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

0 From the Station Biologique , Roscoff SUMMARY 1. Unfertilized, fertilized and activated eggs of Sabellaria alveolata were submitted to cytochalasin B concentrations ranging from 0 1 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. - 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: (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 - 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). 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 cytoch (...truncated)