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.     

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.     

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.     

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.     

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.     

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.     

Subcellular localization of epidermal growth factor receptor in human submandibular gland

The subcellular distribution of the epidermal growth factor receptor (EGFr) was demonstrated in the normal human submandibular gland by means of immunogold cytochemistry. EGFr labelling appeared in both acinar and ductal cells, where strong immunoreactivity was associated with a tubulovesicular system near the basolateral surfaces. In addition, groups of reactive vesicles were highlighted among secretory granules of both serous and mucous cells and at the apex of ductal cells. Basolateral vesicles were interpreted as being a result of EGFr internalization after activation by an exogenous ligand, although the functional meaning of those located apically remains unclear.     

Acute and chronic adrenergic stimulation of submandibular salivary glands. Effects on the endocrine function of epidermal growth factor in mice

Submandibular salivary glands are the major source of epidermal growth factor (EGF) in mice. Acute secretion of EGF from these glands protects the heart against catecholamine-induced injury. Little is known about chronic adrenergic stimulation of salivary glands and the contribution of accumulated EGF to the adaptive hypertrophic response of the heart to such chronic adrenergic stimulation. Here we show that the EGF content of submandibular glands did not recover to normal values 24 h after a single phenylephrine injection or an aggressive encounter. Repeated (twice a day for 2 days) adrenergic stimulation resulted in an almost 90% decrease in EGF content in the submandibular glands. In these conditions, new adrenergic stimulation did not result in an increase in plasma EGF concentration, or in the activation of liver ErbB1 (the EGF receptor). Chronic isoproterenol or phenylephrine administration (7 days) induced atrial natriuretic factor expression in the heart and an increase in both ventricular weight and protein. The surgical removal of submandibular glands (sialoadenectomy) did not affect these adaptive responses of the heart. We conclude that EGF from submandibular glands does not contribute to heart hypertrophy, one of the adaptive responses induced by chronic adrenergic stimulation.     

Immunocytochemical localization of epidermal growth factor during the postnatal development of the submandibular gland of the mouse

The time of appearance and the pattern of localization of epidermal growth factor (EGF) in submandibular glands of mice was studied during postnatal development immunocytochemically. EGF was first detectable in the granular convoluted tubule (GCT) cells in the glands of males at 20 days of age and of females at 30 days of age. Development of GCT cells containing EGF was rapid in males, approaching adult conditions by 45 days of age. In females EGF-containing GCTs developed more slowly and irregularly, and did not reach adult status by 45 days of age. It is concluded that EGF is restricted during postnatal development to the GCT cells, and that these cells and the distribution of EGF are represented dimorphically from their first appearance in the submandibular glands of both sexes.     

Effect of advanced age on the induction by androgen or thyroid hormone of epidermal growth factor and epidermal growth factor mRNA in the submandibular glands of C57BL/6 male mice

We have compared the responsiveness of the submandibular glands of mature (12 month old) and senescent (26-28 month old) male C57BL/6 mice to dihydrotestosterone (DHT) or triiodothyronine (T3) in terms of steady state levels of epidermal growth factor (EGF) protein and EGF mRNA. Northern blot analyses did not disclose any differences with age in the apparent sizes of EGF mRNA species. In untreated animals, submandibular glands of 26-28-month-old mice contained approximately 50% less EGF, and 75% less EGF mRNA than those of 12-month-old males. With advanced age, there was a 20% reduction in the absolute volume of the granular convoluted tubule (GCT) compartment, which is the exclusive site of EGF and EGF mRNA in the gland. In general, GCTs of old mice were composed of smaller cells with fewer secretion granules, but there was considerable cell-to-cell variation. In addition, there was greater variation in the intensity of immunocytochemical staining for EGF in senescent GCT cells, which also gave a lower and more variable in situ hybridization signal for EGF mRNA. After hormonal stimulation for 1 week with either tri-iodothyronine (T3) or dihydrotestosterone (DHT), EGF protein concentration in the glands was induced to the same level at both ages. However, EGF mRNA was 50% less abundant in old hormonally stimulated glands, compared to similarly treated young ones. Although many GCT cells in treated glands of senescent males respond to hormonal stimulation by increases in size and in content of secretion granules, there was cell-to-cell variation in responsiveness, especially after treatment with T3. These findings indicate that the decreases seen in the entire gland in EGF and EGF mRNA are caused by a wide-spread deterioration of the GCT cells themselves, which apparently can be reversed in many but not all GCT cells by stimulation with supraphysiologic doses of either T3 or DHT.     

Circadian variation of epidermal growth factor receptor in mouse liver

The liver undergoes a biochemical and morphological circadian transformation. In this paper, we document circadian variation in the binding parameters of the hepatic epidermal growth factor receptor (EGFR). Liver membranes were prepared from ad libitum fed or fasted male CD2F1 mice killed at different circadian phases at 4 h intervals. Bmax (maximum binding) and Kd (dissociation constant) varied in a rhythmic fashion. The range of change for Bmax along the 24 h time scale was 423%. For Kd, it was 162%. Both peaked late in the dark span, and decreased late in the light span. Fasting and EGF treatment reduced Bmax and the amplitude of circadian variation.     

Pleiotropic effect of Tabby gene on epidermal growth factor-containing cells of mouse submandibular gland

The influence of the Tabby gene on the submandibular gland of the mouse was assessed by comparing the mass of the gland, and its content of granular convoluted tubules, in prepubertal, pubertal, and adult Tabby mice and their wild type brother controls. The Tabby mice showed reduction in mass of the adult submandibular gland, delayed development of the granular convoluted tubules, and reduction in relative content of granular convoluted tubules. Epidermal growth factor (EGF) is released from the granular convoluted tubules of the mouse submandibular gland, and it is known to experimentally influence the development of at least some of the structures that are affected by the Tabby gene. Accordingly, the question of a relationship between the Tabby gene and EGF is raised.     

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。     

Expression of epidermal and nerve growth factor receptors in human thymus and thymomas

We have investigated the immunohistochemical expression of p53 protein in 96 cases of non-Hodgkin's lymphoma using a panel of five antibodies. Positive neoplastic cells were found in 30 (31.2%) cases, which could be divided into two groups according to their patterns of reactivity with the different antibodies; i.e. those positive with both polyclonal and monoclonal antibodies, and those which were stained only by monoclonal antibodies PAb1801 and/or PAb240. Positivity was nuclear in all but six cases in which cytoplasmic staining was found. In view of the hypothesis recently raised that p53 protein induces apoptosis we have compared our results with parallel staining for bcl-2 protein since bcl-2 is believed to be important, at least in lymphomas, in suppression of apoptosis. Staining for bcl-2 protein was performed on 83 cases and it was shown that p53-positive cases accounted for 10 out of 17 (59%) of the bcl-2 -negative lymphomas but only for 15 out of the 66 (23%) bcl-2 -positive cases, suggesting a possible relationship between the expression of these two proteins. Thus, our data show that p53 protein is abnormally expressed in a substantial proportion of non-Hodgkin's lymphomas and bears a significant inverse relationship to bcl-2 protein expression. However the molecular basis of this expression remains to be elucidated.     

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。     

Expression of IGFBPs in the developing mouse submandibular and von Ebner's glands

The expression of insulin-like growth factor binding proteins (IGFBPs) in the developing mouse submandibular and von Ebner’s glands was determined by in situ hybridization and by an immunohistochemical method. In the submandibular glands, IGFBP-2 and IGFBP-4 mRNAs were expressed in the terminal end-buds (TEB) at E13–E17, concomitant with epithelial branching. IGFBP-3 mRNA was expressed in the mesenchyme surrounding the TEB; and IGFBP-5 mRNA, in the ducts. At E17, IGFBP-5 mRNA expression was observed not only in the ducts but also in the TEB. Similarly, IGFBP-4 mRNA expression was observed not only in the TEB but also in the mesenchyme. After birth, IGFBP-4 expression was observed only in the connective tissue and disappeared by P14. That of IGFBP-7 appeared at P1 and was observed in the connective tissue until P21. The IGFBP-5 mRNA expression pattern after birth was the same as that seen at E17, but at P21 IGFBP-5 was immunohistochemically expressed only in the duct. The mRNA level of IGFBP-2 expression at postnatal days was weak, but its protein was detected in the ducts and acini at P14–P21. In von Ebner’s glands, which appeared at the base of the circumvallate papillae at E17, only IGFBP-2 and IGFBP-4 mRNAs were expressed in the ducts and acini. Postnatally, IGFBP-4 was substituted by IGFBP-5 in the same region. Immunohistochemically, IGFBP-5 and IGFBP-2 were expressed in the ducts and acini at P14–P21. Throughout the study, IGFBP-6 was not detected by in situ hybridization, the immunoreactivity for it was observed in the nerve fibers of submandibular and von Ebner’s glands. These data support a role for these molecules as local mediators of salivary growth and differentiation.     

Population and structure of nerve cells in mouse submandibular ganglion

Summary The anatomy of the mouse submandibular ganglion, and population and fine structure of nerve cells were studied by light and electron microscopy. The submandibular ganglion is a plexus containing up to forty ganglia along the main and the smaller excretory ducts of the submandibular gland. Measurements of the volume of nerve cell bodies display a pattern of distribution with two main peaks, suggesting the presence of large and small types of the intraganglionic neuron. The large neurons mainly have axo-dendritic synapses in which the postsynaptic element is a small spine-like process, while axo-somatic synapses are more common in the small neurons. In some small neurons nuclear chromatin is unusually conspicuous, and accumulations of vesicles of fairly uniform size occur within the cell body. Decentralized ganglia contain almost no synapses, showing the absence of definite interneurons.     

Phenotypic expression of human epidermal growth factor in foetal submandibular gland and pleomorphic adenoma of salivary gland

Summary The phenotypic expression of the human epidermal growth factor (EGF) was investigated immunohistochemically in human foetal submandibular glands from the 5th to 10th month of gestation, adult normal submandibular glands and 48 cases of pleomorphic adenomas. In foetal submandibular glands, both the terminal buds and primary ducts at the intermediate stage of gestation were positive for EGF, and in particular, the outer layer cells of primary ducts showed strong EGF-immunoreactivity. EGF-positive cells decreased as the gestational stage advanced and only ductal cells were weakly positive for EGF at the terminal stage of gestation. In the adult normal submandibular gland, weak immunoreactivity for EGF was restricted to ductal cells. However, 41 (86%) of the 48 pleomorphic adenomas had EGF-positive cells which were distributed among the ductal, chondroid and myxoid portion. No EGF-immunoreactivity was detected in the solid portion of pleomorphic adenomas. These results suggest that EGF may play an important role in the growth and differentiation of foetal cells as well as the proliferation of tumour cells in pleomorphic adenomas.