The oxidative stress adaptor p66Shc is required for permanent embryo arrest in vitro

Abstract

Background: Excessive developmental failure occurs during the first week of in vitro embryo development due to elevated levels of cell death and arrest. We hypothesize that permanently arrested embryos enter a stress-induced "senescence-like" state that is dependent on the oxidative stress-adaptor and lifespan determinant protein p66Shc. The aim of this study was to selectively diminish p66Shc gene expression in bovine oocytes and embryos using post-transcriptional gene silencing by RNA-mediated interference to study the effects of p66Shc knockdown on in vitro fertilized bovine embryos.

Results: Approximately 12,000-24,000 short hairpin (sh)RNAi molecules specific for p66Shc were microinjected into bovine germinal vesicle stage oocytes or zygotes. Experiments were comprised of a control group undergoing IVF alone and two groups microinjected with and without p66Shc shRNAi molecules prior to IVF. The amount of p66Shc mRNA quantified by Real Time PCR was significantly (P < 0.001) lowered upon p66Shc shRNAi microinjection. This reduction was selective for p66Shc mRNA, as both histone H2a and p53 mRNA levels were not altered. The relative signal strength of p66Shc immuno-fluorescence revealed a significant reduction in the number of pixels for p66Shc shRNAi microinjected groups compared to controls (P < 0.05). A significant decrease (P < 0.001) in the incidence of arrested embryos upon p66Shc shRNAi microinjection was detected compared to IVF and microinjected controls along with significant reductions (P < 0.001) in both cleavage divisions and blastocyst development. No significant differences in p66Shc mRNA levels (P = 0.314) were observed among the three groups at the blastocyst stage.

Conclusion: These results show that p66Shc is involved in the regulation of embryo development specifically in mediating early cleavage arrest and facilitating development to the blastocyst stage for in vitro produced bovine embryos.

Figure 1

Appearance of 2–4 cell arrested embryos at day-8 post insemination. Morphological appearance of day-8 in vitro produced bovine embryos. Embryos at this time after in vitro fertilization usually reached the blastocyst stage (a), while arrested embryos still appeared as morphologically normal 2–4 cell embryos (b). (Magnification: 400×).

Figure 2

Real Time PCR quantification of p66Shc mRNA levels at the 2-cell stage following RNAi. Real Time PCR analysis of p66Shc (A) and H2a (B) mRNA levels in 35 hpi embryos following control IVF, microinjection of the vehicle and microinjection with 12,000 p66Shc hairpin RNAi molecules. p66Shc mRNA levels decreased in p66Shc RNAi microinjected embryos (*P < 0.001), while Histone H2a mRNA levels showed no significant differences (P = 0.744) among the three groups. This result provided evidence that the down-regulation of p66Shc mRNA levels following p66Shc hairpin RNAi molecule injection is selective for p66Shc transcripts. Real Time PCR analysis of p53 mRNA levels (C) for the three groups was also performed, showing no significant differences (P = 0.98). The experiments were conducted on three different pools of 80–100 embryos (n = 3) and replicated three times (r = 3) for each sample. The data were normalized for the content in one embryo.

Figure 3

p66Shc protein levels following microinjection of p66 shRNA interfering molecules. The distribution of p66Shc in early bovine embryos was assessed (A, B, C and D). Green, red and blue colors in each representative photomicrograph indicate positive staining for the respective primary antibody: p66Shc (FITC), F-actin (Rhodamine phalloidin), and nuclei (DAPI) respectively. Panels A and B represent Uninjected and Injected (Vehicle) controls respectively. Panel C (shRNAi low dose = ~12,000 molecules) and Panel D (shRNA high dose = ~24,000 molecules) display a marked reduction in p66 fluorescence. Panel E represent the no primary antibody controls. Scion Image analysis resulted in the quantification of FITC immunofluorescence (p66Shc) across different groups (F). Relative signal strengths are presented as the mean ± S.E.M. representative of three independent replicates. Bars with different letters represent significant differences in relative signal strength between treatment groups (P ≤ 0.05).

Figure 4

Assessment of cleavage, blastocyst, 2–4 cell embryo arrest and spontaneous embryo cleavage rates following microinjection with p66Shc hairpin RNAi molecules. Cleavage rate (A) and spontaneous cleavage rate (D) were evaluated at day-2 post insemination, while blastocyst rate (B) and arrest rate (C) were evaluated at day 8–9 post insemination. Although cleavage and blastocyst frequencies were significantly decreased (P < 0.001) in the embryos microinjected with the vehicle alone, an even greater significant decrease (P < 0.001) was observed in the p66Shc shRNAi microinjected oocytes (A, B). We observed a significant decrease (P < 0.05) in spontaneous oocyte cleavage rate following microinjection (D). A significant decrease in the frequency of arrested 2–4 cell embryos was achieved only following microinjection of short hairpin RNAi molecules against p66Shc (C). Bars with different letters represent significant differences between groups (P ≤ 0.05).

Figure 5

Real Time PCR quantification of p66Shc mRNA levels at the blastocyst stage following RNAi. Real Time PCR analysis of p66Shc (A) and H2a (B) mRNA levels in day 8–9 blastocysts obtained following control IVF, microinjection of the vehicle and microinjection of 12,000 p66Shc short hairpin shRNAi molecules. No significant differences (P = 0.314) in the p66Shc mRNA levels were measured (A). The quantification of the histone H2a mRNA levels was used as a control, observing no significant differences (P = 0.312) (B). The experiments were conducted on three different pools of 80–100 embryos (n = 3) and replicated three times (r = 3) for each sample. The data were normalized for the content in one blastocyst.

Figure 6

Oocyte microinjection. The short hairpin (sh)RNAi microinjection of a the germinal vesicle (GV) stage bovine oocyte. (A) The oocyte has been localized and is subsequently microinjected with shRNAi molecules (B). The oocyte is being held by the holding pipette and microinjected by a pipette of 4 μm in diameter and beveled at 35°. Care was taken not to strip off the cumulus cells surrounding the GV oocytes and to avoid perforating the germinal vesicle during microinjection.

Figure 7

P66Shc short hairpin (sh)RNAi molecule sequence. The positive strand (A) was designed with a BamH1 site at the 5' end followed by the 19 nucleotide specific for bovine p66Shc (underlined), a loop of 9 non-complementary nucleotides and the reverse complementary sequence of the 19 nucleotides specific for bovine p66Shc (underlined). The negative strand (B) consists of the reverse complementary to the positive one, without the BamH1 site, but with a HindIII restriction site at its 5' end. (C) The represented shRNAi structure that is formed within the oocyte/embryo upon microinjection.