Autoradiographic Visualization of the Mouse Egg's Sperm Receptor Bound to Sperm

The extracellular coat, or zona pellucida, of mammal ian eggs contains species-specific receptors to which sperm bind as a prelude to fertilization. In mice, ZP3, one of only three zona pellucida glycoproreins, serves as sperm receptor. Acrosome-intact, but not acrosome-reacted, mouse sperm recognize and interact with specific O-linked oligosaccharides of ZP3 resulting in sperm-egg binding. Binding, in turn, causes sperm to undergo the acrosome reaction; a membrane fusion event that results in loss of plasma membrane at the anterior region of the head and exposure of inner acrosomal membrane with its associated acrosomal contents. Bound, acrosome-reacted sperm are able to penetrate the zona pellucida and fuse with the egg's plasma membrane (fertilization). In the present report, we examined binding of radioiodinated, purified, egg ZP3 to both acrosome intact and acrosome reacted sperm by whole-mount autoradiography. Silver grains due to bound ~25I-ZP3 were found localized to the acrosomal cap region of heads of acrosome intact sperm, but not to heads of acrosomereacted sperm. Under the same conditions, ~25I-fetuin bound at only background levels to heads of both acrosome-intact and -reacted sperm, and ~25I-ZP2, another zona pellucida glycoprotein, bound preferentially to acrosome-reacted sperm. These results provide visual evidence that ZP3 binds preferentially and specifically to heads of acrosome intact sperm; properties expected of the mouse egg's sperm receptor. S HORTLY after mouse sperm and eggs are combined in vitro, under conditions that support fertilization, sperm attach loosely and nonspecifically to the egg's extracellular coat, or zona pellucida (Hartmann et al., 1972). Such attachment can lead to binding; a tight, relatively speciesspecific association between plasma membrane of the sperm head and sperm receptors of the zona pellucida (Hartmann and Hutchison, 1974; Gwatkin, 1977; Wassarman and Bleil, 1982; Bleil and Wassarman, 1983; Wassarman et al., 1985a, b). Subsequently, bound sperm undergo the acrosome reaction, whereby plasma membrane at the anterior region of the sperm head fuses with outer acrosomal membrane, exposing the inner acrosomal membrane and its associated acrosome contents (Saling et al., 1979; Saling and Storey, 1979; Florman and Storey, 1982; Bleil and Wassarman, 1983). Acrosomereacted sperm that remain bound then penetrate the zona pellucida and reach the perivitelline space, perhaps by using the inner acrosomal membrane-associated protease, acrosin (McRorie and Williams, 1974; Green and Hockaday, 1978; Castellani-Ceresa et al., 1983). A single sperm fuses with the egg's plasma membrane (fertilization). The egg responds to fertilization by releasing cortical granule contents (cortical reaction) into the zona pellucida, altering it such that sperm are unable to bind or penetrate this extracellular coat (zona reaction) (Hartmann et al., 1972; Inou6 and Wolf, 1975; Gwatkin and Williams, 1976; Schuel, 1978; Sato, 1979; Bleil and Wassarman, 1980a). The mouse egg's zona pellucida is composed of only three different glycoproteins, called ZPI, ZP2, and ZP3 that are organized into long, interconnected filaments (Bleil and Wassarman, 1980b; Greve and Wassarman, 1985). ZP3 has been identified as the sperm receptor, primarily on the basis of an in vitro "competition assay," and as inducer of the acrosome reaction (Bleil and Wassarman, 1980a, 1983; Wassarman et al., 1985a, b). ZP3 (Mr 83,000) is synthesized and secreted by growing oocytes and consists of an Mr 44,000 polypeptide chain to which three or four N-linked and an undetermined number of O-linked oligosaccharides are added (Bleil and Wassarman, 1980c; Salzmann et al., 1983; Wassarman et al., 1985b). O-Linked oligosaccharides alone account for the sperm receptor activity of ZP3, whereas its acrosome reactioninducing activity is dependent on polypeptide chain as well (Florman et al., 1984; Florman and Wassarman, 1985). Therefore, in mice, it would appear that a single glycoprotein is responsible for both binding and modification of sperm enabling them to reach and fuse with the egg's plasma membrane. In this report, we present autoradiographic evidence sup© The Rockefeller University Press, 0021-9525/86/04/1363/09 $1.00 The Journal of Cell Biology, Volume 102, April 1986 1363-1371 1363 on A ril 5, 2017 D ow nladed fom Published April 1, 1986