Arp2/3 Complex Regulates Asymmetric Division and
Cytokinesis in Mouse Oocytes
Shao-Chen Sun1, Zhen-Bo Wang2, Yong-Nan Xu1, Seung-Eun Lee1, Xiang-Shun Cui1, Nam-Hyung Kim1*
1Department of Animal Sciences, Chungbuk National University, Cheongju, Republic of Korea, 2State Key Laboratory of Reproductive Biology, Institute of Zoology,
Chinese Academy of Sciences, Beijing, China
Mammalian oocyte meiotic maturation involves oocyte polarization and a unique asymmetric division, but until now, the
underlying mechanisms have been poorly understood. Arp2/3 complex has been shown to regulate actin nucleation and is
widely involved in a diverse range of processes such as cell locomotion, phagocytosis and the establishment of cell polarity.
Whether Arp2/3 complex participates in oocyte polarization and asymmetric division is unknown. The present study
investigated the expression and functions of Arp2/3 complex during mouse oocyte meiotic maturation. Immunofluorescent
staining showed that the Arp2/3 complex was restricted to the cortex, with a thickened cap above the meiotic apparatus,
and that this localization pattern was depended on actin. Disruption of Arp2/3 complex by a newly-found specific inhibitor
CK666, as well as by Arpc2 and Arpc3 RNAi, resulted in a range of effects. These included the failure of asymmetric division,
spindle migration, and the formation and completion of oocyte cytokinesis. The formation of the actin cap and cortical
granule-free domain (CGFD) was also disrupted, which further confirmed the disruption of spindle migration. Our data
suggest that the Arp2/3 complex probably regulates oocyte polarization through its effect on spindle migration,
asymmetric division and cytokinesis during mouse oocyte meiotic maturation.
Citation: Sun S-C, Wang Z-B, Xu Y-N, Lee S-E, Cui X-S, et al. (2011) Arp2/3 Complex Regulates Asymmetric Division and Cytokinesis in Mouse Oocytes. PLoS
ONE 6(4): e18392. doi:10.1371/journal.pone.0018392
Editor: Austin John Cooney, Baylor College of Medicine, United States of America
Received December 16, 2010; Accepted February 28, 2011; Published April 8, 2011
Copyright: ? 2011 Sun et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The funding for this work was provided by Biogreen 21 Program (PJ007124201005, PJ007076201004 and PJ007076201005), RDA, Republic of Korea.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Oocyte polarization results in a unique asymmetric division. The
oocyte is transformed into a highly polarized MII-arrested egg
during mammalian meiotic maturation, which is essential to allow
asymmetric division and the retention of the maternal components
for early development . Disruption of this asymmetry usually
occurs in oocytes that are of low quality or that have experienced
post-ovulatory ageing. Oocyte polarization, which includes spindle
migration, spindle anchoring and cortical reorganization, as well as
asymmetric division, is controlled by microtubule and microfila-
ment cytoskeletons [2,3]. After GVBD (GV breakdown), the
centrally positioned spindle translocates to the cortex of the oocyte
in an actin-dependent way. Furthermore, cortical granules (CGs)
are redistributed to form a CG-free domain (CGFD), microvilli are
lost in the region overlaying the spindle, and microfilaments are
enriched to form an actin cap [4,5,6]. Together, these changes are
referred to as cortical reorganization and polarization. When
cortical polarity becomes intense, the oocyte extrudes the polar
body, leaving a highly polarized egg.
Unlike common ligand-mediated cell polarity, the development of
oocyte polarity and cortical reorganization is independent of the
. Meanwhile, meiotic spindles in oocytes lack true centrosomes,
indicating that specialized mechanisms may be responsible for the off-
centre positioning of the spindles. Until now, the molecular details of
oocyte polarization have been poorly understood.
Arp2/3 complex (actin-related protein 2/3 complex) consists of
Arp2, Arp3 and five other subunits; Arpc1 to Arpc5 [8,9]. The
complex binds to the side of an existing actin filament and initiates
the new filament assembly . ARP2 and ARP3 are actin-related
proteins that nucleate the growth of the new filament, and the
other five proteins link the two actin-related proteins to the mother
filament . Arp2/3 complex is involved in a range of cellular
processes. In many species, inhibition of the activity of the
complex by RNAi or inhibitory antibodies results in the disruption
of cell migration and adhesion [11,12,13], endocytosis [14,15],
and the establishment of cell polarity during mitosis (see reviews
[8,10]). The involvement of Arp2/3 complex in the formation of
new branched actin filaments is dependent upon interactions with
nucleation-promoting factors (NPFs). The NPFs consist of WASP
, N-WASP [17,18], WAVE1 [19,20], WAVE2 [21,22],
WAVE3, and the newly identified compounds, WASH ,
WHAMM  and JMY . Recent work has demonstrated
that Abp1 , Pan1 and cortactin [27,28,29] also activate the
Arp2/3 complex, whilst the NPFs are activated by Cdc42 and Rac
Recent studies using mouse oocytes have shown that the
activators of Arp2/3, Cdc42 and Rac are necessary for oocyte
polarization, spindle formation and migration during meiosis
[33,34,35]. The current study investigated whether the Arp2/3
complex is involved in oocyte polarization during oocyte meiotic
maturation. The complex was found to show a unique expression
pattern and its inhibition by a specific inhibitor and RNAi
PLoS ONE | www.plosone.org1April 2011 | Volume 6 | Issue 4 | e18392
demonstrated that it is indeed involved in this process and the
resulting asymmetric division.
Localization of the Arp2/3 complex during mouse oocyte
The subcellular localization of the Arp2/3 complex at different
stages of meiotic maturation was examined by ARP2 antibody
immunofluorescent staining. As shown in Fig. 1A, actin localized
at the cortex of the oocytes, and an actin cap formed in the area
overlying the chromosomes during the late MI stage and in the
ATI and MII stages. During these stages, ARP2 was concentrated
primarily in the cortex of the oocyte where it co-localized with
actin. We also found ARP2 to be enriched in the actin cap and to
have a greater distribution around the chromosomes.
Localization of ARP2 and actin after cytochalasin B
The relationship between the Arp2/3 complex and actin
dynamics was investigated through the successful disruption of
F-actin by cytochalasin B treatment. ARP2 was found to disperse
to the cytoplasm and no specific localization pattern was observed
in MI stage oocytes (Fig. 1B).
CK666 treatment and RNAi cause disruption of
To further investigate the roles of the Arp2/3 complex during
mouse oocyte meiotic maturation, we employed the newly-found
Arp2/3 specific inhibitor, CK666 . CK-666 targets a pocket
formed between Arp2 and Arp3, preventing the complex from
shifting into an active conformation. As shown in Fig. 2A, after
12 h in culture, most oocytes (78.764.8%, n=219) extruded the
first polar body normally in the control group, but a large
proportion of those in the CK666 treatment group underwent
symmetric division, producing two cells of a similar size
(30.2611.3%, n=123 vs 4.565%, n=120) (p,0.05) (Fig. 2B).
We next employed time-lapse microscopy to examine the dynamic
changes occurring in maturing oocytes and further confirmed that
they tended to divide equally after 12 h in culture (Fig. 2C).
Arpc2 and Arpc3 siRNA injection was used to down-regulate the
expression of the Arp2/3 complex, which successfully depressed the
mRNA level of both Arpc2 and Arpc3 (49.2614.4% vs 100%;
18.3611.8% vs 100%, respectively) (Fig. 2D). Similar results were
observed to those found after treatment with CK666 (Fig. 2A) in
which most oocytes extruded the first polar body normally in the
control group (62.6%66.5%, n=118), but in RNAi group, oocytes
underwent symmetric division (21.666.7%, n=66 vs 1.462.5%,
n=72) (p,0.05) (Fig. 2B).
Figure 1. Localization of Arp2/3 complex in mouse oocytes. (A) Subcellular localization of the Arp2/3 complex during mouse oocyte meiotic
maturation. ARP2 antibody staining was employed to show the subcellular localization of the Arp2/3 complex in mouse oocytes as revealed by
immunofluorescence staining. From the GV to the MII stage, all ARP2 accumulated at the cortex of the oocytes and the region near the cortex. Green,
actin; red, ARP2; blue, chromatin. Bar=20 mm. (B) Subcellular localization of the Arp2/3 complex after CB treatment during mouse oocyte meiotic
maturation. The subcellular localization of ARP2 in mouse oocytes was revealed by immunofluorescence staining. Actin was disrupted during the MI
stage and ARP2 dispersed into the cytoplasm. Green, actin; red, ARP2; blue, chromatin. Bar=20 mm.
Arp2/3 Complex in Oocyte Polarity
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CK666 treatment and RNAi cause failure of spindle
migration and cytokinesis
Since the oocytes described above underwent symmetric
division, the effect of the Arp2/3 complex on spindle migration
was analyzed. The spindle of most of the control oocytes had
formed and moved to the cortex by the late MI stage following
9.5 h of culture (Fig. 3A). However, a large proportion of those
treated with CK666 (59.865.9%, n=140 vs control 40.264.9%,
Figure 2. Effects of CK666 and RNAi treatment on asymmetric division in mouse oocytes. (A) Oocytes underwent symmetric division after
treatment with CK666 and RNAi. (B) Rates of symmetric division when the oocytes were cultured with CK666 in M16 medium, and after RNAi. (C)
Time lapse microscopy of maturing oocytes treated with CK666 after GVBD. These oocytes divided symmetrically. (D) Levels of Arpc2 and Arpc3
mRNA after Arpc2 and Arpc3 siRNA injection. *, significantly different (p,0.05).
Arp2/3 Complex in Oocyte Polarity
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n=150; p,0.05) or RNAi (5267.3%, n=142 vs control
29.2611.9%, n=67) (Fig. 3B) possessed centrally-located spindles.
Thus, the disruption of the Arp2/3 complex caused the failure of
spindle migration. We then employed time-lapse microscopy to
examine the dynamic changes in chromosome migration following
CK666 treatment. In control group, oocyte extruded polar body
normally at 12h culture; While in the CK666 treatment group,
even after a 12 h culture period, the chromosomes were seen to
remain in the central cytoplasm and spindle migration was
disrupted, which further confirmed the involvement of the Arp2/3
complex in this process (Fig. 3C).
CK666 treatment and RNAi cause failure of cytokinesis
After culturing for 12 h, most oocytes in the control group
extruded the polar body and arrested at the MII stage, whilst most
of those in CK666 treatment group arrested at telophase I and
failed to extrude the polar body. Furthermore, the chromosomes
were condensed by the sustained arrest (Fig. 4A) in the latter
group. The rate of TI-arrested oocytes is significantly higher
compared to the control group (27.7613.2%, n=182 vs
17.268.5%, n=126), while the number of oocytes in the MII
stage was significantly reduced after this treatment (29.169.8%,
n=182 vs 43.763.5%, n=126). Similar results were observed in
oocytes after RNAi (TI stage, RNAi 36.668.1%, n=159 vs
control 9.668.9%, n=81; MII stage, RNAi 15.6611.2%, n=159
vs control 56.964.4%, n=81) (Fig. 4B). Time-lapse microscopy
showed that the chromosomes segregated at 9.5 h and reached the
TI stage at 11.5 h. However, the oocytes treated CK666 remained
arrested at this stage even after 16 h in culture and failed to
complete cytokinesis and extrude the polar body (Fig. 4C). The
results therefore indicate that disruption of the Arp2/3 complex
affects completion of cytokinesis and final polar body extrusion.
CK666 treatment and RNAi cause disruption of actin cap
Symmetric division and failure of spindle migration may be due
to the disruption of oocyte polarity. To assess this, we examined
actin cap formation; a feature of oocyte polarization. As shown in
Fig. 5, the chromosomes of the control group had already moved
to the cortex and formed an actin cap by the latter part of MI, but
in the CK666 treatment and RNAi group, they remained in the
centre of the cytoplasm and no actin cap was observed. The
chromosomes segregated at the region of the cortex with the actin
cap in the control oocytes, but in the CK666 and RNAi groups,
they segregated at the central position and cytokinesis was
initiated. In the MII stage of the control group, a small polar
body and a large MII oocyte formed, and the chromosomes were
located under the region of the cortex where the actin cap had
appeared. Conversely, in the CK666 and RNAi groups, the
oocytes formed a 2 cell-like structure with no actin cap. Thus,
actin cap formation was disrupted after CK666 treatment and
CK666 treatment and RNAi cause disruption of the
cortical granule-free domain (CGFD)
Formation of the cortical granule-free domain (CGFD) was
examined as a further feature of oocyte polarization. The cortical
granules were absent near the region of the cortex close to the
Figure 3. Effects of CK666 treatment and RNAi on spindle formation and migration in mouse oocytes. (A) In the control group, the
spindle formed and moved to the cortex during the late MI stage, but remained centrally located after treatment with CK666 and RNAi. Green, a-
tubulin; blue, chromatin. Bar=20 mm. (B) Rate of spindle localization after 9.5 h of culture in oocytes treated with CK666 and RNAi. *, significantly
different (p,0.05). (C) Time lapse microscope image of a maturing oocyte treated with DMSO or CK666 after GVBD. Spindle migration failed to occur
in the oocyte treated by CK666.
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chromosomes during the MI stage in the control group, whilst
CK666 treatment and RNAi resulted in the cortical granules
being distributed uniformly across the entire cortex (Fig. 6).
Similar phenotypes were observed in MII stage oocytes; CGFD
formed in the MII-arrested oocytes of the control group but was
absent in the 2 cell-like MII oocytes of the CK666 and RNAi
groups. The results are evidence that the formation of the cortical
granule-free domain was disrupted after CK666 treatment and
RNAi. Taken together with the disrupted formation of the actin
cap, the inhibition of CGFD formation indicates that a failure in
oocyte polarization occurred.
The present study investigated the expression, localization, and
potential functions of the Arp2/3 complex during mouse oocyte
meiotic maturation. The results demonstrated the relationship
between this complex and actin. In particular, the disruption of the
activity of the Arp2/3 complex by specific inhibitor treatment and
the RNAi approach can affect the formation of the actin cap and
cortical granule-free domain, together with causing disruption to
spindle migration, asymmetric division and completion of
cytokinesis during mouse oocyte meiotic maturation. The study
therefore provides direct evidence of the involvement of the Arp2/
3 complex in oocyte polarization and cytokinesis.
Localization of the Arp2/3 complex showed the
relationship with actin in mouse oocytes
During all stages of mouse oocyte meiotic maturation, the
Arp2/3 complex was mainly found alongside F-actin at the cortex
and was also enriched in the region overlying the actin cap. This
localization pattern is similar to that described by previous studies
in somatic cells or embryo fibroblasts of other species in which the
complex was showed to localize at the membrane together with F-
actin [37,38,39,40]. We observed the enrichment of ARP2 around
the chromosomes towards the end of the MI stage, indicating that
the Arp2/3 complex may be involved in the migration of
chromosomes to the cortex. The complex also enriched at the
area underlying the actin cap from the late MI to the MII stage,
indicating that it may be involved in oocyte polarization. CB
treatment disrupted the specific localization of the Arp2/3
complex that dispersed into the cytoplasm, indicating that possible
relationship between the complex and actin. This observation is
consistent with previous work showing that the complex regulates
actin nucleation (see reviews [8,10]). The results together suggest
that the localization pattern of Arp2/3 is related to actin in mouse
oocytes and indicates that the complex has a possible role on actin-
related processes during oocyte meiotic maturation.
Arp2/3 complex regulates asymmetric division through
its effect on spindle migration in mouse oocytes
The roles of the Arp2/3 complex during mouse oocyte meiotic
maturation were investigated using the newly developed specific
inhibitor CK666 and the RNAi approach. The results demon-
strated that inhibition of Arp2/3 complex activity resulted in
symmetric cell division and the disruption of oocyte polarity.
Multiple processes appear to be involved in the regulation of
asymmetric division, the first of which is spindle positioning,
including spindle migration and anchoring. The second process
involves cortical reorganization and includes the loss of microvilli,
cortical granule exclusion and actin cap formation [2,41]. As the
Figure 4. Effects of CK666 treatment and RNAi on cytokinesis in mouse oocytes. (A) In the control group, the oocytes extruded the polar
body and arrested at the MII stage, whilst the arrest occurred during telophase I and the chromosomes condensed following treatment with CK666
and RNAi. Green, a-tubulin; blue, chromatin. Bar=20 mm. (B) Frequency of telophase I arrested oocytes after 12 h in culture by RNAi. *, significantly
different (p,0.05). (C) Time lapse microscopy of maturing oocytes treated with CK666. The oocytes failed to show the completion of cytokinesis after
16 h culture and remained at the TI stage.
Arp2/3 Complex in Oocyte Polarity
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development of the actin cap and CGFD are the key steps in
oocyte polarization, these events were examined in this study.
Formation of both of these regions was found to be lacking after
treatment with CK666 and RNAi, in contrast, actin and cortical
granules were seen to be distributed uniformly throughout the
cortex. These results are indicative of the disruption of oocyte
polarization, which may have caused the subsequent symmetric
Cortical reorganization was initiated after the spindle migrated to
the cortex. We therefore investigated whether the Arp2/3 complex
was involved in the earlier process of spindle positioning. Previous
studies have shown that Cdc42 and Rac, the activators of the Arp2/
3 complex, regulate spindle formation and polar body extrusion in
mouse oocytes [33,34,35]. Due to this link, we postulated that the
Arp2/3 complex itself is also involved in this process. The results
showed that the disruption of the Arp2/3 complex activity by
treatment with CK666 and RNAi played a major part in the arrest
of the spindle in a central location after 9.5 h in culture; a time by
which the spindles of most oocytes should have moved to the cortex.
Spindle migration was also dependent on actin, suggesting that the
Arp2/3 complex may regulate spindle migration through its
influence on actin nucleation (Fig. 7). However, disruption of the
complex did not affect spindle morphology, indicating that it is
probably not involved in the regulation of spindle formation itself.
Thisfindingisincontrasttoresults frompreviousworkshowing that
spindle formation can be disrupted by deactivation of Cdc42 and
formation through a different pathway that does not involve the
Arp2/3 complex. Recent work in mouse oocytes has shown that
PAK1, a downstream molecule of Cdc42, can regulate spindle
formation, polar body extrusion and localization of MEK in mouse
oocytes , indicating that a Cdc42-PAK1-MEK-MAPK path-
way might exist. Meanwhile, Cdc42 may activate the Arp2/3
complex through the binding of WASP, which results in the
regulation of actin nucleation needed for spindle migration. The
processes of actin cap formation and the subsequent formation of
the CGFD would also appear to be mediated by this latter pathway.
Arp2/3 complex regulates polar body extrusion through
the effect on cytokinesis in mouse oocytes
Polar body extrusion was depended upon cytokinesis, which in
turn was driven by actin. Since the Arp2/3 complex regulates actin
nucleation, we speculated whether the complex also regulates
cytokinesis. Our results showed that after 12 h of culture, the
disruption of the activity of the complex was largely responsible for
the arrested development of oocytes during telophase I, which was
characterized by a failure to extrude the polar body and by the
condensation of the chromosomes. These observations are
indicative of a failure of cytokinesis (Fig. 7). Although Rac and
Cdc42 also mediate polar body extrusion, they do this through the
Figure 5. Effects of CK666 treatment and RNAi on actin cap formation in mouse oocytes. The chromosomes in the control group had
moved to the cortex and an actin cap had formed by the late MI stage, whilst the chromosomes of the CK666 treatment and RNAi oocytes remained
at a central position and no actin cap was observed. The chromosomes segregated at the cortex of the oocyte during TI in the control group. In
oocytes treated with CK666 and RNAi, no actin cap formed, oocytes segregated with the chromosomes at the central position, and cytokinesis was
initiated from this central location. During MII, a small polar body and a large oocyte formed in the control group, whilst CK666 treatment and RNAi
samples formed two cell-like structures. An arrowhead illustrates the actin cap. Green, actin; blue, chromatin. Bar=20 mm.
Arp2/3 Complex in Oocyte Polarity
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regulation of spindle stability and positioning. The regulation of
polar body extrusion appears to be achieved using an Arp2/3
complex mechanism that is distinct from the Rac and Cdc42
mechanism. The functions of the Arp2/3 complex are similar to
those described for Formin-2, JMY and cortactin [43,44,45] in
mouse oocytes, whose disruption also results in the failure of spindle
migration and cytokinesis but has no effect on actual spindle
formation. This suggests that there may be a relationship between
Arp2/3 and Formin-2 that would justify further investigation. In
our results, some oocytes failed to migrate spindle, cytokinesis
occurred in the central oocyte and 2-cell-like MII oocytes formed;
some oocytes succeed in spindle migration, but were arrested at TI
stage and cytokinesis failed, the controdiction may be due to the
knock down efficiency and individual difference.
In conclusion, our results indicate that the Arp2/3 complex
regulates oocyte polarization through wide-ranging effects on
cortical reorganization, spindle migration, asymmetric division
and cytokinesis during mouse oocyte meiotic maturation.
Materials and Methods
Antibodies and chemicals
Mouse monoclonal anti-ARP2 antibody was purchased from
Abcam (Cambridge, UK), whilst Phalloidin-FITC, Lectin-FITC and
mouse monoclonal anti-a-tubulin antibody were obtained from
Sigma (St Louis, MO). Alexa Fluor 488 and 568 goat anti-mouse
antibodies were purchased from Invitrogen (Carlsbad, CA) and CK-
666 was a gift from Prof. Thomas Pollard at Yale University.
Animal care and use were conducted in accordance with the
Animal Research Institute Committee guidelines of Chungbuk
Figure 6. Effects of CK666 treatment and RNAi on cortical granule-free domain formation in mouse oocytes. The cortical granules were
absent in the cortex close to where the chromosomes were located during the MI and MII stages in the control group. Conversely, in the oocytes
treated with CK666 and RNAi, the cortical granules were distributed throughout the entire cortex. Z-stack showed the presence of different scanned
layers. An arrowhead shows the cortical granule-free domain. Green, cortical granules; blue, chromatin. Bar=20 mm.
Arp2/3 Complex in Oocyte Polarity
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National University, Korea (Approval number CB-R28). Mice
were housed in a temperature-controlled room with proper
darkness-light cycles, fed with a regular diet, and maintained
under the care of the Laboratory Animal Unit, Chungbuk
National University, Korea. The mice were killed by cervical
dislocation. This study was specifically approved by the Commit-
tee of Animal Research Institute, Chungbuk National University
(Approval number CB-R28).
Oocyte collection and culture
Germinal vesicle-intact oocytes were collected from the ovaries
of 6- to 8-week-old ICR mice and were cultured in M16 medium
(Sigma) under paraffin oil at 37uC, 5% CO2. Oocytes were
collected for immunostaining and microinjection after a range of
times in culture.
Real-time quantitative PCR analysis
Analysis of Arpc2 and Arpc3 gene expression was measured
by real-time quantitative PCR and the DDCTmethod. Total
RNA was extracted from 50 oocytes using a Dynabead mRNA
DIRECT kit (Invitrogen Dynal AS, Norway), and first strand
cDNA was generated with a cDNA synthesis kit (Takara,
Japan), using Oligo(dT)12–18 primers (Invitrogen). cDNA
fragments of Arpc2 and Arpc3 were amplified using the
The DyNAmo HS SYBR Green qPCR kit (FINNZYMES,
Finland) was used with a DNA Engine OPTICON 2 Continuous
Fluorescence Detector (MJ Research, MA) under the following
conditions: 95uC for 10 sec, and 38 cycles of 95uC for 5 sec, and
50uC for 32 sec.
Cytochalasin B treatment
One mg/ml cytochalasin B (CB) stock was diluted in M16
medium to a final concentration of 10 mg/ml. The oocytes were
cultured in this combined M16 CB medium for 8 h before
collection for immunofluorescence microscopy.
Stock CK666 (50 mM in DMSO) was diluted in M16 medium
to a final concentration of 500 mM. Oocytes were then cultured in
this medium for a range of times and used for immunofluorescence
microscopy. The control group was cultured in the same
concentration of DMSO.
Arpc2 and Arpc3 siRNA injection
Approximately 5–10 pl of Arpc2 and Arpc3 siRNA (Ambion,
TX) was microinjected into the cytoplasm of a fully-grown GV
oocyte using an Eppendorf FemtoJet (Eppendorf AG, Hamburg,
Germany) with a Nikon Diaphot ECLIPSE TE300 inverted
microscope (Nikon UK Ltd., Kingston upon Thames, Surrey, UK)
equipped with a Narishige MM0-202N hydraulic three-dimen-
sional micromanipulator (Narishige Inc., Sea Cliff, NY). After
injection, the oocytes were cultured in M16 medium containing
5 mM milrinone for 24 h, and then washed five times, each for
3 min, in fresh M16 medium. The oocytes were then transferred
to fresh M16 medium and cultured under paraffin oil at 37uC in
an atmosphere of 5% CO2 in air. The control oocytes were
microinjected with 5–10 pl of negative control siRNA. The actin
cap, cortical granules and spindle location were examined using
confocal microscopy. Polar body extrusion was observed using a
Time lapse microscopy
For imaging chromosome dynamics during oocyte maturation,
oocytes injected with tubulin-GFP were incubated with M16
medium containing Hoechst 33342 (5 ng/ml, Sigma) and CK666.
Images were taken by a 206/0.5 objective lens (Carl Zeiss,
Germany) under a computer controlled video microscope (Zeiss
LSM 710 META, Germany). Exposure time was 300 ms every
20 min. The ZEN software (Carl Zeiss) was used to analyze the
resulting video files.
To allow the single staining of ARP2, actin and CGs, oocytes
were fixed in 4% paraformaldehyde in PBS for 30 min at room
temperature and then transferred to a membrane permeabiliza-
tion solution (0.5% Triton X-100) for 20 min. After 1 h in
blocking buffer (1% BSA-supplemented PBS), oocytes were
incubated overnight at 4uC or for 4 h at room temperature
with 1:200 mouse anti-ARP2, 10 mg/ml Phalloidin-FITC, or
Figure 7. Arp2/3 complex was involved in multiple processes associated with oocyte polarization, including spindle migration and
cytokinesis. Disruption of spindle migration caused the spindle to remain in a central position and symmetrical division to occur. Disruption of
cytokinesis caused arrest during telophase I and a failure to extrude the polar body.
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100 mg/ml Lectin-FITC. After three washes in a washing buffer
(0.1% Tween 20 and 0.01% Triton X-100 in PBS), the oocytes
were labeled with 1:100 Alexa Fluor 488 goat-anti-mouse IgG
(for ARP2 staining) for 1 h at room temperature. The samples
were co-stained with propidium iodide (PI) or Hoechst 33342
for 10 min and were then washed three times in washing buffer.
For double staining of ARP2 and actin, oocytes were stained
with anti-ARP2 and Alexa Fluor 568 goat-anti-mouse IgG. They
were then labeled with Phalloidin-FITC for 30 min, washed three
times in PBS containing 0.1% Tween 20 and 0.01% Triton X-100
for 2 min, and stained with Hoechst 33342 (10 mg/ml in PBS) for
The samples were mounted on glass slides and examined with a
confocal laser-scanning microscope (Zeiss LSM 710 META). At
least 30 oocytes were examined for each group.
At least three replicates were performed for each treatment.
Statistical analyses were conducted using an analysis of variance
(ANOVA) and differences between treatment groups were
evaluated with Duncan’s multiple comparison test. Data were
expressed as mean 6 SEM and p,0.05 was considered to be
We gratefully thank Prof. Thomas Pollard for kindly providing the CK666.
We also thank Bo Xiong for helpful discussions and Ying-Hua Li and
Jeong-Seon Jeon for technical assistance.
Conceived and designed the experiments: SCS NHK. Performed the
experiments: SCS ZBW. Analyzed the data: SCS NHK. Contributed
reagents/materials/analysis tools: YNX SEL XSC. Wrote the paper: SCS
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