Pharmacological inhibition of EZH2 disrupts the female germline epigenome
Prokopuk et al. Clinical Epigenetics
Pharmacological inhibition of EZH2 disrupts the female germline epigenome
Lexie Prokopuk 0 1
Kirsten Hogg 0 1
Patrick S. Western 0 1
0 Department of Molecular and Translational Science, Monash University , Clayton, Victoria 3168 , Australia
1 Centre for Reproductive Health, Hudson Institute of Medical Research , Clayton, Victoria 3168 , Australia
Background: Recently discovered drugs that target epigenetic modifying complexes are providing new treatment options for a range of cancers that affect patients of reproductive age. Although these drugs provide new therapies, it is likely that they will also affect epigenetic programming in sperm and oocytes. A promising target is Enhancer of Zeste 2 (EZH2), which establishes the essential epigenetic modification, H3K27me3, during development. Results: In this study, we demonstrate that inhibition of EZH1/2 with the clinically relevant drug, tazemetostat, severely depletes H3K27me3 in growing oocytes of adult female mice. Moreover, EZH2 inhibition depleted H3K27me3 in primary oocytes and in fetal oocytes undergoing epigenetic reprogramming. Surprisingly, once depleted, H3K27me3 failed to recover in growing oocytes or in fetal oocytes. Conclusion: Together, these data demonstrate that drugs targeting EZH2 significantly affect the germline epigenome and, based on genetic models with oocyte-specific loss of EZH2 function, are likely to affect outcomes in offspring.
Germline; Oocyte; Pharmacology; Epigenetic; PRC2; H3K27me3; Inheritance
Although many studies indicate that epigenetic changes
in gametes can influence development and health in
offspring, the mechanisms are poorly understood
(reviewed in [
]). Despite this, new pharmaceuticals
that specifically target epigenetic modifying enzymes are
currently being developed for the treatment of cancer
and other diseases, and these drugs are being
administered to patients of reproductive age. While these
therapies are likely to offer significant improvements in
cancer treatment, their potential impacts on the
germline epigenome, and on offspring, remain unknown.
Altered epigenetic states can result in disease, and
more than 50% of cancers harbour mutations in
chromatin-associated proteins. This has driven recent
interest in the development of drugs that specifically
target epigenetic modifying complexes with the aim of
providing new cancer treatments [
]. A prominent
target is Enhancer of Zeste 2 (EZH2), which is
commonly subject to gain of function mutations or
overexpression in a range of tumours [
catalyses methylation of lysine 27 on histone 3
(H3K27me3), an epigenetic modification that is critical
for repressing developmental genes during
embryogenesis. EZH2 is the catalytic component of polycomb
repressive complex 2 (PRC2), which also contains
embryonic ectoderm development (EED) and
Suppressor of Zeste 12 (SUZ12). Inactivation of any one of
these three protein subunits severely compromises the
enzymatic activity of PRC2 and results in the loss of
Recently developed pharmaceuticals that inhibit
EZH2 include EPZ-6438, also known as the
proprietary product, tazemetostat [
]. Tazemetostat is
currently in stage I/II clinical trials in patients for the
treatment of multiple cancers, including lymphomas,
mesothelioma, myelomas, solid tumours, and
malignant rhabdoid tumours of the kidney and ovary.
Patient cohorts include children and adults of
reproductive age (12 current NIH Clinical Trials 02860286,
02875548, 03028103, 03010982, 02601937, 01897571,
02601950, 03009344, 03213665, 02220842, 03217253,
03155620). Three studies include the recruitment of
children, aged 6 months to 21 years (NIH Clinical
Trials 02601937, 03213665, 03155620).
As H3K27me3 is enriched in developing germ cells
during epigenetic reprogramming and in mature
], it is likely that drugs targeting EZH2
will disrupt the sperm and oocyte epigenome. Current
guidelines for tazemetostat typically recommend
avoiding pregnancy for 30 days post-treatment (NIH
Clinical Trials Register), significantly less time than
the ~ 350 days required for human oocyte growth and
]. Other studies do not stipulate any
form of contraception after treatment with
tazemetostat. As these drugs are systemic and the germline will
be exposed in patients undergoing treatment,
determining whether current treatments affect the germline
epigenome is of significant importance for developing
informed therapeutic guidelines and providing advice
to patients with regard to post-treatment reproductive
management. Moreover, although the use of these
drugs during pregnancy is contra-indicated, there is
little empirical data to support this advice. This is
important, as recent studies have provided examples
of drugs taken during pregnancy that have disrupted
fetal or ovarian development [
]. Of particular
note, the epigenetic modifying drug valproic acid
(Epilim) has been extensively used for the treatment of
epilepsy, bipolar mania and migraine prophylaxis, but
recent evidence of affects in children of women who
took the drug during pregnancy have raised concerns
over its clinical management . While the effects of
these, and other drugs, on the fetus have been of
central concern, their potential impacts on the
germline epigenome have been largely overlooked. These
examples underline the need to understand the
germline effects of epigenetic modifying drugs to allow
appropriate development of informed clinical guidelines.
The developing germline plays a central role in
regulating epigenetic information transmitted by gametes
to offspring. To ensure transmission of appropriate
epigenetic information to offspring, epigenetic
information is reset during germline development [
]. During this period of germline epigenetic
reprogramming, H3K27me3 is highly enriched in primordial
germ cells and in both male and female germ cells
compared to somatic cells of the developing gonad
13, 14, 21, 25
]. Moreover, H3K27me3 undergoes
EZH2-dependent reorganisation while DNA
methylation levels are at their lowest, indicating important
roles for EZH2 and H3K27me3 for setting the
germline epigenome [
]. Consistent with this,
genomewide mapping demonstrated H3K27me3 enrichment
at developmental genes during epigenetic
programming in the mouse fetal germline and in mature sperm
]. Given that many of these genes are not
expressed during germline development, it has been
speculated that these epigenetic modifications are
established to regulate outcomes in offspring [
This concept is consistent with observations that
deletion of Ezh2 in growing oocytes affects
preimplantation development and birth weight in offspring [
Females are born with a finite pool of primordial
follicles, which provide the lifelong oocyte reserve
that supports fertility. Primordial follicles are
continually activated during reproductive life, resulting in
the formation of primary follicles and oocyte growth
]. As activated follicles grow from primary to
pre-ovulatory stages, epigenetic modifications and
maternal factors are established in the oocyte that are
important for offspring development [
maternal factors are produced through co-ordinate
regulation of gene expression in the growing oocyte, a
process that is considered to depend on appropriate
epigenetic regulation throughout this period. An
important example is provided by EZH2, which
performs an essential role in the oocyte that profoundly
affects offspring development and growth [
Here, we show that the EZH1/2-specific inhibitor,
tazemetostat, substantially alters H3K27me3 in the
developing female germline and maturing oocytes.
Tazemetostat severely reduced H3K27me3 in growing
oocytes in vivo, and H3K27me3 was not recovered
after treatment withdrawal for half of the mouse
oocyte growth period. Moreover, tazemetostat
significantly reduced H3K27me3 in developing germ cells
and in primary oocytes. Together, these data
demonstrate that systemic pharmacological inhibition of
EZH2 affects H3K27me3 at all stages of oocyte
development and growth. While tazemetostat and similar
drugs that target epigenetic pathways offer important
therapeutic options to patients, our findings highlight
an urgent need for a greater understanding of their
impacts on the germline and the reassessment of
clinical guidelines to ensure safe management in
patients who may wish to conceive after treatment.
PRC2 and H3K27me3 are enriched in growing oocytes of adult female mice
To determine the potential for PRC2 to catalyse
H3K27me3 in growing oocytes of adult females, we
assessed EZH2, EED, SUZ12 and H3K27me3 enrichment
in postnatal day (PND) 24 ovaries using
immunofluorescent staining. EED, EZH2 and SUZ12 were all detected in
the nucleus of growing oocytes in PND24 ovaries (Fig. 1a–
c), coinciding with H3K27me3 enrichment (Fig. 1d).
EZH2 inhibition potently depletes H3K27me3 in growing oocytes
As tazemetostat and other similar drugs are systemic,
treatment of females of reproductive age is expected to
result in oocyte exposure to these EZH2-inhibiting
drugs. To determine whether pharmacological inhibition
of EZH1/2 affected H3K27me3 enrichment in the adult
female germline, we treated adult females with
tazemetostat. This is an especially important question as
females are born with a finite supply of oocytes and
disruption of oocyte epigenetic state may affect offspring
outcomes. Previous in vivo preclinical studies of
tazemetostat have shown that 220 mg/kg administered twice
daily is required to induce effective tumour regression in
mouse models carrying grafted malignant rhabdoid cells
]. To investigate whether this dose caused depletion of
H3K27me3 in growing oocytes in vivo, 7-week-old adult
female mice were injected subcutaneously with either
220 mg/kg tazemetostat (n = 3) or sham control (0.5%
NaCMC, 0.1% Tween80; n = 3) for 10 days. Notably, we
injected mice once a day, thereby delivering less drug
than in the preclinical cancer model, in which the drug
was administered twice daily at 220 mg/kg by oral
]. Immunofluorescent analysis of H3K27me3
enrichment in secondary follicles revealed that H3K27me3
was depleted by 84% in the oocytes of females injected
with tazemetostat compared to sham injected controls
(P < 0.0001, Student’s t test; Fig. 2). This demonstrated
potent disruption of EZH1/2 function in growing
oocytes of adult females.
Adult oocytes failed to recover H2K27me3 after withdrawal of EZH2 inhibition
To assess whether growing oocytes could recover after
the extensive loss of H3K27me3 resulting from
tazemetostat treatment, we carried out an in vivo recovery
experiment. One cohort of 7-week-old adult female mice
were injected subcutaneously with either 220 mg/kg
tazemetostat (n = 3) or sham control (n = 3) once a day
for 10 days and collected. An additional cohort was
injected with either 220 mg/kg tazemetostat (n = 3) or
sham control (n = 3) once a day for 10 days and then
allowed to recover for a further 10 days with no
treatment. After treatment or treatment/recovery regimes,
immunofluorescence was performed on ovaries and
secondary follicles to assess H3K27me3 enrichment in
oocytes. Consistent with the initial in vivo depletion
experiment in growing oocytes, H3K27me3 was robustly
depleted in growing oocytes after 10 days of EZH2
inhibition compared to sham injected controls (Fig. 3).
However, remarkably, we did not observe any
H3K27me3 rescue in oocytes allowed to recover for
10 days (P < 0.0001, one-way ANOVA with post hoc
Tukey’s multiple comparisons test; Fig. 3). Instead, after
the 10-day recovery period, H3K27me3 was depleted by
a further 50% in oocytes.
Together, these data demonstrate that systemic
tazemetostat treatment at the minimal preclinical dose that
effectively controls tumour growth in mice also results
in severe depletion of H3K27me3 in maturing oocytes.
Moreover, our data indicate that once depleted,
H3K27me3 does not recover in a window corresponding
to half the growth period for mouse oocytes.
EZH2 inhibition depletes H3K27me3 in female fetal germ cells undergoing epigenetic reprogramming
Although use of tazemetostat during pregnancy is
contra-indicated, empirical evidence for this position is
lacking. Previous studies provide examples where similar
drugs have been used in pregnancy [
], raising the
possibility that drugs targeting epigenetic modifying
enzymes may be similarly used in the future. Since
exposure of the fetus would also expose the germline of
future offspring to the drug, we determined the ability of
EZH2 inhibitors to alter H3K27me3 in developing
oocytes. To achieve this, we performed a series of
experiments on germ cells using ex vivo cultured fetal gonads
treated with tazemetostat. Germ cells undergo
significant epigenetic reprogramming during fetal life that
results in removal of existing epigenetic modifications and
the establishment of new sex-specific germline
epigenomes. This developmental period in germ cells is
therefore considered susceptible to modification by agents
that disrupt epigenetic programming and may alter the
germline epigenome in mature gametes. As H3K27me3
is significantly reorganised in germ cells undergoing
epigenetic reprogramming [
], we initially determined the
potential for EZH2 inhibition to affect H3K27me3
enrichment in fetal germ cells during this period.
Female Swiss mice were time-mated to Oct4GFP
transgenic males (129T2svJ) to allow stage-specific
collection of embryonic gonads with Oct4GFP expressed in
germ cells but not in somatic cells [
14, 34, 35
treated E12.5 XX (female) fetal gonads grown in ex vivo
organ culture with tazemetostat or vehicle control
(DMSO) for 24–72 h. At the end of the culture period,
the gonads were dissociated and H3K27me3 levels
measured in individual germ and somatic cells using flow
]; Additional file 1: Figure S1). Robust
depletion of H3K27me3 was observed in germ cells of
XX E12.5 gonads treated for 72 h with 100 nM–5 μM
tazemetostat (Fig. 4a, b; Additional file 1: Figure S1C).
Treatment with 100 nM tazemetostat resulted in
significant depletion of H3K27me3, although slightly less
efficiently than that at higher doses. Tazemetostat was very
well tolerated in culture, with even the highest doses
having no noticeable effect on gonad growth or the
proportions of germ cells at the end of the culture period
(Fig. 4c, Additional file 1: Figures. S1C, S2A-B). This is
consistent with our previous observations that cell
viability was unaffected by a similar EZH2-inhibiting drug,
GSK126, in this culture system .
A major milestone for female germ cell development
is their entry into meiosis, which occurs between E13.5
and E15.5 [
]. To assess the impact of EZH2
inhibitors on female germ cell development, E12.5 gonads
were treated for 72 h allowing sufficient time for germ
cells to enter meiosis. Cell cycle state was assessed by
incorporation of EdU during the final 2 h of gonad culture
and staining germ cells with propidium iodide to
determine individual cell DNA content using flow cytometry
]; Additional file 1: Figure S2). XX germ cells
arrested in meiosis with normal timing and in normal
numbers in E12.5 gonads treated for 72 h with
tazemetostat, indicating that EZH2 inhibition and loss of
H3K27me3 did not prevent E12.5 germ cells from
reaching this key developmental milestone (Fig. 4c). Similarly,
somatic cells proliferated at a normal rate in XX gonads
indicating that EZH2 inhibition did not impact gonad
growth during the treatment period, despite loss of
H3K27me3 (Additional file 1: Figure S2B).
H3K27me3 failed to recover in female fetal germ cells following EZH2 inhibition
Collectively, these data demonstrate that tazemetostat
potently depletes H3K27me3 in XX fetal germ cells as
they undergo epigenetic reprogramming and entry into
meiosis. However, all PRC2 components have been
detected in fetal XX germ cells at E11.5 and E12.5 [
indicating that germ cells may be able to recover
H3K27me3 after drug exposure. To determine whether
H3K27me3 could be re-established after depletion in
fetal germ cells undergoing epigenetic reprogramming,
we cultured E11.5 gonads for 24 h with DMSO,
100 nM tazemetostat or 1 μM tazemetostat; removed
the drug by washing three times with drug-free culture
media; and continued culture for an additional 48 h in
the presence of 100 nM, 1 μM tazemetostat or DMSO
(Fig. 5a). To ensure collection of control and
experimental data at the end of the initial drug depletion
period and after the recovery period, treatments were
carried out on four treatment groups: (1) treatment
with DMSO for 24 h followed by treatment with
DMSO for 48 h, which provided a negative control to
determine the H3K27me3 levels after 72 h culture
without the drug; (2) treatment with tazemetostat for
24 h only, which determined the reduction of
H3K27me3 after 24 h of treatment as a pre-recovery
control; (3) treatment with tazemetostat for 24 h,
followed by treatment with DMSO for 48 h, which
determined the extent to which H3K27me3 recovered
after tazemetostat exposure; and (4) treatment with
tazemetostat for 24 h followed by treatment with
tazemetostat for 48 h, providing a positive control to
determine the H3K27me3 depletion after 72 h of drug
treatment (Fig. 5a). Flow cytometry was used to
determine the levels of H3K27me3 in the germ cells after
each treatment. Germ cell H3K27me3 levels were
depleted to a lesser extent at 100 nM, compared to
1 μM tazemetostat at 24 h, and these levels were
maintained without restoration across the 48-h recovery
period (Fig. 5b). At the higher dose (1 μM), germ cell
H3K27me3 levels were depleted by 54% after 24 h and
continued to decline over the 48-h recovery period
(DMSO; P < 0.01, one-way ANOVA post hoc Tukey’s
multiple comparisons test; Fig. 5b). Together, these
data demonstrate that EZH2 inhibition rapidly, and
potently, depleted germ cells of H3K27me3 and that
recovery of H3K27me3 in fetal germ cells was
dependent on tazemetostat dose.
EZH2 inhibition potently depletes H3K27me3 in primary oocytes
As tazemetostat is currently in clinical trials for
treatment of cancer in patients aged 6 months to
18 years, it is possible that oocytes in the primordial
follicle reserve will be affected by the drug in these
patients. This is of particular relevance as the
primordial follicle reserve provides all oocytes for
the female reproductive life. To assess whether
H3K27me3 was depleted in primary oocytes after
birth, we cultured E18.5 mouse ovaries for 7 days
with either vehicle control (DMSO) or 5 μM TAZ
(Fig. 6). Primordial oocytes of E18.5 ovaries treated
with 5 μM TAZ were significantly less enriched for
H3K27me3 compared to controls (Fig. 6).
Drugs that target epigenetic modifying complexes
hold great promise in oncology and potentially for
treatment of a range of other diseases and disorders.
These drugs are therefore likely to make a significant
contribution to the treatment of human disease and
provide substantial improvements in health outcomes.
From this point of view, drugs targeting epigenetic
modifiers are highly desirable and may be of
significant benefit. However, these drugs also have the
potential to affect epigenetic programming in the
germline, with significant implications for health and
developmental outcomes in offspring. There is
therefore a need to assess the germline impacts of drugs
that inhibit epigenetic modifying proteins to enable
the informed clinical management of these drugs in
patients of reproductive age.
In this study, we investigated the potential for drugs
that inhibit EZH2 to alter H3K27me3 in growing
oocytes, primary oocytes and the developing female
germline. Significantly, tazemetostat prevented H3K27me3
enrichment in growing oocytes of adult females in vivo,
and this effect was relatively stable as H3K27me3 did
not recover after 10 days of drug withdrawal. In
addition, EZH2 inhibition reduced H3K27me3 in the
primary oocyte pool, indicating that the lifelong
primordial follicle reserve is affected by these drugs. Finally,
tazemetostat severely depleted H3K27me3 in
developing oocytes undergoing epigenetic reprogramming and
established that once depleted, H3K27me3 did not
recover in these cells within the window analysed.
Combined, these findings demonstrate that EZH2
inhibitors potently reduce H3K27me3 at all stages of
female germline development.
Previous work, and our own experiments, using a
mouse genetic model in which EZH2 was specifically
deleted in growing oocytes demonstrated that
maternal H3K27me3 was lost in the oocyte. Moreover,
offspring had abnormal preimplantation development
and were born with substantial growth restriction
], Prokopuk et al. unpublished data). Growth
restriction is a risk factor for lifelong health impacts
including metabolic disease [
]. Moreover, de
novo germline mutations in human EZH2 result in
Weaver syndrome, characterised by growth and
skeletal defects and intellectual disability [
these combined studies indicate that altered H3K27me3
in growing oocytes affects outcomes in offspring, it is
likely that maternal exposure to EZH2 inhibitors and
similar epigenetic modifying drugs will have the potential to
impact on offspring growth and development. Although
the specific impacts of EZH2 inhibitors on oocytes and
offspring are yet to be determined, it is clear that much
greater understanding of the potential germline impacts of
these drugs in patients of reproductive age is required.
Understanding the impacts of drugs on both the
parental germline epigenome and on offspring is of
significant importance as a range of studies have shown that
environmental impacts in the parent can affect
outcomes in offspring through epigenetic mechanisms that
are, by and large, poorly understood [
Moreover, although studies of other epigenetic modifying
compounds, such as valproic acid, have demonstrated
direct effects on the unborn fetus and on children of
mothers treated during pregnancy [
], these studies
have not addressed the potential intergenerational or
transgenerational effects of drugs that target other
epigenetic mechanisms in the germline.
In this study, tazemetostat caused severe depletion of
H3K27me3 from growing oocytes after only 10 days of
treatment and H3K27me3 had not recovered 10 days
after treatment of the females had been terminated.
This recovery period is equivalent to approximately
half of the period required for a mouse oocyte to reach
maturity from the primordial follicle stage [
human oocyte takes approximately 350 days to grow
and mature after recruitment from the primordial
follicle pool [
]. However, the current clinical
guidelines recommend not to become pregnant for at
least 30 days after treatment has been terminated.
Consequently, after the termination of treatment,
growing oocytes that were exposed to tazemetostat will
remain for almost 11 months, and offspring conceived
during this period may be also affected. Our findings
in mice indicate that even after drug withdrawal there
is a risk that H3K27me3 levels in oocytes will be
affected for a significant period of oocyte growth and
maturation. Based on oocyte growth and maturation
periods of 21 days for mouse  and 350 days for
], we estimate that a similar withdrawal
period in humans would require at least a full cycle of
oocyte growth to allow the clearance of potentially
affected oocytes. This is significantly longer than the
current 30-day period recommended in most current
tazemetostat clinical trials before patients should
consider becoming pregnant. Therefore, it is possible that
even after tazemetostat treatment has ceased in human
patients, there will be a protracted period during which
the oocyte epigenome will be affected and may have
consequences for offspring.
Our data demonstrate that in growing oocytes,
tazemetostat potently depleted H3K27me3 and H3K27me3
levels continued to decline until at least 10 days after
treatment was withdrawn from the mice. Despite this,
all core components of PRC2 were detected in growing
oocytes, and H3K27me3 was enriched during oocyte
growth demonstrating that PRC2 is normally active in
growing oocytes. Similarly, H3K27me3 was not
recovered in female fetal germ cells after tazemetostat
treatment of fetal ovaries. We have previously shown that
Ezh2 is transcribed, EZH2 protein is present and EZH2
is required for redistributing H3K27me3 in XX germ
cells during epigenetic reprogramming, demonstrating
a functional role for EZH2 during this developmental
]. A possible explanation for the lack of
H3K27me3 recovery after drug withdrawal is that once
inhibited, EZH2 activity cannot recover, possibly
because the drug irreversibly blocks and inhibits activity.
This observation also implies that new EZH2 was either
not synthesised in oocytes or the drug continued to
block its activity throughout the withdrawal period.
Retention of drug activity is unlikely as a previous study
demonstrated an in vivo half-life for tazemetostat of
only 4 h in mice [
], indicating that greater than 90%
drug clearance in the first 24 h after drug withdrawal.
Similarly, tazemetostat was washed out of the fetal
ovarian cultures by changing the media for drug-free
media and washing the wells multiple times. It
therefore appears more likely that once blocked in either
growing or fetal oocytes, EZH2 activity cannot readily
recover. This may affect all stages of oocyte
development and growth as EZH2 inhibition also compromised
H3K27me3 in primary oocytes. It is therefore possible
that tazemetostat and similar drugs may have persistent
effects on all oocytes, including those in the primordial
follicle pool. This is an important consideration when
female children or patients of reproductive age are
treated with the drug and is of direct relevance in
current clinical trials involving patients aged 6 months
to 18 years.
The current clinical guidelines clearly state that use
of EZH2-inhibiting drugs is contra-indicated in
patients during pregnancy. This position is presumably
based on the direct impacts of EZH2 on somatic
development in the fetus, but empirical evidence for this
position is currently lacking. Recent reports of adverse
impacts of the anti-epilepsy drug Epilim (valproic
] have highlighted the importance of a direct
evidence-based position in developing stringent
guidelines for these drugs in pregnancy. In addition to
teratogenic impacts on somatic development in the
fetus, in utero exposure to valproic acid has been
associated with long-term effects on children, including
decreased learning ability, autism and behavioural
]. Similar outcomes in the fetus are likely
for other drugs that inhibit epigenetic modifying
complexes. However, it is also possible that drugs targeting
epigenetic modifiers will cause intergenerational or
transgenerational epigenetic effects in the patients’
children or grandchildren. Using gonad culture and in
vivo treatments, respectively, we have clearly
demonstrated that germline exposure to EZH2
inhibiting drugs severely affects epigenetic programming in
the fetal and adult oocytes, highlighting the potent
ability of these drugs to disrupt epigenetic
programming in the germline and a need to further understand
the impacts of these drugs on the germline.
Although new drugs that target epigenetic modifying
enzymes offer important new therapeutic options for
patients, it is clear that these drugs also have the
capacity to significantly alter the germline epigenome
and that these effects may persist for a significant
period of time. One obvious way to avoid such effects
in patients of reproductive age undergoing treatment
with drugs that target epigenetic modifiers would be to
employ germline preservation methods, such as sperm
and oocyte freezing. However, as gamete preservation
techniques are imperfect and not available to all
patients, it is also clear that significantly more work is
required to determine the molecular and phenotypic
effects of EZH2 inhibitors in the germline and their
potential to adversely affect subsequent children of
Significantly, the potential impacts on the germline of
drugs that target epigenetic modifying enzymes appear
to be largely “off the radar” when the clinical safety
of these drugs is being considered—most attention is
reserved for the potential teratogenicity of the drugs
rather than the potential germline impacts and
subsequent effects on offspring. This study highlights an
urgent need to determine risks of these and similar
treatments for oogenesis and offspring outcomes.
Mouse strains, animal housing, breeding and ethics
Mice were housed at Monash Medical Centre Animal
Facility using a 12-h light-dark cycle. Food and water
were available ad libitum and room temperature was
21–23 °C with controlled humidity. Embryos were
collected from Oct4 [Pou5f1]-eGFP on 129T2svJ
background males crossed with Swiss females.
Females were checked for vaginal plugs daily and
detection of a plug was noted as E0.5. Embryos E12.5 or
older were sexed by the presence (male) or absence
(female) of testis cords in gonads. E11.5 embryos
were genetically sexed using PCR as described [
Gonad collection and organ culture
All culture reagents were purchased from Life
Technologies unless otherwise stated. Embryos were
collected from Swiss females mated to 129T2svJ
Oct4eGFP transgenic males. Gonad plus mesonephros was
cultured on 30 mm organotypic cell culture inserts
(Merck Millipore; PICM03050) in 1200 μL culture
media (250 μM sodium pyruvate, 15 mM Hepes, 1×
non-essential amino acids) (Life Technologies, 11140),
1 mg/ml N-acetylcysteine (Sigma, A9165), 55 μM
βmercaptoethanol (Life Technologies, 21985) and 10%
FCS in DMEM/F12 with Glutamax (Life Technologies,
10565) containing either DMSO (vehicle control) or
tazemetostat (EPZ6438; SelleckChem, S7128). Culture
media also contained 1× penicillin/streptomycin (Life
Technologies, 15070). Gonads were randomly
allocated to each culture treatment condition and
cultured for 48 h in 37 °C/5% CO2 conditions. Culture
media was refreshed daily. Gonads were processed for
flow cytometry, FACS and immunofluorescence (IF).
Gonad collection, dissociation, fixation, antibody
staining and flow cytometry were performed as described
]. Gonad samples stained with rabbit IgG
control antibody were used as negative controls to set
flow cytometry gates for H3K27me3 intensity and
mesonephros or limb samples were used as a germ cell
negative control to gate for eGFP. Cell proliferation
was assessed by dosing cultured gonads with 20 μM
5ethynyl-2 deoxyuridine (EdU) for the final 2 hours of
culture prior to tissue collection, dissociation and
fixation. Cell cycle analysis was carried out as
] with germ cells identified by their
expression of mouse vasa homologue (MVH). Cells
were stained with 20 μg/ml propidium iodide, allowing
quantitation of cellular DNA content. Proliferation was
measured by gating EdU-positive cells against
propidium iodide to identify cells actively in S-phase. Cells
in G1 and G2/M were identified by their DNA content
and the absence of S-phase activity. All flow cytometry
was performed on a FACS Canto instrument and data
were analysed in FlowJo and Graphpad Prism. For all
analyses, at least three biological replicates were
analysed and statistical significance was determined
using one-way ANOVA with Tukey’s multiple
comparison or t test as appropriate. P values < 0.05 were
Tissue fixation and embedding
Gonads were fixed in 4% paraformaldehyde (PFA) in
PBS for immunofluorescence (IF) overnight at 4 °C.
Samples were washed twice in PBS and left in 30%
sucrose in PBS overnight at 4 °C. Samples were then
placed in disposable cryostat moulds (Sakura Finetek,
4565) filled with OCT (Sakura Finetek, 4583) and
frozen in dry ice and stored at − 80 °C.
Immunofluorescence was carried out as previously
]. Eight micron sections were cut from
OCT-embedded gonads fixed in 4% PFA, mounted on
Superfrost Plus slides and dried for 5 min before
immersing in 1× PBS. Sections were then permeabilized
by incubation in 1% Triton X 100 (Sigma, T8787) in
PBS for 10 min at room temperature (RT). Slides
were washed three times for 5 min each in PBS.
Sections were blocked in PBS containing 5% BSA (Sigma,
A9647) and 10% donkey serum (Sigma, D9663) and
incubated for 45 min at RT. Blocking solution was
replaced by PBS containing 1% BSA and appropriately
diluted primary antibodies (rabbit anti-H3K27me3
1:400, Cell Signaling Technology #C36B11; sheep
anti-EED 1:100, R&D #AF5827; rabbit anti-EZH2
1:400, Cell Signaling Technology #D2C9; rabbit
antiSUZ12 1:100, Cell Signaling Technology #D39F6) and
incubated for 1 h at RT. Slides were washed three
times for 5 min in PBS and secondary antibodies
diluted at 1:300 in 1% BSA in 1× PBS (Alexa Fluor,
Life Technologies, Donkey anti Sheep 647 #A21447;
Donkey anti Rabbit 594 #A21207). Secondary
antibody incubation was carried out in a dark
humidity chamber for 1 h at RT. Slides were washed three
times in PBS (5 min each wash) and mounted in
ProLong Gold® containing DAPI (Life Technologies,
#P36931) and left in a dark box overnight to dry. For
control slides, only a secondary antibody was applied.
Confocal images were taken as single optical sections
using a Nikon® C1 inverted confocal microscope. All
pictures were taken at ×80, using a ×40 oil immersion
lens. H3K27me3 mean intensity was measured in at
least three biological replicates using ImageJ ROI
manager to calculate the mean intensity for H3K27me3,
and unpaired t tests were used to statistically analyse
groups, with P ≤ 0.05 considered significant.
In vivo injection of tazemetostat
Seven-week old C57Bl/6J females were
subcutaneously injected with 220 mg/kg/day tazemetostat. Mice
were weighed daily to ensure correct dosage. Dosage
regime involved injecting mice once a day for 9
consecutive days and collecting females on the 10th
day. Females were culled via cervical dislocation and
ovaries were collected and processed for IF.
Additional file 1: Figure S1. Representative flow cytometric scatter plots
demonstrating separation, gating and analysis of germ and somatic cells
derived from gonads cultured with DMSO or tazemetostat. A. Representative
flow cytometric plots of cells analysed from female cultured gonads.
Oct4GFPpositive germ cells and Oct4GFP-negative somatic cells were separated and
gated based on their respective positive or negative expression of Oct4GFP
(left plot). Mean H3K27me3 staining intensity of individual germ (middle plot)
and somatic cells (right plot) was quantified using FlowJo. B. Representative
flow cytometric plots of cells analysed from limb control cells, which do not
express Oct4GFP. This and similar controls facilitated the gating of
Oct4GFPpositive and Oct4GFP-negative populations shown in A and B. C. Average
H3K27me3 staining intensity in germ cells of E12.5 XX gonads cultured with
DMSO, 1 μM and 5 μM tazemetostat for 72 h. Figure S2. Representative flow
cytometric scatter plots demonstrating separation, gating and cell cycle
analysis of germ and somatic cells derived from female E12.5 gonads cultured
with DMSO or tazemetostat for 72 h. A. Oct4GFP positive germ cells and
Oct4GFP negative somatic cells were separated and gated based on their
respective positive or negative expression of Oct4GFP (left plots). The middle
and right-hand plots represent cell cycle analyses based on incorporation of
EdU and propidium iodide (PI) staining quantified using FlowJo (germ cell
cycle-middle plots; somatic cell cycle-right plots). B. Average H3K27me3
staining intensity (left) and cell cycle state (right) in somatic cells from female
E12.5 gonads cultured with DMSO or tazemetostat. ****P < 0.0001, one-way
ANOVA plus post hoc Tukey’s multiple comparisons test; n = 3–5 biological
replicates. (PDF 599 kb)
E: Embryonic day; EED: Embryonic ectoderm development; eGFP: Enhanced
GFP; EPZ: EZH1/2 inhibitor, TAZ, tazemetostat/EPZ-6438; EZH1: Enhancer of
Zeste 1; EZH2: Enhancer of Zeste 2; FACS: Fluorescence-activated cell sorting;
GSK126: EZH1/2 inhibitor, GSK; H3K27me3: Trimethylated lysine 27 on
histone 3; MVH: Mouse vasa homologue (also known as DDX4); Nsd: No
significant difference; PRC2: Polycomb repressive complex 2;
SUZ12: Suppressor of Zeste 12
We thank Monash Animal Research Platform staff for assistance with mouse
care. We thank Jessica Stringer and Karla Hutt for manuscript review and
Ellen Jarred for technical assistance with ovary culture experiments.
This work was supported by National Health and Medical Research Grants
GNT1043939 and GNT1051223 awarded to PSW, funding from the Monash
University Faculty of Medicine, Nursing and Health Sciences funding granted
to PSW and the Victorian Government’s Operational Infrastructure Support
Program. LP is supported by an Australian Postgraduate Award.
Availability of data and materials
All data is available from the corresponding author.
PSW conceived and designed the study, obtained financial support, collected
and analysed the data and drafted the manuscript. LP collected and processed
the samples, optimised and performed experiments, analysed the data and
drafted the manuscript. KH optimised and performed experiments, analysed
data and drafted the manuscript. All authors read and approved the final
K.H and L.P are now located at the Stem Cells and Cancer Division of the
Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia.
No human samples or subjects were used in this study. Animal work was
undertaken in accordance with Monash University Animal Ethics Committee
(AEC) approval MMCA-2015-60.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published
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