iTRAQ-Based Sperm Proteomic Analyses Reveals Candidate Proteins Affecting the Quality of Spermatozoa From Boar on Plateaus

Abstract

Background: Tibetan pigs (TP) exhibit heritable adaptations to their hypoxic environments as a result of natural selection. Whereas, what candidate proteins affecting the sperm quality of boar on plateaus has not been clearly investigated yet. Methods: In this study, to reveal the candidate proteins affecting the quality of spermatozoa from boar on plateaus, we analyzed the sperm quality by Computer-assisted semen analysis (CASA) system and Reactive oxygen species (ROS) levels, compared the sperm proteomes between TP and Yorkshire pigs (YP) raised at high altitudes using the isobaric tags for relative and absolute quantitation (iTRAQ) in combination with the liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomic method, and confirmed the relative expression levels of the four proteins by western blot. Results: The sperm quality of the TP was superior to that of the YP on plateaus. Of 1,555 quantified proteins, 318 differentially expressed proteins (DEPs) were identified. Gene ontology (GO) analysis revealed that the DEPs were predominantly associated with the sorbitol metabolic process, removal of superoxide radicals, cellular response to superoxide, response to superoxide and regulation of the mitotic spindle assembly. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were mainly enriched in pathways involved in the regulation of the actin cytoskeleton, glutathione metabolism, oxidative phosphorylation, and estrogen signaling. And based on the protein-protein interaction (PPI) network analysis, we identified 8 candidate proteins (FN1, EGF, HSP90B1, CFL1, GPX4, NDUFA6, VDAC2, and CP) that might play important roles that affect the sperm quality of boar on plateaus. Moreover, the relative expression levels of the proteins (CFL1, EGF, FN1, and GPX4) were confirmed by western blot. Conclusions: Our results reveal 8 candidate proteins (FN1, EGF, HSP90B1, CFL1, GPX4, NDUFA6, VDAC2, and CP) affecting the sperm quality of boar on plateaus, providing a reference for studies on improving sperm quality and the molecular breeding of boar on plateaus.