Lectin Histochemical Characterization of Glycoconjugates Present in Abomasal Epithelium of the Goat

In the abomasal epithelium of the goat, glycoconjugates have been studied by means of selective histochemical methods. The staining procedures employed were alcian blue (AB) pH 1.0 AB pH 2.5, Periodic acid Schiff (PAS) and AB pH 2.5-PAS. In addition twelve lectins, Triticum vulgaris WGA, Concana valin A, Glycine max SBA, Dolichos biflorus DBA, Arachis hypogea PNA, Solanum tuberosum STL, Datura stramonium DSA, Ulex europeus UEA-I, Bauhinia purpurea BPA, Ricinus communis RCAI, Lotus tetragonolobus LTA, and Limulus polyphemus LPA were applied to detect saccharides residues. According to the results obtained, the nature of glycoconjugates in mucous epithelium was found to change during cell migration from deep to superficial part. The surface mucous cells contained primarily neutral glycoconjugates with vicinal diol groupings and terminal fucose residues. By contrast, the mucous cells in deep pits and in pyloric glands contained acidic sulfated glycoconjugates with terminal sialic acid residues. The α-D-mannose, α-D-glucose, β-D-galactose, N-acetylglucosamine and N-acetylgalactosamine were also found at the pyloric gland cells while they were smaller in amount at the surface mucous cells. The data suggested that changes of epithelial cells were closely correlated with cellular maturation. Key word: lectin, glycoconjugates, abomasum, goat Kasetsart J. (Nat. Sci.) 33 : 234 242 (1999) 1 Department of Anatomy, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, Thailand. 2 Department of Anatomy and Physiology, College of Agriculture and Veterinary Medicine, Nihon University, Kameino 1866, Japan. INTRODUCTION Investigations in many laboratories have revealed that oligosaccharides of cell glycoconjugates have a major influence over developmental and differentiative processes, intercellular recognition, and thus also in many pathological processes, including malignancy (Taatjes and Roth, 1991; Bourillon and Aubery, 1989). A voluminous literature exists concerning the histochemistry of glycoconjgates in gastrointestinal epithelium of various mammals (Ito et al., 1985; 1983; Sato and Spicer, 1980; Sheahan and Jervis, 1976) Recently, lectin has been used as a potent probe for the demonstration of sugar residues in mammalian gastrointestinal mucosa and for discrimination of specific carbohydrate unit alterations, where conventional histochemical technique cannot perform (Aoki et al., 1993; Spicer and Schulte, 1992). Cellular and regional differences in lectin binding to mammalian gastrointestinal cells have been reported (Fischer et al., 1984; Boland et al., 1982) However, little knowledge is available concerning abomasum of the goat. In view of interesting structures and functions of goat stomach and the physiological important of glycoconjugates in this organ, glycoconjugates involved in the abomasal epithelium of this animal have been studied histochemically, employing a wealth of currently available methods of lectin histochemical staining procedures. The results obtained here are believed to involve hitherto unknown aspects of histochemical architecture of the goat stomach and give a clue to thorough recognition of the histophysiological functions performed by goat abomasal epithelium in general. MATERIALS AND METHODS A total of 3 goats of different ages and sexes were sacrificed by exsanguination under deep ether anesthesia. From the donor animals, pyloric part of abomasum has dissected out and then fixed by immersion in any one of the following fixatives. 1). 10% formalin containing 2% calcium acetate (Leppi, 1968) for 24 hrs at 4°C 2). Bouin’s fluid for 24 hrs at 4°C. The tissue specimen was then dehydrated and embedded in paraplast. On sliding microtome, sections were cut at a thickness of 1-2 μm, deparaffinized in xylene, hydrated in grade ethanol series and then subjected to the following histological and histochemical staining procedures. Conventional staining procedures 1). Haematoxylin and eosin for the general observation of histological structures. 2). Periodic acid-Schiff (PAS) for vicinal diol containing glycoconjugates (Pearse, 1968). 3). Alcian blue (AB) pH 1.0 for sulfated glycoconjugates (Lev and Spicer, 1964). 4). AB pH 2.5 for acidic glycoconjugates (Spicer et al., 1967). 5). AB pH 2.5-PAS for demonstrating of acidic and neutral glycoconjugates (Spicer et al., 1967). Lectin staining procedures Briefly, deparaffinized sections were treated with 1% bovine serum albumin (BSA) in 10 mM phosphate buffer saline (PBS) pH 7.4 and then incubated with biotinyl lectins (25 μg/ml, Vector Lab Inc. USA) in0.1% BSA-PBS for 30 min. After rinsing with PBS, the sections were incubated in avidin-biotinyl peroxidase complex (ABC Vector Lab Inc. USA) for 30 min. After briefly rinsing in PBS, the sections were immersed in 3,3diaminobenzidine (DAB, 0.2 mg/ml)-H2O2 (0.005%) for 10 min, rinsed with distilled water, dehydrated and mounted. Control experiments for lectin staining, control procedures were performed: Tissue sections were preincubated in appropriated hapten sugars for each lectin (sucrose for ConA, lactose for RCA-I and PNA, Nacetylglucosamine for WGA and STL, fucose for UEA-I and LTA, and N-acetylgalactosamine for DBA, DSA and SBA) and then incubated in lectin solutions containing the hapten sugars. Non specific staining was also checked by incubation in the ABC and DAB-H2O2 solutions.