Follicle stimulating hormone (FSH) is produced by the anterior pituitary gland and it is a key hormone in the reproductive system. FSH is a heterodimeric glycoprotein composed with two extensively glycosylated protein sobunits (α and β) N-glycosylated in two positions (Asn52 and Asn78 in the FSHα subunit and Asn7 and Asn24 in the FSHβ subunit) giving rise to numerous isoforms of FSH. Glycosylation has been shown to impact on the in vivo activity of FSH by influencing its clearance and its ability to bind and activate receptors. Different FSH glycoforms are used for assisted reproduction. The aim of this study in vitro is to compare the intracellular signaling pattern mediated by two differently glycosylated, recombinant FSH. Gonal-f® (Merck KGaA) is produced using a modified chinese hamster ovary (CHO) cell line while Rekovelle® (Ferring Pharmaceuticals) is produced by the human fetal retinal PER.C6® cell line. Transfected HEK293 cells overexpressing the FSH receptor (FSHR) were treated by increasing FSH doses (0.0-1.0 µM range). The intracellular cAMP and Ca2+ increase, IP1 production, FSHR homomers formation and the evaluation of transcriptional activity of genes dependent on cAMP and pERK1/2 activation, using both CRE- and NFAT-luciferase reporter assay, were evaluated by energy transfer-based methods. Results were compared by two-way ANOVA and Bonferroni post-test (P<0.05). The two differently glycosylated rFSH impact differently on FSHR homomer formation (n=4). No statistically significant difference was evaluated regarding the intracellular accumulation of cAMP (n=6). However, Gonal-F® is more effective than Rekovelle®. Gonal-f® was less potent but more effective than Rekovelle® in activating the CRE-controlled reporter gene (n=5). Furthermore, Rekovelle® induced a biphasic response, at low concentrations it is more potent than Gonal-f® while at high concentrations it seems to inhibit the activation of the reporter gene controlled by CRE. In order to understand the contribution of non-classical FSH-dependent pathways, not necessarily dependent on cAMP, on cell functions the CRE-controlled reporter gene activation was evaluated in the presence or absence of Dynasore or specific PKA, PLC, AKT, MEK-ERK1/2 inhibitors (n=6). The two differently glycosylated rFSH induce a different signal pattern given by the activation of CRE-controlled reporter gene in the presence of inhibitors except in the presence of AKT inhibitor. Rekovelle® is more potent to induce intracellular Ca 2+ release (n=6) than Gonal-F® but no different IP1 production and NFAT-controlled reporter gene activation were detected (n=4). Recombinant FSH used in clinical practice may impact differently intracellular signaling cascades in vitro.
13 May 2023 - 16 May 2023