The function of cyclin B1 (CB1) and cyclin B2 (CB2) during porcine oocyte maturation was investigated by injecting oocytes with their antisense RNAs (asRNAs). At first, protein levels of both cyclin Bs were examined by immunoblotting, revealing that immature oocytes had only CB2, at a level comparable to 1/20 to 1/40 of that detected in first metaphase oocytes. Both cyclin B syntheses were started around germinal vesicle breakdown (GVBD); CB1 and CB2 peaked at the second metaphase and first metaphase, respectively. We obtained a porcine CB2 cDNA fragment, which was 88% homologous with human CB2, by reverse-transcriptase polymerase chain reaction (RT-PCR) using total RNAs of immature porcine oocytes and a primer set of human CB2. Specific asRNAs of CB1 and CB2 were prepared in vitro. Then one, the other, or both were injected into the cytoplasm of immature oocytes. CB1 asRNA inhibited CB1 synthesis specifically; the injected oocytes underwent first meiosis normally but could not arrest at the second meiotic metaphase. CB2 asRNA inhibited CB2 synthesis specifically, but had almost no effect on the maturation of injected oocytes. When both CB1 and CB2 asRNAs were injected, synthesis of both cyclin Bs was inhibited, and GVBD was significantly suppressed but occurred slowly. These results suggest that CB1 is the principal molecule for regulation in mammalian oocyte maturation, whereas CB2 has only an accessory role. They also show that in porcine oocytes, cyclin B synthesis is not necessary for GVBD induction itself, but synthesis of at least one cyclin B, CB1 or CB2, is necessary for GVBD induction in a normal time course.

Abstract. The inhibition of mitogen activated protein kinase (MAPK) activation during porcine oocyte maturation leads to decreased maturation promoting factor (MPF) activity and to the induction of parthenogenetic activation. In the present study, in order to analyze the mechanism underlying the suppression of MPF activity in MAPK-inhibited porcine oocytes, we injected mRNA of SASA-MEK, a dominant negative MAPK kinase, or antisense RNA of c-mos, a MAPK kinase kinase, into immature porcine oocyte cytoplasm. The injection of SASA-MEK mRNA or c-mos antisense RNA inhibited the MAPK activity partially or completely, respectively, decreased the MPF activity slightly or significantly, respectively, and induced parthenogenetic activation in 17.1% or 96.6% of mature oocytes, respectively, although no parthenogenetic activation was observed in the control oocytes. Immunoblotting experiments revealed that cyclin B accumulation in these MAPK-suppressed porcine oocytes was increased significantly after 50 h of culture and that a considerable amount of MPF was converted into inactive pre-MPF by hyperphosphorylation. These results indicate that the inhibition of MAPK activity in porcine oocytes did not promote cyclin B degradation but rather suppressed it; also the decrease in MPF activity in MAPK-suppressed porcine oocytes correlated with the conversion of active MPF into inactive pre-MPF.

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