Glycobiology Advance Access originally published online on June 5, 2007
Glycobiology 2007 17(8):877-885; doi:10.1093/glycob/cwm058
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Expression of two different sulfated fucans by females of Lytechinus variegatus may regulate the seasonal variation in the fertilization of the sea urchin
2 Laboratório de Tecido Conjuntivo, Hospital Universitário Clementino Fraga Filho
3 Programa de Glicobiologia, Instituto de Bioquímica Médica
4 Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Caixa Postal 68041, Rio de Janeiro, RJ 21941-590, Brazil
1 To whom correspondence should be addressed; e-mail: pmourao{at}hucff.ufrj.br
Received on April 3, 2007; revised on May 15, 2007; accepted on May 23, 2007
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Abstract |
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 Top Abstract IntroductionResults and discussionMain conclusionsMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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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-
-L-Fucp-2(OSO3)-1 
3--L-Fucp-4(OSO3)-1 3--L-Fucp-2,4(OSO3)-1 3--L-Fucp-2(OSO3)-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 -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. Key words: acrosome reaction / egg jellies of sea urchins / reproductive cycle of echinoderms / sulfated polysaccharides
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Introduction |
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TopAbstractIntroductionResults and discussionMain conclusionsMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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Most echinoid species have distinct reproductive seasons in which gametes are released under the appropriate environmental conditions. Spawning in different sea urchin species is seasonal despite the wide variations observed in timing and extent of this event (Moore and Lopez 1972
; Ernest and Blake 1981; Lessios 1985). There is evidence that both biotic and abiotic factors, such as temperature, photoperiod, lunar phases, and phytoplanctonic blooms, can influence or even drive the periodicity and extension of echinoid reproductive cycles (Walker and Lesser 1998; Walker et al. 2001). Different spawning patterns and even intraspecific reproductive variability may occur among geographically separated populations. This could be explained by genetic divergence and/or phenotypic adaptation to external stimuli (Lessios 1984; Ventura et al. 2003). Therefore, gamete release in most echinoid species occurs in a restricted period of the year in order to ensure the ideal conditions for fertilization success and offspring survival (Lessios 1985).
Molecular recognition between egg and sperm is critical for the success of fertilization in sea urchins, preventing hybridization in these broadcast-spawning marine invertebrates. This process consists of two major steps: the well-known bindin system, which involves the interaction of the sperm protein bindin and its egg receptor, and the acrosome reaction, a signaling event that precedes the bindin(g) step in the fertilization cascade (Alves et al. 1997, 1998; Vilela-Silva et al. 1999; Hirohashi et al. 2002; Vilela-Silva et al. 2002; Biermann et al. 2004; Mourão 2007).
Previous studies from our laboratory have shown how the jelly coat operates as a species-specific mediator of fertilization. The sperm acrosome reaction is induced when a sperm with the correct receptor type contacts specific sulfated polysaccharides in the egg jelly. This reaction exposes the protein bindin outside of the sperm tip, which then reacts with a matching egg membrane receptor. The sulfated polysaccharides have been isolated from the egg jellies of several sea urchin species and structurally characterized by our group. These polymers have an unusual structure, composed of linear chains of L-fucose or L-galactose units, with well-defined repetitive units. The specific pattern of sulfation and the position of the glycosidic bond vary among the sulfated polysaccharides from each species of sea urchins. This refinement of the saccharide chain and its sulfation pattern regulate the species specificity of this signaling event (Alves et al. 1997, 1998; Vilela-Silva et al. 1999; Hirohashi et al. 2002; Vilela-Silva et al. 2002; Biermann et al. 2004; Mourão 2007). Thus, small structural changes in glycans can modulate an entire system of gamete recognition in sea urchins. These findings represent a new example of highly specific biological signaling activity, one that enables sperm to discriminate chemically among egg cells.
Lytechinus variegatus was the first sea urchin species explored in our studies, in which we originally obtained one single sulfated fucan from its egg jelly, with the regular and repetitive sequence [3--L-Fucp-2(OSO3)-1 3--L-Fucp-4(OSO3)-1 3--L-Fucp-2,4(OSO3)-1 3--L-Fucp-2(OSO3)-1]n (Mulloy et al. 1994) (Figure 1A). However, after recurrent purifications over several years, we have observed the sporadic appearance of another sulfated polysaccharide in its egg jelly. This fact led us to perform a more refined investigation of the possible occurrence of an additional sulfated fucan isotype in L. variegatus egg jelly, as previously reported for Strongylocentrotus purpuratus (Alves et al. 1998) and Strongylocentrotus droebachiensis (Vilela-Silva et al. 2002).
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In this study, we isolated, purified, and determined the structure of this new sulfated fucan isotype. It has a simple structure, composed of 4-sulfated, 3-linked
-fucose units (Figure 1B). Interestingly, the appearance of this distinct sulfated polysaccharide is seasonal and its expression corresponds to a specific period of the reproductive cycle. This raises the possibility that the synthesis of this molecule could be influenced by environmental factors. Furthermore, in contrast to other sulfated fucan isotypes found in other sea urchin species, this sulfated fucan is unable to induce the acrosome reaction. The seasonal limitation on the expression of the egg jelly sulfated fucans with different potencies as inducers of the sperm acrosome reaction has never been observed before. |
Results and discussion |
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TopAbstractIntroductionResults and discussionMain conclusionsMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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Egg jellies of L. variegatus contain two distinct and seasonally variable sulfated fucans
The egg jellies from specimens of L. variegatus were collected monthly over the year. Crude polysaccharides were extracted from a pool of egg jellies obtained each month and then sulfated polysaccharides were purified by anion-exchange chromatography on a diethylaminoethyl (DEAE)-cellulose column (Figure 2A and B). The elution profile from the females collected during the spring, summer, and autumn, were the same, showing two fractions, and were represented in the panel by the one from the summer (Figure 2A). Surprisingly, for females from the winter (June–August in the southern hemisphere), we observed three fractions (Figure 2B).
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) and for sialic acid (
). The NaCl concentration (––) was estimated by conductivity. The fractions indicated by the horizontal bar were pooled, dialyzed against distilled water, and lyophilized. The purified polysaccharides were applied to a 0.5% agarose gel and the electrophoresis was run for 1 h in 0.05 M 1,3-diaminopropane:acetate (pH 9.0). The gel was fixed with 0.1% N-cetyl-N,N,N-trimethylammonium bromide solution. After 12 h, the gel was dried and stained with 0.1% toluidine blue in acetic acid:ethanol:water (0.1:1:5, v/v). SG, sialic acid-rich glycoconjugate; P1 and P2 are the two isotypes of the sulfated fucan.The polysaccharide eluted from the column at a low NaCl concentration corresponds to a sialic acid-rich glycoconjugate (SG in Figure 2), as indicated by the positive sialic acid (closed circles) and metachromatic assays (open squares). A second polysaccharide (denominated as P1), eluted from the column at 1.2 M NaCl, shows a positive metachromatic assay but is nonreactive for sialic acid. These two polysaccharides are regularly found in the egg jellies collected during the year. However, the egg jellies collected during winter present a third sulfated polysaccharide, which elutes from the column at a high NaCl concentration (denominated as P2, Figure 2B).
The purity of the polysaccharides obtained from the anion-exchange chromatography was confirmed by agarose gel electrophoresis, which showed single bands for each compound (Figure 2C). The sialic acid-rich glycoconjugate and the sulfated fucan denominated as P1 showed the same electrophoretic migration irrespective of the season of the year in which the egg jellies were collected. However, the sulfated polysaccharide denominated as P2, which is expressed exclusively during the winter, showed a different pattern of electrophoretic migration, suggesting a distinct structure. Chemical analysis of the purified sulfated polysaccharides P1 and P2 revealed fucose as the only constituent sugar and hereafter are denominated as sulfated fucans. The two sulfated fucan isotypes have a similar average molecular size, estimated as approximately 290 kDa by polyacrylamide gel electrophoresis (not shown).
The sulfated fucan isotype expressed during winter has a single 4-sulfated, 3-linked repetitive unit
The structure of the sulfated fucans was investigated using nuclear magnetic resonance (NMR) spectroscopy. The spectra of the sulfated fucan P1 fit precisely with the ones we reported previously (Mulloy et al. 1994), confirming that this polysaccharide is composed of the repetitive sequence [3--L-Fucp-2(OSO3)-1 3--L-Fucp-4(OSO3)-1 3--L-Fucp-2,4(OSO3)-1 3--L-Fucp-2(OSO3)-1]n (Figure 1A). In contrast, the 1H spectrum of the sulfated fucan P2, which is expressed exclusively during winter, shows a single anomeric signal at 5.1 ppm and a methyl signal at 1.25 ppm (Figure 3A and B), compatible with a linear homopolymer of -fucopyranoside residues. For comparative purposes, we show in Figure 3C the 5.6–4.3 ppm region of the 1H NMR spectrum of sulfated fucan P1, which clearly displays four anomeric signals, in contrast to the simplicity of the 1H spectrum of the sulfated fucan P2.
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We conducted further NMR experiments in order to elucidate the structure of the yet unknown sulfated fucan P2. All sulfated fucan protons could be assigned in the 1H-COSY (correlation spectroscopy) (Figure 4A) and TOCSY (total correlated spectroscopy) (not shown) spectra. The cross-peaks show unambiguously the correlation between the spin systems and the 1H chemical shifts are presented in Table I. The 1H/13C HMQC (heteronuclear multiple quantum coherence) spectrum (Figure 4B) shows six major peaks and was interpreted using the information obtained in the COSY and TOCSY spectra, yielding the 13C-chemical shifts pointed out in Table II. Sulfation position was clearly deduced by the strong downfield chemical shift (approximately 0.7 ppm) of H4 in comparison with nonsulfated protons signals, indicating that sulfation was restricted to this position. Moreover, the strong downshift of C3 indicates 3-linked residues.
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Overall, these data indicate that during winter, L. variegatus females express in their egg jellies a distinct sulfated fucan isotype, composed of 4-sulfated, 3-linked
-L-fucopyranosyl units (Figure 1B), whereas the isotype composed of a regular repetitive tetrasaccharide unit (Figure 1A) is expressed over all seasons of the year.
The two sulfated fucan isotypes differ markedly in their potencies to induce the acrosome reaction in the sea urchin sperm
In order to verify whether the two sulfated fucan isotypes could be involved in the regulation of sea urchin fertilization, we tested their effects as inducers of the acrosome reaction in homologous sperm (Figure 5A and B). The sperm were examined by fluorescent microscopy after staining with rhodamine phalloidin (for actin) + 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) (for the nucleus). Typical reactive and nonreactive sperm are shown in Figure 5B. The sulfated fucan expressed exclusively during winter (isotype P2) did not induce the acrosome reaction in L. variegatus sperm even at the highest concentration tested (Figure 5A). The sperm responded to this sulfated fucan in the same way as in the negative control with the sea water. In contrast, sulfated fucan P1, which is composed of regular tetrasaccharide-repeating units, was a potent inducer of the acrosome reaction, almost as much as the crude homologous egg jelly from the sea urchin (Figure 5A).
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), sulfated fucan P1 (
) dissolved in sea water were incubated with homologous sperm of the sea urchin, and the acrosome reaction was detected using fluorescent phalloidin (see the Methods and materials section); controls in the absence of acrosome reaction inductors (
). Approximately 100–150 sperm were scored per data point. (B) Confocal images of L. variegatus sperm stained with rhodamine phalloidin and DAPI after incubation with egg jelly (100 µg fucose/mL). We simultaneously collected red (phalloidin, for actin), blue (DAPI, for nucleus), and transmitted light channels. The arrows point to the red rod that represents acrosomal filamentous actin, indicating positive acrosome reaction (R); and the red dot in the sperm head indicating no reaction (NR).Previously we reported the occurrence of sulfated fucan isotypes in two sea urchin species, namely S. purpuratus (Alves et al. 1998
) and S. droebachiensis (Vilela-Silva et al. 2002). In these cases, the individual females from each species express only one of the two possible isotypes. Furthermore, in contrast to the present results, the sulfated fucan isotypes have similar potency as inducers of the acrosome reaction in homologous sperm. We were not able to detect seasonal variation in their expression, although it was difficult to collect egg jellies from these species over the entire year. For S. droebachiensis, we observed a geographic variation in the expression of the sulfated fucan isotypes, but again, they showed similar potency as inducers of the acrosome reaction (Vilela-Silva et al. 2002; Biermann et al. 2004).
Individual females express predominantly a single sulfated fucan isotype during winter
The presence of two sulfated fucan isotypes in the egg jelly of L. variegatus during winter was observed when we analyzed a pool of egg jellies obtained from different females (Figure 2B). On the basis of these data, we could not assert whether individual females express a single isotype or a mixture of the two isotypes. We thus proceeded to the purification of the sulfated fucans collected separately from individual females (Figure 6). Of 70 females obtained during summer (December–March in the southern hemisphere), all showed, on anion-exchange chromatography, exclusively the sulfated fucan P1, besides the sialic acid-rich glycoconjugate, as exemplified in Figure 6D. Of 45 females collected during winter (June–August in the southern hemisphere), which spawned enough egg jelly for the analysis of the glycoconjugates by anion-exchange chromatography, 28 expressed predominantly isotype P2 of the sulfated fucan, whereas 17 secreted isotype P1, besides the sialic acid-rich glycoconjugate, as exemplified in Figure 6A–C.
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The amounts of egg jelly spawned by sea urchin females and of their constituent sulfated polysaccharides vary along the year
The natural physiological role of sulfated fucans in sea urchin egg jelly is the induction of the sperm acrosome reaction. The appearance of a biologically inactive isotype of the sulfated fucan in the egg jelly of the sea urchin during winter may be related to regulation of the fertilization process. We tried to evaluate whether the amounts of egg jellies spawned by the sea urchin females and of their constituent sulfated polysaccharides vary along the year. These variations during the reproductive cycles of gametogenesis in the species may indicate some connection with the appearance of the biologically inactive isotype of the sulfated fucan. We analyzed the egg jellies from individual females collected throughout the year. As seen in Figure 7, the dry weight of egg jellies spawned per female (open circles) and the amounts of sulfated polysaccharide (closed circles) varied greatly throughout the year, showing two peaks of high production. We noticed two periods of decreased production of egg jelly and of the sulfated polysaccharides between April and June and between August and October.
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) and of their sulfated polysaccharides (
) during these months are also represented in the panel and the expression of sulfated fucans P1 and P2 is indicated inside rectangles. Winter and summer months indicated are in the southern hemisphere. Water temperature was obtained from Godoy et al. (2002)Interestingly, McCarthy and Young (2002)
have shown that L. variegatus from Florida presents two distinct reproductive seasons during the year. Even though that population originated from the North Atlantic Ocean, its reproductive cycle resembles the occurrence of two peaks in the production of egg jellies and of their sulfated polysaccharides observed for the sea urchins of the same species but collected in the South Atlantic Ocean.
We tried to correlate these variations with some kind of environmental factor. No correlation was observed between the production of egg jelly by the sea urchins and pH, density, salinity, and concentration of O2 in the sea water or the amounts of rain fall in the period (Godoy et al. 2002). The temperature of the sea water (closed lozenges in Figure 7) in the same area where the females of sea urchins were collected did not show a correlation with the sum of sulfated polysaccharides or the amount of egg jelly spawned by the females. Nevertheless, we observed that the appearance of the isotype P2 correlates with the lowest temperatures of the winter months (June–August in the southern hemisphere). Therefore, the temperature of the sea water could be an environmental factor that induces L. variegatus females to express a different sulfated fucan isotype in their egg jelly. However, this proposition requires more direct evidence, and also other environmental factors should be considered.
We cannot rule out the possibility that small amounts of isotype P2 of the sulfated fucan might be present in the egg jellies collected during summer but were undetectable by our methodology owing to the high concentrations of isotype P1. In this case, our results do not reflect the synthesis of a new sulfated fucan by the sea urchin females during winter but rather the inhibition of the production of isotype P1 in some females. In order to rule out this possibility, we applied high concentrations of the egg jelly collected during summer to a DEAE–cellulose column (150 mg instead of 50 mg used in the experiments in Figure 2A). The fractions eluted in the range of NaCl concentrations required to elute isotype P2 from the column were pooled and analyzed by 1H NMR spectroscopy. No isotype P2 of the sulfated fucan was detected (data not shown). This experiment rules out the presence of this polysaccharide in the egg jelly during summer.
Also, it may be possible that there are two populations of L. variegatus; one produces sulfated fucan P1 through the year and other produces P2 only in winter. In this case, the sea urchins collected in different seasons were selected for a particular subpopulation. In fact, many sea urchin species migrate to tide pools prior to spawning. The difference between P1- and P2-producing populations may be their reproductive cycle. In this case, the sulfated fucans act as a reproductive barrier not only for interspecies fertilization but also for intraspecies fertilization. We cannot clarify this aspect because the acrosome reactions were performed exclusively with sperm collected during summer. Whatever the truth is, it will be of interest to investigate the biology of these two sulfated fucans in future studies. Environment cues may change the gene expression that switches sulfate patterns in the polysaccharide. Alternatively, changes in the structure of the sulfated fucan may drive speciation (Biermann et al. 2004).
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Main conclusions |
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TopAbstractIntroductionResults and discussionMain conclusionsMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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The egg jelly of the sea urchin L. variegatus contains a 3-linked sulfated fucan, composed of regular, repetitive tetrasaccharide units, which are defined by a specific pattern of sulfation at 2- and 4-positions. This sulfated fucan induces the acrosome reaction in homologous sperm, a signaling event that precedes the bindin step in the fertilization of the invertebrate. We have now isolated, purified, and determined the structure of a new sulfated fucan isotype from the egg jelly of the same sea urchin. The polysaccharide has a simple structure, composed of 4-sulfated, 3-linked
-fucose units. This sulfated fucan isotype is expressed by females during a narrow period of the year, close to winter, and is inactive as an inducer of the acrosome reaction in homologous sperm. We predict that females expressing this sulfated fucan isotype in their egg jelly are infertile since they are unable to induce the exposure of bindin in the tip of the approaching sperm, as in the case of females that express the preponderant sulfated fucan isotype. In conclusion, we propose that the occurrence of two isotypes of sulfated fucan in the egg jelly of the sea urchin L. variegatus, which differ in their biological activity, may be involved in the periodicity of the reproductive cycle of the invertebrate. |
Materials and methods |
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TopAbstractIntroductionResults and discussionMain conclusionsMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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Extraction of sulfated fucans from the egg jelly of sea urchins
Adults of L. variegatus were collected in Guanabara Bay (Rio de Janeiro, Brazil) and gametes spawned by intracelomic injection of 0.5 M KCl (1 mL/animal). Female gametes were collected in Millipore-filtered (Billenica, MA) sea water and the egg jelly was separated by pH shock, as described previously (SeGall and Lennarz 1979
). The acidic polysaccharides were extracted from the egg jelly by papain digestion and partially purified by ethanol precipitation, as described previously (Albano and Mourão 1986). Both, egg jelly and sulfated polysaccharides, were obtained as dry weight/individual female.
Purification of the sulfated fucans
The crude polysaccharides from the egg jelly (approximately 50 mg) were applied to a DEAE–cellulose column (15x2 cm), equilibrated with 50 mM sodium acetate (pH 5.0), and washed with 50 mL of the same buffer. The column was eluted by linear gradient prepared by mixing 100 mL of 50 mM sodium acetate (pH 5.0) with 100 mL of 3.0 M NaCl in the same buffer. The flow rate of the column was 12 mL/h and fractions of 2.4 mL were collected. Fractions were checked for fucose and sialic acid by the Dubois et al. (1956) reaction and by the Ehrlich assay (Kabat and Mayer 1971), respectively, and also by their metachromasia (Farndale et al. 1986). The NaCl concentration was estimated by conductivity. Fractions containing the sulfated polysaccharides and the sialic acid glycoconjugate were pooled, dialyzed against distilled water, and lyophilized. In some experiments, the sulfated polysaccharides (approximately 15 mg) were purified on a MONO-Q FPLC column (HR5/5) instead of the DEAE–cellulose. The column was equilibrated in 20 mM Tris–HCl (pH 8.0) and developed with a linear gradient of 0–4.0 M NaCl in the same solution. The flow rate of the column was 0.5 mL/min and fractions of 0.5 mL were collected and assayed by metachromasia (Farndale et al. 1986).
Agarose and polyacrylamide gel electrophoresis
Sulfated fucans were analyzed by agarose gel electrophoresis. The sample (approximately 15 µg) was applied to a 0.5% agarose gel and run for 1 h at 110 V in 0.05 M 1,3-diaminopropane-acetate (pH 9.0). The sulfated polysaccharides in the gel were fixed with 0.1% N-cetyl-N,N,N-trimethylammonium bromide solution. After 12 h, the gel was dried and stained with 0.1% toluidine blue in acetic acid/ethanol/water (0.1:5:5, v/v). The average molecular weights of the sulfated fucans were estimated by polyacrylamide gel electrophoresis. Samples (approximately 15 µg) were applied to a 6% polyacrylamide slab gel and run for 1 h at 100 V in 0.06 M sodium barbital (pH 8.6). The gel was stained with 0.1% toluidine blue in 1% acetic acid and washed overnight in 1% acetic acid.
NMR experiments
1H and 13C spectra of the sulfated fucan were recorded using a Bruker DRX 600 apparatus with a triple resonance probe (Bruker Bioscience Corporation, Billerica, MA). About 3 mg of the sample was dissolved in 0.5 mL of 99.9% D2O (Cambridge Isotope Laboratory, Andover, MA). All spectra were recorded at 60 °C with water suppression by presaturation. COSY, TOCSY, and 1H/13C HMQC spectra were recorded using states–time proportion phase incrementation for quadrature detection in the indirect dimension. TOCSY spectra were run with 4096 x 400 points with a spin-lock field of approximately 10 kHz and a mixing time of 80 ms. HMQC spectra were run with 1024 x 256 points and globally optimized alternating phase rectangular pulses for decoupling. Chemical shifts are relative to external trimethylsilylpropionic acid at 0 ppm for 1H and to methanol for 13C.
Chemical analysis
Total fucose was measured by the method of Dische and Shettles (1948). The sugar composition of the sea urchin polysaccharide was determined by paper chromatography in 1-butanol/pyridine/water (3:2:1, v/v) for 48 h after acid hydrolysis of the polysaccharide (5.0 M trifluoroacetic acid for 5 h at 100 °C). The chromatogram was stained with silver nitrate.
Acrosome reaction assays
The method used to score the acrosome reaction using fluorescent phalloidin was slightly modified from Biermann et al. (2004) and Su et al. (2005). Sperm used in these experiments were obtained during summer months (December–March in the southern hemisphere) because of the difficulty of obtaining large quantities of sperm in other periods of the year. The results shown in the figures are averages from three different assays performed with a pool of sperm collected from at least four males. Sperm were collected undiluted, as described previously for the eggs, and stored on ice before dilution. They were then diluted 1:5 in HEPES-buffered (10 mM, pH 7.9, 9 °C) Millipore-filtered sea water. Twenty-five microliters of the sperm suspension were gently mixed with 50 µL of the test solution (egg jelly or purified sulfated fucan in filtered sea water). The hexose content of these solutions was quantified by the phenol–sulfuric acid assay (Dubois et al. 1956). After 5 min in ice, sperm were fixed by the addition of 350 µL of ice-cold 3.7% formaldehyde in Millipore-filtered sea water. All subsequent washing and staining steps were done at room temperature by gently pelleting the sperm in a centrifuge and re-suspending using pipettes with wide-bore tips or vortexing. After at least 30 min in the fixative, sperm were washed with 500 µL phosphate-buffered solution and stained with agitation for at least 2 h with 1 U of rhodamine phalloidin (Molecular Probes R415, Invitrogen Corporation, Carlsbad, CA) in 50 µL of 0.1 M glycine, 1 mg/mL bovine serum albumin, 0.02% sodium azide in phosphate-buffered saline, pH 7.4. Cells were then washed twice in 500 µL of phosphate-buffered saline and incubated with 30 µL of 0.5% DAPI for 6 min. Finally, cells were washed twice in 500 µL of phosphate-buffered saline and then re-suspended in 30 µL of 70% glyceraldehyde in the same solution, mounted in a thin layer, and the coverslip sealed. Sperm were scored blindly using a Zeiss Axioskop 2 plus fluorescent microscope. Photos were acquired from a Zeiss LSM 510 Meta confocal microscope (Jeha, Germany). We simultaneously collected red (phalloidin), blue (DAPI), and transmitted light channels.
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Conflict of interest statement |
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TopAbstractIntroductionResults and discussionMain conclusionsMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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None declared.
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Acknowledgments |
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TopAbstractIntroductionResults and discussionMain conclusionsMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Funda
ão de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), and the International Foundation of Science (IFS, for A.C.E.S.V.-S.). L.P.C. was an MSc student at Departamento de Bioquímica, Instituto de Química, Universidade Federal do Rio de Janeiro. We are grateful to Dr Maria Cristina Mello for revising the manuscript and to Dr Ana Paula Valente for help with the NMR spectra. |
Footnotes |
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* These two authors contributed equally to the work.
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Abbreviations |
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COSY, correlation spectroscopy; DAPI, 4',6-diamidino-2-phenylindole dihydrochloride; DEAE, diethylaminoethyl; HMQC, heteronuclear multiple quantum coherence; NMR, nuclear magnetic resonance; TOCSY, total correlated spectroscopy.
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References |
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TopAbstractIntroductionResults and discussionMain conclusionsMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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