Duan Xing, Liu Jun, Dai Xiao-Xin, Liu Hong-Lin, Cui Xiang-Shun, Kim Nam-Hyung, Wang Zhen-Bo, Wang Qiang, Sun Shao-Chen
College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, China.
Biol Reprod. 2014 Feb 20;90(2):37. doi: 10.1095/biolreprod.113.113522. Print 2014 Feb.
During oocyte meiosis, a spindle forms in the central cytoplasm and migrates to the cortex. Subsequently, the oocyte extrudes a small body and forms a highly polarized egg; this process is regulated primarily by actin. ROCK is a Rho-GTPase effector that is involved in various cellular functions, such as stress fiber formation, cell migration, tumor cell invasion, and cell motility. In this study, we investigated possible roles for ROCK in mouse oocyte meiosis. ROCK was localized around spindles after germinal vesicle breakdown and was colocalized with cytoplasmic actin and mitochondria. Disrupting ROCK activity by RNAi or an inhibitor resulted in cell cycle progression and polar body extrusion failure. Time-lapse microscopy showed that this may have been due to spindle migration and cytokinesis defects, as chromosomes segregated but failed to extrude a polar body and then realigned. Actin expression at oocyte membranes and in cytoplasm was significantly decreased after these treatments. Actin caps were also disrupted, which was confirmed by a failure to form cortical granule-free domains. The mitochondrial distribution was also disrupted, which indicated that mitochondria were involved in the ROCK-mediated actin assembly. In addition, the phosphorylation levels of Cofilin, a downstream molecule of ROCK, decreased after disrupting ROCK activity. Thus, our results indicated that a ROCK-Cofilin-actin pathway regulated meiotic spindle migration and cytokinesis during mouse oocyte maturation.
在卵母细胞减数分裂过程中，纺锤体在中央细胞质中形成并迁移至皮质。随后，卵母细胞排出一个小体并形成高度极化的卵子；这个过程主要由肌动蛋白调节。ROCK是一种Rho-GTPase效应器，参与多种细胞功能，如应力纤维形成、细胞迁移、肿瘤细胞侵袭和细胞运动。在本研究中，我们研究了ROCK在小鼠卵母细胞减数分裂中的可能作用。在生发泡破裂后，ROCK定位于纺锤体周围，并与细胞质肌动蛋白和线粒体共定位。通过RNA干扰或抑制剂破坏ROCK活性会导致细胞周期进程和极体排出失败。延时显微镜观察表明，这可能是由于纺锤体迁移和胞质分裂缺陷，因为染色体分离但未能排出极体，然后重新排列。这些处理后，卵母细胞膜和细胞质中的肌动蛋白表达显著降低。肌动蛋白帽也被破坏，这通过未能形成无皮质颗粒区域得到证实。线粒体分布也被破坏，这表明线粒体参与了ROCK介导的肌动蛋白组装。此外，破坏ROCK活性后，ROCK的下游分子Cofilin的磷酸化水平降低。因此，我们的结果表明，ROCK-Cofilin-肌动蛋白途径在小鼠卵母细胞成熟过程中调节减数分裂纺锤体迁移和胞质分裂。
