AMPK Activity Contributes to G2 Arrest and DNA Damage Decrease via p53/p21 Pathways in Oxidatively Damaged Mouse Zygotes.
AMPK Activity Contributes to G2 Arrest and DNA Damage Decrease via p53/p21 Pathways in Oxidatively Damaged Mouse Zygotes.
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AMPK 活性通过氧化损伤小鼠受精卵中的 p53/p21 途径促进 G2 停滞和 DNA 损伤减少
DOI：
10.3389/fcell.2020.539485
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发表时间：
2020
影响因子：
5.5
通讯作者：
Peng S
中科院分区：
文献类型：
--
作者：
He P;Li Z;Xu F;Ru G;Huang Y;Lin E;Peng S
在受精卵中，G2/M检查点和DNA修复机制对DNA损伤的反应能力取决于不同的外部压力。在我们以前的研究中，我们发现轻度氧化应激诱导体外受精的小鼠受精卵G2/M期延迟，这是由于纺锤体组装检查点的激活。然而，目前尚不清楚G2/M期延迟是否涉及G2阻滞，由G2/M检查点激活触发，以及AMPK，一种高度保守的细胞能量传感器，是否参与小鼠受精卵的G2阻滞和DNA损伤修复。在这里，我们发现，用0.03 mM H2 O2处理的小鼠受精卵在授精后7小时（G1期），进入G2停滞在第一次卵裂。此外，磷酸化的H2 AX，一个特定的DNA损伤和修复标记，可以检测到自早期S期。我们还观察到氧化应激诱导AMPK的磷酸化和活化。氧化应激激活的AMPK在G1期晚期首先定位于小鼠受精卵的细胞质中，然后从S期早期移位到细胞核中。总体而言，大多数激活的AMPK积累在小鼠受精卵的细胞核中，阻滞在G2期。用化合物C和SBI-0206965抑制AMPK活性消除了氧化应激诱导的G2停滞，增加了CDK 1的活性，并降低了细胞周期调节蛋白p53和p21的诱导。AMPK抑制后绕过G2期阻滞加重了氧化应激诱导的M期DNA损伤，增加了囊胚的凋亡率，降低了4-细胞胚胎和囊胚的形成率。我们的研究结果表明，G2/M检查点和DNA修复机制在应对轻度氧化应激诱导的DNA损伤中是有效的。此外，AMPK激活通过p53/p21途径抑制CDK 1活性，在氧化应激诱导的G2期阻滞的调节中起着至关重要的作用，从而促进DNA损伤的修复以及氧化应激损伤胚胎的发育和存活。我们的研究提供了深入了解氧化应激诱导胚胎发育停滞的分子机制，这对于开发新的策略以确保可行的胚胎生成至关重要。
In zygotes, the capacity of G2/M checkpoint and DNA repair mechanisms to respond to DNA damage varies depending on different external stressors. In our previous studies, we found that mild oxidative stress induced a G2/M phase delay in mouse zygotes fertilized in vitro, due to the activation of the spindle assembly checkpoint. However, it is unclear whether the G2/M phase delay involves G2 arrest, triggered by activation of the G2/M checkpoint, and whether AMPK, a highly conserved cellular energy sensor, is involved in G2 arrest and DNA damage repair in mouse zygotes. Here, we found that mouse zygotes treated with 0.03 mM H2O2 at 7 h post-insemination (G1 phase), went into G2 arrest in the first cleavage. Furthermore, phosphorylated H2AX, a specific DNA damage and repair marker, can be detected since the early S phase. We also observed that oxidative stress induced phosphorylation and activation of AMPK. Oxidative stress-activated AMPK first localized in the cytoplasm of the mouse zygotes in the late G1 phase and then translocated to the nucleus from the early S phase. Overall, most of the activated AMPK accumulated in the nuclei of mouse zygotes arrested in the G2 phase. Inhibition of AMPK activity with Compound C and SBI-0206965 abolished oxidative stress-induced G2 arrest, increased the activity of CDK1, and decreased the induction of cell cycle regulatory proteins p53 and p21. Moreover, bypassing G2 arrest after AMPK inhibition aggravated oxidative stress-induced DNA damage at M phase, increased the apoptotic rate of blastocysts, and reduced the formation rate of 4-cell embryos and blastocysts. Our results suggest the G2/M checkpoint and DNA repair mechanisms are operative in coping with mild oxidative stress-induced DNA damage. Further, AMPK activation plays a vital role in the regulation of the oxidative stress-induced G2 arrest through the inhibition of CDK1 activity via p53/p21 pathways, thereby facilitating the repair of DNA damage and the development and survival of oxidative stress-damaged embryos. Our study provides insights into the molecular mechanisms underlying oxidative-stress induced embryonic developmental arrest, which is crucial for the development of novel strategies to ensure viable embryo generation.