褪黑素关联的绵羊附睾头上皮细胞调控精子成熟机制

•The recent researches show that melatonin exist in human seminal plasma and also the melatonin receptor expressing in the epididymis and sperm in Rats, mice, hamsters, guinea pigs, and human. These suggest that sperm maturation associated with melatonin and epididymis epithelial cell in caput epididymis. However, its mechanism of the action and the influence about sperm maturation is not very clear. Within sheep caput epididymis epithelial cell, by using cell isolation and culture, molecular biology, iTRAQ protein spectrum quantitative analysis, etc., the project aims are to research: (1) the dose-dependent manners of melatonin promote sperm maturation in caput epididymis epithelial cells; (2) differential proteomics of melatonin regulating the caput epididymis epithelial cells protein and their exocrine protein; (3) the influences of differentially expressed exocrine protein factors on sperm maturation and vitality in vitro. And then discusses related mechanisms of melatonin regulating caput epididymis epithelial cells of gene and protein expression. These will reveal physiological role of melatonin in sperm development and for the further development and utilization of mammalian reproductive potential.

当前研究显示人的精浆中存在褪黑素，并且在大鼠、小鼠、仓鼠、豚鼠以及人附睾和附睾精子也含有褪黑素受体，推测褪黑素对精子成熟有影响，而其作用机制及对精子成熟的影响机制还不清楚。本项目拟以绵羊附睾头上皮细胞和附睾头精子为研究对象，应用细胞分离和培养、分子生物学、iTRAQ蛋白质谱定量分析等实验方法，研究：①褪黑素促进绵羊精子成熟和活力的浓度；②褪黑素调控绵羊附睾头上皮细胞及其外分泌蛋白的差异蛋白组学；③差异表达外分泌蛋白因子对附睾精子成熟和活力的影响。进而探讨褪黑素调控附睾上皮细胞的基因和蛋白表达的关联机制，这将为揭示褪黑素在精子成熟过程中的生理作用和为进一步开发和利用哺乳动物的繁殖潜能奠定基础。

本项目采集了性成熟绵羊附睾组织，按其解剖学位置分为附睾头、附睾体、附睾尾，系统地检测了褪黑素及相关蛋白在附睾组织及其内精子中的表达差异；成功体外培养绵羊附睾上皮细胞，检测附睾上皮细胞中褪黑素与双氢睾酮合成的相互调控作用及相关机制，及褪黑素在附睾炎体外模型中的抗炎作用机制；通过蛋白质组学方法检测了褪黑素对附睾上皮细胞蛋白质表达的影响。结果显示：在绵羊附睾中，附睾头、附睾体和附睾尾组织及其内精子中褪黑素浓度及相关蛋白表达存在显著差异，说明褪黑素在雄性生殖器官发育及精子进一步成熟过程中起着重要的调节作用。褪黑素主要通过其受体MT1和MT2介导抑制附睾上皮细胞中双氢睾酮的合成及雄激素受体表达，同时，双氢睾酮也通过其受体介导参与附睾上皮细胞中褪黑素合成。蛋白质组学结果显示，褪黑素引起附睾上皮细胞中69中蛋白质发生显著变化，进一步探究发现，褪黑素主要通过抗氧化损伤和抗炎等途径参与了对附睾上皮细胞的功能调节；此外，褪黑素可通过受体介导部分解除LPS诱导的炎症模型。