Comparative analysis of AQP7 expression and cryotolerance in X- and Y-chromosome bearing bovine sperm.

INTRODUCTION: The livestock industry has witnessed a notable increase in the proportion of male calves born following artificial insemination with cryopreserved semen, hinting at a possible association between cryotolerance and inherent structural or functional disparities between X- and Y-chromosome bearing sperm. To delve into this phenomenon, we conducted a comprehensive proteomic analysis on bovine sex-sorted semen. METHODS: Sperm samples were meticulously categorized based on stringent quality parameters. Concurrent sorting of X- and Y-sperm from identical ejaculates was performed, followed by protein extraction for subsequent analysis. Quantitative isobaric tags for relative and absolute quantification (iTRAQ) were employed to identify differentially expressed proteins. RESULTS: iTRAQ pinpointed 20 proteins with differential expression between X- and Y-spermatozoa, including 12 upregulated and 8 downregulated proteins in Y-sperm. Aquaporin 7 (AQP7), significantly upregulated in Y-sperm, was identified as a key candidate. Western blot analysis corroborated elevated AQP7 expression in Y-sperm compared to X-sperm, while Aquaporin 3 (AQP3) showed no significant difference. Immunofluorescence revealed AQP7 localization to the post-acrosomal region, midpiece, and principal piece of Y-sperm with higher fluorescence intensity, whereas AQP3 distribution was comparable between groups. DISCUSSION: These findings imply a potential role for AQP7 in augmenting Y-sperm cryotolerance, providing molecular insights into sex-specific cryopreservation effects on fertility outcomes. Future research should elucidate AQP7's functional mechanisms and explore implications for livestock breeding technologies.