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Cell Cycle 9:23, 1-14; December 1, 2010; © 2010 Landes Bioscience
Phosphorylation of histone H3 serine 10
in early mouse embryos
Active phosphorylation at late S phase and differential
effects of ZM447439 on first two embryonic mitoses
*Correspondence to: Ewa Borsuk; Email: email@example.com
Submitted: 10/20/10; Accepted: 10/22/10
Previously published online: www.landesbioscience.com/journals/cc/article/14023
Aurora kinases belong to the evolutionarily conserved family of
serine-threonine kinases, which play substantial role during mito-
sis.1-3 Yeast have only one such kinase, named Ipl1 in S. cerevisiae
and Ark1 in S. pombe.4,5 In C. elegans and in D. melanogaster two
Aurora kinases, A and B have been discovered,6-9 while mam-
mals have additionally Aurora C. Different stages of cell division
are regulated by members of this family. The main functions of
Aurora A are associated with centrosomes and the mitotic spindle
organization.10 Its activity is indispensable for a proper centro-
some duplication, maturation and separation11-13 as well as for a
bipolar spindle formation.14 During M phase Aurora A activity
is also required for the maintenance of a proper concentration
of cyclin B, the regulatory subunit of MPF. Aurora A stimulates
the synthesis15 and inhibits the degradation of cyclin B.16 Aurora
C is the less characterized. In mammals it has probably evolved
Cell division in mammalian cells is regulated by Aurora kinases. the activity of Aurora A is indispensable for correct
function of centrosomes and proper spindle formation, while Aurora B for chromosome biorientation and separation.
Aurora B is also responsible for the phosphorylation of histone H3 serine 10 (H3S10ph) from G2 to metaphase. Data
concerning the Aurora B activity and H3S10ph in embryonic cells are limited to primordial and maturing oocytes and
advanced pronuclei in zygotes. In the present study we have analyzed H3S10ph in 1- and 2-cell mouse embryos. We
show that H3S10 remains phosphorylated at anaphase and telophase of the second meiotic division, as well as during
the anaphase and telophase of the first and second embryonic mitoses. At late G1 H3S10 is dephosphorylated and
subsequently phosphorylated de novo at late S phase of the first and second cell cycle. these results show that the
H3S10 phosphorylation/dephosphorylation cycle in embryonic cells is different than in somatic cells. the behaviour of
thymocyte G0 nuclei introduced into ovulated oocytes and early 1-cell parthenogenotes confirms that kinases responsible
for de novo H3S10 phosphorylation, most probably Aurora B, are active until G1 of the first cell cycle of mouse embryo.
the inhibition of Aurora kinases by ZM447439 caused abnormalities both in the first and second mitoses. However, the
disturbances in each division differed, suggesting important differences in the control of these mitoses. In ZM447439-
treated mitotic zygotes Mad2 protein remained continuously present on kinetochores, what confirmed that spindle
checkpoint remained active.
Marta teperek-tkacz,1,† Maciej Meglicki,1 Michal pasternak,1 Jacek Z. Kubiak2 and ewa Borsuk1,*
1Department of embryology; Institute of Zoology; Faculty of Biology; University of Warsaw; Warsaw, poland; 2CNRS UMR 6061; Institute of Genetics & Development of Rennes;
University of Rennes 1; Cell Cycle Group; IFR 140 GFAS; Faculty of Medicine; Rennes, France
†present address: the Wellcome trust/Cancer Research UK Gurdon Institute; and Department of Zoology; University of Cambridge; Cambridge, UK
Key words: H3S10 phosphorylation, ZM447439, spindle assembly checkpoint, Mad2, Aurora kinases, karyokinesis
This manuscript has been published online, prior to printing. Once the issue is complete and page numbers have been assigned, the citation will change accordingly.
as a result of aurora B gene duplication. The structure and cell
localization of both kinases is similar in human mitotic cells.17
Moreover, Aurora C seems to be capable of substituting Aurora
B, as it can support the mitotic progression in cells depleted of
Aurora B kinase is a chromosomal passenger protein3,19-22
and is indispensable for chromosome biorientation and proper
segregation during cell division. At metaphase, when all chro-
mosomes are properly bioriented, Cdc20 activates the anaphase
promoting complex/cyclosome (APC/C), which ubiquitinates
securin and cyclin B targeting them for a proteasome-mediated
degradation. Dissociation of cyclin B from CDK1 inactivates
MPF,23 whereas the degradation of securin releases the prote-
ase activity of separase, directly responsible for sister chromatid
separation.24 The proper chromosome alignment is controlled by
a spindle assembly checkpoint (SAC), which prevents anaphase
onset until the last chromosome is correctly bioriented.3,25 Active
2 Cell Cycle Volume 9 Issue 23
oocyte maturation and at least some elements of cytostatic factor
(CSF)61-63 responsible for the MII-arrest, remain active in mouse
zygotes.64 Moreover, the transient metaphase arrest, observed
during the first mitosis and independent of the SAC signalling,
resembles the one occurring in MII oocytes.65
Data concerning the activity of Aurora kinases in mouse
embryos are limited. The H3S10Ph, which was shown to be
the marker of Aurora B activity, was observed by Wang et al.54
and Huang et al.66 in the chromatin of advanced zygotic pro-
nuclei. However, a question remains open whether in mouse
embryos this modification is present like in somatic cells, i.e.,
exclusively at G2 and M phases. The role of H3S10Ph and Aurora
kinases during early mitotic divisions of the mouse embryo is
also unknown. The inhibition of Aurora kinases during oocyte
maturation caused abnormalities of the spindle assembly and
functioning, leading to chromosome dispersion and/or forma-
tion of micronuclei.67,68 Similar inhibition in mitotically dividing
somatic cells blocked them in interphase or produced polyploid
cells,31,32 suggesting different roles of Aurora kinases in meiosis
and mitosis. Here we have analyzed the spatiotemporal local-
ization of H3S10Ph in one- and two-cell mouse embryos. We
show that the cycle of H3S10 phosphorylation/dephosphoryla-
tion differs from the one observed in somatic cells via prolonged
phosphorylation following anaphase. We show that the latter is
due to a sustained kinase, and not a lower phosphatase, activ-
ity. We demonstrate that the inhibition of Aurora kinases activ-
ity caused abnormalities in the first and in the second mitoses
but perturbations were different in each embryonic cycle. We
also confirm that Aurora kinases inhibition removes correction
of microtubule attachments to kinetochores and thus activates
H3S10Ph during the 1st and the 2nd cell cycle of mouse embryo.
The phosphorylation of H3S10, the well known target of Aurora
B, was analyzed in 250 1-cell embryos fixed at different time
points after hCG injection corresponding to all phases of the cell
cycle. The classification of zygotes into G1, S and G2 was based
not only on their age phCG but also on their morphology and
localization of pronuclei. In the group of embryos fixed 19–21
h phCG an intensive, diffused H3S10Ph staining was detected
all over the male and female chromatins in these zygotes, which
were still in the telophase of the 2nd meiotic division or had small
pronuclei located typically for the early G1 (Fig. 1A–A”). During
the progression of G1 phase H3S10 was gradually dephosphor-
ylated (Fig. 1B–B”). In the majority of zygotes fixed 23–24 h
phCG (77%), what corresponds to early/middle S phase, the sig-
nal of H3S10Ph was no longer observed (Fig. 1C–C”). In some
of the zygotes a weak signal of de novo H3S10 phosphorylation
was detected already 24 h phCG. It was observed mainly around
nucleolar precursor bodies of the male pronucleus. In the pro-
nuclei of zygotes fixed 2 h later (Fig. 1D–D”) such signal was
present in the majority of cases. As mentioned above, H3S10Ph
was first observed only in the male pronucleus. Next, it appeared
in both pronuclei (Fig. 1E–E”, arrows) and spread gradually onto
SAC promotes formation of an inhibitory complex of proteins
composed of Mad2, Mad3/BubR1, Bub1, Bub3 and Cdc20,
located on kinetochores. As soon as all chromosomes are properly
attached, SAC is inactivated and inhibitory proteins are displaced
from kinetochores.26-28 Aurora B recognizes improperly attached
chromosomes, phosphorylates the kinetochore-localized proteins
responsible for microtubule capturing and decreases their affin-
ity for microtubule binding.29 Consequently, unattached kineto-
chores are generated which turn on the SAC signalling.30 Aurora
B can also directly activate SAC signalling, by yet not fully elu-
cidated mechanism.19 In cells in which Aurora B was inhibited
Mad2, Bub1 and BubR1 were displaced from kinetochores.31-33
Aurora B activity is also indispensable for a correct chromo-
some-associated localization of separase and for the initiation of
separase-mediated chromatid separation.34 In addition, Aurora
B is responsible for the phosphorylation of proteins engaged in
One of the functions of Aurora B is the cell cycle dependent
phosphorylation of serine 10 of histone H3 (H3S10Ph).4,32,38-43 In
somatic cell nuclei H3S10Ph is detected for the first time in the G2
phase and reaches its maximum in metaphase chromosomes,44-46
accordingly with Aurora B activity.5,47,48 At the onset of anaphase
H3S10 is rapidly dephosphorylated.44-46,49,50 The role of this his-
tone modification is still controversial. Some data suggest that
H3S10 phosphorylation is indispensable for chromosome con-
densation.50,51 On the other hand, the inhibition of H3S10Ph in
mitotic cells40 did not block the process of chromatin condensa-
tion, although often the chromosomes were abnormal. H3S10Ph
is also engaged in the regulation of gene expression during inter-
phase. Phosphorylation of H3S10 at promoters and enhancers
generates a specific mechanism indispensable for transcription
of distinct genes. Kinases, other than Aurora, were shown to be
engaged in this regulation.52,53
The data concerning the phosphorylation of H3S10 in mouse
oocytes and embryos are limited and controversial. According to
Wang et al.54 H3S10Ph is present in the whole chromatin of pri-
mordial fully-grown oocytes and in the chromosomes of matur-
ing oocytes. In contrast, Swain et al.55 did not observe H3S10Ph
before 2 h after GVBD. The presence of H3S10Ph at anaphase
and telophase I differentiate maturing oocytes from mitotic
cells.54,55 No data is available concerning the H3S10 phosphory-
lation during the telophase of the 2nd meiotic division and pronu-
clei formation in mouse zygotes.
The cell cycle organisation is often modified during early
embryonic development. During the first cell cycle of mouse
embryo two haploid pronuclei, developed from sperm and oocyte
chromatins, exist independently in the same cytoplasm. Their
progression through the S phase, the onset of transcription and
the condensation of chromosomes at the beginning of the first
mitosis are slightly asynchronous.56-58 As a result of the unusu-
ally long first mitosis,59 maternal and paternal chromosomes are
segregated into sister blastomeres of 2-cell embryo. In the sec-
ond cell cycle, during which the transition from the maternal
to embryonic control occurs,60 both sets of chromatin are gath-
ered in a single nucleus for the first time. The progression of
the first cell cycle and first mitosis is controlled similarly to the
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dephosphorylation. In NIH3T3 cells already at late anaphase the
intensity of H3S10Ph signal was lower than in metaphase, and in
L929 cells the dephosphorylation started even earlier, because the
intensity of observed signal dropped already in early anaphase.
Consequently, in MEF and NIH3T3 cells a weak or very weak
signal of H3S10 phosphorylation was still present at telophase,
while in L929 cells serine 10 of histone H3 seemed to be com-
pletely dephoshorylated. In all three types of cells no signal of
H3S10 phosphorylation was ever observed at early G1 of the next
the whole chromatin during G2 (Fig. 1E–E”, asterisk). To
verify the changes in the level of the H3S10 phosphorylation,
the signal in the male and female pronuclei was measured
and compared. The measurements were performed only in
these embryos in which both pronuclei were well visible and
their identity was clearly obvious. The quantification of the
H3S10Ph signal (Fig. 2) visualizes graphically tendencies in
the gradual dephosphorylation of H3S10 in both pronuclei
during G1, finished in S phase and followed by de novo phos-
phorylation occurring first in the male pronucleus.
To correlate the reappearance of H3S10Ph with the pro-
gression of S phase, DNA replication was detected simul-
taneously with H3S10 phosphorylation. Sixty six zygotes
microinjected with dU analog were fixed after 20 min.
of culture. In none of analyzed 1-cell embryos, display-
ing replication pattern characteristic for an early/middle S
phase (reviewed in ref. 57; Fig. 3A) any detectable signal
of H3S10Ph has ever been observed (Fig. 3A’). However, it
appeared de novo in embryos, in which the pattern of dU
incorporation was typical for the late S phase (reviewed in
ref. 57; Fig. 3B). The earliest de novo H3S10Ph was detected
in late replicated regions of the chromatin (Fig. 3B–B”,
arrows). At the prophase of the first mitosis a strong signal of
H3S10Ph appeared in forming chromosomes (Fig. 4A–A”)
and increased till metaphase (Fig. 4B–B”). Surprisingly, a
very heavy signal persisted in chromosomes also during ana-
phase and telophase (Fig. 4C–C”) of the first mitosis.
Similar pattern of phosphorylation/dephosphorylation of
H3S10 was observed in the second cell cycle (153 embryos
analysed). It was correlated with the progression of the cell
cycle by simultaneous analysis of H3S10Ph and DNA rep-
lication in 94 embryos (see Sup. Material Figs. S1 and S2).
H3S10Ph in somatic cells. In the first as well as in the
second cell cycle of the mouse embryonic development de
novo phosphorylation of H3S10 began at the end of S phase
and was detected during G2 and the whole M phase includ-
ing ana/telophase. Dephosphorylation of H3S10 started not
earlier than at G1 of both cell cycles.
These results were contradictory to what has been
described for somatic cells, in which the rapid dephos-
phorylation of H3S10 in anaphase/telophase of mitosis
has been demonstrated.44,46,49 To verify that observed dif-
ferences are not due to unspecific labeling by the antibody,
we have analyzed the spatiotemporal pattern of H3S10Ph
in mouse embryonic fibroblasts (MEF) obtained from
13 days old embryo, NIH3T3 cell line of embryonic ori-
gin and L929 cell line originating from adult males. In the
majority of interphase cells of all three cell types H3S10 was not
phosphorylated (Fig. S3A’, S4A’ and S5A’). However, in some
of them the phosphorylated H3S10 was detected in the hetero-
chromatin regions (Fig. S3B’, arrows; Fig. S4B’, arrows and Fig.
S5B’, arrows). Starting with the prophase the signal of H3S10Ph
spread on the whole chromatin and reached its maximum at
metaphase (Fig. S3C’, D’, S4C’, D’ and S5C’, D’). In mitotic
MEFs H3S10 remained highly phosphorylated until telophase,
when the intensity of the signal dropped visibly, suggesting its
Figure 1. H3S10 phosphorylation in zygotes. pn♂-male pronucleus; pn♀-
female pronucleus; n-NpB; IIpb-second polar body. (A–A”) early G1 (18
hphCG), (B–B”) G1/S transition (21 hphCG), (C–C”) early/middle S (23 hphCG),
(D–D”) late S (25 hphCG), (e–e”) late G2 (28 hphCG). During early/middle S
phase H3S10 was dephosphorylated (C’). De novo H3S10 phosphorylation
appeared at the late S, in the condensed chromatin surrounding the NpB of
the male pronucleus (D’, arrow); weak signal of H3S10ph on the decondensed
chromatin at G2 (e’, asterisk). Bar: 20 μm.
4 Cell Cycle Volume 9 Issue 23
phosphorylation is due to the sustained kinase activity or
lowered phosphatase activity, we have analyzed H3S10Ph
in G0 thymocyte nuclei (negative for H3S10Ph in native
state) introduced by cell fusion into the cytoplasm of ovu-
lated oocytes and newly activated, parthenogenetic 1-cell
embryos. After introduction into the cytoplasm of MII
oocytes and early parthenogenotes these nuclei undergo
morphological and physiological changes, which have been
described in details in several papers.72-76
Thymocyte nuclei in ovulated oocytes. Thymocyte nuclei
introduced into the cytoplasm of ovulated oocytes under-
went premature chromosome condensation (PCC). The
chromosomes originating from the thymocyte nuclei
formed separate plates or were scattered in the oocyte cyto-
plasm (Fig. 5A, t+). They became all highly phosphory-
lated, similarly to the oocyte chromosomes (Fig. 5A’). The
H3S10Ph was never detected in the nuclei of non-fused
thymocytes, which remained attached to the oocyte plasma
membrane (Fig. 5A’, t-).
Thymocyte nuclei in parthenogenetic 1-cell embryos. The
majority of oocytes treated with ethanol extruded the sec-
ond polar body 45 min. later and formed a single female
pronucleus 3 h after activation. The thymocyte nuclei
introduced into anaphase II or early telophase II oocytes
underwent nuclear envelope breakdown (NEBD) and PCC.
However, they have never formed chromosomes, but clumps of
chromatin (Fig. 5B, t+), similar to the female, telophase II group
of chromatin (Fig. 5B, tII). The signal of H3S10 phosphoryla-
tion again became always very high in both types of chromatin
Two-three hours after PEG treatment female pronu-
clei (Fig. 5C) were already present, located in the vicin-
ity of the 2nd polar body. The chromatin of thymocyte
origin also formed interphase nuclei resembling female pronuclei
(Fig. 5C, arrow). In both types of nuclei the signal of H3S10Ph
was still present, although it was much weaker than observed at
telophase II (Fig. 5C’).
cell cycle. These results confirmed the specificity of the antibody
used in the embryo study and showed that the H3S10 phosphor-
ylation/dephosphorylation cycle in 1- and 2-cell embryos really
differed from somatic cells.
De novo H3S10 phosphorylation can occur during the
second meiotic division and in the G1 of the first cell cycle.
The H3S10 in the female and male chromatin of early zygotes
remained phosphorylated at least until early G1. However, H3S10
in the female chromosomes at MII was highly phosphorylated,
suggesting that the signal observed on female chromatin/pro-
nucleus could be a manifestation of its slow dephosphorylation
by some, not yet identified phosphatases. To verify whether de
novo H3S10 phosphorylation is possible in MII oocytes and in
early 1-cell embryos, as well as whether the persistence of this
Figure 2. Quantification of H3S10ph level in male and female pronuclei in 1-cell embryos at subsequent phases of cell cycle. Y axis shows H3S10ph
fluorescence signal intensity relative to background values. (m)-male pronuclei, (f)-female pronuclei. error bars show standard errors. Asterisks mark
statistically significant difference in the intensity of fluorescence between male and female pronuclei (p = 0.015; t-test).
Figure 3. DNA replication and H3S10 phosphorylation in zygotes. pn♂-male
pronucleus; pn♀-female pronucleus; n-NpB; IIpb-second polar body. At early
and middle S phase (A) H3S10 remained dephosphorylated (A’). In late S
phase (B) de novo phosphorylation of H3S10 appeared in the pronuclei (B’,
arrows). Bar: 20 μm.
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When fusion occurred at late telophase II the remod-
eling of thymocyte nuclei was less pronounced. They did
not undergo NEBD, their chromatin decondensed and the
nucleoli became well visible. These nuclei always remained
much smaller than female pronuclei (Fig. 5C, t+). The pres-
ence of phosphorylated H3S10 was detected in all thymo-
cyte nuclei, which underwent this type of remodeling (Fig.
5C’). Often the signal of H3S10Ph was heavier in these
nuclei than in the female pronuclei.
These results clearly showed that during completion of the
second meiotic division and transition to interphase, when
gradual dephosphorylation of female chromatin begins, the
cytoplasm retained its ability to continuously phosphorylate
serine 10 of histone H3, demonstrating that kinases respon-
sible for phosphorylation remained unusually active.
Are the DNA replication in pericentric heterochromatin
and the appearance of de novo H3S10Ph in these regions
interdependent? Results presented above showed that in 1-
and 2-cell mouse embryos de novo H3S10 phosphorylation
occurred for the first time in the pericentric heterochro-
matin regions around the time when they were replicated.
It seemed plausible that the presence of H3S10Ph in these
regions is required for the proper replication of their DNA.
To verify this hypothesis the pattern of DNA replication
was analyzed in 19 one-cell and 9 two-cell embryos, treated
with ZM447439 (ZM) an inhibitor of Aurora kinases activ-
ity. As Aurora B was shown to be responsible for H3S10Ph
(see Introduction) ZM is commonly used as an inhibitor
of this process.31,54,68,77 In preliminary experiments zygotes
(26 h phCG) were cultured for 4.5 h in the presence of differ-
ent concentrations of ZM (5, 10 and 20 μM) to optimize the
conditions, under which H3S10Ph will be inhibited. Only in
some embryos treated with ZM in concentration of 5 and 10
μM H3S10 was unphosphorylated (Fig. 6). In the rest, the sig-
nal of H3S10Ph was high and well defined, often very similar
to observed in non treated zygotes. Only the treatment with
ZM in concentration of 20 μM caused almost complete lack
of H3S10 phosphorylation in the majority of embryos (Fig.
6). For this reason in all the experiments presented below this
concentration of ZM was used to inhibit H3S10Ph in 1- and
To verify whether the appearance of H3S10Ph in the peri-
centric heterochromatin is required for the DNA replication in
these regions zygotes at 22 h phCG or 2-cell embryos at 34 h
phCG were placed in the medium containing ZM447439 or
DMSO (control embryos) and cultured for 3 hours. At late S
phase, Dig-11-dUTP was microinjected into their cytoplasm and
embryos were cultured for further 20 min. Immunocytochemical
analysis confirmed the effectiveness of ZM treatment. In zygotes
(Fig. 7B’) and 2-cell embryos (Fig. S6B’) embryos cultured in its
presence no phosphorylation of H3S10 in the pronuclei or blasto-
mere nuclei was observed. The lack of H3S10Ph in the pericentric
heterochromatin had not affected the pattern of DNA replication
in these regions. It was comparable with control embryos (com-
pare Fig. 7A and B; Fig. S6A and B). This result also shows that
1- and 2-cell embryos treated with ZM do not arrest at G1 or
Figure 4. H3S10 phosphorylation during first mitosis. IIpb-second polar body.
(A–A”) prophase, (B–B”) metaphase, (C–C”) telophase. the H3S10 remained
highly phosphorylated during all stages of mitotic division. Bar: 20 μm.
early S. They successfully reach late S phase and replicate hetero-
To verify whether the phosphorylation of H3S10 in the het-
erochromatin regions depends on their replication, the S-phase
entry and progression in 1- and 2-cell embryos was inhibited with
aphidicolin. A total of 77 zygotes at telophase II (19 hphCG)
were cultured in the presence of aphidicolin until the late S phase
(27 hphCG) or late G2 (30 hphCG). To inhibit effectively the
S phase in the second cell cycle 40 late zygotes (30 hphCG)
were transferred into M2 with aphidicolin and cultured until 46
hphCG. In control zygotes at late S phase (27 hphCG) a weak
signal of H3S10Ph was observed around NPBs, mainly in one,
most probably male pronucleus (Fig. 8A’, arrow). Interestingly,
at the same time the signal of H3S10Ph in aphidicolin treated
embryos was stronger than in control and in the majority of cases
was observed in both pronuclei (Fig. 8B’, arrows). At late G2 (30
hphCG) the signal of H3S10Ph in control zygotes was present
in both pronuclei around the NPBs and was much stronger than
at late S phase. In contrast, the intensity of H3S10Ph signal in
aphidicolin treated zygotes had not changed. This shows that the
lack of DNA synthesis in the heterochromatin regions in the first
cell cycle as well as in the second cell cycle (Fig. S7) had not
caused the inhibition of H3S10Ph. In the contrary, in zygotes
it triggered the earlier H3S10Ph in the female pronucleus and
transient increase in the signal intensity.
These results showed no simple and direct dependence
between the appearance of de novo H3S10 phosphorylation in
6 Cell Cycle Volume 9 Issue 23
pericentric heterochromatin of pronuclei and blastomere
nuclei and DNA replication of these regions.
The progression of the 1st and the 2nd mitoses is dif-
ferentially disturbed after treatment with ZM. The role
of H3S10Ph in the condensation of chromosomes and
progression of mitosis in somatic cells is controversial. To
verify whether in embryonic cells this modification is indis-
pensable for the chromosome formation and segregation,
the phosphorylation of H3S10 in 1- and 2-cell embryos at
G2 was inhibited with ZM. They were fixed when control
embryos started to cleave. In the control embryos neither
the first, nor the second mitosis was disturbed by the culture
conditions, whereas numerous abnormalities were observed
in ZM treated embryos. Interestingly, disorders observed in
the first mitosis were different than in the second mitosis.
The first mitosis of ZM-treated zygotes. None of the 121
zygotes treated with ZM cleaved to the 2-cell stage. The
analysis of the spindle formation and chromatids segregation
during the first mitosis revealed serious abnormalities con-
cerning both processes. The initial chromosome condensa-
tion in prophase was not disturbed (Fig. 9A–A”). However,
the organization of the metaphase plate was abnormal.
Always at least a couple of chromosomes were excluded
from the main group (Fig. 9B, arrows) and the mitotic
spindle was usually multipolar (3–4 spindle poles; Fig. 9B’,
arrowheads). Instead of the proper anaphase movement and
separation of sister chromatids to the opposite poles, the dis-
persion of two-chromatid chromosomes along the spindle
was observed (Fig. 9C–C”). Finally, the two-chromatid
chromosomes spread throughout the cytoplasm individually
or in small groups and transformed into multiple micronu-
clei (Fig. 9D–D”).
Twenty five 1-cell embryos were cultured in the presence
of ZM from the G2 of the first cell cycle until the time of the
second cleavage division to test whether they are able to pass the
second mitosis. They were fixed when control embryos reached
the 4-cell stage. All ZM treated embryos remained arrested at
1-cell stage. Numerous micronuclei were present in their cyto-
plasm (data not shown).
The second embryonic mitosis of ZM treated 2-cell embryos.
Similarly to the 1-cell embryos, none of the 2-cell embryos
treated with ZM underwent the second cleavage division.
However, the majority (85%) of 162 blastomeres analyzed
was arrested in the interphase of the 2nd cell cycle (Fig. 10A),
remaining in clear contrast to the behaviour of 1-cell embryos.
In 11.5% of ZM-treated blastomeres condensation of chromatin
was observed (Fig. 10B). However, the proper mitotic chromo-
somes arrangement has never been achieved. Usually abnor-
mally condensed chromosomes clumped into irregular groups
(Fig. 10B, arrows). In 6 blastomeres (3.5%) the presence of two
nuclei has been registered, suggesting the successful karyoki-
nesis (Fig. 10C, arrowheads). Micronuclei, typically appearing
in zygotes treated with ZM, have never been observed in 2-cell
To verify if the majority of the 2-cell embryos treated with
ZM were really arrested at the G2 of the second cell cycle, we
Figure 6. effect of ZM treatment on the level of H3S10 phosphorylation
(H3S10ph). Zygotes at 26 h phCG were cultured for 4.5 h in the pres-
ence of 5, 10 and 20 μM of ZM447439. the intensity of H3S10ph signal
in ZM treated zygotes was compared with control zygotes at similar
stages of development. Signal classified as “strong/distinct” was similar
to observed in control zygotes, while “very weak/none”, was barely
detectable or not detected at all.
Figure 5. H3S10 phosphorylation in thymocyte nuclei fused with ovulated
oocytes and 1-cell parthenogenotes. (A–A”) chromosomes of thymocyte
origin (t+) in the vicinity of metaphase II (mII) chromosomes; (B–B”) activated
oocyte at telophase II (tII), IIpb-second polar body, t+-condensed thymocyte
chromatin. (C–C”) one-cell, G1 parthenogenote: pn-female pronucleus, IIpb-
second polar body, arrow-thymocyte nucleus remodeled into pronucleus-
like nucleus, t+-thymocyte nuclei, which had not undergone pCC. H3S10ph is
concentrated around the nucleoli and in the vicinity of the nuclear envelope
(C’, arrowhead). (t-) examples of non-fused thymocyte nuclei. Bar: 20 μm.
www.landesbioscience.com Cell Cycle 7
have analyzed their ability to enter the
next S phase. Two-cell embryos at late G2,
cultured in the presence or absence of ZM
(control group), were injected with Dig-11-
dUTP. Both groups of embryos were fixed 59
hphCG, when control embryos have already
been at G2 of the third cell cycle. In all control
embryos the presence of Dig-11-dUTP was
detected in the nuclei of sister blastomeres
derived from the one, microinjected with the
dU analog (Fig. 11A’). In contrast, none of
ZM-treated embryos (17/17) revealed any
signs of Dig-11-dUTP incorporation (Fig.
11B’), what confirmed their inability to start
the new round of DNA replication. This
result clearly showed that 2-cell embryos
treated with ZM remained arrested at the G2
of the second cell cycle.
Spindle assemble checkpoint (SAC)
is not inactivated in the first embryonic
mitosis upon ZM treatment. Our results
showed that during the first mitosis of ZM
treated embryos the anaphase movement of
chromosomes and separation of chromatids
were highly aberrant suggesting that chro-
mosomes were not properly attached to the
spindle. In such a case SAC should remain
active. To verify this, the presence of Mad2
protein, one of the core components of
SAC, localized on kinetochores when SAC
is on, was analyzed in ZM treated embryos.
Mad2 is a good marker of SAC activity, as
it detaches from the kinetochores as soon as
SAC is inactivated. One-cell embryos treated
with Nocodazole served as a positive control.
In Nocodazole treated embryos SAC remains
active, what leads to continuous presence of
Mad2 protein on kinetochores.65 In order
to synchronize control and ZM treated
embryos, the metaphase of the first cell cycle
was prolonged by the treatment with a prote-
asome inhibitor MG132 (MG).78,79 We veri-
fied that the presence of MG does not affect
the spindle formation.80 Drugs were added
to the cultured groups of embryos according to the scheme pre-
sented in Figure 12.
In embryos treated with Nocodazole neither the formation
of chromosomes in prophase, nor the nuclear envelope break-
down of pronuclei was disturbed. However, as the polymer-
ization of microtubules was inhibited and the mitotic spindle
was not formed, the maternal and paternal chromosomes
have never formed a single metaphase plate. The presence of
Nocodazole often caused a hyper-condensation of chromosomes
resulting in two irregular groups of chromatin (Fig. 13A). In
all Nocodazole treated embryos, punctuate, strong signal of
Mad2 protein was observed in both groups of chromosomes/
Figure 7. DNA replication in zygotes treated with ZM447439 (ZM). pn♂-male pronucleus; pn♀-
female pronucleus; IIpb-second polar body; arrow-de novo H3S10ph in control embryo. Inhibi-
tion of H3S10 phosphorylation did not change the pattern of late DNA replication. Bar: 20 μm.
Figure 8. H3S10ph in zygotes treated with aphidicolin. pn♂-male pronucleus; pn♀-female pro-
nucleus; n-NpB; arrows-de novo H3S10ph in the condensed chromatin surrounding the NpBs.
At late S phase the H3S10ph in aphidicolin treated zygotes (B’) was present in both pronuclei
and was relatively high in comparison with control group (A’). Bar: 20 μm.
chromatin (Fig. 13A’, arrowheads). In embryos treated with
MG, the chromosomes were properly aligned in the metaphase
plate (Fig. 13B). In none of them Mad2 was found on kineto-
chores (Fig. 13B’), similarly as was recently shown in human
cells,81 indicating that the presence of proteasome inhibitor
had not affected SAC inactivation similarly as in the somatic
cells. In embryos treated with ZM and MG, the two-chroma-
tid chromosomes were scattered throughout the cytoplasm
(Fig. 13C, arrows). A strong signal of Mad2 protein was
observed on the kinetochores of these scattered chromosomes
(Fig. 13C’, arrowheads). This result showed that SAC remained
active during the first embryonic mitosis of ZM+MG132 treated
8 Cell Cycle Volume 9 Issue 23
We have studied the spatiotemporal presence and locali-
sation of phosphorylated serine 10 of histone H3 in the
first and second cell cycle of mouse embryo. Our results
showed that H3S10Ph is present in pronuclei and blasto-
mere nuclei in early G1 phase, but not during early and
middle S phase. The simultaneous analysis of H3S10Ph
and DNA replication revealed that de novo phosphory-
lation of H3S10 in 1- and 2-cell embryos begins in the
pericentric heterochromatin around the time of these
regions replication. Subsequently, with the progression
of G2 phase, H3S10Ph spreads on the whole chromatin
and is present in chromosomes during the first and second
mitosis. This is clearly different from somatic cells,46,82 in
which H3S10 is phosphorylated from G2 to metaphase
while H3S10 dephosphorylation starts with the onset of
anaphase and is completed in telophase. Such pattern of
H3S10Ph was confirmed in the present paper on mouse
somatic cell lines. Although the rate of H3S10 dephos-
phorylation differed slightly among the cell lines, this
process was initiated not later then between anaphase and
It was shown by numerous studies (see Introduction)
that in somatic cells the phosphorylation of H3S10 is
controlled by the activity of Aurora B. In somatic cells
the level of Aurora B is highest during mitosis, then it
drops to undetectable level in G1 and increases again in
S/G2 cells.83 This corresponds to H3S10 phosphoryla-
tion/dephosphorylation pattern during the cell cycle of
these cells. The abundance of Aurora B is regulated by
the Anaphase-Promoting Complex/Cyclosome (APC/C),
an ubiquitin ligase essential for mitotic progression. The
regulators of APC/C are the Cdc20 and Cdh1 proteins,
which activate the complex and determine their substrate
specifity. Cdc20 activates APC/C at the onset of ana-
phase. The complex APC-Cdc20 ubiquitinates cyclin B
and securin allowing mitotic exit and separation of sis-
ter chromatids. Subsequently, at late anaphase, Cdc20 is
replaced by Cdh1, which remains associated with APC/C
until late G1, sustaining its activity on a high level. The
complex APC/C-Cdh1 ubiquitinates Aurora B, what
leads to its proteolytic degradation when cells exit from
mitosis.83 In a consequence of Aurora B degradation,
H3S10 phosphorylation ceases, and it is rapidly dephos-
phorylated by unknown phosphatases.
Observations of 1- and 2-cell mouse embryos (this
paper) as well as meiotically dividing oocytes55 suggest that
both in embryonic cells and in oocytes this mechanism is
modified. In oocytes arrested at prophase of the first mei-
otic division H3S10 is dephosphorylated,55 what suggests
that Aurora B is absent/inactive. This is in agreement
with the presence of active APC/C-Cdh1 at this stage of
oogenesis.84 In maturing oocytes APC/C-Cdh1 is detected until
metaphase I, when it is displaced by APC/C-Cdc20.84 Despite
the presence of APC/C-Cdh1, the phosphorylation of H3S10
Figure 10. Influence of Aurora kinases inhibition on the course of the second
mitosis. (A–C) chromatin; IIpb-second polar body. (A) two-cell embryo arrested
in the interphase of the second cell cycle, (B) chromosomes clumped into irregu-
lar groups; (C) two nuclei (arrowheads) in one blastomere of two-cell embryo.
Nonspecific background signal was artificially increased to show contour of
blastomeres. Bar: 20 μm.
Figure 9. the first embryonic mitosis in ZM treated zygotes. (A–A”)-prophase,
♂-male pronucleus; ♀-female pronucleus. Formation of metaphase plate was
disturbed (B–B”); (B) arrows indicate chromosomes outside the main group form-
ing the metaphase plate; (B’) arrowheads indicate three spindle poles. (C–C”)
chromosomes scattered along the spindle. (D–D”) micronuclei. Bar: 20 μm.
embryos, suggesting that proper attachments between the chro-
mosomes and the spindle could not be created when the activity
of Aurora kinases was inhibited.
www.landesbioscience.com Cell Cycle 9
inhibited the phosphorylation of H3S10, what could suggest that
Aurora B is not the only kinase controlling this process in early
mouse embryos. From all Aurora kinases Aurora C seems to be
a good candidate for this role. This member of Aurora family is
the less characterized, but it is known that it shows some simi-
larities with Aurora B and can control the progression of mitosis
in cells deprived of the latter (see Introduction). It seems thus
possible that Aurora C, rather than Aurora A, which functions
are mainly linked to the formation and function of centrosomes
and the mitotic spindle organization, is involved in the H3S10
phosphorylation in early mouse embryos. We cannot exclude
that, similarly to maturing oocytes, the balance between Aurora
kinases and the protein phosphatases plays an important role in
promoting and maintaining the phosphorylated state of H3S10
during the exit of the second meiotic division as well as the first
and second mitoses. However, the observations of thymocytes
behaviour point that the kinases involved in H3S10 phosphory-
lations and not phosphatases are rather modified in meiotic and
embryonic cell cycles.
Simultaneous analysis of DNA replication and H3S10Ph
localisation confirmed that during the majority of the S phase
period in 1- and 2-cell embryos, serine 10 of histone H3
remained dephosphorylated. De novo phosphorylation of this
residue appeared at the end of the S phase and was detected for
the first time in the late replicated, condensed chromatin regions
surrounding NPBs of pronuclei or nucleoli of blastomere nuclei.
These regions consist mainly of centromeric and pericentric
heterochromatin.87,88 The maintenance of their proper struc-
ture is indispensable for correct function of centromeres,89 the
protein complexes responsible for attachment of spindle micro-
tubules to chromosomes and successful chromosome segrega-
tion during karyokinesis. Disruption of these regions disables
starts immediately after GVBD. This modifi-
cation is maintained at anaphase/telophase I as
well as during the metaphase of the second mei-
otic division.54,55 Aurora kinases inhibition with
ZM447439 followed by rescue experiments con-
firmed Aurora B presence and specific functions
in maturing mouse oocytes.85 Moreover, Aurora
B localized on chromosomes and was enriched at
kinetochores at metaphase I.85 Its presence was
apparently not disturbed by the APC/C-Cdh1
suggesting a protection against polyubiquitina-
tion. It was also shown that the maintenance of
H3S10Ph and proper chromosome condensa-
tion in maturing oocytes depends on the balance
between the activity of Aurora B and protein
phosphatases.55 Our results showed that H3S10
remained phosphorylated during anaphase and
telophase of the second meiosis as well as dur-
ing the first and second mitosis, and in the G1 of
1- and 2-cell embryos. These data suggest that
Aurora B or other kinases, as well as phospha-
tases engaged in this process remained active
not only during the M phase like in somatic
cells and maturing oocytes, but also after tran-
sition to interphase. The presence of phosphorylated H3S10
in recondensed male chromatin supports this suggestion. In
sperm nucleus histones are replaced by protamins. After fertil-
ization they are exchanged for histones deposited in the oocyte
cytoplasm. Most probably histones are modified epigenetically
after being introduced into the male chromatin, what requires
the activity of kinases, acetylases or methyltrasferases engaged
in these processes. The kinases controlling the phosphorylation
of S10 in histone H3 newly introduced into male chromatin
have to be active at least until telophase II. The phosphoryla-
tion of H3S10 occurring in G0 arrested thymocyte nuclei intro-
duced into MII or newly activated oocytes fully confirmed this
hypothesis. When thymocyte nuclei reacted to the new cyto-
plasmic environment by premature chromosome condensation
(PCC; fusion occurring between MII and early TII), H3S10 in
their chromatin became highly phosphorylated. Moreover, this
modification, never detected in non-fused G0 arrested thymocyte
nuclei, appeared also in the nuclei, which did not undergo PCC
but swelled and decondensed (fusion at late TII). This result, as
well as the appearance of de novo H3S10Ph during late S phase
of the first and second cell cycle, collectively demonstrate that
the activity of kinases acting on the chromatin during interphase
and sensitive to ZM, are modified in embryonic vs. somatic cell
cycles. It also strongly suggests that the regulation is not sub-
stantially altered at the phosphatase level. ZM447439 is a low
molecule chemical reagent referred as Aurora kinases inhibitor.31
In the lowest concentrations it inhibits selectively Aurora B activ-
ity. In higher concentrations (1,000 nM and more) it acts also
on Aurora A and C. It has no influence on several other kinases
(Cdk1, Cdk2, Cdk4, PLK1, CHK1) at least not in the concen-
tration below 10 μM (IC50 for these kinases >10 μM).31 In our
studies only ZM in concentration as high as 20 μM effectively
Figure 11. the ability of 2-cell arrested, ZM treated embryos to enter the S phase of the
third cell cycle. IIpb-second polar body. (A–A”) control embryo; (B–B”) ZM treated embryo.
In control, 4-cell embryos the signal of dU incorporation was present in sister blastomeres
derived from dig-11-dUtp injected blastomere of 2-cell stage (A’, arrows). At the same
time all ZM treated embryos remained arrested at 2-cell stage and were unable to enter
the S phase of the third cell cycle. Bar: 20 μm.
10 Cell Cycle Volume 9 Issue 23
the pattern of DNA synthesis in heterochro-
matic regions of pronuclei or blastomere nuclei.
At the same time, the inhibition of replication
had not affected the appearance and the pat-
tern of de novo phosphorylation of H3S10.
These data suggest that either observed spatio-
temporal correlation is of no significance, or the
changes are very subtle and our method cannot
In zygotes, de novo H3S10 phosphorylation
was first detected in the male pronuclei. This
phenomenon is possibly related to the faster
development of the male pronucleus in com-
parison with the female one. It is well known
that pronuclei develop asynchronously. The
male pronucleus appears as a first92 and is also
the first to initiate and terminate DNA replica-
tion as well as RNA synthesis.57 As it is slightly
more advanced in the development all the time,
it most probably sooner achieves the organiza-
tion of pericentric chromatin, allowing H3S10
phosphorylation. This phenomenon may be
related to the progression of DNA replica-
tion. In aphidicolin treated zygotes H3S10Ph
appeared simultaneously in both pronuclei, as if
the inhibition of replication synchronized their
We have shown that the inhibition of Aurora
kinases and H3S10 phosphorylation in 1-cell
embryos resulted in abnormalities in chromo-
some alignment and segregation. Although the
chromosome condensation at prophase was not affected, none of
the zygotes formed normal metaphase plate. Defects concerned
also the separation of sister chromatids at anaphase. Similar
effects were observed when maturing mouse oocytes were treated
with ZM447439.68,85 Overexpression of Aurora B, but not Aurora
A and C, rescued significant number of oocytes from the align-
ment defects caused by ZM treatment.85 In Aurora B-depleted
Drosophila S2 cells, the main mitotic defects concerned not the
proper kinetochores function and chromosome segregation.90 In
S. pombe at the end of the S phase, when the regions of peri-
centric heterochromatin are replicated, the level of H3S10Ph is
transiently increased.91 The spatiotemporal correlation between
DNA replication in pericentric heterochromatin and the appear-
ance of de novo H3S10 phosphorylation in 1- and 2-cell embryos
described in the present paper suggested, that these two events
could be related. However, the lack of H3S10Ph had not affected
Figure 12. Diagram showing the timing (h post hCG injection) of treatment of 1-cell embryos with ZM447439 (ZM), Nocodazole (Noc) and MG132 (MG).
Figure 13. Mad2 localization during the first mitosis of zygotes treated with Nocodazole
(Noc). (A–A”), MG132 (MG) (B–B”), ZM447439 and MG132 (ZM+MG) (C–C”). IIpb-second polar
body. Mad2 remained attached to the kinetochores in embryos treated with Nocodazole
(A’, arrowheads) and treated with ZM and MG132 (C’, arrowheads). In the latter group chro-
mosomes were scattered throughout the cytoplasm (C, arrows). Bar: 20 μm.
www.landesbioscience.com Cell Cycle 11
observed in somatic cells, in which Aurora B activity was inhib-
ited with ZM447439,31 or hesperadin.32 In contrast, the effects of
inhibition of Aurora kinases during first mitosis were comparable
with observed in maturing oocytes i.e., during meiotic divisions.
In both cases the chromosomes spread along the spindle68,85 and
finally formed micronuclei.67 These differences may confirm dif-
ferent regulation of the first and second mitosis also in relation to
Aurora kinases. The progression of the first cell cycle and regula-
tion of the first mitosis can be controlled like the oocyte matura-
tion (see Introduction), while the next ones more like the somatic
cell cycle. The first mitosis lasts almost twice as long as the sec-
ond one.59 This phenomenon is not related to the prolonged pro-
metaphase or SAC activity, but to a transient metaphase arrest
resembling the one in the second meiotic division.65 The molecu-
lar mechanism of this arrest remains unknown. However, a plau-
sible explanation is a specific regulation of APC/C activity, is also
involved in Aurora B regulation. Whether the different effects
of ZM treatment on the first and second mitoses are related to
the prolongation of the first mitosis or to the regulation of gene
expression has to be clarified.
Materials and Methods
Ethical approval for presented study was obtained from the Local
Ethic Committee No. 1 in Warsaw, Poland. Embryos used in all
the experiments were isolated from F1 (C57BL/6x CBA) mouse
females, which were induced to superovulate by injection of 10
IU of PMSG (Folligon, Intervet, Netherlands) and 10 IU of hCG
(Chorulon, Intervet, Netherlands) 48–52 h later. Females were
mated with F1 males immediately after hCG injection.
All chemicals, if not stated differently, were purchased from
Sigma Aldrich (Germany).
Collection of oocytes and embryos. Ovulated oocytes and
zygotes. To obtain ovulated oocytes females were sacrificed 14 or
17 h after hCG. Zygotes at different stages of the first cell cycle
(G1, S, G2 or M) were collected from mated females between 18
and 31 hours post hCG injection. Ovulated oocytes or zygotes
surrounded by cumulus cells were released into hyaluronidase
solution (300 μg/ml). After follicular cells dispersion, oocytes
and zygotes were washed and collected in M2 medium.69
Two-cell embryos. Two-cell embryos at G1, S, G2 and M
phase were collected from females sacrificed between 32 and 49
hours post hCG. Dissected oviducts were cut into small frag-
ments in M2. Released 2-cell embryos were collected in M2 and
either used in other experiments or immediately processed for
Parthenogenetic activation. Ovulated oocytes were activated
parthenogenetically by 8 min. exposure to 8% ethanol solu-
tion.70,71 Subsequently, oocytes were washed in M2 and trans-
ferred to the drops of the same medium under mineral oil. They
were cultured at 37.5°C, 5% CO2 in the air, until the fusion with
Cell fusion. The zonae pellucidae of ovulated or partheno-
genetically activated oocytes were removed with Pronase (0.5%
in Ringers solution). Zona-free MII oocytes or parthenogenotes
at anaphase II or telophase II, were used for the fusion with
initial condensation of chromosomes, but their attachment to
the spindle and anaphase movement.39 Our results also suggest
that in mouse 1-cell embryos H3S10 phosphorylation was not
required for the chromosome condensation. However, the activ-
ity of Aurora kinases was indispensable for their correct attach-
ment to the microtubules and separation of sister chromatids.
We showed that in zygotes, in which the activity of Aurora
kinases was inhibited with ZM447439, Mad2 remained associ-
ated with kinetochores. Similar continuous presence of Mad2
was observed in Nocodazole treated somatic cells32 and zygotes.65
In these cases SAC remained active because the assembly of
microtubules was inhibited, the spindles were not formed and
the kinetochores remained unattached. Here we showed that in
ZM-treated zygotes the formation of spindles was not abolished
and the kinetochores were attached to microtubules. However, as
suggested by the presence of Mad2, these attachments were not
stable. The instability of the spindle should arrest the cell in pro-
metaphase until the proper attachments will form.93 This was not
the case in ZM treated 1-cell embryos. Although the normal ana-
phase movement or separation of chromatids was not observed,
the cells eventually exited mitosis and randomly scattered chro-
mosomes formed multiple nuclei. The cytokinesis has never
occurred and these embryos remained in a 1-cell stage. Somatic
cells treated with ZM are also able to exit from mitosis and they
fail to divide as well.31 However, in contrary to our results, the
kinetochore localization of Mad2 and BubR1 was inhibited and
the separation of sister chromatids took place. The authors con-
cluded that Aurora B activity is not required for kinetochore-
microtubules interactions, but is rather engaged in promoting
correct chromosome alignment. As mentioned above, our results
suggest that in mouse embryos the activity of Aurora kinases is
indispensable for correct sister chromatid separation. The mono-
oriented chromosomes were rather pushed back and forth from
one spindle pole to another what resulted in their spreading along
the spindle. Finally, the spindle was disorganized and multiple
nuclei were formed. This could be the effect of Aurora A inhibi-
tion caused by ZM treatment. It was shown that Aurora A kinase
stabilizes cyclin B.15,16 After inhibition of Aurora A the cyclin
B-associated Cdk1 activity level might have been downregulated
despite that SAC remained active. Moreover, it was reported that
HeLa cells treated with ZM rapidly exited mitosis and degraded
cyclin B.31 The transition to interphase could occur as a result of
decreased level of cyclin B.
Our results showed that the inhibition of Aurora kinases with
ZM exerts different effect on the first and second embryonic
mitosis. The majority of the 2-cell embryos treated with ZM did
not enter mitosis and remained blocked in the interphase of the
second cell cycle. Similar effect was observed after inhibition of
zygotic genome activation (ZGA) by α-amanitin.94 It seems pos-
sible that the phosphorylation of H3S10 in the regulatory regions
is engaged in selective gene activation during ZGA. As the first
cell cycle remains under the maternal control, the inhibition of
H3S10 phosphorylation in zygotes did not inhibit their entry into
mitosis. In rare cases in ZM treated 2-cell embryos two nuclei
were observed in each blastomere, indicating successful karyo-
kinesis without subsequent cytokinesis. Similar reactions were
12 Cell Cycle Volume 9 Issue 23
then permeabilized with 0.5% Triton X-100 in PBS-Mg2+-Ca2+
for 30 min. Blocking was performed in 3% BSA (Bovine Serum
Albumin, fraction V) in PBS-Mg2+-Ca2+ for 45 min. at room
temperature. Incubation with primary antibodies was carried
out overnight at 4°C. The following primary antibodies were
used: rabbit polyclonal anti-H3S10Ph (1:200 in PBS-Mg2+-Ca2+
+ 3% BSA, Upstate); mouse monoclonal anti-digoxygenin
(1:100 in PBS-Mg2+-Ca2+ + 3% BSA, Roche Diagnostics); flu-
orescein-conjugated mouse monoclonal anti-β-tubulin (1:50 in
PBS-Mg2+-Ca2+ + 3% BSA). After incubation with the primary
antibodies, embryos were washed twice in PBS-Mg2+-Ca2+ + 3%
BSA. The primary antibodies were detected with polyclonal sec-
ondary antibodies: goat anti-mouse IgG FITC-conjugated (1:200
in PBS-Mg2+-Ca2+ + 3% BSA, Invitrogen); donkey anti-rabbit
IgG FITC-conjugated (1:200 in PBS-Mg2+-Ca2+ + 3% BSA,
Jackson Immunoresearch Laboratories) or with goat anti-mouse
IgG TRITC-conjugated (1:200 in PBS-Mg2+-Ca2+ + 3% BSA,
Jackson Immunoresearch Laboratories). Incubation with second-
ary antibodies was performed at room temperature for 1 hour.
Subsequently, embryos were washed twice in PBS-Mg2+-Ca2+. In
a double staining procedure (H3S10Ph and DNA replication),
the staining procedure for H3S10Ph was followed with the pro-
cedure for DNA replication. For Mad2 detection blocking was
prolonged to an overnight incubation at 4°C. Furthermore,
blocking as well as the incubations with primary and secondary
antibodies were performed with the addition of 0.05% Tween
20. Primary rabbit antibody against Mad2 (a gift from Dr. Katja
Wassmann, Université Pierre et Marie Curie, Paris, France) was
diluted 1:50. For a primary antibody detection, polyclonal goat
anti-rabbit Alexa 488-conjugated antibody was used (1:200 in
PBS-Mg2+-Ca2+ + 3% BSA, 0.05% Tween 20, Invitrogen).
After a thorough washing in PBS-Mg2+-Ca2+ embryos were
mounted on poly-lysine coated (poly-L-lysine, 10% water solu-
tion) microscope slides in a drop of Citifluor mounting medium
(Citifluor, London, England), Citifluor mounting medium with
3 μg/ml of propidium iodide or Vectashield Mounting Medium
with 1.5 μg/ml propidium iodide (Vector Laboratories), depend-
ing on the experimental variant.
Samples were scanned using confocal microscope (Axiovert
100M, Carl Zeiss, Jena, Germany), using the same laser settings
for comparative analysis between different samples. Images were
analyzed using LSM Image Browser Program and in ImageJ. For
quantitative fluorescence measurements, intensities of fluores-
cence signal were divided by the intensity of the background.
This work was partly financed by a grant from the Ministry of
Science and Higher Education (grant N N303 407936). J.Z.K.
was supported by a grant from ARC (4900).
Supplementary materials can be found at:
thymocytes obtained from the thymuses of the newborn (2–3
days old) males, as described earlier in reference 72. After wash-
ing in M2 without BSA (M2-BSA) oocytes or parthenogenotes
were pretreated with phytohemagglutinin (PHA, 150 μg/ml)
for 3 min. and agglutinated with thymocytes in M2-BSA. Cell
aggregates were transferred for 1 min 30 sec to the polyethylene
glycol solution (PEG, 45% in M2-BSA, MW 2000, Fluka) solu-
tion. After PEG treatment cell aggregates were washed in M2,
and placed in the drops of the same medium. They were cultured
from 30 min up to 2–3 h after PEG treatment.
DNA replication. For the detection of DNA replication, no
more than 2pl of digoxigenin-11-2'-deoxy-uridine-5'-triphosphate
(11-dig-dUTP, 1 mM, Roche Diagnostics), the analog of deoxy-
uridine (dU), was microinjected into the cytoplasm of zygotes
or into one blastomere of 2-cell embryos using Eppendorf 5242
microinjector (Eppendorf-Netheler-Hinz GmbH). Before fixa-
tion injected embryos were incubated for 20–25 min. at standard
culture conditions, in the drops of appropriate medium (depend-
ing on the experimental variant—see below).
Inhibition of DNA replication. Zygotes at 19 hphCG were
placed in drops of M2 medium containing aphidicolin (4 μg/ml
prepared from stock solution in DMSO) and cultured for 8 h or 11
h. To inhibit DNA replication in the second cell cycle late zygotes
(30 hphCG) were placed in M2 with aphidicolin and cultured for
16 h (until 46 hphCG). Control embryos were cultured for the
same time in M2 containing DMSO in equivalent concentration.
ZM447439, MG132 and nocodazole treatment. Depending
on the experimental variant zygotes and 2-cell embryos at differ-
ent stages of cell cycle were treated with: ZM447439 (ZM, Tocris
Bioscience, Ellisville, Missouri, USA)—an inhibitor of Aurora
kinases, MG132 (MG, BIOMOL International)—inhibitor of
proteasome or Nocodazole—inhibitor of microtubule polymer-
ization. Stock solutions of all of them were prepared in 99.9%
DMSO (Methyl Sulfoxide, 99.9%, HPLC grade). Inhibitors
were then diluted in M2 to obtain the final solutions (ZM-5–20
μM; MG-5 μM; Nocodazole-10 μM), in which embryos were
cultured. The final concentration of DMSO was never higher
than 0.25%. Embryos at particular stages of the cell cycle were
placed in drops of M2 containing one (or two) of inhibitors listed
above and cultured for at least 3 hours. Control embryos were
cultured in M2 with DMSO in equivalent concentration.
Cell culture. Mouse embryonic fibroblasts (MEFs), mouse
NIH3T3 and L929 fibroblast cell lines were cultured at 37°C, 5%
CO2 in Dulbecco’s Modified Eagle’s Medium (DMEM #31966;
Invitrogen) containing 10% heat inactivated fetal bovine serum
(FBS, Biochrom, Berlin, Germany), 50 IU/ml of Penicillin and
50 μg/ml of Streptomycin (Penicillin-Streptomycin; Invitrogen).
Cells from exponentially growing culture were passaged onto 35
mm dishes with sterile cover glasses at the bottom (8 x 105 cells/
dish) and cultured for further 48 h.
Fixation and immunostaining. Zonae pellucidae were removed
with 0.5% Pronase. Subsequently, embryos were washed in M2
and fixed with 4% paraformaldehyde in PBS without Mg2+ and
Ca2+ (PBS-Mg2+Ca2+; Biomed, Lublin, Poland) for 20 min. and
www.landesbioscience.com Cell Cycle 13
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