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A serological study of the cell fractions during the embryonic development of liver in chick
Experimental
Cell Research
31, 499-507
499
(1963)
A SEROLOGICAL STUDY OF THE CELL FRACTIONS DURING THE EMBRYONIC DEVELOPMENT OF LIVER IN CHICK’ V. D’AMELIO, Laboratory
of Comparative
V. MUTOLO
Anatomy, of the Centro Received
University Tamori, November
and
E. PIAZZA
of Palermo, and Biological Palermo, Italy
Laboratory
6, 1962
IT seems to be generally agreed that the fundamental process of ontogenesis involves a gene-controlled synthesis of specific proteins. Presumably, the activity of each particular gene is stimulated or repressed in the course of development with consequent stimulation or repression of the synthesis of different molecules. Immunological methods, with their high degree of resolving po\ver and specificity, appear to be well suited for studies of the changes occurring at molecular level in tissues, cells, and cell particles, in the course of morphogenesis. Indeed, interesting results have been obtained on different materials. The extensive investigations regarding the differentiation of the lens have shown that adult lens antigens appear in advance of a morphological differentiation of the lens [3] and in sequential order in the course of development [15]. Of particular interest are the observations related to stage specific antigens which are present in early stages and later disappear [‘L, 141. It has been established that in the case of chick hemoglobin certain embryonic components are found at very early stages, but disappear during the later stages when adult types of hemoglobin are demonstrable [9]. New antigens have been found to appear at certain stages of development in sea urchin [24] and in amphibian embryos [.5, 6, 111. The immunoelectrophoretic analysis of the total homogenates of liver from embryos at different stages of development has shown that some of the adult antigens appear at different periods of incubation [7]. It seems of interest to mention that adult lens antigens are associated with microsomes prepared from early chick embryos 1231. Also the microsomes of the wheat colcoptile have been found to contain organ-specific antigens [SO]. ’ These Ricerche, Foundation
investigations have Research Group for of the U.S.A.
been supported by Grants from the Consiglio Nazionale delle the Problems of Differentiation, and from the National Science
Experimental
Cell Research
31
500
V.
D’Amelio,
V. Mutolo
and E. Piazza
Some recent work has indicated the possibility of localizing specific antigens in mitochondria, microsomes aud cell sap from rat liver [S, 10, 19, 22, 2,5]. These results have prompted us to carry out an immunological study of the subcellular fractions of the chick liver in the course of development. In this way it has been found that the microsomes and the cell sap fraction acquire some adult type antigens during the course of development, whereas from the immunological point of view the mitochondria do not seem to undergo any signific.ant change, at least up to the stage of the 9th day embryo. MATERIAL
AND
METHODS
Livers were obtained from white Leghorn chicks fasted overnight and killed by decapitation. The tissues were homogenized in 0.44 M sucrosein a glasshomogenizer with a teflon pestle (1 g wet wt/2 ml of sucrose).The homogenate was centrifuged at 1000 g for 15 min. The mitochondria were then obtained by centrifuging at 10,000 g for 10 min: the mitochondrial pellet was washed twice. The supernatant obtained after the sedimentation of the mitochondria was centrifuged at 25,000 g for 20 min and the sediment discarded. From the supernatant, the microsomeswere sedimented at 105,000 g for 60 min. The pellet was washed once. The deoxycholate-insoluble microsomal fraction was obtained by a treatment of the microsomal suspensionin 0.3 per cent sodium deoxycholate (final concentration) and centrifugation at 105,000 g for 60 min. After the sedimentation of the microsomes the upper two thirds of supernatant were collected as “ceI1 sap”. Embryonic livers of white Leghorn embryos were removed on different days of incubation (6, 7, 10, 12, 15 and 18 days) and homogenized as described above. The subcellular fractions were prepared by procedures used for adult liver, but mitochondria and microsomes were not washed. Serum albumin was prepared by DEAE fractionation as suggestedby Sober and Peterson [29]. The antisera against adult liver cell fractions and total serum were obtained in rabbits with Freund’s adjuvant technique [12], using the method previously described [lo]. Four rabbits were utilized for each antigen. Proteins were estimated by the method of Lowry et al. [16]; RNA was extracted by Schneider’s method [27] and estimated by the orcinol method [17]. For the immunological tests the double diffusion on agar [20] was used. As a rule, the antigens were used at the sameprotein concentration. RESULTS
Immunological analysis.-Preliminary experiments using the doublediffusion agar tests showed that the cell fractions prepared from adult chick liver represent a suitable material for the study of the antigens at subcellular level. Experimenfol
Cell Research
31
Serology
of chick liver differenfiation
501
A Fig. I.-Agar double diffusion precipitation bryonic liver against an anti-adult mitochondria chondria; (b) 15.day embryonic mitochondria; embryonic mitochondria.
test
of mitochondria isolated from serum (A). 4.5 mg of protein/ml. (c) g-day embryonic mitochondria:
adult and em(a) adult mitoand (d) 7-day
The immunological analysis indicates that the mitochondrial fraction of embryonic liver, starting from the 9th day of development, exhibits the same reaction pattern as the one prepared from the adult liver. Indeed, five groups of precipitation lines Lvere found in the preparations of both adult and embryo (Fig. l), while in preparations of 7-dayold embryos one of these seemed to be absent. Furthermore, Fig. 2 shows that the mitochondria from embryonic, liver, when reacting with a serum anti-chicken serum, give rise to a precipitation line which seems to correspond to serum albumin. Such a reaction has not been observed \vith adult mitochondria. Several precipitation lines arise in the reaction of microsomes from dif-
Fig. Z.-Agar chicken serum mitochondria;
double diffusion test of mitochondria and microsomes with serum against adult (A). 5 mg of protein/ml. (a) chicken serum albumin; (b) adult serum; (c) adult (d) 15-day embryonic microsomes; and (e) 15-day embryonic mitochondria. Experimental
Cell
Research
31
502
V. D’Amelio,
V. Mutolo
and E. Piazza
Fig. 3.-Agar double diffusion precipitation test of microsomes isolated from adult and embryonic liver against an anti-adult microsomes serum (B) and a serum anti-chicken serum (A). 4 mg of protein/ml. (a) adult serum; (b) chicken serum albumin; (c) 7-day embryonic microsomes; (d) IO-day embryonic microsomes; (e) 15-day embryonic microsomes; (f) ZO-day embryonic microsomes; and (g) adult microsomes.
ferent stages of development, already at the 6th day of incubation, with an anti-adult-microsomes serum (Fig. 3). This pattern, however, is not entirely like that of the adult, as new reactions appear in the course of development. In order to identify the missing material, preparations of embryonic liver microsomes have been compared with adult microsomes and with their deoxycholate-insoluble fraction i.e. RNP (Fig. 4). This has shown that the precipitation lines lacking in the reaction of microsomes of the 12-days liver correspond to adult RNP-bound antigens. The membranaceous material, at least from the 12th day of incubation, does not show any difference from the adult, whereas in younger stages some precipitation lines are absent. Parallel experiments have been carried out with the fraction which does not sediment at 105,000 g (so-called “cell sap”) prepared from adult and embryonic liver which was allowed to react with a serum against adult liver cell sap. Again, it was found that the reaction typical of the adult liver arises gradually in the course of development. In fact, as can be seen in Fig. 5, the reaction between cell sap from a liver of 7-day-old embryos and an antiserum against adult liver cell sap, shows that two groups of precipitaExperimental
Cell
Research
31
Fig. 4.-Agar double diffusion precipitation test of adult microsomes, adult RNP, and embryonic microsomes against anti-adult microsome serum (A) and against serum anti-chicken serum (B). 6.35 mg of protein/ml. (a) adult microsomes; (b) adult RNP; (c) 6-day embryonic microsomes; and (tf) 12-day embryonic microsomes.
tion lines typical of the adult liver are missing. One of them makes its appearance after the 16th day and the other after the 15th day of incubation. Chemical crnnlysis.-In Table I and in Fig. 6 are reported the values of a typical experiment on the RNA/protein N ratio of preparations of microsomes from livers of 6-, 12-, and l&day-old embryos, and adult liver. The data show that the ratio gradually decreases in the course of development and a value close to that of adult microsomes is reached at the 18th day. The TABLE
I. RNA/protein
X ratio adult
of preparations cud embryonic
of microsomes livers. RNA/protein
.i-day 12.day l&day Adult Adult 18.day
embryonic embryonic embryonic microsomes RNP embryonic
microsomcs microsomcs microsomes
RNP
and
RNP
from
X
2.91 2.14 1.02 0.x.5 6.9G 5.20
E.zrxrimenfal
Cell
Research
31
504
V. D’Amelio,
V. Mutolo
Fig. 5.-Agar double diffusion precipitation adult cell sap serum (A). 4 mg of protein/ml. (e) g-day embryonic cell sap; and (d) 7-day
and E. Piazza
test of adult and embryonic cell sap against anti(a) adult cell sap; (0) 15.day embryonic cell sap; embryonic cell sap.
d _ 6
0.500~
O.LOO
0.300.
0.200.
0.100.
OJ 6 Days
Fig. Fig.
6.-RNA/protein
N ratio
12 of incubation
18
6. of embryonic
Fig. 7.-UV spectra of adult and embryonic (A) adult microsomes; (B) B-day embryonic and (D) l&day embryonic microsomes. Experimental
Cell Research
31
2LO 260 Wavelength
Fig. microsomes
at different
microsomes microsomes;
280 (mp)
300
7.
days
of incubation.
suspended in 1.0 M NaCl at pH 7.0. (C) 12-day embryonic microsomes;
Serology
of chick liver differentiation
505
RNA/protein N ratio of the fraction insoluble in 0.3 per cent deoxycholate from liver microsomes of It)-day-old embryos differs slightly from the value of the adult tissue. The UV spectrum of microsomal fractions suspended in 1.0 M NaCl at pH 7.0 indicates a decrease of the 260/280 m,u ratio and an increase of the 240/260 rnp ratio (Fig. 7).
DISCUSSION
Our chemical analyses have shown a progressive decrease of the RNA/ protein N ratio in the microsomal fraction from high values at the 6th day of incubation, to the value typical of the adult tissue, at about the 18th day. These observations are in line with the electron microscope studies of several authors [l, 4, 13, 18, 21, 26, 281 indicating that it is typical of the embryonic cells, during the early stages of development, to have numerous free RIVP particles. Our immunological data show that in the earliest stages thus far examined some of reacting substances like the antigens typical of the adult microsomes are apparently missing. These could be both membrane antigens and antigens bound to the RNP particles. As to the former, however, the fact that we have been unable to detect them does not necessarily imply that they do not exist. In fact, as has just been mentioned, in the early stages the free RNP particles are very plentiful and since we have always diluted our preparations to the same protein concentration, the concentration of the membranaceous material may have been too low to give rise to a reaction. This, on the other hand, does not apply in the case of RNP particles, which are plentiful from the earliest stages. Hence the results indicate the actual lack of some of the adult type antigens, i.e. that these particles are different from those of the adult liver. At this point it seems to be of some interest to recall the hypothesis that free particles synthetize cytoplasmic proteins for growth, whilst the attached RNP particles are mainly devoted to the synthesis of secretory proteins [ 1, 13, 18, 21, 261. As to the presence of serum-like material observed in the mitochondria fraction in the liver of 15-day-old embryos, but never in the preparations of adult liver, we think that the most likely explanation is a particularly heavy contamination with microsomes. The immunological analysis of the cell sap indicates a progressive appearance of material like to the antigen of the adult fraction in the course of 33
- 631814
Experimental
Cell
Research
31
506
V. D’Amelio,
V. Mutolo
and E. Piazza
development. These results seem to reflect the microsomal differentiation already discussed. The present investigations suggest that in the subcellular fractions of adult chick liver two sets of antigens may be distinguished, the one present from the earliest stages of development, the other appearing in the course of development and hence to be considered as an expression of the biochemical differentiation of the liver cells. Further work is now in progress to extend a similar immunological analysis to the other organs.
SUMMARY
Mitochondria, microsomes and cell sap from embryonic chick liver have been tested with sera produced against the homologous fractions from adult liver. Since the earliest stages of development considered, each one of the three subcellular fractions was able to give rise to a number of precipitation lines when reacting with the homologous adult antiserum. In the course of development the pattern of precipitation lines progressively approaches that of the adult. However, the microsomal fraction from embryonic liver, which is known to contain a large amount of free ribonucleoprotein particles, does not give rise to any reaction like that obtained with adult liver ribosomes. Chemical analyses of the embryonic microsomes from different stages of development have demonstrated a high RNA/protein ratio in the earliest stages which decreases until the 18th day of incubation when values identical to those of the adult are reached.
REFERENCES 1. BIRBECK, M. S. C. and 2. BURKE, V., SULLIVAN, 3. GATE, G. ten and VAN
MERGER, E. H., N. P., PETRSON, DOORENMALEN,
IVature 189, 558 (1961). H. and WEED, R., .I. infect. W. J., Proc. Koninkl. Ned&.
Dis. 74, 225 (1944). Adad. Weiensch. 53,849
(1950). 4.
5. 6. 7. 8. 9.
10. 11.
12. 13.
CICADA LEONARDI, M., Ric. Sci. 27, 2853 (1957). CLAYTON. R. M., J. Embryol. exptl. Morphol. 1, 25 (1953). COOPER, k. S., j. exptl. Zool. lOi, 397 (i948). CROISILLE, Y., Pathol. Biol. 9, 253 (1961). D’AMELIO, V. and PERLMANN, P., Exptl. Cell Res. 19, 383 (1960). D’AMELIO, V. and SALVO, M. A., Acfa Embryol. Morphol. Exptl. 4, 250 (1961). D’AMELIO. V.. MUTOLO. V. and BARBARINO, A., Expfl. Cell Res. 29, 1 (1963). FLICKING& k. A. and’NAcq G. W., Expti &ZZ Rks. 3, 393 (1952). FREUND, J. and BONANTO, M. V., J. Immunol. 48, 325 (1944). HOWATSON, A. F. and HAM, A. W., Cancer Res. 15, 62 (1955).
Experimental
Cell Research
31
Serology 14. 15.
16. 17. 18. 19.
20. 21. 22. 23. 24. 25.
26. 27. 28. 29. 30.
of chick liver differentiation
KONYUIWO~. B. V.. Biull. eksper. Viol. Med. 44, 96 (1957). LANGMAN, J.‘, J. Embrbol. Ex$l. Morphol. 7, 264 (1959). LOWRY. 0. H.. ROSEBROUGH. N. J.. FARR, A. L. and RANDALL, R. J., J. Riol. Chem. 193,265 (i951). MEJBAUM. W. Z., Phusiol. Chem. 258, 117 (1939). MUNGER, ‘B., Am. J.-Am& 103, 1 (1958). MUTOLO, V. and D’AMELIO, V., Experientia 18, 556 (1962). OCCHTERLONY, o., Acta Pathol. Microbial. Stand. 32, 231 (1953). PALADE, G. E., J. Biophys. Biochem. Cytol. 1, 59 (1953). PERLMA~N, P. and D’~&LIo, V., h’af&e 181, 491 (1958). PERLMANN, P. and I)E VINCENTIIS, M., Expll. Cell Res. 23, 612 (1961). PERLX~NN, P. and GUSTAFSOX, T., Experienfia 4, 481 (1948). PERL~IANN, P., HULTIS, T., D’AMELIO, V. and MORGAN, W. S., Exptl. Cell Res. Suppl. 7, 279 (1959). PORTER, I<. R., J. Histochem. Cytochem. 2, 346 (1954). SCHWIIDER, W., J. Biol. Chem. 161, 293 (1945). SLAUTTERBACK, D. B. and DON I’AWCETT, \V., .I. Biophys. Biochem. Cytol. 5, 441 (1959). SOBEI~, H. A. and PETERSON, E. A., Fed. Proc. 17, 1116 (1958). WRIGIIT, S. T. C., Nature 185, 82 (1960).
Experimenlal
Cell Research
31
Cell Research
31, 499-507
499
(1963)
A SEROLOGICAL STUDY OF THE CELL FRACTIONS DURING THE EMBRYONIC DEVELOPMENT OF LIVER IN CHICK’ V. D’AMELIO, Laboratory
of Comparative
V. MUTOLO
Anatomy, of the Centro Received
University Tamori, November
and
E. PIAZZA
of Palermo, and Biological Palermo, Italy
Laboratory
6, 1962
IT seems to be generally agreed that the fundamental process of ontogenesis involves a gene-controlled synthesis of specific proteins. Presumably, the activity of each particular gene is stimulated or repressed in the course of development with consequent stimulation or repression of the synthesis of different molecules. Immunological methods, with their high degree of resolving po\ver and specificity, appear to be well suited for studies of the changes occurring at molecular level in tissues, cells, and cell particles, in the course of morphogenesis. Indeed, interesting results have been obtained on different materials. The extensive investigations regarding the differentiation of the lens have shown that adult lens antigens appear in advance of a morphological differentiation of the lens [3] and in sequential order in the course of development [15]. Of particular interest are the observations related to stage specific antigens which are present in early stages and later disappear [‘L, 141. It has been established that in the case of chick hemoglobin certain embryonic components are found at very early stages, but disappear during the later stages when adult types of hemoglobin are demonstrable [9]. New antigens have been found to appear at certain stages of development in sea urchin [24] and in amphibian embryos [.5, 6, 111. The immunoelectrophoretic analysis of the total homogenates of liver from embryos at different stages of development has shown that some of the adult antigens appear at different periods of incubation [7]. It seems of interest to mention that adult lens antigens are associated with microsomes prepared from early chick embryos 1231. Also the microsomes of the wheat colcoptile have been found to contain organ-specific antigens [SO]. ’ These Ricerche, Foundation
investigations have Research Group for of the U.S.A.
been supported by Grants from the Consiglio Nazionale delle the Problems of Differentiation, and from the National Science
Experimental
Cell Research
31
500
V.
D’Amelio,
V. Mutolo
and E. Piazza
Some recent work has indicated the possibility of localizing specific antigens in mitochondria, microsomes aud cell sap from rat liver [S, 10, 19, 22, 2,5]. These results have prompted us to carry out an immunological study of the subcellular fractions of the chick liver in the course of development. In this way it has been found that the microsomes and the cell sap fraction acquire some adult type antigens during the course of development, whereas from the immunological point of view the mitochondria do not seem to undergo any signific.ant change, at least up to the stage of the 9th day embryo. MATERIAL
AND
METHODS
Livers were obtained from white Leghorn chicks fasted overnight and killed by decapitation. The tissues were homogenized in 0.44 M sucrosein a glasshomogenizer with a teflon pestle (1 g wet wt/2 ml of sucrose).The homogenate was centrifuged at 1000 g for 15 min. The mitochondria were then obtained by centrifuging at 10,000 g for 10 min: the mitochondrial pellet was washed twice. The supernatant obtained after the sedimentation of the mitochondria was centrifuged at 25,000 g for 20 min and the sediment discarded. From the supernatant, the microsomeswere sedimented at 105,000 g for 60 min. The pellet was washed once. The deoxycholate-insoluble microsomal fraction was obtained by a treatment of the microsomal suspensionin 0.3 per cent sodium deoxycholate (final concentration) and centrifugation at 105,000 g for 60 min. After the sedimentation of the microsomes the upper two thirds of supernatant were collected as “ceI1 sap”. Embryonic livers of white Leghorn embryos were removed on different days of incubation (6, 7, 10, 12, 15 and 18 days) and homogenized as described above. The subcellular fractions were prepared by procedures used for adult liver, but mitochondria and microsomes were not washed. Serum albumin was prepared by DEAE fractionation as suggestedby Sober and Peterson [29]. The antisera against adult liver cell fractions and total serum were obtained in rabbits with Freund’s adjuvant technique [12], using the method previously described [lo]. Four rabbits were utilized for each antigen. Proteins were estimated by the method of Lowry et al. [16]; RNA was extracted by Schneider’s method [27] and estimated by the orcinol method [17]. For the immunological tests the double diffusion on agar [20] was used. As a rule, the antigens were used at the sameprotein concentration. RESULTS
Immunological analysis.-Preliminary experiments using the doublediffusion agar tests showed that the cell fractions prepared from adult chick liver represent a suitable material for the study of the antigens at subcellular level. Experimenfol
Cell Research
31
Serology
of chick liver differenfiation
501
A Fig. I.-Agar double diffusion precipitation bryonic liver against an anti-adult mitochondria chondria; (b) 15.day embryonic mitochondria; embryonic mitochondria.
test
of mitochondria isolated from serum (A). 4.5 mg of protein/ml. (c) g-day embryonic mitochondria:
adult and em(a) adult mitoand (d) 7-day
The immunological analysis indicates that the mitochondrial fraction of embryonic liver, starting from the 9th day of development, exhibits the same reaction pattern as the one prepared from the adult liver. Indeed, five groups of precipitation lines Lvere found in the preparations of both adult and embryo (Fig. l), while in preparations of 7-dayold embryos one of these seemed to be absent. Furthermore, Fig. 2 shows that the mitochondria from embryonic, liver, when reacting with a serum anti-chicken serum, give rise to a precipitation line which seems to correspond to serum albumin. Such a reaction has not been observed \vith adult mitochondria. Several precipitation lines arise in the reaction of microsomes from dif-
Fig. Z.-Agar chicken serum mitochondria;
double diffusion test of mitochondria and microsomes with serum against adult (A). 5 mg of protein/ml. (a) chicken serum albumin; (b) adult serum; (c) adult (d) 15-day embryonic microsomes; and (e) 15-day embryonic mitochondria. Experimental
Cell
Research
31
502
V. D’Amelio,
V. Mutolo
and E. Piazza
Fig. 3.-Agar double diffusion precipitation test of microsomes isolated from adult and embryonic liver against an anti-adult microsomes serum (B) and a serum anti-chicken serum (A). 4 mg of protein/ml. (a) adult serum; (b) chicken serum albumin; (c) 7-day embryonic microsomes; (d) IO-day embryonic microsomes; (e) 15-day embryonic microsomes; (f) ZO-day embryonic microsomes; and (g) adult microsomes.
ferent stages of development, already at the 6th day of incubation, with an anti-adult-microsomes serum (Fig. 3). This pattern, however, is not entirely like that of the adult, as new reactions appear in the course of development. In order to identify the missing material, preparations of embryonic liver microsomes have been compared with adult microsomes and with their deoxycholate-insoluble fraction i.e. RNP (Fig. 4). This has shown that the precipitation lines lacking in the reaction of microsomes of the 12-days liver correspond to adult RNP-bound antigens. The membranaceous material, at least from the 12th day of incubation, does not show any difference from the adult, whereas in younger stages some precipitation lines are absent. Parallel experiments have been carried out with the fraction which does not sediment at 105,000 g (so-called “cell sap”) prepared from adult and embryonic liver which was allowed to react with a serum against adult liver cell sap. Again, it was found that the reaction typical of the adult liver arises gradually in the course of development. In fact, as can be seen in Fig. 5, the reaction between cell sap from a liver of 7-day-old embryos and an antiserum against adult liver cell sap, shows that two groups of precipitaExperimental
Cell
Research
31
Fig. 4.-Agar double diffusion precipitation test of adult microsomes, adult RNP, and embryonic microsomes against anti-adult microsome serum (A) and against serum anti-chicken serum (B). 6.35 mg of protein/ml. (a) adult microsomes; (b) adult RNP; (c) 6-day embryonic microsomes; and (tf) 12-day embryonic microsomes.
tion lines typical of the adult liver are missing. One of them makes its appearance after the 16th day and the other after the 15th day of incubation. Chemical crnnlysis.-In Table I and in Fig. 6 are reported the values of a typical experiment on the RNA/protein N ratio of preparations of microsomes from livers of 6-, 12-, and l&day-old embryos, and adult liver. The data show that the ratio gradually decreases in the course of development and a value close to that of adult microsomes is reached at the 18th day. The TABLE
I. RNA/protein
X ratio adult
of preparations cud embryonic
of microsomes livers. RNA/protein
.i-day 12.day l&day Adult Adult 18.day
embryonic embryonic embryonic microsomes RNP embryonic
microsomcs microsomcs microsomes
RNP
and
RNP
from
X
2.91 2.14 1.02 0.x.5 6.9G 5.20
E.zrxrimenfal
Cell
Research
31
504
V. D’Amelio,
V. Mutolo
Fig. 5.-Agar double diffusion precipitation adult cell sap serum (A). 4 mg of protein/ml. (e) g-day embryonic cell sap; and (d) 7-day
and E. Piazza
test of adult and embryonic cell sap against anti(a) adult cell sap; (0) 15.day embryonic cell sap; embryonic cell sap.
d _ 6
0.500~
O.LOO
0.300.
0.200.
0.100.
OJ 6 Days
Fig. Fig.
6.-RNA/protein
N ratio
12 of incubation
18
6. of embryonic
Fig. 7.-UV spectra of adult and embryonic (A) adult microsomes; (B) B-day embryonic and (D) l&day embryonic microsomes. Experimental
Cell Research
31
2LO 260 Wavelength
Fig. microsomes
at different
microsomes microsomes;
280 (mp)
300
7.
days
of incubation.
suspended in 1.0 M NaCl at pH 7.0. (C) 12-day embryonic microsomes;
Serology
of chick liver differentiation
505
RNA/protein N ratio of the fraction insoluble in 0.3 per cent deoxycholate from liver microsomes of It)-day-old embryos differs slightly from the value of the adult tissue. The UV spectrum of microsomal fractions suspended in 1.0 M NaCl at pH 7.0 indicates a decrease of the 260/280 m,u ratio and an increase of the 240/260 rnp ratio (Fig. 7).
DISCUSSION
Our chemical analyses have shown a progressive decrease of the RNA/ protein N ratio in the microsomal fraction from high values at the 6th day of incubation, to the value typical of the adult tissue, at about the 18th day. These observations are in line with the electron microscope studies of several authors [l, 4, 13, 18, 21, 26, 281 indicating that it is typical of the embryonic cells, during the early stages of development, to have numerous free RIVP particles. Our immunological data show that in the earliest stages thus far examined some of reacting substances like the antigens typical of the adult microsomes are apparently missing. These could be both membrane antigens and antigens bound to the RNP particles. As to the former, however, the fact that we have been unable to detect them does not necessarily imply that they do not exist. In fact, as has just been mentioned, in the early stages the free RNP particles are very plentiful and since we have always diluted our preparations to the same protein concentration, the concentration of the membranaceous material may have been too low to give rise to a reaction. This, on the other hand, does not apply in the case of RNP particles, which are plentiful from the earliest stages. Hence the results indicate the actual lack of some of the adult type antigens, i.e. that these particles are different from those of the adult liver. At this point it seems to be of some interest to recall the hypothesis that free particles synthetize cytoplasmic proteins for growth, whilst the attached RNP particles are mainly devoted to the synthesis of secretory proteins [ 1, 13, 18, 21, 261. As to the presence of serum-like material observed in the mitochondria fraction in the liver of 15-day-old embryos, but never in the preparations of adult liver, we think that the most likely explanation is a particularly heavy contamination with microsomes. The immunological analysis of the cell sap indicates a progressive appearance of material like to the antigen of the adult fraction in the course of 33
- 631814
Experimental
Cell
Research
31
506
V. D’Amelio,
V. Mutolo
and E. Piazza
development. These results seem to reflect the microsomal differentiation already discussed. The present investigations suggest that in the subcellular fractions of adult chick liver two sets of antigens may be distinguished, the one present from the earliest stages of development, the other appearing in the course of development and hence to be considered as an expression of the biochemical differentiation of the liver cells. Further work is now in progress to extend a similar immunological analysis to the other organs.
SUMMARY
Mitochondria, microsomes and cell sap from embryonic chick liver have been tested with sera produced against the homologous fractions from adult liver. Since the earliest stages of development considered, each one of the three subcellular fractions was able to give rise to a number of precipitation lines when reacting with the homologous adult antiserum. In the course of development the pattern of precipitation lines progressively approaches that of the adult. However, the microsomal fraction from embryonic liver, which is known to contain a large amount of free ribonucleoprotein particles, does not give rise to any reaction like that obtained with adult liver ribosomes. Chemical analyses of the embryonic microsomes from different stages of development have demonstrated a high RNA/protein ratio in the earliest stages which decreases until the 18th day of incubation when values identical to those of the adult are reached.
REFERENCES 1. BIRBECK, M. S. C. and 2. BURKE, V., SULLIVAN, 3. GATE, G. ten and VAN
MERGER, E. H., N. P., PETRSON, DOORENMALEN,
IVature 189, 558 (1961). H. and WEED, R., .I. infect. W. J., Proc. Koninkl. Ned&.
Dis. 74, 225 (1944). Adad. Weiensch. 53,849
(1950). 4.
5. 6. 7. 8. 9.
10. 11.
12. 13.
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