Role of p53 and p66(shc) during cellular senescence and early embryonic arrest in the bovine.

摘要

High percentage of embryonic arrest occurs in the first week of bovine embryo development. We hypothesized that arrested embryos enter a "senescence-like state" and that the tumor suppressor protein p53 and the stress-response protein p66shc regulate this early embryonic arrest. We first established a model of cellular senescence in somatic cells culturing primary fetal bovine fibroblasts in 20% O2 and 5% O2 atmospheres until senescence was reached. p53 mRNA gradually decreased in 20% O2 cultured fibroblasts until senescence was reached, whereas p53 protein and p53 phosphorylation on serine 20 significantly increased. Senescence was also associated with higher levels of p66shc, while remaining low in dividing 5% O2 cultured fibroblasts. Using this knowledge, we then investigated early arrest in in vitro produced (IVP) bovine embryos. In our IVP system 13.5 +/- 0.5% of embryos arrest at the 2--4 cell stage. First cleavage occurs between 26 hours post insemination (hpi) and 48 hpi, with later cleaving embryos more likely to arrest. By comparing 2--4 cell embryos collected at 28 hpi with those arrested at the 2--4 cell stage collected at day-8 post insemination we showed significantly higher p66shc levels in arrested embryos. No significant changes in p53 mRNA, protein and phosphorylation levels were detected. Therefore, we suggest that embryonic developmental potential is related to the time of first cleavage and that p66shc, but not p53, plays a significant role in early developmental arrest. We then investigated how different culture environments, the SOF culture versus the co-culture systems, affect oxidative stress and embryonic arrest. Co-cultured embryos in 20% O2 tension showed higher levels of oxidative stress, higher percentage of early embryonic arrest and lower developmental capability compared to SOF cultured embryos under 5% O2 tension. p66shc but not p53, was up-regulated in co-cultured embryos that are more likely to arrest. Finally, using RNA interference, we selectively down-regulated the mRNA levels of p66shc in bovine oocytes and demonstrated a significantly lower percentage of arrest in embryos with down-regulated p66shc mRNA. These results suggest a role of p66shc in early embryonic arrest that we considered a senescent-like state.