BIOLOGY OF REPRODUCTION 85, 524–535 (2011)
Published online before print 18 May 2011.
DOI 10.1095/biolreprod.110.089334
DNA Damage-Sensing Kinases Mediate the Mouse 2-Cell Embryo’s Response
to Genotoxic Stress1
X.F. Mu, X.L. Jin, M.M.J. Farnham, Y. Li, and C. O’Neill2
Human Reproduction Unit, Sydney Centre for Developmental and Regenerative Medicine, Kolling Institute of Medical
Research, Sydney Medical School, University of Sydney, Sydney, New South Wales, Australia
A critical function of cells is the maintenance of their genomic
integrity. A family of phosphoinositide-3-kinase-related protein
kinases, which includes ataxia telangiectasia mutated (ATM) and
ataxia telangiectasia and Rad3 related (ATR) kinases, play key
roles in sensing DNA damage. ATM and ATR were demonstrated
in the cleavage stages of mouse embryo development. Genotoxic
stress was imposed by exposure to ultraviolet (UV) radiation
(causes DNA strand breaks) or cisplatin (causes strand crosslinks). UV irradiation or cisplatin treatment of 2-cell embryos in
the G2 phase of the cell cycle caused DNA damage as defined by
increased phosphorylation of the H2A histone family, member X
(H2AFX; previously H2AX) variant. UV irradiation caused a
stable G2-M arrest, and cisplatin treatment allowed progression
through mitosis followed by activation of a G1-S checkpoint.
Both checkpoints were transformation-related protein 53independent. Caffeine (inhibits both ATM and ATR), but not
KU55933 (ATM-selective inhibitor), reversed the G2-M block
induced by UV, inferring a primary role for ATR in sensing this
form of DNA damage. Caffeine and KU55933 were equally
effective in reversing the cisplatin-induced G1-S block, implicating ATM as the primary sensing enzyme. Breaching of either
checkpoint by treatment with caffeine or KU55933 allowed
embryos to progress through several further cell cycles, yet none
developed to blastocysts. The results show, to our knowledge for
the first time, that the G2-M and G1-S cell-cycle checkpoints in
the early embryo are differentially regulated by ATM and ATR in
response to genotoxic stress and that they act as an initial point
for containment of genomic damage. Under conditions of
extensive or persistent DNA damage, the demise of the embryo
is the ultimate method of protecting genomic integrity.
apoptosis, ataxia telangiectasia and Rad3 related, ataxia
telangiectasia mutated, cell cycle, checkpoint, DNA damage,
early development, embryo, H2AFX, transformation-related
protein 53, tumor suppressor gene
INTRODUCTION
A critical function for all cells is monitoring and
maintaining the integrity of the genome. Structural damage
or alterations to DNA are generally mutagenic, and the
propagation of such damaged cells within a lineage may
compromise the viability of the organism. If DNA damage
1
Supported by grants from the Australian National Health and Medical
Research Council to C.O.
2
Correspondence: FAX: 61 2 9926 6343;
e-mail: [email protected]
Received: 25 October 2010.
First decision: 23 November 2010.
Accepted: 13 April 2011.
Ó 2011 by the Society for the Study of Reproduction, Inc.
eISSN: 1529-7268 http://www.biolreprod.org
ISSN: 0006-3363
524
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occurs within cells of the early embryo, the consequences can
be particularly severe, because their perpetuation can potentially affect all cells in the developing organism, including the
germline. Key to the management of genomic integrity is the
detection of DNA damage. A family of phosphoinositide-3kinase-related protein kinases (PIKKs) plays key roles in
sensing DNA damage [1]. Foremost within this family are the
tumor suppressor proteins ataxia telangiectasia mutated (ATM)
and ataxia telangiectasia and Rad3 related (ATR) kinases. The
aim of the present study was to assess the role that ATM and
ATR play in sensing DNA damage in the early mouse embryo
and the nature of the embryo’s response to this damage.
Recruitment of the PIKK family of kinases to sites of DNA
damage causes activation of their kinase activity, which in turn
activates downstream effectors of cell-cycle regulation or
apoptosis. PIKKs are recruited to sites of damage by specific
partner proteins. For example, ATR-interacting protein forms a
partnership with ATR [2], and the meiotic recombination 11
homolog A complex (formerly known as Mre11) partners with
ATM [3]. The minor histone 2A variant, H2A histone family,
member X (H2AFX), is an important substrate for the PIKKs.
The phosphorylated (serine-129) form of this histone is
commonly referred to as gamma-H2AFX. Its phosphorylation
in response to DNA damage is very efficient and results in
accumulation of H2AFX for many megabase pairs (Mbp) on
either side of the lesion, making immunostaining for gammaH2AFX the most sensitive measure of damage [4, 5]. GammaH2AFX serves as a key component in the DNA damage repair
response by loosening the conformation of DNA and, thus,
facilitating recruitment of the repair enzymes to the lesion. It
also recruits molecules, such as cohesion molecules, that
provide structural integrity during the repair process [4, 5].
This combination of proteins acts to sense the DNA damage
and amplify the DNA damage signal, and this results in the
recruitment of the required effectors.
A canonical response of cells to DNA damage is the
activation of cell-cycle checkpoints [6]. Checkpoints provide a
mechanism whereby cells are prevented from progressing past
specific points within the cell cycle if conditions such as DNA
damage exist. Two important checkpoints are the G1-S and G2M checkpoints. ATM is most commonly implicated in the
activation of the G1-S checkpoint [1], whereas ATR is active in
the induction of the G2-M checkpoint [7]. It is becoming
increasingly recognized, however, that some overlap and
redundancy occurs between the action of these two kinases at
these checkpoints [1]. The checkpoints also provide an
opportunity for repair of DNA before cell division. Where
DNA damage is severe or irreparable, cell death, typically via
apoptosis, may also result. ATM and ATR can mediate this
response by activation of mediators such as transformationrelated protein 53 (TRP53; also known as P53) [8, 9], which
can induce BCL2-associated X protein (BAX)-mediated
apoptosis.
ABSTRACT
SENSING DNA DAMAGE IN EMBRYOS
and KU55933-sensitive) and ATR (caffeine-sensitive and
KU55933-insensitive) in response to defined genotoxic stress
can be assigned.
Most studies of the early embryo to date have used Xirradiation as a source of genotoxic stress; however, exposure
of embryos to chemicals or UV radiation seems to be a form of
stress that the embryo is more likely to encounter, particularly
with the advent of assisted reproductive technologies. In the
present study, 2-cell mouse embryos were subjected to two
different forms of genomic stress: UV irradiation, which
typically induces DNA single-strand breaks and is therefore
expected to be sensed by ATR, and cisplatin (a chemotherapeutic drug), which induces inter- and intrastrand DNA crosslinks and DNA-protein cross-links and is therefore a likely
target for ATM. We provide evidence for different responses
by the embryo to these distinct forms of DNA damage and the
differential involvement of ATR and ATM in the embryo’s
response to each. To our knowledge, the present study provides
the first analysis of the relative roles of key DNA damage
sensor kinases in monitoring the integrity of the embryonic
genome and directing the embryo’s response to this damage.
MATERIALS AND METHODS
Animals
The use of animals in the present study was in accordance with the
Australian Code of Practice for the Care and Use of Animals for Scientific
Purpose and was approved by the Institutional Animal Care and Ethics
Committee. C57BL6J mice were housed and bred in the Gore Hill Research
Laboratory (St Leonards, NSW, Australia) under a 12L:12D photoperiod and
had access to food and water ad libitum. Female mice (age, 4–6 wk) were
superovulated by intraperitoneal injection of 5 IU of equine chorionic
gonadotropin (Folligon; Intervet International), followed 48 h later by 5 IU
of human chorionic gonadotropin (hCG; Chorulon; Intervet). Females were
paired with males of proven fertility. Pregnancy was confirmed by the presence
of a copulation plug the following morning (Day 1).
Mouse Embryo Collection and Culture
Two-cell embryos were cultured in modified-human tubal fluid medium
(mod-HTF) after being flushed from the oviduct with Hepes-buffered modHTF [31]. All components of the media were tissue-culture grade (Sigma
Chemical Co.), and all media contained 3 mg/ml of bovine serum albumin
(Sigma) unless otherwise stated. Embryos were cultured in groups of 10 in 10ll volumes of mod-HTF overlaid by approximately 2 mm of heavy paraffin oil
(Sigma) at 378C in 5% CO2 in air for the periods indicated in individual
experiments. The developmental stage and morphology of embryos were
assessed by visualizing the embryos with an inverted phase-contrast
microscope (Diaphot; Nikon) at 24-h intervals after collection and treatment.
Immunofluorescence
Embryos were washed three times in PBS with 0.1% (w/v) bovine serum
albumin, 0.1% (w/v) Tween-20 (Sigma), and 0.2% (w/v) sodium azide
(washing buffer), then fixed with freshly prepared 2% paraformaldehyde (w/v;
Sigma) in PBS (pH 7.4) for 30 min, and then permeabilized with 2% (w/v)
paraformaldehyde with 0.3% Tween-20 at room temperature for 30 min.
Embryos were washed three times in washing buffer and then blocked in PBS
containing 2% (w/v) bovine serum albumin and 30% (v/v) serum for 3 h. Next,
they were stained overnight at 48C with primary antibodies: rabbit anti-ATM
antibody (sc-7230; Santa Cruz Biotechnology) at 4 lg/ml, goat polyclonal antiATR (sc-1887; Santa Cruz Biotechnology) at 2 lg/ml, and rabbit antipolyclonal gamma-H2AFX (phosphor-serine 139; ab2893; Abcam) and antimouse monoclonal phosphorylated ATM (pATM; phosphorylated serine 1981;
ab36810; Abcam) at 4 lg/ml. Each antibody was prepared in PBS with 2%
(w/v) bovine serum albumin. Equivalent concentrations of nonimmune
immunoglobulin (Ig) G were used as negative controls. Detection with
fluorescently labeled secondary antibodies was achieved by labeling at room
temperature for 1 h.
Whole-section imaging was performed with mercury lamp UV illumination
and epifluorescence on a Nikon Optiphot microscope with a DPlan Apo 40 UV
objective. Images were subjected to deconvolution using Image-Pro Plus with
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Damage to DNA can take many forms, including single- or
double-strand breaks, inter- and intrastrand DNA cross-links,
DNA-protein cross-links, and chemical modification, such as
generation of 8-hydroxydeoxyguanosine residues or polycyclic
aromatic hydrocarbon adducts. ATR is thought to be most
important for sensing single-strand DNA breaks, whereas ATM
is most involved in sensing double-strand breaks [3]. Sensing
of interstrand cross-linking defects may involve ATR, but not
ATM, in somatic cells [10]. A consensus is forming that
implicates considerable, but not complete, overlap in the
functions of ATM and ATR in somatic cells [11]. Some
nonredundant actions, however, are powerfully demonstrated
by the observations that Atr/ embryos are nonviable by the
late blastocyst stage [12, 13] but that Atm/ embryos are
viable [14]. This indicates fundamental functions of Atr in early
development that are not compensated for by the presence of
the Atm gene.
X-irradiation of the zygote or 2-cell mouse embryo induces
a G2-M arrest [15]. This type of arrest was ameliorated by
caffeine, an inhibitor of both ATR and ATM [16]. X-irradiated
embryos that passed the arrested phase showed increased
micronuclei formation and reduced developmental viability
[17–19]. Drug- and ultraviolet light (UV) irradiation-induced
DNA damage has been less widely investigated; however, UV
exposure has been shown to cause increased rates of sister
chromatid exchange [20] and single-strand breaks (hamster)
[21]. Exposure of mouse zygotes to UV radiation resulted in
unscheduled DNA synthesis, which is interpreted as evidence
for activation of DNA repair processes [22]. Exposure of sperm
to UV radiation did not prevent fertilization; however, the
resulting embryos (bovine) tended to block at the 2-cell stage
[23]. X-irradiation of sperm also results in developmental arrest
because of the activation of a Trp53-dependent, S-phase
checkpoint in the resulting mouse zygotes [24, 25]. Embryos
treated in this manner also activated a cyclin-dependent kinase
(CDK) inhibitor 1A-dependent cell-cycle arrest at the morula/
blastocyst stage.
Caffeine is effective in relieving the cell-cycle blocks
induced by a range of genotoxic stresses in vitro. It achieves
this by inhibiting the phosphorylation and activation of
mammalian PIKKs [26], but it is not a global inhibitor of
other protein kinase activities. The PIKK family of protein
kinases also includes mechanistic target of rapamycin (serine/
threonine kinase; MTOR) and protein kinase, DNA-activated,
catalytic polypeptide (PRKDC). Caffeine has relatively poor
selectivity for PRKDC. It does act on MTOR, but a selective
MTOR inhibitor (rapamycin) does not influence checkpoint
blocks induced by irradiation of cells, arguing that MTOR is
not a major caffeine-sensitive effector of these checkpoints
[27]. Caffeine is also a well-known inhibitor of cAMPphosphodiesterase [28], yet specific inhibition of cAMPphosphodiesterase and increased cellular cAMP concentration
actually increase the radiosensitivity of cells, providing no
support for a role of phosphodiesterase inhibition in the relief
of checkpoint action in cells with damaged DNA [29]. Thus, it
is generally recognized that the ability of caffeine to allow cells
with damaged DNA to bypass checkpoints is primarily the
result of its capacity to inhibit ATR and ATM.
Recently, a highly selective competitive inhibitor of ATM
has been developed (KU55933) [30]. This drug has high ATM
specificity with respect to inhibition of other PIKKs, prevents
phosphorylation of selective ATM targets following DNA
damage, and prevents the normal ATM-dependent cell-cycle
checkpoint responses to ionizing radiation [26, 30]. The
selectivity of KU55933 for ATM means that when used in
conjunction with caffeine, the relative roles of ATM (caffeine-
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MU ET AL.
FIG. 1. Immunolocalization and Western
blot analysis of ATM and ATR in the
preimplantation mouse embryo. Embryos
were collected from the reproductive tract
and immediately fixed and stained. In each
case, staining with IgG control antisera was
undetectable, and the microscope and
imaging settings were the same for each
stage of development for a given antibody.
The images shown are representative of at
least five embryos in each of three replicates. A-C) Whole-section images of ATM
staining of 2-cell, 8-cell, and blastocyststage embryos, respectively. D-F) Wholesection images of ATR staining of 2-cell, 8cell, and blastocyst-stage embryos, respectively. G) A single equatorial confocal
section of ATR stained 2-cell embryo. H) A
two-dimensional reconstruction of all sections through the embryo shown in G. Bar
¼ 10 lm. I) Representative Western blot of
ATM and ATR with molecular mass of
markers shown with arrows.
Western Blot Analysis
Western blot analysis was performed as previously described [33]. Fresh
hybrid 2-cell embryos were collected from reproductive tracts 40 h post-hCG,
washed three times in cold PBS, and transferred into 1.5 ll of extraction buffer
supplemented with protease and phosphatase inhibitors (23 PBS, 2% [v/v]
Triton X-100, 24 mM deoxycholic acid, 0.2% [w/v] SDS, 20 mM NaF, 20 mM
Na4P2O7, 2 mM PMSF, 3.08 lM aprotinin, 42 lM leupeptin, and 2.91 lM
Pepstatin A; all from Sigma). The embryos were lysed by three cycles of
freezing in liquid nitrogen and thawing (with vortexing). Protein samples were
diluted with 1 ll of 53 Laemmli buffer (50 mM Tris-HCl, 5 mM
ethylenediaminetetra-acetic acid [pH 8.0], 12.5% [w/v] SDS, 0.05% [w/v]
bromophenol blue, and 25% beta-mercaptoethanol), incubated for 5 min at
958C, and size separated on PhastGel Gradient 4-15 (catalog no. 170678-01;
GE Healthcare) using a PhastSystem electrophoresis apparatus (PhastSystem
separation and control unit; Amersham Pharmacia Biotech). The proteins were
blotted onto polyvinylidene fluoride transfer membrane (Hybond-P; GE
Healthcare) with transfer buffer containing 12 mM Tris (Sigma), 96 mM
glycine (BDH), 0.1% (w/v) SDS, and 10% (v/v) methanol (BDH) using a Phast
Transfer system. The membrane was incubated in 10 ml of blocking buffer
containing 2.5% (w/v) skim milk powder (Diploma). The primary antibodies
were the same as used for immunofluorescence staining at a concentration of
0.4 lg/ml for each. They were detected with 1:5000 horseradish peroxidaseconjugated secondary antibody (Sigma). The membrane was incubated in
SuperSignal West Femto (Pierce) diluted 1:4 and analyzed by chemiluminescence (Las 4000; Fuji), and HiMark Pre-Stained Standards (Invitrogen) were
used to assess molecular mass. Analyses were performed on groups of 90
embryos for each antigen.
Treatments
The ATM inhibitor, 2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one
(KU55933; gift from Dr. Graeme Smith, KuDOS Pharmaceuticals Ltd.) [30];
the ATR and ATM inhibitor, 1,3,7-trimethylxanthine (caffeine; Sigma); the 1o-phosphatidylinositol-3-kinase inhibitor, (2-(4-morpholinyl)-8-phenyl-4H-1benzopyran-4-one (LY294002; Calbiochem); the TRP53 inhibitor, 2-(2-imino4,5,6,7-tetrahydrobenzothiazol-3-yl)-1-p-tolylethanone (pifithrin-a; Sigma);
and cis-Diammineplatinum(II) dichloride (cisplatin; P4394; Sigma). UV
irradiation was achieved by placing embryos in 1 ml of Hepes-buffered modHTF media in an open, 35-mm, plastic Petri dish. UV irradiation (253.7 nm)
equated to a dose of 1.95 J/cm2. Changes in UV dosage are not readily
calibrated or validated in a biological laboratory; hence, it is common to change
exposure by varying the time of exposure to a standard dose. This strategy has
been adopted in this laboratory. After exposure, embryos were transferred to
mod-HTF, with or without other drugs, in 10-ll drops under mineral oil
(Sigma). Cisplatin treatment was performed as a conventional dose-response
study.
TUNEL Assay
The TUNEL assay was used to detect apoptosis (fluorescein; In Situ Death
Detection Kit; Roche). Embryos were fixed and permeabilized as described
above and incubated in the TUNEL reaction mixture supplied with the kit for 1
h at 378C in the dark. Negative controls lacked terminal deoxynucleotidyl
transferase, and positive controls were embryos incubated in DNase I (50 U/ml;
Sigma) for 10 min at 378C to induce DNA strand breaks. Embryos were
washed three times in PBS, stained with 2 0 -(4-ethoxyphenyl)-5-(4-methyl-1piperazinyl)-2,5 0 -bi-1H-benzamidazole trihydrochloride (4 lg/ml; bisBenzimide; Sigma) and then mounted. The total number of nuclei was enumerated by
the bisBenzimide staining, whereas the fluorescein TUNEL staining identified
those nuclei undergoing apoptosis.
DNA Synthesis
Embryos were incubated in media supplemented with 20 lM 5-bromo-2 0 deoxyuridine (BrdU; Sigma). They were then fixed and stained using the
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Sharpstack deconvolution software (Media Cybernetics, Inc.). Optical
sectioning was performed with a Bio-Rad Radiance Confocal microscope
using a Nikon Plan Apo 60X/1.4 oil-emersion objective as previously described
[32]. Images were captured using Lasersharp 2000 software. Microscope and
laser settings were adjusted such that no fluorescence was observed with
nonimmune controls. All test specimens were observed with these same
settings. Quantitative analysis of staining was achieved using the area-ofinterest marking tool within Image-Pro Plus image analysis software and
expressed as arbitrary units of optical density.
SENSING DNA DAMAGE IN EMBRYOS
527
immunolocalization technique described above with a 1:200 dilution of antiBrdU antibody (Sigma).
Statistical Analysis
Statistical analyses were performed with the SPSS software (Version 11.5;
SPSS). The proportion of embryos developing to a given developmental
landmark following culture in vitro or the proportion of embryonic cells was
assessed by binary logistic regression analysis. The proportion developing to a
given landmark or undergoing apoptosis was treated as the dichotomous
dependent variable and the treatment and experimental replicate as covariates in
the model.
The effects of treatments on the number of cells and the number of TUNELstaining cells in blastocysts as well as the intensity of immunostaining were
assessed using univariate regression analysis within the general linear model.
When appropriate, the statistical model incorporated the effect of treatment
with the number of cells as the dependent variable and treatment as fixed-factor
main effects. Full factorial analysis was performed with significant factor
contrasts to assess the difference between individual treatments.
RESULTS
FIG. 2. Development of 2-cell embryos in culture after exposure to
genotoxic stress. Two-cell embryos were collected from the reproductive
tract and immediately exposed to different levels of two genotoxic stress,
UV irradiation and cisplatin treatment. After exposure, the embryos were
washed and placed in culture for (A) 24 h, after which the proportion of
embryos that had developed past the 2-cell stage was recorded, or (B) 48
h, after which the proportion of embryos that developed past the 4-cell
stage was recorded. The results represent three independent replicates,
with at least 30 embryos per treatment per replicate.
with BrdU commenced before 40 h post-hCG, many cells
(.50%) incorporated BrdU, but by 43 h post-hCG, no cells
incorporated BrdU. Based on this analysis, we concluded that
2-cell embryos collected 44 h post-hCG were within the G2
phase, and this was the time point used for further analyses.
We next investigated the capacity of UV irradiation or
cisplatin treatment to induce cell-cycle blocks in the early
embryo. Two-cell embryos (44 h post-hCG) were treated with
increasing exposure times to UV radiation or increasing
cisplatin concentration. Increasing the time of exposure of 2cell mouse embryos to UV radiation caused a progressive
increase in the proportion of embryos that failed to progress
through the cell cycle (2-cell block) 24 h after treatment (P ,
0.001) (Fig. 2A). UV exposure for 4 min prevented all embryos
from undergoing mitosis. Treatment of embryos with an
increasing concentration of cisplatin for 4 h caused only a small
proportion to block at the 2-cell stage, and this proportion
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Immunolocalization of ATM (Fig. 1, A–C) and ATR (Fig.
1, D–F) in embryos collected directly from the reproductive
tract showed that both antigens were present in the 2-cell and 8cell embryos. Blastocysts stained intensely for ATM but
showed negligible ATR staining (Fig. 1F). In whole-embryo
sections, nuclear accumulation of ATM was evident, although
it was less clear whether this was also the case for ATR. Thus,
single equatorial confocal sections of ATR staining were
imaged in fresh 2-cell embryos. These showed some nuclear
accumulation of ATM staining (Fig. 1G), which had a
relatively uniform distribution, except that it was reduced
within the presumptive nucleoli. Cytoplasmic staining showed
a heterogeneous granular pattern, which was more intense in
the cortical regions. A two-dimensional reconstruction of
confocal sections through the depth of the embryo showed
extensive distribution of these ATR-staining granules (Fig.
1H). The nuclear localization of ATM and ATR in embryos
that were assumed to be normal and not subject to genotoxic
stress was surprising. To ensure that it was not an acute
response to the process of collecting and preparing embryos, 2cell embryos were collected again but flushed from the oviduct
with fixative (paraformaldehyde) to minimize any stress
response before fixation. These embryos showed the same
pattern of staining as those subjected to conventional collection
procedures (not shown). Western blot analyses (Fig. 1I) of
extracts of 2-cell embryos (using the same antibodies used for
immunolocalization) showed a single dominant high-molecular-weight band for each of the ATM and ATR antibodies,
confirming their suitability for immunolocalization studies.
The next component of the study was to assess the response
of embryos to two different forms of genotoxic stress. The
design of the experiment was to expose 2-cell embryos to the
genotoxic stressors during the G2 phase of the cell cycle. If the
stress caused a G2-M cell-cycle checkpoint, then a 2-cell block
was expected. If the block occurred at the G1-S checkpoint,
then progression of embryos through the 2-cell stage with a
subsequent block in the 4-cell stage was expected. To
determine the time that 2-cell embryos completed the S phase
and had thus entered the G2 phase, cell-cycle analysis was
performed by BrdU incorporation. Zygotes were collected at
29 h post-hCG and cultured to 50 h post-hCG in media
supplemented with BrdU at progressive times after culture was
initiated. Addition of BrdU commenced 37 h post-hCG and at
1.5-h intervals thereafter (at least 25 embryos were analyzed at
each time point). Immunolocalization of BrdU was performed
in the resulting 2-cell embryos, and nuclei were categorized as
either with or without BrdU incorporation. When incubation
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MU ET AL.
FIG. 3. The phosphorylation of H2AFX in
response to genotoxic stress in the 2-cell
embryo. Immunofluorescence images of 2cell embryos stained for gamma-H2AFX
after their treatment with (A) UV radiation
or (B) cisplatin. Embryos were exposed to
UV radiation for 0, 1, 2, or 4 min and
stained after 1, 2, or 4 h in culture. Cisplatin
treatment was at a concentration of 0, 3, 6,
or 12 lM. The images are representative of
response for each treatment. Nonimmune
sera resulted in no detectable staining. Bar
¼ 10 lm.
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plateaued as the concentration increased (P , 0.001 vs. UV
treatment) (Fig. 2A).
Examination of the proportion of embryos that progressed
past the 4-cell stage after 48 h of culture showed the UVtreated embryos that bypassed the 2-cell block were mainly
blocked at the 4-cell stage (Fig. 2B). A strong cisplatin dosedependent block to progression past the 4-cell stage was
observed. More than 80% of 2-cell embryos did not progress
past the 4-cell stage after treatment with 12 lM cisplatin (Fig.
2B).
Phosphorylation of H2AFX (gamma-H2AFX) is a highly
sensitive marker of damage induced by a range of genotoxic
stressors. Two-cell embryos were immunostained for this
marker at various times after UV irradiation (Fig. 3A) or
cisplatin treatment (Fig. 3B). Untreated 2-cell embryos showed
little detectable gamma-H2AFX. The amount of nuclear
staining increased with the time that embryos were exposed
to UV at each of four assessed time points after treatment (1–4
h). Exposure of embryos to cisplatin also caused an increase in
gamma-H2AFX staining, but this was only observed consistently after 4 h of exposure. The results show that both UV and
cisplatin treatment caused DNA damage in the 2-cell embryo.
Severe genotoxic stress might conceivably cause the acute
onset of cell death by apoptosis. Cohorts of embryos treated
with the highest UV exposure times and the highest
concentration of cisplatin were subjected to TUNEL staining
SENSING DNA DAMAGE IN EMBRYOS
24 h after treatment. No UV-treated or control embryos were
TUNEL positive (n ¼ 25 for each). Cisplatin-treated embryos
that were blocked at the 4-cell stage showed only a small
number of apoptotic cells (5% 6 2% of cells, mean 6 SEM, n
¼ 15 embryos). During TUNEL staining, the nucleus was also
stained with bisBenzimide; this showed that the blocks induced
by both UV irradiation and cisplatin treatment were not
associated with the entrapment of cells at the metaphase stage.
To assess whether the 4-cell block induced by cisplatin
occurred before cells entering the next round of DNA synthesis
(S phase), control or cisplatin (12 lM)-treated embryos were
cultured in the presence of BrdU and then tested for
incorporation by immunolocalization. All cells in control
embryos showed levels of BrdU incorporation consistent with
progression through the S phase, but this was only the case for
7% 6 4% (mean 6 SEM) of cells in embryos treated with
cisplatin (n ¼ 15 embryos in each case). These results show that
G2-stage embryos exposed to UV radiation were blocked
before entering metaphase, and we therefore conclude that UV
irradiation activated a G2-M checkpoint. Cisplatin treatment
FIG. 5. The effect of inhibitors of ATM and ATR on the developmental
blocks induced by UV irradiation and cisplatin treatment of 2-cell
embryos. Embryos were treated with (A) UV radiation (4 min) or (B)
cisplatin (12 lM) in the presence of increasing concentrations of caffeine
or KU55933. After extensive washing, embryos were cultured in media
with the same concentration of caffeine or KU55933 for 24 h (UV
irradiation) or 48 h (cisplatin treatment), and the proportion achieving
developmental landmarks was recorded. The results represent a minimum
of 30 embryos per treatment in each of three replicates.
did not block cells from progressing through metaphase but,
rather, induced a block before the next S phase; therefore, it can
be concluded to have induced a G1-S checkpoint.
To determine whether the embryos at the 2-cell (UV
treatment) and 4-cell (cisplatin treatment) stage were blocked
rather than transiently delayed, their fate was followed over 72
h in culture. None of embryos blocked at the 2-cell stage after
UV exposure progressed past this stage after 72 h of culture.
Most of the embryos exposed to cisplatin were at the 4-cell
stage 24 h after treatment (Fig. 4A). At 48 h (Fig. 4B) or 72 h
(Fig. 4C), this block persisted, with only a small increase in the
number of embryos progressing to the 8-cell stage (P , 0.01)
and none progressing past this stage. Over this time, an
increase (P , 0.01) was observed in the proportion of embryos
showing morphological degeneration (fragmentation). It is
concluded that the G1-S checkpoint activated by cisplatin and
the G2-M checkpoint activated by UV radiation were relatively
stable over at least 72 h.
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FIG. 4. The rate of development of embryos treated with cisplatin over
72 h in culture. Two-cell embryos recovered from the reproductive tract
were immediately exposed to control media or media supplemented with
12 lM cisplatin. After washing, they were cultured in media for 72 h. The
proportion of embryos reaching each developmental landmark after (A) 24
h, (B) 48 h, and (C) 72 h is shown. The results represent a minimum of 90
embryos in each treatment group.
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MU ET AL.
FIG. 6. The effect of ATR and ATM
inhibitors on the activation of H2AFX in
response genotoxic stress. A) Embryos were
exposed to UV radiation for 4 min, treated
with cisplatin (12 lM), or untreated (Control). During exposure, embryos were either
untreated (Vehicle) or cotreated with caffeine (10 mM), KU55933 (20 lM), or a
combination of the two (caffeine þ
KU55933). They were cultured for 4 h and
then stained for gamma-H2AFX. B) The
intensity of nuclear staining was measured,
and the graph shows the mean 6 SEM of
this staining in arbitrary units. **P , 0.001
vs. respective control treatment, *P , 0.01
vs. respective vehicle treatment group. Bar
¼ 10 lm.
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H2AFX is a substrate for ATM and ATR, and the
observation of the induction of gamma-H2AFX staining in
the nuclei of 2-cell embryos exposed to UV radiation and
cisplatin (Fig. 3) implicates the activation of these PIKKs as a
component of the embryo’s response to these stressors. The
role of ATM and ATR in the developmental blocks induced by
UV irradiation or cisplatin treatment was assessed by the use of
their inhibitors, caffeine and/or KU55933. Caffeine caused
dose-dependent (P , 0.001) amelioration of the 2-cell
checkpoint induced by UV irradiation (Fig. 5A) and the 2cell and 4-cell blocks caused by cisplatin (Fig. 5B) (P ,
0.001). KU55933 had no dose-dependent effect on the UV
irradiation-induced 2-cell checkpoint (P . 0.05) but partially
ameliorated the cisplatin-induced 4-cell checkpoint (P ,
0.001). The amelioration of a UV irradiation-induced 2-cell
checkpoint by caffeine, but not by KU55933, implicates a
primary role for ATR in this response. The capacity of
KU55933 to ameliorate the 4-cell checkpoint induced by
cisplatin implicates ATM in this response. The increased
staining of gamma-H2AFX in response to UV irradiation was
ameliorated by treatment with caffeine but not by KU55933
(Fig. 6A). No additive interaction between caffeine and
KU55933 was observed. The accumulation of gammaH2AFX in 2-cell nuclei in response to cisplatin was equally
blocked by caffeine and KU55933, and no further additive
effect of these two inhibitors was found (Fig. 6B). Phosphorylation of H2AFX is a canonical response to DNA damage
sensed by ATR and ATM. The inhibition of this response by
inhibitors of ATR and ATM confirm the role of these PIKKs in
the embryo’s response to DNA damage.
The activation of ATM is associated with its phosphorylation (at serine 1981; pATM) [34]. To date, an equivalent
activation state has not been identified for ATR. Immunolocalization of pATM showed little staining in untreated 2-cell
embryos, but increased staining was found following exposure
to UV radiation or cisplatin (Fig. 7). This increased staining
was characterized by an accumulation of pATM staining within
the nucleus. The increased pATM staining in response to UV
irradiation was largely blocked by treatment with caffeine but
not with KU55933 (Fig. 7). Caffeine and KU55933 reduced the
level of pATM staining after cisplatin treatment (Fig. 7).
SENSING DNA DAMAGE IN EMBRYOS
531
FIG. 7. The phosphorylation of ATM in
response to genotoxic stress in the 2-cell
embryo. Immunofluorescence images of 2cell embryos stained for pATM after their
treatment with UV irradiation or cisplatin in
the presence or absence of ATM and ATR
inhibitors. Embryos were exposed to UV
radiation for 4 min, treated with cisplatin
(12 lM), or untreated (Control). During
exposure, embryos were either untreated
(vehicle) or cotreated with caffeine (10
mM), KU55933 (20 lM), or a combination
of the two (caffeine þ KU55933). They
were cultured for 4 h and then stained for
pATM. Nonimmune control sera resulted in
no detectable staining. Bar ¼ 10 lm.
0.01) and less than the proportion of caffeine-treated embryos
that reached the 4-cell landmark after 24 h of culture (P ,
0.05). No caffeine-treated, UV-exposed embryos achieved the
blastocyst landmark after 72 h of culture, with about half at the
morula stage at this time. Over this culture period, KU55933
was without any beneficial effect on embryo development, and
no additive effect of combined treatment with caffeine and
KU55933 was observed (P . 0.05) (Fig. 9A).
Analysis of the cellular composition of the embryos after 72
h of culture presented a slightly more complex picture (Fig.
9B). It showed that whereas caffeine partially ameliorated the
effects of UV treatment, the resulting embryos, on average, had
many fewer cells (P , 0.01) than control embryos, and many
of these were apoptotic, as evident by TUNEL-staining (P ,
0.01). The cellular analysis confirmed the lack of effect of
KU55933 but showed that combining KU55933 with caffeine
actually slightly reduced the beneficial effect of caffeine on the
number of cells within embryos (P , 0.01). The relative
proportion of cells undergoing apoptosis was similar (P .
0.05) after treatment with both inhibitors, as it was after
caffeine treatment alone, with a large proportion of embryonic
cells undergoing apoptosis.
Cisplatin treatment at the 2-cell stage caused progressively
worsening development over the 72 h of culture after treatment.
No cisplatin-treated embryos developed to morphological
blastocysts (Fig. 10A). Caffeine treatment prevented the
activation of the 2-cell block (P , 0.001) and allowed many
embryos to escape the 4-cell block and reach the 8-cell
landmark after 48 h (P , 0.001), yet none of these embryos
developed to blastocysts by 72 h. Treatment with KU55933 or
a combination of caffeine and KU55933 resulted in development rates that were not different (P . 0.05) from the rate for
treatment with caffeine alone.
After 72 h of culture, cisplatin-treated embryos contained
fewer cells than untreated controls (P , 0.001), and almost all
of these were TUNEL-positive (Fig. 10B). Whereas apoptosis
was not an acute response to the 4-cell checkpoint, its
persistence did result in extensive apoptosis after 72 h.
Caffeine treatment partially relieved the 4-cell block. However,
the total number of cells in the resulting embryos was still
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Both ATM and ATR act in some circumstances via the
activation of TRP53. To test for a role of TRP53, embryos
were subjected to both forms of genotoxic stress in the
presence of a TRP53 inhibitor, pifithrin-a, over a concentration
range known to block TRP53-mediated loss of viability in
embryos [35]. This treatment had no beneficial effect on the
capacity of embryos to bypass the checkpoints induced by
either UV irradiation (Fig. 8A) or cisplatin treatment (Fig. 8B).
The results show that the stable checkpoints induced by UV
irradiation and cisplatin treatment acted in a TRP53-independent manner.
The inhibitor LY294002 is a potent inhibitor of phosphatidylinositol-3-kinase activity, but it is not an effective inhibitor
of the protein kinase activities of ATM and ATR. Two-cell
embryos subjected to UV irradiation or cisplatin treatment were
cotreated with the phosphoinositide-3-kinase inhibitor,
LY294002, at a concentration that blocks phosphatidylinositol-3-kinase activity [36]. This treatment caused a dosedependent augmentation (P , 0.01) of the adverse response
of embryos to cisplatin treatment but had no effect (P . 0.05)
on the block caused by UV irradiation (Fig. 8C). The results
show that a phosphatidylinositol-3-kinase activity was not
responsible for the developmental blocks induced by UV
irradiation or cisplatin treatment. It further showed that a
LY294002-sensitive phosphatidylinositol-3-kinase activity
may play a role in mitigating the effects of some forms of
genotoxic stress.
It was expected that bypassing the cell-cycle checkpoints
induced by UV irradiation or cisplatin treatment would mean
that these embryos would develop further, with marked DNA
damage. The developmental fate of these embryos was
therefore of interest and was studied by monitoring development over 72 h of culture. Caffeine treatment allowed most UV
radiation-treated embryos to escape the 2-cell G2-M block (P ,
0.001), although fewer achieved the 4-cell landmark after 24 h
compared to control embryos (P , 0.01), indicating embryos
were delayed in the 2-cell stage even after caffeine treatment
(Fig. 9A). Many UV-exposed embryos reached the 8-cell
landmark after 48 h culture with caffeine treatment; however,
this proportion was less than that in untreated controls (P ,
532
MU ET AL.
FIG. 8. The effect of TRP53 (pifithrin-a [PFT]) and phosphatidyl-3-kinase
(LY294002) inhibitors on the developmental blocks induced by UV
irradiation and cisplatin treatment of embryos. A) The effect of PFT on the
development of control (UV) or UV radiation-exposed ( þUV) 2-cell
embryos to the 4-cell stage after 24 h. B) The effect of PFT on the
development of control (cisplatin) or cisplatin-treated ( þcisplatin)
embryos to the 8-cell stage after 48 h. C) The effect of LY294002
treatment on the development of 2-cell embryos exposed to UV radiation
(4 min) or treated with cisplatin (3 lM) beyond the 4-cell stage after 48 h
of culture. The results represent at least 60 embryos per treatment from
three replicates.
markedly less than untreated embryos (P , 0.001), and most of
the cells so created were apoptotic after 72 h of culture (Fig.
10B). Treatment with KU55933, by contrast, allowed fewer
cells to accumulate in embryos, but fewer of these were
apoptotic. Embryos cotreated with both caffeine and KU55933
showed a similar response to KU55933 alone. Thus, treatment
of embryos with caffeine and KU55933 allowed embryos to
accumulate more cells than cisplatin controls and allowed more
of these cells to survive (Fig. 10B). Yet, these embryos still had
many fewer total cells than untreated controls, and they were
not capable of development to the blastocyst stage at a normal
rate. Caffeine treatment partially relieved the cell-cycle
checkpoints induced by cisplatin but not the onset of apoptosis,
whereas KU55933 was less effective at relieving cell-cycle
blocks but more effective in reducing the onset of apoptosis.
The results argue for some nonoverlapping roles of ATR and
ATM in mediating the effects of the genotoxic stress induced
by cisplatin.
DISCUSSION
The present study demonstrates, to our knowledge for the
first time, that two DNA damage-sensing kinases, ATM and
ATR, play important yet nonredundant roles in mediating the
embryo’s response to DNA damage. The cell-cycle checkpoints induced by UV irradiation or cisplatin treatment of 2cell embryos were TRP53-independent and generally occurred
Downloaded from www.biolreprod.org.
FIG. 9. The effect of ATM and ATR inhibitors on development over 72 h
after UV irradiation. Embryos were untreated or exposed to UV radiation
(4 min) with or without caffeine and/or KU55933 A) The proportion of
embryos that achieved the 4-cell, 8-cell, and blastocyst stages (landmarks)
after 24, 48, and 72 h, respectively, of culture. B) After 72 h, the resultant
embryos were fixed and stained for cell number and TUNEL. The results
are presented as the mean 6 SEM of at least 30 embryos.
SENSING DNA DAMAGE IN EMBRYOS
FIG. 10. The effect of ATM and ATR inhibitors on development over 72 h
after cisplatin treatment. Embryos were untreated or treated with cisplatin
(12 lM) with or without caffeine and/or KU55933. A) The proportion of
embryos that achieved the 4-cell, 8-cell, and blastocyst stages (landmarks)
after 24, 48, and 72 h, respectively, of culture. B) After 72 h, the resultant
embryos were fixed and stained for cell number and TUNEL. The results
are presented as the mean 6 SEM of at least 30 embryos.
irradiation also caused the nuclear accumulation of activated
ATM (pATM) in 2-cell embryos. Yet, the failure of KU55933
to ameliorate the effects of exposure to UV radiation shows
that the activation of ATM is not sufficient to account for the
developmental block in response to UV irradiation in the 2-cell
embryo. ATR canonically activates checkpoint kinase 1
homolog (Schizosaccharomyces pombe; formerly known as
CHK1) [7], which among other actions blocks cell division
cycle 25 homologs B and C (CDC25B/C), which in turn results
in the failure of cyclin-dependent kinase 1 (CDK1; formerly
known as CDC2) activation. The absence of active CDK1
causes a block at the G2-M checkpoint, and this potential
effector pathway warrants further investigation.
The cisplatin-induced 4-cell checkpoint required the actions
of a KU55933-sensitive ATM. In other cellular systems,
cisplatin was found to preferentially activate ATR rather than
ATM [10]. It is also noteworthy that in somatic cells, the
effects of cisplatin-induced activation of the ATR pathway
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without the acute onset of apoptosis. The presence and nuclear
localization of both ATM and ATR was demonstrated in the
cleavage stages of development. ATM, but not ATR, persisted
at a high level in the blastocyst. The paucity of ATR staining in
blastocysts argues that a future research priority is to assess the
capacity of blastocysts to mount an adequate response to
single-strand DNA breaks (the DNA damage most implicated
in ATR sensing). The TRP53 independence of the response is
consistent with the known latency of TRP53 expression in the
early embryo [35, 37] and the adverse developmental
consequences of breaching this latency [38].
Two different forms of genotoxic insult caused different
responses in the 2-cell embryo. UV irradiation is expected to
induce predominantly single-strand DNA breaks, and this
treatment caused a stable G2-M arrest without the induction of
apoptosis in the 2-cell embryo. Cisplatin treatment typically
causes DNA strand cross-links and DNA-protein cross-linking.
This treatment did not prevent 2-cell embryos from progressing
through mitosis, but it then caused activation of a stable G1-S
checkpoint in the next cell cycle (4-cell stage). GammaH2AFX is a highly sensitive marker of DNA damage [4, 5]. Its
phosphorylation by the PIKK enzymes is most active in
response to strand breaks, yet the reaction can also be activated
by a range of genotoxic stresses, such as the cross-linking
events most likely catalyzed by cisplatin [4, 5]. The more
intense staining of gamma-H2AFX in UV-treated embryos is
consistent with an accumulation of strand breaks after this
treatment. The less intense staining of cisplatin-treated embryos
may not necessarily reflect a lower degree of damage but,
rather, a difference in the sensitivity of this marker to different
forms of damage. The prevention of gamma-H2AFX accumulation by caffeine or KU55933 confirms the relative role of
these two PIKKs in sensing DNA damage in the 2-cell embryo.
This result shows that these forms of genotoxic stress both
caused DNA damage and activated PIKK activity in the 2-cell
embryo. The persistence of the gamma-H2AFX 4 h after
treatment shows that over this time frame, the DNA damage
was not resolved.
Caffeine blocks both ATM and ATR, whereas KU55933 is
a highly selective inhibitor of ATM. The capacity of caffeine,
but not KU55933, to reverse the G2-M block and gammaH2AFX accumulation induced by UV irradiation shows that
these responses were primarily sensed by ATR. By contrast,
both caffeine and KU55933 were equally effective at reversing
the cell-cycle block and gamma-H2AFX accumulation induced
by cisplatin treatment, yet no additive effect of these two
inhibitors was observed. This result argues for an important
role of ATM in mediating the G1-S block. The present study
showed that inhibiting phosphatidylinositol-3-kinase did not
reverse the checkpoints induced by genotoxic stress but, rather,
exacerbated the effects of cisplatin. Phosphatidylinositol-3kinase mediates the actions of survival factors in the
preimplantation embryo [39], and such signaling can partially
mitigate the effects of genotoxic stress in somatic cells [40].
It has long been recognized that functionally, considerable
overlap exists in the actions of ATM and ATR, yet the
embryonic lethality of Atr/ [13], but viability of Atm/ [14],
embryos shows that nonredundant and nonoverlapping roles
for each kinase exist in the early embryo. Our results show the
UV irradiation-induced checkpoint in 2-cell embryos was an
ATR-dependent, ATM-independent G2-M block. This result is
interesting, because studies in somatic cells implicate overlapping roles of ATR and ATM in UV radiation-mediated cellcycle block [41]. Autophosphorylation of ATM can occur
downstream of ATR following UV irradiation in some cells
[42]. This is consistent with our demonstration that UV
533
534
MU ET AL.
The results show that under the conditions of extensive or
persistent DNA damage, the demise of the embryo is the
ultimate method of protecting genomic integrity.
ACKNOWLEDGMENT
We thank Dr. Graeme Smith, KuDOS Pharmaceuticals Ltd., for the
kind gift of KU55933.
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