Haryana Vet. 48 (December, 2009), pp 33-38 Research Article SCANNING AND TRANSMISSION ELECTRON MICROSCOPIC STUDIES ON THE PALATINE OF THE SHEEP

PAWAN KUMA ..RI, GURDIAL SINGH and S. K. NAGPAL Department of Veterinary Anatomy, College of Veterinary Sciences CCS Haryana Agricultural University, Hisar-125 004

ABSTRACT

The ultrastructure of was studied in six young sheep of either sex of local mixed breed. Scanning electron microscopy of the palatine tonsil revealed longitudinal folds, grooves and crypts towards outer surface whereas the cut surface presented reticular epithelium, lymphoid tissue and extent of crypts. Both the epithelia presented microplicae at higher magnification and their ultrastructure highlighted cellular details of strata basale, spinosum, corneum and superficiale. The lymphoid tissue organized mainly into follicles having germinal centre, parafollicular and interfollicular areas was constituted by different sized Iymphocytes, plasma cells, follicular dendritic cells, macrophages and high endothelialvenules.

Key words: SEM, TEM, palatine tonsil, sheep

The palatine tonsil constitutes first line of defence scanned in electron microscope (Leo 435-VP, Japan) because of its mass oflymphoid tissue and is constantly at EM Laboratory, AIIMS, New Delhi. The tissues for exposed to alimentary and airborne antigens (Perry, transmission electron microscopy were secondarily 1994). The tonsillar crypt epithelium in addition to fixed in 1% osmium tetraoxide for 1 h and washed in germinal centre, mantle zone and interfollicular areas, chilled 0.1 M phosphate buffer (pH 7.4). The rest of acts as an additional lymphoid compartment by procedure to make resin blocks was carried out at EM contributing to the production of immunocytes and to Lab. AIIMS, New Delhi. Ultra thin sections of 60-90 the protection of the mucosal surface (Kataura et al., nm were taken on grids and stained with uranyl acetate 1992). The histomorphology and histochemistry of the for 10 min and lead citrate for 5 min followed by palatine tonsil in sheep had been studied earlier (Kumar washing in distilled water. The sections were examined et al., 2008). Hence, the present study has been and photographs were taken in the transmission electron envisaged to explore the ultrastructural details of cells microscope. constituting the palatine tonsil of the sheep. .. RESULTS AND DISCUSSION MATERIALS AND METHODS SEM: The surface' of palatine tonsil (PT) presented longitudinally oriented folds and grooves which were The palatine were collected from 6 young interrupted towards crypt (Fig 1). The crypt appeared sheep of 6-9 months age of either sex of local mixed as deep oval trench from the outer surface however, breed immediately after slaughter. The tissues were it was divided and distinct in longitudinally cut sections washed in chilled 0.1 M phosphate buffer (pH 7.4), reaching up to deeper portion (Fig 2). The crypts fixed in 2% glutaraldehyde for 8·hand rewashed in increased considerably the total epithelial surface area phosphate buffer. The tissues for scanning electron of the tonsil as reported earlier (Howie, 1980). A 295 microscopy were dehydrated in ascending grades of ern' epithelial surface area of crypts had been reported ethanol, critical point dried, sputter coated with gold and in addition to 45 ern' of the epithelium covering the 'Corresponding author oropharyngeal surface of the palatine tonsil in humans

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i'.\ Fig l. SEM showing longitudinal folds and crypt (arrow) towards Fig 3. SEM showing crypt epithelium modifying into reticular outer surface of the palatine tonsil of sheep. (x 40) epithelium (arrow) and lymphoid tissue (L). (x 85)

Fig 2. SEM showing cut surface of palatine tonsil. Note outer Fig 4. SEM of surface epithelium at higher magnification showing surface epithelium (S), crypt and lymphoid tissue (L). microplicae. (x 10K) (x 32) (Slipka and Kotyza, 1987). The outer surface epithelium crypts contained desquamating epithelial cells, lymphoid along the crypt was modified into reticular epithelium cells and degenerating lymphoid cells along with their however, both the surfaces showed various patterns of debris. Lymphoid tissue mainly comprised oflymphoid microplicae (Figs 3, 4) at higher magnification as follicles were separated by connective tissue and reported in horse and this region was comparable to showed different sized and at places high follicle associated epithelium (FAE) of the nasopharyngeal endothelial venule (HEY) entrapping the lymphocytes tonsil (Kumar and Timoney, 2005 b). A large papilla like (Figs 3, 5). The dynamic nature of the tonsillar lymphoid structure and small elevated areas separated by linear tissue, such as changes in number and sizes offollicles, folds and depressions were observed in the palatine may account for the varied shapes and sizes of crypts tonsil of goat (Kumar et al., 2006). The crypt surface (Perry, 1994). showed intact smooth upper surfaces comprising of TEM: The outer surface stratified squamous keratinised closely fitted squamous cells and distorted epithelial epithelium was comprised of strata basale, spinosum cells at disrupted areas (Perry, 1994). Flat, angular and corneum as reported earlier in goat and sheep squamous cells along with a few interspersed closely (Kumar et al., 2006, 2008). Tall columnar cells of packed cells having knob like villi had been reported in . stratum basale were attached with each other by the bovine palatine tonsil (Palmer et al., 2009). The desmosomes and tight junctions. The lateral surfaces

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/ more centric/ eccentric nucleoli (Fig 6). The cells of stratum spinosum were comprised of varying shaped cells with small sized microvilli emanating through out their surface and were separated from each other by wide intercellular spaces (Fig 7). The distribution of cell organelles and inclusions was similar to that of stratum basale cells except that the concentration of polyribosomes and tonofilaments was more. The nuclei with irregular outer surface were less electron-dense than those of stratum basale cells. The chromatin was concentrated mainly towards the inner nuclear membrane. The nucleoli were eccentric and not distinct Fig 5. SEM of lymphoid tissue at higher magnification showing in majority of cells. The cells of stratum corneum high endothelial venule (H) and Iymphocytes. (x 1.85 K) presented small spicule like projections towards the free surface ar.c led to an interlocking arrangement (Fig 8). Only a few cells possessed nuclei passing through degenerativ tnges. The nuclei were pyknotic and containe •..,~chromatin material. The cytoplasm of these cells had predominance oftonofilaments whereas the cell organelles were not distinctly visible. In addition, a few small vacuoles were also observed. Reticular epithelium infiltration by non-epithelial cells was not uniform and presented a patchy reticulated appearance. It was comprised of strata basale, spinosum and superficiale in the region where large number of epithelial cell layers was present, and shared histological Fig 6. TEM showing columnar cells of stratum basale. (x 880) features as described earlier. However, the reticular epithelium was intimately associated with, lymphoid cells and also lacked distinct strata in the regions where only a few cell layers were observed. In these regions, some of the cells were narrow, elongated containing darkly stained, irregular shaped nuclei (Fig 9) and were attached by desmosomes. Their cytoplasm contained large number of polyribosomes, tonofilaments along with a few mitochondria, endoplasmic reticulum and glycogen granules. Their surfaces also showed small finger like microvillus projections which intermingled with those of adjacent cells. However, some of the cells towards the surface of the crypt were large sized Fig 7. TEM showing cells of stratum spinosum. (x 880) having electron lucent nuclei due to small condensation of these cells showed small sized microvilli and the of chromatin material towards the outer nuclear cytoplasm was rich in polyribosomes, tonofilaments, a membrane and possessed a centrally located nucleolus few mitochondria and endoplasmic reticulum (ER). (Fig 9). These cells contained comparatively less Their irregular elongated shaped nuclei had electron- distribution ofpolyribosomes and tonofilarnents but an dense chromatin localized in small patches with one or increased number of small vacuoles. Desmosomes may , 35 \

'.' Fi~. TEM showing cells of stratum corneum. (x 1100) Fig 10. TEM showing lymphocytes and plasma cell (S). (x 880)

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Fig 9. TEM showing superficial cells of reticular epithelium. Fig 11. TEM showing . lymphoid cells and follicular dendritic (x 1400) . ~l~. ~8~ be modulated during epithelial morphogenesis and the and secretory similar to foetal periderm and the stored extent of desmosomal adhesion may be affected by glycogen in the superficial strata may contribute towards external signals: transforming growth factor 15 can required energy source (Breathnach, 1981). The direct increase expression of desmosomes in epithelial cells, transepithelial access of antigen s may result in greater while the reduction in extra cellular Ca2+ concentration influx and recruitment of non-epithelial cells in a can disrupt the initial desmosomal adhesion mediated by particular patch, in contrast to a less strongly stimulated the calcium dependent cadherin family of adhesion area which may have retained stratified squamous non- molecules (Garrod, 1993). keratinised epithelium (Perry, 1994): The reticular epithelium provides a venue for The lymphoid tissue was mainly organized into •. intimate contact between antigens, specialized epithelial lymphoid foiIIicles along with isolated small aggregations cells, intraepitheliallymphocytes and antigen presenting without follicle in the subepithelial propria submucosa. cells, with subsequent antigen uptake and transport Most of follicles had darkly stained corona facing the (Palmer et al., 2009). The epithelial coverings of crypt epithelium, parafoIIicular area and lightly stained palatine tonsil and human foetus may share some of germinal centre and were constituted by lymphocytes, morphological and functional similarities as both were plasma cells, follicular dendritic cells (FDC), reported to be exposed to a hydrophilic environment in macrophages, (HEV s) and a similar way (Perry, 1994). The tonsillar epithelial cells blood capillaries along with a meshwork of fine reticular may exhibit energy demanding functions, both absorptive fibers (Kumar et al., 2008). The germinal centre

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of effective humoral antibody responses (Heinen et al., 1995). Parafollicular area had interdigitating cells, macrophages and high endothelial venules (REVs). The interdigitating cells (IDC) had different shaped nuclei with uniform distribution of fine chromatin material and eccentric nucleolus. Their cytoplasm showed predominance of. lysosomesand phagosomes. These cells possessed very large cytoplasmic processes which were folded and their cytoplasm containedrilaments~ <, Macrophages had characteristic distribution of cell organeIles with a predominance of Iysosomes, phagosomes and engulfed material (Fig 12). Monocyte- Fig 12. TEM showing macrophage with engulfed particles. (x 880) macrophage and dendritic cells help each other in processing and presentation of foreign antigens to the tonsillar lymphocytes (Yamamoto et al., 1988). The reticulated epithelium may provide favorable environment. for the contact between intraepithelial lymphocytes, antigen presenting cells and antigens (Perry, 1994). HEV s had columnar irregularly shaped endothelial cells with distinct basal lamina and a pericytic sheath (Fig 13). A few peripheral weaker layers of basal lamina provided circumferential compartments for migrating lymphocytes (Sunami-Kataoka et al., 2001). The cells with narrow bases, and blunt luminal free surfaces had small cytoplasmic processes and were attached to adjacent cells by desmosomes at irregular irxervals. These processes increased the surface area and might converted a laminar flow into a turbulent-oneand led to increase chances of contacts between endothelial cells and the circulating lymphocytes (Kumar and Timoney 2005 a). However, the narrow elongated nuclei with irregular surface showed smaller clumps or Fig 13. TEM showing high endcthelial venule. (x 880) chromatin localized towards outer nuclear membrane showed-small, medium and large lymphocytes, plasma (Fig 13). The cell organelles were comprised of varying cells and FDC (Figs 10, 11). The cytoplasm of plasma distribution of mitochondria, smooth and rough ER, cells had predominance of rough endoplasmic reticulum Golgi, multi vesicular and membrane bound bodies as whereas the nuclei had irregularly distributed large reported in the horse (Kumar and Timoney, 2005 b). aggregates of darkly stained chromatin. FDC possessed The varying shapes of high endothelial cells have been large number of cytoplasmic processes and their nuclei attributed to migrating lymphocytes (Ohmann, 1980). were also darkly stained because of large aggregates Caveolae as spherical invaginations of the plasma of electron dense chromatin material (Fig 11). FDC membrane and associated vesicles were either' single develop through transformation of fibroblastic reticulum or in clusters in the cytoplasm and attached to the cells during germinal centre formation (Heusermann et . endothelial cell surface as reported in the horse (Kumar al., 1980) and bind antigen-antibody complexes to their and Timoney, 2005 b). These have been reported to be surface for long periods and are essential for generation involved in endocytosis, transcytosis, signal transduction,

37 mechanotransduction, potocytosis and cholesterol cells in palatine tonsil. Adv. Oto-Rhino-Laryngol. 47: 97-100. Kumar, P., Kumar, Pawan and Kumar, Suraj. (2006). Light and trafficking (Stan, 2002). Transvascular and inter- scanning electron microscopic studies on the palatine tonsil endothelial migrations of Iymphocytes from the of the goat. Indian J. Anim. Sci. 76: 1004-1006. circulation to lymphatic parenchyma were observed as Kumar, Pawan, Mahesh, R., Singh, G and Nagpal, S.K. (2008). reported in the horse (Kumar and Timoney, 2005 a, b). Light microscopic studies on the palatine tonsil of sheep. The ultrastructural details of the palatine tonsil will be Haryana Vet. 47: 15-18. Kumar, Pawan and Timoney, 1.F. (2005 a). Histology and ultra- helpful not only to understand the pathogenesis of structure of the equine lingual tonsil. 11.Lymphoid tissue and various diseases where tonsils play crucial role but will associated high endothelial venules. Anat. Histol. Embryo!. also aid in the development ofvaccines targeted to this 34: 98-104. tonsil for efficient vaccine uptake and processing. Kumar, Pawan and Timoney, 1.F. (2005 b). Histology, immuno- histochemistry and ultrastructure of the equine palatine tonsil. Anat. Histo!' Embryol. 34: 192-98. Acknowledgements Ohmann, H.B. (1980). Electron microscopic study of the paracortical post-capillary high endothelial venules in lymph The SEM studies were carried out at the nodes of the normal calf. Cell Tissue Res. 212: 465-474. Sophisticated Instruments Facility for EM at theAIIMS, Palmer, M.V., Thacker, T and Waters, W.R. (2009). Histology, New Delhi. immunohistochemistry and ultrastructure of the bovine pa- latine tonsil with special emphasis on reticular epithelium. REFERENCES Vet. lmmunol. lmmunopathol. 127: 277-285. Perry, M.E. (1994). The specialized structure of crypt epithelium Breathnach, A.S. (1981). Ultrastructure of embryonic skin. Curr. in the human palatine tonsil and its functional significance. Problems Dermatol. 9: 1-28. J. Allat. 185: 111-127. Garrod, D.R. (1993). Desmosomes and hemidesmosomes. Curr. Slipka, 1. and Kotyza, F. (1987). 0 structure a funkci krypt Opinion Cell Bio/. 5: 30-40. patrovych mandli. Ceskoslovenska. Otolaryngologic. 36: Heinen, E., Bosselioir, A. and Bouzahzah, F. (1995). Follicular 209-216. dendritic cells. Origin and function. Curr. Top. Microbiol. Stan, R.V. (2002). Structure and function of endothelial caveolae. Immunol. 201: 15-47. Microsc. Res. Tech. 57: 350-364. Heusennann, U., Zurbom, K.H., Schroeder, L. and Stutte, H.J. Sunami-Kataoka, Y.Akagi, H., Nishizaki, K., Taguchi, T, Murakami, (1980). The origin of dendritic reticulum cell. An experimen- T and Ohtsuka, A. (200 I). Chondroitin sui fate proteoglycan tal enzyme-histochemical and electron microscopic study on at the basal lamina beneath high endothelial cells in human the rabbit . Cell Tissue Res. 209: 279-294. palatine tonsils: A light and electron microscopic study using Howie, A.J. (1980). Scanning and transmission electron micros- the cationic colloidal iron method. Arch. Histol. Cyto/. 64: copy of the epithelium of human palatine tonsil. J. Patho!' 535-543. Yamamoto, Y., Shuichiro, 0., Hiroaki, T, Kazuo, T and Sumiko, 130: 91-97. Kataura, A., Harabuchi, H., Matsuyama, H. and Yamanaka, N. M. (1988). Distribution and morphology of macrophages in (1992). Immunohistological studies on immunocompetent palatine tonsils. Acta Otolaryngol. (Stockh.) 454: 83-95.

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