Oocytes lacking both Cyclin B1 and Cyclin B2 were permanently arrested at the GV stage. (A) Morphological observation and immunofluorescent staining for the ovulated oocytes in the GDF9-Ccnb1^−/−;Ccnb2^−/− female mice. Oocytes were collected from the oviducts 16 h after HCG injection. Control (n = 52), GDF9-Ccnb1^−/−;Ccnb2^-/+ (n = 28), and GDF9-Ccnb1^−/−;Ccnb2^−/− (n = 44) were used. Bars: 40 µm (A, brightfield [BF]); 20 µm (A, immunofluorescence). (B) GVBD was blocked completely in the GDF9-Ccnb1^−/−;Ccnb2^−/− mouse oocytes after culture in vitro. The numbers of oocytes used (n) are shown. Data are presented as mean ± SEM. *, P < 0.05. (C) Rescue of GVBD by Cyclin B2 in the GDF9-Ccnb1^−/−;Ccnb2^−/− oocytes. Microinjection of Ccnb2 mRNA into the GDF9-Ccnb1^−/−;Ccnb2^−/− oocytes overcame the GV arrest, and most oocytes underwent GVBD in the presence of IBMX. Bar, 20 µm. (D) Restoration of MII arrest by exogenous Cyclin B2 in the GDF9-Ccnb1^−/− oocytes. Ccnb2 mRNA was introduced into the GV-intact GDF9-Ccnb1^−/− oocytes, which were released into IBMX-free M2 medium after 2 h incubation in the M2 containing IBMX. At 16 h after GVBD, α-tubulin and DNA staining were performed, and the oocytes also showed three phenotypes similar to the results by Ccnb1 introduction (Fig. S4 H). Bar, 20 µm. The numbers of oocytes used (n) are shown in C and D.