Fig. 4. ERK inactivation in early two-cell-stage embryos induces developmental arrest in the G2 phase, just before M phase in the two-cell stage. (A) Early two-cell-stage embryos were treated with 20 µM U0126 or DMSO, and embryos were observed from E1.0 to E2.0. The zona pellucida was removed before inhibitor treatment. (B) BrdU incorporation into the nucleus was investigated. Early two-cell-stage embryos were cultured in the presence of BrdU and U0126 or BrdU and DMSO (control), fixed, and stained with anti-BrdU antibody. Fluorescence was viewed at the stages indicated with a confocal microscope. (C) Cyclin B1 accumulation in the nucleus and phosphorylation of histone H3 were examined. Early two-cell-stage embryos were cultured in the presence of U0126 or DMSO. Embryos were fixed and stained with anti-cyclin B1 or anti-phospho-histone H3 antibody. Fluorescence was viewed with a confocal microscope at the stages indicated.