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Glycoconjugates in the mucosa of the digestive tract of Cynoscion guatucupa: A histochemical study
ARTICLE IN PRESS Acta histochemica 110 (2008) 76—85
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Glycoconjugates in the mucosa of the digestive tract of Cynoscion guatucupa: A histochemical study Alcira Ofelia Dı´aza,, Alicia Mabel Garcı´ab, Adriana Lı´a Goldemberga a
Departamento de Biologı´a, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata, Funes 3250 31 piso, 7600 Mar del Plata, Argentina b Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas (CONICET), 1033AAJ Buenos Aires, Argentina Received 11 July 2007; received in revised form 13 August 2007; accepted 21 August 2007
KEYWORDS Cynoscion guatucupa; Digestive tract; Mucous cells; Glycoconjugates; Histochemistry; Lectins
Summary This study addresses the histomorphology, the histochemistry and the distribution of glycoconjugates (GCs) in the mucosa of the digestive tract of the weakfish Cynoscion guatucupa. The histological characterization of the buccopharyngeal cavity and the esophagus revealed that they are lined by a stratified epithelium where the mucosa is thrown into longitudinal folds. The stomach is lined with a simple columnar secretory epithelium with tubular glands. GCs were analyzed using a range of histochemical methods. They were identified as oxidizable vicinal diols; sialic acids and some of their chain variants, C7, C8 or C9; sialic acid residues with O-acyl substitution at C8 or C9; carboxyl groups and sulfate groups. Sugar residues in the oligosaccharide side chains of the GCs were investigated using seven biotinylated lectins. Mucous cells from the contents of the buccopharyngeal cavity, esophagus and stomach evidenced neutral, sulphated and sialylated GCs. The latter were substituted mainly in C8. A moderately strong lectin labeling was observed in mucous cells of the three organs studied. Nonetheless, the apical edge of the gastric gland cells showed a strong positive labeling. The presence of different classes of GCs has been associated with different functions, such as lubrication, protection, inhibition of microorganisms proliferation and ionic regulation. & 2007 Elsevier GmbH. All rights reserved.
Introduction Corresponding author. Tel.: +54 223 475 2426;
fax: +54 223 475 3150. E-mail address: [email protected] (A.O. Dı´az).
The digestive tract of fishes shows a marked diversity of morphology and function. This is related both to different feeding habits and to
0065-1281/$ - see front matter & 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.acthis.2007.08.002
ARTICLE IN PRESS Histochemistry of the Cynoscion guatucupa digestive tract taxonomy, as well as to body shape, size and sex (Kapoor et al., 1975). The occurrence of mucus is a common feature in the digestive tract of teleosts. The mucosubstances secreted differ between species and also along the fish alimentary canal (Dı´az et al., 2003). Mucus is a highly hydrated gel that consists approximately of 95% water and 5% mucins, plus minor components like electrolytes (Neutra and Forstner, 1987). Mucins are high molecular weight glycoproteins that form highly glycosylated gels (Schumacher et al., 2004). Mucosubstances are crucial for the maintenance of adequate functioning of epithelial tissues (Niew Amerongen et al., 1995). The distribution and presence of glycoproteins is correlated with different functions, such as lubrication, protection against proteolytic degradation and inhibition of microorganisms (Reid et al., 1988). In addition, an osmotic function is especially important in fishes, in the form of binding and transport of water and various ions (Loretz, 1995). The stripped weakfish, Cynoscion guatucupa Cuvier (Pisces, Perciformes, Scianidae), is a demersal coastal fish of growing commercial value with a wide distribution from 221350 S (Rio de Janeiro, Brazil) to approximately 431S (Argentina) (Cousseau and Perrotta, 2000). The aim of this study was to analyze the carbohydrate composition of mucins in mucussecreting cells of the digestive tract mucosa of C. guatucupa, from the bucopharyngeal cavity to the stomach. Classical and specific mucin and lectin histochemical techniques were used to achieve this aim. Lectins are naturally occurring carbohydratebinding proteins that are not enzymes or antibodies. They are powerful and reliable tools to characterize
77
glycoconjugates (GCs) in tissues. Lectin histochemical studies have demonstrated the utility of lectins as biomarkers of specific secretory functions, structural components and alterations of cell and tissues (Chan and Ho, 1999). The present work introduces for the first time histological and histochemical studies of the mucosa of the digestive tract of C. guatucupa.
Material and methods Live specimens of C. guatucupa (46.473.0 cm length; 879.07181.0 g weight; n ¼ 10) were collected from the coast of Mar del Plata, Argentina (381050 S, 571320 W). Fish were killed by decapitation. The digestive tract was rapidly removed and fixed by immersion in Bouin’s fluid or 10% buffered formalin for light microscope studies. Samples were routinely processed and embedded in paraffin wax. Four micrometer-thick histological sections were cut by microtome, prepared according to standard protocol and then stained using the following techniques: routine hematoxylin and eosin (H–E) stain, Masson trichrome stain for morphology and Mayer mucicarmin for mucin identification. Sections of tissue were also subjected to histochemical procedures for the identification of GCs, as detailed in Table 1. Sections were stained with (1) PAS (periodic acid Schiff’s reagent) to demonstrate periodate-reactive vicinal diols, (2) PA*S (selective periodic acid Schiff reaction): oxidation for 1 h at 4 1C with 0.4 mM periodic acid in approximately 1 M hydrochloric acid is used as a specific reagent for the selective
Table 1. Histochemical procedures for visualizing and identifying GCs in mucous cells of Cynoscion guatucupa digestive tract Procedures
Interpretation of staining reactions
References
1 PAS
GCs with oxidizable vicinal diols and/or glycogen
Mc Manus (1948)
2 PA*S
Sialic acid and some of their chain variants (C7 and/or C9)
Volz et al. (1987)
3 KOH/PA*S
GCs with sialic acid residues
Culling et al. (1976)
4 KOH/PA*/Bh/PAS
Neutral GCs with oxidizable vicinal diols
Volz et al. (1987)
5 PA/Bh/KOH/PAS
Sialic acid residues with O-acyl substitution at C7, C8 or C9 and Oacyl sugars
Reid et al. (1973)
6 AB pH 2.5
GCs with carboxyl groups (sialic acid or urinic acid) and/or with O-sulphate esters
Lev and Spicer (1964)
7 AB pH 2.5/PAS
GCs with carboxyl groups and/or with O-sulphate esters
Mowry (1956)
Symbols: PAS, periodic acid/Schiff; PA*S, periodic acid/Schiff at low temperature and low pH (oxidation with 0.4 mM periodic acid in 1.0 M hydrochloric acid at 4 1C); PA, periodic acid; KOH, saponification; Bh, borohydride; AB, Alcian blue; GCs, glycoconjugates.
ARTICLE IN PRESS 78
(3)
(4)
(5)
(6)
(7)
A.O. Dı´az et al. visualization of sialic acids in the PAS procedure. The selectivity of the reaction is the result of an increase in the rate of the oxidation of the sialic acid residues together with a decrease in the rate of oxidation of neutral sugars, KOH/PA*S (saponification-selective periodic acid Schiff reaction) to allow the characterization of total sialic acids. The saponification with 0.5% potassium hydroxide in 70% ethanol for 30 min at room temperature was performed to deacetylate sialic acid residues and was followed by PA*S, KOH/PA*/Bh/PAS (saponification-selective periodic acid-borohydride reduction-periodic acid Schiff reaction) for the characterization of neutral sugars, PA/Bh/KOH/PAS (periodic acid-borohydride reduction-saponification-periodic acid Schiff reaction): this method was carried out using a 2-h oxidation at room temperature with 1% periodic acid. The aldehydes generated by the initial oxidation are reduced to Schiff-unreactive primary alcohols with sodium borohydride (PA-Bh). Following saponification (KOH), only sialic acids with O-acyl substituents at C7, C8 or C9 (or which had two or three side-chains O-acyl substituents) are PAS positive, AB pH 2.5 (Alcian Blue 8GX pH 2.5): to demonstrate GCs with carboxyl groups (sialic acid or uronic acid) and/or with O-sulfate esters, AB pH 2.5/PAS (Alcian Blue 8GX pH 2.5/periodic acid Schiff staining): to demonstrate both acidic GCs (blue) and periodate-reactive vicinol diols (purple).
Labeling with biotinylated lectins was used to identify specific sugar residues of GCs. Lectin labeling was performed according to the method described by Gimeno et al. (1995). Briefly, paraffin wax sections were mounted on slides coated with poly-L-lysine (Sigma Diagnostics, St. Louis, MO, USA). These were deparaffinized with xylene, then incubated in 0.3% H2O2 in methanol for 30 min at room temperature in order to block endogenous peroxidase activity. They were then hydrated through a graded ethanol series, washed in a phosphate-buffered saline (PBS) 0.01 M, pH 7.2, and incubated for 30 min with biotinylated lectins. Table 2 lists the seven lectins used in this study, their sources and their major sugar specificities. All lectins were employed at a dilution of 30 mg/ml in PBS, except for PNA which was applied at a concentration of 10 mg/ml. Biotinylated lectins were purchased from Vector Laboratories Inc.,
Table 2. Carbohydrate binding specificity of lectins employed in this study Lectin
Acronym
Specificity/ hapten sugar,y
Canavalia ensiformis agglutinin
Con-A
a-D-Man; a-D-Glc
Triticum vulgaris (wheatgerm) agglutinin
WGA
b-D-GlcNAc; NeuNAc
Dolichos biflorus agglutinin
DBA
a-D-GalNAc
Glycine max (soyabean) agglutinin
SBA
a-D-GalNAc; b-DGalNAc
Arachis hypogaea (peanut) agglutinin
PNA
b-D-Gal (b1-3) D-GalNAc
Ulex europaeus agglutinin-I
UEA-I
a-L-Fuc
Ricinus communis agglutinin-I
RCA-I
b-Gal
Goldstein and Hayes (1978). y Fuc, fucose; Gal, galactose; GalNAc, N-acetylgalactosamine; Glc, glucose; GlcNAc, N-acetylglucosamine; Man, mannose; NeuNAc, acetyl neuraminic acid (sialic acid).
Burlingame, CA, USA. Afterwards, sections were incubated for 30 min with ABC reagent, prepared according to manufacturer’s instructions (Vectastain Elite PK 6200 Vector Laboratories Inc, Burlingame, CA, USA). Lectin binding was revealed by incubation with 0.5 mg/mol diaminobenzidine tetrahydrochloride (DAB) (DAKO) in Tris buffer 0.1 M, pH 7.2, plus 0.02% H2O2. All dilutions and thorough washes between steps were carried out using PBS unless otherwise stated. Two types of controls were performed: (1) lectin solution was replaced by PBS and (2) lectin labeling was performed as described above after lectins had been preincubated for 1 h in the presence of the appropriate hapten sugars (0.2 M in PBS), as listed in Table 2, at room temperature. No labeling was detected in control sections. Evaluation of labeling intensities was based on subjective estimates by all authors after examination of two sections per sample of all the animals tested.
Results The results of histochemistry to reveal GCs in the mucosa of the digestive tract of C. guatucupa are summarized in Table 3 and the results of lectin
ARTICLE IN PRESS Histochemistry of the Cynoscion guatucupa digestive tract Table 3.
79
Histochemical reactions of GCs in the mucosa of the digestive tract of Cynoscion guatucupa
Procedures
Mucous cells
Stomach
Buccopharyngeal
PAS PA*S KOH/PA*S KOH/PA*/Bh/PAS PA/Bh/KOH/PAS AB pH 2.5 AB pH 2.5/PAS
Esophagus
3 1 1 3 2 3 3
3 2 3 3 3 3 3
Secretor epithelium
Glands
Cell coat
Cells
Apical edge
Cells
3 1 1 2 2 0 3
3 1 1 3 3 0 3
2 2 2 3 2 0 2
2 2 2 2 2 0 2
Staining intensity: 0, negative; 1, weak; 2, moderate; 3, strong.
Table 4.
Con A WGA DBA SBA PNA UEA I RCA I
Lecting binding in the mucosa of the digestive tract of Cynoscion guatucupa Buccopharyngeal cavity
Esophagus
Cell coat
Cell coat
2 2 2 2 2 2 1
Mucous cells
1 2–3 0–1 1 1 0–1 0
2 2 2 3 3 3 2
Stomach Mucous cells
0–1 0 1–2 2 2 0 0
Secretor epithelium
Glands
Cell coat
Cells
Apical edge
Cells
3 3 3 3 3 3 3
1 1 1 1 1 1 1
0 3 3 3 3 3 3
0 1 2 2 2 0 1
Labeling intensity: 0, negative; 1, weak; 2, moderate; 3, strong.
histochemistry are summarized in Table 4. They are explained in more detail below.
Buccopharyngeal cavity/esophagus The buccopharyngeal cavity was situated in the posterior portion of the head; together with the gill arch it constituted the gill chamber that filters and retains food. The esophagus was short and possessed the thickest wall of the digestive tract. The tunica mucosa of the buccopharyngeal cavity and the esophagus featured a great number of different sized longitudinal folds, separated by well-pronounced furrows in the esophagus. In both organs, the mucosal epithelium was stratified, with high cuboidal cells in the basal layer, cuboidal cells in the intermediate layers and more flattened cells in the superficial layers. A great many large mucous cells with a foamyappearing cytoplasm were visible in between the epithelial layers. The cytoplasm of mucous cells was granular and contained a heavily PAS-reactive material in the buccopharyngeal cavity as well as in
the esophagus (Figure 1A). The PAS positivity disappeared after acetylation, and it was recovered following saponification. Sections subjected to a-amylase, to demonstrate periodate reactive vicinal diols and exclude the presence of glycogen, were positive for the PAS reaction after the same treatment. The mucous cells were alcianophilic (AB positive) and stained violet with the Alcian blue pH 2.5-PAS sequence (Figure 1B and C). In addition, these secretory cells were stained red by the KOH/PA*/Bh/PAS reaction. Mucous cells in the buccopharyngeal cavity reacted weakly to the PA*S and KOH/PA*S, and stained moderately with the PA/Bh/KOH/PAS sequence. Mucous cells in the esophagus were strongly stained red by PA/Bh/KOH/PAS and showed KOH/PA*S positivity. They stained moderately with PA*S. A different type of mucous cell, identified through KOH/PA*/ Bh/PAS, was encountered in this organ. These cells stained more intensely than the rest of the secretory cells with the last technique. Mucous cells in the buccopharyngeal cavity labeled moderately to strongly with WGA and weakly with Con A, SBA and PNA lectins. Mucous
ARTICLE IN PRESS 80
A.O. Dı´az et al.
Figure 1. Histochemistry of the buccopharyngeal cavity, esophagus and stomach of C.guatucupa. (A) Buccopharyngeal cavity. The mucous cells are PAS-reactive (arrow). Scale bar ¼ 60 mm. (B) Esophagus AB pH 2.5. The mucous cells are AB reactive (arrow). Scale bar ¼ 12 mm. (C) Esophagus AB pH 2.5/PAS sequence. The mucous cells are positive (arrow). Scale bar ¼ 40 mm. (D) Stomach. The epithelium (asterisk) and the gastric glands (arrow) are PAS-reactive. Scale bar ¼ 80 mm. (E) Stomach (PA/Bh/KOH/PAS). The epithelial secretory cells (arrow) and the gland cells (arrowhead) are positively stained. Scale bar ¼ 60 mm. (F) Stomach (KOH/PA*/Bh/PAS). The epithelial secretory cells (arrow) and the gland cells (arrowhead) are positive. Scale bar ¼ 60 mm.
cells in the esophagus labeled moderately with SBA and PNA and weakly to moderately with DBA lectins. The adherent mucus gel of the buccopharyngeal cavity and the esophagus was labeled by all the lectins tested (Figure 2A–H and Table 4). Stomach The transition from esophageal to gastric mucosa was abrupt. The stratified epithelium of the esophagus was replaced by the secretory columnar epithelium that lines the whole surface of the stomach. Glands were tubular and opened at the base of the gastric pits. Mucus was abundant in the supranuclear cytoplasmic zone of all the surface secretory cells, and their histochemical properties remained uniform throughout the whole organ. Thus, the simple columnar epithelium that lined the gastric mucosa was strongly PAS positive. It was unreactive with Alcian blue and stained
magenta with the Alcian blue pH 2.5/PAS sequence. The epithelial secretory cells also showed a strong reaction with PA/Bh/KOH/PAS and KOH/PA*/ Bh/PAS, and were stained pale red by the PA*S reaction and the KOH/PA*S sequence (Figure 1D–F). Adherent mucous gel showed similar histochemical characteristics. The epithelium lining the gastric mucosa and its luminal surface labeled for binding of all lectins tested. The cytoplasmic contents of the epithelial cells weakly labeled with these lectins, while the cell coat showed a strong positive labeling for binding of all the lectins (Figure 2I–L). The gastric glands were reactive with the different histochemical methods, showing only minor variation in reactivity. Thus, the epithelial cells which made up the glands displayed a moderate reaction to PAS, PA*S, PA/Bh/KOH/PAS, KOH/PA*/ Bh/PAS and to KOH/PA*S (Figure 1D–F). They labeled
ARTICLE IN PRESS Histochemistry of the Cynoscion guatucupa digestive tract
81
Figure 2. Lectin histochemistry of the buccopharyngeal cavity, esophagus and stomach of C.guatucupa. Buccopharyngeal cavity: (A) PNA. Scale bar ¼ 52 mm. (B) DBA. Scale bar ¼ 42 mm. (C) UEA-I. Scale bar ¼ 52 mm. (D) WGA. Scale bar ¼ 24 mm. Esophagus: (E) UEA-I. Scale bar ¼ 28 mm. (F) DBA. Scale bar ¼ 28 mm. (G) PNA. Scale bar ¼ 78 mm. (H) Con A. Scale bar ¼ 40 mm. Stomach: (I) SBA. Scale bar ¼ 100 mm. (J) PNA. Scale bar ¼ 68 mm. (K) RCA-l. Scale bar ¼ 60 mm. (L) UEA-I. Scale bar ¼ 100 mm. Adherent mucus gel (arrow), mucous cells (asterisk) and gland cells (star).
moderately for binding of DBA, SBA and PNA, and weakly for RCA-I and WGA. The apical edge of the gland cells featured strong positive labeling for binding of the same lectins (Figure 2I–L).
Discussion This study provides the first description of the histology and the histochemistry of GCs in the digestive tract of C. guatucupa. In addition, the
present work is one of a series describing GCs in the alimentary tract of fish such as Micropogonias furnieri (Dı´az et al., 2002), Engraulis anchoita (Dı´az et al., 2003) and Odontesthes bonariensis (Dı´az et al., 2005). Secretion of mucus is a common characteristic of the C. guatucupa organs analyzed in this study. Nonetheless, the morphology of the cell producing mucus varies from mucous cells in the buccopharyngeal cavity and the esophagus to surface secretory epithelium in the stomach. These cells also
ARTICLE IN PRESS 82 display diversity in the presence of GCs, as revealed by their histochemical and lectin-binding properties. The general morphology of the mucosa of the portion of the digestive system studied here is in accordance with the morphologies described for the most part in fishes (Dı´az et al., 2003; Domeneghini et al., 2005). However, in some species, the distribution and characteristics of the mucus secreting cells deviate from the general pattern. This is most commonly seen in the esophagus, where the presence of stratified epithelium with mucous cells usually presents morphological variations. This is the case in the esophagus of Seriola dumerrili (Grau et al., 1992), Anguilla anguilla (Abaurrea-Equisoain and Ostos-Garrido, 1996) and Engraulis anchoita (Dı´az et al., 2003), where two zones are evident: a cranial one with a few layers of squamous stratified epithelium with numerous superficial mucous cells and a caudal zone with that epithelium replaced by a simple columnar epithelium involved in secretion processes. In the study described here, mucous cells from the contents of the buccopharyngeal cavity and the esophagus evidenced neutral GCs, GCs with sialic acids and some of their side chain variants, and carboxylated and sulphated GCs. The fact that mucous cells in the buccopharyngeal cavity and the esophagus stained intensely with PAS and amylase/PAS, indicates that cells must contain neutral hexoses. These results indicate an absence of glycogen. This has been demonstrated in other teleosts, in which mucous cells react in the same way with these histochemical techniques (Sarasquete et al., 1998a, b; Dı´az et al., 2001). The Alcian blue sequence revealed sulphated GCs that could be responsible for an increasing viscosity of the secretions. The visualization of non-substituted and substituted sialic acids at C7, C8 and C9 was confirmed. In the buccopharyngeal cavity, weak reactions for PA*S and KOH/PA*S and moderate reactions for the sequence PA/Bh/KOH/PAS were evidenced. This indicates a scarce quantity of substituted sialic acids in C7, C9, and non-substituted sialic acids and the greatest presence of substituted sialic acids in C8. On the other hand, in the esophagus, PA/Bh/ KOH/PAS and KOH/PA*S staining was strong, while PA*S staining was moderate, thus indicating the presence of total and substituted sialic acids in C7 and C8. Domeneghini et al. (2005) used similar methods and described O-acylated sialic acids at C7 in the mucous cells throughout the alimentary canal of Anguilla anguilla. The presence of sialic acid substituted at C8 has also been confirmed in
A.O. Dı´az et al. the buccopharyngeal cavity and the esophagus of larval stages, juveniles and adults of Sparus auratus (Domeneghini et al., 1998). Because of their negative charge, sialic acids have been proposed to give rise to extended GC molecules and to play a role in the hydration of their immediate environment (Mittal et al., 1994). The presence of neutral GCs was confirmed with the KOH/PA*/Bh/PAS reaction. This also identified two cell types in the esophagus, not distinguished using other methods. Gargiulo et al. (1996) and Scocco et al. (1998) described periodic acid-Schiffstained and Alcian blue-stained goblet cells in the esophagus, but did not describe a second type of secretory cell. Two mucous cell types were described in the esophagus of Oreochromis mossambicus Peters and Oreochromis niloticus (Pasha, 1964; Morrison and Wright, 1999). Both cell types appeared to have different types of secretion. Using the binding of biotinylated lectins to identify terminal sugar residues in GCs, we also found differences between the buccopharyngeal cavity and the esophagus in relation to the plasmalemma and sometimes to the cytoplasm of the secretory cells. The plasmalemma in the buccopharyngeal cavity labeled weakly to moderately for the binding of the lectins. While mucous cells contained D-N-acetylglucosamine, a-D-mannose, and perhaps, a-D-glucose, D-N-acetyl galactosamine and galactosyl(b1-3)N-acetylgalactosamine were also present, although in smaller amounts. In the esophagus, the apical edge of the epithelium also labeled for binding of all the lectins used in this study. The mucous cells in the esophagus showed a uniform and similar labeling intensity with DBA, SBA and PNA. The residues of D-N-acetylgalactosamine were in the a- and b-anomeric form, supporting the overlapping results of the respective lectins (Damjanov, 1987). As in other fishes, the buccopharyngeal cavity mainly acts as a channel for food transit and it is the first barrier against the environment. Mucus is an important layer protecting peripheral epithelia either from mechanical injuries or bacterial invasion (Humbert et al., 1984). In particular, the mucous cells of the epithelium from the buccopharyngeal cavity of C. guatucupa produce neutral mucosubstances that could cooperate in the enzymatic digestion of food and its transformation into chyme, as well as in absorptive functions (Domeneghini et al., 1998). Moreover, the cells secrete sulphoglycoconjugates that lead to an increasing viscosity of secretion (Tibbets, 1997), which could be ascribed a protective role
ARTICLE IN PRESS Histochemistry of the Cynoscion guatucupa digestive tract (Suprasert et al., 1987). Also, a lubricant role is provided by glycoproteins containing O-sulphated esters (Mittal et al., 1994, 1995). The abundant mucous secretion and the varied GCs is, no doubt, a particular characteristic of the esophagus of fishes, and it could be connected to the absence of salivary glands (Scocco et al., 1998). It is well known that salivary mucins serve as a masticatory lubricant that contributes to the formation of a thin film, which maintains the mucosa integrity of the first tract of the digestive system. Digestion in mammals starts in the mouth, due to the presence of secretion from the salivary glands. Considering all the functions carried out by salivary mucins, their importance is evident. Thus, the esophageal mucous cells could very likely substitute for their absence in fishes (Pedini et al., 2004). The ultrastructural characteristics of the esophageal epithelial cells of fish have been related to the possible synthesis of digestive enzymes (Radaelli et al., 2000). In some fish species, digestion begins in the esophagus, continuing in the stomach (Domeneghini et al., 1998). The different components of the GCs found in the esophageal mucous cells (especially the acidic forms) may be related to the desalinization of ingested seawater (Loretz, 1995) and to a general osmoregulatory role of the esophagus. In this work, this hypothesis is supported by the results of lectin labeling, which reveals a number of terminal sugar residues in the apical membrane of the epithelial cells. Moreover, it is well known that in mammals the joining of lectins to the GCs of the cell membrane is a signal of the function of those molecules in the regulation of ion and fluid movements (Spicer and Schulte, 1992). In most teleosts the gastric glands are located in the cardiac and fundic regions, and the glandular cells are, in general, poorly histochemically characterised (Domeneghini et al., 1998). In mammals, the GCs are, for the most part, easily identifiable in the different cell types of the gastric glands (Kantani-Matsumoto and Kataoka, 1989). The simple columnar epithelium lining the C. guatucupa gastric mucosa secretes GCs, mainly present in the supranuclear zone of the cells. GC histochemistry demonstrates the predominant presence of neutral GCs, but also of acidic GCs. The adherent mucus gel showed the same histochemical pattern. The acidic GCs were exclusively of the sialylated type and contained substituted (O-acylated) sialic acid at C8. Similar results have been obtained in the stomach of Sparus aurata, Umbrina cirrosa and Anguilla anguilla (Domeneghini et al., 1998, 2005; Pedini et al.,
83
2001). A comparison on the GC composition of different teleost fish revealed a wide variation among species (Reifel and Travill, 1978; OstosGarrido et al., 1993). For example, the secretory epithelium of the stomach of Solea solea is reported to be histochemically unreactive (Veggetti et al., 1999). These variations could be attributed not only to different feeding habits but also to different taxonomic positions (Gargiulo et al., 1997). The gland cells that make up the gastric glands are called oxyntopeptic cells by most authors because they secrete both enzymes and hydrochloric acid (Ostos-Garrido et al., 1993; Garcı´a Herna ´ndez et al., 2001). As is usual for other teleosts, we found no mucous neck cells in the gastric glands of C. guatucupa, either with histochemical methods or lectin histochemistry. According to Gargiulo et al. (1997), special neck cells are not a typical feature of the fish stomach and have only been found in a few species. They have been observed in species of the genus Oreochromis (Morrison and Wright, 1999; Caceci et al., 1997; Gargiulo et al., 1997) and Seriola dumerili (Grau et al.,1992). In the study reported here, lectin histochemistry showed that b-D-galactose, D-N-acetylgalactosamine, a-D-mannose and perhaps a-D-glucose, D-N-acetylglucosamine, a-L-fucose and galactosyl(b1-3)N-acetylgalactosamine appeared in GCs secreted by the surface columnar epithelium. These same sugars were histochemically demonstrated in the adherent mucous gel, which labeled more intensely. As already shown in other fishes, the gastric pits of C. guatucupa contained GCs with sugar residues analogous to those found at the epithelial surface (Pedini et al., 2005). Furthermore, no decrease in the presence of N-acetylgalactosamine in C. guatucupa was found, as described for Umbrina cirrosa (Pedini et al., 2005) and Tilapia spp. (Scocco et al., 1996). The gastric glands contained D-N-acetylgalactosamine, galactosyl(b1-3)N-acetylgalactosamine, b-D-galactose and D-N-acetylglucosamine. Domeneghini et al. (2005) suggest the possibility that sugars in the gastric mucosa might switch their position in the oligosaccharide branch during the process of secretion. Thus, in gastric glands of adult Anguilla anguilla D-N-acetylgalactosamine and b-D-galactose were identified. On the other hand, D-N-acetylglucosamine and N-acetylgalactosamine were found in Sparus aurata for a limited larval period. If the sugars are present in a subterminal position in the oligosaccharide chain, they will not be available for lectin binding. It can be concluded that the C. guatucupa stomach secretes a variety of acidic and neutral
ARTICLE IN PRESS 84 GCs. Acid GCs prevent damage to the gut epithelium, acting as a lubricant for the stomach contents and as a buffer for the highly acidic gastric juices (Ferraris et al., 1987). The presence of neutral GCs could represent evidence of the absorption and transport of macromolecules through the membranes (Reifel and Travill, 1978; Sarasquete et al., 2001; Pedini et al., 2005).
Acknowledgments This research was partly supported by grant from the Universidad de Mar del Plata (UNMdP) and from the Agencia Nacional de Promocio ´n Cientı´fica y Tecnolo ´gica (ANPCyT) Argentina.
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Reifel CW, Travill AA. Structure and carbohydrate histochemistry of the stomach in eight species of teleosts. J Morphol 1978;158:155–67. Sarasquete C, Gonza ´lez de Canales ML, Arellano JM, Pe ´rez-Prieto S, Garcı´a Rosado E, Borrego JJ. Histochemical study of lymphocystis disease in skin of gilthead seabream, Sparus aurata from the southatlantic coasts of Spain. Histol Histopathol 1998a; 13:37–45. Sarasquete C, Gonza ´lez de Canales ML, Arellano JM, Mun ˜oz Cueto JA, Ribeiro L, Dinis MT. Histochemical study of skin and gills of Senegal sole, Solea senegalensis larvae and adults. Histol Histopathol 1998b;13:727–35. Sarasquete C, Gisbert E, Ribeiro L, Vieira L, Dinis MT. Glycoconjugates in epidermal, branchial and digestive mucous cells and gastric glands of gilthead seabream Sparus aurata, Senegal sole Solea senegalensis and Siberian sturgeon Acipenser baeri development. Eur J Histochem 2001;45:267–78. Schumacher U, Duku M, Katoh M, Jo ¨rns J, Krause WJ. Histochemical similarities of mucins produced by Brunner0 s glands and pyloric glands: a comparative study. Anat Rec Part A 2004;278A:540–50. Scocco P, Menghi G, Ceccarelli P. Glycohistochemistry of the Tilapia spp stomach. J Fish Biol 1996;49:584–93. Scocco P, Accili D, Menghi G, Ceccarelli P. Unusual glycoconjugates in the esophagus of a tilapine polyhybrid. J Fish Biol 1998;53:39–48. Spicer SS, Schulte BA. Diversity of cell glycoconjugates shown histochemically: a perspective. J Histochem Cytochem 1992;40:1–38. Suprasert A, Fujioka T, Yamada K. The histochemistry of glycoconjugates in the colonic epithelium of the chicken. Histochemistry 1987;86:491–7. Tibbets IR. The distribution and function of mucous cells and their secretions in the alimentary tract of Arrhamphus sclerolepis krefftii. J Fish Biol 1997;50: 809–20. Veggetti A, Rowlerson A, Radaelli G, Arrighi S, Domeneghini C. Posthatching development of the gut and lateral muscle in the Sole, Solea solea (L). J Fish Biol 1999;59:44–65. Volz D, Reid PE, Park CM, Owen DA, Dunn WL. A new histochemical method for the selective periodate oxidation of total tissue sialic acids. Histochem J 1987;19:311–8.
www.elsevier.de/acthis
Glycoconjugates in the mucosa of the digestive tract of Cynoscion guatucupa: A histochemical study Alcira Ofelia Dı´aza,, Alicia Mabel Garcı´ab, Adriana Lı´a Goldemberga a
Departamento de Biologı´a, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata, Funes 3250 31 piso, 7600 Mar del Plata, Argentina b Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas (CONICET), 1033AAJ Buenos Aires, Argentina Received 11 July 2007; received in revised form 13 August 2007; accepted 21 August 2007
KEYWORDS Cynoscion guatucupa; Digestive tract; Mucous cells; Glycoconjugates; Histochemistry; Lectins
Summary This study addresses the histomorphology, the histochemistry and the distribution of glycoconjugates (GCs) in the mucosa of the digestive tract of the weakfish Cynoscion guatucupa. The histological characterization of the buccopharyngeal cavity and the esophagus revealed that they are lined by a stratified epithelium where the mucosa is thrown into longitudinal folds. The stomach is lined with a simple columnar secretory epithelium with tubular glands. GCs were analyzed using a range of histochemical methods. They were identified as oxidizable vicinal diols; sialic acids and some of their chain variants, C7, C8 or C9; sialic acid residues with O-acyl substitution at C8 or C9; carboxyl groups and sulfate groups. Sugar residues in the oligosaccharide side chains of the GCs were investigated using seven biotinylated lectins. Mucous cells from the contents of the buccopharyngeal cavity, esophagus and stomach evidenced neutral, sulphated and sialylated GCs. The latter were substituted mainly in C8. A moderately strong lectin labeling was observed in mucous cells of the three organs studied. Nonetheless, the apical edge of the gastric gland cells showed a strong positive labeling. The presence of different classes of GCs has been associated with different functions, such as lubrication, protection, inhibition of microorganisms proliferation and ionic regulation. & 2007 Elsevier GmbH. All rights reserved.
Introduction Corresponding author. Tel.: +54 223 475 2426;
fax: +54 223 475 3150. E-mail address: [email protected] (A.O. Dı´az).
The digestive tract of fishes shows a marked diversity of morphology and function. This is related both to different feeding habits and to
0065-1281/$ - see front matter & 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.acthis.2007.08.002
ARTICLE IN PRESS Histochemistry of the Cynoscion guatucupa digestive tract taxonomy, as well as to body shape, size and sex (Kapoor et al., 1975). The occurrence of mucus is a common feature in the digestive tract of teleosts. The mucosubstances secreted differ between species and also along the fish alimentary canal (Dı´az et al., 2003). Mucus is a highly hydrated gel that consists approximately of 95% water and 5% mucins, plus minor components like electrolytes (Neutra and Forstner, 1987). Mucins are high molecular weight glycoproteins that form highly glycosylated gels (Schumacher et al., 2004). Mucosubstances are crucial for the maintenance of adequate functioning of epithelial tissues (Niew Amerongen et al., 1995). The distribution and presence of glycoproteins is correlated with different functions, such as lubrication, protection against proteolytic degradation and inhibition of microorganisms (Reid et al., 1988). In addition, an osmotic function is especially important in fishes, in the form of binding and transport of water and various ions (Loretz, 1995). The stripped weakfish, Cynoscion guatucupa Cuvier (Pisces, Perciformes, Scianidae), is a demersal coastal fish of growing commercial value with a wide distribution from 221350 S (Rio de Janeiro, Brazil) to approximately 431S (Argentina) (Cousseau and Perrotta, 2000). The aim of this study was to analyze the carbohydrate composition of mucins in mucussecreting cells of the digestive tract mucosa of C. guatucupa, from the bucopharyngeal cavity to the stomach. Classical and specific mucin and lectin histochemical techniques were used to achieve this aim. Lectins are naturally occurring carbohydratebinding proteins that are not enzymes or antibodies. They are powerful and reliable tools to characterize
77
glycoconjugates (GCs) in tissues. Lectin histochemical studies have demonstrated the utility of lectins as biomarkers of specific secretory functions, structural components and alterations of cell and tissues (Chan and Ho, 1999). The present work introduces for the first time histological and histochemical studies of the mucosa of the digestive tract of C. guatucupa.
Material and methods Live specimens of C. guatucupa (46.473.0 cm length; 879.07181.0 g weight; n ¼ 10) were collected from the coast of Mar del Plata, Argentina (381050 S, 571320 W). Fish were killed by decapitation. The digestive tract was rapidly removed and fixed by immersion in Bouin’s fluid or 10% buffered formalin for light microscope studies. Samples were routinely processed and embedded in paraffin wax. Four micrometer-thick histological sections were cut by microtome, prepared according to standard protocol and then stained using the following techniques: routine hematoxylin and eosin (H–E) stain, Masson trichrome stain for morphology and Mayer mucicarmin for mucin identification. Sections of tissue were also subjected to histochemical procedures for the identification of GCs, as detailed in Table 1. Sections were stained with (1) PAS (periodic acid Schiff’s reagent) to demonstrate periodate-reactive vicinal diols, (2) PA*S (selective periodic acid Schiff reaction): oxidation for 1 h at 4 1C with 0.4 mM periodic acid in approximately 1 M hydrochloric acid is used as a specific reagent for the selective
Table 1. Histochemical procedures for visualizing and identifying GCs in mucous cells of Cynoscion guatucupa digestive tract Procedures
Interpretation of staining reactions
References
1 PAS
GCs with oxidizable vicinal diols and/or glycogen
Mc Manus (1948)
2 PA*S
Sialic acid and some of their chain variants (C7 and/or C9)
Volz et al. (1987)
3 KOH/PA*S
GCs with sialic acid residues
Culling et al. (1976)
4 KOH/PA*/Bh/PAS
Neutral GCs with oxidizable vicinal diols
Volz et al. (1987)
5 PA/Bh/KOH/PAS
Sialic acid residues with O-acyl substitution at C7, C8 or C9 and Oacyl sugars
Reid et al. (1973)
6 AB pH 2.5
GCs with carboxyl groups (sialic acid or urinic acid) and/or with O-sulphate esters
Lev and Spicer (1964)
7 AB pH 2.5/PAS
GCs with carboxyl groups and/or with O-sulphate esters
Mowry (1956)
Symbols: PAS, periodic acid/Schiff; PA*S, periodic acid/Schiff at low temperature and low pH (oxidation with 0.4 mM periodic acid in 1.0 M hydrochloric acid at 4 1C); PA, periodic acid; KOH, saponification; Bh, borohydride; AB, Alcian blue; GCs, glycoconjugates.
ARTICLE IN PRESS 78
(3)
(4)
(5)
(6)
(7)
A.O. Dı´az et al. visualization of sialic acids in the PAS procedure. The selectivity of the reaction is the result of an increase in the rate of the oxidation of the sialic acid residues together with a decrease in the rate of oxidation of neutral sugars, KOH/PA*S (saponification-selective periodic acid Schiff reaction) to allow the characterization of total sialic acids. The saponification with 0.5% potassium hydroxide in 70% ethanol for 30 min at room temperature was performed to deacetylate sialic acid residues and was followed by PA*S, KOH/PA*/Bh/PAS (saponification-selective periodic acid-borohydride reduction-periodic acid Schiff reaction) for the characterization of neutral sugars, PA/Bh/KOH/PAS (periodic acid-borohydride reduction-saponification-periodic acid Schiff reaction): this method was carried out using a 2-h oxidation at room temperature with 1% periodic acid. The aldehydes generated by the initial oxidation are reduced to Schiff-unreactive primary alcohols with sodium borohydride (PA-Bh). Following saponification (KOH), only sialic acids with O-acyl substituents at C7, C8 or C9 (or which had two or three side-chains O-acyl substituents) are PAS positive, AB pH 2.5 (Alcian Blue 8GX pH 2.5): to demonstrate GCs with carboxyl groups (sialic acid or uronic acid) and/or with O-sulfate esters, AB pH 2.5/PAS (Alcian Blue 8GX pH 2.5/periodic acid Schiff staining): to demonstrate both acidic GCs (blue) and periodate-reactive vicinol diols (purple).
Labeling with biotinylated lectins was used to identify specific sugar residues of GCs. Lectin labeling was performed according to the method described by Gimeno et al. (1995). Briefly, paraffin wax sections were mounted on slides coated with poly-L-lysine (Sigma Diagnostics, St. Louis, MO, USA). These were deparaffinized with xylene, then incubated in 0.3% H2O2 in methanol for 30 min at room temperature in order to block endogenous peroxidase activity. They were then hydrated through a graded ethanol series, washed in a phosphate-buffered saline (PBS) 0.01 M, pH 7.2, and incubated for 30 min with biotinylated lectins. Table 2 lists the seven lectins used in this study, their sources and their major sugar specificities. All lectins were employed at a dilution of 30 mg/ml in PBS, except for PNA which was applied at a concentration of 10 mg/ml. Biotinylated lectins were purchased from Vector Laboratories Inc.,
Table 2. Carbohydrate binding specificity of lectins employed in this study Lectin
Acronym
Specificity/ hapten sugar,y
Canavalia ensiformis agglutinin
Con-A
a-D-Man; a-D-Glc
Triticum vulgaris (wheatgerm) agglutinin
WGA
b-D-GlcNAc; NeuNAc
Dolichos biflorus agglutinin
DBA
a-D-GalNAc
Glycine max (soyabean) agglutinin
SBA
a-D-GalNAc; b-DGalNAc
Arachis hypogaea (peanut) agglutinin
PNA
b-D-Gal (b1-3) D-GalNAc
Ulex europaeus agglutinin-I
UEA-I
a-L-Fuc
Ricinus communis agglutinin-I
RCA-I
b-Gal
Goldstein and Hayes (1978). y Fuc, fucose; Gal, galactose; GalNAc, N-acetylgalactosamine; Glc, glucose; GlcNAc, N-acetylglucosamine; Man, mannose; NeuNAc, acetyl neuraminic acid (sialic acid).
Burlingame, CA, USA. Afterwards, sections were incubated for 30 min with ABC reagent, prepared according to manufacturer’s instructions (Vectastain Elite PK 6200 Vector Laboratories Inc, Burlingame, CA, USA). Lectin binding was revealed by incubation with 0.5 mg/mol diaminobenzidine tetrahydrochloride (DAB) (DAKO) in Tris buffer 0.1 M, pH 7.2, plus 0.02% H2O2. All dilutions and thorough washes between steps were carried out using PBS unless otherwise stated. Two types of controls were performed: (1) lectin solution was replaced by PBS and (2) lectin labeling was performed as described above after lectins had been preincubated for 1 h in the presence of the appropriate hapten sugars (0.2 M in PBS), as listed in Table 2, at room temperature. No labeling was detected in control sections. Evaluation of labeling intensities was based on subjective estimates by all authors after examination of two sections per sample of all the animals tested.
Results The results of histochemistry to reveal GCs in the mucosa of the digestive tract of C. guatucupa are summarized in Table 3 and the results of lectin
ARTICLE IN PRESS Histochemistry of the Cynoscion guatucupa digestive tract Table 3.
79
Histochemical reactions of GCs in the mucosa of the digestive tract of Cynoscion guatucupa
Procedures
Mucous cells
Stomach
Buccopharyngeal
PAS PA*S KOH/PA*S KOH/PA*/Bh/PAS PA/Bh/KOH/PAS AB pH 2.5 AB pH 2.5/PAS
Esophagus
3 1 1 3 2 3 3
3 2 3 3 3 3 3
Secretor epithelium
Glands
Cell coat
Cells
Apical edge
Cells
3 1 1 2 2 0 3
3 1 1 3 3 0 3
2 2 2 3 2 0 2
2 2 2 2 2 0 2
Staining intensity: 0, negative; 1, weak; 2, moderate; 3, strong.
Table 4.
Con A WGA DBA SBA PNA UEA I RCA I
Lecting binding in the mucosa of the digestive tract of Cynoscion guatucupa Buccopharyngeal cavity
Esophagus
Cell coat
Cell coat
2 2 2 2 2 2 1
Mucous cells
1 2–3 0–1 1 1 0–1 0
2 2 2 3 3 3 2
Stomach Mucous cells
0–1 0 1–2 2 2 0 0
Secretor epithelium
Glands
Cell coat
Cells
Apical edge
Cells
3 3 3 3 3 3 3
1 1 1 1 1 1 1
0 3 3 3 3 3 3
0 1 2 2 2 0 1
Labeling intensity: 0, negative; 1, weak; 2, moderate; 3, strong.
histochemistry are summarized in Table 4. They are explained in more detail below.
Buccopharyngeal cavity/esophagus The buccopharyngeal cavity was situated in the posterior portion of the head; together with the gill arch it constituted the gill chamber that filters and retains food. The esophagus was short and possessed the thickest wall of the digestive tract. The tunica mucosa of the buccopharyngeal cavity and the esophagus featured a great number of different sized longitudinal folds, separated by well-pronounced furrows in the esophagus. In both organs, the mucosal epithelium was stratified, with high cuboidal cells in the basal layer, cuboidal cells in the intermediate layers and more flattened cells in the superficial layers. A great many large mucous cells with a foamyappearing cytoplasm were visible in between the epithelial layers. The cytoplasm of mucous cells was granular and contained a heavily PAS-reactive material in the buccopharyngeal cavity as well as in
the esophagus (Figure 1A). The PAS positivity disappeared after acetylation, and it was recovered following saponification. Sections subjected to a-amylase, to demonstrate periodate reactive vicinal diols and exclude the presence of glycogen, were positive for the PAS reaction after the same treatment. The mucous cells were alcianophilic (AB positive) and stained violet with the Alcian blue pH 2.5-PAS sequence (Figure 1B and C). In addition, these secretory cells were stained red by the KOH/PA*/Bh/PAS reaction. Mucous cells in the buccopharyngeal cavity reacted weakly to the PA*S and KOH/PA*S, and stained moderately with the PA/Bh/KOH/PAS sequence. Mucous cells in the esophagus were strongly stained red by PA/Bh/KOH/PAS and showed KOH/PA*S positivity. They stained moderately with PA*S. A different type of mucous cell, identified through KOH/PA*/ Bh/PAS, was encountered in this organ. These cells stained more intensely than the rest of the secretory cells with the last technique. Mucous cells in the buccopharyngeal cavity labeled moderately to strongly with WGA and weakly with Con A, SBA and PNA lectins. Mucous
ARTICLE IN PRESS 80
A.O. Dı´az et al.
Figure 1. Histochemistry of the buccopharyngeal cavity, esophagus and stomach of C.guatucupa. (A) Buccopharyngeal cavity. The mucous cells are PAS-reactive (arrow). Scale bar ¼ 60 mm. (B) Esophagus AB pH 2.5. The mucous cells are AB reactive (arrow). Scale bar ¼ 12 mm. (C) Esophagus AB pH 2.5/PAS sequence. The mucous cells are positive (arrow). Scale bar ¼ 40 mm. (D) Stomach. The epithelium (asterisk) and the gastric glands (arrow) are PAS-reactive. Scale bar ¼ 80 mm. (E) Stomach (PA/Bh/KOH/PAS). The epithelial secretory cells (arrow) and the gland cells (arrowhead) are positively stained. Scale bar ¼ 60 mm. (F) Stomach (KOH/PA*/Bh/PAS). The epithelial secretory cells (arrow) and the gland cells (arrowhead) are positive. Scale bar ¼ 60 mm.
cells in the esophagus labeled moderately with SBA and PNA and weakly to moderately with DBA lectins. The adherent mucus gel of the buccopharyngeal cavity and the esophagus was labeled by all the lectins tested (Figure 2A–H and Table 4). Stomach The transition from esophageal to gastric mucosa was abrupt. The stratified epithelium of the esophagus was replaced by the secretory columnar epithelium that lines the whole surface of the stomach. Glands were tubular and opened at the base of the gastric pits. Mucus was abundant in the supranuclear cytoplasmic zone of all the surface secretory cells, and their histochemical properties remained uniform throughout the whole organ. Thus, the simple columnar epithelium that lined the gastric mucosa was strongly PAS positive. It was unreactive with Alcian blue and stained
magenta with the Alcian blue pH 2.5/PAS sequence. The epithelial secretory cells also showed a strong reaction with PA/Bh/KOH/PAS and KOH/PA*/ Bh/PAS, and were stained pale red by the PA*S reaction and the KOH/PA*S sequence (Figure 1D–F). Adherent mucous gel showed similar histochemical characteristics. The epithelium lining the gastric mucosa and its luminal surface labeled for binding of all lectins tested. The cytoplasmic contents of the epithelial cells weakly labeled with these lectins, while the cell coat showed a strong positive labeling for binding of all the lectins (Figure 2I–L). The gastric glands were reactive with the different histochemical methods, showing only minor variation in reactivity. Thus, the epithelial cells which made up the glands displayed a moderate reaction to PAS, PA*S, PA/Bh/KOH/PAS, KOH/PA*/ Bh/PAS and to KOH/PA*S (Figure 1D–F). They labeled
ARTICLE IN PRESS Histochemistry of the Cynoscion guatucupa digestive tract
81
Figure 2. Lectin histochemistry of the buccopharyngeal cavity, esophagus and stomach of C.guatucupa. Buccopharyngeal cavity: (A) PNA. Scale bar ¼ 52 mm. (B) DBA. Scale bar ¼ 42 mm. (C) UEA-I. Scale bar ¼ 52 mm. (D) WGA. Scale bar ¼ 24 mm. Esophagus: (E) UEA-I. Scale bar ¼ 28 mm. (F) DBA. Scale bar ¼ 28 mm. (G) PNA. Scale bar ¼ 78 mm. (H) Con A. Scale bar ¼ 40 mm. Stomach: (I) SBA. Scale bar ¼ 100 mm. (J) PNA. Scale bar ¼ 68 mm. (K) RCA-l. Scale bar ¼ 60 mm. (L) UEA-I. Scale bar ¼ 100 mm. Adherent mucus gel (arrow), mucous cells (asterisk) and gland cells (star).
moderately for binding of DBA, SBA and PNA, and weakly for RCA-I and WGA. The apical edge of the gland cells featured strong positive labeling for binding of the same lectins (Figure 2I–L).
Discussion This study provides the first description of the histology and the histochemistry of GCs in the digestive tract of C. guatucupa. In addition, the
present work is one of a series describing GCs in the alimentary tract of fish such as Micropogonias furnieri (Dı´az et al., 2002), Engraulis anchoita (Dı´az et al., 2003) and Odontesthes bonariensis (Dı´az et al., 2005). Secretion of mucus is a common characteristic of the C. guatucupa organs analyzed in this study. Nonetheless, the morphology of the cell producing mucus varies from mucous cells in the buccopharyngeal cavity and the esophagus to surface secretory epithelium in the stomach. These cells also
ARTICLE IN PRESS 82 display diversity in the presence of GCs, as revealed by their histochemical and lectin-binding properties. The general morphology of the mucosa of the portion of the digestive system studied here is in accordance with the morphologies described for the most part in fishes (Dı´az et al., 2003; Domeneghini et al., 2005). However, in some species, the distribution and characteristics of the mucus secreting cells deviate from the general pattern. This is most commonly seen in the esophagus, where the presence of stratified epithelium with mucous cells usually presents morphological variations. This is the case in the esophagus of Seriola dumerrili (Grau et al., 1992), Anguilla anguilla (Abaurrea-Equisoain and Ostos-Garrido, 1996) and Engraulis anchoita (Dı´az et al., 2003), where two zones are evident: a cranial one with a few layers of squamous stratified epithelium with numerous superficial mucous cells and a caudal zone with that epithelium replaced by a simple columnar epithelium involved in secretion processes. In the study described here, mucous cells from the contents of the buccopharyngeal cavity and the esophagus evidenced neutral GCs, GCs with sialic acids and some of their side chain variants, and carboxylated and sulphated GCs. The fact that mucous cells in the buccopharyngeal cavity and the esophagus stained intensely with PAS and amylase/PAS, indicates that cells must contain neutral hexoses. These results indicate an absence of glycogen. This has been demonstrated in other teleosts, in which mucous cells react in the same way with these histochemical techniques (Sarasquete et al., 1998a, b; Dı´az et al., 2001). The Alcian blue sequence revealed sulphated GCs that could be responsible for an increasing viscosity of the secretions. The visualization of non-substituted and substituted sialic acids at C7, C8 and C9 was confirmed. In the buccopharyngeal cavity, weak reactions for PA*S and KOH/PA*S and moderate reactions for the sequence PA/Bh/KOH/PAS were evidenced. This indicates a scarce quantity of substituted sialic acids in C7, C9, and non-substituted sialic acids and the greatest presence of substituted sialic acids in C8. On the other hand, in the esophagus, PA/Bh/ KOH/PAS and KOH/PA*S staining was strong, while PA*S staining was moderate, thus indicating the presence of total and substituted sialic acids in C7 and C8. Domeneghini et al. (2005) used similar methods and described O-acylated sialic acids at C7 in the mucous cells throughout the alimentary canal of Anguilla anguilla. The presence of sialic acid substituted at C8 has also been confirmed in
A.O. Dı´az et al. the buccopharyngeal cavity and the esophagus of larval stages, juveniles and adults of Sparus auratus (Domeneghini et al., 1998). Because of their negative charge, sialic acids have been proposed to give rise to extended GC molecules and to play a role in the hydration of their immediate environment (Mittal et al., 1994). The presence of neutral GCs was confirmed with the KOH/PA*/Bh/PAS reaction. This also identified two cell types in the esophagus, not distinguished using other methods. Gargiulo et al. (1996) and Scocco et al. (1998) described periodic acid-Schiffstained and Alcian blue-stained goblet cells in the esophagus, but did not describe a second type of secretory cell. Two mucous cell types were described in the esophagus of Oreochromis mossambicus Peters and Oreochromis niloticus (Pasha, 1964; Morrison and Wright, 1999). Both cell types appeared to have different types of secretion. Using the binding of biotinylated lectins to identify terminal sugar residues in GCs, we also found differences between the buccopharyngeal cavity and the esophagus in relation to the plasmalemma and sometimes to the cytoplasm of the secretory cells. The plasmalemma in the buccopharyngeal cavity labeled weakly to moderately for the binding of the lectins. While mucous cells contained D-N-acetylglucosamine, a-D-mannose, and perhaps, a-D-glucose, D-N-acetyl galactosamine and galactosyl(b1-3)N-acetylgalactosamine were also present, although in smaller amounts. In the esophagus, the apical edge of the epithelium also labeled for binding of all the lectins used in this study. The mucous cells in the esophagus showed a uniform and similar labeling intensity with DBA, SBA and PNA. The residues of D-N-acetylgalactosamine were in the a- and b-anomeric form, supporting the overlapping results of the respective lectins (Damjanov, 1987). As in other fishes, the buccopharyngeal cavity mainly acts as a channel for food transit and it is the first barrier against the environment. Mucus is an important layer protecting peripheral epithelia either from mechanical injuries or bacterial invasion (Humbert et al., 1984). In particular, the mucous cells of the epithelium from the buccopharyngeal cavity of C. guatucupa produce neutral mucosubstances that could cooperate in the enzymatic digestion of food and its transformation into chyme, as well as in absorptive functions (Domeneghini et al., 1998). Moreover, the cells secrete sulphoglycoconjugates that lead to an increasing viscosity of secretion (Tibbets, 1997), which could be ascribed a protective role
ARTICLE IN PRESS Histochemistry of the Cynoscion guatucupa digestive tract (Suprasert et al., 1987). Also, a lubricant role is provided by glycoproteins containing O-sulphated esters (Mittal et al., 1994, 1995). The abundant mucous secretion and the varied GCs is, no doubt, a particular characteristic of the esophagus of fishes, and it could be connected to the absence of salivary glands (Scocco et al., 1998). It is well known that salivary mucins serve as a masticatory lubricant that contributes to the formation of a thin film, which maintains the mucosa integrity of the first tract of the digestive system. Digestion in mammals starts in the mouth, due to the presence of secretion from the salivary glands. Considering all the functions carried out by salivary mucins, their importance is evident. Thus, the esophageal mucous cells could very likely substitute for their absence in fishes (Pedini et al., 2004). The ultrastructural characteristics of the esophageal epithelial cells of fish have been related to the possible synthesis of digestive enzymes (Radaelli et al., 2000). In some fish species, digestion begins in the esophagus, continuing in the stomach (Domeneghini et al., 1998). The different components of the GCs found in the esophageal mucous cells (especially the acidic forms) may be related to the desalinization of ingested seawater (Loretz, 1995) and to a general osmoregulatory role of the esophagus. In this work, this hypothesis is supported by the results of lectin labeling, which reveals a number of terminal sugar residues in the apical membrane of the epithelial cells. Moreover, it is well known that in mammals the joining of lectins to the GCs of the cell membrane is a signal of the function of those molecules in the regulation of ion and fluid movements (Spicer and Schulte, 1992). In most teleosts the gastric glands are located in the cardiac and fundic regions, and the glandular cells are, in general, poorly histochemically characterised (Domeneghini et al., 1998). In mammals, the GCs are, for the most part, easily identifiable in the different cell types of the gastric glands (Kantani-Matsumoto and Kataoka, 1989). The simple columnar epithelium lining the C. guatucupa gastric mucosa secretes GCs, mainly present in the supranuclear zone of the cells. GC histochemistry demonstrates the predominant presence of neutral GCs, but also of acidic GCs. The adherent mucus gel showed the same histochemical pattern. The acidic GCs were exclusively of the sialylated type and contained substituted (O-acylated) sialic acid at C8. Similar results have been obtained in the stomach of Sparus aurata, Umbrina cirrosa and Anguilla anguilla (Domeneghini et al., 1998, 2005; Pedini et al.,
83
2001). A comparison on the GC composition of different teleost fish revealed a wide variation among species (Reifel and Travill, 1978; OstosGarrido et al., 1993). For example, the secretory epithelium of the stomach of Solea solea is reported to be histochemically unreactive (Veggetti et al., 1999). These variations could be attributed not only to different feeding habits but also to different taxonomic positions (Gargiulo et al., 1997). The gland cells that make up the gastric glands are called oxyntopeptic cells by most authors because they secrete both enzymes and hydrochloric acid (Ostos-Garrido et al., 1993; Garcı´a Herna ´ndez et al., 2001). As is usual for other teleosts, we found no mucous neck cells in the gastric glands of C. guatucupa, either with histochemical methods or lectin histochemistry. According to Gargiulo et al. (1997), special neck cells are not a typical feature of the fish stomach and have only been found in a few species. They have been observed in species of the genus Oreochromis (Morrison and Wright, 1999; Caceci et al., 1997; Gargiulo et al., 1997) and Seriola dumerili (Grau et al.,1992). In the study reported here, lectin histochemistry showed that b-D-galactose, D-N-acetylgalactosamine, a-D-mannose and perhaps a-D-glucose, D-N-acetylglucosamine, a-L-fucose and galactosyl(b1-3)N-acetylgalactosamine appeared in GCs secreted by the surface columnar epithelium. These same sugars were histochemically demonstrated in the adherent mucous gel, which labeled more intensely. As already shown in other fishes, the gastric pits of C. guatucupa contained GCs with sugar residues analogous to those found at the epithelial surface (Pedini et al., 2005). Furthermore, no decrease in the presence of N-acetylgalactosamine in C. guatucupa was found, as described for Umbrina cirrosa (Pedini et al., 2005) and Tilapia spp. (Scocco et al., 1996). The gastric glands contained D-N-acetylgalactosamine, galactosyl(b1-3)N-acetylgalactosamine, b-D-galactose and D-N-acetylglucosamine. Domeneghini et al. (2005) suggest the possibility that sugars in the gastric mucosa might switch their position in the oligosaccharide branch during the process of secretion. Thus, in gastric glands of adult Anguilla anguilla D-N-acetylgalactosamine and b-D-galactose were identified. On the other hand, D-N-acetylglucosamine and N-acetylgalactosamine were found in Sparus aurata for a limited larval period. If the sugars are present in a subterminal position in the oligosaccharide chain, they will not be available for lectin binding. It can be concluded that the C. guatucupa stomach secretes a variety of acidic and neutral
ARTICLE IN PRESS 84 GCs. Acid GCs prevent damage to the gut epithelium, acting as a lubricant for the stomach contents and as a buffer for the highly acidic gastric juices (Ferraris et al., 1987). The presence of neutral GCs could represent evidence of the absorption and transport of macromolecules through the membranes (Reifel and Travill, 1978; Sarasquete et al., 2001; Pedini et al., 2005).
Acknowledgments This research was partly supported by grant from the Universidad de Mar del Plata (UNMdP) and from the Agencia Nacional de Promocio ´n Cientı´fica y Tecnolo ´gica (ANPCyT) Argentina.
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