MURINE SPERM-ZONA BINDING, A FUCOSYL RESIDUE IS REQUIRED FOR A HIGH-AFFINITY SPERM-BINDING LIGAND - A 2ND SITE ON SPERM BINDS A NONFUCOSYLATED, BETA-GALACTOSYL-CAPPED OLIGOSACCHARIDE

An essential initial step in murine fertilization is the binding of ac rosome-intact sperm to specific O-linked oligosaccharides on zona pell ucida glycoprotein 3. While there is agreement on the primary role of O-linked glycans in this process, there is a lack of consensus on both the terminal monosaccharide(s) required for a functional sperm bindin g site and the corresponding protein on the sperm cell surface that re cognizes this ligand. Much current debate centers on an essential role for either a terminal N-acetylglucosaminyl or, alternatively,: a term inal alpha-galactosyl residue, To gain insight into the terminal sacch arides required to form a functional,sperm-binding ligand, dose-respon se curves were generated for a series of related tri-and tetrasacchari des to evaluate their relative effectiveness to competitively inhibit the in vitro binding of murine sperm to zona pellucida-enclosed eggs, A GlcNAc-capped trisaccharide, GlcNAc beta 1,4GlcNAc beta 1,4GlcNAc,wa s inactive (1-72 mu M range), In contrast, a beta 4-galactosyl-capped trisaccharide (Gal beta 1,BGIcNA alpha 1, 4GlcNAc) and an alpha 3-gala ctosyl-capped trisaccharide (Gal alpha 1,3Gal beta 1,4 GlcNAc) inhibit ed sperm-zona binding with low or moderate affinity (ED50 = 42 mu M an d 5.5 mu M, respectively), The addition of an alpha 3-fucosyl residue to each of these two competitive inhibitors, forming Gal beta 1,4[Fuc alpha 1,3] GlcNAc beta 1,4GlcNAc or Gal alpha 1,3Gal beta 1, B[Fuc alp ha 1,3]Glc NAc, resulted in ligands with 85- and 12-fold higher affini ties for sperm, respectively (ED50 = 500 and 430 nM), Thus, the presen ce of a fucosyl residue appears to be obligatory for an oligosaccharid e to bind sperm with high affinity, Last, mixing experiments with pair s of competitive inhibitors suggest that murine sperm-zona binding is mediated by two independent oligosaccharide-binding sites on sperm, Th e first (apparently high affinity) site binds both the alpha 3-galacto syl-capped trisaccharide and the two fucosylated tetrasaccharides. The second (apparently low affinity) site binds a nonfucosylated beta-gal actosyl-capped trisaccharide.