We measured TGFbeta1 and TGFbeta2 secretion by specific ELISA assay, cell growth by DNA assay, and TGFbeta type II receptor expression by RT-PCR. RESULTS: Co-culture resulted in a 20% inhibition of epithelial cell growth, similar to that obtained after TGFbeta treatment (2 ng/ml for 48 hr), but without affecting fibroblast proliferation. This was accompanied by a five- to six-fold increase in epithelial TGFbeta2 secretion. CONCLUSIONS: These results demonstrate for the first time that TGFbeta2 secretion by prostate epithelial cells is under the direct control of a diffusible factor secreted by fibroblasts. They emphasize the role of TGFbeta in stromal-epithelial interactions.

Evaluation of gonadal function in 107 intersex patients by means of serum antimüllerian hormone measurement.

Fetal male sexual differentiation is driven by two testicular hormones: testosterone (synthesized by interstitial Leydig cells) and antimüllerian hormone (AMH; produced by Sertoli cells present in the seminiferous tubules). Intersex states result either from gonadal dysgenesis, in which both Leydig and Sertoli cell populations are affected, or from impaired secretion or action of either testosterone or AMH. Until now, only Leydig cell function has been assessed in children with ambiguous genitalia, by means of testosterone assay. To determine whether serum AMH would help in the diagnosis of intersex conditions, we assayed serum AMH levels in 107 patients with ambiguous genitalia of various etiologies. In XY patients, AMH was low when the intersex condition was caused by abnormal testicular determination (including pure and partial gonadal dysgenesis) but was normal or elevated in patients with impaired testosterone secretion, whereas serum testosterone was low in both groups. AMH was also elevated during the first year of life and at puberty in intersex states caused by androgen insensitivity. In 46,XX patients with a normal male phenotype or ambiguous genitalia, in whom the diagnosis of female pseudohermaphroditism had been excluded, serum AMH levels higher than 75 pmol/L were indicative of the presence of testicular tissue and correlated with the mass of functional testicular parenchyma. In conclusion, serum AMH determination is a powerful tool to assess Sertoli cell function in children with intersex states, and it helps to distinguish between defects of male sexual differentiation caused by abnormal testicular determination and those resulting from isolated impairment of testosterone secretion or action.

Hormonal regulation of the androgen receptor expression in human prostatic cells in culture.

The regulation of the androgen receptor (AR) expression was studied using immunocytochemical and Western blot techniques on separate cultures of epithelial cells (PNT2) and fibroblasts of human prostate. In both cell types, immunocytochemistry revealed both nuclear and cytoplasmic staining. Treatment with DHT (5 x 10(-9) M) increased both the intensity of nuclear staining and the number of cells stained. The increase, observed after DHT treatment was markedly decreased by cyproterone acetate (5 x 10(-7) M), confirming a direct action of DHT via the AR. This autoregulation of AR was confirmed by Western blot, and seems to involve transcription and protein synthesis, since it was suppressed by actinomycin D and cycloheximide. In fibroblasts, known to contain an estrogen receptor, estradiol treatment (5 x 10(-7) M) also increases the AR immunostaining. In addition, coculture studies show that epithelial cells require the presence of fibroblasts for optimal expression of the AR. These results demonstrate that prostate epithelial cells and fibroblasts have retained in culture, an hormonal sensitivity correlated with the presence of specific receptors and can serve as a model for the study of hormone action in this tissue in normal or pathological conditions.

Pharmacological and molecular evidence for the expression of the two steroid 5 alpha-reductase isozymes in normal and hyperplastic human prostatic cells in culture.

BACKGROUND: Whereas the embryological development of the human prostate is clearly dependent on steroid 5 alpha-reductase (5 alpha-R) type 2 expression, the respective expression of the two known isoforms (types 1 and 2) of 5 alpha-R in the adult human prostate remains unclear. METHODS: 5 alpha-R isoform mRNA expression (Northern blots and reverse transcriptase-polymerase chain reaction [RT-PCR]) and enzyme activity were studied in immortalized epithelial cells (NE) and in fibroblasts from normal (NF) or hyperplastic (BPHF) human prostates. RESULTS: 5 alpha-R activity (fmol/microgram DNA/hr) was 1.43 +/- 0.5 in NE, 10.7 +/- 4.7 in NF, and 79 +/- 37 in BPHF. mRNAs for both 5 alpha-R isoforms were expressed in the three cell types, as shown by Northern blot and RT-PCR analysis. LY306089, a selective 5 alpha-R type 1 inhibitor, strongly inhibited 5 alpha-R activity in all cell types (IC50: 10 nM), confirming the predominant expression of 5 alpha-R type 1 in these cells. Finasteride, a 5 alpha-R type 2 inhibitor, was less efficient (IC50: 45, 35, and 65 nM in NE, NF, and BPHF, respectively). In addition, the inhibition by finasteride decreased with serial subculture in NF only, suggesting an effect of age in culture on the expression of 5 alpha-R type 2 in these cells. SKF105657, also a 5 alpha-R type 2 inhibitor, was a poor inhibitor in this system. CONCLUSIONS: These studies demonstrate that human prostate cells in culture express both isoforms of 5 alpha-R and suggest a balance in the expression of the two isoforms as a function of various regulatory factors.

[Mutations of the androgen receptor gene a possible role in prostate cancer]. / Mutations du gène du récepteur des androgènes: rôle possible dans le cancer de la prostate.

Prostate cancer is the second most frequent cancer in men, and the first after 75 years of age. It is androgen dependent and modifications of the androgen receptor (AR) could therefore be involved in its apparition, progression and evolution towards a stage of androgen independence which always occurs. Mutations of the AR have indeed been described. Some result in a more active receptor (constitutive mutations, amplification of the AR gene expression, prevalence of shorter alleles in exon 1) and could therefore be responsible for the progression of the disease in the absence of androgens or the increased cancer risk in some populations. Other mutations, in the ligand binding domain, modify the AR specificity resulting in its activation by weak androgens or even antiandrogens, progestagens or estradiol. These mutations could also explain why prostate cancer progresses in the absence of androgens. On the other hand, mutations resulting in androgen insensitivity could explain why a significant number of prostate cancer remain latent and never develop into an aggressive tumor. Finally, exon 1 polymorphism could be used as a prognostic marker, as it has been shown that the shorter alleles result in a more active AR and are predominant in populations at higher risk for prostate cancer. At any rate, even if AR mutations may be more frequent than initially thought, they remain rare and their significance remains to be determined.

Different phenotypes in a family with androgen insensitivity caused by the same M780I point mutation in the androgen receptor gene.

Mutations in the coding sequence of the androgen receptor (AR) gene result in a wild range of androgen insensitivity (AI) syndromes. Differences in the clinical expression of the same mutation in unrelated patients have been reported. However, this study reports for the first time strikingly different phenotypes among three patients within the same kindred. Two of the patients had a feminine phenotype, suggesting complete AI, but for some pubic hair. The third subject was male with partial AI, perineoscrotal hypospadias, and cryptorchidism. 5 alpha-reductase activity measured in genital skin fibroblasts and binding capacity of the AR were higher in the male than in the two patients with female phenotype. Northern blot analysis of AR messenger RNA revealed a 10-kb band of normal intensity in the three subjects. Molecular analysis of the coding sequence of the AR revealed a unique M780I mutation in exon 6, changing a methionine 780 to isoleucine in the hormone-binding domain. In conclusion, the same mutation of the AR gene in the same family can result in clinical phenotypes characteristic of complete or partial AI. Therefore, the molecular defect of the AR gene may not alone predict the phenotype in families with AI.

The first detection of complete androgen insensitivity with no mutation in the coding sequence of the androgen receptor gene.

We have analyzed the entire nucleotide sequences of complementary DNAs of the androgen receptor gene in two siblings (patients 8044 and 8047) with complete androgen insensitivity. Plasma testosterone was in the normal male range, however, androgen binding capacity was undetectable as measured in skin fibroblasts in both patients. 5alpha-reductase activity was normal in both cases confirming that this enzyme is not involved in the mechanism of androgen insensitivity. Northern blot analysis indicated that mRNA of the AR was normal in size. In addition, no mutation was found in the entire nucleotide sequences of complementary DNAs of the androgen receptor gene. Together, our results reveal an unusual insight into the molecular basis of androgen resistance, and the molecular heterogeneity in this clinical spectrum.

Predominant expression of 5 alpha-reductase type 1 in pubic skin from normal subjects and hirsute patients.

Dihydrotestosterone (DHT), the 5 alpha-reduced metabolite of testosterone, is the active molecule triggering androgen action, and 5 alpha-reductase (5 alpha-R), the enzyme converting testosterone to DHT, is a key step in this mechanism. Skin, like prostate, is a DHT- dependent tissue. Our laboratory demonstrated, many years ago, that 5 alpha-R in external genitalia was not regulated by androgens, whereas it was androgen dependent in public skin. As two genes, 5 alpha-R types 1 and 2, encoding for 5 alpha-R enzymes have been recently cloned, we undertook the present study to determine whether the two enzymes we had postulated on the basis of regulation studies were coincident with the cloned isoforms. The expression of the two isoforms was studied in genital and pubic skin fibroblasts from normal men, normal women, and hirsute patients. Messenger ribonucleic acid analysis, using Northern blot and RT-PCR techniques, indicated that both 5 alpha-R1 and -2 messenger ribonucleic acids are expressed in genital skin as well as in public skin fibroblasts. In contrast, studies using specific inhibitors of 5 alpha-R1 (LY306089) and 5 alpha-R2 (finasteride) showed that 5 alpha-R2 is predominant in pubic skin of normal men, normal women, and hirsute patients. These data raise the question of the possible use of specific 5 alpha-R1 inhibitors in the treatment of idiopathic hirsutism.

Human prostatic cells in culture: different testosterone metabolic profile in epithelial cells and fibroblasts from normal or hyperplastic prostates.

The prostate gland depends on androgens for its development and the maintainance of its differentiated structures and secretory function. We have characterized the metabolic pathways of testosterone in isolated epithelial (NE) and fibroblast cultured cells from normal (NF) and hyperplastic (BPHF) prostates, in order to provide a tool for the study of androgen function in the prostate in defined conditions. In NE, 5 alpha-reductase (5 alpha-R) is the predominant metabolic pathway whereas in NF 17 beta-hydroxysteroid dehydrogenase (17 beta-OHSDHase) is the main activity. However, 5 alpha-R in NF is 5-10-fold higher than in NE. Furthermore, a striking increase in both enzyme activities is observed in fibroblasts from hyperplastic prostates (5 alpha-R x 8; 17 beta-OHSDHase x 250 relative to NF). delta 4-androstenedione could serve as a reservoir for testosterone or could be a tentative protective mechanism directing testosterone metabolism towards an inactive molecule. In conclusion, human epithelial and stromal cells maintain in culture their main metabolic characteristics. The knowledge derived from these studies should facilitate the reconstitution and analysis of their interactions, which in vivo may modify their respective metabolism.

[5-alpha-reductases: physiology and pathology]. / Les 5 alpha-réductases: physiologie et pathologie.

In most androgen target tissues, the first step of androgen action is the 5 alpha-reduction of testosterone to DHT which binds to the androgen receptor with an affinity 3 to 4 fold higher than testosterone. Two genes, encoding two isozymes of 5 alpha-reductase (5 alpha-R) have been cloned. The two isoforms, 5 alpha-R1 and 5 alpha-R 2 are located on chromosomes 5 and 2 respectively and differ in optimal pH, substrate and inhibitor affinities and tissue expression. 5 alpha-R 2 is responsible for sexual differentiation. It is the major form expressed in the prostate where it seems necessary for embryonic growth and development. 5 alpha-reductase deficiency results in androgen insensitivity due to abnormal 5 alpha-R 2. Affected patients are XY individuals with a very peculiar form of male pseudohermaphroditism: they have feminine genitalia at birth and masculinize at puberty. 29 mutations, spanning the whole coding portion of the gene, have been described; correlation between mutations and enzyme activity have led to the suggestion that both the N- and the C-terminal end of the gene are involved in substrate binding, whereas the cofactor binding-site is located in the C-terminus. In contrast to androgen insensitivity due to 5 alpha-reductase deficiency, increased 5 alpha-reductase activity can result in androgen hypersensitivity as described in idiopathic hirsutism or benign prostatic hyperplasia. In these case 5 alpha-R 1 could possibly be involved.

Anti-müllerian hormone in children with androgen insensitivity.

Anti-Müllerian hormone (AMH), also called Müllerian inhibiting substance or factor, is secreted in high amounts by the immature Sertoli cell; it is negatively regulated by testosterone at puberty. In the present study, we measured serum AMH in 20 patients with defects of androgen synthesis or action: 9 with complete androgen insensitivity syndrome, 9 with a partial form, 1 patient with 3 beta-hydroxysteroid dehydrogenase deficiency, and 1 with Leydig cell agenesis. AMH was also determined in 15 control patients with idiopathic male pseudohermaphroditism. The serum AMH concentration was elevated in all testosterone-insensitive or -deficient patients compared with control levels during the first year of life. From 1 yr of age to the onset of puberty, serum AMH levels in patients with androgen insensitivity returned to normal values, but after pubertal development began, AMH levels again rose to extremely high levels in the complete androgen insensitivity syndrome. These results suggest that AMH is negatively regulated by testosterone not only at puberty, but also during the postnatal period. An elevation of serum AMH appears to be an interesting marker of androgen resistance or defect of androgen production in sexually ambiguous male infants.

[Androgen receptor and insensitivity to androgens]. / Récepteur des androgènes et insensibilités aux androgènes.

Androgen insensitivity syndromes are suspected in XY subjects with normal testosterone secretion presenting with absent or severely impaired androgen dependent sexual differentiation. Such clinical features suggest an abnormality of the androgen receptor, necessary step in the transmission of the hormonal message. The androgen receptor is a member of the steroid/thyroid nuclear receptors superfamily. It is a soluble protein of 919 amino acids, divided in independent functional domains responsible for the various functions of the receptor:hormone and DNA binding, and transcriptional activation. The highest concentration of androgen receptor is found, in both sexes, in tissues resulting from primary or secondary sexual differentiation. Cloning of the androgen receptor and use of molecular biology techniques have led to a new classification of androgen insensitivity syndromes. In complete forms (complete androgen insensitivity:CAI) the phenotype is feminine. In receptor negative CAI (Rc-: complete loss of hormone binding), molecular abnormalities include rare, partial or complete, deletions of the gene, or, more frequently, single point mutations in the hormone binding domain, leading to a functionally inactive receptor. Identification and characterization of these mutations provide valuable information regarding the functional importance of specific amino acids of the androgen receptor. In receptor positive CAI (RC+: conserved hormone binding capacity), abnormalities have been reported in the DNA binding domain (deletion of a zinc finger, single point mutations), but also in the hormone binding domain, thus distinguishing between the hormone binding activity and the transcriptional activation activity of this domain. Partial insensitivity syndromes are characterized by an ambiguous and extremely variable phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)

Natural mutagenesis study of the human steroid 5 alpha-reductase 2 isozyme.

The enzyme steroid 5 alpha-reductase utilizes NADPH to reduce the double bonds of a variety of steroid substrates with generalized 3-oxo, delta 4,5 structures. One substrate for this membrane-bound enzyme is testosterone, whose reduction to dihydrotestosterone is required for the embryonic differentiation of the external male genitalia and prostate. There are two 5 alpha-reductase isozymes, designated types 1 and 2, which have different biochemical and pharmacological properties. Inherited deficiencies of 5 alpha-reductase type 2 result in a form of male pseudohermaphroditism in which the external genitalia fail to develop normally. Here, nine additional mutations in the 5 alpha-reductase 2 gene in subjects with 5 alpha-reductase deficiency are described. The biochemical consequences of these mutations, as well as 13 previously identified missense mutations, were characterized by recreating the mutations in an expressible cDNA and transfecting into mammalian cells. Twelve of the 22 mutations inactivated the enzyme. The remaining 10 mutations impaired enzyme function by affecting either substrate or cofactor binding. Almost all mutations decreased the half-life of the protein, and all but one of the impaired enzymes had an altered pH optimum. The mutations provide insight into functional domains in the protein as well as an unusual acidic pH optimum characteristic of the 5 alpha-reductase type 2 isozyme.

A point mutation in the second zinc finger of the DNA-binding domain of the androgen receptor gene causes complete androgen insensitivity in two siblings with receptor-positive androgen resistance.

We have analyzed the nucleotide sequence of complementary and genomic DNAs of the human androgen receptor (AR) gene in two siblings (patients 9006 and 9030) with receptor-positive complete androgen insensitivity (Rec(+)-CAI). Northern analysis indicated that mRNA of the AR was normal in size. However, its expression was relatively reduced in both patients. Consistent with the normal androgen-binding capacity (496 and 552 fmol/mg DNA for patients 9006 and 9030, respectively) but decreased DNA-binding ability (168 fmol/mg DNA) measured in genital skin fibroblasts, no mutation was found in both N-terminal and ligand-binding domains of the AR. However, a single base substitution (G-->A) was found in the second zinc finger of the DNA-binding domain at nucleotide 2372 of the AR cDNA in both cases. This resulted in the replacement of a highly conserved arginine residue (amino acid 614) by a histidine. When the mutated receptor plasmid was cotransfected into PC-3 cells together with the reporter chloramphenicol acetyltransferase gene, chloramphenicol acetyltransferase activity was not induced by 5 alpha-dihydrotestosterone treatment, confirming that the mutation renders the AR nonfunctional and can, therefore, be held responsible for the clinical features in these patients. These results highlight the importance of Arginine-614 in the second zinc finger of the DNA-binding domain of the AR in the protein-DNA interaction.

Fertility in women with late-onset adrenal hyperplasia due to 21-hydroxylase deficiency.

Fertility was evaluated in 53 female patients with late-onset adrenal hyperplasia (LAH) due to 21-hydroxylase deficiency. The majority of patients (n = 33) were seen for isolated postpubertal hirsutism, 9 patients consulted for sterility, and 11 for irregular menstrual cycles. At the time of diagnosis, the ages of patients ranged from 15-40 yr (mean +/- SD, 24.6 +/- 5.2). No patient had major signs of virilization. The plasma 17-hydroxyprogesterone level was higher than normal in all patients (26.8 +/- 18.9 nmol/L; range, 3.4-139.4) and dramatically increased to 140.1 +/- 80.6 nmol/L (range, 35.2-324.2) after ACTH treatment. Plasma androgen levels were high (testosterone, 3.25 +/- 2.03 nmol/L; delta 4-androstenedione, 13.65 +/- 5.60 nmol/L). Plasma basal and LHRH-stimulated values were normal for FSH and high for LH. Basal and TRH-stimulated plasma PRL levels were normal. Among these 53 LAH patients, only 20 desired a pregnancy. These had a total of 38 pregnancies. Ten patients became pregnant before the diagnosis of LAH and without any treatment; they had a total of 18 pregnancies, 12 of which were successful. Moreover, 19 normal pregnancies without any spontaneous abortion were carried to term by 14 of 16 hydrocortisone-treated patients. One patient needed the association of one cure of clomiphene citrate. Hypofertility in LAH patients seems, therefore, to be relative. Its mechanism is hormonal, with anovulation or dysovulation, due to the continuous steroid feedback of adrenal origin on the hypothalamo-pituitary axis. Hydrocortisone is the appropriate treatment in most cases, reducing adrenal androgen overproduction and relieving hypothalamic-pituitary gonadotropin function, thereby making possible cyclic ovarian activity and ovulations.

Isoelectric focusing and 2D electrophoresis of the human androgen receptor.

Nuclear androgen receptors from cultured genital skin fibroblasts were analyzed by non-denaturing isoelectric focusing (IEF) in ultrathin polyacrylamide gels before and after photoaffinity labeling with [3H]methyltrienolone. Both reversibly and covalently labeled receptors focused at pH 5.28 +/- 0.20 when extracted from nuclei with high salt. Lowering of the salt concentration yielded, in both cases, a second species which focused at pH 7.16. This species became predominant when nuclei were sonicated in IEF sample buffer containing no salt, even after extensive nucleic acid digestion. Low salt cytosols from both prostate and foreskin focused as a single peak of pI: 4.93 +/- 0.31 which remained unchanged when KCl was added to the cytosol up to a concentration of 0.6 M. SDS-polyacrylamide gel electrophoresis of photoaffinity labeled receptors revealed labeled proteins with Mw 90-95 kDa. Two-dimensional electrophoresis of photoaffinity labeled nuclear receptors, extracted in low or high salt, showed that the two isoforms (pI 5.28 and 7.16) contain the same steroid-binding subunit with Mw 90-95 kDa. Nuclear receptors from 4 patients with the receptor positive form of the Complete Androgen Insensitivity Syndrome (CAIS, Rc+) were analyzed by non-denaturing IEF: a single species was observed, focusing at pH 6.0 whether in high or low salt conditions. These results indicate that the nuclear androgen receptor is an acidic protein with pI 5.28 and Mw 90-95 kDa under maximum protein dissociation conditions. When extracted under low salt conditions, it can be isolated in a neutral form (pI 7.16) suggesting its association with a nuclear protein. Receptors of (CAIS, Rc+) patients have an abnormal charge and show no pI shift upon lowering of the salt concentration suggesting that this shift could be a significant step in the mechanism of action of androgens.