Anabolic responsiveness of skeletal muscles correlates with androgen receptor protein but not mRNA

Anabolic effects of androgens on skeletal muscle are well documented, but the physiological and biochemical bases of these effects are poorly understood. Skeletal muscles that differ in their androgen responsiveness can be used to examine these mechanisms. We compared androgen receptor mRNA and protein levels of the rat levator ani, a perineal skeletal muscle that depends on androgens for its normal maintenance and function with that of the rat extensor digitorum longus, a limb muscle that does not require androgens. Western immunoblotting indicated that androgen receptor protein is significantly elevated in the levator ani relative to the extensor digitorum longus. Surprisingly, steady state androgen receptor mRNA levels were equivalent in these muscles, as determined by Northern blot analysis and quantitative RT-PCR. These results suggest that androgen responsiveness of skeletal muscles is determined by the level of androgen receptor protein in a particular muscle and that androgen receptor protein content is regulated by translational or post-translational mechanisms.     

EVOLUTION OF β-GLUCURONIDASE REGULATION IN THE GENUS MUS

Despite the central role suggested for regulatory mutations in many evolutionary scenarios, there is relatively little information available about the type and extent of regulatory differences between species, or to what extent differences between species are independent of variation within species. To address this issue we have studied the regulatory system of β-glucuronidase, a gene implicated in a murine androgen-inducible pheromone-signalling system. We examined the changes in β-glucuronidase hormonal regulation which have occurred during the radiation of a group of 12 closely related species of mice by assaying β-glucuronidase activity in six different tissues after treatment with estrogen, and with androgen alone and in combination with either estrogen or growth hormone. We also examined in some detail the extent of variation in regulatory responses within species. We found extensive variation in regulatory phenotypes both within and among the species surveyed, suggesting that many of the species examined are currently polymorphic for various regulatory factors that affect inducibility of β-glucuronidase. The variation we observed reflects changes in the ability of the β-glucuronidase gene to respond to hormonal influences, rather than changes in aspects of the hormonal signalling system exterior to the gene. The marked differences among species in the renal and uterine responses to hormonal induction of β-glucuronidase are not easily related to the phylogeny of the genus Mus. If hormonal induction of the gene for β-glucuronidase is subject to natural selection, it appears to be subject to widely fluctuating selective forces. We review evidence that the apparently disorderly evolution of the hormonal responsiveness of β-glucuronidase does not appear to be a unique property of this regulatory system. In contrast to the evolution of many protein sequences, which are tightly correlated with phylogeny and proceed at a relatively constant rate, some, perhaps many, regulatory phenotypes are in rapid evolutionary flux, providing an extensive range of phenotypes upon which selection can act.     

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.     

Genetic variation in androgen regulation of ornithine decarboxylase gene expression in inbred strains of mice

Previous studies have indicated that androgen regulation of certain gene products in murine kidney is genetically controlled. In the present work, the expression of renal ornithine decarboxylase (ODC) gene(s) was used as a biological marker to study androgen responsiveness of eight inbred strains of mice (A/J, C57BR/cdJ, 129/J, C57L/J, BALB/cJ, SM/J, RF/J, and C57BL/6J). Kidneys of untreated females from these strains did not have significantly different basal ODC activities or ODC mRNA concentrations. However, renal enzyme concentrations in intact male mice exhibited marked strain-dependent variation; three strains (RF/J, SM/J, and C57BR/cdJ) had 5- to 20-fold higher activities than the other five strains. Renal ODC mRNA content showed similar genetic variability in the male mice; animals with highest enzyme activity had higher mRNA levels than those with low activity. These results could not be explained by differences in either serum testosterone levels or renal nuclear androgen receptor content, suggesting that the animals were differentially sensitive to endogenous androgens. To evaluate further the androgen regulation of ODC gene expression, female mice were treated with testosterone-releasing implants for 5-7 days. The two strains (A/J and C57BL/6J) that had low enzyme activity in response to endogenous testosterone in male mice also showed blunted responses to exogenous androgen administration, as measured by the induction of ODC and its mRNA. The relative distribution of the two mRNA species coding for ODC (2.2 and 2.7 kb in size) exhibited strain-dependent variation that did not, however, correlate with the androgen responsiveness. Studies of the mRNA levels in reciprocal F1 hybrids of C57BR/cdJ and C57BL/6J mice suggested that androgen sensitivity of ODC gene expression, at least in these crosses, was inherited in an autosomal dominant manner.     

Molecular analysis of the androgen receptor gene in 52 patients with complete or partial androgen insensitivity syndrome: a collaborative study.

In patients with androgen insensitivity syndrome (AIS), RFLP study of the androgen receptor gene made it possible to analyze whether deletions or mutations could be responsible for abnormalities in androgen responsiveness. We studied RFLPs of DNA from 25 46,XY patients with partial AIS (PAIS), defined as a concentration of androgen receptor in genital-skin fibroblasts less than 340 fmol/mg DNA, and DNA from 27 46,XY patients with complete AIS (CAIS) with no detectable androgen receptor site. DNA samples were digested with BamHI, EcoRI, HindIII and TaqI restriction enzymes and hybridized with three cDNA probes covering the three domains of the androgen receptor. When we had the maternal and an unaffected brother's DNA, we analyzed the two androgen receptor gene polymorphisms described, the HindIII and the exon 1 CAG repeat polymorphisms, in order to distinguish the two maternal X chromosomes, and to detect carriers of AIS. We did not find any large deletion among the 52 patients. We observed a heterozygous mother in 3 of 14 families studied with the HindIII polymorphism, and in 12 of 25 families using the exon 1 CAG repeat polymorphism. This study suggests that in AIS, abnormalities in androgen receptor response could be related to point mutations or microdeletions rather than to gross structural alterations of the androgen receptor gene. Furthermore, unless the point mutation has been described, exon 1 and HindIII polymorphism studies would enable the identification of carriers in 50% of families, and the prenatal diagnosis of AIS.     

Transfection with steroid-responsive reporter constructs shows glucocorticoid rather than androgen responsiveness in cultured Sertoli cells

It remains unclear why it has proven so difficult to identify androgen target genes in cultured Sertoli cells. Given the lack of useful endogenous reporter genes, we studied the androgen and glucocorticoid responsiveness of these cells by transfection with three different steroid-responsive reporter constructs. The constructs were driven by the tyrosine aminotransferase steroid-responsive region (TAT-GRE4x-Luc), the mouse mammary tumor virus promoter (MMTV-Luc) and the Pem homeobox gene proximal promoter respectively (Pem-Luc). These constructs can be activated either by both the glucocorticoid receptor (GR) and the androgen receptor (AR) (TAT-GRE4x-Luc and MMTV-Luc) or selectively by the AR (Pem-Luc). Despite high transfection efficiency (30-40%) none of the constructs could be activated by treatment of the Sertoli cells with testosterone, 5alpha-dihydrotestosterone or synthetic androgens. Even pretreatment with follicle-stimulating hormone to raise AR levels (from 31 up to 82fmol/mg protein) did not result in androgen responsiveness. In contrast, treatment with dexamethasone markedly stimulated TAT-GRE4x-Luc and MMTV-Luc activity. GR levels reached a value of 172fmol/mg protein in the cultured cells and both AR and GR displayed homogeneous distribution by immunocytochemical evaluation. Androgen responsiveness was restored and glucocorticoid responsiveness was increased by cotransfection with AR or GR expression constructs. Under cotransfection conditions, 1nM of testosterone (a concentration that is some 100 times lower than that estimated to be present in the testis) was sufficient to stimulate the TAT-GRE4x-Luc maximally. Our data indicate that cultured Sertoli cells respond better to glucocorticoids than to androgens and that one of the factors limiting androgen responsiveness is the availability of AR. Other factors limiting the transactivation capacity of the (endogenous) AR, however, cannot be excluded.     

Specificity of androgen resistance inMus caroli kidney

Androgen controls the expression of beta-glucuronidase and several other proteins in the kidney of the standard laboratory mouse, Mus musculus. Other species within the genus Mus exhibit a variety of response patterns for kidney beta-glucuronidase and other markers of androgen action. We have investigated the mechanism of androgen action in M. caroli, a Mus species that does not produce beta-glucuronidase in response to testosterone. The failure of testosterone to induce beta-glucuronidase in M. caroli females cannot be overcome by treatment with dihydrotestosterone, with pharmacological doses of testosterone propionate or dihydrotestosterone propionate, or with a variety of potent androgen analogues. All of these compounds induce kidney beta-glucuronidase in M. musculus females and kidney ornithine decarboxylase, submandibular gland renin, and submandibular gland epidermal growth factor in both M. caroli and M. musculus females. Furthermore, kidney androgen receptor proteins from M. caroli and M. musculus animals have the same sedimentation characteristics on sucrose density gradients. These data indicate that androgen resistance in M. caroli is not due to deficient 5 alpha-reductase or aberrant hormone metabolism producing suboptimal levels of functional androgen and is not caused by a defective androgen receptor. They suggest that the resistance of beta-glucuronidase in M. caroli kidney to induction by androgen occurs at the level of the beta-glucuronidase gene.     

An abundant 56 kD protein with low affinity androgen binding: another member of the steroid/thyroid receptor family?

We have found in genital skin fibroblasts an abundant 56 kD protein which appears related to the androgen receptor. The protein is detected in the soluble fraction by two-dimensional polyacrylamide gel electrophoresis as two spots with isoelectric points of 6.7 and 6.5 respectively. Photoaffinity labelling of GSF with 8 nM or 50 nM [3H]-methyltrienolone selectively labels the two protein spots but with an apparently lower affinity than is known for the androgen receptor. The two protein spots stem from one protein as judged from peptide patterns of partial proteolytic digests and the equal labelling of both spots with methyltrienolone. Cells from subjects with mutant androgen receptors generally lack the 56 kD protein and labelling with methyltrienolone fails, but the protein is not the androgen receptor itself. We propose the hypothesis that the 56 kD protein is synthesized from the same gene as the androgen receptor and that androgen may not be its natural ligand.     

Autologous down-regulation of androgen receptor messenger ribonucleic acid

Autoregulation of androgen receptor (AR) mRNA was investigated using Northern blot analysis with AR cDNA fragments as probes. The amount of AR mRNA increased 2- to 10-fold with androgen withdrawal and decreased below control levels after androgen stimulation in rat ventral prostate, coagulating gland, epididymis, seminal vesicle, kidney, and brain, and in a human prostate cancer cell line, LNCaP. In rat ventral prostate, AR mRNA increased 2- to 3-fold within 24 h after castration and remained elevated for 4 days. Treatment with testosterone propionate beginning 24 h after castration reduced ventral prostate AR mRNA 4-fold within 8 h of androgen replacement. Administration of estradiol 24 h after castration had no significant effect on prostatic AR mRNA. Androgens, including testosterone and the synthetic androgen methyltrienolone (R1881), or the antiandrogen cyproterone acetate down-regulated AR mRNA in vitro in LNCaP cells, whereas estradiol was without effect. Administration of testosterone propionate to rats with androgen insensitivity did not decrease AR mRNA. Down-regulation of AR mRNA by androgen is therefore a receptor-mediated process which occurs in vivo in rat tissues that differ in androgen responsiveness and in cultured human prostate cells.     

Unique patterns of androgen regulation of the expression of two genes in murine kidney

The kidneys of androgen stimulated mice exhibit a hypertrophic response but no hyperplasia or concomitant DNA replication. Androgens increase the expression of several genes in mouse kidney. The response of the beta-glucuronidase gene to testosterone in this tissue is characterized by a 1-2 day lag and relatively slow induction kinetics. The gene coding for kidney androgen-regulated protein (KAP) exhibits quite a different response to the hormone when compared on the basis of initial response to a given dose, dose required to produce maximal response, and apparent sensitivity to low levels of androgen-receptor complexes in renal nuclei. The analysis of the accumulation of the mRNAs produced by these two genes suggests that gene-specific differential sensitivity to androgen receptor complexes governs the development of the cellular male phenotype in this tissue.     

Distinguishing seasonal androgen responses from male-male androgen responsiveness-revisiting the Challenge Hypothesis

Androgen levels show strong patterns throughout the year in male vertebrates and play an important role in the seasonal modulation of the frequency, intensity and persistence of aggression. The Challenge Hypothesis (Wingfield, J.C., Hegner, R.E., Dufty, A.M., Ball, G.F., 1990. The "Challenge Hypothesis": Theoretical implications for patterns of testosterone secretion, mating systems, and breeding strategies. Am. Nat. 136, 829-846) predicts that seasonal patterns in androgen levels vary as a function of mating system, male-male aggression and paternal care. Although many studies have addressed these predictions, investigators have often assumed that the ratio of the breeding season maximum and breeding baseline concentrations (termed "androgen responsiveness") reflects hormonal responses due to social stimulation. However, increasing evidence suggests that seasonal androgen elevations are not necessarily caused by social interactions between males. Here, we separate the seasonal androgen response (R(seasonal)) and the androgen responsiveness to male-male competition (R(male-male)) to begin to distinguish between different kinds of hormonal responses. We demonstrate that R(seasonal) and R(male-male) are fundamentally different and should be treated as separate variables. Differences are particularly evident in single-brooded male birds that show no increase in plasma androgen levels during simulated territorial intrusions (STIs), even though R(seasonal) is elevated. In multiple-brooded species, STIs typically elicit a rise in androgens. We relate these findings to the natural history of single- and multiple-brooded species and suggest a research approach that could be utilized to increase our understanding of the factors that determine different types of androgen responses. This approach does not only include R(seasonal) and R(male-male), but also the androgen responsiveness to receptive females (R(male-female)) and to non-social environmental cues (R(environmental)), as well as the physiological capacity to produce and secrete androgens (R(potential)). Through such studies, we can begin to better understand how social and environmental factors may lead to differences in androgen responses.     

Interaction of androgen response elements with the DNA-binding domain of the rat androgen receptor expressed in Escherichia coli

A fragment of the rat androgen receptor (amino acids 533-637) containing the DNA-binding domain was produced in Escherichia coli as a fusion product with protein A of Staphylococcus aureus. The fusion protein was purified on IgG-Sepharose, a method that does not involve the use of denaturing agents. Approximately 4 mg of fusion protein was obtained from 500 ml of bacterial culture. In gel shift assays, the recombinant DNA-binding domain displays an affinity for a fragment of the long terminal repeat of mouse mammary tumor virus and for an intronic fragment of the gene coding for the C3 component of the androgen-regulated rat prostatic binding protein. In a DNase I footprinting assay, the fusion protein protects a sequence in the C3 fragment that has previously been shown to act as a functional androgen response element. Interestingly, a single base pair mutation in the response element, which abolishes androgen inducibility, also destroys the ability to interact with the recombinant androgen receptor DNA-binding domain.     

Putative androgen receptors distinguished in wild-type and testicular-feminized (Tfm) mice.

The reduced level of putative androgen receptor in the mouse mutant, testicular feminization (Tfm), chromatographs on DNA-cellulose differently from the bulk of wild-type receptors. While the elution maximum for extracts of Tfm/Y kidney is in the 180–190 mM NaCl range, wild-type kidney extracts exhibit two maxima of elution at 140–150 mM NaCl and 180–190 mM NaCl, respectively. For hypothalamus-preoptic area, Tfm/Y has one elution maximum at approximately 180 mM NaCl, while the wild-type exhibits a major elution maximum at 140–150 mM NaCl, with a minor peak at approximately 180 mM NaCl. Mixing experiments between wild-type and Tfm/Y cytosols reveal that the different characteristic elution patterns are intrinsic to the binding complexes and are not conveyed simply by other soluble factors. The distinctive pattern for Tfm indicates that the mutation does not cause merely a reduced level of wild-type receptor. Rather the residual receptor of the mutant may be either an abnormal protein or a minor form of wild-type receptor, not readily seen in wild-type tissue due to the presence of more preponderant species. Differences in the elution profiles of androgen receptor species of wild-type kidney with the two bound androgens, testosterone and dihydrotestosterone, are also presented. A model of the androgen receptor system is proposed which includes several binding classes for androgen ligands and metabolites. In light of aromatization of androgens to estrogens and its probable role in some androgenic responses, we include the “estrogen receptor” in this mechanism.