Acinar cell proliferation in the parotid and submandibular salivary glands of the neonatal rat

Abstract. Although the rat salivary glands are deficient in acini at birth, acinar cells proliferate rapidly during the early post-natal period. The pattern of acinar cell proliferation was analysed in the parotid and submandibular glands of neonatal rats from day of birth until day 34. Mitotic and [³H]thymidine ([³H]TdR) labelling indices of the two glands show distinctly different patterns. Analysis of cell division in the rat parotid gland demonstrated a peak of mitotic index at 14 days (2.9 ± 0.4%) and labelling index at 16 days (25.2 ± 2.1%). Submandibular gland acinar cell proliferation reaches a zenith between 7–8 days; labelling index (14.2 ± 1.1%) and mitotic index (2.3 ± 0.3%). Cell proliferation decreases rapidly in both glands after reaching a peak in activity. Gland size increases more rapidly in the submandibular gland which correlates with the earlier shift from cell proliferation to differentiation which occurs in this organ. Circadian rhythms of [³H]TdR incorporation were also investigated in this study. A circadian rhythm of [³H]TdR incorporation into DNA occurs at 15 days after birth with a peak at 06.00 hours in both glands and a trough occurring at 15.00 hours in parotid gland and 18.00 hours in the submandibular gland. Determination of specific activity of DNA (ct/min per μg DNA) on days 8, 10, 12, 13, 14, 15, and 16 after birth at 06.00 and 15.00 hours indicated that a circadian rhythm in [³H]TdR incorporation into DNA began on day 14. The developmental switch from suckling to solid food may be an initiating factor in the sychronization of the circadian rhythm in cell proliferation.     

Effects of double ligation of Stensen's duct on the rabbit parotid gland

Salivary gland duct ligation is an alternative to gland excision for treating sialorrhea or reducing salivary gland size prior to tumor excision. Duct ligation also is used as an approach to study salivary gland aging, regeneration, radiotherapy, sialolithiasis and sialadenitis. Reports conflict about the contribution of each salivary cell population to gland size reduction after ductal ligation. Certain cell populations, especially acini, reportedly undergo atrophy, apoptosis and proliferation during reduction of gland size. Acini also have been reported to de-differentiate into ducts. These contradictory results have been attributed to different animal or salivary gland models, or to methods of ligation. We report here a bilateral double ligature technique for rabbit parotid glands with histologic observations at 1, 7, 14, 30, 60 days after ligation. A large battery of special stains and immunohistochemical procedures was employed to define the cell populations. Four stages with overlapping features were observed that led to progressive shutdown of gland activities: 1) marked atrophy of the acinar cells occurred by 14 days, 2) response to and removal of the secretory material trapped in the acinar and ductal lumens mainly between 30 and 60 days, 3) reduction in the number of parenchymal (mostly acinar) cells by apoptosis that occurred mainly between 14–30 days, and 4) maintenance of steady-state at 60 days with a low rate of fluid, protein, and glycoprotein secretion, which greatly decreased the number of leukocytes engaged in the removal of the luminal contents. The main post- ligation characteristics were dilation of ductal and acinar lumens, massive transient infiltration of mostly heterophils (rabbit polymorphonuclear leukocytes), acinar atrophy, and apoptosis of both acinar and ductal cells. Proliferation was uncommon except in the larger ducts. By 30 days, the distribution of myoepithelial cells had spread from exclusively investing the intercalated ducts pre-ligation to surrounding a majority of the residual duct-like structures, many of which clearly were atrophic acini. Thus, both atrophy and apoptosis made major contributions to the post-ligation reduction in gland size. Structures also occurred with both ductal and acinar markers that suggested acini differentiating into ducts. Overall, the reaction to duct ligation proceeded at a considerably slower pace in the rabbit parotid glands than has been reported for the salivary glands of the rat.     

THE CELL GENERATION CYCLE OF THE ELEVEN-DAY MOUSE EMBRYO

The incorporation of tritiated thymidine into the DNA of erythroblasts, primitive ependymal cells, and mesenchymal cells of 11-day mouse embryos was studied by radioautography at different times between 25 minutes and 18 hours after injection intraperitoneally. There was no labeling of mitotic figures until 1 hour after injection. Following this, mitotic figures were labeled for about 5.5 hours in primitive ependymal cells and mesenchymal cells, and for a longer period in erythroblasts. The percentage of the labeled primitive ependymal cells at various times after injection indicate a periodic migration into and out of the mitotic zone. The cell generation cycle of primitive ependymal cells and mesenchymal cells is similar to some kinds of adult cells. The cycle of the erythroblasts is more like that of the cells of aging mice.     

Ultrastructural changes in the pars distalis of the maturing male rat

Anterior pituitary glands of male rats (2, 3, 5, 8, 12, 25, 36, 52, 56, and 62 days of age) were processed for electron microscopy. During early postnatal stages secretory cells are found in various stages of differentiation and comparatively few secretory granules are seen. Nuclei are mostly irregular, and the nucleo-cytoplasmic ratio is large. Many free ribosomes are present; the endoplasmic reticulum is generally sparse and the Golgi complex small or invisible. Cells are of variable shape, and numerous cytoplasmic processes project into large intercellular spaces. Many electron-dense cells which often contain myelinlike figures are seen. Lysosomes and lysosomal precursors are frequently found in secretory cells, predominantly in somatotrophs, of all immature glands. Mitotic figures are numerous in early stages after brith and decrease in number as the gland grows in size. A gradual increase in cytoplasmic volume with concomitant differentiation of cytoplasmic components as well as accumulation of secretory granules, accompanied by loss of myelin-like figures and decrease in the number of electron-dense cells, is observed as the animal reaches the prepuberal stage. Few lysosomes are seen in cells of mature glands. At 36 days of age all secretory cells seem to have differentiated, and morphological features as well as granule content show little change until puberty is reached. Gonadotrophs attain their characteristic morphology later than other cells. Cilia are observed in all developmental stages but are relatively infrequent in the mature gland. The described ultrastructural characteristics reflect the degree of maturation as well as the functional capacities of secretory cells at particular stages of development.     

Apoptosis and mitosis of parenchymal cells in the duct-ligated rat submandibular gland

Apoptosis and proliferation of parenchymal cells during atrophy of rat submandibular gland induced by double duct ligation were investigated using immunohistochemistry for proliferating cell nuclear antigen (PCNA), terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end labelling (TUNEL) and transmission electron microscopy (TEM). At 2 and 3 days after ligation, increased PCNA positive cells and mitoses were seen in ducts; thereafter PCNA positive cells decreased in number. At 3 and 4 days, the acinar cell population rapidly decreased, with many remaining TUNEL positive acinar cells. During this period, TEM showed typical apoptotic acinar cells that were phagocytosed by adjacent acinar cells or intraepithelial macrophages. After 7 days, most acinar cells had disappeared, leaving prominent residual ducts; a few acinar cells remained, especially at the lobule periphery. Submandibular gland duct ligation thus induced marked depletion of acinar cell by apoptosis and a concurrent short-lived cycle of duct cell proliferation.     

Changes in pancreatic acinar cell nuclear number and DNA content during aging in the rat

Pancreatic acinar cells from rats 5 to 658 days (94 weeks) of age were isolated by enzymatic dissociation and stained with the DNA specific fluorochrome Hoechst 33258. The nuclear DNA content and the incidence of binucleation were estimated in these cells. Total pancreatic weight, RNA, protein and DNA, and the incorporation of 3H-thymidine into pancreatic acinar cell DNA were also estimated in similar animals as measures of pancreatic growth. From 5 to 17 days after birth, 95% of the cells were mononucleate diploid and 5% were binucleate diploid; but during the period of rapid pancreatic growth over the following 39 days, acinar cells became increasingly binucleate. By 56 days after birth, 64% of cells were binucleate with a diploid DNA content per nucleus; and the incidence of binucleation then remained constant. At 28 days of age, 4% of mononucleate cells were tetraploid, increasing to 6% at 658 days of age. At this time 3% of binucleate cells contained dual tetraploid nuclei. There is thus a rapid development towards diploid binucleate acinar cells in the growing, postnatal pancreas; and in the adult pancreas a small proportion of these cells develop tetraploid nuclei.     

Active participation of apoptosis and mitosis in sublingual gland regeneration of the rat following release from duct ligation

Summary This study was designed to establish how mitotic cell proliferation and apoptotic cell death participate in the regeneration of atrophied rat sublingual glands. To induce atrophy to the sublingual gland of rats, the excretory duct was ligated unilaterally near the hilum, and after 1 week of ligation (day 0) the duct ligation was released to enable gland regeneration. The regenerating glands were examined with routine histology, immunohistochemistry for proliferating cell nuclear antigen (PCNA) as a marker of proliferating cells, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) as a marker of apoptotic cells, and transmission electron microscopy. At day 0, a few acini and many ducts remained in the atrophic sublingual glands, and newly formed immature acini were observed at day 3. Thereafter acinar cells progressively matured and increased in number, although the number of ducts decreased. Many PCNA- and some TUNEL-positive cells were seen in acini and ducts during regeneration. The labeling indices for both cell types were statistically significantly different from that of the control at several time points of the regeneration. Apoptotic and mitotic cells were also confirmed to be present in the experimental sublingual glands by electron microscopy. These observations suggest that apoptosis as well as mitosis of duct and acinar cells actively participate in and play important roles in sublingual gland regeneration.     

Density-dependent cell division after cortisol treatment of rat thymus in relation to age involution.

Cellular proliferation, in relation to cell density was investigated in the thymus of control and cortisol treated animals at 6 and 18 weeks of age. It was found that there was very little difference in the response of the two age groups to cortisol treatment. Cell density and cellular proliferation were markedly reduced 2 days after cortisol administration. From 4 days there was a rapid increase in cellular proliferation to triple the control rate. The mitotic index remained above normal until 12days then decreased to control values at 14 days. During this time the cell density of the thymus was being progressively restored. At all stages of regeneration, the mitotic index at first increased to a maximum at the mean cell density then decreased at the highest cell concentrations. A model system is discussed to account for this density dependent control of cellular proliferation in the thymus.     

Mitotic activity and its 24-hour rhythm in the rat pineal gland

Previous studies have yielded equivocal results concerning the 24-hour rhythmicity of mitotic activity in the rat pineal. The aim of the present study was to re-investigate this problem by carrying out three separate 24-hour experiments on alternate days. The results obtained confirm previous findings showing that in the pineal gland of adults mitotic activity is low. On average 22.3 mitotic figures of pinealocytes are seen per pineal gland, corresponding to a mitotic index of 0.2-0.6/1,000 pinealocytes. Mitotic activity is distinctly higher at daytime than at night. The timing of the peaks and troughs differs slightly from experiment to experiment. The majority of observations now indicate that in the rat pineal gland mitotic activity is higher at day time than at night.     

Gastric lipase and pepsinogen during the ontogenesis of rabbit gastric glands

In rabbit stomach, gastric lipase activity level was found to increase from birth to 30 days old (weaning), and then decreased. In contrast, pepsin activity only appeared between 30 to 45 days old, and increased till to the adult level. It was observed that maturation of gastric glands in cardial mucosa was a downward elongation process from the mitotic cell pool. These mitotic cells were always found in the neck of the gastric glands, corresponding to the bottom of the gland at 6 days old and to the mid-zone of the gland in adult. Location of rabbit gastric lipase (RGL) cells in cardial glands varied with age and was found along the pit of the gastric glands at 6 days old. The extent of this cellular location decreased with age, whereas a second RGL cell zone appeared below the mitotic cell area at 18 and 30 days old. At 45 days old, the pepsinogen cells appeared in the bottom of the gland, and consequently the RGL cells were located in the mid-zone of the gastric glands, between mitotic cells (neck of the gland) and pepsinogen cells (lower part of the gland). Ultrastructural study of cardial gastric glands revealed different morphologies of the secretion granules in the cells along the gastric glands. In 6-day-old rabbits, secretory granules were found uniformly electron dense in the bottom of the glands and were RGL-labeled by the immunogold technique. In the medium part of the glands, granules appeared biphasic, with a clear and a dense part, and RGL labeling was confined to the electron-dense part.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell proliferation is not required for the initiation of early cleft formation in mouse embryonic submandibular epithelium in vitro

An X-ray irradiation method was employed to analyse the role of cell proliferation in vitro in the cleft formation of mouse embryonic submandibular epithelium at early stages. When the mid 12-day gland was exposed to 200 rad of X-rays, the growth was severely retarded. In contrast, late 12-day and early 13-day glands grew apparently in a normal fashion, as did the control gland, for up to 40 h. In either case, they formed shallow clefts within 10 h of culture. With 1000 rad irradiation, the mid 12-day gland did not grow at all, but formed clefts within 20 h of culture followed by a rapid degeneration. Under the same conditions, the growth of the late 12-day gland, which was at the stage just before branching, was retarded until 10 h of culture, followed by a slight increase in epithelial size, but cleft formation was also observed within 6-10 h, as in the control gland. When exposed to a dose of 1000 rad of X-rays, the early 13-day and the late 12-day glands exhibited similar radiosensitivity; the initial narrow clefts in the epithelium deepened and new clefts began to form within 6-10 h of culture. [3H]thymidine incorporation studies revealed that a dose of 1000 rad reduced DNA synthesis of mid and late 12-day glands by 72 and 65%, respectively. Histological examination of X-irradiated late 12-day gland showed that mitotic figures were rarely seen in the epithelium at 6 h of culture. Aphidicolin, a specific inhibitor of DNA synthesis, could not halt the cleft formation of the late 12-day gland. In this experiment 89% of DNA synthesis was inhibited. Treatment of an X-ray irradiated late 12-day gland with aphidicolin blocked 92% of the DNA synthesis, but did not prevent cleft formation taking place. These results indicate that neither cell division nor DNA synthesis, is required for the initiation process of the cleft formation of the mouse embryonic submandibular epithelium at early morphogenetic stages in vitro.     

The roles of apoptosis and mitosis in atrophy of the rat sublingual gland

The roles of apoptosis and mitosis of acinar and duct cells in the atrophy of the sublingual gland of rat induced by double duct ligation was investigated using immunohistochemistry for proliferating cell nuclear antigen (PCNA), terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end labeling (TUNEL), and transmission electron microscopy (TEM). Many PCNA-positive duct cells were observed 3 days after duct ligation, and the numbers decreased thereafter. At 3 and 5 days, several TUNEL-positive acinar cells were observed and typical apoptotic acinar cells were identified by TEM. Necrotic acinar cells were also observed ultrastructurally. After 7 days, there were few acini but many ducts, as well as many structures representing transition from acinus to duct. These observations demonstrate that acinar cell loss by apoptosis and duct cell proliferation by mitosis occur in atrophic sublingual glands as well as in other atrophic salivary glands. In addition, it appears that the transition from acinar to duct cell and the necrosis of acinar cells play important roles in the atrophy of the sublingual gland.     

Isoprenaline-induced cell proliferation in mouse salivary glands: the effect of castration

The proliferative response to isoprenaline in the submaxillary and parotid glands of the Balb/c mouse has been studied in the intact male and female, and also in the male castrated one month prior to stimulation. The hyperplastic response of the acinar cells has been monitored by serial measurements of the flash tritiated thymidine labelling index and the mitotic index. Castration caused the atrophy of the granular ducts in the submaxillary gland, and therefore an increased predominance of the acini. At one month after castration the acini occupied an area almost 1.5-fold greater than that of the granular ducts, but this was not as great as in the intact female gland where acini occupied twice the area of the granular ducts. Hyperplasia was induced by a single injection of isoprenaline (0.3 mM/kg body weight). The response of the submaxillary gland in the intact male and intact female was very similar, DNA synthesis commencing 21-24 h after stimulation and mitotic activity first noted after 33-36 h. On the other hand, in the submaxillary gland of the castrated male, DNA synthesis began after only 18-21 h and mitotic activity after only 27-30 h. A metaphase arrest experiment with vincristine confirmed the more prompt response in the castrated animals; between 33-36 h after isoprenaline injection, the rate of entry of cells into mitosis was 4 cells/100 cells/h in the castrated group but only 0.4 cells/100 cells/h in the intact males. Thus castration appears to bestow a unique state of responsiveness upon the submaxillary gland to isoprenaline stimulation. The mechanisms underlying this change are not yet understood, for it is paradoxical that atrophy of a structural component rich in specific protein growth factors can alter the format of isoprenaline-induced hyperplasia in acinar cells that produce secretory glycoproteins.     

Studies of mitochondrial development prior to lactogenesis in the mouse mammary gland

Studies were performed to define mitochondrial development in relation to epithelial cell proliferation and differentiation prior to lactogenesis in the mammary gland of the mouse. Gland weight, total DNA, and total protein, selected as criteria of gland growth and proliferation, and various parameters of mitochondrial development were followed throughout the period. Gland weight and total DNA started increasing about Day 5 of pregnancy and reached maximal values by parturition. Total gland protein began to increase at the same time but did not reach maximal values until Day 4 of lactation. Total mitochondrial protein and the mitochondrial marker enzyme activities, succinate oxidase, succinate-linked ATP formation, and cytochrome oxidase increased gradually during pregnancy with rapid 2- to 3-fold increases occurring during the early days of lactation. Similarly, succinate oxidase activity per unit DNA of isolated mammary parenchymal cells increased gradually from mid-pregnancy to parturition with a precipitous, 2-fold increase occurring during early lactation.     

Characteristics of trophoblast cell reproduction in the connective zone of the rat placenta. II. Determination of the degree of ploidy of mitotic shapes

Morphological and cytophotometric studies have been made on polyploidization of placenta connective zone cells. Measurement of the DNA content in mitotic figures show that within a period of development ranging from day 13 to day 14 the bulk of mitoses (up to 25%) become tetraploid and octaploid. This may suggest that polyploidization of placenta connective zone cells proceeds via incomplete polyploidizing mitoses. Among tetraploid and octaploid mitotic figures, there are those corresponding to all the mitotic stages, from prophase to telophase. Consequently, mitosis in tetraploid and octaploid cells can reach telophase. In such cases polyploidization is likely to follow the acytokinetic mitotic pattern. A question of a certain maximum level of polyploidy that may be reached by cells due to the incomplete mitosis is discussed.     

Pancreatic cell proliferation in normal rats studied by in vivo autoradiography with 3H-thymidine

In vivo 3H-Thymidine autoradiographic investigations of DNA synthesis in acinar, islet and duct cells in the pancreas of normal rats showed that activity was dependent on age. The proliferation of acinar and islet cells, which was high in young animals, decreased exponentially with age; proliferation of the ductal cells on the other hand, increased until the animals became mature. These findings suggest that the physiological regeneration of acinar and islet cells, as well as their replacement after injury in adult animals commences from pancreatic ducts.     

The postnatal development of the submandibular gland of the mouse

Summary The postnatal development of the submandibular gland was investigated in male mice of the Swiss-Webster strain, which were killed at 1, 2, 3, 4, 5, 6, 8, 10, 12, 16 and 20 weeks of age, while the older mice had been weaned at 3 weeks of age. The mean weight of the submandibular gland increases from 9.5 mg at 1 week to 232.9 mg at 20 weeks of age, and the rate of increase is rapid between 3 and 10 weeks of age. The gland's contents of DNA, RNA and protein increase in a similar manner.The changes in the constituent cell types of the gland were studied in radioautographs prepared from Epon-embedded sections of mice given ³H-thymidine and stained with toluidine blue. At 1 week of age, the gland consists of acinar cells (36%), intercalated duct cells (26%), juxta-acinar cells (13%), striated duct cells (12%) and others. The cellular composition of the gland changes little before weaning, but the absolute number of all types of cells increases with age. Between 3 and 4 weeks, juxta-acinar cells disappear and granular convoluted tubule cells appear and increase rapidly in number with age. The rapid expansion of the population size of granular convoluted tubule cells after weaning coincides with the second peak of increased proliferative activity of intercalated duct cells, whereas all the other cell types show a progressive decrease in their proliferative activity with age. In spite of the burst in proliferative activity, there is no corresponding increase in the absolute number of intercalated duct cells. The number of striated duct cells peak at 5 weeks of age and then declines. These findings indicate that the mitoses of intercalated duct cells give rise to granular convoluted tubule cells through a stage of striated duct cells. At 20 weeks of age, the gland consists of granular convoluted tubule cells (47%), acinar cells (28%), intercalated duct cells (12%), striated duct cells (1%) and others.Supported by Public Health Service Research Grant AMDE 19753 from the National Institute of Health. The authors are indebted to Mr. I. Borcsanyi for technical assistance     

Morphometric and fine-structural studies of rat pancreatic acinar cells during early postnatal life

Summary Pancreatic acinar cells of rats obtained at 1,2, 3, 5, 7 and 14 days of age were examined using fine structural and morphometric techniques. From 5 days of age onwards, the acinar cells were analysed twice per day, at 20.00 h and 08.00 h.The present study demonstrates changes in the average volume of the cell, nucleus and cytoplasm, and volume densities of various cytoplasmic organelles during the first two weeks after birth. During early postnatal life, the volume density of rER increases, whereas that of zymogen granules decreases. From 5 days of age onwards, the volume densities of these two organelles differ significantly at 20.00 h and 08.00 h. During the first 2–3 days after birth, inclusion body-like structures appear in the cytoplasm of acinar cells; they contain aggregated zymogen granules and, sometimes, amorphous structures or cytoplasmic organelles. These structures also occur in interstitial cells and cells located in the intercalated region between acinar and ductal epithelial cells. Serum level of -amylase activity is high at birth, compared with other stages during the first three weeks. Degenerating acinar cells and cell debris can be seen in the acinar and ductal lumina during these stages, a feature suggesting holocrine secretion. Cellular polarity appears to be incomplete during the first two or three days after birth.     

An autoradiographic evaluation of the regeneration of corneal epithelium in Bufo marinus.

Epithelial regeneration following scraping of a central portion of corneal epithelium of the toad, Bufo marinus, was studied. Wound closure occurred by migration of a single layer of epithelial cells from adjacent noninjured areas. Autoradiography indicated that following wound closure at two days after scraping, DNA synthesis began in these epithelial cells covering the former scraped area, and was even more pronounced three days after scraping. At four days the former denuded area was covered by an epithelium two to four cells thick and by the fifth day regeneration was complete. These studies also indicated that mitotic time in this organism is rapid.