Prinrrd in Sweden Copyrixhf @ 1975 by Academic Press. Inc. AI/ right 5 of reproduction in any form reserved
Experimental
Cell Research 94 (1975) 1-6
FORMATION OF FERTILIZATION ACID BY SEA URCHIN EGGS DOES NOT REQUIRE SPECIFIC CATIONS M. PAUL and D. EPEL Department of Biology, University of Victoria, Victoria, BC V8W 2Y2, Canada, and Scripps Institution of Oceanography, La Jolla, CA 92037, USA
SUMMARY We have measured the release of fertilization acid from sea urchin eggs in specific cation-free media by activating the eggs with the calcium ionophore A23 187. The fertilization acid is normal in calcium-free sea water and several other substituted media, indicating that specific cations present in sea water are not required for release of the acid. The fertilization acid may result from release of cortical granule contents, but we suggest the possibility that other cellular processes are involved.
Sea urchin eggs release an acid into the surrounding sea water upon fertilization [l]. A variety of mechanisms has been proposed to account for this acid release which is presumed to reflect the metabolic and structural changes accompanying fertilization (for a recent review, see [2]). Chief among these proposals are (1) release of acidic macromolecules from the cortical granules during their discharge into the perivitelline space [3] accompanied by splitting of sulfate from these molecules [4] and, (2) ion exchange reactions between the egg or egg surface and the sea water [S, 61. Nakazawa et al. [6] specifically suggest a Ca2+-H+ exchange reaction between the egg and the medium. This suggestion is supported by the observations that an increased uptake of exogenous calcium was temporally correlated with acid release and that addition of the calcium chelator EGTA to an egg suspension during acid release terminated this process [6].
To directly test this hypothesis one would like to fertilize or artificially activate eggs in the absence of exogenous calcium and determine whether hydrogen ions were still released. Previously this has been impossible, since fertilization could not be carried out in the absence of exogenous calcium nor were available parthenogenetic agents, such as butyric acid, effective in calcium-free media. Steinhardt & Epel [7] have recently demonstrated that sea urchin eggs can be parthenogenetically activated by the calcium ionophore A23187 even in media free of divalent cations. Thus, use of the ionophore A23187 permits a direct test of the Ca?- H+ exchange hypothesis. Our results show that protons are released when eggs are activated in calciumfree sea water and indeed in a number of other unusual media, including isotonic potassium chloride. These results indicate that the formation of acid does not depend on a Ca2+-H+ exchange mechanism and Exptl Cell Res 94 (1975)
Paul and Epel
Fig. 1. Abscissa: time (min) after addition of (A) sperm or(B) ionophore; ordinate: pH.
Fertilization acid release in SW. Five ml 1.6% egg
that the temporal correlation of acid release and calcium uptake is coincidental. We show that the apparent termination of acid release by EGTA, observed by Nakazawa et al., is due to the buffering properties of this chelator. Our measurements of acid release suggest that it is a biphasic process, with an early major phase temporally linked to the cortical reactions, suggestive that this component of the acid may originate from the cortical granules. A second, minor phase may represent some other process such as metabolic ion exchange reactions.
suspension. (A) Eggs inseminated with 25 ~1 10% sperm suspension; (B) eggs activated with 25 ~1 (lower) or 50 ~1 (upper) of 5 mM A23 187.
eliminates problems resulting from differing buffer capacity of the egg suspensions and the various solutions. Rates of acid release were calculated from titration curves and first derivatives of the pH records. Artificial media were prepared as previously described [7] except that choline chloride was substituted for NaCl in 0 Na SW. Prior to each measurement, eggs were suspended in the appropriate medium with three washes by hand centrifugation. The calcium ionophore A23187, prepared as a 5 mM stock solution in DMSO, was added to egg suspensions with an automatic micropipette (Eppendorf) with rapid mixing. Final maximal concentrations of ionophore were 25-50 PM. Appropriate DMSO and ionophore controls were carried out.
RESULTS AND DISCUSSION
In fig. IA we show the pH change in an egg suspension following insemination, and in fig. 2 (dashed line) the rate of acid release. MATERIALS AND METHODS Coincident with the beginning of the cortiAbbreviations: EGTA. ethvleneelvcol-bis-(B-aminoethyl ether)-N,N’-tetralacetic acid: .DMSO, dimethylcal reaction, the pH begins to decrease. The sulfoxide: SW. sea water: 0 Ca SW. 0 K SW. 0 Ma SW. maximum rate of acid release occurs at 0 Na SW’, cakcium-free,‘potassium-free, magneiiumfree. and sodium-free sea waters, resoectivelv. about 1 min, and the release is 70% comGametes of Strongylocentrotus jmrpuraius were obtained by intracoelomic injection of 0.5 M KC1 and plete by 2 min. Release of protons condejellied by gentle agitation and washing in filtered tinues at a reduced rate for an additional SW. Egg volume was determined by centrifugation in a 4-6 min and is essentially complete by 8 calibrated Bauer-Schenk tube. To measure fertilization acid, 5 ml of a 1.6% egg suspension (6.96~ 104eggs/ min. We refer to the period of the early comml), in a water-jacketed vessel (18°C) of 10 ml capacity was stirred with a magnetic bar and stirrer. pH was ponent (O-2 min) as phase I and that of the measured with a Coming combination pH electrode, component after 2 min as phase II. Corning 12 expanded pH meter, and the meter signal recorded on a Sargent SRL potentiometric recorder. The pattern of acid release in eggs acThe amount of acid released was determined at the end tivated with the ionophore A23 187is similar of each experiment by incremental back-titration with a standardized solution of NaOH. This procedure to that of fertilized eggs (cf fig. 1A, B). The ExptI Cell Res 94 (1975)
Sea urchin fertilization
acid
3
Table 1. Fertilization acid titrations Acid Expt
Medium
(pmoles/e&
l-1 -2
SW SW
1.5 1.7
3 2-1” -2” -3 4 -5 -6 -7 -8
0OCaSW Ca SW +5 mM EGTA SW SW SW OCaSW 0 Ca SW+ 10 mM EGTA 0 Ca SW+4 mM EGTA ONaSW OMgSW
1.6 1.9 1.9 1.9 1.9 1.4 I 1.6 0.5 2.2 (I) 1.8 (II)
3-l -2 -3
SW OKSW KCI 0.55 M
2.0 1.8 0.7
Comments
Titrations relatively inaccurate; EGTA buffers so pH change is small No fertilization membrane Phase I O-2 mm Phase II 2-16 min; pH continued to decrease after 16 min
O-l min; lysis began at 1 min
Experiments within each of the three sets were done with aliquots taken from a single stock egg suspension. a Experiment 2-1,2-2, eggs inseminated with sperm; all others, eggs activated with the ionophore A23187.
kinetics of proton release stimulated by A23187 is dependent on the concentrations of both the ionophore and the eggs, since the ionophore is lipid soluble and can be partitioned or sequestered by cells. With 25 PM A23 187 in a 1.6 % egg suspension, both cortical activation (judged by vitelline layer elevation) and acid release begin later than in fertilized eggs (cf fig. lB, lower right pH record, and fig. 1A). With 50 ,uM A23187, both begin earlier than in fertilized eggs (cf fig. 1B, upper left, and 1A ; also, fig. 2). The rate of acid release is also higher in ionophore-activated than fertilized eggs. This difference probably reflects the different modes of activation with these agents. During fertilization, the cortical activation is propagated from the site of sperm fusion, while during A23 187-induced. parthenogenesis, cortical activation occurs uniformly around the egg (direct observation). However, the total amounts of acid released during ionophore activation and fertilization are similar (table 1).
We see the same pattern of proton release in eggs activated with A23187 in 0 Ca SW containing 0.25 mM EGTA (fig. 3). The initial change in pH is an electrode response to ionophore (as indicated by control addition of ionophore to 0 Ca SW); the subsequent change is attributable to the eggs. 12 1
.
Fig. 2. Abscissa: time (mm) after addition of sperm or ionophore; ordinate: rate of acid release; nmoles H+/sec. Five ml 1.6% egg suspension. Rate of fertilization acid release by eggs in A-A, SW upon insemination or O-O, 0 Ca SW upon activation with A23187. Rates were determined by analysing the slopes of pH records similar to those of fig. 1A and tig. 3. Five ml 1.6 % egg suspension. Since the titration of egg suspensions in this pH range is not linear, titration curves were determined in each experiment. Exptl Cell Res 94 (1975)
4
Paul and Epel
drop in pH due to the chelation of calcium ion. When we activated eggs with A23181 in 0 Ca SW containing 10 mM EGTA (fig. 4), there was a striking decrease in acid release as judged by the magnitude of the pH change. The change was only 0.01 pH unit @ii. 4) compared with a change of 0.3 pH unit in 0 Ca SW containing only 0.25 mM EGTA (fig. 3). This observation is consistant with that of Nakazawa et al. [6]. However, that the change in pH was so small was due to the very strong buffering capacity of EGTA in the egg suspension. Fig. 3. Abscissa: time (min) after addition of ionoThis effect on acid release is clear when the phore; ordinate: PH. Fertilization acid release in 0 Ca SW. Five ml 1.6% pH records of eggs activated in the presegg suspension activated by addition of 50 ~1 of 5 mM A23187. The immediate decrease in pH at time 0 is an ence of 4 and 10 mM EGTA are compared effect of ionophore on the pH electrode (see fig. 4). (fig. 4). When we titrated the SW back to the original pH, there was no significant differWhen eggs are activated with ionophore in ence in acid between these eggs and eggs 0 Ca SW, both cortical activation and acid activated in 0 Ca SW containing only 0.25 release occur more rapidly than in normal mM EGTA or in normal SW (table 1). These experiments led us to investigate SW. We observed this rapid activation in all media deficient in divalent cations (see the possible participation of other ions in below, and [7]), and assume it reflects the acid release. The use of the ionophore higher solubility of the ionophore in A23187 permitted the activation of eggs in divalent cation-free media. The amount of other media in which activation by sperm acid released in 0 Ca SW is similar to that would not be feasible. Acid release was observed in all substituted media (table I). in normal SW (table 1). The normal release of fertilization acid in The initial phase was essentially identical 0 Ca SW demonstrates that concurrent uptake of calcium ion is not necessary for release of protons from the eggs and therefore the Ca2+-H+ exchange hypothesis can7.66. 4 rnM EGT* not account for the fertilization acid. The major evidence supporting this hypothesis 7.70 was the observation that proton release 0 * 4 6 8 terminated upon ‘addition of 10 mM EGTA’ Fig. 4. Abscissa: time (mitt) after addition of ionoto the egg suspension [6]. We were unable piore;.ordinate: pH. Ferttlization acid release in 0 Ca SW containing 4 to exactly repeat this experiment because mM or 10 mM EGTA as indicated. Five ml 1.6% egg the procedure was insufficiently described. suspensions activated by addition of 50 ~1 5 mM Additions of 10 ~1 0.025 N NaOH (V). However, we did observe that addition of A23187. Although less acid appears to be formed, the titrations EGTA solution (0.1 M) to an egg suspen- indicate that this effect is due to the buffering capacity the EGTA. Arrow, control addition of ionophore; sion of the same pH (pH 8; 10 mM EGTA of we conclude that the initial sharp decrease in pH at final concentration) causes a precipitous time 0 is an effect of ionophore on the pH electrode.
Sea urchin fertilization
in 0 K SW, 0 Mg SW, and 0 Ca SW. However, acid release continued during phase IT in 0 Mg SW and release of acid was accelerated in 0.55 M KC1 and in isotonic NaCl : KC1 (19 : 1). There was less acid in 0 Na SW. The acceleration of acid release in 0.55 M KC1 and NaCl : KC1 can be attributed to cell lysis, beginning within 60 set of ionophore addition. Lysis did not occur in 0 Mg SW; we are unable to account for the continued release of acid in this medium. It may be related to some kind of ionophore mediated transport. The decreased acid release in 0 Na SW might reflect an inhibition of cortical granule discharge. We observed an impaired cortical reaction in these eggs directly and by cytological examination of eggs fixed in acetic acid-alcohol (3 : l), resuspended in 45% acetic acid, and observed by phase contrast microscopy. This inhibition of the cortical reactions in 0 Na SW may have resulted from toxic effects of choline, the substituent for sodium, since no effects on cortical granule breakdown were observed in 0.55 M KCI, the other sodium-free medium used. Our results indicate that the mechanism underlying formation of fertilization acid does not involve exchange of protons for any specific cation present in SW. However, we have not attempted to measure fertilization acid in non-ionic media, and one could propose that any available cation might suffice for exchange with protons during fertilization acid release. While we cannot exclude this possibility, it seems unlikely since the kinetics of acid release is so similar in all the ion-substituted media we have used. The temporal correlation between the onset of acid release and cortical granule discharge supports the view that the con-
acid
5
tents of these granules (e.g., sulfated mucopolysaccharide) are the source of the acid [3, 41. Acid is also released upon fertilization of Urechis eggs [8]. However, cortical granule discharge does not occur in Urechis eggs at the time of acid release [S, 9, lo], and thus, if the mechanism suggested above to account for fertilization acid in sea urchins is correct, the source of acid must be different in these two kinds of egg. However, we would raise the possibility that the mechanism of acid formation may be similar in sea urchin and Urechis eggs. Consistent with this ate the observations that in both sea urchin eggs [l 1, 121 and Urechis eggs [8], a light-scattering decrease begins concomitant with acid release. Could this represent a conformational surface change, associated with acid release, which happens to correlate in time with the cortical reaction in sea urchin eggs? Or, as another possibility, could acid release reflect a cytoplasmic change, such as activation of a mitochondrial Ca2+-H+ antiport [13] with the ultimate release of protons from the egg? The biphasic nature of the acid release in our measurements raises the possibility of more than one mechanism underlying fertilization acid formation, with phase II representing a second process contributing a minor component of the acid. The cause of fertilization acid in Urechis has not been established. We suggest that the mechanism underlying the sea urchin fertilization acid has also not yet received a satisfactory explanation. We thank R. L. Hamill of the Eli Lillv Co. for his generous gift of the ionophore A23 187,A: De Vries for the loan of eauioment, and E. Baker for technical . . assistance. This work was supported in part by the following nrants: NSF GB-38629 from the NSF to D.E. and ?%?A6946 from the National Research Council of Canada and a faculty research grant from the University of Victoria to M. P. Exptl Cell Res 94 (1975)
6
Paul and Epel REFERENCES
1. Ashbel, R, Boll sot ital biol sper 4 (1929) 492. 2. Giudice, G, Developmental biology of the sea urchin embryo, p. 107. Academic Press, New York (1973). 3. Ishihara, K, Exp cell res 5 1 (1968) 473. 4. Aketa, K, Exp cell res 30 (1963) 93. 5. Mehl, J W & Swann, M M, Exp cell res 22 (1961) 233. 6. Nakazawa, T, Asami, K, Shoger, R, Fujiwam, A & Yasumasu, I, Exp cell res 63 (1970) 143. 7. Steinhardt, R A & Epel, D, Proc natl acad sci US 71(1974) 1915.
8. Paul, M, Dev bio143 (1975) 299. 9. Paul, M, Ph.D. Thesis. Stanford University, Stanford. Calif. (1970). 10. Gould-Somero, M & Holland, L, Am zool 14 (1974) 1299. 11. Epel, D, Pressman, B C, Elsaesser, S & Weaver, A M, The cell cvcle (ed G M Padilla, G L Whitson & I L Cameron) p.‘279. Academic Press, New York (1969). 12. Paul, M & Epel, D, Exp cell res 65 (1971) 281. 13. Lehninger, A L, Biochem j I19 (1970) 129. Received January 22, 1975
Experimental
Cell Research 94 (1975) 1-6
FORMATION OF FERTILIZATION ACID BY SEA URCHIN EGGS DOES NOT REQUIRE SPECIFIC CATIONS M. PAUL and D. EPEL Department of Biology, University of Victoria, Victoria, BC V8W 2Y2, Canada, and Scripps Institution of Oceanography, La Jolla, CA 92037, USA
SUMMARY We have measured the release of fertilization acid from sea urchin eggs in specific cation-free media by activating the eggs with the calcium ionophore A23 187. The fertilization acid is normal in calcium-free sea water and several other substituted media, indicating that specific cations present in sea water are not required for release of the acid. The fertilization acid may result from release of cortical granule contents, but we suggest the possibility that other cellular processes are involved.
Sea urchin eggs release an acid into the surrounding sea water upon fertilization [l]. A variety of mechanisms has been proposed to account for this acid release which is presumed to reflect the metabolic and structural changes accompanying fertilization (for a recent review, see [2]). Chief among these proposals are (1) release of acidic macromolecules from the cortical granules during their discharge into the perivitelline space [3] accompanied by splitting of sulfate from these molecules [4] and, (2) ion exchange reactions between the egg or egg surface and the sea water [S, 61. Nakazawa et al. [6] specifically suggest a Ca2+-H+ exchange reaction between the egg and the medium. This suggestion is supported by the observations that an increased uptake of exogenous calcium was temporally correlated with acid release and that addition of the calcium chelator EGTA to an egg suspension during acid release terminated this process [6].
To directly test this hypothesis one would like to fertilize or artificially activate eggs in the absence of exogenous calcium and determine whether hydrogen ions were still released. Previously this has been impossible, since fertilization could not be carried out in the absence of exogenous calcium nor were available parthenogenetic agents, such as butyric acid, effective in calcium-free media. Steinhardt & Epel [7] have recently demonstrated that sea urchin eggs can be parthenogenetically activated by the calcium ionophore A23187 even in media free of divalent cations. Thus, use of the ionophore A23187 permits a direct test of the Ca?- H+ exchange hypothesis. Our results show that protons are released when eggs are activated in calciumfree sea water and indeed in a number of other unusual media, including isotonic potassium chloride. These results indicate that the formation of acid does not depend on a Ca2+-H+ exchange mechanism and Exptl Cell Res 94 (1975)
Paul and Epel
Fig. 1. Abscissa: time (min) after addition of (A) sperm or(B) ionophore; ordinate: pH.
Fertilization acid release in SW. Five ml 1.6% egg
that the temporal correlation of acid release and calcium uptake is coincidental. We show that the apparent termination of acid release by EGTA, observed by Nakazawa et al., is due to the buffering properties of this chelator. Our measurements of acid release suggest that it is a biphasic process, with an early major phase temporally linked to the cortical reactions, suggestive that this component of the acid may originate from the cortical granules. A second, minor phase may represent some other process such as metabolic ion exchange reactions.
suspension. (A) Eggs inseminated with 25 ~1 10% sperm suspension; (B) eggs activated with 25 ~1 (lower) or 50 ~1 (upper) of 5 mM A23 187.
eliminates problems resulting from differing buffer capacity of the egg suspensions and the various solutions. Rates of acid release were calculated from titration curves and first derivatives of the pH records. Artificial media were prepared as previously described [7] except that choline chloride was substituted for NaCl in 0 Na SW. Prior to each measurement, eggs were suspended in the appropriate medium with three washes by hand centrifugation. The calcium ionophore A23187, prepared as a 5 mM stock solution in DMSO, was added to egg suspensions with an automatic micropipette (Eppendorf) with rapid mixing. Final maximal concentrations of ionophore were 25-50 PM. Appropriate DMSO and ionophore controls were carried out.
RESULTS AND DISCUSSION
In fig. IA we show the pH change in an egg suspension following insemination, and in fig. 2 (dashed line) the rate of acid release. MATERIALS AND METHODS Coincident with the beginning of the cortiAbbreviations: EGTA. ethvleneelvcol-bis-(B-aminoethyl ether)-N,N’-tetralacetic acid: .DMSO, dimethylcal reaction, the pH begins to decrease. The sulfoxide: SW. sea water: 0 Ca SW. 0 K SW. 0 Ma SW. maximum rate of acid release occurs at 0 Na SW’, cakcium-free,‘potassium-free, magneiiumfree. and sodium-free sea waters, resoectivelv. about 1 min, and the release is 70% comGametes of Strongylocentrotus jmrpuraius were obtained by intracoelomic injection of 0.5 M KC1 and plete by 2 min. Release of protons condejellied by gentle agitation and washing in filtered tinues at a reduced rate for an additional SW. Egg volume was determined by centrifugation in a 4-6 min and is essentially complete by 8 calibrated Bauer-Schenk tube. To measure fertilization acid, 5 ml of a 1.6% egg suspension (6.96~ 104eggs/ min. We refer to the period of the early comml), in a water-jacketed vessel (18°C) of 10 ml capacity was stirred with a magnetic bar and stirrer. pH was ponent (O-2 min) as phase I and that of the measured with a Coming combination pH electrode, component after 2 min as phase II. Corning 12 expanded pH meter, and the meter signal recorded on a Sargent SRL potentiometric recorder. The pattern of acid release in eggs acThe amount of acid released was determined at the end tivated with the ionophore A23 187is similar of each experiment by incremental back-titration with a standardized solution of NaOH. This procedure to that of fertilized eggs (cf fig. 1A, B). The ExptI Cell Res 94 (1975)
Sea urchin fertilization
acid
3
Table 1. Fertilization acid titrations Acid Expt
Medium
(pmoles/e&
l-1 -2
SW SW
1.5 1.7
3 2-1” -2” -3 4 -5 -6 -7 -8
0OCaSW Ca SW +5 mM EGTA SW SW SW OCaSW 0 Ca SW+ 10 mM EGTA 0 Ca SW+4 mM EGTA ONaSW OMgSW
1.6 1.9 1.9 1.9 1.9 1.4 I 1.6 0.5 2.2 (I) 1.8 (II)
3-l -2 -3
SW OKSW KCI 0.55 M
2.0 1.8 0.7
Comments
Titrations relatively inaccurate; EGTA buffers so pH change is small No fertilization membrane Phase I O-2 mm Phase II 2-16 min; pH continued to decrease after 16 min
O-l min; lysis began at 1 min
Experiments within each of the three sets were done with aliquots taken from a single stock egg suspension. a Experiment 2-1,2-2, eggs inseminated with sperm; all others, eggs activated with the ionophore A23187.
kinetics of proton release stimulated by A23187 is dependent on the concentrations of both the ionophore and the eggs, since the ionophore is lipid soluble and can be partitioned or sequestered by cells. With 25 PM A23 187 in a 1.6 % egg suspension, both cortical activation (judged by vitelline layer elevation) and acid release begin later than in fertilized eggs (cf fig. lB, lower right pH record, and fig. 1A). With 50 ,uM A23187, both begin earlier than in fertilized eggs (cf fig. 1B, upper left, and 1A ; also, fig. 2). The rate of acid release is also higher in ionophore-activated than fertilized eggs. This difference probably reflects the different modes of activation with these agents. During fertilization, the cortical activation is propagated from the site of sperm fusion, while during A23 187-induced. parthenogenesis, cortical activation occurs uniformly around the egg (direct observation). However, the total amounts of acid released during ionophore activation and fertilization are similar (table 1).
We see the same pattern of proton release in eggs activated with A23187 in 0 Ca SW containing 0.25 mM EGTA (fig. 3). The initial change in pH is an electrode response to ionophore (as indicated by control addition of ionophore to 0 Ca SW); the subsequent change is attributable to the eggs. 12 1
.
Fig. 2. Abscissa: time (mm) after addition of sperm or ionophore; ordinate: rate of acid release; nmoles H+/sec. Five ml 1.6% egg suspension. Rate of fertilization acid release by eggs in A-A, SW upon insemination or O-O, 0 Ca SW upon activation with A23187. Rates were determined by analysing the slopes of pH records similar to those of fig. 1A and tig. 3. Five ml 1.6 % egg suspension. Since the titration of egg suspensions in this pH range is not linear, titration curves were determined in each experiment. Exptl Cell Res 94 (1975)
4
Paul and Epel
drop in pH due to the chelation of calcium ion. When we activated eggs with A23181 in 0 Ca SW containing 10 mM EGTA (fig. 4), there was a striking decrease in acid release as judged by the magnitude of the pH change. The change was only 0.01 pH unit @ii. 4) compared with a change of 0.3 pH unit in 0 Ca SW containing only 0.25 mM EGTA (fig. 3). This observation is consistant with that of Nakazawa et al. [6]. However, that the change in pH was so small was due to the very strong buffering capacity of EGTA in the egg suspension. Fig. 3. Abscissa: time (min) after addition of ionoThis effect on acid release is clear when the phore; ordinate: PH. Fertilization acid release in 0 Ca SW. Five ml 1.6% pH records of eggs activated in the presegg suspension activated by addition of 50 ~1 of 5 mM A23187. The immediate decrease in pH at time 0 is an ence of 4 and 10 mM EGTA are compared effect of ionophore on the pH electrode (see fig. 4). (fig. 4). When we titrated the SW back to the original pH, there was no significant differWhen eggs are activated with ionophore in ence in acid between these eggs and eggs 0 Ca SW, both cortical activation and acid activated in 0 Ca SW containing only 0.25 release occur more rapidly than in normal mM EGTA or in normal SW (table 1). These experiments led us to investigate SW. We observed this rapid activation in all media deficient in divalent cations (see the possible participation of other ions in below, and [7]), and assume it reflects the acid release. The use of the ionophore higher solubility of the ionophore in A23187 permitted the activation of eggs in divalent cation-free media. The amount of other media in which activation by sperm acid released in 0 Ca SW is similar to that would not be feasible. Acid release was observed in all substituted media (table I). in normal SW (table 1). The normal release of fertilization acid in The initial phase was essentially identical 0 Ca SW demonstrates that concurrent uptake of calcium ion is not necessary for release of protons from the eggs and therefore the Ca2+-H+ exchange hypothesis can7.66. 4 rnM EGT* not account for the fertilization acid. The major evidence supporting this hypothesis 7.70 was the observation that proton release 0 * 4 6 8 terminated upon ‘addition of 10 mM EGTA’ Fig. 4. Abscissa: time (mitt) after addition of ionoto the egg suspension [6]. We were unable piore;.ordinate: pH. Ferttlization acid release in 0 Ca SW containing 4 to exactly repeat this experiment because mM or 10 mM EGTA as indicated. Five ml 1.6% egg the procedure was insufficiently described. suspensions activated by addition of 50 ~1 5 mM Additions of 10 ~1 0.025 N NaOH (V). However, we did observe that addition of A23187. Although less acid appears to be formed, the titrations EGTA solution (0.1 M) to an egg suspen- indicate that this effect is due to the buffering capacity the EGTA. Arrow, control addition of ionophore; sion of the same pH (pH 8; 10 mM EGTA of we conclude that the initial sharp decrease in pH at final concentration) causes a precipitous time 0 is an effect of ionophore on the pH electrode.
Sea urchin fertilization
in 0 K SW, 0 Mg SW, and 0 Ca SW. However, acid release continued during phase IT in 0 Mg SW and release of acid was accelerated in 0.55 M KC1 and in isotonic NaCl : KC1 (19 : 1). There was less acid in 0 Na SW. The acceleration of acid release in 0.55 M KC1 and NaCl : KC1 can be attributed to cell lysis, beginning within 60 set of ionophore addition. Lysis did not occur in 0 Mg SW; we are unable to account for the continued release of acid in this medium. It may be related to some kind of ionophore mediated transport. The decreased acid release in 0 Na SW might reflect an inhibition of cortical granule discharge. We observed an impaired cortical reaction in these eggs directly and by cytological examination of eggs fixed in acetic acid-alcohol (3 : l), resuspended in 45% acetic acid, and observed by phase contrast microscopy. This inhibition of the cortical reactions in 0 Na SW may have resulted from toxic effects of choline, the substituent for sodium, since no effects on cortical granule breakdown were observed in 0.55 M KCI, the other sodium-free medium used. Our results indicate that the mechanism underlying formation of fertilization acid does not involve exchange of protons for any specific cation present in SW. However, we have not attempted to measure fertilization acid in non-ionic media, and one could propose that any available cation might suffice for exchange with protons during fertilization acid release. While we cannot exclude this possibility, it seems unlikely since the kinetics of acid release is so similar in all the ion-substituted media we have used. The temporal correlation between the onset of acid release and cortical granule discharge supports the view that the con-
acid
5
tents of these granules (e.g., sulfated mucopolysaccharide) are the source of the acid [3, 41. Acid is also released upon fertilization of Urechis eggs [8]. However, cortical granule discharge does not occur in Urechis eggs at the time of acid release [S, 9, lo], and thus, if the mechanism suggested above to account for fertilization acid in sea urchins is correct, the source of acid must be different in these two kinds of egg. However, we would raise the possibility that the mechanism of acid formation may be similar in sea urchin and Urechis eggs. Consistent with this ate the observations that in both sea urchin eggs [l 1, 121 and Urechis eggs [8], a light-scattering decrease begins concomitant with acid release. Could this represent a conformational surface change, associated with acid release, which happens to correlate in time with the cortical reaction in sea urchin eggs? Or, as another possibility, could acid release reflect a cytoplasmic change, such as activation of a mitochondrial Ca2+-H+ antiport [13] with the ultimate release of protons from the egg? The biphasic nature of the acid release in our measurements raises the possibility of more than one mechanism underlying fertilization acid formation, with phase II representing a second process contributing a minor component of the acid. The cause of fertilization acid in Urechis has not been established. We suggest that the mechanism underlying the sea urchin fertilization acid has also not yet received a satisfactory explanation. We thank R. L. Hamill of the Eli Lillv Co. for his generous gift of the ionophore A23 187,A: De Vries for the loan of eauioment, and E. Baker for technical . . assistance. This work was supported in part by the following nrants: NSF GB-38629 from the NSF to D.E. and ?%?A6946 from the National Research Council of Canada and a faculty research grant from the University of Victoria to M. P. Exptl Cell Res 94 (1975)
6
Paul and Epel REFERENCES
1. Ashbel, R, Boll sot ital biol sper 4 (1929) 492. 2. Giudice, G, Developmental biology of the sea urchin embryo, p. 107. Academic Press, New York (1973). 3. Ishihara, K, Exp cell res 5 1 (1968) 473. 4. Aketa, K, Exp cell res 30 (1963) 93. 5. Mehl, J W & Swann, M M, Exp cell res 22 (1961) 233. 6. Nakazawa, T, Asami, K, Shoger, R, Fujiwam, A & Yasumasu, I, Exp cell res 63 (1970) 143. 7. Steinhardt, R A & Epel, D, Proc natl acad sci US 71(1974) 1915.
8. Paul, M, Dev bio143 (1975) 299. 9. Paul, M, Ph.D. Thesis. Stanford University, Stanford. Calif. (1970). 10. Gould-Somero, M & Holland, L, Am zool 14 (1974) 1299. 11. Epel, D, Pressman, B C, Elsaesser, S & Weaver, A M, The cell cvcle (ed G M Padilla, G L Whitson & I L Cameron) p.‘279. Academic Press, New York (1969). 12. Paul, M & Epel, D, Exp cell res 65 (1971) 281. 13. Lehninger, A L, Biochem j I19 (1970) 129. Received January 22, 1975
