Characterization of the Binding of Recombinant Mouse Sperm Fertilin β Subunit to Mouse Eggs: Evidence for Adhesive Activity via an Egg β1Integrin-Mediated Interaction

Abstract

The sperm protein fertilin (also known as PH-30) is a candidate for mediating the interactions between sperm and egg plasma membranes. Fertilin is a heterodimer. The β subunit, which has a region with homology to the family of integrin ligands known as disintegrins, has been hypothesized to be involved in the binding of sperm to the egg surface. To investigate this hypothesis and determine what role fertilin β plays in fertilization, we have expressed the putative extracellular domain of mouse fertilin β in bacteria as a fusion protein with maltose-binding protein (hereafter referred to as recombinant fertilin β-EC) and used two assays to characterize its binding to mouse eggs. Immunocytochemistry was used to examine the localization of recombinant fertilin β-EC binding. A luminometric assay was also developed to quantify levels of binding of recombinant fertilin β-EC to single eggs. We find that recombinant fertilin β-EC binds to the region of the plasma membrane of the egg to which sperm bind, thus providing the first direct evidence that fertilin β has adhesive properties. Peptides corresponding to the disintegrin domain of fertilin β reduce its binding to eggs, suggesting that this domain is at least partially involved in the recognition of fertilin β by binding sites on the egg. Treatment of zona pellucida-free eggs with chymotrypsin reduces the ability of the eggs to support the binding of recombinant fertilin β-EC, implicating an egg surface protein as a binding site for recombinant fertilin β-EC. Binding of recombinant fertilin β-EC to eggs is also reduced in the absence of divalent cations and is supported by 2.0 mMCa2+, Mg2+, or Mn2+. Furthermore, eggs incubated in recombinant fertilin β-EC prior toin vitrofertilization show reduced levels of sperm binding. Finally, we have examined the possible role of integrins on eggs as receptors for fertilin β, since an anti-α6integrin subunit monoclonal antibody, GoH3, has been shown to inhibit sperm binding (E. A. C. Almeidaet al.(1995)Cell81, 1095–1104). We find that: (a) an increased amount of GoH3 epitope on the egg surface does not correlate with an increased ability of the eggs to bind sperm or recombinant fertilin β-EC; (b) the GoH3 antibody has virtually no inhibitory effect on recombinant fertilin β-EC binding; and (c) recombinant fertilin β-EC binding is reduced in the presence of anti-β1integrin antibodies. These results suggest that a β1-containing integrin participates in the binding of recombinant fertilin β-EC to mouse eggs.