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Introduction
Apoptosis, which is widely observed in different cells of
various organisms, is a unique morphological pattern of cell
death characterized by chromatin condensation, membrane
blebbing, and DNA fragmentation[1]. Apoptosis plays an
important role in the homeostasis of multicellular organisms.
Moreover, abnormal apoptotic function has been associated
with multiple human diseases, including neurodegenerative
disorders and cancers[2]. During normal embryogenesis,
apoptosis functions to remove abnormal or redundant cells
from preimplantation embryos[3,4]; during normal mouse
embryonic development, this occurs only after the blastocyst
stage[5]. Premature apoptosis, such as that triggered by
exposure to teratogens, causes improper embryonic
development[6,7]. We recently reported that several natural
phytochemical compounds, including curcumin and ginkgolides,
were able to prevent or induce apoptosis in embryonic stem
cells and/or embryos[8_10]. The effects of genistein on
human embryo development are unclear. In the present study,
we investigated whether another natural compound, genistein, had cytotoxic effects on embryonic development.
Our study will provide important new insights about the
effect of dietary genistein on embryogenesis. The effect of
genistein on embryo development in vitro must be studied
because it is especially in relation to the dietary use of
iso-flavones (such as genistein) in pregnant women.
Genistein, a natural isoflavone compound found in soy
products, has been shown to inhibit protein tyrosine kinase
and topoisomerase-II activity[11,12], leading to numerous
effects on diverse cell functions. For example, genistein has
been shown to inhibit tumor cell proliferation and induce
tumor cell differentiation[13_16], and may also trigger cell cycle
arrest and apoptosis in some cell
types[17_19], while blocking apoptosis under other
circumstances[20_22]. Recent studies have shown that genistein can cause cell cycle arrest and
developmental injury in embryos via its function as a
tyro-sine kinase inhibitor[23_25]. It is thought that genistein
inhibits epidermal growth factor (EGF) receptor tyrosine kinase
activity, inhibiting the positive effect of EGF on glucose
uptake. Since glucose is the main exogenous energy source
for the early development of the preimplantation embryo,
the genistein-induced inhibition of glucose uptake would be
expected to dramatically affect early embryonic develop-
ment[23]. Genistein has also been suggested to inhibit
tyrosine phosphorylation of the cadherin-catenin complex,
which mediates compaction, adhesive functions, and
embryonic cleavage from the 2 to 4 cell stages in the mouse
embryo[26]. In addition, mitochondria were observed to form
numerous tiny clusters uniformly distributed in the
cytoplasm in genistein-treated embryos, causing G2-stage arrest
in the 2 cell stage of the embryo, suggesting that genistein
may inhibit embryonic development by altering
mitochondrial structure and
function[24]. However, although multiple
studies have addressed the inhibitory effects of genistein
on embryonic development due to the inhibition of tyrosine
kinases or mitochondria-based mechanisms, no previous
work has examined the apoptotic effects of genistein on the
early stages of mammalian embryonic development,
especially during the blastocyst stage.
In the present study, we exposed mouse blastocysts to
genistein and examined apoptosis in the inner cell mass (ICM)
and trophectoderm (TE), and also observed early
postim-plantation embryonic development on culture dishes
in vitro. Finally, the in vivo effects of dietary genistein on cell
apopto-sis and proliferation were investigated in an animal model of
embryogenesis.
Materials and methods
Chemicals and reagents Pregnant mares' serum
gonadotropin (PMSG), sodium pyruvate, genistein, and daidzein
were purchased from Sigma (St Louis, MO, USA). Human
chorionic gonadotropin (hCG) was obtained from Serono (NV
Organon Oss, the Netherlands). The Annexin-V-FLUOS
staining kit and the terminal deoxynucleotidyl transferase-
mediated dUTP nick-end labeling (TUNEL) in
situ cell death
detection kit were obtained from Roche Molecular
Biochemi-cals (Mannheim, Germany) and the CMRL-1066 medium was
from Gibco Life Technologies (Grand Island, NY, USA).
Animals and collection of embryos ICR (a strain of mice)
virgin albino mice (6_8 weeks old) were induced to
superovulate by injection of 5 IU PMSG followed 48 h later by an
injection of 5 IU hCG. The mice were then mated overnight
with a single fertile male of the same strain. Female mice with
vaginal plugs were separated and used for the experiments.
All mice were maintained on breeder chow and kept under a
12 h day/12 h night regimen, with food and water available
ad libitum. All animals received humane animal care, as
outlined in the Guidelines for Care and Use of Experimental
Animals (Canadian Council on Animal Care, Ottawa, Canada,
1984). The day a vaginal plug was found was defined as d 0
of pregnancy. Morulas were obtained by flushing the
fallopian tubes on the afternoon of d 3, and the blastocysts were
obtained by flushing the uterine horn on d 4; in both cases
the flushing solution consisted of CMRL-1066 culture
medium containing 1 mmol/L glutamine and 1 mmol/L sodium
pyruvate.
Genistein treatment The embryos were collected in
uncoated plastic 35 mm culture dishes and washed a minimum
of 3 times. The expanded blastocysts from different females
were pooled and randomly selected for the experiments. The
blastocysts were incubated at 37 oC for 24 h in CMRL-1066
medium with and without the indicated concentrations of
genistein or daidzein, and were then used for further
experiments as described below.
Blastocyst cell counting The blastocysts were
incubated with culture medium containing 0_50 µmol/L genistein.
Twenty-four hours later, the blastocysts were washed with
genistein-free medium, and dual differential
staining[27] was used to facilitate counting of cell numbers in the ICM and
TE. The blastocysts were incubated in 0.4% pronase in
M2 medium containing 0.1% bovine serum albumin
(M2-BSA medium) for the removal of the zona pellucida. The denuded
blastocysts were exposed to 1 mmol/L
trinitrobenzenesul-phonic acid in BSA-free
M2 medium containing 0.1% polyvinylpyrrolidone (PVP) at 4
oC for 30 min, and then washed with
M2 medium[28]. The blastocysts were further treated
with 30 µg/mL anti-dinitrophenol-BSA complex antibody in
M2-BSA at 37 oC for 30 min, and then with
M2 medium supplemented with 10% whole guinea pig serum as a source of
complement, 20 μg/mL bisbenzimide, and 10 µg/mL propidium
iodide (PI) at 37 oC for 30 min. The immunolysed blastocysts
were gently transferred to slides and protected from light
before observation. Under UV light excitation, the ICM cells
(which take up bisbenzimide, but exclude PI) appeared blue,
whereas the TE cells (which take up both fluorochromes)
appeared orange-red. As multinucleated cells are not
common in preimplantation embryos[29], the number of nuclei was
considered to represent an accurate measure of the cell
number.
Detection of cell apoptosis For TUNEL staining, the
embryos were washed in genistein-free medium, fixed,
permeabilized, and subjected to TUNEL labeling using an
in situ cell death detection kit (Roche, Germany) according to
the manufacturer's protocol. Photographic images were
taken using a fluorescence microscope under bright light
(Olympus BX 51, Tokyo, Japan). For Annexin-V staining,
the blastocysts were incubated in 0_50 µmol/L genistein for
24 h, washed with genistein-free culture medium, and then
stained with an Annexin-V-FLUOS staining kit (Roche,
Germany), according to the manufacturer's instructions.
Briefly, the blastocysts were incubated in
M2-BSA for the removal of the zona pellucida, washed well with phosphate
buffer saline (PBS) plus 0.3% BSA, and then incubated for
60 min with a mixture of 100 µL binding buffer, 1 µL
fluorescein isothiocyanate (FITC)-conjugated Annexin-V and 1 µL
PI. After incubation, the embryos were washed and
photographed under a fluorescence microscope. Cells staining
Annexin-V+/PI_ were considered apoptotic, while those
staining Annexin-V+/PI+ were considered necrotic.
Observation of in vitro implantation and postimplantation
development The blastocysts were cultured according to a
modification of the previously reported
method[30]. Briefly, the embryos were cultured in 4 well multidishes at 37 °C. For
group culture, 3 embryos were cultured per well. The basic
medium consisted of CMRL-1066 supplemented with 1
mmol/L glutamine and 1 mmol/L sodium pyruvate, plus 50 IU/mL
penicillin and 50 mg/mL streptomycin (hereinafter called
culture medium). For the treatments, the embryos were cultured
with the indicated doses of genistein for 24 h without serum
supplementation. Thereafter, the embryos were cultured for
3 d in culture medium supplemented with 20% fetal calf serum,
and for 4 d in culture medium supplemented with 20%
heated-inactivated human placental cord serum, for a total culture
time of 8 d from the onset of treatment. The embryos were
inspected daily under a phase contrast microscope (Olympus
IMT-2, Tokyo, Japan), and the developmental stages were
classified according to established
methods[31]. Developmental parameters, such as hatching through the zona
pellucida, attachment to the culture dish, trophoblastic
outgrowth, and differentiation of the embryo proper were
recorded daily. To decrease observer bias, all data were
analyzed using the following previously published
criteria[32]: an implanted blastocyst was defined by attachment to the
culture dish, followed by outgrowth; an early egg cylinder
(EEC) was defined as an embryo that had reached stage 9 or
10 by d 4; a late egg cylinder (LEC) was defined as an embryo
that had reached stage 11, 12, or 13 by d 6 of culture; and an
early somite (ES) was defined as an embryo that had reached
stage 14 or 15 by d 8[32].
Effect of genistein on mouse embryo development
in vivo Female mice were fed a standard diet supplemented with or
without genistein (10 µmol/L) in the drinking water for a total
of 5 d. Twenty-four hours after the initiation of genistein
exposure, the mice were mated overnight with a single fertile
male of the same strain. The blastocysts were obtained by
flushing the uterine horn on d 4 after mating, and cell
apopto-sis and proliferation were analyzed as described above.
Statistics The data were analyzed using one-way ANOVA
and t-tests, and are presented as the mean±SD.
P<0.05 was considered significant.
Results
Effects of genistein on cell apoptosis in mouse
blastocysts To study the apoptotic effects of genistein on embryos,
we treated mouse blastocysts (150 blastocysts in each group)
with 0_50 µmol/L genistein at 37 oC for 24 h, and then
measured cell apoptosis. TUNEL staining revealed that
treatment with 25 and 50 µmol/L genistein dose-dependently
increased apoptosis in the mouse blastocysts (Figure 1A),
and quantitative analyses revealed 4 to 6.7 fold more
apo-ptotic cells in the genistein-treated blastocysts versus the
untreated controls (Figure 1B). For the comparison, we used
similar methods to analyze the cytotoxic effects of daidzein,
another type of soybean isoflavone, on mouse blastocysts.
Interestingly, TUNEL staining revealed that daidzein did not
induce cell apoptosis in mouse blastocysts under our
experimental conditions (Figure 1A, 1B). Additional TUNEL
staining experiments revealed that the effect of genistein on cell
apoptosis was more pronounced in the ICM than in the TE
(Figure 1C), and Annexin-V staining revealed a higher rate of
Annexin-V-positive cells in the ICM of treated blastocysts
versus the controls, but similar rates of positivity in the TE
(Figure 1D). These results indicate that genistein is a potent
inducer of apoptosis in mouse blastocysts, primarily in the
ICM, whereas daidzein does not appear to induce apoptosis
in mouse blastocysts.
Effects of genistein on the blastocyst cell number
To investigate the effect of genistein on cell proliferation in
embryos, we treated mouse blastocysts (120 blastocysts
in each group) with or without genistein (25_50 µmol/L)
for 24 h, and then analyzed proliferation using differential
staining followed by cell counting. Our results revealed that
the blastocysts treated with 25 or 50 µmol/L of
genistein contained fewer cells than the control blastocysts; this effect was more pronounced in the ICM than in the TE
(Figure 2).
Effects of genistein on implantation and postimplantation
development The blastocysts treated with 25 or 50 µmol/L of
genistein (148_173 blastocysts, as indicated) and 148
control blastocysts were assessed for in
vitro implantation and development. Implantation was similar in both the treatment
and control groups (Figure 3A). However, the
genistein-treated blastocysts showed reduced formation of the 2 layer
ICM and ectoplacental cones, and fewer instances of
embryos developing to the advanced egg cylinder stages
(LEC and ES stages) when compared with the control group
(Figure 3A). Morulas exposed to 25 or 50 µmol/L genistein
showed 55%_65% development into blastocysts, whereas
86% of control morulas developed into blastocysts under
our experimental conditions (Figure 3B).
Effect of dietary genistein on mouse blastocyst
development in vivo The ability of genistein to trigger apoptosis
and developmental injury in vitro led us to suspect that it
might have the same activity in vivo. To evaluate this
possibility in an animal model, we fed female mice a standard diet
supplemented with or without genistein (10 µmol/L) in the
drinking water, 24 h prior to mating and during the first 4 d of
embryonic development. An analysis of flushed blastocysts
(280 per group) revealed that dietary genistein significantly
induced apoptosis (Figure 4A) and decreased cell
proliferation (Figure 4B) in mouse blastocysts. Collectively, these
studies suggest that genistein can induce apoptosis and
inhibit the proliferation of embryonic cells, and appears to
cause embryonic developmental injury in
vitro and in vivo, indicating that genistein may have a hazardous effect on
embryonic development.
Discussion
Embryonic development is a complex process during
which chemical injury can lead to abortion or embryonic
malformation. Thus, it is important to examine the possible
teratogenic effects of commonly used plant extracts. We
herein show that genistein can decrease the viability of
mammalian blastocysts via the increased induction of apoptosis
(Figure 1). Our results revealed that treatment of mouse
blastocysts with 25 and 50 µmol/L genistein
dose-depen-dently induced 4 to 6.7 fold increases in apoptosis, as
assessed by TUNEL staining (Figure 1A, 1B). In contrast,
another soy isoflavone, daidzein, had no such effect (Figure
1A, 1B). Annexin-V staining revealed that genistein-induced
cell apoptosis occurred more commonly in the ICM than in
the TE (Figure 1C, 1D). Although the treated blastocysts
could implant on culture dishes in vitro, postimplantation
development was retarded, leading to embryonic death.
Previous reports have shown that genistein can either
induce or prevent apoptosis, depending on the cell type or
experimental conditions[33,34]. For example, genistein induced
apoptosis in immature human
thymocytes[19], leukemia cell
lines[18], human stomach cancer
cells[14], and bladder carcinoma cell
lines[35], but inhibited ionization radiation-induced
apoptosis of human B lymphocyte
precursors[36], taxol-induced apoptosis of a human ovarian tumor cell
line[37], anti-CD3-induced apoptosis of mouse
thymocytes[20], didemnin B-induced apoptosis of human HL-60
cells[22], and UV irradiation-induced apoptosis of a human epidermal carcinoma
cell line[38]. In addition, other studies have demonstrated
that genistein can inhibit proliferation and induce
differentiation in tumor cells[39_41]. In the present work, we used
TUNEL and Annexin-V staining to show that genistein
promotes apoptosis in mouse blastocysts (Figure 1).
During embryonic development, cells are often poised
between proliferation and apoptosis. Studies by several other
groups and our laboratory have demonstrated that genistein
can modulate cell apoptosis, but no previous work has
examined the apoptotic effects of genistein on embryonic cell
death. We herein report that genistein treatment of mouse
blastocysts decreased cell numbers, increased apoptosis,
and triggered developmental delays in postimplantation
mouse embryos in vitro, and further report that dietary
genistein appeared to have a teratogenic effect
in vivo. Taken together, our novel observations in conjunction with the
previous reports suggest that the actions of genistein
depend on the cell type and treatment duration/dosage. Clearly,
additional studies will be required to assess the molecular
mechanism of action by which genistein affects cell
apoptosis.
Although the effect was more pronounced in the ICM,
some genistein-induced cell loss and apoptosis was observed
in the TE as well (Figures 1C, 2). The TE arises from the
trophoblast at the blastocyst stage, and subsequently develops into a sphere of epithelial cells surrounding the
ICM and the blastocoel; these cells contribute to the
placenta and are required for the development of the
mammalian conceptus[42]. Although we observed that genistein
treatment slightly reduced the cell number and increased apoptosis
in the TE of mouse blastocysts in this study (Figures 1C, 1D,
2), embryonic implantation did not appear to be affected
in vitro (Figure 3A). Future work will be required to assess the
effects of genistein on differentiation and giant-cell
formation in vitro and in vivo.
Genistein has been shown to act as an agonist for
estrogen receptor (ER)α and ERβ[43]. The ability of genistein to
protect against mammary carcinomas has been attributed to
its anti-estrogenic effects[44], while other studies
demonstrated that the estrogenic and/or anti-estrogenic activities
of genistein may reduce or enhance estrogen-dependent
tumor growth, depending on the dose and timing of
exposure[45,46]. To test whether genistein treatment could affect
ER expression, we used real time PCR to investigate the
effect of genistein treatment on the expression levels of
ERα and ERβ in mouse blastocysts. Our results revealed no
significant differences in ERα and ERβ expression levels
between the genistein-treated and the control blastocysts
(data not shown), suggesting that genistein-induced cell
apoptosis does not act via the alteration of ERα and
ERβ expression levels in the studied mouse blastocysts.
Researchers recently investigated the effects of
phyto-estrogens such as genistein on the production of the protein
hormone hCG in trophoblasts. Their results revealed that
hCG production was decreased by genistein treatment in the
trophoblasts of term placentas[47]. Another study showed
that genistein affected the production of hCG and the
steroid hormone progesterone in the BeWo and Jeg3
trophoblast cell lines[48]. Importantly, the report noted that
different treatment dosages of genistein could cause different
effects on the production of progestrogen and hCG. The
result showed that a correlation between the effects on the
proliferation and the production of progesterone and hCG at
high concentrations of genistein (>1 mmol/mL). With low
concentrations of genistein treatment, the study found that
a stimulation of the production of hCG and a weak inhibition
of proliferation in both cell lines BeWo and
Jeg3[48]. These results seem to imply that exposure to genistein during
sensitive periods of development may alter reproductive
functions and influence fertility. These reports, in conjunction
with the present findings, suggest that genistein acts as a
teratogen, negatively impacting embryonic development via
the inhibition of trophoblast cell proliferation and hCG
production, the inactivation of tyrosine kinases, and the
induction of cell apoptosis.
In summary, we show for the first time that genistein can
decrease the viability of mammalian blastocysts by inducing
apoptosis in the ICM. Although additional work will be
required to fully elucidate the action mechanisms of genistein,
especially in relation to the dietary use of isoflavones (such
as genistein) in pregnant women, this study provides the
first evidence that genistein could have teratogenic effects
through the induction of apoptosis.
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