Abstract
BackgroundExcessive 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.ResultsApproximately 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.ConclusionThese 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.
Highlights
Excessive developmental failure occurs during the first week of in vitro embryo development due to elevated levels of cell death and arrest
We [1,12,13] and others [11,14] have observed no morphological or biochemical signs of apoptosis during the early 2–4 cell cleavage stages of bovine embryogenesis. It is at this early developmental stage that approximately 14% of all in vitro produced (IVP) bovine embryos permanently arrest in a senescence-like state (Fig. 1) [2,15]
The results presented here demonstrate the successful application of RNA interference (RNAi) in selectively diminishing the oxidative stress adaptor protein p66Shc during in vitro bovine embryo development
Summary
Excessive developmental failure occurs during the first week of in vitro embryo development due to elevated levels of cell death and arrest. BMC Developmental Biology 2007, 7:132 http://www.biomedcentral.com/1471-213X/7/132 of these unable to sustain development following embryo transfer [5] The reasons for this high rate of in vitro embryo demise remains unclear, but it has been proposed as a protective mechanism for preventing further development of abnormal, poor quality embryos. We [1,12,13] and others [11,14] have observed no morphological or biochemical signs of apoptosis during the early 2–4 cell cleavage stages of bovine embryogenesis It is at this early developmental stage that approximately 14% of all in vitro produced (IVP) bovine embryos permanently arrest in a senescence-like state (Fig. 1) [2,15]
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