线粒体膜蛋白prohibitin调控人精子运动的机制研究

Recent findings from our laboratory suggest that high levels of reactive oxygen species (ROS) in human semen may contribute to male infertility and that sperm motility could be the earliest and most sensitive indicator of oxidative damage. We have also shown that prohibitin expression is an indicator of sperm quality in infertile men: it correlates significantly with mitochondrial membrane potential and sperm motility in normospermic, asthenospermic and oligoasthenospermic subjects. Thus we hypothesize that prohibitin may regulate sperm motility via an alteration in mitochondrial membrane potential and increased levels of ROS which is generated in the mitochondria. Therefore, the first objective of this study is to investigate prohibitin expression and sperm motility in relation to the following sperm mitochondrial functions: ROS production, membrane depolarization, respiratoring mitochondria and lipid peroxidation using fluorescence probes and flow cytometry, as well as mitochondrial respiratory chain complex I and III activity using spectrophotometry. The second objective is to determine if the PI3K/AKT signaling pathway is involved in the regulation of mitochondrial function and sperm motility and how it relates to prohibitin expression. We will investigate if there are differences in PI3K and AKT localizations, levels of their phosphorylation and expression in sperm from infertile men (using immunofluorescence staining, phosphoproteomics, and Western blots) in relation to prohibitin expression. The impact of PI3K on AKT phosphorylation, sperm mitochondrial function, sperm motility, and prohibitin expression will further be investigated by exposure of sperm from infertile men to PI3K inhibitors. Finally, to confirm the above findings, the prohibitin knockdown mouse model will be generated using microinjection of lentivirus-coated Phb shRNA to seminiferous tubules of testes. After successfully confirming the efficiency of prohibitin knockdown using RT-PCR, Western blots and immunofluorescence staining in sperm from infected mice, the levels of PI3K and AKT expression and phosphorylation, sperm mitochondrial function and sperm motility will be analyzed. Taken together, this study promises to provide new mechanistic insights on sperm motility regulation mediated by prohibitin and PI3K/AKT signaling pathway, and also new diagnostic targets in the clinical treatment of male infertility.

申请人前期研究表明，人精子运动能力下降是精液活性氧增高所致；弱精症或少弱精症精子运动和线粒体膜电位下降与线粒体膜蛋白prohibitin表达下降密切相关，提示prohibitin调控人精子运动能力。因此本项目将进一步以荧光探针和流式细胞术检测线粒体活性氧水平、膜去极化、呼吸状态和脂质过氧化程度，分光光度法检测呼吸链复合体I和III活性，比较精子线粒体功能变化及与精子运动和prohibitin表达的相关性。免疫荧光、磷酸化蛋白质质谱术和蛋白免疫印迹比较精子PI3K和AKT表达和磷酸化差异及与prohibitin表达相关性；添加PI3K抑制剂验证AKT磷酸化、线粒体功能和精子运动变化。Phb shRNA慢病毒感染小鼠睾丸验证精子PI3K和AKT表达及磷酸化、线粒体功能和精子运动的变化，以明确prohibitin通过PI3K/AKT调控人精子运动的机制，为男性不育诊治提供新的分子靶标和理论依据。

本课题组前期研究结果表明，人精子运动能力对活性氧(ROS)增高导致的氧化应激最易感；弱精症或少弱精症患者精子运动能力和线粒体膜电位的下降与其线粒体膜蛋白prohibitin(PHB)表达下降密切相关。我们推测精子PHB可能通过影响线粒体功能包括ROS产生而调控精子运动能力。因此，本项目进一步检测精子的线粒体功能，包括线粒体ROS和膜电位水平，脂质过氧化水平，以及呼吸链复合体I(MCI)酶活性，比较精子的线粒体功能与其PHB表达和运动能力的相关性；其次，检测分析精子PI3K和AKT表达及其磷酸化位点和水平，以及与精子PHB表达、线粒体功能和运动能力的相关性。在完成上述研究计划并取得预期成果的基础上，进一步检测分析：①精子MCI结构亚单位组装因子NDUFAF1表达，以明确MCI结构完整性；②精子PTEN表达及其磷酸化位点和水平，以明确其对PI3K/AKT信号通路的调控；③精子PHB蛋白磷酸化位点和水平，以及与AKT蛋白及其磷酸化位点和水平的定位关系，以明确磷酸化的精子PHB和AKT蛋白的相互关系。研究结果表明，与正常精子相比，弱精症或少弱精症患者精子的线粒体功能出现明显异常，主要表现在线粒体ROS增高与膜电位水平降低，脂质过氧化水平增高，以及MCI电子传递功能下降。究其原因，可能与精子PHB和MCI结构亚单位组装因子NDUFAF1的表达下降有关，从而影响精子MCI结构和功能的完整性，导致线粒体ROS增高，精子运动能力下降。其次，PI3K/AKT/PTEN信号通路在人精子表达，其磷酸化水平与精子PHB表达、线粒体功能和运动能力密切相关；AKT磷酸化蛋白(pAKT S473)与PHB蛋白在精子线粒体共定位表达，而且PHB磷酸化蛋白(pPHB T258)在精子线粒体表达。提示精子PHB蛋白的磷酸化水平与AKT蛋白的磷酸化有关。同时AKT可能通过调控精子PHB蛋白的磷酸化水平，影响精子MCI结构和功能的完整性，最终调节线粒体ROS水平，从而确保精子的线粒体功能和运动能力。该研究成果为深入理解PI3K/AKT/PTEN信号通路与PHB相互作用而影响精子的线粒体功能和运动能力，提供了新的理论基础。同时基于Crispr-Cas9技术成功构建的phb/floxp小鼠，也为后续深入研究PHB表达及其磷酸化机制创造了有利条件。