Extent of bivalent breakage correlates with aMTOC spreading and MT density. (A) Quantitative analysis of aMTOC number per oocyte in the two Plk4 OE lines (line A, blue squares, and line B, green triangles) in prophase I; *, P = 0.0365 (two-tailed Mann–Whitney test). The mean number of aMTOCs per prophase I oocyte is indicated below the graph for each line. (B) Extent of bivalent breakage increases significantly as meiosis I progresses in oocytes from line A. Percentage of Plk4 OE oocytes from line A with one broken bivalent as a function of the stage of meiosis I (NEBD + 4h00 and NEBD + 6h00); **, P = 0.0047. (C) Effect of 50 nM nocodazole treatment observed at NEBD + 6h00 in living Plk4 OE oocytes from line A, expressing EB3-GFP (upper panels) and in fixed samples (lower panels) labeled for DNA (white) and CREST (pink). The black dotted circles in the upper panels highlight the oocyte contour. Scale bar is 20 µm. (D) Quantification of the relative MT density measured at NEBD around chromosomes in Plk4 OE from line A oocytes injected with EB3-GFP in control conditions or after treatment with 50 or 100 nM nocodazole (noco). The EB3-GFP signal was measured in a Ø20-µm region around the chromosomes; total signal intensity was measured in a Ø70-µm region covering the whole oocyte. Normalized signal intensity is the ratio of local/total intensity measured for individual oocytes. Tukey’s multiple comparison test gives *, P < 0.05 for controls vs. noco 50 nM; **, P < 0.01 for controls vs. noco 100 nM; and *, P < 0.05 for noco 50 nM vs. noco 100 nM. (E) Decreasing MT density with nocodazole reduces the extent of DNA breakage. Percentage of Plk4 OE oocytes from line A presenting one broken bivalent at NEBD + 6h00 as a function of nocodazole concentration (0, 50, and 100 nM, respectively, darker blue histograms). P = 0.1725 for 0 vs. 50 nM, and **, P = 0.0068 for 0 vs. 100 nM. For B and E, the statistical tests used are Fisher’s exact tests. For A, B, and E, n is the number of oocytes. Error bars correspond to SD.