We had previously identified a new mammalian A-type cyclin, cyclin A1, in addition to the already known cyclin A2. Both A-type cyclins are expressed in the mouse testis. Expression of the cyclin A1 gene, Ccna1, is germ cell-specific and restricted to late meiotic prophase spermatocytes while Ccna2 is expressed more broadly, in both somatic cells and in spermatogonia and pre-leptotene spermatocytes. Targeted mutagenesis of Ccna1 resulted in viable progeny but male sterility, while females were fully fertile. Spermatocytes in cyclin A1-deficient mice cannot enter into the first meiotic division, the activation of MPF is deficient and the cells undergo apoptosis. In contrast, cyclin A2-deficient mice are embryonic lethal. We now wish to understand the unique regulation and function of cyclin A1 in spermatocytes, to explore the function of cyclin A2 in the male germ line, and to dissect the regulatory pathways that are driven by cyclin A1 and its catalytic Cdk partners during male meiosis. We will also investigate the extraordinarily robust induction of apoptosis that occurs in the cyclin A1- and Cdk2-deficient models. We will identify regulatory elements and corresponding transcription factors required for the proper in vivo expression of Ccna1. This will involve DNase 1 hypersensitivity mapping, electrophoretic mobility shift assays (EMSA), chromatin immunoprecipitation (ChIP), expression of reporter constructs in transgenic mice, sequence analysis, and a novel transcription factor screen to identify the regulatory network responsible for both activating and repressing its expression. We will also examine the changes in the events in the cell cycle, chromosome dynamics, and phosphorylation status of putative substrates that result in the arrest at diplotene and apoptosis in cyclin A1 and Cdk2-defident spermatocytes. This will also involve a novel phosphorylation screen to identify substrates of cyclin A1-complexes. Finally, we will determine the affect of targeted conditional mutagenesis of cyclin A2 in spermatogonia. These studies will provide new and novel insight into the function of the mammalian A-type cyclins, information that cannot be obtained from simpler model systems such as yeast, which lack this class of cyclins, or Drosophila, which contain only a single A-type cyclin. They will further enhance our understanding of the control of meiosis as it relates to male infertility and possible new targets for contraception.
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