Secretagogue-mediated discharge of nerve growth factor from granular tubules of male mouse submandibular glands: an immunocytochemical study.

Submandibular glands of male mice were stained for nerve growth factor by light microscopic immunocytochemistry. Nerve growth factor (NGF) was present in the granules of granular tubule cells, with the immunoreactive material often concentrated at the periphery of granules. Administration of the alpha-adrenergic agent, phenylephrine, to animals resulted in a marked depletion of NGF-containing granules from granular tubules. Some release also occurred following administration of the beta-adrenergic agent, isoproterenol. Cholinergic stimulation (pilocarpine) did not result in appreciable loss of immunoreactive granules from these cells. In vitro results were not as clear cut, immunocytochemically, as those obtained with intact animals. It is concluded that discharge of NGF from male mouse submandibular glands is mediated predominantly by alpha-adrenergic activation, and that this phenomenon is readily demonstrated in the intact animal.     

Ultrastructure of the secretory response of male mouse submandibular gland granular tubules

The organization and fine structure of granular convoluted tubule cells (GCT) from male mouse submandibular glands have been examined in controls and in animals injected with adrenergic and cholinergic secretagogues. Control submandibular glands exhibited a single population of GCT cells with numerous homogeneous granules filling the apical two-thirds of the cytoplasm. A zone of transition cells, exhibiting characteristics of both GCT and striated duct cells, was found between the agranular intercalated duct and GCT segments. These transition cells possessed apical granules of variable size as well as prominent basal striations. Dramatic changes in the morphology of GCT cells followed administration of the alpha-adrenergic agent, phenylephrine. The extensive degranulation involved formation of "secretory pools" of fused granules and release of secretory material into the lumen. The appearance of numerous smooth vesicles near luminal membranes suggested extensive membrane retrieval. Intracellular membrane-limited aggregates of membrane fragments suggested that much of the retrieved membrane was destined for degradation. Rough endoplasmic reticulum was highly dilated but there was no indication of increased size or activity of the Golgi complex. Ultrastructural evidence indicated that the secretory responses to isoproterenol, a beta-adrenergic agent, and to pilocarpine, a cholinergic agent, were much more modest, but it is clear that some secretory response to these agents does occur. The other cell types of the duct and tubule system did not exhibit comparable morphological changes in response to the agents used.     

Cellular and subcellular colocalization of nerve growth factor and epidermal growth factor in mouse submandibular glands

Immunocytochemical methods have been used to compare the cellular and subcellular distribution of nerve growth factor (NGF) and epidermal growth factor (EGF) in mouse submandibular glands. Rabbit antisera raised against purified proteins were characterized by immunoblot methods and were used to stain sections of salivary glands embedded in plastic. For light microscopy, antibodies were visualized by indirect immunofluorescence. For electron microscopy, thin sections were treated simultaneously with IgG against NGF and EGF coupled to colloidal gold particles of different size. Data indicate that NGF and EGF are present in all granular convoluted tubule cells and in no other cell type within the salivary gland. Ultrastructural analyses indicate that NGF and EGF are evenly distributed together within mature secretory granules, although a population of small granules was identified that is not immunoreactive for either protein. Taken together, the data suggest that granular convoluted tubule cells are homogeneous in the production and storage of NGF and EGF.     

Electron microscopic immunostaining of nerve growth factor: secretagogue stimulated submandibular glands

Immunocytochemical localization of nerve growth factor (NGF) was assessed on thin sections of plastic-embedded male mouse submandibular glands by electron microscopy. Both control and secretagogue-stimulated glands were examined. NGF was localized in granules of both granular convoluted tubule (GCT) cells and transition cells. The latter were intermediate in morphology between GCT cells and striated duct cells. Both large and small granules were immunostained in GCT cells; however, considerable variability in immunostaining intensity was observed in both sizes of granules but especially in the small granules of transition cells. Rough endoplasmic reticulum (RER) in both cell types exhibited NGF immunoreactivity. No Golgi-associated immunostaining was observed. Following alpha-adrenergic stimulation with phenylephrine, NGF-containing granules were sharply reduced because of extensive degranulation. Pools of immunostained secretory material suggested intracellular fusion of NGF-containing granules. Immunostaining was also observed on membrane fragments found within large vacuoles in GCT cells. Evidence of NGF secretion after beta-adrenergic or cholinergic stimulation was less dramatic. In isoproterenol-stimulated GCT cells there was evidence of fusion of small, apical, NGF-stained granules. These cells also possessed heavily immunostained apical membrane blebs. Pilocarpine-stimulated cells exhibited pleomorphic immunostained apical granules but less apical membrane immunostaining. Abundant basal lysosomes appearing in GCT cells after pilocarpine stimulation did not stain for NGF.     

小鼠下颌下腺颗粒曲管细胞的免疫细胞化学观察

本采用免疫细胞化学ABC法，观察到小鼠下颌下腺颗粒曲管细胞呈现降钙素基因相关肽和生长抑素免疫反应阳性，但两在颗粒曲管细胞内分布特点不尽相同，腺泡细胞呈免疫反应阴性，本就下颌下腺中降钙素基因相关肽和生长抑素存在的意义进行了讨论。     

Morphological study of granular convoluted tubules in the submandibular gland of the mouse during the growth of a sarcoma-180 subcutaneous tumor

Histological and cytological changes in the submandibular glands of adult male mice arising during the growth of sarcoma-180 subcutaneous tumors were studied. The submandibular glands of the mice were examined by morphometric analysis at 1, 3, 6, 10, 20, 30 and 64 days after inoculation of the tumor cells. There was a slow increase in the relative cross-sectional area of the granular convoluted tubule (GCT) in the section of the submandibular gland of the animals as the tumors grew. The increased proportional area of the GCT was significantly different from that of the control's from day 30. However, the mean weight of the glands was not increased. The proportional area of the granular cluster in the cells of the GCT of tumor cells in inoculated animals decreased about 5% on the first day and then quickly increased by 16% on the third day in comparison with those of the controls, eventually reaching a maximum of 74% (control, 54%) by day 30. In addition, the average number of granules per GCT cell decreased in the first three days, then increased to normal levels from day 6, going above the normal level from day 20 of the tumor growth. These changes in the glands of tumor-bearing animals disappeared within 20 days after removal of the tumor. These results indicate that the growth of the sarcoma-180 subcutaneous tumor caused morphological changes in the GCT and GCT cells, suggesting an alternation in the requirements of the secretions contained in the granules, such as the epidermal growth factor, during the growth of the tumor.     

In situ hybridization of nerve growth factor mRNA in the mouse submandibular gland

We studied the presence of beta-nerve growth factor (NGF) mRNA in the submandibular gland of the mouse by in situ hybridization using 35S-labeled prepro-beta-NGF antisense RNA. Female and male mice were studied at different stages of postnatal development, ranging from 3 to 12 weeks. Although NGF mRNA was detectable in the granular convoluted tubules of the submandibular gland in all the age and sex groups studied, the abundance of the signal dramatically increased after 5 weeks during the development of the submandibular gland. In addition, a conspicuous sexual dimorphism became increasingly apparent in the 6-, 7-, 10-, and 12-week-old animals, due to the remarkable development of the granular convoluted tubules in the adult male mouse, that expressed abundant NGF mRNA.     

Precocious induction of secretory granules by hormones in convoluted tubules of mouse submandibular glands

Precocious differentiation of convoluted tubules of the submandibular glands of mice was induced by daily injection of thyroxine, insulin, hydrocortisone and 5α-dihydrotestosterone from day 4 after birth. This treatment induced enlargement of the tubules and synthesis of secretory granules in the tubules. Treatment with 5α-dihydrotestosterone alone had no effect on enlargement of the tubules or synthesis of the granules. These findings suggest that, besides androgenic hormones, these three hormones are also involved in synthesis of granules in developing mice.     

Proliferative and structural differences between male and female mouse submandibular glands

Sexual dimorphism has been observed in salivary glands of many species. In this study, evidence for sexual differences in adult mouse submandibular gland is extended beyond parenchymal cell composition, size, and volumes to include patterns of DNA synthesis and complexity of ductal branching. Computer-assisted three-dimensional reconstructions also revealed differences in overall organization of secretory complexes. Consistent with observations by others, granular intercalated duct cells were absent, while striated granular duct cells were low in proportion in the male glands relative to female glands. When the mean of average cell volumes were compared, acinar (AC) cells were smaller than granular duct (GD) cells in the male, but in the female the reverse was true. Furthermore, in addition to differences in average volumes of GD cells, the average volume of AC cells was significantly greater in females than males. The most dramatic evidence for sexual dimorphism was observed following a 90-min labeling with ³H-thymidine. Though all cell types showed DNA replication activity, the intercalated duct (ID) cells were substantially more active than AC and GD cells in the female, while in the maléeA the GD cells, ID cells, and AC cells all showed approximately equal activity. Three-dimensional reconstructions indicated that the female possessed a more highly branched intercalated duct system and that the GD usually terminated within a secretory complex, whereas in males the GD typically passes through a secretory complex and forms a prominent cap-like structure on the opposite side. © 1993 Wiley-Liss, Inc.     

Epidermal growth factor in the submandibular glands of inbred mice

Epidermal growth factor (EGF), an androgen-dependent polypeptide, occurs in high concentration in male mouse submandibular gland. Glands of adult male and female mice of six inbred strains (129/J, C57BL/6J, C58/J, SWR/J, RF/J, A/J) were assayed for EGF by radioimmunoassay. In all strains, the glands of males contained 30 to 500-fold more EGF than those of females. Furthermore, significant differences in EGF content were found among the various strains in both sexes; the highest amount of EGF was present in RF/J and the lowest in C57BL/6J, with a ratio of three in the males and four in the females of the two strains, respectively. Factors that effect EGF levels were analyzed further, using these two strains. EGF was measurable in the glands of mice of both strains at 21 days of age and increased rapidly thereafter, up to 14 weeks of age. Throughout postnatal development, the level of EGF was greater in the glands of RF/J mice than in those of the C57BL/6J animals. Thirty days after castration, the EGF levels were reduced by about 98% in both strains, but the strain difference was not abolished. Testosterone implants (1 mg in Silastic tube) in castrated mice induced EGF levels six- to ten-fold compared to castrates. Even in induced animals, which had similar plasma testosterone levels, as measured by radioimmunoassays, the difference in EGF levels between the two strains was manifest. Such a difference, however, was not seen after the daily administration of 5-alpha-dihydrotestosterone for 3–14 days. Immunocytochemical staining for EGF also indicated a higher concentration of the polypeptide in the glands of RF/J mice than in those of C57BL/6J animals, and confirmed the exclusive localization of EGF in the cells of the granular convoluted tubules (GCT). According to our morphometric analysis, in the glands of male RF/J mice the GCT compartment occupied a greater portion (8% greater, P<0.001) of the gland volume than in C57BL/6J mice. The difference in the relative GCT volumes in the glands of female mice of the two strains was, however, statistically not significant. There was no direct correlation between the amount of EGF and the relative volume of the GCTs in the two strains. The evidence obtained implies that strain differences in submandibular-gland EGF levels are determined genetically.     

Co-localization of the nerve growth factor precursor protein and mRNA in the mouse submandibular gland

Indirect immunofluorescence on frozen sections of the mouse submandibular gland (MSG) and affinity-purified sera directed against synthetic peptides that reproduce sequences of the nerve growth factor (NGF) precursor protein permitted its localization in the basal parts of the cells forming the secretory tubules. In situ hybridization experiments employing 35S-labeled NGF cDNA probe localized the NGF mRNA in the same region. Conversion of proNGF to mature NGF results in an altered localization of the cleaved peptide throughout the cytoplasm of the tubular cells with a preferential concentration at their apical pole.     

Immunocytochemical localization of nerve growth factor,epidermal growth factor,renin and protease A in the submandibular glands of Tfm/Y mice

The submandibular glands of mice with testicular feminization (Tfm/Y) and their normal adult male littermates (Ta/Y) were studied by immunocytochemical techniques for the demonstration of epidermal growth factor (EGF), nerve growth factor (NGF), renin and protease A. In the glands of both the affected and normal males, these polypeptides were restricted to cells of the granular convoluted tubules (GCT), with the exception of protease A, which was also found in small amounts in striated duct cells. Compared to those of Ta/Y males, GCTs were narrower in the glands of Tfm/Y mice and contained a markedly reduced number of cells immunoreactive for EGF, NGF and renin. However, the number of GCT cells that stained for protease A in the glands of Tfm/Y males was not as drastically decreased.     

Functional nerve growth factor receptor in von Recklinghausen neurofibromatosis: an immunocytochemical and short-term culture study

Immunocytochemistry reveals 75 kDa low affinity type nerve growth factor receptor (NGFR) on the cell membrane of human neurofibroma cells of von Recklinghausen disease in vivo and in vitro. NGF-immunoreactivity is detected in the primary and cultured tumor cells. Growth augmentation of cultured neurofibroma cells by exogenous NGF is also confirmed. Phosphotyrosine-immunoreactivity is demonstrated by immunocytochemistry in the in vivo and in vitro neurofibroma cells suggesting possible phosphorylation of tyrosine residue in the NGFR or a cellular protein downstream of signal transduction through the ligand receptor system. These results indicate human neurofibroma cells possess functional NGFR and the growth is potentiated through the NGF-NGFR system in the paracrine and/or autocrine fashion.     

Precocious development of granular convoluted tubules in the mouse submandibular gland induced by thyroxine or by thyroxine and testosterone

The effects of the administration of thyroxine (T₄) and/or testosterone (TP) on the early postnatal differentiation of granular convoluted tubule (GCT) cells of the submandibular glands were studied in Swiss-Webster mice. From birth, mice were injected daily with T₄ up to the time of killing at 15 or 20 days of age. In addition, groups of mice were given one injection of TP eight days before killing. Control animals received vehicles (saline and/or sesame oil). Sections of the glands were stained with toluidine blue, or immunocytochemically by the unlabelled antibody enzyme method, for the localization of protease A, epidermal growth factor, or renin. Supernatants of the gland were analyzed for protease activity (pH 8.5, substrate: tosyl-glyclyl-L-prolyl-L-arginine nitroanilide acetate), or by radioimmunoassay for EGF content. At 15 days of age no GCT cells were present in the glands of control or TP-treated mice. In T₄-injected mice many small GCT cells occurred, while larger and more numerous GCT cells were seen in the glands of mice that received both hormones. TP alone had no effect on levels of EGF or protease activity. In T₄-treated mice, protease levels increased 10-fold and EGF content rose 28-fold. TP administration to T₄-treated mice caused a further threefold, increase in both protease activity and EGF content. At 20 days of age the glands of control mice had a few small GCT cells, and both protease activity and EGF content were substantially increased. TP treatment was again without effect. However, daily injections of T₄ caused both protease activity and EGF levels to increase 20- and 11-fold, respectively. Just as in 15-day-old mice, TP administration to T₄-primed mice resulted in further increases in the two polypeptides. Immunocytochemical staining for protease A and EGF confirmed the chemical data. Renin was detectable immunocytochemically at both ages in the glands of mice that had received T₄, but it was seen in the glands of mice that had received TP alone only at 20 days of age. It is concluded that T₄ caused a precocious induction of GCT cells and their specific products. Although TP alone had no effect on their early differentiation, it acted synergistically with T₄ in inducing GCT cells. There were no obvious sex differences in any of these features in control or treated animals.     

Biological and immunological properties of the nerve growth factor from bovine seminal plasma: comparison with the properties of mouse nerve growth factor

The biological activities of Nerve Growth Factor (NGF) purified from bovine seminal plasma have been compared with those of NGF from mouse submandibular glands in a variety of systems: maintenance of survival in vitro and stimulation of nerve fibre outgrowth from sensory, sympathetic and parasympathetic neurons of the embryonic chick; maintenance of survival in vitro, stimulation of nerve fibre outgrowth and specific induction of tyrosine hydroxylase in neonatal rat sympathetic neurons; stimulation of nerve fibre outgrowth and cellular hypertrophy and specific induction of choline acetyltransferase in pheochromocytoma PC12 cells; induction of tyrosine hydroxylase in bovine adrenal medullary cells; and stimulation of nerve fibre outgrowth from expiants of goldfish retinae. In all cases, the two NGFs had the same effects qualitatively and quantitatively, and with identical dose-dependencies. The results indicate that the wide range of biological effects and target cells delineated in detail for mouse NGF can justifiably be attributed to other NGF proteins, and that they are not exclusively restricted to the mouse NGF molecule. Furthermore, as bovine NGF is free of the renin contaminants so difficult to remove from mouse NGF, the above biological activities can truly be assigned to the NGF molecule.Immunologically, however, mouse and bovine NGFs differ substantially. This is demonstrated by the relatively poor ability of antisera against bovine NGF to inhibit the activity of mouse NGF in vitro, and by the incomplete nature of the immunosympathectomy caused in rats by treatment with antisera to bovine NGF, in contrast to the extensive immunosympathectomy caused in these animals by the administration of comparable quantities of antisera against mouse NGF.Clearly, the biochemical features of the NGF molecules responsible for their biological effectiveness and for their predominant antigenic properties are different.     

小鼠下颌下腺发育中转化生长因子β受体的表达

背景：小鼠的下颌下腺是研究唾液腺的发育的良好模型,转化生长因子β是器官发育和疾病中重要的生长因子,但是在下颌下腺中转化生长因子β受体的表达以及作用机制至今并不明确。 目的：观察胚胎小鼠下颌下腺发育过程中转化生长因子βⅠ型受体和Ⅱ型受体以及p-ERK1/2的表达,揭示转化生长因子β在小鼠涎腺发育中的作用。 方法：取C57BL/6J小鼠胚胎期第14.5天的标本,使用转化生长因子βⅠ型受体和Ⅱ型受体以及p-ERK1/2抗体,对小鼠的下颌下腺进行免疫组化染色。取新生小鼠标本,大体观察下颌下腺,并且使用苏木精-伊红染色观察其形态。 结果与结论：①小鼠出生时,下颌下腺位于下颌骨下方;苏木精-伊红染色发现小鼠下颌下腺的腺泡、导管和闰管细胞也已经分化完成。②在胚胎期第14.5天,转化生长因子βⅠ型和Ⅱ型受体在腺泡上皮和导管上皮内高表达,而腺体上皮细胞外的间充质没有表达。③p-ERK1/2主要也是表达在下颌下腺的上皮细胞中,与转化生长因子βⅠ型受体和Ⅱ型受体在下颌下腺中的表达基本一致。说明在小鼠下颌下腺的发育过程中,转化生长因子β蛋白可能通过与上皮细胞表面的受体结合,激活ERK信号通路来调节涎腺腺泡和导管的发育。     

Effects of pretreatment with androgen or thyrold hormone on androgen-induced proliferation of granular convoluted tubular cells in mouse submandibular glands

The effects of pretreatment with androgen or thyroid hormone on androgen-induced proliferation of granular convoluted tubular cells (GCT cells) in the submandibular glands of ovariectomized female BALB/c or C57BL/6 mice were investigated. The proliferation of GCT cells was estimated by their labeling index. Daily injections of 5α-dihydrotestosterone (DHT) (100 μg/mouse/day) caused a transient increase in the labeling index of GCT cells of ovariectomized 60-day-old BALB/c mice during the first four injections, but injections of thyroxine (T₄) (15 μg/mouse/day) did not. On the other hand, both DHT and T₄ increased the esteroprotease activity, a marker of the differentiation of GCT cells, time dependently. Injections of DHT into ovariectomized 102-day-old BALB/c mice also caused a transient increase in the labeling index of GCT cells. However, pretreatment of ovariectomized 60-day-old BALB/c mice with DHT for 4 or 14 days completely abolished the DHT-induced increase in the labeling index of 102-day-old mice, and pretreatment with T₄ for 14 days reduced this increase. Pretreatment with DHT or T₄ for 14 days did not affect the DHT-induced increase in esteroprotease activity. Pretreatment of ovariectomized 60-day-old C57BL/6 mice with DHT for 14 days also completely abolished the DHT-induced increase in the labeling index of GCT cells at the age of 102 days, but pretreatment with T₄ for 14 days did not affect the increase. These results suggest that the proliferation of mouse GCT cells induced by androgen results in a complete abolition of their proliferative response to androgen, and that the effect of thyroid hormone on the proliferative response of GCT cells to androgen may differ in different strains of mice. © 1993 Wiley-Liss, Inc.     

Subcellular localization of nerve growth factor receptors in identified cells of the rat nucleus basalis magnocellularis: an immunocytochemical study.

The subcellular location of nerve growth factor receptor in the ventromedial portion of rat globus pallidus was investigated with affinity-purified monoclonal 192-IgG following the unlabelled antibody peroxidase-antiperoxidase immunocytochemical procedure. At the light microscopic level, punctate immunoreaction product was observed in the perinuclear region and in the plasma membrane of large, probably cholinergic neurons. Examination in the electron microscope of these neurons confirmed that nerve growth factor receptor-stained cells were basal forebrain cholinergic neurons. Within these cells, immunostaining occurred in the Golgi apparatus, in multivesicular bodies and, occasionally, in rough endoplasmic reticulum cisternae and the nuclear envelope. Moreover, patches of immunoreactivity were observed associated with the outer surface of the plasma membrane of the soma and their proximal dendrites and also with the plasma membrane of distal dendrites showing scarcity of synaptic input. Positive immunostaining was never observed in synaptic clefts, but filled the space between the plasma membranes of immunoreactive neurons and those of thin glial processes in their vicinity. The location of membrane nerve growth factor receptor in close apposition to membranes of neighbouring astrocytes rather than near synaptic complexes, suggests that glial cells may be a physiological source of nerve growth factor.     

Effect of estradiol on ultrastructure of granular ducts in submandibular glands of female rats

The effects of estradiol on the granular ducts in submandibular glands of female albino rats were studied. Twenty-five-milligram pellets of 17 beta-estradiol were implanted subcutaneously in the experimental animals, and their glands, as well as controls, were examined after 2, 4, 7, and 10 wk using light and electron microscopy. During the course of the experiment an increasing proportion of the granules in the granular ducts appeared more lightly stained in the experimental animals. In the estradiol-treated rats the granular ducts increased in relative cross-sectional area at a faster rate than in the controls, which exhibited maturation changes. In addition, the average number of granules per granular duct cell decreased significantly in the treated animals. Our results indicate that estradiol caused a change in the cytology of the granular ducts suggesting an alteration in protein synthesis. These results might occur through a change in structural proteins or in other hormones and growth factors which are known to influence the submandibular gland.     

A granular cell in the proximal intercalated duct of human parotid and submandibular glands

In human parotid and submandibular gland unusual granulated cells are observed at the acinar-intercalated duct junction. These cells, which show a well developed Golgi apparatus, greatly differ from the typical elements of the intercalated ducts mainly due to the presence of abundant secretory granules of unknown nature. A complex substructure distinguishes these granules from those of conventional ductal cells and from those of acinar cells as well. In addition, some differences exist in the morphology of the granules observed in parotid with respect to those of submandibular gland.