A high incidence of permanent embryo arrest occurs during the first week of in vitro development. We hypothesize that this developmental arrest event is regulated by the stress adaptor protein p66shc, a genetic determinant of life span in mammals, which regulates ROS metabolism, apoptosis, and cellular senescence. The aim of this study was to assess the relationship between intracellular oxidative stress levels with the incidence of embryo arrest and the expression of senescent-associated genes in embryos produced under different oxygen tensions. Embryos cultured under 20% oxygen conditions showed approximately 10-fold increase in oxidative stress, 2-fold increase in the percentage of 2- to 4-cell arrest, and significantly lower developmental capabilities compared to embryos cultured under a 5% oxygen environment. Quantification by real-time PCR and by semiquantitative immunofluorescence showed significantly higher p66shc mRNA and protein levels, respectively, in embryos cultured in 20% versus those cultured in 5% oxygen atmosphere. No significant changes in p53 mRNA and protein levels were detected among embryos derived from both oxygen tensions. Taken together, these results demonstrate that p66shc, but not p53, is significantly more abundant in an embryo population that exhibits higher frequencies of embryo arrest and quantities of intracellular ROS. These results further substantiate that p66shc and oxidative stress are associated with a p53-independent embryonic arrest event for in vitro-produced embryos.

译文

在体外发育的第一周内,永久性胚胎停滞的发生率很高。我们假设这种发育停滞事件是由应激衔接蛋白p66shc调节的,p66shc是哺乳动物寿命的遗传决定因素,它调节ROS代谢,凋亡和细胞衰老。这项研究的目的是评估细胞内氧化应激水平与胚胎停滞发生率之间的关系,以及在不同氧气张力下产生的胚胎中衰老相关基因的表达。与在20% 氧环境下培养的胚胎相比,在5% 氧环境下培养的胚胎显示氧化应激增加约10倍,在2-4-细胞阻滞百分比增加2倍,以及显著更低的发育能力。通过实时PCR和半定量免疫荧光定量显示,在20% 培养的胚胎中,与在5% 氧气气氛中培养的胚胎相比,p66shc mRNA和蛋白质水平分别显著更高。在来自两种氧张力的胚胎中,未检测到p53 mRNA和蛋白质水平的显着变化。总之,这些结果表明,在表现出更高的胚胎停滞频率和细胞内ROS数量的胚胎群体中,p66shc而非p53明显更丰富。这些结果进一步证实了p66shc和氧化应激与体外产生的胚胎的p53-independent胚胎停滞事件有关。

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