Journal of Histochemistry and Cytochemistry, Vol. 46, 1185-1188, October 1998, Copyright © 1998, The Histochemical Society, Inc.


BRIEF REPORT

Identification of N- and O-linked Oligosaccharides in the Human Epididymis

María I. Arenasa, Juan F. Madridb, Fermín R. Bethencourta, Benito Frailea, and Ricardo Paniaguaa
a Department of Cell Biology and Genetics, University of Alcalá, Alcalá de Henares, Madrid
b Department of Cell Biology, University of the Basque Country, Vizcaya, Spain

Correspondence to: Ricardo Paniagua, Dept. of Cell Biology and Genetics, U. of Alcalá, 28871 Alcalá de Henares (Madrid), Spain..


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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 46:1185–1188, 1998)

Key Words: glycoconjugates, lectin histochemistry, oligosaccharide side chains, epididymis


  Introduction
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Introduction
Literature Cited

Lectin histochemistry of the epididymis has been carried out in some mammals (Arya and Vanha-Perttula 1986 ; Burkett et al. 1987 ), including humans (Arenas et al. 1996 ). The aim of this study was to determinate the oligosaccharide sequences of glycoconjugates and the nature of their linkages in the human epididymis, using lectin histochemistry and chemical treatments.

The testes and epididymides from 17 men aged 25–70 years, without reproductive disease and showing complete spermatogenesis, were obtained from autopsies 6–10 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 (Leis et al. 1997 ). For light microscopy, either horseradish peroxidase (HRP)- or digoxygenin (DIG)-conjugated lectins were used. For HRP lectins, endogenous peroxidase was blocked with H2O2 and the sections were incubated with the lectins (Sigma; Barcelona, Spain) (Table 1): PNA (25 µg/ml), ConA (20 µg/ml), LTA (25 µg/ml), WGA (10 µg/ml), SBA (18 µg/ml), UEA-I (20 µg/ml), HPA (6 µg/ml), and DBA (15 µg/ml). Peroxidase was developed with diaminobenzidine (DAB) and H2O2. For DIG–lectins, endogenous peroxidase activity was destroyed by pretreatment with H2O2 and the sections were incubated with the lectin–DIG conjugates (Boehringer; Mannheim, Germany): DSA (10 µg/ml), MAA (10 µg/ml), AAA (20 µg/ml), SNA (15 µg/ml), and GNA (60 µg/ml) (Table 1). Sections were incubated further with peroxidase-conjugated anti-DIG Fab fragments. Peroxidase activity was visualized with DAB and H2O2.


 
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Table 1. Labeling intensity and cellular localization of histochemical reactions to lectins in the epithelium of the human epididymisa

For electron microscopy, the samples were fixed in glutaraldehyde–paraformaldehyde and embedded in Lowicryl K4M. Ultrathin sections were incubated with: (a) ConA–gold lectin (10 nm); (b) unlabeled DSA and then floated on a drop with 15-nm gold-labeled ovomucoid; or (c) a lectin–DIG (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 8–16 electron micrographs of 5–12 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 hapten–sugar 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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Figures 1-10. Lectin labeling of the human ductus epididymidis (corpus). Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Light microscopic sections counterstained with H.E. Bars = 20 µm. Figure 1 SNA labeling. Staining is weak in principal cells and intense in mitochondria-rich cells (arrow) and basal cells (arrowhead). Figure 2 SNA labeling decreases after endo-F digestion except for the mitochondria-rich cells (arrow). Figure 3 Negative reaction to SNA after preincubation with the inhibitor Neu5Ac. Figure 4 MAA reaction in the luminal content (L) and columnar cells, but not in the basal cells (arrowhead). Figure 5 MAA labeling disappears after ß-elimination. Figure 6 HPA reaction in microvilli. Figure 7 HPA labeling increases after desulfation. Figure 8 Negative staining to HPA after preincubation with the inhibitor D-N-GalNAc. Figure 9 Figure 10 Electron microscopic sections of principal cells counterstained with uranyl acetate. Bars = 0.5 µm. Figure 9 The electron-dense vesicles (arrow) and, to a lesser degree, the cytosol (arrowhead) label for WGA. Figure 10 The Golgi complex (G), electron-dense vesicles (arrow), and cytosol (arrowhead) label for DSA.

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 (Liu et al. 1991 ). The increased WGA reaction after desialization in the cauda epididymidis suggests addition of Neu5Ac in this region. Sialoglycoconjugates have been found in the human sperm tail (Focarelli et al. 1995 ).

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 Neu5Ac{alpha}2,3Galß1,4GlcNAc 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 sperm–egg interactions (LoLeggio et al. 1994 ). Reactions to DSA suggest O-linked oligosaccharides with Galß1, 4GlcNAc sequences without sialic acid of sulfate groups.

AAA probably binds fucose residues that are {alpha}1, 6-linked to glycoproteins (Yamashita et al. 1985 ). Chemical treatments used here indicate that these residues are in O-linked oligosaccharides. GNA appears to bind terminal mannoses in high-mannose N-linked oligosaccharides, whereas ConA would label trimannosyl cores in complex carbohydrates (Brewer and Bhattacharyya 1988 ). GNA reactivity could be located in lysosomes, whereas ConA reaction would be associated with secreted and/or absorbed glycoproteins. DBA, SBA, and HPA preferentially bind nonreducing GalNAc residues (Spicer and Schulte 1992 ). The increase in HPA and SBA labeling with desialization and desulfation suggests the presence of Neu5AcGalNAc and SO4-GalNAc residues.


  Literature Cited
Top
Summary
Introduction
Literature Cited

Arenas MI, de Miguel MP, Bethencourt FR, Fraile B, Royuela MM, Paniagua R (1996) Lectin histochemistry in the human epididymis. J Reprod Fertil 106:313-320[Abstract]

Arya M, Vanha–Perttula T (1986) Comparison of lectin-staining pattern in testis and epididymis of gerbil, guinea pig, mouse and nutria. Am J Anat 175:449-469[Medline]

Brewer CF, Bhattacharyya L (1988) Concanavalin A interactions with asparagine-linked glycopeptides. The mechanisms of binding of oligomannose, bisected hybrid, and complex type carbohydrates. Glycoconjugate J 5:159-173

Burkett BN, Schulte BA, Spicer SS (1987) Histochemical evaluation of glycoconjugates in the male reproductive tract with lectin-horseradish peroxidase conjugates: II. Staining of ciliated cells, basal cells, flask cells, and clear cells in the mouse. Am J Anat 178:23-29[Medline]

Focarelli R, Giuffrida A, Rosati F (1995) Changes in the sialylglycoconjugate distribution on the human sperm surface during in-vitro capacitation: partial purification of a 20 kDa sialylglycoprotein of capacited spermatozoa. Hum Reprod 10:2755-2759[Abstract]

Leis O, Madrid JF, Ballesta J, Hernández F (1997) N- and O-linked oligosaccharides in the secretory granules of Paneth cells: an ultrastructural cytochemical study. J Histochem Cytochem 45:285-293[Abstract/Free Full Text]

Liu HW, Wang JJ, Chao CF, Muller C (1991) Identification of two maturation-related, wheat-germ-lectin-binding proteins on the surface of mouse sperm. Acta Anat (Basel) 142:165-170[Medline]

LoLeggio L, Williams RM, Jones R (1994) Some effects of zona pellucida glycoproteins and sulfated polymers on the autoactivation of boar sperm proacrosin and activity of ß-acrosin. J Reprod Fertil 100:177-185[Abstract]

Spicer SS, Schulte BA (1992) Diversity of cell glycoconjugates shown histochemically: a perspective. J Histochem Cytochem 40:1-38[Free Full Text]

Yamashita K, Kochibe N, Ohkura T, Ueda I, Kobata A (1985) Fractionation of L-fucose-containing oligosaccharides on immobilized Auleria aurantia lectin. J Biol Chem 260:4688-4693[Abstract]