Possible role for phosphatidylinositol 3-kinase in regulating meiotic maturation of bovine oocytes in vitro

Abstract

In this study 2 phosphatidylinositol 3-kinase (PI 3-kinase)-specific inhibitors, wortmannin and 2-[4-Morpholinyl]-8-phenyl-4H-1-benzopyran-4-one (LY294002), were used to investigate whether PI 3-kinase is involved in the signal transduction that leads to bovine oocyte maturation. Bovine follicular oocytes were cultured in vitro for 24 h in a basic medium consisting of tissue culture medium-199 supplemented with LH, FSH, fetal cow serum, Na-pyruvate and gentamicin. The oocytes were then examined for the stage of meiotic progression and degree of cumulus expansion. In Experiment 1, in cumulus-oocyte complexes (COCs), wortmannin, at any level tested (10 −8 M, 10 −7M or 10 −6M), had no effect on resumption of meiosis as judged by germinal vesicle breakdown and progression to prometaphase I or metaphase I. However, wortmannin significantly (P< 0.01) decreased the proportion of oocytes developing to metaphase II in a dose-dependent manner. In Experiment 2, when denuded oocytes were cultured with wortmannin at 0, 10 −7M and 10 −6 M concentrations, the same pattern of response for COCs was observed, with no effect on meiotic resumption and a significant (P< 0.01) decrease in the proportion of oocytes reaching metaphase II. In Experiment 3, half of the recovered COCs were denuded and both denuded and intact COCs were cultured in the presence of 0, 2.5 × 10 −5 M, 5.0 × 10 −5 M and 7.5 × 10 −5 M LY 294002 before being examined for meiotic progression. Whereas LY294002, at any examined level, had no effect on the percentage of oocytes developing to metaphase I, it significantly (P<0.01) decreased the proportion of metaphase II oocytes when used at 5.0 × 10 −5 or 7.5 × 10 −5 M for both intact COCs and denuded oocytes. In Experiment 4, no significant difference in the degree of cumulus expansion was scored after the COCs were cultured in the presence of wortmannin or LY294002 or in the absence of either treatment. These results provide indirect evidence for a role of PI 3-kinase in the bovine oocyte itself in regulating meiotic progression beyond metaphase I.