ARTICLE ORIGINAL
° E. ERGÜN, °° L. ERGÜN, °° R.N. ASTI and ° A. KÜRÜM
° Department of Histology and Embryology, Faculty of Veterinary Medicine, University of Kirikkale, Turkey
°° Department of Histology and Embryology, Faculty of Veterinary Medicine, University of Ankara, Turkey
Address for correspendence : Department of Histology and Embryology, Faculty of Veterinary Medicine, University of Kirikkale, 71450 Kirikkale, Turkey eergun69@hotmail.com
SUMMARY
The aim of the study was to describe the light and electron microscopic morphology of Paneth cells in the sheep small intestine, as well as their histochemical properties, location and numerical distribution. Samples from the small intestine : duodenum, jejunum and ileum, of 7 healthy adult sheep were studied. The Paneth cells are located in the crypts of Lieberkühn and are more numerous in the base and neck of the crypts than in the higher parts. They are characterised by a basal nucleus, and acidophilic apical granules. Granules are larger in Paneth cells located in the deep portions of crypts. The numerical distribution of Paneth cells is heterogeneous along the small intestine, with higher density in the jejunum, and decreasing densities in the duodenum and ileum. The difference between the three regions of the small intestine is statistically significant (p < 0.01). Although homogeneous by light microscopy, apical granules proved to be of different electron densities by transmission electron microscopy.
KEY-WORDS : Paneth cells - small intestine - sheep.
RÉSUMÉ
Morphologie des cellules de Paneth de l’intestin grêle de mouton en m i c roscopie optique et électronique Par E. ERGÜN, L. ERGÜN, R.N.
ASTI et A. KÜRÜM.
Cette étude a été effectuée dans le but de décrire la morphologie des cellules de Paneth de l’intestin grêle de mouton en microscopie optique et électronique, ainsi que leurs propriétés histochimiques, leur localisation et leur densité numérique. L’intestin grêle : duodénum, jéjunum et iléon, de 7 moutons adultes sains, a servi de matériel d’étude. Les cellules de Paneth sont situées dans les cryptes de Lieberkühn, préférentiellement dans la base et le collet. Elles se caractérisent par un noyau basal et la présence de granulations acidophiles apicales, qui sont plus volumineuses dans les cellules de
Paneth situées en profondeur des cryptes. La répartition des cellules de
Paneth au sein de l’intestin grêle est hétérogène, le jéjunum montrant la plus forte densité, suivi du duodénum et de l’iléon. La différence de densité entre les trois segments de l’intestin grêle est statistiquement significative
(p < 0.01). Les granulations cytoplasmiques, quoique homogènes en microscopie optique, se révèlent de densités différentes en microscopie électronique.
MOTS-CLÉS : cellules de Paneth - intestin grêle - mouton.
The epithelial monolayer that lines the mammalian small intestine is both the route of nutrient absorption and an active barrier between the external environment and the circulation.
Expansion of the surface area for the absorption of nutrients also increases the risk of mucosal colonization by potential pathogens. Lieberkühn crypts (intestinal crypts), which are blind invaginations of the intestinal mucosa are ideal microniches for bacterial growth. Nevertheless, the bacterial density of the small intestines is comparably lower than that of the large intestines [25].
The epithelium of the small intestine is made up of enterocytes, enteroendocrine cells, goblet and Paneth cells. Paneth
Revue Méd. Vét., 2003, 154, 5, 351-355 cells, located in Lieberkühn crypts, originate from the same crypt stem cells that generate all intestinal epithelial cell lineages [3, 14, 25].
Paneth cells are found accumulated in the base of the crypts of the small intestines in many species [2, 3, 14, 29]. They are easily distinguished by their prominent eosinophilic granules
[14, 15, 22, 37]. They are pyramidal shaped cells with their broad base sitting on the basement membrane and narrowing towards the apical end. The irregularly shaped nucleus with its prominent nucleolus occupies a third of the basal cytoplasm [5, 21, 29]. On the apical surfaces of the cells are seen brush borders. In addition to the numerous secretory granules in the apical cytoplasm, the remainder of the cytoplasm

352 ERGÜN (E.) AND COLLABORATORS contains well developed granular endoplasmic reticulum and the Golgi complex [2, 6, 10, 18, 21]. The endoplasmic reticulum is abundant in the paranuclear region of the cell. The
Golgi apparatus is located in a supranuclear position [29].
The function of the Paneth cells has not yet been clearly defined. In recent studies [1, 19, 25, 26, 32], Paneth cells have been shown to contain immunoglobulins, lysozyme, the bacteriolytic enzyme, and the anti-microbial peptide termed cryptidins (defensin/corticostatin-like peptide). The antimicrobial peptides that are actively secreted are effective in the reduction of bacterial density in the small intestines [17, 19,
26, 28, 32, 33]. Paneth cells are also responsible for the elimination of heavy metals and phagocytosis of bacteria
[6, 30]. Their ability to digest intestinal microorg a n i s m s demonstrates that the cell may be a part of the natural defense mechanism of the small intestine [12]. Paneth cells also contain phospholipase-A
2
(enhancing factor) which may be involved in regulating apoptosis. Despite their various functions like the synthesis of lysozyme and the phagocytosis of microorganisms, their ultrastructure is predominantly that of a secretory cell [11].
Most studies on Paneth cells have been performed on laboratory rodents kept under pathogen-free conditions. To date, the secretory activities of the Paneth cells in the natural state have not been investigated satisfactorily [32]. On the other hand, there are any research on the Paneth cells in the sheep.
Samples were obtained from the duodenum, jejunum, and ileum of 7 healthy adult sheep slaughtered under the supervision of a veterinarian at the abattoirs of Ankara Sincan and
Yenikent.
Tissue samples were fixed in 10 % buffered neutral formalin and washed through a series of graded alcohols, methyl benzoate and benzol after which they were blocked in paraplast.
S i x - m i c r o m e t e r-thick sections were stained with Lend r u m ’s phloxine-tartrazine, Masson’s trichrome, Periodic
Acid Schiff (PAS), Performic acid-Alcian blue and Alcian blue (pH 2.5) [7].
The specimens taken for electron microscopy were kept for
24 hours in glutaraldehyde-paraformaldehyde pre-fixing (pH
7.4) according to the Karnovsky’s method [16], rinsed for three hours in a cacodylate buffer and fixed for a second time in a 1 % osmic acid solution for two hours. After storing in a
0.5 % uranyl acetate for two hours, samples were passed through graded alcohols and propylene oxide, embedded in araldite M. Sections of 300-400 Angstroms in thickness, and contrasted according to the method of V E N E A B L E a n d
C O G G E S H A L L [39], and then examined in the Carl Zeiss
EM 9S-2 model transmission electron microscope (Zeiss,
Ober-kochen, Germany).
With the aim of determining the numerical distribution of the Paneth cells, 5 sections each from the three diff e r e n t regions of the intestine were cut and stained with the phloxine-tartrazine. In each section the cells in ten crypts were counted. For the counting only sections cutting across the crypts longitudinally were used [34]. For the statistical analysis of the groups and the significance of the average values between groups the analysis of variance was used whilst the Duncan’s test [36] was used to determine the significance of the difference between groups.
By light microscopy, Paneth cells characterized by acidophilic granules (Figure 1, arrows) were observed in the crypts of Lieberkühn. Whereas the cells were found in the base and neck regions of the crypts, none was found in the villi. The round or oval shaped nuclei (arrowhead) were found in the base of the cells with the granules (arrows) in the apical cytoplasm.
The best demonstration of the Paneth cell was obtained with Lendrum’s phloxine-tartrazine method. The granules of the Paneth cells stained red with the cytoplasm staining yellow (Figure 1, 2). The difference between the granule size and the quantity in the cytoplasm was clearly exhibited. The granular size (Figure 2, arrows) was seen to enlarge towards the bottom of the crypts. Whereas majority of the cells filled with granules was observed to release their granular contents into the lumen of the crypts of Lieberkühn, in some Paneth cells, the number of granule was low. The secretory material
(Figure 3, arrow) in the lumen of the crypts was seen in the same density as the granules in the apicale of the cell.
With the Alcian blue-Performic acid (Figure 4, arrows) and
Periodic Acid Schiff (PAS) (Figure 5, arrows) the granules in the Paneth cells gave positive reactions whilst with the
Alcian blue (pH 2.5) they gave negative reactions. PAS (+) granules were observed to be stable to the enzymatic activity of ptyalin.
By electron microscopy, Paneth cells showed remarkable polarization by locating on the basement membrane of the crypts with their bases (Figure 3). Whilst the irregularly shaped nucleus (Figure 6, N) were found at the base of the cells, the homogeneous granules (Figure 7, arrows) of diff e r e n t electron densities were observed in the apical cytoplasm. The granular endoplasmic reticulum was observed around the nucleus. The microvilli extending to the lumen and the connection complexes were prominent (Figure 8). The area between the nucleus and the apical cytoplasmic granules was rich in Golgi complexes (Figure 7, G).
Results of the cell counts to determine the numerical distribution of Paneth cells and the statistical analysis are shown in
Table I. The differences among the duodenum, jejunum and the ileum were found to be statistically significant (p < 0.01).
The density of the Paneth cells was remarkably decreased towards the ileum. The jejunum had the highest cell density.
Revue Méd. Vét., 2003, 154, 5, 351-355

LIGHT AND ELECTRON MICROSCOPIC MORPHOLOGY OF PANETH CELLS IN THE SHEEP SMALL INTESTINE 353
F
I G U R E
1. — Paneth cells possess a basal nucleus (arrowhead) and acidophilic granules (arrows) in the apical cytoplasm. Duodenum, Phloxinetartrazine. Bar : 15
µ m.
F
IGURE
2. — Granules (arrows) are larger in Paneth cells located in the base of Lieberkühn crypts than in Paneth cells located in the superficial parts of crypts. Duodenum, Phloxine-tartrazine. Bar : 18
µ m.
F
IGURE
3. — Secretory material in the crypt lumen (arrow) was of a similar density to that of secretory granules in the apical region. Ileum. Bar :
3
µ m.
F
IGURE
4. — The Paneth cell granules gave a positive reactions with Alcian blue-performic acid staining (arrows) Duodenum. Bar : 19
µ m.
F
I G U R E
5. — The Paneth cell granules gave a positive reaction with PA S
(arrows) Duodenum. Bar : 18
µ m.
F
IGURE
6. — The irregularly shaped nucleus (N) was found at the base of the cell. Ileum. Bar : 2
µ m.
Revue Méd. Vét., 2003, 154, 5, 351-355

354 ERGÜN (E.) AND COLLABORATORS
F
IGURE
7. — The area between the nucleus and the apical cytoplasmic granules (arrows) was rich in Golgi complexes (G). Ileum. Bar : 1
µ m.
F
IGURE
8. — The microvilli extending to the lumen and the connection complexes were prominent. Ileum. Bar : 0.5
µ m.
Paneth cells characterized by prominent eosinophilic granules have been reported in the crypts of Lieberkühn of the small intestines of various mammalian species, [14, 15, 22,
37]. These were easily distinguished by their unique morphology in the sheep.
In a study on human Paneth cells, DESCHNER [9] reported that the cells were not only limited to the bases of the crypts but also to the entire length of the crypts and even in the villi. In this study, however, no Paneth cells were encountered in the villi of the small intestines of the sheep. On the other hand, results of the study supports the views of
BJERKNES and CHENG [3] and GARABEDIAN et al., [14] that the Paneth cells differentiate as such towards the base of the crypts and hence the finding of the young cells at the top and the matured cells at the bottom of the crypts. It is in line with this that these cells are abundant in the neck and bottom regions of the intestinal crypts.
The size of the apical secretory granules has been reported to be related to their age [3, 14]. MATHAN et al., [22] also found the granular size to be a sign of the maturity of the
Paneth cells. In this study, the granular size was seen to enlarge towards the bottom of the crypts in the sheep. In view of these findings, it can be said that the granules signifies the level of maturity or age of the cells under consideration. In this study evidence that these granules release their contents into the lumen of the crypts of Lieberkühn was observed as reported by various investigators [1, 6, 13, 24].
The Paneth cell granules with different structure and sizes among species are reported to be composed of carbohydrateprotein complexes [6, 10]. Neutral mucin is an essential component of the mouse Paneth cell [23]. Rat and guinea pig
Paneth cells also contain neutral mucin. The Periodic Acid
S c h i ff (PAS) reaction in these is however weaker [20, 27,
37]. There are conflicting reports on the PAS reaction of human Paneth cells [35]. According to LEWIN [20] the intensity of the reactions corresponded with the amount of hexose sugars. Thus, the carbohydrate-protein nature of the
Paneth cell granule appeared to differ from one animal to another [20]. In the sheep, in line with M E R Z E L’S f i n d i n g
[23], a strongly PAS (+) reaction was observed. The absence of glycogen in the granules was demonstrated by the enzymatic activity of ptyalin.
* : p < 0.01
T
ABLE
I. — The numerical distribution of Paneth cells in the small intestine
Revue Méd. Vét., 2003, 154, 5, 351-355

LIGHT AND ELECTRON MICROSCOPIC MORPHOLOGY OF PANETH CELLS IN THE SHEEP SMALL INTESTINE 355
Histochemical studies also confirm the protein nature of the Paneth cell granules [15, 20, 23, 37]. As reported earlier by MERZEL [23] and GLEREAN and DE CASTRO [15], in our findings, the granules gave a positive reaction with performic acid-alcian blue stain. This observation goes to confirm the view that the granules contain disulfide groups of protein.
The ultrastructure of the Paneth cell granules show diff erences among various mammals. In human, bat and the guinea pig it is large and filled with morphologically homogenous material [31]. In rat, the granules are smaller in size but also appear homogenous [2, 31]. In the hamster however, it is of different electron densities [31]. In mouse, the granules have a bipartite substructure showing an electron dense core and an electron lucent halo [31, 38]. In this study, the granules were found to be similar to those of the hamster with its homogeneity and different electron dense granules.
The distribution of the Paneth cells shows considerable variation among mammalian species. The small intestinal epithelium of the cat, dog and pig is devoid of Paneth cells while that of man, monkeys, rats, guinea pigs and ruminants contains abundant numbers of these cells in the crypts [4, 8].
In a study conducted on human S I N G H [34] showed the
Paneth cells to be much more denser in the duodenum and ileum and less dense in the jejunum, but suggested that at the terminal ileum the number of crypts and Paneth cells are decreased due to the increased lymphoid tissue. B E H N K E and MOE [2] reported these cells to show an increase in number from the duodenum towards the ileum in rat [2].
However, CHENG et al., [5] demonstrated in the mouse that
Paneth cell density increases from the duodenum towards the jejunum but in contrast, in the ileum it is less dense than in the duodenum. In this study, the Paneth cells were found not to have a homogenous distribution along the entire length of the small intestine, with the region of highest cell density being the jejunum. The duodenum was found to have the lower number of cells than the jejunum. In the ileum where the Peyer’s plates especially abound, the cell count was lowest. Inasmuch as the distribution in the sheep resembles that of the mouse, no satisfactory explanation can be drawn from the findings in this study as to the differences among the species. That apart, according to RIECKEN and P E A R S E
[27] the number of Paneth cells and their enzyme content may be related to the functional demands in the animal. The function of the Paneth cells needs further investigations.
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