The aim of this study was to understand the molecular mechanisms behind the biological differences of X- and Y-sperm and to screen the sex-specific candidate antigen proteins for sexed semen production. To this end, we investigated differential expression of total membrane proteins of the two sperm types by using high-purity X- and Y-sperm from 20 Holstein bulls and applying the label-free proteomic technique; 1521 proteins were identified. In the X-sperm group, 8 and 23 proteins were significantly up- and down-regulated, respectively. In the X- and the Y-sperm group, 151 and 88 proteins were specifically expressed, respectively. These were overexpressed in the dynamic changes of the actin cytoskeleton, and cell senescence/apoptosis induced by the immune response, and could result in differences in the state, size, and immune sensitivity of the X-/Y-sperm membranes. The prediction of transmembrane structure, subcellular localization, and Western blotting validation results showed that the CLRN3 and SCAMP1 proteins were cell surface specific antigens of X- and Y-sperm, respectively. Our findings help explain the molecular mechanism behind the biological differences of X-/Y-sperm and lay the foundation for application of immunological methods to produce sex-sorted semen and control livestock sex. Data are available via ProteomeXchange with identifier PXD019435.
Keywords: X-/Y-sperm; dairy cattle; membrane proteome; sex-specific antigen.