J. Anat. (1989), 162, pp. 157-168 157 With 23 figures Printed in Great Britain An immunohistochemical study of gastrointestinal endocrine cells in a nectarivorous marsupial, the honey possum (Tarsipes rostratus)* J. YAMADA, K. C. RICHARDSONt AND R. D. WOOLLERt Department of Veterinary Anatomy, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080, Japan, t School of Veterinary Studies, Murdoch University, Western Australia 6150 and t Biological Sciences, Murdoch University, Western Australia 6150 (Accepted 6 May 1988) INTRODUCTION The honey possum, Tarsipes rostratus, is a unique marsupial, feeding almost exclusively on nectar and pollen. It is small (7-9 g in males and 10-12 g in females) and restricted to the sandplain heathlands of the southwestern corner of Australia (Renfree, Russell & Wooller, 1984). The honey possum is highly adapted for its specialised feeding habit, having a head which is dorsoventrally flattened and an elongated snout. The elongate tongue is brush-tipped and is protruded into flowers or pollen presenters to collect nectar and pollen (Richardson, Wooller & Collins, 1986). The alimentary tract is characterised by the presence of a diverticulum of the stomach, a short intestine and the absence of a caecum (Richardson et al. 1986). There have been only three investigations of the gastrointestinal endocrine cells of marsupials using immunohistochemical techniques, Krause, Yamada & Cutts (1985, 1986) on the North American opossum Didelphis virginiana and Yamada, Krause, Kitamura & Yamashita (1987) on the koala Phascolarctos cinereus. Although the honey possum has unique feeding habits, the endocrine cells of its specialised gastrointestinal tract have not yet been investigated. This study, therefore, reports the results of an immunohistochemical examination of the gastrointestinal endocrine cells of honey possums using specific antisera for gastro-entero-pancreatic (GEP) hormones. MATERIALS AND METHODS The eight adult honey possums used in this study (weighing 7-15 g) were taken under licence during a study of the species in the Fitzgerald River National Park, Western Australia. Animals were killed with sodium pentobarbitone, their alimentary tracts removed immediately, Bouin's fixative injected into the lumen and the complete tracts immersed in the same fixative for about 12 hours. After fixation, six segments from the gastrointestinal tract were dissected out (Fig. 1), dehydrated through graded ethanol, cleared in xylene, embedded in paraffin wax and serially sectioned at 6 /,m. Representative deparaffinised and rehydrated sections were stained with haema- toxylin-eosin, periodic acid Schiff-alcian blue (PAS-AB) (pH 2-5) or Masson's trichrome for histological examination. Other sections were treated with methanol containing 0-3 % H202 for 10 minutes to block any endogenous peroxidase. The * Reprint requests to Dr R. D. Wooller. 158 J. YAMADA, K. C. RICHARDSON AND R. D. WOOLLER Table 1. Antisera used Antisera raiseda Code Specificity Dilution Serotoninb Lot 16302 1:10000 Synthetic human cyclic 1:3000 somatostatinc Synthetic human GP-1304 No cross-reaction with CCK-8 1:8000 gastrind Synthetic porcine R-1104 Reacts against entire molecule 1: 1000 motilind Porcine glucagone RPN 1602 Completely cross-reacts with pan- 1:1000 creatic and intestinal glucagons Synthetic porcine GL-5 Reacts with pancreatic glucagon 1:3000 glucagond Synthetic bovine R-3501 1:1000 neurotensind CCK (cholecystokinin)e No cross-reaction with gastrin 1: 1000 Synthetic porcine R-801 Reacts with the C- and N- 1:1000 secretind terminals BPP (bovine pancreatic 615-R-110- Cross-reacts with human pancre- 1:12000 polypeptidef 146-17 atic polypeptide GIP (gastric inhibitory) G/R/34-IIID No cross-reaction with glucagon 1: 10000 peptideg Insulinb 47291 1:1000 a All antisera were raised in rabbits except those against gastrin and insulin which were raised in guinea-pigs. b, e, These antisera were purchased from Immunonuclear Corp., Stillwater; Amersham International plc, Amersham; and Guildhay, Surrey, respectively. C,d,f These antisera were kindly donated by Dr S. Ito, Niigata; Professor N. Yanaihara, Shizuoka; and Dr R. E. Chance, Indianapolis, respectively. sections were then incubated in non-immunised goat or rabbit serum at room temperature for 30 minutes. Subsequently, they were stained immunohistochemically in a three layer procedure using the avidin-biotin-peroxidase complex (ABC) method (Hsu, Raine & Fanges, 1981) to identify specific endocrine cells. Details of the specific antisera used in this study are listed in Table 1. In the first layer, the sections were incubated with specific antisera for individual gastrointestinal hormones for 20 hours at 4 'C. Biotinylated anti-rabbit IgG serum raised in goat or biotinylated anti-guinea- pig IgG serum raised in rabbit was used as the second layer at 1:200 for 30 minutes at room temperature. The ABC was used as the third layer at 1: 200 for 30 minutes at room temperature. The immunoreactions were then visualised using a final solution of 3,3'-diaminobenzidine hydrochloride, according to the methods of Graham & Karnovsky (1966). The specificity of each immunohistochemical reaction was determined as recom- mended by Sternberger (1979), including the replacement of specific antiserum by the same antiserum which had been preincubated with its corresponding antigen. The relative frequency of occurrence of each type of immunoreactive cell was allocated to one of five categories according to their frequency as seen by light microscopy. The relative frequencies and distributions of immunoreactive cells in the gastrointestinal mucosa are summarised in Table 2. In this study, glucagon- immunoreactive cells were classified into two subtypes. Cells detected by one antiserum (GL-5, specific for pancreatic glucagon) were classified as pancreatic glucagon-immunoreactive cells and cells detected by another antiserum (RPN 1603, which completely cross-reacted with pancreatic- and entero-glucagon) were classified as enteroglucagon-immunoreactive cells. Gut endocrine cells of the honey possum 159 00 0 x0 u U +1 +1 00 00 +e + +I + I Io +I +1 00 00 00 fid + en + C,4+ IenI +1 +1 +1 CO + :1 .E O- + =1 00 00 .5 + --- I -I I I + 00 00 00 0. +-d' C) +1 1t+ ._ s) +1+1~~~+ I I C; 0) 00 x0 00 *0 +s,. + I-I CO 10 A40 C) 00 00 Cl I I 0) +l s Hk rA 0 >t I II I I II I I '0 +1 +1 rA CIO U * er C) 00 00 '0 CO. U 000 0 0 0 oz 160 J. YAMADA, K. C. RICHARDSON AND R. D. WOOLLER a f'~~~~~~~~ k~~~~ R / Fig.1.Schemati~~~~Crersntto ofa. atritstnltrc.ndsmpigpotos.fte.oe , anscl ine 1 cm Fig. 1. Schematic representation of a gastrointestinal tract and sampling portions of the honey possum. a, oeophagus; b, cardia, c, diverticulum; d, fundic gland region; e, pyloric gland region; f, proximal duodenum; g, distal duodenum; h, proximal jejunum; i, distal jejunum;j, ileum; k, colon; 1, anus. Scale line, I cm. RESULTS The stomach The stomcardiac gas wterised by the presence of a diverticulum which was connected to the main chamber of the stomach at its cardiac gland portion (Fig. 1). The cardiac glands were restricted to a narrow zone at the cardia and in the diverticulum (Figs. 1, 2). They were short and unbranched tubular glands which consisted mainly of mucus-secreting cells and a few acidophilic parietal cells were also present. The cardiac glands were particularly short or absent in the diverticulum (Fig. 3). Approximately three quarters of the main chamber of the stomach was lined with fundic glands (Fig. 1). These were slightly elongated tubular glands consisting mainly of acidophilic parietal cells (Fig. 4). The gastric pits were relatively deep and lined with mucus-secreting surface epithelium which was strongly PAS-positive. The mucous neck cells and chief cells were very difficult to identify in sections stained with haematoxylin-eosin. Although some small cells were identified in the neck region of the gland, they showed no special staining properties for the mucous neck cells. In sections stained with PAS-AB, the cells from the basal glandular regions were stained a magenta colour and possessed a foamy cytoplasm (Fig. 4). These cells were cuboidal, pyramidal or irregular in shape with their spherical nuclei located near the bases of the cells. The pyloric glands occupied the distal quarter of the main chamber of the stomach and consisted of mucus-secreting cells showing a PAS-positive, but not AB- positive, reaction. The structure and staining properties of the pyloric glands were basically similar to those of the cardiac glands. However, the pyloric glands were more developed than the cardiac glands. In the cardiac gland region, including the diverticulum, endocrine cells were very scarce and serotonin- and somatostatin-immunoreactive cells were detected in only Gut endocrine cells of the honey possum 161 9'6skI 4W~~~~~~4~~.07-ak> yr4- C,~~~%on 'C~~~~~~~. vi \j~~~~~a ,' U~~~~~~~1 Fig. 2. A cardiac region. A long arrow shows stratified squamous epithelium and short arrows show parietal cells. Fig. 3. A mucosa of diverticulum is lined with cardiac glands consisting of mucus-secreting cells. Fig. 4. Fundic glands consisting mainly of parietal cells. Some cells show a magenta colour at the glandular basis (arrowheads). Mucous neck cells are not identified. PAS-AB. x 225. %'¾, '4- .! 4.2 Vi 'r' tt 6 d * Fig. 5. Photomicrograph illustrating the distribution of serotonin-immunoreactive cells in the fundic glands. Figs. 6-8. Photomicrographs illustrating distribution of serotonin (Fig. 6), somatostatin (Fig. 7), and gastrin (Fig. 8) immunoreactive cells in the pyloric glands. These endocrine cells are located in neck portions of the glands. d, duodenum. ABC method. x 90. 162 J. YAMADA, K. C. RICHARDSON AND R. D. WOOLLER :&d4 v W > (t^. ';er io$% ~~~~~~~~~~~~,~~~~ ~ ~ 94~S~ a 9 *Jzeg 10o8;;)St^vwit11~~~~~~W 10) 07 Figs. 9-11. Photomicrographs showing general structure of the duodenum (Fig. 9), ileum (Fig.