The level of MPF was highest in oocytes treated with DEM for 1 h, when it was significantly higher than in untreated oocytes. The level of MPF gradually decreased in oocytes treated with DEM for longer amounts of time. Similar results have been reported in mouse embryos, rat oocytes, and bovine oocytes. DEM is a microtubule-disrupting agent that depolymerizes microtubules and limits microtubule formation. The destruction of spindles by DEM inhibits degradation of cyclin B1, which, in turn, increases MPF activity. Changes in the level of cyclin B1 correlate with changes in MPF activity. In the current study, MII porcine oocytes were bisected to examine changes in the distribution of MPF following DEM treatment. MPF was unevenly distributed in the cytoplasm of mature denuded porcine oocytes, and the level of MPF was high in the karyoplast. These results are consistent with a previous study of mouse oocytes, and indicate that MPF is predominantly associated with the spindle. By contrast, when oocytes were treated with 0.4 mg/ml DEM for 1 h, MPF activity did not markedly differ between the karyoplast and cytoplast, indicating that MPF was homogenously distributed. To further observe the distribution of MPF, cyclin B1 was examined in mature oocytes using immunofluorescence microscopy. In untreated oocytes, cyclin B1 was accumulated around the meiotic spindle, and was lowly detected in the cytoplasm. In oocytes treated with DEM for 1 h, maternal chromosomes were condensed, the level of cyclin B1 was reduced in the nuclear region, but not in the polar body, and cyclin B1 was homogenously distributed in the cytoplasm. In mouse oocytes, a high level of cyclin B is maintained for several hours following spindle disruption by nocodazole. Activation of MPF depends on the association of p34/cdc2 with cyclin B. Basal levels of p34/cdc2 do not substantially change during in vitro maturation of porcine oocytes. However, the level of cyclin B in oocytes tends to increase following in vitro maturation. Clute et al. reported that the localization of cyclin B1 is extremely dynamic during mitosis. The protein is concentrated at centrosomes and spindle microtubules in organisms ranging from yeast to humans, and is rapidly degraded during late metaphase. Therefore, we examined the localization of cyclin B1 in porcine oocytes by performing immunofluorescence microscopy. DEM treatment disrupted spindle microtubules, induced chromosome condensation, and decreased the level of cyclin B1 in the nuclear region. Overall, in DEM-treated oocytes, the level of cyclin B1 was increased and the protein was uniformly distributed in the cytoplasm. Consequently, MPF activity remained high in oocytes following DEM-assisted enucleation. The efficiency of DEM-assisted GSK2118436 enucleation was significantly higher than that of mechanical enucleation. DEM-assisted enucleation is an attractive method because it induces formation of a membrane protrusion containing a mass of condensed chromosomes. This facilitates removal of maternal chromosomes and produces a competent cytoplast for SCNT.