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Endocrine Abstracts (2017) 49 EP1112 | DOI: 10.1530/endoabs.49.EP1112

ECE2017 Eposter Presentations: Reproductive Endocrinology Female Reproduction (62 abstracts)

Biological activity mediated by sfingosine-1-phospate receptors in human primary granulosa cells and immortalized granulosa cell line in vitro

Livio Casarini 1, , Giulia Fornari 1 , Manuela Simoni 1, & Francesco Potì 4

1Unit of Endocrinology, Department Biomedical, 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; 3Azienda Ospedaliero-Universitaria di Modena, Modena, Italy; 4Department of Neurosciences, University of Parma, Parma, Italy.

Introduction: Sphingosine-1-phosphate (S1P) is a sphingolipid mediating proliferative signals in human ovarian follicles, acting through five receptors (S1PR1-5). We characterized the effects of S1PR1 and S1PR3 in vitro, as those mostly expressed in human primary granulosa cells (hGLC) and immortalized, tumor-like granulosa cell line (hGL5).

Methods: The S1P-mediated signaling, as well as the activity of S1PR1- and S1PR3-specific agonists (SEW281 and CYM5541, respectively), was evaluated in hGLC and hGL5 cells. Dimethyl-sulfoxide-, cholera toxin- and phorbol-12-myristate-13-acetate (PMA)-treatments served as controls. The kinetics of pERK1/2, pAKT and pCREB activation were evaluated over 2 h by Western blotting, while total cAMP production was measured by ELISA. The downstream cell viability was measured by MTT assay over 72 h, ±FSH. Differences were significant for P<0.05 (two-way Anova; n=2–12).

Results: Dose-response experiments revealed that 0.1 μM S1P, 1.0 nM SEW2871 and 1.0 nM CYM5541 are the most effective concentrations, in terms of pERK1/2 and pAKT activation. In hGLC, pERK1/2 activation occurs within 5–60 min by S1P, and, in a lesser extent, within 15–30 min by SEW2871 and CYM5541. In hGL5 cells, SEW2871- and CYM5541-dependent pERK1/2 activation is prolonged until 2 h. We describe for the first time S1P- and, to a lesser extent, SEW2871/CYM5541-mediated pCREB activation in both cell models, occurring in spite of no pAKT and cAMP recruitment. All the agonists increased cell viability in hGL5 cells, an effect reverted in the presence of 50 nM FSH. Only S1P mediated anti-apoptotic effects in hGLC, while the treatment by SEW2871 and CYM5541 impacts negatively cell viability.

Discussion: We found S1PRs-mediated cAMP-independent steroidogenic potential and different signaling kinetics in hGLC and hGL5 cells, revealing opposite effects on the downstream cell signaling, depending on the cell model.

Conclusion: Our study suggests that S1PRs play a role in ovarian follicle growth and oocyte maturation.

Volume 49

19th European Congress of Endocrinology

Lisbon, Portugal
20 May 2017 - 23 May 2017

European Society of Endocrinology 

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