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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.

SUBMITTER: Favetta LA

PROVIDER: S-EPMC2220003 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background: Oxidative stress has emerged as an essential factor in the pathogenesis of intestinal ischemia/reperfusion (I/R) injury. The adaptor protein p66Shc is a key regulator of reactive oxygen species (ROS) generation and a mediator of I/R damage in the intestine, but the upstream mechanisms that directly regulate p66Shc expression during intestinal I/R remain largely unknown. Recent studies have suggested that noncoding RNAs, such as circular RNAs (circRNAs), are important players in physiological and pathological processes based on their versatile regulatory roles in gene expression. The aim of this study was to elucidate the contribution of p66Shc to oxidative damage in intestinal I/R and to investigate the regulation of p66Shc by circRNA sponges. Methods: Intestinal I/R was induced in mice via superior mesenteric artery (SMA) occlusion. A miR-339-5p agomir or circ-protein kinase C beta (PRKCB) siRNA was injected intravenously before I/R challenge. In addition, Caco-2 cells were subjected to hypoxia/reoxygenation (H/R) in vitro to simulate an in vivo I/R model. Results: In vitro, p66Shc deficiency significantly reduced H/R-induced ROS overproduction by attenuating mitochondrial superoxide anion (O2 -) levels, suppressing NADPH oxidase activity and enhancing antioxidant enzyme expression. Moreover, miR-339-5p was identified to directly regulate p66Shc expression in the intestine. Furthermore, we found that a circRNA transcribed from the PRKCB gene, named circ-PRKCB, acted as an endogenous miR-339-5p sponge to regulate p66Shc expression. circ-PRKCB silencing or miR-339-5p overexpression significantly downregulated p66Shc expression and attenuated oxidative stress levels and I/R injury in vivo and in vitro. Notably, the increased circ-PRKCB levels and decreased miR-339-5p levels associated with murine intestinal I/R were consistent with those in patients with intestinal infarction. Conclusions: Our findings reveal a crucial role for the circ-PRKCB/miR-339-5p/p66Shc signaling pathway in regulating oxidative stress in the I/R intestine. This pathway may be a potential therapeutic target for intestinal I/R injury.

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

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