Immunohistochemical Study of The
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J. Vet. Sci. (2002), 3(4), 303-314 J O U R N A L O F Veterinary Science Immunohistochemical Study of the Endocrine Cells in the Pancreas of the Carp, Cyprinus carpio (Cyprinidae) Hee-Sun Kong, Jae-Hyun Lee*, Ki-Dae Park, Sae-Kwang Ku1 and Hyeung-Sik Lee2 Laboratory of Histology, College of Veterinary Medicine, Kyungpook National University, Taegu 702-701, Korea 1Pharmacology & Toxicology Lab., Central Research Laboratories, Dong-Wha Pharm. Ind. Co., Anyang 430-017, Korea 2Department of Biology, Faculty of Natural Science Kyungsan University, Kyungsan 712-240, Korea Received J uly 4, 2002 / Accepted November 21, 2002 Abstract8) of n u m e rou s in su lin -, m od e rate glu c ago n - a n d somatostatin-IR cells of low frequency w ere also The re gional distribution and relative frequency of observed in the subepithelial regions of the pancreatic som e e ndocrine ce lls in the pancreas of the carp, duct. In the exocrine regions, insulin-, glucagon-, Cyprinus ca rpio Linnae us, belonging to the fam ily somatostatin- and hPP-IR cells w ere located in the Cyprinidae in the order Cypriniformes, w ere observed inter-acinus regions w ith rare, a few , m oderate and using specific m am m alian antisera against insulin, moderate frequencies, respectively. In conclusion, the glucagon, som atostatin and hum an pancreatic poly- regional distribution and re lative frequency of four pe ptide (hP P ) by peroxidase antiperoxidase (P AP ) major pancreatic endocrine cells, insulin-, glucagon-, m ethod. The pancreas w as divided into four re gions somatostatin- and hP P -IR ce lls, in the pancreas of the (principal and secondary islets, exocrine and pancreatic carp show ed general patterns w hich w ere obse rved in duct regions). In addition, the pancre atic islet re gions other stom achle ss te leost. How ever, some spe cies- w ere further subdivided into three regions (ce ntral, depende nt different distributional patterns and/or m antle and peripheral re gions) and the pancreatic relative frequencies w ere also de monstrated. duct regions w ere subdivide d into tw o regions (epithelial and subepithelial regions). Spherical to Key w ords : Carps, pancreas, hepatopancreas, immuno- spindle or occasionally round to oval shaped im m uno- histochemistry, immunoreactive cells, endocrine cells reactive (IR) cells w ere demonstrated in the pancreatic islets, exocrine and pancreatic duct. In the principal Introduction islet re gions, som e cells w ere also de tected in the other regions, most of insulin- and som atostatin-IR cells w ere The carp, Cyprinus carpio Linnaeus, belonging to the located in the central regions, and glucagon- and family Cyprinidae in the order Cypriniformes is a fresh- hP P-IR cells w ere situate d in the peripheral regions. water stomachless teleost. Although this species was originated In this regions, insulin-IR cells w ere m ost predominant from Asia, now their habitation is distributed throughout cell types and then, glucagon, som atostatin and hPP in the world except for some regions of South America and that order. In the secondary isle t regions, the regional Australia. Among teleost, the pancreas of this stomachless distribution and relative frequency of these four teleost has been widely studied because their unique anatomical types of endocrine cells w ere quite sim ilar to those of and histological profiles, so called hepatopancreas in there, the principal islets except for cell clusters consisted pancreas was dispersed throughout the whole liver parenchyma, of hPP -IR cells that w ere situated in the peripheral to of course some pancreatic parenchyma having large islets m antle regions. In the pancreatic duct regions, all was also demonstrated between liver and mesenteric membrane four major pancreatic endocrine cells were de monstrated [21]. in the inte r-e pithelial cells and/or basal regions of the It is generally known that pancreas of vertebrates is epithelial linning. In addition, cell clusters com posed subdivided into two regions. One is an exocrine region where digestive enzymes are released and the other is an endocrine portion where regulatory hormones such as insulin, *Corresponding author: Dr. Jae-Hyun Lee glucagon, somatostatin and pancreatic polypeptide (PP) are Laboratory of Histology, College of Veterinary Medicine, Kyungpook released into blood vessels. The appearance, regional distribution National University, Taegu 702-701, Korea Tel : +82-53-950-5970, Fax : +82-53-950-5955 and relative frequency of these regulatory hormones secreted E-mail : [email protected] by endocrine cells in the pancreas are well recognized by 304 Hee-Sun Kong, Jae-Hyun Lee, Ki-Dae Park, Sae-Kwang Ku and Hyeung-Sik Lee histochemistry [28], immunofluorescence method [39] and distributional patterns of pancreatic endocrine cells were immunohistochemistry [52]. In addition to four regulatory found in two species of stomach fresh water teleost having hormones mentioned above, peptide YY-, neuropeptide Y- similar feeding habits [34]. Although many studies have [2], and chromogranin family- [22, 43] immunoreactive (IR) elucidated regional distribution and relative frequency of cells were also demonstrated in the vertebrate pancreas. endocrine cells, IR to the antisera against mammalian insulin, The pancreas has been treated as a valuable organ for glucagon, somatostatin and PP, in the pancreas of teleost, endocrine studies and endocrine pancreas was extensively localization of endocrine cells on the pancreas of the carp studied associated with diabetes [23]. Until now, investigations has not yet been reported except for insulin. Regulation of of gastroenteropancraetic (GEP) endocrine cells have been insulin biosynthesis was reported in the carp [20] and the considered to be an important part of a phylogenetic study carp insulin was isolated and crystallized [42]. [11] and the distribution and relative frequency of these In the present study, the regional distribution and endocrine cells in the pancreas were varied with animal relative frequency of some endocrine cells in the pancreas of species and feeding habits. Recently most intensive studies stomachless fresh-water teleost, the carp, Cyprinus carpio have been done on the Pisces because some endocrine cells Linnaeus (Cyprinidae) having unique hepatopancreas, were were demonstrated in the skin, gills and airways [58], and observed using specific antisera against mammalian insulin, the alteration of regional distribution and relative frequency glucagon, somatostatin and PP by peroxidase antiperoxidase of these cells by heavy metal intoxication such as lead was (PAP) method. also demonstrated [41]. In addition, the possibility of using the teleost fish endocrine tissues for treatment hormonal Materials and Methods disorder such as diabetes was suggested [37]. The endocrine pancreas of teleost fish is mainly composed of two types of Experim ental anim als pancreatic islets: 1) one, two or even more multiple large Five adult carp, Cyprinus carpio Linnaeus (Cyprinidae), islets, called principal pancreatic islets and 2) numerous, about 40cm in length, were purchased from a merchant in widely scattered small islets, called secondary pancreatic Taegu, Korea and used in this study without sexual islets [14]. Until now, the appearance, regional distribution distinction. and relative frequency of numerous types of regulatory peptides have been demonstrated in the pancreas of the Histological procedures Pisces. Insulin-, glucagon-, somatostatin- and pancreatic After decapitation, samples from the pancreas were fixed polypeptide (PP)-IR cells, which were major four endocrine in Bouin's solution. After paraffin embedding, 3∼ 4㎛ sections cell types detected in mammalian pancreas, were also were prepared. Representative sections of each tissue were observed in the pancreas of the five species of Osteo- stained with hematoxylin and eosin for light microscopic glossomorpha [3, 4], the gar [16], the southern-hemisphere examination of the normal pancreatic architecture. lampreys [56], the sea bream [1], the lamprey [7, 8], the sea bass [35], the dipnoan fish [47], the cartilaginous fish [15], Im m unohistochem ical procedures the rainbow trout [38], the coho salmon [38], the arctic The each representative sect ion was deparaffinized, lamprey [57], the anglerfish [24], the channel catfish [24], rehydrated and immunostained with the peroxidase anti- the ray [48] and the telecostei [27] by immunohistochemical peroxidase (PAP) method [51]. Blocking of nonspecific and/or electron microscopical methods. In addition, similar reaction was performed with normal goat serum prior to to that of mammals, the appearance of neuropeptide Y and incubation with the specific antisera (Table 1). After rinsed peptide YY-IR cells and/or nerve fibers was also demon- in phosphate buffered saline (PBS; 0.01M, pH 7.4), the strated in the pancreas of the eel [12], the dogfish [9, 10] sections were incubated in secondary antiserum. They were and the spiny dogfish [40]. The ontogenic changes and then washed in PBS buffer and finally the PAP complex changes of distribution and relative frequency of some was prepared. The peroxidase reaction was carried out in a endocrine cells with developmental stages were also well solution 3,3′-diaminobenzidine tetrahydrochloride containing documented in the Japanese flounder [29], the dogfish [9], 0.01% H2O2 in Tris-HCl buffer (0.05M, pH 7.6). After the lamprey [13], the sea bream [46] and the sea bass [5]. immunostained, the sections were lightly counterstained Well corresponding to those of mammals, the regional with Mayer's hematoxylin and the IR cells were observed distribution and relative frequency of endocrine cells within under light microscope the pancreas, and the cell population seemed to be con- siderably variable among species and feeding habits, especially Specificity of antiserum reaction in the case of occurrence in PP cells [55]. Namely, these IR The specificity of each immunohistochemical reaction was cells that were generally demonstrated in teleost, were not determined as recommended by Sternberger [51], including detected in the pancreas of channel catfish and lungfish [18, the replacement of specific antiserum by the same antiserum, 36].