Morphological studies on the synthesis of secretory granules in convoluted tubules of mouse submandibular gland

The synthesis of zymogen-like secretory granules in convoluted tubules of mouse submandibular gland (SMG) was investigated by histometry, light microscopy and electron microscopy. In normal males secretory granules in the SMG increased greatly from 25 days after birth and reached a maximum level 50 days after birth. Castration of adult male mice markedly decreased the level, but it was completely restored by testosterone administration. A parallel was found between change in the granule level and the amount of rough endoplasmic reticulum (RER) in the convoluted tubular cells during development or after various treatments. Development of the Golgi apparatus was also observed in the cells when the granules increased. Both the increase in the granules and in the RER induced by testosterone were prevented by actinomycin D or puromycin. These results indicate that the granule contents are synthesized on the RER under the control of testosterone, and then condensed in the Golgi apparatus.     

Morphological studies on the synthesis of secretory granules in convoluted tubules of mouse submandibular gland.

The synthesis of zymogen-like secretory granules in convoluted tubules of mouse submandibular gland (SMG) was investigated by histometry, light microscopy and electron microscopy. In normal males secretory granules in the SMG increased greatly from 25 days after birth and reached a maximum level 50 days after birth. Castration of adult male mice markedly decreased the level, but it was completely restored by testosterone administration. A parallel was found between change in the granule level and the amount of rough endoplasmic reticulum (RER) in the convoluted tubular cells during development or after various treatments. Development of the Golgi apparatus was also observed in the cells when the granules increase. Both the increase in the granules and in the RER induced by testosterone were prevented by actinomycin D or puromycin. These results indicate that the granule contents are synthesized on the RER under the control of testosterone, and then condensed in the Golgi apparatus.     

Precocious development of granular convoluted tubules in the mouse submandibular gland induced by thyroxine or by thyroxine and testosterone

The effects of the administration of thyroxine (T₄) and/or testosterone (TP) on the early postnatal differentiation of granular convoluted tubule (GCT) cells of the submandibular glands were studied in Swiss-Webster mice. From birth, mice were injected daily with T₄ up to the time of killing at 15 or 20 days of age. In addition, groups of mice were given one injection of TP eight days before killing. Control animals received vehicles (saline and/or sesame oil). Sections of the glands were stained with toluidine blue, or immunocytochemically by the unlabelled antibody enzyme method, for the localization of protease A, epidermal growth factor, or renin. Supernatants of the gland were analyzed for protease activity (pH 8.5, substrate: tosyl-glyclyl-L-prolyl-L-arginine nitroanilide acetate), or by radioimmunoassay for EGF content. At 15 days of age no GCT cells were present in the glands of control or TP-treated mice. In T₄-injected mice many small GCT cells occurred, while larger and more numerous GCT cells were seen in the glands of mice that received both hormones. TP alone had no effect on levels of EGF or protease activity. In T₄-treated mice, protease levels increased 10-fold and EGF content rose 28-fold. TP administration to T₄-treated mice caused a further threefold, increase in both protease activity and EGF content. At 20 days of age the glands of control mice had a few small GCT cells, and both protease activity and EGF content were substantially increased. TP treatment was again without effect. However, daily injections of T₄ caused both protease activity and EGF levels to increase 20- and 11-fold, respectively. Just as in 15-day-old mice, TP administration to T₄-primed mice resulted in further increases in the two polypeptides. Immunocytochemical staining for protease A and EGF confirmed the chemical data. Renin was detectable immunocytochemically at both ages in the glands of mice that had received T₄, but it was seen in the glands of mice that had received TP alone only at 20 days of age. It is concluded that T₄ caused a precocious induction of GCT cells and their specific products. Although TP alone had no effect on their early differentiation, it acted synergistically with T₄ in inducing GCT cells. There were no obvious sex differences in any of these features in control or treated animals.     

Secretagogue-mediated discharge of nerve growth factor from granular tubules of male mouse submandibular glands: An immunocytochemical study

Submandibular glands of male mice were stained for nerve growth factor by light microscopic immunocytochemistry. Nerve growth factor (NGF) was present in the granules of granular tubule cells, with the immunoreactive material often concentrated at the periphery of granules. Administration of the α-adrenergic agent, phenylephrine, to animals resulted in a marked depletion of NGF-containing granules from granular tubules. Some release also occurred following administration of the β-adrenergic agent, isoproterenol. Cholinergic stimulation (pilocarpine) did not result in appreciable loss of immunoreactive granules from these cells. In vitro results were not as clear cut, immunocytochemically, as those obtained with intact animals. It is concluded that discharge of NGF from male mouse submandibular glands is mediated predominantly by α-adrenergic activation, and that this phenomenon is readily demonstrated in the intact animal.     

Three-dimensional reconstruction of adult female mouse submandibular gland secretory structures

Computer-assisted reconstructions of adult female mouse submandibular gland have been used to positionally characterize within the three-dimensional structure likely intermediates in secretory cell replacement. The locations of striated granular duct cells and granular intercalated duct cells are consistent with a role as intermediates between intercalated duct cells and granular duct cells or acinar cells, respectively. Average volumes of the two putative intermediate cell types are also consistent with this role. The reconstructions suggest that, in addition to a "streaming" mechanism for secretory cell replacement, formation of new secretory structures composed of multiple acini and second-order intercalated ducts may also contribute to the cell replacement process.     

Ultrastructural histochemical studies of secretory granule replenishment in rat submandibular granular tubules after cyclocytidine-induced secretion

Rat submandibular glands have been examined electron microscopically at various times after degranulating the granular tubules by injecting cyclocytidine (75 mg/kg i.p.), to study events in the reformation of secretory granules in these cells. The changes were progressive but not synchronous in the cells. The first evidence of recovery was the re-appearance of glycogen particles 6 h after injection. Residual secretory granules were small and located periluminally at that time. More granules were present at 15 h after injection but they were still small and placed periluminally. There was more glycogen in the cells and some was present in aggregates. At 1 day after injection there were more secretory granules and they tended to be larger than previously. The secretory granules increased in size and number progressively thereafter and the cells appeared like normal controls by day 7. During the recovery, fusion profiles were seen between granules from 2 days onwards. Throughout, few Golgi complexes were detected and this may be related with the low glycosylation of the secretory proteins in these cells. The results confirm that the reformation of the secretory granules in granular tubule cells is a slow process that involves fusions of smaller granules.     

Secretagogue-mediated discharge of nerve growth factor from granular tubules of male mouse submandibular glands: an immunocytochemical study.

Submandibular glands of male mice were stained for nerve growth factor by light microscopic immunocytochemistry. Nerve growth factor (NGF) was present in the granules of granular tubule cells, with the immunoreactive material often concentrated at the periphery of granules. Administration of the alpha-adrenergic agent, phenylephrine, to animals resulted in a marked depletion of NGF-containing granules from granular tubules. Some release also occurred following administration of the beta-adrenergic agent, isoproterenol. Cholinergic stimulation (pilocarpine) did not result in appreciable loss of immunoreactive granules from these cells. In vitro results were not as clear cut, immunocytochemically, as those obtained with intact animals. It is concluded that discharge of NGF from male mouse submandibular glands is mediated predominantly by alpha-adrenergic activation, and that this phenomenon is readily demonstrated in the intact animal.     

Morphological study of granular convoluted tubules in the submandibular gland of the mouse during the growth of a sarcoma-180 subcutaneous tumor

Histological and cytological changes in the submandibular glands of adult male mice arising during the growth of sarcoma-180 subcutaneous tumors were studied. The submandibular glands of the mice were examined by morphometric analysis at 1, 3, 6, 10, 20, 30 and 64 days after inoculation of the tumor cells. There was a slow increase in the relative cross-sectional area of the granular convoluted tubule (GCT) in the section of the submandibular gland of the animals as the tumors grew. The increased proportional area of the GCT was significantly different from that of the control's from day 30. However, the mean weight of the glands was not increased. The proportional area of the granular cluster in the cells of the GCT of tumor cells in inoculated animals decreased about 5% on the first day and then quickly increased by 16% on the third day in comparison with those of the controls, eventually reaching a maximum of 74% (control, 54%) by day 30. In addition, the average number of granules per GCT cell decreased in the first three days, then increased to normal levels from day 6, going above the normal level from day 20 of the tumor growth. These changes in the glands of tumor-bearing animals disappeared within 20 days after removal of the tumor. These results indicate that the growth of the sarcoma-180 subcutaneous tumor caused morphological changes in the GCT and GCT cells, suggesting an alternation in the requirements of the secretions contained in the granules, such as the epidermal growth factor, during the growth of the tumor.     

Precocious induction of secretory granules by hormones in convoluted tubules of mouse submandibular glands

Precocious differentiation of convoluted tubules of the submandibular glands of mice was induced by daily injection of thyroxine, insulin, hydrocortisone and 5α-dihydrotestosterone from day 4 after birth. This treatment induced enlargement of the tubules and synthesis of secretory granules in the tubules. Treatment with 5α-dihydrotestosterone alone had no effect on enlargement of the tubules or synthesis of the granules. These findings suggest that, besides androgenic hormones, these three hormones are also involved in synthesis of granules in developing mice.     

Ultrastructure of the submandibular gland in the rabbit

The secretory endpieces of the rabbit submandibular gland are unusual in that they consist of seromucous acini (not demilunes) that empty into serous tubules that in turn drain into intercalated ducts. Seromucous granules consist of a moderately dense spherule in a fibrillogranular matrix. Serous granules contain a feltwork of filaments, which are liberated as a tangled skein during exocytosis. Peculiar granulated cells that have secretory granules of complex morphology are present at each end of the serous tubules. Intercalated ducts are, cytologically speaking, relatively simple, but the duct cells may contain a few oblong secretory granules. Striated ducts are typical in structure, although postfixation with ferrocyanide-reduced osmium reveals significant amounts of glycogen in the basal processes. Modified mitochondria are present in striated duct cells, but their frequency varies from rabbit to rabbit. Such mitochondria contain either an array of parallel, rigid cristae linked by intermembranous bridges, or a bundle of helical filaments within an expanded crista. Interspersed with the striated duct cells, especially near the duct origin, are some highly vacuolated cells with sparse mitochondria. Excretory ducts consisting of stratified columnar (sometimes pseudostratified) epithelium often show bleb formation of the luminal surface of the tall cells.     

Regulation of delayed-hypersensitivity response by the submandibular gland of male mice.

This study was performed to determine whether the removal of submandibular gland (SG) of mice influences the delayed-hypersensitivity (DH) response. The removal of the SG increased the weights of thymus and spleen and enhanced DH response, only in males and not in females. The injection of crude extract of male mouse SG returned the enhanced DH response of SG-ectomized males to the normal level whereas the injections of crude extracts of female or castrated male mouse SG was not affected. These results suggested that SG of male mice contains an endocrine factor(s) to regulate DH response.     

Parenchymal cell proliferation and mechanisms for maintenance of granular duct and acinar cell populations in adult male mouse submandibular gland

To evaluate proliferation as a factor in maintenance of parenchymal cell populations in adult male mouse submandibular glands, a variety of surveys were conducted following a pulse with ³H-thymidine. Striated granular duct (SGD) cells had the highest labeling index, followed by intercalated duct (ID) cells, then acinar (AC) cells, and granular duct (GD) cells had the lowest. These cell types showed from 30% to 60% completion of mitosis by 24 hr, with SGD, AC, and GD showing a likely second wave of mitosis sometime between 2 and 7 days after the pulse. About 40% of the pulse-labeled cells still remained as single cells at 42 days after the pulse. Repeat divisions in daughter cells of the primary labeled cells were very rare. A shift in the pattern of labeled cells at the ID-GD junction indicates that ID and SGD cells in this compartment are differentiating to GD cells. Further comparison of the magnitude of this conversion with the amount of noncompartmental GD cell proliferation provided a basis for calculating that ～70% of GD cell population maintenance occurs by selfproliferation, and the remaining 30% is contributed by differentiation from ID and SGD cells. A similar survey at the ID-acinus junction showed no evidence of conversion of ID cells to AC cells indicating that most, if not all, proliferative activity leading to AC cell population maintenance occurs by self-proliferation. Finally, based in part on structural changes at the ID-GD junction during the survey period, a pattern of cell conversion described as “in situ differentiation” is proposed. When this pattern is carried to fruition, this explains several structural features of the secretory complex typical to the male pattern submandibular gland. The proposed mechanism is supported by a three-dimensionally reconstructed sequence of likely intermediate structures. © 1993 Wiley-Liss, Inc.     

Ultrastructure of primary squamous cell carcinoma of the submandibular gland

Primary squamous cell carcinoma of the submandibular gland is a rare tumor. In this report, the histological and ultrastructural features of a case of primary squamous cell carcinoma arising in the left submandibular gland is presented. Light microscopically, the tumor consisted of well differentiated keratinizing squamous cell nests. Ultrastructurally, the tumor cells were oval or spindle-shaped, and several tumor cells had intracytoplasmic desmosome-like structures, resembling intercellular desmosomes. The majority of the tumor cells contained a large number of intermediate filaments (tonofilaments). Intercellular desmosomes were well developed. No secretory granules were found. These ultrastructural features may enable us to distinguish primary squamous cell carcinoma from mucoepidermoid carcinoma which is often misdiagnosed as squamous cell carcinoma.     

Ultrastructure of the secretory epithelium,nerve fibers,and capillaries in the mouse sweat gland

The ultrastructure of the mouse sweat gland was examined, in support of neurological studies of sweat glands and their relationships to the autonomic nervous system. It was found that the mouse sweat gland is similar to that of the rat and has only one type of secretory cell. Many nerve fibers are entwined with the secretory tubule and contain accumulations of round, clear vesicles, some microtubules, but apparently no neurofilaments. Cholinesterase is found in the clefts between nerve fibers and their ensheathing Schwann cells. The nerve fibers tend to run parallel with capillaries, but have no close association with either the capillaries or the secretory epithelium. Capillaries provide an abundant blood supply to the sweat gland and are fenestrated. The relationships between cellular elements of the sweat gland provide no direct evidence of the mechanisms involved in neurogenic sweating, although it seems likely that effector substances are diffusely distributed.     

Changes with senescence in the fine structure of the granular convoluted tubule of the submandibular gland of the mouse

Cells of the granular convoluted tubules (GCTs) of the submandibular glands of senescent male mice show structural changes indicative of functional decline. In order to define the nature of these age-related changes more clearly, the fine structure of GCT cells of 12- and 28-month-old males was compared. In old mice, there was cell-to-cell variation in the extent of these changes, with some cells of senescent males appearing no different from those of young adults. In affected cells the most striking alterations were seen in secretion granules and lysosomal elements. Secretion granules varied greatly in size, with some GCT cells having only very fine apical granules. Secondary lysosomes and large lipofuscin granules were frequent in the basal cytoplasm. Very large dense bodies (3-5 μm) occurred in many cells. These possibly represent intracellular pools of released secretory materials, as they were occasionally seen in continuity with the luminal contents. Structures whose appearance was intermediate between the very large dense bodies and lipofuscin granules were common, suggesting crinophagic activity. There was an apparent decrease in numbers of polysomes and in the extent of the Golgi apparatus. These fine structural changes are consistent with impairments with advanced age in synthesis and posttranslational processing of secretory products by affected GCT cells. In addition to cell-to-cell variation in any one male, there was also interanimal variation in the degree and extent of these senescent changes.     

The functional organization of the parasympathetic secretory innervation of the submandibular gland

1. In cats the hypoglossal and chorda-lingual nerves were cut and their central ends sewn to the peripheral end of the latter nerve. At monthly intervals the sensitivity of the submandibular gland to a secretory agent was then estimated.

2 When the supersensitivity resulting from the decentralization of the gland had been lowered to a constant level acute experiments were made. Electrical stimulation of both nerves caused secretion, which was abolished by ganglion blocking drugs and atropine.

3. Simultaneous stimulation of the nerves at frequencies subliminal for each nerve did not cause any secretion.

4. Stimulation of one nerve at a subliminal frequency did not increase a flow of saliva evoked by stimulation of the other nerve. However, as soon as the secretory threshold was reached, the flow was markedly accelerated, provided it was submaximal.

5. In many cases stimulation of the regenerated chorda fibres at a high frequency caused a flow corresponding to the maximal secretory capacity of the gland. In some experiments this was true for the hypoglossal fibres also.

6. The functional organization of the parasympathetic secretory innervation of the submandibular gland of the cat is discussed in the light of these observations.

     

The development of the granular convoluted duct in the rat submandibular gland

The development of the granular convoluted duct in the submandibular gland of male rats, 4 to 12 weeks of age, was investigated. During this period, the average weight of the gland increased from 213 to 526 mg, the total DNA and RNA contents doubled, and the protein content tripled. Radioautographs were prepared from Epon embedded sections of the gland of the rats given ³H-thymidine and stained with toluidine blue. The glands of 4-week-old rats consisted mainly of acinar cells (45%), intercalated ductal cells (20%) and striated ductal cells (16%). A few granular convoluted ductal cells were seen in the striated duct close to the intercalated duct. The frequency (and absolute number) of granular convoluted ductal cells increased linearly from 1% (3 × 10⁶) at four weeks to 26% (68 × 10⁶) at eight weeks, while the calculated number of striated ductal cells remained stationary. The absolute number of acinar cells and intercalated ductal cells nearly doubled between four to eight weeks of age. The proliferative activity of all cell types declined with age but between six and ten weeks of age the rate of proliferation of ductal cells was relatively higher than the rate of proliferation of the acinar cells. Morphologically the size and number of granules in the granular convoluted ductal cells increased with age. Based on the above data it is concluded that the granular convoluted ductal cells developed from that segment of the striated ductal cells which is in close proximity with the intercalated ductal cells. The heterogeneity of the granules in the granular convoluted ductal cells observed from six weeks of age might denote the functional diversity of the cells.