Fine structure of transforming‐type granules in mucous cells in the early postnatal rat parotid gland when processed by rapid freezing followed by freeze‐substitution fixation

The present study was designed to clarify the more precise ultrastructural feature of granules, especially mucous granules in the early postnatal rat parotid gland by using rapid freezing followed by freeze‐substitution fixation. The parotid gland of Wistar rats (aged 0–10 days) was removed under anesthesia and immediately underwent cryofixation followed by substitution with osmium tetroxide. After fixation, the samples were embedded in Epon‐Araldite, cut into ultrathin section, and then examined by transmission electron microscopy. Electron microscopy showed that the mucous granules of samples treated by freeze‐substitution fixation had low electron density and were almost spherical in shape with a clear limiting membrane. By Day 5, granules that were a little more electron dense than mucous granules, granules with a more electron dense portion at their periphery, and heterogeneous granules with an internal highly electron dense portion were found. Moreover, cells containing both homogeneous granules with a high electron density similar to that of mature serous granules and heterogeneous granules were observed. These findings demonstrated that the morphology of the transforming‐type mucous granules by chemical fixation in the previous study was an artifact and, as a result, strongly suggested that on the sequential morphological changes of transitional mucous/serous granules by rapid freezing method in the present study, the mucous cells in the early postnatal rat parotid gland undergo transformation to serous cells. Anat Rec 260:387–391, 2000. © 2000 Wiley‐Liss, Inc.     

Morphological and histochemical changes in the secretory granules of mucous cells in the early postnatal mouse parotid gland

It has previously been known that the developing parotid glands in humans and rats contain mucous cells in their terminal clusters and acini, but these cells disappear within a short period of time. Using rat parotid glands, IKEDA and AIYAMA (1997, 1999) suggested that the mucous cells might change into serous cells in the early postnatal period, but it is uncertain whether mucous cells appear only in the developing parotid gland of a few species such as humans and rats, or whether the cell transformation actually occurs. To clarify these points, the present study investigated the developing mouse parotid glands. Light microscopy showed cells with secretory granules that stained extensively with PAS and alcian blue in the terminal clusters of a 1-day-old mouse parotid gland. Mucous cell numbers in the terminal clusters and the acini reached a peak on day 5 and decreased on day 7. By day 10, the mucous cells had disappeared altogether. Thus, the presence of mucous cells in the developing mouse parotid gland was confirmed. Electron microscopy showed granules of low-electron-density and bipartite granules in the mucous cells. Bipartite granules and highly electron-dense granules sometimes co-existed in a single cell. Immuno-electron microscopy revealed a positive reaction for amylase to the low-electron-density granules and the low-electron-density portions of the bipartite granules, in addition to the highly electron-dense granules and the electrondense cores of the bipartite granules. No mucous cells with nuclei displaying characteristics of apoptosis were recognizable. Lectin histochemistry both at the light and electron microscopic levels showed that the secretory granules in the mouse parotid gland mucous cells had sugar residues similar to those of the mature serous granules. These findings demonstrate that mucous cells appear in the early postnatal mouse parotid gland, and that almost all of these cells may be converted into serous cells.     

Developmental changes of sugar residues and secretory protein in mucous cells of the early postnatal rat parotid gland.

Mucous cells have been identified in the terminal portions of the early postnatal parotid gland in human and rat, although mature parotid gland acini are composed of serous cells or seromucous cells. Previously, Ikeda et al. demonstrated that mucous cells are present in the rat parotid gland on days 1 to 8 after birth and that the secretory granules within these mucous cells share some histochemical characteristics with mature serous cells. However, it is still not clear whether the mucous cells change into serous cells as the gland develops. The purpose of this study was to determine whether the mucous cells that appear in the early postnatal rat parotid gland change into serous cells. Parotid glands were obtained from male or female Wistar rats (aged 0-14 days and adults). Fixed tissue sections were reacted with soybean agglutinin (SBA) and wheat germ agglutinin (WGA) to detect glycoconjugates, or were stained using an anti-neonatal submandibular gland protein B1 (SMG-B1) antibody to identify serous acinar cells. The sections were observed by transmission electron microscopy. Electron microscopy revealed that cells with characteristics intermediate between those of mucous and serous cells (transitional cells) appeared around day 8 and that the nuclei of these cells did not show chromatin condensation, a characteristic of apoptotic cells. Lectin histochemistry showed that the mucous cells had the same sugar residues as the serous cells, which appeared after day 10. Immunohistochemistry with an anti-SMG-B1 antibody gave a positive reaction not only in the cells with highly electron-dense granules but also in the electron-dense cores of bipartite or tripartite granules in the transitional cells. Cells with morphological characteristics intermediate between those of mucous and serous cells (transitional cells) appearing in the early postnatal rat parotid gland begin to produce B1-immunoreactive protein common to serous acinar cells during development of the gland.     

The fine structure of testicular interstitial cells in the mouse. A comparison after conventional double fixation versus rapid freezing followed by freeze substitution

The interstitial cells in the testis of the mouse have been studied both after conventional double fixation with glutaraldehyde followed by osmium tetroxide and after rapid freezing and cryosubstitution with osmium tetroxide-acetone by light, scanning and transmission electron microscopy. Differences have been found especially concerning the preservation of lipids and the appearance of mitochondria. Varying proportions and different aspects of rough and smooth endoplasmic reticulum were observed corresponding after both preparation methods thus confirming their real existence. Numerous specific contacts are found between macrophages and Leydig cells, indicating a functional interdependence between these two cell types. Though structural preservation is superior after freeze substitution in the outermost zone, artifacts caused by the initial impact, by ice crystal formation, and during substitution must be considered.     

Changes in the number and distribution of myoepithelial cells in the rat parotid gland during postnatal development

The mature rat parotid gland shows hardly any cell bodies of myoepithelial cells around the acini, only a few cell processes being visible. However, in the early postnatal period, the rat parotid gland shows many myoepithelial cell bodies around the acini, including the intercalated ducts. In order to clarify the reason for the disappearance of myoepithelial cells from the area around the acinus during postnatal development, changes in the number and distribution of myoepithelial cells in the rat parotid gland were examined histochemically and chronologically, with particular reference to cell proliferation and cell death. From day 7 to day 14, many myoepithelial cells showing a positive reaction with anti-actin antiserum were found around the acini and intercalated ducts, but thereafter the number of such cells decreased gradually, particularly around the acini, and had almost disappeared after day 35. BrdU/PCNA-positive myoepithelial cells surrounding the acini were easily detected on day 14, but disappeared by day 21, whereas BrdU/PCNA-positive acinar cells remained numerous even after day 21. TUNEL/ISEL staining showed no positive myoepithelial cells throughout the observation period. Transmission electron microscopy also demonstrated no myoepithelial cells with chromatin condensation characteristic of apoptosis through the observation period. These findings suggest that the main reason for the disappearance of myoepithelial cells from the area around the acinus during postnatal development is the large difference between the number of myoepithelial cells and that of acinar cells, because the acinar cells retain their proliferative activity even after myoepithelial cells have become quiescent.     

The fine structure of secretory granules in submandibular glands of the rat during early postnatal development

The secretory end-pieces of the submandibular gland of rats during the first week of postnatal development are studied with regard to the fine structure of the secretion granules in these end-pieces. The terminal ends of the secretory ducts during this period consist of two types of cells; one cell is an acinar-type and the other is a duct-type found in the gland of adult rats. The secretion granules of the acinar-type cells are similar in appearance to those of the acinar cells in the gland of adult rats, and the structure of these granules remains the same throughout the week. However, granules widely different in appearance are present in the duct-type cells, and their structure varies in different cells as well as within a single cell at different stages of development. These granules contain unusual substructures which are not found in the secretion granules of adult rats, suggesting that the granules are transitory. Granules containing short tubular profiles are predominant in the gland of one day-old rats. A large number of granules in three day-old rats contain elongated tubules. More granules of widely different substructures are present in the gland of seven day-old rats than in the gland of younger rats. The matrix of the granules in seven day-old rats is of higher density than that of the granules in younger rats. In the dense matrix of these granules, less dense tubules form fingerprint-like or somewhat more irregular patterns.     

Developmental changes in the fine structure and histochemical properties of mucous cells in the parotid gland of the infant Japanese macaque

Mucous cells have been known to occur in the terminal portions of the parotid gland in a few species of mammals during a limited period of their development. The aim of this study was to examine the occurrence and features of mucous cells in the parotid gland of the infant Japanese macaque. Light microscopy revealed that mucous cells in the macaque parotid gland were present in the terminal clusters and acini at postnatal day 15, were less prevalent at day 30, and continued to decrease in number over 3 months. Mucous cells were no longer recognized in the parotid gland in 6-month-old macaques. Electron microscopy showed that the mucous cells contained electron-lucent secretory granules and bipartite or tripartite secretory granules. By 3 months of age, there was a scarcity of mucous cells and a concomitant increase in transitional cells. These transitional cells were intermediate in structure between mucous and serous cells, and contained three types of granules: electron-lucent, bipartite or tripartite, and electron-dense. None of the cells showed apoptotic figures. Lectin histochemistry indicated that the mucous cells in the early postnatal period had sugar residues identical in nature to those seen in the granules from mature serous cells in the glands of 3-month-old macaques. Immunohistochemistry using an antibody against human alpha-amylase showed a weakly positive reactivity in the secretory granules of the mucous cells, starting from day 15. In the transitional cells, the electron-dense granules showed a stronger immunoreactivity than either the electron-lucent granules or the heterogeneously structured granules. These results suggest that the secretory granules of mucous cells have characteristics in common with those of serous cells, and that during the transitional period the mucous granules change from the initial electron-lucent to hetorogenous forms, finally becoming the electron-dense granules. The mucous cells in the parotid gland of the juvenile Japanese macaque are therefore suggested to be converted into serous cells.     

Differentiation of myoepithelial cells in the developing rat parotid gland

Parotid glands of rats were prepared for light and electron microscopy and for the histochemical demonstration of myofibrils and alkaline phosphatase (AkPase) activity. Through 18 days in utero, the epithelial cells of the developing gland remain relatively undifferentiated. At 20 days in utero, a few cells in the outer layer of the terminal buds and adjacent segments of ducts acquire a cilium, the initial indication that they are differentiating into myoepithelial cells (MEC). Up until the time of birth, the only additional characteristics of MEC that the outer cells develop are to flatten against the underlying cells, begin to send out processes, and produce a few dilated cisternae of rough endoplasmic reticulum. Myofibrils and AkPase activity are first detected at the light microscopic level at five days after birth, around both the developing acini and intercalated ducts. Progressive increases in AkPase activity and in the size and number of myofibrils continue until the acini and intercalated ducts are invested with well-differentiated MEC at 15 days. Subsequently, as the acini undergo maturation during the weaning period (18-25 days), the MEC cease to surround the acini and assume the adult pattern of investing only the intercalated ducts. The pattern of MEC differentiation in the parotid gland differs from those in the sublingual and submandibular glands of the rat in several important respects. They begin to differentiate last, yet mature almost as early as do the MEC of the sublingual gland; they begin to differentiate prior to, rather than simultaneously with, the secretory cells; and their distribution changes as the acinar cells become mature.     

Melatonin release by exocytosis in the rat parotid gland

Several beneficial effects on oral health are ascribed to melatonin. Due to its lipophilic nature, non‐protein‐bound circulating melatonin is usually thought to enter the saliva by passive diffusion through salivary acinar gland cells. Recently, however, using transmission electron microscopy (TEM), melatonin was found in acinar secretory granules of human salivary glands. To test the hypothesis that granular located melatonin is actively discharged into the saliva by exocytosis, i.e. contrary to the general belief, the β‐adrenergic receptor agonist isoprenaline, which causes the degranulation of acinar parotid serous cells, was administered to anaesthetised rats. Sixty minutes after an intravenous bolus injection of isoprenaline (5 mg kg^?1), the right parotid gland was removed; pre‐administration, the left control gland had been removed. Samples were processed to demonstrate melatonin reactivity using the immunogold staining method. Morphometric assessment was made using TEM. Gold particles labelling melatonin appeared to be preferentially associated with secretory granules, occurring in their matrix and at membrane level but, notably, it was also associated with vesicles, mitochondria and nuclei. Twenty‐six per cent of the total granular population (per 100 μm² per cell area) displayed melatonin labelling in the matrix; three‐quarters of this fraction disappeared (P < 0.01) in response to isoprenaline, and melatonin reactivity appeared in dilated lumina. Thus, evidence is provided of an alternative route for melatonin to reach the gland lumen and the oral cavity by active release through exocytosis, a process which is under the influence of parasympathetic and sympathetic nervous activity and is the final event along the so‐called regulated secretory pathway. During its stay in granules, anti‐oxidant melatonin may protect their protein/peptide constituents from damage.     

Acinar degranulation in the rat parotid gland induced by neuropeptides

Vasoactive intestinal peptide (VIP, 4 microg kg(-1) min(-1)), substance P (3 microg kg(-1) min(-1)) and neurokinin A (2.5 microg kg(-1) min(-1)) were infused intravenously for 30 min in anaesthetized rats and the effects of these peptides on the parotid gland were examined. VIP reduced the numerical density of parotid acinar secretory granules, storing proteins, by 29 % and the glandular amylase activity by 33 %. Substance P reduced the number of secretory granules by 18 % but the amylase activity was unchanged. These results make VIP and substance P likely contributors to the parasympathetic non-adrenergic, non-cholinergic (NANC)-evoked parotid acinar degranulation. Neurokinin A, on the other hand, caused no reduction in granular number but reduced the glandular amylase activity by 19 %, indicating vesicular protein secretion.     

Regeneration of the parotid gland in the rat

Summary Parotid gland regeneration was studied in rats in which all of the left gland and part (50–60%) of the right had been removed. In young rats weighing 60–100 g, regeneration occurred in 19% of cases, as judged by the restoration of the weight of the organ. The weight of the glands did not always reach the initial level, however, and averaged 80% of the weight in control animals.The number of cases with gland regeneration increased in adult rats when the capsule was left open after the operation. Histological investigation showed that regeneration does not occur by growth starting at the injured surface, but rather by the proliferation of the secretory epithelium (regenerative hypertrophy) of the acini over the whole organ.(Presented by Active Member AMN SSSR V. V. Parin) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 50, No. 10, pp. 113–118, October, 1960     

Changes in the secretory acinar cells of the rat parotid gland during aging

The secretory acinar cells of parotid glands from rats of varying ages have been examined by electron microscopy to determine what age-related changes occur in these cells. The most prominent change noted in these cells is the progressive increase in the amount of lipofuscin granules with age. Lipofuscin granules are membrane-bound structures consisting of lipids, other subcomponents, and a matrix. In addition, these cells contain lipid droplets that are not associated with any other components and tend to accumulate at the base of the cells in older rats. Also, many acinar cells in the glands of old rats contain altered secretory granules which appear to be in the process of degeneration. The accumulation of lipid and degenerating secretory granules appears to be related to the reduced level of cellular secretory activity in the glands of older rats. It is possible that these two types of inclusions contribute to the formation of lipofuscin granules. Lipofuscin and degenerating secretory granules are associated with acid phosphatase, which is demonstrated cytochemically, indicating that these granules are lysosomal structures.     

Intercalated duct cells in the rat parotid gland may behave as tissue stem cells

This study was conducted to determine whether intercalated duct cells in the rat parotid gland have the properties of tissue stem cells. After induction of cellular proliferation by repeated administration of isoproterenol (IPR), a β-adrenergic agonist, proliferation activity in acinar, intralobular, and intercalated ductal cells was quantified using Ki-67 immunohistochemistry. The total number of each type of component cell in a gland was also estimated in the course of IPR treatment. IPR was found to induce proliferation of acinar and intercalated duct cells, but not intralobular duct cells. The total number of acinar cells in a gland on day 5 of IPR treatment was 1.6 times of that at day 0 (baseline). In contrast, the total numbers of intercalated and intralobular duct cells did not change from baseline, indicating a high possibility that the proliferated intercalated duct cells differentiated into acinar cells. On days 2 to 3 of IPR treatment, intercalated duct cells with amylase-positive secretory granules were recognized in a region very close to the acini, and were suspected of being transitional cells from intercalated duct to acinar cells. This quantitative study indicates that intercalated duct cells may have the properties of tissue stem cells upon IPR stimulation.     

Proliferative activity by cell type in the developing rat parotid gland

Background: In contrast to the considerable amount of research that has been done on the proliferative activity of the several types of parenchymal cells in the developing submandibular glands of rodents, systematic studies of cellular proliferation in the developing parotid gland have been confined to the acinar cells. The purpose of the present study was to attempt to fill this knowledge gap. Methods: Tritiated thymidine was parenterally administered to Sprague-Dawley rats at ages representative of the pre- and postnatal development of the parotid gland, and glands were harvested for autoradiography 90 min after injection. Mitotic activity among all cell types was verified by electron microscopy. Results: At all ages, the % labeled cells was much greater among the acini than any other cell type, including well-differentiated cells at 25 and 40 days. However, there were only small alterations in the proportions of cells comprised by the major cell types. Conclusions: Current theories on the histogenesis of salivary glands and their neoplasms are based on the renewing population model, in which both normal differentiated cells and neoplastic cells arise from undifferentiated stem cells in the ducts. However, these results suggest that most of the migration and redifferentiation in the developing rat parotid gland must be in the opposite direction, i.e., the acinar cells redifferentiate into ductal cells. They also indicate that until there are precise data on the rates of cell death among the several cell types, it remains more appropriate for salivary glands to be categorized as an expanding, rather than renewing, population. © 1995 Wiley-Liss, Inc.