The surface proteome of rabbit spermatozoa

Cell surface proteins, targeted by approximately 70 % of current pharmaceuticals, offer promising prospects for therapeutic and biotechnological advancements. The recent identification of cell surface proteins, differentially expressed by rabbit spermatozoa, could support technologies to enable sperm sex selection. However, a more detailed knowledge of the rabbit sperm plasma membrane proteome is crucial to such developments. Hence, the primary objective of this study was to conduct a shotgun proteomic (LC-MS/MS) analysis of New Zealand White rabbit spermatozoa protein lysates enriched in cell surface proteins isolated through biotinylation. This approach was designed to provide an overall characterization of this proteome and so determine an expanded list of protein candidates with potential for rabbit sperm sexing. The most promising targets were identified through functional annotation (UniProt and eggNOG-mapper v.2.1.9) and topology prediction (DeepTMHMM v.1.0.13). Additionally, a statistical overrepresentation test (PANTHER 18.0) and analysis of protein-protein interactions (STRING v.12.0) were conducted. Among the 859 detected proteins, 803 had Gene Ontology information, with 574 predicted as globular proteins, 152 as transmembrane proteins, and 133 possessing only a signal peptide. The combined data identified 107 proteins as potential cell surface targets, including three transmembrane proteins encoded by the X chromosome (ADGRG2, ATP6AP2, and VSIG1). Furthermore, two proteins (BCAP31 and PGRMC1), previously identified as putative rabbit X-targets, were recognized. This study enhances our comprehension of rabbit spermatozoa proteomics. Further validation of the utility of these five proteins to differentiate between X- and Y-sperm will determine their suitability for integration into sperm sexing technologies.