Embryos lacking maternal Ikbkap exhibited progressive developmental delays and failed to develop into blastocysts. (A,B) MII oocytes were fertilized in vitro, and zygotes at the pronuclear stage were collected. Zygotes were immunolabeled with H3K9me3 antibody to visualize female pronuclei (green), and chromosomes were counterstained with DAPI (blue). Shown are representative confocal images (A) and incidence of digynic zygotes from in vitro fertilized Ctrl (n = 52) and CKO (n = 144) oocytes. (B) n indicates the number of oocytes analyzed from at least two independent experiments. At least three Ctrl and three CKO females were used for each experiment. (C,D) MII oocytes isolated from superovulated Ctrl and CKO females were fertilized in vitro, and the embryos were cultured in vitro until the blastocyst stage. Their developmental stages were determined by morphology. Shown are the percentages of embryos at different stages (C) and the representative images of embryos at 120 hours post-fertilization (D). Abnormal embryos included those that were at the 1-cell stage (arrows) or 2-cell stage (arrowheads) or those exhibiting abnormal morphology. 47 Ctrl and 121 CKO embryos were examined at each time point. Data are presented as mean ± SEM of two independent experiments. Three mice per group were used for each experiment. *p < 0.05; **p < 0.01.