An Immunohistochemical Study of Gastrointestinal Endocrine Cells in a Nectarivorous Marsupial, the Honey Possum (Tarsipes Rostratus)*

Home , Honey possum

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 haematoxylin-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

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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

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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

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four out of eight animals examined (Table 2). These endocrine cells were small, round or oval in shape, and made luminal contact via their apical cytoplasm. They were found in the glands or basal portions of the folds. In the fundic glands, moderate numbers of serotonin- and small numbers of somatostatin-immunoreactive cells were found in all animals examined (Table 2; Fig. 5). They were distributed throughout the glands and were either round or irregular in shape; their luminal contact was not clearly defined. In the pyloric glands, relatively numerous gastrin-immunoreactive cells and small numbers of serotonin- and somatostatin-immunoreactive cells were detected mainly in the neck portions of the glands (Table 2; Figs. 6-8). These were round or pyramidal in shape and established luminal contact by their apical cytoplasmic processes. The intestine The intestine of the honey possum is relatively short (a mean length of 11 9 cm) and simple, without a caecum (Richardson et al. 1986). Since there is no obvious landmark between small and large intestines, the latter was identified macroscopically by its mesenteric attachments and microscopically by the absence of intestinal villi. In the proximal small intestine (duodenum and proximal jejunum), the intestinal glands (crypts of Leiberkuhn) could not be clearly identified (Fig. 9) as reported in the South American marsupial, Marmosa robinsoni (Barnes, 1977). The striated border, which was PAS-positive, covered the surface of the epithelium to the bottom of the crypts. In the distal small intestine (distal jejunum and ileum), however, the intestinal glands were clearly identified (Fig. 10). The large intestine was characterised by the absence of intestinal villi and the presence of shallow folds and numerous goblet cells as in other mammals (Fig. 11). In the proximal small intestine, serotonin-, somatostatin-, gastrin-, motilin- and gastric inhibitory peptide (GIP)-immunoreactive cells were identified (Table 2; Figs. 12, 20). Moderate numbers of serotonin- but only small numbers of motilin- immunoreactive cells were found in all segments of the gastrointestinal tract of all Gut endocrine cells of the honey possum16 163

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animals. Three other types of endocrine cells were few in number and not found in all animals, except for gastrin-immunoreactive cells detected in the proximal jejunum. These endocrine cells were spindle-shaped in the villi and oval, pyramidal or spindle- shaped at the base of the villi. Their apical cytoplasmic processes reached the lumen.

Serotonin- and motilin-immunoreactive cells were distributed throughout the mucosal

epithelium, but the former were most prominent in the basal portion of the intestinal villi. Gastrin-immunoreactive cells were fewest in the proximal duodenum and increased in number distally along the small intestine. Gastrin-immunoreactive cells were mainly observed in the apical half of the intestinal villi, while somatostatin- and

GIP-immunoreactive cells were located mainly in the basal half. The duodenal glands

(Brunner's glands) of honey possums were poorly developed and were confined to a

narrow zone in the initial portion of the duodenum (Fig. 17). The glands were absent 164 J. YAMADA, K. C. RICHARDSON AND R. D. WOOLLER

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in one out of eight animals examined. In sagittal sections of the initial duodenum, the duodenal glands were found only on one side of the submucosa in three out of eight animals (Fig. 18). Serotonin- and somatostatin-immunoreactive cells were each identified in the duodenal glands of one out of eight animals (Table 2; Fig. 19). In the distal small intestine, enteroglucagon- and neurotensin-immunoreactive cells were identified, in addition to the endocrine cells in the proximal small intestine, but GIP-immunoreactive cells were absent from these segments (Table 2; Figs. 13-15, 21, 22). Small numbers of serotonin- gastrin- and enteroglucagon-immunoreactive cells were detected in all animals, while a few somatostatin-, motilin- and neurotensin- immunoreactive cells were found in all ilea. Neurotensin-immunoreactive cells were found in the apical halves of the villi, but enteroglucagon-immunoreactive cells were found mainly in the basal halves of the villi and in the intestinal glands. The cellular morphology and distribution of the other endocrine cells in the distal small intestine were similar to those in the proximal small intestine. Serotonin-, somatostatin-, enteroglucagon- and neurotensin-immunoreactive cells were observed in the large intestine (Table 2; Figs. 16, 23). Small numbers of serotonin- and enteroglucagon-immunoreactive cells were detected in all animals, but somatostatin- and neurotensin-immunoreactive cells were found in only three out of eight animals and two out of eight animals, respectively. These endocrine cells were located exclusively in the intestinal glands and were oval, pyramidal or spindle- shaped. Some serotonin-immunoreactive cells were also found in the stratified squamous epithelium of the transitional zone between the rectum and the anus. Secretin-, pancreatic glucagon-, CCK- and BPP-immunoreactive cells were not detected at any site along the gastrointestinal tract of the honey possum. Gut endocrine cells of the honey possum 165

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DISCUSSION Krause et at. (1985) detailed the quantitative distribution of gastrointestinal endocrine cells over the whole length of the gastrointestinal tract of the North American opossum. They reported 10 types of gastrointestinal endocrine cells, immunoreactive for serotonin (5-hydroxytryptamine, 5-HT), somatostatin, gastrin, motilin, pancreatic glucagon, neurotensin, gastric inhibitory peptide (GIP), cholecystokinin (CCK), secretin and bovine pancreatic polypeptide (BPP). They found that the numbers and distribution of gastrointestinal endocrine cells in the opposum was basically similar to that reported for most eutherians (Kitamura, Yamada, Yamashita & Yanaihara, 1982; Kawano, Yamashita, Yamada & Kitamura, 1983; Calingasan et at. 1984; Kitamura, Yamada. Calingasan & Yamashita, 1984, 1985; Yamada et at. 1984; Ohara, Kitamura, Yamada & Yamashita, 1986; Itoh et at. 1987), except that neurotensin-immunoreactive cells (Helmstaedter, Taugner, Feurle & Forssmann, 1977; Sundler et at. 1977) were distributed not only in the distal small intestine but also in the colon of the opossum (Krause et at. 1985). This finding was made in the opossum using antisera for eutherian gastro-entero-pancreatic (GEP) hormones, although there are some structural differences between the neurohypophysial hormones of metatherians and eutherians (Chauvet, Hurpet, Chauvet & Acher, 1984). Comparing the cell types identified in the North American opossum (Krause et at. 1985) and the honey possum, CCK-, secretin-, pancreatic glucagon- and BPP- immunoreactive cells were not identified in the honey possum yet were present in the opossum, while enteroglucagon-immunoreactive cells were not tested for in the opossum. The presence of enteroglucagon-immunoreactive cells in the opossum was expected, since all mammals so far examined possess this cell type. The distribution of each endocrine cell type detected in both marsupials was similar, but their frequency of occurrence was lower in the honey possum than in the opossum. In particular, the somatostatin- and gastrin-immunoreactive cells of the honey possum were not as numerous as reported for eutherians (Tobe, Chen, Henmi & Fukuchi, 1976; Alumets, Sundler & Hakanson, 1977; Lehy, Gress & Fereira De Castro, 1979; Kitamura et at. 166 J. YAMADA, K. C. RICHARDSON AND R. D. WOOLLER 1982; Kawano et al. 1983; Calingasan et al. 1984; Kitamura et al. 1984, 1985; Yamada et al. 1984; Ohara et al. 1986; Itoh et al. 1987). The lower number of the types and frequency of gastrointestinal endocrine cells in the honey possum might have a phylogenetic or a functional basis. The opossum (Didelphis) has a generalised morphology which is thought to resemble the ancestral proto-mammalian condition (Lee & Cockburn, 1984), whereas the honey possum is a highly specialised marsupial whose origins are still unclear (Renfree et al. 1984). The honey possum is one of very few mammalian terrestrial nectarivores. The negative immunohistochemical results for CCK, secretin, pancreatic glucagon and BPP in the gastrointestinal endocrine cells of the honey possum could therefore be explained by structural differences in these hormones between the North American opossum and the Australian honey possum, because some differences in neurohypophysial peptides between American and Australian marsupials have been reported (Chauvet et al. 1984). Alternatively, however, the absence of some, and decrease in frequency of other, gastrointestinal endocrine cells in the honey possum may be the result of loss during adaptation to its highly unusual diet. Ingestion solely of pollen and nectar may have resulted in a reduced need for complex motility patterns and secretory control of its gastrointestinal tract, and an overall reduction in digestive capacity. Comparative investigations of other marsupials with different feeding habits might resolve this point. Interestingly, a similar reduction in type and frequency of gastrointestinal endocrine cells has also been recorded in the New Holland honeyeater Phylidonyris novaehollandiae, a nectarivorous bird from Southern Australia (Richardson, Yamada & Wooller, submitted for publication). It is likely that their similar feeding habit, nectarivory, accounts for these similar findings in the honey possum and the honeyeater. In conclusion, the pattern of gastrointestinal endocrine cells found in the honey possum may reflect, in part, its nectarivorous diet. However, further comparative morphological and functional studies of nectarivorous birds, especially honeyeaters, as well as nectarivorous bats, should be undertaken to clarify the functional and phylogenetic significance of these findings in the gastrointestinal endocrine cells of the honey possum.

SUMMARY The distribution and relative frequency of occurrence of gastrointestinal endocrine cells exhibiting immunoreactivity to eleven peptides and one amine were examined immunohistochemically in the gastrointestinal mucosa of the adult honey possum which feeds almost exclusively on nectar and pollen. Seven types of endocrine cells, immunoreactive for serotonin, somatostatin, gastrin, motilin, enteroglucagon, neurotensin and gastric inhibitory peptide (GIP), were detected in the gastrointestinal mucosa of the honey possum. In the cardiac gland region, including the diverticulum, endocrine cells were very scarce, and few serotonin- and somatostatin-immunoreactive cells were identified in 4 out of 8 honey possums. Moderate numbers of serotonin- and a few somatostatin-immunoreactive cells were found in the fundic glands. In the pyloric glands, moderate numbers of gastrin-immunoreactive cells and a few serotonin- and somatostatin-immunoreactive cells were detected. Along the length of the small intestine, serotonin-immunoreactive cells were the dominant endocrine cell type but small numbers of somatostatin-, gastrin-, motilin-, enteroglucagon-, neurotensin- and GIP-immunoreactive cells, all with different distribution patterns and relative frequencies of occurrences, were also found. In the large intestine, a small Gut endocrine cells of the honey possum 167 number of serotonin- and enteroglucagon-immunoreactive cells were found in all animals, but a few somatostatin- and neurotensin-immunoreactive cells were detected in some animals. The Western Australian Department of Conservation and Land Management kindly allowed us to collect the animals used in this study, which was supported by the Ministry of Education, Science and Culture, Japan and the Murdoch University Special Research Grant. We thank Ms E. Watkins and Mr G. Griffiths for their excellent histological and photographic assistance.

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