唾液酸酶（NEU1/NEU3）影响精子获能机理及其在男性不育病因中的研究

Like all mammlian cells, sperm are covered with glycocalyx, and most of these glycoconjugates carry terminal sialic acids. Sialic acids (Sias) mediate many biological functions, including molecular recognition during development, immune response, and fertilization. A Sia-rich glycocalyx coats the surface of sperm, allowing them to survive as allogeneic cells in the female reproductive tract despite female immunity reaction. As our recent reported the sheded Sia monosaccharides released from capacitated sperm and measured sialidase activity during mouese and human sperm capacitation in vitro, and we showed the presence of two sialidases (neuraminidases NEU1 and NEU3) on mice and human sperm. Inhibiting sialidase activity interferes could lead to inhibit the binding of sperm with the zona pellucida of the ovum. A pilot study of human sperm samples for the presence of sialidases NEU1 and NEU3 identified a lack of one or both sialidases in sperm of some male idiopathic subfertility cases. The results might contribute new insights into the dynamic remodeling of the sperm glycocalyx prior to fertilization. Here, we will test the hypothese that " the activity of NEU1/NEU3 on sperm play some role in capacitaion during sperm fertilzation and investigate the potential relevance between NEU1/NEU3 activity and human idiopathic male subfertility. This project will test how NEU1/3 release from sperm, study the role of NEU1/3 for the fate of sperm during fertilzation and explore the relevance between sialidase and human subfertility.Based on our study, hope it might benefit to learn more about fertilization by focusing on sialic acid and pathogenesis of male sterility.

精子获能是自然受精的必要关键事件，糖基化修饰（包括添加与脱落）在精子获能的现象及机理尚未明了。唾液酸是存在于精子膜表面丰富的糖基化修饰且呈动态变化。我们最近的发现证实：小鼠及人的体外获能过程中，精子膜上唾液酸酶（NEU1/NEU3）的释放参与了剪切精子表面的唾液酸复合物（-脂、蛋白质）导致单个唾液酸分子的脱落；同时，唾液酸酶活性影响精子的获能；在部分男性不育患者，精子唾液酸酶的表达缺失或减少。基于此，我们提出实验假说：唾液酸酶的表达异常与活性通过影响人精子获能的能力而使男性生育能力降低或导致不育。本课题拟以精子质膜流动性，顶体膜激活诱导的钙离子内流及蛋白激酶A介导的精子获能机理为主线，探讨唾液酸酶活性影响精子获能的分子机理；另一方面,明确唾液酸酶表达与活性是否是男性不育的可能病因。旨在为精子获能机理的阐释和男性不育因素的探讨展示新的研究视角，丰富糖分子参与的受精过程的生理及疾病的机理理解。

背景：精子获能是精卵识别、成功受精的前提，精子表面蛋白的糖基丰富，但糖蛋白的糖基化修饰在精子获能中的生物学效应及机理尚未明了。唾液酸是存在于精子膜表面丰富的糖基化修饰且呈动态变化。我们发现：与正常育龄期男性相比，部分男性不育患者的精子唾液酸酶（NEU1/NEU3）的表达水平及定位存在明显差异。.主要研究内容：NEU1/NEU3在正常和不明原因性男性不育患者精子中的表达和定位差异；NEU1/NEU3与精子活性、精液检查数据的相关性；NEU1/NEU3在精子获能中的功能效应；NEU1/NEU3影响精子获能的分子机理。.重要结果：免疫荧光染色显示NEU1/NEU3在正常和不明原因性男性不育患者精子中的定位存在明显差异；蛋白印迹、流式细胞术和ELISA检测结果显示，NEU1/NEU3在正常男性精子中的表达水平和酶活性显著高于不明原因性男性不育患者精子；精子蛋白磷酸化水平检测、钙离子内流效率等实验显示，NEU1/NEU3可明显促进精子获能；免疫共沉淀和免疫荧光双染共定位实验显示，在精子中NEU1和IRβ相互结合，NEU3和脂筏蛋白CAV1相互结合，并引起下游信号通路的改变。本项目研究成果已启动临床转化并获得相应资助。.科学意义：本项目研究成果启动临床转化，为临床上不明原因性男性不育提供新的诊断参考指标。项目实验数据进一步揭示了精子获能的效应机理和影响因素，促进了对精子获能的认识，为后续相关研究提供新的思路。