Proc. Nat. Acad. Sci. USA Vol. 71, No. 5, pp. 1915-1919, May 1974

Activation of Sea-Urchin Eggs by a Calcium Tonophore (fertilization/membrane conductance/respiration/protein synthesis/DNA synthesis) RICHARD A. STEINHARDT* AND DAVID EPELt * Departinent of Zoology, University of California, Berkeley, Calif. 94720; and t Scripps Institution of Oceanography, La Jolla, California 92037 Communicated by Daniel Mazia, February 13, 1974

ABSTRACT Micromolar amounts of the divalent iono- in normal fertilization. We extended our investigations of the phore A23187 can activate echinoderm eggs. The activa- ionophore activation, comparing the changes of membrane tions by ionophore A23187 were examined in terms of membrane elevation, the program of membrane conduc- conductance, respiration, protein synthesis, and DNA syn- tance changes, the respiratory burst, and the increases thesis with those resulting from normal fertilization. For these in protein and DNA synthesis which normally accompany experiments we confined ourselves to A23187 which is re- activation by sperm. In all these respects activation by the specific for divalent cation transport as ionophore was fairly normal although subsequent cleav- portedly (5) opposed age and embryonic development was limited. to X-537A which transports monovalent cations to some lonophore A23187 activations of the cortex of Lytechinus extent (6, 7). pictus and Strongylocentrotus purpuratus eggs were A23187 activations of L. pictus and Strongylocentrotus compared in various ionic media and were found to be purpuratus eggs were compared in various ionic media and completely independent of the ionic composition of the showed independence from the ionic external solution. Respiration and protein synthesis of surprisingly complete L. pictus eggs in singly substituted ionic media also in- composition of the external solution. For these tests the dicated that these activations were independent of ex- cortical reaction proved the most conclusive since it could ternal sodium, calcium, or magnesium. These results be done in the absence of any divalent cations. Measurements suggest that the ionophore acts by releasing intracellular of respiration and protein synthesis in singly substituted ionic Ca++. Consistent with this interpretation is the finding that eggs preloaded with 45Ca show a 20-fold increase in media also indicated that these activations were independent 45Ca-efflux when activated by ionophore A23187 or fertiliza- of external Na+, Ca±±, and Mg++ tion. Measurements of the "free" and "bound" calcium Since the activation of eggs by A23187 did not depend on and magnesium in homogenates of the unfertilized eggs external ions, the ionophore may act by releasing divalent show that most of the Mg++ is already available in the in stores. soluble form, whereas Ca++ is sequestered but available for ions, most probably Ca++, sequestered intracellular release. Preloaded eggs in fact were found to release large quantities We propose that both normal fertilization and iono- of 45Ca as a consequence of ionophore activation or normal phore activation affect the metabolism of the egg by re- fertilization. leasiiig Ca + + sequestered in intracellular stores. Determinations of the "free" and "bound" calcium and An increase of the intracellular concentrations of calcium ion magnesium were made from homogenates of the unfertilized at fertilization may play a key role in the promotion of cell egg. These showed that most of the calcium is bound while synthesis which follows the activation of eggs. Mazia (1) most of the magnesium is already freely available, suggesting observed an increase in Ca++ in the ultrafiltrates of ho- that the ionophore activates by its effect on calcium stores. mogenates of Arbacia eggs which had just been fertilized. MATERIALS AND METHODS Similar estimates of increases in intracellular "free" Ca++ have since been made following the activation of other species Handling of Gametes. The sheddings of L. pictus and S. of echinoderm egg (2) and by other methods (3). purpuratus gametes were induced by intracoelomic injection If intracellular calcium activity is central to activation, of 0.5 M KCl. Jelly was removed mechanically by passing the then we should be able to parthenogenetically activate eggs eggs once through a silk mesh and washing. Experiments were with divalent cation-transporting ionophorous antibiotics. done at 16-18' using filtered seawater (SW), except in iron- These compounds promote transport of divalent cations substitution experiments, where artificial SW was used (see through lipid barriers (4, 5). We found that micromolar below). amounts of the ionophores X-537A (Hoffman-LaRoche) and Ionophores. A23187 was obtained from R. L. Hamill, Eli A23187 (Lilly) could activate Lytechinus pictus eggs and Lilly Co., Indianapolis, Ind. For use with L. pictus embryos, cause the development of new potassium conductance just as the 5 mM stock solution was prepared in one part N,N- dimethyl formamide (DMIF) to three parts ethanol. DMF- Abbreviations: SW, seawater; TCA, trichloroacetic acid; D)MIF, .A/,NV-dimethyl formamide; M\e2SO, dimethyl sulfoxide; EDTA, ethanol and dimethyl sulfoxide (Me2SO) had no activating ethylenediaminetetraacetic acid; 0 Na+ SW, artificial SW with effects at the concentrations used. For use with S. purpuratus glycerol substituted for NaCl; 0 MIg++ SW, artificial SW with embryos A23187 was dissolved in Me2SO since these eggs will NaCl substituted for M\1gCI2; 0 Ca+± SW, artificial SW with lyse if activated or fertilized in the presence of ethanol. When NaCl substituted for CaCl2, + 2 mM ethyleneglycol-bis(O-amino- diluting in seawater, the solutions were continuously stirred ethylether)N,N '-tetraacetic acid. to insure good mixing, since the ionophore is very insoluble 1915 Downloaded by guest on September 27, 2021 1916 Cell Biology: Steinhardt and Epel Proc. Nat. Acad. Sci. USA 71 (1974)

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0 -10 _ mV -20 -30 -40- -50 Minutes after addition FIG. 2. Changes in respiration following ionophore A23187 0 2 4 6 8 10 12 14 16 18 20 activation or fertilization of 10% suspension of L. pictus eggs. Minutes after addition 0-O, fertilized; A--A, 7.51AM A23187. FIG. 1. Membrane potential of L. pictus eggs following activa- tion with 18 MM ionophore A23187. In this and all subsequent RESULTS figures, time 0 refers to addition of A23187 or sperm. Physiological and biochemical activation

in water and there is some precipitation. Final concentrations Cortical Reactions. Exposure of L. pictus or S. purpuratus given in the text are therefore maximum estimates. A23187 eggs to micromolar amounts of A23187 resulted in rapid dis- should be kept in the dark, since we observed loss of activity charge of the cortical granules and elevation of fertilization when exposed to light. membranes. Using L. pictus eggs (less than 1% suspension), the time from the addition of the ionophore to the beginning Artificial Sea Waters. Artificial seawater had the com- of elevation of the fertilization membranes (latency period) position: 460 mM NaCl, 55 mM MgCl2, 11 mM CaCl2, 10 depended on the concentration of A23187. The latency period mM KCl, and 2.5 mM NaHCO3, adjusted to pH 8.0 with with sperm is 25-30 sec. We found that 0.5 MAM A23187 did NaOH. 0 Na+ SW had 920 mM glycerol substituted for the not activate more than 30% of the eggs, the first elevation NaCl. 0 Mg++ SW had NaCl substituted for the MgC12. 0 occuring after a 4-min latency period. Membrane elevation Ca++ SW had NaCl substituted for CaCl2 plus 2 mM ethylene- began at 30 sec in 5,uM and at 10-15-sec in 50 uM concen- glycol-bis(,-aminoethylether)N,N'-tetraacetic acid (EGTA). tration. At the higher concentrations of ionophore, membrane Isotonic KCl contained 2 mM ethylenediaminetetraacetic elevations were synchronous and proceeded simultaneously acid (EDTA). at all points on the egg surface instead of starting at one point Membrane Conductance. Electrophysiological measure- and propagating as is the case with sperm activation. Similar ments were made by the methods of Steinhardt, Lundin, and results were obtained with S. purpuratus eggs. Using these Mazia (8). eggs, we found that the activation by A23187 also depended on egg concentration, suggesting that the drug is sequestered Respiration. Respiration was measured polarographically by the eggs. Using initiation of membrane elevation in 50% using a Yellow Springs model 5331 oxygen electrode inserted of the eggs as a criterion, the latency period in 2 MAM A23187 into a 1.3-ml water-jacketed chamber maintained at 180. was 20 sec in 0.04% suspension, 45 sec in 0.2% suspension, The rate of 02 consumption of the 10% egg suspension was and 120 see in 0.8% suspension. calculated from the slope of the recorder tracing, using a value of 5.6 ml of 02 per ml for 02-saturated seawater at 180. Membrane Conductance. The A23187 activation of the program of membrane conductance changes appeared to be Protein Synthesis. Transport and incorporation into protein nearly identical with normal fertilization (Fig. 1). First a of labeled amino acid during 5-min pulses were measured as transient Na+-dependent depolarization develops, followed described earlier (9), using 0.2% egg suspensions. Incorpora- shortly by a permanent K+-sensitive hyperpolarization which tion into protein is expressed as percent incorporation, using is accounted for by new potassium conductance (8). The the ratio "dpm in trichloroacetic acid (TCA) insoluble per initial depolarization is absent in 0 Na+ SW. The final inside dpm in TCA soluble + TCA-insoluble." negative values of membrane voltages were sensitive to DNA Synthesis. A 0.1% suspension of L. pictus eggs was external K+ and fell off in the expected manner, going below incubated in 0.2 MCi/ml of [3H]thymidine (methyl label, 72 12 mV at concentrations of K+ above 120 mM. Ci/mM, Cal Atomic). Following activation by ionophore or Respiration. The effect of A23187 on 02 uptake was measured 2-ml were collected at the indicated sperm, samples times, polarographically, testing over the range 0.75-75 MM with washed free of exogenous isotope with two sea-water washes, 10% suspensions of L. pictus eggs. At this higher concentra- and then extracted in 5 ml of 10% TCA. The TCA extracts tion of eggs, no activation was seen at 0.75 MM, but at 1.5 were heated at 600 for 10 min, the pellets washed twice with MAM A12387 the peak 02 uptake during the respiratory burst TCA, dissolved in 1 ml of NCS, and counted in a toluene- was nearly at the maximal rate. As with the cortical reaction, based solution scintillation as above. by liquid counting however, the latency period depended on the concentration Measurement of Ca++ and Mg++. Eggs were extracted as of ionophore. The respiratory burst in normal fertilization described in the text, and ion content was analyzed on a Perkin- occurs within 2 min of sperm addition. With 25-75MAM A23187, Elmer 403 Atomic Absorption Spectrometer using a Ca++- this peak was at 3 min; at 7.5 MAM, 4 min; at 1.5 MAM, 7 min; Zn++ or Mg++-hollow cathode lamp. with no activation of respiration occurring at lower concen- Downloaded by guest on September 27, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Calcium Activation of Eggs 1917

.2 50 40- .3030 E 20 - 10 10 20 30 40 50 60 Minutes after addition 0 20 40 60 80 100 120 Minutes after addition FIG. 4. Changes in DNA synthesis following ionophore of FIG. 3. Changes in protein synthesis following ionophore A23187 activation or fertilization of 0.1% suspension L. pictus A23187 activation or fertilization of 0.2% suspension of L. pictus eggs. O-O, fertilized; A- - -A, .5M)M A23187. eggs. O-O, fertilized; 0 0, 0.5 AM A23187; A A, 5,AM A23187. Comparisons of ionophore activation in different ionic media trations. Fig. 2 shows the similar patterns of 02 uptake during To test whether A23187 required the presence of any l)articu- A23187 activation and fertilization. lar ion in the external medium in order to activate, we ex- posed L. pictus eggs to ionophore in different ionic media. Protein Synthesis. The effect of A23187 on protein syn- Our comparisons include membrane elevation, respiration. thesis was measured by ["4C]leucine incorporation during 5- and protein synthesis. min pulses over a 2-hr period. Fig. 3 shows that up until the time of entry into the first prophase following 0.5 MAM or 5 Membrane Elevation. Membrane elevation was observed in MiM A23187 activation, the rate of protein synthesis was as all media tested, 0 Ca++ SW, 0 Mg++ SW, 0 Na+ SW, 0 K+ high as that resulting from normal fertilization. After this SW, and 0.55 M KCl plus 2 mM EDTA. This was also true period, however, the rate of protein synthesis in ionophore for S. purpuratus eggs. These results indicate that no single activated eggs fell behind fertilized eggs. ion or combination of ions is required for ionophore activa- tion of membrane elevation. DNA Synthesis. DNA synthesis during the first cell cycle The test of membrane elevation in 0.55 M KCl plus 2 mM was determined by measuring [3H]thymidine incorporation EDTA was the only indicator of activation we could use in into hot TCA-insoluble material. The ionophore-activated the total absence of divalent cations. Eggs lyse in absence of DNA synthesis during the first hour was, as expected, close divalents if stirred, and stirring is required for other metabolic to 50% of the diploid fertilized controls (Fig. 4). [3H]Thymi- measurements (see below). Membrane potentials can be mea- dine transport into eggs is increased 10-fold by 30 min from sured without stirring, but not in the absence of calcium. We the addition of sperm or 5MuM A23187. could not test activation in the total absence of salts since Cytological Observations. Microscopic observations of living eggs lyse and quickly die in isotonic non-electrolyte media. eggs during the first few hours following A23187 activation Respiration. A23187 activated the burst of 02 uptake in the revealed nuclear membrane breakdown and formation of absence of calcium or magnesium (Fig. 5). The O0 uptake in clear areas suggestive of asters or mitotic apl)aratus. Around 0 Ca++ or 0 Mg++ media was slightly less than the fertilized the time of first cleavage of the fertilized egg controls, surface sea-water control. This slight reduction might be an effect in some changes resembling cleavage were seen ionophore- of the ionic media directly on respiration rather than on the treated cells. Furrowing was generally unsuccessful but some 2-celled "embryos," containing one nucleus per cell, were seen. No further cleavage activity was observed. More detailed observations of mitotic activity were made with cells fixed in Carnoy's fluid and examined by phase con- 300 trast microscopy. L. pictus eggs were activated with 0.5 or 250- 5 MM A23187 and fixed between 80 and 180 min. Events were 200- similar in both concentrations. In general, mitotic events and behind fertilized control em- 150- were asynchronous lagged C'j bryos. For example, control eggs were in anaphase or telo- 100 phase at 80-min l)ost-fertilization, whereas, A23187-activated cells were in prolphase or in a "prometaphase-like" stage. later than usual, such that Mitotic spindle material appeared 02 4 6 8 0 2 4 6 8 when the nuclear membranes broke down between 80- and 100 mill, the chromosomes were deposited directly into "cyto- Minutes after addition material. FIG. 5. Changes in respiration following ionophore A23187 l)lasm" or into small areas of clear spindle-like activation (7.5 ,.AM) or fertilization of L. pictus eggs in singly Fibrous monasters were occasionally seen at 100 and 125 min, substituted ionic media. Fertilization in 0 Ca++ SW or 0 Mg++ either associated with highly-condensed metaphase chromo- SW were not possible, so comparisons with fertilization could somes or with newly-reformed nuclei. By 145 and 180 min, not be made. (A) O- O, artificial SW; 0 0, 0 Mg++ SW; most cells were in telophase or interphase with one nucleus, A A, 0 Ca++ SW. (B) O-O, fertilized in 0 Na+ SW; but occasional binucleate cells were seen. A hA A23187 in 0 Na + SW. Downloaded by guest on September 27, 2021 1918 Cell Biology: Steinhardt and Epel Proc. Nat. Acad. Sci. USA 71 (1974) TABLE 1. Protein synthesis in singly-substituted ionic media

Percent at Activator and incorporation type of seawater (SW) 30 min 60 min

C'S 200 Sperm, artificial SW 18.8 18.7 A23187, ASW 20.2 16.4 A23187,0 Ca++ SW 32.0 15.2 A23187,0 Mg++ SW 24.4 23.3 A23187, 0 Na+ SW 11.9 10.8 Unfertilized 1.5 -60 -40 -20 0 20 Time (minutes) Eggs were activated with sperm or ionophore (5 pAM) and FIG. 6. 45Ca efflux following ionophore A23187 activation or pulsed for 5 min with ['Hjvaline 30 and 60 min later. The de- fertilization of 5% suspension of L. pictus eggs. O-O, un- pression of incorporation in 0 Na+ SW is most likely a general fertilized; aJ-fo, fertilized; A /A, 5 jgd A23187. The K effect on cell synthesis and not an effect on activation. In ex- values of 45Ca efflux refer to the fraction X 103 of the radioactive periments using choline-substituted 0 Na + SW, protein synthesis calcium released during the preceding 10-min period. In order of embryos was reduced 59%. to calculate the fraction released, we need the amount of isotope remaining in the eggs for each period. This is determined by taking ice-cold Dounce homogenizer in a variety of media (Table the 45Ca remaining at the end of the experiments. Efflux is then 2). The extracts were immediately centrifuged at 37,000 X added back to the midpoint of each period to give total 4"Ca g for 30 min and separated into "soluble" and "insoluble" for each point. The minus time values refer to the efflux before fractions. The supernatant fractions were analyzed directly treatment. At time zero the eggs were divided into three batches by atomic absorption, and the insoluble fractions were ex- and treated with A23187, sperm, or a DMF-ethanol blank. (The tracted overnight in concentrated HNO3, neutralized, and numbers on the ordinate have been multiplied by 103.) then analyzed. As seen (Table 2), 70-80% of the magnesium is found in the soluble fraction in all the homogenation activation of respiration but we could not test this point since media. This contrasts markedly with calcium, which ranges fertilization is not possible in either 0 Ca++ or 0 Mg++ arti- from 9% soluble in 0.55 M KCl, pH 6, to 47% soluble in ficial seawaters. In 0 Na+ SW, the ionophore activation 0.60 M sucrose. These results indicate that whereas magne- actually stimulated more 02 uptake than sperm activation, sium is generally available, the amount of calcium present but respiration was considerably reduced in that media for in the soluble fraction depends on the conditions of homog- both types of activation. The possibility that the respiratory enation. This suggests that calcium is usually in the bound burst reflects energy used to pump out Na+ is currently being state but in a form that can be transfered to the soluble phase. investigated. DISCUSSION Protein Synthesis. A23187-activated protein synthesis in Every parameter of normal activation with sperm that we the absence of calcium, magnesium, or sodium is shown in examined is also observed when eggs are exposed to divalent Table 1. Ionophore activations in 0 Na + SW show a reduced transporting ionophore. Membrane elevation, membrane rate. However, this reduction of rate is also seen with nor- conductance changes, the respiratory burst, and increases in mally fertilized eggs after they are put in 0 Na+ media (legend, protein and DNA synthesis are all initiated in the usual Table 1). fashion by micromolar concentrations of A23187. However, differences develop as time goes on. Protein synthesis falls Divalent cations behind in the ionophore-activated eggs after 40 min, and mitosis is delayed and was not successfully completed. Ca++ Since the data above is consistent with an Efflux. Monasters eventually formed in most cells and subsequent ionophore-stimulated release of intracellular stores of calcium, we tested for the release of preloaded 45Ca by A23187 and by TABLE 2. "Free" Calcium and Magnesium in normal fertilization. A 6.8% suspension of L. pictus eggs was S. purpuratus eggs preloaded with 45Ca (18 uCi/ml) for 1 hr at 4°. The preloaded eggs were washed four times with sea water and then their % Free % Free efflux was followed until they reached low equilibrium levels. Homogenation media Mg ++ * Ca + +t The eggs were then divided into three batches and treated. The blank remained at low levels of "'Ca 0.55 M KCl, pH 6 47 9 DMF/ethanol 0.55 M KCl, pH 7 62 22 efflux, returning to equilibrium values within the next 30 0.25 M KCl, min. Both the A23187-activated eggs and fertilized eggs 0.40 M sucrose, pH 7 61 43 showed a 20-fold increase in calcium efflux which remained 0.60 M\I sucrose, pH 7 56 47 high over the 30-min l)eriod sampled after activation (Fig. 6). 0.55 M KCl, 0.005 M EDTA, pH 7 51 19 "Free" and "Bound" Calcium and Magnesium in the Un- fertilized Egg. Since A23187 can bind to and transport Mg++ Values determined by atomic absorption; pH 7 extracts buf- as well as Ca++, we examined the proportions of these ions fered with 0.05 M\ imidazole. which were already available as "free" cation. S. purpuratus * Total MNg + + is 17.9 mMI. eggs were washed twice in 0.55 KCl and then disrupted in an t Total Ca++ is 7.1 mIM. Downloaded by guest on September 27, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Calcium Activation of Eggs 1919

events were similar to those seen for other parthenogenetic eggs would discount that possibility. Most of the Mg++ is agents when cleavage is unsuccessful (10). already available, whereas the low levels of free Ca++ in the We were unable to obtain normal embryonic development, unfertilized egg make calcium the likely candidate for release even when we washed L. pictus eggs in fresh sea water 5 min by divalent ionophore. after A23187 activation. However, we have made no con- We thank D. Mazia, T. Schroeder, P. Reed, C. Mehard, B. centrated efforts to promote normal development with secon- Volcani, and R. Hamill for helpful discussion and E. Baker for dary treatments, such as hypertonic seawater. technical assistance. This work was supported in part by grants These physiological, biochemical, and cytological observa- from the National Science Foundation and the Population tions point to a fairly normal activation by ionophore with Council. lesions emerging as development proceeds. Since it is dif- 1. Mazia, D. (1937) J. Cell. Comp. Physiol. 10, 291-304. ficult to obtain parthenogenetic development with L. pictus 2. Nigon, V. & Do, F. (1963) C. R. H. Acad. Sci. 257, 2178- eggs, it is not clear whether these lesions result from the 2180. ionophore per se or are characteristic of parthenogenetic 3. Clothier, G. & Timourian, H. (1972) Exp. Cell Res. 75, 105- of these 110. activation eggs. 4. Pressman, B. C. (1972) in The Role of Membranes in Meta- A23187 can bind to and transport Ca++ and Mg++, but bolic Regulation, eds. Mehlman, M. A. & Hanson, R. W. not K+, ions through lipid phases (5). Like X-537A (11), (Academic Press, New York), pp. 149-164. A23187 has been shown to act across cell membranes and 5. Reed, P. W. & Lardy, H. A. (1972) in The Role of Mem- show dependence (12) or independence (13) of the ionic com- branes in Metabolic Regulation, eds. Mehlman, M. A. & Hanson, R. W. (Academic Press, New York), pp. 111-132. position of external solutions. A23187 activation of sea urchin 6. Lardy, H. A., Graven, S. N. & Estrada, 0. (1967) Fed. Proc. eggs is independent of ions in the external solution and sug- 26, 1355-1360. gests divalent ion release from subcellular stores. Since Ca++ 7. Henderson, P. J. F. McGivan, J. D. & Chappell, J. B. ion activity was previously shown to increase with fertiliza- (1969) Biochem. J. 111, 521-535. tion (1), we conclude that most probably intracellular calcium 8. Steinhardt, R. A., Lundin, L. & Mazia, D. (1971) Proc. Nat. Acad. Sci. USA 68, 2426-2430. release is the normal activating agent in fertilization and in 9. Epel, D. (1972) Exp. Cell Res. 72, 74-89. activation by A23187. 10. von Ledebur-Villiger, M. (1972) Exp. Cell Res. 72, 285-308. Our results showing a 20-fold increase in 45Ca effiux with 11. Levy, J. V., Cohen, J. A. & Inesi, G. (1973) Nature 242, 461- ionophore activation or normal fertilization support the cal- 463. 12. Foreman, J. C., Mongar, J. L. & Gomperts, B. P. (1973) cium hypothesis of activation. A23187 could theoretically Nature 245, 249-251. activate by Mg++ movements. However, our data on free 13. Schroeder, T. E. & Strickland, D. E. (1974) Exp. Cell Res. and bound Ca++ and Mg++ in homogenates of unfertilized 83, 139-142. Downloaded by guest on September 27, 2021