Low levels of mouse sperm chromatin fragmentation delay embryo development.
Low levels of mouse sperm chromatin fragmentation delay embryo development.
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DOI：
10.1093/biolre/ioad106
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发表时间：
2023-11-15
影响因子：
3.6
通讯作者：
中科院分区：
文献类型：
--
作者：
我们以前证明，氯化锰诱导精子双链DNA断裂的过程中，我们称之为精子染色质片段化。在这里，我们测试了双链DNA断裂的水平是否与MnCl2的浓度相关，并将其与另一种导致单链DNA断裂的试剂H2O2进行了比较。我们发现，这两种方法都有诱导DNA断裂的浓度依赖性的方式的优势。用不同浓度的H2O2或MnCl2处理小鼠精子，通过脉冲场凝胶电泳、碱性和中性彗星试验评估DNA损伤。卵母细胞被注入处理过的精子，用胚胎镜分析得到的胚胎，以检测胚胎发育的细微变化。我们证实，H2O2处理诱导主要是单链DNA断裂和氯化锰诱导主要是双链DNA断裂，表明不同的损伤机制。这些精子被注射到卵母细胞中，然后用配备有延时记录的胚胎镜观察所产生的胚胎的发育。我们发现，畸变在早期胚胎发育的第2天，即使是最低水平的DNA损伤和胚胎畸变的水平与H2O2或氯化锰的浓度。低水平的H2O2造成显着更多的畸变在胚胎发育中比低水平的氯化锰，即使通过彗星试验测量的DNA损伤水平是相似的。这些数据表明，即使是低水平的精子DNA损伤也会导致胚胎发育的延迟和停滞。用剂量太低而不能引起容易观察到的DNA断裂的DNA损伤剂处理小鼠精子，当用于使未处理的卵子受精时，会引起显著的胚胎发育问题。
We previously demonstrated that MnCl2 induces double-stranded DNA breaks in sperm in a process that we term as sperm chromatin fragmentation. Here, we tested if the levels of double-stranded DNA breaks were corelated to the concentration of MnCl2, and we compared this to another agent that causes single-stranded DNA breaks, H2O2. We found that both methods have the advantage of inducing DNA breaks in a concentration-dependent manner. Mouse sperm were treated with varying concentrations of either H2O2 or MnCl2, and the DNA damage was assessed by pulse-field gel electrophoresis, and the alkaline and neutral comet assays. Oocytes were injected with either treated sperm and the resulting embryos analyzed with an embryoscope to detect subtle changes in embryonic development. We confirmed that H2O2 treatment induced primarily single-stranded DNA breaks and MnCl2 induced primarily double-stranded DNA breaks, indicating different mechanisms of damage. These sperm were injected into oocytes, and the development of the resulting embryos followed with an embryoscope equipped with time lapse recording. We found that aberrations in early embryonic development by day 2 with even the lowest levels of DNA damage and that the levels of embryonic aberrations correlated to the concentration of either H2O2 or MnCl2. Low levels of H2O2 caused significantly more aberrations in embryonic development than low levels of MnCl2 even though the levels of DNA damage as measured by comet assays were similar. These data demonstrate that even low levels of sperm DNA damage cause delays and arrests in embryonic development. Treatment of mouse sperm with doses of DNA damaging agents that are too low to cause easily visualized DNA breaks cause significant embryonic development problems when used to fertilize untreated eggs.
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