-2851$/ 2) 9H W H U L Q D U \  6FLHQFH J. Vet. Sci. (2004), / 5 (2), 87–95 An immunohistochemical study of the gastrointestinal endocrine cells in the ddY mice Sae-kwang Ku, Hyeung-sik Lee 1, *, Jae-hyun Lee 2 Pharmacology & Toxicology Lab., Central Research Laboratories, Dong-Wha Pharm. Ind. Co., Anyang 430-017, Korea 1 Department of Herbal Biotechnology, Daegu Haany University, Daegu 712-715, Korea 2 Department of Histology, College of Veterinary Medicine, Kyungpook National University, Daegu 702-701, Korea The distributions and frequencies of some endocrine cells in the gastrointestinal (GI) tract of ddY mice were studied with immunohistochemical method using 7 types of antisera against bovine chromogranin (BCG), serotonin, gastrin, cholecystokinin (CCK)-8, somatostatin, glucagon and human pancreatic polypeptide (HPP). All of 7 types of immunoreactive (IR) cells were identified. Most of IR cells in the intestinal portion were generally spherical or spindle in shape (open typed cell) while cells showing round in shape (close typed cell) were found in the intestinal gland and stomach regions occasionally. Their relative frequencies were varied according to each portion of GI tract. BCG-IR cells were demonstrated throughout whole GI tract except for the cecum and they were most predominant in the fundus and pylorus. Serotonin-IR cells were detected throughout whole GI tract and they were most predominant cell types in this species of mice. Gastrin- IR cells were restricted to the pylorus and CCK-8-IR cells were demonstrated in the pylorus, duodenum and jejunum with numerous frequencies in the pylorus. Somatostatin-IR cells were detected throughout whole GI tract except for the cecum and rectum and they showed more numerous frequencies in the stomach regions. In addition, glucagon- IR cells were restricted to the fundus, duodenum and jejunum with rare frequencies, and HPP-IR cells were restricted to the rectum only with rare frequency. In conclusion, some strain-dependent unique distributional patterns of gastrointestinal endocrine cells were found in GI tract of ddY mice. Key words: Gastrointestinal tract, endocrine cell, ddY mouse, immunohistochemistry, PAP method Introduction The ddY mouse is an closed colony albino mouse. From non-inbred dd of Institute of Infectious Diseases, University of Tokyo, 1953 [39], most suitable one among 6 strains was tested for the culture of Clonorchis sinensis [16]. Now it is widely distributed and one of the most widely used inbred mouse strains in Japan and other countries. This strain is particularly well known for the induction of osteoporosis by ovariectomy [42] and sciatic neurectomy [30]. Gastrointestinal endocrine cells dispersed in the epithelia and gastric glands of the digestive tract synthesized various kinds of gastrointestinal hormones and played an important role in the physiological functions of the alimentary tract [2]. Until now, the investigation of gastrointestinal endocrine cells is considered to be an important part of a phylogenic study [5]. In addition, the regional distributions and relative frequencies of these endocrine cells were varied with animal species and feeding habits [34]. Many studies have elucidated the regional distribution and relative frequency of different endocrine cells in the gastrointestinal (GI) tract of the various vertebrates including various species of rodents, and also the researches or data processing about gastrointestinal endocrine cells in the mouse strains have been widely executed. In the Rodentia, the localization of endocrine cells in the GI tract of the Manchurian chipmunk [23] and gerbil [26] was demonstrated, and the distribution of the endocrine cells in the intestinal tract was also detected in the Korean tree squirrel [22,24]. And also endocrine cells in the GI tract of homozygous obese mouse were investigated [36,37] and Pinto et al . [27] showed that the gastrointestinal endocrine cells in genetically diabetic (db/ db) mouse had quite different distributional patterns compared to those of nondiabetic control (db/+) mouse. In addition, the changes of regional distribution and relative frequency of some gastrointestinal endocrine cells in mice with ageing were also reported [31-33] and the regional distribution and relative frequency of gastrointestinal endocrine cells in hairless [21] and C57BL/6 [20] were also *Corresponding author Tel: 82-53-819-1436; Fax: 82-53-819-1574 E-mail: endohist@dhu.ac.kr 88 Sae-kwang Ku et al. demonstrated. Although many studies have elucidated the regional distribution and relative frequency of different endocrine cells in the GI tract of the various vertebrates including various species and strains of rodents, the reports dealing with the endocrine cells in GI tract of ddY mice were seldom in spite of their biological, physiological and anatomical differences from the other rodents and usefulness in many research fields. The objective of this study was to clarify the regional distribution and relative frequency of the endocrine cells in the GI tract of ddY mice by specific immunohistochemistry using 7 types of antisera against bovine chromogranin (BCG), serotonin, gastrin, cholecystokinin (CCK)-8, somatostatin, glucagon and human pancreatic polypeptide (HPP). Materials and Methods Five adult female ddY mice (6-wk old, 24-26 g body weight upon receipt) were acquired from Japan SLC (Shizuoka, Japan) and used in this study after acclimatization for one week. Animals were allocated 5 per polycarbonate cage in a temperature (20-25 o C) and humidity (30-35%) controlled room during acclimatization periods. Light: dark cycle was 12hr: 12hr and feed (Samyang, Korea) and water were supplied ad libitum . After anesthetizing, the GI tract of mouse was divided into 8 portions according to general classification of mammalian GI tract [29]. For inducing gastric and/or intestinal empty, animals were fasted about 24 hours. After phlebotomization, samples from the fundus, pylorus, duodenum, jejunum, ileum, cecum, colon and rectum were fixed in Bouin's solution. After paraffin embedding, 3-4 µ m serial sections were prepared. Representative sections of each tissue were stained with hematoxylin and eosin for light microscopic examination of the normal gastrointestinal architecture. The each representative section was deparaffinized, rehydrated and immunostained with the peroxidase-anti peroxidase (PAP) method [38]. Blocking of nonspecific reaction was performed with normal goat serum prior to incubation with the specific antisera (Table 1). After rinsing in phosphate buffered saline (PBS; 0.01 M, pH 7.4), the sections were incubated in secondary antiserum. They were then washed in PBS buffer and finally the PAP complex was prepared. The peroxidase reaction was carried out in a solution 3,3'-diaminobenzidine tetrahydrochloride containing 0.01% H 2 O 2 in Tris-HCl buffer (0.05 M, pH 7.6). After immunostaining, the sections were lightly counterstained with Mayer's hematoxylin and the immunoreactive (IR) cells were observed under light microscope. The specificity of each immunohistochemical reaction was determined as recommended by Sternberger [38], including the replacement of specific antiserum by the same antiserum, which had been preincubated with its corresponding antigen. The relative frequency of IR cell was placed into one of five categories, not detected ( − ), rare (±; mean values were below 2/one field), a few (+; mean values were below 5/one field); moderate (++; mean values were below 10/one field) and numerous (+++; mean values were up to 20/one field), according to their observed mean numbers as seen under one filed of light microscope (×200) and the observation of each regions of GI tract was conducted as triplet by 3 histologists. Results In this study, all of seven kinds of the IR endocrine cells were detected with the antisera against BCG, serotonin, gastrin, CCK-8, somatostatin, glucagon and HPP in the GI tract of ddY mice (Table 2). According to the location of the GI tract, different regional distributions and relative frequencies of these IR cells were observed and these differences are shown in Table 2. The regional distribution and relative frequency of gastrointestinal endocrine cells were varied with the GI tract, and some peculiar distributional patterns were found in ddY mice. Most of these IR cells in the intestinal portions were generally spherical or spindle in shape (open typed cell), while occasionally round in shape (close typed cell) cells were also found in the intestinal gland and stomach regions. BCG-IR cells BCG-IR cells were observed throughout the whole GI tract except for the cecum and they showed highest Table 1. Antisera used in this study Antisera raised 1 Code Source Diluton BCG 2 805398 Dia Sorin, Stillwater, Minnesota, USA 1 : 1,000 Serotonin BO68082C BioGenex Lab., San Ramon, CA, USA 1 : 20 Gastrin PUO190796 BioGenex Lab., San Ramon, CA, USA 1 : 20 CCK-8 2 750257 Dia Sorin, Stillwater, Minnesota, USA 1 : 500 Somatostatin PUO421295 BioGenex Lab., San Ramon, CA, USA 1 : 20 Glucagon 927604 Dia Sorin, Stillwater, Minnesota, USA 1 : 2,000 HPP 2 A610 DAKO Corp., Carpinteria, CA, USA 1 : 600 1 All antisera were raised in rabbits; 2 BCG: bovine SP-1/chromogranin, CCK-8: cholecystokinin-8, HPP: human pancreatic polypeptide Gastrointestinal endocrine cells of ddY mouse 89 frequencies in the fundus and pylorus (Table 2). Close typed cells with rare frequencied open typed cells were dispersed in the basal portions of the mucosa, between chief and parietal cells, of the fundus with numerous frequency (Fig. 1a, b). In the pylorus, close typed or occasionally open typed BCG-IR cells were situated in the basal portions of the gastric mucosa especially in the pyloric gland regions with numerous frequency but no cells were observed in the upper part of the pyloric mucosa and epithelial lining (Fig. 1c, d). In the duodenum, close typed cells were located in the intestinal glands, which were located in the basal portion of duodenal mucosa and open typed cells were located in the inter-epithelial cell regions with moderate frequency (Fig. 1e). In the jejunum and ileum, open typed BCG-IR cells having long cytoplasmic process which was extended to the lumen, were mainly located in the inter-epithelial cell regions with moderate or a few frequencies, respectively and close typed cells were restricted to the intestinal gland Table 2. Regional distributions and relative frequencies of the gastrointestinal endocrine cells in the gastrointestinal tract of the ddY mice Fundus Pylorus Duo 1 Jej 1 Ileum Cecum Colon Rectum BCG 2 +++ 3 +++ ++ ++ + - + ± Serotonin + +++ +++ +++ ++ ++ +++ +++ Gastrin-+++ CCK-8 2 -++++± Som 2 +++++++-±- Glucagon±-±± HPP 2 ± 1 Duo: duodenum, Jej: jejunum; 2 BCG: bovine SP-1/chromogranin, Som: somatostatin, HPP: human pancreatic polypeptide, CCK-8: cholecystokinin-8; 3 Relative frequencies; +++: numerous, ++: moderate, +: a few, ± : rare, -: not detected. F ig. 1. BCG-IR cells in the in the GI tract of ddY mice. They were demonstrated in the fundus (a, b), pylorus (c, d), duodenum ( e), j ejunum (f), ileum (g), colon (h) and rectum (i) but no cells were detected in the cecum. Scale bars = 40 µ m. PAP method. 90 Sae-kwang Ku et al. regions (Fig. 1f, g). In the colon, open typed BCG-IR cells were demonstrated in the inter-epithelial cell regions with a few frequency (Fig. 1h) and close typed cells were demonstrated in the intestinal gland regions of the rectum with rare frequency (Fig. 1i). In the cecum, no BCG-IR cells were detected in this study. Serotonin-IR cells Serotonin-IR cells were observed throughout the whole GI tract with variable relative frequencies in each portion of the GI tract and they were predominant cell type in this strain of mice (Table 2). In the fundus, open and close typed cells were dispersed in the whole gastric mucosa, between chief and parietal cells, with a few frequency (Fig. 2a). Although some open typed cells were also observed in the relatively upper parts of pyloric gastric mucosa, most of cells were mainly situated in the basal portions with numerous frequency and they were poly-morphic but close typed appearances (Fig 2b~d). In the small intestine, serotonin-IR cells were demonstrated in the inter-epithelial cells or intestinal glands, which were located in the basal portion of mucosal layer with moderate to numerous frequencies, respectively. Open typed cells were restricted to the inter-epithelial cell regions while most of close typed cells were found in the intestinal gland regions (Fig. 2eg). Similar to that of the small intestine, open and close typed serotonin-IR cells were widely dispersed in the mucosa of the large intestine (Fig. 2h~j) with moderate to numerous frequencies, respectively. Gastrin- and CCK-8-IR cells Gastrin-IR cells were restricted to the pylorus with numerous frequency (Table 2). Close typed gastrin-IR cells were exclusively located in the basal portion of pyloric gastric mucosa but occasionally open typed cells were situated in that regions mixed with close type cells (Fig. 3a, b). CCK-8-IR cells were detected in the pylorus, duodenum and jejunum with numerous, a few and rare frequencies, respectively (Table 2). In the pylorus, CCK-8-IR cells were located in the basal portion of pyloric gastric mucosa with numerous frequency and most of these cells were close typed cells but occasionally open typed cells were situated in that regions mixed with close-typed cells similar to that of gastrin-IR cells (Fig. 3c, d). Open and close typed cells were F ig. 2. Serotonin-IR cells in the in the GI tract of ddY mice. These IR cells were located in the epithelium and gastric or intestinal gla nd r egions of the fundus (a), pylorus (b~d), duodenum (e), jejunum (f), ileum (g), cecum (h), colon (i) and rectum (j). Scale bars = 40 µ m . P AP method. Gastrointestinal endocrine cells of ddY mouse 91 demonstrated in the inter-epithelial cell and intestinal gland regions of the duodenum (Fig. 3e) and open typed CCK-8- IR cells were restricted to the inter-epithelial cell regions of the jejunum (Fig. 3f). However, no gastrin- and CCK-8-IR cells were observed in the remaining portions of the GI tract of this strain of mice. Somatostatin-IR cells Somatostatin-IR cells were demonstrated throughout the whole GI tract except for the cecum and rectum, and they showed highest frequencies in the fundus and pylorus (Table 2). They were dispersed in the whole gastric mucosa, between chief and parietal cells (Fig. 4a). However, close typed cells were exclusively located in the basal portion of pyloric gastric mucosa but occasionally open typed cells were situated in that regions mixed with close type cells (Fig. 4b). In the duodenum (Fig. 4c, d), jejunum (Fig. 4e, f) and ileum (Fig. 4g), open typed cells having long cytoplasmic processes were located in the inter-epithelial cell regions with a few frequencies, respectively. However, no close typed cells were demonstrated in that portion of the GI tract. In the colon, open typed somatostatin-IR cells were detected in the inter-epithelial cell regions with rare frequency (Fig. 1h). Glucagon-IR cells Close typed glucagon-IR cells were restricted to the fundus, duodenum and jejunum with rare frequencies (Table 2). In the fundus, they were located in the gastric mucosa between chief and parietal cells (Fig. 5a). In the duodenum (Fig. 5b) and jejunum (Fig. 5c), close typed glucagon-IR cells were demonstrated in the basal regions of the epithelial lining. HPP-IR cells Open typed HPP-IR cells were restricted to the inter- epithelial cell regions of the rectum (Fig. 6a, b) with rare frequency (Table 2). Discussion It is generally accepted that the endocrine cells in the alimentary tracts appeared remarkably different depending on the regional distribution, relative frequency, cell types with animal species and each regional part of the GI tract. In addition, many studies have elucidated the regional distribution and relative frequency of different endocrine cells in the GI tract of the various vertebrates including various species of rodents. Also the researches or data processing about gastrointestinal endocrine cells in the mice strains have been widely executed [20,21,27,37]. The gastrointestinal endocrine cells were generally divided into two types, one was round to spherical shaped close typed cells which were located in the stomach regions, and the other was spherical to spindle shaped open typed cells which were situated in the intestinal regions. In this study, close typed cells were mainly located in the gastric or intestinal gland regions whereas most of open typed cells were found in the epithelial regions of ddY mice. Chromogranin (CG) belongs to a family of large anionic proteins (CG A, B and secretogranin), the members of which are known to be present in the secretory granules of a broad spectrum of amine and peptide-producing cells of adrenal medulla and gastrointestinal endocrine system, as well as in some neurons of the peptidergic and catecholaminergic nervous system of several mammals [12, 28]. CGs have been found to occur in large variety of endocrine organs and cells outside the adrenal medulla, and they have been claimed as common “markers” of all neuroendocrine cells [4,11]. Although, the distributional patterns of these CG-IR cells in the GI tract of Rodentia F ig. 3. Gastrin- and CCK-8-IR cells in the GI tract of ddY mic e. G astrin-IR cells were restricted to the pylorus (a, b), and CCK- 8- I R cells were demonstrated in the pylorus (c, d), duodenum ( e) a nd jejunum (f). Scale bars = 40 µ m. PAP method. 92 Sae-kwang Ku et al. were seldom, Hawkins et al. [14] reported that CGA-IR cells were demonstrated throughout the whole GI tract of 7 species of laboratory animals including mouse. In the present study, BCG-IR cells were detected throughout the whole GI tract of ddY mice except for the cecum. These results were, well corresponded to those of previous studies [12,14,28]. However, it is considered that single use of BCG is not suitable as a marker of endocrine cells because the relative frequencies of BCG-IR cells were not detected or lower than those of serotonin- and other IR cells in case of some regions. If mixed or concomitantly immunostained with other types of CGs, it is considered that CGs are suitable as marker of other endocrine cells in this strain of mouse similar to that of C57BL/6 mouse [20]. Serotonin, consisted of monoamines, was widely distributed in nervous system and in gastric epithelial endocrine cells [9]. The main functions of serotonin were inhibition of gastric acid secretion and contraction of smooth muscle in the GI tract [13]. El-Salhy et al . [9] reported that serotonin-IR cells were detected throughout the GI tract of all species and established in the GI tract at the early stage of vertebrate evolution. In addition, these IR cells were detected in the whole alimentary tract including esophagus of low vertebrates [19]. Serotonin-IR cells were detected in the whole GI tract of the gerbil [26], common tree shrew [41], Philippine carabao [1], Manchurian chipmunk [25], rat [15] and several strains of mice [20,21, 33]. In the present study, serotonin-IR cells were detected throughout the whole GI tract and they were most predominant endocrine cell types in ddY mice. These results were considered as similar to most of other mammals [1,15, 20,21,25,26,31,41]. However, it is considered that serotonin- IR cells in this strain of mouse showed somewhat higher relative frequency than those of other mammals especially other rodents [20,21]. It is generally accepted that gastrin and CCK-8 originated from the same ancestor and in the human duodenum a large fraction of these cells, besides reacting with non-C terminal CCK antibodies and C-terminal gastrin/CCK antibodies, also show immunoreactivity with C-terminal gastrin-34 antibodies, colocalised with CCK in a variable portion of secretory granules [35]. Gastrin secretion by intestinal G cell promotes gastric acid secretion, and CCK secretion by intestinal I cell stimulates the secretion of pancreatic enzyme secretion. In present study, gastrin-IR cells were restricted to the pylorus and CCK-8-IR cells were demonstrated in the pylorus, duodenum and jejunum of ddY mice and these results were well corresponded to those of hairless mice [21] and C57BL/6 mice [20]. Generally, it is well known that gastrin- and CCK-8-IR cells were located in the gastric mucosa and whole small intestinal tract in mammals [18,41]. However, Lee et al ., [22] reported that gastrin/CCK- IR cells were abundant in the pyloric gland region but scarce in the duodenum and no cells were found in the other gastrointestinal regions of the Korean tree squirrel. In the GI tract of gerbil [26], gastrin-IR cells were restricted to the pylorus and CCK-8-IR cells were located in the pylorus and duodenum similar to that of Korean tree squirrel. Although somewhat different aspects were also demonstrated, these results were well corresponded to those of present study. However, in the GI tract of Manchurian chipmunk, gastrin- IR cells were demonstrated from the fundus to ileum and CCK-8-IR cells were detected from the duodenum to ileum [23]. These differences were considered that it might be due F ig. 4. Somatostatin-IR cells in the GI tract of ddY mice. Somatostatin-IR cells were demonstrated in the fundus (a), pylorus (b), duodenu m ( c, d), jejunum (e, f), ileum (g) and colon (h) but no cells were detected in the cecum and rectum. Scale bars = 40 µ m. PAP method. Gastrointestinal endocrine cells of ddY mouse 93 to the differences of the antisera tested or the methods and/or species differences used in the each study [6,7,40]. Somatostatin, consisting of 14 amino acids, was first isolated from hypothalamus of sheep and can be divided into straight form and cyclic form [3]. This substance inhibits the secretion of the other neuroendocrine hormones [17]. It is known that somatostatin-IR cells show the widest distribution in the whole GI tract except for the large intestine of all vertebrate species investigated, including the primitive agnathans with serotonin-IR cells [10]. However, somewhat species-dependent variations on the distributional pattern of these IR cells have been reported. In the GI tract of Manchurian chipmunk, they were detected throughout the whole GI tract and showed the highest frequencies in the pylorus [25] but they were restricted to the pylorus of the gerbil [26]. In mice strains, somatostatin-IR cells decrease in the duodenum of NMRI mice with age [31] and in the antrum of diabetic mouse regardless of their obesity [8,37]. In hairless [21] and C57BL/6 mice [20], somatostatin-IR cells were demonstrated from fundus to ileum and showed highest frequency in the fundus. In the present study, somatostatin-IR cells were detected throughout the whole GI tract except for cecum and rectum. These results were somewhat similar to those of other mouse strains [8,20,21, 37] and the Manchurian chipmunk [25] but quite different from those of the gerbil [26]. Glucagon is synthesized in the A cells of the pancreas and regulates serum glucose levels. These IR cells have been demonstrated in various mammals. They were demonstrated in the whole GI tract of the common tree shrew [41] and musk shrew [17] but Baltazar et al . [1] persisted that these IR cells were only detected in the intestinal tract of the Philippine carabao and Lee et al . [22] reported that they were restricted to the cardia and fundus of the Korean tree squirrel. In addition, glucagon-IR cells were detected in the stomach and small intestine of Manchurian chipmunk [23] and they were restricted to the fundus with relatively low frequencies in hairless mice [21]. In addition, they were restricted to the fundus, ileum and colon [20]. Collectively it is considered that the distributional patterns of glucagon-IR cells in the GI tract of the mammals show species-dependent variation. Especially, appearances of these IR cells in large intestine were also reported in mouse [31,32]. However, no glucagon-IR cells were demonstrated in the GI tract of the gerbil [26]. In the present study, glucagon-IR cells were restricted to the fundus, duodenum and jejunum with rare F ig. 5. Glucagon-IR cells in the GI tract of ddY mice. Close typ ed g lucagon-IR cells were restricted to the fundus (a), duodenum ( b) a nd jejunum (c). Scale bars = 40 µ m. PAP method. F ig. 6. HPP-IR cells in the GI tract of ddY mice. Open typ ed H PP-IR cells were restricted to the rectum (a, b). Scale bars = 4 0 µ m. PAP method. 94 Sae-kwang Ku et al. frequencies, respectively. These findings were somewhat different from those of previous studies [18,20-23,26,31,32, 41] and these differences were considered as species and/or strain-dependent variations. Since PP was isolated from insulin extraction of pancreas at 1961, the regional distribution of PP-IR cells in the mammalian species was relatively well known but species- dependant differences existed among the mammals [1,22, 25,26,41]. These IR cells were demonstrated from the fundus to the jejunum of the Manchurian chipmunk [25] and restricted to the fundus of C57BL/6 mice [20] but no cells were detected in the GI tract of gerbil [26] and hairless mouse [21]. In the present study, somewhat differed from that of other rodents, HPP-IR cells were restricted to the rectum with rare frequency. In conclusion, the regional distribution and relative frequency of the GI tract endocrine cells in the ddY mice is similar to that of other rodents. However, strain-dependent unique distributional patterns of gastrointestinal endocrine cells were also found in ddY mice especially to the CCK-8-, glucagons- and PP-IR endocrine cells. References 1. 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In the Rodentia, the localization of endocrine cells in the GI tract of the Manchurian. typed cells were restricted to the intestinal gland Table 2. Regional distributions and relative frequencies of the gastrointestinal endocrine cells in the gastrointestinal tract of the ddY mice Fundus. species of rodents. Also the researches or data processing about gastrointestinal endocrine cells in the mice strains have been widely executed [20,21,27,37]. The gastrointestinal endocrine cells