Our initial efforts to elucidate the genetic program controlling mitosis and meiosis in germ cells have focused on four classes of genes known to be involved with cell cycle control: the cyclins, the cyclin-dependent kinases (Cdks), and the mammalian homologues of the fission yeast phosphatase Cdc25 and tyrosine kinase Wee1. We have demonstrated remarkable cell-cycle specificity of expression of several of these genes and have established the existence of sex-specific differences in the lineage specificity of expression of several genes. We have furthermore recently identified a germ-line specific gene, Cyclin Al, a member of a class that has yet to be identified in simpler eukaryotes. These observations have underscored the fact that there exist unique regulatory features of meiosis that are not found in less complex eukaryotes nor in mitotic lineages, and which differ between the male and female germ cells. In this proposal, we will investigate the role of the cyclin A~s in particular, in meiosis. We will test the hypothesis that CycA1 functions during the first meiotic division of spermatocytes by examining the effects on the progression of meiosis in male mice carrying a null mutation in the Cyclin A1 gene produced by targeted mutagenesis and homologous recombination in ES cells and the generation of chimeric mice from the targeted cells. To examine the role of cyclin A2 during meiotic maturation of oocytes, we will interfere with the function of Cyclin A2 by injection of antisense oligonucleotides and possibly antibodies into germinal vesicle intact oocytes and monitoring the progression of meiosis. The potential catalytic partners of Cyclin A1 and Cyclin A2 in testicular and ovarian cells will be identified by two strategies. In the first, co-immunoprecipitation with antibodies to Cyclin A1 and cyclin A2 and to cyclin dependent kinases (Cdk~s) known to be expressed in these cells followed by immunoblot analysis will identify whether such potential interacting Cdk~s actually associate with CycA1 or A2 in vivo. Second, potential new catalytic partners will be identified by using the yeast 2-hybrid screen with CycA1 and CycA2 cDNAs as the baits to screen testicular and ovarian cDNA libraries, respectively. Finally, we will begin to explore the role of potential activators of the CycA/Cdk complexes, namely, the Cdc25 family of dual specificity phosphatases, during gametogenesis. These studies will provide insight as to the in vivo functions of the cyclin A~s and their interacting proteins in mammalian cells in general and in germ cells in particular.
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