Effects of cytochalasin B on meiosis and development of fertilized and activated eggs of Sabellaria alveolata L. (Polychaete Annelid)

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 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:

• The fact that the regulative treatment of any two-step activating method gives rise to cytasters.

• 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. (1971 a, 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.

Sand tube blocks of Sabellaria 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/μ1, 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–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, 1971 a).

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. 1A). This process, however, does not affect more than a small percentage of the eggs, since a 2 h treatment of 2 μg/ml gives no more than 6% modified eggs, this proportion decreasing to 0·4% when 0·2 μg/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.

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 μg/ml, concentrations of 0·3 μg/ml or more completely stop it.

At low concentrations (0·3–0·5 μg/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 μg/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.

The pattern of changes just described applies also to eggs treated after the first polar body extrusion. Nevertheless, at the end of telophase, 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.

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, 1973b).

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 μg/ml cytochalasin B the lobes completely regress in 1–2 min. On the other hand, when eggs are removed from a 0·5 μg/ml solution and washed carefully, some 15 min before first cleavage, the polar lobe develops normally 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. 1D). 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 μg/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 μg/ml. With moderate concentrations of about 2 μg/ml, we sometimes observed that first cleavage could not be completed, giving rise to binucleate eggs which, as a rule, will nevertheless segment further.

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 μg/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 μg/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 μg/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 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.

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.

Treatment with 0·5 μg/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.

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.

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 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, 1971 a,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 μg/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.

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 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 Bo veri’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 (la₂–ld₂) of mosaic embryos do not usually undergo more than two successive divisions (Costello, 1945).

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, pronuclei 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 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.

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 of Loligo (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). Micro-surgical 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 μg/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 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.

1. Des œufs non fécondés, fécondés et activés de Sabellaria alveolata ont été soumis à des doses de cytochalasine B allant de 0,1 à 20 μg/ml. Leur évolution a été étudiée tant in vivo qu’après réalisation de montages au carmin acétique.

2. L’émission des globules polaires, la cytodiérèse et la formation du lobe polaire sont complètement inhibées par des doses très faibles de cytochalasine B (0,3à 0,5 μg/ml).

3. La réalisation de caryotypes démontre que les processus chromosomiques méiotiques et mitotiques ne sont en aucune manière affectés par la drogue. En particulier, on peut obtenir une évolution normale d’embryons polyploïdes à partir d’œufs fécondés et traités, tandis que les œufs activés et traités restent toujours bloqués en monaster. Cette situation est assez paradoxale dans la mesure où une inhibition du processus d’émission des globules polaires laisse subsister dans l’œuf deux ou quatre centrosomes. Ces observations suggèrent l’existence d’un mécanisme régulateur contrôlant le nombre de centrioles efficaces à l’issue de la méiose. Elles démontrent également que l’évolution abortive des œufs activés ne saurait dépendre de leur état haploïde ou polyploïde.

4. L’application de doses modérées de cytochalasine B pendant la méiose permet l’obtention de larves nageuses. Bien que le développement du lobe polaire n’apparaisse pas affecté, celles-ci présentent souvent des anomalies au niveau des structures soumises à son contrôle. De telles observations suggèrent soit que la cytochalasine B altère irréversiblement quelque élément décisif de la zone corticale, soit que les processus d’activation que nous avons décrits antérieurement et qui débutent lors de la formation du lobe polaire s’exercent réellement sur des matériaux spécifiques dont la localisation s’effectue au cours du processus de maturation.