effects of cytochalasin b on meiosis and developmen otf ...solution of pure cacl2 (700 m-osmole). in...

/ . Embryol. exp. Morph. Vol. 31, 1, pp. 61-74,1974 6 1

Printed in Great Britain

Effects of cytochalasin B on meiosisand development of fertilized and activated eggs of

Sabellaria alveolata L. (Polychaete Annelid)

By G. PEAUCELLIER,1 P. GUERRIER1 AND J. BERGERARD1

From the Station Biologique, Roscoff

SUMMARY1. Unfertilized, fertilized and activated eggs of Sabellaria alveolata were submitted to

cytochalasin B concentrations ranging from 01 to 20/tg/ml. Their behaviour was studiedeither /// vivo or in acetocarmine squash preparations.

2. Polar body extrusion, cytokinesis and polar lobe formation are completely inhibited bycytochalasin B concentrations as low as 0-3-0-5 /*g/ml.

3. Caryotype determinations demonstrate that chromosomal meiotic and mitotic processesare not affected by the drug. Thus, polyploid embryos usually developed from fertilized eggswhilst they did not from activated ones. This is related to the contrasting behaviour of meioticand cleavage centres. While the latter duplicates at each cycle, the former cannot replicateat the end of meiosis. This leads to an abortive monastral stage even if inhibition of polarbody extrusion has provided the egg with two or four centres. These observations suggest theexistence of an internal mechanism regulating the number of effective centrioles at the end ofmeiosis. They demonstrate also that the main cause of developmental failure in activated eggscannot be related to ploidy.

4. Eggs treated throughout meiosis with moderate drug concentrations developed intoswimming larvae. However, frequent developmental abnormalities affecting lobe dependentstructures were obtained even if polar lobe formation was unimpaired. This suggests eitherthat cytochalasin B has irreversibly affected some decisive cortical element or that previouslydescribed activating processes, which begin with polar lobe formation, are actually exertedon specific materials segregated during meiosis.

INTRODUCTION

In a study of the ability of the egg of Sabellaria alveolata to develop partheno-genetically, we found a technique which elicits all the early processes usuallybrought about by fertilization but without ensuing cleavage. These processes,which include the extrusion of polar bodies, lead only to the formation of amonaster, instead of the normal first cleavage spindle, so that development doesnot proceed any further.

Such a situation is frequently explained by assuming that, after completionof meiosis, there is no more than one centre in the oocyte, which is unable toreplicate (Tyler, 1941). This assumption fits well with two observations:

1 Authors' address: Station Biologique, Place Georges-Teissier, 29211 Roscoff, France.

62 G. PEAUCELLIER AND OTHERS

(a) The fact that the regulative treatment of any two-step activating methodgives rise to cytasters.

(b) The fact that, in species where fertilization normally induces the achieve-ment of meiosis, one cannot obtain parthogenetic cleavage unless one polarbody fails to form so that its spindle functions as the first cleavage spindle(Tyler, 1941; Sachs, 1971; Motomura, 1954).

The drug cytochalasin B, which seems to be rather innocuous to fundamentalcell metabolism (Spooner, Yamada & Wessels, 1971; Prescott, Myerson &Wallace, 1972; Zigmond & Hirsch, 1972; Raff, 1972) appeared an ideal tool fortesting such an hypothesis, by preventing the extrusion of polar bodies. Indeed,since the pioneer work of Carter (1967), the specific effect of this substance on cyto-kinesis has been well known. (See also recent reviews and discussions by Carter(1972), Estensen, Rosenberg & Sheridan (1972), Forer, Emmersen & Behnke(1972), Wessels et al. (1971a, b); Holtzer & Sanger (1972)). Furthermore,Longo (1972) successfully used this drug to inhibit the formation of polar bodiesin the egg of the surf clam Spisula solidissima. In the course of the present work,we tested first the effect of cytochalasin B on unfertilized and fertilized eggsbefore applying it to activated eggs. In this way it was possible to demonstratea difference in behaviour between meiotic and cleavage centres. Several otherfeatures were noted which it is worth while to report.

MATERIALS AND METHODS

Sand tube blocks of Sabellaha were collected in the vicinity of Roscoff andmaintained in running sea-water. In these conditions, animals remain in goodcondition for many weeks. Shedding occurs spontaneously as soon as wormsare extracted from their individual tubes. Therefore before putting them inbowls of filtered sea-water, they were first washed with running sea-water andtap water in order to eliminate the possibility of sperm contamination ofoocytes. By this treatment, the number of naturally fertilized eggs does notexceed a few per thousand.

Egg shedding is stopped after 15 min by removing the laying females whilethe eggs wait another 45 min to ensure that they have all completed the pre-maturation process to reach the stable state of waiting oocyte (i.e. metaphaseof the first meiotic division). Successful artificial fertilization (about 80%) isobtained with a final sperm concentration (spectrophotometric determinationat 460 nm) of about 15000 sperm//tl, using pooled gametes from differentindividuals.

Parthenogenetic activation resulted from a 30 min treatment in a hypotonicsolution of pure CaCl2 (700 m-osmole). In such conditions about 50 % of theeggs are activated, but this percentage is only an average since it can vary from90 to 10%, according to the experiment.

Cytochalasin B (I.C.I., Macclesfield, Cheshire, U.K.) was prepared as a0-l%(w/v) stock solution in dimethyl sulphoxide (DMSO) and stored at

Cytochalasin on a mosaic embryo 63- 20 °C. For experimental use this solution was added to a culture of eggs infiltered sea-water at concentrations referred to in the text. Controls developednormally in a 2 % solution of DMSO, a concentration which corresponds tothe highest one used in the present work.

For accurate chromosome counting, cleaving eggs were treated for 30 minwith a 0-15 % colchicine solution. The eggs, fixed for 30 min to 1 h in Carnoy'sfluid, were stained for at least 3 h in acetocarmine. Cytological studies wereperformed either on whole mounts or on squashes for caryotype determinations.Living eggs were also studied by the hanging drop technique, free or compressedas previously described (Guerrier, 1971a).

RESULTS

I. Effects on unfertilized eggs

Cytochalasin B seems not to be very harmful to the egg. However, in someeggs we found that cytoplasmic extrusions developed in the perivitelline space.These appear to remain bound by a membrane, as there is no yolk dispersionin the perivitelline space and as they can be resorbed more or less completelyafter returning the egg to sea-water. Such protuberances may appear at anypoint around the egg surface and develop to about half the egg volume (Fig. 1 A).This process, however, does not affect more than a small percentage of the eggs,since a 2 h treatment of 2 /^g/ml gives no more than 6 % modified eggs, thisproportion decreasing to 0-4 % when 0-2 /*g/ml is applied for the same length oftime. The same blebbing phenomenon can also affect fertilized eggs, where it isespecially widespread and evident during the time of polar body extrusion.

II. Effects on fertilized eggs

A. First maturation division

Eggs were transferred to various solutions of cytochalasin B, 10-15 min beforethe usual time for polar body extrusion. While this process is not affected at0-1 /*g/ml, concentrations of 0-3 jLtg/ml or more completely stop it.

At low concentrations (0-3-0-5 /^g/ml), the first maturation spindle takes itsusual position at the animal pole and there is often an indication of the pro-tuberance which usually precedes polar body extrusion. However, this protrusionis not quite characteristic for it is much wider than usual. Moreover, it regressesrapidly or degenerates into cytoplasmic blebbing. As a result, the two sets ofdyads remained in the egg cytoplasm. As in normal development, there is nopronucleus formation at this stage.

With higher concentrations, ranging from 5 to 20 ̂ g/ml, anaphase of firstmaturation division does not take place in the normal position but right in thecentre of the egg. No other modification of chromosomal processes is observed,nor is there any indication of animal pole flattening or of the meiotic pro-tuberance.


B

25//m

D

Cytochalasin on a mosaic embryo 65

B. Second maturation division

The pattern of changes just described applies also to eggs treated after the firstpolar body extrusion. Nevertheless, at the end oftelophase, astral rays vanish whilepronuclei appear as in normal development. As far as we can tell from thecytological techniques used in this study, it seems that the two sets of maternalchromosomes usually fuse in the same pronucleus while sperm chromosomesgive rise to the male pronucleus.

In eggs treated before the onset of the first maturation division, two spindlesdevelop when controls are engaged in the second maturation division. Thesespindles are more or less parallel to each other but may present differentorientations relative to the egg surface. As illustrated on Fig. 1C, each spindlecarries a set of dyads which are engaged simultaneously in the process of ana-phase. Chromosomes then fade away and seem again to give rise only to onemale and one female pronucleus.

C. Early cleavage

During the overall pronuclear stage the acetocarmine stain is unable to revealthe existence of any astral figure. However, when pronuclear membranes breakdown, we must stress that one always obtains a single effective cleavage spindle.

Depending on whether the eggs have been treated before or after the extrusionof the first polar body, the metaphase plate exhibits 80 or 48 chromosomes,which appeared to be normally duplicated. This corresponds to pentaploidy ortriploidy (Peaucellier, 19736).

In normal development a polar lobe develops at the vegetal pole of the egg,long before the indication of the first cleavage furrow. During cytochalasin Btreatment we do not observe any attempt of the egg to produce such a forma-tion. This holds true not only for eggs treated from the onset of meiosis but alsofor those which were only treated from 10 to 15 min before the usual time forpolar lobe occurrence. In addition, when eggs with developing polar lobes areexposed to 0-5/*g/ml cytochalasin B the lobes completely regress in 1-2 min.On the other hand, when eggs are removed from a 0-5 /*g/ml solution and washedcarefully, some 15 min before first cleavage, the polar lobe develops normally

Fig. 1. Acetocarmine squashes from Sabellaria aheolata eggs. Living egg diameter isabout 60 /tm. Swelling through preparative treatment is about twofold. (A) Cyto-plasmic protrusion in a virgin oocyte I, after a 2 /*g/ml cytochalasin B treatment.(B) Fertilized egg treated with 0-5 /*g/ml throughout meiosis before returning tosea-water: pentaploid anaphase of first cleavage with polar lobe occurrence.(C) Activated egg treated with 0-5/*g/mI throughout meiosis: two simultaneousanaphases corresponding to the usual process of second polar body formation andto an unusual new division of first polar body material. (D) Fertilized egg treatedwith 0-5/tg/ml throughout meiosis and early cleavage: second cleavage anaphase.(E) Untreated activated egg: haploid monaster block. (F) Activated treated egg:tetraploid monaster block.

5 EMB 31


but with a slight delay (10-15 min) when compared with the controls (Fig. 1B).This effect is less regular when eggs are submitted to higher concentrations.

First cleavage furrowing reacts in a quite similar way, leading to the pro-duction of binucleate eggs. Other cell processes seem to be unaffected and astralfigures double at each cycle. Thus, one can obtain a second cleavage tetrapolaranaphase without ensuing cytokinesis (Fig. ID). This phenomenon proceedsquite regularly but, after several hours have elapsed, eggs tend to cytolyse,unless treatment has been previously stopped.

Finally, when eggs treated with 0-5 /*g/ml during the meiotic period are washedand returned to sea-water, they cleave normally, despite their polyploid state.As we mentioned before, their development is only slightly delayed relative tothe controls. When higher concentrations were used, cleavage also resumed butwith frequent abnormalities. Thus, abortion and abnormal cleavage are quitecommon, with concentrations ranging from 10 to 20/tg/ml. With moderateconcentrations of about 2 /*g/ml, we sometimes observed that first cleavagecould not be completed, giving rise to binucleate eggs which, as a rule, willnevertheless segment further.

D. Larval morphogenesis

In controls, swimming trochophores appear about 10 h after fertilization.Forty hours later they are fitted with two complete sets of post-trochal bristles,while short apical cilia have replaced the apical tuft (Fig. 2A), this last eventtaking place at about the 36th h of development.

Eggs maintained in solutions leading to an inhibition of cytokinesis (0-5 /*g/mlor more) cytolyse in a few hours, but swimming larvae differentiate in moredilute solutions. Eggs returned to normal sea-water after a treatment limited tothe meiotic period always give rise to swimming larvae about 10 h afterfertilization, except when very high concentrations, of the order of 20 /^g/ml areused, where the percentage of living larvae is quite low. Even at this early stage,various anomalies can be recognized, which are more easily studied on larvae50 h old.

From the observations made at this latter stage it appears that there is alwaysa significant rate of abnormal morphogenesis after treatment with the drug.Thus, eggs which were treated only during the meiotic period with 0-5 /*g/ml andreturned to normal sea-water did not give more than about 20 % of larvaebearing post-trochal bristles, despite the fact that cleavage of these embryosseemed to proceed normally (Fig. 2B, C, D). Similarly, eggs treated with thesame concentration for a short length of time just at the beginning of the meioticphase and which are returned to normal conditions about 40 min before thefirst cleavage, do not give more than 40 % of successfully differentiated larvae.With higher concentrations fewer larvae remain alive, the rate of abnormalitiesincreasing gradually with the concentration. Whatever the level of abnormalitiesencountered may be, such larvae remain quite active. One cannot estimate their


Fig. 2. Larvae from control and treated eggs of Sabellaria alveolata. (A) Normaltrochophore 68 h old. (B) Slightly abnormal 68 h larva obtained after a 0-5 /tg/mltreatment throughout meiosis. (C) Abnormal 42 h trochophore bearing an apicaltuft but lacking the overall post-trochal region (same experiment). (D) Abnormal68 h larva from the same experiment.

further viability, however, since rearing is a most uncertain and time-consumingtask, even with normal larvae (Wilson, 1968).

The range of observed deviations from normal morphogenesis is rather large:lack of certain parts, doubling of others, all phenomena which can be observedon larvae bearing post-trochal bristles. Nevertheless, it appears that post-trochalstructures are reduced or lacking in the greatest part of the population. Fig. 2Cillustrates a rather frequent anomaly. This trochophore of 42 h still bears anapical tuft which, normally, would have been replaced by the shortest apicalcilia. Furthermore, it is deprived of the post-trochal region and, if we consideronly this feature, looks like a lobeless embryo (NovikofF, 1940). Hence, it isnoteworthy that, in most cases, we are not dealing merely with a simpledeformation of normally occurring structures, but rather with the result ofa highly modified pattern of differentiation.

5-2


III. Effects on activated eggs

A. Steps of normal activation

First indications of a successful activation do not appear before the eggs arereturned to sea water. Meiosis proceeds as in fertilized eggs but leads to theformation of a single pronucleus of normal aspect. When the pronuclear mem-brane disappears, the polar lobe develops and chromosomes condense.

The next step is supposed to lead to the formation of the first cleavage spindle.However, in these conditions, we noticed only the constitution of a single astralfigure which bears the haploid set of chromosomes (Fig. 1E). At high magni-fication these appeared to be normally duplicated. The embryo does not de-velop further and one cannot observe the so-called monastral cycles so frequentlydescribed in other species. The time schedule of these processes correspondsaccurately to that observed in normal development, zero time being no longerrelated to fertilization but to the cessation of the treatment inducing partheno-genesis.

B. Effect of the drug

Treatment with 0-5 /*g/ml gives quite similar results to those described forfertilized eggs. The first maturation spindle is normally situated at the animalpole but first polar body extrusion is inhibited. When treatment is maintained,two spindles develop which may take various positions. Then, chromosomes aregrouped again in a single tetraploid pronucleus. When treatment was stoppedbefore the second maturation cleavage or initiated after the first polar bodyextrusion, eggs were obtained which carried one polar body and a diploidnucleus.

Such eggs were always returned to sea-water. In every case, disappearance ofthe pronucleus was accompanied by the development of a single astral figurewhich was fitted with diploid or tetraploid sets of normally duplicated chromo-somes (Fig. 1F). Here again, development appears to be blocked at the monasterstage.

DISCUSSION

Some peculiar features observed during this study need to be discussed. Theyrelate to the nuclear, astral and cytoplasmic mechanisms at work duringmeiosis, mitosis and cytokinesis, or to the important problem of what factorscontrol the early steps of differentiation in the mosaic embryo.

I. Cytokinesis and polar lobe formation

The data obtained show unequivocally that, in Sabellaria alveolata as invarious other species tested so far (Carter, 1972), cytochalasin B affects cleavagecytokinesis. It also impedes polar body extrusion as shown by Longo (1972) onthe egg of Spisula solidissima. Similarly, it is effective in preventing polar lobe


formation as was first described by Raff (1972) on the egg of Ilyanassa obsoleta.Our own data indicate that polar lobe development and meiotic or mitoticcytokinesis exhibit the same sensitivity with respect to cytochalasin B. More-over, it seems that Sabellaria eggs respond to the drug in a quite similar mannerto the eggs of the sea-urchin (Schroeder, 1969, 1972) and of the squid Loligo(Arnold & Williams-Arnold, 1970). However, they react differently fromXenopus eggs (Bluemink, 1971a, b; Hammer, Sheridan & Estensen 1971) ormammalian cells in culture (Carter, 1967; Krishan & Ray-Chaudhuri, 1969;Estensen, 1971; Krishan, 1972) which always show a clear indication offurrowing.

Such discrepancies might be related to differences in the degree of perme-ability of the plasma membrane with respect to cytochalasin B. The recentmicroinjection experiments performed by De Laat, Luchtel & Bluemink (1973)on the egg of Xenopus seem, indeed, to demonstrate that furrowing is actuallysensitive to cytochalasin B from the onset of cytokinesis, but that this drugwould normally enter the egg only at the time when a brief increase in perme-ability is produced, some few minutes after furrow induction.

One can then suppose that the egg of Sabellaria is readily permeable to cyto-chalasin B from the early beginning of cytokinesis, which prevents furrowdevelopment.

The mechanism of polar body extrusion seems to exhibit the same sensitivityto cytochalasin B, since polar body protuberance and meiotic furrowing aresimultaneously inhibited. Our data differ on this point from those obtained byLongo (1972) on the egg of Spisula, since this author did not observe anyinhibition of the polar body protuberance even at a concentration of 10 /*g/ml.However, as already suggested by Longo, it might be possible that the animalpole meiotic protuberance found in Spisula depends merely on a lower viscosityof the animal pole cortex, a situation which could also account for the pro-trusions we observed at this stage on the egg of Sabellaria.

However, it would seem that the meiotic furrow constriction which developsat the base of the polar body protuberance is the result of a mechanism in everysense identical with that of cleavage cytokinesis.

II. Mitotic apparatus

Our data confirm that even very high concentrations of cytochalasin B haveno direct effect on the mitotic apparatus. Thus, the size and time of appearanceof meiotic and cleavage spindles are not modified by the drug.

However, indirect effects are quite interesting. Thus, the formation of twoindependent spindles after the first telophase of fertilized, meiosis-treated eggs,indicates that the two centrioles of the first meiotic spindle are able to duplicate,although the one normally trapped in the first polar body usually does notdevelop.

In activated eggs, the use of cytochalasin B demonstrates that none of the


meiotic spindles remains able to give rise to the cleavage spindle. It seems likelythat this might require activating treatments which modify more thoroughly theschedule of normally occurring meiotic processes, as we have found usinghypertonic sea-water (Peaucellier, 1973 a).

On the other hand, it is noteworthy that the number of centres which remainin the treated egg at the end of meiosis has no effect on the number of astersthat will appear at time of first cleavage, since fertilized eggs have a normaldicentric spindle whilst activated eggs are only provided with a monaster. Thisstrongly contrasts with the effect of cytochalasin B on cleavage divisions where thelack of cytokinesis does not preclude the normal doubling of asters, leading tomultipolar figures.

Dealing with fertilized eggs, one can suppose, in accordance with Boveri'stheory (1906), that the sperm aster inhibits the development of any aster ofmaternal origin, though paternal origin of first cleavage centres has not beenproven so far in Sabellaria alveolata (Faure-Fremiet, 1924). However, the con-sistent appearance of a monaster in activated eggs cannot be explained byBoveri's theory, since, after cytochalasin B-induced inhibition of the extrusionof one or both polar bodies, two or four centres might remain in the egg. Theseshould be able to allow the development of several asters, even if we supposethat centrioles involved in meiosis sooner or later lose their ability to replicate,as seems to be the case for various freshwater gastropods (Raven, 1958, 1964).

The most likely interpretation accounting for such results would be that, in theabsence of induced paired cytasters, development is only possible from the spermintroduced centrioles. However, this remains to be tested further. An alternativeand non-exclusive hypothesis might be that there exists, in the egg, a mechanismresponsible for the regulation of the number of effective centrioles. This couldresult merely from the complete disappearance of the maternal centrioles at thetime when pronuclei develop, as seems to be the case in the sea-urchin egg(Sachs & Anderson, 1970). In this last species, centrioles reappear under theinfluence of pronuclei, when these are about to rupture. Thus, our own resultsmight suggest that one cannot obtain more asters than pronuclei present atthis stage. Such an interpretation remains rather speculative, since cytologicaltechniques used so far do not allow more than the observation of asters. Itfollows that one cannot decide whether meiotic centres have actually disappearedor whether some of them have simply lost their ability to induce astral configura-tions. The existence of a similar cytoplasmic regulatory mechanism could alsoexplain why trochal cells (Ia2-ld2) of mosaic embryos do not usually undergomore than two successive divisions (Costello, 1945).

III. Nuclear phenomena

In our experiments, cytochalasin B appeared unable to directly affect suchprocesses. Specifically, the division from tetrads to dyads and then the formationof single chromosomes is effected as normally during meiosis; likewise, pro-

Cytochalasin on a mosaic embryo 71nuclei are formed. Moreover, DNA synthesis seems to take place at this stage,as in normal development (Pasteels & Lison, 1951; Alfert & Swift, 1953) since,when chromosomes reappear, they seem to be typically duplicated. Similarly,treatments during early cleavage apparently do not affect mitotic cycles.Nevertheless some indirect effects can be described. Thus, the lack of extrusionof the first polar body allows the division of both sets of dyads, whilst in normalconditions dyads from the first polar body do not cleave.

On the other hand, treatment throughout meiosis gives rise to polyploid eggs,but this situation does not preclude pronuclear chromosomal duplication. Thisimplies that the egg is able to synthesize up to 2\ times the normal quantityof chromatin it usually does at this stage and that polyploidy is neither anobstacle to cleavage, nor to further differentiation, since some of the resultinglarvae appeared quite normal. With activated eggs, cytochalasin B allows theformation of diploid and tetraploid embryos which, however, do not go beyondthe monaster stage, confirming that haploidy is not the main cause of develop-mental failure.

IV. Morphogenetic processes

The regular occurrence of a normal percentage of swimming larvae, aftertreatment of fertilized eggs throughout meiosis with moderate cytochalasin Bconcentrations, confirms that this drug has no noticeable harmful effect on theoverall egg metabolism.

However, polyploidy which results from the lack of extrusion of polar bodiesappears unlikely to explain the important level of morphogenetic abnormalitiesfound in our material as well as in the egg ofLoligo (Arnold & Williams-Arnold,1970).

A great deal of experimental work has been accomplished on spiralianembryos, as reviewed by Raven (1966), Cather (1971), Guerrier (1971 a). Micro-surgical experiments have been performed on the egg of Sabellaria, stressingthe importance of regional differences in controlling major features of develop-ment (Hatt, 1932; Novikoff, 1938, 1940; Guerrier, 1970). Thus, first polar lobeexcision gives rise to larvae lacking post-trochal region, bristles and apical tuft.But similar larvae are also obtained frequently after treatment with cytochalasinB. This suggests that some kind of alteration has occurred at the level of thepolar lobe.

However, this structure seems to function quite normally after a 0-5 /*g/mltreatment applied throughout meiosis. Moreover, it is noteworthy that the samekind of abnormality develops even after a rather short exposure, limited to thebeginning of the meiotic period. The simplest explanation for these results isthat the deviation was induced long before polar lobe formation. In this con-nexion, it may be advisable to take into account some incidental experimentsreported by Hatt (1932) and which still need to be confirmed. By isolating thepresumptive polar lobe region at the time of first meiotic division, this author


seemed to have shown that definitive settlement of developmental capacities isnot completed at this stage. Thus the possibility must be considered that somedecisive phenomenon of ooplasmic segregation precedes the actual activatingprocesses which seem to proceed from the time of first polar lobe occurrence(Guerrier, 1971 b). If this were the case, then perhaps cytochalasin B couldmodify this pattern by interfering with the early meiotic cytoplasmic streamingmovements, as already described for Loligo with somewhat higher drug con-centration (Arnold & Williams-Arnold, 1970). However, such an interpretationis not completely satisfactory since we have shown by centrifugation thatdevelopment of lobe-dependent structures was also impaired after an abnormalequatorial cleavage, despite the fact that cytoplasmic materials were equallydistributed between the two resulting blastomeres (Guerrier, 1970).

Accounting for these difficulties, an alternative hypothesis would be that thisdrug had irreversibly affected some decisive element located in the membraneor in the cortical layer of the egg.

These last conclusions deserve to be tested further by carrying out moresurgical experiments on the uncleaved egg and by studying carefully the indivi-dual history of each treated egg.

RESUME

Action de la cytochalasine B sur la meiose et le developpement d'ceufs fecondes etactives de Sabellaria alveolata (Annelide polychete)

1. Des ceufs non fecondes, fecondes et actives de Sabellaria alveolata ont ete soumis a desdoses de cytochalasine B allant de 0,1 a 20/^g/ml. Leur evolution a ete etudiee tant in vivoqu'apres realisation de montages au carmin acetique.

2. L'emission des globules polaires, la cytodierese et la formation du lobe polaire sontcompletement inhibees par des doses tres faibles de cytochalasine B (0,3a 0,5 /ig/ml).

3. La realisation de caryotypes demontre que les processus chromosomiques meiotiqueset mitotiques ne sont en aucune maniere affectes par la drogue. En particulier, on peutobtenir une evolution normale d'embryons polyploides a partir d'oeufs fecondes et traites,tandis que les oeufs actives et traites restent toujours bloques en monaster. Cette situation estassez paradoxale dans la mesure ou une inhibition du processus d'emission des globulespolaires laisse subsister dans l'oeuf deux ou quatre centrosomes. Ces observations suggerentl'existence d'un mecanisme regulateur controlant le nombre de centrioles efficaces a Tissue dela meiose. Elles demontrent egalement que 1'evolution abortive des oeufs actives ne sauraitdependre de leur etat haploiide ou polyploide.

4. L'application de doses moderees de cytochalasine B pendant la meiose permet l'obtentionde larves nageuses. Bien que le developpement du lobe polaire n'apparaisse pas affecte,celles-ci presentent souvent des anomalies au niveau des structures soumises a son controle.De telles observations suggerent soit que la cytochalasine B altere irreversiblement quelqueelement decisif de la zone corticale, soit que les processus d'activation que nous avonsdecrits anterieurement et qui debutent lors de la formation du lobe polaire s'exercent reelle-ment sur des materiaux specifiques dont la localisation s'effectue au cours du processus dematuration.


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{Received 1 June 1973, revised 17 August 1973)

effects of cytochalasin b on meiosis and developmen otf ...solution of pure cacl2 (700 m-osmole). in...

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