Abstract
PurposeEstradiol (E2) modulates testicular functions including steroidogenesis, but the mechanisms of E2 signaling in human testis are poorly understood. GPER-1 (GPR30), a G protein-coupled membrane receptor, mediates rapid genomic and non-genomic response to estrogens. The aim of this study was to evaluate GPER-1 expression in the testis, and its role in estradiol dependent regulation of steroidogenesis in isolated rat Leydig cells and human testis.Materials and MethodsIsolated Leydig cells (LC) from adult rats and human testicular tissue were used in this study. Expression and localization studies of GPER-1 were performed with qRT-PCR, immunofluorescence, immunohistochemistry and Western Blot. Luteinizing Hormone (LH) -stimulated, isolated LC were incubated with estradiol, G-1 (GPER-1-selective agonist), and estrogen receptor antagonist ICI 182,780. Testosterone production was measured with radioimmunoassay. LC viability after incubation with G-1 was measured using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay.ResultsGPER-1 mRNA is abundantly expressed in rat LC and human testis. Co-localization experiments showed high expression levels of GPER-1 protein in LC. E2-dependent activation of GPER-1 lowers testosterone production in isolated rats LCs and in human testis, with statistically and clinically significant drops in testosterone production by 20–30% as compared to estradiol-naïve LC. The exposure to G-1 does not affect viability of isolated LCs.ConclusionsOur results indicate that activation of GPER-1 lowers testosterone levels in the rat and human testis. The expression of GPER-1 in human testis, which lack ERα, makes it an exciting target for developing new agents affecting testosterone production in men.
Highlights
Estrogens are necessary for maintaining structural and functional integrity of the male reproductive tract, [1] but little is known regarding the effects of estradiol on steroidogenesis
Our results indicate that activation of GPER-1 lowers testosterone levels in the rat and human testis
High-resolution Z-stack imaging with 3D deconvolution indicates that in Leydig Cells (LC) GPER-1 is expressed predominantly within the cytoplasm and most likely within the endoplasmic reticulum, which is consistent with data found in breast cancer cell lines (Figure 1B). [16,17]
Summary
Estrogens are necessary for maintaining structural and functional integrity of the male reproductive tract, [1] but little is known regarding the effects of estradiol on steroidogenesis. The cellular response to estrogens is mediated through the welldescribed nuclear estrogen receptors a and b (ERa, ERb), which function as ligand-dependent transcription factors; ligand-activated estrogen receptors bind to estrogen response elements (ERE) in the genome and modulate gene expression in many tissues, including those of the male reproductive tract. The identification of G-1, the first synthetic agonist for GPER-1[12] allowed us to differentiate between the effects of GPER-1 and ER, as G-1 binds with high selectivity to GPER-1
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