FIG. 2.
PR-Set7 knockout embryos arrest at late G2 or M phase prior to the eight-cell stage and exhibit decreased H4K20me1, increased H3S10P, and apoptosis. (A) Microscopic analysis of embryos at 2.5 dpc that were collected and transferred to in vitro culture for a day. (Top) Embryos having eight or more cells developed into blastocysts after 1 day of culture. (Bottom) Those having less than eight cells did not develop into blastocysts and degenerated. (B) Genotyping results of in vitro-cultured embryos shown in panel A. Embryos that developed into blastocysts were wt (+/+) or heterozygous (+/−). All the embryos that had less than eight cells at 2.5 dpc were homozygous (−/−). (C) Immunofluorescence analysis using anti-H4K20me1 and anti-H3S10P antibodies. (Left) A 2.5-dpc embryo wt for PR-Set7 exhibits variable levels of H4K20me1 in each blastomere, and the H3S10P signal is negative. (Right) PR-Set7 KO embryos arrest with nearly undetectable levels of H4K20me1 in the blastomeres but with clear staining in the polar body (PB; arrow). H3S10P shows speckled or diffuse staining in all blastomeres. (D) TUNEL assay performed on 2.5-dpc embryos after in vitro culture for a day. Presumptive PR-Set7 KO embryos showed positive signals (white arrows) as opposed to other embryos that showed few, weak signals and developed into blastocysts. (E) Comparison of homozygous null (PR-Set7−/−) versus wt (PR-Set7+/+) 2.5-dpc embryos in an in situ caspase assay after in vitro culture for a day. The PR-Set7−/− embryo shows strong caspase-3 and caspase-7 activity.