ARTICLE |
Correspondence to: Francisco J. Sáez, Universidad del País Vasco, Departamento de Biología Celular e Histología, Facultad de Medicina y Odontología, B&ogr; Sarriena s/n, E-48940 Leioa (Vizcaya), Spain. E-mail: gcpsacrf@lg.ehu.es
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Summary |
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Previous works have shown that glycoconjugates with terminal fucose (Fuc) are located in the primordial germ cells (PGCs) of some mammals and might play a role in the migration and adhesion processes during development. The aim of this work was to identify the terminal Fuc moieties of Xenopus PGCs by means of three Fuc-binding lectins: from asparagus pea (LTA), gorse seed (UEA-I), and orange peel fungus (AAA). The histochemical procedures were also carried out after deglycosylation pretreatments: ß-elimination with NaOH to remove O-linked oligosaccharides; incubation with PNGase F to remove N-linked carbohydrate chains; and incubation with (1,2)- and
(1,6)-fucosidase. The PGCs were always negative for LTA and UEA-I, two lectins that have the highest affinity for Fuc
(1,2)-linked. However, the PGCs were strongly labeled with AAA, which preferentially binds to Fuc with
(1,3) or
(1,4) linkages and to Fuc
(1,6)-linked to the proximal N-acetylglucosamine. There was fainter labeling with AAA when the sections were preincubated with
(1,6)-fucosidase, but the labeling remained strong when the sections were pretreated with
(1,2)fucosidase. When the ß-elimination procedure was carried out, the PGC labeling with AAA was slight. If the PNGase F incubation was performed, the PGCs remained moderately positive for AAA. These data suggest that the Xenopus PGCs have Fuc moieties in O- and N-linked oligosaccharides, including Fuc
(1,6) linked to the innermost GlcNAc, and that the Fuc was not in
(1,2)-linkage. (J Histochem Cytochem 51:239243, 2003)
Key Words: N-linked oligosaccharides, O-linked oligosaccharides, fucosidase, ß-elimination, peptide N glycosidase F, Xenopus embryogenesis, germ cells
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Introduction |
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In the embryos of most vertebrate species, primordial germ cells (PGCs) arise early in development and must migrate to the genital ridges (
On the other hand, one of the major difficulties in study of the PGCs in most animals is the identification and isolation of these cells, owing to the absence of good markers. This is specially true for Xenopus embryos (
The aim of the present work was to characterize, by use of Fuc-binding lectins, fucosylated glycans as a first approach to determine the possible role of glycoconjugates in Xenopus PGC migration and adhesion and to investigate if some of these lectins might be used as PGC markers.
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Materials and Methods |
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Sample Preparation
Xenopus eggs obtained after injection of 1000 U hCG (Profasi HP; Serono, Madrid, Spain) were fertilized in vitro by gently rubbing with testis fragments. The embryos were reared in darkness in 1:10 normal amphibian medium (NAM;
Lectin Histochemistry
In this study, horseradish peroxidase (HRP)- and digoxigenin (DIG)-conjugated lectins were employed to stain semithin sections of epoxy-embedded embryos by the methodology previously described ((1,2)-fucosidase and
(1,6)-fucosidase (Sigma Química).
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The intensity of the staining was quantified by two independent observers that classified the PGC labeling into four arbitrary categories: no labeling (0), weak (1), moderate (2), and strong (3). This quantification is useful because it enabled us to compare the PGC labeling without the pretreatments and combined with them.
Deglycosylation Pretreatments
The enzymatic pretreatment using PNGase F to remove the N-linked oligosaccharides was performed as described previously (
To remove Fuc, (1,2)- and
(1,6)-fucosidases were employed. The sections were incubated with the enzyme in a 1:200 dilution in 250 mM sodium phosphate buffer, pH 5.0, for 2 days at 37C in a moist chamber.
Controls
The following controls were used: (a) substitution of the lectins, anti-DIG antibody, and enzymes by the buffer alone; (b) preincubation of the lectins with 0.2 M Fuc (Sigma Química) (
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Results |
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The control sections were always negative.
The binding patterns of each lectin observed in stages 43, 4647, and 48 were very similar. Then de-glycosylative pretreatments were performed only with the stage 4647 embryos. These results are shown in Table 2. No lectin can be employed as a marker of the PGCs because the PGCs and the surrounding mesentery cells showed similar lectin binding patterns.
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The PGCs were negative for LTA (Fig 1) and UEA-I (Fig 2) with all the pretreatments employed. AAA labeled the plasma membrane and the cytoplasm of the PGCs and the surrounding mesentery cells (Fig 3a). This labeling was notably decreased by the ß-elimination pretreatment (Fig 3b). However, after incubation with PNGase F, labeling with AAA was slightly decreased and remained moderate (Fig 3c).
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When the sections were previously incubated with (1,2)fucosidase, AAA labeled the PGCs (Fig 3d). However, when the incubation was carried out with
(1,6)fucosidase pretreatment, they were scarcely positive.
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Discussion |
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One of the major difficulties in research on Xenopus PGC migration is the absence of a specific marker for these cells, a problem that led some groups to study germ-cell migration in other animals (
In this study, LTA and UEA-I showed a different binding pattern from that of AAA, in spite of the fact that all of them bind Fuc. It has been previously reported that several lectins with affinity for the same sugar could show different labeling patterns ((1,6)-linked to the proximal GlcNAc of the N-linked glycans, and to terminal Fuc with an
(1,3) or
(1,4) linkage (
(1,2) linkage (
The analysis of the results obtained with LTA and UEA-I in the present work in the Xenopus PGCs allows us to suggest the absence of (1,2)-linked Fuc in these cells. This was previously suggested by using UEA-I by
(1,2)-linked Fuc (
(1,2)-linked Fuc-containing oligosaccharides but has no reactivity for the oligosaccharides with terminal GalNAc
(1,3)[Fuc
(1,2)]Gal (
(1,2)-fucosylated glycans different from those recognized by LBA and SBA. Therefore, from these data it could be inferred that no fucosylated glycans with
(1,2) linkage are in the PGCs of Xenopus. The results with AAA suggest that
(1,3),
(1,4), or
(1,6)-linked Fuc is in the glycoconjugates of the PGCs. To verify this, lectin histochemistry was performed again after incubation with
(1,2)fucosidase, and the labeling pattern was not modified, supporting the proposition that AAA did not label
(1,2)-linked Fuc. Moreover, the staining was notably decreased when the O-linked oligosaccharides were removed (ß-elimination pretreatment) and remained almost invariable if N-linked oligosaccharides were removed (PNGase F incubation). These data suggest that some Fuc moieties were in O-linked oligosaccharides, although others could be in N-linked oligosaccharides. Perhaps Fuc with an
(1,6) linkage to GlcNAc-Asn, which is present only in N-linked chains, could be responsible for the faint staining after ß-elimination, whereas Fuc with
(1,3) or
(1,4) could be present in O-linked chains and could remain after PNGase F pretreatment. Preincubation of the samples with
(1,6)fucosidase resulted in fainter labeling of the PGC compared with cells without pretreatment, supporting the concept that some Fuc residues have an
(1,6) linkage. If we consider that
(1,6) linked Fuc can be mostly linked to the GlcNAc-Asn core of the N-linked chains, it would be considered contradictory that the staining with AAA after
(1,6)fucosidase incubation was slighter than after PNGase F digestion, because it would be expected that the change in lectin staining should be very similar after both pretreatments. However, the moderate staining with AAA lectin after PNGase F incubation could be explained if we assume that removal of N-linked glycans could result in unmasking of other O-linked sugar chains with Fuc moieties that were not accessible to the lectin without pretreatment. The unmasking of sugar residues after oligosaccharide removal has been previously described in other tissues (
(1,6)fucosidase incubation because this enzyme removes one sugar moiety from the glycan, but not all the N-linked oligosaccharide chain.
Most of the vertebrate species studied have shown scarce or null reactivity in their PGCs for the Fuc-binding lectins ((1,2) Fuc. However, a previous work with human salivary glands from secretors and nonsecretors provided evidence of affinity for LTA but not UEA-I for
(1,4)-linked Fuc (
(1,4)-linked Fuc (
In summary, in this work we have shown by lectin histochemistry that the Xenopus PGCs have Fuc moieties, mainly in O-linked oligosaccharides, and probably with (1,3) or
(1,4) linkages. Moreover, the Fuc localized in N-linked chains could be attributed to
(1,6) linkages to the innermost GlcNAc. The absence of
(1,2)-linked Fuc can be inferred.
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Acknowledgments |
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Supported by grants from the University of the Basque Country (EA137/97 and G10/99). EA was supported by fellowships from the University of the Basque Country and the Spanish Government (Ministerio de Educación, Cultura y Deporte). Ms M. Portuondo and Ms C. Otamendi contributed to sample preparation.
Received for publication July 5, 2002; accepted October 10, 2002.
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