Role of gonadal hormones in development of the sexual phenotypes

Summary 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 secretion 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 characteristic 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.The original work described in this review was supported by grant AM 03892 from the National Institutes of Health     

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.     

Inhibition of androgen binding in human foreskin fibroblasts by antiandrogens

Antiandrogen effects on androgen receptor binding and androgen metabolism were studied in cultured human newborn foreskin fibroblasts. Three different antiandrogens were tested in this system: (a) cyproterone acetate (CA); (b) RU23908; and (c) R2956. CA and R2956 were equipotent inhibitors of androgen binding to its intracellular receptor. The magnitude of this action was nearly twice as great against the endogenous androgen ligands, dihydrotestosterone (DHT) or testosterone (T), than with the synthetic ligand, methyltrienolone (R1881). Whereas the relative binding affinities of CA and R2956 were approximately 5-10 times less than T or DHT, RU23908 was another order of magnitude less effective as an inhibitor of androgen binding. The lower relative binding affinity determined for RU23908 could not be explained on the basis of a requirement for metabolic activation. Subcellular fractionation studies and sucrose density gradient analysis further confirmed the rank order of antiandrogenic potency. None of the antiandrogens influenced the rate or profile of metabolites from cellular metabolism of T or DHT. We propose that cultured human genital skin fibroblasts may serve as a valuable system for the future evaluation of antiandrogens in intact ells under physiologic conditions.     

Virilization of the male pouch young of the tammar wallaby does not appear to be mediated by plasma testosterone or dihydrotestosterone.

Virilization of the male urogenital tract of all mammals, including marsupials, is mediated by androgenic hormones secreted by the testes. We have previously demonstrated profound sexual dimorphism in the concentrations of gonadal androgens in pouch young of the tammar wallaby Macropus eugenii during the interval when the urogenital sinus virilizes. To provide insight into the mechanisms by which androgens are transported from the testes to the target tissues, we measured testosterone and dihydrotestosterone in plasma pools from tammar pouch young from the day of birth to Day 150. Plasma testosterone levels were measurable (0.5-2 ng/ml) at all times studied, but there were no differences between males and females. These low concentrations of plasma testosterone appear to be derived from the adrenal glands and not the testes. Plasma dihydrotestosterone levels in plasma pools from these animals were also low and not sexually dimorphic. We conclude that virilization of the male urogenital tract cannot be explained by the usual transport of testosterone or dihydrotestosterone in plasma but may be mediated by the direct delivery of androgens to the urogenital tract via the Wolffian ducts. Alternatively, circulating prohormones may be converted to androgens in target tissues.     

The influence of 4-hydroxy-4-androstene-3,17-dione on androgen metabolism and action in cultured human foreskin fibroblasts

4-hydroxy-4-androstene-3,17-dione (4-OHA) has been shown to be a potent inhibitor of aromatase activity. It is effective in the control of estrogen-dependent processes in female subjects and may potentially be useful in the treatment of estrogen-dependent processes in men. Human foreskin fibroblasts grown in cell culture provide a model to investigate the effects of 4-OHA on extraglandular aromatase activity as well as the ability of the compound to influence androgen receptor binding and the 5 alpha-reduction of testosterone (T). Initial experiments were carried out to determine the potency of 4-OHA in genital skin fibroblasts by incubating cells with 4-OHA over a range of concentrations. When aromatase activity was determined at a substrate concentration close to the apparent Km of the enzyme, a 44% inhibition of enzyme activity occurred at a mean concentration of 5 nM 4-OHA. Enzyme kinetic studies analyzed by Eadie-Hofstee plots demonstrated competitive inhibition by 4-OHA with a mean apparent Ki of 2.7 nM. When 5 alpha-reductase activity was determined in the presence of 200 nM [3H]T, in the absence or presence of 4-OHA, a 50% inhibition of enzyme activity occurred at an inhibitor concentration of 3 microM. In androgen receptor binding studies, 4-OHA possessed 1% of the affinity of dihydrotestosterone (DHT) for [3H]DHT binding sites. In summary: 4-OHA is a potent and specific inhibitor of aromatase activity in human genital skin fibroblasts, the affinity of the enzyme for 4-OHA being greater than its affinity for the substrate, androstenedione. The influence of 4-OHA on 5 alpha-reductase activity and androgen receptor binding is minimal.     

Up-regulation of androgen receptor binding in male rat fat pad adipose precursor cells exposed to testosterone: study in a whole cell assay system

Binding of androgens to adipocytes has previously been evaluated using cytosol fractions without taking into account nuclear binding, although the latter is suggested to be close to the physiological site of action. In the present study, performed in differentiated fat pad adipose precursor cells, we describe a simple, reliable and reproducible androgen binding assay in a system with intact cells. Tritiated and unlabeled methyltrienolone (R1881) were used to define specific and unspecific androgen binding. Triamcinolone acetonide was added to prevent the binding of R1881 to other types of receptors. Differentiated adipose precursor cells contain a homogeneous class of high affinity androgen binding sites, and binding is saturable and reversible. Binding apparently occurs at one site, with a Kd in the range of physiological androgen concentration (about 4 nM). Competition studies indicate that the receptor is specific for R1881, testosterone and dihydrotestosterone, which have approximately the same affinity, while progesterone, estradiol and dexamethasone show much lower affinity. Androgen binding was markedly enhanced after cellular exposure to R1881 and testosterone but not dihydrotestosterone, and this increase was dependent on protein synthesis, suggesting the formation of new receptors by these androgens. In conclusion, fully differentiated adipocytes contain a specific, high affinity receptor, the density of which is dependent on androgens.     

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.     

Phenotypic feminization in a genetic male dog caused by nonfunctional androgen receptors

A dog raised as a female pup did not have utero-ovarian structures when ovariohysterectomy was attempted at 6 mo of age. Three months later, the dog exhibited male-like behavior, and 2 symmetrical testicular-shaped structures arose bilateral to the vulva. Intersex cases such as this one may involve chromosomal abnormalities, defects in testicular secretions or androgen insensitivity in tissues of the sex accessories. Serum concentrations of testosterone and dihydrotestosterone were measured in the intersex and in intact normal male (control; n=7) dogs. In addition, 5alpha-reductase enzyme activity was determined in the gonadal tissue of the intersex dog and control dogs. Androgen receptors were studied in cultured fibroblasts from the genital skin of the intersex and control dogs. The cytogenetic studies identified the animal's karyotype to be 78 XY. All complete cells from the gonadal tissue showed an XY sex chromosome complement. Serum testosterone concentrations before and after challenge with hCG were not different between the intersex and control dogs (6.2 vs 5.72 +/- 1.8 ng/ml and 9.5 vs 8.69 +/- 2.39 ng/ml before and after hCG challenge, respectively). Similarly serum dihydrotestosterone concentrations were not different between the intersex and control dogs before and after challenge with hCG (158 vs 162 +/- 1.4 pg/ml and 270 vs 278.71 +/- 45.98 pg/ml before and after hCG challenge, respectively). The 5alpha-reductase enzyme activity of the gonads was not different between the intersex and control dogs (105 pmol/h/mg protein vs 110 +/- 2.4 pmol/h/mg protein). The Bmax values of binding in the control dogs fibroblast strains were 25 +/- 3.5 fmol/mg protein. However, specific binding of dihydrotestosterone was virtually undetectable in the fibroblasts cultured from the intersex dog. These results exclude chromosomal abnormalities and deficient secretion of testosterone and dihydrotestosterone as causative factors. Because fibroblasts cultured from genital skin lacked any ability to specifically bind dihydrotestosterone, it is suggested that nonfunctional androgen receptors in some tissues of the sex accessories contributed to the feminine phenotype of this particular dog.     

The 56 kDa protein of human genital skin fibroblasts is identical to that radiolabelled by [3H]dihydrotestosterone 17 beta-bromoacetate

Analysis of soluble proteins from human genital skin fibroblasts by two-dimensional polyacrylamide gel electrophoresis reveals an abundant protein doublet of mol. wt 56,000 with isoelectric points (pI) of 6.7 and 6.5. This protein is absent in non-genital skin fibroblasts as well as in genital skin fibroblasts of most patients with complete forms of androgen insensitivity. The protein specifically binds androgen. A protein of similar estimated molecular weight (58,000) from human genital skin fibroblasts has recently been found to be covalently radiolabelled by the affinity ligand dihydrotestosterone 17 beta-bromoacetate (DHT-BA). In the present study these proteins have been found to be indistinguishable on one- and two-dimensional gel electrophoresis. Antibodies raised against the 56 kDa pI 6.7/6.5 protein also recognized the protein covalently radiolabelled by DHT-BA. A third protein of estimated mol. wt 59,000 has been found to be associated with several steroid hormone receptor complexes but has no known ligand binding activity. This protein was found to be clearly separable from the 56/58 kDa protein on two-dimensional gel electrophoresis as it has a more acidic pI of approximately 5.4. Furthermore, antibodies against the 59 kDa protein do not recognize the 56 kDa species, and vice versa.     

Androgens and the development of the vagina

Today it is generally held that the vagina develops from sinovaginal bulbs and that the lower third of the definitive vagina is derived from the urogenital sinus. Here we show that the entire vagina arises by downward growth of Wolffian and Müllerian ducts, that the sinovaginal bulbs are in fact the caudal ends of the Wolffian ducts, and that vaginal development is under negative control of androgens. We designed a genetic experiment in which the androgen receptor defect in the Tfm mouse was used to examine the effects of androgens. Vaginal development was studied by 3D reconstruction in androgen-treated female embryos and in complete androgen-insensitive littermates. In androgen-treated females, descent of the genital ducts was inhibited, and a vagina formed in androgen-insensitive Tfm embryos as it does in normal females. By immmunohistochemical localization of the androgen receptor in normal mouse embryos, we demonstrated that the androgen receptor was expressed in Wolffian duct and urogenital sinus-derived structures, and was entirely absent in the Müllerian duct derivatives. We conclude that the Wolffian ducts are instrumental in conveying the negative control by androgens on vaginal development. The results are discussed under evolutionary aspects at the transition from marsupial to eutherian mammals.     

Clinical, biochemical and morphologic diagnostic markers in an infant male pseudohermaphrodite patient with compound heterozygous mutations (G115D/R246W) in SRD5A2 gene

A patient with male pseudohermaphroditism and clinical diagnosis of partial androgen insensitivity in the neonatal period was studied at pubertal age for a molecular diagnosis. Hormone studies were conducted at baseline and under hCG stimulation for testosterone and dihydrotestosterone determinations at 2 months of age. Gonadectomy was performed at 4 months. At the age of 13 years genital skin fibroblasts were studied for androgen binding and 5alpha-reductase activity and peripheral blood DNA was available for androgen receptor (AR) and 5alpha-reductase (SRD5A2) gene analysis. Exons 1-8 of AR gene and exons 1-5 of SRD5A2 gene were sequenced. AR gene coding sequences were normal. SRD5A2 gene analysis revealed two heterozygote mutations (G115D and R246W), with the mother carrying the G115D and the father the R246W mutations. The compound heterozygote mutations in SRD5A2 gene explained an extremely low 5alpha-reductase enzyme activity in genital skin fibroblasts. Revision of hormonal data from the neonatal period revealed an increased testosterone-to-dihydrotestosterone ratio at the end of an hCG stimulation test, which concurred with the molecular diagnosis. Testis morphology at 4 months of age was normal. Clinical and biochemical differential diagnosis between partial androgen insensitivity syndrome and 5alpha-reductase enzyme deficiency is difficult in the neonatal period and before puberty. Our results show that in our patient the testosterone-to-dihydrotestosterone ratio would have adequately orientated the diagnosis. The two mutations in SRD5A2 gene have been described in patients of different lineages, though not in combination to date. Testis morphology showed that, during early infancy, the 5alpha-reductase deficiency may not have affected interstitial or tubular development.     

Why boys will be boys: two pathways of fetal testicular androgen biosynthesis are needed for male sexual differentiation

Human sexual determination is initiated by a cascade of genes that lead to the development of the fetal gonad. Whereas development of the female external genitalia does not require fetal ovarian hormones, male genital development requires the action of testicular testosterone and its more potent derivative dihydrotestosterone (DHT). The "classic" biosynthetic pathway from cholesterol to testosterone in the testis and the subsequent conversion of testosterone to DHT in genital skin is well established. Recently, an alternative pathway leading to DHT has been described in marsupials, but its potential importance to human development is unclear. AKR1C2 is an enzyme that participates in the alternative but not the classic pathway. Using a candidate gene approach, we identified AKR1C2 mutations with sex-limited recessive inheritance in four 46,XY individuals with disordered sexual development (DSD). Analysis of the inheritance of microsatellite markers excluded other candidate loci. Affected individuals had moderate to severe undervirilization at birth; when recreated by site-directed mutagenesis and expressed in bacteria, the mutant AKR1C2 had diminished but not absent catalytic activities. The 46,XY DSD individuals also carry a mutation causing aberrant splicing in AKR1C4, which encodes an enzyme with similar activity. This suggests a mode of inheritance where the severity of the developmental defect depends on the number of mutations in the two genes. An unrelated 46,XY DSD patient carried AKR1C2 mutations on both alleles, confirming the essential role of AKR1C2 and corroborating the hypothesis that both the classic and alternative pathways of testicular androgen biosynthesis are needed for normal human male sexual differentiation.     

The 56 kDa androgen binding protein is an aldehyde dehydrogenase.

We have described a 56 kDa protein from genital skin fibroblasts that specifically binds androgen and that is generally not expressed in genital skin fibroblasts from patients with androgen insensitivity due to genetic defects of the androgen receptor. We have isolated a partial cDNA clone for the 56 kDa protein from an expression library of genital skin fibroblasts. In vitro translation of message selected with this clone faithfully produces the 56 kDa protein which can be immuneprecipitated with an anti-56 kDa antiserum. Northern blots probed with this clone show a 2.2 kb message, which parallels the expression of the 56 kDa protein. The sequence of this 998bp clone is identical to human liver aldehyde dehydrogenase 1, the cytoplasmic isoenzyme. On activity gels of genital skin fibroblast cytosol covalently labelled with androgen, aldehyde dehydrogenase activity comigrates with the single band labelled specifically with androgen. Thus, the 56 kDa androgen binding protein is an aldehyde dehydrogenase, which is prominently expressed in normal genital skin fibroblasts, but not in non-genital skin fibroblasts.     

Differential effects of prenatal testosterone timing and duration on phenotypic and behavioral masculinization and defeminization of female sheep

The process of sexual differentiation leaves genetically female individuals at risk of being masculinized by exogenous androgens. Previous research with sheep indicates that exposure to excess testosterone from Gestational Day (GD) 30 to GD 90 of the 147-day gestation masculinizes and defeminizes behavior as well as genitalia. Lower doses and shorter durations produce animals with varying degrees of genital virilization and alterations of the hypothalamic-pituitary-gonadal axis, but to our knowledge, the effects on complex behavior and its prediction by the amount of external virilization have not been explored. Previous research in rodents has suggested that sexual differentiation of the central nervous system and the external genitalia can be dissociated. Therefore, we hypothesized that the extent of virilization of external genitalia would not be predictive of the lack of female-typical, or the presence of male-typical, mating behavior. To test this hypothesis, we compared control females, females exposed to exogenous testosterone from GD 30 to GD 90 (T60 females) that have virilized genitalia, and females exposed to testosterone from GD 60 to GD 90 (T30 females) that have female-typical genitalia. Both natural behavioral estrus in the flock and hormonally controlled behavioral tests were used to explore reproductive behavior. The T60 and T30 females exhibited more masculinized reproductive behavior than the controls; however, the T30 females also exhibited feminine behavior. Neither testosterone-treated group was receptive or was mounted at rates comparable to those of controls. These data illustrate that variation in the timing or duration of exposure to prenatal testosterone during a critical period for masculinization can have variable effects on defeminization and that the effects of testosterone on genitalia are not entirely predictive of behavior.     

Expression of the androgen receptor and 5α-reductase type 2 in the developing human fetal penis and urethra

Normal penile development is dependent on testosterone, its conversion via steroid 5 alpha-reductase type 2 to dihydrotestosterone, and a functional androgen receptor (AR). The goal of this study was to investigate the distribution of AR and 5 alpha-reductase type 2 in the developing human fetal external genitalia with special emphasis on urethra formation. Twenty fetal genital specimens from normal human males (12-20 weeks gestation) were sectioned serially and stained by avidin-biotinylated peroxidase complex method with antigen retrieval. Stained sections throughout male genital development documented the expression of AR and 5 alpha-reductase type 2 in the phallus. Between 12 and 14 weeks of gestation, AR was localized to epithelial cells of the urethral plate in the glans, the tubular urethra of the penile shaft, and stromal tissue surrounding the urethral epithelium. In the fetal penis between 16 and 20 weeks gestation, the density of AR expression was greatest in urethral epithelial cells versus the surrounding stromal tissues. There was a characteristic pattern of AR expression in the glandular urethral epithelium between 16 and 20 weeks gestation. AR expression was greater along the ventral aspect of the glandular urethra than along the dorsal aspect of the urethral epithelium. The expression of 5 alpha-reductase type 2 was localized to the stroma surrounding the urethra, especially along the urethral seam area in the ventral portion of the remodeling urethra. These anatomical studies support the hypothesis that androgens are essential for the formation of the ventral portion of the urethra and that abnormalities in either the AR or 5 alpha-reductase type 2 can explain the occurrence of hypospadias.