Histochemical Characterization of Convict Cichlid (Amatitlania nigrofasciata) Intestinal Goblet Cells

Sezgi Arman* and Sema İşisağ Üçüncü

Department of Biology, Ege University, 35100 Bornova-Izmir, Turkey

ABSTRACT

In the present study, some histochemical features of goblet cells of the intestine of convict cichlid (A. nigrofasciata) were described. In order to reveal the main histological construction, transverse sections of different parts of intestine were firstly stained with hematoxylin eosin (H-E). Sections of anterior, mid and posterior intestinal segments were also treated with different staining methods of alcian blue (AB) at pH 2.5, aldehyde fuchsin (AF), periodic acid-Schiff (PAS), KOH/PAS and PAS/AB (pH 2.5) and investigated. Goblet cells of anterior intestine were colored strongly by AB (pH 2.5), AF and PAS; and moderately by PAS/AB (pH 2.5) (AB dominant). In the mid part of the gastrointestinal tract, goblet cells were also stained strongly with AB, PAS and PAS/AB (pH 2.5) (AB dominant), however, their reactions to KOH/PAS treatment were recorded as negative. Because of numerous supranuclear vacuoles of the epithelial cells, only a few goblet cells could be differentiated in posterior parts of alimentary channel, with their weakly reaction to AB (pH 2.5), PAS and PAS/AB (pH 2.5). Moreover, these cells were not stained with AF and KOH/PAS. According to their affinities, goblet cells oriented in anterior and the mid intestine were mainly classified as acidic (AB-positive) and neutral (PAS-positive). Statistical analysis were confirmed that, the numbers of acidic and neutral cells of per unite square of epithelial area were significantly different.

Article Information

Received 08 October 2015

Revised 09 September 2016

Accepted 20 September 2016

Available online 30 January 2017

Authors’ Contributions

SA designed the study, performed experimental work and analyzed the data. SİÜ helped in microscopic examinations. SA and SİÜ were involved in manuscript write up.

Key words

Goblet cell, Intestine, Glycoconjugate, Glycoprotein, Amatitlania nigrofasciata.

* Corresponding author: sezgiarman@gmail.com

0030-9923/2016/0004-1161 $ 8.00/0

Copyright 2016 Zoological Society of Pakistan

DOI: http://dx.doi.org/10.17582/journal.pjz/2017.49.2.445.453

INTRODUCTION

Inspite of some morphological and functional alterations due to feeding habits, body form, weight and sex (Grosh and Das, 1987; Boglione et al., 1992; Murray et al., 1994; Cinar and Şenol, 2006), the main histological organization of the alimentary channel of teleosts is similar. In order to determine the complicated activities of intestine, understanding of the coordinated interactions of cells within different tissue layers is required. In addition to their essential functions noted as absorption and transportation, goblet cells, the main secretory units of the intestinal mucosa, have some important defense mechanism against irritants, microbial attachments and invasion (Phalipon et al., 2002; Bruno et al., 2005; Strugnel and Wijburg, 2010; Hasnain et al., 2013; Kim and Khan, 2013).

Mucoid substance of goblet cells is mainly responsible for hydrating and protecting the intestinal mucosa. It is composed of both organic and inorganic materials such as lipids, proteins, glycoproteins, nucleic acids, salt and water (Hasnain et al., 2010, 2013; Bosi and Dezfuli, 2015). As a physiological barrier, mucus is an essential layer of the innate immune system and plays an important role in the regulation and protection of intestinal mucosa. The functional integrity of the intestinal mucosal epithelial cells depends on the regulation of the mucus layer, the intercellular tight junction, epithelial cells, and host innate and adaptive immune response (Kim and Ho, 2010).

Methods to identify the contents of goblet cells may be helpful for expanding the data on complex intestinal functions. The distribution and histochemical properties of goblet cells of gastrointestinal tract of some teleosts have previously been investigated by various histochemical techniques (Sarasquete et al., 2001; Díaz et al., 2003; Leknes, 2010, 2015). However, the reports are still limited and further studies are needed.

The present study is based on histochemical demonstration of glycoproteinic contents of the intestine of a popular aquarium fish Amatitlania nigrofasciata, which has also been used for behavioral studies (Townshend and Wootton, 1985; Budaev et al., 1999; Grant et al., 2002; Foam et al., 2005). For comparison of the content of mucosubstances of different parts of digestive tract, the cells oriented in anterior, mid and posterior parts of intestine were identified by their specific staining properties. Some statistical data related to goblet cell distribution was also evaluated.

MATERIALS AND METHODS

All experiments were performed in accordance with the guidelines for animal research established by the Local Ethics Committee of Animal Experiments at Ege University (Certificate number: 000018).

Fish maintenance

Ten specimens of adult A. nigrofasciata were obtained from commercial dealers. Fishes were maintained in well aerated glass aquaria at 26±2°C, 14 h light:10 h dark photoperiod and fed twice a day with Artemia sp. for two weeks before the experiment. Specimens were euthanatized with overdose of MS-222 (Sigma). The whole intestine was removed and equally divided into three segments as anterior, mid and posterior.

Histochemistry

Tissue samples were fixed in Bouin’s fluid for 24 h at 4ºC, rinsed in distilled water, dehydrated in ethanol, treated with xylene and embedded in paraffin. In order to demonstrate the general morphology of the intestine, 5 µm-thick transverse sections were stained with hematoxylin-eosin (H-E). To identify different contents of the goblet cells, the staining methods of alcian blue (AB) at pH 2.5 for acidic glycoconjugates with carboxylated and sulfate esters; aldehyde fuchsin (AF) for glycoconjugates with sulfate; periodic acid (0.5%)-Schiff reagent (PAS) for neutral glycoconjugates; potassium hydroxide/PAS (KOH/PAS) for glycoconjugates with sialic acid residues; and PAS/AB (pH 2.5) to distinguish between neutral and acidic glycoconjugates (Çınar and Şenol, 2006) were performed (Presnell et al., 1997). Slides were examined and photographed by Leica DM300 microscope equipped with a Leica digital camera (DFC290) and Zeiss Axio Scope. A1 equipped with Zeiss AxioCam ERc5s.

Goblet cells: differentiation and counting

By using the grid plugin of Image J software, AB-positive and PAS-positive cell populations in per unit (mm2) of the epithelial area of randomly choosen from five to ten microphotographs of both anterior and the mid intestine were examined. For each specimen, AB-positive and PAS-positive goblet cells in random 100 grids were counted. The total grid area was presented in mm2.

Statistical analysis

The analysis were performed with SPSS 20.0 software and the data was evaluated by 2-tailed t-test for independent samples (p ≤ 0.05).

RESULTS

The intestinal wall of A. nigrofasciata is composed of the four layers called as serosa (S), muscularis externa (ME), submucosa (SM) and mucosa (M) (Fig. 1). Serosa, the outmost layer of the intestine was composed of a thin layer of simple squamous epithelium. Muscularis externa was consisted of smooth muscle fibers. In all of the intestinal segments investigated, numerous eosinophilic granule-containg cells and blood vessels were observed at the loose connective tissue forming submucosa. The mucosa was displayed of single layer of characteristic columnar epithelium (E) and lamina propria (LP). More folds (villi) were observed in the mucosa of the anterior part than those of the mid and posterior intestine, and the height of villi was gradually decreased towards mid and posterior. The epithelial cell layers of the anterior intestine were consisted mainly of columnar enterocytes, sac-shaped goblet and a few APUD cells (Figs. 1, 2). Goblet cells which were characterized with their typical shape, basally located nucleus and scarce organelles, were abundant in the epithelial layer of the anterior intestine. The middle intestine was exhibited similar histological features to the anterior intestine. The most striking feature of the enterocytes of posterior intestine was noted as the presence of large and clear supranuclear vacuoles. Although most of the posterior enterocytes were exhibited a number of heterogen vacuoles, some of them had fusioned, giant and homogenous ones (Fig. 3). Only a few and small goblet cells could be observed in this segment, moreover, APUD cells were not seen. The dense brush border of enterocytes was easily observed in all of the segments investigated (Figs. 1, 2, 3), but Paneth cells were not identified.

The differences in reaction intensities of the goblet cells of three intestinal segments are given in Table I.

Anterior and mid intestine

The strong reaction of goblet cells with AB at pH 2.5 indicated acidic glycoprotein content of the mucus (Figs. 4a, 5a).

Since mucus consists of glycoproteins with oxidizable vicinal diols and/or glycogen, goblet cells were stained as magenta in color with PAS method (Figs. 4b, 5b). Reactions to PAS/AB (pH 2.5) staining of the goblet cells of these parts were slightly different, while AB was dominant for both of them. When compared to anteriorly oriented ones which were moderately stained, mid intestinal cells showed strong reaction (Figs. 4d, 5d).

In the anterior intestine, goblet cells were strongly stained with AF, whereas mid intestinal ones were moderately stained (Figs. 4c, 5c). The content of the AF-positive, purple cells was composed of sulfated glycoproteins.

The anteriorly embedded goblet cells were weakly stained as pale pink by KOH/PAS method, the cells of mid intestine were however, not colored. The low affinity to KOH/PAS proved that the mucous content lacked sialic acid residues.

Table I.- Reaction intensities of goblet cells of anterior, mid and posterior intestine of A. nigrofasciata.

| Ant. Int. | Mid. Int. | Post. Int.
AB (pH 2.5) | +++ | +++ | ±
AF | +++ | ++ | -
PAS | +++ | +++ | ±
KOH/PAS | + | - | -
PAS/AB/ (pH 2.5) | ++ | +++ | ±

Degree of intensity: -, negative; ±, poorly; +, weakly; ++, moderately, +++, strongly stained. AB, alcian blue; AF, aldehyde fuchsin; KOH/PAS, potassium hydroxide/ periodic acid-Schiff’s reagent; PAS, periodic acid-Schiff’s reagent; PAS/AB, periodic acid-Schiff’s/alcian blue.

Posterior intestine

Only a few, small goblet cells which could rarely be observed, were poorly stained AB at pH 2.5 (Fig. 6), PAS and PAS/AB (pH 2.5), however they did not react with AF and KOH/PAS methods. Because of scarce distribution of posteriorly oriented goblet cells, only anterior and mid intestine data was statistically compared (Table II). One way-ANOVA analysis indicated that the number of acidic and neutral goblet cells in the anterior and mid intestine were significantly different. Both of the acidic and neutral cells of mid intestine were higher in number than anteriorly oriented ones.

Table II.- Comparison of acidic and neutral goblet cells per mm2 of anterior and mid intestine of A. nigrofasciata.

| Mean | SD | SEM | Sig. (2-tailed) | t-value
Acidic goblet cells (AB-Positive)
Anterior intestine | 1392.00 | 806.99 | 80.69 | 0.001 | 3.345
Mid intestine | 1616.00 | 691.46 | 69.14 | 0.000 | 3.614
Neutral goblet cells (PAS-Positive)
Anterior intestine | 1060.00 | 578.04 | 57.80 | 0.001 | 3.345
Mid intestine | 1272.00 | 654.13 | 65.413 | 0.000 | 3.614

SD, standard deviation; SEM, standard error of mean. p≤ 0.05 by 2-tailed t-test.

DISCUSSION

Although the alimentary canal shows striking differences in its morphology and function due to variation of habitats and feeding behavior, the histological structure of intestine of different species comprises of four layers which is common in all fishes. Several authors have shown that post-gastric parts of alimentary canal of fishes are responsible for nutrient absorption (Kapoor et al., 1975; Fänge and Grove, 1979; Morrison, 1987; Murray et al., 1996; Cinar and Şenol, 2006; Leknes, 2010). The histochemical characterization of this part was generally associated with feeding habits (Martin and Blaber, 1984; Grosh and Das, 1987; Murray et al., 1996). Consequently, the numbers and types of the cells located in post-gastric parts were differed due to the feeding behavior (Tyler, 1972; Kapoor et al., 1975; Martin and Blaber, 1984; Anderson, 1986; Kuperman and Kuz’mina, 1994; Murray et al., 1994, 1996). Intestinal parts can also be distinguished by villi sizes which were decreased in length, distinctly (Albrecht et al., 2001; Cinar and Şenol, 2006).

Main histological organization of the intestine of A. nigrofasciata is considerably similar to other teleosts (Wallace et al., 2005) except the abundancy of vacuolar enterocytes of posterior intestine in Ictalurus punctatus, Krementz and Chapman (1975) formerly noted that there were large and clear vacuoles present in the apical cytoplasm of epithelial cells of the posterior half of intestine after feeding. Enterocytes with supranuclear vacuoles were also reported in the larvae of Sparus aurata (Calzada et al., 1998), Pelteobagrus fulvidraco (Yang et al., 2010), and the juveniles of Culter alburnus (Cao et al., 2011). While their function is still unclear, some studies indicate that they may be associated with nutrition type or protein digestion (Smith and Sepúlveda, 1989; Cyrino et al., 2008; Cao et al., 2011). It was also assumed that the supranuclear vacuoles of the posterior enterocytes of A. nigrofasciata also might be involved in intracellular digestion.

The essential function of the gastrointestinal epithelium is to regulate counteractions between the external environment and the body (Loretz, 1995; Domeneghini et al., 2005). As concluded by Delashoub et al. (2010), intestinal digestive activity depends on goblet cell secretions, proteolytic action of pancreatic juice and/or intracellular digestion. Goblet cells as common structural elements of the alimentary tract and histochemical properties of the mucoid substance have been investigated in several teleosts (Domeneghini et al., 1998; Pedini et al., 2001; Bucke, 1971; Groman, 1982; Tibbetts, 1997; Díaz et al., 2003; Leknes, 2011).

Various glycoproteins (neutral, acidic and sulfated) in mucoid substance of different species had special functions such as lubrication, regulation of viscosity, entrapping of food particles, buffering the fluids at the epithelial surface, preclusion of proteolytic epithelial damage, antimicrobial activity and immunological defense (Reid et al., 1988; Yashpal et al., 2007; Díaz et al., 2008; Zhu, 2015). Neutral glycoproteins were involved mainly in absorption and transportation of molecules through the membranes (Reifel and Travill, 1979; Sarasquete et al., 2001; Pedini et al., 2005; Díaz et al., 2010). As a lubricative and protective secretion, acidic glycoproteins were associated with protection of epithelial damage against the high acidic gastric juices. Sulfated glycoproteins were also lubricative and usually related with the protection against microorganisms (Ferraris et al., 1987; Mittal et al., 2002; Park et al., 2003; Díaz et al., 2010; Leknes, 2015).

Domeneghini et al. (2005) have shown the presence of neutral and acidic glycoconjugates in intestinal mucus cells of Anguilla anguilla. The intestinal goblet cells of Sparus aurata were also shown to secrete both neutral and acidic glycoconjugates, especially sulfated forms (Domeneghini et al., 1998). Leknes (2011) has noted an ingredient composed of sulfated and neutral mucins in Hyphessobrycon anisitsi.

In this study, positive reactions of the goblet cells of anterior and mid intestine of A. nigrofasciata to AB, PAS and AF staining methods indicated intense acidic and neutral content, which is an evidence for relatively less sulfated glycoprotein.

PAS/AB (pH 2.5) staining, showed densely acidic glycoconjugates both in the anterior and mid intestinal goblet cells. The fact that the cells stained dominantly with AB at pH 2.5, showed occurrence of glycoproteins with carboxyl groups and/or with sulfate esters. However, negative reaction to KOH/PAS staining was evaluated as absence of sialic acid residues in this content. Presence of glycoproteins with sialic acid residues was correlated with protection against microorganism (Suprasert et al., 1987; Díaz et al., 2010).

It has also been noted that only anterior intestinal goblet cells exhibited too low affinity to KOH/PAS staining. Neutral glycoconjugates were also observed in the anterior and mid intestine of A. nigrofasciata.

As it was mentioned above, goblet cells were rarely distributed in posterior intestine of A. nigrofasciata. Several authors have shown that goblet cells increased in number in the posterior intestine, due to ease of defecation (Martin and Blaber, 1984; Cataldi et al., 1987; Grau et al., 1992; Murray et al., 1996; Domeneghini et al., 1998; Veggetti et al., 1999; Pedini et al., 2001; Cinar and Bilgin, 2001; Cinar and Şenol, 2006; Banan Khojasteh et al., 2009). At this point, this kind of disagreement could only be explained by abundance of vacuolar enterocytes.

Although, only two parts of intestine could be compared, the results presented in this study are in accordance with those reported in literature. The number of acidic and neutral goblet cells of the mid part of intestine was higher than those in anterior part.

It is concluded that the distribution of goblet cells and glycoconjugate composition of the gut of A. nigrofasciata depends on the intestinal region.

ACKNOWLEDGMENTS

We would like to thank Dr. Esra Akat for her contributions and technical support throughout the study.

Statement of conflict of interest

Authors have declared no conflict of interest.

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