Xu Yongjie, Han Qiu, Ma Chaofeng, Wang Yaling, Zhang Pengpeng, Li Cencen, Cheng Xiaofang, Xu Haixia
College of Life Science, Xinyang Normal University, Xinyang, China.
Institute for Conservation and Utilization of Agro-Bioresources in Dabie Mountains, Xinyang Normal University, Xinyang, China.
Front Cell Dev Biol. 2021 Jul 16;9:652809. doi: 10.3389/fcell.2021.652809. eCollection 2021.
Sperm cells are of unique elongated structure and function, the development of which is tightly regulated by the existing proteins and the posttranslational modifications (PTM) of these proteins. Based on the phylogenetic relationships of various swine breeds, Yorkshire boar is believed to be distinctly different from Duroc boar. The comprehensive differential proteomics and phosphoproteomics profilings were performed on spermatozoa from both Yorkshire and Duroc boars. By both peptide and PTM peptide quantification followed by statistical analyses, 167 differentially expressed proteins were identified from 1,745 proteins, and 283 differentially expressed phosphopeptides corresponding to 102 unique differentially phosphorylated proteins were measured from 1,140 identified phosphopeptides derived from 363 phosphorylated proteins. The representative results were validated by Western blots. Pathway enrichment analyses revealed that majority of differential expression proteins and differential phosphorylation proteins were primarily concerned with spermatogenesis, male gamete generation, sperm motility, energy metabolism, cilium morphogenesis, axonemal dynein complex assembly, sperm-egg recognition, and capacitation. Remarkably, axonemal dynein complex assembly related proteins, such as SMCP, SUN5, ODF1, AKAP3, and AKAP4 that play a key regulatory role in the sperm physiological functions, were significantly higher in Duroc spermatozoa than that of Yorkshire. Furthermore, phosphorylation of sperm-specific proteins, such as CABYR, ROPN1, CALM1, PRKAR2A, and PRKAR1A, participates in regulation of the boar sperm motility mainly through the cAMP/PKA signal pathway in different breeds, demonstrating that protein phosphorylation may be an important mechanism underlying the sperm diversity. Protein-protein interaction analysis revealed that the 14 overlapped proteins between differential expression proteins and differential phosphorylation proteins potentially played a key role in sperm development and motility of the flagellum, including the proteins ODF1, SMCP, AKAP4, FSIP2, and SUN5. Taken together, these physiologically and functionally differentially expressed proteins (DEPs) and differentially expressed phosphorylated proteins (DPPs) may constitute the proteomic backgrounds between the two different boar breeds. The validation will be performed to delineate the roles of these PTM proteins as modulators of Yorkshire and Duroc boar spermatozoa.
精子细胞具有独特的细长结构和功能，其发育受到现有蛋白质及其翻译后修饰（PTM）的严格调控。基于各种猪品种的系统发育关系，约克夏公猪被认为与杜洛克公猪明显不同。对约克夏和杜洛克公猪的精子进行了全面的差异蛋白质组学和磷酸化蛋白质组学分析。通过肽段和PTM肽段定量，随后进行统计分析，从1745种蛋白质中鉴定出167种差异表达蛋白质，从363种磷酸化蛋白质衍生的1140种已鉴定磷酸肽中测量到283种对应于102种独特差异磷酸化蛋白质的差异表达磷酸肽。代表性结果通过蛋白质免疫印迹法进行了验证。通路富集分析表明，大多数差异表达蛋白质和差异磷酸化蛋白质主要与精子发生、雄配子生成、精子运动、能量代谢、纤毛形态发生、轴丝动力蛋白复合体组装、精卵识别和获能有关。值得注意的是，在精子生理功能中起关键调节作用的轴丝动力蛋白复合体组装相关蛋白质，如SMCP、SUN5、ODF1、AKAP3和AKAP4，在杜洛克精子中显著高于约克夏精子。此外，精子特异性蛋白质如CABYR、ROPN1、CALM1、PRKAR2A和PRKAR1A的磷酸化，在不同品种中主要通过cAMP/PKA信号通路参与公猪精子运动的调节，表明蛋白质磷酸化可能是精子多样性的重要机制。蛋白质-蛋白质相互作用分析表明，差异表达蛋白质和差异磷酸化蛋白质之间的14种重叠蛋白质可能在精子发育和鞭毛运动中起关键作用，包括ODF1、SMCP、AKAP4、FSIP2和SUN5等蛋白质。综上所述，这些生理和功能上差异表达的蛋白质（DEPs）和差异表达的磷酸化蛋白质（DPPs）可能构成了两种不同公猪品种之间的蛋白质组学背景。将进行验证以阐明这些PTM蛋白质作为约克夏和杜洛克公猪精子调节剂的作用。
