Murine Sperm-Zona Binding, A Fucosyl Residue Is Required for a High Affinity Sperm-binding Ligand

An essential initial step in murine fertilization is the binding of acrosome-intact sperm to specificO-linked oligosaccharides on zona pellucida glycoprotein 3. While there is agreement on the primary role ofO-linked glycans in this process, there is a lack of consensus on both the terminal monosaccharide(s) required for a functional sperm binding site and the corresponding protein on the sperm cell surface that recognizes this ligand. Much current debate centers on an essential role for either a terminalN-acetylglucosaminyl or, alternatively, a terminal α-galactosyl residue. To gain insight into the terminal saccharides required to form a functional sperm-binding ligand, dose-response curves were generated for a series of related tri- and tetrasaccharides to evaluate their relative effectiveness to competitively inhibit thein vitro binding of murine sperm to zona pellucida-enclosed eggs. A GlcNAc-capped trisaccharide, GlcNAcβ1,4GlcNAcβ1,4GlcNAc,was inactive (1–72 μm range). In contrast, a β4-galactosyl-capped trisaccharide (Galβ1,4GlcNAcβ1, 4GlcNAc) and an α3-galactosyl-capped trisaccharide (Galα1,3Galβ1,4 GlcNAc) inhibited sperm-zona binding with low or moderate affinity (ED50 = 42 μm and 5.3 μm, respectively). The addition of an α3-fucosyl residue to each of these two competitive inhibitors, forming Galβ1,4[Fucα1,3] GlcNAcβ1,4GlcNAc or Galα1,3Galβ1, 4[Fucα1,3]Glc NAc, resulted in ligands with 85- and 12-fold higher affinities for sperm, respectively (ED50 = 500 and 430 nm). Thus, the presence of a fucosyl residue appears to be obligatory for an oligosaccharide to bind sperm with high affinity. Last, mixing experiments with pairs of competitive inhibitors suggest that murine sperm-zona binding is mediated by two independent oligosaccharide-binding sites on sperm. The first (apparently high affinity) site binds both the α3-galactosyl-capped trisaccharide and the two fucosylated tetrasaccharides. The second (apparently low affinity) site binds a nonfucosylated β-galactosyl-capped trisaccharide.