Knockdown of p66Shc ameliorates H₂O₂-induced apoptosis and developmental arrest of early bovine embryos.

<p>Groups of zygotes (n = 50/r, r = 3) were either: non-injected controls, injected with negative control siRNAs, or injected with p66Shc siRNA molecules (RNAi-E). At the 9–16 cell stage, all groups were subjected to 50 µM H₂O₂ treatment for 1 hr. After an overnight incubation, embryos were fixed and stained with a mixture of TUNEL labeling stain (green) and propidium iodide (red) to assess their apoptotic status. (A) Representative negative control embryo stained in the absence of active TUNEL enzyme. (B) Representative positive control embryo subjected to DNase treatment before TUNEL stain. (C) Representative apoptotic 9–16 cell embryo displaying TUNEL positive stain in conjunction with nuclear condensation and fragmentation. (D) Representative non-apoptotic 9–16 cell embryo displaying TUNEL negative staining in conjunction with intact nuclear staining. (E) Summary of the incidence of apoptosis following RNAi microinjection and H₂O₂ treatments. While embryos injected with negative siRNA molecules exhibited a greater proportion of apoptosis, embryos injected with RNAi-E exhibited a significantly reduced incidence of apoptosis. (F) The effect of p66Shc knockdown on the frequency of blastocyst formation following H₂O₂ treatment. Groups of embryos (non-injected, negative control siRNA- injected, or RNAi-E injected, were subjected to a 1 hr treatment of 50 µM H₂O₂ at the 2–4 cell stage after which they were allowed to develop until the 8th day of culture (192 hpi) at which point the frequency of blastocyst development was assessed. A significantly greater proportion of embryos injected with the p66Shc-specific siRNA molecules (RNAi-E) successfully developed into blastocysts compared to those injected with a negative control siRNA. The proportion of blastocysts amongst the RNAi-E group was comparable to that of the non-injected control group. Different letters above the histogram bars represent significant differences (p<0.05). Scale bar, 50 µM.