Table_2_Comparative Proteomics and Phosphoproteomics Analysis Reveal the Possible Breed Difference in Yorkshire and Duroc Boar Spermatozoa.XLSX

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与功能，其发育过程受现有蛋白质及这些蛋白质的翻译后修饰（posttranslational modifications, PTM）严格调控。基于不同猪品种的系统发育关系，约克夏公猪与杜洛克公猪被认为存在显著差异。本研究对约克夏与杜洛克公猪的精子开展了全面的差异蛋白质组学与磷酸化蛋白质组学图谱分析。通过对肽段及翻译后修饰肽段进行定量并结合统计分析，本研究从1745个鉴定到的蛋白质中筛选得到167个差异表达蛋白；从363个磷酸化蛋白质衍生的1140个鉴定到的磷酸化肽段中，检测到283个差异表达磷酸化肽段，对应102个独特的差异磷酸化蛋白质。代表性结果通过蛋白质免疫印迹（Western blot）得到验证。通路富集分析显示，大部分差异表达蛋白与差异磷酸化蛋白主要参与精子发生、雄配子生成、精子活力、能量代谢、纤毛形态发生、轴丝动力蛋白复合物组装、精卵识别及精子获能等生物学过程。值得注意的是，与轴丝动力蛋白复合物组装相关的蛋白质，如在精子生理功能中发挥关键调控作用的SMCP、SUN5、ODF1、AKAP3及AKAP4，在杜洛克精子中的表达量显著高于约克夏精子。此外，精子特异性蛋白质的磷酸化修饰，如CABYR、ROPN1、CALM1、PRKAR2A及PRKAR1A，主要通过环腺苷酸/蛋白激酶A（cAMP/PKA）信号通路参与调控不同品种公猪的精子活力，这表明蛋白质磷酸化可能是导致不同品种公猪精子多样性的重要机制。蛋白质相互作用分析显示，差异表达蛋白与差异磷酸化蛋白之间存在14个重叠蛋白质，这些蛋白质可能在精子发育及鞭毛活力调控中发挥关键作用，包括ODF1、SMCP、AKAP4、FSIP2及SUN5。综上，这些具有生理与功能差异的差异表达蛋白（differentially expressed proteins, DEPs）与差异磷酸化蛋白（differentially phosphorylated proteins, DPPs）或可构成两种不同公猪品种间的蛋白质组学差异背景。后续将通过验证实验阐明这些翻译后修饰蛋白作为调控因子在约克夏与杜洛克公猪精子功能中的作用。