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Introduction
Quantum dots (QD) are colloidal nanocrystalline
semiconductors that have unique light-emitting properties and
can be used as a novel luminescent material. Typical QD are
1_12 nm in diameter and contain a small number of atoms in
a discrete cluster[1]. QD absorb irradiated energy at
wavelengths greater than their lowest energy transition, and then
convert the irradiated energy to a narrow bandwidth
emission. QD are ideal for development as luminescent probes due to
the advantages of broadband excitation, narrow bandwidth
emission, high intensity of emitted light, resistance to
quenching, and photochemical stability. These properties
make QD applicable for biochemical assays, in particular
immunofluorescence staining. A previous study showed that
CdSe-core QD induce cell death[2,3], but the regulatory
mechanisms underlying this effect have not been elucidated.
Apoptosis, which is widely observed in different cells of
various organisms, is the process of programmed cell death
involving several morphological patterns, including
chromatin condensation, membrane blebbing, and DNA
fragmentation[4]. Apoptosis plays an important role in
embryogenesis and homeostasis of multicellular organisms. The
impairment of apoptotic function has been associated with
several human diseases, including neurodegenerative
disorders and cancer[5]. Although apoptosis contributes to
normal embryonic development[6_8], teratogens can induce
abnormal apoptosis during early embryogenesis, leading to
disruption of the developmental
process[9_13]. We recently reported the capacity of several apoptotic inducers to
trigger apoptosis in embryonic stem cells and/or embryos of
ICR Swiss Webster mice[10,11,14_16]. The effects of CdSe-core
QD on human embryonic development are unclear. Our
findings provide important new insights regarding the use of
CdSe-core QD as biological tracers in mouse embryos.
To examine the effect of CdSe-core QD on
pre-implantation development, we exposed mouse blastocysts to
CdSe-core QD and examined apoptosis and cell proliferation in the
inner cell mass (ICM) and trophectoderm (TE). We also
investigated the effect of CdSe-core QD on the development
of morulas into blastocysts. The effect of CdSe-core QD on
post-implantation development was investigated by means
of in vivo analyses of transferred embryos.
Materials and methods
Chemicals and reagents Pregnant mare's serum
gonadotropin (PMSG) and sodium pyruvate 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 TUNEL
in situ cell death detection kit were obtained from Roche
(Mannheim, Germany) and the CMRL-1066 medium was from
Gibco Life Technologies (Grand Island, NY, USA).
QD preparation Nanocrystals comprising a CdSe core
and a ZnS shell were synthesized by Prof Chuan-hsin LU
and coworkers at the Department of Chemical Engineering,
National Taiwan University. Briefly, appropriate amounts of
trioctylphosphine oxide (TOPO), cadmium oxide, and
tetradecylphosphonic acid were heated to 180
oC under zargon, and dried and degassed under a vacuum. The
reaction temperature was then increased to 330
oC; selenium (Se) precursor solution in trioctylphosphine (TOP) was injected
into the reaction flask, and the mixture was allowed to cool to
240 oC. Zn and S stock solutions prepared with
bis(trimethylsilyl)sulfide in TOP, along with a dimethyl zinc
solution, were added drop-wise with vigorous stirring until a
final mole ratio of 1:4 (Cd/Se:Zn/S) was achieved in the
reaction. The reaction mixture was cooled to room
temperature, and the nanocrystals were precipitated with
anhydrous methanol, collected by centrifugation, and
washed 3 times with anhydrous methanol for the removal of
residual TOPO and the unreacted reagents. The precipitate
was dissolved in anhydrous chloroform or tetrahydrofuran
for the experiments. For water solubilization, the CdSe QD
were surface coupled with mercaptoacetic acid and then
suspended in phosphate-buffered saline (PBS) (the
modification was performed by Prof Ruoh-chyu RUANN and
coworkers at the Department of Chemical and Materials
Engineering, National Central University, Taiwan). A
particle sizer was used to measure the CdSe QD, which were
found to be approximately 3.5 nm in diameter.
Animals and collection of embryos ICR mice were
purchased from the National Laboratory Animal Center (Taiwan).
This research was also approved by the Animal Research
Ethics Board of Chung Yuan Christian University (Taiwan).
ICR virgin albino mice (6_8 weeks old) were induced to
superovulate by an injection of 5 IU PMSG followed by an
injection of 5 IU hCG 48 h later. The mice were then mated
overnight with a single fertile male of the same strain. The
female mice with vaginal plugs were separated and used
for the experiments. All of the 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 of the 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 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.
QD treatment The embryos were collected in uncoated
plastic 35 mm culture dishes and washed at least 3 times.
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 0_500 nmol/L QD, and were then used for
further experiments as described later.
Blastocyst cell counting To investigate the effect of
CdSe-core QD on cell proliferation in embryos, we treated
the mouse blastocysts (180 blastocysts in each group) with
or without CdSe-core QD and then analyzed proliferation
using differential staining. The blastocysts were incubated
with culture medium containing 0_500 nmol/L QD for 24 h,
washed with QD-free medium, and then dual differential
staining was used to facilitate counting of cell numbers in the
ICM and TE[17]. 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
trinitro-benzenesulphonic acid in BSA-free
M2 medium containing 0.1% polyvinylpyrrolidone at 4
oC for 30 min, and then washed with
M2 medium[18]. The blastocysts were further
treated with 30 µg/mL antidinitrophenol-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 bisbenzimidine, but exclude PI)
appeared blue, whereas the TE cells (which take up both
fluorochromes) appeared orange-red. As multinucleated cells
are not common in pre-implantation
embryos[19], the number of nuclei was considered to represent an accurate measure
of the cell number.
Detection of cell apoptosis To study the apoptotic
effects of CdSe-core QD on embryos, we treated mouse
blastocysts (200 blastocysts in each group) with 0_500 nmol/L
CdSe-core QD at 37 oC for 24 h, and then measured cell
apoptosis. For terminal deoxynucleotidyl
transferase-mediated digoxigenin-dUTP nick-end labeling (TUNEL) staining,
the embryos were washed in QD-free medium, fixed, permeabilized, and subjected to TUNEL labeling using an
in situ cell death detection kit (Roche Molecular Biochemicals,
Mannheim, Germany) according to the manufacturer's
protocol. Photographic images were taken using a
fluorescence microscope under bright-field illumination (Olympus
BX 51, Tokyo, Japan). For Annexin V staining, the
blastocysts were incubated in 0_500 nmol/L QD for 24 h, washed
with QD-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 PBS plus 0.3% BSA, and then
incubated for 60 min with a mixture of 100 µL binding buffer,
1 µL fluorescein isothiocyanate-conjugated Annexin V, and
1 µL PI. After incubation, the embryos were washed and
photographed using a fluorescence microscope under
fluorescent illumination. The cells staining Annexin
V+/PI_ were considered apoptotic, while those staining Annexin
V+/PI+ were considered necrotic.
Observation of in vitro implantation and
post-implantation development The blastocysts were cultured according
to a modification of the previously reported
method[11]. 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 QD 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[20]. 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[9]: 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 (ESS) was defined as an embryo
that had reached stage 14 or 15 by d
8[9].
Blastocyst development following the embryo
transfer To examine the ability of expanded blastocysts to implant
and develop in vivo, the generated embryos were transferred
to recipient mice. ICR females (white skin color) were mated
with vasectomized males (C57BL/6J; black skin color; from
the National Laboratory Animal Center, China) to produce
pseudopregnant dams as recipients for the embryo transfer.
To ensure that all fetuses in the pseudopregnant mice came
from the embryo transfer (white color) and not from
fertilization by C57BL/6J (black color), we examined the skin color of
the fetuses at d 18 post-coitus. To assess the impact of QD
on post-implantation growth in vivo, the blastocysts were
exposed to 0 and 500 nmol/L QD for 24 h, and then 8 embryos
were transferred in parallel to the paired uterine horns of d 4
pseudopregnant mice. The surrogate mice were killed on d
18 post-coitus, and the frequency of implantation was
calculated as the number of implantation sites per number of
embryos transferred. The incidence rates of resorbed and
surviving fetuses were calculated as the number of
resorp-tions or surviving fetuses, respectively, per number of
implantations. The weights of the surviving fetuses and
placentae were measured immediately after dissection.
Statistics The data were analyzed using one-way ANOVA
and t-tests, and are presented as the mean±SD. A
P-value <0.05 was considered significant.
Results
Effect of CdSe-core QD on cell apoptosis in mouse
blastocysts TUNEL staining revealed that 250 and 500 nmol/L
CdSe-core QD induced apoptosis in mouse blastocysts in a
dose-dependent manner (Figure 1A). Quantitative analyses
revealed that 250 and 500 nmol/L CdSe-core QD increased
the number of apoptotic cells in blastocysts 4.2- and
6.6-fold, respectively, above the controls (Figure 1B). Annexin
V and PI staining revealed a higher ratio of Annexin
V+/PI_ cells in the ICM of blastocysts exposed to QD than in the
ICM of the controls. The ratio of Annexin
V+/PI_ cells was similar in the TE and controls, suggesting that the TE is
unaffected by exposure to QD (Figure 1C). These results
show that CdSe-core QD potently induce apoptosis in the
ICM of mouse blastocysts.
Effect of CdSe-core QD on blastocyst cell number
The effect of CdSe-core QD on cell proliferation was investigated
by differential staining. The results showed that
blastocysts exposed to 250 or 500 nmol/L of CdSe-core QD
contained fewer cells than the control blastocysts, an effect that
was most pronounced in the ICM (Figure 2).
Effect of CdSe-core QD on implantation and
post-implantation development The blastocysts treated with 125, 250,
or 500 nmol/L CdSe-core QD (240, 200, and 250 blastocysts,
respectively) showed reduced formation of the 2-layer ICM
and ectoplacental cones. Also, fewer embryos developed to
the advanced egg cylinder stages (LEC and ESS stages)
compared to the controls (Figure 3A). When morulas were
exposed to 500, 250, or 125 nmol/L CdSe-core QD, 41%, 55%,
and 78.7% developed into blastocysts, respectively. By
comparison, 85% of control morulas developed into
blastocysts (Figure 3B).
Determination of blastocyst developmental potential by
embryo transfer The effect of CdSe-core QD on
post-implantation development was investigated by transferring
mouse blastocysts exposed to CdSe-core QD into a host
mother and examining the embryos 13 d post-transfer (d 18
post-coitus). The implantation ratios for the CdSe-core
QD-pretreated blastocysts and controls were ~65% (130 of 200
embryos in 25 recipients) and ~70% (140 of 200 embryos in
25 recipients), respectively (Figure 4A). The proportion of
implanted embryos that failed to develop normally was
significantly higher for the CdSe-core QD-pretreated
blastocysts (105 of 130 implanted embryos; 80.8%) compared to
the controls (50 of 140 implanted embryos; 35.7%). The
embryos that implanted but fail to develop subsequently
resorbed. The embryos pretreated with CdSe-core QD had a
higher resorption rate and a lower surviving fetus rate than
the controls (Figure 4A). There was no difference in
placental weight between the CdSe-core QD-pretreated and the
control groups (Figure 4B). By comparison, the fetal weight
was lower in the CdSe-core QD-pretreated group versus the
controls (477±61 mg vs 583±58 mg). Fetal weight is an
important indicator of successful embryonic and
fetal development. Of the fetuses in the CdSe-core QD-pretreated group, only
13% weighed over 600 mg. By comparison, 35% of the
control fetuses exceeded this threshold (Figure 4C). Collectively,
these results show that exposure to CdSe-core QD at the
blastocyst stage reduces post-implantation development potential.
ZnS coating decreases the cytotoxic effect of CdSe QD
on blastocysts The capacity of a ZnS coating on CdSe QD to
reduce the cytotoxicity of CdSe QD on embryos was investigated. TUNEL assays and a proliferation analysis
revealed that ZnS-coated CdSe QD had no significant
cytotoxic effect on blastocysts (Figures 1,2). Furthermore,
ZnS-coated CdSe QD did not inhibit post-implantation
development (Figure 3). Collectively, these findings show that CdSe
QD coated with ZnS have no significant cytotoxic effect on
embryonic development.
Discussion
The induction of cell death by CdSe-core QD under
certain conditions correlates with the release of free
Cd2+ from the CdSe lattice. This effect is significantly reduced by
adding a ZnS coating to the CdSe-core
QD[2]. Photoluminescent semiconductor QD are novel nanometer-size probes for the
bioimaging of immunostained cells[21], and fluorescent QD
probes are useful bioimaging tools for tracing target cells
over time in mouse models[22]. Fluorescent QD probes have
the potential for development as biotracers for human
disease diagnosis. For this reason, it is essential to fully
investigate the cytotoxic capacity of QD. In the present study we
show that CdSe-core QD-induced apoptosis and the
inhibition of embryonic development is effectively reduced by a
ZnS coating (Figures 1-3). Our results suggest that QD are
likely to have latent cytotoxicity in the event of disruption of
the ZnS coatings in vivo.
Cadmium (Cd) is a significant environmental and
occupational toxic metal. Cd can have pro- or anti-apoptotic
effects, depending on the cell type, dosage, and exposure
period. Cd induces apoptosis in T
lymphocytes[23], pig proximal tubule epithelial cells (LLC-PK1
cells)[24], canine proximal
tubules[25], and rat testicular
tissue[26]. The induction of apoptosis by Cd occurs by
caspase-dependent[27] and
-independent[28] pathways.
Cd2+ also blocks apoptosis induced by a variety of
agents[29]. Recent studies show that
Cd2+ activates Ca2+/calmodulin-dependent protein kinase II in
mouse mesangial cells and triggers both apoptotic and
necrotic cell death[30]. Cd can also
induce reactive oxygen species (ROS) generation (ie oxidative stress) and trigger
apoptosis via a caspase-dependent
pathway[31]. However, the mechanisms underlying Cd-induced apoptosis, cell
cytotoxicity, and carcinogenesis remain unclear.
Se is an important dietary essential trace element for
human life. There are at least 8 Se-containing proteins,
including glutathione peroxidase and thioredoxin reductase.
Dietary Se supplementation can protect cells from oxidative
injury[32] and inhibit
apoptosis[33,34]. Conversely, Se can also
trigger cell apoptosis[34,35]. Further investigation is required
to unravel the mechanisms underlying the protective and
apoptotic effect of Se.
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 nanoparticle
fluorescence biotracers. The present study shows for the first time
that CdSe-core QD decrease the viability of mammalian
blastocysts by inducing apoptosis (Figure 1). Our results reveal
that apoptosis in mouse blastocysts exposed to 250 and 500
nmol/L CdSe-core QD increases 4.2- and 6.6-fold,
respectively, above the controls in a dose-dependent manner. CdSe-core
QD at 125 nmol/L had no cytotoxic effect, as assessed by
TUNEL staining (Figure 1A,1B). Annexin V/PI staining
revealed significant CdSe-core QD-induced apoptosis in the
ICM, but not in the TE (Figure 1C). While the blastocysts
exposed to CdSe-core QD maintained the capacity to implant
in vitro, post-implantation development was retarded,
leading to embryonic death.
During embryonic development, cells are often poised
between proliferation and apoptosis. While our previous
studies showed that CdSe-core QD induced apoptosis, no
studies have examined the apoptotic effects of CdSe-core
QD during embryonic development. In the present study,
we show that the exposure of mouse blastocysts to
CdSe-core QD decreases cell number, induces apoptosis, delays
pre-implantation development in vitro, and inhibits
post-implantation development, perhaps due to a teratogenic
effect.
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[36]. CdSe-core QD did not
reduce cell numbers or induce apoptosis in the TE, and
implantation in vitro was unaffected (Figures 1_3). Further
work will be required to assess the effect of CdSe-core QD
on differentiation and giant-cell formation in
vitro and in vivo.
Mitochondria acts as important signaling conduits
during programmed cell death, and loss of mitochondrial
integrity can be promoted or inhibited by many key regulators of
apoptosis[37,38]. Our previous study showed that CdSe-core
QD induced the loss of mitochondrial membrane potential
and the mitochondrial release of cytochrome c in IMR-32
cells in a dose-dependent manner[3]. That study also
revealed a key role for ROS in CdSe-core QD-induced apoptosis
in mitochondria, and that c-Jun N-terminal kinase (JNK) is an
upstream regulator of the mitochondria-dependent apoptotic
pathway[3]. These results indicate that the mechanism
underlying CdSe-core QD-induced apoptosis involves ROS
generation, JNK activation, and mitochondrial-dependent
processes in IMR-32 cells. Our previous study also showed
that CdSe-core QD-induced apoptosis was associated with
reduced protein levels of heat shock protein 90 (HSP90) and
the downstream targets, Ras, Raf-1, extracellular
signal-regulated kinase-1 (ERK-1), and ERK-2
[3]. These findings suggest that a CdSe-core QD-induced decrease in HSP90
expression leads to an increase in the proteasome-dependent
degradation of Ras and Raf-1, with the decrease in Raf-1
levels resulting in the subsequent downregulation of ERK-1
and ERK-2. Details of the regulatory mechanisms
underlying CdSe-core QD-induced apoptosis in blastocysts are
unclear and require further investigation.
Cd-induced cytotoxicity is associated with inflammation,
fibrosis, organ dysfunction[39,40] and the development of
various cancers[41]. Studies in Hep G2 cells implicate Cd in
caspase-dependent apoptosis[31]. The cytotoxicity of CdSe
QD correlates with the release of free
Cd2+ from the CdSe lattice, which appears to be associated with surface
oxidation[2]. The addition of a ZnS coating to CdSe QD
significantly reduces any associated
cytotoxicity[2]. For this reason, the capacity of a ZnS coating to reduce CdSe-core QD
cytotoxicity during embryogenesis was investigated. Our study
shows that a ZnS coating effectively reduces CdSe
QD-induced cytotoxicity in blastocysts. We propose that the ZnS
coating prevents CdSe-core QD-induced cell death and
cytotoxicity by blocking surface oxidation and the subsequent
release of Cd2+ ions. Further studies are required to
comprehensively assess the mechanism by which a ZnS coating
prevents CdSe-core QD-induced cell death and cytotoxicity.
In summary, the present study shows for the first time
that CdSe-core QD decrease the viability of mammalian
blastocysts by inducing apoptosis in the ICM. This is the first
evidence that CdSe-core QD have a teratogenic effect via
the induction of apoptosis. Further work is required to fully
elucidate the mechanisms underlying CdSe-core QD-induced
cell death and cytotoxicity and the relationship between these
processes and teratogenicity.
Acknowledgments
We thank Dr Chuan-hsin LU (Department of Chemical
Engineering, National Taiwan University, Taiwan) and Dr
Ruoh-chyu RUAAN (Department of Chemical and Materials
Engineering, National Central University, Taiwan) for
providing the modified CdSe quantum dots.
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