Morphology and Glycan Composition of the Mandibular Glands in the White-Eared Opossum (Didelphis albiventris)

Abstract

The white-eared opossum, Didelphis albiventris, is an opportunistic and omnivorous marsupial, whose diet ranges from wild fruits to eggs and birds. Salivary glycoproteins play a key role in the protection of the oral cavity and the formation of the food bolus. Despite the importance of salivary glycoproteins, their detailed investigation in the white-eared is lacking. This study investigated the morphology and glycan composition of the mandibular salivary glands of the white-eared opossum for the first time. Histological and histochemical investigations were conducted on tissue fragments fixed with 4% PBS-buffered paraformaldehyde and embedded in Paraplast. The pattern of glycoproteins was investigated using traditional histochemical methods (PAS, Alcian Blue pH 2.5, and High-Iron Diamine staining) and lectin histochemistry. The glandular parenchyma consisted of acinar secretory units and a duct system characterized by abundant striated ducts. Secretory acini secrete neutral glycans and non-sulfated acid glycans. Mannosylated N-linked glycans terminating in α2,6-sialic acid and fucose are expressed in the secretory acini, containing intraluminal α2,3-sialylated O-linked glycans. The epithelial lining of the striated and interlobular ducts also shows O-linked glycans with terminal Galβ1, 3GalNAc, and αGalNAc residues. Finally, the epithelium and lumen of interlobular ducts are enriched with additional GalNAc-terminated O-linked glycans with the appearance of lactosaminated glycans and the disappearance of α2,3-sialylated glycans. These results suggest that the saliva produced by the mandibular gland of the white-eared opossum consists of a species-specific pattern of glycoproteins, to whose composition the ductal system also contributes. The observed glycan composition is probably related to the diet of the white-eared opossum and its adaptations to the environment and food availability. These results indicate that the mandibular salivary gland of the white-eared opossum Didelphis albiventris has specific histological and molecular characteristics compared to other marsupial species, suggesting that diet and habitat, but not the taxonomic group, influence the mandibular gland features.

Keywords: glycans; lectins; marsupials; salivary glands.

Figure 1

Didelphis albiventris, lateral (A) and ventral (B) view of the neck and head regions. Note the mandibular (1) and parotid (2) salivary glands, masseter muscle (3), zygomaticus muscle (4), sternohyoid muscle (5), mandibular lymph nodes (asterisks) and external jugular vein (arrow). Scale bar = 1 cm.

Figure 2

Didelphis albiventris, mandibular salivary gland. The connective tissue capsule (c) gives off septa (s) that divide the mandibular gland into lobules (lo). Mucous (ma) and serous acini (sa) constituted the lobules. Note the serous demilune (arrowheads) surrounding mucous acini (ma). Note also the abundance of the striated ducts (sd) and interlobular ducts (id) that drain glandular secretion and the presence of blood vessels (asterisks) in the interlobular connective tissue. Masson's Trichrome (A,B); Haematoxylin and eosin staining (C, D). Scale bar: A = 50 µm, B and C = 20 µm, D = 100 µm.

Figure 3

Didelphis albiventris, mandibular salivary gland. Note the intercalated ducts (ic), striated ducts (sd), and interlobular ducts (id). Serous (sa) and mucous (ma) acini, and myoepithelial cells (mc) are showed. The blood vessels such as arteries (a) and veins (v) are also observed in the interlobular connective tissue. Masson's Trichrome staining. Scale bar: A and C = 20 µm, B = 50 µm.

Figure 4

Didelphis albiventris, PAS (A) and Alcian blue pH 2.5 (AB 2.5) (B) staining of the mandibular gland. (A) Note the strong PAS‐positivity (magenta) of the secretory acini and the faint staining of intraluminal content of the striated (arrows) and interlobular ducts (double arrows) whose epithelium was PAS negative. The inset shows a detail of the PAS positive reaction in the secretory acini. (B) AB 2.5 staining (azure) was observed in the secretory epithelium and in a minor extent in the lumen of the duct. The inset shows a detail of the AB positive reaction in the secretory acini. ct, connective tissue that surrounds the interlobular duct. The nuclei were stained with Hematoxylin in A and with fast red in B. Asterisk, blood vessel. Scale bar: A = 100 µM; B = 80 µM; inset of A,B = 20 µM.

Figure 5

Didelphis albiventris, reactivity pattern of Con A (A) and RCA₁₂₀ (B) with the mandibular gland. (A) Con A binding sites are present in the secretory acini (a), apical surface of the epithelium lining of the striated ducts (arrows), and in the epithelium of the interlobular ducts (double arrow). (B) RCA reactivity was observed in the lumen content of the interlobular ducts (double arrows) and in the connective tissue. ct, connective tissue (stroma); asterisks, blood vessels. Scale bar: A, B = 50 µm.

Figure 6

Didelphis albiventris, binding pattern of PNA (A), DBA (B), SBA (C), and AAL (D) in the mandibular gland. (A) PNA affinity is observed in the apical surface of the striated ducts (arrows), in the apical cytoplasm of interlobular ducts and in the interlobular connective tissue. (B) Moderate DBA reaction in the epithelium of the striated (arrows) and interlobular (double arrows) ducts. The latter ducts contained a faintly visible positive substance. (C) SBA binding sites are visible in the epithelium of the interlobular ducts (double arrows), which contain a barely reactive substance and are surrounded by a well visible stroma (ct). (D) AAL strongly reacts with the secretory acini (a) and the apical surface of the striated ducts (arrows). A barely AAL reaction is visible in the interlobular ducts (double ducts). ct, connective tissue; asterisks, blood vessels. Scale bar: A, B, C, D = 50 µm.

Figure 7

Didelphis albiventris, MAL II (A) and SNA (B) binding pattern in the mandibular gland. (A) MAL II reactivity is present in the lumen (arrowhead) and the lateral surface (red double arrows) of the secretory acini (a) and in the striated ducts (arrows). The inset shows a detail of the positive reaction of the acini. (B) SNA binding sites are faintly observed in the secretory acini (a), and weakly in the striated (arrows) and apical cytoplasm of the interlobular (double arrows) ducts. Note the strong staining of the stroma. ct, connective tissue; asterisks, blood vessels. Scale bar: A, B = 50 µm; inset of A = 15 µm.