BRIEF REPORT |
Correspondence to: Ricardo Paniagua, Dept. of Cell Biology and Genetics, U. of Alcalá, 28871 Alcalá de Henares (Madrid), Spain..
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Summary |
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The oligosaccharide sequences of glycoconjugates in the human normal epididymis and the nature of linkages were studied with lectin histochemistry. The usual terminal sequences of oligosaccharide side chains in epithelial cell secretions were Neu5Ac2,3Galß1,3GalNAc; SO4Galß1,3GalNAc; and Galß1,4GlcNAc, and they were mainly found in O-linked glycoproteins. The lectin pattern of mitochondria-rich cells differed from that of principal cells. (J Histochem Cytochem 46:11851188, 1998)
Key Words: glycoconjugates, lectin histochemistry, oligosaccharide side chains, epididymis
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Introduction |
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Lectin histochemistry of the epididymis has been carried out in some mammals (
The testes and epididymides from 17 men aged 2570 years, without reproductive disease and showing complete spermatogenesis, were obtained from autopsies 610 hr after death and processed for light and electron microscopy. To evaluate postmortem changes, three epididymides were obtained from testicular tumor surgery and processed in the same way.
The lectin histochemical procedures applied have been previously reported in detail (
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For electron microscopy, the samples were fixed in glutaraldehydeparaformaldehyde and embedded in Lowicryl K4M. Ultrathin sections were incubated with: (a) ConAgold lectin (10 nm); (b) unlabeled DSA and then floated on a drop with 15-nm gold-labeled ovomucoid; or (c) a lectinDIG (MAA, SNA, AAA, SBA, PNA, WGA, UEA-I, HPA, or GNA), incubated further in sheep anti-DIG monoclonal antibody, and floated on a drop of donkey anti-sheep 15-nm gold-labeled IgG (BioCell; Cardiff, UK). To evaluate staining intensity of each lectin, the average number of colloidal gold particles per µm2 was calculated for each subject and epididymal zone in 816 electron micrographs of 512 ultrathin sections.
The carbohydrates that could be masked by sialic acid were exposed with HCl (paraffin sections) or neuraminidase (ultrathin sections). Removal of sulfate esters was carried out by treatment with HCl and thereafter with Ba(OH)2 in paraffin sections or before embedding (ultrathin sections). Elimination of O-linked oligosaccharides (ß-elimination) was carried out by incubation with NaOH in paraffin sections or before embedding. Hydrolysis of N-linked oligosaccharides was done by treatment of both section types with endo-ß-acetylglucosaminidase F/peptide N-glycosidase F (endo-F) (Boehringer). Hydrolysis of terminal glucose residues was done in both section types by treatment with glucose oxidase (Sigma).
The control procedures were substitution of conjugated and unconjugated lectins by their buffers and preincubation of each lectin with its haptensugar inhibitor (Sigma).
Comparison of surgical and autopsy specimens showed no histochemical changes. The results of lectin histochemistry are summarized in Table 1 and illustrated in Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10. The exact location and intensity of lectin labeling in epididymal cells were evaluated by electron microscopy. No labeling was found in control sections for all lectins and treatments used.
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Lectin reactions in the epididymis could be related to structural oligosaccharides (microvilli and cytosol) and/or glycoprotein secretion (electron-dense vesicles and luminal content). The decreased WGA reactions after endo-F digestion and ß-elimination suggest that terminal GlcNAc residues are in both N- and O-linked oligosaccharides. WGA receptors and GlcNAc residues have been reported over the sperm head (
SNA labeling was absent in the lumen, weak in principal cells, and more intense in mitochondria-rich cells. Because this reactivity was maintained with ß-elimination, the labeled residues are probably present in both N- and O-linked oligosaccharides. The disappearance of MAA labeling after ß-elimination in the cells and lumen of the ductus epididymidis suggests secretion of Neu5Ac2,3Galß1,4GlcNAc in O-linked glycoproteins.
Chemical treatments for PNA suggest the presence of three different chains (Galß1,3GalNAc, Neu5AcGalß1, 3GalNAc, and SO4-Galß1,3GalNAc) in O-linked oligosaccharides. Sulfate has been implicated in gamete adhesion, induction of the acrosome reaction, and spermegg interactions (
AAA probably binds fucose residues that are 1, 6-linked to glycoproteins (
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Literature Cited |
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