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。     

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.     

Androgen regulation of the cellular Distribution of the true tissue kallikrein mK1 in the submandibular gland of the mouse.

The kallikrein gene family encodes for at least four different proteases in the mouse submandibular gland (SMG): mK1 (true tissue kallikrein), mK9, mK13, and mK22. These enzymes and many other biologically active proteins are synthesized by the granular convoluted tubule (GCT), a specialized segment of the SMG duct system. The GCT is under multihormonal regulation by androgens, thyroid hormones, and adrenocortical hormones. Androgens suppress synthesis of mK1 in the SMG but enhance expression of the other three kallikreins. We prepared an antibody with limited immunoreactivity for mK1 and used it to examine the effects of androgen status on the distribution of this isozyme in the SMGs of developing and mature mice by immunoperoxidase staining for the light microscope and immunogold labeling for the electron microscope. In prepubertal mice, every immature GCT cell contains mK1, confined to an accumulation of small granules in the subluminal cytoplasm. In mature mice, not every GCT cell contains mK1, and in those cells that do there is considerable intergranular variation in the intensity of staining for mK1. GCT cells containing mK1 are much more abundant in the glands of females than of males, resulting in a peculiar sexually dimorphic mosaic distribution of this isozyme in the mature SMG. Castration of adult males increases the number of GCT cells expressing mK1. Administration of androgen to intact or castrated males or to intact females reduces the number of cells staining for mK1. In all cases, immunogold labeling for mK1 is confined to secretory granules. No fine structural differences were noted between cells that were positively or negatively stained for mK1. Therefore, although GCT cells appear to be composed of a uniform population of cells on the basis of morphology alone, they are not homogeneous in their content of secretory proteins. These results indicate that androgen regulation of GCT cells is more complex than has been appreciated to date.     

Immunocytochemical localization of renin in the submandibular gland of the mouse.

Renin was localized in the submandibular gland of the adult mouse at light and electron microscopic levels by the unlabeled antibody enzyme method of Sternberger. At the light microscopic level, renin was confined to the granular convoluted tubule (GCT) segment of the gland with considerable variation among GCT cells in intensity of staining. Some GCT cells failed to stain for renin. The pattern of staining was the same in the gland of male and female mice, but in the glands of females GCT segments were smaller and less numerous. At the electron microscopic level, staining for renin was also confined to the GCT cells, and was localized exclusively to the secretory granules. The intensity of staining of the secretory granules within a given GCT cell varied; some cells contained only minimally reactive or negative secretory granules. All other organelles within the GCT cell, except condensing vacuoles, failed to stain.     

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.     

四川短尾唾液腺的组织学观察

利用生物显微技术观察和研究了四川短尾鼩(Anourosorex squamipes)唾液腺的组织结构。结果表明,腮腺属纯浆液腺,有闰管和分泌管,无颗粒曲管;颌下腺属混合腺,以混合性腺泡为主,有少量浆液性腺泡和黏液性腺泡,有闰管、颗粒曲管和分泌管;舌下腺属纯黏液腺,有闰管和分泌管,无颗粒曲管,但在分泌管上存在有颗粒曲管细胞。     

Thyroxine accelerates the differentiation of granular convoluted tubule cells and the appearance of epidermal growth factor in the submandibular gland of the neonatal mouse

Summary The effects of the administration of thyroxine (T4) on the postnatal cytodifferentiation of granular convoluted tubule (GCT) cells of the submandibular gland (SMG) of Lewiss-Webster mice were studied by light and electron microscopy. From birth, mice of both sexes were injected daily with T4 (sc 0.4 g/g BW) and were sacrificed 24 h after the last injection at 7, 9, 11, 14 and 21 days of age. Control mice received vehicle only. In control mice, granulated striated duct (SD) cells were first detected at 9 days and 7 days of age by light- and electron microscopy, respectively. Furthermore, a few scattered granulated SD cells were observed by light microscopy as early as day 7 in T4-treated mice of both sexes. At 21 days of age, in mice given T4, GCT cells were larger and more numerous and the Golgi apparatus, rough endoplasmic reticulum, and secretion granules were more abundant. In control mice, immunocytochemical staining for epidermal growth factor-(EGF) was first detectable at day 21 at the light- and electron-microscopic levels. However, positively stained cells were first observed in T4-treated mice by light- and electron-microscopic immunocytochemistry at 14 and 11 days of age, respectively. Moreover, in the 21-day-old T4-treated mice, the number of immunoreactive GCT cells, as well as the intensity of the staining per cell, was markedly increased as compared to controls. EGF immunostaining was restricted to GCT cells, and by immuno-electron-microscopy was only seen in apical secretory granules in granulated SD cells and GCT cells. There were no sex differences in the differentiation of the duct system under any conditions. It is concluded that T4 stimulates the biosynthesis of EGF by an acceleration of the differentiation of the GCT precursor cells to mature cells.Supported in part by grant no. MT-5730 from the Medical Research Council of CanadaHolder of a fellowship from the Medical Research Council of CanadaScholar of the Fonds de la Recherche en Santé du Québec     

Distribution of nerve growth factor in the submandibular gland of the male and female mouse

Summary Nerve growth factor (NGF) was localized in the mouse submandibular gland by means of indirect immunofluorescence applied to 0.5 mthick sections of freeze-dried, plastic-embedded tissue. The antibody to NGF (I_gG-fraction) was raised in rabbits immunized with pure 2.5 S NGF from submandibular glands of adult male mice.In the male gland anti-NGF bound selectively to the secretory granules was present in the cells of the granular ducts. Immunoreactive granules extended from the perinuclear region toward the apical pole. In the female gland immunoreactive cells and granules were considerably less abundant than in males. Immunofluorescence was confined to individual secretory cells located in the wall of the granular striated duct.In the present study no support was found for the hypothesis suggesting that immunoreactive NGF is formed within the secretory granules during their transport from the perinuclear region to the apical pole.     

Cellular basis for the differential response of mouse kallikrein genes to hormonal induction.

The expression of many mouse kallikrein genes in the salivary gland is sexually dimorphic and inducible in females by administration of testosterone or thyroxine. Induction is slow (3-7 days) and is accompanied by the non-uniform differentiation of the cell type expressing these genes from striated duct (SD) cells (female) to granular convoluted tubule (GCT) cells (male). One kallikrein gene, mGK-6, is expressed at an apparently constant total level in male and female and is not induced by either hormone. In situ hybridization histochemistry shows that all kallikrein genes analyzed exhibit uniform cellular distribution of expression in the SD cells of the female. The hormonally mediated differentiation of some, but not all, of these cells has different effects on kallikrein gene expression--mGK-6 is repressed while other kallikreins are induced--leading to non-uniform distribution of expression.     

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.     

An Unusual Sexually Dimorphic Mosaic Distribution of a Subset of Kallikreins in the Granular Convoluted Tubule of the Mouse Submandibular Gland Detected by an Antibody with Restricted Immunoreactivity

The granular convoluted tubule of the mouse submandibular gland contains a wide variety of biologically active proteins, including several kallikreins. The tubule is under multihormonal regulation, and is sexually dimorphic, being larger in males than in females. Correspondingly, levels of its various protein secretory products are more abundant in males than in females. However, isoelectric focussing studies show that the true tissue kallikrein, mK1, is more abundant in the female than in the male submandibular gland. In this study, an antiserum was prepared with restricted immunoreactivity for mouse mK1, and possibly other kallikrein family members of low abundance in the mouse submandibular gland, and used for the immunocytochemical staining of the granular convoluted tubule cells in the submandibular gland of adult male and female mice, by indirect enzyme-labeled and immunogold-labeled antibody methods for light and electron microscopy, respectively. The distribution of immunoreactive tubule cells showed an unusual sexual dimorphism. In males only a few scattered slender tubule cells were strongly stained, while the more typical large tubule cells were only occasionally weakly positive, and many of them were not stained. By contrast, in females slender tubule cells were not seen, and about two thirds of the more typical tubule cells showed moderate to strong immunostaining. Immunoelectron microscopy revealed that immunostaining was confined to the secretion granules in granular convoluted tubule cells in both sexes. The slender tubule cells of males had many strongly stained small apical secretion granules and occasional basal infoldings; in the weakly positive larger more typical tubule cells not all secretion granules were positive, and there was intergranular variation in the intensity of staining of positive granules. In females, although more tubule cells were stained, intergranular variations in staining intensity were also noted. In both sexes, many tubule cells did not contain any secretion granules that showed immunogold labeling for kallikreins. These findings establish that, in contrast to the situation for the majority of granular convoluted tubules proteins, mK1 and possibly other minor kallikrein family members are more abundant in the granular convoluted tubules of female mice, and that there is considerable variation in the content of these kallikreins not only between different tubule cells, but also in individual secretion granules in any given tubule cell in either sex.     

Developmental and androgenic regulation of the immunocytochemical distribution of mK1, a true tissue kallikrein, in the granular convoluted tubule of the mouse submandibular gland.

The action of androgens on the immunocytochemical distribution of mK1, a true tissue kallikrein, was examined in the submandibular gland (SMG) of developing and adult mice by indirect enzyme-labeled and immunogold-labeled antibody methods for light and electron microscopy, respectively. In both sexes at 3 weeks of age, essentially all of the immature granular convoluted tubule (GCT) cells were uniformly immunostained. At 4 weeks of age (the onset of puberty), morphological differences between the two sexes appeared in the GCTs, in which some cells became immunonegative. Thereafter, the immunonegative GCT cells became more abundant in the SMG of males than of females and considerable intercellular variation in staining intensity for mK1 was seen, especially in males. A few slender GCT cells with strong immunoreactivity appeared in GCT segments only in males. Castration of males resulted in an increase in the number of immunopositive GCT cells, whereas administration of dihydrotestosterone (DHT) decreased the number of immunopositive GCT cells in the SMGs of both sexes. Slender GCT cells immunoreactive for mK1 were seen in females treated with DHT for 6 days. However, there were no immunostained slender GCT cells in female SMGs after injection of DHT for 2 weeks. Immunoelectron microscopy disclosed this type of cell in male SMGs, which closely resembles immature GCT cells of prepubertal mice, with a few small secretory granules uniformly labeled with gold particles, a sparse Golgi apparatus and RER, and basal infoldings. In mature male SMGs and in SMGs of DHT-treated females and castrated males, typical GCT cells had a well-developed Golgi apparatus and a net-like RER but few to no basal infoldings, whereas in the female gland equivalent cells had moderately developed RER and some basal infoldings. These results suggest that mK1 is one of the enzymes characteristically present in immature GCT cells and that its synthesis is inhibited in part by androgens, resulting in decreased numbers of immunopositive cells.     

Transduction of TAT-HA-beta-galactosidase fusion protein into salivary gland-derived cells and organ cultures of the developing gland, and into rat submandibular gland in vivo.

We have studied the transduction of TAT-HA-beta-galactosidase fusion protein into two cell lines of rat salivary gland origin, A5 and C6-21, into cells of fetal mouse submandibular glands in organ culture, and into rat submandibular gland after retrograde duct injection, using a histochemical method to demonstrate beta-galactosidase activity. Transduction of the fusion protein into A5 and C6-21 cells was concentration- and time-dependent. Therefore, the intensity of the beta-galactosidase staining, which was cytoplasmic, was less after 1 hr of exposure compared to exposures up to 24 hr. However, the fusion protein was transduced into 100% of both types of cultured cells. When explants of mouse fetuses at 13 days of gestation were exposed to the fusion proteins, both epithelial and mesenchymal cells were stained for the enzyme, with a conspicuous accumulation of the reaction product at perinuclear cytoplasmic regions. The histochemical staining of the mesenchymal cells was more intense compared to that seen in epithelial cells. TAT-HA-beta-galactosidase fusion protein was also delivered to rat submandibular glands by retrograde duct injection. Histochemical staining for beta-galactosidase activity of cryostat sections prepared from the injected glands revealed that the transduction of the fusion protein was also time- and dose-dependent. In the glands of rats sacrificed from 10 min to 1 hr after the retrograde injection, essentially all acinar and duct cells showed cytoplasmic staining. The intensity of the staining then declined, and was not seen in the glands of rats killed 24 hr after the injection of the fusion proteins. These results indicate that a full-length, active TAT fusion protein can be targeted to salivary gland cells both in vitro and in vivo to analyze physiological, developmental, and pathophysiological processes.     

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.     

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     

Cytochemical correlates of structural sexual dimorphism in glandular tissues of the mouse

Summary Circulating androgens are known to effect a sexual dimorphism of the submandibular gland and kidney of the mouse. Enzyme histocytochemical differences that correlate with these structural changes have been the subject of much study, especially in the kidney. In the present study, emphasis was placed on the hypogonadic effects of diabetes mellitus on the submandibular gland and kidney of C57BL/KsJ db/db inbred mice with an autosomal recessive disease resembling maturity onset human diabetes mellitus. These glands of adult diabetic mice of both sexes were compared with those of unafflicted heterozygous littermates. The mitochondrial cytochrome oxidase and peroxisomal and cytoplasmic catalase were studied in their submandibular glands and kidneys. The parasympathetic innervation of the submandibular glands was studied by a histochemical method for acetylcholinesterase. The extensive differentiation of striated ducts of the submandibular gland into granular tubules in the postpubertal male mouse was readily evident with the cytochrome oxidase procedure. This differentiation resulted in ductal staining patterns characteristic of the sexes. Alteration of these patterns suggested that demasculinization or feminization was occuring in the male diabetic mice and that masculinization or virilization (defeminization) was occurring in the female diabetics. Similarly, in kidney, study of the parietal epithelium of Bowman's capsule revealed feminization in the male diabetics and masculinization in the female diabetics. With the catalase procedure, a dramatic sexual dimorphism was observed in the kidneys of the heterozygous unafflicted mice. Peroxisomal staining of epithelial cells of the proximal convoluted tubules was much more intense in the outer medulla of the male than of the female. In kidneys of the diabetics, the staining patterns again suggested that feminization of the male and masculinization of the female kidneys had occurred. On the other hand, neither a sexual dichotomy nor effects due to diabetes could be observed in the characteristic catalase staining observed in the luminal epithelial cells of submandibular gland distal ducts. The parasympathetic innervation of the submandibular gland, as revealed by the acetylcholinesterase method, was also markedly sexually dimorphic in the unafflicted mice. This was due to the more extensive innervation of the larger granular ducts characteristic of male than of the smaller striated ducts of the female. As a result of diabetes, the innervation and duct size decreased in the submandibular gland of the male, suggesting feminization, whereas they increased in the female suggesting masculinization. These changes were consistent with those observed in submandibular with the cytochrome oxidase procedure. Attempts were made to interrelate all of the enzyme histochemical changes observed in submandibular gland and kidney with the weights of these glands, sex, gonadal weights, diabetic status and urinary protein excretion. Generally, significant differences were recorded which suggested that the feminization of the submandibular gland and kidney in the diabetic male mice, and their masculinization in the female diabetics, were due to the hypogonadism of the disease.This investigation was supported by NIH research grants DE 02668, DE 04730, DE 00014 and RR 05333