Immuno‐Targeting of <scp>CLRN3</scp> and <scp>SCAMP1</scp> as a Potential Sex Specific Marker in Bovine Spermatozoa

Sex-specific proteins are unique markers found on either X- or Y-chromosome bearing spermatozoa, acting like molecular signatures, and can be used as a key target to distinguish X- or Y-bearing cells. Towards this goal, by reviewing the literature, a total of 6 proteins were selected from the selected, of which 2, CLRN3 and SCAMP1, were finally chosen for the immunoprecipitation study. For the finally selected proteins, predictions of subcellular localisation and transmembrane topology were generated using online tools such as BUSCA, DeepLoc 2.0, DeepTMHMM, and TMHMM. Additionally, several online platforms like SEMMA 2.0 and IEDB were employed to identify potential immunogenic targets. Following this, commercially available antibodies against the 2 identified proteins were procured for subsequent use in multiple immunoprecipitation-based techniques, such as Western blotting, immunocytochemistry, and ELISA (Enzyme-linked immunosorbent assay). Targeting these proteins revealed their binding intensity on spermatozoa. In this study, the CLRN3 antibody targeting X-spermatozoa showed stronger interaction with X-sorted samples than the Y-specific SCAMP1 antibody in the cell-based ELISA. Results from the study suggest targeting sex-specific surface proteins could enable the development of an immune-based sperm-sorting technique in bovines. Overall, the results support the potential of CLRN3 as an X-specific surface marker that needs further validation for the development of a novel immune-based sperm sorting technique.