Skeletal muscle differentiation: role of dehydroepiandrosterone sulfate

Dehydroepiandrosterone (DHEA) and its sulfonated form dehydroepiandrosterone sulfate (DHEAS) are the main circulating steroid hormones and many epidemiological studies show an inverse relationship between DHEA/DHEAS levels and muscle loss for which the primary cause is the accelerated protein breakdown. The aim of this work was to determine whether DHEA/DHEAS supplementation in differentiating C2C12 skeletal muscle cells might influence the expression of the atrophy-related ubiquitin ligase, MuRF-1, and thereby impact key molecules of the differentiation program. DHEA is the prohormone crucial for sex steroid synthesis, and DHEAS is thought to be its reservoir. However, our preliminary experiments showed that DHEAS, but not DHEA, is able to influence MuRF-1 expression. Therefore, we treated differentiating C2C12 cells with various concentrations of DHEAS and analyzed the expression of MuRF-1, Hsp70, myosin heavy chain (MHC), myogenin, and the activity of creatine kinase. We observed that DHEAS at physiological concentrations downregulates MuRF-1 expression and affects muscle differentiation, as shown by the increased levels of MHC, which is a sarcomeric protein that undergoes MuRF-1-dependent degradation, and also by an increase in creatine kinase activity and myogenin expression, which are two other well-known markers of differentiation. Moreover, we found that DHEAS might have a protective effect on differentiating cells as suggested by the augmented levels of Hsp70, a member of heat shock proteins family that, besides its cytoprotective action, seems to have a regulatory role on key atrophy genes such as MuRF-1. In conclusion, our data shed light on the role of DHEAS at physiologic concentrations in maintaining muscle mass.     

The effects of dexamethasone and dehydroepiandrosterone (DHEA) on cytokines and receptor expression in a human osteoblastic cell line: potential steroid-sparing role for DHEA

Osteoporosis and associated fractures are the most common and debilitating complication of glucocorticoid use. The use of alternative anti-inflammatory agents without the deleterious skeletal effects of glucocorticoids is needed. Dehydroepiandrosterone (DHEA) may have immunomodulatory as well as positive effects on bone. For our further understanding of the mechanisms of action of DHEA, as a steroid-sparing agent, we investigated and compared the effects of dexamethasone (DEX) and DHEA on the regulation of the downstream effector pathway of osteoclastogenesis; RANKL/OPG and a range of inflammatory/pro-resorbing cytokines and receptors using a human clonal osteoblastic cell line. The cells were treated with DEX, DHEA, and androstenedione (ANDI). The mRNA expression of RANKL and OPG was determined by real-time PCR after overnight incubation. The regulation of a broad spectrum of cytokines by DEX and DHEA was also investigated using a human cytokine/growth factor and receptor gene array consisting of 268 cytokine-related cDNAs. To confirm some of the gene expression changes, protein production was measured by ELISA. RANKL expression and RANKL/OPG ratio were increased by DEX. This effect was reversed by co-treatment with both DHEA or ANDI. Several pro-inflammatory/resorptive cytokines including IL-6, IL-4, IFN-gamma, macrophage inhibitory factor (MIF) were down-regulated not only by DEX but also by DHEA. In contrast to DEX, DHEA did not lead to suppression of growth factors including vascular endothelial growth factor (VEGF), fibroblast growth factor-5 (FGF-5), insulin-like growth factor-binding protein3 (IGF-BP3). Several new target genes previously documented to influence bone formation were up-regulated by DHEA such as Notch 2, insulin receptor, thrombin receptor (PAR1). The data suggest that DHEA has immunomodulatory properties without the catabolic effects on bone remodeling, observed with glucocorticoid use. DHEA may thus prove useful as a steroid-sparing agent in the management of inflammatory disorders such as SLE or rheumatoid arthritis. Further in vivo studies are indicated.     

Dehydroepiandrosterone inhibits intracellular calcium release in beta-cells by a plasma membrane-dependent mechanism

Both dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS) affect glucose stimulated insulin secretion, though their cellular mechanisms of action are not well characterized. We tested the hypothesis that human physiological concentrations of DHEA alter insulin secretion by an action initiated at the plasma membrane of beta-cells. DHEA alone had no effect on intracellular calcium concentration ([Ca(2+)](i)) in a rat beta-cell line (INS-1). However, it caused an immediate and dose-dependent inhibition of carbachol-induced Ca(2+) release from intracellular stores, with a 25% inhibition at zero. One nanometer DHEA. DHEA also inhibited the Ca(2+) mobilizing effect of bombesin (29% decrease), but did not inhibit the influx of extracellular Ca(2+) evoked by glyburide (100 microM) or glucose (15 mM). The steroids (androstenedione, 17-alpha-hydroxypregnenolone, and DHEAS) had no inhibitory effect on carbachol-induced intracellular Ca(2+) release. The action of DHEA depended on a signal initiated at the plasma membrane, since membrane impermeant DHEA-BSA complexes also inhibited the carbachol effect on [Ca(2+)](i) (39% decrease). The inhibition of carbachol-induced Ca(2+) release by DHEA was blocked by pertussis toxin (PTX). DHEA also inhibited the carbachol induction of phosphoinositide generation, with a maximal inhibition at 0.1 nM DHEA. Furthermore, DHEA inhibited insulin secretion induced by carbachol in INS-1 cells by 25%, and in human pancreatic islets by 53%. Taken together, this is the first report showing that human physiological concentrations of DHEA decrease agonist-induced Ca(2+) release by a rapid, non-genomic mechanism in INS-1 cells. Furthermore, these data provide evidence consistent with the existence of a specific plasma membrane DHEA receptor, mediating this signal transduction pathway by pertussis toxin-sensitive G-proteins.     

Prolactin, progesterone, and dexamethasone coordinately and adversely regulate glucokinase and cAMP/PDE cascades in MIN6 beta-cells

Islet cells undergo major changes in structure and function to meet the demand for increased insulin secretion during pregnancy, but the nature of the hormonal interactions and signaling events is incompletely understood. Here, we used the glucose-responsive MIN6 beta-cell line treated with prolactin (PRL), progesterone (PRG), and dexamethasone (DEX, a synthetic glucocorticoid), all elevated during late pregnancy, to study their effects on mechanisms of insulin secretion. DEX alone or combined with PRL and PRG inhibited insulin secretion in response to 16 mM glucose-stimulating concentrations. However, in the basal state (3 mM glucose), the insulin levels in response to DEX treatment were unchanged, and the three hormones together maintained higher insulin release. There were no changes of protein levels of GLUT2 or glucokinase (GK), but PRL or PRG treatment increased GK activity, whereas DEX had an inhibitory effect on GK activity. alpha-Ketoisocaproate (alpha-KIC)-stimulated insulin secretion was also reduced by DEX alone or combined with PRL and PRG, suggesting that DEX may inhibit distal steps in the insulin-exocytotic process. PRL treatment increased the concentration of intracellular cAMP in response to 16 mM glucose, suggesting a role for cAMP in potentiation of insulin secretion, whereas DEX alone or combined with PRL and PRG reduced cAMP levels by increasing phosphodiesterase (PDE) activity. These data provide evidence that PRL and to a lesser extent PRG, which increase in early pregnancy, enhance basal and glucose-stimulated insulin secretion in part by increasing GK activity and amplifying cAMP levels. Glucocorticoid, which increases throughout gestation, counteracts only glucose-stimulated insulin secretion under high glucose concentrations by dominantly inhibiting GK activity and increasing PDE activity to reduce cAMP levels. These adaptations in the beta-cell may play an important role in maintaining the basal hyperinsulinemia of pregnancy while limiting the capacity of PRL and PRG to promote glucose-stimulated insulin secretion during late gestation.     

Effects of ketamine-xylazine and isoflurane on insulin sensitivity in dehydroepiandrosterone sulfate-treated minipigs (Sus scrofa domestica)

Isoflurane and ketamine-xylazine (KX) combinations are widely used veterinary anesthetics, KX being the particularly common agent for immobilizing swine. Results of previous studies indicate that KX and xylazine suppress insulin release. The steroid hormones, dehydroepiandrosterone (DHEA) and its sulfated form, dehydroepiandrosterone-sulfate (DHEAS), have variable effects on insulin sensitivity in animals. We evaluated the effect of DHEAS on plasma glucose and insulin concentrations in female Yucatan swine under KX and isoflurane anesthesia. A 2 x 2 factorial design was used. Twenty-four 17-week-old gilts were randomly assigned to receive vehicle (placebo) or DHEAS as part of an ongoing study. The KX was given intramuscularly to all animals prior to blood sample collection at weeks two and four. At week three, all animals received isoflurane by inhalation. During KX anesthesia, mean insulin concentration in DHEAS-treated and control groups approximated half the postisoflurane values (P < 0.001). While under isoflurane, the DHEAS group had significantly higher mean plasma insulin concentration and mean insulin-to-glucose ratio, compared with values for controls (P < 0.05). These findings are consistent with changes in insulin values following DHEAS treatment observed previously in nonanesthetized swine. The effect of DHEAS treatment was absent in animals under KX anesthesia. These results suggest that KX significantly decreases plasma insulin concentration and blunts DHEAS-associated insulin resistance in female minipigs.     

Antioxidant activity of dioscorea and dehydroepiandrosterone (DHEA) in older humans

Dioscorea is a yam steroid extract used in commercial steroid synthesis and consumed by people. DHEA is a steroid which declines with age, but without known activity. This study was designed to determine whether dioscorea supplementation could increase serum dehydroepiandrosterone sulfate (DHEAS) in humans and modulate lipid levels in older people. The subjects were selected volunteers aged 65–82 years. The serum DHEAS level, lipid peroxidation and lipid profile were assessed. Three weeks of dioscorea supplementation had no affect on serum DHEAS level. However DHEA intake of 85 mg/day increased serum DHEA levels 100.3 %. DHEA and dioscorea significantly reduced serum lipid peroxidation, lowered serum triglycerides, phospholipid and increased HDL levels. Both DHEA and the steroid yam extract, dioscorea, have significant activities as antioxidant to modify serum lipid levels.     

Glucocorticoids stimulate the release of a viral tumor marker (MMTV gp52) while inhibiting tumor cell growth

The influence of glucocorticoid treatments on the release of mouse mammary tumor virus (MMTV) envelope antigen (gp52) has been studied in C3H mammary tumor cell cultures and compared to treatment-mediated effects on tumor cell growth. Simultaneous assessment of extracellular viral antigen levels and tumor cell growth has indicated that both are coordinately affected by glucocorticoid treatment. While gp52 release is stimulated by treatment, this effect is accompanied by an inhibition of tumor cell growth. These stimulatory and inhibitory effects are mediated by dexamethasone (DEX) in a dose-dependent fashion, and both effects are more pronounced with the synthetic glucocorticoids DEX or triamcinolone acetonide (TA). Quantitation of media gp52 levels by RIA revealed the following hierarchy of glucocorticoid enhancement: TA greater than DEX greater than prednisolone greater than hydrocortisone greater than triamcinolone. A similar order of activity was observed in terms of inhibition of cell growth. The ability of TA to enhance gp52 release was 2.4-2.7 times greater than DEX, a previously proven stimulator of MMTV expression. Cell density of B9 mammary tumor cells was reduced 73% following 72 h of 10(-8) MTA treatment while C3H Mm5mt/cl mammary tumor cells were reduced by 53%. Hormone-mediated changes in in vitro gp52 release suggest that hormones might also influence plasma levels of MMTV gp52 as a systemic marker for the presence and status of murine mammary tumors. Coordinate stimulatory and inhibitory effects suggest that glucocorticoids may play a complex role in murine mammary tumorigenesis and subsequent mammary disease.     

Inhibitory action of sphingosine, sphinganine and dexamethasone on glucose uptake: studies with hydrogen peroxide and phorbol ester

The mechanism of the inhibitory action of glucocorticoids on glucose uptake is incompletely understood. Treatment with corticosteroids of cells in which glucose uptake is stimulated at insulin postbinding and postreceptor sites may clarify the site of the steroid inhibitory action. Hydrogen peroxide, which has been shown to stimulate the insulin receptor tyrosine kinase, and phorbol myristate acetate (PMA) which stimulates protein kinase C were, therefore, used as stimulators of glucose transport in this study. These studies demonstrate that dexamethasone and the sphingoid bases, sphinganine and sphingosine, inhibit glucose uptake that has been stimulated at either the receptor kinase or protein kinase C level in both 3T3-L1 and 3T3-C2 cells. These data confirm glucocorticoid inhibitory action at a post binding level and support the suggestion that some corticosteroid inhibitory effects may be mediated by an action on sphingolipid metabolism.     

Glucocorticoids modulate neurotransmitter-induced glycogen metabolism in cultured cortical astrocytes

Glucocorticoids (GC) are considered as key modulators of glycogen homeostasis in peripheral tissues, but their role in the central nervous system has only partially been characterized. Exposure of primary cultures of cortical astrocytes to dexamethasone (DEX), a synthetic glucocorticoid, results in the reduction of noradrenaline (NA)-induced glycogen synthesis in a concentration-dependent manner with a IC50 of 4.88 nm and a maximum inhibition of 51%. Such an effect is mediated via glucocorticoid receptors (GRs), since it is mimicked by the glucocorticoid analogue RU28362 (100 nm) and prevented by the GR antagonist RU38486 (1 micro m). DEX does not act through alteration of signal transduction mechanisms, as cAMP formation induced by noradrenergic stimulation was unchanged. Moreover, glycogen synthesis was inhibited to the same extent when DEX was applied either together or only after a brief NA application. Neither [3H]2-deoxyglucose uptake nor lactate release was altered by DEX in the presence of NA, demonstrating that inhibition of glycogen synthesis is not a consequence of reduced glucose utilization or availability. Interestingly, enhancement of glycogen synthase activity induced by NA was reduced in the presence of DEX (-27%). These results suggest that GC could have a significant influence on neuroenergetics as they could modulate activity-related changes in brain glycogen metabolism.     

DHEAS prevents pro-metastatic and proliferative effects of 17ß-estradiol on MCF-7 breast cancer cells

It is generally assumed that circulating dehydroepiandrosterone sulfate (DHEAS) can be desulfated and further metabolized to estrogen, which is of concern for all patients with estrogen-responsive breast cancer. We addressed this issue by comparing the effects of DHEAS, its desulfated form DHEA, and 17ß-estradiol on human metastatic, estrogen-responsive MCF-7 breast cancer cells.Physiological concentrations of DHEAS promoted phosphorylation of Erk1/2, whereas DHEA and 17ß-estradiol failed to stimulate Erk1/2 phosphorylation, indicating that the sulfated steroid acts as an autonomous hormone. Exposure of MCF-7 cells to 17ß-estradiol stimulated cell proliferation and the expression of pro-metastatic and pro-invasive elements such as claudin-1, matrix metalloproteinase 9 (MMP9), and the CC chemokine ligand 2 (CCL2). In contrast, treatment with DHEAS did not stimulate these responses but prevented all of the actions of 17ß-estradiol, and as a consequence cell migration and invasion were completely inhibited.The results of this study not only challenge the assumption that DHEAS poses a danger as an endogenous source of estrogen, they rather favor the idea that keeping DHEAS levels within a physiological range might be supportive in treating estrogen-responsive breast cancer.     

Molecular targets for 17α-ethynyl-5-androstene-3β,7β,17β-triol, an anti-inflammatory agent derived from the human metabolome

HE3286, 17α-ethynyl-5-androstene-3β, 7β, 17β-triol, is a novel synthetic compound related to the endogenous sterol 5-androstene-3β, 7β, 17β-triol (β-AET), a metabolite of the abundant adrenal steroid dehydroepiandrosterone (DHEA). HE3286 has shown efficacy in clinical studies in impaired glucose tolerance and type 2 diabetes, and in vivo models of types 1 and 2 diabetes, autoimmunity, and inflammation. Proteomic analysis of solid-phase HE3286-bound bead affinity experiments, using extracts from RAW 264.7 mouse macrophage cells, identified 26 binding partners. Network analysis revealed associations of these HE3286 target proteins with nodes in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for type 2 diabetes, insulin, adipokine, and adipocyte signaling. Binding partners included low density lipoprotein receptor-related protein (Lrp1), an endocytic receptor; mitogen activated protein kinases 1 and 3 (Mapk1, Mapk3), protein kinases involved in inflammation signaling pathways; ribosomal protein S6 kinase alpha-3 (Rsp6ka3), an intracellular regulatory protein; sirtuin-2 (Sirt2); and 17β-hydroxysteroid dehydrogenase 1 (Hsd17β4), a sterol metabolizing enzyme.     

Antidepressants attenuate the dexamethasone-induced decrease in viability and proliferation of human neuroblastoma SH-SY5Y cells: A involvement of extracellular regulated kinase (ERK1/2)

Excessive glucocorticoid levels in depressed patients have been associated with atrophic changes in some brain regions, but only few studies suggest that some antidepressants can interfere with deleterious effect of glucocorticoids on neuronal cells. The aim of the present study was to examine the effect of dexamethasone (DEX), a synthetic glucocorticoid and some antidepressants from different chemical groups (imipramine, desipramine, amitriptyline, citalopram, fluoxetine, reboxetine and tianeptine) on SH-SY5Y cells cultured in the medium containing steroid-free serum. DEX in concentrations from 1 to 100 μM did not change LDH release but exposure to 10 μM and 100 μM DEX for 24, 48 and 72 h caused a significant reduction in cell viability and proliferation as confirmed by MTT reduction and BrdU ELISA assays, respectively. Twenty four-hour incubation of cells with antidepressants (0.05–10 μM) and DEX (10 μM) showed that imipramine, amitriptyline, desipramine, citalopram and fluoxetine at concentrations from 0.1 up to 1 μM, reboxetine (0.1 μM) and tianeptine (0.05 μM) prevented the DEX-induced decreases in cell viability and proliferation rate. The protective effects of antidepressants were ameliorated by inhibitors of MAPK/ERK1/2, but not PI3-K/Akt pathway as shown for imipramine, fluoxetine and reboxetine. Moreover, Western blot analysis showed the decrease in the activated form of ERK1/2 (p-ERK) after DEX treatment and this effect was inhibited by imipramine. Thus, the reduction in SH-SY5Y cell viability caused by DEX appears to be related to its antiproliferative activity and some antidepressant drugs in low concentrations attenuate this effect by mechanism which involves the activation of MAPK/ERK1/2 pathway.     

Phorbol esters reduce gonadotrope responsiveness to protein kinase C activators but not to Ca2+-mobilizing secretagogues. Does protein kinase C mediate gonadotropin-releasing hormone action?

The demonstration that activators of the Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C), such as phorbol esters and diacylglycerols, can provoke luteinizing hormone (LH) release from pituitary gonadotropes, suggests a possible role for protein kinase C in stimulus-release coupling. We now report that administration of phorbol myristate acetate (PMA) to pituitary cell cultures causes a sustained reduction in Triton X-100-extracted protein kinase C activity. Further, phorbol ester- and diacylglycerol-stimulated LH release, as well as inhibition by PMA of gonadotropin-releasing hormone (GnRH)-stimulated inositol phosphate production, were reduced by pretreatment with PMA. The effects of phorbol ester pretreatment on PMA-stimulated LH release and protein kinase C activity were dose-dependent, sustained (greater than or equal to 24 h) and specific (no measurable effect with 4 alpha-phorbol didecanoate). The effect on PMA-stimulated LH release was apparently Ca2+-independent. In pituitary cell cultures with reduced protein kinase C activity, the gonadotropes have reduced responsiveness to PMA but release a similar proportion of cellular LH in response to Ca2+-mobilizing secretagogues (GnRH and A23187) as do control cells. The normal responsiveness to GnRH of cells with reduced responsiveness to protein kinase C activators calls into question the requirement for this enzyme for GnRH-stimulated LH release.     

Conversion of dehydroepiandrosterone sulfate at physiological plasma concentration into estrogens in MCF-7 cells

Metabolism of dehydroepiandrosterone (DHEA), its sulfate (DHEAS), and androstene-3,17-dione (delta(4)) was performed at their physiological plasma concentrations in MCF-7 cell cultures (1 microM, 10 and 2 nM, respectively). Final metabolic products of these steroids were separated by HPLC-radioactive flow detection and identified by LC/MS or MS/MS. Typical and specific mass fragmentation spectra identified the presence of estrone (E(1)), 17beta-estradiol (E(2)), delta(4), DHEA, 5-androstene-3beta,17beta-diol (delta(5)), and testosterone as principal DHEAS metabolites. Other steroids, such as androstenedione, androsterone, and DHEA fatty acid esters at very low concentrations (from pM to nM), were also obtained after steroid incubation. This highly specific method allowed us to conclude whether a metabolite and enzymatic activity of interest were present in MCF-7 cells or not. We also showed that DHEAS at its physiological plasma concentration may be converted into estrogens and estrogen-like compounds in breast cancer cells. The estrogenic action of DHEAS on breast cancer cells was also measured by bioluminescence in a stably transfected human breast cancer MCF-7 cell line with a reporter gene that allowed expression of the firefly luciferase enzyme under the control of an estrogen regulatory element.     

Reduced plasma dehydroepiandrosterone sulfate levels are significantly correlated with fatigue severity in patients with primary biliary cirrhosis

Fatigue is a common debilitating complication of primary biliary cirrhosis (PBC), the pathophysiologic mechanism of which is poorly understood. Recently, the neuroactive steroid dehydroepinadrosterone sulfate (DHEAS) was reported to be implicated in Chronic Fatigue Syndrome in the absence of liver disease. The present study was undertaken to analyse fatigue scores and their relationship with disease severity and circulating levels of DHEAS as well as its precursors DHEA and pregnenolone in PBC patients with (n=15) or without fatigue (n=10) compared to control subjects (n=11). Fatigue was assessed using the fatigue impact scale (FIS) including cognitive, physical and psychosocial subclasses. Steroids were measured by radioimmunoassay or gas chromatography/mass spectrometry. Plasma concentrations of DHEAS were significantly reduced in PBC patients with fatigue as compared to controls, while those of its precursors DHEA and pregnenolone remained within the control range. Plasma levels of DHEAS in PBC patients were significantly correlated with fatigue severity as reflected by total FIS scores including total (rp=-0.42; p=0.018), as well as the cognitive (rp=-0.37; p=0.03), physical (rp=-0.48; p=0.006) and psychosocial (rp=-0.35; p=0.04) subclasses of fatigue scores. No correlation of fatigue scores was observed with indices of liver function. These findings suggest that reduced levels of the neurosteroid DHEAS may contribute to fatigue in patients with PBC; substitutive therapy using DHEAS or its precursor DHEA could be beneficial in the management of fatigue in patients with low levels of DHEAS.     

Down regulation of CYP 1A1 by glucocorticoids in trout hepatocytes in vitro

Summary Short-term culture of rainbow trout (Onchorhynchus mykiss) hepatocytes was used to examine the effect of dexamethasone (DEX) on microsomal CYP 1A1 protein content and 7-ethoxyresorufin-O-deethylase (EROD) activity in vitro. Hepatocytes prepared by controlled collagenase digestion and plated at a density of 0.25 × 10⁶ cells/cm² in plastic culture dishes precoated with trout skin extract (7.6 μg skin protein/cm²) to facilitate cell attachment were maintained at 16° C. Cells were treated with DEX (10⁻⁹ to 10⁻⁷ M) or vehicle (dimethyl sulfoxide, DMSO) at 24 h. Microsomal CYP 1A1 protein content and EROD activities were measured at 72 h. Both CYP 1A1 protein as measured by Western blots using CYP 1A1 specific anti-sera and EROD activity were significantly lower in DEX (10⁻⁸ to 10⁻⁷ M)-treated hepatocytes compared to untreated (control) or DMSO-treated cells. The effect was dose dependent in that a gradual decrease of CYP 1A1 protein and EROD activities were seen with increasing doses of DEX (10⁻⁸ to 10⁻⁷ M). DEX at 10⁻⁹ M was ineffective. Concomitant addition of 10⁻⁶ M RU486, a type II specific glucocorticoid receptor antagonist, to hepatocytes treated with 10⁻⁷ M DEX abolished the DEX effect. RU486 at 10⁻⁸ M was ineffective. Spironolactone (10⁻⁸ to 10⁻⁶ M), a type I specific glucocorticoid receptor antagonist, did not counteract the DEX effect. RU486 or spironolactone (10⁻⁶ M) alone had no effect on CYP 1A1 under similar conditions. DEX thus down regulates CYP 1A1 in fish cultured hepatocytes and this regulation is mediated through the type II glucocorticoid receptor(s).     

Direct regulating effects of transforming growth factor beta on the Leydig cell steroidogenesis in primary culture

The effect of transforming growth factor beta on testicular steroidogenesis was studied by using a model of immature porcine Leydig cells cultured in a chemically defined medium. Leydig cells were cultured in the presence of human or porcine purified TGF beta and the following parameters were measured: cell proliferation, LH/hCG binding, and hCG-stimulated steroid hormone productions (DHEA, DHEAS and testosterone). Whereas TGF beta from the two sources had no effect on Leydig cell multiplication, it markedly inhibited LH/hCG-stimulated DHEA and DHEAS in a time- and dose-dependent manner. The maximal inhibitory effect of this peptide on LH/hCG binding (65% decrease), hCG-stimulated DHEA (77% decrease) and DHEAS (92% decrease) productions was observed with 2 ng/ml for 48 h of treatment. In contrast, TGF beta exerted a biphasic effect on hCG-stimulated testosterone production: stimulating (110% increase) until 2 ng/ml and inhibiting (35% decrease) for higher concentrations. [125I]TGF beta was cross-linked to Leydig cells using disuccinimidyl suberate; cells affinity labelled with [125I]TGF beta exhibit a major labelled band of approx 280 kDa, which has the properties expected from a TGF beta receptor. These data demonstrate that TGF beta is a direct potent regulator of Leydig cell steroidogenic function and its effects are probably mediated via a specific receptor.