This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution

and sharing with colleagues.

Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party

websites are prohibited.

In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information

regarding Elsevier’s archiving and manuscript policies areencouraged to visit:

http://www.elsevier.com/authorsrights

Author's personal copy

Tissue and Cell 46 (2014) 70– 77

Contents lists available at ScienceDirect

Tissue and Cell

j our nal homep a ge: www.elsev ier .com/ locate / t i ce

Quantification of endocrine cells and ultrastructural study of insulingranules in the large intestine of opossum Didelphis aurita(Wied-Neuwied, 1826)

Daiane Cristina Marques dos Santosa,∗, Marli do Carmo Cupertinoa,Maria do Carmo Queiroz Fialhoa, Alfredo Jose Afonso Barbosab,Cláudio Cesar Fonsecac, Sirlene Souza Rodrigues Sartorid,Sérgio Luis Pinto da Mattaa

a Department of General Biology, Federal University of Vic osa (UFV), Avenida Peter Henry Rolfs, s/n. Campus Universitário, Vic osa 36570-000, MG, Brazilb Laboratory of Digestive and Neuroendocrine Pathology, Federal University of Minas Gerais (UFMG), Belo Horizonte, MG, Brazilc Department of Veterinary Medicine, Federal University of Vic osa (UFV), Vic osa, MG, Brazild Department of Animal Biology, Federal University of Vic osa (UFV), Vic osa, MG, Brazil

a r t i c l e i n f o

Article history:Received 6 August 2013Received in revised form14 November 2013Accepted 14 November 2013Available online 20 November 2013

Keywords:Insulin-immunoreactive endocrine cellsArgyrophil endocrine cellsArgentaffin endocrine cellsHistochemistryImmunohistochemistryTransmission electron microscopy

a b s t r a c t

This study aimed to investigate the distribution of argyrophil, argentaffin, and insulin-immunoreactiveendocrine cells in the large intestine of opossums (Didelphis aurita) and to describe the ultrastructure ofthe secretory granules of insulin-immunoreactive endocrine cells. Fragments of the large intestine of 10male specimens of D. aurita were collected, processed, and subjected to staining, immunohistochemistry,and transmission electron microscopy. The argyrophil, the argentaffin, and the insulin-immunoreactiveendocrine cells were sparsely distributed in the intestinal glands of the mucous layer, among other celltypes of the epithelium in all regions studied. Proportionally, the argyrophil, the argentaffin, and theinsulin-immunoreactive endocrine cells represented 62.75%, 36.26%, and 0.99% of the total determinedendocrine cells of the large intestine, respectively. Quantitatively, there was no difference between theargyrophil and the argentaffin endocrine cells, whereas insulin-immunoreactive endocrine cells wereless numerous. The insulin-immunoreactive endocrine cells were elongated or pyramidal, with roundednuclei of irregularly contoured, and large amounts of secretory granules distributed throughout the cyto-plasm. The granules have different sizes and electron densities and are classified as immature and mature,with the mature granules in predominant form in the overall granular population. In general, the granuleis shown with an external electron-lucent halo and electron-dense core. The ultrastructure pattern in thegranules of the insulin-immunoreactive endocrine cells was similar to that of the B cells of pancreaticislets in rats.

© 2013 Elsevier Ltd. All rights reserved.

1. Introduction

The digestive system has a crucial role in controlling energyhomeostasis through its action on the digestion and absorption ofingested nutrients. This regulatory mechanism is developed in partby endocrine cells (Drucker, 2007).

These cells are distributed in the stomach and intestines of ver-tebrates, 1% of which are present in the intestinal epithelia andrepresent the largest population of hormone-producing cells in the

∗ Corresponding author. Tel.: +55 31 3899 2515/+55 31 3899 3361;fax: +55 31 3899 2549.

E-mail addresses: daiane.santos@ufv.br, biomarques@vicosa.ufv.br(D.C.M.d. Santos).

body (Rehfeld, 1998; Sternini et al., 2008). They control the secre-tion, absorption, motility, and proliferation of epithelial cells (Rindiet al., 2004; Drucker, 2007).

In addition, these cells have essential roles in the regulationof food intake, in the homeostasis of glucose through its actionson peripheral target organs (Drucker, 2007), in immunity, and inthe maintenance of the integrity of the intestinal mucosal barrier(Moran et al., 2008; Zhang et al., 2012). In pathological condi-tions, they are also related to severe gastrointestinal diseases,such as postinfections, irritable bowel syndrome, enteric infections,inflammatory bowel diseases (Moran et al., 2008), and neoplasms(Polak et al., 1993).

The endocrine cells can be classified according to their mor-phology (Fujita and Kobayashi, 1977; Dayal et al., 1987; Sjölundet al., 1983), ability to absorb certain salts (Grimelius and Wilander,

0040-8166/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.tice.2013.11.004

Author's personal copy

D.C.M.d. Santos et al. / Tissue and Cell 46 (2014) 70– 77 71

1980), the morphology and content of their secretory granules(Polak et al., 1993), and the presence of specific marker molecules(Rindi et al., 2004; Schönhoff et al., 2004). According to morphol-ogy, endocrine cells are considered to be open type (there is acytoplasmic prolongation that reaches the luminal surface) andclosed type (there is no communication with the lumen) (Fujitaand Kobayashi, 1977). According to the capacity to absorb silversalts, endocrine cells are considered argentaffin (when the silversalts are directly absorbed and reduced) or argyrophil (when theabsorbed salts are reduced by the action of an exogenous substancereductive) (Grimelius and Wilander, 1980).

Insulin immunoreactivity has been located in extrapancreaticregions, such as the prostate (Stahler et al., 1988), the nephron(Coutinho et al., 1985), the central nervous system (Devaskar et al.,2002), the retina (Meimaridis et al., 2003), the submandibulargland (Égéa et al., 2000), and the intestine (Coutinho et al., 1984;Bendayan and Park, 1991; Kendzierski et al., 2000; Freitas-Ribeiroet al., 2011, 2012; Basile et al., 2012; Santos et al., 2013).

Fonseca et al. (1998) observed the existence of glucagon-immunoreactive cells in the gastric mucosa of Didelphis albiventriswith ultrastructural characteristics similar to the glucagon-producing cells of the pancreatic islets. However, there areno studies in the literature related to insulin-immunoreactiveendocrine cells in the large intestine of marsupials, which high-lights the necessity and importance of this research.

The didelphids have gained prominence as a model in ontoge-nesis research because of the simultaneous differentiation of thedigestive system when the animal is still in the intramarsupialperiod (Krause et al., 1989; Fonseca et al., 2002b). Furthermore,the marsupials (Didelphis) show special features that put them inevidence for research, among which are the following: the origin, inwhich they share common ancestry with the eutherian mammals,considered as morphologically primitive mammals (MacAlester,1994); the short gestation period (Samoto et al., 2006); and thefacility of access to embryos, wherein embryonic development iscompleted outside the uterus.

Opossums (Didelphis aurita) are nocturnal, scansorial, and veryagile (Loretto and Vieira, 2005; Cunha and Vieira, 2005). Theyare found in the eastern part of Brazil, in Alagoas, in the northof Rio Grande do Sul, in the west of Mato Grosso do Sul, inthe southeastern part of Paraguay, and in the Misiones Province,Argentina (Cerqueira and Lemos, 2000; Cerqueira and Tribe,2008).

Immunohistochemical studies are focused on endocrine cellsin the pancreas, stomach, and small intestine of the marsupials(Krause et al., 1985, 1989; Barbosa et al., 1987, 2006; Takagi et al.,1990; Fonseca et al., 2002a; Freitas-Ribeiro et al., 2011, 2012;Basile et al., 2012), and there is only one study referring to theendocrine cells in the large intestine of D. aurita (Santos et al., 2013),however, no describe the ultrastructure of insulin-immunoreactiveendocrine cells.

Therefore, this study aimed to investigate the distributionof argyrophil, argentaffin, and insulin-immunoreactive endocrinecells in the large intestine of opossums D. aurita and to describe theultrastructure of the secretory granules of insulin-immunoreactiveendocrine cells.

2. Materials and methods

2.1. Animals and ethical aspects

Ten male specimens of D. aurita were used in this study (weight,1.03 ± 0.19 kg; crown-rump and snout-rump corporal lengths,32.70 ± 3.59 and 46.05 ± 12.50 cm, respectively; and perimeter andthoracic height, 22.70 ± 1.57 and 11.35 ± 0.78 cm, respectively).

The animals were considered adults by having complete dentition(Macedo et al., 2006).

Catches were authorized by the Brazilian Institute of Environ-ment and Renewable Natural Resources (IBAMA, license 23204-1)in a small Atlantic forest area in Minas Gerais, Brazil (S20◦45′14′′,W42◦52′55′′). The study was approved by the Institutional EthicsCommittee for Animal Research (approval protocol 11/2010).

Hooklike traps with dimensions of 75 cm × 31 cm × 31 cm wereused, with bananas and eggs as bait. After capture, the animalswere kept in captivity for 24 h, receiving water and food used in thecapture, a diet similar to their natural feed (Carvalho et al., 2005).The animals were anesthetized with sodium thiopental, 30 mg/mL,using an intraperitoneal dose of 60 mg/kg. The animals were euth-anized under anesthesia through intracardiac administration of0.25% potassium chloride, depending on the body weight.

2.2. Biometry and histological processing

After exposure of the abdominal cavity, the intestine wasremoved and placed in a flume, moistened with saline, and thenmeasured using a caliper, with no distention of the organ.

The intestinal regions, cecum, colon (ascending, transverse,and descending), and rectum were identified by flexures andintestinally delimited by lashing. Three fragments were collected(proximal, middle, and distal) in each region, for a total of 15 frag-ments for each animal.

2.3. Histochemistry and immunohistochemistry

Fragments of the material collected were fixed in 10% bufferedformalin for 24 h, dehydrated in increasing series of ethanol, clearedin xylene, embedded in paraffin, and sectioned (5 �m thick) atintervals of 50 �m in a rotary microtome (Leica Multicut 2045;Reichert-Jung Products, Germany).

The sections were stained according to the technique ofGrimelius and Wilander (1980) and modified Masson-Fontana(Barbosa et al., 1984) for the detection of argyrophil and argentaffinendocrine cells, respectively.

For the detection of insulin-immunoreactive endocrinecells, a monoclonal antibody produced by Bethyl Labo-ratories (lote no. A90-117p-4) was used, and an indirectimmunoperoxidase technique (Sternberger, 1979) was per-formed. We used sections of pancreas from the same speciesprocessed under the same conditions as positive and negativecontrols.

The photographic documentation of the preparations was per-formed under a CX31 light microscope (Olympus, Tokyo, Japan)with an SC020 digital camera (Olympus, Tokyo, Japan). The argy-rophil, the argentaffin, and the insulin-immunoreactive endocrinecells were quantified using the software Image-Pro Plus 3.5 (MediaCybernetics, Inc., Rockville, Maryland, USA), analyzing 10 fieldsof 78,500 �m2 of the mucosa, and the results were converted incells/mm2.

Were considered positive argyrophil and argentaffin endocrinecells that showed cytoplasmic granules impregnated by silver salts,and insulin-immunoreactive endocrine cells, with the cytoplasm ofgranular appearance of brown staining.

2.4. Transmission electron microscopy

Fragments of the ascendant and descendant colon were fixedin 2.5% glutaraldehyde for 24 hs, postfixed in 1% osmium tetrox-ide for 2 h, and then dehydrated in increasing series of acetone andembedded in Epon 812 resin. Semifine sections (500 nm thick) wereobtained using rotary microtome (Leica Multicut 2045; Reichert-Jung Products, Germany) and submitted to plastic-free technique

Author's personal copy

72 D.C.M.d. Santos et al. / Tissue and Cell 46 (2014) 70– 77

Fig. 1. Tunica mucosa of the large intestine of the opossum Didelphis aurita. ((A), (D), (G), (J) and (M)) Argyrophil endocrine cells. Grimelius. ((B), (E), (H), (K) and (N))Argentaffin endocrine cells. Modified Fontana-Masson. ((C), (F), (I), (L) and (O)) Insulin-immunoreactive endocrine cells. Indirect immunoperoxidase. (A), (B) and (C) = ceco;(D), (E) and (F) = ascending colon; (G), (H) and (I) = transverse colon; (J), (K) and (L) = descending colon; (M), (N) and (O) = rectum. Arrow = endocrine cell; Gc = goblet cell;Lp = lamina propria; Mm = muscularis mucosae.

(Lane and Europa, 1965). Afterward, an indirect immunoperoxi-dase technique (Sternberger, 1979) was performed to identify theinsulin-immunoreactive endocrine cells. In this technique, a poly-clonal antibody produced by DakoCytomation (A0564) was used.

The blocks who presented the identified cells were delimitedby the method described by Fonseca et al. (1998); subsequently,ultrathin sections were obtained with the assistance of ultrami-crotome (Du Pont-Sorvall, Porter-Blum MT2-B). These sectionswere contrasted with 2% uranyl acetate and 0.2% lead citrateand observed in transmission electron microscope (Zeiss EM109).

The granules of insulin-immunoreactive endocrine cells werequantified per unit area in �m2 and qualified according to the elec-tron density in immature and mature granules (Fukuma, 1974). Inaddition, the diameter, the area, and the volume of these granuleswere calculated. These analyses were performed using the softwareImage-Pro Plus 3.5 (Media Cybernetics, Inc., Rockville, Maryland,USA).

2.5. Statistical analysis

The results were represented as mean ± standard deviation. Thenormality of data distribution was verified using the Shapiro–Wilktest. Considering the results of this test, the Kruskal–Wallis testor the Dunn test was used for multiple comparisons. Results wereconsidered significant if p < 0.05. All tests were performed usingthe GraphPad Prism 5.0 statistical software program (GraphPadSoftware, Inc., La Jolla, CA, USA).

3. Results and discussion

The argyrophil, the argentaffin, and the insulin-immunoreactiveendocrine cells were sparsely distributed along the intestinalglands, among other cell types of the epithelium in all regionsstudied (Fig. 1).

These cells were elongated, pyramidal, or rounded. Theyhad ovoid nucleus and cytoplasm with large amounts of secretory

Author's personal copy

D.C.M.d. Santos et al. / Tissue and Cell 46 (2014) 70– 77 73

Table 1Total argyrophil endocrine cells for intestinal area (mm2) in the tunica mucosa ofthe large intestine of the opossum D. aurita.

Proximal Middle Distal

Cecum 26.94 ± 8.62aA 30.11 ± 9.17aAB 34.23 ± 7.08aA

Colon Ascending 43.67 ± 9.16aBC 39.73 ± 9.04aAB 35.65 ± 8.21aA

Transverse 50.54 ± 9.27aBD 48.92 ± 12.97aA 37.53 ± 12.02aA

Descending 40.44 ± 11.47aACD 31.64 ± 9.06aAB 29.21 ± 7.99aAB

Rectum 28.75 ± 5.99aAC 26.77 ± 7.13aB 16.86 ± 4.30bB

Mean followed by the same letter, minuscule on the line and uppercase in the col-umn, do not differ (p > 0.05) by the Kruskal–Wallis and Dunn’s multiple comparisontests.

Table 2Total argentaffin endocrine cells for intestinal area (mm2) in the tunica mucosa ofthe large intestine of the opossum D. aurita.

Proximal Middle Distal

Cecum 21.71 ± 6.46aAB 15.49 ± 5.11abA 12.00 ± 3.55bAB

Colon Ascending 24.18 ± 6.91aAB 20.63 ± 4.60aA 25.62 ± 6.41aA

Transverse 27.32 ± 9.74aA 19.95 ± 10.50aA 24.09 ± 11.92aAB

Descending 23.17 ± 10.94aAB 18.63 ± 10.37aA 26.41 ± 14.64aAB

Rectum 17.02 ± 2.96aB 15.69 ± 2.39aA 10.96 ± 4.21bB

Mean followed by the same letter, minuscule on the line and uppercase in the col-umn, do not differ (p > 0.05) by the Kruskal–Wallis and Dunn’s multiple comparisontests.

granules, particularly in the perinuclear portion. They were locatedat the basal, middle and apical regions of the intestinal glands(Fig. 1).

Most of the endocrine cells were open type, and have apicalprolongations, long and narrow, reaching the luminal surface. Oth-ers were closed type, without apparent communication with thelumen, and generally located at the base of the intestinal glands.The basal portion of these cells presents itself, will often, dilatedand in contact with the basal portion of adjacent cells.

The number of argyrophil and insulin-immunoreactiveendocrine cells did not differ between the portions analyzedin the region of the cecum (Tables 1 and 3). However, argentaffinendocrine cells were greater in number in the proximal portiontoward distal portion (Table 2).

The ascending, transverse, and descending colon showedno difference in the number of argyrophil, argentaffin, andinsulin-immunoreactive endocrine cells in the analyzed portions(Tables 1–3).

In the rectum, the number of argyrophil, argentaffin, andinsulin-immunoreactive endocrine cells was higher in the proximaland medial portions (Tables 1 and 2). In the same region, there wasno difference in the number of insulin-immunoreactive endocrinecells between the portions analyzed (Table 3).

In the proximal portion, the number of argyrophil endocrinecells in the region of the cecum was lower than that in the ascendingand transverse colon; and in the rectum, the number of these cellswas lower than that in the transverse colon (Table 1). In this same

Table 3Total insulin-immunoreactive endocrine cells for intestinal area (mm2) in the tunicamucosa of the large intestine of the opossum D. aurita.

Proximal Middle Distal

Cecum 0.61 ± 0.99aA 0.31 ± 0.33aA 0.54 ± 1.02aA

Colon Ascending 0.89 ± 1.07aA 0.38 ± 0.24aA 1.09 ± 1.13aA

Transverse 1.20 ± 1.40aA 0.61 ± 0.85aA 0.86 ± 0.85aA

Descending 0.74 ± 0.93aA 0.59 ± 1.01aA 1.01 ± 0.95aA

Rectum 0.31 ± 0.38aA 0.26 ± 0.58aA 0.19 ± 0.26aA

Mean followed by the same letter, minuscule on the line and uppercase in the col-umn, do not differ (p > 0.05) by the Kruskal–Wallis and Dunn’s multiple comparisontests.

portion, the number of argentaffin endocrine cells was higher inthe transverse colon compared with the rectum (Table 2).

In the middle portion, the number of argyrophil endocrine cellsin the region of the transverse colon was greater than that in therectum (Table 1).

In the distal portion, the number of argyrophil endocrine cellsin the rectum was smaller when compared with the cecum andascending and transverse colon (Table 1). In this same portion, thenumber of argentaffin endocrine cells was higher in the ascendingcolon compared with the rectum (Table 2).

The proximal, middle and distal portions of all regions studiedshowed no difference in insulin-immunoreactive endocrine cells(Table 3), wherein these cells were evenly distributed throughoutthe large intestine.

There was no difference in the average number of argy-rophil and argentaffin endocrine cells, and the number of thesecells was predominant in relation to insulin-immunoreactiveendocrine cells. Proportionally, argyrophil, argentaffin, and insulin-immunoreactive endocrine cells represented 62.75%, 36.26%, and0.99%, respectively, of the total number of endocrine cells deter-mined in the large intestine (Table 4).

In the region of the cecum, 47.32 cells/mm2 were quanti-fied. Of these, 64.31% were argyrophil endocrine cells, 34.66%were argentaffin endocrine cells, and 1.03% were insulin-immunoreactive endocrine cells. In the ascending, transverse, anddescending colon, 63.85 cells/mm2. Of these, 61.97% were argy-rophil endocrine cells, 36.74% were argentaffin endocrine cells, and1.29% were insulin-immunoreactive endocrine cells. In the rectum,38.94 cells/mm2. Of these, 61.96% were argyrophil endocrine cells,37.39% were argentaffin endocrine cells, and 0.65% were insulin-immunoreactive endocrine cells (Table 4).

4. Ultrastructure of insulin-immunoreactive endocrinecells

The insulin-immunoreactive endocrine cells were distributedthroughout the epithelium of the large intestine of D. aurita, locatedat the base of the intestinal glands, and usually did not havecontact with the lumen, which were classified as closed type(Fig. 2).

These cells were elongated or pyramidal, with rounded nucleiof irregularly contoured, and large amounts of secretory granules,and distributed throughout the cytoplasm, generally concentratedin the apical and basal poles of the cell. These cells were mor-phologically distinct from other non-insulin-producing cells of theintestinal epithelium (Fig. 2).

The granules showed different sizes and electron densities andwere classified as immature and mature. Generally, the gran-ules showed external electron-lucent halo and electron-dense core(Fig. 2).

The mature granules of these cells had central or eccentricelectron-dense core surrounded by broad electron-lucent halo. Theimmature granules were similar to mature granules, but the corewas less electron dense and amorphous or presented homogeneouselectron-dense core and narrow electron-lucent halo (Fig. 2).

The mature granules were predominant and represented 65% ofthe total of secretory granules, with a diameter of 0.56 ± 0.07 �m,an area of 0.25 ± 0.06 �m2, and a volume of 0.09 ± 0.04 �m3. Theimmature granules represented 35% of the total secretory granules,with a diameter of 0.45 ± 0.07 �m, an area of 0.16 ± 0.05 �m2, anda volume of 0.05 ± 0.02 �m3 (Table 5).

The insulin-immunoreactive endocrine cells had 1.75 ± 0.44secretory granules/�m2, whereas mature granules had 1.13 ± 0.35secretory granules/�m2, and immature granules had 0.63 ± 0.52secretory granules/�m2 (Table 5).

Author's personal copy

74 D.C.M.d. Santos et al. / Tissue and Cell 46 (2014) 70– 77

Table 4Total number and percentage of argyrophil, argentaffin and insulin-immunoreactive endocrine cells for intestinal area (mm2) in the tunica mucosa of the large intestine ofthe opossum D. aurita.

Argyrophil Argentaffin Insulin-immunoreactive

N (mm2) % N (mm2) % N (mm2) %

Cecum 30.43 ± 8.29 64.31 16.40 ± 5.04 34.66 0.49 ± 0.78 1.03Colons 39.70 ± 9.91 61.97 23.33 ± 9.07 36.74 0.82 ± 0.94 1.29Rectum 24.13 ± 5.81 61.96 14.56 ± 3.19 37.39 0.25 ± 0.41 0.65Mean 31.42 ± 8.00a 62.75 18.10 ± 5.77a 36.26 0.52 ± 0.71b 0.99

Mean followed by the same letter, in the line, do not differ (p > 0.05) by the Kruskal–Wallis and Dunn’s multiple comparison tests.

Fig. 2. Ultrastructure of insulin-immunoreactive endocrine cells of the large intestine of the opossum D. aurita. (A) Secretory granules distributed throughout the cytoplasmof the cell. (B) Details of mature and immature insulin granules. (C) Cell of the intestinal epithelium that is not insulin-producing. n = cell nucleus; m = mature granule;i = immature granule. Transmission electron microscopy.

In the present study, the argyrophil and the argentaffinendocrine cells were located in the epithelium of the mucous layerin all regions and portions of the large intestine of D. aurita, most ofthem being open type. The cells of the closed type were restrictedat the base of the glands. Open- and closed-type argyrophil andargentaffin endocrine cells were also observed in the large andsmall intestine of the same species (Freitas-Ribeiro et al., 2011,2012; Basile et al., 2012; Santos et al., 2013), in the stomach of otheropossums (Didelphis virginiana) (Krause et al., 1986), throughoutthe digestive tract of deer (Muntiacus muntjak) (Adnyane et al.,2011), in the stomach and small and large intestines of babirusa(Babyrousa babyrussa) (Agungpriyono et al., 2000), and in the smalland large intestines of rodents (Spalax leucodon) (Yaman et al.,2012).

Functionally, the endocrine cells are intimately linked to theenteric nervous system and can act as local regulators in thedigestive processes (Grube, 1986). In accordance with Polak et al.

Table 5Morphometry of secretory granules of insulin-immunoreactive endocrine cells ofthe large intestine of the opossum D. aurita.

Granules % Diameter (�m) Area (�m2) Volume (�m3) N (�m2)

Immature 35 0.45 ± 0.07 0.16 ± 0.05 0.05 ± 0.02 1.13 ± 0.35Mature 65 0.56 ± 0.07 0.25 ± 0.06 0.09 ± 0.04 0.63 ± 0.52

(1993), the cytoplasmic prolongations are typical of cells that haveparacrine functions, as it increases their contact with neighboringcells. The endocrine cells observed in the large intestine of D. auritacertainly may act through endocrine, paracrine, or autocrine mech-anisms, in particular as a paracrine on those that showed dilatationin the basal portion.

There was no difference between the number of argyrophiland argentaffin endocrine cells in the intestine of D. aurita. Inthe small intestine of adult and postpubertal animals of thesame species, there was a predominance of argyrophil endocrinecells, particularly in the duodenum, the region that presentedthe highest number of these cells (Freitas-Ribeiro et al., 2011).Rodrigues (2005), working with the small intestine of capybara(Hydrochaeris hydrochaeris), also reported a greater number of argy-rophil endocrine cells compared with argentaffin endocrine cells,whereas Bressan et al. (2004) found no difference in the number ofargyrophil and argentaffin endocrine cells in the cecum this samespecies.

Differences in the distribution pattern of endocrine cellsin the intestinal segments are related to their physiologi-cal functions in region or intestinal segments. According toKendzierski et al. (2000), the endocrine cells are mainly involvedin controlling the release of digestive enzymes in the smallintestine and in controlling intestinal motility in the largeintestine.

Author's personal copy

D.C.M.d. Santos et al. / Tissue and Cell 46 (2014) 70– 77 75

In the present study, the colon showed the highest proportion ofboth argyrophil and argentaffin endocrine cells, unlike the hypoth-esis established by Kitamura et al. (1982) and Ito et al. (1987), whofound that high concentrations of endocrine cells are present inthe terminal regions of each intestinal segment, that is, in the largeintestine, in the rectum, with a higher proportion of endocrine cellsinvolved in the feedback control of intestinal motility and secre-tion.

In the intestinal regions, the argentaffin endocrine cells werepredominant in the proximal portion of the cecum and the proxi-mal and medial portions of the rectum. Argyrophil endocrine cellsalso showed predominance the proximal portion of the rectum.According to Sjölund et al. (1983), the serotonin-producing cells areargentaffin. Serotonin is known to stimulate the contraction of thesmooth muscles of the gastrointestinal tract, the secretion of waterand electrolytes, and the stimulation of the myenteric neurons bypromoting vasodilatory responses in the submucosa (Vanner, 2000;Ahlman and Nilsson, 2001). From these results, it can be inferredthat the control of secretion and motility in the large intestine ofD. aurita is most effective in the initial and middle portions of eachregion.

The total number of argyrophil endocrine cells in the largeintestine of D. aurita was 94.26 ± 8.02 cells/mm2. Argyrophil cellsrepresent a heterogeneous population of endocrine cells, pre-senting a wide variety of peptides and biogenic amines as a productof secretion (Polak et al., 1993). Fonseca et al. (2002a) identifiedargyrophil cells in the colon of D. albiventris in different stagesof development and found an average of 45.8 ± 6.2 to 84.4 ± 23.9cells/mm2.

However, on a smaller proportion of cells, insulin-immunoreactive endocrine cells were distributed in all regions andportions of the large intestine of D. aurita, located at the base of theintestinal glands, and usually did not have contact with the lumen,which is considered as closed type. These cells are elongated orpyramidal with rounded nuclei, besides having large amounts ofsecretory granules distributed throughout the cytoplasm. Basileet al. (2012) also described these morphological characteristics inthese cells in different regions of the small intestine of the samespecies.

A high proportion of argyrophil and argentaffin endocrine cellsin relation to insulin-immunoreactive endocrine cells from thelarge intestine of D. aurita were observed. In the small intestine ofthe same species, Freitas-Ribeiro et al. (2011, 2012) and Basile et al.(2012) also found lower proportions of insulin-immunoreactiveendocrine cells in relation to argyrophil and argentaffin endocrinecells. Such cells were not located in the stomach and small andlarge intestines of buffalo (Bubalus bubalis) and deer (M. muntjak)(Baltazar et al., 1998; Adnyane et al., 2011).

Explaining the differences in the patterns of the distribution andfrequency of endocrine cells, Forssmann et al. (1969) found thatdifferent types of these cells often vary according to the area ofinvestigation, and some of them are only found in specific regions.Accordingly, Yaman et al. (2012) considered that endocrine cellsappear remarkably different depending on the animal species.

The intestine of the opossums (D. virginiana) has similar fea-tures to the pancreas for the presence of endocrine cells (Krauseet al., 1986), although the mechanism of action of these hormonesis distinct. Intestinal insulin has autocrine or paracrine functions ofgreat importance in the control of cell division, secretion of pep-tides, absorption, and intestinal motility intestinal (Bendayan andPark, 1991; Kendzierski et al., 2000; Basile et al., 2012).

The secretory granules of insulin-immunoreactive endocrinecells observed in this study have ultrastructural features similarto those of the B cells of pancreatic islets in rats. Thus, the synthesisof this hormone should follow the same route: pancreatic insulinis initially synthesized as a single-chain precursor (preproinsulin),

which is rapidly converted as a proinsulin in the endoplasmicreticulum. The proinsulin is transported to the Golgi complex,where it is packaged into secretory granules. The proinsulin is con-verted into insulin and stored, awaiting release by specific stimuli(Steiner, 2000; Pfützner and Forst, 2011).

In this study, these granules showed different sizes and electrondensities relative to other types of endocrine cells. Ultrastruc-ture studies confirmed the heterogeneous nature of endocrinecells by demonstrating that the granules differ from one celltype to the other in relation to the size, shape, electron density(Forssmann et al., 1969), and distribution in the cytosol (Polak et al.,1993). Secretory granules of the glucagon in the gastric mucosa ofopossums and in cell culture in rats have large electron-dense coreand narrow electron-lucent peripheral region (Fonseca et al., 1998;El-Naggar, 2000). Forssmann et al. (1969) described that the secre-tory granules of serotonin-producing cells in the intestine of ratshave high density and polymorphs.

The secretory granules of insulin-immunoreactive endocrinecells were classified as mature or immature. According Fukuma(1974), there is a direct relationship between the polymorphismand the ultrastructural maturation of insulin granules. In thepresent study, the mature granules showed a central or eccen-tric electron-dense core surrounded with electron-lucent halo ofvarying size between the core and the limiting membrane. Thesecharacteristics were similar to mature secretory granules of the Bcells of pancreatic islets in rats (Grube, 1986; Tahir et al., 1992;El-Naggar, 2000).

The immature granules analyzed showed narrow electron-lucent halo and rounded electron-dense core, or a nucleus withlower density of varying size, and varied space between the limitingmembrane. In the cultured B cells of pancreatic islets described byEl-Naggar (2000), immature secretory granules have a fluffy cen-tral core surrounded with either a narrow electron-lucent halo ora well-fitting limiting membrane. Fukuma (1974) described twoforms of immature secretory granules. The first form was similar tothe mature one, but with an amorphous less-dense core, and wasconsidered the predominant one in the cultured islets. The secondform, rarely observed, had a homogeneous dense core and a narrowspace between the core and the limiting membrane.

These forms of immature secretory granules are considered pre-cursors of the mature form, often observed near the Golgi complexof the B cells when stimulated by glucose. The maturation wasattributed to the condensation of secretion material on a cen-tral core accompanied by the resolution of the peripheral zone(Fukuma, 1974).

In this study, the mature forms of the granules of insulin-immunoreactive endocrine cells were predominant, representing65% of secretory granules, whereas the immature granules repre-sent 35% of the total secretory granules. El-Naggar (2000) found aproportion of 44% of mature granules and 56% of immature granulesin the cultured B cells of pancreatic islets of rats.

The average diameter of the immature and mature granules ofinsulin was 0.50 ± 0.07 �m. This value was similar to that foundby Fukuma (1974) in the cultured B cells of pancreatic isletsof rats (0.50 ± 0.11 �m) and higher compared with the diame-ter of granules of the B cells of pancreatic islets in cultured mice(0.27 ± 0.05 �m) (El-Naggar, 2000).

5. Conclusion

The insulin-immunoreactive endocrine cells as well as theargyrophil and argentaffin endocrine cells were distributed in allregions and portions of the large intestine of D. aurita.

There was no difference in the average number of argy-rophil and argentaffin endocrine cells in the large intestine, which

Author's personal copy

76 D.C.M.d. Santos et al. / Tissue and Cell 46 (2014) 70– 77

was higher compared with insulin-immunoreactive endocrinecells.

The ultrastructural pattern of granule of the insulin-immunoreactive endocrine cells in the large intestine of D.aurita was similar to that of B cells of pancreatic islets in rats.

References

Adnyane, I.K., Zuki, A.B., Noordin, M.M., Agungpriyono, S., 2011. Immunohistochem-ical study of endocrine cells in the gastrointestinal tract of the barking deer,Muntiacus muntjak. Anat. Histol. Embryol. 40, 365–374.

Agungpriyono, S., Macdonald, A.A., Leus, K.Y., Kitamura, N., Adnyane, I.K., Goodall,G.P., Hondo, E., Yamada, J., 2000. Immunohistochemical study on the distribu-tion of endocrine cells in the gastrointestinal tract of the babirusa, Babyrousababyrussa (Suidae). Anat. Histol. Embryol. 29, 173–178.

Ahlman, H., Nilsson, O., 2001. The gut as the largest endocrine organ in the body.Ann. Oncol. 2, 63–68.

Baltazar, E.T., Kitamura, N., Hondo, E., Yamada, J., Maala, C.P., Simborio, L.T.,1998. Immunohistochemical study of endocrine cells in the gastrointestinaltract of the Philippine Carabao (Bubalus bubalis). Anat. Histol. Embryol. 27,407–411.

Barbosa, A.J., Castro, L.P., Margarida, A., Nogueira, M.F., 1984. A simple and econom-ical modification of the Masson-Fontana method for staining melanin granulesand enterochromaffin cells. Stain Technol. 59, 193–196.

Barbosa, A.J., Nogueira, J.C., Penna, F.J., Polak, J.M., 1987. Distribution ofenteroglucagon- and polypeptide YY-immunoreactive cells in the gastrointesti-nal tract of the white-belly opossum (Didelphis albiventris). Histochemistry 88,37–40.

Barbosa, A.J.A., Nogueira, J.C., Fonseca, C.C., 2006. Células endócrinas (APUD) do sis-tema digestivo do gambá Didelphis albiventris. In: Cáceres, N.C., Monteiro-Filho,E.L.A. (Eds.), Os Marsupiais do Brasil: Biologia, ecologia e evoluc ão. UFMS, CampoGrande, pp. 89–98.

Basile, D.R.S., Novaes, R.D., Marques, D.C.S., Fialho, M.C.Q., Neves, C.A., Fonseca, C.C.,2012. Analysis of the morphology and distribution of argentaffin, argyrophiland insulin-immunoreactive endocrine cells in the small intestine of the adultopossum Didelphis aurita (Wied-Neuwied, 1826). Tissue Cell 44, 301–307.

Bendayan, M., Park, I.S., 1991. Presence of extrapancreatic islets of Langerhans in theduodenal wall of the rat. Diabetologia 34, 604–606.

Bressan, M.S., Fonseca, C.C., Menin, E., Paula, T.A.R., 2004. Identificac ão equantificacão de gânglios nervosos, células argentafins, argirófilas e imunor-reativas à serotonina no ceco de capivara (Hydrochoerus hydrochaeris). Rev. Ceres51, 729–739.

Carvalho, F.M.V., Fernandez, F.A.S., Nessimian, J.L., 2005. Food habits of sympatricopossums coexisting in small Atlantic Forest fragments in Brazil. Mamm. Biol.70, 366–375.

Cerqueira, R., Lemos, B., 2000. Morphometric differentiation between neotropi-cal black-eared opossums, Didelphis marsupialis and D. aurita (Didelphimorphia,Didelphidae). Mammalian 64, 319–327.

Cerqueira, R., Tribe, C.J., 2008. Genus Didelphis linnaeus, 1758. In: Gardner, A.L.(Ed.), Mammals of South America. Marsupials, Xenarthrans, Shrews, and Bats,1. Chicago University Press, Chicago, pp. 17–25.

Coutinho, H.B., Sewell, H.F., Coutinho, V.B., 1985. Immunocytochemical demonstra-tion of insulin in the mesonephros and metanephros of the Brazilian opossum(Didelphis albiventris). Ann. Anz. 159, 97–103.

Coutinho, H.B., Sewell, H.F., Smith, D.I., Coutinho, V.B., Pinheiro, P.B., 1984. Demon-stration of insulin in the pancreas of the Didelphis albiventris (opossum) byimmunocytochemical techniques. Ann. Anz. 157, 167–175.

Cunha, A.A., Vieira, M.V., 2005. Age, season, and vertical use of the Atlantic rain-forest by the common opossum, Didelphis aurita—Wied, 1826. Acta Theriol. 50,551–560.

Dayal, Y., DeLellis, R.A., Wolf, H.J., 1987. Hyperplastic lesions of the gastrointestinalendocrine cells. Am. J. Surg. Pathol. 1, 87–101.

Devaskar, S.U., Singh, B.S., Carnaghi, L.R., Rajakumar, P.A., Giddings, S.J., 2002. InsulinII gene expression in rat central nervous system. Regul. Pept. 48, 55–63.

Drucker, D.J., 2007. The role of gut hormones on glucose homeostasis. J. Clin. Invest.1, 24–32.

Égéa, J.-C., HirtZ, C., Gross, R., Lajoix, A.-D., Traskawka, E., Ribes, G., Deville de Périère,D., 2000. Preproinsulin I and II mRNA expression in adult rat submandibularglands. Eur. J. Oral Sci. 108, 292–296.

El-Naggar, M.M., 2000. Ultrastructural immunogold study on the various cell typesof cultured pancreatic islets of adult rats. Folia Morphol. 59, 253–262.

Fonseca, C.C., Nogueira, J.C., Barbosa, A.J., 1998. Ultrastructural pattern of glucagonproducing-cells in the gastric mucosa of the developing opossum Didelphisalbiventris (Marsupialia). Ann. Anat. 180, 477–480.

Fonseca, C.C., Nogueira, J.C., Barbosa, A.J.A., 2002a. Argyrophilic and glucagon-immunoreactive cells in the Ileum and colon of the developing opossumDidelphis albiventris (Marsupialia). Cells Tissues Organs 170, 29–33.

Fonseca, C.C., Nogueira, J.C., Barbosa, A.J.A., 2002b. Diâmetro das ilhotas pancreáticasdo gambá Didelphis albiventris em desenvolvimento intramarsupial. Arch. Vet.Sci. 7, 129–134.

Forssmann, W.G., Orci, L., Pictet, R., Renold, A.E., Rouiller, C., 1969. The endocrinecells in the epithelium of the gastrointestinal mucosa of the rat. An electronmicroscope study. J. Cell Biol. 40, 692–715.

Freitas-Ribeiro, G.M., Fonseca, C.C., Rodrigues, S.S., Matta, S.L.P., Neves, C.A., 2011.Quantification of argyrophillic, argentaffin and insulin immunoreactive cells inthe small intestine in the opossum Didelphis aurita (Wied-Neuwied, 1826). ActaSci. Biol. Sci. 33, 479–485.

Freitas-Ribeiro, G.M., Fonseca, C.C., Sartori, S.S.R., Loures-Ribeiro, A., Neves, C.A.,2012. Endocrine cells and nerve ganglia of the small intestine of the opossumDidelphis aurita Wied-Neuwied, 1826 (Mammalia: Didelphidae). An. Acad. Bras.Cienc. 84, 747–757.

Fujita, T., Kobayashi, S., 1977. Structure and function of gut endocrine cells. Int. Rev.Cytol. 6, 187–233.

Fukuma, M., 1974. Electron microscopic studies on the granule release from the ratpancreatic B-cells in organ culture. J. Electron Microsc. 23, 167–183.

Grimelius, L., Wilander, E., 1980. Silver stains in the study of endocrine cells of thegut and pancreas. Invest. Cell Pathol. 3, 3–12.

Grube, D., 1986. The endocrine cells of the digestive system: amines, peptides, andmodes of action. Anat. Embryol. 175, 151–162.

Ito, H., Yamada, J., Yamashita, T., Hashimoto, Y., Kudo, N., 1987. An immunohisto-chemical study on the distribution of endocrine cells in the gastrointestinal tractof the pig. Jpn. J. Vet. Sci. 49, 105–114.

Kendzierski, K.S., Pansky, B., Budd, G.C., Saffran, M., 2000. Evidence for biosynthesisof preproinsulin in gut of rat. Endocrine 13, 353–359.

Kitamura, N., Yamada, J., Yamashita, T., Yanaihara, N., 1982. Endocrine cells in thegastrointestinal tract of the cat. Biomed. Res. 3, 612–622.

Krause, W.J., Cutts 3rd, J.H., Cutts, J.H., Yamada, J., 1989. Immunohistochemical studyof the developing endocrine pancreas of the opossum (Didelphis virginiana). ActaAnat. 135, 84–96.

Krause, W.J., Yamada, J., Cutts, J.H., 1985. Quantitative distribution of enteroen-docrine cells in the gastrointestinal tract of the adult opossum, Didelphisvirginiana. J. Anat. 140, 591–605.

Krause, W.J., Yamada, J., Cutts, J.H., 1986. Enteroendocrine cells in the developingopossum stomach. J. Anat. 148, 47–56.

Lane, B.P., Europa, D.L., 1965. Diferential staining of ultrathin sections ofepon-embedded tissues for light microscopy. J. Histochem. Cytochem. 13,579–582.

Loretto, D., Vieira, M.V., 2005. The effects of reproductive and climatic seasons onmovements in the black-eared opossum (Didelphis aurita Wied-Neuwied, 1826).J. Mammal. 86, 287–293.

MacAlester, A.L., 1994. História Geológica da vida. E. Blucher, São Paulo, pp. 173.Macedo, J., Loretto, D., Vieira, M.V., Cerqueira, R., 2006. Classes de desenvolvi-

mento em marsupiais: um método para animais vivos. Mastozool. Neotrop. 13,133–136.

Meimaridis, D.G., Morse, D.E., Pansky, B., Budd, G.C., 2003. Insulin immunoreactivityin the fetal and neonatal rat retina. Neurosci. Lett. 118, 116–119.

Moran, G.W., Leslie, F.C., Levison, S.E., McLaughlin, J.T., 2008. Enteroendocrine cells:Neglected players in gastrointestinal disorders? Therap. Adv. Gastroenterol. 1,51–60.

Pfützner, A., Forst, T., 2011. Elevated intact proinsulin levels are indica-tive of Beta-cell dysfunction, insulin resistance, and cardiovascular risk:impact of the antidiabetic agent pioglitazone. J. Diabetes Sci. Technol. 1,784–793.

Polak, J.M., Bishop, A.E., Barbosa, A.J.A., Bloom, S.R., 1993. Hormônios gastrointesti-nais. In: Dani, R., Paula Castro, L. (Eds.), Gastroenterologia Clínica. GuanabaraKoogan, Rio de Janeiro, pp. 1446–1465.

Rehfeld, J.F., 1998. The new biology of gastrointestinal hormones. Physiol. Rev. 78,1087–1102.

Rindi, G., Leiter, A.B., Kopin, A.S., Bordi, C., Solcia, E., 2004. The “normal” endocrinecells of the gut changing concepts and new evidences. Ann. N.Y. Acad. Sci. 1014,1–12.

Rodrigues, S.S., 2005. Aspectos anátomo-histológicos e neuro-endócrinos dointestino delgado da capivara Hydrochoerus hydrochaeris Linnaeus, 1766 (Mam-malia, Rodentia, Hydrochaeridae). Dissertac ão de Mestrado. Programa dePós-Graduac ão em Medicina Veterinária, Universidade Federal de Vic osa, pp.95.

Samoto, V.Y., Miglino, M.A., Ambrosio, C.E., Pereira, F.T.V., Lima, M.C., Carvalho, A.F.,2006. Opossum (Didelphis sp.) mammary gland morphology associated to themarsupial model. Biota Neotrop. 6, 1–12.

Santos, D.C.M., Cupertino, M.C., Novaes, R.D., Soares, I.A.C., Fonseca, C.C., Matta, S.L.P.,Sartori, S.S.R., 2013. Morphologic characterization and distribution of endocrinecells in the large intestine of the opossum Didelphis aurita (Wied-Neuwied,1826). Tissue Cell 45, 338–349.

Schönhoff, S.E., Giel-Moloney, M., Leiter, A.B., 2004. Minireview: development anddifferentiation of gut endocrine cells. Endocrinology 145, 2639–2644.

Sjölund, K., Sandén, G., Hakanson, R., Sundler, F., 1983. Endocrine cellsin human intestine: an immunocytochemical study. Gastroenterology 85,1120–1130.

Stahler, M.S., Pansky, B., Budd, G.C., 1988. Immunocytochemical demonstration ofinsulin-like immunoreactivity in the rat prostate gland. Prostate 13, 189–198.

Steiner, D.F., 2000. New aspects of proinsulin physiology and pathophysiology. J.Pediatr. Endocrinol. Metab. 13, 229–239.

Sternberger, L.A., 1979. Immunocytochemistry, second ed. Ed. John Wiley & Sons,New York.

Sternini, C., Anselmi, L., Rozengurt, E., 2008. Enteroendocrine cells: a site of ‘taste’in gastrointestinal chemosensing. Curr. Opin. Endocrinol. Diabetes Obes. 15,73–78.

Tahir, M., Elayat, A.A., Jalalah, S., El-Naggar, M.M., 1992. Isolated pancreatic islets ofthe rat: an ultrastructural study. Acta Anat. 145, 93–100.

Author's personal copy

D.C.M.d. Santos et al. / Tissue and Cell 46 (2014) 70– 77 77

Takagi, C., Yamada, J., Krause, W.J., Kitamura, N., Yamashita, T., 1990. An immuno-histochemical study of endocrine cells in the proximal duodenum of eightmarsupial species. J. Anat. 168, 49–56.

Vanner, S., 2000. Myenteric neurons activate submucosal vasodilator neu-rons in guinea pig ileum. Am. J. Physiol. Gastrointest. Liver Physiol. 279,380–387.

Yaman, M., Bayrakdar, A., Tarakci, B.G., 2012. Existence of serotonin andneuropeptides-immunoreactive endocrine cells in the small and large intestinesof the mole-rats (Spalax leucodon). Tissue Cell 44, 257–263.

Zhang, W.J., Duan, J.Z., Lei, N., Xing, H., Shao, Y., Yang, G.B., 2012. Cellular bases forinteractions between immunocytes and enteroendocrine cells in the intestinalmucosal barrier of rhesus macaques. Cell Tissue Res. 350, 135–141.