Shotgun Proteomics-based distinctive fingerprint of sperm-surface revealed over- representation of proteins driving spermatogenesis, motility, and fertility

Abstract

Background Numerous distinct secretagogues such as (glyco) proteins including the GPI-anchored proteins are added to the sperm surface during their transit through the male reproductive tract (MRT). This remodelling of the sperm surface is critical for sperm maturation, survival and function in the female reproductive tract (FRT). This study aimed to characterize the proteins present on the buffalo sperm surface. Results A buffalo sperm surface-specific proteomic fingerprint was generated using shotgun proteomics (LC-MS/MS). The protein informatics platform, Proteome Discoverer (v2.2) identified 1342, 678, and 982 distinct proteins and isoforms (P < 0.05, FDR < 0.01) in the salt-extracted, PI-PLC treated and capacitated samples, respectively. Overall, 1695 unique proteins (minimum 2 peptides) with ≥ 1 high-quality PSM/s and their isoforms (proteoforms) were identified. Descriptive statistical analysis indicated that these buffalo-specific proteoforms exhibit remarkable heterogeneity in their molecular weight, pI, distribution of expression from the genome and their functional roles in the MRT and the FRT. Subsequent analysis and a thorough literature search revealed that the fertility-related, reproduction-specific proteoforms constituted more than 50% (873) of the identified sperm-surface proteome (1695). Discussion These identified proteoforms are unique to buffalo since a buffalo-specific database, NCBI reference proteome (translated from the latest chromosome level genome assembly, (UOA_WB_1) was used as the search space. These proteoforms were mapped to 252 buffalo-specific proteins implicated in the regulation of various aspects of male reproductive physiology across multiple species. Besides, more than 200 orphan, buffalo-specific proteins and their isoforms (undefined locus, uncharacterized, P < 0.05, FDR < 0.01) were also identified using our computational strategy. This allowed us to consider these novel proteins for considered for mapping their ontology terms. This led to the elucidation of the biological functions of these hitherto unreported, buffalo-specific proteins by extrapolation of function from their sequence orthologs in more several ruminant and non-ruminant (e.g. Primates and Rodents) mammalian. These uncharacterized proteins constitute an extensive, yet unexplored, reproduction-specific sperm-surface proteome repertoire. Conclusions The proteomic signature driving the buffalo sperm production, maturation, survival and function discovered in this study is unparalleled vis-à-vis the depth identification of fertility-related and reproduction-specific cell-surface proteins. These results would facilitate advances in understanding the functional roles of proteins implicated in mammalian sperm function.