Mammalian fertilization requires a cascade of interactions between sperm and the egg's zona pellucida (ZP). O-linked glycans on mouse glycoprotein ZP3 have been implicated in mediating one step of the fertilization process, the firm adhesion of acrosome-intact sperm to the ZP. Experiments to identify structural requirements of a sperm-binding glycan have demonstrated that a Lewis X (Lex)-containing glycan (Galβ4[Fucα3]GlcNAc-R) was a potent, competitive inhibitor of in vitro sperm-ZP binding (Johnston et al. J Biol Chem 1998; 273: 1888–1895). However, those experiments did not define the particular step in the fertilization pathway that was blocked. The experiments described herein test the hypothesis that Lex-containing glycans are specific, competitive inhibitors of the binding of Alexa Fluor 568 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 Lewis A (Lea)-capped glycan (Galβ3[Fucα4]GlcNAc) was also a potent inhibitor (IC50 ∼150–200 nM), but at saturation, it reduced Alexa568-ZP3 binding by only 30%. In contrast, nonfucosylated glycans with nonreducing GlcNAcβ4 or Galβ4 residues did not compete; neither did sialyl-Lex (Neu5Acα 3Galβ4[Fucα3]GlcNAc-Lewis X) nor sulfo-Lex (3′-O-SO3-Lewis X). However, at saturation, Galα3Galβ4GlcNAcβ3Galβ4Glc reduced Alexa568-ZP3 binding by ∼70% but with moderate apparent affinity (IC50 ∼3000 nM). Fluorescence microscopy revealed that Alexa568-labeled Lex-Lac-BSA, Lea-Lac-BSA, and ZP3 bound to the same sperm surface domains. However, Lea-Lac did not inhibit binding of Alexa568-Lex-Lac-BSA, and Lex-Lac did not inhibit binding of Alexa568-Lea-Lac-BSA. Finally, Lex-Lac and Lea-Lac had an additive inhibitory effect on Alexa568-ZP3 binding. Thus, Lex is a ligand for a major class of ZP3 binding sites on mouse sperm, whereas Lea binding defines a different but less-abundant class of sites.
You have requested a machine translation of selected content from our databases. This functionality is provided solely for your convenience and is in no way intended to replace human translation. Neither BioOne nor the owners and publishers of the content make, and they explicitly disclaim, any express or implied representations or warranties of any kind, including, without limitation, representations and warranties as to the functionality of the translation feature or the accuracy or completeness of the translations.
Translations are not retained in our system. Your use of this feature and the translations is subject to all use restrictions contained in the Terms and Conditions of Use of the BioOne website.