基于UCP2调节线粒体功能和ROS损伤探索精子活力低下的可能机制

Oxidative damage caused by motichondrial dysfunction has been demonstrated as a leading cause of asthenospermia. Generally, mature sperm lacks an endogenous antioxidant system. Mitochondrial uncoupling protein 2 (UCP2) plays a key role in regulating mitochondrial function and ROS production in numerous tissues. Our previous studies in mice and macaque sperm also suggested UCP2 levels are correlated positively with sperm motility. Based on the previous conclusions, a hypothesis has been proposed that UCP2 may play an important protective role against oxidative stress damage to human sperm, and oxidative damage caused by abnormal expression of UCP2 in sperm may be one of the main pathogenesis of asthenospermia. We are planning to investigate the hypothesis via the following three aspects. ① The differences of UCP2 expression in high and low motility sperm would be compared. And the correlation between UCP2 levels and sperm motility related factors will also be analysed. ② An model of UCP2 regulation will be established to explore whether ROS levels, mitochondrial functions and sperm motility would be changed correspondingly with UCP2 regulated by a specific inhibitor or activator. ③Sperms with low/high motility will be exposed firstly with the UCP2 inhibitor or activator, and then be treated with mitoQ, a ROS specific scavenger, or H2O2, or vice versa, in aiming to test whether high motility sperm owns a UCP2-ROS mutual regulating system and determine its roles in regulating sperm motility. This study will help to expand our knowledge about sperm physiology and male infertility, and to explore new therapeutic method for sperm quality improvement.

线粒体功能异常引起的氧化损伤是造成精子活力下降的重要原因，以往一般认为成熟精子缺乏有效的内源性抗氧化体系。课题组前期发现，RNAi沉默小鼠睾丸UCP2表达后，精子活力下降；同时，猕猴低活力精子UCP2基因/蛋白表达又“恰巧”低于高活力精子。由此提出假说：UCP2是精子自身内源性抗氧化调节的关键；UCP2异常是导致精子活力下降的重要原因。拟从三方面验证：①分析UCP2表达与精子活力低下相关因素的关联性；②分析UCP2功能阻断/激活对精子ROS损伤、线粒体功能和运动的影响，以验证“UCP2的确可引起ROS水平和精子活力的变化”；③在上述干预基础的上引入ROS干预：正常或低活力精子依次调节UCP2，再调节ROS水平，或反之；分析干预过程中精子运动参数的变化，以验证UCP2-ROS调节体系的存在，并明确其与精子活力的关系。本研究将有利于拓展对精子生理与男性不育的认识和探索改善精子活力的新措施。

由于弱精子症是男性不育的重要原因，探索精子活力低下的机制是当前生殖医学和男科学研究的焦点之一。线粒体功能异常引起的氧化损伤是造成精子活力下降的重要原因，以往一般认为成熟精子缺乏有效的内源性抗氧化体系。课题组前期发现，RNAi沉默小鼠睾丸UCP2表达后，精子活力下降；同时，猕猴低活力精子UCP2基因/蛋白表达又“恰巧”低于高活力精子。基于前期的研究，我們提出假説：UCP2是精子自身内源性抗氧化调节的关键；UCP2异常是导致精子活力下降的重要原因。对此进行了以下三部分研究：①分析UCP2表达与精子活力低下相关因素的关联性；②UCP2干预对精子线粒体ROS(mROS)水平,线粒体功能以及精子活力的影响；③ UCP2与mROS之间可能存在的调控机制。结果: ①UCP2在成熟精子定位表达于精子颈部即线粒体鞘部，并在成熟精子线粒体部高表达，正常生育男性精子中UCP2表达显著高于弱精子症患者，且与弱精子症患者的精子相比，其mROS水平显著降低，线粒体膜电位及ATP含量显著上升；②UCP2抑制后mROS显著上升， 精子活力显著下降，且具有浓度依赖性;线粒体膜电位和ATP含量没有显著差异。即 UCP2干预对精子mROS水平及精子活力有影响, 且UCP2可能通过调控成熟精子中mROS含量，进而影响精子活力。③上调mROS后(H2O2处理), 精子活力显著下降，mROS显著上升，线粒体膜电位显著下降，UCP2表达显著上升。增加mROS清除后(NAC处理), mROS显著下降，线粒体膜电位及UCP2表达没有显著差异。即精子中存在着UCP2-mROS相互调控系统：精子中mROS水平上升可反馈性增加UCP2的表达，而UCP2可以加速精子中mROS的清除。本项目实施有助于探讨精子运动功能障碍的形成机制及影响因素，从而进一步拓展我们对精子生理和男性不育的认识，也为今后探讨改善弱精子症患者的精子活力的新方法提供了重要实验依据。