Fig. 4. Early mitotic defects leading to apoptosis. (A) Embryo development in culture. One-cell embryos were cultured to 72 hours post-hCG and categorized according to cell number. Line 1 is shown. n=number of embryos. (B) Autoradiogram and (C) quantification of TRC upregulation. Two-cell embryos from Line 1 were radiolabeled at various timepoints post-cleavage. At each timepoint, TRC expression in 3-5 embryos derived from Tg mice were normalized to TRC expression in an equivalent number of embryos derived from Ntg mice (relative expression=1). Early, Mid and Late indicate embryos radiolabeled at 6, 12 and 21 hours post-cleavage; ^*P<0.05; n=number of experiments. + indicates Spindlin, an abundant maternal protein. (D) Embryo development in vivo. Cleavage-stage embryos were flushed at 72 hours post-hCG and categorized according to cell number. Lines 1, 12 and 21 are shown. n=number of embryos. (E) Immunofluorescence images of DAPI-stained embryos in D. One confocal section of an embryo is shown per panel, with green indicating DAPI-stained nuclei. Line 1 is shown. Tg embryos have ectopic nuclei. Arrow, large ectopic nucleus; arrowhead, small ectopic nucleus. Scale bar: 20 µm. (F) Percentages of embryos in D with ectopic nuclei. Embryos were categorized according to whether large (white bars) or small (gray bars) ectopic nuclei were observed. Lines 1, 12 and 21 are shown. ^*P<0.05; ^**P<0.0001; n=number of embryos. (G) Apoptosis at 120 hours post-hCG. One-cell embryos were cultured to 120 hours post-hCG and TUNEL stained. One embryo is shown per panel, with green indicating DAPI-stained nuclei and red indicating TUNEL staining. Line 1 is shown. Only 7% (2/28) of embryos derived from Tg mice versus 94% (29/31) of embryos derived from Ntg littermates formed a blastoceol cavity by 120 hours post-hCG. Scale bar: 40 µm.