Estradiol Regulates mRNA Levels of Estrogen Receptor Beta 4 and Beta 5 Isoforms and Modulates Human Granulosa Cell Apoptosis

Abstract

Estrogen receptor beta (ERβ) plays a critical role in granulosa cell (GC) functions. The existence of four human ERβ splice isoforms in the ovary suggests their differential implication in 17β-estradiol (E2) actions on GC apoptosis causing follicular atresia. In this study, we investigated whether E2 can regulate ERβ isoforms expression to fine tune its apoptotic activities in human GC. For this purpose, we measured by RT-qPCR the expression of ERβ isoforms in primary culture of human granulosa cells (hGCs) collected from patients undergoing in vitro fertilization, before and after E2 exposure. Besides, we assessed the potential role of ERβ isoforms on cell growth and apoptosis after their overexpression in a human GC line (HGrC1 cells). We confirmed that ERβ1, ERβ2, ERβ4, and ERβ5 isoform mRNAs were predominant over that of ERα in hGCs, and found that E2 selectively regulates mRNA levels of ERβ4 and ERβ5 isoforms in these cells. In addition, we demonstrated that overexpression of ERβ1 and ERβ4 in HGrC1 cells increased cell apoptosis by 225% while ERβ5 or ERβ2 had no effect. Altogether, our study revealed that E2 may influence GC fate by specifically regulating the relative abundance of ERβ isoforms mRNA to modulate the balance between pro-apoptotic and non-apoptotic ERβ isoforms.

Estradiol Signaling at the Heart of Folliculogenesis: Its Potential Deregulation in Human Ovarian Pathologies

“…ERβ expression begins in the GC of immature follicles and reaches maximum levels in maturing and preovulatory follicles [ 6 , 28 ]. Analyses of ERβ isoform mRNA in human also show that all ERβ isoforms are expressed in the ovary [ 29 ], in GC [ 6 , 30 ], as well as in various forms of ovarian cancers [ 31 , 32 ], including GC tumors [ 33 ]. In addition, ERβ1 and ERβ2 mRNA are differently expressed across the luteal phase with a maximal expression observed in the mid-luteal phase for ERβ1 and in the early luteal phase for ERβ2 [ 34 ].…”

“…The precise molecular mechanisms regulating ERβ alternative splicing are largely unknown. Still, E2 is described to specifically up-regulate the expression of ERβ4 and ERβ5 mRNA in human GC of antral follicles [ 30 ]. ERβ originates from multiple putative promoters (5′-untranslated first exons 0K, 0N, and E1) [ 35 , 36 ] with the same final transcript ( Figure 1 ).…”

“…The observed E2-induced GC apoptosis may be the result of activation of different ER subtypes, especially ERβ isoforms [ 67 , 68 , 69 , 70 ]. Indeed, various studies reveal differential effects of ERβ isoforms on cell proliferation and/or apoptosis, such as ERβ2 and ERβ5 that increase prostate or glioma tumor proliferation [ 68 , 71 ], while ERβ1 and ERβ4 induce GC apoptosis [ 30 ].…”

“…Two studies report an increase in both ERα ( ESR1 ) and ERβ ( ESR2 ) expression in mural GC of PCOS women [ 93 , 99 ], while a third one described the opposite [ 100 ]; these discrepancies can stem from the difference in the ovarian stimulation protocol used. These investigations did not quantify the expression levels of each ERβ isoform present in luteinized GC [ 30 ]. Therefore, further data on the respective expression level of ERβ isoforms in GC from PCOS and non-PCOS women will be helpful to better understand E2 signaling in PCOS.…”

“…E2 may also contribute to GC fate in PCOS, as depicted in rhesus monkeys, high concentrations of E2 inducing atresia of antral follicles [ 3 ]. In addition, E2 specifically regulates the expression of ERβ isoforms, of which two can trigger apoptotic events in GC [ 30 ]. E2, by regulating specific ERβ isoform expression in GC of PCOS, might participate in their follicular fate.…”