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1
* Asamushi Marine Biological Station, Asamushi, Aomori 039-3501, Japan;
Department of Biochemistry, University of Padova, 35121 Padova, Italy; and
Laboratory of Cell Biology, Stazione Zoologica ‘A. Dohrn’ Villa Comunale, I-80121, Napoli, Italy
1Correspondence: Laboratory of Cell Biology, Stazione Zoologica ‘A. Dohrn’, Villa Comunale, I-80121, Napoli, Italy. E-mail: santella{at}alpha.szn.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: Ca2+ response at fertilization • nicotinic acid adenine nucleotide phosphate • inositol 1,4,5-trisphosphate • starfish oocytes
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3)
. Maturation is
induced by the hormone 1-methyladenine (1-MA) (4)
, whose
unidentified receptor transduces the signal via a G-protein-linked
pathway (5
6
7)
. The first indication of meiosis
reinitiation (maturation) is the breakdown of the germinal vesicle
(GVBD): after the mixing of its contents with the cytoplasm, the
oocytes undergo monospermic fertilization, which normally occurs
between the time of GVBD and that of the first polar body formation
(8)
. 1-MA also increases the sensitivity of the oocytes to
inositol 1,4,5-trisphosphate (InsP3)
(9)
and induces profound structural changes in their
endoplasmic reticulum (ER). The transient fragmentation of the latter
induced by fertilization has been linked to the release of
Ca2+ since it can be mimicked by the injection of
InsP3 into the oocyte (10
, 11)
.
Because the injection of InsP3 in sea urchin eggs
elicits a cortical granule exocytosis similar to that following sperm
activation (12)
, the suggestion has been
InsP3 is the mediator of
Ca2+ release at fertilization (13)
.
However, oocytes from several species also respond to the other
Ca2+-mobilizing messengers such as cyclic
ADP-ribose (cADPr) (14)
and nicotinic acid adenine
nucleotide phosphate (NAADP+) (15)
.
Immature and mature starfish oocytes differ in their sensitivity to
InsP3 (9)
and
NAADP+, i.e., the Ca2+
response elicited by the latter became significantly larger after
maturation. In addition, the response to NAADP+,
unlike the responses to InsP3 and cADPr, appeared
to be linked to external Ca2+ (15)
.
This is in line with results on sea urchin egg homogenates showing that
the release of Ca2+ induced by
NAADP+ was antagonized by L-type
Ca2+ channel blockers whereas that induced by
InsP3 and cADPr was not (16)
. The
NAADP+-sensitive Ca2+
stores have not yet been fully characterized, but it is commonly
accepted they are independent of those sensitive to ryanodine and
InsP3 (17
18
19
20)
. Thus, the three
Ca2+ messengers may have specific roles at
fertilization (21)
. The experiments presented here suggest
a triggering role of NAADP+ in the
Ca2+ response, in line with previous suggestions
from our laboratory (15)
and recent results of Cancela et
al. (22
, 23)
. The Ca2+ wave at
fertilization would be initiated by NAADP+, then
propagated by InsP3. The results have also shown
that the Ca2+ response induced by
InsP3 at fertilization is profoundly affected by
the removal of the nucleus.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Microinjections
The calcium fluorescent dye, OR Green 488 BAPTA-1 coupled
to a 10 kDa dextran (OGBD, Molecular Probes, Inc. Eugene, OR) was
injected into the cytoplasm of the oocyte. The concentration of the dye
in the injecting pipette (diameter of the tip is 1 µm) was adjusted
to 5 mg/ml with injection buffer (IB, 450 mM potassium chloride, 10 mM
HEPES, pH 7.0). The agonists were also delivered through the pipette.
The volume of injected dye and agonists corresponded to 1–2% of the
total cell volume: thus, the final concentration of injected substances
in the cellular environment was 50- to 100-fold lower than in the
micropipette. Injection of the dye itself did not inhibit maturation
and fertilization.
The 100 mM stock solution of both InsP3 and NAADP+ (Sigma) in IB was prepared and kept frozen. The concentrations of InsP3 and NAADP+ in the injecting pipette were 5 µM and 1 mM, respectively, diluted from the stock solution using IB before use, producing final concentrations in the cell of 50–100 nM and 10–20 µM, respectively. For experiments with caged compounds, the solution in the pipette contained 3 µM caged InsP3 (Calbiochem, La Jolla, CA) or 100 µM caged NAADP+ (Molecular Probes) in IB. Thus, final concentrations in the oocyte were: 30–60 nM for InsP3 and 1–2 µM for NAADP+. For experiments with Ca2+-free sea water (CaFSW) injected with caged compounds, oocytes were transferred for 3 min in a solution containing (470 mM NaCl, 10.1 mM KCl, 35.9 mM MgCl2, 17.5 mM MgSO4, 2.5 mM NaHCO3, 2 mM EGTA, pH 8.0).
Photolysis of caged NAADP+ and InsP3 and
Ca2+ imaging
Cytosolic Ca2+ changes were measured at
1.0 or 1.2 s intervals using a Confocal Laser Scanning Microscope
Olympus FVX-ZM-IL (Olympus Optical Co., LTD., Japan), an UplanApo
20x/0.70 objective, laser power 20%, and confocal aperture no. 2.
Photolysis of the caged compounds was performed by manually opening the
UV shutter for 5 s. Fluorescence images were acquired with a
Fluoview Personal Confocal Microscope System, version 1.2, and
processed with a MetaMorph Imaging System (Universal Imaging
Corporation, West Chester, PA). To exclude variations of fluorescent
intensity, the signals were corrected for variations in dye
concentration by normalizing fluorescence (F) against baseline
fluorescence (F0). This permitted reliable
information on Ca2+ changes from baseline values
(24)
. The region of interest (ROI) to measure the
fluorescence level was 1/10th of the oocyte diameter and positioned as
shown in Fig. 1B
in experiments with NAADP+ (see Fig. 2B
for experiments with InsP3 and Fig. 8
for fertilization experiments).
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NAADP+ response in verapamil and SKF 96365
experiments-treated oocytes.
Verapamil was purchased from Sigma, prepared as a 20 mM stock
solution in DMSO, and kept at room temperature. SKF 96365 (Tocris
Cookson Ltd., Bristol, UK) was prepared as a 10 mM stock solution in
distilled water and kept frozen until use. Chemicals were dissolved in
ASW just before use. Mature oocytes were incubated in seawater
containing different concentrations of verapamil or SKF 96365. They
were injected during incubation with a mixture of 100 µM caged
NAADP+ and 5 mg/ml OGBD 10 kDa in the pipette.
After 10 min of incubation with the inhibitors, photolysis by UV
irradiation for 10 s and fluorescent measurements were performed
using a computer-controlled photomultiplier system (Olympus
IMT2, equipped by Olympus OSP-3), described in detail
elsewhere (15)
. The recorded F was divided by the
F0 to normalize the fluorescent intensities in
each experiment as described above.
Transmission electron microscopy
Samples were fixed in 1% glutaraldehyde in sea water for 1 h and postfixed in 1% OsO4 in sea water for
1 h. The samples were dehydrated in a graded alcohol series and
embedded in Epon 812. Sections were stained with 2% uranyl acetate and
0.2% lead citrate, then examined with a Philips 400 transmission
electron microscope.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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had shown that
injection of NAADP+ into oocytes matured by 1-MA
induced an elevation of intracellular Ca2+ that
failed to decay within the time of experimental observation (3–5 min).
The effect of NAADP+ has now been analyzed using
confocal microscopy (Fig. 1)
. When the injection was made in the center
of the oocyte, a negligible Ca 2+ elevation was
observed in the first few seconds around the point of injection (Fig. 1A
). A much stronger Ca2+ increase was
observed a little later (after 12 s) in the cortical region of the
oocyte, showing that the stores sensitive to
NAADP+ were located essentially, if not
exclusively, in the region beneath the plasma membrane.
Ca2+ then gradually spread to the remainder of
the cytoplasm, likely due to its direct diffusion or a
Ca2+-induced Ca2+ release
(CICR) phenomenon acting on stores sensitive to
InsP3 and/or cADPr. The graph of the relative
fluorescence of the Ca2+ indicator shown in Fig. 1B
offers a numerical equivalent of the pseudocolor in the
cortical region of the oocyte. It shows that the relative fluorescence
increased in 12 s to a value of 1.10 ± 0.15 arbitrary units
(n=12), decreased a few seconds later to a lower value, then
increased more gradually to a plateau of 1.31 ± 0.31 arbitrary
units that was maintained for the 5 min of the experimental
observation. Figure 1C
shows that the
Ca2+ increase in the cortical region induced a
normal elevation of the fertilization envelope.
Injected InsP3 has been repeatedly shown to
promote a Ca2+ elevation in mature oocytes from
several species. When comparing the Ca2+
responses to NAADP+ and to
InsP3, however, clear differences became
apparent. At variance with NAADP+,
InsP3 injected in the center of the oocyte
promoted an immediate and large elevation of Ca2+
around the point of injection (Fig. 2
). The Ca2+ spread to the cortical region a few
seconds after the injection, but in sharp contrast with
NAADP+, the Ca2+ response
induced by InsP3 had already covered the entire
oocyte (0.93±0.10 arbitrary units, n=18) 12 s after
the injection. At further variance with NAADP+,
the Ca2+ increase induced by
InsP3 decayed after this time: 90 s after
the injection, the Ca2+ level had decreased
significantly (see the confocal images in Fig. 2A
and graph
in Fig. 2B
). As expected, injected
InsP3 promoted normal elevation of the
fertilization envelope (Fig. 2C
).
The effects of NAADP+ and
InsP3 were also analyzed using the caged variants
of the compounds (Fig. 3
). The uncaging reaction was performed 5 min after the injection, when
caged NAADP+ and InsP3 had
presumably spread to the entire cytoplasm. In the case of
NAADP+, the irradiation failed to mobilize
Ca2+ in the center of the oocyte (Fig. 3A
, B
), whereas a massive increase of Ca2+
occurred in the cortical region without the delay observed when
injecting uncaged NAADP+ (see Fig. 1A
). Later on, Ca2+ increased in the
center of the cell as well to reach its highest level 20 s after
photolysis (see open squares in Fig. 1B
). The massive
liberation of Ca2+ in the cortical region of the
oocyte promoted the normal elevation of the fertilization envelope, as
shown in the transmitted light microscopy image of Fig. 1C
.
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When caged InsP3 was injected, a
Ca2+ increase was also immediately observed in
the cortical region (Fig. 4A
); however, the increase in the center of the oocyte reached
the highest level only a few seconds after the irradiation, i.e., much
more rapidly than with NAADP+ (see Fig. 4A
and graph in panel B). As expected, the
elevation of the fertilization envelope was entirely normal (Fig. 4C
).
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Previous work in our laboratory with a photomultiplier
(15)
had shown that the Ca2+
response induced by NAADP+ was affected by
external Ca2+. This was now confirmed by showing
that the uncaging of NAADP+ failed to elicit the
Ca2+ response and promote elevation of the
fertilization envelope (Fig. 5A
, B
) in oocytes kept in Ca2+-free sea
water containing 2 mM EGTA. The photoactivation of caged
InsP3 in these oocytes (Fig. 6A
) induced instead a Ca2+ increase
similar to that observed in oocytes irradiated in normal sea water (see
Fig. 4A
for comparison), even if the emptying of the
InsP3-sensitive Ca2+ store
was not sufficient to trigger elevation of the fertilization envelope
(Fig. 6C
). The electron micrograph in Fig. 6D
shows that in the oocytes kept in
Ca2+-free sea water, the cortical
granules remained positioned in a subplasma membrane location without
fusing with the plasma membrane.
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Experiments were performed to establish whether the external
Ca2+ involved in the response to
NAADP+ penetrated into the cell.
Voltage-dependent channels (essentially L-type, documented in oocytes
from various invertebrates; see refs 25
26
27
) were explored
using the inhibitor verapamil. Capacitative channels, which have been
demonstrated in vertebrate oocytes (28
29
30)
but so far not
in invertebrate oocytes, were also explored with the inhibitor SKF
96365, even if it was deemed unlikely that Ca2+
would penetrate through channels of this type, since
NAADP+ (see above) failed to empty the internal
Ca2+ stores. Figure 7
shows that the Ca2+ response to the uncaging of
NAADP+ was completely inhibited by both
compounds. The result supports a role for L-type channels, but is
inconclusive for the capacitative channels not only because the
internal Ca2+ stores were not emptied under the
conditions of the experiment, but also because of the poor channel
specificity of the inhibitor SKF 96365 (see, for instance, ref
31
).
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Ca2+ response induced by fertilization
The addition of spermatozoa to mature oocytes induced the expected
spreading of the calcium wave from the point of sperm interaction to
the antipode (Fig. 8A
). This is shown graphically in Fig. 8B
, which
documents the traveling of the wave across the cell by placing the ROI
(see scheme) over different locations in the cell. As expected, sperm
interaction induced a normal elevation of the fertilization envelope
(Fig. 8C
).
Ca2+ responses to NAADP+,
InsP3, and spermatozoa in enucleated oocytes
As mentioned, the sensitivity of oocytes to
InsP3 (9)
and
NAADP+ (15)
changes during the
maturation process, which is characterized by the disappearance of the
nuclear envelope and the subsequent intermixing of the nucleoplasm with
the cytoplasm (8)
. Although information on the role played
by the individual nuclear components in preparing the cell for
fertilization is limited, recent work has indicated that MPF (a complex
of a cdc2 kinase and a cyclin B regulatory subunit that governs the
progression through meiosis and mitosis) may be an important actor. For
instance, MPF is involved in the oscillatory response of
Ca2+ at fertilization (32
33
34)
.
Since the final activation of MPF in starfish oocytes occurs in the
nucleus (35)
, it was decided to study the
Ca2+ response to fertilization (but also to
NAADP+ and InsP3) in
enucleated oocytes. The response to NAADP+ is
shown in Fig. 9A
. The injection induced the same cortical increase of
Ca2+ seen in control oocytes (see Fig. 1A
): it failed to decay during the 90 s of the
observation (see graph in Fig. 9A
) and the elevation of the
fertilization envelope occurred normally (Fig. 9B
). This was
also confirmed by electron microscopy experiments, which showed that
the injection of NAADP+ induced a normal cortical
granule exocytosis induced by (Fig. 9C
). The response of
enucleated oocytes to InsP3 (see graph in
Fig. 10
A) differed substantially from that seen with
NAADP+. The rise in Ca2+
after the injection eventually reached about the same height seen in
control oocytes (0.94±0.2, arbitrary units, n=13; compare
Fig. 2
and Fig. 10
). However, the time necessary to reach the plateau
was about twice as long as in the control (15 ± 3.3 s vs.
7.1 ± 1.4 s in the control, Fig. 2A
). In
addition, Ca2+ stayed at the peak level for only
a few seconds in the control (Fig. 2B
), but remained there
for 
30 s in the case of enucleated oocytes (Fig. 10A
):
The difference was particularly striking in the case of the cortical
layer. One additional difference between the two cell types was the
decay of the Ca2+ level after the peak, which was
only abortive in the controls (Fig. 2B
) and instead reached
baseline level in the enucleated oocytes (Fig. 10A
). Most
important, despite the elevation of Ca2+ in the
cortical region and at sharp variance with
NAADP+, no elevation in the fertilization
envelope occurred (Fig. 10B
). The failure of
InsP3 to induce cortical exocytosis was confirmed
by electron microscopy (Fig. 10C
), which showed that the
cortical granules were normally positioned beneath the plasma membrane
but failed to fuse with it to initiate the exocytotic process. Figure 10C
shows an abortive elevation of the fertilization
envelope in a limited region of the plasma membrane without obvious
indications of fusion with the cortical granules.
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The Ca2+ response of enucleated oocytes to
fertilization also differed dramatically from that seen in the
controls. The initial Ca2+ wave near the point of
sperm interaction (Fig. 11
A) reached a level slightly lower than that observed in the
control (see Fig. 8B
; for a general overview of the
Ca2+ responses in starfish oocytes, see
Table 1
). The propagation of the Ca2+ wave to the
remainder of the cell to reach the antipode was significantly slowed
(Fig. 11A
). The histograms in Fig. 11B
show that
the speed of wave propagation was decreased by more than 50% with
respect to oocytes containing the nucleus (the decrease was highly
significant, P=0.001). However, elevation of the
fertilization envelope occurred normally as previously observed, see
(36)
and Fig. 11C
.
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Role of MPF in the Ca2+ response during maturation and
fertilization
The results of the experiments on enucleated oocytes could have
been due to the absence of activated MPF, which undergoes final
activation in the nucleus, after two activating steps in the cytoplasm
(35)
. The role of MPF was explored by using a specific
inhibitor, roscovitine (37)
. Unfortunately, oocytes
incubated with the concentration of roscovitine routinely used to block
MPF resumed meiosis normally, even if with a significant delay. Once
meiosis had occurred, all Ca2+ responses were
normal. At variance with this, when roscovitine (even at very low
concentrations, i.e., 10 µM final) was directly injected into the
cytoplasm or the nucleus of oocytes meiosis resumption was blocked.
A. pectinifera starfish oocytes, as other oocytes
(38
), probably have low permeability to
roscovitine. Since no changes in the
Ca2+-sensitive receptors occurred in the absence
of meiosis, it was impossible to assess the role of MPF in the
Ca2+ response. Thus, even if nuclear components
may be essential to the Ca2+ response at
fertilization, the role of MPF remains undefined.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. They initiate at the point of
sperm interaction and spread gradually to the antipode of the cell
(40
, 41)
. Their shape varies with the species. For
instance, in cnidarians, echinoderms, fish, and frogs
(42)
, the wave travels across the cytoplasm as a single
Ca2+ transient whereas in ascidians and mammals,
the spiking is repetitive (43
, 44)
. According to a popular
view, Ca2+ signals are initiated and propagated
by InsP3 (45
, 46)
generated by a
conventional G-protein-linked pathway (12)
or by the
phosphorylation of PLC
by tyrosine kinases (47
, 48)
.
The initial liberation of InsP3, and thus of
Ca2+, is assumed to occur in the cortical region
of the oocyte near the point of sperm interaction. A regenerative
mechanism would propagate the wave to the remainder of the cell
(essentially a CICR phenomenon) by activating additional
InsP3 channels (and the other
Ca2+-mobilizing receptors present in oocytes,
i.e., the cADPr/ryanodine channels; ref 40
). The most
recent addition to the family of Ca2+-mobilizing
second messengers is NAADP+, which acts on
Ca2+ stores different from those sensitive to
InsP3 and cADPr. NAADP+ is
also active in starfish oocytes (15)
, where it liberates
Ca2+ from a pool confined to the vicinity of the
plasma membrane (or even physically associated with it) and somehow
related to extracellular Ca2+. Since the
Ca2+ mobilization by NAADP+
was abolished if no Ca2+ was in the external
medium (15)
, it must be concluded that
NAADP+ directly opens plasma membrane
Ca2+ channels. If NAADP+
would instead release Ca2+ from a cortical store,
which would in turn be followed by the activation store-operated
Ca2+ entry, a cortical fluorescent rim should be
visible immediately on uncaging NAADP+ in oocytes
kept in Ca2+-free sea water.
Ca2+ penetration into oocytes under the influence
of NAADP+ had been already been indicated by
experiments with inhibitors of L-type Ca2+
channels (15)
. The results with channel inhibitors
reported here have shown that both verapamil and SKF 96365 abolished
the response to NAADP+. Whereas L-type channels
are likely to have been operative under the experimental conditions,
those operated by the emptying of the stores, if existing in these
oocytes, must instead be assumed to have been inactive (see above). The
inhibitory effect of SKF 96365 could thus reflect its poor channel
specificity, but the remote possibility that a new type of channel,
different from that activated by the emptying of the cellular
Ca2+ stores but still somehow sensitive to SKF
96365, was the target of NAADP+ could still be
considered.
Membrane electroporation (49)
has been used to investigate
the role of external Ca2+ in the fertilization
process, showing that voltage pulsation induced localized exocytosis of
the contents of cortical granules and the development of a partial
fertilization envelope. The process was promoted by the penetration of
Ca2+ through the voltage-induced pores
(50)
. That fertilization is associated with the
penetration of Ca2+ into the egg was originally
suggested by Jaffe and co-workers (51
, 52)
and confirmed
by others (53
, 54)
. Later work has added weight to the
proposal by showing that the electrophysiological response of the
plasma membrane of sea urchin eggs at fertilization was shaped by the
influx of Ca2+ through voltage-dependent L-type
channels (55)
. The observation that the mobilization of
the Ca2+ pool sensitive to
NAADP+ appeared to involve penetration of
Ca2+ from the external medium elicited our
interest in investigating whether, at variance with the prevailing idea
that InsP3 is the exclusive mediator of the
Ca2+ response at fertilization,
NAADP+ also had a role, possibly by initiating
the Ca2+ wave. In fact, a role for
NAADP+ had been postulated by others in the
fertilization of sea urchin eggs and ascidian oocytes (17
, 21)
.
The work presented here has documented clear differences between the
Ca2+ responses to NAADP+
and to InsP3, confirming that the mechanism of
action of NAADP+ differs from that of the other
two Ca2+-related messengers (18
, 56)
. In particular, we have shown that the
Ca2+ store sensitive to
NAADP+ was located exclusively in the cortical
layer of the oocytes (as suggested by previous work in our laboratory;
ref 15
), whereas that sensitive to
InsP3 was present throughout the cell. The
Ca2+ elevation induced by the injection of
NAADP+ lasted minutes, but faded away much more
rapidly in the case of InsP3. The shape of the
Ca2+ transient induced by
NAADP+ in the cortical region of the cell also
had features that were not observed in that induced by
InsP3. In the case of
NAADP+, the initial peak was followed by a rapid
partial decline, which was almost immediately reversed by a slower
phase of Ca2+ increase that lasted minutes: Thus,
the cortical response to NAADP+ had a dual
character. Since the removal of Ca2+ from the
external medium abolished both the rapid and slow phases of the
responses (15)
, it was not possible to decide whether only
one of the two was directly linked to external
Ca2+ and, if so, which one.
In agreement with previous findings in sea urchin eggs (57
, 58)
and starfish oocytes (41)
, the present results
have shown that the activation of InP3 receptors,
though not involved in its initiation, is essential for the normal
propagation of the calcium wave at fertilization. However, they have
also shown that the uncaging of injected InsP3 in
Ca2+-free sea water failed to trigger cortical
granules exocytosis, in contrast to previous suggestions that the
liberation of Ca2+ from intracellular stores was
sufficient for elevation of the fertilization envelope (59
, 60)
. Evidently, even if large amounts of
Ca2+ are available in the cytosol as the result
of emptying of the intracellular stores, the exocytotic process still
required the influx of Ca2+ into the cell linked
to the activation of the cortical
NAADP+-sensitive Ca2+
stores. This is reminiscent of the situation prevailing during
neurosecretion, where Ca2+ is necessary for the
release of neurotransmitters (61
, 62)
, and is at variance
with the claim that external Ca2+ is only
necessary for the compensatory endocytosis that follows the exocytotic
process in fertilized sea urchin eggs (63)
. The
experiments presented here have shown that in starfish oocytes (but in
agreement with most secretory cells), external
Ca2+ is instead directly involved in the
exocytotic process as well (64)
. Since the removal of the
nucleus failed to affect the Ca2+ response and
the cortical exocytotic process induced by fertilization or
NAADP+ injection, but abolished the exocytotic
response to InsP3, it is reasonable to suggest
that Ca2+ release at fertilization is initiated
by the activation of NAADP+ receptors. The
InsP3 receptors would instead be involved in the
propagation of the Ca2+ wave once
NAADP+ has initiated it.
The effects of the removal of the nucleus on the propagation of the Ca2+ wave open interesting perspectives for future investigations. At this preliminary stage, the most interesting aspect is the clear demonstration that the speed of wave propagation after the injection of InsP3 and the subsequent exocytosis of cortical granules depend on the intermixing of the nucleoplasm and the cytoplasm. The sharp contrast with the effects of NAADP+, which induced instead a normal Ca2+ response and a normal exocytotic process in enucleated oocytes, is certainly worth stressing. It is hoped that future work will identify the nuclear component(s) responsible for the alteration of the oocyte response to InsP3.
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ACKNOWLEDGMENTS |
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Received for publication February 19, 2001.
Revision received June 22, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-subunit of starfish G protein and possible existence of the subsequent effector in the cytoplasm. Mol. Biol. Cell 4,1027-1034[Abstract]
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