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Distribution and developmental changes in ghrelin-immunopositive cells in the gastrointestinal tract of African ostrich chicks
Regulatory Peptides 154 (2009) 97–101
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Regulatory Peptides j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / r e g p e p
Distribution and developmental changes in ghrelin-immunopositive cells in the gastrointestinal tract of African ostrich chicks J.X. Wang, K.M. Peng ⁎, H.ZH. Liu, H. Song, X. Chen, M. Liu College of Animal Science and Veterinary Medicine, Huazhong Agricultural University, South Lake, Wuhan 430070, PR China
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Article history: Received 15 September 2008 Received in revised form 6 January 2009 Accepted 3 February 2009 Available online 14 February 2009 Keywords: African ostrich Ghrelin Gastrointestinal tract Immunohistochemistry
a b s t r a c t Ghrelin, the endogenous ligand for the growth hormone secretagogue receptor (GHS-R), has been found in the gastrointestinal tract of many vertebrates, but little is known about its distribution in the gastrointestinal tract of African ostrich chicks. In the present study, the distribution, morphological characteristics, and developmental changes of ghrelin-producing cells in the gastrointestinal tract of African ostrich chicks were investigated using immunohistochemistry. Ghrelin-immunopositive (ghrelin-ip) cells were found to be localized in the mucous membrane of the entire gastrointestinal tract, but not in the myenteric plexus. The greatest number of ghrelin-ip cells was found in the proventriculus, and the ghrelin-ip cell density gradually decreased from the proventriculus to the rectum. Interestingly, from postnatal day 1 to day 45 in the proventriculus, and from postnatal day 1 to day 90 in the gizzard and small intestine, there was a steady increase in the number of ghrelin-ip cells. By day 45 in the proventriculus and day 90 in the gizzard and small intestine, the number of cells reached a plateau and remained steady. These results clearly demonstrate that ghrelin-ip cells exist and the number of ghrelin-ip cells increases with age in the African ostrich gastrointestinal tract from postnatal day 1 to day 90; ghrelin may be involved in gastrointestinal tract development. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Ghrelin is a brain-gut peptide that has been isolated as an endogenous ligand for the growth hormone secretagogue receptor (GHS-R) from the rat stomach [1]. This peptide consists of 28 amino acids, in which the third serine residue is n-octanoylated; this side chain is essential to its biological activity [1]. Ghrelin is predominantly produced by the X/A-like endocrine cells in the oxyntic mucosa of the stomach, which is the major source of circulating ghrelin [2–4]. Ghrelinimmunopositive (ghrelin-ip) cells have been found to be localized in the mucous membranes of the stomach, duodenum, ileum, cecum, and colon, but not in the myenteric plexus, and they can be classified into open- and closed-type cells [5]. The number of ghrelin-ip cells in the fetal stomach increases as the stomach grows [6]. In mammals, ghrelin stimulates growth hormone (GH) release, regulates food intake, energy balance, body weight, gastric motility and acid secretion, endocrine pancreas functions, and glucose metabolism [1,7]. In addition to mammals, ghrelin has also been identified in non-mammalian species. In the chicken, ghrelin consists of 26 amino acids. The serine residue at position 3 (Ser3) is conserved between the chicken and mammalian species, as is its acylation by either n-octanoic or n-decanoic acid [8]. Ghrelin-ip cells have been found to be localized in the mucous ⁎ Corresponding author. Tel.: +86 27 8728 6970; fax: +86 27 87280408. E-mail address: [email protected] (K.M. Peng). 0167-0115/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2009.02.005
membranes of the proventriculus, duodenum, ileum, cecum, and colon, but not in the myenteric plexus, and they can be classified into open- and closed-type cells [9]. The number of ghrelin-ip cells in the adult chicken is also greater than that in the hatching chicken [10]. Ghrelin also stimulates GH release in chickens [8,11]; however, in contrast to its action in mammals, ghrelin inhibits feeding, particularly in neonatal chicks when injected intracerebroventricularly [12–14]. Ghrelins have been identified in other avian species, such as the duck, goose, emu, and turkey [15]. However, there have been no studies on the distribution and function of ghrelin in African ostrich chicks. In general, to understand or hypothesize regarding the physiological role of newly identified peptides, it is important to know the morphological characteristics of the producing cell and its distribution. Therefore, in this study, the distribution and morphological characteristics and developmental changes of ghrelin cells in the gastrointestinal tract were studied in detail using immunohistochemistry (IHC). 2. Materials and methods 2.1. Animals Neonatal day 1, 45, 90, and 334 female African ostriches were used in the present study. African ostrich chicks (24 females) were obtained from a standard ostrich farm in Guangdong Province, China, on postnatal day 1 (newly hatched chicks) and were transported within
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10 h to a battery house, where feed and water were made available ad libitum. The 24 birds were divided into 4 groups (6 ostriches per group) on the basis of their body weight (BW), to equalize BW and the variance among groups. All of the birds were maintained in a heated room with slatted plastic flooring and were fed a starter diet for postnatal days 1–334, which was formulated according to the specifications of the Elsenburg Ostrich Feed Database [16]. All procedures were approved by the Animal Care and Welfare Committee of our Institute. 2.2. Tissue preparation On postnatal days 1, 45, 90, and 334 (control group), the birds were weighed, deeply anesthetized with 10% urethane (Caoyang Secondary
Chemical Plant, Shanghai, China) at a dose of 1 g/kg BW, and perfused, initially with 1000 mL of 0.85% normal saline (containing 0.075% sodium citrate) and thereafter with 1500 mL of 4% paraformaldehyde phosphate-buffered solution (0.1 mol/L, pH 7.4) at 4 °C. The abdomen was cut open. Segments of their gastrointestinal tracts, approximately 1 cm in length, were quickly removed and opened along their longitudinal axes. The segments were postfixed for more than 24 h with 4% paraformaldehyde. The specimens were obtained from the following portions of the African ostrich gastrointestinal tracts: the proventriculus (3 cm proximal to the pylorus), the gizzard, the duodenum (5 cm distal to the pylorus), the jejunum (3 cm proximal to Meckel's diverticulum), the ileum (10 cm proximal to the ileocecal junction), the cecum (5 cm from the ileocecal junction), the colon (5 cm distal to the cecocolonic junction), and the rectum (1 cm from
Fig. 1. Microphotographs of ghrelin-immunopositive cells in the gastrointestinal tract. (A) A large number of ghrelin cells (arrows) were found in the proventriculus. (B) Almost all of the Ghrelin-ip cells in the proventriculus were round-shaped cells, so called closed-type cells(arrow). (C) Few Ghrelin-ip cells in the proventriculus were Opened-type (arrow) in contact with the lumen.(D) Closed-type ghrelin cells (arrow) were found in the gizzard. (E) Closed-type ghrelin cells (arrow) were found in the crypt of the duodenum.(F) Openedtype ghrelin-ip cells (arrows) in the villi of the duodenum. (G) Closed-type ghrelin cells (arrows) were found in the crypt of the jejunum (H) Ghrelin-ip cells with long processes (arrow) were found in villi of the jejunum. (I) Opened-type ghrelin-ip cells (arrow) in villi of the ileum. (J) Opened-type ghrelin-ip cells (arrows) in contact with the lumen were found in the villi of the cecum. (K) Microphotograph of absorption test in the proventriculus. (L) Microphotograph of absorption testing in the duodenum. MU, mucosa; SL, smooth muscle layer; VI, villi; CY, crypt ;LU, lumen. (A, K,L) bar; 100 μm. (B,C,D, E,F,G,H, I, J) bar; 10 μm.
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the anus). After immersion, the tissues were embedded in paraffin. Serial sections (5 μm) were cut on a Leica microtome (Nussloch Gmbh, Germany), mounted on polylysine-coated slides (Boster Corporation, China).
range test where appropriate. Differences of p b 0.05 were considered significant.
2.3. Immunohistochemistry
3.1. Distribution of ghrelin-immunopositive cells
Immunohistochemical detection of ghrelin cells using rabbit antighrelin was carried out by the streptavidin-biotin-peroxidase complex (SABC) method. The anti-ghrelin serum used in this study was a synthetic human ghrelin (gssflspehqrvqqrkeskkppaklqpr), which is different from the related rat and mouse sequence by two amino acids. The anti-ghrelin antiserum recognizes the n-octanoylated Ser3 epitope and the anti-ghrelin antiserum recognizes the C-terminal region of rat ghrelin. Immunohistochemical staining was performed according to the following procedure. The sections were deparaffinized with xylene and rehydrated with decreasing concentrations of ethanol, then treated with 3% hydrogen peroxide (H2O2) to block endogenous peroxidase for 10 min at room temperature. After rinsing with distilled water, the sections were incubated with a pH 6.0 citrate buffer and placed in a microwave oven until the water boiled to fully expose the antigen. After rinsing with phosphate-buffered saline (PBS), the sections were incubated with 5% normal goat serum for 20 min. The sections were then diluted 1:100 with PBS and incubated with rabbit anti-ghrelin primary antibody (BA1619; Boster Corporation) for 12 h in a humid chamber at 4 °C. After washing with PBS for 6 min, a second incubation with biotin-conjugated anti-rabbit IgG serum (SA1022; Boster) was carried out for 20 min, and this was followed by further washing with PBS. Finally, the sections were incubated for 20 min with an SABC solution prepared according to the manufacturer's instructions. After washing with PBS for 20 min, the sections were reacted in a diaminobenzidine-tetrachloride kit (DAB kit, AR1022) for 30 min to detect immunostaining. After washing with distilled water, the sections were dehydrated with a graded ethanol series, cleared in xylene, mounted with a coverslip, and viewed under a light microscope (BH-2; Olympus, Japan). All of the incubations were carried out in a humid chamber at room temperature. Control sections were prepared using the same method, omitting the primary antibody. To examine the specificity of rabbit anti-human ghrelin antiserum, the diluted antiserum (1:100) was incubated with human ghrelin (5 μg/ml) at room temperature for 10 h, and mixtures were centrifuged at 12,000 rpm for 25 min at 4 °C. The supernatant was used as the primary antiserum for absorption tests.
On postnatal days 1, 45, 90, and 334, female African ostrich ghrelin-ip cells were found in the mucosal layer, but not in the myenteric plexus, in all of the examined regions (Figs. 3A–D, 4A–D). Ghrelin-ip cells were most dense in the proventriculus, and low densities of ghrelin cells were found in the other examined regions (the duodenum, jejunum, ileum, and gizzard). Very few ghrelin-ip cells were found in the cecum, colon, and rectum on postnatal days 1, 45, 90, and 334. Ninety-day-old female African ostriches were used to investigate the distribution of ghrelin-ip cells in various tissues. It was found that ghrelinip cells existed in all regions of the gastrointestinal tract: the proventriculus (Fig. 1A–C), the gizzard (Fig. 1D), the duodenum (Fig. 1E,F), the jejunum (Fig. 1G,H), the ileum (Fig. 1I), the cecum (Fig. 1J), the colon, and the rectum (data not shown). In the proventriculus, most of the ghrelin-ip cells were observed at the base of glandular lobuli, and a few ghrelin-ip cells were observed in the basal zone of proventriculus plicae (Fig. 1A). In the gizzard, ghrelin-ip cells were scattered in the glandular epithelia of crypts (Fig. 1D). In the duodenum, jejunum, ileum, cecum, colon, and rectum, ghrelin-ip cells were scattered in the epithelia of crypts (Fig.1E, G) and villi (Fig. 1F, H, I, J). There were virtually no differences in the immunoreactivities of ghrelin cells in the various regions of the gastrointestinal tract in the African ostrich. Two types of ghrelin cells were found; i.e., closed-type cells and cells with triangular or elongated
3. Results
2.4. Morphometric analysis The densities of ghrelin cells in various regions of the African ostrich gastrointestinal tract were estimated. After taking digital photographs under a light microscope (BH-2; Olympus) with a digital camera (COOLPIX4500; Nikon, Japan), the number of ghrelin cells in each section was counted and the area of the mucosal layer in each section was measured using a computerized image analysis program, HMIAS-2000 High-definition Chromatic Color Medical Science Figure Analysis Program (Qianping, Wuhan, China). The ghrelin cell density was calculated as the number of ghrelin-ip cells per unit area. The percentage of open-type ghrelin cells in the gastrointestinal segment was also calculated. First, the total number of all ghrelin cells (closedtype and open-type cells) in each section was counted using digital photography as mentioned above, and then the percentage of opentype ghrelin cells to total ghrelin cells was calculated. 2.5. Statistical analyses Results are expressed as means ± standard errors on the mean (means± S.E.). Statistical analysis was done using analysis of variance statistics software (SAS Institute, 2000) with Duncan's multiple
Fig. 2. (A). Histogram showing the densities of ghrelin cells (cells/mm2) in the 90 dayold African ostrich gastrointestinal tract. Ghrelin-immunopositive cells were present throughout the gastrointestinal mucosa, from the proventriculus to rectum, (B) Histogram showing the percentages of opened-type cells to all immunopositive cells in the 90 day-old African ostrich gastrointestinal tract. The percentage of openedtype ghrelin cells gradually increased in the direction from the gizzard to the large intestine. a–hDifferent letters within the same column indicate significant differences among segments according to Duncan's multiple range (P ≤ 0.05).PR, proventriculus; GI, gizzard; DU, duodenum; JE, jejunum ; IL, ileum; CE, cecum; CO, colon; RE, rectum.
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J.X. Wang et al. / Regulatory Peptides 154 (2009) 97–101 Table 1 Ghrelin-ip cell densities (cells/mm2) in the stomach and small intestine of African ostrich chickens on postnatal days 1, 45, 90, and 334. Day (d) Cell density (cells/mm2) Proventriculus Gizzard 0 45 90 334
9.00 ± 2.83bA 29.60 ± 9.13aA 24.40 ± 3.71aA 26.60 ± 4.39aA
Duodenum
Jejunum
Ileum
0.80 ± 0.45cD 6.00 ± 1.58cB 10.40 ± 2.07cA 3.4 ± 0.56cC 2.00 ± 0.71bC 13.00 ± 1.00bB 12.2 ± 1.48bcB 4.20 ± 0.84bcC 4.40 ± 0.55aD 18.60 ± 1.14aB 15.40 ± 2.19aC 5.00 ± 0.71abD 5.00 ± 0.71aD 18.80 ± 1.79aB 14.4 ± 1.14abC 6.00 ± 1.00abD
All of the data are expressed as means ± S.E. a–c Different letters within the same column indicate significant differences among ages according to Duncan's multiple range (P ≤ 0.05). A–DDifferent letters within the same column indicate significant differences among segments according to Duncan's multiple range (P ≤ 0.05).
apical cytoplasmic processes in contact with the lumen (open-type cells) (Fig. 1F, H, I,J). Few ghrelin-ip cells were round-shaped, so-called closedtype cells (Fig. 1E, G). The results of absorption tests using antiserum absorbed with 5 μg/ml ghrelin showed complete disappearance of immunoreactivity in the gastrointestinal tract (Fig. 1 K, L). 3.2. Morphometric analysis Fig. 3. Representative photomicrograph showing the mucosa in the proventriculus of African ostrich on postnatal days 1, 45, 90, and 334 (stained with immunohistochemistry). Ghrelin-ip cell (arrows) could be observed at every time point. Panels A, B, C, and D correspond to days 1, 45, 90, and 334 respectively. MU, mucosa. (A), bar; 100 μm. (B, C, D) bar; 250 μm.
shapes and with their apical cytoplasmic process in contact with the lumen (open-type cells), indicating that ghrelin cells can be classified into two cell types in the entire gastrointestinal tract. These two types of cells showed similar staining, and no marked differences in immunoreactivities were found in the various regions of the gastrointestinal tract. The more numerous ghrelin-ip cells in the proventriculus and gizzard were small and round-shaped, so-called closed-type cells (Fig. 1B, D). Few ghrelin-ip cells were elongated-shaped with apical cytoplasmic processes in contact with the lumen, so-called open-type cells (Fig. 1C). On the other hand, in the duodenum, jejunum, ileum, cecum, colon, and rectum, the more numerous ghrelin-ip cells had triangular or elongated shapes with their
Morphometric analysis revealed that ghrelin-ip cells were localized preferentially in the proventriculus (P b 0.05); in the duodenum, jejunum, ileum, gizzard, cecum, colon, and rectum, the cell density gradually decreased (P b 0.05) (Fig. 2A). The percentages of open-type cells in various regions of the gastrointestinal tract gradually increased from the gizzard to the large intestine (P b 0.05), being particularly high in the ileum, cecum, colon, and rectum; more than 60% of the ghrelin-ip cells were open-type cells (Fig. 2B). 3.3. Developmental changes in ghrelin-immunopositive cells in the African ostrich stomach and small intestine Immunohistochemistry revealed that the number of ghrelin-ip cells increased gradually from postnatal day 1. In the proventriculus, the number of ghrelin-ip cells increased gradually after birth until postnatal day 45(Fig. 3B) and peaked on day 45 (P b 0.05); in the gizzard and small intestine, the number of ghrelin-ip cells increased gradually after birth until postnatal day 90(Fig. 4C) and peaked on day 90 (P b 0.05) (Table 1). There were virtually no differences in the number of ghrelin-ip cells from postnatal day 90 to postnatal day 334 in the stomach and small intestine (Table 1). By day 45 in the proventriculus and day 90 in the gizzard and small intestine, the number of cells reached a plateau and remained steady. The number of ghrelin-ip cells in the stomach and small intestine was highest in the jejunum and proventriculus on postnatal day 1 (P b 0.05), but highest in the proventriculus on days 45, 90, and 334 (P b 0.05) (Table 1). 4. Discussion 4.1. Assumptions regarding the structure of ostrich ghrelin
Fig. 4. Representative photomicrograph showing the villi in the duodenum of African ostrich on postnatal days 1, 45, 90, and 334 (stained with immunohistochemistry). Ghrelin-ip cell (arrows) could be observed at every time point. Panels A, B, C, and D correspond to days 1, 45, 90, and 334 respectively. VI, villi (A, B, C, D) bar; 100 μm.
Recently, Kojima and Kangawa [15] reported that the amino acid sequences of mammalian ghrelins are well conserved. This structural conservation and the universal requirement for acyl-modification of the third residue indicate that this NH2-terminal region is of central importance to the activity of the peptide, and NH2-terminal cores with acyl-modification sites are well conserved among all vertebrate ghrelins. In this study, we performed absorption tests to evaluate the specificity of the immunostaining, and found that immunoreactivity was absorbed completely. So rabbit anti-synthetic human ghrelin was used for the detection of ghrelin-immunopositive cells, and ghrelin-ip cells were found in the African ostrich gastrointestinal tract. These results indicate that the antiserum cross-reacts with
J.X. Wang et al. / Regulatory Peptides 154 (2009) 97–101
ostrich ghrelin. It is natural to assume that the structure of ostrich ghrelin is identical to the structure of human ghrelin, that ostrich ghrelin is also evolutionally conserved in its basic structure, and that the first seven amino acids and n-octanoylated modification on Ser3 of ostrich ghrelin are conserved; however, the structure of ostrich ghrelin remains to be further studied.
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ip cells peaked on days 45 and 90 respectively. The gastrointestinal tract of African ostrich gradually develops from postnatal day 1 to 72, in relation to the body growth of growing ostriches [24]. All these data suggest that ghrelin may be closely related to the development of the African ostrich. The present research has established a basis for further studies on the effects of ghrelin in the development of the African ostrich and associated regulatory mechanisms.
4.2. Distribution features of ghrelin-immunopositive cells Acknowledgements Ghrelin-ip cells have been found in the stomach, duodenum, jejunum, ileum, and colon in many animals [2,5,9,10,17]. In agreement with these results, as summarized above, ghrelin cells were found in the African ostrich gastrointestinal mucosa from the proventriculus to the rectum. The present study shows that the largest number of ghrelin-ip cells was in the proventriculus and the next-largest number was in the duodenum; very small numbers of ghrelin cells were present in the cecum, colon, and rectum. These results are in agreement with distribution patterns reported for the human and chicken digestive tracts [5,9]. However, a large number of ghrelin-ip cells were found in the jejunum and ileum, and ghrelin-ip cells were also found in the gizzard, which is not in agreement with the distribution patterns reported for human and chicken digestive tracts [5,9].These results indicate that ostrich ghrelin is not different from other animals in its distribution. In 2007, Wang et al. [18] reported that the digestive ducts of African ostrich chicks are more developed than in other avian species; in particular, they have a developed gizzard and jejunum. It is possible that the large number of ghrelin-ip cells in the jejunum, ileum, and gizzard are related to the more developed digestive ducts. Ahmed and Harvey [19] suggested that the presence of ghrelin-ip cells in the chicken gastrointestinal tract may act as a gastrointestinal hormone, in addition to its function as a neuropeptide. The different distribution of ostrich ghrelin may result in diverse functions of ghrelin. The functions of ghrelin in the African ostrich is also an area for further study. It has been well established that gastrointestinal endocrine cells consist of two cell types; i.e., open-type cells, which are in contact with the glandular lumen, and closed-type cells, which do not have a luminal connection [20]. As demonstrated in the present study, ghrelinproducing cells exist as both open-type and closed-type cells in the African ostrich, similar to rats and chickens [6,10]. The open-type cells are thought to be functionally regulated by receiving luminal information such as information on nutrients and pH, while closed-type cells are functionally modulated by hormones, neuronal stimulation, or mechanical distension [20–23]. The presence of these two ghrelin cell types in the gastrointestinal tract suggests that ghrelin secretion from various regions of the gastrointestinal tract is regulated by many stimulants and that each region of the gastrointestinal tract has a different physiological effect. 4.3. Developmental changes in ghrelin-immunopositive cells in the African ostrich The most interesting result obtained in the present study was with regard to the number of ghrelin-ip cells, which increased gradually after postnatal day 1. In the proventriculus, the number of ghrelin-ip cells increased gradually after birth until postnatal day 45 and peaked on day 45; in the gizzard and small intestine, the number of ghrelin-ip cells increased gradually after birth until postnatal day 90 and peaked on day 90. In the rat, the ghrelin concentration of the fetal stomach is also very low and gradually increases after birth until 5 wk of age [6]. These results reveal that the developmental pattern of ghrelin-ip cells was similar in the ostrich to that of the rat and chicken [6,10]. The number of ghrelin-ip cells increased with age, but the time to reach peak numbers was different. A possible explanation is that the timing of ghrelin-ip cell development differs among species. In the proventriculus, gizzard, and small intestine, the number of ghrelin-
We would like to thank Dr. Liu Huazhen of Department of Anatomy, Histology and Embryology, College of Animal Science and Veterinary Medicine, Huazhong Agricultural University for her valuable comments on the experiments. This study was supported by the National Natural Science Foundation Project of China, No.30471249 and No.39970547. References [1] Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K. Ghrelin is a growth hormone-releasing acylated peptide from stomach. Nature 1999;402:656–60. [2] Date Y, Kojima M, Hosoda H, Sawaguchi A, Mondal MS, Suganuma T, et al. Ghrelin, a novel growth hormone-releasing acylated peptides, is synthesized in a distinct endocrine cell type in the gastrointestinal tracts of rats and humans. Endocrinology 2000;141:4255–61. [3] Ariyasu H, Takaya K, Tagami T, Ogawa Y, Hosoda K, Akamizu T, et al. Stomach is a major source of circulating ghrelin, and feeding state determines plasma ghrelinlike immunoreactivity levels in humans. J Clin Endocrinol Metab 2001;86:4753–8. [4] Dornonville de la Cour C, Bjorkqvist M, Sandvik AK, Bakke I, Zhao CM, Chen D, et al. A-like cells in the rat stomach contain ghrelin and do not operate under gastrin control. Regul Pept 2001;99:141–50. [5] Sakata I, Nakamura K, Yamazaki M, Matsubara M, Hayashi Y, Kangawa K. Ghrelinproducing cells exist as two types of cells, closed- and opened type cells, in the rat gastrointestinal tract. Peptides 2002;23:531–6. [6] Hayashida T, Nakahara K, Mondal1 MS, Date Y, Nakazato M, Kojima M. Ghrelin in neonatal rats: distribution in stomach and its possible role. J Endocrinol 2002;173:239–45. [7] Ueno H, Yamaguchi H, Kangawa K, Nakazato M. Ghrelin: a gastric peptide that regulates food intake and energy homeostasis. Regul Pept 2005;126:11–9. [8] Kaiya H, Van der Geyten S, Kojima M, Hosoda H, Kitajima Y, Matsumoto M, et al. Chicken ghrelin: purification, cDNA cloning, and biological activity. Endocrinology 2002;143:3454–63. [9] Neglia S, Arcamone N, Esposito V, Gargiulo G, de Girolamo Paolo. Presence and distribution of ghrelinimmunopositive cells in the chicken gastrointestinal tract. Acta Histochem 2005;107:3–9. [10] Wada R, Sakata I, Kaiya H, Nakamura K, Hayashi Y, Kangawa K, et al. Existence of ghrelin-immunopositive and -expressing cells in the proventriculus of the hatching and adult chicken. Regul Pept 2003;111:123–8. [11] Baudet ML, Harvey S. Ghrelin-induced GH secretion in domestic fowl in vivo and in vitro. J Endocrinol 2003;179:97–105. [12] Furuse M, Tachibana T, Ohgushi A, Ando R, Yoshimatsu T, Denbow DM. Intracerebroventricular injection of ghrelin and growth hormone releasing factor inhibits food intake in neonatal chicks. Neurosci Lett 2001;301:123–6. [13] Geelissen SME, Swennen Q, Van der Geyten S, Kühn ER, Kaiya H, Kangawa K, et al. Darras. Peripheral ghrelin reduces food intake and respiratory quotient in chicken. Domest Anim Endocrinol 2006;30:108–16. [14] Saito ES, Kaiya H, Takagi T, Yamasaki I, Denbow DM, Kangawa K, et al. Chicken ghrelin and growth hormone-releasing peptide-2 inhibit food intake of neonatal chicks. Eur J Pharmacol 2002;453:75–9. [15] Kojima M, Kangawa K. Ghrelin: structure and function. Physiol Rev 2005;85:495–522. [16] Brand TS. Elsenburg Ostrich Feed Databases. Elsenburg, South Africa: Elsenburg Agricultural Research Centre; 2000. [17] Hosoda H, Kojima M, Matsuo H, Kangawa K. Ghrelin and des-acyl ghrelin: two major forms of rat ghrelin peptide in gastrointestinal tissue. Biochem Biophys Res Commun 2000;279:909–13. [18] Wang JX, Peng KM, Du AN, Tang L, Wei L, Jin EH, et al. Histological study on the digestive ducts of African ostrich chicks. J Zool 2007;42(3):131–5 [in Chinese]. [19] Ahmed S, Harvey S. Ghrelin: a hypotalamic GH-releasing factor in domestic fowl (Gallus domesticus). J Endocrinol 2002;172:117–25. [20] Solcia E, Rindi G, Buffa R, Fiocca R, Capella C. Gastric endocrine cells: types, function and growth. Regul Pept 2000;93:31–5. [21] Larsson L, Goltermann N, de Magistris L, Rehfeld JF, Schwartz TW. Somatost-atin cell processess as pathways for paracrine secretion. Science 1979;205:1393–5. [22] Lewin KJ. The endocrine cells of the gastrointestinal tract. The normal endocrine cells and their hyperplasias. Pathol Annu 1986;21 Pt1:1–27. [23] Solcia E, Capella C, Vassallo G, Buffa R. Endocrine cells of the gastric mucosa. Int Rev Cytol 1975;42:223–86. [24] Iji PA, van der Walt JG, Brand TS, Boomker EA, Booyse D. Development of the digestive tract in the ostrich (Struthio camelus). Arch Anim Nutr 2003;57(3):217–28.
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Distribution and developmental changes in ghrelin-immunopositive cells in the gastrointestinal tract of African ostrich chicks J.X. Wang, K.M. Peng ⁎, H.ZH. Liu, H. Song, X. Chen, M. Liu College of Animal Science and Veterinary Medicine, Huazhong Agricultural University, South Lake, Wuhan 430070, PR China
a r t i c l e
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Article history: Received 15 September 2008 Received in revised form 6 January 2009 Accepted 3 February 2009 Available online 14 February 2009 Keywords: African ostrich Ghrelin Gastrointestinal tract Immunohistochemistry
a b s t r a c t Ghrelin, the endogenous ligand for the growth hormone secretagogue receptor (GHS-R), has been found in the gastrointestinal tract of many vertebrates, but little is known about its distribution in the gastrointestinal tract of African ostrich chicks. In the present study, the distribution, morphological characteristics, and developmental changes of ghrelin-producing cells in the gastrointestinal tract of African ostrich chicks were investigated using immunohistochemistry. Ghrelin-immunopositive (ghrelin-ip) cells were found to be localized in the mucous membrane of the entire gastrointestinal tract, but not in the myenteric plexus. The greatest number of ghrelin-ip cells was found in the proventriculus, and the ghrelin-ip cell density gradually decreased from the proventriculus to the rectum. Interestingly, from postnatal day 1 to day 45 in the proventriculus, and from postnatal day 1 to day 90 in the gizzard and small intestine, there was a steady increase in the number of ghrelin-ip cells. By day 45 in the proventriculus and day 90 in the gizzard and small intestine, the number of cells reached a plateau and remained steady. These results clearly demonstrate that ghrelin-ip cells exist and the number of ghrelin-ip cells increases with age in the African ostrich gastrointestinal tract from postnatal day 1 to day 90; ghrelin may be involved in gastrointestinal tract development. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Ghrelin is a brain-gut peptide that has been isolated as an endogenous ligand for the growth hormone secretagogue receptor (GHS-R) from the rat stomach [1]. This peptide consists of 28 amino acids, in which the third serine residue is n-octanoylated; this side chain is essential to its biological activity [1]. Ghrelin is predominantly produced by the X/A-like endocrine cells in the oxyntic mucosa of the stomach, which is the major source of circulating ghrelin [2–4]. Ghrelinimmunopositive (ghrelin-ip) cells have been found to be localized in the mucous membranes of the stomach, duodenum, ileum, cecum, and colon, but not in the myenteric plexus, and they can be classified into open- and closed-type cells [5]. The number of ghrelin-ip cells in the fetal stomach increases as the stomach grows [6]. In mammals, ghrelin stimulates growth hormone (GH) release, regulates food intake, energy balance, body weight, gastric motility and acid secretion, endocrine pancreas functions, and glucose metabolism [1,7]. In addition to mammals, ghrelin has also been identified in non-mammalian species. In the chicken, ghrelin consists of 26 amino acids. The serine residue at position 3 (Ser3) is conserved between the chicken and mammalian species, as is its acylation by either n-octanoic or n-decanoic acid [8]. Ghrelin-ip cells have been found to be localized in the mucous ⁎ Corresponding author. Tel.: +86 27 8728 6970; fax: +86 27 87280408. E-mail address: [email protected] (K.M. Peng). 0167-0115/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2009.02.005
membranes of the proventriculus, duodenum, ileum, cecum, and colon, but not in the myenteric plexus, and they can be classified into open- and closed-type cells [9]. The number of ghrelin-ip cells in the adult chicken is also greater than that in the hatching chicken [10]. Ghrelin also stimulates GH release in chickens [8,11]; however, in contrast to its action in mammals, ghrelin inhibits feeding, particularly in neonatal chicks when injected intracerebroventricularly [12–14]. Ghrelins have been identified in other avian species, such as the duck, goose, emu, and turkey [15]. However, there have been no studies on the distribution and function of ghrelin in African ostrich chicks. In general, to understand or hypothesize regarding the physiological role of newly identified peptides, it is important to know the morphological characteristics of the producing cell and its distribution. Therefore, in this study, the distribution and morphological characteristics and developmental changes of ghrelin cells in the gastrointestinal tract were studied in detail using immunohistochemistry (IHC). 2. Materials and methods 2.1. Animals Neonatal day 1, 45, 90, and 334 female African ostriches were used in the present study. African ostrich chicks (24 females) were obtained from a standard ostrich farm in Guangdong Province, China, on postnatal day 1 (newly hatched chicks) and were transported within
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10 h to a battery house, where feed and water were made available ad libitum. The 24 birds were divided into 4 groups (6 ostriches per group) on the basis of their body weight (BW), to equalize BW and the variance among groups. All of the birds were maintained in a heated room with slatted plastic flooring and were fed a starter diet for postnatal days 1–334, which was formulated according to the specifications of the Elsenburg Ostrich Feed Database [16]. All procedures were approved by the Animal Care and Welfare Committee of our Institute. 2.2. Tissue preparation On postnatal days 1, 45, 90, and 334 (control group), the birds were weighed, deeply anesthetized with 10% urethane (Caoyang Secondary
Chemical Plant, Shanghai, China) at a dose of 1 g/kg BW, and perfused, initially with 1000 mL of 0.85% normal saline (containing 0.075% sodium citrate) and thereafter with 1500 mL of 4% paraformaldehyde phosphate-buffered solution (0.1 mol/L, pH 7.4) at 4 °C. The abdomen was cut open. Segments of their gastrointestinal tracts, approximately 1 cm in length, were quickly removed and opened along their longitudinal axes. The segments were postfixed for more than 24 h with 4% paraformaldehyde. The specimens were obtained from the following portions of the African ostrich gastrointestinal tracts: the proventriculus (3 cm proximal to the pylorus), the gizzard, the duodenum (5 cm distal to the pylorus), the jejunum (3 cm proximal to Meckel's diverticulum), the ileum (10 cm proximal to the ileocecal junction), the cecum (5 cm from the ileocecal junction), the colon (5 cm distal to the cecocolonic junction), and the rectum (1 cm from
Fig. 1. Microphotographs of ghrelin-immunopositive cells in the gastrointestinal tract. (A) A large number of ghrelin cells (arrows) were found in the proventriculus. (B) Almost all of the Ghrelin-ip cells in the proventriculus were round-shaped cells, so called closed-type cells(arrow). (C) Few Ghrelin-ip cells in the proventriculus were Opened-type (arrow) in contact with the lumen.(D) Closed-type ghrelin cells (arrow) were found in the gizzard. (E) Closed-type ghrelin cells (arrow) were found in the crypt of the duodenum.(F) Openedtype ghrelin-ip cells (arrows) in the villi of the duodenum. (G) Closed-type ghrelin cells (arrows) were found in the crypt of the jejunum (H) Ghrelin-ip cells with long processes (arrow) were found in villi of the jejunum. (I) Opened-type ghrelin-ip cells (arrow) in villi of the ileum. (J) Opened-type ghrelin-ip cells (arrows) in contact with the lumen were found in the villi of the cecum. (K) Microphotograph of absorption test in the proventriculus. (L) Microphotograph of absorption testing in the duodenum. MU, mucosa; SL, smooth muscle layer; VI, villi; CY, crypt ;LU, lumen. (A, K,L) bar; 100 μm. (B,C,D, E,F,G,H, I, J) bar; 10 μm.
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the anus). After immersion, the tissues were embedded in paraffin. Serial sections (5 μm) were cut on a Leica microtome (Nussloch Gmbh, Germany), mounted on polylysine-coated slides (Boster Corporation, China).
range test where appropriate. Differences of p b 0.05 were considered significant.
2.3. Immunohistochemistry
3.1. Distribution of ghrelin-immunopositive cells
Immunohistochemical detection of ghrelin cells using rabbit antighrelin was carried out by the streptavidin-biotin-peroxidase complex (SABC) method. The anti-ghrelin serum used in this study was a synthetic human ghrelin (gssflspehqrvqqrkeskkppaklqpr), which is different from the related rat and mouse sequence by two amino acids. The anti-ghrelin antiserum recognizes the n-octanoylated Ser3 epitope and the anti-ghrelin antiserum recognizes the C-terminal region of rat ghrelin. Immunohistochemical staining was performed according to the following procedure. The sections were deparaffinized with xylene and rehydrated with decreasing concentrations of ethanol, then treated with 3% hydrogen peroxide (H2O2) to block endogenous peroxidase for 10 min at room temperature. After rinsing with distilled water, the sections were incubated with a pH 6.0 citrate buffer and placed in a microwave oven until the water boiled to fully expose the antigen. After rinsing with phosphate-buffered saline (PBS), the sections were incubated with 5% normal goat serum for 20 min. The sections were then diluted 1:100 with PBS and incubated with rabbit anti-ghrelin primary antibody (BA1619; Boster Corporation) for 12 h in a humid chamber at 4 °C. After washing with PBS for 6 min, a second incubation with biotin-conjugated anti-rabbit IgG serum (SA1022; Boster) was carried out for 20 min, and this was followed by further washing with PBS. Finally, the sections were incubated for 20 min with an SABC solution prepared according to the manufacturer's instructions. After washing with PBS for 20 min, the sections were reacted in a diaminobenzidine-tetrachloride kit (DAB kit, AR1022) for 30 min to detect immunostaining. After washing with distilled water, the sections were dehydrated with a graded ethanol series, cleared in xylene, mounted with a coverslip, and viewed under a light microscope (BH-2; Olympus, Japan). All of the incubations were carried out in a humid chamber at room temperature. Control sections were prepared using the same method, omitting the primary antibody. To examine the specificity of rabbit anti-human ghrelin antiserum, the diluted antiserum (1:100) was incubated with human ghrelin (5 μg/ml) at room temperature for 10 h, and mixtures were centrifuged at 12,000 rpm for 25 min at 4 °C. The supernatant was used as the primary antiserum for absorption tests.
On postnatal days 1, 45, 90, and 334, female African ostrich ghrelin-ip cells were found in the mucosal layer, but not in the myenteric plexus, in all of the examined regions (Figs. 3A–D, 4A–D). Ghrelin-ip cells were most dense in the proventriculus, and low densities of ghrelin cells were found in the other examined regions (the duodenum, jejunum, ileum, and gizzard). Very few ghrelin-ip cells were found in the cecum, colon, and rectum on postnatal days 1, 45, 90, and 334. Ninety-day-old female African ostriches were used to investigate the distribution of ghrelin-ip cells in various tissues. It was found that ghrelinip cells existed in all regions of the gastrointestinal tract: the proventriculus (Fig. 1A–C), the gizzard (Fig. 1D), the duodenum (Fig. 1E,F), the jejunum (Fig. 1G,H), the ileum (Fig. 1I), the cecum (Fig. 1J), the colon, and the rectum (data not shown). In the proventriculus, most of the ghrelin-ip cells were observed at the base of glandular lobuli, and a few ghrelin-ip cells were observed in the basal zone of proventriculus plicae (Fig. 1A). In the gizzard, ghrelin-ip cells were scattered in the glandular epithelia of crypts (Fig. 1D). In the duodenum, jejunum, ileum, cecum, colon, and rectum, ghrelin-ip cells were scattered in the epithelia of crypts (Fig.1E, G) and villi (Fig. 1F, H, I, J). There were virtually no differences in the immunoreactivities of ghrelin cells in the various regions of the gastrointestinal tract in the African ostrich. Two types of ghrelin cells were found; i.e., closed-type cells and cells with triangular or elongated
3. Results
2.4. Morphometric analysis The densities of ghrelin cells in various regions of the African ostrich gastrointestinal tract were estimated. After taking digital photographs under a light microscope (BH-2; Olympus) with a digital camera (COOLPIX4500; Nikon, Japan), the number of ghrelin cells in each section was counted and the area of the mucosal layer in each section was measured using a computerized image analysis program, HMIAS-2000 High-definition Chromatic Color Medical Science Figure Analysis Program (Qianping, Wuhan, China). The ghrelin cell density was calculated as the number of ghrelin-ip cells per unit area. The percentage of open-type ghrelin cells in the gastrointestinal segment was also calculated. First, the total number of all ghrelin cells (closedtype and open-type cells) in each section was counted using digital photography as mentioned above, and then the percentage of opentype ghrelin cells to total ghrelin cells was calculated. 2.5. Statistical analyses Results are expressed as means ± standard errors on the mean (means± S.E.). Statistical analysis was done using analysis of variance statistics software (SAS Institute, 2000) with Duncan's multiple
Fig. 2. (A). Histogram showing the densities of ghrelin cells (cells/mm2) in the 90 dayold African ostrich gastrointestinal tract. Ghrelin-immunopositive cells were present throughout the gastrointestinal mucosa, from the proventriculus to rectum, (B) Histogram showing the percentages of opened-type cells to all immunopositive cells in the 90 day-old African ostrich gastrointestinal tract. The percentage of openedtype ghrelin cells gradually increased in the direction from the gizzard to the large intestine. a–hDifferent letters within the same column indicate significant differences among segments according to Duncan's multiple range (P ≤ 0.05).PR, proventriculus; GI, gizzard; DU, duodenum; JE, jejunum ; IL, ileum; CE, cecum; CO, colon; RE, rectum.
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J.X. Wang et al. / Regulatory Peptides 154 (2009) 97–101 Table 1 Ghrelin-ip cell densities (cells/mm2) in the stomach and small intestine of African ostrich chickens on postnatal days 1, 45, 90, and 334. Day (d) Cell density (cells/mm2) Proventriculus Gizzard 0 45 90 334
9.00 ± 2.83bA 29.60 ± 9.13aA 24.40 ± 3.71aA 26.60 ± 4.39aA
Duodenum
Jejunum
Ileum
0.80 ± 0.45cD 6.00 ± 1.58cB 10.40 ± 2.07cA 3.4 ± 0.56cC 2.00 ± 0.71bC 13.00 ± 1.00bB 12.2 ± 1.48bcB 4.20 ± 0.84bcC 4.40 ± 0.55aD 18.60 ± 1.14aB 15.40 ± 2.19aC 5.00 ± 0.71abD 5.00 ± 0.71aD 18.80 ± 1.79aB 14.4 ± 1.14abC 6.00 ± 1.00abD
All of the data are expressed as means ± S.E. a–c Different letters within the same column indicate significant differences among ages according to Duncan's multiple range (P ≤ 0.05). A–DDifferent letters within the same column indicate significant differences among segments according to Duncan's multiple range (P ≤ 0.05).
apical cytoplasmic processes in contact with the lumen (open-type cells) (Fig. 1F, H, I,J). Few ghrelin-ip cells were round-shaped, so-called closedtype cells (Fig. 1E, G). The results of absorption tests using antiserum absorbed with 5 μg/ml ghrelin showed complete disappearance of immunoreactivity in the gastrointestinal tract (Fig. 1 K, L). 3.2. Morphometric analysis Fig. 3. Representative photomicrograph showing the mucosa in the proventriculus of African ostrich on postnatal days 1, 45, 90, and 334 (stained with immunohistochemistry). Ghrelin-ip cell (arrows) could be observed at every time point. Panels A, B, C, and D correspond to days 1, 45, 90, and 334 respectively. MU, mucosa. (A), bar; 100 μm. (B, C, D) bar; 250 μm.
shapes and with their apical cytoplasmic process in contact with the lumen (open-type cells), indicating that ghrelin cells can be classified into two cell types in the entire gastrointestinal tract. These two types of cells showed similar staining, and no marked differences in immunoreactivities were found in the various regions of the gastrointestinal tract. The more numerous ghrelin-ip cells in the proventriculus and gizzard were small and round-shaped, so-called closed-type cells (Fig. 1B, D). Few ghrelin-ip cells were elongated-shaped with apical cytoplasmic processes in contact with the lumen, so-called open-type cells (Fig. 1C). On the other hand, in the duodenum, jejunum, ileum, cecum, colon, and rectum, the more numerous ghrelin-ip cells had triangular or elongated shapes with their
Morphometric analysis revealed that ghrelin-ip cells were localized preferentially in the proventriculus (P b 0.05); in the duodenum, jejunum, ileum, gizzard, cecum, colon, and rectum, the cell density gradually decreased (P b 0.05) (Fig. 2A). The percentages of open-type cells in various regions of the gastrointestinal tract gradually increased from the gizzard to the large intestine (P b 0.05), being particularly high in the ileum, cecum, colon, and rectum; more than 60% of the ghrelin-ip cells were open-type cells (Fig. 2B). 3.3. Developmental changes in ghrelin-immunopositive cells in the African ostrich stomach and small intestine Immunohistochemistry revealed that the number of ghrelin-ip cells increased gradually from postnatal day 1. In the proventriculus, the number of ghrelin-ip cells increased gradually after birth until postnatal day 45(Fig. 3B) and peaked on day 45 (P b 0.05); in the gizzard and small intestine, the number of ghrelin-ip cells increased gradually after birth until postnatal day 90(Fig. 4C) and peaked on day 90 (P b 0.05) (Table 1). There were virtually no differences in the number of ghrelin-ip cells from postnatal day 90 to postnatal day 334 in the stomach and small intestine (Table 1). By day 45 in the proventriculus and day 90 in the gizzard and small intestine, the number of cells reached a plateau and remained steady. The number of ghrelin-ip cells in the stomach and small intestine was highest in the jejunum and proventriculus on postnatal day 1 (P b 0.05), but highest in the proventriculus on days 45, 90, and 334 (P b 0.05) (Table 1). 4. Discussion 4.1. Assumptions regarding the structure of ostrich ghrelin
Fig. 4. Representative photomicrograph showing the villi in the duodenum of African ostrich on postnatal days 1, 45, 90, and 334 (stained with immunohistochemistry). Ghrelin-ip cell (arrows) could be observed at every time point. Panels A, B, C, and D correspond to days 1, 45, 90, and 334 respectively. VI, villi (A, B, C, D) bar; 100 μm.
Recently, Kojima and Kangawa [15] reported that the amino acid sequences of mammalian ghrelins are well conserved. This structural conservation and the universal requirement for acyl-modification of the third residue indicate that this NH2-terminal region is of central importance to the activity of the peptide, and NH2-terminal cores with acyl-modification sites are well conserved among all vertebrate ghrelins. In this study, we performed absorption tests to evaluate the specificity of the immunostaining, and found that immunoreactivity was absorbed completely. So rabbit anti-synthetic human ghrelin was used for the detection of ghrelin-immunopositive cells, and ghrelin-ip cells were found in the African ostrich gastrointestinal tract. These results indicate that the antiserum cross-reacts with
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ostrich ghrelin. It is natural to assume that the structure of ostrich ghrelin is identical to the structure of human ghrelin, that ostrich ghrelin is also evolutionally conserved in its basic structure, and that the first seven amino acids and n-octanoylated modification on Ser3 of ostrich ghrelin are conserved; however, the structure of ostrich ghrelin remains to be further studied.
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ip cells peaked on days 45 and 90 respectively. The gastrointestinal tract of African ostrich gradually develops from postnatal day 1 to 72, in relation to the body growth of growing ostriches [24]. All these data suggest that ghrelin may be closely related to the development of the African ostrich. The present research has established a basis for further studies on the effects of ghrelin in the development of the African ostrich and associated regulatory mechanisms.
4.2. Distribution features of ghrelin-immunopositive cells Acknowledgements Ghrelin-ip cells have been found in the stomach, duodenum, jejunum, ileum, and colon in many animals [2,5,9,10,17]. In agreement with these results, as summarized above, ghrelin cells were found in the African ostrich gastrointestinal mucosa from the proventriculus to the rectum. The present study shows that the largest number of ghrelin-ip cells was in the proventriculus and the next-largest number was in the duodenum; very small numbers of ghrelin cells were present in the cecum, colon, and rectum. These results are in agreement with distribution patterns reported for the human and chicken digestive tracts [5,9]. However, a large number of ghrelin-ip cells were found in the jejunum and ileum, and ghrelin-ip cells were also found in the gizzard, which is not in agreement with the distribution patterns reported for human and chicken digestive tracts [5,9].These results indicate that ostrich ghrelin is not different from other animals in its distribution. In 2007, Wang et al. [18] reported that the digestive ducts of African ostrich chicks are more developed than in other avian species; in particular, they have a developed gizzard and jejunum. It is possible that the large number of ghrelin-ip cells in the jejunum, ileum, and gizzard are related to the more developed digestive ducts. Ahmed and Harvey [19] suggested that the presence of ghrelin-ip cells in the chicken gastrointestinal tract may act as a gastrointestinal hormone, in addition to its function as a neuropeptide. The different distribution of ostrich ghrelin may result in diverse functions of ghrelin. The functions of ghrelin in the African ostrich is also an area for further study. It has been well established that gastrointestinal endocrine cells consist of two cell types; i.e., open-type cells, which are in contact with the glandular lumen, and closed-type cells, which do not have a luminal connection [20]. As demonstrated in the present study, ghrelinproducing cells exist as both open-type and closed-type cells in the African ostrich, similar to rats and chickens [6,10]. The open-type cells are thought to be functionally regulated by receiving luminal information such as information on nutrients and pH, while closed-type cells are functionally modulated by hormones, neuronal stimulation, or mechanical distension [20–23]. The presence of these two ghrelin cell types in the gastrointestinal tract suggests that ghrelin secretion from various regions of the gastrointestinal tract is regulated by many stimulants and that each region of the gastrointestinal tract has a different physiological effect. 4.3. Developmental changes in ghrelin-immunopositive cells in the African ostrich The most interesting result obtained in the present study was with regard to the number of ghrelin-ip cells, which increased gradually after postnatal day 1. In the proventriculus, the number of ghrelin-ip cells increased gradually after birth until postnatal day 45 and peaked on day 45; in the gizzard and small intestine, the number of ghrelin-ip cells increased gradually after birth until postnatal day 90 and peaked on day 90. In the rat, the ghrelin concentration of the fetal stomach is also very low and gradually increases after birth until 5 wk of age [6]. These results reveal that the developmental pattern of ghrelin-ip cells was similar in the ostrich to that of the rat and chicken [6,10]. The number of ghrelin-ip cells increased with age, but the time to reach peak numbers was different. A possible explanation is that the timing of ghrelin-ip cell development differs among species. In the proventriculus, gizzard, and small intestine, the number of ghrelin-
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