Abstract:
Commercial gonadotropin-releasing hormone (GnRH) antagonists di er 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 L T2 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, -catenin activation and mouse luteinizing-hormone -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 L T2 cells. Cetrorelix inhibited the 3 EC50 GnRH-activated calcium
signaling at concentrations of 1 nM–1 M, demonstrating higher potency than Ganirelix and Teverelix, whose inhibitory doses fell within the 100 nM–1 M 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 L T2 and transfected HEK293 cells treated with 100 nM
GnRH. Although immunostainings suggested that Teverelix could be less e ective than Cetrorelix
and Ganirelix in inhibiting 1 M GnRH-induced -catenin activation, Lhb gene expression increase
occurring upon L T2 cell treatment by 1 M GnRH was similarly inhibited by all antagonists.
To conclude, this study has demonstrated Cetrorelix-, Ganirelix- and Teverelix-specific biased e ects
at the intracellular level, not a ecting the e cacy of antagonists in inhibiting Lhb gene transcription.
