Sulfated fucans from the egg jellies of the closely related sea urchins Strongylocentrotus droebachiensis and Strongylocentrotus pallidus ensure species-specific fertilization.

Sulfated polysaccharides from egg jelly are the molecules responsible for inducing the sperm acrosome reaction in sea urchins. This is an obligatory event for sperm binding to, and fusion with, the egg. The sulfated polysaccharides from sea urchins have simple, well defined repeating structures, and each species represents a particular pattern of sulfate substitution. Here, we examined the egg jellies of the sea urchin sibling species Strongylocentrotus droebachiensis and Strongylocentrotus pallidus. Surprisingly, females of S. droebachiensis possess eggs containing one of two possible sulfated fucans, which differ in the extent of their 2-O-sulfation. Sulfated fucan I is mostly composed of a regular sequence of four residues ([4-alpha-l-Fucp-2(OSO3)-1-->4-alpha-l-Fucp-2(OSO3)-1-->4-alpha-l-Fucp-1-->4-alpha-l-Fucp-1]n), whereas sulfated fucan II is a homopolymer of 4-alpha-l-Fucp-2(OSO3)-1 units. Females of S. pallidus contain a single sulfated fucan with the following repeating structure: [3-alpha-l-Fucp-2(OSO3)-1-->3-alpha-l-Fucp-2(OSO3)-1-->3-alpha-l-Fucp-4(OSO3)-1-->3-alpha-l-Fucp-4(OSO3)-1]n. The egg jellies of these two species of sea urchins induce the acrosome reaction in homologous (but not heterologous) sperm. Therefore, the fine structure of the sulfated alpha-fucans from the egg jellies of S. pallidus and S. droebachiensis, which differ in their sulfation patterns and in the position of their glycosidic linkages, ensures species specificity of the sperm acrosome reaction and prevents interspecies crosses. In addition, our observations allow a clear appreciation of the common structural features among the sulfated polysaccharides from sea urchin egg jelly and help to identify structures that confer finer species specificity of recognition in the acrosome reaction.     

Structure of the sulfated alpha-L-fucan from the egg jelly coat of the sea urchin Strongylocentrotus franciscanus: patterns of preferential 2-O- and 4-O-sulfation determine sperm cell recognition.

The egg jelly coats of sea urchins contains sulfated polysaccharides responsible for inducing the sperm acrosome reaction which is an obligatory event for sperm binding to, and fusion with, the egg. Here, we extend our study to the sea urchin Strongylocentrotus franciscanus. The egg jelly of this species contains a homofucan composed of 2- O -sulfated, 3-linked units which is the simplest structure ever reported for a sulfated fucan. This polysaccharide was compared with other sulfated alpha-L-fucans as inducers of acrosome reaction in conspecific and heterospecific sperm. Although all these fucans are linear polymers composed of 3-linked alpha-L-fucopyranosyl units, they differ in the proportions of 2-O- and 4-O-sulfation. The reactivity of the sperm of each species is more sensitive to the egg jelly sulfated fucan found in their own species. The reactivity of the sperm does not correlate with the charge density of the fucan, but with the proportion of 2-O- and 4-O-sulfation. The pattern of sulfation may be an important feature for recognition of fucans by the sperm receptor contributing to the species-specificity of fertilization.     

Females of the sea urchin Strongylocentrotus purpuratus differ in the structures of their egg jelly sulfated fucans

The egg jelly coats of sea urchins contain sulfated fucans which bind to a sperm surface receptor glycoprotein to initiate the signal transduction events resulting in the sperm acrosome reaction. The acrosome reaction is an ion channel regulated exocytosis which is an obligatory event for sperm binding to, and fusion with, the egg. Approximately 90% of individual females of the sea urchin Strongylocentrotus purpuratus spawned eggs having only one of two possible sulfated fucan electrophoretic isotypes, a slow migrating (sulfated fucan I), or a fast migrating (sulfated fucan II) isotype. The remaining 10% of females spawned eggs having both sulfated fucan isotypes. The two sulfated fucan isotypes were purified from egg jelly coats and their structures determined by NMR spectroscopy and methylation analysis. Both sulfated fucans are linear polysaccharides composed of 1-->3-linked alpha-L-fucopyranosyl units. Sulfated fucan I is entirely sulfated at the O -2 position but with a heterogeneous sulfation pattern at O -4 position. Sulfated fucan II is composed of a regular repeating sequence of 3 residues, as follows: [3-alpha-L-Fuc p - 2,4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)- 1]n. Both purified sulfated fucans have approximately equal potency in inducing the sperm acrosome reaction. The significance of two structurally different sulfated fucans in the egg jelly coat of this species could relate to the finding that the sperm receptor protein which binds sulfated fucan contains two carbohydrate recognition modules of the C-type lectin variety which differ by 50% in their primary structure.      

Horseradish peroxidase. XXV. An electron spin resonance study of the low temperature photochemical reaction of compound I.

The insoluble acrosome granule content of sea urchin sperm consists of a single 30,500 dalton protein named bindin. Bindin mediates species-specific recognition and adhesion of sperm to the egg surface. Bindin from $\text{Strongylocentrotus purpuratus}$ (Sp) and $\text{Strongylocentrotus franciscanus}$ (Sf) have tyrosine as their single N-terminal amino acid. The pI of Sp bindin is 6.62 and of Sf 6.59. Amino acid analysis reveals almost identical composition between the two species for 16 amino acids. Only two (or three) amino acids, Pro and Asx, show large species differences. Tryptic peptide maps of the two species of bindin show very similar patterns with 24 spots of identical correspondence.     

The structure of sulfated polysaccharides ensures a carbohydrate-based mechanism for species recognition during sea urchin fertilization

The evolution of barriers to inter-specific hybridization is a crucial step in the fertilization of free spawning marine invertebrates. In sea urchins, molecular recognition between sperm and egg ensures species recognition. Here we review the sulfated polysaccharide-based mechanism of sperm-egg recognition in this model organism. The jelly surrounding sea urchin eggs is not a simple accessory structure; it is molecularly complex and intimately involved in gamete recognition. It contains sulfated polysaccharides, sialoglycans and peptides. The sulfated polysaccharides have unique structures, composed of repetitive units of alpha-L-fucose or alpha-L-galactose, which differ among species in the sulfation pattern and/or the position of the glycosidic linkage. The egg jelly sulfated polysaccharides show species-specificity in inducing the sperm acrosome reaction, which is regulated by the structure of the saccharide chain and its sulfation pattern. Other components of the egg jelly do not possess acrosome reaction inducing activity, but sialoglycans act in synergy with the sulfated polysaccharide, potentiating its activity. The system we describe establishes a new view of cell-cell interaction in the sea urchin model system. Here, structural changes in egg jelly polysaccharides modulate cell-cell recognition and species-specificity leading to exocytosis of the acrosome. Therefore, sulfated polysaccharides, in addition to their known functions as growth factors, coagulation factors and selectin binding partners, also function in fertilization. The differentiation of these molecules may play a role in sea urchin speciation.     

Carbohydrate‐based species recognition in sea urchin fertilization: another avenue for speciation?

Spawning marine invertebrates are excellent models for studying fertilization and reproductive isolating mechanisms. To identify variation in the major steps in sea urchin gamete recognition, we studied sperm activation in three closely related sympatric Strongylocentrotus species. Sperm undergo acrosomal exocytosis upon contact with sulfated polysaccharides in the egg-jelly coat. This acrosome reaction exposes the protein bindin and is therefore a precondition for sperm binding to the egg. We found that sulfated carbohydrates from egg jelly induce the acrosome reaction species specifically in S. droebachiensis and S. pallidus. There appear to be no other significant barriers to interspecific fertilization between these two species. Other species pairs in the same genus acrosome react nonspecifically to egg jelly but exhibit species-specific sperm binding. We thus show that different cell-cell communication systems mediate mate recognition among very closely related species. By comparing sperm reactions to egg-jelly compounds from different species and genera, we identify the major structural feature of the polysaccharides required for the specific recognition by sperm: the position of the glycosidic bond of the sulfated alpha-L-fucans. We present here one of the few examples of highly specific pure-carbohydrate signal transduction. In this system, a structural change in a polysaccharide has far-reaching ecological and evolutionary consequences.     

Insertion of a short repetitive sequence (D881) in a sea urchin gene: A typical interspersed repeat?

Summary A comparison has been made between the Sp88 gene regions of the DNAs of the sea urchinsStrongylocentrotus purpuratus (Sp.) andStrongylocentrotus drobachiensis (Sd.). Examination of the 3 terminal part of the transcribed region revealed a short repetitive sequence present in Sd. but absent from Sp. A 12-nucleotide sequence present once in Sp. is almost perfectly duplicated at both ends of the repeat in Sd., suggesting that a mobile repeat was inserted in the Sd. genome. Other members of this family of repeated sequences occur in many interspersed locations in the genomes of both species. Except for the insertion duplication, the inserted sequence lacks direct or reverse repeats.     

Chemical characterization of the component of the jelly coat from sea urchin eggs responsible for induction of the acrosome reaction.

Jelly coat, a multicomponent extracellular matrix surrounding the sea urchin egg, induces the acrosome reaction in sperm. The jelly coats of the four species studied, Arbacia punctulata, Strongylocentrotus purpuratus, Strongylocentrotus drobachiensis, and Lytechinus variegatus, were found to be very similar in chemical composition. A sialoprotein (approximately 20% of the mass of the jelly coat) and a fucose sulfate polysaccharide (approximately 80%) are the major macromolecular components of the jelly coat. The acrosome reaction inducing capacity resides solely in the fucose sulfate polysaccharide. Induction of the acrosome reaction ranges from highly species specific to nonspecific. Thus, A. punctulata and S. drobachiensis sperm are induced to undergo the acrosome reaction only with their homologous jelly coat, while S. purpuratus sperm react equally well with homologous or L. variegatus jelly coat, but not with A. punctulata jelly coat. L. variegatus sperm seem to be relatively nonspecific in response. Species-specific induction of the acrosome reaction resides solely in the fucose sulfate polysaccharide, suggesting that there must be structural differences in this polysaccharide in the various species. Therefore, in some species, fertilization appears to involve sperm-egg recognition at the level of the jelly coat as well as at the level of sperm-egg receptors.     

A Unique 2-Sulfated ��-Galactan from the Egg Jelly of the Sea Urchin Glyptocidaris crenularis: CONFORMATION FLEXIBILITY VERSUS INDUCTION OF THE SPERM ACROSOME REACTION*

Sulfated polysaccharides from the egg jelly of sea urchins act as species-specific inducers of the sperm acrosome reaction, which is a rare molecular mechanism of carbohydrate-induced signal-transduction event in animal cells. The sea urchin polysaccharides differ in monosaccharide composition (l-fucose or l-galactose), glycosylation, and sulfation sites, but they are always in the α-anomeric configuration. Herein, structural analysis of the polysaccharide from the sea urchin Glyptocidaris crenularis surprisingly revealed a unique sulfated β-d-galactan composed by (3-β-d-Galp-2(OSO₃)-1→3-β-d-Galp-1)_n repeating units. Subsequently, we used the G. crenularis galactan to compare different 2-sulfated polysaccharides as inducers of the acrosome reaction using homologous and heterologous sperm. We also tested the effect of chemically over-sulfated galactans. Intriguingly, the anomeric configuration of the glycosidic linkage rather than the monosaccharide composition (galactose or fucose) is the preferential structural requirement for the effect of these polysaccharides on sea urchin fertilization. Nuclear magnetic resonance and molecular dynamics indicate that sulfated α-galactan or α-fucan have less dynamic structural behavior, exhibiting fewer conformational populations, with an almost exclusive conformational state with glycosidic dihedral angles Φ/Ψ = −102°/131°. The preponderant conformer observed in the sulfated α-galactan or α-fucan is not observed among populations in the β-form despite its more flexible structure in solution. Possibly, a proper spatial arrangement is required for interaction of the sea urchin-sulfated polysaccharides with the specific sperm receptor.The evolution of barriers to inter-specific hybridization is a crucial step in the fertilization of free-spawning marine invertebrates. In sea urchins the molecular recognition between sperm and egg ensures species recognition. The jelly coat surrounding sea urchin eggs is not a simple accessory structure; it is considerably complex on a molecular level and intimately involved in gamete recognition. It contains sulfated polysaccharides, sialoglycans, and peptides.Structural changes in the sulfated polysaccharide from the egg jelly of sea urchins modulate cell-cell recognition and species specificity leading to exocytosis of the acrosomal vesicle, the acrosome reaction. This is a crucial event for the recognition between male and female gametes, leading to the fertilization success, and is also what prevents intercrosses. The sulfated polysaccharide from the egg jelly recognizes its specific receptor present in the sperm. Apart from the sialoglycans that act in synergy with the sulfated polysaccharides, other components of the egg jelly do not possess acrosome reaction-inducing activity (1). The sulfated polysaccharide-mediated mechanism of sperm-egg recognition co-exists with that of bindin and its receptor in the egg (2–4).The sulfated polysaccharides from sea urchin show species-specific structures composed of repetitive units (mono-, tri-, and tetrasaccharides) that differ in the monosaccharide backbone (l-fucose or l-galactose), glycosidic linkage (3- or 4-linked), and sulfation (2- and/or 4-sulfation). However, they are always in the α-enantiomeric configuration (4, 5). Previous studies from our laboratory have demonstrated that sea urchin-sulfated polysaccharides induce the acrosome reaction in a species-specific way. In some cases the sperm from a certain species of sea urchin recognizes the sulfated polysaccharide containing a similar structure from a different species. For example, the egg jelly from Strongylocentrotus franciscanus contains a 2-sulfated, 3-linked α-fucan, but the sperm from this species recognizes a heterologous 2-sulfated, 3-linked α-galactan from Echinometra lucunter (6).We now extended our studies to the sulfated polysaccharides of the sea urchin Glyptocidaris crenularis (7). Surprisingly, we observed that this species contains a unique sulfated β-d-galactan composed of repetitive disaccharide units alternating 2-sulfated and non-sulfated 3-linked units. This polymer is markedly distinct from all other sea urchin-sulfated polysaccharides described so far that are composed of units on α-l-configuration. Furthermore, this sea urchin does not contain sialoglycans, which are commonly found in the echinoderm egg jelly.We used this new sulfated β-galactan to investigate the acrosome reaction in a further molecular detail using homologous and heterologous sperm. We tested three 2-sulfated polysaccharides that differ in their conformation (α or β) and monosaccharide composition (galactose or fucose) as inducers of the sperm acrosome reaction. We aimed to establish the structure versus biological activity of the echinoderm polysaccharides, including structural features at a conformational level.     

Involvement of Wheat Germ Agglutinin (WGA)-Binding Protein in the Induction of the Acrosome Reaction of the Sea Urchin, Strongylocentrotus intermedius. I. WGA Affects the Ion Fluxes Associated with the Acrosome Reaction

The lectin wheat germ agglutinin (WGA) inhibited the egg jelly-induced acrosome reaction (AR) of sperm of the sea urchin, Strongylocentrotus intermedius . Fluorescein-conjugated WGA applied to sperm bound to the acrosomal region, to the midpiece, and to the tip of the flagellum. These effects were not observed in the presence of N-acetly-D-glucosamine. When the egg jelly was replaced by artificial AR inducers such as A23187 or nigericin, the AR was not inhibited by WGA. Results obtained using a Ca²⁺ indicator fura-2, a pH indicator 2',7'-bis(carboxyethyl)carboxyfluorescein (BCECF) and a membrane potential sensitive dye 3,3'-dipentyl 2,2'-dioxacarbocyanine [diO-C₅(3)] showed that WGA suppresses the egg jelly-induced influx of Ca²⁺ and slightly suppresses the efflux of H⁺ caused by the egg jelly, whereas the depolarization of the plasma membrane by the egg jelly is remarkably amplified by the treatment with WGA. These results suggest that WGA affects the regulatory system of the ion fluxes associated with the AR. The target protein of WGA (WGA-binding protein) was a membrane glycoprotein of 260 kD under non-reducing condition.     

Regulation of the starfish sperm acrosome reaction by cGMP, pH, cAMP and Ca2+

In the starfish, Asterias amurensis, three components in the jelly coat of eggs, namely acrosome reaction-inducing substance (ARIS), Co-ARIS and asterosap, act in concert on homologous spermatozoa to induce the acrosome reaction (AR). Molecular recognition between the sperm surface molecules and the egg jelly molecules must underlie signal transduction events triggering the AR. Asterosap is a sperm-activating molecule, which stimulates rapid synthesis of intracellular cGMP, pH and Ca(2+). This transient elevation of Ca(2+) level is caused by a K(+)-dependent Na(+)/Ca(2+) exchanger, and the increase of intracellular pH is sufficient for ARIS to induce the AR. The concerted action of ARIS and asterosap could induce elevate intracellular cAMP levels in starfish sperm and the sustained increase in [Ca(2+)], which is essential for the AR. The signaling pathway induced by these factors seems to be synergistically regulated to trigger the AR in starfish sperm.     

Response of isolated sperm plasma membranes from sea urchin to egg jelly

The acrosome reaction in sea urchin sperm is induced by a glycoprotein jelly surrounding the egg and is accompanied by changes in ion permeability of sperm plasma membrane. In an attempt to learn what membrane components are involved in the response to jelly, we have begun to reassemble sperm membrane components into artificial membranes and assay for permeability changes mimicking those that occur in sperm. Jelly in sea water at concentrations that induce the acrosome reaction did not significantly change 45Ca2+ uptake of sonicated unilamellar vesicles made with soybean lipid only (ratio jelly:control uptake = 1.08 +/- 0.36 SD, n = 21). Experiments with pure lipid planar bilayers made with soybean lipid or a lipid extract from sperm and held at various voltages, also did not reveal substantial permeability changes at comparable jelly concentrations. Thus, jelly by itself does not change the conductance of a pure lipid bilayer. In contrast, significant (P----0.0005, t test for two sample means) 45Ca2+ uptake was observed with vesicles made by cosonicating soybean phospholipids and Strongylocentrotus purpuratus sperm membranes isolated by the method of Cross, N. L. [1983, J. Cell Sci. 59, 13-25] (ratio jelly: control uptake = 1.51 +/- 0.75, n = 20, 16 positive out of 20 experiments). The calcium uptake response of the mixed vesicles was also species-specific: it did not occur with jelly from Arbacia punctulata (ratio Arbacia jelly: control = 1.18 +/- 0.51; ratio Strongylocentrotus jelly: control = 1.71 +/- 0.97, n = 10; P----0.025, paired t statistic). Vesicles made with soybean lipid and an octyl glucoside extract of sperm membranes also responded to jelly with increased 45Ca2+ uptake. Our results indicate that we have the starting conditions to isolate and characterize the sperm membrane components that participate in the egg jelly induced permeability changes.     

Flagellasialin: a novel sulfated alpha2,9-linked polysialic acid glycoprotein of sea urchin sperm flagella

A novel alpha2,9-linked polysialic acid (polySia)-containing glycoprotein of sea urchin sperm flagella was identified and named "flagellasialin." Flagellasialin from Hemicentrotus pulcherrimus shows a diverse relative molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of 40-80 kDa. Flagellasialin is a 96-amino acid, threonine-rich, heavily O-glycosylated (80-90% by weight) glycoprotein with a single transmembrane segment at its C-terminus and no apparent cytosolic domain. Of 12 extracellular Thr residues, eight are O-glycosylated and three are nonglycosylated. Flagellasialin is highly expressed in the testis but cannot be detected in the ovary. The amino acid sequences of flagellasialin from three sea urchin species (H. pulcherrimus, Strongylocentrotus purpuratus, and Strongylocentrotus franciscanus) are identical, but some species differences exist in the three core glycan structures to which the sulfated alpha2,9-linked polyNeu5Ac chain is linked. Finally, the treatment of sperm with a specific antibody against the alpha2,9-linked polyNeu5Ac structure results in the elevation of intracellular Ca(2+) and inhibition of sperm motility and fertilization, implicating flagellasialin as a regulator of these critical processes.     

Expression of two different sulfated fucans by females of Lytechinus variegatus may regulate the seasonal variation in the fertilization of the sea urchin

The egg jellies of sea urchins contain sulfated polysaccharides with unusual structures, composed of linear chains of l-fucose or l-galactose with well-defined repetitive units. The specific pattern of sulfation and the position of the glycosidic bond vary among sulfated polysaccharides from different species. These polysaccharides show species specificity in inducing the acrosome reaction, which is a critical event for fertilization. Females of the sea urchin Lytechinus variegatus spawn eggs containing a sulfated fucan with the repetitive sequence [3-alpha-L-Fucp-2(OSO(3))-1 --> 3-alpha-L-Fucp-4(OSO(3))-1 --> 3-alpha-L-Fucp-2,4(OSO(3))-1 --> 3-alpha-L-Fucp-2(OSO(3))-1](n). We now observe that, close to winter, a period of decreased fertility for the sea urchin, the females synthesize a distinct sulfated fucan with a simple structure, composed of 4-sulfated, 3-linked alpha-fucose residues. This sulfated fucan is inactive when tested in vitro for the acrosome reaction using homologous sperm. The amount of egg jellies spawned by females (and their constituent sulfated polysaccharides) varied greatly throughout the year. Apparently, there is a correlation between the temperature of the sea water and the expression of the 4-sulfated, 3-linked sulfated fucan. Overall, we described the occurrence of two isotypes of sulfated fucan in the egg jelly of the sea urchin L. variegatus, which differ in their biological activity and may be involved in the periodicity of the reproductive cycle of the invertebrate.     

N-Linked Oligosaccharides of Sea Urchin Egg Jelly Induce the Sperm Acrosome Reaction

The sea urchin egg jelly coat (EJ) induces the acrosome reaction (AR) of sperm. We previously demonstrated that a fraction of EJ containing two glycoproteins of 82- and 138-kDa possess the AR inducing activity (8). Here we show that Peptide-N-Glycosidase-F treatment of EJ followed by precipitation and washing in 70% ethanol results in a substantial loss of AR inducing activity in the ethanol insoluble material. When a PNGase-F digest of EJ is chromatographed on a Sepacryl-200 gel filtration column, an AR inducing fraction elutes within the partitioning volume. Acrosome reaction inducing activity of undigested EJ does not elute within the partitioning volume. The chromatographed AR inducing fraction of the PNGase-F digest reacts strongly in the phenol-sulfuric assay demonstrating carbohydrate is present; silver stained gels do not detect the presence of protein. Harsh alkaline hydrolysis of EJ in an excess of NaBH₄, preserves a substantial amount of AR inducing activity. These data show that N-linked oligosaccharides released from EJ by PNGase-F digestion are capable of inducing the sperm acrosome reaction.     

Characterization of a monoclonal antibody that induces the acrosome reaction of sea urchin sperm

A monoclonal antibody, J18/29, induces the acrosome reaction (AR) in spermatozoa of the sea urchin Strongylocentrotus purpuratus. J18/29 induces increases in both intracellular Ca2+ and intracellular pH similar to those occurring upon induction of the AR by the natural inducer, the fucose sulfate-rich glycoconjugate of egg jelly. Lowering the Ca2+ concentration or the pH of the seawater inhibits the J18/29-induced AR, as does treatment with Co2+, an inhibitor of Ca2+ channels. The J18/29-induced AR is also inhibited by verapamil, tetraethylammonium chloride, and elevated K+. All these treatments cause similar inhibition of the egg jelly-induced AR. J18/29 reacts with a group of membrane proteins ranging in molecular mass from 340 to 25 kD, as shown by immunoprecipitation of lysates of 125I-labeled sperm and Western blots. The most prominent reacting proteins are of molecular masses of 320, 240, 170, and 58 kD. The basis of the multiple reactivity appears to reside in the polypeptide chains of these proteins, as J18/29 binding is sensitive to protease digestion but resistant to periodate oxidation. There are approximately 570,000 sites per cell for J18/29 binding. J18/29 is the only reagent of known binding specificity that induces the AR; it identifies a subset of sperm membrane proteins whose individual characterization may lead to the isolation of the receptors involved in the triggering of the AR at fertilization.     

Involvement of Wheat Germ Agglutinin (WGA)-Binding Protein in the Induction of the Acrosome Reaction of the Sea Urchin, Strongylocentrotus intermedius. II. Antibody against WGA-Binding Protein Induces the Acrosome Reaction

We obtained a polyclonal antibody against the WGA-binding protein (WGAbp) of Strongylocentrotus intermedius sperm, which is a membrane glycoprotein of 260 kD under non-reducing condition. Anti-WGAbp antibody induced increases in both intracellular Ca²⁺ ([Ca²⁺]i) and intracellular pH (pHi), resulting in the onset of the AR. The increases in [Ca²⁺]i and pHi required extracellular Ca²⁺ and Na⁺, respectively, and were suppressed by the pretreatment with WGA, resulting in the inhibition of the AR. Anti-WGAbp antibody-induced AR was inhibited also by lowered extracellular pH. elevated K⁺, removal of Na⁺ from seawater and the treatment with verapamil, a Ca²⁺ channel inhibitor. These inhibitory conditions are identical with those of the egg jelly-induced AR. Monovalent Fab fragments from anti-WGAbp antibody also induced the AR at relatively high concentration. These results suggest that the WGAbp on the sperm plasma membrane is involved in the regulation of Ca²⁺ influx and Na⁺/H⁺ exchange associated with the AR of S. intermedius sperm. It is a strong candidate for the receptor of the AR-inducing substance in the egg jelly.     

Novel conserved structural domains of acrosome reaction-inducing substance are widespread in invertebrates

In the starfish Asterias amurensis, acrosome reaction inducing substance (ARIS) is the main factor responsible for allowing sperm to recognize the egg jelly and begin the acrosome reaction (AR). ARIS is a large proteoglycan-like molecule, and its pentasaccharide repeat, Fragment 1 (Fr. 1), is responsible for inducing AR. Here, we investigated the primary structure of ARIS for the first time in order to improve our understanding of its functionality. Electrophoretic analysis revealed that ARIS is a complex of three proteins, all of which are modified by the Fr. 1 sugar chain. Sequencing indicated that there are two novel, conserved domains in all three ARIS proteins: ARIS N-terminus (AR-N) and ARIS C-terminus (AR-C) domains. We also found that other echinoderms possess ARIS proteins that are capable of inducing the AR for homologous sperm, indicating that ARIS proteins may be a ubiquitous component for echinoderm fertilization. Moreover, we identified ARIS-like genes from Ctenophora to Protochordata.     

Pretreatment effects of jelly components on the sperm acrosome reaction and histone degradation in the starfish, Asterina pectinifera.

Acrosome reaction (AR) and histone degradation (HD) of Asterina pectinifera sperm are induced by co-operation of ARIS and a diffusible fraction (M8) of egg jelly. Once sperm are treated with ARIS or M8 separately for several minutes, they do not undergo the AR in response to the egg jelly. Preincubation of sperm with M8 at 0 degrees C is not effective to block the jelly-induced AR whereas inhibitory effects of ARIS remain at 0 degrees C. Jelly-induced HD is inhibited by pretreatment of sperm with ARIS but is not affected by the incubation with M8. The blockage of the jelly-induced reactions, both AR and HD, by ARIS- or M8-pretreatment can be bypassed by ionophores, A23187 and monensin.     

High molecular mass egg fucose sulfate polymer is required for opening both Ca2+ channels involved in triggering the sea urchin sperm acrosome reaction.

A linear fucose sulfate polymer (FSP), >10(6) 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 Ca(2+) 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 Ca(2+) channels decreases with increasing time of hydrolysis. Hydrolyzed FSP of approximately 60 kDa blocks intact FSP from inducing the AR. At 44 microg/ml hydrolyzed FSP, Ca(2+) entry into sperm is almost equal to that occurring in 3.8 microg/ml intact FSP; however the AR is not induced. The shape of the [Ca(2+)](i) increase curve and use of the Ca(2+) channel blockers nifidipine and Ni(2+) indicate that hydrolyzed FSP opens the second Ca(2+) channel, but not the first, and thus does not induce the AR. The giant size of intact FSP is required to open both Ca(2+) channels involved in triggering the AR.