DEVELOPMENTAL
BIOLOGY
Potassium
48, 466-472
(1976)
Is Not Compartmentalized within Urchin Egg of Biological
Sciences, Accepted
Sea
R. ROBINSON
KENNETH Department
the Unfertilized
Purdue
University,
September
West Lafayette,
Indiana
47907
lo,1975
Virtually all of the potassium in the unfertilized eggs of the purple sea urchin Strongylocentrotus purpuratus is in a single compartment and is exchangeable with extracellular potassium. This conclusion is based on an analysis of ‘2K uptake and efllux experiments and is in conflict with some claims of other investigators.
mechanism for compartmentalizing potassium. One is forced to the conclusion that the inexchangeable ion must be in membrane-bound organelles and that the activity of potassium in these must be the same as in the cytoplasm. This in turn implies that 70-80% of the interior of the cells must be occupied by these compartments. The difficulty with this is that zones that contain organelles that might sequester potassium (mitochondria, yolk, and pigment) occupy only about 60% of a centrifuged egg (Harvey, 1956, p. 130). Since these zones are unlikely to be more than 50% packed with organelles, only 30% of the volume of these eggs is available for potassium compartmentation. Also, there is no precedent for such compartmentation in small cells. In the most nearly comparable case in which potassium exchange has been studied, that of the Fucus egg, there is clearly only one potassium compartment (Robinson and Jaffe, 1973). In view of these facts, it would be quite remarkable indeed if the bulk of the potassium in sea urchin eggs were inexchangeable. There would not seem to be any mechanism to account for it. Therefore, it was decided to reinvestigate the exchangeability of potassium in the unfertilized eggs of Strongylocentrotus purpuratus.
INTRODUCTION
On the basis of isotope exchange experiments, it has been suggested by several investigators that potassium in unfertilized sea urchin eggs exists in two states (Chambers et al., 1948; Tyler and Monroy, 1959; Tupper, 1973, 1974). According to this view, a small fraction (20-30%) is free in solution in the cytoplasm and its exchange with external potassium is limited by the plasma membrane. The remainder is said to be somehow compartmentalized so that it does not exchange (or exchanges slowly) with the free potassium and therefore not with external potassium. Presumably, this compartmentalized potassium is either bound to proteins or sequestered in impermeable organelles. In any case, most of this potassium is said to become decompartmentalized after fertilization of the egg and to become exchangeable with external potassium. Recently, the two-compartment theory has been challenged on kinetic grounds (see the report of the 1974 La Jolla Conference on Membrane Changes in Fertilization and Egg Maturation in Developmental Biology (1975) 42,20). Further, it is difficult to reconcile these ideas with the findings of Steinhardt et al. (1971). They found, using potassium-selective microelectrodes, that there was no change in the MATERIALS AND METHODS activity of potassium when Lytechinus eggs were fertilized. This would seem to Gametes of the purple sea urchin Stronrule out any sort of protein binding as a gylocentrotus purpuratus were obtained 466
Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
BRIEF NOTES
by injection of 0.5 M KCl. The eggs were collected in 15°C sea water (kept at 15 i 0.5”C throughout the experiments), washed several times, and filtered through silk mesh to remove foreign matter. Sperm was kept “dry” on ice until it was used. The animals used in the experiments reported here were supplied by Pacific BioMarine Co. of Venice, California, in January and February, 1975. J2K was obtained from ICN of Cleveland, Ohio. At the start of the experiment about 1 mCi of a2K was added to 300 ml of artificial sea water (ASW) to make 42KASW stock solution. For each uptake experiment, the eggs from a single female were suspended in ASW at about twice the desired density; 200 ml of this suspension was added to each of three beakers, one of which was used for continuous labeling, one for pulse labeling, and one for total potassium determination. Two hundred millimeters of “2K-ASW stock was then added to one beaker of eggs and stirred at 60 rpm. The concentration of eggs was O.l-0.2%‘. At intervals, 10 ml of this suspension were taken out; extracellular 42K was removed by gently (lo-1OOg) hand-centrifuging the eggs, pouring off the supernatant, and resuspending them in unlabeled ASW. After three such washes, taking a total of 2-3 min, the eggs were evenly distributed in concentrically ringed planchets, dried, and counted in a low-background gas-flow planchet counter. Unlabeled ASW (200 ml) was added to a second of the three beakers and it was stirred as the first. When the rate of uptake of 42K was to be measured, lo-ml samples were removed and the eggs were allowed to settle. Five milliliters of supernatant were then removed and 5 ml of 42K-ASW was added. After 15 min with occasional stirring, the eggs were washed free from the extracellular 42K, dried, and counted as above. The third beaker also had 200 ml of
467
ASW added and was stirred. For total potassium measurements, 10 ml of egg suspension was removed and washed as above with K-free ASW. The eggs were then put in 10 ml of distilled water and the potassium content was determined with an atomic absorption spectrophotometer. To do efflux experiments, eggs were put in a2K-ASW at the appropriate density. This egg suspension was then divided into two beakers, one of which was used to monitor uptake as above. After the desired preloading time, the eggs in the other beaker were allowed to settle, most of ,‘2KASW was aspirated off, and the eggs were washed by hand centrifugation as before. The eggs were then resuspended in ASW and 10 ml samples were removed at desired times, washed once, dried, and counted. The number of eggs in the suspensions was determined by removing 1 ml and distributing the eggs in a dish of known size. Several micrographs were taken and the eggs in the photograph were counted. The total number of eggs per milliliter could then be calculated. Counts of eggs were also made after they had gone through the washing procedure and no loss could be detected as long as the eggs were viable. The viability of the eggs was checked at the beginning of an experiment and periodically thereafter. The eggs from all three vessels were checked for elevation of the fertilization membrane, cleavage, and formation of swimming blastulae after insemination with freshly prepared sperm. The eggs used were always initially 98--1000/r fertilizable. Standards were prepared by adding 0.02 ml of 42K-ASW to planchets containing the same amount of eggs and sea water as the samples. The size of the eggs was determined by measuring the diameter of about 50 eggs under high (400x1 magnification. In all experiments, the average diameter was 80 pm, so the volume was 2.7 x 10e7cm3 and the area was 2.0 x 10e4cm2.
468
DEVELOPMENTAL
BIOLOGY
The composition of ASW was: NaCl, 430 d; MgCL, 55 rniV; CaC12, 10 mM; KCl, 10 miV; Na2S0,, 28 mM; NaHC03, 2 mM. Its pH was 8.0 2 0.2. RESULTS
AND
ANALYSIS
The data in Fig. 1 show the fraction of the total internal potassium that exchanged with external labeled potassium as a function of exposure time to 42K. In both experiments, the fraction exchanged continued to increase until the eggs lost the capacity to respond to fertilization, reaching about 45% in each case. During this time, the total internal potassium, Kr, did not change. In experiment A, four determinations of K, at 3l/2, 7, 13l/2, and 27 hr gave an average of 45.3 pmoles/egg with no value differing from the average by more than 0.5 pmoles/egg. Two determinations in experiment B averaged 44.6 pmoles/egg. The influx was measured by giving eggs a 15-min pulse of 42K at various times during both experiments and it did not change during either. In experiment A, J = 1.26 * 0.14 pmoles/cm*-set (eight measurements) and in B, J = 1.54 2 0.13 pmoles/cm2-set (three measurements). 06I
VOLUME
48. 1976
The loss of developmental capacity occurred quite rapidly and was accompanied by an obvious increase in turbidity, presumably due to proliferation of bacteria. When fertilized, these eggs did not form a membrane and most did not cleave. This was first noticed at the arrows labeled b in Fig. 1. Three or four hours before these drastic changes, the first sign of decline was noticed as a greatly reduced or absent fertilization membrane (arrows a in Fig. 1); however, these eggs still cleaved and 80-85% developed into apparently normal blastula. It is clear that the decrease in tracer potassium seen in both experiments is correlated with the general and rapid deterioration of the eggs. The course of these changes is similar to that observed by Tyler et al. (1938); they concluded that at some point the eggs became susceptible to bacterial attack which caused some eggs to lyse, providing nutrients for further bacterial growth. Such an autocatalytic process would explain the precipitous decline of the eggs in the present experiments. In the further analysis of the data in Fig. 1, only the points prior to the decrease in fraction exchanged were used. If the potassium in the eggs is in a single 0.6 05!b KT 0403o*-
FIG. 1. The uptake of’2K in unfertilized Strongylocentrotusp~rpurutus eggs expressed as a fraction of the total potassium exchanged. Curves A and B each show the results of an experiment on the eggs from a single female. The arrows a indicate the time in each experiment at which samples of the eggs first failed to form a fertilization membrane upon insemination; however, the eggs in these samples still cleaved and most developed into swimming blastulae. Egg samples inseminated at the times indicated by the arrows b largely failed to develop at all. It is clear that prior to failure of the eggs, the internal potassium continues to potassium, was 45 pmoles/egg in both cases. The exchange with external potassium. K r, the total internal curves shown are theoretical plots of K(t)/Kr, assuming various values for f, the hypothesized inexchangeable fraction.
469
BRIEF NOTES
compartment (or if the exchange between the cytoplasm and any intracellular compartments is rapid compared to the exchange across the plasma membrane) the fraction exchanged, K(t)/K,, is given by K(t)/K, = (1 - exp (-JAtlK,)), where J is the influx of potassium in pmoles/cm2, KT is the total internal potassium in picomoles, K(t) is the amount of 42K in an egg at time t divided by the specific activity of the 42K-ASW, and t is the exposure time in seconds. If some fraction f of the total potassium is inexchangeable, the appropriate equation to describe K(t)/K, is K(t)/K, = (1 - f) (1 - exp (-JAtIK, (1 - fl)). The curves shown in Fig. 1 are computer-generated plots of this equation using the appropriate values of J and KT for A and B. It is clear from these curves that any inexchangeable fraction is not greater than 15% of the total. Of course, if there were a second internal compartment that ex-
changed slowly with the cytoplasmic compartment compared to the exchange between cytoplasm and exterior but not infinitely slowly, it could be larger than 15%. The above equation for the fraction of total potassium exchanged can be rewritten as ln[1-F]
= In [l - (1 - f) (1 - exp (-JAt/K,
and for
f =
11 - f)))l
0,
In [l -F]
= -et.
Thus, if t is plotted against In 11 (K(t)/K,)], a straight line will result if and only if all of the internal potassium is in a single compartment. The data from Fig. 1 are plotted in this way in Fig. 2. The straight lines shown are the least-squares best fit lines. Since
.6 -
IO
15 Exposure
Time
20 (Hrs)
25
30
35
20 ( Hrs)
25
30
I 35
.60 0
5
IO
15 Exposure
FIG. 2. Semilogarithmic
Time
plots of the data from Fig. 1; A is from curve A and B from curve B of Fig. 1. If it is assumed that all of the potassium is exchangeable, the points should lie on straight lines and the bestfitting straight lines are shown. The dashed lines are drawn assuming that some fraction fis inexchangeable.
470
DEVELOPMENTAL
J = -KT A
Ah (1 - VW/W) At
BIOLOGY
VOLUME
48, 1976
ter, the fraction of radioactivity eggs after a time t is
’
42K(t) = exp (-JAt/K, 42K#J
the slopes of these lines can be used to calculate the fluxes. This yields 1.18 and 1.43 pmole/cm2-set for A and B, respectively. These values compare well with the earlier-mentioned numbers of 1.26 and 1.54 pmoles/cm2-set calculated directly from flux measurements. The dashed lines in Fig. 2 are theoretical plots of In (1 - (K(t)&)) assuming various values for f, the inexchangeable fraction. Again, it can be seen that any inexchangeable fraction is small, certainly no more than lo-20% of the total. Another approach to the problem of finding the second potassium compartment is to compare the ef?lux of labeled potassium to uptake. If the eggs are preloaded with 42K and then washed in unlabeled sea wa-
left in the
(1 - f)),
where f is the fraction of the total potassium that is inexchangeable. Thus, In-=-42K(t) 42KO
JA KT (1 - fl ”
A plot oft against In (42K(t)/42K,) will produce a straight line (regardless of the size off, whose slope depends on f. If the uptake of 42K is plotted as in Fig. 2, the initial slope will be - (K,/JA), and hence, a comparison of uptake and efflux thus graphed should give a measure of f. An experiment of this sort was done, using eggs from a single female. In Fig. 3A are shown the results of the influx measure-
.6 A 1 IO
I 15 Exposure
Time
I 20 ( Hrs)
I 25
I 30
I 25
I 30
.8 -
.6 I 5
I IO Wosh
I 15 Time
I 20 (Hrs)
FIG. 3. A comparison of 4ZK uptake (A) and eMux (B) from eggs from a single female. The eggs for the etflux experiment were preloaded with 4pK for 3’/2 hr before washing was started. The solid line in B has the same slope as the line in A and is the expected result if there is no inexchangeable potassium, while the dashed lines indicate the expected results if various fractions of the potassium are inexchangeable. Clearly, the solid line best fits the data points.
BRIEF
ments. As before, the plot is linear but the flux J was considerably smaller at 0.73 pmoles/cm*-set, even though the eggs were normal in their response to fertilization and had a normal amount of total potassium. The efflux results are given in Fig. 3B. The straight line is not fitted to data points; rather, it is taken from Fig. 3A and is the expected result if there is no inexchangeable potassium. The dashed lines indicate the expected result if various fractions of the potassium are inexchangeable. It is obvious that the data best fit the f = 0 line. Indeed, the true best fit would be a line lying slightly above the f = 0 line, which represents the physically impossible circumstance of greater than 100% exchangeable potassium. DISCUSSION
The data presented above show that at least 80-90s of potassium in the unfertilized eggs of Strongylocentrotus purpuratus exchanges as if it were in a single compartment limited only by the plasma membrane. No strong evidence of any inexchangeable potassium could be found. This is in general agreement with the results of Steinhardt et al. (1971) who found that the activity of potassium in the cytoplasm of Lytechinus eggs was about what one would expect if the concentration were calculated by dividing the total potassium by the amount of cell water. It is also easier to reconcile a single compartment model with the centrifugation studies mentioned in the Introduction. However, a question arises as to the basis of the conflict between the conclusion drawn in this paper and earlier contrary claims. The data presented in Chambers and Chambers (1949) and Tyler and Monroy (1959) are plainly insufficient for the sort of analysis that would allow one to draw conclusions about potassium compartmentation; indeed, Chambers and Chambers did not make any claim of two potassium compartments in their 1949 pa-
471
NOTES
per. Such a suggestion was only made in two earlier abstracts (Chambers et al., 1948; Chambers, 1949) which lacked supporting data. If the potassium exchange data of Tupper (Tupper, 1973, Figs. 4B, 5B; Tupper, 1974, Fig. 4A) are replotted in semilogarithmic form as in Fig. 2 of this paper, it can be seen that the case for a second inexchangeable compartment is not conclusive. Further, those experiments were done with Strongylocentrotus eggs, and species differences cannot be ruled out. Another factor that needs to be considered in evaluating earlier potassium-exchange experiments is that many of them were done on batches of eggs taken from several females. In the results reported here, a twofold difference in potassium flux between the eggs from two different females was seen; thus, mixing eggs from different females could easily lead to complicated exchange kinetics. This work BMS72-02389
was supported by N.S.F. A02 to Dr. L. F. Jaffe.
Grant
No.
REFERENCES CHAMBERS, E. L. (1949). The uptake and loss of 42K in the unfertilized and fertilized eggs of Strongylocentrotus purpuratus and Arbacia punctulata. Biol. Bull. 97, 251-252. CHAMBERS, E. L., and CHAMBERS, R. (1949). Ion exchanges and fertilization in echinoderm eggs. Amer. Natur. 83, 269-284. CHAMBERS, E. L., WHITE, W., JEUNG, N., and BROOKS, S. C. (1948). Penetration and effects of low temperature and cyanide on penetration of radioactive potassium into the eggs of Strongylocentrotus purpuratus and Arbacia punctulata. Biol. Bull. 95, 252-253. HARVEY, E. B. (1956). “The American Arbacia and Other Sea Urchins,” Princeton Univ. Press, Princeton, N. J. ROBINSON, K. R., and JAFFE, L. F. (1973). Ion movements in a developing fucoid egg. Deuelop. Biol. 35, 349-361. STEINHARDT, R. A., LUNDIN, L., and MAZIA, D. (1971). Bioelectric responses of the echinoderm egg to fertilization. Proc. Nat. Acad. Sci. USA 68, 2426-2430. TUPPER, J. (1973). Potassium exchangeability, potassium permeability and membrane potential: some observations in relation to protein synthesis in the
472
DEVELOPMENTAL
BIOLOGY
early echinoderm embryo. Develop. Biol. 32, 140154. TUPPER, J. (1974). Inhibition of increased potassium permeability following fertilization of the echinoderm embryo: Its relationship to the initiation of protein synthesis and potassium exchangeability. Develop. Biol. TYLER, A., and
38, 332-345.
MONROY, A. (1959). Changes in the
VOLUME
48,
1976
rate of transfer of potassium across the membrane upon fertilization of eggs ofArbaciu punctulatu. J. Exp. 2001. 142, 675-690. TYLER, A., RICCI, N., and
HOROWITZ, N. H. (1938). The respiration and fertilization life of Arbaciu eggs under sterile and non-sterile conditions. J. Exp. 2001. 79, 129-143.
BIOLOGY
Potassium
48, 466-472
(1976)
Is Not Compartmentalized within Urchin Egg of Biological
Sciences, Accepted
Sea
R. ROBINSON
KENNETH Department
the Unfertilized
Purdue
University,
September
West Lafayette,
Indiana
47907
lo,1975
Virtually all of the potassium in the unfertilized eggs of the purple sea urchin Strongylocentrotus purpuratus is in a single compartment and is exchangeable with extracellular potassium. This conclusion is based on an analysis of ‘2K uptake and efllux experiments and is in conflict with some claims of other investigators.
mechanism for compartmentalizing potassium. One is forced to the conclusion that the inexchangeable ion must be in membrane-bound organelles and that the activity of potassium in these must be the same as in the cytoplasm. This in turn implies that 70-80% of the interior of the cells must be occupied by these compartments. The difficulty with this is that zones that contain organelles that might sequester potassium (mitochondria, yolk, and pigment) occupy only about 60% of a centrifuged egg (Harvey, 1956, p. 130). Since these zones are unlikely to be more than 50% packed with organelles, only 30% of the volume of these eggs is available for potassium compartmentation. Also, there is no precedent for such compartmentation in small cells. In the most nearly comparable case in which potassium exchange has been studied, that of the Fucus egg, there is clearly only one potassium compartment (Robinson and Jaffe, 1973). In view of these facts, it would be quite remarkable indeed if the bulk of the potassium in sea urchin eggs were inexchangeable. There would not seem to be any mechanism to account for it. Therefore, it was decided to reinvestigate the exchangeability of potassium in the unfertilized eggs of Strongylocentrotus purpuratus.
INTRODUCTION
On the basis of isotope exchange experiments, it has been suggested by several investigators that potassium in unfertilized sea urchin eggs exists in two states (Chambers et al., 1948; Tyler and Monroy, 1959; Tupper, 1973, 1974). According to this view, a small fraction (20-30%) is free in solution in the cytoplasm and its exchange with external potassium is limited by the plasma membrane. The remainder is said to be somehow compartmentalized so that it does not exchange (or exchanges slowly) with the free potassium and therefore not with external potassium. Presumably, this compartmentalized potassium is either bound to proteins or sequestered in impermeable organelles. In any case, most of this potassium is said to become decompartmentalized after fertilization of the egg and to become exchangeable with external potassium. Recently, the two-compartment theory has been challenged on kinetic grounds (see the report of the 1974 La Jolla Conference on Membrane Changes in Fertilization and Egg Maturation in Developmental Biology (1975) 42,20). Further, it is difficult to reconcile these ideas with the findings of Steinhardt et al. (1971). They found, using potassium-selective microelectrodes, that there was no change in the MATERIALS AND METHODS activity of potassium when Lytechinus eggs were fertilized. This would seem to Gametes of the purple sea urchin Stronrule out any sort of protein binding as a gylocentrotus purpuratus were obtained 466
Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
BRIEF NOTES
by injection of 0.5 M KCl. The eggs were collected in 15°C sea water (kept at 15 i 0.5”C throughout the experiments), washed several times, and filtered through silk mesh to remove foreign matter. Sperm was kept “dry” on ice until it was used. The animals used in the experiments reported here were supplied by Pacific BioMarine Co. of Venice, California, in January and February, 1975. J2K was obtained from ICN of Cleveland, Ohio. At the start of the experiment about 1 mCi of a2K was added to 300 ml of artificial sea water (ASW) to make 42KASW stock solution. For each uptake experiment, the eggs from a single female were suspended in ASW at about twice the desired density; 200 ml of this suspension was added to each of three beakers, one of which was used for continuous labeling, one for pulse labeling, and one for total potassium determination. Two hundred millimeters of “2K-ASW stock was then added to one beaker of eggs and stirred at 60 rpm. The concentration of eggs was O.l-0.2%‘. At intervals, 10 ml of this suspension were taken out; extracellular 42K was removed by gently (lo-1OOg) hand-centrifuging the eggs, pouring off the supernatant, and resuspending them in unlabeled ASW. After three such washes, taking a total of 2-3 min, the eggs were evenly distributed in concentrically ringed planchets, dried, and counted in a low-background gas-flow planchet counter. Unlabeled ASW (200 ml) was added to a second of the three beakers and it was stirred as the first. When the rate of uptake of 42K was to be measured, lo-ml samples were removed and the eggs were allowed to settle. Five milliliters of supernatant were then removed and 5 ml of 42K-ASW was added. After 15 min with occasional stirring, the eggs were washed free from the extracellular 42K, dried, and counted as above. The third beaker also had 200 ml of
467
ASW added and was stirred. For total potassium measurements, 10 ml of egg suspension was removed and washed as above with K-free ASW. The eggs were then put in 10 ml of distilled water and the potassium content was determined with an atomic absorption spectrophotometer. To do efflux experiments, eggs were put in a2K-ASW at the appropriate density. This egg suspension was then divided into two beakers, one of which was used to monitor uptake as above. After the desired preloading time, the eggs in the other beaker were allowed to settle, most of ,‘2KASW was aspirated off, and the eggs were washed by hand centrifugation as before. The eggs were then resuspended in ASW and 10 ml samples were removed at desired times, washed once, dried, and counted. The number of eggs in the suspensions was determined by removing 1 ml and distributing the eggs in a dish of known size. Several micrographs were taken and the eggs in the photograph were counted. The total number of eggs per milliliter could then be calculated. Counts of eggs were also made after they had gone through the washing procedure and no loss could be detected as long as the eggs were viable. The viability of the eggs was checked at the beginning of an experiment and periodically thereafter. The eggs from all three vessels were checked for elevation of the fertilization membrane, cleavage, and formation of swimming blastulae after insemination with freshly prepared sperm. The eggs used were always initially 98--1000/r fertilizable. Standards were prepared by adding 0.02 ml of 42K-ASW to planchets containing the same amount of eggs and sea water as the samples. The size of the eggs was determined by measuring the diameter of about 50 eggs under high (400x1 magnification. In all experiments, the average diameter was 80 pm, so the volume was 2.7 x 10e7cm3 and the area was 2.0 x 10e4cm2.
468
DEVELOPMENTAL
BIOLOGY
The composition of ASW was: NaCl, 430 d; MgCL, 55 rniV; CaC12, 10 mM; KCl, 10 miV; Na2S0,, 28 mM; NaHC03, 2 mM. Its pH was 8.0 2 0.2. RESULTS
AND
ANALYSIS
The data in Fig. 1 show the fraction of the total internal potassium that exchanged with external labeled potassium as a function of exposure time to 42K. In both experiments, the fraction exchanged continued to increase until the eggs lost the capacity to respond to fertilization, reaching about 45% in each case. During this time, the total internal potassium, Kr, did not change. In experiment A, four determinations of K, at 3l/2, 7, 13l/2, and 27 hr gave an average of 45.3 pmoles/egg with no value differing from the average by more than 0.5 pmoles/egg. Two determinations in experiment B averaged 44.6 pmoles/egg. The influx was measured by giving eggs a 15-min pulse of 42K at various times during both experiments and it did not change during either. In experiment A, J = 1.26 * 0.14 pmoles/cm*-set (eight measurements) and in B, J = 1.54 2 0.13 pmoles/cm2-set (three measurements). 06I
VOLUME
48. 1976
The loss of developmental capacity occurred quite rapidly and was accompanied by an obvious increase in turbidity, presumably due to proliferation of bacteria. When fertilized, these eggs did not form a membrane and most did not cleave. This was first noticed at the arrows labeled b in Fig. 1. Three or four hours before these drastic changes, the first sign of decline was noticed as a greatly reduced or absent fertilization membrane (arrows a in Fig. 1); however, these eggs still cleaved and 80-85% developed into apparently normal blastula. It is clear that the decrease in tracer potassium seen in both experiments is correlated with the general and rapid deterioration of the eggs. The course of these changes is similar to that observed by Tyler et al. (1938); they concluded that at some point the eggs became susceptible to bacterial attack which caused some eggs to lyse, providing nutrients for further bacterial growth. Such an autocatalytic process would explain the precipitous decline of the eggs in the present experiments. In the further analysis of the data in Fig. 1, only the points prior to the decrease in fraction exchanged were used. If the potassium in the eggs is in a single 0.6 05!b KT 0403o*-
FIG. 1. The uptake of’2K in unfertilized Strongylocentrotusp~rpurutus eggs expressed as a fraction of the total potassium exchanged. Curves A and B each show the results of an experiment on the eggs from a single female. The arrows a indicate the time in each experiment at which samples of the eggs first failed to form a fertilization membrane upon insemination; however, the eggs in these samples still cleaved and most developed into swimming blastulae. Egg samples inseminated at the times indicated by the arrows b largely failed to develop at all. It is clear that prior to failure of the eggs, the internal potassium continues to potassium, was 45 pmoles/egg in both cases. The exchange with external potassium. K r, the total internal curves shown are theoretical plots of K(t)/Kr, assuming various values for f, the hypothesized inexchangeable fraction.
469
BRIEF NOTES
compartment (or if the exchange between the cytoplasm and any intracellular compartments is rapid compared to the exchange across the plasma membrane) the fraction exchanged, K(t)/K,, is given by K(t)/K, = (1 - exp (-JAtlK,)), where J is the influx of potassium in pmoles/cm2, KT is the total internal potassium in picomoles, K(t) is the amount of 42K in an egg at time t divided by the specific activity of the 42K-ASW, and t is the exposure time in seconds. If some fraction f of the total potassium is inexchangeable, the appropriate equation to describe K(t)/K, is K(t)/K, = (1 - f) (1 - exp (-JAtIK, (1 - fl)). The curves shown in Fig. 1 are computer-generated plots of this equation using the appropriate values of J and KT for A and B. It is clear from these curves that any inexchangeable fraction is not greater than 15% of the total. Of course, if there were a second internal compartment that ex-
changed slowly with the cytoplasmic compartment compared to the exchange between cytoplasm and exterior but not infinitely slowly, it could be larger than 15%. The above equation for the fraction of total potassium exchanged can be rewritten as ln[1-F]
= In [l - (1 - f) (1 - exp (-JAt/K,
and for
f =
11 - f)))l
0,
In [l -F]
= -et.
Thus, if t is plotted against In 11 (K(t)/K,)], a straight line will result if and only if all of the internal potassium is in a single compartment. The data from Fig. 1 are plotted in this way in Fig. 2. The straight lines shown are the least-squares best fit lines. Since
.6 -
IO
15 Exposure
Time
20 (Hrs)
25
30
35
20 ( Hrs)
25
30
I 35
.60 0
5
IO
15 Exposure
FIG. 2. Semilogarithmic
Time
plots of the data from Fig. 1; A is from curve A and B from curve B of Fig. 1. If it is assumed that all of the potassium is exchangeable, the points should lie on straight lines and the bestfitting straight lines are shown. The dashed lines are drawn assuming that some fraction fis inexchangeable.
470
DEVELOPMENTAL
J = -KT A
Ah (1 - VW/W) At
BIOLOGY
VOLUME
48, 1976
ter, the fraction of radioactivity eggs after a time t is
’
42K(t) = exp (-JAt/K, 42K#J
the slopes of these lines can be used to calculate the fluxes. This yields 1.18 and 1.43 pmole/cm2-set for A and B, respectively. These values compare well with the earlier-mentioned numbers of 1.26 and 1.54 pmoles/cm2-set calculated directly from flux measurements. The dashed lines in Fig. 2 are theoretical plots of In (1 - (K(t)&)) assuming various values for f, the inexchangeable fraction. Again, it can be seen that any inexchangeable fraction is small, certainly no more than lo-20% of the total. Another approach to the problem of finding the second potassium compartment is to compare the ef?lux of labeled potassium to uptake. If the eggs are preloaded with 42K and then washed in unlabeled sea wa-
left in the
(1 - f)),
where f is the fraction of the total potassium that is inexchangeable. Thus, In-=-42K(t) 42KO
JA KT (1 - fl ”
A plot oft against In (42K(t)/42K,) will produce a straight line (regardless of the size off, whose slope depends on f. If the uptake of 42K is plotted as in Fig. 2, the initial slope will be - (K,/JA), and hence, a comparison of uptake and efflux thus graphed should give a measure of f. An experiment of this sort was done, using eggs from a single female. In Fig. 3A are shown the results of the influx measure-
.6 A 1 IO
I 15 Exposure
Time
I 20 ( Hrs)
I 25
I 30
I 25
I 30
.8 -
.6 I 5
I IO Wosh
I 15 Time
I 20 (Hrs)
FIG. 3. A comparison of 4ZK uptake (A) and eMux (B) from eggs from a single female. The eggs for the etflux experiment were preloaded with 4pK for 3’/2 hr before washing was started. The solid line in B has the same slope as the line in A and is the expected result if there is no inexchangeable potassium, while the dashed lines indicate the expected results if various fractions of the potassium are inexchangeable. Clearly, the solid line best fits the data points.
BRIEF
ments. As before, the plot is linear but the flux J was considerably smaller at 0.73 pmoles/cm*-set, even though the eggs were normal in their response to fertilization and had a normal amount of total potassium. The efflux results are given in Fig. 3B. The straight line is not fitted to data points; rather, it is taken from Fig. 3A and is the expected result if there is no inexchangeable potassium. The dashed lines indicate the expected result if various fractions of the potassium are inexchangeable. It is obvious that the data best fit the f = 0 line. Indeed, the true best fit would be a line lying slightly above the f = 0 line, which represents the physically impossible circumstance of greater than 100% exchangeable potassium. DISCUSSION
The data presented above show that at least 80-90s of potassium in the unfertilized eggs of Strongylocentrotus purpuratus exchanges as if it were in a single compartment limited only by the plasma membrane. No strong evidence of any inexchangeable potassium could be found. This is in general agreement with the results of Steinhardt et al. (1971) who found that the activity of potassium in the cytoplasm of Lytechinus eggs was about what one would expect if the concentration were calculated by dividing the total potassium by the amount of cell water. It is also easier to reconcile a single compartment model with the centrifugation studies mentioned in the Introduction. However, a question arises as to the basis of the conflict between the conclusion drawn in this paper and earlier contrary claims. The data presented in Chambers and Chambers (1949) and Tyler and Monroy (1959) are plainly insufficient for the sort of analysis that would allow one to draw conclusions about potassium compartmentation; indeed, Chambers and Chambers did not make any claim of two potassium compartments in their 1949 pa-
471
NOTES
per. Such a suggestion was only made in two earlier abstracts (Chambers et al., 1948; Chambers, 1949) which lacked supporting data. If the potassium exchange data of Tupper (Tupper, 1973, Figs. 4B, 5B; Tupper, 1974, Fig. 4A) are replotted in semilogarithmic form as in Fig. 2 of this paper, it can be seen that the case for a second inexchangeable compartment is not conclusive. Further, those experiments were done with Strongylocentrotus eggs, and species differences cannot be ruled out. Another factor that needs to be considered in evaluating earlier potassium-exchange experiments is that many of them were done on batches of eggs taken from several females. In the results reported here, a twofold difference in potassium flux between the eggs from two different females was seen; thus, mixing eggs from different females could easily lead to complicated exchange kinetics. This work BMS72-02389
was supported by N.S.F. A02 to Dr. L. F. Jaffe.
Grant
No.
REFERENCES CHAMBERS, E. L. (1949). The uptake and loss of 42K in the unfertilized and fertilized eggs of Strongylocentrotus purpuratus and Arbacia punctulata. Biol. Bull. 97, 251-252. CHAMBERS, E. L., and CHAMBERS, R. (1949). Ion exchanges and fertilization in echinoderm eggs. Amer. Natur. 83, 269-284. CHAMBERS, E. L., WHITE, W., JEUNG, N., and BROOKS, S. C. (1948). Penetration and effects of low temperature and cyanide on penetration of radioactive potassium into the eggs of Strongylocentrotus purpuratus and Arbacia punctulata. Biol. Bull. 95, 252-253. HARVEY, E. B. (1956). “The American Arbacia and Other Sea Urchins,” Princeton Univ. Press, Princeton, N. J. ROBINSON, K. R., and JAFFE, L. F. (1973). Ion movements in a developing fucoid egg. Deuelop. Biol. 35, 349-361. STEINHARDT, R. A., LUNDIN, L., and MAZIA, D. (1971). Bioelectric responses of the echinoderm egg to fertilization. Proc. Nat. Acad. Sci. USA 68, 2426-2430. TUPPER, J. (1973). Potassium exchangeability, potassium permeability and membrane potential: some observations in relation to protein synthesis in the
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DEVELOPMENTAL
BIOLOGY
early echinoderm embryo. Develop. Biol. 32, 140154. TUPPER, J. (1974). Inhibition of increased potassium permeability following fertilization of the echinoderm embryo: Its relationship to the initiation of protein synthesis and potassium exchangeability. Develop. Biol. TYLER, A., and
38, 332-345.
MONROY, A. (1959). Changes in the
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rate of transfer of potassium across the membrane upon fertilization of eggs ofArbaciu punctulatu. J. Exp. 2001. 142, 675-690. TYLER, A., RICCI, N., and
HOROWITZ, N. H. (1938). The respiration and fertilization life of Arbaciu eggs under sterile and non-sterile conditions. J. Exp. 2001. 79, 129-143.
