Cell Differentiation in the Terminal Tubule of Fetal R a t Submandibular Gland in Organ Culture MYRA BLUESTEIN RUFO AND TIBOR BARKA Department of Anatomy, Mount Sinai School o f Medicine of the City University of New York, Fifth Avenue and 100th Street, New York, Nezu York 10029
ABSTRACT
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. I n 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 P 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 perinatal cytodifferentiation of the terminal tubule of rodent submandibular gland has been the subject of many finestructural, histochemical, and quantitative studies in the past few years (Fukuda, '67; Dvofak, '69; Yohro, '70; Kim et al., '70; Strum, '71; Yamashina and Barka, '72, '73; Bressler, '73; Chang, '74; Cutler and Chaudhry, '74). To summarize briefly, "terminal tubule" is the term introduced by Jacoby and Leeson ('59) to refer to the immature secretory unit of rat submandibular gland through the first six weeks of postnatal life. A few days before birth two separate cell types arise from undifferentiated precursor cells in the terminal tubules; these are known respectively as terminal tubular cells and cells with polymorphic granules (Dvozak, '69) or proacinar cells (Yamashina and Barka, '73). Peroxidase activity has been localized in the cisternae of the rough endoplasmic reticulum and nuclear envelope and in the secretory granules of proacinar cells in the glands of fetal and newborn rats. Terminal ANAT. REC., 184: 301-310.
tubular cells, on the other hand, have not been shown to contain peroxidase (Strum, '71; Yamashina and Barka, '72, '73). For this reason, the cells with polymorphic granules are believed to represent direct precursors of acinar cells. As the acinar cells mature the composition and ultrastructure of the secretory granules are altered, The secretory granules become electrolucent and flocculent with a great reduction in peroxidase activity that is demonstrable electron histochemically. However, a strong peroxidase activity in the endoplasmic reticulum and nuclear envelope characterizes the mature acinar cells (Yamashina and Barka, '72). The present study was undertaken to compare the differentiation of submandibular gland in organ culture with development as known in vivo. Using peroxidase activity as a marker for proacinar cell differentiation, the objective of this study has been to define a system in which to test the effects of drugs and hormones on Received May 21, '75.Accepted July 29, '75.
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the differentiation of the fetal gland in vitro. MATEHIALS AND METHODS
The culture medium employed was a modified version of that used by Parsa et al. ('69), consisting of 87% Eagle's Minimum Essential Medium, 10% horse serum, and 3% chick embryo extract to which 0.01 M HEPES buffer and 1% AntibioticAntimycotic Solution (GIBCO) were added. The initial pH of the medium was 7.2-7.4. Morphological observations. Submandibular glands were removed from Sprague-Dawley rat fetuses ranging in age from 16 to 19 days, and carefully trimmed of extraneous connective tissue. Individual glands were transferred in pairs to Falcon plastic organ culture dishes, and positioned on a strip of lens paper (Ross Optical) supported by a wire mesh grid. The wells of the culture dishes contained medium sufficient to moisten the lens paper strip. Cultures were maintained for variable periods ranging from two to seven days, in an atmosphere of 5% COz in air at 37°C and 95% humidity. The medium was changed every 48 hours. At daily intervals, several explants were fyred 30 minutes in half-strength Karnovsky's fixative ('65), rinsed in 0.1 M cacodylate buffer (pH 7.4) containing 7% sucrose and incubated 60 minutes in the medium of Graham and Karnovsky ('66) for the demonstration of peroxidase activity. (Yamashina and Barka, '72). Following rinses in buffer and post-fixation in cacodylate-buffered 1% osmium tetroxide (pH 7.4) for 30 minutes the tissues were dehydrated in ethanol and embedded in Epon 812. One micron Epon sections were stained with toluidine blue for light microscopic observations. Thin sections, cut on an LKB Ultratome, were stained with uranyl acetate and lead citrate (Reynolds, ' 6 3 ) , and examined in a JEM lOOB electron microscope. 'H-thymidine incorporation. In a separate experiment submandibular gland rudiments were removed in pairs from 22 17-day old rate fetuses and cultured according to the method described above. The explants were allowed to grow undisturbed for 18 hours. On the morning of
day 2, tritiated thymidine, in a final concentration of 2 Ci/ml, ( Schwarz-Mann Bioresearch, specific activity 18 ,.&i/mrnole), was added to the first group of cultures (each group contained 4-6 explants). After 60 minutes these explants were fixed 30 minutes in half-strength Karnovsky's fixative. The cultures were rinsed in 0.1 M cacodylate buffer (pH 7.4) containing 7% sucrose, incubated for the demonstration of peroxidase activity as described above, dehydrated and embedded in Epon. This procedure was repeated for successive groups of cultures on days 3, 4, and 5. One micron Epon sections were exposed to Kodak NTB-2 emulsion for eight days, developed, (Kopriwa and Leblond, '62), and stained with toluidine blue. Counts were made on approximately 3,000 cells of the terminal tubules from each group. Connective tissue cells and cells of the developing sublingual gland were excluded from the counts. RESULTS
Day I . At the time of explanation, cultures from 17 day old fetuses consisted of a few cords of undifferentiated epithelial cells surrounded by loose mesenchymal tissue (fig. 1 ) . In some of the cords, the beginning of lumen formation could be observed. While pseudoperoxidase activity was present in red blood cells, at this stage no peroxidase activity was demonstrable in the epithelial cells. Day 2. After 24 hours in culture, an extensive branching of the terminal knobs had occurred (fig. 2). While some diversity in staining characteristics and nuclei could be observed, no specific secretory granules were yet present within the epithelial cells. Figure 3 shows the relative frequencies of the four cell types in the terminal tubules over the culture period. Figure 4 indicates the labelling index, (percent of labelled nuclei) for the total tubule cell population, By day 2, approximately one-third of the cells were labelled. Day 3 . By the equivalent of day 19 in utero, undifferentiated cells still predominated. In addition, two specific cell types could be distinguished in approximately equal numbers : terminal tubule cells, containing granules stained by toluidine blue; and proacinar cells, containing
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Fig. 1 Photomicrograph of 1 $L thick Epon-embedded section of 17-day old fetal r a t submandibular gland a t the time of explanation (day l ) , incubated for the demonstration of peroxidase activity, and stained with toluidine blue. The glandular rudiment consists of a few cords of undifferentiated epithelial cells embedded in mesenchyme. Lumen formation is occasionally seen as in the large cord in the lower part of the micrograph. Pseudoperoxidase activity is visible in red blood cells (arrow). x 400. Fig. 2 Photomicrograph of cultured 17-day old fetal gland after 24 hours in vitro (day 2 ) , treated as in figure 1. Branching of the terminal epithelial knobs has occurred. Prospective ductal ( D ) and secretory ( S ) units may frequently be discerned. Neither specific secretory granules nor peroxidase activity have yet appeared within the epithelial cells. x 400.
peroxidase-positive granules (fig. 3 ) . At the light microscopic level, peroxidase activity was also diffusely visible in the cytoplasm of some of the proacinar cells. Electron micrographs from this stage showed the presence of polyhedral cells of three types in the secretory tubules : undifferentiated cells devoid of secretory granules (figs. 5, 6 ) cells containing dense granules which did not react for peroxidase (figs. 6, 7)-terminal tubular cells; and cells containing peroxidase-positive secretory granules as well as peroxidase activity in the endoplasmic reticulum and nuclear membranes (fig. 7)-proacinar cells. In many proacinar cells the granules were not uniformly reactive for peroxidase, nor were they uniform in size. After 48 hours in culture, the proportion of cells incorporating 3H-thymidine had dropped to approximately 25% (fig. 4 ) .
All four cell types present in the explants were synthesizing DNA, as indicated in fig. 8 which depicts the labelling indices for the five days of culture. Day 4. At 72 hours, the architecture of the gland had become evident. Ducts could clearly be distinguished from the secretory units, and lumen formation was apparent throughout the explant. Terminal tubular cells and proacinar cells were abundant; in some cultures, a few acinar cells were seen (fig. 3 ) . By day 4, about 15% of all cells were incorporating 3H-thymidine (fig. 4). Each of the four cell types present continued to synthesize DNA; differences in proliferative activity among the various cell types did not appear to be significant (fig. 8). Day 5. After 96 hours in vitro, many portions of the gland contained acinar cells. At the light microscopic level these
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cells about 3 0 % , and acinar cells about 5% on the average, but with considerable differences from culture to culture, (fig. 3). On this last day of the culture period, about 12% of the total tubule cell population was incorporating 3H-thymidine (fig. 4 ) . There was a great variability in the proliferative activity among the different cell types by this time (fig. 8), a further reflection of the fact that the cultures did not develop uniformly through the fifth day. DISCUSSION
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Fig. 4 The labelling index for the total tubule cell population during a 5-day culture period. The labeling index is based on counts of 3,000 nuclei from daily intervals of 4-6 cultures each. Means and S.D. are shown.
cells could be distinguished by the staining properties of their secretory granules; these granules stained pink with toluidine blue, and revealed no peroxidase activity (fig. 9). Also present in the tubules at day 5 were undifferentiated cells, terminal tubular cells, proacinar cells, and some "transitional cells"-intermediate between proacinar and acinar forms (fig. 9). Transitional cells were characterized by secretory granules with a diminished peroxidase activity. By day 5, terminal tubular cells accounted for about 20% of the cells in the secretory endpieces of the gland; proacinar
Although there have been several light and electron microscopic investigations of the postnatal differentiation of the submandibular gland (Jacoby and Leeson, '59; Leeson and Jacoby, '59; Yohro, '70; Kim et aI., '70; Strum, '71; Yamashina and Barka, '72; Bressler, '73; Chang, '74, Cutler and Chaudhry, '74) relatively few studies have examined the prenatal development in vivo (DvoEak, '69; Yamashina and Barka, '73). While in vitro studies of fetal salivary gland have been more numerous (Borghese, '50; Grobstein, '53; Lawson, '70; Spooner and Wessels, '72; Ash et al., '73), these have concentrated on the histomorphogenesis of the gland rather than cytodifferentiation within the secretory units. In this study, a basic assumption has been made, that with respect to differentiation a day in vitro corresponds to a day in vivo. Thus, the period of culture beginning on day 17 in utero and continuing for five days would extend through the
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Fig. 5 Electron micrograph of a portion of a cultured 17-day old fetal rat submandibular gland after 48 hours in vitro (day 3 ) , incubated for the demonstration of peroxidase activity, stained with uranyl acetate and lead citrate. This field depicts a portion of a terminal tubule consisting of undifferentiated cells ( U ) , bordering a lumen (Lu). x 9,500.
date of birth, or twenty-first gestation day. There is a precedent for this assumption in other organ culture studies of exocrine glands (Parsa et al., '69; Lawson, ' 7 0 ) , and the present observations appear to warrant the supposition: in vitro differentiation of submandibular gland from day 17 in utero correlated well with in vivo observations of other investigators for the period from day 17 to day 21 of gestation. The chronologic age at which the terminal tubular cells, proacinar cells and acinar cells initially appeared corresponded closely to those recorded by Dvo?ak ('69) and Yamashina and Barka ('73); Dvo?ak described the presence of terminal tubular cells on day 19 of gestation, and of cells with polymorphic granules, or proacinar cells, on day 21. Yamashina and Barka observed the concurrent appearance of terminal tubular cells and peroxidase-containing proacinar cells on the eighteenth gestation day. Neither of these investigators re-
ported the occurrence of acinar cells before day 21. In our study, terminal tubular cells and proacinar cells were first seen on day 3 of culture, which corresponds to the nineteenth gestation day in vivo; on the following day the first acinar cells were seen in some cultures. The frequencies of terminal tubular, proacinar and acinar cells at day 5 of culture resembled those obtained by Chang ('74) for the 2day old animal. Since no data are available regarding the relative frequencies of these cell types in the newborn (twenty-first gestation day), speculation regarding precocity in some of the cultures is unwarranted; sequential appearance of cell types in culture, however, resembled closely, though not exactly, the in vivo timetable. The results shown in figure 4 indicate a gradual decline in the number of cells incorporating 3H-thymidine over the culture period. This decline may be attributed to a progressive loss of viability of
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Fig. 6 Electron micrograph of a portion of a cultured 17-day old fetal gland, treated as in figure 5. Low power view depicting several terminal tubular cells ( T ) and proacinar cells ( P ) . The heterogeneiy of granules in these cells is evident. Some undifferentiated cells are also present (U).x 5,700.
cells in the cultures; such a view is supported by results of other attempts to culture exocrine glands. McEvoy et al. ('73) have shown that in explants of fetal rat pancreas differentiation of the islets occurs at the expense of the exocrine portion, and that after a few days in vitro the cells of the exocrine pancreas cease to develop.
However, the labeling index of 12% obtained for the fourth day in vitro, corresponding to date of birth, compares favorably with the labelling index of 7.9% found by Chang ('74) for the overall cell population of the gland of the 2-day old animal. Data on the proliferative activity of the
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Fig. 7 Electron micrograph of a portion of a cultured 17-day old fetal gland, incubated and stained as in figure 6. Two proacinar cells ( P ) , containing many peroxidase-positive secretory granules are seen. The granules are heterogeneous with respect to size and the amount of reaction product for peroxidase. Reaction product is also visible in the rough endoplasmic reticulum of one of these cells. Also present are two terminal tubular cells ( T ) ; their granules and cytoplasm are devoid of peroxidase activity. At the periphery of the tubule, an undifferentiated cell is seen. x 8,000.
cells of the secretory units during days 2 through 5, (fig. 8), indicate that all four cell types were dividing throughout the culture period. This is expected in view of the fact that these are precursor cells, undergoing transformation. The relationships among the various cell types during the perinatal period in vivo have been studied quantitatively (Chang, '74) and morphologically (Dvofak, '69; Strum, '71; Yamashina and Barka, '72). The results of these studies indicated that the earliest undifferentiated cells give rise to terminal tubular and proacinar cells; proacinar cells in turn differentiate to form acinar cells, and the latter continue to divide as the gland grows. Whereas the absolute number of proacinar cells decreases during the first two weeks of postnatal life, the absolute number of terminal tubular cells increases;
these latter cells are believed to contribute to the duct system and perhaps to the acinar cell populaion of the mature gland. Many factors in the present study prohibit further speculation on the relationships of these cell types: the brevity of the culture period; the lack of uniformity among the cultures; the small number of cultures examined. Figures 3 and 9 do indicate, however, that events occurring in vitro resemble those reported in vivo: the timing and sequence of appearance of the various cell types, and the mitotic behaviour of these cell types during the culture period, are in accordance with information obtained in vivo. The appearance of peroxidase-containing proacinar cells in particular, and their gradual transformation into acinar cells, support the observations of Yamashina and Barka ('73).
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Fig, 8 The labelling indices of individual epithelial cell types in the terminal tubules during a 5-day culture period. At least 2,000 nuclei were counted from each of 4-6 cultures. Means and S.D. are shown.
Fig. 9 Photomicrograph of a cultured 17-day old fetal gland after 96 hours in vitro (day 5 ) , treated as in figure 1. Terminal tubule cells ( T ) , proacinar cells (P), and acinar cells (A) may be identified by the presence of specific granules. A n occasional transitional cell, characterized by granules partially reactive for peroxidase (arrowhead), may be observed. x 1,600.
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a new technique. J. Histochem. Cytochem., 14: 291303. Grobstein, C. 1953 Analysis in vitro of the early organization of the rudiment of the mouse subma;idibular gland. J. Morph., 93: 19-44. Jacoby, F., and C. R. Leeson 1959 The postnatal development of the rat submaxillary gland. J. Anat., 93: 201-216. Karnovsky, M. J. 1965 A formaldehyde-glutaraldehyde fixative of high osmolality for use i n electron microscopy. J. Cell Biol., 27: 137A138A. Kim, S. K., S. S. Han and C. E. Nasjleti 1970 The fine structure of secretory granules in subACKNOWLEDGMENTS mandibular glands of the rat during early postnatal development. Anat. Rec., 168:463The authors thank Drs, R. S. Bressler 476. and W. W. L. Chang for critical assistance Kopriwa, B. M., and C. P. Leblond 1962 Imin preparation of the manuscript, and Mrs. provements in the coating technique of radioautography. J. Histochem. Cytochem., 10: 269Tana Ross for technical assistance. 284. This investigation was supported by U.S. K. A. 1970 Morphogenesis and funcPublic Health Service Research Grants Lawson, tional differentiation of the rat parotid gland CA17038 and CA11155 from the National in vivo and in vitro. J. Embryol. Exp. Morph., Cancer Institute. 24: 411-424. Leeson, C. R., and F. Jacoby 1959 An electronmicroscopic study of the r a t submaxillary gland LITERATURE CITED during its postnatal development and in the Ash, J. F., B. S. Spooner and N. K. Wessels 1973 adult. J. Anat., 93: 287-295. Effects of papaverine and calcium-free medium McEvoy, R. C., 0. D. Hegre, R. J. Leonard and on salivary gland morphogenesis. Dev. Biol., A. Lazarow 1973 Fetal r a t pancreas: differ33: 463469. entiation of the acinar cell component in vivo Borghese, E. 1950 The development in vitro of and in vitro. Diabetes, 22: 584-589. the submandibular and sublingual glands of Parsa, I., W. H. Marsh and P. J. Fitzgerald 1969 Mus musculus. J. Anat., 84: 287-302. Pancreas acinar cell differentiation I. MorphoBressler, R. S. 1973 Fine structure of the diflogic and enzymatic comparisons of embryonic ferentiating acini in submandibular glands of rat pancreas and pancreatic anlage grown in isoproterenol-treated rats. Am. J. Anat., 138: organ culture. Am. J. Pathol., 57: 457-469. 431-447. Reynolds, E. S. 1963 The use of lead citrate at Chang, W. W. L. 1974 Cell population changes high pH as a n electron-opaque stain i n electron during acinus formation in the postnatal rat microscopy. J. Cell Biol., 17: 208-212. submandibular gland. Anat. Rec., 178: 187- Spooner, B. S., and N. K. Wessels 1972 A n 201. analysis of salivary gland morphogenesis: role Cutler, L. S., and A. P. Chaudhry 1973 Interof cytoplasmic microfilaments and microtubcellular contacts a t the epithelial-mesenchyules. Dev. Biol., 27: 38-54. mal interface during the prenatal development Strum, J. M. 1971 Unusual peroxidase-positive of the rat submandibulir gland. Dev: Biol., granules in the developing rat submaxillary 33: 229-240. gland. J. Cell Biol., 51: 575-579. Dvofak, M. 1969 The secretory cells of the Yamashina, S., and T. Barka 1972 Localization submaxillary gland in the perinatal period of of peroxidase activity in the developing subdevelopment in the rat. 2. Zellforsch., 99: mandibular gland of normal and isoproterenol346-356. treated rats. J. Histochem. Cytochem., 20: 855Fukuda, M. 1967 Histochemical studies on the 872. rat submaxillary gland during postnatal devel1973 Development of endogenous peropment. Histochemie, 8: 342-354. oxidase in fetal rat submandibular gland. J. Histochem. Cytochem., 21: 42-50. Graham, C. J., and M. J. Karnovsky 1966 The early stages of absorption of injected horse Yohro, T., 1970 Development of secretory units of mouse submandibular gland. Z. Zellforsch., radish peroxidase in the proximal tubules of 110: 173-184. mouse kidney: ultrastructural cytochemistry by
In conclusion, it appears that organ culture is a useful technique in the short-term study of cytodifferentiation of the secretory unit of fetal rat submandibular gland. The pattern of differentiation in these cultures resembles the in vivo situation, and thus provides a convenient system for studies of drug and hormonal effects on the fetal gland, studies which might prove difficult or impossible to perform in vivo.
ABSTRACT
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. I n 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 P 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 perinatal cytodifferentiation of the terminal tubule of rodent submandibular gland has been the subject of many finestructural, histochemical, and quantitative studies in the past few years (Fukuda, '67; Dvofak, '69; Yohro, '70; Kim et al., '70; Strum, '71; Yamashina and Barka, '72, '73; Bressler, '73; Chang, '74; Cutler and Chaudhry, '74). To summarize briefly, "terminal tubule" is the term introduced by Jacoby and Leeson ('59) to refer to the immature secretory unit of rat submandibular gland through the first six weeks of postnatal life. A few days before birth two separate cell types arise from undifferentiated precursor cells in the terminal tubules; these are known respectively as terminal tubular cells and cells with polymorphic granules (Dvozak, '69) or proacinar cells (Yamashina and Barka, '73). Peroxidase activity has been localized in the cisternae of the rough endoplasmic reticulum and nuclear envelope and in the secretory granules of proacinar cells in the glands of fetal and newborn rats. Terminal ANAT. REC., 184: 301-310.
tubular cells, on the other hand, have not been shown to contain peroxidase (Strum, '71; Yamashina and Barka, '72, '73). For this reason, the cells with polymorphic granules are believed to represent direct precursors of acinar cells. As the acinar cells mature the composition and ultrastructure of the secretory granules are altered, The secretory granules become electrolucent and flocculent with a great reduction in peroxidase activity that is demonstrable electron histochemically. However, a strong peroxidase activity in the endoplasmic reticulum and nuclear envelope characterizes the mature acinar cells (Yamashina and Barka, '72). The present study was undertaken to compare the differentiation of submandibular gland in organ culture with development as known in vivo. Using peroxidase activity as a marker for proacinar cell differentiation, the objective of this study has been to define a system in which to test the effects of drugs and hormones on Received May 21, '75.Accepted July 29, '75.
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the differentiation of the fetal gland in vitro. MATEHIALS AND METHODS
The culture medium employed was a modified version of that used by Parsa et al. ('69), consisting of 87% Eagle's Minimum Essential Medium, 10% horse serum, and 3% chick embryo extract to which 0.01 M HEPES buffer and 1% AntibioticAntimycotic Solution (GIBCO) were added. The initial pH of the medium was 7.2-7.4. Morphological observations. Submandibular glands were removed from Sprague-Dawley rat fetuses ranging in age from 16 to 19 days, and carefully trimmed of extraneous connective tissue. Individual glands were transferred in pairs to Falcon plastic organ culture dishes, and positioned on a strip of lens paper (Ross Optical) supported by a wire mesh grid. The wells of the culture dishes contained medium sufficient to moisten the lens paper strip. Cultures were maintained for variable periods ranging from two to seven days, in an atmosphere of 5% COz in air at 37°C and 95% humidity. The medium was changed every 48 hours. At daily intervals, several explants were fyred 30 minutes in half-strength Karnovsky's fixative ('65), rinsed in 0.1 M cacodylate buffer (pH 7.4) containing 7% sucrose and incubated 60 minutes in the medium of Graham and Karnovsky ('66) for the demonstration of peroxidase activity. (Yamashina and Barka, '72). Following rinses in buffer and post-fixation in cacodylate-buffered 1% osmium tetroxide (pH 7.4) for 30 minutes the tissues were dehydrated in ethanol and embedded in Epon 812. One micron Epon sections were stained with toluidine blue for light microscopic observations. Thin sections, cut on an LKB Ultratome, were stained with uranyl acetate and lead citrate (Reynolds, ' 6 3 ) , and examined in a JEM lOOB electron microscope. 'H-thymidine incorporation. In a separate experiment submandibular gland rudiments were removed in pairs from 22 17-day old rate fetuses and cultured according to the method described above. The explants were allowed to grow undisturbed for 18 hours. On the morning of
day 2, tritiated thymidine, in a final concentration of 2 Ci/ml, ( Schwarz-Mann Bioresearch, specific activity 18 ,.&i/mrnole), was added to the first group of cultures (each group contained 4-6 explants). After 60 minutes these explants were fixed 30 minutes in half-strength Karnovsky's fixative. The cultures were rinsed in 0.1 M cacodylate buffer (pH 7.4) containing 7% sucrose, incubated for the demonstration of peroxidase activity as described above, dehydrated and embedded in Epon. This procedure was repeated for successive groups of cultures on days 3, 4, and 5. One micron Epon sections were exposed to Kodak NTB-2 emulsion for eight days, developed, (Kopriwa and Leblond, '62), and stained with toluidine blue. Counts were made on approximately 3,000 cells of the terminal tubules from each group. Connective tissue cells and cells of the developing sublingual gland were excluded from the counts. RESULTS
Day I . At the time of explanation, cultures from 17 day old fetuses consisted of a few cords of undifferentiated epithelial cells surrounded by loose mesenchymal tissue (fig. 1 ) . In some of the cords, the beginning of lumen formation could be observed. While pseudoperoxidase activity was present in red blood cells, at this stage no peroxidase activity was demonstrable in the epithelial cells. Day 2. After 24 hours in culture, an extensive branching of the terminal knobs had occurred (fig. 2). While some diversity in staining characteristics and nuclei could be observed, no specific secretory granules were yet present within the epithelial cells. Figure 3 shows the relative frequencies of the four cell types in the terminal tubules over the culture period. Figure 4 indicates the labelling index, (percent of labelled nuclei) for the total tubule cell population, By day 2, approximately one-third of the cells were labelled. Day 3 . By the equivalent of day 19 in utero, undifferentiated cells still predominated. In addition, two specific cell types could be distinguished in approximately equal numbers : terminal tubule cells, containing granules stained by toluidine blue; and proacinar cells, containing
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Fig. 1 Photomicrograph of 1 $L thick Epon-embedded section of 17-day old fetal r a t submandibular gland a t the time of explanation (day l ) , incubated for the demonstration of peroxidase activity, and stained with toluidine blue. The glandular rudiment consists of a few cords of undifferentiated epithelial cells embedded in mesenchyme. Lumen formation is occasionally seen as in the large cord in the lower part of the micrograph. Pseudoperoxidase activity is visible in red blood cells (arrow). x 400. Fig. 2 Photomicrograph of cultured 17-day old fetal gland after 24 hours in vitro (day 2 ) , treated as in figure 1. Branching of the terminal epithelial knobs has occurred. Prospective ductal ( D ) and secretory ( S ) units may frequently be discerned. Neither specific secretory granules nor peroxidase activity have yet appeared within the epithelial cells. x 400.
peroxidase-positive granules (fig. 3 ) . At the light microscopic level, peroxidase activity was also diffusely visible in the cytoplasm of some of the proacinar cells. Electron micrographs from this stage showed the presence of polyhedral cells of three types in the secretory tubules : undifferentiated cells devoid of secretory granules (figs. 5, 6 ) cells containing dense granules which did not react for peroxidase (figs. 6, 7)-terminal tubular cells; and cells containing peroxidase-positive secretory granules as well as peroxidase activity in the endoplasmic reticulum and nuclear membranes (fig. 7)-proacinar cells. In many proacinar cells the granules were not uniformly reactive for peroxidase, nor were they uniform in size. After 48 hours in culture, the proportion of cells incorporating 3H-thymidine had dropped to approximately 25% (fig. 4 ) .
All four cell types present in the explants were synthesizing DNA, as indicated in fig. 8 which depicts the labelling indices for the five days of culture. Day 4. At 72 hours, the architecture of the gland had become evident. Ducts could clearly be distinguished from the secretory units, and lumen formation was apparent throughout the explant. Terminal tubular cells and proacinar cells were abundant; in some cultures, a few acinar cells were seen (fig. 3 ) . By day 4, about 15% of all cells were incorporating 3H-thymidine (fig. 4). Each of the four cell types present continued to synthesize DNA; differences in proliferative activity among the various cell types did not appear to be significant (fig. 8). Day 5. After 96 hours in vitro, many portions of the gland contained acinar cells. At the light microscopic level these
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cells about 3 0 % , and acinar cells about 5% on the average, but with considerable differences from culture to culture, (fig. 3). On this last day of the culture period, about 12% of the total tubule cell population was incorporating 3H-thymidine (fig. 4 ) . There was a great variability in the proliferative activity among the different cell types by this time (fig. 8), a further reflection of the fact that the cultures did not develop uniformly through the fifth day. DISCUSSION
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Fig. 4 The labelling index for the total tubule cell population during a 5-day culture period. The labeling index is based on counts of 3,000 nuclei from daily intervals of 4-6 cultures each. Means and S.D. are shown.
cells could be distinguished by the staining properties of their secretory granules; these granules stained pink with toluidine blue, and revealed no peroxidase activity (fig. 9). Also present in the tubules at day 5 were undifferentiated cells, terminal tubular cells, proacinar cells, and some "transitional cells"-intermediate between proacinar and acinar forms (fig. 9). Transitional cells were characterized by secretory granules with a diminished peroxidase activity. By day 5, terminal tubular cells accounted for about 20% of the cells in the secretory endpieces of the gland; proacinar
Although there have been several light and electron microscopic investigations of the postnatal differentiation of the submandibular gland (Jacoby and Leeson, '59; Leeson and Jacoby, '59; Yohro, '70; Kim et aI., '70; Strum, '71; Yamashina and Barka, '72; Bressler, '73; Chang, '74, Cutler and Chaudhry, '74) relatively few studies have examined the prenatal development in vivo (DvoEak, '69; Yamashina and Barka, '73). While in vitro studies of fetal salivary gland have been more numerous (Borghese, '50; Grobstein, '53; Lawson, '70; Spooner and Wessels, '72; Ash et al., '73), these have concentrated on the histomorphogenesis of the gland rather than cytodifferentiation within the secretory units. In this study, a basic assumption has been made, that with respect to differentiation a day in vitro corresponds to a day in vivo. Thus, the period of culture beginning on day 17 in utero and continuing for five days would extend through the
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Fig. 5 Electron micrograph of a portion of a cultured 17-day old fetal rat submandibular gland after 48 hours in vitro (day 3 ) , incubated for the demonstration of peroxidase activity, stained with uranyl acetate and lead citrate. This field depicts a portion of a terminal tubule consisting of undifferentiated cells ( U ) , bordering a lumen (Lu). x 9,500.
date of birth, or twenty-first gestation day. There is a precedent for this assumption in other organ culture studies of exocrine glands (Parsa et al., '69; Lawson, ' 7 0 ) , and the present observations appear to warrant the supposition: in vitro differentiation of submandibular gland from day 17 in utero correlated well with in vivo observations of other investigators for the period from day 17 to day 21 of gestation. The chronologic age at which the terminal tubular cells, proacinar cells and acinar cells initially appeared corresponded closely to those recorded by Dvo?ak ('69) and Yamashina and Barka ('73); Dvo?ak described the presence of terminal tubular cells on day 19 of gestation, and of cells with polymorphic granules, or proacinar cells, on day 21. Yamashina and Barka observed the concurrent appearance of terminal tubular cells and peroxidase-containing proacinar cells on the eighteenth gestation day. Neither of these investigators re-
ported the occurrence of acinar cells before day 21. In our study, terminal tubular cells and proacinar cells were first seen on day 3 of culture, which corresponds to the nineteenth gestation day in vivo; on the following day the first acinar cells were seen in some cultures. The frequencies of terminal tubular, proacinar and acinar cells at day 5 of culture resembled those obtained by Chang ('74) for the 2day old animal. Since no data are available regarding the relative frequencies of these cell types in the newborn (twenty-first gestation day), speculation regarding precocity in some of the cultures is unwarranted; sequential appearance of cell types in culture, however, resembled closely, though not exactly, the in vivo timetable. The results shown in figure 4 indicate a gradual decline in the number of cells incorporating 3H-thymidine over the culture period. This decline may be attributed to a progressive loss of viability of
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Fig. 6 Electron micrograph of a portion of a cultured 17-day old fetal gland, treated as in figure 5. Low power view depicting several terminal tubular cells ( T ) and proacinar cells ( P ) . The heterogeneiy of granules in these cells is evident. Some undifferentiated cells are also present (U).x 5,700.
cells in the cultures; such a view is supported by results of other attempts to culture exocrine glands. McEvoy et al. ('73) have shown that in explants of fetal rat pancreas differentiation of the islets occurs at the expense of the exocrine portion, and that after a few days in vitro the cells of the exocrine pancreas cease to develop.
However, the labeling index of 12% obtained for the fourth day in vitro, corresponding to date of birth, compares favorably with the labelling index of 7.9% found by Chang ('74) for the overall cell population of the gland of the 2-day old animal. Data on the proliferative activity of the
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Fig. 7 Electron micrograph of a portion of a cultured 17-day old fetal gland, incubated and stained as in figure 6. Two proacinar cells ( P ) , containing many peroxidase-positive secretory granules are seen. The granules are heterogeneous with respect to size and the amount of reaction product for peroxidase. Reaction product is also visible in the rough endoplasmic reticulum of one of these cells. Also present are two terminal tubular cells ( T ) ; their granules and cytoplasm are devoid of peroxidase activity. At the periphery of the tubule, an undifferentiated cell is seen. x 8,000.
cells of the secretory units during days 2 through 5, (fig. 8), indicate that all four cell types were dividing throughout the culture period. This is expected in view of the fact that these are precursor cells, undergoing transformation. The relationships among the various cell types during the perinatal period in vivo have been studied quantitatively (Chang, '74) and morphologically (Dvofak, '69; Strum, '71; Yamashina and Barka, '72). The results of these studies indicated that the earliest undifferentiated cells give rise to terminal tubular and proacinar cells; proacinar cells in turn differentiate to form acinar cells, and the latter continue to divide as the gland grows. Whereas the absolute number of proacinar cells decreases during the first two weeks of postnatal life, the absolute number of terminal tubular cells increases;
these latter cells are believed to contribute to the duct system and perhaps to the acinar cell populaion of the mature gland. Many factors in the present study prohibit further speculation on the relationships of these cell types: the brevity of the culture period; the lack of uniformity among the cultures; the small number of cultures examined. Figures 3 and 9 do indicate, however, that events occurring in vitro resemble those reported in vivo: the timing and sequence of appearance of the various cell types, and the mitotic behaviour of these cell types during the culture period, are in accordance with information obtained in vivo. The appearance of peroxidase-containing proacinar cells in particular, and their gradual transformation into acinar cells, support the observations of Yamashina and Barka ('73).
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Undifferentiated cells Terminal tubule cells Proacinar cells Acinar cells
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Fig, 8 The labelling indices of individual epithelial cell types in the terminal tubules during a 5-day culture period. At least 2,000 nuclei were counted from each of 4-6 cultures. Means and S.D. are shown.
Fig. 9 Photomicrograph of a cultured 17-day old fetal gland after 96 hours in vitro (day 5 ) , treated as in figure 1. Terminal tubule cells ( T ) , proacinar cells (P), and acinar cells (A) may be identified by the presence of specific granules. A n occasional transitional cell, characterized by granules partially reactive for peroxidase (arrowhead), may be observed. x 1,600.
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a new technique. J. Histochem. Cytochem., 14: 291303. Grobstein, C. 1953 Analysis in vitro of the early organization of the rudiment of the mouse subma;idibular gland. J. Morph., 93: 19-44. Jacoby, F., and C. R. Leeson 1959 The postnatal development of the rat submaxillary gland. J. Anat., 93: 201-216. Karnovsky, M. J. 1965 A formaldehyde-glutaraldehyde fixative of high osmolality for use i n electron microscopy. J. Cell Biol., 27: 137A138A. Kim, S. K., S. S. Han and C. E. Nasjleti 1970 The fine structure of secretory granules in subACKNOWLEDGMENTS mandibular glands of the rat during early postnatal development. Anat. Rec., 168:463The authors thank Drs, R. S. Bressler 476. and W. W. L. Chang for critical assistance Kopriwa, B. M., and C. P. Leblond 1962 Imin preparation of the manuscript, and Mrs. provements in the coating technique of radioautography. J. Histochem. Cytochem., 10: 269Tana Ross for technical assistance. 284. This investigation was supported by U.S. K. A. 1970 Morphogenesis and funcPublic Health Service Research Grants Lawson, tional differentiation of the rat parotid gland CA17038 and CA11155 from the National in vivo and in vitro. J. Embryol. Exp. Morph., Cancer Institute. 24: 411-424. Leeson, C. R., and F. Jacoby 1959 An electronmicroscopic study of the r a t submaxillary gland LITERATURE CITED during its postnatal development and in the Ash, J. F., B. S. Spooner and N. K. Wessels 1973 adult. J. Anat., 93: 287-295. Effects of papaverine and calcium-free medium McEvoy, R. C., 0. D. Hegre, R. J. Leonard and on salivary gland morphogenesis. Dev. Biol., A. Lazarow 1973 Fetal r a t pancreas: differ33: 463469. entiation of the acinar cell component in vivo Borghese, E. 1950 The development in vitro of and in vitro. Diabetes, 22: 584-589. the submandibular and sublingual glands of Parsa, I., W. H. Marsh and P. J. Fitzgerald 1969 Mus musculus. J. Anat., 84: 287-302. Pancreas acinar cell differentiation I. MorphoBressler, R. S. 1973 Fine structure of the diflogic and enzymatic comparisons of embryonic ferentiating acini in submandibular glands of rat pancreas and pancreatic anlage grown in isoproterenol-treated rats. Am. J. Anat., 138: organ culture. Am. J. Pathol., 57: 457-469. 431-447. Reynolds, E. S. 1963 The use of lead citrate at Chang, W. W. L. 1974 Cell population changes high pH as a n electron-opaque stain i n electron during acinus formation in the postnatal rat microscopy. J. Cell Biol., 17: 208-212. submandibular gland. Anat. Rec., 178: 187- Spooner, B. S., and N. K. Wessels 1972 A n 201. analysis of salivary gland morphogenesis: role Cutler, L. S., and A. P. Chaudhry 1973 Interof cytoplasmic microfilaments and microtubcellular contacts a t the epithelial-mesenchyules. Dev. Biol., 27: 38-54. mal interface during the prenatal development Strum, J. M. 1971 Unusual peroxidase-positive of the rat submandibulir gland. Dev: Biol., granules in the developing rat submaxillary 33: 229-240. gland. J. Cell Biol., 51: 575-579. Dvofak, M. 1969 The secretory cells of the Yamashina, S., and T. Barka 1972 Localization submaxillary gland in the perinatal period of of peroxidase activity in the developing subdevelopment in the rat. 2. Zellforsch., 99: mandibular gland of normal and isoproterenol346-356. treated rats. J. Histochem. Cytochem., 20: 855Fukuda, M. 1967 Histochemical studies on the 872. rat submaxillary gland during postnatal devel1973 Development of endogenous peropment. Histochemie, 8: 342-354. oxidase in fetal rat submandibular gland. J. Histochem. Cytochem., 21: 42-50. Graham, C. J., and M. J. Karnovsky 1966 The early stages of absorption of injected horse Yohro, T., 1970 Development of secretory units of mouse submandibular gland. Z. Zellforsch., radish peroxidase in the proximal tubules of 110: 173-184. mouse kidney: ultrastructural cytochemistry by
In conclusion, it appears that organ culture is a useful technique in the short-term study of cytodifferentiation of the secretory unit of fetal rat submandibular gland. The pattern of differentiation in these cultures resembles the in vivo situation, and thus provides a convenient system for studies of drug and hormonal effects on the fetal gland, studies which might prove difficult or impossible to perform in vivo.
