(Received for publication, October 12, 1994; and in revised form, January 9, 1995)
From the
Fertilization of mature mouse oocytes triggered highly
repetitive Ca oscillations lasting 2-3 h.
However, immature oocytes generated only two or three oscillations,
which ceased within 1 h. Development of repetitive Ca
transients to sperm occurred late in oocyte maturation and was
dependent on cytoplasmic modifications that were independent of cell
cycle progression from metaphase I to metaphase II. Immature oocytes
released significantly less Ca
from stores than
mature oocytes in response to ionomycin and thapsigargin. Ryanodine had
no effect on intracellular Ca
in maturing oocytes but
stimulated an increase in Ca
in mature oocytes. The
ability of ryanodine to increase Ca
levels was,
however, strain-dependent.
Preincubation of oocytes with
thapsigargin or ryanodine significantly attenuated the normal
fertilization Ca response, causing a decrease in the
number and the rate of rise of the transients. The inhibition of
sperm-induced Ca
transients by ryanodine was
independent of its ability to cause an immediate Ca
increase. Low concentrations of ryanodine had no effect on
resting Ca
levels but inhibited Ca
oscillations at fertilization. Similarly Ca
oscillations were blocked in oocytes from a strain of mouse that
showed no immediate Ca
increase with ryanodine. These
results suggest that modifications in Ca
stores and
ryanodine-sensitive Ca
release mechanisms during
oocyte maturation play an important role in Ca
oscillations at fertilization.
In mammalian oocytes a series of highly repetitive
Ca transients lasting several hours are triggered at
fertilization(1, 2, 3) . The Ca
transients act both to initiate completion of meiosis and to
block polyspermy by stimulating cortical granule
exocytosis(4, 5) . Precisely how these Ca
oscillations are generated is not clear.
Ca oscillations, like those observed at fertilization, occur in a
wide variety of cell types (6, 7, 8) and are
mediated by two families of Ca
channels on the
endoplasmic reticulum(9) . The generation of Ca
oscillations is normally stimulated by an agonist-induced
increase in inositol trisphosphate (InsP
). (
)Ca
released in response to InsP
acts, by positive feedback, to stimulate further Ca
release through the InsP
receptor (10) or the
ryanodine receptor(11) . The ability of Ca
to
stimulate further Ca
release from intracellular
stores is known as Ca
-induced Ca
release (CICR). InsP
-induced Ca
release is present in both mouse and hamster oocytes since
microinjection of InsP
triggers Ca
oscillations(12, 13) . Additionally an antibody
to the InsP
receptor inhibits Ca
oscillations induced by InsP
, Ca
injection, and sperm (14) . This suggests that both
InsP
- and Ca
-induced Ca
release in oocytes are mediated through the InsP
receptor(15) .
In mouse oocytes there is also evidence
for the presence of a ryanodine receptor. Ryanodine binds to the
ryanodine receptor and blocks it in an open state that has low
conductance(16) . Its effects are complex, with activation and
inhibition of CICR through the ryanodine receptor being reported
depending on experimental conditions(17) . The ability of
ryanodine to raise Ca levels in mouse oocytes is
controversial, with Swann finding that ryanodine triggers
Ca
release, inhibits agonist-induced oscillations,
and enhances CICR (18) whereas Kline and Kline find no
effect(19) .
The ability to undergo normal fertilization is acquired during oocyte maturation(20) . Immature mouse oocytes arrested at prophase of the first meiotic division are morphologically identified by a germinal vesicle (GV)(20) . In vivo, maturation is stimulated by gonadotropins but can occur in vitro by removal of the immature oocyte from the follicle into culture medium(21) . GV breakdown (GVBD) occurs about 2 h after the resumption of meiosis. At 8-10 h the first polar body is extruded, and then the oocyte arrests at metaphase II until fertilization stimulates the completion of meiosis and entry into the first mitotic cell cycle. In addition to the events of meiotic maturation, cytoplasmic modifications such as the ability to release cortical granules (22, 23) and decondense the sperm nucleus (24) are necessary for normal fertilization.
Recent
observations suggest that an important aspect of cytoplasmic maturation
is modifications in Ca homeostasis. GV stage oocytes
show spontaneous Ca
oscillations, which decrease
following GVBD(25) . Mature oocytes, but not immature oocytes,
show Ca
waves in response to a
Ca
-releasing factor from sperm(26) .
Additionally, during maturation there is an increase in sensitivity to
InsP
-induced Ca
release (27) and
an increase in the amount of Ca
released by
ionomycin(28) , suggesting that Ca
stores
increase in preparation for fertilization.
The aim of the present
study was to examine how Ca homeostasis is modified
during oocyte maturation by examining the responses to ionomycin,
thapsigargin, ryanodine, and sperm. We find that immature oocytes do
not show highly repetitive Ca
transients during
fertilization, and the development of such transients occurs late in
oocyte maturation. This modification in the mechanism of sperm-induced
Ca
release is correlated with an increase in
sensitivity to ionomycin, thapsigargin, and ryanodine.
Figure 1:
Comparison of sperm-induced
Ca oscillations in mature and immature oocytes. a, representative Ca
recording from a mature
oocyte loaded with Indo-1. The oocyte continued to show Ca
oscillations for 3.5 h. Arrows indicate the pacemaker
Ca
triggering further Ca
release. b, the first transient in a mature oocyte. Arrow indicates a shoulder during the rise phase. Note the
superimposed spikes during the peak. c,
representative Ca
recording in an immature oocyte
showing that the Ca
transients do not continue. Inset, a magnified trace of the second and third transient and
subsequent pacemaker Ca
that fails to initiate a
further transient. d, the first transient in an immature
oocyte. Arrow indicates a shoulder during the rise phase. Note
the absence of spikes during the peak. The timebar is 5 min.
The
pattern of Ca oscillations in response to sperm was
markedly different in immature oocytes where there were a maximum of
three much smaller transients, which ceased after 1 h (Fig. 1c) (n = 15). Similar to mature
oocytes the first transient showed a shoulder in its rising phase but
was shorter in duration and had no superimposed spikes (Fig. 1d). A common feature in oocytes in which
oscillations ceased was that the pacemaker increase in basal
Ca
occurred but failed to initiate a complete
transient (Fig. 1c, inset). To determine the
state of filling of the Ca
stores once oscillations
had ceased, 10 µM ionomycin was added to the oocytes after
chelation of extracellular Ca
with 10 mM EGTA. Ionomycin did not trigger a Ca
transient
in these oocytes (Fig. 2, n = 4), suggesting
that fertilization had depleted intracellular Ca
stores.
Figure 2:
Depletion of Ca stores
in an immature fertilized oocyte. Once sperm-induced Ca
transients had ceased, 10 mM EGTA (openbar) and 10 µM ionomycin (solidbar) were added sequentially. No Ca
increase was recorded.
Figure 3:
The development of continuous oscillations
in response to sperm during maturation. a, at 10.5 h after
release from the follicle, oocytes that had extruded the first polar
body showed an attenuated response to sperm. Continuous Ca oscillations following fertilization were observed both in b, oocytes at 12 h, and c, metaphase I-arrested
oocytes at 14 h after meiotic resumption.
During in vitro maturation some oocytes arrest spontaneously at metaphase I. These
arrested oocytes when fertilized 14 h after the resumption of meiosis
generated continuous Ca oscillations similar to
mature ovulated oocytes (Fig. 3c). Therefore, the
mature oocyte-like response to sperm does not require progression from
metaphase I to metaphase II but is dependent on cytoplasmic
modifications, which occur about 12 h after release from the follicle.
Figure 4:
Comparison of thapsigargin and
ionomycin-induced Ca transients in immature and
mature oocytes. a, GV stage (a, b) and mature (c,
d) oocytes were placed in Ca
-free medium
containing 3 mM EGTA. The Ca
increase was
recorded following addition of 10 µM thapsigargin (a,
c) or 10 µM ionomycin (b, d). The responses
of mature oocytes were greater than immature oocytes. The timebar represents 5 min. b, the area under the
transient. c, peak ratio when thapsigargin (openbars) or ionomycin (hatchedbars) were
added to oocytes at the GV stage (GV), 2-4 h after GVBD (GVBD), or to mature oocytes (mature). * (p < 0.05, Student's unpaired t test) and** (p < 0.01), significantly different from corresponding GV stage
oocyte. ++ (p < 0.01), significantly different
from GVBD stage oocytes.
Figure 5:
The effects of ryanodine on intracellular
Ca in mature and immature oocytes. Ryanodine (1
mM) was added extracellularly at the indicated time (solidbar) to mature (a) and immature (b)
oocytes. Ryanodine caused an increase in Ca
in mature
(but not immature) oocytes. Oocytes were loaded with Fluo-3 AM, and
therefore fluorescence intensity is
arbitrary.
Figure 6:
Fertilization-induced Ca
changes in mature oocytes treated with thapsigargin or ryanodine.
Preincubation of oocytes with 10 µM thapsigargin had a
variable effect on Ca
transients at fertilization. In
some oocytes, the first transient was reduced but oscillations
continued (a) while in others, continuous oscillations were
inhibited (b). c, preincubation with 1 mM ryanodine inhibited repetitive Ca
oscillations
in all oocytes studied.
The first four
sperm-induced Ca transients in mature oocytes treated
with thapsigargin and ryanodine were compared with controls with
respect to the rate of rise (Fig. 7a), duration (Fig. 7b), and peak ratio (Fig. 7c).
The first transient showed a distinct shoulder (see Fig. 1, b and d); therefore, the rise time before and after
this shoulder was calculated. In mature oocytes the duration of the
first transient was longer and the peak ratio greater than subsequent
transients (Fig. 7, b and c). However, the
rise time of the transients became progressively faster (Fig. 7a). In mature oocytes preincubated with
ryanodine or thapsigargin the rate of rise and peak ratio were reduced
for all transients.
Figure 7:
Analysis of the fertilization-induced
Ca transients in mature and immature oocytes. The
rise time (a), the duration (b), and the peak ratio (c) of the first four transients following fertilization in
both mature and immature oocytes in controls or after treatment with 10
µM thapsigargin (Tg) or 1 mM ryanodine (Ry) are shown. Transients are in sequential order:
,
first transient; &cjs2106;, second transient; &cjs2110;, third
transient,
,fourth transient. The first transient had a distinct
shoulder during its rising phase; therefore, the rate of rise is
measured before and after this shoulder.
In immature oocytes the rise time, duration, and
peak ratio of the sperm-induced Ca transients were
found to be less than in mature oocytes (Fig. 7). Thapsigargin
preincubation abolished the shoulder of the first transient and greatly
shortened the duration of all the oscillations. Only one sperm-induced
transient was observed in immature oocytes preincubated with 1 mM ryanodine, and this was of shorter duration and smaller peak
ratio. This suggests that ryanodine had an inhibitory effect on the
immature oocyte response to sperm without inducing an immediate rise in
intracellular Ca
. The possibility that immature
oocytes have a ryanodine receptor was further examined in spontaneously
oscillating GV stage oocytes since ryanodine binding is influenced by
the open state of the channel and Ca
concentration(17) . 1 mM ryanodine induced an
immediate Ca
rise superimposed on the spontaneous
oscillations in 71% (n = 7) of oocytes studied (Fig. 8).
Figure 8:
Ryanodine induces a Ca
rise in GV stage oocytes undergoing spontaneous oscillations. 1 mM ryanodine (solidbar) added to GV stage oocytes
undergoing spontaneous oscillations induced a rise in
Ca
. Spontaneous oscillations occurred superimposed on
the ryanodine-induced Ca
transient.
Figure 9:
The effect of ryanodine on Ca levels in MF1 outbred mice. a, 1 mM ryanodine
did not raise Ca
levels when added to mature MF1
oocytes; b, normal sperm-induced Ca
transients at fertilization in MF1 oocytes; c, 1 mM ryanodine preincubation during dye loading blocked the normal
sperm-induced Ca
transients.
We have investigated the ability of oocytes to generate
Ca oscillations in response to sperm at different
times during oocyte maturation. In agreement with previous
studies(1, 2, 3) , we have found that
fertilization of mature mouse oocytes triggered a series of 20-40
Ca
transients continuing for 2-3 h. In immature
oocytes the response to sperm was attenuated, with the generation of
only 2-3 transients that ceased within 1 h. The ability to
generate multiple Ca
transients occurred late during
oocyte maturation, after first polar body formation. This transition
was associated with an increase in the amount of Ca
released in response to ionomycin and thapsigargin in addition to
an increase in the sensitivity to ryanodine. These results show that
modifications in Ca
homeostasis are an integral part
of oocyte maturation.
An increase in
intracellular Ca at fertilization is known to
stimulate cell cycle re-entry and cortical granule exocytosis. The
attenuated Ca
response of oocytes 10-12 h after
meiotic resumption may explain the low rate of oocyte activation
induced by sperm and parthenogenetic stimuli in these
oocytes(40) . Similarly the ability to undergo global cortical
granule release does not occur until late in maturation between
metaphase I and metaphase II(41) , suggesting that this
response may require the maturation of intracellular Ca
stores. Together these data suggest that the changes in
Ca
homeostasis during oocyte maturation are an
important mechanism for normal fertilization to occur. Further, the
late onset of these changes would prevent premature oocyte activation
and cortical granule exocytosis.
A common
feature of the first transient was a shoulder during its rising phase
suggesting there are two Ca release mechanisms or
stores, triggered at differing levels of cytoplasmic
Ca
. This shoulder has been observed using the
Ca
-sensitive protein aequorin in both mouse and human
oocytes at fertilization (2, 42) but not previously
using conventional dyes. The subsequent transients had a faster rate of
rise and did not show a distinct shoulder during the rising phase,
suggesting that Ca
release from the second store had
undergone sensitization. This is consistent with previous findings that
demonstrate that fertilization leads to an increased sensitivity of
CICR induced by Ca
injection(43) . In
immature oocytes, similar to mature oocytes, the first transient was
larger and longer lasting than subsequent oscillations. However,
oscillations did not continue and had a smaller peak ratio, a shorter
duration, and a slower rate of rise compared with mature oocytes. The
possible reasons for this attenuated response are discussed in the
following sections.
The increased thapsigargin-sensitive
Ca store appears to be important for fertilization
since preincubation with thapsigargin decreased the rate of rise, the
duration, and the number of oscillations generated by sperm. The
ability of thapsigargin to inhibit repetitive Ca
oscillations was correlated with its effect on the first
Ca
transient, which was normal in
thapsigargin-treated oocytes that continued to oscillate but was not
present in oocytes where oscillations ceased (Fig. 6). A similar
observation that thapsigargin is able to block continuous oscillations
in only some oocytes has previously been made (44) and
suggested to be due to the time between thapsigargin addition and
fertilization, which in the present study was kept constant. We
therefore suggest that sensitivity to thapsigargin varies among
oocytes.
The sensitivity of InsP-induced Ca
release is directly correlated with an increase in Ca
stores (45) . Therefore, the observed increase in
Ca
stores seen during oocyte maturation may explain
the increased sensitivity to InsP
during the maturation of
hamster oocytes(27) . Ca
store size is also
known to regulate the sensitivity of Ca
release from
ryanodine- and caffeine-sensitive Ca
stores(46, 47) . While there is no evidence for
caffeine-sensitive stores in mouse oocytes, ryanodine-sensitive release
mechanisms have been reported(18) .
Further support for a
ryanodine-sensitive Ca release mechanism in both
mature and immature oocytes is provided by the observation that
ryanodine blocks sperm-induced Ca
oscillations. This
is independent of the ability of ryanodine to induce an immediate
increase in Ca
. The addition of 100 µM ryanodine to mature oocytes and 1 mM ryanodine to
immature oocytes has no immediate effect on Ca
but
inhibits sperm-induced oscillations. The presence of a ryanodine
receptor in oocytes is controversial since Kline and Kline (19) report no Ca
increases following
ryanodine addition, in contrast to the present findings and those of
Swann(18) . Results presented here may explain the discrepancy
between these studies and those of Kline and Kline (19) because
we have shown that the ability of ryanodine to cause an immediate
Ca
rise is strain-dependent. Ryanodine stimulates an
immediate Ca
rise in oocytes from an F1 hybrid as
used here and by Swann (18) and not in oocytes from an outbred
MF1 strain, similar to the outbred CF1 strain used by Kline and
Kline(19) . However, in both strains ryanodine inhibited
sperm-induced Ca
oscillations. Therefore, results
presented here with ryanodine and those of Miyazaki et al.(14, 51) suggest that both ryanodine and
InsP
receptors are essential for the generation of
Ca
oscillations at fertilization in the mouse.