GnRH Antagonists Produce Differential Modulation of the Signaling Pathways Mediated by GnRH Receptors


Int J Mol Sci. 2019 Nov; 20(22): 5548

Samantha Sperduti,1,2 Silvia Limoncella,1 Clara Lazzaretti,1,3 Elia Paradiso,1,3 Laura Riccetti,1 Sara Turchi,1 Ilaria Ferrigno,1 Jessika Bertacchini,4 Carla Palumbo,4 Francesco Potì,5 Salvatore Longobardi,6 Robert P. Millar,7 Manuela Simoni,1,2,8,9 Claire L. Newton,7 and Livio Casarini1,2
1Unit of Endocrinology, Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Via P. Giardini 1355, 41126 Modena, Italy
2Center for Genomic Research, University of Modena and Reggio Emilia, Via G. Campi 287, 41125 Modena, Italy
3International PhD School in Clinical and Experimental Medicine (CEM), University of Modena and Reggio Emilia, Via G. Campi 287, 41125 Modena, Italy
4Department of Biomedical, Metabolic Science and Neuroscience, University of Modena and Reggio Emilia, Via G. Campi 287, 41125 Modena, Italy
5Department of Medicine and Surgery, Unit of Neurosciences, University of Parma, Via Volturno 39/E, 43125 Parma, Italy
6Global Clinical Development, R and D Merck KGaA, Frankfurter Strasse 250, 64293 Darmstadt, Germany
7Centre for Neuroendocrinology and Department of Immunology, Faculty of Health Sciences, University of Pretoria, Lynnwood Road and Roper Street, P.O. Box 2034, Pretoria 0001, South Africa
8Unit of Endocrinology, Department of Medical Specialties, Azienda Ospedaliero-Universitaria, Via P. Giardini 1355, 41126 Modena, Italy
9Physiologie de la Reproduction et des Comportements (PRC), Institut National de la Recherche Agronomique (INRA), Centre National de la Recherche Scientifique (CNRS), Institut Français du Cheval et de l’Equitation (IFCE), Université de Tours, 37380 Nouzilly, France


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.


Gonadotropin-releasing hormone (GnRH)
luteinizing hormone (LH)
follicle-stimulating hormone (FSH)
assisted reproduction techniques (ART)
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International Journal of Molecular Sciences