Independent activation of MAP kinase and MPF during the initiation of meiotic maturation in pig oocytes

Abstract

Mitogen-activated protein (MAP) kinase is universally activated during oocyte maturation in all vertebrates studied to date. Its role in the resumption of meiosis and in the activation of maturation-promoting factor (MPF) remains unclear, especially in domestic species such as the pig. This study aimed to clarify the temporal and causal relationships between MAP kinase and MPF during meiotic maturation, particularly during the resumption of meiosis. Pig oocytes were matured synchronously in culture by treatment with cycloheximide. Kinase activities were analysed using a sensitive in vitro double-kinase assay and the specific MAP kinase pathway inhibitor U0126. MAP kinase and MPF were activated simultaneously at the time of germinal vesicle breakdown (GVBD; 6 h after removal of cycloheximide); they reached significant activity at 7 h (P < 0.05). The activities increased in parallel during GVBD (6-10 h) and peaked when the oocytes entered metaphase I (MI; 10 h). Whereas MAP kinase remained stable at peak activity thereafter, MPF activity significantly declined during the MI-MII transition (16-20 h) but increased to a second peak at MII (22 h). MAP kinase activity in denuded and cumulus-cell enclosed oocytes was completely inhibited by 20 and 80 mmicro mol U0126 l(-1), respectively. Oocytes without detectable MAP kinase activity underwent normal GVBD in terms of nuclear morphology and timing, although later meiotic stages were abnormal. The kinetics of MPF activity during GVBD were unaffected by U0126. This study has demonstrated that MAP kinase is activated simultaneously with MPF at GVBD, but that its activation is not essential for the activation of MPF nor for the resumption of the first meiosis in pig oocytes.