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.

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.

Steroid 5 alpha-reductase deficiency in a 65-year-old male pseudohermaphrodite: the natural history, ultrastructure of the testes, and evidence for inherited enzyme heterogeneity.

We report a 65-yr-old male pseudohermaphrodite with steroid 5 alpha-reductase deficiency in whom there was no medical intervention before, during, or after puberty, enabling us to observe the natural history of this condition. The affected subject has an android build, with more facial and body hair than in previously described affected adults. Although the subject was raised as a girl, a male gender identity evolved with the events of puberty, but social factors have delayed the complete expression of a male gender role. Plasma levels of dihydrotestosterone and the in vivo conversion of radiolabeled testosterone to dihydrotestosterone were decreased. There was an elevated urinary etiocholanolone to androsterone ratio, typical of the syndrome. Characterization of 5 alpha-reductase enzyme activity in cultured genital skin fibroblasts demonstrated a pattern of enzyme activity distinctly different from three previously described families with this condition. There was decreased enzyme affinity for testosterone and NADPH. Also, the stability of the enzyme to elevated temperature was not protected by NADPH, resulting in rapid disappearance of enzyme activity after inhibition of protein synthesis with cycloheximide. Electron microscopic evaluation of the testes was carried out.

Characterization of the testicular abnormality in 5 alpha-reductase deficiency.

The testes of five phenotypic women (from four families) with 5 alpha-reductase deficiency were studied. In one of the patients, the enzyme deficiency was similar in the testis and epididymis and in fibroblasts cultured from the labia majora. In testes from four of the patients, the concentrations of the 5 alpha-reduced steroids dihydrotestosterone and 3 alpha-androstanediol were less than 10% of those in normal subjects. We conclude that the testis is involved in 5 alpha-reductase deficiency. Impaired spermatogenesis was evident in testicular biopsies from all five subjects, and in two, sperm production, as estimated in testicular homogenates, was less than 10% of normal. The extent to which spermatogenic arrest is due to 5 alpha-reductase deficiency or testicular maldescent is not clear.

Acute adrenal insufficiency: recognition, management, and prevention.

Acute adrenal insufficiency may present only with nonspecific symptoms and signs. Hyperpigmentation is not a feature of secondary adrenal insufficiency and is absent in patients with primary adrenal failure of recent or acute onset. Similarly, characteristic electrolyte disturbances may be obscured by concomitant vomiting and diarrhea as well as by parenteral electrolyte replacement. A high index of suspicion must therefore be maintained to make the diagnosis of acute adrenal insufficiency in patients without a recognized history of autoimmune adrenal insufficiency or of other diseases or therapeutic regimens known to result in pituitary-adrenal failure. Timely intervention with volume and glucocorticoid replacement rapidly reverses all symptoms and signs of adrenal insufficiency. Guidelines are presented for glucocorticoid replacement in the treatment of adrenal crisis as well as for the prevention of acute adrenal insufficiency in patients with known or suspected pituitary-adrenal disease. In addition, recommendations are given for the simultaneous diagnosis and treatment of adrenal insufficiency in patients without previously established disease.

Genetic control of extraglandular aromatase activity in the chicken.

Feminization of feathers in the Sebright cock is the result of increase in the activity of skin aromatase. This increased estrogen synthesis is the consequence of an autosomal dominant mutation that causes an increase in the specific androgen-binding cytochrome P450 oxidase involved in the reaction. Since this oxidase appears to be kinetically indistinguishable from the activity in control ovary we believe that the mutation causes an increased steady-state level of normal enzyme. The mechanism by which the mutation acts is unknown, but its presence implies that in normal birds an allele of the mutation limits the activity of the enzyme in all tissues other than ovary.

Polyglandular autoimmune syndromes.

Dysfunction of multiple endocrine glands may develop as the result of hypopituitarism, various infiltrative disorders, or an organ-specific autoimmune mechanism. When dysfunction of two or more endocrine glands occurs in association with circulating organ-specific antibodies directed against the involved glands, the term polyglandular autoimmune syndrome is applied. Characteristics of polyglandular autoimmunity include specific patterns of disease association and frequently a family history of similar involvement. The principal endocrine components of these syndromes are adrenal insufficiency, autoimmune thyroid disease, insulin-dependent diabetes mellitus, and premature gonadal failure. In addition, primary hypoparathyroidism is a key feature of one form of polyglandular autoimmunity that occurs in children. Several nonendocrine organ-specific autoimmune disorders are also associated with polyglandular autoimmunity, of which pernicious anemia is the most frequent. The underlying abnormality responsible for polyglandular autoimmunity is most likely a defect in T suppressor cell function, but there is evidence that aberrant expression of HLA DR antigens also plays an important role in the pathogenesis of these disorders.

Hirsutism.

Hirsutism is a common clinical problem. The pathogenesis of hirsutism in most women is not clearly defined. In this review, the characteristic endocrinologic findings in women with hirsutism are described, and the disorder's evaluation and treatment are discussed.

5-Azacytidine and sodium butyrate induce expression of aromatase in fibroblasts from chickens carrying the henny feathering trait but not from wild-type chickens.

Male chickens with the henny feathering trait have a female feathering pattern. In two henny-feathered breeds, the Sebright bantam and the Golden Campine, the synthesis of estrogen is increased as a consequence of increased activity of aromatase, a cytochrome P-450 enzyme that converts androgen to estrogen. The activity of the enzyme is elevated in tissue slices and in cultured fibroblasts from heterozygous and homozygous birds of both breeds. In contrast, aromatase activity is very low in extraglandular tissues from control chickens and is undetectable in fibroblasts cultured from these tissues. The current studies show that two agents known to alter gene expression--5-azacytidine and sodium butyrate--markedly induce expression of aromatase activity in Sebright and Campine fibroblasts but have no effect on aromatase activity in fibroblasts from wild-type chickens. Induction of aromatase is specific since two other microsomal enzymes in chicken fibroblasts--one, a component of the aromatase enzyme complex and the other a cytochrome P-450 oxidase distinct from the aromatase--are not significantly affected by these agents. Further study of this unique mutation should provide insight into the mechanisms by which genes are switched to an uninducible state during differentiation.

Solubilization and partial purification of aromatase from chicken ovary.

Aromatase, the cytochrome P-450 that converts androgen to estrogen, has been solubilized from chicken ovarian microsomes with the nonionic detergent Emulgen 913. Following chromatography on gel filtration, anion exchange, dye affinity, and hydrophobic media, ovarian aromatase is purified up to 27-fold with 10-15% recovery. Separation of the cytochrome P-450 aromatase from NADPH cytochrome P-450 reductase is achieved during the purification. The partially purified enzyme is stable for as long as 6 months when frozen in liquid nitrogen in buffer containing dithiothreitol, glycerol, Emulgen and 150 mM KCl.

Inhibition of steroid 5 alpha-reductase from human skin fibroblasts by 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one.

The capacity of 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one (DMAA) to inhibit competitively steroid 5 alpha-reductase has been confirmed in human foreskin fibroblasts. The inhibitor works equally well in homogenates and in intact cells, is rapidly reversible, does not influence the amount of the enzyme, is equally effective in inhibiting the 5 alpha-reduction of testosterone, 4-androstene-3,17-dione, 20 alpha-hydroxy-4-pregnen-3-one and cortisol, and appears to be weakly bound to serum protein. The inhibitor is particularly effective (apparent Ki of approximately 3 nM) at the optimal pH of the enzyme (pH 5.5) and has no effect in the alkaline range (pH 9.0). DMAA appears to be a steroid that will be useful in the investigation of 5 alpha-reduction of steroid hormones in intact animals and in embryos.

Androgen resistance syndromes.

Hereditary defects that impede androgen action cause resistance to the hormone both during embryogenesis and in later life and hence usually cause developmental defects of the male urogenital tract. In genetic males such defects produce a phenotypic spectrum ranging from infertile but otherwise normal men to individuals with varying degrees of ambiguous genitalia to phenotypic women. These disorders can be classified on the basis of the step in androgen action that is impeded by the individual mutations. 5 alpha-Reductase deficiency is an autosomal recessive enzyme defect that impairs the conversion of testosterone to dihydrotestosterone. The internal male genital tract virilizes normally, but the external genitalia are predominantly female in character. The syndrome is the result of one of several mutations that impair the function of the 5 alpha-reductase enzyme. A variety of disorders influence the androgen receptor that mediates the action of both testosterone and dihydrotestosterone. At least four phenotypic variants can be distinguished: complete testicular feminization, incomplete testicular feminization, the Reifenstein syndrome, and the infertile male syndrome, each of which is inherited as an X-linked trait. Absence of receptor binding is found commonly in complete testicular feminization, but qualitative and/or less severe quantitative defects in receptor function can be associated with all four variants. A third type of disorder, receptor positive resistance, also causes variable defects in male development and is associated with normal 5 alpha-reductase activity and normal androgen receptor. The underlying defect is presumed to lie at the intranuclear site or sites of action of the hormone-receptor complex.

Characterization of the increased estrogen synthesis in skin fibroblasts from the Sebright bantam.

We have utilized the Sebright bantam chicken as a model system to explore the regulation of estrogen formation in peripheral tissues. As the result of a gene mutation, Sebright males develop a female feathering pattern associated with an increase in estrogen synthesis in skin and in fibroblasts cultured from skin. To provide insight into the mechanisms by which estrogen synthesis is increased we examined several parameters of the aromatase reaction in homogenates from control ovaries, Sebright ovaries and fibroblasts grown from Sebright skin: the pH optima; the apparent Km's for testosterone, 19-nortestosterone, androstenedione, 16-hydroxyandrostenedione, and NADPH; the apparent Ki's for 4-hydroxyandrostenedione, aminoglutethimide, dehydroepiandrosterone, and 19-hydroxytestosterone; and the inactivation of the reaction by heating. No qualitative differences were identified in the enzyme from Sebright and control birds.

Role of gonadal hormones in development of the sexual phenotypes.

Male and female embryos develop in an identical fashion during the initial portion of gestation. If the indifferent gonad differentiates into an ovary (or if no gonad is present), a female phenotype is formed. Male phenotypic differentiation, however, requires the presence of an endocrinologically active testis. Two secretions of the fetal testis, Müllerian inhibiting substance and testosterone, are responsible for male development. Studies of single gene mutations that interfere with androgen action indicate that testosterone itself is responsible for virilization of the Wolffian duct system into the epididymis, vas deferens, and seminal vesicle, whereas the testosterone metabolite dihydrotestosterone induces development of the prostate and male external genitalia. Thus, impairment of dihydrotestosterone formation results in a characteristics phenotype consisting of predominantly female external genitalia but normally virilized Wolffian ducts. The molecular mechanisms by which testosterone and dihydrotestosterone act during fetal development appear to involve the same high affinity receptor, a protein that transports both testosterone and dihydrotestosterone to the nucleus of target cells. When this receptor is either absent, deficient, or structurally abnormal, the actions of both testosterone and dihydrotestosterone are impaired, and the resulting developmental anomalies involve both internal and external genital structures.

Increased estrogen formation and aromatase activity in fibroblasts cultured from the skin of chickens with the Henny feathering trait.

The henny feathering trait in chickens leads to a marked increase in the conversion of androgen to estrogen in skin and other peripheral tissues with the result that feathers of affected males are feminized. To gain insight into the mechanisms responsible for this increased estrogen synthesis, we studied the conversion of testosterone to estrogen in fibroblasts cultured from the skin of control chickens and from two breeds carrying the henny feathering trait, the Sebright bantam and the Campine. Estrogen synthesis was measured in suspensions of intact fibroblasts and in cell-free fibroblast extracts by two assays: 1) direct measurement of 17 beta-estradiol formation from [1,2,6,7-3H]testosterone, and 2) assessment of 3H2O release from [1 beta-3H]testosterone. Both assays gave comparable results. Estrogen formation was as much as several hundred-fold higher in fibroblasts cultured from skin of chickens carrying the henny feathering trait compared to that observed in fibroblasts from skin of control chickens. The current data indicate that increased estrogen formation in skin of chickens with the henny feathering trait is due to an enhanced activity of the aromatase complex of enzymes responsible for estrogen synthesis. The molecular basis for this increased activity is unclear.

The endocrine control of male phenotypic development.

Male and female embryos develop in an identical fashion during the initial portion of gestation. If the indifferent gonad differentiates into an ovary (or if no gonad is present), a female phenotype is formed. Male phenotypic differentiation, however, requires the presence of an endocrinologically active testis. Two secretions of the foetal testis, Mullerian-inhibiting substance and testosterone, are responsible for male development. Testosterone itself is responsible for virilization of the Wolffian duct system into the epididymis, vas deferens, and seminal vesicle, whereas dihydrotestosterone induces development of the prostate and male external genitalia. Thus, impairment of dihydrotestosterone formation results in a characteristic phenotype consisting of predominantly female external genitalia but normally virilized Wolffian ducts. The molecular mechanisms by which testosterone and dihydrotestosterone act during foetal development appear to involve the same high affinity receptor, a protein that transports both testosterone and dihydrotestosterone to the nucleus of target cells. When this receptor is either absent, deficient, or structurally abnormal, the actions of both testosterone and dihydrotestosterone are impaired, and the resulting developmental anomalies involve both internal and external genital structures.