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Swelling of the nuclei of Asterias eggs
204 SWELLING
OF THE
NUCLEI
A. MARSHAK Marine
Bidogkal
OF ASTE&IAS
EGGS1
and R. P. BENOIT
Laboratory,
Woods Hole, Mass.,
U.S.A.
Received November 6, 1957
IN a previous paper [l] it was reported that the swelling of nuclei of Bsterias eggs in 4.8 M glycerol varies with pH, being minimal at pH 7.4. In that investigation, a phosphate-citrate (McIlvalne) buffer was used in the pH range 7.0-7.4 and a Verona1 buffer in the pH range 7.5-8.0. It therefore seemed possible that the observed changes in nuclear swelling might be attributable not to the differences in pH but to specific effects of the ions or molecules of the buffer’s used. The investigations have therefore been extended in an effort to resolve this problem. Asterias eggs were treated as previously described by immersion in solutions of 4.8 M glycerol containing trishydroxy methylaminomethane (tris), glycine ethyl ester, and Verona1 buffers. The pH range covered was 7.1-8.0 the intervals being approximately 0.1 pH unit. With the tris and Verona1 buffers minima in the mean nuclear diameters (n = 20 in all cases) were found at pH 7.4, and with the glycine ethyl ester the minimum was at pH 7.35. In Table I the mean nuclear diameters at the pH intervals where minima were found and those of the adjacent intervals are given together with the standard deviations of the means. In all but two instances the differences between the means of adjacent intervals are on the border line of being statistically significant. Thus 6 of the total of 12 differences have a probability of occurring by chance of 0.06 or less, and 10 of the 12 differences have a probability of occurring by chance of 0.13 or less. The data therefore suggest that there is an effect of pH on nuclear swelling, a minimum being found at pH 7.35-7.42, but they do not conclusively prove the existence of this effect. Although with these buffers, the minimum was found at approximately the same pH as that previously observed with combination of MacIlvaine and Verona1 buffers, the magnitude of the effect is very much less, suggesting the operation of other factors than pH in producing this phenomenon. To obtain further information on the nature of the other factor or factors comparison was made of the effects of MacIlvaine and of phosphate buffers on nuclear swelling in glycerol with results as shown in Tables II and III. In the MacIlvaine-buffered solutions minimum swelling was observed at pH 7.4 as in previous experiments. However, in the phosphate-buffered solutions there was no nuclear swelling at pH greater than 7.0. The appearance of the cells and nuclei in the latter solutions was markedly different from the appearance in MacIlvaine-buffered solutions or those buffered with tris, glycine ethyl ester, or veronal. The cytoplasm was very granular while the nucleus was hyaline except for the nucleolus. Nuclei extruded from their cells at pH 7.1 or higher were irregular in outline suggesting a “soft” and partially collapsed condition. There was thus a striking difference in the behavior of the buffers containing citrate as well as phosphate as compared with the buffers containing only phosphate. The results indicated 1 This work was supported Experimental
Cell Research 14
by a grant from the American
Cancer Society.
Swelling of nuclei TABLE
I. Summary
of differences in mean nuclear diameters at pH 7.2-7.5 and their standard errors. All in 4.8 M glycerol.
Buffer Tris (0.065 M)
Expt.
pH
m
am
1=
7.20 7.42 7.48 7.60
15.4 12.9 13.2 15.7
0.77 1.00 0.83 0.96
7.22 7.42 7.48
18.1 16.6 18.7
1.08 1.18 0.92
7.25 7.35 7.42
18.4 16.3 19.5
0.54 0.83 0.46
7.25 7.35 7.42
19.3 18.4 20.5
0.35 0.45 0.39
7.31 7.39 7.52
20.5 18.9 20.5
0.45 0.73 0.60
7.31 7.39 7.52
19.9 17.8 20.3
0.99 1.25 0.92
2
Glycine ethyl ester (0.065 ,If)
1
2
Verona1 (0.325 di
1
2
All measurements are in m = mean am = standard deviation d - difference between ad = standard deviation
d
ad
d/ad
2.5
1.26
2.0
2.5
1.27
2.0
1.5
1.61
0.9
2.1
1.42
1.5
2.0
0.99
2.0
3.2
0.95
3.4
0.9
0.55
1.6
2.1
0.59
3.4
1.6
0.85
1.9
1.6
0.94
1.7
-2.1
1.59
1.3
2.5
1.55
1.6
ocular micrometer scale units. of the mean
means of adjacent intervals of the difference
a Measurements in this experiment were taken with a different set of lenses than those used in the subsequent experiments. Absolute values are therefore not comparable to those of other series. an interference of the nuclear swelling by phosphate possibly by dissolution of some nuclear constituents and inhibition of the phosphate effect by citrate. Whether or
not the effect of the citrate is the result of possible competition with phosphate for nuclear constituents cannot be determined from these data although it is suggestive that the nuclear diameter is reduced by a factor of 2 when the ratio of phosphate to citrate is increased by a factor of 2. As shown in Table IV, there is in the phosphate buffered solutions, at pH 7.4, a range of phosphate concentrations (6.5 x 10-s - 6.5 x 10-l) in which the effect of phosphate is minimal, i.e., the swelling in this range was greater than at lower and Experimental
Cell Research 14
A. Marshak and R. P. Benoit II. Mean nuclear diameters in 0.065 M Macllvaine glycerol.
TABLE
Molarity citrate Molarity phosphate PH Mean diameter Fraction control $+ 1
TABLE
III.
0.009 0.113 7.20 13.0
0.006 0.118 7.40 10.1
l/20
4118
15120
control 9.6
Mean nuclear diameters in 0.065 Mphosphate glycerol.
PH Mean diameter Fraction s control +l TABLE
0.011 0.107 6.98 20.4
buffer in 4.8 M
6.90 13.2 7120
7.10 9.6 18120
buffer in 4.8 M
7.19 9.5
7.30
7.39
10.6
9.0
v3
6/10
16/20
control 9.6
IV. Mean nuclear diameters in relation to phosphate concentration in 4.8 M glycerol.
Phosphate concentration x 6.5 Mean nuclear diameter Standard deviation of mean ad/d
10-s 12.4
10-h 14.9
0.75
10-6 13.8 0.90
0.82 2.25
0.90
buffer
10-6 12.3 0.73 2.24
higher concentrations. From these observations we conclude that phosphate plays a significant role in determining the capacity of the nucleus to swell and the extent of the swelling which may occur in hypertonic glycerol solutions. Whether, as the data of Table IV suggests, this may be through phosphate involvement with metabolic activity of nuclear constituents as, for example, by its relation to ATP or other organic phosphate and pyrophosphate compounds remains to be determined. Although this suggests possible similarity to mitochondrial swelling, comparison of the action of Ca++ and Mg++ on the swelling of these nuclei in glycerol have shown no difference in the effects of these ions. REFERENCE 1. MARSHAK,
A., Expfl. Celf Research12, 599 (1957).
Experimental
Cell Research 14
OF THE
NUCLEI
A. MARSHAK Marine
Bidogkal
OF ASTE&IAS
EGGS1
and R. P. BENOIT
Laboratory,
Woods Hole, Mass.,
U.S.A.
Received November 6, 1957
IN a previous paper [l] it was reported that the swelling of nuclei of Bsterias eggs in 4.8 M glycerol varies with pH, being minimal at pH 7.4. In that investigation, a phosphate-citrate (McIlvalne) buffer was used in the pH range 7.0-7.4 and a Verona1 buffer in the pH range 7.5-8.0. It therefore seemed possible that the observed changes in nuclear swelling might be attributable not to the differences in pH but to specific effects of the ions or molecules of the buffer’s used. The investigations have therefore been extended in an effort to resolve this problem. Asterias eggs were treated as previously described by immersion in solutions of 4.8 M glycerol containing trishydroxy methylaminomethane (tris), glycine ethyl ester, and Verona1 buffers. The pH range covered was 7.1-8.0 the intervals being approximately 0.1 pH unit. With the tris and Verona1 buffers minima in the mean nuclear diameters (n = 20 in all cases) were found at pH 7.4, and with the glycine ethyl ester the minimum was at pH 7.35. In Table I the mean nuclear diameters at the pH intervals where minima were found and those of the adjacent intervals are given together with the standard deviations of the means. In all but two instances the differences between the means of adjacent intervals are on the border line of being statistically significant. Thus 6 of the total of 12 differences have a probability of occurring by chance of 0.06 or less, and 10 of the 12 differences have a probability of occurring by chance of 0.13 or less. The data therefore suggest that there is an effect of pH on nuclear swelling, a minimum being found at pH 7.35-7.42, but they do not conclusively prove the existence of this effect. Although with these buffers, the minimum was found at approximately the same pH as that previously observed with combination of MacIlvaine and Verona1 buffers, the magnitude of the effect is very much less, suggesting the operation of other factors than pH in producing this phenomenon. To obtain further information on the nature of the other factor or factors comparison was made of the effects of MacIlvaine and of phosphate buffers on nuclear swelling in glycerol with results as shown in Tables II and III. In the MacIlvaine-buffered solutions minimum swelling was observed at pH 7.4 as in previous experiments. However, in the phosphate-buffered solutions there was no nuclear swelling at pH greater than 7.0. The appearance of the cells and nuclei in the latter solutions was markedly different from the appearance in MacIlvaine-buffered solutions or those buffered with tris, glycine ethyl ester, or veronal. The cytoplasm was very granular while the nucleus was hyaline except for the nucleolus. Nuclei extruded from their cells at pH 7.1 or higher were irregular in outline suggesting a “soft” and partially collapsed condition. There was thus a striking difference in the behavior of the buffers containing citrate as well as phosphate as compared with the buffers containing only phosphate. The results indicated 1 This work was supported Experimental
Cell Research 14
by a grant from the American
Cancer Society.
Swelling of nuclei TABLE
I. Summary
of differences in mean nuclear diameters at pH 7.2-7.5 and their standard errors. All in 4.8 M glycerol.
Buffer Tris (0.065 M)
Expt.
pH
m
am
1=
7.20 7.42 7.48 7.60
15.4 12.9 13.2 15.7
0.77 1.00 0.83 0.96
7.22 7.42 7.48
18.1 16.6 18.7
1.08 1.18 0.92
7.25 7.35 7.42
18.4 16.3 19.5
0.54 0.83 0.46
7.25 7.35 7.42
19.3 18.4 20.5
0.35 0.45 0.39
7.31 7.39 7.52
20.5 18.9 20.5
0.45 0.73 0.60
7.31 7.39 7.52
19.9 17.8 20.3
0.99 1.25 0.92
2
Glycine ethyl ester (0.065 ,If)
1
2
Verona1 (0.325 di
1
2
All measurements are in m = mean am = standard deviation d - difference between ad = standard deviation
d
ad
d/ad
2.5
1.26
2.0
2.5
1.27
2.0
1.5
1.61
0.9
2.1
1.42
1.5
2.0
0.99
2.0
3.2
0.95
3.4
0.9
0.55
1.6
2.1
0.59
3.4
1.6
0.85
1.9
1.6
0.94
1.7
-2.1
1.59
1.3
2.5
1.55
1.6
ocular micrometer scale units. of the mean
means of adjacent intervals of the difference
a Measurements in this experiment were taken with a different set of lenses than those used in the subsequent experiments. Absolute values are therefore not comparable to those of other series. an interference of the nuclear swelling by phosphate possibly by dissolution of some nuclear constituents and inhibition of the phosphate effect by citrate. Whether or
not the effect of the citrate is the result of possible competition with phosphate for nuclear constituents cannot be determined from these data although it is suggestive that the nuclear diameter is reduced by a factor of 2 when the ratio of phosphate to citrate is increased by a factor of 2. As shown in Table IV, there is in the phosphate buffered solutions, at pH 7.4, a range of phosphate concentrations (6.5 x 10-s - 6.5 x 10-l) in which the effect of phosphate is minimal, i.e., the swelling in this range was greater than at lower and Experimental
Cell Research 14
A. Marshak and R. P. Benoit II. Mean nuclear diameters in 0.065 M Macllvaine glycerol.
TABLE
Molarity citrate Molarity phosphate PH Mean diameter Fraction control $+ 1
TABLE
III.
0.009 0.113 7.20 13.0
0.006 0.118 7.40 10.1
l/20
4118
15120
control 9.6
Mean nuclear diameters in 0.065 Mphosphate glycerol.
PH Mean diameter Fraction s control +l TABLE
0.011 0.107 6.98 20.4
buffer in 4.8 M
6.90 13.2 7120
7.10 9.6 18120
buffer in 4.8 M
7.19 9.5
7.30
7.39
10.6
9.0
v3
6/10
16/20
control 9.6
IV. Mean nuclear diameters in relation to phosphate concentration in 4.8 M glycerol.
Phosphate concentration x 6.5 Mean nuclear diameter Standard deviation of mean ad/d
10-s 12.4
10-h 14.9
0.75
10-6 13.8 0.90
0.82 2.25
0.90
buffer
10-6 12.3 0.73 2.24
higher concentrations. From these observations we conclude that phosphate plays a significant role in determining the capacity of the nucleus to swell and the extent of the swelling which may occur in hypertonic glycerol solutions. Whether, as the data of Table IV suggests, this may be through phosphate involvement with metabolic activity of nuclear constituents as, for example, by its relation to ATP or other organic phosphate and pyrophosphate compounds remains to be determined. Although this suggests possible similarity to mitochondrial swelling, comparison of the action of Ca++ and Mg++ on the swelling of these nuclei in glycerol have shown no difference in the effects of these ions. REFERENCE 1. MARSHAK,
A., Expfl. Celf Research12, 599 (1957).
Experimental
Cell Research 14