Genetic Consequences of Blocking Polar Body I with Cytochalasin B in Fertilized Eggs of the Pacific Oyster, Crassostrea gigas: I. Ploidy of Resultant Embryos

6 Pages·1992·2.5 MB·English

by  X Guo

Reference: Biiil. Bull. 183: 381-386. (December. 1992)
Genetic Consequences of Blocking Polar Body I with
B
Cytochalasin in Fertilized Eggs of the Pacific Oyster,
Cmssostrea gigas: I. Ploidy of Resultant Embryos
XIMING GUO1*, KENNAENTDH KCEONONPEETRH2**, WCIHLLEIWAM K. HERSHBERGER1,
K. 1
[SchoolofFisheries, U'II-10. UniversityofWashington, Seattle, Washington 98195, ami
2Coast Oyster Company, Quilcene, Washington 98376
Abstract. Theeffect ofblockingpolarbody I (FBI)with gas, and many other marine mollusks, mature eggs are
cytochalasin B (CB)ontheploidy ofembryoswasstudied arrested at prophase ofmeiosis I (Ahmed, 1973; Lu, 1986;
in the Pacific oyster, Cmssostrea gigus. To block the re- Strathmann, 1987). Only after fertilization or activation,
lease ofPB1, fertilized eggs were treated with CB (1.0 fig/ do the eggs complete meiosis I and II, releasingtwo polar
ml) for 15 min beginning at 5 min post-fertilization at bodies. Delayed meiosis in eggs ofthe Pacific oyster pro-
25C. The CB treatment and itscontrol were repeated in videsa unique opportunityformanipulation ofboth polar
three crosses. Ploidy of8-h-old embryos was determined body I (PB1) and polar body II (PB2). Although it has
with karyological analysis. been well established that blocking the release ofPB2 re-
In control groups, the majority ofthe cells(89.3%) had sults in triploidy (reviewed by Beaumont and Fairbrother,
a diploid number of20 chromosomes, although sponta- 1991), it is still uncertain what ploidy will result from
neous haploids (0.7%), triploids (1.3%) and aneuploids blocking PB1.
(8.7%)werealsoencountered. In CB-treated groups, only Stanley el al. (1981) first reported that cytochalasin B
4.5% ofthe cells remained as diploid. and the majority (CB)treatmentappliedduringmeiosisI resultedintriploid
were either triploid (15.6%), tetraploid (19.4%) or aneu- and tetraploid embryos in the American oyster, Crassos-
ploid (57.6%). Despite variation among the three crosses, trcti virginica, although onlytriploidssurvivedto8 months
contingency Chi-square analysis showed that the occur- of age. Similarly, both triploid and tetraploid embryos
rence oftriploids, tetraploids and aneuploids had a sig- were produced from blocking PB1 in the blue mussel
nificant (P = 0.0001 ) dependence on the CB treatment. Mytilus edulis(Yamamoto and Sugawara, 1988) and the
The majority ofthe aneuploids fell into two groups con- Pacificabalone, Haliotisdiscushannai(Arai elal., 1986).
taining either 23-25 or 35-37 chromosomes. The pro- In the Pacific oyster, however, only triploids were found
duction oftriploids, tetraploidsand aneuploids in specific whenthermal shockswereapplied duringmeiosis I (Quil-
distributions suggeststhatblocking PB1 complicates sub- let and Panelay, 1986). In a later study, blocking FBI
sequent chromosome segregation. with CB in the Pacific oyster produced tetraploids, not
triploids(Stephens and Downing, 1988; Stephens, 1989).
Introduction In the Pacific oyster, production ofeither meiosis I tri-
In most vertebrates, eggs usually matureafterthecom- ploids or tetraploids is ofinterest. Triploid oysters have
pletion ofmeiosis I. In the Pacific oyster, Crassostreagi- become an important part ofthe oyster culture industry
because oftheir sterility (Allen, 1988; Allen el al, 1989).
Meiosis I triploids may grow better than meiosis II tri-
Received 3 March 1992;accepted 31 July 1942. ploids (Stanley el al., 1984) that are currently produced
Lab*orCaotrorreys.pRountdgienrgsaUuntihvoerrs,itpyr.esBeonxtBa-dd8r,esPso:rtHNaosrkriisn,SNheelwlfJiesrhseRyes0e8a3r49c.h in commercial hatcheries. Tetraploids may be mated to
** Present address: 24888 Taree Drive NE. Kingston. Washington diploids to produce all triploid progeny. However, avail-
98346. abledataare inconclusive, ifnot conflicting, on theques-
381
382 X. GUO ET AL
tion ofwhat ploidy is m blocking PB1. The placed by 1:1 (v/v) glacial acetic acid and absolute meth-
lack ofdetailed ploidy >!'early embryos may be anol solution.
partly responsible ^repancy. This study used Embryo suspensions were dropped onto slidesand air-
extensive karyologu .sistodetermine the ploidyof dried. Slides were stained with Leishman's stain (Benn
8-h-old Pacific nbryos following blocking FBI and Perle, 1986). The stock solution was made by dis-
with CB. It wasanticipated that resultsofthisstudywould solving 150mgLeishman'sstain(Sigma)in 100 ml meth-
clarify some oftheconfusion concerningthegeneticcon- anol. The staining solution was prepared by mixing one
sequences ofblocking PB1. partoftMhestocksolution with three partsphosphatebuffer
(0.025 KH2PO4. pH 6.8). The slides were stained for
Materials and Methods 10-15 min.
Only metaphase plates which showed no evidence of
Gametepreparation artificial chromosome losswere counted. Only one meta-
Gameteswereobtained bystrip-spawningsexuallyma- phase plate perembryo was scored and used to represent
twuarsedPiascsiefcitceodysitnetros.2T1 oofosbetaawianteerg.gsT,hfeemeaglgesugsopneandsailotniswsause tmheattapehmabsreyow.ereEmrbarrey.oAsmwiinthimmuomreoft5h0anmeotnaephcaosuenptlaabtlees
first passed through a 100jum screen (Research Nets Inc.) from 50embryoswerecounted forcontrol and treatment
to remove large tissue debris. Eggs about 50 /urn in di- groups ofall replicates. Diploid Pacific oysters have 20
ameter were collected onto a 20 jum screen. Broken eggs chromosomes (Ahmed and Sparks. 1967; Ahmed, 1973).
and other smaller debris were washed off. The eggs were In this study, ploidies were classified asthe following: 10,
then re-suspended in 1 1 ofseawater, ready forfertilization. haploid; 20, diploid: 29-30. triploid; 39-40, tetraploid:
To prepare sperm suspensions, 1 g of male gonadal 49-50, pentaploid. All otherswereconsideredtobeaneu-
tissue wascompletely suspended in 99 gofseawater. The ploid.
large tissue debris were removed by passing the sperm
suspension through a 20 ^m screen. For fertilization. 5 Data analysis
ml ofthesperm suspension wasadded toone million eggs
esumsbpreynodecdulitnur1e1woefrseeaawlaltceor.ndFuecrttieldizaatti2on5, Ct.reatment, and amFoonrgbtohtehthcroenetrcorlossaensdwatsreatteesdtedgrwoiutphs,Chhi-osmqougaerneeihtoy-
mogeneity analysis. Differences between the control and
CB treatment treatedgroupswerethen tested bycontingencyChi-square
analysis. The null hypothesis was that the occurrence of
To block the release of PB1, 1.0 mg CB (Sigma) dis- ploidies was independent ofCB treatment.
solved in 1.0 ml dimethyl sulfoxide (DMSO) was added
to 1 1 egg suspension at 5 min post-fertilization for 15 Results
min. CB was removed by collecting and rinsing treated
eggs on a 20 ^m screen. Eggs were then returned to 0.1% In thecontrolgroups, PB1 wasreleased around 17 min
DMSO in seawater for 30 min to remove residual CB. In post-fertilization, and PB2 was released around 35 min
the control groups, fertilized eggs were treated with only post-fertilization. In the CB-treated groups, PB1 was not
0.1% DMSO. The embryos were stocked at a density of observed at the time expected, but one polar body was
20embryos/ml. Theexperimentwasrepeated threetimes released in most ofthe eggs between 35 and 45 min post-
using three pairs ofoysters as parents. fertilization. The first mitosis in the treated groups ap-
peared to be normal, except apparently lesssynchronized
Determination < -'oidv than that in the control groups.
At 8 h post-fertilization, all embryos developed to
Ploidy of i tant embryos in both control and swimming trochophores, and the number of swimmers
treated group med by karyological analysis. hatched was high in both treated and control groups(95-
Samples for ko . >!ysis were taken at 8 h post- 100%). However, abnormal trochophoreswerenoticed in
fertilization. Embr \ treated with 0.01% col- the CB-treated groups. The abnormal trochophores were
chicine for 30 min colchicine waMs removed, eitherdeformed orconstantly swam in acircularmotion.
nine parts of a hypotomc solution (0.075 KC1) was In thecontrol groups, the karyological analysis revealed
addedtoonepartembryo ^pension for25-30 min. The that 89.3% ofall cells examined had exactly 20 chromo-
hypotonic solution was then removed as completely as somes (Table I). Only 8.7%. could be classified as aneu-
possible, and sampleswere fixed in a solution ofone part ploids, ofwhich, about half(4.7%) had 19 chromosomes,
glacial acetic acid and three parts ofabsolute methanol one quarter (2.0%-) had 15-18 chromosomes, and one
(v/v). About 15 min before staining, the fixative was re- quarter (2.0%) had 21 chromosomes (trisomics). Spon-
PLOIDV OF EMBRYOS 383
taneous haploids and triploids were also observed in the cross, CB treatment induced 26%- triploids, 24% tetra-
control groups at a low frequency (0.7% and 1.3%, re- ploids and 45%- aneuploids. compared to 1 1%, 6%, and
spectively). No tetraploids or pentaploids were found 72% ofrespective ploidies induced in cross 3 (Table I).
among the 150 cells counted from the control groups. Thedistribution ofpooled chromosome counts forthe
Compared with the control groups, all three CB-treated control and for the treated groups from the three crosses
groups had dramatic increases in the proportion ofpoly- is shown in Figure 2. As expected, only one peak (at 2n
ploid and aneuploid cells; only about 4.5% of all cells = 20 chromosomes) was present in the control groups
remained as diploid (Table I). The majority ofcells were (Fig. 2A). Inthetreatedgroups, however, fourpeakscould
either triploid (15.6%), tetraploid (19.4%) or aneuploid be identified: triploids, tetraploids, and two aneuploid
(57.6%) (Fig. 1). A low percentage ofhaploids and pen- peaks, corresponding to chromosome counts of 23-25
taploidswerealso recorded in theCB-treatedgroups. The and 35-37 (Fig. 2B).
mitotic index ofCB-treated groups was noticeably lower
Discussion
than control groups.
For both the control and the treated groups, the ho- This study provides an estimate of the frequency of
mogeneity among crosses was accepted at a significance spontaneousaneuploidy in 8-h-oldembryosofthe Pacific
level of95% forthe frequencies ofdiploids, triploids, tet- oyster under our experimental conditions. It is unlikely
raploids and aneuploids. Homogeneity was not tested for that the aneuploidy in the control groups was caused by
haploids and pentaploids, where at least onecross had no DMSO, although this study cannot rule out such a pos-
occurrence of the ploidy tested (Table I). Because the sibility. DMSO iswidely used with cultured cellsandem-
numberofhaploids and pentaploids observed in all three bryos, and has no known effects on chromosome segre-
crosses was low (0-4%), homogeneity amongcrosses was gation. Comparable data have not been reported. Since
assumed. Consequently, data from all three crosses were karyological techniques tend to create chromosome losses
used in a contingency Chi-square analysis to test the null and overestimate the occurrence ofaneuploidy, the 8.7%
hypothesis that the occurrence ofploidies is independent aneuploids observed in this study may be considered as
ofthe CB treatment. When all ploidies were analyzed in an upper limit ofaneuploidy in the 8-h-old embryos. On
one Chi-square test (df = 5), the null hypothesis was re- theother hand, since the probability ofcreatingtrisomics
jected at a confidence level of95%. When ploidies were by artifacts is low, the 2% trisomics observed here may
tested separately (df = 1), however, the null hypothesis provide a conservative low limit.
was accepted for haploids and pentaploids (Table I). On The formation ofaneuploid gametes in mollusks may
the other hand, the occurrence ofdiploids, triploids, tet- be common, particularly under environmental stress
raploids. and aneuploidsshowed asignificant (P= 0.0001) (Dixon, 1982). Gaffney et al. (1990) suggested that het-
dependence on whether or not the eggs were treated with erozygosity deficiency, a common phenomenon in mol-
CB for blocking PB1. lusks. could be caused by the occurrence ofaneuploids.
Although the homogeneity among crosses was statis- Injuvenile Pacific oysters, aneuploid cellswereestimated
tically accepted, the CB-treated groups from different torangefrom4%to 36% (Thiriot-Quievreux elal, 1988).
crosses had noticeable differences in the distribution of Frequency ofspontaneous aneuploids could be very dif-
the different ploidies induced. For example, in the first ferent at different stages ofthe life cycle.
Table I
Percentage<>/Jittcrcniploidiesobservedfromnormalfertilization (Control) andblockingFBI (Treated) in thePacificoyster
Cross Group Sample size In 3n 4n An
1
384 X. GUO ET AL.
B
*
I
IHrJir
Figure 1. Melaphase plates oftriploid (A), tetraploid (B) and aneuploid (C. D) cells produced from
blockingpolarbody I withcytochalasin Bin the fertilized eggsofthe Pacificoyster.
Results from this study suggest that spontaneous hap- PB1 has been previously suggested, based on thedifferent
loids and triploids might occur in the Pacific oyster. fluorescence intensity ofthe two polar bodies (Stephens,
Spontaneous triploids have been reported in amphibians 1989).
and fish and e probably caused by fertilization of un- Thedetection oftriploidsand tetraploids from blocking
reduced ova b ierm (Fankhauser, 1945; Thor- PB1 in this study agrees with results from the American
gaard aiui i nloids could be caused by the oyster(Stanley el at.. 1981), the blue mussel (Yamamoto
failure of sperr s incorporation. Again, this and Sugawara, 1988), and the Pacific abalone(Arai eta/..
study cannot o i the possibility that the 1986). Also, resultsofthisstudydonotconflictwith stud-
haploids and triploi e control groups were in- ies in which only triploid juveniles were observed from
duced by the DMSU. blocking PB1 (Stanley ct a/.. 1984; Downing and Allen.
ItisclearthattheCBuvatment applied duringmeiosis 1987). The lack oftetraploids atjuvenile or adult stages
I waseffective in preventingpolarbody release at 17 min was probably due to poor viability oftetraploids (Stanley
post-fertilization, a time corresponding to the release of ct al.. 1981; Stephens, 1989; Guo. 1991). On the other
FBI. That the polar body released at 40 min post-fertil- hand, results of this study differ from other studies, in
ization (afterCB treatment) is PB2 ratherthan thedelaved which only triploid embryos (Quillet and Panelay. 1986)
PLOIDY OF EMBRYOS 385
150-, A: Control
(n=150)
100-
50-
10 20 30 40 50
ChromosomeNumber
50-
B: CBTreated
(n=169)
40-
30-
u
o"
386 X. GUO ET AL.
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