Morphological and Histochemical Investigations of Esophagogastric Tract of a Lizard, Laudakia Stellio (Agamidae, Linnaeus 1758)

Acta Biologica Hungarica 62(4), pp. 376–387 (2011) DOI: 10.1556/ABiol.62.2011.4.4

MORPHOLOGICAL AND HISTOCHEMICAL INVESTIGATIONS OF ESOPHAGOGASTRIC TRACT OF A LIZARD, LAUDAKIA STELLIO (AGAMIDAE, LINNAEUS 1758)

YÜCEL BAŞIMOĞLU KOCA1* and BEYHAN GÜRCÜ2

1 Department of Biology, Faculty of Sciences and Art, Adnan Menderes University, Aydin, Turkey 2 Department of Biology, Faculty of Sciences and Art, Celal Bayar University, Manisa, Turkey

(Received: September 14, 2010; accepted: January 18, 2011)

Histological structures of esophagus and stomach tissue samples of Lacerta stellio have been studied, and glycosaminoglycan (GAG) distribution has been histochemically determined. Histologically, esophagus and stomach of L. stellio are composed of four layers: mucosa, submucosa, muscularis mucosae and serosa. Mucosa of esophagus is covered by simple columnar ciliated epithelium with many mucous secreting goblet cells and contains branched tubular glands. Stomach of L. stellio is composed of fundus (oral and aboral) and pylorus regions. Mucosa is covered by columnar epithelium. Fundic glands are branched tubular glands while pyloric glands are usually simple tubular glands. In both regions of the stomach, glands are subdivided into three areas as base, neck and isthmus. Both in the esophagus and stomach, muscular layer is in the form of smooth muscle having inner circular and outer longitudinal layers. According to the results obtained by Alcian Blue (pH 5.8)/Periodic Acid Schiff staining, stomach is similar to esophagus in that neutral mucins and hyaluronic acid (HA) are dominant in isthmus and neck regions of gland tissue of stomach. In the base of the stomach, only neutral mucins have been observed. HA has been observed to be dominant in all other regions of both stomach and esophagus, along with some but not much sulphated GAGs.

Keywords: Histology – histochemistry – esophagus – stomach – L. stellio

INTRODUCTION

The digestive tract of reptiles shows a marked diversity in its morphology and function. Although the alimentary canal differs considerably among species, it shows some basic structural similarities. For a given species, the overall morphology is chieﬂy related to the nature of the food, feeding habits, body size, shape and sex [36]. There is some disagreement in the literature about the non-mammalian vertebrates oesophogogastric tract [41]. In non-mammalian vertebrates, esophagus epithelium differs among species depending on the diet. While single layered columnar epithelium with brush border has been reported for esophagus, stomach and intestine of different species,

*Corresponding author; e-mail: [email protected]

Chalcalburnus tarichi has been reported to contain simple layered cuboidal epithelium during its development [11, 25, 37, 44]. In fishes, lamina epithelialis differs among species. For example, in Umbrina cirrosa L., it is a stratified epithelium with many goblet cells composed of cuboidal cells in the base, columnar cells in the middle and squamous cells on the surface [27, 28]. In rainbow trout, anterior and middle sections of esophagus are covered by multi-layered cuboidal epithelium with many goblet cells. The lower section of esophagus is characterized with single layered columnar epithelium [9]. In water turtle, lamina epithelialis has a multi-layered, squamous epithelial structure containing a thick keratin layer in order to provide protection against any trauma caused by shell foods such as stone and silica rich coral [4]. It is composed of a single layered prismatic epithelium with kinocilium in lizard [18, 20], and multi-layered prismatic epithelium in snake [19]. In mammalian eosophagus covered with squamous epithelia [43]. The stomach is anatomically and histologically subdivided into two distinct regions: a fundic region that constitutes most of the stomach, and a small pyloric region in reptiles [1, 24, 32]. Histochemical studies have demonstrated that in vertebrates there are differences between fundic and pyloric glands in the qualitative expresion of neutral, sulphated and carboxylated mucins [35] and in the lectin binding pattern in mucous neck cells [10, 12]. The mucous neck cells of the gasric glands appeared phylogenetically first in amphibians [38]. They have also been found in the stomach of reptiles and mammals, but not in fishes, birds [38] or in most sauri- ans [13]. The main products of the exocrine cells in the oesophagogastric mucosa are mucins. The type of mucins produced along the alimentary tract varies with the region, cell type, developmental stage and species in relation to different functions, such as protection against gastric juice or lubrication during the passage of food [11, 26, 38], barrier against penetration of bacteria and infective agents and, probably, to phylogenetic relationships [38]. Mucins are large, extracellular glycoproteins, which consist of highly glycosylated carbohydrates N-acetylglucosamine and N-acetylgalactosamine, fucose, galactose, and sialic acid (N-acetylneuraminic acid) and also contain traces mannose and sulfate [6]. Glycosaminoglycans (GAGs), also called acid mucopolysaccharides and contains N-acetylgalactosamine (GalNAc) or N-acetylglucosamine (GlcNAc), are linear polymers and have various important biological roles such as cell proliferation, cell migration, differentiation, cell-matrix interaction, and tissue damage repair [29, 40]. No studies have been found that used Alcian blue staining technique by preparing five different MgCl2 concentrations to study histological structures of esophagus and stomach of L. stellio (starred agama) and to determine GAG distribution. Thus, our aim was to study histological structure of esophagus and stomach tissue samples of L. stellio and determine glycosaminoglycan (GAG) distribution in epithelial cell basal layer, connective tissue and tunica muscularis layers, and epithelial mucins types.

Acta Biologica Hungarica 62, 2011 378 YÜCEL BAŞIMOĞLU KOCA and BEYHAN GÜRCÜ

MATERIALS AND METHODS

Ten (5 male, 5 female) L. stellio caught in Kalfaköy-Aydın/Turkey were brought to the laboratory alive, knocked out by ether anesthesia and esophagus (middle part) and stomach (fundic and pyloric parts) tissue samples were taken. Tissues were fixed in Saint Marie (SM) [42] fixative solution for 24 hours and embedded in paraffin after passed through alcohol and xylene series. Tissues were then sectioned at a thickness of 5 micron by a Rotary microtom (RM 2145). Sections were stained by hematoxylin- eosin to determine the general structure, by Gomori trichome to determine the connective tissue and by 0.1% Alcian Blue 8 GX prepared in five different concentrations (0.025, 0.06, 0.3, 0.65, 0.9 M MgCl2) buffered (pH 5.8) with sodium acetate (Table 1) to determine the GAGs distribution. Periodic Acid-Schiff (PAS) used to determine the neutral mucins. GAGs were identified by critical electrolyte concentrations at which the polyanions changed from binding AB to MgCl++. In this method, at low molarities of MgCl++ (e.g. 0.06 M) both carboylated (i.e. hyaluronic acid) and

Fig. 1. Histologic structure of oesophagus of L. stellio. L; Lumen, p; plica circulares, m; mucosa, E; simple columnar epithelium, ; cilia, x; mucus cell, g; branched tubular glands, lp; lamina propria, *; muscularis mucosa, ●; submucosa, ◘; circular muscle layer, ▪; longitudinal muscle layer, ; serosa. A; Hematoxylin-eosin, ×10 – B; Gomori trichrome, ×20. C; PAS, ×40, D; Gomori trichrome ×100

Acta Biologica Hungarica 62, 2011 Morphology and histochemistry of the lizard esophagus 379 sulfated acid mucins (i.e. chondroitin sulfate, dermatan sulfate, heparan sulfate and keratan sulfate) were stained, whereas at higher molarities (e.g. 0.3, 0.65, 0.9 M) only sulfated acid mucins were stained (Table 1) [16, 33]. Although it does not show the total amount of GAGs, the change in dying gives an opinion about the type of GAGs. Results obtained were evaluated by comparisons with the results of the histochemical reactions obtained in various sources [5].

RESULTS

Esophagus

L. stellio esophagus is composed of four layers: tunica mucosa (lamina epithelialis, lamina propria, muscularis mucosae), tunica submucosa, tunica muscularis and serosa (Fig. 1A, B). Mucosa is covered by simple columnar ciliated epithelium with many mucus secreting goblet cells and contains branched tubular glands (Fig. 1C, D). Muscle ﬁbers in muscularis mucosa are arranged circular in the inner part and longitudinal in the outer part (Fig. 1A, B). Tunica muscularis of esophagus is in the form of smooth muscle having inner circular and outer longitudinal layers. Serosa layer is composed of a thin loose connective tissue (Fig. 1B). Based on the results of AB (pH 5.8)/PAS, it was found that glycoproteins were dominant in mucus secreting goblet cells (Table 2, Fig. 2), especially when 0.025 M and 0.06 M MgCl2 concentrations were compared, and the most dominant GAG type in mucus secreting goblet cells was hyaluronic acid, with a little chondroitin sulphate and dermatane sulphate and very very little heparine, heparan sulphate and keratan sulphate, when 0.06 M, 0.3 M, 0.65 M and 0.9 M MgCl2 concentrations were compared (Table 2, Fig. 2). When epithelial basal layer, connective tissue and muscle layer of esophagus were compared, dominant GAG type, especially in muscle layer, was HA. It was noticed that Ch-S and DS, which are sulphated GAGs, were dominant in basal layer and connective tissue compared to muscle tissue and very little HP, HS and KS were present at all regions (Table 3, Fig. 2).

Table 1 Staining speciﬁcities of AB-PAS sequences at different electrolyte concentrations

Stain Interpretation of reaction Reference

AB pH 5.8 in 0.025 M MgCl2 Sulphated GPs, All acid mucins 5, 33 AB pH 5.8 in 0.06 M MgCl2 All acid mucins AB pH 5.8 in 0.3 M MgCl2 Weakly and strongly sulphated mucins AB pH 5.8 in 0.65 M MgCl2 Heparin, Heparan Sulphate and Keratan Sulphate AB pH 5.8 in 0.9 M MgCl2 Keratan Sulphate PAS GPs with oxidizable vicinal diols and/or glycogen 5, 45

AB – Alcian Blue; GPs – Glycoproteins; PAS – Periodic acid Schiff’s.

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Table 2 GAG staining speciﬁcations in mucosal layers of oesophagus and stomach of L. stellio. Reaction intensities indicated with increased plus signs

MgCl2 concentrations 0.025 0.06 0.3 0.65 0.9 MgCl2 MgCl2 MgCl2 MgCl2 MgCl2 Regions

OESOPHAGUS Goblet cell +++++ +++ ++ + +

Superﬁcial cell +++++ +++++ +++ ++ +

Foveolar cell +++++ +++++ +++ ++ +

Fundus-Oral Mucous neck cell +++++ +++++ +++ + +

Superﬁcial cell +++++ ++++ +++ + +

Foveolar cell +++++ ++++ +++ + ±

STOMACH Mucous neck cell +++++ ++++ +++ + ± Fundus-Aboral

Superﬁcial cell +++++ ++++ +++ +++ ++

Foveolar cell +++++ ++++ +++ +++ ++ Pylor Mucous neck cell +++++ ++++ +++ + ±

Stomach

L. stellio stomach is anatomically composed of fundic (oral and aboral) and pyloric regions. Histologically, it has four layers (from inner to outer): mucosa, submucosa, muscularis and serosa (Fig. 3A–C). Mucosa is covered by simple columnar epithelium with many mucous cells and the fundus contains branched tubular glands while the pyloric region usually contains simple tubular glands. Moreover, in both regions of the stomach, glands are subdivided into three areas as base, neck and isthmus. Submucosal layer which is in the form of loose connective tissue is separated from lamina propria by thin muscularis mucosae (Fig. 3A–C). Muscle ﬁbers in muscular layer are arranged circularly in the inner part and longitudinaly in the outer part. Serosa layer is composed of loose connective tissue covered by squamous epithelium (Fig. 3A–C). AB stainings were evaluated for glands (base, neck and isthmus), basal layer, submucosa (connective tissue) and tunica muscularis in two different regions of the stomach. When oral and aboral regions of the fundus and pylorus were compared in

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Fig. 2. Histochemistry of the oesophagus of L. stellio. AB (pH 5.8). Two different concentrations of MgCl2 (0.025 M, 0.9 M MgCl2)/PAS

Fig. 3. Histologic structure of stomach (fundus; oral-aboral, pylor) of L. stellio. 1; Tunica mucosa (E; epithelium, Sg; secretory glands-branched tubuler glands, Lp; lamina propria, *; muscularis mucosae), 2; tunica submucosa, 3; tunica muscularis, : tunica serosa. A; Fundus (Oral)-Gomori trichrome, ×20, B; Fundus (Aboral)-Gomori trichrome, ×20, C; Pylor-Hematoxylin-eosin, ×20 terms of AB/PAS stainings, it was observed that glycoproteins (sulfated and carboxylated sialomucins), neutral mucins and GAG types were similar. It was also observed that neutral mucins and hyaluronic acid (HA) were dominant in the superﬁcial cells, foveolar cells and mucous neck cells and, very little heparine (HP), heparin sulphate (HS) and keratan sulphate but mainly chondroitin sulphate (Ch-S) and dermatan sul-

Acta Biologica Hungarica 62, 2011 382 YÜCEL BAŞIMOĞLU KOCA and BEYHAN GÜRCÜ phate (DS) were also present. AB staining was not seen in the base region, which indicates that neutral mucins were dominant in this region and there were no GAGs. When sulphated GAG content of fundus oral and fundus aboral were compared with the pylorus, it was detected that Ch-S and DS were dominant in fundus and KS was dominant in superﬁcial cells of pyloric glands (Table 2). When epithelial basal layer, connective tissue and muscle layer of fundus and pylorus were compared, it was noticed that the dominant GAG type was HA, especially in muscle layer. Ch-S and DS, which are sulphated GAGs, were found to be more dominant in epithelial basal layer and connective tissue compared to muscle tissue and, little HP, HS and KS were present in all regions (AB–Five different MgCl2 concentrations/PAS-images) (Table 3, Fig. 4).

Fig. 4. Histochemistry of the stomach of L. stellio. AB (pH 5.8). Two different concentrations of MgCl2 (0.025 M, 0.9 M MgCl2)/PAS, ×100

When compared, stomach is similar to esophagus in that neutral mucins and HA (carboxylated mucin) were dominant in the isthmus and neck regions of gland tissue of stomach. In the base of the stomach, only neutral mucins were observed. GAG contents of all the other regions of both stomach and esophagus were similar.

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Table 3 Staining gradation of GAGs in oesophagus and stomach of L. stellio according to AB sequences at different critical electrolyte concentrations. Reaction intensities indicated with increased plus signs

MgCl concentrations 2 0.025 0.06 0.3 0.65 0.9

MgCl MgCl MgCl MgCl MgCl Regions 2 2 2 2 2

Basal layer ++++ ++++ +++ ++ +

Connective tissue ++++ ++++ +++ ++ +

OESOPHAGUS Tunica muscularis ++++ ++++ ++ + +

Basal layer ++++ ++++ +++ ++ +

Connective tissue ++++ ++++ +++ ++ +

Tunica muscularis ++++ ++++ ++ + + Fundus-Oral

Basal layer ++++ ++++ +++ ++ +

Connective tissue ++++ ++++ +++ ++ +

STOMACH Tunica muscularis ++++ ++++ ++ + + Fundus-Aboral

Basal layer ++++ ++++ +++ ++ +

Connective tissue ++++ ++++ +++ ++ + Pylor Tunica muscularis ++++ ++++ ++ + +

DISCUSSION

The oesophagogastric tract of L. stellio presents the general features of most non- mammalian vertebrates. Microscopic examination of the esophagus and stomach mucosa of several investigated species of reptiles have showed a considerable varia- tion in their histological structure [18–20, 38]. The reptile esophagus differs from the mammalian. Mammalian eosophagus is covered with squamous epithelia whereas reptilian epithelia contain ciliated columnar epithelium (squamousepithelia) and non-ciliated mucus cells. The mucosal lining of the esophagus is composed of ciliated columnar cells in the proximal (craniad) and mid-portions of the esophagus, although cilia were sparse to absent in the distal esophagus of American alligator (Alligator mississippiensis) [43]. In the mammalian

Acta Biologica Hungarica 62, 2011 384 YÜCEL BAŞIMOĞLU KOCA and BEYHAN GÜRCÜ esophagus the wall and muscle layers are thinner, with only two muscle layers repre- sented (inner circular and outer longitudinal) and the proximal one-third to one-half is formed of skeletal muscle, while the crocodilian esophagus is composed entirely of smooth muscle. An entirely smooth muscled organ is reported to exist in the duck- billed platypus, amphibians, birds and other reptiles [21]. However, more recent work has found striated muscle in the pharyngeal region of the esophagus in bullfrogs and African clawed frogs [46] and in the proximal esophagus of some birds [15]. In our study, it has been determined that esophagus and stomach of L. stellio are composed of t. mucosa, t. submucosa, t. muscularis and serosa layers histologically. Both in esophagus and in stomach, muscularis mucosa layer is in the form of smooth muscle having inner circular and outer longitudinal layers. Mucosal layer of esophagus of L. stellio is covered by simple columnar ciliated epithelium with many mucous secreting goblet cells and contains branched tubular glands. The mucous secreting cells stained strongly with PAS and AB in 0.06 and 0.3 M MgCl++ and lower affinity with AB in 0.65 and 0.9 M MgCl++, suggesting that they secrete both neutral and acidic, especially carboxylated mucin. Biologically it is very important that HA forms up a barrier against penetration of bacteria and infective agents [30] along with providing lubrication and decreasing effects of impacts [14]. The mucous, especially acidic mucins, may be important for lubrication the mucosa and allowing the passage food particle, in addition to protect the mucosa from the sharp object. Both neutral and acidic mucins in covering epithelium of esophagus were detected in other reptiles [1, 23], crocodiles [43] and many birds [34]. Mucosal layer of stomach is covered by simple columnar epithelium with many mucous cells and the fundus region contains branched tubular glands while the pylorus region usually contains simple tubular glands. Moreover, in both sections of the stomach, glands are subdivided into three areas as base, neck and isthmus. Muscularis mucosa separates lamina propria from submucosal layer characterized by loose connective tissue. In the fundic part of the stomach, mucus was secreted by superficial, foveolar and mucous neck cells. The mucous secreting superficial and foveolar cells stained strongly with PAS and AB in 0.06 and 0.3 M MgCl++ and have a lower affinity for AB in 0.65 and 0.9 M MgCl++, revealing that these cells secrete mostly neutral mucins, carboxylated and strongly sulphated mucins in addition to a small amount of weakly sulphated mucins. While, the mucus secreted by mucous neck cells contained both neutral and strongly sulphated acidic mucins as revealed by AB staining in five different MgCl++ concentrations. The mucous contents of pyloric glands is similar to the mucous contents of fundic glands. But, mucus secreted by superficial and foveolar cells in pyloric glands contained mostly all acid mucins as revealed by our histochemical results. The possible functions of gastric mucus are probably to protect the epithelium from pathogenic organisms and against digestion enzymes, bicarbonate, hormones, hydrochloric acid, intrinsic factor and acids produced by the stomach itself. Mucus and bicarbonate protect the stomach from the irritation of gastric secretion [2]. On the other hand, the production of sulphated mucin may be related to the fluid balance between external and internal enviroments as suggested by Smith [36]. Both neutral and acidic mucins in the stomach in L. stellio have been found in

Acta Biologica Hungarica 62, 2011 Morphology and histochemistry of the lizard esophagus 385 amphibian (Rana ridibunda) [22, 31], and also in the snake (Elaphe climacophora), in turtle (Clemmys japonica) [38], and in European eel (Anguilla anguilla) [8]. Mucus is a thick secretion composed of water, electrolyte, proteins, GPs, lipids and GAGs. Although GPs in mucus structure have been extensively studied, little is known about GAG compounds of mucus. These molecules regulate, on one hand, stability of cells and fibrous components; on the other hand, water and salt balance of the body and mucus secretions and fluidity of synovial fluid as they are negatively charged [14]. Mucus protects the tunica mucosa from chemicals, bacteria, parasites and acids by forming a slippery surface through the digestive tract and forms a diffu- sion barrier against different ions; moreover, it protects epithelium against mechani- cal damages [7, 17, 39]. In vertebrate, mucins are similar in digestion system in terms of their macromolecular properties and polymeric structures; however, they differ in intestine, esophagus and stomach depending on whether they are neutral or acidic mucins (carboxylated and sulphated mucins, sulphated sialomucins). These different carbohydrate structures may affect specific functions of mucus at different regions of digestion system [11]. Absorption and macromolecular transport [3] are among functions of mucus which is forming a physical barrier between mucosa and environmental fac- tors [23]. We may say that epithelium has very important functions such as providing a selective permeability barrier between channel contents and body tissues, facilitating transport and digestion of food, being involved in the absorption of digested foods and generating hormones that affect the activity of the system. We believe that mucus secreted by mucous cells is important for the digestive system with respect to its functions such as protecting the epithelium against damages and the organism against bacterial invasion along with lymphoid nodules in lamina propia and submucosal layer.

REFERENCES

1. Ahmed, Y. A., El-Hafez, A. A. E., Zayed, A. E. (2009) Histological and histochemical studies on the esophagus, stomach and small intestines of Varanus niloticus. J. Vet. Anat. 2, 35–48. 2. Allen, A., Flemström, G. (2005) Gastroduodenal mucus bicarbonate barrier: protection against acid and pepsin. Am. J. Physiol. Cell Physiol. 288, C1–C19. 3. Arellano, J. M., Storch, V., Sarasquete, C. (2002) Ultrastructural study on the intestine of Senegal sole, Solea senegalensis. J. Appl. Ichthyol. 18, 154–158. 4. Aughey, E., Frye, L. F. (2001) Comparative Veterinary Histology. Manson Publishing Ltd., pp. 105– 107. 5. Bancroft, J. D., Cook, H. C. (1994) Manual of Histological Techniques and Their Diagnostic Application. Churchill Livingstone. London, p. 457. 6. Bansil, R., Turner, B. S. (2006) Mucin structure, aggregation, physiological functions and biomedical applications. Current Opinion in Colloid&Interface Science 11, 164–170. 7. Domeneghini, C., Ponnelli, R. S., Veggetti, A. (1998) Gut glycoconjugates in Sparus aurata L. (Pisces, Teleostei). A comparative histochemical study in larval and adult ages. Histol. Histopathol. 13, 359–372.

Acta Biologica Hungarica 62, 2011 386 YÜCEL BAŞIMOĞLU KOCA and BEYHAN GÜRCÜ

8. Domeneghini, C., Arrighi, S., Radaelli, G., Bosi, G., Veggetti, A. (2005) Histochemical analysis of glycoconjugate secretion in the alimentary canal Anguilla anguilla L. Acta Histochem. 106, 477– 487. 9. Ezeasor, D. N. (1984) Light and electron microscopic studies on the esophageal epithelium of the Rainbow Trout, Salmo gairdneri. Anat. Anz. 155, 71–83. 10. Ferri, D., Liquori, G. E. (1994) Immunohistochemical investigations on the pyloric glands of the ruin lizard (Podarcis sicula campestris de Betta). Acta Histochem. 96, 96–103. 11. Ferri, D., Liquori, G. E., Natale, L., Santarelli, G., Scillitani, G. (2001) Mucin histochemistry of the digestive tract of the red-legged frog Rana aurora aurora. Acta Histochem. 103, 225–237. 12. Ferri, D., Liquori, G. E. (1992) Characterization of secretory cell glycoconjugates in the alimentary tract of the ruin lizard (Podarcis sicula campestris de Betta) by means of lectin histochemistry. Acta Histochem. 93, 341–349. 13. Gabe, M., Saint-Girons, H. (1972) Contribution al’histologie de l’estomac des lepidossauriens (Reptilis). Zool. Jb. 89, 572–599. 14. Gandhi, N. S., Mancera, R. L. (2008) The structure of glycosaminoglycans and their interaction with proteins. Chem. Biol. Drug Des. 72, 455–482. 15. Geyikoglu, F., Temelli, A., Ozkaral, A. (2002) Muscle fiber types of the tunica muscularis externa at the upper part of the sparrow (Passer domesticus) esophagus. Turk. J. Zool. 26, 217–221. 16. Goldstein, C. D., Jankiewicz, J. J., Desmond, M. E. (1986) Identification of glycosaminoglycans in the chondrocranium of the chick embryo before and at the onset of chondrogenesis. J. Embryol. Exp. Morphol. 93, 29–49. 17. Humbert, W. R., Kirsch, R., Meister, M. F. (1984) Scanning electron microscopic study of the oesophageal mucous layer in the eel, Anguilla anguilla L. J. Fish Biol. 25, 117–122. 18. Imai, M., Shibata, T., Izumi, T. (1992) Histological and histochemical investigations on Japanese lizard esophagus. Okajimas Folia Anat. Jpn. 69, 25–34. 19. Imai, M., Shibata, T., Moriguchi, K. (1991) Pepsinogen granules in the esophageal epithelium of the rock snake. Okajimas Folia Anat. Jpn. 68, 231–234. 20. Imai, M., Shibata, T., Moriguchi, K., Hayama, H. (1991) Glands distributed in the lamina propria mucosae of the oesophagus in the gecko and Japanese lizard. Okajimas Folia Anat. Jpn. 68, 289– 293. 21. Ingelfinger, F. J. (1958) Esophageal motility. Physiol. Rev. 38, 533–584. 22. Liquori, G. E., Mastrodonato, M., Zizza, S., Ferri, D. (2007) Glycoconjugate histochemistry of the digestive tract of Triturus carnifex (Amphibia, Caudata). J. Mol. Hist. 38, 191–199. 23. Loumbourdis, N. S. (2003) Mucosubstance histochemistry in the esophagus of the lizard Agama stellio stellio. Amphibia-Reptilia 24, 224–229. 24. Lupa, H. (1977) Histology of the digestive tract. In: Gans, C., Parsons, T. S. (ed.). Biology of the Reptilia. Academic Press, London, pp. 225–302. 25. Morrison, C. M., Wright, J. R. (1999) A study of the histology of the digestive tract of the Nile tilapia. J. Fish Biol. 54, 597–606. 26. Oinuma, T., Kawano, J. I., Suganuma T. (1991) Glycoconjugate histochemistry of Xenopus laevis fundic gland with special reference to mucouse neck cells during development. Anat. Rec. 230, 502–512. 27. Pedini, V., Scocco, P., Radaelli, G., Fagioli, O., Ceccarelli, P. (2001) Carbohydrate histochemistry of the alimentary canal of the shi drum, umbrina cirrosa L. Anat. Histol. Embryol. 30, 345–349. 28. Pedini, V., Dall’Aglio, C., Parillo, F., Scocco, P. (2004) A lectin histochemical study of the oesophagus of shi drum. J. Fish Biol. 64, 625–631. 29. Prabhakar, V., Capila, I., Sasisekharan, R. (2009) Glycosaminoglycan characterization methodolo- gies: probing biomolecular interactions. Meth. Mol. Biol. 534, 331–340. 30. Russell, A. L. (1999) Glycosaminoglycan (GAG) deficiency in protective barrier as an underlying, primary cause of ulcerative colitis, Crohn’s disease, interstitial cystitis and possibly Reiter’s syn- drome. Medical Hypothesis 52, 297–301.

Acta Biologica Hungarica 62, 2011 Morphology and histochemistry of the lizard esophagus 387

31. Sancar-Bas, S., Kaptan, E., Sengezer-Inceli, M., Sezen, A., Us, H. (2009) Glycoconjugate histochemistry in the Fundic stomach and small intestine of the frog (Rana ridibunda). IUFS J. Biol. 68, 93–104. 32. Sartori, S. S. R., Nogueira, K. O. P. C., Rocha, A. S. R., Neves, C. A. (2010) Morphology of the stomach of the tropical house gecko Hemidactylus mabouia (Squamata: Gekkonidae). Acta Zoologica, 1–8. 33. Scott, J. E. (1996) Alcian blue. Now you see it, now you don’t. Eur. J. Oral Sci. 104, 2–9. 34. Selvan, P. S., Ushakumary, S., Ramesh, G. (2008) Studies on the histochemistry of the proventriculus and gizzard of post-hatch guinea fowl (Numida meleagris). Inter. J. Poultry Scien. 7, 1112–1116. 35. Sheahan, D. G., Jervis, H. R. (1976) Comparative histochemistry of gastrointestinal mucosubstances. Am. J. Anat. 146, 103–131. 36. Smith, L. S. (1989) Digestive functions in teleost fish. In: J. E. Halvert (ed.), Fish Nutrition. Academic Press, San Diego, pp. 331–421. 37. Starck, J. M., Beese, K. (2002) Structural flexibility of the small intestine and liver of garter snakes in response to feding and fasting. J. Exp. Biol. 205, 1377–1388. 38. Suganuma, T., Katsuyama, T., Tsukahara, M., Tatematsu, M., Sakakura, Y., Murata, F. (1981) Comparative histochemical study of alimentary tracts with special reference to the mucous neck cell of the stomach. Am. J. Anat. 161, 219–238. 39. Suprasert, A., Fujioka, T., Yamad, K. (1987) The histochemistry of glycoconjugates in the colonic epithelium of the chicken. Histochemistry 86, 491–497. 40. Suzuki, S., Mizutani, A., Koike, Y., Kato, M., Yoshida, K., Kimata, K. (1991) Glycosaminoglycan chains of proteoglycans: approaches to the study of their structure and function. Pure Appl. Chem. 63, 545–554. 41. Taylor, P. M., Tyler, M. J. (1986) Pepsin in the toad Bufo marinus. Comp. Biochem. Physiol. 84, 669–672. 42. Tuckett, F., Morris-Kay, G. (1988) Alcian blue staining of glycosaminoglycans in embrionic material: Effect of different fixatives. Histochem. J. 20, 174–182. 43. Uriona, T. J., Farmer, C. G., Dazely, J., Clayton, F., Moore, J. (2005) Structure and function of the esophagus of the American alligator (Alligator mississippiensis). J. Exp. Biol. 208, 3047–3053. 44. Ünal, G., Çetinkaya, O., Kankaya, E., Elp, M. (2001) Histological study of the organogenesis of the digestive system and swim bladder of the Chalcalburnus tarichi Palas, 1811 (Cyprinidae). Turk. J. Zool. 25, 217–228. 45. Yamabayashi, S. (1987) Periodic acid-Sciff-Alcian blue: A method for the differential staining of glycoproteins. Histochem. J. 19, 565–571. 46. Yoshida, M. (2001) A light microscope study of the distribution of muscle in the frog esophagus and stomach. J. Smooth Muscle Res. 37, 95–104.

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