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Egg Sialoglycans Increase Intracellular pH and Potentiate the Acrosome Reaction of Sea Urchin Sperm

2002; Elsevier BV; Volume: 277; Issue: 10 Linguagem: Inglês

10.1074/jbc.m110661200

1083-351X

Noritaka Hirohashi, Victor D. Vacquier,

Reproductive biology and impacts on aquatic species

Sea urchin egg jelly (EJ) triggers sperm acrosome reaction (AR), an exocytotic event required for membrane fusion of the gametes. Purified fucose sulfate polymer (FSP) in EJ is one inducer of the AR. Binding of FSP to its receptor regulates opening of two distinct calcium channels and also elevates intracellular pH (pHi). EJ also contains sialic acid-rich glycans (sialoglycans (SG)) that were isolated by β-elimination followed by DEAE chromatography. In the presence of limiting amounts of FSP, the SG fraction markedly potentiates the AR; however, by itself SG has no activity. The SG fraction increases the pHi of sperm without increasing intracellular Ca2+. The SG-induced increase in pHi is not blocked by nifedipine or high K+, whereas the FSP-induced pHi increase is sensitive to both these agents. Treatment of the SG fraction with neuraminidase or mild metaperiodate that specifically cleaves the glycerol side chain of sialic acid abolishes the AR potentiation and ability of SG to elevate pHi. These data are the first to show that there are at least two pathways to induce sperm pHiincrease and that egg surface sialic acid plays a role in triggering the sperm AR. Sea urchin egg jelly (EJ) triggers sperm acrosome reaction (AR), an exocytotic event required for membrane fusion of the gametes. Purified fucose sulfate polymer (FSP) in EJ is one inducer of the AR. Binding of FSP to its receptor regulates opening of two distinct calcium channels and also elevates intracellular pH (pHi). EJ also contains sialic acid-rich glycans (sialoglycans (SG)) that were isolated by β-elimination followed by DEAE chromatography. In the presence of limiting amounts of FSP, the SG fraction markedly potentiates the AR; however, by itself SG has no activity. The SG fraction increases the pHi of sperm without increasing intracellular Ca2+. The SG-induced increase in pHi is not blocked by nifedipine or high K+, whereas the FSP-induced pHi increase is sensitive to both these agents. Treatment of the SG fraction with neuraminidase or mild metaperiodate that specifically cleaves the glycerol side chain of sialic acid abolishes the AR potentiation and ability of SG to elevate pHi. These data are the first to show that there are at least two pathways to induce sperm pHiincrease and that egg surface sialic acid plays a role in triggering the sperm AR. acrosome reaction egg jelly fucose sulfate polymer intracellular Ca2+ intracellular pH sea urchin receptor for egg jelly-1 2′,7′-bis-(2-carboxyl)-5-(and-6-)-carboxyfluorescein DEAE-purified sialoglycans electrodialyzed sialoglycans high pH anion-exchange deionized water N-glycolylneuraminic acid seawater hydrazine The acrosome reaction (AR)1 of animal spermatozoa is necessary for sperm penetration of the extracellular matrices of the egg and fusion of the gamete plasma membranes. The mechanism underlying the AR represents a potential target for novel methods of contraception. Sea urchin spermatozoa are model cells for studying the signal transduction pathways leading to the AR. Molecules in the egg's jelly coat (EJ) bind to sperm receptors and trigger the AR by activating ion fluxes that result in a net influx of Ca2+and Na+ and net efflux of H+ and K+(1Schackmann R.W. Eddy E.M. Shapiro B.M. Dev. Biol. 1978; 65: 483-495Crossref PubMed Scopus (168) Google Scholar, 2Schackmann R.W. Shapiro B.M. Dev. Biol. 1981; 81: 145-154Crossref PubMed Scopus (109) Google Scholar, 3Darszon A. Labarca P. Nishigaki T. Espinosa F. Physiol. Rev. 1999; 79: 481-510Crossref PubMed Scopus (281) Google Scholar). The macromolecular fraction of EJ is composed of sialoglycoproteins (4Kitazume S. Kitajima K. Inoue S. Troy F.A. Cho J.W. Lennarz W.J. Inoue Y. J. Biol. Chem. 1994; 269: 22712-22718Abstract Full Text PDF PubMed Google Scholar, 5Suzuki N. Zool. Sci. 1995; 12: 13-27Crossref PubMed Scopus (90) Google Scholar) and a fucose sulfate polymer (FSP (6SeGall G.K. Lennarz W.J. Dev. Biol. 1979; 71: 33-48Crossref PubMed Scopus (203) Google Scholar)). The FSPs of several sea urchin species are linear polymers of 1 → 3-linked α-l-fucose with species-specific patterns ofO-sulfation (7Vilela-Silva A.C. Alves A.P. Valente A.P. Vacquier V.D. Mourao P.A. Glycobiology. 1999; 9: 927-933Crossref PubMed Scopus (113) Google Scholar). The pattern of sulfation is what makes FSP a species-specific inducer of the AR (8Alves A.P. Mulloy B. Diniz J.A. Mourao P.A. J. Biol. Chem. 1997; 272: 6965-6971Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 9Alves A.P. Mulloy B. Moy G.W. Vacquier V.D. Mourao P.A. Glycobiology. 1998; 8: 939-946Crossref PubMed Scopus (92) Google Scholar). FSP induces the sequential activation of two sperm calcium channels. The first channel, which is transiently opened, is blocked by dihydropyridines (10Guerrero A. Darszon A. J. Biol. Chem. 1989; 264: 19593-19599Abstract Full Text PDF PubMed Google Scholar). The second channel, which is insensitive to dihydropyridines, allows a sustained increase in intracellular Ca2+([Ca2+]i) that triggers the AR (11Gonzalez-Martinez M.T. Galindo B.E. de De La Torre L. Zapata O. Rodriguez E. Florman H.M. Darszon A. Dev. Biol. 2001; 236: 220-229Crossref PubMed Scopus (56) Google Scholar). The second channel may be a store-operated channel that is dependent for activation on the increase in intracellular pH (pHi (11Gonzalez-Martinez M.T. Galindo B.E. de De La Torre L. Zapata O. Rodriguez E. Florman H.M. Darszon A. Dev. Biol. 2001; 236: 220-229Crossref PubMed Scopus (56) Google Scholar, 12Guerrero A. Garcia L. Zapata O. Rodriguez E. Darszon A. Biochim. Biophys. Acta. 1998; 1401: 329-338Crossref PubMed Scopus (17) Google Scholar)). The EJ-induced increase in sperm pHi occurs by activation of a voltage-dependent Na+/H+ exchanger (12Guerrero A. Garcia L. Zapata O. Rodriguez E. Darszon A. Biochim. Biophys. Acta. 1998; 1401: 329-338Crossref PubMed Scopus (17) Google Scholar, 13Lee H.C. J. Biol. Chem. 1985; 260: 10794-10799Abstract Full Text PDF PubMed Google Scholar, 14Gonzalez-Martinez M.T. Guerrero A. Morales E. de De La Torre L. Darszon A. Dev. Biol. 1992; 150: 193-202Crossref PubMed Scopus (47) Google Scholar). Sea urchin sperm receptor for egg jelly-1 (suREJ1 (15Moy G.W. Mendoza L.M. Schulz J.R. Swanson W.J. Glabe C.G. Vacquier V.D. J. Cell Biol. 1996; 133: 809-817Crossref PubMed Scopus (220) Google Scholar)) is a single transmembrane segment, 1450-amino acid glycoprotein, localized to the flagellar and acrosomal plasma membranes. suREJ1 binds FSP to induce the AR (16Vacquier V.D. Moy G.W. Dev. Biol. 1997; 192: 125-135Crossref PubMed Scopus (135) Google Scholar) and monoclonal antibodies to suREJ1 induce the AR, and purified suREJ1 neutralizes FSP as an inducer of the AR (15Moy G.W. Mendoza L.M. Schulz J.R. Swanson W.J. Glabe C.G. Vacquier V.D. J. Cell Biol. 1996; 133: 809-817Crossref PubMed Scopus (220) Google Scholar). Sea urchin sperm suREJ3 is a 2681-amino acid orphan receptor that localizes exclusively to the cell membrane covering the sperm acrosomal vesicle. suREJ1 and suREJ3 share significant homology with each other and with the human polycystin-1 family (the protein mutated in autosomal dominant polycystic kidney disease (17Mengerink K.J. Moy G.W. Vacquier V.D. J. Biol. Chem. 2002; 277: 943-948Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar)). Although ligands binding suREJ3 remain unknown, it is reasonable to assume that suREJ3 is somehow involved in AR induction. EJ contains several high molecular weight components. Although purified FSP, devoid of amino acids, induces all the ion channel events triggering the AR (6SeGall G.K. Lennarz W.J. Dev. Biol. 1979; 71: 33-48Crossref PubMed Scopus (203) Google Scholar, 9Alves A.P. Mulloy B. Moy G.W. Vacquier V.D. Mourao P.A. Glycobiology. 1998; 8: 939-946Crossref PubMed Scopus (92) Google Scholar, 16Vacquier V.D. Moy G.W. Dev. Biol. 1997; 192: 125-135Crossref PubMed Scopus (135) Google Scholar), earlier data (18Ishihara K. Dan J.C. Dev. Growth Differ. 1970; 12: 179-188Crossref PubMed Scopus (21) Google Scholar, 19Shimizu T. Kinoh H. Yamaguchi M. Suzuki N. Dev. Growth Differ. 1990; 32: 473-487Crossref Scopus (31) Google Scholar, 20Keller S.H. Vacquier V.D. Dev. Growth Differ. 1994; 36: 551-556Crossref Scopus (13) Google Scholar, 21Keller S.H. Vacquier V.D. Dev. Biol. 1994; 162: 304-312Crossref PubMed Scopus (41) Google Scholar) suggested that other EJ macromolecules may also be involved in secondary or redundant signal transduction pathways leading to the AR. For example, protease treatment of EJ, which has no effect on FSP, was found to decrease the AR inducing activity of EJ by as much as 50% (18Ishihara K. Dan J.C. Dev. Growth Differ. 1970; 12: 179-188Crossref PubMed Scopus (21) Google Scholar, 19Shimizu T. Kinoh H. Yamaguchi M. Suzuki N. Dev. Growth Differ. 1990; 32: 473-487Crossref Scopus (31) Google Scholar). Treatment of EJ with sodium metaperiodate decreased AR activity but left FSP intact (18Ishihara K. Dan J.C. Dev. Growth Differ. 1970; 12: 179-188Crossref PubMed Scopus (21) Google Scholar). Treatment of EJ with peptide-N-glycosidase-F releases N-linked oligosaccharides from EJ and decreases its AR activity (20Keller S.H. Vacquier V.D. Dev. Growth Differ. 1994; 36: 551-556Crossref Scopus (13) Google Scholar). These results, and other conflicts in the literature (21Keller S.H. Vacquier V.D. Dev. Biol. 1994; 162: 304-312Crossref PubMed Scopus (41) Google Scholar), caused us to revisit the question of the role of the oligosaccharide fraction of EJ in AR induction. Here we present data showing that β-elimination of total EJ, followed by DEAE chromatography and electrodialysis, separates the sialic acid-rich high molecular mass glycans from FSP. This fraction has no AR activity of its own. However, it greatly potentiates the FSP-induced AR, indicating that it works through a receptor pathway that is different from the FSP pathway. The sialoglycan fraction appears to elevate sperm pHi, suggesting that its ultimate target might be the Na+/H+ exchanger responsible for the pHi increase (3Darszon A. Labarca P. Nishigaki T. Espinosa F. Physiol. Rev. 1999; 79: 481-510Crossref PubMed Scopus (281) Google Scholar). Treatment of sialoglycans with neuraminidase or mild metaperiodate that specifically destroys sialic acid renders the fraction inactive. This is the first direct evidence for the involvement of sialic acid as a ligand in the induction of the animal sperm AR. Fura-2 AM, BCECF-AM, and nigericin were from Molecular Probes. Rhodizonic acid disodium and l-ascorbic acid were from Aldrich. Ionomycin and NeuAc were from Calbiochem. 1,9-Dimethylmethylene blue was from Serva. Heparin, Vibrio cholerae neuraminidase, l-fucose, nifedipine, and DEAE-cellulose were from Sigma. Sepharose CL-6B was from Amersham Biosciences. Affi-Gel Hz was from Bio-Rad. Strongylocentrotus purpuratus sperm were collected as undiluted semen by intercoelomic injection of 0.5m KCl and kept on ice. Eggs were spawned into beakers filled with seawater. Preparation of crude EJ was described (22Hirohashi N. Vacquier V.D. J. Biol. Chem. 2002; 277: 1182-1189Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Preparation and fractionation of EJ oligo/polysaccharides were performed as described (22Hirohashi N. Vacquier V.D. J. Biol. Chem. 2002; 277: 1182-1189Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Briefly, crude EJ, dialyzed against deionized water, was subjected to β-elimination (0.05 nKOH, 1 m NaBH4, 45 °C for 24 h), diluted with 4× of acetate buffer (50 mm sodium acetate, pH 5.0), and the pH adjusted to 5.0 by addition of 2 macetic acid. The solution containing EJ oligo/polysaccharides was applied to a DEAE-cellulose column equilibrated in the above acetate buffer. The column (1.4 × 6 cm) was then washed with 10× acetate buffer. Bound materials were eluted with a 0–3 m NaCl gradient in acetate buffer. Each 3-ml fraction was tested for negatively charged polysaccharides by the metachromatic assay (23Terho T.T. Hartiala K. Anal. Biochem. 1971; 41: 471-476Crossref PubMed Scopus (436) Google Scholar). The fractions containing sialoglycans eluted between 0 and 1 mNaCl. They were pooled and sialoglycans precipitated by addition of 3× 95% ethanol. FSP eluted between 1.5 and 2.5 m NaCl and was precipitated by adding 1× 95% ethanol. Both sialoglycan and FSP precipitates were then sedimented by centrifugation at 12,000 ×g for 20 min. The supernatants were decanted, and the centrifuge tubes were air-dried. For FSP, the pellet was dissolved in deionized water (dH2O) and dialyzed overnight against dH2O at 4 °C. For some experiments (Figs. Figure 4, Figure 5, Figure 6, Figure 7), the sialoglycan fraction was further purified by electrodialysis. For electrodialysis, the sample was placed in a Centricon YM-10 cartridge (10-kDa cut-off; Amicon), and the cartridge was placed in the electrodialysis apparatus (Centrilutor, Amicon) and filled with 50 mm sodium acetate, pH 5.0. The sample was subjected to 1 watt for 4 h at room temperature. After electrodialysis, sialoglycans remaining in the cartridge were recovered and dialyzed against dH2O. The Sepharose CL-6B column (1.2 × 60 cm) was equilibrated with Millipore filtered seawater. The sample was applied, and 1-ml fractions were collected. The fractions were tested in the metachromatic assay for negatively charged polysaccharides, the thiobarbituric acid assay for sialic acid (24Aminoff D. Biochem. J. 1961; 81: 384-392Crossref PubMed Scopus (1443) Google Scholar), the Dische-Shettle cysteine assay for fucose (25Dische Z. Shettles L.B. J. Biol. Chem. 1951; 192: 579-582Abstract Full Text PDF PubMed Google Scholar), and the sperm AR potentiation assay. Sialogycans in the concentration range equivalent to 0.1–1 mg of heparin/ml (metachromatic assay) were placed in acetate buffer (10 mm sodium acetate, 4 mmCaCl2, pH 5.5) and V. cholerae neuraminidase (0.3 units/mg of sialoglycans) added, and the reaction tube was incubated at 37 °C for 24 h. The reaction was stopped by addition of 10× quenching buffer (25 mm Tris, 192 mm glycine, 10 mm EDTA, pH 8.3), and the released sialic acid was removed by electrodialysis using Centricon YM-10 cartridges. Sialogycans were treated with NaIO4 to truncate specifically the glycerol side chain of sialic acid (26Lenten L.V. Ashwell G. J. Biol. Chem. 1971; 246: 1889-1894Abstract Full Text PDF PubMed Google Scholar). Briefly, the sialoglycans were treated with 2 mmNaIO4 in phosphate-buffered saline for 30 min on ice in the dark. This reaction mixture was immediately used for cross-linking to Affi-Gel Hz. For biological assays of mild metaperiodate treated sialoglycans, the resulting aldehyde of sialic acid was reduced with 10 mm NaBH4 in phosphate-buffered saline for 2 h on ice in the dark. The reaction mixture was then dialyzed overnight against seawater at 4 °C. A 50-μl aliquot of Affi-Gel Hz beads was washed twice with 20× dH2O. The beads were sedimented by centrifugation at 12,000 × g for 30 s, and the supernatant was discarded. Fifty μl of solution containing mild periodate-treated sialogycans was mixed continuously with the beads for 30 min at room temperature (total volume 100 μl). The beads were sedimented by centrifugation, and the supernatant was recovered. The beads were resuspended in 50 μl of high salt buffer (2 m NaCl in 10 mm sodium acetate, pH 5.0) and centrifuged, and the supernatant was recovered. The beads were then resuspended in 50 μl of acetate buffer and heated to 100 °C for 10 min, and the supernatant was recovered after centrifugation. The supernatants recovered after different washes were tested in the metachromatic assay and the thiobarbituric acid assay. Sperm intracellular Ca2+ and pH were measured as described (22Hirohashi N. Vacquier V.D. J. Biol. Chem. 2002; 277: 1182-1189Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Briefly, fresh, undiluted semen was suspended in 4× dye loading buffer (450 mm NaCl, 9 mm KCl, 48 mmMgSO4, 6 mm NaHCO3, 1 mm CaCl2, 10 mm HEPES, 50 μg/ml soybean trypsin inhibitor, pH 7.0). The suspension was gently mixed with dimethyl sulfoxide (Me2SO, final concentration 0.6%) containing either fura-2 AM or BCECF-AM at a final concentration of 12 μm, and the tube was incubated on ice at least 8 h before washing. To remove free dye, sperm were sedimented by centrifugation in a swinging bucket rotor at 430 × gfor 7 min. The supernatants were removed, and 9× dye loading buffer was added, followed by gently mixing until the pellet of sperm was completely resuspended. This washing procedure was repeated 3 times. The final cell pellet was resuspended in 4× dye loading buffer without soybean trypsin inhibitor and stored on ice in the dark. For the measurements, 50 μl of fura-2 loaded sperm (2 × 108cells/ml) or 10 μl of BCECF loaded sperm were placed in an 11-mm diameter round bottomed-glass tube containing 1.5 ml of 10 mm HEPES-buffered-Millipore filtered seawater, pH 8.0 (HEPES-SW). The tube was mounted in a temperature-controlled cell holder (16 °C) in a FluoroMax-2 fluorometer (JOBIN YVON-SPEX), and the fluorescence at excitation/emission wavelength pairs of 380/500 and 340/500 nm (for fura-2) and 490/510 and 440/510 nm (for BCECF) were recorded while continuously stirred with a micromagnetic bar. Fura-2 signals were calibrated to the ratio of 340/380 nm and presented as the increased [Ca2+]i/K d value or Δ[Ca2+]i/K d (27Kao J.P.Y. Methods Cell Biol. 1994; 40: 155-181Crossref PubMed Scopus (277) Google Scholar) obtained after 5 min of recording. The calibration of pHi was performed as described (22Hirohashi N. Vacquier V.D. J. Biol. Chem. 2002; 277: 1182-1189Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Monosaccharide analyses were done by Glycobiology Core Resource, Glycobiology Research and Training Center (University of California, San Diego). The DEAE-fractionated (dSG) and electrodialyzed (eSG) sialoglycan fractions were hydrolyzed. The hydrolysates were then analyzed by high pH anion-exchange (HPAE) chromatography on a Dionex CarboPac PA-10 or PA-1 column with pulsed amperometoric detector. Standard mixtures of monosaccharides were run in parallel to calibrate HPAE retention times and to allow qualifications. The AR was evaluated by scoring the number of acrosome intact and reacted sperm under a phase contrast microscope at 1200× magnification (16Vacquier V.D. Moy G.W. Dev. Biol. 1997; 192: 125-135Crossref PubMed Scopus (135) Google Scholar). Briefly, 10 μl of sperm suspension that was freshly diluted 50-fold in ice-cold HEPES-SW was mixed in a 1.5-ml centrifuge tube with 30 μl of HEPES-SW containing the test compound at 16 °C. After 5 min sperm were fixed with 40 μl of 6% glutaraldehyde in HEPES-SW. For the AR potentiation assay, the concentration of FSP resulting in 10–20% AR was determined. FSP at this concentration was then mixed with sialoglycans in the range of 0–25 μg of heparin equivalent/ml. These mixtures were then tested in the AR assay. EJ was subjected to β-elimination which released theO-linked oligosaccharides and FSP from total EJ. The EJ oligosaccharides were then separated from FSP by DEAE chromatography. Each eluted fraction was tested in the metachromatic assay for negatively charged glycans such as sulfated glycosaminoglycans (28Farndale R.W. Buttle D.J. Barrett A.J. Biochim. Biophys. Acta. 1986; 883: 173-177Crossref PubMed Scopus (2907) Google Scholar) and sulfated fucans (9Alves A.P. Mulloy B. Moy G.W. Vacquier V.D. Mourao P.A. Glycobiology. 1998; 8: 939-946Crossref PubMed Scopus (92) Google Scholar). The first peak appeared in the fractions at∼0.7 m NaCl (Fig.1 A). Monosaccharide compositional analysis of this sharp peak showed that it contained two major constituents: N-glycolylneuraminic acid (NeuGc; 78.5% of total sugar) and fucose (12.3% of total sugar; TableI). The high molar ratio of NeuGc to fucose suggests the presence of polysialic acid in this fraction (4Kitazume S. Kitajima K. Inoue S. Troy F.A. Cho J.W. Lennarz W.J. Inoue Y. J. Biol. Chem. 1994; 269: 22712-22718Abstract Full Text PDF PubMed Google Scholar). The metachromatic assay is a novel method for the detection of sulfated glycosaminoglycans, so the first peak was examined for uronic acid. Neither glucuronic acid nor galacturonic acid was detected (Table I). This first peak is hereafter referred to as the DEAE-purified sialoglycans (dSG). The second broad peak eluting at 1.6–2.5m NaCl is FSP as determined in previous experiments (6SeGall G.K. Lennarz W.J. Dev. Biol. 1979; 71: 33-48Crossref PubMed Scopus (203) Google Scholar, 9Alves A.P. Mulloy B. Moy G.W. Vacquier V.D. Mourao P.A. Glycobiology. 1998; 8: 939-946Crossref PubMed Scopus (92) Google Scholar,16Vacquier V.D. Moy G.W. Dev. Biol. 1997; 192: 125-135Crossref PubMed Scopus (135) Google Scholar). The elution profile of the FSP peak has a shoulder, which probably represents the two isotypes of FSP present in S. purpuratusEJ that differ in sulfate content (9Alves A.P. Mulloy B. Moy G.W. Vacquier V.D. Mourao P.A. Glycobiology. 1998; 8: 939-946Crossref PubMed Scopus (92) Google Scholar). Fractions 16–20, representing the dSG peak, and fractions 40–75, representing the FSP peak, were pooled, and both pools were separated by electrophoresis on a 4–15% SDS-polyacrylamide gel that was stained with 0.1% toluidine blue (Fig.1 B). Samples were equalized on the basis of heparin equivalents as determined by the metachromatic assay. FSP appeared as a smear in the high molecular mass range, whereas no staining appeared in the dSG lane. Because toluidine blue stains the fragmented FSP (22Hirohashi N. Vacquier V.D. J. Biol. Chem. 2002; 277: 1182-1189Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), the lack of stain in the dSG lane does not represent the degradation of FSP.Table IMonosaccharide composition analyses of EJ sialoglycan fractionsMonosaccharidedSGeSGmolar ratio1-aThe error margins in the values are ±10%.Fucose1.001-bArbitrarily set to 1.00.1.001-bArbitrarily set to 1.00.Mannose/xylose1-cThe peak in the HPAE profile may consist of a mixture of mannose and xylose. Mannose and xylose elute at the same retention time under these conditions.0.300.22Galactose0.060.05GlcNAc0.220.08GalNAc0.160.06Neu5AcND1-dND, not detected.NDNeu5Gc6.395.85GlcA1-eGlcA, glucuronic acid; GalA, galacturonic acid.NDNDGalA1-eGlcA, glucuronic acid; GalA, galacturonic acid.NDNDThe only monosaccharide present in FSP is fucose (9Alves A.P. Mulloy B. Moy G.W. Vacquier V.D. Mourao P.A. Glycobiology. 1998; 8: 939-946Crossref PubMed Scopus (92) Google Scholar,21Keller S.H. Vacquier V.D. Dev. Biol. 1994; 162: 304-312Crossref PubMed Scopus (41) Google Scholar).1-a The error margins in the values are ±10%.1-b Arbitrarily set to 1.00.1-c The peak in the HPAE profile may consist of a mixture of mannose and xylose. Mannose and xylose elute at the same retention time under these conditions.1-d ND, not detected.1-e GlcA, glucuronic acid; GalA, galacturonic acid. Open table in a new tab The only monosaccharide present in FSP is fucose (9Alves A.P. Mulloy B. Moy G.W. Vacquier V.D. Mourao P.A. Glycobiology. 1998; 8: 939-946Crossref PubMed Scopus (92) Google Scholar,21Keller S.H. Vacquier V.D. Dev. Biol. 1994; 162: 304-312Crossref PubMed Scopus (41) Google Scholar). The specific activity of purified FSP to induce the AR was examined in comparison with total EJ. Sugar concentrations of EJ and FSP were determined by the phenol-sulfuric acid method and are presented as hexose equivalents. Although sperm obtained from different batches differ in sensitivity of response to EJ (16Vacquier V.D. Moy G.W. Dev. Biol. 1997; 192: 125-135Crossref PubMed Scopus (135) Google Scholar), EJ is always a better inducer of the AR than purified FSP (Fig.2 A). In this experiment, 10 μg of hexose/ml FSP was needed to achieve 50% AR, whereas only 0.5 μg/ml total EJ induced 50% AR. Although purified FSP is an AR inducer, these data suggest that EJ contains other components that enhance AR induction. The dSG fraction was tested for AR inducing activity, alone or in combination with FSP (Fig. 2 B). EJ at 3 μg of hexose/ml induced 98% AR, whereas 1% AR occurred with only seawater as the negative control. FSP at 3 μg/ml induced 20% AR, whereas 3 μg/ml dSG induced 2%. However, when FSP and dSG (3 μg/ml each) were combined, the AR reached 95%. Decreasing both FSP and dSG to 1.5 μg/ml each resulted in 90% AR. A 5-fold increase in FSP to 15 μg/ml induced 92% AR; however, the dSG at 15 μg/ml yielded only 1% AR. These data clearly show that the dSG fraction potentiates the FSP-induced AR. The dSG fraction was chromatographed on Sepharose CL-6B. Fractions were tested in the metachromatic assay, and the peak was found to have an average mass of ∼100 kDa (Fig.3 A). Because the dSG fraction contains fucose at 12.3% mol of total monosaccharides (Table I), fucose content in each fraction was also determined by the Dische-Shettles cysteine method. The peak for fucose elution was identical to the peak determined by the metachromatic assay (Fig.3 B). Sepharose CL-6B fractions were further tested in the AR potentiation assay in the presence of limiting amount of FSP. The maximum activity for AR potentiation corresponded to the peak determined by the metachromatic assay (Fig. 3 C). The dSG fraction was subjected to further purification by electrodialysis. After 4 h of electrodialysis, 23% total sialic acid was removed (data not shown). A molar ratio between fucose and NeuGc was ∼1:6 (Table I). The electrodialyzed sialoglycan fraction (eSG) was tested in the AR potentiation assay. The AR specific activity of the eSG increased ∼4-fold relative to the dSG fraction when both fractions were normalized to heparin equivalents (Fig.4 A). To be specific, the concentrations of dSG or eSG that are required for 50% AR were 13.5 or 3.0 μg/ml, respectively. eSG was chromatographed on Sepharose CL-6B. Two peaks, one representing the metachromatic assay and the other sialic acid, closely overlapped but were not perfectly superimposable (Fig. 4 B). The eSG fractions were treated with V. cholerae neuraminidase. After a 24-h incubation, the reaction mixture was rechromatographed on Sepharose CL-6B. The peaks of metachromasia and sialic acid were now superimposable (Fig.4 C). The small peak at fraction 57 is monosialic acid released by the neuraminidase. However, the majority of sialic acid remained associated with the original glycans. The reason that the two peaks are not perfectly superimposable (Fig. 4 B) may be because of the heterogeneous sialylation of the SG molecules (4Kitazume S. Kitajima K. Inoue S. Troy F.A. Cho J.W. Lennarz W.J. Inoue Y. J. Biol. Chem. 1994; 269: 22712-22718Abstract Full Text PDF PubMed Google Scholar). The time course of sialic acid release (Fig. 4 D) shows maximum release (30% of total) by 1 h. These results show that ∼70% of total sialic acid in the eSG is neuraminidase-resistant. The parallel decreases in both metachromasia and sialic acid suggest that the glycans detected by the metachromatic assay contain sialic acid (Fig.4 D). The eSG fraction was treated with 2 mm NaIO4(mild metaperiodate) to remove the glycerol side chain and form an aldehyde in the sialic acid (26Lenten L.V. Ashwell G. J. Biol. Chem. 1971; 246: 1889-1894Abstract Full Text PDF PubMed Google Scholar). Following the treatment, the aldehyde group of eSG was used to covalently couple it to Affi-Gel Hz (beads containing hydrazine groups). Using the thiobarbituric acid assay, 86.2% of total sialic acid bound to the beads. However, in the absence of NaIO4 oxidation, 34.4% bound to the beads (Fig.4 E). Using the metachromatic assay, 81.2% of total negatively charged glycans bound to the beads, whereas in the no NaIO4 control, only 11.3% bound to the beads. These data show that the metachromasia-positive glycans responsible for AR potentiation contain sialic acid. To understand the underlying mechanism of AR potentiation by sialoglycans, we first examined the effect on sperm [Ca2+]i. The eSG alone, when added to fura-2 loaded-sperm, had no effect on [Ca2+]i (Fig.5 A). FSP at 0.25 μg/ml induced 30% of the sperm to AR; these sperm showed a moderate increase in [Ca2+]i compared with that of total EJ. FSP mixed with eSG increased the percentage of AR in a concentration-dependent manner (0–10 μg/ml). However, this mixture did not significantly alter the [Ca2+]i in comparison to FSP alone. Increases in [Ca2+]i were quantified and are represented as Δ[Ca2+]i/K d as described under “Experimental Procedures.” Purified FSP caused a concentration-dependent increase in [Ca2+]i (Fig. 5 B). However, eSG did not increase [Ca2+]i either in the presence or absence of FSP. These results show that sialoglycans do not up-regulate sperm Ca2+ channels. Next, we examined the effect on sperm pHi, because an increase in pHi is a crucial event in AR induction (3Darszon A. Labarca P. Nishigaki T. Espinosa F. Physiol. Rev. 1999; 79: 481-510Crossref PubMed Scopus (281) Google Scholar, 29Garbers D.L. Annu. Rev. Biochem. 1989; 58: 719-742Crossref PubMed Scopus (187) Google Scholar). The eSG alone increased sperm pHi in a concentration-dependent manner (Fig.6 A). The pHi change produced by FSP was ∼3-fold greater than that produced by eSG. The FSP-induced pHi increase is known to be blocked by Ca2+ channel blockers such as nifedipine (22Hirohashi N. Vacquier V.D. J. Biol. Chem. 2002; 277: 1182-1189Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) or extracellular high K+ (10Guerrero A. Darszon A. J. Biol. Chem. 1989; 264: 19593-19599Abstract Full Text PDF PubMed Google Scholar). In fact, 50 μmnifedipine, which completely blocks the opening of the first sperm Ca2+ channel (11Gonzalez-Martinez M.T. Galindo B.E. de De La Torre L. Zapata O. Rodriguez E. Florman H.M. Darszon A. Dev. Biol. 2001; 236: 220-229Crossref PubMed Scopus (56) Google Scholar), inhibited the FSP-induced pHiincrease (Fig. 6 B). The FSP-induced pHi increase was also inhibited by 50 mm K+. Three hundred mm Ni2+, which blocks the second sperm Ca2+ channel (11Gonzalez-Martinez M.T. Galindo B.E. de De La Torre L. Zapata O. Rodriguez E. Florman H.M. Darszon A. Dev. Biol. 2001; 236: 220-229Crossref PubMed Scopus (56) Google Scholar), also inhibited the FSP-induced pHi increase (Fig. 6 B). In contrast, nifedipine and 50 mm K+ were only moderately effective in blocking the eSG-induced pHi increase (Fig. 6 B). However, Ni2+ completely blocked the eSG-induced pHi increase. These data show that EJ sialoglycans induce sperm pHi increases by a different pathway than the one used by FSP.Figure 6Sialoglycans increase sperm pHi. A, changes in sperm pHi were measured at various concentrations of FSP and eSG. The increased pHi(ΔpHi) values obtained 5 min after addition of FSP (●) or eSG (○) are presented. B, 2.5 μg of heparin/ml FSP or 25 μg of heparin/ml eSG were tested in the absence or presence of 50 μm nifedipine (Nif), 50 mm KCl (K+), and 300 μm NiCl2(Ni2+). The columns show mean ± S.E. from three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The eSG fractions, treated either with neuraminidase or mild metaperiodate, were tested in the AR potentiation assay. The non-treated eSG fraction (plus FSP) potentiated the AR in a concentration-dependent manner, and a maximum percentage AR was obtained at 80 μg of heparin/ml (Fig.7 A). The potentiation activity of the eSG was completely lost when it was treated with either neuraminidase or mild metaperiodate (Fig. 7 A). The percentage AR was constant at 15 ± 5% in either neuraminidase or mild periodate-treated eSGs up to 160 μg/ml, showing that neither sample negatively affects AR induction. These results demonstrate that a glycerol side chain of NeuGc residues is essential for potentiation activity. Finally, we tested the effects of the treated eSGs on sperm pHi. The non-treated eSG at 50 μg of heparin/ml caused a 0.05 unit increase in pHi (Fig. 7 B). In contrast, the eSG fractions treated with either neuraminidase or mild metaperiodate were completely ineffective at increasing pHi. In conclusion, sialoglycans, consisting mainly NeuGc, increase sperm pHi by an unknown mechanism that is independent of the FSP-regulated [Ca2+]i increasing pathway. These sialoglycans markedly potentiate the FSP-induced AR. Approximately 80% of the mass of S. purpuratus EJ is FSP, and 20% composes the sialoglycoproteins (6SeGall G.K. Lennarz W.J. Dev. Biol. 1979; 71: 33-48Crossref PubMed Scopus (203) Google Scholar). The purified FSP alone will induce the increase in intracellular Ca2+ and pH needed to trigger the AR (9Alves A.P. Mulloy B. Moy G.W. Vacquier V.D. Mourao P.A. Glycobiology. 1998; 8: 939-946Crossref PubMed Scopus (92) Google Scholar, 16Vacquier V.D. Moy G.W. Dev. Biol. 1997; 192: 125-135Crossref PubMed Scopus (135) Google Scholar, 22Hirohashi N. Vacquier V.D. J. Biol. Chem. 2002; 277: 1182-1189Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), but it is much less active than whole EJ (Fig. 2 A). Treatment of EJ with mild periodate, which has no effect on FSP, decreases the AR activity (18Ishihara K. Dan J.C. Dev. Growth Differ. 1970; 12: 179-188Crossref PubMed Scopus (21) Google Scholar). These results suggest that other EJ components, especially periodate-sensitive glycans, must be involved in AR induction through an ancillary pathway or as co-factors that facilitate the FSP-induced AR. β-Elimination of whole EJ degrades the protein components and releases O-linked oligosaccharides. Sialoglycans can be completely separated from FSP by DEAE chromatography (Fig. 1). Monosaccharide analysis of the SG fraction shows that its molar ratio of fucose to sialic acid is ∼1:6 with small amounts of other monosaccharides (Table I). Other workers (30Hotta K. Kurokawa M. Isaka S. J. Biol. Chem. 1970; 245: 6307-6311Abstract Full Text PDF PubMed Google Scholar) have also found that the oligosaccharides in EJ are rich in sialic acid and that only NeuGc and not NeuAc is present. S. purpuratus EJ contains a unique polysialic acid, [Neu5Gcα2→5-O-glycolylNeu5Gc]n, which isO-glycosidically linked to a 250-kDa glycoprotein (4Kitazume S. Kitajima K. Inoue S. Troy F.A. Cho J.W. Lennarz W.J. Inoue Y. J. Biol. Chem. 1994; 269: 22712-22718Abstract Full Text PDF PubMed Google Scholar). Also, a disaccharide, Fucp1→4NeuGc, was found in the acid hydrolysate of Pseudocentrotus depressus EJ (31Hotta K. Kurokawa M. Isaka S. J. Biol. Chem. 1973; 248: 629-631Abstract Full Text PDF PubMed Google Scholar). Although some of these investigators had tried to induce the AR with fractionated SG, until now no one had reported attempts to use the SG mixed with limiting amounts of FSP to look for possible synergistic effects on AR induction. This paper demonstrates for the first time that the SG fraction is a potent potentiator of the FSP-induced AR. When tested alone, no AR inducing activity was found in the SG fraction (Fig. 2 B), showing that FSP is a necessary component. The AR potentiation activity was found in glycans of an apparent molecular mass of 100 kDa that are strongly negatively charged and contain fucosyl residues (Fig. 3). Further purification of dSG by electrodialysis removed 23% of the total sialic acid and resulted in a 4-fold increase in AR potentiation activity (Fig. 4 A). The evidence that sialic acid is a constituent of the metachromasia-positive glycans was demonstrated by Sepharose CL-6B chromatography of eSG after digestion with V. cholerae neuraminidase (Fig. 4, B and C). This enzyme is known to cleave a unique EJ polysialic acid (32Inoue S. Lin S.L. Inoue Y. Groves D.R. Thomson R.J. von Itzstein M. Pavlova N.V. Li S.C. Li Y.T. Biochem. Biophys. Res. Commun. 2001; 280: 104-109Crossref PubMed Scopus (5) Google Scholar). Mild metaperiodate treatment of the eSG fraction immobilized the metachromasia-positive glycans to hydrazine beads (Fig. 4 E). The sialyl residues of the eSG fraction are crucial for the AR potentiation, because the above treatments, which specifically destroy sialyl residues, caused a loss of biological activity (Fig.7 A). The eSG fraction had no effect on the [Ca2+]i of fura-2-loaded sperm (Fig. 5). However, eSG increased pHi even in the absence of FSP (Fig.6 A). These data suggest that the SG-induced AR potentiation signaling pathway is different from the FSP-induced pathway. This hypothesis was further supported by the differential sensitivities of the FSP and eSG responses to the Ca2+ channel blocker, nifedipine, and high extracellular K+ (Fig. 6 B). The eSG-induced pHi increase was abolished by neuraminidase or mild metaperiodate treatment, suggesting that sialyl residues of sialoglycans are responsible for the AR potentiation. Our previous studies (15Moy G.W. Mendoza L.M. Schulz J.R. Swanson W.J. Glabe C.G. Vacquier V.D. J. Cell Biol. 1996; 133: 809-817Crossref PubMed Scopus (220) Google Scholar, 16Vacquier V.D. Moy G.W. Dev. Biol. 1997; 192: 125-135Crossref PubMed Scopus (135) Google Scholar) showed that suREJ1 is a receptor for FSP. Antibodies showed that suREJ1 localizes to the entire flagellum as well as the acrosomal region (33Trimmer J.S. Trowbridge I.S. Vacquier V.D. Cell. 1985; 40: 697-703Abstract Full Text PDF PubMed Scopus (77) Google Scholar). suREJ3, a homologue of suREJ1, localizes only to the acrosomal region (17Mengerink K.J. Moy G.W. Vacquier V.D. J. Biol. Chem. 2002; 277: 943-948Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Because suREJ3 also has extracellular carbohydrate recognition domains (17Mengerink K.J. Moy G.W. Vacquier V.D. J. Biol. Chem. 2002; 277: 943-948Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 34Mengerink K.J. Moy G.W. Vacquier V.D. Zygote. 2000; 8: 28-30Crossref Google Scholar), we speculate that it might be a receptor for SG. The eSG fraction increased pHi by 0.08 units at maximum concentration, whereas a pHi increase of 0.23 units occurred in FSP (Fig.6 A). If SG binds to suREJ3 to evoke the pHiincrease, this small increase (compared with FSP) may be due to a local pHi increase that might occur only in the acrosomal region. Two different sperm plasma membrane Ca2+ channels mediate the FSP-induced AR (11Gonzalez-Martinez M.T. Galindo B.E. de De La Torre L. Zapata O. Rodriguez E. Florman H.M. Darszon A. Dev. Biol. 2001; 236: 220-229Crossref PubMed Scopus (56) Google Scholar). Our data suggest that there may be two distinct channel/exchanger systems to regulate the pHi increase occurring during AR induction. Several parallel signaling pathways may underlie AR induction by whole EJ (3Darszon A. Labarca P. Nishigaki T. Espinosa F. Physiol. Rev. 1999; 79: 481-510Crossref PubMed Scopus (281) Google Scholar). To our knowledge, this is the first demonstration for a role of sialic acid residues in the induction of the animal sperm AR. Structure studies of SG and molecular cloning of its receptor will increase our understanding of this unique signal transduction pathway. We thank Drs. B. Hayes, A. Varki, J. Esko, and H. van Halbeek for helpful discussions. We thank Drs. Meredith Gould, Sawako Oshima, and Jose Stephano for the collection of gametes. We thank Dr. B. Galindo and Sheryl Huffman for technical suggestions and discussions.