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PubMed1979

Brain regional levels of neurotransmitter amines as neurochemical correlates of sex-specific ontogenesis in the rat.

Brain regional levels of three neurotransmitter amines - serotonin (5-HT), norepinephrine (NE), dopamine (DA) - were measured in young rats prior to weaning to determine the extent to which modifications in levels of amines might reflect alterations in the sex steroid hormonal environment during the first postnatal week in the life of the rat. Sex-related levels of DA, NE, and 5-HT were found in some brain regions of the 12-day-old rat. Male midbrain DA exceeded the corresponding female value while female hypothalamic NE levels were greater than those of the male. Levels of 5-HT in the corpus striatum and the midbrain of males were greater than those of the female. Castration of the male on day 1 or testosterone propionate (TP) administration to the newborn female resulted in modifications of levels of midbrain 5-HT which reflected feminization of the castrated males and masculinization of the TP-treated females. Castration on day 1, or diethylstilbestrol given on days 2, 4, and 6, resulted in apparent feminization of NE levels in the hypothalamus of 12-day-old male rats. Thus, it appears that regional levels of hypothalamic NE and midbrain 5-HT in the 12-day-old rat may reflect the course of brain organizational activity which becomes recognizable in the adult as sex-specific behavior.

Animals↗
PubMed1999

Phytoestrogens inhibit growth and MAP kinase activity in human aortic smooth muscle cells.

-Estrogens are known to induce cardioprotective effects by inhibiting smooth muscle cell (SMC) growth and neointima formation. However, the use of estrogens as cardioprotective agents is limited by carcinogenic effects in women and feminizing effects in men. If noncarcinogenic and nonfeminizing estrogenlike compounds, such as natural phytoestrogens, afford cardioprotection, this would provide a safe method for prevention of cardiovascular disease in both men and women. Therefore, we evaluated and compared in human aortic SMCs the effects of phytoestrogens (formononetin, genistein, biochanin A, daidzein, and equol) on 2.5% fetal calf serum-induced proliferation (3H-thymidine incorporation and cell number), collagen synthesis (3H-proline incorporation), and total protein synthesis (3H-leucine incorporation) and on PDGF-BB (25 ng/mL)-induced migration (modified Boydens chambers). Moreover, the effects of phytoestrogens on PDGF-BB (25 ng/mL)-induced mitogen-activated protein kinase (MAP kinase) activity in SMCs was also studied. Phytoestrogens inhibited proliferation, collagen and total protein synthesis, migration, and MAP kinase activity in a concentration-dependent manner and in the following order of potency: biochanin A>genistein>equol>daidzein>formononetin. In conclusion, our studies provide the first evidence that in human aortic SMCs phytoestrogens inhibit mitogen-induced proliferation, migration and extracellular matrix synthesis and inhibit/downregulate MAP kinase activity. Thus, phytoestrogens may confer protective effects on the cardiovascular system by inhibiting vascular remodeling and neointima formation and may be clinically useful as a safer substitute for feminizing estrogens in preventing cardiovascular disease in both women and men.

Adult↗
PubMed1969

The conversion of testosterone to 5 alpha-androstan-17 beta-ol-3-one (dihydrotestosterone) by skin slices of man.

The conversion of testosterone-1,2-(3)H to dihydrotestosterone by slices of human skin obtained from various anatomical sites in 112 normal subjects and three individuals with the syndrome of testicular feminization has been measured under standardized conditions. Very low rates of dihydrotestosterone formation were observed in sites obtained from the mons or from miscellaneous areas of the trunk and limbs of the control subjects. The mean rates of conversion were very high, however, in slices of skin obtained from several perineal sites (labia majora, scrotum, prepuce, and clitoris). Furthermore, as measured here, the rate of dihydrotestosterone formation by prepuce rises during the 3 months after birth and then falls progressively thereafter, reaching a level in the adult that is almost as low as that observed in the slices of nonperineal skin from all ages. In the patients with testicular feminization dihydrotestosterone formation by slices of skin obtained from the mons was within the normal range, whereas the rates observed in labia majora were lower than the average values obtained in the normal subjects.

Adolescent↗
PubMed1983

Familial incomplete male pseudohermaphroditism associated with impaired nuclear androgen retention. Studies in cultured skin fibroblasts.

The androgen resistance syndromes are generally felt to be due to quantitative or qualitative abnormalities of the androgen receptor. Some patients with testicular feminization have no demonstrable fibroblast cytosol androgen binding, whereas others have androgen binding in cultured fibrobalsts that is thermolabile or fails to be stabilized by sodium molybdate. I describe here familial incomplete testicular feminization associated with reduced nuclear androgen retention. Fibroblasts, cultured from pubic skin biopsies of two phenotypic female 46XY siblings, were assayed for whole cell and nuclear uptake of [(3)H]dihydrotestosterone in dispersed, intact cells. Whole cell binding of [(3)H]dihydrotestosterone at 22 degrees C in the patients' fibroblasts was in the normal range. However, no high affinity, saturable binding of [(3)H]dihydrotestosterone was demonstrable in crude nuclear pellets prepared from the patients' fibroblasts incubated at 37 degrees C with the hormone. Incubating the patients' cells with [(3)H]methyltrienolone or examining the nuclear uptake of [(3)H]dihydrotestosterone in these cells at 22 degrees C did not alter these findings. Although cytosol from the patients' cells revealed a quantitatively diminished 8S peak for [(3)H]dihydrotestosterone after centrifugation on sodium molybdate-containing sucrose gradients, there was no peak of (3)H in the 4S region from 0.3 M KCl nuclear extracts of the patients' cells after they had been incubated with [(3)H]dihydrotestosterone at 37 degrees C. Although whole cell binding studies at 37 degrees C showed minimally diminished androgen binding in the patients' cells compared with binding at 22 degrees C, Griffin (1979. J. Clin. Invest.64: 1624-1631.) has demonstrated thermolability of the androgen receptors in fibroblasts also cultured from these patients. The observations with intact cells coupled with the diminished cytosol 8S peak of [(3)H]dihydrotestosterone on sucrose gradients indicate that these patients have cytosol androgen receptors that are qualitatively abnormal physicochemically, the physiologic consequence of which is failure of nuclear androgen localization. Thus, although the underlying defect in the pathogenesis of the androgen resistance in these patients appears to reside in the androgen receptor, the crucial biologic manifestation of the molecular lesion is impaired nuclear androgen retention. These experiments, therefore, suggest that assessment of nuclear [(3)H]dihydrotestosterone uptake is an effective indicator of the functional integrity of the androgen receptor system in patients with various forms of androgen insensitivity and provides additional insights to those obtained by thermolability or cytosol sucrose gradient studies.

Adolescent↗
PubMed1992

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.

Amino Acid Sequence↗
PubMed2006

Embryonic exposure to the fungicide vinclozolin causes virilization of females and alteration of progesterone receptor expression in vivo: an experimental study in mice.

BACKGROUND: Vinclozolin is a fungicide that has been reported to have anti-androgenic effects in rats. We have found that in utero exposure to natural or synthetic progesterones can induce hypospadias in mice, and that the synthetic progesterone medroxyprogesterone acetate (MPA) feminizes male and virilizes female genital tubercles. In the current work, we selected a relatively low dose of vinclozolin to examine its in utero effects on the development of the genital tubercle, both at the morphological and molecular levels. METHODS: We gave pregnant dams vinclozolin by oral gavage from gestational days 13 through 17. We assessed the fetal genital tubercles from exposed fetuses at E19 to determine location of the urethral opening. After determination of gonadal sex, either genital tubercles were harvested for mRNA quantitation, or urethras were injected with a plastic resin for casting. We analyzed quantified mRNA levels between treated and untreated animals for mRNA levels of estrogen receptors alpha and beta, progesterone receptor, and androgen receptor using nonparametric tests or ANOVA. To determine effects on urethral length (males have long urethras compared to females), we measured the lengths of the casts and performed ANOVA analysis on these data. RESULTS: Our morphological results indicated that vinclozolin has morphological effects similar to those of MPA, feminizing males (hypospadias) and masculinizing females (longer urethras). Because these results reflected our MPA results, we investigated the effects of in utero vinclozolin exposure on the mRNA expression levels of androgen, estrogen alpha and beta, and progesterone receptors. At the molecular level, vinclozolin down-regulated estrogen receptor alpha mRNA in females and up-regulated progesterone receptor mRNA. Vinclozolin-exposed males exhibited up-regulated estrogen receptor alpha and progesterone receptor mRNA, effects we have also seen with exposure to the synthetic estrogen, ethinyl estradiol. CONCLUSION: The results suggest that vinclozolin virilizes females and directly or indirectly affects progesterone receptor expression. It also affects estrogen receptor expression in a sex-based manner. We found no in vivo effect of vinclozolin on androgen receptor expression. We propose that vinclozolin, which has been designated an anti-androgen, may also exert its effects by involving additional steroid-signaling pathways.

Analysis of Variance↗
PubMed1982

On the primary site of action of estrogens and androgens in the regulation of hepatic prolactin receptors.

The aim of the present study was to identify the primary sites of action of estrogens and androgens in the regulation of hepatic PRL receptors in the rat. Implantation of estradiol benzoate in the pituitary region of male rats caused a feminization (i.e. an increase) of PRL receptors to a concentration typical of that found in female rats. Estrogen implanted in the paraventricular region of male rats was less effective in causing such a feminization. Subcutaneous implantation of estrogen did not affect the PRL receptor concentration. It is concluded that estrogen induces PRL receptors via an action at the hypothalamo-pituitary level, possibly directly on the pituitary. The PRL receptor-suppressive action of sc injected testosterone was also exerted by the synthetic androgen R1881 (17 beta-hydroxy-17 alpha-methylestra-4,9,11-trien-3-one). However, anterior hypothalamic deafferentation rendered both male and female rats insensitive to this action of R1881. It is concluded that an intact hypothalamo-pituitary unit is required for the PRL receptor-suppressive action of R1991. It is possible that the site of action of androgens is in the rostral hypothalamus or in adjacent areas of the brain.

Animals↗
PubMed1985

The development of a male pseudohermaphroditic rat using an inhibitor of the enzyme 5 alpha-reductase.

Incomplete masculinization of the external genitalia occurred in male Sprague-Dawley rats treated with a potent inhibitor of enzyme 5 alpha-reductase at the critical period of sexual differentiation in utero. The studies were performed using the 5 alpha-reductase inhibitor, 4-methyl-4-aza-5-pregnan-3-one-20[s] carboxylate, one of a series of aza steroids known to competitively inhibit the enzyme 5 alpha-reductase. The degree of inhibition of male external genital development was dependent upon the dose of the inhibitor, and at a dose of 36 mg/kg X day, there was complete feminization of the external genitalia of the male animal with a urogenital sinus and a pseudovagina. These studies provide conclusive evidence for the hypothesis that 5 alpha-reductase activity and dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one) formation are essential for normal differentiation of male external genitalia. Epididymidis, vasa deferentia, and seminal vesicles were present at all doses of the inhibitor, suggesting testosterone dependency. However, confirmation of the testosterone dependency of Wolffian ductal differentiation awaits further studies, particularly comparison studies with the rabbit and dog, since Wolffian ductal differentiation in the rat, unlike the rabbit and dog, is not abolished with the antiandrogen, cyproterone acetate. The presence of prostatic buds, despite complete external genital feminization, was unexpected and suggests that these structures may have different thresholds of response for dihydrotestosterone. Prostatic differentiation may have a much lower threshold, requiring less dihydrotestosterone for differentiation.

5-alpha Reductase Inhibitors↗
PubMed1985

Imprinting of growth hormone secretion, body growth, and hepatic steroid metabolism by neonatal testosterone.

The influence of endogenous sex steroids and exogenous testosterone treatment on pulsatile GH secretion, body weight, longitudinal bone growth, and hepatic steroid metabolism was studied in male and female adult rats. Blood samples were obtained from the tip of the tail, and maximum and minimum GH levels were determined in individual rats to evaluate pulse heights and baseline levels. Longitudinal bone growth was measured using the intravital marker tetracycline, and hepatic steroid metabolism was evaluated by determining the enzyme activities of 16 alpha-hydroxylase and 5 alpha-reductase. Neonatal, but not prepubertal, gonadectomy of male rats suppressed maximum and mean plasma GH levels during adult life. The body weight and the rate of longitudinal bone growth were also decreased. Testosterone treatment neonatally reversed all of these effects. Neonatal gonadectomy of male rats also caused an elevation of minimum plasma GH levels, an effect, however, which was not reversed by testosterone replacement during neonatal life. Neonatally gonadectomized females treated with testosterone neonatally or during adult life increased their maximum and decreased their minimum GH levels. Their longitudinal bone growth was increased. The body weight of these rats was increased by neonatal, but not adult, testosterone treatment. There was no effect of neonatal ovariectomy on plasma GH levels in 3- to 4-month-old female rats. However, neonatal ovariectomy did increase the maximum and mean plasma GH levels immediately postpubertally, suggesting that the effect of the ovaries on GH secretion differs among mature female rats of different ages. Prepubertal gonadectomy of male rats feminized their hepatic steroid metabolism by decreasing 16 alpha-hydroxylase and increasing 5 alpha-reductase activities. Neonatal gonadectomy caused an even more pronounced feminization, which was partly reversed in rats given testosterone replacement therapy neonatally. In neonatally gonadectomized female rats, treatment with testosterone during adult life increased 16 alpha-hydroxylase and decreased 5 alpha-reductase to levels seen in intact male rats. The present results indicate that neonatally secreted testicular androgens imprint the high amplitude pulses characteristic of GH secretion in adult male rats. Neonatal androgens also stimulate somatic growth and partially account for the masculinized hepatic steroid metabolism in the adult animal. It is proposed that imprinting of the GH secretory pattern contributes to the influence of neonatal testicular androgens on body growth and hepatic steroid-metabolizing enzymes.

Animals↗
PubMed1985

Hypothalamo-pituitary regulation of cytochrome P-450(15) beta apoprotein levels in rat liver.

The regulation of the sexually differentiated steroid sulfate 15 beta-hydroxylase, cytochrome P-450(15) beta of female rat liver has been investigated. Specific antibodies raised to isozyme P-450(15) beta were used with the Western blot technique to quantitate the specific levels of P-450(15) beta in liver microsomes. The method demonstrated that the levels of the protein are about 16-fold higher in female than in male microsomes and also showed that the specific microsomal content of P-450(15) beta is controlled by GH. Hypophysectomy of female animals resulted in a decrease of P-450(15) beta to male levels. Continuous infusion of human GH, mimicking the female pattern of GH secretion in intact male animals, caused an elevation of the P-450(15) beta level to that of the female. The same dose of human GH in hypophysectomized male or female animals raised the P-450(15) beta level 8-fold or 50% of that seen in normal females. Infusion of ovine PRL to intact male rats had no effect on P-450(15) beta levels, whereas infusion of rat GH caused a 4-fold increase. Thus, the regulation of P-450(15) beta by GH is mainly associated with the somatogenic properties of the hormone. Furthermore, sc injection of rat GH every 12 h, mimicking the male pattern of GH secretion, had no effect on P-450(15) beta levels, demonstrating the importance of the GH secretory pattern in regulation of the specific protein levels. Postpubertal castration of male animals did not influence the microsomal P-450(15) beta content, whereas neonatal castration led to a feminization of the P-450(15) beta content in the adult male rat. Administration of estradiol valerate to male animals caused complete feminization of P-450(15) beta levels, whereas administration of androgen to female animals caused a decrease to male levels. Before 21 days of age, the P-450(15) beta level was slightly higher in male than in female rats. At 35 days, however, the P-450(15) beta level in female rats had increased almost 100-fold, whereas the levels in males increased only slightly. These changes are concomitant with the development of the sexual differentiation of the GH secretory pattern, supporting the role of GH in P-450(15) beta regulation. In conclusion, isozyme P-450(15) beta is a GH-regulated enzyme specific for female rats. The low level of the protein in males is probably explained by neonatal androgenic programming of the GH secretory pattern.

Androgens↗
PubMed1994

The sensitivity of hepatic CYP2C gene expression to baseline growth hormone (GH) bioactivity in dwarf rats: effects of GH-binding protein in vivo.

Hepatic mRNA transcripts for the steroid-metabolizing enzymes cytochrome P4502C11 (male specific) and P4502C12 (female specific) differ in abundance by 10- to 20-fold in male and female rats and are regulated by their different patterns of GH secretion. This sex difference is also found in dwarf rats with low GH secretion, implying that these transcripts may be very sensitive to low level GH exposure. This has now been characterized in normal and dwarf rats. Continuous i.v. infusion of recombinant human (h) GH (0, 3, 12, and 48 micrograms/day) in both dwarf and normal male rats caused a dose-dependent decrease in P4502C11 and an increase in P4502C12, so that the 2C11/2C12 ratio fell from 17.9 +/- 1.3 to 1.5 +/- 1.0 in normal males and from 6.5 +/- 0.9 to 0.4 +/- 0.3 in dwarf males (0 vs. 48 micrograms hGH/day); over this dose range of hGH, body weight gain, total hepatic insulin-like growth factor-I mRNA levels, and plasma GHBP levels were largely unaffected. These effects of hGH were pattern dependent. The 2C11/2C12 ratio in dwarf males was feminized (from 11.9 +/- 1.3 to 0.08 +/- 0.03) by continuous infusion of hGH (36 micrograms/day), whereas a pulsatile infusion (3-min pulses every 3 h) of the same daily hGH dose was much less effective. Neither continuous nor pulsatile hGH affected P4502C11 or P4502C12 transcripts in dwarf females, although pulsatile hGH infusion caused a significant weight gain. To test whether baseline GH levels could be modified by circulating GH-binding protein (GHBP), hGH infusions were given with and without recombinant hGHBP in different patterns. Pulsatile infusions of recombinant hGHBP (42 micrograms/day, i.v.) did not prevent the feminizing effect of continuously infused hGH (36 micrograms/day, sc) in dwarf males (2C11/2C12 ratios were 0.08 +/- 0.01 and 0.09 +/- 0.01 for hGH vs. hGH plus hGHBP, respectively). This suggested that intermittent complex formation with GHBP did not prevent continuous access of hGH to the hepatic GH receptors. Furthermore, pulses of hGH complexed with GHBP significantly reduced the 2C11/2C12 ratio in dwarf males (from 21.5 +/- 3.9 with pulsatile hGH alone to 9.2 +/- 2.5 with pulses of hGH plus hGHBP), indicating that GHBP prolongs the exposure to hGH. Thus, 2C11/2C12 expression is very sensitive to basal GH levels in dwarf rats, and GHBP can alter hepatic gene expression by modifying the pattern of GH exposure.

Animals↗
PubMed2003

Expression of functional estrogen receptors in human fetal male external genitalia.

It is generally assumed that male genital development is determined by androgens on a default program leading to female genitalia. Female genitalia virilization is due to high levels of androgens, whereas feminization is linked to reduction or lack of fetal androgen. Excess androgen determines sex reversion in female, whereas excess estrogen does not cause male feminization. In the present study, we investigate the presence of androgen receptors (AR) and estrogen receptors (ER) in human fetal penile tissue and in a cellular model of human fetal penile smooth muscle cells (hfPSMC). By immunohistochemistry, we showed the presence of ER and AR in the developing penile tissue of male fetuses. Besides the presence of AR, hfPSMC showed ERalpha/beta as demonstrated by RT-PCR, Western blot, and binding techniques. These receptors are functionally active because cell stimulation with 17beta-estradiol increased progesterone receptor B expression and inhibited hfPSMC growth, both effects being reversed by tamoxifen. Conversely, cell proliferation was stimulated by R1881 and testosterone, an effect enhanced by letrozole. These findings are the first demonstration of the presence of functional ER in differentiating male external genitalia and indicate a possible novel inhibitory role of estrogens in the regulation of the development of these sex structures.

Aromatase Inhibitors↗
PubMed2005

Salutary effects of combining early very low-dose systemic estradiol with growth hormone therapy in girls with Turner syndrome.

CONTEXT: Optimizing pubertal estrogen replacement in girls with Turner syndrome is important. OBJECTIVE: The study objective was to test the hypotheses that physiological estradiol replacement administered early with GH will preserve height potential as much as if administered late and that it will bring about a greater height gain than standard oral estrogen therapy combined with GH. DESIGN: The study was randomized to early or late estrogen treatment; follow-up was at 3.5 yr or later. SETTING: This was a multicenter outpatient study. PATIENTS: Turner syndrome girls 12.0-12.9 yr (n = 7) or 14.0-14.9 yr (n = 7) of age who began GH before age 12.0 yr were the patients. The girls were matched to National Cooperative Growth Study registry patients who began GH and oral conjugated estrogen at similar ages and were similarly followed to adult or near-adult height. INTERVENTIONS: Depot estradiol, 0.2 mg/month i.m., was given initially and gradually increased; GH was 0.05 mg/kg daily. MAIN OUTCOME VARIABLE: Adult or near-adult height was the main outcome variable. RESULTS: Depot estradiol treatment resulted in height significantly taller than predicted at 12 yr of age (P < 0.02). All height potential was gained in the first 2 yr of the study, during which the early group grew 3.5 cm more than the late group, which was receiving GH alone (P < 0.01). The early depot estradiol group also gained 5.9 cm more height after starting estrogen than did the early National Cooperative Growth Study group (P < 0.05). Although feminization proceeded slowly on the lowest dose of estradiol, it advanced normally thereafter. CONCLUSIONS: These results suggest that very low-dose parenteral estradiol permits relatively age-appropriate feminization without interfering with the effect of GH on the enhancement of height potential.

Body Height↗
PubMed1982

The frequency of androgen receptor deficiency in infertile men.

To ascertain the frequency of androgen resistance as the cause of male infertility and to determine whether endocrine abnormalities are a universal feature of the disorder, we measured the androgen receptor in fibroblasts cultured from the genital skin of 28 unrelated phenotypically normal men with idiopathic azoospermia or oligospermia. The amounts of androgen receptor were compared with those in genital skin fibroblasts from a variety of other subjects, including 10 men with azoospermia of known cause, 5 normal men, 28 subjects with disorders of androgen formation of metabolism of known cause, and 28 persons with documented disorders of the androgen receptor (testicular feminization and Reifenstein syndrome). The mean androgen receptor Bmax (amount of high affinity binding) was 12 fmol/mg protein or greater in 10 infertile men with azoospermia of known cause and in 6 infertile men with mild oligospermia. In fibroblasts from 1 to 4 individuals with severe oligospermia of unknown cause (less than 1 million/ml) and 8 of 18 subjects with idiopathic azoospermia, the androgen receptor Bmax was less than 12 fmol/mg protein. The mean value in these 9 men was not significantly different from that in subjects with testicular feminization or Reifenstein syndrome. Serum concentrations of testosterone and LH were normal in 6 of these 9 infertile men, and plasma production rates of testosterone were elevated in only 2 of the 6 men studied in whom the Bmax values in genital skin fibroblasts were less than 12 fmol/mg protein. We conclude that androgen resistance may be the cause of a significant fraction (40% or more) or idiopathic male infertility due to azoospermia or severe oligospermia, and that this disorder may not be manifested by a functional defect in the pituitary-testicular axis.

Biopsy↗
PubMed1983

Dihydrotestosterone stimulates 5 alpha-reductase activity in pubic skin fibroblasts.

In vivo, the 5 alpha-reduction of testosterone (T) to dihydrotestosterone (DHT) is androgen dependent in pubic skin but not in the skin of the external genitalia. The aim of the present study was to determine whether pubic skin fibroblasts (PSF) had retained this androgen dependency. PSF were prepared from explants of skin from normal subjects (four men, three women) and three patients with complete form of the testicular feminization syndrome. Culture medium containing 5% fetal calf serum and DHT was added 24 h after subculture (day 1) and renewed every other day. 5 alpha-Reductase was assayed on day 4 or day 8 by incubation of intact cell monolayers with [3H]T (2 nM), extraction of the medium, and chromatography of the metabolites; DNA was assayed in the cell pellets; 5 alpha-reductase was expressed as fmol/micrograms DNA . h. Controls were untreated plates from the same subcultures. DHT had no effect on cell DNA, whereas it resulted in a dose-dependent increase in 5 alpha-reductase activity. In seven PSF strains tested, DHT (10(-7) M) increased 5 alpha-reductase activity 2- to 4-fold over the control levels. This effect was abolished by the simultaneous addition of cyproterone acetate (2 X 10(-6) M) and was not observed in PSF from testicular feminization syndrome patients, suggesting that it was indeed mediated via the androgen receptor. T but not estradiol or cortisol also increased 5 alpha-reductase activity in PSF. The effect of androgens was suppressed by protein synthesis inhibitors. These data provide strong evidence that PSF respond to androgens via a receptor mediated mechanism, and that 5 alpha-reductase can be used as a marker of androgen action in pubic skin in vitro as well as in vivo.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1990

Estrogen and androgen production rates in two brothers with Reifenstein syndrome.

Defects of the androgen receptor in 46,XY individuals cause aberrant virilization that varies from a female phenotype to men with minor defects. More severely affected individuals also develop gynecomastia associated with enhanced estradiol secretion by the testis. However, the degree of breast development does not correlate with the rate of estrogen production, leading us to propose that feminization is a function of the degree of androgen resistance as well as the rate of estrogen formation. To test this hypothesis we measured estrogen and androgen formation in two brothers with perineoscrotal hypospadias and severe gynecomastia (the Reifenstein phenotype) due to a mutation that impairs androgen receptor function. Rates of estradiol production (60 and 70 micrograms/day) were elevated, but were not as high as in previously studied men with a similar phenotype. We conclude that the variable degree of feminization in this disorder cannot be explained by androgen resistance alone.

Adult↗
PubMed1991

Androgen resistance associated with a mutation of the androgen receptor at amino acid 772 (Arg----Cys) results from a combination of decreased messenger ribonucleic acid levels and impairment of receptor function.

Analysis of the nucleotide sequence of the coding segment of the androgen receptor gene in a patient (N105) with the receptor-negative form of complete testicular feminization has revealed a single substitution (CGC----TGC) at nucleotide 2476. This alteration results in the conversion of an arginine at amino acid 772 to a cysteine. Introduction of this mutation into an androgen receptor cDNA and transfection of the mutant cDNA into COS cells result in the production of a receptor protein with an alteration in the apparent Kd of ligand binding (3 nM) compared to that of the normal androgen receptor (0.5 nM). The mutant receptor protein predicted for patient N105 also demonstrates thermal instability of ligand binding that is not associated with quantitative or qualitative changes in the immunoreactive androgen receptor protein. When assayed in cotransfection experiments using a mouse mammary tumor virus-chloramphenicol acetyl transferase reporter system, the N105 receptor protein appears to be about a tenth as active as the control receptor. These functional characteristics do not appear sufficient to account for the phenotype of complete testicular feminization and do not explain the profound deficiency of androgen receptor in cultured skin fibroblasts. Quantitative S1 nuclease protection assays reveal that the level of androgen receptor mRNA in fibroblasts from patient N105 is markedly reduced. These results suggest that the phenotype in patient N105 is due to two effects of the nucleotide substitution at residue 2476: the replacement of a crucial amino acid (772) in the hormone-binding domain that impairs the function of any receptor molecules formed and a decrease in the level of androgen receptor mRNA.

Androgen-Insensitivity Syndrome↗
PubMed1994

Human female phenotypic development: role of fetal ovaries.

Morphogenesis of external genitalia was studied in human fetuses obtained from cases of medical termination of pregnancies and cases of spontaneous abortions. Eighty-two fetuses, 16 mm crown rump (CR) length (6 weeks gestation) to 240 mm CR length (26 weeks gestation), were studied. Fetal sex was determined with the help of chorionic villus of fetal skin biopsies in cases of fetuses less than 70 mm CR length. Growth and differentiation up to 50 mm CR length (9 weeks gestation) was identical for both sexes. After this there was rapid growth and differentiation in males. But in females subsequent growth until 180-200 mm CR-length stage (20 weeks gestation), was restricted to growth of labia majora. After this there was rapid ventral outgrowth of the region of perineum between the clitoris and anus, which brought the urethral and vaginal orifices to the surface. There was also further growth of labia. This process of feminization of external genitalia was completed by the 26th week of gestation. Histology revealed that ovary and testes could be clearly identified at 60 mm CR length stage. Maturation of fetal testes progressed rapidly, but there was little change in the histological appearance of ovaries until 160-180 mm CR-length stage (18-20 weeks of gestation). Follicular growth started after this. Feminization of urogenital sinus started after follicular growth started in the fetal ovaries. So from the temporal profile of events, it appears that it may be initiated by fetal ovarian steroids.

Abortion, Induced↗