LIGHT MICROSCOPIC OBSERVATIONS OF VALLATE PAPILLAE IN PRENATAL AND POSTNATAL PERIODS OF RABBIT (ORYCTOLAGUS CUNICULUS F. DOMESTICA)

Mirosława Kulawik¹, Szymon Godynicki², Hieronim Frąckowiak^1
1 Institute of Zoology, Department of Animal Anatomy, Poznań University of Life Sciences
² Department of Histology and Embryology, Institute of Zoology, Poznań University of Life Sciences

ABSTRACT

This study was conducted on 87 tongues of the rabbits Oryctolagus cuniculus f. domestica,of both sexes, which were collected at day 15, 18, 20, 22 and 26 of prenatal development (E) and at day 1, 15 and 30 of postnatal period (P). Samples of lingual tissue with the vallate papillae were prepared for observation under a light microscope (LM). The Masson-Goldner staining or HE were applied in this study. Light microscopic study revealed that the first primordia of vallate papillae were thickenings of the epithelium. The thickenings were observed in the period from E15 to E20. Starting from E22 the lamina propria of mucosa formed the body of developing vallate papillae. It was surrounded by a circular primary epithelial streak. From E26 the epithelium covering primordia of vallate papillae grew into the body of papillae, forming secondary epithelial streaks. The furrow of vallate papillae started to form from E22. The taste buds were observed from P1 in places where the primary epithelial streak cleaved, forming the furrow of the papilla. They were identified both in the epithelium covering the body of papilla and in the epithelium of the outer wall from the side of the furrow. Occasionally taste buds were observed in the epithelium covering the vallate papillae dorsally, where they accompany openings of excretory ducts of lingual glands. The epithelium covering the developing vallate papillae changed in the investigated periods from a three-layer epithelium into nonkeratinized stratified squamous epithelium.

Key words: Light microscope, morphogenesis, rabbit, tongue, vallate papillae.

INTRODUCTION

The morphology of the tongue in vertebrates varies between species. That is why many researchers study them, using light (LM) and scanning (SEM) or transmission electron microscopes (TEM). The structure of papillae of the tongue was described in adult: bat [4], common opossum [23], koala [16], Zaedyus pichiy [5], red-eared turtle [20], japanese badgers [28] and many other species. These studies give information about an animal’s environment, habits and diet. They are also important in taxonomic relationships.

Few studies are devoted to the development of the lingual papillae in sheep [26], pig [24, 25, 26], cat [27], mouse [2, 12, 14], rat [13], red sokoto goats [11]. Fujimoto [9] described morphogenesis of the foliate papillae in the rabbit. Authors described also development dynamics of vallate papillae [18]. The lack in scientific literature of a description of the morphogenesis of the vallate papillae of the tongue in the rabbit induced us to conduct this research. This work describes the LM features of morphogenesis of vallate papillae from day 15 of prenatal development to day 30 of postnatal period. The rabbit is a popular laboratory animal, and is often bred and kept at home similarly to dog and cat. It is essential to know how the structure of the tongue changes during prenatal and postnatal period of rabbit.

MATERIALS AND METHODS

The material for study consisted of 87 tongues of the rabbits Oryctolagus cuniculus f. domestica of both sexes. Tongues were collected at day 15, 18, 20, 22 and 26 of prenatal development (E) and at day 1, 15 and 30 of postnatal period (P). The age of fetuses was determined on the basis of the CRL (Crown-Rump-Length) value [7].

For observations using a LM, tongues were fixed in 10% buffered formalin. Tissue samples with vallate papillae were dehydrated in a graded series of ethanol (30-100%), embedded in paraplast® and sliced in three planes, i.e. in the sagittal, transverse and horizontal planes into sections with thickness from 3 to 5 µm. The samples were cut using a rotary microtome Leica RM 2055 (Germany). The Masson-Goldner and HE staining were applied in this study. The specimens were examined under a light microscope Jenaval (Carl Zeiss Jena, Germany). Studies on animals were conducted with approval by the Local Ethics Committee, permission N. 4/2000.

RESULTS

At E15 two round and symmetrically located primordia of vallate papillae in the form of epithelial thickenings were observed on the root of the tongue. They were formed by up to 8 layers of epithelial cells. The epithelial thickenings extended into the mesenchyma. The surface of the tongue in places where epithelial thickenings were found was even (Fig. 1).

From prenatal day 18 to 20 the epithelial thickenings were formed by up to 10-13 layers of cells. The surface of the tongue in sites of the epithelial thickenings was uneven (Fig. 2).

Starting from E22, the structure of developing vallate papillae in the rabbit changes dramatically. The body of primordium of vallate papilla was surrounded by a circular primary epithelial streak. The epithelium covering primordia of vallate papillae was composed of 3 layers of cells. The basal layer was formed by high, cuboidal cells with large oval nuclei. The intermediate layer was formed by lower cells with round nuclei and the superficial layer was composed of flattened cells. High and light superficial cells were found in the site of primary epithelial streak. Some of them had two nuclei. The first symptoms of the formation of the papillary furrow were observed at the surface of the tongue, in the site of the circular primary epithelial streak. In this site a slight depression was observed (Fig. 3).

At E26 the circular primary epithelial streaks elongated more in some places and extended into the underlying muscles of the tongue. Primordia of vallate papillae were covered by the epithelium composed of 4-5 layers of cells, which became flattened towards the surface. Secondary epithelial streaks, ingrowing into the body of primordia were observed to appear in the analyzed period. The developing vallate papillae were round in outline and surrounded by shallow circular depression (Figs. 4, 5).

From P1 it was observed that the circular primary epithelial streaks became partly cleaved,  resulting in  closed spaces   not contacting with the surface of the tongue. These spaces were lined with squamous epithelial cells. The taste buds could be seen in the epithelium of the cleaving primary epithelial streaks. Primary epithelial streaks, which were elongated more in some places, formed an excretory ducts of the lingual glands. Developing vallate papillae were covered by nonkeratinized stratified squamous epithelium. The surface of the vallate papillae was grooved (Figs. 6, 7).

At day 15 of postnatal period, each round in shape vallate papilla was surrounded by a distinct furrow, formed as a result of cleavage of the primary epithelial streak surrounding the body of the papilla. The outer wall of the papillae was around the furrow of vallate papillae. The epithelium covering papillae was a nonkeratinized, stratified squamous epithelium. The epithelium formed numerous secondary epithelial streaks, differing in length, which grew into connective tissue. In the epithelium covering vallate papillae from the side of the furrow and in the epithelium of the outer wall of the papillae taste buds were found (Fig. 8).

At P30 some secondary epithelial streaks which were varied in shape extended as far as the bases of the papillae. Openings of the lingual glands were visible on the bottom of the furrows of the vallate papillae. Moreover, it was shown that some excretory ducts of these glands opened directly on the surface of the papillae, where they were accompanied by taste buds. Numerous and densely arranged taste buds were found both in the epithelium of vallate papillae and in the epithelium of the outer wall of papillae from the side of the furrow. The surface of the vallate papillae was grooved (Fig. 9).

DISCUSSION

The investigation using LM has shown that the first symptoms of the formation of vallate papillae were epithelial thickenings. Their shape and structure were different from the vallate papillae described previously in adult rabbits [17]. In the periods from E15 to E20, the primordia of vallate papillae were similar to those described in day 12 of prenatal development in mice by Jitpukdeebodintra et al. [14]. Ahpin et al. [2] also described in mice at E13 a similar structure of the primordia of vallate papillae.

Starting from E22, the structure of developing vallate papillae in the rabbit had a similar form to the primordia of these papillae during morphogenesis, which were observed in the golden syrian hamster [21], cat [27] and mouse [14].

The epithelium covering the developing vallate papillae in the rabbit changes from a 3-layer epithelium into the nonkeratinized, stratified squamous epithelium. In the hamster, Miller and Chaudhry [21] observed in the initial period of development of vallate papillae, i.e. at E13.5, also a thick epithelium composed of 2-3 cell layers. Changes in epithelium structure means that its thickness is changing. The dynamics of growth in thickness of epithelium and height of developing vallate papillae were examined by Kulawik and Godynicki [18].

The process of formation of the circular furrow of vallate papilla in the rabbit runs similarly to that described in the scientific literature in the pig and cat [24, 27]. This process is analogous to development of the furrows of foliate papillae. It was described by Fujimoto et al. [9].

The distribution of taste buds in the epithelium of vallate papillae is a feature that changes in accordance with species.  The rabbit taste buds are located both in the epithelium of vallate papillae and in the epithelium of the outer wall of papillae from the side of the furrow. Taste buds in the golden syrian hamster are situated similarly [21]. In turn, in the cat [27] and pig [25] taste buds were observed solely in the epithelium covering vallate papillae at their lateral surfaces. Moreover, the rabbit taste buds are sporadically observed in the epithelium covering vallate papillae on the dorsal side. Similar observations in the goat were reported by Kumar et al. [19].

The vallate papillae in the rabbit were round in contour. A similar shape of papillae was observed in the bat [4] and formosan serow [3]. For comparison, in the Zaedyus pichiy [5], blind mole rat [15] and Dasypus hybridus [6], the vallate papillae found on the tongues were oval in shape. Agungpriyono et al. [1] showed on the tongue of a lesser mouse deer, only in one individual, two types of papillae: elongated and round-shaped types.

The vallate papillae, being gustatory papillae, begin to form earlier than mechanical papillae. A significant role in such a mechanism of development is played by sensory nerves [8, 29]. It was also confirmed experimentally by denervation of taste papillae [10, 22].

These findings revealed that the morphogenesis of vallate papillae of the rabbit tongue is a complicated process, and showed unique features of developing papillae.

CONCLUSIONS

1. The first primordia of vallate papillae in the form of epithelial thickenings appeared at E15.
2. Starting from E22, lamina propria of the mucosa forms the body of developing vallate papillae.
3. The furrow of vallate papillae begins to form at E22, as a result of cleavage of the primary epithelial streak.
4. The epithelium covering the developing vallate papillae in the period from E22 to P30 changes from a 2-3 cell layers epithelium into a nonkeratinized, stratified squamous epithelium.

ACKNOWLEDGMENT

Financial support by KBN, grant No 5P06D01719

REFERENCES

Accepted for print: 3.07.2013

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