Cell differentiation in the terminal tubule of fetal rat submandibular gland in organ culture

Submandibular glands from 17-day-old rat fetuses were maintained in organ culture for five days in a medium consisting of Eagle's MEM (87%), horse serum (10%), and chick embryo extract (3%). Each day of the culture period explants were incubated for the demonstration of peroxidase activity and processed for light and electron microscopic observations. In some experiments cultures were exposed to ³H-thymidine one hour prior to fixation and incubation for the demonstration of peroxidase activity. Labelling index was determined using radioautographs of 1 μ Epon-embedded sections. At the time of explantation the submandibular gland rudiment consisted of undifferentiated epithelial cells arranged in cords. On day 3 of culture two additional cell types could be distinguished: terminal tubule cells and proacinar cells. The proacinar cells were characterized by peroxidase activity in their granules and cytoplasm. By day 4 acinar cells begin to appear. On the fifth day of culture the four cell types of the terminal tubule were present in the following proportions: undifferentiated cells, 44%; terminal tubule cells, 19%; proacinar cells, 31%; acinar cells, 6%. These results indicate that the cytodifferentiation of the secretory unit of rat submandibular gland in vitro is comparable to the differentiation in vivo.     

Cell differentiation in the terminal tubule of fetal rat submandibular gland in organ culture.

Submandibular glands from 17-day-old rat fetuses were maintained in organ culture for five days in a medium consisting of Eagle's MEM (87%), horse serum (10%), and chick embryo extract (3%). Each day of the culture period explants were incubated for the demonstration of peroxidase activity and processed for light and electron microscopic observations. In some experiments cultures were exposed to 3H-thymidine one hour prior to fixation and incubation for the demonstration of peroxidase activity. Labelling index was determined using radioautographs of 1 mu Epon-embedded sections. At the time of explantation the submandibular gland rudiment consisted of undifferentiated epithelial cells arranged in cords. On day 3 of culture two additional cell types could be distinguished: terminal tubule cells and proacinar cells. The proacinar cells were characterized by peroxidase activity in their granules and cytoplasm. By day 4 acinar cells begin to appear. On the fifth day of culture the four cell types of the terminal tubule were present in the following proportions: undifferentiated cells, 44%; terminal tubule cells, 19%; proacinar cells, 31%; acinar cells, 6%. These results indicate that the cytodifferentiation of the secretory unit of rat submandibular gland in vitro is comparable to the differentiation in vivo.     

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.     

The effects of 5-bromodeoxyuridine and isoproterenol on the postnatal differentiation of rat submandibular gland

The effects of 5-bromodeoxyuridine (BrdU) on the postnatal differentiation of rat submandibular gland and on the isoproterenol-induced changes of differentiation were studied. The rats were injected with BrdU for three consecutive days, beginning at two days of age. The total dose of BrdU was 0.9 mg/g body weight. BrdU caused a severe retardation of growth up to 15 days of age. A rapid growth of the animals between 15 and 22 days indicated a recovery from the effect of BrdU. The growth of the submandibular gland was retarded similarly with a corresponding decrease in DNA, RNA and protein content. Incorporation of tritiated thymidine into the submandibular gland was not altered in the BrdU-treated animals at one and three days after the last injection of the analog. At days 15 and 22 the rate of thymidine incorporation was greater in the submandibular gland of BrdU-treated rats as compared to littermate controls. Isoproterenol stimulated thymidine incorporation into the submandibular glands of two-week-old rats. This stimulation was not observed in rats which received BrdU at age 7–9 days, prior to the administration of isoproterenol. Electron microscopic observations, including a quantitative analysis of the frequency distribution of the various cell types in the terminal tubules and developing acini, indicated a retardation of acinar cell differentiation in the glands of BrdU-treated rats. In addition, there was an increase in the number and size of the secretory granules in the terminal tubule cells. BrdU treatment, however, caused no obvious pathologic alterations in the submandibular gland. Administration of isoproterenol for five days, beginning at five days of age, caused an apparent acceleration of the differentiation of acinar cells. In the glands of isoproterenol-treated rats the acinar cells were enlarged and were filled with homogenous secretory granules. Pretreatment with BrdU partially inhibited the effects of isoproterenol on differentiation and hypertrophy of the submandibular gland. With the given dose of BrdU, approximately 5% of thymine was replaced by bromouracil in the DNA of the submandibular gland. Such a replacement would be consistent with a direct effect of BrdU on the cytodifferentiation in the submandibular gland. However, because of the severe retardation of growth of the BrdU-treated rats, indirect effects can not be excluded.     

Cell death during development of intercalated ducts in the rat submandibular gland

Programmed cell death, or apoptosis, occurs during the development of many tissues and organs in almost all multicellular organisms. Although apoptosis of salivary gland cells has been demonstrated in several pathological conditions, the role of apoptosis in the postnatal development of the salivary glands is unknown. We have studied the development of the rat submandibular gland (SMG) during its transition from the perinatal stage to the mature adult stage. Terminal tubule or Type I cells, which synthesize the secretory protein SMG-C, are prominent in the perinatal acini and are believed to form the intercalated ducts of the adult gland. Between 25 days and 30 days after birth, the number of Type I cells and their SMG-C immunoreactivity markedly decreased. Apoptotic cells in association with the developing intercalated ducts were labeled with the Terminal Deoxyribonucleotidyl Transferase-Mediated dUTP Nick End Labeling (TUNEL) method. Between 25 and 40 days of age, from 50 to 80% of the apoptotic cells in cryostat sections of the SMG were closely associated with the intercalated ducts. Electron microscopy showed that the Type I cells became vacuolated, their secretory granules were reduced in size and number, and the amount of rough endoplasmic reticulum was decreased. Cellular debris resembling apoptotic bodies was phagocytosed by macrophages and adjacent intercalated duct cells. These observations suggest that the loss of Type I cells and reduction of SMG-C immunoreactivity during development of the intercalated ducts of the adult rat SMG is due, at least in part, to apoptosis.     

Transient occurrence of 27 kDa heat-shock protein in the terminal tubule cells during postnatal development of the rat submandibular gland.

It has been suggested that the 27 kDa heat-shock protein (Hsp27) plays a role at crucial cellular checkpoints for proliferation, apoptosis, and differentiation. We examined the immunolocalization of Hsp27 in the rat submandibular gland during postnatal development, wherein acinar cells proliferate and differentiate at earlier postnatal periods. At 2 weeks of age, weak Hsp27 immunoreactivity was distributed diffusely over all gland components. At 3 weeks, Hsp27 immunoreactivity disappeared in most parts of the acini and ducts, but was intensely accumulated in a small cell population located in the acinar center. This population was composed mostly of terminal tubule (TT) type I cells. At 4 weeks, the Hsp27-immunopositive cell population in the acinar center was composed primarily of immature (type II) acinar cells, partly of immature (granulated) intercalated duct (ID) cells, and occasionally of apoptotic cells. After 5 weeks, all acinar components became mature and were no longer immunoreactive for Hsp27. When acinar cell differentiation was accelerated by administration of isoproterenol to 3-week-old rats for 7 days, the number of Hsp27-positive cells was significantly lower than in the control gland at 4 weeks, confirming that Hsp27 expression is downregulated in mature acinar cells. These results suggest that at around 3-4 weeks in postnatal development, the centroacinar TT cells stop proliferating and begin to differentiate into acinar and ID cells, and occasionally undergo apoptosis. Hsp27 is transiently expressed in the centroacinar TT cells during this critical period, and thus may play a role in their differentiation into the immediate descendants.     

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 3-H-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 X 10-6) at four weeks to 26% (68 X 10-6) 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.     

胎儿颌下腺内神经肽免疫组织化学研究

目的 :对胎儿颌下腺内神经肽进行定性和定位研究 ,以探讨其功能属性。方法 :采用免疫组织化学ABC方法。结果 :第 16～ 18周 ,胎儿颌下腺呈P物质 (SP)、血管活性肠肽 (VIP)及神经肽Y (NPY)免疫反应性 ,为弱阳性～中等阳性 ,主要分布于导管上皮胞质内 ,细胞核为阴性。至 2 2～ 2 4周左右 ,SP和VIP免疫反应强度达到强阳性反应 ,随后反应强度减弱。NPY无明显增龄变化。结论 :(1)人胎颌下腺内含有SP、VIP及NPY等神经肽类物质 ;(2 )这些神经肽共存于颌下腺导管上皮细胞内 ,上述结果为深入研究颌下腺分泌功能属性提供了形态学资料     

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.     

Ultrastructure of the secretory response of male mouse submandibular gland granular tubules

The organization and fine structure of granular convoluted tubule cells (GCT) from male mouse submandibular glands have been examined in controls and in animals injected with adrenergic and cholinergic secretagogues. Control submandibular glands exhibited a single population of GCT cells with numerous homogeneous granules filling the apical two-thirds of the cytoplasm. A zone of transition cells, exhibiting characteristics of both GCT and striated duct cells, was found between the agranular intercalated duct and GCT segments. These transition cells possessed apical granules of variable size as well as prominent basal striations. Dramatic changes in the morphology of GCT cells followed administration of the alpha-adrenergic agent, phenylephrine. The extensive degranulation involved formation of "secretory pools" of fused granules and release of secretory material into the lumen. The appearance of numerous smooth vesicles near luminal membranes suggested extensive membrane retrieval. Intracellular membrane-limited aggregates of membrane fragments suggested that much of the retrieved membrane was destined for degradation. Rough endoplasmic reticulum was highly dilated but there was no indication of increased size or activity of the Golgi complex. Ultrastructural evidence indicated that the secretory responses to isoproterenol, a beta-adrenergic agent, and to pilocarpine, a cholinergic agent, were much more modest, but it is clear that some secretory response to these agents does occur. The other cell types of the duct and tubule system did not exhibit comparable morphological changes in response to the agents used.     

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.     

Transient occurrence of 27 kDa heat‐shock protein in the terminal tubule cells during postnatal development of the rat submandibular gland

It has been suggested that the 27 kDa heat‐shock protein (Hsp27) plays a role at crucial cellular checkpoints for proliferation, apoptosis, and differentiation. We examined the immunolocalization of Hsp27 in the rat submandibular gland during postnatal development, wherein acinar cells proliferate and differentiate at earlier postnatal periods. At 2 weeks of age, weak Hsp27 immunoreactivity was distributed diffusely over all gland components. At 3 weeks, Hsp27 immunoreactivity disappeared in most parts of the acini and ducts, but was intensely accumulated in a small cell population located in the acinar center. This population was composed mostly of terminal tubule (TT) type I cells. At 4 weeks, the Hsp27‐immunopositive cell population in the acinar center was composed primarily of immature (type II) acinar cells, partly of immature (granulated) intercalated duct (ID) cells, and occasionally of apoptotic cells. After 5 weeks, all acinar components became mature and were no longer immunoreactive for Hsp27. When acinar cell differentiation was accelerated by administration of isoproterenol to 3‐week‐old rats for 7 days, the number of Hsp27‐positive cells was significantly lower than in the control gland at 4 weeks, confirming that Hsp27 expression is downregulated in mature acinar cells. These results suggest that at around 3–4 weeks in postnatal development, the centroacinar TT cells stop proliferating and begin to differentiate into acinar and ID cells, and occasionally undergo apoptosis. Hsp27 is transiently expressed in the centroacinar TT cells during this critical period, and thus may play a role in their differentiation into the immediate descendants. Anat Rec 264:358–366, 2001. © 2001 Wiley‐Liss, Inc.     

Immunochemical analysis of laminin during postnatal development of the rat submandibular gland.

Anti-laminin serum was used to investigate distribution patterns and chain composition of laminin during postnatal development of rat submandibular gland. Immunohistochemical analysis showed that in glands of newborn rats laminin was not uniformly present around growing acini. Staining was frequently weak or absent in clefts formed between adjacent cells. This irregular staining pattern decreased progressively over the subsequent periods, and in 30-day-old animals immunoreactivity was observed only at the periphery of glands. Immunoblot analysis showed that laminin was composed of bands corresponding to the alpha 1, beta 1 and gamma 1 polypeptides. The correlation between the pattern of laminin expression and gland maturation suggests a role of laminin in the functional maturation of acinar cells.     

Effects of irradiation on the submandibular gland of the rat

Summary Single-dose cervical irradiation by cobalt 60 in rats induced lasting functional disturbances of the submandibular gland which were excessive when compaired with the relative integrity of the gland as seen under the light microscope. Enzyme histochemical and ultrastructural studies revealed severe damage shortly after exposure with appearance of karyolytic bodies and autophagosomes accompanied by increased hydrolase activity. Mitochondrial alterations were concomitant with diminished ductal oxidative enzyme activity. Although most of these alterations resolved rapidly as a result of acinar and ductal cell repair and regeneration originating in the intercalated ducts, secretory abnormalities were still observed two months after exposure as evidenced by the accumulation of granules in acinar cells and the heterogeneity of ductal cell granules. These anomalies, comparable to those observed in sialadenoses, probably result from persistent alterations of intralobular nerve endings.The authors wish to thank M.F. Baucher, A. Lesot and M. Tacnet for their technical assistance     

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.     

Perivascular nerves in the rat submandibular salivary gland.

Perivascular nerves in the rat submandibular salivary gland have been studied using a variety of histochemical procedures coupled with electron microscopy. Two principal nerve types, adrenergic and cholinergic, appear to predominate and are localized principally around arterioles. Venules are rarely innervated. The possibility that a non-adrenergic non-cholinergic nerve population might influence blood flow is discussed critically in the light of anatomical and physiological findings.