Histochemical study on the localization of glucose-6-phosphate dehydrogenase induced by androgen or thyroxine in the convoluted tubules of mouse submandibular gland

Summary The effects of 5-dihydrotestosterone (DHT) and thyroxine (T₄) on glucose-6-phosphate dehydrogenase (G-6-PDH) activity in mouse submandibular gland were investigated histochemically. A strong positive histochemical reaction for G-6-PDH was observed in the excretory ducts of untreated male and female mice, with a slight reaction in the basal portion of the convoluted tubules (striated ducts) of males. Administraition of DHT to female mice increased G-6-PDH activity specifically in the convoluted tubules. T₄ increased the enzyme activity in the tubules more than DHT. The induction of G-6-PDH activity by T₄ in adrenalectomized mice suggests that T₄ has a direct effect on the submandibular gland.     

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     

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.     

Sexually dimorphic duct system of the submandibular gland in mouse with testicular feminization mutation (Tfm/Y).

The X-linked testicular feminization mutation (Tfm/Y) in the mouse is characterized by androgen insensitivity of the target cells. The aim of this study was to examine sexually dimorphic development of the submandibular gland of Tfm/Y mutant mice in comparison with those of wild-type male, wild-type female and heterozygous Tfm female mice. In either 30- or 90-day-old wild-type male mice, the granular convoluted tubules (GCT) of the glands were more developed, and the relative occupied areas (ROA) of GCT were superior to those of the age-matched wild-type and heterozygous Tfm females. In androgen-insensitive Tfm/Y mice, the glandular structures rather resembled the female glands, showing lower values of the ROA of the GCT. Sex differences in the mitotic rate were observed at 30 days of age, being significantly higher in the wild-type male GCT than in the female GCT. Thereafter, the mitotic rate of the wild-type male GCT declined to the female levels by 90 days of age. The mitotic rate of GCT in Tfm/Y mutants was as low as those of the females during observation periods. An other three regions, the acini, the intercalated ducts and the excretory striated ducts, were not significantly different in either the ROA or the mitotic rate among wild-type males and females, and Tfm/Y. On the other hand, either the ROA or the mitotic activity of GCT of the glands in Tfm/Y mutants was completely unaffected by 5 alpha-dihydrotestosterone (DHT).(ABSTRACT TRUNCATED AT 250 WORDS)     

Kallikrein localization in rodent salivary glands and kidney with the immunoglobulin-enzyme bridge technique.

Kallikrein has been localized in rodent kidney and salivary glands by means of an immunoglobulin-enzyme bridge technique. In sections of kidney, anti-kallikrein antibodies bound to the apical region of certain distal tubule segments in the cortex, to reabsorption droplets of proximal convoluted tubules, and to certain duct segments in the papilla. In salivary glands of both male and female rats and mice, and apical rim of most striated duct cells of submandibular, parotid and sublingual glands and granular tubules of submandibular glands exhibited immunoreactivity. Granular intercalated duct cells in female submandibular glands also displayed immunostaining for kallikrein. Phenylephrine administration resulted in loss of immunoreactive granules from the granular convoluted tubule cells of male mouse submandibular gland. This response was paralleled by a biochemically demonstrable decrease in kallikrein-like tosylarginine methyl ester (TAME) esterase activity.     

Immunocytochemical localization of epidermal growth factor in mouse submandibular gland.

The cellular and subcellular localization of epidermal growth factor in the submandibular glands of male and female adult mice was established by immunoperoxidase techniques. In light microscopic preparations epidermal growth factor was found exclusively in the granular convoluted tubules of the gland. The intensity of staining for epidermal growth factor varied from cell to cell, and some cells apparently were negative. The pattern of staining was similar in the glands of male and female mice; however, the granular convoluted tubules are androgen-responsive, and thus more extensive and composed of larger cells in males. In thin sections epidermal growth factor was most heavily concentrated in the secretion granules of the granular convoluted tubule cells. Within a given cell there was variation in intensity of staining of individual secretion granules, with some granules appearing minimally reactive or negative. The only other cell component with deposits of reaction product was the ribosomes.     

Cytochemical localization of peroxidase activity in the submandibular salivary gland of the hamster

Summary Peroxidase activity has been localized to duct cells of the submandibular salivary gland of the hamster using a 3,3-diaminobenzidine (DAB)-H₂O₂ medium. In cryostat sections of glutaraldehyde-fixed tissue the enzyme activity is found in the proximal part of the duct system of the gland. In Epon sections studied in the light microscope or thin sections studied in the electron microscope the peroxidase activity is observed in cytoplasmic granules in cells of the convoluted tubules of the ducts. No activity is seen in the acini or in cells of the intralobular striated ducts. The submandibular gland of the rat was negative with respect to peroxidase reaction. The findings are discussed with special reference to the possible correlation between peroxidase activity and iodine metabolism in salivary glands.     

Relationship between expression of epidermal growth factor and simian virus 40 T antigen in a line of transgenic mice

The pattern of expression of the simian virus 40 (SV40) T antigen gene and resultant dysplasia were re-examined in a line of transgenic mice in which the T antigen gene was under the control of the SV40 early promoter. We found that T antigen expression in the kidney, and resulting dysplastic lesions, occurred exclusively in the distal convoluted tubules and the ascending limbs of Henle. Epidermal growth factor (EGF) expression in the kidney of normal mice was similarly immunolocalized. The correlation between high EGF immunoreactivity in normal mouse tissues and T antigen expression in the transgenic counterpart was also seen in the choroid plexus epithelium and in the submandibular glands of male mice. T antigen was not found in the submandibular gland of transgenic females. Similarly, EGF was only rarely detected in the normal female submandibular gland. In contrast to the correlation between T antigen expression in the transgenic mice and EGF expression in the corresponding tissues of the normal mice, within the dysplastic lesions of the transgenic mice EGF expression was severely diminished. Adenocarcinomas of the male submandibular gland from another line of transgenic mice that expresses theInt-1 transgene, showed similarly reduced levels of immunostaining for EGF. Thus, reduced expression of EGF might be a general feature of dysplasia and tumorigenesis in those tissues that normally express EGF.     

Immunocytochemical demonstration of nerve growth factor and histofluorescence of catecholaminergic nerves in the salivary glands of diabetic mice

Summary Nerve growth factor (NGF) was localized in the submandibular, sublingual, and parotid salivary glands of male and female diabetic mice and their normal littermates by immunoperoxidase staining usingp-phenylenediamine-pyrocatechol as a chromogen for the cytochemical demonstration of peroxidase activity. In the normal male submandibular gland, immunoreactive NGF was localized in the apical regions of granular, intercalated and collecting duct cells, while in the normal female submandibular gland, NGF was present throughout the cytoplasm of granular duct cells. The localization of NGF in the diabetic male and female submandibular glands was similar and resembled that of the normal female. NGF immunoreactivity was also observed in the striated duct cells in the sublingual and parotid glands of all four types of mice.The sympathetic innervation of the submandibular glands of normal and diabetic mice was demonstrated using glyoxylic acid-induced histofluorescence. The pattern of sympathetic innervation and the intensity of catecholamine fluorescence was consistently different in the four types of mice. In the normal male submandibular gland the fluorescence was very intense, particularly in nerves adjacent to the granular ducts. In the normal female submandibular gland, the fluorescence was weak, while in the diabetic male and female the fluorescence was moderate.The correlation between the intensity of the immunocytochemical staining for NGF and the catecholamine fluorescence adjacent to the granular ducts suggests a trophic influence of the NGF-containing granular ducts on their sympathetic innervation.     

Occurrence and Nuclear Localization of cAMP Response Element- Binding Protein in the Post-natal Development of the Rat Submandibular Gland

Cyclic AMP response element-binding protein (CREB) is a 43-kDa polypeptide that binds a cAMP response element located at the 5 promoter region of cAMP regulatory genes. The spatial and temporal distribution of CREB in the post-natal development of the rat submandibular gland was investigated using immunohistochemistry with a specific antibody. At birth, cells of the terminal tubules and ducts in the submandibular gland showed a nuclear CREB immunoreactivity of moderate intensity. At 1–2 weeks after birth, an intense CREB immunoreactivity was localized primarily to acinar cells. When the r352;-adrenergic agonist isoproterenol was administered to 2-week-old rats, a twofold transient increase in the number of immunoreactive acinar cells was induced. Beginning 3 weeks after birth, CREB immunoreactivity shifted from acini to the duct system and showed a clear localization in the cells of the intercalated ducts and distal portions of striated ducts, where the granular convoluted tubule develops after 4 weeks. Immunopositive materials were localized exclusively in the nuclei of both acinar and ductal immunoreactive cells. After the development of the granular convoluted tubules, CREB immunoreactivity was absent in the tubule cells and was gradually reduced in intensity over the entire gland. In order to examine a hypothesis that CREB is involved in the initial differentiation of the granular convoluted tubular cells, testosterone was administered to hypophysectomized adult rats. Whereas the tubular cells of hypophysectomized rats showed a complete regression, and no CREB immunoreactivity was found in any acinar or duct cells, administration of testosterone for a few days induced an intense CREB immunoreactivity in the nuclei of duct cells, followed by their differentiation into the granular convoluted tubular cells. These results suggested that CREB is involved not only in the growth and differentiation of acinar ce lls that are regulated by r352;-adrenergic nerves but also in those of the duct system, and especially in the androgen-regulated differentiation of the granular convoluted tubular cells, during the post-natal development of the rat submandibular gland.     

Effect of tuftsin on enzyme activity in the energy metabolism of lymphocytes

The cytochemical technique was used to measure the activity of succinate dehydrogenase (SDH), lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G-6-PDH) of peripheral blood lymphocytes of mice and rats given intraperitoneal injections of an endogenous immunostimulant tuftcin (Tre-Lys-Pro-Arg) in a dose of 0.3 mg/kg. A significant decrease of SDH activity was observed both in mice and rats 4 and 6 hours following injection, respectively. In mice, that activity returned to normal in 12, while in rats in 24 hours. An opposite action was produced by tuftcin on G-6-PDH, causing the maximum elevation of the enzyme activity in rat lymphocytes 6 hours after peptide administration. The decrease to the initial level was observed in 24 hours. Tuftcin did not affect the activity of LDH. The data obtained indicate that the immunological effect of tuftcin is coupled with the changes in the activity of Krebs cycle enzymes (SDH) and pentose phosphate cycle enzymes (G-6-PDH).     

Proliferative response of mouse submandibular gland to androgen but not to thyroid hormone

Hormone-induced differentiation and proliferation of cells were investigated in the submandibular gland of castrated female mice, by determining the esteroprotease activity and 3H-thymidine labelling index, respectively. Injections of 5 alpha-dihydrotestosterone (4 micrograms/g body weight/day) or L-thyroxine (0.5 microgram/g body weight/day) induced a significant increase in the activity of esteroprotease, which has been shown to be localized in the convoluted tubule cells of the submandibular gland. Injections of the above-mentioned dose of 5 alpha-dihydrotestosterone for 3 days induced a 43-fold increase in the labelling index of the convoluted tubule cells, but injections of the above-mentioned dose of L-thyroxine for any duration did not induce a significant increase in the labelling index. The present result suggests that hormones which induce differentiation of cells in mouse submandibular gland do not necessarily induce cell proliferation.     

Fine structural localization of acetylcholinesterase activity in rat submandibular gland

Using the indirect thiocholine method, the ultrastructural localization of acetylcholinesterase (AChE) activity in the normal rat submandibular gland was studied. Cytochemical demonstration of AChE is based on coupling the hydrolysis of acetylthiocholine iodide to the precipitation of heavy metal salts. AChE-associated reaction product was selectively revealed in the perinuclear space and in the endoplasmic reticulum of the intercalated duct cells, in some cells of granular convoluted tubules, and in the striated duct epithelium, as well as in the myoepithelial cells. Although AChE activity generally occurred inside the cells, electron-dense precipitates were shown in intercellular space and in the stroma of the gland. Fine localization of AChE activity was also found in nerve bundles, predominantly between axons and between axons and Schwann cell. Our observations indicate that AChE is synthesized in the epithelium of the ducts and in the myoepithelial cells of the salivary gland. It is not known yet whether this enzyme is released from the intracytoplasmic membrane system into the extracellular space and then transported to the regions of the gland innervation. Conceivably AChE synthesized in the submandibular gland cells could also be considered an inhibitory modulator of the regulatory functions of biologically active polypeptides.     

Hormonal regulation of chymotrypsin-like esteroproteases in the mouse submandibular gland

Summary The activities of chymotrypsin-like esteroproteases in the mouse submandibular gland were additively induced by 5-dihydrotestosterone (DHT) and triiodothyronine (T₃). Zymograms showed that there are many isozymes whose activities are regulated by DHT and/or T₃. Some isozymes seemed to be hormone-independent. Histochemical studies revealed that all these isozymes are localized in the granular convoluted tubules.     

Postnatal development of trypsin-like esteroproteases in mouse submandibular gland

The development of trypsin-like esteroproteases in the submandibular gland of mice was studied, using a newly synthesized naphthyl ester (tosyl-L-lysine alpha-naphthyl ester) for the preparation of zymograms and for histochemical demonstration of the enzyme. Esteroprotease activities were first detected spectrophotometrically on day 15 after birth; then increased markedly after day 20. A sex difference in esteroprotease activity appeared on day 25. Zymograms prepared after isoelectric focusing in polyacrylamide slab gels showed that the glands of neonatal mice contained esteroproteases with a rather different composition from that of adult mice. The adult type isozymes appeared first on day 15, and their activities increased markedly after day 20. Histochemical studies revealed that the isozymes of neonatal mice were derived from mast cells. A few striated ducts were first stained on day 15, and the sex difference of the granular tubules became obvious on day 25. These results indicate that the development of trypsin-like esteroproteases faithfully reflects the differentiation of granular tubules in the mouse submandibular gland, except in the neonatal period.     

Histochemical observations on the occurrence of glycolytic and pentose phosphate cycle enzymes in the hepatopancreas and their possible relation to eyestalk factor(s) in the crab Scylla serrata (Forskal).

Histochemical studies were carried out on some of the glycolytic enzymes viz. phosphorylase, aldose, alpha-glycerophosphate dehydrogenase (alpha-GPDH) and lactic dehydrogenase (LDH) and a key enzyme of the pentose phosphatase cycle, glucose-6-phosphate dehydrogenase (G-6-PDH), in the hepatopancreas of Scylla serrata (Forskal). 1. Weak activities of phosphorylase and aldolase and strong-activities of alpha-GPDH and LDH were noticed mainly in the brush border of the tubules and R-cell cytoplasm. A trace activity of G-6-PDH was noticed in the brush border. 2. Bilateral eyestalk removal results in inhibition of both phosphorylase and aldolase. However, enhanced activities of alpha-GPDH and LDH were noticeable 4 h after the operation. The G-6-PDH activity remained unaltered till 24 h. 3. Injection of eyestalk extract into both intact and destalked crabs activated all the enzymes.     

Proliferative Response of Mouse Submandibular Gland to Androgen But Not to Thyroid Hormone

Abstract. Hormone-induced differentiation and proliferation of cells were investigated in the submandibular gland of castrated female mice, by determining the esteroprotease activity and ³H-thymidine labelling index, respectively. Injections of 5α-dihydrotestosterone (4 μg/g body weight/day) or l -thyroxine (0.5 μg/g body weight/day) induced a significant increase in the activity of esteroprotease, which has been shown to be localized in the convoluted tubule cells of the submandibular gland. Injections of the above-mentioned dose of 5α-dihydrotestosterone for 3 days induced a 43-fold increase in the labelling index of the convoluted tubule cells, but injections of the above-mentioned dose of l -thyroxine for any duration did not induce a significant increase in the labelling index. the present result suggests that hormones which induce differentiation of cells in mouse submandibular gland do not necessarily induce cell proliferation.     

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.     

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.     

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.