Participation of PI3-kinase/Akt signalling in insulin stimulation of p34cdc2 activation in zebrafish oocyte: Phosphodiesterase 3 as a potential downstream target

Abstract

Exposure of fully grown oocytes to growth factors (insulin/IGFs) initiates various signalling cascades that culminate to final stages of oocyte maturation. Regulation of signalling pathways during growth factor-induced meiosis resumption in fish is not well characterized. Here we studied the participation of PI3K/Akt signalling pathway during recombinant human insulin (rh-insulin)-induced meiotic maturation in zebrafish (Danio rerio) oocytes. Priming of defolliculated oocytes in vitro with rh-insulin promotes germinal vesicle breakdown (GVBD) in a dose- and time-dependent manner, an effect sensitive to translation but not transcription inhibition. More than 80% of the oocytes underwent GVBD due to 0.8IU/ml rh-insulin within 10h of incubation and the kinetics of p34cdc2 kinase activation corresponded well with GVBD data. PI3K inhibitors, wortmannin and LY294002 blocked insulin, but not 17α, 20β-DHP-induced GVBD. Immunoblot analyses of oocyte extract revealed that phospho-PI3K (p85α) was up regulated within 30–60min of insulin stimulation followed by phospho-Akt (Ser473) at 60–120min. Though PI3K/Akt phosphorylation was largely unaffected, pre-incubation with phosphodiesterase (PDE) inhibitors, IBMX and cilostamide, but not rolipram completely blocked rh-insulin-induced p34cdc2 activation and GVBD. These results suggest that PDE3 may be one potential downstream target to PI3K/Akt signalling necessary for rh-insulin-induced GVBD in zebrafish.