Immunocytochemical methods were employed to demonstrate endocrine cells, containing peptides and
serotonin, in the gut of the impala. Cells immunoreactive to serotonin, somatostatin, gastrin, cholecystokinin,
glucagon, neurotensin, secretin, glucose-dependant insulinotropic peptide and motilin were detected.
Antisera raised to substance P and pancreatic polypeptide failed to stain any cells. The distribution
of these peptide-containing cells is more in line with the situation in sheep than other ruminants. In
contrast, the distribution and abundance of serotonin cells in the gut of the impala parallels the situation
seen in game herbivores.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. 
Adobe Acrobat X Pro was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.mn201

De Vos, Valerius

Schoeman, J.H.

Van Aswegen, G.

The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. 
Adobe Acrobat X Pro was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.Immunocytochemical methods were employed to demonstrate endocrine cells, containing peptides and
serotonin, in the gut of the impala. Cells immunoreactive to serotonin, somatostatin, gastrin, cholecystokinin,
glucagon, neurotensin, secretin, glucose-dependant insulinotropic peptide and motilin were detected.
Antisera raised to substance P and pancreatic polypeptide failed to stain any cells. The distribution
of these peptide-containing cells is more in line with the situation in sheep than other ruminants. In
contrast, the distribution and abundance of serotonin cells in the gut of the impala parallels the situation
seen in game herbivores.mn201

UPSpace at the University of Pretoria

Onderstepoort Journal of Veterinary Research , 65:31 - 35 (1998) Distribution of endocrine cells in the gut of the impala (Aepyceros melampus) J.H. SCHOEMAN1, V. DE VOS2 and G. VAN ASWEGEN3* ABSTRACT SCHOEMAN, J.H., DEVOS, V. & VAN ASWEGEN, G. 1996. Distribution of endocrine cells in the gut of the impala. Onderstepoort Journal of Veterinary Research, 65:31-35 Immunocytochemical methods were employed to demonstrate endocrine cells, containing peptides and serotonin, in the gut ofthe impala. Cells immunoreactive to serotonin, somatostatin, gastrin, cholecysto-kinin, glucagon, neurotensin, secretin, glucose-dependant insulinotropic peptide and motilin were de-tected . Antisera raised to substance P and pancreatic polypeptide failed to stain any cells. The distribu-tion of these peptide-containing cells is more in line with the situation in sheep than other ruminants. In contrast, the distribution and abundance of serotonin cells in the gut of the impala parallels the situa-tion seen in game herbivores. Keywords: Aepyceros melampus, endocrine cells, gut peptides, impala, serotonin INTRODUCTION Impala are distributed in woodlands of southern Af-rica (Skinner & Smithers 1990). They are grazers and browsers (Kay, Engelhardt & White 1979) and their diet may vary depending on the habitat and season (Skinner & Smithers 1990). In spite of their abun-dance in southern Africa (Skinner & Smithers 1990) , and particularly in the Kruger National Park (Nicholls, Viljoen, Knight & Van Jaarsveld 1996), little is known of the digestive physiology of this species. Recently, considerable attention has been focused on the neuroendocrine modulation of the functions of the gut (Sundler, Bottcher, Ekblad & Hakanson 1989). Some gut endocrine functions have been * Author to whom correspondence is to be directed 1 Department of Biological Sciences, Technikon Pretoria, Preto-ria , 0002 South Africa 2 Kruger National Park, Skukuza, 1350 South Africa 3 Department of Pharmacology, Potchefstroom University for Christian Higher Education, Potchefstroom, 2520 South Af-rica Accepted for publication 11 December 1997- Editor elucidated, for example, secretin evoking pancreatic secretion and motilin which elicits gut motility (Holst & Schmidt 1994). Endocrine/paracrine modulation of gut secretion, absorption and mucosal blood flow is not yet fully understood (Holst & Schmidt 1994). As research progresses the evidence for the integration of nervous and endocrine control of digestive func-tions is increasing (cf. Cooke 1987; Sundler eta/. 1989). The gut endocrine system is complex, and acts in various ways, viz. autocrine, endocrine, para-crine and neuroendocrine processes (cf. Rehfeldt 1990). The field of gut regulatory peptides is there-fore exciting and comparative studies, involving spe-cies other than those traditionally employed in the laboratory, may contribute to a better understanding of gut physiology. Reports on the gut endocrine cells of some ruminants have contributed to this effort, including bovine spe-cies (Kitamura, Yamada, Calingasan & Yamashita 1985; Weyrauch, Schnorr & Glaser 1989), the sheep (Calingasan, Kitamura, Yamada, Oomori & Yamashita 1984), the lesser mouse deer (Tragulus javanicus) (An-gungpriyono, Yamada, Kitamura, Yamamoto, Said, Sigit & Yamashita 1994) and the fallow deer (Oama dama L.) (Ceccarelli, Pedini & Gargiulo 1995). 31 Distribution of endocrine cells in the gut of the impala (Aepyceros melampus) In this investigation the distribution of endocrine cells TABLE 1 Primary antisera used containing bioactive peptides and serotonin was demonstrated in the gut of the impala, and compared to the situation in other ruminants. MATERIALS AND METHODS During a culling expedition samples of tissue were promptly taken from six adult impala including the abomasum, duodenum, jejunum, ileum, caecum, co-lon and rectum. Fixation and processing All tissue samples were rinsed vigorously in Ringer's solution fo r 30 s to remove sand and other particles from the luminal surface. For immunoperoxidase staining, the samples were fixed in Bouin's fluid for 12 h at room temperature. The tissue blocks were dehydrated in ethanol and embedded in paraffin wax. Sections (5 Jlm) were cut and floated on slides pre-treated with poly-L-Iysine (Huang, Gibson, Facer, Gu & Polak 1983). lmmunoperoxidase staining for mucosal endocrine cells Paraffin sections were dewaxed and treated with 0,3 % hydrogen peroxide in methanol for 30 min to block all endogenous peroxidase activity. The sec-tions were hydrated through a series of ethanols and transferred to 0,05 MTris-saline. To reduce non-specific staining, the sections were incubated with 10 % normal swine serum (Burns 1979}. Either the indirect peroxidase or the peroxidase anti-perox-idase (PAP) (Sternberger 1979) methods were employed to identify immunoreactivity for the bio-active peptides and serotonin. The reaction sites were revealed by the method of Graham & Karnov-sky (1966). Details of the primary antisera employed are listed in Table 1 . Controls for immunocytochemistry As positive controls, sections of tissues from other species (swine and dog), known to contain the pep-tides in question, were employed. Negative controls consisted of substitution of non-immune rabbit se-rum for the primary antibody and pre-absorption of the diluted antibody with 1 0 nmol or at least 20 Jig per mQ of the homologous peptide or serotonin . RESULTS All the antisera were absorbed by their parent anti-gens. No immunoreactivity was observed for the an-tisera for pancreatic polypeptide and substance P (Fig. 1) . 32 Antisera Somatostatin 14 (synthetic)b Gastrin (porcine)• CCK (synthetic midportion)b Glucagon (porcine pancreatic)• Secretin (natural porcine)• Neurotensinb Neurotensin• GIP (natural porcine)b Motilin (porcine)c Serotonind 1 Pancreatic polypeptide (bovine) b Substance P (synthetic porcine) a - purchased from MILAB b - supplied by Prof. J.M. Polak, London c - purchased from Quandrologic Inc. Code 744 B35 280 B27 B37 810 -378 M37B -Lance d - supplied by Prof. J.M. Lander, Chapel Hill e - supplied by Prof. C. Shaw, Belfast 1 - purchased from Lilly Research Laboratories Dilution 1:1000 1:1000 1:1000 1:2000 1:2000 1:1000 1 :500d 1:1000 1:1000 1:500 1:2000 Except for the caecum and rectum , somatostatin immunoreactive cells were observed in all the regions of the gut. They were particularly numerous in the pyloric region (Fig. 2). Some cells had basal cytoplas-mic extentions. Cells immunoreactive for gastrin were abundant in the pyloric region (Fig . 3) , less abundant in the duo-denum and sparse in the small intestine. They were absent from the large bowel. In all the regions where these cells occurred, none of them reached the lu-men. Cholecystokinin (CCK)-immunoreactive cells were confined to the small intestine (Fig. 4), and their numbers declined from the proximal duodenum to the ileum. Very few of these cells were seen to reach the lumen. No glucagon immunoreactive cells were observed in the abomasum. In the entire small intestine they were quite sparse, but in the duodenum, colon and rec-tum they were slightly more abundant. They were confined to the basal part of the mucosa (Fig . 5) . Antiserum 810 failed to demonstrate endocrine cells. At a dilution of 1 :500 the antiserum supplied by Prof. C. Shaw stained a few cells immunoreactive for neurotensin in jejunum and ileum. They were seen in the crypts and villi. The antiserum to secretin stained cells in the mucosa of the duodenum only (Fig. 6). They were present in the crypts and villi, and many of them were of the open type. Similarly, the distribution of glucose-dependant insulinotropic peptide (GIP)-immuno-reactivity (Fig. 7) was limited to the duodenal mucosa. The GIP-endocrine cells were preferentially located towards the basal part of the mucosa. Cells immunoreactive to motilin were sparsely scat-tered in the duodenum and jejunum (Fig. 8). The anti-serum to serotonin (5HT) demonstrated endocrine J.H. SCHEOMAN, V. DEVOS & G. VAN ASWEGEN 40-r-------,-------.--------.-------,-------,-------,-------,-------.-------~ 30 ··················· ·····················I ······ ····· · ··· ··· ··+······· - ··· -- ······· · ·· -~- --· · ····· · ··· ·· .... .. . j ...... . . 20 1 0 0 Somatos ta ti n Gastrin CCK Glucagon Neurotensin Secretin GIP Mot ilin 5HT ~Fu nd u s D Py lorus §Duodenum • Jejenum • Ileum [!Zl Colon CSJ Rectum FIG. 1 Histogram showing the relative abundance of cells per 0,25 mm2 cells in all the regions of the gut under investigation. They were relatively scarce in the fundus, somewhat more abundant in the jejunum and ileum, but mark-edly numerous in the pyloric region. If compared to the small intestine, their numbers progressively in-creased in the colon and rectum (Fig. 9). In there-gions of the gut where they were sparse, they were randomly distributed, but as their numbers increased they were increasingly confined to the basal part of the mucosa. FIG. 2 Somatostatin cells in the basal part of the pyloric region . Arrow head pointing towards the lumen 200 x Indirect peroxidase FIG. 3 Gastrin cells in the pyloric region 200 x Indirect peroxidase FIG. 4 CCK-immunoreactive cells in the jejunum 350 x PAP FIG. 5 Glucagon cells in the ileum 350 x PAP FIG. 6 A cell immunoieactive to secretin in the duodenum 350x PAP FIG. 7 A GIP-immunoreactive cell in the duodenum 350x PAP FIG. 8 A motilin cell in the jejunum 350x PAP FIG. 9 Cells immunoreactive to 5HT in the rectum 200 x Indirect peroxidase DISCUSSION The distribution and abundance of somatostatin and gastrin endocrine cells in the gut of the impala are in line with the general situation seen in smaller ru-minants, such as goat and sheep (Calingasan eta/. 1984; Weyrauch, Blahser & Perschbacher 1987), the lesser mouse deer (Angungpriyono eta/. 1994) and the fallow deer (Ceccarelli eta/. 1995). In contrast to the situation in the lesser mouse deer (Angungpri-33 Distribution of endocrine cells in the gut of the impala (Aepyceros melampus) yono eta/. 1994), the GIP- and secretin-containing cells of the impala are confined to the proximal small bowel only, which is in agreement with findings on the cow (Kitamura eta/. 1985) and confirms the pres-ence of the typical mammalian entero-pancreatic axis of the impala (Sundler et al. 1989) in this region of the gut. In contrast to sheep (Calingasan eta/. 1984), but in line with the situation in the lesser mouse deer (An-gungpriyono eta/. 1994) and cow (Kitamura eta/. 1985), endocrine cells containing motilin were re-stricted to the proximal small intestine of the impala. CCK-endocrine cells seem to be preferentially located in most of the regions of the small intestine of the impala and small ruminants (Calingasan eta/. 1984; Ceccarelli eta/. 1995). The paucity of neurotensin cells seen in the impala is typical for herbivores (Sundler, Hakanson, Hammer, Alumets, Carraway, Leeman & Zimmerman 1977) and is in agreement with the situation in sheep (Calingasan eta/. 1984). In contrast to the findings on other ruminants (Caling-asan eta/. 1984; Kitamura eta/. 1985; Angungpriyono eta/. 1994} it seems that either endocrine cells im-munoreactive for substance P and pancreatic poly-peptide do not occur in the gut of the impala, or their sequences are so different that they escaped detec-tion by the antisera employed. The topographical distribution and abundance of serotonin endocrine cells in gut of the impala follow the situation in the fallow deer (Ceccarelli eta/. 1995) more closely than that of the lesser mouse deer (An-gungpriyono eta/. 1994). These cells seem to be markedly more abundant in game herbivores, if com-pared to their domesticated counterparts. Judging from their abundance, it seems that serotonin plays an important role in the digestive physiology of the impala and the fallow deer (Ceccarelli eta/. 1995). It is known that serotonin, together with other gut pep-tides, modulates the digestive processes (Gonella 1981). The abundance of serotonin cells, and its ac-tion in the gut of the impala, may allow it to thrive on a harsh and much varied seasonal and habitat diet (ct. Pietersen 1991). Cross-reaction between sub-stance P and serotonin (Rawdon & Andrew 1994) is not considered in this study, because no substance P immunoreactivity was observed. The absence of glucagon endocrine cells in the stom-ach of the impala was also seen in the fallow deer (Ceccarelli et a/. 1995), and cow (Kitamura et a/. 1985), whereas the distribution of these cells in the intestine of the impala is parallel to the findings on glicentin in sheep (Calingasan eta/. 1984}. The pos-sibility that the antiserum employed to detect glu-cagon cross-reacted with glicentin, should not be ignored. The absence of glucagon immunoreactive cells, notably in the fundus of the impala and cow, is not surprizing, because they are known to occur in 34 the fundic regions of carnivores (cat and dog) (Lars-son, Holst, Hakanson & Sundler 1975; Grimelius, Holst, Buffa, Polak, Pearse & Solcia 1976). In omni-vores (rat and man) these cells are sparsely distrib-uted (Grimelius eta/. 1976}. The situation in the impala and cow therefore seems to be typical for herbivores as in the case of the elephant (Van Aswegen, Schoe-man, De Vos & Van Noorden 1994). One of the main features of the topographical distri-bution of endocrine cells in gut of the impala is that most were situated in the basal part of the mucosa and therefore implicate a paracrine function (Lars-son, Golterman, De Magistris, Rehfeld & Schwartz 1979). This study is the first to report on the gut endocrine cells of an African game ruminant. Furthermore, it shows that the distribution patterns of the endocrine cell types in the gut of the impala resemble those of sheep more closely than other ruminants, but are different enough to conclude that they are unique for the species, particularly the abundance and distribu-tion of serotonin endocrine cells. The influence of diet on the gut endocrine cells of the impala is not con-sidered here, because it may be very difficult to as-certain, as its diet varies from one habitat or season to another (Skinner & Smithers 1990). ACKNOWLEDGEMENTS The authors wish to thank Mrs Riana Kriel for typing the manuscript, Miss M. van Aswegen for preparing the figures and Mr 0 . Bergh for his assistance dur-ing field expeditions. 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Distribution of endocrine cells in the gut of the impala (Aepyceros melampus)

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Distribution of endocrine cells in the gut of the impala (Aepyceros melampus)

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