Porcine Sperm Bind to Specific 6-Sialylated Biantennary Glycans to Form the Oviduct Reservoir1

Abstract

After mating, many female mammals store a subpopulation of sperm in the lower portion of the oviduct, forming a reservoir. The reservoir lengthens sperm lifespan, regulates sperm capacitation, controls polyspermy, and selects normal sperm. It is believed that sperm bind to glycans on the oviduct epithelium to form the reservoir, but the specific adhesion molecules that retain sperm are unclear. Herein, using a glycan array to test 377 glycans for their ability to bind porcine sperm, we found two glycan motifs in common among all glycans with sperm-binding ability: the Lewis X trisaccharide and biantennary structures containing a mannose core with 6-sialylated lactosamine at one or more termini. Binding to both motifs was specific; isomers of each motif did not bind sperm. Further work focused on sialylated lactosamine. Sialylated lactosamine was found abundantly on the apical side of epithelial cells collected from the oviduct isthmus, among N-linked and O-linked glycans. Sialylated lactosamine bound to the head of sperm, the region that interacts with the oviduct epithelium. After capacitation, sperm lost affinity for sialylated lactosamine. Receptor modification may contribute to release from the reservoir so that sperm can move to the site of fertilization. Sialylated lactosamine was required for sperm to bind oviduct cells. Simbucus nigra agglutinin or an antibody specific to sialylated lactosamine with a preference for Neu5Acalpha2-6Gal rather than Neu5Acalpha2-3Gal reduced sperm binding to oviduct isthmic cells, as did occupying putative receptors on sperm with sialylated biantennary glycans. These results demonstrate that sperm binding to oviduct 6-sialylated biantennary glycans is necessary for normal adhesion to the oviduct.

Enhanced fertility prediction of cryopreserved boar spermatozoa using novel sperm function assessment

“…The final concentration of PI was 32.9 lM and of FITC-PNA was 28.2 ng/mL. Spermatozoa were subsequently fixed with 0.4% paraformaldehyde in PBS (a concentration that does not permeabilize spermatozoa (Kadirvel et al, 2012) at 0, 30 and 60 min after thawing and evaluated. A total of 300 spermatozoa were evaluated using a Zeiss AxioCamHRc (Zeiss Microscopy, LLC, Thornwood, NY, USA) at 4009 magnification.…”

“…Epithelial cells were concentrated by centrifugation (200 g for 30 s) and disrupted by pipetting. Disrupted cells were incubated in dmTALP (Kadirvel et al, 2012) at 39°C for 1.5-2 h to allow cell reaggregation. Three-dimensional aggregates with a diameter of~150 lm were selected, washed through three consecutive droplets of dmTALP and placed into a final droplet in groups of 10 for coincubation with spermatozoa.…”

“…Spermatozoa were thawed at 50°C for 20 s, held at 37°C for 7 min and centrifuged on a Percoll cushion containing noncapacitating dmTALP (dmTALP in which BSA was replaced with an equal mass of PVP and bicarbonate was replaced with an equimolar amount of HEPES; Kadirvel et al, 2012), hereafter referred to as TALPNC. Following a second wash by centrifugation, spermatozoa were held in TALPNC and counted by hemocytomter.…”

“…Samples were centrifuged on a Percoll cushion containing dmTALP (Kadirvel et al, 2012). The sperm pellet was washed in dmTALP and the resulting pellet was held in TALP and spermatozoa counted by hemocytomter.…”

Combined albumin and bicarbonate induces head-to-head sperm agglutination which physically prevents equine sperm–oviduct binding

“…In pigs, the major inhibitory effect could be mimicked by biantennary structures containing a mannose core with six sialylated lactosamines at one or more termini. In pigs, as in cattle, binding to carbohydrate moieties was specific; different isomers of the specific motif did not bind sperm (Kadirvel et al 2012).…”