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Both fibroblast growth factor 2 (FGF2) and luteinizing hormone (LH) have been reported to regulate androgen production in Leydig cells in progenitor Leydig cells. The objective of the present study is to examine the regulation of androgen production in rat immature Leydig cells (ILCs). ILCs were isolated from 35-day-old rat testes and cultured in DMEM/F12 medium with LH (1 ng/ml) or FGF2 (10 ng/ml). 5alpha-Androstane-3alpha, 17beta-diol (3alpha-DIOL), the primary androgen in ILCs, and testosterone (T) were measured by Radioimmuno assay. The results showed the LH stimulated androgen production in ILCs, and FGF2 did not. However, FGF2 decreased the LH-stimulated androgen production. Real-time PCR and enzyme assay showed that FGF2 decreased levels of several steroidogenic enzymes, inhibited the expressions of steroidogenic acute regulatory (StAR) protein and steroidogenic factor 1 (Nr5a1) in LH-stimulated ILCs. FGF2-mediated inhibition of Nr5a1gene expression may be the mechanism through which FGF2 inhibits LH-stimulated androgen production.

The free radical nitric oxide (NO), generated through the oxidation of L-arginine to L-citrulline by NO synthases (NOSs), has been shown to inhibit steroidogenic pathways. NOS isoforms are known to be present in rat and human testes. Our study examined the sensitivity of Leydig cells to NO and determined whether NOS activity resides in Leydig cells or in another cell type such as the testicular macrophage. The results showed a low level of L-[14C]arginine conversion in purified rat Leydig cell homogenates. Administration of the NOS inhibitor L-N(G)-nitro-arginine methyl ester (L-NAME), or the calcium chelator ethylenebis (oxyethylenenitrilo)tetraacetic acid (EGTA), had no effect on L-[14C]citrulline accumulation. Increased intracellular Ca2+ concentrations that were induced by a calcium ionophore, or the addition of luteinizing hormone (LH), failed to affect NO formation in intact cells that were cultured in vitro. Introduction of a high concentration of the NO precursor L-arginine did not decrease testosterone (T) production, and NOS inhibitors did not increase T biosynthesis. However, exposing Leydig cells to low concentrations of the NO donor S-nitrosoglutathione (GSNO) induced a dramatic blockade of T production under basal and LH-stimulated conditions. DNA array assays showed a low level of expression of endothelial NOS (eNOS), while the neuronal and inducible isoforms of NOS (nNOS and iNOS) were below detection levels. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses confirmed these findings and demonstrated the presence of high iNOS messenger RNA (mRNA) levels in activated testicular macrophages that produced large amounts of NO. These data suggest that, while T production in rat Leydig cells is highly sensitive to NO and an endogenous NO-generating system is not present in these cells, NOS activity is more likely to reside in activated testicular macrophages.

Abstract: As one of the most abundant polybrominated diphenylethers (PBDEs) detected in adipose tissue and breast milk of humans, 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) is considered as a potential endocrine disruptor. The objective of this study is to explore whether environment-related level of BDE-47 could affect the androgen production in rat Leydig cells. Rat adult Leydig cells (ALCs) were treated with 10−8 to 10−4 M BDE-47 in vitro, the production of testosterone (T) and steroidogenic acute regulatory (StAR) protein level were determined. BDE-47 significantly increased basal T production and steroidogenic acute regulatory protein (StAR) level of ALCs after treatment with 10−4 M BED-47. Overall, LH (0.1ng/ml) stimulated T production in ALCs by 6 folds, however it did not increase T production in BDE-47-treated ALCs when compared to untreated ALC. Both 8-Br-cAMP (for cAMP signaling) and 22R-hydroxycholesterol (22-diol, for P450 cholesterol side chain cleavage enzyme P450scc activity) significantly increased T production in ALCs treated with BDE-47 from 10−7 to 10−5 M. The results of this study indicate that environment-related level of BDE-47 in vitro increased T production in a dose-dependent manner. The stimulated effects of BDE-47 on StAR and P450scc might play key roles in BDE-47-mediated stimulation of T production. [Copyright &y& Elsevier]