The experiments proposed here address the interdependency of genetic events of meiotic prophase with the regulation of the meiotic cell cycle during mammalian spermatogenesis. This work is inspired by recent observations that meiotic metaphase can be rapidly and precociously induced in midprophase mouse spermatocytes in culture. The fundamental hypothesis to be tested is that the spermatocyte develops competency to condense chiasmate bivalent metaphase chromosomes concomitant with the appearance of the tripartite synaptonemal complex, and that competence requires both effective chromosome pairing and function of the synaptonemal complex. Key to this work is the availability of a novel system for induction of meiotic metaphase in spermatocytes in vitro. To test this hypothesis, zygotene and early pachytene spermatocytes will be used in a competency assay and genetic mutants will be used to assess the role of chromosome pairing and the synaptonemal complex. This work will also test the hypothesis that spermatocyte competence to condense metaphase chromosomes also depends on accumulation and activation of proteins that mediate the cell-cycle transition. Roles of critical regulators of chromosome condensation and the cell cycle, topoisomerase Il, MPF, MAPK, and phosphatases, will be assessed by biochemical assays to determine presence and activity of these proteins in noncompetent, competent, and meiotic metaphase cells. Additionally, various methods, including transfer of spermatocyte nuclei into maturing mouse oocytes, will be used to induce pachytene spermatocytes to undergo meiotic divisions in order to determine when competence to segregate chromosomes during the meiotic divisions arises. Segregation will be monitored by a fluorescence in situ hybridization (FISH) assay that reliably assesses fidelity of chromosome segregation. The results of these experiments will provide new and crucial information about mechanisms regulating the spermatogenic cell cycle, as well as relate cell cycle to meiotic chromosome segregation. The latter is of importance in the etiology of aneuploidy, a major contributor to human early pregnancy failure.
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