Mechanisms regulating the selection of antral ovarian follicles are poorly understood and supposed to rely on low estrogen levels, decline of follicle-stimulating hormone (FSH) levels and receptor (FSHR) expression on the surface of granulosa cells. These concepts are challenged in-vitro, where apoptosis of human granulosa cells (hGLC) and transfected cell lines is induced by high doses of FSH or FSHR overexpression, while estrogens induce anti-apoptotic signals via nuclear and a G protein-coupled estrogen-receptor (GPER). Therefore, in-vitro data suggest that antral follicle selection may be driven by underestimated, FSH-FSHR-dependent apoptotic signals due to transiently maximized FSHR expression and overload of cAMP signalling, prevailing on estrogen-dependent signals. In this study, we demonstrate how estrogens can rescue the dominant follicle from FSHR-mediated death.
Experiments: 10 nM FSH induces high intracelular levels of cAMP (FSH 160×10−3±26×10−3 vs basal 5×10−3±9×10−3 bioluminescence resonance energy transfer (BRET)-changes; Mann-Whitneys U-test; P<0.05; n=5; mean±S.D.) and apoptosis in cultured hGLC under conditions where GPER levels are depleted by siRNA. This result was confirmed in transfected HEK293 cells overexpressing FSHR. Using BRET and photo-activated localization microscopy (PALM), we also demonstrate that FSHR forms heteromers with GPER at the cell surface. The role of FSHR-GPER heteromers may be relevant to inhibit FSH-induced death signals, since increasing GPER expression levels in HEK293 cells coexpressing FSHR results in displacement of the Gαs-protein to FSHR (IC50=0.221±0.002; r2=0.546; nonlinear regression; n=3; mean±S.D.), blockade of FSH-induced cAMP production (FSH 0−10−3±6−10−3 vs basal 1−10−3±9−10−3 BRET-changes; Mann-Whitneys U-test; P<0.05; n=5; mean±S.D.) and inhibition of apoptosis. Interestingly, in HEK293 cells coexpressing GPER/FSHR, FSH-induced activation of the anti-apoptotic AKT-pathway via a Gβγ-dependent mechanism, as demonstrated by Western blotting in cells treated using the inhibitor gallein. Critically, inhibition of both FSH-induced cAMP production (FSH 152×24−3±24×10−3 vs basal 5−10−3±9−10−3 BRET changes; Mann-Whitneys U-test; P<0.05; n=4; mean±S.D.) and apoptosis was lost when FSHR is coexpressed together with a mutant GPER, unable to heteromerize with FSHR (r2=0.7×10−3; P=0.86; linear regression). GPER-FSHR coexpression is confirmed in secondary follicles from paraffin-embedded tissues of human ovary by immunohistochemistry, suggesting that FSHR-GPER heterodimers could be physiologically relevant in-vivo for inhibiting cAMP-linked apoptosis. We demonstrate that death signals in atretic follicles are delivered through overexpressed FSHR and inhibited by FSHR-GPER heteromerization, activating anti-apoptotic pathways. This finding unveils a novel working model of the physiology of dominant follicle selection and the relationship between FSH and estrogens.
18 - 21 May 2019
European Society of Endocrinology