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Recombinant Porcine Zona Pellucida Glycoproteins Expressed in Sf9 Cells Bind to Bovine Sperm but Not to Porcine Sperm

2005; Elsevier BV; Volume: 280; Issue: 21 Linguagem: Inglês

10.1074/jbc.m414242200

ISSN

1083-351X

Autores

Naoto Yonezawa, Katsuyasu Kudo, Hirotomo Terauchi, Saeko Kanai, Naoto Yoda, Masaru Tanokura, Kosuke Ito, Kin‐ichiro Miura, Toshiyuki Katsumata, Minoru Nakano,

Tópico(s)

Reproductive Biology and Fertility

Resumo

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 α-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. 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 α-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. 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 (1Litscher E.S. Wassarman P.M. Trends Glycosci. 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The zona consists of three glycoproteins (ZPGs) 1The 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. that are designated ZPA, ZPB, and ZPC in order of the sizes of their respective cDNAs (4Harris J.D. Hibler D.W. Fontenot G.K. Hsu K.T. Yurewicz E.C. Sacco A.G. DNA Seq. 1994; 4: 361-393Crossref PubMed Scopus (318) Google Scholar). In the pig, these glycoproteins were formerly known as ZP1, ZP3α, and ZP3β, respectively (4Harris J.D. Hibler D.W. Fontenot G.K. Hsu K.T. Yurewicz E.C. Sacco A.G. DNA Seq. 1994; 4: 361-393Crossref PubMed Scopus (318) Google Scholar). 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 (5Florman H.M. Wassarman P.M. Cell. 1985; 41: 313-324Abstract Full Text PDF PubMed Scopus (477) Google Scholar, 6Chen J. Litscher E.S. Wassarman P.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6193-6197Crossref PubMed Scopus (124) Google Scholar); the nonreducing terminal residues, such as α-Gal (7Bleil J.D. Wassarman P.M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6778-6782Crossref PubMed Scopus (297) Google Scholar), β-GlcNAc (8Lopez L.C. 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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 (13Jungnickel M.K. Sutton K.A. Florman H.M. Cell. 2003; 114: 401-404Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 14Hoodbhoy T. Dean J. Reproduction. 2004; 127: 417-422Crossref PubMed Scopus (123) Google Scholar) and discussed in recent papers (15Rankin T.L. Coleman J.S. Epifano O. Hoodbhoy T. Turner S.G. Castle P.E. Lee E. Gore-Langton R. Dean J. Dev. Cell. 2003; 5: 33-43Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 16Dell A. Chalabi S. Easton R.L. Haslam S.M. Sutton-Smith M. Patankar M.S. Lattanzio F. Panico M. Morris H.R. Clark G.F. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15631-15636Crossref PubMed Scopus (85) Google Scholar, 17Shi S. Williams S.A. Seppo A. Kurniawan H. Chen W. Ye Z. Marth J.D. Stanley P. Mol. Cell. Biol. 2004; 24: 9920-9929Crossref PubMed Scopus (81) Google Scholar). 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 (18Sacco A.G. Yurewicz E.C. Subramanian M.G. Matzat P.D. Biol. Reprod. 1989; 41: 523-532Crossref PubMed Scopus (100) Google Scholar, 19Berger T. Davis A. Wardrip N.J. Hedrick J.L. J. Reprod. Fertil. 1989; 86: 559-565Crossref PubMed Scopus (56) Google Scholar, 20Yurewicz E.C. Pack B.A. Armant D.R. Sacco A.G. Mol. Reprod. Dev. 1993; 36: 382-389Crossref PubMed Scopus (56) Google Scholar, 21Yonezawa N. Aoki H. Hatanaka Y. Nakano M. Eur. J. Biochem. 1995; 233: 35-41Crossref PubMed Scopus (75) Google Scholar, 22Yonezawa N. Mitsui S. Kudo K. Nakano M. Eur. J. Biochem. 1997; 248: 86-92Crossref PubMed Scopus (42) Google Scholar, 23Yurewicz E.C. Sacco A.G. Gupta S.K. Xu N. Gage D.A. J. Biol. Chem. 1998; 273: 7488-7494Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). ZPB loses this activity when its N-linked carbohydrate chains are removed by N-glycanase digestion (21Yonezawa N. Aoki H. Hatanaka Y. Nakano M. Eur. J. Biochem. 1995; 233: 35-41Crossref PubMed Scopus (75) Google Scholar, 22Yonezawa N. Mitsui S. Kudo K. Nakano M. Eur. J. Biochem. 1997; 248: 86-92Crossref PubMed Scopus (42) Google Scholar). Neutral N-linked carbohydrate chains that are released from the ZPB/ZPC mixture on N-glycanase digestion retain their sperm binding activities (24Noguchi S. Hatanaka Y. Tobita T. Nakano M. Eur. J. Biochem. 1992; 204: 1089-1100Crossref PubMed Scopus (78) Google Scholar). Of these carbohydrate chains, the triantennary and tetraantennary complex type chains (Fig. 1A) bind more strongly than biantennary complex type chains (25Kudo K. Yonezawa N. Katsumata T. Aoki H. Nakano M. Eur. J. Biochem. 1998; 252: 492-499Crossref PubMed Scopus (55) Google Scholar). 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 (26Yurewicz E.C. Pack B.A. Sacco A.G. Mol. Reprod. Dev. 1991; 30: 126-134Crossref PubMed Scopus (101) Google Scholar). 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 (27Yonezawa N. Fukui N. Kuno M. Shinoda M. Goko S. Mitsui S. Nakano M. Eur. J. Biochem. 2001; 268: 3587-3594Crossref PubMed Scopus (30) Google Scholar) and that a high mannose type chain containing 5 Man residues (Fig. 1B) has sperm binding activity (28Amari S. Yonezawa N. Mitsui S. Katsumata T. Hamano S. Kuwayama M. Hashimoto Y. Suzuki A. Takeda Y. Nakano M. Mol. Reprod. Dev. 2001; 59: 221-226Crossref PubMed Scopus (51) Google Scholar). Moreover, the nonreducing terminal α-Man residues are essential for this activity (28Amari S. Yonezawa N. Mitsui S. Katsumata T. Hamano S. Kuwayama M. Hashimoto Y. Suzuki A. Takeda Y. Nakano M. Mol. Reprod. Dev. 2001; 59: 221-226Crossref PubMed Scopus (51) Google Scholar). Thus, differences in the structures of the carbohydrate chains involved in sperm binding may explain the species-selective recognition of sperm by the ZPGs. Recombinant forms of the ZPGs have been expressed in yeast, insect, and mammalian cells, as well as in bacteria (29Gahlay G.K. Srivastava N. Govind C.K. Gupta S.K. J. Reprod. 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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. 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 (4Harris J.D. Hibler D.W. Fontenot G.K. Hsu K.T. Yurewicz E.C. Sacco A.G. DNA Seq. 1994; 4: 361-393Crossref PubMed Scopus (318) Google Scholar). 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 (36Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar). 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 × 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 × 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 × 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 (37Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). 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 (38Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44939) Google Scholar), 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 (25Kudo K. Yonezawa N. Katsumata T. Aoki H. Nakano M. Eur. J. Biochem. 1998; 252: 492-499Crossref PubMed Scopus (55) Google Scholar, 39Noguchi S. Yonezawa N. Katsumata T. Hashizume K. Kuwayama M. Hamano S. Watanabe S. Nakano M. Biochim. Biophys. Acta. 1994; 1201: 7-14Crossref PubMed Scopus (56) Google Scholar) 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. α-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 α-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 α-mannosidase. To investigate the effect of α-mannosidase itself on sperm-zona binding, an α-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 (27Yonezawa N. Fukui N. Kuno M. Shinoda M. Goko S. Mitsui S. Nakano M. Eur. J. Biochem. 2001; 268: 3587-3594Crossref PubMed Scopus (30) Google Scholar, 40Brackett B.G. Oliphant G. Biol. Reprod. 1975; 12: 260-274Crossref PubMed Scopus (964) Google Scholar) or with modified Krebs-Ringer bicarbonate solution for the porcine sperm binding assay (24Noguchi S. Hatanaka Y. Tobita T. Nakano M. Eur. J. Biochem. 1992; 204: 1089-1100Crossref PubMed Scopus (78) Google Scholar, 41Toyoda Y. Chang M.C. J. Reprod. Fertil. 1974; 36: 9-22Crossref PubMed Scopus (321) Google Scholar). 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 × 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 × 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 × 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 (24Noguchi S. Hatanaka Y. Tobita T. Nakano M. Eur. J. Biochem. 1992; 204: 1089-1100Crossref PubMed Scopus (78) Google Scholar), and the carbohydrate chains were fluorescently labeled with 2-aminopyridine, as described previously (24Noguchi S. Hatanaka Y. Tobita T. Nakano M. Eur. J. Biochem. 1992; 204: 1089-1100Crossref PubMed Scopus (78) Google Scholar). 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). 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 (42Williams Z. Wassarman P.M. Biochemistry. 2001; 40: 929-937Crossref PubMed Scopus (50) Google Scholar, 43Zhao M. Gold L. Ginsberg A.M. Liang L.F. Dean J. Mol. Cell. Biol. 2002; 22: 3111-3120Crossref PubMed Scopus (37) Google Scholar). 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

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