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首页> 外文期刊>Molecular human reproduction. >Double-stranded DNA breaks hidden in the neutral Comet assay suggest a role of the sperm nuclear matrix in DNA integrity maintenance
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Double-stranded DNA breaks hidden in the neutral Comet assay suggest a role of the sperm nuclear matrix in DNA integrity maintenance

机译：隐藏在中性彗星实验中的双链DNA断裂表明精子核基质在DNA完整性维持中的作用

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We used a mouse model in which sperm DNA damage was induced to understand the relationship of double-stranded DNA (dsDNA) breaks to sperm chromatin structure and to the Comet assay. Sperm chromatin fragmentation (SCF) produces dsDNA breaks located on the matrix attachment regions, between protamine toroids. In this model, epididymal sperm induced to undergo SCF can religate dsDNA breaks while vas deferens sperm cannot. Here, we demonstrated that the conventional neutral Comet assay underestimates the epididymal SCF breaks because the broken DNA ends remain attached to the nuclear matrix, causing the DNA to remain associated with the dispersion halo, and the Comet tails to be weak. Therefore, we term these hidden dsDNA breaks. When the Comet assay was modified to include an additional incubation with sodium dodecyl sulfate (SDS) and dithiothreitol (DTT) after the conventional lysis, thereby solubilizing the nuclear matrix, the broken DNA was released from the matrix, which resulted in a reduction of the sperm head halo and an increase in the Comet tail length, exposing the hidden dsDNA breaks. Conversely, SCF-induced vas deferens sperm had small halos and long tails with the conventional neutral Comet assay, suggesting that the broken DNA ends were not tethered to the nuclear matrix. These results suggest that the attachment to the nuclear matrix is crucial for the religation of SCF-induced DNA breaks in sperm. Our data suggest that the neutral Comet assay identifies only dsDNA breaks that are released from the nuclear matrix and that the addition of an SDS treatment can reveal these hidden dsDNA breaks.
机译：我们使用了其中诱导了精子DNA损伤的小鼠模型，以了解双链DNA（dsDNA）断裂与精子染色质结构和Comet分析之间的关系。精子染色质片段化（SCF）在鱼精蛋白环面之间的基质附着区域产生dsDNA断裂。在此模型中，被诱导经历SCF的附睾精子可以重新连接dsDNA断裂，而输精管精子则不能。在这里，我们证明了常规中性彗星测定法低估了附睾SCF断裂，因为断裂的DNA末端仍然附着在核基质上，导致DNA保持与分散晕相关，而彗星尾巴却很弱。因此，我们称这些隐藏的dsDNA断裂。在常规裂解后，如果将Comet分析修改为包括用十二烷基硫酸钠（SDS）和二硫苏糖醇（DTT）额外孵育，从而溶解核基质，则破碎的DNA从基质中释放出来，从而降低了精子头晕和彗星尾巴长度的增加，暴露出隐藏的dsDNA断裂。相反，SCF诱导的输精管精子具有小的晕圈和长尾巴，这是通过常规的中性彗星实验确定的，这表明断裂的DNA末端没有束缚在核基质上。这些结果表明，对核基质的附着对于精子中SCF诱导的DNA断裂的重新连接至关重要。我们的数据表明，中性彗星试验只能识别从核基质中释放的dsDNA断裂，并且添加SDS处理可以揭示这些隐藏的dsDNA断裂。

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