- Email: [email protected]
Changes in nucleic acid and protein content during development of the rat submandibular gland
Archs
oral
Bid. Vol. 18,pp. 1325-
1336,
1973.Pergamon Press.Printedin GreatBritain.
CHANGES IN NUCLEIC ACID AND PROTEIN CONTENT DURING DEVELOPMENT OF THE RAT SUBMANDIBULAR GLAND L. MENAKER* Department
and S. A. MILLER
of Nutrition and Food Science, Massachusetts Institute Cambridge, Massachusetts 02139, U.S.A.
of Technology,
Summary-The DNA, RNA and protein content of the submandibular gland during early development has been investigated and correlated with histological changes seen during that time. The sequential appearance of DNA and RNA concentration peaks at days 17 and 19 respectively has been demonstrated. Three separate periods of protein accumulation were shown to be associated with the changing cellular populations in the gland during development. INTRODUCTION STUDIES of growth
and development have progressed from those concerned mainly with the description of physical parameters to more recent studies investigating the biochemical basis responsible for these outward changes. Many workers (ENESCO and LEBLOND, 1962 ; WINICK and NOBLE, 1965 ; MILLER, 1969) realized that development involved the adaptation of the essentially parasitic foetus to the environmental demands of the free-living organism. This adaptive process is, to a great extent, associated with the onset of regulatory processes controlled through enzymatic reactions. The most basic mechanisms underlying growth and development must, therefore, be associated with the synthesis of enzymes specifically, and the synthesis of proteins generally. It must be concluded that investigation of the biochemistry of protein synthesis during the neonatal period is essential to the understanding of growth and development of the organism. The present set of experiments, part of a larger study of the development of protein synthesis, was designed to determine the interrelationships between the cellular components most intimately involved in protein synthesis (RNA, DNA and protein) in a specific organ system, the rat submandibular gland (SMG). MATERIALS
AND
METHODS
Animals
Timed-pregnant, Sprague-Dawley (CD) rats (Charles River Strain) were obtained 5 days postconception (Charles River Breeding Laboratories, Wilmington, Mass.). Pregnant rats were housed in plastic breeding tubs containing sterilized wood shavings and given both tap water and a standard rat-mouse chow ad libitum. Pups were selected for randomization from litters born within a specified 4-hr time period. Lights were kept on a 12-hr on-off cycle by an automatic timer. Temperature was maintained at 70” i 3°F and 50 & 10 per cent relative humidity. Animals were weaned at 21 days after birth and transferred to plastic tubs containing a maximum of 4 pups. * Present address: University of Alabama Medical Center, School of Dentistry, 1919 Seventh Ave. S., Birmingham, Alabama 35233, U.S.A. 1325
L. MENAKERAND
1326
S. A. MILLER
Materials Indole and phenol reagents were purchased from Fisher Scientific Company (Fair Lawn, N.J.). Bovine serum albumin (crystallized) and calf thymus deoxyribonucleic acid were products of General Biochemicals (Chagrin Falls, Ohio). All other chemicals used were reagent grade. Methods Animals were decapitated. The paired SMG were quickly excised, placed on dry ice and immediately transfered to a - 40°C freezer. After a sufficient number of glands were collected, they were pooled and analysed for RNA, DNA and protein. RNA content of the SMG was determined by the procedure of SCHMIDTand THANNHAUSER (1945) as modified by HUTCHISONand MUNRO (1961) and MUNRO and FLECK (1966). DNA content of the SMG was estimated using the procedure of CERRIOTTI(1952). Tissue protein was measured by the method of LOWRYet al. (1951). For light microscopy, tissue was fixed in 10 per cent buffered formalin. Samples approximately 2 mm in thickness were washed and dehydrated by routine procedures, embedded in “Paraplast” blocks and cut to 6 pm in thickness. After 24 hr, the sections were stained with haematoxylin and eosin and viewed under the light microscope. Samples were prepared every day from 3 days before to 24 days after birth, in addition to days 27, 35 and 50. EXPERIMENTAL
RESULTS
Weight measurements As a measure of the normality of the experimental population, body weight was monitored throughout each study, and compared to data gathered from previous studies of the rat in our laboratory. Birth weights of approximately 5 *5 g and weaning o-5or 0.45 0.40 Y 0 $ $ $ 2 d
0,350.30 0.25 -
Days
of total body weight. Each point gland grows at a rate relatively faster than the whole rat from -2 to 5 days of age. Growth is more in line with the average for the entire animal until approximately day 27, and thereafter represents a decreasing proportion of total weight. FIG. la. The submandibular
gland as a percentage
represents at least 16 gland pairs. The submandibular
NUCLEIC
ACIDS AND
PROTEIN
1327
IN RAT SMO DEVELOPMENT
200 180 c E” 160< E ‘E f
-z 0 3
140IZO-
6O60 -
I
I
I
I
I
I
I
Birth
Age, FIG.
days
lb. Total submandibular gland weight from three days before birth to day 50 postpartum. Each point represents at least 32 glands.
(21 days old) weights of about 45-50 g were considered consistent with our past experience. SMG were collected and weighed from 3 days before birth to day 50 post-partum. Figure la shows the percentage of total body weight which the gland represents. Figure lb represents the total weight of the gland per animal over this period. As can be seen, SMG increased in weight at a rate greater than that for the whole body at a time just before birth and for approximately 5 days after. From about day 27 on, the gland represented a constantly decreasing proportion of total body weight. RNA content Tissue RNA was estimated from 2 days prior to parturition up to day 50 after birth. As expected, total RNA content of the gland constantly increased during the neonatal period reaching approximately 1.60 mg/gland at day 50. However, as seen in Fig. 2a, the rate of increase of RNA concentration (mg/g wet weight of tissue) was not a linear function. The concentration of RNA varied in such a way as to result in relative peaks at specific times during development. Specific peaks were seen at 5, 12 and 19 days of age. Figure 2b shows the amount of RNA per cell (RNA/DNA) during this same period. Just before and just after birth there is a spurt in RNA synthesis per cell. After day 34, acinar cell proliferation began and the relative content of RNA diminished. However, synthesis of RNA leads to an accumulation of this material in ever increasing relative amounts so that by day 35 the RNA/DNA ratio reached 1.5. DNA content Tissue DNA was determined for samples from 2 days before birth through 50 days post-partum. As was true with total gland RNA, DNA constantly increased over this
L. MENAKER
1328
3
II -21
AND S. A. MILLER
I 20
I 5 Time,
I 30
days
FIG. 2a. The major peak in RNA concentration
in the developing rat submandibular gland occurs around day 19. “Step-peaks” lead up to and follow this main peak. Each point represents duplicate homogenates of at least 8 glands. Numbers in parentheses represent days of age. 225 t 200 =
;:;,?,--"1..
: 0.25 1 II I 13 135
I 15
I IO Age,
I 20
I 25
I 30
I 35
days
FIG. 2b. Ratio of RNA to DNA from three days before birth to 35 days post-partm.
RNA content rapidly increases just prior to, and just after, birth. From day 7 onward, RNA accumulation constantly increases as cell division slows.
period, reaching a maximum of 1.25 mg/gland at day 50. Again, when plotted per gram of tissue, specific peaks in concentration appeared. These occurred at days 4, 9 and 17 (see Fig. 3). Protein content The content of protein of the SMG was measured from -3 days until +50 days. A sequential representation of the total protein per gland is shown in Fig. 4. Total
NUCLEIC
ACIDS
AND
PROl’fiIN
IN RAT
Time,
SMG
1329
DEVELOPMENT
days
FIG.
3. The major peak in DNA concentration in the developing rat submandibular gland occurs around day 17. “Step-peaks” lead up to and follow this main peak. Each point represents duplicate homogenates of at least 8 glands. Numbers in parentheses represent days of age.
protein seems to be associated with 3 distinct periods of [accretion: day -3 to 8, day 9 to 20, and day 21 to 50. Each segment fits the mathematical form of an exponential function with the equation y = aebx where a and b are constants, x is the age in days and y is the total protein per gland. The linear regression and resulting 64 r
-3-It2
4
6
IO
1
I5
20
Age,
days
I
25
I
28
35I”
- 501
Birth
FZG. 4. Total glandular protein showed three different rates of accretion during development: days -3 to 8,9 to 20 and 21 to 50. An approximation of these rates is shown by the solid line.
1330
L. MENAKER AND S. A. MILLER
FIG. 5. Semi-log plot of the total protein content of the submandibular gland vs. age with curve fitting by linear regression. Total protein data was fit to the exponential function y = a~?, where a and b are constants, y is the total protein in mg, and x is the age in days. Linear regression by the method of least squares resulted in estimates of the fit (correlation coefficient v) for each period.
correlation coefficients (Fig. 5) support the idea of 3 separate periods of protein accretion, each fitting an exponential curve differing only in slope and point of origin. The existence of 3 separate periods of protein accumulation is further supported by a plot of the amount of protein estimated per cell (Fig. 6). Developmental changes in the SMG again seem to be associated with 3 distinct periods in the neonate: days -3 to 8, days 9 to 20 and days 21 to 50.
t1 Birth
FIG. 6. Protein concentration per cell in the used as an index of tissue cellularity. Protein development, days -3 to 8, 9 to 20 and 21 cates of a minimum
rat submandibular gland. Total DNA was content per DNA displayed three peaks in to 50. Each point represents at least dupliof 8 pooled glands.
NUCLEIC
ACIDS
AND
PROTEIN
IN RAT
SMG DEVELOPMENT
1331
Light microscopy In general, our observations of the SMG support the findings reported by JACOBY and LEESON (1959). We are in even more agreement with DVORAK (1969), who looked at the perinatal histology of the gland during the last 3 days of gestation and the first 6 days after birth. Briefly, at -3 days of age, the rat submandibular gland is made up of approximately 75 per cent parenchymal and 25 per cent stromal cells, Of the parenchymal population, about 90 per cent are terminal tubules and the remainder intralobular ducts. The terminal tubule represents the precursor of the acinar cell and at -3 days displayed a dark staining, basally located nucleus (see Fig. 7a). The terminal tubule consisted of about 6 cells surrounding a large central lumen. The intralobular duct (see Fig. 7a) was composed of lighter staining columnar cells with a centrally located nucleus. These cells were relatively similar in appearance to the cells comprising the duct of the adult (see Fig. 7f). At days 1-2, there appeared to occur a narrowing of the central lumen of the terminal tubule coincidental to a condensation, or shrinking in size, of the entire terminal tubule (see Fig. 7b). As early as day 3, the terminal buds started appearing as outcroppings from the terminal tubules (see Fig. 7~). By days 5-6 the terminal buds were becoming more numerous while the terminal tubules from which they evolved seemed to have reached their peak concentration (see Fig. 7d). The difference between terminal bud and acinar cell depended solely on the subjective interpretation of the observer. Perhaps, in the future, histochemical procedures will permit more accurate identification of the transition. Acinar cells continued to increase, and terminal tubules to decrease, so that, by days 18-21, acinar cells were by far the most dominant cell type and had reached very nearly the numbers (see Fig. 7e) present at day 50 (see Fig. 7f). The proliferation of ductal tissue started about the third week and by the fourth week became even more apparent. This trend continued up to the last day sampled (day 50). DISCUSSION Since the animals used in the experiments were bred in a commercial laboratory, precise control over gestation time was possible only within rrtl2 ,hr. However, a 4-hr time interval (f2 hr) was set for the acceptance of a litter into the experiment; this interval may still have contributed to some of the variability observed in the determination of nucleic acids and protein. However, an investigation of those biochemical components intimately involved with protein synthesis did reveal certain basic interrelationships. The appearance of one major peak in DNA (day 17) and one for RNA (day 19) with “step-peaks” around these maxima supports the sequential appearance of cellular concentrations of DNA and RNA as previously reported by MILLER (1970) for other tissues. The appearance of peaks in DNA, RNA and protein content per unit tissue is consistent with the “spurt” type of development associated with perinatal growth rather than the “linear-expansion” type of growth associated with the post-weaning period. Evidence for 3 biochemically separate phases during
1332
L. MENAKER AND S. A. MILLER
development is supported by the appearance of distinct protein accretion periods in the perinatal animal. This is further emphasized when viewed on a per cell basis. The increase in the relative weight of the SMG (in relation to total body weight), noted about the time of birth, was supported at the biochemical level by the concomitant accumulation of cellular protein in the gland. Also observed in our laboratory (MENAKERand MILLER, unpublished) were changes in glandular RNase activity. Peaks in RNase activity occurred during the times of maximal cellular differentiation of the terminal buds into acinar cells and suggests an anabolic or “conservation” role of the enzyme at this time. A peak in DNA concentration at day 17 also correlates with the time of near maximal proliferation of acinar cells. A schematic representation of the cellular composition of the gland and associated changes in its protein content appears in Fig. 8. This representation of
Time,
FIG. 8. Schematic representation
days
of total cell protein and cell type in the neonatal rat submandibular gland.
histological observation and biochemical data suggested a specific cellular contribution to the total protein content of the gland during the perinatal period. At the earliest times observed in the study, the gland consisted of terminal tubules and terminal buds. Theoretically, these produced one set of proteins. As formation of acinar cells from the buds took place, both cell types contributed to the total protein of the gland, giving rise to a new rate in the accumulation of protein (Figs. 4-6, 8). A third phase was encountered coincidental to the proliferation of ductal cells, resulting in yet another change in the rate of accumulation of total protein (Figs. 4-6, 8). As mentioned previously, speaks in the submandibular RNase activity seemed to be associated with the period of maximal differentiation of terminal buds into acinar cells. It has been shown (MENAKERand MILLER, unpublished) that a single
NUCLEIC
ACIDS
AND PROTEIN
IN RAT
SMG DEVELOPMENT
1333
peak in alkaline RNase activity occurred between days 5 and 12 and represented by far the major contribution to total RNase activity in the gland throughout the developmental period. Its cyclicity seemed better controlled during this time than at the other times tested. These points led us to conclude that alkaline RNase plays a greater role than the acidic enzyme during early gland development. Because of the associated cellular proliferation at this time, we have hypothesized that the enzyme was involved in developmental, anabolic activities through the rapid recycling of hydrolysed nucleotides available for new messenger synthesis. Confirmation of the hypothesis was obtained by the report from our laboratory (MENAKER and MILLER, unpublished) that the SMG possessed an RNase inhibitor whose presence was observed at a time just after maximal RNase activity. The effect of food deprivation at this specific time (around day 9-l I), has been further associated with the loss of polyribosomal aggregates. Our observations further support the hypothesis that, in the SMG, diet has its first effects on protein synthesis by lowering ribonuclease inhibitor activity. This allows for increasing releasable RNase activity which then results in degradation of m-RNA and subsequent polyribosome breakdown. Prohferation of acinar tissue starts around day 4 and continues up to about day 21 in this gland. It is within this period that we have shown DNA concentration to reach peak value (day 17). It was also about this time that, as discussed above, dietary regulation of protein synthesis occurred through its effect on ribonuclease activity. These facts, taken together, point to a possible “critical” time in the development of the SMG, about the second postnatal week in the rat. It is possible that dietary insult during this critical period could lead to permanent, irreversible changes with subsequent loss of functional capabilities. It is this possible “stunting” that may have clinical repercussions for the entire oral cavity milieu, Acknowledgement-This work was done in partial fulfillment for the degree of Sc.D. by L. MENAKER at the Massachusetts Institute of Technology. It was supported by funds provided by NIDR Grant No. DE02174 and by a Grant-in-Aid from Ross Laboratories, Inc., Columbus, Ohio. L. Menaker was the recipient of a postdoctoral traineeship under NIDR Training Grant No. DE105 at M.I.T. Contribution Number 1920 from the Department of Nutrition and Food Science, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Mass. 02139, U.S.A. R&me-La teneur en DNA, RNA et en proteine de la glande sous-mandibulaire a et& Ctudiee au tours du premier developpement et reliee aux changements histologiques vus au tours de cette periode. L’apparitionsequentielledecretes de concentration de DNA et de RNA au 176 et au 196 jour respectivement a Cte demontree. Trois periodes separees d’accumulation de prottine sont montrees etre associees au changement de populations cellulaires presentes dans la glande au tours du developpement. Zusammenfassung-Der DNA, RNA und Protein Gehalt der sumandibularen Driise zu Beginn der Entwicklung wurde getestet und verglichen mit histologischen VeranderA.O.B.18/10-1
1334
L. MENAKERAND S. A. MILLER ungen, die zur gleichen Zeit festgestellt wurden. Das periodische Erscheinen von DNA nnd RNA in h(ichster Konzentration an den Tagen 17 sowie 19 wurde dargestellt. Drei gesonderte Perioden der Proteinansammlung sind in Verbindung mit der w&rend der Entwicklung in der Drtise gegenwartigen wechselhaften Zellenanzahl gezeigt.
REFERENCES CER~O~, G. 1952. A microchemical determination of desoxyribonucleic acid. J. biol. Chem. 198, 297-303. DVORAK, M. 1969. The secretory cells of the submaxillary gland in the perinatal period of development in the rat. Z. Zellforsch. 99, 346-356. ENESCO,M. and LEBLOND,C. P. 1962. Increase in cell number as a factor in the growth of the organs and tissues of the young male rat. J. Embryol. exp. Morph. 10, 530-562. HUTCHISON,W. C. and MUNRO,H. N. 1961. The determination of nucleic acids in biological materials. Analyst 86,768-813. JACOBY,F. and LEESON,C. R. 1959. The post-natal development of the rat submaxillary gland. J. Anat. 93, 201-216. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L. and RANDALL,R. 1951. Protein measurement with folin phenol reagent. J. biol. Chem. 193, 265-275. MILLER, S. A. 1969. In: Mammalian Protein Metabolism (edited by MUNRO, H. N.), Vol. III, pp. 183-233. Academic Press, New York. MILLER, S. A. 1970. Nutrition in the neonatal development of protein metabolism. Fed. Proc. 29, 1497-1501. MUNRO, H. N. and FLECK, A. 1966. Recent developments in the measurement of nucleic acids in biological materials. Anulyst 91, 78-88. SCHMIDT,G. and THANNHAUSER, S. J. 1945. Method for the determination of desoxyribonucleic acid and phosphor-proteins in animal tissues. J. biol. Chem. 161,83-89. WINICK, M. and NOBLE,A. 1965. Quantitative changes in DNA, RNA and protein during prenatal and postnatal growth in the rat. Develop. Biol. 12,451466.
NUCLEICACIDSAND
PRGTEININRATSMGDEb'ELOPMENT
PLATE 1 OVERLEAP
1335
L. MENAKERAND
1336
S. A. MILLER
PLATE 1.
Haematoxylin
and eosin stained rat submandibular
gland from -2
to 50 days of age.
FIG. 7a. Submandibular gland at 2 days before birth. Parenchymal cells predominate at this age. A typical terminal tubule can be seen (arrow). An intralobular duct is seen at the upper right. x 108 FIG. 7b. Gland from l-day-old
rat. Central lumen of the terminal tubule as well as the entire terminal tubule has condensed. x 270
FIG. 7c. Three-day-old rat submandibular gland. Terminal buds (arrows) have become more numerous at this age and appear as outcroppings from terminal tubules. x 270 FIG. 7d. Six-day-old rat submandibular gland. Terminal buds seen to be greatly proliferating in relation to terminal tubules. x 270 FIG. 7e. Twenty-day-old rat submandibular gland. True acini predominate. Acinar cells show their typical mutinous accumulation and demonstrate basally located nuclei. x 338 FIG. 7f. Fifty-day-old rat submandibular gland. Acinar cells demonstrate slightly more accumulation of typical mutinous product. Intralobular duct shown by arrow. Gland not much different from that seen in the 20-day-old animal. x 338
PLATE 1 A.O.B. f.p. 1336
oral
Bid. Vol. 18,pp. 1325-
1336,
1973.Pergamon Press.Printedin GreatBritain.
CHANGES IN NUCLEIC ACID AND PROTEIN CONTENT DURING DEVELOPMENT OF THE RAT SUBMANDIBULAR GLAND L. MENAKER* Department
and S. A. MILLER
of Nutrition and Food Science, Massachusetts Institute Cambridge, Massachusetts 02139, U.S.A.
of Technology,
Summary-The DNA, RNA and protein content of the submandibular gland during early development has been investigated and correlated with histological changes seen during that time. The sequential appearance of DNA and RNA concentration peaks at days 17 and 19 respectively has been demonstrated. Three separate periods of protein accumulation were shown to be associated with the changing cellular populations in the gland during development. INTRODUCTION STUDIES of growth
and development have progressed from those concerned mainly with the description of physical parameters to more recent studies investigating the biochemical basis responsible for these outward changes. Many workers (ENESCO and LEBLOND, 1962 ; WINICK and NOBLE, 1965 ; MILLER, 1969) realized that development involved the adaptation of the essentially parasitic foetus to the environmental demands of the free-living organism. This adaptive process is, to a great extent, associated with the onset of regulatory processes controlled through enzymatic reactions. The most basic mechanisms underlying growth and development must, therefore, be associated with the synthesis of enzymes specifically, and the synthesis of proteins generally. It must be concluded that investigation of the biochemistry of protein synthesis during the neonatal period is essential to the understanding of growth and development of the organism. The present set of experiments, part of a larger study of the development of protein synthesis, was designed to determine the interrelationships between the cellular components most intimately involved in protein synthesis (RNA, DNA and protein) in a specific organ system, the rat submandibular gland (SMG). MATERIALS
AND
METHODS
Animals
Timed-pregnant, Sprague-Dawley (CD) rats (Charles River Strain) were obtained 5 days postconception (Charles River Breeding Laboratories, Wilmington, Mass.). Pregnant rats were housed in plastic breeding tubs containing sterilized wood shavings and given both tap water and a standard rat-mouse chow ad libitum. Pups were selected for randomization from litters born within a specified 4-hr time period. Lights were kept on a 12-hr on-off cycle by an automatic timer. Temperature was maintained at 70” i 3°F and 50 & 10 per cent relative humidity. Animals were weaned at 21 days after birth and transferred to plastic tubs containing a maximum of 4 pups. * Present address: University of Alabama Medical Center, School of Dentistry, 1919 Seventh Ave. S., Birmingham, Alabama 35233, U.S.A. 1325
L. MENAKERAND
1326
S. A. MILLER
Materials Indole and phenol reagents were purchased from Fisher Scientific Company (Fair Lawn, N.J.). Bovine serum albumin (crystallized) and calf thymus deoxyribonucleic acid were products of General Biochemicals (Chagrin Falls, Ohio). All other chemicals used were reagent grade. Methods Animals were decapitated. The paired SMG were quickly excised, placed on dry ice and immediately transfered to a - 40°C freezer. After a sufficient number of glands were collected, they were pooled and analysed for RNA, DNA and protein. RNA content of the SMG was determined by the procedure of SCHMIDTand THANNHAUSER (1945) as modified by HUTCHISONand MUNRO (1961) and MUNRO and FLECK (1966). DNA content of the SMG was estimated using the procedure of CERRIOTTI(1952). Tissue protein was measured by the method of LOWRYet al. (1951). For light microscopy, tissue was fixed in 10 per cent buffered formalin. Samples approximately 2 mm in thickness were washed and dehydrated by routine procedures, embedded in “Paraplast” blocks and cut to 6 pm in thickness. After 24 hr, the sections were stained with haematoxylin and eosin and viewed under the light microscope. Samples were prepared every day from 3 days before to 24 days after birth, in addition to days 27, 35 and 50. EXPERIMENTAL
RESULTS
Weight measurements As a measure of the normality of the experimental population, body weight was monitored throughout each study, and compared to data gathered from previous studies of the rat in our laboratory. Birth weights of approximately 5 *5 g and weaning o-5or 0.45 0.40 Y 0 $ $ $ 2 d
0,350.30 0.25 -
Days
of total body weight. Each point gland grows at a rate relatively faster than the whole rat from -2 to 5 days of age. Growth is more in line with the average for the entire animal until approximately day 27, and thereafter represents a decreasing proportion of total weight. FIG. la. The submandibular
gland as a percentage
represents at least 16 gland pairs. The submandibular
NUCLEIC
ACIDS AND
PROTEIN
1327
IN RAT SMO DEVELOPMENT
200 180 c E” 160< E ‘E f
-z 0 3
140IZO-
6O60 -
I
I
I
I
I
I
I
Birth
Age, FIG.
days
lb. Total submandibular gland weight from three days before birth to day 50 postpartum. Each point represents at least 32 glands.
(21 days old) weights of about 45-50 g were considered consistent with our past experience. SMG were collected and weighed from 3 days before birth to day 50 post-partum. Figure la shows the percentage of total body weight which the gland represents. Figure lb represents the total weight of the gland per animal over this period. As can be seen, SMG increased in weight at a rate greater than that for the whole body at a time just before birth and for approximately 5 days after. From about day 27 on, the gland represented a constantly decreasing proportion of total body weight. RNA content Tissue RNA was estimated from 2 days prior to parturition up to day 50 after birth. As expected, total RNA content of the gland constantly increased during the neonatal period reaching approximately 1.60 mg/gland at day 50. However, as seen in Fig. 2a, the rate of increase of RNA concentration (mg/g wet weight of tissue) was not a linear function. The concentration of RNA varied in such a way as to result in relative peaks at specific times during development. Specific peaks were seen at 5, 12 and 19 days of age. Figure 2b shows the amount of RNA per cell (RNA/DNA) during this same period. Just before and just after birth there is a spurt in RNA synthesis per cell. After day 34, acinar cell proliferation began and the relative content of RNA diminished. However, synthesis of RNA leads to an accumulation of this material in ever increasing relative amounts so that by day 35 the RNA/DNA ratio reached 1.5. DNA content Tissue DNA was determined for samples from 2 days before birth through 50 days post-partum. As was true with total gland RNA, DNA constantly increased over this
L. MENAKER
1328
3
II -21
AND S. A. MILLER
I 20
I 5 Time,
I 30
days
FIG. 2a. The major peak in RNA concentration
in the developing rat submandibular gland occurs around day 19. “Step-peaks” lead up to and follow this main peak. Each point represents duplicate homogenates of at least 8 glands. Numbers in parentheses represent days of age. 225 t 200 =
;:;,?,--"1..
: 0.25 1 II I 13 135
I 15
I IO Age,
I 20
I 25
I 30
I 35
days
FIG. 2b. Ratio of RNA to DNA from three days before birth to 35 days post-partm.
RNA content rapidly increases just prior to, and just after, birth. From day 7 onward, RNA accumulation constantly increases as cell division slows.
period, reaching a maximum of 1.25 mg/gland at day 50. Again, when plotted per gram of tissue, specific peaks in concentration appeared. These occurred at days 4, 9 and 17 (see Fig. 3). Protein content The content of protein of the SMG was measured from -3 days until +50 days. A sequential representation of the total protein per gland is shown in Fig. 4. Total
NUCLEIC
ACIDS
AND
PROl’fiIN
IN RAT
Time,
SMG
1329
DEVELOPMENT
days
FIG.
3. The major peak in DNA concentration in the developing rat submandibular gland occurs around day 17. “Step-peaks” lead up to and follow this main peak. Each point represents duplicate homogenates of at least 8 glands. Numbers in parentheses represent days of age.
protein seems to be associated with 3 distinct periods of [accretion: day -3 to 8, day 9 to 20, and day 21 to 50. Each segment fits the mathematical form of an exponential function with the equation y = aebx where a and b are constants, x is the age in days and y is the total protein per gland. The linear regression and resulting 64 r
-3-It2
4
6
IO
1
I5
20
Age,
days
I
25
I
28
35I”
- 501
Birth
FZG. 4. Total glandular protein showed three different rates of accretion during development: days -3 to 8,9 to 20 and 21 to 50. An approximation of these rates is shown by the solid line.
1330
L. MENAKER AND S. A. MILLER
FIG. 5. Semi-log plot of the total protein content of the submandibular gland vs. age with curve fitting by linear regression. Total protein data was fit to the exponential function y = a~?, where a and b are constants, y is the total protein in mg, and x is the age in days. Linear regression by the method of least squares resulted in estimates of the fit (correlation coefficient v) for each period.
correlation coefficients (Fig. 5) support the idea of 3 separate periods of protein accretion, each fitting an exponential curve differing only in slope and point of origin. The existence of 3 separate periods of protein accumulation is further supported by a plot of the amount of protein estimated per cell (Fig. 6). Developmental changes in the SMG again seem to be associated with 3 distinct periods in the neonate: days -3 to 8, days 9 to 20 and days 21 to 50.
t1 Birth
FIG. 6. Protein concentration per cell in the used as an index of tissue cellularity. Protein development, days -3 to 8, 9 to 20 and 21 cates of a minimum
rat submandibular gland. Total DNA was content per DNA displayed three peaks in to 50. Each point represents at least dupliof 8 pooled glands.
NUCLEIC
ACIDS
AND
PROTEIN
IN RAT
SMG DEVELOPMENT
1331
Light microscopy In general, our observations of the SMG support the findings reported by JACOBY and LEESON (1959). We are in even more agreement with DVORAK (1969), who looked at the perinatal histology of the gland during the last 3 days of gestation and the first 6 days after birth. Briefly, at -3 days of age, the rat submandibular gland is made up of approximately 75 per cent parenchymal and 25 per cent stromal cells, Of the parenchymal population, about 90 per cent are terminal tubules and the remainder intralobular ducts. The terminal tubule represents the precursor of the acinar cell and at -3 days displayed a dark staining, basally located nucleus (see Fig. 7a). The terminal tubule consisted of about 6 cells surrounding a large central lumen. The intralobular duct (see Fig. 7a) was composed of lighter staining columnar cells with a centrally located nucleus. These cells were relatively similar in appearance to the cells comprising the duct of the adult (see Fig. 7f). At days 1-2, there appeared to occur a narrowing of the central lumen of the terminal tubule coincidental to a condensation, or shrinking in size, of the entire terminal tubule (see Fig. 7b). As early as day 3, the terminal buds started appearing as outcroppings from the terminal tubules (see Fig. 7~). By days 5-6 the terminal buds were becoming more numerous while the terminal tubules from which they evolved seemed to have reached their peak concentration (see Fig. 7d). The difference between terminal bud and acinar cell depended solely on the subjective interpretation of the observer. Perhaps, in the future, histochemical procedures will permit more accurate identification of the transition. Acinar cells continued to increase, and terminal tubules to decrease, so that, by days 18-21, acinar cells were by far the most dominant cell type and had reached very nearly the numbers (see Fig. 7e) present at day 50 (see Fig. 7f). The proliferation of ductal tissue started about the third week and by the fourth week became even more apparent. This trend continued up to the last day sampled (day 50). DISCUSSION Since the animals used in the experiments were bred in a commercial laboratory, precise control over gestation time was possible only within rrtl2 ,hr. However, a 4-hr time interval (f2 hr) was set for the acceptance of a litter into the experiment; this interval may still have contributed to some of the variability observed in the determination of nucleic acids and protein. However, an investigation of those biochemical components intimately involved with protein synthesis did reveal certain basic interrelationships. The appearance of one major peak in DNA (day 17) and one for RNA (day 19) with “step-peaks” around these maxima supports the sequential appearance of cellular concentrations of DNA and RNA as previously reported by MILLER (1970) for other tissues. The appearance of peaks in DNA, RNA and protein content per unit tissue is consistent with the “spurt” type of development associated with perinatal growth rather than the “linear-expansion” type of growth associated with the post-weaning period. Evidence for 3 biochemically separate phases during
1332
L. MENAKER AND S. A. MILLER
development is supported by the appearance of distinct protein accretion periods in the perinatal animal. This is further emphasized when viewed on a per cell basis. The increase in the relative weight of the SMG (in relation to total body weight), noted about the time of birth, was supported at the biochemical level by the concomitant accumulation of cellular protein in the gland. Also observed in our laboratory (MENAKERand MILLER, unpublished) were changes in glandular RNase activity. Peaks in RNase activity occurred during the times of maximal cellular differentiation of the terminal buds into acinar cells and suggests an anabolic or “conservation” role of the enzyme at this time. A peak in DNA concentration at day 17 also correlates with the time of near maximal proliferation of acinar cells. A schematic representation of the cellular composition of the gland and associated changes in its protein content appears in Fig. 8. This representation of
Time,
FIG. 8. Schematic representation
days
of total cell protein and cell type in the neonatal rat submandibular gland.
histological observation and biochemical data suggested a specific cellular contribution to the total protein content of the gland during the perinatal period. At the earliest times observed in the study, the gland consisted of terminal tubules and terminal buds. Theoretically, these produced one set of proteins. As formation of acinar cells from the buds took place, both cell types contributed to the total protein of the gland, giving rise to a new rate in the accumulation of protein (Figs. 4-6, 8). A third phase was encountered coincidental to the proliferation of ductal cells, resulting in yet another change in the rate of accumulation of total protein (Figs. 4-6, 8). As mentioned previously, speaks in the submandibular RNase activity seemed to be associated with the period of maximal differentiation of terminal buds into acinar cells. It has been shown (MENAKERand MILLER, unpublished) that a single
NUCLEIC
ACIDS
AND PROTEIN
IN RAT
SMG DEVELOPMENT
1333
peak in alkaline RNase activity occurred between days 5 and 12 and represented by far the major contribution to total RNase activity in the gland throughout the developmental period. Its cyclicity seemed better controlled during this time than at the other times tested. These points led us to conclude that alkaline RNase plays a greater role than the acidic enzyme during early gland development. Because of the associated cellular proliferation at this time, we have hypothesized that the enzyme was involved in developmental, anabolic activities through the rapid recycling of hydrolysed nucleotides available for new messenger synthesis. Confirmation of the hypothesis was obtained by the report from our laboratory (MENAKER and MILLER, unpublished) that the SMG possessed an RNase inhibitor whose presence was observed at a time just after maximal RNase activity. The effect of food deprivation at this specific time (around day 9-l I), has been further associated with the loss of polyribosomal aggregates. Our observations further support the hypothesis that, in the SMG, diet has its first effects on protein synthesis by lowering ribonuclease inhibitor activity. This allows for increasing releasable RNase activity which then results in degradation of m-RNA and subsequent polyribosome breakdown. Prohferation of acinar tissue starts around day 4 and continues up to about day 21 in this gland. It is within this period that we have shown DNA concentration to reach peak value (day 17). It was also about this time that, as discussed above, dietary regulation of protein synthesis occurred through its effect on ribonuclease activity. These facts, taken together, point to a possible “critical” time in the development of the SMG, about the second postnatal week in the rat. It is possible that dietary insult during this critical period could lead to permanent, irreversible changes with subsequent loss of functional capabilities. It is this possible “stunting” that may have clinical repercussions for the entire oral cavity milieu, Acknowledgement-This work was done in partial fulfillment for the degree of Sc.D. by L. MENAKER at the Massachusetts Institute of Technology. It was supported by funds provided by NIDR Grant No. DE02174 and by a Grant-in-Aid from Ross Laboratories, Inc., Columbus, Ohio. L. Menaker was the recipient of a postdoctoral traineeship under NIDR Training Grant No. DE105 at M.I.T. Contribution Number 1920 from the Department of Nutrition and Food Science, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Mass. 02139, U.S.A. R&me-La teneur en DNA, RNA et en proteine de la glande sous-mandibulaire a et& Ctudiee au tours du premier developpement et reliee aux changements histologiques vus au tours de cette periode. L’apparitionsequentielledecretes de concentration de DNA et de RNA au 176 et au 196 jour respectivement a Cte demontree. Trois periodes separees d’accumulation de prottine sont montrees etre associees au changement de populations cellulaires presentes dans la glande au tours du developpement. Zusammenfassung-Der DNA, RNA und Protein Gehalt der sumandibularen Driise zu Beginn der Entwicklung wurde getestet und verglichen mit histologischen VeranderA.O.B.18/10-1
1334
L. MENAKERAND S. A. MILLER ungen, die zur gleichen Zeit festgestellt wurden. Das periodische Erscheinen von DNA nnd RNA in h(ichster Konzentration an den Tagen 17 sowie 19 wurde dargestellt. Drei gesonderte Perioden der Proteinansammlung sind in Verbindung mit der w&rend der Entwicklung in der Drtise gegenwartigen wechselhaften Zellenanzahl gezeigt.
REFERENCES CER~O~, G. 1952. A microchemical determination of desoxyribonucleic acid. J. biol. Chem. 198, 297-303. DVORAK, M. 1969. The secretory cells of the submaxillary gland in the perinatal period of development in the rat. Z. Zellforsch. 99, 346-356. ENESCO,M. and LEBLOND,C. P. 1962. Increase in cell number as a factor in the growth of the organs and tissues of the young male rat. J. Embryol. exp. Morph. 10, 530-562. HUTCHISON,W. C. and MUNRO,H. N. 1961. The determination of nucleic acids in biological materials. Analyst 86,768-813. JACOBY,F. and LEESON,C. R. 1959. The post-natal development of the rat submaxillary gland. J. Anat. 93, 201-216. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L. and RANDALL,R. 1951. Protein measurement with folin phenol reagent. J. biol. Chem. 193, 265-275. MILLER, S. A. 1969. In: Mammalian Protein Metabolism (edited by MUNRO, H. N.), Vol. III, pp. 183-233. Academic Press, New York. MILLER, S. A. 1970. Nutrition in the neonatal development of protein metabolism. Fed. Proc. 29, 1497-1501. MUNRO, H. N. and FLECK, A. 1966. Recent developments in the measurement of nucleic acids in biological materials. Anulyst 91, 78-88. SCHMIDT,G. and THANNHAUSER, S. J. 1945. Method for the determination of desoxyribonucleic acid and phosphor-proteins in animal tissues. J. biol. Chem. 161,83-89. WINICK, M. and NOBLE,A. 1965. Quantitative changes in DNA, RNA and protein during prenatal and postnatal growth in the rat. Develop. Biol. 12,451466.
NUCLEICACIDSAND
PRGTEININRATSMGDEb'ELOPMENT
PLATE 1 OVERLEAP
1335
L. MENAKERAND
1336
S. A. MILLER
PLATE 1.
Haematoxylin
and eosin stained rat submandibular
gland from -2
to 50 days of age.
FIG. 7a. Submandibular gland at 2 days before birth. Parenchymal cells predominate at this age. A typical terminal tubule can be seen (arrow). An intralobular duct is seen at the upper right. x 108 FIG. 7b. Gland from l-day-old
rat. Central lumen of the terminal tubule as well as the entire terminal tubule has condensed. x 270
FIG. 7c. Three-day-old rat submandibular gland. Terminal buds (arrows) have become more numerous at this age and appear as outcroppings from terminal tubules. x 270 FIG. 7d. Six-day-old rat submandibular gland. Terminal buds seen to be greatly proliferating in relation to terminal tubules. x 270 FIG. 7e. Twenty-day-old rat submandibular gland. True acini predominate. Acinar cells show their typical mutinous accumulation and demonstrate basally located nuclei. x 338 FIG. 7f. Fifty-day-old rat submandibular gland. Acinar cells demonstrate slightly more accumulation of typical mutinous product. Intralobular duct shown by arrow. Gland not much different from that seen in the 20-day-old animal. x 338
PLATE 1 A.O.B. f.p. 1336