Aglycone Analysis and Quantitative Tracking of Correlated Terminal Glycan Pair on Spermatozoa

Mammalian sperm glycans directly mediate several key life events. However, previous studies have not focused on two key factors that regulate these processes, the terminal glycan pattern and the anchoring sites. Herein, we group the capping monosaccharide sialic acid (Sia) and its capping substrates galactose/<i>N</i>-acetylgalactosamine (Gal/GalNAc) into a "correlated terminal glycan pair" (glycopair) and, for the first time, reveal the differences in the aglycone pattern of this pair on spermatozoa using glyco-selective in situ covalent labeling techniques. Sia is mainly found in glycoproteins, whereas terminal Gal/GalNAc is mainly found in glycolipids. We quantitatively track the dynamic changes of the glycopair during sperm epididymal migration and find that the Sia capping ratio decreases with the increased expression of the glycopair; caudal upswim spermatozoa also show a lower Sia capping ratio than down spermatozoa. We thus propose two new parameters reflecting the terminal glycoforms of spermatozoa, which can well distinguish the maturity of spermatozoa. By fluorescence imaging of the glycopair in different regions of the sperm, we find that different parts of the sperm contribute differently to the overall glycan changes.