猪精液蛋白OPN和CRISP1表达差异与种公猪繁殖力的相关性及其机制研究

The improvement of boar reproductive performance is one of the scientific focuses in animal husbandry. The sperm fertilization ability, which closely related to the roles of semen proteins, is one of the important indicators reflecting male animal reproductive ability. The relationships and mechanism between boar semen proteins and sperm fertilization ability or boar breeding performance are eagerly need to be elucidated. This project intends to use statistical methods for selecting two animal groups (high breeding and low breeding group) as the main research objects from a large boar group based on a criterion of qualified conventional semen indexes but different breeding performance. Then with two-dimensional electrophoresis, immune signatures and ELISA technologies to analyze and quantify the semen proteins (e.g., OPN, CRISP1, etc.) of the two group animals mentioned above. And then joint with boar breeding achievements, a correlation model related sperm protein and reproductive performance will be set up. On the other hand, the expression and distribution of the objective proteins were detected in boar reproductive organs, seminal plasma and sperm. And the effect of objective proteins in the process of fertilization was studied by using artificial insemination, in vitro fertilization and related technologies, so as to explore and elucidate the effect and mechanism of objective protein actions with the sperm fertilization ability. This project can provide protein markers for accurate evaluation of semen fertilization capacity, improving the efficiency of artificial insemination and boar reproduction; also it can provide important reference basis for boar breeding; At the same time, it can also provide reference for the research and treatment of infertility in human reproduction.

提高种公猪繁殖性能是畜牧科学研究的焦点之一，精子受精能力是体现公畜繁殖性能的重要指标，并与精液蛋白的作用密切相关。猪精液蛋白与精子受精能力和公猪繁殖性能的关系与作用机制亟待阐释。本项目拟利用统计学方法从大群体公猪中筛选出常规精液指标合格、但繁殖性能存在差异的两个群体（高繁组和低繁组）为主要研究对象；通过双向电泳、免疫印记、ELISA等技术对两组公猪精液中的蛋白（如OPN、CRISP1等）进行分析和定量，再联合公猪繁殖成绩建立相关精液蛋白与繁殖性能的相关性模型；然后通过检测目标蛋白在公猪生殖器官、精清和精子的表达分布，并利用体外受精、人工授精等技术研究目标蛋白在受精过程中的作用，探索并阐释目标蛋白对精子受精能力的影响及作用机制。本项目研究可为准确评定精液受精能力提供蛋白标记、提高人工授精和种公猪繁殖效率；还可为种公猪育种提供重要参考依据；同时也可为人类生殖不孕不育的研究和治疗提供参考。

提高种公猪繁殖能力是畜牧科学研究的焦点问题之一，而猪精液蛋白含量与精子受精能力及公猪繁殖能力之间的关系与调控作用尚有待于研究和阐释。本项目通过对长白种公猪繁殖成绩大数据统计分析，建立了一套评价种公猪繁殖能力的数学模型，并依据该数学模型和体外受精试验，筛选出3头高繁殖能力和3头低繁殖能力种公猪来研究其精液差异蛋白。利用高通量iTRAQ技术对上述6头种公猪的精子蛋白和精清蛋白分别进行定量，筛选出了一批不同繁殖能力种公猪的精液差异蛋白，选取部分差异蛋白进行ELISA定量验证，一致性达到100%。我们主要关注OPN、CRISP1，SPACA4和IZUMO2这4个蛋白，进行后续研究。首先，对原种猪场的在役种公猪的精液蛋白OPN、CRISP1、SPACA4和IZUMO2分别进行定量检测，并与其相应公猪的繁殖成绩数据进行相关性分析，发现精子及精清OPN蛋白含量与参配母猪窝产活仔数均呈显著正相关（r=0.577和0.513），精子CRISP1蛋白含量与参配母猪窝产总仔数显著正相关（r=0.998），精子SPACA4和IZUMO2蛋白含量与参配母猪的繁殖指标亦存在相关关系。然后，Western blot、免疫组化或免疫荧光原位杂交检测发现，上述蛋白均特异分布于成年公猪睾丸和精子上，在体外受精液中添加上述蛋白抗体均会造成受精率和卵裂率的下降。上述试验结果表明，精液中的特定蛋白含量与公猪的繁殖能力存在相关关系，这些蛋白可通过直接调控精子的受精能力而影响公猪的繁殖能力。最后，利用真核表达系统成功获得重组OPN纯化蛋白，并改进了猪人工授精技术，为在精液中添加OPN蛋白来提高精液受精能力和人工授精技术效果奠定了基础。总之，本项目为评定公猪繁殖性能和精液受精能力提供了可靠的蛋白标记，为高繁殖能力种公猪选育、提高精液受精能力和人工授精技术效果提供了重要参考依据。