CD147调节的CatSper非依赖性钙离子内流对人精子功能的作用及机制研究

Calcium signaling plays a central role in regulating various sperm functions that are essential for successful fertilization. Although CatSper was demonstrated as a main channel in mediating calcium mobilization and sperm functions, the involvement of CatSper-independent calcium influx in regulating sperm functions remains largely unknown. Our early studies have shown that the expression of CD147 in asthenozoospermic sperms was significantly lower than that of in normal sperms. Furthermore, rCD147 treatment significantly induced the acrosome reaction (AR). More importantly, the CD147-induced calcium influx in sperm is CatSper-independent. These observations prompt us to hypothesize that CD147 regulates sperm functions via inducing CatSper-independent calcium influx. To examine this hypothesis, we propose to i) demonstrate the role of CD147 in human sperm motility, capacitation and fertilization ability through rCD147 mediating CatSper-independent calcium influx. ii) determine the CD147 interacted protein by co-immunoprecipitation and Mass spectrum analysis and further confirm their interaction and investigate how CD147 induces CatSper-independent calcium mobilization in sperm. iii) explore the applying possibility of CD147 in vitro fertilization (IVF) or the treatment of asthenozoospermia and other patients such as CatSper mutant sperm. These results will shed lights on the treatment of CatSper mutated patients and asthenozoospermia patients.

钙离子动员在精子功能乃至成功受孕中具有关键作用。虽然CatSper依赖的钙离子动员被认为是调节精子运动的主要钙信号，但CatSper非依赖的钙离子调节对精子的功能也有重要作用。我们前期的研究发现，CD147作为跨膜糖蛋白，在弱精症患者精子的表达显著低于正常精子并与精子活力呈正相关。使用重组人CD147（rCD147）处理精子后，可诱发人精子顶体反应；提高弱精症患者精子的活力。此外，rCD147可诱发精子CatSper非依赖的钙离子内流。因此，我们推测CD147通过CatSper非依赖的钙离子内流调控人精子的功能。本课题拟研究CD147介导的CatSper非依赖钙离子内流对人精子活力、获能、顶体反应和受精能力的作用；确定精子中CD147的结合蛋白及其调节CatSper非依赖钙离子内流的分子机制。通过该研究，为CD147对CatSper突变患者和弱精症患者的治疗及其在IVF中的应用提供线索。

精子在女性生殖道游动，经历获能、超活化以及顶体反应等过程与卵子相遇并受精，在这些过程均需钙离子的参与和调节。CatSper 通道家族是精子特异的钙离子通道，但是CatSper非依赖的钙离子动员在精子功能中的作用及机制仍有待阐明。本项目在前期工作的基础上，发现CD147定位于人精子的头部和颈部；获能后，部分CD147从精子的颈部转移到精子的头部，表明CD147在精子获能前后可能发挥着不同的作用。在弱精症患者的精子中，CD147的表达显著低于正常男性并与精子活力呈正相关，而用重组CD147（rCD147）蛋白处理精子可以显著提高弱精症患者的精子活力以及诱导顶体反应，同时rCD147治疗显著提高弱精症患者精子与透明质酸结合，表明CD147可以改善弱精症患者精子的受精能力。此外，我们发现胞外型CD147与精子膜表面CD147存在相互作用并介导钙离子内流。有趣的是，用CatSper 抑制剂NNC55-0396(NNC)预处理人精子后无法抑制rCD147诱导的钙离子内流，rCD147仍然可以诱导CatSper1敲除小鼠精子胞内钙离子的升高，表明CD147引发的钙离子动员并不依赖CatSper的功能。综上所述，我们的研究初步揭示了CD147调节的CatSper非依赖性钙离子内流在人精子功能的作用及机制。本项目为男性弱精症的治疗提供了潜在的生物学靶点，也为CD147在IVF中的应用奠定良好的理论基础。