The successful union of the parental genomes during mammalian fertilization requires intracellular architectural changes. In particular, microtubule- mediated motility is essential for the apposition of the sperm and egg nuclei, for the proper maturation of the ovulated oocyte and for the completion of the fertilization process at first division. This application proposes to investigate five question about microtubule organization and dynamics. 1. Are the dynamics of the microtubules participating in mammalian fertilization unusual? This might be expected due to metaphase arrest, and since the first mitotic spindle is organized without centrioles. 2. What are the in vivo functions of kinetochore proteins? Will microinjected kinetochore antibodies interfere with the anaphase separation of the chromosomes? Will they influence spindle organization at metaphase or spindle formation at exogenous centrioles? 3. Is the centrosome maternally inherited in this mammal? 4. When and where do centrioles arise during early embryogenesis? At which developmental stage will the embryonic cytoplasm tolerate the introduction of exogenous centrioles? 5. Is alpha-tubulin post-translationally modified by acetylation or detyrosination, and are these changes correlated with microtubule stability or age? The hypotheses to be tested are that: 1. while the metaphase-arrested oocyte appears dormant, its microtubules are highly dynamic; 2. kinetochore antibodies will prevent prometaphase congression and anaphase chromosome separation if introduced prior to metaphase; 3. the centrosomes in this mammal are maternally inherited; 4. centrioles appear late in embryogenesis and the oocyte cytoplasm has a centriole disassembly factor; and 5. the microtubules involved in pronuclear union are post- translationally detyrosinated and acetylated, and these modifications are correlated with the generation of differing classes of stable microtubules. This research will advance knowledge about the structural changes leading to genomic union, a central step in reproduction. By addressing fundamental questions regarding the union of the parental genomes at fertilization, this research may contribute to new approaches for treating infertility, designing contraceptive strategies and for avoiding or screening for birth defects.
