Seminal plasma and cryopreservation alter ram sperm surface carbohydrates and interactions with neutrophils

Spermatozoa deposited vaginally must navigate the physical, chemical and immune barriers of the cervix to reach the site of fertilisation. Characteristics that favour successful cervical transit remain largely unknown beyond the obvious factors of motility and viability. Epididymal and cryopreserved ram spermatozoa demonstrate poor cervical transit, for unknown reasons. We hypothesised that seminal plasma exposure and cryopreservation alter the surface sugars of these sperm populations and, consequently, their interaction with immune cells, both potential factors for successful cervical transit. The carbohydrate profiles of epididymal, ejaculated and frozen-thawed ram spermatozoa were assessed by flow cytometry and western blotting using lectins for galactose, sialic acid, N-acetylglucosamine and mannose. Seminal plasma exposure and cryopreservation caused significant changes to the relative amounts of surface sugars detected by flow cytometry and lectin blotting. Immune cell interaction was characterised using a neutrophil-binding assay. Seminal plasma acted as a robust protective mechanism, limiting binding of spermatozoa, whereas the media used for cryopreservation caused a significant disruption to opsonin-mediated binding. We were unable to demonstrate a link between changes to surface sugars and neutrophil susceptibility. Seminal plasma and cryopreservation clearly alter the sperm glycocalyx, as well as the interaction of spermatozoa with immune cells.