0020-71 IX 79 1201497580200.0
TISSUE SPECIFIC AND PROTEIN
CHANGES
CONTENT
POSTNATAL
IN DNA, RNA
DURING
DEVELOPMENT
LATE
FETAL
AND
IN THE RAT
ANDREAS KISTLER Biological
Pharmaceutical
Research
Department.
F. Hoffmann-La
Roche & Co., Ltd, 4002 Basle,
Switzerland (Received
23 April
1979)
Abstract-l. The levels of DNA, RNA, and protein in brain, heart, kidney, lung, liver and spleen have been studied in the rat during late fetal, peri- and postnatal development. 2. The protein/DNA ratio increased with age in all tissues tested, supporting the view that the ratio of cytoplasm to nucleus increases with differentiation. 3. A tissue specific pattern of the RNA/DNA ratio was observed with, however, highest and lowest values at quite different developmental stages. 4. The possible relationships between the developmental changes in the macromolecular constituents and morphological, biochemical and functional changes in the various tissues have been discussed.
nucleus have been reported for the whole body from day 13 of gestation up to the age of 95 days (Enesco & Leblond, 1962). In addition, the increase in cell number as a factor in the growth of the organs has been described in the young male rat (Enesco & Leblond, 1962). However, during the fetal, peri- and early postnatal development of the rat there is scanty information on quantitative changes in the main macromolecular components DNA, RNA, and protein in the various tissues, except in the brain. Therefore, we have determined the changes in the macromolecular constituents in six organs during the late fetal and early postnatal development in the rat.
INTRODUCTION During late fetal and early postnatal development of the rat changes in endocrine functions, in the environment and in nutrition may affect the differentiation of the tissues. For example, in developing rat liver major changes in the enzymic pattern coincide with major changes in endocrine functions (Greengard, 1970) and the capacity of the liver to degrade fatty acids is higher during the suckling period when the high-fat content milk diet is being consumed than at other stages of life (Bailey & Lockwood, 1973). Whereas the heart begins its function at an early stage of development some other tissues become functional at late fetal stages or at birth like the lung. Thus, the development of the biochemical and morphological differentiation of the cells is tissue and stage specific. It is generally believed that the rati+mass of cytoplasm to mass of nucleus-increases with ditlerentiation (Davidson & Leslie, 1950). Either morphological or biochemical studies may reveal information on changes in cell size. Whereas the microscopical descriptions are largely qualitative in nature, unless one is using the quantitative morphometric method described recently (Weibel, 1969), the biochemical information is quantitative. However, since homogenates of whole organs are usually employed in biochemical studies instead of separation of the various cell types, the biochemical data represents the mean composition of a mean cell which does not exist in situ. Therefore, combined biochemical and morphometric studies should be performed in order to characterize growth and differentiation. From the technical point of view biochemical measurements are less time consuming than quantitative morphometric studies and may serve to pinpoint interesting stages during the differentiation of a particular tissue. In rats the changes in the number of nuclei (estimated from the DNA content) and the weight per
MATERIALS AND METHODS F&albino rats, outbred stock, were obtained from the Institute of Biological and Medical Research (Fiillinsdorf, BL). Adult female rats (age 2-3 months) were mated overnight and females which had a vaginal plug present were considered to be at day I of gestation. These animals have a gestation period of 22 days. The organs of the animals were rapidly removed, weighed and homogenized in distilled water. According to the size up to 10 organs were pooled for one determination. Four independent determinations representing animals from four different fitters were performed at each stage. Proteins and nucleic acids were precipitated overnight in ice-cold 5% trichloroacetic acid. The precipitate was washed twice with 5% trichloroacetic acid, once with ethanol + lOoA potassium acetate and once with ethanolether (3: 1) in order to remove lipids. Nucleic acids were extracted with 10% (w/w) perchloric acid at 70°C for 30 min. The protein fraction was dissolved in I N sodium hydroxide. DNA and RNA were determined according to. Burton (1956) and Cerriotti (1955) using herring sperm DNA and yeast RNA as standards respectively. Protein was measured by Rutter’s (1967) modification of the method of Lowry et al. (1951). using bovine serum albumin as a standard. 975
ANDREAS KISTLER
976 RESULTS
AND
DISCUSSION
The development of the ectodermal brain, the mesodermal heart (ventricles only) and kidney and of the lung, liver and spleen, organs of entodermal origin, were followed during rat development. In the rat brain and liver, biochemical differentiation proceeds extensively during the last few days of gestation and during early postnatal life. In order to be able to compare our results with reported work, we chose fetal stages between day 15 and 21 of gestation, neonatal pups (14 hr after parturition) and pups at 4, 12, and 29 days of postnatal age. For comparison the values for adult rats (> 100 day-old) were also determined. The variations in the organ weights expressed as per cent of body weight during development are given in Fig. I. Large differences in the pattern of the relative organ weights between various tissues were found during the late fetal period. However, the relative weight of the brain, lung, kidney, and heart remained constant during the early postnatal period (until day 12) implying that these organs grew proportionally to the body weight. The relative weight of all the tissues investigated decreased with further development. Table 1 summarizes the developmental changes in the amount of macromolecular tissue constituents. In adult rats the concentration of DNA was lowest in all tissues, except in the lung which showed almost the same level during the whole development. In contrast to the DNA, the concentration of protein was highest in the adult tissues except in the spleen. In all tissues a decrease in the RNA concentration was observed during the late fetal period. More or less the same levels of RNA were found during postnatal life. In the brain, heart and kidney there was an almost continuous increase in the protein/DNA ratio. A similar increase in this ratio was found for the other three tissues tested but with some fluctuations around or just before birth (Fig. 2). This data supports the view
AGE IN DAYS
Fig. 2. RNA/DNA and protein/DNA ratio during development of the rat. Points represent mean values of four independent determinations. Vertical bars indicate the standard error.
that the ratio of cytoplasm (main component protein) to nucleus (main component DNA) increases with differentiation. Tissue-specific patterns in the RNA/DNA ratio with, however, highest and lowest values at quite different stages were observed (Fig. 2). High RNA/DNA ratios may indicate cells in an active synthetic period although the increase in this ratio after postnatal day 12 in the heart, kidney, liver and spleen stems mainly from a decrease in DNA content per g tissue (Table I) and may represent larger cells. In order to get information on the growth of the different organs, the total amount of DNA, RNA and protein per organ was estimated and plotted against the time of development (Fig. 3). In all tissues, the highly proliferative phase before birth was followed by a less steep increase in DNA content and a diversification of the RNA and protein content. In contrast, Winick & Noble (1965) noted the same or a slower proliferative activity during fetal than during early postnatal development. In the liver there appeared a transient drop in the total cell number at birth-estimated from the total DNA content-whereas in the other tissues a continuous increase in the cell number is suggested. Next, the developmental changes in the various tissues and possible correlations with morphological, biochemical, and functional changes will be discussed. Brain
15 17 19 21
4
t_Gestat~ont--_Postnatal~~ AGE IN DAYS
Fig. I. Changes in the relative organ weights during development of the rat. Points represent mean values of four independent determinations. Vertical bars indicate the standard error.
Virtually the same changes in the amount of DNA and protein as reported earlier (Balizs et al., 1968; Brasel er al., 1970; Croskerry et al., 1973) were found in the developing rat brain. Our study confirmed the postnatal changes in RNA content reported by Adams (1966). In addition, we found a marked increase in the RNA/DNA ratio between gestational day 19 and birth (Fig. 2) which stems mainly from a retarded increase in RNA compared with DNA between gestational day 17 and 19 followed by a faster increase in RNA than in DNA (Fig. 3).
t_ 0.002 * 0.59 & 0.41 & 0.5
_+ 0.1 -r_0.15 _t 0.37 _t 2.8
&-0.2 k 0.46 & 1.30 & 5.7
f: 0.001 rf: 1.36 & 1.8 zfr6.0
0.024 7.00 6.28 66.2
1.2 3.48 6.44 67.2
1.0 5.07 9.03 56.1
0,004 9.10 14.9 109.8
0.17 f: 0.01
0.044 11.1 12.9 90.6
12.0 5.16 5.97 51.8
0.9 0.24 0.25 2.2
-f 0.006 f 0.4 Ifr0.3 k 1.0
* ): f &
rfr0.6 & 1.02 + 1.32 * 10.4
2.5 6.73 6.64 57.9
0.006 0.11 0.12 1.6
* 0.3 -I_0.32 J- 0.52 & 5.1
k & t_ +
4.0 2.84 4.10 49.1
0.076 5.44 4.28 45.8
0.56 & 0.09
17
* & f f
10.0 5.26 4.09 44.4
0.9 0.28 0.26 1.4
1.0 0.10 0.12 2.3
0.7 10.3 8.44 68.0
0.124 11.4 9.00 81.4 f f k f
0.02 0.3 0.45 4.8
& 0.002 jc 0.3 f 0.23 Ifr 3.2
46.0 + 1.0 5.96 + 0.12 4.99 * 0.12 45.7 rfr0.9
* 4: f _t
* 0.002 1_ 0.15 + 0.02 * 2.0
10.0 2.48 2.85 46.4
0.112 5.14 3.04 52.5
1.45 f 0.04
19
5.0 9.42 5.68 93.7
0.325 4.22 6.84 76.2
117 4.91 3.50 46.1 0.018 0.17 0.04 2.4
+ 1.0 & 0.72 _t 0.14 J- 8.5
+ _t k &
-t_ 11 * 0.12 rf: 0.06 + 0.7
+ 1.0 + 0.25 + 0.14 ;t: 2.0
31.1 5.91 4.87 62.5
0.9 OJ7 0.20 8.0
oIJO7 0.05 0.08 Lf
& * + It
* f. + _t
19.1 3.14 3.33 70.8
0.188 3.44 2.75 50.0
4.3 j; 0.2
21
*Each value represents the mean + standard error of four independent determinations.
DNA (mg/gf RNA (mdgf Protein (mg/gf
DNA (mg/g) RNA (mg/g) Protein (mg/g) Liver Fresh weight (g) DNA (m&r/g) RNA (mg/g) Protein (mg/g) Spleen Fresh weight (mg)
DNA (me/g) RNA (mg/g) Protein (mg/g) Kidney Fresh weight (mg) DNA (mgip) RNA (meJg) Protein (mg/g) Lung Fresh weight (mg)
Body weight (g) Brain Fresh weight (g) DNA (mg/g) RNA (mg/g) Protein (mg/gf Heart Fresh weight (mgf
15
Gestation
+ + + 4 + I f + + f + f
116 5.56 4.03 55.0 0.306 3.18 8.30 94.0 12.1 8.35 5.87 85.3
0.9 1.12 0.64 10.2
0.021 0.35 0.12 3.7
9 0.21 0.08 1.9
)_ 2.0 &-0.28 * 0.19 f 5.1
so.2 5.44 4.35 64.1
1.1 0.05 0.16 0.7
0.006 0.09 0.17 0.8
f f + +
+ + + +
28.2 2.98 3.26 66.8
0.235 3.02 3.65 49.5
5.3 * 0.2
Neonatal
Age (days)
22.0 14.5 6.09 115.9
0.319 4.42 8.52 120.0
0.007 0.55 0.21 2.1
15 0.21 0.11 1.6
2.0 0.12 0.10 1.0
2.0 0.16 0.19 2.7
0.012 0.05 0.05 0.7
Ifr 1.1 + 0.8 f 0.35 k 6.7
t f + f
& + + f
+ f f *
82.0 6.94 4.01 89.9 181 5.26 3.32 62.3
f + * k
+ f f f
42.0 3.41 3.44 92.9
0.366 2.27 2.34 54.7
8.1 k 0.1
4
f + + f i + _t _t + f + f
0.705 3.54 7.34 130.0 128 13.5 5.16 103.7
* & + 5
244 6.13 4.23 93.0 49s 4.94 3.03 64.8
& + * 5
16 0.4 0.35 2.8
0.076 0.13 0.18 3.7
33 0.03 0.07 0.8
20 0.14 0.05 1.4
9 0.10 0.03 2.4
* 0.01 _t 0.07 ) 0.13 &- 1.2
118 3.39 2.64 100.5
1.02 1.50 2.26 61.0
22.5 k 1.8
Postnatal 12
320 12.2 6.65 103.4
3.24 2.32 8.42 124.4
812 4.52 2.81 62.2
775 4.14 4.90 117.0
302 1.94 2.39 106.4
1.52 1.74 2.42 75.8
0.03 0.07 0.05 0.8
k & + &
+ + + +
+ + + +
f 5 + +
37 0.1 0.11 5.8
0.25 0.01 0.12 4.4
102 0.09 0.06 2.2
48 0.08 0.03 1.4
f 25 + 0.06 ) 0.07 -I_4.0
f f + +
71 +4
29
Table 1. Body weight, organ weight and changes in the macromolecular constituents per g tissue during development in the rat*
* + * F
& ) + +
rt: SO ) 2.07 + 0.18 + 3.4
10.3 1.00 7.33 146.8 610 6.71 5.84 110.4
1.7 0.23 0.23 3.3
+ 180 It: 0.22 _t 0.13 + 3.4
1240 5.46 3.31 108.6
5
ir
s 3 5’
3.36 i: 0.11 4.22 + 0.05 118.1 f 3.3
1850 + 270
-I 5’ 5 a 3 B 0
0.06 0.02 0.01 1.8 70 0.02 0.07 5.1
710 + 0.98 * 1.78 + 138.4 &
1.90 1.39 1.66 74.2
289 f 37
Adult
978
ANDREAS
KISTLER
n+ IO/LUNG
&vi
nch l! Gestot~on
+----
~~,,naro: --+
AGE IN DAYS
Fig. 3. Changes in total DNA, RNA, and protein content per organ during development Points represent mean values of four independent determinations. Berthold & Lim (1976 a,b) reported changes in the metabolism and nucleocytoplasmic transport of rat brain RNA during postnatal growth. However, no data is available on RNA metabolism during late fetal and perinatal development of the rat brain, a period which appears to involve marked changes in RNA meta~lism, Heart
The process of differentiation of cardiac myocytes during development involves the continuing synthesis and organization of contractile proteins into characteristic cross-striated myofibrils (Manasek, 1968). Only a slight increase in the prote~n/DNA ratio was observed until postnatal day 12 (Fig. 2). However, the structural differentiation appears to be accomplished before, since at 1 week of postnatal development the combined mitochondrial and myofibrillar volume expressed as per cent of the sarcoplasm (82.3%; Hagopian et al., 1975) reached the adult level (83.4%; Page & McCallister, 1973). The respective volume percentage in the fetal ventricle (19-21 days of gestation) was 47.2 (Anversa et af., 1975). The decrease in the RNA/ DNA ratio during the prenatal and early postnatal development may correlate with the successive attainment of myofibrillar differentiation.
of the rat
Kidney In contrast to the well documented morphogenesis of the metanephros (Du Bois, 1969) little is known about the biochemical differentiation of the kidney. The great heterogenicity of cells with different functions in the kidney parenchyma makes it difficult to correlate biochemical parameters with structure and function. Using histochemical approaches Fisher & Gruhn (1959) reported that the maturation of succinic dehydrogenase was complete in all zones of the kidney by about the 30th postnatal day. The decrease in the RNA/DNA ratio of 30% between birth and postnatal day 4 (Fig. 2) suggests a marked shift in the biosynthetic activity during this period. After day 4 there was a gradual increase in both the RNA/DNA and the protein/DNA ratio implying an increase in cell size during this period of differentiation.
LW Recently, the postnatal lung growth of the rat has been investigated using quantitative stereological techniques,, autoradiographic and morphological analysis(Burri et al., 1974; Kauffman et al., 1974; Burri, 1974). Three growth phases during postnatal lung
979
Tissue development in the rat development were postulated: (a) a short phase of lung expansion between days 1 and 4, (b) a phase of intense tissue proliferation, (c) a long period of equilibrated growth starting between days 13 and 21. A decrease of 32% in the volume density of lung tissue between days 1 and 4 was noted (Burri et al., 1974), however, the number of nuclei per cm3 lung tissue decreased only slightly during this and the following period (Kauffman et al., 1974). This correlates with the small decrease in DNA concentration (mg/g lung) during the postnatal growth (Table 1). Our finding that the RNA/DNA and protein/DNA ratio remained almost constant during postnatal development (Fig. 2) suggests that the cell size may not change markedly during this period of differentiation, which is in agreement with the morphometric data. It is noteworthy that the formation of the bulk of alveoli between days 4 and 13 by a rapid outgrowth of secondary septa from the primary septa present at birth (Burri, 1974) does not affect the biochemical parameters measured. The marked decrease in the RNA/DNA ratio during the late fetal development suggests changes in the biosynthetic activity of lung cells during this period which deserves further biochemical and morphological investigation. Liver
Loss of hematopoietic tissue is a major cellular event during perinatal differentiation of liver and has been quantitatively described using morphometric methods (Greengard et al., 1972). The large decrease in DNA concentration (mg/g liver) during late fetal life (Table 1) is largely due to the diminished number of hematopoietic cells and to a lesser extent to a coincident decrease in the number of parenchymal cells per cm3 which results from an increase in the mean volume of these cells (Greengard et al., 1972). This increase in cell volume is reflected by the rise of the RNA/DNA and protein/DNA ratio during the late fetal period (Fig. 2). It is noteworthy that from gestational day 19 to postnatal day 12 a continuous decrease in the relative liver weight from 8.6% to 3.1% was observed (Fig. 1). Enzymic differentiation in the developing liver appears to occur during three developmental periods: (a) during the late fetal, (b) the neonatal, (c) the late suckling period (Greengard, 1970). The biochemical differentiation of the hepatocytes during the first two clusters may be obscured by changes in the hematopoietic cells. However, the increase in the relative liver weight and in the RNA/DNA and protein/D.NA ratio between postnatal day 12 and 29 correlates with the increase of several enzymes during the third postnatal week. Spleen
The marked increase in the relative spleen weight during the late fetal and early postnatal period (Fig. 1) may reflect the rising importance of the spleen as hematopoietic tissue during this period. On the other hand only scanty information is available on the developmental differentiation of this tissue. In a combined biochemical and morphometric study on the changes in the liver during amphibian metamorphosis, Kistler & Weber (1975) found a close
correlation between changes in the macromolecular constituents and quantitative morphological parameters. This study supports the reported morphometric changes in the developing rat lung and liver. Thus, combined biochemical and morphometric studies appear to be useful to characterize growth and differentiation and to relate biochemical changes to the cellular events in developing tissues. Numerous physiological conditions change in the course of the biological maturation. It is well established that the infant animals have a different, usually greater, sensitivity to drugs than adult animals (Done, 1966). The different phases of maturation of the various tissues revealed in this study may, therefore, serve to select interesting stages for studying developmental pharmacology and toxicology.
REFERENCES ADAMS D.
H. (1966) The relationship between cellular nucleic acids in the developing rat cerebral cortex. Biochem. J. 98, 63G64O.
ANVERSAP.,
VITALI-MAZZAL. & LOUD A. V. (1975) Morphometric and autoradiographic study of developing ventricular and atrial myocardium in fetal rats. Lab. Invest. 33, 696-705. BAILEYE. & LCICKW~~DE. A. (1973) Some aspects of fatty acid oxydation and ketone body formation and utilization during development of the rat. Enzyme l&239-253. BALAZSR., KovAcs S., TEICHGRABER P., CKKS W. A. & EAYRSJ. T. (1968). Biochemical effects of thyroid deficiency on the developing brain. J. Neurochem. 15, 1335-1349. BERTHOLD W. & LIM L. (1976a) The metabolism of highmolecular-weight ribonucleic acid, including polyadenylated species, in the developing rat brain. B&hem. J. 154, 5 17-527.
BERTHOLD W. & LIM L. (1976b) Nucleecytoplasmic relationships of high-molecular-weight ribonucleic acid, including polyadenylated species, in the developing rat brain. Biochem. J. 154, 529-539. BRASELA., EHRENKRANZ R. A. & WINICK M. (1970) DNA polymerase activity in rat brain during ontogeny. Devl. Biol. 23, 424432.
BURRIP. H. (1974) The postnatal growth of the rat lung. III. Morphology. Anat. Rec. 180, 77-98. BURRI P. H.. DBALYJ. & WEIBELE. R. (1974) The postnatal growth of the rat lung. 1. Morphometry. Anat. Rec. 178, 7 1I-730. BURTON K. (1956) A study of the conditions and mechanism of the diphenylamin reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J. 62, 3 15-323.
CERRIO~ G. (1955) Determination of nucleic acids in animal tissues. J. biol. Chem. 214, 59-70. CROSKERRY P. G., Sham G. K., SHEPARDB. J. & FREEMAN K. B. (1973) Postnatal brain DNA in the nbrmal and growth hormone-treated rat. Braif Res. 52, 413-418. DAVIDSONJ. N. & LESLIEI. (1950) A new approach in the biochemistry of growth and development. Nature 165, 49-53. DONE A. K. (1966) Perinatal pharmacology. Ann. Reo. Pharmac. 6, 189-208.
Du BOISA. M. (1969) The embryonic kidney. In The Kidney (Edited by ROUILLER D. & MULLERA. F.) Vol. I, pp. 1-59. Academic Press, New York. ENE~COM. & LEBL~NDC. P. (I 962) Increase in cell number as a factor in the growth of the organs and tissues of the young male rat. J. Embry. exp. Morph. 10, 53C-562.
980
ANDREASKISTLER
FISHERE. R. & GRUHN J. (1959) Maturation of succinic dehydrogenase and cytochrome oxidase in neonatal rat kidney. Proc. Sot. exp. Biol. Med. 101, 781-784. GREENGARD0. (1970) The developmental formation of enzymes in rat liver. In Biochemical Actions of Hormones (Edited by LITTWACKG.) Vol. I, pp. 53-87. Academic Press New York. GREENGARD O., FEDERMAN M. & KNOXW. E. (1972) Cytomorphometry of developing rat liver and its application to enzymic differentiation. J. Cell Biol. 52, 261-272. HAGOPIANM., ANVERSAP. & LOUDA. V. (1975) Quantitative radioautographic localization of newly synthesized protein in the postnatal rat heart. J. Molec. Cell. Cardiol. 7, 357-367.
KAUFFMANS. L., BURRI P. J. & WEIBELE. R. (1974) The postnatal growth of the rat lung. II. Autoradiography. Anat. Rec. 180, 63-76.
KISTLERA. & WEBERR. (1975) A combined biochemical and morphometric study on tissue changes in Xenopus larvae during induced metamorphosis. Molec. Cell. Endocr. 2. 261-288.
LOWRY
0. H., ROSEBROUGH N. J., FARR A. L. & RANDALL R. J. (1951) Protein measurement with the folin phenol reagent. /. biol. Chem. 193, 265-275. MANASEKF. J. (1968) Embryonic development of the heart. I. A light and electron microscopic study of myocardial development in the early chick embryo. J. Morph. 125, 329. PAGEE. & MCCALLISTER L. P. (1973) Quantitative electron microscopic description of heart muscle cells. Am. J. Cardiol. 31, 172-l 81. RUTTERW. J. (1967) Protein determination in embryos. In Methods in Developmental Biology (Edited by WILT F. H. & WEASELSN. K.). pp. 671-683. C’rowell. New York.
WEIBELE. R. (1969) Stereological principles for morphometry in electron microscopic cytology. Inf. Rev. eytol. 26, 235’302.
WINICK M. & NOBLEA. (1965) Quantitative changes in DNA, RNA, and protein during prenatal and postnatal growth in the rat. Devl. Biol. 12, 451466.
TISSUE SPECIFIC AND PROTEIN
CHANGES
CONTENT
POSTNATAL
IN DNA, RNA
DURING
DEVELOPMENT
LATE
FETAL
AND
IN THE RAT
ANDREAS KISTLER Biological
Pharmaceutical
Research
Department.
F. Hoffmann-La
Roche & Co., Ltd, 4002 Basle,
Switzerland (Received
23 April
1979)
Abstract-l. The levels of DNA, RNA, and protein in brain, heart, kidney, lung, liver and spleen have been studied in the rat during late fetal, peri- and postnatal development. 2. The protein/DNA ratio increased with age in all tissues tested, supporting the view that the ratio of cytoplasm to nucleus increases with differentiation. 3. A tissue specific pattern of the RNA/DNA ratio was observed with, however, highest and lowest values at quite different developmental stages. 4. The possible relationships between the developmental changes in the macromolecular constituents and morphological, biochemical and functional changes in the various tissues have been discussed.
nucleus have been reported for the whole body from day 13 of gestation up to the age of 95 days (Enesco & Leblond, 1962). In addition, the increase in cell number as a factor in the growth of the organs has been described in the young male rat (Enesco & Leblond, 1962). However, during the fetal, peri- and early postnatal development of the rat there is scanty information on quantitative changes in the main macromolecular components DNA, RNA, and protein in the various tissues, except in the brain. Therefore, we have determined the changes in the macromolecular constituents in six organs during the late fetal and early postnatal development in the rat.
INTRODUCTION During late fetal and early postnatal development of the rat changes in endocrine functions, in the environment and in nutrition may affect the differentiation of the tissues. For example, in developing rat liver major changes in the enzymic pattern coincide with major changes in endocrine functions (Greengard, 1970) and the capacity of the liver to degrade fatty acids is higher during the suckling period when the high-fat content milk diet is being consumed than at other stages of life (Bailey & Lockwood, 1973). Whereas the heart begins its function at an early stage of development some other tissues become functional at late fetal stages or at birth like the lung. Thus, the development of the biochemical and morphological differentiation of the cells is tissue and stage specific. It is generally believed that the rati+mass of cytoplasm to mass of nucleus-increases with ditlerentiation (Davidson & Leslie, 1950). Either morphological or biochemical studies may reveal information on changes in cell size. Whereas the microscopical descriptions are largely qualitative in nature, unless one is using the quantitative morphometric method described recently (Weibel, 1969), the biochemical information is quantitative. However, since homogenates of whole organs are usually employed in biochemical studies instead of separation of the various cell types, the biochemical data represents the mean composition of a mean cell which does not exist in situ. Therefore, combined biochemical and morphometric studies should be performed in order to characterize growth and differentiation. From the technical point of view biochemical measurements are less time consuming than quantitative morphometric studies and may serve to pinpoint interesting stages during the differentiation of a particular tissue. In rats the changes in the number of nuclei (estimated from the DNA content) and the weight per
MATERIALS AND METHODS F&albino rats, outbred stock, were obtained from the Institute of Biological and Medical Research (Fiillinsdorf, BL). Adult female rats (age 2-3 months) were mated overnight and females which had a vaginal plug present were considered to be at day I of gestation. These animals have a gestation period of 22 days. The organs of the animals were rapidly removed, weighed and homogenized in distilled water. According to the size up to 10 organs were pooled for one determination. Four independent determinations representing animals from four different fitters were performed at each stage. Proteins and nucleic acids were precipitated overnight in ice-cold 5% trichloroacetic acid. The precipitate was washed twice with 5% trichloroacetic acid, once with ethanol + lOoA potassium acetate and once with ethanolether (3: 1) in order to remove lipids. Nucleic acids were extracted with 10% (w/w) perchloric acid at 70°C for 30 min. The protein fraction was dissolved in I N sodium hydroxide. DNA and RNA were determined according to. Burton (1956) and Cerriotti (1955) using herring sperm DNA and yeast RNA as standards respectively. Protein was measured by Rutter’s (1967) modification of the method of Lowry et al. (1951). using bovine serum albumin as a standard. 975
ANDREAS KISTLER
976 RESULTS
AND
DISCUSSION
The development of the ectodermal brain, the mesodermal heart (ventricles only) and kidney and of the lung, liver and spleen, organs of entodermal origin, were followed during rat development. In the rat brain and liver, biochemical differentiation proceeds extensively during the last few days of gestation and during early postnatal life. In order to be able to compare our results with reported work, we chose fetal stages between day 15 and 21 of gestation, neonatal pups (14 hr after parturition) and pups at 4, 12, and 29 days of postnatal age. For comparison the values for adult rats (> 100 day-old) were also determined. The variations in the organ weights expressed as per cent of body weight during development are given in Fig. I. Large differences in the pattern of the relative organ weights between various tissues were found during the late fetal period. However, the relative weight of the brain, lung, kidney, and heart remained constant during the early postnatal period (until day 12) implying that these organs grew proportionally to the body weight. The relative weight of all the tissues investigated decreased with further development. Table 1 summarizes the developmental changes in the amount of macromolecular tissue constituents. In adult rats the concentration of DNA was lowest in all tissues, except in the lung which showed almost the same level during the whole development. In contrast to the DNA, the concentration of protein was highest in the adult tissues except in the spleen. In all tissues a decrease in the RNA concentration was observed during the late fetal period. More or less the same levels of RNA were found during postnatal life. In the brain, heart and kidney there was an almost continuous increase in the protein/DNA ratio. A similar increase in this ratio was found for the other three tissues tested but with some fluctuations around or just before birth (Fig. 2). This data supports the view
AGE IN DAYS
Fig. 2. RNA/DNA and protein/DNA ratio during development of the rat. Points represent mean values of four independent determinations. Vertical bars indicate the standard error.
that the ratio of cytoplasm (main component protein) to nucleus (main component DNA) increases with differentiation. Tissue-specific patterns in the RNA/DNA ratio with, however, highest and lowest values at quite different stages were observed (Fig. 2). High RNA/DNA ratios may indicate cells in an active synthetic period although the increase in this ratio after postnatal day 12 in the heart, kidney, liver and spleen stems mainly from a decrease in DNA content per g tissue (Table I) and may represent larger cells. In order to get information on the growth of the different organs, the total amount of DNA, RNA and protein per organ was estimated and plotted against the time of development (Fig. 3). In all tissues, the highly proliferative phase before birth was followed by a less steep increase in DNA content and a diversification of the RNA and protein content. In contrast, Winick & Noble (1965) noted the same or a slower proliferative activity during fetal than during early postnatal development. In the liver there appeared a transient drop in the total cell number at birth-estimated from the total DNA content-whereas in the other tissues a continuous increase in the cell number is suggested. Next, the developmental changes in the various tissues and possible correlations with morphological, biochemical, and functional changes will be discussed. Brain
15 17 19 21
4
t_Gestat~ont--_Postnatal~~ AGE IN DAYS
Fig. I. Changes in the relative organ weights during development of the rat. Points represent mean values of four independent determinations. Vertical bars indicate the standard error.
Virtually the same changes in the amount of DNA and protein as reported earlier (Balizs et al., 1968; Brasel er al., 1970; Croskerry et al., 1973) were found in the developing rat brain. Our study confirmed the postnatal changes in RNA content reported by Adams (1966). In addition, we found a marked increase in the RNA/DNA ratio between gestational day 19 and birth (Fig. 2) which stems mainly from a retarded increase in RNA compared with DNA between gestational day 17 and 19 followed by a faster increase in RNA than in DNA (Fig. 3).
t_ 0.002 * 0.59 & 0.41 & 0.5
_+ 0.1 -r_0.15 _t 0.37 _t 2.8
&-0.2 k 0.46 & 1.30 & 5.7
f: 0.001 rf: 1.36 & 1.8 zfr6.0
0.024 7.00 6.28 66.2
1.2 3.48 6.44 67.2
1.0 5.07 9.03 56.1
0,004 9.10 14.9 109.8
0.17 f: 0.01
0.044 11.1 12.9 90.6
12.0 5.16 5.97 51.8
0.9 0.24 0.25 2.2
-f 0.006 f 0.4 Ifr0.3 k 1.0
* ): f &
rfr0.6 & 1.02 + 1.32 * 10.4
2.5 6.73 6.64 57.9
0.006 0.11 0.12 1.6
* 0.3 -I_0.32 J- 0.52 & 5.1
k & t_ +
4.0 2.84 4.10 49.1
0.076 5.44 4.28 45.8
0.56 & 0.09
17
* & f f
10.0 5.26 4.09 44.4
0.9 0.28 0.26 1.4
1.0 0.10 0.12 2.3
0.7 10.3 8.44 68.0
0.124 11.4 9.00 81.4 f f k f
0.02 0.3 0.45 4.8
& 0.002 jc 0.3 f 0.23 Ifr 3.2
46.0 + 1.0 5.96 + 0.12 4.99 * 0.12 45.7 rfr0.9
* 4: f _t
* 0.002 1_ 0.15 + 0.02 * 2.0
10.0 2.48 2.85 46.4
0.112 5.14 3.04 52.5
1.45 f 0.04
19
5.0 9.42 5.68 93.7
0.325 4.22 6.84 76.2
117 4.91 3.50 46.1 0.018 0.17 0.04 2.4
+ 1.0 & 0.72 _t 0.14 J- 8.5
+ _t k &
-t_ 11 * 0.12 rf: 0.06 + 0.7
+ 1.0 + 0.25 + 0.14 ;t: 2.0
31.1 5.91 4.87 62.5
0.9 OJ7 0.20 8.0
oIJO7 0.05 0.08 Lf
& * + It
* f. + _t
19.1 3.14 3.33 70.8
0.188 3.44 2.75 50.0
4.3 j; 0.2
21
*Each value represents the mean + standard error of four independent determinations.
DNA (mg/gf RNA (mdgf Protein (mg/gf
DNA (mg/g) RNA (mg/g) Protein (mg/g) Liver Fresh weight (g) DNA (m&r/g) RNA (mg/g) Protein (mg/g) Spleen Fresh weight (mg)
DNA (me/g) RNA (mg/g) Protein (mg/g) Kidney Fresh weight (mg) DNA (mgip) RNA (meJg) Protein (mg/g) Lung Fresh weight (mg)
Body weight (g) Brain Fresh weight (g) DNA (mg/g) RNA (mg/g) Protein (mg/gf Heart Fresh weight (mgf
15
Gestation
+ + + 4 + I f + + f + f
116 5.56 4.03 55.0 0.306 3.18 8.30 94.0 12.1 8.35 5.87 85.3
0.9 1.12 0.64 10.2
0.021 0.35 0.12 3.7
9 0.21 0.08 1.9
)_ 2.0 &-0.28 * 0.19 f 5.1
so.2 5.44 4.35 64.1
1.1 0.05 0.16 0.7
0.006 0.09 0.17 0.8
f f + +
+ + + +
28.2 2.98 3.26 66.8
0.235 3.02 3.65 49.5
5.3 * 0.2
Neonatal
Age (days)
22.0 14.5 6.09 115.9
0.319 4.42 8.52 120.0
0.007 0.55 0.21 2.1
15 0.21 0.11 1.6
2.0 0.12 0.10 1.0
2.0 0.16 0.19 2.7
0.012 0.05 0.05 0.7
Ifr 1.1 + 0.8 f 0.35 k 6.7
t f + f
& + + f
+ f f *
82.0 6.94 4.01 89.9 181 5.26 3.32 62.3
f + * k
+ f f f
42.0 3.41 3.44 92.9
0.366 2.27 2.34 54.7
8.1 k 0.1
4
f + + f i + _t _t + f + f
0.705 3.54 7.34 130.0 128 13.5 5.16 103.7
* & + 5
244 6.13 4.23 93.0 49s 4.94 3.03 64.8
& + * 5
16 0.4 0.35 2.8
0.076 0.13 0.18 3.7
33 0.03 0.07 0.8
20 0.14 0.05 1.4
9 0.10 0.03 2.4
* 0.01 _t 0.07 ) 0.13 &- 1.2
118 3.39 2.64 100.5
1.02 1.50 2.26 61.0
22.5 k 1.8
Postnatal 12
320 12.2 6.65 103.4
3.24 2.32 8.42 124.4
812 4.52 2.81 62.2
775 4.14 4.90 117.0
302 1.94 2.39 106.4
1.52 1.74 2.42 75.8
0.03 0.07 0.05 0.8
k & + &
+ + + +
+ + + +
f 5 + +
37 0.1 0.11 5.8
0.25 0.01 0.12 4.4
102 0.09 0.06 2.2
48 0.08 0.03 1.4
f 25 + 0.06 ) 0.07 -I_4.0
f f + +
71 +4
29
Table 1. Body weight, organ weight and changes in the macromolecular constituents per g tissue during development in the rat*
* + * F
& ) + +
rt: SO ) 2.07 + 0.18 + 3.4
10.3 1.00 7.33 146.8 610 6.71 5.84 110.4
1.7 0.23 0.23 3.3
+ 180 It: 0.22 _t 0.13 + 3.4
1240 5.46 3.31 108.6
5
ir
s 3 5’
3.36 i: 0.11 4.22 + 0.05 118.1 f 3.3
1850 + 270
-I 5’ 5 a 3 B 0
0.06 0.02 0.01 1.8 70 0.02 0.07 5.1
710 + 0.98 * 1.78 + 138.4 &
1.90 1.39 1.66 74.2
289 f 37
Adult
978
ANDREAS
KISTLER
n+ IO/LUNG
&vi
nch l! Gestot~on
+----
~~,,naro: --+
AGE IN DAYS
Fig. 3. Changes in total DNA, RNA, and protein content per organ during development Points represent mean values of four independent determinations. Berthold & Lim (1976 a,b) reported changes in the metabolism and nucleocytoplasmic transport of rat brain RNA during postnatal growth. However, no data is available on RNA metabolism during late fetal and perinatal development of the rat brain, a period which appears to involve marked changes in RNA meta~lism, Heart
The process of differentiation of cardiac myocytes during development involves the continuing synthesis and organization of contractile proteins into characteristic cross-striated myofibrils (Manasek, 1968). Only a slight increase in the prote~n/DNA ratio was observed until postnatal day 12 (Fig. 2). However, the structural differentiation appears to be accomplished before, since at 1 week of postnatal development the combined mitochondrial and myofibrillar volume expressed as per cent of the sarcoplasm (82.3%; Hagopian et al., 1975) reached the adult level (83.4%; Page & McCallister, 1973). The respective volume percentage in the fetal ventricle (19-21 days of gestation) was 47.2 (Anversa et af., 1975). The decrease in the RNA/ DNA ratio during the prenatal and early postnatal development may correlate with the successive attainment of myofibrillar differentiation.
of the rat
Kidney In contrast to the well documented morphogenesis of the metanephros (Du Bois, 1969) little is known about the biochemical differentiation of the kidney. The great heterogenicity of cells with different functions in the kidney parenchyma makes it difficult to correlate biochemical parameters with structure and function. Using histochemical approaches Fisher & Gruhn (1959) reported that the maturation of succinic dehydrogenase was complete in all zones of the kidney by about the 30th postnatal day. The decrease in the RNA/DNA ratio of 30% between birth and postnatal day 4 (Fig. 2) suggests a marked shift in the biosynthetic activity during this period. After day 4 there was a gradual increase in both the RNA/DNA and the protein/DNA ratio implying an increase in cell size during this period of differentiation.
LW Recently, the postnatal lung growth of the rat has been investigated using quantitative stereological techniques,, autoradiographic and morphological analysis(Burri et al., 1974; Kauffman et al., 1974; Burri, 1974). Three growth phases during postnatal lung
979
Tissue development in the rat development were postulated: (a) a short phase of lung expansion between days 1 and 4, (b) a phase of intense tissue proliferation, (c) a long period of equilibrated growth starting between days 13 and 21. A decrease of 32% in the volume density of lung tissue between days 1 and 4 was noted (Burri et al., 1974), however, the number of nuclei per cm3 lung tissue decreased only slightly during this and the following period (Kauffman et al., 1974). This correlates with the small decrease in DNA concentration (mg/g lung) during the postnatal growth (Table 1). Our finding that the RNA/DNA and protein/DNA ratio remained almost constant during postnatal development (Fig. 2) suggests that the cell size may not change markedly during this period of differentiation, which is in agreement with the morphometric data. It is noteworthy that the formation of the bulk of alveoli between days 4 and 13 by a rapid outgrowth of secondary septa from the primary septa present at birth (Burri, 1974) does not affect the biochemical parameters measured. The marked decrease in the RNA/DNA ratio during the late fetal development suggests changes in the biosynthetic activity of lung cells during this period which deserves further biochemical and morphological investigation. Liver
Loss of hematopoietic tissue is a major cellular event during perinatal differentiation of liver and has been quantitatively described using morphometric methods (Greengard et al., 1972). The large decrease in DNA concentration (mg/g liver) during late fetal life (Table 1) is largely due to the diminished number of hematopoietic cells and to a lesser extent to a coincident decrease in the number of parenchymal cells per cm3 which results from an increase in the mean volume of these cells (Greengard et al., 1972). This increase in cell volume is reflected by the rise of the RNA/DNA and protein/DNA ratio during the late fetal period (Fig. 2). It is noteworthy that from gestational day 19 to postnatal day 12 a continuous decrease in the relative liver weight from 8.6% to 3.1% was observed (Fig. 1). Enzymic differentiation in the developing liver appears to occur during three developmental periods: (a) during the late fetal, (b) the neonatal, (c) the late suckling period (Greengard, 1970). The biochemical differentiation of the hepatocytes during the first two clusters may be obscured by changes in the hematopoietic cells. However, the increase in the relative liver weight and in the RNA/DNA and protein/D.NA ratio between postnatal day 12 and 29 correlates with the increase of several enzymes during the third postnatal week. Spleen
The marked increase in the relative spleen weight during the late fetal and early postnatal period (Fig. 1) may reflect the rising importance of the spleen as hematopoietic tissue during this period. On the other hand only scanty information is available on the developmental differentiation of this tissue. In a combined biochemical and morphometric study on the changes in the liver during amphibian metamorphosis, Kistler & Weber (1975) found a close
correlation between changes in the macromolecular constituents and quantitative morphological parameters. This study supports the reported morphometric changes in the developing rat lung and liver. Thus, combined biochemical and morphometric studies appear to be useful to characterize growth and differentiation and to relate biochemical changes to the cellular events in developing tissues. Numerous physiological conditions change in the course of the biological maturation. It is well established that the infant animals have a different, usually greater, sensitivity to drugs than adult animals (Done, 1966). The different phases of maturation of the various tissues revealed in this study may, therefore, serve to select interesting stages for studying developmental pharmacology and toxicology.
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H. (1966) The relationship between cellular nucleic acids in the developing rat cerebral cortex. Biochem. J. 98, 63G64O.
ANVERSAP.,
VITALI-MAZZAL. & LOUD A. V. (1975) Morphometric and autoradiographic study of developing ventricular and atrial myocardium in fetal rats. Lab. Invest. 33, 696-705. BAILEYE. & LCICKW~~DE. A. (1973) Some aspects of fatty acid oxydation and ketone body formation and utilization during development of the rat. Enzyme l&239-253. BALAZSR., KovAcs S., TEICHGRABER P., CKKS W. A. & EAYRSJ. T. (1968). Biochemical effects of thyroid deficiency on the developing brain. J. Neurochem. 15, 1335-1349. BERTHOLD W. & LIM L. (1976a) The metabolism of highmolecular-weight ribonucleic acid, including polyadenylated species, in the developing rat brain. B&hem. J. 154, 5 17-527.
BERTHOLD W. & LIM L. (1976b) Nucleecytoplasmic relationships of high-molecular-weight ribonucleic acid, including polyadenylated species, in the developing rat brain. Biochem. J. 154, 529-539. BRASELA., EHRENKRANZ R. A. & WINICK M. (1970) DNA polymerase activity in rat brain during ontogeny. Devl. Biol. 23, 424432.
BURRIP. H. (1974) The postnatal growth of the rat lung. III. Morphology. Anat. Rec. 180, 77-98. BURRI P. H.. DBALYJ. & WEIBELE. R. (1974) The postnatal growth of the rat lung. 1. Morphometry. Anat. Rec. 178, 7 1I-730. BURTON K. (1956) A study of the conditions and mechanism of the diphenylamin reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J. 62, 3 15-323.
CERRIO~ G. (1955) Determination of nucleic acids in animal tissues. J. biol. Chem. 214, 59-70. CROSKERRY P. G., Sham G. K., SHEPARDB. J. & FREEMAN K. B. (1973) Postnatal brain DNA in the nbrmal and growth hormone-treated rat. Braif Res. 52, 413-418. DAVIDSONJ. N. & LESLIEI. (1950) A new approach in the biochemistry of growth and development. Nature 165, 49-53. DONE A. K. (1966) Perinatal pharmacology. Ann. Reo. Pharmac. 6, 189-208.
Du BOISA. M. (1969) The embryonic kidney. In The Kidney (Edited by ROUILLER D. & MULLERA. F.) Vol. I, pp. 1-59. Academic Press, New York. ENE~COM. & LEBL~NDC. P. (I 962) Increase in cell number as a factor in the growth of the organs and tissues of the young male rat. J. Embry. exp. Morph. 10, 53C-562.
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ANDREASKISTLER
FISHERE. R. & GRUHN J. (1959) Maturation of succinic dehydrogenase and cytochrome oxidase in neonatal rat kidney. Proc. Sot. exp. Biol. Med. 101, 781-784. GREENGARD0. (1970) The developmental formation of enzymes in rat liver. In Biochemical Actions of Hormones (Edited by LITTWACKG.) Vol. I, pp. 53-87. Academic Press New York. GREENGARD O., FEDERMAN M. & KNOXW. E. (1972) Cytomorphometry of developing rat liver and its application to enzymic differentiation. J. Cell Biol. 52, 261-272. HAGOPIANM., ANVERSAP. & LOUDA. V. (1975) Quantitative radioautographic localization of newly synthesized protein in the postnatal rat heart. J. Molec. Cell. Cardiol. 7, 357-367.
KAUFFMANS. L., BURRI P. J. & WEIBELE. R. (1974) The postnatal growth of the rat lung. II. Autoradiography. Anat. Rec. 180, 63-76.
KISTLERA. & WEBERR. (1975) A combined biochemical and morphometric study on tissue changes in Xenopus larvae during induced metamorphosis. Molec. Cell. Endocr. 2. 261-288.
LOWRY
0. H., ROSEBROUGH N. J., FARR A. L. & RANDALL R. J. (1951) Protein measurement with the folin phenol reagent. /. biol. Chem. 193, 265-275. MANASEKF. J. (1968) Embryonic development of the heart. I. A light and electron microscopic study of myocardial development in the early chick embryo. J. Morph. 125, 329. PAGEE. & MCCALLISTER L. P. (1973) Quantitative electron microscopic description of heart muscle cells. Am. J. Cardiol. 31, 172-l 81. RUTTERW. J. (1967) Protein determination in embryos. In Methods in Developmental Biology (Edited by WILT F. H. & WEASELSN. K.). pp. 671-683. C’rowell. New York.
WEIBELE. R. (1969) Stereological principles for morphometry in electron microscopic cytology. Inf. Rev. eytol. 26, 235’302.
WINICK M. & NOBLEA. (1965) Quantitative changes in DNA, RNA, and protein during prenatal and postnatal growth in the rat. Devl. Biol. 12, 451466.
