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

Abstract

BackgroundMature 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.ResultsThe 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.ConclusionsThis study offers an in-depth proteomic mapping of mature boar spermatozoa that will enable comparative and discovery research for the improvement of male fertility.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-4442-2) contains supplementary material, which is available to authorized users.

The Proteome of Pig Spermatozoa Is Remodeled During Ejaculation

“…This shortage evidences the yet incomplete functional and structural annotation of the Sus scrofa protein-coding genes (16). Surprisingly, the total number of identified proteins was substantially lower than the 2,728 proteins identified recently by Feugang et al (15) using a shotgun approach. The difference in the number of identified proteins between both studies is related to the methodological differences used for peptide detection and data processing for protein identification and validation, including cutoff data acquisition, algorithms and search databases.…”

“…3C), the 21 hits found were mainly attributed to the extracellular region (8, 38%) and cell parts (7, 34%). DISCUSSION Sperm proteomes have been reported for mouse (19), human (20), monkey (21), equine (22) and most relevant livestock species including pig (15,23,24). However, to improve the understanding of complex regulate mechanisms involved in sperm function or to identify molecular causes of male infertility, the sperm proteome should be updated as the annotation of the protein-coding genome of each species has increased over time (3), as has recently been the case for pigs (16).…”

“…In addition, the FDR differed substantially between the studies. Feugang et al (15) set the FDR Ͻ10% while we used an FDR Ͻ1%, which substantially improved the confidence for the identified proteins. For the sake of comparing results between studies, if we had used an FDR Ͻ10%, the number of identified proteins in our study would have been 2,203, near the number found by Feugang et al (15).…”

“…Feugang et al (15) set the FDR Ͻ10% while we used an FDR Ͻ1%, which substantially improved the confidence for the identified proteins. For the sake of comparing results between studies, if we had used an FDR Ͻ10%, the number of identified proteins in our study would have been 2,203, near the number found by Feugang et al (15). The quantitative approach used, together with the opportunity to access the latest updates of the porcine proteome, allowed us to identify and quantify a proportionally higher number of (9), using frozen-thawed bovine sperm samples subjected to Percoll-based gradient centrifugation enrichment suggested the possibility that the mammalian ejaculated sperm population could be heterogeneous in protein composition, a bold consideration considering they used a species where the ejaculate is delivered as a single volume without fractions.…”

“…A recent study (14) suggested differences in the proteome of spermatozoa in the different boar ejaculate fractions. The present study aimed to update the porcine sperm proteome (15), taking advantage of the increased annotation of protein-coding genes (16). In addition, it aimed to identify and measure, using isobaric tags for relative and absolute quantification (iTRAQ) 1 , eventual putative differences in protein composition between mature spermatozoa retrieved from the cauda epididymis and from the most representative ejaculate fractions, specifically the sperm peak (first 10 ml of the sperm-rich ejaculate fraction, SRF), the remaining SRF and the post-SRF.…”