ECE2014 Poster Presentations Female reproduction (54 abstracts)
The FSHR polymorphism p.N680S mediates different response kinetics to FSH in vitro
Livio Casarini 1, , Valeria Moriondo 1, , Marco Marino 1, , Francesca Adversi 3 , Francesco Capodanno 3 , Chiarina Grisolia 4 , Antonio La Marca 5 , Giovanni Battista La Sala 3, & Manuela Simoni 1,
1Department of Biomedic, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy; 2Center for the Genomic Research, University of Modena and Reggio Emilia, Modena, Italy; 3Department of Obstetrics and Gynecology, Arcispedale S.Maria Nuova, IRCCSS, Azienda Santa Maria Nuova, Reggio Emilia, Italy; 4Azienda Ospedaliero-Universitaria, Policlinico di Modena, Modena, Italy; 5Mother-Infant Department, Institute of Obstetrics and Gynecology, University of Modena and Reggio Emilia, Modena, Italy; 6Department of Mother-Infant and Adult Medical and Surgical Sciences, University of Modena and Reggio Emilia, Modena, Italy.
Introduction: FSH acts on its receptor (FSHR) resulting in signal transduction activation, gene expression and steroidogenesis. The FSHR common SNP p.N680S is a marker of gonadal response in vivo. However, in vitro dose–response experiments failed to demonstrate the molecular basis thereof so far. In this study, we systematically investigated whether p.N680S mediates different kinetics of FSH response in vitro.
Design: We evaluated the activation kinetics of cAMP, phERK1/2, phCREB by ELISA and western blotting in FSHR homozygous, primary, human granulosa lutein cells (hGLC-680N, -680S) stimulated by 50 nM r-FSH for up to 2 h (short-term stimulation). Following short-term stimulation the expression of target genes was evaluated by real-time PCR after 12 h, and progesterone production kinetics over 24 h. Specific inhibitors/agonists (U0126, PMA) were used in the presence and in the absence of FSH.
Results: Intracellular cAMP increased within 5–10 min in hGLC-680N, reaching the plateau in about 45 min. cAMP increase was delayed in hGLC-680S, reaching the plateau in 120 min, revealing different activation kinetics (Mann–Whitney U test; P<0.05; n=4). r-FSH-dependent cAMP stimulation kinetics resulted in different ERK1/2 and CREB phosphorylation, reaching maximal levels in 5–30 min in hGLC-680N, whereas, in hGLC-680S, these were weaker and steady over 2 h (Mann–Whitney U test; P<0.05; n=3). hGLC-680N stimulation resulted in higher expression levels of AREG and StAR (Mann–Whitney U test; P<0.05; n=4) and in subsequently different progesterone production kinetics, achieving overall higher levels in hGLC-680N vs -680S (Mann–Whitney U test; P<0.05; n=3). Interestingly, the different kinetics of progesterone production between hGLC-680N and -680S were interchanged by selective phospho-ERK1/2 blockade/activation through specific inhibitor/agonist, revealing a short-term cross-talk mediated by ERK1/2.
Conclusions: This study demonstrates for the first time in vitro, how FSHR p.N680S mediates different response to FSH, resulting in different kinetics of cAMP, phERK1/2 and phCREB activation, and progesterone production.
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Endocrine Abstracts
ISSN 1470-3947 (print) | ISSN 1479-6848 (online)
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