Mutagenesis vol. 20 no. 1 pp. 65--75, 2005
doi:10.1093/mutage/gei010
Aneuploidy in mouse metaphase II oocytes exposed in vivo and in vitro
in preantral follicle culture to nocodazole
Fengyun Sun1,2, Ilse Betzendahl1, Francesca Pacchierotti3,
Roberto Ranaldi3, Johan Smitz4, Rita Cortvrindt4
and Ursula Eichenlaub-Ritter1,
1
Institute of Genetechnology/Microbiology, Faculty of Biology, University
of Bielefeld, D-33501 Bielefeld, Germany, 3Section of Toxicology and
Biomedical Sciences, ENEA CR Casaccia, I-00060 Rome, Italy and
4
EggCentris NV, Kranenberg 6, B-1731 Zellik, Belgium, Free University
Brussels, Laarbeeklaan 101, B-1090 Brussels, Belgium
Aneuploidy tests are important in evaluating genetic
hazards especially when chemical exposures are suspected
to affect the fidelity of chromosome segregation in oocytes
and embryos. In the current study, a newly established
method, mouse preantral follicle culture, was employed to
grow oocytes in vitro within follicles. The sensitivity of
in vitro grown follicle enclosed oocytes was compared with
oocytes maturing in vivo in the ovary. In both the cases,
oocytes were exposed to the cytostatic chemical, nocodazole, from the time of hormonally stimulated resumption of
meiosis. The in vivo study revealed a significant decrease in
the number of ovulated mouse oocytes and an increase in
meiosis I-arrested and hyperploid metaphase II oocytes at
a single i.p. dose of 70 mg/kg body weight of nocodazole.
A significant increase was also observed in the number
of meiosis I-arrested and hyperploid mouse oocytes from
preantral follicle culture, when they were cultured in the
presence of >30 nM nocodazole during the final stages of
maturation. This concentration is slightly lower than that
previously shown to induce nondisjunction in denuded
mouse oocytes or in cultured human lymphocytes. The
higher sensitivity of the in vitro matured oocytes from preantral follicle culture than that of denuded oocytes may be
related to a synergistic adverse influence of nocodazole on
the oocyte, on somatic cell integrity and on cell--cell communication, which possibly also affects ovulation in vivo.
When expressed in molarity relative to the mouse weight,
the effective dose of the acute exposure in vivo is 3--4 orders
of magnitude higher than the lowest effective concentration
employed continuously in vitro. Reduced bioavailability of
nocodazole to the target cells due to its poor water solubility
may contribute to this difference. Preantral follicle culture
can be helpful in analysing mechanisms in chemically
induced aneuploidy in mammalian oogenesis, and in predicting the consequences of chemical exposures in vivo.
Introduction
Aneuploidy resulting from chromosome nondisjunction at
female meiosis imposes a significant impact on human
health (Hook, 1983; Parry et al., 1996; Aardema et al., 1998;
Eichenlaub-Ritter, 1998, 2002, 2003). The incidence of
aneuploidy has been estimated to reach at least 5% and
possibly up to 20% in clinically recognized conceptions
(Hassold and Hunt, 2001). Disturbances in chromosome
segregation in oocytes and aneuploidy of the embryo are
responsible for subfertility as they can cause implantation
failure or congenital abnormalities leading to spontaneous
abortion in humans. The adverse influence of chemicals on
oogenesis and folliculogenesis is also likely to contribute to
subfertility when they interfere with the differentiation and
survival of the follicle or with ovulation. Thus, there is a need
to study the consequences of chemical exposures during oogenesis in experimental animals as well as in suitable in vitro
models to identify mechanisms of action, targets and thresholds
for risk assessment.
Nocodazole,methyl [5-(2-thienylcarbonyl)-1H-benzimidazol2-yl] carbamate, a synthetic anti-tumour drug interfering with
microtubule assembly (De Brabander et al., 1976; Heath and
Rethoret, 1981), competes with colchicine for the same binding
site on the Arg-390 of b-tubulin (Sackett and Varma, 1993) and
directly affects microtubule polymerization and microtubuledependent processes (Wilson et al., 1974; Heath and Heath,
1978; Heath and Rethoret, 1981; Mejillano et al., 1996).
Perturbation of the microtubule polymerization can activate
the ‘spindle checkpoint’ (Nicklas, 1997; Gardner and Burke,
2000; Nasmyth, 2001; Musacchio and Hardwick, 2002; Shonn
et al., 2003), leading to a persistent or transient cell cycle
arrest. Anti-microtubule agents like nocodazole may also trigger apoptosis as a consequence of checkpoint activation
(Decordier et al., 2002), which relates to the efficacy of these
agents in cancer treatment (Sasaki et al., 2002; Masuda et al.,
2003). However, nocodazole is also a potent mitotic aneugen
which induces mitotic nondisjunction at low nanomolar concentrations, e.g. in cultured human lymphocytes (Elhajouji
et al., 1995, 1997; Decordier et al., 2002; Kirsch-Volders
et al., 2003).
We previously used denuded mouse oocytes maturing in
in vitro experiments (further referred to as ‘denuded oocytes’)
to analyse the activity of aneugens, including nocodazole,
on spindle formation and chromosome segregation during
mammalian oogenesis (Eichenlaub-Ritter and Boll, 1989;
Shen et al., 2005). We showed that a 1 h exposure to nocodazole at micromolar concentrations during meiosis I resulted
in the complete depolymerization of the oocyte spindle and
meiotic block (Eichenlaub-Ritter and Boll, 1989). After the
removal of nocodazole, recovery of oocytes and their progression to metaphase II hyperploidy was dramatically increased.
Low concentrations of nocodazole, in the nanomolar range, do
not completely depolymerize microtubules but may alter the
morphology and stability of the spindle in mitotically dividing
cells (Jordan et al., 1992; Vasquez et al., 1997). Similarly,
when denuded mouse oocytes matured in the presence
To
2
whom correspondence should be addressed. Tel: 149 521 106 6907; Fax: 149 521 106 6015; Email: [email protected]
Present address: The Jackson Laboratory, Bar Harbor, ME 04609, USA
Mutagenesis vol. 20 no. 1 ß UK Environmental Mutagen Society 2005; all rights reserved.
65
F.Sun et al.
of 20--40 nM nocodazole, at low concentrations, which
may also be relevant for the in vivo situation of potential
exposures to the cytostatic chemical in humans, the pole-topole distance of the metaphase I and II spindle decreased
significantly (Shen et al., 2005). However, only some of
the denuded oocytes exposed to 30 and 40 nM nocodazole
were meiotically blocked. Nondisjunction was significantly
increased at a concentration of 40 nM nocodazole, suggesting
the existence of a biological threshold for the aneugen
(Shen et al., 2005). In the in vivo studies, a single dose of 35
mg/kg body weight (b.w.) of nocodazole administered to mice
1 h after mating resulted in a high incidence of chromosome
malsegregation at meiosis II, detectable as chromosomal aberrations in one-cell embryos and as an increase in the embryonic
death (Generoso et al., 1989). To date, there are no comparable
experimental studies on the consequences of in vivo exposures
to nocodazole for nondisjunction in oocytes before ovulation
during meiosis I.
The relative sensitivity of different cell types to aneugens
can be influenced by differential susceptibility to mitotic/
meiotic slippage (Dai et al., 2004), by differential response to
the induction of apoptosis (e.g. in mitotic and meiotic cells
of the gonads; Seaman et al., 2003; Kim and Tilly, 2004) or
by a gender-specific expression of cellular defence mechanisms, the so called ‘checkpoints’ in cell cycle progression
(Hunt and Hassold, 2002). Differential susceptibility to aneugens in chronic versus acute exposures and in species-specific
responses have to be considered in aneuploidy research
(Eichenlaub-Ritter et al., 2002; Witt et al., 2003). However,
we still do not know much about the differential and synergistic
effects of chemicals on germ cells and their somatic compartments, respectively, which might contribute to the differences
in the biological threshold concentrations in vivo and in vitro
and are therefore of importance in hazard and risk assessments.
To study these chemical effects on oogenesis, we employed
a new in vitro model of oocyte maturation, preantral follicle
culture, in which oocytes from prepubertal mice are cultured
together with the supporting somatic cell compartment
(Cortvrindt and Smitz, 2002; Sun et al., 2004). Initially, follicles with two layers of granulosa (follicle) cells (preantral) and
some theca cells were mechanically isolated from the ovary of
12--14-day-old mice and cultured in the presence of recombinant gonadotrophins for 12 days (Cortvrindt and Smitz, 2002;
Sun et al., 2004). Under the culture conditions, granulosa and
theca cells proliferate, the follicle cells differentiate into mural
and cumulus granulosa cell layers, and an antral cavity is
formed in the majority of the follicles. Oocytes grown in vitro
reach meiotic competence. After adding recombinant human
chorionic gonadotrophin (rHCG) and recombinant epidermal
growth factor (rEGF) on day 12 of the culture, cumulus follicle
cells mucify, in vitro, and the metaphase II oocyte is ovulated
in vitro after a final period of culture for 16 h (Figure 1). Under
these conditions, normal metaphase II spindles are formed in
the oocytes (Hu et al., 2001), and the chromosomes segregate
with high fidelity, a prerequisite for the in vitro model for
aneugen tests (Sun et al., 2004). We used this in vitro culture
system to expose mouse oocytes within follicles to low concentrations of nocodazole during the last stages of maturation
in culture. Comparing the data with recent observations on
nocodazole-exposed, in vitro maturing follicle-cell denuded
oocytes, it is possible to determine whether nocodazole influences the functionality of the somatic compartment and indirectly affects the survival and maturation of the oocyte during
66
the last stages of oogenesis in vitro. Apart from chromosomal
constitution, we analysed mucification of cumulus/in vitro
ovulation, meiotic progression, spindle formation and chromosome behaviour. In parallel, we tested the aneugenic effect of
nocodazole on the first meiotic division of mouse oocytes
in vivo by injecting mice with the cytostatic chemical at the
time of hormonally stimulated resumption of oocyte maturation in vivo (Mailhes, 1995). The study showed that nocodazole
induced nondisjunction in vivo and in vitro, and suggests that
the influence of nocodazole on the somatic compartment is
likely to contribute to adverse effects during ovulation and
the fidelity of meiotic chromosome segregation.
Materials and methods
Chemicals
Nocodazole (CAS no. 31430-18-9) was purchased from Sigma (Germany).
For in vitro studies, the stock solution was prepared at a concentration of
10 mM in dimethyl sulfoxide (DMSO). The stock solution was diluted to
suitable concentrations just before use. The final concentration of DMSO in
the medium was 0.1%. For in vivo studies, nocodazole was dissolved in DMSO
at a concentration adjusted to inject the mice with 5 ml/kg b.w., the highest
advisable dose of DMSO for animal treatment.
In vitro experiments
Preantral follicle culture. Female mice (12--14 days old) were F1 hybrids
(C57B1/6J 3 CBA/Ca) from mice originally obtained from Harlan
Winkelmann (Borchen, Germany). They were reared and maintained in the
departmental animal facilities, housed in a temperature- (23--25 C) and lightcontrolled room (12L:12D; 7 a.m.--7 p.m.), and fed with food and water
ad libitum.
In the present study, 2316 preantral follicles from a total of 40 prepubertal
mice were obtained for culture. The isolation medium was composed of L15
Leibovitz-glutamax (GIBCO, Invitrogen, Germany), 10% foetal calf serum
(GIBCO, Invitrogen, Germany) and antibiotics (Sigma, Germany). Mechanical
dissection of the ovary and isolation of the early preantral follicles was done as
previously described. The current study was different from the previous cytogenetic study (Sun et al., 2004) in that 96 well culture plates (Corning, USA),
instead of dishes with individual microdroplets of culture medium covered with
a thick oil layer, were used as the culture vessel, and the medium was refreshed
on every fourth and not on every second day of culture (Cortvrindt and Smitz,
2002). Each follicle was contained within 50 ml of medium, covered by 30 ml of
mineral oil. Microwell culture prevents the cross talk among the cultured
follicles through the oil overlay by the lipophilic steroids, and reduces the
amount of dilution of lipophilic components into the oil phase. Thus, some
nocodazole may segregate by diffusion into the oil phase during the last 16 h of
culture, but this should not cause an extensive dilution of nocodazole in
the medium due to the restricted surface area and the short exposure period.
The culture medium consisted of a-minimal essential medium with glutamax
(GIBCO, Invitrogen, Germany), and was supplemented with 10 mIU/ml
recombinant luteinizing hormone (rLH, Serono, Germany, kindly donated),
100 mIU/ml recombinant follicular stimulating hormone (rFSH, Serono,
Germany, kindly donated), foetal calf serum (GIBCO, Invitrogen, Germany)
and insulin--transferrin--selenium mixture (Sigma, Germany). All follicles were
placed in an incubator at 37 C with 5% CO2 in air and 100% humidity.
Follicles with two layers of granulosa cells (100--130 mM diameter) were
initially cultured for 12 days with refreshments of 20 ml of medium on days
4, 8 and 12. The spent medium was pooled for each plate and kept at 80 C for
later measurement of steroid production. The pattern of follicle development
and oocyte growth in the plate culture was similar to that reported for the
microdroplet culture on dishes (Sun et al., 2004). Steroid production in pooled,
spent medium before nocodazole exposure was in the normal range (data not
shown). The survival of follicles at day 12 of the culture was over 90%. At the
end of the 12 day culture period, plates were randomly divided into control,
solvent control and nocodazole-treated groups. Nocodazole was added to the
last refreshment of medium on day 12 (Figure 1). The follicle-like structures
cultured in wells were simultaneously stimulated for in vitro ovulation by rEGF
(Promega, Germany) and rHCG (Serono, Germany, kindly donated) at a final
concentration of 1.5 IU/ml rHCG and 5 ng/ml rEGF in the medium. Nocodazole was added at the same time as the in vitro ovulatory stimulus, at the
appropriate concentrations, to have 20, 30 or 40 nM nocodazole present during
the 16 h of culture before oocyte harvest. In response to the in vitro ovulatory
stimulus the cumulus granulosa cells surrounding the oocyte mucify, which can
Aneuploidy in mouse metaphase II oocytes
(A) In vitro protocol
Prepubertal, 12-14 day old mice
Control
Solvent
d4
d8
Refreshment of medium
d0
12-day follicle culture in
rLH/rFSH containing medium
(A⬘)
d12
Noc
rHCG
rEGF
16 h
•
•
•
•
In vitro Ovulation/Mucification
Spindle Formation
Chromosome Congression
Chromosomal Constitution
30µm
40µm
150µm
50µm
45µm
e⬘
e
200µm
a
PB
d0
b
200µm
d13
50µm
d4
c
d8
GV
40µm g
GVBD
f
d13
40µm
d
(B) In vivo protocol
Prepubertal, 28 day old mice
PMSG
d12
h
Solvent
48 h
• In vivo Ovulation
• Chromosomal Constitution
Noc
HCG
17 h
Fig. 1. Experimental design of in vitro and in vivo tests. (A) Preantral follicles were isolated from 12--14-day-old prepubertal mice and cultured for 12 days in
the presence of rLH and rFSH with the medium refreshed on days 4, 8 and 12. At day 12 rHCG and rEGF were added to the culture medium and oocytes
matured in vitro within the follicles for a further 16 h (control). In the other experimental groups, solvent or nocodazole was added when the culture medium
was refreshed for the last time. In vitro ovulation/mucification, spindle formation, chromosome congression and chromosomal constitution were then analysed.
(A0 ) Representative features of follicles and oocytes grown and matured in preantral follicle culture: (a) Preantral follicles with a small GV oocyte obtained
from a 12-day-old prepubertal mouse with two to three layers of granulosa cells and some theca cells (arrows) attached to the surrounding basal membrane;
(b) attachment of follicle, outgrowth of theca cells (arrow), and initiation of proliferation of theca and granulosa cells surrounding the growing GV-stage
oocyte on day 4 of the culture; (c) increased numbers of somatic cells containing a growing oocyte on day 8 of the culture; (d) follicle with antral-like cavity
(translucent area, arrow) separating differentiated outer mural granulosa cells (arrowhead) from inner cumulus granulosa cells surrounding the in vitro
grown GV-stage oocyte on day 12 of the culture; (e) in vitro ovulation on day 13 with mature oocyte (arrow, out of focus) surrounded by mucified, highly expanded
cumulus cells (larger magnification shown in e0 ) with cumulus mass containing a mature PB-stage oocyte (inset) 16 h after maturation in vitro, following
hormonal stimulation on day 12; (f) oocyte--cumulus complex that failed to mucify with cumulus firmly attached to the oocyte, which may contain a GV-stage
(g) or GVDB-stage (h) oocyte without polar body, which are still arrested at meiosis I on day 13 of the culture. (B) Folliculogenesis was primed by i.p. injection of
PMSG in prepubertal 28-day-old mice, followed 48 h later by stimulation with HCG and in vivo maturation for 17 h after an i.p. injection of DMSO as solvent
control or nocodazole. The number of ovulated oocytes per female and the chromosomal constitution of these in vivo matured oocytes were analysed.
be assessed using a dissection microscope prior to oocyte harvest. A schematic
diagram of the experimental protocol of the in vitro and in vivo exposure is
provided in Figure 1.
Analysis of meiotic progression. Although maturation status can usually be
assessed by the presence of a germinal vesicle (GV), the resolution of the
nucleus (germinal vesicle breakdown, GVBD) or the presence of a polar body
(PB), sometimes PBs cannot unambiguously be identified in all oocytes. Therefore, denuded oocytes were transferred individually into a Petri dish for vital
staining of chromosomes with Hoechst 33342 for 20--30 min (Sigma,
Germany). Nuclear staining was assessed with a Zeiss (Axiovert 10) inverted
microscope, equipped with a mercury lamp and a heated stage. On the basis of
the fluorescence patterns of the chromatin, the oocytes were then assigned to
GV oocytes, which were maturation incompetent or meiotically blocked and
therefore still arrested in the dictyate stage at the end of the culture period. For
oocytes resuming maturation, meiotic progression was determined by analysing the number of GVBD oocytes and PB oocytes. GVBD oocytes had resolved
their nuclear membrane and characteristically possessed one or two sets of
condensed chromosomes but had not emitted a PB. Oocytes with PB had
two spatially separated sets of chromosomes in the ooplasm and in the PB
compartment, and visibly had undergone cytokinesis. Oocytes were pooled
according to their meiotic stages, and oocytes resuming maturation (GVBD
and PB oocytes) were spread separately for chromosome analysis.
Chromosomal analysis. The spreading procedure was performed according
to a modified Tarkowski (1966) method (Sun et al., 2004). C-banding was
performed as previously described (Eichenlaub-Ritter and Betzendahl, 1995).
Cytogenetic analysis was performed with a phase contrast microscope
(Zeiss) at 10003 final magnification. First, nuclear maturation was determined.
GVBD oocytes either contained bivalents, and accordingly reached meiosis I
during maturation, or contained two sets of dyads (metaphase II chromosomes).
PB oocytes contained metaphase II chromosomes in the haploid range with or
without separated chromatids (predivision). The PB oocytes, in which chromosomal number could unambiguously be determined, were analysed further for
ploidy (e.g. euploid oocytes had 20 metaphase II chromosomes, Figure 2A).
Oocytes with PB with 420 metaphase II chromosomes, or exceeding the
67
F.Sun et al.
B
A
G
G⬘
C
D
E
H
F
H⬘
I
I⬘
Fig. 2. (A--F) Typical patterns of chromosome constitution of oocytes with or without nocodazole treatment. (A) Euploid oocyte with 20 metaphase II
chromosomes in control. (B) Hyperploid oocyte with 25 metaphase II chromosomes and one chromatid (arrow) from the 30 nM nocodazole group. (C) Hyperploid
oocyte with one extra chromatid (arrow) from the 30 nM nocodazole group. (D) Diploid oocyte with 40 metaphase II chromosomes, which matured in the presence
of 40 nM nocodazole. (E) Hyperploid oocyte with 22 metaphase II chromosomes (upper part) and corresponding hypoploid PB with 18 metaphase II chromosomes
(lower part) from the 30 nM nocodazole group. (F) Hypoploid oocyte with 19 metaphase II chromosomes (upper part) and hyperploid polar body with
21 metaphase II chromosomes (lower part) from the 20 nM nocodazole group. Scale bars in A--F; 5 mm. (G--I) Spindle morphology and chromosome behaviour
in oocytes matured in the presence or absence of nocodazole. (G) Control oocyte with normal metaphase II spindle and aligned chromosomes (G0 ).
(H) Oocyte with aberrant spindle with unaligned chromosomes (H0 ) from the 30 nM nocodazole group. (I) Presumably polyploid GVBD oocyte with two
spindles and two sets of chromosomes (I0 ) from the 30 nM nocodazole group. Scale bars in G--H, 10 mm.
68
Aneuploidy in mouse metaphase II oocytes
euploid chromosome constitution with their overall content of dyads plus
bivalents or chromatids (Figure 2B and C), were recorded as hyperploid. The
number of oocytes with metaphase II chromosomes in the diploid range was
recorded over the total number of oocytes with or without PB (GVBD) with
countable metaphase II chromosomes (from 17 to 40) to assess the incidence of
polyploidy. Premature separation of chromatids (predivision) prior to anaphase
II was also analysed (Figure 2B and C), irrespective of ploidy (including
oocytes with euploid, hyperploid or hypoploidy constitution), in the oocytes
with PB. Cases in which premature separation of chromatids was observed, but
ploidy could not be unequivocally determined (one oocyte from the control,
and the solvent control, and two oocytes from the 20 nM nocodazole group with
two sets of chromatids), were also recorded.
Immunofluorescence. Oocytes were processed for spindle immunofluorescence as described previously (Eichenlaub-Ritter and Betzendahl, 1995;
Hu et al., 2001; Sun et al., 2004). In short, the zona pellucida was removed
by brief pronase (Boehringer Mannheim, Germany) treatment. Oocytes were
extracted in a microtubule-stabilizing solution at 37 C. After attachment to the
slides, fixation in methanol at 20 C and rehydration in phosphate-buffered
saline, the spindle was labelled by a mouse monoclonal anti-a-tubulin antibody
(Sigma) and an anti-mouse FITC-conjugated secondary antibody (Sigma).
Chromosomes were stained by 4,6-diaminidino-2-phenylindole (DAPI, Sigma)
or propidium iodide (Sigma). Spindle images were recorded with a Zeiss
Axiophot fluorescence microscope equipped with sensitive charge coupled
device (CCD) camera (SensiCam, PCO CCD imaging, Kelheim, Germany).
In vivo experiments
These experiments were approved by the internal ethical committee of ENEA
and officially authorized by the Italian Ministry of Health.
Three groups of 4-week-old female MF1 mice (Harlan, Italy) were used in
this set of experiments. They were maintained in the institutional animal
facilities, housed in a temperature-(21 C) and light-controlled room
(12L:12D; 7 a.m.--7 p.m.), and fed with food (Teklad Global Diet 2018, Harlan,
Italy) and water ad libitum.
These mice were superovulated by an i.p. injection of 7.5 IU of pregnant
mare serum (PMSG, Crono-gest, Intervet, The Netherlands) to stimulate folliculogenesis. After 48 h, 5 IU of HCG (Chorulon, Intervet, The Netherlands)
was injected to stimulate ovulation. At the time of HCG injection, one group
was i.p. treated with 5 ml/kg b.w. of DMSO to serve as the solvent control.
This was the highest advisable dose of DMSO for animal treatment. A second
group was administered 35 mg/kg b.w. of nocodazole in DMSO and a third
group 70 mg/kg b.w. of nocodazole in DMSO (Figure 1).
In vivo ovulated oocytes were collected 17 h after the treatment from the
ampulla of the oviducts.
Chromosomal analysis. After the removal of cumulus cells with a brief
digestion with hyaluronidase, GVBD and PB oocytes were spread on a slide
according to the mass harvest method of Mailhes and Yuan (1987). Chromosomes were stained by C-banding (Salamanca and Armendares, 1974) and
analysed under a phase contrast microscope (Zeiss, Germany) at 10003 final
magnification according to the criteria similar to those adopted for in vitro
experiments. The frequencies of oocytes containing a full set of bivalents
(metaphase I-arrested oocytes), of oocytes containing a diploid or nearly diploid number of dyads (polyploid oocytes) and of oocytes displaying premature
separation of chromatids or premature anaphase II were measured from all
analysable oocytes, including GVBD and PB oocytes. The number of metaphase II oocytes with PBs, with 420 chromosomes (actual observed range:
21--25) was also recorded over the total number of metaphase II oocytes in
which chromosomal number could unequivocally be determined to evaluate the
induction of nondisjunction.
Statistical analysis
Data on cytoplasmic progression, nuclear maturation and chromosomal
constitution after in vitro and in vivo experiments were compared between
each treated group and the appropriate control or solvent control by the x2 -test
with Yates correction. The x2 -test with Yates correction was also used to
statistically evaluate the percentage of follicles which did not show mucification by cumulus cells. Trend analysis compared the increase in the percentage
of follicles failing to mucify with increasing nocodazole doses. Student’s t-test
was used to compare differences between the mean number of oocytes ovulated
in the control and the nocodazole-treated mice.
Results
Development of early preantral follicles in vitro
In the current study, 96 well plates were used for preantral
follicle culture. When the medium was refreshed for the last
Table I. Mucification of cumulus cell-enclosed oocyte complexes 16 h
after hormonal stimulation and the treatment of antral follicles with 20,
30 or 40 nM nocodazole (noc)
Control
Solvent control
20 nM noc
30 nM noc
40 nM noc
Number of
antral follicles
Failure in
mucification/in vitro
ovulation (%)
185
106
125
131
117
12
6
10
12
16
(6.5)
(5.7)
(8.0)
(9.2)
(13.7)
time, including the addition of a combination of rHCG and
rEGF, nocodazole was also added to give a final concentration
of 20, 30 or 40 nM in medium. DMSO was used as a solvent
(0.1% in solvent control). There was no visible influence of
nocodazole on follicle or oocyte survival at 16 h after exposure.
We also analysed progesterone in pooled, spent culture
medium before the addition of nocodazole and at the end of
oocyte maturation, but we did not find any pronounced effect
(data not shown). However, the percentage of follicles with
mucification, expansion and detachment of cumulus around
oocytes (Figure 1f), was reduced in the treated groups. There
was a slight increase in the percentage of follicles without
mucification (Table I) in the 30 nM nocodazole group. The
numbers of follicles failing to mucify increased further
with maturation in the presence of 40 nM nocodazole.
Although differences between the treated groups and the control did not reach statistical significance, a statistically significant (P 5 0.05) trend for an increase in the percentage of
follicles without mucification with increasing nocodazole concentration was detected.
Cytoplasmic maturation of nocodazole-exposed oocytes
from follicle culture
Less than 10% of the oocytes in any group failed to resume
maturation (Table II, left panel), and most of the oocytes
(490%), including both controls and treated groups, were
competent to resume meiotic maturation to either resolve the
nucleus (GVBD) or also emit a PB (Figure 1). Vital staining
with Hoechst 33342 revealed that solvent alone had no effect
on meiotic progression of oocytes compared with the control
group. However, the proportion of oocytes with PB decreased
from 470% in the control and solvent control to 67.8, 61.7 and
40.9% in the 20, 30 and 40 nM nocodazole groups, respectively. Concomitantly, the percentage of oocytes at GVBD
stage increased. Decrease in PB oocytes was significant at
30 and 40 nM nocodazole (Table II; P 5 0.005 and 0.001,
respectively).
Nuclear maturation of oocytes from follicle culture treated
with nocodazole
Cytogenetic analysis of oocytes with GVBD and PB showed
that nuclear maturation of oocytes in the solvent control was
comparable to that in the untreated control group. In contrast,
in the treated groups, the percentage of oocytes arrested at
meiosis I stage with bivalent chromosomes increased, from
between 16 and 20% in the controls to 440% at the highest
concentration of nocodazole. Correspondingly, the overall percentage of oocytes with metaphase II chromosomes decreased.
69
F.Sun et al.
Table II. Meiotic and nuclear maturation, and chromosomal aberrations of in vitro grown mouse oocytes from preantral follicle culture matured within
follicles in the presence of 20--40 nM nocodazole (noc) for the last 16 h following the in vitro ovulatory stimulus
Maturation arrest
Meiotic development
Total
Arrested No. of
With
number with
maturing GVBD
of
GV (%) oocytes (%)
oocytes
Control
Solvent
control
20 nM noc
30 nM noc
40 nM noc
Nuclear maturation
With
PB (%)
Chromosomal aberrations
No. of
GVBD
and PB
oocytes
MI oocytes Polyploidy
Predivision
with
MII with
No. of
Polyploids No. of
bivalents
chrom.
MII GVBD (%)
MII
(%)
and PB
oocytes (%)
oocytes
with PBa
Aneuploidy
116
136
19 (16.4)
29 (21.3)
187
220
14 (7.5)
10 (4.5)
173
210
40 (23.1) 133 (76.9)
49 (23.3) 161 (76.7)
314
244
117
25 (8.0)
22 (9.0)
7 (6.0)
289
222
110
93 (32.2) 186 (67.8)
186
85 (38.3) 137 (61.7) 155
65 (59.1) 45 (40.9) 53
97
107
43 (23.1)
143
42 (27.1)
113
22 (41.5) 31
No. of Hyperploids
MII
(%)
oocytes
with PB
7 (7.2)
2 (1.9)
91
106
1 (1)
1 (0.9)
90
105
14 (9.8)
15 (13.3)
9 (29)
131
98
22
4 (3.1)
3 (2.7)
2 (9.1)
129
98
22
0 (0)
0 (0)
5 (3.9)
12 (12.2)
5 (22.7)
GV, germinal vesicle; GVBD, germinal vesicle breakdown; PB, polar body. MII with chrom., metaphase II oocytes with chromatids/precocious anaphase II.
x2 : significant difference to control: P 5 0.005; P 5 0.001.
a
Including PB oocytes with two sets of chromatids in haploid range.
Unlike in denuded untreated or solvent-exposed oocytes
analysed in previous studies, which tend to have only a few
oocytes progressing through first anaphase in the absence of
cytokinesis (e.g. 0.4% in control and 0.9% in DMSO/solvent
control; Shen et al., 2005), a consistently higher percentage
of polyploid, ‘diploid’ meiosis II oocytes with two sets of
metaphase II chromosomes was previously observed in
oocytes from preantral follicle culture (e.g. 5.7% and 6.8% of
diploids in the untreated control in two sets of experiments;
Sun et al., 2004). Currently, the number of diploids was also
higher compared with the standard values in in vitro matured
denuded oocytes, and varied between replicate experiments,
with inconsistent and insignificant differences between the
control and the solvent control. However, the percentage of
such polyploid oocytes increased in this study in a dose-related
fashion from a total of 7.2 and 1.9% in the control and
the solvent control, respectively, to 29% in the group treated
with the highest concentration of nocodazole (P 5 0.005)
(Figure 2D).
Chromosome nondisjunction and predivision in
nocodazole-exposed oocytes from follicle culture
No hyperploids were found in the control and the solvent
control. The percentage of hyperploid oocytes (Figure 2E)
increased to 3.9, 12.2 and 22.7% in the mouse oocytes retrieved
from follicles cultured in the presence of 20, 30 and 40 nM
nocodazole, respectively (Table II, right panel). The increase
compared with controls reached significance in the 30
and 40 nM nocodazole groups (P 5 0.005 and 0.001, respectively). Precocious separation of chromatids was comparatively rare among oocytes, which had resumed maturation
(Table II). From the oocytes with predivision, one oocyte
with complete chromatid separation and two sets of chromatids
were found in each of the control and solvent groups. Two
oocytes in the 20 nM nocodazole group contained a single
chromatid and two had all chromatids separated into two sets.
In the 30 nM nocodazole group, two oocytes had premature
centromere separation of all chromatids and one oocyte
possessed a single chromatid (Figure 2C). In the 40 nM
nocodazole group, one oocyte exhibited premature centromere
separation of all chromatids and one contained a single
chromatid.
70
Table III. Spindle morphology and chromosome behaviour in metaphase II
oocytes from control, solvent control and nocodazole (noc)-exposed follicles.
Control
Solvent control
20 nM noc
30 nM noc
Number
of oocytes
Total
aberrant
meiosis II
oocytes (%)
Oocytes
with
aberrant
spindle (%)
Oocytes with
displaced or
dispersed
chromosomes (%)
87
96
116
121
8
8
38
58
6
5
34
53
7
6
33
50
(9.2)
(8.3)
(32.8)
(47.9)
(6.9)
(5.2)
(29.3)
(43.8)
(8.0)
(6.3)
(28.4)
(41.3)
x2 -test: significant difference to control: P 5 0.001.
Spindle morphology and chromosome congression in
nocodazole-exposed metaphase II oocytes from
follicle culture
The spindle apparatus plays a key role in the fidelity of
chromosome segregation in both mitosis and meiosis. It is considered as a major target for disturbances by aneugens. In the
current study, the influence of nocodazole on the morphology
of metaphase II spindles and on chromosome alignment was
therefore analysed in controls and the 20 and 30 nM nocodazole groups (Table III). In the control, most oocytes possessed
normal spindles (Figure 2G and G0 ). The solvent alone did not
increase the percentage of metaphase II oocytes with aberrant
spindles. However, nocodazole induced an increase in the
number of aberrant oocytes at 20 and 30 nM concentration
(Table III), and the percentage of oocytes with aberrant
spindles (Figure 2H) and/or unaligned, dispersed or displaced
chromosomes (Figure 2H0 ) was also significantly increased in
both the dose groups. Nearly 50% of all meiosis II oocytes with
PB in the 30 nM nocodazole group were abnormal indicating
a high susceptibility to second meiotic errors (Table III).
In addition, some highly aberrant GVBD oocytes with two
spindles and two sets of chromosomes were also observed
(Figure 2I and I0 ) in the treated groups.
Ovulation, maturation and nondisjunction of oocytes
exposed to nocodazole in vivo
Nocodazole interfered with the process of ovulation significantly reducing the mean number of oocytes ovulated and
Aneuploidy in mouse metaphase II oocytes
Table IV. Results of the experiments testing nocodazole (noc) effects on mouse oocytes after in vivo exposure
Mean
number
of oocytes/
female
5 ml/kg DMSO
35 mg/kg noc
70 mg/kg noc
33.6 6 1.6
33.8 6 1.5
27.7 6 3.0
Nuclear maturation
Chromosomal aberrations
No. of
GVBD
and PB
oocytes
MI oocytes
with
bivalents
(%)
Polyploidy
441
329
264
0 (0)
1 (0.3)
18 (6.8)
409
328
233
No. of
MII GVBD
and PB
oocytes
Predivision
Polyploids
(%)
1 (0.2)
0 (0)
2 (0.9)
Aneuploidy
No. of
MII GVBD
and PB
oocytesa
Oocytes with
single
chromatid
(%)
Oocytes with
pre-mature
ana II (%)
No. of MII
oocytes
with PB
Hyperploids
(%)
409
306
216
24 (5.9)
11 (3.6)
6 (2.8)
9 (2.4)
3 (1.0)
3 (1.4)
230
194
110
0 (0)
0 (0)
8 (7.3)
MI, meiosis I; MII, oocytes with metaphase II chromosomes; GVBD, germinal vesicle breakdown; PB, polar body; Predivision, meiosis II oocytes with
premature segregation of individual chromatids (single chromatids) or premature anaphase II.
Significantly lower than the corresponding solvent control value: Student’s t-test P 5 0.05.
Significantly higher than the corresponding solvent control value: x2 -test P 5 0.001.
a
Excluding oocytes in which chromatids could not be individually recognized on all chromosomes.
harvested per female at the highest dose tested (Table IV).
Nocodazole (35 mg/kg b.w.) had no apparent effect on ovulation, nuclear maturation and aneuploidy induction. A dose of
70 mg/kg b.w. of nocodazole induced a significant increase
in the percentage of meiosis I-arrested oocytes compared with
the solvent control (P 5 0.001). Notably, at the same dose,
nocodazole significantly increased the frequency of hyperploid
oocytes compared with the solvent control (P 5 0.001) from
0 to 7.3%. The frequency of oocytes with premature centromere separation of all (premature anaphase II) or nearly all
dyads (predivision) decreased dose-dependently in nocodazoletreated mice, although differences between the treated groups
and the control did not reach statistical significance. If the
decrease in oocytes with chromatids and premature anaphase
II was actually nocodazole-related, it could be a secondary
consequence of a transient arrest at the metaphase I stage,
thus shortening the time spent by the oocytes at metaphase II,
when, in the absence of fertilization, degeneration may start
with relaxation of chromatid cohesion at the centromeres.
Discussion
Influence of nocodazole on meiotic progression and oocyte
nuclear maturation
Chemicals with aneugenic potential can interfere with oocyte
maturation, both in vivo and in vitro, and can result in a meiotic
delay or an arrest. Nocodazole was shown to induce a cell cycle
arrest at high concentrations (Eichenlaub-Ritter and Boll,
1989; Everett and Searle, 1995; Brunet et al., 2003; Wassmann
et al., 2003). Mitotic slippage in response to nocodazole
exposure can cause polyploidy and susceptibility to apoptosis
or secondary nondisjunction (Verdoodt et al., 1999). In the
current study, a low dose of nocodazole, resembling pharmacological exposure levels, was used to analyse the aneugenic
activity on oocytes from preantral follicle culture. Indeed,
nocodazole influenced both oocyte cytoplasmic and nuclear
maturation. There was a dose-related arrest at meiosis I,
demonstrated by increased numbers of oocytes in GVBD and
in oocytes containing bivalents. This was similar to the results
of the in vivo assay which also had significant increases in the
number of oocytes ovulated in meiosis I. Nocodazole also
interfered with cytoplasmic maturation and caused ‘meiotic
slippage’ in preantral follicle culture, as can be deduced from
the increased polyploidy suggesting an asynchrony in the
formation of a first PB and anaphase I progression. There was
a significant increase in the number of polyploid oocytes with
two sets of metaphase II chromosomes in preantral follicle
culture but not in the in vivo study with ovulated oocytes.
Since the number of ovulated oocytes decreased in vivo, it is
conceivable that oocytes with this type of disturbance were
possibly retained within the ovary. Likewise, oocytes from
follicle culture could be particularly susceptible to the uncoupling of cytokinesis from anaphase I, or the concentration of
nocodazole at the target site achieved in vivo by a single i.p.
injection was below the threshold for induction of this effect.
Susceptibility of spindle formation to nocodazole-induced
disturbances
Microtubule depolymerizing agents can disrupt microtubuledependent processes by altering microtubule dynamics at low
concentrations (Jordan and Wilson, 1990; Jordan et al., 1992;
Toso et al., 1993; Dhamodharan et al., 1995; Tanaka et al.,
1995). This can suppress the dynamic instability by decreasing
microtubule elongation and shortening velocities both in vivo
and in vitro (Vasquez et al., 1997). Suppression of dynamic
instability of kinetochore microtubules (Mitchison et al., 1986;
Hamaguchi et al., 1987; Mitchison, 1989), especially of microtubules at centromeres (Andrews et al., 2004; Bringmann et al.,
2004; Lan et al., 2004), or steric hindrance of kinetochore/
microtubule attachment might lead to a disruption of the
balance of forces in the spindle and result in the shortening of
the spindle (Nicklas, 1988). Wendell et al. (1993) showed that
kinetochore microtubule number was reduced by about 25% in
cells arrested in mitosis by 2 nM vinblastine. Using polarizing
microscopy, we found that 20 nM nocodazole caused a reduction in the overall spindle length, an indicator for reduction in
the microtubular lattice of the spindle of denuded, nocodazoleexposed mouse oocytes (Shen et al., 2005). About 33 nM
nocodazole was sufficient to induce monopolar spindles in
somatic cells (Jordan et al., 1992). In accordance, the current
study shows that a high number of oocytes treated with 20 nM
nocodazole in preantral follicle culture had aberrant metaphase
II spindles, although we did not find typical monopolar configurations. Since oocytes possess multiple microtubule organizing centres instead of two centriolar centrosomes (for review
see Eichenlaub-Ritter et al., 2004), formation of a monopolar
71
F.Sun et al.
spindle may be a rather rare event in oogenesis. There was no
increase in first meiotic nondisjunction at this low nocodazole
concentration. However, the severe spindle aberrations at
meiosis II and the failure to assemble chromosomes at the
spindle equator at metaphase II in the group exposed to only
20 nM nocodazole can contribute to chemically induced
second meiotic error, after fertilization of the oocyte. Indeed,
numerical chromosome aberrations including hypodiploidy,
hyperdiploidy, haploidy and triploidy have been reported in
one-cell embryos when metaphase II oocytes of hormonally
unstimulated mice were exposed to 35 mg/kg b.w. of nocodazole in vivo (Generoso et al., 1989). The same paper did not
report cytogenetic observations after in vivo exposure of meiosis I oocytes, but a uterine content analysis did not suggest
that nocodazole caused the loss of conceptuses after treatment
at this stage. This observation agrees with our finding that
35 mg/kg b.w. of nocodazole also did not induce aneuploidy
in metaphase II oocytes collected after gonadotrophin-induced
ovulation. Currently, we cannot determine whether hormonal
stimulation influenced the response in the current in vivo study,
but altogether these observations suggest that metaphase II
spindles might be especially sensitive to nocodazole-induced
alterations. In addition, accumulation of damage during a
chronic exposure spanning the whole period of meiotic
resumption, as in the follicle culture, could contribute to
second meiotic disturbances, while a transiently high concentration in earlier prometaphase I induced by the acute exposure
at meiotic resumption by the single i.p. in vivo administration
may have a less severe, reversible effect.
Chromosomal aberrations in metaphase II oocytes
Nocodazole increased the number of oocytes with hyperploidy
dose-dependently in metaphase II oocytes after a single dose
in vivo as well as after low dose-exposure in follicle culture
during the last 16 h of oocyte maturation. An aneugenic
activity of nocodazole has been observed in many cell types,
including yeast Saccharomyces cerevisiae (Wood, 1982;
Zimmermann et al., 1984; Mayer and Goin, 1987), human
lymphocytes cultured in vitro (Correll and Ford, 1987; Sandhu
et al., 1988; Elhajouji et al., 1995, 1997; Decordier et al.,
2002), meiotically dividing crane-fly spermatocytes (Ladrach
and LaFountain, 1986), PtK1 cells (Cimini et al., 2001) and
human lung diploid fibroblasts (MRC-5 cells) (Cimini et al.,
2002). Nocodazole induced a dramatic increase of aneuploidy
in denuded, young and old metaphase II oocytes of CBA/Ca
mice exposed transiently for only 1 or 2 h to micromolar
concentrations (Eichenlaub-Ritter and Boll, 1989) and in mouse
oocytes of (C3H/HeH 3 101/H)F1 hybrids or homozygous
Rb(16.17)7Bnr mice (Everett and Searle, 1995). Moreover, it
has been demonstrated that nocodazole led to remarkably high
frequencies of embryonic lethality as well as varied numerical
chromosome abnormalities with changes in ploidy when
35 mg/kg b.w. of nocodazole was administered once at the
time of sperm entry (Generoso et al., 1989). A 16 h continuous
exposure to relatively low concentrations of 40 nM nocodazole
induced a significant rise in the hyperploidy frequency in
follicle-cell denuded metaphase II oocytes of the MF1 mouse
exposed throughout in vitro maturation (Shen et al., 2005). The
present study not only fully confirmed the aneugenic potential
of nocodazole at meiosis I but also provided evidence that
continuous exposures of the oocyte within a follicle throughout
nuclear maturation may be especially critical for a loss of
fidelity of chromosome separation at meiosis II. In addition,
72
increases in polyploidy were pronounced in follicle-enclosed
oocytes (here 29% polyploids with 40 nM nocodazole) but not
so much in isolated, denuded mouse oocytes (4.4% in the
40 nM nocodazole group; Shen et al., 2005).
Cell--cell signalling and chemically induced aneuploidy in
follicle-enclosed oocytes
In the present study, aneuploidy rose significantly at 30 nM
nocodazole in the follicle culture, while a significant increase
in aneuploidy of nocodazole-exposed denuded oocytes
occurred only at 40 nM nocodazole (Shen et al., 2005). This
indicates that exposure of the somatic compartment with the
oocyte may impair the health and function of the follicle
directly and indirectly (e.g. by also disturbing granulosa and
theca cell function). It may thus synergistically affect the
quality of the enclosed oocyte. There are distinct differences
in the regulation of meiotic resumption in the denuded oocyte
and follicle-enclosed oocyte maturation. In the case of denuded
oocytes, maturation is spontaneous because inhibiting signals
from follicle cells and components of follicular fluid are
removed by the isolation (Edwards, 1965). In contrast, follicleenclosed oocyte maturation in vivo is accompanied by the
generation of a positive stimulus of the membrana granulosa
and the cumulus cell origin in the presence of functional
heterologous gap junctional communication with the oocyte
(e.g. Downs, 1995; Byscov et al., 1997; Albertini et al., 2001;
Matzuk et al., 2002; Su et al., 2003; Ashkenazi et al., 2004).
It is clear that oocytes are not passive but have an active role in
follicular function (e.g. Matzuk et al., 2002; Su et al., 2003).
They actually resume maturation before gap junctional communication ceases. One kind of cellular process that extends
through the zona pellucida from the follicle cell to the
oolemma, termed transzonal projection, contains microtubules
(Albertini and Barrett, 2003). It is conceivable that nocodazole
exerts some effect on the cross-talk between the compartments
and may influence the maturation of the enclosed oocyte directly as well as indirectly through alteration of general follicle
function. There was no evidence for disturbed hormonal
homeostasis by nocodazole but a pronounced cell cycle arrest
and disturbance in meiosis II spindle formation could partially
result from disturbed cell-to-cell communication.
Dose-response to nocodazole in vitro and in vivo,
and in somatic cells
For many years, it has been commonly accepted that risk
assessments of genotoxic chemicals are based on linear models
for extrapolating low dose effects from experimental data. The
underlying assumption is the absence of a threshold for the
induction of mutations (Speit et al., 2000). However, recent
data indicate that biologically meaningful threshold effects
exist for some types of mutagenic events, e.g. aneuploidyinducing chemicals that interact with non-DNA targets
(Kirsch-Volders et al., 2000; Elhajouji et al., 1995, 1997).
Most studies on thresholds were performed with somatic cells
in the past. Little is known about the relative risk of aneuploidy
induction in somatic versus germ cells, or on gender differences in susceptibility. A recent study on aneuploidy induced
by nocodazole in denuded mouse oocytes suggested that the
biological threshold for induction of nondisjunction could be
similar in female meiosis and cultured human lymphocytes
(Eichenlaub-Ritter et al., 2002; Kirsch-Volders, et al., 2003).
This was in agreement with the notion that the dose-response
to aneugens is similar between somatic and meiotic cells
Aneuploidy in mouse metaphase II oocytes
(Parry et al., 2000), a concept that may have to be critically
re-evaluated.
Preantral follicle culture has been previously used to demonstrate the sensitivity of follicle-enclosed oocytes to aneugens
(Sun et al., 2004). In the current study, 20 nM nocodazole
significantly increased the number of aberrant meiosis II
oocytes, while only 9.1% of in vitro matured denuded mouse
oocytes had unaligned chromosomes after maturation to metaphase II in presence of 20 nM nocodazole in the medium (Shen
et al., 2005). As shown here, the percentage was about three
times higher (28.4%) in mouse oocytes exposed to the same
concentration during the last hours of maturation in preantral
follicle culture. Similarly, only 21.7% of the denuded oocytes
that matured in the 30 nM nocodazole group had displaced
chromosomes (Shen et al., 2005), while 440% of the in vitro
grown and matured oocytes from preantral follicle culture
showed this aberration after exposure to the same nocodazole
dose during the last 16 h of culture. The actual effective dose to
induce spindle aberrations in metaphase II oocytes of cultured
follicles may be even lower, but we did not test doses in this
range. Hyperploidy was significantly increased by 30 nM
nocodazole in the present study. Since paracrine signalling at
the resumption of meiosis involves gap junctional coupling and
nocodazole has been reported to inhibit the formation/function
of gap junctions, e.g. in Novikoff hepatoma cells (Johnson
et al., 2002), disruption of these critical activities can indirectly
affect the spindle function and the fidelity of chromosome
segregation and contribute to the high susceptibility to meiotic
errors at oogenesis in follicle culture.
The experiments involving follicle-enclosed oocytes unequivocally showed that nocodazole induced perturbations of
nuclear maturation and aneuploidy also after in vivo exposure
of the mouse. However, these effects were only observed at the
highest tested dose (70 mg/kg b.w.). Doses of 35 and 70 mg/kg
b.w., expressed in molarity relative to the mouse weight, correspond to 115 3 103 and 230 3 103 nM nocodazole, respectively. These acute doses are between 103 and 104 times higher
than the continuous in vitro effective nocodazole concentrations. Biodistribution and bioavailability play an important role
in in vivo studies. In our experiments, evidence was provided at
autopsy that nocodazole bioavailability to oocytes was reduced
by precipitates observed in the peritoneum after the administration of both tested doses. Nocodazole has poor solubility
in water; thus it is conceivable that the DMSO solution
re-precipitated, once diluted in the body fluids. The amount
of precipitates was inversely correlated to the volume of DMSO
injected, but doses higher than 5 ml/kg b.w. of DMSO could
not be used because of their toxicity.
These observations contribute to explain the differences
between aneugenic concentrations of nocodazole measured
in vitro and in vivo. In contrast to quantitative effects, results
observed after in vitro and in vivo exposure show a good
qualitative agreement, in terms of both perturbations of nuclear
maturation and aneuploidy induction. Notably, in both experimental systems, several oocytes were detected with severe
aneuploidies (more than two supernumerary chromosomes).
The in vitro and in vivo models to test the aneugenic effects
of chemicals on mouse oocytes thus appear to be complementary, with the former providing a sensitive tool to test for and
measure threshold concentrations, characterize effects of continuous exposures and investigate mechanisms of action (e.g.
Eichenlaub-Ritter et al., 1996; Yin et al., 1998a,b; Mailhes
et al., 2002, 2004; Cukurcam et al., 2004; Sun et al., 2004;
Van Wemmel et al., in press), and the latter demonstrating that
similar effects are also induced in the intact animal, albeit at
much higher treatment doses. In the future, experiments
designed to expose animals to low protracted chemical doses
could contribute to move from hazard identification to risk
assessment of aneugenic exposures.
Preantral follicle culture appears to be a sensitive method to
detect aneugens after chronic low dose exposure, as suggested
by this and other in vitro studies (Sun et al., 2004; Van Wemmel
et al., 2005), comparable to in vivo observations in the mouse
(Everett and Searle, 1995). In comparison with mitotically
dividing somatic cells, meiotically dividing oocytes from preantral follicle culture seem to be more sensitive to aneugens
when they reside in the follicular compartment. Therefore, in
risk assessment, extrapolation of findings from somatic cells to
meiotic cells should be viewed with caution (Pratt and Barron,
2003).
Acknowledgement
The work has been supported by EU (QCRT-2000-00058).
References
Aardema,M.J., Albertini,S., Arni,P., Henderson,L.M., Kirsch-Volders,M.,
Mackay,J.M., Sarrif,A.M., Stringer,D.A. and Taalman,R.D. (1998)
Aneuploidy: a report of an ECETOC task force. Mutat. Res., 410, 3--79.
Albertini,D.F. and Barrett,S.L. (2003) Oocyte-somatic cell communication.
Reprod. Suppl., 61, 49--54.
Albertini,D.F., Combelles,C.M., Benecchi,E. and Carabatsos,M.J. (2001)
Cellular basis for paracrine regulation of ovarian follicle development.
Reproduction, 121, 647--653.
Andrews,P.D., Ovechkina,Y., Morrice,N., Wagenbach,M., Duncan,K.,
Wordeman,L. and Swedlow,J.R. (2004) Aurora B regulates MCAK at the
mitotic centromere. Dev. Cell, 6, 253--268.
Ashkenazi,H., Cao,X., Motola,S., Popliker,M., Conti,M. and Tsafriri,A. (2004)
EGF-like growth factors: endogenous mediators of the ovulatory response?
Endocrinology, 146, 77--84.
Bringmann,H., Skiniotis,G., Spilker,A., Kandels-Lewis,S., Vernos,I. and
Surrey,T. (2004) A kinesin-like motor inhibits microtubule dynamic
instability. Science, 303, 1519--1522.
Brunet,S., Pahlavan,G., Taylor,S. and Maro,B. (2003) Functionality of the
spindle checkpoint during the first meiotic division of mammalian oocytes.
Reproduction, 126, 443--450.
Byskov,A.G., Yding Andersen,C., Hossaini,A. and Guoliang,X. (1997)
Cumulus cells of oocyte-cumulus complexes secrete a meiosis-activating
substance when stimulated with FSH. Mol. Reprod. Dev., 46, 296--305.
Cimini,D., Howell,B., Maddox,P., Khodjakov,A., Degrassi,F. and Salmon,E.D.
(2001) Merotelic kinetochore orientation is a major mechanism of
aneuploidy in mitotic mammalian tissue cells. J. Cell Biol., 153, 517--527.
Cimini,D., Fioravanti,D., Salmon,E.D. and Degrassi,F. (2002) Merotelic
kinetochore orientation versus chromosome mono-orientation in the
origin of lagging chromosomes in human primary cells. J. Cell Sci., 115,
507--515.
Correll,A.T. and Ford,J.H. (1987) Chromosome flexion: potential for assessing
the state of spindle assembly. Mutat. Res., 190, 137--143.
Cortvrindt,R.G. and Smitz,J.E. (2002) Follicle culture in reproductive
toxicology: a tool for in-vitro testing of ovarian function? Hum. Reprod.
Update, 8, 243--254.
Cukurcam,S., Sun,F., Betzendahl,I., Adler,I.-D. and Eichenlaub-Ritter,U.
(2004) Trichlorfon predisposes to aneuploidy and interferes with spindle
formation in in vitro maturing mouse oocytes. Mutat. Res., 564, 165--178.
Dai,W., Wang,Q., Liu,T., Swamy,M., Fang,Y., Xie,S., Mahmood,R.,
Yang,Y.M., Xu,M. and Rao,C.V. (2004) Slippage of mitotic arrest and
enhanced tumor development in mice with BubR1 haploinsufficiency.
Cancer Res., 64, 440--445.
De Brabander,M.J., Van de Veire,R.M., Aerts,F.E., Borgers,M. and
Janssen,P.A. (1976) The effects of methyl (5-(2-thienylcarbonyl)-1Hbenzimidazol-2-yl) carbamate (R 17934; NSC 238159), a new synthetic
antitumoral drug interfering with microtubules, on mammalian cells
cultured in vitro. Cancer Res., 36, 905--916.
73
F.Sun et al.
Decordier,I., Dillen,L., Cundari,E. and Kirsch-Volders,M. (2002) Elimination
of micronucleated cells by apoptosis after treatment with inhibitors of
microtubules. Mutagenesis, 17, 337--344.
Dhamodharan,R., Jordan,M.A., Thrower,D., Wilson,L. and Wadsworth,P.
(1995) Vinblastine suppresses dynamics of individual microtubules in living
interphase cells. Mol. Biol. Cell, 6, 1215--1229.
Downs,S.M. (1995) The influence of glucose, cumulus cells, and metabolic
coupling on ATP levels and meiotic control in the isolated mouse oocyte.
Dev. Biol., 167, 502--512.
Edwards,R.G. (1965) Maturation in vitro of mouse, sheep, cow, pig, rhesus
monkey and human ovarian oocytes. Nature, 208, 349--351.
Eichenlaub-Ritter,U. (1998) Genetics of oocyte ageing. Maturitas, 30,
143--169.
Eichenlaub-Ritter,U. (2002) Ageing and aneuploidy in oocytes. Ernst Schering
Res. Found. Workshop, (41), 111--136.
Eichenlaub-Ritter,U. (2003) Aneuploidy in ageing oocytes and after toxic
insult. In Trounson,A.O. and Gosden,R.G. (eds), Biology and Pathology of
the Oocyte. Cambridge University Press, Cambridge, pp. 220--257.
Eichenlaub-Ritter,U. and Betzendahl,I. (1995) Chloral hydrate induced spindle
aberrations, metaphase I arrest and aneuploidy in mouse oocytes.
Mutagenesis, 10, 477--486.
Eichenlaub-Ritter,U. and Boll,I. (1989) Age-related non-disjunction, spindle
formation and progression through maturation of mammalian oocytes.
Prog. Clin. Biol. Res., 318, 259--269.
Eichenlaub-Ritter,U., Baart,E., Yin,H. and Betzendahl,I. (1996) Mechanisms
of spontaneous and chemically-induced aneuploidy in mammalian oogenesis: basis of sex-specific differences in response to aneugens and the
necessity for further tests. Mutat. Res., 372, 279--294.
Eichenlaub-Ritter,U., Shen,Y. and Tinneberg,H.R. (2002) Manipulation of the
oocyte: possible damage to the spindle apparatus. Reprod. Biomed. Online,
5, 117--124.
Eichenlaub-Ritter,U., Vogt,E., Yin,H. and Gosden, R. (2004) Spindles,
mitochondria and redox potential in ageing oocytes. Reprod. Biomed.
Online, 8, 45--58.
Elhajouji,A., Van Hummelen,P. and Kirsch-Volders,M. (1995) Indications for
a threshold of chemically-induced aneuploidy in vitro in human lymphocytes. Environ. Mol. Mutagen., 26, 292--304.
Elhajouji,A., Tibaldi,F. and Kirsch-Volders,M. (1997) Indication for thresholds of chromosome non-disjunction versus chromosome lagging induced
by spindle inhibitors in vitro in human lymphocytes. Mutagenesis, 12,
133--140.
Everett,C.A. and Searle,J.B. (1995) Pattern and frequency of nocodazole
induced meiotic nondisjunction in oocytes of mice carrying the ‘tobacco
mouse’ metacentric Rb(16.17)7Bnr. Genet. Res., 66, 35--43.
Gardner,R.D. and Burke,D.J. (2000) The spindle checkpoint: two transitions,
two pathways. Trends Cell Biol., 10, 154--158.
Generoso,W.M., Katoh,M., Cain,K.T., Hughes,L.A., Foxworth,L.B.,
Mitchell,T.J. and Bishop,J.B. (1989) Chromosome malsegregation and
embryonic lethality induced by treatment of normally ovulated mouse
oocytes with nocodazole. Mutat. Res., 210, 313--322.
Hamaguchi,Y., Toriyama,M., Sakai,H. and Hiramoto,Y. (1987) Redistribution
of fluorescently labeled tubulin in the mitotic apparatus of sand dollar eggs
and the effects of taxol. Cell Struct. Funct., 12, 43--52.
Hassold,T. and Hunt,P. (2001) To err (meiotically) is human: the genesis of
human aneuploidy. Nature Rev. Genet., 2, 280--291.
Heath,I.B. and Heath,M.C. (1978) Microtubules and organelle movements in
the rust fungus Uromyces phaseoli var. vignae. Cytobiologie, 16, 393--411.
Heath,I.B. and Rethoret,K. (1981) Nuclear cycle of Saprolegnia ferax.
Cell Sci., 49, 353--367.
Hook,E.B. (1983) Prenatal diagnosis: the population at risk. Hosp. Pract., 18,
239--246.
Hu,Y., Betzendahl,I., Cortvrindt,R., Smitz,J. and Eichenlaub-Ritter,U. (2001)
Effects of low O2 and ageing on spindles and chromosomes in mouse
oocytes from pre-antral follicle culture. Hum. Reprod., 16, 737--748.
Hunt,P.A. and Hassold,T.J. (2002) Sex matters in meiosis. Science, 296,
2181--2183.
Johnson,R.G., Meyer,R.A., Li,X.R., Preus,D.M., Tan,L., Grunenwald,H.,
Paulson,A.F., Laird,D.W. and Sheridan,J.D. (2002) Gap junctions assemble
in the presence of cytoskeletal inhibitors, but enhanced assembly requires
microtubules. Exp. Cell Res., 275, 67--80.
Jordan,M.A. and Wilson,L. (1990) Kinetic analysis of tubulin exchange at
microtubule ends at low vinblastine concentrations. Biochemistry, 29,
2730--2739.
Jordan,M.A., Thrower,D. and Wilson,L. (1992) Effects of vinblastine,
podophyllotoxin and nocodazole on mitotic spindles. Implications for the
role of microtubule dynamics in mitosis. J. Cell Sci., 102, 401--416.
74
Kim,M.R. and Tilly,J.L. (2004) Current concepts in Bcl-2 family member
regulation of female germ cell development and survival. Biochim. Biophys.
Acta, 1644, 205--210.
Kirsch-Volders,M., Aardema,M. and Elhajouji,A. (2000) Concepts of
threshold in mutagenesis and carcinogenesis. Mutat. Res., 464, 3--11.
Kirsch-Volders,M., Vanhauwaert,A., Eichenlaub-Ritter,U. and Decordier,I.
(2003) Indirect mechanisms of genotoxicity. Toxicol. Lett., 140--141,
63--74.
Ladrach,K.S. and LaFountain,J.R.,Jr (1986) Malorientation and abnormal
segregation of chromosomes during recovery from colcemid and nocodazole. Cell Motil. Cytoskeleton, 6, 419--427.
Lan,W., Zhang,X., Kline-Smith,S.L., Rosasco,S.E., Barrett-Wilt,G.A.,
Shabanowitz,J., Hunt,D.F., Walczak,C.E. and Stukenberg,P.T. (2004)
Aurora B phosphorylates centromeric MCAK and regulates its localization
and microtubule depolymerization activity. Curr. Biol., 14, 273--286.
Mailhes,J.B. (1995) Important biological variables that can influence the
degree of chemical-induced aneuploidy in mammalian oocyte and zygotes.
Mutat. Res., 339, 155--176.
Mailhes,J.B. and Yuan,Z.P. (1987) Cytogenetic technique for mouse
metaphase II oocytes. Gamete Res., 18, 77--83.
Mailhes,J.B., Hilliard,C., Lowery,M. and London,S.N. (2002) MG-132, an
inhibitor of proteasomes and calpains, induced inhibition of oocyte
maturation and aneuploidy in mouse oocytes. Cell Chromosome, 1, 2.
Mailhes,J.B., Mastromatteo,C. and Fuseler,J.W. (2004) Transient exposure to
the Eg5 kinesin inhibitor monastrol leads to syntelic orientation of
chromosomes and aneuploidy in mouse oocytes. Mutat. Res., 559, 153--167.
Masuda,A., Maeno,K., Nakagawa,T., Saito,H. and Takahashi,T. (2003)
Association between mitotic spindle checkpoint impairment and susceptibility to the induction of apoptosis by anti-microtubule agents in human
lung cancers. Am. J. Pathol., 163, 1109--1116.
Matzuk,M.M., Burns,K.H., Viveiros,M.M. and Eppig,J.J. (2002) Intercellular
communication in the mammalian ovary: oocytes carry the conversation.
Science, 296, 2178--2180.
Mayer,V.W. and Goin,C.J. (1987) Aneuploidy induced by nocodazole or ethyl
acetate is suppressed by dimethyl sulfoxide. Mutat. Res., 187, 31--35.
Mejillano,M.R, Shivanna,B.D. and Himes,R.H. (1996) Studies on the
nocodazole-induced GTPase activity of tubulin. Arch. Biochem. Biophys.,
336, 130--138.
Mitchison,T.J. (1989) Polewards microtubule flux in the mitotic spindle:
evidence from photoactivation of fluorescence. J. Cell Biol., 109, 637--652.
Mitchison,T., Evans,L., Schulze,E. and Kirschner,M. (1986) Sites of
microtubule assembly and disassembly in the mitotic spindle. Cell, 45,
515--527.
Musacchio,A. and Hardwick,K.G. (2002) The spindle checkpoint: structural
insights into dynamic signalling. Nature Rev. Mol. Cell Biol., 3, 731--741.
Nasmyth,K. (2001) Disseminating the genome: joining, resolving, and
separating sister chromatids during mitosis and meiosis. Annu. Rev.
Genet., 35, 673--745.
Nicklas,R.B. (1988) The forces that move chromosomes in mitosis. Annu. Rev.
Biophys. Biophys. Chem., 17, 431--449.
Nicklas,R.B. (1997) How cells get the right chromosomes. Science, 275,
632--637.
Parry,J.M., Parry,E.M., Bourner,R., Doherty,A., Ellard,S., O’Donovan,J.,
Hoebee,B., de Stoppelaar,J.M., Mohn,G.R., Onfelt,A., Renglin,A.,
Schultz,N., Soderpalm-Berndes,C., Jensen,K.G., Kirsch-Volders,M.,
Elhajouji,A., Van Hummelen,P., Degrassi,F., Antoccia,A., Cimini,D.,
Izzo,M., Tanzarella,C., Adler,I.D., Kliesch,U., Hess,P. et al. (1996)
The detection and evaluation of aneugenic chemicals. Mutat. Res., 353,
11--46.
Parry,J.M., Jenkins,G.J., Haddad,F., Bourner,R., Parry,E.M. (2000) In vitro
and in vivo extrapolations of genotoxin exposures: consideration of factors
which influence dose-response thresholds. Mutat. Res., 464, 53--63.
Pratt,I.S. and Barron,T. (2003) Regulatory recognition of indirect genotoxicity
mechanisms in the European Union. Toxicol. Lett., 140--141, 53--62.
Sackett,D.L. and Varma,J.K. (1993) Molecular mechanism of colchicine
action: induced local unfolding of beta-tubulin. Biochemistry, 32,
13560--13565.
Salamanca,F. and Armendares,S. (1974) C-bands in human metaphase
chromosomes treated with barium hydroxide. Ann. Genet., 17, 135--136.
Sandhu,S.S., Gudi,R. and Athwal,R.S. (1988) A genetic assay for aneuploidy:
quantitation of chromosome loss using a mouse/human monochromosomal
hybrid cell line. Mutat Res., 201, 423--430.
Sasaki,J., Ramesh,R., Chada,S., Gomyo,Y., Roth,J.A. and Mukhopadhyay,T.
(2002) The anthelmintic drug mebendazole induces mitotic arrest and
apoptosis by depolymerizing tubulin in non-small cell lung cancer cells.
Mol. Cancer Ther., 1, 1201--1209.
Aneuploidy in mouse metaphase II oocytes
Seaman,F., Sawhney,P., Giammona,C.J. and Richburg,J.H. (2003) Cisplatininduced pulse of germ cell apoptosis precedes long-term elevated apoptotic
rates in C57/BL/6 mouse testis. Apoptosis, 8, 101--108.
Shen,Y., Betzendahl,I., Sun,F., Tinneberg,H-R. and Eichenlaub-Ritter,U.
(2005) Non-invasive method to assess genotoxicity of nocodazole
interfering with spindle formation in mammalian oocytes. Reprod.
Toxicol., doi:10.1016/j.reprotox.2004.09.007.
Shonn,M.A., Murray,A.L. and Murray,A.W. (2003) Spindle checkpoint
component mad2 contributes to biorientation of homologous chromosomes.
Curr. Biol., 13, 1979--1984.
Speit,G., Autrup,H., Crebelli,R., Henderson,L., Kirsch-Volders,M., Madle,S.,
Parry,J.M., Sarrif,A.M. and Vrijhof,H. (2000) Thresholds in genetic
toxicology—concluding remarks. Mutat. Res., 464, 149--153.
Su,Y.Q., Denegre,J.M, Wigglesworth,K., Pendola,F.L., O’Brien,M.J. and
Eppig.J.J. (2003) Oocyte-dependent activation of mitogen-activated protein
kinase (ERK1/2) in cumulus cells is required for the maturation of the
mouse oocyte-cumulus cell complex. Dev. Biol., 263, 126--38.
Sun,F.Y., Betzendahl,I., Shen,Y., Cortvrindt,R., Smitz,J. and EichenlaubRitter,U. (2004) Preantral follicle culture as novel in vitro assay in
reproductive toxicology testing in mammalian oocytes. Mutagenesis, 19,
13--25.
Tanaka,E., Ho,T. and Kirschner,M.W. (1995) The role of microtubule
dynamics in growth cone motility and axonal growth. J. Cell Biol., 128,
139--155.
Tarkowski,A.K. (1966) An air-drying method for chromosome preparation
from mouse eggs. Cytogenetics, 5, 394--400.
Toso,R.J., Jordan,M.A., Farrell,K.W., Matsumoto,B. and Wilson,L. (1993)
Kinetic stabilization of microtubule dynamic instability in vitro by
vinblastine. Biochemistry, 32, 1285--1293.
Van Wemmel,K., Cortvrindt,R., Eichenlaub-Ritter,U. and Smitz,J.J. (2005)
Influence of diazepam on folliculogenesis, steroidogenesis and oocyte
quality assessed by an in vitro follicle bioassay. Reprod. Toxicol., in press.
Vasquez,R.J., Howell,B., Yvon,A.M., Wadsworth,P. and Cassimeris,L. (1997)
Nanomolar concentrations of nocodazole alter microtubule dynamic
instability in vivo and in vitro. Mol. Biol. Cell, 8, 973--985.
Verdoodt,B., Decordier,I., Geleyns,K., Cunha,M., Cundari,E. and KirschVolders,M. (1999) Induction of polyploidy and apoptosis after exposure to
high concentrations of the spindle poison nocodazole. Mutagenesis, 14,
513--520.
Wassmann,K., Niault,T. and Maro,B. (2003) Metaphase I arrest upon
activation of the Mad2-dependent spindle checkpoint in mouse oocytes.
Curr. Biol., 13, 1596--1608.
Wendell,K.L., Wilson,L. and Jordan,M.A. (1993) Mitotic block in HeLa cells
by vinblastine: ultrastructural changes in kinetochore-microtubule attachment and in centrosomes. J. Cell Sci., 104, 261--724.
Wilson,L., Bamburg,J.R., Mizel,S.B., Grisham,L.M. and Creswell,K.M.
(1974) Interaction of drugs with microtubule proteins. Fed. Proc., 33,
158--166.
Witt,K.L., Hughes,L.A., Burka,L.T., McFee,A.F., Mathews,J.M., Black,S.L.
and Bishop,J.B. (2003) Mouse bone marrow micronucleus test results do not
predict the germ cell mutagenicity of N-hydroxymethylacrylamide in the
mouse dominant lethal assay. Environ. Mol. Mutagen., 41, 111--20.
Wood,J.S. (1982) Mitotic chromosome loss induced by methyl benzimidazole2-yl-carbamate as a rapid mapping method in Saccharomyces cerevisiae.
Mol. Cell. Biol., 2, 1080--1087.
Yin,H., Cukurcam,S., Betzendahl,I., Adler,I.D. and Eichenlaub-Ritter,U.
(1998a) Trichlorfon exposure, spindle aberrations and nondisjunction in
mammalian oocytes. Chromosoma, 107, 514--522.
Yin,H., Baart,E., Betzendahl,I. and Eichenlaub-Ritter,U. (1998b) Diazepam
induces meiotic delay, aneuploidy and predivision of homologues and
chromatids in mammalian oocytes. Mutagenesis, 13, 567--580.
Zimmermann,F.K., Mayer,V.W. and Scheel,I. (1984) Induction of aneuploidy
by oncodazole (nocodazole), an anti-tubulin agent, and acetone. Mutat.
Res., 141, 15--18.
Received on December 1, 2004; revised on January 12, 2005;
accepted on January 13, 2005
75