Possible involvement of calmodulin in maturation and activation of Chaetopterus eggs

DEVELOPMENTAL

BIOLOGY

99.1-6

(1983)

Possible Involvement of Calmodulin in Maturation Activation of Chaetopterus Eggs ALAN G. CARROLL*” *Department

of Biology,

Indiana Washington,

AND WILLIAM

and

R. ECKBERG~$

University-Purdue University, Indianapolis, Indiana 46223; j-Department of Zoology, D. C. 20059; and *Marine Biological Laboratory, Woods Hole, Massachusetts 02543 Received

June

28, 1982; accepted

in revised

form

March

Howard

University,

31. 1983

We report the isolation of calmodulin from oocytes of Chaetopterus pergamentaceus. The identification of this protein is based on (1) activation of beef heart CAMP phosphodiesterase, (2) heat stability, (3) sensitivity to chlorpromazine, and (4) electrophoretic mobility identical to that of porcine brain calmodulin after sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of either Ca2+ or EGTA. We treated oocytes with chlorpromazine and W-7 to investigate the involvement of calmodulin in meiosis initiation and egg activation. Very low concentrations of chlorpromazine inhibited germinal vesicle breakdown (GVBD). This effect was shown to be dependent upon bright indirect light, since the drug was much less effective at GVBD inhibition under conditions of very low illumination. Higher concentrations of chlorpromazine and W-7 (100 PM) inhibited GVBD and activated eggs with intact germinal vesicles as determined by fertilization envelope formation and the onset of ameboid activity. Neither egg activation nor inhibition of calmodulin stimulation of phosphodiesterase activity in vitro was affected by light. These results are consistent with a role for calmodulin in egg activation and GVBD, but suggest that chlorpromazine in bright light may prevent GVBD by some mechanism other than calmodulin inhibition.

INTRODUCTION

in mediating the action of calcium on enzymes and, in turn, on cell function (Cheung, 1980; Klee et al, 1980). Calmodulin has been found in all eukaryotic cells examined, including the eggs of sea urchins, starfish, and amphibians (Head et aZ., 1979; Dor&e, 1980; Cartaud et ah, 1980; Meijer and Guerrier, 1981; Wasserman and Smith, 1981). Furthermore, calmodulin has been implicated in meiosis initiation (Moreau et al., 1980; Wasserman and Smith, 1981) and in egg activation (Baker and Whitaker, 1980; Steinhardt and Alderton, 1982; A. G. Carroll, unpublished observations). As part of an effort to determine the roles of calcium and calmodulin in GVBD and fertilization, we investigated the effects of the calmodulin inhibitors, chlorpromazine (CPZ) (Weiss and Levin, 1978) and W-7 (Hidaka et al, 1980), and the inactive CPZ analog, chlorpromazine sulfoxide (CPZSO) (Weiss and Levin, 1978), on GVBD and subsequent events in the polychaete Chaetopterus pergamentaceus. GVBD occurs spontaneously after the oocyte contacts natural seawater in this species, but does not occur in artificial seawater unless supplemented with excess KC1 (Ikegami et ah, 1976). In this medium, GVBD is followed by “differentiation without cleavage,” a partial morphogenesis without cytokinesis originally described by Lillie (1902) and more recently by Brachet and Donini-Denis (1978), Eckberg (1981b), and Eckberg and Kang (1981). Our results indicate that CPZ and W-7 inhibited GVBD, and,

Maturation of eggs, characterized by germinal vesicle breakdown (GVBD),2 is a process that often requires calcium. This is reflected in the failure of GVBD in the absence of calcium in some species (Goldstein, 1953; Allen, 1953; Ikegami et al, 1976; Brachet and Donini-Denis, 1978; Dub& et al., 1982), and in the increase in intracellular free calcium after stimulation of GVBD (Do&e et al., 1978; Moreau et al, 1978,198O; Wasserman et al., 1980). Calcium is also believed to be a critical part of the activation reaction of the egg after fusion with the sperm (Jaffe, 1980; Epel, 1980; Gilkey, 1981). In most species that have been investigated, sufficient calcium for GVBD and activation may be derived from the internal stores of the egg (Steinhardt et al, 1974; Moreau et al, 1978; Schmidt et al, 1982; Eckberg and Carroll, 1982). The mechanism of calcium’s action on GVBD and egg activation has not been elucidated. Studies of other systems have shown that calmodulin is frequently involved ’ To whom all correspondence should be addressed: Department of Biology, IUPUI, Box 647, Indianapolis, Ind. 46223. ‘Abbreviations used: GVBD, germinal vesicle breakdown: CPZ, chlorpromazine; W-7, N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide; CPZSO, CPZ sulfoxide; CFSW, calcium-free artificial seawater; EGTA, ethyleneglycol-his@-aminoethyl ether)N,N’-tetraacetic acid.

1 0012-1606/83 Copyright All rights

$3.00

0 1983 by Academle Press. Inc of reproduction in anv form rearrwd

2

DEVELOPMENTAL

at the same time, activated no effect. MATERIALS

BIOLOGY

eggs, whereas CPZSO had

AND

METHODS

Experimental animuls. Mature specimens of Chaetopterus pergamentaceus were obtained from the Marine Resources Division, Marine Biological Laboratory, Woods Hole, Massachusetts, and Pacific Bio-Marine Laboratories, Inc., Venice, California, and handled as described previously (Eckberg, 1981a). All natural and artificial seawaters used were buffered with 10 mMTrisHCl, pH 8.0. Artificial seawater and calcium-free artificial seawater (CFSW) were made according to MBL formulae (Cavanaugh, 1964). Potassium-supplemented artifical seawaters with and without calcium were made as described by Ikegami et al. (1976). Primary oocytes were obtained by shedding into CFSW (Eckberg and Carroll, 1982). Calnwdulin assay. We determined calmodulin activity in homogenates of oocytes or in purified preparations of protein by the calmodulin-dependent activation of 33’-cyclic nucleotide phosphodiesterase (Sharma and Wang, 1979). One unit of calmodulin activity (U) was the amount of calmodulin that yields 50% of maximal activation. Puri,fication of calmdulin, Settled eggs in CFSW were washed in 10 ~010.2 M NaCl, 20 mlM KCl, 5 mM MgClz, 5 mM EGTA, 20 mM BES, pH 7.2. Packed eggs were homogenized in 10 vol of the same buffer and frozen. Calmodulin was purified by the method of Kakiuchi et al. (1981) with affinity chromatography on CAPP-Sepharose (Jamieson and Vanaman, 1979). Porcine brain calmodulin was prepared by the method of Sharma and Wang (1979). Protein was assayed by the dye-binding method (Bradford, 1976) using bovine serum albumin as the standard. Electrophoresis. SDS-polyacrylamide gel electrophoresis was performed according to the procedure of Laemmli (1970). The stacking gel contained 5% acrylamide monomer, and the separating gel contained 15% acrylamide. Gel and buffer contained either 0.1 mlM Ca2+ or 0.5 mM EGTA. Molecular weight standards included bovine albumin (66,000), ovalbumin (43,000), PMSFtreated trypsinogen (24,000), @lactoglobulin (18,400), and lysozyme (14,300). In viva inhibitor analysis. A 10 mM stock solution of chlorpromazine (CPZ, Sigma), CPZSO, or W-7 (kind gifts of Milton Cormier, University of Georgia) in dimethylsulfoxide (DMSO), was prepared fresh before each experiment and diluted with seawater to the appropriate experimental concentration. Seawater pH was not affected. DMSO had no effect on the eggs at the highest concentrations used. Oocytes that had been washed in

VOLUME

99, 1983

CFSW’were placed in Syracuse dishes containing inhibitor in the appropriate seawater and examined for GVBD and egg activation (fertilization envelope elevation and ameboid activity). Activation defined this way represents the early phases of differentiation without cleavage (Brachet and Donini-Denis, 1978; Eckberg, 1981b). Because a limited and variable amount of GVBD occurred before eggs were washed in CFSW, all data were normalized by the following formula: S = (P - N) f (M - N) X 100, where S = normalized percent, P = observed percentage, M = maximum percentage (natural seawater control), and N = basal level (CFSW control). In vitro inhibitor treatment. Inhibition of calmodulin activity in vitro by CPZ was determined by incubating various concentrations of CPZ with 10 units of calmodulin in the phosphodiesterase assay system described above. The 15,, of CPZ was the concentration of CPZ needed to reduce the calmodulin stimulation of phosphodiesterase activity by 50%. Experiments with ultraviolet exposure were performed with a General Electric F18T8-BLB longwave black light fluorescent bulb at 45 cm from the sample. RESULTS

Characterization Chaetopterus

of Calmodulin Eggs

from

A boiled homogenate of Chaetopterus oocytes was assayed for calmodulin activity with beef heart calmodulin-dependent phosphodiesterase. The specific activity of the homogenate was approximately 240 U/mg protein. Protein purified from the homogenate by CAPP-Sepharose affinity chromatography also stimulated phosphodiesterase activity. The specific activities of the purified Chaetopterus protein and porcine brain calmodulin were similar, 0.14 and 0.09 U/rig, respectively. Comparison of the homogenate activity and affinity-purified protein suggests that calmodulin constitutes about 0.17% of the homogenate protein. Electrophoretic comparison of the Chaetopterus protein and pig calmodulin is shown in Fig. 1. Both proteins showed similar mobilities and the characteristic calcium-dependent shift in mobility in SDS-polyacrylamide gels. CPZ inhibited phosphodiesterase activation by both pig brain calmodulin and purified Chaetopterus protein (Table 1). Because light-induced CPZ free radicals can have altered inhibitary properties (Akera and Brody, 1968; Lee et al, 1976) and because CPZ affected Chaetopterus oocytes differently in light and darkness, we assayed the effect of CPZ on the Chaetopterus protein in vitro under different lighting conditions. Lighting conditions had very little effect on CPZ inhibition of

CARROLL

AND ECKBERG

Calmodulin

in

3

Chaetopterus

45K

24K

18.4K

O3

14.3K

0.3

1

2

3

FIG. 1. SDS-polyacrylamide gel electrophoresis topterm protein (2,4) and porcine brain calmodulin of EGTA (1, 2) and calcium (3, 4).

phosphodiesterase activation tein in vitro (Table 1).

FIG. 2. Inhibition of GVBD in natural sunlight (v), CPZ in dim light (m), W-7 in dim light (A).

4

by the Chaetopterus

TABLE 1 ON CALMODULIN

Calmodulin source

Lighting conditions

Pig brain Pig brain Chaetopterus Chaetopterw

uv lightb Dim light” uv light Dim light

ACTIVITY

300

seawater by CPZ in indirect in dim light (O), and CPZSO

Effects of Calmodulin Inhibitors on Egg Activation

pro-

The calmodulin inhibitors, CPZ and W-7, both inhibited GVBD with an Iso of about 100 PM, CPZSO had no effect (Fig. 2). CPZ was more effective in bright indirect sunlight than in dim artificial light, with an I50 of about 2 &f, Light alone had no effect on the eggs. CPZ inhibited GVBD when induced by 60 n-&KC1 in artificial seawater at all calcium concentrations tested (Fig. 3).

OF CPZ

30 INHIBITOR

of purified Chae(1,3) in the presence

Eflects of Calmodulin Inhibitors on GVBD

EFFECT

MICROMOLAR

CPZ and W-7 activated eggs, whereas CPZSO was again ineffective (Fig. 4). Furthermore, CPZ was equally effective at activating eggs in light and darkness. The activation response varied somewhat with the dose of the drug. At the highest concentrations used, CPZ and W-7 induced violent ameboid contractions of the oocyte which eventually (within 1 hr) resulted in cytolysis. 1

IN VITRO 0

a I,”

(PM)

W

57 68 54 65

a Amount of CPZ required to reduce activation of calmodulin-dependent CAMP phosphodiesterase by 50%. * Assay tubes were exposed to a General Electric F18T8-BLB longwave black light fluorescent bulb at 45 cm during incubation. ‘All manipulations were performed in the presence of indirect light from not more than three 60-W tungsten light bulbs.

0

I 0.1 CALCIUM

r 1 blILLIMOLAR)

FIG. 3. Calcium dependence of GVBD in 60 mM potassium-supplemented artificial seawater in the presence (m) and absence (0) of 0.1 mM CPZ in indirect sunlight.

4

DEVELOPMENTAL BIOLOGY

VOLUME 99, 1983

The primary effect of CPZ on GVBD in bright light is evidently not due to calmodulin inhibition. First, the large difference in CPZ effect on GVBD in the light and dark was not matched by a similar in vitro change in calmodulin inhibition. Second, CPZ inhibition of GVBD in the light occurred at concentrations much lower that those needed to inhibit calmodulin activity in vitro. In addition, photo-oxidized CPZ-free radicals inhibit at least one enzyme, Na+-K+-ATPase, at much lower concentrations than native CPZ (Akera and Brody, 1968). Thus, the effect of CPZ in bright light probably involves an interaction with a protein other than calmodulin.

a 20-

Eflect of Calmodulin 0.3

3 MtCR0ti40L~R

30 INHIBITOR

300

FIG. 4. Activation of eggs in natural seawater by CPZ in indirect sunlight (‘I), CPZ in dim light (m), W-7 in dim light (a), and CPZSO in dim light (A).

Egg activation composition of in KSW became free conditions tivated eggs in lutions of NaCl,

by CPZ was independent of the ionic the medium. Oocytes treated with CPZ activated even under nominally calcium(Fig. 5). In other experiments, CPZ aceither natural seawater or isotonic sosucrose, or glycine. DISCUSSION

Presence of Calm&din

in Eggs

Inhibitors

on Egg Activation

The activation of Chuetopterus oocytes by CPZ and W-7 suggests that calmodulin plays a role in the maintenance of the unfertilized egg in meiotic arrest until fertilization. This egg activation was similar to that observed by Lillie and others after treatment with excess KC1 (Lillie, 1902; Ikegami et al, 1976; Eckberg, 1981a; Eckberg and Kang, 1981), or ionophore A23187 (Brachet and Donini-Denis, 1978). We believe that the effect of calmodulin inhibitors is due to the inhibition of calmodulin and not to membrane destabilization. Dor&e et al. (1982) treated starfish oocytes with 1 mM CPZ and 10 mM W-7 but did not report lysis or activation. Further, while CPZ and W-7 lysed red blood cells, W-7 was effective at much lower concentrations than CPZ (Kobayashi et aL, 1979). It seems likely, therefore, that egg activation by CPZ is not due to partial lysis

We have isolated calmodulin from Chaetopterus oocytes. Calmodulin was expected in Chaetopterus oocytes since oocytes of other species have calmodulin (Head et aL, 1979; Doree, 1980; Cartaud et aL, 1980; Meijer and Guerrier, 1981; Wasserman and Smith, 1981). Our identification of calmodulin was based on (1) activation of calmodulin-dependent, beef heart CAMP phosphodiesterase, (2) heat stability, (3) sensitivity to CPZ, and (4) electrophoretic mobility identical to that of pig brain calmodulin in calcium and in EGTA. Efect

of Calmodulin

Inhibitors

on GVBD

The GVBD inhibition data obtained under dim light are consistent with a calmodulin effect. The Iho’s of CPZ and W-7 for GVBD in the dark are similar. Comparative, in vitro studies of the Iso of these compounds on three different calmodulin-activated enzymes, show that CPZ and W-7 have similar effectiveness despite substantially different strucutres (Hidaka et ah, 1980). The similarity of action, combined with the lack of effect of the CPZ analog, CPZSO, suggests that GVBD may be dependent on calmodulin-protein interactions.

0IIL

0:1 CALCIUM

1 (MILLMOLAR)

FIG. 5. Calcium dependence of Chaetopterus egg activation in 60 mM potassium-supplemented artifical seawater in the presence (B) and absence (0) of 0.1 mM CPZ in indirect sunlight.

CARROLL

AND ECKBERC

or non-specific membrane effects. The violent ameboid contractions observed at the highest drug concentrations may have been due to disruption of the oocyte cytoskeletal organization as has been reported for mammalian (&born and Weber, 1980) and sea urchin (Hagstrom and Lonning, 1973) cells and may also be dependent on calmodulin. Unlike Chaetopterus, anti-calmodulin drugs inhibit egg activation in sea urchins as determined by the cortical reaction (Baker and Whitaker, 1980; Steinhardt and Alderton, 1982; A. G. Carroll, unpublished observations). This difference may, however, only reflect the fact that vitelline layer elevation in Chaetopterus does not involve a cortical reaction comparable to that in the sea urchin (Eckberg, 1981a). We conclude that, while calmodulin is likely to be involved both in the stimulation of GVBD and in the maintenance of the Chaetopterus egg in an unactivated state, the precise role of calmodulin in these events is not clear. This work was supported in part by NIH RR08016 to W.R.E. and in part by grants from Indiana University Foundation and Purdue University to A.G.C. REFERENCES AKERA, T., and BRODY, T. M. (1968). Inhibition of brain sodium- and potassium-stimulated adenosine triphosphatase activity by chlorpromazine free radical. Mol. Ph,urmacoL 4, 600-612. ALLEN, R. D. (1953). Fertilization and artificial activation in the egg of the surfclam, @n&la solidtisiwu~ BioL BulL 105, 213-239. BAKER, P. F., and WHITAKER, M. J. (1980). Trifluoperazine inhibits exocytosis in sea-urchin eggs. J. PhysioL (London) 298. BRACHET, J., and DONINI-DENIS, S. (1978). Studies on maturation and differentiation without cleavage in Chaetopterusvariqwdatus. Effects of ions, ionophores, sulfhydril reagents, colchicine and cytochalasin B. D$erentiation 11, 19-37. BRADFORD, M. (19’76). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal. Biochem 72, 248-254. CARTAUD, A., OZON, R., WALSH, M. P., HAIECH, J., and DEMAILLE, J. G. (1980). Xenopus lo.evis oocyte calmodulin in the process of meiotic maturation. J. BioL Ch,em. 255, 9404-9408. CAVANAUGH, G. M. (1964). “Formulae and Methods V.” Marine Biological Laboratory, Woods Hole, Mass. CHEUNG, W. Y. (1980). Calmodulin plays a pivotal role in cellular regulation. Science 207, 19-2’7. DORBE, M. (1980). Calmodulin content does not change following hormone-induced meiosis reinitiation in starfish oocytes. Experientia 36, 932-933. DORBE, M., MOREAU, M., and GUERRIER, P. (1978). Hormonal control of meiosis: in vitro induced release of calcium ions from the plasma membrane. Exp. Cell Res. 115, 251-260. DOR$E, M., PICARDO,A., CAVADORE,J. C., LEPEUCH, C., and DEMAILLE, J. G. (1982). Calmodulin antagonists and hormonal control of meiosis in starfish oocytes. Exp. Cell Res. 139, 135-144. DUB& F., DUFRESNE-DUB& L., and GUERRIER, P. (1982). Sperm de-

Calmodulin

in

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Chaetopterus

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in Barnes con&do (Mollusca, Pelecypoda) oocytes does germinal vesicle breakdown. J. Ezp. ZOOL 221,383-387. R. (1981a). An ultrastructural analysis of cytoplasmic in Chadopterus pe-rgamentaceus. BioL Bull 160, 228-

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ECKBERG, W. R. (1981b). The effects of cytoskeleton inhibitors on cytoplasmic localization in Chuetqpterus pergamentacews. Di&rentiation

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ECKBERG, W. R., and CARROLL, A. G. (1982). Sequestered calcium triggers oocyte maturation in Chaetopterus. Cell Lh&+-ren 11, 155-160. ECKBERG, W. R., and KANG, Y.-H. (1981). A cytological analysis of differentiation without cleavage in cytochalasin B- and colchicinetreated embryos of Chaetopterus pggamentaceus. werentiation 19, 154-160.

EPEL, D. (1980). Ionic triggers in the fertilization Ann

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GOLDSTEIN, L. (1953). A study of the mechanism of activation and nuclear breakdown in the Chaetopterus egg. BioL Bull 105.87-102. HAGSTROM, B. E., and LBNNING, S. (1973). The sea urchin egg as a testing object in toxicology. Acta PharmacoL ToxicoL 32(Suppl. 1). l-49. HEAD, J. F., MADER, S., and KAMINER, B. (1979). Calcium-binding modulator protein from the unfertilized egg of the sea urchin Arbucia punctulata, J. Cell BioL 80, 211-218. HIDAKA, H., YAMAKI, T., NAKA, M., TANAKA, T., HAYASHI, H., and KOBAYASHI, R. (1980). Calcium-regulated modulator protein interacting agents inhibit smooth muscle calcium-stimulated protein kinase and ATPase. Mol. Pharnw.coL 17, 66-72. IKEGAMI, S., OKADA, T. S., and KOIDE, S. S. (1976). On the role of calcium ions in oocyte maturation in the polychaete Chaetqpterus pergamentaceus.

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JAFFE, L. F. (1980). Calcium explosions as triggers of development. Ann. N. Y. Acad Sci 339. 86-101. JAMIESON, G. A., and VANAMAN, T. C. (1979). Calcium-dependent affinity chromatography of calmodulin on an immobilized phenothiazine. B&hem.

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KAKIUCHI, S., SOBUE, K., YAMAZIKI, R., KAMBAYASHI, J., SAKON, M., and KOSAKI, G. (1981). Lack of tissue specificity of calmodulin: a rapid and high-yield purification method. FEBS Lett. 126,203-207. KLEE, C. B., CROUCH,T. H., and RICHMAN, P. G. (1980). Calmodulin. Annu. Rev. B&hem. 49,489-515. KOBAYASHI, R., TAWATA, M., and KIDAKA, H. (1979). Ca’+ regulated modulator protein interacting agents: inhibition of Ca’+-Mga+ATPase of human erythrocyte ghost. B&hem Biophys. Res. Cornmun. 88, 1037-1045. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685.

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6

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SCHMIDT, T., PATTON, C., and EPEL, D. (1982). Is there a role for the Ca2’ influx during fertilization of the sea urchin egg? Deu. BioL 90, 284-290. SHARMA, R. K., and WANG, J. H. (1979). Preparation and assay of the Cal+-dependent modulator protein. Advan C&k Nucl Res. 10,187198. STEINHARDT, R. A., and ALDERTON, J. M. (1982). Calmodulin confers calcium sensitivity on secretory exocytosis. Nature (London) 295, 154-155. STEINHARDT, R. A., EPEL, D., CARROLL, E. J., and YANAGIMACHI, R.

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(1974). Is calcium ionophore a universal activator for unfertilized eggs? Nature (London) 252,41-43. WASSERMAN, W. J., PINTO, L. H., O’CONNOR, C. M., and SMITH, L. D. (1980). Progesterone induces a rapid increase in Ca2+ of Xenopus laevis oocytes. Proc. Nat. Acad. Sci USA 77,1534-1536. WASSERMAN, W. J., and SMITH, L. D. (1981). Calmodulin triggers the resumption of meiosis in amphibian oocytes. J. Cell Biol. 89, 389394. WEISS, B., and LEVIN, R. M. (1978). Mechanism for selectively inhibiting the activation of cyclic nucleotide phosphodiesterase and adenylate cyclase by antipsychotic agents. Advan Cyclic NULL Res. 9, 285303.

WEISS, B., and WALLACE, J. L. (1989). Mechanisms and pharmacological implications of altering calmodulin activity. In “Calcium and Cell Function, Vol. I., Calmodulin” (W. Y. Cheung, ed.), pp. 329-379. Academic Press, New York.