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Intracellular pH controls protein synthesis rate in the sea urchin egg and early embryo
DEVELOPMENTAL
BIOLOGY
Intracellular
68,396-406
(1979)
pH Controls Protein Synthesis Rate in the Sea Urchin Egg and Early Embryo
J.L. GRAINGER, M.M. Department
of Zoology,
WINKLER,~. University
S.&IEN,
of California,
AND R. A. STEINHARDT' Berkeley,
California
94720
Received July 5, 1978; accepted in revised form September 14, 1978 Direct comparisons between intracellular pH and protein synthesis in the sea urchin egg and early embryo show that pH controls protein synthesis rate in a highly sensitive and reversible manner. The entire increase and maintenance of protein synthesis at fertilization or parthenogenetic activation could be accounted for by a permanent increase in intracellular pH. However, unfertilized eggs whose intracellular pH has been raised artifXally by ammonia take at least 30 min longer to reach the rate of protein synthesis seen in fertilized eggs. This time lag for ammonia activation and the decrease in protein synthesis rate during mitosis suggest that other unknown factors can also influence protein synthesis rate during fertilization and early embryogenesis.
plasmic pH to the rate of protein synthesis under identical experimental conditions. With the additional tool of passively lowering or raising the intracellular pH by exposure of unfertilized or fertilized eggs to penetrating weak acids or bases, we have been able to examine the interrelationship between intracellular pH and protein synthesis. Finally, we have extended our manipulations of intracellular pH to see to what extent pH can be solely responsible for variations in protein synthesis and chromosome condensation during the cell cycle.
INTRODUCTION
Fertilization of the sea urchin egg sets in motion a series of ionic and metabolic changes associated with the activation of the repressed, unfertilized egg. Among these changes is a 5 to 30-fold stimulation of protein synthesis starting from 5 to 10 min after fertilization (H&in, 1952; Epel, 1967; Fry and Gross, 1970; Humphreys, 1971; Reiger and Kafatos, 1977). Since this increase in protein synthesis could be elicited by the penetration of weak bases into the unfertilized egg, it was postulated that intracellular pH controlled the rate of protein synthesis and that normal fertilization resulted in the elevation of the egg’s intracellular pH (Steinhardt and Mazia, 1973; Epel et al., 1974; Johnson et al., 1976; Lopo and Vacquier, 1977; Winkler and Grainger, 1978). The postulated increase in intracellular pH at fertilization and in activation by weak bases has been recently demonstrated by direct measurement with intracellular pH microelectrodes (Shen and Steinhardt, 1978). The intracellular pH microelectrode has now allowed us to directly compare cyto’ To whom correspondence
should be addressed.
MATERIALS
Copyright All rights
0 1979 by Academic heas, of reproduction in any form
Inc. reserved.
METHODS
Handling of gametes. The shedding of gametes from the sea urchin Lytechinus pi&us was induced by intracoelomic injection of 0.5 M KCl. The sperm was collected dry and stored at 4°C. The eggs were washed once in seawater, pH 5, to remove the jelly, and then washed twice more in Millipore-filtered seawater (0.45 pm). All experiments were carried out at 16-18’C. Preloading unfertilized eggs. The method of preloading unfertilized eggs was modified from Epel (1967). Packed, dejellied eggs, 0.75 ml, were preloaded in 15 ml of seawater containing 5 &i/ml [2,3-3H]-
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GRAINGER ET
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pH Control of Protein Synthesis
valine (Schwarz/Mann, 26 Ci/mmole) for 45 min. The eggs were then washed three times in ice-cold seawater and 0.05 ml of packed eggs was added to 50 ml of seawater containing sperm or various amounts of NH&l or Na-acetate. Two-milliliter samples were taken at the indicated times and the eggs were dissolved in SDS (final concentration 2.5% with 0.1 mg/ml BSA). The macromolecules were then precipitated with an equal volume of ice-cold 20% TCA containing 2 mg/ml unlabeled valine. The precipitates were collected on glass-fiber filters and washed three times with ice-cold 5% TCA containing 1 mg/ml unlabeled valine, once with distilled water, and twice with methanol. The filters were air-dried, 5 ml of NCS Omnifluor was added and the samples were counted in a Beckman LS1OOCscintillation counter. Pulse-labeling unfertilized, fertilized, or activated eggs. The pulse-labeling procedure was modified from Hogan and Gross (1971). Packed, dejellied eggs, 0.25 ml, were resuspended in 250 ml of seawater containing either sperm or the designated amount of NH&l or Na-acetate. Four-milliliter samples were taken every 10 min. Immediately after taking the sample, the eggs were centrifuged and the seawater was aspirated to a final volume of 0.2 ml. [2,3-3H]Valine, 1 PCi, in 20 ~1 seawater was added and the eggs were incubated at 17°C for 5 min with constant stirring. The uptake of [3H]valine was linear in unfertilized, fertilized, NH&lactivated, and Na-acetate-treated eggs over this time interval. The eggs were then washed three times with ice-cold seawater containing 0.5 mg/ml unlabeled valine. After the final wash, the eggs were dissolved in SDS (2.5% with 0.1 mg/ml BSA) and then precipitated with an equal volume of 20% TCA containing 2 mg/ml unlabeled valine. After lhr at 2”C, the tubes containing both the TCA-soluble and -insoluble counts were vortexed and a fixed aliquot was removed to determine the total uptake. Aquasol, 5 ml, was added to this aliquot
397
and the samples were counted as above. The efficiency of counting in Aquasol was equal to the efficiency obtained with NCS Omnifluor. The remaining precipitates were collected on glass-fiber filters and processed as described above for preloaded eggs. The relative rates of protein synthesis were determined by dividing the counts accumulated into TCA-insoluble material over the 5-min pulse by the total counts accumulated in the eggs over this time interval. IntracellularpH measurement. Intracellular pH (pHJ was measured with Thomas type H’-sensitive microelectrodes with recessed tips (Thomas, 1976). These electrodes gave a linear response of slope 56-59 mV/pH unit over the range pH 2-9 and had less than a &3-mV drift when calibrated before and after each experiment in 100 n&f phosphate buffers (pH 6.6 and 7.6). The response times of the pH microelectrodes used in this study were 30 set or less and were unaffected by the presence of high protein concentrations (50 mg/ml BSA). In our experiments, two microelectrodes penetrated the egg, which was firmly attached to the dish with poly-lysine. Measurements of the egg membrane potential (E,) were made with conventional microelectrodes filled with 3 M KCl. This electrode was placed first in the egg by negative capacitance, The pH-sensitive microelectrode was then placed in the egg and did not cause a depolarization of E, greater than 2 mV. These electrodes recorded the membrane potential and a voltage proportional to pHi. Because of the high resistance of the pH microelectrode, its voltage was fed into a high-impedance ( 1015 a) amplifier (Model F-23, W-P Instruments, New Haven, Conn.), while the membrane potential was fed into a Getting preamplifier (Model 5, Getting Instruments, Palo Alto, Calif.). Both potentials were displayed continuously on separate channels of a Gould Brush Mark 220 chart recorder. To obtain pHi, the membrane potential was sub-
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tracted from the pH microelectrode record. Each pH measurement was repeated at least twice with essentially identical results before any conclusions were drawn. In order to determine that both microelectrodes were implanted in the egg with minimal membrane damage, a current pulse was passed through the conventional microelectrode periodically and the corresponding membrane potential deflection was monitored by the pH-sensitive microelectrode. Excessive membrane damage or vesication at the electrode tip resulted in a loss of coupling between the two microelectrodes.
Comparisons between amino acid incorporation andpH measurements. There are substantial variations in the rate of amino acid incorporation in both unfertilized and fertilized eggs from different females. This variation makes exact quantitative comparisons of rate and intracellular pH more difficult since experiments on the measurement of pH require many attempts involving eggs from several females over long periods of time. However, good estimates of the relative magnitude of the effect of cytoplasmic pH on rates of protein synthesis can be made, since there is much less variation in pH measurements in eggs from different females. Cytology. Samples of eggs were taken at indicated times and the eggs were fixed in EtOH:acetic acid (3:l) overnight and then stained with 2% orcein in 75% acetic acid. The presence of condensed chromosomes or mitotic spindles was then scored. RESULTS
Relationship between the Initial Change in the Intracellular pH and the Onset of the Increase in the Rate of Protein Synthesis Shen and Steinhardt (1978) have demonstrated that the intracellular pH in sea urchin eggs increases after fertilization and NH&l activation. Following this increase, there is an acceleration in the rate of protein synthesis as well as the initiation of
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DNA synthesis and chromosome condensation cycles (Mazia, 1974; Epel et al., 1974). However, the increase in the rate of protein synthesis following NH&l activation occurs later than the increase observed after fertilization (Epel et al., 1974). If the increase in the intracellular pH is related to the increase in the rate of protein synthesis, then the onset of the change of the intracellular pH after NH&l activation should be delayed when compared to fertilization. We have examined more closely the timing between the onset of the initial change in the intracellular pH with this initial increase in the rate of protein synthesis by measuring the change in the intracellular pH directly with pH microelectrodes and determining the exact timing of the acceleration in the rate of protein synthesis after fertilization or NH&l activation. At fertilization, the increase in the intracellular pH begins l-2 min after sperm-egg fusion, while in eggs activated with 10 m&f NH&l, pH 8, there is a 3- to 4-min lag between the addition of weak base and the observed increase in intracellular pH (Fig. 1A). If the change in intracellular pH of the egg initiates the change in the rate of protein synthesis, one would expect to find a 2-min lag in the onset of protein synthesis with NH&l-activated eggs. Using preloaded eggs, the first detectable increase in accumulation of radioactivity in fertilized eggs occurs at 6-7 min after fertilization, while the first detectable increase in NH&l-activated eggs occurs between 9 and 10 min after the addition of 10 r&f NH&l, pH 8 (Fig. 1B). Thus, for fertilization, there is a 4- to 6-min interval between the initial change in intracellular pH and the onset of protein synthesis acceleration. If this stimulation of protein synthesis is directly related to the pH change, then the rate of protein synthesis in eggs activated with NH&l should increase 2 min later than normal fertilization or 8-9 min after the initial exposure of these eggs to NH.&l. The close agreement between our observed val-
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while eggs fertilized from the same batches reached their similar and maximum rates at 30 min after fertilization. Therefore, in eggs whose pH has been raised artificially by ammonia, it takes at least 30 min longer to reach the rate of protein synthesis seen in fertilized eggs. However, the pH change 18 22 26 30 34 in 10 n&f NH&l (Fig. 1) is only a few Time (mm) minutes slower than in fertilization and reaches higher levels within 12 min. Thus the time lag for ammonia stimulation of protein synthesis cannot be entirely explained by the slight delay in intracellular pH increase. This suggests that either other E IO 0 factors contribute to the acceleration of 6 protein synthesis at fertilization or that am2 monia has toxic effects. In an experiment 2 6 IO 14 18 22 26 30 34 to discriminate between these two possibilTime (min.) ities, we fertilized and 10 min later added FIG. 1. (A) The change in intracellular pH (pHJ NH&l and we found that 10 m.M NH&l, following fertilization or NH&l activation. The eggs pH 8, did not retard protein synthesis in were either fertilized (A) or activated with 10 r&f the first 30 min compared to the fertilized NH&l, pH 8 (O), and the increase in pHi was contincontrol. Since the NH&l does not have an uously monitored with H+-sensitive microelectrodes as described under Materials and Methods. These inhibitory effect within the time interval continuous pHi recordings were made under experi- where the biggest difference in protein synmental conditions identical to those of the correspond- thesis rates develop between ammoniaing protein synthesis measurements. These records stimulated and fertilized eggs, we conclude are typical of over 20 pHi recordings of fertilization and 10 pHi recordings of NH&l activation. (B) The that a factor or factors other than intracelincrease in the rate of protein synthesis following lular pH, also plays a role at fertilization. fertilization or NH&l activation. Eggs were preloaded Control of the Rate of Protein Synthesis by and then placed in SW (unfertilized, l ), SW containing Manipulating the Intracellular pH sperm (fertilized, A), or SW containing 10 m&f NH&l, A
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pH 8 (NH&l activated, 0). Samples were taken every 1 min for the first 10 mm and every 2 min thereafter and processed as described under Materiala and Methods. The relative rates of protein synthesis were calculated as the change in cpm incorporated over tune.
ues of 9-10 min and the predicted values implies that the initial increase in the rate of protein synthesis is directly related to the onset in the change of the intracellular PH. Maximum Rates in Fertilized and NH&lActivated Eggs In three experiments using the pulse-label technique, unfertilized eggs reached the maximum rate of protein synthesis at 60-120 min after exposure to 10 mA4 NH&l
In unfertilized eggs, the intracellular pH and the rate of protein synthesis are low compared to fertilized or NH&l-activated eggs (Epel et al., 1974;Shen and Steinhardt, 1978). If the low intracellular pH of the unfertilized eggs is in some way inhibiting the rate of protein synthesis, then lowering the intracellular pH of fertilized or NH&lactivated eggs should lead to a decrease in the rate of protein synthesis. We have examined this question by first lowering the intracellular pH of NH&l-activated eggs by washing out the NH&l after the initial pH and rate of protein synthesis increase, and second, by using the weak acid, Naacetate to artificially lower the intracellular PH.
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The addition of 10 mM NH&l, pH 8, to unfertilized eggs caused an increase in the intracellular pH (Fig. IA). The NH&l enters the eggs by simple diffusion. The uncharged form of the weak base diffuses into the egg and picks up a H+ ion from the cytoplasm, resulting in an increase in the intracellular pH (Winkler and Grainger, 1978). Due to the equilibrium established between the charged and uncharged forms of the weak base inside the egg, a certain proportion of the intracellular NH&l is uncharged. When the NH&l on the outside is removed, the uncharged base will diffuse out of the egg and the intracellular pH should drop. As measured using intracellular pH microelectrodes, the pH does drop after NH&l is washed out @hen and Steinhardt, 1978). Even after extensive washing, however, the intracellular pH does not reach the unfertilized level, but levels off between the unfertilized and activated pH. Upon examining the rates of protein synthesis under these same circumstances, we found that the rate of protein synthesis increases when the intracellular pH increases and remains high as long as the eggs remain in NH&l and the intracellular pH remains high (Fig. 2). As soon as the NH&l is washed out, the intracellular pH and the rate of protein synthesis both drop. They both stabilize above the unfertilized but below the maximally activated levels. Why the intracellular pH does not drop to the unfertilized level when the NH,Cl is washed out is not clear. But the fact that both the intracellular pH and the rate of protein synthesis drop to an intermediate level is further evidence that intracellular pH is correlated with the rate of protein synthesis. In order to directly determine the effect of lowering the intracellular pH on the rate of protein synthesis, we used the weak acid, Na-acetate, to lower the intracellular pH. As the weak base, NH&l, the uncharged form of the weak acid diffuses across the plasma membrane. The weak acid gives up protons as it enters the cytoplasm, causing
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Time (min.) FIG. 2. The rate of protein synthesis in NH&lactivated eggs. Unfertilized eggs were added to SW, pH 8 (O), or SW containing 5 m&f NH&l, pH 8 (O), at t = 0. Samples were taken every 10 min and the rate of protein synthesis was determined by the pulselabel technique. At 120 min, the eggs were washed twice in SW to remove the NH&l and resuspended in SW at the original concentration (O.l%, closed arrow). Other samples were taken every 10 min, fixed in EtOH:acetic acid (3:1), and stained in orcein to score for chromosome condensation. Time of maximal chromosome condensation was at 110 min (open arrow).
a decrease ir. the intracellular pH. If we adjust the pH of the seawater containing 10 mM Na-acetate to pH 6.5, then enough of the acetate is in the uncharged form to penetrate the plasma membrane and lower the intracellular pH. When seawater containing 10 mM Na-acetate, pH 6.5, is added to fertilized eggs after the initial pH increase has been completed, the intracellular pH drops to the unfertilized level within 25 min (Fig. 3A). Immediately after the addition of Na-acetate, while the intracellular pH is dropping, the rate of protein synthesis stops at its present level (Fig. 3B). After the intracellular pH has returned to the unfertilized level, the rate drops off linearly until it reaches the unfertilized rate. One could argue that the Na-acetate is poisoning the eggs, but as will be shown below, the effects of lowering the intracellular pH are totally reversible by removing the acetate stimulus (Fig. 3B, arrow 2). Thus, using this weak acid, we can lower the intracellular pH of a permanently activated egg
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Time (mln) FIG. 3. (A) The effect of Na-acetate on pH, of fertilized eggs. Eggs were fertilized at tt = 0, and at 20 min after fertilization (arrow), the egg was perfused with SW containing 10 m&f Na-acetate, pH 6.5. (B) The effect of Naacetate on the rates of protein synthesis and cell division. Unfertilized control (0); fertilized control (0); fertilized with Na-acetate treatment as described below (A). Unfertilized eggs were added to SW containing sperm at t = 0 and time points were taken as described under Materials and Methods and Fig. 2. At 30 min after fertilization, one batch of eggs (A) was resuspended in SW containing 10 m&f Na-acetate, pH 6.5 (arrow 1). At 130 min, this same batch of eggs was washed twice in SW, pH 8, and resuspended in SW at the original concentration (O.l%, arrow 2). Samples were also taken every 15 mm, fixed in EtOH:acetic acid (3:1), stained in orcein, and scored for nuclear envelope breakdown, chromosome condensation, mitotic spindle formation, and cell division.
and shut down the protein synthesis machinery. This implies that in order to maintain a high rate of protein synthesis, the egg must maintain an alkaline pH.
Relationship of the Magnitude of the Change in the Intracellular pH to the Rate of Protein Synthesis Since the rate of protein synthesis can be altered by raising or lowering the intracellular pH, we want to know the sensitivity of the protein synthetic machinery to changes in intracellular pH. The determination of the effect of small changes in pH
in fertilized or NH&l-activated eggs becomes complex due to the fact that the rates are constantly changing. However, in unfertilized eggs, the rate of protein synthesis remains constant, and thus small changes in the rate induced by small changes in the intracellular pH can be observed without the complication of the fluctuations seen in fertilized or NH&l-activated eggs. The addition of 10 m.M Na-acetate, pH 6.5, to unfertilized eggs lowers the intracellular pH from 6.85 to 6.67, a drop in the intracellular pH of ca. 0.2 units (Fig. 4A).
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Time (min.) FIG. 4. (A) The effect of Na-acetate on pHi of unfertilized eggs. At t = 0, the eggs were perfused with SW containing 10 mM Na-acetate, pH 6.5. Between 26 and 33 min (arrows) after the addition of Na-acetate, the acetate was removed by perfusing with SW, pH 8. (B) The effect of lowering pHi on the rate of protein synthesis in preloaded unfertilized eggs. Unfertilized eggs were preloaded and placed into SW, pH 8 (O), or SW containing 10 m&f Na-acetate, pH 6.5 (0). Samples were taken every 10 min and processed as described in Fig. 1. (C) The effect of lowering and raising pHi of unfertilized eggs. Unfertilized eggs were either placed in SW, pH 8 (0) or in SW containing 10 mM Na-acetate, pH 6.5, at t = 0 (0). Samples were taken every 10 min and the rate of protein synthesis was determined by the pulse-label technique. In this batch of eggs which were already at a very low rate of protein synthesis, acetate addition did not show a repression of protein synthesis since we are already below the threshold for accurate rates with the pulse-label technique. At 40 min after acetate addition, the eggs were washed twice in SW and resuspended in SW, pH 8, at the original concentration (arrow). With removal of the acetate, the pH rose as in Fig. 4A, beyond the unfertilized level and amino acid incorporation increased to three- to fourfold the unfertilized rate.
When the acetate is washed out of the eggs, the intracellular pH increases. Instead of returning to the unfertilized level, it overshoots the unfertilized pH slightly, stabilizing at pH 6.98, 0.13 pH unit above the previous unfertilized level. Upon examining the rates of protein synthesis under these same conditions, two points are evident. First, the rate of protein synthesis in acetate-treated eggs is reduced sixfold compared to the control unfertilized eggs (Fig. 4B). Second, upon removing the acetate, the rate of protein synthesis increases (Fig. 4C). However, it does not return to the unfertilized level, but increases to a higher rate, after which it remains constant. Within 20 min after the acetate is washed out, both the intracellular pH and the rate of protein synthesis are greater than the unfertilized levels. In this case, the final effect of removing the acetate, thus raising
the intracellular pH by 0.13 unit, results in an increase in the rate of protein synthesis by three- to fourfold. Therefore, small changes in the intracellular pH lead, in a reversible manner, to large changes in the rate of protein synthesis.
Effects of Altering the Intracellular pH on Protein Synthesis and Chromosome Condensation Both the timing and the magnitude of the increase and the decrease in the rates of protein synthesis as seen in fertilized eggs can be induced by activating eggs with NH&l (Fig. 2). As shown in Fig. 5, the rate of protein synthesis increases after fertilization and decreases as the eggs approach mitosis. The maximal rate obtained during the first cycle was consistently 30x the unfertilized rate in our measurements in preloaded eggs (Fig. 1B) or pulse-labeled
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FIG. 5. The cyclic changes in the rate of protein synthesis in fertilized eggs. At t = 0, eggs were placed in SW (0) or in SW containing sperm (0). The rate of protein synthesis was determined using the pulse-label technique. At 15-min intervals, samples were taken for cytology and scored for cell division.
eggs (Fig. 5). At mitosis the rate of protein synthesis reaches its lowest level, 60% of the maximal rate, and then accelerates rapidly until the second division. At this time, the rate levels off again and then continues to cycle through at least one more cell division. The difference in sharpness in the rate decrease between the first and second division is probably due to slight asynchrony in the population of fertilized eggs. After the addition of NH&l to unfertilized eggs, the rate of protein synthesis increases and decreases as in fertilized eggs (Fig. 2). In these experiments, 5 mM NHdCl, pH 8, remained in the seawater for 2 hr. The maximal rate obtained is 30x the unfertilized rate as in fertilized eggs. There is a drop in the rate to 60% of this maximal value when the chromosomes are maximalIy condensed, corresponding to metaphase in the fertilized egg. Thus, in the continuous presence of NH&l, the variations in the rates of protein synthesis are similar to those seen in fertilized eggs (see also Fig. 8).
Do changes in intracellular pH correspond to the variations of protein synthesis observed during the cell cycle? In our longest continuous recordings of intracellular pH of fertilized eggs, we do see a dip of 0.15 pH unit preceding, in the expected manner, the subsequent decrease in the rate of protein synthesis (Fig. 6). Both intracellular pH and protein synthesis remain above the unfertilized levels and the magnitude of the decline of both pH and protein synthesis during the cell cycle roughly correspond to what we expect from our previous analysis of the effect of intracellular pH on the rate of protein synthesis. However, with the continuous exposure of unfertilized eggs to NH&l, the intracellular pH remains consistently high without the small decline observed in fertilized eggs as they approach cell division (Fig. 6). Yet the protein synthesis rate declines at the appropriate time in these NH&l-activated eggs (Fig. 2), even though the pH remains constant at high alkaline levels. Therefore, we conclude that the correlation between the intracellular pH and protein synthesis can be overridden by another factor or factors at points in the cell cycle. Lowering the intracellular pH by adding mM NH&
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FIG. 6. Measurement of the changes in pHi after fertilization (0) or activation with 5 m&f NH&l, pH 8 (0). Nuclear envelope breakdown occurred at 62 min in the fertilized eggs and at 70 min in the NH&lactivated eggs.
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Na-acetate can shut down a previously activated fertilized egg completely and reversibly. As described above, if Na-acetate is added to fertilized eggs after the initial pH increase has been completed, the intracellular pH and the rate of protein synthesis drop to the unfertilized levels (Figs. 3A and B). The normal variation in the rate of protein synthesis is stopped and the eggs are prevented from entering mitosis. No nuclear envelope breakdown, chromosome condensation, or mitotic spindle formation occurs. When the acetate is washed out (Fig. 3B, arrow 2)) the intracellular pH rises and the rate of protein synthesis accelerates until it reaches the control fertilized level. This is followed by the breakdown of nuclear envelope, chromosome condensation, mitotic spindle formation, and cell division. Thus, the addition of Na-acetate and the lowering of the intracellular pH reversibly block the cyclic changes in protein synthesis and mitotic events in fertilized eggs. The intracellular pH must remain continuously above a certain level in order to get the normal rates of protein synthesis and the normal cycling of these rates with the cell cycle. When eggs are exposed to 10 mM NH&l, pH 8, for only 15 min, a normal increase in the rate of protein synthesis is observed (Fig. 7). The rate than decreases as in fertilized eggs, but instead of reaccelerating to a higher level, it remains at 40% of the maximal rate. No activation of chromosome cycles is observed. Thus, this brief exposure to NH&l, which results in only a partial increase of pH (Shen and Steinhardt, 1978), only stimulates one burst of protein synthesis and is not sufficient to induce the cyclic variation in protein synthesis or chromosome condensation. In an attempt to determine if this effect was due to the lower intracellular pH after the NH&l was removed from the eggs we activated the eggs with 10 mM NH&l for 15 min, washed out the NH&l, and then placed the eggs in a low concentration of weak base. The short exposure to a high
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FIG. 7. The effect of a short pulse of NH&l on the cycling of the rate of protein synthesis. Eggs were placed in SW containing 10 mM NH&l, pH 8, at t = 0. After 15 min, the NH&l-activated eggs were washed twice in SW and resuspended in SW, pH 8. The rate of protein synthesis was monitored by the pulse-label technique. The three plots are of three independent determinations of the change in the rate of protein synthesis.
concentration of weak base would cause the increase in initial pH and rate of protein synthesis increase, and the addition of a lower concentration of weak base would maintain the high intracellular pH and rate of protein synthesis as seen in fertilized eggs. Under these conditions, the rate of protein synthesis remains high and the observed increases and decreases in the rate exactly match those of fertilized eggs (Fig. 8). Thus, this is further evidence that the timing of the cyclic variation in the rate of protein synthesis can be maintained if the intracellular pH remains high. DISCUSSION
We have shown that the level of intracellular pH directly correlates with the rate of protein synthesis during fertilization or parthenogenetic activation with NH&l. This conclusion is supported by three lines of evidence. First, the onset of an increase in the intracelhilar pH is directly coupled with the onset of the stimulation of protein synthesis in both fertilization and NHdCl
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Time (min) FIG. 8. The effect of the maintenance of a high pHi on the cycling of the rate of protein synthesis. Eggs were placed in SW, pH 8 (O), or SW containing 10 mA4 NH&i, pH 8, for 15 min (O), after which they were washed twice in SW and resuspended at the original concentration in 0.5 mM NH&l, pH 8. The arrows indicate the times of the drop in the rate of protein synthesis found in fertilized eggs.
activation. Second, the rate of protein synthesis can be slowed down to the repressed, unfertilized rate by simply lowering the intracellular pH of fertilized eggs back down to the unfertilized level. Finally, these stimulations or inhibitions of protein synthesis rate are controlled in a completely reversible manner by the passive manipulations of intracellular pH with weak acids or weak bases. The rate of protein synthesis is highly sensitive to intracellular pH. Since we are comparing intracellular pH measurements made on small numbers of eggs to the pooled results from the large numbers of eggs used in amino acid incorporation experiments, it is difficult to make exact quantitative comparisons. Nevertheless, we can easily estimate the relative magnitude of the effect of cytoplasmic pH on rates of protein synthesis. A 0.2-pH-unit decrease reduces amino acid incorporation 6-fold, a 0.13-unit increase leads to a 3- to 4-fold stimulation. By calculation with these factors relating pH and protein synthesis rate as seen by amino acid incorporation, we would predict a 27- to 64-fold increase in protein synthesis at fertilization where the
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cytoplasmic pH increases from 6.84 f 0.02 to 7.27 + 0.03 (Shen and Steinhardt, 1978). Our observed rate at fertilization, 30x the unfertilized rate, falls within this range. Therefore, within the intracellular pH range of 6.7 to 7.3, this high sensitivity could account for the entire stimulation at fertilization. At higher pH, however, we do not observe further stimulation of protein synthesis rate since NH&l-activated eggs do not exceed the rate attained in fertilized eggs although the intracellular pH in NH&l-activated eggs reaches 7.65 + 0.05 (Shen and Steinhardt, 1978). What are the molecular mechanisms responsible for this high sensitivity to pH? Humphreys (1971) has shown that mRNA availability controlled the increase in protein synthesis at fertilization. The fact that we always observe a lag time between the changes in intracellular pH and the changes in protein synthesis rate is consistent with Humphreys’ interpretation since such a lag rules out a direct effect of pH on the enzymatic steps of peptide elongation. We are currently investigating to what extent these lags can be explained by mRNA availability and whether additional regulatory mechanisms sensitive to pH remain to be uncovered. During the cell cycle there is a cycle in the rate of protein synthesis. We have shown that intracellular pH must be sustained at a high degree of alkalinity, well above that of the unfertilized egg, in order to maintain normal rates and normal cycling of protein synthesis during cell cycles. Furthermore, since these cycles in protein synthesis rates are seen in NH&l-activated eggs whose intracellular pH is clamped at high values, we have evidence that other factors can influence protein synthesis rate later on in the cell cycle. Finally, we conclude that the entire increase and maintenance of protein synthesis at fertilization or parthenogenetic activation could be accounted for by a permanent increase in the intracellular pH. How-
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ever, since the timing differences observed in protein synthesis rate between ammoniaactivated and fertilized eggs cannot be explained on the basis of intracellular pH, we believe other unknown factors can also contribute to the derepression of protein synthesis at fertilization. One possible candidate is the intracellular calcium released at fertilization or some change induced by that calcium release (Steinhardt et al., 1977).
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HUMPHREYS, T. (1971). Measurement of messenger
RNA entering polysomes upon fertilization of sea urchin eggs. Develop. Biol. 26, 201-208. JOHNSON, J. D., EPEL, D., and PAUL, M. (1976). Intracellular pH and activation of sea urchin eggs after fertilization. Nature (London) 262,661-664. LOPO, A., and VACQUIER, V. D. (1977). The rise and fall of intracellular pH of sea urchin eggs after fertilization. Nature (London) 269, 590-592. MAZIA, D. (1974). Chromosome cycles turned on in unfertilized sea urchin eggs exposed to NH40H. Proc. Nat. Acad. Sci. USA 71,690-693. REGIER, J. C., and KAFATOS, F. C. (1977). Absolute This investigation was supported by USPHS Grant rate of protein synthesis in sea urchins with specific GM 13882 to Dr. Daniel Mazia and NSF Grant PCM activity measurements of radioactive leucine and 77-042690 to R.A.S. S.S.S. was supported by NIH leucyl t-RNA. Develop. Biol. 57,270-283. Grant CA-21711. SHEN, S. S., and STEINHARDT, R. A. (1978). Direct measurement of intracellular pH during metabolic REFERENCES derepression at fertilization and ammonia activation of the sea urchin egg. Nature (London) 272, EPEL, D. (1967). Protein synthesis in sea urchin eggs: A “late” response to fertilization. hoc. Nat. Acad. 253-254. STEINHARDT, R. A., and MAZIA, D. (1973). DevelopSci. USA 57.889-906. ment of K+ conductance and membrane potential in EPEL, D., STEINHARDT, R. A., HUMPHREYS, T., and unfertilized sea urchin eggs after exposure to MAZIA, D. (1974). An analysis of the partial metabolic derepression of sea urchin eggs by ammonia: NH,OH. Nature (London) 241,400-401. The existence of independent pathways. Develop. STEINHARDT, R., ZUCKER, R., and SCHATTEN, G. (1977). Intracellular calcium release at fertilization Biol. 40,245-255. in the sea urchin egg. Develop. Biol. 58, 185-196. FRY, B. J., and GROSS, P. R. (1970). Patterns and rates of protein synthesis in sea urchin embryos. I. Uptake THOMAS, R. C. (1976). Construction and properties of and incorporation of amino acids during the fist recessed-tip microelectrodes for sodium and chlocleavage cycle. Develop. Biol. 21, 105-124. ride ions and pH. In “Ion and Enzyme Electrodes in HOGAN, B., and GROSS, P. R. (1971). The effect of Biology and Medicine” (M. Kessler, L. C. Clark, Jr., protein synthesis inhibition on the entry of messenD. W. Lubbers, I. A. Silver, and W. Simon, eds.), pp. ger RNA into the cytoplasm of sea urchin embryos. 141-148. University Park Press, Baltimore. J. Cell Biol. 49, 692-701. WINKLER, M. M., and GRAINGER, J. L. (1978). Mechanism of action of NH&l and other weak bases in HULTIN, T. (1952). Incorporation of “Nlabelled glytine and alanine into the proteins of developing sea the activation of sea urchin eggs. Nature (London) urchin eggs. Exp. Cell Res. 3,494-496. 273, 236-238.
BIOLOGY
Intracellular
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pH Controls Protein Synthesis Rate in the Sea Urchin Egg and Early Embryo
J.L. GRAINGER, M.M. Department
of Zoology,
WINKLER,~. University
S.&IEN,
of California,
AND R. A. STEINHARDT' Berkeley,
California
94720
Received July 5, 1978; accepted in revised form September 14, 1978 Direct comparisons between intracellular pH and protein synthesis in the sea urchin egg and early embryo show that pH controls protein synthesis rate in a highly sensitive and reversible manner. The entire increase and maintenance of protein synthesis at fertilization or parthenogenetic activation could be accounted for by a permanent increase in intracellular pH. However, unfertilized eggs whose intracellular pH has been raised artifXally by ammonia take at least 30 min longer to reach the rate of protein synthesis seen in fertilized eggs. This time lag for ammonia activation and the decrease in protein synthesis rate during mitosis suggest that other unknown factors can also influence protein synthesis rate during fertilization and early embryogenesis.
plasmic pH to the rate of protein synthesis under identical experimental conditions. With the additional tool of passively lowering or raising the intracellular pH by exposure of unfertilized or fertilized eggs to penetrating weak acids or bases, we have been able to examine the interrelationship between intracellular pH and protein synthesis. Finally, we have extended our manipulations of intracellular pH to see to what extent pH can be solely responsible for variations in protein synthesis and chromosome condensation during the cell cycle.
INTRODUCTION
Fertilization of the sea urchin egg sets in motion a series of ionic and metabolic changes associated with the activation of the repressed, unfertilized egg. Among these changes is a 5 to 30-fold stimulation of protein synthesis starting from 5 to 10 min after fertilization (H&in, 1952; Epel, 1967; Fry and Gross, 1970; Humphreys, 1971; Reiger and Kafatos, 1977). Since this increase in protein synthesis could be elicited by the penetration of weak bases into the unfertilized egg, it was postulated that intracellular pH controlled the rate of protein synthesis and that normal fertilization resulted in the elevation of the egg’s intracellular pH (Steinhardt and Mazia, 1973; Epel et al., 1974; Johnson et al., 1976; Lopo and Vacquier, 1977; Winkler and Grainger, 1978). The postulated increase in intracellular pH at fertilization and in activation by weak bases has been recently demonstrated by direct measurement with intracellular pH microelectrodes (Shen and Steinhardt, 1978). The intracellular pH microelectrode has now allowed us to directly compare cyto’ To whom correspondence
should be addressed.
MATERIALS
Copyright All rights
0 1979 by Academic heas, of reproduction in any form
Inc. reserved.
METHODS
Handling of gametes. The shedding of gametes from the sea urchin Lytechinus pi&us was induced by intracoelomic injection of 0.5 M KCl. The sperm was collected dry and stored at 4°C. The eggs were washed once in seawater, pH 5, to remove the jelly, and then washed twice more in Millipore-filtered seawater (0.45 pm). All experiments were carried out at 16-18’C. Preloading unfertilized eggs. The method of preloading unfertilized eggs was modified from Epel (1967). Packed, dejellied eggs, 0.75 ml, were preloaded in 15 ml of seawater containing 5 &i/ml [2,3-3H]-
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pH Control of Protein Synthesis
valine (Schwarz/Mann, 26 Ci/mmole) for 45 min. The eggs were then washed three times in ice-cold seawater and 0.05 ml of packed eggs was added to 50 ml of seawater containing sperm or various amounts of NH&l or Na-acetate. Two-milliliter samples were taken at the indicated times and the eggs were dissolved in SDS (final concentration 2.5% with 0.1 mg/ml BSA). The macromolecules were then precipitated with an equal volume of ice-cold 20% TCA containing 2 mg/ml unlabeled valine. The precipitates were collected on glass-fiber filters and washed three times with ice-cold 5% TCA containing 1 mg/ml unlabeled valine, once with distilled water, and twice with methanol. The filters were air-dried, 5 ml of NCS Omnifluor was added and the samples were counted in a Beckman LS1OOCscintillation counter. Pulse-labeling unfertilized, fertilized, or activated eggs. The pulse-labeling procedure was modified from Hogan and Gross (1971). Packed, dejellied eggs, 0.25 ml, were resuspended in 250 ml of seawater containing either sperm or the designated amount of NH&l or Na-acetate. Four-milliliter samples were taken every 10 min. Immediately after taking the sample, the eggs were centrifuged and the seawater was aspirated to a final volume of 0.2 ml. [2,3-3H]Valine, 1 PCi, in 20 ~1 seawater was added and the eggs were incubated at 17°C for 5 min with constant stirring. The uptake of [3H]valine was linear in unfertilized, fertilized, NH&lactivated, and Na-acetate-treated eggs over this time interval. The eggs were then washed three times with ice-cold seawater containing 0.5 mg/ml unlabeled valine. After the final wash, the eggs were dissolved in SDS (2.5% with 0.1 mg/ml BSA) and then precipitated with an equal volume of 20% TCA containing 2 mg/ml unlabeled valine. After lhr at 2”C, the tubes containing both the TCA-soluble and -insoluble counts were vortexed and a fixed aliquot was removed to determine the total uptake. Aquasol, 5 ml, was added to this aliquot
397
and the samples were counted as above. The efficiency of counting in Aquasol was equal to the efficiency obtained with NCS Omnifluor. The remaining precipitates were collected on glass-fiber filters and processed as described above for preloaded eggs. The relative rates of protein synthesis were determined by dividing the counts accumulated into TCA-insoluble material over the 5-min pulse by the total counts accumulated in the eggs over this time interval. IntracellularpH measurement. Intracellular pH (pHJ was measured with Thomas type H’-sensitive microelectrodes with recessed tips (Thomas, 1976). These electrodes gave a linear response of slope 56-59 mV/pH unit over the range pH 2-9 and had less than a &3-mV drift when calibrated before and after each experiment in 100 n&f phosphate buffers (pH 6.6 and 7.6). The response times of the pH microelectrodes used in this study were 30 set or less and were unaffected by the presence of high protein concentrations (50 mg/ml BSA). In our experiments, two microelectrodes penetrated the egg, which was firmly attached to the dish with poly-lysine. Measurements of the egg membrane potential (E,) were made with conventional microelectrodes filled with 3 M KCl. This electrode was placed first in the egg by negative capacitance, The pH-sensitive microelectrode was then placed in the egg and did not cause a depolarization of E, greater than 2 mV. These electrodes recorded the membrane potential and a voltage proportional to pHi. Because of the high resistance of the pH microelectrode, its voltage was fed into a high-impedance ( 1015 a) amplifier (Model F-23, W-P Instruments, New Haven, Conn.), while the membrane potential was fed into a Getting preamplifier (Model 5, Getting Instruments, Palo Alto, Calif.). Both potentials were displayed continuously on separate channels of a Gould Brush Mark 220 chart recorder. To obtain pHi, the membrane potential was sub-
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tracted from the pH microelectrode record. Each pH measurement was repeated at least twice with essentially identical results before any conclusions were drawn. In order to determine that both microelectrodes were implanted in the egg with minimal membrane damage, a current pulse was passed through the conventional microelectrode periodically and the corresponding membrane potential deflection was monitored by the pH-sensitive microelectrode. Excessive membrane damage or vesication at the electrode tip resulted in a loss of coupling between the two microelectrodes.
Comparisons between amino acid incorporation andpH measurements. There are substantial variations in the rate of amino acid incorporation in both unfertilized and fertilized eggs from different females. This variation makes exact quantitative comparisons of rate and intracellular pH more difficult since experiments on the measurement of pH require many attempts involving eggs from several females over long periods of time. However, good estimates of the relative magnitude of the effect of cytoplasmic pH on rates of protein synthesis can be made, since there is much less variation in pH measurements in eggs from different females. Cytology. Samples of eggs were taken at indicated times and the eggs were fixed in EtOH:acetic acid (3:l) overnight and then stained with 2% orcein in 75% acetic acid. The presence of condensed chromosomes or mitotic spindles was then scored. RESULTS
Relationship between the Initial Change in the Intracellular pH and the Onset of the Increase in the Rate of Protein Synthesis Shen and Steinhardt (1978) have demonstrated that the intracellular pH in sea urchin eggs increases after fertilization and NH&l activation. Following this increase, there is an acceleration in the rate of protein synthesis as well as the initiation of
VOLUME 68,1979
DNA synthesis and chromosome condensation cycles (Mazia, 1974; Epel et al., 1974). However, the increase in the rate of protein synthesis following NH&l activation occurs later than the increase observed after fertilization (Epel et al., 1974). If the increase in the intracellular pH is related to the increase in the rate of protein synthesis, then the onset of the change of the intracellular pH after NH&l activation should be delayed when compared to fertilization. We have examined more closely the timing between the onset of the initial change in the intracellular pH with this initial increase in the rate of protein synthesis by measuring the change in the intracellular pH directly with pH microelectrodes and determining the exact timing of the acceleration in the rate of protein synthesis after fertilization or NH&l activation. At fertilization, the increase in the intracellular pH begins l-2 min after sperm-egg fusion, while in eggs activated with 10 m&f NH&l, pH 8, there is a 3- to 4-min lag between the addition of weak base and the observed increase in intracellular pH (Fig. 1A). If the change in intracellular pH of the egg initiates the change in the rate of protein synthesis, one would expect to find a 2-min lag in the onset of protein synthesis with NH&l-activated eggs. Using preloaded eggs, the first detectable increase in accumulation of radioactivity in fertilized eggs occurs at 6-7 min after fertilization, while the first detectable increase in NH&l-activated eggs occurs between 9 and 10 min after the addition of 10 r&f NH&l, pH 8 (Fig. 1B). Thus, for fertilization, there is a 4- to 6-min interval between the initial change in intracellular pH and the onset of protein synthesis acceleration. If this stimulation of protein synthesis is directly related to the pH change, then the rate of protein synthesis in eggs activated with NH&l should increase 2 min later than normal fertilization or 8-9 min after the initial exposure of these eggs to NH.&l. The close agreement between our observed val-
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while eggs fertilized from the same batches reached their similar and maximum rates at 30 min after fertilization. Therefore, in eggs whose pH has been raised artificially by ammonia, it takes at least 30 min longer to reach the rate of protein synthesis seen in fertilized eggs. However, the pH change 18 22 26 30 34 in 10 n&f NH&l (Fig. 1) is only a few Time (mm) minutes slower than in fertilization and reaches higher levels within 12 min. Thus the time lag for ammonia stimulation of protein synthesis cannot be entirely explained by the slight delay in intracellular pH increase. This suggests that either other E IO 0 factors contribute to the acceleration of 6 protein synthesis at fertilization or that am2 monia has toxic effects. In an experiment 2 6 IO 14 18 22 26 30 34 to discriminate between these two possibilTime (min.) ities, we fertilized and 10 min later added FIG. 1. (A) The change in intracellular pH (pHJ NH&l and we found that 10 m.M NH&l, following fertilization or NH&l activation. The eggs pH 8, did not retard protein synthesis in were either fertilized (A) or activated with 10 r&f the first 30 min compared to the fertilized NH&l, pH 8 (O), and the increase in pHi was contincontrol. Since the NH&l does not have an uously monitored with H+-sensitive microelectrodes as described under Materials and Methods. These inhibitory effect within the time interval continuous pHi recordings were made under experi- where the biggest difference in protein synmental conditions identical to those of the correspond- thesis rates develop between ammoniaing protein synthesis measurements. These records stimulated and fertilized eggs, we conclude are typical of over 20 pHi recordings of fertilization and 10 pHi recordings of NH&l activation. (B) The that a factor or factors other than intracelincrease in the rate of protein synthesis following lular pH, also plays a role at fertilization. fertilization or NH&l activation. Eggs were preloaded Control of the Rate of Protein Synthesis by and then placed in SW (unfertilized, l ), SW containing Manipulating the Intracellular pH sperm (fertilized, A), or SW containing 10 m&f NH&l, A
‘:::fffyf
,
pH 8 (NH&l activated, 0). Samples were taken every 1 min for the first 10 mm and every 2 min thereafter and processed as described under Materiala and Methods. The relative rates of protein synthesis were calculated as the change in cpm incorporated over tune.
ues of 9-10 min and the predicted values implies that the initial increase in the rate of protein synthesis is directly related to the onset in the change of the intracellular PH. Maximum Rates in Fertilized and NH&lActivated Eggs In three experiments using the pulse-label technique, unfertilized eggs reached the maximum rate of protein synthesis at 60-120 min after exposure to 10 mA4 NH&l
In unfertilized eggs, the intracellular pH and the rate of protein synthesis are low compared to fertilized or NH&l-activated eggs (Epel et al., 1974;Shen and Steinhardt, 1978). If the low intracellular pH of the unfertilized eggs is in some way inhibiting the rate of protein synthesis, then lowering the intracellular pH of fertilized or NH&lactivated eggs should lead to a decrease in the rate of protein synthesis. We have examined this question by first lowering the intracellular pH of NH&l-activated eggs by washing out the NH&l after the initial pH and rate of protein synthesis increase, and second, by using the weak acid, Naacetate to artificially lower the intracellular PH.
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The addition of 10 mM NH&l, pH 8, to unfertilized eggs caused an increase in the intracellular pH (Fig. IA). The NH&l enters the eggs by simple diffusion. The uncharged form of the weak base diffuses into the egg and picks up a H+ ion from the cytoplasm, resulting in an increase in the intracellular pH (Winkler and Grainger, 1978). Due to the equilibrium established between the charged and uncharged forms of the weak base inside the egg, a certain proportion of the intracellular NH&l is uncharged. When the NH&l on the outside is removed, the uncharged base will diffuse out of the egg and the intracellular pH should drop. As measured using intracellular pH microelectrodes, the pH does drop after NH&l is washed out @hen and Steinhardt, 1978). Even after extensive washing, however, the intracellular pH does not reach the unfertilized level, but levels off between the unfertilized and activated pH. Upon examining the rates of protein synthesis under these same circumstances, we found that the rate of protein synthesis increases when the intracellular pH increases and remains high as long as the eggs remain in NH&l and the intracellular pH remains high (Fig. 2). As soon as the NH&l is washed out, the intracellular pH and the rate of protein synthesis both drop. They both stabilize above the unfertilized but below the maximally activated levels. Why the intracellular pH does not drop to the unfertilized level when the NH,Cl is washed out is not clear. But the fact that both the intracellular pH and the rate of protein synthesis drop to an intermediate level is further evidence that intracellular pH is correlated with the rate of protein synthesis. In order to directly determine the effect of lowering the intracellular pH on the rate of protein synthesis, we used the weak acid, Na-acetate, to lower the intracellular pH. As the weak base, NH&l, the uncharged form of the weak acid diffuses across the plasma membrane. The weak acid gives up protons as it enters the cytoplasm, causing
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Time (min.) FIG. 2. The rate of protein synthesis in NH&lactivated eggs. Unfertilized eggs were added to SW, pH 8 (O), or SW containing 5 m&f NH&l, pH 8 (O), at t = 0. Samples were taken every 10 min and the rate of protein synthesis was determined by the pulselabel technique. At 120 min, the eggs were washed twice in SW to remove the NH&l and resuspended in SW at the original concentration (O.l%, closed arrow). Other samples were taken every 10 min, fixed in EtOH:acetic acid (3:1), and stained in orcein to score for chromosome condensation. Time of maximal chromosome condensation was at 110 min (open arrow).
a decrease ir. the intracellular pH. If we adjust the pH of the seawater containing 10 mM Na-acetate to pH 6.5, then enough of the acetate is in the uncharged form to penetrate the plasma membrane and lower the intracellular pH. When seawater containing 10 mM Na-acetate, pH 6.5, is added to fertilized eggs after the initial pH increase has been completed, the intracellular pH drops to the unfertilized level within 25 min (Fig. 3A). Immediately after the addition of Na-acetate, while the intracellular pH is dropping, the rate of protein synthesis stops at its present level (Fig. 3B). After the intracellular pH has returned to the unfertilized level, the rate drops off linearly until it reaches the unfertilized rate. One could argue that the Na-acetate is poisoning the eggs, but as will be shown below, the effects of lowering the intracellular pH are totally reversible by removing the acetate stimulus (Fig. 3B, arrow 2). Thus, using this weak acid, we can lower the intracellular pH of a permanently activated egg
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Time (mln) FIG. 3. (A) The effect of Na-acetate on pH, of fertilized eggs. Eggs were fertilized at tt = 0, and at 20 min after fertilization (arrow), the egg was perfused with SW containing 10 m&f Na-acetate, pH 6.5. (B) The effect of Naacetate on the rates of protein synthesis and cell division. Unfertilized control (0); fertilized control (0); fertilized with Na-acetate treatment as described below (A). Unfertilized eggs were added to SW containing sperm at t = 0 and time points were taken as described under Materials and Methods and Fig. 2. At 30 min after fertilization, one batch of eggs (A) was resuspended in SW containing 10 m&f Na-acetate, pH 6.5 (arrow 1). At 130 min, this same batch of eggs was washed twice in SW, pH 8, and resuspended in SW at the original concentration (O.l%, arrow 2). Samples were also taken every 15 mm, fixed in EtOH:acetic acid (3:1), stained in orcein, and scored for nuclear envelope breakdown, chromosome condensation, mitotic spindle formation, and cell division.
and shut down the protein synthesis machinery. This implies that in order to maintain a high rate of protein synthesis, the egg must maintain an alkaline pH.
Relationship of the Magnitude of the Change in the Intracellular pH to the Rate of Protein Synthesis Since the rate of protein synthesis can be altered by raising or lowering the intracellular pH, we want to know the sensitivity of the protein synthetic machinery to changes in intracellular pH. The determination of the effect of small changes in pH
in fertilized or NH&l-activated eggs becomes complex due to the fact that the rates are constantly changing. However, in unfertilized eggs, the rate of protein synthesis remains constant, and thus small changes in the rate induced by small changes in the intracellular pH can be observed without the complication of the fluctuations seen in fertilized or NH&l-activated eggs. The addition of 10 m.M Na-acetate, pH 6.5, to unfertilized eggs lowers the intracellular pH from 6.85 to 6.67, a drop in the intracellular pH of ca. 0.2 units (Fig. 4A).
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Time (min.) FIG. 4. (A) The effect of Na-acetate on pHi of unfertilized eggs. At t = 0, the eggs were perfused with SW containing 10 mM Na-acetate, pH 6.5. Between 26 and 33 min (arrows) after the addition of Na-acetate, the acetate was removed by perfusing with SW, pH 8. (B) The effect of lowering pHi on the rate of protein synthesis in preloaded unfertilized eggs. Unfertilized eggs were preloaded and placed into SW, pH 8 (O), or SW containing 10 m&f Na-acetate, pH 6.5 (0). Samples were taken every 10 min and processed as described in Fig. 1. (C) The effect of lowering and raising pHi of unfertilized eggs. Unfertilized eggs were either placed in SW, pH 8 (0) or in SW containing 10 mM Na-acetate, pH 6.5, at t = 0 (0). Samples were taken every 10 min and the rate of protein synthesis was determined by the pulse-label technique. In this batch of eggs which were already at a very low rate of protein synthesis, acetate addition did not show a repression of protein synthesis since we are already below the threshold for accurate rates with the pulse-label technique. At 40 min after acetate addition, the eggs were washed twice in SW and resuspended in SW, pH 8, at the original concentration (arrow). With removal of the acetate, the pH rose as in Fig. 4A, beyond the unfertilized level and amino acid incorporation increased to three- to fourfold the unfertilized rate.
When the acetate is washed out of the eggs, the intracellular pH increases. Instead of returning to the unfertilized level, it overshoots the unfertilized pH slightly, stabilizing at pH 6.98, 0.13 pH unit above the previous unfertilized level. Upon examining the rates of protein synthesis under these same conditions, two points are evident. First, the rate of protein synthesis in acetate-treated eggs is reduced sixfold compared to the control unfertilized eggs (Fig. 4B). Second, upon removing the acetate, the rate of protein synthesis increases (Fig. 4C). However, it does not return to the unfertilized level, but increases to a higher rate, after which it remains constant. Within 20 min after the acetate is washed out, both the intracellular pH and the rate of protein synthesis are greater than the unfertilized levels. In this case, the final effect of removing the acetate, thus raising
the intracellular pH by 0.13 unit, results in an increase in the rate of protein synthesis by three- to fourfold. Therefore, small changes in the intracellular pH lead, in a reversible manner, to large changes in the rate of protein synthesis.
Effects of Altering the Intracellular pH on Protein Synthesis and Chromosome Condensation Both the timing and the magnitude of the increase and the decrease in the rates of protein synthesis as seen in fertilized eggs can be induced by activating eggs with NH&l (Fig. 2). As shown in Fig. 5, the rate of protein synthesis increases after fertilization and decreases as the eggs approach mitosis. The maximal rate obtained during the first cycle was consistently 30x the unfertilized rate in our measurements in preloaded eggs (Fig. 1B) or pulse-labeled
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FIG. 5. The cyclic changes in the rate of protein synthesis in fertilized eggs. At t = 0, eggs were placed in SW (0) or in SW containing sperm (0). The rate of protein synthesis was determined using the pulse-label technique. At 15-min intervals, samples were taken for cytology and scored for cell division.
eggs (Fig. 5). At mitosis the rate of protein synthesis reaches its lowest level, 60% of the maximal rate, and then accelerates rapidly until the second division. At this time, the rate levels off again and then continues to cycle through at least one more cell division. The difference in sharpness in the rate decrease between the first and second division is probably due to slight asynchrony in the population of fertilized eggs. After the addition of NH&l to unfertilized eggs, the rate of protein synthesis increases and decreases as in fertilized eggs (Fig. 2). In these experiments, 5 mM NHdCl, pH 8, remained in the seawater for 2 hr. The maximal rate obtained is 30x the unfertilized rate as in fertilized eggs. There is a drop in the rate to 60% of this maximal value when the chromosomes are maximalIy condensed, corresponding to metaphase in the fertilized egg. Thus, in the continuous presence of NH&l, the variations in the rates of protein synthesis are similar to those seen in fertilized eggs (see also Fig. 8).
Do changes in intracellular pH correspond to the variations of protein synthesis observed during the cell cycle? In our longest continuous recordings of intracellular pH of fertilized eggs, we do see a dip of 0.15 pH unit preceding, in the expected manner, the subsequent decrease in the rate of protein synthesis (Fig. 6). Both intracellular pH and protein synthesis remain above the unfertilized levels and the magnitude of the decline of both pH and protein synthesis during the cell cycle roughly correspond to what we expect from our previous analysis of the effect of intracellular pH on the rate of protein synthesis. However, with the continuous exposure of unfertilized eggs to NH&l, the intracellular pH remains consistently high without the small decline observed in fertilized eggs as they approach cell division (Fig. 6). Yet the protein synthesis rate declines at the appropriate time in these NH&l-activated eggs (Fig. 2), even though the pH remains constant at high alkaline levels. Therefore, we conclude that the correlation between the intracellular pH and protein synthesis can be overridden by another factor or factors at points in the cell cycle. Lowering the intracellular pH by adding mM NH&
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FIG. 6. Measurement of the changes in pHi after fertilization (0) or activation with 5 m&f NH&l, pH 8 (0). Nuclear envelope breakdown occurred at 62 min in the fertilized eggs and at 70 min in the NH&lactivated eggs.
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Na-acetate can shut down a previously activated fertilized egg completely and reversibly. As described above, if Na-acetate is added to fertilized eggs after the initial pH increase has been completed, the intracellular pH and the rate of protein synthesis drop to the unfertilized levels (Figs. 3A and B). The normal variation in the rate of protein synthesis is stopped and the eggs are prevented from entering mitosis. No nuclear envelope breakdown, chromosome condensation, or mitotic spindle formation occurs. When the acetate is washed out (Fig. 3B, arrow 2)) the intracellular pH rises and the rate of protein synthesis accelerates until it reaches the control fertilized level. This is followed by the breakdown of nuclear envelope, chromosome condensation, mitotic spindle formation, and cell division. Thus, the addition of Na-acetate and the lowering of the intracellular pH reversibly block the cyclic changes in protein synthesis and mitotic events in fertilized eggs. The intracellular pH must remain continuously above a certain level in order to get the normal rates of protein synthesis and the normal cycling of these rates with the cell cycle. When eggs are exposed to 10 mM NH&l, pH 8, for only 15 min, a normal increase in the rate of protein synthesis is observed (Fig. 7). The rate than decreases as in fertilized eggs, but instead of reaccelerating to a higher level, it remains at 40% of the maximal rate. No activation of chromosome cycles is observed. Thus, this brief exposure to NH&l, which results in only a partial increase of pH (Shen and Steinhardt, 1978), only stimulates one burst of protein synthesis and is not sufficient to induce the cyclic variation in protein synthesis or chromosome condensation. In an attempt to determine if this effect was due to the lower intracellular pH after the NH&l was removed from the eggs we activated the eggs with 10 mM NH&l for 15 min, washed out the NH&l, and then placed the eggs in a low concentration of weak base. The short exposure to a high
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FIG. 7. The effect of a short pulse of NH&l on the cycling of the rate of protein synthesis. Eggs were placed in SW containing 10 mM NH&l, pH 8, at t = 0. After 15 min, the NH&l-activated eggs were washed twice in SW and resuspended in SW, pH 8. The rate of protein synthesis was monitored by the pulse-label technique. The three plots are of three independent determinations of the change in the rate of protein synthesis.
concentration of weak base would cause the increase in initial pH and rate of protein synthesis increase, and the addition of a lower concentration of weak base would maintain the high intracellular pH and rate of protein synthesis as seen in fertilized eggs. Under these conditions, the rate of protein synthesis remains high and the observed increases and decreases in the rate exactly match those of fertilized eggs (Fig. 8). Thus, this is further evidence that the timing of the cyclic variation in the rate of protein synthesis can be maintained if the intracellular pH remains high. DISCUSSION
We have shown that the level of intracellular pH directly correlates with the rate of protein synthesis during fertilization or parthenogenetic activation with NH&l. This conclusion is supported by three lines of evidence. First, the onset of an increase in the intracelhilar pH is directly coupled with the onset of the stimulation of protein synthesis in both fertilization and NHdCl
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Time (min) FIG. 8. The effect of the maintenance of a high pHi on the cycling of the rate of protein synthesis. Eggs were placed in SW, pH 8 (O), or SW containing 10 mA4 NH&i, pH 8, for 15 min (O), after which they were washed twice in SW and resuspended at the original concentration in 0.5 mM NH&l, pH 8. The arrows indicate the times of the drop in the rate of protein synthesis found in fertilized eggs.
activation. Second, the rate of protein synthesis can be slowed down to the repressed, unfertilized rate by simply lowering the intracellular pH of fertilized eggs back down to the unfertilized level. Finally, these stimulations or inhibitions of protein synthesis rate are controlled in a completely reversible manner by the passive manipulations of intracellular pH with weak acids or weak bases. The rate of protein synthesis is highly sensitive to intracellular pH. Since we are comparing intracellular pH measurements made on small numbers of eggs to the pooled results from the large numbers of eggs used in amino acid incorporation experiments, it is difficult to make exact quantitative comparisons. Nevertheless, we can easily estimate the relative magnitude of the effect of cytoplasmic pH on rates of protein synthesis. A 0.2-pH-unit decrease reduces amino acid incorporation 6-fold, a 0.13-unit increase leads to a 3- to 4-fold stimulation. By calculation with these factors relating pH and protein synthesis rate as seen by amino acid incorporation, we would predict a 27- to 64-fold increase in protein synthesis at fertilization where the
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cytoplasmic pH increases from 6.84 f 0.02 to 7.27 + 0.03 (Shen and Steinhardt, 1978). Our observed rate at fertilization, 30x the unfertilized rate, falls within this range. Therefore, within the intracellular pH range of 6.7 to 7.3, this high sensitivity could account for the entire stimulation at fertilization. At higher pH, however, we do not observe further stimulation of protein synthesis rate since NH&l-activated eggs do not exceed the rate attained in fertilized eggs although the intracellular pH in NH&l-activated eggs reaches 7.65 + 0.05 (Shen and Steinhardt, 1978). What are the molecular mechanisms responsible for this high sensitivity to pH? Humphreys (1971) has shown that mRNA availability controlled the increase in protein synthesis at fertilization. The fact that we always observe a lag time between the changes in intracellular pH and the changes in protein synthesis rate is consistent with Humphreys’ interpretation since such a lag rules out a direct effect of pH on the enzymatic steps of peptide elongation. We are currently investigating to what extent these lags can be explained by mRNA availability and whether additional regulatory mechanisms sensitive to pH remain to be uncovered. During the cell cycle there is a cycle in the rate of protein synthesis. We have shown that intracellular pH must be sustained at a high degree of alkalinity, well above that of the unfertilized egg, in order to maintain normal rates and normal cycling of protein synthesis during cell cycles. Furthermore, since these cycles in protein synthesis rates are seen in NH&l-activated eggs whose intracellular pH is clamped at high values, we have evidence that other factors can influence protein synthesis rate later on in the cell cycle. Finally, we conclude that the entire increase and maintenance of protein synthesis at fertilization or parthenogenetic activation could be accounted for by a permanent increase in the intracellular pH. How-
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DEVELOPMENTAL BIOLOGY
ever, since the timing differences observed in protein synthesis rate between ammoniaactivated and fertilized eggs cannot be explained on the basis of intracellular pH, we believe other unknown factors can also contribute to the derepression of protein synthesis at fertilization. One possible candidate is the intracellular calcium released at fertilization or some change induced by that calcium release (Steinhardt et al., 1977).
VOLUME 68,1979
HUMPHREYS, T. (1971). Measurement of messenger
RNA entering polysomes upon fertilization of sea urchin eggs. Develop. Biol. 26, 201-208. JOHNSON, J. D., EPEL, D., and PAUL, M. (1976). Intracellular pH and activation of sea urchin eggs after fertilization. Nature (London) 262,661-664. LOPO, A., and VACQUIER, V. D. (1977). The rise and fall of intracellular pH of sea urchin eggs after fertilization. Nature (London) 269, 590-592. MAZIA, D. (1974). Chromosome cycles turned on in unfertilized sea urchin eggs exposed to NH40H. Proc. Nat. Acad. Sci. USA 71,690-693. REGIER, J. C., and KAFATOS, F. C. (1977). Absolute This investigation was supported by USPHS Grant rate of protein synthesis in sea urchins with specific GM 13882 to Dr. Daniel Mazia and NSF Grant PCM activity measurements of radioactive leucine and 77-042690 to R.A.S. S.S.S. was supported by NIH leucyl t-RNA. Develop. Biol. 57,270-283. Grant CA-21711. SHEN, S. S., and STEINHARDT, R. A. (1978). Direct measurement of intracellular pH during metabolic REFERENCES derepression at fertilization and ammonia activation of the sea urchin egg. Nature (London) 272, EPEL, D. (1967). Protein synthesis in sea urchin eggs: A “late” response to fertilization. hoc. Nat. Acad. 253-254. STEINHARDT, R. A., and MAZIA, D. (1973). DevelopSci. USA 57.889-906. ment of K+ conductance and membrane potential in EPEL, D., STEINHARDT, R. A., HUMPHREYS, T., and unfertilized sea urchin eggs after exposure to MAZIA, D. (1974). An analysis of the partial metabolic derepression of sea urchin eggs by ammonia: NH,OH. Nature (London) 241,400-401. The existence of independent pathways. Develop. STEINHARDT, R., ZUCKER, R., and SCHATTEN, G. (1977). Intracellular calcium release at fertilization Biol. 40,245-255. in the sea urchin egg. Develop. Biol. 58, 185-196. FRY, B. J., and GROSS, P. R. (1970). Patterns and rates of protein synthesis in sea urchin embryos. I. Uptake THOMAS, R. C. (1976). Construction and properties of and incorporation of amino acids during the fist recessed-tip microelectrodes for sodium and chlocleavage cycle. Develop. Biol. 21, 105-124. ride ions and pH. In “Ion and Enzyme Electrodes in HOGAN, B., and GROSS, P. R. (1971). The effect of Biology and Medicine” (M. Kessler, L. C. Clark, Jr., protein synthesis inhibition on the entry of messenD. W. Lubbers, I. A. Silver, and W. Simon, eds.), pp. ger RNA into the cytoplasm of sea urchin embryos. 141-148. University Park Press, Baltimore. J. Cell Biol. 49, 692-701. WINKLER, M. M., and GRAINGER, J. L. (1978). Mechanism of action of NH&l and other weak bases in HULTIN, T. (1952). Incorporation of “Nlabelled glytine and alanine into the proteins of developing sea the activation of sea urchin eggs. Nature (London) urchin eggs. Exp. Cell Res. 3,494-496. 273, 236-238.