The overall objective of this research is to understand how cell cycle regulatory molecules function to control the meiotic cell divisions in germ cells. Germ cell development and meiosis require a highly orchestrated series of unique cell cycle events. These cells undergo several proliferative mitoses, a unique DNA replication for meiosis, meiotic recombination, the meiotic reduction divisions, and, differentiation. Cell division is regulated, in part, by specific protein kinase complexes. In mitosis the key complexes comprise a regulatory subunit, cyclin, and a catalytic subunit, cyclin dependent kinase (Cdk). In cyclin/Cdk complexes, the cyclin directs kinase activity to specific substrates. To date, there is little data on the identity and/or function of the cyclins and Cdks during meiosis, especially with regard to the meiotic divisions that take place during spermatogenesis. The applicant's previous research has shown that a novel cyclin, Cyclin Al (CycAl) is expressed during mouse spermatogenesis in a pattern that suggests it is important for specific functions in the meiotic cell cycle. In the male mouse, CycAl is present only in germ cells. More intriguingly, CycA1 is expressed during the first meiotic division, but not the second meiotic division. This narrow window of expression suggests a specific function during the unique first division event. In addition, in whole testis extracts, immunoprecipitates of CycAl have histone kinase activity. However, the question remains as to the specific meiotic cell types in which this activity is present, and indeed which Cdk is responsible for the activity. In fact, the applicant has observed that the peak expression of Cdkl (formerly Cdc2) during meiosis appears not to coincide with peak CycAl expression, suggesting a more specific link between CycAl and a different Cdk. Further, it remains unknown if CycAl interacts with additional proteins, as shown for other cyclins. The hypothesis to be tested here is that cyclin Al plays a key role in the regulation of meiosis in males by directing protein kinase activity to meiotic substrates (as yet unidentified). The experiments outlined in this proposal will test this hypothesis by characterizing the CycAl-associated kinase activity, determining the Cdk partners of CycAl in meiotic cells, and examining other CycAl-associated proteins.
Specific Aim 1 is to examine the mechanism of CycAl activity during meiosis in the mouse by determining the Cdk catalytic subunits of CycAl. The applicant will immunoprecipitate CycAl from lysates of isolated germ cells undergoing meiosis from both prepuberal and adult male animals. Co-precipitated candidate Cdks will be detected by Western blot analyses, and kinase activity will be determined using histone H1 as the substrate.
Specific Aim 2 will determine the role of CycAl in multimeric kinase complexes during specific stages of meiosis by identifying other proteins in addition to Cdks, with which CycAl interacts. Two different experimental approaches will be used: a) co-immuno-precipitation to identify candidate proteins such as those known to bind the related CycA2/Cdk complexes in mitotic cells (e.g., p107, p19Skp1, or p45Skp2; and b) identification of interacting proteins using the yeast two-hybrid screen.
Specific Aim 3 will examine CycAl function during meiosis in the rat by studying the expression and activity of CycAl during spermatogenesis. The applicant proposes to will extend studies on meiotic functions of CycAl in the mouse by examining CycAl expression and activity in the rat, taking advantage of established model systems (not easily feasible in the mouse) to obtain cells at specific meiotic stages. Systems to be used will include the vitamin A withdrawal/replacement model of synchronous spermatogenesis and isolation of spermatogenic tubule segments. CycAl, Cdkl, and Cdk2 expression will be determined using immunohistochemistry. Co-immunoprecipitated proteins will be examined as Aim 1.
