The Surface Proteome of Bovine Unsexed and Sexed Spermatozoa.

Current sperm sexing methods are costly and largely restricted to cattle, while immunological techniques targeting sex-specific membrane proteins may offer more economical alternatives. To advance these methods, understanding the proteomic differences between the cell membranes of X- and Y-chromosome-bearing spermatozoa is essential. This study aimed to characterize the cell surface proteome of bovine sperm and identify potential targets for sperm sexing through LC-MS/MS analysis. Cell surface protein lysates were extracted from unsexed, X-sperm (BX), and Y-sperm (BY) samples via biotinylation. Promising targets were identified through functional annotation (UniProt, eggNOG-mapper v.2.1.7) and topology prediction (DeepTMHMM v.1.0.13). Additionally, statistical overrepresentation (PANTHER 18.0) and orthology analyses were performed. Excluding contaminants, 130 proteins were detected, of which 64 proteins were detected in the BX samples and not in the BY samples. Of these, five transmembrane proteins stood out as potential X-sperm targets (ADAM2, ATP11C, DG1, MCT1, and PMCA4). They were identified as potential cell surface targets, based on GO terms and topology predictions, detected in at least two replicates of the BX samples, and shown to share orthology with other livestock species. These findings enhance our understanding of bovine sperm proteomics; however, further validation is required to confirm the utility of these five proteins in sperm sexing technologies.