Characterization of the binding of recombinant mouse sperm fertilin beta subunit to mouse eggs: evidence for adhesive activity via an egg beta1 integrin-mediated interaction.
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 beta 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 beta plays in fertilization, we have expressed the putative extracellular domain of mouse fertilin beta in bacteria as a fusion protein with maltose-binding protein (hereafter referred to as recombinant fertilin beta-EC) and used two assays to characterize its binding to mouse eggs. Immunocytochemistry was used to examine the localization of recombinant fertilin beta-EC binding. A luminometric assay was also developed to quantify levels of binding of recombinant fertilin beta-EC to single eggs. We find that recombinant fertilin beta-EC binds to the region of the plasma membrane of the egg to which sperm bind, thus providing the first direct evidence that fertilin beta has adhesive properties. Peptides corresponding to the disintegrin domain of fertilin beta reduce its binding to eggs, suggesting that this domain is at least partially involved in the recognition of fertilin beta 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 beta-EC, implicating an egg surface protein as a binding site for recombinant fertilin beta-EC. Binding of recombinant fertilin beta-EC to eggs is also reduced in the absence of divalent cations and is supported by 2.0 mM Ca2+, Mg2+, or Mn2+. Furthermore, eggs incubated in recombinant fertilin beta-EC prior to in vitro fertilization show reduced levels of sperm binding. Finally, we have examined the possible role of integrins on eggs as receptors for fertilin beta, since an anti-alpha6 integrin subunit monoclonal antibody, GoH3, has been shown to inhibit sperm binding (E. A. C. Almeida et al. (1995) Cell 81, 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 beta-EC; (b) the GoH3 antibody has virtually no inhibitory effect on recombinant fertilin beta-EC binding; and (c) recombinant fertilin beta-EC binding is reduced in the presence of anti-beta1 integrin antibodies. These results suggest that a beta1-containing integrin participates in the binding of recombinant fertilin beta-EC to mouse eggs.
Evans
J P
JP
Center for Research on Reproduction and Women's Health, University of Pennsylvania, Philadelphia 19104, USA.
Kopf
G S
GS
Schultz
R M
RM
D051722:ADAM Proteins; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology; D002448:Cell Adhesion; D017931:DNA Primers; D000939:Epitopes; D005260:Female; D000072196:Fertilins; D019012:Integrin beta1 / Q000502:physiology*; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008345:Manganese / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010063:Ovum / Q000502:physiology*; D010446:Peptide Fragments / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D013084:Sperm-Ovum Interactions / Q000187:drug effects; D013094:Spermatozoa / Q000502:physiology*; D015044:Zona Pellucida / Q000502:physiology
D000911:Antibodies, Monoclonal; D002413:Cations, Divalent; D017931:DNA Primers; D000939:Epitopes; D019012:Integrin beta1; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D008345:Manganese; D051722:ADAM Proteins; C000606458:Adam2 protein, mouse; D000072196:Fertilins; D008666:Metalloendopeptidases; D008274:Magnesium; D002118:Calcium