METHODS AND RESULTS: Four rounds of Cell-SELEX were performed, where the DNA aptamer library was incubated with X-sorted semen. Subsequently, affinity-depicting aptamers were sequenced. Fourteen out of thirty-four identified sequences were tested for specificity towards X-sorted sperm. The magnetic bead-based experiment revealed that the aptamers interacted preferentially with the acrosome region (sperm head) of sperm. We investigated the specificity of aptamers for X-spermatozoa by conducting real-time PCR assays targeting sex-specific genes. Aptamers X8, X19, X22 & X27 exhibited higher affinity for X-sorted sperm than Y in bovine semen. Western blot analysis showed an interaction between the aptamer and sperm protein at 35 kDa. The results underwent further analysis using in-gel mass spectrometry (MS) that predicted SPACA1 protein as the potential/putative aptamer binding protein on the sperm surface. Competitive western blot further confirmed aptamers' differential specificity for X-sperm.
CONCLUSIONS: The current work helps in creating new knowledge for the separation of Y-chromosome-bearing sperm from XY sperm populations that have the potential to produce good-quality cattle (cows). The work also generates proof of concept (POC) of using aptamers, which is cost-effective and could be used in sorting sex-specific spermatozoa without causing any ill effect on sperm.