SAC activation is not the cause of metaphase I arrest in Ccnb3^−/− oocytes. (A) Left: Chromosome spreads were prepared 3 h (early prometaphase I) and 6 h (metaphase I) after GVBD, then stained with Hoechst (blue), CREST (green), and anti-MAD2 (red). Scale bar, 5 µm. Right: Quantification of MAD2 signal intensity relative to CREST; each point is the mean relative intensity averaged across centromeres in an oocyte. n: number of oocytes from three independent experiments. P values are from t tests. Number and means of oocytes stained 3 h after GVBD: 23 Ccnb3^+/− (mean: 0.794) and 19 Ccnb3^−/− (mean: 0.77); 6 h after GVBD: 26 Ccnb3^+/− (mean: 0.302) and 20 Ccnb3^−/− (mean: 0.325). (B) Oocytes were treated with reversine to override a potential SAC arrest. Control oocytes were treated with nocodazole from 6 h after GVBD onward, whereas Ccnb3^−/− oocytes were allowed to arrest without nocodazole. 6 h 40 min after GVBD, reversine was added, PB extrusion was scored visually (graph on the right, n: number of oocytes scored per genotype. 0% of oocytes extruded PBs, except oocytes treated with nocodazole and reversine, which extruded PBs in 85.29% of oocytes analyzed), and all oocytes were spread 20 h after GVBD. Kinetochores were stained with CREST (green) and chromosomes with Hoechst (blue). Insets show typical chromosome figures observed. Scale bar, 5 µm. n: the number of spreads from three independent experiments that allowed to unambiguously distinguish bivalents from univalents; percentage of oocytes with same phenotype is indicated.