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Acta histochemica 110 (2008) 134—142

0065-1281/$ - sdoi:10.1016/j.

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A comparative immunohistochemical studyof endocrine cells in the digestive tractof two frugivorous bats: Artibeus cineriusand Sturnira lilium

Clarice Machado Dos Santosa,�, Aparecida Alves Do Nascimentob, AdrianoLucio Peracchic, Daniela Diasa, Thatiana Paz Ribeiroa, Armando Salesb

aPostgraduate Program in Animal Biology, UFRRJ, Seropedica, RJ, BrazilbAnimal Biology Department, Histology and Embryology Area, UFRRJ, Seropedica, RJ, BrazilcLaboratory of Mastozoology, UFRRJ, Seropedica, RJ, Brazil

Received 30 July 2007; received in revised form 14 September 2007; accepted 1 October 2007

KEYWORDSEndocrine cells;Immunohisto-chemistry;Gastrointestinaltract;Artibeus cinerius;Sturnira lilium

ee front matter & 2007acthis.2007.10.002

ing author. Laboratoriod. BR 465, Km 07, CEP 2ess: claricemachado@y

SummaryThe purpose of the present study was to examine the serotonin (5-hydroxytrypta-mine, 5-HT), gastrin (GAS), cholecystokinin (CCK) and glucagon (GLUC) endocrinecells in the gastrointestinal tract of frugivorous Phillostomidae bats, Sturnira liliumand Artibeus cinerius, to clarify the correlation between distribution of cell typesand their relative frequency, with feeding habits. Five portions of the gastro-intestinal tract – fundus, pilorus, and three parts of the intestine, I, II and III – wereexamined. Most of the immunoreactive cells in the stomach and intestine were oftriangular, oval or piriform shape. Serotonin-immunoreactive cells were mostcommonly found in the S. lilium intestine I (66.679.9) and the A. cinerius intestineIII (35718). Gastrin-immunoreactive cells were the most abundant cell type in thepyloric glands of both species. They were more numerous in A. cinerius(126.9727.4) than in S. lilium (75.871.8). CCK-immunoreactive cells were foundin the alimentary tract epithelia at moderate frequencies in both species. GLUC-immunoreactive cells were detected at very low or low frequencies. This studysuggests that there is a correlation between endocrine cell distribution andfrequency, and the feeding habits of the bats.& 2007 Elsevier GmbH. All rights reserved.

Elsevier GmbH. All rights reserved.

de Histologia e Embriologia, Instituto de Veterinaria, sala 85, Universidade Federal Rural do Rio3.890-000, Seropedica, Brazil.ahoo.com.br (C.M. Dos Santos).

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Figure 1. Schematic drawing of the digestive tractillustrating the regions sampled from A. cinereus andS. lilium (modified from Komori et al., 2000). Stomach,fundic region, 2. Stomach, pyloric region, 3. IntestineI – duodenum, 4. Intestine II, jejunum/ileum, 5. IntestineIII – large intestine and rectum.

Comparative immunohistochemical study of endocrine cells in the digestive tract 135

Introduction

Gastrointestinal (GI) endocrine cells are dis-persed along the epithelia and glands of thegastrointestinal tube (GIT). They produce varioustypes of hormones and play important roles in thephysiological functions of the GIT (Bell, 1978).

Secretin, gastrin (GAS) and cholecystokinin (CCK)were the first gut hormones to be described.Currently, more than 30 genes coding for GIhormones are recognized, along with a multitudeof bioactive peptides, which make the gut thelargest endocrine organ in the body (Ahlman andNilsson, 2001). In recent years, the field of GIhormones has expanded rapidly and has become animportant domain in gastroenterology (Huang andWu, 2005). Some GI dysfunctions are related to GIhormones (Milutinovic et al., 2003). The investiga-tion of endocrine cells is considered an importantpart of GI hormone studies and can provide baselinedata for basic research into gastroenterology(Huang and Wu, 2005).

The Chiroptera order is surpassed in number ofspecies only by rodents. It comprises 17 families,177 genera and nearly 930 species (Wilson andReeder, 1993). The Chiroptera GIT is a veryattractive GI model, not only for understandingthe digestive tube’s evolution in general, but alsofor investigation of the relationship between thedistribution and frequency of gut endocrinecells and animal feeding habits. This is possiblebecause bats have very varied diets. Carnivorous,insectivorous, frugivorous, nectarivorous and san-guivorous species can be compared (Yamada et al.,1988).

More than 15 different endocrine cell types havebeen described in bats’ digestive tracts (Yamadaet al., 1984, 1988; Ashihara et al., 1999; Komoriet al., 2000; Santos et al., in press). These studieshave revealed some inter-species differences in thefrequency, cells types and regional distributions ofdifferent endocrine cells in the GIT of bats. Theyhighlight the need to pay much more attention tothe classificatory position of specific bat speciesand to compare the possible effects of feedinghabits on the distribution and frequency of gutendocrine cells.

The purpose of the present study was toexamine, for the first time, the serotonin,GAS, CCK and glucagon (GLUC) endocrinecells in the GIT of frugivorous Phillostomidae bats,Sturnira lilium and Artibeus cinerius, using im-munohistochemistry, to elucidate the correlationbetween the distribution of these cells, theirrelative frequency and the feeding habits of thebats.

Materials and methods

Six animals were studied; three A. cinerius (twomales and one female) and three S. lilium (twomales and one female). The animals were collectedaccording to Brazilian laws (license 042/2005-Process number 02001, 007915/01/72 /IBAMA). Thebats were caught at night with mist nets and handnets in the Tingua Reserve in the municipality ofNova Iguac-u, Rio de Janeiro State, Brazil. The batswere sacrificed with sodium thiopentone at a dose of100mg/kg. The GIT were removed and fixed withBouin’s fluid for 6 h. After fixation, the tubes wereseparated into the five regions as shown in Figure 1.The tissues were dehydrated through a graded seriesof ethanol solutions and embedded in Paraplastusing routine protocols. Five-micrometer thick sec-tions were cut by microtome and mounted on glassslides precoated with 0.1% poly-L-lysine (SigmaChemical Co., Saint Quentin Fallavier, France).

Primary antisera

The primary antisera were used for both thespecificity controls and immunolocalization of cells

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C.M. Dos Santos et al.136

immunoreactive to regulatory peptides and bio-genic amine. They were: rabbit polyclonal anti-serotonin (5-hydroxytryptamine, 5-HT) (S 5545-Sigma-Aldrich, Inc.), rabbit polyclonal anti-GAS(G 0785-Sigma-Aldrich, Inc.), rabbit polyclonalanti-colecystokinin (CCK) (C 2581-Sigma-Aldrich,Inc.) and mouse monoclonal anti-GLUC (G 2654-Sigma-Aldrich, Inc.). We refer to the endocrinecells immunoreactive to GLUC antiserum with codeG 2654 as enteroglucagon-immunoractive cellssince the antiserum showed a cross-reaction withpancreatic GLUC and enteroglucagon.

Single antigen immunohistochemistry

The sections were dewaxed and rehydrated byroutine protocols. They were incubated withmethanol containing 3% H2O2 for 15min to blockany endogenous peroxidase. The sections werethen incubated with a 1:100 dilution of bovineserum albumin (B4287; Sigma) in phosphate buf-fered saline (PBS), for 30min. Subsequently, theywere labeled immunohistochemically using a threelayered avidin–biotin–peroxidase complex (ABC)method (Hsu et al., 1981) to identify specificendocrine cells. The sections were first incubatedovernight at 4 1C with the primary antisera againstindividual GI hormones, at the following dilutions:1:8,000 for serotonin (5-HT); 1:1,000 for GAS;1:8,000 for CCK and 1:2,000 for GLUC. Sectionswere then incubated with biotinylated anti-horseserum, diluted 1:200, for 30min, then withavidin–biotin complex (ABC), diluted 1:200, for30min (both from PK 6200, Vector Lab. Inc.).Subsequently, the peroxidase label was revealed byreaction with 3, 30-diaminobenzidine tetrahy-drochloride (DAB) (Dakocytomation 003222) pre-pared according to kit instructions. All steps wereperformed at room temperature unless otherwisespecified. All dilutions and thorough washes be-tween stages were performed in PBS unless other-wise specified. The slides were finally rinsedseveral times with deionized water, dehydratedthrough a series of ethanol solutions and methyl-cyclohexanes, and mounted using Entelan (Merck).

Controls

In the present study, the immunohistochemicallocalization of regulatory peptides and biogenicamine (serotonin/5-HT) in the endocrine cells wasinvestigated by use of polyclonal antiserum. Themethod control was demonstrated by the usualspecificity tests, that included: (1) omission of theprimary antiserum, (2) replacement of the primary

antiserum with non-immune serum, (3) dilutionprofiling of the primary antiserum using doublingdilutions on serial sections, (4) assessment of theinfluence of the salt content (up to 0.5M) of thebuffer, and (5) complement-deprived antisera(Heyderman, 1979; Van Noorden, 1986; Burry,2000).

Observation, photomicrographyand cell count

Three samples from each of the six bats bat wereobserved using an Olympus Dx-41 photomicroscopeand representative images captured. The relativefrequency of immunoreactive cells in each regionwas calculated as the number of immunoreactivecells per unit area (0.25mm2) of tissues using acomputerized image analyzer (Image Pro-Plus soft-ware). The frequency of occurrence of immuno-reactive cells is expressed as mean7SD (standarddeviation) per unit area.

Results

Serotonin/5-HT-, GAS- and GLUC-immunoreac-tive cells were identified in the stomach, andserotonin/5-HT-, CCK- GLUC-immunoreactive cellswere identified in the intestine of A. cinerius andS. lilium. The regional distribution and frequencyof the different types of endocrine cells variedaccording to their location in the GIT. Thesedifferences are shown in Table 1. No positivelabeling was seen in any of the negative controlsections.

Serotonin/5-HT-immunoreactivity

Serotonin/5-HT-immunoreactive cells, detectedthroughout the whole GIT of the two bat species,were the most commonly seen GI endocrine cells.They were found primarily in the basal half of thefundic and pyloric glands of A. cinerius, illustratedin Figure 2A. These serotonin/5-HT-immunoreac-tive cells were triangular or oval in shape, and werealso detected in the submucosa among the compo-nents of the connective tissue, seen in Figure 2B. InS. lilium, the serotonin/5-HT-immunoreactive cellsin the stomach were distributed mainly in themedial and basal portion of the fundic glands, seenin Figure 2C, and were frequently oval andtriangular in shape, as illustrated in Figure 2D. Inthe pylorus region, they were concentrated mainlyin the apical to medial region of the gland, seen inFigure 2E. Their relative frequency varied along the

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Table 1. Distribution and relative frequency of endocrine cells of A. cinerius* and S. lilium (cells/mm2, mean7SD)

Region Serotonin Gastrin CCK Enteroglucagon

Stomach-fundus 20.571.7*/12.273.4 0/0 0/0 0.9170.3/�Stomach-pylorus 16.675/14.672.4 126.9727/75.875.7 0/0 0/0Intestine I 19.677.5/66.679.9 0/0 7.271.8/24.176.1 0/5.072Intestine II 18.676.3/31.979 0/0 8.271.9/22.878.5 1.771/3.771Intestine III 35715/23.970.6 0/0 14.275.8/22.475.5 3.771/ 2.070.5

Comparative immunohistochemical study of endocrine cells in the digestive tract 137

intestine. These cells were at their highest densityin the intestine III of A. cinerius (35718 cells/0.25mm2) and intestine I of S. lilium (66.679.9/0.25mm2). They were mostly found in the intest-inal epithelium and intestinal glands. These endo-crine cells were piriform in shape and frequentlyhad apical cytoplasmic process directed towardsthe glandular lumen, and were therefore classifiedas an open type (Figure 2F). In S. lilium sparseserotonin/5-HT-immunoreactive cells were de-tected in the Brunner’s glands, located in thesubmucosa of the pyloro-duodenal junction.

Gastrin immunoreactivity

GAS-immunoreactive cells were most abundantin the pyloric glands in both species of bats. In S.lilium, their distribution was irregular, and theywere clustered in groups located in the medialregions of the pyloric gland, seen in Figures 3Aand B. In contrast, the distribution of GAS-immunoreactive cells in A. cinerius was moreuniform, seen in Figure 3C. These endocrinecells were frequently oval in shape, illustrated inFigure 3D. They were distributed in the pyloricgland from the middle to apical portions, and theywere more numerous in A. cinerius (126.9727.4/0.25mm2) than in S. lilium (75.871.8/0.25mm2).They were also identified in the duodenal gland.

CCK immunoreactivity

CCK-immunoreactive cells were detected in bothspecies of bat, distributed throughout the entireintestine. Their frequency was comparable in bothspecies, and they were most abundant in theintestine III (14.275.8/0.25mm2 in A. cineriusand 22.876.5/0.25mm2 in S. lilium). Theywere detected in the intestinal glands and theepithelial components both species. Immunoreac-tive cells varied in shape from oval to piriform, asseen in Figures 4A and B. CCK-immunoreactiveendocrine cells were not detected in the duodenalgland.

Glucagon immunoreactivity

Oval-shaped GLUC-immunoreactive cells weredetected in the medial to basal glands of thefundic regions (0.9170.3) of A. cinerius, illustratedin Figure 5A. Their occurrence was low in theintestine and varied according to the species. InA. cinerius, their distribution was restricted tointestines II and III, and cells were most often ovalin shape, seen in Figure 5B, while in S. liliumGLUC-immunoreactive were detected in all threeintestinal regions.

Discussion

Recent advances in immunohistochemistry havemade it possible to demonstrate the presence of anumber of different types of endocrine cells in theGI tract of mammals. More than 15 different kindsof endocrine cells have been described in chirop-teran GI tracts. Nine were described in thesanguivorous Desmodus rotundus (Yamada et al.,1984), eleven in the insectivorous Pipistrellusabramusi and Plecotus auritus (Yamada et al.,1988), four in Molossus molossus and Lonchorhinaaurita (Santos et al., in press), eight in thenectarivorous and frugi-nectarivorous, Anoura cau-difer and Carollia perspicillata (Ashihara et al.,1999), ten in the piscivorous Noctilio leporinus(Komori et al., 2000) and four in the pancreasof the frugivorous bat Rousettus aegytiacus(Michelmore et al., 1998). These studies showedinter-species differences and have suggested acorrelation between endocrine cell distributionand feeding habits.

The present study mapped four types of endo-crine cells in the GI tract of the frugivorous batsA. cinerius and S. lilium: endocrine cells immunor-eactive for serotonin, GAS, CCK and GLUC. Thegeneral pattern of distribution of the endocrinecells in the GI tract of A. cinerius and S. lilium wassimilar to the findings reported in other mammalianspecies. However, when compared with other bats,the specific distribution of cells immunoreactive toGAS and CCK was wider, whereas the distribution of

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Figure 2. Serotonin/5-HT-immunoreactive cells. (A) Fundic glands of A. cinerius, serotonin/5-HT-immunoreactive cellsin the basal half of the glands (arrows). Bar ¼ 50 mm. (B) Pyloric gland of A. cinerius with serotonin/5-HT-immunoreactive cells in connective tissue in the submucosa (arrows). Bar ¼ 50 mm. (C) Fundic glands of S. lilium; withserotonin/5-HT-immunoreactive cells distributed in the medial to basal region of the gland (arrows). Bar ¼ 25 mm.(D) Fundic gland of S. lilium showing serotonin/5-HT-immunoreactive cells with oval (arrowhead) and triangular (arrow)cell shape. Bar ¼ 50 mm. (E) Pyloric gland of S. lilium with serotonin/5-HT-immunoreactive cells in the apical andmedial portion of the gland (arrows). Bar ¼ 50 mm. (F) Intestine I of A. cinerius with serotonin/5-HT-immunoreactivecells in the intestinal epithelium and intestinal glands (arrows). Bar ¼ 50 mm.

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serotonin/5-HT- and enteroglucagon-immunoreac-tive cells was more restricted. It was interesting tolocalize the presence of CCK in all regions of theintestine of A. cinerius and S. lilium.

Serotonin (5-HT), a regulatory amine of mucosalenterochromaffin (EC) cells, plays an importantrole in the control of GI smooth muscle contractionand epithelial secretion (Fink et al., 2006). EC cells

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Figure 3. Gastrin-immunoreactive cells. (A) and (B) Pyloric gland of S. lilium. Note the distribution of gastrin-immunoreactive cells is irregular in the medial regions of the gland. Bar ¼ 50 mm. (C) Pyloric gland of A. cinerius. Notethe uniform distribution gastrin-immunoreactive cells. Bar ¼ 50 mm. (D) Oval-shaped gastrin-immunoreactive cells inpyloric gland of A. cinerius (arrows). Bar ¼ 50 mm.

Comparative immunohistochemical study of endocrine cells in the digestive tract 139

constitute the largest endocrine cell population inthe GI tract and produce over 90% of all theserotonin/5-HT synthesized in the body (Ahlmanand Nilsson, 2001). Moderate numbers of seroto-nin/5-HT-immunoreactive cells were evenly dis-tributed in all regions of the stomach and intestine,and showed similar distribution and frequency inboth species of bats. This observation furtherdemonstrates that serotonin/5-HT-IR cells have awider distribution than other types of GI endocrinecells in the GITof vertebrates. In S. lilium, as in thePhyllostomid bats A. caudifer and C. perspicillata,these cells were less abundant in the fundic glands.The abundance of serotonin/5-HT immunoreactiveopen type cells indicates that these cells probablysecrete serotonin/5-HT by the paracrine pathway(Ashihara et al., 1999). These cells were alsofound in the duodenal gland of S. lilium, as hasbeen reported in the insectivorous P. abramus(Yamada et al., 1988), M. molossus (Santos et al.,in press), nectarivorous A. caudifer, frugi-nectar-ivorous C. perspicillata (Ashihara et al., 1999) and

sanguivorous D. rotundus (Yamada et al., 1984),but these cells have not been identified inpiscivorous N. leporinus (Komori et al., 2000), orin the frugivorous A. cinerius (Yamada et al., 1988).

GAS is a linear peptide hormone produced by Gcells of the duodenum and in the pyloric antrum ofthe stomach (Tzaneva, 2003). GAS stimulates ECcells to release histamine. The increased histamineand the direct stimulation by GAS cause parietalcells to increase hydrochloric acid secretion in thestomach (Larsson, 2000). Previous investigationshave revealed that in bats, GAS production mightplay some other roles within the pylorus itself(Mennone et al., 1986). The GAS-immunoreactivecells in this study were restricted in distribution tothe middle to apical portions of the pyloric glandsin both species of bat. The distribution pattern ofthe GAS-immunoreactive endocrine cells in thepyloric glands may facilitate a quick response tothe luminal ingesta (Nisa et al., 2005). A remark-able difference in the distribution of GAS-immu-noreactive cells is seen between bat species with

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Figure 4. Cholecystokinin (CCK)-immunoreactive cells. (A) Intestine I of A. cinerius. Note that CCK-immunoreactivecells are dispersed in the interepithelial region and intestinal glands (arrows). Bar ¼ 50 mm. (B) Intestine II of S. lilium,with CCK-immunoreactive cells in the intestinal glands (arrows). Bar ¼ 50 mm.

Figure 5. Enteroglucagon-immunoreactive cells. (A) Fundic region of A. cinerius with enteroglucagon-immunoreactivecells distributed in medial portions of the gland (arrows). Bar ¼ 50 mm. (B) Intestine III of A. cinerius, withenteroglucagon-immunoreactive oval cells in the intestinal gland (arrows). Bar ¼ 50 mm.

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different feeding habits. In this study, the highestdensity of GAS-immunoreactive cells in the pylorusmay be related to the frugivorous feeding habits ofS. lilium and A. cinerius. Mennone et al. (1986)suggested that the GAS endocrine cells playing animportant role in regulating the digestive functionin frugivorous bats. These cells have been de-scribed in small numbers in the intestine ofinsectivorous (Yamada et al., 1988), piscivorous(Komori et al., 2000), frugi-nectarivorous andnectarivorous (Ashihara et al., 1999) bats. Theywere absent in the intestine samples examined inthe present study.

CCK plays a key role in facilitating digestionwithin the small intestine. It is secreted frommucosal epithelial cells in the first segment of thesmall intestine (duodenum) and stimulates releaseof digestive enzymes from the pancreas and bile

from the gallbladder into the small intestine. It isreported that CCK-like peptides are widely dis-tributed among the lower vertebrates and thesefamilies of peptides were established early inanimal evolution (Crim and Vigna, 1983). TheseCCK-immunoreactive cells were observed scatteredalong the entire length of the intestine of frugivor-ous bats studied here. A similar distribution hasbeen reported in vampire (Yamada et al, 1984) andinsectivorous bats (Yamada et al, 1988, Santos etal., in press). However, CCK-immunoreactive cellshave not been detected in piscivorous (Komoriet al., 2000), nectarivorous and frugi-nectarivorousbats (Ashihara et al., 1999). Komori et al (2000)suggested that these results reflect differences infeeding habits or may reflect a real absence.However, S. lilium, A. cinerius, A. caudifer,C. perspicillata and L. aurita belong to the same

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Comparative immunohistochemical study of endocrine cells in the digestive tract 141

family of Phyllostomoidea and occasionally havesimilar feeding resources.

The GLUC gene is expressed along the GIT byhighly specialized gut endocrine cells, designated Lcells. The majority of L cells are classically thoughtto be located in the distal gut, predominantly in theileum and colon. Their most reported action is toincrease glycemia, counteracting the effects ofinsulin. GLUC-immunoreactive cells have beendemonstrated in various mammals, and it isconsidered that the distribution pattern of thesecells in the GIT of mammals show species-depen-dent variation (Ku et al., 2003). In the presentstudy, the distribution of enteroglucagon-immunor-eactive cells was similar to that of frugi-nectar-ivorous bats reported by Ashihara et al. (1999). Therelative frequency of enteroglucagon-immunoreac-tive cells was moderate, similar to the findingsreported in nectarivorous, frugi-nectarivorous(Ashihara et al., 1999) and insectivorous bats(Yamada et al, 1988).

In the study reported here, species-specificdifferences in the distribution of endocrine cellswere observed along the gut of bats with similarfeeding habits. However, the differences betweenthese two species were less pronounced than thosereported for bats with distinct feeding habits.Therefore, these data suggest a correlation bet-ween the distribution and frequency of endocrinecells and the feeding habits of these animals.

Acknowledgments

We thank Ilza Lucas Coelho Meirelles for hercollaboration in technical assistance and the Officefor Improvement of University Personnel (Coorde-nac-ao de Aperfeic-oamento de Pessoal de NıvelSuperior – CAPES) for financial support.

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