氧化应激影响精子组蛋白转换和遗传稳定的分子机制

Histone to protamine transition is the key mechanism of DNA integrity and genetic stability in human sperms.It has been found that male infertility is tightly associated with abnormality in histone to protamine transition. Our previous work suggested that abnormal histone transition was linked with increased oxidative stress, and this linkage is more significant in males with low activity of antioxidant gene NRF2.Biochemical analysis shows that both protamines and enzymes involved in histone epigenetic regulation are rich in thiols, which can be damaged by reacitve oxygen species(ROS). Therefore, it is hypothezied that oxidative stress might affect histone transition and genetic stability via modification of epigenetic enzymes and protamines. In this project, both population based study and NRF2 knockout mice will be used for mechanism investigation. We will analyze the oxidative damage of ROS on protamine and epigenetic enzymes using Biotin-switch and LC-MS-MS methods. In populations, we will analyze the association of antioxidative gene polymorphisms, ROS and histone transition abnormality. In animal models, we will analyze the effects of NRF2 gene,ROS and antioxidant drugs on sperm histone transition and genetic stability. Our study aims to clarify the mechanisms of damage on sperm histone transition by oxidative stress and to investigate the effects of NRF2 activity on histone transition. Therefore, this project will provide important evidence for spermatogenesis and male infertility.

组蛋白到鱼精蛋白的转换是保护精子DNA完整和维持遗传稳定的特有机制，其异常与男性不育的发生密切相关。我们的前期工作发现，活性氧物质ROS的增加与组蛋白转换异常有关联，抗氧化基因NRF2活性低下者异常更加明显。我们认为氧化应激可能通过损伤组蛋白表观修饰酶和鱼精蛋白来影响组蛋白转换，造成精子遗传稳定的失衡。为证实该假说，并阐明氧化应激影响组蛋白转换的机制，本项目拟在生化、小鼠模型和人群三个层次开展研究：通过Biotin标记和LC-MS分析ROS对表观酶类和鱼精蛋白的氧化作用；在NRF2敲除小鼠模型上验证NRF2基因、ROS和抗氧化药物对精子组蛋白转换和遗传稳定的影响；在人群中分析抗氧化基因多态、ROS和组蛋白转换异常的关联性。本项目的实施将阐明氧化应激损伤精子组蛋白转换的分子机制，并探讨NRF2靶向调控在组蛋白转换异常所致的男性不育中的应用，可进一步拓展男性不育的理论基础和临床治疗的实践依据。

本项目采用小鼠模型和人群样本，分析氧化应激损伤精子组蛋白到鱼精蛋白转换的分子机制和对精子表观遗传稳定性的影响。研究发现吸烟等环境ROS可致精子的表观遗传物质包括DNA甲基化和组蛋白结合发生改变。与来源于同一个体的低质量精子相比，高质量精子呈现低甲基化、低组蛋白滞留等表观遗传特征。在人群样本中，检测到吸烟和IkBa基因功能多态协同影响精子质量，同时发现精液ROS与精子组蛋白到鱼精蛋白转换的异常相关。在小鼠模型中，建立了香烟烟雾提取物诱发小鼠弱精症模型的方法，探讨了香烟烟雾中所含外源活性氧物质ROS对小鼠精子活力的影响，并在Nrf2缺陷小鼠模型中证实Nrf2基因缺失和外源ROS导致精子质量降低和受精潜能降低，推测与精子表观遗传稳定性改变相关。