The goal of fertilization is the union of the parental genomes within the cytoplasm of an activated egg. To accomplish this several movements must occur which culminate in the apposition and fusion of the male and female pronuclei at the egg center. This revised application is focused on two central questions: how is motility regulated?; and how are microtubules organized at fertilization? Seven questions will be posed about the regulation of egg-mediated motility during fertilization and the dynamics of microtubule organization: 1. Are phosphorylation and dephosphorylation necessary for egg-mediated motility during fertilization, and is the first cell cycle regulated differently than later cell cyles? This will be performed by localizing phosphorylated epitopes, and investigating the effects of kinase and phosphatase inhibition on motility. 2. What are the temporal and spatial patterns of the intracellular calcium release at fertilization, and is there a requirement for increased nuclear calcium? Confocal calcium ion detection provides improved imaging of the fertilization transient, and the effects of regionally blocking the newly discovered increase in nuclear calcium will be explored by reversible chelation. 3. Is the increase in centrosome size observed during fertilization dependent on newly synthesized proteins? The impact of arresting protein synthesis on the centrosome will be studied. 4. Is the centrosome contributed exclusively by the sperm, and is its apparent size due to an unfurling of compact material? The paternal contribution to centrosome volume will be investigated during polyspermy by quantitative confocal laser scanning microscopy. 5. Is the bulk of the centrosomal material maternally-derived and attracted to the sperm during fertilization? This will be tested by studying centrosome behavior during polyspermy, fertilization of egg fragments, artificial activation, parthenogenesis, refertilization and fertilization after artificial activation. 6. Is the centrosome cycle regulated similarly to the chromosome cycle? The possibility of uncoupling the two cycles with disulfide-reducing agents, sulfhydryl oxidizers, and protease inhibitors will be investigated. 7. Is gamma- tubulin the key microtubule organizing component of the centrosome and is it paternally contributed at fertilization? This will be explored using a novel antibody to the conserved region of gamma-tubulin in gametes during fertilization, parthenogenesis, and after microtubule disruption. By investigating the mechanisms leading to the union of the parental genomes at fertilization, this research addresses a central question in reproduction, and may contribute to new approaches for treating infertility, designing contraceptive approaches, and for the screening and avoidance of birth defects.
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