氟对小鼠精子ATP生成途径的影响及分子损伤机理研究

The prevalence and hazardness of fluorosis have got more attention than ever before. Numerous documents constantly demonstrate the reproductive toxicity of fluoride. A significant reduction in sperm mobility and hyperactivation in sperm of animals exposed to fluoride was observed. Notably, high-level sperm mobility is essential to successful fertilization. Our previous study, by applying gene expression profiling analysis, found that fluoride significantly affacted expressions of a group of genes related to ATP-generating in sperm. Therefore, the present study is designed mainly focusing on ATP metabolism in sperm of mice treated with different doses of fluoride for four spermatogenic cycles. After the observation of sperm ultrastructure through transmission electron microscope, glycolysis and mitochondrial respiration models are established to analyze the ATP-generating pathway applying micro-video recorder and chemiluminescence assay, followed by the assessment of sperm mitochondrial respiration using oxygen electrode method. Gene mutation, mRNA expression and protein level of key molecules involved in sperm ATP metabolism are also detected by PCR-SSCP and PCR-DS, Realtime PCR and Western blotting, respectively. The findings of this study will supply the direct evidence for fluoride-induced low sperm motility and further clarity the molecular mechanism underlying sperm lesion resulting from fluoride, thereby providing scientific basis for clinical diagnosis and target drug design.

氟中毒的广泛性与危害性越来越引起人们的密切关注，氟的生殖毒性也得到了肯定。大量实验证实氟可导致动物精子活力和超激活运动显著降低，而高水平的精子活动力对于正常受精至关重要。本课题组前期对氟中毒小鼠精子进行了全基因组表达谱芯片分析，结果发现诸多与ATP生成相关的基因发生改变。因此，本项目着重对精子动力来源-ATP展开研究，通过建立不同剂量氟暴露动物模型，及精子无氧酵解和有氧呼吸模型，应用电镜技术、显微录像与化学发光法、线粒体呼吸功能测定仪、PCR-SSCP与PCR-DS、荧光定量PCR和蛋白免疫印迹技术等，分别检测精子超微结构、ATP生成途径、精子线粒体呼吸状态，以及ATP代谢关键分子在DNA、RNA和蛋白质水平上的变化，综合分析氟对精子ATP的影响及分子损伤机理，以探求氟致精子活动力降低的动力依据，进一步阐明氟的精子毒性机制，从而为实施科学诊断和靶标药物设计提供依据。

氟中毒的广泛性与危害性越来越引起人们的密切关注，氟的生殖毒性也得到了肯定。研究证实氟中毒可致精子质量下降，尤其值得关注的是精子的活力、超激活运动受到严重影响。 而精子运动能力对于正常的受精至关重要。因此，本项目从精子运动的动力基础入手，通过氟中毒小鼠精子无氧酵解和有氧呼吸条件下ATP分析及相关分子检测，发现氟中毒精子总ATP和有氧呼吸模型中ATP含量显著降低，且相关分子COX2和MTCYB mRNA表达量显著下降，MTCYB基因发生点突变，透射电镜观察到氟组精子线粒体形状和大小不规则，部分线粒体发生肿胀，且嵴间产生空泡，提示了氟主要通过降低和影响线粒体有氧呼吸发挥毒性作用，从而找到了氟致精子活动力降低的动力依据，为进一步阐明氟的精子毒性机制提供了理论基础。本项目研究成果在SCI源期刊上发表论文7篇，中文核心期刊论文1篇，申请专利1项。其中《Chemosphere》2篇，《Environ Toxicol Phar》1篇，《Biol Trace Elem Res》3篇, 《Arch Toxicol》1篇。在此基础上，借助深度测序和生物信息学分析研究了氟中毒小鼠精子miRNA表达的变化，发现低氟与高氟同时导致31个已知miRNA发生改变，预测靶基因进行GO和KEGG分析之后提示涉及蛋白酶抑制、免疫应答、质膜完整性、MAPK信号通路、泛素介导的蛋白酶水解、TGF-beta通路、Wnt通路、p53通路、细胞凋亡，以及钙信号通路等，为进一步研究氟的精子毒性开拓了新的思路。