Cyclin B3 is required for the metaphase-to-anaphase I transition in oocytes. (A) Live-cell imaging of meiotic maturation. β-Tubulin-GFP and H2B-RFP mRNA, used to visualize spindle and chromosomes, were injected into GV-stage oocytes, which were then induced to enter meiosis I. Selected time frames are shown, with overlay of DIC, GFP, and RFP channels of collapsed z-sections (11 section, 3-µm steps) from a representative video. Time after GVBD is indicated as hours:minutes. n: number of oocytes analyzed. Percentage of oocytes of the observed phenotype from two independent experiments is indicated. White asterisks indicate PBs. Scale bar, 20 µm. Related to Videos 1 and 2. (B) Chromosome spreads 6 h (corresponding to metaphase I) and 16 h (corresponding to metaphase II in controls) after GVBD. Kinetochores were stained with CREST (green) and chromosomes with Hoechst (blue). Insets show typical chromosome figures observed; for better visualization, chromosomes are shown in grayscale. Schematics of metaphase I bivalents or metaphase II univalent chromosomes are shown to aid interpretation. Scale bar, 5 µm. n: number of oocytes analyzed in three independent experiments. (C) Whole-mount immunofluorescence staining of cold-treated spindles. Microtubules were stained with anti-tubulin antibody (green), kinetochores with CREST (red), and chromosomes with Hoechst (blue). Spindle poles typical of metaphase I and metaphase II are indicated with arrows in controls. Scale bar, 5 µm. n: number of oocytes analyzed in three independent experiments.