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Effects of cytochalasin B on meiosis
and development of fertilized and activated eggs of
Sabellaria alveolata L. (Polychaete Annelid)
By G. PEAUCELLIER, 1 P. GUERRIER 1 AND J. BERGERARD 1
From the Station Biologique, Roscoff
SUMMARY
1. Unfertilized, fertilized and activated eggs of Sabellaria alveolata were submitted to
cytochalasin B concentrations ranging from 01 to 20/tg/ml. Their behaviour was studied
either /// vivo or in acetocarmine squash preparations.
2. Polar body extrusion, cytokinesis and polar lobe formation are completely inhibited by
cytochalasin B concentrations as low as 0-3-0-5 /*g/ml.
3. Caryotype determinations demonstrate that chromosomal meiotic and mitotic processes
are not affected by the drug. Thus, polyploid embryos usually developed from fertilized eggs
whilst they did not from activated ones. This is related to the contrasting behaviour of meiotic
and cleavage centres. While the latter duplicates at each cycle, the former cannot replicate
at the end of meiosis. This leads to an abortive monastral stage even if inhibition of polar
body extrusion has provided the egg with two or four centres. These observations suggest the
existence of an internal mechanism regulating the number of effective centrioles at the end of
meiosis. They demonstrate also that the main cause of developmental failure in activated eggs
cannot be related to ploidy.
4. Eggs treated throughout meiosis with moderate drug concentrations developed into
swimming larvae. However, frequent developmental abnormalities affecting lobe dependent
structures were obtained even if polar lobe formation was unimpaired. This suggests either
that cytochalasin B has irreversibly affected some decisive cortical element or that previously
described activating processes, which begin with polar lobe formation, are actually exerted
on specific materials segregated during meiosis.
INTRODUCTION
In a study of the ability of the egg of Sabellaria alveolata to develop parthenogenetically, we found a technique which elicits all the early processes usually
brought about by fertilization but without ensuing cleavage. These processes,
which include the extrusion of polar bodies, lead only to the formation of a
monaster, instead of the normal first cleavage spindle, so that development does
not proceed any further.
Such a situation is frequently explained by assuming that, after completion
of meiosis, there is no more than one centre in the oocyte, which is unable to
replicate (Tyler, 1941). This assumption fits well with two observations:
1
Authors' address: Station Biologique, Place Georges-Teissier, 29211 Roscoff, France.
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G. PEAUCELLIER AND OTHERS
(a) The fact that the regulative treatment of any two-step activating method
gives rise to cytasters.
(b) The fact that, in species where fertilization normally induces the achievement of meiosis, one cannot obtain parthogenetic cleavage unless one polar
body 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 fundamental
cell metabolism (Spooner, Yamada & Wessels, 1971; Prescott, Myerson &
Wallace, 1972; Zigmond & Hirsch, 1972; Raff, 1972) appeared an ideal tool for
testing 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 cytokinesis 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 bodies
in 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 eggs
before applying it to activated eggs. In this way it was possible to demonstrate
a difference in behaviour between meiotic and cleavage centres. Several other
features were noted which it is worth while to report.
MATERIALS AND METHODS
Sand tube blocks of Sabellaha were collected in the vicinity of Roscoff and
maintained in running sea-water. In these conditions, animals remain in good
condition for many weeks. Shedding occurs spontaneously as soon as worms
are extracted from their individual tubes. Therefore before putting them in
bowls of filtered sea-water, they were first washed with running sea-water and
tap water in order to eliminate the possibility of sperm contamination of
oocytes. By this treatment, the number of naturally fertilized eggs does not
exceed a few per thousand.
Egg shedding is stopped after 15 min by removing the laying females while
the eggs wait another 45 min to ensure that they have all completed the prematuration process to reach the stable state of waiting oocyte (i.e. metaphase
of the first meiotic division). Successful artificial fertilization (about 80%) is
obtained with a final sperm concentration (spectrophotometric determination
at 460 nm) of about 15000 sperm//tl, using pooled gametes from different
individuals.
Parthenogenetic activation resulted from a 30 min treatment in a hypotonic
solution of pure CaCl2 (700 m-osmole). In such conditions about 50 % of the
eggs are activated, but this percentage is only an average since it can vary from
90 to 10%, according to the experiment.
Cytochalasin B (I.C.I., Macclesfield, Cheshire, U.K.) was prepared as a
0-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 in
filtered sea-water at concentrations referred to in the text. Controls developed
normally in a 2 % solution of DMSO, a concentration which corresponds to
the highest one used in the present work.
For accurate chromosome counting, cleaving eggs were treated for 30 min
with a 0-15 % colchicine solution. The eggs, fixed for 30 min to 1 h in Carnoy's
fluid, were stained for at least 3 h in acetocarmine. Cytological studies were
performed either on whole mounts or on squashes for caryotype determinations.
Living eggs were also studied by the hanging drop technique, free or compressed
as previously described (Guerrier, 1971a).
RESULTS
I. Effects on unfertilized eggs
Cytochalasin B seems not to be very harmful to the egg. However, in some
eggs we found that cytoplasmic extrusions developed in the perivitelline space.
These appear to remain bound by a membrane, as there is no yolk dispersion
in the perivitelline space and as they can be resorbed more or less completely
after returning the egg to sea-water. Such protuberances may appear at any
point 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, this
proportion decreasing to 0-4 % when 0-2 /*g/ml is applied for the same length of
time. The same blebbing phenomenon can also affect fertilized eggs, where it is
especially 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 before
the usual time for polar body extrusion. While this process is not affected at
0-1 /*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 its
usual position at the animal pole and there is often an indication of the protuberance which usually precedes polar body extrusion. However, this protrusion
is not quite characteristic for it is much wider than usual. Moreover, it regresses
rapidly or degenerates into cytoplasmic blebbing. As a result, the two sets of
dyads remained in the egg cytoplasm. As in normal development, there is no
pronucleus formation at this stage.
With higher concentrations, ranging from 5 to 20 ^g/ml, anaphase of first
maturation division does not take place in the normal position but right in the
centre of the egg. No other modification of chromosomal processes is observed,
nor is there any indication of animal pole flattening or of the meiotic protuberance.
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G. PEAUCELLIER AND OTHERS
B
D
25//m
Cytochalasin on a mosaic embryo
65
B. Second maturation division
The pattern of changes just described applies also to eggs treated after the first
polar body extrusion. Nevertheless, at the end oftelophase, astral rays vanish while
pronuclei appear as in normal development. As far as we can tell from the
cytological techniques used in this study, it seems that the two sets of maternal
chromosomes usually fuse in the same pronucleus while sperm chromosomes
give rise to the male pronucleus.
In eggs treated before the onset of the first maturation division, two spindles
develop when controls are engaged in the second maturation division. These
spindles are more or less parallel to each other but may present different
orientations relative to the egg surface. As illustrated on Fig. 1C, each spindle
carries a set of dyads which are engaged simultaneously in the process of anaphase. Chromosomes then fade away and seem again to give rise only to one
male and one female pronucleus.
C. Early cleavage
During the overall pronuclear stage the acetocarmine stain is unable to reveal
the existence of any astral figure. However, when pronuclear membranes break
down, we must stress that one always obtains a single effective cleavage spindle.
Depending on whether the eggs have been treated before or after the extrusion
of the first polar body, the metaphase plate exhibits 80 or 48 chromosomes,
which appeared to be normally duplicated. This corresponds to pentaploidy or
triploidy (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 B
treatment we do not observe any attempt of the egg to produce such a formation. This holds true not only for eggs treated from the onset of meiosis but also
for those which were only treated from 10 to 15 min before the usual time for
polar lobe occurrence. In addition, when eggs with developing polar lobes are
exposed 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 washed
carefully, some 15 min before first cleavage, the polar lobe develops normally
Fig. 1. Acetocarmine squashes from Sabellaria aheolata eggs. Living egg diameter is
about 60 /tm. Swelling through preparative treatment is about twofold. (A) Cytoplasmic 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 to
sea-water: pentaploid anaphase of first cleavage with polar lobe occurrence.
(C) Activated egg treated with 0-5/*g/mI throughout meiosis: two simultaneous
anaphases corresponding to the usual process of second polar body formation and
to an unusual new division of first polar body material. (D) Fertilized egg treated
with 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.
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EMB 31
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G. PEAUCELLIER AND OTHERS
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 production of binucleate eggs. Other cell processes seem to be unaffected and astral
figures double at each cycle. Thus, one can obtain a second cleavage tetrapolar
anaphase without ensuing cytokinesis (Fig. ID). This phenomenon proceeds
quite 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 washed
and returned to sea-water, they cleave normally, despite their polyploid state.
As we mentioned before, their development is only slightly delayed relative to
the controls. When higher concentrations were used, cleavage also resumed but
with frequent abnormalities. Thus, abortion and abnormal cleavage are quite
common, with concentrations ranging from 10 to 20/tg/ml. With moderate
concentrations of about 2 /*g/ml, we sometimes observed that first cleavage
could not be completed, giving rise to binucleate eggs which, as a rule, will
nevertheless 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 event
taking place at about the 36th h of development.
Eggs maintained in solutions leading to an inhibition of cytokinesis (0-5 /*g/ml
or more) cytolyse in a few hours, but swimming larvae differentiate in more
dilute solutions. Eggs returned to normal sea-water after a treatment limited to
the meiotic period always give rise to swimming larvae about 10 h after
fertilization, except when very high concentrations, of the order of 20 /^g/ml are
used, 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 larvae
50 h old.
From the observations made at this latter stage it appears that there is always
a 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 and
returned to normal sea-water did not give more than about 20 % of larvae
bearing post-trochal bristles, despite the fact that cleavage of these embryos
seemed to proceed normally (Fig. 2B, C, D). Similarly, eggs treated with the
same concentration for a short length of time just at the beginning of the meiotic
phase and which are returned to normal conditions about 40 min before the
first cleavage, do not give more than 40 % of successfully differentiated larvae.
With higher concentrations fewer larvae remain alive, the rate of abnormalities
increasing gradually with the concentration. Whatever the level of abnormalities
encountered may be, such larvae remain quite active. One cannot estimate their
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Cytochalasin on a mosaic embryo
Fig. 2. Larvae from control and treated eggs of Sabellaria alveolata. (A) Normal
trochophore 68 h old. (B) Slightly abnormal 68 h larva obtained after a 0-5 /tg/ml
treatment throughout meiosis. (C) Abnormal 42 h trochophore bearing an apical
tuft but lacking the overall post-trochal region (same experiment). (D) Abnormal
68 h larva from the same experiment.
further viability, however, since rearing is a most uncertain and time-consuming
task, 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 observed
on larvae bearing post-trochal bristles. Nevertheless, it appears that post-trochal
structures are reduced or lacking in the greatest part of the population. Fig. 2C
illustrates a rather frequent anomaly. This trochophore of 42 h still bears an
apical tuft which, normally, would have been replaced by the shortest apical
cilia. Furthermore, it is deprived of the post-trochal region and, if we consider
only this feature, looks like a lobeless embryo (NovikofF, 1940). Hence, it is
noteworthy that, in most cases, we are not dealing merely with a simple
deformation of normally occurring structures, but rather with the result of
a highly modified pattern of differentiation.
5-2
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III. Effects on activated eggs
A. Steps of normal activation
First indications of a successful activation do not appear before the eggs are
returned to sea water. Meiosis proceeds as in fertilized eggs but leads to the
formation of a single pronucleus of normal aspect. When the pronuclear membrane 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 astral
figure which bears the haploid set of chromosomes (Fig. 1E). At high magnification these appeared to be normally duplicated. The embryo does not develop further and one cannot observe the so-called monastral cycles so frequently
described in other species. The time schedule of these processes corresponds
accurately to that observed in normal development, zero time being no longer
related to fertilization but to the cessation of the treatment inducing parthenogenesis.
B. Effect of the drug
Treatment with 0-5 /*g/ml gives quite similar results to those described for
fertilized eggs. The first maturation spindle is normally situated at the animal
pole but first polar body extrusion is inhibited. When treatment is maintained,
two spindles develop which may take various positions. Then, chromosomes are
grouped again in a single tetraploid pronucleus. When treatment was stopped
before the second maturation cleavage or initiated after the first polar body
extrusion, eggs were obtained which carried one polar body and a diploid
nucleus.
Such eggs were always returned to sea-water. In every case, disappearance of
the pronucleus was accompanied by the development of a single astral figure
which was fitted with diploid or tetraploid sets of normally duplicated chromosomes (Fig. 1F). Here again, development appears to be blocked at the monaster
stage.
DISCUSSION
Some peculiar features observed during this study need to be discussed. They
relate to the nuclear, astral and cytoplasmic mechanisms at work during
meiosis, mitosis and cytokinesis, or to the important problem of what factors
control 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 in
various other species tested so far (Carter, 1972), cytochalasin B affects cleavage
cytokinesis. It also impedes polar body extrusion as shown by Longo (1972) on
the egg of Spisula solidissima. Similarly, it is effective in preventing polar lobe
Cytochalasin on a mosaic embryo
69
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 mitotic
cytokinesis exhibit the same sensitivity with respect to cytochalasin B. Moreover, it seems that Sabellaria eggs respond to the drug in a quite similar manner
to the eggs of the sea-urchin (Schroeder, 1969, 1972) and of the squid Loligo
(Arnold & Williams-Arnold, 1970). However, they react differently from
Xenopus eggs (Bluemink, 1971a, b; Hammer, Sheridan & Estensen 1971) or
mammalian cells in culture (Carter, 1967; Krishan & Ray-Chaudhuri, 1969;
Estensen, 1971; Krishan, 1972) which always show a clear indication of
furrowing.
Such discrepancies might be related to differences in the degree of permeability of the plasma membrane with respect to cytochalasin B. The recent
microinjection experiments performed by De Laat, Luchtel & Bluemink (1973)
on the egg of Xenopus seem, indeed, to demonstrate that furrowing is actually
sensitive to cytochalasin B from the onset of cytokinesis, but that this drug
would normally enter the egg only at the time when a brief increase in permeability is produced, some few minutes after furrow induction.
One can then suppose that the egg of Sabellaria is readily permeable to cytochalasin B from the early beginning of cytokinesis, which prevents furrow
development.
The mechanism of polar body extrusion seems to exhibit the same sensitivity
to cytochalasin B, since polar body protuberance and meiotic furrowing are
simultaneously inhibited. Our data differ on this point from those obtained by
Longo (1972) on the egg of Spisula, since this author did not observe any
inhibition 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 animal
pole meiotic protuberance found in Spisula depends merely on a lower viscosity
of the animal pole cortex, a situation which could also account for the protrusions we observed at this stage on the egg of Sabellaria.
However, it would seem that the meiotic furrow constriction which develops
at the base of the polar body protuberance is the result of a mechanism in every
sense identical with that of cleavage cytokinesis.
II. Mitotic apparatus
Our data confirm that even very high concentrations of cytochalasin B have
no direct effect on the mitotic apparatus. Thus, the size and time of appearance
of meiotic and cleavage spindles are not modified by the drug.
However, indirect effects are quite interesting. Thus, the formation of two
independent 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 not
develop.
In activated eggs, the use of cytochalasin B demonstrates that none of the
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G. PEAUCELLIER AND OTHERS
meiotic spindles remains able to give rise to the cleavage spindle. It seems likely
that this might require activating treatments which modify more thoroughly the
schedule of normally occurring meiotic processes, as we have found using
hypertonic sea-water (Peaucellier, 1973 a).
On the other hand, it is noteworthy that the number of centres which remain
in the treated egg at the end of meiosis has no effect on the number of asters
that will appear at time of first cleavage, since fertilized eggs have a normal
dicentric spindle whilst activated eggs are only provided with a monaster. This
strongly contrasts with the effect of cytochalasin B on cleavage divisions where the
lack of cytokinesis does not preclude the normal doubling of asters, leading to
multipolar figures.
Dealing with fertilized eggs, one can suppose, in accordance with Boveri's
theory (1906), that the sperm aster inhibits the development of any aster of
maternal origin, though paternal origin of first cleavage centres has not been
proven so far in Sabellaria alveolata (Faure-Fremiet, 1924). However, the consistent appearance of a monaster in activated eggs cannot be explained by
Boveri's theory, since, after cytochalasin B-induced inhibition of the extrusion
of one or both polar bodies, two or four centres might remain in the egg. These
should be able to allow the development of several asters, even if we suppose
that 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 the
absence of induced paired cytasters, development is only possible from the sperm
introduced centrioles. However, this remains to be tested further. An alternative
and non-exclusive hypothesis might be that there exists, in the egg, a mechanism
responsible for the regulation of the number of effective centrioles. This could
result merely from the complete disappearance of the maternal centrioles at the
time when pronuclei develop, as seems to be the case in the sea-urchin egg
(Sachs & Anderson, 1970). In this last species, centrioles reappear under the
influence of pronuclei, when these are about to rupture. Thus, our own results
might suggest that one cannot obtain more asters than pronuclei present at
this stage. Such an interpretation remains rather speculative, since cytological
techniques used so far do not allow more than the observation of asters. It
follows that one cannot decide whether meiotic centres have actually disappeared
or whether some of them have simply lost their ability to induce astral configurations. The existence of a similar cytoplasmic regulatory mechanism could also
explain why trochal cells (Ia 2 -ld 2 ) of mosaic embryos do not usually undergo
more than two successive divisions (Costello, 1945).
III. Nuclear phenomena
In our experiments, cytochalasin B appeared unable to directly affect such
processes. Specifically, the division from tetrads to dyads and then the formation
of single chromosomes is effected as normally during meiosis; likewise, pro-
Cytochalasin on a mosaic embryo
71
nuclei 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 extrusion
of the first polar body allows the division of both sets of dyads, whilst in normal
conditions 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. This
implies that the egg is able to synthesize up to 2\ times the normal quantity
of chromatin it usually does at this stage and that polyploidy is neither an
obstacle to cleavage, nor to further differentiation, since some of the resulting
larvae appeared quite normal. With activated eggs, cytochalasin B allows the
formation of diploid and tetraploid embryos which, however, do not go beyond
the monaster stage, confirming that haploidy is not the main cause of developmental failure.
IV. Morphogenetic processes
The regular occurrence of a normal percentage of swimming larvae, after
treatment of fertilized eggs throughout meiosis with moderate cytochalasin B
concentrations, confirms that this drug has no noticeable harmful effect on the
overall egg metabolism.
However, polyploidy which results from the lack of extrusion of polar bodies
appears unlikely to explain the important level of morphogenetic abnormalities
found in our material as well as in the egg ofLoligo (Arnold & Williams-Arnold,
1970).
A great deal of experimental work has been accomplished on spiralian
embryos, as reviewed by Raven (1966), Cather (1971), Guerrier (1971 a). Microsurgical experiments have been performed on the egg of Sabellaria, stressing
the importance of regional differences in controlling major features of development (Hatt, 1932; Novikoff, 1938, 1940; Guerrier, 1970). Thus, first polar lobe
excision gives rise to larvae lacking post-trochal region, bristles and apical tuft.
But similar larvae are also obtained frequently after treatment with cytochalasin
B. This suggests that some kind of alteration has occurred at the level of the
polar lobe.
However, this structure seems to function quite normally after a 0-5 /*g/ml
treatment applied throughout meiosis. Moreover, it is noteworthy that the same
kind of abnormality develops even after a rather short exposure, limited to the
beginning of the meiotic period. The simplest explanation for these results is
that the deviation was induced long before polar lobe formation. In this connexion, it may be advisable to take into account some incidental experiments
reported by Hatt (1932) and which still need to be confirmed. By isolating the
presumptive polar lobe region at the time of first meiotic division, this author
72
G. PEAUCELLIER AND OTHERS
seemed to have shown that definitive settlement of developmental capacities is
not completed at this stage. Thus the possibility must be considered that some
decisive phenomenon of ooplasmic segregation precedes the actual activating
processes which seem to proceed from the time of first polar lobe occurrence
(Guerrier, 1971 b). If this were the case, then perhaps cytochalasin B could
modify this pattern by interfering with the early meiotic cytoplasmic streaming
movements, as already described for Loligo with somewhat higher drug concentration (Arnold & Williams-Arnold, 1970). However, such an interpretation
is not completely satisfactory since we have shown by centrifugation that
development of lobe-dependent structures was also impaired after an abnormal
equatorial cleavage, despite the fact that cytoplasmic materials were equally
distributed between the two resulting blastomeres (Guerrier, 1970).
Accounting for these difficulties, an alternative hypothesis would be that this
drug had irreversibly affected some decisive element located in the membrane
or in the cortical layer of the egg.
These last conclusions deserve to be tested further by carrying out more
surgical experiments on the uncleaved egg and by studying carefully the individual history of each treated egg.
RESUME
Action de la cytochalasine B sur la meiose et le developpement d'ceufs fecondes et
actives de Sabellaria alveolata (Annelide polychete)
1. Des ceufs non fecondes, fecondes et actives de Sabellaria alveolata ont ete soumis a des
doses de cytochalasine B allant de 0,1 a 20/^g/ml. Leur evolution a ete etudiee tant in vivo
qu'apres realisation de montages au carmin acetique.
2. L'emission des globules polaires, la cytodierese et la formation du lobe polaire sont
completement 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 meiotiques
et mitotiques ne sont en aucune maniere affectes par la drogue. En particulier, on peut
obtenir 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 est
assez paradoxale dans la mesure ou une inhibition du processus d'emission des globules
polaires laisse subsister dans l'oeuf deux ou quatre centrosomes. Ces observations suggerent
l'existence d'un mecanisme regulateur controlant le nombre de centrioles efficaces a Tissue de
la meiose. Elles demontrent egalement que 1'evolution abortive des oeufs actives ne saurait
dependre de leur etat haploiide ou polyploide.
4. L'application de doses moderees de cytochalasine B pendant la meiose permet l'obtention
de 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 quelque
element decisif de la zone corticale, soit que les processus d'activation que nous avons
decrits anterieurement et qui debutent lors de la formation du lobe polaire s'exercent reellement sur des materiaux specifiques dont la localisation s'effectue au cours du processus de
maturation.
Cytochalasin on a mosaic embryo
73
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{Received 1 June 1973, revised 17 August 1973)