Carbohydrate-Mediated Binding and Induction of Acrosomal Exocytosis in a Sperm-Somatic Cell Adhesion Model. (2009)

Initial murine and porcine sperm-oocyte binding is primarily dependent on the interaction of carbohydrate sequences presented on the zona pellucida (ZP) with one or more lectin-like egg binding proteins (EBP) localized to the plasma membrane overlying the acrosome. Recent findings indicate that the EBP associated with mouse and boar spermatozoa mediates binding to their respective oocytes by interacting with tri- and tetraantennary complex type N-glycans expressed on the ZP. Mouse spermatozoa bind with high affinity to rabbit erythrocytes (REs), presumably via the interaction of their EBP with branched polylactosamine type sequences expressed at a high density on this somatic cell type. Therefore if identical carbohydrate ligands mediate initial gamete binding in the mouse and the pig as previously proposed, then these branched polylactosamine sequences should also bind to the lectin-like domain of the EBP associated with boar sperm. By employing this logic, we hypothesized that boar spermatozoa should also bind with high affinity to REs. Mouse spermatozoa were used as positive controls to test this hypothesis. Bull spermatozoa were selected as negative controls based on the proposed affinity of their EBP for a high mannose sequence (Man5GlcNAc2) that is associated with bovine but not porcine ZP. As anticipated, co-incubation of capacitated boar spermatozoa with REs resulted in the immediate high affinity binding between these two very different cell types, which we recorded on still images and video. This interaction was restricted to the acrosomal regions of boar sperm head. No binding was observed between capacitated bull spermatozoa and REs. Unlike murine sperm, acrosomal exocytosis was induced in boar sperm immediately after binding. The triggering of this reaction following contact with a somatic cell type is a novel observation. Mild periodate oxidation specifically degrades carbohydrate sequences by converting vicinal hydroxyl groups on sugars to aldehydes. REs were subjected to this chemical treatment to determine if their interaction with boar spermatozoa depends on carbohydrate recognition. Boar spermatozoa did not bind to oxidized REs, nor did they undergo acrosomal exocytosis in the presence of these cells. The branched polylactosaminoglycan type sequences expressed on REs are primarily terminated with Galα1-3Gal β1-4GlcNAc sequences. Exhaustive digestion of these somatic cells with α-galactosidase did not affect boar sperm binding or the induction of acrosomal exocytosis in this artificial adhesion system. These results indicate that the EBP on boar spermatozoa recognizes glycans terminated with either Galα1-3Galβ1-4GlcNAc or Galβ1-4GlcNAc sequences. Previous studies confirm that the murine EBP also accommodates these terminal sequences. In summary, these observations are consistent with the proposal that the EBP on boar and mouse spermatozoa mediates carbohydrate dependent binding by recognizing tri- and tetraantennary N-glycans expressed on their respective ZP. These results also indicate a potential role for carbohydrate recognition in the induction of acrosomal exocytosis in boar spermatozoa during fertilization. [Supported by the Mission Enhancement Program in Reproductive Biology and Medicine funded by the state of Missouri (G.F.C.), by USDA-NRI grant #2007-01319 (P.S.) and by seed funding from the F21C Program of UM (P.S.)]. (poster)