Table Of ContentReference: Blot Bull, 188: 136-14? IApril. 1945)
Microtubule
Arrays During Ooplasmic Segregation
Medaka
in the Fish Egg (Oryzias latipes)
V. C. ABRAHAM1, A. L. MILLER2. AND R. A. FLUCK1,*
^Biology Department. Franklin and Marshall College. Lancaster, Pennsylvania 17604-3003: ami
2,Marine Biological Laboratory. Woods Hole. Massachusetts (12543
We
Abstract. used indirect immunofluorescence to Introduction
study microtuhule arrays in the medaka egg between fer-
tilization (normalized time. T,,, = 0)andthefirstcleavage Ooplasmic segregation in the medaka egg consists of
(Tn = 1.0). Eggs were fixed at various times after fertil- the approximately simultaneous streaming of ooplasm
ization andexamined with conventional fluorescence mi- toward theanimal pole, the saltatory movement ofparcels
croscopy, laserscanning confocal microscopy, and three- toward the animal pole and vegetal pole, and the move-
dimensional fluorescence microscopy. Soon aftertheeggs ment ofoil droplets toward the vegetal pole (Abraham el
were fertilized (T,, = 0.02). we saw microtiibules oriented ul.. 1993a; Webband Fluck. 1993). These movementsare
perpendicular to the plane ofthe plasma membrane but roughly simultaneous and are essentially completed dur-
wneonseapwaraalnlealrtroaythoefpmliacsrmoatumbeumlbersanorei.enLtaetderm(oTr,,e=o0r.0l8e)s,s ainngimtahle pfiorlsteciesllinchyicbliet.edStbryeaemyitoncghaolfaosionplDasamndtotwhaursdptrhe-e
parallel to the plasma membrane but having no apparent sumably requires microfilaments(Webband Fluck. 1993),
preferred orientation with respect to the animal-vegetal whereas saltatory movements and the movement of oil
axis ofthe egg. In the interpolar regions ofthe egg, this droplets toward the vegetal pole are both inhibited by
network increased in density by T,, = 0.24 and remained microtubule poisonsand thus presumably require micro-
a constant feature ofthe ooplasm until the first cleavage. tubules (Abraham el a/.. 1993a). All ofthese movements
From Tn = 0.30 to 0.76 the polar regions ofthe egg con- can be easily observed in the optically clear medaka egg,
tained dense arrays of organized microtubules. At the in which a thin (^15 ^m thick) peripheral layer of oo-
animal pole, microtubules radiated from a site near the plasm surrounds a large, central yolk vacuole.
pronuclei; while at the vegetal pole, an array of parallel In a number of diverse animals, including annelids
microtubules was present. Injection of the weak (K.,, (Astrow el ai. 1989). ctenophores (Houliston ct al.. 1993).
= 1.5 nl\I) calcium buffer 5,5'-dibromo-BAPTA disrupted echmoderms (Harris el al., 1980), ascidians (Sawada and
the radial pattern of microtubules near the animal pole Schatten. 1988). and amphibians (Elinson and Rowning,
but had no apparent effect on the parallel array ofmicro- 1988: Houliston and Elinson. 1991; Schroederand Card.
tubules nearthe vegetal pole. Because this buffer has pre- 1992; Elinson and Palacek. 1993). a network of micro-
viouslybeen showntosuppressazoneofelevatedcytosolic tubules that forms during the first cell cycle is required
calcium at the animal pole and to disrupt ooplasmic seg- forthe movement ofspecificcomponentsoftheooplasm.
regation in this egg, the results of the present study ( I ) includingthe pronuclei. In the present study, wedescribe
are consistent with a model in which microtubules are thedevelopment ofsuch an array ofmicrotubulesduring
required for ooplasmic segregation in the medaka egg, the first cell cycle ofthe medaka egg. Previous studies of
and(2)suggest that the normal function ofa microtubule- microtubules in teleost embryoseither have provided very
organizing center at the animal pole ofthe egg requires a little information (Beams el al.. 1985) or have been done
zone ofelevated calcium. on later stages ofdevelopment (Strahle and Jesuthasan,
1993; Solnica-Kre/el and Driever. 1994).
We
Received 2(1June 1994; accepted II Januan, 1995. also pursued the hypothesis that gradients of cy-
* Towhom correspondence should he addressed tosolic free Ca:< organize the multimolecular assemblies
136
MICROTUBULES IN THE MEDAKA EGG 137
involved in ooplasmic segregation in the medaka egg. with TBS. We performed two kinds ofantibody controls:
Zones ofelevated calcium are present at the animal and omission ofthe primary antibody and omission ofboth
vegetal polesofthe medakaeggduringsegregation (Fluck the primary and the secondary antibodies. To stain the
el a/., 1992). Moreover, injection of5.5'-dibromo-BAPTA nuclei, we incubated the eggs for 30 min in a solution of
(hereafterreferredtoasdihromo-BAPTA), aweakcalcium Hoechst 33258 ( 10 /ug ml ') in TBS.
buffer that dissipates cytosolic calcium gradients (Pethig The eggs were mounted between a coverglass and a
ct nl.. 1989; Speksnijder et at.. 1989; Fluck ct <//., 1992), microscope slideasdescribed previously (Abraham ct til .
inhibits ooplasmic segregation in this egg (Fluck ct ill . 1993a) and examined by one ofthree methods. Conven-
1994). In the present study we monitored the effects of tional epifluorescence microscopy was performed with a
this bufferon the spatiotemporal pattern ofmicrotubules Nikon Optiphot microscope coupled to a Dage-MTI SIT
and on pronuclear movements. A preliminary account of camera and video monitor; in later replicates, the SIT
these findings hasbeen published (Abraham ct ill.. 1993b). camera was coupled to a Dage-MTI DSP-2000 image
processor. In either case, the image on the monitor was
Materials and Methods photographed through a Ronchi grating (Rolyn Optics
Co.. Covina. California: Inoue, 1981). Some eggs were
Procedures
examinedwithalaserscanningconfocal microscope(Zeiss
Methods for removing gonads from breeding medaka LSCM 410) at the Marine Biological Laboratory.
and forthe in vitrofertilization ofeggshavebeen described We also acquired and processed images at the Science
previously (Abraham ct <//.. 1993a). Some eggs were in- and Technology Center at Carnegie-Mellon University.
cubated in 100 n\lcolchicine (dissolved in buffered saline Imagesateach focal plane wereacquired on a multimode
mM
s0to.el6muptmeiroMna:tuMr1ge1S1(Om24.0;U-52Nma5CMCl);HbE5e.Pf3Eo7rSe,tpheHyKC7w.1e3;r)ef1o.f0rermt1i.lhiMzaetCd.arColo:m: sbmuoirncgrchoh.sipcP,oepncenoosly(elBdvioaClnoiCgaiD)caeclqaumDieeprtpaeec(dtPiwhoiontthomSaeyt5srt7ie6emssX,L3td8I.4n.c.TT,hucoPsimotptns-,-
Using a high-pressure microinjection system, we in- Arizona). Three-dimensional fluorescence microscopy
mM
jected enough 50 dibromo-BAPTA (tetrapotassium used the nearest neighbor algorithm to correct forout-of-
mM
salt: containing 5 HEPES, pH 7.2, and sufficient focus fluorescence (Biological Detection Systems, Inc.)
cCoanCcle2nttroatsieton[Ctao22+.]7fri,mc aMt (1F0l0unc.k\fc)ttaoi,ra1is9e92)t.heIncjyetcotsioolnisc wTihtehftinhaelptrhroeger-admimAenNsAioLnaYlZrEepr(eBsieonmteadtiicoanlwaIsmaggeinnegraRteed-
were made within 10 arc toward the vegetal pole from source, Mayo Foundation, Rochester, Minnesota) on the
ONYX
the equator within 6 min after fertilization. Control eggs SGI. Inc.. workstation.
received a comparable volume (ca. 1.5 nl) of 150m/U The results summarized herein represent 1 1 replicate
KG. 5 m.U HEPES. pH 7.2. experiments in which we fixed eggs at various intervals
The procedures for indirect immunofluorescence were after fertilization and three replicate experiments in which
those developed by Card (1991) for the study of micro- we examined the effects ofdibromo-BAPTA on micro-
tubules in Xcnopus lucvis eggs. At regular intervals after tubules. In all, we have examined a total of 1 17 eggs from
fertilization, eggs were transferred to fixative solution at 17 females, including 20 eggs into which we injected di-
room temperature (3.7% formaldehyde, 0.25% glutaral- bromo-BAPTA and 8 eggs into which we injected KC1.
dehymdMe,0.2% Triton X-100. 5 nWEGTA, 1 mA/MgCU,
80 potassium PIPES. pH 6.8). After 4 h. the eggs Chemicals
were dechorionated with fine forceps and post-fixed in Formaldehyde and glutaraldehyde were obtained from
absolute methanol (-20C)overnight. The eggswerethen Electron Microscopy Sciences (Fort Washington, Penn-
wNaaCslh;ed2 mwitMh KpCh1o;sp8hamtMe-NbuaffHer:ePdO4sa;l2inme.(MPBKSH::P12O84,mp.HU sdryildvea.niab)o:viTnreitsonerXu-m10a0l.buNmoinni.deHtoePc-h4s0t, s3o32d5i8u,mabnodrochoyl--
7.2) and incubated in 100 m.M sodium borohydride (in chicine from Sigma (St. Louis. Missouri); 5.5'-dibromo-
PBS) for 6 h. The eggs were then washedmwMith cold Tris- BAPTA from Molecular Probes (Eugene. Oregon); anti-
bs7l.ui4fd;tee0s.re(1d%ThsNaolominnaiesd(eSTtcBiSPe-:n4t0i1f)5i;5c,trmaSAnw/sefNdeaersCrbelod;rtoo1.0LaNbe-wTeTJkreircss-heCyaI)m.;bpienHr- Oratnr-dgtaubnruholonindaTameniktnnieib-kocadoyn(jMfuarglovametreInd,CNgPoea(ntCnossaytnlavtaiMn-iemaso)au.,seCaIlgifGornfirao)m:
cubated with a monoclonal mouse anti--tubulin anti-
body (ICN, DM1A; diluted 1:250 with TBS containing
Results
2% bovine serum albumin); washed with TBS for 24 h;
incubated with the secondary antibody (rhodamine-con- The various events that constitute ooplasmic segrega-
jugated goat anti-mouse IgG. diluted 1:25 with TBS con- tion in the fertilized medaka egg have already been de-
taining 2%> bovine serum albumin): and washed for 24 h scribed in detail (Abraham ct ii/.. 1993a) and are only
ABRAHAM
138 V. C. 1:1 II.
summarized below. To indicate the relative temporal po- movements, and oil droplet movement a network of
sition of these events, we have used a normalized time microtubules was present throughout the ooplasm, and
(7",,) scale in which the time between fertilization and the the network in the equatorial region was denser than at
beginning ofcytokinesis is 1 unit. In the range of room 7",, =t 0.08 (Fig. IE). The network did not yet appear to
temperatures in which the experiments were done (20- have a preferred orientation.
25C). 1 unit of normalized time corresponds to 75- To test for autofluorescence. we examined fixed eggs
1 10 min. Fertilization in the medaka egg is followed by that were incubated with noantibodiesandthatwerepro-
acorticalgranulereactionandacontraction, duringwhich cessed for viewing up to the first wash with TBS. We saw
the ooplasm and its contents appear to be pulled toward no fluorescence at all in these eggs (data not shown). We
theanimal pole. Thesecond meioticdivision iscompleted alsoexaminedeggsthatwere incubated withthesecondary
by T,, = 0.12 and is followed clo~sely by a second animal- antibody but not with the primary antibody. In these eggs.
pole-directed contraction at Tn 0.15. This second con- we saw difluse fluorescence but no linear elements or
traction heralds the beginning of ooplasmic segregation punctate sources offluorescence (Fig. IF). In eggs incu-
in the form ofstreaming ofooplasm toward the animal bated with 100 JJ.M colchicine before fixation and incu-
pole, saltatory movements of parcels toward both polar bated with both the primary and secondary antibodies,
regions,and movementofoildroplets(aclassofooplasmic we saw diffuse fluorescence but no microtubules or any
inclusions) ofvarious sizes toward the vegetal pole. At Tn punctate sources offluorescence (data not shown).
= 1.0. theblastodiscdividesintotwoblastomeres. Bythis The pattern ofmicrotubules in eggs fixed at Tn = 0.16
timetheoil dropletshave formed acrude ringaround the is summarized in Fig. 3A.
vegetal pole. Tn = 0.24. 0.3, 0.4. 0.76. In ooplasm nearthe equator,
the networkofmicrotubuleswasdenserthan at T,, = 0.16
Microtubule network dyiuimic.\ during ooplasmic (Fig. 2A; compare with Fig. IE) but still showed no pre-
segregation ferred orientation with respect to the animal-vegetal axis.
We identified three regions ofthe medaka egg on the In contrast, oriented microtubule networks were present
basis of the spatiotemporal pattern of microtubules in near the vegetal pole and animal pole by T,, = 0.24 and
them (Figs. 1-3): (1) a region within 30 (=300//m) arc T,, = 0.3. respectively. Near the vegetal pole, an array of
of the animal pole: (2) a region within 60 (sa600^m) (mostly) parallel microtubuleswas present (Fig. 2B). This
arc on both sides ofthe equator: and (3) a region within "vegetal mat" extended about 30 arc (300 A<m) in all
30 (=300 ^m) arc ofthe vegetal pole. In all regions of directions from the vegetal pole and covered at least 30%
the egg except the animal pole, all the microtubules were ofthe area ofthe vegetal hemisphere. The orientation of
within 2-3 ftm ofthe surface ofthe egg and were visible microtubules was uniform throughout the vegetal mat.
in a single optical section. Beyond theedgesofthe mat, the networkofmicrotubules
Tn = 0.02. O.OS. ami 0.16. The earliest time at which abruptly lost its orientation and became the network
we fixed eggs was Tn = 0.02; that is, immediately after characteristic ofinterpolar ooplasm. Using the z-section-
the contraction that follows the cortical granule reaction. ing function of the LSCM 410, we determined that the
Though we searched the ooplasm everywhere on these thickness ofthe microtubule network in the vegetal mat
eggs (and all ofthe ooplasm is peripheral, see Introduc- ( =2.7 ^/m)wasthesameasthethicknessoftheunoriented
tion), we saw no microtubules parallel to the surface of microtubule network just outside the mat. The two net-
theeggbut saw instead a punctate pattern offluorescence works appeared to be continuous with each other and
(Fig. 1A). Analysisoftheseeggsby three-dimensional flu- were in the same optical section, suggesting that the two
orescence microscopy suggests that the sources of fluo- networks are located at the same depth in the ooplasm.
rescencewere microtubulesoriented perpendiculartothe Neartheanimal pole, microtubuleswere oriented more
surface ofthe egg (Fig. IB). This punctate pattern was a or less along meridian linesand appeared to radiate from
prominent feature ofeggs fixed at 7",, = 0.02 and0.08 and a microtubule-organizing center nearthe maleand female
=
was less prominent at later stages. pronuclei, which were 38.6 6.7 i/rn (X SD, /; 5)
In eggs fixed at T,, = 0.08,wesawaverysparse network below the surface ofthe blastodisc at T,, = 0.76 (Fig. 2D-
ofmicrotubules oriented parallel to the surface ofthe egg I). This network of oriented microtubules extended ap-
but havingnoapparent preferred orientation with respect proximately 30 arc (300 /urn) in all directions from the
to the animal-vegetal axis ofthe egg. This network was animal pole.
roughly confined to the animal hemisphere, and we saw The pattern of microtubule distribution in eggs fixed
no microtubules in most ofthe vegetal hemisphere (Fig. at T,, = 0.30-0.76 is summarized in Fig. 3B.
1C, D). Tn = 1.0. In these eggs, the blastodisc has begun to
In eggs fixed at T,, = 0.16 by which time ooplasmic divide into twoblastomeres. and essentiallyall oil droplets
segregation has begun in the form ofstreaming, saltatory haveaggregated in a crude ring nearthe vegetal pole. Each
M1CROTUBULES IN THE MEDAKA EGG 139
Figure 1. Formation ofanetworkofmicrotubulesinthe fertilizedegg. A,C, D, E,andFwereobtained
by conventional epifluorescence microscopy and B by three-dimensional fluorescence microscopy. Scale
bars, lOfim. A, C, D, and E are printed at the same magnification, B at another, and F at another. (A)
Ooplasm near the equator ofan egg fixed at Tn = 0.02. Characteristic ofthis stage ofdevelopment are a
punctate pattern offluorescence throughout the ooplasm and the absence ofunambiguous microtubular
elementsorientedparalleltotheplasmamembrane.Thedarkstructures(arrowheads)areooplasmicinclusions.
(B) Three-dimensional rendering ofoptical sections of ooplasm near the equator at Tn = 0.024. Using a
lOOx objective lens, we made 33 optical sectionsat stepsof0.158nm. beginningatthesurfaceoftheegg.
Visiblein this reconstruction are both the punctate pattern at the surfaceoftheegg(the topofthe image)
andlinearelementsthatareoriented perpendiculartothesurfaceoftheegg(arrowheads). Twoedgesofthe
reconstruction are marked byawhitedashed line. (C) Ooplasm neartheanimal poleofan egg fixed at ?
= 0.08. Note the presence ofunambiguous microtubules in the field (arrowheads). (D) Ooplasm nearthe
vegetal pole ofan egg fixed at /" = 0.08. This image was obtained from the same egg as Fig. 2C. No
unambiguous microtubules oriented parallel to the plasma membrane can be seen. (E) Ooplasm nearthe
equator ofan egg fixed at Tn = 0.16. The animal-vegetal axis runs from lower left to upper right. This
unoriented network isdenserthan that found at theequatorofan eggfixed at Tn = 0.08 (not shown). (F)
In eggs not incubated with the primary antibody after fixation (T,, = 0.4), we saw only weak and diffuse
fluorescencebutsawneitheranyfilamentousstructuresnorapunctatepatternoffluorescence.Theseimages
wereobtainedbystandardepifluorescence microscopy.
Figure 2. Microtuhule arrays in eggs fixed at Ta = 0.4-1.0. All imageswere obtained by conventional
epifluorescence microscopy. Scale bars: A, 10^m; D, 25 ^m. A-Cwere printed at one magnification. D-I
atanother.(A)Equatorialooplasm ofaneggfixedat Tn =0.4.Theanimal-vegetalaxisrunsfrom lowerleft
toupper right. The microtubule network here isdenserthan in eggsfixed at Tn = 0.16(compare with Fig.
1C)and still has no apparent preferred orientation. (B) The vegetal mat ofparallel microtubules in an egg
fixedat Tn = 0.4. Practicallyall the microtubulesshareasingleorientation in this mat, which wesawat Tn
= 0.24-0.76. (C) Ooplasm at the vegetal pole ofan egg fixed at Tn = 1.0. The parallel organization of
microtubules near the vegetal pole isgone. (D and E) The same microscopic field ofadouble-stained egg
140
M1CROTUBULES IN THE MEDAKA EGG 141
B
Tn = 0.16 Tn = 0.4 Blastomere Tn =1.0
MTOCs
MTOC
Animal
Hemisphere
Equator=
Oil Vegetal
Droplet Hemisphere
Unoriented
Vegetal Mat MTs
Vegetal
Figure3. Summaryofthespatialdistributionofmicrotubulesinmedakaduringthefirstcellcycle.The
diagramsweredrawn approximately toscalewith respect tothe sizeofthe microtubule-organizingcenter,
thevegetal mat,andoildroplets;butthethicknessofindividual microtubuleshasbeengreatlyexaggerated
to demonstrate the arraysclearly. The diameter ofthe egg is about 1140^m. (A) Microtubules in an egg
fixedat /" = 0.16.Anunorientednetworkofmicrotubules,denserneartheanimalpole,ispresentthroughout
theegg. (B) Microtubules in aneggfixed at T = 0.4. Anarrayofmicrotubulesthat radiatefrom azonein
theblastodiscnearthepronuclei formsby Tn = 0.3andpersistsuntilatleast Tn = 0.76,whileatthevegetal
pole,amatofparallel microtubules formsby Tn = 0.24 and persists until at least Tn = 0.76. Microtubules
in interpolar ooplasm have no preferred orientation at any time. Note that most ofthe oil droplets from
theanimal hemispherehavemovedintotheequatorial regionbythistime. (C) Microtubulesinaneggfixed
at Tn = 1.0. Two microtubule-organizing centers, one at each pole ofthedividingblastodisc, are present.
Thedensityofmicrotubulesininterpolarooplasmhasdecreased,andtheparallelorganizationofmicrotubules
atthevegetal pole hasbeen lost.
ofthese blastomeres hada microtubule-organizingcenter crotubules near the animal pole was disrupted (Fig. 4C,
(data not shown) similar to the one present in the blas- D). The only apparent effect ofdibromo-BAPTA on the
todisc from Tn = 0.30 to at least T,, = 0.76. The parallel microtubule arrays in theequatorial region wasadecrease
organization ofthe microtubule network nearthe vegetal in the density ofmicrotubules nearthe injection site (Fig.
pole was lost by this stage (Fig. 2C). The pattern of mi- 5B, C).
crotubules in eggs fixed at T,, 1.0 is summarized in Dibromo-BAPTA also inhibited the movement ofthe
Fig. 3C. pronuclei. At Tn ^ 0.5, the distance between the male
and female pronuclei was as follows: uninjected control
Mwiitchro5t,u5b'u-ldeinbertowmoor-kBsAPinTAeggs injected wegegs,in1j.e8cted3.K0C/1^,m 1(.X7 S3D.,4n^m= 1(/0;e=ggs4);eeggggss);inetgogswhiinctho
which we injected dibromo-BAPTA, 40.4 28.6 ^m (n
The effects of injected KC1 and dibromo-BAPTA on = 5 eggs). A one-way analysis of variance and post-hoc
microtubule networks are summarized in Figures 4 and Student's/-testsrevealedthattheBAPTA-treatedeggsdif-
5. The microtubule networks in eggs into which we in- fered significantly (P = 0.038) from the other two treat-
jected KC1 were indistinguishable from those found in ments.
uninjected eggs fixed at the same time (T,, = 0.76 [from
57 min to 84 min after fertilization, depending on the Discussion
temperature]; Fig. 4A [compare with Fig. 2B], 5A [com-
pare with Fig. 2A]). The parallel organization ofmicro- The pattern of microtubules in the fertilized medaka
tubules in the vegetal mat was notdisrupted bydibromo- egg varied both temporally and spatially during the first
BAPTA (Fig. 4B). However, the injection of dibromo- cell cycle. Temporal changes included an increase in the
BAPTA hadprofoundeffectsonthemicrotubulenetworks density of microtubules throughout the ooplasm and
near the animal pole, where the radiating pattern ofmi- changes in the degree oforientation of microtubules in
(Tn = 0.4) isshown, usingeithera filterto show the fusing Hoechst-stained pronuclei (D) orone to show
rhodamine-stainedmicrotubules(E). Microtubulesradiatefromaregion nearthepronuclei,roughlyfollowing
meridianlines.(F-I)Thesearephotographsoffourquadrantsaround,andjustoutside,theregioncontaining
thepronuclei. Notethestrongorientation ofmicrotubulesalongmeridian lines. Thepronucleiaretolower
rightin F, lowerleftinG, upperright in H, upperleftin I.
142 V. C. ABRAHAM ET AL
Figure4. The effect ofdibromo-BAPTA on microtubule arrays in polarooplasm. All eggswere fixed
at /" = 0.50. A and B areconfocal imagesobtained usingtheZeisslaserscanningconfocal microscope; C
and Dwereobtainedbystandardepifluorescence microscopy. Scale bar = 10^m; A and Bareatthesame
magnification, Cand Dat another. (A and B) Injection ofneither KC1 (A) nordibromo-BAPTA (B; fixed
32 min afterfertilization andabout 26min afterinjection ofdibromo-BAPTA) hadanyapparenteffecton
the parallel array ofmicrotubules in the vegetal mat. (C) Microtubules are oriented along meridian lines
neartheanimal poleofthiscontrol (uninjected)egg. The pronuclei areto the right oftheregion shown in
thisphotograph. (D) Injection ofdibromo-BAPTA disrupted theradial patternofmicrotubulesintheblas-
todisc.Thiseggwasfixedabout 52 minafterfertilization and about46 min aftertheinjection ofdibromo-
BAPTA. Notetheabsenceofradiallyorientedmicrotubulesinthisphotograph,which isofthesameregion
oftheeggastheoneshown in Fig. 4C.
the polar regions ofthe egg. The striking feature ofthe are present in fertilized eggs ofa number ofanimals, in-
region near the animal pole was the development ofan cludingannelids(Astrowelat., 1989). ctenophores(Hou-
array of microtubules that radiated from near the pro- liston el a/., 1993), echinoderms (Harris el al, 1980), as-
nuclei to approximately 30 arc from the animal pole. cidians (Sawada and Schatten. 1988), and amphibians
Though microtubules were initially (T,, = 0.08) present (Houliston and Elinson, 1991; SchroederandCard, 1992;
near the animal pole as a sparse unoriented network, a Elinson and Palacek, 1993), and are associated with the
dense network of radially oriented microtubules had sperm aster and sometimes with the female pronucleus
formed by Tn = 0.3. Such radial arrays of microtubules as well.
MICROTUBULES IN THE MEDAKA EGG 143
el a/.. 1978; Scharf and Gerhart, 1983; Vincent et al.,
1987; Gerhart et al.. 1989; Elinson and Rowning, 1988:
Elinson and Palacek, 1993). A similar array of parallel
microtubules is also present at the vegetal pole ofthe ze-
brafish (Danio rerio) egg (see Fig. 9C in Strahle and Je-
suthasan, 1993). Although there is no evidence for the
occurrence ofa cortical rotation in the medaka egg, em-
bryos of some primitive fishes that undergo holoblastic
cleavage (species in the Superorder Chondrostei within
the Actinopterygii, for example the sturgeon. Acipenser
giildenstlidti) do form a gray crescent and thus perhaps
do undergo a cortical rotation (reviewed by Bolker, 1993;
see also Clavert. 1962: Ginsburg and Dettlaff. 1991). In
amphibian eggs, the plane ofthe first cleavage is parallel
to the microtubules near the vegetal pole; and in both
amphibians and primitive fishes, the plane of the first
cleavagebisectsthegraycrescentalongameridian (Bolker.
1993). We are currently pursuing the question ofthe re-
lationship between the orientation ofmicrotubules in the
vegetal mat and that oftheembryonicaxesin the medaka
egg. In both Xenupux laevis and the medaka. the parallel
orientation ofthe microtubules in the vegetal pole region
is transient. In .V. laevix. this mat begins to form at T,,
= 0.55 and disappears after Tn = 0.8 (Elinson and Pa-
lacek. 1993). In medaka eggs, it is present by Tn = 0.24
and is still present at T,, = 0.76; but by the beginning of
the first cleavage, the network has lost its parallel orga-
nization.
In sharp contrast to the strongly oriented networks of
microtubules in polar ooplasm, interpolar ooplasm con-
tained a network ofcrisscrossed microtubules having no
apparent preferred orientation. Microtubuleswere present
here by Tn = 0.08 and subsequently increased in density
through Tn = 0.24.Thelack oforientation ofmicrotubules
in this region ofthe medaka eggs contrasts with the sit-
uation in the zebrafish egg. in which microtubules are
oriented along the animal-vegetal axis during both the
6ar0rFamiyigsnurienafte5e.qruafteTorhrteiilailezfafoteiocotpnslaaosfnmdK..aCAb1lolauntedg5gds4iwmbeirrnoemaffoit-exreBdAinPajtTeAcTtnioon=n om0fi.c7dr6iobt(ruaobbmuoolu-et Sfiorlstniceclal-KcryeclzeelanadndepDirbioelvyer(.St1r9a9h4l)e.and Jesuthasan, 1993;
BAPTA), and all images were obtained using the Zeiss laser scanning Because microtubule poisons inhibit the movement of
confocal microscope.Scalebar. 10^m.(A)Ineggsintowhichweinjected oil droplets toward the vegetal pole ofthe medaka egg. it
K.C1. the array ofmicrotubuleswas similarto uninjected controls (not has been suggested that these droplets are transported
osfhodwin)b.roThmeo-aBniAmPaTl-Aveagpeptaelaraexdistroundsecfrreoamseleftthteodreignhsti.t(yBoafndmiCc)roItnjuebcutlieosn along microtubules by microtubule-based motors (Abra-
neartheinjectionsite(B:theanimal-vegetalaxisrunsfrom righttoleft) ham et al.. 1993a). Further, because oil droplets move
relative to thedensity observed at theantipode ofthe injection site(C; toward the vegetal pole roughly along meridians, we pre-
theanimal-vegetal axis runs from bottom to top). dictedthata network ofmicrotubules, also oriented more
or less along meridians, would extend from the animal
pole to nearthe vegetal pole. Although such a pattern did
The mat ofparallel microtubules at the vegetal pole of develop near the animal pole by T,, = 0.3. a time when
the medaka egg is reminiscent of the one found at the oil droplets have begun to move toward the vegetal pole,
vegetal poleofamphibian eggs, where it isinvolved in the we saw no such preferred orientation ofmicrotubules in
cortical rotation that forms the gray crescent and thus mostoftheinterpolarooplasm, suggestingthatoildroplets
determines the dorsal-ventral axis ofthe embrvo (Manes move toward the vegetal pole along a biochemically dis-
144 V. C. ABRAHAM ET AL
tincUGard. 1991; Wolfftfa/., 1992) subsetofinterpolar pole. In a number ofspecies, including the medaka, the
microtubules that are oriented along meridian lines. movement of the pronuclei is inhibited by microtubule
The movement ofmost oil droplets toward the vegetal poisons (Hiramoto et al.. 1984; Sawada and Schatten,
poleofthemedakaeggbeginsat T,, = 0.15-0.30(Catalone 1989; Abraham et al.. I993a). suggesting that microtu-
and Fluck. 1994). The results ofthe present study, which bules are necessary for these movements.
show that microtubules are present before oil droplets Theparallel arrayofmicrotubulesnearthevegetal pole
begin to move toward the vegetal pole, suggest that this was apparently resistant to the effects ofthe buffer, sug-
movement is triggered by some factor otherthan the ap- gesting that microtubules in this region ofthe egg differ
pearance ofmicrotubules. We have also shown that even in some way from those in other regions ofthe egg. This
though the movement of oil droplets is "frozen" at the possible difference could be probed by microinjecting a
antipode ofthe site at which dibromo-BAPTA is injected microtubule poison, for example demecolcine, directly
into the egg (Fluck el at., 1994), many microtubules are into the vegetal ooplasm.
present in this region ofthe ooplasm. Factors that could The suggestion that the medaka egg has two indepen-
initiate the movement ofoil droplets toward the vegetal dent microtubule networks isconsistent with the situation
pole include the formation ofan appropriate subtype of in A laevis. in which two independent networks of mi-
microtubules,theactivationofamicrotubule-based motor crotubules are present during the first cell cycle: one is
such askinesin (Brady et al.. 1990; Schnapp et al . 1992), neartheanimal poleand isassociated with thepronuclei,
fulfillment ofa requirement that a minimum number of while the other is the parallel array ofmicrotubules near
microtubules be associated with an oil droplet before it the vegetal pole (Elinson and Rowning. 1988; Houliston
begins to move, or a gel-sol transition of the ooplasm and Elinson. 1991; Elinson and Palacek. 1993; Sardet et
(Janson and Taylor, 1993) that frees the droplets and en- al.. 1994). Moreover, in both amphibian eggs (Houliston
ables them to move toward the vegetal pole. and Elinson. 1991; Elinson and Palacek, 1993) and me-
We have also demonstrated in the present study that dakaeggs(Webband Fluck, unpub. obs.), vegetal cortical
injection of dibromo-BAPTA into medaka eggs affects arrays of microtubules form in parthenogenetically acti-
thepattern ofmicrotubules in theegg. Thisrelatively weak vated eggs. Using the "Colcemid-UV" method (Hiramoto
calcium buffer (KD = 1.5 M; Pethig el al.. 1989) is be- et al.. 1984: Webb and Fluck. 1993), we are currently
lieved to act asa shuttle buffer, one that bindsCa:+ at the pursuing the question ofthe existence ofindependent ar-
high end of a [Ca2+] gradient and releases it at the low rays of microtubules in the animal pole region, vegetal
end; in other words, the buffer facilitates the diffusion of pole region, and interpolarooplasm. Ourdataat thistime
Ca:+ within the cell (Speksnijder et al.. 1989). The time do not permit usto say whether microtubulesare contin-
between injection ofdibromo-BAPTA and fixation ofthe uous from one region ofthe egg to another.
eggs in the present study was sufficient for the buffer to
diffuse to both the animal pole and vegetal pole, to dis- Acknowledgments
sinihpiabtietctyhteosofloircmaCtai:o4n gorfadtiheentbslansetaodristcheanpdoletsh,eamnodvet-o Supported by NSF DCB-9017210 and NSF MCB-
ment ofoil droplets toward the vegetal pole (Fluck et al.. 9a3n1d6A1L25M,toanRdAgFr,anNtsSfFroBmIRFra9n2k1l1i8n5&5 MtaorsLhiaolnellCoFl.leJgaef'fse
1992, 1994).
diOburromroe-suBlAtsPTsuAggoenstootphlatasamticlesaestgrsegoamteioonfinthteheefmfeecdtaskoaf HHuafcfknamgalne FScuhnodlatroPVrCoAg.raLmouainsdKHearrrryprWov.idaenddtMecahrnyicaBl.
eggarecaused by itsdisruption ofmicrotubule formation assistance in the use of the laser scanning confocal mi-
acanldciourmg-anbiiznadtiinogn,rewghuilcathorayreprcootnetirnosll(eWdeibsyenabenrugm,be1r972o;f cBrioolsocgoipcealinLtahbeoCreanttorrayl.MiTcarmoiskcaopeWeFabciblitayssaitsttehde Mianritnhee
S1c9h94l)i.waFaectilailt..ate1d98d1i;ffKuseiiotnhtehleoalr.y. p1r9e8d3i;ctsLitehuavtincaeltciau/.m, pBroebpaGroaltdioenr aasnsdisetexdamiinntahteiopnreopfaroanteionbaotfchFiogfureeggs3,. Tahned
buffer concentrations similar to those found effective in research atCarnegie-Mellon University wassupported by
National Science Foundation and Technology Center
t[1hC9e8a92)p+.r]ferseAeennitnesxtthauemdpymlieacctroofbmyoaldapirrsostirepaianntgientgh(atSzpoeinskessrneiosjfpdoecnrystieovlseoallit.co. girmaangteDaIcRqu-i8s9i2ti0o1n,18d.atWaeprtohcaensksinRgo,bbainnd 3L.-DDerBeiparseiseontfao-r
c1h9a80n)g,eswhiinc[hCrae:g+u]l,raeteeisntthheisacrtainvigteyiosfcparlomtoedinuslitnha(tCihnetuernagc.t atinodn;iamnagdeGarceqguoisriytiMon.. LaRocca for technical assistance
with microtubules (Ishikawa el a/.. 1992).
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