papel de los glicoconjugados en la migraciÓn de las cgps en anuros

8/4/2019 PAPEL DE LOS GLICOCONJUGADOS EN LA MIGRACIN DE LAS CGPs EN ANUROS

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J. Embryol. exp. M orph. 82, 119-129 (1984)Printed in Great Britain Th e Company of Biologists Limited 1984

The role of the glycoconjugates in the migration ofanuran amphibian germ cellsByM. DELBOS1, J.-D. GIPOULOUX 1, N. SAIDI2

1 Laboratoire de Biologie Animale, Universite de Bordeaux 133405 TalenceCedex, France

2 Faculte des Sciences de Oujda, Maroc, France

SUMMARY1. The presence of a large amount of glycoconjugates on the anuran amphibian germ cellswas demonstrated using fluorescein isothiocyanate lectins binding specifically to D-galactoseand at a lower level, by other lectins b inding specifically to iV-acetyl-galactosamine.2. Glycoconjugates including D-galactose were found near the pseudopodial expansionsand in the extracellular space, between germ cells and follicular cells. They were alsodisseminated in the cytoplasm.3. The injection of PNA lectin (from Arachis hypogea) into the endoderm inhibited themigration of 90 % of the germ cells. This inhibition was lectin-concentration dependent.Ultrastructural study of germ cells, the migration of which was inhibited, showed that they

were degenerating. These results suggest that glycoconjugates are related to the migratoryactivity of germ cells.

RESUME1. Les cellules germinales primordiales des amphibiens anoures sont caract6risees parl'abondance de glycoconjugues, reVeles par des lectines fluorescentes toutes affines du D-galactose et a un moindre degr de la iV-acetyl-galactosamine.2. Les glycoconjugu6s porteurs de D-galactose sont presents au niveau des expansionspseudopodiales et dans l'espace extracellulaire separant les cellules germinales des cellulesfolliculaires.3. Les glycoconjugues sont en relation avec l'activite migratoire des cellules germinales.L'injection de lectine PNA (extraite de Arachis hypogea) entraine une inhibition de la migra-tion de 90 % des cellules germinales. L 'etude ultrastructurale des cellules germinales dont lamigration est inhibe montre qu'elles subissent une lyse importante.

INTRODUCTIONIt is possible by treating histological sections with lectins linked to fluorescein-

isothiocyanate to show up lectin-binding sites and hence the glycosidic chains towhich they are specifically bound. For example, Johnson & Smith (1976) haveshown that lectins extracted from soybean (SB A) and from Canavalia ensiformis(Con A) bind to Xenopus laevis and Xenopus mulleri associated cells, and both Far-geix, Didier, Guillot & Damez (1980), and Didier, Fargeix & Didier (1980) havedemonstrated the presence of glycosidic chains on the surface of avian germ cells.


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1 2 0 M . D E L B O S , J . - D . G I P O U L O U X A N D N . S A I D IPrevious studies (Delbos, Saidi & Gipouloux, 1982), employing a variety oflectins which bind to different sugars, allow the conclusion that glycosidic

residues on the surface of germ cells have functional importance. The timecourse of this labelling suggests that these glycoside residues are involved inmigration. Similar studies with lectins like Con A, have an inhibitory effect onthe morph ogen esis of amphibian em bryos since the addition, in vitro, of Con A toamphibian neural crest cells and to chick neural crest cells stops the migration ofthese cells (M oran , 1974a,b; Boucaut, Bernard & Aubery, 1977; Boucaut, 1978).In order to know if the migratory activity of germ cells is perturbed in vivo bythe action of lectins possessing a particular affinity for these cells, we injectedthese lectins into the endoderm of early embryos during the migration of germcells. The results were observed by optical microscopy and by transmissionelectron microscopy.

MATERIALS AND METHODSExpe riments were carried out on Rana dalmatina embryos, between stages 26

and 30 (Cambar & Marrot, 1954), on Bufo bufo, between stages Ills and III10(Cambar & Gipouloux, 1956), on Xenopus laevis between stages 30 and 47(Nieuwkoop & Faber, 1956).

Fluorescein-isothiocyanate lectins (FITC) were used for the cytochemicaldetection of membrane glycoconjugates. The lectins used were extracted fromArachis hypogea (PNA), from soybean (SBA), from Phaseolus vulgaris ( PHA) ,from Phytolacca americana (PWM), from Lens culinaris (LC A ), from Triticumvulgaris (WGA) and Ulex europeus (UEAF). They were provided by Sigma.

The samples were fixed in Bouin-Hollande solution for 2h, dehydrated, thenem bed ded in paraffin. Th e sections were 5 fim thick. Sections were trea ted withfluorescent lectins according to the Avrameas technique (Avrameas, 1969).After rehy dration sections were incubated at 25 C with lectins at a concentrationof 500/ig/ml in 0-01 M - P B S (pH 6-8 ). For control animals, incubation was m adein 0-01 M - P B S alone (pH6-8). The specificity of each lectin was checked bycarrying out an incubation with the lectin plus its inhibitory sugar. T he observa-tion was made with a Zeiss microscope fitted with an excitation filter BG 12.

In order to visualize the sugars of the surface and the intracellular sugars,glycosylated ferritin was used and sections observed with electron microscope.The samples were fixed with 6% glutaraldehyde in cacodylate buffer 0-1 M(pH 7-4) for l h . Sections were treated with PNA solution (250/ig/ml) or SB A(250/ig/m l) in 0-1 M-cacodylate buffer (pH 7-4 ) for 2 h . Th e samples were trans-ferred to buffer, then incubated in a lactosyl-ferritin solution at a concentrationof 250/ig/ml in PBS for 3h. Some samples were treated with lectin and 0-2 M-lactose together, in order to prevent the reaction. Then, they were post-fixed in2 % osmic acid in cacodylate buffer for 1 h at 0 C. They were dehydrated andembedded in Epon. Sections were observed without staining.


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Glycoconjugates in germ cell migration ofanuran amph ibia 121The other embryos were prepared for ultrastructural study: fixation involved1 h in 6 % glutaraldehyde, 1 h in 2 % osmic acid. The specimens were em bed ded

in Epon. Sections were stained by uranyl acetate and lead citrate and observedwith a Philips 201 electron microscope .The lectin injections (15 \A, about 7 /ig lectin per em bryo) we re m ad e, each dayinto the end ode rma l mass of the embryos with a microp ipette during the time ofthe m igration of germ cells, according to the following sch em e.

LO T I : 100 em bryo s, injection of PN A each 24 hLO T II : 100 em bryo s, injection of PN A each 48 hLOT II I : 100 embryos, injections of PNA each 48 h, then each24 h after 3 days

L O T IV : 100 em bryo s, injection of SBA each 24 hLO T V : 100 em bryo s, injection of PBS each 24 hLO T VI : 100 emb ryos, injection of PN A+ galacto se each 24 hL O T V II : 100 em bryo s, no injection (control animals)

At the stage during which germ-cell migration was complete in controlanim als, on e part of the em bryos wasfixedwith Bouin liquid and em bedd ed withparaffin wax. Sections were stained with Groat haematoxylin.

R E S U L T SI. Presence of glycoconjugates on the germ cellsSelective labelling of an uran germ cells is obtained with P NA and SB A whichboth bind to galactose and to a lesser degree, with PHA which binds to galac-tosamine (Figs 1, 2, 3, 4). The lectins extracted from Ulex (UEAF binding tofucose), from Triticum (WGA binding to JV-acetyl-galactosamine) and fromLens (LCA binding to mannose and glucose) do not show a specific reaction withgerm cells (Figs 5, 6).Th e ultrastructu ral study gives some information on the precise localization of

specific carbohydrates on germ cells. The observations were made on Xenopuslaevis em bryos (stage 47) and by com parison, on Rana dalmatina embryos (stage39), at stages during which fluorescence is the most intensive. The use of thedouble staining with PNA lectin and lactosyl-ferritin reveals the presence ofman y spherical granules of 9-15 nm d iame ter (abo ut the d iameter of ferritingranules) (Fig. 7). These granules are distributed regularly on the pseudopodiaof the germ cells, collected in clusters (Figs 8, 9) in the extracellular spaceseparating germ cells from adjacent follicular cells, but also distributed ratheruniformly in the cytoplasm of the germ cells (Fig. 12).In Xenopus emb ryos trea ted by lactosyl-ferritin without lectin, or by PN A withgalactose or lactose, we do not observe ferritin granule dep osits. Epiderm al cellsor pronephric duct cells of the embryos treated at the same time by PNA andlactosyl-ferritin, as contro l ce lls, show no specific labelling (Figs 10, 11).


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1 2 2 M . D E L B O S , J . - D . G I P O U L O U X A N D N . S A I D I

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Glycoconjugates in germ cell migration ofanuran amphibia 123II. Injection of PNA

Co unting of germ cells in the gen ital ridges shows notab le differences betw eencontrol and treated animals. In embryos treated by PNA injections, the numberof germ cells reaching genital ridges is very low. Genital ridges are practicallydevoid of germ cells (Fig. 13). Ectopic germ cells are observed easily in theintestine part (Figs 15, 16). Numbers of germ cells were obtained by countingtheir nuclei, which have a characteristic shape and coloration.We observed the same number of germ cells in untreated gonads or thosetreated by regular injections of PBS (or PNA with galactose) - control animals(Fig. 14 ). Co untin g of germ cells in the different classes of trea ted animals gave

the following results (Table I).Ex am ination of germ cells staying in the en dode rm reveals that the se cells aredegenerating.

DISCUSSIONUsing some fluorescent lectins, we have demonstrated the particular abun-dance of certain glycoconjugates on germ cells. The most intense fluorescencewas obtained with PN A which binds to galactose and to a lesser degree with SB Aand PHA which both bind to iV-acetyl-galactosamine.The importance of glycoproteins in the migration of germ cells has previouslybeen suggested by many au thors (Cuminge & Dubois, 1974; Moran, 19746; Lee,Karasanyi & Nagele, 1978). Thus, anuran germ cells seem to be characterizedby the presence of a large amount of galactose and ./V-acetyl-galactosamine.Ho we ver, th e observed fluorescence seemed to be located in both cytoplasm andcell me mb rane.W e have tried to localize these glycosidic residues by electron microscopy. T he

great specificity of lectins for carbohydrates has been used for ultrastructural

Fig. 1. Intragonadal primordial germ cells after incubation with FITC-labelled PNAand galactose: no fluorescence is observed (x50 0). pgc, primordial germ cell; pd ,pronephric duct. (Rana dalmatina, st. 39).Fig. 2. Intragonadal primordial germ cells after incubation with FITC-labelled PNA(x500). (Rana dalmatina, st. 39). pgc, primordial germ cells; pd , pronephric duct.Fig. 3. Intragonadal p rimordial germ cells after incubation with FITC-labelled PNA(X1200). (Bufo, IIIio)- pgc, primordial germ cell; pd , pronephric duct.Fig. 4. Intragonadal primordial germ cell after incubation with PBS alone (Controlanimals). No fluorescence is observed (x500). pgc, primordial germ cell; pd ,pronephric duct.Fig. 5. Intragonadal primordial germ cells after incubation with FITC-labelledUEAF (x500). pgc, primordial germ cell; pd , pronephric duct.Fig. 6. Intragonadal germ cell after incubation with FITC-labelled LCA (x500).pgc, primordial germ cell; pd , pronephric duct.


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1 2 4 M . D E L B O S , J . - D . G I P O U L O U X A N D N . S A I D Idetection of membrane and intracellular glycosidic sites (Nicolson, 1974,1976,1978). Lectins are not electron opaque but have the advantage of possessing atleast two attachment sites for sugars. Thus, it is possible to attach them toelectron-dense molecules like ferritin.


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Glycoconjugates in germ cell migration of anuran amphibia 125

Fig. 12. Intragonadal germ cell cytoplasm (X6000).

W e observed th at the ferritin granules bound to the receptor sites of PN A w eresituated in the cytoplasm of germ cells and on their cellular surface. This factconfirms our observations using fluorescence microscopy. Furthermore, we ob-served a uniform distribution of ferritin g ranules on the pseudopo dial expansionsof germ cells and in the ex tracellular spaces sepa rating germ cells from adjacentfollicular cells. The absence of ferritin deposits after treatment by PNA andgalactose confirms the specificity of the reaction.Previous work has dem onstrated (Delbos, Gu enno un & Gipouloux , 1981) theexistence of an abund ant e xtracellular matrix particularly near the p seudo podialexpa nsions. Th ese results suggest that the extracellular matrix, which com prisescollagen fibres and glycosam inoglycans associated w ith glycoconjugates of which

Fig. 7. Presence of ferritin granules (arrow) in extracellular space and in thecytoplasm of primordial germ cell (x80 000). pgc, primordial germ ce ll;/c , follicularcell.Fig. 8. Presence of ferritin granules (arrow) in clusters near the membranes of thegerm cells (x51000). pgc, primordial germ cell.Fig. 9. Same aspect of ferritin granules (arrow) (X 78000).Fig. 10. Aspect of the epidermal cells in embryos treated by lactosyl-ferritin. Nodeposits of ferritin granules (x 40 000).Fig. 11. Aspect of the pronephric duct cells in embryos after the same treatment(x 40 000).

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126 M . D E L B O S , J . - D . G I P O U L O U X A N D N . S A I D I

Fig. 13. No germ cell is observed in genital ridges of embryos treated by PNAinjections (x300). gr , genital ridge; ip, intestinal part.Fig. 14. Germ cells are observed in genital ridges of embryos treated by injectionsof PBS or PNA with galactose (x 300 ). gr , genital ridge; ip , intestinal part.Fig. 15. Aspect of the germ cells observed in intestinal part (arrow) of embryostreated by PNA injections (x l2 0) .Fig. 16. Germ cells observed in intestinal part (arrow) (x300).

Table 1. PGC number

ControlsTreated with PNA 24 hPNA48/24hPNA 48 hSB A 24 h

Intragonadal PGC20 0-573-72 0-545-20 2-605-33 3-179-20 1-24

Intraendodermal PGC12-0 1-327-80 3-175-33 1-325-22 2-18

Total20 0-5715-72 1-1213-0 5-2010-66 4-4114-42 3-3 9


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Glycoconjugates in germ cell migration o f anuran amphibia 127100 r

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0 0Fig. 17. Percen tage of gonadal primordial germ cells in treated embryos comparedwith untreated ones. 1: PNA (24h); 2: PNA 48/24h; 3: PNA 48h; 4: SBA 24h;5: untreated embryos (physiological liquid).the most abundant would be D-galactose, constitutes an available substrate forthe movement of cells in migration (Lee et al. 1978). In addition, D-galactosecould be an energy source together with glycogen for the active migration ofgerm cells.

After injection of PNA into the endodermal mass of embryos, the totalnumber of germ cells observed in embryos treated by SBA and PNA is alwayslower than th at observed in controls. Th ere are two possible reasons for this: onone hand, the difficulty of identifying intraendodermal germ cells, and on theoth er the deg ene ration of a great num ber of germ cells which remained 'endode r-mal'. A statistical study reveals that differences between populations ofintragon adal germ cells of control animals and of embryos trea ted by injectionsof PNA each day, are greatly significant. Fig. 17 gives the percentage ofintragonadal germ cells observed in the different classes of treated embryos incomparison with the number of germ cells observed in controls. Furthermore,ultrastructural observa tion of intraend ode rma l germ cells reveals that these cellsare degenerating.The effect of lectins on migration seems clear since inhibition of migration is


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128 M. D E L B O S , J . - D . G I P O U L O U X AND N. S A I D Ilectin-concentration dependent. Furthermore, a combination of galactose andPNA breaks the inhibition produced by lectin alone. The observations reportedin this present paper suggest that the inhibitory action of PNA on germ cellmigration is produced either on the germ cell membrane or on the extracellularmatrix adjoining germ cells, masking the galactose residues of the glycocon-jugates which play a role in migration. It is possible that PNA (with two receptorsites of galactose) associates simultaneously with membrane galactosyl residues ofgerm cells, and with those of the extracellular matrix, preventing their migration.

In summary, the observations reported are consistent with an important roleof glycoconjugates in the migration of amphibian germ cells.

REFERENCESAVRAMEAS, S. (1969). Coupling of enzymes to proteins. Use of the conjugates for the detectionof antigens and antibodies. Immunochemistry 6, 43-52.BOUCAUT, J. C , BERNARD, B. & AUBERY, M. (1977). Influence de la Concanavaline A sur ledeveloppement des blastulas de Pleurodeles waltlii (Amphibien U rodele). C.r. hebd. Seanc.Acad. ScL, Paris 285, s6rie D, 193-196.BOUCAUT, J. C. (1978). Effet de la Concanavaline A sur la morphogenese chez Pleurodeleswaltlii. Mem. Soc. Zool. France 41, 117-122.CAMBAR, R. & MARROT, BR. (1954). Table chronologique du developpement de la Grenouilleagile Rana dalmatina. Bull. biol. Fr. Belg. 88 (2), 168-177.CAMBAR, R. & GIPOULOUX, J.-D. (1956). Table chronologique embryonnaire et larvaire du

Crapaud commun Bufo bufo. Bull. biol. Fr. Belg. 90 (2), 198-217.CUMINGE, D . & DUBOIS, R. (1974). Role des glycoproteines dans la migration chimiotactiquedes cellules germinales de poulet, d'apres les resultats obtenus avec la concanavaline A enculture organotypique. C. r. hebd. Seanc. Acad. Sci. Paris 279, 995-999.DELBOS, M., GUENNOUN, S. & GIPOULOUX, J.-D. (1981). Caracteres ultrastructuraux etcytochimiques des cellules germinales primordiales des Amphibiens A noures. ArchsAnat.microsc. Morph. exp. 70, 117-128.DELBOS, M., SAIDI, N. & GIPOULOUX, J.-D. (1982). Detection au moyen de lectinesfluorescentes de chaines osidiques specifiques des cellules germinales primordiales d'Am-phibiens Anoures. Archs Anat. microsc. Morph. exp. 71 No 2, 89-98.DIDIER, E., FARGEIX, N. & DIDIER, P. (1980). Extracellular polyanions in the splanchnopleuralmesoderm of the chick embryo. Biol. Cell 38, II A.FARGEIX, N., DIDIER, E., GUILLOT, J. & DAMEZ, M. (1980). Utilisation de diverses lectinesfluorescentes dans l'e"tude des cellules germinales en migration chez l'embryon d'Oiseau. C.r. hebd. Seanc. Acad. ScL, Paris 290, serie D, 999-1002.JOHNSON, K. E. & SMITH, E. P. (1976). The binding of Concanavalin A to dissociatedembryonic amphibian cells. Expl Cell Res. 101, 63-70.LEE, H ., KARASANYI, N. & N A G E L E , R . G. (1978). The role of the cell surface in the m igrationof primordial germ cells in early chick embryos: effects of concanavalin A. / . Embryol. exp.Morph. 46, 5-20.MORAN, D. (1974a). The inhibitory effects of Concanavalin A on the development of theamphibian embryo. / . exp. Zool. 188, 361-365.MORAN, D. (19746). The action of concanavalin A on migrating and differentiation neuralcrest cells. Expl Cell Res. 86, 365-373.NICOLSON, G. L. (1974). The interactions of lectins with animal cell surfaces. Int. Rev. Cytol.39, 89-190.NICOLSON, G. L. (1976). Transmembrane control of the receptors on normal and tumor cells.I - Cytoplasmic influence over cell surface components. Biochem. Biophys. Acta 457,57-108.


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Glycoconjugates in germ cell migration of anuran amphibia 129NICOLSON, G. L. (1978). Ultrastructural localization of lectin receptors. In: AdvancedTechniques in Biological Electron Microscopy, (ed. J. K. K 6ehler), vol 2, pp. 1-38. Berlin:Springer Verlag.NIEUWKOOP, P. D. & FABER, J. (1956). Normal Table o/Xenopus laevis. A msterdam: North-Holland Publ. Co.

{Accepted 3 M arch 1984)


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papel de los glicoconjugados en la migraciÓn de las cgps en anuros

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