FIG. 1.

The effect of the stage of embryo development at the initiation of culture on the subsequent expression of TRP53 and rate of embryo development. B6 or hybrid embryos were collected at either the zygote or 2-cell stage and cultured in vitro for 96 or 72 h, respectively. A) The proportion of embryos developing to morphological blastocysts was recorded; the total number of embryos cultured is shown above each column. *P < 0.01. B) The relative intensity of TRP53 staining assessed by immunolocalization and visualization by single equatorial confocal images. The images in each panel are representative of three replicate experiments. Magnification for all images is ×300. F2, B6CBF2 embryos. C) To test that the reduced development of B6 zygotes cultured in vitro compared to hybrid embryos was a consequence of TRP53 expression, embryos were treated to increasing concentration of the TRP53 inhibitor Pifithrin-alpha. The number of embryos in each treatment from three replicates is shown above each bar. Solid bars, B6; shaded bars, hybrid; p, level of significance of the effect of Pifithrin-alpha on B6 zygotes. D) A cohort of embryos from each group, together with a cohort collected fresh from the uterus on Day 3.5 postcoitum, were subjected to quantitative RT-PCR for the relative levels of Trp53/Actb mRNA. The results are the mean and SEM for at least nine determinations for each group.