The grass lizard, Takydromus wolteri Fischer, belonging to the Laceridae in the order of Lacertinae, lives in eastern part of Russia, China and Korea and is considered to be a primitive palearctic lacertid, most closely related to Lacerta vivipara. They have a distinct white ventrolateral band running from the tip of the snot to the groin. In Korea, numbers and habitats of this grass lizard have dramatically decreased because of pollution and immigration of foreign species of amphibians and reptiles having similar feeding habits.

Gastrointestinal endocrine cells are dispersed in epithelia and glands of the gastrointestinal tract and synthesize various types of hormones and play an important role in physiological functions of the gastrointestinal tract. The study of gastrointestinal endocrine cells is considered to be important for phylogenetic purpose. In addition, distribution patterns and the relative frequency of these endocrine cells vary with animal species and feeding habits. Although many studies have elucidated distribution patterns and the relative frequency of different types of endocrine cells in the gastrointestinal tract of various vertebrates, immunohistochemical studies on reptilia have received little attention until now. Studies have been performed on reptile species because their phylogenetic tree is found between amphibians and mammals and reports show characteristic distribution patterns and a relative frequency of gastrointestinal endocrine cells and have been compared with those of other vertebrates. Many studies have dealt with the identification of regulatory peptides in the gastrointestinal tract in reptile species using silver techniques, radioimmunochemical or immunohistochemical methods. Distribution patterns and relative frequency of these endocrine cells in reptile species varied with species and feeding habits. For example, different from amphibians and mammals, cells positive for insulin have been demonstrated in intestinal parts in reptiles. However, gastrointestinal endocrine cells have not been studied yet in gastrointestinal tract of the grass lizard, Takydromus wolteri Fischer. Therefore, we monitored these cells in the grass lizard using specific immunohistochemical methods and different antibodies raised against bovine Sp-1/chromogranin (BCG), serotonin, somatostatin, gastrin, cholecystokinin (CCK)-8, glucagon, insulin, human pancreatic polypeptide (HPP) and secretin, and the results were compared with those obtained in other reptile species.

Five adult grass lizards (45–50 mm in length) of the Laceridae, Takydromus wolteri Fischer, were captured around Kyungpook, Korea. Both males and females were used in our study. After phlebotomy from the head, samples of six parts of the gastrointestinal tract (from proximal to distal: esophagus, fundus, pylorus, duodenum, small and large intestine) were fixed in Bouin’s solution, according to. After paraffin embedding, serial sections (3–4 ?m thick) were prepared. Sections were deparaffinized, rehydrated and stained with hematoxylin and eosin for light microscopic examination of the normal alimentary architecture. Other sections were used for immunostaining using the peroxidase anti-peroxidase (PAP) method. Blocking of nonspecific peroxidase reactions was performed with normal goat serum prior to incubation with the specific antibodies. After rinsing in phosphate buffered saline (PBS; 0.01 M, pH 7.4), sections were incubated with secondary antibodies (goat anti-rabbit IgG or goat anti-guinea pig IgG, dilution, 1:200; Sigma, St. Louis, MO, USA). Sections were then washed in PBS buffer and finally incubated with PAP complex (dilution, 1:200; Sigma). The peroxidase reaction was carried out using a solution 3,3?-diaminobenzidine tetrahydrochloride containing 0.01% H₂O₂ in Tris-HCl buffer (0.05 M, pH 7.6). After immunostaining, sections were analysed with the use of a light microscope.

Specificity of the immunohistochemical staining methods was determined as recommended by, including preincubation of the antibodies with their corresponding antigens. The relative frequency of each type of endocrine cell was scored using 5 categories: (?), not present; (±), rare occurrence; (+), only a few cells present; (++), moderate amounts of cells present; (+++), numerous amounts of cells present.

Nine types of endocrine cells were detected with the antibodies against BCG, serotonin, somatostatin, gastrin, CCK-8, glucagon, insulin, HPP and secretin. The distribution patterns and relative frequencies of these endocrine cells in the gastrointestinal tract of the grass lizard are shown in Table 2. In addition, most endocrine cells were spindle shaped (open cell type), whereas cells that were spherical in shape (closed cell type) were occasionally found in stomach and large intestine.

BCG-positive cells were detected throughout the whole gastrointestinal tract including esophagus and most predominant frequencies were detected in the stomach. Cells stained for BCG showing a spherical to spindle shape were dispersed throughout the esophageal mucosa and especially in esophageal glands. In addition, polymorphic cells were demonstrated in epithelia and gastric glands of the stomach. Spindle-shaped BCG-positive cells having relatively short cytoplasmic processes were detected in epithelia of the small intestine. Spherical-shaped BCG-positive cells were observed in the large intestinal mucosa and appeared to be crowded in some regions.

Serotonin-positive cells were found in various relative frequencies throughout the entire gastrointestinal tract including the esophagus and showed the highest frequency in the pylorus. They were the most predominant cell types found in this study. Spindle-shaped cells having relatively short cytoplasmic process were dispersed throughout esophageal mucosa but they were not detected in other gastrointestinal epithelia. Cells showing a polymorphic shape were demonstrated in regions of gastric glands. Spindle-shaped cells having relatively long cytoplasmic processes ending in the lumen were detected in epithelia of small intestine but spherical-shaped cells were found in the large intestinal mucosa.

Cells stained for somatostatin were demonstrated throughout the entire gastrointestinal tract except for esophagus and large intestine and were most predominant in pylorus and duodenum. Spherical-shaped cells were located in regions of gastric glands but spindle-shaped somatostatin-positive cells having relatively long cytoplasmic processes were detected in epithelia of small intestine.

Gastrin-positive cells were restricted to pylorus and duodenum and they showed spherical to spindle-shaped morphology in regions of gastric glands in the pylorus, but they were not found in epithelia of the pylorus. Spindle-shaped gastrin-positive cells having relatively long cytoplasmic process were detected in epithelia of the duodenum.

Figure 4. Gastrin- and CCK-8-positive cells in the gastrointestinal tract of the grass lizard, Takydromus wolteri. Note that gastrin-positive cells are restricted to pylorus (a) and duodenum (b), and CCK-8-positive cells to pylorus (c, d), duodenum (e) and small intestine (f; arrowhead). Scale bar, 33 ?m.

CCK-8-positive cells were observed from pylorus to small intestine and showed the highest frequency in pylorus. Spherical to spindle-shaped cells were located in regions of gastric glands but they were not found in other epithelia. Spindle-shaped cells with cytoplasmic processes were detected in epithelia of small intestine.

Glucagon-positive cells were restricted to duodenum and small intestine. Spindle-shaped cells were detected in the epithelia of small intestine. These cells were crowded in some parts of the duodenum.