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Developmental regulation of lectin-binding patterns in Paracentrotus lividus gonads, gametes, and early embryos
Acta histochem. 92, 179-189 (1992) Gustav Fischer Verlag lena
Dipartimento di Biologia Animale ed Ecologia Marina dell'Universita', Messina*, and Istituto di Anatomia Comparata dell'Universita', Genova (Ita1ia)
Developmental regulation of lectin-binding patterns in Paracentrotus lividus gonads, gametes, and early embryos By ANTONIETTA CONTINI*, CARLA FALUGI and SALVATORE FASULO* With 5 Figures (Received April 9, 1991)
Summary By use of several lectins (ConA, WGA, SBA, GS I, PNA), a study was carried on gametes and developing embryos of the sea urchin Paracentrotus lividus. to investigate developmental changes in cell surface, leading to changements in cell-environment interactions. ConA, WGA, and SBA, with high affinity, bind to the vitelline membrane of unfertilized eggs, while PNA labelling at the same site is weak; GS I-binding is only present in the cytoplasm and cortical region of the unfertilized eggs. Immediately after fertilization, no ConA-binding is present in the membrane, while WGA- and SBA-binding molecules are located in the fertilization layer. In zygotes, 40 min after fertilization, ConA affinity sites were again present in both cytoplasm and cortical region. During cleavages and gastrulation, ConA binds to the blastomere cytoplasm and cortical region, to the intercellular matrix, and to the cytoplasm of mesenchyme cells. WGA binds to the cortical region of cleaving blastomeres, including the hyaline layer, up to the unhatched blastula. Then it labels the gastrula inner and outer surfaces. SBA binds to the blastomere membranes; no GS 1- and PNA-binding was detected during embryonic development. Sperms are bound by all the lectins, except GS I. Mannose and glucose conjugates are the most represented throughout the whole development of P. lividus, and their origin and locations are developmentally regulated. Galacto-residues are scarcely represented or are masked by other terminal sugars (e.g. sialic acid), and become functional during particular developmental events (cell movements).
Key words: development, lectins, glycoconjugates, sea urchin.
1. Introduction Cell surface glycoconjugates are known to play an important role in the regulation of cell interactions during development. Ascidian development was thoroughly studied by this point of view by O'DELL et al. (1973), ORTOLANI et al. (1977), ROSATI et al. (1978) who, by use of severallectins, found significant changements in oligosaccharide residues, mainly at the cell surfaces. Cell-surface oligosaccharides were also shown involved in the control of differentiation and growth in mammals (SKUTELSKY and BAYER 1983, MANN 1988). The authors found that every specific cell differentiation resulted in a specific oligosaccharide change on the cell surface, so changing the cell ability to interact with its environment in a specific manner. MOSCONA (1974) showed that the agglutination properties of ConA on cultured embryonic cells differ significantly, depending on embryonic stage, suggesting the possibility that ConA receptors are correlated with cell motility. In chick early development, an increase was found in surface sialic acid residues, suggesting an interaction between cell surface and cell environ12*
180
A. CONTINI, C. FALUGI and S. FASULO
ment differentiation. Lectin changes, during heart differentiation in mammals, were found to playa role in maintaining and modulating calcium ions fluxes, so that the glycocalyx may be hypothesized as implied in the control over numerous cell reactions (see MANN [1988] for review). A very studied model from this point of view is represented by echinoderm early development (see GIUDICE [1986] for review), where relevant transformations take place during short time lapses; sperm-egg reception is due to a glycoprotein (binding, GLABE et al. 1982); ConA affinity sites were found on the vitelline membrane (SCHMELL et al. 1977) and their implication in sperm-egg interactions was shown; by computer image analysis, they were localized in vivo on the outer side of the vitelline membrane (FALUGI et al. 1984); furthermore, it was shown that ConA inhibits the dispersion of the cortical granule contents (VACQUIER and O'DELL 1975); the saccharidic residues of the vitelline membrane proteins were characterized by analytical methods (GLABE and VACQUIER 1977 a, b); by agglutination experiments carried out with several lectins on free embryonic cells, glycoconjugates bound by ConA were detected on the cell surface, and those bound by WGA in intracellular sites (KRACH et al. 1974); and in vivo experiments were performed by injecting lectins in early embryos, blastula, and gastrula stages (SPIEGEL and SPIEGEL 1985; SOLURSH 1985). Although a great number of relevant findings on localizations and possible functions of glycoconjugates during echinoderm development is available in literature, these works were performed in different moments of the development of different echinoderm species, by use of different lectins. Moreover, most of the studies on pluricellular stages were performed on cultured disaggregated cells, devoided, by enzyme activity, of at least a part of their extracellular matrix. In this work, an attempt is made to investigate the origin and nature of different glycoconjugates during the early development of the sea urchin Paracentrotus lividus, both in vivo and in sections, by use of a panel of 5 labelled lectins. We found that mannose and glucose conjugates are present during the whole development from ovarian egg to pluteus; that their origin and distribution is developmentally regulated, accordingly with their functions, and that galacto-conjugates are mainly present during segmentation and morphogenetic movements.
2. Materials and methods Paraeentrotus lividus gametes were obtained in the laboratory from adult specimens, collected in the Gulf of Genova. Mature eggs were maintained in finger bowls containing sea water pasteurized and aerated overnight, and fertilized by sperms diluted according to the usual procedures. Some eggs were artificially activated by use of A 23187 Ca++ or Ca2+-ionophore, and some eggs were devoided of the inner cytoplasms by needle handling in Ca**-free sea water, to obtain ghosts. Part of gametes and embryos were treated by lectins diluted 11100 in sea water in vivo, for I h at 5°C, in the dark. Part of them were also embedded in historesin (BOH), cut 3 Ilm, and dried at room temperature for 2d. Lectins were employed to label oligosaccharidic residues: fluorescein isothiocyanate (ATC) labelled concanavalin A (ConA, Sigma. USA), from Coneanavalia ensiformis. with high affinity to carbohydrate residues of the nature of mannose and glucose; weat germ agglutinin (WGA) , labelled by either mc or horseradish peroxidase (HRP), with affinity to sialic acid and N-acetyl-O-glucosamine, HRP-Iabelled soy bean agglutinin (SBA) , with affinity to N-acetylgalactosamine and O-galactose; HRP-Iabelled Griffonia simplieifolia f (GS f), with affinity to cx-O galactose; and peanut agglutinin (PNA), labelled with either ATC or HRP, with affinity to galactose; 1-3-galactose-N-acetylgalactosarnine. All the last lectins were obtained from Kem-en-tee, OK. Slides carrying sections were washed in PBS (phosphate buffered saline, pH = 7.4) for 30 min. Lectins, diluted 11100 in PBS were put on sections, and incubated for I h in the dark and cold (5°C), in a humid staining-tray. The sections treated by lectins labelled with fluorescein were mounted with III = v/v glycerol/PBS and
Developmental regulation of lectin-binding patterns
181
observed with a Leitz microscope, equipped with an UV apparatus, and a filter set for fluorescein revelation. Fluorescence revealed the presence of lectin-binding sites. Slides treated with lectins labelled with HRP were stained by use of 30% diaminobenzidine (DAB) and 0.01 % H20 2 in PBS. The oxidation of the benzidine moiety by the peroxide-peroxidase reaction produces a brown staining at the sites of peroxidase activity. Slides were rinsed in buffer,dehydrated by alcohol, clarified with xylol, and mounted in balsam. Specificity controls were performed by incubating sections with lectins preadsorbed with the hapten sugars (respectively
3. Results In ovaries, ConA-binding was mainly present around immature oocytes, that were weakly stained at every stage of differentiation in tiny cytoplasmic granulations and around the nuclear envelope, while a strong fluorescence was present at the vitelline membrane, and in the tissue surrounding eggs. In controls performed by ConA preadsorbed with
Fig. I. P. lividus ovary. a FITC-ConA-binding sites; b toluidin blue staining of an ovarian oocyte. 311m sections; bar. t!, 10 11m.
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A. CONTINI, C. FALUGI and S. FASULO
Fig. 2. FlTC-WGA-binding sites in P. lividus ovary. Fluorescence is mainly located in forming egg envelopes. Moreover, some fluorescence is also present in cytoplasms and in ovarian tissues around maturing eggs. 3 f.tm sections; Bar !d 50 f.tm (a) and 20 f.tm (b).
In mature unfertilized eggs, ConA, WGA, SBA bound with high affinity to the outer surface of the vitelline membrane, with homogeneous distribution in fixed materials; mature egg ghosts showed the same distribution of ConA-affinity sites (Fig. 3b) on the vitelline membrane. Controls showed that ConA affinity sites in the vitelline membrane were still revealed after
183
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Fig. 3. FITC-ConA-binding sites in P. lividus eggs and embryos. a unfertilized egg, 10 min after in toto treatment, showing clusters of receptors; b a ghost of vitelline membrane obtained 5 min after ConA treatment; c in toto egg activated by A 23187 Ca ionophore; d 2 blastomeres embryo, 3 !!m section; e,f gastrula stages, 3 !!m sections. Bars ~ 50 !!m.
blastomeres appeared less stained, in a thin region (Fig. Sa). SBA-affinity sites were located on blastomere surfaces and in the extracellular matrix, while the cytoplasmic staining was weak, localized in the basal portion of the cytoplasm. GS 1- and PNA-labelling was weak and generally negative, also after sialidase treatment.
184
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Fig. 4. Lectin-binding sites in P. lividus eggs. a HRP-GS I-binding sites in unfertilized eggs; b HRP-SBAbinding sites in unfertilized egg; c HRP-WGA-binding sites in unfertilized eggs. Residues of the jelly-coat are also stained (arrows); d FITC-ConA-binding sites in zygote, 40 min after fertilization; e HRP-WGA-binding sites in the same stage;jHRP-SBA-binding sites in the same stage. 311m sections; bar ~ 100 11m (a, e,/), 50 11m (c, d), and 20 11m (b).
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Fig. S. Lectin-binding sites in P. lividus embryos and sperms. a HRP-WGA-binding sites in 4 blastomeres embryo; b HRP-SBA-binding in the same stage; c HRP-WGA-binding in early gastrula; d FITC-ConA-labelling in pluteus; e FITC-ConA-labelling in unreacted sperms;fHRP-WGA-staining in the same sample; g HRP-SBAstiaining in the same sample; h HRP-PNA-binding in the same sample; i negative HRP-GS I-staining in sperms.
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A. CONTINI, C. FALUGI and S. FASULO
Table 1: Lectin binding at different stages of P. Lividus development Lectin
ConA WGA GSI PNA SBA
oocyt.
++ ++
± ±
+
unfert. egg
fert. egg
segm.
gastr.
plut.
sperm
++ +
++ ++
5min
40 min
++ ++
+
++ ++
++ +
++ ++
+
+
+
+
+
± ±
+ +
++ strong binding; + binding; - no binding; ± scarce binding, or in different sites. Table 2: Binding of lectins in controls Treatment
before fertil
after fertil
ConNa-D-Glucose ConNa-D-mannose ConA PNNsialidase PNA SBNN-ac-a-D-galam SBA
+ +
++
WGAN-ac-~-D-glucam
WGA
++
+
++
±
+ +
++
+ +
++
ConAJa-D-glucose ConA preadsorbed with o:-D-glucose; ConAJa-D-rrumnose eonA preadsorbed with o:-Dmannose; PNNsialidase PNA binding on sections pretreated by sialidase; SBNN-ac-a-D-galam SBA preadsorbed with N-acetyl-o:-D-galactosamine; WGNN-ac-f3-D-glucam WGA preadsorbed with N-acetyl-~-D glucosamine. For other symbols, see Table I.
Since the stage of mesenchymal blastula and during gastrulation, ConA labelled the external surface of the embryos and the basal lamina of ectodennal cells. In gastrula stage, the extracellular matrix present in the mesenchyme migrating pathway was also stained, and affinity sites were present in the cytoplasm of some of the moving cells (Figs. 3e, f); binding to ECM was prevented in controls treated with ConA preadsorbed with lX-Dmannose. WGA and SBA bound to the external surface of embryos and, in a weaker way, to the ectodennal and mesenchymal cells, including the extracellular matrix (Fig.5b, arrow); no GS 1- and very weak PNA-binding was present during further development, while ConA-binding was present in all the cells of plutei and in the matrix around skeletal elements (Fig.5b, arrow); WGA bound to the matrix around skeletal elements in late gastrula and pluteus stages. During segmentation and gastrulation, ConA-binding was prevented by lX-D-mannose preabsorption. Lectins-binding results are shown in Table 1, control results in Table 2. Spenns were bound by all the lectins (mainly CanA and WGA), except GS I (Fig.5, Table 1).
4. Discussion In ovaries, the distribution of ConA- and WGA-binding sites shows that the corresponding glycoconjugates are produced by the cytoplasm of both oocytes and ovarian cells, that co-work in building egg
Developmental regulation of lectin-binding patterns
187
envelopes. Controls show that, during oogenesis, glycoconjugates present mannose, glucose, and sialic acid residues, while galacto-residues are masked by terminal sialic acid, and are probably inactive. In mature eggs, the positive staining of GS 1- and SBA-binding sites shows the liberation of previously masked galacto-residues, and the activation of their function (e.g. movement of the egg towards the genital pores). Furthermore, the "clustering" tendency of ConA receptors, found in unfertilized eggs, shows that the vitelline layer, although it is made up of a fibre network containing glycoproteins (GLABE and VACQUIER 1977 a), and has a certain fluidity, allowing rapid lateral movements of the glycoproteins bound by ConA. ConA-binding sites transiently disappear, after both fertilization by sperms and egg activation with A 23187 Ca**-ionophore. This finding is related to the fact that proteases released from the cortical granules cause destruction of sperm receptor glycoproteins (VACQUIER et al. 1972b), thus confirming the function as sperm receptors for the ConA-binding sites located in the vitelline layer. The results of lectin-binding on sections show that ConA-binding sites undergo significant changes during development, as they are first present in unfertilized eggs, disappear after cortical reaction, are again present 40 min after, with cytoplasmic origin, change localization and quantity at every stage, and are mainly present during segmentation and cell migration up to the metamorphosing pluteus, where their presence is maximum. These findings agree with those of OPPENHEIMER and MEYER (l982b), who, working on sea urchin embryo cultured cells, described temporal changes in ConA agglutination. In particular, ConA-binding sites are found in the skeletal spicules, where WGA-binding sites are also found around the skeletal elements. Such location may correspond to the presence of a large calcium-binding protein involved in spiculogenesis, found by IWATA and NAKANO (1986). Since the first developmental stages, the attachment of cells to the hyaline layer may be due to its nature as glycoconjugates (CITKOWITZ 1971, 1972); SPIEGEL et al. (1989) described extracellular matrix fibres, linking microvilli, to the hyaline layer and fertilization envelope. The microvillar region and the perivitelline material are labelled by use of ConA, WGA, and SBA: this fact shows that the linking complex may be formed by glycoconjugates able to modulate adhesion (to maintain the embryo in the correct position within the envelopes) and disaggregation (to allow changements in position of cells during segmentation movements). The trend to disaggregation is a property of ConA-binding molecules (MOSCONA 1974); furthermore, the ability of galacto-compounds to inhibit reaggregation is known (SCHNEIDER et al. 1978). In this light, the fact (shown by the SBA-Iabelling) that galacto-conjugates are present during segmentation indicates a slight displacement towards disaggregation of the equilibrium between aggregation and disaggregation, but this latter is controlled and modulated by the presence of glucose-conjugates, and by the absence of PNA-binding galacto-conjugates. Actually, aggregation and disaggregation are needed for cell movements during segmentation and gastrulation. Generally, morphogenetic movements are caused by the presence of glycoproteins in the extracellular matrix. For instance, fibronectin, that is a heavy MW galacto-protein of ECM is known to be involved in cell shape maintenance and changements, besides in cell movements. Its presence may correspond to the SBA pattern found in P. /ividus. as antigens that cross-react with antibodies against human plasma fibronectin have been detected between blastomeres and on the outer (SPIEGEL et al. 1980) and inner surfaces of gastrula (SPIEGEL et al. 1983), while KATOW et al. (1982) found fibronectin-like immunoreactivity only in the migratory pathway. At the same way, the distribution of lectins-binding sites found in this work in part agrees with the in vivo findings of other authors: LALLIER (1972) found that ConA injection into the blastocoele displays an inhibitory effect on embryonic echinoderm cell adhesion, limited to detachment of filopodia and retraction of the archenteron; SPIEGEL and SPIEGEL (1985), by in vivo injection of WGA in gastrulae, on the contrary, caused a binding only on the surface of the cells of the primary mesenchyme ring. Furthermore, our controls obtained by pre-adsorbing ConA with haptens agree with the result obtained by KATOW and SOLURSH (1982) by pretreating embryos in vivo with a-D-mannoside, while OPPENHEIMER and MEYER (I982b) showed that glucose compounds are responsible for cell adhesion. In conclusion, throughout P. lividus development, ConA- and WGA-binding sites were always found, while SBA-binding was only present during segmentation and gastrulation. ConA showed affinity for different saccharidic residues respectively before and after fertilization. PNA- and GS I-binding sites were seldom present, in correspondence with particular developmental events. This shows the prominent production in this species of mannose- (mainly after fertilization) and glucose-conjugates, in comparison with galacto-conjugates, that are present at the cell surfaces and in the ECM mainly during cell movements.
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Acknowledgments We would particularly like 10 thank Prof. Dr. G. ZACCONE for his constructive comments on the manuscript, his valuable suggestions, and critical reading of the paper. This work was supported by MURST grants (40%, 60%).
References CITKOWITZ, E., The hyaline layer: Its isolation and role in echinoderm development. Develop. BioI. 24, 348-362 (1971). - Analysis of the isolated hyaline layer of sea urchin embryos. Develop. BioI. 27,494-503 (1972). FALUGI, c., GRATTAROLA, M., PARODI, G., e PRESTIPINO, G., Visualizzazione di recettori di membrana in uova di riccio di mare mediante tecniche di analisi di immagine in fluorescenza. Atti Conge. Biofis. 1984, 114 (1984). GIUDICE, G., The Sea Urchin Embryo. A Developmental Biological System. Springer, BerlinlHeidelbergi New Yorkffokyo 1986. GLABE, C. G., GRABEL, L. B. VACQUIER, V. D., and ROSEN, S. D., Carbohydrate specificity of sea urchin sperm binding: A cell surface lectin mediating sperm-egg interaction. J. Cell BioI. 94, 123-128 (1982). and VACQUIER, V. D., Isolation and characterization of the vitelline layer of sea urchin eggs. J. Cell BioI. 75, 410-421 (1977 a). - The surfaces of sea urchin eggs possess a species-specific receptor for binding. J. Cell BioI. 75, 56 (l977b). IWATA, M., and NAKANO, E., A large calcium-binding protein associated with the larval spicules of the sea urchin embryo. Cell Different. 19, 229-236 (1986). KATOW, H., and SOLURSH, M., In situ distribution of concanavalin A-binding sites in mesenchyme blastulae and early gastrulae of the sea urchin Lytechinus pictus. Exp. Cell Res. 139, 171-180 (1982). - YAMADA, K. M., and SOLURSH, M., Occurrence of fibronectin on the primary mesenchyme cell surface during migration in sea urchin embryos. Differentiation 22, 120-124 (1982). KRACH, S. W., GREEN, A., NICOLSON, G. L., and OPPENHEIMER, S. B., Cell surface changes occurring during sea urchin embryonic development monitored by quantitative agglutination with plant lectins. Exp. Cell Res. 84, 191-198 (1974). LALLIER, R., Effects of concanavalin A on the development of sea urchin eggs. Exp. Cell Res. 72, 157-163 (1972). MANN, P. L., Membrane oligosaccharides: Structure and function during differentiation. Internat. Rev. Cytol. 112,67-95 (1988). MOSCONA, A. A., Surface specifications of embryonic cells: lectin receptors, cell recognition, and specific cell ligands. In: MOSCONA, A. A. (ed.), The Cell Surface in Development, vol. 1. J. Wiley & Sons, New York/LondonlSydneyfforonto 1974, pp. 67-100. O'DELL, D. S., ORTOLANI, G., and MONROY, A., Increased binding of radioactive concanavalin A during maturation of Ascidia eggs. Exp. Cell Res. 83, 408-411 (1973). OPPENHEIMER, S. B., and MEYER, J. T., Carbohydrate specificity of sea urchin blastula adhesion component. Exp. Cell Res. 139, 472-476 (l982b). ORTOLANI, G., O'DELL, D. S., and MONROY, A., Localized binding of Dolichos lectin to the early ascidian embryo. Exp. Cell Res. 106,402-404 (1977). ROSATI, F., DE SANTIS, R., and MONROY, A., Studies on fertilization in the ascidians. II. Lectin binding to gametes of Ciona intestinalis. Exp. Cell Res. 116,419-427 (1978). SCHMELL, E., EARLES, B. J., BREAUX, c., and LENNARZ, W. J., Identification of a sperm receptor on the surface of the eggs of the sea urchin Arbacia punctulata. J. Cell BioI. 72, 35-46 (1977). SCHNEIDER, E. G., NGUYEN, H. T., and LENNARZ, W. J., The effect of tunicamicin, an inhibitor of protein glycosilation, on embryonic development in the sea urchin. J. BioI. Chern. 253, 2348-2355 (1978). SKUTELSKY, E., and BAYER, E. A., Cell-type-related segregation of surface galactosyl-containing components at an early developmental stage in hemopoietic bone marrow cells in the rabbit. J. Cell BioI. 96, 184-190 (1983).
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SOLURSH, M., Migration of sea urchin primary mesenchyme cells. In: BROWDER, L. W. (ed.), Developmental Biology. A Comprehensive Synthesis, vol. 2. Plenum Press, New York/London 1985. pp. 391-432. SPIEGEL, E., and BURGER, M. M., Cell adhesion during gastrulation. A new approach. Exp. Cell Res. 139, 377-382 (1982). - and SPIEGEL, M., Fibronectin in the developing sea urchin embryo. J. Cell BioI. 80, 309-313 (1980). - - Fibronectin and laminin in the extracellular matrix and basement membrane of sea urchin embryos. Exp. Cell Res. 144,47-55 (1983). HOWARD, L., and SPIEGEL, M., Elongated microvilli support the sea urchin embryo concentrically within the perivitelline space until hatching. Roux's Arch. Develop. BioI. 198,85-191 (1989). and SPIEGEL, M., Cell-cell interactions during sea urchin morphogenesis. In: BROWDER, L. W. (ed.), Developmental Biology, a Comprehensive Synthesis, vol. 2. Plenum Press, New York/London 1985, pp. 195-239. VACQUIER, V. D., LEGNER, M., and EPEL, D., Protease activity establishes the block to polyspermy in sea urchin eggs. Nature 240,352-353 (1972 b). and O'DELL, D. S., Concanavalin A inhibits the dispersion of the cortical granule contents of sand dollar eggs. Exp. Cell Res. 90, 465-468 (1975). Author's address: A. CONTINI, Dip. BioI. Animale ed Ecol. Marina, Papardo, S. Agata, 1- 981 66 Messina, Italy.
Dipartimento di Biologia Animale ed Ecologia Marina dell'Universita', Messina*, and Istituto di Anatomia Comparata dell'Universita', Genova (Ita1ia)
Developmental regulation of lectin-binding patterns in Paracentrotus lividus gonads, gametes, and early embryos By ANTONIETTA CONTINI*, CARLA FALUGI and SALVATORE FASULO* With 5 Figures (Received April 9, 1991)
Summary By use of several lectins (ConA, WGA, SBA, GS I, PNA), a study was carried on gametes and developing embryos of the sea urchin Paracentrotus lividus. to investigate developmental changes in cell surface, leading to changements in cell-environment interactions. ConA, WGA, and SBA, with high affinity, bind to the vitelline membrane of unfertilized eggs, while PNA labelling at the same site is weak; GS I-binding is only present in the cytoplasm and cortical region of the unfertilized eggs. Immediately after fertilization, no ConA-binding is present in the membrane, while WGA- and SBA-binding molecules are located in the fertilization layer. In zygotes, 40 min after fertilization, ConA affinity sites were again present in both cytoplasm and cortical region. During cleavages and gastrulation, ConA binds to the blastomere cytoplasm and cortical region, to the intercellular matrix, and to the cytoplasm of mesenchyme cells. WGA binds to the cortical region of cleaving blastomeres, including the hyaline layer, up to the unhatched blastula. Then it labels the gastrula inner and outer surfaces. SBA binds to the blastomere membranes; no GS 1- and PNA-binding was detected during embryonic development. Sperms are bound by all the lectins, except GS I. Mannose and glucose conjugates are the most represented throughout the whole development of P. lividus, and their origin and locations are developmentally regulated. Galacto-residues are scarcely represented or are masked by other terminal sugars (e.g. sialic acid), and become functional during particular developmental events (cell movements).
Key words: development, lectins, glycoconjugates, sea urchin.
1. Introduction Cell surface glycoconjugates are known to play an important role in the regulation of cell interactions during development. Ascidian development was thoroughly studied by this point of view by O'DELL et al. (1973), ORTOLANI et al. (1977), ROSATI et al. (1978) who, by use of severallectins, found significant changements in oligosaccharide residues, mainly at the cell surfaces. Cell-surface oligosaccharides were also shown involved in the control of differentiation and growth in mammals (SKUTELSKY and BAYER 1983, MANN 1988). The authors found that every specific cell differentiation resulted in a specific oligosaccharide change on the cell surface, so changing the cell ability to interact with its environment in a specific manner. MOSCONA (1974) showed that the agglutination properties of ConA on cultured embryonic cells differ significantly, depending on embryonic stage, suggesting the possibility that ConA receptors are correlated with cell motility. In chick early development, an increase was found in surface sialic acid residues, suggesting an interaction between cell surface and cell environ12*
180
A. CONTINI, C. FALUGI and S. FASULO
ment differentiation. Lectin changes, during heart differentiation in mammals, were found to playa role in maintaining and modulating calcium ions fluxes, so that the glycocalyx may be hypothesized as implied in the control over numerous cell reactions (see MANN [1988] for review). A very studied model from this point of view is represented by echinoderm early development (see GIUDICE [1986] for review), where relevant transformations take place during short time lapses; sperm-egg reception is due to a glycoprotein (binding, GLABE et al. 1982); ConA affinity sites were found on the vitelline membrane (SCHMELL et al. 1977) and their implication in sperm-egg interactions was shown; by computer image analysis, they were localized in vivo on the outer side of the vitelline membrane (FALUGI et al. 1984); furthermore, it was shown that ConA inhibits the dispersion of the cortical granule contents (VACQUIER and O'DELL 1975); the saccharidic residues of the vitelline membrane proteins were characterized by analytical methods (GLABE and VACQUIER 1977 a, b); by agglutination experiments carried out with several lectins on free embryonic cells, glycoconjugates bound by ConA were detected on the cell surface, and those bound by WGA in intracellular sites (KRACH et al. 1974); and in vivo experiments were performed by injecting lectins in early embryos, blastula, and gastrula stages (SPIEGEL and SPIEGEL 1985; SOLURSH 1985). Although a great number of relevant findings on localizations and possible functions of glycoconjugates during echinoderm development is available in literature, these works were performed in different moments of the development of different echinoderm species, by use of different lectins. Moreover, most of the studies on pluricellular stages were performed on cultured disaggregated cells, devoided, by enzyme activity, of at least a part of their extracellular matrix. In this work, an attempt is made to investigate the origin and nature of different glycoconjugates during the early development of the sea urchin Paracentrotus lividus, both in vivo and in sections, by use of a panel of 5 labelled lectins. We found that mannose and glucose conjugates are present during the whole development from ovarian egg to pluteus; that their origin and distribution is developmentally regulated, accordingly with their functions, and that galacto-conjugates are mainly present during segmentation and morphogenetic movements.
2. Materials and methods Paraeentrotus lividus gametes were obtained in the laboratory from adult specimens, collected in the Gulf of Genova. Mature eggs were maintained in finger bowls containing sea water pasteurized and aerated overnight, and fertilized by sperms diluted according to the usual procedures. Some eggs were artificially activated by use of A 23187 Ca++ or Ca2+-ionophore, and some eggs were devoided of the inner cytoplasms by needle handling in Ca**-free sea water, to obtain ghosts. Part of gametes and embryos were treated by lectins diluted 11100 in sea water in vivo, for I h at 5°C, in the dark. Part of them were also embedded in historesin (BOH), cut 3 Ilm, and dried at room temperature for 2d. Lectins were employed to label oligosaccharidic residues: fluorescein isothiocyanate (ATC) labelled concanavalin A (ConA, Sigma. USA), from Coneanavalia ensiformis. with high affinity to carbohydrate residues of the nature of mannose and glucose; weat germ agglutinin (WGA) , labelled by either mc or horseradish peroxidase (HRP), with affinity to sialic acid and N-acetyl-O-glucosamine, HRP-Iabelled soy bean agglutinin (SBA) , with affinity to N-acetylgalactosamine and O-galactose; HRP-Iabelled Griffonia simplieifolia f (GS f), with affinity to cx-O galactose; and peanut agglutinin (PNA), labelled with either ATC or HRP, with affinity to galactose; 1-3-galactose-N-acetylgalactosarnine. All the last lectins were obtained from Kem-en-tee, OK. Slides carrying sections were washed in PBS (phosphate buffered saline, pH = 7.4) for 30 min. Lectins, diluted 11100 in PBS were put on sections, and incubated for I h in the dark and cold (5°C), in a humid staining-tray. The sections treated by lectins labelled with fluorescein were mounted with III = v/v glycerol/PBS and
Developmental regulation of lectin-binding patterns
181
observed with a Leitz microscope, equipped with an UV apparatus, and a filter set for fluorescein revelation. Fluorescence revealed the presence of lectin-binding sites. Slides treated with lectins labelled with HRP were stained by use of 30% diaminobenzidine (DAB) and 0.01 % H20 2 in PBS. The oxidation of the benzidine moiety by the peroxide-peroxidase reaction produces a brown staining at the sites of peroxidase activity. Slides were rinsed in buffer,dehydrated by alcohol, clarified with xylol, and mounted in balsam. Specificity controls were performed by incubating sections with lectins preadsorbed with the hapten sugars (respectively
3. Results In ovaries, ConA-binding was mainly present around immature oocytes, that were weakly stained at every stage of differentiation in tiny cytoplasmic granulations and around the nuclear envelope, while a strong fluorescence was present at the vitelline membrane, and in the tissue surrounding eggs. In controls performed by ConA preadsorbed with
Fig. I. P. lividus ovary. a FITC-ConA-binding sites; b toluidin blue staining of an ovarian oocyte. 311m sections; bar. t!, 10 11m.
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Fig. 2. FlTC-WGA-binding sites in P. lividus ovary. Fluorescence is mainly located in forming egg envelopes. Moreover, some fluorescence is also present in cytoplasms and in ovarian tissues around maturing eggs. 3 f.tm sections; Bar !d 50 f.tm (a) and 20 f.tm (b).
In mature unfertilized eggs, ConA, WGA, SBA bound with high affinity to the outer surface of the vitelline membrane, with homogeneous distribution in fixed materials; mature egg ghosts showed the same distribution of ConA-affinity sites (Fig. 3b) on the vitelline membrane. Controls showed that ConA affinity sites in the vitelline membrane were still revealed after
183
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Fig. 3. FITC-ConA-binding sites in P. lividus eggs and embryos. a unfertilized egg, 10 min after in toto treatment, showing clusters of receptors; b a ghost of vitelline membrane obtained 5 min after ConA treatment; c in toto egg activated by A 23187 Ca ionophore; d 2 blastomeres embryo, 3 !!m section; e,f gastrula stages, 3 !!m sections. Bars ~ 50 !!m.
blastomeres appeared less stained, in a thin region (Fig. Sa). SBA-affinity sites were located on blastomere surfaces and in the extracellular matrix, while the cytoplasmic staining was weak, localized in the basal portion of the cytoplasm. GS 1- and PNA-labelling was weak and generally negative, also after sialidase treatment.
184
A.
CONTINI, C. FALUGI
and S.
FASULO
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Fig. 4. Lectin-binding sites in P. lividus eggs. a HRP-GS I-binding sites in unfertilized eggs; b HRP-SBAbinding sites in unfertilized egg; c HRP-WGA-binding sites in unfertilized eggs. Residues of the jelly-coat are also stained (arrows); d FITC-ConA-binding sites in zygote, 40 min after fertilization; e HRP-WGA-binding sites in the same stage;jHRP-SBA-binding sites in the same stage. 311m sections; bar ~ 100 11m (a, e,/), 50 11m (c, d), and 20 11m (b).
Developmental regulation of lectin-binding patterns
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Fig. S. Lectin-binding sites in P. lividus embryos and sperms. a HRP-WGA-binding sites in 4 blastomeres embryo; b HRP-SBA-binding in the same stage; c HRP-WGA-binding in early gastrula; d FITC-ConA-labelling in pluteus; e FITC-ConA-labelling in unreacted sperms;fHRP-WGA-staining in the same sample; g HRP-SBAstiaining in the same sample; h HRP-PNA-binding in the same sample; i negative HRP-GS I-staining in sperms.
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A. CONTINI, C. FALUGI and S. FASULO
Table 1: Lectin binding at different stages of P. Lividus development Lectin
ConA WGA GSI PNA SBA
oocyt.
++ ++
± ±
+
unfert. egg
fert. egg
segm.
gastr.
plut.
sperm
++ +
++ ++
5min
40 min
++ ++
+
++ ++
++ +
++ ++
+
+
+
+
+
± ±
+ +
++ strong binding; + binding; - no binding; ± scarce binding, or in different sites. Table 2: Binding of lectins in controls Treatment
before fertil
after fertil
ConNa-D-Glucose ConNa-D-mannose ConA PNNsialidase PNA SBNN-ac-a-D-galam SBA
+ +
++
WGAN-ac-~-D-glucam
WGA
++
+
++
±
+ +
++
+ +
++
ConAJa-D-glucose ConA preadsorbed with o:-D-glucose; ConAJa-D-rrumnose eonA preadsorbed with o:-Dmannose; PNNsialidase PNA binding on sections pretreated by sialidase; SBNN-ac-a-D-galam SBA preadsorbed with N-acetyl-o:-D-galactosamine; WGNN-ac-f3-D-glucam WGA preadsorbed with N-acetyl-~-D glucosamine. For other symbols, see Table I.
Since the stage of mesenchymal blastula and during gastrulation, ConA labelled the external surface of the embryos and the basal lamina of ectodennal cells. In gastrula stage, the extracellular matrix present in the mesenchyme migrating pathway was also stained, and affinity sites were present in the cytoplasm of some of the moving cells (Figs. 3e, f); binding to ECM was prevented in controls treated with ConA preadsorbed with lX-Dmannose. WGA and SBA bound to the external surface of embryos and, in a weaker way, to the ectodennal and mesenchymal cells, including the extracellular matrix (Fig.5b, arrow); no GS 1- and very weak PNA-binding was present during further development, while ConA-binding was present in all the cells of plutei and in the matrix around skeletal elements (Fig.5b, arrow); WGA bound to the matrix around skeletal elements in late gastrula and pluteus stages. During segmentation and gastrulation, ConA-binding was prevented by lX-D-mannose preabsorption. Lectins-binding results are shown in Table 1, control results in Table 2. Spenns were bound by all the lectins (mainly CanA and WGA), except GS I (Fig.5, Table 1).
4. Discussion In ovaries, the distribution of ConA- and WGA-binding sites shows that the corresponding glycoconjugates are produced by the cytoplasm of both oocytes and ovarian cells, that co-work in building egg
Developmental regulation of lectin-binding patterns
187
envelopes. Controls show that, during oogenesis, glycoconjugates present mannose, glucose, and sialic acid residues, while galacto-residues are masked by terminal sialic acid, and are probably inactive. In mature eggs, the positive staining of GS 1- and SBA-binding sites shows the liberation of previously masked galacto-residues, and the activation of their function (e.g. movement of the egg towards the genital pores). Furthermore, the "clustering" tendency of ConA receptors, found in unfertilized eggs, shows that the vitelline layer, although it is made up of a fibre network containing glycoproteins (GLABE and VACQUIER 1977 a), and has a certain fluidity, allowing rapid lateral movements of the glycoproteins bound by ConA. ConA-binding sites transiently disappear, after both fertilization by sperms and egg activation with A 23187 Ca**-ionophore. This finding is related to the fact that proteases released from the cortical granules cause destruction of sperm receptor glycoproteins (VACQUIER et al. 1972b), thus confirming the function as sperm receptors for the ConA-binding sites located in the vitelline layer. The results of lectin-binding on sections show that ConA-binding sites undergo significant changes during development, as they are first present in unfertilized eggs, disappear after cortical reaction, are again present 40 min after, with cytoplasmic origin, change localization and quantity at every stage, and are mainly present during segmentation and cell migration up to the metamorphosing pluteus, where their presence is maximum. These findings agree with those of OPPENHEIMER and MEYER (l982b), who, working on sea urchin embryo cultured cells, described temporal changes in ConA agglutination. In particular, ConA-binding sites are found in the skeletal spicules, where WGA-binding sites are also found around the skeletal elements. Such location may correspond to the presence of a large calcium-binding protein involved in spiculogenesis, found by IWATA and NAKANO (1986). Since the first developmental stages, the attachment of cells to the hyaline layer may be due to its nature as glycoconjugates (CITKOWITZ 1971, 1972); SPIEGEL et al. (1989) described extracellular matrix fibres, linking microvilli, to the hyaline layer and fertilization envelope. The microvillar region and the perivitelline material are labelled by use of ConA, WGA, and SBA: this fact shows that the linking complex may be formed by glycoconjugates able to modulate adhesion (to maintain the embryo in the correct position within the envelopes) and disaggregation (to allow changements in position of cells during segmentation movements). The trend to disaggregation is a property of ConA-binding molecules (MOSCONA 1974); furthermore, the ability of galacto-compounds to inhibit reaggregation is known (SCHNEIDER et al. 1978). In this light, the fact (shown by the SBA-Iabelling) that galacto-conjugates are present during segmentation indicates a slight displacement towards disaggregation of the equilibrium between aggregation and disaggregation, but this latter is controlled and modulated by the presence of glucose-conjugates, and by the absence of PNA-binding galacto-conjugates. Actually, aggregation and disaggregation are needed for cell movements during segmentation and gastrulation. Generally, morphogenetic movements are caused by the presence of glycoproteins in the extracellular matrix. For instance, fibronectin, that is a heavy MW galacto-protein of ECM is known to be involved in cell shape maintenance and changements, besides in cell movements. Its presence may correspond to the SBA pattern found in P. /ividus. as antigens that cross-react with antibodies against human plasma fibronectin have been detected between blastomeres and on the outer (SPIEGEL et al. 1980) and inner surfaces of gastrula (SPIEGEL et al. 1983), while KATOW et al. (1982) found fibronectin-like immunoreactivity only in the migratory pathway. At the same way, the distribution of lectins-binding sites found in this work in part agrees with the in vivo findings of other authors: LALLIER (1972) found that ConA injection into the blastocoele displays an inhibitory effect on embryonic echinoderm cell adhesion, limited to detachment of filopodia and retraction of the archenteron; SPIEGEL and SPIEGEL (1985), by in vivo injection of WGA in gastrulae, on the contrary, caused a binding only on the surface of the cells of the primary mesenchyme ring. Furthermore, our controls obtained by pre-adsorbing ConA with haptens agree with the result obtained by KATOW and SOLURSH (1982) by pretreating embryos in vivo with a-D-mannoside, while OPPENHEIMER and MEYER (I982b) showed that glucose compounds are responsible for cell adhesion. In conclusion, throughout P. lividus development, ConA- and WGA-binding sites were always found, while SBA-binding was only present during segmentation and gastrulation. ConA showed affinity for different saccharidic residues respectively before and after fertilization. PNA- and GS I-binding sites were seldom present, in correspondence with particular developmental events. This shows the prominent production in this species of mannose- (mainly after fertilization) and glucose-conjugates, in comparison with galacto-conjugates, that are present at the cell surfaces and in the ECM mainly during cell movements.
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Acknowledgments We would particularly like 10 thank Prof. Dr. G. ZACCONE for his constructive comments on the manuscript, his valuable suggestions, and critical reading of the paper. This work was supported by MURST grants (40%, 60%).
References CITKOWITZ, E., The hyaline layer: Its isolation and role in echinoderm development. Develop. BioI. 24, 348-362 (1971). - Analysis of the isolated hyaline layer of sea urchin embryos. Develop. BioI. 27,494-503 (1972). FALUGI, c., GRATTAROLA, M., PARODI, G., e PRESTIPINO, G., Visualizzazione di recettori di membrana in uova di riccio di mare mediante tecniche di analisi di immagine in fluorescenza. Atti Conge. Biofis. 1984, 114 (1984). GIUDICE, G., The Sea Urchin Embryo. A Developmental Biological System. Springer, BerlinlHeidelbergi New Yorkffokyo 1986. GLABE, C. G., GRABEL, L. B. VACQUIER, V. D., and ROSEN, S. D., Carbohydrate specificity of sea urchin sperm binding: A cell surface lectin mediating sperm-egg interaction. J. Cell BioI. 94, 123-128 (1982). and VACQUIER, V. D., Isolation and characterization of the vitelline layer of sea urchin eggs. J. Cell BioI. 75, 410-421 (1977 a). - The surfaces of sea urchin eggs possess a species-specific receptor for binding. J. Cell BioI. 75, 56 (l977b). IWATA, M., and NAKANO, E., A large calcium-binding protein associated with the larval spicules of the sea urchin embryo. Cell Different. 19, 229-236 (1986). KATOW, H., and SOLURSH, M., In situ distribution of concanavalin A-binding sites in mesenchyme blastulae and early gastrulae of the sea urchin Lytechinus pictus. Exp. Cell Res. 139, 171-180 (1982). - YAMADA, K. M., and SOLURSH, M., Occurrence of fibronectin on the primary mesenchyme cell surface during migration in sea urchin embryos. Differentiation 22, 120-124 (1982). KRACH, S. W., GREEN, A., NICOLSON, G. L., and OPPENHEIMER, S. B., Cell surface changes occurring during sea urchin embryonic development monitored by quantitative agglutination with plant lectins. Exp. Cell Res. 84, 191-198 (1974). LALLIER, R., Effects of concanavalin A on the development of sea urchin eggs. Exp. Cell Res. 72, 157-163 (1972). MANN, P. L., Membrane oligosaccharides: Structure and function during differentiation. Internat. Rev. Cytol. 112,67-95 (1988). MOSCONA, A. A., Surface specifications of embryonic cells: lectin receptors, cell recognition, and specific cell ligands. In: MOSCONA, A. A. (ed.), The Cell Surface in Development, vol. 1. J. Wiley & Sons, New York/LondonlSydneyfforonto 1974, pp. 67-100. O'DELL, D. S., ORTOLANI, G., and MONROY, A., Increased binding of radioactive concanavalin A during maturation of Ascidia eggs. Exp. Cell Res. 83, 408-411 (1973). OPPENHEIMER, S. B., and MEYER, J. T., Carbohydrate specificity of sea urchin blastula adhesion component. Exp. Cell Res. 139, 472-476 (l982b). ORTOLANI, G., O'DELL, D. S., and MONROY, A., Localized binding of Dolichos lectin to the early ascidian embryo. Exp. Cell Res. 106,402-404 (1977). ROSATI, F., DE SANTIS, R., and MONROY, A., Studies on fertilization in the ascidians. II. Lectin binding to gametes of Ciona intestinalis. Exp. Cell Res. 116,419-427 (1978). SCHMELL, E., EARLES, B. J., BREAUX, c., and LENNARZ, W. J., Identification of a sperm receptor on the surface of the eggs of the sea urchin Arbacia punctulata. J. Cell BioI. 72, 35-46 (1977). SCHNEIDER, E. G., NGUYEN, H. T., and LENNARZ, W. J., The effect of tunicamicin, an inhibitor of protein glycosilation, on embryonic development in the sea urchin. J. BioI. Chern. 253, 2348-2355 (1978). SKUTELSKY, E., and BAYER, E. A., Cell-type-related segregation of surface galactosyl-containing components at an early developmental stage in hemopoietic bone marrow cells in the rabbit. J. Cell BioI. 96, 184-190 (1983).
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SOLURSH, M., Migration of sea urchin primary mesenchyme cells. In: BROWDER, L. W. (ed.), Developmental Biology. A Comprehensive Synthesis, vol. 2. Plenum Press, New York/London 1985. pp. 391-432. SPIEGEL, E., and BURGER, M. M., Cell adhesion during gastrulation. A new approach. Exp. Cell Res. 139, 377-382 (1982). - and SPIEGEL, M., Fibronectin in the developing sea urchin embryo. J. Cell BioI. 80, 309-313 (1980). - - Fibronectin and laminin in the extracellular matrix and basement membrane of sea urchin embryos. Exp. Cell Res. 144,47-55 (1983). HOWARD, L., and SPIEGEL, M., Elongated microvilli support the sea urchin embryo concentrically within the perivitelline space until hatching. Roux's Arch. Develop. BioI. 198,85-191 (1989). and SPIEGEL, M., Cell-cell interactions during sea urchin morphogenesis. In: BROWDER, L. W. (ed.), Developmental Biology, a Comprehensive Synthesis, vol. 2. Plenum Press, New York/London 1985, pp. 195-239. VACQUIER, V. D., LEGNER, M., and EPEL, D., Protease activity establishes the block to polyspermy in sea urchin eggs. Nature 240,352-353 (1972 b). and O'DELL, D. S., Concanavalin A inhibits the dispersion of the cortical granule contents of sand dollar eggs. Exp. Cell Res. 90, 465-468 (1975). Author's address: A. CONTINI, Dip. BioI. Animale ed Ecol. Marina, Papardo, S. Agata, 1- 981 66 Messina, Italy.