invertebrate Reproduction and /Jevelopment, 44:2-3 (2003) 131-137 131 8alaban, Philadelphia/Rehovot 0168-8170(03($05.0002003 8alaban

Effects of cytochalasin B on fertilization and ploidy in the Pacifie oyster Crassostrea gigas

CHRISTOPHE LEDU· and HELEN MCCOMBlE·· Laboratoire de Génétique et Pathologie. institut Français pour la Recherche et Exploitation de la Mer (iFREMFR), 17390 La Tremblade, France Tel. +33 (5) 46369836; Fax +33 (5) 46363751.. email: hmccombi@ifremerfr

Received 23 June 2003; Accepted 22 September 2003

Summary The effects ofcytochalasin B on fertilization and ploidy in Crassostrea gigas were examined in a controlled experiment. This enabled us to better understand why variable ploidy results have been found with the retention ofpolar bodies in previous studies on bivalve mollusks, and what the interaction with fertilization may be. By looking at the early development of Crassas/rea gigas embryos with sequential sampies stained using Hoechst 33258, a fluorescent DNA-specific dye, we compared normal evolution of fertilized eggs with that obtained with cytochalasin B treatrnent. In treated eggs, embryos with both three and four pronuclei were found after the second meiotic division. When embryos had three pronuclei, these were identified as the female pronucleus plus two chromatin groups arising from the retention of the two polar bodies. The fourth pronucleus present in sorne embryos was the male pronucleus. The absence ofthe male pronucleus in the embryos with only three pronuc1ei was interpreted as gynogenetic development due to the inhibitory effects of cytochalasin B on penetration of the sperm into the eggs. This result explains the findings of several previous studies where different ploidies resulted from double polar body inhibition in bivalve species. Implications ofsuch treatments for post-embryo­ genic development are discussed.

Key words: Polyploidy, polar body, cytochalasin B, fertilization, Crassostreagigas

Introduction 1991; Nell, 2002). The principle ofthis treatmenl is the Cytoehalasin B (CB) treatments have been used to inhibition of the first or second polar body (PB 1 or produce triploids in the Pacifie cupped oyster Crassa­ PB2) during the early stages of embryonie develo­ s/rea gigas and other commercial shellfish species pment (Longo, 1972). Either of these methods can be where they offer advantages for aquaculture in termsof used to produce triploids in C. gigas (e.g., Downing sterility and enhanced growth over their diploid and Alien, 1987; Yamamoto et al., 1988; Gérard et al., counterparts (reviews in Beaumont and Fairbrother, 1999). However, bloeking the expulsion of the first

'Current address: Laboratoire d'Aquaculture tropicale, Centre Océanographique de la Pacifique (COP), Institut Français pour :~ Recherche .et Exploitation de la Mer (IFREMER), BP 7004, 98719 Taravao, Tahiti, French Polynesia. Corre<'oqnd:'lg author. 132 c redll and fi ;'vkCombie I/RD 44 (2003) /3/-/37 polar body in different bivalves has been seen to pro­ Matcrials and Methods duce not only triploids (3n) but also tetraploids (4n) and in some cases pentaploids (5n) or numerous aneu­ Preparation ofgametes ploids ofvarying chromosome number (Yamamoto et Oysters collected from the Marennes-Oléron basin al., 1988; Guo et al., 1992a; Gérard et al., 1999; Yang (Charente-Maritime, France) were matured under et al., 2000a). Diverse ploidies issued from a single controlled conditions in the IFREMER hatchery at treated batch are presently explained by disordered La Tremblade (Charente-Maritime, France) for chromosome segregation (Komura et al., 1990; Guo et 2 months. The gametes were removed from the ripe a\.; 1992b; Longo et al., 1993; Yang et al., 2000b), or animais (10 fema\es, 6 males) by stripping the gonads. accidentai retention ofboth polar bod ies (Yamamoto et The oocytes were then filtered on 100 !lm screen and al., 1988; Komura et al., 1990), but it has also been the spermatozoids on 25 !lm and the male and female ~l1n suggested that a treatment to suppress PB 1 might affect gametes pooled separately in 1 filtered seawater. A ploidy through effects on fertilization and first water bath was prepared at 25°C for the fertilization (Diter and Dufy, 1990; Gendreau and Grizel, 1990). It and treatments. is known that cytochalasin B inhibits polymeri­ zation (Maclean-Fletcher and Pollard, 1980) and that ce treatments and embryo sampling this could lead to the non-penetration ofspermatozoids Ali treatments and samplings were made at precise (Longo, 1978) in addition to the targeted retention of points in time after the sperm and oocytes were mixed polar bodies in polyploidy induction experiments. The (t = 0 min). The experiment comprised a control with principle objectives of our present study were to no CB (Batch 1) and two separate CB treatments examine the effects of the chemical on fertilization, to (Batches 2 and 3, started at t =2 and t = 3 min, respect­ see whether non-penetrating sperm could nevertheless ively). The three batches each had 15 million oocytes activate eggs and to observe subsequent inhibition of with spermatozoids (oocyte:spennatozoid ratio=1:200) polar bodies for the potential effects on ploidy. in 1 L filtered seawater at 25°C. CB (Sigma C-6762), ln previous studies, the retention of both polar dissolved in dimethylsulfoxide (DMSO, Lab-Osi bodies produced mostly tetraploid and pentaploid lar­ D-770) was added to each of the treatment beakers to vae in mussels Mytilus galloprovincialis (Scarpa et al., produce final concentrations of0.5 mg/1. The CR treat­ 1993). In the dwarf surf clam Mulinia lateralis ments lasted 25 min in order to block the expulsion of (Peruzzi and Guo, 2002), triploids, tetraploids and both polar bodies. The CB was then removed by pentaploids were found. Embryos from similar treat­ filtration on a 10 ~lm screen and the embryos rinsed in ments on C gigas (Cooper and Guo, 1989) and the DMSO (1 ml/L) in seawater for 15 min. They were Japanese pearl oyster Pinctada fucata martensii subsequcntly re-filtered and put in c1ean filtercd (Komura et al., 1990) were pentaploid. seawater. Tetraploids are valued in aquaculture for the pro­ Samples for embryo study were taken at fertili­ duction of triploids via 2nx 4n crosses, which in con­ zation and every following 5 min until 80 min post­ trast to chemical methods give 100% triploids (Chour­ fertilization. One ml was taken at each interval and was rout ct al., 1986; Guo et al., 1996) and are now used in immediately fixed in in a glucosamine­ hatchery production of trip10id oysters (Nell, 2002). acetate buffer (Dubé et al., 1985). The present experiment was also designed to explain why high numbcrs of tetraploids can some­ times be seen when both polar bodies are blocked, Embryo coloration and observation rather than uniquely the expected pentaploicls given by The sampled embryos were colored with Hoechst the addition of four genomes from the female (female 33258 (Sigma B-2883), a fluorochrome, and examined pronucleus plus the content oftwo non-expulsed polar by epifluorescence using an Olympus BH-2 UV micro­ bod ies, the second twice the size of the tirst) and one scope. One hundred embryos were studied per sample from the male. Our study of CB-treated embryos for their state of development: expulsion of polar showed evidence for the expected inhibitory effect of bodies and division. Polar body counting (Beau­ CB on tèrtilization. Following this, our examination of mont, 1986) aliows the inhibitory action of the CR to the meioses and the tirst metaphase allowed us to be assessed in a manner corresponding to the ploidy examine in c1etail the effects of the treatment on devel­ level. opment and how it affected subsequent p10idy of the emhrvos. C. Ledu and fi. McCombie /!RD 44 (2003J 131-137 133

Results In both the CB treatments, the retenti on of the two polar bodies witl1in the embryos eould be obscrved The control provides an example of normal diploid resulting in two extra nuclei. The number of nuclei development (r'igs. 1a-g). Expulsion of the tirst PB should be four (male pronucleus, female pronucleus, started between 10 and 15 min after fertilization PB 1 and PB2) (Fig. 2a). At 50 min, 21 % of batch 2 (Fig. 1b), followed by the second PB during meiosis Il (Fig. le and d). Expulsed polar bodies ean be readily and 16 % of batch 3 showed this type of development. observed because they remain stuck to the outside of However, there were more cases in both batches where the cmbryo from expulsion until afler the first cleav­ there were only three nuclei and no expulsed polar age. The spermatozoid decondenses gradually (Fig. 1d) bodies (26% and 25% in batches 2 and 3 respectively; and tùses with the female pronucleus for karyogamy Fig. 2b). The missing nucleus was identified as the (Fig. 1e). The tirst mitosis then takes place (Fig. 1t), male pronucleus, given that the male amI female pro­ followed by division (Fig. 1g), which began between nuc!ei can be easi Iy distinguished because the female 45 and 50 minutes in our control. These events took pronuc\eus is found unuer the polar bodies at the edge place more slowly in the presence of cytochalasin B; of the cell (Fig. 2a and b). meiosis Il appeared 5-10 min later than in the control. Karyogamy took place with either the three or four M itosis and , where observed, were about pronuclei, after the removal of the CB. Finally, whilst 20 min later than the control. in the control, first cell division had started by 50 min,

a) b) c)

d) e) f)

Fig. 1. Normal diploid embryo dcvclopmcnl \VitiJ timl~ post-ferlili­ zntion until appearance of each stage. (a) meiosis 1 mctaphase; (b) expulsion ofPr1 l, la -15 min; (c) and (d) expulsion ofPB2, 25~30 min: (c) karyogamy. 35-40 min; (t) first mitosis 40-45 min: ----f.gClL) --'(g) cell division \Vilh formation ofp()lai~ lobe -1550 min. 134 C. Ledit and fl. McCombie IIRD 44 (2003) 131 ~I 37

b)

Fig. 2. Embryos resulting from the retention of both Polar Bodies (PBs) (a) with male pronucleus; (b) without male pronucleus. in the treatments, the tirst divided eells were only seen Discussion at 70 min implying that the treatment eould have had Our results show that early fertilization, embryo an effect on capaeity for, or speed of, subsequent mito­ development and ploidy outcome are markedly differ­ sis and division. Delayed mitosis in the treated batehes ent between treated and untreated C. gigas eggs. Under could be partly explained by the delay in meiosis II normal fertilization conditions in marine invertebrates, mentioned above, although the difference with the the membranes of the spermatozoid and oocyte fuse. control had become greater by mitosis. Based on the The sperm then achieves its entry into the oocyte via a amount of genetic material, specimens with four pro­ cytoplasmic bridge which is created by actin polymeri­ nuclei and no released polar bodies (Fig. 2a) would zation (Colwin and Colwin, 1963; Longo and Scarpa, develop as pentaploids and those with three (Fig. 2b) 1991). Cytochalasin B inhibits actin polymerization as tetraploids. (Maclean-Fletcher and Pollard, 1980), and therefore, if The two CH treatments were not markedIy different applied close to fertilization, could prevent sperm from in timing of development or numbers of pronuclei. penetrating the egg as has already been demonstated in Very few expulsed PBs were observed, maximum 8% molluscs and echinoderms (Longo, ]978; Schatten and in batch 2 and 5% in batch 3. These started to be seen Schatten, ]981). In the present experiment, the CB just after the removal of the CB, and though they were treatments were made at 2 or 3 min after mixing sperm probably delayed PB 1s might also have been PB2s and oocytes, meaning that etfects on the fertilization judging by the control time seale where sorne eggs mechanism are likely. In previous studies aiming to were still releasing either PB 1 or PB2 at this time. retain PB 1 (or both PBs) in bivalves (e.g., Yamamoto Although the two types of development shown in et al., 1988; Cooper and Guo, 1989; Scarpa et al., Fig. 2 were the most common at 50 min, others were 1993; Guo et al., 1992a; Gérard et aL, 1999; Yang et also seen. The l'est of the samples showed a variety of al., 2000a), the added sperm inhibition effect may have formations present at low frequencies (<;5%), sorne of been partial or absent because the treatment was later which were difficult to interpret. There were examples relative to sperm penetration and embryogenic devel­ with five or more pronuclei, which were observee! both opment. This would explain the absence of tetraploid in the treatments and control. The most likely reason C gigas embryos in the double retention experiment of for this is polyspermy (multiple fertilization) (Togo and Cooper and Guo (J 989). ln addition, the development Morisawa, 1999). Tripolar segregation at meiosis II ofCrassostrea sp. eggs is asynchronous (Longwell and and the formation oftwo nuclei were also occasionally Stiles, ]968; Desrosiers et al., ] 993), meaning that in observed. Tripolar segregation can lead ta expulsion of our study some eggs would have been ready to be a PI32 containing variable amounts ofgenetic material fertilized before the CS treatment was applied but that (Guo et al., 1992b). The most extreme formation that for others the penetration of the spermatozoids would we observed was the expulsion ofail chromatin groups have becn inhibited by the CB. In the latter case, the in a single polar body. There were some also unfer­ eggs woule! be activatee! but unfertilized, leading to tilizee! eggs in al! samples which remainecl al meta­ gynogeneticdevelopment. This effect would have been I·,·,,,~ ,,~ ~.-_I phase of ll1eiosis 1(Fig. 1a) enhanced by our lise of strioncd (1()(,\ltpç .. C Ledu and H. McCombie / lRD 44 (2003) 131-137 135 females which could have increased variability in states to establish if the early cell divisions we observed of devclopment. occurred in embryos where feliilization had bcen In a previous study with early CB treatment to inhibited. Detailed study of the centrosomes them­ retain P131 for tri plo id induction, Yamamoto et al. selves, using more precise techniques like fluorcscent (1988) suggested that the percentage of tetraploid and immunocytochemical marking (Stephano and GOllid pentaploid C. gigas embryos that they subsequently 2000), could be used to establish whether or not a observed had been generated by the double retention of maternaI meiotic centrosome could play this important PB 1 and PB2. Diter and Dufy (1990), working on role in mitosis. Rudi/apes philippinarum and Gendreau and Grizel Sorne previous authors have also noted five or (1990), working on Os/rea edulis, suggested that tetra­ variable numbers of pronuclei in studies on other ploidy arose from inhibition of the first mitosis due to species where bath polar bodies were retaincd (Longo, ahsence ofthe spennatozoid inhibited by the CB. None ]972; Komura et al., ]990; Scarpa et al., ]993). Other of these studies examined embryogenesis in detail. In types of development we observed may result from our experiment, with double polar body inhibition, the abnormal segregation following the inhibition of PB] resulting embryos would be pentaploids if fertilized as observed by Guo et al. (] 992b) or from polyspcrmy. and tetraploids if gynogenetic. This is in aceordance A further study of the subsequent stages of devel­ with Guo et al.'s (]993) observations of 4n C. gigas opment will help to understand the impOliance of the cmbryos produced by double PB retention and fertili­ events we have observed for future developmcnt and zation with UV-inactivated sperm, which gave 4n survival of induced polyploid C. gigas. At 4 h post­ gynogens, and with the Sn embryos observed in the fertilization, we did observe some tetraploid and penta­ same species by Cooper and Guo (]989) where viable ploid mitotic metaphases on microscope preparations sperm was used. ]n 2002, Peruzzi and Guo suggested made from the embryos of the CR treatments, which that the 4n/5n embryo mixture they obtained with underline that mitotic divisions continue beyond the double P8 inhibition on Mulinia la/eralis could have early embryonic stages. However, we know of no been due ta chromosome loss from some Sn embryos reports of viable surviving pentaploid progenies in or accidentai release ofPB2 associated with abnormal bivalves. This could be due to physical problems of segregation, although embryogenic events were not cell division associatcd with large nucleus to cell recorded. Very few PBs were released in our treated volume ratio (Guo and Allen, ]994). Tetraploids in­ samples, and the identification ofthe male pronucleus duced directly from diploid bivalves have been seen to or its absence using epifluoresence provides a clear survive to post-rnetamorphic stages in several bivalve explanation for a mixture of4nJ5n ploidies. Our results species (Scarpa et al., ]993; Allen et al., 1994; Peruzzi do not preclude a subsequent effect on first mitosis due and Guo, 2002). For aquaculture, this method avoids to inhibition of sperm penetration. In fact, we ob­ selecting fertile triploid females otherwise required as scrved, as in Scarpa et al. (1993), that the first division maternai parents in a cross ta make tetraploids (Guo was later (70 min) in the treated embryos than in the and Allen, ]994), which might select for undesired control (50 min), which may also be a consequence of fertility in future triploid progeny. Exploitation of fer­ spermatie inhibition. Scarpa et al. (1992) suggested tilization inhibition combined with polar body retention that the absence of sperm in gynogenetic induction could also provide a means ofproducing tetraploid and cou Id prevent l'irst mitosis, c1eavage and subsequent diploid gynogens for selection programs and genetic clevelopmcnt because of the absence of a paternal studies. centrosome and Diter and Dufy (1990) that the first mitotic division and cleavage would be delayed by this means but that development could resume later. How­ Acknowledgements ever, Hei Ibrunn (1925) suggested for Cummingia (Mollusca) and Washitani-Nemoto et al. (1994) for We are indebted to André Gerard for initiating this As/erina pec/imfera (Echinodermata) that the inhibi­ project. We would also like to thank the technical staff tion ofpolar body expulsion in unfertilized eggs couId at Ifremer La Tremblade for the care and preparation of compensate for this lack of centrosome due to the the animals used in this stlldy and Pascal Phelipot for presence of meiotic centrosomes which would allow praetieal assistance ctllring the induction experiment. normal mitosis and first cleavage to take place. This Finally we thank Pierre Boudry, Carol McCombic and would explain why we obscrved continued cmbryo A lexanrlra Leitao who gave us useful advice on the dGvclopn~ellt, although further study will be nccessary preparation of this 113nllscript. 136 c. Ledu und H. A1cCambie / IRD ·14 (2003) 131-J37

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