Fig. 4. Changes in the methylation levels of H3/K9 during early pre-implantation development in the mouse. Embryos that were prepared by in vitro fertilization (IVF), parthenogenesis (Partheno) and androgenesis (Andro) were examined for H3/K9 methylation using immunocytochemistry. The diploid parthenogenetic embryos were prepared by activating MII-stage oocytes by treatment with 10 mM Sr²⁺ in CZB for 10 minutes, followed by an additional 6-hour incubation in CZB that contained 5 µg/ml cytochalasin B to prevent extrusion of the polar bodies from the oocytes. The diploid androgenetic embryos were prepared by collecting the one-cell embryos with two pronuclei after in vitro fertilization of the enucleated oocytes. (A) Immunofluorescent confocal micrographs of mouse embryos. The two-, four- and eight-cell embryos were collected 30, 40 and 50 hours, respectively, after in vitro fertilization or parthenogenetic activation. As not all of the nuclei in some of the four-cell embryos could be aligned, only three nuclei are shown. (B) Semi-quantification of methylated histone H3/K9 in the pre-implantation mouse embryos. Mouse one-, two-, four- and eight-cell embryos were collected 12, 20, 40 and 50 hours, respectively, after in vitro fertilization or parthenogenetic activation. The diploid parthenogenetic two-cell embryos were used as control embryos in each experiment, and the averaged value of the fluorescence intensity in the control embryos was arbitrarily set at 100%. The fluorescence intensity observed for each sample was expressed relative to this value. Data were accumulated from at least two independent experiments, and were expressed on the basis of the blastomere. The numbers of blastomeres examined were: 8, 18, 32 and 47 for the androgenetic one-, two-, four- and eight-cell embryos, respectively; 22, 66, 47 and 70 for the parthenogenetic one-, two-, four- and eight-cell embryos, respectively; and 53, 90, 56 and 76 for the in vitro-fertilized one-, two-, four- and eight-cell embryos, respectively. The columns and bars represent mean±s.e.m.