203 PI3-K PATHWAY IS ONE OF THE KEY REGULATORS OF STEROIDOGENESIS OF CUMULUS CELLS OF BOVINE CUMULUS–OOCYTE COMPLEXES MATURATED IN DEFINED MEDIUM IN THE PRESENCE OF FSH

Abstract

The aim of the present study was to access the function of PI3-K pathway tested by the use of its inhibitor, LY294002, in maturation medium of bovine cumulus-oocyte complexes (COC) in steroid concentration in the medium, key enzymes of steroidogenic pathway and gonadotrophin receptors of cumulus cells. This study was performed in defined medium without serum and albumin (MIV B) in absence or presence of 10 ng mL–1 of FSH. Androstenedione 10–7 M was used as a precursor of steroidogenesis. Bovine COC (n = 35–40/well) collected from ovaries obtained at abattoirs of Brasilia (DF, Brazil) were cultivated in 400 μL of medium MIV B; MIV B + 100 μmol mL–1 of LY294002; MIV B added to 10 ng mL–1 of FSH; or MIV B added to 10 ng mL–1 of FSH + 100 μmol mL–1 of LY294002 for 22 to 24 h. After culture, COC were mechanically denuded and cumulus cells from 20 COC were isolated by centrifugation to determine the gene expression of LHR, FSHR, CYP11A1, CYP19A1, and HSD17B1 by PCR real time. Cumulus cells of immature COC were also collected and analysed as the calibrator group. Student–Newman–Keuls was performed as statistical test. The culture medium was collected after culture to determine progesterone and 17β-oestradiol concentration by quimioluminescence method and to calculate E2/P4 ratio. Two-way ANOVA, followed by Bonferroni test, and t-test were performed to determine the statistical significance. The MIVB enhanced LHR, FSHR, CYP11A1, CYP19A1, and HSD17B1 and LY294002 inhibited the expression of all genes (P < 0.05). MIVB + FSH decreased the expression of all genes (P < 0.05) except CYP11A1. LY294002 in the presence of 10 ng mL–1 of FSH did not affect the gene expression of FSHR, CYP11A1, and HSD17B1; however, it increased expression of LHR and CYP19A1. Oestradiol and progesterone production was increased by supplementation of FSH in MIVB (P < 0.05), but the E2/P4 ratio did not differ between treatments. LY294002 decreased oestradiol and E2/P4 ratio in absence or presence of FSH (P < 0.05), but did not alter progesterone concentration in MIVB+FSH. The inhibitor of PI3-K decreased the expression of steroidogenic proteins and steroid production in absence of FSH. The supplementation of FSH increased steroid production and decreased gene expression of steroidogenic enzymes, except CYP11A1. The inhibition of PI3-K in presence of FSH increases LHR and CYP19A1 expression. This fact suggests a strong role of the PI3-K pathway in the regulation of LHR and CYP19A1 expression.The authors thank FAP-DF (193.000.577/2009), CNPq, CAPES, Sabin, and Ponte Alta abattoir, Brasilia.