<em>In vitro</em> analysis of FSHR internalization and signalling mediated by two different FSH preparations

Lara Baschieri; Clara Lazzaretti; Elisa Mascolo; Carmela Perri; Samantha Sperduti; Claudia Fusco; Manuela Simoni; Livio Casarini

doi:10.1530/endoabs.99.P561

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Endocrine Abstracts (2024) 99 P561 | DOI: 10.1530/endoabs.99.P561

ECE2024 Poster Presentations Reproductive and Developmental Endocrinology (45 abstracts)

In vitro analysis of FSHR internalization and signalling mediated by two different FSH preparations

Lara Baschieri ^1,2 , Clara Lazzaretti ¹ , Elisa Mascolo ¹ , Carmela Perri ^1,2 , Samantha Sperduti ^1,3 , Claudia Fusco ¹ , Manuela Simoni ^1,3,4 & Livio Casarini ^1,3

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¹Unit of Endocrinology - University of Modena and Reggio Emilia, Department of Biomedical, Metabolic and Neural Sciences, Modena, Italy; ²University of Modena and Reggio Emilia, International Ph.D. School in Clinical and Experimental Medicine (CEM), Modena, Italy; ³University of Modena and Reggio Emilia, Center for Genomic Research, Modena, Italy; ⁴Azienda Ospedaliero-Universitaria di Modena, Department of Medical Specialties, Modena, Italy

Follicle stimulating hormone (FSH) is produced by the anterior pituitary gland and is a key hormone in the reproductive system. FSH is a heterodimeric glycoprotein composed with two extensively glycosylated protein subunits (α and β) N-glycosylated in two positions (Asn⁵² and Asn⁷⁸in the FSHα subunit and Asn⁷ and Asn²⁴ in the FSHβ subunit). Due to different glycosylation pattern, several FSH isoforms may differently act on the receptor (FSHR). We compared in vitro the trafficking mediated by two FSH commercial preparations, characterized by different glycosylation pattern, Gonal-f® (Merck KGaA) and Rekovelle® (Ferring Pharmaceuticals). HEK293 cells transiently overexpressing FSHR were treated by increasing doses of FSH (nM-µM range) and with/without Dynamin inhibition (Dynasore). We evaluated cAMP production, interaction between FSHR and endosomal specific markers, and CRE-controlled reporter gene activation using luciferase and energy transfer-based methods. Statistics: Mann Whitney’s U-test (P<0.05; n=4-5). We found no different interaction between FSHR and the marker of early endosome Rab5, in cells treated with Gonal-f® or Rekovelle®, regardless the presence of Dynasore (P>0.05; n=5). Dynamin inhibition was linked to increased Gonal-f®-, but not Rekovelle®-induced FSHR-Rab7 interaction, indicating preparation-specific routing to the degradation pathway (P<0.05; n=5). Gonal-F® induced more pronounced cAMP production at 1 µM concentration, while at 10 nM Rekovelle® induced a significantly higher cAMP production, regardless of Dynasore treatment (P<0.05; n=4). Finally, CRE reporter gene activation in absence of Dynasore is higher at 60 nM concentration of Gonal-F®, compared to Rekovelle® (P<0.05; n=4). To conclude, Gonal-f® and Rekovelle® mediate different FSHR internalization and signaling.