Protamine 2 deficiency leads to sperm DNA damage and embryo death in mice
Protamine 2 deficiency leads to sperm DNA damage and embryo death in mice
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DOI：
10.1095/biolreprod.102.015115
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发表时间：
2003-07-01
影响因子：
3.6
通讯作者：
Eddy, EM
中科院分区：
文献类型：
--
作者：
Cho, C;Jung-Ha, H;Eddy, EM
在生精细胞中胞质分裂是不完全的，每个干细胞的后代形成克隆合胞体。因此，在减数分裂后表达的基因中的杂合突变影响合胞体内的所有单倍体精子细胞。先前，我们已经发现，在小鼠中破坏鱼精蛋白I（PRM1）或鱼精蛋白2（PRM2）基因的一个拷贝导致相应蛋白质的量减少，PRM2的异常加工，以及雄性嵌合体不能将来自129基因型胚胎干细胞的突变或野生型等位基因传递给下一代。尽管人们认为鱼精蛋白对于精子核的致密化和保护DNA免受损伤至关重要，但这尚未得到实验证明。为了验证嵌合体未能将129基因型传递给后代是由于精子DNA的组织和完整性发生了改变这一假设，我们使用了单细胞DNA电泳（彗星）试验、超微结构分析和胞浆内精子注射（ICSI）程序。彗星试验证明，PRM 2单倍不足的精子比例与DNA受损的精子频率之间存在直接相关性。超微结构分析显示染色质致密化减少。PRM2缺陷精子的ICSI导致大多数中期II停滞的小鼠卵子激活，但很少有能够发育到囊胚阶段。这些发现表明，发育失败是因为父亲DNA受损，而PRM2对维持精子染色质的完整性至关重要。
Cytokinesis is incomplete in spermatogenic cells, and the descendants of each stem cell form a clonal syncytium. As a result, a heterozygous mutation in a gene expressed postmeiotically affects all of the haploid spermatids within a syncytium. Previously, we have found that disruption of one copy of the gene for either protamine I (PRM1) or protamine 2 (PRM2) in the mouse results in a reduction in the amount of the respective protein, abnormal processing of PRM2, and inability of male chimeras to transmit either the mutant or wild-type allele derived from the 129-genotype embryonic stem cells to the next generation. Although it is believed that protamines are essential for compaction of the sperm nucleus and to protect the DNA from damage, this has not been proven experimentally. To test the hypothesis that failure of chimeras to transmit the 129 genotype to offspring was due to alterations in the organization and integrity of sperm DNA, we used the single-cell DNA electrophoresis (comet) assay, ultrastructural analysis, and the intracytoplasmic sperm injection (ICSI) procedure. Comet assay demonstrated a direct correlation between the fraction of sperm with haploinsufficiency of PRM2 and the frequency of sperm with damaged DNA. Ultrastructural analysis revealed reduced compaction of the chromatin. ICSI with PRM2-deficient sperm resulted in activation of most metaphase II-arrested mouse eggs, but few were able to develop to the blastocyst stage. These findings suggest that development fails because of damage to paternal DNA and that PRM2 is crucial for maintaining the integrity of sperm chromatin.