17 beta-estradiol activates rapid signaling pathways involved in rat pachytene spermatocytes apoptosis through GPR30 and ER alpha.

Aim of the present study was to investigate whether estrogens were able to directly activate rapid signaling pathways controlling spermatogenesis in rat pachytene spermatocytes (PS). Classically, estrogens act by binding to estrogen receptors (ERs) alpha and beta. Recently, it has been demonstrated that rapid estrogen action can also be activated through the G-protein-coupled receptor (GPR)-30. Herein, we demonstrated that rat PS express ER alpha, ER beta and GPR30. Treatment of PS with estradiol (E2), the selective GPR30 agonist G1 and the selective ER alpha agonist PPT determined activation of ERK1/2 which are part of GPR30 signaling cascade. ERK1/2 activation in response to E2 and G1 was correlated to an increased phosphorylation of c-Jun. All treatments failed to induce these responses in the presence of EGFR inhibitor AG1478, ERK inhibitor PD98059 and ER inhibitor ICI182780. mRNA expression of cell cycle regulators cyclin A1 and B1 was downregulated by E2 and G1 while an up-regulation of proapoptotic factor Bax was observed in the same conditions. These data demonstrate that E2, working through both ER alpha and/or GPR30, activates in PS the rapid EGFR/ERK/c-Jun pathway, modulating the expression of genes involved in the balance between cellular proliferation and apoptosis.

Chimento A ，Sirianni R ，Delalande C ，Silandre D ，Bois C ，Andò S ，Maggiolini M ，Carreau S ，Pezzi V ... -  《-》

The novel estrogen receptor, G protein-coupled receptor 30, mediates the proliferative effects induced by 17beta-estradiol on mouse spermatogonial GC-1 cell line.

Sirianni R ，Chimento A ，Ruggiero C ，De Luca A ，Lappano R ，Andò S ，Maggiolini M ，Pezzi V ... -  《ENDOCRINOLOGY》

17β-Estradiol activates GPER- and ESR1-dependent pathways inducing apoptosis in GC-2 cells, a mouse spermatocyte-derived cell line.

In mammals, spontaneous apoptosis is observed particularly in differentiating spermatogonia and in spermatocytes. 17β-Estradiol (E2) in primary rat pachytene spermatocytes (PS) binds estrogen receptor α (ESR1) and GPER to activate EGFR/ERK/c-Jun pathway leading to up regulation of proapoptotic factor bax. Aim of this study was to clarify the effector pathway(s) controlling spermatocytes apoptosis using as model GC-2 cells, an immortalized mouse pachytene spermatocyte-derived cell line, which reproduces primary cells responses to E2. In fact, in GC-2 cells we observed that ESR1 and GPER activation caused rapid ERK and c-Jun phosphorylation, bax up-regulation, events associated with apoptosis. We further investigated the apoptotic mechanism demonstrating that E2, as well as ESR1 and GPER specific agonists, induced sustained ERK, c-Jun and p38 phosphorylation, Cytochrome c release, caspase 3 and endogenous substrate Poly (ADP-ribose) polymerase (PARP) activation and increased expression of cell cycle inhibitor p21. When ESR1 or GPER expression was silenced, E2 was still able to decrease cell proliferation, only the concomitant silencing abolished E2 effect. These results indicate that GC-2 cells are a valid cell model to study E2-dependent apoptosis in spermatocytes and show that E2, activating both ESR1 and GPER, is able to induce an ERK1/2, c-Jun and p38-dependent mitochondrion apoptotic pathway in this cell type.

Chimento A ，Sirianni R ，Casaburi I ，Ruggiero C ，Maggiolini M ，Andò S ，Pezzi V ... -  《-》

Gper and ESRs are expressed in rat round spermatids and mediate oestrogen-dependent rapid pathways modulating expression of cyclin B1 and Bax.

Spermatogenesis is a precisely controlled and timed process, comprising mitotic divisions of spermatogonia, meiotic divisions of spermatocytes, maturation and differentiation of haploid spermatids giving rise to spermatozoa. It is well known that the maintenance of spermatogenesis is controlled by gonadotrophins and testosterone, the effects of which are modulated by a complex network of locally produced factors, including oestrogens. However, it remains uncertain whether oestrogens are able to activate rapid signalling pathways directly in male germ cells. Classically, oestrogens act by binding to oestrogen receptors (ESRs) 1 and 2. Recently, it has been demonstrated that rapid oestrogen action can also be mediated by the G-protein-coupled oestrogen receptor 1 (Gper). The aim of the present study was to investigate ESRs and Gper expression in primary cultures of adult rat round spermatids (RS) and define if oestradiol (E2) is able to activate, through these receptors, pathways involved in the regulation of genes controlling rat RS apoptosis and/or maturation. In this study, we demonstrated that rat RS express ESR1, ESR2 and Gper. Short-time treatment of RS with E2, the selective Gper agonist G1 and the selective ESR1 and ERβ agonists, 4,4',4"-(4-propyl-[1H]pyrazole-1,3,5-triyl) trisphenol (PPT) and 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN), respectively, determined activation of Extra-cellular signal-regulated kinase (ERK1/2) through the involvement of epidermal growth factor receptor transactivation. In addition, we investigated the effects of ESRs and Gper pathway activation on factors involved in RS maturation. Expression of cyclin B1 mRNA was downregulated by E2, G1 and PPT, but not by DPN. A concomitant and inverse regulation of the pro-apoptotic factor Bax mRNA expression was observed in the same conditions, with DPN being the only one determining an increase in this factor expression. Collectively, these data demonstrate that E2 activates, through ESRs and Gper, pathways involved in the regulation of genes controlling rat RS apoptosis and differentiation such as cyclin B1 and Bax.

Chimento A ，Sirianni R ，Zolea F ，Bois C ，Delalande C ，Andò S ，Maggiolini M ，Aquila S ，Carreau S ，Pezzi V ... -  《-》

Involvement of estrogen receptor variant ER-alpha36, not GPR30, in nongenomic estrogen signaling.

Kang L ，Zhang X ，Xie Y ，Tu Y ，Wang D ，Liu Z ，Wang ZY ... -  《-》