High Molecular Mass Egg Fucose Sulfate Polymer Is Required for Opening Both Ca2+ Channels Involved in Triggering the Sea Urchin Sperm Acrosome Reaction
2002; Elsevier BV; Volume: 277; Issue: 2 Linguagem: Inglês
10.1074/jbc.m108046200
ISSN1083-351X
AutoresNoritaka Hirohashi, Victor D. Vacquier,
Tópico(s)Reproductive biology and impacts on aquatic species
ResumoA linear fucose sulfate polymer (FSP), >106 daltons, is a major component of sea urchin egg jelly. FSP induces the sperm acrosome reaction (AR), an exocytotic process required for animal fertilization. Two Ca2+channels activate during AR induction, the first opens 1 s after FSP addition, and the second opens 5 s after the first. Mild acid hydrolysis of FSP results in a linear decrease in polymer size. The ability of FSP to induce the AR and activate sperm Ca2+channels decreases with increasing time of hydrolysis. Hydrolyzed FSP of ∼60 kDa blocks intact FSP from inducing the AR. At 44 μg/ml hydrolyzed FSP, Ca2+ entry into sperm is almost equal to that occurring in 3.8 μg/ml intact FSP; however the AR is not induced. The shape of the [Ca2+]i increase curve and use of the Ca2+ channel blockers nifidipine and Ni2+ indicate that hydrolyzed FSP opens the second Ca2+ channel, but not the first, and thus does not induce the AR. The giant size of intact FSP is required to open both Ca2+ channels involved in triggering the AR. A linear fucose sulfate polymer (FSP), >106 daltons, is a major component of sea urchin egg jelly. FSP induces the sperm acrosome reaction (AR), an exocytotic process required for animal fertilization. Two Ca2+channels activate during AR induction, the first opens 1 s after FSP addition, and the second opens 5 s after the first. Mild acid hydrolysis of FSP results in a linear decrease in polymer size. The ability of FSP to induce the AR and activate sperm Ca2+channels decreases with increasing time of hydrolysis. Hydrolyzed FSP of ∼60 kDa blocks intact FSP from inducing the AR. At 44 μg/ml hydrolyzed FSP, Ca2+ entry into sperm is almost equal to that occurring in 3.8 μg/ml intact FSP; however the AR is not induced. The shape of the [Ca2+]i increase curve and use of the Ca2+ channel blockers nifidipine and Ni2+ indicate that hydrolyzed FSP opens the second Ca2+ channel, but not the first, and thus does not induce the AR. The giant size of intact FSP is required to open both Ca2+ channels involved in triggering the AR. acrosome reaction egg jelly fucose sulfate polymer intact FSP hydrolyzed FSP FSP fragments generated by 5 h hydrolysis receptor for egg jelly polycystin-1 artificial seawater 2′,7′-bis-(2-carboxyl)-5-(and-6-)-carboxyfluorescein dihydropyridine carbohydrate recognition domain The sperm acrosome reaction (AR)1 is required for animal fertilization and is a potential target for the development of novel methods of non-hormonal contraception. Sea urchin spermatozoa are ideal for studying signal transduction underlying the animal sperm AR because they can be obtained as pure cells in vast quantities at low cost. The AR is triggered when sperm contact the jelly layer surrounding the egg (EJ). Morphologically, the AR involves the exocytosis of the acrosomal vesicle and the polymerization of actin to form the acrosomal process; both events are required for sperm to bind to and fuse with eggs. Physiologically, the AR requires the influx of Ca2+ and Na+ and the efflux of H+ and K+ions (1Schackmann R.W. Shapiro B.M. Dev. Biol. 1981; 81: 145-154Google Scholar, 2Darszon A. Labarca P. Nishigaki T. Espinosa F. Physiol. Rev. 1999; 79: 481-510Google Scholar). There are two plasma membrane Ca2+ channels involved in AR induction: the first is receptor-operated and opens 1 s after sperm contact EJ, the second opens 5 s after the first in response to increased intracellular pH (pHi). The second channel can also transport Mn2+ (2Darszon A. Labarca P. Nishigaki T. Espinosa F. Physiol. Rev. 1999; 79: 481-510Google Scholar, 3Guerrero A. Darszon A. J. Biol. Chem. 1989; 264: 19593-19599Google Scholar, 4Gonzalez-Martinez M.T. Galindo B.E. De La Torre L. Zapata O. Rodriguez E. Florman H.M. Darszon A. Dev. Biol. 2001; 236: 220-229Google Scholar). The Ca2+ channel blocker nisoldipine does not block Mn2+ movement through the second channel but does block the first channel and hence also blocks the AR (3Guerrero A. Darszon A. J. Biol. Chem. 1989; 264: 19593-19599Google Scholar).Eighty percent of the mass of sea urchin EJ is a fucose sulfate polymer (FSP) of >1 million daltons (5SeGall G.K. Lennarz W.J. Dev. Biol. 1979; 71: 33-48Google Scholar). Purified FSP, having no amino acid content, induces the AR (5SeGall G.K. Lennarz W.J. Dev. Biol. 1979; 71: 33-48Google Scholar, 6Vacquier V.D. Moy G.W. Dev. Biol. 1997; 192: 125-135Google Scholar). However, oligosaccharides of EJ glycoproteins substantially potentiate the FSP-induced AR, suggesting there is more than one receptor system regulating ion channels that trigger the AR (7Keller S.H. Vacquier V.D. Dev. Biol. 1994; 162: 304-312Google Scholar). 2N. Hirohashi and V. D. Vacquier, manuscript in preparation. 2N. Hirohashi and V. D. Vacquier, manuscript in preparation. FSP is a linear polymer of α-l-1,3-fucose with a species-specific pattern of sulfation of the fucosyl residues (8Alves A.P. Mulloy B. Moy G.W. Vacquier V.D. Mourao P.A. Glycobiology. 1998; 8: 939-946Google Scholar). The sulfation pattern is responsible for FSP's species-specific induction of the AR (9Alves A.P. Mulloy B. Diniz J.A. Mourao P.A. J. Biol. Chem. 1997; 272: 6965-6971Google Scholar, 10Vilela-Silva A.C. Alves A.P. Valente A.P. Vacquier V.D. Mourao P.A. Glycobiology. 1999; 9: 927-933Google Scholar). FSP is also a potent inhibitor of human blood coagulation through its high affinity binding to heparin cofactor II (11Pereira M.S. Mulloy B. Mourao P.A. J. Biol. Chem. 1999; 274: 7656-7667Google Scholar).Receptor for egg jelly-1 (REJ1) is a 1450-amino acid glycoprotein located in the plasma membrane over the sea urchin sperm acrosomal vesicle and also on the sperm flagellum. Available data support the hypothesis that REJ1 is at least one of the sperm receptors for FSP (6Vacquier V.D. Moy G.W. Dev. Biol. 1997; 192: 125-135Google Scholar,12Moy G.W. Mendoza L.M. Schulz J.R. Swanson W.J. Glabe C.G. Vacquier V.D. J. Cell Biol. 1996; 133: 809-817Google Scholar). Purified REJ1 neutralizes the AR activity of EJ (12Moy G.W. Mendoza L.M. Schulz J.R. Swanson W.J. Glabe C.G. Vacquier V.D. J. Cell Biol. 1996; 133: 809-817Google Scholar). An affinity column of REJ1 binds only FSP when crude EJ is applied (6Vacquier V.D. Moy G.W. Dev. Biol. 1997; 192: 125-135Google Scholar). Monoclonal antibodies to REJ1 induce Ca2+ influx into sperm (13Trimmer J.S. Schackmann R.W. Vacquier V.D. Proc. Natl. Acad Sci. U. S. A. 1986; 83: 9055-9059Google Scholar) and induce the AR (14Trimmer J.S. Ebina Y. Schackmann R.W. Meinhof C.G. Vacquier V.D. J. Cell Biol. 1987; 105: 1121-1128Google Scholar). Approximately 1000 residues of REJ1 consist of a domain named “the REJ module,” which is found in only one other protein family, the polycystin-1s (PKD1; Refs. 12Moy G.W. Mendoza L.M. Schulz J.R. Swanson W.J. Glabe C.G. Vacquier V.D. J. Cell Biol. 1996; 133: 809-817Google Scholar and 15Wu G. Somlo S. Mol. Genet. Metab. 2000; 69: 1-15Google Scholar). PKD1 is mutated in 85% of autosomal dominant polycystic kidney disease, the most frequent human genetic disease (16Arnaout M.A. Annu. Rev. Med. 2001; 52: 93-123Google Scholar). The polycystin-1 proteins are a new class of signaling molecules whose function remains to be clarified. Recent work shows that they may be regulators or subunits of nonselective cation channels (17Gonzalez-Perret S. Kim K. Ibarra C. Damiano A.E. Zotta E. Batelli M. Harris P.C. Reisin I.L. Arnaout M.A. Cantiello H.F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1182-1187Google Scholar). The “REJ module” homology between REJ1 and PKD1 is restricted to extracellular regions. Both proteins have carbohydrate binding, C-type lectin domains (12Moy G.W. Mendoza L.M. Schulz J.R. Swanson W.J. Glabe C.G. Vacquier V.D. J. Cell Biol. 1996; 133: 809-817Google Scholar, 18Weston B.S. Bagneris C. Price R.G. Stirling J.L. Biochim. Biophys. Acta. 2001; 1536: 161-176Google Scholar). Ligands for PKD1 remain unknown.FSP is the only ligand known to bind to a protein containing a REJ module. Because FSP is a giant polymer, we studied what effect reduction of its size might have on sperm physiology. Mild acid hydrolysis of purified FSP randomly cleaves the large polymer to smaller size. Treatment of sperm with fragmented FSP triggers the opening of the second but not the first Ca2+ channel and also fails to induce the AR. Further results suggest that intact and fragmented FSP compete for the same binding site(s) and that the binding of fragmented FSP greatly desensitizes the AR response. The giant size of intact FSP is therefore required for the normal opening of both sperm Ca2+ channels involved in AR induction.DISCUSSIONLinear polymers of α-l-1,3-sulfofucose are only known in echinoderms (10Vilela-Silva A.C. Alves A.P. Valente A.P. Vacquier V.D. Mourao P.A. Glycobiology. 1999; 9: 927-933Google Scholar), the single invertebrate phylum leading to the evolution of vertebrates (29Mengerink K.J. Vacquier V.D. Glycobiology. 2001; 11: 37-43Google Scholar). In addition to egg jelly coats, these polymers are also found in the extracellular matrix of the adult body wall in echinoderms (30Ribeiro A.C. Vieira R.P. Mourao P.A. Mulloy B. Carbohydr. Res. 1994; 255: 225-240Google Scholar). The egg FSPs mediate signal transduction in sperm and are also potent inhibitors of human blood coagulation (11Pereira M.S. Mulloy B. Mourao P.A. J. Biol. Chem. 1999; 274: 7656-7667Google Scholar,31Farias W.R. Valente A.P. Pereira M.S. Mourao P.A. J. Biol. Chem. 2000; 275: 29299-29307Google Scholar). Their biosynthesis has not been studied, and glycosidases that degrade them have not been described. The mouse sperm AR is also triggered by carbohydrate components of the egg's extracellular matrix; however these are oligosaccharide chains of unknown structure of the glycoprotein ZP3 (32Wassarman P.M. Cell. 1999; 96: 175-183Google Scholar). Also of interest is the fact that the sulfation pattern of FSP is responsible for its species-specific induction of the AR (8Alves A.P. Mulloy B. Moy G.W. Vacquier V.D. Mourao P.A. Glycobiology. 1998; 8: 939-946Google Scholar, 9Alves A.P. Mulloy B. Diniz J.A. Mourao P.A. J. Biol. Chem. 1997; 272: 6965-6971Google Scholar, 10Vilela-Silva A.C. Alves A.P. Valente A.P. Vacquier V.D. Mourao P.A. Glycobiology. 1999; 9: 927-933Google Scholar). Sulfation pattern is a relatively unknown structural mechanism to confer specificity on a cell-cell interaction leading to signal transduction and physiological activation. It is also unusual that a pure carbohydrate, completely lacking amino acids (6Vacquier V.D. Moy G.W. Dev. Biol. 1997; 192: 125-135Google Scholar), should induce signal transduction leading to exocytosis in animal cells.In the present study, the fragmentation of purified FSP was achieved by mild acid hydrolysis, creating a size-heterogeneous population of fragments (Fig. 1A). Glycosidic bond cleavage was confirmed by an increase in reducing sugar with time (Fig. 1B). By 30 min of hydrolysis, gel analysis shows that FSP is still relatively large; however, it has already lost much of its ability to induce Ca2+ influx (Fig. 2B). By 2 h of hydrolysis about 13% of the sulfate groups are lost (Fig. 1D); however the percent AR induction has decreased from 80 to 20% (Fig.2A). By 5 h of hydrolysis gel filtration shows that FSP chromatographs as a broad peak with a relative mass of 60 kDa (Fig.1C). We cannot distinguish whether the small amount of sulfate loss, or the decrease in polymer size, are singly, or in combination, responsible for the loss of biological activity of hFSP. However, physically braking FSP by sonication at pH 8.0 yields the same data presented in Figs. 1A and 4A, suggesting that loss of polymer size is responsible for the characteristics of hFSP described herein (date not shown). In starfish, the giant pentasaccharide repeat polymer of EJ contains two sulfate groups per repeat. Solvolysis of the polymer shows that the loss of one sulfate per repeat greatly decreases its ability to induce the AR of starfish sperm (33Koyota S. Wimalasiri K.M. Hoshi M. J. Biol. Chem. 1997; 272: 10372-10376Google Scholar).hFSP samples showed a marked decrease in the ability to induce Ca2+ influx after 30 min of hydrolysis (Fig.2B), yet the polymer size was still relatively large (Fig.1A). The concentration dependences of iFSP and 5h-hFSP to stimulate increases in Ca2+ and pHi and to induce the AR show that iFSP is always a more potent inducer than 5h-hFSP (Fig. 3). 5h-hFSP blocked iFSP from inducing the AR, and this inhibitory effect was restored by an excess amount of iFSP, suggesting that both types of FSP bind the same site(s) (Fig. 4, A andB). Although 22 μg/ml hFSP induced a final [Ca2+]i elevation equivalent to 0.5 μg/ml iFSP, AR did not occur in the hFSP-treated cells (Fig. 5). As previously documented by others, nisoldipine blocks the first Ca2+channel but not the second (3Guerrero A. Darszon A. J. Biol. Chem. 1989; 264: 19593-19599Google Scholar). Our experiments show that the hFSP-induced increase in Ca2+ is not inhibited by 50 μm nifedipine, suggesting that hFSP up-regulates the second channel but not the first (Fig. 6B). This hypothesis is supported by the data showing that Ni2+, which blocks the second sperm Ca2+ channel but not the first (4Gonzalez-Martinez M.T. Galindo B.E. De La Torre L. Zapata O. Rodriguez E. Florman H.M. Darszon A. Dev. Biol. 2001; 236: 220-229Google Scholar), completely blocks the 5h-hFSP-induced increase in Ca2+(Fig. 7).In regard to the mechanism of AR induction in different animal species, the egg ligands and their sperm receptors appear to be highly variable and evolutionarily unrelated (34Vacquier V.D. Science. 1998; 281: 1995-1998Google Scholar). However, the intracellular mechanism of the animal sperm AR appears to be conserved in that elevation of Ca2+ and pHi are required in all species (2Darszon A. Labarca P. Nishigaki T. Espinosa F. Physiol. Rev. 1999; 79: 481-510Google Scholar). For example, planar lipid bilayer experiments show that sea urchin and mouse sperm possess a readily detectable, Ca2+-selective, sperm-specific, high conductance, multistate, voltage-dependent channel with similar voltage dependence (35Beltran C. Darszon A. Labarca P. Lievano A. FEBS Lett. 1994; 338: 23-26Google Scholar, 36Lopez-Gonzalez I. De La Vega-Beltran J.L. Santi C.M. Florman H.M. Felix R. Darszon A. Dev. Biol. 2001; 236: 210-219Google Scholar).In the NH2-terminal extracellular portion of both proteins called the “REJ module,” sea urchin REJ1 and human PKD1 have structural motifs identifying them as “C-type lectin-like,” carbohydrate-binding proteins (12Moy G.W. Mendoza L.M. Schulz J.R. Swanson W.J. Glabe C.G. Vacquier V.D. J. Cell Biol. 1996; 133: 809-817Google Scholar, 18Weston B.S. Bagneris C. Price R.G. Stirling J.L. Biochim. Biophys. Acta. 2001; 1536: 161-176Google Scholar). Ligands for PKD1 are unknown, but the available evidence suggests that FSP binds REJ1 with high affinity (6Vacquier V.D. Moy G.W. Dev. Biol. 1997; 192: 125-135Google Scholar). REJ1 has two carbohydrate recognition domains (CRDs) that could bind FSP (12Moy G.W. Mendoza L.M. Schulz J.R. Swanson W.J. Glabe C.G. Vacquier V.D. J. Cell Biol. 1996; 133: 809-817Google Scholar). REJ1 has only one transmembrane segment at its extreme COOH terminus with only 15 residues being putatively cytoplasmic; therefore, REJ1 cannot be a pore-forming ion channel subunit. Because some (but not all) monoclonal antibodies to REJ1 induce Ca2+ elevations and the AR, REJ1 must be a regulator of sperm Ca2+ channels (12Moy G.W. Mendoza L.M. Schulz J.R. Swanson W.J. Glabe C.G. Vacquier V.D. J. Cell Biol. 1996; 133: 809-817Google Scholar, 13Trimmer J.S. Schackmann R.W. Vacquier V.D. Proc. Natl. Acad Sci. U. S. A. 1986; 83: 9055-9059Google Scholar). The location of REJ1 over the acrosomal vesicle supports its role in AR regulation.Given a molecular mass of ∼60 kDa in 5h-hFSP and an average molar ratio of 1.1 sulfate groups per fucose residue (8Alves A.P. Mulloy B. Moy G.W. Vacquier V.D. Mourao P.A. Glycobiology. 1998; 8: 939-946Google Scholar), the average 5h-hFSP fragment has about 220 fucosyl residues. This would seem large enough to bridge many CRDs of many REJ1 proteins. In addition to binding the plasma membrane receptor REJ1, egg FSP also has affinity for bindin, the protein released from the acrosomal vesicle that species-specifically attaches the sperm to the sea urchin egg (37Vacquier V.D. Swanson W.J. Hellberg M.E. Dev. Growth Differ. 1995; 37: 1-10Google Scholar). Studies on how the size of FSP affects its affinity to bindin have shown that the large size is again important. Little binding occurred below an FSP average size of 15 kDa. In addition, sulfate groups were essential for the binding of FSP to bindin (38DeAngelis P.L. Glabe C.G. J. Biol. Chem. 1987; 262: 13946-13952Google Scholar). Why is such a giant size of FSP required for the normal physiological response of opening both Ca2+ channels? As with other CRD-containing proteins (39Weis W.I. Taylor M.E. Drickamer K. Immunol. Rev. 1998; 163: 19-34Google Scholar), the two CRDs of REJ1 should recognize terminal sugar residues of oligosaccharides; therefore, perhaps only the first and last residues bind REJ1. 5h-hFSP must bind REJ1 receptors because its AR-inhibiting activity is overridden by an excess amount of iFSP, and it can also induce Ca2+ increases in sperm.Our data, and the data of others (4Gonzalez-Martinez M.T. Galindo B.E. De La Torre L. Zapata O. Rodriguez E. Florman H.M. Darszon A. Dev. Biol. 2001; 236: 220-229Google Scholar), show that mere elevation of [Ca2+]i to a certain concentration is not what induces the AR; it is the pathway leading to induction that is important. It could be that hFSP uncouples components of the pathway, a phenomenon that can be demonstrated in these cells by other means. For example, seawater is ∼10 mm Ca2+. Treatment of sperm with EJ in 2 mm Ca2+ makes the cells refractory to AR induction and to increases in [Ca2+]i when the Ca2+ is returned to 10 mm (40Guerrero A. Darszon A. Biochim. Biophys. Acta. 1989; 980: 109-116Google Scholar, 41Guerrero A. Garcia L. Zapata O. Rodriguez E. Darszon A. Biochim. Biophys. Acta. 1998; 1401: 329-338Google Scholar). This AR inactivation is hypothesized to result from uncoupling the linkage between the sperm EJ receptors and Ca2+ channels. Pretreatment of sperm with 5h-hFSP causes a marked decrease in the AR response, yet [Ca2+]iincreases to reasonably high levels. Desensitization of the AR response by hFSP is thus different from AR inactivation. This might be due to opening of the second Ca2+ channel before the first channel opens, the order of opening being crucial to the AR. Normally, the first channel causes rapid increases in [Ca2+]i, whereas the second is responsible for the sustained high level of [Ca2+]i. The first channel may be activated by the binding of FSP to REJ1 (6Vacquier V.D. Moy G.W. Dev. Biol. 1997; 192: 125-135Google Scholar), whereas the second channel, which is normally activated by the up-regulation of the first channel, has characteristics of a store-operated channel (4Gonzalez-Martinez M.T. Galindo B.E. De La Torre L. Zapata O. Rodriguez E. Florman H.M. Darszon A. Dev. Biol. 2001; 236: 220-229Google Scholar). That binding of hFSP to the cell surface bypasses the first channel and up-regulates the second channel suggests that the sensor regulating the second channel could involve unidentified cell surface receptors. The sperm acrosome reaction (AR)1 is required for animal fertilization and is a potential target for the development of novel methods of non-hormonal contraception. Sea urchin spermatozoa are ideal for studying signal transduction underlying the animal sperm AR because they can be obtained as pure cells in vast quantities at low cost. The AR is triggered when sperm contact the jelly layer surrounding the egg (EJ). Morphologically, the AR involves the exocytosis of the acrosomal vesicle and the polymerization of actin to form the acrosomal process; both events are required for sperm to bind to and fuse with eggs. Physiologically, the AR requires the influx of Ca2+ and Na+ and the efflux of H+ and K+ions (1Schackmann R.W. Shapiro B.M. Dev. Biol. 1981; 81: 145-154Google Scholar, 2Darszon A. Labarca P. Nishigaki T. Espinosa F. Physiol. Rev. 1999; 79: 481-510Google Scholar). There are two plasma membrane Ca2+ channels involved in AR induction: the first is receptor-operated and opens 1 s after sperm contact EJ, the second opens 5 s after the first in response to increased intracellular pH (pHi). The second channel can also transport Mn2+ (2Darszon A. Labarca P. Nishigaki T. Espinosa F. Physiol. Rev. 1999; 79: 481-510Google Scholar, 3Guerrero A. Darszon A. J. Biol. Chem. 1989; 264: 19593-19599Google Scholar, 4Gonzalez-Martinez M.T. Galindo B.E. De La Torre L. Zapata O. Rodriguez E. Florman H.M. Darszon A. Dev. Biol. 2001; 236: 220-229Google Scholar). The Ca2+ channel blocker nisoldipine does not block Mn2+ movement through the second channel but does block the first channel and hence also blocks the AR (3Guerrero A. Darszon A. J. Biol. Chem. 1989; 264: 19593-19599Google Scholar). Eighty percent of the mass of sea urchin EJ is a fucose sulfate polymer (FSP) of >1 million daltons (5SeGall G.K. Lennarz W.J. Dev. Biol. 1979; 71: 33-48Google Scholar). Purified FSP, having no amino acid content, induces the AR (5SeGall G.K. Lennarz W.J. Dev. Biol. 1979; 71: 33-48Google Scholar, 6Vacquier V.D. Moy G.W. Dev. Biol. 1997; 192: 125-135Google Scholar). However, oligosaccharides of EJ glycoproteins substantially potentiate the FSP-induced AR, suggesting there is more than one receptor system regulating ion channels that trigger the AR (7Keller S.H. Vacquier V.D. Dev. Biol. 1994; 162: 304-312Google Scholar). 2N. Hirohashi and V. D. Vacquier, manuscript in preparation. 2N. Hirohashi and V. D. Vacquier, manuscript in preparation. FSP is a linear polymer of α-l-1,3-fucose with a species-specific pattern of sulfation of the fucosyl residues (8Alves A.P. Mulloy B. Moy G.W. Vacquier V.D. Mourao P.A. Glycobiology. 1998; 8: 939-946Google Scholar). The sulfation pattern is responsible for FSP's species-specific induction of the AR (9Alves A.P. Mulloy B. Diniz J.A. Mourao P.A. J. Biol. Chem. 1997; 272: 6965-6971Google Scholar, 10Vilela-Silva A.C. Alves A.P. Valente A.P. Vacquier V.D. Mourao P.A. Glycobiology. 1999; 9: 927-933Google Scholar). FSP is also a potent inhibitor of human blood coagulation through its high affinity binding to heparin cofactor II (11Pereira M.S. Mulloy B. Mourao P.A. J. Biol. Chem. 1999; 274: 7656-7667Google Scholar). Receptor for egg jelly-1 (REJ1) is a 1450-amino acid glycoprotein located in the plasma membrane over the sea urchin sperm acrosomal vesicle and also on the sperm flagellum. Available data support the hypothesis that REJ1 is at least one of the sperm receptors for FSP (6Vacquier V.D. Moy G.W. Dev. Biol. 1997; 192: 125-135Google Scholar,12Moy G.W. Mendoza L.M. Schulz J.R. Swanson W.J. Glabe C.G. Vacquier V.D. J. Cell Biol. 1996; 133: 809-817Google Scholar). Purified REJ1 neutralizes the AR activity of EJ (12Moy G.W. Mendoza L.M. Schulz J.R. Swanson W.J. Glabe C.G. Vacquier V.D. J. Cell Biol. 1996; 133: 809-817Google Scholar). An affinity column of REJ1 binds only FSP when crude EJ is applied (6Vacquier V.D. Moy G.W. Dev. Biol. 1997; 192: 125-135Google Scholar). Monoclonal antibodies to REJ1 induce Ca2+ influx into sperm (13Trimmer J.S. Schackmann R.W. Vacquier V.D. Proc. Natl. Acad Sci. U. S. A. 1986; 83: 9055-9059Google Scholar) and induce the AR (14Trimmer J.S. Ebina Y. Schackmann R.W. Meinhof C.G. Vacquier V.D. J. Cell Biol. 1987; 105: 1121-1128Google Scholar). Approximately 1000 residues of REJ1 consist of a domain named “the REJ module,” which is found in only one other protein family, the polycystin-1s (PKD1; Refs. 12Moy G.W. Mendoza L.M. Schulz J.R. Swanson W.J. Glabe C.G. Vacquier V.D. J. Cell Biol. 1996; 133: 809-817Google Scholar and 15Wu G. Somlo S. Mol. Genet. Metab. 2000; 69: 1-15Google Scholar). PKD1 is mutated in 85% of autosomal dominant polycystic kidney disease, the most frequent human genetic disease (16Arnaout M.A. Annu. Rev. Med. 2001; 52: 93-123Google Scholar). The polycystin-1 proteins are a new class of signaling molecules whose function remains to be clarified. Recent work shows that they may be regulators or subunits of nonselective cation channels (17Gonzalez-Perret S. Kim K. Ibarra C. Damiano A.E. Zotta E. Batelli M. Harris P.C. Reisin I.L. Arnaout M.A. Cantiello H.F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1182-1187Google Scholar). The “REJ module” homology between REJ1 and PKD1 is restricted to extracellular regions. Both proteins have carbohydrate binding, C-type lectin domains (12Moy G.W. Mendoza L.M. Schulz J.R. Swanson W.J. Glabe C.G. Vacquier V.D. J. Cell Biol. 1996; 133: 809-817Google Scholar, 18Weston B.S. Bagneris C. Price R.G. Stirling J.L. Biochim. Biophys. Acta. 2001; 1536: 161-176Google Scholar). Ligands for PKD1 remain unknown. FSP is the only ligand known to bind to a protein containing a REJ module. Because FSP is a giant polymer, we studied what effect reduction of its size might have on sperm physiology. Mild acid hydrolysis of purified FSP randomly cleaves the large polymer to smaller size. Treatment of sperm with fragmented FSP triggers the opening of the second but not the first Ca2+ channel and also fails to induce the AR. Further results suggest that intact and fragmented FSP compete for the same binding site(s) and that the binding of fragmented FSP greatly desensitizes the AR response. The giant size of intact FSP is therefore required for the normal opening of both sperm Ca2+ channels involved in AR induction. DISCUSSIONLinear polymers of α-l-1,3-sulfofucose are only known in echinoderms (10Vilela-Silva A.C. Alves A.P. Valente A.P. Vacquier V.D. Mourao P.A. Glycobiology. 1999; 9: 927-933Google Scholar), the single invertebrate phylum leading to the evolution of vertebrates (29Mengerink K.J. Vacquier V.D. Glycobiology. 2001; 11: 37-43Google Scholar). In addition to egg jelly coats, these polymers are also found in the extracellular matrix of the adult body wall in echinoderms (30Ribeiro A.C. Vieira R.P. Mourao P.A. Mulloy B. Carbohydr. Res. 1994; 255: 225-240Google Scholar). The egg FSPs mediate signal transduction in sperm and are also potent inhibitors of human blood coagulation (11Pereira M.S. Mulloy B. Mourao P.A. J. Biol. Chem. 1999; 274: 7656-7667Google Scholar,31Farias W.R. Valente A.P. Pereira M.S. Mourao P.A. J. Biol. Chem. 2000; 275: 29299-29307Google Scholar). Their biosynthesis has not been studied, and glycosidases that degrade them have not been described. The mouse sperm AR is also triggered by carbohydrate components of the egg's extracellular matrix; however these are oligosaccharide chains of unknown structure of the glycoprotein ZP3 (32Wassarman P.M. Cell. 1999; 96: 175-183Google Scholar). Also of interest is the fact that the sulfation pattern of FSP is responsible for its species-specific induction of the AR (8Alves A.P. Mulloy B. Moy G.W. Vacquier V.D. Mourao P.A. Glycobiology. 1998; 8: 939-946Google Scholar, 9Alves A.P. Mulloy B. Diniz J.A. Mourao P.A. J. Biol. Chem. 1997; 272: 6965-6971Google Scholar, 10Vilela-Silva A.C. Alves A.P. Valente A.P. Vacquier V.D. Mourao P.A. Glycobiology. 1999; 9: 927-933Google Scholar). Sulfation pattern is a relatively unknown structural mechanism to confer specificity on a cell-cell interaction leading to signal transduction and physiological activation. It is also unusual that a pure carbohydrate, completely lacking amino acids (6Vacquier V.D. Moy G.W. Dev. Biol. 1997; 192: 125-135Google Scholar), should induce signal transduction leading to exocytosis in animal cells.In the present study, the fragmentation of purified FSP was achieved by mild acid hydrolysis, creating a size-heterogeneous population of fragments (Fig. 1A). Glycosidic bond cleavage was confirmed by an increase in reducing sugar with time (Fig. 1B). By 30 min of hydrolysis, gel analysis shows that FSP is still relatively large; however, it has already lost much of its ability to induce Ca2+ influx (Fig. 2B). By 2 h of hydrolysis about 13% of the sulfate groups are lost (Fig. 1D); however the percent AR induction has decreased from 80 to 20% (Fig.2A). By 5 h of hydrolysis gel filtration shows that FSP chromatographs as a broad peak with a relative mass of 60 kDa (Fig.1C). We cannot distinguish whether the small amount of sulfate loss, or the decrease in polymer size, are singly, or in combination, responsible for the loss of biological activity of hFSP. However, physically braking FSP by sonication at pH 8.0 yields the same data presented in Figs. 1A and 4A, suggesting that loss of polymer size is responsible for the characteristics of hFSP described herein (date not shown). In starfish, the giant pentasaccharide repeat polymer of EJ contains two sulfate groups per repeat. Solvolysis of the polymer shows that the loss of one sulfate per repeat greatly decreases its ability to induce the AR of starfish sperm (33Koyota S. Wimalasiri K.M. Hoshi M. J. Biol. Chem. 1997; 272: 10372-10376Google Scholar).hFSP samples showed a marked decrease in the ability to induce Ca2+ influx after 30 min of hydrolysis (Fig.2B), yet the polymer size was still relatively large (Fig.1A). The concentration dependences of iFSP and 5h-hFSP to stimulate increases in Ca2+ and pHi and to induce the AR show that iFSP is always a more potent inducer than 5h-hFSP (Fig. 3). 5h-hFSP blocked iFSP from inducing the AR, and this inhibitory effect was restored by an excess amount of iFSP, suggesting that both types of FSP bind the same site(s) (Fig. 4, A andB). Although 22 μg/ml hFSP induced a final [Ca2+]i elevation equivalent to 0.5 μg/ml iFSP, AR did not occur in the hFSP-treated cells (Fig. 5). As previously documented by others, nisoldipine blocks the first Ca2+channel but not the second (3Guerrero A. Darszon A. J. Biol. Chem. 1989; 264: 19593-19599Google Scholar). Our experiments show that the hFSP-induced increase in Ca2+ is not inhibited by 50 μm nifedipine, suggesting that hFSP up-regulates the second channel but not the first (Fig. 6B). This hypothesis is supported by the data showing that Ni2+, which blocks the second sperm Ca2+ channel but not the first (4Gonzalez-Martinez M.T. Galindo B.E. De La Torre L. Zapata O. Rodriguez E. Florman H.M. Darszon A. Dev. Biol. 2001; 236: 220-229Google Scholar), completely blocks the 5h-hFSP-induced increase in Ca2+(Fig. 7).In regard to the mechanism of AR induction in different animal species, the egg ligands and their sperm receptors appear to be highly variable and evolutionarily unrelated (34Vacquier V.D. Science. 1998; 281: 1995-1998Google Scholar). However, the intracellular mechanism of the animal sperm AR appears to be conserved in that elevation of Ca2+ and pHi are required in all species (2Darszon A. Labarca P. Nishigaki T. Espinosa F. Physiol. Rev. 1999; 79: 481-510Google Scholar). For example, planar lipid bilayer experiments show that sea urchin and mouse sperm possess a readily detectable, Ca2+-selective, sperm-specific, high conductance, multistate, voltage-dependent channel with similar voltage dependence (35Beltran C. Darszon A. Labarca P. Lievano A. FEBS Lett. 1994; 338: 23-26Google Scholar, 36Lopez-Gonzalez I. De La Vega-Beltran J.L. Santi C.M. Florman H.M. Felix R. Darszon A. Dev. Biol. 2001; 236: 210-219Google Scholar).In the NH2-terminal extracellular portion of both proteins called the “REJ module,” sea urchin REJ1 and human PKD1 have structural motifs identifying them as “C-type lectin-like,” carbohydrate-binding proteins (12Moy G.W. Mendoza L.M. Schulz J.R. Swanson W.J. Glabe C.G. Vacquier V.D. J. Cell Biol. 1996; 133: 809-817Google Scholar, 18Weston B.S. Bagneris C. Price R.G. Stirling J.L. Biochim. Biophys. Acta. 2001; 1536: 161-176Google Scholar). Ligands for PKD1 are unknown, but the available evidence suggests that FSP binds REJ1 with high affinity (6Vacquier V.D. Moy G.W. Dev. Biol. 1997; 192: 125-135Google Scholar). REJ1 has two carbohydrate recognition domains (CRDs) that could bind FSP (12Moy G.W. Mendoza L.M. Schulz J.R. Swanson W.J. Glabe C.G. Vacquier V.D. J. Cell Biol. 1996; 133: 809-817Google Scholar). REJ1 has only one transmembrane segment at its extreme COOH terminus with only 15 residues being putatively cytoplasmic; therefore, REJ1 cannot be a pore-forming ion channel subunit. Because some (but not all) monoclonal antibodies to REJ1 induce Ca2+ elevations and the AR, REJ1 must be a regulator of sperm Ca2+ channels (12Moy G.W. Mendoza L.M. Schulz J.R. Swanson W.J. Glabe C.G. Vacquier V.D. J. Cell Biol. 1996; 133: 809-817Google Scholar, 13Trimmer J.S. Schackmann R.W. Vacquier V.D. Proc. Natl. Acad Sci. U. S. A. 1986; 83: 9055-9059Google Scholar). The location of REJ1 over the acrosomal vesicle supports its role in AR regulation.Given a molecular mass of ∼60 kDa in 5h-hFSP and an average molar ratio of 1.1 sulfate groups per fucose residue (8Alves A.P. Mulloy B. Moy G.W. Vacquier V.D. Mourao P.A. Glycobiology. 1998; 8: 939-946Google Scholar), the average 5h-hFSP fragment has about 220 fucosyl residues. This would seem large enough to bridge many CRDs of many REJ1 proteins. In addition to binding the plasma membrane receptor REJ1, egg FSP also has affinity for bindin, the protein released from the acrosomal vesicle that species-specifically attaches the sperm to the sea urchin egg (37Vacquier V.D. Swanson W.J. Hellberg M.E. Dev. Growth Differ. 1995; 37: 1-10Google Scholar). Studies on how the size of FSP affects its affinity to bindin have shown that the large size is again important. Little binding occurred below an FSP average size of 15 kDa. In addition, sulfate groups were essential for the binding of FSP to bindin (38DeAngelis P.L. Glabe C.G. J. Biol. Chem. 1987; 262: 13946-13952Google Scholar). Why is such a giant size of FSP required for the normal physiological response of opening both Ca2+ channels? As with other CRD-containing proteins (39Weis W.I. Taylor M.E. Drickamer K. Immunol. Rev. 1998; 163: 19-34Google Scholar), the two CRDs of REJ1 should recognize terminal sugar residues of oligosaccharides; therefore, perhaps only the first and last residues bind REJ1. 5h-hFSP must bind REJ1 receptors because its AR-inhibiting activity is overridden by an excess amount of iFSP, and it can also induce Ca2+ increases in sperm.Our data, and the data of others (4Gonzalez-Martinez M.T. Galindo B.E. De La Torre L. Zapata O. Rodriguez E. Florman H.M. Darszon A. Dev. Biol. 2001; 236: 220-229Google Scholar), show that mere elevation of [Ca2+]i to a certain concentration is not what induces the AR; it is the pathway leading to induction that is important. It could be that hFSP uncouples components of the pathway, a phenomenon that can be demonstrated in these cells by other means. For example, seawater is ∼10 mm Ca2+. Treatment of sperm with EJ in 2 mm Ca2+ makes the cells refractory to AR induction and to increases in [Ca2+]i when the Ca2+ is returned to 10 mm (40Guerrero A. Darszon A. Biochim. Biophys. Acta. 1989; 980: 109-116Google Scholar, 41Guerrero A. Garcia L. Zapata O. Rodriguez E. Darszon A. Biochim. Biophys. Acta. 1998; 1401: 329-338Google Scholar). This AR inactivation is hypothesized to result from uncoupling the linkage between the sperm EJ receptors and Ca2+ channels. Pretreatment of sperm with 5h-hFSP causes a marked decrease in the AR response, yet [Ca2+]iincreases to reasonably high levels. Desensitization of the AR response by hFSP is thus different from AR inactivation. This might be due to opening of the second Ca2+ channel before the first channel opens, the order of opening being crucial to the AR. Normally, the first channel causes rapid increases in [Ca2+]i, whereas the second is responsible for the sustained high level of [Ca2+]i. The first channel may be activated by the binding of FSP to REJ1 (6Vacquier V.D. Moy G.W. Dev. Biol. 1997; 192: 125-135Google Scholar), whereas the second channel, which is normally activated by the up-regulation of the first channel, has characteristics of a store-operated channel (4Gonzalez-Martinez M.T. Galindo B.E. De La Torre L. Zapata O. Rodriguez E. Florman H.M. Darszon A. Dev. Biol. 2001; 236: 220-229Google Scholar). That binding of hFSP to the cell surface bypasses the first channel and up-regulates the second channel suggests that the sensor regulating the second channel could involve unidentified cell surface receptors. Linear polymers of α-l-1,3-sulfofucose are only known in echinoderms (10Vilela-Silva A.C. Alves A.P. Valente A.P. Vacquier V.D. Mourao P.A. Glycobiology. 1999; 9: 927-933Google Scholar), the single invertebrate phylum leading to the evolution of vertebrates (29Mengerink K.J. Vacquier V.D. Glycobiology. 2001; 11: 37-43Google Scholar). In addition to egg jelly coats, these polymers are also found in the extracellular matrix of the adult body wall in echinoderms (30Ribeiro A.C. Vieira R.P. Mourao P.A. Mulloy B. Carbohydr. Res. 1994; 255: 225-240Google Scholar). The egg FSPs mediate signal transduction in sperm and are also potent inhibitors of human blood coagulation (11Pereira M.S. Mulloy B. Mourao P.A. J. Biol. Chem. 1999; 274: 7656-7667Google Scholar,31Farias W.R. Valente A.P. Pereira M.S. Mourao P.A. J. Biol. Chem. 2000; 275: 29299-29307Google Scholar). Their biosynthesis has not been studied, and glycosidases that degrade them have not been described. The mouse sperm AR is also triggered by carbohydrate components of the egg's extracellular matrix; however these are oligosaccharide chains of unknown structure of the glycoprotein ZP3 (32Wassarman P.M. Cell. 1999; 96: 175-183Google Scholar). Also of interest is the fact that the sulfation pattern of FSP is responsible for its species-specific induction of the AR (8Alves A.P. Mulloy B. Moy G.W. Vacquier V.D. Mourao P.A. Glycobiology. 1998; 8: 939-946Google Scholar, 9Alves A.P. Mulloy B. Diniz J.A. Mourao P.A. J. Biol. Chem. 1997; 272: 6965-6971Google Scholar, 10Vilela-Silva A.C. Alves A.P. Valente A.P. Vacquier V.D. Mourao P.A. Glycobiology. 1999; 9: 927-933Google Scholar). Sulfation pattern is a relatively unknown structural mechanism to confer specificity on a cell-cell interaction leading to signal transduction and physiological activation. It is also unusual that a pure carbohydrate, completely lacking amino acids (6Vacquier V.D. Moy G.W. Dev. Biol. 1997; 192: 125-135Google Scholar), should induce signal transduction leading to exocytosis in animal cells. In the present study, the fragmentation of purified FSP was achieved by mild acid hydrolysis, creating a size-heterogeneous population of fragments (Fig. 1A). Glycosidic bond cleavage was confirmed by an increase in reducing sugar with time (Fig. 1B). By 30 min of hydrolysis, gel analysis shows that FSP is still relatively large; however, it has already lost much of its ability to induce Ca2+ influx (Fig. 2B). By 2 h of hydrolysis about 13% of the sulfate groups are lost (Fig. 1D); however the percent AR induction has decreased from 80 to 20% (Fig.2A). By 5 h of hydrolysis gel filtration shows that FSP chromatographs as a broad peak with a relative mass of 60 kDa (Fig.1C). We cannot distinguish whether the small amount of sulfate loss, or the decrease in polymer size, are singly, or in combination, responsible for the loss of biological activity of hFSP. However, physically braking FSP by sonication at pH 8.0 yields the same data presented in Figs. 1A and 4A, suggesting that loss of polymer size is responsible for the characteristics of hFSP described herein (date not shown). In starfish, the giant pentasaccharide repeat polymer of EJ contains two sulfate groups per repeat. Solvolysis of the polymer shows that the loss of one sulfate per repeat greatly decreases its ability to induce the AR of starfish sperm (33Koyota S. Wimalasiri K.M. Hoshi M. J. Biol. Chem. 1997; 272: 10372-10376Google Scholar). hFSP samples showed a marked decrease in the ability to induce Ca2+ influx after 30 min of hydrolysis (Fig.2B), yet the polymer size was still relatively large (Fig.1A). The concentration dependences of iFSP and 5h-hFSP to stimulate increases in Ca2+ and pHi and to induce the AR show that iFSP is always a more potent inducer than 5h-hFSP (Fig. 3). 5h-hFSP blocked iFSP from inducing the AR, and this inhibitory effect was restored by an excess amount of iFSP, suggesting that both types of FSP bind the same site(s) (Fig. 4, A andB). Although 22 μg/ml hFSP induced a final [Ca2+]i elevation equivalent to 0.5 μg/ml iFSP, AR did not occur in the hFSP-treated cells (Fig. 5). As previously documented by others, nisoldipine blocks the first Ca2+channel but not the second (3Guerrero A. Darszon A. J. Biol. Chem. 1989; 264: 19593-19599Google Scholar). Our experiments show that the hFSP-induced increase in Ca2+ is not inhibited by 50 μm nifedipine, suggesting that hFSP up-regulates the second channel but not the first (Fig. 6B). This hypothesis is supported by the data showing that Ni2+, which blocks the second sperm Ca2+ channel but not the first (4Gonzalez-Martinez M.T. Galindo B.E. De La Torre L. Zapata O. Rodriguez E. Florman H.M. Darszon A. Dev. Biol. 2001; 236: 220-229Google Scholar), completely blocks the 5h-hFSP-induced increase in Ca2+(Fig. 7). In regard to the mechanism of AR induction in different animal species, the egg ligands and their sperm receptors appear to be highly variable and evolutionarily unrelated (34Vacquier V.D. Science. 1998; 281: 1995-1998Google Scholar). However, the intracellular mechanism of the animal sperm AR appears to be conserved in that elevation of Ca2+ and pHi are required in all species (2Darszon A. Labarca P. Nishigaki T. Espinosa F. Physiol. Rev. 1999; 79: 481-510Google Scholar). For example, planar lipid bilayer experiments show that sea urchin and mouse sperm possess a readily detectable, Ca2+-selective, sperm-specific, high conductance, multistate, voltage-dependent channel with similar voltage dependence (35Beltran C. Darszon A. Labarca P. Lievano A. FEBS Lett. 1994; 338: 23-26Google Scholar, 36Lopez-Gonzalez I. De La Vega-Beltran J.L. Santi C.M. Florman H.M. Felix R. Darszon A. Dev. Biol. 2001; 236: 210-219Google Scholar). In the NH2-terminal extracellular portion of both proteins called the “REJ module,” sea urchin REJ1 and human PKD1 have structural motifs identifying them as “C-type lectin-like,” carbohydrate-binding proteins (12Moy G.W. Mendoza L.M. Schulz J.R. Swanson W.J. Glabe C.G. Vacquier V.D. J. Cell Biol. 1996; 133: 809-817Google Scholar, 18Weston B.S. Bagneris C. Price R.G. Stirling J.L. Biochim. Biophys. Acta. 2001; 1536: 161-176Google Scholar). Ligands for PKD1 are unknown, but the available evidence suggests that FSP binds REJ1 with high affinity (6Vacquier V.D. Moy G.W. Dev. Biol. 1997; 192: 125-135Google Scholar). REJ1 has two carbohydrate recognition domains (CRDs) that could bind FSP (12Moy G.W. Mendoza L.M. Schulz J.R. Swanson W.J. Glabe C.G. Vacquier V.D. J. Cell Biol. 1996; 133: 809-817Google Scholar). REJ1 has only one transmembrane segment at its extreme COOH terminus with only 15 residues being putatively cytoplasmic; therefore, REJ1 cannot be a pore-forming ion channel subunit. Because some (but not all) monoclonal antibodies to REJ1 induce Ca2+ elevations and the AR, REJ1 must be a regulator of sperm Ca2+ channels (12Moy G.W. Mendoza L.M. Schulz J.R. Swanson W.J. Glabe C.G. Vacquier V.D. J. Cell Biol. 1996; 133: 809-817Google Scholar, 13Trimmer J.S. Schackmann R.W. Vacquier V.D. Proc. Natl. Acad Sci. U. S. A. 1986; 83: 9055-9059Google Scholar). The location of REJ1 over the acrosomal vesicle supports its role in AR regulation. Given a molecular mass of ∼60 kDa in 5h-hFSP and an average molar ratio of 1.1 sulfate groups per fucose residue (8Alves A.P. Mulloy B. Moy G.W. Vacquier V.D. Mourao P.A. Glycobiology. 1998; 8: 939-946Google Scholar), the average 5h-hFSP fragment has about 220 fucosyl residues. This would seem large enough to bridge many CRDs of many REJ1 proteins. In addition to binding the plasma membrane receptor REJ1, egg FSP also has affinity for bindin, the protein released from the acrosomal vesicle that species-specifically attaches the sperm to the sea urchin egg (37Vacquier V.D. Swanson W.J. Hellberg M.E. Dev. Growth Differ. 1995; 37: 1-10Google Scholar). Studies on how the size of FSP affects its affinity to bindin have shown that the large size is again important. Little binding occurred below an FSP average size of 15 kDa. In addition, sulfate groups were essential for the binding of FSP to bindin (38DeAngelis P.L. Glabe C.G. J. Biol. Chem. 1987; 262: 13946-13952Google Scholar). Why is such a giant size of FSP required for the normal physiological response of opening both Ca2+ channels? As with other CRD-containing proteins (39Weis W.I. Taylor M.E. Drickamer K. Immunol. Rev. 1998; 163: 19-34Google Scholar), the two CRDs of REJ1 should recognize terminal sugar residues of oligosaccharides; therefore, perhaps only the first and last residues bind REJ1. 5h-hFSP must bind REJ1 receptors because its AR-inhibiting activity is overridden by an excess amount of iFSP, and it can also induce Ca2+ increases in sperm. Our data, and the data of others (4Gonzalez-Martinez M.T. Galindo B.E. De La Torre L. Zapata O. Rodriguez E. Florman H.M. Darszon A. Dev. Biol. 2001; 236: 220-229Google Scholar), show that mere elevation of [Ca2+]i to a certain concentration is not what induces the AR; it is the pathway leading to induction that is important. It could be that hFSP uncouples components of the pathway, a phenomenon that can be demonstrated in these cells by other means. For example, seawater is ∼10 mm Ca2+. Treatment of sperm with EJ in 2 mm Ca2+ makes the cells refractory to AR induction and to increases in [Ca2+]i when the Ca2+ is returned to 10 mm (40Guerrero A. Darszon A. Biochim. Biophys. Acta. 1989; 980: 109-116Google Scholar, 41Guerrero A. Garcia L. Zapata O. Rodriguez E. Darszon A. Biochim. Biophys. Acta. 1998; 1401: 329-338Google Scholar). This AR inactivation is hypothesized to result from uncoupling the linkage between the sperm EJ receptors and Ca2+ channels. Pretreatment of sperm with 5h-hFSP causes a marked decrease in the AR response, yet [Ca2+]iincreases to reasonably high levels. Desensitization of the AR response by hFSP is thus different from AR inactivation. This might be due to opening of the second Ca2+ channel before the first channel opens, the order of opening being crucial to the AR. Normally, the first channel causes rapid increases in [Ca2+]i, whereas the second is responsible for the sustained high level of [Ca2+]i. The first channel may be activated by the binding of FSP to REJ1 (6Vacquier V.D. Moy G.W. Dev. Biol. 1997; 192: 125-135Google Scholar), whereas the second channel, which is normally activated by the up-regulation of the first channel, has characteristics of a store-operated channel (4Gonzalez-Martinez M.T. Galindo B.E. De La Torre L. Zapata O. Rodriguez E. Florman H.M. Darszon A. Dev. Biol. 2001; 236: 220-229Google Scholar). That binding of hFSP to the cell surface bypasses the first channel and up-regulates the second channel suggests that the sensor regulating the second channel could involve unidentified cell surface receptors. We thank Drs. B. Hayes, A. Varki, J. Esko, and H. van Halbeek for helpful discussions. We thank Dr. B. Galindo and Sheryl Huffman for technical suggestions and discussions.
Referência(s)