Gamete Biology

Lewis X-Containing Glycans Are Specific and Potent Competitive Inhibitors of the Binding of ZP3 to Complementary Sites on Capacitated, Acrosome-Intact Mouse Sperm

Abstract
Mammalian fertilization requires a cascade of interactions between sperm and the egg's zona pellucida (ZP). O-linked glycans on mouse ZP3 have been implicated as mediating one step, firm adhesion of acrosome-intact sperm to the ZP. Experiments to identify structural requirements of a sperm-binding glycan demonstrated that Lewis X (Le^x) -containing glycans (Gal4[Fuc3]GlcNAc-R) were potent competitive inhibitors for in vitro sperm-ZP binding (Johnston, D. S., et al (1998) J Biol Chem 273:1888-95). However, those experiments did not define the particular step in the fertilization pathway that was blocked. The experiments described herein test the hypothesis that Le^x-containing glycans are specific competitive inhibitors of the binding of fluorochrome (Alexa₅₆₈)-labeled ZP3 to sperm and, thus, bind the same sperm surface sites as ZP3. Dose response analyses demonstrated that these glycans are potent inhibitors (IC₅₀~180 nM) which at saturation reduced Alexa₅₆₈-ZP3 binding by ~70%. A Le^a-capped (Gal3[Fuc4]GlcNAc) glycan was also a potent inhibitor (IC₅₀~150-200 nM) but at saturation, reduced Alexa₅₆₈-ZP3 binding by only 30%. In contrast, non-fucosylated glycans with nonreducing GlcNAc4- or Gal4- residues did not compete, neither did sialyl-Le^x nor sulfo-Le^x. However, Gal3Gal4GlcNAc3Gal4Glc, at saturation, reduced Alexa₅₆₈-ZP3 binding by ~70% but with moderate apparent affinity (IC₅₀~3000 nM). Fluorescence microscopy revealed that Alexa₅₆₈-labeled Le^x-Lac-BSA, Le^a-Lac-BSA and ZP3 bound to the same sperm surface domains. However, Le^a-Lac did not inhibit binding of Alexa₅₆₈-Le^x-Lac-BSA and Le^x-Lac did not inhibit binding of Alexa₅₆₈-Le^a-Lac-BSA. Lastly, Le^x-Lac and Le^a-Lac had an additive inhibitory effect on Alexa₅₆₈-ZP3 binding. Thus, Le^x is a ligand for a major class of ZP3 binding sites on mouse sperm while Le^a binding defines a different but less abundant class of sites.

Key words: Gamete Biology • Fertilization • Sperm

This article has been cited by other articles:

				S. A. Williams, L. Xia, R. D. Cummings, R. P. McEver, and P. Stanley Fertilization in mouse does not require terminal galactose or N-acetylglucosamine on the zona pellucida glycans J. Cell Sci., April 15, 2007; 120(8): 1341 - 1349. [Abstract] [Full Text] [PDF]

				J. Holgersson and J. Lofling Glycosyltransferases involved in type 1 chain and Lewis antigen biosynthesis exhibit glycan and core chain specificity Glycobiology, July 1, 2006; 16(7): 584 - 593. [Abstract] [Full Text] [PDF]

				W. F. Hanna, C. L. Kerr, J. H. Shaper, and W. W. Wright Lewis X-Containing Neoglycoproteins Mimic the Intrinsic Ability of Zona Pellucida Glycoprotein ZP3 to Induce the Acrosome Reaction in Capacitated Mouse Sperm Biol Reprod, September 1, 2004; 71(3): 778 - 789. [Abstract] [Full Text] [PDF]