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. 2020 May 27;11:2652. doi: 10.1038/s41467-020-16488-y

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — a MII cytoplasm supports Ndc80-independent spindle bipolarization. An Ndc80^f/f Zp3-Cre oocyte in metaphase II (MII) and an Ndc80^f/f Zp3-Cre oocyte at the GV stage (MI) were fused. EGFP-Map4 (microtubules, green) and H2B-mCherry (chromosomes, magenta) are shown. Bipolar spindle formation around MI chromosomes (magnified) was observed in 10 of 10 oocytes from three independent experiments. Time after NEBD of the MI nucleus (h). See also Supplementary Movie 8. b Upregulation of Prc1 in MII and artificial increase in Prc1 levels in MI. Western blotting of oocytes at MI (0 and 6 h after NEBD), and at MII (16 h). The Prc1 level at 0 h was artificially increased (+Prc1). Fifty oocytes were used in each sample. A full scan image is provided in the Source Data file. c Live imaging of oocytes expressing increased Prc1. EGFP-Map4 (microtubules, green) and H2B-mCherry (chromosomes, magenta) were monitored. Spindles were reconstructed in 3D. Time after NEBD. Three independent experiments were performed. See also Supplementary Movie 9. d Increased Prc1 accelerates spindle bipolarization. Spindles reconstructed in 3D were categorized (see Methods). e Acceleration of bipolar spindle establishment. Time at which a stable bipolar spindle was established are plotted (n = 9, 9 oocytes). The mean +/− SD are shown. ****p = 1.3E−06. f MII-like spindle shapes. Oocytes were immunostained for microtubules (green), kinetochores (ACA, magenta) and DNA (Hoechst33342, blue) at metaphase I (6 h after NEBD) and metaphase II (16 h after NEBD). Aspect ratios (length/width) were plotted (n = 19, 8, 11 oocytes). Boxes show the 25th to 75th percentiles and whiskers show the 1st to 99th percentiles. ****p = 2.54E−05. n.s., not significant. g Increased Prc1 increases aneuploidy in eggs. Oocytes at metaphase II were immunostained for kinetochores (ACA, magenta) and counterstained for DNA (Hoechst33342, blue). Arrowheads indicate a misaligned chromatid in an aneuploid egg. The number of kinetochores were counted (n = 33, 40 oocytes from three independent experiments). Mean +/− SD are presented. ***p = 0.0001. Statistical values were obtained from two-tailed unpaired Student’s test. Scale bars, 10 μm.