The success of nuclear transplantation with mammalian oocytes depends critically on the potential of oocytes activation, which mainly caused to prevent the re-accumulation of maturation promoting factor (MPF). This study was conducted to compare the effect of combined treatment of lonomycin with a Hl-histone kinase inhibitor (dimethylaminopurine, DMAP) or cdc2 kinase inhibitor (sodium pyrophosphate, SPP) on activation of bovine oocytes. In vitro matured bovine oocytes with the first polar body (PB) and dense cytoplasm were assigned to 3 experimental groups. For activation treatment, oocytcs were exposed to 5 M lonomycin for 5 min (Group 1), and followed by 1.9 mM dimethylaminopurine (DMAP) for 3 h (Group 2) or followed by 2 mM sodium pyrophosphate (SPP) for 3 h (Group 3). The activation effects in the three treatments and the control group (untreated) were judged by the extrusion of the second PB and formation of a pronucleus (PN). Differences among groups were analysed using one-way ANOVA after arc-sine transformation of proportional data. All three treatments led to high activation rates (90% to 95%), with significant difference from the control. However, the extrusion of the second PB and the rate of PN formation differed remarkably among treatments. In Group I and 3, about 95% of the oocytes had extruded the second polar body, but one PN had formed in a higher proportion of oocytes in Group 3 than in Group 1 (90% vs. 5%). In experiment 2, the rates of cleavage and development into blastocysts in Group 1 were significantly lower than those of Group 2 and 3 (8.7% and 0% vs. 50.5% and 11.6%, and 44.6% and 7.2%, respectively, P<0.05). In experiment 3, ~80% of parthenotes in Group 1 were developed with haploid chromosomal sets. However, when ionomycin was followed immediately by DMAP (Group 2). only 20% of parthenotes were haploid. In Group 3, combined treatment with ionomycin and SPP, the appearance of abnormal chromosomal tracts was significantly (P〈0.05) reduced and the proportion of haploid parthenotes was increased to 85% (17/20) than in Group 2. These results demonstrate that SPP acted as a cdc2 kinase inhibitor and formed the haploidy in oocyte activation. Thus, the present study suggests that cdc2 kinase inhibitor, such as sodium pyrophosphate, may have an effective role in oocyte activation for the production of cloned embryos/animals by nuclear transplantation.

In oocytes from different species, MPF, a complex of Cdk1 and cyclin B, is the master regulator of cell cycle. The activity of MPF is regulated by the phosphorylations mediated by Wee1B kinase and Cdc25B phosphatase. Although a regulation of MPF activity by these inhibitory phosphorylations are well established, a dogma in the cell cycle is that MPF activity is regulated by the dynamics of cyclin B during the metaphase II (MII) arrest (also known as CSF arrest). However, growing evidences suggest that Wee1B-mediated Cdk1 phosphorylation is also critical to trigger the progression of cell cycle during the onset of anaphase. Therefore, in the present study, we investigated the role of Cdc25B phosphatase during MII arrest. Cdc25B is present in MII arrested oocytes as a hyperphosphorylated form and disruption of its function either by antibody or siRNA injection induces the progression of cell cycle to interphase. Moreover, the hyperphosphorylated form, which has been known as an active form of Cdc25B, is dephosphorylated during the anaphase onset. Interestingly, this dephosphrylation occurred ahead of cyclin B degradation. Conversely, overexpression of Cdc25B prevents metaphase to anaphase transition induced by calcium stimulation. Therefore, our findings provide novel paradigm in cell cycle that MPF activity during metaphase arrest is regulated by the balance between Cdk1 inhibitory kinases, Wee1, and the counteracting phosphatases, Cdc25. When cells exit from metaphase, Cdc25 is inactivated and Wee1 is reactivated and thereby Cdk1 kinase activity is rapidly and transiently decreased. This initial decrease of Cdk1 activity is further promoted by the proteolytic degradation of cyclin B, which ensures irreversible progression of cell cycle to interphase. Thus, the concerted effort of phosphorylation/dephosphorylation of Cdk1 and synthesis/degradation of cyclin B play roles in fine-tuning the activity of Cdk1 during metaphase to anaphase transition.