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.     

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.     

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.     

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.     

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.     

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.     

Non-specific secretory supersensitivity in rat parotid gland following neonatal sympathetic denervation

Newborn rats were surgically sympathectomized by extirpation of the left superior cervical ganglion. After 9 weeks the parotid glands of both sides were used for secretory studies. Isoprenaline, dopamine, and the dibutyryl analogue of cAMP (DBcAMP) caused an increase in amylase release, which was significantly higher in the denervated glands. Also carbamylcholine was more effective in the denervated gland; the concentration-response curve was shifted to the left, and the maximal output of amylase was increased. Neonatal sympathetic denervation induces supersensitivity for both adrenergic and cholinergic agonists as well as for DBcAMP. This may be due to compensatory mechanisms involving both up-regulation of receptors as well as amplification of the intracellular mediation.     

Granule types and their morphological changes in terminal cluster and acinar cells in the late pre- and early postnatal rat sublingual gland

The developmental characteristics of serous cells appearing in the rat sublingual gland from the late prenatal to the early postnatal period were investigated in this study. Particular attention was paid to the morphological changes observed in the secretory granules at the histochemical and ultrastructural level. On prenatal day 18, granules with homogeneous high electron density (Type I granules), and mottled granules (Type II granules) with heterogeneous electron density appeared in the narrow luminar cytoplasm of cells constituting the terminal clusters. On prenatal day 19, these granules decreased in number and were replaced by bipartite granules (Type III granules) composed of a highly electron-dense core and a more electron-lucent rim. Pronase treatment almost completely digested the Type I and II granules and the electron-dense core of the Type III granules, although some of the Type I and II granules in serous demilunes at a later stage were insufficiently digested. On prenatal day 19.5, homogeneous granules of low electron density (Type IV granules) appeared in the terminal clusters and acini, and increased in number daily, making up 92.8% of the total granules on postnatal day 28. The granule morphology on electron microscopy, Alcian blue, and periodic acid-Schiff staining strongly suggested that Type I and II granules were serous granules, Type IV granules were mucous granules, and Type III granules were transforming-type granules. None of the secretory cells showed chromatin condensation, which is a characteristic of apoptosis. These findings suggest that the developing rat sublingual gland from the late prenatal to early postnatal period has numerous serous granules in the terminal clusters and acini, and that the majority of granules are replaced by mucous granules through transforming-type granules. In addition, because apoptotic figures of secretory cells could not be detected, it appears that most of the serous cells in the developing rat sublingual gland might have changed to mucous cells.     

N-linked protein glycosylation in the rat parotid gland during aging

N-Linked protein glycosylation was examined in vitro in dispersed rat parotid acinar cells from young adult (3-6 months) and aged (22-24 months) rats. A small decrease in general protein production was observed with cells from aged animals (approximately 20% lower incorporation of [14C]leucine into 10% CCl3 COOH insoluble protein during continuous pulse labeling). Incorporation of [3H]mannose into N-linked glycoproteins by aged cells was further reduced (approximately 35%). Similarly microsomal membranes from parotid glands of aged animals showed approximately 50% reduction in the synthesis of mannosylphosphoryl dolichol, a key intermediate in the dolichol pathway of protein N-glycosylation. Man-P-Dol synthase, the microsomal enzyme responsible for production of this saccharide-lipid, displayed no change in apparent Km for GDP-mannose when preparations from aged animals were utilized, but did show approximately 50% reduction in Vmax. Following beta-adrenoreceptor activation, cells from both young adult and aged glands showed increased N-linked protein glycosylation almost to the same extent (approximately 2-fold). The data suggested that in aged rat parotid cells there is a basal reduction of activity in the pathway responsible for asparagine-linked protein glycosylation, but that following exocytotic stimuli this pathway responds in a manner comparable to cells from young adult glands.     

Dissociation of rat parotid gland.

Rat parotid gland was dissociated by sequential collagenase and hyaluronidase digestions, chelation with ethylenediaminetetraacetic acid, and mild shearing force to yield predominantly single cells. The isolated acinar cells retained their morphologic characteristics and their amylase activity. The functional integrity of the isolated cells was assessed by measuring their secretory response to isoproterenol, epinephrine, and carbamylcholine and by their ability to incorporate radioactively labeled leucine and thymidine. The discharge of amylase from the dissociated cells was not effected by isoproterenol or norepinephrine and the response to carbamylcholine was minimal. The data indicate a destruction or perturbation of hormone receptors during the dissociation procedure. The maintenance of the cells in culture for up to 18 hours failed to restore the responsiveness of the isolated parotid gland acinar cells to isoproterenol. The isolated cells incorporated 14C-leucine into proteins at a linear rate between 30 and 180 minutes. Chromatographic and electrophoretic profiles of newly synthesized proteins indicated that all major proteins synthesized in vivo were also synthesized by the isolated cells. The isolated cells incorporated tritiated thymidine into DNA. Furthermore, stimulation of DNA synthesis by isoproterenol in vivo was reflected by a higher rate of thymidine incorporation by the isolated cells as compared with controls. The dissociated parotid gland cells offer a convenient system for studying various cellular processes, particularly the synthesis of macromolecules with high specific activity. However, some functions, notably the response to beta- adrenergic agonists, are lost during the dissociation procedure.     

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.     

Tuberoinfundibular peptide of 39 residues in the embryonic and early postnatal rat brain

Tuberoinfundibular peptide of 39 residues (TIP39) was identified as the endogenous ligand of parathyroid hormone 2 receptor. We have recently demonstrated that TIP39 expression in adult rat brain is confined to the subparafascicular area of the thalamus with a few cells extending laterally into the posterior intralaminar thalamic nucleus (PIL), and the medial paralemniscal nucleus (MPL) in the lateral pontomesencephalic tegmentum. During postnatal development, TIP39 expression increases until postnatal day 33 (PND-33), then decreases, and almost completely disappears by PND-125. Here, we report the expression of TIP39 during early brain development. TIP39-immunoreactive (TIP39-ir) neurons in the subparafascicular area first appeared at PND-1. In contrast, TIP39-ir neurons were detectable in the MPL at embryonic day 14.5 (ED-14.5), and the intensity of their labeling increased thereafter. We also identified TIP39-ir neurons between ED-16.5 and PND-5 in two additional brain areas, the PIL and the amygdalo-hippocampal transitional zone (AHi). We confirmed the specificity of TIP39 immunolabeling by demonstrating TIP39 mRNA using in situ hybridization histochemistry. In the PIL, TIP39 neurons are located medial to the CGRP group as demonstrated by double immunolabeling. All TIP39-ir neurons in the AHi and most TIP39-ir neurons in the PIL disappear during early postnatal development. The adult pattern of TIP39-ir fibers emerge during postnatal development. However, fibers emanating from PIL can be followed in the supraoptic decussations towards the hypothalamus at ED-18.5. These TIP39-ir fibers disappear by PND-1. The complex pattern of TIP39 expression during early brain development suggests the involvement of TIP39 in transient functions during ontogeny.     

Neuropeptides and disuse of the rat parotid gland.

Rats were fed a liquid diet with the aim of decreasing nervous reflex activity in the parotid glands. In rats killed after 21 days on this diet the glands were atrophied and the total amounts of the neuropeptides, substance P, vasoactive intestinal peptide and calcitonin gene-related peptide, were lower than in control glands.     

Investigation of the capillaries of the rat parotid gland during a 3-h secretory cycle

Principles governing changes in diameter of the lumen and area of the endothelium of capillaries in the parotid salivary gland of rats with an experimentally established 3-h secretory cycle, and also during circulatory ischemia of the organ for 5 min, were studied. The diameter of the lumen and area of the endothelium of the capillaries changed very little with the phase of the secretory cycle. Occlusion of the common carotid artery for 5 min likewise did not change the diameter of the lumen or the area of the endothelium of the capillaries. The results suggest that the increase in the transcapillary blood flow during secretion can be explained by the recruiting of more capillaries into the circulation.Laboratory of Electron Microscopy, N. I. Pirogov Second Moscow Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR V. V. Kupriyanov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 80, No. 7, pp. 104–106, July, 1975.     

The secretory nerves of the parotid gland of the dog

1. Secretory nerve fibres to the parotid gland of the dog have been found in the adventitia of the internal maxillary artery. In dogs under chloralose anaesthesia the course of these fibres from the mandibular nerve to the gland was determined by electrical stimulation at different sites. Secretion induced by these nerves was abolished by atropine but not by hexamethonium.

2. In dogs in light barbiturate anaesthesia parotid secretion was evoked reflexly by pouring citric acid on the tongues. After division of the auriculo-temporal nerve and the nerves on the artery this secretion disappeared in about half the experiments and was greatly reduced in the others.

3. The maximal secretory rate which could be obtained reflexly from the normally innervated parotid gland was first estimated in light barbiturate anaesthesia. Chloralose was then given and the auriculo-temporal nerve and the nerves on the artery were electrically stimulated together; in about half the experiments this could produce secretion at a rate as high as that obtained reflexly.

4. By separate and simultaneous stimulation of different sets of nerve fibres convergence of the secretory fibres to the glandular cells could be demonstrated.

5. It is concluded that a large number of post-ganglionic, cholinergic secretory fibres to the canine parotid gland run on the internal maxillary artery. In some dogs a small number of fibres seem to have escaped detection. Possibly they may reach the gland by way of the facial nerve.

     

Developmental changes of sugar occurrence and distribution in the rat submandibular and sublingual glands

 The developmental expression of salivary glycoconjugates was investigated in the rat submandibular and sublingual glands by conventional and lectin histochemistry. By the time of the first differentiation of secretory structures, in spite of similar morphological features, a different histochemical reactivity was detected, accounting for a relevant content of neutral glycoconjugates in the submandibular gland and the occurrence of both neutral and acidic glycoconjugates in the sublingual one. The use of lectins allowed the main changes of secretory components to be noted around gestational day 18. DBA and WGA lectins seemed to act as pre- and post-natal development markers while Con A lectin was indicative of post-natal differentiation. Taken together, data from lectin histochemistry indicated the transitional occurrence of glycoconjugates, probably involved in temporally restricted functions, as well as the co-existence of different secretory components that might also reflect maturational changes of single products. Accepted: 31 July 1998