Cell TissueRes. 198, 363-371 (1979)
Cell and Tissue Research 9 by Springer-Verlag 1979
The Postnatal Development of the Submandibular Gland of the Mouse* R. Srinivasan and W.W.L. Chang Department of Anatomy, Mount Sinai School of Medicine of the City Universityof New York, New York, N.Y. 10029, U.S.A.
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 3Hthymidine and stained with toluidine blue. At i week of age, the gland consists of acinar cells (36~o), intercalated duct cells (26~), juxta-acinar cells (13 ~o), striated duct cells ( 1 2 ~ ) 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 o f 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 ~o), intercalated duct cells (12~), striated duct cells (1~o) and others. Key words: Postnatal development - Submandibular gland - Mouse. Dr. R. Srinivasan, Department ofAnatomy, Mount Sinai Schoolof Medicine, 100th Street and Fifth Avenue, New York, New York 10029, U.S.A. * Supported by Public Health ServiceResearch Grant AMDE 19753 from the National Institute of Health. The authors are indebted to Mr. I. Borcsanyifor technical assistance
Send offprint requests to:
0302-766X/79/0198/0363/$01.80
364
R. Srinivasan and W.W.L. Chang
The p o s t n a t a l d e v e l o p m e n t o f the s u b m a n d i b u l a r glands has been investigated extensively in the r a t (Sreebny et al., 1955; J a c o b y a n d Leeson, 1959; L e e s o n a n d J a k o b y , 1959; D v o r a k , 1969; C u t l e r a n d C h a u d h r y , 1974; Y a m a s h i n a a n d M i z u h i r a , 1976, a n d others), a n d changes in cell p o p u l a t i o n kinetics d u r i n g the p o s t n a t a l p e r i o d have been a n a l y z e d (Chang, 1974; S r i n i v a s a n a n d C h a n g , 1975; A l v a r e s a n d Sesso, 1975). I n the mouse, the p o s t n a t a l d e v e l o p m e n t o f the g l a n d has been e x p l o r e d m o r p h o l o g i c a l l y ( C a r a m i a , 1966; Y o h r o , 1970; G r e s i k a n d M a c R a e , 1975). T h e present investigation was carried o u t (1) to o b t a i n i n f o r m a t i o n o n the p o p u l a t i o n kinetics o f v a r i o u s cell types d u r i n g the p o s t n a t a l d e v e l o p m e n t in the mouse, a n d (2) to c o m p a r e these d a t a with those o b t a i n e d previously in the r a t (Chang, 1974; S r i n i v a s a n a n d C h a n g , 1975).
Materials and Methods Male mice of the Swiss-Webster strain were housed in an air-conditioned room and given Purina Laboratory Chow and water adlibitum. Eleven groups of mice 1, 2, 3, 4, 5, 6, 8,10,12,16 and 20 weeks of age were used. The animals in the older age groups were weaned at 3 weeks of age. Each group consisted of five mice except for the 1-week age group in which 10 mice were used. One hour before killing, each animal received 1 laCi per gram body weight of 3H-thymidine (New England Nuclear, Boston, Mass.; specific activity 20 Ci per m mole). The submandibular glands were carefully separated from the sublingual glands and connective tissue and weighed. One gland per animal (except for two pooled glands in the 1-week age group) was used for the determination of DNA, RNA and protein contents by the methods previously described by Barka et al. (1973). The other gland was minced into small pieces, 1-3 mm in each dimension, fixed in Karnovsky's fixative for 2 h and, after washing, post-fixed in 1% osmium tetroxide in 0.1 M phosphate buffer (pH7.2) for 1 h. After dehydration, the tissues were embedded in Epon 812. Radioautographs were prepared from one ~tm thick sections mounted on precleaned glass slides by the dipping method of Kopriwa and Leblond (1962) using NTB-2 emulsion (Eastman-Kodak, Rochester, New York) and were developed after 5 weeks of exposure. They were stained with toluidine blue. In the gland of each animal, approximately 3,000 cells were classified according to cell type and 3Hthymidine-labeling. The frequency and labeling index (percentage of 3H-thymidine-labeled cells) of various types of cells were analyzed in all of the 11 age groups. On the basis of the frequency of individual cell types and the total DNA content of the gland, and on the assumption that each diploid nucleus contains 6.58 • 10-4 mg DNA, the absolute numbers of individual cell types per gland were estimated. The value was corrected for the number of cells in the phase of DNA synthesis (Srinivasan et al., 1973).
Results
Chemical Findings I n m a l e mice, the m o d e o f a g e - d e p e n d e n t i n c r e m e n t in the weight o f the s u b m a n d i b u l a r g l a n d is similar to t h a t in the b o d y weight (Fig. 1). T h e m e a n b o d y weight increases f r o m 3.2 g at 1 week to 42.3 g at 20 weeks o f age, while the m e a n weight o f the s u b m a n d i b u l a r g l a n d increases f r o m 9.5 m g at 1 week to 232.9 m g at 20 weeks. T h e rate o f i n c r e m e n t is r a p i d between 3 a n d 10 weeks a n d a p p e a r s to be greatest in the week following weaning. B o t h the b o d y a n d the g l a n d weights reach a p l a t e a u between 10 a n d 20 weeks o f age. The t o t a l contents o f D N A , R N A a n d p r o t e i n in the s u b m a n d i b u l a r g l a n d increases with age in a similar m a n n e r (Fig. 2), the rate o f i n c r e m e n t being greatest
Development of Submandibular Gland _1
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between 3 and 6 weeks of age. Between 1 week and 20 weeks, the DNA content of the gland rises from 0.05 mg to 0.56 mg, the RNA content from 0.08 mg to 2.00 mg, and the protein content from 1.22 mg to 40.90 mg.
Morphological Observations The cells of the submandibular gland of the mouse vary with age (Figs. 3-7). In male mice older than 4 weeks of age, several acini are continuous with the intercalated duct (ICD) whose cells are flattened or low cuboidal and contain no cytoplasmic granules. However, in animals up to 2 weeks of age, a group of cuboidal cells is present at the junction between the acini and the ICD; these cells are characterized by small darkly stained granules in the supranuclear cytoplasm and by basophilic cytoplasm mostly at the basal half of the cells (Figs. 3, 4). They are morphologically and topographically similar to the so-called terminal tubule cells of the rat submandibular gland during the early postnatal period (Srinivasan and Chang, 1973; Chang, 1974). They are also called juxta-acinar cells (Shear, 1969) or granular intercalated duct cells (Gresik and MacRae, 1975). Because of their location, the term juxta-acinar (JA) cells seems preferable. Away from the acini, the ICD is continuous with either the striated duct (SD) or the granular convoluted tubule (GCT) depending on the age of the animal. At or before 3 weeks of age, the ICD is connected directly to the SD, whose columnar cells are characterized by a large rounded vesicular nucleus nearly at the center of the
Figs. 3--7. Photomicrographs of Epon embedded, 1 I~m thick sections stained with toluidine blue Fig. 3. Submandibular gland of 1-week-old male mouse showing acinar cells (.4) containing pale secretory granules and juxta-acinar cells (J.4). Several acinar cells have pale stained secretory granules with a darkly stained core. x 1200 Fig. 4. Submandibular gland of 3-week-old male mouse showing acinar cells (A), intercalated duct cells
(ICD), striated duct cells (SD) and juxta-acinar cells (JA). Note absence of granular convoluted tubule cells, x 1200 Fig. 5. Gland of 4-week-old. Note granular convoluted cells x 1200
(GCT) and intercalated duct cells (1CD).
Fig. 6. Gland of 8-week-old. Number and size of granules in granular convoluted tubule cells are increased (compare with Fig. 5). x 1200 Fig. 7. Gland of 20-week-old. Note tremendous increase in number of GCT cells and heterogeneity of granules within and among GCT cells, x 1200
Developmentof SubmandibularGland
367
cells, basal striations and the absence of secretory granules in the cytoplasm (Figs. 3,4). Following weaning at 3 weeks of age, darkly stained granules appear in the apical portion of some SD cells located close to the ICD cells (Fig. 5); with the accumulation of granules, these SD cells are transformed into GCT cells (Srinivasan and Chang, 1975). Thus, in male mice older than 3 4 weeks, the ICD is continuous with the GCT. The frequency of GCT cells increases with age (see below); the number and size of the granules per GCT cell also increase and the heterogeneity of the granules becomes obvious in older age groups (Figs. 6, 7). On the other hand, typical SD cells become less noticeable in the glands of older mice.
Age-dependent Changes of Various Types of Cells In the gland from one-week old mice, the cells of the forming acini constitute 36% of the total cell population (Fig. 8). There are, in addition, JA cells (13 % ), ICD cells (26%), SD cells (12%) and miscellaneous cells (13%) including fibroblasts, mast cells, lymphocytes, endothelial cells, pericytes and myoepithelial cells. The relative frequency distribution of various cell types changes little before weaning except for a slight increase in the frequency of JA cells and ICD cells at 2 weeks of age (Fig. 8). However, the estimated absolute number of all the cell types present increases with age (Fig. 9). A drastic change in cellular composition occurs following weaning. Between 3 and 4 weeks, JA cells disappear, and GCT cells appear and increase rapidly in number (Fig. 8). In the 4-week old gland, GCT cells occupy 29% of the total cell population. Thereafter, there is a steady increase in the relative and absolute number of GCT cells, the population size of which seems to reach a plateau (47%) between 16 and 20 weeks of age. The estimated number of GCT cells in the gland is 8.6 • 1 0 6 at 4 weeks of age, and the number increases progressively to 4.24 x 107 in the 20-week old mouse (Fig. 9). The increase in the GCT cell population is accompanied by a decrease in the relative number of SD cells and ICD cells. From the 4th week of postnatal life onward, the frequency of SD cells decreases, and after 10 weeks of age, the SD cells make up approximately i% of the total cell population. The calculated absolute number of SD cells increases from 8 x 105 at 1 week to 46 x 105 at 5 weeks, the increase being greatest between 4 and 5 weeks of age. Thereafter, their number decreases steadily to 11 x 105 at 10 weeks and to 7 x 10s at 20 weeks of age. The frequency of ICD cells is highest (30%) in the 2-week old mouse; it decreases progressively thereafter, to 16% at 6 weeks, and slowly to 13% at 16--20 weeks. However, the estimated absolute number of ICD cells rises from 1.8 X 10 6 at 1 week to 11.8 x 10 6 at 6 weeks, and above that, the number of ICD cells appears to be stabilized (Fig. 9). The frequency of acinar cells is high (36 %-38 %) in the first 4 weeks of life and then decreases steadily to about 28 % at 12-20 weeks, the greatest decrease being observed between 4 and 5 weeks of age (Fig. 8). However, the estimated absolute number of acinar cells increses progressively from 2.5 x 10 6 at 1 week to 23.4 x 10 6 at 6 weeks, and between 6 and 20 weeks, the acinar cell population seems to be stabilized (Fig. 9).
368
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Proliferative Activity of Various Types of Cells (Fig. 10) The labeling index of the entire gland estimated at one hour after the administration of 3H-thymidine declines sharply from 8.9% at 1 week to 0.5% at 3 weeks of age. A second small peak (2.5 %) in proliferative activity occurs at 4 weeks. Thereafter, the labeling index decreases progressively to less than 1% at 8 weeks of age and then remains low (Fig. 10). The analysis of the proliferative activity of individual types of cells revealed that the second peak is due solely to the increased proliferative activity of ICD cells (Fig. 10). Following a sharp decline in the labeling index in the first 3 weeks of life, the ICD cells have a spurt in proliferative activity at 4-5 weeks. Thereafter, the labeling index of ICD cells decreases steadily but remains higher than that of any other cells type.
Development of Submandibular Gland
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Fig. 10. A Proliferative activityof the male mousesubmandibular gland from 1 weekto 20 weeksof age. B Proliferative activity of the individual cell typesin the male mouse submandibular gland from 1 week to 20 weeks of age
Acinar cells and JA cells show a similar trend in their proliferative activity in the first 3 weeks of age, i.e., a sharp reduction. After 4 weeks, the acinar cells and G C T cells show a similar steady decrease in their labeling index. The limited proliferative capacity of the SD cells also decreases slowly from 1 week to 8 weeks, after which age there are hardly any labeled SD cells present.
Discussion The postnatal development of the submandibular gland of the mouse is almost similar to that of the rat (Jacoby and Leeson, 1959; Chang, 1974; Srinivasan and Chang, 1975). In both, the definitive acini begin to appear at birth from the rudimentary secretory units known as terminal tubules (Jacoby and Leeson, 1959)
370
R. Srinivasan and W.W.L. Chang
or primary acini (Mueller, 1968). The acinar phase of glandular development is more rapid in the mouse than in the rat; it seems to be completed by 6 weeks in the mouse, and by 8 weeks in the rat (Srinivasan and Chang, 1975). During the development of acini from terminal tubules, the terminal tubule cells or JA cells appear secretory, but the nature of the secretory product and the function and eventual fate of JA cells have not been clarified (see Chang, 1974; Gresik and MacRae, 1975). Several investigators (Caramia, 1966; Gresik and MacRae, 1975) have demonstrated the persistence of few JA cells in the adult female mouse, whereas in the male, these disappear soon after weaning. In the male rat, JA cells decrease in number postnatally, but persist at the frequency of 3~o even at 12 weeks of age. The reason for these differences is unknown. It appears, however, that the presence and number of JA cells are inversely related to the appearance and degree of development of GCT cells. The changes in cellular composition after weaning are much more drastic in the male mouse than in the male rat. In the mouse, the rapid development of GCT cells coincides with the second peak of increased proliferative activity of ICD cells. These findings as well as the changes in the absolute number of ICD and SD cells indicate that the daughter cells of ICD cell mitoses should give rise to GCT cells through a stage of SD cells, in accordance with the hypothesis presented previously in the development of the submandibular gland of the rat (Srinivasan and Chang, 1975). However, in the rat, the development of GCT cells following weaning is a rather slow process and is not associated with any apparent increase in the proliferative activity ofductal cells, although there is a"shift" in mitotic activity from acinar cells to ductal cells during this period (Jacoby and Leeson, 1959; Srinivasan and Chang, 1975). In addition, the population size of GCT cells seems to become stabilized earlier in the male mouse than in the male rat and is much greater in the former than in the latter for a given age. The quantitative difference in the population size of GCT cells in two species of rodents of the same sex may reflect a difference in the relative amounts of various biologically active peptides in the glands. Among these are nerve growth factor (Levi-Montalcini, 1964), epidermal growth factor (Cohen, 1962), renin (Takeda et al., 1969), kallikrein (Erdos et al., 1968) and proteases (Ekfors and Hopsu-Havu, 1971).
References Alvares, E.P., Sesso, A.: Cell proliferation, differentiation and transformation in the rat submandibular gland during early postnatal growth. A quantitative and morphological study. Arch. Histol. Jap. 38, 177-208 (1975) Barka, T., Chang, W., van der Noen, H.: Stimulation of DNA synthesis by isoproterenol in rat submandibular gland during postnatal growth. Cell Tissue Kinet. 6, 135--146 (1973) Caramia, F.: Ultrastructure of mouse submandibular gland. I. Sexual differences. J. Ultrastruct. Res. 16, 505-523 (1966) Chang, W.W.L.: Cell population changes during acinus formation in the postnatal rat submandibular gland. Anat. Rec. 178, 187-201 (1974) Cohen, S.: Isolation of a mouse submaxillary gland protein accelerating incisor eruption an eyelid opening in the newborn animal. J. Biol. Chem. 237, 1555-1562 (1962) Cutler, L.S., Chaudhry, A.P.: Cytodifferentiation of the acinar cells of the rat submandibular gland. Develop. Biol. 41, 31-41 (1974)
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Dvorak, M.: The secretory cells of the submaxillary gland in the perinatal development in the rat. Z. Zellforsch. 99, 346--356 (1969) Ekfors, T.O., Hopsu-Havu, V.K.: Immunofluorescent localisation of trypsin-like estero - peptidases in the mouse submandibular gland. Histochem. J. 3, 415-420 (1971) Erdos, E,G., Tague, L.L., Miwa, L.: Kallikrein in granules of the submaxillary gland. Biochem. Pharmacol. 17, 667-674 (1968) Gresik, E.W., MacRae, E.K.: The postnatal development of the sexually dimorphic duct system and of amylase activity in the submandibular glands of mice. Cell Tissue Res. 157, 411-422 (1975) Jacoby, R., Leeson, C.R.: The postnatal development of the rat submaxillary gland. J. Anat. (Lond) 93, 201-216 (1959) Kopriwa, B.M., Leblond, C.P.: Improvements in the coating technique for radioautography. J. Histochcm. Cytochem. 10, 269-284 (1962) Leeson, C.R., Jacoby, F.: An electron microscopic study of the rat submaxillary gland during its postnatal development and in the adult. J. Anat. 93, 287-295 (1959) Levi-Montalcini, R.: The nerve growth factor. Ann. N.Y. Acad. Sci. 118, 149-170 (1964) Mueller, H.B.: Die Speicheld~sen junger Ratten nach Parasympathektomie. Z. Zellforsch. 88, 80-104 (1968) Shear, M.: Ultrastructural studies of the intercalated ducts in rat parotid glands. S. Aft. J. Med. Sci. 34, 21-27 (1969) Sreebny, L.M., Meyer, J., Bachem, E., Weinmann, J.P.'. Postnatal changes in proteolytic activity and in the morphology of the submaxillary gland in male and female albino rats. Growth 19, 57-74 (1955) Srinivasan, R., Chang, W.W.L., van der Noen, H., Barka, T.: The effect of isoproterenol on the postnatal differentiation and growth of the rat submandibular gland. Anat. Rec. 117, 243-253 (1973) Srinivasan, R., Chang, W.W.L.: The development of the granular convoluted duct in the rat submandibular gland. Anat. Rec. 182, 29-40 (1975) Takeda, T., Debusk, J., Grollman, A.: Physiologic role of renin like constituent of submaxillary gland of the mouse. Amer. J. Physiol. 216, 1196-1198 (1969) Yamashina, S., Mizuhira, V.: Postnatal development of acinar cells in rat submandibular glands as revealed by electron microscopic staining for carbohydrates. Am. J. Anat. 146, 211-256 (1976) Yohro, T.: Development of secretory units of mouse submandibular gland. Z. Zcllforsch. 110, 173-186 (1970)
Accepted February 7, 1979
Cell and Tissue Research 9 by Springer-Verlag 1979
The Postnatal Development of the Submandibular Gland of the Mouse* R. Srinivasan and W.W.L. Chang Department of Anatomy, Mount Sinai School of Medicine of the City Universityof New York, New York, N.Y. 10029, U.S.A.
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 3Hthymidine and stained with toluidine blue. At i week of age, the gland consists of acinar cells (36~o), intercalated duct cells (26~), juxta-acinar cells (13 ~o), striated duct cells ( 1 2 ~ ) 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 o f 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 ~o), intercalated duct cells (12~), striated duct cells (1~o) and others. Key words: Postnatal development - Submandibular gland - Mouse. Dr. R. Srinivasan, Department ofAnatomy, Mount Sinai Schoolof Medicine, 100th Street and Fifth Avenue, New York, New York 10029, U.S.A. * Supported by Public Health ServiceResearch Grant AMDE 19753 from the National Institute of Health. The authors are indebted to Mr. I. Borcsanyifor technical assistance
Send offprint requests to:
0302-766X/79/0198/0363/$01.80
364
R. Srinivasan and W.W.L. Chang
The p o s t n a t a l d e v e l o p m e n t o f the s u b m a n d i b u l a r glands has been investigated extensively in the r a t (Sreebny et al., 1955; J a c o b y a n d Leeson, 1959; L e e s o n a n d J a k o b y , 1959; D v o r a k , 1969; C u t l e r a n d C h a u d h r y , 1974; Y a m a s h i n a a n d M i z u h i r a , 1976, a n d others), a n d changes in cell p o p u l a t i o n kinetics d u r i n g the p o s t n a t a l p e r i o d have been a n a l y z e d (Chang, 1974; S r i n i v a s a n a n d C h a n g , 1975; A l v a r e s a n d Sesso, 1975). I n the mouse, the p o s t n a t a l d e v e l o p m e n t o f the g l a n d has been e x p l o r e d m o r p h o l o g i c a l l y ( C a r a m i a , 1966; Y o h r o , 1970; G r e s i k a n d M a c R a e , 1975). T h e present investigation was carried o u t (1) to o b t a i n i n f o r m a t i o n o n the p o p u l a t i o n kinetics o f v a r i o u s cell types d u r i n g the p o s t n a t a l d e v e l o p m e n t in the mouse, a n d (2) to c o m p a r e these d a t a with those o b t a i n e d previously in the r a t (Chang, 1974; S r i n i v a s a n a n d C h a n g , 1975).
Materials and Methods Male mice of the Swiss-Webster strain were housed in an air-conditioned room and given Purina Laboratory Chow and water adlibitum. Eleven groups of mice 1, 2, 3, 4, 5, 6, 8,10,12,16 and 20 weeks of age were used. The animals in the older age groups were weaned at 3 weeks of age. Each group consisted of five mice except for the 1-week age group in which 10 mice were used. One hour before killing, each animal received 1 laCi per gram body weight of 3H-thymidine (New England Nuclear, Boston, Mass.; specific activity 20 Ci per m mole). The submandibular glands were carefully separated from the sublingual glands and connective tissue and weighed. One gland per animal (except for two pooled glands in the 1-week age group) was used for the determination of DNA, RNA and protein contents by the methods previously described by Barka et al. (1973). The other gland was minced into small pieces, 1-3 mm in each dimension, fixed in Karnovsky's fixative for 2 h and, after washing, post-fixed in 1% osmium tetroxide in 0.1 M phosphate buffer (pH7.2) for 1 h. After dehydration, the tissues were embedded in Epon 812. Radioautographs were prepared from one ~tm thick sections mounted on precleaned glass slides by the dipping method of Kopriwa and Leblond (1962) using NTB-2 emulsion (Eastman-Kodak, Rochester, New York) and were developed after 5 weeks of exposure. They were stained with toluidine blue. In the gland of each animal, approximately 3,000 cells were classified according to cell type and 3Hthymidine-labeling. The frequency and labeling index (percentage of 3H-thymidine-labeled cells) of various types of cells were analyzed in all of the 11 age groups. On the basis of the frequency of individual cell types and the total DNA content of the gland, and on the assumption that each diploid nucleus contains 6.58 • 10-4 mg DNA, the absolute numbers of individual cell types per gland were estimated. The value was corrected for the number of cells in the phase of DNA synthesis (Srinivasan et al., 1973).
Results
Chemical Findings I n m a l e mice, the m o d e o f a g e - d e p e n d e n t i n c r e m e n t in the weight o f the s u b m a n d i b u l a r g l a n d is similar to t h a t in the b o d y weight (Fig. 1). T h e m e a n b o d y weight increases f r o m 3.2 g at 1 week to 42.3 g at 20 weeks o f age, while the m e a n weight o f the s u b m a n d i b u l a r g l a n d increases f r o m 9.5 m g at 1 week to 232.9 m g at 20 weeks. T h e rate o f i n c r e m e n t is r a p i d between 3 a n d 10 weeks a n d a p p e a r s to be greatest in the week following weaning. B o t h the b o d y a n d the g l a n d weights reach a p l a t e a u between 10 a n d 20 weeks o f age. The t o t a l contents o f D N A , R N A a n d p r o t e i n in the s u b m a n d i b u l a r g l a n d increases with age in a similar m a n n e r (Fig. 2), the rate o f i n c r e m e n t being greatest
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between 3 and 6 weeks of age. Between 1 week and 20 weeks, the DNA content of the gland rises from 0.05 mg to 0.56 mg, the RNA content from 0.08 mg to 2.00 mg, and the protein content from 1.22 mg to 40.90 mg.
Morphological Observations The cells of the submandibular gland of the mouse vary with age (Figs. 3-7). In male mice older than 4 weeks of age, several acini are continuous with the intercalated duct (ICD) whose cells are flattened or low cuboidal and contain no cytoplasmic granules. However, in animals up to 2 weeks of age, a group of cuboidal cells is present at the junction between the acini and the ICD; these cells are characterized by small darkly stained granules in the supranuclear cytoplasm and by basophilic cytoplasm mostly at the basal half of the cells (Figs. 3, 4). They are morphologically and topographically similar to the so-called terminal tubule cells of the rat submandibular gland during the early postnatal period (Srinivasan and Chang, 1973; Chang, 1974). They are also called juxta-acinar cells (Shear, 1969) or granular intercalated duct cells (Gresik and MacRae, 1975). Because of their location, the term juxta-acinar (JA) cells seems preferable. Away from the acini, the ICD is continuous with either the striated duct (SD) or the granular convoluted tubule (GCT) depending on the age of the animal. At or before 3 weeks of age, the ICD is connected directly to the SD, whose columnar cells are characterized by a large rounded vesicular nucleus nearly at the center of the
Figs. 3--7. Photomicrographs of Epon embedded, 1 I~m thick sections stained with toluidine blue Fig. 3. Submandibular gland of 1-week-old male mouse showing acinar cells (.4) containing pale secretory granules and juxta-acinar cells (J.4). Several acinar cells have pale stained secretory granules with a darkly stained core. x 1200 Fig. 4. Submandibular gland of 3-week-old male mouse showing acinar cells (A), intercalated duct cells
(ICD), striated duct cells (SD) and juxta-acinar cells (JA). Note absence of granular convoluted tubule cells, x 1200 Fig. 5. Gland of 4-week-old. Note granular convoluted cells x 1200
(GCT) and intercalated duct cells (1CD).
Fig. 6. Gland of 8-week-old. Number and size of granules in granular convoluted tubule cells are increased (compare with Fig. 5). x 1200 Fig. 7. Gland of 20-week-old. Note tremendous increase in number of GCT cells and heterogeneity of granules within and among GCT cells, x 1200
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cells, basal striations and the absence of secretory granules in the cytoplasm (Figs. 3,4). Following weaning at 3 weeks of age, darkly stained granules appear in the apical portion of some SD cells located close to the ICD cells (Fig. 5); with the accumulation of granules, these SD cells are transformed into GCT cells (Srinivasan and Chang, 1975). Thus, in male mice older than 3 4 weeks, the ICD is continuous with the GCT. The frequency of GCT cells increases with age (see below); the number and size of the granules per GCT cell also increase and the heterogeneity of the granules becomes obvious in older age groups (Figs. 6, 7). On the other hand, typical SD cells become less noticeable in the glands of older mice.
Age-dependent Changes of Various Types of Cells In the gland from one-week old mice, the cells of the forming acini constitute 36% of the total cell population (Fig. 8). There are, in addition, JA cells (13 % ), ICD cells (26%), SD cells (12%) and miscellaneous cells (13%) including fibroblasts, mast cells, lymphocytes, endothelial cells, pericytes and myoepithelial cells. The relative frequency distribution of various cell types changes little before weaning except for a slight increase in the frequency of JA cells and ICD cells at 2 weeks of age (Fig. 8). However, the estimated absolute number of all the cell types present increases with age (Fig. 9). A drastic change in cellular composition occurs following weaning. Between 3 and 4 weeks, JA cells disappear, and GCT cells appear and increase rapidly in number (Fig. 8). In the 4-week old gland, GCT cells occupy 29% of the total cell population. Thereafter, there is a steady increase in the relative and absolute number of GCT cells, the population size of which seems to reach a plateau (47%) between 16 and 20 weeks of age. The estimated number of GCT cells in the gland is 8.6 • 1 0 6 at 4 weeks of age, and the number increases progressively to 4.24 x 107 in the 20-week old mouse (Fig. 9). The increase in the GCT cell population is accompanied by a decrease in the relative number of SD cells and ICD cells. From the 4th week of postnatal life onward, the frequency of SD cells decreases, and after 10 weeks of age, the SD cells make up approximately i% of the total cell population. The calculated absolute number of SD cells increases from 8 x 105 at 1 week to 46 x 105 at 5 weeks, the increase being greatest between 4 and 5 weeks of age. Thereafter, their number decreases steadily to 11 x 105 at 10 weeks and to 7 x 10s at 20 weeks of age. The frequency of ICD cells is highest (30%) in the 2-week old mouse; it decreases progressively thereafter, to 16% at 6 weeks, and slowly to 13% at 16--20 weeks. However, the estimated absolute number of ICD cells rises from 1.8 X 10 6 at 1 week to 11.8 x 10 6 at 6 weeks, and above that, the number of ICD cells appears to be stabilized (Fig. 9). The frequency of acinar cells is high (36 %-38 %) in the first 4 weeks of life and then decreases steadily to about 28 % at 12-20 weeks, the greatest decrease being observed between 4 and 5 weeks of age (Fig. 8). However, the estimated absolute number of acinar cells increses progressively from 2.5 x 10 6 at 1 week to 23.4 x 10 6 at 6 weeks, and between 6 and 20 weeks, the acinar cell population seems to be stabilized (Fig. 9).
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Proliferative Activity of Various Types of Cells (Fig. 10) The labeling index of the entire gland estimated at one hour after the administration of 3H-thymidine declines sharply from 8.9% at 1 week to 0.5% at 3 weeks of age. A second small peak (2.5 %) in proliferative activity occurs at 4 weeks. Thereafter, the labeling index decreases progressively to less than 1% at 8 weeks of age and then remains low (Fig. 10). The analysis of the proliferative activity of individual types of cells revealed that the second peak is due solely to the increased proliferative activity of ICD cells (Fig. 10). Following a sharp decline in the labeling index in the first 3 weeks of life, the ICD cells have a spurt in proliferative activity at 4-5 weeks. Thereafter, the labeling index of ICD cells decreases steadily but remains higher than that of any other cells type.
Development of Submandibular Gland
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Acinar cells and JA cells show a similar trend in their proliferative activity in the first 3 weeks of age, i.e., a sharp reduction. After 4 weeks, the acinar cells and G C T cells show a similar steady decrease in their labeling index. The limited proliferative capacity of the SD cells also decreases slowly from 1 week to 8 weeks, after which age there are hardly any labeled SD cells present.
Discussion The postnatal development of the submandibular gland of the mouse is almost similar to that of the rat (Jacoby and Leeson, 1959; Chang, 1974; Srinivasan and Chang, 1975). In both, the definitive acini begin to appear at birth from the rudimentary secretory units known as terminal tubules (Jacoby and Leeson, 1959)
370
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or primary acini (Mueller, 1968). The acinar phase of glandular development is more rapid in the mouse than in the rat; it seems to be completed by 6 weeks in the mouse, and by 8 weeks in the rat (Srinivasan and Chang, 1975). During the development of acini from terminal tubules, the terminal tubule cells or JA cells appear secretory, but the nature of the secretory product and the function and eventual fate of JA cells have not been clarified (see Chang, 1974; Gresik and MacRae, 1975). Several investigators (Caramia, 1966; Gresik and MacRae, 1975) have demonstrated the persistence of few JA cells in the adult female mouse, whereas in the male, these disappear soon after weaning. In the male rat, JA cells decrease in number postnatally, but persist at the frequency of 3~o even at 12 weeks of age. The reason for these differences is unknown. It appears, however, that the presence and number of JA cells are inversely related to the appearance and degree of development of GCT cells. The changes in cellular composition after weaning are much more drastic in the male mouse than in the male rat. In the mouse, the rapid development of GCT cells coincides with the second peak of increased proliferative activity of ICD cells. These findings as well as the changes in the absolute number of ICD and SD cells indicate that the daughter cells of ICD cell mitoses should give rise to GCT cells through a stage of SD cells, in accordance with the hypothesis presented previously in the development of the submandibular gland of the rat (Srinivasan and Chang, 1975). However, in the rat, the development of GCT cells following weaning is a rather slow process and is not associated with any apparent increase in the proliferative activity ofductal cells, although there is a"shift" in mitotic activity from acinar cells to ductal cells during this period (Jacoby and Leeson, 1959; Srinivasan and Chang, 1975). In addition, the population size of GCT cells seems to become stabilized earlier in the male mouse than in the male rat and is much greater in the former than in the latter for a given age. The quantitative difference in the population size of GCT cells in two species of rodents of the same sex may reflect a difference in the relative amounts of various biologically active peptides in the glands. Among these are nerve growth factor (Levi-Montalcini, 1964), epidermal growth factor (Cohen, 1962), renin (Takeda et al., 1969), kallikrein (Erdos et al., 1968) and proteases (Ekfors and Hopsu-Havu, 1971).
References Alvares, E.P., Sesso, A.: Cell proliferation, differentiation and transformation in the rat submandibular gland during early postnatal growth. A quantitative and morphological study. Arch. Histol. Jap. 38, 177-208 (1975) Barka, T., Chang, W., van der Noen, H.: Stimulation of DNA synthesis by isoproterenol in rat submandibular gland during postnatal growth. Cell Tissue Kinet. 6, 135--146 (1973) Caramia, F.: Ultrastructure of mouse submandibular gland. I. Sexual differences. J. Ultrastruct. Res. 16, 505-523 (1966) Chang, W.W.L.: Cell population changes during acinus formation in the postnatal rat submandibular gland. Anat. Rec. 178, 187-201 (1974) Cohen, S.: Isolation of a mouse submaxillary gland protein accelerating incisor eruption an eyelid opening in the newborn animal. J. Biol. Chem. 237, 1555-1562 (1962) Cutler, L.S., Chaudhry, A.P.: Cytodifferentiation of the acinar cells of the rat submandibular gland. Develop. Biol. 41, 31-41 (1974)
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Dvorak, M.: The secretory cells of the submaxillary gland in the perinatal development in the rat. Z. Zellforsch. 99, 346--356 (1969) Ekfors, T.O., Hopsu-Havu, V.K.: Immunofluorescent localisation of trypsin-like estero - peptidases in the mouse submandibular gland. Histochem. J. 3, 415-420 (1971) Erdos, E,G., Tague, L.L., Miwa, L.: Kallikrein in granules of the submaxillary gland. Biochem. Pharmacol. 17, 667-674 (1968) Gresik, E.W., MacRae, E.K.: The postnatal development of the sexually dimorphic duct system and of amylase activity in the submandibular glands of mice. Cell Tissue Res. 157, 411-422 (1975) Jacoby, R., Leeson, C.R.: The postnatal development of the rat submaxillary gland. J. Anat. (Lond) 93, 201-216 (1959) Kopriwa, B.M., Leblond, C.P.: Improvements in the coating technique for radioautography. J. Histochcm. Cytochem. 10, 269-284 (1962) Leeson, C.R., Jacoby, F.: An electron microscopic study of the rat submaxillary gland during its postnatal development and in the adult. J. Anat. 93, 287-295 (1959) Levi-Montalcini, R.: The nerve growth factor. Ann. N.Y. Acad. Sci. 118, 149-170 (1964) Mueller, H.B.: Die Speicheld~sen junger Ratten nach Parasympathektomie. Z. Zellforsch. 88, 80-104 (1968) Shear, M.: Ultrastructural studies of the intercalated ducts in rat parotid glands. S. Aft. J. Med. Sci. 34, 21-27 (1969) Sreebny, L.M., Meyer, J., Bachem, E., Weinmann, J.P.'. Postnatal changes in proteolytic activity and in the morphology of the submaxillary gland in male and female albino rats. Growth 19, 57-74 (1955) Srinivasan, R., Chang, W.W.L., van der Noen, H., Barka, T.: The effect of isoproterenol on the postnatal differentiation and growth of the rat submandibular gland. Anat. Rec. 117, 243-253 (1973) Srinivasan, R., Chang, W.W.L.: The development of the granular convoluted duct in the rat submandibular gland. Anat. Rec. 182, 29-40 (1975) Takeda, T., Debusk, J., Grollman, A.: Physiologic role of renin like constituent of submaxillary gland of the mouse. Amer. J. Physiol. 216, 1196-1198 (1969) Yamashina, S., Mizuhira, V.: Postnatal development of acinar cells in rat submandibular glands as revealed by electron microscopic staining for carbohydrates. Am. J. Anat. 146, 211-256 (1976) Yohro, T.: Development of secretory units of mouse submandibular gland. Z. Zcllforsch. 110, 173-186 (1970)
Accepted February 7, 1979
