Cell Cycle Regulation In Oogenesis

Child Health And Human Development

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Abstract

The long-term goal of the laboratory is to understand how the cell cycle events of meiosis are coordinated with the developmental events of gametogenesis. Animal oocytes undergo a highly conserved developmental arrest in prophase of meiosis I. The maintenance of the prophase I arrest requires the silencing of Cdk1 activity. Drosophila oocytes inhibit the accumulation of the mitotic Cyclins, the activating subunits of Cdk1, via a poorly defined post-transcriptional mechanism. We have demonstrated that the translational repressor Bruno inhibits translation of the mitotic cyclin, cyclin A, during prophase of meiosis I. In the absence of Bruno, ovarian cysts enter meiosis but rapidly accumulate high levels of mitotic Cyclins and return to the mitotic cycle. Based on our results we propose a model in which Bruno and the anaphase promoting complex/cyclosome act together to restrict the accumulation of the mitotic Cyclins, and thus Cdk1 activity, during the prophase I arrest of the Drosophila oocyte. During the mitotic cycle once per cell cycle DNA replication is achieved because two sequential steps in the process of DNA replication have opposite requirements for Cdk activity. While the formation of prereplication complexes (pre-RCs) (or licensing) requires the presence of low Cdk activity, the initiation of DNA replication is triggered by high Cdk activity. During the mitotic cycle, low Cdk activity is achieved in G1 after the completion of mitosis and the destruction of the mitotic cyclins. The endocycle is a variant cell cycle in which cells become polyploid by undergoing successive rounds of DNA replication without an intervening mitosis. Although endocycling cells do not undergo mitosis, they must pass through an obligate Gap phase during which Cdk activity is low. How Cdk oscillations are achieved during an endocycle is poorly understood. Over the last year we have shown that the oscillations of the cyclin-dependent kinase inhibitor (CKI) Dacapo (Dap) help introduce the Gap phase during the endocycle. Specifically, Dacapo oscillations promote entry into the Gap phase, and the formation of pre-Replication Complexes (pre-RC), through the cyclic inhibition of Cyclin E/Cdk2 kinase activity. In dacapo mutants, endocycling cells have reduced levels Double-Parked (Dup/Cdt1), a pre-RC component that is required to load the MCM2-7 complex onto chromatin. Consistent with low levels of Dup/Cdt1, dacapo nurse cells have dramatically lower levels of chromatin bound MCM2-7 complex, although the total available nucleoplasmic pool of MCM2-7 is equivalent to that found in wild-type cells. In support of the hypothesis that dacapo nurse cells assemble fewer pre-RCs during the Gap phase, mutant nurse cells exhibit reduced rates of BrdU incorporation relative to wild-type. Moreover dacapo nurse cells have increased levels of DNA damage, as measured by gamma-H2Av staining, consistent with the inability to complete genomic replication. Our data suggest a model in which Dacapo inhibits Cyclin E/Cdk2 activity during the Gap phase and thus promotes the efficient licensing of DNA replication origins. Intriguingly, a similar role has been proposed for the CKI Sic1 in promoting replication origin licensing in late G1 in S. cerevisiae. In yeast, hypomorphic alleles of cdt1 are synthetically lethal with a sic1 deletion. Similarly, in Drosophila dacapo and dup/Cdt1 genetically interact revealing a general role for dacapo in promoting the assembly of pre-RCs during the endocycle.