Mouse Sublingual Glands Research Articles

Altered Rheological Properties of Saliva with Aging in Mouse Sublingual Gland

Mucin in saliva plays a critical role in the hydration and lubrication of the oral mucosa by retaining water molecules, and its impaired function may be associated with hyposalivation-independent xerostomia. Age-dependent effects on salivary gland function and rheological properties of secreted saliva are not fully understood as aging is a complex and multifactorial process. We aimed to evaluate age-related changes in the rheological properties of saliva and elucidate the underlying mechanism. We performed ex vivo submandibular gland (SMG) and sublingual gland (SLG) perfusion experiments to collect saliva from isolated glands of young (12 wk old) and aged (27 mo old) female C57BL/6J mice and investigate the rheological properties by determining the spinnbarkeit (viscoelasticity). While fluid secretion was comparable in SMG and SLG of both mice, spinnbarkeit showed a significant decrease in SLG saliva of aged mice than that of young mice. There were no significant differences in GalNAc concentration between young and aged SLG saliva. Liquid chromatography/tandem mass spectrometry analysis of SLG saliva revealed that (Hex)1 (HexNAc)1 (NeuAc)1 at m/z 793.31 was the most abundant O-glycan structure in SLG saliva commonly detected in both mice. Lectin staining of salivary gland tissue showed that SLG stained strongly with Maackia amurensis lectin II (MAL II) while Sambucus nigra agglutinin (SNA) stained little, if any, SLG. The messenger RNA expression of St3gal1 that encodes an α-2,3 sialic acid sialyltransferase SIAT4-A showed a decrease in SLG of aged mice, confirmed by a Western blot analysis. Lectin blot analysis in SLG saliva revealed that the relative signal intensity detected by MAL II was significantly lower in aged SLG. Our results suggest that spinnbarkeit decreases in SLG of aging mice due to downregulation of sialic acid linked to α-2,3 sialic acid sialyltransferase expression.

Response of the mouse sublingual gland to spaceflight.

The ultrastructure and immunohistochemistry of secretory proteins of sublingual glands were studied in mice flown on the US space shuttles Discovery [Space Transportation System (STS)-131] and Atlantis (STS-135). No differences in mucous acinar or serous demilune cell structure were observed between sublingual glands of ground (control) and flight mice. In contrast, previous studies showed autophagy and apoptosis of parotid serous acinar cells in flight mice. The expression of parotid secretory protein (PSP) in sublingual demilune cells of STS-131 flight mice was significantly increased compared with ground (control) mice but decreased in STS-135 flight mice. Similarly, expression of mucin (MUC-19) in acinar cells and expression of the type II regulatory subunit of protein kinase A (PKA-RII) in demilune cells were increased in STS-131 flight mice and decreased in STS-135 flight mice, but not significantly. Demilune cell and parotid protein (DCPP) was slightly decreased in mice from both flights, and nuclear PKA-RII was slightly increased. These results indicate that the response of salivary glands to spaceflight conditions varies among the different glands, cell types, and secretory proteins. Additionally, the spaceflight environment, including the effects of microgravity, modifies protein expression. Determining changes in salivary proteins may lead to development of non-invasive methods to assess the physiological status of astronauts.

Aquaporin 5 distribution pattern during development of the mouse sublingual salivary gland.

Aquaporin 5 (AQP5) is important in salivary fluid secretion, and has been found in acinar cells of salivary glands in several species. Recently, studies have shown the AQP5 transcript and protein expression patterns as well as the temporal-spatial protein distribution during development of the mouse submandibular salivary gland. The AQP5 distribution pattern of the closely located sublingual gland (SLG) is, however, not well known. Thus, in this study, the Aqp5 RNA expression pattern and the temporal-spatial distribution of AQP5 protein in prenatal development and in adult mouse SLG was investigated. SLGs from embryonic day 14.5 (E14.5) to 18.5 and postnatal days 0 (P0), 25, and 60 were examined using real time PCR and immunohistochemistry. The Aqp5 transcript was detected from E13.5 and was found to increase towards birth and in young adults. The protein was first detected in a scattered pattern in the canalicular stage and became more organized in the luminal membrane of the acinar cells towards birth. During all postnatal developmental stages studied, AQP5 was localized in the luminal and lateral membrane of acinar cells. AQP5 was also detected in the intercalated duct and additional apical membrane staining in the entire intralobular duct was found in the terminal bud stage. These results indicate that AQP5 plays a role during embryonic salivary gland development.

Hypophysectomy and hormonal therapy modulate mK1-immunoreactive duct cells in the mice sublingual glands

The immunocytochemical localization of a true tissue kallikrein, mK1, in mouse sublingual glands (SLGs) was examined following hypophysectomy and hormonal replacement therapy. In the glands of intact mice (14 weeks of age), mK1 was detected in the striated ducts (SDs). Full-fledged granular cells were scattered in the SDs of male mice (but not in those of female mice), showing a cellular mosaic distribution of mK1 with some being positive and others being negative. mK1 was also detected in transitional-type granular cells, though the secretory granules were too small and scarce to be visible by a light microscopy. Hypophysectomy in male mice resulted in the atrophy and loss of secretory granules in many SD cells. Granulation recovered after the repeated injection of 5alpha-dihydrotestosterone (DHT), 3,5,3'-triiodo-L: thyronine (T3), and dexamethasone (Dex), given either alone or in combination to the hypophysectomized mice. The concomitant injection of DHT and T3, with or without Dex, resulted in the reappearance of the full-fledged granular cells, only some of which were mK1-positive. Electron microscopy revealed mK1 to be present exclusively in the secretory granules of these mK1-positive cells, and no ultrastructural differences were observed between mK1-positive and mK1-negative full-fledged granular cells. These results show that the differentiation of the granular cell phenotype in the mouse SLG duct system requires the concomitant action of androgen and thyroid hormone and retards mK1 synthesis.

Differential accumulation of IgA ASC in sublingual vs. submaxillary salivary glands is directly correlated with the level of CCL28 expression. (40.4)

Abstract Optimal function of the immune system is highly dependent on efficient homing and accumulation of leukocytes. The homing of leukocytes has been shown to be dependent on interactions between leukocyte expressed chemokine receptors and adhesion molecules, and tissue expressed chemokines and adhesion molecule ligands. The homing and accumulation of IgA antibody secreting B cells (ASC) to mucosal tissues is thought to be highly dependent upon leukocyte expressed chemokine receptors CCR9 and CCR10 interacting with the chemokines CCL25 and CCL28 respectively. CCL28 has been shown to be involved in the homing of IgA ASC to the gastrointestinal tract and the lactating mammary gland, but has not been demonstrated to play a role, in vivo, in the accumulation of IgA ASC to other mucosal tissues. CCL28 has been shown to be expressed in mouse salivary glands. Here we show that CCL28 is expressed at higher levels in mouse sublingual salivary glands than in submaxillary salivary glands. This higher level of expression in the sublingual gland correlates directly with greater numbers of IgA ASC in the sublingual gland compared with the submaxillary gland. These results demonstrate differential accumulation of IgA ASC into mouse salivary glands and suggest that this difference in lymphocyte accumulation is at least in part the result of differential expression of CCL28.

Identification of a human ortholog of the mouseDcppgene locus, encoding a novel member of the CSP-1/Dcpp salivary protein family

Salivary fluid, the collective product of numerous major and minor salivary glands, contains a range of secretory proteins that play key defensive, digestive, and gustatory roles in the oral cavity. To understand the distinct protein "signature" contributed by individual salivary glands to salivary secretions, we studied a family of proteins shown by in vitro mRNA translation to be abundantly expressed in mouse sublingual glands. Molecular cloning, Southern blotting, and restriction fragment length polymorphism analyses showed these to represent one known and two novel members of the common salivary protein (CSP-1)/Demilune cell and parotid protein (Dcpp) salivary protein family, the genes for which are closely linked in the T-complex region of mouse chromosome 17. Bioinformatic analysis identified a putative human CSP-1/Dcpp ortholog, HRPE773, expressed predominantly in human salivary tissue, that shows 31% amino acid identity and 45% amino acid similarity to the mouse Dcpp query sequence. The corresponding human gene displays a similar structure to the mouse Dcpp genes and is located on human chromosome 16 in a region known to be syntenic with the T-complex region of mouse chromosome 17. The predicted mouse and human proteins both display classical NH(2)-terminal signal sequences, putative jacalin-related lectin domains, and potential N-linked glycosylation sites, suggesting secretion via sublingual saliva into the oral cavity where they may display antimicrobial activity or provide a defensive coating to enamel. Identification of a human CSP-1/Dcpp ortholog therefore provides a key tool for investigation of salivary protein function in human oral health and disease.

Localization of FGF-6 and FGFR-4 during prenatal and early postnatal development of the mouse sublingual gland

A number of fibroblast growth factors (FGFs) are involved in regulatory mechanisms of the salivary gland development. However, the role of FGF-6 unique in myogenic cells has not been elucidated in the developing sublingual gland. In the present study, temporo-spatial expression of FGF-6 and its receptor (FGFR)-4, in conjunction with some related histo-chemical properties, were investigated in the sublingual gland of the prenatal and early postnatal mice. The earliest expression of both FGF-6 and FGFR-4 was detected in immature acinar cells at gestational day 17 (GD17). The staining intensity increased gradually and some acinar cells showed a distinct staining at postnatal day 0 (PD0). The immunopositive cells had a relatively round profile and were assumed to be acinar cells. The positive staining decreased thereafter and disappeared completely by PD11. To confirm the identity of cells positive for FGF-6, double immunolabeling with anti-alphasmooth muscle actin (alphaSMA) and anti-FGF-6 antibodies was performed. The positive staining of alphaSMA, a marker of myoepithelial cells, was detected in the flattened cells surrounding the acini but not in the cells positive for FGF-6. The staining properties of secretory granules in acinar cells were also examined with periodic acid-Shiff (PAS) and alcian blue (AB). PAS-positive granules abundant in the late gestational stages (GD17 to PD0) began to be replaced with AB-positive mucous granules at early neonatal days (PD0-3), when the FGF-6/FGFR-4 expression was the strongest. These findings suggest that FGF-6/FGFR-4 might be involved in the changes of secretory granule content of acinar cells in the sublingual gland during the late gestational and early neonatal stages.

Role of thyroid hormone in the initiation of EGF (epidermal growth factor) expression in the sublingual gland of the postnatal mouse

The effect of triiodo-L-thyronine (T3) and propylthiouracil (PTU) on the initiation of epidermal growth factor (EGF) expression in the sublingual glands (SLGs) of postnatal mice was investigated by indirect enzyme-labeled and immunogold antibody methods for light and electron microscopy, respectively. In normal males, EGF immunoreactivity first appeared in a few scattered granular cells of striated ducts (SDs) at 5 weeks of age, and the immunoreactive cells had increased in number at 6 weeks of age. No EGF expression was observed in the glands of females at any ages examined. When T3 (1 mg/kg body weight) was given to males every other day for 2 weeks before examination, EGF expression began earlier; the immunoreactive granular cells were first detected at 4 weeks of age, and at later ages they were markedly increased in number compared to those of normal males. Moreover, T3 was capable of inducing EGF in the female glands. After T3 was administered to females in the same manner as in males, a few immunoreactive cells were first detected at 5 weeks of age, and increased numbers were detected at later ages. By contrast, when PTU (1 mg/kg body weight) was given to male mice every other day for 2 weeks before examination, the EGF-immunoreactive cells were markedly decreased in number compared to those of normal males of the same age. Electron microscopy revealed that many SD cells contained secretory granules, and that these cells constituted the granular striated tubule (GST) in a portion of SDs, but they were undetectable by light microscopy, because their secretory granules were minimal in size and few in number. Gold-labeling of EGF was confined to the secretory granules of scattered granular cells, whose secretory granules were far larger in size and more abundant than those of the GST cells. These results suggest that thyroid hormone is essential to differentiation of the cellular phenotype of GST precursor cells into typical granular cells (detectable by light microscopy) that express EGF in the mouse SLG, showing a close resemblance to the submandibular granular convoluted tubule cells.

Morphological changes in mouse sublingual gland parenchyma subjected to chorda tympani resection.

Morphological changes in the mouse sublingual gland parenchyma subjected to parasympathetic nerve block were investigated. Mice were subjected to unilateral resection of the chorda tympani, near its point of joining with the lingual nerve. After 1, 2, 3, 5, 10 or 20 weeks, the mice were killed and their sublingual glands were removed and processed for light and electron microscopy. Two weeks after resection, the space between the adjoining lobules of the glands on the treated side began to be expanded, and by 10 weeks were 10 times the size of the spaces in the glands of the untreated mice. Three weeks after resection, the lobule area decreased to about 72% of the area of glands in the untreated mice and the acinus area to about 52%. However, no significant difference was seen between the numbers of acini in each group. Electron microscopy showed that the glands on the treated side contained fewer secretory granules than the glands in the untreated mice, though there was no difference in size. Neither the lobules of the glands on the treated side nor those of the glands of the untreated mice contained many TUNEL-positive cells. These findings suggest that following parasympathetic nerve resection, mouse sublingual gland acinar cells undergo atrophy with a reduction size rather than cell death.

Ultrastructural study of hormonally responsive striated duct cells in the mouse sublingual gland.

In semithin sections stained with Heidenhain's iron hematoxylin, a few scattered granular cells were observed in the striated ducts (SDs) of sublingual glands (SLGs) of the mouse; they were seen normally only in the glands of adult males. However, it was shown by electron microscopy that many SD cells, other than these granular cells, had apical secretory granules, thus forming a granular striated tubule (named the GST in this study) in a portion of SD segments in both sexes. Sublingual GST cells had very small dense secretory granules near the apical surface, with the nucleus in the apical one-third to one-half of the cell; small Golgi apparatus; sparse rough endoplasmic reticulum (RER); and well-developed basal infoldings. However, some granular cells in male GSTs had abundant large dense secretory granules in the apical two-thirds of the cell, a basal nucleus, and modest basal infoldings. Such granular SD cells disappeared after castration in males. Granular SD cells could be induced in the GSTs of females by the injection of 5 alpha-dihydrotestosterone (DHT), triiodothyronine (T(3)), and/or dexamethasone (Dex); given simultaneously, these hormones acted synergistically in this induction. These results indicate a close similarity between the duct systems of the SLG and those of the submandibulan gland (SMG) of the mouse: granular SD cells of the GST in the SLG resemble GCT cells in the SMG in expressing some of the same biologically active polypeptides, in being sexually dimorphic, and in being under the same multihormonal regulation.

Characterization of a UDP-GalNAc:Polypeptide N-Acetylgalactosaminyltransferase That Displays Glycopeptide N-Acetylgalactosaminyltransferase Activity

We report the cloning, expression, and characterization of a novel member of the mammalian UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (ppGaNTase) family that transfers GalNAc to a GalNAc-containing glycopeptide. Northern blot analysis revealed that the gene encoding this enzyme, termed ppGaNTase-T6, is expressed in a highly tissue-specific manner. Significant levels of transcript were found in rat and mouse sublingual gland, stomach, small intestine, and colon; trace amounts were seen in the ovary, cervix, and uterus. Recombinant constructs were expressed transiently in COS7 cells but demonstrated no transferase activity in vitro against a panel of unmodified peptides, including GTTPSPVPTTSTTSAP (MUC5AC). However, when incubated with the total glycosylated products obtained by action of ppGaNTase-T1 on MUC5AC (mainly GTT(GalNAc)PSPVPTTSTT(GalNAc)SAP), additional incorporation of GalNAc was achieved, resulting in new hydroxyamino acids being modified. The MUC5AC glycopeptide failed to serve as a substrate for ppGaNTase-T6 after modification of the GalNAc residues by periodate oxidation and sodium borohydride reduction, indicating a requirement for the presence of intact GalNAc. This suggests that O-glycosylation of multisite substrates may proceed in a specific hierarchical manner and underscores the potential complexity of the processes that regulate O-glycosylation.

Allele-specific expression of the PSP gene in the mouse sublingual glands.

There are two known alleles of the mouse parotid secretory protein (PSP) gene: Pspa and Pspb. Pspa is carried by DBA/2J mice and Pspb is carried by C57BL/6J mice. Eighty-eight mice derived from a F1(C57BL/6J x DBA/2J) to DBA/2J backcross were analysed for PSP mRNA expression in the sublingual glands. Expression was found in heterozygous mice only. This indicates that only Pspb is expressed in this tissue. Furthermore, it maps the allele-specific sublingual gland determinant within 3.4 cM of Psp. Previous analysis of Pspb identified an enhancer-like region in position -4.6 to -3.1 kb that was necessary for transgene expression in the sublingual glands. Here it is shown that the corresponding region in Pspa enhances transgene expression in the sublingual glands as efficiently. The implications for regulation of PSP mRNA expression in the sublingual glands are discussed.

CDNA cloning, sequencing and in situ localization of a transcript specific to both sublingual demilune cells and parotid intercalated duct cells in mouse salivary glands

A cDNA clone derived from mouse sublingual gland was isolated from a λ-phage cDNA library. Northern blot hybridization indicated that the transcript from which it was derived was approx. 700 nucleotides in length. This mRNA encoded a protein of about 20 kDa, as determined by hybrid selection and cell-free translation. Conceptual translation of the cDNA clones showed that p20 is 170 amino acids in length. The putative protein is hydrophobic in nature, is neither a mucin-like protein nor does its amino acid sequence or composition resemble the other known mouse proteins. However, the amino acid sequence of p20 suggests that it may be from a gene or gene family homologous to rat common salivary protein 1. The p20 mRNA also appears to share a non-random degree of sequence homology with the cysteine-rich domains of bovine and porcine submandibular mucins. The p20 mRNA is abundant in the mouse sublingual gland, and its expression is approx. nine times greater than in the parotid gland. In situ hybridizations localized the p20 mRNA exclusively in the demilune cells of the sublingual gland and in the intercalated duct cells of the parotid gland. It is detectable in the neonatal and adult submandibular gland at very low levels, but is absent from liver, heart, brain, thymus, spleen, lens and lacrimal glands.

Molecular cloning and antibody screening for the mucln cDNA clone from a mouse sublingual gland cDNA library: Chih-Hsin Hsieh, Los Angeles: University of Southern California, 1991

Molecular cloning and antibody screening for the mucln cDNA clone from a mouse sublingual gland cDNA library: Chih-Hsin Hsieh, Los Angeles: University of Southern California, 1991

Detection of nerve growth factor mRNA in rodent salivary glands with digoxigenin- and 33P-labeled oligonucleotides: effects of castration and sympathectomy.

Nerve growth factor (NGF) is a protein highly expressed in the male mouse submandibular gland. We have applied a non-radioactive in situ hybridization method using digoxigenin-labeled NGF oligonucleotides, and have found the highest amounts of NGF mRNA in the secretory striated ducts of the male mouse submandibular gland. Scattered strongly positive cells were found in male mouse sublingual glands. Weakly labeled cells were seen in female mouse and in male rat submandibular gland striated duct cells. Using 33P as an alternative to 32P and 35S, we demonstrated a 1.3 KB NGF mRNA in salivary glands of male mice by Northern blot hybridization. Using 33P we detected NGF mRNA in male mouse submandibular glands by in situ hybridization but with a signal that, compared with the non-radioactive method, had a very low resolution. Castration of male mice almost abolished both the 1.3 KB NGF mRNA seen with Northern blots and the NGF mRNA labeling in submandibular glands 4 weeks after the operation, whereas levels were increased 6 hr and 2 days after sympathectomy. We conclude that hybridization with digoxigenin-labeled NGF oligonucleotides is a good tool to study the expression and regulation of NGF mRNA in male mouse submandibular glands.

Iodothyronine 5'-deiodinase is present in mouse sublingual gland.

Homogenates from male ddy mouse sublingual glands were incubated with 100 nM [125I]rT3 in the presence of 2 mM dithiothreitol (DTT). Metabolites were analyzed by HPLC or Dowex-50 minicolumn assay. 3,3'-Diiodothyronine and I- were the only appreciable products. Iodide release from rT3 was compatible with the enzymatic nature. The Km and maximum velocity from three separate determinations were 326, 356, and 629 nM and 29.8, 42.1, and 31.3 pmol I- release/mg protein.min, respectively. The deiodinase activity was DTT dependent and had higher affinity for rT3 than for T4 or T3. 6-Propyl-2-thiouracil inhibited the deiodination, which was competitively overcome by DTT. Sublingual 5'-deiodinase activity was approximately 80% of that in the liver, while the submandibular gland showed no deiodination. Our results show the presence of 5'-deiodinase (type I) in mouse sublingual gland for the first time. Selective localization and abundance of the enzyme suggest a previously unrecognized role of the sublingual gland in thyroid hormone physiology.

Iodothyronine 5'-deiodinase is present in mouse sublingual gland

Iodothyronine 5'-deiodinase is present in mouse sublingual gland

Biochemical properties of epidermal growth factor in the mouse sublingual gland.

Biochemical properties of epidermal growth factor in the mouse sublingual gland.

Epidermal growth factor, renin, and protease in hormonally responsive duct cells of the mouse sublingual gland.

In the striated ducts of the sublingual glands of normal adult male, but not female, Swiss-Webster mice a few scattered cells have apical secretion granules. These sublingual duct cells resemble the granular convoluted tubule (GCT) cells of the submandibular glands of adult female mice, in that they are smaller than submandibular GCT cells of adult males, and contain fewer apical granules, and prominent basal striations. These cells stain immunocytochemically for epidermal growth factor (EGF), renin, and protease A. Such granular striated duct cells could be induced in the sublingual glands of adult female mice by treatment with either testosterone propionate or thyroxine; the two hormones given simultaneously acted synergistically in this induction.

Ultrastructural localization of acidic glycoconjugates with the low iron diamine method.

The present study has applied the low iron diamine (LID) method at the ultrastructural level to demonstrate acid glycoconjugates. We have examined rat epiphyseal cartilage, human bone marrow, rat tracheal glands, and mouse sublingual glands stained with LID prior to embedment. The LID staining appeared to require postosmication for adequate visualization at the electron microscope level. Thiocarbohydrazide-silver proteinate (TCH-SP) staining of thin sections variably enhanced LID reactive sites. LID-TCH-SP stained carboxyl and sulfate groups of glycosaminoglycans in the extracellular cartilage matrix, secretory granules, and expanded Golgi saccules of chondrocytes. In human bone marrow, LID-TCH-SP variably stained the cytoplasmic granules, known to contain sulfated glycosaminoglycans, and the external surface of the plasma membrane of leukocytes. Moderately strong LID staining was observed in secretory granules in mucous tubules of rat tracheal glands, known to contain sulfated glycoproteins, and in acinar cells of mouse sublingual glands, known to contain a sialoglycoprotein. The lack of sulfated glycoconjugates in acinar cells of the mouse sublingual gland was confirmed by their failure to stain with the high iron diamine method. Thus these studies indicate that the LID and LID-TCH-SP methods are useful for the ultrastructural localization of carboxylated and sulfated glycoconjugates in extracellular and intracellular sites.