Calcium influx-mediated signaling is required for
complete mouse egg activation
Yi-Liang Miaoa, Paula Steinb, Wendy N. Jeffersona, Elizabeth Padilla-Banksa, and Carmen J. Williamsa,1
aReproductive Medicine Group, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National
Institutes of Health, Research Triangle Park, NC 27709; andbDepartment of Biology, University of Pennsylvania, Philadelphia, PA 19104
Edited by John J. Eppig, The Jackson Laboratory, Bar Harbor, ME, and approved February 1, 2012 (received for review July 28, 2011)
Mammalian fertilization is accompanied by oscillations in egg
cytoplasmic calcium (Ca2+) concentrations that are critical for com-
pletion of egg activation. These oscillations are initiated by Ca2+
releasefrom inositol 1,4,5-trisphosphate (IP3)-sensitive intracellular
stores. We tested the hypothesis that Ca2+influx across the plasma
membrane was a requisite component of egg activation signaling,
and not simply a Ca2+source for store repletion. Using intracyto-
plasmic sperm injection (ICSI) and standard in vitro fertilization
tion of meiosis II. However, even if multiple oscillations in intracel-
lular Ca2+occurred, in the absence of Ca2+influx, the fertilized eggs
pronuclei. Additional experiments using the Ca2+chelator, BAPTA/
AM, demonstrated that Ca2+influx is sufficient to support polar
body emission and pronucleus formation after only a single
sperm-induced Ca2+transient, whereas BAPTA/AM-treated ICSI or
fertilized eggs cultured in Ca2+-free medium remained arrested in
metaphase II. Inhibition of store-operated Ca2+entry had no effect
on ICSI-induced egg activation, so Ca2+influx through alternative
channels must participate in egg activation signaling. Ca2+influx
appears to be upstream of CaMKIIγ activity because eggs can be
parthenogenetically activated with a constitutively active form of
CaMKIIγ in the absence of extracellular Ca2+. These results suggest
that Ca2+influx at fertilization not only maintains Ca2+oscillations
by replenishing Ca2+stores, but also activates critical signaling
pathways upstream of CaMKIIγ that are required for second polar
oscillations in the egg that persist for several hours and terminate
around the time of pronucleus formation. This pattern of Ca2+
oscillations is essential for events of “egg activation,” the com-
plex series of events that occurs between the time of sperm-egg
plasma membrane fusion and cleavage to the two-cell stage (1).
Successful egg activation accomplishes conversion of these two
gametes into a single embryo capable of implantation and full-
term development. In mammals, the pattern of Ca2+oscillations
that occurs at fertilization is responsible for driving the early
developmental program, and in the absence of appropriate Ca2+
signaling at fertilization, the embryo will fail to implant and/or
develop to term (1).
The sperm protein responsible for initiating Ca2+oscillations
at fertilization is a testis-specific phospholipase C, PLCζ, which
is released from the sperm head after sperm-egg plasma mem-
brane fusion (2). The first Ca2+transient experienced by the
egg is a result of PLC-mediated generation of inositol 1,4,5-tri-
sphosphate (IP3) and IP3-mediated Ca2+release from the en-
doplasmic reticulum (ER). The intracellular cytoplasmic Ca2+
level then rises to 1–3 μM and persists close to this level for
several minutes before returning to baseline. The return to
baseline is likely mediated by the combined actions of sarco-
plasmic/ER Ca2+-ATPase (SERCA) pumps that move Ca2+
back into the ER and plasma membrane Ca2+ATPase (PMCA)
pumps that move Ca2+out of the cell. Once initiated, repetitive
Ca2+oscillations persist for several hours. Persistence of the
Ca2+oscillations depends on Ca2+influx to replenish Ca2+stores
universal feature of fertilization in mammals is that the
fertilizing sperm evokes a series of repetitive calcium (Ca2+)
(3, 4). There is an absolute requirement for Ca2+/calmodulin-
dependent protein kinase II gamma (CaMKIIγ) signaling
downstream of Ca2+oscillations to accomplish egg activation
in vivo (5). CaMKIIγ activity leads to proteasome-mediated
degradation of cyclin B and decreases in maturation promoting
factor (MPF), and mitogen-activated protein kinase (MAPK)
activities that trigger resumption of meiosis (1).
Although it has been clear for many years that Ca2+oscil-
lations are important for initiating mammalian development, less
is known regarding how the Ca2+oscillations are transduced into
actions by downstream effectors that are responsible for de-
velopment. In mouse eggs, CaMKIIγ activity oscillates with only
a slight lag period after initiation of each Ca2+oscillation, sug-
gesting that the Ca2+transients each generate discrete waves of
CaMKIIγ activity (6). Similarly, protein kinase C (PKC) trans-
location to the egg plasma membrane, indicative of PKC acti-
vation, occurs with a temporal pattern similar to the timing of
Ca2+oscillations (7, 8). The intermittent elevations in Ca2+
provide a digital mechanism for generating graded responses by
downstream effectors at the same time as avoiding down-regu-
lation due to hyperactivation.
There is now evidence from somatic cell culture systems that
alterations in subplasma membrane Ca2+levels via store-oper-
ated Ca2+entry (SOCE) are responsible for triggering specific
Ca2+-dependent signaling pathways (9). These signaling path-
ways are not activated in response to alterations in bulk in-
tracellular cytoplasmic Ca2+in the absence of Ca2+influx. These
observations led us to hypothesize that Ca2+influx across the
plasma membrane contributes to downstream signaling required
for egg activation. In this study, we used both intracytoplasmic
sperm injection (ICSI) and in vitro fertilization (IVF) of zona
pellucida-free eggs as methods of fertilization in conjunction
with biochemical manipulations of Ca2+influx and efflux to
document a critical role for Ca2+influx through plasma mem-
brane channels in the initiation of development at fertilization.
Effects of Modulating Plasma Membrane Ca2+Fluxes on Events of
ICSI-Induced Egg Activation. In somatic cells, SOCE can be
inhibited specifically by incubation in medium containing 1 μM
gadolinium (Gd3+) (10). This treatment results in a gradual
rundown of induced Ca2+oscillations despite the presence of
extracellular Ca2+because depleted intracellular stores cannot
be refilled. At higher micromolar concentrations, Gd3+is no
longer specific for SOCE and inhibits Ca2+influx via additional
channel types, whereas millimolar Gd3+also inhibits PMCA
pumps and simultaneously prevents Ca2+efflux and influx. This
Author contributions: Y.-L.M. and C.J.W. designed research; Y.-L.M., P.S., and E.P.-B. per-
formed research; Y.-L.M., P.S., W.N.J., and C.J.W. analyzed data; and Y.-L.M., P.S., W.N.J.,
and C.J.W. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
www.pnas.org/cgi/doi/10.1073/pnas.1112333109 PNAS Early Edition
| 1 of 6
technique, known as “Ca2+insulation,” causes induced Ca2+
oscillations to persist indefinitely, even in the absence of extra-
cellular Ca2+(11, 12). Of note, Gd3+is not soluble in the
presence of phosphate or bicarbonate, so these experiments are
generally carried out in Hepes-buffered saline solution (HBSS)
or HBSS supplemented with 2 mM Ca2+(HBSS/Ca).
To determine the effects of modulating Ca2+influx and efflux
on egg activation, we treated eggs that had undergone ICSI (ICSI
because HBSS does not support standard IVF and because ICSI
allowed us to control the timing of fertilization within a precise
time window. Similar to eggs fertilized by IVF, untreated ICSI
eggs exhibited repetitive low frequency Ca2+oscillations (Fig.
1A). These eggs resumed meiosis and emitted the second polar
body by 1 h after ICSI (Fig. 1B) and reached the pronuclear stage
by 6 h after ICSI (Fig. 1C). As expected, ICSI eggs that were
placed immediately after microinjection into Ca2+-free medium
(HBSS) generated only one or two Ca2+transients (Fig. 1D).
Although these eggs completed anaphase, they failed to undergo
spindle rotation or polar body emission (Fig. 1E). In addition,
neither maternal nor paternal DNA formed normal-appearing
pronuclei (Fig. 1F).
In the presence of extracellular Ca2+, ICSI eggs required
treatment with 200 μM Gd3+(HBSS/Ca/LGd) to completely
block Ca2+entry required for subsequent Ca2+oscillations, al-
though there was a small increase in baseline Ca2+over time and
the oscillation frequency was somewhat reduced at 50–100 μM
Gd3+(Fig. 1G and Fig. S1). Only one or two sperm-induced Ca2+
ICSI, they had completed anaphase but failed to undergo spindle
rotation and enter telophase (Fig. 1H). Even after continued
culture for 6 h, the DNA remained condensed and no pronuclei
formed (Fig. 1I). Insulating ICSI eggs from extracellular Ca2+
using 5 mM Gd3+(HBSS/HGd) significantly increased the am-
plitude and lengthened the time of the initial Ca2+transient,
likely due to inhibitory effects on Ca2+efflux via PMCA pumps
(Fig. 1J, Table S1, and Fig. S2). The initial transient was followed
by repetitive intracellular Ca2+oscillations of somewhat lower
oscillation frequency compared with controls, but the mean area
S1). Ca2+-insulated ICSI eggs completed anaphase by 1 h after
ICSI (Fig. 1K); however, despite the repetitive Ca2+oscillations,
they failed to undergo spindle rotation and emit a second polar
body and, as a result, formed three rather than two pronuclei by
6 h after ICSI (Fig. 1L).
a slower decline in MAPK activity; reduction in activity of both
kinases is required for completion of meiosis and pronuclear
formation (13). To determine whether Ca2+influx influenced the
activities of these kinases, we used a dual kinase assay to measure
MPF and MAPK activities in individual ICSI eggs in which Ca2+
fluxes were modulated. As expected, in control ICSI eggs MPF
reduced by 4 h after ICSI (Fig. 1 M and N). Neither MPF nor
MAPK activities were significantly different from ICSI egg con-
trols when ICSI eggs were cultured without extracellular Ca2+or
were cultured in Gd3+to block Ca2+influx and/or efflux, at either
time point (Fig. 1 M and N).
To determine whether modulating Ca2+influx or efflux af-
fected cortical granule (CG) exocytosis, ICSI eggs were cultured
for 1 h under the conditions described above and then stained for
CGs. The extent of CG exocytosis was similar in all groups, al-
though there was a subtle difference in the distance of some CGs
from the plasma membrane in the groups where Ca2+influx was
prevented (Fig. S3 A–E). There was no difference among the
treatment groups in alterations of the zona pellucida in response
to CG exocytosis as indicated by the degree of proteolytic
cleavage of zona pellucida protein ZP2 to ZP2f (Fig. S3F).
To rule out the possibility that the abnormalities in egg acti-
vation under Ca2+insulation conditions was a result of Gd3+
toxicity, we washed the ICSI eggs free of Gd3+after 1.5 h and
placed them into standard embryo culture medium. The majority
(13/21; 62%) of the ICSI eggs continued to exhibit Ca2+oscil-
lations (Fig. 2 A and B). Almost all control eggs emitted a second
polar body by 1 h after ICSI, whereas the Ca2+-insulated ICSI
eggs remained arrested in anaphase 1.5 h after ICSI. If washed
free of Gd3+after treatment for 1.5 h, the ICSI eggs emitted
a second polar body and formed two pronuclei (Fig. 2 C and D).
These embryos progressed to the blastocyst stage almost as well
as controls (Fig. 2D). However, if the ICSI eggs were washed free
of Gd3+after treatment for 3 h, they failed to emit a polar body,
formed three pronuclei, and cleaved (Fig. 2 C and D). These
embryos failed to progress beyond the two-cell stage; this finding
was expected because of their abnormal chromosome comple-
ment. We also tested for Gd3+toxicity by parthenogenetically
activating eggs in the presence of Gd3+-containing media. MII-
alterations in intracellular Ca2+for 1 h after ICSI; spindle morphology/DNA
ICSI. Fractions on tracings indicate the number of ICSI eggs/total to exhibit
indicated pattern;see also Table S3. (A–C) HBSS/Ca. (D–F) HBSS. (G–I) HBSS/Ca/
LGd. (J–L) HBSS/HGd. (M and N) MPF and MAPK assays on single MII eggs and
ICSI eggs cultured 1 or 4 h in the indicated media. For these kinase assays,
HBSS/HGd contained no lactate and only 2 mM Gd3+. Data expressed as mean
± SEMof fourexperiments; oneeggevaluated pergroupinfour independent
experiments. *P < 0.05 compared with MII, ANOVA. (Scale bar: 20 μm.)
Effects of inhibiting Ca2+fluxes on Ca2+oscillations, cell cycle re-
2 of 6
| www.pnas.org/cgi/doi/10.1073/pnas.1112333109 Miao et al.
arrested eggs were microinjected with cRNA encoding a consti-
tutively active form of CaMKIIγ (CA-CaMKIIγ), which can ac-
tivate mouse eggs in the absence of Ca2+oscillations (5, 14).
More than 90% of all injected eggs (n ≥ 40 per group) cultured
for 6 h in HBSS/Ca/LGd or HBSS/HGd formed pronuclei,
suggesting that the Gd3+did not have toxic side effects.
Effects of Buffering Intracellular Ca2+on Events of ICSI-Induced Egg
Activation. Based on the above findings, we predicted that even if
repetitive cytoplasmic Ca2+oscillations were blocked, second
polar body emission would occur after ICSI as long as Ca2+in-
flux from the extracellular medium was allowed. The Ca2+
chelator 1,2-bis (o-aminophenoxy) ethane-N,N,N′,N′-tetra-acetic
acid acetoxymethyl ester (BAPTA/AM) has been used to ma-
nipulate the extent of Ca2+oscillations that occur after IVF of
zona pellucida-free mouse eggs (15, 16). We first tested various
concentrations and times of exposure to BAPTA/AM to de-
termine the treatment that would limit the egg to having a single
Ca2+transient after ICSI. The single transient pattern was al-
most always observed in ICSI eggs loaded with 1 or 2 μM
BAPTA/AM for 60 min, whether or not there was Ca2+in the
extracellular medium, whereas only a single extremely low am-
plitude Ca2+transient was observed in ICSI eggs loaded with 5
μM BAPTA/AM (Fig. 3 A, D, G, and J and Fig. S4). Staining of
the spindles and F-actin demonstrated that a single Ca2+tran-
sient was sufficient to induce meiosis resumption followed by
polar body emission and pronucleus formation as long as ex-
tracellular Ca2+was present (Fig. 3 D–F). In contrast, BAPTA/
AM-treated ICSI eggs that exhibited a single Ca2+transient but
were cultured in the absence of extracellular Ca2+failed to re-
sume meiosis and instead formed a second spindle in the region
of the sperm DNA (Fig. 3 G–I). The spindle morphology ob-
served in these ICSI eggs was identical to that in ICSI eggs
treated with 5 μM BAPTA/AM (Fig. 3 J–L). Despite preloading
with 1 μM BAPTA/AM to inhibit subsequent Ca2+oscillations,
>60% of the ICSI eggs placed into Ca2+-containing medium
after ICSI emitted a second polar body and formed two pronu-
clei, whereas none of the ICSI eggs placed into Ca2+-free me-
dium did so (Fig. 3M). ICSI eggs in the 5 μM BAPTA/AM group
rarely formed pronuclei; pronuclei formed only if they were cul-
tured in Ca2+-containing medium (Fig. 3M).
Measurements of MPF and MAPK activities were consistent
with these findings. One hour after ICSI, MPF levels decreased
significantly in control ICSI eggs and were slightly lower than in
MII eggs in both of the BAPTA/AM-treated groups (Fig. 3N).
Six hours after ICSI, MPF levels were significantly reduced in
control ICSI eggs and in eggs treated with 1 μM BAPTA/AM
then cultured in Ca2+-containing medium, whereas in BAPTA/
AM-treated ICSI eggs cultured in Ca2+-free medium, the MPF
levels had returned to a level similar to that in MII eggs (Fig.
3O). At the 6-h time point, MAPK levels in BAPTA/AM-treated
eggs cultured in Ca2+-containing medium were slightly lower
than in MII eggs but not as low as in control ICSI eggs, whereas
MAPK levels in BAPTA/AM-treated eggs cultured in Ca2+-free
medium were similar to MII eggs (Fig. 3O). Taken together with
the data presented regarding effects of controlling Ca2+fluxes
with Gd3+, the results of the BAPTA/AM experiments support
the idea that movement of Ca2+across the plasma membrane is
required for spindle rotation and second polar body emission. In
addition, assuming there is at least one ICSI-induced Ca2+
transient, Ca2+influx can substitute for subsequent Ca2+oscil-
lations to drive cell cycle resumption during egg activation.
Ca2+Influx and Events of IVF-Induced Egg Activation. Although most
aspects of ICSI-induced and IVF-induced events of egg activation
are essentially identical, there are subtle differences in the Ca2+
oscillatory patterns observed (17) and an obvious difference in
events that occur during IVF in the egg’s outer cortex during
sperm-egg fusion. To determine whether Ca2+influx was required
to support polar body emission after standard IVF, we performed
a series of experiments in which zona pellucida-free eggs were
fertilized quickly by using a high sperm concentration and then
observed under different culture conditions for their ability to
resume meiosis and emit a polar body. Ca2+-oscillatory patterns of
eggs placed after sperm-egg fusion into HBSS/Ca or HBSS/HGd
(Ca2+insulation) were similar to those observed after ICSI, with
the caveat that the first Ca2+transient occurred before imaging
could be started (Fig. 4 A and C). More than 90% (30/33) of
fertilized eggs cultured in Ca2+-containing medium emitted
a second polar body after 1 h in culture (Fig. 4B and Table S2).
Significantly fewer (20/56; 36%) of the Ca2+-insulated fertilized
eggs emitted a second polar body, whereas the majority (34/56;
61%) remained arrested in anaphase II (Fig. 4D). In a second set
of IVF experiments, eggs were loaded with 1 μM BAPTA/AM
before IVF and then cultured in the presence or absence of
extracellular Ca2+. These eggs exhibited a single Ca2+transient
eggs after washing out 5 mM Gd3+. (A and B) Representative
tracings of ICSI eggs cultured in HBSS/HGd for 1.5 h and then
transferred to HBSS/Ca. Fraction indicates number of ICSI
eggs/total to exhibit the indicated pattern. (C) Chromatin
configuration after washing ICSI eggs free of 5 mM Gd3+after
1.5 or 3 h of treatment; see Table S3 for egg numbers. ICSI
eggs were cultured in (a–c) HBSS/Ca for 1.5 (a), 3 (b), or 6 h (c);
HBSS/HGd for 1.5 h (d); (e and f) HBSS/HGd were cultured for
1.5 h then HBSS/Ca for 1.5 h (e) or 3 h (f); (g) HBSS/HGd for
3 h; (h and i) HBSS/HGd for 3 h then HBSS/Ca for 1.5 (h) or 3 h
(i). PB2, second polar body; Sp, sperm DNA. (Scale bar: 20 μm.)
(D) Effects of washing out 5 mM Gd3+after 1.5 or 3 h on
embryo development. Graph shows the percentage of ICSI
eggs to reach each embryo stage at the specified times after
ICSI. n = 37–40 per group from three independent experi-
ments. *P < 0.0001, Fisher’s exact test. Ctr, no Gd3+treatment;
2PN, two pronuclei; 3PN, three pronuclei.
Ca2+oscillation patterns and development of ICSI
Miao et al.PNAS Early Edition
| 3 of 6
(Fig. 4 E and G). The majority (21/30; 70%) of the BAPTA/AM-
loaded eggs cultured with Ca2+emitted polar bodies, whereas
55% (16/29) of those cultured without Ca2+remained arrested in
metaphase II (Fig. 4 F and H), and a small number either entered
anaphase or emitted a second polar body (Table S2). These
experiments were limited by our inability to precisely time transfer
of the fertilized eggs after sperm-egg fusion but before the first
Ca2+transient, so we could not be certain that Ca2+influx was
completely inhibited in all cases in the relevant groups. However,
both experiments demonstrated that the majority of the fertilized
eggs exhibited essentially the same responses as ICSI eggs cultured
under the same conditions and suggest that the requirement for
Ca2+influx applies to both IVF and ICSI.
Store-Operated Calcium Entry and Calcium Signaling at Fertilization.
Mouse eggs express STIM1, the ER Ca2+sensor, and there is
evidence that STIM1 could mediate SOCE at fertilization (18).
ORAI1, a Ca2+pore-forming protein that mediates SOCE, is also
expressed in mouse eggs (19). To test whether egg activation sig-
first documented the efficacy of two different SOCE inhibitors in
preventing Ca2+entry into mouse eggs in response to Ca2+store
depletion. Thapsigargin, a potent and specific inhibitor of SERCA
pumps, is widely used to cause depletion of ER Ca2+stores by
preventing reuptake from the cytoplasm. Freshly ovulated meta-
tern, cell cycle resumption, and MPF and MAPK activities in ICSI eggs. MII eggs
were loaded with 0, 1, or 5 μM BAPTA/AM for 60 min. The eggs then un-
derwent ICSI and were observed for Ca2+oscillatory patterns, cell cycle re-
sumption, and MPF and MAPK activities in CZB medium (contains 2 mM Ca2+)
(33) or Ca2+-free CZB. ICSI eggs were stained for actin, tubulin, and DNA either
1 (B, E, H, and K) or 6 h (C, F, I, and L) after ICSI; see Table S3 for egg numbers.
(A–C) ICSI eggs cultured in CZB; (D–F) 1 μM BAPTA/AM-treated ICSI eggs
cultured in CZB; (G–I) 1 μM BAPTA/AM-treated ICSI eggs cultured in Ca2+-free
CZB; (J–L) 5 μM BAPTA/AM-treated ICSI eggs cultured in CZB. (M) ICSI eggs
were cultured for 6 h under the indicated conditions and then evaluated for
pronucleus (PN) formation. Data expressed as mean ± SEM of three experi-
ments. Groups with different letters (a, b, and c) are significantly different,
P < 0.05, ANOVA. (N and O) MPF and MAPK assays of MII eggs and BAPTA/
AM-treated ICSI eggs. Data expressed as the mean ± SEM of four experiments.
*P < 0.05 compared with MII eggs, ANOVA. (Scale bar: 20 μm.)
Effects of BAPTA/AM and extracellular Ca2+on Ca2+oscillatory pat-
(A–D) Fertilized eggs were observed for alterations in intracellular Ca2+for
1 h and spindle morphology was determined 1.5 h after IVF. (A and B) HBSS/
Ca. (C and D) HBSS/HGd. (E–H) One micromolar BAPTA/AM-loaded fertilized
eggs cultured in CZB (E and F) or Ca2+-free CZB (G and H). Tracings are
representative of 18–23 eggs per group. Images shown represent the most
common spindle morphology in that group; see also Table S2.
Effects of blocking Ca2+influx on chromatin configuration after IVF.
4 of 6
| www.pnas.org/cgi/doi/10.1073/pnas.1112333109Miao et al.
phase II-arrested eggs were depleted of Ca2+by incubation in
10 μM thapsigargin in Ca2+-free medium for 1 h, and then SOCE
was observed after adding 5 mM Ca2+into the extracellular me-
dium. In somatic cells, 1–2 μM Gd3+is sufficient to inhibit SOCE;
however, 10 μM Gd3+was required for complete inhibition of
SOCE in Ca2+-depleted eggs under these conditions (Fig. S5A).A
second SOCE inhibitor, Synta66 (20), also completely blocked
SOCE in Ca2+-depleted eggs when used at 2 μM (Fig. S5A). To
test whether SOCE was required for egg activation signaling, we
treated ICSI eggs with 10 μM Gd3+or 2 μM Synta66 and recorded
alterations in cytoplasmic Ca2+. These SOCE inhibitors did not
persistent Ca2+oscillations (Fig. S5A) and by quenching of the
fura 2 signal when 1 mM Mn2+was added to the incubation me-
dium after the first Ca2+transient (Fig. S5B) (21). In addition,
ICSI eggs incubated in 10 μM Gd3+and 2 μM Synta66 emitted
a second polar body and formed pronuclei to a similar extent as
SOCE may occur at fertilization, it is not required for egg activa-
tion and that alternate Ca2+influx channels can support the req-
Calcium Influx and CaMKIIγ Signaling at Fertilization. To determine
whether Ca2+influx across the plasma membrane is upstream
or downstream of CaMKIIγ during egg activation signaling,
we tested whether extracellular Ca2+was required for CA-
CaMKIIγ–induced parthenogenetic activation. MII eggs were in-
jected with cRNA encoding CA-CaMKIIγ and then cultured for
6 h in the presence or absence of extracellular Ca2+. More than
90% of the CA-CaMKIIγ cRNA-injected eggs formed a second
polar body and pronucleus, whether or not Ca2+was present in
the culture medium, whereas controls did not (Fig. S5C). Because
injection of this cRNA elicits normal activation, bypassing the
need for Ca2+influx, we conclude that Ca2+influx across the
plasma membrane is not required for egg activation provided
there is sufficient CaMKIIγ activity. These findings suggest that
CaMKIIγ activation is positioned downstream of Ca2+influx
during sperm-induced egg activation.
In this study, we used the Ca2+insulation technique to demon-
strate that bulk oscillations in cytoplasmic Ca2+alone are not
sufficient to fully activate eggs in the absence of Ca2+influx. In-
stead, cytoplasmic Ca2+oscillations fully support pronucleus for-
mation, but Ca2+influx via Gd3+-sensitive plasma membrane
channels is required to mediate spindle rotation and second polar
body emission at fertilization. These data indicate an absolute
requirement for Ca2+influx to transmit signals required for com-
plete egg activation rather than simply serving as a source of Ca2+
torefill depleted intracellular stores. This observation is important
because it provides information regarding spatially restricted
control of egg activation signaling in the subplasma membrane
region that will alter the prevailing view that global cytoplasmic
calcium changes directly drive all downstream Ca2+-dependent
signaling molecules to carry out egg activation. A schematic in-
dicating how this information extends the current view of signaling
during egg activation is shown in Fig. 5.
The second method used for separating the effects of Ca2+in-
flux from the effects of sperm-mediated Ca2+release from IP3-
sensitive intracellular storeswaschelating intracellular Ca2+levels
with BAPTA/AM. These experiments showed that one Ca2+
transient in response to ICSI was sufficient to cause cell cycle re-
Ca2+influx can substitute for global cytoplasmic Ca2+oscillations
to drive cell cycle resumption. This finding is consistent with
a previous report that chelating intracellular Ca2+levels suffi-
ciently to allow only a single Ca2+transient during standard IVF
causes resumption of meiosis in a high percentage of the eggs
(16). Lawrence et al. (15) demonstrated that at least two Ca2+
transients are required to induce pronuclear formation in eggs
treated after fertilization with BAPTA/AM to block subsequent
Ca2+transients. This slight discrepancy from our findings is likely
a result of the high concentration of BAPTA/AM (5 μM) used in
these experiments, which in our hands effectively suppressed in-
tracellular Ca2+alterations and prevented cell cycle resumption,
evenwithextracellular Ca2+present.Additionofthe BAPTA/AM
Ca2+influx,probably explains the successfulpronucleusformation
despite the high BAPTA/AM concentration used in their study.
The results presented here shed light on previous observations
that antibody-mediated ligation of an extracellular mouse egg
membrane-associated protein resulted in reorganization of cor-
tical actin and cell cycle resumption in the absence of measurable
Ca2+oscillations (22). Furthermore, the results are consistent
with experiments in which MPF and MAPK inhibitors were used
to induce mouse egg activation (23). Our data suggest that in
both cases, Ca2+influx from the culture medium across the
plasma membrane supported the observed activation events. Our
findings are also consistent with the previous observation that
eggs can be activated by Ca2+influx induced by repetitive cell
membrane electroporation in Ca2+-containing medium in the
absence of Ca2+release from internal stores (24). Studies in
which this electroporation method was used to show that more
than one Ca2+transient was required for complete partheno-
genetic egg activation appear to conflict with our results (25).
However, the electroporation experiments were done in the
absence of sperm, and our interpretation of the different findings
is that the sperm provides a unique signal that is more effective
in inducing downstream signaling cascades important for egg
activation than Ca2+influx via electroporation.
Numerous types of regulated Ca2+channels support Ca2+
influx across plasma membranes (26). Although some Ca2+in-
flux via SOCE channels probably occurs during egg activation, it
is not likely to be required based on our findings that two dif-
ferent SOCE inhibitors could not block Ca2+oscillations or egg
activation in response to sperm. This finding is consistent with
previous observations that SOCE is inactivated during meiosis in
Xenopus oocytes (27, 28). Instead, our data suggest that one or
more alternate Ca2+-permeable channels are opened in response
to the initial Ca2+transient, either directly in response to Ca2+
oscillations activate CaMKIIγ, causing decreases in MPF and MAPK activities
that promote cell cycle resumption. The first Ca2+transient increases
CaMKIIγ activity enough to drive the metaphase to anaphase transition
and causes exocytosis of sufficient CGs to induce the block to polyspermy.
The first Ca2+transient also triggers Ca2+influx, providing Ca2+to refill ER
stores necessary for persistence of the oscillations. This Ca2+influx acti-
vates subplasma membrane signaling required to support spindle rotation
and second polar body (PBII) emission, and may help retain CGs at the
plasma membrane (PM). Additional Ca2+oscillations or Ca2+influx are
required for pronucleus (PN) formation. CaMKIIγ activity can bypass the
need for Ca2+oscillations and Ca2+influx to support PBII emission and PN
formation, but not CG exocytosis. Solid arrows indicate well-documented
direct or indirect connection; dashed arrows indicate less well character-
Working model of Ca2+influx signaling during egg activation. Ca2+
Miao et al.PNAS Early Edition
| 5 of 6
sensitization or indirectly in response to an activated signaling
cascade, (e.g., one involving PLC or PKC activation). There are
many candidate Ca2+channels that could serve this function. It
is not likely that Ca2+influx occurs via voltage-gated channels
because mouse eggs only experience very small changes in
membrane potential at fertilization (29). Transient receptor
potential (TRP) channels are good candidates because they are
widely expressed in nonexcitable cells and have important roles
in modulating Ca2+influx (30). Of note, some TRP channels can
be sensitized by Ca2+or by PKC-dependent phosphorylation. In
addition, canonical TRPC channels can be activated by diac-
ylglycerol (31), which is generated by PLC activity at fertilization
and can be detected at the egg plasma membrane (8).
In summary, the results presented herein indicate that Ca2+in-
flux not only maintains Ca2+oscillations by replenishing Ca2+
stores, but also provides an important spatially restricted Ca2+
signal required for complete egg activation at fertilization. These
studies provide evidence that bulk intracellular Ca2+oscillations
do not directly activate all downstream signaling pathways re-
quired for egg activation. Instead, the Ca2+oscillations trigger
Ca2+influx back into the egg, likely by activating Ca2+channels
in addition to those normally activated by Ca2+store depletion.
This Ca2+influx is required to activate the downstream signaling
molecules including CaMKIIγ that are essential for sperm-in-
duced resumption of meiosis and embryo development and are
particularly important in driving the cortical actin-based functions
of spindle rotation and polar body emission. Understanding how
Ca2+influx affects egg activation is important for improving
laboratory practices in clinical assisted reproductive technologies
such as standard IVF and ICSI. In addition, this knowledge is
particularly relevant to the growing field of fertility preservation
because it is essential to design egg cryopreservation methods
that prevent even low levels of Ca2+influx from occurring pre-
maturely and leading to egg activation in the absence of the
Materials and Methods
Generation of Camk2g Mutant Construct and in Vitro Transcription. Detailed
procedures are described in SI Materials and Methods.
Animals and Chemicals. Detailed information is provided in SI Materials
Egg Collection, Treatments, and Imaging. Detailed protocols are described in SI
Materials and Methods.
MPF and MAPK Assays. MPF and MAPK activities were determined in a single
assay as described (32).
Immunoblotting, Fluorescence, and Immunofluorescence Microscopy. Detailed
protocols are described in SI Materials and Methods.
Data Analysis. Data were analyzed by using Prism software (Graphpad).
Analyses used are indicated in figure legends.
ACKNOWLEDGMENTS. We thank Jurrien Dean (National Institute of Di-
abetes and Digestive and Kidney Diseases) for the mZP2 antibody and Glaxo
Smith Kline for the gift of Synta66; Jim Putney and Gary Bird (National
Institute on Environmental Health Sciences) for critical reading of the
manuscript and advice throughout this project; and Grace Kissling (National
Institute on Environmental Health Sciences) for assistance with statistical
analyses. This work was supported by the Intramural Research Program of
the National Institutes of Health, National Institute of Environmental Health
Sciences, Grant Z01-ES102985.
1. Ducibella T, Schultz RM, Ozil JP (2006) Role of calcium signals in early development.
Semin Cell Dev Biol 17:324–332.
2. Swann K, Saunders CM, Rogers NT, Lai FA (2006) PLCzeta(zeta): A sperm protein that
triggers Ca2+ oscillations and egg activation in mammals. Semin Cell Dev Biol 17:
3. Igusa Y, Miyazaki S (1983) Effects of altered extracellular and intracellular calcium
concentration on hyperpolarizing responses of the hamster egg. J Physiol 340:
4. Kline D, Kline JT (1992) Repetitive calcium transients and the role of calcium in exo-
cytosis and cell cycle activation in the mouse egg. Dev Biol 149:80–89.
5. Backs J, et al. (2010) The gamma isoform of CaM kinase II controls mouse egg acti-
vation by regulating cell cycle resumption. Proc Natl Acad Sci USA 107:81–86.
6. Markoulaki S, Matson S, Abbott AL, Ducibella T (2003) Oscillatory CaMKII activity in
mouse egg activation. Dev Biol 258:464–474.
7. Halet G, Tunwell R, Parkinson SJ, Carroll J (2004) Conventional PKCs regulate the
temporal pattern of Ca2+ oscillations at fertilization in mouse eggs. J Cell Biol 164:
8. Yu Y, Halet G, Lai FA, Swann K (2008) Regulation of diacylglycerol production and
protein kinase C stimulation during sperm- and PLCzeta-mediated mouse egg acti-
vation. Biol Cell 100:633–643.
9. Di Capite J, Ng SW, Parekh AB (2009) Decoding of cytoplasmic Ca(2+) oscillations
through the spatial signature drives gene expression. Curr Biol 19:853–858.
10. Luo D, Broad LM, Bird GS, Putney JW, Jr. (2001) Signaling pathways underlying
muscarinic receptor-induced [Ca2+]i oscillations in HEK293 cells. J Biol Chem 276:
11. Bird GS, Putney JW, Jr. (2005) Capacitative calcium entry supports calcium oscillations
in human embryonic kidney cells. J Physiol 562:697–706.
12. Kwan CY, Takemura H, Obie JF, Thastrup O, Putney JW, Jr. (1990) Effects of MeCh,
thapsigargin, and La3+ on plasmalemmal and intracellular Ca2+ transport in lacrimal
acinar cells. Am J Physiol 258:C1006–C1015.
13. Ducibella T, Fissore R (2008) The roles of Ca2+, downstream protein kinases, and
oscillatory signaling in regulating fertilization and the activation of development.
Dev Biol 315:257–279.
14. Knott JG, et al. (2006) Calmodulin-dependent protein kinase II triggers mouse egg
activation and embryo development in the absence of Ca2+ oscillations. Dev Biol 296:
15. Lawrence Y, Ozil JP, Swann K (1998) The effects of a Ca2+ chelator and heavy-metal-
ion chelators upon Ca2+ oscillations and activation at fertilization in mouse eggs
suggest a role for repetitive Ca2+ increases. Biochem J 335:335–342.
16. Gardner AJ, Williams CJ, Evans JP (2007) Establishment of the mammalian membrane
block to polyspermy: Evidence for calcium-dependent and -independent regulation.
17. Sato MS, Yoshitomo M, Mohri T, Miyazaki S (1999) Spatiotemporal analysis of [Ca2+]i
rises in mouse eggs after intracytoplasmic sperm injection (ICSI). Cell Calcium 26:
18. Gómez-Fernández C, et al. (2009) Relocalization of STIM1 in mouse oocytes at fer-
tilization: Early involvement of store-operated calcium entry. Reproduction 138:
19. Lopez-Guerrero AM, Pozo-Guisado E, Gomez-Fernandez C, Alvarez IS, Martin-
Romero FJ (2012) Calcium signalling in mouse oocyte maturation: The roles of STIM1,
ORAI1 and SOCE. Mol Hum Reprod, 10.1093/molehr/gar071.
20. Ng SW, di Capite J, Singaravelu K, Parekh AB (2008) Sustained activation of the ty-
rosine kinase Syk by antigen in mast cells requires local Ca2+ influx through Ca2+
release-activated Ca2+ channels. J Biol Chem 283:31348–31355.
21. Chiavaroli C, Bird G, Putney JW, Jr. (1994) Delayed “all-or-none” activation of inositol
1,4,5-trisphosphate-dependent calcium signaling in single rat hepatocytes. J Biol
22. Tutuncu L, Stein P, Ord TS, Jorgez CJ, Williams CJ (2004) Calreticulin on the mouse egg
surface mediates transmembrane signaling linked to cell cycle resumption. Dev Biol
23. Phillips KP, et al. (2002) Inhibition of MEK or cdc2 kinase parthenogenetically acti-
vates mouse eggs and yields the same phenotypes as Mos(-/-) parthenogenotes. Dev
24. Ozil JP, Swann K (1995) Stimulation of repetitive calcium transients in mouse eggs.
J Physiol 483:331–346.
25. Ducibella T, et al. (2002) Egg-to-embryo transition is driven by differential responses
to Ca(2+) oscillation number. Dev Biol 250:280–291.
26. Parekh AB, Putney JW, Jr. (2005) Store-operated calcium channels. Physiol Rev 85:
27. Machaca K, Haun S (2000) Store-operated calcium entry inactivates at the germinal
vesicle breakdown stage of Xenopus meiosis. J Biol Chem 275:38710–38715.
28. Arredouani A, Yu F, Sun L, Machaca K (2010) Regulation of store-operated Ca2+ entry
during the cell cycle. J Cell Sci 123:2155–2162.
29. Jaffe LA, Cross NL, Picheral B (1983) Studies of the voltage-dependent polyspermy
block using cross-species fertilization of amphibians. Dev Biol 98:319–326.
30. Gees M, Colsoul B, Nilius B (2010) The role of transient receptor potential cation
channels in Ca2+ signaling. Cold Spring Harb Perspect Biol 2:a003962.
31. Hardie RC (2007) TRP channels and lipids: From Drosophila to mammalian physiology.
J Physiol 578:9–24.
32. Svoboda P, Stein P, Hayashi H, Schultz RM (2000) Selective reduction of dormant
maternal mRNAs in mouse oocytes by RNA interference. Development 127:
33. Chatot CL, Lewis JL, Torres I, Ziomek CA (1990) Development of 1-cell embryos from
different strains of mice in CZB medium. Biol Reprod 42:432–440.
6 of 6
| www.pnas.org/cgi/doi/10.1073/pnas.1112333109Miao et al.