Delay in maturation of the submandibular gland in Chagas disease correlates with lower DNA synthesis

It has been demonstrated that the acute phase of Trypanosoma cruzi infection promotes several changes in the oral glands. The present study examined whether T. cruzi modulates the expression of host cell apoptotic or mitotic pathway genes. Rats were infected with T. cruzi then sacrificed after 18, 32, 64 or 97 days, after which the submandibular glands were analyzed by immunohistochemistry. Immunohistochemical analyses using an anti-bromodeoxyuridine antibody showed that, during acute T. cruzi infection, DNA synthesizing cells in rat submandibular glands were lower than in non-infected animals (p < 0.05). However, after 64 days of infection (chronic phase), the number of immunolabeled cells are similar in both groups. However, immunohistochemical analysis of Fas and Bcl-2 expression did not find any difference between infected and non-infected animals in both the acute and chronic stages. These findings suggest that the delay in ductal maturation observed at the acute phase of Chagas disease is correlated with lower expression of DNA synthesis genes, but not apoptotic genes.     

Epidermal growth factor-like material in rat submandibular gland.

By using an antiserum specific for mouse epidermal growth factor (EGF), only the granular convoluted tubule (GCT) cells revealed immunochemical staining in rat submandibular glands. There was no regular sexual difference in the frequency or size of immunoreactive cells. Extracts of gland contained an antigen which showed a complete cross-reactivity with mouse EGF in radioimmunoassays. The relative amounts of EGF, determined by a heterologous radioimmunoassay, were not significantly different in the glands of rats of the two sexes. Administration of testosterone caused an increase, in both sexes, in the number of GCT cells stained for EGF and in the amount of EGF in the gland. There was no significant sexual differeence in these two parameters after androgen treatment.     

In situ localization of mRNA for epidermal growth factor in the submandibular gland of the mouse

Epidermal growth factor (EGF) is a polypeptide originally isolated from the mouse submandibular gland, where it is localized immunocytochemically in cells of the granular convoluted tubules (GCT). cDNAs encoding the precursor of mouse submandibular EGF have been cloned (Scott et al. Science 221:236, 1983; Gray et al. Nature 303:722, 1983). A fragment of one of these clones, pmegf10, containing the EGF coding region, was tritium-labeled by nick-translation and used as a probe for in situ hybridization to EGF mRNA. A specific hybridization signal for EGF mRNA was seen only in mature or developing GCT cells. The intensity of the signal was stronger in glands of intact males than in females or in castrated males. In glands of castrates treated with testosterone, or of intact females treated with triiodothyronine (T3), the signal was comparable to that in intact males. In glands of males treated with T3 the intensity of the signal was stronger than in untreated males. A weak to moderate signal was seen in developing GCT cells of 20-day-old males but not females. Hybridization for 3 days gave a stronger signal than that for 1 day. No signal was seen in either sex at 10 days of age, or in control preparations exposed to labeled DNA of pBR322. The presence of EGF mRNA exclusively in GCT cells provides strong evidence that these cells are the only site of synthesis of EGF in the submandibular gland. In situ hybridization with this cDNA probe will provide a sensitive method to determine possible cellular sites of EGF production outside of the submandibular gland.     

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.     

Altered secretion and processing of epidermal growth factor in adrenergic-induced growth of the rat submandibular gland

The granular convoluted tubule (GCT) cells of the submandibular glands represent a major production site for epidermal growth factor (EGF). This study investigates EGF production in the submandibular glands in relation to beta-adrenergic stimulation. Rats were treated with isoproterenol (beta-agonist), which caused up to a 400% increase in submandibular tissue weight after 3 weeks. The weight increase coincided with marked morphologic changes, with degranulation and an apparent decrement in the number of the GCT cells. Immunostaining against EGF revealed a reduction in the number of EGF-immunoreactive cells. Concomitantly, the glandular contents of 6-kDa EGF decreased from 12.86+/-3.42 nmol/gland (mean+/-S.E.M.) in controls to 0.26+/-0.03 nmol/gland. EGF mRNA levels, expressed relative to total RNA levels, only tended to be reduced after 3 weeks as judged from RT-PCR and in situ hybridization (ISH). The isoproterenol-treated rats had increased output of EGF in the saliva, but the salivary secretion of protein was also increased. In both glandular tissue and saliva, gel filtration revealed partially processed high molecular weight forms of EGF in the isoproterenol-treated rats. These data indicate that isoproterenol treatment leads to a hyperstimulatory state of the GCT cells, which then causes depletion of the cellular stores of mature EGF, and most likely due to a shortened posttranslational transit, incomplete peptide processing.     

Growth hormone and epidermal growth factor in salivary glands of giant and dwarf transgenic mice.

Epidermal growth factor (EGF) in rat salivary glands is regulated by testosterone, thyroxin, and growth hormone (GH). Salivary glands of 45-day-old giant and dwarf male and female transgenic mice were examined histologically and by immunohistochemistry (IHC) for EGF. Male giants showed no significant differences from wild-type (WT) parotid and submandibular glands. However, their sublingual glands expressed EGF diffusely and strongly in granular cells within the striated ducts, where they were not found in WT mice. Submandibular gland ducts of female WT were different, having individual granular cells strongly positive for EGF and distributed sporadically along the striated duct walls. Neither female GH-antagonist dwarf mice nor GH-receptor knockout mice had any granular cells expressing EGF in any gland. Obvious presence of granular duct cells in the sublingual glands of giant male mice suggests GH-upregulated granular cell EGF expression. Furthermore, absence of granular duct cells from all glands in female GH-antagonist and GH-receptor knockout transgenic mice suggests that GH is necessary for the differentiation of the granular cell phenotype in female salivary glands.     

Immunocytochemical localization of renin in the submandibular gland of the mouse during postnatal development.

The localization of renin in the developing mouse submandibular gland was studied immunocytochemically using the unlabelled antibody-enzyme method of Sternberger ('74). Bouin-fixed submandibular glands of mice of both sexes were examined at 5-day-intervals from birth (day 0) to 50 days of age. At all stages studied, only granular convoluted tubule (GCT) cells stained immunocytochemically for renin; such cells were first seen in glands of 30-day-old males and of 30-day-old females. The size and number of renin-containing GCT cells increased rapidly in males, attaining adult status by 50 days of age. In females, differentiation of GCT cells immunoreactive for renin was slower and less regular than in males, and at 50 days of age the GCT segment had not yet reached adult conditions with respect to the distribution of renin. Renin appears in GCT cells at later ages than other GCT cell products (e.g., EGF and amylase), suggesting the existence of independent developmental control for the expression of various biologically active substances in the GCTs.     

Age-Related Changes in EGF and Protease in Submandibular Glands of C57BL/6J Mice1,4

The submandibular glands of male C57BL/6J mice were studied cytologically and chemically at the following ages (months): 1–1.5, 6–8, 12–13, 28–32. The relative proportion of granular convoluted tubules (GCT) as well as the size and content of secretion granules of GCT cells, progressively increased throughout the first year of life. Correspondingly, the concentration within the glands of two GCT cell products, epidermal growth factor (EGF) and protease, also steadily increased. In senescent glands, GCTs formed relatively less of the gland parenchyma and were composed of shorter cells with reduced amounts of secretory granules. The concentration of EGF was reduced to 17% of its peak value at one year, while protease activity declined to 50% of its peak value. These morphologic and chemical findings imply a functional impairment in submandibular glands of the mouse with senescence.     

Epidermal growth factor secretion by submandibular glands is not perturbed in the early phase of streptozotocin-induced diabetes in mice

In rodents, diabetes mellitus is accompanied by a decreased concentration of epidermal growth factor (EGF) in plasma and urine and by a reduced number of EGF receptors on the surface of target cells. A combination of these two abnormalities may reduce the effects of EGF and could be implicated in some complications of diabetes. The aim of the present work was to investigate the possible implication of the major source of EGF in the organism, the submandibular glands, upon the reduced EGF concentration in blood and urine. Firstly, we measured the content of mice submandibular gland EGF by radioimmunoassay and by morphometric determinations of the relative volume occupied by granular convoluted tubules in the gland and by the EGF-containing granules in the cells at the light and electron microscopical levels respectively. We found no differences in the EGF content of submandibular glands of streptozotocin-treated diabetic animals compared to control ones. Secondly, we estimated stimulus-secretion coupling by EGF-secreting cells, present in an enriched preparation of granular convoluted tubules (GCT) perifused in thermoregulated chambers. Phenylephrine was the only agent tested that was capable of stimulating EGF secretion. The stimulation was dose-dependent and similar in streptozotocin-diabetic and control mice. Thus, the EGF deficiency observed in diabetic mice is related neither to a defect of EGF content in the submandibular glands nor to an abnormal EGF secretion by the glands.     

Autoradiographic localization of dihydrotestosterone binding in the major salivary glands and other androgen-responsive organs of the mouse

Mouse submandibular glands show an androgen-dependent sexual dimorphism, reflected in higher concentrations in males than in females of bioactive peptides, such as epidermal growth factor (EGF), nerve growth factor, and renin in the cells of the granular convoluted tubules (GCT). Biochemical studies have demonstrated androgen receptors in submandibular gland and other androgen-responsive organs in mouse. We have determined the cellular localization of these receptors using steroid autoradiography. Fifteen adult gonadectomized male mice were injected intravenously with 0.13 microgram or 0.26 microgram [3H]-dihydrotestosterone (SA 135 Ci/mM); some animals were pre-treated with cyclocytidine to stimulate secretion by GCT cells. Animals were killed 15 min, 1, 2, or 3 hr after isotope injection. Steroid autoradiographs were prepared, and some were stained immunocytochemically for EGF. Of the different cell types of submandibular gland, the acinar cells most frequently and intensely concentrated [3H]-DHT; GCT cells also concentrated the hormone, as did a small number of striated duct cells. In the other major salivary glands, the only cells that concentrated the androgen were interlobular striated duct cells in sublingual gland. In prostate, anterior pituitary, and brain a large number of cells concentrated androgen, as has been previously reported. Androgen binding by the GCT cells was a predictable finding, since androgen-induced alterations in composition and form of these cells are well documented. The intense androgen concentration by the acinar cells was an unexpected finding and suggests a hitherto unknown androgen regulation of these cells. An incidental finding was intense concentration of [3H]-DHT in the nuclei of the endothelial cells of the post-capillary venules of the cervical lymph nodes.     

雄性大鼠颌下腺雄激素受体表达及镉和丙酸睾酮对其的影响

目的探测雄性大鼠颌下腺内雄激素受体(AR)表达及细胞分布特征,了解氯化镉和丙酸睾酮对AR表达的影响。方法35只雄性Wistar大鼠分为3组:对照(C)组、镉(Cd)组和镉加丙酸睾酮(Cd T)组。利用免疫组化SABC法和图像分析系统作AR表达检测及平均光密度(AOD)值定量分析。结果AR在浆液性腺泡(AC)、颗粒曲管(GCT)、纹状管(SD)和排泄管等细胞内均有不同程度的表达,定位以细胞质为主。其中GCT细胞内AR的AOD值最高,SD次之,AC细胞最低。Cd处理24h后,三种细胞内AR的AOD值均见下降,7d时达最低水平,此后有所增加,但到30d时仍低于对照组(P<0.05)。Cd T处理后,GCT细胞内AR的AOD值明显增加。与3、7、15d时Cd组相比,差别均有显著性。AC和SD细胞内AR的AOD值虽高于相应时间点的Cd组,但两者比较仅在7d时差别有显著性。结论雄性大鼠颌下腺内广泛存在AR,镉可致颌下腺GCT、AC、SD细胞AR表达减少,补充雄激素可明显增加GCT细胞AR表达,但对AC和SD细胞AR表达的影响较小。     

Effects of castration and sex steroids on sexually dimorphic development of the mouse submandibular gland

The aims of this study were to characterize sexual dimorphism in the submandibular glands of young adult mice and to determine how sex differences arise during postnatal development. In the mouse submandibular glands, prominent sexual dimorphism was observed at 30 days of age, when the male gland was superior in both the relative occupied area (ROA) and the mitotic rate of the granular convoluted tubules (GCT) to those of the female. By neonatal castration, this sexual dimorphism was abolished, and the intraglandular structures of castrated males were similar to those of normal females. In castrated mice of both sexes, daily treatment with testosterone and 5 alpha-dihydrotestosterone for 10 days from 20 days induced only the ROA of the GCT to increase to the normal male levels but not those of the other three regions of the glands, the acini, intercalated ducts and excretory striated ducts. Testosterone responsiveness of the glands, considering both the glandular weight gain and the mitotic rate of the GCT, was significantly higher in castrated males than in castrated females. On the other hand, 17 beta-estradiol had no effect on the glands of castrated mice. Therefore, the present study suggests that the testicular hormones are responsible for the masculine development of GCT of the glands, but not the ovarian hormones, and that there is a sex difference in the responsiveness of the glands to testosterone, which is more effective in males than in females.     

Immunohistochemical localization of S100A1 and S100A6 in postnatally developing salivary glands of rats

 S100 proteins are calcium-binding proteins of the EF-hand superfamily and are involved in the regulation of a number of cellular processes. The present study deals with the immunohistochemical expression of S100A1 and S100A6 in the rat submandibular and sublingual glands during postnatal development from day 0 to 12 weeks. Expression of S100A1 was particularly concentrated in pillar and transition cells in the granular convoluted tubule (GCT) and in striated duct cells of the submandibular gland age 4 weeks to maturity. None or only weak staining for S100A1 was observed in the duct segment at 0–5 days. On the contrary, immunostaining of S100A6 was present in proacinar cells in undifferentiated submandibular gland at age 3 days to 2 weeks. S100A6 immunoreactivity in rat submandibular gland coexisted with chromogranin reactivity. It is possible that S100A6 regulates secretion of chromogranin in proacinar cells. Secretion of growth factors and biologically active peptides in the GCT are regulated by calcium signals, and S100A1 may be involved in the secretory mechanism of granular cells. S100A1 and S100A6 are potentially of great importance in secretory functions of granular cells and proacinar cells, as well as in rat salivary glands. Accepted: 14 July 1998     

Erythropoietin expressed in granular convoluted tubule cells of mice submandibular glands under hypoxia, anemia, and nephrectomy

Immunohistochemically detectable erythropoietin-like substance(Epo) in granular convoluted tubule(GCT) cells of submandibular glands (SMG's) was examined in mice in which hemolytic anemia had been induced by phenylhydrazine (ph), and in mice subjected to hypoxia, nephrectomy, or testosterone (TP) injections. Staining for Epo was negative in GCT cells of SMG's in normal mice, while positive staining occurred in GCT cells of the anemic mice and mice subjected to hypoxia or nephrectomy. A positive Epo reaction was also revealed at the luminal borders of distal tubules, and in cells of proximal and distal tubules in the kidney, and in some hepatic and spleen cells, of mice that had received combination regimens producing anemia and hypoxia, or had been nephrectomized. Increased staining of Epo was found in GCT cells of SMG's, and in proximal and distal kidney tubules of mice given the combination treatment plus TP injections. The detection of Epo in GCT cells suggests these extrarenal cells to be sites of accumulation or biosynthesis of the protein under certain specific conditions such as hemolytic anemia and hypoxia.     

Postnatal developmental changes in submandibular glands of rats and mice

The submandibular glands of mice and rats are not fully developed at birth. In early postnatal life, differentiation of acini takes place before that of granular convoluted tubule (GCT) cells. The latter develop from striated duct cells, and first appear in both species around 15 days of age. In mice their full development gets under way by 20 days of age and is rapid in males and slow in females, resulting in a clear sexual dimorphism in adults. In rats, GCT development is more protracted, and accelerates around 40 days of age, with no sexual dimorphism seen at any time. The course of postnatal development of several GCT cell products is correlated with the cytodifferentiation of these cells. Reliable data are available for the development of amylase, proteases (including kallikrein), renin, epidermal growth factor, and nerve growth factor. Preliminary information exists for a glucagon-like substance. Cytodifferentiation of GCT cells is under hormonal control. Androgens alone can not precociously induce GCT cells, but thyroid hormones can do so, acting either alone or synergistically with androgens.     

Comparative image analysis of EGF immunoreaction in rat submandibular gland using 3,3'-diaminobenzidine with metal enhancer substrate

We have shown the efficacy of image analysis using 3,3'-diaminobenzidine (DAB) with a metal enhancer substrate for demonstrating quantitative differences in the amount of epidermal growth factor (EGF) in the submandibular gland from normal, castrated, and testosterone propionate (TP) treated castrated rats. Immunohistochemical determination of EGF visualized by DAB-nickel reagent was performed with image analysis using a computed image analyzer system (ACAS 570). Immunohistochemistry for EGF disclosed positive staining in granular convoluted tubule cells in the tissue sections from each experimental group. Using the tools of a competent data program installed in the ACAS 570 software, we measured quantitative differences among the experimental glands examined. Castration was shown to elicit a significant reduction in the EGF-positive area and staining intensity, and administration of TP to the castrated animals restored these parameters to levels greater than those of normal rats. Our study demonstrates that a simple, inexpensive, commercially available metal enhancer substrate can be applied accurately to the computer assisted quantification of histochemical hormone-induction studies.     

Expression of epidermal growth factor in salivary adenoid cystic carcinoma

This study used an immunohistochemical technique to assess the expression of epidermal growth factor (EGF) in 40 specimens of salivary adenoid cystic carcinoma (ACC), 7 specimens of labial glands adjacent to mucocele, and 5 specimens of normal submandibular glands. In normal submandibular glands, immunohistochemically detectable EGF was demonstrated in all ductal segments, including intercalated, striated, and excretory duct cells. No EGF positive staining was found in acinar compartments. including serous and mucous acinar cells. In degenerated labial glands adjacent to mucocele, no EGF staining was detected in the remaining acinar and ductal cells. In salivary ACCs, positive EGF immunostaining was observed in one of the 5 (20%) ACCs with a solid pattern and in 13 of the 35 (37.1%) ACCs with a tubular-cribriform pattern. The overall EGF expression rate in 40 salivary ACCs was 35%. Positive EGF staining was predominantly found in tubular structures in the tubular ACCs and in duct-like structures in large cribriform patterns or in the stroma of the cribriform ACCs. There was no significant correlation between EGF expression in salivary ACCs and any of the clinicopathological parameters including patient age and sex, cancer location, TNM status, clinical stage, histologic type, perivascular or perineural invasion, focal necrosis of tumor, and cellular atypia. We conclude that the duct segments of the normal submandibular gland are the sites of EGF synthesis and secretion. In degenerated labial glands adjacent to mucocele, EGF synthesis is completely inhibited. Furthermore, EGF is mainly biosynthesized in cells forming tubular or duct-like structures in tubular or cribriform salivary ACCs, and EGF may play a biologic role, particularly as a mitogen in salivary ACC growth.     

Expression of transforming growth factor beta 1 in the submandibular gland of the rat

We employed immunocytochemical and in situ hybridization techniques to study the expression of transforming growth factor beta 1 (TGF-beta 1) in rat submandibular gland. Immunoreactivity for TGF-beta 1 was observed in the cells of granular convoluted tubules (GCTs), striated ducts, and excretory ducts, whereas it was absent in the intercalated ducts and secretory acini in both male and female rats. Immunoelectron microscopy revealed the ultrastructural localization of TGF-beta 1 in the secretory granules of GCT cells. On the other hand, signals for rat TGF-beta 1 mRNA were abundant in the GCT and striated duct cells but were lacking in the excretory duct cells. These results provided evidence for the production of TGF-beta 1 in the GCTs and striated ducts of rat submandibular gland.     

Histological and histoquantitative study of the rat parotid gland after Trypanosoma cruzi infection

The present study deals with the morphology of the rat parotid gland and its changes after Trypanosoma cruzi infection. The glands of control and infected animals were analyzed by histologic and histoquantitative methods. After 18 days of infection with T. cruzi, a significant reduction of the density of the volume of the acini and duct system, as well as a significant increase in the amount of connective tissue was noted. In addition, these animals displayed an increase in the number of cells undergoing mitosis. In the 45 day infected rats, there was return to the normal pattern. It is suggested that in the infected animals the decrease in body weight could be responsible for retarded sexual maturity, leading to the lower level of testosterone. It can be assumed that decreased levels of epidermal growth factor (EGF) and neural growth factor (NGF) caused by the lack of testosterone in infected animals also contribute to the atrophy of the parotid gland and to the proliferation of the connective tissue.     

An immunohistochemical study of secretagogue-induced secretion of epidermal growth factor in the submandibular salivary glands of mice

Routine histological and indirect immunofluorescence techniques were used to examine the histological details of changes in the distribution of epidermal growth factor (EGF) in the submandibular salivary glands of mice during secretion. Comparisons were made bewteen glands of normal mice and those of mice given one of a number of secretagogues at various times prior to sampling. Normal submandibular salivary glands in male mice had an extensive system of convoluted granular tubules (CGT), the cells of which contained EGF. When adrenaline of alpha-phenylephrine was administered, the CGT cells degranulated, and there was a concomitant loss of intracellular EGF-positive immunofluorescence. The excretory ducts were engorged with immunofluorescent material, indicating secretion of EGF into saliva, while the ductal cells themselves remained EGF-negative. The degranulation response could be blocked by phentolamine, but not by propranolol, and no changes in EGF distribution followed the administration of pilocarpine. It was concluded that EGF is secreted, at least partly into the saliva, following an alpha-adrenergic response, and that this occurs with degranulation of the cells of the CGT.