Medycyna Wet. 2008, 64 (1) 64
Praca oryginalna Original paper
It is now well known that various kind of endocrine cells are present in the gastrointestinal tract of birds and mammals. Studies using immunocytochemical methods have been carried out on the gastrointestinal tract in chicks (1, 3, 4, 6, 8, 12) more than other avian species. In contrast to the relatively huge body of know-ledge of the immunohistochemical features of the ga-strointestinal tract of the fowl, very little is known about the ostrich gastrointestinal tract (2). Bezuidenhout et al. (2) examined peptide-storing endocrine cells of the gastrointestinal tract of the ostrich (Struthio camelus). However, no information concerning the occurrence of gastrointestinal tract endocrine cells in subspecies of ostrich (Struthio camelus camelus) is available. Thus, the present study investigated the distribution and relative frequency of some mediators commonly produced by the gastrointestinal tract of various avian species (3-6, 16) in the gastrointestinal tract endocrine cells of the Struthio camelus camelus, by using immu-nohistochemistry. The data obtained will be useful for a better understanding of the digestive function of the gastrointestinal tract of this animal.
Material and methods
Animal and tissue samples. Six adult ostrich of male were used. Birds with a body mass of 45-60 kg were anaesthetized by injecting pentobarbitone sodium. The left carotid artery was cannulated at the base of the neck and allowed to exsanguinate. Tissue samples were taken from
the proventriculus, gizzard, duodenum, ileum and colon and fixed in 4% neutral-buffered formalin for 24 hr. They were then dehydrated through graded ethanol and embedded in paraffin. Seven µm-thick sections were obtained and pro-cessed for immunohistochemical staining.
Immunohistochemistry. PAP (Peroxidase-Anti-Peroxi-dase) method. Immunohistochemical staining was carried out by using the peroxidase-antiperoxidase (PAP) method. Blocking of endogenous peroxidase was carried out with 0.008% hydrogen peroxidase (H2O2) in methanol for 5 mi-nutes (11). In order to block unspecific binding, incubation with normal goat serum in 0.1 M phosphate buffered saline (PBS), pH 7.2 (Dilution 1 : 10) was performed. Sections were incubated for 16-20 hours at 4°C with rabbit IgG antibodies against serotonin (Zymed Lab., 18.0077), calci-tonin gene-related peptide (Chemicon, AB5920), somato-statin-14 (Chemicon, AB1976), vasoactive intestinal poly-peptide (Chemicon, AB982), neurotensin (Chemicon, AB5496) and cholecystokinin (Chemicon, AB1973). Anti-bodies were diluted to 1 : 200, 1 : 1000, 1 : 500, 1 : 1000, 1 : 500 and 1 : 200 in PBS containing 0.25% sodium azide and 2.5% bovine serum albumin. Sections were then incu-bated in goat anti-rabbit IgG (Dako, Z0421, Denmark) followed by rabbit peroxidase anti-peroxidase complex (Zymed Lab., 61.2003, San Francisco), both at a dilution of 1 : 50 in PBS, for 1 hour at room temperature. Sections were washed in PBS for 30 minutes following incubation and finally immersed in glucose oxidise-DAB-nickel ammonium sulphate substrate (9) for 10 minutes. After washing in distilled water and counterstaining with eosin,
Immunohistochemical study on the endocrine cells
in the gastrointestinal tract of the ostrich
BERRIN GENÇER TARAKÇI, MINE YAMAN, ALI BAYRAKDAR, IHSAN YAMAN*
Department of Histology, *Department of Pathology, Faculty of Veterinary Science, University of Firat, 23119 Elazig/Turkey
Gençer Tarakçi B., Yaman M., Bayrakdar A., Yaman I.
Immunohistochemical study on the endocrine cells in the gastrointestinal tract of the ostrich Summary
The study carried out an immunohistochemical investigation on the regional distribution and relative frequency of endocrine cells in the gastrointestinal tract of the ostrich, (Struthio camelus camelus) using antibodies against serotonin, somatostatin-14, and vasoactive intestinal polypeptide (VIP), calcitonin gene related peptide (CGRP), neurotensin, cholecystokinin (CCK). Serotonin immunoreactive cells were observed in all regions of the gastrointestinal tract except the gizzard. They were particularly abundant in the duode-num and ileum, whereas the presence of somatostatin 14 positive cells was restricted to the proventricularis and gizzard. Substance P immunoreactive cells were predominant in the stomach. By contrast, VIP, CGRP, neurotensin and CCK immunoreactive cells were not detected in the ostrich.
Keywords: immunohistochemistry, ostrich, serotonin, somatostatin-14, substance P, calcitonin gene related peptide, vasoactive intestinal polypeptide, neurotensin, cholecystokinin
Medycyna Wet. 2008, 64 (1) 65 the sections were dehydrated and coverslips mounted with
an aqueous permanent mounting medium.
The specificity of each immunohistochemical reaction was determined as recommended by Sternberger (10) by using (including the replacement of) specific antiserum pre-incubated with its corresponding antigen. Sections were examined with a light microscope and photographs were taken. For semi-quantitative analysis, the average number of positive cells in the microscopic field was determined through identifying and counting these cells on 5 micro-scopic fields (magnification × 40).
Results and discussion
Serotonin, somatostatin-14 and substance P immu-noreactive cells were found in the gastrointestinal tract of the ostrich and CGRP, VIP, neurotensin and CCK immunoreactive cells were not detected in any region of the ostrich gastrointestinal tract. Nevertheless, CGRP and VIP immunoreactivty were observed in the nerve fibers of gastrointestinal tract. Most of these immunoreactive cells were generally round to spheri-cal-shaped close type cells and spherical to spindle-shaped open type cells situated in the stomach regions and intestinal gland regions.
Serotonin immunoreactive cells were observed along the whole length of the gastrointestinal tract except for the gizzard region. The cells were numerous in the duodenum and ileum. They were mostly close type cells and some of the cells had cytoplasm projections in the glands (fig. 1). There were only a few in the proventriculus and colon.
Somatostatin immunoreactive cells were detected only in the stomach. They were a few in the gizzard, but the cells were numerous in the proventricularis. Immunoreactive cells with sitoplasmic projections were embedded in deep glands (fig. 2) in the gizzard. Substance P immunoreactive cells were numerous in the gizzard and in the proventricularis. Moreover, some nerve fibers immunoreactive to substance P were also found in the gizzard and in the proventricularis. In the intestine, immunoreactive endocrine cells were only noted in the duodenum and occurred in the epi-thelium of the villi and crypts (fig. 3).
CGRP and VIP, neurotensin and CCK immuno-reactive endocrine cells were not detected for the whole length of the gastrointestinal tract. On the other hand, CGRP and VIP immunoreactivity were observed in the nerve fibers in every section (fig. 4).
Fig. 3. Substance P immunoreactive cell in the duodenum of ostrich (Struthio camelus camelus) (arrow). × 200
Fig. 1. Serotonin immunoreactive cells in the ileum of ostrich
(Struthio camelus camelus (arrows). × 200 Fig. 2. Somatostatin containing cells in the gizzard of ostrich(Struthio camelus camelus) (arrows). × 200
Fig. 4. VIP immunoreactive nerve fibers in the ileum of ostrich (Struthio camelus camelus) (arrows). × 200
Medycyna Wet. 2008, 64 (1) 66
The distribution pattern of endocrine cells in the gastrointestinal tract was identical in the 6 tested animals (tab. 1). Although the basic pattern of distri-bution corresponded closely with the re-sults of previous studies on avian (5, 8, 15, 16), some characteristic differences were noted in the present study.
Serotonin immunoreactive cells appe-ared numerous in the upper small inte-stine, but very few in the proventriculus. They were missing in the gizzard. This data is substantially in agreement with that observed in the gastrointestinal tract of the chick (8, 16) and duck (5, 15).
The presence of somatostatin con-taining cells in the proventriculus and gizzard is in accordance with the findings of other studies on the avian gastrointes-tinal tract (13-16). Most somatostatin immunoreactive cells were detected in the proventriculus and only a few immu-noreactive cells were found in the giz-zard. Somatostatin was not detected in the intestine. This is in agreement with previous reports for other avian species (5, 7, 16), but differs to that found by Bezuidenhout (2) who reported that these cells were numerous in the small intestine of the ostrich (Struthio came-lus). The absence of somatostatin immu-noreactive cells from the intestine in the present study might be due to differen-ces in the antisera tested and/or the spe-cies investigated.
Substance P immunoreactive cells were numerous in the proventriculus and gizzard. Similar results have been repor-ted in quail proventriculus (6). Conver-sely, substance P was not detected in the proventriculus of the chicken (7, 16), duck (5) and ostrich (2). In the present study, substance P immunoreactive cells were also observed in the duodenum. This data corresponds to the findings re-ported for ducks and ostriches. Contrary to our results, immunoreactive cells were detected in the intestine of chicks (7, 16). Moreover, the study also found substan-ce P immunoreactive nerve fibers in the proventriculus and gizzard of the ostrich. These results are in agreement with others obtained for the chicken gut (3). These data confirm the double distribu-tion of this peptide in endocrine cells as well as in the nervous system.
No endocrine cells showing CGRP and VIP immunoreactivity were
detec-Tab. 1. Distribution and relative frequency of serotonin, neurotensin, soma-tostatin-14, calcitonin gene-related peptide (CGRP), cholecystokinin (CCK), vasoactive intestinal polypeptide (VIP) and substance P (SP) immuno-reactive endocrine cells in the gastrointestinal tract ostrich (Struthio camelus camelus), (n = 5). Results are expressed as mean ± standard deviation
s n a g r O Animals 1 h c ir t s O Ostirch2 Ostirch3 Ostirch4 Ostirch5 Mean t c a rt l a n it s e t n i o rt s a G s u l u c ir t n e v o r P n i n o t o r e S 30 35 32 35 32 32.2±3.0 n i s n e t o r u e N 0 0 0 0 0 0 4 1 n it a t s o t a m o S 32 30 35 28 35 32.0±3.0 P R G C 0 0 0 0 0 0 K C C 0 0 0 0 0 0 P I V 0 0 0 0 0 0 P S 29 27 28 30 25 27.8±1.9 d r a z zi G n i n o t o r e S 0 0 0 0 0 0 n i s n e t o r u e N 0 0 0 0 0 0 4 1 n it a t s o t a m o S 6 5 7 6 9 6.6±1.5 P R G C 0 0 0 0 0 0 K C C 0 0 0 0 0 0 P I V 0 0 0 0 0 0 P S 29 25 28 22 25 25.8±2.7 e n it s e t n i ll a m S m u n e d o u D n i n o t o r e S 25 20 23 27 22 23.4±2.7 n i s n e t o r u e N 0 0 0 0 0 0 4 1 n it a t s o t a m o S 0 0 0 0 0 0 P R G C 0 0 0 0 0 0 K C C 0 0 0 0 0 0 P I V 0 0 0 0 0 0 P S 20 23 18 22 25 21.6±2.7 m u e lI n i n o t o r e S 27 24 26 27 25 25.8±1.3 n i s n e t o r u e N 0 0 0 0 0 0 4 1 n it a t s o t a m o S 0 0 0 0 0 0 P R G C 0 0 0 0 0 0 K C C 0 0 0 0 0 0 P I V 0 0 0 0 0 0 P S 0 0 0 0 0 0 e n it s e t n i e g r a L n i n o t o r e S 2 3 4 2 1 2.4±1.1 n i s n e t o r u e N 0 0 0 0 0 0 4 1 n it a t s o t a m o S 0 0 0 0 0 0 P R G C 0 0 0 0 0 0 K C C 0 0 0 0 0 0 P I V 0 0 0 0 0 0 P S 0 0 0 0 0 0
Medycyna Wet. 2008, 64 (1) 67 ted in the gastrointestinal tract of the ostrich. On the
other hand, CGRP and VIP immunoreactivity were observed in nerve fibers throughout the gastrointesti-nal tract. The absences of these immunoreactive cells suggest that there are no endocrine cells containing these peptides. These results are in agreement with previous reports (5, 13).
Neurotensin immunoreactivities were recorded in the proventriculus, pylorus, rectum and seca of the chicken (7), proventriculus of the ostrich (2), pigeon, quail and duck (14). The present study failed to detect neurotensin immunoreactivty or CCK in the gastro-intestinal tract of the ostrich (Struthio camelus came-lus). Other studies have also reported similar data in the gastrointestinal tract of ostriches (Struthio came-lus) (2).
In conclusion, the results obtained indicated diffe-rences between the ostrich and other avian species concerning the relative frequency and distribution of some endocrine cells in the gastrointestinal tract. Whether or not this may be attributed to phylogenetic differences is uncertain, because all the observations reported have been carried out on species phylogene-tically equally distant from ostrich. These variations could also be due to antisera employed, feeding habits and probably to unknown functions of the cells.
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Authors address: Berrin Gençer Tarakçi, Department of Histology, Faculty of Veterinary Science, University of Firat, 23119 Elazig/Turkey; e-mail: btarakci@firat.edu.tr
