The Reproductive Medicine Center, Peking University Shenzhen Hospital, Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center, Shenzhen, China.
State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China.
J Cell Physiol. 2020 Jul;235(7-8):5541-5554. doi: 10.1002/jcp.29469. Epub 2020 Jan 26.
Cell division cycle protein, CDC6, is essential for the initiation of DNA replication. CDC6 was recently shown to inhibit the microtubule-organizing activity of the centrosome. Here, we show that CDC6 is localized to the spindle from pro-metaphase I (MI) to MII stages of oocytes, and it plays important roles at two critical steps of oocyte meiotic maturation. CDC6 depletion facilitated the G2/M transition (germinal vesicle breakdown [GVBD]) through regulation of Cdh1 and cyclin B1 expression and CDK1 (CDC2) phosphorylation in a GVBD-inhibiting culture system containing milrinone. Furthermore, GVBD was significantly decreased after knockdown of cyclin B1 in CDC6-depleted oocytes, indicating that the effect of CDC6 loss on GVBD stimulation was mediated, at least in part, by raising cyclin B1. Knockdown of CDC6 also caused abnormal localization of γ-tubulin, resulting in defective spindles, misaligned chromosomes, cyclin B1 accumulation, and spindle assembly checkpoint (SAC) activation, leading to significant pro-MI/MI arrest and PB1 extrusion failure. These phenotypes were also confirmed by time-lapse live cell imaging analysis. The results indicate that CDC6 is indispensable for maintaining G2 arrest of meiosis and functions in G2/M checkpoint regulation in mouse oocytes. Moreover, CDC6 is also a key player regulating meiotic spindle assembly and metaphase-to-anaphase transition in meiotic oocytes.
细胞分裂周期蛋白 CDC6 对于 DNA 复制的起始至关重要。最近的研究表明，CDC6 可抑制中心体的微管组织活性。在这里，我们发现 CDC6 从减数分裂前期 I（MI）到卵母细胞的 MII 期定位于纺锤体，并且在卵母细胞减数成熟的两个关键步骤中发挥重要作用。在含有米力农的 GVBD 抑制培养系统中，CDC6 耗竭通过调节 Cdh1 和 cyclin B1 的表达和 CDK1（CDC2）磷酸化，促进 G2/M 转换（GVBD）。此外，在 CDC6 耗竭的卵母细胞中敲低 cyclin B1 后，GVBD 明显减少，表明 CDC6 缺失对 GVBD 刺激的影响至少部分是通过提高 cyclin B1 介导的。CDC6 的敲低还导致 γ-微管蛋白的异常定位，导致纺锤体缺陷、染色体排列不齐、cyclin B1 积累和纺锤体装配检查点（SAC）激活，导致显著的 pro-MI/MI 阻滞和 PB1 挤出失败。这些表型也通过实时活细胞成像分析得到了证实。结果表明，CDC6 对于维持减数分裂的 G2 阻滞和在小鼠卵母细胞中 G2/M 检查点调节中发挥作用是必不可少的。此外，CDC6 也是调节减数分裂纺锤体组装和减数分裂中期到后期过渡的关键因素。
