Polycomb function during oogenesis
is required for mouse embryonic
Eszter Posfai,1,2Rico Kunzmann,1,2,7Vincent Brochard,3,4,7Juliette Salvaing,3,4Erik Cabuy,1
Tim C. Roloff,1Zichuan Liu,1Mathieu Tardat,1Maarten van Lohuizen,5Miguel Vidal,6
Nathalie Beaujean,3,4and Antoine H.F.M. Peters1,8
1Friedrich Miescher Institute for Biomedical Research (FMI), CH-4058 Basel, Switzerland;
of Basel, CH-4056 Basel, Switzerland;
De ´veloppement et Reproduction, F-78350 Jouy-en-Josas, France;
Maisons Alfort, France;5Division of Molecular Genetics, Centre for Biomedical Genetics, The Netherlands Cancer Institute
(NKI), 1066 CX Amsterdam, The Netherlands;
Cientı ´ficas (CSIC), 28040 Madrid, Spain
2Faculty of Sciences, University
3Institut National de la Recherche Agronomique (INRA), UMR1198, Biologie du
4Ecole Nationale Ve ´te ´rinaire d’Alfort (ENVA), F-94700
6Centro de Investigaciones Biolo ´gicas, Consejo Superior de Investigaciones
In mammals, totipotent embryos are formed by fusion of highly differentiated gametes. Acquisition of totipotency
concurs with chromatin remodeling of parental genomes, changes in the maternal transcriptome and proteome,
and zygotic genome activation (ZGA). The inefficiency of reprogramming somatic nuclei in reproductive cloning
suggests that intergenerational inheritance of germline chromatin contributes to developmental proficiency after
natural conception. Here we show that Ring1 and Rnf2, components of Polycomb-repressive complex 1 (PRC1),
serve redundant transcriptional functions during oogenesis that are essential for proper ZGA, replication and cell
cycle progression in early embryos, and development beyond the two-cell stage. Exchange of chromosomes
between control and Ring1/Rnf2-deficient metaphase II oocytes reveal cytoplasmic and chromosome-based
contributions by PRC1 to embryonic development. Our results strongly support a model in which Polycomb acts
in the female germline to establish developmental competence for the following generation by silencing
differentiation-inducing genes and defining appropriate chromatin states.
[Keywords: Polycomb-repressive complex 1; maternal effect; intergenerational inheritance; epigenetic memory; nuclear
transfer; intra-S-phase checkpoint]
Supplemental material is available for this article.
Received January 24, 2012; revised version accepted March 21, 2012.
In mammals, fusion of two dimorphic gametes generates
a totipotent embryo that has the ability to form all dif-
ferent cell types of the embryonic and extraembryonic
lineages. Initially, both parental genomes are transcrip-
tionally silent, and early embryonic events are controlled
by ‘‘maternal’’ transcripts and proteins, stored during
oogenesis, and provided to the embryo (Tadros and
Lipshitz 2009). However, the role of potentially inherited
germline chromatin states is largely unknown.
Classical work on genomic imprinting shows that DNA
methylation established in oocytes confers intergenera-
tional epigenetic inheritance (Gill et al. 2012). For certain
repetitive sequences and many genes, however, DNA
methylation is reprogrammed in early embryos (Lane
et al. 2003; Blewitt et al. 2006; Smallwood et al. 2011).
Nuclear transfer experiments revealed the capacity of the
cytoplasm of metaphase II (M-II) oocytes and mitotic one-
cell embryos to reprogram chromatin states of somatic
nuclei, thereby promoting embryonic development (Egli
et al. 2007; Inoue et al. 2008). Nonetheless, reprogram-
ming of germ cell nuclei is more effective than that of
somatic cell nuclei (Hochedlinger and Jaenisch 2003),
suggesting that germ cell chromatin is more compatible
with the reprogramming abilities of oocytes. This may
be due to the fact that mammalian germline chromatin
is prepatterned for early embryonic development, as in
zebrafish and Caenorhabditis elegans (Arico et al. 2011;
Lindeman et al. 2011).
Several recent studies on mammalian systems have
suggested the existence of intergenerational (between) or
transgenerational (across multiple) epigenetic inheri-
tance of acquired traits (Anway et al. 2005; Anderson
et al. 2006; Carone et al. 2010; Ng et al. 2010). Other
studies indicate that proper chromatin regulation in the
7These authors contributed equally to this work.
Article published online ahead of print. Article and publication date are
online at http://www.genesdev.org/cgi/doi/10.1101/gad.188094.112.
920GENES & DEVELOPMENT 26:920–932 ? 2012 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/12; www.genesdev.org
germline is required for gene regulation or other chroma-
tin-based processes in the next generation (Blewitt et al.
2006; Chong et al. 2007; Puschendorf et al. 2008). The
later studies suggest the existence of so-called ‘‘intrinsic’’
(nonacquired) intergenerational epigenetic programs that
support early embryonic development in mammals (Gill
et al. 2012). Here we study whether Ring1/Rnf2 and
Polycomb-repressive complex 1 (PRC1) constitute such
an intrinsic program that is essential for mediating trans-
mission of epigenetic information between generations.
Polycomb group (PcG) proteins are evolutionarily con-
served transcriptional repressors that were originally
identified in Drosophila as factors required for the main-
tenance but not establishment of transcriptional silenc-
ing of homeotic genes during embryonic development
(Ju ¨rgens 1985). More recently, PcG proteins have been
implicated in more dynamic modes of gene silencing
during development, dosage compensation, and genomic
imprinting and in tumorigenesis (Sparmann and van
Lohuizen 2006; Schuettengruber and Cavalli 2009).
Mammalian PcG proteins function in at least two
catalyze monoubiquitination of H2A (H2A119ub1) and
trimethylation of H3K27 (H3K27me3) (Simon and Kingston
2009). It has been shown that methylation by the PRC2
components E(Z)/EZH2 and recognition of H3K27me3 by
ESC/EED are required for propagation of the repressed
state (Hansen et al. 2008; Margueron et al. 2009), provid-
ing, in principle, a mechanism for epigenetic inheritance.
H3K27me3 is further thought to contribute to chromatin
targeting of canonical PRC1 complexes containing differ-
ent Cbx and Pcgf proteins (Gao et al. 2012; Morey et al.
2012; Tavares et al. 2012). Finally, PRC1 complexes may
repress transcription by compacting chromatin and/or
blocking RNA polymerase elongation, the latter possibly
through H2A119ub (Simon and Kingston 2009).
In mouse embryonic stem cells (ESCs), PcG proteins
and their associated histone modifications occupy genes
encoding transcription and signaling factors required
later during development (Boyer et al. 2006; Mikkelsen
et al. 2007; Endoh et al. 2008; Ku et al. 2008; Mohn et al.
2008). Similar genes are marked by H3K27me3 in pluri-
potent inner cell mass (ICM) cells from blastocyst stage
embryos (Dahl et al. 2010). However, it is unknown
whether PcG-mediated gene silencing in pluripotent ICM
cells and ESCs is newly established during preimplanta-
tion embryonic development or originates from PcG-
based repression in the germline. Compatible with the
second option, mature oocytes and spermatozoa contain
H3K27me3. In sperm, H3K27me3 marks genes that serve
developmental functions, reminiscent of Polycomb-bind-
ing profiles in somatic cells types (Hammoud et al. 2009;
Brykczynska et al. 2010).
In mice, zygotic deficiency of the core PRC1 compo-
nent Rnf2 (Ring1b) results in embryonic lethality during
gastrulation (Valk-Lingbeek et al. 2004). In contrast, the
Rnf2 paralog Ring1 (Ring1a) is not essential (del Mar
Lorente et al. 2000). Recently, we showed that various
PRC1 components are expressed in oocytes and mater-
nally provided to the embryo. As shown by immunoflu-
orescence analyses, Rnf2 is required for propagation and
establishment of global patterns of repressive chromatin
on maternal and paternal genomes, respectively, in early
embryos (Puschendorf et al. 2008). However, maternal
deficiency for Rnf2 does not aggravate the developmental
defects observed in embryos zygotically deficient for
Rnf2, suggesting no major role for maternally provided
PRC1 for early embryonic development (Terranova et al.
2008). Then again, Ring1, although lowly expressed, may
compensate for the loss of Rnf2 function during oogenesis
and early embryonic development, as observed in ESCs
(Endoh et al. 2008).
Here we address the maternal function of PRC1 in early
embryogenesis by deleting Rnf2 and Ring1 in growing
oocytes. We show that Ring1 does indeed compensate for
Rnf2 deficiency during oogenesis. Genetic ablation of
both paralogs results in loss of chromatin-bound PRC1 in
oocytes, induction of massive transcriptional misregula-
tion during oocyte growth, and a developmental arrest
at the two-cell stage of embryogenesis. Importantly, by
performing nuclear transfer experiments, we dissect the
components underlying this strong maternal effect. Our
data indicate that PRC1 functions during oogenesis to
specify maternal contributions in the cytoplasm as well
as on maternal chromosomes, both of which contribute
to the developmental competence of preimplantation
Maternal Ring1/Rnf2-deficient embryos do not develop
beyond the two-cell stage
To investigate the function of maternal PRC1 in early
embryogenesis, we deleted Ring1 and Rnf2 in developing
oocytes. To generate Ring1/Rnf2 double homozygous
mutant (dm) oocytes, we intercrossed animals that were
constitutively deficient for Ring1 with mice carrying
floxed alleles of Rnf2 (Rnf2F/F) and a transgenic allele of
Cre recombinase, which is specifically expressed in grow-
ing oocytes under the control of the Zona pellucida 3
promoter (Zp3-cre) (Supplemental Fig. S1A,B). We used
a Prm1-cre transgene, expressed in late haploid sperma-
tids, to generate Ring1/Rnf2 dm sperm (Supplemental Fig.
S1A,B). We subsequently fertilized Ring1/Rnf2 dm oocytes
withRing1/Rnf2 dmsperm toobtain embryosdeficientfor
both maternal (m?) and zygotic (z?) expression of both
paralogs (Ring1m?z?/Rnf2m?z?). We observed that devel-
opment of Ring1m?z?/Rnf2m?zembryos was abrogated at
the two-cell stage. Similarly, Ring1/Rnf2 dm oocytes fertil-
ized with wild-type sperm (Ring1m?z+/Rnf2m?z+embryos)
also arrested at the two-cell stage, suggesting a maternal
origin of the developmental phenotype (Fig. 1A,B).
PRC1 function during oogenesis is required
for early embryogenesis
The early arrest of Ring1m?z+/Rnf2m?z+embryos could
reflect a function of PRC1 during oogenesis, or alterna-
tively, maternally provided Ring1 and Rnf2 transcripts
and proteins may be required during early embryogenesis.
Role of Polycomb during oogenesis
GENES & DEVELOPMENT921
To address these possibilities, we first analyzed transcript
and protein expression of PRC1 components in growing
oocytes and early embryos (Supplemental Fig. S1C–F). We
found that in contrast to early embryos, where only Rnf2
mRNA is detected, growing oocytes express both Ring1
and Rnf2 (Supplemental Fig. S1C). Furthermore, in oo-
cytes, expression of either paralog is sufficient for nuclear
localization of PRC1 core components (e.g., Cbx2 and
Bmi1) and Rybp, a Rnf2-interacting protein (Gao et al.
2012; Hisada et al. 2012; Tavares et al. 2012), and for
supporting early embryogenesis (Supplemental Fig. S1D).
Interestingly, in Rnf2 single-mutant oocytes, we observed
increased Ring1 protein levels, while transcript levels
were unaltered, arguing for a post-transcriptional compen-
sation mechanism operating during oocyte growth, as in
Rnf2 mutant ESCs (Supplemental Fig. S1D; Endoh et al.
2008). These results suggest that Ring1-supported PRC1
function during oocyte growth enables Rnf2m?z+embryos
to develop beyond the two-cell stage.
We subsequently tested whether reconstitution of
PRC1 function in Ring1m?z+/Rnf2m?z+embryos could
alleviate their two-cell arrest. After microinjection of
Rnf2 mRNA into early zygotes, we observed nuclear lo-
calization of myc-tagged Rnf2 in control and Ring1m?z+/
Rnf2m?z+late zygotes (Fig. 2A) and two-cell embryos
(data not shown) as well as reappearance of Cbx2 and
Bmi1 (Fig. 2A; Supplemental Fig. S2A–D). All three PRC1
members showed wild-type-like chromatin localization
patterns as described before (Puschendorf et al. 2008),
arguing for reconstitution of a de novo chromatin-bound
PRC1 complex. Nonetheless, irrespective of the amount
of Rnf2 mRNA injected, we never observed a develop-
mental rescue of Ring1m?z+/Rnf2m?z+embryos (Fig. 2B;
Supplemental Fig. S2E). Thus, although we cannot exclude
the possibility that chromatin localization of reconstituted
PRC1 occurred too late during pronuclear formation in
mutant zygotes, the data suggest that Ring1/Rnf2 function
is likely required during oocyte growth to ensure proper
early embryonic development (Fig. 1C).
Maternal Ring1/Rnf2 deficiency delays meiotic
maturation and embryonic development
To dissect the cause of the embryonic arrest, we studied
cell cycle progression and transcription in mutant oo-
cytes and embryos. In contrast to embryogenesis, Ring1/
Rnf2 double deficiency in growing oocytes did not ma-
jorly impair oogenesis (Fig. 1B; Supplemental Fig. S3A).
Ring1?/?/Rnf2F/F/Zp3-cre and control females generated
similar numbers of phenotypically normal germinal ves-
icle (GV) oocytes (Fig. 1B). Meiotic maturation was af-
fected, however, with delays in GV breakdown and in
alignment of chromosomes during the first and second
meiotic divisions, possibly due to impaired spindle for-
mation (Supplemental Fig. S3B,C). Nonetheless, Ring1/
Rnf2 dm oocytes complete meiosis, as we isolated equiv-
Zp3-cre and control littermates (Fig. 1B).
Upon fertilization, the formation of maternal and
paternal pronuclei was delayed in Ring1m?z+/Rnf2m?z+
zygotes compared with control embryos (Fig. 3A; Supple-
mental Fig. S3D). Correspondingly, the first cleavage
interference contrast images of Ring1m?z+/Rnf2m+z+(further used as control; C) and Ring1m?z+/Rnf2m?z+(DM) embryos at embryonic
day 3.5. (B) Average number of Ring1?/?(C) and Ring1/Rnf2 dm (DM) oocytes and Ring1?/?(C) and Ring1m?z+/Rnf2m?z+(DM) embryos
isolated per female mouse at the indicated developmental stages. The number of female mice analyzed is shown in brackets. (C)
Cartoons illustrating developmental potential of embryos with different maternal and/or zygotic deficiencies for Ring1 and Rnf2. (m?)
Maternal deficiency; (z?) zygotic deficiency; (z+) zygotic proficiency from either paternal or both parental origins. Coloring of (pro)nuclei
indicates wild-type alleles of Ring1 and Rnf2 ([red] maternal; [blue] paternal; [green] maternal and paternal). The right panel shows
microinjection of Rnf2 mRNA. Yellow and white cytoplasms indicate the presence or absence of Ring1/Rnf2 proteins, respectively.
Loss of maternal Ring1 and Rnf2 impairs early embryonic development beyond the two-cell stage. (A) Differential
Posfai et al.
922 GENES & DEVELOPMENT
division was delayed (Supplemental Fig. S3E), and slightly
fewer mutant embryos entered the two-cell stage in vitro
and in vivo (Fig. 1B). Finally, development of Ring1m?z+/
Rnf2m?z+embryos ceased before the second cleavage
division, as we failed to detect signs of chromatin con-
densation, spindle formation, genome-wide acquisition of
phosphorylation at histone H3 Ser 10 (a marker of late
G2/M-phase chromatin), or nuclear localization of the
M-phase marker CyclinB1 (Supplemental Fig. S3F,G;
Ohashi et al. 2001).
Defective replication and S-phase checkpoint
activation in Ring1m?z+/Rnf2m?z+embryos
To further delineate the time point of cell cycle arrest, we
studied DNA replication in Ring1m?z+/Rnf2m?z+em-
(a deoxyribonucleotide analog) revealed that Ring1m?z+/
Rnf2m?z+zygotes entered S phase with a delay and in a
less synchronous fashion compared with control em-
bryos. Notably, the majority of Ring1m?z+/Rnf2m?z+
embryos also entered the second S phase but did not exit
it, as BrdU incorporation continued even up to the time
when control embryos were engaged in the third round of
replication (Supplemental Fig. S4A). In line with this,
quantification of EdU incorporation (another deoxyribo-
nucleotide analog) showed that DNA synthesis was
markedly impaired in most Ring1m?z+/Rnf2m?z+two-cell
embryos compared with control embryos (Fig. 3B,C).
To address whether impaired replication would be due
to replication fork stalling, we stained two-cell embryos
with anti-Ser 139-phospho H2AX (gH2AX) antibody, a
marker of DNA damage known to accumulate in re-
sponse to replication stress (Smith et al. 2010). In control
two-cell embryos, gH2AX labeling changed dynamically
during cell cycle progression (Supplemental Fig. S4B).
While G1/early S-phase embryos exhibited only a few
gH2AX foci, the number increased drastically by mid/late
S phase, declined again by G2 phase, and disappeared
completely before M phase. Mitotic chromatin was heavily
labeled with gH2AX, as reported before (Ziegler-Birling
et al. 2009). In Ring1m?z+/Rnf2m?z+embryos, we observed
similar gH2AX patterns at G1/early S and mid-S phases
85% (n = 53) of control embryos showed a G2-phase-
like gH2AX pattern, almost 80% (n = 27) of Ring1m?z+/
Rnf2m?z+embryos still showed an S-phase-like gH2AX
pattern. These results support the notion that most
Ring1m?z+/Rnf2m?z+embryos do not finish S phase.
To test whether sustained gH2AX in Ring1m?z+/
Rnf2m?z+embryos would activate S-phase checkpoint
kinases, we stained embryos for the phosphorylated
forms of Chk1 and Chk2 and of proteins phosphorylated
by Atr/Atm kinases (Supplemental Fig. S4D–F). As con-
trols, we used g-irradiated and hydroxyurea (HU)-treated
control embryos that displayed a strong and intermediate
activation, respectively, of all checkpoint proteins exam-
ined and a corresponding increase in gH2AX levels.
Interestingly, in mid-two-cell (S-phase) control embryos,
we did not detect checkpoint kinase activation, although
gH2AX was abundant. In contrast, the kinases were
activated in 16 out of 19 Ring1m?z+/Rnf2m?z+late two-
cell stage embryos. Thus, these findings suggest that the
prolonged S-phase in Ring1m?z+/Rnf2m?z+embryos is in
part due to reduced DNA synthesis, triggering an intra-
S-phase checkpoint response that may underlie the two-
cell arrest observed in these embryos.
Zygotic genome activation (ZGA) is severely impaired
in Ring1m?z+/Rnf2m?z+two-cell embryos
In mice, major ZGA occurs in two-cell embryos and is
essential for progression beyond the two-cell stage. To
examine whether ZGA is affected, we carried out ge-
nome-wide expression profiling of control (Ring1m?z+/
Rnf2m+z+) and Ring1m?z+/Rnf2m?z+late two-cell embryos
cultured with or without the transcriptional elongation
inhibitor a-amanitin (Fig. 4A). We found 3676 probe sets
that were a-amanitin-sensitive in control embryos, repre-
senting de novo activated genes. In contrast, only 909 probe
sets were a-amanitin-sensitive in Ring1m?z+/Rnf2m?z+
embryos, with 92 being inappropriately de novo acti-
vated. Together, ZGA is severely impaired in Ring1m?z+/
Rnf2m?z+embryos. We speculate that this impairment
contributes to the developmental arrest.
embryonic development. (A) Microinjection of myc-tagged
Rnf2 mRNA into Ring1m?z+/Rnf2m?z+zygotes leads to de novo
PRC1 complex formation. Immunofluorescence analyses of
control and Ring1m?z+/Rnf2m?z+zygotes microinjected with
water or with myc-tagged Rnf2 mRNA. Embryos were stained
with anti-myc antibody to detect injected myc-tagged Rnf2, and
with anti-Cbx2 and anti-Bmi1 antibodies to visualize reconsti-
tution of chromatin-bound PRC1. For some embryos, focal
planes of two parental pronuclei were merged into one image.
Bar, 20 mm. (B) Microinjection of 25 ng of myc-tagged Rnf2
mRNA into early zygotes does not alleviate the developmental
arrest at the two-cell stage. Diagram shows developmental
potential of control and Ring1m?z+/Rnf2m?z+embryos at em-
bryonic day 3.5 that had been microinjected at the early zygote
stage with water or Rnf2 mRNA.
PRC1 function during oogenesis is required for
Role of Polycomb during oogenesis
GENES & DEVELOPMENT 923
Genome-wide transcriptional misregulation
in Ring1/Rnf2 dm oocytes
To elucidate the mechanism underlying aberrant replica-
tion and ZGA in Ring1m?z+/Rnf2m?z+embryos, we sub-
sequently investigated the effect of Ring1/Rnf2 deficiency
on transcription during oogenesis. We observed that
transcription is properly shut down in Ring1/Rnf2 dm GV
oocytes, as assessed by immunofluorescence staining for
RNA polymerase II (RNAPII) (Abe et al. 2010) as well as
microinjected BrUTP, a ribonucleotide analog incorpo-
rated into nascent RNA (Supplemental Fig. S5A,B). To
examine gene-specific expression defects during oocyte
growth, we determined genome-wide mRNA levels in
GV oocytes, which naturally store the majority of tran-
scripts produced during the growing phase for subsequent
meiotic maturationandearly embryogenesis. We observed
that 2563 probe sets were misexpressed in Ring1/Rnf2 dm
oocytes, 60% of which wereup-regulated.Incontrast, only
165 and 92probe setswere misregulatedin Ring1 andRnf2
single mutants, respectively, suggesting, at least for some
Supplemental Fig. S6A,B). Single-gene analyses confirmed
the mRNA profiling results (Fig. 6A, below; Supplemental
Figs. S6C, S7A). These findings demonstrate that Ring1
and Rnf2 serve similar, mostly redundant, gene regulatory
functions during oogenesis.
Aberrant maternal transcripts are transmitted
to the embryo
To address whether transcripts misregulated in Ring1/
Rnf2 dm GV oocytes may contribute to the two-cell
embryonic arrest, we compared transcriptomes of Ring1/
Rnf2 dm GV oocytes and Ring1m?z+/Rnf2m?z+two-
cell embryos. Among the 989 probe sets up-regulated in
Ring1m?z+/Rnf2m?z+embryos, just 24 were only de novo
transcribed in the embryo (being a-amanitin-sensitive). In
contrast, 953 had been expressed in Ring1/Rnf2 dm GV
oocytes, with 273 even being up-regulated in Ring1/Rnf2
of transcripts up-regulated in double-mutant two-cell em-
bryos are indeed inherited from the Ring1/Rnf2 dm oocyte.
In contrast, among 2767 probe sets down-regulated in
Ring1m?z+/Rnf2m?z+embryos, only 34 showed reduced
transcript levels in Ring1/Rnf2 dm oocytes. Instead, 641
probe sets were zygotically activated in Ring1m?z+/
Rnf2m?z+and control embryos, while the remaining
1793 were part of the ZGA program in control embryos
only. These data argue that reduced transcript levels in
to activate gene expression in the course of ZGA (Fig. 4C).
In sum, Ring1/Rnf2 deficiency in oocytes effectively
alters the transcriptome of Ring1m?z+/Rnf2m?z+two-cell
embryos by providing extra maternal transcripts while
Genes up-regulated in Ring1/Rnf2 dm GV oocytes
are likely Polycomb targets
Gene ontology (GO) analyses indicated that genes up-
regulated in Ring1/Rnf2 dm oocytes are overrepresented
for developmental gene functions, similar to genes bound
by PRC1 and PRC2 proteins in ESCs and differentiated
somatic cells (Fig. 5A; Supplemental Table S1; Boyer et al.
2006). Additionally, transcriptome analyses showed that
a significant number of genes are commonly misregu-
lated in Ring1/Rnf2 dm GV oocytes and in mouse ESCs
deficient for the PcG components Eed and Rnf2 (P-value <
2 310?16) (Supplemental Table S2; Leeb et al. 2010).
Furthermore, significantly more of the up-regulated genes
Ring1m?z+/Rnf2m?z+embryos. (A) Ring1m?z+/Rnf2m?z+
zygotes stained with DAPI show a delay in pro-
nuclear formation at 5 h post-insemination. For some
embryos, focal planes of two parental pronuclei were
merged into one image. Bars, 20 mm. (B) Timing of
cell cycle phases of two-cell embryos and definitions
for early, mid, late, and very late two-cell stages. (C)
Quantification of DNA synthesis in control and
Ring1m?z+/Rnf2m?z+two-cell and four-cell embryos
cultured in the presence of EdU for the indicated
intervals. EdU was quantified by measuring total
fluorescent signal in both nuclei of two-cell embryos
or in two randomly chosen nuclei of a four-cell
embryo (indicated by two adjacent bars).
Aberrant cell cycle progression in
Posfai et al.
924 GENES & DEVELOPMENT
than down-regulated genes were bound by Rnf2 in ESCs
(P-value = 4.9 3 10?8versus P-value = 0.00192) (Supple-
mental Table S3; Endoh et al. 2008).
Finally, genes marked by H3K27me3 in human sperm,
mouse ESCs, or mouse embryonic fibroblasts (MEFs)
(Mikkelsen et al. 2007; Hammoud et al. 2009) are more
frequently up-regulated in Ring1/Rnf2 dm oocytes than
genes lacking H3K27me3 on their promoters in these
cells (Fig. 5B). Together, these data support the notion
that many genes up-regulated in Ring1/Rnf2 dm GV
oocytes are direct targets of PRC1 during oogenesis,
elucidating for the first time a function for PRC1 in the
Constitutive and dynamic gene repression by PRC1
during oogenesis and embryogenesis
To investigate during which developmental periods
Ring1 and Rnf2 regulate transcription, we compared the
expression status of probe sets misregulated in Ring1/
Rnf2 dm fully grown GV oocytes to the temporal expres-
sion pattern of genes at subsequent stages of normal
oogenesis (Supplemental Fig. S1A; Zeng et al. 2004; Pan
et al. 2005; Zeng and Schultz 2005). We classified loci as
either not expressed or expressed early, late, or stably
during normal oogenesis. We observed that 44% of probe
sets up-regulated in Ring1/Rnf2 dm GV oocytes are not
expressed during normal oogenesis and that 28% are only
active at early stages of oogenesis (Supplemental Fig. S6D).
GO term studies revealed a clear overrepresentation of
developmental gene functions among up-regulated tran-
scripts that are normally never expressed during oogen-
esis (Supplemental Fig. S6E; Supplemental Table S4).
Quantitative PCR (qPCR) analyses confirmed that tran-
script levels of three lineage markers normally expressed
during embryogenesis (Eomes, Gata4, and Pax6) are up-
regulated in Ring1/Rnf2 dm oocytes from the primary
follicle stage onward, concurrent with Zp3-cre expres-
sion driving deletion of Rnf2 (Fig. 6A; Supplemental Fig.
S7A). Functions in the cytoplasm, organelles, and apo-
ptosis were overrepresented for up-regulated genes that
normally become repressed during oocyte growth (Sup-
plemental Fig. S6E; Supplemental Table S4), indicating
special physiological functions for PRC1-mediated gene
repression during oogenesis as well. In all, the majority
of up-regulated transcripts are normally repressed by
PRC1 throughout oogenesis or become repressed during
Furthermore, we correlated expression states of probe
sets during normal oogenesis and preimplantation em-
bryogenesis (Fig. 5C; Supplemental Fig. S1A, S6D; Zeng
et al. 2004; Zeng and Schultz 2005). These analyses
indicate that Ring1/Rnf2 repress different gene sets either
constitutively or dynamically during oogenesis and likely
early embryogenesis. Hox genes are examples of consti-
tutively repressed loci, while genes like Sox2, Klf4, Eomes,
Analyses of ZGA by transcriptome profiling of untreated, a-amanitin-treated control, and Ring1m?z+/Rnf2m?z+late two-cell embryos.
Venn diagram shows overlap among a-amanitin-sensitive probe sets ($1.5-fold in untreated vs. a-amanitin-treated; P-value < 0.05) in
control (dark-green) and Ring1m?z+/Rnf2m?z+(light green) embryos. (B) Analyses of gene expression in wild-type, Ring1 mutant, Rnf2
mutant, and Ring1/Rnf2 dm GV oocytes. Venn diagrams show overlap among probe sets either up-regulated or down-regulated in Rnf2
mutant, Ring1 mutant, and Ring1/Rnf2 dm GV oocytes compared with wild type ($1.5-fold; P-value < 0.05). (C) Origin of transcripts
misregulated in Ring1m?z+/Rnf2m?z+late two-cell embryos. (Left diagram) For probe sets up-regulated in Ring1m?z+/Rnf2m?z+versus
control two-cell embryos, the majority was expressed in control and Ring1/Rnf2 dm GV oocytes (blue), with some up-regulated in Ring1/
Rnf2 dm GV oocytes (dark red). Only a minority is de novo transcribed (a-amanitin-sensitive) in Ring1m?z+/Rnf2m?z+two-cell embryos
(light green). (Right diagram) For probe sets down-regulated in Ring1m?z+/Rnf2m?z+versus control two-cell embryos, the majority is not de
novo transcribed (a-amanitin-sensitive) to a level observed in control embryos (light and dark green). Only a few probe sets were down-
regulated in Ring1/Rnf2 dm GV oocytes (light red). For all comparisons, a $1.5-fold difference with a P-value of <0.05 was used.
Aberrant gene expression in Ring1/Rnf2 dm GV oocytes and impaired ZGA in Ring1m?z+/Rnf2m?z+two-cell embryos. (A)
Role of Polycomb during oogenesis
GENES & DEVELOPMENT925
Gata4, and Cebpa, marking lineage specification during
preimplantation development, are repressed by PRC1 in
oocytes (Supplemental Figs. S6C, S7A).
Translational repression of Ring1/Rnf2 controlled
We subsequently investigated the fate of transcripts up-
regulated in Ring1/Rnf2 dm GV oocytes. We analyzed
protein expression of key developmental regulators Eomes,
Gata4, and Pax6 in oocytes, zygotes, and two-cell em-
bryos (Fig. 6B; Supplemental Fig. S7B). Consistent with
their role in blastocyst and embryonic differentiation, we
did not detect protein expression for any gene in control
oocytes and early embryos. Despite elevated transcript
levels in Ring1/Rnf2 dm GVoocytes (Fig. 6A; Supplemental
Fig. S7A), we also failed to detect these proteins in Ring1/
Rnf2 dm GV and M-II oocytes. In Ring1m?z+/Rnf2m?z+
zygotes and two-cell embryos, however, we detected
nuclear localization of Eomes, Gata4, and Pax6 proteins,
indicating translation of these aberrant maternal mes-
sengers only upon fertilization. These data indicate that
the impairment of Ring1/Rnf2-mediated transcriptional
repression during oogenesis is at least in part function-
ally suppressed via translational repression of aberrant
maternal transcripts, a widely conserved gene regulatory
mechanism functioning during oogenesis and maternal-
to-embryonic transition in a variety of species (Stitzel
and Seydoux 2007; Chen et al. 2011). The delay in
translation of aberrant transcripts may explain the
timing of the arrest during early embryogenesis and
not during oogenesis.
Both cytoplasmic and chromatin contributions defined
by Ring1 and Rnf2 in oocytes are needed for proper
Finally, we asked whether the developmental arrest of
Ring1m?z+/Rnf2m?z+embryos is mediated by abnormal
transcripts andproteinspresent inthe cytoplasm of Ring1/
Rnf2 dm oocytes, by inheritance of maternal chromo-
somes present in an aberrantly programmed chromatin
state, or by a combination of these two.
To test this, we exchanged maternal pronuclei (matPN)
between control and Ring1m?z+/Rnf2m?z+early zygotes,
thereby constructing diploid hybrid embryos (Fig. 7A).
Control experiments showed that embryos obtained by
exchanging matPNs between either control zygotes or
Ring1m?z+/Rnf2m?z+zygotes largely recapitulated the de-
velopmental phenotypes observed in naturally generated
embryos (Figs. 1B, 7B; Supplemental Fig. S8A), under-
scoring the technical feasibility of the transfers (as
demonstrated previously in Egli et al. 2007). Importantly,
87% of hybrid embryos composed of double-mutant
cytoplasm and a control matPN (cyto-DM/matPN-C)
failed to develop into morulae or blastocysts (P-value =
3.315 3 10?12). Furthermore, development of 69% of
hybrid embryos reconstructed with control cytoplasm
and a double-mutant matPN (cyto-C/matPN-DM) was
impaired (P-value = 7.085 3 10?8) (Fig. 7B). In both
conditions, we ruled out that the reductions in develop-
mental potential were due to the cytoplasm fraction
transferred along with a control or mutant matPN (Sup-
plemental Fig. S7B,C). These data reveal the importance
of PRC1 in defining proper cytoplasmic and possibly
nuclear contributions for embryonic development.
However, since pronuclear formation is likely con-
trolled by maternal cytoplasmic factors, our experiments
do not exclude the possibility that the poor developmen-
tal outcome of cyto-C/matPN-DM embryos is due to
aberrant cytoplasm present in mutant early zygotes,
affecting pronuclear formation and impairing the devel-
opmental potential of matPN-DMs before their transfer
into the cytoplasm of control embryos. To circumvent
this possibility, we exchanged M-II chromosomes between
control and Ring1/Rnf2 dm M-II oocytes, followed by
parthenogenetic activation of reconstructed oocytes (Fig.
7C; Supplemental Fig. S8).
target genes in Ring1/Rnf2 dm oocytes. (A) GO analysis of genes
up-regulated (red) or down-regulated (green) in Ring1/Rnf2 dm
GV oocytes. Fold overrepresentation indicates the observed
percentage of up-regulated genes in a particular GO category
over the percentage expected on the basis of all GO-annotated
genes on the array. P-values indicate significance of enrichment.
(B) Box plot showing log2 fold change of expression in Ring1/
Rnf2 dm over control GV oocytes for all probe sets (black) and
for probe sets classified as promoter H3K27me3-positive (dark
gray) or H3K27me3-negative (light gray) in chromatin immu-
noprecipitation coupled with deep sequencing (ChIP-seq) stud-
ies in human sperm (1:1 orthologs) (Hammoud et al. 2009),
mouse ESCs, and MEFs (Mikkelsen et al. 2007). (C) Percent of
probe sets detected (ON) or not detected (OFF) during normal
oogenesis (Pan et al. 2005) and early embryonic development
(Zeng et al. 2004; Zeng and Schultz 2005) among not changed,
up-regulated, or down-regulated probe sets in Ring1/Rnf2 dm GV
oocytes compared with control ($1.5-fold; P-value < 0.05).
Transcriptional misregulation of canonical PRC1
Posfai et al.
926 GENES & DEVELOPMENT
We observed that 87% of unmanipulated and 78% of
reconstructed (cyto-C/M-II-C) control embryos developed
to the morula and blastocyst stages following parthenoge-
netic activation (Fig. 7D; Supplemental Fig. S8A). Compa-
rable with the pronuclei exchange experiments, 98% of
hybrid oocytes composed of double-mutant cytoplasm and
control M-II chromosomes (cyto-DM/M-II-C) failed to
develop into morulae and blastocysts (P-value < 2.2 3
10?16), confirming the importance of proper cytoplasm for
Furthermore, 50% of hybrid embryos reconstructed
with control cytoplasm and double-mutant M-II chromo-
somes (cyto-C/M-II-DM) failed to develop into morulae
and blastocysts. Intriguingly, morphological analyses of
fertilization. (A) Quantitative real-time PCR analysis of Eomes transcripts in growing oocytes (from primary and secondary follicles),
GV oocytes, zygotes, and two-cell embryos that are wild type or single or double deficient for maternal Ring1 and/or Rnf2. Transcript
levels were normalized to LnmB1 control. Error bars indicate standard deviation based on two to three biological replicates. (B)
Immunofluorescence staining for Eomes in control and Ring1/Rnf2 dm GV and M-II oocytes, zygotes, and two-cell embryos. Numbers
of oocytes or embryos analyzed at each stage are indicated in brackets. Bar, 20 mm.
Transcripts of the developmental regulator Eomes, derepressed in Ring1/Rnf2 dm oocytes, are only translated after
defines maternal cytoplasmic and chromatin contri-
butions required for embryonic development. (A)
Cartoon illustrating generation of hybrid embryos
by exchanging matPN between early zygotes with
different genotypes. Wild-type paternal pronuclei
(patPN) are not exchanged. (B) Diagram showing
developmental potential (scored at embryonic day
4.5) of reconstructed zygotes. Control groups include
(1) control (Ring1m?z+) zygote containing a matPN
from another control zygote (cyto-C/matPN-C) and
(2) Ring1m?z+/Rnf2m?z+zygote containing a matPN
from another Ring1m?z+/Rnf2m?z+zygote (cyto-DM/
matPN-DM). Experimental groups include (1) matPN
of control zygote transferred
Rnf2m?z+zygote (cyto-DM/matPN-C) and (2) matPN
of Ring1m?z+/Rnf2m?z+zygote transferred into
control zygote (cyto-C/matPN-DM). Numbers of
reconstructed embryos analyzed are shown in
brackets. (C) Cartoon illustrating generation of
hybrid embryos by exchanging M-II chromosomes
between M-II oocytes with different genotypes.
M-II oocytes were activated to produce parthenoge-
netic embryos. (D) Diagram showing developmental
potential (scored at embryonic day 4.5) of partheno-
genetically activated reconstructed M-II oocytes.
Control group includes (enucleated) control (Ring1
mutant) M-II oocyte containing chromosomes from
another control M-II oocyte (cyto-C/M-II-C). Experi-
mental groups include (1) chromosomes of control
M-II oocyte transferred into (enucleated) Ring1/Rnf2
dm M-II oocyte (cyto-DM/M-II-C) and (2) chromo-
somes of Ring1/Rnf2 dm M-II oocyte transferred into
(enucleated) control M-II oocyte (cyto-C/M-II-DM).
Ring1/Rnf2 expression during oogenesis
Numbers of reconstructed embryos analyzed are shown in brackets. Note that parthenogenic activation causes a developmental arrest
more frequently at the zygotic than at the two-cell stage compared with embryonic activation by sperm fusion. (E,F) Immunofluorescence
staining for H2AK119ub1 in wild-type GVand MII oocytes and in Ring1/Rnf2 dm GVoocytes. Numbers of oocytes analyzed are indicated
Role of Polycomb during oogenesis
GENES & DEVELOPMENT927
cyto-C/M-II-DM- versus cyto-C/M-II-C-arrested zygotes
showed that equal percentages of mutant and control
reconstructed embryos failed to be activated. However,
three times more cyto-C/M-II-DM than control embryos
arrested at the one-cell stage after having formed pronu-
clei (P-value = 0.0002) (data not shown), revealing a strong
detrimental effect of double-mutant chromosomes on
early development, even prior to ZGA. These data may
reflect structural alterations in chromatin organization of
Ring1/Rnf2 dm M-II oocytes.
Together, our data indicate that Ring1/Rnf2-mediated
gene regulation during oogenesis is essential to provide
oocytes with proper maternal cytoplasmic factors that
support preimplantation development. Furthermore,
the chromosomal transfer data argue that Ring1 and
Rnf2 program maternal chromatin to sustain preimplan-
tation development. Importantly, the low percentage of
cyto-C/matPN-DM and cyto-C/M-II-DM embryos pro-
gressing to the blastocyst stage suggests that an aberrant
chromatin state inherited from Ring1/Rnf2 dm oocytes
can only be partially reprogrammed in early embryos by
wild-type maternal cytoplasmic factors.
In this study, we demonstrate that two PRC1 core com-
ponents, Ring1 and Rnf2, serve redundant gene regulatory
functions during mouse oogenesis that are essential for
early embryogenesis. Loss of Ring1/Rnf2 function in oo-
cytes causes a multitude of developmental defects. Impor-
tantly, reconstitution of chromatin-bound PRC1 in ma-
ternal mutant zygotes does not alleviate the developmental
arrest at the two-cell stage. Instead, we show that maternal
cytoplasmic and chromosomal contributions defined by
PRC1 during oogenesis confer intergenerational control
of early embryonic development.
In Ring1m?z+/Rnf2m?z+two-cell embryos, we observed
a prolonged S phase with reduced DNA synthesis rates
and activation of checkpoint kinases. Although PRC1 has
been linked to the DNA damage response (Liu et al. 2009),
the two-cell arrest is unlikely to be due to checkpoint
activation alone, since this was shown to only delay cell
cycle progression in two-cell embryos but not arrest
development, even if repair of DNA damage is incom-
plete (Shimura et al. 2002; Yukawa et al. 2007). Instead,
impairment of ZGA is known to cause a two-cell arrest
(Aoki et al. 1997), which is indeed majorly affected in late
Ring1m?z+/Rnf2m?z+two-cell embryos. We speculate
that both attenuation of cell cycle and defective ZGA
cause the developmental failure of Ring1m?z+/Rnf2m?z+
We found that PRC1-mediated repression is needed to
limit transcriptional activity in growing oocytes. Many of
the genes up-regulated in Ring1/Rnf2 dm oocytes are
regulated by PcG proteins in a variety of somatic cells,
including ESCs. For example, homeotic genes and lineage
specification factors such as Eomes, Gata4, Gata6, and
Krt8 are repressed by PRC1 in oocytes and ESCs. The
transcriptional up-regulation of such genes in Ring1/Rnf2
dm oocytes that normally control embryogenesis may
reflect the developmentally primed state of oocytes (sim-
ilar to ESCs) and underscores the importance of Polycomb
function during oogenesis in suppressing aberrant gene
activation, presumably driven by a variety of inducing
signals. Furthermore, several genes controlled by PRC1
during oogenesis are marked by H3K27me3 in human
spermatozoa and mouse round spermatids and sperma-
tozoa (Hammoud et al. 2009; Brykczynska et al. 2010; S
Erkek, M Hisano, M Stadler, and AHFM Peters, unpubl.).
Thus, our data show for the first time that PRC1 controls
repression of classes of genes in the female germline
similar to those in soma.
To determine whether changes in the pool of maternal
transcripts in the cytoplasm of fully grown GV oocytes,
resulting from transcriptional misregulation in Ring1/
Rnf2-deficient growing oocytes, would underlie the ob-
served embryonic defects, we performed nuclear transfer
experiments. We observed that cytoplasm of Ring1/Rnf2
maternally deficient oocytes or zygotes indeed triggered
a robust early developmental arrest in reconstructed
embryos. Protein analyses of three differentiation-induc-
ing factors transcriptionally up-regulated in Ring1/Rnf2
dm oocytes demonstrated that such ectopic transcripts
were only translated after fertilization. These data argue
that translational repression mechanisms are not per-
turbed in Ring1/Rnf2 dm growing oocytes and that aber-
rant messages are recruited for translation during meiotic
maturation or after fertilization, concomitant with the
timing of the appearance of phenotypic defects. Similarly,
Ring1/Rnf2 deficiency in oocytes may cause, directly or
indirectly, down-regulation of maternal factors required
in the nucleus, PcG proteins in oocytes are essential
factors to define the proper dormant maternal cytoplasmic
program that is executed during subsequent meiotic
maturation and early embryonic development. Likewise,
PcG proteins are required in ESCs for defining the proper
cytoplasm that enables reprogramming of nuclei of differ-
entiated cells upon cellular fusion (Pereira et al. 2010).
Thus, similar cytoplasmic feedback mechanisms may
operate at other stages of development to reinforce
chromatin-based repressive mechanisms in suppressing
changes in cell identity otherwise induced by stochastic
variations in transcriptional regulatory circuitries.
Our chromosome transfer experiments also strongly
suggest that intergenerational transmission of chromatin
states specified in oocytes by PRC1 is required for full
developmental competence of early embryos. In support
of this concept, comparison of wild-type expression states
between oocytes and early embryos shows that 37% of
genes up-regulated in Ring1/Rnf2-deficient GV oocytes
are normally repressed throughout oogenesis and early
embryogenesis. Furthermore, another 12% of up-regu-
lated genes become repressed during the course of normal
oocyte growth and remain repressed during preimplan-
tation development. Thus, these data strongly suggest
that PRC1 drives transcriptional repression all through
the reproductive life cycle of gametogenesis and early
embryogenesis, until expression is induced later during
Posfai et al.
928 GENES & DEVELOPMENT
Our data challenge the classical paradigm, based on
studies in Drosophila, which states that Polycomb func-
tions in maintaining transcriptionally repressed states
that were established by gene-specific transcriptional
repressors during embryonic development. Instead, our
chromosomal transfer data raise the intriguing possibility
that for a number of genes, PcG function is already
required in the mammalian germline to safeguard the
repressed state during early embryogenesis. In other words,
besides possible de novo establishment of a PRC1-con-
trolled transcriptional repressive state by sequence-specific
transcription factors and/or noncoding RNAs during
embryogenesis (Margueron and Reinberg 2011), inheri-
tance of germline prepatterned chromatin states is likely
required for faithful gene repression in the early embryo.
We therefore propose that Ring1/Rnf2 and PRC1 consti-
tute an intrinsic intergenerational epigenetic program,
which is essential for mediating transmission of epige-
netic information between generations.
What is the molecular entity of such an intrinsic inter-
generational epigenetic program? In somatic cells, ‘‘Poly-
comb-repressed’’ chromatin is generally characterized by
the presence of PRC1 and PRC2 proteins, H2AK119ub1
H3K27me3, and, at times, long noncoding RNAs and the
absence of elongating RNAPII and associated active
chromatin marks (Stock et al. 2007; Ku et al. 2008; Leeb
PRC1 and PRC2 proteins are dissociated from chromatin
in fully grown GV oocytes and during meiotic maturation
inheritance. Only upon fertilization do PRC1 components
become reloaded ontochromatin (Puschendorfet al.2008).
The significance of H2AK119ub1 in epigenetic memory
is also unclear, given that global down-regulation of this
mark during mitosis by the deubiquitinase Ubp-M/USP16
is required for mitotic progression (Joo et al. 2007) and that
the E3 ligase activity of Rnf2 is dispensable for higher-
order chromatin compaction and silencing of Hox genes
is present on chromatin, while the immunofluorescence
signal is absent in Ring1/Rnf2 dm GVoocytes, indicating
that Ring1 and Rnf2 are the main enzymes responsible for
H2AK119ub1 in growing oocytes (Fig. 7E). Intriguingly,
we failed to detect H2AK119ub1 on chromosomes of
wild-type M-II oocytes (Fig. 7F), arguing that this modi-
fication may not be involved in intergenerational trans-
mission of PRC1-mediated gene repression. In contrast,
germline PRC2-mediated H3K27me3 may function in
the intergenerational epigenetic memory of PRC1. The
chromodomain of the maternally provided PRC1 compo-
nent Cbx2 binds to H3K27me3 (Bernstein et al. 2006;
Puschendorf et al. 2008). Consistently, PRC1 binding to
euchromatin in zygotes is Ezh2-dependent and correlates
with levels of H3K27me3 (Puschendorf et al. 2008).
It is currently unclear what changes in chromatin
structed embryos carrying chromosomes from double-
to genes misregulated in mutant oocytes, having acquired
active chromatin characteristics (modifications and pro-
teins) or aberrant repressive chromatin (e.g., H3K9 and
DNA methylation) at up-regulated or down-regulated
genes, respectively. Other PRC1 targets, however, with
unaltered expression in mutant oocytes, may still be
marked by H3K27me3, thereby potentially reducing pro-
spective deleterious effects on embryonic development.
In agreement, we found that chromatin-localized levels of
PRC2 components (Ezh2, Eed, and Suz12) were unchanged
in Ring1/Rnf2 dm growing oocytes. Likewise, H3K27me3
was present in growing and GV Ring1/Rnf2 dm oocytes
(data not shown).
It is well appreciated that the cytoplasm of oocytes has
a remarkable capacity to reprogram chromatin. We found
that only 18% of cyto-C/matPN-DM and 30% of cyto-
C/M-II-DM reconstructed embryos (vs. 70%–80% of con-
trol embryos) were able to develop to the blastocyst stage,
arguing that aberrant chromatin inherited from Ring1/
Rnf2-deficient oocytes can only be in part reprogrammed
by wild-type maternal cytoplasmic factors. Qualitative
differences in the kind of cytoplasm used and the duration
efficiency, possibly explaining why more cyto-C/M-II-DM
than cyto-C/matPN-DM reconstructed embryos devel-
oped into morulae and blastocysts. For example, it is
thought that upon M-phase entry and nuclear membrane
breakdown, nuclear factors are released in the cytoplasm
that would promotereprogramming (Egli et al. 2007; Inoue
et al. 2008; Egli and Eggan 2010). However, the extent to
which epigenetic reprogramming and thus modulation
of intrinsic intergenerational epigenetic programs occur
during normal early embryonic development remains to
Materials and methods
Mice maternally and/or zygotically deficient for Rnf2 were gen-
erated using Zp3-cre and Prm1-cre transgenes to mediate de-
letion in growing oocytes or maturing spermatids, respectively.
We further used a constitutive mutant allele of Ring1 (for details,
see the Supplemental Material).
Collection, in vitro fertilization (IVF), and culture of mouse
oocytes and embryos
Oocytes/embryos were harvested from 5- to 12-wk-old females
in M2 (Sigma) or FHM medium (Chemicon) at the indicated time
points after hCG injection: GV oocyte, 46 h after PMSG, 2.5 mM
milrinone in medium; M-II oocyte, 14 or 18 h; late zygotes, 26 h;
early two-cell, 36 h; mid-two-cell, 42 h; late two-cell, 48 h; and
blastocyst stage, 94 h. For meiotic maturation experiments, GV
oocytes were transferred into M16 medium (Sigma) without
milrinone and harvested at indicated time points. For IVF, sperm
capacitation was carried out in human tubular fluid (HTF)
in capacitation medium for 2 h, and embryos were cultured in
FHM or KSOM + AA (Chemicon) in a humidified atmosphere of
5% CO2in air until required. Meiotically incompetent growing
oocytes (primary [diameter 50–60 mm] or secondary [diameter
>60 mm]) were collected from 12- to 14-d-old mice. Ovaries were
dissected in Ca2+- and Mg2+-free CZBT medium (CMF-CZBT)
Role of Polycomb during oogenesis
GENES & DEVELOPMENT 929
with 1 mg/mL collagenase (Worthington Biochemical Corp.) and
0.2 mg/mL DNase I (Sigma) and dissociated by repeated pipetting.
For inhibition of de novo transcription, 24 mg/mL a-amanitin was
added at 6 h post-fertilization (hpf) (after IVF) to culture medium.
For positive controls of checkpoint activation, control embryos
were subjected to 10 Gy of g-radiation or placed into culture
medium containing 0.02 M HU (Sigma, H8627) at 24 hpf.
Immunofluorescence stainings of GV and M-II oocytes and
embryos were carried out as described before (Puschendorf et al.
Ovaries from 12- to 14-d-old mice were frozen in Tissue-Tek
O.C.T. compound (Sakura Finetek) on dry ice. Ten-micrometer-
thick cryosections were cut with Microm HM355S. Cryosec-
tions were fixed on slides with 2% PFA in PBS (pH 7.4) for 10 min
on ice, permeabilized in 0.1% Triton-X100 in 0.1% sodium
citrate for 15 min, and blocked for 30 min at room temperature
in 0.1% Tween-20 in PBS containing 2% BSA and 5% normal
goat serum. Incubation with primary and secondary antibodies
as well as mounting were the same as for embryos.
Microscopy and image analysis
Immunofluorescence stainings were analyzed using a laser scan-
ning confocal microscope LSM510 META (Zeiss) and LSM510
software. Either a Z-series of 1.3-mm slices or one confocal slice
through the maximal radius of each (pro)nucleus was scanned.
Images were analyzed using Imaris (Bitplane) software and
exported as TIFF files. For data presentation, in case the planes
of maximal radius of maternal and paternal pronuclei were in
different focal planes, separate images of both pronuclei were
merged into a single image using Photoshop.
Differential interference contrast images were recorded with
a 2.45 Zeiss Z1 microscope.
Quantification of Rnf2 signal was done using ImageJ software
by summing fluorescent intensities of all Z-slices into one plane
and quantifying total fluorescent signal. Nuclear fluorescent
signal was corrected for background levels (cytoplasmic signal).
DNA replication analysis by BrdU or EdU incorporation
Time intervals for culture in the presence of BrdU (500 mM;
Sigma) and EdU (100 nM; Invitrogen Click-iT Alexa Fluor 488)
are indicated in Supplemental Figure S4A and Figure 3C, respec-
tively. Embryos were fixed at the end of each indicated interval.
For BrdU analysis, standard immunofluorescence protocol was
used with the addition of a denaturing step (25 min. at room
temperature, 4 M HCl in PBS/0.1% Triton X-100) and a neutral-
izing step (0.1 M Tris-HCl at pH 8.5) after permeabilization.
EdU was detected according to the manufacturer’s instructions
(Invitrogen Click-iTAlexa Fluor 488). Quantificationof BrdU sig-
nals was performed by eye (+ for strong, +/? for weak, and ? for
no BrdU incorporation), while EdU signals were quantified using
Microinjection of Rnf2 mRNA into early zygotes
N-terminally myc-tagged Rnf2 (NM_011277) cloned into a
pcDNA3.1-polyA vector (Yamagata et al. 2005) was in vitro
transcribed using the mMessage mMachine T7 kit (Ambion,
AM1344). Two picoliters to 4 pL of mRNA in nuclease-free water
(Ambion, AM9937) (0, 0.1, 1, 10, and 50 ng/mL for quantification
experiments; 0, 2, 25, and 50 ng/mL for developmental experi-
ments) was microinjected into early zygotes using the Eppendorf
FemtoJet injector system.
Microinjection of BrUTP into oocytes
GVoocytes were injected with 2–4 pL of 100 mM BrUTP (Sigma)
in TE and cultured in M16 (Sigma) with milrinone for 2 h before
Pronuclear and M-II chromatin transfer experiments
matPN were exchanged between 24 and 28 h post-hCG zygotes,
and M-II chromatin was exchanged in 13-h post-hCG M-II
oocytes in M2 medium + 5 mg/mL Cytochalasin B using an in-
verted microscope with micromanipulators (Olympus-Narishige
Micromanipulators MO-188, Nikon, and Burleigh PiezoDrill
system). For pronuclear transfers, the polar bodies were removed,
and the smaller matPN were aspirated and subsequently rein-
jected into the cytoplasm of receiver embryos from which the
matPN had been previously removed. For M-II transfers, chro-
matin and spindle were aspired and injected into previously
enucleated oocytes and allowed to recover for 15 min in FHM.
For parthenogenetic activation, oocytes were placed into Ca++-
free CZB medium containing 10 mM strontium chloride (CZB-
Sr) and 5 mg/mL Cytochalasin Bfor 5–6 h. Embryos were cultured
in FHM under mineral oil at 37°C under 5% CO2.
Quantitative real-time RT–PCR
Oocytes or embryoswere pooled from several mice, and RNAwas
isolated from batches of five to 50 using the PicoPure RNA
Isolation kit (KIT0202), adding 100 ng of Escherichia coli rRNA
as carrier and a bacterial probe set as spike (GeneChipEukaryotic
Poly-A RNA Control kit). Reverse transcription of RNA corre-
sponding to 20–25 oocytes or embryos was done using random
primers and SuperScript III Reverse Transcriptase (Invitrogen).
cDNA corresponding to 0.4 oocytes or embryos was used for
each qPCR reaction using SYBR Green PCR Master mix (Applied
Biosystem) and ABI Prism 7000 Real-Time PCR machine. Mea-
surements were performed on at least two biological replicates
from independent isolations and were normalized against endog-
enous LnmB1 and to exogenous bacterial spike gene Thr (data not
Expression profiling of late two-cell embryos and GV oocytes
and data analysis
IVF and in vitro cultured (in the presence of a-amanitin [as
described before] or not) late two-cell embryos from several mice
were harvested at 35 hpf in batches of 40 embryos, three bio-
logical replicates per genotype/treatment condition. GV oocytes
were pooled from several mice in batches of 50 oocytes, three
biological replicates per genotype. RNA was isolated using the
PicoPure RNA Isolation kit (KIT0202, Stratagene). RNA quality
was assessed with the Agilent 2100 Bioanalyzer and RNA 6000
Pico Chip. RNA was converted into OmniPlex WTA cDNA
libraries and amplified by WTA PCR using the TransPlex Whole
Transcriptome Amplification kit (WTA1, Sigma) following the
manufacturer’s instructions with minor modifications. cDNA
was purified using the GeneChip cDNA Sample Cleanup module
(Affymetrix). The labeling, fragmentation, and hybridization of
cDNA were performed according to Affymetrix’s instructions
(GeneChip Whole Transcription Sense Target Labeling technical
manual, revision 2) with minor modifications. Samples were
hybridized to Mouse Gene 1.0 arrays from Affymetrix. Expression
data are available at NCBI Gene Expression Omnibus (GEO).
Microarray quality control and analysis were carried out in R
2.10.0 and Bioconductor 2.5. Briefly, array quality was assessed
using the ‘‘ArrayQualityMetrics’’ package. GV oocyte raw data
Posfai et al.
930 GENES & DEVELOPMENT
were normalized with RMA using the ‘‘affy’’ package, and dif-
ferentially expressed genes were identified using the empirical
Bayes method (F test) (LIMMA) and P-values adjusted for false
discovery rate (FDR) with the Benjamini and Hochberg correc-
tion. Probe sets with a log2 average contrast signal of at least 3,
a P-value of <0.05, and an absolute linear fold change of at least
1.5-fold were selected. Two-cell raw data were normalized by the
vsnrma function of the ‘‘vsn’’package. Probe sets with an average
contrast signal of at least 4.5, a P-value of <0.05, and an absolute
linear fold change of at least 1.5-fold were selected.
P-values for enriched GO terms were obtained using GO Stat
(http://gostat.wehi.edu.au). All overrepresented and underrepre-
sented GO terms with a P-value of <0.05 were considered.
Clustering criteria 2 was used, meaning that GO categories that
do not differ by two or more genes are shown together. The raw
expression data of GVoocytes and two-cell embryos are available
at GEO (GSE23033 and GSE28710).
We thank Arie Otte (Swammerdam Institute for Life Sciences,
University of Amsterdam, The Netherlands) for providing antisera,
and Kazuo Yamagata (CDB, RIKEN, Japan) for the pcDNA3.1-
polyA vector. We are grateful to Laurent Gelman (microscopy
and imaging), Stephane Thiry (functional genomics), Michael
Stadler (bioinformatics), Fred Zilbermann, and the FMI animal
facility for excellent assistance, and Serap Erkek for computa-
tional support. We thank Peter de Boer and members of the
Peters laboratory for critical reading of the manuscript, and
Dirk Schu ¨beler and coworkers for fruitful discussions. Research
in the Peters laboratory has been supported by the Novartis
Research Foundation, the Swiss National Science Foundation
(31003A_125386 and NRP 63–Stem Cells and Regenerative
Medicine), SystemsX.ch (Cell Plasticity), the Japanese Swiss
Science and Technology Cooperation Program, the European
Network of Excellence ‘‘The Epigenome,’’ and the EMBO YIP
program. E.P., R.K., J.S., N.B., and A.H.F.M.P. conceived and
designed the experiments. E.P., R.K., V.B., J.S., E.C., Z.L., and
M.T. performed the experiments. E.P., R.K., T.C.R., J.S., N.B., and
A.H.F.M.P. analyzed the data. M.v.L. provided conditionally
deficient Rnf2 mice and antibodies. M.V. provided Ring1-
deficient mice and antibodies. E.P. and A.H.F.M.P. wrote the
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