Building Fidelity at the Beginning of Life

Abstract

Accurate chromosome segregation depends on the formation of a functional bipolar spindle. While somatic cells rely on centrosomes to organize spindle assembly, mammalian oocytes lack canonical centrosomes and must assemble their meiotic spindles through acentrosomal mechanisms. Following fertilization, embryos progressively establish the centrosome-dependent spindle assembly program characteristic of somatic cells. How these mechanisms differ between mammalian species and how the transition from acentrosomal to centrosomal spindle assembly occurs during early embryogenesis remain incompletely understood. This thesis investigates the mechanisms of spindle assembly and chromosome segregation in mammalian oocytes and early embryos, with a particular focus on species-specific pathways and the establishment of the centrosomal system after fertilization. Comparative analyses revealed that mammalian oocytes employ distinct acentrosomal spindle assembly strategies. Mouse and equine oocytes predominantly rely on acentriolar microtubule-organizing centers (aMTOCs), which fragment and self-organize to establish the spindle poles. In contrast, human and bovine oocytes depend primarily on chromosome-associated microtubule organization, with the fibrous corona surrounding kinetochores acting as a major site of microtubule nucleation and organization. Despite these species-specific differences, conserved pathways, including RanGTP-mediated microtubule nucleation and the augmin complex, contribute to spindle formation across the species examined. These findings suggest that mammalian oocytes combine a conserved core acentrosomal machinery with species-specific microtubule-organizing structures. This thesis further demonstrates that fertilization does not trigger an immediate switch from acentrosomal to centrosomal spindle assembly. In non-rodent mammals, centrioles are inherited from the sperm and provide the foundation for establishing the embryonic centrosomal system. Super-resolution imaging of bovine zygotes revealed distinct architectures of the sperm-derived proximal and distal centrioles, with the proximal centriole retaining more canonical structural features and the distal centriole displaying extensive remodeling. Functionally, the first embryonic divisions represent a transitional period during which centrosomal and acentrosomal mechanisms coexist. In bovine embryos, centrosomes become essential for spindle stability and chromosome segregation from the second mitotic division onward, with their absence resulting in spindle defects and developmental arrest. In contrast, equine embryos retain aMTOC-mediated spindle organization for a longer developmental period, demonstrating that the transition toward centrosome-dependent mitosis occurs with species-specific timing. Live-cell imaging of equine oocytes and IVF-derived zygotes, together with super-resolution STED microscopy of bovine zygotes, enabled direct investigation of these dynamic and structural changes. Collectively, this work establishes that mammalian spindle assembly is governed by both conserved principles and species-specific adaptations. Oocytes employ diverse acentrosomal mechanisms to construct bipolar spindles, whereas early embryos progressively transition toward centrosome-dependent mitosis rather than undergoing an abrupt switch at fertilization. These findings provide a comparative mechanistic framework for understanding how the microtubule-organizing machinery is reorganized during the oocyte-to-embryo transition and how faithful chromosome segregation is maintained during the earliest stages of mammalian development.