OR24-04 Ovarian Follicle Survival Is Determined by Follicle-Stimulating Hormone Receptor (FSHR) and Estrogen Receptor (GPER) Heteromers
Authors: Casarini Livio , Lazzaretti Clara , Paradiso Elia , Limoncella Silvia , Riccetti Laura , Sperduti Samantha , Melli Beatrice , Marcozzi Serena , Anzivino Claudia , Sayers Niamh S , Czapinski Jakub , Brigante Giulia , Potì Francesco , Marca Antonio La , Villani Maria T , Klinger Francesca G , Fanelli Francesca , Rivero-Müller Adolfo , Hanyaloglu Aylin C , Simoni Manuela 
Published: May 2020

Abstract
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. 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 nuclears 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. Here we demonstrate how FSHR/GPER physical interaction rescue ovarian follicles from FSH-mediated death. 10 nM FSH induces high intracellular levels of cAMP, measured by bioluminescence resonance energy transfer (BRET), 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, photo-activated localization microscopy (PALM) and bioinformatics prodiction, we also demonstrate FSHR/GPER heteromers 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 co-expressing FSHR results in displacement of the Gαs-protein to FSHR, blockade of FSH-induced cAMP production and inhibition of apoptosis. However, 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. Inhibition of both FSH-induced cAMP production and apoptosis was lost when FSHR is coexpressed together with a mutant GPER, unable to heteromerize with FSHR, as well as in KO HEK293 cells unable to produce a molecular complex associated with GPER inhibiting cAMP. 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. Most importantly, FSHR and GPER co-expression correlates in hGLC from FSH-normo-responder women undergoing assisted reproduction, while it is not in hGLC from FSH-poor-responders, where increasing FSHR mRNA levels do not correspond to increasing GPER mRNA levels. 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.