RNA酶T2对人精子运动作用机制的研究

Adequate sperm motility is requisite for mammalian fertilization, whereas poor sperm motility (asthenozoospermia) is a cause of male infertility in clinical, occupying almost 20% in total cases. Recent researches indicated that RNASET2 (Ribonuclease T2) was significantly relevant to cell metabolic activities such as cell motility and migration. In addition, our experiments also demonstrated that both human spermatozoa and seminal plasma were rich in RNASET2, and purified RNase T2 had a significant effect on the sperm motility. Thus, here we will investigate the RNASET2 effects on male fertility by the followings: 1)Detecting the corresponding signal pathways and sperm proteins phosphorylation modulated by RNASET2 in order to clarify the molecular mechanism of RNASET2 effects on human sperm motility; 2)Employing the qualitative and quantitative methods to examine the RNASET2 levels in spermatozoa and seminal plasma from both normal fertile male and asthenozoospermia, and then to compare the results with each others from two different groups and try to verify the abnormal RNASET2 levels in spermatozoa or seminal plasma correlated with asthenozoospermia. Meanwhile, developing a relevant test kit in order to improve the diagnosis and treatment for asthenozoospermia in clinical; 3)Establishing transgenic mouse model, whose RNase T2 gene was conditionally overexpressed in its epididymis and then to measure the effects of increasing expressed RNASET2 on the sperm motility and fertility in mouse epididymis, and finally, it will provide the sufficient experimental evidences and reliable theoretical basis for a study of novel male contraceptive drugs regarding the RNASET2 as a targeting molecule.

哺乳类动物受精成功与否的一个重要指标是雄性精子必须具备良好的运动力，而临床众多男性不育患者中弱精子症可占其中20%左右。最近研究表明RNA酶T2 与细胞运动、迁移等代谢活动密切相关；此外，实验也证实人精子和精浆中均含RNA酶T2且纯化的RNA酶T2体外对人精子运动具有明显的抑制效应。因此，本课题将通过1）检测人精子运动中RNA酶T2介导的相关细胞信号传导通路及对精子蛋白磷酸化的影响，首次分析它在精子运动中作用的分子机制；2）对正常或弱精子症患者精子和精浆中RNA酶T2进行定性、定量检测并比较分析，尝试明确该蛋白与弱精子症发病的相关性及研发检测试剂盒以提高临床上因弱精子症所导致男性不育的诊治水平。3）构建附睾RNA酶T2条件性过表达转基因小鼠模型并进行验证，检测RNA酶T2表达增加后对小鼠精子运动及受精功能的影响，为最终以该蛋白作为靶分子的男性新型节育药物的研制提供可靠的理论基础和充分依据。

附睾是精子功能成熟的场所，即在附睾环境中，精子的膜脂成分、蛋白组成和蛋白修饰发生改变，从而使精子获得运动和受精的能力。精子附睾成熟异常与弱精子症发生以及男性不育密切相关。在过去研究中，我们首次发现人精子和精浆中含有RNA酶T2（RNASET2），并且其与弱精子症存在一定的相关性，而精液中的RNA酶T2主要来源于附睾头部组织分泌。因此，我们推测RNASET2可能参与精子成熟过程，从而影响精子运动能力。为了进一步明确RNASET2与弱精子症发生的相关性及其影响精子运动的分子机制，本课题通过临床精液样本收集(205例)、免疫荧光染色及流式细胞分析精子RNASET2含量、RNA酶T2蛋白分离纯化、体外精子运动抑制实验、信号分子检测，以及条件性基因敲入小鼠构建，全面探索了RNASET2在精子运动中的功能和作用机制。研究结果显示：（1）在男性精子中，RNA酶T2含量与精子运动能力呈负相关，即在弱精子症患者精子中RNA酶T2含量显著上升；（2）RNA酶T2蛋白可以在体外有效抑制精子运动，并且该蛋白可能通过与精子中AKAP4蛋白相互作用，抑制精子中PKA-PI3K-Ca2+信号传导通路；（3）我们构建了RNase T2条件基因敲入（Conditional knock-in）小鼠，即Rosa26-RNASET2；Lcn5 cre小鼠，该小鼠可在附睾头部过量表达RNASET2蛋白，表现为雄性不育，并且具有弱精子症和畸形精子症的特征。通过以上研究，我们进一步了解人精子或精浆中RNA酶T2的生物学功能，即附睾分泌的RNASET2可能参与精子成熟过程；异常高表达的RNASET2可能过度抑制精子运动能力，从而造成弱精子症。我们的研究结果将为探索精子成熟及精子运动不良或弱精子症发生的分子机制提供了重要的实验依据。