In-depth proteomic analysis of boar spermatozoa through shotgun and gel-based methods

Jean M. Feugang, Shengfa F. Liao, Scott T. Willard, Peter L. Ryan

Mature spermatozoa contain numerous epididymal and seminal plasma proteins, which full identification through high-throughput technologies may allow for a better understanding of the sperm biology. Therefore, we conducted a global proteomic analysis of boar spermatozoa through shotgun and gel-based methodologies. The total proteins were extracted from mature spermatozoa and subjecsted to proteome analyses. Functional analyses of gene ontology representations and pathway enrichments were conducted on the shotgun dataset, followed by immunology and gene expression validations. Shotgun and gel-based approaches allowed the detection of 2728 proteins and 2123 spots, respectively. Approximately 38% and 59% of total proteins were respectively fully and partially annotated, and 3% were unknown. Gene ontology analysis indicated high proportions of proteins associated with intracellular and cytoplasm localizations, protein and nucleic acid binding, hydrolase and transferase activities, and cellular, metabolic, and regulation of biological processes. Proteins associated with phosphorylation processes and mitochondrial membranes, nucleic acid binding, and phosphate and phosphorous metabolics represented 77% of the dataset. Pathways associated with oxidative phosphorylation, citrate cycle, and extra-cellular matrix-receptor interaction were significantly enriched. Protein complex, intracellular organelle, cytoskeletal parts, fertilization and reproduction, and gap junction pathway were significantly enriched within the top 116 highly abundant proteins. Nine randomly selected protein candidates were confirmed with gel-based identification, immunofluorescence detection, and mRNA expression. This study offers an in-depth proteomic mapping of mature boar spermatozoa that will enable comparative and discovery research for the improvement of male fertility.