We had previously identified a new mammalian A-type cyclin, cyclin A1, in addition to the already known cyclin A2. We showed that cyclin A1 is testis-specific and by targeted mutagenesis, that it is essential for spermatogenesis--spermatocytes arrest at the diplotene to metaphase 1 transition. As knockouts of the more ubiquitously expressed cyclin A2 resulted in early embryonic lethality, we generated a conditional knockout model (A2CKO) and discovered unexpected and important functions of cyclin A2 in hematopoietic and embryonic stem cells. We have now also generated A2CKO models lacking cyclin A2 expression in undifferentiated type A spermatogonia, which results in a severe disruption of spermatogenesis and sterility.
In Aim 1 of this renewal, we will determine the effects of loss of cyclin A2 function in different stages of spermatogonial development and in primordial germ cells (PGCs). We will determine which cell types require cyclin A2 for proliferation, where in the cell cycle the arrest occurs, and the fate of spermatogonia lacking cyclin A2 (Aim 1a). We will then extend these observation by ablating cyclin A2 in PGCs and in embryonic and post- natal gonocytes (Aim 1b) and in later stages of post-natal spermatogonial differentiation (Aim 1c), using our floxed Ccna2 mice and selected Cre deleter lines. We will begin to address the fundamental question of why higher organisms have evolved two A-type cyclins by asking if cyclin A1 and cyclin A2 are functionally redundant, using both genetic and biochemical approaches. We previously showed that loss of cyclin A1 results in a very specific and completely penetrant arrest of meiotic prophase spermatocytes just before the first meiotic division and have begun to identify key regulatory regions of the Ccna1 gene. We therefore propose to knock-in cyclin A2 coding sequences into the Ccna1 locus and examine the effects on meiotic progression, that is, can cyclin A2 function in place of cyclin A1 if expressed at the correct time? Finally, to further address the function of cyclin A1 in spermatocytes and to extend assessment of functional redundancy, in Aim 3 we will identify candidate testicular substrates of cyclin A1 and cyclin A2 complexed with their catalytic partners CDK1 and CDK2. This will be accomplished by fractionating lysates from spermatocytes and immature testes and subjecting them to phosphorylation by active cyclin A1- and A2-CDK complexes in which the ATP-binding pocket of the CDK is mutated so that it can readily accept modified ATP analogues, which endogenous kinases cannot (the """"""""Shokat"""""""" strategy). These studies will provide novel insights 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. Our findings will enhance our understanding of the control of meiosis as it relates to male infertility, possibly suggest new targets for contraception, and provide critical insight into the unique properties of cell cycle regulation in stem cells, which i highly relevant in both normal development and in oncogenesis.
Successful completion of the experiments outlined in this proposal will enhance our understanding of the function of an important class of cell cycle regulators, the mammalian A-type cyclins. Our particular focus is on their function in the male germ line, but the reagents we have generated and the discoveries we make will be useful to investigators in other fields, particularly in the field of stem cell biology and oncogenesis. Our findings will provide insight into causes of unexplained human infertility and alternatively, may identify novel targets for contraception.
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