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.

Broadcast spawning echinoderms are a model system for studying molecular mechanisms of fertilization and the evolution of mating barriers. In marine species without temporal or spatial segregation of spawning events, molecular recognition of egg and sperm surfaces is critical to prevent hybridization. Knowing which steps confer species specificity will further our understanding of the evolution of reproductive isolation and ultimately of speciation and biodiversity. Environmental spawning cues and sperm attractants have not been found to be species-specific in sea urchins (1). Species specificity must therefore be achieved during subsequent gamete interactions. Once released, the sperm must find and interact with an egg of the correct species. An obligatory event for sperm binding to, and fusion with, the egg is the induction of the acrosome reaction in the sperm, an exocytosis of lytic and binding proteins from a vesicle at the tip of the sperm head. This is a signal transduction event linked to ion fluxes, membrane depolarization, and internal pH changes, but the signal transduction pathway of which remains to be elucidated (2,3).
The sea urchin egg is surrounded by a transparent jelly coat, which contains molecules inducing physiological changes in sperm (4). A major macromolecule of the egg jelly coat, the one responsible for inducing the sperm acrosome reaction, is a sulfated polysaccharide (5)(6)(7). We have demonstrated (5-7) that these compounds have simple, repeating structures and that each species represents a particular pattern of sulfate substitution. The sulfated polysaccharides are species-specific as inducers of the sperm acrosome reaction (5) and represent an unusual simple example of ligand-induced signal transduction leading to exocytosis (5,8).
We also reported two structurally distinct sulfated ␣-L-fucans in the egg jelly of the sea urchin Strongylocentrotus purpuratus (6). Approximately 90% of individual females of this species spawn eggs with only one of two possible fucans. Both purified sulfated ␣-L-fucans have equal potency in inducing the acrosome reaction in homologous sperm. The reason that eggs from this species possess two sulfated fucan isotypes remains unknown.
For our initial demonstration that sulfated polysaccharides are species-specific inducers of the acrosome reaction, we used polysaccharides from distantly related species expressing marked interspecies structural variation (5). More recently, we evaluated the finer specificity of recognition in the acrosome reaction with egg jelly sulfated fucans containing the same backbone of 3-linked ␣-L-fucopyranosyl units, but with different proportions of 2-O-and 4-O-sulfation (7). Although we observed a less strict species specificity in sperm recognition of * This work was supported in part by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (FNDCT, PADCT, and PRONEX), the Financiadora de Estudos e Projetos, and the Fundaçã o de Amparo à Pesquisa do Estado do Rio de Janeiro. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18   Here, we extend our studies to two new sea urchins, the closely related species Strongylocentrotus droebachiensis and Strongylocentrotus pallidus, which both have a circumarctic distribution. The egg jellies of these sea urchins contain sulfated ␣-fucans with new structures. Our results show expanded possibilities for structural variation among sulfated ␣-L-fucans from echinoderms and possible biological and evolutionary implications of these unique polysaccharides. Detailed structural characterizations also help evaluate the therapeutic potential of sulfated polysaccharides, as already demonstrated for the anticoagulant activity of sulfated fucans (9) and sulfated galactans (10).

EXPERIMENTAL PROCEDURES
Extraction-Mature females of S. droebachiensis and S. pallidus were collected near Friday Harbor, WA. Atlantic S. droebachiensis females were collected in Bergen, Tromsø, and Svalbard, Norway. Eggs were spawned into filtered sea water after intracelomic injection of 0.55 M KCl. Egg jelly was isolated by pouring eggs repeatedly through nylon mesh, prepared as a 20,000 ϫ g supernatant, and stored at Ϫ20°C or lyophilized after dialysis against distilled water (8). The acidic polysaccharides were extracted from the jelly coat by papain digestion and partially purified by ethanol precipitation as described previously (11).
Purification-The crude polysaccharides (10 mg) from the egg jelly coats were applied to a Mono Q FPLC 1 column (HR5/5; Amersham Biosciences, Inc.) equilibrated with 20 mM Tris-HCl (pH 8.0). The col-umn was washed with 10 ml of the same buffer and then eluted by a linear gradient of 0 -4.0 M NaCl in the same buffer. The flow rate of the column was 0.45 ml/min, and fractions of 0.5 ml were collected. Fractions were checked for fucose and sialic acid by the Dubois reaction (12) and by the Ehrlich assay (13), respectively, and by their metachromasia (14). The NaCl concentration was estimated by conductivity. Fractions containing the sulfated ␣-L-fucan and the sialic acid glycoconjugate were pooled, dialyzed against distilled water, and lyophilized.
Chemical Analyses-Total fucose was measured by the method of Dische and Shettles (15). After acid hydrolysis of the polysaccharide (5.0 M trifluoroacetic acid for 5 h at 100°C), sulfate was measured by the BaCl 2 /gelatin method (16). The presence of hexoses and 6-deoxyhexoses in the acid hydrolysates was estimated by paper chromatography in 1-butanol/pyridine/water (3:2:1, v/v) for 48 h and by gas-liquid chromatography-mass spectrometry of derived alditols (17).
Desulfation and Methylation of the Fucans-Desulfation of the sulfated fucans was performed by solvolysis in dimethyl sulfoxide as described previously for desulfation of other types of polysaccharides (19,20). Sulfate esters located at different sites of the fucose residues may have variable susceptibility to the desulfation reaction (5-7). In addition, the desulfation reaction simultaneously reduced the molecular mass of the polysaccharide. It is necessary to have a balance between removal of sulfate ester and decrease in the polysaccharide chain. For these reasons, we obtained a totally desulfated fucan in some experiments and a partially desulfated fucan in others.
The native and desulfated fucans (5 mg of each) were subjected to three rounds of methylation as described previously (21), with the modifications suggested by Patankar et al. (22). The methylated polysaccharides were hydrolyzed in 6 M trifluoroacetic acid for 5 h at 100°C and reduced with borohydride, and the alditols were acetylated with acetic anhydride/pyridine (1:1, v/v) (17). The alditol acetates of the methylated sugars were dissolved in chloroform and analyzed in a gas chromatography-mass spectrometer.
NMR Experiments-1 H and 13 C spectra of the native and desulfated fucans were recorded using a Bruker DRX 600 apparatus with a triple resonance probe. About 3 mg of each sample was dissolved in 0.5 ml of 99.9% D 2 O Cambridge Isotope Laboratory. All spectra were recorded at 60°C with HOD suppression by pre-saturation. COSY, TOCSY, and 1 H/ 13 C HMQC spectra were recorded using states-time proportion phase incrementation for quadrature detection in the indirect dimension. TOCSY spectra were run with 4096 ϫ 400 points with a spin-lock field of ϳ10 kHz and a mixing time of 80 ms. HMQC spectra were run with 1024 ϫ 256 points and globally optimized alternating phase rectangular pulses for decoupling. NOESY spectra were run with a mixing time of 100 ms. Chemical shifts are relative to external trimethylsilylpropionic acid at 0 ppm for 1 H and to methanol for 13 C.
Fertilization-Sea urchins were induced to spawn by intracelomic injection of 0.55 M KCl. Sperm were collected undiluted from the gonophores and stored on ice, whereas eggs were released into filtered seawater at ambient water temperature. Freshly diluted sperm were added to 480-l aliquots of gently washed 5% (v/v) egg suspensions in 24-well tissue culture plates. A 1:4 dilution of sperm at each of five steps, starting with a 1:10,000 dilution of sperm, covered the range from near zero to 100% fertilization for intraspecific crosses. Fertilization success was assessed by counting the proportion of eggs (out of 200 -300 eggs/well) with an elevated fertilization envelope or of eggs that were cleaving. The concentration of sperm, which differs among species and individuals, was determined later by 10 counts of fixed sperm suspensions in a hemocytometer. The percentages of fertilization were calculated by back-transformation from logistic regressions for multiple male/female combinations crossed over a range of sperm concentrations.
To obtain egg jelly for acrosome-reacting sperm, a 5-10% suspension of eggs was poured through Nitex mesh several times. This stripped the eggs of their soluble jelly; the supernatant was pipetted off after the dejellied eggs had settled. Carrying out the final sperm dilution step in conspecific egg jelly water induced the sperm acrosome reaction and is referred to as "pre-reaction with conspecific egg jelly."

RESULTS AND DISCUSSION
Egg Jelly of the Sea Urchin S. droebachiensis (but Not S. pallidus) Possesses Two Isotypes of Sulfated Fucans-Agarose gel electrophoresis in 1,3-diaminopropane acetate buffer followed by toluidine blue staining showed that egg jelly isolated from individual females of East Pacific S. droebachiensis contained either a slow (sulfated fucan I) or fast (sulfated fucan II) migrating fucan isotype (Fig. 1A). Of 22 individual females, 9 had eggs with sulfated fucan I, and 13 had eggs with sulfated fucan II. Surprisingly, nine individual females of the same species, but collected in the Atlantic Ocean, contained only the slow migrating sulfated fucan (isotype I) (Fig. 1B).
Small differences in the electrophoretic mobility of sulfated fucans I and II (Fig. 1, A and B) could reflect intermediate sulfation degrees, variation in the molecular mass of the polymers (11,23), or even interaction of the sulfated fucan with other macromolecules (24) because the agarose gel electrophoresis was performed with crude egg jelly. These aspects were further investigated using Mono Q FPLC of mixed samples of egg jellies from a large number of S. droebachiensis females. Egg jellies from 31 Pacific females showed two distinct fractions of sulfated fucans ( Fig. 2A), whereas egg jellies from 19 Atlantic females contained a single fraction eluted at lower NaCl concentration (Fig. 2B). A peak rich in sialic acid was eluted completely by 0.7 M NaCl from the two samples and termed "sialic acid-rich glycoconjugate" in analogy with similar compounds described in other species of sea urchins (25).
The absence of intermediate fractions between sulfated fucans I and II suggests that females of S. droebachiensis synthesize either type of fucan with a defined sulfation pattern. If the difference between the sulfated fucans from females of S. droebachiensis were a consequence of temporal variation in the sulfation or related to the stage of oogenesis, one would expect to see intermediate fractions between sulfated fucans I and II upon agarose gel electrophoresis (Fig. 1, A and B) and anionexchange chromatography (Fig. 2, A and B).
Seven individual females of the sea urchin S. pallidus from the Pacific coast, collected at the same site as the S. droebachiensis females used in the experiment in Fig. 1A, contained a single sulfated fucan isotype (Fig. 1C). Mono Q FPLC of a mixed sample of egg jellies from 25 S. pallidus females confirmed the occurrence of a single sulfated fucan (Fig. 2C) eluting at high NaCl concentration, like sulfated fucan II from S. droebachiensis, in addition to the sialic acid-rich glycoconjugate.
Overall, these results indicate that spawned eggs from individual females of the sea urchin S. droebachiensis have one of two possible sulfated fucan isotypes. This polymorphism was observed only in one population. In contrast, all assayed fe- FIG. 3. Agarose gel electrophoresis of the purified sulfated ␣-fucans from S. droebachiensis and S. pallidus from the Atlantic and Pacific Oceans. A mixed sample and purified sulfated fucans I and II from S. droebachiensis as well as the purified fucan from S. pallidus (15 g of each) were applied to a 0.5% agarose gel, and electrophoresis was carried out and the gel was stained as described in the legend of Fig. 1. M, mixture of sulfated fucans I and II; SF, sulfated fucan.  Fig. 2, A and B). Sulfated ␣-fucan from S. pallidus was purified as shown in Fig. 2C. males of the sea urchin S. pallidus contained a single type of sulfated fucan.
Sulfated ␣-Fucans from S. droebachiensis Are Linear 4-Linked Polysaccharides, but Differ in the Extent of Their 2-O-Sulfation-Both sulfated fucans (purified as in Fig. 2, A and B) migrated on agarose gels (Fig. 3) identically as crude egg jelly (shown in Fig. 1, A and B). The slow and fast migrating sulfated fucans were eluted at low and high NaCl concentrations, respectively. Chemical analysis of the purified sulfated fucans (Table I) Table III are based on the interpretations of TOCSY, COSY, and HMQC spectra.
NMR spectra of desulfated fucan I show a single anomeric signal (Fig. 4B) with a strong downfield shift (ϳ11 ppm) of C-4 ( Fig. 5B and Table III), compatible with a linear homopolymer of 4-linked ␣-fucopyranoside residues. NMR spectra of native sulfated fucan I contain four anomeric signals in near-equal proportions by integration (Figs. 4A and 5A). TOCSY and COSY spectra confirmed that the four anomeric signals of native sulfated fucan I correspond to four spin systems, each consistent with ␣-fucose. The spin systems can be traced, giving the values in Table III. Strong downshifts (approximately Ϫ0.65 ppm) of H-2 of residues A and B relative to H-2 of residues C and D indicate that two of the residues are sulfated at C-2. Thus, sulfated ␣-fucan I from S. droebachiensis is mostly a tetrasaccharide repeat unit consisting of 4-linked residues, two sulfated at the O-2-position and two that are unsulfated.
The order of the four residues can be easily deduced. The only possible array is two consecutive 2-O-sulfated residues followed by two unsulfated residues. If the 2-O-sulfated and unsulfated units alternate, the fucan would contain a disaccharide instead of a tetrasaccharide repeating structure. Our proposition was confirmed by the NOESY spectrum (Fig. 6). As in the NOESY spectra of other fucans from echinoderms (5,26,27), NOEs between protons of different units can be seen, and they were used to reveal the sequence (besides, of course, NOEs on other protons in the same residue). In sulfated ␣-fucan I   Fig. 7A. The presence of minor random components in sulfated ␣-fucan I cannot be ruled out. For example, small amounts of three consecutive 2-O-sulfated fucose units followed by three unsulfated residues may occur in the polysaccharide. In this case, the additional structures are either undetectable due to their low proportions or cannot be discriminated by the NMR spectra. Nevertheless, the near-equal proportions by integration of the four anomeric signals (Figs. 4A and 5A) indicate these additional structures cannot account for a substantial proportion of the sulfated fucan structure.
The structure of sulfated ␣-fucan II from S. droebachiensis was investigated using the same methodologies. Methylation of native sulfated fucan II yielded 3-methylfucose, whereas 2,3di-O-methylfucose was obtained from totally desulfated fucan II (Table II). Clearly, this indicates a linear homopolymer composed of 4-linked and 2-O-sulfated fucopyranoside residues, the structure of which was confirmed by NMR analysis (Figs. 4 (C and D) and Fig. 5 (C and D)). The 1 H spectrum of sulfated ␣-fucan II resulting from desulfation processes shows a reduction in intensity of the anomeric residue at 5.30 ppm and a corresponding increase at 5.05 ppm. 3 Again, the chemical shifts were based on the interpretations of TOCSY, COSY (data not shown), and HMQC spectra (Fig. 5, C and D). The chemical shifts of the desulfated residues from fucans I and II are similar, indicating that both polysaccharides have the same saccharide backbone. But, in contrast with sulfated fucan I, sul-3 Different samples of desulfated fucan II were used for methylation and NMR analyses. Totally desulfated fucan II was employed for methylation analysis (Table II), whereas a partially desulfated preparation was used for NMR analysis (Figs. 4D and 5D).

FIG. 5. 1 H/ 13 C HMQC spectra of native (A and C) and desulfated (B and D) ␣-fucan I (A and B) and ␣-fucan II (C and D) from S. droebachiensis.
The assignment was based on TOCSY and COSY spectra. The values of chemical shifts in Table III are Table III).

The Sulfated ␣-Fucan from S. pallidus Has a 3-Linked Tetrasaccharide Repeating Unit Defined by a Specific Pattern of Sulfation at the 2-O-and 4-O-Positions-
The sulfated fucan from S. pallidus that eluted from an anion-exchange chromatography column at high NaCl concentration (Fig. 2C) contains fucose as the only sugar with a high content of sulfate ester (Table I), but has a slower mobility upon agarose gel electrophoresis than the two sulfated fucans from S. droebachiensis (Fig. 3). The electrophoretic mobility of sulfated polysaccharides in 1,3-diaminopropane acetate buffer depends on the structure of the glycan, which forms a complex with the diamino groups (20,28). Thus, the retarded electrophoretic mobility of the sulfated fucan from S. pallidus is a preliminary indication of its distinctive polysaccharide structure.
As in the case of the polysaccharides from S. droebachiensis, the structure of this new sulfated fucan was determined by NMR analysis. The native sulfated fucan showed four anomeric residues in near-equal proportions by integration (Figs. 8A and 9A), whereas after desulfation, a single anomeric signal was seen (Figs. 8B and 9B), as already observed for sulfated fucan I from S. droebachiensis (Figs. 4 (A and B) and 5 (A and B)). But, in the case of desulfated fucan from S. pallidus, a strong downfield shift (ϳ8 ppm) of C-3 (Table IV, values shown in italic type), and not of C-4, is compatible with a 3-linked polysaccharide. The NMR spectra of the native sulfated fucans from the two species of sea urchins also differ significantly. For S. pallidus, strong downshifts of H-2 of residues A and B (Ϫ0.50 ppm) and H-4 of residues C and D (Ϫ0.70 ppm) ( Fig.  10A and Table IV) indicate that two of the four residues are 2-O-sulfated and that the other two are 4-O-sulfated. Minor structural components, which may occur in this sulfated ␣-fucan (such as those indicated by arrows in Fig. 8A), do not account for Ͼ5% of the total signals in the anomeric region based on integration of the peaks in this region of the 1 H spectrum. In addition, the proportions of these minor components (but not those of the A-D spin systems) vary among different preparations of sulfated ␣-fucan.
The order of the four residues can be easily deduced, as already discussed for sulfated fucan I from S. droebachiensis. The only possible array is two consecutive 2-O-sulfated residues followed by two 4-O-sulfated residues. Again, if the 2-Oand 4-O-sulfated units alternate, the fucan would contain a disaccharide instead of a tetrasaccharide repeating structure. There is no indication of disulfated units in the TOCSY spectrum. Although only one inter-residue NOE could be unambiguously identified in the NOESY spectrum (Fig. 10B), it was enough to confirm the proposed structure. Thus, it was possible to identify NOEs from H-1 of residue A to H-4 of residue D, whereas H-1 of residue B, C, or D does not have any interresidue NOEs. These NOEs are in agreement with the repeat- ing unit of this sulfated fucan as -B-A-D-C- (Fig. 7C).
Overall, the NMR analyses indicated that the sulfated fucan from S. pallidus is composed mostly of a regular sequence of four residues, as follows:  (Fig. 7C). As in the case of sulfated ␣-fucan I from S. droebachiensis, we cannot rule out the occurrence of minor random components in the sulfated ␣-fucan from S. pallidus. In this case, the additional structures are either undetectable due to their low proportions or cannot be discriminated by the NMR spectra.
Summary of Variants of Sulfated ␣-L-Fucans from Sea Urchin Egg Jelly-A variety of sulfated fucans have been described in marine algae (29 -31). These compounds are among the most abundant and widely studied of all sulfated polysaccharides of non-mammalian origin. The algal fucans have com-plex, heterogeneous structures. Their regular repeating sequences are not easily deduced; even high-field NMR is at the limit of its resolution, and complete description of their structure is not available at present (9,27). Recently, we isolated and characterized several sulfated ␣-L-fucans from echinoderms, mostly from sea urchin egg jelly. In contrast to the algal fucans, these sea urchin polysaccharides have simple, linear structures composed of well defined repeating units of oligosaccharides (5-7).

Structural Features in the Sea Urchin Polysaccharides That Confer Finer Specificity of Recognition in the Sperm Acrosome
Reaction-Sulfated polysaccharides from sea urchin egg jelly are responsible for inducing the sperm acrosome reaction, which is an obligatory event for fertilization (5)(6)(7). Shortly after fertilization, the sulfated ␣-fucan disappears (32), which indicates that it has no further role in embryo development. These polysaccharides are species-specific as inducers of the sperm acrosome reaction and may represent one of the barriers that prevent interspecific fertilization.
We have now fully characterized eight sulfated polysaccharides from the egg jellies of seven species of sea urchins (Fig. 7). We can now formulate questions such as follows. What are the common structural features among these polysaccharides? Can we identify the structures that confer finer specificity of recognition in the acrosome reaction?
Clearly, as we examine the eight structures shown in Fig. 7, the common feature shared by these polysaccharides is always the occurrence of 2-O-sulfation at the first unit of the oligosaccharide repeating sequence. In this way, the sea urchin S. franciscanus, which contains a sulfated fucan composed exclusively of the common 2-O-sulfated ␣-L-fucose unit (Fig. 7G), has a less strict species specificity in sperm recognition of sulfated polysaccharide. The potency of acrosome reaction induction clearly depends on the extent of 2-O-sulfation in the chain of 3-linked ␣-fucose units (7).
As a distinctive feature for a different polysaccharide backbone, the sea urchin E. lucunter synthesizes sulfated ␣-L-galactan (Fig. 7H) instead of sulfated ␣-L-fucan (5). However, the majority of the sea urchin species contain sulfated ␣-fucans with increased complexity due to variable 2-O-and 4-O-sulfation of their oligosaccharide repeating units as well as 133 or 134 glycosidic linkage. In the case of a species enriched in 4-O-sulfated units, as exemplified by S. purpuratus (Fig. 7, D  and E), a more strict species specificity is observed than in S. franciscanus, and the sperm react only with homologous polysaccharide or, to a lesser extent, with heterologous 3-linked The two new species of sea urchins we have now studied allow a more in depth analysis concerning the species specificity of sulfated ␣-fucans as inducers of the acrosome reaction in echinoderms. The sulfated ␣-fucans from these species contain two consecutive 2-O-sulfated fucose residues, which alternate either with two unsulfated or 2-O-sulfated residues (in S. droebachiensis) or with two 4-O-sulfated fucose units (in S. pallidus). Therefore, analysis of the species specificity of the acrosome reaction between these two species will definitively demonstrate that the arrangement of the oligosaccharide repeating unit determines the sperm reactivity.
In our previous studies, we quantified the proportion of sperm that underwent the acrosome reaction after incubation with sulfated polysaccharides using microscopic examination (5-7). This approach is not possible in the case of the new species of sea urchins due to the extremely pointed tip of S. droebachiensis sperm. We overcame this limitation by measuring fertilization successes among three species of Strongylocentrotus (Table V). We were able to identify the contribution of the sperm acrosome reaction to the interspecific fertilization of these species by comparison of the ratio of fertilization success after and before pre-reaction of the sperm with conspecific egg jelly. For conspecific fertilization, this ratio is ϳ1.0, as expected, but increases up to 3.67 and 6.67 in the heterospecific crosses. This indicates that the induction of the sperm acrosome reaction by the egg jelly sulfated fucan is the major limitation for interspecific fertilization between S. droebachiensis and S. pallidus. Sperm of S. pallidus are slightly more potent than those of S. droebachiensis in achieving heterospecific fertilization without pre-activated sperm, indicating a slightly lower species specificity (Table V). We cannot determine whether this is a consequence of differences in the position of the glycosidic linkage (3-linked in S. pallidus and 4-linked in S. droebachiensis) or in the sulfation pattern of the tetrasaccharide repeating unit.
For S. purpuratus eggs, we still did not detect fertilization after pre-reaction of the sperm with conspecific egg jelly. Therefore, additional steps of gamete interaction, in addition to induction of the sperm acrosome reaction, prevent interspecific fertilization of S. purpuratus eggs by S. droebachiensis or S. pallidus sperm. For example, the binding of sperm to the eggs could be prevented by divergent evolution of the protein bindin (see Ref. 33 and references therein).
Overall, the experiments summarized in Table V indicate that the sulfated ␣-fucans from the egg jellies of S. pallidus and S. droebachiensis induce the acrosome reaction in homologous (but not heterologous) sperm. This was confirmed by recent assays of acrosomal exocytosis using immunofluorescence microscopy and anti-bindin antibody. 4 Again, the immunological staining of sperm after incubation with the purified sulfated ␣-fucans demonstrated that the egg jelly polysaccharides induce the acrosome reaction in homologous (but not heterologous) sperm. This is the major limitation for interspecific fertilization between these two species of sea urchins. It is interesting, and suggestive of adaptation, that these two closely related species, which co-occur over a huge geographic range, show such a strong specificity early in the cascade of gamete recognition events.
Two Sulfated ␣-Fucan Isotypes in a Single Species of Sea Urchin-We have extended to S. droebachiensis our observation in S. purpuratus (6) that individual females spawn eggs possessing only one of two sulfated ␣-L-fucan isotypes (Fig. 1, A  and B). As in S. purpuratus, both S. droebachiensis isotypes induce the acrosome reaction with similar potency in homologous sperm, as revealed by the immunofluorescence microscopy assay. It appears that in S. droebachiensis, one of the isotypes does not occur or occurs at lower frequencies in a population from a different ocean. Additional studies with a larger number 4 C. H. Biermann, unpublished data.  The two sulfated ␣-fucan isoforms of S. droebachiensis have well defined sulfation patterns and are not a consequence of variable degrees of sulfation (Fig. 7, A and B). The inheritance of such sulfation patterns is unknown. We expect that they are produced by site-specific sulfotransferases by analogy with the extensive studies on the biosynthesis of mammalian glycosaminoglycans. Sulfated fucan II requires a single sulfotransferase, but sulfated fucan I requires two sulfotransferases, one that recognizes the first ␣-fucose residue of the repeating sequence and a second that recognizes the 2-O-sulfated fucose unit and sulfates the second residue. 5 Of course, we cannot exclude unique metabolic pathways, as reported for the biosynthesis of a sulfated ␣-L-galactan from ascidians (34,35). For example, an alternative to explain the presence of either sulfated fucan I or II in separate females of S. droebachiensis is to postulate that, in both types of females, all fucose residues become 2-O-sulfated, but in females containing sulfated fucan I, specific sulfatases remove the sulfate esters from the third and fourth residues.
Another noteworthy observation is that S. droebachiensis and A. lixula, unrelated sea urchin species from the Arctic and tropical Atlantic Oceans, respectively, synthesize sulfated ␣-fucans with the same repeating structure (Fig. 7A). Our recent experiments (not shown) with immunological staining of S. droebachiensis sperm with anti-bindin antibody after incubation with the purified polysaccharides indicate that A. lixula sulfated fucan is indeed equivalent to S. droebachiensis sulfated fucan I in its physiological activity in vitro. According to phylogenetic analysis, these two species diverged ϳ200 million years ago (36). The species S. droebachiensis, S. pallidus, and S. purpuratus diverged 3.5 million years ago (37), but their egg jelly sulfated fucans are markedly different. Therefore, the genes involved in the biosynthesis of the sulfated fucans and their sperm receptors (8) did not evolve in concordance with the evolutionary distance between these echinoderms, but were possibly driven to diverge by natural selection where several species co-occur. a The values are the percentage of eggs fertilized at a sperm concentration of 200 sperm/l before (Ϫ) and after (ϩ) pre-reaction with conspecific egg jelly.