Up-regulation of Cyclin B2 during GVBD in the GDF9-Ccnb1^−/− oocytes. (A) Western blot of Cyclin B2 in the GDF9-Ccnb1^−/− oocytes. Cyclin B2 displayed an apparent increase in the GDF9-Ccnb1^−/− oocytes during GVBD, but it showed weak expression after PBE. 150 oocytes were used in each lane. (B) Nuclear import of Cyclin B2 in mouse oocytes during GVBD. GV-stage oocytes were microinjected with Ccnb2-Venus and H2B-mCherry cRNAs, and after 2 h incubation in IBMX, the oocytes were released from IBMX to undergo GVBD. For both the GDF9-Ccnb1^−/− oocytes (n = 17) and control oocytes (n = 15), Cyclin B2–Venus fluorescence was concentrated in the nucleus at 10–20 min before GVBD. The time is shown (h:min). Bars, 20 µm. (C) Ratio of nuclear versus cytoplasmic Cyclin B2–Venus fluorescence in oocytes. The nuclear and cytoplasmic Cyclin B2–Venus fluorescence intensities were measured every 5 min from 30 min before GVBD to 15 min after GVBD in the control and GDF9-Ccnb1^−/− oocytes. Then, the ratio of nuclear versus cytoplasmic signal was calculated. The numbers of oocytes used (n) are shown. *, P < 0.05. (D) Western blot of Cyclin B2 in the Cyclin B2–knockdown oocytes. Following microinjection of Ccnb2 MO, Cyclin B2 was decreased markedly in oocytes. After microinjection, the oocytes were incubated in IBMX for 24 h. 150 oocytes were used in each lane. (E) Cyclin B2 interference in the GDF9-Ccnb1^−/− oocytes induced severe GV arrest. GVBD rate in the GDF9-Ccnb1^−/− oocytes after Cyclin B2 interference. After microinjection with Ccnb2 MOs, the microinjected GDF9-Ccnb1^−/− oocytes were released into M2 medium to undergo GVBD following 24 h incubation in IBMX. GVBD was severely suppressed after Cyclin B2 interference (23.35% ± 4.2%) compared with the noninjected GDF9-Ccnb1^−/− oocytes (87.18% ± 0.6%). GVBD rates were scored at 3 h incubation in M2 medium. Data are presented as mean ± SEM. ***, P < 0.0001.