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Effects of Cytochalasin B on Meiosis and Development of Fertilized and Activated Eggs of Sabellaria Alveolata L

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Effects of Cytochalasin B on Meiosis and Development of Fertilized and Activated Eggs of Sabellaria Alveolata L /. Embryol. exp. Morph. Vol. 31, 1, pp. 61-74,1974 61 Printed in Great Britain Effects of cytochalasin B on meiosis and 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 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 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: 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 method gives 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 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 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 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 pre- maturation 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 pro- tuberance 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 pro- tuberance. 64 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 ana- phase. 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).
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