Histochemical Demonstration of Two Subtypes of O‐linked Oligosaccharides in the Rat Gastric Glands

Alonso

Díaz‐Flores

et al. 2010

The Anatomical Record

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Identification of glycans in amphibian testis has shown the existence of N-acetylgalactosamine (GalNAc)-containing carbohydrates. Labeling of the sperm acrosome with GalNAc-binding lectins has allowed the identification of GalNAc-containing glycans in this organelle. Futhermore, this specific labeling of the acrosome has allowed the study of acrosomal biogenesis by lectin histochemistry. However, the testis of Xenopus laevis has never been analyzed by lectin histochemistry to locate GalNAc-containing glycoconjugates. The aim of this work was to elucidate the expression of GalNAc in glycoconjugates of Xenopus testis using five specific lectins. The results showed that most of the lectins labeled the interstitium with variable intensity. However, labeling of the different spermatogenetic germ cell types showed different labeling patterns. Some lectins produced weak or very weak staining in germ cells, for example, horse gram Dolichos biflorus agglutinin, which labeled most of the germ cell types, and lima bean Phaseolus lunatus agglutinin, which weakly labeled only spermatogonia, but did not stain other germ cells. By contrast, Maclura pomifera lectin (MPL) moderately labeled all germ cell types, except mature sperm. Labeling with other lectins was seen only at later stages, suggesting variations involved in the spermatogenetic development. Thus, snail Helix pomatia agglutinin labeled spermatids, but neither spermatogonia nor spermatocytes, while soybean Glycine max agglutinin (SBA) labeled from preleptotene spermatocytes to later stages. The periphery of the acrosome was labeled with MPL and SBA, but no specific labeling of the acrosomal content was seen with any lectin. Thus, the GalNAc-binding lectins that have been used as acrosomal markers in some amphibians cannot be used in Xenopus testis, suggesting that acrosomal glycoconjugates in amphibians are species specific.

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Identification of N‐Acetylgalactosamine in Carbohydrates of Xenopus laevis Testis

Alonso

Díaz‐Flores

et al. 2010

The Anatomical Record

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“…However, a reduction of labelling intensity after each pretreatment is to be expected, which is not evident with some lectins in our work. These amazing results are also common, and have been explained by assuming that each deglycosylation method unmasks some glycans which were previously inaccessible to the lectin (Alonso et al 2001;Gómez-Santos et al 2007;Sáez et al 2001b;Desantis et al 2006;Parillo et al 2009). Another possible explanation is that lectins could be labelling not only glycoproteins, but also glycolipids.…”

Section: Discussionmentioning

confidence: 86%

“…Incubation with recombinant PNGase F to remove N-linked glycans was performed as described (Sáez et al 2000a;Gómez-Santos et al 2007). …”

Section: Methodsmentioning

confidence: 99%

Identification of fucosylated glycoconjugates in Xenopus laevis testis by lectin histochemistry

Madrid

Hernández

et al. 2010

Histochem Cell Biol

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Glycoconjugates play roles in many physiological and pathological processes. Previous works have shown important functions mediated by glycans in spermatogenesis, and the carbohydrate composition of testis has been studied by several approaches, including lectin-histochemical methods. However, the testis of Xenopus laevis, an animal model extensively employed in biochemical, cell and developmental research, has not yet been analysed. The aim of this work was to carry out a histochemical study of the fucose (Fuc)-containing glycoconjugates of Xenopus testis by means of lectins, combined with deglycosylation pretreatments. Four Fuc-binding lectins were used: orange peel (Aleuria aurantia) lectin (AAL), gorse seed (Ulex europaeus) agglutinin-I (UEA-I), fresh water eel (Anguilla anguilla) agglutinin (AAA), and asparagus pea (Lotus tetragonolobus) agglutinin (LTA), each recognizing different forms of fucosylated glycans. Labelling with UEA-I, which preferably binds Fucalpha(1,2) containing oligosaccharides, did not show any appreciable staining. LTA, specific for Fucalpha(1,3), and AAA, which binds Fucalpha(1,2), labelled spermatocytes and spermatids, but no labelling was seen when the histochemical procedure was carried out after either beta-elimination (which removes O-linked oligosaccharides) or incubation with PNGase F (which removes N-linked oligosaccharides), suggesting that fucosylated glycans are of both N- and O-linked types. AAL, which has its highest affinity to Fucalpha(1,6), but also recognizes Fucalpha(1,2) and Fucalpha(1,3), labelled the whole testis, and the staining remained when the histochemical method was performed after either beta-elimination or incubation with PNGase F. Labelling with AAL could be explained by the fact that this lectin could be binding to diverse fucosylated glycans in N- and O-glycans, and even in glycolipids. The importance of these glycans is discussed.

“…β‐elimination was carried out following the technique previously described (Sáz et al,2000a) for five, seven, and twelve days, (Valbuena et al,2010). To remove N‐linked glycans, incubation with recombinant PNGase F was performed as previously described (Gómez‐Santos et al,2007; Sáez et al,2000a).…”

Section: Methodsmentioning

confidence: 99%

Galactosides in glycoconjugates of Xenopus laevis testis shown by lectin histochemistry

Microscopy Res & Technique

Alonso

Madrid

et al. 2011

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The implication of galactosides and other glycoconjugates on spermatogenesis has been previously reported. Glycans show such a complex structure that it makes them very difficult to analyze. Lectin histochemistry is a helpful tool for the study of glycan composition. Lectin histochemistry can be combined with deglycosylation pretreatments to explore the glycan type to which carbohydrates are linked. The aim of the present work was the localization of galactose (Gal)-containing glycoconjugates in the testis of Xenopus laevis, a species widely used in cell, molecular and developmental biology. Gal specific lectins BPL, PNA, BSI-B4, MAA-I, and RCA-I, were used in combination with deglycosylation procedures. Except for BPL, all the lectins were reactive for several testicular tissues. Some of the lectins showed a different reactivity depending on the stage of spermatogenic development, suggesting that cell glycoconjugates are modified during spermatogenesis. The surface of primary spermatocytes was strongly labeled with lectins from peanut (PNA) and castor bean (RCA-I), which agrees with the presence of galactosyl-glycolipids reported in the cell membrane of mammalian spermatocytes. The acrosome was unexpectedly negative to all the lectins tested, whereas the acrosome of mammals and other amphibians has shown a high expression of glycoconjugates, including galactosides. The results obtained after deglycosylation by β-elimination or incubation with PNGase F, which respectively remove O- and N-linked oligosaccharides, allowed us to elucidate the nature of the labeled glycans. The strong expression of galactosides at the cell surface of spermatocytes and spermatids suggests the involvement of these glycans in cell adhesion mechanisms during spermatogenesis.