Steroid 5 alpha-reductase 2 deficiency.

In the 20 yr since it was established that impairment of dihydrotestosterone formation is the cause of a rare form of human intersex, a wealth of information has accumulated about the genetics, endocrinology, and variable phenotypic manifestations, culminating in the cloning of cDNAs encoding two 5 alpha-reductase genes and documentation that mutations in the steroid 5 alpha-reductase 2 gene are the cause of 5 alpha-reductase deficiency. Perplexing and difficult problems remain unresolved, e.g. whether the variability in manifestations is due to variable expressions of steroid 5 alpha-reductase 1 or to effects of testosterone itself. It is also imperative to establish whether defects in steroid 5 alpha-reductase 2, perhaps in the heterozygous state, are responsible for a portion of cases of sporadic hypospadias, to determine whether 5 alpha-reductase plays a role in progesterone action in women, and to elucidate the relation between androgen action and gender role behavior.

Testicular feminization associated with a thermolabile androgen receptor in culutred human fibroblasts.

Evidence for a qualitative abnormality in the androgen receptor was obtained by studies of temperature sensitivity. The binding of [(3)H]dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one) was studied in monolayers of cultured genital skin fibroblasts from genetic males with abnormal sexual differentiation resulting from androgen resistance. Binding in cells from eight patients with a female phenotype (complete and incomplete testicular feminization) fell from half-normal levels at the usual assay temperature of 37 degrees C to levels <20% of normal when cells were incubated at 42 degrees C. This thermal inactivation was rapidly reversed when the assay temperature was lowered to 37 degrees C, was not associated with altered dihydrotestosterone metabolism, and was also demonstrable with [(3)H]methyltrienolone as the binding ligand. Binding increased to overlap the normal range when the assay temperature was lowered to 26 degrees C. The patients with receptor-deficient testicular feminization include three pairs of siblings; the pedigrees in two of these families are compatible with X-linkage. Only minor changes in the amount of binding at elevated temperatures were observed in cells from 10 control subjects and from 2 male pseudohermaphrodites with normal levels of androgen receptors. In 10 patients with androgen resistance and partial receptor deficiency associated with a predominantly male phenotype (Reifenstein syndrome and infertile men), dihydrotestosterone binding also did not change consistently with elevated temperature. Binding was approximately half-normal at 37 degrees C and either increased or decreased slightly at 42 degrees C. The thermal instability in receptor-deficient testicular feminization represents a new molecular defect associated with hereditary male pseudohermaphroditism that appears to be caused by an alteration in the tertiary structure of the androgen receptor protein.

Impaired stimulation of 25-hydroxyvitamin D-24-hydroxylase in fibroblasts from a patient with vitamin D-dependent rickets, type II. A form of receptor-positive resistance to 1,25-dihydroxyvitamin D3.

We describe studies of the molecular defect in 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] action in cultured skin fibroblasts from a patient previously reported to have vitamin D-dependent rickets, type II. Binding of [3H]1,25-(OH)2D3 in fibroblast cytosol was normal with a Bmax (amount of high affinity binding) of 26 fmol/mg protein and a half-maximal saturation of 0.2 nM. Nuclear binding of [3H]1,25-(OH)2D3 following whole cell uptake was 1.5 fmol/micrograms DNA in patient fibroblasts compared with a range of 0.5-2.9 fmol/micrograms DNA in five control strains. The size of the [3H]1,25-(OH)2D3-receptor complex on sucrose density gradients, 3.8 S, was the same as in normal cells. This patient, therefore, appeared to have a receptor-positive form of resistance to 1,25-(OH)2D3. To document resistance to 1,25-(OH)2D3 in the fibroblasts we developed a method for detection of 1,25-(OH)2D3 action in normal skin fibroblasts. Following treatment of normal cell monolayers with 1,25-(OH)2D3 there was more than a 20-fold increase of 25-hydroxy-vitamin D-24-hydroxylase (24-hydroxylase) activity. Treatment of 10 control cell strains with 1,25-(OH)2D3 for 8 h increased the formation of 24,25-dihydroxy-vitamin D3 from 25-hydroxyvitamin D3 in cell sonicates from less than 0.02 to 0.11-0.27 pmol/min per mg protein. When cells from the patient with vitamin D-dependent rickets, type II were treated with 1,25-(OH)2D3 in a similar manner, maximal 24-hydroxylase activity was only 0.02 pmol/min per mg protein, less than a fifth the lower limit of normal. 24-Hydroxylase activity in fibroblasts from the parents of the patient increased normally following treatment with 1,25-(OH)2D3. We conclude that impaired induction of 24-hydroxylase in the presence of normal receptor binding is evidence for postreceptor resistance to the action of 1,25-(OH)2D3.

Hereditary male pseudohermaphroditism associated with an unstable form of 5 alpha-reductase.

The properties of 5alpha-reductase have been compared in genital skin fibroblasts cultured from five patients from three families (Los Angeles, Dallas, and Dominican Republic) in which hereditary male pseudohermaphroditism has been established to result from deficient conversion of testosterone to dihydrotestosterone. Despite the fact that 5alpha-reductase was immeasurable in a homogenate of epididymis removed from one of the Los Angeles patients, 5alpha-reductase activity was normal in intact fibroblasts and fibroblast extracts from both patients from the Los Angeles family. Although the apparent K(m) for testosterone was also near normal, the apparent K(m) for NADPH in these mutants is elevated some 40-fold above normal. Furthermore, the enzyme is not protected against denaturation at 45 degrees C by concentrations of NADPH that stabilize normal 5alpha-reductase, and in intact fibroblasts from these patients (but not from controls), enzyme activity decreases promptly when protein synthesis is inhibited. We conclude that the mutation in this family results in an unstable enzyme. In contrast 5alpha-reductase activity in fibroblast extracts from a patient from the Dominican Republic family is similar to that previously described in two members of the Dallas family, namely total enzyme activity is low at the optimal pH for the normal reaction, and the apparent K(m) for testosterone is some 20-fold higher than that of the controls. We conclude that the mutations in the Dallas and Dominican Republic families are similar and result in low activity of the enzyme as the result of a decreased affinity for testosterone.Thus, two distinct types of mutations can produce male pseudohermaphroditism due to deficient dihydrotestosterone formation.

Dihydrotestosterone binding by cultured human fibroblasts. Comparison of cells from control subjects and from patients with hereditary male pseudohermaphroditism due to androgen resistance.

Dihydrotestosterone binding was measured in culture fibroblasts from 14 control subjects and from 12 patients with five different types of hereditary male pseudohermaphroditism. Two assays of binding were used--an intact monolayer assay and density gradient centrifugation of cell extracts. In the intact monolayer assay of normal cells the uptake of [3H]dihydrotestosterone consisted of two components. The first was a high affinity component that exhibited saturation at approximately 1 nM dihydrotestosterone. The second was a low affinity component that was not saturable with concentrations of steroid up to 5 nM. Twice the number of high affinity binding sites were present in fibroblasts grown from genital skin (foreskin, labia majora, and scrotum) as from nongenital sites (37 vs. 14 fmol/mg protein). In the density gradient assay in 5-10% sucrose, the major peak of dihydrotestosterone binding was in the 8S region in low molarity buffer and in the 4S region in 0.5 M KCl. High affinity binding was normal in cells from two patients with familial incomplete male pseudohermaphroditism, type 2, an autosomal recessive defect in which dihydrotestosterone formation is deficient, and in cells from a patient with male pseudohermaphroditism due to 17 beta-hydroxysteroid dehydrogenase deficiency, an autosomal recessive defect of testosterone synthesis. High affinity binding was low by both methods in fibroblasts from five patients with complete testicular feminization. Furthermore, binding by both methods was also low in cells from three subjects with familial incomplete male pseudohermaphroditism, type 1, a presumed X-linked recessive disorder of androgen resistance, and in fibroblasts grown from a subject with the incomplete form of testicular feminization. The finding that dihydrotestosterone binding is abnormal in two forms of hereditary androgen resistance in addition to complete testicular feminization suggests either that these disorders are the result of allelic mutations affecting the function of the androgen-binding protein or that normal dihydrotestosterone binding requires the participation of more than one gene product.

Amino acid substitutions in the hormone-binding domain of the human androgen receptor alter the stability of the hormone receptor complex.

We have investigated the basis of androgen resistance in seven unrelated individuals with complete testicular feminization or Reifenstein syndrome caused by single amino acid substitutions in the hormone-binding domain of the androgen receptor. Monolayer-binding assays of cultured genital skin fibroblasts demonstrated absent ligand binding, qualitative abnormalities of androgen binding, or a decreased amount of qualitatively normal receptor. The consequences of these mutations were examined by introducing the mutations by site-directed mutagenesis into the androgen receptor cDNA sequence and expressing the mutant cDNAs in mammalian cells. The effects of the amino acid substitutions on the binding of different androgens and on the capacity of the ligand-bound receptors to activate a reporter gene were investigated. Substantial differences were found in the responses of the mutant androgen receptors to incubation with testosterone, 5 alpha-dihydrotestosterone, and mibolerone. In several instances, increased doses of hormone or increased frequency of hormone addition to the incubation medium resulted in normal or near normal activation of a reporter gene by cells expressing the mutant androgen receptors. These studies suggest that the stability of the hormone receptor complex is a major determinant of receptor function in vivo.

Complete testicular feminization caused by an amino-terminal truncation of the androgen receptor with downstream initiation.

We have characterized the molecular defect causing androgen resistance in two 46,XY siblings with complete testicular feminization. Although binding studies in genital skin fibroblasts showed a reduced Bmax, an increased dissociation rate of ligand, and an 8S peak of dihydrotestosterone binding on sucrose density gradient centrifugation, no immunoreactive androgen receptor (AR) was detected in immunoblots using anti-NH2-terminal antibodies, suggesting an abnormal amino terminus. Sequence analysis of the AR gene revealed a point mutation CAG-->TAG (Gln-->Stop) at nucleotide 340. In vitro mutagenesis studies suggest the synthesis of the mutant AR is initiated downstream of the termination codon at reduced levels and that each molecule is functionally impaired. These results define a novel mechanism causing androgen resistance: the combination of decreased amount and functional impairment of AR caused by an abnormality within the amino terminus of the receptor. These findings suggest that domains important to the in vivo function of the receptor reside within the amino terminus and that disruption of these domains can occur with only subtle effects on receptor binding. Identification of this mutation made it possible to identify the mutant allele within the family and to ascertain antenatally that it was not present in a 46,XY fetal sibling of the proband at 9 wk gestation.

Molecular basis of androgen resistance in a family with a qualitative abnormality of the androgen receptor and responsive to high-dose androgen therapy.

We have examined the nature of the mutant androgen receptor in a family with a severe defect in virilization associated with a qualitative defect in receptor function. The androgen receptor gene in this family contains two structural alterations: a single nucleotide substitution at position 2444 in exon 5 (adenosine----guanosine) that converts tyrosine 761 to a cysteine residue and a shortened glutamine homopolymeric segment in exon 1 that encodes 12 rather than the usual 20-22 glutamines. A family study was performed using polymerase chain reaction amplification of the glutamine-rich segment, and it was shown that the sister of the proband does not carry the mutant allele. The effects of these two mutations on the function of the androgen receptor were studied by introducing the changes, individually and in combination, into cDNAs encoding the normal human androgen receptor and analyzing the receptor protein produced after transfection of the cDNAs into eukaryotic cells. The presence of a cysteine residue at position 761 causes rapid dissociation of dihydrotestosterone from the receptor protein. Marked thermolability of the transfected receptor protein, however, was demonstrable only upon introduction of an androgen receptor cDNA containing both the partial deletion of the glutamine homopolymeric segment and a cysteine residue at position 761. Likewise, the ability of the receptor to stimulate a reporter gene is strikingly diminished only when both alterations are present, suggesting that the shortened glutamine homopolymeric segment amplifies the impairment of receptor function caused by the tyrosine to cysteine substitution.

A mutation in the DNA-binding domain of the androgen receptor gene causes complete testicular feminization in a patient with receptor-positive androgen resistance.

Androgen resistance is associated with a wide range of quantitative and qualitative defects in the androgen receptor. However, fibroblast cultures from approximately 10% of patients with the clinical, endocrine, and genetic features characteristic of androgen resistance express normal quantities of apparently normal androgen receptor in cultured genital skin fibroblasts (receptor-positive androgen resistance). We have analyzed the androgen receptor gene of one patient (P321) with receptor-positive, complete testicular feminization and detected a single nucleotide substitution at nucleotide 2006 (G----C) within the second "zinc finger" of the DNA-binding domain that results in the conversion of the arginine residue at position 615 into a proline residue. Introduction of this mutation into the androgen receptor cDNA and transfection of the expression plasmid into eukaryotic cells lead to the synthesis of a receptor protein that displays normal binding kinetics but is inactive in functional assays of receptor activity. We conclude that substitution mutations in the DNA-binding domain of the androgen receptor are one cause of "receptor-positive" androgen resistance.

Mutations in the ligand-binding domain of the androgen receptor gene cluster in two regions of the gene.

We have analyzed the nucleotide sequence of the androgen receptor from 22 unrelated subjects with substitution mutations of the hormone-binding domain. Eleven had the phenotype of complete testicular feminization, four had incomplete testicular feminization, and seven had Reifenstein syndrome. The underlying functional defect in cultured skin fibroblasts included individuals with absent, qualitative, or quantitative defects in ligand binding. 19 of the 21 substitution mutations (90%) cluster in two regions that account for approximately 35% of the hormone-binding domain, namely, between amino acids 726 and 772 and between amino acids 826 and 864. The fact that one of these regions is homologous to a region of the human thyroid hormone receptor (hTR-beta) which is a known cluster site for mutations that cause thyroid hormone resistance implies that this localization of mutations is not a coincidence. These regions of the androgen receptor may be of particular importance for the formation and function of the hormone-receptor complex.

A mutation that causes lability of the androgen receptor under conditions that normally promote transformation to the DNA-binding state.

Dihydrotestosterone-receptor complexes formed in human fibroblast cytosol prepared at 0 degrees C in the presence of sodium molybdate can be readily transformed to the DNA-binding state by heating at 25 degrees C. Under these conditions 50-70% of dihydrotestosterone-receptor complexes bind to DNA. We describe here studies of the transformation process in cytosols derived from normal cells and from fibroblasts propagated from subjects with syndromes of androgen resistance. In contrast to the situation with dihydrotestosterone, normal testosterone-receptor complexes are unstable under in vitro transforming conditions. Although equal amounts of hormone-receptor complex are formed at 0 degrees C, only 15% of testosterone-receptor complexes remain stable and acquire DNA-binding capacity after warming. This instability is not reversible upon lowering the temperature and is corrected by low concentrations (0.25 microM) of the protease inhibitor leupeptin. We have also identified two cousins with androgen resistance whose androgen-receptor complexes exhibit similar in vitro transformation lability with both dihydrotestosterone and testosterone. Phenotypic evidence in these subjects indicates that dihydrotestosterone-mediated processes are more completely impaired than are testosterone-mediated events. These findings suggest that dihydrotestosterone may amplify the androgenic signal at its targets not only by its higher affinity for the receptor but also by its more efficient conversion to the DNA-binding state and that such amplification may be less critical in target tissues in which testosterone suffices for androgenic effect. This offers one possible explanation of how a mutation that affects a single receptor protein may differentially impair the actions of two binding ligands of the receptor.

A single nucleotide substitution introduces a premature termination codon into the androgen receptor gene of a patient with receptor-negative androgen resistance.

Mutations of the androgen receptor that impair the action of 5 alpha-dihydrotestosterone and testosterone result in abnormal male sexual development. The definition of the organization of the androgen receptor gene has permitted us to examine its structure in nine patients with androgen resistance that exhibit absent 5 alpha-dihydrotestosterone binding in cultured fibroblasts (receptor-negative androgen resistance). Using labeled probes specific for each individual coding exon, we find no gross rearrangements, insertions, or deletions of the androgen receptor gene in these patients. To analyze the genetic defect in these receptor-negative patients, we used the polymerase chain reaction to amplify each individual exon of the androgen receptor gene in nine affected patients. In all patients, the size of each amplified exon segment was identical to that in normal individuals. The nucleotide sequence of the entire coding region of the androgen receptor was determined in one of these patients. A single nucleotide substitution was identified that results in a premature termination codon in exon 6 at amino acid 794. S1 nuclease protection assays demonstrated that normal levels of androgen receptor mRNA are present in skin fibroblasts of this patient. Transfection of a mutated androgen receptor cDNA containing a termination codon at position 794 into eukaryotic cells resulted in formation of a normal amount of receptor protein, as indicated by immunoblotting, but the expressed protein does not bind 5 alpha-dihydrotestosterone. These findings suggest that the presence of a premature termination codon at amino acid 794 of the androgen receptor is the cause of androgen resistance in this patient.

Molecular genetics of steroid 5 alpha-reductase 2 deficiency.

Two isozymes of steroid 5 alpha-reductase encoded by separate loci catalyze the conversion of testosterone to dihydrotestosterone. Inherited defects in the type 2 isozyme lead to male pseudohermaphroditism in which affected males have a normal internal urogenital tract but external genitalia resembling those of a female. The 5 alpha-reductase type 2 gene (gene symbol SRD5A2) was cloned and shown to contain five exons and four introns. The gene was localized to chromosome 2 band p23 by somatic cell hybrid mapping and chromosomal in situ hybridization. Molecular analysis of the SRD5A2 gene resulted in the identification of 18 mutations in 11 homozygotes, 6 compound heterozygotes, and 4 inferred compound heterozygotes from 23 families with 5 alpha-reductase deficiency. 6 apparent recurrent mutations were detected in 19 different ethnic backgrounds. In two patients, the catalytic efficiency of the mutant enzymes correlated with the severity of the disease. The high proportion of compound heterozygotes suggests that the carrier frequency of mutations in the 5 alpha-reductase type 2 gene may be higher than previously thought.

High dose androgen therapy in male pseudohermaphroditism due to 5 alpha-reductase deficiency and disorders of the androgen receptor.

We describe the clinical and biochemical features of six men with male pseudohermaphroditism due to androgen resistance. Each of the subjects had male-gender behavior but incomplete virilization. The underlying defects in androgen metabolism were defined by studies of the 5 alpha-reductase enzyme and the androgen receptor in fibroblasts cultured from biopsies of genital skin. Four of the six have 5 alpha-reductase deficiency, and two have defects of the androgen receptor (the Reifenstein syndrome). The responses of these men to androgen treatment were assessed by monitoring nitrogen balance, plasma luteinizing hormone (LH) values, and clinical parameters of virilization including penile growth, potency and ejaculatory volume, muscle bulk, and growth of body and facial hair. In all of the subjects with 5 alpha-reductase deficiency and one man with the Reifenstein syndrome significant response occurred, as evidence by nitrogen retention, lowered plasma LH levels, and improved virilization, with doses of parenteral testosterone esters that raised plasma testosterone levels above the normal male range and brought plasma dihydrotestosterone levels into the normal male range. The subject who did not respond with clinical virilization nevertheless showed nitrogen retention in response to acute testosterone administration. This patient had a profound deficiency of the androgen receptor, whereas the man with a receptor defect who did respond clinically to therapy had normal amounts of a qualitatively abnormal receptor. We conclude that high dose androgen therapy may be of benefit in improving virilization, self-image, and sexual performance in subjects with 5 alpha-reductase deficiency who have male-gender behavior and in some subjects with defects of the androgen receptor.

Amino acid substitutions in the DNA-binding domain of the human androgen receptor are a frequent cause of receptor-binding positive androgen resistance.

In some subjects with genetic and endocrine evidence of androgen resistance, no defect is demonstrable in the binding of androgen to its receptor in cultured genital skin fibroblasts. We have defined the molecular defect in the androgen receptor in four unrelated subjects in this category (termed receptor positive) with the phenotype of compete or incomplete testicular feminization. In these patients we detected amino acid substitutions in either exon 2 or exon 3, which encodes the DNA-binding domain of the androgen receptor. In one patient with incomplete testicular feminization, two separate mutations were present in exon 3. Introduction of these amino acid substitutions into the androgen receptor-coding segment leads to the expression of receptor proteins that bind ligand in a normal fashion but do not activate the transcription of the androgen-responsive mouse mammary tumor virus promoter. Mobility shift assays using androgen receptor fusion proteins produced in E. coli indicate that these mutations impair binding of the receptor to specific DNA sequences. In the subject with incomplete testicular feminization, a Ser-Gly substitution at amino acid residue 595 is able to partially restore DNA-binding activity to a mutant receptor protein that carries an Arg-Pro substitution at position 615. These findings indicate that mutations in amino acid residues crucial to the binding of the androgen receptor to target DNA sequences are a common cause of receptor-binding positive androgen resistance and that variable impairment of DNA binding can lead to distinctive phenotypes.

Definition of the human androgen receptor gene structure permits the identification of mutations that cause androgen resistance: premature termination of the receptor protein at amino acid residue 588 causes complete androgen resistance.

We have isolated and characterized the gene encoding the human androgen receptor. The coding sequence is divided into eight coding exons and spans a minimum of 54 kilobases. The positions of the exon boundaries are highly conserved when compared to the location of the exon boundaries of the chicken progesterone and human estrogen receptor genes. Definition of the intron/exon boundaries has permitted the synthesis of specific oligonucleotides for use in the amplification of segments of the androgen receptor gene from samples of total genomic DNA. This technique allows the analysis of all segments of the androgen receptor gene except a small region of exon 1 that encodes the glycine homopolymeric segment. Using these methods we have analyzed samples of DNA prepared from a patient with complete androgen resistance and have detected a single nucleotide substitution at nucleotide 1924 in exon 3 of the androgen receptor gene that results in the conversion of a lysine codon into a premature termination codon at amino acid position 588. The introduction of a termination codon into the sequence of the normal androgen receptor cDNA at this position leads to a decrease in the amount of mRNA encoding the human androgen receptor and the synthesis of a truncated receptor protein that is unable to bind ligand and is unable to activate the long terminal repeat of the mouse mammary tumor virus in cotransfection assays.

Inactivation of the luteinizing hormone/chorionic gonadotropin receptor by an insertional mutation in Leydig cell hypoplasia.

We previously identified a nonsense mutation (Cys545Stop) in the paternal human LH/CG receptor (hLHR) allele in a family with two 46,XY children afflicted with Leydig cell hypoplasia. This mutation abolished the signal transduction capability of the affected hLHR. We have now examined all coding exons and the transcript of both alleles of the hLHR gene of the affected children. A 33-bp in-frame insertion was found in the maternal hLHR allele. This insertion occurred between nucleotide 54 and 55 and might be the result of a partial gene duplication. Genomic DNA-PCR showed that this defective maternal hLHR allele was inherited by the two affected children. However, examination of the inheritance of the 935-A/G polymorphism of the hLHR by genomic- and RT-PCR indicated that the maternal hLHR allele was not expressed in cultured fibroblasts of the patients. The effect of the in-frame insertion on the biological activity of the hLHR was examined by expressing the mutated hLHR construct, generated by site-directed mutagenesis, in HEK 293 cells. The expression of the mRNA for the mutant hLHR in HEK 293 cells was not affected. Response of cells expressing the mutated hLHR to hCG stimulation was impaired as demonstrated by reduced intracellular cAMP biosynthesis. This change in signal transduction was the result of a profound reduction in hormone binding at the cell surface due to altered expression and processing of the mutated receptor. We conclude that Leydig cell hypoplasia in this family is the result of compound heterozygous loss-of-function mutations of the hLHR gene.

Transformation of the androgen receptor to the deoxyribonucleic acid-binding state: studies in homogenates and intact cells.

Transformation of the [3H]dihydrotestosterone-receptor complex to the DNA-binding state was studied in intact monolayers and in homogenates of cultured human fibroblasts and mouse L-cells. When homogenates of either cell type were prepared in low salt buffer, incubated at 0 C with [3H]dihydrotestosterone, chromatographed on DNA-Sepharose, and eluted with a NaCl gradient, the receptor complex was eluted at 25 mM NaCl (peak A). After incubation of the homogenate at 25 C for 20 min, peak A decreased in amplitude. The major peak of the receptor from human fibroblasts eluted at 100 mM NaCl, while that from L-cells eluted at 170 mM NaCl (peak B). Flow-through fractions contained only minimal amounts of transformable dihydrotestosterone-receptor complex under the same conditions. Furthermore, isolated peak A could be converted to peak B by the same warming process. The appearance of peak B was prevented when 10 mM, but not 1 mM, sodium molybdate was present during the homogenization process. Unoccupied receptor was recovered exclusively in peak A both at 0 C and after incubation at 25 C. When intact fibroblast and L-cell monolayers were incubated with [3H]dihydrotestosterone at 37 C, all receptor in both cytosol and nuclear extract was recovered in peak B. In sucrose density gradient centrifugation, peak A was 6-8S in size, and peak B was 4.6S. These findings suggest that peak A corresponds to the nontransformed and peak B to the transformed states of the androgen receptor; the transformation reaction may be the consequence of a dissociation of a macromolecular complex into subunits; and sodium molybdate acts to stabilize the macromolecular complex.

Transformation of human androgen receptors to the deoxyribonucleic acid-binding state.

The transformation of [3H]dihydrotestosterone-androgen receptor complexes to the nuclear binding state was studied in fibroblasts cultured from human genital skin, using binding of the androgen-receptor complex to DNA-cellulose as a measure of transformation. Transformation of cytosolic dihydrotestosterone-receptor complexes proceeded slowly at 0 C, and heating to 25 C caused rapid transformation that was essentially complete within 15 min. Sodium molybdate (up to 10 mM) slowed the rate of transformation in a concentration-dependent fashion; low concentrations of molybdate (0.5-1 mM) prevented transformation at 0 C, but transformation of up to 70% of the total cytosolic hormone-receptor complexes could be achieved in the presence of high concentrations of molybdate by appropriately prolonged heating at 25 C. Molybdate appeared to exhibit a dual effect, increasing the total amount of androgen-receptor complex recoverable in cytosol and prolonging the time course of heat-induced transformation. Treatment of cytosolic dihydrotestosterone-receptor complexes with 0.5 M NaCl caused a degree of transformation similar to that seen after heat treatment but was unaffected by 10 mM molybdate. Transformation was accompanied by disappearance of the 7S form of the receptor on sucrose density gradients, but there was no reciprocal increase in a smaller size binding moiety on low ionic strength sucrose density gradients. The heat-transformed receptor could be detected, however, as a 3S binding peak on gradients containing 0.3 M NaCl, suggesting that transformation was accompanied by a structural alteration of the hormone-receptor complex.

Intranuclear binding of [3H]dihydrotestosterone by cultured human fibroblasts.

The uptake of [1,2,4,5,6,7-3H]dihydrotestosterone into whole cells and nuclei has been assessed in fibroblasts grown from genital skin of 10 controls and 9 subjects with hereditary male pseudohermaphroditism due to androgen resistance. The cells were exposed to hypotonic buffer and ruptured by passage through a 25-gauge needle, and the nuclei were purified by sedimentation through 2.1 M sucrose. Uptake of the hormone into nuclei reached an apparent plateau in 45 min, was saturable at 1 nM dihydrotestosterone, and was not detectable in the presence of excess nonradioactive hormones. Over a wide range of uptake by intact cells from control subjects and from subjects with androgen resistance due to 5alpha-reductase deficiency or receptor deficiency, nuclear uptake averaged about half of the total cell uptake. Furthermore, in cells from two unrelated 46,XY phenotypic females with androgen resistance but normal 5alpha-reductase activity and normal whole cell dihydrotestosterone binding, uptake into the nucleus was also normal. In these subjects, the defect in androgen action must be at some terminal phase of androgen action.