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)     

Nerve growth factor-like immunoreactivities in rodent salivary glands and testis

Summary A series of polyclonal affinity-purified antibodies against mouse submandibular-gland nerve growth factor (NGF) are described. Using the submandibular gland of the male mouse and indirect immunofluorescence, the specificity and sensitivity of affinity-purified immunoglobulins and various other fractions from the immunized animals have been tested. It will be shown that affinity-purification schemes, including pre-purification of protein A-fractionated immunoglobulins to remove antibodies that bind to unrelated hydrophilic and hydrophobic proteins, significantly enhance the signal-to-noise ratio and specificity of the antibodies. The antibodies effectively detect NGF-like immunoreactivity in both fresh and fixed glandular tissue. Optimal fixation procedures are described. Fluorescence intensities are linearly correlated to log antibody concentration. By use of the best antibody fractions and optimal fixation protocols, the distribution of NGF-like immunoreactivity is described in eight different salivary glands (rat and mouse, male and female, submandibular and sublingual glands). In addition to the well-known large numbers of immunoreactive cells in the submandibular gland of the male mouse, immunoreactive cells were found in the sublingual gland of male mice and in the submandibular and sublingual glands of female mice. One antibody revealed a weak specific fluorescence also in the submandibular gland of the male mouse. In a survey of genital organs of male mice, one antibody revealed fluorescence in the germ cell line. We conclude that several polyclonal affinity-purified antibodies have been characterized that show a strong NGF-dependent binding to the secretory granules of tubular cells in the submandibular gland of male mice. These antibodies should make it possible to locate endogenous and perturbed NGF levels immunocytochemically, e.g., in the peripheral and central nervous system, where NGF concentrations may be several orders of magnitude lower than in the salivary glands.     

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.     

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.     

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.     

Morphological changes in the submandibular glands and in the X zone of the adrenal gland following ovariectomy in mice

Summary Morphological changes in submandibular glands of female mice following ovariectomy were studied morphometrically by light microscopy and ultrastructurally by electron microscopy. The X zone of the adrenal gland was examined in order to assess possible changes that might be expected to occur after ovariectomy.In submandibular glands, 1 to 4 weeks after ovariectomy, no changes were observed in percentages of the acinar, intercalated duct, and granular convoluted tubular areas occupying photomicrographs. However, an increase in the granular content of both intercalated duct and granular convoluted tubular cells was recognized. By contrast, the glandular picture 4 months after ovariectomy changed remarkably, resembling that of the male mouse both morphometrically and in terms of fine structure. In the adrenal cortex of control female mice, the X zone became thinner with aging. As compared with this, the X zone of ovariectomized mice at any time after the operation was thicker than that of controls.These observations suggest that the absence of ovarian hormones in the ovariectomized mouse may lead to prolonged functioning of X zone cells, which in turn may cause masculinization of the submandibular gland.     

Sex-related expression of sialic acid acceptor sugars in the mouse submandibular gland. Simultaneous visualization by confocal laser scanning microscopy.

The novel combination of sialidase digestion with simultaneous PNA and DBA binding yielded marked differences on sialoglycoconjugate occurrence and distribution in the mouse submandibular gland acinar cells of the two sexes. Striking differences in the structure of terminal disaccharides within stored secretory sialoglycoconjugates were also found. High content of sialic acid, characterized by the terminal sequence sialic acid-alpha-N-acetylgalactosamine, was established to only occur in the male acini where secretory cells appeared to be differently stained; indeed, some cells exhibited codistribution of sialic acid-alpha-N-acetylgalactosamine and sialic acid-beta-galactose terminal disaccharides, whereas other ones exclusively contained one of the two kinds of terminal sequences. In the female acinar cells, the secretory products were found to be almost exclusively composed by glycoconjugates having sialic acid subtended to beta-galactose without appreciable differences between acinar cells. Our finding of such extensive differences in the acinar cells of male and female mice adds new insights into the submandibular gland sexual dimorphism, commonly attributed to the androgen responsiveness of the granular convoluted tubule portion of the gland.     

Immunocytochemical localization of nerve growth factor (NGF) in the submandibular gland of adult mice by light and electron microscopy

Summary Nerve growth factor (NGF) was localized in the submandibular gland of adult male mice by a direct immunocytochemical method using highly purified antibodies against NGF coupled to horseradish peroxidase. In light microscopic sections the reaction product was entirely confined to the cells of the secretory tubules. The acinar part of the gland was free of reaction product. This finding was confirmed by electron microscopy. Within the cells NGF was localized exclusively in the apical secretory granules.No reaction was observed in the rough endoplasmic reticulum, the Golgi region or in the granules of the basal part of the cells. This observation favours the assumption that NGF is derived from a precursor molecule and that the precursor is transformed into immunologically active NGF within the secretory granules during their transport from the basal to the apical part of the tubular cells. Stimulation of the submandibular gland with carbachol (2 mg/kg) led to a massive release of the content of the secretory granules, including NGF, into the salivary duct.We wish to thank Dr. C. Rufener, Geneva, for communicating to us a new method of antibodyperoxidase coupling and Mrs. M. Durand-Wenger for her excellent technical assistance. This study was supported by the Swiss National Foundation for Scientific Research (Grant Nr. 3.432.74)     

Steroid regulation of kidney histidine decarboxylase and ornithine decarboxylase levels in mouse kidney: effects of the mutation testicular feminization, Tfm

1. Testosterone represses kidney histidine decarboxylase levels in both normal male and female mice. Tfm/Y mutant mice lack an androgen receptor and are phenotypically female. It has been suggested that the testosterone induction of HDC levels in these animals is a result of aromatisation to oestrogens in the absence of the androgen receptor; the oestrogens then induce the enzyme. 2. It is shown that the induction of HDC in Tfm/Y mice is specific to testosterone and not other androgens and can be mimiced by low doses of beta-oestradiol in normal female mice. 3. Analysis of Tfm/+ mice indicates that the testosterone induction effect is a function of individual kidney cells.     

Cytochemical studies of GERL and its role in secretory granule formation in exocrine cells

Synopsis the structure and cytochemistry of GERL was studied in several different exocrine secretory cells, including the exorbital lacrimal gland, parotid, lingual serous (von Ebner's), submandibular, and sublingual salivary glands, and exocrine pancreas of the rat; the lacrimal, parotid and pancreas of the guinea-pig; and the lacrimal gland of the monkey. GERL was morphologically and cytochemically similar in all cell types studied. It was located in the inner Golgi region and consisted of cisternal and tubular portions. Immature secretory granules were in continuity with GERL through multiple tubular connections. Modified cisternae of endoplasmic reticulum, with ribosomes only on one surface, closely paralleled parts of GERL. GERL and immature granules were intensely reactive for acid phosphatase activity, while the inner Golgi saccules were reactive for thiamine pyrophosphatase and nucleoside diphosphatase activities. In the rat exorbital lacrimal and parotid glands, reaction product for endogenous peroxidase, a secretory enzyme, was present in the endoplasmic reticulum, Golgi saccules, immature and mature secretory granules. GERL was usually free of reaction product or contained only a small amount. The widespread occurrence of GERL in secretory cells, and its intimate involvement with the formation of granules, suggest that it is an integral component of the secretory process.     

The influence of fixation on the carbohydrate cytochemistry of rat salivary gland secretory granules

Synopsis A series of studies was performed to assess the optimum fixation conditions for staining of carbohydrate-containing constituents of rat salivary gland secretory granules. In the parotid and submandibular salivary glands of the rat, the reactivity of secretory granules, at both the light and electron microscopic level, with routine stains and with cytochemical reagents was highly dependent upon the nature of the fixative employed. At the light microscopic level, secretory granules in rat parotid gland were periodic acid-Schiff (PAS) positive if fixed with buffered formalin fixatives. However, if the gland was fixed with lipid-solvent-containing fixatives, or with formalin at a very acid pH (as in Bouin's fixative), the PAS reactivity of the granules was lost. In the submandibular gland of rats, the acinar cells and granular tubules behaved similarly after such fixation in terms of their PAS reactivity, particularly in males; acinar cells of the female submandibular gland stained only lightly with PAS. At the fine structural level, the morphology of secretory granule constituents depended on the buffer used (cacodylate, phosphate or collidine) and on whether or not tissue was post-osmicated. Post-osmication considerably reduced the reaction of secretory granule components with stains for carbohydrates.The experimental evidence indicated that the carbohydrate-containing components of both parotid and submandibular gland secretory granules were not typical long-chain neutral or acidic mucins, but were rather glycolipids or lipophilic glycoproteins that were solubilized by lipid solvents or at acidic pH and were lost or destroyed in the presence of strong oxidants.     

Effect of autonomic agents on the amount of androgen-dependent granules in convoluted tubular cells of the mouse submandibular gland.

The convoluted tubular cells of the male mouse submandibular gland contain many serous-like granules in their apical cytoplasm. The autonomic regulation of the secretory process of the contents of these granules was studied by the following two methods: (1) immunochemical method using an antiserum specific to the granular components; and (2) histometric observations using light and electron microscopes. The results obtained by these two methods were well in agreement. When male mice were administered either phenylephrine or norepinephrine, the amount of granules in the glands significantly decreased. These two adrenergic stimulators were very effective, whereas synephrine was less effective. When mice were injected with a beta-adrenergic agent(isoproterenol) or a parasympathomimetic agent (pilocarpine), the amount of granules in the glands did not change. The alpha-adrenergic blockers phenoxybenzamine and phentolamine almost completely neutralized the effect of alpha-adrenergic agents on the glands, whereas another alpha-blocker (ergotamine) was less effective. These facts suggest that the secretion of the granular components is mediated by way of alpha-adrenergic receptor sites in the glands.     

Cell-specific expression of kidney androgen-regulated protein messenger RNA is under multihormonal control

Kidney androgen-regulated protein (KAP) gene expression is under androgenic control in the epithelial cells of the proximal convoluted tubule in the mouse kidney. In Tfm/Y androgen receptor-deficient mice, KAP mRNA was detected by in situ hybridization in a subpopulation of these cells only in the S3 segment of the proximal tubules in the outer medulla. Treatment of Tfm/Y animals with testosterone caused a partial induction of KAP mRNA levels, while dihydrotestosterone had no effect. These data suggested that the androgen receptor-independent induction of KAP gene expression in these animals was mediated by an estrogenic metabolite of testosterone, since dihydrotestosterone cannot be aromatized to an estrogenic form. Estrogen treatment of Tfm/Y mice caused an increase in KAP gene expression similar to that observed with testosterone. However, ovariectomy of normal female mice did not eliminate KAP gene expression in the S3 cells and, in fact, resulted in a slight increase. Adrenalectomy in combination with castration had no effect on KAP mRNA levels in S3 cells. However, hypophysectomy alone completely eliminated this cell-specific component of KAP gene expression. These results indicate that KAP gene expression is subject to cell-specific regulation in different segments of the proximal tubule and that this regulation is mediated by hormones of both gonadal and pituitary origin.     

Cellular compartmentation of lysozyme and alpha-amylase in the mouse salivary glands. Immunogold approaches at light and electron microscopy level

The research was planned to study the subcellular distribution of enzymatic secretory products within the secretory structures of the mouse major salivary glands at light and electron microscopy level by immunogold silver stain (IGSS) technique and double-sided post-embedding immunogold binding and silver amplification in order to speculate about their compartmentation. In particular, we experimented the above immunogold labeling approaches to localize the lysozyme and to verify its distribution patterns in relation to another secretion enzyme, alpha-amylase. Co-presence of lysozyme and alpha-amylase was observed in the convoluted granular tubule cells of the submandibular gland and in the demilunar cells of the sublingual gland as well as in the electron-dense regions of the mottled secretory granules in the parotid gland. Exclusive binding patterns of lysozyme were observed in the acinar cells of the submandibular and sublingual glands where alpha-amylase did not occur.     

Histochemical and biochemical determination of calcium in salivary glands of cat

Although feline salivary glands have been used in investigations on secretion and microlithiasis and both processes involve calcium, nothing is known about its distribution in these glands. Therefore we have demonstrated the presence of calcium by a histochemical technique using glyoxal bis(2-hydroxyanil) and a biochemical technique using dry ashing. The histochemical technique stained serous acinar cells weakly and rarely found mucous acinar cells strongly in the parotid gland, mucous acinar cells moderately to strongly and serous acinar cells weakly in the sublingual gland, and central and demilunar acinar cells moderately to strongly in the submandibular gland. The biochemical technique revealed less calcium in the parotid than in the submandibular and sublingual glands. Both techniques revealed a decrease of calcium in submandibular and sublingual glands following parasympathetic stimulation. The histochemical distribution of calcium, which corresponds to that of acinar secretory glycoprotein, and the loss of calcium following parasympathetic stimulation, which causes release of secretory granules, indicate the presence of calcium in secretory granules. The concentration of calcium in the different types of acinar cell corresponds to the acidity of the secretory glycoprotein and suggests that calcium is present as a cationic shield to allow the condensation of polyionic glycoprotein in secretory granules.     

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.     

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.     

Secretory and basal cells of the epithelium of the tubular glands in the male Mullerian gland of the caecilian Uraeotyphlus narayani (Amphibia: Gymnophiona)

Caecilians are exceptional among the vertebrates in that males retain the Mullerian duct as a functional glandular structure. The Mullerian gland on each side is formed from a large number of tubular glands connecting to a central duct, which either connects to the urogenital duct or opens directly into the cloaca. The Mullerian gland is believed to secrete a substance to be added to the sperm during ejaculation. Thus, the Mullerian gland could function as a male accessory reproductive gland. Recently, we described the male Mullerian gland of Uraeotyphlus narayani using light and transmission electron microscopy (TEM) and histochemistry. The present TEM study reports that the secretory cells of both the tubular and basal portions of the tubular glands of the male Mullerian gland of this caecilian produce secretion granules in the same manner as do other glandular epithelial cells. The secretion granules are released in the form of structured granules into the lumen of the tubular glands, and such granules are traceable to the lumen of the central duct of the Mullerian gland. This is comparable to the situation prevailing in the epididymal epithelium of several reptiles. In the secretory cells of the basal portion of the tubular glands, mitochondria are intimately associated with fabrication of the secretion granules. The structural and functional organization of the epithelium of the basal portion of the tubular glands is complicated by the presence of basal cells. This study suggests the origin of the basal cells from peritubular tissue leukocytes. The study also indicates a role for the basal cells in acquiring secretion granules from the neighboring secretory cells and processing them into lipofuscin material in the context of regression of the Mullerian gland during the period of reproductive quiescence. In these respects the basal cells match those in the epithelial lining of the epididymis of amniotes.     

Quantitation and localization of acinar cell-specific mucin in submandibular glands of mice during postnatal development

Summary In this study, antiserum to acinar cell-specific mucin was utilized to determine whether mucin could be detected in the mouse submandibular gland prior to cytodifferentiation of acinar cells. Results from radioimmunoassay indicated that mucin occurs in submandibular glands from newborn mice, i.e., before the appearance of mature acinar cells. Additionally, mucin quantitated in various stages of development was found to be antigenically identical to adult mucin. After sections of glands were treated with immunohistochemical reagents, we observed that the mature acinar cell-specific mucin was present in secretory terminal-tubule cells and in proacinar cells of newborn animals. The present findings suggest that in young animals, the proacinar cells are an immediate precursor of acinar cells and that the secretory terminal-tubule cells may represent an earlier stage in development of acinar cells. In adult female glands, mucin was also detected in the granular intercalating-duct cells. This latter observation is consistent with the hypothesis that these cells are an intermediate in the acinar cell replacement process.     

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