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Uptake and incorporation of leucine and thymidine in developing sea urchin eggs
8 406
Experimenfal
UPTAKE
AND
THYMIDINE
INCORPORATION IN DEVELOPING
THE EFFECT
1968 by Academic Press Inc.
Cell Research 51,
OF LEUCINE SEA URCHIN
406-412
(1968)
AND EGGS
OF HEXAHOMOSERINE’
G. BELLEMARE, J. PINARD, A. AUBIN and G. H. COUSINEAU Laboratoire
de Biologic
Mol&zulaire,
UniversitB
de Montr&d,
Montrt%zl, P.Q., Canada
Received September26, 1967
SIIORTI~Y after fertilization, or parthenogenetic activation, of sea urchin eggs there is a rapid increase in the incorporation of labelled amino acids into proteins, and this activity continues until the mesenchpme blastula stage [3, 8, 91. Although RNA metabolism is remarkably low during the early period of development the rate of DNA synthesis increases with cell division [5, 71. Our attention was arrested lately by the possibility that these values might have to be qualified in some n-ay. Indeed, these considerations came from observations made recently by l’iatigorsky and m’hiteley [13] and Mitchison and Cummins [lo] who reported on the uptake of uridine, valine and cytidine in developing eggs and found that there is a permeability change during development, e.g., the permeability rises sharply after fertilization and reaches a maximum before the first cleavage, the permeation rate then becomes constant until the fifth cleavage. Moreover, because of a permeability differential, Gross and Fry [6I suggested that “the fraction of total uptake incorporated into macromolecules might reflect more accurately protein synthesis”, and these authors found that there is a close relationship betiveen changes in the rate of permeability and amino acid incorporation into protein during the first cell cycle. Their data suggest that penetration may limit the number of counts incorporated into protein. Thus, since incorporation of radioactive precursors into macromolecules is a direct function of those labelled molecules present at any one time in the cell pool, we decided to reinvestigate the “active syntheses” observed during development of sea urchin eggs and to qualify these values, if necessary, by those obtained from penetration data. \Ve therefore chose to study 1 This work was supported in part by grant no. 211 from the Jane Coffin Childs Jlemorial Fund for Medical Research, grant no. DRG-918AT from the Damon Runyon Memorial Fund for Cancer Research and by grant no. A-3624 from the National Research Council of Canada. Experimental
Cell Research 51
Leucine and thymidine
in developing sea urchin
eggs
407
not only normal development but also development affected by the presence of hexahomoserine (d-amino-a-hydroxycaproic acid) which is known to be a potent inhibitor of protein synthesis [‘L, 111. MATERIAL
AND
METHODS
Eggs and sperms from the sea urchin Sfrongyiocenfrotus purpuratus (Pacific Biomarine Supply Company, Venice, Calif.) were obtained by injection of 0.53 M KC1 [12]. The eggs, with jelly coats removed, were washed several times with Milliporefiltered sea water and tested for fertilization. Only those batches that gave more than 95 per cent membrane elevation were used in these experiments [5]. The eggs, with and without hexahomoserine, were fertilized and allowed to develop at 15°C with gentle agitation. Aliquots of the cell suspensions were taken before and at different intervals after fertilization and placed in the presence of a radioactive precursor for 30 min. At the end of this period (pulse) the eggs or embryos from one sample were separated into two batches and gently centrifuged. One batch was repeatedly washed with filtered sea water containing loo-fold excess unlabelled precursor, spun down, and the cells taken up in 10 ml of Bray’s solution for counting in a liquid scintillation system (Nuclear Chicago Mark 1). The other batch was similarly treated and at the same time, but in this case the reaction was stopped by addition of cold TCA (5 per cent final concentration) containing excess unlabelled precursor (results obtained from hot TCA treatment of the samples [l, 31 did not differ appreciably from those obtained from the cold TCA treatments). The acidinsoluble material was washed with cold TCA and filtered sea water, and finally taken up in Bray’s for counting. In this way it was possible to measure total incorporation and total uptake (from which incorporation values were later subtracted). In other experiments, both batches were processed directly on Millipore filter papers and these were placed in Bray’s for counting. Results from one or the other method of sample preparation were identical. 14C-i-oL-Leucine (27.5 mc/mM) and 14C-2-thymidine (37.9 mc/mM) were purchased from the Radiochemical Centre, Amersham, UK. Hexahomoserine
concentration
experiments
The eggs approx. 20,00O/ml, were fertilized and allowed to develop in varying concentrations of hexahomoserine for 8 h (young blastula stage) at 15°C with gentle agitation. At this time 0.02 ml of W-1-DL-leucine was added to each sample (final volume 4.0 ml) and left in contact with the embryos for 30 min. At the end of the pulse the reactions were stopped and samples prepared for uptake and incorporation measurements. The final concentration of the radioactive precursor was 476 mpg/ml. RESULTS
Results from a number of drug concentration experiments are shown in Table 1. We can see that by increasing the concentration of hexahomoserine, Experimental
Cell Research 51
G. Bellemare, J. Pinard,
408
A. Aubin and G. H. Cousineau
under the experimental conditions used, the net effect of the drug is twofold, namely, a reduction of leucine uptake \vith concomitant decrease of leucine incorporation into protein and an unexpectedly high rate of protein synthesis as measured by the percent ratio af incorporation over uptake. However, since \\-e have made no measurements of the leucine pool at this time-which may possibly decrease under the influence of hexahomoserine, e.g., by leakage through the inhibited cell membrane-the incorporation/uptake ratio cannot be used with total confidence as a measure of the net rate of protein synthesis, and the unexpectedly high rate of protein synthesis in the hexahomoserine-treated embryos may possibly represent a calculation artifact. On the other hand, we have measured the radioactivity in the supernatants before washing, and these values do not increase appreciably after addition of the supernatants from the sea water washes. Also, normal development (judged by phase-microscopy) and DNA labelling (see below) observed in the presence of hexahomoserine indicate that protein synthesis as such is not impaired by this analogue. In all the experiments presented below the final concentration of hexahomoserine was maintained at 100 lug/ml. TABLE
1. Effect of hexahomoserine concentration and incorporation
on leucine uptake
Eggs (2O,OOO/ml) were allowed to develop in varying concentrations of hexahomoserine and pulsed for 30 min with 14C-l-DL-leucine 8 h after fertilization. The labelled precursor had a specific activity of 27.5 mc/mM and was added at a final concentration of 476 mpg/ml. Each incubation flask contained a total volume of 4.0 ml. At the end of the pulse the embryos were washed and the samples prepared for counting of total uptake and incorporation as described in text
Hexahomoserine in pg/ml
Control 10 20 30 50 75 100 250 500
Experimental
Cell Research 51
Uptake CPM
286,113 279,146 137,923 106,329 50,059 39,746 30,614 16,412 11,309
Incorporation CPM
71,006 69,190 45,137 39.596 33,084 27,141 22,012 11,649 8,840
“/ Incorporation 0 Uptake
25 25 33 37 66 68 72 71 79
Leucine
Uptake
and thymidine
and incorporation
in developing
sea urchin
eggs
409
of 14C-leucine
These tests were done in the following way: Eggs were pre-treated for 15 min with hexahomoserine (100 lug/ml), fertilized and allowed to develop as previously described. At 0, 1.5, 3, 5 and 8 h after fertilization 3.98 ml aliquots were taken and placed into 0.02 ml of l”C-1-leucine at 476 m,ug/ml and 0.1 PC/ml. The samples were left in contact for 30 min with the radioactive precursor. Data from Fig. 1 show the results of such experiments. Setting uptake values obtained from 1.5h-old embryos at 100, non-fertilized eggs (control and hexahomoserine-treated samples) exhibit a noticeable uptake of labelled precursor, e.g., 17 and 2 per cent or 23,000 and 3000 counts, respectively. after fertilization the uptake profile changes drastically, and the increase continues during early development. At the beginning of blastulation (8 h post-fertilization) the uptake of leucine in controls has started to decrease while that in treated embryos has reached a plateau. In all of our experiments the general profile of leucine uptake in the hexahomoserine-treated embryos is certainly less important than the activity observed in controls but, nevertheless, it is significant, e.g., from 23,000 counts (eggs-controls) to 195,000 counts (blastulae-controls) and from 3000 to 27,000 counts (hesahomoserine-eggs and -blastulae, respectively).
Uptake
und incorporation
of 14C-2-thymidine
Identical experiments mere done using 14C-2-thymidine as the labelled precursor (Fig. 2). 0.02 ml of this pyrimidine nucleoside (specific activity 37.9 mc/mM and 20 PC/ml) was used in a total volume of 4.0 ml (20,800 eggs/ml), i.e., 0.1 PC/ml and 640 mpg/ml. Samples were treated as usual and washed with excess unlabelled thymidine. Results show that uptake and incorporation values, up to 8 h after fertilization, for both control and hexahomoserine embryos are fairly equivalent and, therefore, so are the rates of thymidine incorporation into DNA. Although we have included the rates of DNA synthesis in unfertilized eggs care must be taken in evaluating these data. Indeed these results ensue from extremely low counts, e.g., 17 over 340 (CPM-controls) and 20 over 447 (CPM-hexahomoserine), and the value for the net rate of DNA synthesis is approx. 6 per cent in both cases. Perhaps we cannot disregard the rates obtained from unfertilized eggs but a note of caution is necessary. Experimental Cell Research 51
410
G. Bellemare, J. Pinard,
a
A. Auhin and G. H. Cousineau
b
t
Fig. l.-4.0 ml suspensions contained 20,000 cells /ml and 476 mpg/ml of %-l-DL-leucine (0.1 PC/ml) for a 30 min pulse at the intervals indicated. Treatment of samples described in text. All the values presented are calculated on the basis of the control uptake (at 1.5 h post-fertilization) set at 100. Hexahomoserine concentration was 100 pg/ml. Abscissa: Time (h); a, control; b, hexahomoserine; c, y0 incorporation/uptake; ordinate: % incorporation-uptake; uptake (control) value at 1.5 h = 100%. m, Uptake; m, incorporation; m, control; m, hexahomoserine.
uptake and incorporation in sea urchin eggs Fig. 2.-Thirty minute-measure of W-2-thymidine and embryos (20,80O/ml). %-Thymidine at a specific activity of 37.9 mc/mM and 20 PC/ml and used at 640 mpg/ml (0.1 PC/ml) in a total volume of 4.0 ml. Uptake and incorporation data presented were obtained by calculation from counts of 1.5 h embryos (controls) set at 100. Treated samples developed in the presence of 100 pg/ml of hexahomoserine. Abscissa: Time (h); a, % uptake; b, % incorporation; e, % incorporation/uptake; ordinate: % incorporation-uptake; uptake (control) and incorporation (control).values at 1.5 h = 100 %. [EIl, control; a, hexahomoserine. Experimental
Cell Research 51
Leucine and thymidine
in developing sea urchin eggs
411
From first cell division to 3 h after fertilization (4-&l stage) we have consistently found a slight increase in the rate of thymidine incorporation into DNA for controls and experimcntals. This observation is taken from the following ratios: controls, 500 over 9000 ( 5.5 per cent at 1.5 h) and 1300 over 20,000 (6.5 per cent at 3 h); experimentals, 700 over 16,000 (4.4 per cent at 1.5 h) and 900 over 18,000 (5.0 per cent at 3 h). Fig. 2 may be summarized as follows. The rate of DNA synthesis from 1.5 to 3 h after fertilization, i.e., 2- and 4-cell stage, respectively, increases but very slightly. Measurements taken at 5 h after fertilization (64-cell stage) show no more than a 4-fold augmentation in the thymidine incorporation rate into DNA. Activity increases therefrom to the young blastula stage. DISCUSSION
Many authors have reported on the variation of precursor molecule incorporation in developing sea urchin eggs. Amongst others, Giudice, Vittorelli and Monroy [4] have studied the uptake of amino acids and have found this activity to increase up to the 64-cell stage when the rate of uptake became constant. This plateau was seen to be maintained until the mesenchyme blastula stage when a second increase occurred. Data from their work show that rates for methionine incorporation (defined as the TCA-insoluble fraction calculated as percent of the total uptake) increased from fertilization, peaked out at the 64-cell stage, and then declined. Different results were obtained when they measured incorporation rates for leucine. In this case the rates were found to remain constant from fertilization to about the mesenthyme blastula stage. In our experiments we have defined uptake as the taking up of exogenous molecules into the cell pool, incorporation as precursor molecules incorporated into acid-insoluble material, and incorporation rates as the ratio of incorporation over uptake. Our leucine results agree well with those of Giudice, Vittorelli and Monroy [4], i.e., we observed a rapid increase in the rate of incorporation from fertilization, and this rate was maintained constant until the blastula stage when it declined. Before fertilization leucine uptake appeared somewhat important, but after fertilization and up to the morula stage the activity increased tremendously. The uptake then decreased at blastulation. The pattern of the hexahomoserine effect is rather interesting. The drug seems to reduce leucine uptake and incorporation while it enhances, in some way, the net rate of protein synthesis. These results, as mentioned previously, may represent a calculation artifact. Experimental
Cell Research 51
G. Bellemare, J. Pinard,
412
A. Aubin and G. H. Cousinecur
Thus our data for the leucine experiments indicate that general incorporation is low relative to net uptake. The same observations are noticed with the thymidine experiments, but to a greater degree. Indeed, the uptake of thymidine by control, unfertilized, eggs read 340 counts while 25,000 counts were obtained for control blastulae, and values for incorporation ranged from 17 counts in unfertilized eggs to 10,000 counts in blastulae. The presence of hexahomoserine had little effect on these values. In conclusion we wish to underline the importance of testing precursor uptake when measuring rates of incorporation into macromolecules. This is true of incorporation experiments in normal systems but especially is it important when attention is directed at knowing the rates of incorporation of precursor molecules in cells affected by exogenous chemicals. In this context we have also done experiments in which developing sea urchin eggs have been treated with actinomycin D and deuterium oxyde [14], and the data obtained from such experiments she\\-, in part, drastic effects on cell permeability with concomitant influence on precursor incorporation into macromolecules. REFERENCES 1. BERG, W. E., Exptl Cell Res. 40, 469 (1965). DITTMER, K., Ann. N. Y. Acad. Sci. 52, 1274 (1950). EPEL, D., Proc. Nat1 Aead. Sci. 57, 899 (1967). GIUDICE, G., VITTORELLI, WI. L. and MONROY, A., Acta Embryol. Morphol. GROSS, P. R. and COUSINEAU, G. H., Expfl Cell Res. 33, 368 (1964). GROSS, P. R. and FRY, B. J., Science 153, 749 (1966). 7. HULTIN, T., Arkiu Kemi 5, 267 (1953).
2. 3. 4. 5. 6.
8. ~ 9. --
Exptl5,
Deuelop. Biol. 10, 305 (1964). Exptl Cell Res. 25, 405 (1961).
10. MITCHISON, J. M. and CUMMINS, J. E., J. Cell Sci. 1, 35 (1966). 11. PAcB, E., GINGRAS, R. and GAUDRY, R., Can. J. Res. 27, 364 (1949). 12. PALMER, L., Physiol. Zool. 10, 352 (1937). 13. PIATIGORSKY, J. and \VHITELEY, A. H., Biochim. Biophys. Acta 108, 404 (1965). 14. PINARD, J., BELLEMARE, G. and COUSINEAU, G. H., In preparation.
Experimental
Cell Research 51
113 (1962).
Experimenfal
UPTAKE
AND
THYMIDINE
INCORPORATION IN DEVELOPING
THE EFFECT
1968 by Academic Press Inc.
Cell Research 51,
OF LEUCINE SEA URCHIN
406-412
(1968)
AND EGGS
OF HEXAHOMOSERINE’
G. BELLEMARE, J. PINARD, A. AUBIN and G. H. COUSINEAU Laboratoire
de Biologic
Mol&zulaire,
UniversitB
de Montr&d,
Montrt%zl, P.Q., Canada
Received September26, 1967
SIIORTI~Y after fertilization, or parthenogenetic activation, of sea urchin eggs there is a rapid increase in the incorporation of labelled amino acids into proteins, and this activity continues until the mesenchpme blastula stage [3, 8, 91. Although RNA metabolism is remarkably low during the early period of development the rate of DNA synthesis increases with cell division [5, 71. Our attention was arrested lately by the possibility that these values might have to be qualified in some n-ay. Indeed, these considerations came from observations made recently by l’iatigorsky and m’hiteley [13] and Mitchison and Cummins [lo] who reported on the uptake of uridine, valine and cytidine in developing eggs and found that there is a permeability change during development, e.g., the permeability rises sharply after fertilization and reaches a maximum before the first cleavage, the permeation rate then becomes constant until the fifth cleavage. Moreover, because of a permeability differential, Gross and Fry [6I suggested that “the fraction of total uptake incorporated into macromolecules might reflect more accurately protein synthesis”, and these authors found that there is a close relationship betiveen changes in the rate of permeability and amino acid incorporation into protein during the first cell cycle. Their data suggest that penetration may limit the number of counts incorporated into protein. Thus, since incorporation of radioactive precursors into macromolecules is a direct function of those labelled molecules present at any one time in the cell pool, we decided to reinvestigate the “active syntheses” observed during development of sea urchin eggs and to qualify these values, if necessary, by those obtained from penetration data. \Ve therefore chose to study 1 This work was supported in part by grant no. 211 from the Jane Coffin Childs Jlemorial Fund for Medical Research, grant no. DRG-918AT from the Damon Runyon Memorial Fund for Cancer Research and by grant no. A-3624 from the National Research Council of Canada. Experimental
Cell Research 51
Leucine and thymidine
in developing sea urchin
eggs
407
not only normal development but also development affected by the presence of hexahomoserine (d-amino-a-hydroxycaproic acid) which is known to be a potent inhibitor of protein synthesis [‘L, 111. MATERIAL
AND
METHODS
Eggs and sperms from the sea urchin Sfrongyiocenfrotus purpuratus (Pacific Biomarine Supply Company, Venice, Calif.) were obtained by injection of 0.53 M KC1 [12]. The eggs, with jelly coats removed, were washed several times with Milliporefiltered sea water and tested for fertilization. Only those batches that gave more than 95 per cent membrane elevation were used in these experiments [5]. The eggs, with and without hexahomoserine, were fertilized and allowed to develop at 15°C with gentle agitation. Aliquots of the cell suspensions were taken before and at different intervals after fertilization and placed in the presence of a radioactive precursor for 30 min. At the end of this period (pulse) the eggs or embryos from one sample were separated into two batches and gently centrifuged. One batch was repeatedly washed with filtered sea water containing loo-fold excess unlabelled precursor, spun down, and the cells taken up in 10 ml of Bray’s solution for counting in a liquid scintillation system (Nuclear Chicago Mark 1). The other batch was similarly treated and at the same time, but in this case the reaction was stopped by addition of cold TCA (5 per cent final concentration) containing excess unlabelled precursor (results obtained from hot TCA treatment of the samples [l, 31 did not differ appreciably from those obtained from the cold TCA treatments). The acidinsoluble material was washed with cold TCA and filtered sea water, and finally taken up in Bray’s for counting. In this way it was possible to measure total incorporation and total uptake (from which incorporation values were later subtracted). In other experiments, both batches were processed directly on Millipore filter papers and these were placed in Bray’s for counting. Results from one or the other method of sample preparation were identical. 14C-i-oL-Leucine (27.5 mc/mM) and 14C-2-thymidine (37.9 mc/mM) were purchased from the Radiochemical Centre, Amersham, UK. Hexahomoserine
concentration
experiments
The eggs approx. 20,00O/ml, were fertilized and allowed to develop in varying concentrations of hexahomoserine for 8 h (young blastula stage) at 15°C with gentle agitation. At this time 0.02 ml of W-1-DL-leucine was added to each sample (final volume 4.0 ml) and left in contact with the embryos for 30 min. At the end of the pulse the reactions were stopped and samples prepared for uptake and incorporation measurements. The final concentration of the radioactive precursor was 476 mpg/ml. RESULTS
Results from a number of drug concentration experiments are shown in Table 1. We can see that by increasing the concentration of hexahomoserine, Experimental
Cell Research 51
G. Bellemare, J. Pinard,
408
A. Aubin and G. H. Cousineau
under the experimental conditions used, the net effect of the drug is twofold, namely, a reduction of leucine uptake \vith concomitant decrease of leucine incorporation into protein and an unexpectedly high rate of protein synthesis as measured by the percent ratio af incorporation over uptake. However, since \\-e have made no measurements of the leucine pool at this time-which may possibly decrease under the influence of hexahomoserine, e.g., by leakage through the inhibited cell membrane-the incorporation/uptake ratio cannot be used with total confidence as a measure of the net rate of protein synthesis, and the unexpectedly high rate of protein synthesis in the hexahomoserine-treated embryos may possibly represent a calculation artifact. On the other hand, we have measured the radioactivity in the supernatants before washing, and these values do not increase appreciably after addition of the supernatants from the sea water washes. Also, normal development (judged by phase-microscopy) and DNA labelling (see below) observed in the presence of hexahomoserine indicate that protein synthesis as such is not impaired by this analogue. In all the experiments presented below the final concentration of hexahomoserine was maintained at 100 lug/ml. TABLE
1. Effect of hexahomoserine concentration and incorporation
on leucine uptake
Eggs (2O,OOO/ml) were allowed to develop in varying concentrations of hexahomoserine and pulsed for 30 min with 14C-l-DL-leucine 8 h after fertilization. The labelled precursor had a specific activity of 27.5 mc/mM and was added at a final concentration of 476 mpg/ml. Each incubation flask contained a total volume of 4.0 ml. At the end of the pulse the embryos were washed and the samples prepared for counting of total uptake and incorporation as described in text
Hexahomoserine in pg/ml
Control 10 20 30 50 75 100 250 500
Experimental
Cell Research 51
Uptake CPM
286,113 279,146 137,923 106,329 50,059 39,746 30,614 16,412 11,309
Incorporation CPM
71,006 69,190 45,137 39.596 33,084 27,141 22,012 11,649 8,840
“/ Incorporation 0 Uptake
25 25 33 37 66 68 72 71 79
Leucine
Uptake
and thymidine
and incorporation
in developing
sea urchin
eggs
409
of 14C-leucine
These tests were done in the following way: Eggs were pre-treated for 15 min with hexahomoserine (100 lug/ml), fertilized and allowed to develop as previously described. At 0, 1.5, 3, 5 and 8 h after fertilization 3.98 ml aliquots were taken and placed into 0.02 ml of l”C-1-leucine at 476 m,ug/ml and 0.1 PC/ml. The samples were left in contact for 30 min with the radioactive precursor. Data from Fig. 1 show the results of such experiments. Setting uptake values obtained from 1.5h-old embryos at 100, non-fertilized eggs (control and hexahomoserine-treated samples) exhibit a noticeable uptake of labelled precursor, e.g., 17 and 2 per cent or 23,000 and 3000 counts, respectively. after fertilization the uptake profile changes drastically, and the increase continues during early development. At the beginning of blastulation (8 h post-fertilization) the uptake of leucine in controls has started to decrease while that in treated embryos has reached a plateau. In all of our experiments the general profile of leucine uptake in the hexahomoserine-treated embryos is certainly less important than the activity observed in controls but, nevertheless, it is significant, e.g., from 23,000 counts (eggs-controls) to 195,000 counts (blastulae-controls) and from 3000 to 27,000 counts (hesahomoserine-eggs and -blastulae, respectively).
Uptake
und incorporation
of 14C-2-thymidine
Identical experiments mere done using 14C-2-thymidine as the labelled precursor (Fig. 2). 0.02 ml of this pyrimidine nucleoside (specific activity 37.9 mc/mM and 20 PC/ml) was used in a total volume of 4.0 ml (20,800 eggs/ml), i.e., 0.1 PC/ml and 640 mpg/ml. Samples were treated as usual and washed with excess unlabelled thymidine. Results show that uptake and incorporation values, up to 8 h after fertilization, for both control and hexahomoserine embryos are fairly equivalent and, therefore, so are the rates of thymidine incorporation into DNA. Although we have included the rates of DNA synthesis in unfertilized eggs care must be taken in evaluating these data. Indeed these results ensue from extremely low counts, e.g., 17 over 340 (CPM-controls) and 20 over 447 (CPM-hexahomoserine), and the value for the net rate of DNA synthesis is approx. 6 per cent in both cases. Perhaps we cannot disregard the rates obtained from unfertilized eggs but a note of caution is necessary. Experimental Cell Research 51
410
G. Bellemare, J. Pinard,
a
A. Auhin and G. H. Cousineau
b
t
Fig. l.-4.0 ml suspensions contained 20,000 cells /ml and 476 mpg/ml of %-l-DL-leucine (0.1 PC/ml) for a 30 min pulse at the intervals indicated. Treatment of samples described in text. All the values presented are calculated on the basis of the control uptake (at 1.5 h post-fertilization) set at 100. Hexahomoserine concentration was 100 pg/ml. Abscissa: Time (h); a, control; b, hexahomoserine; c, y0 incorporation/uptake; ordinate: % incorporation-uptake; uptake (control) value at 1.5 h = 100%. m, Uptake; m, incorporation; m, control; m, hexahomoserine.
uptake and incorporation in sea urchin eggs Fig. 2.-Thirty minute-measure of W-2-thymidine and embryos (20,80O/ml). %-Thymidine at a specific activity of 37.9 mc/mM and 20 PC/ml and used at 640 mpg/ml (0.1 PC/ml) in a total volume of 4.0 ml. Uptake and incorporation data presented were obtained by calculation from counts of 1.5 h embryos (controls) set at 100. Treated samples developed in the presence of 100 pg/ml of hexahomoserine. Abscissa: Time (h); a, % uptake; b, % incorporation; e, % incorporation/uptake; ordinate: % incorporation-uptake; uptake (control) and incorporation (control).values at 1.5 h = 100 %. [EIl, control; a, hexahomoserine. Experimental
Cell Research 51
Leucine and thymidine
in developing sea urchin eggs
411
From first cell division to 3 h after fertilization (4-&l stage) we have consistently found a slight increase in the rate of thymidine incorporation into DNA for controls and experimcntals. This observation is taken from the following ratios: controls, 500 over 9000 ( 5.5 per cent at 1.5 h) and 1300 over 20,000 (6.5 per cent at 3 h); experimentals, 700 over 16,000 (4.4 per cent at 1.5 h) and 900 over 18,000 (5.0 per cent at 3 h). Fig. 2 may be summarized as follows. The rate of DNA synthesis from 1.5 to 3 h after fertilization, i.e., 2- and 4-cell stage, respectively, increases but very slightly. Measurements taken at 5 h after fertilization (64-cell stage) show no more than a 4-fold augmentation in the thymidine incorporation rate into DNA. Activity increases therefrom to the young blastula stage. DISCUSSION
Many authors have reported on the variation of precursor molecule incorporation in developing sea urchin eggs. Amongst others, Giudice, Vittorelli and Monroy [4] have studied the uptake of amino acids and have found this activity to increase up to the 64-cell stage when the rate of uptake became constant. This plateau was seen to be maintained until the mesenchyme blastula stage when a second increase occurred. Data from their work show that rates for methionine incorporation (defined as the TCA-insoluble fraction calculated as percent of the total uptake) increased from fertilization, peaked out at the 64-cell stage, and then declined. Different results were obtained when they measured incorporation rates for leucine. In this case the rates were found to remain constant from fertilization to about the mesenthyme blastula stage. In our experiments we have defined uptake as the taking up of exogenous molecules into the cell pool, incorporation as precursor molecules incorporated into acid-insoluble material, and incorporation rates as the ratio of incorporation over uptake. Our leucine results agree well with those of Giudice, Vittorelli and Monroy [4], i.e., we observed a rapid increase in the rate of incorporation from fertilization, and this rate was maintained constant until the blastula stage when it declined. Before fertilization leucine uptake appeared somewhat important, but after fertilization and up to the morula stage the activity increased tremendously. The uptake then decreased at blastulation. The pattern of the hexahomoserine effect is rather interesting. The drug seems to reduce leucine uptake and incorporation while it enhances, in some way, the net rate of protein synthesis. These results, as mentioned previously, may represent a calculation artifact. Experimental
Cell Research 51
G. Bellemare, J. Pinard,
412
A. Aubin and G. H. Cousinecur
Thus our data for the leucine experiments indicate that general incorporation is low relative to net uptake. The same observations are noticed with the thymidine experiments, but to a greater degree. Indeed, the uptake of thymidine by control, unfertilized, eggs read 340 counts while 25,000 counts were obtained for control blastulae, and values for incorporation ranged from 17 counts in unfertilized eggs to 10,000 counts in blastulae. The presence of hexahomoserine had little effect on these values. In conclusion we wish to underline the importance of testing precursor uptake when measuring rates of incorporation into macromolecules. This is true of incorporation experiments in normal systems but especially is it important when attention is directed at knowing the rates of incorporation of precursor molecules in cells affected by exogenous chemicals. In this context we have also done experiments in which developing sea urchin eggs have been treated with actinomycin D and deuterium oxyde [14], and the data obtained from such experiments she\\-, in part, drastic effects on cell permeability with concomitant influence on precursor incorporation into macromolecules. REFERENCES 1. BERG, W. E., Exptl Cell Res. 40, 469 (1965). DITTMER, K., Ann. N. Y. Acad. Sci. 52, 1274 (1950). EPEL, D., Proc. Nat1 Aead. Sci. 57, 899 (1967). GIUDICE, G., VITTORELLI, WI. L. and MONROY, A., Acta Embryol. Morphol. GROSS, P. R. and COUSINEAU, G. H., Expfl Cell Res. 33, 368 (1964). GROSS, P. R. and FRY, B. J., Science 153, 749 (1966). 7. HULTIN, T., Arkiu Kemi 5, 267 (1953).
2. 3. 4. 5. 6.
8. ~ 9. --
Exptl5,
Deuelop. Biol. 10, 305 (1964). Exptl Cell Res. 25, 405 (1961).
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