叶酸缺乏引起的Rad54启动子甲基化异常导致精子DNA损伤的研究

Human sperm DNA damage is a common cause of male infertility. A variety of environmental stress can lead to sperm DNA damage, but the mechanism of sperm damage is not clear. Researchers pay attention to the epigenetic effects. As a methyl group donor, Folic acid regulates the process of DNA methylation. In the early we confirmed that folic acid concentration was associated with sperm DNA damage in 198 infertile males, and the whole genome methylation sequencing of the patients of low folic acid was performed. We found that the methylation of the Rad54 promoter region was significantly increased and the result was proved in mice model. Rad54 plays an important role in DNA double bond cleavage (DSB) repair pathway. Therefore, we hypothesize that male folic acid deficiency enhances the methylation of the spermatogenic cells and inhibits the gene expression, then increases the sperm DNA damage by blocking the DNA double bond cleavage pathway. In order to prove this, firstly we will detect the effect of different folic acid uptake on DNA methylation in mice. The effect of Rad54 on DSB repair pathway will be studied in spermatocytes. Finally, the mechanism of gene function and protection will be studied in GC-2 cell model. The completion of this study not only broaden the knowledge of epigenetic regulation of sperm DNA damage, but also give clinical reference to improve the male fertility.

人精子DNA损伤是引起男性不育的常见原因。多种环境压力都可导致精子DNA损伤增加，但损伤机制并不清楚，其中表观遗传作用引人关注。叶酸作为甲基基团供体，影响DNA甲基化过程。前期我们在198名不育男性中证实叶酸浓度与精子DNA损伤程度相关，进一步对低叶酸者进行全基因组甲基化测序，筛查到Rad54启动子区甲基化显著升高，并在小鼠模型中验证，而Rad54在DNA双键断裂(DSB)修复通路发挥重要作用。因此我们提出假设：男性叶酸缺乏会增强生精细胞Rad54启动子甲基化而抑制其基因表达，通过阻碍DNA双键断裂修复通路而造成精子DNA损伤增加。为证明此，本课题拟在小鼠中检测不同叶酸摄入对DNA甲基化影响，再分离精母细胞研究Rad54对DSB修复通路影响，并通过构建GC-2细胞模型进行基因功能和保护机制的研究。本研究的完成，不仅拓宽表观遗传调控精子DNA损伤机制的认识，还对临床提高男性生育力有借鉴意义。

【背景】 精子DNA损伤是男性不育的常见原因之一，多种原因可导致精子DNA损伤。体内叶酸缺乏可能影响精子DNA甲基化，研究其导致精子DNA损伤增加的机制。.【研究内容】 本研究C57雌性小鼠随机分成3组，分别用低叶酸（0.3 mg/kg）的饲料、正常饲料（2 mg/kg）和高叶酸（20 mg/kg）的饲料喂养后与正常饲料喂养的雄性小鼠合笼。F1代8周时分别检测小鼠血清叶酸浓度，确定模型是否构建成功；分别检测附睾精液参数，用精子染色体结构分析（SCSA）法检测精子DNA碎片化指数（DFI）；提取精子DNA并检测Rad54基因启动子区甲基化水平；分别检测各组小鼠睾丸中Rad54蛋白和γ-H2AX蛋白的表达；F1代雄鼠与正常雌鼠交配，统计F1代生育后代情况。细胞实验分别用无叶酸培养基、叶酸浓度分别为4 ng/ml、20 ng/ml、200 ng/ml的培养基构建GC-2细胞模型，测序法检测各组Rad54基因启动子区甲基化水平；检测Rad54和γ-H2AX的表达水平；并用5 mmol/L的H2O2处理后检测各组对外界损伤刺激的敏感性。.【结果】 检测8周龄F1代雄鼠血清叶酸浓度，低叶酸组明显低于正常组和叶酸补充组，低叶酸组小鼠精子浓度、精子活力低于正常组和叶酸补充组，免疫荧光和WB结果可以看出低叶酸组Rad54表达低于正常组和叶酸补充组，低叶酸组γ-H2AX的表达高于正常组和叶酸补充组，低叶酸组精子DFI明显高于正常组和叶酸补充组，低叶酸组Rad54基因启动子区甲基化水平高于正常组和叶酸补充组。细胞实验中Rad54的表达在无叶酸组明显低于4 ng/ml、20 ng/ml、200 ng/ml叶酸组，DNA双键断裂标志性蛋白γ-H2AX在无叶酸组明显高于4 ng/ml、20 ng/ml、200 ng/ml叶酸组，Rad54基因启动子区甲基化水平在无叶酸组明显增高。在受到H2O2处理后，γ-H2A荧光焦点数和Rad51的表达在低叶酸中显著增高。.【意义】 叶酸缺乏可以使DNA双建断裂修复（DSB）通路相关基因Rad54启动子区甲基化水平增加，使Rad54蛋白的表达降低，使DSB修复通路受到影响，在精子发生的过程中更易受到外界损伤刺激的影响，使成熟精子DFI增加。