iTRAQ-based proteomic analysis of sperm reveals candidate proteins that affect the quality of spermatozoa from boars on plateaus

Abstract

Background Tibetan pigs (TP) exhibit heritable adaptations to their hypoxic environments as a result of natural selection. However, candidate proteins that affect the sperm quality of boars on plateaus have not yet been clearly investigated. Methods In this study, to reveal the candidate proteins that affect the quality of spermatozoa of boars on plateaus, we analyzed the sperm quality using computer-assisted semen analysis (CASA) system and reactive oxygen species (ROS) levels. We also compared the proteomes of 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 blotting. Results The sperm quality of the TP was superior to that of the YP on plateaus. Of the 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. 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 and affect the sperm quality of boars on plateaus. Moreover, the relative expression levels of four proteins (CFL1, EGF, FN1, and GPX4) were confirmed by western blot analysis. Conclusions Our study revealed 8 candidate proteins (FN1, EGF, HSP90B1, CFL1, GPX4, NDUFA6, VDAC2, and CP) that affect the sperm quality of boar on plateaus and provide a reference for further studies on improving sperm quality and the molecular breeding of boars on plateaus.

Applications of Omics Technology for Livestock Selection and Improvement

“…Proteomics has also enabled the identification of candidate protein markers of fertility for molecular breeding. The LC-MS/ MS analysis of pig sperm revealed eight fertility-related proteins over-represented in Tibetan pigs having heritable adaptation to hypoxic environments (Zhao et al, 2021). In another study, analysis of seminal plasma proteins by LC-MS/MS found a consistent correlation of 1,343 proteins with fertility (Willforss et al, 2021).…”

“…However, the application of proteomics in livestock research has been limited in the past due to its high cost and lack of optimized protocols for various cell types in different species (Baykalir et al, 2018). Nevertheless, with advancements in new analytical methods and computational tools for the analysis of proteomic data, reports on proteomics studies in animal science are increasing for understanding the animal health status and production and reproduction efficiency (Zhao et al, 2021;Kaya et al, 2022;Ye et al, 2022).…”

Chlorogenic Acid Improves Quality of Chilled Ram Sperm by Mitigating Oxidative Stress

“…The lysate was centrifuged at 12,000× g for 5 min at 4 °C to obtain the supernatant. G250 staining solution (detergent compatibility, 300 µL) was added to the sample (10 µL), the absorbance was measured at 595 nm, and the protein concentration was obtained according to the standard curve [ 32 , 33 ].…”

Integration of omics studies indicates that species-dependent molecular mechanisms govern male fertility

“…3A, P < 0.05). Also, VDAC2, ENO1, and GPX4 proteins were used as the spermatogenesis markers [36][37][38] which was uniquely clustered in fertility related transcriptomes from bovine spermatozoa (Fig. 1B).…”