Participation of cAMP-dependent protein kinase and MAP kinase pathways during Anabas testudineus oocyte maturation

Abstract

Possible involvement of cyclic nucleotide dependent protein kinase (PKA) and MAP kinase (MAPK) pathways during oocyte maturation in Anabas testudineus was investigated. Pre-incubation with phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (IBMX), inhibited 17α, 20β-DHP-induced GVBD dose dependently. PKA inhibitor, H89 could induce resumption of meiosis independent of 17α, 20β-DHP, in dose and duration dependent manner. The maximum response was obtained with the dose of 10μM of H89 and 95% of cells underwent GVBD within 18h. Moreover, stimulation with 17α, 20β-DHP inhibited endogenous PKA activity significantly within first hour and this effect was attenuated by PDE inhibitor IBMX at all time points. The pattern of PKA inhibition corresponded well with kinetics of histone H1 kinase activation and p34cdc2 phosphorylation. These results suggest physiological relevance of cAMP/PKA signaling in perch oocytes undergoing G2/M transition. MAPK was demonstrated as two distinct isoforms (ERK1 and ERK2) which resolved in the range of 42–44kDa in immunoblot. Though total protein content did not show significant variation, H89 stimulation was able to stimulate phosphorylation of ERK1/2 from 5h onwards and the strongest response was observed between 10 and 18h. MEK inhibitor, U0126 completely blocked PKA inhibition induced MAPK activation and GVBD. In addition, inhibition of endogenous PKA by a more selective peptide inhibitor [PKI-(6–22)-amide] was sufficient to resume GVBD and MAPK activation in intact perch oocytes. Also, significant ERK1/2 phosphorylation could be stimulated in cell-free extracts of perch oocytes supplemented with PKI-(6–22)-amide. The results suggest an interaction between cAMP/PKA and MAPK pathways in mediating meiosis resumption in perch oocyte.