Diethylstilbestrol (DES)-induced cell cycle delay and meiotic spindle disruption in mouse oocytes during in-vitro maturation
Alp Can and Olcay Semiz
Department of Histology-Embryology, Ankara University School of Medicine, Sihhiye, 06339 Ankara, Turkey
Abstract
Due to the growing amount of data related to the
deleterious effects of the synthetic oestrogenic compound,
diethylstilbestrol (DES), on the female reproductive system, we
tested the potential effects of this compound on mouse oocytes.
Controlled time- and dose-dependent in-vitro experiments were
carried out on isolated cumulus–oocyte–complexes (COCs) to examine
the meiotic spindle assembly and chromosome distribution. 
-tubulin, chromosomes and F-actin were labelled
and detected by confocal laser scanning
microscope. COCs were exposed to varying doses of DES (5–30 µmol/l)
from the germinal vesicle (GV) stage to the end of metaphase II
(MII) when meiosis I and meiosis II is normally completed. Exposure
to DES during meiosis I caused a dose-dependent inhibition of cell
cycle progression. In comparison with controls, fewer oocytes
reached metaphase I (MI) at low doses (5 µmol/l) of DES, while none
of the oocytes reached MI in high doses (30 µmol/l). When COCs were
exposed to high doses of DES during meiosis II, fragmentation of
first meiotic spindle was detected, whereas lower doses caused
loosening of the first and the second meiotic spindles. No
microtubular abnormalities were detected either in GV-stage oocytes
or in cumulus cells. The above data
demonstrate that one mode of action of DES on mouse oocytes is a
severe yet reversible deterioration of meiotic spindle microtubule
organization during maturation.
Key Words: confocal microscopy • diethylstilbestrol • in-vitro maturation • microtubule • spindle
Introduction
Disruption of normal structure and function has been at the centre
of toxicological studies focusing on reproductive toxicants for many years. Vast
numbers of compounds have been reported to cause detrimental outcome
during development of the gametes, fetus or neonate. For example,
many man-made or generated chemicals used in household products,
including pesticides and plasticizers, pharmaceuticals, and dietary
supplements, as well as some naturally occurring substances such as
phyto-oestrogens found in plants, are adversely affecting
reproductive tract development and function (McLachlan, 1985
).
In developmental exposures, chemicals usually exert their effects
only at a specific time during maturation and differentiation and
then they disappear. Moreover, they may display a reversible
interaction pattern depending on the type of chemical, duration and
most importantly the critical timing of exposure (Can and Albertini, 1997a,b).
If the agent ceases before the `vulnerability interval' ends, the
adverse developmental effects might be partially or totally overcome
(Can and Albertini,
1997b).
Therefore, assessing the developmental toxicity of suspected agents
is often problematic and requires several precisely-timed
dose-response experiments associated with sophisticated analytical
procedures.
The profound effects of synthetic oestrogens, in particular, on
the developing reproductive tract have been previously demonstrated by
prenatal exposure to diethylstilbestrol (DES) (Herbst and Bern, 1981),
a non-steroidal compound functionally (but not structurally) similar
to natural oestradiol. Historically, DES had been prescribed to
women with high-risk pregnancies to prevent spontaneous abortions
and other complications of pregnancy. In following years, many
health problems have been reported implicating DES in both female
reproductive system abnormalities, e.g. vaginal adenocarcinoma,
abnormal pregnancies, reduction in fertility and immune system
disorders (see Newbold, 1999 for review). Although DES is no longer
used clinically to prevent miscarriage, a major concern remains that
when DES-exposed women age and reach the time at which the incidence
of reproductive organ cancers
normally increase, they will show a much higher incidence of cancer than unexposed individuals.
Further, the possibility of second generation effects has been
suggested (Turusov et al., 1992;
Newbold et al., 1998),
which puts still another generation at risk for developing problems
associated with the DES treatment of their grandmothers. In
emergency situations, e.g. post-coital conceptions, DES is still
used as the morning-after pill. Thus, the DES episode continues to
be a serious health concern and remains a reminder of the potential
toxicities that can
be caused by hormonally active chemicals.
Studies of DES-induced adverse effects are primarily focused on
its teratogenicity and carcinogenicity. Some in-vivo animal models
were suggested (Newbold, 1999)
to investigate these effects in which DES was shown to target the
ovaries causing series ovarian developmental failures including
early depletion of follicles (Sangvai et al.,
1997)
multi-ovular follicles (Iguchi et al., 1990)
and lower implantation rates (Pal et al., 1997).
Although several complex mechanisms are involved in ovarian follicular
and stromal development and are not easy to differentiate, further
approaches would help to elucidate the cellular response to DES.
Since in-vivo models do not enable testing to observe whether
toxicity occurs due to the impairment of hypothalamic/pituitary axis
or by direct action on target tissues/cells, we used a unique 2-cell
model to monitor the detrimental effect of DES on oogenesis. In the
present model, as we have previously shown (Can and Albertini, 1997a),
we screened the meiotic cell division with regard to the formation
of meiotic spindle and chromosome distribution during in-vitro
maturation of mouse cumulus–oocyte–complexes (COCs). Controlled
exposure of COCs to varying doses of DES and during different stages
of meiotic progression beginning from the germinal vesicle (GV)
stage to the end of metaphase II (MII) enabled the analysis of the
effects of DES on meiotic spindle formation and function. The
formation of first meiotic spindle was tested during the first 8 h
of treatment, whereas the second treatment period of 10 h enabled us
to test the effect of DES after the first meiotic spindle had been
assembled. The results show that DES is a potent yet reversible
disrupter of meiosis in mouse oocyte through actions that perturb
both cell cycle progression and microtubular organization.
Materials and methods
Collection and culture of mouse COCs
Ovarian follicular development was stimulated by i.p. injection of 5
IU pregnant mare's serum gonadotrophin (PMSG; Sigma
Chemical Co, St. Louis, MO, USA) in 19–21
day-old female Balb-C mice. Animals were killed after 48 h of
injection and COCs were collected (n = 1655) from isolated
ovaries and by follicular puncture. Upon
isolation, they were cultured for different time intervals (see
below) in minimal essential medium (MEM; Sigma Chemical Co)
supplemented with Earle's salts, 2 mmol/l L-glutamine,
0.23 mmol/l pyruvate, 100 IU/ml penicillin, 100 µg/ml streptomycin
and 3% bovine serum albumin (BSA) in a humidified atmosphere of 5%
CO2 at
DES experiments
Diethylstilbestrol (DES) (purity 99.7%, Mr
268,4) (Sigma Chemical Co) was dissolved in dimethylsulphoxide
(DMSO) as a 10 mmol/l stock solution and freshly prepared working
solutions of 5, 15 and 30 (in mmol/l final concentrations) were used
to treat COCs during different stages of oocyte maturation. DMSO
concentration of treated and control samples never exceeded 0.1% v/v and at a given concentration, no adverse
effects of DMSO on oocyte maturation were observed.
Three different sets of experiments were carried out using three different
doses of DES at specific stages (Table I). One group of
COCs was treated during initial stages of in-vitro maturation for
7–8 h between the GV stage and MI. A second group was exposed to DES
during an 8–18 h interval between MI and metaphase II (MII). In the final set of experiments, COCs were exposed to
low (5 µmol/l) or high doses (30 µmol/l) of DES for 8 h,
subsequently washed twice in control medium and then transferred to
control fresh media for the remaining 10 h of culture to test the
reversibility of DES under these conditions. In all
experiments, groups of control COCs were associated; therefore, the
meiotic status of oocytes was evaluated immediately before or after
the interval of DES exposure.
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Fixation and staining of oocytes
After removal of cumulus cells by gentle pipetting, control and
treated oocytes were fixed and extracted, for 30 min at ),
washed three times in a blocking and aldehyde-reducing solution of
phosphate-buffered saline (PBS) containing 2% BSA, 2% powdered milk,
2% normal goat serum, 0.1 mol/l glycine and 0.01% Triton X-100 and
then stored at
Multiple fluorescence labelling has been performed to evaluate the
organization of meiotic spindle microtubules, microtubule organizing
centres (MTOCs), F-actin and chromosomes. For visualization of
microtubules, optimal results were obtained using a 1:50 dilution of
a rat monoclonal antibody, YOL1:34 (Kilmartin et al., 1982),
specific for -tubulin. After treatment with primary antibody,
samples were incubated with fluorescein-conjugated anti-rat secondary
antibodies (Jackson ImmunoResearch Laboratories, West Group, PA,
USA). Primary and secondary antibodies were diluted in blocking
buffer (see above) and applied for 90 min at )
which was filled with a 1:1 glycerol/PBS medium containing 25 mg/ml
sodium azide as anti-fading reagent.
Confocal microscopy
Labelled oocytes were examined and images were recorded using a
Zeiss LSM-510 confocal laser scanning microscope (Germany) equipped
with 488 nm Argon ion, 543 nm green He–Ne, 633 nm red He–Ne lasers
and a x63 Zeiss Plan-Apo objective.
Single and z axis optical sections were collected by LSM-510 Software (Germany) running on a
Siemens-Nixdorf PC. The final three dimensional (3D) reconstructions
and distance measurements were performed using Zeiss 3D LSM Software
(Germany).
Results
Effects of DES on meiotic cell cycle progression
The effects of DES on meiotic cell cycle progression were evaluated by
treating COCs for different time intervals during in-vitro meiotic
maturation process. COCs were exposed to various doses of DES for
either 0–8 or 8–18 h of culture (Table I), during which in-vitro
oocyte maturation in mice is normally completed. In all experiments,
oocytes were fixed and meiotic stage was determined by analysing the
chromosome distribution pattern and F-actin decoration. Results were
summarized in Table II in which the percentages
in each meiotic stage are indicated.
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In general, DES caused a dose-dependent inhibition of
cell cycle progression compared to control cells (Table II). Of all isolated GV-stage
oocytes, 17% did not resume meiosis in control groups. In none of
any DES group was the number of oocytes that remained at GV-stage
significantly different
from in controls. Therefore, all GV oocytes remained both in control
and DES-treated cultures were considered as incompetent to resume
meiosis and excluded from the study.
In the 5 µmol/l DES group during the first half of meiotic division
(meiosis I), many cells (48%) remained in prometaphase, whereas the
rest somehow reached to MI (38%) or even to anaphase I (14%).
Compared with controls, this group showed a slight delay in
progression and was blocked mostly around prometaphase I. In the 15
µmol/l DES group, only 39% of cells displayed a proper chromosome
alignment consistent with either prometaphase I (18%) or MI (21%).
The rest of the cells (61%) had irregular and variable chromosome patterns
implying a moderate to severe impairment of chromosome alignment in
this group. To test the extreme effects of DES in this period, 30
µmol/l of DES was used. In this group, almost all cells (97%)
displayed abnormally condensed chromosomal patterns that are not
compatible with any of the meiotic stages. Typically in these
oocytes a compact mass of chromosomal material was located close to
the centre of the cell and a failure to form normal chromosomal
bivalents was noted (see Figures 1 and 2, for details). Only a
small portion of cells (3%) showed a prometaphase I pattern.
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In the second half of meiotic division (meiosis II), where
the transition from MI to MII lasts for ~8–10 h in mice, COCs were
exposed to DES in the same doses used in meiosis I. In low doses of
DES (5 µmol/l), cells reached different stages of maturation ranging
from prometaphase I to MII (27%) with minor alterations in
chromosome distribution. Compared with controls, DES-treated
cells were predominantly found in anaphase I and telophase I during
which normal cells could be barely detected. This indicates that the
machinery at certain cell cycle check-points is impaired by DES and,
therefore, the transition from MI to MII takes longer than in
controls. It also implies that DES, directly or indirectly,
interacts with cytokinesis, since anaphase and particularly
telophase are crucial steps for the accomplishment of asymmetric
cell division that results in polar body extrusion. Intermediate
doses of DES (15 µmol/l) impaired meiotic progression more significantly, since 31% of cells
remained at MI, just a few in anaphase I (1%) while the rest (68%)
were diagnosed as abnormal. Those included compaction of chromosome
bivalents and occasionally fragmentation of the chromosome set into
two pieces implying an improper chromosome separation. High doses of
DES (30 µmol/l) caused the most dramatic arrest of meiosis. Few
oocytes (11%), in this group, showed normal prometaphase or
metaphase figures while the majority of oocytes (89%) arrested
during or after prometaphase I. Chromosomes were either located in
the centre of ooplasm as a condensed spot or dispersed throughout
the ooplasm. The latter finding indicates that the integrity of the
first meiotic spindle was totally lost due to DES exposure, since
the `pieces of chromosomes' dispersed into cytoplasm after they had
been successfully organized in a bipolar metaphase plane prior to
DES exposure.
Effects of DES exposure on spindle formation during MI and MII
After preliminary results showing abnormal redistribution of oocyte
chromosomes due to DES exposure, we performed multilabel fluorescent
staining to determine whether the effects of DES on meiotic
maturation affected spindle microtubule integrity. For this purpose,
antibodies raised against total -tubulin were used to visualize
the entire meiotic spindle(s) microtubules elongating from poles to
kinetochores, as well as MTOCs.
Control MI spindles displayed the typical barrel shape and were located
at the periphery of the cell (Figure 1A). Chromosomes were
lined up on mid-plate, where they were tightly bound to kinetochore
microtubules. The long axis of spindle which transverses the spindle
poles was always seen perpendicular to plasma membrane. The mean
distance from pole-to-pole was found to be 26.29 µm in control MI
oocytes. Control MII spindles, on the other hand, were seen to be
slightly smaller than MI spindles regarding the microtubule mass
(Figure 1B) and interpolar distance
(22.50 µm), yet they maintained a barrel shape appearance. They
were often located close to the polar body and slightly rotated compared
with MI spindles, so that the long axis became parallel to plasma
membrane. These spindle dynamics are normal events that are required
for proper meiotic progression (Albertini, 1992).
In 5 µmol/l DES-treated cells, the prominent feature of the MI
spindle was the dilation of spindle mass particularly at the pole
regions indicating that spindle poles responsible for the integrity
of spindle halves were becoming loosened. Another supporting sign of
this result was the displacement of outer spindle microtubules,
resulting in a loss in the integrity of spindle laterally (Figure ). Consistent with this,
few chromosomes had migrated from the mid-plate either towards the
poles or outside the spindle region. Apart from this slightly altered
MI appearance, 48% of cells displayed normal prometaphase I and 14%
anaphase I figures. When oocytes were treated with 5 µmol/l DES
during meiosis II, more dramatic alterations appeared regarding the
spindle assembly and chromosome distribution. An unusual number of
cells was detected in transitional stages from MI to MII. Loosening
and elongation of spindles (mean interpolar distance = 37.49 µm)
were frequently noted in all spindles (Figure 1D). Tubulin mass was significantly low, evidenced by the
loss of many microtubules due to DES exposure. Chromosomes were
localized arbitrarily between poles and the mid-plate, a critical
point, which may prospectively give rise to the non-disjunction of
chromosomes in later stages.
Intermediate doses of DES (15 µmol/l) were administered next to
see more severe effects of DES on spindle formation. The profound
outcome was the formation of several abnormal oocytes (61%) bearing
significantly deteriorated
spindles. It was difficult to consider each modified spindle in one
group, therefore a series of common malformations is documented in
Figure 1E, Figures 2A, B and C from an abnormal
prometaphase I (Figure 2A), to MI figures
(Figure 1E). The formation of
abnormal spindles largely depended on the stage during which a given
cell had been maximally exposed to DES. In cells exhibiting MI
figures, spindles were found similar to the ones detected in the 5
µmol/l DES group (Figure 1E). When cells were treated
after the MI spindle had been assembled, they were totally unable to
form the second meiotic spindle and, therefore, failed to extrude
polar body. In these examples, extremely scattered spindle
microtubule forms were noted (Figures ) some of which were
associated with clumps of chromosomes (Figure ) rather than individual
chromosome bivalents. Measurement of interpolar distance was not
applicable to those spindles. Occasionally, a few telophase I figures
were recognized by the existence of a cellular outgrowth (Figure 2B) that gives rise to polar
body. However, the location of the telophase spindle was not correct
which may result in an aneuploid gamete formation.
For testing the extreme doses of DES on cultured mouse oocytes, 30
µmol/l DES was administered before and after the formation of the
first meiotic spindle. As expected, a more drastic rate of abnormal
oocytes was detected in this group. Almost no proper alignment of
chromosomes or spindle microtubules was observed during meiosis I.
The most prominent finding was the apparent loss of spindle tubulin
that resulted in a total or partial loss of spindles (Figure 1G and
Figure 2D). The remaining tubulin
mass, if any, was not able to orient the chromosomes properly, therefore
most cells contained small masses of compacted tubulin without a
filamentous appearance and intermingled with a clump of chromosomes.
Occasionally, distinct chromosome bivalents were noticed after z
axis 3D image reconstruction renderings (Figure 1G). However, even in these
examples, spindle pieces did not have a filamentous appearance,
implying that the primary defect was mainly confined to the failure
of microtubule assembly consequently giving rise to chromosome
misalignment. When cells were treated with 30 µmol/l DES during
meiosis II, the existing spindles were consistently fragmented into
several small pieces randomly distributed throughout the ooplasm
(Figure 1H). Occasionally, severe
reduction in the first meiotic spindle microtubules was noted
(Figure ). The spindle fragments or
leftover miniature spindles after DES were most likely dysfunctional,
since cells could not extrude any polar body at all.
Recovery of oocytes after DES exposure
To determine whether the effects of DES exposure on cell cycle progression
are reversible, oocytes were treated with 5 µmol/l and 30 µmol/l DES
for 8 h then washed and transferred to control culture medium for an
additional 10 h (see Table I, for experiment design). In
18 h of total culture, most untreated cells progressed to MII (92%).
Quite interestingly, a small portion of oocytes (15%) exposed to 5
µmol/l DES were able to progress up to MII (Figure 3A) while a larger portion
of cells (25%) accumulated in anaphase I or telophase I (Figure 3B). The remaining 60% of
cells were at prometaphase I and MI. In all stages, cells possessed
intact spindle microtubules associated with proper chromosome
alignment. Cells were only able to reach MI (91%), yet had a normal
spindle and well-organized MTOCs (Figure ), when recovered from 30
µmol/l DES during meiosis I. No obvious signs of cell death were
noted in DES-treated cultures. Taken together, these data show that actions of DES are reversible in a
dose-dependent fashion. This is also evidenced by the above
observations in which oocytes managed to recover from the cell cycle
blocking effect of DES, during the post-dosing period (10 h).
However, it seems that recovering cells needed more time to complete
meiosis.
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Effects of DES on GV-stage oocytes and cumulus cells
During observations of DES-exposed oocytes, few GV stage oocytes and
oocyte-enclosing cumulus cells were encountered and served as
positive controls for both DES treatments and staining procedures. Interesting,
none of the GV stage oocyte or cumulus cell displayed signs of
microtubule disassembly or chromatin/chromosome abnormality (Figure
4A). In GV stage oocytes, a
well-developed cytoplasmic microtubule network was recognized as
delicate bundles of microtubules overlapping each other (Figure 4B). This microtubular
lattice was found throughout the ooplasm except where the germinal
vesicle is located (Figure ). As expected for GV stage
oocytes, each non-dividing cumulus cell exhibited well- preserved
cytoplasmic microtubules throughout the cytoplasm (Figures 4A and D).
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Discussion
The present studies were designed to evaluate the effects of the
synthetic oestrogenic compound, DES, on the process of meiotic maturation
in cultured mouse oocytes, as a possible model for determining the
mode of action of oestrogenic agents on meiosis in mammalian germ
cells. Although examining the in-vitro actions of oestrogenic agents
on oocytes may not accurately reflect their in-vivo activity, there
is mounting evidence to suggest that DES and related compounds
impair reproductive function in mammals (Halling and Forsberg, 1990)
including human (Pal et al., 1997)
by a potential action that interferes with meiosis at key stages of
germ cell nuclear maturation. In view of the established
anti-microtubule effects of DES in purified microtubules (Sharp and
Parry, 1985;
Sato et al., 1987)
and in cultured cells (Sakakibara et al., 1991),
it was anticipated that this agent would disrupt the process of
meiotic maturation in mammalian oocytes and would eventually give
rise to aneuploidy. Given the multi-step nature of meiosis and the
stage-specific involvement of the cytoskeleton in general and
microtubules in particular (Albertini, 1992),
the present studies were undertaken, for the first time in the
literature, to evaluate the actions of DES on oocyte maturation.
This study shows that DES interferes with the mechanism whose primary
function is to build the asymmetric cell division machinery that is
unique to mammalian oocytes. Two major consequences of DES exposure
have been revealed. First, DES exposure is shown to
interfere with meiotic cell cycle progression by delaying advancement
to MI and by inhibiting the MI spindle formation during meiosis I.
These effects are stage and cell specific since both the initial
phase of meiotic maturation, involving GV, germinal vesicle
breakdown (GVBD), chromatin condensation and cumulus cells are
unaffected even at relatively high concentrations of DES. Secondly,
severe disturbances in chromosome alignment are observed that are
most likely related, in an indirect way, to the effects of DES on
the maintenance of spindle structure, as has been observed by others
(Tsutsui and Barret, 1997).
Collectively, those results led us to conclude that the microtubule-depolymerizing
effect of DES is somehow restricted to meiotic microtubules, at
least in tested doses. The resistance of interphase
microtubules found either in GV-stage oocytes or cumulus cells
suggests that meiosis is more susceptible to the adverse effects of
DES and, therefore, might ultimately result in a series of genetic
defects in oocytes, e.g. aneuploidy or fertilization failure without
grossly altering the surrounding cells and tissues.
The COC is an excellent 2-cell system for investigating the physical
and functional relationship between the oocyte and the surrounding
cumulus cells. The reason that COCs were used instead
of denuded oocytes in present experiments is to simulate the in-vivo
conditions to some extent, a closed 2-cell system where an exogenous
agent should trespass the first checkpoint (i.e. cumulus cells) in
order to reach the enclosed oocyte mainly by the route of transzonal
processes and intercellular junctions. The latter are known
to provide a dynamic intercommunication (Buccione et al., 1990)
presumably facilitating two-way trafficking of metabolic products
and reagents. The recovery of DES-exposed cumulus-enclosed oocytes,
as presented in this study, support this assumption. However,
DES-exposed denuded oocytes could not be reversed from abnormal
meiotic stages, then one could postulate that the cumulus cells play
a regulatory role in the uptake and discharge of environmental
agents.
Effective concentrations (EC) of DES required for induction of
microtubule disruption in 50% of cells in two breast cancer cell lines have been
reported as 48 and 50 µmol/l (Aizu Yokota et al., 1994),
whereas the lethal dose at which 50% of cells are no longer viable
(LD50), was reported as 19–25 µmol/l in prostate cancer cells (Robertson et al.,
1996).
An increase in the number of apoptotic nuclei was detected when the
same cells were treated with 15 or 30 µmol/l DES. On the other hand,
the in-vivo dose for DES was reported to be 1–5 µg daily when
injected in neonatal or 10 day old rats and mice (Halling and
Forsberg, 1990;
Iguchi et al.,
1990).
Since the form and duration of exposure, and even the animals' age,
are potential variables for the acute toxicity of DES, it is
difficult to compare in-vivo and in-vitro doses. Taken together, it
is likely that the optimum effective but not
lethal dose for in-vitro DES treatment depends on the type of cell
and the stage of cell cycle, at least in isolated and cultured
cells.
Higher concentrations of DES led to progressive and differential alterations
in the organization of both MI and MII meiotic spindles. Despite
causing a dramatic decrease in overall microtubule mass in meiosis
I, during which the first meiotic spindle is formed, loss of spindle
pole integrity dominates during meiosis II, where the existing MI
spindle undergoes fragmentation to form several groups of
microtubules and chromosomes. The characteristic miniature spindle
fragments observed following exposure to 30 µmol/l DES points
further to an action of this compound on spindle pole components
that must in part be related to the organization and function of
centrosomal material. One explanation for this structural effect
could be direct interference with -tubulin, the tubulin isoform known to be
exclusively situated within MTOCs or centrosomes (Joshi, 1994).
These results collectively support concerns on the role of DES as a
causative agent of aneuploidy in mammalian oocytes.
In view of its purported microtubule depolymerizing activity, it
was somewhat surprising that microtubule depolymerization was not
observed in non-dividing somatic cumulus cells and more interestingly
in GV-stage oocytes. It is noteworthy that differences in the
selective impairment of spindle microtubules in response to DES
could be due either to cell cycle specific alterations in
microtubule dynamics (Kuriyama and Borisy, 1981;
Robertson et al., 1996)
or the concentration of the drug used. Mitotic spindle microtubules
vary in their susceptibility to different anti-microtubule drugs.
For instance, while vinblastin, colchicine or nocodazole
depolymerize all microtubule subtypes (Jordan et al., 1992),
MBC, a commonly used fungicide, exhibit selective disruption of astral
and interpolar spindle microtubules in mitotic cells without showing
deleterious effects on interphase or kinetochore microtubules (Can and Albertini, 1997b).
One possible explanation for the selective impairment of
microtubules is a difference in mode of action between several
compounds because of differences in cellular uptake or in their
conversion to active metabolites, which may have altered binding
activity to tubulins. Participation of two distinct forms of tubulin
dimers ( and ß) in microtubule assembly could explain
the differential impairment of certain microtubule populations due
to various agents. For instance, a post-transitional change of -tubulin, e.g. acetylation, has been shown to
cast stable microtubules that are significantly resistant to depolymerising agents
(Salmon et al., 1984).
Furthermore, it was demonstrated that differences in acetylation
rate of different microtubule populations in a meiotic spindle alter
the turn-over rate of microtubule assembly (Webster and Borisy, 1989)
which in turn makes meiotic microtubules more susceptible to agents.
Diverse microtubule responses to several oestrogenic
agents, e.g. DES, 17ß-oestradiol, E-dienestrol, bisphenol-A or to
their metabolitic components, such as diethylbestrol oxide and
indenestrol-A, examined in vivo and in vitro give
support to the dynamic instability model of microtubule polymerization,
a phenomenon in which minute changes in time or concentration derive
critical outcomes.
Since a hormone receptor–second messenger system is known to be
the most common pathway of steroid hormone efficacy, it is possible
that cell cycle delay in oocytes due to DES exposure is a
receptor-mediated action. However, studies on the microtubule disrupting
effect of DES via oestrogen receptors failed to demonstrate that
oestrogen receptor-positive cells were more prone to DES-induced toxicity
(Aizu Yokota et al., 1994).
Supporting evidence comes from a microbial study testing the effect
of indenestrol-A and B, well-known DES metabolic products, on the
oestrogen receptors (Metzler and Pfeiffer, 1995).
This study showed that the cytotoxic activity of those end-products
was a result of a direct action on microtubule polymerization rather
than a receptormediated activity, although indenestrols are known to
have strong binding affinities for oestrogen receptors. Moreover,
this study also demonstrated that, in cell-free systems, the ability
of oestrogenic substances to interact with microtubules was not
correlated with hormonal activity. The in-vitro maturation model
used in this study lends credence to the finding that no hormonal
milieu was used in our culture media to mimic the maturation
process, suggesting that DES toxicity is independent from the
hormonal environment of oocytes. However, future research should be
of interest on tracking the DES-binding receptors and target
molecules including other cytoskeletal proteins.
The observation that meiotic cell cycle progression was delayed at
low DES doses and that most cells arrest at anaphase and telophase in
meiosis I, suggests that DES acts at this critical juncture of
meiosis. In both mitotic and meiotic systems, anaphase is known to
depend on the ubiquitin-mediated proteolysis of cyclins, the
regulatory subunit of maturation-promoting factor (MPF) (Murray and
Hunt, 1993).
DES could prevent the resumption of anaphase or telophase by
inhibitory cyclin degradation. This is a plausible explanation for
the action of DES since protease inhibitors have been shown to
arrest somatic cells at metaphase by inhibiting cyclin degradation
(Sherwood et al., 1993).
It has been shown that compounds arrest mitosis by activation of
a mechanism that detects errors in spindle assembly (Murray and
Hunt, 1993).
Conditions that influence microtubule stability, spindle pole
function, or microtubule motors have been shown to cause mitotic
arrest in yeast due to a negative feedback control on M-phase
progression. The present studies have shown that DES influences both
microtubule stability and spindle pole organization in mouse oocytes
making it likely that meiotic cell cycle impairment in this system
is also subject to negative feedback regulation. Clearly, further
studies on cyclin proteolysis and spindle organization will be
needed to ascertain the mechanisms whereby DES exerts its effects on
mouse oocytes.
Acknowledgments
We thank Professor David F.Albertini for providing the YOL 1:34 antibody. This study was financially supported by NATO-CRG 951282 to AC for his travel expenses, by TUB0TAK-SBAG AYD 164 and 240, Ankara University Research Fund 98090009 for consumables and by DPT 97K120560 and DPT 98K120730 for the assembly and advancement of confocal microscopy unit.
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