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Originally published In Press as doi:10.1074/jbc.M414242200 on March 23, 2005

J. Biol. Chem., Vol. 280, Issue 21, 20189-20196, May 27, 2005
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Recombinant Porcine Zona Pellucida Glycoproteins Expressed in Sf9 Cells Bind to Bovine Sperm but Not to Porcine Sperm*

Naoto Yonezawa{ddagger}§, Katsuyasu Kudo{ddagger}, Hirotomo Terauchi{ddagger}, Saeko Kanai{ddagger}, Naoto Yoda§, Masaru Tanokura¶, Kosuke Ito¶, Kin-ichiro Miura||, Toshiyuki Katsumata**, and Minoru Nakano{ddagger}§{ddagger}{ddagger}

From the {ddagger}Graduate School of Science and Technology and the §Department of Chemistry, Faculty of Science, Chiba University, Inage-ku, Chiba 263-8522, the Graduate School of Agriculture and Life Science, the University of Tokyo, Hongo, Tokyo 113-8657, the ||Institute for Biomolecular Science, Gakushuin University, Mejiro, Tokyo 171-8588, and the **College of Liberal Arts and Sciences, Tokyo Medical and Dental University, Ichikawa, Chiba 272-0827, Japan

Received for publication, December 17, 2004 , and in revised form, March 23, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The zona pellucida, which surrounds the mammalian oocyte, consists of the ZPA, ZPB, and ZPC glycoproteins and plays roles in species-selective sperm-egg interactions via its carbohydrate moieties. In the pig, this activity is conferred by tri- and tetraantennary complex type chains; in cattle, it is conferred by a chain of 5 mannose residues. In this study, porcine zona glycoproteins were expressed as secreted forms, using the baculovirus-Sf9 insect cell system. The sperm binding activities of the recombinant proteins were examined in three different assays. The assays clearly demonstrated that recombinant ZPB bound bovine sperm weakly but did not bind porcine sperm; when recombinant ZPC was also present, bovine sperm binding activity was greatly increased, but porcine sperm still was not bound. The major sugar chains of ZPB were pauci and high mannose type chains that were similar in structure to the major neutral N-linked chain of the bovine zona. In fact, the nonreducing terminal {alpha}-mannose residues were necessary for the sperm binding activity. These results show that the carbohydrate moieties of zona glycoproteins, but not the polypeptide moieties, play an essential role in species-selective recognition of porcine and bovine sperm. Moreover, Asn to Asp mutations at either of two of the N-glycosylation sites of ZPB, residue 203 or 220, significantly reduced the sperm binding activity of the ZPB/ZPC mixture, whereas a similar mutation at the third N-glycosylation site, Asn-333, had no effect on binding. These results suggest that the N-glycans located in the N-terminal half of the ZP domain of porcine ZPB are involved in sperm-zona binding.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mammalian oocytes are surrounded by a transparent envelope called the zona pellucida, which is involved in several critical aspects of fertilization. Its functions include species-selective recognition of sperm, blocking of polyspermy, and protection of the oocyte and embryo until implantation (1–3). The zona consists of three glycoproteins (ZPGs)1 that are designated ZPA, ZPB, and ZPC in order of the sizes of their respective cDNAs (4). In the pig, these glycoproteins were formerly known as ZP1, ZP3{alpha}, and ZP3{beta}, respectively (4).

Studies of the murine zona pellucida have established that the carbohydrate moieties of the ZPGs play an essential role in sperm binding. Mouse sperm were proposed to bind to the O-linked carbohydrate chains linked to Ser-332 and Ser-334 of ZPC (5, 6); the nonreducing terminal residues, such as {alpha}-Gal (7), {beta}-GlcNAc (8, 9), {alpha}-Fuc (10), {alpha}-Man (11), and {beta}-Gal (12), are thought to mediate mouse sperm binding. Nevertheless, the suspected level of importance of the carbohydrate moieties in mouse sperm binding has declined because of the results of recent studies using genetically engineered mice that lack a glycosyltransferase, recent structural data on N- and O-linked carbohydrate chains of the mouse zona pellucida, and the determination of glycosylation sites on the mouse ZPGs, as outlined in recent reviews (13, 14) and discussed in recent papers (15–17). Studies of the porcine and bovine zonae pellucidae underline the importance of the zona glycans in sperm-egg interactions, as described below.

In the pig, ZPB and the ZPB/ZPC mixture have sperm binding activities (18–23). ZPB loses this activity when its N-linked carbohydrate chains are removed by N-glycanase digestion (21, 22). Neutral N-linked carbohydrate chains that are released from the ZPB/ZPC mixture on N-glycanase digestion retain their sperm binding activities (24). Of these carbohydrate chains, the triantennary and tetraantennary complex type chains (Fig. 1A) bind more strongly than biantennary complex type chains (25). Conversely, it has been reported that O-linked carbohydrate chains, but not N-linked chains, specifically released from the ZPB/ZPC mixture inhibit sperm-egg binding (26). Therefore, both N- and O-linked carbohydrate chains are thought to act as ligands for sperm binding.

Recent studies of the bovine zona pellucida reveal that ZPB has the highest sperm binding activity among the three bovine ZPGs (27) and that a high mannose type chain containing 5 Man residues (Fig. 1B) has sperm binding activity (28). Moreover, the nonreducing terminal {alpha}-Man residues are essential for this activity (28). Thus, differences in the structures of the carbohydrate chains involved in sperm binding may explain the species-selective recognition of sperm by the ZPGs.



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  		FIG. 1.
N-Linked neutral carbohydrate chains active in sperm binding. In the pig, the triantennary and tetraantennary complex type chains, in which Fuc is attached to the innermost GlcNAc residue, shown in A, have sperm binding activity (25). In cattle, the high mannose type chain that possesses 5 Man residues, shown in B, has sperm binding activity (28).

 
Recombinant forms of the ZPGs have been expressed in yeast, insect, and mammalian cells, as well as in bacteria (29–35). However, the species selectivities of the sperm binding activities of these recombinant ZPGs have not been studied with respect to their carbohydrate structures. In this study, we expressed the porcine ZPGs as secreted, glycosylated forms using the baculovirus-Sf9 insect cell system, and we examined the sperm binding activities of the recombinant glycoproteins (rZPGs). Glycosylated rZPGs bound to bovine sperm but not to porcine sperm, suggesting that the carbohydrate moieties play an essential role in species selectivity of sperm-zona interactions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cloning, Expression, and Purification of rZPGs—cDNAs encoding the secreted, mature polypeptides of porcine ZPA, ZPB, and ZPC were obtained by reverse transcriptase-PCR using pig ovary poly(A)+ RNA as the template. The polypeptides of porcine ZPA, ZPB, and ZPC, which were expressed in this study correspond to the regions from Ile-36 to Arg-641, from Asp-137 to Arg-466, and from Asp-28 to Arg-348, respectively (4). The translation initiation Met is numbered as 1. The poly(A)+ RNA was isolated from pig ovary according to the method of Chomczynski and Sacchi (36). The 5'-sense primer for ZPA contained an XhoI site, and the 5'-sense primers for ZPB and ZPC contained BamHI sites. The 3'-antisense primer for ZPA contained a stop codon and XhoI site, and the 3'-antisense primers for ZPB and ZPC contained stop codons and BamHI sites. The PCR products were electrophoresed on 1% agarose gels, and the bands of expected sizes were recovered from the gels and ligated to pGEM-T Easy (Promega, Madison, WI). The DNA sequences of the PCR products were confirmed by DNA sequencing. The cDNAs were subcloned into the baculovirus transfer vector pBACgus-6 (Novagen, Madison, WI) to obtain the recombinant proteins as secreted proteins with N-terminal S and His tags using the XhoI and BamHI sites that were incorporated into the primers.

Plasmid DNA preparations that contained individual cDNAs (0.25 µg) were transfected along with 0.1 µg of BacVector-2000 virus DNA (Novagen) into Sf9 cells using Eufectin (Novagen), according to the manufacturer's protocol. Recombinant plaques were identified and purified by the plaque assay, according to the protocol supplied with the BacVector-2000 DNA kit (Novagen). Sf9 cells were routinely propagated in Sf-900 II serum-free medium (Invitrogen). Several purified plaques were examined for expression and secretion of the recombinant proteins. Sf9 cells (1.8 x 106 cells) were attached to the flask, infected with the recombinant virus from each purified plaque at a multiplicity of infection of 5–10, and cultured in 2.5 ml of Sf-900 II serum-free medium for 48 h at 27 °C. S protein-agarose (10 µl of suspension; Novagen), which was prewashed with phosphate-buffered saline (PBS), was mixed with 500 µl of the 48-h culture supernatant and shaken gently at room temperature for 30 min. The recombinant proteins bound to the S protein-agarose through their N-terminal S tags. After this period of incubation, the agarose beads were washed three times with PBS, followed each time by centrifugation. The pellet that contained the agarose beads was prepared for SDS-PAGE.

For large scale protein production, the Sf9 cells (200 ml of 1.0 x 106 cells/ml of stock) were infected with the recombinant virus at a multiplicity of infection of 10. When two proteins were expressed simultaneously, the two recombinant viruses were added to the Sf9 cells at a multiplicity of infection of 5. After 48 h of culture in suspension, the medium was centrifuged at 800 x g for 10 min to remove the cells, and the supernatant was filtrated through a 0.45-µm filter. The filtrate was sonicated and then stored at 4 °C.

The filtered and sonicated supernatants were subjected to metal chelation column chromatography using His-Bind resin (Novagen), which was equilibrated with 5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl (pH 7.9) at a flow rate of 0.5 ml/min at 4 °C. After washing the column with 10 column volumes of the equilibration buffer, the bound protein was eluted with 6 column volumes of 60 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl (pH 7.9) followed by 6 column volumes of 1 M imidazole, 0.5 M NaCl, 20 mM Tris-HCl (pH 7.9).

Construction of Mutated Porcine ZPB Genes by PCR—Mutations were introduced into porcine ZPB cDNA by two rounds of PCR. To mutate Asn-203 to Asp, the following pairs of primers were used for the first PCR: N203D mutation sense primer (5'-GTGTCTCGCGATGTGACCTCACCTCC-3') and the 3' antisense primer for ZPB described above, and the 5'-sense primer for ZPB described above and an EcoRI antisense primer (5'-GGGGAATTCATTGACTAGCATGGGCC-3'). Base substitutions (C to A and C to T, respectively) present at the fifth and eighth bases of the EcoRI antisense primer served to generate an EcoRI site. PCR products were electrophoresed on agarose gels, purified from the gels, and used as templates for the second PCR, which used the 5'-sense and 3'-antisense primers for ZPB. As a result, the PCR products that contained the desired N203D mutation did not have an EcoRI site. After treatment with EcoRI, those PCR products that were not digested were recovered from the agarose gel and ligated to pGEM-T Easy. The entire DNA sequence including the mutation was confirmed by DNA sequencing.

To mutate Asn-220 or Asn-333 of ZPB to Asp, N220D or N333D sense primers (5'-CTGGCCTTCAGAGATGACAGTGAATG-3' or 5'-GGCTCCTACTACGATGCTAGTGAC-3', respectively) were used in place of the N203D sense primer. The underlined bases identify the replacement Asp codon.

Electrophoresis, Immunoblot Analysis, and Lectin Blot Analysis of rZPGs—SDS-PAGE was performed under reducing conditions, according to the Laemmli protocol (37). The proteins were separated on 9–15% polyacrylamide gels and either visualized by silver staining or transferred to Immobilon-P membranes (Millipore, Bedford, MA), according to the method of Towbin (38), for the immunoblot and lectin blot analyses. For the immunoblot analysis, the membranes were blocked with 3% bovine serum albumin (BSA) in Tris-buffered saline (TBS; 500 mM NaCl and 20 mM Tris-HCl (pH 7.5)) for 1 h. The membranes were incubated for 2 h with rabbit anti-porcine ZPA, rabbit anti-porcine ZPB, and rabbit anti-porcine ZPC polyclonal antibodies (25, 39) that were diluted 1/200, 1/2,000, and 1/2,000, respectively, in TBS plus 1% BSA. After washing the membranes three times for 15 min each with TBS that contained 0.05% Tween 20 (T-TBS), the membranes were incubated for 1.5 h with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody that was diluted 1/1,000 in TBS plus 1% BSA. After washing the membranes three times for 15 min each with T-TBS, the blots were developed using an Immunostain kit (Seikagaku Kogyo, Tokyo, Japan). For the lectin blots, the membranes were blocked with T-TBS for 1 h and then incubated for 2 h with 1 µg/ml either horseradish peroxidase-conjugated lectin or biotin-conjugated lectin in T-TBS that contained 1 mM MgCl2 and 1 mM CaCl2. The following horseradish peroxidase-conjugated lectins were used in this study: concanavalin A (ConA), Lens culinaris agglutinin (LCA), and Ricinus communis agglutinin (RCA120). The following biotin-conjugated lectins were used: Datura stramonium agglutinin (DSA), Phaseolus vulgaris agglutinin (PHA)-L4, Amaranthus candatus agglutinin (ACA), and Galanthus nivalis agglutinin (GNA). ACA and GNA were purchased from EY Laboratories (San Mateo, CA), and the remaining lectins were from Seikagaku Kogyo. After washing the membranes three times for 15 min each with T-TBS that contained the metal ions, the peroxidase-conjugated lectins were developed as described above. The membranes that contained the biotin-conjugated lectins were incubated for an additional hour with 0.5 µg/ml horseradish peroxidase-conjugated streptavidin (Sigma) in T-TBS that contained the metal ions and were then washed three times for 15 min each with T-TBS that contained the metal ions followed by color development as described above.

Glycopeptidase F Digestion of rZPGs—The digestion of each rZPG (about 0.4 µg) with glycopeptidase F (Takara, Shiga, Japan) was performed under denaturing conditions, according to the manufacturer's protocol. In addition, 10 mM o-phenanthroline was added to the solution. An aliquot that corresponded to 0 min of digestion was taken from the solution before the addition of the enzyme. Digestion was started by adding 1 milliunit of glycopeptidase F. Aliquots were taken at 1 and 5 min and at 24 h, and digestion was terminated by boiling.

{alpha}-Mannosidase Digestion of a Mixture of Recombinant Porcine ZPB (rZPB) and Recombinant Porcine ZPC (rZPC)—The pH of the rZPB/rZPC mixture was adjusted to 6.5 with 25 mM citrate, 25 mM phosphate, NaOH (pH 4.5). Jack bean {alpha}-mannosidase (Seikagaku Kogyo) dissolved in citrate/phosphate buffer was added to the rZPB/rZPC solution at 5 milliunits/µg of protein. Protease inhibitor mixture (Roche Applied Science) was added to the mixture, according to the manufacturer's protocol. After digestion at 37 °C for 9 h, the buffer was exchanged for PBS using Amicon Ultra-4 (Millipore) spin filters. Aliquots (0.5 µg) of the digests were electrophoresed on SDS gels and transferred to Immobilon-P membranes. The membranes were then subjected to lectin blotting with GNA or to immunoblotting with anti-porcine ZPB antibody.

Control (sham) digestions were performed, as above, except that citrate/phosphate buffer was used in place of {alpha}-mannosidase. To investigate the effect of {alpha}-mannosidase itself on sperm-zona binding, an {alpha}-mannosidase digestion reaction was carried out in the absence of rZPB/rZPC and then used in competitive inhibition assays.

Sperm-Agarose Bead Binding Assay—The fraction that was eluted from the His-Bind resin by washing with 60 mM imidazole was incubated with S protein-agarose, as described above. As a control, the Sf9 cells were cultured without recombinant viruses; the culture supernatant was subjected to His-Bind column chromatography and then mixed with S protein-agarose. The agarose beads were washed with Brackett and Oliphant (BO) solution for the bovine sperm binding assay (27, 40) or with modified Krebs-Ringer bicarbonate solution for the porcine sperm binding assay (24, 41). Frozen bovine sperm were thawed and washed twice in BO solution without BSA, which was prewarmed to 38.5 °C. The bovine sperm were then capacitated by incubating the sperm in BO solution that contained BSA for 30 min at 38.5 °C in 2% CO2. The sperm concentration was calculated from a standard curve of percentage transmittance at 400 nm versus cells/ml, which was determined by hemacytometer counting of the sperm. About 20 beads that were coated with each recombinant protein were transferred to BO solution that contained BSA for one assay, and bovine sperm were then added to the solution to give a final density of 2.0 x 106 cells/ml. After a 2-h incubation at 38.5 °C in 2% CO2, the beads were transferred to fresh BO solution that contained BSA using a siliconized pipette with a bore size of ~1.2 times the diameter of the beads. The beads were then transferred to PBS that contained 3% glutaraldehyde and fixed for 40 min at room temperature. To visualize the sperm that were bound to the beads, the beads were transferred to PBS that contained 10 µg/ml 4',6-diamidino-2-phenylindole and incubated for 10 min at room temperature. Finally, the beads were transferred onto slide glasses and covered with cover glasses. The sperm that bound to the beads were counted under the fluorescence microscope. This assay was repeated at least three times for each recombinant protein, to ensure the reproducibility. For the assays of frozen and freshly ejaculated porcine sperm, modified Krebs-Ringer bicarbonate solution was used instead of BO solution, and the incubation was performed at 37 °C in 5% CO2.

Competitive Inhibition Assay—Solubilized bovine zona (0.1 µg/50 µl PBS) was added to each well of a 96-well plate (Nalge Nunc, Rochester, NY), which was then incubated at 4 °C overnight. After rinsing with PBS, the wells were blocked with 3% BSA in TBS at 37 °C for 2 h. Frozen bovine sperm were washed and capacitated as described above, and 50-µl aliquots (2 x 105 sperm) were incubated with the solubilized bovine zona or each recombinant for 30 min at 38.5 °C in 2% CO2. The amount of inhibitor was adjusted to 0.2 µg. The wells were rinsed three times with PBS, and the preincubated sperm solutions were transferred into the wells. After incubation for 2 h at 38.5 °C in 2% CO2, the wells were washed three times with BO solution. PBS (50 µl) was added to each well, and the sperm that were bound to the wells were recovered by pipetting vigorously 20 times. The number of sperm in 0.1 µl of the suspension was counted using a hemacytometer. For the assay of frozen porcine sperm, wells were coated with solubilized porcine zona, modified Krebs-Ringer bicarbonate solution was used instead of BO solution, and incubation of sperm was performed at 37 °C under 5% CO2.

Indirect Immunofluorescence Staining of Sperm-binding rZPGs— The frozen bovine sperm were washed and capacitated as described above. Sperm (50-µl aliquots of 2 x 106/ml) were incubated with solubilized bovine zona (0.2 µg) or each of the recombinant proteins (0.2 µg) in BO solution for 30 min at 38.5 °C in 2% CO2. The sperm were washed three times with BO solution, followed each time by centrifugation. The sperm were suspended in PBS and transferred to cover glasses. The sperm were fixed with 3.7% formaldehyde in PBS for 30 min at 37 °C. After rinsing with PBS, the cover glasses were blocked with 3% BSA in TBS for 30 min at 37 °C. The proteins that bound to sperm were detected using the mixture of anti-porcine ZPA antibody (1/100 diluted), anti-porcine ZPB antibody (1/1,000 diluted), and anti-porcine ZPC antibody (1/1,000 diluted) as the primary antibodies, and fluorescein-conjugated goat anti-rabbit IgG antibody (1/1,000 diluted; Wako Chemicals, Tokyo, Japan) as the secondary antibody. The sperm were observed under a fluorescence microscope.

Mass Spectrometric Analysis of Carbohydrate Chains—rZPB was purified from the culture supernatant, as described above. The rZPB (40 µg) was desalted by dialysis against water and then lyophilized. The release of N-linked carbohydrate chains was performed by hydrazinolysis (24), and the carbohydrate chains were fluorescently labeled with 2-aminopyridine, as described previously (24). The molecular masses of the pyridylaminated carbohydrate chains were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) using Voyager-DE STR (PerkinElmer Life Sciences), and with 2,5-dihydroxybenzoic acid as the matrix.

Statistical Analysis—The Mann-Whitney U test was used to determine whether the data on sperm binding to the recombinant proteinbeads were significantly different from the data on sperm binding to the control beads, as described above (i.e. p < 0.05). Welch's t test was applied to determine whether the levels of sperm binding to the rZPB-beads were significantly different from the levels of sperm binding to the rZPB/rZPC-beads (i.e. p < 0.05). Welch's t test was also applied to determine whether the rZPGs had significant inhibitory activity for sperm-zona binding (i.e. p < 0.05) and to determine whether the inhibitory activities were significantly different among rZPB alone, the rZPB/rZPC, and the rZPB mutant/rZPC mixture (i.e. p < 0.05).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of Recombinant Porcine Proteins rZPA, rZPB, and rZPC in Insect Cells Infected with Recombinant Baculoviruses—ZPGs are synthesized as membrane proteins, processed N-terminal to their transmembrane regions, and then secreted as mature polypeptides without their transmembrane regions (42, 43). Porcine ZPA, ZPB, and ZPC proteins have putative furin processing sites from Arg-638 to Arg-641, from Arg-463 to Arg-466, and from Arg-345 to Arg-348, respectively (4). (The translation initiator residue, Met, is designated as residue 1.) Mutations in the putative furin processing sites of murine and human ZPGs abolish ZPG secretion (42–44). Therefore, it seems likely that porcine ZPGs are also processed at the putative processing sites.

In this study, S- and His-tagged recombinant polypeptides corresponding to porcine ZPA, ZPB, and ZPC regions Ile-36 to Arg-641, Asp-137 to Arg-466, and Asp-28 to Arg-348, respectively, were expressed in Sf9 cells. The N termini of the resulting rZPA and rZPB proteins correspond to those reported previously for mature, native porcine ZPA and ZPB (pZPA and pZPB, respectively, and 45 and 46, respectively). The resulting rZPC, however, is 5 amino acids shorter than mature, native porcine ZPC (pZPC) at the N terminus. This discrepancy is a result of the fact that, when we designed this study, the available data indicated that the N terminus of mature pZPC was Asp-28 (46). Only later was the N terminus of pZPC revealed to be a pyroglutamate at position 23 (47). The C termini of mature pZPB and pZPC were recently reported to be Ala-462 and Ser-332, respectively (48). Thus, rZPB and rZPC are 4 and 17 amino acid residues longer at their C termini than their native counterparts. The C terminus of mature pZPA is yet to be determined.



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  		FIG. 2.
Immunoblot analysis of recombinant porcine ZPGs expressed in Sf9 cells. Recombinant proteins rZPA, rZPB, and rZPC were expressed in Sf9 cells and secreted into the culture medium. An rZPB/rZPC mixture was expressed by simultaneous infection of Sf9 cells with the two corresponding recombinant viruses. A, recombinant proteins rZPA, rZPB, and rZPC were collected from the culture supernatants using metal chelation column chromatography and detected by SDS-PAGE (lanes 1, 3, and 5, respectively) or by immunoblot analysis using antibodies specific for each zona glycoprotein (lanes 2, 4, and 6, respectively). B, rZPA, rZPB, rZPC, and the rZPB/rZPC mixture were immobilized on S protein-agarose beads, as revealed by SDS-PAGE (lanes 1, 2, 3, and 4, respectively). The rZPB and rZPC bands in the mixture are indistinguishable because of similar molecular masses. Detection of rZPB and rZPC by specific antibodies (lanes 5 and 6, respectively) shows that the mixture contains equivalent amounts of rZPB and rZPC. Molecular mass markers are indicated (in kDa) at the left of each panel.

 
The apparent molecular masses of rZPA, rZPB, and rZPC, as determined by SDS-PAGE, were similar to the molecular masses predicted from their encoded amino acid sequences, and immunoblots with antibodies specific for each of the ZPGs confirmed the presence of the eluted proteins (Fig. 2A). The absorbance at 280 nm of the eluted fractions was used to estimate the yield of each protein; about 20 µg of each rZPG was obtained from 200 ml of culture medium.

Sperm Binding Activities of rZPA, rZPB, and rZPC—First, we performed a sperm-bead binding assay. The rZPGs were immobilized on S protein-agarose beads via their N-terminal S tags, and the binding of each rZPG to the agarose beads was confirmed by SDS-PAGE (Fig. 2B). Frozen or freshly ejaculated porcine sperm were incubated with the rZPG-coated beads, and the sperm that bound to each bead were counted. Neither frozen nor freshly ejaculated porcine sperm exhibited significant binding to rZPA-, rZPB-, or rZPC-coated beads (Fig. 3A). Bovine sperm, on the other hand, exhibited significant binding to rZPB-coated beads, but not to rZPA- or to rZPC-coated beads.

A mixture of rZPB and rZPC, which was prepared by coinfection of Sf9 cells with rZPB and rZPC viruses, was also immobilized on S-agarose beads, as described above. Binding of rZPB/rZPC to the beads was demonstrated by SDS-PAGE and immunoblot analysis of the beads (Fig. 2B). Beads coated with rZPB/rZPC bound bovine sperm but not porcine sperm, and the level of binding was higher than that of beads coated with rZPB alone (Fig. 3A). The binding affinity of sperm to beads coated with rZPB/rZPC was much weaker than the affinity for bovine eggs in an examination using pipetting. No sperm remained bound to beads coated with rZPB/rZPC when we washed the beads 10 times with fresh BO solution after incubating the beads with bovine sperm, following our procedure for the competitive inhibition assay using bovine eggs (27). By contrast, an average of 40 sperm remained bound to each bovine egg after 10 transfers to fresh BO solution (Fig. 3B). Therefore, in this study, we washed the beads using a single transfer to fresh BO solution, as described under "Materials and Methods." Nevertheless, the number of sperm bound to one bead was still low (Fig. 3, A and B).



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  		FIG. 3.
Sperm binding to agarose beads coated with rZPGs. A, the number of sperm that bound to agarose beads coated with rZPA, rZPB, rZPC, or the rZPB/rZPC mixture is shown. The sperm samples included frozen-thawed porcine sperm (open bars), freshly ejaculated porcine sperm (hatched bars), and frozen-thawed bovine sperm (solid bars). As a control, Sf9 cells were cultured without recombinant virus, and the culture supernatant was added to the His-Bind resin, as described under "Materials and Methods." Approximately 60 beads were examined for each sample, and the data are shown as the means ± S.D. B, 32 bovine sperm remained bound to the bovine egg after 10 transfers to fresh BO solution by pipetting (bovine egg), whereas 8 sperm remained bound to the agarose bead coated with rZPB/rZPC after a single transfer to fresh BO solution by pipetting (rZPB/rZPC-bead). This figure shows sperm bound to egg or bead in one plane of focus. Sperm were visualized by fluorescently staining with 4',6-diamidino-2-phenylindole.

 
We also examined the inhibitory effects of the porcine rZPGs on bovine sperm-bovine zona binding and porcine sperm-porcine zona binding (Fig. 4). In the presence of solubilized porcine zona, binding of porcine sperm to plastic wells coated with porcine zona was reduced to about 30%, but none of the porcine rZPGs, nor rZPB/rZPC, significantly inhibited this binding. In contrast, rZPB/rZPC significantly inhibited the binding of bovine sperm to plastic wells coated with bovine zona. Bovine sperm-bovine zona binding was inhibited significantly by rZPB, although the effect was much weaker than that of rZPB/rZPC.

The sperm binding activities of the porcine rZPGs were also examined by indirect immunofluorescence detection of rZPG-bound sperm (Fig. 5). Solubilized, native porcine zona bound to the acrosomal region of porcine sperm (Fig. 5A), and solubilized, native bovine zona bound to the acrosomal region of bovine sperm (Fig. 5B), as shown by fluorescent staining. Only faint fluorescent staining was observed for rZPA, rZPB, rZPC, and rZPB/rZPC in the acrosomal and postacrosomal regions of porcine sperm (Fig. 5A), indicating that little binding occurred. On the other hand, rZPB and rZPB/rZPC exhibited significant binding to the acrosomal and postacrosomal regions of bovine sperm (Fig. 5B). Compared with the solubilized bovine zona, binding of rZPB and rZPB/rZPC to the acrosomal region was weaker, and binding to the postacrosomal region was stronger. Staining of the bovine sperm postacrosomal region was clearly evident for rZPB and rZPB/rZPC, whereas this region was only faintly stained for the solubilized bovine zona.

Glycosylation of the rZPGs—The carbohydrate moieties of the rZPGs were analyzed by digestion with glycopeptidase F. The mobility of rZPB on SDS-PAGE increased as digestion progressed (Fig. 6), and three bands with higher mobilities appeared, indicating that rZPB has three N-linked chains. Similarly, digestion with glycopeptidase F revealed that rZPC has three N-linked chains as well. Thus, rZPB and rZPC have the same number of N-linked chains as their native counterparts. Although glycopeptidase F digestion also revealed that rZPA contains N-linked chain(s), the bands were not sufficiently resolved to allow determination of the number of N-linked chains. The change in apparent Mr of the rZPB protein after glycopeptidase F digestion was estimated at 3,000. In a previous study, glycopeptidase F digestion of endo-{beta}-galactosidase-treated pZPB decreased its apparent Mr by 7,000 (21). Thus, the carbohydrate chains of rZPB are smaller than those of native pZPB.



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  		FIG. 4.
Inhibitory effect of rZPGs on sperm-zona binding. Solubilized bovine or porcine zona were adsorbed to each well of a 96-well plate (0.1 µg/well) for bovine or porcine sperm binding assays, respectively. Bovine or porcine sperm (2 x 105) were incubated with 0.2 µg of solubilized zona (ZP), rZPA, rZPB, rZPC, or rZPB/rZPC for 30 min, and then transferred to the wells. After incubation for 2 h, the wells were washed, and 50 µl of PBS was added to each well. The sperm that bound to the zona were recovered from the wells by vigorous pipetting. The number of sperm in 0.1 µl of the suspension was counted using a hemacytometer. The number of sperm binding to the zona in the absence of inhibitors is designated as 100%. Assays were repeated at least three times, and the data are shown as the means ± S.D. Open bars, bovine sperm-bovine zona binding; Shaded bars, porcine sperm-porcine zona binding.

 



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  		FIG. 5.
Indirect immunofluorescence staining of sperm-bound rZPGs. Suspensions of porcine sperm (A) and bovine sperm (B) (50-µl aliquots of 2 x 106/ml) were incubated with 0.2 µg of solubilized porcine (A) or bovine (B) zona for 30 min or with 0.2 µg of rZPA, rZPB, rZPC, or rZPB/rZPC for 30 min. The proteins that bound to sperm were detected using a mixture of anti-pZPA, anti-pZPB, and anti-pZPC antibodies as the primary antibodies, and fluorescein-conjugated goat anti-rabbit IgG antibody as the secondary antibody. The sperm were observed using fluorescence microscopy. As a control, the sperm were incubated without solubilized zona or rZPGs and then treated with the antibodies, as described above. Phase, phase-contrast image; fluorescence, fluorescence image.

 



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  		FIG. 6.
Glycopeptidase F digestion of rZPGs. The rZPA, rZPC, rZPB, rZPBN203D, rZPBN220D, and rZPBN333D proteins were digested with glycopeptidase F for 0, 1, or 5 min, or 24 h, and the shifts in the mobilities of the recombinant proteins on SDS-PAGE were examined. rZPA that was digested for 24 h migrated faster than the undigested rZPA (0 min), indicating that rZPA contains N-glycan(s), although the number of N-glycans was not verified in this study. The rZPC and rZPB samples yielded four bands (indicated by bars) after 1 min of digestion, indicating that both rZPC and rZPB contain three N-glycans, as is also the case for the native pZPC and pZPB, respectively. On the other hand, rZPBN203D, rZPBN220D, and rZPBN333D proteins yielded three bands (indicated by bars) after 1 min of digestion.

 
We compared the carbohydrate structures of the recombinant and native ZPGs using seven different lectins. All three rZPGs were recognized by ConA, LCA, and GNA (Fig. 7), but not by DSA, ACA, RCA120, or PHA-L4 (data not shown). The native pZPA, pZPB, and pZPC proteins were recognized by all of the tested lectins, except GNA (Fig. 7 and data not shown).

Although we attempted to examine the carbohydrate structures of rZPB by two-dimensional sugar mapping analysis, the amounts of pyridylaminated chains obtained were insufficient for analysis because of the low yields of the recombinant proteins. Therefore, we examined the molecular masses of the pyridylaminated chains of rZPB using MALDI-TOF MS. Major peaks were observed at m/z = 659.63, 1137.51, 1157.49, and 1295.48. These peaks were assigned to Man-GlcNAc-GlcNAc-pyridylamino (PA) (calculated mass of 664), Man3-GlcNAc-(Fuc-)GlcNAc-PA (1,135), Man4-GlcNAc-GlcNAc-PA (1,151), and Man4-GlcNAc-(Fuc-)GlcNAc-PA (1,297), respectively. This result is consistent with our findings that the rZPGs are recognized by ConA, LCA, and GNA (Fig. 7) and that the molecular masses of the N-linked chains of rZPB are small. Thus, rZPB contains pauci and high mannose type chains that have {alpha}-Man residues at their nonreducing termini. These structures are not found in the native porcine ZPB/ZPC mixture (24).

Effect of {alpha}-Mannosidase Treatment on the Sperm Binding Activity of rZPB/rZPC—The rZPB/rZPC mixture was treated with {alpha}-mannosidase to examine the role of the nonreducing terminal {alpha}-Man residues in sperm binding. After {alpha}-mannosidase treatment, binding of GNA to rZPB/rZPC was markedly reduced, as revealed by lectin blotting (Fig. 8A), and the inhibitory activity of rZPB/rZPC was almost completely abolished (Fig. 8B). In the control (sham) reaction, rZPB/rZPC incubated in the absence of {alpha}-mannosidase exhibited a small decrease in inhibitory activity compared with that of rZPB/rZPC stored at –80 °C, probably because of denaturation during the incubation. In the absence of rZPB/rZPC, {alpha}-mannosidase did not inhibit sperm-zona binding.



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  		FIG. 7.
Lectin blot analysis of rZPGs. Native porcine ZPGs (lane 1 in each panel) and recombinant porcine ZPGs (lane 2 in each panel) were subjected to lectin blot analysis with ConA, LCA, and GNA. ConA and LCA recognized the native glycoproteins, whereas GNA did not, as expected from the reported structures of the major N-linked chains (24). On the other hand, the rZPGs were recognized by GNA as well as by ConA and LCA.

 
Sperm Binding Activity of Mixtures of rZPC and Mutant rZPBs—To eliminate N-linked glycosylation at specific sites on rZPB, we made three separate Asn to Asp site-directed mutations in the polypeptide sequence. These mutations, which were located at Asn-203, Asn-222, and Asn-333 of ZPB, resulted in mutant rZPBs, referred to in this study as rZPBN203D, rZPBN220D, and rZPBN333D, respectively. Asp substitutions, rather than Gln substitutions, were selected to imitate N-glycanase digestion at each N-glycosylation site.

The mutant rZPBs were secreted into the culture medium, and the yields of rZPB and the three mutant rZPBs were similar. The mutant rZPBs and the rZPB mutant/rZPC mixtures were partially purified from the culture media by nickel-chelating gel chromatography (data not shown, and Fig. 9A for rZPB mutant/rZPC). The number of N-linked chains of rZPB mutants was determined using glycopeptidase F (Fig. 6). Two bands with greater mobilities appeared as the digestion of the rZPB mutants with glycopeptidase F progressed (Fig. 6), indicating that the rZPB mutants had lost one of their three N-linked chains.

Because the sperm binding activity of rZPB is weak to begin with, direct comparison of the mutant rZPB and non-mutant rZPB activities was problematic. Therefore, we examined, instead, the inhibitory effects of the rZPB mutant/rZPC mixtures on bovine sperm-bovine zona binding. rZPB mutants exhibited a slightly greater mobility on SDS gels than did rZPC (Fig. 9A). Expression of rZPB mutants and rZPC was confirmed by immunoblotting with anti-pZPB antibody and anti-pZPC antibody, respectively (data not shown). The rZPBN333D/rZPC mixture inhibited the binding of bovine sperm to bovine zona as well as solubilized bovine zona (Fig. 9B). On the other hand, rZPBN220D/rZPC did not inhibit, significantly, the spermzona binding (Fig. 9B). The rZPBN203D/rZPC mixture inhibited the sperm-zona binding significantly, but the inhibitory effect was much weaker than that of rZPB/rZPC (Fig. 9B).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The sperm binding activity of pZPB has recently been reported to require the presence of a trace amount of pZPC protein (23). Our rZPB, however, has bovine sperm binding ability independent of rZPC, suggesting that sperm binding by native ZPB may not require ZPC after all. We could not address the role of rZPC in porcine sperm binding by rZPB, however, because rZPB does not bind porcine sperm in our system. On the other hand, our finding that the rZPB/rZPC mixture has a much higher level of sperm binding activity than does rZPB alone is consistent with reports that ZPC promotes the sperm binding activity of ZPB in the native porcine zona (20, 23).



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  		FIG. 8.
Effect of {alpha}-mannosidase treatment on the inhibitory activity of rZPB/rZPC. A, rZPB/rZPC was incubated with (+) or without (–) {alpha}-mannosidase as described under "Materials and Methods." The aliquots were subjected to SDS-PAGE and transferred to Immobilon-P membranes. A large amount of nonreducing terminal {alpha}-Man was removed by the {alpha}-mannosidase digestion, as shown by lectin blot analysis using GNA. The amount of rZPB/rZPC was not reduced by proteolytic degradation during the digestion, as shown by immunoblotting with anti-pZPB antibody (Anti-ZPB). B, bovine sperm were incubated with 0.2 µg of solubilized zona (ZP), rZPB/rZPC, {alpha}-mannosidase-treated rZPB/rZPC (+{alpha}-Mannosidase), or sham-{alpha}-mannosidase-treated rZPB/rZPC (–{alpha}-Mannosidase) for 30 min. The assay was performed as described in Fig. 4. The number of sperm binding to the zona in the absence of inhibitors is designated as 100%. Assays were performed at least three times, and the data are shown as the means ± S.D. The effect of {alpha}-mannosidase itself on sperm-zona binding was examined by preincubating sperm with 1 milliunit of {alpha}-mannosidase that had been incubated without ZPGs ({alpha}-Mannosidase alone).

 



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  		FIG. 9.
Inhibitory effect of rZPB mutant/rZPC mixtures on sperm-zona binding. Mixtures rZPC and rZPB, rZPBN203D, rZPBN220, or rZPBN333D were expressed by simultaneous infection of Sf9 cells with the two corresponding recombinant viruses. A, the recombinant proteins were collected from the culture supernatant using metal chelation column chromatography and detected by SDS-PAGE (lanes 1, 2, and 3: rZPBN203D/rZPC, rZPBN220D/rZPC, and rZPBN333D/rZPC, respectively). (For the SDS-PAGE analysis of rZPB/rZPC, see Fig. 2B.) B, the inhibitory effect of each rZPB mutant/rZPC mixture on sperm-zona binding was determined by preincubating bovine sperm with 0.2 µg of solubilized zona (ZP), rZPB/rZPC, rZPBN203D/rZPC, rZPBN220D/rZPC, or rZPBN333D/rZPC for 30 min as described in Fig. 4. The number of sperm binding to the zona in the absence of inhibitors is designated as 100%. These assays were performed at least three times, and the data are shown as the means ± S.D.

 
Notably, neither rZPB nor rZPB/rZPC bound to porcine sperm, but both of these protein preparations bound to bovine sperm. The major neutral N-linked chains in the porcine zona pellucida are di-, tri-, and tetraantennary complex type chains (24), whereas the neutral N-linked chain fraction of the bovine zona pellucida contains a high mannose type chain of 5 Man residues (49). Thus, the carbohydrate structures of the neutral N-linked chains differ markedly between the porcine and bovine zona (Fig. 1).

The neutral complex type chains of the native porcine zona pellucida bind porcine sperm (24), and the tri- and tetraantennary chains have greater activity than the biantennary chains (25), although there is some controversy over whether O- or N-linked chains are sperm ligands in the pig, as mentioned in the Introduction. The polyvalence of the nonreducing termini of the complex type chains may be important for sperm binding activity, as reported previously for murine ZPGs (50). The high mannose type chain of the bovine zona pellucida binds bovine sperm, and the nonreducing terminal {alpha}-Man residues are essential for this activity (28). In the sperm-agarose bead binding assay, the binding affinity of bovine sperm for rZPB/rZPC-coated beads was very low, as revealed by pipetting. After this series of experiments, we performed two additional assays to obtain more convincing data on the sperm binding activities of rZPGs. Because the binding of bovine sperm to agarose beads coated with native bovine ZPB via biotin was also very weak in our previous study (27), the higher affinity for the egg zona pellucida may need the clustering of functional carbohydrate chains, and the low affinity for beads might be a result of the small amount of protein that was immobilized on the beads. In addition, the low affinity of sperm for beads observed in this study might be the result of the low valence of the terminal {alpha}-Man in the rZPGs compared with the native high mannose type chain with 5 Man residues found in the native bovine zona (see below). The presence of additional binding mechanisms involving sperm and the egg zona pellucida should also be considered.

The rZPGs yielded distinct bands on SDS-PAGE compared with the relatively broad bands of native porcine ZPGs that were purified after digestion with endo-{beta}-galactosidase (see Fig. 7). The shift in the mobility of rZPB on SDS-PAGE after glycopeptidase F digestion was smaller than that noted for the native pZPB, which was purified after digestion with endo-{beta}-galactosidase (Fig. 6). These observations suggest that the carbohydrate moieties of the recombinants are less heterogeneous in structure and are smaller than those of the native porcine ZPGs.

The finding that the rZPGs were recognized by ConA, LCA, and GNA, and the MALDI-TOF MS results, are consistent with the types of glycosylation normally found in recombinant glycoproteins expressed in Sf9 cells. The major carbohydrate structures of Sf9-expressed recombinant proteins are N-linked chains that consist mainly of high mannose type chains and pauci mannose type chains, with or without Fuc residues linked to the innermost GlcNAc residue (51, 52). The finding that the rZPGs were not recognized by DSA, RCA120, or PHA-L4 suggests that the recombinants do not contain detectable amounts of complex type chains. In addition, the rZPGs may not have O-linked chains because they are not recognized by ACA. The native porcine zona pellucida does not possess a detectable amount of high mannose type chains that are recognized by GNA, but the zona contains di-, tri-, and tetraantennary complex type N-linked chains with Fuc linked to the innermost GlcNAc, and the zona also has O-linked chains (21, 24). Therefore, the lectin binding activities of the native porcine ZPGs (Fig. 7) match their known structures.

The result that {alpha}-mannosidase digestion of rZPB/rZPC almost abolished its bovine sperm binding activity clearly demonstrates the essential role of nonreducing terminal {alpha}-Man residues of rZPB/rZPC. Thus, although the rZPB/rZPC mixture that is glycosylated in Sf9 cells possesses the terminal {alpha}-Man residues that are essential for bovine sperm binding, it does not possess complex type chains necessary for porcine sperm binding, and therefore it binds bovine, rather than porcine, sperm.

We showed previously that the 111-amino acid N-terminal fragment of pZPB generated by lysylendoprotease digestion is N-glycosylated at Asn-203 and Asn-220 and exhibits sperm binding activity (22). In addition, we have shown that the 17-amino acid fragment that is N-glycosylated at Asn-333 does not have sperm binding activity (22). From these observations, we were not able to determine the relative importance of the polypeptide moiety versus the number of N-glycans in determining the sperm binding activity of each fragment. In the present study, the inhibitory activity of rZPB/rZPC for spermzona binding was not affected by the Asn-333 to Asp mutation (Fig. 9B), clearly demonstrating that the N-glycans linked to Asn-333 are not necessary for sperm binding activity.

In the previous study, we also showed that of the three N-glycosylation sites of ZPB, the triantennary and tetraantennary complex type chains are localized mainly at Asn-220, whereas the biantennary chains are present at all three N-glycosylation sites (25). Considering the localization of the triantennary and tetraantennary chains, we surmised that the N-linked glycan on Asn-220 in the N-terminal fragment of ZPB is involved in sperm binding rather than the N-linked glycan on Asn-203 (25). In the present study, the inhibitory effect of rZPB/rZPC on sperm-zona binding was greatly diminished by the Asn-203 to Asp mutation and almost abolished by the Asn-220 to Asp mutation (Fig. 9B). Taken together, these results suggest that Asn-220 is the most important of the three N-glycosylation sites of ZPB for the sperm binding activity of ZPB/ZPC. In addition, the tri- and tetraantennary chains, which are the N-linked chains of the porcine zona pellucida that are active in sperm binding, are localized at the site. However, we cannot rule out the possibility that the loss of sperm binding activity caused by the Asn to Asp mutation in rZPB was caused by a change in tertiary structure rather than a loss of glycosylation. The effects of the amino acid substitutions on the interaction between rZPB and rZPC and on the tertiary structure of rZPB are important issues that will be addressed in future experiments.

In mice, intense efforts have determined the potential carbohydrate binding specificity of mouse sperm using native ZPGs, rZPGs, glycans of known structures, or neoglycoconjugates as sperm-binding ligands or inhibitors of sperm-egg binding. The nonreducing terminal residues, such as {alpha}-Gal (7), {beta}-GlcNAc (8, 9), {alpha}-Fuc in the context of Lewisx (10), {alpha}-Man (11), and {beta}-Gal (12), are thought to mediate mouse sperm binding. The report that deletion of the {alpha}-1,3-galactosyltransferase gene does not affect murine sperm-egg binding indicates that {alpha}-Gal is not an essential ligand for sperm binding (53). Murine sperm lacking {beta}-1,4-galactosyltransferase even show enhanced binding to murine eggs compared with wild type sperm, which indicates that the nonreducing terminal {beta}-GlcNAc is not essential for sperm recognition (54). Recent structural analysis of murine ZPGs has shown that both O- and N-linked chains lack Lewisx type structures and that they lack {alpha}-Fuc in the Lewisx structures (16, 55, 56). O-Linked chains on the mouse ZPC have been proposed to be sperm ligand carbohydrate chains (5). Subsequently, the sperm ligand O-linked chains were shown to be linked to Ser-332 and Ser-334 (6). Nevertheless, a recent structural analysis using mass spectrometry showed that Ser-332 and Ser-334 are not O-glycosylated (57). Transgenic mice lacking tetraantennary complex type chains were generated by deleting the N-acetylglucosaminyltransferase V gene, and the mice were fertile (58). Mouse eggs with a zona pellucida that has only high mannose type chains as N-linked chains and lacks complex and hybrid type chains were fertilized using mice with conditional knock-out of the N-acetylglucosaminyltransferase I gene (17). Thus, the in vivo studies performed using transgenic mice lacking each glycosyltransferase gene and the mass spectrometric analyses of mouse ZPGs do not support the proposed sperm binding sites on the mouse zona pellucida. Therefore, the essential role of the carbohydrate moiety in murine sperm-egg binding is now being debated.

Alternatively, it was proposed that mouse sperm recognize the supramolecular structure of the zona matrix but not the carbohydrate structures, based on the data obtained from the mice rescued using human ZP2 (ZPA according to the nomenclature used in this paper) and ZP3 (ZPC) (15; for reviews, see Refs. 13 and 14). Recent studies do not completely exclude the involvement of carbohydrate chains in mouse sperm-egg binding, as the {alpha}-Man in the high mannose type chains is still a possible sperm ligand residue because it has not been ruled out in studies of transgenic mice or carbohydrate structures. Given that in vitro experiments examining mouse sperm-egg binding suggest the involvement of multiple receptors on sperm or ligands on the zona pellucida (59), the redundant sperm binding sites might compensate for the lack of some of the sperm-binding sugar residues; therefore; the transgenic mice may be fertile. Consequently, the question as to whether the carbohydrate moieties of ZPGs are essential for mouse sperm-egg binding has not been answered yet; however, it should be answered using genetically engineered mice.

In conclusion, this study strongly suggests that the carbohydrate moieties of the ZPGs are essential for the species-selective recognition of bovine and pig sperm; however, the issue of whether the carbohydrate moieties of mouse ZPGs are essential for mouse sperm binding remains controversial. Furthermore, we have shown that recombinant ZPGs expressed in Sf9 cells are useful for examining the mechanism of the interaction between sperm and the zona pellucida.


   FOOTNOTES
 
* This work was supported by grants-in-aid for Scientific Research, and the National Project on Protein Structural and Functional Analyses from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger}{ddagger} To whom correspondence should be addressed: Dept. of Chemistry, Faculty of Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan. Tel.: 81-43-290-2794; Fax: 81-43-290-2874; E-mail: mnakano{at}faculty.chiba-u.jp.

1 The abbreviations used are: ZPG, zona pellucida glycoprotein; ACA, Amaranthus candatus agglutinin; BO, Brackett and Oliphant; BSA, bovine serum albumin; ConA, concanavalin A; DSA, Datura stramonium agglutinin; Fuc, fucose; GNA, Galanthus nivalis agglutinin; LCA, Lens culinaris agglutinin; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; PA, pyridylamino; PBS, phosphate-buffered saline; PHA, Phaseolus vulgaris agglutinin; pZPA, native porcine ZPA; pZPB, native porcine ZPB; pZPC, native porcine ZPC; RCA120, Ricinus communis agglutinin; rZPA, recombinant porcine ZPA; rZPB, recombinant porcine ZPB; rZPC, recombinant porcine ZPC; rZPB/rZPC, mixture of rZPB and rZPC; rZPG, recombinant ZPG; TBS, Tris-buffered saline. Back



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