Molecular Human Reproduction vol.2 no.12 pp. 895-901, 1996
The oxidizing agent tertiary butyl hydroperoxide induces
disturbances in spindle organization, c-meiosis, and aneuploidy in
mouse oocytes
Juan J.Tarin1'4, Francisco J.Vendrell2, Jorge Ten2, Raquel Blanes2, Jonathan Van Blerkom1 and
Antonio Cano3
department of Molecular, Cellular and Developmental Biology, Porter Biosciences Building, Campus Box 347, Boulder,
Colorado 80309-0347, USA, department of Biochemistry and department of Paediatrics, Obstetrics, and Gynaecology,
Faculty of Medicine, University of Valencia, Valencia, Spain
4
To whom correspondence should be addressed at Department of Paediatrics, Obstetrics and Gynaecology, Faculty of
Medicine, University of Valencia, Avda, Blasco Ibanez 17, 46010, Valencia, Spain
It has been recently proposed that a concomitant generation of oxidative stress of oocytes with increasing
maternal age may be a major factor responsible for the age-related increase in aneuploid conceptions. As a
preliminary step in the testing of this hypothesis, we need to confirm that oxidative stress in itself can induce
errors in chromosome segregation. In order to achieve this goal, germinal vesicle (GV)-stage mouse oocytes
from unstimulated ICR and (C57BLXCBA) F1 hybrid female mice were matured in vitro for 9 h for metaphase
I (Ml) oocytes or 16 h for metaphase II (Mil) oocytes in the presence of varying concentrations of the oxidizing
agent tertiary-butyl hydroperoxide (tBH). Mil oocytes from (C57BLXCBA) F, hybrid mice were fixed and
C-banded for karyotyping analysis. Ml and Mil oocytes from ICR mice were fixed and stained with the DNAfluorescent probe 4',6-diamidino-2-phenylindole (DAPI) to detect abnormalities in chromosomal distribution.
Meiosis I and meiosis II spindles from ICR mice were visualized by confocal immunofluorescence microscopy.
Data from these experiments demonstrate that in-vrtro exposure of mouse oocytes to tBH during meiosis I
reduces the length (pole-to-pole distance) and width (diameter at the equator of the spindle) of meiosis I and
meiosis II spindles. This reduction is associated with an increase in the percentage of oocytes showing
chromosome scattering and clumping on the Mil plate, and of aneuploidy (hyperhaploidy) in Mil oocytes.
However, tBH at the concentrations used in the present study has only a minimal negative effect on the
frequency of meiotic maturation. These results suggest that oxidative stress during meiotic maturation
in vitro may induce chromosomal errors that are undetectable in the living oocyte and whose developmental
consequences may become manifest after fertilization.
Key words: aneuploidy/meiosis/mouse oxidative stress/spindleAertiary butyl hydroperoxide
Introduction
While numerous hypotheses have been proposed to account
for the maternal age-associated increase in the frequency of
chromosomally abnormal human fetuses, none has been able
to provide a definitive aetiology (for reviews, see Bond and
Chandley, 1983; Hassold and Jacobs, 1984; Gaulden, 1992;
Hassold et al, 1993). To shed light on this topic, a mechanism
based on the 'free radical theory of ageing' has been recently
suggested (Tarin, 1995). This hypothesis suggests concomitant
generation of oxidative stress in oocytes with increasing
maternal age as a major cause responsible for the increased
frequency of aneuploidy in oocytes of women during the latter
stages of their reproductive life. One of the fundamental pillars
of this hypothesis is based on the fact that many physical and
chemical agents, which are directly or indirectly involved
in intracellular free radical production or oxidative stress
generation, or both, are potential inducers of aneuploidy. These
agents include ionizing (X-rays or y-rays) and non-ionizing
(UV) radiation, quinones, heavy metal ions (e.g. mercury, lead,
and cadmium), anaesthetics (e.g. nitrous oxide, chloroform,
halothane, and ethanol), reduced glutathione-(GSH)-conjugat© European Society for Human Reproduction and Embryology
ing (e.g. diethyl maleate and l-chloro-2,4-dinitrobenzene) and
GSH-oxidizing [e.g. diamide, thimerosal, and tertiary-butyl
hydroperoxide (tBH)] agents, carbamates (e.g. carbaryl),
organo-mercurial agents (e.g. methyl mercury), inducers of
mixed-function oxidase reaction (e.g. phenobarbital), cigarette
smoke, methylxanthines (e.g. caffeine), and oestrogens (e.g.
natural and synthetic oestrogens) (Kaufman, 1982; Lynch and
Parry, 1993) (for reviews, see Bond and Chandley, 1983;
Onfelt, 1986). Most of these studies have been performed,
however, in cultured somatic cells or in oocytes retrieved from
females exposed to these oxidizing agents. Although studies
on somatic cells are informative, extrapolation to the female
germ cell line may be inappropriate. Furthermore, the in-vivo
environment may conceal, counteract or dilute the effects of
these agents on oocytes. For instance, there is no indication
as to whether the observed damage of oocytes in vivo is due
to a direct interaction of these agents with the female gamete
or with their neighbouring granulosa cells.
In the present study, we examined the effects of experimentally-induced oxidative stress on the chromosomal normality
of the mouse oocyte during maturation in vitro. Fully-grown
895
J.J.Tarfn et al.
immature mouse oocytes were cultured in the presence of
the oxidizing agent tBH and the normality of chromosomal
distribution on the metaphase spindle and ploidy were determined. tBH was selected for these studies because it is well
established that its metabolism generates directly reactive
oxygen species (Ochi and Miyaura, 1989), decreases GSH/
oxidized glutathione (GSSG) ratio (Bellomo et al, 1982),
increases free cytosolic Ca 2+ concentration (Bellomo et al,
1984), and collapses the mitochondrial membrane potential,
resulting in a decrease in ATP concentration and acidification
of the cytoplasm (Masaki et al, 1989). This information is of
special relevance because any of these variables may affect
the dynamic equilibrium of microtubules (assembling-disassembling of tubulins) and so induce mistakes in chromosome
segregation (Tarin, 1995, 1996). Fully-grown immature mouse
oocytes were selected for analysis in order to determine
whether acute exposure to an oxidizing agent such as tBH
affects nuclear maturation in general, and chromosomal
normality in particular. If developmental processes during
meiotic maturation can be perturbed by an agent which is
known to induce oxidative stress, the findings may also be
relevant to the in-vitro maturation and fertilization of immature
human oocytes which occur under conditions that may also
induce oxidative stress.
Material and methods
Oocyte collection
Fully-grown germinal vesicle (GV)-stage oocytes were obtained from
unstimulated 6-8 week old ICR and (C57BLXCBA) F, hybrid female
mice by tearing the ovaries apart in medium M2 (Quinn et al., 1982)
supplemented with bovine serum albumin (BSA) 4 mg/ml. GV
oocytes were denuded of granulosa cells (if present) by repeated
passage through a micropipette. Oocytes were cultured for 9 h for
metaphase I (MI) oocytes or 16 h for metaphase II (Mil) oocytes in
the presence or the absence of varying concentrations of tBH (Sigma
Chemical CO, St. Louis, MO, USA) in 200 ul (from ICR females)
or 1 ml (F, hybrid mice) of medium M16 (Whittingham, 1971)
supplemented with 4 mg BSA/ml at 37"C in an humidified atmosphere
of 5% CO2 in air. No mineral oil was used to cover the surface of
the culture medium owing to its ability to alter the composition of the
medium by absorbing and/or transferring certain types of compounds
(Miller and Pursel, 1987).
Chromosomal analysis
In-vitro matured (IVM) Mil oocytes from (C57BLXCBA) F, hybrid
mice were prepared for karyotyping. Oocytes were exposed to
hypotonic 0.9% sodium citrate solution at 4°C for 20 min to 3 h and
fixed in methanol:glacial acetic acid (3:1) at -20°C (Dyban, 1983).
Chromosomes were C-banded (Salamanca and Armendares, 1974) to
ensure the unequivocal identification of whole chromosomes and
single chromatids. Criteria for removing a cell from the cytogenetic
study were: insufficient C-banding to discriminate between whole
chromosomes and single chromatids, overlapping or clumped chromosomes that precluded an accurate count, and excessive chromosome
scatter. The frequency of aneuploidy was calculated as twice the
frequency of hyperhaploidy because an unknown proportion of
hypohaploid cells may arise from technical artefact during slide
preparation.
Chromosomal distribution of IVM MI and Mil oocytes from
896
ICR female mice was determined by fluorescence microscopy after
exposure of oocytes for 5 min to 150 uM 4',6-diamino-2-phenylindole (DAPI; Sigma Chemical Co.) in medium M2. Oocytes were
fixed before staining with 3% formaldehyde in phosphate-buffered
saline (PBS, pH 7.2) for 5 min and washed overnight at 4°C in
medium M2. Anomalies in chromosomal distribution [colchicinemeiosis (c-meiosis), a partial or complete inactivation of the spindle
mechanism resembling that caused by the action of colcichine]
were classified into three subgroups: (i) chromosome clustering; (ii)
chromosome scattering; or (iii) the presence of two DNA-containing
polar bodies with no chromosomes inside the oocyte.
Immunostaining and confocal laser scanning microscopy
analysis of oocytes
IVM MI and Mil oocytes from ICR females were fixed at room
temperature for 10 min in 3.7% formaldehyde in microtubule stabilization buffer (80 mM potassium PIPES, 5 mM EGTA, 1 mM MgCl2,
0.2% Triton X-100, and 0.5 uM taxol, pH 6.8) (Gard, 1991). Before
fixation, zonae pellucidae were removed in medium M2 containing
0.5% pronase (B grade; Calbiochem, Los Angeles, CA, USA).
Oocytes were then allowed to recover for 30 min in culture medium.
After fixation, oocytes were stored in medium M2 at 4°C for a
minimum of 24 h. Prior to antibody staining, oocytes were rinsed
overnight at 4°C in Tris-buffered saline (TBS: 155 mM NaCl, 10 mM
Tris-HCI, and 0.1% NP-40, pH 7.4). Oocytes were incubated in
mouse anti (3-tubulin antibody [immunoglobulin (Ig)G, diluted 1:100
in TBS containing 2% BSA and 1% dimethyl sulphoxide (DMSO)
(TBS-BSA)] that was kindly provided by DrM.Klymkowsky. Oocytes
were then rinsed in TBS-BSA, followed by incubation in fluorescein
isothiocyanate (FITC)-conjugated rabbit anti-mouse IgG antibody
(Sigma Immuno Chemicals; diluted 1:50 with TBS-BSA) and wash
in TBS-BSA. Each antibody and its respective rinse was applied for
1 h at 37°C. After washing, oocytes were incubated overnight at
room temperature in the SlowFade Light Antifade Kit (Molecular
Probes, Eugene, OR, USA) to retard photobleaching. Specimens were
examined with a confocal laser scanning microscope (Molecular
Dynamics, Sunnyvale, CA, USA) fitted to a Nikon Diaphot microscope
using a X60 objective. Each spindle was examined at intervals of
1 urn, and projections of varying thicknesses were obtained by
compiling multiple consecutive images after electronic processing
with a 3-D Gaussian filter and a 3-D Gradient filter (Image Space
software, Molecular Dynamics). Only those spindles which were
laying in a horizontal plane were evaluated.
Statistical analysis
One- and two-way analysis of variance (ANOVA) tests and Student's
r-test were applied for comparisons of means. In order to stabilize
variances, proportions were transformed to arcsine before carrying
out comparisons of the means. When the one-way ANOVA test
showed statistically significant differences, the student NewmanKeuls (SNK) test was used to discriminate between groups. The j}
test was applied for comparisons of frequencies. Significance was
defined as P =sO.O5. The entire statistical analysis was carried out
using the Statistical Package for Social Sciences (SPSS).
Results
•
The chromosomal constitution of mouse oocytes matured
in vitro in the presence of varying concentrations of tBH is
shown in Table I. The levels of aneuploidy (2 Xhyperhaploidy)
were significantly (P =s0.05) higher in oocytes exposed to tBH
when compared to control oocytes. Most hyperhaploid oocytes
Oxidative stress and aneuploidy
Table I. Chromosome constitution of metaphase II (Mil) mouse oocytes matured in vitro in the presence of varying concentrations of tertiary-butyl
hydroperoxide (tBH)
t-BH (uM)
± 2.2°
± 2.7
± 2.8
±4.8
120 (27.2)b
109(26 9)
95 (21.8)
98(21.6)
Chromosome number
18
19
2
3
2
5
5
8
8
9
20
112
92
82
81
Aneuploidy
21
1
5
3
2
22
i —
83.0
78.2
78.2
71.2
No. Mil oocytes analysed
I
23
23
23
23
In-vitro maturation (%)
40
I
0.0
0.05
0.5
5.0
n
1
2 (1.7)c
10 (9.2)
6 (6.3)
6(6.1)
•Values are means ± SE.
''Percentages shown in parentheses.
°Value significantly different from combined tBH-treated groups (P =s0.05).
n = no. experiments.
showed a complement of 21 chromosomes. A diploid oocyte
with 40 chromosomes was also detected in the 0.05 uM
tBH group.
The percentage of oocytes displaying a normal distribution
of Mil chromosomes on the meiosis II spindle (Figure 1)
decreased significantly (P =£0.0001) as the tBH concentration
increased from 0 to 5 \LM (Table II). This drop was due to an
increase in the proportion of oocytes showing chromosome
scattering (Figure 2) and/or chromosome clustering (Figure 3).
Several oocytes showed an abnormally large first polar body
or absence of chromosomes in the cytoplasm together with
two DNA-containing polar bodies (Figure 4). However, these
anomalies were not associated with or related to the tBH
concentrations used in these experiments. No apparent anomalies in chromosomal distribution were detected in MI oocytes
after 9 h of exposition to 0.0, 0.05, 0.5, and 5 ^M tBH (data
not shown).
Table III shows the length (pole-to-pole distance) and width
(diameter at the equator of the spindle) of meiosis I and
meiosis II spindles after in-vitro maturation in the presence of
5 |iM tBH. As the Bartlett's test of sphericity showed that the
variables, length and width of the spindles were independent,
i.e. they were not correlated, we had no reason for using
multivariate analysis of variance (MANOVA) for the analysis
of these data and, therefore, two-way ANOVA was applied.
Both the meiosis stage at which the spindle was measured and
oocyte treatment with tBH were significant factors modifying
the length and width of spindles. Meiosis I spindles were
significantly longer (P =£0.0005) and wider (P =£0.0005) than
meiosis II spindles, whereas tBH caused a shrinking of both
meiosis I and meiosis II spindles. The shrinking effect of tBH
on meiotic spindles was indicated by a reduced pole-to-pole
distance (P =£0.002) and decreased diameter at the equator of
the spindle (P =£0.0005) when compared to control oocytes.
Meiosis I and II spindles showed the characteristic anastral
barrel-shaped morphology of mouse oocytes (Figures 5 and
6A). No apparent differences in spindle shape were observed
between control and tBH-treated oocytes. Two gross morphological abnormalities were, however, detected after tBH treatment: a dipolar spindle was present in a MTI oocyte (Figure
6B) and a completely disorganized spindle in a MI oocyte
(Figure 7). A tripolar spindle was also observed in a control
Mil oocyte. In some MI and Mil oocytes, a few small asters
were scattered throughout the cytoplasm (Figure 8). The
presence as well as the number of these microtubular structures
were not associated with the fact that oocytes were matured
in vitro in the presence or the absence of tBH (data not shown).
Discussion
The experiments reported here demonstrate that exposure of
mouse oocytes to tBH during in-vitro maturation from the GV
stage reduces the length and width of the meiosis I and meiosis
II spindles, increases the percentage of oocytes showing
chromosome scattering and clumping on the MH plate, and
induces aneuploidy in Mil oocytes. These results are, therefore,
in agreement with previous studies showing an increased
frequency of hyperhaploidy (OnfSlt, 1987a) and c-mitosis
(Onfglt, 1987b) in an established cell line (V79) of Chinese
hamster lung cells when treated with tBH for 30 min and 3 h
respectively. In our study, however, no apparent disturbances
in chromosomal distribution were detected in MI oocytes after
9 h of exposition to tBH. This may be due to the fact that the
transition between prometa-/meta-/anaphase I seems to be very
brief (Eichenlaub-Ritter et al., 1986), so that it is quite unusual
to find all bivalents precisely positioned in the equatorial plane
such as happens in arrested MH oocytes. Thus, these results
agree with the conclusion drawn by Eichenlaub-Ritter et al.
(1986) that 'the positioning of bivalents in the dynamic process
of maturation does not seem to be a suitable parameter
to reveal predisposition to non-disjunction'. In contrast, the
presence of c-meiosis on the MTI plate suggests that factors)
intrinsic or extrinsic to the oocyte is(are) affecting spindle
microtubules and so may cause(s) aneuploidy in ME oocytes
if also present and active during the first meiotic division.
Nonetheless, discrepancies between the percentages of aneuploidy and the percentages of c-meiosis induced by different
concentrations of tBH were found in the present study. In fact,
whereas percentages of aneuploidy were more or less constant,
the percentages of c-meiosis on the Mil plate showed a
significant increase as the concentration of tBH rose from 0.05
to 5 ^M. Such discrepancies may be explained, at least in
part, by several factors. Firstly, they may be due to intrinsic
differences between oocytes from (C57BL/XCBA) F, and ICR
mice in their homeostatic response when exposed to an
oxidizing agent such as tBH. Oocytes from the hybrid strain
may activate a defence mechanism against the toxic effects of
tBH at much lower concentrations than oocytes from ICR
897
JJ.Tarfn et al.
Figures 1-4. Metaphase n (MOO) mouse oocytes after fixation and exposure to 4',6-diarmdino-2-phenylindote (DAPI) for assessment of
chromosome distributioo by fluorescence microscopy. Oocytes were matured in vitro from the germinal vesicle (GV) stage in the presence
or the absence of the oxidizing agent tertiary-butyl hydroperoxide (tBH). Figure 1. Normal distribution of chromosomes on the Mil spindle.
Figure 2. Several tBH-treated oocytes showed chromosome scattering (arrow) on the Mil plate. Figure 3. Several tBH-treated oocytes
showed chromosome clustering on the Mil plate. Figure 4. A few oocytes exhibited two DNA-containing polar bodies (PB) with no DNA
staining inside the oocyte cytoplasm.
females. The existence of such a cellular defence mechanism
has been previously suggested by Onfe'lt (1987b) after observing a biphasic response for c-mitosis in V79 Chinese hamster
lung cells exposed to varying concentrations of tBH. Secondly,
Mil chromosomes and/or the meiosis II spindle may be more
sensitive to the disrupting effects of tBH than MI chromosomes
and/or the meiosis I spindle, and so may be differentially
affected when exposed to tBH. And thirdly, tBH may be an
agent far more active for inducing c-meiosis than aneuploidy.
This idea is supported by studies using V79 Chinese hamster
lung cells in which a concentration of 1.4X 10~s M tBH induced
10% c-mitosis (Onfe'lt, 1987b) whereas a similar percentage
898
of aneuploidy was not reached until the tBH concentration
was increased to 3.4X10" 5 M (Onfe'lt, 1987a).
In-vivo ageing of mouse oocytes for ~15 h after ovulation
is associated with shrinkage of the Mil spindle, change in
spindle morphology towards a more fusiform shape, appearance
of large asters scattered throughout the oocyte, and formation
of astral microtubule fibres pointing away from the centrosomes
into the cytoplasm (Eichenlaub-Ritter et at., 1986). Further
ageing of oocytes, however, decreases the number of cytoplasmic microtubule organizing centres (MTOCs) and increases
the critical cytoplasmic concentration for tubulin polymerization around the remaining MTOCs (Webb et aL, 1986). The
Oxidative stress and anouploidy
Thble D. Chromosomal distribution of metaphase II (Mil) mouse oocytes matured in vitro in the presence of varying concentrations of tertiary-butyl
hydroperoxide (tBH)
No. Mil oocytes analysed
Chromosomal distribution
Normal
Abnormally large first polar body
C-meiosis
Chromosome clustering
Chromosome scattering
No chromosomes + two polar bodies
Control
0.05 HM tBH
0.5 UM tBH
5 nMtBH
182
178
169
185
181 (99.5)m-b
1 (0.5)
166 (93.3)
1 (0.6)
120 (92.3)
-
150(81.1)
1 (0.5)
6 (3.4)
3 (1.6)
2(1.1)
5 (3.0)
8 (4.7)
17 (9.2)
15(8.1)
2(1.1)
-
-
•Percentages shown in parentheses.
'Value significantly different among groups (P «0.0001).
Tbble i n . Length and width of meiosis I and mciosis II spindles of mouse oocytes matured in vitro in the presence of 5 uM tertiary-butyl hydroperoxide
(tBH)
Meiosis I spindle (Jim)
Control
tBH
Meiosis II spindle (um)
n
Length
Width
n
Length
Width
17
15
26.99 ± 1.18"
25.24 ± 0.75
16.21 ± 0.33
15.02 ± 0.29
19
16
23.14 ± 0.42
20.68 ± 0.49
14.08 ± 0.32
12.71 ± 0.31
•Values are means ± SE.
Two-way analysis of variance:
Treatment
Meiotic division
TreatmentXmeiotic division
Length
P "S0.002
P «0.0005
NS
Width
P S5O.OOO5
P =50.0005
NS
NS = not significant.
presence of such changes led Eichenlaub-Ritter et al. (1986)
to conclude that the first alterations in the microtubular
cytoskeleton seem to be caused mainly by a redistribution of
microtubules from the spindle into the cytoplasm. In oocytes
treated with tBH, it appears that such a redistribution of
microtubules does not account for the shrinkage of meiosis I
and meiosis II spindles. A bias in the dynamic equilibrium of
microtubules towards the disassembling of tubulins seems to
be a more convincing explanation. In fact, neither astral
microtubules at the centrosomes of the spindles nor large asters
scattered throughout the oocyte were visualized. Only in some
tBH-treated as well as in some control oocytes, were a few
small asters noticed in the cytoplasm.
Although the mechanism by which tBH modifies the organization of the meiotic spindles and induces c-meiosis and
aneuploidy in mouse oocytes remains to be elucidated, it
appears that it is not caused by the tBH molecule in itself. In
fact, it has been shown that tBH has no direct effect on
microtubule assembly in vitro (Oliver et al., 1976). In contrast,
tBH can inhibit both microtubule assembly and depolymerization of assembled cytoplasmic microtubules in peripheral blood
polymorphonuclear leucocytes exposed to the plant lectin
concanavalin A (Oliver et al., 1976, 1977). The effects of tBH
on spindle microtubules in vivo are more likely to be due to
perturbations of the balance between cellular sulphydryl
groups (SH) and disulphide bonds (SS). In fact, other GSHoxidizing agents such as diamide and thimerosal, or even
GSH-conjugating agents such as diethyl maleate and 1-chloro2,4-dinitrobenzene, may also damage the cell-division apparatus (Onfelt, 1987b; Cheek et al., 1993; Lynch and Parry,
1993). tBH appears to affect the balance between cellular SH
groups and SS bonds by oxidizing GSH to GSSG when
metabolized by GSH peroxidase. As mentioned above, a
decrease in the intracytoplasmic GSH/GSSG ratio and/or any
of its downstream effects (rises in intracellular Ca 2+ ions and
collapse of the mitochondrial membrane potential with the
resulting decrease in ATP concentration and acidification of
cytoplasm) may account for the partial or complete blockage
of the spindle mechanism which normally directs chromosome
distribution and/or segregation.
The present study demonstrates that although tBH can
induce c-meiosis and aneuploidy in IVM mouse oocytes, it
has only a minimal negative effect on the resumption of
arrested meiosis or the nuclear events associated with the
maturation of the mouse oocyte in vitro, including GV breakdown and polar body expulsion. These findings have special
relevance to the laboratory conditions associated with in-vitro
maturation and fertilization of human oocytes. Our results
suggest that aneuploidy may be iatrogenically induced in
human oocytes matured and fertilized in vitro, if the conditions
that may promote oxidative stress, including exposure to high
atmospheric O2 concentrations and/or visible light, and the
presence of transition metals and/or prooxidant substances in
the culture medium are not controlled or minimized. The
effects of an acute exposure in vitro to an oxidizing agent
during meiosis I on chromosomal normality of oocytes may
be undetectable by light microscopy but become manifest after
fertilization.
There is evidence suggesting that reducing agents and/or
reactive oxygen species scavengers are able to neutralize
899
J.J.Tarin ef a/.
Figures 5-8. Confocal laser scanning microscopic images of metaphase I (MI) (Figures 5 and 7) and MH (Figures 6A, 6B and 8) spindles
of mouse oocytes matured in vitro from the GV stage in the presence (Figures 6A, 6B, 7 and 8) or me absence (Figure S) of tertiary-butyl
hydroperoxide (tBH). Each image is a reconstruction of consecutive 1 urn scans. Figure 5. Notice the characteristic anastral barrel-shape of
MI spindles of mouse oocytes. Figure 6. (A) shows the characteristic anastral barrel-shape of Mil spindles of mouse oocytes. (B) shows a
tripolar spindle observed in a Mil oocyte. Figure 7. A completely disorganized spindle was found in a MI oocyte matured in vitro in the
presence of 5 uM tBH. Figure 8. An Mil oocyte with five small asters scattered throughout the cytoplasm and its respective spindle laying
in a oblique plane.
endogenous (Fraga et al., 1991), drug-induced (Emerit et al,
1983; Weitberg, 1987; He and Yasumoto, 1994), or gamma
irradiation-induced (Gaziev et al., 199S) oxidative damage to
DNA and/or chromosomes. Aneuploidy induced by oxidative
stress may be also prevented by dietary intake of antioxidants
and/or by supplementing culture media with antioxidants.
Further work is in progress to determine the exact mechanism
by which oxidative stress induces mistakes in chromosome
segregation and to also ascertain whether aneuploidy induced
900
by oxidative stress can be prevented by the presence of
antioxidants.
Acknowledgements
We thank Drs R.Gil, C.L6pez, and M.Gregori, Department of Pathological Anatomy, Faculty of Medicine, University of Valencia, Valencia,
Spain, for technical assistance. This study was supported in part by
a grant to A.C. from 'Ministerio de Sanidad y Consumo' (FIS 93/
0680) and a grant to J.V.B. from the National Institutes of Health
(HD-31907).
Oxidative stress and aneuploidy
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Received on August 16, 1996; accepted on November 4, 1996
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