Interaction of mouse sperm with purified sperm receptors covalently linked to silica beads.
We describe a solid-phase assay that has permitted further analysis of zona pellucida glycoprotein, ZP3, as sperm receptor and acrosome reaction-inducer during fertilization in mice. The assay employs silica beads that contain epoxy groups to which purified, mouse oocyte ZP3 is covalently linked (ZP3-beads). ZP3-beads were characterized, e.g. by whole-mount autoradiography and flow cytofluorometry, incubated with capacitated mouse sperm under a variety of conditions, and the extent of sperm binding determined by light microscopy. Results of experiments presented suggest the following: (1) sperm bind specifically to ZP3-beads, but not to silica beads either exposed to 2-aminoethanol or derivatized with oocyte ZP2, fetuin or bovine serum albumin. (2) In nearly all cases, only one sperm binds per ZP3-bead and binding occurs via the sperm head. (3) The extent of sperm binding to ZP3-beads is dependent on ZP3 and sperm concentrations, as well as on incubation time and temperature. (4) Sperm binding to ZP3-beads is unaffected by antibodies directed against ZP3, but is inhibited in a reversible manner by treatment of ZP3-beads with galactose oxidase. (5) Only acrosome-intact sperm bind to ZP3-beads but, once bound, sperm can undergo the acrosome reaction, which results in their release from ZP3-beads. (6) Islet-activating protein and 3-quinuclidinyl benzilate, two inhibitors of the zona pellucida-induced acrosome reaction, prevent sperm bound to ZP3-beads from undergoing the acrosome reaction. These results confirm and extend previous studies of sperm-egg interaction in mice, and suggest that the solid-phase assay will be useful for both cellular and biochemical analyses of mammalian fertilization.
Vazquez
M H
MH
Department of Cell and Developmental Biology, Roche Institute of Molecular Biology, Roche Research Center, Nutley, NJ 07110.
Phillips
D M
DM
Wassarman
P M
PM
D000177:Acrosome / Q000378:metabolism; D000818:Animals; D000906:Antibodies; D001665:Binding Sites; D004527:Egg Proteins; D005434:Flow Cytometry; D005692:Galactose Oxidase / Q000378:metabolism; D006023:Glycoproteins / Q000276:immunology / Q000378:metabolism*; D008297:Male; D008562:Membrane Glycoproteins; D051379:Mice; D008855:Microscopy, Electron, Scanning; D010063:Ovum / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D012822:Silicon Dioxide; D013094:Spermatozoa / Q000378:metabolism* / Q000648:ultrastructure; D013696:Temperature; D015044:Zona Pellucida / Q000378:metabolism*; D000072216:Zona Pellucida Glycoproteins
D000906:Antibodies; D004527:Egg Proteins; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; D000072216:Zona Pellucida Glycoproteins; C000606520:Zp2 protein, mouse; C000606521:Zp3 protein, mouse; D012822:Silicon Dioxide; D005692:Galactose Oxidase