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The effect of cell size and DNA content on the cellular regulation of DNA synthesis in haploid and diploid embryos
Experimental
THE EFFECT ON THE
13
Cell Research 43, 13-l 9 (1966)
OF CELL SIZE AND DNA CONTENT
CELLULAR REGULATION OF DNA SYNTHESIS HAPLOID AND DIPLOID EMBRYOS
IN
C. F. GRAHAM The Zoology Department,
Oxford, England
Received December 21, 1965
THE normal growth and differentiation of amphibian haploid embryos, during early embryonic development, provide favourable conditions for testing the effects of cell size and nuclear content of DNA on the initiation and duration of the S phase (nomenclature from [15] after [12]). Compared with their diploid counterparts, late haploid embryos generally contain twice the number of cells with half the diploid cell volume and therefore with half the diploid cell mass [6]. Support has been obtained for the hypothesis that the initiation of DNA synthesis is dependent on absolute cell mass in cells in tissue culture [13]. If absolute cell mass, by itself, is an important factor in the initiation of this synthesis in embryonic frog cells, then haploid cells should enter the S phase at a different time in the cell cycle to diploid cells. Haploid and diploid cells also provide a situation in which the effect of the quantity of DNA inside the cell nucleus on the duration of the S phase can be studied in identical cell types from the same species. In previous studies the duration of the S phase of cells containing different amounts of DNA has been compared in two situations. Either the comparison has been between different cell types from the same species, in which changes in the duration of the S phase may also be due to differences in the nucleotide metabolism of the cells [S], or it has been between similar cells from different strains [l] and species [18], in which the differences in the duration of the S phase could also be due to differences in the genetic constitution of the nucleus. If the amount of DNA in the nucleus effects the time that the cell is engaged in DNA synthesis, then the S phase of haploids should be a different length to that of diploids. The labelling of cells of the endoderm of diploid embryos with tritiated thymidine has been described [9] and this paper reports similar labelling experiments on haploid neurulae. Experimental
Cell Research 43
14
C. F. Graham
MATERIALS
AND
METHODS
Rearing of embryos.-Adult females of Xenopus laevis (Daudin) were induced to lay eggs by the injection of gonadotrophic hormones [4]. The freshly layed eggs were fertilized in a macerated testis suspension and reared at 20°C + I” in modified Barth X saline solution until Stage 9 [14] and then in l/10 modified Barth X solution [4]. Production of hapZoids.-The eggs were irradiated with UV at the animal pole for 30 set between 5 and 15 min after fertilization. The egg nucleus breaks down after this treatment and androgenetic haploids develop [IO]. Fifty-six embryos, which were controls from all the matings which were used, were UV’ed in this way. Their survival was similar to that obtained by Gurdon, and all those surviving until the post neurula stage showed the haploid syndrome. To test if this dose of UV effects the cytoplasm of the egg, 65 eggs were UV’ed 60 min after fertilization, when the egg nucleus is near the centre of the egg and protected from the UV rays. These all developed as normal diploids and this dose of UV does not affect the subsequent development of the embryos. In all the cell counting experiments, the diploids were also UV’ed in this way so that both haploid and diploid embryos would be similarly influenced by any slight effect of the UV on cell division. LabelZing of nuclei.-The figures for the percentage of labelled cells in diploid embryos are those obtained previously [9]. The haploids were treated similarly except that the jelly coats were not removed prior to the injection of the tritiated thymidine because they had been dissolved at the animal pole by the UV irradiation. Autoradiographs.-Sections of the haploid embryos were fixed, embedded and sectioned as described previously for diploid embryos 191. Autoradiographs were prepared using Ilford K2 dipping emulsion. Grain counting.-Counts were made on median longitudinal sections. The percentage of labelled nuclei was determined by scoring 200 nuclei from each of three embryos. The percentage of labelled metaphases and anaphases was determined by scoring a total of fifty from the three embryos. A nucleus was scored as labelled if the number of grains over it exceeded by two or more the number of grains over an adjacent area of equal size in the cytoplasm (the background count was very low and never exceeded 4 grains per nuclear area in the cytoplasm). The mitotic index was measured by counting the number of metaphase and anaphase figures in a total of 1000 cells. During the course of an experiment some embryos may begin to degenerate. To exclude these from the final results, six embryos were injected with label and only three normal embryos were chosen for the counting. It has been argued previously that the injection of small quantities of tritiated thymidine into the amphibian embryo does not affect cell division or the survival of the embryo [9]. The early degeneration of embryos in these experiments is probably due to an effect of haploidy which is believed to affect a small proportion of embryos during early development. Cell counting.-The total numbers of cells in haploid and diploid embryos were counted in a dispersion of the embryo on a haemocytometer slide [3]. Between six and ten embryos were collected at each stage and left for g hr in 1 per cent citric acid. After this time the jelly coats were carefully dissected away and the embryos were transferred to a 300 ~1 capacity homogenizer with a loose fitting plunger. The Experimental
Cell Research 43
15
Cell size and DNA synthesis
homogenizer was then filled up with 1 per cent orcein in 45 per cent acetic acid and the embryos broken up into single cells and nuclei with ten strokes of the plunger. The number of cells and free nuclei were counted in five samples of each dispersion and several dispersions were made up for each stage. The relative numbers of cells in the endoderm of haploid and diploid embryos were compared by counting the number of cells in a fixed volume of the endoderm using a graticule microscope eyepiece and 8 ,U sections of the embryo.
RESULTS Cell counts The first and second cleavage divisions of haploid embryos are slightly slower than in diploids and at 4 hr after fertilization there are slightly fewer cells around the equator of the haploid embryo. At this stage there are too few cells in the embryo to obtain consistent counts on the haemocytometer slide. The counts obtained with the haemocytometer slide are presented in Table I. Eight hours after fertilization there are about one and a half times as many cells in haploid embryos as in diploid embryos. Between 12 hr after fertilization (Stage ll), and 24 hr after fertilization (Stage 20) the haploid I. Cell counts of whole embryos of Xenopus
TABLE
laevis.
Stage
Ploidy of
Number of
Cell
Haploid/ diploid
embryo
matings
counts
ratio
8
9
Haploid Diploid
2
336 240
1.400
12
11
Haploid Diploid
3
1257 641
1.960
16
14
Haploid Diploid
2
936 511
1.831
20
18
Haploid Diploid
2
793 402
1.972
24
20
Haploid Diploid
1
475 225
2.14
Hours
postfertilization
The counts are the total number of cells counted in a given area on a haemocytometer slide. For each figure, six suspensions containing between six and ten embryos were counted. The number of embryos used was the same for haploids and diploids at each developmental stage, but fewer embryos were used at later developmental stages. Experimental
Cell Research 43
16
C. F. Graham
embryos contain slightly less than twice as many cells as the diploid embryos. The volume of haploid embryos appears to be the same as that of the diploid embryos at least up to 24 hr after fertilization, although at later stages the haploid embryos are smaller in size [ll]. The haploid cells of the neurula must therefore be about half the volume of the diploid cells. TABLE II. Cell counfs in the endoderm of Xenopus Hours postfertilization
laevis embryos.
Ploidy of embryos
Cell counts
9
Haploid Diploid
203 200
1.015
12.5
11
Haploid Diploid
254 131
1.938
20.5
19
Haploid Diploid
466 238
1.957
8
Stage
Cells were counted in a fixed volume of the endoderm scope eyepiece and 8 p sections of the embryo.
Haploid/ diploid ratio
at each stage, using a graticule
micro-
These results show that cells of haploid embryos on average divide at the same speed as those of diploid embryos until 4 hr after fertilization, apart from a slight delay in the first and second cleavage divisions. Between 4 and 12 hr after fertilization they divide once more than diploid cells, and then divide at the same speed as diploid cells at least up to 24 hr after fertilization. In some haploid embryos, such as those of the salamander, not all the body tissues contain twice as many cells as diploid embryos [5]. The number of cells in the endoderm of haploid and diploid embryos was compared by counting the number of cells in a given area of sections of haploid and diploid embryos. These figures are presented in Table II. Eight hours after fertilization there are almost exactly the same numbers of cells in the endoderm of haploid and diploid embryos. However between 124 and 204 hr after fertilization there are twice as many cells in the haploid endoderm. The endoderm cells of haploids, therefore, divide once more than those of diploids slightly later in development than do the cells of the embryo as a whole. At 12) and 204 hr after fertilization the numerical ratio between haploid and diploid cells is the same. Both cell types must be dividing at the same rate and presumably take the same time to pass through the whole cell cycle, during this period. Experimental
Cell Research 43
17
Cell size and DNA synthesis Incorporation
of 3H-thymidine
If label is injected into the embryo at Stage 11, its uptake into the cells of the endoderm can be compared in haploids and diploids to find out if the duration of the S phase is related to the amount of DNA in each cell nucleus. Labelled thymidine was injected into the archenteron at Stage 11 and its uptake into the cells is plotted in Fig. 1. After the cells have been exposed to label for 1 hr, a relatively short time in relation to the whole cycle, the percentage of labelled cells in haploid and diploid embryos is almost exactly the same (Fig. 1). The percentage of labelled cells a short time after the injection of label is proportional to the percentage of the whole cell cycle occupied by the S phase although it does not equal the percentage of the cell cycle occupied by the S phase [ 171. Since the speed of division of haploid and diploid cells has been shown to be the same at this stage, the duration of the S phase in haploid and diploid cells is almost exactly the same. The time, after the injection of label, at which 50 per cent of the metaphase and anaphase figures are labelled, is similar in the haploid and diploid
Fig. 1. Fig. 1.-O nuclei.
-0,
Percentage
of labelled
Fig. 2. haploid
nuclei.
0 -0,
Percentage
Fig. 2.- l - 0 , Percentage of labelled metaphases and anaphases in haploids. of labelled metaphases and anaphases in diploids. 2 - 661806
Experimental
of labelled
diploid
o - 0, Percentage
Cell Research 43
18
C. F. Graham
experiments (Fig. 2). This is the time taken by nuclei near the end of the S phase to pass through the G2 phase and prophase and enter the metaphase and anaphase stages of mitosis, and it reflects the length of the G, phase [9]. Clearly the G2 phase has a similar length in the two cell types. The mitotic index in diploid embryos is 4.5 per cent at Stage 14 and in haploid embryos it is 3.1 per cent. This difference is probably not significant. DISCUSSION
During cleavage the DNA in diploid embryos is synthesised extremely rapidly. Until 8 hr post-fertilization, cell division proceeds at about the same speed in the endoderm of haploid and diploid embryos, but the rate of DNA synthesis of haploids over this period must be half that of their diploid counterparts. Between 8 and 12 hr after fertilization the rate of cell division in the endoderm of diploid embryos slows dramatically. During this period haploid cells divide once more than diploid cells and must pass through the cell cycle at about twice the rate of diploid cells, since the number of cells in the diploid endoderm roughly doubles during this period [9]. As the cells of the endoderm divide, during early development, so the cell volume and mass decrease. There is little increase in the total volume occupied by the endoderm. This situation is in complete contrast to adult cells in which cell volume and mass increase steadily during interphase [12]. If there was a critical cell mass for the initiation of DNA synthesis, then this would have to decrease as early development proceeds. However, in the neurula, the duration of the Gl, S, G2, and M phases is similar in haploid and diploid embryos. Since haploid cells are on the average half the size and mass and are at the same stage of development as their diploid counterparts, the initiation of the S and M phases must occur in them when the haploid cells are half the size of diploid cells. Although there is a striking correlation between cell mass and the initiation of DNA synthesis in cells in culture [13], absolute cell size or cell mass cannot be the only factor in the initiation of the S phase in the embryonic cells of Xenopus. When different cell lines from various species of mammals are compared, it is found that there is a tendency for cells with the greatest amounts of DNA to have an extended S phase [2, 81. In plants, the duration of the S phase is longer in the species with the greatest amounts of DNA in the nucleus [18]. It is not clear, in these cases, whether the differences in the S phase are due to the quantity of DNA in the nucleus or to other factors; in one species, the mouse, cells containing the same amount of DNA and the same genes may have different S phases in morphologically distinct proliferating epithelia [19] and at different temperatures [16]. Experimental
Cell Research 43
19
Cell size and DNA synthesis
In the haploid and diploid cells discussed in this work, the cells are at the same temperature and of the same type; in these conditions, cells with DNA contents which differ by a multiple of two, have exactly the same duration of the S phase. This striking constancy probably reflects a controlled temporal pattern of replication of DNA along the chromosomes [7] which would probably be the same whatever the ploidy of the nucleus. A similar independence of the duration of the S phase and the amount of DNA in the nucleus has been found in three strains of Tetrahymena [ 1 ].
SUMMARY
The synthesis of DNA in haploid and diploid neurulae of Xenopus laevis has been studied using tritiated thymidine as a specific marker of DNA synthesis. Haploid cells, which are half the volume and half the mass of their diploid counterparts, have an S phase which occupies the same relative position in the cell cycle and lasts for the same period as in diploid cells. In these embryonic endoderm frog cells, cell mass can not be the only factor initiating DNA synthesis and the duration of DNA synthesis is not changed by doubling the number of chromosome sets and the amount of DNA in the nucleus. The author would like to thank Dr J. B. Gurdon for helpful discussion of this work. The support of this research by the Medical Research Council is gratefully acknowledged. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
13. 14. 15. 16. 17. 18. 19.
CAMERON, I. L. and STONE, G. E., Expfl Cell. Res. 36, 510 (1964). DEFENDI, V. and MANSON, L. A., Nature Land. 198, 359 (1963). DEUCHAR, E. M., J. Embryol. expfl Morph. 6, 223 (1958). ELSDALE, T. R., GURDON, J. B. and FISCHBERG, M., J. Embryol. Expil Morphol. 8,437(1960). FANKHAUSER, G., J. Morphol. 62, 393 (1938). FANKHAUSER, G., Quart. Rev. Biol. 20, 20 (1945). GERMAN, J., J. Cell. Biol. 20, 37 (1964). GOLDFEDER, A., Nature Land. 207, 612 (1965). GRAHAM, C. F. and MORGAN, R., Dev. Biol. In press. (1966). GURDON, J. B., Quart. J. Microscop. Sci. 101, 299 (1960). HERTWIG, G., Arch. mikr. Anat. 81, 87 (1913). HOWARD, A. and PELC, S. R., Heredity, Suppl. 6, 261 (1953). KILLANDER, D. and ZETTERBERG, A., Exptl Cell Res. 40,12 (1965). NIEUWKOOP, P. D. and FABER, J., in Normal table of Xenopus laevis (Daudin). Amsterdam, North-Holland Publishing Co., 1956. QUASTLER, H., in L. F. LAMERTON and R. J. M. Fry (eds), Cell Proliferation, p. 18. Blackwell’s Scientific Publications, Oxford, 1963. SHERMAN, F. G., QUASTLER, H. and WIMBER, D. R., Exptl Cell Res. 25, 114, (1961). &KEN, J. E., in D. M. PRESCOTT (ed.), Methods in Cell Physiology, vol. 1, p. 387. Academic Press, New York, 1964. Van’t HOFF, J., Expfl Cell Res. 39, 48 (1965). WOLFSBERG, M. F., Exptl Cell Res. 35, 119 (1964).
Experimental
Cell Research 43
THE EFFECT ON THE
13
Cell Research 43, 13-l 9 (1966)
OF CELL SIZE AND DNA CONTENT
CELLULAR REGULATION OF DNA SYNTHESIS HAPLOID AND DIPLOID EMBRYOS
IN
C. F. GRAHAM The Zoology Department,
Oxford, England
Received December 21, 1965
THE normal growth and differentiation of amphibian haploid embryos, during early embryonic development, provide favourable conditions for testing the effects of cell size and nuclear content of DNA on the initiation and duration of the S phase (nomenclature from [15] after [12]). Compared with their diploid counterparts, late haploid embryos generally contain twice the number of cells with half the diploid cell volume and therefore with half the diploid cell mass [6]. Support has been obtained for the hypothesis that the initiation of DNA synthesis is dependent on absolute cell mass in cells in tissue culture [13]. If absolute cell mass, by itself, is an important factor in the initiation of this synthesis in embryonic frog cells, then haploid cells should enter the S phase at a different time in the cell cycle to diploid cells. Haploid and diploid cells also provide a situation in which the effect of the quantity of DNA inside the cell nucleus on the duration of the S phase can be studied in identical cell types from the same species. In previous studies the duration of the S phase of cells containing different amounts of DNA has been compared in two situations. Either the comparison has been between different cell types from the same species, in which changes in the duration of the S phase may also be due to differences in the nucleotide metabolism of the cells [S], or it has been between similar cells from different strains [l] and species [18], in which the differences in the duration of the S phase could also be due to differences in the genetic constitution of the nucleus. If the amount of DNA in the nucleus effects the time that the cell is engaged in DNA synthesis, then the S phase of haploids should be a different length to that of diploids. The labelling of cells of the endoderm of diploid embryos with tritiated thymidine has been described [9] and this paper reports similar labelling experiments on haploid neurulae. Experimental
Cell Research 43
14
C. F. Graham
MATERIALS
AND
METHODS
Rearing of embryos.-Adult females of Xenopus laevis (Daudin) were induced to lay eggs by the injection of gonadotrophic hormones [4]. The freshly layed eggs were fertilized in a macerated testis suspension and reared at 20°C + I” in modified Barth X saline solution until Stage 9 [14] and then in l/10 modified Barth X solution [4]. Production of hapZoids.-The eggs were irradiated with UV at the animal pole for 30 set between 5 and 15 min after fertilization. The egg nucleus breaks down after this treatment and androgenetic haploids develop [IO]. Fifty-six embryos, which were controls from all the matings which were used, were UV’ed in this way. Their survival was similar to that obtained by Gurdon, and all those surviving until the post neurula stage showed the haploid syndrome. To test if this dose of UV effects the cytoplasm of the egg, 65 eggs were UV’ed 60 min after fertilization, when the egg nucleus is near the centre of the egg and protected from the UV rays. These all developed as normal diploids and this dose of UV does not affect the subsequent development of the embryos. In all the cell counting experiments, the diploids were also UV’ed in this way so that both haploid and diploid embryos would be similarly influenced by any slight effect of the UV on cell division. LabelZing of nuclei.-The figures for the percentage of labelled cells in diploid embryos are those obtained previously [9]. The haploids were treated similarly except that the jelly coats were not removed prior to the injection of the tritiated thymidine because they had been dissolved at the animal pole by the UV irradiation. Autoradiographs.-Sections of the haploid embryos were fixed, embedded and sectioned as described previously for diploid embryos 191. Autoradiographs were prepared using Ilford K2 dipping emulsion. Grain counting.-Counts were made on median longitudinal sections. The percentage of labelled nuclei was determined by scoring 200 nuclei from each of three embryos. The percentage of labelled metaphases and anaphases was determined by scoring a total of fifty from the three embryos. A nucleus was scored as labelled if the number of grains over it exceeded by two or more the number of grains over an adjacent area of equal size in the cytoplasm (the background count was very low and never exceeded 4 grains per nuclear area in the cytoplasm). The mitotic index was measured by counting the number of metaphase and anaphase figures in a total of 1000 cells. During the course of an experiment some embryos may begin to degenerate. To exclude these from the final results, six embryos were injected with label and only three normal embryos were chosen for the counting. It has been argued previously that the injection of small quantities of tritiated thymidine into the amphibian embryo does not affect cell division or the survival of the embryo [9]. The early degeneration of embryos in these experiments is probably due to an effect of haploidy which is believed to affect a small proportion of embryos during early development. Cell counting.-The total numbers of cells in haploid and diploid embryos were counted in a dispersion of the embryo on a haemocytometer slide [3]. Between six and ten embryos were collected at each stage and left for g hr in 1 per cent citric acid. After this time the jelly coats were carefully dissected away and the embryos were transferred to a 300 ~1 capacity homogenizer with a loose fitting plunger. The Experimental
Cell Research 43
15
Cell size and DNA synthesis
homogenizer was then filled up with 1 per cent orcein in 45 per cent acetic acid and the embryos broken up into single cells and nuclei with ten strokes of the plunger. The number of cells and free nuclei were counted in five samples of each dispersion and several dispersions were made up for each stage. The relative numbers of cells in the endoderm of haploid and diploid embryos were compared by counting the number of cells in a fixed volume of the endoderm using a graticule microscope eyepiece and 8 ,U sections of the embryo.
RESULTS Cell counts The first and second cleavage divisions of haploid embryos are slightly slower than in diploids and at 4 hr after fertilization there are slightly fewer cells around the equator of the haploid embryo. At this stage there are too few cells in the embryo to obtain consistent counts on the haemocytometer slide. The counts obtained with the haemocytometer slide are presented in Table I. Eight hours after fertilization there are about one and a half times as many cells in haploid embryos as in diploid embryos. Between 12 hr after fertilization (Stage ll), and 24 hr after fertilization (Stage 20) the haploid I. Cell counts of whole embryos of Xenopus
TABLE
laevis.
Stage
Ploidy of
Number of
Cell
Haploid/ diploid
embryo
matings
counts
ratio
8
9
Haploid Diploid
2
336 240
1.400
12
11
Haploid Diploid
3
1257 641
1.960
16
14
Haploid Diploid
2
936 511
1.831
20
18
Haploid Diploid
2
793 402
1.972
24
20
Haploid Diploid
1
475 225
2.14
Hours
postfertilization
The counts are the total number of cells counted in a given area on a haemocytometer slide. For each figure, six suspensions containing between six and ten embryos were counted. The number of embryos used was the same for haploids and diploids at each developmental stage, but fewer embryos were used at later developmental stages. Experimental
Cell Research 43
16
C. F. Graham
embryos contain slightly less than twice as many cells as the diploid embryos. The volume of haploid embryos appears to be the same as that of the diploid embryos at least up to 24 hr after fertilization, although at later stages the haploid embryos are smaller in size [ll]. The haploid cells of the neurula must therefore be about half the volume of the diploid cells. TABLE II. Cell counfs in the endoderm of Xenopus Hours postfertilization
laevis embryos.
Ploidy of embryos
Cell counts
9
Haploid Diploid
203 200
1.015
12.5
11
Haploid Diploid
254 131
1.938
20.5
19
Haploid Diploid
466 238
1.957
8
Stage
Cells were counted in a fixed volume of the endoderm scope eyepiece and 8 p sections of the embryo.
Haploid/ diploid ratio
at each stage, using a graticule
micro-
These results show that cells of haploid embryos on average divide at the same speed as those of diploid embryos until 4 hr after fertilization, apart from a slight delay in the first and second cleavage divisions. Between 4 and 12 hr after fertilization they divide once more than diploid cells, and then divide at the same speed as diploid cells at least up to 24 hr after fertilization. In some haploid embryos, such as those of the salamander, not all the body tissues contain twice as many cells as diploid embryos [5]. The number of cells in the endoderm of haploid and diploid embryos was compared by counting the number of cells in a given area of sections of haploid and diploid embryos. These figures are presented in Table II. Eight hours after fertilization there are almost exactly the same numbers of cells in the endoderm of haploid and diploid embryos. However between 124 and 204 hr after fertilization there are twice as many cells in the haploid endoderm. The endoderm cells of haploids, therefore, divide once more than those of diploids slightly later in development than do the cells of the embryo as a whole. At 12) and 204 hr after fertilization the numerical ratio between haploid and diploid cells is the same. Both cell types must be dividing at the same rate and presumably take the same time to pass through the whole cell cycle, during this period. Experimental
Cell Research 43
17
Cell size and DNA synthesis Incorporation
of 3H-thymidine
If label is injected into the embryo at Stage 11, its uptake into the cells of the endoderm can be compared in haploids and diploids to find out if the duration of the S phase is related to the amount of DNA in each cell nucleus. Labelled thymidine was injected into the archenteron at Stage 11 and its uptake into the cells is plotted in Fig. 1. After the cells have been exposed to label for 1 hr, a relatively short time in relation to the whole cycle, the percentage of labelled cells in haploid and diploid embryos is almost exactly the same (Fig. 1). The percentage of labelled cells a short time after the injection of label is proportional to the percentage of the whole cell cycle occupied by the S phase although it does not equal the percentage of the cell cycle occupied by the S phase [ 171. Since the speed of division of haploid and diploid cells has been shown to be the same at this stage, the duration of the S phase in haploid and diploid cells is almost exactly the same. The time, after the injection of label, at which 50 per cent of the metaphase and anaphase figures are labelled, is similar in the haploid and diploid
Fig. 1. Fig. 1.-O nuclei.
-0,
Percentage
of labelled
Fig. 2. haploid
nuclei.
0 -0,
Percentage
Fig. 2.- l - 0 , Percentage of labelled metaphases and anaphases in haploids. of labelled metaphases and anaphases in diploids. 2 - 661806
Experimental
of labelled
diploid
o - 0, Percentage
Cell Research 43
18
C. F. Graham
experiments (Fig. 2). This is the time taken by nuclei near the end of the S phase to pass through the G2 phase and prophase and enter the metaphase and anaphase stages of mitosis, and it reflects the length of the G, phase [9]. Clearly the G2 phase has a similar length in the two cell types. The mitotic index in diploid embryos is 4.5 per cent at Stage 14 and in haploid embryos it is 3.1 per cent. This difference is probably not significant. DISCUSSION
During cleavage the DNA in diploid embryos is synthesised extremely rapidly. Until 8 hr post-fertilization, cell division proceeds at about the same speed in the endoderm of haploid and diploid embryos, but the rate of DNA synthesis of haploids over this period must be half that of their diploid counterparts. Between 8 and 12 hr after fertilization the rate of cell division in the endoderm of diploid embryos slows dramatically. During this period haploid cells divide once more than diploid cells and must pass through the cell cycle at about twice the rate of diploid cells, since the number of cells in the diploid endoderm roughly doubles during this period [9]. As the cells of the endoderm divide, during early development, so the cell volume and mass decrease. There is little increase in the total volume occupied by the endoderm. This situation is in complete contrast to adult cells in which cell volume and mass increase steadily during interphase [12]. If there was a critical cell mass for the initiation of DNA synthesis, then this would have to decrease as early development proceeds. However, in the neurula, the duration of the Gl, S, G2, and M phases is similar in haploid and diploid embryos. Since haploid cells are on the average half the size and mass and are at the same stage of development as their diploid counterparts, the initiation of the S and M phases must occur in them when the haploid cells are half the size of diploid cells. Although there is a striking correlation between cell mass and the initiation of DNA synthesis in cells in culture [13], absolute cell size or cell mass cannot be the only factor in the initiation of the S phase in the embryonic cells of Xenopus. When different cell lines from various species of mammals are compared, it is found that there is a tendency for cells with the greatest amounts of DNA to have an extended S phase [2, 81. In plants, the duration of the S phase is longer in the species with the greatest amounts of DNA in the nucleus [18]. It is not clear, in these cases, whether the differences in the S phase are due to the quantity of DNA in the nucleus or to other factors; in one species, the mouse, cells containing the same amount of DNA and the same genes may have different S phases in morphologically distinct proliferating epithelia [19] and at different temperatures [16]. Experimental
Cell Research 43
19
Cell size and DNA synthesis
In the haploid and diploid cells discussed in this work, the cells are at the same temperature and of the same type; in these conditions, cells with DNA contents which differ by a multiple of two, have exactly the same duration of the S phase. This striking constancy probably reflects a controlled temporal pattern of replication of DNA along the chromosomes [7] which would probably be the same whatever the ploidy of the nucleus. A similar independence of the duration of the S phase and the amount of DNA in the nucleus has been found in three strains of Tetrahymena [ 1 ].
SUMMARY
The synthesis of DNA in haploid and diploid neurulae of Xenopus laevis has been studied using tritiated thymidine as a specific marker of DNA synthesis. Haploid cells, which are half the volume and half the mass of their diploid counterparts, have an S phase which occupies the same relative position in the cell cycle and lasts for the same period as in diploid cells. In these embryonic endoderm frog cells, cell mass can not be the only factor initiating DNA synthesis and the duration of DNA synthesis is not changed by doubling the number of chromosome sets and the amount of DNA in the nucleus. The author would like to thank Dr J. B. Gurdon for helpful discussion of this work. The support of this research by the Medical Research Council is gratefully acknowledged. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
13. 14. 15. 16. 17. 18. 19.
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