Deciphering the molecular basis of equine sperm-oviduct binding: a stepwise exclusion approach

Abstract

In several mammalian species, the molecular interaction between the apical part of the sperm plasma membrane and oviduct epithelium, to establish a sperm reservoir at the utero-tubal junction and oviductal isthmus, is based on Ca²⁺-dependent carbohydrate-lectin recognition. Additionally, disulfide reductants markedly reduce the affinity of spermatozoa for carbohydrate moieties on bovine oviduct epithelium. In the horse, the nature of this binding is not fully understood. Using an oviduct explant model, we previously demonstrated that equine sperm-oviduct binding is not regulated by a Ca²⁺-dependent lectin (electrostatic) interaction (Leemans et al., 2016, Reproduction 151:313-30). In the current study, we aimed to further investigate the nature of equine sperm-oviduct binding. For all experiments, oviduct epithelial cells were collected and cultured as described previously by Nelis et al. (2014, Reproduction Fertility Development 26:954-66). After overnight incubation, oviduct explants 〈200 µm diameter were selected and washed in non-capacitating (100 mM NaCl, 4.7 mM KCl, 1.2 mM MgCl₂, 5.5 mM glucose, 22 mM HEPES, 2.4 mM sodium lactate, 1.0 mM pyruvate and 0.05% polyvinylpyrrolidone; pH=7.4 and 280–300 mOsm/kg) sperm medium, before 5 oviduct explants were transferred to 45 µL droplets of non-capacitating medium. Next, fresh Hoechst-33342 stained spermatozoa were added (after Percoll^Ⓡ centrifugation) in a volume of 5 µL to a final sperm concentration of 2 × 10⁶ sperm cells/mL in each droplet. After 2 h co-incubation, density of sperm binding to oviduct explants was determined using a fluorescent microscope in five 400x magnification fields. In the first experiment, we identified the expression of carbohydrate moieties at the apical region of the sperm plasma membrane and oviduct epithelium using fluorescent (FITC) labeled lectins. N-acetylglucosamine (WGA-FITC) moieties were highly expressed on the apical part of the sperm plasma membrane, whereas N-acetylgalactosamine (DBA-FITC and HPA-FITC), N-acetylneuraminic acid (sialic acid; SNA-FITC) and D-mannose or D-glucose (Con A-FITC, LCA-FITC and PSA-FITC) were highly abundant on the oviduct epithelium. In a second experiment, the role of these highly expressed carbohydrates in sperm-oviduct interaction was assessed by pre-incubating stallion spermatozoa for 30 min with WGA lectin (0-5-50 µg/ml); and oviduct explants for 30 min with DBA, HPA, SNA, Con A, LCA and PSA lectins (0-5-50 µg/ml for each lectin). Subsequently, oviduct explants and spermatozoa were washed twice in non-capacitating medium before co-incubation. None of the lectins reduced sperm binding to oviduct explants, except WGA pre-incubation of sperm. This result is likely related to the concentration-dependent effect of WGA on sperm motility. In a third experiment, a possible covalent disulfide bridge-based sperm-oviduct interaction was evaluated by pre-incubating stallion spermatozoa and oviduct explants individually with D-penicillamine (0-0.25-0.5 mM) and dithiothreitol (0-0.5 mM; lowest non-toxic concentration), two disulfide bridge reducing conditions. No effect on density of sperm binding after oviduct co-incubation was observed. Additionally, spermatozoa did not release from pre-established sperm-oviduct binding under similar conditions during 2, 10 and 20 h incubation. Additional investigations on the nature of sperm-oviduct interaction will improve understanding of the biology of oviductal selection of stallion spermatozoa.