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Revista chilena de anatomía

versión impresa ISSN 0716-9868

Rev. chil. anat. v.16 n.1 Temuco  1998

http://dx.doi.org/10.4067/S0716-98681998000100009 

LIGHT AND SCANNING ELECTRON MICROSCOPIC STUDY OF THE VALLATE PAPILLAE OF THE OPOSSUM (Didelphis albiventris)

ESTUDIO A LOS MICROSCOPIOS DE LUZ Y ELECTRONICO DE BARRIDO DE LAS PAPILAS VALADAS DEL OPOSUM (Didelphis albiventris)

* Marcelo Martinez,
** Francisco E. Martinez
** Patrícia F. F. Pinheiro
** Camila C. D. Almeida
** Heraldo Lorena Guida
*** II-Sei Watanabe

* Department of Morphology and Pathology, Federal University of São Carlos, (UFSCar), São Carlos/SP and Department of Anatomy, Institute of Biosciences, State University of São Paulo, (UNESP), Rubião Júnior, Botucatu/SP, Brazil.
** Department of Anatomy, Institute of Biosciences, State University of São Paulo, (UNESP), Rubião Júnior, Botucatu/SP, Brazil.
***Department of Anatomy, Institute of Biomedical Sciences, University of São Paulo, (USP), São Paulo/SP, Brazil. The paper was supported by CNPq.

SUMMARY: The morphological characteristics of the epithelium and the connective tissues of the vallate papillae of opossum were observed by light and scanning electron microscopy. The NaOH cell maceration method were used to visualize the connective tissue architecture. The posterior dorsum of the tongue of opossum possessed 2 vallate papillae. The histological sections showed that the covering epithelium of the vallate papillae is keratinized stratified squamous type with associated taste buds and small vallum. These gustatory structures showed circular or oval shapes. SEM images demonstrated the epithelial cell surfaces with microridges, distinct intercellular borders and taste pores. The dense connective tissue is composed by a meshwork of collagen fibers and showed papillae with different shapes. The architecture of the lamina propria of the vallate papillae is pine-cone-like shape.

KEY WORDS: 1. Epithelium; 2. Connective tissue; 3. Collagen fibers; 4. SEM.

INTRODUCTION

The mucous membrane of the dorsum of the tongue of marsupials frequently possess filiform, fungiform, foliate and vallate papillae (KRAUSE & CUTTS 1982). The lingual papillae is related to specific functions like gustation and lining. The morphological aspects of these important structures are variable according to the analysed species as demonstrated by FERNANDEZ et al. (1978); STEFLIK et al. (1983); IWASAKI & MIYATA (1989); KOBAYASHI et al. (1987, 1988, 1989); KOBAYASHI (1990, 1992); AGUNGPRIYONO et al. (1995); WATANABE et al. (1992, 1995); MORAIS et al. (1996). KRAUSE & CUTTS (1982) related the tongue morphology of opossum Didelphis virginiana using light and scanning electron microscopics method.

Previously in Didelphis albiventris, MARTINEZ et al. (1995b) and MARTINEZ et al. (1997) described the characteristics of the hard palatine mucosa.

Taste buds are found in the epithelial layer of the tongue mucosa of foliate, fungiform and vallate papillae (KOBAYASHI et al., 1987; KOBAYASHI et al. 1989; KOBAYASHI & WANICHANON 1992). WATANABE et al. (1997) studied the Von Ebner`s gland of the Cebus apella tongue by scanning electron microscopy. According to FAHRENHOLZ (1937), these glands are related to gustation because their secretions wash out the taste buds (BARALDI & BOURN, 1953) removing the alimentary debris.

The purpose of the present paper is to examine the histological and ultrastructural features of the vallate papillae of opossum and to compare to other species described in the literature.

MATERIAL AND METHOD

A total of 10 adult opossum, Didelphis albiventris, of both sexes were used. The tongues were washed with distilled water and dissected. For light microscopy, 4 specimens were fixed in Bouin's solution for 12 h, dehydrated in a graded series of ethanol and embedded in paraffin routinely. The frontal or sagittal sections of five micrometers thickness were stained with haematoxylin and eosin and Van Gieson-orcein.

For observations on the dorsum of the tongue we used 3 tongues and the conventional SEM method. For studies of the lamina propria by SEM, 3 specimens were fixed with modified Karnovsky solution, containing 2.5% glutaraldehyde, 2.0% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2. Then, the specimens were rinsed and immersed in a 10% aqueous solution of NaOH for 4-7 at room temperature according to the method of OHTANI (1987, 1992). The samples were rinsed in distilled water and post-fixed with 1% osmium tetroxide solution for 2 h. After immersion in a 1.0% aqueous solution of tannic acid for 1 h and 30 minutes (MURAKAMI, 1974), they were rinsed again in distilled water for several hours and dehydrated in an increasing series of ethanol. The samples were dried in a critical point using CO2 liquid (Balzers CPD-010), coated with gold ions in a SEM coating (Balzers MED - 010) and examined under a Philips FEM 515 SEM at 15 Kv.



Fig. 1. Light micrograph of tongue mucosa of opossum showing the vallate papillae. Epithelial layer (**), lamina propria (*) and vallum (arrows). H.E. X10.



Fig. 2. Shows at higher magnification, the connective tissue papillae (*) of the lamina propria (**). H.E. X20.



Fig. 3. Frontal section of the vallate papillae showing several taste buds (arrows) and the vallum (*). Orcein-Van Gieson. X20.



Fig. 4. Shows the taste buds at higher magnification (arrow). Orcein-Van Gieson. X40.

RESULTS

Light microscopy

The dorsum of the opossum tongue possessed two vallate papillae situated on the posterior third. These papillae had oval shape. The frontal histological sections showed that these structures were lined with keratinized stratified squamous epithelium, composed by basal, spinosum, granulosum and corneum layers (Figs. 1, 2). The underlying lamina propria is formed by dense connective tissue (Figs. 2, 3). This lamina propria showed connective tissue papillae of different sizes and with general finger-like shape (Figs. 1, 2). Fig. 1 also demonstrated the small vallum of the papillae. On the lateral part of the stratified epithelium several taste buds with circular or oval shapes can be noted. (Figs. 3, 4).

Scanning electron microscopy

Scanning electron microscopic observations confirmed that the vallate papillae of opossum have oval form, small vallum (Fig. 5) and are lined with keratinized stratified squamous epithelium. The corneum layer possessed numerous desquamating cells (Fig. 5). The epithelial cells showed polygonal shape, distinct intercellular border and microridges (Fig. 6). These microridges were not so developed. The vallate papillae possessed taste pores with circular shape (Fig. 6).

When the epithelial layer was removed, the architecture of the connective tissue was observed. The lamina propria of the vallate papillae were pine-cone-like (Fig. 7). These structure were composed by several connective tissue papillae with different shapes (Fig. 8). The pine-cone-like papillae are surronded by walls of connective tissue (Fig. 7). At higher magnification it is possible to note that the papillae were constituted by collagen fibers disposed in several directions forming a complicated meshwork (Fig. 9).

DISCUSSION

The present paper described the morphological features of the vallate papillae of the opossum Didelphis albiventris.

Several methodologies of macerations were developed and described in the literature for scanning electron microscopy. HORSTMAMN (1954) used acetic acid, KLEIN-SZANTO & SCHROEDER (1977) used EDTA, LOW & MCLUGAGE (1984) used ultrasonication and KOBAYASHI et al. (1987) employed HCL to remove the epithelial tissue. Our work used NaOH maceration method to remove the epithelial layer permiting the visualization of the connective tissue of the vallate papillae. This technique introduced by OHTANI (1987, 1992) eliminates the celular elements more effectively than any other method and preserve the integrity of the lamina propria.

The mucosa of the oral cavity can be divided into three parts: masticatory, lining and specialized mucosae. The masticatory mucosa includes hard palate and gingiva; the lining mucosa corresponds to lip, cheeck and soft palate while the dorsum of the tongue is the specialized mucosa. Several investigators have been studied the ultrastructural differences between these mucosae. KOBAYASHI et al. (1987) in the newborn dog tongue, KOBAYASHI et al. (1988) in the cat papillae, WATANABE et al. (1992) in the armadillo tongue, NAKANO (1991, 1992), MARTINEZ et al. (1995a,b) in the armadillo lip, MARTINEZ et al. (1997) in the opossum hard palate.

The number of vallate papillae is variable between the species. The posterior third of the dorsum of the opossum tongue possessed a pair of vallate papillae. This quantity is also observed in insectivores (SONNTAG, 1923), in great anteater (KUBOTA et al., 1962), in armadillo (MORAIS & WATANABE, 1988). According to KRAUSE & CUTTS (1982), the opossum Didelphis virginiana showed three vallate papillae with triangular arrangement. Others species like several primates, including man, (KUBOTA & HAYAMA, 1964) or elephants (KUBOTA, 1967) can have four or more vallate papillae.

The vallate papillae of the opossum is lined by keratinized stratified squamous epithelium. The superficial cells had polygonal shape with distinct borders and microridges. Similar characteristics were reported in opossum Didelphis virginiana (KRAUSE & CUTTS, 1982), horse and cow (CHAMORRO et al. 1986), armadillo (MORAIS et al. 1988), rat snake (IWASAKI & KUMAKURA, 1994) and lesser mouse deer (AGUNGPRIYONO et al., 1995). These microridges must protect the superficial cells keeping more quantity of mucous between adjacent cells (ANDREWS, 1976).

Our results demonstrated numerous taste buds on the lateral epithelial layer of the vallate papillae. These results coincide with those reported in the opossum Didelphis virginiana by KRAUSE & CUTTS and in the lesser mouse by AGUNGPRIYONO et al. KOBAYASHI & WANICHANON described taste buds in the lateral epithelium of the vallate papillae of the treeshrew. Taste buds are also observed in fungiform, foliate papillae (KOBAYASHI, 1992; AGUNGPRIYONO et al.), hard palate, soft palate, oropharynx, glosso-palatine arches and epiglottis (MÄRK, 1940; KAPLICK, 1953).



Fig. 5. SEM image of the vallate papillae of opossum tongue. Shows desquamating cells (big arrow), vallum (*) and filiform papillae (small arrow). X265.



Fig.6. SEM image of the polygonal epithelial cells with distinct borders (arrow head), microridges and taste pores (arrow). X2750.



Fig.7. NaOH treated specimen. General view of the connective tissue of the vallate papillae with pine-cone-like shape and vallum (*). X300.



Fig.8. Shows the numerous connective tissue papillae. (*) X900.


Fig.9. At higher magnification, it shows the meshwork of collagen fibers of the connective tissue of the vallate papillae. (*) X1800.

Our findings described that the connective tissue of the vallate papillae is pine-cone-like shape with several small connective tissue papillae surrounded by walls of connective tissue. Similar results were obtained by KOBAYASHI & WANICHANON in the lingual papillae of treeshrew. However, the vallate papillae of this specie did not showed surrounded by walls of connective tissue but by numerous connective tissue papillae of different shapes and arrangements.

The lamina propria of the vallate papillae of opossum is composed by a meshwork of collagen fibers and agreed with the results of KOBAYASHI & WANICHANON, WATANABE et al. (1995) and MORAIS et al. (1996) in the vallate papillae of treeshrew, Macaca fuscata and Dasypus novemcinctus, respectively. The lamina propria contain blood vessels and sensory nerve fibers. The connective tissue papillae enlarge the adhesion between connective and epithelial tissues and distribute the nerve fibers closer to the epithelial layer (KOBAYASHI, 1990).

According to KUBOTA (1988), there is an intimate relationship between habitat, the feeding habits and the development of the vallate papillae. In the process of species evolution, the number of the vallate papillae increased like occured in primates. Primitive monkeys that live only in forest have three vallate papillae while other groups of monkeys have 7 or 8 vallate papillae. These species extend their habitat to the plain. So, there is a correlation with the opossum that is a primitive animal.

RESUMEN: Las características morfológicas del epitelio y tejido conjuntivo de las papilas valadas del oposum fueron observadas a través de los microscopios de luz y electrónico de barrido. Fue utilizado el método de maceración de las células con NaOH para visualizar la arquitectura del tejido conjuntivo. El dorso posterior de la lengua del oposum posee dos papilas valadas. Las secciones histológicas mostraron que la cubierta epitelial de estas papilas es del tipo escamoso, estratificado, queratinizado y asociadas con yemas gustativas y papilas pequeñas. Estas estructuras gustativas mostraron formas circulares u ovales. Imágenes al SEM mostraron la superficie de las células epiteliales con microcrestas, diferentes de los márgenes intercelulares y poros gustativos. El tejido conjuntivo denso está compuesto por unas redes de fibras colágenas y mostró papilas de diferentes formas. La arquitectura de lámina propia de las papilas valadas es de forma de un cono.

PALABRAS CLAVE: 1. Epitelio; 2. Tejido conjuntivo; 3. Fibras colágenas; 4. SEM.

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Correspondence to:
Prof. Dr. Marcelo Martinez
Departamento de Morfologia e Patologia
Universidade Federal de São Carlos
Rod. Washington Luís Km 235 s/n
CEP: 13.565.905,
São Carlos / SP
BRASIL

Tel: (016) 2608330
Fax: (016) 2608326
Email: martinez@power.ufscar.br

Recibido : 01-12-1997
Aceptado : 22-04-1998

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