(Received for publication, June 18, 1996, and in revised form, October 9, 1996)
From the Departamento de Bioquímica,
Instituto de Ciências Biomédicas, Universidade Federal do
Rio de Janeiro, Caixa Postal 68041, Rio de Janeiro, RJ,
21941-590, Brazil, the § National Institute for Biological
Standards and Control, Blanche Lane, South Mimms, Potters Bar,
Hertfordshire, EN6 3QG, United Kingdom, and the
¶ Laboratório de Ultraestrutura Celular Hertha Mayer,
Instituto de Biofísica Carlos Chagas Filho, Universidade
Federal do Rio de Janeiro,
Rio de Janeiro, RJ 21949-900 Brazil
We have characterized the fine structure of
sulfated polysaccharides from the egg jelly layer of three species of
sea urchins and tested the ability of these purified polysaccharides to
induce the acrosome reaction in spermatozoa. The sea urchin
Echinometra lucunter contains a homopolymer of 2-sulfated,
3-linked -L-galactan. The species Arbacia
lixula and Lytechinus variegatus contain linear sulfated
-L-fucans with regular tetrasaccharide
repeating units. Each of these sulfated polysaccharides induces the
acrosome reaction in conspecific but not in heterospecific spermatozoa.
These results demonstrate that species specificity of fertilization in
sea urchins depends in part on the fine structure of egg jelly sulfated
polysaccharide.
Successful fertilization by free-spawning organisms such as sea urchins can occur only if a series of constraints are overcome before the sperm ever makes contact with the egg (1). First, males and females must synchronize the time of release of their gametes (2). Once spawned, the sperm must find and interact with an egg of the correct species. A further event necessary for successful fertilization is induction of the acrosome reaction in the sperm (3, 4), which involves fusion of the acrosomal vesicle membrane with the plasma membrane. This results in exocytosis of the vesicle contents, which include proteases and bindin. Concomitantly, actin in the subacrosomal region of the sperm polymerizes and causes extension of the tip of the sperm. As a consequence of these two events, bindin is localized to the outside of the tip of the process where it can then interact with an egg protein (3, 4).
The acrosome reaction is induced when the sperms contact the egg jelly layer. The sea urchin egg is surrounded by a transparent gelatinous layer composed mainly of sulfated fucan, sialoprotein, and other glycoproteins or peptides (5). Previous attempts to identify the acrosome reaction inducer in sea urchin egg jelly have suggested that all the activity resides in the sulfated fucan (6, 7). In addition, these authors suggested that the jelly coat preparations of some species of sea urchins are totally species-specific and induce the acrosome reaction only in homologous sperm. On the basis of these observations, it was suggested that the specificity of induction of the acrosome reaction might reside in structural differences in the carbohydrate linkages and/or location and degree of sulfation of the polysaccharide (6, 7). Recent studies suggest that a glycoprotein or peptide, tightly associated with the sulfated fucan, was also involved in acrosome reaction induction (8-11).
In this study, we isolated, purified, and characterized the fine structure of the sulfated polysaccharides from the egg jelly coat of three species of sea urchins. These compounds have simple, well-defined repeating structures that from each species present a particular pattern of sulfate substitution. Purified polysaccharide from the egg jelly induces the acrosome reaction in sperm from the same species of sea urchin and not from different species. This result is of considerable significance for the study of fertilization processes since it is the first time that fully described carbohydrate structures have been shown to regulate part of the process at such a specific level.
Sulfated Polysaccharides from the Jelly Coat of Sea Urchin
ExtractionMature species of sea urchins were collected in Guanabara Bay (Rio de Janeiro, Brazil) and gametes were isolated by intracelomic injection of 0.5 M KCl (~5 ml/specimen). Eggs were collected in a solution containing 450 mM NaCl, 9 mM KCl, 48 mM MgSO4·7H2O, 10 mM CaCl2, and 6 mM NaHCO3. The egg jelly was separated by pH shock, as described previously (6). The acidic polysaccharides were extracted from the jelly coat by papain digestion and partially purified by cetylpyridinium chloride and ethanol precipitation as described (12).
PurificationThe crude polysaccharides (~100 mg) from the
jelly coat of the sea urchins were applied to a DEAE-cellulose column
(15 × 2 cm), equilibrated with 50 mM sodium acetate
buffer (pH 5.0), and washed with 250 ml of the same buffer. The column
was eluted in three different steps. Initially, the column was eluted
by a linear gradient prepared by mixing 50 ml of 50 mM
sodium acetate buffer (pH 5.0) with 50 ml of 1.0 M NaCl in
the same buffer. Then the column was washed with 100 ml of the sodium
acetate buffer containing 1.0 M NaCl. Finally, the column
was eluted by a linear gradient prepared by mixing 100 ml of 1.0 M NaCl with 100 ml of 5.0 M NaCl, both in the
same sodium acetate buffer. The flow rate of the column was 15 ml/h,
and fractions of 3.5 ml were collected in the different elution steps.
Fractions were checked for fucose (or galactose) and sialic acid by the
Dubois et al. reaction (13) and by the Ehrlich assay (14),
respectively, and by their metachromasia (15). The NaCl concentration
was estimated by conductivity. Fractions containing the sulfated
-L-fucan or the sulfated
-L-galactan were
pooled, dialyzed against distilled water, and lyophilized.
The DEAE-cellulose-purified sulfated polysaccharide (~40 mg) was applied to a Sephacryl S-400 column (90 × 1.5 cm) and eluted with 50 mM sodium acetate buffer (pH 5.0) at a flow rate of 8 ml/h. Fractions of 1.5 ml were collected and assayed by the reaction of Dubois et al. (13) and by their metachromasia (15). The column was calibrated using blue dextran and cresol red as markers of Vo and Vt, respectively.
Chemical AnalysesTotal galactose was measured by the method of Dubois et al. (13) and total fucose measured by the method of Dische and Shettles (16). After acid hydrolysis of the polysaccharides (5.0 M trifluoroacetic acid for 5 h at 100 °C), sulfate was measured by the BaCl2/gelatin method (17). The percentages of hexoses and 6-deoxyhexoses in the acid hydrolysates were estimated by paper chromatography in 1-butanol:pyridine:water (3:2:1, v/v) for 48 h and by gas-liquid chromatogaphy of derived alditol acetates (18). Optical rotations were measured with a digital polarimeter (Perkin-Elmer model 243-B).
Oxidation with L-Fucose DehydrogenaseFucose obtained by acid hydrolysis of the sulfated fucans (5.0 M trifluoroacetic acid for 5 h at 100 °C), and authentic samples of D- or L-fucose (20 µg of each) were incubated with 0.2 units of porcine liver L-fucose dehydrogenase, as described (19).
Oxidation with D-Galactose OxidaseGalactose obtained by acid hydrolysis of the sulfated galactan (see above) and authentic samples of D- or L-galactose (20 µg of each) were incubated with 1 unit of Dactylium dendroides D-galactose oxidase, as described (20).
Agarose Gel ElectrophoresisSulfated polysaccharides were
analyzed by agarose gel electrophoresis as described (12). Purified
sulfated -L-fucan or sulfated
-L-galactan
(~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 buffer (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).
Desulfation of the sulfated polysaccharides was performed by solvolysis in dimethyl sulfoxide, as described previously for desulfation of other types of polysaccharides (12, 20). The native and desulfated polysaccharides (~5 mg) were subjected to three rounds of methylation as described (21), with the modifications suggested by Patankar et al. (22). The methylated polysaccharides were hydrolyzed with 6 M trifluoroacetic acid for 5 h at 100 °C, reduced with borohydride, and the alditols were acetylated with acetic anhydride:pyridine (1:1, v/v) (18). The alditol acetates of the methylated sugars were dissolved in chloroform and analyzed in a gas chromatography/mass spectrometry unit.
NMR Spectroscopy1H spectra were recorded at 500 MHz and 13C spectra at 125 MHz using a Varian Unity 500 spectrometer. The polysaccharide sample (~15 mg) was converted to the sodium salt by passage through a column 10 × 1 cm of DOWEX 50-X8 Na+ form, and all samples were dissolved in approximately 0.7 ml of 99.8% D2O. The spectra were recorded at 60 °C with suppression of the HOD signal by presaturation. 13C-spectra were recorded with full proton decoupling. Two dimensional double-quantum filtered COSY, TOCSY,1 and NOESY experiments were performed using pulse sequences supplied by Varian. TOCSY spectra were run with a spin-lock field of about 10 kHz and a mixing time of 80 ms; NOESY spectra were run with a mixing time of 100 ms. All chemical shifts were relative to internal or external trimethylsilylpropionic acid.
Effects of the Sulfated Polysaccharides as Inducers of the Acrosome ReactionThe effects of the various sulfated polysaccharides as inducers of the acrosome reaction in conspecific and heterospecific spermatozoa were assayed essentially as described (6). Sperms of each species were prepared by intracelomic injection of 0.5 M KCl (12). The reaction mixtures contain ~107 sperms and ~100 µg/ml (as galactose or fucose content) various sulfated polysaccharides in 200 µl of filtered sea water. After incubation at 20 °C for 3 min, an equal volume of cold 6% glutaraldehyde in sea water was added, and the acrosome reaction was monitored by direct counting, using transmission electron microscopy to identify the distinct morphological changes characteristic of the acrosome reaction, of at least 100 sperms for each point.
Sulfated
polysaccharides were purified from the jelly coat of three species of
sea urchins. Purification was achieved by anion exchange chromatography
on a DEAE-cellulose column (Fig. 1,
A-C). A peak rich in sialic acid was completely
eluted by ~1.0 M NaCl. A second peak, eluted at higher
salt concentration, corresponds to the sulfated polysaccharide. The
purity of these polysaccharides was confirmed by gel filtration
chromatography on Sephacryl S-400 (not shown).
We found that egg jelly coats of the species Lytechinus variegatus and Arbacia lixula contain sulfated L-fucans as has also been reported for other sea urchin species (6, 7), but surprisingly, the jelly coat of the sea urchin Echinometra lucunter contains a sulfated L-galactan (Table I).2 These sulfated polysaccharides have a similar molecular mass, as indicated by their migration on polyacrylamide gel electrophoresis (not shown). The variation in electrophoretic mobilities observed among sulfated polysaccharides from different species of sea urchins (Fig. 1D) may be accounted for in part by slight differences in the sulfate:sugar molar ratio, but this variation may also reflect other important structural differences (24), as discussed below.
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Methylation of
the native sulfated L-galactan from E. lucunter
yields 4,6-di-O-methylgalactose, whereas
2,4,6-tri-O-methylgalactose is the predominant methyl ether
derivative from the desulfated L-galactan (Table
II). This indicates a linear polysaccharide composed of
3-linked and 2-sulfated L-galactopyranoside residues, which
structure was confirmed by the 1H and 13C NMR
spectra. The 1H spectrum (Fig. 2,
A-C) was assigned by means of COSY and TOCSY spectra (not shown). On desulfation of the L-galactan,
alterations in chemical shifts of proton signals are consistent with
2-sulfation: 0.57 ppm for H2,
0.09 for H3, and
0.03 for H4 (Fig.
2, A-C), confirming that C2 is the position of
sulfation. The 13C spectrum of the native polysaccharide
(Fig. 2D) contains 6 resonances (indicating that the sample
is a homopolymer): an anomeric signal at 97.16 ppm, unsubstituted C-6
at 63.8 ppm, glycosylated or sulfated carbons at 76.1 and 75.9 ppm, and
two other ring carbons at 73.9 and 69.1 ppm. In the 13C
spectrum of the desulfated L-galactan (Fig. 2E),
a single substituted carbon at 77.25 ppm is observed, as expected.
Tentative assignments indicated in Fig. 2E are based on
comparison with literature values (25).
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The Egg Jelly of the Sea Urchins A. lixula and L. variegatus Contain Linear Sulfated
The structure of the sulfated
-L-fucan from the egg jelly coat of L. variegatus was described in our previous study (24). This sulfated
-L-fucan is essentially a linear polymer, composed of a
regular repeating sequence of 4 residues, as follows:
[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 (24).
The structure of the sulfated -L-fucan from the sea
urchin A. lixina has not previously been investigated. As
for the polysaccharide from L. variegatus, we observed that
the high-field 1H NMR spectrum of the sulfated
L-fucan from A. lixula contains four anomeric
residues in equal proportions by integration (Fig. 3A). Double quantum filtered COSY and TOCSY
spectra (not shown) confirm that the four anomeric residues correspond
to four spin systems, each consistent with
-fucose. The spin systems
can be traced, giving the values of Table III. Strong
downfield shifts of H2 of residues A and B relative to H2 of C and D
indicate that two of the residues are sulfated at C2. Thus, the
sulfated
-L-fucan from A. lixina also has a
tetrasaccharide repeat unit but consists of two residues sulfated at
the O-2 position and two unsulfated residues.
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The order of the four residues was determined by the NOESY spectrum
(Fig. 4). As in the NOESY spectra of the other fucan
from the sea urchin L. variegatus (24) or from the sea
cucumber (24, 26), cross-peaks can be seen from H1 of each residue to
protons on one and only one of the other residues (besides, of course, nOes to other protons in the same residue). This suggests that the four
residues make up a linear tetrasaccharide repeating unit, as in the
case for the other echinoderm fucans we have studied (24, 26). The
pattern of the nOes is, however, different. In the fucans from the sea
urchin L. variegatus (24) and from a sea cucumber (26)
studied previously, the major inter-residue nOes were between H1 and H3
of the next residue. In the sulfated L-fucan from A. lixula, nOes can be seen from each H1 to one of the H6 signals,
from H1 of residues A and C to the envelope containing signals from H4
of residues A and B (4.01 ppm), and from H1 of residues B and D to the
overlapping H4 signals of residues C and D (3.92 ppm) (Fig. 4). This
pattern of nOes is indicative of 14 linkages (27) rather than the
1
3 linkages previously seen in other similar compounds (24, 26) and
indicates the repeating 1
4-linked structure -B-D-C-A- in which two
consecutive 2-O-sulfated residues are followed by two
unsulfated residues to give the structure shown in Fig.
5A.
Methylation analysis (Table II) confirms the occurrence of 14
linkages in the sulfated L-fucan from A. lixula;
62% of 2,3-di-O-methylfucose and 38% of 3-methylfucose
were formed from the native polysaccharide. Although the proportions of
the methylated derivatives are not exactly as expected, they are
consistent with a polysaccharide composed of 4-linked fucose residues,
half of them sulfated at the O-2 position and half
unsulfated units.3
The 1H spectrum of the polysaccharide resulting from successive desulfation processes (Fig. 3, B and C) shows a reduction in the intensity of anomeric residues at 5.2-5.3 ppm and a corresponding increase in intensity at 5.04 ppm, indicating that the fully desulfated polysaccharide is a homopolymer of identical fucose residues, as expected from our proposed structure.
The 13C spectrum of the native -L-fucan from
A. lixula shows 4 signals of anomeric carbons (Fig.
3D). Most of the ring carbons resonate at about 69-71
ppm and overlap heavily. The 13C spectrum of the desulfated
polysaccharide (Fig. 3E) contains one major anomeric signal
at 103.1 ppm; again, most of the ring carbons overlap heavily. The
13C spectra corroborate our proposition that the sulfated
polysaccharide from A. lixula is composed of
-L-fucose units with a single type of linkage and a
tetrasaccharide repeat unit defined by a specific pattern of
sulfation.
Overall, the combination of chemical analysis, specific optical
rotation, methylation experiments and NMR spectroscopy has allowed us
to determine the fine structure of the sulfated polysaccharides isolated from the egg jelly coat of three species of sea urchins. The
sea urchin E. lucunter contains a homopolymer of sulfated -L-galactose, composed of 2-sulfated and 4-linked
-L-galactopyranosyl units (No. 2 in Fig.
5A). The sulfated
-L-fucans from A. lixula and L. variegatus are essentially linear
polymers, composed of a regular tetrasaccharide repeat unit defined by
the pattern of O-sulfation. The specific pattern of
sulfation varies in the two species. The
-L-fucan from
L. variegatus consists of two residues sulfated at the
O-2 position, one sulfated at both O-2 and -4 positions, and one residue sulfated at the O-4 position (No.
3 in Fig. 5A). The sulfated
-L-fucan from A. lixula consists of two
residues sulfated at the O-2 position and two unsulfated
residues (No. 1 in Fig. 5A). The fucose residues
are 3-linked in L. variegatus and 4-linked in A. lixula (compare Nos. 1 and 3 in Fig.
5A).
Once we had isolated, purified, and characterized the
fine structure of the sulfated polysaccharides from the egg jelly layer of three species of sea urchins, we were in a position to test the
ability of these polysaccharides to induce the acrosome reaction in
conspecific and heterospecific spermatozoa. Transmission electron microscopy can be used to differentiate sperms that have undergone the
acrosome reaction from those that have not, by clear and unambiguous morphological differences, so that the extent of the acrosome reaction
among a sample of sperm may be monitored by direct counting. Effectively, we observed a species-specific induction of the acrosome reaction in the sperms of the three sea urchin species (Fig.
5B). Thus, the sulfated -L-galactan from the
egg jelly coat of E. lucunter induces the acrosome reaction
exclusively in sperm of this species (No. 2 in Fig.
5B). Even the similar sulfated
-L-galactan from the ascidian Herdmania monus, which is also linear but
composed of 4-linked and 3-sulfated
-L-galactose units
(28), does not induce the acrosome reaction in this species of sea
urchin.
The acrosome reaction in sperms of L. variegatus is induced
exclusively by the sulfated -L-fucan from the egg jelly
coat of this species (No. 3 in Fig. 5B). Sulfated
-L-fucans from A. lixula or from the sea
cucumber Ludwigothurea grisea, although composed of regular
repeating tetrasaccharide units but with a different sulfation pattern
(compare Nos. 1, 3, and 4 in Fig. 5A), do not induce acrosome reaction in sperms of L. variegatus (No. 3 in Fig. 3B). We also
observed a conspecific but not heterospecific induction of the acrosome
reaction by the sulfated
-L-fucan from A. lixula (No. 1 in Fig. 5B).
Further purification of the sulfated -L-fucan from
L. variegatus on a gel filtration column yields a single and
narrow peak (Fig. 6), which induces the acrosome
reaction in sperms of this species of sea urchin at the same extent
reported in Fig. 5B. This experiment indicates that a
sulfated polysaccharide itself, and not a contaminant present in the
preparation, is in fact the inducer of acrosome reaction.
Specificity in fertilization is the result of a series of interactions between molecules located on the surfaces of the egg and of the sperm. This is especially relevant in free-spawning organisms and constitutes a barrier to prevent interspecific crosses, and consequently the formation of hybrids. It may be that the induction of acrosome reaction by sulfated polysaccharides from the egg jelly coat is the first level of recognition during fertilization in sea urchin species while the more specific interaction of egg receptor with sperm serves as a second level of recognition (4).
Our results indicate that the acrosome reaction in sea urchin is mediated by sulfated polysaccharide and in fact is regulated by the structure of the saccharide chain and its sulfation pattern. This constitutes an unusually clear-cut example of a biological event regulated by sulfated polysaccharides. Variations in the structure of these polymers among the species of sea urchins may represent one of the barriers which prevent interspecific crosses.
Approximately 900 species of sea urchins have been described (29). To act as a recognition molecule for the acrosome reaction in such a large number of species, the sulfated polysaccharides from the egg jelly must have a wide capacity for structural variation. In fact, an array of sulfated esters in the eight possible sulfation positions of the tetrasaccharide repeat units of a linear sulfated L-fucan (as those reported in Fig. 5) allows 255 combinations. Different positions of the glycosidic linkages (2-, 3-, or 4-linked), as we reported for the sulfated L-fucans from the sea urchins L. variegatus and A. lixula, increase the possible combinations to 765. In addition, the presence of sulfated L-galactan in some other species, for example E. lucunter, expands the variation possibilities for sulfated polysaccharides from the egg jelly of sea urchins.
Recent studies have suggested that glycoproteins or peptides, tightly associated with the sulfated polysaccharides of the egg jelly coat, are in fact the inducers of the acrosome reaction in sea urchins (8-11). It is unlikely that glycoproteins or peptides would resist the drastic protease digestion conditions we have used to extract sulfated polysaccharides from the egg jelly or would remain associated with the sulfated polysaccharides at the NaCl concentration we used to elute these polymers from the DEAE-cellulose column (Fig. 1A-C). But, if this is the case, and the sulfated polysaccharides are not the inducers of the acrosome reaction but the "carriers" of glycoproteins or peptides, which directly induce the process, our proposition is still valid. That is, there is a species-specific variation in the structure of the sulfated polysaccharide from the sea urchin egg jelly coat, and these polymers are either directly involved in the induction of the acrosome reaction or are specific "carriers" of acrosome reaction-inducing molecules. We believe the relative importance of these glycoproteins or peptides and of the sulfated polysaccharides as inducers of the acrosome reaction in sea urchins requires further investigation.
We thank Adriana A. Eira for technical assistance and Maria Cristina H.P. Lima (Central Analítica, NPPN, UFRJ) for the mass spectrometry analysis.