Fig. 4. Overexpression of XCds1 delays meiosis reinitiation. (A) Immature oocytes were uninjected or injected with 10 ng of mRNA for XCds1-WT, XCds1-KD or LacZ and cultured for 12 hours. Western blots show the expression levels of endogenous XCds1 (uninjected and LacZ), introduced XCds1-WT or -KD proteins in addition to endogenous XCds1, and ß-galactosidase (ßgal) derived from control LacZ mRNA. (B) 12 hours after the injection, immature oocytes of A were treated with progesterone to induce maturation and then scored for the percentage of GVBD at the indicated times. Oocytes were uninjected (open triangles), or injected with mRNA for XCds1-WT (solid circles), XCds1-KD (open circles) or control LacZ (open squares). (C) XCds1-overexpressed immature oocytes were dissected into cytoplasmic (C) and nuclear (N) fractions, followed by western blot analysis with anti-XCds1 antibody. (D) Before or after overexpression of XCds1-WT, the GV was removed from some of the immature oocytes. Then, XCds1 was recovered with immunoprecipitation from enucleated (-) or nucleated (+) oocytes (100 each), and was assayed for kinase activity using GST-XCdc25C(254-316)-WT as a substrate (top). Phosphorylation at Ser287 was detected by immunoblots with anti-phospho human Cdc25C (Ser-216) antibody. Immunoblots with anti-XCds1 indicate protein amounts of XCds1 (bottom).
