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Reprod Fertil Dev ; 28(3): 337-48, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25482090


Analysis of glycan chains of glycoconjugates is difficult because of their considerable variety. Despite this, several functional roles for these glycans have been reported. N-Glycans are oligosaccharides linked to asparagine residues of proteins. They are synthesised in the endoplasmic reticulum (ER) in a unique way, and later modified in both the ER and Golgi apparatus, developing different oligosaccharide chains. An essential role for complex N-glycans in mammalian spermatogenesis has been reported. The aim of the present study was to analyse the N-glycans of the Xenopus laevis testis by means of lectin histochemistry. Five lectins were used that specifically recognise mannose-containing and complex glycans, namely Galanthus nivalis agglutinin (GNA) from snowdrops, concanavalin A (Con A) from the Jack bean, Lens culinaris agglutinin (LCA) from lentils and Phaseolus vulgaris erythroagglutinin (PHA-E) and P. vulgaris leukoagglutinin (PHA-L) from the common bean. GNA and Con A labelled the interstitium and most of the germ cell types, whereas LCA and PHA-E showed affinity only for the interstitium. A granular cytoplasmic region was labelled in spermatogonia and spermatocytes by GNA and PHA-L, whereas GNA and LCA labelled a spermatid region that is probably associated with the centriolar basal body of the nascent flagellum. There was no specific labelling in the acrosome. Some unexpected results were found when deglycosylative pretreatments were used: pre-incubation of tissue sections with peptide N glycosidase F, which removes N-linked glycans, reduced or removed labelling with most lectins, as expected. However, after this pretreatment, the intensity of labelling remained or increased for Con A in the follicle (Sertoli) and post-meiotic germ cells. The ß-elimination procedure, which removes O-linked glycans, revealed new labelling patterns with GNA, LCA and PHA-L, suggesting that some N-glycans were masked by O-glycans, and thus they became accessible to these lectins only after removal of the O-linked oligosaccharides. The functional role of the glycan chains identified could be related to the role of N-glycans involved in mammalian spermatogenesis reported previously.

Imuno-Histoquímica/métodos , Lectinas de Plantas , Polissacarídeos/análise , Testículo/química , Xenopus laevis/metabolismo , Animais , Concanavalina A , Masculino , Lectinas de Ligação a Manose , Fito-Hemaglutininas
J Anat ; 221(4): 318-30, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22881213


Carbohydrate chains of glycoprotein and glycosphingolipids are highly diverse molecules involved in many cell functions, including cell recognition, adhesion and signalling. Sialylated glycans are of special interest because the terminal position of sialic acid (NeuAc) in glycans linked by different ways to subterminal monosaccharides has been shown to be involved in several biological processes, as occurs with gangliosides, which have been reported as being essential in spermatogenesis in mammals. Some glycan-binding proteins, the lectins, which specifically recognize glycan sequences, have been extensively used to characterize tissue and cell carbohydrates by means of cytochemical techniques. The aim of the present work was to determine the presence of NeuAc by means of histochemical techniques in the testis of Xenopus laevis, an animal model widely used in cell and molecular biology research. However, considering that some NeuAc-binding lectins are capable of binding to N-acetylglucosamine (GlcNAc), other GlcNAc-binding lectins were also assayed. The results showed that NeuAc is mainly expressed in the interstitium, and only a weak labelling in the male germ cells was observed. Most NeuAc was located in O-linked oligosaccharides, but some masked NeuAc in N-glycans were identified in primary and secondary spermatogonia and spermatocytes. By contrast, GlcNAc was widely expressed in all germ cell types. Deglycosylative pre-treatments suggest that both N- and O-glycans and/or glycolipids could be responsible for this labelling. In addition, GlcNAc in O-linked oligosaccharides has been identified in spermatogonial cells. The acrosome of spermatids was always negative. Variations of glycan expression have been found in different cell types, suggesting that glycosylation is modified during spermatogenetic development.

Ácido N-Acetilneuramínico/análise , Polissacarídeos/análise , Testículo/química , Animais , Histocitoquímica/métodos , Lectinas , Masculino , Xenopus laevis
Microsc Res Tech ; 74(8): 778-87, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21563271


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 ß-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.

Galactosídeos/análise , Glicoconjugados/análise , Histocitoquímica/métodos , Lectinas/análise , Testículo/química , Xenopus laevis/metabolismo , Animais , Galactosídeos/metabolismo , Glicoconjugados/metabolismo , Lectinas/metabolismo , Masculino , Espermátides/química , Espermátides/metabolismo , Espermatócitos/química , Espermatócitos/metabolismo , Testículo/citologia , Testículo/metabolismo
Anat Rec (Hoboken) ; 294(2): 363-71, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21235011


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.

Acetilgalactosamina/metabolismo , Glicoconjugados/metabolismo , Testículo/metabolismo , Xenopus laevis/metabolismo , Acetilgalactosamina/análise , Acrossomo/metabolismo , Animais , Metabolismo dos Carboidratos , Glicoconjugados/química , Histocitoquímica/métodos , Lectinas , Masculino , Espermatozoides/metabolismo
Histochem Cell Biol ; 134(2): 215-25, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20617442


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.

Fucose , Glicoconjugados/análise , Lectinas , Testículo/química , Animais , Fucose/análise , Fucose/metabolismo , Glicoconjugados/metabolismo , Histocitoquímica/métodos , Masculino , Lectinas de Plantas , Espermátides/química , Espermatócitos/química , Espermatogênese , Xenopus laevis
Anat Rec (Hoboken) ; 291(9): 1097-105, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18521902


The origin of the acrosome is controversial, because of both its lysosomal nature and at the moment of its appearance, which seems to be species-specific. Considering the amazing organization shown by the acrosome of some urodele amphibians, HPA-colloidal gold cytochemistry was used to analyze the biogenesis of the acrosome in the urodele Pleurodeles waltl at electron microscopy level. The results showed that HPA-labeling is useful to label the acrosome and its precursor vesicles and, consequently, HPA-histochemistry could be used as a marker of acrosomal content. Labeling of the Golgi apparatus and precursor vesicles was seen in primary spermatocytes and round (stage I) spermatids, thus contributing solid evidence for the beginning of acrosome biogenesis before meiosis. In both primary spermatocytes and round spermatids, an enigmatic vesicle, probably related to the biosynthesis of the neck piece or the tail, was also labeled. Labeling in elongating spermatids (stage II-IV), showed a homogeneous distribution of colloidal gold particles in the acrosomal cap, but the perforatorium was not positive to the lectin. However, in mature (stage V-VI) spermatids, a regional distribution of labeling in the acrosome was seen, with the apical knob showing a stronger labeling than the lateral barb, and the lateral barb showing a stronger labeling than the principal piece of the acrosomal cap. This regional distribution of the labeling suggests that the acrosome develops several domains with different glycoconjugate compositions.

Acrossomo/fisiologia , Pleurodeles/fisiologia , Espermátides/fisiologia , Espermatócitos/fisiologia , Acrossomo/ultraestrutura , Animais , Lectinas , Masculino , Espermátides/citologia , Espermatócitos/citologia , Testículo/fisiologia