Sulfated Polysaccharides from the Egg Jelly Layer Are Species-specific Inducers of Acrosomal Reaction in Sperms of Sea Urchins

Abstract

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 α-L-galactan. The species Arbacia lixula and Lytechinus variegatus contain linear sulfated α-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. 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 α-L-galactan. The species Arbacia lixula and Lytechinus variegatus contain linear sulfated α-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. INTRODUCTIONSuccessful 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 (1Tyler A. Tyler B.S. Boolootian R. Physiology of Echinodermata. Interscience Publication, New York1966: 683-741Google Scholar). First, males and females must synchronize the time of release of their gametes (2Starr M. Himmelman J.H. Therriault J.C. Mar. Ecol. Prog. Ser. 1992; 79: 275-287Crossref Google Scholar). 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 (3Trimmer J.S. Vacquier V.D. Annu. Rev. Cell Biol. 1986; 2: 1-26Crossref PubMed Scopus (72) Google Scholar, 4Foltz K.R. Lennarz W.J. Dev. Biol. 1993; 158: 46-61Crossref PubMed Scopus (76) Google Scholar), 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 (3Trimmer J.S. Vacquier V.D. Annu. Rev. Cell Biol. 1986; 2: 1-26Crossref PubMed Scopus (72) Google Scholar, 4Foltz K.R. Lennarz W.J. Dev. Biol. 1993; 158: 46-61Crossref PubMed Scopus (76) Google Scholar).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 (5Suzuki N. Zool. Sci. 1990; 7: 355-370Google Scholar). Previous attempts to identify the acrosome reaction inducer in sea urchin egg jelly have suggested that all the activity resides in the sulfated fucan (6SeGall G.K. Lennarz W.L. Dev. Biol. 1979; 71: 33-48Crossref PubMed Scopus (202) Google Scholar, 7SeGall G.K. Lennarz W.L. Dev. Biol. 1981; 86: 87-93Crossref PubMed Scopus (85) Google Scholar). 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 (6SeGall G.K. Lennarz W.L. Dev. Biol. 1979; 71: 33-48Crossref PubMed Scopus (202) Google Scholar, 7SeGall G.K. Lennarz W.L. Dev. Biol. 1981; 86: 87-93Crossref PubMed Scopus (85) Google Scholar). Recent studies suggest that a glycoprotein or peptide, tightly associated with the sulfated fucan, was also involved in acrosome reaction induction (8Shimizu T. Kinoh H. Yamaguchi M. Suzuki N. Dev. Growth & Differ. 1990; 32: 473-487Crossref Scopus (31) Google Scholar, 9Mikami-Takei K. Kosakai M. Isemura M. Suyemitsu T. Ishihara K. Schmid K. Exp. Cell Res. 1991; 192: 82-86Crossref PubMed Scopus (11) Google Scholar, 10Keller S.H. Vacquier V.D. Dev. Biol. 1994; 162: 304-312Crossref PubMed Scopus (41) Google Scholar, 11Bonnell B.S. Keller S.H. Vacquier V.D. Chandler D.E. Dev. Biol. 1994; 162: 313-324Crossref PubMed Scopus (39) Google Scholar).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.CONCLUSIONSpecificity 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 (4Foltz K.R. Lennarz W.J. Dev. Biol. 1993; 158: 46-61Crossref PubMed Scopus (76) Google Scholar).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 (29Barnes R.D. Invertebrate Zoology. W. B. Saunders Co., Philadelphia, PA1980: 1001-1102Google Scholar). 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 (8Shimizu T. Kinoh H. Yamaguchi M. Suzuki N. Dev. Growth & Differ. 1990; 32: 473-487Crossref Scopus (31) Google Scholar, 9Mikami-Takei K. Kosakai M. Isemura M. Suyemitsu T. Ishihara K. Schmid K. Exp. Cell Res. 1991; 192: 82-86Crossref PubMed Scopus (11) Google Scholar, 10Keller S.H. Vacquier V.D. Dev. Biol. 1994; 162: 304-312Crossref PubMed Scopus (41) Google Scholar, 11Bonnell B.S. Keller S.H. Vacquier V.D. Chandler D.E. Dev. Biol. 1994; 162: 313-324Crossref PubMed Scopus (39) Google Scholar). 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. INTRODUCTIONSuccessful 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 (1Tyler A. Tyler B.S. Boolootian R. Physiology of Echinodermata. Interscience Publication, New York1966: 683-741Google Scholar). First, males and females must synchronize the time of release of their gametes (2Starr M. Himmelman J.H. Therriault J.C. Mar. Ecol. Prog. Ser. 1992; 79: 275-287Crossref Google Scholar). 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 (3Trimmer J.S. Vacquier V.D. Annu. Rev. Cell Biol. 1986; 2: 1-26Crossref PubMed Scopus (72) Google Scholar, 4Foltz K.R. Lennarz W.J. Dev. Biol. 1993; 158: 46-61Crossref PubMed Scopus (76) Google Scholar), 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 (3Trimmer J.S. Vacquier V.D. Annu. Rev. Cell Biol. 1986; 2: 1-26Crossref PubMed Scopus (72) Google Scholar, 4Foltz K.R. Lennarz W.J. Dev. Biol. 1993; 158: 46-61Crossref PubMed Scopus (76) Google Scholar).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 (5Suzuki N. Zool. Sci. 1990; 7: 355-370Google Scholar). Previous attempts to identify the acrosome reaction inducer in sea urchin egg jelly have suggested that all the activity resides in the sulfated fucan (6SeGall G.K. Lennarz W.L. Dev. Biol. 1979; 71: 33-48Crossref PubMed Scopus (202) Google Scholar, 7SeGall G.K. Lennarz W.L. Dev. Biol. 1981; 86: 87-93Crossref PubMed Scopus (85) Google Scholar). 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 (6SeGall G.K. Lennarz W.L. Dev. Biol. 1979; 71: 33-48Crossref PubMed Scopus (202) Google Scholar, 7SeGall G.K. Lennarz W.L. Dev. Biol. 1981; 86: 87-93Crossref PubMed Scopus (85) Google Scholar). Recent studies suggest that a glycoprotein or peptide, tightly associated with the sulfated fucan, was also involved in acrosome reaction induction (8Shimizu T. Kinoh H. Yamaguchi M. Suzuki N. Dev. Growth & Differ. 1990; 32: 473-487Crossref Scopus (31) Google Scholar, 9Mikami-Takei K. Kosakai M. Isemura M. Suyemitsu T. Ishihara K. Schmid K. Exp. Cell Res. 1991; 192: 82-86Crossref PubMed Scopus (11) Google Scholar, 10Keller S.H. Vacquier V.D. Dev. Biol. 1994; 162: 304-312Crossref PubMed Scopus (41) Google Scholar, 11Bonnell B.S. Keller S.H. Vacquier V.D. Chandler D.E. Dev. Biol. 1994; 162: 313-324Crossref PubMed Scopus (39) Google Scholar).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.