Cdc6 amd replication competence in xenopus oocytes

The early division cycles of an embryo following fertilization rely on the oocyte's ability to replicate DNA. During meiosis, oocytes temporarily lose their ability to replicate DNA. After a single round of pre-meiotic S-phase, oocytes enter meiosis and rapidly arrest at prophase of meiosis I. During this G2 arrest (immature oocyte), that can last months or years depending on the animal species, oocytes are unable to replicate DNA. Upon hormonal stimulation, arrested oocytes resume meiosis, reestablish DNA replication competence in meiosis I shortly after germinal vesicle breakdown (GVBD), but repress replication until fertilization. How oocytes lose and regain replication competence during meiosis are important questions underlying the production of functional gametes that will support the early development of the embryo.

This study focuses on determining why immature oocytes are unable to replicate their DNA. Since the elongation machinery is known to be present and functional in the immature oocyte, we hypothesized that the ability of an oocyte to replicate its DNA is controlled at the initiation step. The initiation of DNA replication in mitotic cells requires the assembly and activation of pre-replication complexes (pre-RCs). During assembly, the Origin Recognition Complex (ORC), Cdc6, Cdtl and the Mem proteins bind to the DNA in a sequential order. Following assembly, phosphorylation by two kinases, Cdk2/Cyclin E and Cdc7/Dbf4, leads to activation of the pre-RC.

We show here that immature oocytes are unable to replicate due to three defects which prevent pre-RC assembly and thus, replication ability: (1) absence of Cdc6 protein; (2) differential localization of pre-RC components; and (3) phosphorylation of the Mcm4 protein. Next we describe experiments, which illustrate that Cdc6 protein synthesis and GVBD are the only limiting events for the development of replication competence during meiosis.

Since Cdc6 seems to be critical for replication competence, we also focused experiments on determining the mechanisms that regulate Cdc6 expression in the immature oocyte. It appears that Cdc6 is absent from the immature oocyte because it is not being synthesized and is also very unstable in the immature oocyte. The Cdc6 message is present in the immature oocyte, but is repressed via sequences in its 3'UTR, which are most likely cytoplasmic polyadenylation elements (CPEs).