3 P66^SHC, BUT NOT P53, IS INVOLVED IN EARLY ARREST OF IN VITROPRODUCED BOVINE EMBRYOS

Abstract

High embryo losses occur in the first week of bovine embryo development, before the activation of the embryonic genome, with a high percentage of embryo death and arrest. Arrested embryos appear morphologically normal and do not exhibit any characteristic sign of apoptosis, including DNA fragmentation. We hypothesized that these embryos enter a senescence-like state and that both the cell cycle regulatory protein p53 and the stress-related protein p66^shc, which are involved in the onset of senescence in somatic cells, are responsible for this early embryonic arrest of development. The aim of this study was to characterize the expression of p53 and p66^shc in 2–4 cell-arrested bovine embryos. Our experimental model consists of in vitro-produced bovine embryos, co-cultured with oviductal cells. In our in vitro production system 86.8 ± 41.4% of embryos cleave, 13.5 ± 0.5% arrest at the 2–4 cell stage and 24.5 ± 0.7% develop to the blastocyst stage. Cleavage occurs between 26 hours post-insemination (hpi) and 48 hpi. Embryos that cleave by 28 hpi show only 0.6 ± 0.3% of 2–4 cell arrest and 41.2 ± 2.1% of blastocyst development, whereas 14.2 ± 0.9% of later-cleaving embryos arrest at the 2–4 cell stage and only 26.5 ± 1.7% develop into blastocysts. We compared 2–4 cell embryos collected at 28 hpi with those arrested at the 2–4 cell stage and collected at Day 8 post-insemination. Quantification by Real Time PCR showed significantly higher p66^shc mRNA levels (P < 0.001), but no changes in p53 mRNA levels (P = 0.860) in arrested embryos v. 28-hpi embryos (n = 3 pools of 100 embryos each). We obtained the same pattern of p53 and p66^shc mRNA expression when we compared 28-hpi embryos with later-cleaving embryos (28 hpi to 48 hpi, n = 3 pools of 100 embryos each), and higher p66^shc mRNA levels (P < 0.050), or similar p53 mRNA levels (P = 0.960). We also confirmed higher p66^shc protein levels (P < 0.001), but no changes in p53 protein levels (P = 1.000), in later cleaving embryos compared with 28 hpi-cleaving embryos by semi-quantitative immunocytochemistry (n = 70). Statistical analysis was carried out using 2-sample t-test or the equivalent nonparametric test (Mann-Whitney test). Taken together, these results demonstrate that the developmental potential of in vitro-produced embryos is related to the time of first cleavage and that p66^shc, but not p53, plays a role in early developmental arrest of in vitro-produced bovine embryos. Further experiments are required to investigate the functional role of p66^shc in early embryo arrest. Funded by NSERC, CIHR, OGS and OMAFRA.