Changes to sperm surface carbohydrates following exposure to seminal plasma and freezing

Abstract

Epididymal, ejaculated and frozen–thawed spermatozoa are exceptionally different sperm types. When placed directly into the uterus all sperm types display high fertility. However, when forced to traverse the ovine cervix (following vaginal or cervical AI), epididymal and frozen–thawed ram spermatozoa display dramatically lower fertility than fresh ejaculated spermatozoa. We suggest that changes to the outer carbohydrate-rich glycoprotein coat of spermatozoa represent a potential reason for altered cervical transit ability, as this is the primary interface with the female environment. As such, we used lectin binding to investigate modifications of the carbohydrate portion of sperm glycoconjugates in response to seminal plasma exposure and freezing. Fresh and frozen ejaculated spermatozoa and fresh epididymal spermatozoa were subjected to a swim up to remove loosely associated glycoproteins and remnants of freezing diluent. Spermatozoa were stained with fluorescein isothiocyanate (FITC) conjugated lectins (LPA, PNA, WGA and ConA), specific for the sugars of interest (sialic acid, galactose, N-acetylglucosamine andmannose, respectively) and counter-stained with propidium iodide (PI). Median FITC fluorescence of the PI negative (live) population was compared amongst treatments using a restricted maximum likelihood approach. The results provide strong evidence that both exposure to seminal plasma and freezing are processes which alter the outermost surface of spermatozoa. Seminal plasma exposure significantly increased N-acetylglucosamine (WGA; p<0.001) on the sperm membrane. Conversely, freezing led to a significant decrease in available galactose (PNA; p<0.01) and N-acetylglucosamine (WGA; p<0.001) as well as increased availability of mannose (ConA; p<0.001), typically a carbohydrate located proximal to the membrane in glycoconjugates. The observed changes lend support to the theory that while exposure to seminal plasmamay orchestrate meaningful alteration of the sperm surface, the freezing process further modifies this important region, possibly to its detriment. This is the first evidence that industry standard freezing of ram semen leads to large and significant changes in selected sperm surface carbohydrates. Further research is required to fully elucidate the consequenceof these changesandestablishpossiblemeans to prevent or repair the damage to the glycoprotein coat of ram spermatozoa. Research support by AWI and NSWSMBA Trust.