Conservation of methylation reprogramming in
mammalian development: Aberrant
reprogramming in cloned embryos
Wendy Dean*, Fa ´tima Santos*, Miodrag Stojkovic†, Valeri Zakhartchenko†, Jo ¨rn Walter‡§, Eckhard Wolf†,
and Wolf Reik*¶
*Laboratory of Developmental Genetics and Imprinting, Developmental Genetics Program, Babraham Institute, Cambridge CB2 4AT, United Kingdom;
†Institute of Molecular Animal Breeding, Gene Centre, Ludwig-Maximilian University, Munich, Germany;‡Max-Planck-Institut fu ¨r Molekulare Genetik,
Ihnestrasse 73, 14195 Berlin, Germany; and§Universita ¨t des Saarlandes, Genetik, 66041 Saarbru ¨cken, Germany
Communicated by Shirley M. Tilghman, Princeton University, Princeton, NJ, October 3, 2001 (received for review August 9, 2001)
Mouse embryos undergo genome-wide methylation reprogram-
ming by demethylation in early preimplantation development,
wide reprogramming is conserved in several mammalian species
and ask whether it also occurs in embryos cloned with the use of
highly methylated somatic donor nuclei. Normal bovine, rat, and
pig zygotes showed a demethylated paternal genome, suggesting
active demethylation. In bovine embryos methylation was further
reduced during cleavage up to the eight-cell stage, and this
reduction in methylation was followed by de novo methylation by
the 16-cell stage. In cloned one-cell embryos there was a reduction
in methylation consistent with active demethylation, but no fur-
ther demethylation occurred subsequently. Instead, de novo meth-
ylation and nuclear reorganization of methylation patterns resem-
bling those of differentiated cells occurred precociously in many
cloned embryos. Cloned, but not normal, morulae had highly
methylated nuclei in all blastomeres that resembled those of the
fibroblast donor cells. Our study shows that epigenetic reprogram-
ming occurs aberrantly in most cloned embryos; incomplete repro-
gramming may contribute to the low efficiency of cloning.
in a number of key genome functions (1, 2). These include roles
in imprinting, X chromosome inactivation, genome stability,
silencing of retrotransposons, and inactivation of genes in can-
cers. Whether methylation also has a role in regulating gene
expression during development is still debated (2, 3). In the
mouse, mutations in the genes for the maintenance methyltrans-
ferase, Dnmt1 (4), or the two de novo methyltransferases,
Dnmt3a and -b (5), result in genome demethylation and lethality
at postimplantation stages or after birth, possibly involving
Genomic methylation patterns in somatic differentiated cells
are generally stable and heritable by virtue of maintenance
methylation by Dnmt1 (2, 4). However, in the mouse there are
are reprogrammed genome wide (7). The first occurs in primor-
dial germ cells of both sexes and leads to rapid demethylation of
imprinted genes and single copy sequences, followed by de novo
methylation in male and female germ cells several days later (8).
This reprogramming of methylation patterns is essential for
imprinting and may be important for the erasure of acquired
epigenetic modifications (7, 8).
A similar reprogramming cycle occurs in mouse preimplan-
tation embryos. Only hours after fertilization, before DNA
replication in the fertilized embryo, the paternal genome un-
dergoes genome-wide demethylation (9, 10), which is likely to
occur by active demethylation, but the mechanisms are un-
known. The maternal genome in contrast does not become
actively demethylated in the zygote. Instead sequential stepwise
demethylation occurs during the first cleavage divisions (11–13);
pigenetic modification of DNA by methylation in mammals
occurs predominantly at CpG dinucleotides and is involved
this demethylation is likely to be caused by exclusion from the
nucleus of Dnmt1o (14). As a result of these two demethylation
novo methylation is thought to occur at some stage soon after
implantation (11). In contrast to the reprogramming that occurs
during germ cell development, however, imprinted genes are not
after implantation (15). The oocyte form of Dnmt1 has recently
been identified as being important for the maintenance of
imprinted gene methylation. This form is generally excluded
from the nucleus in preimplantation embryos, except in eight-
cell embryos. This nuclear localization for one cell cycle is
apparently important for the maintenance of imprinted methyl-
ation at the eight-cell stage (14).
All information about methylation reprogramming in mam-
malian preimplantation development is currently limited to the
mouse. However, in zebrafish (16) and in Xenopus (I. Stancheva,
O. El-Maarri, J. Walter, and R. Meehan, personal communica-
tion) there is no demethylation in early embryos, thus raising the
question of whether demethylation is restricted to mammals.
This is a particularly important question in light of recent
successes in cloning of various mammalian species (17–19).
Although cloning is possible now in mammals, the rate of success
of obtaining live young is very low, and it has been proposed that
epigenetic reprogramming of somatic donor nuclei is important
for attaining totipotency (15, 17–21). Recently a study on bovine
cloned preimplantation embryos that made use of methylation
analysis by bisulfite sequencing (21) found that some DNA
sequences were more highly methylated in cloned versus normal
morulae. Here we show that methylation reprogramming is
conserved in eutherian mammals, but that reprogramming of
methylated somatic donor nuclei occurs aberrantly in many
cloned preimplantation embryos, thus explaining how hyper-
methylation arises in cloned embryos on a genome-wide scale.
Materials and Methods
Collection of Mammalian Oocytes and Embryos. Mouse fertilized
oocytes and embryos were collected from superovulated females
on appropriate days for cleavage-stage embryos according to
standard procedures (22). Embryos used were derived from a
cross of (C57BL?6J ? CBA?Ca) F1females mated to (C57BL?
6J ? CBA?Ca) F1males. The day after mating is termed day 1.
Rat fertilized oocytes were collected from naturally mated
Wistar rats at ?12 h after fertilization. Fertilized pig oocytes
were obtained from artificially inseminated gilts that had been
superovulated. Fertilized oocytes were collected and centrifuged
Abbreviation: ICM, inner cell mass.
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§1734 solely to indicate this fact.
November 20, 2001 ?
vol. 98 ?
to visualize the pronuclei before fixation. The zonae were
removed with acidic Tyrode’s solution before further processing.
Bovine fertilized oocytes and embryos were obtained from in
vitro matured oocytes harvested from ovaries obtained from the
abattoir. Matured oocytes were fertilized in vitro and subse-
quently cultured before fixation.
Cloning of Bovine Embryos. The nuclear transfer procedure was
that described in ref. 23. Non-starved fetal fibroblasts were used
as nuclear donor cells.
Indirect Immunofluorescence. Fertilized oocytes and early preim-
plantation embryos were washed in PBS, fixed for 15 min in 4%
paraformaldehyde in PBS, and permeabilized with 0.2% Triton
X-100 in PBS for 15 min at room temperature. For the detection
with 2 M HCl at room temperature for 30 min and subsequently
neutralized for 10 min with 100 mM Tris?HCl buffer (pH 8.5)
after permeabilization. After extensive washing with 0.05%
Tween-20 in PBS, all samples were blocked overnight at 4°C in
1% BSA?0.05% Tween-20 in PBS. Anti-5-methyl-cytosine an-
tibodies (24) were detected by a secondary antibody coupled
with either Cy3 or Texas-Red, respectively (Jackson ImmunoRe-
search). DNA was stained with either the intercalating dye
YOYO-1 iodide (Molecular Probes) at 100 nM or 5 ?g?ml
4?,6-diamidino-2-phenylindole and mounted in 50% glycerol in
Digital Imaging Microscopy. Observations were performed with an
Olympus BX40 epifluorescence microscope. Images were re-
corded digitally with a high-resolution charge-coupled device
camera (F-View) and ANALYSIS 3.0 image analysis software (SIS
GmbH, Mu ¨nster, Germany). Greyscale images were pseudocol-
ored after capture by separate filter sets for YOYO-1, CY3?
Texas Red, and 4?,6-diamidino-2-phenylindole and merged with
ADOBE PHOTOSHOP 5.0 software.
Confocal Microscopy. Digital optical sections from preimplanta-
tion embryos (blastocysts) were recorded with a confocal laser
scanning Ultraview microscope (Perkin–Elmer). For each wave-
length a z series of 0.2 ?m slices were scanned and exported as
projected with IMAGEJ 1.19Z and pseudocolored with ADOBE
Conservation of Methylation Reprogramming. Genome-wide de-
methylation can be examined comprehensively with the use of
immunostaining with a 5-methyl cytosine antibody (10, 13, 24).
Here we used immunostaining of interphase nuclei in mamma-
lian preimplantation embryos. Interphase nuclei were chosen
because they give an overall impression of genome-wide meth-
ylation, and normal nuclear organization is retained. Normal
bovine, pig, and rat zygotes showed demethylation of the larger
(male) pronucleus, as in the mouse (Fig. 1). Because the sperm
genome is highly methylated in all of these species (25), this
finding shows that genome-wide demethylation of the paternal
genome is conserved in eutherian mammals. We next examined
bovine cleavage stage embryos in comparison with the mouse
(Fig. 2). From the two-cell to the eight-cell stage there was a
further reduction in methylation, consistent with passive de-
methylation occurring during DNA replication, as demonstrated
de novo methylation in bovine embryos from the eight-cell to the
16-cell stage, which resulted in a finely granular staining pattern
in many nuclei (Fig. 2 Aj and Ak, and Fig. 3Bc). This pattern
resembled that of the DNA itself, suggesting that the nuclear
methylation pattern reflects at least in part the distribution of
interphase chromosomes in the nucleus (Fig. 3B). In contrast,
mouse 16-cell embryos continued to remain demethylated, and
genome-wide de novo methylation occurred approximately four
(Fig. 2Af), regardless of whether in vivo developed or in vitro
cultured embryos were analyzed. As a result, bovine blastocysts
show considerably higher levels of methylation specifically in
trophectodermal cells (Fig. 2B). Thus although the basic events
of active demethylation in the zygote, and passive demethylation
during cleavage divisions, followed by de novo methylation,
appear to be conserved in mammals, their timing with respect to
developmental events can apparently be different.
Aberrant Reprogramming in Clones. Because the three basic meth-
ylation reprogramming events appeared to be conserved in
mammalian preimplantation embryos, it was important to de-
termine the extent to which a highly methylated somatic nucleus
would be reprogrammed during cloning. Bovine fetal fibroblast
nuclei were used as donors for cloned embryos (23); in previous
and parallel experiments these experiments typically resulted in
and b), rat (c and d), pig (e and f), and cow (g and h). DNA staining is in blue.
The larger of the two pronuclei is the male, except in cows, where they are of
the same size. In all cases embryos were collected and fixed before DNA
replication. (Scale bar, 20 ?m.)
Dean et al.
November 20, 2001 ?
vol. 98 ?
no. 24 ?
rates of development to blastocysts in the range of 30–50%, and
in live births in 2–5% (23). The fibroblast nuclei were uniformly
highly methylated but with a pattern that was concentrated in
much fewer but larger and more intense foci than in normal
16-cell embryos (Fig. 3Bi). The large intense foci have been
cells (26). Because there are fewer foci than there are centro-
meres, this finding means that centromeres of different chro-
mosomes are probably colocalized in specific areas. On intro-
duction of fibroblast nuclei into enucleated oocytes and
activation, pseudopronuclei are formed in the zygote (Fig. 3A).
These stained much less intensely than the female pronucleus in
the normal zygote, consistent with loss of methylation from the
somatic nucleus in the reconstituted zygote (Fig. 3A). The
comparison at the two-cell stage shows this point very clearly.
Whereas there was substantial staining in both nuclei in the
normal embryo, staining was considerably reduced in cloned
two-cell embryos (Fig. 3 Ab and Ah). Because the zygotic
considerable demethylation must have occurred.
After the two-cell stage, however, cloned embryos did not
appear to undergo further demethylation, in contrast to the
normal ones (Fig. 3). Instead, in cloned four-cell and eight-cell
embryos there was heterogeneity between individual embryos,
with their methylation patterns falling into two groups. In
approximately half of the embryos all nuclei stained relatively
dimly; they were comparable to cloned two-cell embryos. Strik-
ingly, however, in the other half all nuclei stained very brightly
and had clearly undergone de novo methylation by the four-cell
or eight-cell stage and thus were ahead of schedule (Fig. 3). This
precocious de novo methylation was associated with a charac-
teristic change in morphology of the methylation pattern. In
some of the cloned four-cell embryos, the finely granular pattern
that is characteristic of the normal 16-cell embryo was observed;
however, in others there was a significant change in the meth-
ylation pattern of the nucleus. This altered pattern persisted in
all subsequent stages evaluated; was characterized by methyl-
ation foci that were much fewer, larger, and more intense; and
was reminiscent of the donor fibroblast nuclei (Fig. 3 Bd–Bf).
Although we cannot exclude the possibility that these changes
are brought about by demethylation of specific regions and de
novo methylation of others, we feel it is more likely that the
change in pattern is brought about by a different intranuclear
organization of the DNA, because similar changes can be seen
when we stain for DNA only (Fig. 3B).
As a result of the aberrant reprogramming events in cloned
embryos, particularly of precocious de novo methylation, all
nuclei in cloned morulae stained very brightly, and the pattern
of staining was very similar to that of fibroblast donors (Fig.
3Bh). In contrast, normal morulae were much more heteroge-
neous in their levels of staining, and many nuclei stained only
dimly (Fig. 3Bg).
[(Inset) DNA stained to identify two pronuclei, green]. Thereafter the remaining decline in signal occurs in a stepwise fashion up to the morula stage (e). The
ICM, but not the trophectoderm, has undergone de novo methylation by the blastocyst stage (f). Bovine zygotes also show loss of methylation from one
with highly and moderately methylated nuclei (k) such that at the blastocyst stage (l) the ICM contains highly methylated nuclei and the trophectoderm
moderately methylated ones. (B) To better define the location of the methylated nuclei images are presented with the methylation signal (red) and the merged
image of the DNA (blue) superimposed on the methylation signal (pink). This superimposition of images clearly shows that in the mouse the ICM has become
remethylated, but in bovine nuclei both ICM and trophectoderm are methylated.
Demethylation and remethylation are conserved during preimplantation development. (A) Normal mouse (a–f) and bovine (g–l) embryos were stained
www.pnas.org?cgi?doi?10.1073?pnas.241522698Dean et al.
Our study shows that methylation reprogramming is conserved
in eutherian mammals and that somatic nuclei undergo some
genome-wide reprogramming events in cloned embryos, but that
in most cloned embryos aspects of reprogramming are aberrant
(Fig. 3). Rapid demethylation of the paternal genome in the
zygote is conserved in the eutherian mammals tested. The
mechanism of this presumably active demethylation process
remains unknown; recent studies in the mouse that made use of
an MBD2 knockout have excluded this protein as a candidate in
vivo (27). However, because there is no early demethylation in
Xenopus and zebrafish, organisms without imprinting, the
observed conservation in mammals lends further support to
the suggestion that paternal demethylation is related to im-
Further loss of methylation during cleavage-stage divisions is
consistent with passive demethylation (of predominantly the
maternal genome) occurring as in the mouse. Thus Dnmt1 is
presumably absent or excluded from the nucleus, which is similar
to the finding in mouse embryos. However, substantial de novo
methylation occurs in bovine embryos at the 8–16-cell stage.
This finding is remarkable for two reasons. One is that it
coincides with the major wave of transcriptional activation of the
embryonic genome (29), suggesting the possibility that de novo
methylation enzymes such as Dnmt3a or -b are activated at that
at which in the mouse Dnmt1 enters the nucleus for one cell
cycle, being important at this stage for maintenance of imprinted
methylation (14). The early de novo methylation in bovine
embryos leads to relatively higher levels of methylation in
trophectoderm cells of the blastocyt than in the mouse. How-
ever, de novo methylation does occur in mouse blastocysts but is
limited to ICM cells. This finding explains why mouse embryonic
stem cells are relatively highly methylated (5). Thus the first
embryos. (a) Zygotes 12 h after fertilization [(Inset) staining of the DNA; n ? 25, number of embryos analyzed]. (b) Two-cell embryos stain intensely (n ? 10).
(c and d) Four-cell embryos (n ? 15; c) and eight-cell embryos (d) show reduced staining (n ?10). (e) Ten- to sixteen-cell embryos undergo a dramatic increase
in methylation (n ?10). ( f) Morulae (?24 cells) maintain the high-intensity signal (n ? 10). (g–l) Cloned embryos. (g) Cloned one-cell embryos stain faintly (n ?
10). (Inset) DNA staining. (Lower Inset) Fibroblast donor nuclei. (h) Two-cell cloned embryos (n ? 15). (i and j) In four- (i) and eight-cell (j) clones there are two
populations of embryos with high and low staining, respectively (n ? 7?14), in each group. (k) Ten to sixteen cells (n ? 20). (l) Morula stage (n ? 10). (Scale bar,
50 ?m.) (B) Organization of methylation patterns in normal and cloned bovine embryos. (a–c) Normal bovine embryos at four- to 16-cell stage. (d–f) Cloned
bovine embryos at the 4- to 16-cell stage. Note the heterogeneous de novo methylation and fine granular staining in normal 16-cell embryos (c) and the
precocious de novo methylation and organization into fewer and more intense foci in cloned 4- to 16-cell embryos (d–f). (Insets) Merged images of methylation
(g) Normal morula stains heterogeneously. (Inset) Nuclei indicate the two patterns of staining. (h) Cloned morula stains homogeneously. (i) Methylation
organization in fetal fibroblast donor cells. (Scale bar, 20 ?m.) All insets are enlarged ?3.
Aberrant methylation patterns in cloned bovine preimplantation embryos. (Aa–Af) Anti-5-methyl cytosine immunofluorescence of normal bovine
Dean et al.
November 20, 2001 ?
vol. 98 ?
no. 24 ?
differentiation event in mammalian embryos (that of trophec- Download full-text
toderm and ICM cells) and the resulting loss of totipotency (of
Because in cattle extraembryonic tissues in the placenta are
required for a much longer time (more than 270 days) than in the
mouse (15 days), their higher methylation level may confer
added stability on the differentiated state.
Somatic donor nuclei appear to be considerably demethylated
in the recipient oocyte within hours of their introduction and
activation. This demethylation may occur by the same putative
active demethylation mechanism by which the paternal genome
is demethylated at fertilization, which may suggest that an
important trigger for demethylation is remodeling of chromatin
by factors present in the oocyte cytoplasm. Presence of the
chromatin factor imitation switch (ISWI) has recently been
shown to be important for remodeling in cloned Xenopus
embryos (30). If active demethylation occurs in the somatic
nucleus in clones, this raises the question of whether differential
methylation in imprinted genes will be protected against de-
methylation, or whether clones could have altered imprinting
patterns. Mice cloned from embryonic stem cells can have
altered imprinting patterns (31) because embryonic stem donor
cells appear to be epigenetically unstable (32). Imprinting de-
fects could lead to postimplantation failure of cloned embryos,
which often show characteristic placental abnormalities (17–19),
or to perinatal failures associated with abnormal functioning of
the cardiovascular system in particular (17–19), which are also
characteristic of altered imprinted gene expression.
Cloned embryos seemed to lack further passive demethyl-
ation, and a large proportion became precociously methylated de
novo at the four- and eight-cell stages. Although the bovine
Dnmts have not been extensively characterized, by analogy with
the mouse the oocyte form of Dnmt1 may be excluded from the
nucleus, whereas the somatic form of Dnmt1 will have been
introduced with the somatic nucleus and may remain associated
with it. This pattern of exclusion and association may result in
increased levels of Dnmt1, and passive demethylation may
therefore not occur in clones. De novo methylation is likely to be
caused by Dnmt3a and -b; whether these enzymes are present in
the oocyte cytoplasm or are made from embryonic transcripts is
not known. It is striking that precocious de novo methylation
occurred in cloned embryos mainly at the four-cell stage, at the
same stage at which precocious transcriptional activation of
somatic donor nuclei in cloned bovine embryos has been ob-
served (33). On the other hand, many somatic tissues continue
to express Dnmt3a and -b, so it is possible that transcription of
these genes is not silenced in transferred somatic nuclei.
Precocious de novo methylation and precocious nuclear reor-
ganization in cloned embryos may be independent events. On
the other hand, de novo methylation in cloned embryos at the
four-cell stage was accompanied by nuclear reorganization,
whereas in normal embryos de novo methylation at the 16-cell
stage was followed by nuclear reorganization at the morula to
blastocyst stage. It is therefore possible that genome-wide
methylation, nuclear reorganization, and differentiation are
intricately linked and lead to stable programs of gene expression
and repression (34). Perturbation of the normal timing of these
events, as observed in most cloned embryos here, may lead to
aberrant development, which would be consistent with the very
substantial losses of cloned embryos during preimplantation and
early postimplantation development (17–19). Cloned mouse
fetuses had normal patterns of X chromosome inactivation,
suggesting that this aspect of epigenetic reprogramming was
successful in embryos that survived (35). Our results show that
global epigenetic reprogramming of somatic nuclei is aberrant in
most preimplantation cloned embryos; the effects on expression
of individual genes, particularly imprinted ones, have yet to be
addressed. Our study strongly supports the view that correct
epigenetic reprogramming is necessary for successful and nor-
mal development of clones.
Note Added in Proof. A complementary analysis of metaphase chro-
mosomes in cloned embryos using the 5-methyl cytosine antibody has
been published by Bourc’his et al. (36).
We thank A. Niveleau for providing the antibody against 5-methyl
cytosine and P. Lipp for expert help with confocal microscopy. This work
was funded by Biotechnology and Biological Sciences Research Council
Grant GTH 12511.
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