/ . Embryol. exp. Morph. Vol. 60, pp. 71-82, 1980 71Printed in Great Britain © Company of Biologists Limited 1980

The effect of microtubule- andmicrofilament-disrupting drugs on preimplantation

mouse embryos

By G. S1RACUSA,1 D. G. WHITT1NGHAM2 AND M. DE FEL1C11

From the University of Rome and the MRC Mammalian Development Unit,University College, London

SUMMARY

The sensitivity of early preimplantation mouse embryos to drugs which disrupt micro-filament function (cytochalasin B-CB and cytochalasin D-CD) and microtubule assembly(colchicine, colcemid, vinblastine and griseofulvin) was examined. CD inhibited cleavage at aconcentration 35-fold lower than CB (3 x 10~7 M v. 1 x 10~5 M). Treatment of 2-cell embryosfor 6 h with 1 x 10~5 M CB or 1 x 10~6 M CD or continuous exposure to lower concentrationsof CB or CD did not affect development to the blastocyst stage in vitro. Vinblastine inhibitedcleavage at a concentration tenfold lower than colcemid or colchicine (1 x 10~8 M v. 1 x 10~7

M). The continuous presence of colcemid at 10~8 M did not affect the development of 2-cellembryos to the blastocyst stage, but development was reduced with vinblastine at I x 10~" Mand completely inhibited with colchicine at 1 x 10~8 M. The drugs produced similar responseswhen 2-cell embryos were treated for 6 h with concentrations that inhibited cleavage. Com-plete inhibition of cleavage was obtained after only a 2 h exposure to 2 x 10~7 M colchicine. Asimilar concentration of lumicolchicine did not affect cleavage or blastocyst formation.Embryos were less sensitive to griseofulvin; the first cleavage division was unaffected byconcentrations as high as 3X10~ 4 M and only 50% of 2-cell embryos failed to cleave in1 x 10 ' and 3x 10 4M griseofulvin. At these concentrations a small proportion of 1-cellembryos and the majority of the 2-cell embryos showed unequal cytoplasmic division probablycaused by the formation of multipolar spindles. The continuous exposure of 2-cell embryos to3 x 10'5 M griseofulvin did not affect blastocyst formation.

INTRODUCTION

The cell cytoskeleton consists of a complex cytoplasmic arrangement ofmicrotubules and microfilaments which are believed to be involved in manydifferent cell functions, such as cell movement, cell division, endo- and exo-cytosis, surface receptor mobility, etc. (see Allison, 1973). It has recently beensuggested that it might also play a role in the maintenance of the meiotic blockin the unfertilized mouse oocyte (Siracusa, Whittingham, Codonesu & DeFelici, 1978). Drugs affecting the cytoskeleton are currently being used to

^Authors' address: Istituto di Istologia e Embriologia Generate, Universitadi Roma, Via A.Scarpa 14, 00161 Roma, Italy.

"Author's address: MRC Mammalian Development Unit, Wolfson House, UniversityCollege London, 4 Stephenson Way, London NW1 2HE.

72 G. SIRACUSA, D. G. WHITTINGHAM AND M. DE FELICI

investigate the role of microtubules and microfilaments in these various cellularactivities. However, at high concentrations the drugs can produce non-specificeffects such as the inhibition of membrane transport (Mizel & Wilson, 1972;Wilson, 1975). Thus it is important to determine the lowest effective concen-tration of the drugs which will produce cytoskeletal disruption with the mini-mum of side effects and to know whether normal cytoskeletal functions areresumed when the drugs are removed. Experiments were designed to study theeffects of microfLlament- and microtubule-disrupting drugs on the cleavage ofpreimplantation mouse embryos in vitro. Six compounds were examined; fourwhich interfere with microtubule assembly (colchicine, colcemid, vinblastineand griseofulvin) and two which impair microfilament function (cytochalasin Band D). The reversibility of action of the drugs was also studied.

MATERIALS AND METHODS

Embryo collection

Adult female albino mice (MF1, Olac, U.K.) were superovulated with intra-peritoneal injections of 5 i.u. pregnant mares' serum gonadotrophin (PMSGFolligon, Intervet) given at 10.00 to 13.00 h and 5 i.u. of human chorionicgonadotrophin (hCG-Chorulon, Intervet) given approximately 48 h later. Thefemales were mated with albino males of the same strain. One-cell embryos atthe pronuclear stage and 2-cell embryos were flushed from the oviducts of matedfemales approximately 28 h and 51 h after the injection of hCG. Furtherdetails on the experimental treatments are given in the Results section. Theembryos were cultured in 10 /i\ droplets of medium (Whittingham, 1971) underparaffin oil, at 37 °C, in a humidified atmosphere of 5 % CO2 in air.

Chemicals

Vinblastine sulphate, colchicine, demecolcine (colcemid), griseofulvin, cyto-chalasin B (CB) and cytochalasin D (CD) were purchased from Sigma. Con-centrated stock solutions of colchicine, colcemid and vinblastine were preparedin triple distilled water, and kept frozen in small aliquots in the dark; eachaliquot was thawed and used only once. Griseofulvin and the cytochalasins weredissolved in dimethyl sulphoxide (DMSO) (BDH Analar) at 01 M and 1 mg/mlrespectively; the stock solutions were also kept frozen in the dark, but were usedseveral times after freezing and thawing without any apparent loss of potency.DMSO had no detrimental effect on cleavage when added to control groups inconcentrations of up to 1-5%.

Lumicolchicine was prepared from colchicine by a modification of a methoddescribed by Mizel & Wilson (1972). An aqueous solution of colchicine at 2 x10~5 M was irradiated in a quartz cuvette with two long wave u.v. (366 mm) lamps(Birchover Instruments Ltd.). The transformation of colchicine into its analoglumicolchicine was monitored every 10 min with a spectrophotometer by

Effects of drugs on preimplantation mouse embryos 73

100 r

10 10~9 10"8 10"

Drug concentration (M)

Fig. 1. The effect of microtubuie-disrupting drugs on the first cleavage of mouseembryos, a, colchicine; b, colcemid; c, vinblastine. Within each graph different sym-bols refer to different experiments. Totals of 213 (a), 112 (b) and 180 embryos (c)were used. The incidence of cleavage in 107 additional control embryos was 95-3 %.

measuring the absorbance at 350 nm (colchicine) and 261 nm (lumicolchicine).The ultraviolet irradiation was stopped when the 350 nm peak disappeared andthe 261 nm peak ceased to increase.

RESULTS

Dose-response experiments

First cleavage. Zygotes were removed and incubated in various drugs approxi-mately 28 h post hCG; the following morning the number of 1- and 2-cell

74 G. SIRACUSA, D. G. WHITTINGHAM AND M. DE FELICI

Table 1. A comparison of the effects of colchicine and lumicolchicine on first andsecond cleavage divisions, and blastocyst formation. Average of two experiments;19-20 embryos per treatment

% cleaved% cleaved to > 4-cell % blastocysts

Treatment to 2-cell (from 2-cell) (from 2-cell)

Control 100 100 95Colchicine 2 X10" 7 M 0 0 0Lumicolchicine 2 X10~7M 100 100 84

Table 2. Effect of griseofulvin on first and second cleavage divisions, andblastocyst formation. Average of three experiments. First cleavage: 55 embryosper treatment; second cleavage: 36 embryos per treatment

Griseofulvinconcentration (M)

03xl0- 5

io-4

3xlO-4

io-3

* 9%X The

% cleavedto 2-cell

100100100*loot

precipitates

% cleavedto ^ 4-cell

(from 2-cell)

9410086J42J

and t 31 % had blastomeres of unequal size.majority with blastomeres of unequal size.

% blastocysts(from 2-cell)

8186190

embryos was recorded. Vinblastine appeared to be the most powerful of the fourantimicrotubular agents tested (Fig. lc): a concentration of 10~8 M completelyprevented the zygotes from undergoing the first cleavage. Colcemid and col-chicine were tenfold less effective than vinblastine.

Fragmentation occurred in some of the embryos exposed to concentrationsthat only partially inhibited cleavage. This phenomenon was also observed withthe cytochalasins. The fragmented embryos were excluded from the calculationsbecause in this aberrant form of cytokinesis the embryos were neither blocked atthe 1-cell stage nor had they progressed to the 2-cell stage.

Lumicolchicine ( 2 X 1 0 ~ 7 M ) had no effect on the first cleavage division(Table 1).

Concentrations of griseofulvin up to 3 x 10~5 M did not affect the first mitoticdivision (Table 2). In the presence of 1 x 10~4 M and 3 x 10~4 M griseofulvin allthe zygotes completed cytokinesis, but in 9 and 31 % of the cases respectively,the mitosis yielded three daughter cells of unequal size. This probably resultedfrom the formation of tripolar spindles. Higher concentrations could not be used

Effects of drugs on preimplaniation mouse embryos 75

100 r

50

^ 0 L

50

0 L

10"8 10"7 10"6 10"

Drug concentration (M)

10"

Fig. 2. The effect of cytochalasin B (a) and cytochalasin D (b) on the first cleavage ofmouse embryos. Within each graph different symbols refer to separate experiments.A total of 121 (a) and 135 (b) embryos were used. The incidence of cleavage in 51additional control embryos was 92%.

because the griseofulvin slowly precipitated out of the medium during incu-bation.

The effect of cytochalasin B and cytochalasin D is shown in Fig. 2. CD pre-vented the first cleavage at a concentration (3 x 10~7 M) approximately 35-foldlower than CB (1 x 10~5M).

Second cleavage and blastocyst formation. Two-cell embryos removed approxi-mately 51 h post hCG were incubated in the various drugs. At approximately72 h post hCG, the number of embryos that had reached at least the 4-cell stagewas recorded. After an additional 48 h in the continuous presence of the drugthe number of embryos at the blastocyst stage was recorded. The results ob-tained for the second cleavage (Tables 1-4) were similar to those for the firstcleavage. Concentrations of the drugs that were ineffective in preventing thefirst and second cleavage divisions did not significantly affect the development of

76 G. SIRACUSA, D. G. WHITTINGHAM AND M. DE FELICI

Table 3. Effect of continuous incubation of 2-cell mouse embryos in micro tubule-disrupting drugs on the second cleavage division andb/astocyst formation. Averageof two experiments; 46 embryos per treatment

Treatment

ControlColcemid 10"8 MColcemid 10~7 MVinblastine lO"9 MVinblastine 10~8 MColchicine 10~8 MColchicine 10~7 M

% cleavedto 2s 4-cell

91984

919

9313

% blastocysts

8574

059

000

Table 4. Effect of continuous incubation of 2-cell mouse embryos in microfilament-disrupting drugs on the second cleavage division andblastocyst formation. Averageof two experiments; 40 embryos per treatment

Treatment

ControlCytochalasin B 10"6 MCytochalasin B lO"5 MCytochalasin D 3 x 10~8 MCytochalasin D 3 x 10~7 M

% cleavedto 2s 4-cell

9592

09517

% blastocysts

88650

652

2-cell embryos to the blastocyst stage, with the exception of colchicine andgriseofulvin. Colchicine 1 x 10~8 M had no effect on the first two cleavagedivisions (Fig. 1 and Table 3) but prevented blastocyst formation (only 65 %reached the 8-cell stage); lmuicolchicine had no effect on cleavage and blasto-cyst formation. Most of the 2-cell embryos which cleaved in the presence oflx lO" 4 and 3xlO~4M griseofulvin (86 and 42% respectively, Table 2)showed blastomeres of unequal size, No blastocysts were formed in the presenceof 3 x 10~4 M griseofulvin, and only 19 % in 1 x 10~4 M.

Recovery experiments

Two-cell embryos removed approximately 51 h post hCG were treated for6 h with effective inhibitory concentrations of the drugs, and then washed andtransferred to control medium to observe further development. At transfer,88 % (range 67-100 %) of the control embryos had cleaved to 4-cell stage, whileall the treated embryos were at the 2-cell stage. The next morning the treated

Effects of drugs on preimplantation mouse embryos 11

Table 5. In vitro development to the blastocyst stage of 2-cell mouse embryostreated for 6 h with microfilament- or microtubule-disrupting drugs. Average ofthree experiments {except colchicine, 1 expt.)

Treatment

ControlCytochalasin B 10~5 MCytochalasin D 10~6MControlColcemid 3 x 10~7 MVinblastine 3 x 10~8 MColchicine 3 x 10~7 M

% blastocysts

878582

9068180

No. of embryos

474845

67656726

Table 6. In vitro development to the blastocyst stage of 2-cell mouse embryostreated for various times with 3 x 10~7 M colchicine. Average of two experiments;34-38 embryos per treatment

Duration oftreatment % blastocysts

Control15 min30 minl h

2 h

4 h

839278

23

0

0

embryos still lagged one division behind the control embryos (4 cells v. 8 cells).At approximately 120 h post hCG the number of blastocysts was scored.

Embryos treated with CB or CD appeared to recover completely, and wereable to develop into blastocysts in similar proportion to the controls (Table 5).Development after a 6 h treatment with the microtubule-disruptive drugs wasless satisfactory. Only 68% of the embryos treated with 3 x 10~7 M colcemid and18 % of those treated with 3 x 10~8 M vinblastine reached the blastocyst stage v.90 % of the controls (Table 5). Colchicine treatment was irreversible, the 2-cellembryos treated with 3 x 10~7 M colchicine for 2h or more failed to resumecleavage (Table 6).

DISCUSSION

In this study we investigated the sensitivity of early preimplantation mouseembryos to microfilament- and microtubule-disruptive drugs, and the reversi-bility of such treatments. Two drugs which interfere with the function of micro-filaments, cytochalasin B and D, were examined. The mechanism of action of thedrugs has been only partially clarified. Apparently they cause a state of hyper-

6 EMB 60

78 G. SIRACUSA, D. G. WHITTINGHAM AND M. DE FELICI

contraction and displacement of the cortical mesh of microfilaments, and thisprobably entails detachment of the connexions between microfilaments and thecell membrane (Miranda, Godman, Deitch & Tanebaum, 1974 a). This postu-lated detachment may explain many of the effects of the cytochalasins, includingthe inhibition of cell division. The effect of the cytochalasins appears to berapidly reversible, since the microfilament web is reconstituted in the cortexwithin 1 h after withdrawal of CD (Miranda, Godman & Tanebaum, 19746). CDis more powerful than CB, and unlike CB it does not interfere with hexose up-take in most cell lines (Miranda et al. 1974a). Our experiments have confirmedthat mole for mole CD is more potent than CB in preventing cell division(Fig. 2 and Table 4). Complete inhibition of the first cleavage in mouse embryoswas obtained with a concentration of CD approximately 35-fold lower than CB(3 x 10~7 M v. 1 x 10~5 M). A similar concentration of CB (4 /*g/ml) was foundto be the lowest that totally prevented the morula to blastocyst transition ofmouse embryos (Granholm & Brenner, 1976). As already mentioned, at con-centrations immediately below the minimally effective ones, a proportion of theembryos underwent fragmentation. This phenomenon was also observed withmicrotu bule-disruptive drugs, and it is probably due to the disorderly activity ofa partly disrupted cytoskeleton.

It has been shown previously (Snow, 1973,1975;Niermerko, 1975; Tarkowski,Witkowska & Opas, 1977) that preimplantation mouse embryos treated for6-12 h with 5-10 /*g/ml CB at the 1- or 2-cell stage, can develop to the blastocyststage and beyond; the treatment induces polyploidy in most of the embryos.Similar findings of complete reversibility of CB effects have also been reportedfor a variety of cell types (Yamada, Spooner & Wessels, 1971; Spooner, Yamada& Wessels, 1971; Schaeffer, Schaeffer & Brick, 1973, etc.). We confirm that a6 h treatment with 1 x 10~5M CB (~ 5/£g/ml) is well tolerated by the 2-cellmouse embryos (Table 5) and have extended the observation to show that CDis equally effective at a concentration of 1 x 10~6M (~ 0-5/tg/ml). No attemptwas made to study whether CD causes any of the latent effects which becomevisible during post-blastocyst development after CB treatment of preimplanta-tion mouse embryos (Granholm & Brenner, 1976). The greater specificity of theaction of CD and the lower concentrations needed to obtain the same effecton cell cleavage as CB, makes it a more suitable choice for experiments in whichminimal damage to the embryo is essential, such as diploidization after removalof one pronucleus (Hoppe & Ulmensee, 1977).

The mechanism of action of three of the drugs which interfere with micro-tubular function (colchicine, colcemid and vinblastine) is well established: theybind to tubulin and prevent the polymerization of tubulin to form micro-tubules. Since most microtubules are in equilibrium with a soluble pool oftubulin, this results in the dissolution of microtubules (see review by Wilson,Anderson, Grisham & Chin, 1975). Vinblastine is the most potent drug in thegroup in preventing cleavage. The minimal effective concentration of vin-

Effects of drugs on preimplantation mouse embryos 79blastine (1 x 10~8 M) is tenfold lower than the concentration of colchicine orcolcemid required to obtain the same effect (Fig. 1 and Table 3). However, theeffect of colcemid was far more reversible than that of vinblastine or colchicine(Table 5). After a 6 h treatment with effective inhibitory concentrations of thedrugs, the development of 2-cell embryos to blastocysts was reduced from 75 %of controls with colcemid 3 x 10~7 M (~ 0-1 /tg/ml), to 20% with vinblastine3 X 1 0 ~ S M (~ 0-03 /tg/ml), to complete developmental arrest with colchicine3 x 10"7 M (~ 0-1 jug/mV). A short treatment with colcemid (1-5 h) was foundto be non toxic for rabbit eggs undergoing in vitro fertilization (Bomsel-Helmreich, 1965); the treatment produced triploid embryos by suppressing theextrusion of the second polar body.

Colchicine is known to be more 'toxic' than its derivative colcemid, and theaction of colcemid on cultured cells can be reversed more easily (see for instanceKleinfeld & Sisken, 1966; Daniels, 1975). To examine whether the irreversibilityof action of colchicine on mouse embryos was due to some non-specific toxiceffect, preimplantation embryos were also treated with lumicolchicine. Lumi-colchicine is a structural isomer of colchicine (obtained by u.v. irradiation ofcolchicine) which does not bind to tubulin or disrupt microtubules (Wilson et al.1974), but retains the non-specific properties of colchicine, such as the interactionwith cell membranes (Stadler & Franke, 1974) and the inhibition of nucleosidetransport (Mizel & Wilson, 1972). By comparing the effects of colchicine andlumicolchicine, specific (i.e. due to microtubule depolymerization) and non-specific effects of colchicine can therefore be discriminated (Mizel & Wilson,1972;Obikaef a/. 1978).

The present studies show that zygotes can cleave to 2-cell, and 2-cell embryoscan develop normally to blastocysts in the continuous presence of 2 x 10~7 Mlumicolchicine, whereas embryos treated similarly with colchicine (2x 10~ 7 M)

failed to divide or develop to the blastocyst stage (Table 1). Thus, the irreversi-bility of colchicine is related to its direct action on microtubules and not to anynon-specific side effects. This finding agrees with the observation that thebinding of tritium-labelled colchicine to purified tubulin at 37 °C is almostirreversible (Wilson et al. 1974); the colchicine-tubulin complex dissociationhalf-life is 36 h (Garland & Teller, 1973). Our results also show that the minimumperiod of exposure to 3 x 10~7 M colchicine, which allows the alkaloid to bind toa sufficient number of tubulin molecules to prevent cleavage, is 2 h (Table 6). Thisagrees with the biochemical findings which show a similar slow rate of col-chicine binding to purified tubulin (Wilson et al. 1975).

The fourth drug examined for its action on microtubular function wasgriseofulvin (a mould metabolite). Unlike the other microtubular-disruptivedrugs tested, its mechanism of action is not completely understood. Although1- and 2-cell mouse embryos appeared to be quite resistant to the action ofgriseofulvin, the second cleavage division was more sensitive than the first forcleavage inhibition and the incidence of unequal cytoplasmic division. Con-

6-2

80 G. SIRACUSA, D. G. WHITTINGHAM AND M. DE FELICI

centrations up to 3 X 1 0 ~ 4 M did not prevent the first cleavage division butcleavage was inJiibited in 50 % of 2-cell embryos treated with concentrations ofgriseofulvin between 1 x 10~4 and 3 x 10~4 M. Only a few of the embryos under-going first cleavage and the majority of those undergoing second cleavageshowed unequal cytoplasmic division when treated with 1 x 10~4 M or higherconcentrations of griseosulvin. The unequal cytoplasmic divisions were probablycaused by the formation of multipolar mitoses similar to those described inother cells treated with griseofulvin (Grisham, Wilson & Bensch, 1973; Adair,1974; Weber, Wehland & Herzog, 1976). Although the 2-cell embryos showedan apparent increase in sensitivity to griseofulvin, they were unaffected by thecontinuous presence of 3 x 10~5 M griseofulvin during culture to the blastocyststage (Table 2).

Other types of cells appear to be much more sensitive to the action of griseo-fulvin. Concentrations of 2-4 x 10~5 M griseofulvin cause 50 % mitotic arrest inmouse 3T3 cells (Weber et ah 1976) and in HeLa cells (Grisham et ah 1973) andcomplete metaphase block in Chinese hamster Y79 cells (Adair, 1974). At1 x 10~5 M griseofulvin there is a rapid but reversible disappearance of themeiotic spindle in Pectinaria oocytes (Malawista, Sato & Bensch, 1968). Morerecently the depolymerization of microtubules by griseofulvin has been shownby immunofluorescence microscopy in 3T3 cells at 5 x 10~5 M (Weber et ah 1976)and human fibroblasts at 5 x 10~4 M (Spiegelman, Lopata & Kirschner, 1979).The relative resistance of early mouse embryos to griseofulvin may be due to itslow permeability. This will now be investigated with the use of labelled griseo-fulvin.

Apart from the possible permeability barrier to the drug, a further explanationfor the low sensitivity of mouse embryos to griseofulvin can be postulated, basedupon recent observations on the regeneration of the microtubular system afterthe removal of the drug. According to Spiegelman et ah (1979) there appear tobe two types of microtubule-organizing centres: a single primary initiation site,which is probably the centriole, and multiple secondary sites. Since the secondarysites recover from griseofulvin treatment more slowly than the primary sitesSpiegelman et ah (1979) suggest that griseofulvin has a differential effect on thetwo types of initiation centres. Thus it is tempting to speculate that a connexionexists between the relative resistance of mouse embryos to griseofulvin and thelack of centrioles in mouse embryos up to the morula stage (Szollosi, 1972). Suchspeculation is further supported by the observation that plant cells, which alsolack centrioles, are much less sensitive of griseofulvin than animal cells (Deysson,1964).

This work was performed under C.N.R. Research Project 'Biology of Reproduction'(Grant, no. 79.01175.85), C.N.R. Grant CT 79.01010.04, and was also supported in part bythe Ford Foundation (Grant no. 790.0659).

Effects of drugs on preimplantation mouse embryos 81

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(Received 2 January 1980, revised 1 May 1980)