Here, we histochemically investigated developmental lectin-binding pattern changes in the Weber’s and von Ebner’s glands, which are posterior lingual glands present around the rat posterior cervical nipple.
In rodents, four types of papillae exist on the dorsal surface of the tongue: fungiform, circumvallate, foliate, and filiform papillae [32]. In humans, the cardinal papillae are circular bulges with 8-9 papillae [33]. However, in mice and rats, a single circumvallate papilla exists on the posterior midline of the dorsal surface of the tongue. This gustatory papilla contains taste buds [12, 34]. In addition, the von Ebner’s glands open onto the base of the fornix and lobulated papillary sulcus, and the Weber's glands are present laterally and posteriorly to the von Ebner's glands. The Weber's glands are mucous glands [35, 36]; however, the presence of serous cells has been suggested in humans, rats, and mice [31, 35]. Here, the Weber's glands contained mucous cells and fewer serous and serous semilunar cells. These findings are consistent with previous findings in rats [8, 35], humans [2, 37], and other animals [2, 8].
In the present study, on embryonic day 18, the epithelial cells of the mesenchyme in the posterior part of the tongue were continuous with the tongue epithelium and could be considered the Weber's gland primordium. These cells showed serous cell morphology. Hamosh et al. [5] reported that the development of the Weber's glands preceded that of the von Ebner’s glands, and secretory granules were present by embryonic day 20. These findings are in close agreement with those of the present study. Regarding the development of the palatine glands, which are considered to be muciparous like Weber's glands, Shinzato et al. [38] reported that in rats, epithelial incision began on embryonic day 17, terminal cells were formed on embryonic day 18, and mucous cells appeared on embryonic day 21. This change is almost identical to the development of the Weber’s glands demonstrated in the present study.
Here, von Ebner’s glands were not formed on embryonic day 18; however, from embryonic day 20, the epithelium of the fornix papillary sulcus started infiltrating into the mesenchyme. Hamosh et al. [5] showed that in Sprague Dawley rats, the epithelial growth from the fornix and lobulated papillae on embryonic day 19–20 was noted as the von Ebner’s glands began to develop, and no terminal formation or production of secretory granules were observed until 3–4 days after birth. The results of the present study are in close agreement with that of these findings.
UEA I, SBA, RCA, PNA, and WGA bind to serous cells, whereas RCA and DBA bind to mucous cells [18, 39]. Therefore, since the lectin-binding pattern in salivary glands differs depending on the species, location, and salivary characteristics, we investigated the binding of lectins in the posterior lingual gland of rats. We found that SBA, WGA, and PNA bind to serous cells, and DBA binds to mucous cells in adult rats. Therefore, we used these as indicators of the development of the posterior lingual glands in rats. SBA bound to mucous cells in the terminal part of the Weber's glands by 14 days of age, but not after 21 days of age; WGA bound to mucous cells in the terminal part of the von Ebner’s glands by 10 days of age, but not after 21 days of age; SBA bound to mucous cells in the terminal part of the von Ebner’s glands after 1 day of age, whereas DBA bound to mucous cells in the terminal part of the Weber's glands by 21 days of age. In the von Ebner’s gland endings, lectin-binding was observed after postnatal day 1, whereas in the Weber’s gland endings, lectin-binding was identified on embryonic day 18. PNA was found to bind to the Weber's gland endings by postnatal day 21, but not on postnatal day 28, and bind to the von Ebner’s gland endings after postnatal day 1. In the Weber's glands, binding of not only DBA but also the serous markers SBA, WGA, and PNA were observed from the time of cell emergence; however, binding of lectins other than DBA disappeared with maturation. On the other hand, the von Ebner’s glands showed binding to the SBA, DBA, PNA and DBA lectins at the time of cell emergence; however, DBA binding disappeared with maturation. The loss of binding coincided with the change in food intake from a liquid to a solid diet.
Schulte et al. [18] examined the salivary glands of rats aged 12–14 weeks and found that SBA, DBA, and PNA showed strong binding to serous cells of the posterior lingual gland, and SBA and DBA showed moderate binding to mucous cells. Further, in the serous semilunar cells found in mucous cells, the lectin-binding pattern was similar to that of serous cells [18]. The authors also reported that SBA, DBA, and PNA showed moderate binding in serous cells, while the parotid gland, which is also serous in nature, showed weak binding in conduits [18]. These findings for DBA and PNA are consistent with those of the present study; however, the results regarding SBA are different. WGA also highlighted reactions in many of the serous terminal cells of the posterior lingual gland, as well as in mucous cells. Furthermore, PNA reacted with most of the serous terminal cells and only with serous cells in areas with a mixture of mucous and serous cells, but not in the conduit epithelium of the mucous glands.
These findings of lectin binding are partially different from those of the present study, which may be due to lectins having species-dependent binding patterns.
Regarding sugar residues, SBA binds strongly to α/β-N-acetyl-d-galactosamine and weakly to α-d-galactosamine, WGA binds strongly to β-N-acetyl-d-galactosamine and sialic acid, DBA binds to α- N-acetyl-galactosamine, and PNA binds strongly to β-d-galactosyl-(1–3)-N-acetyl-galactosamine and weakly to α-d-galactosamine. [24–29, 39–43]. Thus, α-N-acetyl-galactosamine is present in rat posterior lingual gland mucus terminal cells, and α/β-N-acetyl-d-galactosamine and α-d-galactosamine, β-N-acetyl-d-galactosamine, sialic acid, β-d-galactosyl-(1–3)-N-acetyl-galactosamine, and α-d-galactosamine were present in the von Ebner’s gland terminal cells.
The examination of the distribution of the sugar residues will provide the histological structure at the monosaccharide level and highlight the properties and functionality of the different species and sites.
Here, terminal cells in the Weber's glands on embryonic day 18 and the von Ebner's glands on postnatal day 1 bound both plasma cells and mucus cells before the lectin-binding pattern became identical to that of adult animals. This suggests that the Weber’s glands in late embryonic stages initially assume serous properties before forming mucous cells. It is possible that both the von Ebner’s and Weber’s glands function as mixed glands for some time after the onset of posterior lingual gland formation in the late embryonic period.
In the Weber's glands in rats, undifferentiated cells appear first and form serous cells that differentiate into intermediate-type cells, then into mucous cells. In the present study, we propose that the change in lectin-binding properties observed in the Weber's glands corresponds with this phenomenon.
In our study, we found that the posterior lingual gland in rats aged 21–28 days showed the same lectin-binding pattern as that of adult animals. Furthermore, rats were weaned at 21 days of age and kept on solid feed thereafter. Thus, the change in diet may have caused a change in the properties of salivary gland cells. After 10 days of age, the incisors erupt, and the rats can ingest solid food. During this period, the von Ebner’s glands secrete serous saliva and Weber's glands secrete mucous saliva. Mucous saliva is involved in food mass formation, and such changes in feeding behavior are thought to be closely related to changes in salivary components, i.e., the function of salivary secretory cells. Future studies should investigate these changes in weaning time and feed.
The von Ebner’s glands secrete serous saliva, which cleans the taste pits of the taste buds in the groove epithelium of the papillae and lobe papillae, thereby maintaining and renewing taste receptors [12, 37]. Here, morphologically mature taste buds with taste pits were observed in the sulcus epithelium of the dorsal papillae on postnatal day 1, and the number of these buds increased thereafter. The appearance of mature postnatal taste buds in the epithelium of the papillary sulcus is consistent with the findings of previous reports [44, 45]. By postnatal day 3, The von Ebner's glands opening to the floor of the papillary sulcus was not mature; however, the Weber's glands opening to the mucosa behind the tongue showed morphological and histochemical characteristics of serous cells, suggesting that they secrete serous saliva. Therefore, until the von Ebner’s glands mature, Weber's glands are thought to renew taste receptors by cleaning the taste pits of the taste buds in the papillary sulcus epithelium, compensating for the function that the von Ebner’s glands perform. Not only do the secretory products of the von Ebner’s glands influence the taste response, but also the glyco-chemical properties of each taste bud [46, 47].
We identified cases in which lectins bound to the plasma membrane and cytoplasm. The binding of lectins on the plasma membrane is thought to be due to the sugar chains that make up the plasma membrane. In contrast, lectin-binding in the cytoplasm is thought to be due to the binding of sugars to proteins formed intracellularly, which are modified by intracellular organelles. Here, we did not examine the intracellular aspects of lectins; however, future studies should examine this.