BIOLOGY OF REPRODUCTION 58, 807-813 (1998)
Spontaneous and Fertilization-Induced Ca2 + Oscillations in Mouse Immature
Germinal Vesicle-Stage Oocytes'
Man-Qi Deng,
2
Xiu-Ying Huang, Tie-Shan Tang, and Fang-Zhen Sun
Institute of Developmental Biology, Chinese Academy of Sciences, Beijing 100080, People's Republic of China
ABSTRACT
Mouse germinal vesicle (GV)-stage oocytes not only show
Ca2+ oscillations in response to fertilization but also exhibit
spontaneous Ca2+ oscillations during meiotic maturation in
vitro. Spontaneous Ca2+ oscillations were entirely suppressed by
microinjection of heparin (25 RIM final intracellular concentration), an antagonist of inositol trisphosphate (IP3) receptor,
whereas fertilization-induced Ca2+ oscillations were only partially inhibited by heparin even at a high dosage of 600 ,iM.
Inhibition of endogenous IP3 generation by antagonizing phospholipase C using U73122 (20 tIM final concentration) also
failed to suppress the generation of fertilization-induced Ca2+
transients, suggesting that the two types of Ca2+ oscillations do
not have the same dependence on IP3-induced Ca2+ release. In
addition, spontaneous Ca2+ oscillations require the presence of
intact GV whereas fertilization-induced Ca2+ oscillations are independent of the GV but require cytoplasm, since enucleation
eliminated only spontaneous Ca2+ oscillations but not fertilization-induced Ca2+ oscillations. These results suggest that IP3induced Ca2+ release is the primary mechanism responsible for
spontaneous Ca2+ oscillations. Sperm-induced Ca2+ oscillations,
however, may employ more complex mechanisms during fertilization.
whether the observed results were additive effects of both
the fertilization-induced Ca 2+ oscillations and the spontaneous Ca 2+ oscillations. It is speculated that spontaneous
Ca 2+ oscillations may act as a signal to enhance meiotic
maturation [14, 15]. Inositol-induced Ca 2+ release (IICR) is
suggested to be the primary mechanism underlying the
spontaneous Ca 2+ oscillations in GV oocytes [13], but there
are discrepant reports on the role of IICR in fertilizationinduced Ca 2+ oscillations [16-19]. Some authors claim that
IICR is the sole source of Ca 2+ release and is essential for
Ca 2+ oscillations at fertilization [8, 10, 18]; however, there
is some evidence suggesting that multiple stores of Ca 2+
may be released during fertilization [16, 17, 19, 20]. The
purpose of this study was 1) to define the contribution of
IICR to fertilization-induced Ca 2+ oscillations in GV oocytes in comparison with spontaneous Ca 2+ oscillations,
and 2) to determine whether the two types of Ca 2' oscillations are associated with the nucleus as well as nucleus
progression.
MATERIALS AND METHODS
All reagents used were purchased from Sigma Chemical
Co. (St. Louis, MO) unless stated otherwise.
INTRODUCTION
It is well established that mature mammalian metaphase
II (MII) eggs produce long-lasting Ca 2+ oscillations during
fertilization [1-9]. Unlike mature MII eggs, immature germinal vesicle (GV)-stage oocytes (GV oocytes) not only
show Ca 2+ oscillations in response to fertilization [10-12]
but also exhibit a series of spontaneous Ca 2 + oscillations
after isolation from follicles [10, 13, 14]. The spontaneous
Ca2 + oscillations last for 2-3 h despite the occurrence of
GV breakdown (GVBD) [13], and blocking GVBD using
dibutyryl (db) cAMP fails to suppress the spontaneous Ca 2+
oscillations [11, 13]. As to the Ca 2+ response of GV oocytes to fertilization, there are only scattered and discrepant
reports. Some authors contend that GV oocytes do not acquire the ability to generate repetitive Ca 2 + transients in
response to fertilization but only produce 2-3 Ca 2+ spikes,
which usually cease within 1 h [12]; other authors claim
that immature GV oocytes can produce repetitive Ca 2+ oscillations [10, 11]. Since spontaneous Ca 2+ oscillations last
for several hours, it is quite possible that subsequent fertilization-induced Ca 2+ responses may overlap extensively
with spontaneous Ca 2 + oscillations during the period of an
experiment. Although it was reported that fertilization-induced Ca2 + oscillations in hamsters are higher in amplitude
than spontaneous Ca 2+ oscillations [10], it is uncertain
Accepted October 24, 1997.
Received December 17, 1996.
'This work was supported by the National Natural Science Foundation
of China (No. 39670376) and the Rockefeller Foundation of the United
States.
2
Correspondence: FAX: (010)62551951;
e-mail: dengmq@public.east.cn.net
807
Preparation of Gametes
Immature GV-stage oocytes were retrieved from 4- to 6wk-old ICR mice, 48 h after i.p. injection of 10 IU eCG.
Ovaries were removed and repeatedly punctured with a fine
forceps to release oocytes. Naked oocytes and cumulus cellenclosed oocytes were collected separately, and the cumulus cells were removed by repeatedly pipetting through a
fine-bore pipette. Oocytes, approximately 65-70 txm in diameter and with an intact GV, were collected and thoroughly washed in medium H6 containing 4 mg/ml BSA
[21]. In most experiments, 0.1 mg/ml dbcAMP was included in the medium to prevent spontaneous maturation of
oocytes [22]. For experiments requiring in vitro fertilization, zonae pellucidae were removed by brief treatment
with acidic Tyrode's solution [23]. The zona-free oocytes
were quickly removed from the acidic Tyrode's solution
and washed extensively in H6. After several rinses, the
zona-free oocytes were transferred to a thin glass-bottom
chamber containing T6 (and dbcAMP if required). To prevent movement of oocytes during sperm addition and measurement of intracellular free calcium ([Ca2 +]i), zona-free
oocytes were allowed to attach to the bottom in T6 without
BSA. After the oocytes stuck tightly to the bottom coverslip, a small amount of T6 containing BSA was added to
bring the medium to approximately 15 mg/ml BSA. For in
vitro fertilization, spermatozoa collected from the cauda epididymides of the same strain of male mice and capacitated
in T6 [21] containing 15 mg/ml BSA [24] for 1.5 h were
added to the chamber with a mouth pipette. To reduce the
chance of polyspermy, oocytes were allowed to bind only
3-4 spermatozoa each. In order to preclude the interference
808
DENG ET AL.
TABLE 1. Characteristics of spontaneous and fertilization-induced Ca 2 ' oscillations in mouse GV oocytes.a
Types of Ca2 +
oscillation
Number of
oocytes used
Ca2 ' at baseline
(nM)
Ca2 at peak
(nM)
16
90.2 + 13.6
282.4 + 23.1c
115.6 + 18.7
3.3 + 1.4
15.2 + 3.3
17
101.3 + 12.9
401.4 + 2 6 .8d
83.4 + 13.8
3.8 + 3.1
18.3 + 8.2
Spontaneous Ca2 +
oscillation
Fertilization-induced
Ca2+ oscillation
Peak duration
(sec)
Ca2 + rise interval
(min)
Ca2+ rise
number in 1.5 hb
a Data are presented as means ± SD.
b
Ca2+ rise to as high as 20 I.M higher than baseline was counted.
Means with different superscripts are different within columns (p < 0.05).
',d
of spontaneous Ca 2+ oscillations with the subsequent fertilization-induced Ca 2+ oscillations, and to separate temporally the two kinds of Ca 2+ oscillations, spermatozoa
were not added until the spontaneous Ca 2 + oscillations had
ceased for 1 h.
To determine whether oocytes had been fertilized, the
oocytes were examined under phase contrast microscopy
for evidence of penetration by a spermatozoon.
Measurement of [Ca2 +]
Oocytes were cultured in H6 containing 4-6 p.M fura2/AM (Molecular Probes Inc., Eugene, OR) for 30 min at
37°C. After several rinses, the fura-2-loaded oocytes were
transferred to a chamber containing medium covered with
light mineral oil. The chamber was placed in a well on the
stage of a Nikon Diaphot 200 inverted epifluorescence microscope (Nikon Instruments, Garden City, NY) for imaging. Fura-2 fluorescence was imaged through a Nikon CFFluor X10 objective and intensified CCD camera (Miracal
Life Science, Cambridge, UK), and [Ca 2+]i was determined
by calculating the ratio of fura-2 fluorescence at 510 nm,
excited by UV light alternately at 340 and 380 nm from a
xenon arc lamp. Excitation wavelengths were alternated by
a rotating chopper mirror attached to a stepper-motor, which
was driven in synchrony with the video signal from the
camera, to switch wavelength at the end of each video
frame. The resulting video signals were combined by an
"image" digital image processor (Miracal Image Analysis
System) using a lookup table to implement the formula of
Grynkiewicz et al. [25]. The [Ca 2+]i image was recorded
every 5-10 sec for at least 1.5 h in each experiment. The
chamber was maintained at 37C by a thermostatic controller.
Inhibition of Phospholipase C (PLC) Using U73122
U73122 dissolved in ethanol (4 mM in stock solution)
was added to T6 containing oocytes undergoing the sponFIG. 1. Pattern of spontaneous Ca2 + oscillations in GV oocytes measured in H6
with or without dbcAMP. Spontaneous
Ca2 + oscillations were unaffected by the
occurrence of GVBD and the progression
from prophase I to the MI stage in vitro
(A). Inhibiting GVBD using dbcAMP (0.1
mg/ml) had no effect on spontaneous Ca2 +
oscillations (B).
taneous Ca 2+ oscillations. After the spontaneous Ca 2 + oscillations were inhibited by U73122, spermatozoa were
added to determine whether fertilization could elicit a new
series of Ca 2+ oscillations. The control experiment was carried out using U73343, an inactive analogue of U73122.
Micromanipulation
Microinjection. In experiments that did not involve fertilization, zona-intact oocytes were used. In order to define
the contribution of IICR to spontaneous and fertilizationinduced Ca 2+ oscillations, inositol trisphosphate (IP3) receptor antagonist-low molecular heparin (Mr 4000)-was
pressure-injected into the cytoplasm of the GV oocytes in
H6 free of Ca 2+. The heparin-injected oocytes were transferred to Ca2 +-containing H6 for measurement of the spontaneous Ca 2+ oscillations. The basic injection solution was
composed of 0.9% NaCl, 100 tM EDTA, and 1.0 mM Tris
buffered at pH 7.2. The volume injected was 5-10 pl (total
volume of GV oocyte was estimated at 180 pl). After the
spontaneous Ca 2+ oscillations were completely suppressed
by heparin, capacitated spermatozoa were added to see if
fertilization could induce additional Ca 2+ oscillations.
Enucleation of GV oocytes. To define the role of an intact GV in spontaneous Ca 2+ oscillations and fertilizationinduced Ca 2+ oscillations, enucleation was carried out in
some experiments. Fura-2-loaded GV-intact oocytes undergoing spontaneous Ca 2 + oscillations were collected and
transferred to the manipulation medium M2 [23] supplemented with cytochalasin B (5 ,ug/ml), sucrose (2.5 mg/ml),
and 0.1 mg/ml dbcAMP. Inclusion of cytochalasin B and
sucrose in M2 can make the oocytes more elastic and shrink
slightly, increasing the survival rate after micromanipulation [23]. After several minutes of treatment, the oocyte
was held with a holding pipette and the zona pellucida was
slit with a fine glass needle. Enucleation was achieved by
sucking the GV through the slit with a polished injection
pipette (inner diameter slightly larger than that of the intact
CALCIUM OSCILLATIONS IN MOUSE GV OOCYTES
809
FIG. 2. Fertilization-induced Ca2+ oscillations in oocytes undergoing spontaneous
2+
Ca
oscillations. Within a single oocyte,
the fertilization-induced Ca2+ oscillations
were usually higher in amplitude than before fertilization (A). Suppression of GVBD
using dbcAMP during in vitro fertilization
had no effect on fertilization and fertilization-induced Ca2+ oscillations (B).
GV). The karyoplasts and their corresponding cytoplasts
were paired with each other and transferred to the same
drop of T6 containing dbcAMP. [Ca 2*]i was measured as
described above. The karyoplasts and cytoplasts were inseminated, if required, and Ca 2+ responses to fertilization
were compared.
Statistical comparison of the Ca 2+ rise parameters were
made by t-test.
RESULTS
Spontaneous Ca2
'
Oscillations in Mouse GV Oocytes
GV oocytes exhibited a long series of Ca 2+ oscillations
after release from antral follicles, regardless of whether
they were naked oocytes at isolation or oocytes deprived
of surrounding cumulus cells afterwards. Oocytes exhibiting spontaneous Ca 2+ oscillations had an mean interval of
3.3
1.4 min (means + SD, n = 25). The mean baseline
[Ca 2 +]i was 90.2
13.6 nM with an average peak (Ca 2+)i
of 282.4 + 53.1 nM. The mean duration of Ca 2+ rise was
115.6 ± 38.7 sec (Table 1). The Ca 2+ oscillations persisted
for 2-3 h in most oocytes, and in some oocytes could last
for more than 6 h. It was observed that GVBD usually
occurred within 2 h after release from follicles, GVBD per
se had no direct influence on spontaneous Ca 2 + oscillations,
and the measured oocytes were still exhibiting spontaneous
Ca2 ' oscillations after GVBD (Fig. 1A, n = 18/25). In
some groups of experiments, GV oocytes were collected
and manipulated in medium containing 0.1 mg/ml dbcAMP,
and [Ca2+]i was also measured in dbcAMP-containing medium. Although maturation progression was blocked at the
GV stage during the period of the experiment, the occurrence and timing of spontaneous Ca2+ oscillations were unaffected (Fig. B, n = 38/38), suggesting that blocking mat-
uration progression had no effect on spontaneous cytoplasmic Ca 2 ' oscillations.
Fertilization-InducedCa
Oocytes
2 +
Oscillations in GV-Stage
Pattern of fertilization-induced Ca2+ oscillations in oocytes that were undergoing spontaneous Ca oscillations.
Oocytes undergoing spontaneous Ca2+ oscillations were inseminated in T6 with or without dbcAMP. A series of higher-amplitude Ca 2+ oscillations was induced soon after
sperm addition (Fig. 2A). The amplitude and frequency varied between different oocytes, but within a single oocyte,
the Ca2 ' oscillations after fertilization were usually higher
in amplitude than before fertilization. Comparison of the
Ca2 + increases above baseline showed that the Ca 2+ peak
induced by fertilization was 58.0 + 25.2 nM (means + SD,
n = 23) higher than that of spontaneous Ca2 ' oscillations
after subtraction of their basal Ca 2 ' level (136.2
36.1 vs.
78.2 + 22.1, p < 0.05). Inhibiting GVBD by using
dbcAMP had no effect on the occurrence and timing of in
vitro fertilization or the fertilization-induced Ca2 ' oscillations (Fig. 2B).
Pattern of fertilization-induced Ca2+ oscillations in GV
oocytes that had ceased spontaneous Ca oscillations. To
rule out possible interference of spontaneous Ca 2+ oscillations by fertilization-induced Ca 2+ oscillations, GV oocytes
were allowed to undergo spontaneous Ca 2+ oscillations in
dbcAMP-containing medium, and sperm were added after
the spontaneous Ca2 + oscillations had ceased for more than
1 h. It was observed that fertilization elicited a new series
of Ca 2+ oscillations (n = 17, Fig. 3A). The characteristics
of spontaneous and fertilization-induced Ca 2+ oscillations
in GV oocytes are summarized in Table 1. In a parallel
2 +
2 +
FIG. 3. Fertilization-induced Ca2+ oscillations in GV oocytes that had ceased spontaneous Ca2+ oscillations. In dbcAMP-containing medium, fertilization induced a
2+
new series of Ca
oscillations after the
cessation of spontaneous Ca2+ oscillations
(A). Control oocytes cultured in vitro for
the same period of time underwent GVBD
and progressed to Ml, and showed similar
Ca2* oscillations after fertilization.
810
TABLE 2.
DENG ET AL.
2+
Comparison of fertilization-induced Ca
Stage
GV oocytesa
b
MI oocytes
Number of
oocytes used
10
11
oscillations in GV-intact oocytes and in IVM MI oocytes.
2+
Ca at baseline
(nM)
103.2
106
13.2
6.2
Ca2+ at peak
(nM)
301.1
302.8
37.1
36.8
Peak duration
(sec)
Ca 2 ' rise
number in 1.5 h
Ca2 + rise interval
(min)
35.4
82.3
80.1 + 8.3
4.1
5.3
17.7
16.5
3.1
3.3
8.7
7.0
a GV oocytes were cultured in dbcAMP containing T6.
b GV oocytes from the same pool were allowed to undergo maturation in vitro and were cultured to MI stage.
experiment, GV oocytes cultured in T6 for the same period
of time all underwent GVBD and progressed to the metaphase (MI) stage (n = 11), as demonstrated by the disappearance of the nucleolus, without extrusion of the first
polar body. The characteristics of Ca 2+ oscillations for in
vitro-matured MI oocytes in response to fertilization were
comparable to those for GV-intact oocytes despite nucleus
progression from prophase I to MI (Table 2). There was no
significant difference between GV-intact and MI oocytes in
Ca 2+ peak level, duration, and periodicity.
the fertilization-induced Ca 2+ oscillations (Table 2). This
suggests that spontaneous Ca 2+ oscillations are dependent
on the activation of PLC, while at fertilization, the generation of Ca 2+ oscillations is independent of PLC for the
first few Ca 2+ transients. Nevertheless, the maintenance of
fertilization-induced Ca 2+ oscillations seemed dependent on
PLC since long-lasting fertilization-induced Ca 2+ oscillations were not observed.
Effect of Enucleation on Spontaneous Ca Oscillations
and Fertilization-Induced Ca Oscillations in GV
Oocytes
2 +
2 +
Inhibitory Effect of Heparin on Spontaneous Ca2 +
Oscillations and Fertilization-Induced Ca Oscillations
2 +
Oocytes undergoing spontaneous Ca2+ oscillations were
injected with heparin. Spontaneous Ca 2+ oscillations in GV
oocytes were suppressed completely after heparin injection
(25 M final intracellular concentration). In the control
group, injection of basal medium or de-N-sulfate heparin at
the same concentration had no effect on Ca 2+ oscillations
(Table 3). In contrast, heparin failed to suppress completely
the fertilization-induced Ca 2+ rises. It was observed that
fertilization induced a new series of Ca 2 + oscillations after
inhibition of spontaneous Ca 2+ oscillations by heparin (25
IM). Increasing heparin to 200 IM did not block the occurrence of fertilization-induced Ca2+ oscillations but reduced the Ca 2+ rise numbers (Table 3). Heparin was unable
to suppress all the Ca2+ rises during fertilization, and we
observed that even when heparin was increased to as high
as 600 jiM, oocytes still displayed 1-3 Ca 2+ rises after
fertilization (Table 3).
Inhibition of PLC Using U73 122 and Its Effect on
Spontaneous and Fertilization-Induced Ca2+ Oscillations
in GV Oocytes
Oocytes undergoing spontaneous Ca 2+ oscillations all
ceased to oscillate within 15 min after exposure to U73122
(10 plM in final concentration, n = 25), but subsequent
insemination induced an additional series of Ca2 + oscillations (Fig. 4). Increasing U73122 to 20 jiM, a commonly
accepted dose for inhibition of PLC, did not show an inhibitory effect on fertilization-induced Ca 2+ oscillations
(Table 2). Even when U73122 was increased to as high as
40 tM, fertilization still induced 2.3 + 2.5 Ca 2+ transients
(mean + SD, n = 17). In the control experiment, exposure
to U73343, an inactive analogue of the PLC inhibitor
U73122, had no effect on spontaneous Ca 2+ oscillations or
FIG. 4. Effect of U73122 on spontaneous
2+
and fertilization-induced Ca oscillations
Ca2+
Spontaneous
in mouse GV oocytes.
oscillations were completely suppressed
by 40 [tM U73122 whereas subsequent
2+
fertilization induced a new series of Ca
oscillations.
Spontaneous Ca2+ oscillations in enucleated oocytes
and isolated GVs. Oocytes undergoing spontaneous Ca2+
oscillations were enucleated using micromanipulators, and
the produced karyoplasts and cytoplasts were paired with
each other in the same chamber. It was found that the number of spontaneous Ca 2+ transients were significantly reduced in cytoplasts compared with karyoplasts (Table 4).
Spontaneous Ca 2+ oscillations ceased abruptly in most cytoplasts (n = 8/11), and only a few of them showed 1-3
Ca2+ transients (n = 3/11), whereas oscillations remained
persistent in the karyoplasts (Table 4), indicating that the
presence of an intact GV is required for the maintenance
of spontaneous Ca 2+ oscillations in cytoplasm.
Fertilization-induced Ca2 + responses in karyoplasts and
cytoplasts. The karyoplasts and the cytoplasts showed a
similar pattern of Ca 2+ oscillations in response to fertilization, and there was no difference between karyoplasts and
cytoplasts in the Ca 2+ peak, peak duration, and periodicity
(Table 4). Interestingly, however, the isolated GVs with
much less cytoplasm attached showed fewer and smaller
Ca 2+ rises in response to fertilization (Table 4). This indicates that the Ca 2+ oscillations are dependent primarily on
cytoplasm and that the GV does not play a role in cytoplasmic Ca 2 oscillations at fertilization.
DISCUSSION
Immature GV Oocytes Have Acquired the Ability to
Generate Repetitive Ca2 + Oscillations in Response to
Fertilization
Repetitive Ca 2+ oscillations in mature MII eggs during
fertilization have been widely reported in several species of
mammals [1-6]. However, intracellular Ca 2+ LCa2+]i responses of immature GV-stage oocytes to fertilization are
CALCIUM OSCILLATIONS IN MOUSE GV OOCYTES
TABLE 3. Effect of heparin and U73122 on spontaneous and fertilization-induced Ca2+ oscillations.
Treatment
Number of
oocytes used
Ca2 + rise
number in 1.5 hde
15"
12 b
17b
12a
15 b
25 a
22a
25 b
28 b
0
12.7 + 5.3g
2.5 ± 1.8 h
11.2 + 3.3
13.3 + 3.8
0f
17.2 + 5.3
19.7 + 6.4
18.3 + 8.2
Heparin 25 M
Heparin 200 M
Heparin 600 FLM
de-N-sulfate
Heparinc
U73122 10 M
U73343 10 M
U73122 20 M
U73343 20 M
less studied, and the reported results are inconsistent [1012]. Some authors reported that fertilization cannot induce
repetitive Ca2+ oscillations in immature GV-stage oocytes,
but instead induces only 2-3 [Ca 2+]i spikes that usually
cease within 1 h [12]. They proposed that the development
of repetitive Ca 2+ transients in response to fertilization occurs late in oocyte maturation and is dependent on cytoplasmic modifications that are independent of cell cycle
progression from MI to MII [12]. In contrast, other authors
reported that immature GV oocytes show multiple Ca2+
transients at fertilization but that the first Ca2+ transient is
much smaller [11]. Our experiments demonstrated that, like
mature MII eggs, immature GV oocytes are able to produce
Ca2 ' oscillations lasting for several hours after fertilization
but are lower in amplitude. The reason for the higher amplitude of the fertilization-induced Ca 2+ oscillations compared with the spontaneous Ca 2+ oscillations was unclear.
It may be that fertilization sensitizes Ca 2+ stores and releases more [Ca 2+]i compared with spontaneous Ca2+ oscillations.
The observed fertilization-induced Ca 2+ oscillations cannot be attributed to incomplete cessation of spontaneous
Ca 2+ oscillations, since the interval between spontaneous
Ca 2+ oscillations and fertilization-induced Ca2+ oscillations
was long enough to prevent the possible overlap of the two
types of Ca2 + oscillations. In addition, the subsequent new
Component
Karoplast
Cytoplast
Karoplastsc
series of Ca2+ oscillations was indeed induced by fertilization, because unpenetrated GV oocytes showed no Ca 2+ rise
(data not shown). It is known that GV oocytes frequently
become polyspermic upon fertilization [26]. However, polyspermy does not seem to have an obvious influence on the
Ca 2+ oscillation pattern, since there was no significant difference in the Ca 2+ oscillations between single-sperm insemination and polyspermic insemination in mouse oocytes
(data not shown).
Different Dependence on IICR of Spontaneous Ca2 +
Oscillations and Fertilization-Induced Ca2 + Oscillations
" Oocytes undergoing spontaneous Ca2 + oscillations were injected with
heparin (25 M) or bathed in U73122 (20 M).
b Fertilization-induced Ca2 ' responses in the heparin-injected or U73122treated oocytes.
c Injection of de-N-sulfate heparin or addition of U73343 as a control
experiment.
2+
d Ca
changes were measured for 1.5 h after heparin injection or addition
of U73122.
e Data are presented as means + SD; the interval between heparin injection and Ca2 + measurement was within 15 min.
f Ca2 ' rise number was counted 15 min after addition of U73122, allowing uniform distribution of the drugs.
g,h Means with different superscripts are different within columns (p <
0.05).
TABLE 4.
811
Two mechanisms have been proposed to be responsible
for Ca 2+ oscillations in a wide variety of cells. One is IP3induced Ca 2+ release (IICR), and the other is Ca 2+-induced
Ca 2 + release [8, 9, 27]. There has been no disagreement
with the hypothesis that spontaneous Ca 2 + oscillations in
oocytes are regulated mainly by IICR, but there are discrepant reports on the role of IICR in the fertilization-triggered Ca2 + response in different species [6, 8, 10, 16-20].
Although there is evidence indicating that mouse oocytes
possess both IP3 and ryanodine receptors [28, 29], Ca2+
release from the ryanodine-gated store is not essential for
fertilization-induced Ca 2+ oscillations [29, 30], and therefore IICR is suggested to be the only mechanism responsible for fertilization-induced Ca 2+ oscillations [11, 18,
29, 30]. Microinjection of heparin, an IP3-receptor antagonist, blocks activation and both IP3- and Ca 2+-induced
Ca2+ release in intact frog oocytes and homogenates [3133]; in addition, injection of a function-blocking monoclonal antibody to IP3 receptor blocks fertilization-induced
Ca 2+ oscillations as well as propagating Ca 2+ waves in
hamster eggs [18, 34] and also blocks both early and late
events of mouse egg activation [35]. In contrast, heparin
abolishes the egg's response to IP3 but does not prevent
the fertilization-induced Ca 2 + transient in sea urchin and
pig eggs [16, 17, 19, 20], suggesting that IP3 receptor does
not contribute substantially to the Ca2+ transient at fertilization. It is not clear why the antibody blocks fertilizationinduced Ca 2 + release although the commonly used IP3 receptor antagonist heparin fails to do so. This is paradoxical
because the antibody and heparin are both shown to be
equally effective at inhibiting IP3-induced Ca2+ release
[34]. However, it should be noted that the antibody not only
suppresses fertilization-induced Ca 2+ oscillations but also
thimerosal-induced Ca2+ oscillations [36], whereas heparin
inhibits only the IP3-induced Ca 2+ rise but does not affect
the thimerosal-induced oscillations ([13] and our unpublished results). It is clear that fertilization and thimerosal
stimulate Ca 2+ oscillations by separate mechanisms in
mouse oocytes [37]. The explanation for this discrepancy
may be that heparin blocks the IP3-binding site of the IP3
Effect of enucleation on spontaneous and fertilization-induced Ca2 + oscillations.
Number of
oocytes used
11,
12 b
11 a
12b
8b
Ca2 + at
baseline (nM)
97.7
109.9
106
107.9
72.5
a Spontaneous Ca2 + oscillations after enucleation.
2
± 42.2
+ 22.1
+ 25.1
+ 43.4
+ 23.8
Ca2 + rise
number in 1.5 h
7.5
20.1
1.2
17
2.1
b Fertilization-induced Ca + oscillations.
c Karoplasts with much few cytoplasm attached.
de Means with different superscripts are different within columns (p < 0.01).
_
+
_
+
3.0d
5.9 d
1.20
7.8 d
1.3e
Ca2 + at peak
(nM)
223.4
293.8
310
329.6
150.2
t 82.4
± 58.8d
± 115.3
71.9
± 21.5 e
Peak duration
(sec)
81.5
35.2
66.8
7.3
54.5 ± 18.6
66.7 ± 4.7
124.9
17.6
DENG ET AL.
812
+2
channelreceptor but that the antibody blocks the Ca
failed to
forming domain [38, 39]. Since heparin injection
+2
in
response
Ca
fertilization-induced
the
entirely suppress
sea urchins, pigs [16, 17, 19, 20], and the mice in our experiment, the contribution of IICR to fertilization-induced
Ca 2+ oscillations remains to be further defined. Our experiments in the mouse suggest that IICR is not the only source
of Ca+2 release at fertilization and that the heparin-insensitive Ca 2 + release pathway may be used during fertilization. This is also supported by evidence that inhibition of
endogenous generation of IP3 by antagonizing PLC-induced hydrolysis of PIP2 and production of IP3, using
U73122, was unable to suppress the generation of fertilization-induced Ca 2+ oscillations [8]. This is somewhat inconsistent with the previous report on mature MII eggs [40].
In the above experiment, the authors added U73122 after
+2
oscillations were observed and
fertilization-induced Ca
+2
abolished. However, the
the successive Ca transients were
2+
generation of the first few Ca transients were never inin our experiment.
hibited even at the high dose of 40 M 2+
transients at ferCa
of
generation
the
that
This suggests
tilization may be independent of PLC activation but that
maintenance of Ca 2+ oscillations requires activation of
PLC. The incomplete inhibition by U73122 of fertilizationinduced Ca 2+ oscillations seems not to be due to incomplete
inhibition since the U73122 used in our experiment entirely
2+
suppressed spontaneous Ca oscillations as well as ace2+
not shown). Acetylcholine-induced Ca transients 2(data
+
tylcholine is known to stimulate Ca spiking through PLC
activation [37], indicating that U73122 was effective in inhibiting PLC. These results lead us to conclude that 2+spontaneous Ca 2+ oscillations and fertilization-induced Ca oscillations are modulated by different mechanisms. Spontaprimarily on the IICR,
neous Ca 2 + oscillations are depend
2+
whereas fertilization-induced Ca oscillations may involve
a much more complex intracellular mechanism than was
previously expected.
Fertilization-nduced Ca Oscillations Are Mainly
Dependent on Cytoplasmic Maturation but Not Nuclear
Progression
2+
Mouse oocytes gradually acquire the ability to support
Ca 2+ oscillations during an early stage of growth and maturation [14]. GV oocytes collected from young 2+mice (16
days after birth) begin to show spontaneous Ca oscillations after release from follicles [14]. But how the spontaneous Ca2+ oscillations are initiated and what their biological role is in oocyte maturation are totally unknown.
fails to influAlthough inhibition of GVBD using dbcAMP
2+
reence the occurrence of spontaneous Ca oscillations,
2+
oscilCa
abolishes
oocytes
from
GV
intact
moval of the
lations in the cytoplasm. This suggests that spontaneous
Ca 2 + oscillations depend on the presence of an intact GV.
Spontaneous Ca 2+ oscillations still occurred in karyoplasts
but ceased abruptly in cytoplasts, indicating that the trigger
of spontaneous Ca 2+ oscillations may originate in the maturing nucleus and propagate to the cytoplasm, although the
exact Ca2+ signaling pathways from nucleus to cytoplasm
are unknown at present. In contrast, fertilization-induced
Ca2+ oscillations do not rely on the presence of an intact
GV, since nucleated and enucleated oocytes produced a
similar pattern of Ca2+ oscillations in response to fertiliza2+
tion. Furthermore, the pattern of fertilization-induced Ca
unafremained
oocytes
GV
oscillations in the immature
fected by the presence or absence of nuclear progression
+2
from GVBD to MI during Ca measurement,2 +and in vitrocultured MI oocytes showed a pattern of Ca oscillations
similar to that of GV-intact oocytes at2+fertilization. This
oscillations are
suggests that fertilization-induced Ca
mainly dependent on cytoplasmic maturation but independent of nuclear progression.
REFERENCES
2+
1. Cuthbertson KSR, Whittingham DG, Cobbold PH. Free Ca increas1981;
Nature
activation.
oocyte
mouse
during
es in exponential phases
294:754-757.
2. Miyazaki S, Hashimoto N, Yoshimoto Y, Kishimoto T, Igusa Y, Hiramoto Y. Temporal and spatial dynamics of the periodic increase in
intracellular free calcium at fertilization of golden hamster eggs. Dev
Biol 1986; 118:259-267.
3. Kline D, Kline JT. Repetitive calcium transients and the role of calcium in exocytosis and cell cycle activation in the mouse egg. Dev
Biol 1992; 149:80-89.
4. Sun FZ, Hoyland J, Huang X, Mason W, Moor RM. A comparison
of intracellular changes in porcine eggs after fertilization and electroactivation. Development 1992; 115:947-956.
5. Fissore RA, Dobrinsky JR, Balise JJ, Duby RT, Robl JM. Patterns of
2
intracellular Ca concentration in fertilized bovine eggs. Biol Reprod
1992; 47:960-969.
inositol trisphosphate, and thimerosal6. Fissore RA, Robl JM. Sperm,
2+
induced intracellular Ca elevations in rabbit eggs. Dev Biol 1993;
159:122-130.
7. Jaffe LE The role of calcium explosions, waves and pulses in activating eggs. In: Metz CB, Monroy A (eds.), Biology of Fertilization.
New York: Academic Press; 1985: 127-165.
8. Miyazaki S. Cell signaling at fertilization of hamster eggs. J Reprod
Fertil Suppl 1990; 42:163-175.
9. Whitaker M, Swann K. Lighting the fuse at fertilization. Development
1993; 117:1-12.
10. Fujiwara T, Nakada K, Shirakawa H, Miyazaki S. Development of
inositol trisphosphate-induced calcium release mechanism during maturation of hamster oocytes. Dev Biol 1993; 156:69-79.
11. Mehlmann L, Kline D. Regulation of intracellular calcium in the
mouse egg: calcium release in response to sperm or inositol trisphosphate is enhanced after meiotic maturation. Biol Reprod 1994; 51:
1088-1098.
DG. Ionomycin, thapsigargin, ryano12. Jones K, Carroll J, Whittingham
2+
dine, and sperm induced Ca release increase during meiotic maturation of mouse oocytes. J Biol Chem 1995; 270:6671-6677.
13. Carroll J, Swann K. Spontaneous cytosolic calcium oscillations driven
by inositol trisphosphate occur during in vitro maturation of mouse
oocytes. J Biol Chem 1992; 267:11196-11201.
DG, Whitaker M. Spatiotemporal
14. Carroll J, Swann K, Whittingham
2
dynamics of intracellular [Ca +]i oscillations during the growth and
meiotic maturation of mouse oocytes. Development 1995; 120:35073517.
15. Homa ST, Carroll J, Swann K. The role of calcium in mammalian
oocyte maturation and egg activation. Hum Reprod 1993; 8:12741281.
16. Rakow TL, Shen SS. Multiple stores of calcium are released in the
sea urchin egg during fertilization. Proc Natl Acad Sci USA 1990;
87:9285-9286.
17. Crossley I, Whalley T, Whitaker MJ. Guanosine 5-thiotriphosphate
may stimulate phosphoinositide messenger production in sea urchin
eggs by a different route than the fertilizing sperm. Cell Regul 1991;
2:121-133.
Y. Essential role of the+
18. Miyazaki S, Shirakawa H, Nakada K, Honda
2
2+
release channel in Ca
inositol 1,4,5-trisphosphate receptor/Ca
2
Dev
eggs.
mammalian
waves and Ca oscillations at fertilization of
Biol 1993; 158:62-78.
19. Sun FZ, Bradshaw JP, Galli C, Moor RM. Changes in intracellular
concentration in bovine oocytes following penetration by spermatozoa. J Reprod Fertil 1994; 101:713-719.
20. Lee HC, Aarhus R, Walseth TE Calcium mobilization by dual receptors during fertilization of sea urchin eggs. Nature 1993; 261:352355.
21. Nasr-Esfahani M, Johnson MH, Aitken RJ. The effect of iron and iron
chelators on the in vivo block to development of the mouse preimplantation embryo: BAT6 a new medium for improved culture of
mouse oocytes in vitro. Hum Reprod 1990; 5:997-1003.
CALCIUM OSCILLATIONS IN MOUSE GV OOCYTES
22. Cho WK, Stem S, Biggers JD. Inhibitory effect of dibutyryl cAMP
on mouse oocyte maturation in vitro. J Exp Zool 1974; 187:383-386.
23. Hogan B, Costantini F, Lacy E. Manipulating the Mouse Embryo. A
Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor
Laboratory; 1986.
24. Quinn P, Barros C, Whittingham DG. Preservation of hamster oocytes
to assay the fertilizing capacity of human spermatozoa. J Reprod Fertil
1982; 66:161-168.
25. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem
1985; 260:3440-3450.
26. Ducibella T Mammalian egg cortical granules and the cortical reaction. In: Wasserman PM (ed.), Elements of Mammalian Fertilization.
Boca Raton, FL: CRC Press; 1991: 205-230.
27. Berridge MJ, Galione A. Cytosolic calcium oscillations. FASEB J
1988; 2:3074-3082.
28. Swann K. Different triggers for calcium oscillations in mouse eggs
involve a ryanodine-sensitive calcium store. Biochem J 1992; 287:
79-84.
29. Ayabe T, Kopf G, Schultz RM. Regulation of mouse egg activation:
presence of ryanodine receptors and effects of microinjected ryanodine and cyclic ADP ribose on uninseminated and inseminated eggs.
Development 1995; 121:2233-2244.
30. Kline JT, Kline D. Regulation of intracellular calcium in the mouse
egg: evidence for inositol trisphosphate-induced calcium release, but
not calcium-induced calcium release. Biol Reprod 1994; 50:193-203.
31. Delisle S, Welsh MJ. Inositol trisphosphate is required for the propagation of calcium waves in Xenopus oocytes. J Biol Chem 1992;
267:7963-7966.
813
32. Nuccitelli R, Yim DL, Smart T. The sperm-induced Ca2 wave following fertilization of the Xenopus egg requires the production of
Ins(1,4,5)P3. Dev Biol 1993; 158:200-212.
33. Galione A, McDougall A, Busa WB, Willmott N, Gillot I, Whitaker
M. Ewdundant mechanisms of calcium-induced calcium release underlying calcium waves during fertilization of sea urchin eggs. Science
1993; 261:348-352.
34. Miyazaki S, Yuzaki M, Nakada K, Shirakawa H, Hakanishi S. Nakada
S, Mikoshiba K. Block of Ca2+ wave and Ca 2 + oscillation by antibody
to the inositol 1,4,5-trisphosphate receptor in fertilized hamster eggs.
Science 1992; 257:251-155.
35. Xu Z, Kopf GS, Schultz RM. Involvement of inositol 1,4,5-trisphosphate-mediated Ca 2+ release in early and late events of mouse egg
activation, Development 1994; 120:1851-1859.
36. Miyazaki S, Shirakawa H, Nakada K, Honda Y, Yuzaki M, Nakada
S, Mikoshiba K. Antibody to the inositol trisphosphate receptor blocks
thimerosal-enhanced Ca2+-induced Ca 2+ release and Ca 2+ oscillations
in hamster eggs. FEBS Lett 1992; 309:180-184.
37. Cheek TR, McGuinness OM, Vincent C, Moreton RB, Berridge MJ.
Fertilization and thimerosal stimulate similar calcium spiking patterns
in mouse oocytes but by separate mechanisms. Development 1993;
119:179-189.
38. Furuichi T, Kohda K, Miyazaki A, Mikoshiba K. Intracellular channels. Curr Opin Neurobiol 1994; 4:294-303.
39. Swann K. Soluble sperm factors and Ca 2+ release in eggs at fertilization. Rev Reprod 1996; 1:33-39.
40. Dupont G, Mcguinness OM, Johnson MH, Berridge MJ, Borgese E
Phospholipase C in mouse oocytes: characterization of (and isoforms
and their possible involvement in sperm-induced Ca2 + spiking. Biochem J 1996; 316:583-591.