Activation of bovine oocytes by protein synthesis inhibitors: new findings on the role of MPF/MAPKs†.

Abstract

The present study evaluated the mechanism by which protein synthesis inhibitors activate bovine oocytes. The aim was to analyze the dynamics of MPF and MAPKs. MII oocytes were activated with ionomycin (Io), ionomycin+anisomycin (ANY) and ionomycin+cycloheximide (CHX) and by in vitro fertilization (IVF). The expression of cyclin B1, p-CDK1, p-ERK1/2, p-JNK, and p-P38 were evaluated by immunodetection and the kinase activity of ERK1/2 was measured by enzyme assay. Evaluations at 1, 4, and 15hours postactivation (hpa) showed that the expression of cyclin B1 was not modified by the treatments. ANY inactivated MPF by p-CDK1Thr14-Tyr15 at 4hpa (P<0.05), CHX increased pre-MPF (p-CDK1Thr161 and p-CDK1Thr14-Tyr15) at 1hpa and IVF increased p-CDK1Thr14-Tyr15 at 17hours postfertilization (hpf) (P<0.05). ANY and CHX reduced the levels of p-ERK1/2 at 4hpa (P<0.05) and its activity at 4 and 1hpa, respectively (P<0.05). Meanwhile, IVF increased p-ERK1/2 at 6hpf (P<0.05); however, its kinase activity decreased at 6hpf (P<0.05). p-JNK in ANY, CHX, and IVF oocytes decreased at 4hpa (P<0.05). p-P38 was only observed at 1hpa, with no differences between treatments. In conclusion, activation of bovine oocytes by ANY, CHX, and IVF inactivates MPF by CDK1-dependent specific phosphorylation without cyclin B1 degradation. ANY or CHX promoted this inactivation, which seemed to be more delayed in the physiological activation (IVF). Both inhibitors modulated MPF activity via an ERK1/2-independent pathway, whereas IVF activated the bovine oocytes via an ERK1/2-dependent pathway. Finally, ANY does not activate the JNK and P38 kinase pathways.