Summary Classically, follicle-stimulating hormone receptor (FSHR)-driven cAMP-mediated signaling boosts human ovarian follicle growth and oocyte maturation. However, contradicting in vitro data suggest a different view on physiological significance of FSHR-mediated cAMP signaling. We found that the G-protein-coupled estrogen receptor (GPER) heteromerizes with FSHR, reprogramming cAMP/death signals into proliferative stimuli fundamental for sustaining oocyte survival. In human granulosa cells, survival signals are missing at high FSHR:GPER ratio, which negatively impacts follicle maturation and strongly correlates with preferential Gαs protein/cAMP-pathway coupling and FSH responsiveness of patients undergoing controlled ovarian stimulation. In contrast, FSHR/GPER heteromers triggered anti-apoptotic/proliferative FSH signaling delivered via the Gβγ dimer, whereas impairment of heteromer formation or GPER knockdown enhanced the FSH-dependent cell death and steroidogenesis. Therefore, our findings indicate how oocyte maturation depends on the capability of GPER to shape FSHR selective signals, indicating hormone receptor heteromers may be a marker of cell proliferation.

Follicle-stimulating hormone (FSH) supports spermatogenesis acting via its receptor (FSHR), which activates trophic effects in gonadal Sertoli cells. These pathways are targeted by hormonal drugs used for clinical treatment of infertile men, mainly belonging to sub-groups defined as hypogonadotropic hypogonadism or idiopathic infertility. While, in the first case, fertility may be efficiently restored by specific treatments, such as pulsatile gonadotropin releasing hormone (GnRH) or choriogonadotropin (hCG) alone or in combination with FSH, less is known about the efficacy of FSH in supporting the treatment of male idiopathic infertility. This review focuses on the role of FSH in the clinical approach to male reproduction, addressing the state-of-the-art from the little data available and discussing the pharmacological evidence. New compounds, such as allosteric ligands, dually active, chimeric gonadotropins and immunoglobulins, may represent interesting avenues for future personalized, pharmacological approaches to male infertility.

Recombinant follicle-stimulating hormone (FSH) (follitropin alfa) and biosimilar preparations are available for clinical use. They have specific FSH activity and a unique glycosylation profile dependent on source cells. The aim of the study is to compare the originator (reference) follitropin alfa (Gonal-f®)- with biosimilar preparations (Bemfola® and Ovaleap®)-induced cellular responses in vitro . Gonadotropin N-glycosylation profiles were analyzed by ELISA lectin assay, revealing preparation specific-patterns of glycan species (Kruskal-Wallis test; p < 0.05, n = 6) and by glycotope mapping. Increasing concentrations of Gonal-f® or biosimilar (1 × 10 −3 -1 × 10 3 ng/ml) were used for treating human primary granulosa lutein cells (hGLC) and FSH receptor (FSHR)-transfected HEK293 cells in vitro . Intracellular cAMP production, Ca 2+ increase and β-arrestin 2 recruitment were evaluated by BRET, CREB, and ERK1/2 phosphorylation by Western blotting. 12-h gene expression, and 8- and 24-h progesterone and estradiol synthesis were measured by real-time PCR and immunoassay, respectively. We found preparation-specific glycosylation patterns by lectin assay (Kruskal-Wallis test; p < 0.001; n = 6), and similar cAMP production and β-arrestin 2 recruitment in FSHR-transfected HEK293 cells (cAMP EC 50 range = 12 ± 0.9–24 ± 1.7 ng/ml; β-arrestin 2 EC 50 range = 140 ± 14.1–313 ± 18.7 ng/ml; Kruskal-Wallis test; p ≥ 0.05; n = 4). Kinetics analysis revealed that intracellular Ca 2+ increased upon cell treatment by 4 μg/ml Gonal-f®, while equal concentrations of biosimilars failed to induced a response (Kruskal-Wallis test; p < 0.05; n = 3). All preparations induced both 8 and 24 h-progesterone and estradiol synthesis in hGLC, while no different EC 50 s were demonstrated (Kruskal-Wallis test; p > 0.05; n = 5). Apart from preparation-specific intracellular Ca 2+ increases achieved at supra-physiological hormone doses, all compounds induced similar intracellular responses and steroidogenesis, reflecting similar bioactivity, and overall structural homogeneity.

Commercial gonadotropin-releasing hormone (GnRH) antagonists differ by 1–2 amino acids and are used to inhibit gonadotropin production during assisted reproduction technologies (ART). In this study, potencies of three GnRH antagonists, Cetrorelix, Ganirelix and Teverelix, in inhibiting GnRH-mediated intracellular signaling, were compared in vitro. GnRH receptor (GnRHR)-transfected HEK293 and neuroblastoma-derived SH-SY5Y cell lines, as well as mouse pituitary LβT2 cells endogenously expressing the murine GnRHR, were treated with GnRH in the presence or absence of the antagonist. We evaluated intracellular calcium (Ca2+) and cAMP increases, cAMP-responsive element binding-protein (CREB) and extracellular-regulated kinase 1 and 2 (ERK1/2) phosphorylation, β-catenin activation and mouse luteinizing-hormone β-encoding gene (Lhb) transcription by bioluminescence resonance energy transfer (BRET), Western blotting, immunostaining and real-time PCR as appropriate. The kinetics of GnRH-induced Ca2+ rapid increase revealed dose-response accumulation with potency (EC50) of 23 nM in transfected HEK293 cells, transfected SH-SY5Y and LβT2 cells. Cetrorelix inhibited the 3 × EC50 GnRH-activated calcium signaling at concentrations of 1 nM–1 µM, demonstrating higher potency than Ganirelix and Teverelix, whose inhibitory doses fell within the 100 nM–1 µM range in both transfected HEK293 and SH-SY5Y cells in vitro. In transfected SH-SY5Y, Cetrorelix was also significantly more potent than other antagonists in reducing GnRH-mediated cAMP accumulation. All antagonists inhibited pERK1/2 and pCREB activation at similar doses, in LβT2 and transfected HEK293 cells treated with 100 nM GnRH. Although immunostainings suggested that Teverelix could be less effective than Cetrorelix and Ganirelix in inhibiting 1 µM GnRH-induced β-catenin activation, Lhb gene expression increase occurring upon LβT2 cell treatment by 1 µM GnRH was similarly inhibited by all antagonists. To conclude, this study has demonstrated Cetrorelix-, Ganirelix- and Teverelix-specific biased effects at the intracellular level, not affecting the efficacy of antagonists in inhibiting Lhb gene transcription.