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Histochemical analysis of glycoconjugate secretion in the alimentary canal of Anguilla anguilla L.
ARTICLE IN PRESS Acta histochemica 106 (2005) 477—487
www.elsevier.de/acthis
Histochemical analysis of glycoconjugate secretion in the alimentary canal of Anguilla anguilla L. Cinzia Domeneghinia,, Silvana Arrighia, Giuseppe Radaellib, Giampaolo Bosia, A. Veggettic a
Department of Veterinary Sciences and Technologies for Food Safety, Faculty of Veterinary Medicine, University of Milan, via Trentacoste n.2, Milan 20134, Italy b Department of Experimental Veterinary Sciences, Faculty of Veterinary Medicine, University of Padua, Italy c Department of Morphology, Physiology and Animal Production, Faculty of Veterinary Medicine, University of Bologna, Italy Received 26 January 2004; received in revised form 13 July 2004; accepted 15 July 2004
KEYWORDS Anguilla anguilla; Alimentary canal; Mucous cells; Glycoconjugates; Histochemistry; Lectins
Summary Conventional histochemical methods as well as lectin-binding techniques were used to study glycoconjugates that are present in the alimentary canal of the European eel (Anguilla anguilla). Specimens from pharynx, oesophagus, stomach and intestine were collected from adult (‘‘silver eel’’ stage) females. Alcian Blue pH 2.5/PAS and High Iron Diamine/Alcian Blue pH 2.5 reactions were performed to stain neutral and acidic glycoconjugates. In addition, lectin histochemistry was applied to identify acidic glycoconjugates containing O-acylated sialic acids. Finally, the presence of sugar residues in the oligosaccharide side chains of glycoconjugates were investigated by using biotinylated lectins. Acidic and neutral glycoconjugates were found to be secreted throughout the alimentary canal, the acidic glycoconjugates appeared to be either sialylated or sulphated. Sialylated glycoconjugates were identified to contain sialic acid substituted at carbon in position 7 (C7). Sulphated glycoconjugates were particularly abundant in the distal intestine and were not present in the secretory products of the gastric mucosa, which contained a variety of sugar residues (D-Nacetyl-galactosamine, b-D-galactose, a-D-mannose, a-L-fucose, D-N-acetyl-glucosamine). Lectin binding was observed in mucous cells of pharynx, oesophagus and intestine, and particularly some monosaccharides (D-N-acetyl-galactosamine and b-Dgalactose) were abundantly present. & 2004 Elsevier GmbH. All rights reserved.
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E-mail address: [email protected] (C. Domeneghini). 0065-1281/$ - see front matter & 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.acthis.2004.07.007
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Introduction
Material and methods
The abundant production of mucus by various epithelial tissues of the eel covering both internal and external body surfaces is well known, and has predominantly protective functions. In fish species, digestive mucosubstances, which are predominantly composed of glycoconjugates, are probably implicated in other physiological processes including lubrication, increasing digestive efficiency, promotion of macromolecular absorption, buffering of intestinal fluid, prevention of proteolytic damage to the epithelium and defense against bacteria and other pathogens. In addition, an osmotic function is especially important in fish, in the form of binding and transportation of water and various ions (Gupta, 1989; Smith, 1989; Loretz, 1995). Thus, fish gastrointestinal epithelial surfaces and their secreted glycoconjugates have a fundamental role in mediating relationships between the external environment and the body. In the present study, we focussed on epithelial cells that synthesize glycoconjugates in the gastrointestinal tract of the adult European eel (Anguilla anguilla L.), in order to characterize glycoconjugate secretion and identify possible correlations with specific functional roles of the alimentary canal. The histochemical analysis of glycoconjugates was achieved by applying conventional histochemical reactions. In addition, the presence of various sugar residues in the gut glycoconjugates was investigated by the use of a panel of biotinylated lectins (Spicer and Schulte, 1992; Spicer, 1993). The importance of glucosidic residues for structural and functional properties of glycoprotein moieties is well known (Atuma et al., 2001). The present study is one of a series of investigations of glycoconjugates in the alimentary tract of fish, such as Ictalurus melas (Domeneghini et al., 1992), Sparus aurata (Domeneghini et al., 1998), Acipenser transmontanus (Domeneghini et al., 1999a, 2002), Solea solea (Veggetti et al., 1999) and pantex, a hybrid sparid fish (Domeneghini et al., 1999b). Other authors have described the histochemical nature of mucous cells of the gut of other fish species (Reifel and Travill, 1977, 1978, 1979; Gona, 1979; Elbal and Agulleiro, 1986; Ostos-Garrido et al., 1993; Pajak and Danguy, 1993; Scocco et al., 1996, 1997, 1998; Tibbets, 1997; Arellano et al., 1999; Park and Kim, 2001; Pedini et al., 2001), highlighting remarkable differences between species.
Ten adult female Anguilla anguilla individuals were obtained in April, and were used in the present study. Body length was about 40 cm (‘‘silver eel’’ stage). Fish were killed with an overdose of MS222 (Sandoz, Milan, Italy) anaesthesia, and the alimentary tract specimens were collected immediately afterwards. Several samples of the oesophagus (proximal, medium and distal), stomach (cardiac, fundic and pyloric zones) and intestine (proximal, near the pyloric valve, and distal) were fixed for 24 h at 4 1C in one of the following fixatives: (i) 10% neutral buffered formaldehyde; (ii) 10% formol-calcium; (iii) B4G fixative (6% mercuric chloride and 0.1% glutaraldehyde in 1% sodium acetate), according to Kantani-Matsumoto and Kataoka (1989). Samples were then paraffinembedded after dehydration in a graded series of ethanol. Dewaxed sections (4 mm-thick) obtained from samples fixed in all the three fixatives were stained with haematoxylin and eosin (H&E) as well as with Azan trichromic stain to show general morphology. Sections obtained from samples fixed in B4G fluid were treated with Lugol’s iodine prior to staining. Sections obtained from tissue fixed in 10% neutral buffered formaldehyde were stained with:
diastase/periodic acid Schiff (D/PAS) to demonstrate neutral glycoconjugates and exclude the presence of glycogen. Alcian Blue 8GX pH 2.5/periodic acid-Schiff (AB/ PAS) staining, to demonstrate both acidic glycoconjugates (blue) and periodate-reactive vicinal diols (purple) (Mowry, 1963). high iron diamine/Alcian Blue 8GX pH 2.5 (HID/ AB) staining, to differentiate sulphated (brownish-black) from carboxylated, nonsulphated (blue stained) acidic glycoconjugates (Spicer, 1965; Reid et al., 1989). Astra Blue pH 2.5 (Astra Blue 6GLL; Bioptica, Milan, Italy) to visualize the sialylated glycoconjugates (azure stain) (Elie and Lecluse,1986).
Removal of terminal sialic acids in order to confirm their presence in carboxylated nonsulphated glycoconjugates (Culling et al., 1974) was performed utilizing neuraminidase (sialidase) with or without previous KOH treatment (saponification). Sections were incubated at 37 1C for 18 h in a 0.8 IU/ml solution of neuraminidase (Neu) from Clostridium perfringens (Sigma, Milan, Italy) in 0.1 M sodium acetate buffer, pH 5.5, containing 10 mM CaCl2. Saponification was performed by immersing
ARTICLE IN PRESS Glycoconjugate histochemistry in the eel alimentary canal sections in a 0.5% solution of KOH in 70% ethanol for 15 min at room temperature prior to enzymatic digestion. The presence of sialic acid in carboxylated nonsulphated glycoconjugates was determined by loss (or strong reduction) of AB reactivity after KOH-Neu-AB/PAS treatment. Sections obtained from tissue fixed in 10% formol-calcium were used for further histochemical identification of sialylated glycoconjugates (Culling et al., 1974, 1976; Culling and Reid, 1980). Glycoconjugates containing sialic acid O-acylated at different carbon positions were identified applying the following staining methods:
KOH/PAS; saponification with 0.5% KOH in 70% ethanol for 30 min at room temperature was performed to deacetylate sialic acid residues and was followed by PAS staining. PBT (periodate-borohydride-technique)/KOH/ PAS; PAS staining of preexisting vicinal diols was abolished by periodate oxidation followed by sodium borohydride reduction. KOH/PAS staining was subsequently obtained as a red colour by saponification followed by standard PAS staining. PATS (periodic acid-thionin-Schiff)/KOH/PAS to demonstrate PAS-positive glycoconjugates in blue, whereas other KOH/PAS-positive glycoconjugates appear in purple, with combinations being violet.
Sections obtained from samples fixed with either 10% neutral buffered formaldehyde or B4G fixative were used for lectin histochemistry (Spicer and Schulte, 1988). Biotinylated lectins were purchased from Vector Laboratories (Labtek, Milan, Italy). Table 1 shows the specific names of lectins, their common names and acronyms, as well as binding specificity of the sugar residues. The hapten sugars used in controls (see below) are also listed. After hydration, sections were incubated in a 0.3% solution of H2O2 in methanol for 30 min at room
Table 1. sugars
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temperature to inhibit endogenous peroxidase activity. Sections were then washed in 0.1 M phosphate-buffered saline (PBS), pH 7.6, and incubated with biotinylated lectins, at a concentration of 10 mg/ml in 10 mM HEPES buffer, pH 7.5, for 2 h at room temperature. Sections were then washed again in PBS and subsequently treated with an Avidin–Biotin–peroxidase Complex (ABC; Vector) for 1 h at room temperature. After washing in PBS, sites of lectin binding were visualized by treating sections for 5–6 min at room temperature with a freshly-prepared solution of 0.25 mg/ml 3,30 -diaminobenzidine tetrahydrochloride (DAB; Sigma) and 0.003% H2O2 in PBS. Subsequently, sections were rinsed with tap water, cleared and mounted in Eukitt (Bioptica). Negative controls for lectin binding included: (i) incubation in the buffer medium without the respective lectin, (ii) omission of ABC treatment, (iii) incubation in the presence of lectins to which the respective hapten sugars (see Table 1) were added at a concentration of 0.2 M. All these controls completely abolished all staining. As positive controls, sections of mammal (bovine, swine) alimentary canals were tested in parallel, and the expected positive results were obtained in mucous cell populations. In addition, as sialic acid is commonly linked to the specific sugar recognized by DBA lectin (Menghi and Materazzi, 1994; Gheri et al., 1995), a further control was performed to remove sialic acids before staining with DBA lectin. This was performed by incubating adjacent sections for 18 h at 37 1C in a solution of sodium acetate buffer 0.1 M, pH 5.5, containing 0.1 IU/ml sialidase (neuraminidase from Clostridium perfringens; Sigma) and 10 mM CaCl2 prior to staining with DBA lectin. The removal of sialic acids was confirmed in adjacent sections by the lack of staining with AB. Sections were observed and photographed with a BX50 photomicroscope (Olympus, Tokyo, Japan). Evaluation of staining intensities was based on subjective estimates by all authors after
Biotinylated lectins applied in the present study, with their preferential binding specificities and hapten
Lectin latin name (common name)
Acronym
Sugar specificity
Hapten sugar
Dolichos biflorus (horse gram) Ricinus communis (caster bean) Triticum vulgaris (wheat germ) Glycine maximus (soybean)
DBA RCA-I WGA SBA
N-acetyl-galactosamine Galactose N-acetyl-glucosamine N-acetyl-galactosamine
Ulex europaeus (gorse seed) Canavalia ensiformis (Jack bean)
UEA-I Con-A
a-D-N-acetyl-galactosamine b-D-galactose D-N-acetyl-glucosamine a-D-N-acetyl-galactosamine4 b-D-N-acetyl-galactosamine a-L-fucose a-D-mannose4a-D-glucose
Fucose Mannose
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examination of two sections per sample of all the animals tested.
binding. In addition, basal cells showed RCA lectin binding.
Results
Stomach
Allison (1987) showed that staining with lectins is highly variable in different species and organs, and is affected by fixation procedures. We have found here that fixation in B4G fixative was generally more effective (in terms of maximum staining with minimum background) for lectin binding in eel alimentary canal tissues than fixation in 10% neutral buffered formaldehyde, and so all results have been obtained with tissues that were fixed with B4G fixative. D/PAS staining showed that glycogen was not present in mucous cells in all the parts of the alimentary canal examined. The chemical character of mucous cells is described for each part of the alimentary canal.
The transition from oesophageal to gastric mucosa was gradual, with a progressive replacement of stratified epithelium by secretory columnar cells, which were organized as a simple surface epithelium. Ample glandular regions could be detected in the cardiac and fundic portions of the eel stomach, whereas the pyloric zone was devoid of gastric glands. Gastric glands were tubular and opened at the base of the gastric pits. Glandular cells did not stain, except for supranuclear granular PAS-positive secretory matter. Surface columnar epithelium was secretory throughout the stomach. Mucus was abundantly present in the supranuclear cytoplasm of all surface epithelial cells, and its histochemical properties were uniform all over the stomach. AB/PAS positivity was visualized by an intense purple-violet staining in surface columnar cells (Fig. 2a). HID/AB staining resulted in AB positivity only, and this was confirmed by positivity for Astra Blue (Fig. 2b). In addition, the mucous content of cells stained blue with PATS/KOH/PAS (Fig. 2c) and red with PBT/KOH/PAS (Fig. 2d). The adherent mucous gel showed the same histochemical characteristics as the surface columnar cells. SBA lectin labelling was very intense and present in adherent mucous gel and plasma membranes of surface columnar cells, as well as in gastric glands (Fig. 2e). RCA lectin bound strongly to gastric glands. RCA lectin labelling was also very intense in the basal part of the surface columnar cells all over the stomach (Fig. 2f). Several surface columnar cells and the adherent mucous gel showed weak to strong binding with DBA lectin (Fig. 2g) and ConA lectin (Fig. 2h). Neuraminidase pretreatment slightly enhanced the intensity of DBA lectin binding. UEA lectin binding was limited to the adherent mucous gel. Moderate staining was observed with WGA lectin in plasma membranes of surface columnar cells.
Pharynx/oesophagus Oesophageal mucosa was organized in longitudinal folds of different sizes. Folds were also present, albeit smaller, in the pharynx. In both organs, surface epithelium of the mucosa was stratified, with small cuboidal cells in the basal layer, columnar cells in intermediate layers and more flattened cells in the superficial layer. Numerous rounded mucous cells with flattened basal nuclei were located in all layers of the epithelium (Fig. 1a). Mucous cells were intensely AB/PASpositive and were stained dark violet (Fig. 1b). Adherent mucous gel showed the same histochemical characteristics (see also below). With HID/AB staining, cells were in part brownish-black, in part azure stained, and in part a mixed staining was present (Fig. 1c). The AB-positive mucous cells were also Astra Blue-positive, and stained red with PBT/KOH/PAS, as well as blue with PATS/KOH/PAS (Fig. 1d). Sialidase digestion abolished AB staining at pH 2.5 only when saponification was performed before. This latter result was also found in both the gastric and intestinal tracts (see below). DBA-lectin binding was present with moderate to heavy intensity all over the epithelium, mostly in the surface and intermediate layers (Fig. 1e). A similar pattern was obtained as well when using SBA lectin (Fig. 1f). However, DBA staining was present in the entire cytoplasm of the positive cells, whereas SBA staining was limited to the peripheral cytoplasm and plasma membrane. Neuraminidase treatment slightly increased DBA lectin
Intestine The gross anatomy of the eel intestine was uniform, as its diameter did not change throughout its length. Nevertheless, the folded mucosa gradually changed its micro-anatomical aspect proximodistally, as the number of mucous cells scattered among enterocytes increased towards the distal
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Figure 1. Histology and glycoconjugate histochemistry of the oesophagus in Anguilla anguilla. (a) Morphological aspect of mucosal surface stratified epithelium. Large and roundish mucous cells are present with saccular shape and flattened basal nuclei (arrowheads). Azan staining. Magnification, 250. (b) Mucous cells and adherent mucous gel (thin arrows) are homogeneously stained with AB/PAS. Magnification, 150. (c) Mucous cells show heterogeneous staining with HID/ AB: some cells are diamine positive (arrows), others are AB positive (arrowheads), and some are diamine and AB positive (asterisks). Magnification, 250. (d) Mucous cells and adherent mucous gel (thin arrows) are thionin positive. lp, Lamina propria. PATS/KOH/PAS staining. Magnification, 240. (e) DBA lectin staining is present all over the epithelium (ep) with variable intensity, mostly located in superficial and intermediate layers of the surface epithelium. l, Lumen, lp, lamina propria. B4G-fixed tissue. Magnification 150. (f) SBA lectin staining shows a localization pattern in the superficial epithelium (ep) that is similar to that of DBA lectin staining, but the staining is localized in the peripheral cytoplasm (thin arrows). lp, Lamina propria. B4G-fixed tissue. Magnification, 150.
intestine, and histochemical properties of their secretory products varied in some aspects. Most mucous cells of the proximal intestine stained violet with AB/PAS (Fig. 3a), but others were blue only, showing variable contents of acid and neutral glycoconjugates, or acid glycoconjugates only. Very few mucous cells showed PAS staining only. HID/AB staining resulted a mixed blue–black stain (Fig. 3b). Numerous mucous cells were also stained azure by Astra Blue. PATS/KOH/PAS staining demonstrated a prevalent blue stain in mucous cells (Fig. 3c), and these cells stained also red by PBT/KOH/PAS. KOH
treatment prior to sialidase treatment resulted in loss of AB positivity after AB/PAS staining, so that PAS staining was detected only in both mucous cells and adherent mucous gel (Fig. 3d). Histochemical stining of the distal intestine mucous cells was very similar, but HID/AB staining revealed a larger amount of sulphated glycoconjugates than in the proximal intestine. DBA lectin binding was present in both adherent mucous gel and mucous cells (Fig. 3e). Enzymatic digestion enhanced this staining weakly (Fig. 3f). In the distal intestine (Fig. 3g), DBA-labelled mucous
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Figure 2. Glycoconjugate histochemistry of the stomach in Anguilla anguilla. (a)–(f) Fundic region; (g) and (h) pyloric region. (a) Columnar secretory cells of surface epithelium show a uniform mixture of AB and PAS staining (asterisks). Faint PAS-positive material is present (thin arrows) in the gastric glands (gg). AB/PAS staining. Magnification, 150. (b) Mucosal surface epithelium is weakly Astra Blue positive (arrows). Magnification, 150. (c) Mucous content in surface epithelium is thionin positive after PATS/KOH/PAS staining. Arrowheads indicate unstained gastric glands. Magnification, 150. (d) Mucous content of surface epithelium is KOH/PAS-positive after PBT/KOH/PAS staining. Magnification, 150. (e) SBA lectin binding is evident in mucosal surface epithelium and gastric pits (arrowheads) as well as in gastric glands (gg). lp, Lamina propria. B4G-fixed tissue. Magnification, 150. (f) The basal zone of surface columnar cells–(arrowheads) as well as epithelial cells of the gastric glands (asterisks) are RCA–lectin positive. gg, Gastric glands. B4G-fixed tissue. Magnification, 250. (g) DBA lectin staining is present in numerous surface columnar cells (asterisks) as well as in the adherent mucous gel (arrowheads). lp, Lamina propria. B4G-fixed tissue. Magnification, 250. (h) ConA lectin binding is evident in the apical plasma membrane (arrows) as well as in the basal zone (arrowheads) of surface columnar cells. B4G-fixed tissue. Magnification, 400.
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Figure 3. Glycoconjugate histochemistry of the intestine in Anguilla anguilla. (a)–(f) Proximal intestine; (g) and (h) distal intestine. (a) Mucous cells show a mixed AB/PAS staining. Very few mucous cells show PAS positivity only (arrowheads). Magnification, 250. (b) Several mucous cells appear HID-positive, other mucous cells are AB positive (arrows) after HID/AB staining. lp, Lamina propria. Magnification, 250. (c) Mucous cells are thionin positive after PATS/KOH/PAS staining. Magnification, 400. (d) Mucous cells and adherent mucous gel (thin arrows) appear PAS positive after KOH/sialidase/AB/PAS staining. Magnification, 250. (e) DBA lectin binding is present in the adherent mucous gel (arrowheads) and, more weakly, in mucous cells (arrows). Magnification, 250. (f) Pretreatment with neuraminidase enhances DBA lectin binding in mucous cells (arrows). Staining of the adherent mucous gel (arrowheads) is not modified by pretreatment. Magnification, 150. (g) DBA lectin binding is present in mucous cells (arrows), which are more numerous and more intensely stained in the distal intestine than in the proximal intestine. Staining is present in the adherent mucous gel as well (arrowheads). Magnification, 250. (h) Neuraminidase pretreatment slightly increases DBA staining in the adherent mucous gel (arrowheads) and mucous cells (arrows). Magnification, 250.
cells were more numerous and more intensely stained than in the proximal intestine. Similarly, neuraminidase pretreatment weakly increased DBA staining in this part as well (Fig. 3h). With SBA
lectin, staining was evident in adherent mucous gel as well as in some mucous cells. No staining was found with UEA-I, Con A, WGA and RCA lectins.
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Discussion Mucous cells were present throughout the alimentary canal of Anguilla anguilla, but with different morphologies (goblet cells in the pharynx, oesophagus and intestine, and surface columnar cells in the stomach) and a limited variety in expression of glycoconjugates as revealed by their histochemical and lectin-binding properties. In the eel, stratified surface epithelium of both pharynx and oesophagus was very rich in large roundish mucous cells. Histochemical examination of these mucous cells revealed the presence of a mixture of neutral and acidic glycoconjugates, the latter belonging to both the sialylated and the sulphated type. The fact that Alcian Blue staining was abolished or strongly reduced by sialidase treatment only when previous saponification was performed indicates the presence of substituted sialic acids in sialylated glycoconjugates, since nonsubstituted sialic acids have been described as being susceptible to sialidase digestion only (Culling et al., 1974; Scocco et al., 1998). This is the case not only in eel pharynx–oesophagus, but also in stomach and intestine. We were also able to identify sialylated glycoconjugates that contain O-acylated sialic acids at the carbon in position 7 (C7) because they were stained red with PBT/KOH/PAS and blue with PATS/KOH/PAS. Diaz et al. (2001) used similar methods and described either C7- or C9-substituted sialic acids in the gill mucous cells of another fish species. The eel oesophageal mucous cells mainly contained b-D-galactose and N-acetyl-D-galactosamine. Nacetyl-D-galactosamine was localized differently in the cytoplasm of mucous cells, in its a- (entire cytoplasm) versus b- (peripheral cytoplasm) forms. Spicer and Schulte (1992) have shown that lectin binding in the peripheral cytoplasm of mammalian mucous cells is related to regulation of fluid movements. Yamamoto and Hirano (1978) have shown that eel oesophageal epithelial cells have ultrastructural aspects that are characteristic of cells highly active in reabsorbing water and solutes. Along the entire stomach surface in the eel, the mucosal epithelium secretes glycoconjugates, which were most clearly present in the supranuclear zone of epithelial cells. Their chemical composition was homogeneous. Carbohydrate histochemistry demonstrated the presence of predominantly neutral glycoconjugates but also acidic glycoconjugates. Acidic glycoconjugates were exclusively of the sialylated type and contained sialic acid substituted (O-acylated) at C7. Similar results have been reported in the stomach of Sparus
C. Domeneghini et al. aurata and Umbrina cirrosa (Domeneghini et al., 1998; Pedini et al., 2001). In contrast, a significant variety in glycoconjugate composition has been described in other species (Reifel and Travill, 1978; Sis et al., 1979; Ostos-Garrido et al., 1993). In other fish species like the sole (Veggetti et al., 1999), surface gastric epithelium was histochemically unreactive, whereas the gastric adherent mucous gel was only slightly AB positive. Mucous neck cells like those typically present in mammalian gastric glands (Suganuma et al., 1981, 1984; KantaniMatsumoto and Kataoka, 1989) were not found in the eel. Mucous neck cells were also not found in the sea bream (Domeneghini et al., 1998). In the eel, gastric glands were localized in cardiac and fundic regions of the stomach (and not in the pyloric zone, as has been found in most teleosts; Smith, 1989) and showed PAS staining in the supranuclear cytoplasm of glandular cells, demonstrating secretory neutral glycoconjugates to be limited to this cytoplasmic zone. As is usual for fish species, one cell type only was detected within gastric glands, presumably responsible for the synthesis of both hydrochloric acid and pepsinogens, and thus named ‘‘oxyntopeptic cells’’ (Ferri and Herna ´ndez-Blazquez, 1984; Ostos-Garrido et al., 1993; Garcı´a Herna ´ndez et al., 2001). Lectin histochemistry showed that D-N-acetyl-galactosamine, b-D-galactose, a-D-mannose (and perhaps, aD-glucose), D-N-acetyl-glucosamine were present in the glycoconjugates secreted by the surface columnar epithelium. Neuraminidase pretreatment slightly affected the intensity of DBA staining thus confirming the possible terminal position of sialic acid residues. The same sugars (D-N-acetyl-galactosamine, b-D-galactose, a-D-mannose, D-N-acetylglucosamine) were histochemically demonstrated in the adherent mucous gel as well, which in addition contained a-L-fucose. It is conceivable that this latter sugar, not found in surface columnar secretory cells, occupies a terminal position only after secretion to form the adherent mucous gel on top of the gastric mucosa. Surprisingly, gastric glands that were histochemically unreactive with the exception of a slight PAS positivity at the level of supranuclear cytoplasmic areas were shown to contain D-N-acetyl-galactosamine and b-D-galactose. Possibly, these sugars occupy a subterminal position in oligosaccharide branches, and so the histochemical methods applied did not reveal them. Besides the protection of the mucosa against acidic contents of the fish stomach (Morrison and Wright, 1999), mucus produced by surface columnar cells and to some extent by gastric glands may assist transport processes associated with gastric secretion.
ARTICLE IN PRESS Glycoconjugate histochemistry in the eel alimentary canal At the level of the intestine, the number of mucous cells increased towards the distal part. The large number of mucous cells in the rectum may be related to an increased need of lubrication for the ejection of faeces, and has been found in other fish species as well (Grau et al., 1992; Murray et al., 1996; Domeneghini et al., 1998; Veggetti et al., 1999; Pedini et al., 2001). The mucous cells detected in the eel intestine synthesized both neutral and acidic glycoconjugates. The acidic glycoconjugates were composed of both sialylated and sulphated glycoconjugates. In sialylated glycoconjugates, those containing sialic acid substituted at C7 were detected. This result is in agreement with to the data found in the eel pharynx–oesophagus and stomach (the present study), and it may be that sialic acid substituted at C7 is a ‘‘marker’’ of the eel mucous cells. A similar study conducted on sea bream (Domeneghini et al., 1998) showed that glycoconjugates of gut mucous secretions contained sialic acids substituted at C8. This diversity may have a taxonomical significance or may be related to feeding habits; further studies are necessary to elucidate this aspect. Within the mucous cells of the eel distal intestine, sulphated glycoconjugates predominated over sialylated glycoconjugates. In Scophthalmus maximus (Segner et al., 1994), Pleuronectes americanus (Murray et al., 1996), Sparus aurata (Domeneghini et al., 1998), and Solea solea (Veggetti et al., 1999), sulphated glycoconjugates are also abundant in mucous cells of the rectum. However, Reifel and Travill (1979) and Pedini et al. (2001) observed a chemically uniform secretory activity along the entire intestine of different teleost species, as well as a predominance of sialo-glycoconjugates. The sulpho-glycoconjugates which are abundant in mucous secretions of the distal intestine in the eel and other teleost species are presumably related to their increasing viscosity (Tibbets, 1997), and may thus possibly function in regulating the transfer of proteins or peptides, as well as in trapping of bacteria (and other pathogens). This appears to be related to the species-specific ability of distal intestine enterocytes to ingest proteins for both digestive (Sire and Vernier, 1992; Abaurrea et al., 1993; Segner et al., 1994; Garcı´a Herna´ndez et al., 2001) and defensive purposes (Rombout and Van Der Berg, 1989; Dorin et al., 1993). Finally, the special chemical characteristics of mucous secretions in the distal intestine fit well with another function that is related to the fluid balance between the external and internal environments (Smith, 1989; Loretz, 1995). Lignot et al. (2002) have recently shown that aquaporin 3, a putative water channel protein, is present in mucous cells of
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the distal intestine in the European eel. In the eel, an osmoregulatory action is performed at the intestinal level (Baldisserotto and Mimura, 1995; Trischitta et al., 1996), possibly linked with the complex life cycle of the eel, during which different salinities are encountered. We hypothesize that neuromodulators and hormonal messengers in the eel distal intestine (Domeneghini et al., 2000) may functionally sustain the intestinal osmoregulatory roles, regulating the quantity and quality of glycoconjugates secreted by epithelial cells. Lectin histochemistry revealed the presence of DN-acetyl-galactosamine in both mucous cells and adherent mucous gel, which includes the brush border in the intestinal mucosa. The demonstration of only one sugar residue (N-acetyl-galactosamine) in the eel intestine glycoconjugates strongly differentiates this species from sea bream (Domeneghini et al., 1998) and rainbow trout (Pajak and Danguy, 1993). Comparison of the results described here for the eel alimentary canal with those obtained in other teleosts is complicated by the very diversified habitats and feeding habits among these fish, the distant taxonomic positions of the species studied, and the different staining techniques used. As in other fish species, the gut mucosubstances in the eel are probably strictly related to physico-chemical environmental conditions as well as to nature of food (above all, dimensions of food particles) and frequency of food intake. In the eel gut, the composition of glycoconjugate secretions is less variable than in other fish species studied so far, perhaps because of a relationship between the diversity of secretory glycoconjugates and the salinity of the environment in which the fish species lives. So, in the silver eel the chemical composition of glycoconjugates along the entire alimentary canal is less varied than in a salt-water fish like sea bream. In the sea bream, the pharynx–oesophagus especially shows a high level of histological and histochemical complexity (Domeneghini et al., 1998), probably linked to the fact that marine fish drink salt water for osmotic balance. The osmotic load may be less for fresh water fish, and as a consequence the variety of gut sugar residues may be smaller.
Acknowledgements This research was supported by grants of the University of Milan. Authors are very grateful to Prof. Anthea Rowlerson for her kind assistance in English revision.
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ARTICLE IN PRESS Glycoconjugate histochemistry in the eel alimentary canal Murray HM, Wright GM, Goff GP. A comparative histological and histochemical study of the post-gastric alimentary canal from three species of pleuronectid, the atlantic halibut, the yellowtail flounder and the winter flounder. J Fish Biol 1996;48:187–206. Ostos-Garrido MV, Nun ˜ez-Torres MI, Abaurrea-Equisoain MA. Histological, histochemical and ultrastructural analysis of the gastric mucosa in Oncorhynchus mykiss. Aquaculture 1993;115:121–32. Park JY, Kim IS. Histology and mucin histochemistry of the gastrointestinal tract of the mud loach, in relation to respiration. J Fish Biol 2001;58:861–72. Pajak B, Danguy A. Characterization of sugar moieties and oligosaccharide sequences in the distal intestinal epithelium of the rainbow trout by means of lectin histochemistry. J Fish Biol 1993;43:709–22. Pedini V, Scocco P, Radaelli G, Fagioli O, Ceccarelli P. Carbohydrate histochemistry of the alimentary canal of the shi drum, Umbrina cirrosa L. Anat Histol Embryol 2001;30:345–9. Reid PE, Owen DA, Fletcher K, Rowan RE, Reimer CL, Rouse GJ, Park CM. The histochemical specificity of high iron diamine-Alcian blue. Histochem J 1989;21: 501–4. Reifel CW, Travill AA. Structure and carbohydrate histochemistry of the esophagus in ten teleostean species. J Morphol 1977;152:303–14. Reifel CW, Travill AA. Structure and carbohydrate histochemistry of the stomach in eight species of teleosts. J Morphol 1978;158:155–67. Reifel CW, Travill AA. Structure and carbohydrate histochemistry of the intestine in ten teleostean species. J Morphol 1979;162:343–60. Rombout JHWM, Van Der Berg AA. Immunological importance of the second gut segment of the carp. I. Uptake and processing of antigens by epithelial cells and macrophages. J Fish Biol 1989;35:13–22. Scocco P, Menghi G, Ceccarelli P. Glycohistochemistry of the Tilapia spp stomach. J Fish Biol 1996;49:584–93. Scocco P, Menghi G, Ceccarelli P. Histochemical differentiation of glycoconjugates occurring in the tilapine intestine. J Fish Biol 1997;51:848–57. Scocco P, Accili D, Menghi G, Ceccarelli P. Unusual glycoconjugates in the oesophagus of a tilapine polyhybrid. J Fish Biol 1998;53:39–48. Segner H, Storch V, Reinecke M, Kloas W, Hanke W. The development of functional digestive and metabolic
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www.elsevier.de/acthis
Histochemical analysis of glycoconjugate secretion in the alimentary canal of Anguilla anguilla L. Cinzia Domeneghinia,, Silvana Arrighia, Giuseppe Radaellib, Giampaolo Bosia, A. Veggettic a
Department of Veterinary Sciences and Technologies for Food Safety, Faculty of Veterinary Medicine, University of Milan, via Trentacoste n.2, Milan 20134, Italy b Department of Experimental Veterinary Sciences, Faculty of Veterinary Medicine, University of Padua, Italy c Department of Morphology, Physiology and Animal Production, Faculty of Veterinary Medicine, University of Bologna, Italy Received 26 January 2004; received in revised form 13 July 2004; accepted 15 July 2004
KEYWORDS Anguilla anguilla; Alimentary canal; Mucous cells; Glycoconjugates; Histochemistry; Lectins
Summary Conventional histochemical methods as well as lectin-binding techniques were used to study glycoconjugates that are present in the alimentary canal of the European eel (Anguilla anguilla). Specimens from pharynx, oesophagus, stomach and intestine were collected from adult (‘‘silver eel’’ stage) females. Alcian Blue pH 2.5/PAS and High Iron Diamine/Alcian Blue pH 2.5 reactions were performed to stain neutral and acidic glycoconjugates. In addition, lectin histochemistry was applied to identify acidic glycoconjugates containing O-acylated sialic acids. Finally, the presence of sugar residues in the oligosaccharide side chains of glycoconjugates were investigated by using biotinylated lectins. Acidic and neutral glycoconjugates were found to be secreted throughout the alimentary canal, the acidic glycoconjugates appeared to be either sialylated or sulphated. Sialylated glycoconjugates were identified to contain sialic acid substituted at carbon in position 7 (C7). Sulphated glycoconjugates were particularly abundant in the distal intestine and were not present in the secretory products of the gastric mucosa, which contained a variety of sugar residues (D-Nacetyl-galactosamine, b-D-galactose, a-D-mannose, a-L-fucose, D-N-acetyl-glucosamine). Lectin binding was observed in mucous cells of pharynx, oesophagus and intestine, and particularly some monosaccharides (D-N-acetyl-galactosamine and b-Dgalactose) were abundantly present. & 2004 Elsevier GmbH. All rights reserved.
Corresponding author. Tel.: +39 2 5031 5770; fax: +39 2 5031 5746.
E-mail address: [email protected] (C. Domeneghini). 0065-1281/$ - see front matter & 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.acthis.2004.07.007
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Introduction
Material and methods
The abundant production of mucus by various epithelial tissues of the eel covering both internal and external body surfaces is well known, and has predominantly protective functions. In fish species, digestive mucosubstances, which are predominantly composed of glycoconjugates, are probably implicated in other physiological processes including lubrication, increasing digestive efficiency, promotion of macromolecular absorption, buffering of intestinal fluid, prevention of proteolytic damage to the epithelium and defense against bacteria and other pathogens. In addition, an osmotic function is especially important in fish, in the form of binding and transportation of water and various ions (Gupta, 1989; Smith, 1989; Loretz, 1995). Thus, fish gastrointestinal epithelial surfaces and their secreted glycoconjugates have a fundamental role in mediating relationships between the external environment and the body. In the present study, we focussed on epithelial cells that synthesize glycoconjugates in the gastrointestinal tract of the adult European eel (Anguilla anguilla L.), in order to characterize glycoconjugate secretion and identify possible correlations with specific functional roles of the alimentary canal. The histochemical analysis of glycoconjugates was achieved by applying conventional histochemical reactions. In addition, the presence of various sugar residues in the gut glycoconjugates was investigated by the use of a panel of biotinylated lectins (Spicer and Schulte, 1992; Spicer, 1993). The importance of glucosidic residues for structural and functional properties of glycoprotein moieties is well known (Atuma et al., 2001). The present study is one of a series of investigations of glycoconjugates in the alimentary tract of fish, such as Ictalurus melas (Domeneghini et al., 1992), Sparus aurata (Domeneghini et al., 1998), Acipenser transmontanus (Domeneghini et al., 1999a, 2002), Solea solea (Veggetti et al., 1999) and pantex, a hybrid sparid fish (Domeneghini et al., 1999b). Other authors have described the histochemical nature of mucous cells of the gut of other fish species (Reifel and Travill, 1977, 1978, 1979; Gona, 1979; Elbal and Agulleiro, 1986; Ostos-Garrido et al., 1993; Pajak and Danguy, 1993; Scocco et al., 1996, 1997, 1998; Tibbets, 1997; Arellano et al., 1999; Park and Kim, 2001; Pedini et al., 2001), highlighting remarkable differences between species.
Ten adult female Anguilla anguilla individuals were obtained in April, and were used in the present study. Body length was about 40 cm (‘‘silver eel’’ stage). Fish were killed with an overdose of MS222 (Sandoz, Milan, Italy) anaesthesia, and the alimentary tract specimens were collected immediately afterwards. Several samples of the oesophagus (proximal, medium and distal), stomach (cardiac, fundic and pyloric zones) and intestine (proximal, near the pyloric valve, and distal) were fixed for 24 h at 4 1C in one of the following fixatives: (i) 10% neutral buffered formaldehyde; (ii) 10% formol-calcium; (iii) B4G fixative (6% mercuric chloride and 0.1% glutaraldehyde in 1% sodium acetate), according to Kantani-Matsumoto and Kataoka (1989). Samples were then paraffinembedded after dehydration in a graded series of ethanol. Dewaxed sections (4 mm-thick) obtained from samples fixed in all the three fixatives were stained with haematoxylin and eosin (H&E) as well as with Azan trichromic stain to show general morphology. Sections obtained from samples fixed in B4G fluid were treated with Lugol’s iodine prior to staining. Sections obtained from tissue fixed in 10% neutral buffered formaldehyde were stained with:
diastase/periodic acid Schiff (D/PAS) to demonstrate neutral glycoconjugates and exclude the presence of glycogen. Alcian Blue 8GX pH 2.5/periodic acid-Schiff (AB/ PAS) staining, to demonstrate both acidic glycoconjugates (blue) and periodate-reactive vicinal diols (purple) (Mowry, 1963). high iron diamine/Alcian Blue 8GX pH 2.5 (HID/ AB) staining, to differentiate sulphated (brownish-black) from carboxylated, nonsulphated (blue stained) acidic glycoconjugates (Spicer, 1965; Reid et al., 1989). Astra Blue pH 2.5 (Astra Blue 6GLL; Bioptica, Milan, Italy) to visualize the sialylated glycoconjugates (azure stain) (Elie and Lecluse,1986).
Removal of terminal sialic acids in order to confirm their presence in carboxylated nonsulphated glycoconjugates (Culling et al., 1974) was performed utilizing neuraminidase (sialidase) with or without previous KOH treatment (saponification). Sections were incubated at 37 1C for 18 h in a 0.8 IU/ml solution of neuraminidase (Neu) from Clostridium perfringens (Sigma, Milan, Italy) in 0.1 M sodium acetate buffer, pH 5.5, containing 10 mM CaCl2. Saponification was performed by immersing
ARTICLE IN PRESS Glycoconjugate histochemistry in the eel alimentary canal sections in a 0.5% solution of KOH in 70% ethanol for 15 min at room temperature prior to enzymatic digestion. The presence of sialic acid in carboxylated nonsulphated glycoconjugates was determined by loss (or strong reduction) of AB reactivity after KOH-Neu-AB/PAS treatment. Sections obtained from tissue fixed in 10% formol-calcium were used for further histochemical identification of sialylated glycoconjugates (Culling et al., 1974, 1976; Culling and Reid, 1980). Glycoconjugates containing sialic acid O-acylated at different carbon positions were identified applying the following staining methods:
KOH/PAS; saponification with 0.5% KOH in 70% ethanol for 30 min at room temperature was performed to deacetylate sialic acid residues and was followed by PAS staining. PBT (periodate-borohydride-technique)/KOH/ PAS; PAS staining of preexisting vicinal diols was abolished by periodate oxidation followed by sodium borohydride reduction. KOH/PAS staining was subsequently obtained as a red colour by saponification followed by standard PAS staining. PATS (periodic acid-thionin-Schiff)/KOH/PAS to demonstrate PAS-positive glycoconjugates in blue, whereas other KOH/PAS-positive glycoconjugates appear in purple, with combinations being violet.
Sections obtained from samples fixed with either 10% neutral buffered formaldehyde or B4G fixative were used for lectin histochemistry (Spicer and Schulte, 1988). Biotinylated lectins were purchased from Vector Laboratories (Labtek, Milan, Italy). Table 1 shows the specific names of lectins, their common names and acronyms, as well as binding specificity of the sugar residues. The hapten sugars used in controls (see below) are also listed. After hydration, sections were incubated in a 0.3% solution of H2O2 in methanol for 30 min at room
Table 1. sugars
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temperature to inhibit endogenous peroxidase activity. Sections were then washed in 0.1 M phosphate-buffered saline (PBS), pH 7.6, and incubated with biotinylated lectins, at a concentration of 10 mg/ml in 10 mM HEPES buffer, pH 7.5, for 2 h at room temperature. Sections were then washed again in PBS and subsequently treated with an Avidin–Biotin–peroxidase Complex (ABC; Vector) for 1 h at room temperature. After washing in PBS, sites of lectin binding were visualized by treating sections for 5–6 min at room temperature with a freshly-prepared solution of 0.25 mg/ml 3,30 -diaminobenzidine tetrahydrochloride (DAB; Sigma) and 0.003% H2O2 in PBS. Subsequently, sections were rinsed with tap water, cleared and mounted in Eukitt (Bioptica). Negative controls for lectin binding included: (i) incubation in the buffer medium without the respective lectin, (ii) omission of ABC treatment, (iii) incubation in the presence of lectins to which the respective hapten sugars (see Table 1) were added at a concentration of 0.2 M. All these controls completely abolished all staining. As positive controls, sections of mammal (bovine, swine) alimentary canals were tested in parallel, and the expected positive results were obtained in mucous cell populations. In addition, as sialic acid is commonly linked to the specific sugar recognized by DBA lectin (Menghi and Materazzi, 1994; Gheri et al., 1995), a further control was performed to remove sialic acids before staining with DBA lectin. This was performed by incubating adjacent sections for 18 h at 37 1C in a solution of sodium acetate buffer 0.1 M, pH 5.5, containing 0.1 IU/ml sialidase (neuraminidase from Clostridium perfringens; Sigma) and 10 mM CaCl2 prior to staining with DBA lectin. The removal of sialic acids was confirmed in adjacent sections by the lack of staining with AB. Sections were observed and photographed with a BX50 photomicroscope (Olympus, Tokyo, Japan). Evaluation of staining intensities was based on subjective estimates by all authors after
Biotinylated lectins applied in the present study, with their preferential binding specificities and hapten
Lectin latin name (common name)
Acronym
Sugar specificity
Hapten sugar
Dolichos biflorus (horse gram) Ricinus communis (caster bean) Triticum vulgaris (wheat germ) Glycine maximus (soybean)
DBA RCA-I WGA SBA
N-acetyl-galactosamine Galactose N-acetyl-glucosamine N-acetyl-galactosamine
Ulex europaeus (gorse seed) Canavalia ensiformis (Jack bean)
UEA-I Con-A
a-D-N-acetyl-galactosamine b-D-galactose D-N-acetyl-glucosamine a-D-N-acetyl-galactosamine4 b-D-N-acetyl-galactosamine a-L-fucose a-D-mannose4a-D-glucose
Fucose Mannose
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examination of two sections per sample of all the animals tested.
binding. In addition, basal cells showed RCA lectin binding.
Results
Stomach
Allison (1987) showed that staining with lectins is highly variable in different species and organs, and is affected by fixation procedures. We have found here that fixation in B4G fixative was generally more effective (in terms of maximum staining with minimum background) for lectin binding in eel alimentary canal tissues than fixation in 10% neutral buffered formaldehyde, and so all results have been obtained with tissues that were fixed with B4G fixative. D/PAS staining showed that glycogen was not present in mucous cells in all the parts of the alimentary canal examined. The chemical character of mucous cells is described for each part of the alimentary canal.
The transition from oesophageal to gastric mucosa was gradual, with a progressive replacement of stratified epithelium by secretory columnar cells, which were organized as a simple surface epithelium. Ample glandular regions could be detected in the cardiac and fundic portions of the eel stomach, whereas the pyloric zone was devoid of gastric glands. Gastric glands were tubular and opened at the base of the gastric pits. Glandular cells did not stain, except for supranuclear granular PAS-positive secretory matter. Surface columnar epithelium was secretory throughout the stomach. Mucus was abundantly present in the supranuclear cytoplasm of all surface epithelial cells, and its histochemical properties were uniform all over the stomach. AB/PAS positivity was visualized by an intense purple-violet staining in surface columnar cells (Fig. 2a). HID/AB staining resulted in AB positivity only, and this was confirmed by positivity for Astra Blue (Fig. 2b). In addition, the mucous content of cells stained blue with PATS/KOH/PAS (Fig. 2c) and red with PBT/KOH/PAS (Fig. 2d). The adherent mucous gel showed the same histochemical characteristics as the surface columnar cells. SBA lectin labelling was very intense and present in adherent mucous gel and plasma membranes of surface columnar cells, as well as in gastric glands (Fig. 2e). RCA lectin bound strongly to gastric glands. RCA lectin labelling was also very intense in the basal part of the surface columnar cells all over the stomach (Fig. 2f). Several surface columnar cells and the adherent mucous gel showed weak to strong binding with DBA lectin (Fig. 2g) and ConA lectin (Fig. 2h). Neuraminidase pretreatment slightly enhanced the intensity of DBA lectin binding. UEA lectin binding was limited to the adherent mucous gel. Moderate staining was observed with WGA lectin in plasma membranes of surface columnar cells.
Pharynx/oesophagus Oesophageal mucosa was organized in longitudinal folds of different sizes. Folds were also present, albeit smaller, in the pharynx. In both organs, surface epithelium of the mucosa was stratified, with small cuboidal cells in the basal layer, columnar cells in intermediate layers and more flattened cells in the superficial layer. Numerous rounded mucous cells with flattened basal nuclei were located in all layers of the epithelium (Fig. 1a). Mucous cells were intensely AB/PASpositive and were stained dark violet (Fig. 1b). Adherent mucous gel showed the same histochemical characteristics (see also below). With HID/AB staining, cells were in part brownish-black, in part azure stained, and in part a mixed staining was present (Fig. 1c). The AB-positive mucous cells were also Astra Blue-positive, and stained red with PBT/KOH/PAS, as well as blue with PATS/KOH/PAS (Fig. 1d). Sialidase digestion abolished AB staining at pH 2.5 only when saponification was performed before. This latter result was also found in both the gastric and intestinal tracts (see below). DBA-lectin binding was present with moderate to heavy intensity all over the epithelium, mostly in the surface and intermediate layers (Fig. 1e). A similar pattern was obtained as well when using SBA lectin (Fig. 1f). However, DBA staining was present in the entire cytoplasm of the positive cells, whereas SBA staining was limited to the peripheral cytoplasm and plasma membrane. Neuraminidase treatment slightly increased DBA lectin
Intestine The gross anatomy of the eel intestine was uniform, as its diameter did not change throughout its length. Nevertheless, the folded mucosa gradually changed its micro-anatomical aspect proximodistally, as the number of mucous cells scattered among enterocytes increased towards the distal
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Figure 1. Histology and glycoconjugate histochemistry of the oesophagus in Anguilla anguilla. (a) Morphological aspect of mucosal surface stratified epithelium. Large and roundish mucous cells are present with saccular shape and flattened basal nuclei (arrowheads). Azan staining. Magnification, 250. (b) Mucous cells and adherent mucous gel (thin arrows) are homogeneously stained with AB/PAS. Magnification, 150. (c) Mucous cells show heterogeneous staining with HID/ AB: some cells are diamine positive (arrows), others are AB positive (arrowheads), and some are diamine and AB positive (asterisks). Magnification, 250. (d) Mucous cells and adherent mucous gel (thin arrows) are thionin positive. lp, Lamina propria. PATS/KOH/PAS staining. Magnification, 240. (e) DBA lectin staining is present all over the epithelium (ep) with variable intensity, mostly located in superficial and intermediate layers of the surface epithelium. l, Lumen, lp, lamina propria. B4G-fixed tissue. Magnification 150. (f) SBA lectin staining shows a localization pattern in the superficial epithelium (ep) that is similar to that of DBA lectin staining, but the staining is localized in the peripheral cytoplasm (thin arrows). lp, Lamina propria. B4G-fixed tissue. Magnification, 150.
intestine, and histochemical properties of their secretory products varied in some aspects. Most mucous cells of the proximal intestine stained violet with AB/PAS (Fig. 3a), but others were blue only, showing variable contents of acid and neutral glycoconjugates, or acid glycoconjugates only. Very few mucous cells showed PAS staining only. HID/AB staining resulted a mixed blue–black stain (Fig. 3b). Numerous mucous cells were also stained azure by Astra Blue. PATS/KOH/PAS staining demonstrated a prevalent blue stain in mucous cells (Fig. 3c), and these cells stained also red by PBT/KOH/PAS. KOH
treatment prior to sialidase treatment resulted in loss of AB positivity after AB/PAS staining, so that PAS staining was detected only in both mucous cells and adherent mucous gel (Fig. 3d). Histochemical stining of the distal intestine mucous cells was very similar, but HID/AB staining revealed a larger amount of sulphated glycoconjugates than in the proximal intestine. DBA lectin binding was present in both adherent mucous gel and mucous cells (Fig. 3e). Enzymatic digestion enhanced this staining weakly (Fig. 3f). In the distal intestine (Fig. 3g), DBA-labelled mucous
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Figure 2. Glycoconjugate histochemistry of the stomach in Anguilla anguilla. (a)–(f) Fundic region; (g) and (h) pyloric region. (a) Columnar secretory cells of surface epithelium show a uniform mixture of AB and PAS staining (asterisks). Faint PAS-positive material is present (thin arrows) in the gastric glands (gg). AB/PAS staining. Magnification, 150. (b) Mucosal surface epithelium is weakly Astra Blue positive (arrows). Magnification, 150. (c) Mucous content in surface epithelium is thionin positive after PATS/KOH/PAS staining. Arrowheads indicate unstained gastric glands. Magnification, 150. (d) Mucous content of surface epithelium is KOH/PAS-positive after PBT/KOH/PAS staining. Magnification, 150. (e) SBA lectin binding is evident in mucosal surface epithelium and gastric pits (arrowheads) as well as in gastric glands (gg). lp, Lamina propria. B4G-fixed tissue. Magnification, 150. (f) The basal zone of surface columnar cells–(arrowheads) as well as epithelial cells of the gastric glands (asterisks) are RCA–lectin positive. gg, Gastric glands. B4G-fixed tissue. Magnification, 250. (g) DBA lectin staining is present in numerous surface columnar cells (asterisks) as well as in the adherent mucous gel (arrowheads). lp, Lamina propria. B4G-fixed tissue. Magnification, 250. (h) ConA lectin binding is evident in the apical plasma membrane (arrows) as well as in the basal zone (arrowheads) of surface columnar cells. B4G-fixed tissue. Magnification, 400.
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Figure 3. Glycoconjugate histochemistry of the intestine in Anguilla anguilla. (a)–(f) Proximal intestine; (g) and (h) distal intestine. (a) Mucous cells show a mixed AB/PAS staining. Very few mucous cells show PAS positivity only (arrowheads). Magnification, 250. (b) Several mucous cells appear HID-positive, other mucous cells are AB positive (arrows) after HID/AB staining. lp, Lamina propria. Magnification, 250. (c) Mucous cells are thionin positive after PATS/KOH/PAS staining. Magnification, 400. (d) Mucous cells and adherent mucous gel (thin arrows) appear PAS positive after KOH/sialidase/AB/PAS staining. Magnification, 250. (e) DBA lectin binding is present in the adherent mucous gel (arrowheads) and, more weakly, in mucous cells (arrows). Magnification, 250. (f) Pretreatment with neuraminidase enhances DBA lectin binding in mucous cells (arrows). Staining of the adherent mucous gel (arrowheads) is not modified by pretreatment. Magnification, 150. (g) DBA lectin binding is present in mucous cells (arrows), which are more numerous and more intensely stained in the distal intestine than in the proximal intestine. Staining is present in the adherent mucous gel as well (arrowheads). Magnification, 250. (h) Neuraminidase pretreatment slightly increases DBA staining in the adherent mucous gel (arrowheads) and mucous cells (arrows). Magnification, 250.
cells were more numerous and more intensely stained than in the proximal intestine. Similarly, neuraminidase pretreatment weakly increased DBA staining in this part as well (Fig. 3h). With SBA
lectin, staining was evident in adherent mucous gel as well as in some mucous cells. No staining was found with UEA-I, Con A, WGA and RCA lectins.
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Discussion Mucous cells were present throughout the alimentary canal of Anguilla anguilla, but with different morphologies (goblet cells in the pharynx, oesophagus and intestine, and surface columnar cells in the stomach) and a limited variety in expression of glycoconjugates as revealed by their histochemical and lectin-binding properties. In the eel, stratified surface epithelium of both pharynx and oesophagus was very rich in large roundish mucous cells. Histochemical examination of these mucous cells revealed the presence of a mixture of neutral and acidic glycoconjugates, the latter belonging to both the sialylated and the sulphated type. The fact that Alcian Blue staining was abolished or strongly reduced by sialidase treatment only when previous saponification was performed indicates the presence of substituted sialic acids in sialylated glycoconjugates, since nonsubstituted sialic acids have been described as being susceptible to sialidase digestion only (Culling et al., 1974; Scocco et al., 1998). This is the case not only in eel pharynx–oesophagus, but also in stomach and intestine. We were also able to identify sialylated glycoconjugates that contain O-acylated sialic acids at the carbon in position 7 (C7) because they were stained red with PBT/KOH/PAS and blue with PATS/KOH/PAS. Diaz et al. (2001) used similar methods and described either C7- or C9-substituted sialic acids in the gill mucous cells of another fish species. The eel oesophageal mucous cells mainly contained b-D-galactose and N-acetyl-D-galactosamine. Nacetyl-D-galactosamine was localized differently in the cytoplasm of mucous cells, in its a- (entire cytoplasm) versus b- (peripheral cytoplasm) forms. Spicer and Schulte (1992) have shown that lectin binding in the peripheral cytoplasm of mammalian mucous cells is related to regulation of fluid movements. Yamamoto and Hirano (1978) have shown that eel oesophageal epithelial cells have ultrastructural aspects that are characteristic of cells highly active in reabsorbing water and solutes. Along the entire stomach surface in the eel, the mucosal epithelium secretes glycoconjugates, which were most clearly present in the supranuclear zone of epithelial cells. Their chemical composition was homogeneous. Carbohydrate histochemistry demonstrated the presence of predominantly neutral glycoconjugates but also acidic glycoconjugates. Acidic glycoconjugates were exclusively of the sialylated type and contained sialic acid substituted (O-acylated) at C7. Similar results have been reported in the stomach of Sparus
C. Domeneghini et al. aurata and Umbrina cirrosa (Domeneghini et al., 1998; Pedini et al., 2001). In contrast, a significant variety in glycoconjugate composition has been described in other species (Reifel and Travill, 1978; Sis et al., 1979; Ostos-Garrido et al., 1993). In other fish species like the sole (Veggetti et al., 1999), surface gastric epithelium was histochemically unreactive, whereas the gastric adherent mucous gel was only slightly AB positive. Mucous neck cells like those typically present in mammalian gastric glands (Suganuma et al., 1981, 1984; KantaniMatsumoto and Kataoka, 1989) were not found in the eel. Mucous neck cells were also not found in the sea bream (Domeneghini et al., 1998). In the eel, gastric glands were localized in cardiac and fundic regions of the stomach (and not in the pyloric zone, as has been found in most teleosts; Smith, 1989) and showed PAS staining in the supranuclear cytoplasm of glandular cells, demonstrating secretory neutral glycoconjugates to be limited to this cytoplasmic zone. As is usual for fish species, one cell type only was detected within gastric glands, presumably responsible for the synthesis of both hydrochloric acid and pepsinogens, and thus named ‘‘oxyntopeptic cells’’ (Ferri and Herna ´ndez-Blazquez, 1984; Ostos-Garrido et al., 1993; Garcı´a Herna ´ndez et al., 2001). Lectin histochemistry showed that D-N-acetyl-galactosamine, b-D-galactose, a-D-mannose (and perhaps, aD-glucose), D-N-acetyl-glucosamine were present in the glycoconjugates secreted by the surface columnar epithelium. Neuraminidase pretreatment slightly affected the intensity of DBA staining thus confirming the possible terminal position of sialic acid residues. The same sugars (D-N-acetyl-galactosamine, b-D-galactose, a-D-mannose, D-N-acetylglucosamine) were histochemically demonstrated in the adherent mucous gel as well, which in addition contained a-L-fucose. It is conceivable that this latter sugar, not found in surface columnar secretory cells, occupies a terminal position only after secretion to form the adherent mucous gel on top of the gastric mucosa. Surprisingly, gastric glands that were histochemically unreactive with the exception of a slight PAS positivity at the level of supranuclear cytoplasmic areas were shown to contain D-N-acetyl-galactosamine and b-D-galactose. Possibly, these sugars occupy a subterminal position in oligosaccharide branches, and so the histochemical methods applied did not reveal them. Besides the protection of the mucosa against acidic contents of the fish stomach (Morrison and Wright, 1999), mucus produced by surface columnar cells and to some extent by gastric glands may assist transport processes associated with gastric secretion.
ARTICLE IN PRESS Glycoconjugate histochemistry in the eel alimentary canal At the level of the intestine, the number of mucous cells increased towards the distal part. The large number of mucous cells in the rectum may be related to an increased need of lubrication for the ejection of faeces, and has been found in other fish species as well (Grau et al., 1992; Murray et al., 1996; Domeneghini et al., 1998; Veggetti et al., 1999; Pedini et al., 2001). The mucous cells detected in the eel intestine synthesized both neutral and acidic glycoconjugates. The acidic glycoconjugates were composed of both sialylated and sulphated glycoconjugates. In sialylated glycoconjugates, those containing sialic acid substituted at C7 were detected. This result is in agreement with to the data found in the eel pharynx–oesophagus and stomach (the present study), and it may be that sialic acid substituted at C7 is a ‘‘marker’’ of the eel mucous cells. A similar study conducted on sea bream (Domeneghini et al., 1998) showed that glycoconjugates of gut mucous secretions contained sialic acids substituted at C8. This diversity may have a taxonomical significance or may be related to feeding habits; further studies are necessary to elucidate this aspect. Within the mucous cells of the eel distal intestine, sulphated glycoconjugates predominated over sialylated glycoconjugates. In Scophthalmus maximus (Segner et al., 1994), Pleuronectes americanus (Murray et al., 1996), Sparus aurata (Domeneghini et al., 1998), and Solea solea (Veggetti et al., 1999), sulphated glycoconjugates are also abundant in mucous cells of the rectum. However, Reifel and Travill (1979) and Pedini et al. (2001) observed a chemically uniform secretory activity along the entire intestine of different teleost species, as well as a predominance of sialo-glycoconjugates. The sulpho-glycoconjugates which are abundant in mucous secretions of the distal intestine in the eel and other teleost species are presumably related to their increasing viscosity (Tibbets, 1997), and may thus possibly function in regulating the transfer of proteins or peptides, as well as in trapping of bacteria (and other pathogens). This appears to be related to the species-specific ability of distal intestine enterocytes to ingest proteins for both digestive (Sire and Vernier, 1992; Abaurrea et al., 1993; Segner et al., 1994; Garcı´a Herna´ndez et al., 2001) and defensive purposes (Rombout and Van Der Berg, 1989; Dorin et al., 1993). Finally, the special chemical characteristics of mucous secretions in the distal intestine fit well with another function that is related to the fluid balance between the external and internal environments (Smith, 1989; Loretz, 1995). Lignot et al. (2002) have recently shown that aquaporin 3, a putative water channel protein, is present in mucous cells of
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the distal intestine in the European eel. In the eel, an osmoregulatory action is performed at the intestinal level (Baldisserotto and Mimura, 1995; Trischitta et al., 1996), possibly linked with the complex life cycle of the eel, during which different salinities are encountered. We hypothesize that neuromodulators and hormonal messengers in the eel distal intestine (Domeneghini et al., 2000) may functionally sustain the intestinal osmoregulatory roles, regulating the quantity and quality of glycoconjugates secreted by epithelial cells. Lectin histochemistry revealed the presence of DN-acetyl-galactosamine in both mucous cells and adherent mucous gel, which includes the brush border in the intestinal mucosa. The demonstration of only one sugar residue (N-acetyl-galactosamine) in the eel intestine glycoconjugates strongly differentiates this species from sea bream (Domeneghini et al., 1998) and rainbow trout (Pajak and Danguy, 1993). Comparison of the results described here for the eel alimentary canal with those obtained in other teleosts is complicated by the very diversified habitats and feeding habits among these fish, the distant taxonomic positions of the species studied, and the different staining techniques used. As in other fish species, the gut mucosubstances in the eel are probably strictly related to physico-chemical environmental conditions as well as to nature of food (above all, dimensions of food particles) and frequency of food intake. In the eel gut, the composition of glycoconjugate secretions is less variable than in other fish species studied so far, perhaps because of a relationship between the diversity of secretory glycoconjugates and the salinity of the environment in which the fish species lives. So, in the silver eel the chemical composition of glycoconjugates along the entire alimentary canal is less varied than in a salt-water fish like sea bream. In the sea bream, the pharynx–oesophagus especially shows a high level of histological and histochemical complexity (Domeneghini et al., 1998), probably linked to the fact that marine fish drink salt water for osmotic balance. The osmotic load may be less for fresh water fish, and as a consequence the variety of gut sugar residues may be smaller.
Acknowledgements This research was supported by grants of the University of Milan. Authors are very grateful to Prof. Anthea Rowlerson for her kind assistance in English revision.
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