THE DEVELOPMENT OF THE MUCOUS MEMBRANE OF THE TONGUE WITH SPECIAL EMPHASIS ON THE DEVELOPMENT OF FUNGIFORM PAPILLAE IN THE PRENATAL LIFE OF THE RABBIT

Mirosława Kulawik
Institute of Zoology, Department of Animal Anatomy, Poznań University of Life Sciences

ABSTRACT

Investigations were conducted on 74 tongues, collected from rabbits being in the 15^th, 18^th, 20^th, 22^nd and 26^th day of prenatal life. On the basis of observations under a light microscope the development of the mucous membrane on the dorsum of the apex and body of the tongue was examined, with special emphasis on the development of fungiform papillae.

The first symptoms of the formation of the primordia of fungiform papillae in the form of thickenings in the epithelium were found on the dorsum of the apex and on the dorsum of the body of the tongue at day 18 and next at day 20 of prenatal life. At day 22 of prenatal life primordia of fungiform papillae already have a connective tissue core and are covered with a two-layer epithelium. In that period primordia of fungiform papillae are well-marked on the surface of the dorsum of the apex and body of the tongue. Next at day 26 of intrauterine life primordia of fungiform papillae are bigger and more distinctly visible. They are covered by the epithelium consisting of 3-4 layers of cells. In the same period, in the epithelium of the primordia of fungiform papillae taste buds were found.

At day 15, 18 and 20 of intrauterine life the mesenchymal tissue was observed under the epithelium, found on the well-marked basement membrane.

At day 22 of prenatal life in the rabbit in the connective tissue forming the lamina propria of the mucosa of the tongue delicate collagen fibers and elastic fibers were observed. At day 26 of prenatal life in the rabbit collagen fibers are seen as more marked, while elastic fibers are still very delicate. The lamina propria of the lingual mucosa starting from day 22 of intrauterine life forms protrusions, which are the connective tissue core of the primordia of fungiform papillae.

Results of histochemical studies showed at day 15, 18 and 20 of prenatal life the presence of glycogen in the cytoplasm of cells in the epithelium and the mesenchymal tissue. At day 22 of prenatal life glycogen was already identified in the light-coloured stained superficial cells of the epithelium, whereas at day 26 of prenatal life no glycogen was detected in the examined structures of the lingual mucosa in the rabbit.

Key words: rabbit, tongue, mucous membrane, fungiform papillae, development.

INTRODUCTION

In available literature reports may be found on the presence of fungiform papillae on the tongue of some amphibian species, e.g. Rana catesbeiana [27], and in reptiles, e.g. Alligator mississippiensis [32]. On the basis of information obtained from literature it is also known that in some reptiles fungiform papillae were not found on the tongue, as e.g. is the case with Elaphe climacophora [16] and Elaphe quadrivirgata [14]. Fungiform papillae were no shown on the tongue of investigated bird species, e.g. the chicken [2, 13], goose [17] and penguin [21]. Hence, these papillae are not a permanent element of the structure of the mucous membrane of the tongue. Fungiform papillae belong to the group of gustatory papillae, which are found on the tongue in the rabbit along with foliate and vallate papillae [1, 5].

Studies conducted so far on the mucosa of the tongue covered various animal species, among which many papers concerned laboratory animals such as the rat [33], guinea pig [12] and mouse [18]. In the whole range of these publications there are no reports on the development of the mucous membrane in the rabbit, which – being one of the most popular laboratory animals [5, 36] deserves to have the knowledge supplemented in this respect. Numerous literature reports have been devoted to its anatomical [22, 34] and physiological features [9, 10, 20], used not only in laboratories for experimental purposes, but also in breeding practice since the rabbit as a commercial animal supplies meat, wool and skins [11].

MATERIAL AND METHODS

Experimental material consisted of 74 tongues of domestic rabbits, Oryctolagus cuniculus f. domestica, of both sexes. Tongues for analyses were collected from rabbits being in day 15, 18, 20, 22 and 26 of prenatal life. Material for the examinations came from a slaughterhouse. The age of the animals was determined on the basis of the CRL value (Crown-Rump-Length), characterizing the development in the rabbit [7]. Tongues for analyses under a light microscope were fixed in 10% neutralized formalin or in Bouin’s solution. Tissues for analyses collected from the apex and body of the tongue were dehydrated in series of alcohols with gradually increasing concentrations (from 50% to 96%), embedded in paraffin and sliced in three planes, i.e. median, transverse and dorsal, into series slices with the thickness of 3 to 5 ľm. The following staining methods were applied in the study: Masson-Goldner, orcein, resorcin-fuchsin and PAS, along with control sample stained using amylase and dimedone [3, 30, 39].

RESULTS

Day 15 of prenatal life

As a result of observations under a light microscope it was shown that at day 15 of prenatal life in the rabbit no fungiform papillae or their primordia are found on the dorsum of the tongue. The dorsum of the apex and body of the tongue is covered with a two-layer epithelium. High cylindrical cells of the basal layer of the epithelium have large oval or round nuclei. The superficial layer of the epithelium consists of lower cells. Their nuclei are oval or flattened. Cells in the stage of mitosis are found in the epithelium. Under the epithelium on a well-marked basement membrane the mesenchymal tissue is found. Its cells have large oval nuclei and slight amounts of cytoplasm (Fig. 1).

In the cytoplasm of cells in the epithelium and the mesenchymal tissue irregularly distributed PAS-positive granules were observed. Negative results of PAS staining, obtained after a prior α-amylase treatment and positive results after the application of dimedone blocking showed the presence of glycogen (Fig. 2).

Day 18 of prenatal life

At day 18 of prenatal life in the rabbit, on the dorsum of the apex and on the dorsum of the body of the tongue in its anterior part places were observed, in which the epithelium is thickened, sometimes forming protruding structures on the surface of the tongue. They usually consist of approx. 4-5 layers of cells. Cells of the basal layer in these thickened parts are high and have oval nuclei. The closer to the surface, the lower the cells become and the more rounded or flattened their nuclei become. Some epithelial thickenings extend slightly into the mesenchymal tissue (Fig. 3). Thickenings of the epithelium are distributed irregularly. Apart from the observed thickenings, the epithelium covering the dorsum of the apex and the dorsum of the body of the tongue is composed of 2-3 layers of cells.

In the cytoplasm of epithelial cells on the dorsum of the tongue and in the mesenchymal tissue glycogen was detected (Fig. 3), similarly as it was the case in the previous analyzed period.

Day 20 of prenatal life

Examinations under a light microscope showed that at day 20 of prenatal development in the rabbit irregularly distributed epithelial thickenings are still found on the dorsum of the apex and on the dorsum of the body of the tongue. Their highest concentration is located at the end of the apex of the tongue. On the body of the tongue thickenings of the epithelium are located in its anterior part. The number of the layers of epithelial cells in epithelial thickenings found on the dorsum of the apex of the tongue ranges from 4 to 6, whereas on the body of the tongue it ranges from 5 to 6. These structures sometimes extend into the mesenchymal tissue and at times they protrude distinctly over the surface of the tongue (Fig. 4). Apart from the thickenings, the epithelium covering the dorsum of the apex and body of the tongue consists of 2-3 layers of cells, similarly as in the previous investigated period in the life of the rabbit.

Below the epithelium, which is found on the distinctly visible basement membrane, the mesenchymal tissue was still observed.

Also in this prenatal developmental stage glycogen was detected, distributed irregularly in the cytoplasm of the cells in the epithelium and the mesenchymal tissue. No connective tissue fibers were shown.

Day 22 of prenatal life

At day 22 of intrauterine life in the rabbit as a result of observations of histological specimens under a light microscope on the dorsum of the apex and of the body of the tongue primordia of filiform papillae were shown, between which larger primordia of fungiform papillae are located (Fig. 5). The arrangement of primordia of fungiform papillae on the dorsum of the apex and of the body of the tongue is similar to the distribution of epithelial thickenings, the presence of which was shown for day 18 and 20 of prenatal life. Moreover, at day 22 of prenatal life primordia of fungiform papillae were located laterally to the forming the torus of the tongue, where are arranged linearly, along with the longitudinal axis of the tongue. The epithelium covering the primordia of fungiform papillae consists of two layers of cells. The basal layer is formed by cylindrical cells with large oval nuclei, while the superficial layer is created by flat cells with flattened nuclei. Sometimes on the surface of the primordia of fungiform papillae cells are found, the cytoplasm of which is only slightly stained. These cells are always observed on the junctions of adjacent primordia of lingual papillae. No mitoses were observed in the epithelium covering the primordia of fungiform papillae on their dorsal side.

Below the basement membrane the lamina propria of the mucous membrane is found, which forms protrusions, constituting the connective tissue core of the primordia of lingual papillae. In the apical part of some primordia of fungiform papillae their connective tissue core is widened (Fig. 5). Primordia of fungiform papillae visible in the dorsal sections are round, similarly to their connective tissue core. At the investigated day 22 of prenatal development the first delicate collagen fibers and elastic fibers were identified. In the lamina propria of the mucous membrane blood vessels are visible. Surface cells of the epithelium, the cytoplasm of which is only slightly stained, contain glycogen, as it was shown through histochemical reactions.

Day 26 of prenatal life

Primordia of fungiform papillae are distributed irregularly on the dorsum of the apex and of the body of the tongue, in the arrangement similar to that observed at day 22 of intrauterine development. Primordia of fungiform papillae widen in their apical part. The epithelium covering these primordia consists of 3-4 layers of cells. Cells in the basal layer are high and have oval nuclei. In the layers located higher the cells become lower and their nuclei are round or slightly flattened. In the superficial layer cells are flat and have strongly flattened nuclei (Fig. 6). In the epithelium covering the primordia of fungiform papillae on their dorsal side taste buds were found in the investigated period. Dorsal sections of these specimens showed that primordia of fungiform papillae and their connective tissue core are round in outline.

The lamina propria mucosae protrudes, forming connective tissue cores for primordia of lingual papillae, to which blood vessels are directed. As it was shown in this study, collagen fibers, more distinct than in the previous period, are present in the lamina propria of the mucosa. Elastic fibers are still very delicate (Fig. 7). Along with the atrophy of lightly stained superficial cells of the epithelium, histochemical reactions detecting the presence of glycogen give negative results.

DISCUSSION

On the basis of observations under a light microscope the obtained results made it possible to follow the dynamics of developmental changes in the mucous membrane of the tongue in the period from day 15 to day 26 of prenatal life in the rabbit. The study focused on the development and morphology of primordia of fungiform papillae.

On the basis of observations of specimens sliced in the median, transverse and dorsal planes it was shown that already at day 15 of prenatal life in the rabbit the tongue is developed on the basis of its primordia, and its parts, i.e. the apex, body and root, may be distinguished. The epithelium covering the dorsum of the examined apex and body of the tongue has two layers at day 15 of prenatal life in the rabbit. No primordia of fungiform papillae are observed on the tongue at day 15 of prenatal life in the rabbit. The mesenchymal tissue is located under the epithelium, which is found on a well-visible basement membrane. The lamina propria of the mucosa is not yet formed.

At day 18 and day 20 of prenatal development in the rabbit, the dorsum of the apex and the dorsum of the body of the tongue are covered by the epithelium consisting of 2-3 layers of cells. Additionally, thickenings of the epithelium were observed, formed by 4-5 layers of cells at day 18 and by 4-6 layers of cells at day 20 of prenatal development. The observed thickenings of the epithelium are probably the first forms of primordia of fungiform papillae. Descriptions of morphogenesis of lingual papillae may be found in available literature concerning the mouse, rat and human based on observations under a scanning electron microscope. As a result of these investigations it was shown that at early developmental stages domed structures are found on the surface of the tongue, which were defined as primordia of the fungiform papillae [15, 19, 38]. In studies on the very early development of fungiform papillae there are practically no reports in literature which would compare the results obtained using a scanning electron microscope with the results of observations under a light microscope. The histological structure of the primordia of fungiform papillae, as they are called by the authors of the studies [15, 19, 38], is not known. It is not known whether they are only thickenings of the epithelium as in this study or whether they are primordia of fungiform papillae, which already have their connective tissue core. There are also no reports in literature indicating that on the tongue during the morphogenesis of lingual papillae structures were observed, consisting entirely of epithelial cells, which were referred to in this study as thickenings of the epithelium, which are probably early primordia of fungiform papillae.

At day 15, 18 and 20 of prenatal development in the rabbit, in the cytoplasm of epithelial cells as well as the mesenchymal tissue irregularly distributed glycogen was detected. Glycogen was also shown in places where at day 18 and 20 of prenatal life thickenings of the epithelium were observed. At day 22 of prenatal development in the rabbit glycogen is found only in light cells of the superficial layer of the epithelium, while at day 26 of prenatal development no glycogen was detected. Along with the atrophy of cells with slightly stained cytoplasm reactions detecting the presence of glycogen give negative results. On the basis of available literature it is known that glycogen is present also in epithelial cells of other organs of the alimentary tract during their prenatal development. Its presence was shown in the esophagus in the mouse [4], the stomach in the mouse [28] and in the small intestine in the sheep [35]. Mackenzie [24] in his study reported the presence of glycogen in the epithelium of the oral cavity in the human up to approx. the fifth month of prenatal development, when its disappearance was observed in the areas which are beginning to cornify. That author suggested that glycogen in the epithelium of the oral cavity serves the function of energy storage for further metabolic activity, connected e.g. with the cornification process. However, conclusive theories on the role of glycogen in the developmental period of the above mentioned organs as well as glycogen shown in this study on the tongue are still to be formulated.

At day 22 of prenatal life in the rabbit, on the basis of observations under a light microscope results were obtained showing the presence of primordia of fungiform papillae on the dorsum of the apex and of the body of the tongue, with numerous primordia of filiform papillae neighbouring those primordia. Primordia of fungiform papillae are higher than the primordia of filiform papillae and they protrude distinctly over the surface of the tongue. They are distributed over the area similar to that occupied in the previous examined periods by thickenings of the epithelium. Moreover, at day 22 of prenatal development the presence of primordia of fungiform papillae, arranged linearly, was shown on the sides of the dorsum of the body of the tongue. The largest concentration of primordia of fungiform papillae is observed on the apex of the tongue, similarly as in the rat [19], mouse [15], goat [23], cat [26, 29] and Petaurista leucogenys [6].

Available literature sources reported that in the studies on the morphogenesis of lingual papillae the focus was on factors stimulating their development. Several studies concentrated on the innervation of gustatory papillae. Whitehead and Kachele [37] showed that in the formation of gustatory papillae a considerable role is played by sensory innervation. Studies consisting in the denervation of fungiform papillae conducted by Nakashima et al. [25] in the rabbit confirmed the dependency between the existence of fungiform papillae and taste buds found in their epithelium and innervation. Those authors showed that denervation leads to the atrophy of taste buds and a transformation of the structure of fungiform papillae into filiform-like papillae, in which changes were observed leading to the cornification of the epithelium, which is characteristic of filiform papillae on their anterior and posterior sides. Similar results connected with the atrophy of taste buds after denervation were obtained in the rat [8]. Studies by those authors indicate that also in the complex process of the formation and development of fungiform papillae and their taste buds in the rabbit innervation plays an important role.

At day 22 of prenatal life in the connective tissue marking the area of the lamina propria of the mucosa the first collagen and elastic fibers are observed. The lamina propria of the mucosa forms connective tissue prominences, which are cores for primordia of fungiform papillae. The earliest forms of primordia of fungiform papillae, the so-called thickenings of the epithelium, initiating the morphogenesis of fungiform papillae arranged on the sides of the dorsum of the body of the tongue, are probably formed between day 20 and day 22 of prenatal development, since in the earlier periods they are not observed. Such a situation needs to be clarified through further supplementary studies, covering the above mentioned period in the prenatal life in the rabbit.

The obtained results lead to a question how the thickenings of the epithelium were reorganized into primordia of fungiform papillae, which have a connective tissue core. Obviously an important role in the development of gustatory papillae is played by the nerves [8, 25, 37] and the occurring interactions between the epithelium and the mesenchymal tissue, as it was indicated by Mackenzie [24] and Sanders [31].

The epithelium covering primordia of fungiform papillae at day 22 of prenatal life has two layers. In some places on its surface light stained cells are found. At day 26 of prenatal life in the rabbit, primordia of fungiform papillae are covered by the epithelium consisting of 3-5 layers of cells. In that period it was also observed that primordia of fungiform papillae are distinctly marked on the surface of the tongue.

The lamina propria of the mucosa on the dorsum of the apex and of the body of the tongue in the rabbit starting from day 22 of prenatal life forms protrusions, which are connective tissue cores for the primordia of lingual papillae found there. In the area of the lamina propria of the mucosa blood vessels are found, which extend also into the connective tissue core of lingual papillae. Also starting from day 22 of prenatal life in the rabbit collagen fibers and very delicate elastic fibers were shown in the lamina propria of the lingual mucosa. Next, at day 26 of prenatal life elastic fibers are still very delicate. The delicate elastic fibers were also observed on the connective tissue cores of primordia of fungiform papillae. Available literature does not provide any information on the morphogenesis of the lingual mucosa, which would include the formation of elastic fibers of the tongue in the mucosa. However, information may be found on the distribution of elastic fibers in different animals during their postnatal life [21].

Changes within the mucous membrane of the tongue and its superficial structures, such as fungiform papillae, during the period from day 15 to day 26 of prenatal development in the rabbit indicate the complexity of this process, which is continued in the postnatal life.

CONCLUSIONS

1. The first primordia of fungiform papillae in the form of epithelial thickenings appear on the dorsum of the apex and on the dorsum of the body of the tongue at day 18 of prenatal life in the rabbit.

2. Starting from day 22 of prenatal life in the rabbit primordia of fungiform papillae have the connective tissue core.

3. The epithelium covering the developing fungiform papillae in the period from day 22 to day 26 of prenatal life in the rabbit changes from a 2-layer epithelium into the epithelium composed of 3–5 layers of cells.

4. Collagen fibers and elastic fibers are identified in the lamina propria of the mucosa starting from day 22 of prenatal life in the rabbit.

5. In the period from day 15 to day 20 of prenatal life in the rabbit glycogen is found in the cytoplasm of epithelial cells and in the mesenchymal tissue on the dorsum of the apex and on the dorsum of the body of the tongue, while at day 22 of prenatal life glycogen is found only in the light superficial cells of the epithelium.

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