Cdc42 activation couples spindle positioning to first polar body formation in oocyte maturation.
ABSTRACT During vertebrate egg maturation, cytokinesis initiates after one pole of the bipolar metaphase I spindle attaches to the oocyte cortex, resulting in the formation of a polar body and the mature egg. It is not known what signal couples the spindle pole positioning to polar body formation. We approached this question by drawing an analogy to mitotic exit in budding yeast, as asymmetric spindle attachment to the appropriate cortical region is the common regulatory cue. In budding yeast, the small G protein Cdc42 plays an important role in mitotic exit following the spindle pole attachment . We show here that inhibition of Cdc42 activation blocks polar body formation. The oocytes initiate anaphase but fail to properly form and direct a contractile ring. Endogenous Cdc42 is activated at the spindle pole-cortical contact site immediately prior to polar body formation. The cortical Cdc42 activity zone, which directly overlays the spindle pole, is circumscribed by a cortical RhoA activity zone; the latter defines the cytokinetic contractile furrow . As the RhoA ring contracts during cytokinesis, the Cdc42 zone expands, maintaining its complementary relationship with the RhoA ring. Cdc42 signaling may thus be an evolutionarily conserved mechanism that couples spindle positioning to asymmetric cytokinesis.
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ABSTRACT: Polar body formation in eggs proceeds through two extreme asymmetric divisions, requiring precise coordination between spindle position and the polarized acto-myosin cortex. Two new studies appearing in this issue of Developmental Cell implicate the small GTPases Ran and Rac in cortical polarity of the mouse egg.Developmental Cell 03/2007; 12(2):174-6. · 12.86 Impact Factor
- Biology of Reproduction 07/2011; · 4.03 Impact Factor
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ABSTRACT: Asymmetric meiotic divisions in mammalian oocytes rely on the eccentric positioning of the spindle and the remodelling of the overlying cortex, resulting in the formation of small polar bodies. The mechanism of this cortical polarization, exemplified by the formation of a thick F-actin cap, is poorly understood. Cdc42 is a major player in cell polarization in many systems, however the spatio-temporal dynamics of Cdc42 activation during oocyte meiosis, and its contribution to mammalian oocyte polarization, have remained elusive. In this study, we investigated Cdc42 activation (Cdc42-GTP), dynamics and role during mouse oocyte meiotic divisions. We show that Cdc42-GTP accumulates in restricted cortical regions overlying meiotic chromosomes or chromatids, in a Ran-GTP-dependent manner. This polarized activation of Cdc42 is required for the recruitment of N-WASP and the formation of F-actin-rich protrusions during polar body formation. Cdc42 inhibition in MII oocytes resulted in the release of N-WASP into the cytosol, a loss of the polarized F-actin cap, and a failure to protrude the second polar body. Cdc42 inhibition also resulted in central spindle defects in activated MII oocytes. In contrast, emission of the first polar body during oocyte maturation could occur in the absence of a functional Cdc42/N-WASP pathway. Therefore, Cdc42 is a new protagonist in chromatin-induced cortical polarization in mammalian oocytes, with an essential role in meiosis II completion, through the recruitment and activation of N-WASP, downstream of the chromatin-centered Ran-GTP gradient.Developmental Biology 02/2013; · 3.87 Impact Factor
Current Biology 16, 214–220, January 24, 2006 ª2006 Elsevier Ltd All rights reservedDOI 10.1016/j.cub.2005.11.067
Cdc42 Activation Couples Spindle Positioning to
First Polar Body Formation in Oocyte Maturation
Chunqi Ma,1He ´le ´ne A. Benink,4Daye Cheng,1,2
Ve ´ronique Montplaisir,1Ling Wang,1,2Yanwei Xi,1,2
Pei-Pei Zheng,1,2William M. Bement,4and
X. Johne ´ Liu1,2,3,*
1Ottawa Health Research Institute (OHRI)
1053 Carling Avenue
Ottawa, K1Y 4E9
2Department of Biochemistry, Microbiology
3Department of Obstetrics and Gynecology
University of Ottawa
Ottawa, K1N 6N5
4Department of Zoology
University of Wisconsin-Madison
1117 West Johnson Street
Madison, Wisconsin 53706
after one pole of the bipolar metaphase I spindle at-
taches to the oocyte cortex, resulting in the formation
of a polar body and the mature egg. It is not known
lar body formation. We approached this question by
drawing an analogy to mitotic exit in budding yeast,
as asymmetric spindle attachment to the appropriate
cortical region is the common regulatory cue. In bud-
ding yeast, the small G protein Cdc42 plays an impor-
tant role in mitotic exit following the spindle pole at-
tachment . We show here that inhibition of Cdc42
activation blocks polar body formation. The oocytes
initiate anaphase but fail to properly form and direct
a contractile ring. Endogenous Cdc42 is activated at
to polar body formation. The cortical Cdc42 activity
zone, which directly overlays the spindle pole, is cir-
cumscribed by a cortical RhoA activity zone; the latter
defines the cytokinetic contractile furrow . As the
RhoA ring contracts during cytokinesis, the Cdc42
ship with the RhoA ring. Cdc42 signaling may thus be
an evolutionarily conserved mechanism that couples
spindle positioning to asymmetric cytokinesis.
Results and Discussion
To investigate whether Cdc42 has a role in first polar
body formation during oocyte maturation, we employed
highly specific inhibitors to block individual members
of the Rho family GTPases: RhoA, Rac1, and Cdc42.
Injection of dominant-negative Cdc42 (HA-Cdc42T17N)
caused no visible changes in oocyte morphology nor af-
fected progesterone-induced GVBD (Figure 1A), in spite
of the fact that both were expressed at high levels
(Figure 1D). Contrary to a previous report , we did
not observe consistent acceleration of progesterone-
induced GVBD in oocytes injected with HA-Cdc42T17N
tionofC3toxin mRNA,onthe other hand, causeddepig-
of oocytes with cytochalasin B. The depigmentation in-
terfered with assessment of GVBD in intact oocytes
(Figure 1A). Nonetheless, it was evident that C3-injected
oocytes, as well as oocytes treated with cytochalasin B,
also responded to progesterone by undergoing GVBD,
as determined upon fixing and bisecting the treated
oocytes (data not shown).
We wished to determine whether Cdc42T17Naffected
the transient inactivation of maturation-promoting fac-
tor (MPF) following GVBD; this transient inactivation of
MPF is thought to be important for the completion of
meiosis I . To analyze MPF dynamics in control oo-
individual oocytes at GVBD, 1 hr or 3 hr following GVBD.
Extracts were prepared and analyzed for MPF activity.
As shown in the top panel of Figure 1E, the transient
inactivation of MPF was evident in both control oocytes
(lane 3) and in oocytes injected with Cdc42T17N(lane 7).
Similarly, both groups of oocytes exhibited similar deg-
radation and resynthesis of cyclin B2 (middle panel).
Accumulation of the APC/C activator xFzy  at GVBD
was also normal in oocytes injected with Cdc42T17N
(bottom panel). These results indicated that inhibition
of Cdc42 did not affect APC/C activation or the biphasic
pattern of MPF activity.
Although inhibition of Cdc42 had no apparent effect
on GVBD or the biphasic pattern of MPF activity, analy-
sis ofchromosome morphology revealedthatwhilecon-
trol oocytes (97% or 261/268 in seven experiments) and
HA-Rac1T17N-injected oocytes (98% or 122/125 in three
experiments) had completed meiosis, with a ‘‘flower’’
pattern of metaphase II chromosome array in the pres-
ence of the first polar body, oocytes injected with HA-
Cdc42T17N(97% or 178/184 in six experiments) had not
emitted the first polar body, but had a similar ‘‘flower’’
pattern of chromosome arrays that were bigger and
contained approximately twice as many distinguishable
chromosomes (Figure 1B). Significantly, the single
metaphase spindle in Cdc42T17Noocytes was bipolar
and could be seen asymmetrically attached to the
oocyte cortex, similar to metaphase II spindles found
in control oocytes or oocytes injected with HA-Rac1T17N
(Figure 1C). On the other hand, no chromosome arrays
could be seen from the animal pole (or anywhere else
on the oocyte surface) in C3-injected oocytes, nor could
we detect the presence of the first polar body (data not
shown). These results were similar to those obtained
earlier by others in oocytes treated with cytochalasin B
. These similarities strongly suggested that the meta-
phase spindle in C3-injected oocytes failed to translo-
cate/anchor to the oocyte cortex. We examined the
oocytes after bisecting them and found bipolar spindles
deeply imbedded in oocytes injected with C3 or treated
with cytochalasin B (Figure 1C for example). However,
we could detect spindles in less than 10% of the
Figure 1. Cdc42T17NInhibited First Polar Body Formation
(CB, 5 mg/ml) was used, it was added 2 hr before the addition of progesterone. Oocytes injected with Cdc42T17Nor Rac1T17NmRNA were incu-
bated for at least 6 hr before the addition of progesterone. Oocytes injected with C3 mRNA were immediately placed in medium containing pro-
(B) Oocytes treated as described in (A) were fixed, stained with Sytox green, and viewed from the animal pole under a dissecting fluorescence
microscope. First polar body (PB) is indicated.
(C) Control oocytes and oocytes injected with Cdc42T17Nor Rac1T17NmRNA were coinjected with tubulin-Oregon green 514 conjugate (Molec-
ular Probes, 5nl per oocyte)beforethe addition of progesterone. Following overnight incubation, the oocytes werefixed, stained with propidium
iodide (red), and viewed following lateral bisection of the oocytes. Pictures are representative confocal images viewed from the cutting surface.
C3 mRNA-injected oocytes and CB-treated oocytes were fixed, bisected, and costained with anti-tubulin antibodies (red) and Sytox green. A
dashed line indicates the oocyte cortex in each image.
(D) Uninjected (control) oocytes and oocytes injected with mRNA for HA-Cdc42T17Nor HA-Rac1T17Nwere incubated overnight. Extracts were
prepared and analyzed by SDS-PAGE followed by immunoblotting with antibodies against HA.
(E) Uninjected oocytes (control, lanes 1 to 4) and oocytes injected with HA-Cdc42T17NmRNA (lanes 5 to 8) were incubated overnight before the
addition of progesterone. Individual oocytes were lysed at GVBD or at the indicated times following GVBD. GV oocytes (without progesterone)
were lysed at the same time as the GVBD 3 hr oocytes. Extracts were subjected to MPF assays (top panel) or immunoblotting with antibodies
against cyclin B2 (middle panel) or xFZY (lower panel). Shown is a representative of three independent experiments.
Cdc42 in Oocyte Maturation
examined oocytes. We attributed the rare occurrence of
spindle detection in these experiments to the intrinsi-
cally ‘‘hit-or-miss’’ nature of the technique (the spindle
would be detectable only if it happened to be within 50
mm or so of the cutting surface due to the limitation of
the confocal imaging), rather than indicating the lack of
To precisely determine at which point in the process
of oocyte maturation Cdc42 activity was required, we
monitored chromosome changes in live oocytes incu-
bated with Hoechst dye, as previously done in mouse
oocytes . As shown in Figure 2A, control oocytes ex-
hibited a single chromosome array (metaphase I) before
chromosome separation (anaphase I). Within a few min-
utes, individual chromosomes became unidentifiable,
indicating that oocytes had entered telophase or cytoki-
nesis. Well-formed metaphase II chromosome array
could be seen less than 3 hr after GVBD, in the presence
of the first polar body. Cdc42T17N-injected oocytes
exhibited similar metaphase I spindles and also clearly
underwent chromosome separation (anaphase I). Re-
markably, following a period of separation of the two
chromatin masses, a single metaphase spindle formed
and became stable (175 min). No polar body was emit-
ted, and the single metaphase spindle contained ap-
proximately twice as many identifiable chromosomes
as the metaphase II spindle in control oocytes. It should
formed with the use of an epifluorescence microscope;
as a result, one of the two separating chromosome
Figure 2. Cdc42T17NInhibits Contractile Ring Formation
(A) En face series from time-lapse experiments showing the typical chromosomal changes in control oocytes (top) and oocytes injected with
Cdc42T17N(bottom). Time lapses (in minutes) are from the first appearance of a maturation spot (i.e, GVBD = 0). PB, first polar body.
(B) Time-lapse experiments showing the F-actin (green) contractile ring in relationship with the spindle (red) in control oocytes (first row, en face
series; second row, Z series). Similar experiments (third row, en face series; fourth row, Z series) reveal the lack of contractile ring in oocytes
injected with Cdc42T17N, despite the correct spindle positioning and anaphase initiation. Time lapses (minutes) are from the first appearance
of a maturation spot (i.e., GVBD = 0).
arrays was often out of focus. We have analyzed several
dozen oocytes (about one-third were control oocytes
and two-thirds were oocytes injected with Cdc42T17N)
from four donor frogs. Although anaphase initiation
could be seen as early as 110 min and as late as 140
min following GVBD, we observed no significant differ-
ence in the timing of anaphase in control oocytes versus
oocytes injected with Cdc42T17N(data not shown). The
other striking feature was that anaphase was very
brief—lasting no more than 1–2 min (Figure 2A and
cytokinesis and that the oocytes had achieved a ‘‘meta-
phase II’’ arrest with a tetraploid chromosome comple-
ment. To eliminate the possibility that the Hoechst dye
and/or the UV altered chromosome behavior in these
time-lapse experiments, we also analyzed a large num-
ber of control and Cdc42T17N-injected oocytes fixed at
different times following GVBD. These analyses con-
firmed that Cdc42T17Ndid not affect anaphase initiation
but blocked the first polar body emission (see
Figure S1 in the Supplemental Data available with this
The apparent failure of Cdc42T17N-injected oocytes to
initiate/complete cytokinesis prompted us to examine
whether Cdc42T17Ninhibited formation or contraction
of the actomyosin-based cytokinetic contractile ring.
We performed time-lapse experiments with oocytes
coinjected with Alexa phalloidin and rhodamine-tubulin
(Figure 2B). In control oocytes, F-actin did not accumu-
late in the spindle region until about 115 min after GVBD
(Figure 2B). The F-actin was initially found in a broad
region around and over the spindle and then narrowed
as it contracted inward beneath the forming polar
body (Figure 2B, top two series). From the spindle mor-
phology, it was clear that the contractile ring formed at
the end of anaphase, as indicated by the lack of micro-
115 min). The metaphase II spindle became visible at
140 min, or about 20 min after the severing of the polar
body (122min). In Cdc42T17N-injected oocytes, although
F-actin accumulation was evident at the proper time, it
was far less abundant than in control cells. Further,
rather than closing downward underneath the spindle
and driving polar body emission, the actin ring closed
over the top of the spindle (Figure 2B, bottom two se-
ries), indicating that Cdc42 activity is required not only
for normal actin accumulation but also for proper direc-
tion of the force of the contractile ring.
To determine whether Cdc42 activation occurred at
the time of polar body formation, we utilized the GFP-
wGBD probe previously developed for visualization of
Cdc42 activity in Xenopus oocytes and eggs .
Cdc42 activity was undetectable in GV oocytes (data
not shown) or GVBD oocytes until several minutes be-
patch of increased Cdc42 activity appeared at the
animal pole overlayingthe
(Figure 3A, 51:50). This patch increased in intensity
and, within 2–4 min after the appearance of the patch,
cytokinesis ensued, as the cap spread downward in
concert with formation of the polar body and eventually
surrounded the entire polar body as it was pinched off
(Figure 3A, 63:00). This process is better appreciated
in the Z series of another oocyte, taken at higher magni-
fication (Figure 3B). Importantly, it is apparent that the
point at which the Cdc42 activity starts to go up corre-
sponds to the point where the spindle looks smallest
(Figure3A,51:20). Itlooks smallest because atthat point
it is finally oriented and anchored more or less perpen-
dicular to the oocyte cortex. In contrast, in the earlier
time points, it is still tilted at various angles. Cdc42 acti-
vation was never observed to occur in association with
spindles that were more than 15º off axis (7/7 experi-
ments). Cdc42 activity remained high on the polar
body for at least 10 min post-polar body emission but
eventually subsided (data not shown). In oocytes in-
jected with Cdc42T17N, Cdc42 activation was greatly di-
minished (Figure S2), consistent with our previous find-
ing that Cdc42T17Npartially inhibits Cdc42 activation
during wound healing in Xenopus oocytes . Further-
more, the remaining GFP-wGBD signals were more
diffused and disappeared much sooner than those in
control oocytes (Figure S2).
We have recently demonstrated that a microtubule-
dependent RhoAactivity zone directs cytokinetic furrow
formation in several models, including Xenopus polar
body emission . To determine the relationship be-
tween Cdc42 activity and RhoA activity during polar
body formation, active Rho and Cdc42 were simulta-
neously imaged in living oocytes . As previously re-
ported , RhoA activity first appeared as a circular
zone in the cortical region around the metaphase I spin-
dle (Figure 4A, 3:40). Cdc42 activity, in contrast, was lo-
calized to the inside of the circular RhoA activity zone,
with little or no overlap. (The small dot at 00:00, and in
all en face pictures, represents nonspecific enrichment
of both rGBD and wGBD probes associated with the cy-
toplasm in the region of the spindle, which serves as
a convenient indicator of the spindle position; see ).
The RhoA activity zone then contracted and eventually
ended at midbody at the completion of cytokinesis
(11:20) . As the RhoA zone contracted, the Cdc42
activity patch spread down, trailing the RhoA activity
zone, to eventually cover the whole polar body
(Figure 4A, Z series).
Thus, in the presence of a dominant-negative
polar metaphase I spindle, translocate and anchor the
spindle to the animal pole cortex, and initiate anaphase,
but they fail to properly form and direct the actomyosin-
based contractile ring and consequently fail to emit the
first polar body. As active Cdc42 is first observed in
the cortex in close association with one pole of the
metaphase I spindle (Figure 3B), it is tempting to specu-
be responsible for Cdc42 activation, analogous to the
budding yeast mitotic exit signaling in which the spin-
dle-pole-based Tem1 (a Ras-like GTPase) is activated
by the cortex-based Lte1 (a putative Tem1 guanine nu-
cleotide exchange factor) when the daughter’s spindle
pole contacts the bud cortex. In this regard, it is worth
noting that a Cdc42-specific activator, xGef , is
found to be a binding partner for a known spindle-pole-
associated protein, CPEB .
How does Cdc42 regulate contractile ring formation
and contraction? Based on results from the wound-
healing model [9, 12] we suspect that Cdc42 activity
Cdc42 in Oocyte Maturation
Figure 3. Spindle-Associated Activation of Cdc42 during Meiosis I in Frog Oocytes
(A) En face series from 4D movie showing metaphase I spindle (red) and GFP-wGBD (green). An increase in wGBD signal is apparent
(arrowheads, 51:20) coincident with metaphase I spindle assuming perpendicular orientation relative to the cortex.
(B) Double-label Z series of wGBD (green) and metaphase I spindle (red) during the formation of the first polar body. Active Cdc42 is initially ap-
parent as a patch over the metaphase I spindle (00:00; equivalent to 51:20 in [A]) and then spreads downward from its concentration above the
spindle (arrow, 02:00). Active Cdc42 ultimately surrounds first polar body (16:40). All times are in min:sec from the start of imaging.
promotes formation of a region of high actin turnover,
which could abet the function of a region of more stable
actin templated by the RhoA zone. That is, a bordering
region of rapid actin turnover could both provide more
F-actin for incorporation into the contractile ring and re-
lax the cortex on the polar body itself. This relaxation, in
turn, could help ensure that the closing contractile array
is directed downward rather than over the top of the
We propose (Figure 4B) that RhoA is controlled by aster
microtubules emanating from the spindle pole ap-
proaching the cortex, as previously suggested . This
notion is consistent with studies of polar body emission
in clams  as well as demonstrations of microtubule-
plus end-associated RhoA Gefs . As the spindle
pole makes tight contact with the overlaying cortex,
Cdc42 is activated by an activator associated with the
spindle pole (microtubule-minus ends). The differential
activation of RhoA and Cdc42 by microtubules, together
with a possible direct inhibition of RhoA by Cdc42 ,
temporal relationship of RhoA and Cdc42 activity zones
during polar body formation. The complementary
relationship between Cdc42 and RhoA reported here in
healing in which Cdc42 activity and RhoA activity form
concentric but nonoverlapping zones, driving wound
closure . Further, segregation of the RhoA and
Cdc42 zones during wound healing is microtubule de-
pendent, suggesting the existence of a mechanism for
microtubule-dependent control of rho GTPase activity
zones that is broadly conserved in asymmetric cell divi-
sion as well as wound repair.
We conclude that Cdc42, together with RhoA, couples
lar body formation during Xenopus oocyte maturation.
Our previous study  has indicated that microtubule-
dependent RhoA activity defines the cytokinetic con-
tractile ring. In this study, we provide the first evidence
that spindle pole-cortex contact activates cortical Cdc42.
The Cdc42 activity zone directly overlays the spindle
pole on the inside of the RhoA activity ring. The comple-
mentary nature of the two GTPases thus explains how
spindle pole attachment to the cortex is mechanistically
coupled to the formation, and the contraction, of the
cytokinetic contractile ring during polar body formation.
We believe that in contrast to the role of RhoA in cytoki-
nesis, which is likely universal , the function of Cdc42
Figure 4. Coordination of RhoA and Cdc42 in Polar Body Formation
(A) Double-label 4D movie series showing eGFP-rGBD (indicating active RhoA, red) and GFP-wGBD (indicating active Cdc42, green). RhoA ac-
tivity first appears as a zone around a patch of Cdc42 activity (3:40). As RhoA zone contracts to form the first polar body, Cdc42 activity spreads
to cover the polar body (more evident in the Z series, 11:20). Note the lack of colocalization of rGBD and wGBD in any of the pictures. All times in
min:sec from the start of imaging.
(B) The schematic (top, en face view; bottom, lateral view) depicts the relationship between RhoA activity zone (red) and Cdc42 activity zone
(green) and the possible microtubule-mediated activation of the two small GTPases. Brown circles represent spindle poles (microtubule orga-
nizing centers). Black lines are microtubules, with their plus ends (+) and minus ends (2) indicated. Purple bars are separating chromosomes in
late anaphase or telophase.
Cdc42 in Oocyte Maturation
in cytokinesis discovered here is unique for polar body
formation and perhaps some other forms of asymmetric
cell division (such as budding yeast mitosis).
The Supplemental Data for this article, including Supplemental
Experimental Procedures and figures, can be found online at http://
We thank the following colleagues for reagents: Dr. Nathalie La-
marche for Cdc42 and Rac cDNA, Dr. Alan Hall for C3 cDNA, Dr.
Thierry Lorca for anti-xFZY, and Dr. James Maller for anti-cyclin
B2. Dr. Andrew Ridsdale provided invaluable advice on confocal im-
aging at OHRI in Ottawa. This work was supported by operating
grants from the Canadian Institute of Health Research (MT15381)
and from National Cancer Institute of Canada (to X.J.L.) and from
the National Institute of Health (to W.M.B.). X.J.L. holds a Premier’s
Research Excellence Award from the province of Ontario.
Received: September 27, 2005
Revised: November 22, 2005
Accepted: November 24, 2005
Published: January 23, 2006
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