ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Endocrine Abstracts (2019) 63 P644 | DOI: 10.1530/endoabs.63.P644

Dominance of ovarian follicles is determined by follicle-stimulating hormone receptor (FSHR) and G protein-coupled estrogen receptor (GPER) heteromers

Livio Casarini1,2, Clara Lazzaretti1,2,3, Elia Paradiso1,2,3, Laura Riccetti1, Samantha Sperduti1, Serena Marcozzi4, Niamh Sayers5, Giulia Brigante1,6, Francesco Potì7, Antonio La Marca8,9, Barbara Valli10, Angela Falbo10, Maria Teresa Villani10, Francesca Gioia Klinger4, Francesca Fane11,12, Aylin Carla Hanyaloglu5 & Manuela Simoni12,6


1Unit of Endocrinology, Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy; 2Center for Genomic Research, University of Modena and Reggio Emilia, Modena, Italy; 3International Doctorate School (PhD) in Clinical and Experimental Medicine, University of Modena and Reggio Emilia, Modena, Italy; 4Histology and Embryology Section, Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy; 5Institute of Reproductive and Developmental Biology, Imperial College London, London, UK; 6Department of Medical Specialties, Azienda Ospedaliero-Universitaria di Modena, Modena, Italy; 7Unit of Neurosciences, Department of Medicine and Surgery, University of Parma, Parma, Italy; 8Mother-Infant Department, University of Modena and Reggio Emilia, Modena, Italy; 9Clinica EUGIN, Modena, Italy; 10Department of Obstetrics and Gynaecology, Fertility Center, ASMN. Azienda Unità Sanitaria Locale – IRCCS di Reggio Emilia, Reggio Emilia, Italy; 11Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy; 12Center for Neuroscience and Neurotechnology, Modena, Italy.


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-Whitney’s 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-Whitney’s 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-Whitney’s 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.

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