Table Of ContentReference: Biol Bull 186: 254-262. (June. 1994)
Calcium Buffer Injections Inhibit Ooplasmic
Medaka
Segregation in Eggs
R. A. FLUCK.1 *. A. L. MILLER2, V. C. ABRAHAM1, AND L. F. JAFFE2
1Biology Department. Franklin andMarshall College, Lancaster. Pennsylvania 17604-3003, and
-Marine Biological Laboratory. Woods Hole, Massachusetts 02543
Abstract. Injection ofthe weak (KD = 1.5 pAl)calcium ofthe rhizoid of fucoid embryos (Berger and Brownlee,
buffer 5,5'-dibromo-BAPTA into fertilized medaka eggs 1993), near the tip ofgrowing pollen tubes (Miller et al.,
inhibited the formation of the blastodisc at the animal 1992b), near the vegetal pole offertilized Xenopits laevis
pole, the movement of oil droplets toward the vegetal eggs(Millerel al.. 1991), and neartheanimal and vegetal
pole, and cytokinesis. These inhibitory actions were de- poles of fertilized medaka eggs (Ruck et al., 1992b). In
pendent upon the concentration of the buffer but were medaka eggs, these zones are present throughout the pe-
independent offree [Ca:+] in the injectate. Because this riod ofooplasmic segregation (Fluck et al., 1992b).
bufferhas previously been shown to substantially suppress Regarding the second criterion that artificial induc-
zones ofelevated calcium at the animal and vegetal poles tion ofa cytosolic calcium gradient will organize devel-
ofthe medaka egg, the resultsofthe present study suggest opmental localization there is only indirect evidence.
that these zones are necessary for normal segregation of This evidence includes the occurrence oflocalization in
the ooplasm and its inclusions in the medaka egg. fucoid eggs(Robinson and Cone, 1980)andascidian eggs
(Jeffery, 1982; Bates and Jeffery, 1988) exposed to a gra-
dient ofthe calcium ionophore A23187. Other indirect
Introduction
evidence comes from the medaka egg, in which additional
Nearly 20 years ago, cytosolic calcium gradients were axes ofooplasmic segregation can be induced by "strong
postulated to organize developmental localization in eggs pricking" ofthe egg (Sakai, 1964), an action that might
ofplants and animals (Jaffe et al.. 1974; for reviews, see create a zone ofelevated cytosolic [Ca2+] at the wound.
Jaffe and Nuccitelli, 1977; and Jaffe, 1986). Proofofthis The present study addresses the third criterion that
theory should have three parts (Gilkey et al.. 1978): (1) dispersion ofa cytosolic calcium gradient will inhibit lo-
evidence of the existence ofcytosolic calcium gradients calization. We have pursued this question in the large
in systems undergoing localization; (2) evidence that ar- (diameter = 1.2 mm) and remarkably clear medaka fish
tificial induction ofacytosolic calcium gradient will cause egg. In this egg, which consists of a thin (about 25 /^m
localization; and (3)evidence that dispersion ofacytosolic thick) peripheral layerofcytoplasm bounded byaplasma
calcium gradient will inhibit localization. Evidence for membrane and separated from a large yolk vacuole by a
the existence ofcytosolic calcium gradients in eggs was yolk membrane, ooplasmic segregation consists of the
at first indirect, based on studies suggesting that, in de- roughly simultaneous streaming of the bulk of the oo-
veloping fucoid eggs, rhizoids grow in the direction of plasm and its inclusions toward the animal pole, the
high [Ca:+ (Robinson and Jaffe, 1975; Nuccitelli, 1978). movement of oil droplets toward the vegetal pole, and
]
Sincethen suchgradientshavebeen directlydemonstrated the saltatory movement of some inclusions toward the
in a number ofdeveloping systems in which zones ofel- vegetal pole (Sakai, 1965; Iwamatsu, 1973; Abraham et
evated cytosolic [Ca:+] have been identified: near the tip al.. 1993a). Duringand afterooplasmicsegregation, zones
ofelevatedcytosolic [Ca~+] arepresent at both theanimal
and vegetal poles ofthe egg (Fluck et al., 1992b).
*ReTcoeiwvehdom17cOocrtroebsepron1d9e9n3c;easchcoeupltdedbe10adMdarrescshed.1994. Injection ofthe calcium buffer 5,5' dibromo-BAPTA
Abbreviations: BCECF, 2,7-bis-(2-carboxyethyl)-5-(and 6-)-carboxy- (referred to hereafter as dibromo-BAPTA) substantially
tluorescein. dissipates these polar zones ofelevated [Ca:+] (Fluck et
254
CALCIUM AND OOPLASMIC SEGREGATION 255
a/.. 1992h). The buffer also blocks the development of Three types ofcontrols were used. First, each batch of
fucoid eggs(Speksnijderct a/.. 1989); suppresses high cal- eggs included at least one egg into which we injected no
cium zones in, and inhibits the growth of, pollen tubes fluids. A second type ofcontrol egg received 1.0-1.6 nl
(Millerctill.. 1992b); inhibitscell cyclecontraction waves of 5mA/ HEPES, pH 7.2. containing 50 or 100mA/
(Miller el at.. 1992a) and cytokinesis (Miller ct ul.. 1993; K:SOj or 125 mM KC1. A third type ofcontrol egg re-
Snow and Nuccitelli, 1993) in A", laevisembryos; and in- ceived 1.4nl of a solution of 5,5'-dimethyl-BAPTA, a
(hSibuiltls=ivAa",nlcilicuvl.i.x n1u99c3l)e.arThmiesmrbelraatinveelyvewseicalkecfaulsicoinum//;buvfifterro B=A0P.T1A5-mftiMyM;pe50bumffMer5w,i5t'h-adihmiegthheyrla-fBfAinPiTtAy,fotretcraalpcoituamss(iKumD
(KD 1.5 H.M; Pethig ct a/.. 1989) is believed to act as a salt; 5 HEPES, pH 7.2; and sufficient CaCl, to set
shuttle buffer, one that binds Ca2+ at the high end of a [Ca2+]free at 0.2
[Ca2+] gradient and releases it at the low end ofthe gra-
dient. In other words, the buffer facilitates the diffusion Dc1t'ctennining the amount ofbuffer to inject
ofCa2' within the cell. In the present study, we demon-
strate that injection of dibromo-BAPTA into fertilized According to facilitmateMd diffusion theory, a final con-
medaka eggs inhibits ooplasmic segregation. A prelimi- centration of about 2 dibromo-BAPTA in the oo-
nctaruly..ac1c9o92uan)t.ofthese findings has been published (Fluck pM[olCaraes2+om]vsienhro,tuhl2e.d7mibmecArs/oumfdfoiilcabierrnotrmatnoog-edBi(AsSsPpiTepkaAtsenhiazjsodnebereseelnoafls.eh.loe1wv9na8t9)et.do
Materials and Methods dissipate zones ofelevated [Ca2+] at the poles ofthe fer-
tilized medaka egg (Fluck et al.. 1992b). We thus mi-
Biological material croinjectedenough 50 mA/or 100 mA/dibromo-BAPTA
(YaMmeatmhootdos,fo1r96r7e;moKviircnhgegnoannaddsWefsrto,m1b9r7e6e;dRiuncgkm,ed19a7k8a; 0to.5a-c7h.i0evmeA/f.inaWlebuefsfteirmactoendcetnhtartatthieonascciensstihbeleooooppllaassmmiocf
(AusYbearma(ahAmbaormtaohct,aaml1.9.e6l71a;9/9.A.3ab)1r,9a93phara)em,paearlniadnlg..fee1rgt9gi9sl3ifazoi)rngheaexvgpegesrbie/m/e;ennvitdtaerl-o s0v.ho2ol6uw-mn1.et9wh6aatsnli2n7oj.fe6cbtnualftfe(eFrvl.uoclAkumenteusaml.bi.ne1rt9h9io2sfb)riananvngedsettihhgauavtseoirnsnjoehcadtveee-d
scribed previously. Gonads, gametes, and zygotes were tectable effect on the development of the medaka egg
placed in the same buffered saline solution used in other (Ridgway et al.. 1977; Gilkey, 1983; Yoshimoto et al..
studies of the fertilized medaka egg (Fluck el al.. 1991, 1985, 1986; Fluck etui.. 1991, 1992b); thus a purely me-
1992b; AbmraMham ct al.. 199m3aM): 1 1 1 mMNamClM; 5.37 mM csheaenmiscal effect ofthe injectate on development ofthe egg
KC1; 1.0 CaCl-,, 0.6 MgSO4; 5 HEPES, unlikely.
pH 7.3.
Microinjection
Preparation ofmicropipettes We usedalow-pressure method (Hiramoto, 1962; Kie-
Glass micropipettes (0.8-mm diameter, Drummond hart, 1982; Fluck et al.. 1991, 1992b) in Series I and a
high-pressure method (Narashige IM-200 microinjection
Smceinetnteidf.ic1C-om.,mBdrioaommeatlelr,, NPaernanssyhlivgaeniU.aS,.AS.e.riIensc.I,; Gorrefeinla-- system, Fluck et al.. 1992b) in Series II and III. In both
vale, New York, Series II and III)were madewith a Nara- methods, we front-loaded the micropipettes. The pipettes
spihpiegtetePsNw-e3remibcervoeelleedcattroadneapnugllleero.fT2h0eCtiwpisthofatNhaermaischriog-e winetroecvaelgiebtraabtleed boiylinajnedctimnegaassumrailnlgvothleumdeiaomfettheersoolfutitohne
EG-4 microgrinder; this process was monitored with an spherical droplets.
audio system (Miller el al.. 1993). Tip diameter was ap- Batchesoftwoto six eggswere fertilized simultaneously
proximately 5 nm at the widest part ofthe bevel. in buffered saline solution, transferred to closely fitting
holes drilled in a transparent plastic holder, and injected
Buffer solutions injected s3e0qumeinntiaalfltyerwfietrhtiflliuziadtiosno;on12a.f5ter f6er.t5ilmiiznat.ioXn (ranSgeD,,5N-
mM mM
Solutions of 50 (eggs in Series I-III) or 100 = 104eggs). Everybatch included at least onecontrol egg
(some eggs in Series I) dibromo-BAPTA (Molecular into which no fluid was injected.
PrmobMes, Eugene, Oregon; tetrapotassium salt). Cadi, and The method for injecting fluid into the thin peripheral
5 HEPES (Sigma Chemical Co., St. Louis. Missouri, layer of ooplasm ofthe medaka egg has been described
titrated to pH 7.2 with KOH) were prepared in which elsewhere (Ridgway et al.. 1977; Gilkey. 1983; Fluck et
[Ca2+]rreewas set at 0.2 pM(Series I), 0.14 //A/(Series II), ul., 1991) and will only be summarized here. The micro-
or0.3 nM(Series III). Thesevaluesof[Ca2+]frccarewithin pipette, held in a micromanipulator, was advanced far
therangereported fortheooplasm ofthefertilized medaka enough topenetratethechorion (shell)and plasma mem-
egg(Schantz, 1985). brane but not the yolk membrane, which roughly con-
256 R. A FLUCK ET AL.
Figure 1. Diffusion offluorescein around the medaka egg. The approximate position ofthe injection
siteismarked with anarrowhead. (A) By 5 min afterinjection ofthedye. it had spread more than 50 arc
in all directionsaround theegg. (B) By 18 min, itwas presentabout90 arc from the injection site. (C)At
74 min, it had spread tothe antipodeofthe injection site. Scale bar, 250^m.
formed to the shape ofthe micropipette. When fluid was where tn = 1.0 represents the time at which cytokinesis
injected intothe ooplasm, it distended theyolk membrane begins.
near the tip ofthe micropipette, thus visually confirming
that fluid had been injected into the ooplasm and not the Injection of fluorescein into eggs
yolk vacuole. As the pipette was slowly withdrawn, the
yolk membrane returned to its normal position, causing To estimate both the size of the area over which an
the injectate to be spread quickly over a large area ofthe injectatespreadsimmediatelyafterinjection andthetime
ooplasm. Fluid was injected either equatorially (within required for fluorescein to diffuse around the egg, we in-
3300 aarrcc oofftthheeveeqgueattaolrp)oloer).near the vegetal pole (within jseocltveedd i1n.51-02.00mnlMofK5:0SO/x4A./5flmuoMresHcEePinES(,sopdiHum7.2s;alSt,igdimsa-
Dibromo-BAPTA was injected into the yolk vacuole Chemical Co.) into eggs. Using epi-illumination and a
offoureggs. Sucheggswerepenetratedwitha micropipette SIT camera (Dage/MTI. model 66DX), we examined
in the usual manner, except thatthetipofthe pipettealso these eggs within 5 min after injection and at regular in-
penetrated theyolk membrane. Theinjectatethusdid not tervals thereafter for up to 3 h.
distendtheyolk membrane butdid transientlychangethe
optical properties ofthe yolk near the tip ofthe pipette, Results
confirming that fluid had been injected. Movement offluorescein around the egg
We injected dibromo-BAPTA into eggs in three series
ofexperiments: I (winter 1990, in which we successfully Within 5 min after injection, fluorescein was present
injected butter into the ooplasm of45 eggs from 12 fe- in ooplasm within 50 arc (or 530 /um. assuming an egg
males), II (winter 1992, 27 eggs from 4 females), and III diameter of 1 190 /um) in all directions from the injection
(summer 1992, 20 eggs from 4 females). In addition, we site (Fig. 1); this is equivalent to the molecule having
injected solutions ofKiSOj or KC1 into 15 eggs; injected spread initially into an area equal to 13% ofthe total sur-
dimethyl-BAPTA into 10eggsfrom two females; injected face ofthe egg and thus into a volume equal to 13% of
dibromo-BAPTA into the yolk vacuole of 4 eggs; and the total volume ofthe ooplasm. The molecule spread as
monitored thedevelopment of44 uninjected control eggs. faras90 arcin lessthan 15 minand reached theantipode
ofthe injection site in about 75 min.
Observation ofthe effects ofbuffer injection
Ooplasmic movements in at leastoneeggineach batch Immediate localizedeffects ofdibromo-BAPTA
were recorded bytime-lapse video microscopy (Abraham An immediate reaction ofthe eggs to injection ofdi-
et al., 1993a), while other eggs were observed at regular bromo-BAPTA was an apparent expansion of the yolk
intervalswith a stereomicroscope. Room temperature was membrane near the injection site, which caused the yolk
18-19C for Series I and II and 23-24C for Series III; vacuole to bulge into the ooplasm (Fig. 2A). This bulge
at these temperatures, the first cleavage begins, respec- usually subsided within 10 min in eggs receiving low
tively, 130-140 min and 85-95 min after fertilization. In concentrations of buffer (final cytosolic concentration
ordertocomparedata from experimentsatdifferenttem- < 4 mM}. But in some of these eggs and in most eggs
peratures, we have reported our results not only as "min- receiving higher concentrations ofbuffer, the bulge per-
utesafterfertilization" but alsoastn. ornormalized time. sisted andthe yolk membrane overthebulgelysed (Table
CALCIUM AND OOPLASM1C SEGREGATION 257
I), releasing yolk into the ooplasm or, more often, into concentrations > 2.6 mMinhibited the movement ofoil
the perivitelline space. To determine whether [dibromo- droplets toward the vegetal pole and slowed the growth
BAPTA] and [Ca2+]free significantly affected egg lysis, the oftheblastodisc(Table I; Fig. 2C, E-G). In addition, small
probability ofegg lysis was analyzed using a generalized aggregatesofooplasm often formed outsidetheblastodisc
additive model with [Ca2+]trec as a factor variable and a proper and on the side ofthe egg into which buffer had
slightly nonlinear, nonparametric relationship, with 1.5 been injected (Fig. 2E, G). The specificeffectsofthebuffer
degrees offreedom (intermediate between a linear and a on oil droplet movement varied with the siteofinjection.
quadratic relationship) between final buffer concentration Injection near the equator strongly inhibited oil droplet
and the probability oflysis (function gam ofthe S-PLUS movementthroughoutthecontralateral halfoftheembryo
statistical package, S-PLUS Reference Manual, Version and caused the retention ofoil droplets within the blas-
3.0, Statistical Services, Inc., Seattle, Washington). An todisc at the animal pole (Fig. 2C, E, G). Injection near
analysis ofvariance indicated that P values for the effect thevegetal poleresultedintheaccumulation ofoildroplets
of [Ca2+]rrcc and [dibromo-BAPTA] on lysis were 0.012 neartheequatoroftheegg(Fig. 2F), theresultofdroplets
and 3.4 X 10~7, respectively. in the vegetal hemisphere having floated to the top ofthe
Another immediate response to the injection of di- egg and those in the animal hemisphere having moved
bromo-BAPTA (final cytosolic concentration > 1 mM). normally toward the equator.
which made a convenient marker of the injection site,
was the movement ofnearby oil droplets away from the Effects ofdibromo-BAPTA on cytokinesis and
injection site and toward the top ofthe egg (Figs. IB, C); embryonic axis formation
Tthheisdrmooplveetmseanptpebaergeadnsiimmmpelyditaotfelloyatutpootnheintjoepctoifotnhoefetghg.e Dibromo-BAPTA delayed or blocked cytokinesis in a
bpuafrfiesrona,ntdhewansoremsaselntmioalvleymoevnetr woiftohiilndr1o5plmeitns.toFwoarrcdotmh-e gcornocuepntorfaetgigosn-defpreonmdetnhtemsaanmneerf.emFaolreeaxanmdplien,jewctheednwointeh
differing amounts ofbuffer within a few minutes ofeach
vegetal pole during ooplasmic segregation continues for
more than 140 min at 18-19C and more than 95 min other were examined 170 min after fertilization (18-
at 23-24C (Sakai, 1965; Abraham et al., 1993a). These 19C), the eggs had developed to the following stamgeMs:
immediate localized responses were not due to the me- uninjected control, hadcompleted firstcleavage:0m.6M4
chanical effects ofinjection because we saw no such re- dibromo-BAPTA. was similar to control; 1.1 di-
sMpoornesoevseri,n tchoentyrowlereegglsessrepcreiovmiinngeenitthienreKgCgs1ionrtoKw2hSiOc4h. b2.r0ommoM-BdAiPbTrAo,mow-aBsAPfToAr,mihnagdtnwotobuengeuqnuacyltobkliansetsiosm.erTehse;
we injected dimethyl-BAPTA. Instead, these early re- last egg in this group formed two incomplete furrows by
4.5 h after fertilization but did not go on to form an em-
csepnotnrsaetsiowneroefldikieblyroamroe-sBpAonPsTeAtonetahre tinhietiailnljyechtiigohn csoitne-. abrmyoounnitcsaxoifs,buwfhfeerredaisd tfhoermtwaon eegmgbsrythoanticreacxeiisv.edThleowienr-
toAihnosjepseluciatnmiesitdminagflilstuc2hooa7rnt.ecs6e(cn1entl)irni(anFatjleinucodctnek(d2oe)fbtutabfhluf.ef,efrae1cir9cn9eii2stnibsa)itlb,lhlyeewseovpoorcpelaalundameseasmstoiffsamoratothanees c2hei4ibivetigingogsnb(uoSffefrceiyretscookIniacnneedsnitsrIIa)wtariesocnemsiov>rine2g.p62r.mo6Mn-3o..u6nFcomerdMexidanimepbglrgeos,mrooe--f
BAPTA, 9 did not divide and the other 12 divided from
ac5fo0tnemcrenMitnrjdeactitibioornno.omfFoo-2r.B7AePmxTaMmApltteoh,raociuhngiheeovgugetsartnehceeesiotvoiipmnlagatseedmn,ofuitnghahel 4of0emgigns rteocmeiovriengt>ha4nm3Mh abfutfefrert,hencoonnetruonlsdedriwd;enwthecrmyetaMos-
winoiutiladl cboencaebnoturtat2ionmwMithin 50 arc ofthe injection site dkiinbesriosm(oT-abBlAePIT;AFigd.id2En-oGt).foErmmbarnyoesmbrrecyeoinviicnga>xi2s..6
1
Controls
Effects ofdibromo-BAPTA on ooplasmic segregation Eggsinjectedwith KC1(125 mM, 6embryos)orK2SO4
Dibromo-BAPTA inhibitedooplasmicsegregation and (50 mM, 6 embryos; 100 mM. 3 embryos) neareitherthe
thesubsequent development ofthe embryos in aconcen- equator (12 embryos) or the vegetal pole (3 embryos) de-
tration-dependent manner (Table I and Fig. 2B-G), and veloped normally. There was neither an immediate re-
theseeffectswereindependent of[Ca2+]freein theinjectate. sponsetotheinjectate noranapparenteffecton ooplasmic
Except for causing the early movement of oil droplets segregation, cell division, or formation ofthe embryonic
(described above) and a slight delay in the onset ofcy- axis. Moreover, injecting dibromo-BAPTA into the yolk
tokinesis (see below), low concentrations of dibromo- vacuole had no apparent effect on the embryos.
BAPTA (<2.0 mM) had no substantial effect on either In contrast to the pronounced effects of >2.6 mM
mM
the formation of the blastodisc or the movement of oil dibromo-BAPTA on segregation, the effects of2.6
dropletstowardthevegetal pole(Fig. 2H). However, buffer dimethyl-BAPTA were slight or apparently absent. As
258 R. A. FLUCK /;/' II.
Figure 2. Effect ofdibromo-BAPTA on segregation and cytokinesis in the medakaegg. Abbreviations:
AP. animal pole; VP. vegetal pole. (A) Immediate local response to injection of2.6 m.l/dibromo-BAPTA
(free[Ca:*] =0.2nM).Thisphotographwastaken7 minafterinjectingenoughdibromo-BAPTAtoachieve
a final concentration of2.6m\l in the ooplasm. The egg is still in the injection chamber, the channelsof
which can be seen at the lower left and upper right (*). Note the bulge (white arrowhead) in the egg near
the injection she. Thedark massnearthecenteroftheimage isan accumulation ofoildropletsonthetop
oftheegg.thatis. toward the viewer. Thebulgeeventuallysubsided, butafter 177 min,whenan uninjected
controleggwasatthefour-cell stage,thisegghadnotyetdivided. Scalebar, 250pM. (B)Segregation in an
CALCIUM AND OOPLASMIC SEGREGATION 259
Table
I
Inhibition ofooplasmicsegregation andcytokinesis in themedaka eggbytheinjection of5,5'-dibromo-BAPTA
Numberofembryos
[FreeCa2+]
MA/a
260 R. A. FLUCK ET AL
to form an embryonic axis and appeared to develop the range 0.14-0.30 ^M, with the probability oflysis in-
normally. creasingwith [Ca:+]. Ourchoice of[Ca2+] in the injectate
wasbased primarilyon astudy ofthemedakaegginwhich
Discussion cytosolic [Ca2*] was measured with a calcium microelec-
trode (Schantz, 1985; see his Table 1). The occurrence of
The final ooplasmic concentration ofdibromo-BAPTA lysis in ourstudy suggeststhat in the future we should set
required to block ooplasmic segregation and cell division [Ca2+] intheinjectate lower, perhapsto 50 nvl/or 100 nM,
in the medaka embryo (ca. 2.6 mA/) was comparable to to reduce the probability ofembryo lysis.
that required to inhibit cell division in fucoid eggs (Spek- Two cytoskeletal systems one involving microfila-
snijder el ai, 1989), inhibit cell cycle contraction waves ments and another involving microtubules are involved
and cytokinesis in A", laevis eggs (Miller cl ai, 1992a; in ooplasmic segregation in the medaka egg. Formation
Miller et ai, 1993; Snow and Nuccitelli, 1993), and dis- of the blastodisc at the animal pole ofthe medaka and
sipate cytosolic calcium gradients near the animal and otherteleostembryosisinhibitedbycytochalasins(Katow,
vegetal poles ofthe medaka egg(Fluck et at.. 1992b). Ac- 1983;Ivanenkovf/rv/.. 1987;Webband Fluck, 1993)and
cording to facilitated diffusion theory, the primary effect DNase I (Ivanenkov et ai, 1987) and thus presumably
ofsuch buffers is to dissipate cytosolic Ca2* gradients by involves theaction ofmicrofilaments, whereas the move-
facilitating the diffusion ofCa2+ away from zones ofel- ment ofoil droplets and the saltatory movement ofother
evated cytosolic [Ca:+]. Thus, when high calcium regions inclusionstowardtheanimal and vegetal polesoftheme-
begin to emerge within a cell, as they do at the animal daka egg are both inhibited by microtubule poisons
andvegetal polesofthe fertilized medakaegg, buffermol- colchicine, colcemid, nocodazole and thus presumably
ecules pick up Ca:f there and diffuse to low calcium re- involve microtubules (Abraham et ai. 1993a; Webb and
gions, where the bound Ca2' is then released. The results Fluck, 1993). Moreover, amicrotubule-organizing center
of the present study suggest that these polar zones are (MTOC) is present in the animal pole region ofthe me-
necessary for the normal segregation ofooplasm and its daka egg by tr, = 0.24, and the convergent array of mi-
inclusions in thisegg. However, it istheoretically possible crotubules near this center is disrupted by the injection
that the inhibition of segregation is caused by either an ofdibromo-BAPTA (Abraham el ai, I993b). Theassem-
increase in [Ca:' at sites away from the poles or the dis- bly/disassembly and the function ofboth microfilaments
]
sipation ofthe cytosolic calcium gradients. and microtubulesarecontrolled by a numberofcalcium-
Facilitated diffusion theory predictsthat theCa:+ buffer binding regulatory proteins (Weisenberg, 1972; Schliwa
will be relatively ineffectiveasashuttlebufferifitsaffinity et ai, 1981; Keith el ai. 1983; Mooseker, 1985; Stossel
for Ca2+ is too high. Such a buffer, for example 5,5'-di- et til-. 1985; Bray. 1992. pp. 147-148. 342). Facilitated
methyl-BAPTA (KD = 0.15 n.M), will become saturated diffusion theory predicts that calcium buffer concentra-
with Ca2+ at the high end of the Ca2+ gradient but will tions similar to those found effective in the present study
not release Ca2+ when it diffuses away from this region. act by dissipating zones ofcytosolic [Ca2+]frec in the mi-
Theresultsofthreestudies, which have reporteddimethyl- cromolar range (Speksnijder et ai. 1989). An example of
BAPTA to be from 2.7- to 18-fold less effective than di- a protein that is responsive to changes in [Ca2+]free in this
bromo-BAPTA (Speksnijder et ai. 1989: Snowand Nuc- range is calmodulin (Cheung, 1980), a protein that reg-
citelli, 1993;Sullivan et ai. 1993), areconsistent with this ulates the activity ofproteins that interact with both mi-
prediction. The results ofthe present study, thatdimethyl- crofilaments (Wolenski et ai, 1993) and microtubules
BAPTA had far less effect on ooplasmic segregation and (Ishikawa et ai. 1992).
cytokinesis in the medaka than did dibromo-BAPTA, are Oil droplet movement in the animal hemisphere ofthe
consistent with these three previous studies and with this eggwas more strongly inhibited when bufferwas injected
prediction ofthe theory. near the equator versus near the vegetal pole. One inter-
Facilitated diffusion theory also predicts that the effect pretation ofthese results is that structures/events in the
ofa shuttle buffer should be independent of[Ca2+ in the animal pole region, forexample the MTOC. are essential
]
injectate (Speksnijder et ai, 1989). In other words, the for oil droplet movement and that these are disrupted by
effect ofthe buffer is not to clamp cytosolic [Ca2+ at any dibromo-BAPTA. Given thecircumferenceofthe medaka
]
particular value, but ratherto facilitate calcium diffusion egg (about 3700 /urn), buffer injected near the equator
and thus dissipate calcium gradients in the cytoplasm. would reach theanimal poleoftheeggsoonerthan buffer
Our data are also consistent with this aspect oftheory, at injected near the vegetal pole. To estimate the time re-
least as far as effects on ooplasmic segregation and cyto- quired fordibromo-BAPTA todiffuse around the medaka
kinesis are concerned. However, we did find that imme- egg,we used published valuesforthediffusion coefficients
diatelocalized responsestothe injectate herniation and of molecules similar to dibromo-BAPTA (fura-2 in
subsequent lysis, the movement ofoil droplets to the top skeletal muscle. 3.6 x 10~7 cm2 s~', Baylor and Holling-
oftheegg werea function of[Ca24] in the injectate over worth, 1988) and fluorescein (BCECF in fibroblasts. 2
CALCIUM AND OOPLASMIC SEGREGATION 261
X 10 6 cm2 s"1, Kao et a/., 1993). Assuming that the cy- published) suggest that it will besuccessful in the medaka
toplasm of the medaka egg is more similar to the cyto- as well.
plasm ofa fibroblast than to that ofa skeletal muscle, we
corrected the diffusion constant offura-2 upward to 5.9 Acknowledgments
X 1(T7 cm2 s"1 (Kushmerick and Podolsky, 1969; Kao et
nl.. 1993) and then estimated the times required formol- Supported by NSF DCB-9017210 to RAF, NSF DCB-
ecules with these diffusion coefficients to diffuse around 9103569 to LFJ, NSF BIR 921 1855 to LFJ and ALM,
theegg(Einstein. 1956). Thepredicted values for BCECF and by grants from Franklin and Marshall College's
were consistent with the rate ofmovement offluorescein Hackman Scholars Program and Harry W. and Mary B.
around the egg (Fig. 1), suggesting that our assumptions Huffnagle Fund to VGA. We thank Albert Gough and
arevalid. Weestimatethatthetimesrequired fordibromo- Rick Moog for valuable discussions about diffusion, Tim
BAPTA to diffuse 90 and 180 arc from the injection Hesterberg for his help in statistical analysis, Jane Mc-
site would thus be about 35 min and >3 h, respectively. Laughlin for technical assistance, George Rosenstein for
In otherwords, buffer injected nearthe equator ofan egg his aid in geometry, and Tamika Webb for helping to
would reach the animal pole while segregation ofthe oil improve the text ofthe manuscript.
dropletswasongoing, but buffer injected nearthe vegetal
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