Androgen conversion in osteoarthritis and rheumatoid arthritis synoviocytes--androstenedione and testosterone inhibit estrogen formation and favor production of more potent 5alpha-reduced androgens

In synovial cells of patients with osteoarthritis (OA) and rheumatoid arthritis (RA), conversion products of major anti-inflammatory androgens are as yet unknown but may be proinflammatory. Therefore, therapy with androgens in RA could be a problem. This study was carried out in order to compare conversion products of androgens in RA and OA synoviocytes. In 26 OA and 24 RA patients, androgen conversion in synovial cells was investigated using radiolabeled substrates and analysis by thin-layer chromatography and HPLC. Aromatase expression was studied by immunohistochemistry. Dehydroepiandrosterone (DHEA) was converted into androstenediol, androstenedione (ASD), 16alphaOH-DHEA, 7alphaOH-DHEA, testosterone, estrone (E1), estradiol (E2), estriol (E3), and 16alphaOH-testosterone (similar in OA and RA). Surprisingly, levels of E2, E3, and 16alpha-hydroxylated steroids were as high as levels of testosterone. In RA and OA, 5alpha-dihydrotestosterone increased conversion of DHEA into testosterone but not into estrogens. The second androgen, ASD, was converted into 5alpha-dihydro-ASD, testosterone, and negligible amounts of E1, E2, E3, or 16alphaOH-testosterone. 5alpha-dihydro-ASD levels were higher in RA than OA. The third androgen, testosterone, was converted into ASD, 5alpha-dihydro-ASD, 5alpha-dihydrotestosterone, and negligible quantities of E1 and E2. 5alpha-dihydrotestosterone was higher in RA than OA. ASD and testosterone nearly completely blocked aromatization of androgens. In addition, density of aromatase-positive cells and concentration of released E2, E3, and free testosterone from superfused synovial tissue was similar in RA and OA but estrogens were markedly higher than free testosterone. In conclusion, ASD and testosterone might be favorable anti-inflammatory compounds because they decrease aromatization and increase anti-inflammatory 5alpha-reduced androgens. In contrast, DHEA did not block aromatization but yielded high levels of estrogens and proproliferative 16alpha-hydroxylated steroids. Androgens were differentially converted to pro- and anti-inflammatory steroid hormones via diverse pathways.     

Androgen conversion in osteoarthritis and rheumatoid arthritis synoviocytes – androstenedione and testosterone inhibit estrogen formation and favor production of more potent 5α-reduced androgens

In synovial cells of patients with osteoarthritis (OA) and rheumatoid arthritis (RA), conversion products of major anti-inflammatory androgens are as yet unknown but may be proinflammatory. Therefore, therapy with androgens in RA could be a problem. This study was carried out in order to compare conversion products of androgens in RA and OA synoviocytes. In 26 OA and 24 RA patients, androgen conversion in synovial cells was investigated using radiolabeled substrates and analysis by thin-layer chromatography and HPLC. Aromatase expression was studied by immunohistochemistry. Dehydroepiandrosterone (DHEA) was converted into androstenediol, androstenedione (ASD), 16αOH-DHEA, 7αOH-DHEA, testosterone, estrone (E1), estradiol (E2), estriol (E3), and 16αOH-testosterone (similar in OA and RA). Surprisingly, levels of E2, E3, and 16α-hydroxylated steroids were as high as levels of testosterone. In RA and OA, 5α-dihydrotestosterone increased conversion of DHEA into testosterone but not into estrogens. The second androgen, ASD, was converted into 5α-dihydro-ASD, testosterone, and negligible amounts of E1, E2, E3, or 16αOH-testosterone. 5α-dihydro-ASD levels were higher in RA than OA. The third androgen, testosterone, was converted into ASD, 5α-dihydro-ASD, 5α-dihydrotestosterone, and negligible quantities of E1 and E2. 5α-dihydrotestosterone was higher in RA than OA. ASD and testosterone nearly completely blocked aromatization of androgens. In addition, density of aromatase-positive cells and concentration of released E2, E3, and free testosterone from superfused synovial tissue was similar in RA and OA but estrogens were markedly higher than free testosterone. In conclusion, ASD and testosterone might be favorable anti-inflammatory compounds because they decrease aromatization and increase anti-inflammatory 5α-reduced androgens. In contrast, DHEA did not block aromatization but yielded high levels of estrogens and proproliferative 16α-hydroxylated steroids. Androgens were differentially converted to pro- and anti-inflammatory steroid hormones via diverse pathways.     

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.     

Effect of Interleukin-1beta and Dehydroepiandrosterone on the Expression of Lumican and Fibromodulin in Fibroblast-Like Synovial Cells of the Human Temporomandibular Joint

Several epidemiological studies have reported that temporomandibular disorders (TMDs) are more prevalent in women than in men. It has recently been proposed that sex hormones such as estrogen, testosterone and dehydroepiandrosterone (DHEA) are involved with the pathogenesis of TMDs. Although studies have investigated the relationship between estrogen and testosterone and the restoration of TMDs, the relationship between DHEA and TMDs is unknown. The synovial tissue of the temporomandibular joint (TMJ) is made up of connective tissue with an extracellular matrix (ECM) composed of collagen and proteoglycan. One proteoglycan family, comprised of small leucine-rich repeat proteoglycans (SLRPs), was found to be involved in collagen fibril formation and interaction. In recent years, the participation of SLRPs such as lumican and fibromodulin in the internal derangement of TMJ has been suggested. Although these SLRPs may contribute to the restoration of the synovium, their effect is still unclear. The purpose of this study was to investigate the effect of DHEA, a sex hormone, on the expression of lumican and fibromodulin in human temporomandibular specimens and in cultured human TMJ fibroblast-like synovial cells in the presence or absence of the pro-inflammatory cytokine interleukin-1beta (IL-1beta). In the in vivo study, both normal and osteoarthritic (OA) human temporomandibular synovial tissues were immunohistochemically examined. In the in vitro study, five fibroblast-like synoviocyte (FLS) cell lines were established from human TMJ synovial tissue of patients with osteoarthritis. The subcultured cells were then incubated for 3, 6, 12 or 24 h with/without IL-1beta (1 ng/mL) in the presence or absence of DHEA (10 μM). The gene expression of lumican and fibromodulin was examined using the real-time polymerase chain reaction (PCR) and their protein expression was examined using immunofluorescent staining. We demonstrated that the expression of lumican differs from that of fibromodulin in synovial tissue and furthermore, that IL-1beta induced a significant increase in lumican mRNA and immunofluorescent staining in FLS compared to cells without IL-1beta. DHEA plus IL-1beta induced a significant increase in fibromodulin, but not in lumican mRNA, compared to DHEA alone, IL-1beta alone and in the absence of DHEA and IL-1beta. In immunofluorescent staining, weaker fibromodulin staining of FLS cells was observed in cells cultured in the absence of both DHEA and IL-1beta compared to fibromodulin staining of cells cultured with DHEA alone, with DHEA plus IL-1beta, or with IL-1beta alone. These results indicate that DHEA may have a protective effect on synovial tissue in TMJ by enhancing fibromodulin formation after IL-1beta induced inflammation. DHEA enhancement of fibromodulin expression may also exert a protective effect against the hyperplasia of fibrous tissue that TGF-beta1 induces. In addition lumican and fibromodulin are differentially expressed under different cell stimulation conditions and lumican and fibromodulin may promote regeneration of the TMJ after degeneration and deformation induced by IL-1beta.Key words: Temporomandibular joint, dehydroepiandrosterone, lumican, fibromodulin, small leucine rich repeat proteoglycan     

Testosterone inhibits 11-ketotestosterone-induced spermatogenesis in African catfish (Clarias gariepinus).

Male fish produce 11-ketotestosterone as a potent androgen in addition to testosterone. Previous experiments with juvenile African catfish (Clarias gariepinus) showed that 11-ketotestosterone, but not testosterone, stimulated spermatogenesis, whereas testosterone, but not 11-ketotestosterone, accelerated pituitary gonadotroph development. Here, we investigated the effects of combined treatment with these two types of androgens on pituitary gonadotroph and testis development. Immature fish were implanted for 2 wk with silastic pellets containing 11-ketotestosterone, testosterone, 5alpha-dihydrotestosterone, or estradiol-17beta; cotreatment groups received 11-ketotestosterone in combination with one of the other steroids. Testicular weight and pituitary LH content were higher (two- and fivefold, respectively) in the end control than in the start control group, reflecting the beginning of normal pubertal development. Treatment with testosterone or estradiol-17beta further increased the pituitary LH content four- to sixfold above the end control levels. This stimulatory effect on the pituitary LH content was not modulated by cotreatment with 11-ketotestosterone. However, the stimulatory effect of 11-ketotestosterone on testis growth and spermatogenesis was abolished by cotreatment with testosterone, but not by cotreatment with estradiol-17beta or 5alpha-dihydrotestosterone. Also, normal pubertal testis development was inhibited by prolonged (4 wk) treatment with testosterone. The inhibitory effect of testosterone may involve feedback effects on pituitary FSH and/or on FSH receptors in the testis. It appears that the balanced production of two types of androgens, and the control of their biological activities, are critical to the regulation of pubertal development in male African catfish.     

Rapid androgen actions on calcium signaling in rat sertoli cells and two human prostatic cell lines: similar biphasic responses between 1 picomolar and 100 nanomolar concentrations

Androgen-induced calcium fluxes and gap junctional intercellular communication (GJIC) were studied in three different cell types. A transient (2-3 min duration) increase in intracellular calcium levels was observed within 20-30 sec of androgen addition, which was followed by a plateau phase with steroid concentrations higher than 1 nM. The kinetics of the calcium responses were similar in immature rat Sertoli cells, which contain normal nuclear receptors; the human prostatic tumor cell line, LNCaP, which contains a mutated nuclear receptor; and the human prostatic cell line, PC3, which does not contain a nuclear receptor. The human A431 tumor cell line did not respond to androgens. Concentrations of testosterone and the synthetic androgen, R1881, between 1-1000 pM induced transient calcium increases with ED(50) values near 1 pM and 1 nM, whereas dihydrotestosterone (DHT) was not active at these concentrations. At concentrations higher than 1 nM, testosterone, R1881, and DHT were equipotent in stimulating an increase in calcium that lasted for more than 10 min, with ED(50) values between 5 and 20 nM. Testosterone covalently bound to albumin was also active, whereas 11 related androstane compounds as well as progesterone and estradiol-17beta were inactive at 1000 nM. The calcium response induced by the three androgens (10 nM) was abolished in all cell types by hydroxyflutamide (1000 nM) and finasteride (1000 nM), but not by cyproterone acetate (1000 nM). The calcium response was also abolished in the absence of extracellular calcium and strongly inhibited by the presence of verapamil. Exposure of the responsive cells to brief (150-sec) pulses of androgens generated calcium responses that were similar to those after continuous exposure. After exposure of Sertoli cells for only 30 sec to 100 nM testosterone, the calcium response lasted for at least 50 min. Although nuclear binding of androgens could be demonstrated, there was no evidence for tight binding to the plasma membrane under similar conditions. When protein synthesis was inhibited, an enhancement of GJIC between rat Sertoli cells, but not between LNCaP cells or PC3 cells, was observed within 15 min of the addition of 10 nM testosterone. Because nuclear androgens are not present in PC3 cells and many functional properties of the responsive system are different from the nuclear receptor in all three cell types, we postulate the existence of an alternative cell surface receptor system with biphasic response characteristics (high and low affinity). The calcium signals are probably coupled to the regulation of gap junctional efficiency between Sertoli cells. The low-affinity receptors may convey complementary androgen signals at elevated local levels such as in the testis, when nuclear receptors are (over)saturated.     

Studies on the regulation of the concentration of androgens and androgen receptors in nuclei of prostatic cells.

Experiments were performed to assess the effect of intracellular androgen metabolism and the availability of cytoplasmic receptors on the concentration of androgens and androgen receptors in nuclei of prostatic cells. It was found that androgens are incorporated into the nucleus by a regulated, selective process which appears to limit the type and amount of androgen transported across the nuclear membrane. The metabolic conversion of testosterone to dihydrotestosterone which takes place in cytoplasm does not reduce transport and, very likely, affects only the ratio of testosterone and dihydrotestosterone transferred into the nucleus. In vivo, when the intranuclear concentration of androgens approaches 250 nM (8 pmol per mg DNA), an apparent concentration ceiling is reached even in the presence of a downward concentration gradient that would be expected to promote further transport across the nuclear membrane. This finding strongly suggests that in vivo the nuclear membrane acts as a barrier to the passage of androgens and, therefore, mitigates against the possibility that passive diffusion is an important mechanism of afferent transport of androgens into the nucleus. The ability of the nucleus to concentrate testosterone and dihydrotestosterone was clearly demonstrated in vivo when cytoplasmic concentrations of androgens of approximately 20 nM were accompanied by intranuclear concentrations in the vicinity of 250 nM. Since the measured concentration of testosterone and dihydrotestosterone in prostate of several species fall within the 5-20 nM range, it is evident that androgen concentrations in the nucleus as high as 250 nM may be typical of the physiological steady state. At the latter concentration the nucleus contains 60 000 androgen molecules: in approximate terms one third of this total is bound to a large molecular weight component of the nucleus, one third is bound to a 3.3 S receptor and one third is free or loosely bound. Since 60 000 androgen molecules and 20 000 receptor molecules appear in the nucleus before transport stops, it seems that the quantity of 4.4 S cytoplasmic receptor estimated at 174 plus or minus 24 pmol per mg protein (equivalent to about 8000 molecules per cell) is insufficient to account for the total influx of androgens and androgen receptors into the nucleus. Thus, although these results support the view that cytoplasmic receptors and the capacity to transport androgens are closely linked phenotypic markers of intracellular steroid hormone action, they suggest that the control of androgen concentration in the nucleus is achieved in a more intricate fashion than simply through a dependence on the presumed translocation of 4.4 S androgen-receptor complex into the nucleus.     

Androgen receptor in nuclei of rat testis.

Testis nuclei of hypophysectomized rats selectively accumulate labeled testosterone and 5alpha-dihydrotestosterone following the injection of tritiated testosterone in vivo. Testosterone and 5alpha-dihydrotestosterone are bound to macromolecules in nuclei and can be extracted with 0.5 M KCl. Accumulation of protein bound radioactive androgens in nuclei of isolated seminiferous tubules is similar to that of whole testis. The relative amounts of testosterone and dihydrotestosterone in purified nuclei were similar to the relative amounts bound to cytoplasmic receptors, suggesting that cytoplasmic androgen-receptor complexes may be transported into the nuclei. Binding of labeled androgen is saturable and inhibited by prior injection of unlabeled testosterone or cyproterone acetate. Nuclear binding sites are destroyed by the proteolytic enzyme pronase, but not by DNase. Like the cytoplasmic androgen-receptor complexes in rat testis, nuclear androgen-protein complexes are heat labile and dissociate slowly at 0 degrees C. androgens fail to accumulate in testis nuclei of the Stanley-Gumbreck androgen insensitive rat, a species lacking cytoplasmic androgen receptors in testis and other androgen target tissues.     

IL-16 as an anti-inflammatory cytokine in rheumatoid synovitis

T lymphocytes are a major component of the inflammatory infiltrate in rheumatoid synovitis, but their exact role in the disease process is not understood. Functional activities of synovial T cells were examined by adoptive transfer experiments in human synovium-SCID mouse chimeras. Adoptive transfer of tissue-derived autologous CD8+ T cells induced a marked reduction in the activity of lesional T cells and macrophages. Injection of CD8+, but not CD4+, T cells decreased the production of tissue IFN-gamma, IL-1beta, and TNF-alpha by >90%. The down-regulatory effect of adoptively transferred CD8+ T cells was not associated with depletion of synovial CD3+ T cells or synovial CD68+ macrophages, and it could be blocked by Abs against IL-16, a CD8+ T cell-derived cytokine. In the synovial tissue, CD8+ T cells were the major source of IL-16, a natural ligand of the CD4 molecule that can anergize CD4-expressing cells. The anti-inflammatory activity of IL-16 in rheumatoid synovitis was confirmed by treating synovium-SCID mouse chimeras with IL-16. Therapy for 14 days with recombinant human IL-16 significantly inhibited the production of IFN-gamma, IL-1beta, and TNF-alpha in the synovium. We propose that tissue-infiltrating CD8+ T cells in rheumatoid synovitis have anti-inflammatory activity that is at least partially mediated by the release of IL-16. Spontaneous production of IL-16 in synovial lesions impairs the functional activity of CD4+ T cells but is insufficient to completely abrogate their stimulation. Supplemental therapy with IL-16 may be a novel and effective treatment for rheumatoid arthritis.     

Androgen binding to ammonium sulfate precipitates of human adipose tissue cytosols

Although androgens are believed to influence the distribution of human adipose tissue and have been detected in human fat, receptors for these sex hormones have yet to be identified. These studies demonstrate that a high-affinity, limited-capacity binding component for the synthetic androgen methyltrienolone (R1881) exists in ammonium sulfate precipitates of human adipose tissue cytosols. The equilibrium dissociation constant (Kd = 0.1 to 0.4 nmol/L, n = 6) and the number of binding sites (2 to 26 fmol/mg protein, n = 22) are consistent with those reported for androgen receptors in rat prostate, human prostatic carcinoma, MCF-7 cells, and baboon myocardium. The relative steroid-binding specificities of the human adipose tissue androphile (R1881 approximately 5 alpha-dihydrotestosterone greater than testosterone greater than estradiol approximately progesterone much greater than dexamethasone) are similar, but not identical, to those reported for androgen receptors in rat prostate (R1881 greater than 5 alpha-dihydrotestosterone approximately testosterone greater than estradiol greater than progesterone much greater than cortisol) and baboon myocardium (R1881 greater than 5 alpha-dihydrotestosterone greater than testosterone greater than progesterone greater than estradiol much greater than cortisol). The function of the androgen-binding component in human adipose tissue is not known.     

Natural and recombinant interleukin 1 receptor antagonist does not inhibit human T-cell proliferation induced by mitogens, soluble antigens or allogeneic determinants.

Interleukin 1 receptor antagonist (IL-1ra) has been found in glycosylated form in the urine of febrile patients or of children with rheumatoid arthritis, and in the supernatant of monocytes cultured in the presence of immune complexes. It blocks competitively the binding of IL-1 alpha and beta to their receptors. Produced amongst others by mononuclear cell lines and matured monocytes and alveolar macrophages, it prevents prostaglandin E2 and collagenase production by fibroblasts and synovial cells. In mice, IL-1ra improves survival after lethal endotoxemia. In this study, both natural and recombinant human IL-1ra (rhIL-1ra) were tested in an allogeneic T-cell reaction, and in mitogen- or antigen-induced lymphocyte proliferation. Neither the natural nor the rhIL-1ra blocked T-cell proliferation, but rhIL-1ra abolished the effect of exogenous IL-1 beta. This was not due to a loss of bioactivity of IL-1ra in culture, as the IL-1ra of the supernatant still completely inhibited 125I-IL-1 alpha binding to EL 4-6.1 cells and markedly reduced PGE2 production during antigen presentation. We conclude that IL-1ra alone, even at high concentrations, is not sufficient to block human T-cell proliferation in vitro.     

Androgens and bone

Testosterone is the major gonadal sex steroid produced by the testes in men. Testosterone is also produced in smaller amounts by the ovaries in women. The adrenal glands produce the weaker androgens dehydroepiandrosterone, dehydroepiandrosterone sulfate, and androstenedione. These androgens collectively affect skeletal homeostasis throughout life in both men and women, particularly at puberty and during adult life. Because testosterone can be metabolized to estradiol by the aromatase enzyme, there has been controversy as to which gonadal sex steroid has the greater skeletal effect. The current evidence suggests that estradiol plays a greater role in maintenance of skeletal health than testosterone, but that androgens also have direct beneficial effects on bone. Supraphysiological levels of testosterone likely have similar effects on bone as lower levels via direct interaction with androgen receptors, as well as effects mediated by estrogen receptors after aromatization to estradiol. Whether high doses of synthetic, non-aromatizable androgens may, in fact, be detrimental to bone due to suppression of endogenous testosterone (and estrogen) levels is a potential concern that warrants further study.     

Synovial tissue macrophage as a source of the chemotactic cytokine IL-8

Cells of the synovial microenvironment may recruit neutrophils (PMN) and lymphocytes into synovial fluid, as well as lymphocytes into the synovial tissues, of arthritic patients. We have investigated the production of the chemotactic cytokine IL-8 by using sera, synovial fluid, synovial tissue, and macrophages and fibroblasts isolated from synovial tissues from 75 arthritic patients. IL-8 levels were higher in synovial fluid from rheumatoid (RA) patients (mean +/- SE, 14.37 +/- 5.8 ng/ml), compared with synovial fluid from osteoarthritis patients (0.135 +/- 17 ng/ml) (p less than 0.05) or from patients with other arthritides (5.52 +/- 5.11 ng/ml). IL-8 from RA sera was 8.44 +/- 2.33 ng/ml, compared with nondetectable levels found in normal sera. IL-8 levels from RA sera and synovial fluid were strongly positively correlated (r = 0.96, p less than 0.05). Moreover, RA synovial fluid chemotactic activity for PMN in these fluids was inhibited 40 +/- 5% upon incubation with neutralizing polyclonal antibody to IL-8. Synovial tissue fibroblasts released only small amounts of constitutive IL-8 but could be induced to produce IL-8 by stimulation with either IL-1 beta, TNF-alpha, or LPS. In contrast, unlike normal PBMC or alveolar macrophages, macrophages isolated from RA synovial tissue constitutively expressed both IL-8 mRNA and antigenic IL-8. RA synovial macrophage IL-8 expression was not augmented by incubation with either LPS, TNF-alpha, or IL-1 beta. Immunohistochemical analysis of synovial tissue showed that a greater percentage of RA macrophages than osteoarthritis macrophages reacted with anti-IL-8. Whereas macrophages were the predominant cell for immunolocalization of IL-8, less than 5% of synovial tissue fibroblasts were positive for immunolocalized IL-8. These results suggest that macrophage-derived IL-8 may play an important role in the recruitment of PMN in synovial inflammation associated with RA.     

Sex-specific occurrence of androgen receptors in rat brain.

The metabolism and binding of [1, 2, 6, 7-3H] testosterone in male and female rat brain has been studied in an attempt to find an explanation for the relative androgen unresponsiveness characterizing the female hypothalamo-pituitary axis involved in regulation of hepatic steroid metabolism. The most significant sex differences in the pattern of [3H] testosterone metabolites recovered from several brain regions (including pituitary, pineal gland, and hypothalamus) after intraperitoneal administration of [3H] testosterone were the predominance of testosterone and androstenedione in male brain compared to the quantitative importance of 5alpha-androstane-3alpha, 17beta-diol, 5alpha-androstane-3beta, 17beta-diol, epitestosterone, and dihydroepitestosterone in female brain. One possible explanation for the androgen unresponsiveness of female rats is, therefore, the faster metabolism of testosterone to inactive compounds in female brain. Experiments both in vivo and in vitro showed the presence of high affinity, low capacity binding sites for [3H] testosterone in male pituitary, pineal gland, and hypothalamus (Kd values in the region of 1 X 10(-10) to 1 X 10(-9) M and number of binding sites 1.0 to 1.4 X 10(-14) mol per mg of protein). The steroid - macromolecular complexes generally had a pI of 5.1, were excluded from Sephadex G-200, were heat-labile, and were sensitive to protease. Competition experiments indicated the following order of ligand affinities: testosterone is greater than 5alpha-dihydrotestosterone and estradiol is greater than androstenedione is greater than corticosterone. No steroid-binding proteins of similar nature were found in pituitary, pineal gland, or hypothalamus from female rats. On the basis of these results it is suggested that the androgen unresponsiveness of female rats referred to above relates to the absence of receptor protein for androgens in female rat brain. In support of this hypothesis, 28-day-old female rats, which are known to be affected by androgens with regard to liver enzyme activities, were shown to contain receptor proteins for androgen in the brain. In conclusion, the relative androgen unresponsiveness of the female hypothalamo-pituitary axis is probably explained by the absence of receptor proteins for androgen in female hypothalamus and pituitary. The fast metabolism of testosterone in female rat brain also serves to decrease the availability of active androgen to potential receptor sites. It may be speculated that the presence of androgen receptors in male brain is the result of neonatal programming ("imprinting") by testicular androgen.     

Androgen binding profiles of two distinct nuclear androgen receptors in Atlantic croaker (Micropogonias undulatus)

In the present study, the binding affinities of 28 androgens for two nuclear androgen receptors (AR), termed AR1 and AR2, in Atlantic croaker (Micropogonias undulatus) brain and ovarian tissues, respectively, were determined using competitive binding assays. The 5alpha-reduction of steroids, in general, increased the metabolite's binding affinity for AR2 while decreasing it for AR1. In addition, few androgens bound to AR1 with high affinity and modifications to the basic 3-ketone,4-ene,17beta-hydroxy structure of testosterone usually reduced its binding affinity for AR1. However, androgens with ketone groups at the 3- and 17-position bound with high affinity to AR1 provided that the androgen had either a 5alpha-reduced A-ring or a third ketone group at the 11-position. This suggests that there may be several high affinity conformations that AR1 can occupy depending upon whether an androgen possesses a ketone or a hydroxyl group at the 17-position. The binding of androgens to AR2 showed a more predictable pattern, 5alpha-reduced steroids bound better than 4-ene steroids and any changes to the basic 3-keto,17-hydroxy motif of 5alpha-dihydrotestosterone lowered the binding affinity of a steroid. However, these structural changes often caused only minor decreases in binding affinity, such that AR2 has a broader affinity for androgens and a greater affinity than AR1 for structurally diverse androgens. Widely different androgen binding affinities of AR1 and AR2 suggest that these two nuclear androgen receptors may mediate the physiological actions of different androgens in teleosts.     

Direct measurement of androgen receptors in cultured sertoli cells

Cytoplasmic and nuclear forms of macromolecules with the properties of androgen receptors have been demonstrated by direct labeling techniques in cultured Sertoli cells. The cytoplasmic form was excluded from Sephadex G-200 and could be distinguished from androgen binding protein (ABP) on the basis of size, heat stability, relative electrophoretic mobility, and binding complex dissociation rate. When cultured Sertoli cells were incubated with ³H-testosterone, a time- and temperature-dependent accumulation of label into the nuclear fraction was observed, 46% of which crystallized as authentic testosterone. Specific binding was saturable with an apparent association constant of 0.4nM^?1. Approximately 30% of the nuclear bound hormone was extracted within 1 h by 0.4M KCl and 34% of this was associated with macromolecular species as measured by gel filtration. Unlabeled androgens and to some degree progestogens competed with ³H-testosterone for binding sites. These data constitute direct evidence that Sertoli cells contain androgen receptors.     

Demonstration of a nuclear androgen receptor in erythroblasts obtained by culture of human bone marrow

The new techniques of culture of bone marrow have shown that androgens and 5 beta steroids exert a direct effect on erythroid precursor cells from human or animal bone marrow. By contrast, the mechanisms of the intracellular actions of those compounds are poorly understood. Tritiated methyltrienolone (R 1881), a synthetic androgen that highly binds to androgen receptor, has been used for the study of a binding activity in nuclear extracts of cultured erythroblasts from human bone marrow. The nuclear extracts contain binding sites saturable at low concentrations of 3H-R 1881 (20-30 nM). Scatchard analysis revealed that the R 1881-nuclear complex has a dissociation constant (Kd) of 25-50 nM, and the number of binding sites was 235-370 fmoles/mg protein. The complex sedimented on 5-20% sucrose gradient in the 3.9 S region and 5 beta dihydrotestosterone compete strongly with R 1881 for binding sites. This binding component has characteristics of high affinity, low-capacity, sedimentation behaviour, and specificity commonly attributed to "androgen receptors".     

Influence of 5 alpha-reduced androgens on oestradiol secretion by granulosa cells of pregnant mare serum gonadotrophin treated immature rats

The effect of aromatizable androgens (testosterone and androstenedione) and naturally occurring 5 alpha-androstane, 3 alpha 17 beta-diol and 5 alpha-androstane, -3 beta, 17 beta-diol on oestradiol secretion by granulosa cells isolated from preovulatory follicles of PMSG-primed immature rats was investigated. The amount of oestradiol secreted by granulosa cells in the absence of exogenous aromatizable androgen in a 4h incubation was negligible. However, the addition of testosterone or androstenedione resulted in concentration dependent increases in oestradiol secretion. The 5 alpha-reduced androgens inhibit oestradiol and oFSH-stimulated oestradiol secretion by the granulosa cells in the presence of exogenous testosterone. The least potent of the androgens tested in causing this effect being the 5 alpha-androstane-3 alpha, 17 beta-diol. This result suggests that the naturally occurring 5 alpha-reduced androgens have a direct effect on androgen-aromatizing enzymes. The effect of these androgens may have an important connotation with respect to the control of the onset of puberty and regulation of ovarian oestradiol secretion within the microenvironment of an ovarian follicle.     

Testicular responsiveness to a single hCG dose in patients with testicular feminization

The suggestion that androgens may regulate testosterone (T) production in rat Leydig cells by a receptor-mediated feed-back mechanism, led us to investigate whether in vivo the absence of testicular androgen receptors, as it occurs in testicular feminization (TF), may modify the characteristic testicular response observed in men and prepubertal children after a single dose of hCG. Subjects consist of: 1) six normal men, 2) two adult patients with the complete form of androgen insensitivity syndrome (TF), 3) 12 normal prepubertal boys, 4) one prepubertal boy with the same form of TF. Each subject received i.m. a single dose of hCG 3500 IU/m2 b.s. and blood samples were collected basally and 2, 4, 24, 48, 72 and 96 hours after the hormonal stimulus. Serum levels of T, 17 alpha hydroxyprogesterone (17OHP) and 17 beta estradiol (E2) were measured at each collection time. In normal men a significant increase in T (M +/- SE) was observed at 4 h (758.6 +/- 135 ng/dl, P less than 0.05) and a more significant increase at 48 h (1082 +/- 60.3 ng/dl, P less than 0.001). E2 and 17OHP peaked significantly at 24 h (81.5 +/- 9.6 pg/ml and 460.7 +/- 90.9 ng/dl respectively). This response pattern is characteristic of the testicular desensitization which occurs in normal man after a single hCG dose. The same response pattern has been observed in the two TF adult patients suggesting that human testicular desensitization in vivo does not depend on androgen receptors.(ABSTRACT TRUNCATED AT 250 WORDS)