Naillat et al. Clin Epigenet (2021) 13:132
https://doi.org/10.1186/s13148-021-01116-3
RESEARCH
Oxygen concentration affects de novo
DNA methylation and transcription in in vitro
cultured oocytes
Florence Naillat1,2*
  , Heba Saadeh1,3
, Joanna Nowacka‑Woszuk1,4
, Lenka Gahurova1,5
, Fatima Santos1
,
Shin‑ichi Tomizawa1,6
 and Gavin Kelsey1,7*
 
Abstract 
Background:  Reproductive biology methods rely on in vitro follicle cultures from mature follicles obtained by
hormonal stimulation for generating metaphase II oocytes to be fertilised and developed into a healthy embryo.
Such techniques are used routinely in both rodent and human species. DNA methylation is a dynamic process that
plays a role in epigenetic regulation of gametogenesis and development. In mammalian oocytes, DNA methylation
establishment regulates gene expression in the embryos. This regulation is particularly important for a class of genes,
imprinted genes, whose expression patterns are crucial for the next generation. The aim of this work was to establish
an in vitro culture system for immature mouse oocytes that will allow manipulation of specific factors for a deeper
analysis of regulatory mechanisms for establishing transcription regulation-associated methylation patterns.
Results:  An in vitro culture system was developed from immature mouse oocytes that were grown to germinal
vesicles (GV) under two different conditions: normoxia (20% oxygen, 20% ­O2) and hypoxia (5% oxygen, 5% ­O2). The
cultured oocytes were sorted based on their sizes. Reduced representative bisulphite sequencing (RRBS) and RNAseq
libraries were generated from cultured and compared to in vivo-grown oocytes. In the in vitro cultured oocytes,
global and CpG-island (CGI) methylation increased gradually along with oocyte growth, and methylation of the
imprinted genes was similar to in vivo-grown oocytes. Transcriptomes of the oocytes grown in normoxia revealed
chromatin reorganisation and enriched expression of female reproductive genes, whereas in the 5% ­O2 condition,
transcripts were biased towards cellular stress responses. To further confirm the results, we developed a functional
assay based on our model for characterising oocyte methylation using drugs that reduce methylation and transcrip‑
tion. When histone methylation and transcription processes were reduced, DNA methylation at CGIs from gene bod‑
ies of grown oocytes presented a lower methylation profile.
Conclusions:  Our observations reveal changes in DNA methylation and transcripts between oocytes cultured in vitro
with different oxygen concentrations and in vivo-grown murine oocytes. Oocytes grown under 20% ­O2 had a higher
correlation with in vivo oocytes for DNA methylation and transcription demonstrating that higher oxygen concentra‑
tion is beneficial for the oocyte maturation in ex vivo culture condition. Our results shed light on epigenetic mecha‑
nisms for the development of oocytes from an immature to GV oocyte in an in vitro culture model.
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Open Access
*Correspondence: Florence.naillat@oulu.fi; gavin.kelsey@babraham.ac.uk
1
Epigenetics Program, Babraham Institute, Cambridge CB22 3AT, UK
2
Diseases Network Research Unit, Faculty of Biochemistry and Molecular
Medicine, Oulu University, Oulu, Finland
Full list of author information is available at the end of the article
Page 2 of 17Naillat et al. Clin Epigenet (2021) 13:132
Introduction
Oocytes contain not only the genetic information, but
also the epigenome, which is dynamically changing
during oocyte growth, and both need to be transmitted
correctly to the next generation. De novo DNA
methylation starts at primary follicles and finishes at
metaphase II oocytes (M2) [1, 2] to produce functional,
mature oocytes which are a prerequisite for proper
embryonic development [3].
DNA is methylated at CpG-dinucleotides, and differentially
methylated CpGs form domain on genomic
DNA which can be either hyper- or hypomethylated.
In the M2 oocytes, methylation establishment has been
associated with transcription units delimiting these
hyper- and hypomethylated domains [3, 4]. As well,
groups of CpGs define CpG islands (CGIs) which are
found throughout the genome. Most of the CGIs are
unmethylated but in specific cell type; for example, in
the germ cells, these CGIs are characterised by either
low or high methylation levels. Some of these CGIs
are associated with imprinted germ line differentially
methylated regions (igDMRs). Accurate methylation
of igDMRs is crucial for the proper embryonic development
and for mono-allelic gene expression during
development. Depending on the sex of the gamete,
methylation of the igDMRs will follow an either paternal
or maternal pattern [5]. When DNA methylation
fails to be established or maintained on the igDMRs,
consequences may be severe for the embryos; embryonic
defects are often seen as a result [6].
Assisted reproduction technology (ART) is a common
technique in human infertility treatments. It is
based on retrieving late stage M2 oocytes, which are
fertilised with sperm in a culture dish to generate an
embryo. It is important to understand the mechanisms
regulating oocyte methylation using in  vitro models.
There is evidence that in in  vitro, culture conditions,
such as oxygen concentration, pH, temperature, and
medium composition, have an effect on oocyte epigenome,
but this connection has not yet been carefully
analysed [7]. The oxygen concentration in the ovaries
varies from 2 to 8% depending on the species [8], and
ART techniques are either carried out at 5% oxygen
or 20% oxygen [9]. Studies of methylation of selected
imprinted genes in children born from ART have been
inconclusive. The data either suggest a change in methylation
at imprinted genes which is correlated with a
higher risk in developing imprinted disorders, or no
correlation is observed between the methylation of
imprinted loci and imprinted diseases [10–17]. Similar
controversial results have been published in relation
to super-ovulation. Super-ovulated oocytes from
mice and humans show that Peg1 and H19 imprinted
genes were differentially methylated in super-ovulated
oocytes compared to normally ovulating females, suggesting
a link between methylation of imprinted genes
and super-ovulation [18], whereas other studies reveal
no effect on the methylation of the imprinted genes by
super-ovulation [19, 20].
Oocytes are an ideal system to study de novo methylation
establishment and its relation to transcription,
because replication does not occur in these cells.
Recent studies have generated mouse oocyte methylomes
to understand the mechanisms behind de novo
methylation establishment [3, 21, 22]. The relation
between methylation and transcription starts to be
unveiled in mouse oocytes [4] highlighting a correlation
between gene bodies hypermethylation and transcription
[3, 4]. However, less compact DNA associated
with low amount of chromatin remodellers will establish
methylation first independent of transcription [23].
Dnmt3a with its co-factor Dnmt3L and Dnmt1 methyltransferases
establish methylation in oocytes [22];
however, the methylation events are also regulated by
histone post-translational modifications. To understand
further the interactions between histone posttranslational
modifications, de novo methylation, and
oocyte transcription factors, an in vitro oocyte culture
model needs to be developed. This model will allow
manipulation of such factors for following their implications
during oocyte growth, and such model should
operate as early as possible, in primary follicles.
The main aim was to develop an in vitro culture model
for molecular studies where epigenetic mechanisms will
be analysed to identify and understand their intricate
regulations. Towards this goal, first a murine in  vitro
follicle culture model needed to be optimised for developing
immature mouse oocytes (primary oocytes) to germinal
vesicle oocytes (GV). Second by manipulating the
culture conditions, here two different oxygen concentrations,
at normoxia (20% ­O2), a higher oxygen concentration
than the physiological one, and 5% oxygen (5% ­O2),
closer to the ovarian oxygen concentration, we analysed
whether methylation and transcription are correctly preserved
in in vitro-grown oocytes as in vivo oocytes. Our
data describe for the first time how oxygen levels affect
oocyte growth specifically when the oocyte culture starts
from follicles containing immature oocytes.
Keywords:  Oocyte, Mouse, DNA methylation, Transcription, In vitro culture, Normoxia, 5% oxygen
Page 3 of 17Naillat et al. Clin Epigenet (2021) 13:132	
Results
Optimisation of follicle‑free oocyte culture model
To set up an oocyte in vitro model from immature to germinal-vesicle
(GV) oocytes and to be able to manipulate
oocytes sufficiently early during their development and
maturation, we adopted the protocol from Honda et al.
[24], which took advantage of maturing arrested primordial
oocytes from dissociated ovaries of 1-week-old mice.
In this method, oocytes are allowed to grow in folliclefree
cultures, but initially supported by theca-like cells in
the primary culture, and we considered that this method
would have advantages in allowing manipulations that
would be difficult to achieve in follicle cultures. Although
oocytes grown by this method have been assessed as
completing meiosis upon induction and to establish DNA
methylation correctly for the limited number of igDMRs
tested, their wider epigenetic fidelity has not been evaluated.
We sought to optimise this oocyte culture system
further to produce a larger quantity of GV stage oocytes
for experiments. We evaluated mouse strain and age at
commencement of culture (data not shown), settling on
7-day-old ­F1(C57BL6/Babr background) females. We
included fatty acid-free bovine serum albumin (fafBSA),
epidermal growth factor (EGF), and follicle stimulating
hormone (FSH) in the culture medium, as these factors
have been shown to improve oocyte development and
maturation [25–27]. We also considered the effect of oxygen
tension. Human oocytes in vivo are in contact with
3–5% oxygen [8], while in other mammals the oxygen
concentration has been reported as being between 2 and
8% [28]. Most follicle cultures for mouse oocytes have
been carried out under normoxia, with only a few studies
using lower oxygen levels, such as 5% ­O2 [9]. In this
study, we compared 20% ­O2 and 5% ­O2 (Fig. 1a).
To evaluate the performance of the cultures, and for
subsequent molecular analyses, we size-selected oocytes
and categorised them into four classes by their diameters:
I, < 40 µm; II, 41–49 µm; III, 51–59; µm and IV, 60–70 µm
(Fig. 1a). The progression of de novo DNA methylation
has been described in in  vivo oocytes of similar size
classes [23], which serves as a reference. In the optimised
normoxia cultures, oocytes achieved a maximum diameter
after 14 days, whereas in 5% ­O2, size IV was attained
in only 9 days and after these 9 days, the oocytes started
to degenerate. Under both conditions, oocytes developed
into GV-like oocytes similar to in vivo oocytes (Fig. 1b),
but the maximum sizes attained differed. An in vivo fully
grown GV measures 70–80 µm [29], whereas in 20% ­O2,
only 7% of the oocytes reached a size of 65 µm and in
5% ­O2 10% of oocytes achieved a size of 70 µm (Fig. 1c,
p < 0.0001, 7 independent oocyte cultures for both conditions).
The contingency graph describing the growth
model showed that the number of oocytes under 20%
­O2 decreased slowly from class I–IV, while they grew,
whereas in 5% ­O2 the oocytes maintained their number
at a plateau, when the oocytes reached the size of
50–60 µm (from class II–III) and then slowly decreased
in number to reach class IV (Fig. 1d , n = 7 independent
oocyte cultures for both conditions).
During the final step of oogenesis, the oocyte nucleus
is subject to large-scale chromatin modifications that
correlate with transcriptional silencing. Oocytes that
present uncondensed chromatin (NSN, non-surrounded
nucleolus) are transcriptionally active, while oocytes with
dense chromatin around the nucleolus are silent (SN,
surrounded nucleolus); moreover, the transition to SN is
required for developmental competence [30]. To define
the pattern of chromatin organisation, the in vitro-grown
oocytes were stained with DAPI and compared with
in vivo GVs collected from 23- to 25-day-old females (3
independent oocyte cultures for both conditions, total of
oocytes n = 68 for 5% ­O2 and n = 62 for 20% ­O2). Under
either culture condition, IV-class oocytes comprised
both NSN and SN GVs (Fig. 1d); however, the SN oocytes
grown in 20% ­O2 had a bigger nucleolus than in 5% ­O2.
In terms of the SN and NSN proportions, oocytes grown
in 5% ­O2 condition were closer to those of in  vivo GV
oocytes (75% SN, compared with 71% in vivo GV, n = 31
oocytes from 23- to 25-day-old females; [30]), but only
40% of oocytes grown in 20% ­O2 were in the SN chromatin
organisation.
Characterisation of DNA methylation in in vitro‑grown
oocytes
DNA methylation acquisition in the oocyte correlates
with increasing diameter during oogenesis [23]. Therefore,
the DNA methylation level of the oocytes grown
under the two culture conditions was compared with
the methylation of in vivo size-selected oocytes. We collected
oocytes representing the starting populations
and the largest oocytes at the end of the cultures which,
for practical purposes, corresponded to ≤ 50  µm and
55–65  µm for the 20% ­O2 cultures, and ≤ 40  µm and
60–70 µm in the 5% ­O2 cultures. Pools of ~ 250 for the
class IV to ~ 500 oocytes for class I and II were collected
in replicate and processed for reduced representation
bisulphite sequencing (RBBS), which preferentially samples
CG-rich regions in the genome, including CGIs [31].
RRBS data from the cultured oocytes were compared to
published data from in vivo growing oocytes (I–IV [23]
and GV [3]). When the replicates were merged depending
on their size within the experimental groups, coverage
was sufficient to evaluate between 56.4 and 60.1% of
CGIs (≥ 5 CpG sites per CGI and ≥ 5 reads per CpG; CGI
annotation based on [32]). Principal component analysis
(PCA) indicated that replicates clustered together
Page 4 of 17Naillat et al. Clin Epigenet (2021) 13:132
(Additional file  1: Fig. S1). Furthermore, PCA demonstrated
a separation along PC1 of the in vivo groups corresponding
to developmental stage. Both 20% ­O2 and 5%
­O2 samples also separated on PC1 according to oocyte
size, indicative of a de novo methylation process similar
to in vivo, but the 5% ­O2 samples also separated on
PC2 from the other groups due to the effect of 5% ­O2.
As previously reported [3, 4], total methylation of CpGs
increased from 3.2% in group II in vivo oocytes to 10% in
group IV and 26.3% at the fully grown GV stage (Fig. 2a).
The in vitro-cultured oocytes also exhibited increases in
methylation with size: from 5 to 8% for the 20% ­O2 cultures,
and 9–21% for the 5% ­O2 cultures (Fig. 2a). These
results suggest that growth in 5% ­O2 is more suitable for
in  vivo-like DNA methylation establishment than 20%
­O2.
We next considered the fidelity of DNA methylation
in the in vitro-grown oocytes. Similar to most cell types,
the majority of CGIs are unmethylated in oocytes, but
fully grown GV oocytes in vivo have a defined number
0
10
20
30
40
50
60
70
80
I II III IV
5% O2
< 40 µm 50-59 µm 60-70 µmSize 41-49 µm
In vivo
10%22%27%43%
7%13%51% IV
III
II
I
*
A B
20% O2 1 14
Oocyte I II III IV
1 9
DAYS
5% O2
20% O2
****
N S N
IV5%O2IV20%O2
SN
C
E
category
Oocyte category
Numberofoocytes
5% O2
20% O2
60-70 µm
NSN SN
75%
25%
40%
60%
D
29%
Fig. 1  Characterisation of follicle-free cultured oocytes. a Schematic representation of the in vitro oocyte culture model. The time of collection
of cultured oocytes under 20% ­O2 was day 21 (14 days after start of the culture), and for the oocytes grown under 5% ­O2, it was on day 16 (9 days
after the starting the culture). b Bright-field photomicrograph of in vitro-grown oocyte cultured for 9 days under 5% ­O2; *indicates oocyte of 70 µm
diameter, scale bar 35 µm. c Graph of the proportions of oocytes of the various size categories after 14 days of culture (20% ­O2) and 9 days of culture
(5% ­O2). Combined data from 7 independent cultures. ****p < 0.001 (ANOVA test). d Contingency graph summarising the growth of the cultured
oocytes under 20% ­O2 and 5% ­O2. Combined data from 7 independent cultures. e DAPI staining of GV-like oocytes grown in 20% ­O2 and 5% ­O2.
Pie charts summarising the number of IV oocytes grown under 20% ­O2 and 5% ­O2 classified as non-surrounding nucleus (NSN) and surrounding
nucleus (SN) (3 independent oocyte cultures for both conditions, total of oocytes n = 68 for 5% ­O2 and n = 62 for 20% ­O2, and n = 31 GV oocytes
aged of 23–25 days old). Scale bar 20 µm
Page 5 of 17Naillat et al. Clin Epigenet (2021) 13:132	
of highly methylated CGI [3, 21, 22]: in the in vivo GV
dataset we used as reference 4.1% of CGIs (n = 661) are
hypermethylated (> 75%). In comparison, 1.2% (n = 169)
of the CGIs informative in our RRBS datasets of the
IV-sized oocytes from the 5% ­O2 cultures were classed
as highly methylated, and just 0.06% (n = 9) of CGIs in
oocytes grown in 20% ­O2 (Fig. 2b). On the other hand,
631 CGIs (4.6%) had methylation > 25% in the IV oocytes
grown in 20% ­O2, and 1022 (7.45%) in 5% ­O2, which
might indicate that the CGIs normally fully methylated
in  vivo are partially methylated in the in  vitro-grown
oocytes. To evaluate the CGIs subject to de novo methylation
more closely, we selected those that had ≥ 40%
methylation in at least one sample, which were 1530 of
the 13,710 CGIs for which RRBS data were available in
size III–IV and GV in vivo oocytes, and IV 20% ­O2 and
IV 5% ­O2 oocytes (Additional file 1: Fig. S2). When the
CGIs > 40% methylated in at least one sample including
in vivo samples were clustered with the CGIs > 50%
from GV stage oocytes, we observed that ~ 40% of the
CGIs were common between GV and both in  vitro
conditions (Fig. 2c). With the exception of a single CGI
found in the sample 5% ­O2, all CGIs ≥ 40% methylated
in in  vitro-grown oocytes were found in > 25% methylated
in GVs (Additional file  1: Fig. S3A). To confirm
that CGIs > 40% methylated in at least one sample were
a correct cut-off from the total CGIs for further analysis,
we correlated CGIs > 40% methylated in at least one
sample with CGIs of GV, IV in  vivo, III in  vivo, IV 5%
­O2 and 20% ­O2. The correlation coefficient between GV
and IV in vivo was lower than the correlation coefficient
between IV in vivo and III in vivo, confirming the results
observed by Gahurova et al. [23]. The correlation coefficient
between IV in vivo and 20% ­O2 was better than IV
in vivo and 5% ­O2 confirming the PCA plot (Additional
file 1: Fig. S3B). From unsupervised hierarchical clustering
analysis of the 1530 CGIs, six major clusters were
apparent (Fig.  3a). Cluster 1 generally comprised CGIs
methylated in all samples; cluster 2 had methylated CGIs
in in vitro conditions and Class III in vivo, but lacking the
methylation in class IV and GV in vivo samples; clusters
3 and 4 were dominated by CGIs showing progressive
C
5% O2
B
Methylation%ofCGIs
III IVII
M
II
I20%
O
2
I5%
O2
IV
5%
O2
IV
20%
O
2
80
90
100
75-100%
50-75%
25-50%
0-25%
G
V
in vivo
GV
CGIs
over 50%
20% O2
CGIs over 40%
CGIs over 40%
A
Totalmethylation%
III IVII
in vivo
I20%
O
2
I5%
O2IV
5%
O2
IV
20%
O
2
GV
Fig. 2  DNA methylation landscape in in vivo and in vitro oocytes. a Comparison of the total methylation level, b percentage of methylation at CGI
between in vivo and in vitro oocytes (for II n = 16,473, III n = 16,151, IV n = 16,177, I 20% ­O2 n = 17,441, IV 20% ­O2 n = 17,170, I 5% ­O2 n = 17,256,
IV 5% ­O2 n = 16,009). c Venn diagram showing the common CGIs that were affected in GV, 20% ­O2 and 5% ­O2 oocytes with different levels of
methylation
Page 6 of 17Naillat et al. Clin Epigenet (2021) 13:132
BA
GV
IV
III
IV
in vivo
IV
5%
O
2
20%
O2
0 20 60 100
% of methylation
1
2
3
4
5
6
%ofCGIs
Promoter Intragenic Intergenic
IV 20% O2
IV 5% O2IV
III
GV Control
C
0 20 40 60 80 100
% of methylation
GV
sperm
III in vivo
IV in vivo
II in vivo
MII
I 20% O2
I 5% O2
IV 5% O2
IV 20% O2
Fig. 3  Differentially methylated CGIs. a Heatmap of over 40% of methylation in at least one sample (n = 1530) comparing IV 20% ­O2, 5% ­O2-grown
oocytes with size-selected III, IV, and GV in vivo oocytes. b Distribution of CGIs over 40% of methylation in at least one sample (n = 1530) located
overlapping promoter, intragenic, and intergenic regions. c Bean plot of the methylation level of the 23 igDMRs between in vivo and cultured
oocytes
Page 7 of 17Naillat et al. Clin Epigenet (2021) 13:132	
methylation in  vivo samples but lacking methylation in
the 5% grown oocytes; clusters 5 contained CGIs methylated
predominantly in the in vitro-grown oocytes; and
cluster 6 grouped all the intermediate methylated CGIs
in all samples except GV in vivo samples (Fig. 3b). For all
of the samples, CGIs > 40% methylated were predominantly
intragenic in location, compared with unmethylated
CGIs (Fig.  3B), consistent with the dependence of
CGI methylation on transcription events in oocytes [3,
4].
Amongst the genomic elements for which de novo
DNA methylation in oocytes does have a critical role are
the igDMRs of imprinted genes. Oocytes grown either in
5% ­O2 or 20% ­O2 demonstrated a gain of methylation at
igDMRs comparable to the corresponding sized in vivo
oocytes (Fig. 3c), with the oocytes grown in 5% ­O2 showing
a higher mean methylation level (58.1% compared
with 42.9%), suggesting greater progression towards the
complete methylation characteristic of fully grown GV
oocytes. Together, these results confirm that DNA methylation
starts to be established in the in  vitro cultures,
with critical elements gaining methylation appropriately,
but there are also subsets of CGIs displaying anomalous
methylation.
Transcriptome analysis of in vitro‑grown oocytes
To investigate gene expression patterns in the oocytes
grown in  vitro, we generated deep, strand-specific
libraries in duplicate from the same size populations of
in vitro-grown oocytes as used in the methylation analysis.
PCA of these RNA-seq libraries, in comparison with
in vivo reference datasets [23], indicated that the in vitrogrown
oocytes from either ­O2 concentration separated
from in vivo growing oocytes and were closer to GVs on
PC1, but were separated from all in vivo groups on PC2
(Additional file 1: Fig. S4).
To explore the gene expression differences between
in  vitro-grown and in  vivo oocytes, we used DESeq to
compare the in  vitro RNA-seq data with in  vivo GVs.
The global gene expression correlations between the IV
5% ­O2 oocytes and GVs, and between the IV 20% ­O2
oocytes and GVs, were r = 0.883 and r = 0.917, respectively
(Fig.  4a, b); furthermore, there were 890 differentially
expressed genes between IV 5% ­O2 and in vivo
GVs, and 2990 between IV 20% ­O2 and GVs (Fig. 4a, b,
p < 0.05). Gene Ontology (GO) analysis using Panther
revealed that the differentially expressed genes were most
enriched in biological processes related to the unfolded
protein response, response to starvation, and endoplasmic
reticulum stress for IV 5% ­O2 oocytes, and terms
related to female gamete and chromatin organisation
for IV 20% ­O2 oocytes (Fig. 4c). We also interrogated the
expression of oocyte-specific genes important for follicle
development, reproduction, and early development [33].
In the 20% ­O2-grown oocytes, there was a greater abundance
of transcripts for key oocyte transcription factors
Spermatogenesis And Oogenesis Specific Basic HelixLoop-Helix
1 and 2 (Sohlh1 and Sohlh2), whereas 5%
­O2-grown oocytes had reduced abundance of Folliculogenesis-Specific
BHLH Transcription Factor (Figla) and
LIM Homeobox  8 (Lhx8) transcripts where both transcription
factors co-express in oocyte for its maturation,
as well as for the oocyte markers Zona pellucida genes,
Zp1, Zp2, and Zp3 (Fig. 4d). We also evaluated expression
of Dnmt3A, Dnmt3Ll and Dnmt1 which are key
enzymes for regulating the establishment of methylation
on DNA in the RNA-seq datasets. Transcripts for
all three genes had reduced abundance in the 5% ­O2 and
20% ­O2-grown oocytes, with a greater reduction in the
5% ­O2-grown oocytes (Fig. 5a, p < 0.05 for Dnmt1 gene,
isoform Dnmt1o oocyte specific). For Dnmt3a, the principal
catalytically active de novo methyltransferase in
mouse oocytes, we also examined protein abundance
and localisation by immunofluorescence, comparing IV
5%O2 oocytes (SN and NSN) with in vivo post-natal D15
growing oocytes and GV oocytes. Dnmt3a protein was
observed both in the cytoplasm and nucleus in the NSN
oocytes at D15, whereas in SN GV oocytes it was concentrated
on chromatin, accompanied by an increase in total
protein abundance (Fig. 5b, c). In the NSN oocytes from
the 5% ­O2 cultures, Dnmt3a was predominantly cytoplasmic
and retained some cytoplasmic localisation even in
the SN-like oocytes (Fig. 5b, C).
Finally, because of the role of transcription events in
determining DNA methylation in growing oocytes [4,
21], we integrated results from the RRBS and RNA-seq
from the cultured oocytes. After stratifying intragenic
CGIs for level of DNA methylation attained, we detected
a positive correlation between gene expression level and
methylation, particularly for the oocytes grown in 20%
­O2 (Additional file 1: Fig. S5), suggesting that CGIs more
advanced in de novo methylation are located within more
highly expressed genes. Therefore, although the in vitrogrown
oocytes do not completely recapitulate the DNA
methylation profile of in vivo counterparts, the de novo
methylation mechanism is likely to follow the same
mechanistic principles.
Testing elements of the de novo methylation mechanism
in the in vitro culture system
To investigate the utility of the oocyte culture system
and to investigate mechanisms of DNA methylation
establishment, we conducted an experiment using two
drugs: tranylcypromine hydrochloride [34] and lithium
chloride (LiCl) [35]. Tranylcypromine hydrochloride is a
non-selective monoamine oxidase inhibitor (MAO) and
Page 8 of 17Naillat et al. Clin Epigenet (2021) 13:132
can be used to inhibit an amine oxidase reaction essential
for the activity of the histone lysine demethylases of
the KDM1 family [36, 37], which have activity towards
H3K4me2. Both Kdm1a and Kdm1b have roles in promoting
normal de novo DNA methylation in growing
oocytes [38, 39], in part by demethylation of H3K4me2
which is antagonistic to Dnmt3a binding and activity
[40]. LiCl was chosen because it blocks activity of the
oocyte-specific transcription factor Foxo3a by inhibiting
its phosphorylation and translocation to the nucleus
[35]. In view of the greater apparent fidelity of methylation
in oocytes grown in 20% ­O2, we selected to culture
the oocytes under normoxia. To evaluate the efficacy of
MAO treatment, we performed immunofluorescence
for H3K4me2 and H3K4me3 on oocytes after 14 days of
culture, observing that H3K4me2 and H3K4me3 staining
was reduced (Additional file 1: Fig. S6A, n = 10 oocytes
for control and treated samples). For the LiCl-treated
5%O
Enrichment
Female gamete
Chromatin organisation
Amino acid biosynthesis
Respiratory electron transport
2.8 10
3.2 10
1.29 10
4.5 10
-3
-7
-5
-5
1 2 3 4 50
4- 3- 2- 1--5 0
Enrichment
Notch pathway 9.96 10
Unfolded protein 9.96 10
Response to starvation 6.12 10
Response to ER stress 2.46 10
Positive regulation of organic
acid transport 1.74 10
-3
-3
-3
-2
-3
1 2 3 4 50
4- 3- 2- 1--5 0
D
GV
12.5
10
7.5
5
2.5
0
-2.5
-5
Highlighted differentially
expressed genes
-5 -2.5 0 2.5 5 7.5 10 12.5
R=0.883
A
logFpkmFpkmnormalisedtoIVinvivo
GV
12.5
10
7.5
5
2.5
0
-2.5
-5
Highlighted differentially
expressed genes
R=0.917
-5 -2.5 0 2.5 5 7.5 10 12.5
logFpkm
IV
p< 0.05
C
5%O2
GV
B
log Fpkm log Fpkm
−5 0 5
Va lu e
n=890 n=2 990
% OIV % O220
OIV % 2205%OIV 2
Geneontology
%
O
IV
%
O
2
20
O
%5
O
%5
2
IV
0
1
2
3
4
II
IV
III in vivo
*
*
*
*
OIV % 220
5%OIV 2
Sohlh1
Sohlh2
Fig. 4  Gene expression profiles of grown IV oocytes. a Scatter plot and correlation coefficient of log Fpkm values of 890 transcripts in GV and
5% ­O2, and of 2990 transcripts in GV and 20% ­O2. b Heatmap of the transcripts from the IV 5%O2, IV 20% ­O2, and GV. c Gene ontology of the
differentially expressed genes between GV and 5% ­O2 and GV and 20% ­O2. d Gene expression of selected oocyte genes in in vivo, IV 20% ­O2 and IV
5%O2. *p > 0.05 Student’s t test comparing GV and the cultured oocytes
Page 9 of 17Naillat et al. Clin Epigenet (2021) 13:132	
oocytes, the nuclear staining of Phospho-FOXO3A, that
is seen in the untreated oocytes, is much diminished
(Additional file 1: Fig. S6B, n = 10 oocytes for control and
treated samples).Along with the staining of the cultured
oocytes, GV oocytes (n = 8 for each condition MAO,
LiCl, and control) were stained to observe the correct
localisation of proteins of interest to assure that the 20%
­O2 had no effect on the cultured oocytes (Additional
file 1: Fig. S6C).
To quantify the effect of MAO and LiCl treatments on
DNA methylation, we collected oocytes after 14 days of
culture, grown under 20% ­O2, in the absence or presence
of the inhibitors in triplicate pools of between 70 and 110
oocytes, and processed the oocytes for whole-genome
methylation analysis by low-cell post-bisulphite adaptortagging
(PBAT) [38, 41]. After alignment and removal of
reads from duplicates between 16,798,787 and 45,690,308
uniquely mapped reads were obtained per library. (One
LiCl-treated sample was discarded because of low coverage,
Additional file 2: Table S1.) For the clustering analysis,
we included in this analysis, the Class IV in  vivo
oocyte from Gahurova et  al. [23] to identify how the
samples will group, as the class IV oocytes is our aimed
size and present the maximum level of methylation. The
PBAT libraries clustered according to the treatment
(Additional file 1: Fig. S7). For most subsequent analysis,
replicates were pooled.
First, we assessed the effects of the inhibitors on overall
DNA methylation in the oocytes and observed a decrease
in CpG methylation in the three different conditions
from 39.7% (MAO), 28.8% (LiCl) to 19.6% (MAO + LiCl)
compared to 46.6% in in vivo IV oocytes, and 38% in 20%
Dnmt3a DAPI Dnmt3a DAPI
D15invivoNSNGVSN
5%O5%O2IV
FpkmnormalisedtoGV
GV
IV
III
II
IV 20% O2
IV 5% O2
A B
in vivo
in vivo
in vivo
0
1
2
3
4
D15 in vivo
IV 5% O2 NSN
IV 5% O 2 SN
GV
***
**
*
CTCF/DAPI
C
*
*
Fig. 5  DNA methyl transferase expression in grown oocytes. a Dnmt1, Dnmt3a, and Dnmt3l expression compared to GV. *p > 0.05 Student’s t test GV
compared to cultured oocytes. b Immunostaining of Dnmt3a in different size oocytes.  c Quantification of immunofluorescence intensity as seen in
(b). Corrected total cell fluorescence (CTCF) was calculated for each signal then normalised to DAPI CTCF. Bars represent mean values, and error bars
show SEM (2-way ANOVA test; *p < 0.05; **p < 0.01, ***p < 0.001)
Page 10 of 17Naillat et al. Clin Epigenet (2021) 13:132
­O2 condition (Fig. 6A). As one of the treatments affects
the activity of Kdm1 enzymes, we included the PBAT
methylation datasets of Kdm1a and Kdm1b M2 knockout
oocytes from Stewart et al. [38] in the analysis. Treatments
of MAO or MAO + LiCl showed a reduced CpG
methylation with a pattern close to Kdm1b (data not
shown). However, here we compared oocytes from different
sizes (Class IV in vitro oocytes 20% ­O2 with M2
ovulated oocytes) and could bias the results. Focussing
on CGIs that normally become methylated in oocytes, we
then access the total CGIs methylation which showed a
reduction in highly methylated CGIs from 1.3% (n = 76)
in 20% ­O2 CON to nearly 0% in MAO (n = 32), LiCl
(n = 8), and MAO + LiCl (n = 2). The number of methylated
CGI < 25% was increasing in all treated samples
and was of 10% between 20% ­O2 CON and MAO + LiCl
(Fig. 6B). Then, we used the same strategy as for analysing
the RBBS data, and a cut-off of > 40% methylation was
used on the PBAT data set. This resulted in 1138 CGIs
which clustered within 3 groups and were mostly hypomethylated
in the cultured oocytes under the inhibitory
drugs (Fig.  6c). They failed to properly methylate, and
the proportion of CGIs localised on the promoter and
intergenic regions increased compared to oocyte 20%
­O2 CON. Most of the methylation is observed on the
intragenic regions in GV oocytes [4], and the cultured
oocytes presented a significant decreased in CGIs at the
intragenic regions from 48% in 20% ­O2 CON to 40% in
cultured oocytes with Mao + LiCl (Fig. 6d). We also specifically
evaluated effects on igDMRs, as it has previously
been described that genetic ablation of Kdm1b in
oocytes [32] impairs de novo methylation of igDMRs.
The heatmap of methylation difference of the igDMRs
by comparing 20% ­O2 CON with each drug presented a
various aberrant igDMRs methylation for all the cultured
oocytes (Fig. 6e). For example, Igf2r was highly methylated
(70.1%) in IV 20% O2 CON and the cultured oocytes
under treatment reduced their methylation (MAO
56.5%, LiCl 38.2%, and MAO + LiCl 33.9%, respectively,
Additional file 1: Fig. S7). These results suggest that the
igDMRs did not gain a correct level of DNA methylation
but instead obtained various levels of intermediate
methylation.
Finally, we sought to assess the extent to which the
treatments in  vitro mimicked the ablation of Kdm1a
and Kdm1b in vivo [38]. We took advantage of the
published PBAT data from M2 oocytes of Kdm1a and
Kdm1b knock-out mice [38]. We compared the defined
hypermethylated domains (CGI over 75% methylation)
of M2 oocytes knock-out mice, parameters
defined in Stewart et al. [38], with our data at IV 20%
­O2 CON oocytes > 25% methylation and the 3 other
in  vitro conditions (MAO, LiCl, and MAO + LiCl)
over 10% of methylation. We have chosen a methylation
threshold of ≥ 25% methylation for 20% ­O2 CON
(n = 1433) as most of the CGIs had an intermediate
methylation. For the other in  vitro conditions MAO,
LiCl, and MAO + LiCl, the methylation threshold was
chosen ≥ 10% methylation to gain the maximum number
of CGIs in the different culture conditions (MAO
n = 1983, LiCl n = 2243, and MAO + LiCl n = 1817).
We have generated Venn diagrams to identify the overlapping
CGIs between the effect of the in vitro conditions
and the in  vivo knock-out mice dataset. While
comparing the CGIs in hypermethylated domains with
in  vivo CGIs of Kdm1a knock-out mouse hypermethylated
domains, only 12 CGIs were common with all
the in  vitro conditions. When same comparison was
carried out with the in vivo CGIs of Kdm1b knock-out
mouse hypermethylated domains, only one CGI was
common (Fig. 7a–c). In the case of the comparison with
hypermethylated domains Kdm1a knock-out mouse,
the 12 common CGIs are located mostly on intragenic
regions (75%) and the remaining 25% are located on
the promoter (Additional file  3: Table  S2). However,
in the case of non-common CGIs, we could observe
an increase of CGIs located on intergenic regions for
all conditions compared to Kdm1a and Kdm1b M2
knock-out oocytes (Additional file  3: Table  S2). Interestingly,
the CGIs in hypermethylated domains of 20%
­O2 CON compared with hypermethylated domains of
Kdm1a knockout oocytes (n = 96) and the CGIs from
20% ­O2 CON compared with hypermethylated domains
of Kdm1b knock-out oocytes (n = 98) were all common
(Additional file 3: Table S2). These results suggest that
the drugs against Kdm1 enzyme activity slowed down
the gain of methylation on CGIs in grown oocytes.
(See figure on next page.)
Fig. 6  CGI methylation under four different conditions using oocyte in vitro model. a Bean plots representing total methylation in IV in vivo, IV 20%
­O2, MAO, LiCl, MAO + LiCl-grown oocytes and Kdm1a, Kdm1b knock-out and WT M2 oocytes, (χ2
test, p < 0.05). b Percentage of methylation at CGIs
between in vivo and cultured oocytes (for IV in vivo n = 5707, IV 20% O2 CON n = 5713, MAO n = 5713, LiCl n = 5713, MAO + LiCl n = 5713, χ2
test,
p < 0.05), c Heat map of the CGIs over 40% of methylation in at least one sample between the conditions, (n = 1138; χ2
test, p < 0.05). d Distribution
of CGIs over 40% of methylation in at least one sample (n = 1138) located overlapping promoter, intragenic, and intergenic regions (χ2
test, p < 0.05).
e Heatmap representing the quantitation of the methylation difference between the IV 20% ­O2 CON compared to each cultured in vitro oocytes of
the 23 igDMRs (χ2
test, p < 0.05)
Page 11 of 17Naillat et al. Clin Epigenet (2021) 13:132	
B
p< 0.05
A
Kdm
1A
KO
Kdm
1b
KO
WT
p<0.05
%ofmethylation
0
20
40
60
80
100
IV
in
vivo
IV
20%
O
2
C
O
N
M
AO
LiC
l
M
AO
+
LiCl
D
0 20 60 100
% of methylation
CGIs (n=1 138)
p< 0.05
M
AO
MAO+LiCl
LiCl
C
IV 20% O2
CON
Methylation%ofCGIs
100
90
80
70
60
75-100%
50-75%
25-50%
0-25%
IV
in
vivo
IV
20%
O
2
C
O
N
M
AO
LiC
l
M
AO
+
LiCl
n=5 713
%CGIs
100
80
60
40
0
20
p< 0.05 n=1 138
Promoter Intragenic
Intergenic
E
OAM
lCiL+OAM
lCiL
IV20%O2
CON
Kcnq1ot1
Igf2r/Air
Mcts2
Zfp777
Zac1
Zim2
Peg3
Nespas
Slc38a4
Gnas Exon1a
Impact
Nap1l5
Zfp787
Cdh15
Snurf/Snrpn
Nnat
Peg13
Peg10
Inpp5fv2
AK008011
Grb10
U2af1-Rs1
Mest
O%02VI2
OAM
lCiL
lCiL+OAM
CON
0 6030
% of methylation
Page 12 of 17Naillat et al. Clin Epigenet (2021) 13:132
Discussion
Reproductive biology and regenerative medicine fields
have made great breakthroughs to understand how
to preserve fertility. Reconstitution of gametogenesis
in vitro is crucial for improving and preserving fertility.
To be able to reconstitute mature gametes, different
alternatives have been explored to generate live
offspring based on producing gametes from pluripotent
stem cells [42] or from embryonic mouse germ cells
[43]. However, it is essential to prove the safety of such
procedures before they become applied in humans by
analysing and understanding the effect of the cultures
on the epigenetic profile of the gametes. Towards this
goal, we have analysed the effect of oxygen concentration
(5% ­O2 vs. 20% ­O2) on de novo methylation establishment
and transcription in in  vitro-grown GV-like
oocytes from an in  vitro oocyte culture starting from
immature oocytes.
A B
9
11
4
5
10
12
2
6
8
7
1
3
20
40
60
80
%ofmethylation
M
AO
M
AO+LiCl
LiCl
IV 20%
O2
CON
Kdm
1A
KO
C
IV
20%
O
2
C
O
N
IV
20%
O
2
C
O
N
Fig. 7  Identification of common CGIs between Kdm1s knock-out mouse M2 oocytes and in vitro cultured oocytes under treatments inhibiting
methylation and transcription. a Venn diagrams showing the common and non-common CGIs between the hypermethylated domains defined in
Kdm1a oocytes and the CGIs methylated > 25% in normoxia or 20% ­O2 CON and > 10% in MAO, LiCl, and MAO + LiCl. b Venn diagrams showing the
common and non-common CGIs between the hypermethylated domains defined in Kdm1b oocytes and the CGIs methylated > 25% in normoxia
or 20% ­O2 CON and > 10% in MAO, LiCl, and MAO + LiCl. c Heatmap representing the methylation level of 12 common CGIs between Kdm1a M2
oocytes and the CGIs methylated > 25% in normoxia or 20% ­O2 CON and > 10% in MAO, LiCl, and MAO + LiCl
Page 13 of 17Naillat et al. Clin Epigenet (2021) 13:132	
Most of the follicle culture techniques are using superovulated
oocytes (M2) [44] when they are carried on
animal and human oocytes. In these culture methods,
all follicles are grown with the cumulus cells around the
oocytes, and the connections between the oocyte and
cumulus cells are intact. In our model, few granulosa
cells were attached to the immature oocytes due to the
ovaries digestion step and the free theca cells resulting
from the ovarian digestion formed colonies at the bottom
to the Petri dish as described by Honda et al. [24].
By adding identified factors (fafBSA, EGF, and FSH) in
the culture medium, known to improve oocyte development
and maturation [25–27], class IV in vitro oocytes
were collected. A small amount of class IV grown oocytes
under 20% ­O2 (7%) and 5% ­O2 (10%) reached the size
of GV in vivo oocytes which was the aimed size for our
study. The oocytes that reached class IV under both oxygen
conditions demonstrate that they complete the transition
from the NSN to SN chromatin configuration to be
mature oocytes [24].
Methylation in in  vivo mouse oocytes was first identified
to be crucial in intragenic CGI using RRBS [3]
and then more recently using whole genome bisulphite
sequencing via PBAT method on low amount of sample
amount [21, 22, 38]. Veselovska has been demonstrating
that methylation happens at the same time as transcription
is occurring [4] and methylation of the last
CGIs depends on the chromatin state [23]. However,
the detail of the genomic distribution of methylation
in in vitro mouse oocytes has not yet been clearly analysed.
Here, we provide DNA methylation maps of mouse
grown oocytes from immature to GV stage obtained after
9–14  days in culture under 5% ­O2 and 20% ­O2 condition.
We observed that DNA methylation on CGIs was
increased in all conditions as we could identify the methylation
wave happening in the in vitro conditions. However,
the hypermethylated CGIs regions were altered in
the class IV in vitro oocytes in both conditions due to the
oxygen concentration, 5% and 20% ­O2. We can hypothesise
that a longer period of culture (up to 14  days) in
relation to the oxygen concentration might affect the
methylation status of the cytosine base and a demethylation
wave might be triggered towards hydroxymethylation,
resulting in hydroxymethyl cytosine and even
further to the formyl cytosine. Therefore, such study
needs to be carried out to portray the importance of
reduced oxygen tension in the establishment of demethylation
wave in in vitro-grown oocytes.
Studies exploring the influence of the duration of the
culture on M2 oocytes in close contact with cumulus
cells demonstrated that the blastocyst formation rate,
meiosis resumption, and expression of key oocyte genes
are increased [45], but their effects on imprinted genes
are currently not fully known. In our study, the two different
oxygen conditions (20% vs. 5% ­O2) did not affect
the temporal acquisition of correct imprinted DNA
methylation patterns in the denuded oocytes, as the
methylation of the imprinted genes was closer to the
class IV in vivo oocytes as the igDMR methylation was
restored in in vitro 5% and 20% ­O2 oocytes. This result is
in line with previous studies using in vitro culture model
or super-ovulation method demonstrating that imprinted
DNA methylation acquisition in oocytes is a robust
mechanism and is not influenced by culture conditions
[19, 44]. Imprinted genes are regulated by the methylation
status of the imprinting control region (ICR), and
when the methylation of the ICR failed due to the loss
of transcription, it results in aberrant methylation of the
igDMRs [46]. This is what we observe when we manipulated
methylation and transcription processes at the same
time by inhibiting Kdm1 function and phosphorylation
of Foxo3 transcription factor (MAO + LiCl treatment).
The igDMR methylation was completely altered underlining
that the establishment of methylation on imprinted
genes might be dependent on the transcription.
To further investigate the facets of methylation and
transcription, we generated and compared the methylomes
and transcriptomes of in  vitro-grown oocytes
to in  vivo size selected and class IV in  vivo oocytes.
While DNA methylation establishment at igDMRs in
the oocyte remained unaltered, the overall distribution
of CGI methylation occurred in a similar pattern
that in in vivo oocytes during the oocyte growth; however,
we observed that the methylation in intergenic
region increased. Recent study shows that one-sixth
of all de novo DNA methylation is linked to transcription
initiated at long-terminal repeats (LTRs) [47] and
most of the LTRs are localised in intergenic regions.
We can hypothesise that if the LTR-initiated transcripts
are activated at a wrong time, this will impact
the methylome in oocytes leading to aberrant methylation.
Transcription analysis revealed that in 20% ­O2
culture condition, transcripts were enriched in female
gamete genes and chromatin organisation, whereas
the 5% ­O2 condition increased expression of stress
response genes. Such results suggest that the lower
oxygen concentration towards hypoxia might lead to
an exhaustion of metabolites in the oocytes resulting
in mechanical stress and/or increased of reactive oxygen
species, and normoxia condition to a better availability
of the 3D chromatin conformation. However,
the Dnmts transcripts (Dnmt1, Dnmt3A, Dnmt3L)
were reduced in both oxygen conditions 20% and 5%
­O2. De novo DNA methylation is established by the
Dnmt3A/L complex, and such enzymes are controlled
by their availability at the subcellular localisation and
Page 14 of 17Naillat et al. Clin Epigenet (2021) 13:132
by their interactions with the chromatin. They regulate
the methylation at the hypomethylated and hypermethylated
domains in oocytes [48]. We observed
in our in  vitro oocyte model that the localisation of
Dnmt3A protein was altered in 5% ­O2-grown oocytes.
Dnmt3A was observed in the cytoplasm in class IV
in vitro oocytes, whereas it should have been localised
onto the chromatin as in in vivo class IV oocytes. Such
mis-localisation will lead to reduce catalytic activity
of the enzyme and might explain the lower methylation
level observed at the hypermethylated domain in
grown oocytes.
The clinical implication of low oxygen concentration
in human embryos in assisted reproductive technology
has been analysed mostly at the level of embryo quality
and morphology, pregnancy rate, live birth/ongoing
pregnancy [49–51]. These studies show that in human
low oxygen concentration might have some beneficial
effect in the clinical outcomes of in  vitro culture
such as better pregnancy rate. So far, no studies have
been conducted on analysing epigenetic marks such
as DNA methylation in cultured human oocytes incubated
for few hours in 5% ­O2 or in cultured blastocysts
after 4–6 days incubation at 5% ­O2, due to the difficulty
to obtain the samples. Metadata analysis on human
cohorts has hypothesis that changes in DNA methylation
at specific genes during early embryo development
might result in metabolic diseases in adult life such as
obesity and diabetes [52]. To be able to answer such
hypothesis, more studies need to be carried out to help
us in understanding how DNA methylation is fully
established in cultured oocytes and if any culture conditions
during fertility treatments might affect the epigenome
of the embryo. Therefore, whether the culture
of human oocytes and embryos in low concentrations
of oxygen can actually improve the clinical outcomes of
ART is a question that still requires more data.
Conclusions
In conclusion, the comparison of our in  vitro oocyte
models from immature towards class IV oocytes demonstrates
that genomic DNA methylation is altered
during the oocyte growth under low concentration of
oxygen. Importantly, we show that CGI methylation
of igDMRs is fully conserved, whereas the CGI methylation
on gene bodies is disturbed due to alteration
in culture conditions, here the oxygen concentration.
Therefore, from our results further study on embryos
produced from immature grown oocytes under different
conditions is needed to clarify whether oocytederived
DNA methylation at igDMRs and DMRs is fully
conserved and if the future embryos develop properly.
Material and methods
In vitro oocyte culture
The oocyte culture established by Honda et al. [24] based
on stem cell factor and ES medium was optimised. In
brief, the ovaries, aged of 7  days old, from C57 Black6
background breed with SV129 background mice were
collected and digested with trypsin (Sigma) and collagenase
I (Sigma) at 37 °C for 15 min. The trypsin-collagenase
action was stopped with foetal bovine serum
(Sigma) and centrifuged for 5 min at 1600 rpm. The pellet
was suspended into warm medium and plated onto
a gelatine coated plate. The oocyte culture was performed
under two different conditions: 20% ­O2 and 5%O2
(Fig. 1a). The culture medium was changed every 24 h for
the 5% ­O2 as the colour of the medium changed rapidly to
maintain the integrity of the medium composition, and
every 48 h for the 20% ­O2 condition. The grown oocytes
were collected depending on their size and the culture
condition and were kept in—80 °C until further process.
To reach the appropriate growth size, the oocytes were
grown for 9 days under 5% ­O2 and for 14 days under 20%
­O2. The size of the oocytes has been grouped into classes:
< 40 µm class I, 41–49 µm class II, 50–59 µm class III,
and 60–70 µm class IV (Fig. 1a). For 5% ­O2, the size of the
oocytes was collected at < 40 µm considered as class I and
60–70 µm class IV, and for 20% ­O2, the selected size of
the oocyte was < 49 µm class II and 55–65 µm considered
to be class IV as the diameter of these oocytes reached
65 µm.
Oocytes were cultured with 100 µM tranylcypromine
(Sigma), a working concentration which has been shown
to inhibit KDM1, and cause epigenetic changes at the
promoters of specific genes [53] and with 15  mM/ml
LiCl (Sigma) [35] under 20% ­O2 condition. The control
oocytes (CON) were cultured without any drugs under
20% ­O2 condition. The oocyte cultures were stopped
when the oocytes reached the size of 55–65  µm. The
cultured oocytes were frozen at − 80  °C until further
experiments.
Preparation of bisulphite sequencing libraries
For RBBS, the oocytes were collected in pools of between
250 and 500 oocytes depending on the size of the oocyte
and were carried in replicate for each condition 20% ­O2
and 5% ­O2 at two different oocyte size (class I and IV).
Libraries for RRBS were prepared as described previously
by Smallwood et  al. [3]. Genomic DNA from
approximately 300 cultured oocytes was extracted using
QIAamp DNA Micro Kit (Qiagen) and was spiked with
10 fg of unmethylated lambda phage DNA (Promega) followed
by MspI digestion (Fermentas), and the libraries
were generated according to Smallwood et al. [3]. Libraries
runs, in duplicates, were single-ended, and 100 base
Page 15 of 17Naillat et al. Clin Epigenet (2021) 13:132	
pairs (bps) in length were sequenced on Illumina HiSeq
2500 platform.
Post Bisulfite Amplification Treatment (PABT) libraries
were generated as described previously by Peat et al. [41]
for whole genome sequencing. 70–100 cultured oocytes
under different treatments (Tranylcypromine, LiCl, Tra-
nycyltramine + LiCl, and control also called 20% ­O2
CON) with a size of 55–65 μm were used to generate the
libraries in triplicate. Libraries runs were single-ended,
and 100 bps in length were sequenced on Illumina HiSeq
2500 platform.
Preparation of RNA sequencing libraries
Total RNA from 280 cultured oocytes for each condition
20% ­O2 and 5% ­O2 at two different oocyte size (class IV)
was extracted using trizol method (Sigma) according to
the manufacturer’s instructions, and the total RNA was
purified using the RNA clean and Concentrator-5 kit
(Zymo) followed by depletion of ribosomal RNA with
Ribo-Zero magnetic kit (Illumina) to remove the ribosomal
RNA. The total RNA-seq libraries were constructed
according to Veselovska et al. [4]. Libraries runs, in duplicate,
were sequenced as paired-ended, 100 bps in length
on Illumina HiSeq 2500 platform.
Library mapping
Sequence alignment mapped onto the mouse genome
(mm10) and methylation calls were performed using Bismark
[54]. CpGs with read depth lower than 5 were discarded.
We estimated bisulphite conversion rates using
reads that uniquely aligned to the lambda phage genome
(conversion rate over 94%). Raw reads from RNA-seq
were aligned against mouse genome (mm10) and mapped
using TopHat.
Data analysis
DNA methylation and RNA analysis were done using
the SeqMonk software package (v.1.41; Babraham Institute).
To score CGI methylation, a list of 23,021 CGIs
were obtained from a previous report [32]. The definition
of methylated and unmethylated CGIs was according
to Smallwood et al. [3]. A list of 23 DMRs was obtained
from Proudhon et al. [55]. Methylation data were compared
to RRBS data from size-selected in  vivo oocytes
(40–45 µm, 50–55 µm, 60–65 µm) [23] and the data from
the GV and M2 from Smallwood et  al. [3]. CGIs were
used for methylation analyses if the minimum number
of reads to count a position/minimum number of positions
to count a probe was 5/5 for CGIs. CGI was annotated
as (1) promoter if they overlapped a TSS defined as
− 1 Kb + 500 bp, (2) intragenic if they overlapped a gene,
and 3) intergenic if they did not overlap a promoter or
intragenic regions.
PBAT methylation data were compared to data from
GV oocytes, Kdm1a-, Kdm1b- knock-out, and wild-type
M2 mouse oocytes [21, 22, 38]. The methylation level
reported for a sample was the average methylation across
samples from the generated biological triplicate samples.
The minimum number of reads to count a position/minimum
number of positions to count a probe was 5/3 for
CGIs, followed by Chi-square test of p < 0.05 and probes
which did not cover the requirements were discarded.
The comparison with the PBAT dataset of Kdm1a and
Kdm1b knock-out M2 oocytes, the strategy developed by
Stewart et al. [4], was applied to the dataset to generate
hypomethylated and hypermethylated domains.
For RNA-seq analysis, we used the annotation [4]
that is transcripts oocyte-specific annotation. We then
uploaded into SeqMonk as an oocyte-specific annotation
track. Reads over transcripts were merged across
exons correcting for feature length from the annotation
track, quantitated using RNA-seq quantitation pipeline,
and log2-transformed. Differentially expressed genes
were called using DESeq with significance threshold of
p < 0.05. Panther program was used for the gene ontology
using Benjamini-corrected p < 0.05 significance cutoff.
For gene-level analysis, the expression level of unique
transcripts was ranked by expression levels (calculated
as FPKM values) in each library. RNA-seq data from GV
oocytes were from Veselovska et al. [4].
Whole‑mount immunofluorescence
Cultured oocytes under 5% ­O2, 20% ­O2, and in vivo D15
and GV oocytes from C57 Black6 background breed
with SV129 background mice were collected, fixed in 4%
paraformaldehyde for 15 min, and washed three times in
PBS, and the immunofluorescence protocol was according
to Santos et  al. [56]. The oocytes were incubated
with Dnmt3a (Abcam), H3K4me2/3 (Abcam), phosphoFoxo3a
(Molecular probe), Fig alpha (Santa Cruz), or
Alexa Fluor antibodies (Molecular probes) and were
counterstained with DAPI (Sigma). The pictures were
taken with Leica LSM confocal microscope. To accurately
compare fluorescent intensity between oocytes,
the laser power was set based upon the oocyte with the
strongest signal and the same setting was used to evaluate
all oocytes. Signal quantification was performed using
NIH Image J software; corrected total cell fluorescence
(CTCF) was calculated as CTCF = Integrated density—
(Area of selected region X Mean fluorescence of background
readings) [57]. CTCF was calculated for Dnmt3a
and DAPI signals; Dnmt3a was normalised to the DAPI
signal for each oocyte nucleus. Greater than 10 oocytes
pooled for each condition were examined in 3 separate
experiments.
Page 16 of 17Naillat et al. Clin Epigenet (2021) 13:132
Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s13148-​021-​01116-3.
Additional file 1. Supplementary materials (Figures S1–S8).
Additional file 2: Table S1. Sequencing output for PBAT libraries.
Additional file 3: Table S2. CGIs overlapping promoter, intragenic and
intergenic regions of methylated non-common CGIs after the comparison
with the hypermethylated domains defined in Kdm1a oocytes and the
CGIs methylated >25% in normoxia or 20% O2 CON, and >10% in MAO,
LiCl and MAO+LiCl. PA promoter, Intra intragenic and Inter intergenic.
Acknowledgements
We would like to acknowledge the essential contributions of the Kristina
Tabbada in the Sequencing Facility at the Babraham Institute, Felix Krueger
and Anne Segonds-Pichon from Babraham bioinformatic group for sequence
mapping GEO deposition and helping in statistical test analysis, and Wendi
Dean for helpful discussion.
Authors’contribution
FN and GK conceptualized the study. FN, JNW, LK, FS and ST performed the
experiments, FN and HS performed the bioinformatics analysis. FN and GK
analyzed the data and revised the manuscript. All authors approved the final
version of manuscript.
Funding
This work was supported by grants for FN from the Academy of Finland
Profiling funding to the University of Oulu Profi3 (311934), Academy of Finland
post-doctoral Fellowship (243014583), Foundations’Post Doc Pool (Svenska
Kulturfonden), and Finnish Cultural Foundation (Pekka ja Jukka-Pekka Lylykarin
rahasto); for JNW Polish Ministry of Science and Higher Education postdoctoral
fellowship (Mobility Plus Programme, No: 609/MOB/2011/0); and for
G.K. by the UK Biotechnology and Biological Sciences Research Council and
Medical Research Council (Grants G0800013 and MR/K011332/1).
Availability of data and materials
Mapped sequence data from Bismark software have been deposited in the
Gene Expression Omnibus database (GEO) under accession code GSE164864.
Declarations
Ethical approval and consent to participate
All mouse experimental procedures were approved by the Animal Welfare and
Ethical Review Body at the Babraham Institute and performed under licences
issued by the Home Office (UK) in accordance with the Animals (Scientific
Procedures) Act 1986.
Competing interests
The authors declare that they have no competing interests.
Author details
1
 Epigenetics Program, Babraham Institute, Cambridge CB22 3AT, UK. 2
 Diseases
Network Research Unit, Faculty of Biochemistry and Molecular Medicine, Oulu
University, Oulu, Finland. 3
 Department of Computer Science, King Abdullah II
School of Information Technology, The University of Jordan, Amman, Jordan.
4
 Department of Genetics and Animal Breeding, Poznan University of Life
Sciences, Poznan, Poland. 5
 Laboratory of Early Mammalian Development,
Department of Molecular Biology and Genetics, University of South Bohemia,
37005 České Budějovice, Czech Republic. 6
 School of Medicine, Yokohama
City University, Yokohama, Japan. 7
 Centre for Trophoblast Research, University
of Cambridge, Cambridge CB2 3EG, UK.
Received: 12 March 2021 Accepted: 15 June 2021
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