男性附睾精子成熟微环境蛋白深度解析及其临床意义研究

Epididymal sperm maturation critically regulates male fertility. After testicular spermatozoon interacts with epididymal fluid proteins (sperm-milieu), it acquires its ability to move to, bind to, fuse with, and penetrate the egg (fertilizing potential). Now, diminished/arrested maturation of spermatozoa has become one main cause of male infertility. Therefore the researches on sperm maturation proteins are helpful to improve the level of diagnosis and treatment of male reproduction. In our previous study, we preliminarily identified proteins in sperm-milieu and achieved some results. Here, we will apply isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with multidimensional liquid chromatography-tandem mass spectrometry to deeply detect the protein profiling of continuous and progressive sperm-milieu. The aim is to discovery new epididymal proteins related to sperm motility and fertilization. From basic research to clinical application, our goals are: ①In the semen samples of asthenozoospermia, the quantifications of sperm motility proteins on both human ejaculated spermatozoa and seminal plasma, and their rescues for sperm motility ability are all determined to evaluate their clinical values for diagnosis and treatment; ②In the semen samples of infertility patients who were in vitro fertilization (IVF) failure but intracytoplasmic sperm injection (ICSI) success, the quantifications of sperm-egg fertilization proteins on both human ejaculated spermatozoa and seminal plasma are performed to evaluate the clinical guiding significance of therapeutics scheme (IVF or ICSI); ③In the semen samples of donors aged 30-70 years, the quantification of sperm maturation proteins on both human ejaculated spermatozoa and seminal plasma are detected to assess their availability using as the evaluation targets for human sperm quality. Our work will provide the new potential biomarkers and clues for the diagnosis and treatment of male infertility, male contraception and reproductive health, which will have a great significance in theory and clinical application.

附睾精子成熟是男性生育调控的重要环节。精子由睾丸产生后，通过与附睾微环境蛋白相互作用发育成熟而获得受精潜能。目前精子成熟障碍已是引起男性不育的主要原因之一，因此研究精子成熟蛋白有助于男性生育能力的临床诊疗。我们前期工作初步鉴定了精子成熟微环境蛋白并取得部分成果，本研究将应用高通量iTRAQ技术深度解析附睾微环境中连续变化的蛋白质分子构成，着力发现与精子运动、受精相关的新蛋白。从基础研究结合临床应用角度：①通过筛查弱精症精液中的精子运动蛋白并用其改善弱精运动指标评估其临床诊疗价值；②通过监测IVF失败ICSI成功不育症患者精液中的受精相关蛋白评价其用于IVF或ICSI治疗方案选择的临床指导意义；③通过测定不同年龄阶段(30-70岁)精液中的精子成熟蛋白探讨其作为男性精子质量评价分子的可行性。本研究将为男性不育诊疗、节育避孕和生殖健康研究提供了新的靶点和思路，具有重要的理论意义和临床应用价值。

本项目是在研究团队前期近十年工作基础上，深度阐述精子成熟微环境重要蛋白，为精子功能调控、男性不育症分子水平评估及临床诊疗提供坚实数据基础和科学理论依据。. 本项目主要完成以下工作：. ① 精子成熟微环境蛋白表达谱深度解析：采用同位素iTRAQ标记、多维色谱分离与高分辨质谱仪联用策略深度鉴定男性精子成熟微环境蛋白，数据质量与国际生殖数据库具备可比性。. ② 精子成熟微环境区段蛋白动态变化规律揭示：通过附睾不同区段精子成熟微环境蛋白质组学研究，提高了附睾精子成熟微环境蛋白定量精度及低丰度蛋白鉴定水平。. ③ 精子成熟微环境和精子成熟之间潜在关系阐述：综合比对本研究与国际同行精子微环境蛋白质组数据间关联，前瞻性揭示人精子成熟微环境蛋白功能及作用网络。. ④ 从经典精子成熟微环境到广义精子成熟微环境探究：Ⅰ.精子睾丸发生微环境；Ⅱ.精子附睾成熟微环境（经典）；Ⅲ. 精子体外精浆微环境；Ⅳ. 精子卵周卵泡液微环境。. ⑤ 男性弱精等不育症患者精子运动蛋白解析及改善弱精运动临床诊疗价值评估：弱精子症、少弱精子症和死精子症与生殖结局密切相关，通过蛋白质组学技术阐释男性不育症重要精子成熟蛋白，同时为无创方式发现潜在标志物提供可能。. ⑥ IVF失败ICSI补救受孕不育症患者精子成熟蛋白用于指导治疗方案选择：通过IVF和补救ICSI精子蛋白质组间对比发现精子顶体蛋白表达减弱及自发顶体百分率上升，因而提前检测精子蛋白缺陷对治疗方案选择的具有临床指导意义。. ⑦ 精子成熟蛋白评价不同年龄阶段精子质量及改善生育潜能的可行性探讨：PATE可潜在评估不同年龄精子样本质量；PRDX2可提高弱精症患者精子前向运动能力同时可改善冷冻保存精子质量。. ⑧ 精子成熟蛋白CMTM4在男性不育中的功能及临床意义研究：CMTM蛋白在不育症患者、老年人和NOA睾丸活检中表达降低。利用CRISPR-Cas9技术敲除小鼠Cmtm4基因表现为精子畸形和低生育力，蛋白质组学揭示与精子运动和顶体反应有关蛋白下调，CMTM4可能成为精子质量评估、男性不育诊断治疗潜在靶分子。.