绵羊精子细胞核质转运蛋白KPNA4的研究

Study on Nuclear-plasma Transporter KPNA4 of Sheep Sperm Cell

旨在对绵羊附睾头、体和尾部的精子进行蛋白质组学分析,获得差异表达蛋白,对数据进行功能富集分析,挖掘精子发生/成熟关键蛋白质。本研究选择12月龄左右健康的3只雄性湖羊为试验动物,分离附睾并按区域收集精子,3组样本(附睾头部组、附睾体部组和附睾尾部组),每组3个生物学重复,共计9例绵羊精子细胞样本。基于TMT标定定量蛋白质组学分析和R语言等工具,在获取的差异表达蛋白中进行GO和KEGG富集分析,并利用蛋白质免疫印迹(Western bolt)、免疫荧光(immunofluorescence)和流式细胞术(flow cytometry)试验验证结果的可靠性。从22841个唯一性肽中鉴定到差异蛋白质616种,其中,尾vs头组鉴定出309个差异表达蛋白(上调213个,下调96个);尾vs.体组鉴定出167个差异表达蛋白(上调107个,下调60个);体vs头组鉴定出140个差异表达蛋白(上调88个,下调52个)。根据差异倍数与蛋白质功能,筛选出可能与精子成熟、核质物质转运相关的关键蛋白-KPNA4。本研究揭示了绵羊附睾不同部位精子的特点与差异,这些数据为研究雄性绵羊的生殖机制和精子成熟提供了丰富的资源。

The study aimed to analyze the protein expression of sperm in epididymis at 3 different regions(caput,corpus and cauda)and obtain differentially expressed proteins using proteomics in sheep.The functional enrichment analysis on the data was performed to explore proteins regulating spermatogenesis and maturation.In this study,3 healthy male Hu sheep around 12 months of age were selected as experimental animals,the epididymis were isolated and sperms were collected by regions.There were 3 groups of samples(caput group,corpus group and cauda group),3 biological replicates in each group,totaling 9 samples of sheep sperm.Based on TMT-calibrated quantitative protein technology and R software,GO and KEGG enrichment analysis were performed on differentially expressed proteins,and the reliability of the results was verified by Western blot,immunofluorescence and flow cytometry.A total of 616 differential proteins were identified from 22841 unique peptides.Among them,cauda vs.caput group had 309 differentially expressed proteins(up:213,down:96),cauda vs corpus group had 167 differentially expressed proteins(up:107,down:60),corpus vs caput group had 140 differentially expressed proteins(up:88,down:52).According to the fold change and protein function,the key protein KPNA4,which may be related to sperm maturation and nuclear material transport,was screened out.This study reveals the characteristics and differences of sperm from different regions of the sheep epididymis.These data provide a rich resources for studying the reproductive mechanism and sperm maturation in male sheep.