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 α-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 β-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 α-adrenergic activation, and that this phenomenon is readily demonstrated in the intact animal.     

Epidermal growth factor and insulin-like growth factor I are localized in different compartments of salivary gland duct cells. lmmunohistochemical evidence

Immunohistochemical methods were used to map EGF (epidermal growth factor) and IGF-I (insulin-like growth factor I; somatomedin C) immunoreactivities in salivary glands of adult rodents. Epidermal growth factor is, as is NGF (nerve growth factor), limited in distribution to the granules in granular duct cells in the submandibular gland. Insulin-like growth factor I is, in contrast, cytoplasmic and has a much more widespread distribution. It is seen in intercalated, striated and granulated duct cells as well as in apical parts of excretory duct cells. The parotid and the palatine salivary glands, lacking EGF immunoreactivity, have their IGF-I immunoreactivity similarly distributed as the submandibular gland. Isoproterenol treatment of adult male rats results in rapid and extensive growth of the submandibular and the parotid glands, which double their weights in just a few days. Isoproterenol causes release of granules from the submandibular granular duct cells and decrease in frequency of EGF immunoreactive cells. However, there is no or only minor concomitant changes in the distribution and intensity of the IGF-I immunoreactivity in these duct cells. Our results indicate that the trophic peptides EGF (and NGF) and IGF-I are localized in different compartments in salivary gland duct cells and that divergent pathways control their release.     

Epidermal growth factor and insulin-like growth factor I are localized in different compartments of salivary gland duct cells. Immunohistochemical evidence

Immunohistochemical methods were used to map EGF (epidermal growth factor) and IGF-I (insulin-like growth factor I; somatomedin C) immunoreactivities in salivary glands of adult rodents. Epidermal growth factor is, as is NGF (nerve growth factor), limited in distribution to the granules in granular duct cells in the submandibular gland. Insulin-like growth factor I is, in contrast, cytoplasmic and has a much more widespread distribution. It is seen in intercalated, striated and granulated duct cells as well as in apical parts of excretory duct cells. The parotid and the palatine salivary glands, lacking EGF immunoreactivity, have their IGF-I immunoreactivity similarly distributed as the submandibular gland. Isoproterenol treatment of adult male rats results in rapid and extensive growth of the submandibular and the parotid glands, which double their weights in just a few days. Isoproterenol causes release of granules from the submandibular granular duct cells and decrease in frequency of EGF immunoreactive cells. However, there is no or only minor concomitant changes in the distribution and intensity of the IGF-I immunoreactivity in these duct cells. Our results indicate that the trophic peptides EGF (and NGF) and IGF-I are localized in different compartments in salivary gland duct cells and that divergent pathways control their release.     

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.     

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.     

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.     

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.     

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 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.     

小鼠下颌下腺NGF基因表达的性别差异研究

实验应用地高辛标记人β神经生长因子ＤＮＡ探针原位杂交组化技术，研究小鼠下颌下腺神经生长因子（ＮＧＦ）表达的性别差异。实验结果显示，雄性和雌性小鼠下颌下腺ＮＧＦ基因表达的部位基本相同，原位杂交反应产物都选择性分布于纹状管和颗粒曲管上皮细胞中，但雄性小鼠原位杂交信号强，雌性小鼠杂交信号较弱，原位杂交阳性小管在雄性小鼠以粗大的颗粒曲管为主，在雌性小鼠以细小的纹状管为主，颗粒曲管较少，而且在单位面积中雄性     

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.     

雄激素对发育中小鼠下颌下腺神经生长因子合成的影响

光镜下观察了发育中小鼠下颌下腺内神经生长因子(nerve growth factor,NGF)的免疫反应定位及雄激素对它的影响.在生后发育阶段NGF阳性染色定位于颗粒纡曲导管(granular convoluted tubule,GCT)细胞,并且随着该导管发育的进展而增加;已经证明的成年小鼠下颌下腺NGF表达的性差异在性成熟前已可见到.给发育中小鼠雄性激素促进下颌下腺中GCT的发育和NGF的合成.这些结果表明在小鼠下颌下腺发育过程中雄激素可能对NGF合成起过重要作用.     

生长抑素和神经生长因子免疫反应细胞在人胎下颌下腺的分布

本文采用免疫细胞化学ＡＢＣ法，对２０例胎龄为１６～３５周的人胎下颌下腺进行了观察，结果发现：人胎下颌下腺纹状管有生长抑素和神经生长因子免疫反应阳性的上皮细胞，但两种免疫反应阳性物质在纹状管上皮细胞内分布特点不同。腺泡细胞呈免疫反应阴性。推测下颌下腺纹状管上皮细胞中的生长抑素和神经生长因子对下颌下腺复杂的内分泌活动可能有一定的调节作用     

小鼠神经生长因子的细胞内免疫组织化学阳性反应定位

用免疫组织化学方法对小鼠神经组织和非神经组织中神经生长因子的分布进行了定位观察。实验结果表明 :(1)神经生长因子免疫反应主要局限于神经组织 ,尤其是神经元 ;非神经组织除颌下腺的颗粒曲管外基本上都呈阴性反应 ;(2 )在神经元 ,神经生长因子定位于细胞核与细胞质但以胞核为主 ,少量位于胞质靠近细胞膜的部位 ;(3 )雄性小鼠颌下腺颗粒曲管细胞的NGF主要定位于分泌颗粒的界膜区。神经生长因子在胞内定位的精确性和分布的多样性 ,为进一步研究其作用机制和信号传导提供了重要线索     

Expression and localization of cell growth factors in the salivary gland: a review

The salivary gland secretes not only digestive enzymes but also various cell growth factors. Especially in rodents, granular ducts which develop between striated and intercalated ducts, are known to secrete epidermal growth factor (EGF) and nerve growth factor (NGF). Out of newly discovered growth factors, we have examined hepatocyte growth factor (HGF), transforming growth factor-beta (TGF-beta), insulin-like growth factor I (IGF-I), and basic fibroblast growth factor (FGF-2) and have demonstrated their expression and localization in the rat submandibular gland. HGF and TGF-beta show a very similar distribution pattern to EGF, i.e., exclusive localization in the secretory granules of granular duct cells. These factors are suggested to function as i) exocrine salivary factors, or ii) endocrine factors after reabsorption. In contrast, FGF-2 shows a different pattern of cellular and subcellular localization from EGF and others. FGF-2 is localized in cytoplasm of striated and excretory duct cells and pillar cells in granular ducts, but not in secretory granules of granular duct cells. Since the receptor for FGF also present in the same cells as its ligand, FGF is suggested to function in the salivary gland as an autocrine/paracrine factor. The possible physiological roles of salivary growth factors in the digestive organs are discussed based on our own data and an extensive literature.     

生后小鼠颌下腺神经生长因子基因表达的发育变化

为了探讨在发育过程中小鼠颌下腺神经生长因子基因表达的变化，应用原位杂交组织化学技术和图像分析方法对新生至２月龄ＩＣＲ雄性小鼠颌下ＮＧＦｍＥＮＡ基因表达的变化，应用原位杂交研究。结果显示：新生小儿颌下腺未见ＮＧＦｍＲＮＡ杂交信号，８－１０ｄ龄小鼠颌下腺ＮＧＦ原位杂交信号呈阴性至弱阳性。     

人胎海马生长抑素和神经生长因子免疫阳性神经元的研究

本文采用免疫细胞化学ABC法对20例胎龄为16～36周的人胎儿海马进行了观察。证明人胎海马存在着生长抑素和神经生长因子免疫阳性神经元，两者均散在分布于海马皮质的深层，为多极神经元；免疫反应阳性颗粒均匀地分布于神经元胞质中，生长抑素阳性细胞中的颗粒比神经生长因子阳性细胞中的颗粒粗大。本文结果提示：人胎海马中存在的上述两类阳性神经元可能在中枢神经系统的发育中起着重要作用。     

Immunocytochemical ultrastructural study of hypothalamic neurons containing corticotropin-releasing factor in normal and adrenalectomized rats

The neurons of the rat hypothalamus which secrete corticotropin-releasing factor were studied by using a pre-embedding immunocytochemical staining technique that improves both the penetration of immunoreagents within the tissue and the preservation of the ultrastructural morphology of labeled structures. Comparison was made between the subcellular location of corticotropin-releasing factor-41 in perikarya of the paraventricular nucleus and axons of the median eminence, both in intact and adrenalectomized animals either untreated or 24 h after the intracerebral injection of colchicine. Morphometric analysis of the numerical density and of the diameter of corticotropin-releasing factor immunoreactive neurosecretory granules in axons of the median eminence of rats not treated with colchicine, indicated that the main modifications induced by adrenalectomy concerned (1) the differential repartition of labeled granules within the preterminal and terminal axonal portions of the median eminence, and (2) the enlargement of the diameter of labeled granules contained in these axons (from 98 nm to 165 nm). In the hypothalamus of intact and adrenalectomized rats, colchicine treatment increased the number of corticotropin-releasing factor-immunoreactive granules in the neuronal perikarya and reduced their number in the axons, but both these variations were much more marked in adrenalectomized rats. Although the corticotropin-releasing factor immunoreactive granules that accumulated in the perikarya after colchicine treatment were slightly smaller than those in the corresponding axons, the diameter of perikaryal-labeled granules was larger in adrenalectomized than in intact animals (129 nm vs 93 nm). These findings fit the idea that adrenalectomy markedly stimulates both the synthesis and axonal excretion of secretory granules in the hypothalamic neurons secreting corticotropin-releasing factor. They also indicate that suppression of circulating corticosteroids induces qualitative modifications in these neurons leading to the visualization of larger neurosecretory granules, which may reflect differential synthesis and granular packing of synergistic peptides other than corticotropin-releasing factor and/or changes in the process of intragranular maturation of hormonal material.     

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.