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Geminin stabilizes Cdt1 during meiosis in Xenopus oocytes - PubMed

  • ️Thu Jan 01 2009

Geminin stabilizes Cdt1 during meiosis in Xenopus oocytes

Yadushyla Narasimhachar et al. J Biol Chem. 2009.

Abstract

During the mitotic cell cycle, Geminin can act both as a promoter and inhibitor of initiation of DNA replication. As a promoter, Geminin stabilizes Cdt1 and facilitates its accumulation leading to the assembly of the pre-replication complex on DNA. As an inhibitor, Geminin prevents Cdt1 from loading the mini-chromosome maintenance complex onto pre-replication complexes in late S, G(2), and M phases. Here we show that during meiosis Geminin functions as a stabilizer of Cdt1 promoting its accumulation for the early division cycles of the embryo. Depletion of Geminin in Xenopus immature oocytes leads to a decrease of Cdt1 protein levels during maturation and after activation of these oocytes. Injection of exogenous recombinant Geminin into the depleted oocytes rescues Cdt1 levels demonstrating that Geminin stabilizes Cdt1 during meiosis and after fertilization. Furthermore, Geminin-depleted oocytes did not replicate their DNA after meiosis I indicating that Geminin does not act as an inhibitor of initiation of DNA replication between meiosis I and meiosis II.

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Figures

FIGURE 1.
FIGURE 1.

Geminin and Cdt1 protein levels increase during meiotic maturation. Stage VI or immature oocytes were induced to undergo maturation by exposure to progesterone. Samples were collected at the indicated times and analyzed for Geminin and Cdt1 expression by immunoblot analysis.

FIGURE 2.
FIGURE 2.

Cycloheximide treatment decreases Geminin and Cdt1 levels. Western blot analysis of Cdc27, cyclin B1, Geminin, and Cdt1 proteins at the indicated times during meiotic maturation. A, in the absence of CHX; B, in the presence of CHX. The Mcm4 protein was probed as a loading control.

FIGURE 3.
FIGURE 3.

Absence of Geminin results in a decrease of Cdt1 levels during maturation. Immunoblot analysis of Cdc27, cyclin B1, Geminin, and Cdt1 during meiotic maturation is at the indicated times in Geminin sense oligo-injected oocytes (A). B, Geminin antisense oligo-injected oocytes. The Mcm4 protein is a loading control.

FIGURE 4.
FIGURE 4.

Geminin stabilizes Cdt1 during maturation. Western analysis of Cdc27, cyclin B1, Geminin, and Cdt1 proteins at the indicated times. A, during meiotic maturation. B, in the presence of exogenous Geminin during maturation. C, in the presence of CHX. D, in the presence of CHX and exogenous Geminin. The Mcm4 protein served as a loading control. Asterisk indicates exogenous Geminin.

FIGURE 5.
FIGURE 5.

Depletion of Geminin does not induce DNA replication between MI and MII. Geminin sense or antisense oligo-injected stage VI oocytes were stimulated with progesterone to enter maturation, and samples were collected at the indicated times. A, to measure the MPF activity via H1 kinase assay, 10 oocytes were homogenized and centrifuged, and the supernatant was supplemented with histone-1 and [γ-32P]ATP and incubated for 45 min at room temperature. Phosphorylation of histone H1 was followed by SDS-PAGE and PhosphorImager analysis. B, for replication assay, Geminin-deficient or control (sense injected or uninjected) oocytes were injected with [α-32P]dCTP at the time of GVBD either in the presence or absence of CHX and/or aphidicolin. Replicated DNA was analyzed 4 h after GVBD.

FIGURE 6.
FIGURE 6.

Geminin stabilizes Cdt1 after activation. Geminin sense or antisense oligos injected in in vitro matured oocytes were injected with CaCl2. At the indicated times samples were collected and analyzed for cyclin B1, Geminin, and Cdt1 expression by Western analysis. CSF is a positive control; Mcm4 is a loading control, and the asterisk indicates exogenous Geminin. Zero time point corresponds to mature oocyte (240 min after GVBD). A and B, activation in the absence of exogenous Geminin. C, activation in the presence of exogenous Geminin. Experiments carried out in A and B are from the same batch of oocytes, whereas that of C is on a different batch of oocytes. BSA, bovine serum albumin.

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