Identification of a ZP3-binding protein on acrosome-intact mouse sperm by photoaffinity crosslinking.

Abstract

During the process of fertilization in mammals, sperm bind in a relatively species-specific manner to the zona pellucida (ZP) ofovulated eggs. ZP3, a glycoprotein found in the mouse egg zona pellucida, serves as receptor for sperm during gamete adhesion. We report here that a Mr 56,000 protein found on mouse sperm has properties expected for a sperm component that recognizes and binds to ZP3. This sperm protein is radiolabeled preferentially by a photoactivatable heterobifunctional crosslinker ("Denny-Jaffee reagent") covalently linked to purified ZP3, binds very tightly to ZP3-affinity columns, and is localized to heads of acrosome-intact but not acrosome-reacted sperm. These and other findings suggest that this protein may be a "ZP3-binding protein" that, together with the sperm receptor, supports species-specific binding of mouse sperm to unfertilized eggs.Mammalian eggs are encompassed by a thick extracellular coat called the zona pellucida (ZP) to which sperm bind in a relatively species-specific manner during the course of fertilization (1,2). Sperm bind by their head to sperm receptors present in the ZP. In mice, ZP glycoprotein ZP3 (Mr 83,000) serves as the primary receptor to which acrosome-intact sperm bind (by the plasma membrane overlying their heads) during gamete adhesion (3-6). Sperm bound to ZP3 then undergo the acrosome reaction, a form of exocytosis, which results in loss of plasma membrane overlying the anterior region of the sperm head (4-7). Acrosome-reacted sperm remain bound to the ZP by interacting with another ZP glycoprotein, ZP2 (Mr 120,000), which serves as a secondary sperm receptor (6,8). Bound sperm can then penetrate the ZP, probably by using a proteinase associated with the inner acrosomal membrane, and fuse with egg plasma membrane to form a zygote.ZP3 plays at least two roles during the fertilization process in mice. It serves as primary sperm receptor and as acrosome reaction-inducer. The sperm receptor function of ZP3 apparently is solely dependent on a specific size class of serine/ threonine-(O-) linked oligosaccharides (Mr 3900) present on the glycoprotein (3-6, 9, 10). The oligosaccharides have a galactose residue at their nonreducing terminus (in alinkage with the penultimate sugar) that is essential for sperm receptor function (10,11). The acrosome reaction-inducer function of ZP3 is also dependent on the glycoprotein's O-linked oligosaccharides, but its polypeptide chain plays a role as well (10,12). Therefore, the sperm plasma membrane protein that binds to ZP3 apparently does so by binding to specific carbohydrate determinants on ZP3.Here, we describe results of experiments aimed at identifying the mouse sperm protein that recognizes and binds to ZP3 ("ZP3-binding protein") during gamete adhesion. Purified ZP3 was covalently modified with a radiolabeled, photoactivatable, heterobifunctional crosslinker ("Denny-Jaffee reagent") (13-15), incubated with acrosome-intact and acrosome-reacted sperm, and then subjected to photolysis. Electrophoretic analyses of d...

Sperm arylsulfatase A binds to mZP2 and mZP3 glycoproteins in a nonenzymatic manner

“…The validity of identifying OBF13 stained sperm as AR sperm was first shown by comparing the number of AR sperm assessed by OBF13 reactivity with that obtained by Coomassie blue staining (a more widely used method; Bleil & Wassarman 1990). Supplementary Figure S1 (see section on supplementary data given at the end of this article) indicates that the numbers acquired from the two methods were the same.…”

“…Briefly, capacitated PGC sperm were incubated (30 min, 37 8C, 5% CO 2 ) with six ZP equivalent amounts of solubilized ZP and each individual ZP glycoproteins per microliter of the sperm suspension. The acrosome status of the sperm was determined by Coomassie blue staining (Bleil & Wassarman 1990). Capacitated sperm untreated or treated with ovalbumin or fetuin, which were also subjected to the same electroelution as the ZP glycoproteins, served as negative controls (Carmona et al 2002b), whereas sperm treated with 10 M A23187 were a positive control.…”

Reassessing the role of protein-carbohydrate complementarity during sperm-egg interactions in the mouse

“…A wide variety of sperm surface components have been implicated in binding to the ZP, but only a few have been suggested to function as receptors for ZP3 (Leyton and Saling, 1989;Bleil and Wassarman, 1990). Of these, β1,4-galactosyltransferase (GalT) appears to satisfy the criteria expected of a ZP3 receptor (Shur et al, 2006).…”

Cloning of rat sp56, the homologue of mouse sperm ZP3 receptor–sp56

“…Mouse sp56 was first identified to be responsible for recognition of and binding to mZP3 [5]. Spermegg binding is inhibited in the presence of Anti-sp56 antibody [2,7].…”

“…These candidates include b -1 , 4 g a l a c t o s y l t r a n s f e r a s e [ 3 ] , a 9 5 -K D phosphotyrosine-bearing protein with hexokinase activity [4], and mouse sp56 [5,6]. Mouse sp56 was first identified to be responsible for recognition of and binding to mZP3 by crosslinking and affinity chromatography [5]. The affinity purified sp56 has coordinates of 40 KD under non-reducing conditions and 56 KD under reducing conditions when subjected to 2-D SDS-PAGE [6].…”