Endocrine Abstracts

Introduction: Follicle-stimulating hormone (FSH) is a glycoprotein that support reproduction by regulating ovarian follicular growth and development. Recent studies demonstrated that proliferative signals mediating folliculogenesis are mediated by the G protein-coupled estrogen receptor (GPER) expressed in ovarian tissues throughout the follicular phase. In granulosa cells, GPER forms heteromers with FSHR, reprogramming cAMP-induced death signals to AKT-dependent, anti-apoptotic/proliferative events, fundamental to sustain oocyte survival. Since GPER is mainly located in the endoplasmic reticulum, we may hypothesize it modulates FSH signals via heteromerization and retention of FSHR in the cytoplasm.

Aim: In this study, we analysed FSHR/GPER heteromers trafficking through early and late endosomes and its impact on FSH-mediated signalling from endocytic compartments.

Methods: HEK293 cells were transfected with FSHR- and GPER-coding plasmids and treated by 10 nM FSH, in the presence or absence of receptor internalization blockade by Dynasore. cAMP production and FSHR interaction with specific endosomal markers, i.e. Ras-related proteins (Rab) 5, 7 and 11, were evaluated at different time-points (0-20 min) by bioluminescence resonance energy transfer (BRET). Results were compared by Kruskal-Wallis test and Dunn’s post-hoc test (P<0.05; n=5) and showed as means±SEM.

Results: FSH treatment of FSHR only-expressing cells resulted in the receptor internalization mediated by β-arrestins, and in FSHR localization into Rab7-positive endosomes, addressing it to lysosomes. No FSH-induced compartmentalization of the receptor into early- and recycling-endosomes (Rab5 and Rab11 markers) was found. Conversely, FSHR/GPER co-expression reduces the basal FSHR trafficking through Rab5 and Rab11-marked endosomes, prevents FSH-induced FSHR-Rab7 interaction and drives FSHR internalization mainly through β-arrestin recruitment (FSHR- vs FSHR/GPER-expressing cells; P<0.05). This event might be essential for FSH-induced signalling modulation. Indeed, as previously described, the FSH treatment of FSHR-expressing cells induces intracellular cAMP increase, while it does not in FSHR/GPER co-expressing cells (AUC FSHR=85.51±8.4 vs AUC FSHR+GPER=30.6±7.4; P<0.05). Interestingly, in FSHR/GPER co-expressing cells where internalization was inhibited by Dynasore, FSH induces a cAMP response, oppositely to what demonstrated in Dynasore-untreated cells (AUC Dynasore-treated FSHR+GPER=101.1±16.11; P<0.05) and suggesting the requirement of the heteromer internalization to inhibit cAMP production.

Conclusion: In conclusion, these results suggest that GPER blocks FSHR-mediated cAMP production via compartmentalization of receptors into specific endosomes. These data strengthen the existence of FSHR membrane partners modulating its mode of action, possibly impacting ovarian physiology.