Spindle Architecture and Chromosome Segregation in Mouse Oocyte Meiosis

Heath, Carissa Marie

doi:https://doi.org/10.21985/n2-8xf1-bd50

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Spindle Architecture and Chromosome Segregation in Mouse Oocyte Meiosis

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Oocyte meiosis is a specialized, but error prone, form of cell division that is poorly understood. Errors during meiosis often result in aneuploidy, or abnormal chromosome number, that impacts human health and fertility. Aneuploidy is the leading cause of miscarriages and birth defects, such as Down's syndrome in which cells have three copies of chromosome 21. Oocytes of most species lack centrioles, and use acentriolar microtubule organizing centers (MTOCs) to build a bipolar spindle. Due to the acentriolar nature of these cells, basic mechanisms of meiosis differ from mitosis and warrant further investigation. Understanding basic mechanisms of meiosis could help determine the underlying basis for aneuploidy. During my thesis work I used the mouse as a model to study mammalian oocyte meiosis. This work revealed new, previously unappreciated aspects of oocyte meiosis. I used high-resolution microscopy and morpholino microinjection to elucidate the structure of the acentriolar anaphase spindle and characterize the role of the chromokinesin Kif4 during oocyte meiosis. I also demonstrated that acentriolar anaphase spindles are organized differently than mitotic cells. Specifically I found that lateral microtubule bundles run alongside chromosomes throughout anaphase and that chromosomes eventually move past the poles. I also found that the microtubule bundles attached to kinetochores (k-fibers), which help pull chromosomes to the poles, depolymerize in late anaphase. The chromosomes segregate past the point of k-fiber depolymerization, indicating that other mechanisms contribute to late anaphase chromosome movement. I also found evidence that acentriolar MTOCs break down in late stages of anaphase. Some components either leave the MTOCs or fragment into smaller pieces as chromosomes segregate. Based on these observations, I suggest that acentriolar anaphase uses a K-fiber independent secondary mechanism of chromosome segregation. To establish Kif4's role in oocytes, I studied its localization and function. I found that Kif4 localizes to metaphase chromosome arms and that this localization requires Aurora kinase B/C activity. I also found that Kif4 localizes to structures at the midzone that coincide with anaphase spindle microtubule bundles and regulates anaphase spindle length, as its depletion results in over-elongated anaphase spindles. I determined that other known midzone proteins, PRC1 and MgcRacGAP, localize to mouse oocyte anaphase spindle midzones and may exist in the same structures as Kif4. Through my studies, I determined that Kif4's localization in mouse oocytes is consistent with its identity as a chromokinesin, and that it is an active participant in anaphase spindle regulation. Altogether, my work has generated unprecedented insight into anaphase spindle architecture and regulation.

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