Androgen receptor antagonist suppresses exercise-induced hypertrophy of skeletal muscle

The physiological importance of the increase in androgen receptors in exercise-induced muscle hypertrophy was investigated in rats. Together with training rat gastrocnemius muscles by electrical stimulation every other day for 2 weeks, male rats were administered the androgen receptor antagonist, oxendolone. The androgen receptor antagonist effectively decreased the wet mass of the prostate, an androgen target organ, and did not significantly affect body mass. The increase in muscle mass induced by electrical stimulation was effectively suppressed by the androgen receptor blockade. The mean degree of muscle hypertrophy in the antagonist-treated group was significantly lower than that in the control group (102.30% vs 107.41%, respectively;P=0.006). This result suggests that the androgen pathway has a significant effect in exercise-induced muscle hypertrophy and emphasizes the importance of the increase in the number of androgen receptors in exercised muscle.     

Role of the androgen receptor in male sexual differentiation.

The two androgens responsible for all aspects of male sexual differentiation are testosterone and dihydrotestosterone. The action of both these steroids is mediated by a specific intracellular receptor, the androgen receptor, which is a member of the nuclear receptor superfamily. The androgen receptor gene has been cloned and is located on the X chromosome at Xq11-12. Mutations of this gene have been found in subjects with both complete and partial androgen insensitivity. In a study of 27 subjects with the androgen insensitivity syndrome, we have identified mutations in 14, using a rapid mutation screening assay. The same technique has also been used to determine carrier status in an affected family. We have also identified a mutation in two brothers who show perineal hypospadias as the only evidence of undervirilisation. Familial severe hypospadias should therefore be included as part of the phenotypic spectrum of partial androgen insensitivity. The study of naturally occurring mutations of the androgen receptor gene is providing further information on the function of the androgen receptor and its role in normal male sexual differentiation.     

Sequence variation in the androgen receptor gene is not a common determinant of male sexual orientation.

To test the hypothesis that DNA sequence variation in the androgen receptor gene plays a causal role in the development of male sexual orientation, we have (1) measured the degree of concordance of androgen receptor alleles in 36 pairs of homosexual brothers, (2) compared the lengths of polyglutamine and polyglycine tracts in the amino-terminal domain of the androgen receptor in a sample of 197 homosexual males and 213 unselected subjects, and (3) screened the the entire androgen receptor coding region for sequence variation by PCR and denaturing gradient-gel electrophoresis (DGGE) and/or single-strand conformation polymorphism analysis in 20 homosexual males with homosexual or bisexual brothers and one homosexual male with no homosexual brothers, and screened the amino-terminal domain of the receptor for sequence variation in an additional 44 homosexual males, 37 of whom had one or more first- or second-degree male relatives who were either homosexual or bisexual. These analyses show that (1) homosexual brothers are as likely to be discordant as concordant for androgen receptor alleles; (2) there are no large-scale differences between the distributions of polyglycine or polyglutamine tract lengths in the homosexual and control groups; and (3) coding region sequence variation is not commonly found within the androgen receptor gene of homosexual men. The DGGE screen identified two rare amino acid substitutions, ser205-to-arg and glu793-to-asp, the biological significance of which is unknown.     

Intracellular partitioning of androgen receptor immunoreactivity in the brain of the male syrian hamster: Effects of castration and steroid replacement

The effect of castration and steroid replacement on the intracellular partitioning of the androgen receptor in the brain of the male Syrian hamster was determined using immunocytochemistry. Androgen receptors were visualized using the PG-21 antibody (G. S. Prins) on 40-μm coronal brain sections from hamsters perfused with 4% paraformaldehyde with or without 0.4% glutaraldehyde. Control studies confirmed antibody specificity in gonad-intact and castrate males. In the normal adult male, androgen receptor immunocytochemistry reveals intense staining confined to the cell nucleus. Castration caused a gradual increase in cytoplasmic labelling within 2 weeks, accompanied by a reduction in nuclear staining intensity in androgen receptor-containing neurons throughout the brain. Cytoplasmic androgen receptor staining was eliminated after treatment of orchidectomized males for only 8 h with exogenous testosterone. Likewise, long-term exposure to testosterone and dihydrotestosterone, a nonaromatizable androgen, maintained nuclear androgen receptor immunoreactivity. However, exposure to low physiologic concentrations of estrogen was not effective in this regard. In addition, we determined that nuclear androgen receptor immunoreactivity decreases in response to inhibitory short-day photoperiod, but without an increase in cytoplasmic immunostaining. This appears to be due to the decrease in androgen production by the testis, rather than a direct photoperiodic effect, because testosterone supplementation to short-day males restored the intensity of nuclear androgen receptor immuno-reactivity to levels comparable to those in the intact male. These findings are compatible with a new model for the intracellular localization of androgen receptors, in which a subset of unoccupied receptors is located in the cell cytoplasm in the absence of ligand. They further demonstrate the repartitioning of such cytoplasmic receptors, thereby confirming and extending previous observations using biochemical techniques on the regulation of neuronal androgen receptors. © 1993 John Wiley & Sons, Inc.     

Disruption of androgen receptor signaling in males by environmental chemicals

Androgen-disruptors are environmental chemicals in that interfere with the biosynthesis, metabolism or action of endogenous androgens resulting in a deflection from normal male developmental programming and reproductive tract growth and function. Since male sexual differentiation is entirely androgen-dependent, it is highly susceptible to androgen-disruptors. Animal models and epidemiological evidence link exposure to androgen disrupting chemicals with reduced sperm counts, increased infertility, testicular dysgenesis syndrome, and testicular and prostate cancers. Further, there appears to be increased sensitivity to these agents during critical developmental windows when male differentiation is at its peak. A variety of in vitro and in silico approaches have been used to identify broad classes of androgen disrupting molecules that include organochlorinated pesticides, industrial chemicals, and plasticizers with capacity to ligand the androgen receptor. The vast majority of these synthetic molecules act as anti-androgens. This review will highlight the evidence for androgen disrupting chemicals that act through interference with the androgen receptor, discussing specific compounds for which there is documented in vivo evidence for male reproductive tract perturbations. This article is part of a Special Issue entitled 'Endocrine disruptors'.     

Cultured human skin fibroblasts: a model for the study of androgen action

Human skin may be considered as a target organ for androgens, as are male sex accessory organs, since all events involved in testosterone action have been observed in this tissue. As a corollary, the mechanism of androgen action can be studiedin vitro in cultured skin fibroblasts. The advantages of this system are that studies can be performed with intact human cells under carefully controlled conditions, differentiated genetic and biochemical characteristics of the cells are faithfully preserved and the biological material is renewable from a single biopsy specimen. The metabolism of androgens, in particular the 5α-reduction of testosterone to the active metabolite, dihydrotestosterone, the intracellular binding of androgen to its specific receptor protein and its subsequent translocation to the nucleus have been studied in skin fibroblasts. The intracellular androgen receptor content of genital skin fibroblasts is higher than that from nongenital skin sites. In addition, the androgen receptor has been characterized as a specific macromolecule with properties of high affinity and low capacity similar to that of other steroid hormone receptors. The pathophysiology of three genetic mutations which alter normal male sexual development and differentiation has been identified in the human skin fibroblast system. In 5α-reductase deficiency, an autosomal recessive disorder in which dihydrotestosterone formation is impaired, virilization of the Wolffian ducts is normal but the external genitalia and urogenital sinus derivatives are female in character. At least two types of X-linked disorders of the androgen receptor exist such that the actions of both testosterone and dihydrotestosterone are impaired and developmental abnormalities may involve both Wolffian derivatives and the external genitalia as well. These two forms of androgen insensitivity result from either the absence of androgen receptor binding activity (receptor(−)form) or apparently normal androgen receptor binding with absence of an appropriate biological response (receptor (+) form). In addition, studies with human skin fibroblasts may also be of value in defining the cellular mechanisms underlying the broad spectrum of partial defects in virilization. In summary, we have correlated our studies of the molecular mechanism of androgen action in human genital skin fibroblasts with those of other investigators as these studies contribute to our understanding of male sexual development and differentiation.     

A floxed allele of the androgen receptor gene causes hyperandrogenization in male mice.

We previously generated a conditional floxed mouse line to study androgen action, in which exon 3 of the androgen receptor (AR) gene is flanked by loxP sites, with the neomycin resistance gene present in intron 3. Deletion of exon 3 in global AR knockout mice causes androgen insensitivity syndrome, characterized by genotypic males lacking normal masculinization. We now report that male mice carrying the floxed allele (AR(lox)) have the reverse phenotype, termed hyperandrogenization. AR(lox) mice have increased mass of androgen-dependent tissues, including kidney, (P < 0.001), seminal vesicle (P < 0.001), levator ani muscle (P = 0.001), and heart (P < 0.05). Serum testosterone is not significantly different. Testis mass is normal, histology shows normal spermatogenesis, and AR(lox) males are fertile. AR(lox) males also have normal AR mRNA levels in kidney, brain, levator ani, liver, and testis. This study reaffirms the need to investigate the potential phenotypic effects of floxed alleles in the absence of cre in tissue-specific knockout studies. In addition, this androgen hypersensitivity model may be useful to further investigate the effects of subtle perturbations of androgen action in a range of androgen-responsive systems in the male.     

Effect of castration and administration of testosterone on cytosol and nuclear androgen receptor in mouse submandibular gland

The effect of castration or administration of testosterone propionate on the subcellular distribution of androgen receptor in mouse submandibular gland was investigated. Within 10 h after castration of male mice, most of the androgen receptor in nuclei was significantly reduced, the androgen receptor in cytosol increased and the increased cytosol receptor retained for at least 40 h. A single injection of testosterone propionate to female mice resulted in the translocation of cytosol androgen receptor to the nuclei by 30 min. The nuclear receptor level remained for at least 24 h and the cytosol receptor was replenished by 24-72 h. These results reveal that the endocrine manipulations such as castration and testosterone injection cause the change in the subcellular distribution of androgen receptor from mouse submandibular gland in both sexes.     

Neural Control of Muscle Androgen Receptors

The number of cytosolic androgen receptors in rat skeletal muscle increases following denervation and disuse. This increase was postulated to represent altered intracellular distribution and consequent diminished sensitivity of skeletal muscle to androgens. To test this hypothesis, we measured total (homogenate) androgen receptor levels after denervation. Total (homogenate) androgen receptor binding did not change in response to denervation of leg muscles from adult male rats. An increase in cytosolic receptor number with no increase in total (homogenate) receptor levels supports the hypothesis of altered intracellular distribution of androgen receptors in denervated muscle. Cytosolic androgen receptor binding in muscle from male rats increased by 40% after denervation, whereas in females the increase was 17%. These increases could not be altered by endocrine manipulations of males or females.     

Photoaffinity labelling of androgen receptors with 17 beta-hydroxy-17 alpha-[3H]methyl-4,9,11-estratrien-3-one

The synthetic androgen 17 beta-hydroxy-17 alpha-[3H]methyl-4,9,11-estratrien-3-one (R1881) has been used as photoaffinity label to characterize androgen receptors in calf uterus and rat prostate. Polyacrylamide gel electrophoresis under denaturing conditions showed that the DNA-binding form of the androgen receptor in calf uterus cytosol is a protein with a molecular mass of 98 kD. In rat prostate cytosol an androgen receptor with a molecular mass of 46 kD could be photoaffinity labelled with R1881. The photoaffinity labelling procedure described here provides a method for studying the hormone binding domain of androgen receptors in partial purified preparations.     

Effects of ATD on male sexual behavior and androgen receptor binding: a reexamination of the aromatization hypothesis

The aromatization hypothesis asserts that testosterone (T) must be aromatized to estradiol (E2) to activate copulatory behavior in the male rat. In support of this hypothesis, the aromatization inhibitor, ATD, has been found to suppress male sexual behavior in T-treated rats. In our experiment, we first replicated this finding by peripherally injecting ATD (15 mg/day) or propylene glycol into T-treated (two 10-mm Silastic capsules) or control castrated male rats. In a second experiment, we bilaterally implanted either ATD-filled or blank cannulae into the medial preoptic area (MPOA) of either T-treated or control castrated male rats. With this more local distribution of ATD, a lesser decline in sexual behavior was found, suggesting that other brain areas are involved in the neurohormonal activation of copulatory behavior in the male rat. To determine whether in vivo ATD interacts with androgen or estrogen receptors, we conducted cell nuclear androgen and estrogen receptor binding assays of hypothalamus, preoptic area, amygdala, and septum following treatment with the combinations of systemic T alone. ATD plus T, ATD alone, and blank control. In all four brain areas binding of T to androgen receptors was significantly decreased in the presence of ATD, suggesting that ATD may act both as an androgen receptor blocker and as an aromatization inhibitor. Competitive binding studies indicated that ATD competes in vitro for cytosol androgen receptors, thus substantiating the in vivo antiandrogenic effects of ATD. Cell nuclear estrogen receptor binding was not significantly increased by exposure to T in the physiological range. No agonistic properties of ATD were observed either behaviorally or biochemically. Thus, an alternative explanation for the inhibitory effects of ATD on male sexual behavior is that ATD prevents T from binding to androgen receptors.     

Regulation of sexual odor preference by sex steroids in the posterodorsal medial amygdala in female rats

Our previous study in male rats demonstrated that bilateral administration of flutamide, an androgen receptor (AR) antagonist, into the posterodorsal medial amygdala (MePD) increased the time sniffing male odors to as high as that sniffing estrous odors, eliminating the preference for estrous odors over male odors. This made us speculate that under blockade of AR in the MePD, testosterone-derived estrogen acting on the same brain region arouses interest in male odors which is otherwise suppressed by concomitant action of androgen. In cyclic female rats, endogenous androgen has been thought to be involved in inhibitory regulation of estrogen-activated sexual behavior. Thus, in the present study, we investigated the possibility that in female rats the arousal of interest in male odors is also normally regulated by both estrogen and androgen acting on the MePD, as predicted by our previous study in male rats. Implantation of either the estrogen receptor blocker tamoxifen (TX) or a non-aromatizable androgen 5α-dihydrotestosterone (DHT) into the MePD of ovariectomized, estrogen-primed female rats eliminated preference for male odors over estrous odors by significantly decreasing the time sniffing male odors to as low as that sniffing estrous odors. The subsequent odor discrimination tests confirmed that the DHT and TX administration did not impair the ability to discriminate between male and estrous odors. These results suggest that in estrous female rats estrogen action in the MePD plays critical roles in the expression of the preference for male odors while androgen action in the same brain region interferes with the estrogen action.     

Ontogeny of an 8S androgen binding protein in rat liver cytosol

Studies were performed to elucidate the ontogeny of a single class of androgen binding protein in male rat liver cytosol which exhibits characteristics of a ligand specific, high affinity (Kd = 2.3 nM), 8S-receptor capable of nuclear translocation. Detectable levels of receptor first appear at 45 days of age in the male and reach maximum concentration at 65 days. Barely detectable levels are seen in females throughout the duration of study (80 days). Gonadectomy in both sexes (65 days) and androgen treatment of oophorectomized females do not alter the normal development of sexual differentiation of the high affinity androgen receptor. After neonatal castration (2 days) and DES replacement however, receptor sites do not undergo differentiation and adult males exhibit female levels. Conversely, neonatal androgen replacement in 2-day castrates partially restores the level of binding sites to control males values (TP, 71%; DHT, 51%). Neonatal castration without replacement retards but does not fully eliminate sexual differentiation of levels of receptor sites in adult males. Likewise, neonatal androgen treatment in females results in a partial masculinization of binding sites. Following hypophysectomy, levels of receptor sites in females are similar to intact or hypophysectomized males; sexual differences in the adult are abolished. These studies suggest that sexual differentiation of specific liver cytosol androgen binding sites in the adult may be partially programmed at birth by testicular androgen and furthermore, adult sexual dimorphism is maintained through an inhibitory influence of the pituitary in the female.     

Androgeninsensitivität

Mutations of the androgen receptor gene cause a spectrum of androgen insensitivity phenotypes ranging from women with female external genitalia through patients with genital ambiguities to men with male genitalia but infertility. The CAG repeat in the first exon is important for transactivation of target genes of the androgen receptor and is thought to modulate androgen-dependent processes. Expansion of this repeat is the cause of X-linked spinobulbar muscular atrophy.     

Androgen receptor expression in human tissues: an immunohistochemical study.

The cellular localization of the human androgen receptor was visualized immunohistochemically using a mouse monoclonal antibody (MAb) F39.4, directed against a fragment of the N-terminal domain of the androgen receptor. The nuclear immunoreactivity of various human tissues with F39.4 was generally consistent with earlier biochemical and autoradiographic data. However, previously suggested androgen receptor expression in thyroid, pancreatic, gastrointestinal, and bladder tissues was not confirmed immunohistochemically. Stratified squamous epithelia of vagina and cervix showed selective immunostaining of the basal cell layer, whereas in the preputial epithelium the intensity of immunoreactivity decreased gradually with maturation. In contrast, glandular epithelia of the sweat glands, male accessory sex organs, and female breast showed nearly exclusive F39.4 staining of the inner cylindric layer. In the testis, Sertoli cells, peritubular myoid cells, and interstitial cells were immunoreactive with MAb F39.4. Expression of the androgen receptor by smooth muscle tissue was largely confined to the male reproductive organs. The specificity and sensitivity of this simple and rapidly performed immunohistochemical technique in the detection of the human androgen receptor at the cellular and subcellular level makes it worthwhile to study tissue androgen receptor expression by immunohistochemistry in physiological and pathological states.     

Glucocorticoids, androgens, testis mass, and the energetics of vocalization in breeding male frogs

Male advertisement vocalization in frogs is known to be one of the energetically most expensive activities of ectothermic vertebrates. Glucocorticoids have marked effects on energy metabolism, and, generally, plasma concentrations of glucocorticoids increase during the course of prolonged exercise bouts. Androgen concentrations are also known to vary considerably among breeding male frogs. Intraspecific and interspecific comparisons were used to test for a relationship among androgen concentration, corticosterone concentration, testis mass, and the energetics of vocalization in natural populations of calling male frogs. The results of this study indicate that: (1) intraspecific variation in androgen and corticosteroid concentrations in breeding male frogs is positively correlated as a result of both interindividual variation in the amount of performed vocalization and the relationship between calling effort of an individual male and the level of calling in other males, (2) interspecific variation in corticosteroid concentration of calling male frogs is correlated with the relative energy expended in the species-specific vocalization, and (3) when differences in testis mass are controlled for, vocalization effort is correlated with androgen concentration among species of breeding male frogs. These findings are in contrast to some recent work reported from laboratory experiments on calling frogs.     

Androgen receptor in rat liver: Hormonal and developmental regulation of the cytoplasmic receptor and its correlation with the androgen-dependent synthesis of α2u-globulin

The cytoplasmic receptor for 5α-dihydrotestosterone has been identified in the rat liver and partially characterized. The receptor is a protein with a sedimentation coefficient of 3.5 S and binds both androgens (5α-dihydrotestosterone and testosterone) and estradiol-17β with high affinity. At saturating concentration, for every mole of estradiol there seem to be three moles of 5α-dihydrotestosterone bound to the receptor. Whereas estradiol stronly inhibits the uptake of 5α-dihydrotestosterone by the receptor, the presence of 5α-dihydrotestosterone only weakly interferes with estradiol binding.The level of the androgen receptor activity in the hepatic cytosol was found to follow closely the level of the urinary output of α-_2u-globulin, an androgen-dependent protein of hepatic origin. Immature and senile male as well as female rats, which do not normally produce α_2u-globulin, also lacked androgen receptor activity in their hepatic cytosol. Castration of the adult male rats results in a gradual drop of the urinary output of α_2u-globulin as well as of the hepatic androgen receptor activity. Androgen treatment of immature and senile male rats does not induce α_2u-globulin or any receptor activity. Administration of estradiol to adult male rates results in complete inhibition of both α_2u-synthesis as well as complete loss of the cytosol androgen receptor activity in these animals. These results strongly indicate that the hepatic the hepatic androgen receptor activity. Androgen treatment of immature and senile male rats does not induce α_2u-globulin or any receptor activity. Administration of estradiol to adult male rats results in complete inhibition of boty α_2u-synthesis as well as complete loss of the cytosol androgen receptor activity in these animals. These results strongly indicate that the hepatic androgen receptor is an inducible protein whose synthesis is regulated by its own ligands, the androgens acting as the positive and the estradiol as the negative signals.