The aim of the present study was to determine the mechanisms involved in estrogen actions in cultured rat Sertoli cells. RT-PCR detected transcripts for the estrogen receptors ESR1 and ESR2 in cultured immature Sertoli cells and in the testis of 15-, 28-, and 120-day-old rats. The expression of ESR1 and ESR2 was confirmed in Sertoli cells by immunofluorescence and Western blot. Immunohistochemistry with cryosections of testes from immature and adult rats revealed that ESR1 is present in Sertoli, Leydig, and some peritubular myoid cells, and ESR2 is present in multiple cell types, including germ cells. Treatment of Sertoli cells with 17beta-estradiol (E(2)) induced a translocation of ESR1 and ESR2 to the plasma membrane and a concomitant phosphorylation of MAPK3/1. Both effects reached a maximum after 10 min and were blocked by PP2, an inhibitor of the SRC family of protein tyrosine kinases, and by the antiestrogen ICI 182,780 (ICI). MAPK3/1 phosphorylation was also decreased in the presence of AG 1478, an inhibitor of the epidermal growth factor receptor (EGFR) kinase, and in the presence of MAP2K1/2 inhibitor UO126. Treatment with E(2) for 24 h increased the incorporation of [methyl-(3)H]thymidine, which was blocked by ICI. These results indicate that E(2) activates an SRC-mediated translocation of estrogen receptors to the plasma membrane, which results in the activation of EGFR and the mitogen-activated protein kinase signaling pathway. In addition, activation of ESR1 and/or ESR2 by E(2) is involved in proliferation of immature Sertoli cells. The estrogen actions in Sertoli cells might be a key step mediating cellular events important for spermatogenesis and fertility.

Estrogens play key roles in the development and maintenance of male reproductive function and fertility. In this review, we briefly describe the localization and function of estrogen receptors ESR1 and ESR2 (also known as ERa and ERb, respectively) and the expression of G protein-coupled estrogen receptor-1 (GPER, formerly known as GPR30) in efferent ductules and epididymis. The efferent ductules present the highest levels of ESR1 and ESR2 in the male reproductive system, and represent a major target of estrogen action. In efferent ductules, ESR1 has a crucial role in the regulation of fluid reabsorption, and in the epididymis the receptor helps to maintain fluid osmolality and pH. ESR1 expression in the epididymal epithelium shows considerable variation among species, but differences in laboratory techniques may also contribute to this variation. Here we report that Esr1 mRNA and protein are higher in corpus than in other regions of the rat epididymis. The mRNA level for Gper was also higher in corpus. Although ESR1 is expressed constitutively in efferent ductules and down-regulated by estrogen, in the epididymis, both testosterone (T) and estradiol (E2) may regulate its expression. T and E2 are, respectively, higher and lower in the corpus than in the initial segment/caput and cauda regions. It is important to determine the expression of GPER, ESR1, androgen receptor, and their respective cofactors in specific cell types of this tissue, as well as the intracellular signaling pathways involved in efferent ductules and epididymis. These studies will help to explain the consequences of exposures to environmental endocrine disruptors and provide potential targets for the development of a male contraceptive.

The aim of the present study was to investigate the expression and signaling of the G protein-coupled estrogen receptor 1 (GPER) in cultured immature rat Sertoli cells--in which we have previously described the classical estrogen receptors (ESR1 and ESR2). Expression of GPER in cultured Sertoli cells from 15-day-old rats was detected by RT-PCR and immunoassays. Gper transcripts also were present in testes from 5-, 15-, and 120-day-old rats. Short-term treatment of Sertoli cells with 17beta-estradiol (E2), the GPER agonist G-1, or the ESR antagonist ICI 182,780 (ICI) rapidly activated MAPK3/1 (ERK1/2), even after down-regulation of ESR1 and ESR2, suggesting a role for GPER in the rapid E2 action in these cells. MAPK3/1 phosphorylation induced by ICI or G-1 was blocked by pertussis toxin, selective inhibitor of the SRC family of protein tyrosine kinases, metalloprotease inhibitor, MAP2K1/2 inhibitor, and epidermal growth factor receptor (EGFR) kinase inhibitor. Furthermore, E2, but not G-1, induced up-regulation of cyclin D1 in the Sertoli cells. This effect was blocked by ICI. E2 and G-1 decreased BAX and increased BCL2 expression and these effects were blocked by MAP2K1/2 inhibitor and EGFR kinase inhibitor. The pretreatment with ICI did not block the effect of E2. Taken together, these results indicate that in Sertoli cells 1) GPER-mediated MAPK3/1 activation occurs via EGFR transactivation through G protein beta gamma subunits that promote SRC-mediated metalloprotease-dependent release of EGFR ligands, which bind to EGFR and lead to MAPK3/1 phosphorylation; 2) E2-ESRs play a role in Sertoli cell proliferation; and 3) E2-GPER may regulate gene expression involved with apoptosis. ESR and GPER may mediate actions important for Sertoli cell function and maintenance of normal testis development and homeostasis.

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