DEVELOPMENTAL
147,440-444 (1991)
BIOLOGY
Aggregation
of ,&I ,4-Galactosyltransferase on Mouse Sperm Induces the Acrosome Reaction MARYBETHMACEK,LINDAC.LOPEZ,ANDBARRY
Department of Biochemistry
D. SHUR~
and Molecular Biology, University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030 Accepted July 8, 1991
P-1,4-Galactosyltransferase (GalTase) is present on the surface of mouse sperm, where it functions during fertilization by binding to oligosaccharide residues in the egg zona pellucida. The specific oligosaccharide substrates for sperm GalTase reside on the glycoprotein ZP3, which possesses both sperm-binding and acrosome reaction-inducing activity. A variety of reagents that perturb sperm GalTase activity inhibit sperm binding to the zona pellucida, including UDP-galactose, N-acetylglucosamine, a-lactalbumin, and anti-GalTase Fab fragments. However, none of these reagents are able to cross-link GalTase within the membrane nor are they able to induce the acrosome reaction. On the other hand, intact anti-GalTase IgG blocks sperm-zona binding as well as induces the acrosome reaction. Anti-GalTase IgG induces the acrosome reaction by aggregating GalTase on the sperm plasma membrane, as shown by the inability of anti-GalTase Fab fragments to induce the acrosome reaction unless cross-linked with goat anti-rabbit IgG. These data suggest that zona pellucida oligosaccharides induce the acrosome reaction by clustering GalTase on the sperm surface. o 1991 Academic
Press, Inc.
INTRODUCTION
Gamete recognition and adhesion are mediated by carbohydrate-binding proteins on the sperm surface that have high affinity and specificity for complex glycoconjugates in the extracellular coat of the egg (Macek and Shur, 1988). Examples of sperm surface carbohydrate-binding proteins that have been suggested to participate in gamete recognition include lectins, glycosyltransferases, and glycosidases. The ligands for these receptors are usually specific oligosaccharide chains bound to large proteoglycan-like glycoconjugates, as on sea urchin eggs (Ruiz-Bravo and Lennarz, 1986), or on more conventional complex-type glycoproteins, as within the murine (Bleil and Wassarman, 1980) and porcine (Sacco et aZ., 1989) zonae pellucidae. The molecular basis for species specificity of sperm-egg adhesion is not well-defined. Such specificity could result from entirely distinct classes of species-specific gamete receptors, or rather, from subtle differences in egg coat oligosaccharide sequences and/or conformation that are recognized by closely related sperm surface receptors (Shur, 1989a). Among mammals, gamete recognition is best understood in the mouse. Sperm binding activity of the mu1 To whom correspondence should be addressed at Box 11’7, Department of Biochemistry and Molecular Biology, M.D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX ‘77030. 0012-1606191 $3.00 Copyright All rights
0 1991 by Academic Press, Inc. of reproduction in any form reserved.
440
rine zona pellucida is conferred by oligosaccharide chains that reside on the glycoprotein ZP3 (Florman and Wassarman, 1985). A number of mouse sperm proteins have been suggested to serve as receptors for the zona pellucida (Shur, 1989a). However, the most compelling evidence suggests that gamete binding is mediated by the sperm surface enzyme P-1,4-galactosyltransferase (GalTase) that recognizes terminal N-acetylglucosamine (GlcNAc) residues on the zona (for review, see Shur, 198913). This notion is supported by the fact that a variety of reagents that perturb GalTase activity inhibit sperm-zona binding, including anti-GalTase IgG and Fab fragments, competitive substrates, substrate analogs, the sugar donor substrate UDP-galactose, and the modifier protein a-la&albumin, as do glycosidase digestions and pregalactosylation of the zona (Shur and Hall, 1982a,b; Lopez et aZ., 1985). Biochemical and immunofluorescence evidence shows that GalTase is localized to the plasma membrane overlying the dorsal aspect of the acrosome, where sperm initiate binding to the zona (Lopez et al., 1985). Sperm GalTase has been purified to apparent homogeneity and competitively inhibits sperm-zona binding (Shur and Neely, 1988), analogous to the inhibition of sperm-zona binding by solubilized ZP3 oligosaccharides (Florman and Wassarman, 1985). Recent studies show that sperm GalTase selectively binds to ZP3, and that the sperm receptor activity of ZP3 is dependent upon its interaction with GalTase (Miller et ab, 1990). Thus, at least one
MACEK,
LOPEZ,
AND SHUR
aspect of gamete recognition involves the binding of sperm GalTase to ZP3 oligosaccharides within the zona pellucida. Sperm binding to the zona pellucida induces the acrosome reaction, an activity that is also conferred by ZP3 (Bleil and Wassarman, 1983). Therefore, solubilized intact ZP3 has both sperm-binding and acrosome-reaction-inducing activities. However, ZP3 glycopeptides or isolated oligosaccharides are unable to induce the acrosome reaction, although they still retain the ability to inhibit sperm binding (Florman et aZ., 1984). These results imply that ZP3 induces the acrosome reaction by virtue of multiple sperm-binding oligosaccharides bound to a protein core, which are able to cross-link sperm surface receptors, such as GalTase. Alternatively, ZP3 may induce the acrosome reaction via structures distinct from sperm-binding oligosaccharides. Recent observations are consistent with the former possibility, since ZP3 glycopeptides that inhibit sperm-zona binding can elicit the acrosome reaction after being cross-linked with anti-ZP3 antibodies (Leyton and Saling, 1989). Coincident with the acrosome reaction, GalTase is redistributed to the lateral aspect of the sperm head where it may participate in secondary aspects of sperm adhesion to the zona pellucida (Lopez and Shur, 1987). Redistribution of sperm GalTase is one example of the migration of sperm surface components that occurs during various aspects of spermatogenesis, capacitation, and the acrosome reaction (Myles and Primakoff, 1984). During the course of these earlier studies, we found that pretreatment of sperm with anti-GalTase IgG induced the acrosome reaction. This raised the possibility that zona pellucida oligosaccharides induce the acrosome reaction by aggregating GalTase on the sperm surface. This possibility was examined in studies reported here, in which we show that monovalent reagents that perturb GalTase activity and inhibit sperm-zona binding do not induce the acrosome reaction, whereas the acrosome reaction can be induced by directly crosslinking sperm GalTase within the plasma membrane. MATERIALS
441
GalTase Induces the Acrosme Reaction
AND METHODS
All reagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise noted. Anti-GalTase IgG was prepared by immunizing rabbits with affinity-purified bovine milk GalTase and made monospecific by application on a GalTase affinity column as previously described (Lopez et ah, 1985). The resulting monospecific anti-GalTase IgG was identical to that used previously to detect GalTase on the sperm surface and to inhibit sperm-zona binding in a dose-dependent manner (Lopez et ah, 1985). Affinity-purified bovine
GaiTassma)ctivity 1
2,964
26 -
M
1
2
3
4
5
FIG. 1. Anti-GalTase IgG selectively recognizes GalTase on mouse sperm. This figure illustrates the purification of mouse sperm GalTase as described by Shur and Neely (1988). Lanes 1,4, and 5 (-1 pg protein/lane) show, respectively, the total detergent-solubilized sperm extract, the protein profile applied to the cu-lactalbumin affinity column, and the protein eluted from the affinity column that possesses GalTase activity after renaturation. The GalTase activity is shown for each gel piece taken from a parallel unstained lane. For details of the sperm GalTase purification, see Shur and Neely (1988). Anti-GalTase IgG detects a protein in the detergent-solubilized sperm extract (30 pg protein/lane) that comigrates with the purified GalTase protein (lane 2); normal rabbit IgG shows background reactivity (lane 3). The bound IgG was detected with HRP-conjugated goat antirabbit IgG. Western immunoblotting was performed as described in Scully et al. (1987).
milk GalTase was used as immunogen due to limiting quantities of affinity-purified mouse sperm GalTase. However, the mouse sperm and bovine milk GalTases are highly homologous. They share 82% amino acid sequence identity; are both P-1,4-specific GalTases; have similar molecular weight, substrate specificity, and kinetics; and inhibit sperm-zona binding to similar degrees (Shur and Neely, 1988; Shaper et al, 1988). Furthermore, the monospecific anti-GalTase IgG specifically recognizes GalTase on mouse sperm by a variety of criteria, including immunoprecipitation and inhibition of GalTase enzyme activity, immunoprecipitation of a single iodinated cell surface protein of -60 kDa, and Western immunoblotting of a single protein of -60 kDa, the same molecular weight as affinity-purified sperm GalTase (Fig. 1; Lopez et aZ., 1985; Shur and Neely, 1988). Anti-GalTase Fab fragments were prepared using immobilized papain as described by the manufacturer (Pierce Chemical Co., Rockford, IL). The purity of the Fab preparation was assessed by SDS-polyacrylamide
442
DEVELOPMENTAL
BIOLOGY
gel electrophoresis. HS-21 monoclonal antibody was kindly supplied by Dr. Don Wolf (Oregon Primate Research Center). All IgG and Fab preparations were supplemented with 1 mg/ml bovine serum albumin (final concentration) to improve stability and limit nonspecific adsorption to sperm. Sperm preincubation and analysis of the acrosome reaction. Sperm were collected from the caudae epididymides of one to two, lo-week-old CD-l mice (Charles River Breeders, Wilmington, MA) and dispersed in 5-10 ml of a modified Krebs-Ringer bicarbonate solution containing sodium pyruvate (1 m&f), sodium lactate (25 mM), glucose (5.56 m&f), and bovine serum albumin (20 mg/ ml). Particulate material was removed by filtration through a Nitex cloth, and the sperm (-2.5 X 107/ml) were capacitated for 30 min at 37°C as described previously (Lopez et ah, 1985). After capacitation, the sperm were washed by centrifugation and resuspended in 1 ml Krebs-Ringer solution, from which 25-~1 droplets were removed and added to 25 ~1 of the various test reagents (2x final concentration in Hepes-buffered saline (150 mMNaC1, 10 mMHepes, pH 7.2)) or buffer, as described in the text. Some samples received calcium ionophore A23187 (10 PM final) (Sigma) or solubilized zonae pellucidae (2 zonae/Fl final) as positive controls for the acrosome reaction. All sperm suspensions were incubated for 45 min at 37°C. In some instances when sperm were incubated with Fab fragments, 30 ~1 of the sperm suspension was pelleted by centrifugation and resuspended in 200 ~1 Krebs-Ringer solution to which 2.0 pug goat anti-rabbit IgG (Vector Laboratories, Inc., Burlingame, CA) was added to cross-link the bound Fab fragments and incubated for another 30 min at 37°C. After incubation with the various test reagents, 0.51.0 ml of Krebs-Ringer solution was added (in some instances the suspension was incubated for another 45 min at 37”C), and the sperm were pelleted by centrifugation, washed in another 0.5 ml of Krebs-Ringer solution, and resuspended in 25-50 ~1 Hepes-buffered saline. Sperm samples were applied to glass slides for analysis of the acrosome reaction. Following these procedures, sperm motility was low in all incubations (5-20s forward motile sperm). However, the degree of motility was similar between control and experimental incubations and was unrelated to the degree of acrosome reactions (i.e., incubation with GlcNAc produced the lowest motility but produced control levels of acrosome reactions, see Fig. 2). The acrosomal status of treated sperm was determined by indirect immunofluorescence using the mouse monoclonal antibody HS-21 as described (Lopez and Shur, 1987; Shur and Neely, 1988). HS-21 antibody reacts only with acrosome-intact sperm; loss of anti-HS-21 reactivity is indicative of acrosome-reacted
VOLUME
147, 1991
FIG. 2. The acrosome reaction is induced by cross-linking sperm surface GalTase. Sperm were capacitated for 30 min and incubated in the indicated reagents for an additional 45 min at 37°C as described under Materials and Methods. In this figure, the final concentration of the reagents were as follows: A23187 (10 r&f); UDP-galactose (2.0 mM); GlcNAc (50 mm; chitotriose (10 m&f); a-lactalbumin (10 mg/ ml); preimmune and anti-GalTase IgG (200 PgYml); preimmune and anti-GalTase Fab (1.6 mg/ml). After incubation in the indicated reagents, the acrosomal status was determined by the presence or absence of HS-21 immunoreactivity. All incubations were conducted in duplicate for each experiment, and at least 100 sperm in a minimum of four fields were scored for each incubation. The error bars represent the range within each pair of duplicates. Data shown in this figure are representative of two to four replicate experiments, each producing similar results. The range between experiments varied from +0.5+7.0% under experimental conditions, and was +lO% for ionophoretreated controls.
sperm (Wolf et al, 1985). At least 100 sperm in a minimum of four fields were scored for each sample. RESULTS
AND DISCUSSION
Monovalent reagents that perturb GalTase activity do not induce the acrowme reaction, Sperm were incubated in medium containing a variety of reagents that perturb GalTase activity, each of which has been shown previously to inhibit sperm-zona binding in a dose-dependent manner (Shur and Hall, 1982a,b; Lopez et al, 1985), and the effects on the acrosome reaction were determined. As shown in Fig. 2, neither the sugar donor substrate UDP-galactose (2.0 mM final concentration), the competitive substrates N-acetylglucosamine (50 mM) and N-acetylchitotriose (10 mM), nor the substrate modifier protein cy-lactalbumin (10 mg/ml) had any significant
MACEK,
LOPEZ,
AND SHUR
GalTase Induces th.e Acrosme Reaction
443
els, although they inhibit sperm-zona binding in a dosedependent manner (1.6 and 3.2 mg/ml final concentration assayed, which inhibits sperm-zona binding by 59 and 76%) respectively (Lopez et a& 1985) (Fig. 2). Thus, multivalency is required for anti-GalTase antibodies to induce the acrosome reaction. This was confirmed by sequentially treating sperm with anti-GalTase Fab fragments followed by goat anti-rabbit IgG to cross-link the bound Fab fragments. Under these conditions, acrosome reactions were induced to levels similar to that produced by intact anti-GalTase IgG (Fig. 2). For these studies, controls included sperm preincubated in buffer (no Fab) followed by goat anti-rabbit IgG, sperm preincubated with preimmune Fab followed by goat anti-rabbit IgG, and sperm preincubated with Fab (either antiGalTase or preimmune) followed by buffer (no goat anti-rabbit IgG). In all instances, the level of acrosomereacted sperm was similar to that produced by Fab fragments alone (Fig. 2). Collectively, these results show that the mouse acrosome reaction can be induced by aggregating GalTase on Monospecific anti-GalTase IgG induces the acrosorne the sperm surface. These results have been confirmed by reaction. Monospecific anti-GalTase IgG has been others using anti-GalTase IgG to induce the acrosome shown previously to inhibit sperm-zona binding in a reaction in mouse sperm as assayed by CTC fluorescence dose-dependent manner (Lopez et al., 1985). When sperm (H. Florman, personal communication). Reagents that were incubated in 200 pg/ml (final concentration) monoare not able to aggregate GalTase, but which are still specific anti-GalTase IgG, a concentration that inhibits able to inhibit sperm zona binding by perturbing Galsperm-zona binding by -60% relative to control IgG, Tase activity, do not induce the acrosome reaction. 75% of the sperm were acrosome-reacted compared These results are reminiscent of the ability of ZP3 glywith 18% in buffer-treated controls and 32% in preimcopeptides to inhibit sperm-zona binding, presumably mune IgG-treated sperm. The effects of the anti-Galby competing for the GalTase binding site (Miller et al, Tase IgG were grossly dose dependent (i.e., 200 pg/ml 1990), without inducing the acrosome reaction (Florman produced an additional 43% acrosome-reacted sperm et al, 1984). However, intact ZP3, with multiple oligosacabove preimmune IgG controls, 33 pg/ml produced 11% charide chains, inhibits sperm-zona binding and inabove controls, 20 pg/ml produced no significant differduces the acrosome reaction, results that parallel the ence from controls). The increased percentage of acro- effects of anti-GalTase IgG. The ability of anti-ZP3 antisome-reacted sperm in the presence of preimmune IgG, bodies to induce the acrosome reaction in sperm preinas well as in the presence of Fab fragments (see below) cubated with “monovalent” ZP3 glycopeptides is consiswas reproducibly higher than in control sperm, but was tent with this notion (Leyton and Saling, 1989). always significantly lower than that produced by idenHow cross-linking surface GalTase induces the acrotical concentrations of anti-GalTase IgG. Incubation some reaction awaits further study. Analysis of the priwith whole serum (1:l dilution), rather than isolated mary sequence of GalTase obtained by molecular clonanti-GalTase IgG, had an even more dramatic effect on ing, along with appropriate biochemical studies, shows the extent of acrosome reaction (89% reacted). GalTase to be an integral transmembrane protein on Anti-GalTase IgG induces the acrosmne reaction by ag- sperm (Shaper et aZ., 1988; Lopez et al, 1985; 1989; Shur gregating GalTase. As shown above, anti-GalTase IgG and Neely, 1988). Clustering of transmembrane recepinduced the acrosome reaction, whereas monovalent re- tors on a variety of cell types elicits metabolic responses agents that also inhibit sperm-zona binding did not. appropriate to the responding cell (O’Brien et al, 1987). Since the anti-GalTase IgG is bivalent, the possibility Similarly, a variety of sperm surface components may was tested that anti-GalTase IgG induced the acrosome be able to induce the acrosome reaction if aggregated by reaction by aggregating sperm surface GalTase. In this multivalent ligands. However, during fertilization, the regard, monovalent Fab fragments of anti-GalTase IgG physiologically relevant inducer of the acrosome reacdid not induce the acrosome reaction above control lev- tion is ZP3, thus ensuring the appropriate temporal effect on the extent of the acrosome reaction. Previous studies have shown that these reagent levels inhibit sperm-zona binding from >60 to >90% of control levels (Shur and Hall, 1982a,b; Lopez et a& 1985). The control reagents, which do not inhibit sperm-zona binding, included UDP-glucose, glucose, and lysozyme at concentrations equivalent to their respective experimental reagents. Experiments in which the concentration of UDP-galactose was varied (0.02-2.0 mJY), or 1 mM MnCl, was included to stimulate enzymatic activity, or sperm were incubated in a combination of UDP-galactose, MnCl, and N-acetylglucosamine (conditions that generate optimal enzyme activity, Shur and Neely, 1988) produced control levels of acrosome reactions (data not shown). In each experiment, treatment with the ionophore A23187 served as a positive control and induced acrosome reactions 49-71s above control levels (58% in Fig. 2). In a parallel experiment, solubilized zonae pellucidae (2 zonae/pl final) induced the acrosome reaction in 52% of treated sperm compared to 16% acrosomereacted sperm in controls (Lopez and Shur, 1987).
444
DEVELOPMENTAL
BIOLOGY
specificity. The basal level of acrosome-reacted sperm present in the epididymis may reflect spurious aggregation of GalTase residues by multivalent glycoconjugates (Shur and Hall, 1982a). In any event, it appears that aggregation of GalTase moieties within the plasma membrane by multiple ZP3 oligosaccharides or by antibodies results in fusion and vesiculation of the plasma membrane and outer acrosomal membrane, culminating in the acrosome reaction. Similarly, in sea urchins, specific monoclonal antibodies are able to induce the acrosome reaction by aggregating the appropriate receptors on sperm (Trimmer et CAL,1987). Evidence suggests that ZP3 induces the acrosome reaction by activating guanine nucleotide-binding proteins (Endo et CAL,1988), which usually associate with membrane receptors having multiple transmembrane domains (O’Dowd et aZ., 1988). However, there are examples where G-proteins can be activated directly by surface receptors having one transmembrane spanning segment, such as the IGF-II/Man-6-P receptor (Okamoto et aL, 1990). Future studies will address whether the cytoplasmic tail of GalTase activates G-proteins directly, analogous to the IGF-II/Man-6-P receptor, or indirectly, eventually leading to exocytosis of the acrosome. The authors are grateful to Dr. Don Wolf for supplying HS-21 antibody, to Dr. Harvey Florman for repeating the studies of anti-GalTase IgG effects on the mouse sperm acrosome reaction, and to Dr. David J. Miller and Ms. Deborah Mansfield for critical reading of the manuscript. This work was supported by NIH Grant HD 23479 to B.D.S. REFERENCES BLEIL, J. D., and WASSARMAN, P. M. (1980). Mammalian sperm-egg interaction: Identification of a glycoprotein in mouse egg zonae pellucidae possessing receptor activity for sperm. Cell 20,873~882. BLEIL, J. D., and WASSARMAN, P. M. (1983). Sperm-egg interactions in the mouse: Sequence of events and induction of the acrosome reaction by a zona pellucida glycoprotein. Dev. BioL 95,317-324. ENDO, Y., LEE, M. A., and KOPF, G. S. (1988). Characterization of an islet-activating protein-sensitive site in mouse sperm that is involved in the zona pellucida-induced acrosome reaction. Dev. BtiL 129,12-24. FLORMAN, H. M., BECHTOL, K. B., and WASSARMAN, P. M. (1984). Enzymatic dissection of the functions of the mouse egg’s receptor for sperm. Deu. BioL 106,243-255. FLORMAN, H. M., and WASSARMAN, P. M. (1985). O-linked oligosaccharides of mouse egg ZP3 account for its sperm receptor activity. CeU 41,313-324. LEYTON, L., and SALING, P. (1989). Evidence that aggregation of mouse sperm receptors by ZP3 triggers the acrosome reaction. J. CeU BioL 108.2163-2168. LOPEZ, L. C., BAYNA,
E. M., LITOFF, D., SHAPER, N. L., SHAPER, J. H., and SHUR, B. D. (1985). The receptor function of mouse sperm surface galactosyltransferase during fertilization. J. Cell BioL 101, 1501-1510.
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LOPEZ, L. C., MAILLET, C. M., OLESZKOWICZ,K., and SHUR, B. D. (1989). Cell surface and golgi pools of j3-1,4-galactosyltransferase are differentially regulated during embryonal carcinoma cell differentiation. Mol. CelL BioL 9,2370-2377. LOPEZ, L. C., and SHUR, B. D. (1987). Redistribution of the mouse egg receptor on sperm following the acrosome reaction. J. Cell BioL 105, 1663-1670. MACEK, M. B., and SHUR, B. D. (1988). Protein-carbohydrate complementarity in mammalian gamete recognition. Gamete Res. 20,93109. MILLER, D. J., MACEK, M. B., and SHUR, B. D. (1990). ZP3 recognizes galactosyltransferase on mouse spermatozoa. J. Cell BioL 111,489a. MYLES, D. G., and PRIMAKOFF, P. (1984). Localized surface antigens of guinea pig sperm migrate to new regions prior to fertilization. J. Cell BioL 99, 1634-1641.
O’BRIEN, R. M., SOOS, M. A., and SIDDLE, K. (1987). Monoclonal antibodies to the insulin receptor stimulate the intrinsic tyrosine kinase activity by cross-linking receptor molecules. EMBO .I 6,4003-4010. O’DOWD, B. F., HNATOWICH, M., REGAN, J. W., LEADER, W. M., CARON, M. G., and LEFKOWITZ, R. J. (1988). Site-directed mutagenesis of the cytoplasmic domains of the human beta 2-adrenergic receptor. Localization of regions involved in G protein-receptor coupling. .I BioL Chem. 263, 15,985-15,992. OKAMOTO, T., KATADA, T., MURAYAMA, Y., UI, M., OGATA, E., and NISHIMOTO, I. (1990). A simple structure encodes G protein-activating function of the IGF-II/mannose-6-phosphate receptor. Cell 62,709717. RUIZ-BRAVO, N., and LENNARZ, W. J. (1986). Isolation and characterization of proteolytic fragments of the sea urchin sperm receptor that retain species specificity. Den BioL 118,202-208. SACCO, A. G., YUREWICZ, E. C., SUBRAMA.NIAN, M. G., and MATZAT, P. D. (1989). Porcine zona pellucida: Association of sperm receptor activity with the oc-glycoprotein component of the M, = 55,000 family. BioL Reprod 41, 523-532. SCULLY, N. F., SHAPER, J. H., and SHUR, B. D. (1987). Spatial and temporal expression of cell surface galactosyltransferase during mouse spermatogenesis and epididymal maturation. Dev. BioL 124, 111-124. SHAPER, N. L., HOLLIS, G. F., DOUGLAS, J. G., KIRSCH, I. R., and SHAPER, J. H. (1988). Characterization of the full length cDNA for murine @-1,4-galactosyltransferase. Novel features at the 5’-end predict two translational start sites at two in-frame AUGs. J. BioL Chem. 263,10,420-10,428. SHUR, B. D. (1989a). Glycoconjugates as mediators of cellular interactions during development. Cum: Opin Cell Biology 1,905-912. SHUR, B. D. (1989b). Expression and function of cell surface galactosyltransferase. Biochim. Biophys. Actu 988,389-409. SHUR, B. D., and HALL, N. G. (1982a). Sperm surface galactosyltransferase activities during in vitro capacitation. J. CeU BioL 95, 567573. SHUR, B. D., and Hw, N. G. (1982b). A role for mouse sperm surface galactosyltransferase in sperm binding to the egg zona pellucida. J. Cell BioL 95,574-579.
SHUR, B. D., and NEELY, C. A. (1988). Plasma membrane association, purification and characterization of mouse sperm fl-1,4-galactosyltransferase. .I BioL Chem. 263,17,706-17,714. TRIMMER, J. S., EDNA, Y., SCHACKMANN, R. W., MEINHOF, C.-G., and VAWUIER, V. D. (1987). Characterization of a monoclonal antibody that induces the acrosome reaction of sea urchin sperm. J. CeUBioL 105,1121-1128. WOLF, D. P., BOLDT, J., BYRD, W., and BECHTOL, K. B. (1985). Acrosoma1 status evaluation in human ejaculated sperm with monoclonal antibodies. BioL Reprod 32,1157-1162.
147,440-444 (1991)
BIOLOGY
Aggregation
of ,&I ,4-Galactosyltransferase on Mouse Sperm Induces the Acrosome Reaction MARYBETHMACEK,LINDAC.LOPEZ,ANDBARRY
Department of Biochemistry
D. SHUR~
and Molecular Biology, University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030 Accepted July 8, 1991
P-1,4-Galactosyltransferase (GalTase) is present on the surface of mouse sperm, where it functions during fertilization by binding to oligosaccharide residues in the egg zona pellucida. The specific oligosaccharide substrates for sperm GalTase reside on the glycoprotein ZP3, which possesses both sperm-binding and acrosome reaction-inducing activity. A variety of reagents that perturb sperm GalTase activity inhibit sperm binding to the zona pellucida, including UDP-galactose, N-acetylglucosamine, a-lactalbumin, and anti-GalTase Fab fragments. However, none of these reagents are able to cross-link GalTase within the membrane nor are they able to induce the acrosome reaction. On the other hand, intact anti-GalTase IgG blocks sperm-zona binding as well as induces the acrosome reaction. Anti-GalTase IgG induces the acrosome reaction by aggregating GalTase on the sperm plasma membrane, as shown by the inability of anti-GalTase Fab fragments to induce the acrosome reaction unless cross-linked with goat anti-rabbit IgG. These data suggest that zona pellucida oligosaccharides induce the acrosome reaction by clustering GalTase on the sperm surface. o 1991 Academic
Press, Inc.
INTRODUCTION
Gamete recognition and adhesion are mediated by carbohydrate-binding proteins on the sperm surface that have high affinity and specificity for complex glycoconjugates in the extracellular coat of the egg (Macek and Shur, 1988). Examples of sperm surface carbohydrate-binding proteins that have been suggested to participate in gamete recognition include lectins, glycosyltransferases, and glycosidases. The ligands for these receptors are usually specific oligosaccharide chains bound to large proteoglycan-like glycoconjugates, as on sea urchin eggs (Ruiz-Bravo and Lennarz, 1986), or on more conventional complex-type glycoproteins, as within the murine (Bleil and Wassarman, 1980) and porcine (Sacco et aZ., 1989) zonae pellucidae. The molecular basis for species specificity of sperm-egg adhesion is not well-defined. Such specificity could result from entirely distinct classes of species-specific gamete receptors, or rather, from subtle differences in egg coat oligosaccharide sequences and/or conformation that are recognized by closely related sperm surface receptors (Shur, 1989a). Among mammals, gamete recognition is best understood in the mouse. Sperm binding activity of the mu1 To whom correspondence should be addressed at Box 11’7, Department of Biochemistry and Molecular Biology, M.D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX ‘77030. 0012-1606191 $3.00 Copyright All rights
0 1991 by Academic Press, Inc. of reproduction in any form reserved.
440
rine zona pellucida is conferred by oligosaccharide chains that reside on the glycoprotein ZP3 (Florman and Wassarman, 1985). A number of mouse sperm proteins have been suggested to serve as receptors for the zona pellucida (Shur, 1989a). However, the most compelling evidence suggests that gamete binding is mediated by the sperm surface enzyme P-1,4-galactosyltransferase (GalTase) that recognizes terminal N-acetylglucosamine (GlcNAc) residues on the zona (for review, see Shur, 198913). This notion is supported by the fact that a variety of reagents that perturb GalTase activity inhibit sperm-zona binding, including anti-GalTase IgG and Fab fragments, competitive substrates, substrate analogs, the sugar donor substrate UDP-galactose, and the modifier protein a-la&albumin, as do glycosidase digestions and pregalactosylation of the zona (Shur and Hall, 1982a,b; Lopez et aZ., 1985). Biochemical and immunofluorescence evidence shows that GalTase is localized to the plasma membrane overlying the dorsal aspect of the acrosome, where sperm initiate binding to the zona (Lopez et al., 1985). Sperm GalTase has been purified to apparent homogeneity and competitively inhibits sperm-zona binding (Shur and Neely, 1988), analogous to the inhibition of sperm-zona binding by solubilized ZP3 oligosaccharides (Florman and Wassarman, 1985). Recent studies show that sperm GalTase selectively binds to ZP3, and that the sperm receptor activity of ZP3 is dependent upon its interaction with GalTase (Miller et ab, 1990). Thus, at least one
MACEK,
LOPEZ,
AND SHUR
aspect of gamete recognition involves the binding of sperm GalTase to ZP3 oligosaccharides within the zona pellucida. Sperm binding to the zona pellucida induces the acrosome reaction, an activity that is also conferred by ZP3 (Bleil and Wassarman, 1983). Therefore, solubilized intact ZP3 has both sperm-binding and acrosome-reaction-inducing activities. However, ZP3 glycopeptides or isolated oligosaccharides are unable to induce the acrosome reaction, although they still retain the ability to inhibit sperm binding (Florman et aZ., 1984). These results imply that ZP3 induces the acrosome reaction by virtue of multiple sperm-binding oligosaccharides bound to a protein core, which are able to cross-link sperm surface receptors, such as GalTase. Alternatively, ZP3 may induce the acrosome reaction via structures distinct from sperm-binding oligosaccharides. Recent observations are consistent with the former possibility, since ZP3 glycopeptides that inhibit sperm-zona binding can elicit the acrosome reaction after being cross-linked with anti-ZP3 antibodies (Leyton and Saling, 1989). Coincident with the acrosome reaction, GalTase is redistributed to the lateral aspect of the sperm head where it may participate in secondary aspects of sperm adhesion to the zona pellucida (Lopez and Shur, 1987). Redistribution of sperm GalTase is one example of the migration of sperm surface components that occurs during various aspects of spermatogenesis, capacitation, and the acrosome reaction (Myles and Primakoff, 1984). During the course of these earlier studies, we found that pretreatment of sperm with anti-GalTase IgG induced the acrosome reaction. This raised the possibility that zona pellucida oligosaccharides induce the acrosome reaction by aggregating GalTase on the sperm surface. This possibility was examined in studies reported here, in which we show that monovalent reagents that perturb GalTase activity and inhibit sperm-zona binding do not induce the acrosome reaction, whereas the acrosome reaction can be induced by directly crosslinking sperm GalTase within the plasma membrane. MATERIALS
441
GalTase Induces the Acrosme Reaction
AND METHODS
All reagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise noted. Anti-GalTase IgG was prepared by immunizing rabbits with affinity-purified bovine milk GalTase and made monospecific by application on a GalTase affinity column as previously described (Lopez et ah, 1985). The resulting monospecific anti-GalTase IgG was identical to that used previously to detect GalTase on the sperm surface and to inhibit sperm-zona binding in a dose-dependent manner (Lopez et ah, 1985). Affinity-purified bovine
GaiTassma)ctivity 1
2,964
26 -
M
1
2
3
4
5
FIG. 1. Anti-GalTase IgG selectively recognizes GalTase on mouse sperm. This figure illustrates the purification of mouse sperm GalTase as described by Shur and Neely (1988). Lanes 1,4, and 5 (-1 pg protein/lane) show, respectively, the total detergent-solubilized sperm extract, the protein profile applied to the cu-lactalbumin affinity column, and the protein eluted from the affinity column that possesses GalTase activity after renaturation. The GalTase activity is shown for each gel piece taken from a parallel unstained lane. For details of the sperm GalTase purification, see Shur and Neely (1988). Anti-GalTase IgG detects a protein in the detergent-solubilized sperm extract (30 pg protein/lane) that comigrates with the purified GalTase protein (lane 2); normal rabbit IgG shows background reactivity (lane 3). The bound IgG was detected with HRP-conjugated goat antirabbit IgG. Western immunoblotting was performed as described in Scully et al. (1987).
milk GalTase was used as immunogen due to limiting quantities of affinity-purified mouse sperm GalTase. However, the mouse sperm and bovine milk GalTases are highly homologous. They share 82% amino acid sequence identity; are both P-1,4-specific GalTases; have similar molecular weight, substrate specificity, and kinetics; and inhibit sperm-zona binding to similar degrees (Shur and Neely, 1988; Shaper et al, 1988). Furthermore, the monospecific anti-GalTase IgG specifically recognizes GalTase on mouse sperm by a variety of criteria, including immunoprecipitation and inhibition of GalTase enzyme activity, immunoprecipitation of a single iodinated cell surface protein of -60 kDa, and Western immunoblotting of a single protein of -60 kDa, the same molecular weight as affinity-purified sperm GalTase (Fig. 1; Lopez et aZ., 1985; Shur and Neely, 1988). Anti-GalTase Fab fragments were prepared using immobilized papain as described by the manufacturer (Pierce Chemical Co., Rockford, IL). The purity of the Fab preparation was assessed by SDS-polyacrylamide
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gel electrophoresis. HS-21 monoclonal antibody was kindly supplied by Dr. Don Wolf (Oregon Primate Research Center). All IgG and Fab preparations were supplemented with 1 mg/ml bovine serum albumin (final concentration) to improve stability and limit nonspecific adsorption to sperm. Sperm preincubation and analysis of the acrosome reaction. Sperm were collected from the caudae epididymides of one to two, lo-week-old CD-l mice (Charles River Breeders, Wilmington, MA) and dispersed in 5-10 ml of a modified Krebs-Ringer bicarbonate solution containing sodium pyruvate (1 m&f), sodium lactate (25 mM), glucose (5.56 m&f), and bovine serum albumin (20 mg/ ml). Particulate material was removed by filtration through a Nitex cloth, and the sperm (-2.5 X 107/ml) were capacitated for 30 min at 37°C as described previously (Lopez et ah, 1985). After capacitation, the sperm were washed by centrifugation and resuspended in 1 ml Krebs-Ringer solution, from which 25-~1 droplets were removed and added to 25 ~1 of the various test reagents (2x final concentration in Hepes-buffered saline (150 mMNaC1, 10 mMHepes, pH 7.2)) or buffer, as described in the text. Some samples received calcium ionophore A23187 (10 PM final) (Sigma) or solubilized zonae pellucidae (2 zonae/Fl final) as positive controls for the acrosome reaction. All sperm suspensions were incubated for 45 min at 37°C. In some instances when sperm were incubated with Fab fragments, 30 ~1 of the sperm suspension was pelleted by centrifugation and resuspended in 200 ~1 Krebs-Ringer solution to which 2.0 pug goat anti-rabbit IgG (Vector Laboratories, Inc., Burlingame, CA) was added to cross-link the bound Fab fragments and incubated for another 30 min at 37°C. After incubation with the various test reagents, 0.51.0 ml of Krebs-Ringer solution was added (in some instances the suspension was incubated for another 45 min at 37”C), and the sperm were pelleted by centrifugation, washed in another 0.5 ml of Krebs-Ringer solution, and resuspended in 25-50 ~1 Hepes-buffered saline. Sperm samples were applied to glass slides for analysis of the acrosome reaction. Following these procedures, sperm motility was low in all incubations (5-20s forward motile sperm). However, the degree of motility was similar between control and experimental incubations and was unrelated to the degree of acrosome reactions (i.e., incubation with GlcNAc produced the lowest motility but produced control levels of acrosome reactions, see Fig. 2). The acrosomal status of treated sperm was determined by indirect immunofluorescence using the mouse monoclonal antibody HS-21 as described (Lopez and Shur, 1987; Shur and Neely, 1988). HS-21 antibody reacts only with acrosome-intact sperm; loss of anti-HS-21 reactivity is indicative of acrosome-reacted
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FIG. 2. The acrosome reaction is induced by cross-linking sperm surface GalTase. Sperm were capacitated for 30 min and incubated in the indicated reagents for an additional 45 min at 37°C as described under Materials and Methods. In this figure, the final concentration of the reagents were as follows: A23187 (10 r&f); UDP-galactose (2.0 mM); GlcNAc (50 mm; chitotriose (10 m&f); a-lactalbumin (10 mg/ ml); preimmune and anti-GalTase IgG (200 PgYml); preimmune and anti-GalTase Fab (1.6 mg/ml). After incubation in the indicated reagents, the acrosomal status was determined by the presence or absence of HS-21 immunoreactivity. All incubations were conducted in duplicate for each experiment, and at least 100 sperm in a minimum of four fields were scored for each incubation. The error bars represent the range within each pair of duplicates. Data shown in this figure are representative of two to four replicate experiments, each producing similar results. The range between experiments varied from +0.5+7.0% under experimental conditions, and was +lO% for ionophoretreated controls.
sperm (Wolf et al, 1985). At least 100 sperm in a minimum of four fields were scored for each sample. RESULTS
AND DISCUSSION
Monovalent reagents that perturb GalTase activity do not induce the acrowme reaction, Sperm were incubated in medium containing a variety of reagents that perturb GalTase activity, each of which has been shown previously to inhibit sperm-zona binding in a dose-dependent manner (Shur and Hall, 1982a,b; Lopez et al, 1985), and the effects on the acrosome reaction were determined. As shown in Fig. 2, neither the sugar donor substrate UDP-galactose (2.0 mM final concentration), the competitive substrates N-acetylglucosamine (50 mM) and N-acetylchitotriose (10 mM), nor the substrate modifier protein cy-lactalbumin (10 mg/ml) had any significant
MACEK,
LOPEZ,
AND SHUR
GalTase Induces th.e Acrosme Reaction
443
els, although they inhibit sperm-zona binding in a dosedependent manner (1.6 and 3.2 mg/ml final concentration assayed, which inhibits sperm-zona binding by 59 and 76%) respectively (Lopez et a& 1985) (Fig. 2). Thus, multivalency is required for anti-GalTase antibodies to induce the acrosome reaction. This was confirmed by sequentially treating sperm with anti-GalTase Fab fragments followed by goat anti-rabbit IgG to cross-link the bound Fab fragments. Under these conditions, acrosome reactions were induced to levels similar to that produced by intact anti-GalTase IgG (Fig. 2). For these studies, controls included sperm preincubated in buffer (no Fab) followed by goat anti-rabbit IgG, sperm preincubated with preimmune Fab followed by goat anti-rabbit IgG, and sperm preincubated with Fab (either antiGalTase or preimmune) followed by buffer (no goat anti-rabbit IgG). In all instances, the level of acrosomereacted sperm was similar to that produced by Fab fragments alone (Fig. 2). Collectively, these results show that the mouse acrosome reaction can be induced by aggregating GalTase on Monospecific anti-GalTase IgG induces the acrosorne the sperm surface. These results have been confirmed by reaction. Monospecific anti-GalTase IgG has been others using anti-GalTase IgG to induce the acrosome shown previously to inhibit sperm-zona binding in a reaction in mouse sperm as assayed by CTC fluorescence dose-dependent manner (Lopez et al., 1985). When sperm (H. Florman, personal communication). Reagents that were incubated in 200 pg/ml (final concentration) monoare not able to aggregate GalTase, but which are still specific anti-GalTase IgG, a concentration that inhibits able to inhibit sperm zona binding by perturbing Galsperm-zona binding by -60% relative to control IgG, Tase activity, do not induce the acrosome reaction. 75% of the sperm were acrosome-reacted compared These results are reminiscent of the ability of ZP3 glywith 18% in buffer-treated controls and 32% in preimcopeptides to inhibit sperm-zona binding, presumably mune IgG-treated sperm. The effects of the anti-Galby competing for the GalTase binding site (Miller et al, Tase IgG were grossly dose dependent (i.e., 200 pg/ml 1990), without inducing the acrosome reaction (Florman produced an additional 43% acrosome-reacted sperm et al, 1984). However, intact ZP3, with multiple oligosacabove preimmune IgG controls, 33 pg/ml produced 11% charide chains, inhibits sperm-zona binding and inabove controls, 20 pg/ml produced no significant differduces the acrosome reaction, results that parallel the ence from controls). The increased percentage of acro- effects of anti-GalTase IgG. The ability of anti-ZP3 antisome-reacted sperm in the presence of preimmune IgG, bodies to induce the acrosome reaction in sperm preinas well as in the presence of Fab fragments (see below) cubated with “monovalent” ZP3 glycopeptides is consiswas reproducibly higher than in control sperm, but was tent with this notion (Leyton and Saling, 1989). always significantly lower than that produced by idenHow cross-linking surface GalTase induces the acrotical concentrations of anti-GalTase IgG. Incubation some reaction awaits further study. Analysis of the priwith whole serum (1:l dilution), rather than isolated mary sequence of GalTase obtained by molecular clonanti-GalTase IgG, had an even more dramatic effect on ing, along with appropriate biochemical studies, shows the extent of acrosome reaction (89% reacted). GalTase to be an integral transmembrane protein on Anti-GalTase IgG induces the acrosmne reaction by ag- sperm (Shaper et aZ., 1988; Lopez et al, 1985; 1989; Shur gregating GalTase. As shown above, anti-GalTase IgG and Neely, 1988). Clustering of transmembrane recepinduced the acrosome reaction, whereas monovalent re- tors on a variety of cell types elicits metabolic responses agents that also inhibit sperm-zona binding did not. appropriate to the responding cell (O’Brien et al, 1987). Since the anti-GalTase IgG is bivalent, the possibility Similarly, a variety of sperm surface components may was tested that anti-GalTase IgG induced the acrosome be able to induce the acrosome reaction if aggregated by reaction by aggregating sperm surface GalTase. In this multivalent ligands. However, during fertilization, the regard, monovalent Fab fragments of anti-GalTase IgG physiologically relevant inducer of the acrosome reacdid not induce the acrosome reaction above control lev- tion is ZP3, thus ensuring the appropriate temporal effect on the extent of the acrosome reaction. Previous studies have shown that these reagent levels inhibit sperm-zona binding from >60 to >90% of control levels (Shur and Hall, 1982a,b; Lopez et a& 1985). The control reagents, which do not inhibit sperm-zona binding, included UDP-glucose, glucose, and lysozyme at concentrations equivalent to their respective experimental reagents. Experiments in which the concentration of UDP-galactose was varied (0.02-2.0 mJY), or 1 mM MnCl, was included to stimulate enzymatic activity, or sperm were incubated in a combination of UDP-galactose, MnCl, and N-acetylglucosamine (conditions that generate optimal enzyme activity, Shur and Neely, 1988) produced control levels of acrosome reactions (data not shown). In each experiment, treatment with the ionophore A23187 served as a positive control and induced acrosome reactions 49-71s above control levels (58% in Fig. 2). In a parallel experiment, solubilized zonae pellucidae (2 zonae/pl final) induced the acrosome reaction in 52% of treated sperm compared to 16% acrosomereacted sperm in controls (Lopez and Shur, 1987).
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specificity. The basal level of acrosome-reacted sperm present in the epididymis may reflect spurious aggregation of GalTase residues by multivalent glycoconjugates (Shur and Hall, 1982a). In any event, it appears that aggregation of GalTase moieties within the plasma membrane by multiple ZP3 oligosaccharides or by antibodies results in fusion and vesiculation of the plasma membrane and outer acrosomal membrane, culminating in the acrosome reaction. Similarly, in sea urchins, specific monoclonal antibodies are able to induce the acrosome reaction by aggregating the appropriate receptors on sperm (Trimmer et CAL,1987). Evidence suggests that ZP3 induces the acrosome reaction by activating guanine nucleotide-binding proteins (Endo et CAL,1988), which usually associate with membrane receptors having multiple transmembrane domains (O’Dowd et aZ., 1988). However, there are examples where G-proteins can be activated directly by surface receptors having one transmembrane spanning segment, such as the IGF-II/Man-6-P receptor (Okamoto et aL, 1990). Future studies will address whether the cytoplasmic tail of GalTase activates G-proteins directly, analogous to the IGF-II/Man-6-P receptor, or indirectly, eventually leading to exocytosis of the acrosome. The authors are grateful to Dr. Don Wolf for supplying HS-21 antibody, to Dr. Harvey Florman for repeating the studies of anti-GalTase IgG effects on the mouse sperm acrosome reaction, and to Dr. David J. Miller and Ms. Deborah Mansfield for critical reading of the manuscript. This work was supported by NIH Grant HD 23479 to B.D.S. REFERENCES BLEIL, J. D., and WASSARMAN, P. M. (1980). Mammalian sperm-egg interaction: Identification of a glycoprotein in mouse egg zonae pellucidae possessing receptor activity for sperm. Cell 20,873~882. BLEIL, J. D., and WASSARMAN, P. M. (1983). Sperm-egg interactions in the mouse: Sequence of events and induction of the acrosome reaction by a zona pellucida glycoprotein. Dev. BioL 95,317-324. ENDO, Y., LEE, M. A., and KOPF, G. S. (1988). Characterization of an islet-activating protein-sensitive site in mouse sperm that is involved in the zona pellucida-induced acrosome reaction. Dev. BtiL 129,12-24. FLORMAN, H. M., BECHTOL, K. B., and WASSARMAN, P. M. (1984). Enzymatic dissection of the functions of the mouse egg’s receptor for sperm. Deu. BioL 106,243-255. FLORMAN, H. M., and WASSARMAN, P. M. (1985). O-linked oligosaccharides of mouse egg ZP3 account for its sperm receptor activity. CeU 41,313-324. LEYTON, L., and SALING, P. (1989). Evidence that aggregation of mouse sperm receptors by ZP3 triggers the acrosome reaction. J. CeU BioL 108.2163-2168. LOPEZ, L. C., BAYNA,
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LOPEZ, L. C., MAILLET, C. M., OLESZKOWICZ,K., and SHUR, B. D. (1989). Cell surface and golgi pools of j3-1,4-galactosyltransferase are differentially regulated during embryonal carcinoma cell differentiation. Mol. CelL BioL 9,2370-2377. LOPEZ, L. C., and SHUR, B. D. (1987). Redistribution of the mouse egg receptor on sperm following the acrosome reaction. J. Cell BioL 105, 1663-1670. MACEK, M. B., and SHUR, B. D. (1988). Protein-carbohydrate complementarity in mammalian gamete recognition. Gamete Res. 20,93109. MILLER, D. J., MACEK, M. B., and SHUR, B. D. (1990). ZP3 recognizes galactosyltransferase on mouse spermatozoa. J. Cell BioL 111,489a. MYLES, D. G., and PRIMAKOFF, P. (1984). Localized surface antigens of guinea pig sperm migrate to new regions prior to fertilization. J. Cell BioL 99, 1634-1641.
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SHUR, B. D., and NEELY, C. A. (1988). Plasma membrane association, purification and characterization of mouse sperm fl-1,4-galactosyltransferase. .I BioL Chem. 263,17,706-17,714. TRIMMER, J. S., EDNA, Y., SCHACKMANN, R. W., MEINHOF, C.-G., and VAWUIER, V. D. (1987). Characterization of a monoclonal antibody that induces the acrosome reaction of sea urchin sperm. J. CeUBioL 105,1121-1128. WOLF, D. P., BOLDT, J., BYRD, W., and BECHTOL, K. B. (1985). Acrosoma1 status evaluation in human ejaculated sperm with monoclonal antibodies. BioL Reprod 32,1157-1162.
