猪精液NAGase活力对受精过程中精子与受体识别的影响及分子机制

N-acetyl-β-D-glucosaminidase (NAGase) was found in sperm plasma and acrosome of many animal species. Previous studies showed that NAGase participated in the process of human and mouse sperm penetration into the oocyte zona pellucida , however, the biochemical mechanism of NAGase activity on swine sperm quality and fertilization ability are not clear. Based on the preliminary achievement of the purified Durock semen NAGase and a variety of effectors screened out to regulate the enzyme activity, we will further study the effect of NAGase activity on sperm motion parameters, acrosome integrity, mitochondrial activity and fertility rate. In addition, we will present to reveal the biochemical pathway of NAGase activity involved in recognition between sperm and receptor proteins of spermatozoa, and the molecular mechanism of cAMP/IP3 pathway in NAGase activity regulating sperm quality and sperm receptor recognition. These results will further elucidate the biochemical mechanism of the sperm physiological activity, and thus provide a basis for developing the effective way to improve pig semen quality, fertilization rate, and reproductive performance of swine.

N-酰-β-D-氨基葡萄糖苷酶（NAGase）存在于多种动物精浆和精子的顶体中，有报道其参与人、鼠精卵识别的初始阶段穿越透明带，但有关NAGase活力对猪精子的质量和受精作用影响的生化机制未见报道。本课题组已分离纯化获得杜洛克猪精液NAGase纯酶并筛选出多种可调控酶活性的效应物。本研究项目拟在前期研究的基础上，用已筛选的效应物调控NAGase活力，研究NAGase活力对精子运动参数、精子运动相关基因cAMP-蛋白激酶和磷蛋白磷酸酶(PPs)γ2表达、顶体完整性、线粒体活性和受精率的影响，探讨NAGase活力影响精子与受体识别的生化途径，阐明cAMP/IP3通路在猪精液NAGase活力影响精子质量及精子与受体识别过程中的作用机理。研究结果将深入阐明NAGase对精子生理活性的调控机制，为探索改善猪精液品质、提高受精率和繁殖性能的有效途径提供科学理论依据。

N-乙酰-β-D-氨基葡萄糖苷酶（NAGase）存在于多种动物精浆和精子的顶体中，有报道其参与人、鼠精卵识别的初始阶段—穿越透明带。深入研究阐明NAGase对精子生理活性的调控机制，在改善猪精液品质、提高受精率和繁殖性能等方面研究具有重要的学术理论价值和应用前景。.本项目研究内容包括3个部分：NAGase在猪体内的分布，精液稀释剂成分和NAGase抑制剂对精子运动性能、顶体完整性和线粒体活性的影响，NAGase抑制剂处理后精子差异蛋白的筛选与鉴定。.本研究分析了NAGase在猪组织和生殖系统中的分布情况，结果表明NAGase在猪生殖系统mRNA水平以及蛋白水平上的广泛表达，是猪精液含有大量NAGase的重要原因。通过分析精液稀释剂成分和NAGase抑制剂对精子运动性能、顶体完整性和线粒体活性的影响，发现NAGase 活力与精子运动性能和与线粒体活性正相关，而对精子质膜完整性无显著影响。经乙酰胺处理抑制NAGase活力后，通过双向电泳和LC-MS-MS分析成功筛选鉴定出精子中与精卵结合相关的差异蛋白，并进行了原核表达，为后续研究NAGase影响精卵结合的生化机制提供了基础。