The successful completion of fertilization requires motility mediated by the egg cytoskeleton and architectural changes in the organization of the sperm and egg nuclei. The long term objectives of this research are to understand the structural and motile events necessary for fertilization. To accomplish these goals, the two specific aims of this research are: I. to investigate centrosomal activity, inheritance, and composition as well as the manner in which this pericentriolar material directs microtubule organization and II. to explore nuclear organization by determining the composition of the nuclear lamina, matrix and kinetochore antigens, and by investigating the alterations in nuclear architecture occurring during pronuclear formation, syngamy, and the first cell cycle. The hypothesis that the sperm introduces the dominant microtubule organizing center at fertilization, which is both capable of propagation and attracting stored maternal centrosomal material, will be tested by the use of immunofluorescence, time-lapse video, and transmission and high voltage electron microscopy. The theory that microfilaments, but not microtubules, are responsible for effecting changes in centrosomal shape will be tested with cytoskeletal inhibitors. Whether DNA and protein synthesis, but not RNA synthesis, is required for centrosome duplication will also be tested with specific inhibitors. Centrosome composition may include vimentin, topoisomerase I, phosphoproteins and a cyclic AMP-dependent protein kinase, and this will be studied with monoclonal antibodies. Nuclear lamins in sea urchin embryos, eggs and sperm will be characterized by electrophoresis and immunoblotting to determine whether these proteins change during development and the cell cycle. The hypothesis that the nuclear matrix is exposed only in embryos coordinate with the nuclear accumulation of small nuclear ribonucleoproteins (snRNPs) will be tested, and kinetochore antigens will be traced throughout fertilization with characterized antibodies. The theory that pronuclear membrane fusion during syngamy is dependent on intact disulfide bonds will be investigated with disulfide reducing agents. In addition the strict correlation between the presence of lamins and the nuclear envelope will be elucidated. 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 avoidance or screening of birth defects.
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