Accurate chromosome segregation is critical to ensure genomic integrity during cell division. This process is facilitated by the kinetochore, a multiprotein structure that is assembled on centromeric regions of chromosomes. The kinetochore establishes a mechanical link between the chromosomes and spindle microtubules and modulates cell cycle progression by regulating spindle assembly checkpoint (SAC). Defects in this process result in an aneuploidy, leading to miscarriages, infertility and various genetic disorder such as Down’s syndrome. Although the numerous kinetochore proteins have been identified and studied, the mechanisms that engaged in kinetochore assembly and chromosome segregation are poorly understood. Here we investigated the function of kinetochore protein Zwint-1 on homologous chromosome segregation during oocyte meiotic maturation. We found that Zwint-1 was localized at the kinetochore during meiotic maturation. Knockdown of Zwint-1 caused premature polar body extrusion, indicating acceleration of meiosis I. Interestingly, Zwint-1 knockdown impaired the recruitment of Mad2 at the kinetochores. However, BubR1 localization at the kinetochores was not affected by Zwint-1 knockdown, suggesting that Zwint-1 selectively regulates the recruitment of SAC components into the kinetochores. We also found that Zwint-1 knockdown abrogated chromosome alignment and segregation, thereby resulting in a high incidence of aneuploidy. These chromosomal defects were mostly due to the abnormal kinetochore-microtubule (kMT) attachments. Intriguingly, chromosome misalignment mediated by SAC inactivation was repaired, when anaphase onset was delayed by treating oocytes with proteasome inhibitor MG132. However, surprisingly, chromosomal defects following Zwint-1 knockdown were not restored by delaying anaphase onset. This result suggests that chromosomal defects induced by Zwint-1 knockdown are less likely associated with the failure of SAC activation. In addition, we observed that Aurora B/C kinase activity was not affected by Zwint-1 knockdown. Nevertheless, the meiotic defects induced by Zwint-1 knockdown were similar to those observed in Aurora B/C inhibition, suggesting that Zwint-1 is a downstream effector of Aurora B/C kinase during meiosis. Consistent with this, in Zwint-1 knockdown oocytes chromosomal defects following Aurora B/C inhibition were not restored when Aurora B/C inhibitor was removed, whereas the defects were well rescued in control oocytes after removing Aurora B/C inhibitor. This result suggests that the role of Aurora B/C kinases that correct erroneous kMT attachment is primarily regulated by Zwint-1. Collectively, our results demonstrated for the first time that Zwint-1 is an essential downstream effector of Aurora B/C kinase that corrects erroneous kMT attachment and regulates SAC activity, which ensures accurate homologous chromosome segregation during oocyte meiosis.

Spc25 is a component of the Ndc80 complex which consists of Ndc80, Nuf2, Spc24, and Spc25. Previous work has shown that Spc25 is involved in regulation of kinetochore microtubule attachment, localization of Ndc80, and the spindle assembly checkpoint in mitosis. The role of Spc25 in meiosis remains unknown. Here, we report its expression, localization and functions in mouse oocyte meiosis. The Spc25 mRNA level gradually increased from the GV to MI stage, but decreased by MII during mouse oocyte meiotic maturation. Immunofluorescent staining showed that Spc25 was restricted to the germinal vesicle, and associated with chromosomes during all stages after GVBD. Overexpression of Spc25 resulted in oocyte meiotic arrest, chromosome misalignment and spindle disruption. Conversely, Spc25 RNAi resulted in precocious polar body extrusion and caused severe chromosome misalignment and aberrant spindle formation. Spc25 RNAi affected Ndc80 localization, but Ndc80 RNAi did not affect Spc25 localization.Survivin MO caused Ndc80 dispersion but did not affect localization of Spc25. Our data suggest that Spc25 is required for chromosome alignment, spindle formation, and spindle checkpoint activity through the regulation of Ndc80, but that Spc25 function is independent of survivin during meiosis.