NAADPⴙ initiates the Ca2ⴙ response during
fertilization of starfish oocytes
DMITRI LIM,† KEIICHIRO KYOZUKA,* GIOVANNI GRAGNANIELLO,†
ERNESTO CARAFOLI,‡ AND LUIGIA SANTELLA‡,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
We have explored the role of the recently discovered second messenger nicotinic acid adenine nucleotide phosphate (NAADPⴙ) in Ca2ⴙ swings
that accompany the fertilization process in starfish
oocytes. The injection of NAADPⴙ deep into the cytoplasm of oocytes matured by the hormone 1-methyladenine (1-MA), mobilized Ca2ⴙ exclusively in the cortical
layer, showing that the NAADPⴙ-sensitive Ca2ⴙ pool is
restricted to the subplasma membrane region of the
cell. At variance with this, InsP3 initiated the liberation
of Ca2ⴙ next to the point of injection in the center of
the cell. The initial cortical Ca2ⴙ liberation induced by
NAADPⴙ was followed by a spreading of the Ca2ⴙ wave
to the remainder of the cell and by a massive cortical
granule exocytosis similar to that routinely observed on
injection of InsP3. A striking difference in the responses to NAADPⴙ and InsP3 was revealed by the
removal of the nucleus from immature oocytes, i.e.,
from oocytes not treated with 1-MA. Whereas the Ca2ⴙ
response and the cortical granule exocytosis induced by
NAADPⴙ were unaffected by the removal of the nucleus, the Ca2ⴙ response promoted by InsP3 was significantly slowed. In addition, the cortical granule exocytosis was completely abolished. When enucleated
oocytes were fertilized, the spermatozoon still promoted the Ca2ⴙ wave and normal cortical exocytosis,
strongly suggesting that the Ca2ⴙ response was mediated by NAADPⴙ and not by InsP3. InsP3-sensitive Ca2ⴙ
stores may mediate the propagation of the wave initiated by NAADPⴙ since its spreading was strongly
affected by removal of the nucleus.—Lim, D., Kyozuka,
K., Gragnaniello, G., Carafoli, E., Santella, L. NAADPⴙ
initiates the Ca2ⴙ response during fertilization of starfish oocytes. FASEB J. 15, 2257–2267 (2001)
ABSTRACT
Key Words: Ca2⫹ response at fertilization 䡠 nicotinic acid
adenine nucleotide phosphate 䡠 inositol 1,4,5-trisphosphate
䡠 starfish oocytes
Immature starfish oocytes (Asterina pectinifera) arrested at the prophase of the first meiotic division still
contain the nucleus (germinal vesicle): although they
may incorporate multiple sperms, they cannot be fertilized and do not form the fertilization envelope (1–3).
Maturation is induced by the hormone 1-methylad0892-6638/01/0015-2257 © FASEB
enine (1-MA) (4), whose unidentified receptor transduces the signal via a G-protein-linked pathway (5–7).
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
(17–20). 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
1
Correspondence: Laboratory of Cell Biology, Stazione
Zoologica ‘A. Dohrn’, Villa Comunale, I-80121, Napoli, Italy.
E-mail: [email protected]
2257
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.
MATERIALS AND METHODS
Preparation of gametes
Starfish (A. pectinifera) were collected during the breeding
season in the Mutsu Bay (Aomori, Japan) and kept in
circulating sea water (16°C). Fully grown prophase-arrested
oocytes containing a large nucleus were dissected from the
ovaries in artificial sea water (ASW; 460 mM NaCl, 10.1 mM
KCl, 9.2 mM CaCl2, 35.9 mM MgCl2, 17.5 mM MgSO4, 2.5
mM NaHCO3, pH 8.0), washed several times with it, and kept
for 30 min before use. Oocytes in which the rupture of the
envelope of the nucleus (GVBD) occurred spontaneously
during this period were discarded. All experiments were
performed in artificial sea water. When mature oocytes were
needed, maturation (GVBD) was promoted by adding the
hormone 1-MA (Sigma Chemical Co., St. Louis, MO) at a
concentration of 1 ␮M (final concentration). When enucleated oocytes were needed, the nucleus was removed from the
oocyte using fine needles under a stereo microscope. These
oocytes were then induced to undergo maturation as indicated above. Male gametes were dissected dry and kept at ⫹
4°C during the day. For fertilization experiments, 1 ␮l of dry
sperm was suspended in 2 ml of artificial sea water; 40 ␮l of
this suspension was added to 1 ml of the oocyte suspension to
obtain a final sperm dilution of 1:50000.
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 ␮m) 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 sub-
stances 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
␮M 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 ␮M, respectively. For experiments with caged compounds, the solution in the pipette
contained 3 ␮M caged InsP3 (Calbiochem, La Jolla, CA) or
100 ␮M caged NAADP⫹ (Molecular Probes) in IB. Thus, final
concentrations in the oocyte were: 30 – 60 nM for InsP3 and
1–2 ␮M 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 NAADPⴙ and InsP3 and Ca2ⴙ imaging
Cytosolic Ca2⫹ changes were measured at 1.0 or 1.2 s intervals
using a Confocal Laser Scanning Microscope Olympus FVXZM-IL (Olympus Optical Co., LTD., Japan), an UplanApo
20⫻/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).
NAADPⴙ 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
Figure 1. Ca2⫹ release induced by the
injection of NAADP⫹ in a mature oocyte. A) Confocal laser scanning imaging of Ca2⫹ release induced by injection of NAADP⫹ in the center of the
oocyte. The response began in the
cortical region after a delay of 12 s,
then spread to the remainder of the
cell. B) Ca2⫹ release in the cortical
region of the same oocyte (filled
squares) and in the center (open
squares). The regions of interest
(ROIs) were positioned as shown in
the scheme. C) Elevation of the fertilization envelope (arrow) imaged by
transmitted light microscope. Bar ⫽
30 ␮m.
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LIM ET AL.
Figure 2. Ca2⫹ release in a mature
oocyte induced by injection of InsP3.
A) Confocal laser scanning imaging of
InsP3 induced Ca2⫹ release. The injection was made in the center of the
oocyte. Ca2⫹ propagated immediately
from the point of injection. B) Ca2⫹
increase in the cortex (filled squares)
and in the center (open squares) of
the oocyte. ROIs positioned as in Fig.
1. C) Elevation of the fertilization envelope (arrow). Bar ⫽ 30 ␮m.
oocytes were incubated in seawater containing different concentrations of verapamil or SKF 96365. They were injected
during incubation with a mixture of 100 ␮M 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.
RESULTS
NAADPⴙ and InsP3 induced Ca2ⴙ release in
mature oocytes
Previous work in our laboratory (15) 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.
Figure 3. Ca2⫹ release induced by the
uncaging of caged NAADP⫹ in a mature oocyte. A) Confocal laser scanning imaging of Ca2⫹ increase after
uncaging of injected NAADP⫹. B) The
Ca2⫹ response in the cortical region
occurred immediately after the irradiation (filled squares) and spread
within 20 s to the center of the cell
(open squares). ROIs positioned as in
Fig. 1. C) Elevation of the fertilization
envelope after uncaging of NAADP⫹
(arrow). Bar ⫽ 30 ␮m.
THE ROLE OF NAADP⫹ AT FERTILIZATION
2259
Figure 4. Ca2⫹ release induced by the
uncaging of caged InsP3 in a mature
oocyte. A) Confocal laser scanning imaging of Ca2⫹ increase after uncaging
of the injected InsP3. B). Ca2⫹ in the
cortical (filled squares) and central
(open squares) regions of the oocyte
increased within 3– 6 s after the irradiation. ROIs positioned as in Fig. 1. C)
Elevation of the fertilization envelope
(arrow) after uncaging of InsP3 (arrow). Bar ⫽ 30 ␮m.
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 with-
out 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.
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).
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
Figure 5. Absence of cytosolic Ca2⫹
increase after photoactivation of
caged NAADP⫹ in Ca2⫹-free sea water. A) Confocal laser scanning imaging of Ca2⫹ after uncaging of injected
NAADP⫹. Before this, the oocyte was
transferred in CaFSW containing 2
mM EGTA and kept in it for 3 min. B)
The graph shows that no Ca2⫹ increase occurred. ROIs positioned as in
Fig. 1. C) The fertilization envelope
failed to elevate after the uncaging
reaction. Bar ⫽ 30 ␮m.
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LIM ET AL.
Figure 6. Ca2⫹ release induced by the
uncaging of InsP3 in a mature oocyte
in Ca2⫹free sea water. A) Confocal
laser scanning imaging of Ca2⫹ increase after uncaging of injected
InsP3. Before this, the oocyte was incubated for 3 min in CaFSW containing 2
mM EGTA. B) The graph shows the
Ca2⫹ increase in the cortical region
(filled squares) and the center of the
cell (open squares). ROIs positioned
as in Fig. 1. C). Despite the Ca2⫹
increase after uncaging of InsP3, the
fertilization envelope failed to elevate.
Bar ⫽ 30 ␮m. The electron micrograph in panel D shows that the cortical granules (CG) failed to fuse with
the plasma membrane. Bar ⫽ 1 ␮m.
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.
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 25–27) were explored using the
inhibitor verapamil. Capacitative channels, which have
been demonstrated in vertebrate oocytes (28 –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).
Ca2ⴙ response induced by fertilization
The addition of spermatozoa to mature oocytes induced the expected spreading of the calcium wave
THE ROLE OF NAADP⫹ AT FERTILIZATION
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).
Figure 7. Effect of SKF 96365 and verapamil on NAADP⫹induced Ca2⫹ release in Asterina oocytes. Mature oocytes (1 h
of exposure to 1-MA) were injected with caged NAADP⫹ and
treated with different concentrations of SKF 96365 (filled
square) or verapamil (filled circle) for 10 min. NAADP⫹ was
then released by UV irradiation for 5 s. All measurements
were performed in triplicate and the data are expressed as the
mean ⫾ sd. Additional details are found in Materials and
Methods.
2261
Figure 8. Intracellular Ca2⫹ increase
after fertilization of a control mature
oocyte. A) Confocal laser scanning images of the Ca2⫹ wave after fertilization. B) The Ca2⫹ wave propagated
from the entry point of the sperm
(filled squares) to the center of the
oocyte (open squares) and to the antipode (open circles). ROIs were positioned as shown in the scheme. C)
Elevation of the fertilization envelope
(arrow). Bar ⫽ 30 ␮m.
Ca2ⴙ responses to NAADPⴙ, 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 (32–34). 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. 10A) 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
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Figure 9. Ca2⫹ release and elevation of the fertilization
envelope induced by the injection of NAADP⫹ into an
enucleated mature oocyte. The oocyte was enucleated as
described in Materials and Methods and incubated for 50 min
in ASW containing 1 ␮M 1-MA. The NAADP⫹ injection was
performed in the center of the cell. A) Ca2⫹ increase in the
cortex (filled squares) and in the center (open squares) of
the oocyte. B) Elevation of the fertilization envelope (arrow)
imaged by transmitted light microscope. Bar ⫽ 30 ␮m. C)
Electron micrograph of an enucleated oocyte after injection
of NAADP⫹. The fertilization envelope elevated from the
surface of the oocyte (arrow) after the extrusion of the
cortical granules. Bar ⫽ 1 ␮m.
The FASEB Journal
LIM ET AL.
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.
Role of MPF in the Ca2ⴙ 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 ␮M
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
Figure 10. Ca2⫹ release and absence of cortical granules
exocytosis on injection of InsP3 into an enucleated mature
oocyte. The oocyte was enucleated as described in Materials
and Methods and allowed to mature for 50 min in ASW
containing 1 ␮M 1-MA. InsP3 was injected in the center of the
oocyte. A) Ca2⫹ increase in the cortex (filled squares) and in
the center (open squares) of the cell. B) The fertilization
envelope failed to elevate after the InsP3 injection. Bar ⫽ 30
␮m. C) Electron micrograph of the oocyte showing the
abortive elevation of the fertilization envelope and the absence of cortical granules exocytosis (arrow). Bar ⫽ 1 ␮m.
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.
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. 11A) reached a level slightly lower
than that observed in the control (see Fig. 8B; for a
general overview of the Ca2⫹ responses in starfish
THE ROLE OF NAADP⫹ AT FERTILIZATION
Figure 11. Ca2⫹ wave and elevation of the fertilization envelope after fertilization of an enucleated mature oocyte. The
immature oocyte was enucleated and incubated for 50 min in
ASW containing 1 ␮M 1-MA. The Ca2⫹ increase shown in
graph A was measured at the sperm entry point (filled
squares), in the center of the oocyte (open squares), and at
the opposite side (open circles). B) The speed of wave
propagation of an enucleated mature oocyte was decreased by
more than 50% with respect to oocytes containing the nucleus. C) The fertilization envelope elevated normally (arrow)
in an enucleated oocyte. Bar ⫽ 30 ␮m.
2263
TABLE 1. Intracellular Ca2⫹ release induced by NAADP⫹, InsP3, and sperm in mature oocytes (control) and oocytes enucleated before the
addition of 1-MA
Ca2⫹ increase mean ⫾ sd
Elevation of the
fertilization envelope
1.10 ⫾ 0.15, 1.31 ⫾ 0.31 (n⫽12) a
0.72 ⫾ 0.12 (n⫽12)
0.93 ⫾ 0.10 (n⫽18)
0.93 ⫾ 0.11 (n⫽6)
1.57 ⫾ 0.16 (n⫽12)
1.07 ⫾ 0.18 (n⫽15)
0.94 ⫾ 0.12 (n⫽13)
1.12 ⫾ 0.30 (n⫽10)
0.13 ⫾ 0.09 (n⫽7)
0.88 ⫾ 0.05 (n⫽6)
⫹
⫹
⫹
⫹
⫹
⫹
⫺
⫹
⫺
⫺
Stimulus
Control oocytes
Enucleated oocytes
Oocytes in Ca2⫹-free sea water
Injected NAADP⫹
Uncaging of injected
Injected InsP3
Uncaging of injected
Sperm
Injected NAADP⫹
Injected InsP3
Sperm
Uncaging of injected
Uncaging of injected
NAADP⫹
InsP3
NAADP⫹
InsP3
a
The two values for Ca2⫹ increase given for the injection of NAADP⫹ refer to the first and second Ca2⫹ peak after the injection (see page
Results for details).
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.
DISCUSSION
Ca2⫹ waves are a distinctive feature of the fertilization
process (39). 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
2264
Vol. 15
October 2001
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
The FASEB Journal
LIM ET AL.
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
THE ROLE OF NAADP⫹ AT FERTILIZATION
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.
This work was made possible by the financial contribution
of the Italian Ministry of University and Scientific Research
(MURST-PRIN 1998 and 2000), the Telethon Foundation
(grant no 963), the National Research Council of Italy
(Target Project on Biotechnology), and the Armenise-Harvard Foundation. The help of the Service for marine animal
breeding of the Stazione Zoologica, Naples, is also gratefully
acknowledged.
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Received for publication February 19, 2001.
Revised for publication June 22, 2001.
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