218 SELECTIVE REDUCTION OF p66^shc mRNA IN BOVINE OOCYTES/EMBRYOS BY RNA INTERFERENCE

Abstract

High embryo loss occurs during the first week of in vitro bovine embryo development with a high percentage of embryo death and arrest. These early stages of development are regulated by stored maternal mRNAs that accumulate in the oocyte cytoplasm during its growth phase. In our in vitro production (IVP) system approximately 13.5% of embryos arrest at the 2–4-cell stage, displaying no characteristics of apoptosis. We hypothesized that these arrested embryos enter an oxidative stress-induced senescence-like state. We previously showed that elevated levels of reactive oxygen species and increased levels of the stress-adaptor protein p66^shc were associated with this early embryonic arrest. The aim of this study was to selectively down-regulate p66^shc mRNA levels in bovine oocytes, using post-transcriptional gene silencing by RNA interference (RNAi), to study the effects of p66^shc mRNA “knock-down” on early arrest of IVP bovine embryos. Approximately 12,000 short hairpin (sh) RNAi molecules specific for p66^shc were microinjected into bovine germinal vesicle (GV) oocytes. Experiments consisted of a control group undergoing IVF alone, and two groups microinjected with and without p66^shc shRNAi molecules. Quantification of p66^shc mRNA by real-time PCR was used to assure down-regulation of p66^shc mRNA in 2-cell embryos collected at 35 hours post-insemination (hpi). The amount of p66^shc mRNA detected in control IVF, vehicle-injected, and p66^shc RNAi-injected groups was 0.92 ± 0.02 × 10^-5 pg, 1.2 ± 0.20 × 10^-5 pg, and 0.46 ± 0.06 × 10^-5 pg, respectively. This statistically significant (P < 0.001) reduction in p66^shc mRNA levels by 54% upon p66^shc shRNAi microinjection was selective for p66^shc mRNA, as both histone H2a and p53 mRNA levels were not altered. Percentage of 2–4-cell arrest was evaluated at Day 8 post-insemination and related to p66^shc mRNA down-regulation. While there were no significant differences in the percentage of 2–4-cell arrested embryos between the control (13.3 ± 0.8%) and vehicle-microinjected (10.8 ± 0.7%) embryos, there was a significant decrease (P < 0.001) in the incidence of arrest in p66^shc shRNAi-microinjected embryos (0.9 ± 0.9%). Quantification by real time PCR in blastocysts from the three groups showed no significant differences in p66^shc mRNA levels (P = 0.314) among control IVF, vehicle, or p66^shc hairpin RNAi microinjected at 0.59 ± 0.1 × 10^-5 pg, 0.57 ± 0.2 × 10^-5 pg, and 0.51 ± 0.2 × 10^-5 pg, respectively. This suggests that p66^shc down-regulation by RNAi might be temporary. Thus, using this novel approach of RNAi and microinjection of oocytes at the GV stage, we were able to selectively down-regulate the expression of p66^shc mRNA and correlate this down-regulation with a significant decrease in 2–4 cell arrest. These data reinforce our hypothesis that p66^shc is involved in a stress-induced pathway that executes a senescent-like embryonic arrest at the 2–4-cell stage, thereby postulating the possible role of p66^shc as a new molecular marker for developmental competence. Statistics performed using ANOVA with Fisher LSD test for multiple comparisons.

This work was funded by NSERC, CIHR, OGS, and OMAFRA.