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Experimentally induced changes in fructose-1,6-diphosphatase activity in embryonic chick liver and heart cells in vitro
DEVELOPhlENTAL
BIOLOGY
Experimentally phosphatase
11,
335-351
Induced Changes in Fructose-l ,6-diActivity in Embryonic Chick Liver and Heart Cells in Vitro1 Y. KURODA
Department
(1965)
of Biology,
AND
T.
NAGATANI
Faculty
of Science,
Osaka
Accepted
February
8, 1965
University,
Osaka;
Japan
INTRODUCTION
Cellular differentiation in embryonic development represents processesthat lead to specialization of cells, evidenced by their distinctive actual and potential functions. The actual functions, or phenotypic properties, include specialized physiological activities, energy requirements, and specific enzymatic patterns, One aspect of cellular differentiation is the different enzymatic patterns that arise in cells in the course of development. For example, liver cells contain glucose-6-phosphatase (Cori and Cori, 1952), fructose diphosphatase and fructokinase (Perske et al., 1957); heart cells contain tricarboxylic acid cycle enzymes (Gothoskar et al., 1961), succinic dehydrogenase and cytochrome oxidase (Leslie and Yarnell, 1960), and kidney cells contain alkaline phosphatase, adenosine triphosphatase, succinic dehydrogenase, cytochrome oxidase, n-aminooxidase, and glucose-6-phosphatase (Burlington, 1959). It is known that when cells are grown in. vitro, their enzymatic patterns may become considerably changed, usually in the direction of loss of their specificities. An exception to this is the case of retinal glutamine synthetase (glutamotransferase activity), which, in the neural retina of the chick embryo, has a characteristic developmental pattern marked by a sharp activity rise in the later stages of development; this sharp rise can be reproduced in vitro and also elicited precociously in cultures of retinas from much earlier embryos (Moscona and Hubby, 1963; Kirk and Moscona, 1963). The present paper deals with changes in fructose-1,6-diphosphatase ‘This National
investigation was Institutes of Health,
supported United
by research States Public 335
grant Health
RF-00058 Service.
from
the
336
KURODA
AND
NAGATANI
activity in embryonic chick liver and heart cells cultivated under different conditions: in monolayer cultures, as cell-aggregate cultures, in organ cultures, and as grafts on the chorioallantoic membrane of chick embryos. The choice of this enzyme was based on the preliminary finding that its activity in the embryonic liver was considerably higher than in the heart. MATERIALS
AND
METHODS
Livers and hearts were obtained from chick embryos at ages ranging from 6 days’ incubation to hatching and from chicks 3 weeks old. The preparation of cell suspensions and cultivation in vitro of cells and tissue fragments were as previously described (Moscona and Hubby, 1963). The standard culture medium used consisted of Eagle’s basal medium with 1 mM L-glutamine, 10% bovine serum, 2% chick embryo extract, and penicillin-streptomycin at concentrations of 50 units and 50 pg/ml, respectively. Monolayer cultures. Small fragments of liver and heart from S-day chick embryos were dissociated into single cells in the standard culture medium, by flushing lo-15 times through the tip of a fine pipette, after preincubation in calcium- and magnesium-free salt solution for 15 minutes and in 1%trypsin (Difco, 1:250) for 15 minutes at 37°C. Cell suspensions were diluted with culture medium to a final cell concentration of 1.5 X 10” cells per milliliter. Twenty milliliters of diluted cell suspension was dispensed into square bottles and incubated at 38°C. The medium was changed once a week. After various periods of cultivation in monolayer, the cells were washed 3 times in Tyrode’s solution, scraped off, and assayed for enzyme activity. For morphological examination of monolayer-cultured cells, the cell suspensions containing 4 x 10’ cells per milliliter of medium were dispensed into small square bottles ( 15 X 15 X 35 mm), each with a coverslip ( 12 x 30 mm). The culture medium was changed every 2 days. Each series of cultures consisted of 16 bottles. The cells on the coverslips were fixed in Carnoy’s fluid every 2 days and stained in Harris’s alum hematoxylin and eosin for microscopic examination. Cell-aggregate cultures. The dissociated cells obtained from livers and hearts of lo-day chick embryos were dispersed in culture medium as above. Three-milliliter aliquots of cell suspension containing 3.0 x 106 cells were distributed into 25-ml erlenmeyer flasks, which were rotated on a gyratory shaker with a constant speed of 70 r-pm at 38°C for various lengths of time. The culture medium was changed every
ENZYME
ACTIVITY
IN
CULTURED
CELLS
337
2 days. The harvested aggregates were washed 3 times with Tyrode’s solution and assayed for enzyme activity. Samples of cell aggregates from each flask were fixed in Carnoy’s fluid, sectioned, and stained in Harris’s alum hematoxylin and eosin. Organ cultures. Livers and hearts from ll- and B-day chick embryos were cut up into fragments l-3 mm in diameter in Tyrode’s solution. Approximately 10 fragments were suspended in 8 ml culture medium in a TjO-ml erlenmeyer flask, which was rotated at 70 rpm at 38°C for various lengths of time. The culture medium was changed every second or third day. The harvested cultures were rinsed 3 times with Tyrode’s solution and assayed for enzyme activity. Three cultures in each series were harvested at each interval. Some harvested fragments were prepared for histological examination. Gaits to the chorioallantoic membrane. Livers and hearts from loday chick embryos were cut up into fragments l-3 mm in diameter in Tyrode’s solution. These were placed on the chorioallantoic membrane of lo-day chick embryos; the eggs were incubated at 38°C. The grafts were harvested at “-day intervals; they were rinsed 3 times with Tyrode’s solution and assayed for enzyme activity. Some of the grafts were prepared for histological study. Enzynze assay p~ocedztrc. The assay procedure for fructose-1,6-diphosphatase activity was a slight modification of the method of Piigell and McGilvery (1952). The crude lactic extract was prepared by homogenizing the samples for 2 minutes in 4 times their weight of 0.05 hf lactate buffer (pH 4.0) containing 0.001 hl cysteine. The homogenates were centrifuged 30 minutes at 1000 g (3000 rpm) at O”C, and the resulting residue was discarded. To test tubes (12 ml) containing 0.1 ml of 0.03 M cysteine, 0.1 ml of 0.025 N MgSO,, 0.1 ml of 0.005 JK! MnSO,, and 0.4 ml of 0.05 M borate buffer (pH 9.5), 0.2 ml of the enzyme solution was added to make a total volume of 1 ml; the test tubes were cooled for 5 minutes at O”C, then 0.1 ml of 0.05 M fructose-1,6-diphosphate (Sigma, sodium salt) was added to the reaction mixture. The test tubes were incubated at 38°C for 30 minutes, then placed in ice. The tubes were then centrifuged for 10 minutes at 1000 g (3000 rpm), and 1.0 ml of 0.4 N trichloroacetic acid was immediately added to each tube. Control reaction mixtures were prepared for each assay and consisted of all the above ingredients except the enzyme solution. Inorganic phosphate liberated in the reaction mixture was determined by the method of Fiske and Subbarow (1925).
338
KURODA
AND
NAGATANI
Protein was estimated with Folin’s reagent after the method of Lowry et al. ( 1951). The specific activity of the enzyme was defined as the number of micromoles of inorganic phosphate liberated per minute per gram of protein under the above assay conditions. RESULTS
Fructose-1,6-diphosphatase
Activity
Fructose-1,6-diphosphatase hearts from chick embryos
activity in freshly dissected livers of various ages is shown in Table 1. TABLE
FRUCTOSE-1,6-DIPHOSPHATASE
in Embryonic
Liver and Heart
1
ACTIVITY IN THE CHICK EMBRYOS
LIVER
AND
Liver Age of embryo
6 8 11 14 16 18 20 3
Enzyme activity’ (rmole P/min/gm
wet weight)
days days days days days days days weeks
(L Average
THE
HEART
OF
Heart Specific activity” (jmmle P/min/gm protein)
4.22 4.47 4.16 4.22 4.05 3.70 3.70 3.64 values
and
of determinations
263.8 186.3 115.6 41.6 33.9 37.2 28.6 24.3 on independent
Enzyme activity(~mole P/min/gm wet weight)
Specific activity(pmole P/min/gm protein)
0.30 0.53 0.47 0.36 0.44 0.47 0.30 0.46 triple
29.6 35.6 23.6 14.0 12.5 11.9 11.8 5.9 samples.
In the liver, enzyme activity per gram of wet weight was approximately ten times that in the heart. No striking changes in activity were found in the liver, although the values diminished somewhat in the later stages of development. However, specific activity was high in livers from 6day embryos and decreased markedly until the fourteenth day; thereafter it showed a slight decline, which continued after hatching. Specific activity in the heart was lower than in the liver throughout embryonic development and after hatching. Fructose-1,6-diphosphakutase Cell-Aggregate Cultures Dissociated in monolayer
Activity in Cell Cultures of Liver and Heart
and
liver and heart cells from S-day embryos were cultured and harvested after different periods of cultivation.
ENZYME
ACTIVITY
IN
CULTURED
339
CELLS
Cells from equivalent bottles were pooled, and enzyme determined. The results are shown in Figs. 1 and 2.
0 6
2
4
6 -DAYS
IN MONOLAYER
0
2
4 -DAYS
IN AGGREGATE
8
FIG. 1. Specific activity and cell-aggregate cultures values.
II
14 -
AGE
IN
of fructose-1,6-diphosphatase of embryonic chick liver,
The liver cells in monolayer cultures time of approximately 56 hours. Under was a rapid fall in enzyme activity decrease that continued until 10 days generation time was approximately 42 enzyme was maintained at low levels monolayer. Suspensions of dissociated liver and embryos were swirled in gyrating flasks
DAYS -
activity
was
CULTURE CULTURE 20
41
ih monolayer cultures, compared with the in uioo
proliferated with a generation these culture conditions there after 2 days, then a gradual of cultivation. In heart cells hours; specific activity of the for 9 days of cultivation in heart cells from lo-day chick to form aggregates that were
KURODA
340
AND
NAGATANI
200 -
t 0
“““‘~““”
0 6
8
FIG. 2. Specific activity and cell-aggregate cultures values.
4 -
2 0 II
DAYS IN MONOLAYER CULTURE 3 DAYS IN ASQREGATE CULTURE 14 -
AGE
IN
of fructose-l,B-diphosphatase of embryonic chick heart,
DAYS-20
41
in monolayer cultures, compared with the irz viuo
cultured continuously in the gyrating flasks with changes of medium every 2 days. The data for enzyme activities are summarized in Figs. 1 and 2. In liver cells specific activity of fructose-1,6-diphosphatase fell rapidly 2 days after dissociation. After 2 days in flask cultures specific activity increased gradually and reached levels higher than in the intact liver in embryos after a corresponding period of incubation. These high levels of enzyme activity were nevertheless lower than the initial values in the liver tissue at the time of its removal from embryos. Aggregates of heart cells showed a decline in specific activity of the enzyme 3 or 4 days after the beginning of cultivation, and a rise after that time. In the aggregates of heart cells, enzyme activity reached a high level never detected in the intact tissue in the embryo. Fructose-l,6-~~~hosphatnse Activity in Organ Cultures of Embryonic Liver and Heart and in Grafts on the ChoTioa~~ntoic Membrane Fragments of liver and heart from 11-day and M-day chick embryos were maintained as “organ cultures” in flasks with standard
ENZYME
ACTIVITY
IN
CULTURED
341
CELLS
medium and assayed for enzyme activity. The specific activity of the enzyme in liver fragments from embryos of the two ages used decreased rapidly during the first 2 days of cultivation and gradually during further cultivation, as shown in Fig. 3.
01
“““““‘1
6
FIG. embryonic with the
6
liver, values.
2 II
3. Specific activity chick in viva
2
0 0
and
4 4 14-AGE
”
6 IN
”
*
O-DAYS IN ORGAN CUU. DAYS ON C. A.YEMBRANE DAYS20 41
of fructose-1,6-diphosphatase grafts on the chorioallantoic
in organ membrane,
cultures of compared
This decrease in specific activity in cultures of liver fragments coincided with the gradual decline in enzyme activity per wet weight of liver. In cultures of heart fragments, specific activity decreased 2 days after isolation, then rose to a higher level, as in heart cell aggregates during their later period of cultivation (Fig. 4). In 4-day cultures of fragments from H-day embryonic heart, the specific activity of the enzyme was three times that in the freshly dissected tissue.
342
KURODA
AND
NAGATANI
--IN VIVO h
0 2
0 6
6
FIG. 4. Specific activity embryonic chick heart, and with the in vivo values.
4
II
4 I4
A
. ’ ’ ’ ’ ’ ’ O--DAYS IN ORGAN CULT. 6--DAYS ON C.A.MEMSRANE AGE IN DAYS SO 41 ’
’
of fructose-l,&diphosphatase grafts on the chorioallantoic
in organ membrane,
cultures of compared
Fragments of liver and heart from lo-day chick embryos were grafted to the chorioallantoic membrane of lo-day chick embryos and assayed for enzyme activity after various periods of incubation. In the liver grafts, enzyme activity declined rapidly during the first 2 days and gradually during the further 4 days of incubation (Fig. 3). In grafts of heart fragments there was a rise in specific activity, as in cell aggregates and organ cultures of heart. After 6 days on the chorioallantoic membrane, the specific activity of the enzyme in heart reached values 4.7 times higher than the initial activity in IO-day embryonic fresh heart (Fig. 4). Changes in Morphology of Liuer and Heart Cultured in Monolayer
Cells
In l-day monolayer cultures of cells dissociated from liver of S-day chick embryos most of the cells were rounded; few were fibroblastlike (Fig. 5). After 2 days in monolayer the majority of the cells stretched out and elongated on the glass surface, although some round cells were still present (Fig. 6). Numerous mitotic figures were observed. After 3 days in monolayer cultures the number of the elongated cells increased considerably (Fig. 7).
ENZYME
FIGS. FIG. FIG. FIG.
FIG. FIG. FIG.
5-10.
5.
Magnification:
Liver Liver 7. Liver 8. Heart 9. Heart 10. Heart 6.
ACTIVITY
cells cultured cells cultured cells cultured cells cultured cells cultured cells cultured
IN
CULTURED
CELLS
X 100. in in in in in
monolayer monolayer monolayer monolayer monolayer in monolayer
for for for for for for
1 2 3 1 3
day. days. days. day. days. 10 days.
343
344
FIGS.
KURODA
11-16.
Magnification:
Section 12. Section culture. 13. Section culture. FIG. 14. Section days in culture. FIG.
FIG. days in FIG. days in
11.
AND
NAGATANI
X 200.
of lo-day embryonic chick liver. of an aggregate from lo-day embryonic
chick
liver
after
3
of an aggregate
from
lo-day
embryonic
chick
liver
after
5.
of an aggregate
from
lo-day
embryonic
chick
liver
after
7.
ENZYME
ACTIVITY
IN
CULTURED
CELLS
345
In l-day monolayer cultures of cells dissociated from hearts of $-day chick embryos most of the cells were fibroblast-like; only a few were rounded (Fig. 8). After 3 days in culture, the number of the fibroblast-like cells increased considerably and they tended to form .clusters (Fig. 9). D uring 10 days in culture cell proliferation continued and cell size increased (Fig. 10). The conditions in monolayer cultures favored, therefore, the elongated cells, resulting in progressive decrease in the number of the round cells in both liver and heart cell cultures.
Histological
Observations
on Aggregates
of Liver
and Heart Cells
Liver from lo-day chick embryos showed typical parenchymal, sinusoid, and endothelial structures (Fig. 11)) as previously reported for aggregates of ‘i-day embryonic liver (Moscona, 1961). In cross sections, eight to ten cells were arranged radially around a bile capillary. Aggregates obtained from dissociated liver cells after 3 days’ rotation in gyrating flasks consisted predominantly of parenchymal tissue; the sinusoids were considerably narrower than they had been originally (Fig. 12). The nuclei in the parenchymal cells were larger than in the intact tissue. In aggregates rotated for 5 days in gyrating flasks, the parenchymal structure persisted although some of the cells showed signs of degeneration (Fig. 13). After 7 days the parenchymal structure of the aggregates was still recognizable (Fig. 14). The cells appeared smaller, were densely packed, and on the surface of the aggregates were arranged in a layer. These observations indicate that the hepatic parenchyma of lo-day embryos was reconstructed in aggregates of dissociated liver cells and that, in this respect, the aggregates maintained their basic histological structure during the period of cultivation. In the heart tissue of lo-day chick embryos the myocardium consisted of striated muscle strands (Fig. 15). In aggregates obtained from dissociated heart cells after 3 days, myocardial tissue was present (Fig. 16) and persisted for 5 days.
Section of 10-&y embryonic chick heart. 1% Section of an aggregate from lo-day embryonic chick heart after 3 dnys in culture. FIG.
FIG.
15.
FIGS.
17-24.
Magnification:
X 200.
ENZYME
Histological
Observations
ACTIVITY
IN
CULTURED
on Liver and Heart
347
CELLS
Fragment
Cultures
In liver fragments from l&day chick embryos cultured for 2 days, the parenchymal structure was maintained, although the cells showed a reduced cytoplasmic content and a denser packing (Fig. 17). After 46 days in culture, hepatic parenchyma was still present, but in the peripheral parts of the fragments the sinuses increased in volume (Fig. 18). In liver fragments from 18-day chick embryos cultured in gyrating flasks for 2 days, the parenchymal structure of the tissue was maintained (Fig. 19) except for the central portions, where degenerative changes were frequent. However, after 4 days of culture, the parenchymal structure was still retained throughout most of the fragments (Fig. 20). Thus, liver parenchyma persisted in the cultured liver fragments from both lo-day and l&day chick embryos. Heart fragments cultured for 2 days appeared healthy, although spaces appeared within the tissue fragments. Mitotic figures were frequent near the periphery of the fragments. After 4 days of culture, the basic structure of the myocardium persisted (Fig. 21), and even after 7 days numerous myofibrils were present (Fig. 22). Histological Observations on Liver and Heart to the Chorioallantoic Membrane
Grafts
In 4-day grafts of liver fragments from lo-day chick embryos, the parenchymal structure with bile ducts was maintained; as in normal differentiation, abundant mitochondria appeared in the cytoplasm of FIG.
17.
Section
of a fragment
of lo-day
embryonic
chick
liver
cultmed
for
2 days. FIG.
18.
Section
of a fragment
of lo-day
embryonic
chick
liver
cultured
for
4 days. FIG. 2 days.
19.
Section
of a fragment
of 18-day
embryonic
chick
liver
cultured
for
FIG.
20.
Section
of a fragment
of 18-day
embryonic
chick
liver
cultured
for
4 days. FIG. 4 days.
21.
Section
of a fragment
of lo-day
embryonic
chick
heart
cultured
for
FIG.
22.
Section
of a fragment
of IO-day
embryonic
chick
heart
cultured
for
23. 24.
Section Section
of a liver of a liver
7 days. FIG. FIG.
graft graft
on the on the
chorioallantoic chorioallantoic
membrane membrane
for for
4 days. 7 days.
348
KURODA
AND
NAGATANI
the parenchymal cells (Fig. 23). Characteristic differentiation continued in these grafts. At 7 days numerous blood cells were observed in the sinuses (Fig. 24). In a heart fragment from a IO-day chick embryo grafted to the chorioallantoic membrane for 4 days, the myocardial structure was maintained. DISCUSSION
The activity of fructose-1,6-diphosphatase declined rapidly in cells from embryonic chick liver cultured in monolayer. In embryonic heart cells grown in monolayer cultures the activity of the enzyme remained low. Under the conditions employed, both the dissociated liver and heart cells proliferated to form populations of predominantly fibroblast-like cells. The decrease in enzyme activity in these monolayer-cultured liver and heart cells might be related to the transition to a predominantly fibroblast-like population structure, or to metabolic modifications in the cells resulting from their dispersed state; these modifications might range from “leakage” (Eagle and Piez, 1962) to more complex adaptations. It is of interest here that chondrocytes dissociated from chick embryonic femora when cultured in monolayer decline in their cartilage-forming activity and also in their ability to aggregate effectively ( Kuroda, 1963, 196413); these changes are accompanied by increase in the number of fibroblast-like cells and reduction of rounded cells (Kuroda, 1964a), a situation similar to that observed in this study. The possibility exists, therefore, that there may be a causal relationship between the decline in characteristic products under conditions of monolayer cultures, the transition to a population predominating in nonspecific fibroblast-like cells, and changes in surface properties of the cells. It is of interest that while the dissociated liver cells lost most of their fructose-1,6-diphosphatase specific activity, there was a recovery of enzyme activity if the cells were allowed to aggregate. In such aggregates parenchymatous structures were formed and proceeded to develop into hepatic tissue. Reconstruction and organization of hepatic architecture by the cells might be a factor in the recovery of enzymatic activity. Significantly, although activities remained low at completion of aggregation, they rose toward the initial high level, during further cultivation of the aggregates, as their histological organization and differentiation progressed.
ENZYME
ACTIVITY
IN
CULTURED
CELLS
349
Similarly, the formation of myocardial structures in aggregates of dissociated heart cells may also be correlated with the recovery of enzyme activity in the aggregated state. Here too, there was a delay in recovery in enzyme activity in the initial phase of the development of the aggregates. It was reported ( Moscona and Hubby, 1963) that the precocious rise in retinal glutamotransferase activity in culture could be elicited in aggregates of retinal cells as well as in intact retinal tissue. However, the rate of increase in aggregates was slower than in organ cultures maintained for a comparable length of time. Here too, there appears to be a correspondence between the degree of histological organization and the expression by cells of their characteristic enzymatic patterns. The speculation suggests itself that the recovery of specialized metabolic and functional activities by cells in aggregates not only is a function of their close association, but may also be correlated with completion of their histoformative organization. Such notions are supported by the finding that in cultured liver fragments which retained their parenchymal structure the enzymatic change corresponded to that found in normal liver development, i.e., a decline in fructose-1,6-diphosphatase activity. It seems of interest that in aggregates of heart cells, and also in fragment cultures and in grafts of heart tissue, the specific activity of fructose-1,6-diphosphatase reached a higher level than in normal heart development. This raises a number of questions; of particular interest to us is the question whether in the heart in situ this enzyme might not be controlled by suppressor or inhibitory factors that become less effective when heart tissue is isolated in culture. SUMMARY
Fructose-1,6-diphosphatase activity in liver and heart of chick embryos was assayed in freshly isolated tissue, after cultivation of the cells in monolayers, in cell aggregates, in tissue fragment cultures, and in grafts on the chorioallantoic membrane. In the liver, specific activity of this enzyme was high in 6-day embryos, decreased rapidly until the fourteenth day, then decreased slightly until after hatching. In the heart, specific activity was low throughout embryonic development and after hatching. In monolayer cultures of dissociated liver cells from a-day embryos specific activity decreased rapidly after 2 days, and gradually during
KURODA
AND
NAGATANI
further cultivation. In monolayer-cultured heart cells specific activity was maintained at an initial lower level. After 1 day of cultivation fibroblast-like cells and round cells were observed in monolayer cultures of liver and heart cells. After several days in monolayer cultures, fibroblast-like cells predominated over round cells. In aggregates of dissociated liver cells specific activity declined rapidly after 2 days, then increased gradually to near the initial levels. In aggregates of heart cells specific activity declined in the first 2 or 3 days, then rose gradually to a level above that found in the intact heart tissue in viuo. In aggregates of liver and of heart cells, the characteristic histological patterns of liver and heart tissues were reconstructed. In fragment cultures of liver tissue, specific activity of the enzyme decreased rapidly after 2 days, and gradually during further cultivation In fragment cultures of heart, specific activity decreased after 2 days, then rose to a high level. In grafts of liver tissue to the chorioallantoic membrane there was a gradual decline in the specific activity of the enzyme; in grafts of heart tissue specific activity rose during 6 days of incubation. Histological observations on liver fragments in culture and in chorioallantoic grafts showed that their parenchymal structure was retained for 6 days. In heart fragments in culture and in chorioallantoic grafts the myocardial structure was retained and abundant myofibrils were present. The authors wish to express their appreciation to Professor Hideo Kikkawa for his advice and criticism throughout the course of this work. Grateful acknowledgment is made to Professor A. A. Moscona, Department of Zoology, University of Chicago, for reading the manuscript. REFERENCES H. ( 1959). Enzyme patterns in cultured kidney cells. Am. J* BURLINGTON, Physiol. 197, 68-70. CORI, G. T., and CORI, C. F. ( 1952). Glucose-6-phosphatase of the liver in glycogen storage disease. J. Biol. C&m. 199, 661-667. EAGLE, H., and PIEZ, K. ( 1962). The population-dependent requirement by cultured mammalian cells for metabolites which they can synthesize. J. Exptl. Med. 116, 29-43. FISKE, C. H., and SUBBAROW, Y. (1925). Th e calorimetric determination of phosphorus. J. Biol. Chem. 66, 375-400.
ENZYivlE
ACTIVITY
IN
CULTURED
351
CELLS
B. P., RAINA, P. N., and RA~IAKRISHNAK, C. V. (1961). Activities of the tricarboxylic acid cycle enzymes in fresh, nutritionally depleted and cultivatedexplantsof chickandrat heart tissues, Exptl. Cell Res. 24, 272-279. KIKK, D. L., and MOSCONA, A. A. (1963). Synthesi\ : of exp erimentnlly induced GOTHOSKAR,
glutamine
synthetase
( glutamotransferase
activity
) in
embryonic
chick
retina
in vitro. Develop. Biol. 8, 341-357. KUHODA, Y. (1963). Changes in aggregation and diflerentiation of cartilage cells grown in monolayer cultures. Erptl. Cell Ras. 30, 446448. KURODA, Y. (1964a). Studies on cartilage cells in tiitro. I. Morphology and growth of cartilage cells in monolayer cultures. Exptl. Cell Res. 35, 326-336. KURODA, Y. (19641~). Studies on cartilage cells in vitro. II. Changes in aggregation and in cartilage-forming activity of cells maintained in monolaver cultures.
Exptl. Cell Res. 35, 337-348. LESLIE, I., and YAHNELL, hI. ( 1960). Succinic dehydrogenase and cytochrome oxidase activities in cell cultures. J. Biophys. Biochem. Cytol. 7, 265-272. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265275. MOXOKA, A. ( 1961). Rotation-mediated histogenetic aggregation of dissociated cells. Erptl. Cell Res. 22, 455-475. Mosc:o~-a, A. A., and HUBBY, J. L. ( 1963). Experimentally induced changes in glutamotransferase activity in embryonic tissue. Develop. Biol. 7, 192-206. PERSKE, \V. F., PARKS, R. E., and WALKER, D. L. (1957). Metabolic differences between hcpatic parenchymal cells and a cultured cell line from liver. Science 125, 1290-1291. P~GELL, B. M., and ~ICGILVERY, R. 1%‘. ( 1952). The proteolytic activation of fructose-1,6-phosphatase. J. Bid. Chem. 197, 293-302.
BIOLOGY
Experimentally phosphatase
11,
335-351
Induced Changes in Fructose-l ,6-diActivity in Embryonic Chick Liver and Heart Cells in Vitro1 Y. KURODA
Department
(1965)
of Biology,
AND
T.
NAGATANI
Faculty
of Science,
Osaka
Accepted
February
8, 1965
University,
Osaka;
Japan
INTRODUCTION
Cellular differentiation in embryonic development represents processesthat lead to specialization of cells, evidenced by their distinctive actual and potential functions. The actual functions, or phenotypic properties, include specialized physiological activities, energy requirements, and specific enzymatic patterns, One aspect of cellular differentiation is the different enzymatic patterns that arise in cells in the course of development. For example, liver cells contain glucose-6-phosphatase (Cori and Cori, 1952), fructose diphosphatase and fructokinase (Perske et al., 1957); heart cells contain tricarboxylic acid cycle enzymes (Gothoskar et al., 1961), succinic dehydrogenase and cytochrome oxidase (Leslie and Yarnell, 1960), and kidney cells contain alkaline phosphatase, adenosine triphosphatase, succinic dehydrogenase, cytochrome oxidase, n-aminooxidase, and glucose-6-phosphatase (Burlington, 1959). It is known that when cells are grown in. vitro, their enzymatic patterns may become considerably changed, usually in the direction of loss of their specificities. An exception to this is the case of retinal glutamine synthetase (glutamotransferase activity), which, in the neural retina of the chick embryo, has a characteristic developmental pattern marked by a sharp activity rise in the later stages of development; this sharp rise can be reproduced in vitro and also elicited precociously in cultures of retinas from much earlier embryos (Moscona and Hubby, 1963; Kirk and Moscona, 1963). The present paper deals with changes in fructose-1,6-diphosphatase ‘This National
investigation was Institutes of Health,
supported United
by research States Public 335
grant Health
RF-00058 Service.
from
the
336
KURODA
AND
NAGATANI
activity in embryonic chick liver and heart cells cultivated under different conditions: in monolayer cultures, as cell-aggregate cultures, in organ cultures, and as grafts on the chorioallantoic membrane of chick embryos. The choice of this enzyme was based on the preliminary finding that its activity in the embryonic liver was considerably higher than in the heart. MATERIALS
AND
METHODS
Livers and hearts were obtained from chick embryos at ages ranging from 6 days’ incubation to hatching and from chicks 3 weeks old. The preparation of cell suspensions and cultivation in vitro of cells and tissue fragments were as previously described (Moscona and Hubby, 1963). The standard culture medium used consisted of Eagle’s basal medium with 1 mM L-glutamine, 10% bovine serum, 2% chick embryo extract, and penicillin-streptomycin at concentrations of 50 units and 50 pg/ml, respectively. Monolayer cultures. Small fragments of liver and heart from S-day chick embryos were dissociated into single cells in the standard culture medium, by flushing lo-15 times through the tip of a fine pipette, after preincubation in calcium- and magnesium-free salt solution for 15 minutes and in 1%trypsin (Difco, 1:250) for 15 minutes at 37°C. Cell suspensions were diluted with culture medium to a final cell concentration of 1.5 X 10” cells per milliliter. Twenty milliliters of diluted cell suspension was dispensed into square bottles and incubated at 38°C. The medium was changed once a week. After various periods of cultivation in monolayer, the cells were washed 3 times in Tyrode’s solution, scraped off, and assayed for enzyme activity. For morphological examination of monolayer-cultured cells, the cell suspensions containing 4 x 10’ cells per milliliter of medium were dispensed into small square bottles ( 15 X 15 X 35 mm), each with a coverslip ( 12 x 30 mm). The culture medium was changed every 2 days. Each series of cultures consisted of 16 bottles. The cells on the coverslips were fixed in Carnoy’s fluid every 2 days and stained in Harris’s alum hematoxylin and eosin for microscopic examination. Cell-aggregate cultures. The dissociated cells obtained from livers and hearts of lo-day chick embryos were dispersed in culture medium as above. Three-milliliter aliquots of cell suspension containing 3.0 x 106 cells were distributed into 25-ml erlenmeyer flasks, which were rotated on a gyratory shaker with a constant speed of 70 r-pm at 38°C for various lengths of time. The culture medium was changed every
ENZYME
ACTIVITY
IN
CULTURED
CELLS
337
2 days. The harvested aggregates were washed 3 times with Tyrode’s solution and assayed for enzyme activity. Samples of cell aggregates from each flask were fixed in Carnoy’s fluid, sectioned, and stained in Harris’s alum hematoxylin and eosin. Organ cultures. Livers and hearts from ll- and B-day chick embryos were cut up into fragments l-3 mm in diameter in Tyrode’s solution. Approximately 10 fragments were suspended in 8 ml culture medium in a TjO-ml erlenmeyer flask, which was rotated at 70 rpm at 38°C for various lengths of time. The culture medium was changed every second or third day. The harvested cultures were rinsed 3 times with Tyrode’s solution and assayed for enzyme activity. Three cultures in each series were harvested at each interval. Some harvested fragments were prepared for histological examination. Gaits to the chorioallantoic membrane. Livers and hearts from loday chick embryos were cut up into fragments l-3 mm in diameter in Tyrode’s solution. These were placed on the chorioallantoic membrane of lo-day chick embryos; the eggs were incubated at 38°C. The grafts were harvested at “-day intervals; they were rinsed 3 times with Tyrode’s solution and assayed for enzyme activity. Some of the grafts were prepared for histological study. Enzynze assay p~ocedztrc. The assay procedure for fructose-1,6-diphosphatase activity was a slight modification of the method of Piigell and McGilvery (1952). The crude lactic extract was prepared by homogenizing the samples for 2 minutes in 4 times their weight of 0.05 hf lactate buffer (pH 4.0) containing 0.001 hl cysteine. The homogenates were centrifuged 30 minutes at 1000 g (3000 rpm) at O”C, and the resulting residue was discarded. To test tubes (12 ml) containing 0.1 ml of 0.03 M cysteine, 0.1 ml of 0.025 N MgSO,, 0.1 ml of 0.005 JK! MnSO,, and 0.4 ml of 0.05 M borate buffer (pH 9.5), 0.2 ml of the enzyme solution was added to make a total volume of 1 ml; the test tubes were cooled for 5 minutes at O”C, then 0.1 ml of 0.05 M fructose-1,6-diphosphate (Sigma, sodium salt) was added to the reaction mixture. The test tubes were incubated at 38°C for 30 minutes, then placed in ice. The tubes were then centrifuged for 10 minutes at 1000 g (3000 rpm), and 1.0 ml of 0.4 N trichloroacetic acid was immediately added to each tube. Control reaction mixtures were prepared for each assay and consisted of all the above ingredients except the enzyme solution. Inorganic phosphate liberated in the reaction mixture was determined by the method of Fiske and Subbarow (1925).
338
KURODA
AND
NAGATANI
Protein was estimated with Folin’s reagent after the method of Lowry et al. ( 1951). The specific activity of the enzyme was defined as the number of micromoles of inorganic phosphate liberated per minute per gram of protein under the above assay conditions. RESULTS
Fructose-1,6-diphosphatase
Activity
Fructose-1,6-diphosphatase hearts from chick embryos
activity in freshly dissected livers of various ages is shown in Table 1. TABLE
FRUCTOSE-1,6-DIPHOSPHATASE
in Embryonic
Liver and Heart
1
ACTIVITY IN THE CHICK EMBRYOS
LIVER
AND
Liver Age of embryo
6 8 11 14 16 18 20 3
Enzyme activity’ (rmole P/min/gm
wet weight)
days days days days days days days weeks
(L Average
THE
HEART
OF
Heart Specific activity” (jmmle P/min/gm protein)
4.22 4.47 4.16 4.22 4.05 3.70 3.70 3.64 values
and
of determinations
263.8 186.3 115.6 41.6 33.9 37.2 28.6 24.3 on independent
Enzyme activity(~mole P/min/gm wet weight)
Specific activity(pmole P/min/gm protein)
0.30 0.53 0.47 0.36 0.44 0.47 0.30 0.46 triple
29.6 35.6 23.6 14.0 12.5 11.9 11.8 5.9 samples.
In the liver, enzyme activity per gram of wet weight was approximately ten times that in the heart. No striking changes in activity were found in the liver, although the values diminished somewhat in the later stages of development. However, specific activity was high in livers from 6day embryos and decreased markedly until the fourteenth day; thereafter it showed a slight decline, which continued after hatching. Specific activity in the heart was lower than in the liver throughout embryonic development and after hatching. Fructose-1,6-diphosphakutase Cell-Aggregate Cultures Dissociated in monolayer
Activity in Cell Cultures of Liver and Heart
and
liver and heart cells from S-day embryos were cultured and harvested after different periods of cultivation.
ENZYME
ACTIVITY
IN
CULTURED
339
CELLS
Cells from equivalent bottles were pooled, and enzyme determined. The results are shown in Figs. 1 and 2.
0 6
2
4
6 -DAYS
IN MONOLAYER
0
2
4 -DAYS
IN AGGREGATE
8
FIG. 1. Specific activity and cell-aggregate cultures values.
II
14 -
AGE
IN
of fructose-1,6-diphosphatase of embryonic chick liver,
The liver cells in monolayer cultures time of approximately 56 hours. Under was a rapid fall in enzyme activity decrease that continued until 10 days generation time was approximately 42 enzyme was maintained at low levels monolayer. Suspensions of dissociated liver and embryos were swirled in gyrating flasks
DAYS -
activity
was
CULTURE CULTURE 20
41
ih monolayer cultures, compared with the in uioo
proliferated with a generation these culture conditions there after 2 days, then a gradual of cultivation. In heart cells hours; specific activity of the for 9 days of cultivation in heart cells from lo-day chick to form aggregates that were
KURODA
340
AND
NAGATANI
200 -
t 0
“““‘~““”
0 6
8
FIG. 2. Specific activity and cell-aggregate cultures values.
4 -
2 0 II
DAYS IN MONOLAYER CULTURE 3 DAYS IN ASQREGATE CULTURE 14 -
AGE
IN
of fructose-l,B-diphosphatase of embryonic chick heart,
DAYS-20
41
in monolayer cultures, compared with the irz viuo
cultured continuously in the gyrating flasks with changes of medium every 2 days. The data for enzyme activities are summarized in Figs. 1 and 2. In liver cells specific activity of fructose-1,6-diphosphatase fell rapidly 2 days after dissociation. After 2 days in flask cultures specific activity increased gradually and reached levels higher than in the intact liver in embryos after a corresponding period of incubation. These high levels of enzyme activity were nevertheless lower than the initial values in the liver tissue at the time of its removal from embryos. Aggregates of heart cells showed a decline in specific activity of the enzyme 3 or 4 days after the beginning of cultivation, and a rise after that time. In the aggregates of heart cells, enzyme activity reached a high level never detected in the intact tissue in the embryo. Fructose-l,6-~~~hosphatnse Activity in Organ Cultures of Embryonic Liver and Heart and in Grafts on the ChoTioa~~ntoic Membrane Fragments of liver and heart from 11-day and M-day chick embryos were maintained as “organ cultures” in flasks with standard
ENZYME
ACTIVITY
IN
CULTURED
341
CELLS
medium and assayed for enzyme activity. The specific activity of the enzyme in liver fragments from embryos of the two ages used decreased rapidly during the first 2 days of cultivation and gradually during further cultivation, as shown in Fig. 3.
01
“““““‘1
6
FIG. embryonic with the
6
liver, values.
2 II
3. Specific activity chick in viva
2
0 0
and
4 4 14-AGE
”
6 IN
”
*
O-DAYS IN ORGAN CUU. DAYS ON C. A.YEMBRANE DAYS20 41
of fructose-1,6-diphosphatase grafts on the chorioallantoic
in organ membrane,
cultures of compared
This decrease in specific activity in cultures of liver fragments coincided with the gradual decline in enzyme activity per wet weight of liver. In cultures of heart fragments, specific activity decreased 2 days after isolation, then rose to a higher level, as in heart cell aggregates during their later period of cultivation (Fig. 4). In 4-day cultures of fragments from H-day embryonic heart, the specific activity of the enzyme was three times that in the freshly dissected tissue.
342
KURODA
AND
NAGATANI
--IN VIVO h
0 2
0 6
6
FIG. 4. Specific activity embryonic chick heart, and with the in vivo values.
4
II
4 I4
A
. ’ ’ ’ ’ ’ ’ O--DAYS IN ORGAN CULT. 6--DAYS ON C.A.MEMSRANE AGE IN DAYS SO 41 ’
’
of fructose-l,&diphosphatase grafts on the chorioallantoic
in organ membrane,
cultures of compared
Fragments of liver and heart from lo-day chick embryos were grafted to the chorioallantoic membrane of lo-day chick embryos and assayed for enzyme activity after various periods of incubation. In the liver grafts, enzyme activity declined rapidly during the first 2 days and gradually during the further 4 days of incubation (Fig. 3). In grafts of heart fragments there was a rise in specific activity, as in cell aggregates and organ cultures of heart. After 6 days on the chorioallantoic membrane, the specific activity of the enzyme in heart reached values 4.7 times higher than the initial activity in IO-day embryonic fresh heart (Fig. 4). Changes in Morphology of Liuer and Heart Cultured in Monolayer
Cells
In l-day monolayer cultures of cells dissociated from liver of S-day chick embryos most of the cells were rounded; few were fibroblastlike (Fig. 5). After 2 days in monolayer the majority of the cells stretched out and elongated on the glass surface, although some round cells were still present (Fig. 6). Numerous mitotic figures were observed. After 3 days in monolayer cultures the number of the elongated cells increased considerably (Fig. 7).
ENZYME
FIGS. FIG. FIG. FIG.
FIG. FIG. FIG.
5-10.
5.
Magnification:
Liver Liver 7. Liver 8. Heart 9. Heart 10. Heart 6.
ACTIVITY
cells cultured cells cultured cells cultured cells cultured cells cultured cells cultured
IN
CULTURED
CELLS
X 100. in in in in in
monolayer monolayer monolayer monolayer monolayer in monolayer
for for for for for for
1 2 3 1 3
day. days. days. day. days. 10 days.
343
344
FIGS.
KURODA
11-16.
Magnification:
Section 12. Section culture. 13. Section culture. FIG. 14. Section days in culture. FIG.
FIG. days in FIG. days in
11.
AND
NAGATANI
X 200.
of lo-day embryonic chick liver. of an aggregate from lo-day embryonic
chick
liver
after
3
of an aggregate
from
lo-day
embryonic
chick
liver
after
5.
of an aggregate
from
lo-day
embryonic
chick
liver
after
7.
ENZYME
ACTIVITY
IN
CULTURED
CELLS
345
In l-day monolayer cultures of cells dissociated from hearts of $-day chick embryos most of the cells were fibroblast-like; only a few were rounded (Fig. 8). After 3 days in culture, the number of the fibroblast-like cells increased considerably and they tended to form .clusters (Fig. 9). D uring 10 days in culture cell proliferation continued and cell size increased (Fig. 10). The conditions in monolayer cultures favored, therefore, the elongated cells, resulting in progressive decrease in the number of the round cells in both liver and heart cell cultures.
Histological
Observations
on Aggregates
of Liver
and Heart Cells
Liver from lo-day chick embryos showed typical parenchymal, sinusoid, and endothelial structures (Fig. 11)) as previously reported for aggregates of ‘i-day embryonic liver (Moscona, 1961). In cross sections, eight to ten cells were arranged radially around a bile capillary. Aggregates obtained from dissociated liver cells after 3 days’ rotation in gyrating flasks consisted predominantly of parenchymal tissue; the sinusoids were considerably narrower than they had been originally (Fig. 12). The nuclei in the parenchymal cells were larger than in the intact tissue. In aggregates rotated for 5 days in gyrating flasks, the parenchymal structure persisted although some of the cells showed signs of degeneration (Fig. 13). After 7 days the parenchymal structure of the aggregates was still recognizable (Fig. 14). The cells appeared smaller, were densely packed, and on the surface of the aggregates were arranged in a layer. These observations indicate that the hepatic parenchyma of lo-day embryos was reconstructed in aggregates of dissociated liver cells and that, in this respect, the aggregates maintained their basic histological structure during the period of cultivation. In the heart tissue of lo-day chick embryos the myocardium consisted of striated muscle strands (Fig. 15). In aggregates obtained from dissociated heart cells after 3 days, myocardial tissue was present (Fig. 16) and persisted for 5 days.
Section of 10-&y embryonic chick heart. 1% Section of an aggregate from lo-day embryonic chick heart after 3 dnys in culture. FIG.
FIG.
15.
FIGS.
17-24.
Magnification:
X 200.
ENZYME
Histological
Observations
ACTIVITY
IN
CULTURED
on Liver and Heart
347
CELLS
Fragment
Cultures
In liver fragments from l&day chick embryos cultured for 2 days, the parenchymal structure was maintained, although the cells showed a reduced cytoplasmic content and a denser packing (Fig. 17). After 46 days in culture, hepatic parenchyma was still present, but in the peripheral parts of the fragments the sinuses increased in volume (Fig. 18). In liver fragments from 18-day chick embryos cultured in gyrating flasks for 2 days, the parenchymal structure of the tissue was maintained (Fig. 19) except for the central portions, where degenerative changes were frequent. However, after 4 days of culture, the parenchymal structure was still retained throughout most of the fragments (Fig. 20). Thus, liver parenchyma persisted in the cultured liver fragments from both lo-day and l&day chick embryos. Heart fragments cultured for 2 days appeared healthy, although spaces appeared within the tissue fragments. Mitotic figures were frequent near the periphery of the fragments. After 4 days of culture, the basic structure of the myocardium persisted (Fig. 21), and even after 7 days numerous myofibrils were present (Fig. 22). Histological Observations on Liver and Heart to the Chorioallantoic Membrane
Grafts
In 4-day grafts of liver fragments from lo-day chick embryos, the parenchymal structure with bile ducts was maintained; as in normal differentiation, abundant mitochondria appeared in the cytoplasm of FIG.
17.
Section
of a fragment
of lo-day
embryonic
chick
liver
cultmed
for
2 days. FIG.
18.
Section
of a fragment
of lo-day
embryonic
chick
liver
cultured
for
4 days. FIG. 2 days.
19.
Section
of a fragment
of 18-day
embryonic
chick
liver
cultured
for
FIG.
20.
Section
of a fragment
of 18-day
embryonic
chick
liver
cultured
for
4 days. FIG. 4 days.
21.
Section
of a fragment
of lo-day
embryonic
chick
heart
cultured
for
FIG.
22.
Section
of a fragment
of IO-day
embryonic
chick
heart
cultured
for
23. 24.
Section Section
of a liver of a liver
7 days. FIG. FIG.
graft graft
on the on the
chorioallantoic chorioallantoic
membrane membrane
for for
4 days. 7 days.
348
KURODA
AND
NAGATANI
the parenchymal cells (Fig. 23). Characteristic differentiation continued in these grafts. At 7 days numerous blood cells were observed in the sinuses (Fig. 24). In a heart fragment from a IO-day chick embryo grafted to the chorioallantoic membrane for 4 days, the myocardial structure was maintained. DISCUSSION
The activity of fructose-1,6-diphosphatase declined rapidly in cells from embryonic chick liver cultured in monolayer. In embryonic heart cells grown in monolayer cultures the activity of the enzyme remained low. Under the conditions employed, both the dissociated liver and heart cells proliferated to form populations of predominantly fibroblast-like cells. The decrease in enzyme activity in these monolayer-cultured liver and heart cells might be related to the transition to a predominantly fibroblast-like population structure, or to metabolic modifications in the cells resulting from their dispersed state; these modifications might range from “leakage” (Eagle and Piez, 1962) to more complex adaptations. It is of interest here that chondrocytes dissociated from chick embryonic femora when cultured in monolayer decline in their cartilage-forming activity and also in their ability to aggregate effectively ( Kuroda, 1963, 196413); these changes are accompanied by increase in the number of fibroblast-like cells and reduction of rounded cells (Kuroda, 1964a), a situation similar to that observed in this study. The possibility exists, therefore, that there may be a causal relationship between the decline in characteristic products under conditions of monolayer cultures, the transition to a population predominating in nonspecific fibroblast-like cells, and changes in surface properties of the cells. It is of interest that while the dissociated liver cells lost most of their fructose-1,6-diphosphatase specific activity, there was a recovery of enzyme activity if the cells were allowed to aggregate. In such aggregates parenchymatous structures were formed and proceeded to develop into hepatic tissue. Reconstruction and organization of hepatic architecture by the cells might be a factor in the recovery of enzymatic activity. Significantly, although activities remained low at completion of aggregation, they rose toward the initial high level, during further cultivation of the aggregates, as their histological organization and differentiation progressed.
ENZYME
ACTIVITY
IN
CULTURED
CELLS
349
Similarly, the formation of myocardial structures in aggregates of dissociated heart cells may also be correlated with the recovery of enzyme activity in the aggregated state. Here too, there was a delay in recovery in enzyme activity in the initial phase of the development of the aggregates. It was reported ( Moscona and Hubby, 1963) that the precocious rise in retinal glutamotransferase activity in culture could be elicited in aggregates of retinal cells as well as in intact retinal tissue. However, the rate of increase in aggregates was slower than in organ cultures maintained for a comparable length of time. Here too, there appears to be a correspondence between the degree of histological organization and the expression by cells of their characteristic enzymatic patterns. The speculation suggests itself that the recovery of specialized metabolic and functional activities by cells in aggregates not only is a function of their close association, but may also be correlated with completion of their histoformative organization. Such notions are supported by the finding that in cultured liver fragments which retained their parenchymal structure the enzymatic change corresponded to that found in normal liver development, i.e., a decline in fructose-1,6-diphosphatase activity. It seems of interest that in aggregates of heart cells, and also in fragment cultures and in grafts of heart tissue, the specific activity of fructose-1,6-diphosphatase reached a higher level than in normal heart development. This raises a number of questions; of particular interest to us is the question whether in the heart in situ this enzyme might not be controlled by suppressor or inhibitory factors that become less effective when heart tissue is isolated in culture. SUMMARY
Fructose-1,6-diphosphatase activity in liver and heart of chick embryos was assayed in freshly isolated tissue, after cultivation of the cells in monolayers, in cell aggregates, in tissue fragment cultures, and in grafts on the chorioallantoic membrane. In the liver, specific activity of this enzyme was high in 6-day embryos, decreased rapidly until the fourteenth day, then decreased slightly until after hatching. In the heart, specific activity was low throughout embryonic development and after hatching. In monolayer cultures of dissociated liver cells from a-day embryos specific activity decreased rapidly after 2 days, and gradually during
KURODA
AND
NAGATANI
further cultivation. In monolayer-cultured heart cells specific activity was maintained at an initial lower level. After 1 day of cultivation fibroblast-like cells and round cells were observed in monolayer cultures of liver and heart cells. After several days in monolayer cultures, fibroblast-like cells predominated over round cells. In aggregates of dissociated liver cells specific activity declined rapidly after 2 days, then increased gradually to near the initial levels. In aggregates of heart cells specific activity declined in the first 2 or 3 days, then rose gradually to a level above that found in the intact heart tissue in viuo. In aggregates of liver and of heart cells, the characteristic histological patterns of liver and heart tissues were reconstructed. In fragment cultures of liver tissue, specific activity of the enzyme decreased rapidly after 2 days, and gradually during further cultivation In fragment cultures of heart, specific activity decreased after 2 days, then rose to a high level. In grafts of liver tissue to the chorioallantoic membrane there was a gradual decline in the specific activity of the enzyme; in grafts of heart tissue specific activity rose during 6 days of incubation. Histological observations on liver fragments in culture and in chorioallantoic grafts showed that their parenchymal structure was retained for 6 days. In heart fragments in culture and in chorioallantoic grafts the myocardial structure was retained and abundant myofibrils were present. The authors wish to express their appreciation to Professor Hideo Kikkawa for his advice and criticism throughout the course of this work. Grateful acknowledgment is made to Professor A. A. Moscona, Department of Zoology, University of Chicago, for reading the manuscript. REFERENCES H. ( 1959). Enzyme patterns in cultured kidney cells. Am. J* BURLINGTON, Physiol. 197, 68-70. CORI, G. T., and CORI, C. F. ( 1952). Glucose-6-phosphatase of the liver in glycogen storage disease. J. Biol. C&m. 199, 661-667. EAGLE, H., and PIEZ, K. ( 1962). The population-dependent requirement by cultured mammalian cells for metabolites which they can synthesize. J. Exptl. Med. 116, 29-43. FISKE, C. H., and SUBBAROW, Y. (1925). Th e calorimetric determination of phosphorus. J. Biol. Chem. 66, 375-400.
ENZYivlE
ACTIVITY
IN
CULTURED
351
CELLS
B. P., RAINA, P. N., and RA~IAKRISHNAK, C. V. (1961). Activities of the tricarboxylic acid cycle enzymes in fresh, nutritionally depleted and cultivatedexplantsof chickandrat heart tissues, Exptl. Cell Res. 24, 272-279. KIKK, D. L., and MOSCONA, A. A. (1963). Synthesi\ : of exp erimentnlly induced GOTHOSKAR,
glutamine
synthetase
( glutamotransferase
activity
) in
embryonic
chick
retina
in vitro. Develop. Biol. 8, 341-357. KUHODA, Y. (1963). Changes in aggregation and diflerentiation of cartilage cells grown in monolayer cultures. Erptl. Cell Ras. 30, 446448. KURODA, Y. (1964a). Studies on cartilage cells in tiitro. I. Morphology and growth of cartilage cells in monolayer cultures. Exptl. Cell Res. 35, 326-336. KURODA, Y. (19641~). Studies on cartilage cells in vitro. II. Changes in aggregation and in cartilage-forming activity of cells maintained in monolaver cultures.
Exptl. Cell Res. 35, 337-348. LESLIE, I., and YAHNELL, hI. ( 1960). Succinic dehydrogenase and cytochrome oxidase activities in cell cultures. J. Biophys. Biochem. Cytol. 7, 265-272. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265275. MOXOKA, A. ( 1961). Rotation-mediated histogenetic aggregation of dissociated cells. Erptl. Cell Res. 22, 455-475. Mosc:o~-a, A. A., and HUBBY, J. L. ( 1963). Experimentally induced changes in glutamotransferase activity in embryonic tissue. Develop. Biol. 7, 192-206. PERSKE, \V. F., PARKS, R. E., and WALKER, D. L. (1957). Metabolic differences between hcpatic parenchymal cells and a cultured cell line from liver. Science 125, 1290-1291. P~GELL, B. M., and ~ICGILVERY, R. 1%‘. ( 1952). The proteolytic activation of fructose-1,6-phosphatase. J. Bid. Chem. 197, 293-302.