/ . Embryol. exp. Morph. Vol. 60, pp. 71-82, 1980
Printed in Great Britain © Company of Biologists Limited 1980
71
The effect of microtubule- and
microfilament-disrupting drugs on preimplantation
mouse embryos
By G. S1RACUSA, 1 D. G. WHITT1NGHAM 2 AND M. DE FEL1C1 1
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 microfilament function (cytochalasin B-CB and cytochalasin D-CD) and microtubule assembly
(colchicine, colcemid, vinblastine and griseofulvin) was examined. CD inhibited cleavage at a
concentration 35-fold lower than CB (3 x 10~7 M v. 1 x 10~5 M). Treatment of 2-cell embryos
for 6 h with 1 x 10~5 M CB or 1 x 10~6 M CD or continuous exposure to lower concentrations
of CB or CD did not affect development to the blastocyst stage in vitro. Vinblastine inhibited
cleavage 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-cell
embryos to the blastocyst stage, but development was reduced with vinblastine at I x 10~" M
and completely inhibited with colchicine at 1 x 10~8 M. The drugs produced similar responses
when 2-cell embryos were treated for 6 h with concentrations that inhibited cleavage. Complete inhibition of cleavage was obtained after only a 2 h exposure to 2 x 10~7 M colchicine. A
similar concentration of lumicolchicine did not affect cleavage or blastocyst formation.
Embryos were less sensitive to griseofulvin; the first cleavage division was unaffected by
concentrations as high as 3 X 1 0 ~ 4 M and only 50% of 2-cell embryos failed to cleave in
1 x 10 ' and 3x 10 4 M griseofulvin. At these concentrations a small proportion of 1-cell
embryos and the majority of the 2-cell embryos showed unequal cytoplasmic division probably
caused by the formation of multipolar spindles. The continuous exposure of 2-cell embryos to
3 x 10' 5 M griseofulvin did not affect blastocyst formation.
INTRODUCTION
The cell cytoskeleton consists of a complex cytoplasmic arrangement of
microtubules and microfilaments which are believed to be involved in many
different cell functions, such as cell movement, cell division, endo- and exocytosis, surface receptor mobility, etc. (see Allison, 1973). It has recently been
suggested that it might also play a role in the maintenance of the meiotic block
in the unfertilized mouse oocyte (Siracusa, Whittingham, Codonesu & De
Felici, 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, University
College 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 cellular
activities. However, at high concentrations the drugs can produce non-specific
effects such as the inhibition of membrane transport (Mizel & Wilson, 1972;
Wilson, 1975). Thus it is important to determine the lowest effective concentration of the drugs which will produce cytoskeletal disruption with the minimum of side effects and to know whether normal cytoskeletal functions are
resumed when the drugs are removed. Experiments were designed to study the
effects of microfLlament- and microtubule-disrupting drugs on the cleavage of
preimplantation mouse embryos in vitro. Six compounds were examined; four
which interfere with microtubule assembly (colchicine, colcemid, vinblastine
and griseofulvin) and two which impair microfilament function (cytochalasin B
and 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 intraperitoneal injections of 5 i.u. pregnant mares' serum gonadotrophin (PMSG
Folligon, Intervet) given at 10.00 to 13.00 h and 5 i.u. of human chorionic
gonadotrophin (hCG-Chorulon, Intervet) given approximately 48 h later. The
females were mated with albino males of the same strain. One-cell embryos at
the pronuclear stage and 2-cell embryos were flushed from the oviducts of mated
females approximately 28 h and 51 h after the injection of hCG. Further
details on the experimental treatments are given in the Results section. The
embryos were cultured in 10 /i\ droplets of medium (Whittingham, 1971) under
paraffin oil, at 37 °C, in a humidified atmosphere of 5 % CO2 in air.
Chemicals
Vinblastine sulphate, colchicine, demecolcine (colcemid), griseofulvin, cytochalasin B (CB) and cytochalasin D (CD) were purchased from Sigma. Concentrated stock solutions of colchicine, colcemid and vinblastine were prepared
in triple distilled water, and kept frozen in small aliquots in the dark; each
aliquot was thawed and used only once. Griseofulvin and the cytochalasins were
dissolved in dimethyl sulphoxide (DMSO) (BDH Analar) at 01 M and 1 mg/ml
respectively; the stock solutions were also kept frozen in the dark, but were used
several times after freezing and thawing without any apparent loss of potency.
DMSO had no detrimental effect on cleavage when added to control groups in
concentrations of up to 1-5%.
Lumicolchicine was prepared from colchicine by a modification of a method
described by Mizel & Wilson (1972). An aqueous solution of colchicine at 2 x
10~5 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 analog
lumicolchicine was monitored every 10 min with a spectrophotometer by
Effects of drugs on preimplantation mouse embryos
100
73
r
10
10~9
10"8
10"
Drug concentration (M)
Fig. 1. The effect of microtubuie-disrupting drugs on the first cleavage of mouse
embryos, a, colchicine; b, colcemid; c, vinblastine. Within each graph different symbols 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 and
the 261 nm peak ceased to increase.
RESULTS
Dose-response experiments
First cleavage. Zygotes were removed and incubated in various drugs approximately 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 and
second cleavage divisions, and blastocyst formation. Average of two experiments;
19-20 embryos per treatment
% cleaved
to 2-cell
% cleaved
to > 4-cell
(from 2-cell)
% blastocysts
(from 2-cell)
Control
Colchicine 2 X 1 0 " 7 M
100
0
100
0
95
0
Lumicolchicine 2 X10~ 7 M
100
100
84
Treatment
Table 2. Effect of griseofulvin on first and second cleavage divisions, and
blastocyst formation. Average of three experiments. First cleavage: 55 embryos
per treatment; second cleavage: 36 embryos per treatment
Griseofulvin
concentration (M)
0
3xl0-5
io-4
4
3xlOio- 3
% cleaved
to 2-cell
100
100
100*
loot
% cleaved
to ^ 4-cell
(from 2-cell)
94
100
86J
42J
% blastocysts
(from 2-cell)
81
86
19
0
precipitates
* 9% and t 31 % had blastomeres of unequal size.
X T h emajority with blastomeres of unequal size.
embryos was recorded. Vinblastine appeared to be the most powerful of the four
antimicrotubular agents tested (Fig. lc): a concentration of 10~8 M completely
prevented the zygotes from undergoing the first cleavage. Colcemid and colchicine were tenfold less effective than vinblastine.
Fragmentation occurred in some of the embryos exposed to concentrations
that only partially inhibited cleavage. This phenomenon was also observed with
the cytochalasins. The fragmented embryos were excluded from the calculations
because in this aberrant form of cytokinesis the embryos were neither blocked at
the 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 mitotic
division (Table 2). In the presence of 1 x 10~4 M and 3 x 10~4 M griseofulvin all
the zygotes completed cytokinesis, but in 9 and 31 % of the cases respectively,
the mitosis yielded three daughter cells of unequal size. This probably resulted
from the formation of tripolar spindles. Higher concentrations could not be used
Effects of drugs on preimplaniation mouse embryos
100
75
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 of
mouse 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 51
additional control embryos was 92%.
because the griseofulvin slowly precipitated out of the medium during incubation.
The effect of cytochalasin B and cytochalasin D is shown in Fig. 2. CD prevented the first cleavage at a concentration (3 x 10~7 M) approximately 35-fold
lower than CB (1 x 1 0 ~ 5 M ) .
Second cleavage and blastocyst formation. Two-cell embryos removed approximately 51 h post hCG were incubated in the various drugs. At approximately
72 h post hCG, the number of embryos that had reached at least the 4-cell stage
was recorded. After an additional 48 h in the continuous presence of the drug
the number of embryos at the blastocyst stage was recorded. The results obtained for the second cleavage (Tables 1-4) were similar to those for the first
cleavage. Concentrations of the drugs that were ineffective in preventing the
first 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 tubuledisrupting drugs on the second cleavage division andb/astocyst formation. Average
of two experiments; 46 embryos per treatment
Treatment
% cleaved
to 2s 4-cell
% blastocysts
Control
Colcemid 10" 8 M
Colcemid 10~7 M
Vinblastine lO" 9 M
Vinblastine 10~8 M
Colchicine 10~8 M
Colchicine 10~7 M
91
98
4
91
9
93
13
85
74
0
59
0
0
0
Table 4. Effect of continuous incubation of 2-cell mouse embryos in microfilamentdisrupting drugs on the second cleavage division andblastocyst formation. Average
of two experiments; 40 embryos per treatment
Treatment
Control
Cytochalasin
Cytochalasin
Cytochalasin
Cytochalasin
B 10" 6 M
B lO" 5 M
D 3 x 10~8 M
D 3 x 10~7 M
% cleaved
to 2s 4-cell
% blastocysts
95
92
0
95
17
88
65
0
65
2
2-cell embryos to the blastocyst stage, with the exception of colchicine and
griseofulvin. Colchicine 1 x 10~8 M had no effect on the first two cleavage
divisions (Fig. 1 and Table 3) but prevented blastocyst formation (only 65 %
reached the 8-cell stage); lmuicolchicine had no effect on cleavage and blastocyst formation. Most of the 2-cell embryos which cleaved in the presence of
l x l O " 4 and 3xlO~ 4 M griseofulvin (86 and 42% respectively, Table 2)
showed blastomeres of unequal size, No blastocysts were formed in the presence
of 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 for
6 h with effective inhibitory concentrations of the drugs, and then washed and
transferred to control medium to observe further development. At transfer,
88 % (range 67-100 %) of the control embryos had cleaved to 4-cell stage, while
all 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 embryos
treated for 6 h with microfilament- or microtubule-disrupting drugs. Average of
three experiments {except colchicine, 1 expt.)
Treatment
% blastocysts
Control
Cytochalasin B 10~5 M
Cytochalasin D 10~ 6 M
Control
Colcemid 3 x 10~7 M
Vinblastine 3 x 10~8 M
Colchicine 3 x 10~7 M
87
85
82
90
68
18
0
No. of embryos
47
48
45
67
65
67
26
Table 6. In vitro development to the blastocyst stage of 2-cell mouse embryos
treated for various times with 3 x 10~7 M colchicine. Average of two experiments;
34-38 embryos per treatment
Duration of
treatment
% blastocysts
83
92
78
23
0
0
Control
15 min
30 min
lh
2h
4h
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 were
able to develop into blastocysts in similar proportion to the controls (Table 5).
Development after a 6 h treatment with the microtubule-disruptive drugs was
less satisfactory. Only 68% of the embryos treated with 3 x 10~7 M colcemid and
18 % 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-cell
embryos treated with 3 x 10~7 M colchicine for 2 h or more failed to resume
cleavage (Table 6).
DISCUSSION
In this study we investigated the sensitivity of early preimplantation mouse
embryos to microfilament- and microtubule-disruptive drugs, and the reversibility of such treatments. Two drugs which interfere with the function of microfilaments, cytochalasin B and D, were examined. The mechanism of action of the
drugs has been only partially clarified. Apparently they cause a state of hyper6
EMB
60
78
G. SIRACUSA, D. G. WHITTINGHAM AND M. DE FELICI
contraction and displacement of the cortical mesh of microfilaments, and this
probably entails detachment of the connexions between microfilaments and the
cell membrane (Miranda, Godman, Deitch & Tanebaum, 1974 a). This postulated detachment may explain many of the effects of the cytochalasins, including
the inhibition of cell division. The effect of the cytochalasins appears to be
rapidly reversible, since the microfilament web is reconstituted in the cortex
within 1 h after withdrawal of CD (Miranda, Godman & Tanebaum, 19746). CD
is more powerful than CB, and unlike CB it does not interfere with hexose uptake in most cell lines (Miranda et al. 1974a). Our experiments have confirmed
that 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 embryos
was 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 found
to be the lowest that totally prevented the morula to blastocyst transition of
mouse embryos (Granholm & Brenner, 1976). As already mentioned, at concentrations immediately below the minimally effective ones, a proportion of the
embryos underwent fragmentation. This phenomenon was also observed with
microtu bule-disruptive drugs, and it is probably due to the disorderly activity of
a partly disrupted cytoskeleton.
It has been shown previously (Snow, 1973,1975;Niermerko, 1975; Tarkowski,
Witkowska & Opas, 1977) that preimplantation mouse embryos treated for
6-12 h with 5-10 /*g/ml CB at the 1- or 2-cell stage, can develop to the blastocyst
stage and beyond; the treatment induces polyploidy in most of the embryos.
Similar findings of complete reversibility of CB effects have also been reported
for a variety of cell types (Yamada, Spooner & Wessels, 1971; Spooner, Yamada
& Wessels, 1971; Schaeffer, Schaeffer & Brick, 1973, etc.). We confirm that a
6 h treatment with 1 x 10~ 5 M CB (~ 5/£g/ml) is well tolerated by the 2-cell
mouse embryos (Table 5) and have extended the observation to show that CD
is equally effective at a concentration of 1 x 10~6M (~ 0-5/tg/ml). No attempt
was made to study whether CD causes any of the latent effects which become
visible during post-blastocyst development after CB treatment of preimplantation mouse embryos (Granholm & Brenner, 1976). The greater specificity of the
action of CD and the lower concentrations needed to obtain the same effect
on cell cleavage as CB, makes it a more suitable choice for experiments in which
minimal damage to the embryo is essential, such as diploidization after removal
of one pronucleus (Hoppe & Ulmensee, 1977).
The mechanism of action of three of the drugs which interfere with microtubular function (colchicine, colcemid and vinblastine) is well established: they
bind to tubulin and prevent the polymerization of tubulin to form microtubules. Since most microtubules are in equilibrium with a soluble pool of
tubulin, this results in the dissolution of microtubules (see review by Wilson,
Anderson, Grisham & Chin, 1975). Vinblastine is the most potent drug in the
group in preventing cleavage. The minimal effective concentration of vin-
Effects of drugs on preimplantation mouse embryos
79
8
blastine (1 x 10~ M) is tenfold lower than the concentration of colchicine or
colcemid required to obtain the same effect (Fig. 1 and Table 3). However, the
effect of colcemid was far more reversible than that of vinblastine or colchicine
(Table 5). After a 6 h treatment with effective inhibitory concentrations of the
drugs, 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 vinblastine
3 X 1 0 ~ S M (~ 0-03 /tg/ml), to complete developmental arrest with colchicine
3 x 10"7 M (~ 0-1 jug/mV). A short treatment with colcemid (1-5 h) was found
to be non toxic for rabbit eggs undergoing in vitro fertilization (BomselHelmreich, 1965); the treatment produced triploid embryos by suppressing the
extrusion of the second polar body.
Colchicine is known to be more 'toxic' than its derivative colcemid, and the
action of colcemid on cultured cells can be reversed more easily (see for instance
Kleinfeld & Sisken, 1966; Daniels, 1975). To examine whether the irreversibility
of action of colchicine on mouse embryos was due to some non-specific toxic
effect, preimplantation embryos were also treated with lumicolchicine. Lumicolchicine is a structural isomer of colchicine (obtained by u.v. irradiation of
colchicine) which does not bind to tubulin or disrupt microtubules (Wilson et al.
1974), but retains the non-specific properties of colchicine, such as the interaction
with cell membranes (Stadler & Franke, 1974) and the inhibition of nucleoside
transport (Mizel & Wilson, 1972). By comparing the effects of colchicine and
lumicolchicine, specific (i.e. due to microtubule depolymerization) and nonspecific 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 embryos
can develop normally to blastocysts in the continuous presence of 2 x 10~7 M
lumicolchicine, whereas embryos treated similarly with colchicine (2x 1 0 ~ 7 M )
failed to divide or develop to the blastocyst stage (Table 1). Thus, the irreversibility of colchicine is related to its direct action on microtubules and not to any
non-specific side effects. This finding agrees with the observation that the
binding of tritium-labelled colchicine to purified tubulin at 37 °C is almost
irreversible (Wilson et al. 1974); the colchicine-tubulin complex dissociation
half-life is 36 h (Garland & Teller, 1973). Our results also show that the minimum
period of exposure to 3 x 10~7 M colchicine, which allows the alkaloid to bind to
a sufficient number of tubulin molecules to prevent cleavage, is 2 h (Table 6). This
agrees with the biochemical findings which show a similar slow rate of colchicine binding to purified tubulin (Wilson et al. 1975).
The fourth drug examined for its action on microtubular function was
griseofulvin (a mould metabolite). Unlike the other microtubular-disruptive
drugs tested, its mechanism of action is not completely understood. Although
1- and 2-cell mouse embryos appeared to be quite resistant to the action of
griseofulvin, the second cleavage division was more sensitive than the first for
cleavage inhibition and the incidence of unequal cytoplasmic division. Con6-2
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 but
cleavage was inJiibited in 50 % of 2-cell embryos treated with concentrations of
griseofulvin between 1 x 10~4 and 3 x 10~4 M. Only a few of the embryos undergoing first cleavage and the majority of those undergoing second cleavage
showed unequal cytoplasmic division when treated with 1 x 10~4 M or higher
concentrations of griseosulvin. The unequal cytoplasmic divisions were probably
caused by the formation of multipolar mitoses similar to those described in
other cells treated with griseofulvin (Grisham, Wilson & Bensch, 1973; Adair,
1974; Weber, Wehland & Herzog, 1976). Although the 2-cell embryos showed
an apparent increase in sensitivity to griseofulvin, they were unaffected by the
continuous presence of 3 x 10~5 M griseofulvin during culture to the blastocyst
stage (Table 2).
Other types of cells appear to be much more sensitive to the action of griseofulvin. Concentrations of 2-4 x 10~5 M griseofulvin cause 50 % mitotic arrest in
mouse 3T3 cells (Weber et ah 1976) and in HeLa cells (Grisham et ah 1973) and
complete metaphase block in Chinese hamster Y79 cells (Adair, 1974). At
1 x 10~5 M griseofulvin there is a rapid but reversible disappearance of the
meiotic spindle in Pectinaria oocytes (Malawista, Sato & Bensch, 1968). More
recently the depolymerization of microtubules by griseofulvin has been shown
by 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 its
low permeability. This will now be investigated with the use of labelled griseofulvin.
Apart from the possible permeability barrier to the drug, a further explanation
for the low sensitivity of mouse embryos to griseofulvin can be postulated, based
upon recent observations on the regeneration of the microtubular system after
the removal of the drug. According to Spiegelman et ah (1979) there appear to
be two types of microtubule-organizing centres: a single primary initiation site,
which is probably the centriole, and multiple secondary sites. Since the secondary
sites recover from griseofulvin treatment more slowly than the primary sites
Spiegelman et ah (1979) suggest that griseofulvin has a differential effect on the
two types of initiation centres. Thus it is tempting to speculate that a connexion
exists between the relative resistance of mouse embryos to griseofulvin and the
lack of centrioles in mouse embryos up to the morula stage (Szollosi, 1972). Such
speculation is further supported by the observation that plant cells, which also
lack 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 by
the 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)