From the School of Cell and Molecular Biosciences,
The Medical School, Framlington Place, University of Newcastle upon
Tyne, Tyne and Wear NE2 4HH, United Kingdom, the
§ University Department of Pharmacology, University of
Oxford, Mansfield Road, Oxford OX1 3QT, United Kingdom
Received for publication, October 21, 2002, and in revised form, January 17, 2003
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ABSTRACT |
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Both the inositol 1,4,5-trisphosphate
(InsP3) and ryanodine receptor pathways
contribute to the Ca2+ transient at fertilization in sea
urchin eggs. To date, the precise contribution of each pathway has been
difficult to ascertain. Evidence has accumulated to suggest that the
InsP3 receptor pathway has a primary role in causing
Ca2+ release and egg activation. However, this was recently
called into question by a report implicating NO as the primary egg
activator. In the present study we pursue the hypothesis that NO is a
primary egg activator in sea urchin eggs and build on previous findings that an NO/cGMP/cyclic ADP-ribose (cADPR) pathway is active at fertilization in sea urchin eggs to define its role. Using a
fluorescence indicator of NO levels, we have measured both NO and
Ca2+ at fertilization and establish that NO levels rise
after, not before, the Ca2+ wave is initiated and that this
rise is Ca2+-dependent. By inhibiting the
increase in NO at fertilization, we find not that the Ca2+
transient is abolished but that the duration of the transient is
significantly reduced. The latency and rise time of the transient are
unaffected. This effect is mirrored by the inhibition of cGMP and
cADPR signaling in sea urchin eggs at fertilization. We
establish that cADPR is generated at fertilization, at a time
comparable to the time of the rise in NO levels. We conclude that NO is
unlikely to be a primary egg activator but, rather, acts after the
initiation of the Ca2+ wave to regulate the duration of the
fertilization Ca2+ transient.
In sea urchins the immediate consequence of sperm-egg fusion is
the generation of a single large transient elevation in cytosolic free
calcium ([Ca2+]i), which is absolutely
required for egg activation (1-3). Sea urchin eggs contain both
InsP31 and
ryanodine receptor channels. The microinjection of either InsP3, ryanodine, or the ryanodine receptor agonist cADPR
mobilizes [Ca2+]i and activates eggs (4-6). The
presence of these receptors in sea urchin eggs has been demonstrated
immunologically (7, 8), and a sea urchin ryanodine receptor has now
been cloned (9). Both these channel types contribute to the
Ca2+ transient at fertilization. Studies have shown that
microinjection of the InsP3 receptor antagonist heparin in
conjunction with antagonists of the ryanodine receptor, such as
ruthenium red and the cADPR inhibitor 8-amino-cADPR, block the
Ca2+ transient at fertilization (10, 11). Injection with
heparin alone, unless used at relatively high concentrations (12), does not abolish the fertilization Ca2+ transient (10, 11, 13).
cADPR and ryanodine receptor antagonists block the transient only in
the presence of InsP3 antagonists (10, 11), if at all
(14).
The precise roles of the InsP3 and ryanodine receptor
pathways at fertilization in sea urchins have been more difficult to ascertain. There is evidence to suggest that the InsP3
receptor pathway has a primary role in causing Ca2+ release
and egg activation. InsP3 is produced by members of the phosphatidylinositol phospholipase C (PLC) enzyme family and has been shown to be generated at fertilization (15-18). Chemical
inhibitors of PLC, pentosan polysulfate and U73122, can abolish
fertilization Ca2+ transients in sea urchin eggs (12, 17).
Molecular inhibition studies, using dominant negative PLC Nonetheless an NO-mediated pathway has been proposed to be the primary
mechanism for egg activation (23). In sea urchins the external
application of NO to intact eggs and egg homogenates generates
Ca2+ transients, although this doesn't lead to egg
activation (24, 25). Eggs can be activated when microinjected with an
NO donor (23). Ca2+ elevation in eggs by NO has been
demonstrated to require the operation of a cGMP-mediated synthesis of
cADPR (24, 25), and levels of cGMP (26, 18) and cADPR (18) are known to rise at fertilization in sea urchin eggs. Kuo and coworkers (23) found
that microinjection of an NO scavenger into eggs abolished the
fertilization Ca2+ transient and prevented egg activation.
Microinjection of recombinant nitric-oxide synthase (NOS) and
calmodulin was sufficient to fully activate eggs, indicating that NOS
substrates were present. The measurement of an increase in endogenous
NO in fertilized eggs very soon after sperm-egg fusion using a
fluorescent indicator dye supported the proposition that an NO rise
might trigger the Ca2+ wave and egg activation; NO and
Ca2+ were not measured simultaneously, so the precise
temporal relationship between the NO rise and the Ca2+
transient could only be inferred (23). It was pointed out that initiation of the Ca2+ transient by NO was unlikely to be
due to the activation of a pathway mediated by cADPR, because
antagonists of this pathway alone cannot block fertilization. A more
recent study of two separate chordate species, where NO and
Ca2+ were measured simultaneously, could detect no NO
increases either prior to, or after, the generation of the
Ca2+ transient, or obtain abolition of the transient with
NO-signaling inhibitors (27). We, and others, were also concerned that
the NO indicator dye used by Kuo and co-workers (23),
diaminofluorescein (DAF-2), might also report the post-fertilization pH
increase that occurs in sea urchins (2). The fluorescent product of the
reaction of DAF-2 with NO is said to exhibit pH-dependent fluorescence intensity (28).
This report contributes to the debate on the relative roles of the
InsP3 and ryanodine receptor pathways at fertilization in
sea urchin eggs. We have focused on the specific role of NO and the
cADPR-regulated ryanodine receptor pathway. We have examined the
temporal relationship between NO and Ca2+ in sea urchin
eggs at fertilization by simultaneously measuring these signaling
components. By using specific inhibitors we establish that an
NO/cGMP/cADPR pathway plays a major role in regulating the later phase
of the fertilization Ca2+ transient, supporting the view
that the general role of the NO pathway is to enhance and sustain
Ca2+ signals independently of other signaling pathways
(25). We have been unable to confirm an early role for NO in initiating the events that lead to egg activation in the sea urchin at fertilization.
Collection of Sea Urchin Eggs--
Eggs were obtained by
ovulating female Psammechinus miliaris (Marine Biological
Station, Millport, Isle of Cumbrae, Scotland) or Lytechinus
pictus (Marinus, Long Beach, CA) with an intracoelomic injection
of 0.5 M KCl solution. The jelly was removed by several passages through Nitex mesh, and the eggs were suspended in
artificial sea water (ASW: 410 mM NaCl, 39 mM
MgCl2, 15 mM MgSO4, 2.5 mM NaHCO3, 10 mM CaCl2,
10 mM KCl, 1 mM EDTA, at pH 8.0). Unless otherwise stated, treatments and their corresponding controls were
carried out on the same batches of eggs.
Imaging of Intracellular [Ca2+]i and
NO--
To visualize NO, we examined the specificity of two available
fluorescent indicators, DAF-2, and the more recently available dye
DAF-FM. We compared changes in fluorescence of the two dyes in eggs at
fertilization, with and without the addition of 100 µM
5-(N,N-dimethyl)amiloride hydrochloride
(DMA) to block the Na+/H+ antiporter and thus
prevent pH increases (29, 30). DAF-2 responses were largely abolished
in eggs fertilized in DMA (DAF-2 fluorescence levels 200 s
post-fertilization were on average 9 ± 0.7% higher than resting
levels (n = 6), whereas in the presence of DMA the
increase was just 2 ± 0.6% (n = 5)). DAF-FM
responses were not abolished by DMA treatment. We confirmed that DAF-FM loaded into unfertilized sea urchin eggs reported NO increases by the
external application of 5.7 mM of the NO donor sodium
nitroprusside (which caused a 6 ± 2% fluorescence increase above
resting levels after 5 min in four eggs) and that the fluorescence did
not increase with the addition of 10 mM NH4Cl
(fluorescence levels fell 2 ± 0.5% below resting after 5 min in
four eggs), which is known to cause an elevation in cytosolic pH in sea
urchin eggs (31). Due to its specificity, DAF-FM was our indicator of
choice for measuring NO changes.
Eggs of P. miliaris were transferred to
poly-D-lysine (10 mg/ml)-coated glass coverslips and
maintained at 16 °C in ASW. Fura-2 dextran, pentapotassium salt
(10,000 Mr, 10 mM in the
pipette, Molecular Probes) in a buffer consisting of 0.5 M
KCl, 20 mM PIPES, 0.1 mM EGTA at pH 6.8, was
pulsed into eggs with drawn borosilicate glass micropipettes
(GC150F-10, Clark Electromedical Instruments) using gas pressure, to a
final concentration of ~50-100 µM. Injection volumes
did not exceed 1.5% of the cell volume. Following injection, eggs were
incubated in ASW containing 50 µM DAF-FM DA for 15 min, followed by a 30-40 min post-incubation step in ASW, or a 15-min post-incubation step in ASW followed by 15 min in low sodium ASW (50 mM NaCl, 360 mM choline Cl, 39 mM
MgCl2, 15 mM MgSO4, 2.5 mM KHCO3, 10 mM CaCl2,
10 mM KCl, 1 mM EDTA at pH 8.0) supplemented with 100 µM DMA with fertilization being carried out in
this medium. For DAF-2 experiments, 50 µM DAF-2 DA was
added externally to eggs, and the procedure followed was then the same
as that for DAF-FM loading in the absence of DMA. Oxyhemoglobin (Sigma,
H-0267, lot 20K7618; 46% purity, assayed spectroscopically) was
co-loaded with fura-2 dextran, whereas dibromo-BAPTA was loaded into
cells previously injected with fura-2 dextran, prior to incubation with DAF-FM followed by 100 µM DMA. Because inhibiting the
fertilization pH changes was found to result in enhanced NO production
and Ca2+ transients of longer duration, experiments using
oxyhemoglobin and dibromo-BAPTA were performed in the presence of DMA,
so that differences in levels of NO produced and the Ca2+
transient generated due to the antagonists were more readily apparent
when compared with controls. All work was carried out at 16 °C, and
all post-injection steps were performed under red light. Eggs
were illuminated on a stage of a Nikon Diaphot 300 microscope using a
100-watt mercury lamp light source and a UV-F 20× numerical aperture
0.8 objective (Nikon). Simultaneous Ca2+ and NO levels were
measured using narrow band-pass filters (350, 380, and 490 nm, Chroma)
housed in a Lambda 10-2 filter wheel (Sutter Instrument Co.), and a
polychroic beamsplitter (61000v2bs, Chroma) with emission filter
(61000v2m, Chroma). Images were collected using a charge-coupled device
camera (Photometrix Coolsnap fx, Roper Scientific). Both the filter
wheel and acquisition by the charge-coupled device camera were
controlled by Metafluor software version 4.0 (Universal Imaging Corp.,
West Chester, PA). Free cytosolic Ca2+ concentration was
determined by ratioing average fluorescence intensity values calculated
from the images excited at 350 and 380 nm, and standard
CaCl2 solutions were used to calibrate the system with
corrections for viscosity (32). Images were processed by IDL software
(Research Systems Inc.) on an Indigo 2 workstation (Silicon Graphics).
Treatment of Eggs with cGMP, 8-Bromo-cADPR, Nicotinamide, and
(Rp)-cAMP-S--
All compounds were introduced into
eggs of L. pictus using the same injection buffer and
procedure as above. The amount injected never exceeded 1% of cell
volume. For treatments involving cGMP, eggs were double-injected, first
with fura-2 dextran (final concentration of ~20 µM) or
fura-2 dextran and Treatment of Eggs with 8-Amino-cADPR--
Eggs of L. pictus were prepared as above and microinjected with a mixture of
fura-2 pentapotassium salt and 8-NH2-cADPR (final intracellular concentration of ~10 µM for both
compounds) in injection buffer (0.5 M KCl, 20 mM PIPES, at pH 6.7). Experiments were performed at
20-22 °C. Free cytosolic Ca2+ concentration was
determined by ratioing fluorescence intensities at 340 and 380 nm using
an emission wavelength of 510 nm. Ratio images were obtained using a
fluorometric imaging system (monochromator, TILL Photonics, Germany)
and Ionvision software (Improvision Ltd., UK) as described previously
(33).
Radioreceptor Assay of cADPR Levels--
Eggs of L. pictus (0.8-1.5 ml) were dejellied, washed twice in ASW, and made
to a final volume of 20 ml with ASW. 350 µl of sperm was added, and
the solution continuously mixed. At given time points, 2-ml aliquots
were removed and centrifuged for 6 s at 6000 rpm. Excess ASW was
removed, and eggs were treated with 3 M perchloric acid
(1:1 w/v). The precipitated proteins were removed by centrifugation,
and the supernatants were neutralized with 2 M
KHCO3. To remove contaminating nucleotides that weakly interfere with [32P]cADPR binding, tissue samples were
treated with NADglycohydrolase (0.25 unit/ml), nucleotide
pyrophosphatase (1.75 units/ml), alkaline phosphatase (50 units/ml),
and apyrase (5 units/ml) for 4 h, as described previously (34).
Acid extracts were stored at Materials--
5,5'-Dibromo-BAPTA tetrapotassium salt and fura-2
dextran pentapotassium salt were purchased from Molecular Probes.
4,5-Diaminofluorescein diacetate (DAF-2 DA),
3-amino-4-aminomethyl-2',7'-difluorofluorescein diacetate (DAF-FM DA),
sodium nitroprusside, and adenosine cyclic 3',5'-monophosphorothioate,
Rp isomer
((Rp)-cAMP-S) were purchased from
Calbiochem. 8-Bromo-cADPR was synthesized as described previously (36).
All other chemicals were from Sigma.
Image and Statistical Analyses--
Average pixel intensity
values for 350-, 380-, and 490-nm excitation wavelengths were
determined from a region of interest bordering the outside of labeled
eggs using Metafluor image analysis software and background subtracted
using values determined from a specified background region. DAF-2 and
DAF-FM intensity values were baseline-corrected using the measured
resting values recorded prior to insemination. Statistical analyses
used a Student t test (one-tail, unpaired). Data were
expressed as the means ± S.E. for n eggs tested.
Levels of NO, as Measured with DAF-FM, Rise after the Initiation of
the Ca2+ Wave in Fertilized Sea Urchin Eggs--
DAF-FM is
said to be a more faithful indicator of NO above pH 5.8 (28) and has
been used to image NO production in several cell types, including rat
neuronal cells and smooth muscle cells (37, 38). We confirmed the
specificity of DAF-FM in sea urchin eggs at fertilization (see
"Experimental Procedures"). Using DAF-FM, in combination with the
ratiometric Ca2+ indicator dye fura-2, we were able to
simultaneously measure NO and Ca2+ levels in intact eggs at
fertilization. Simultaneous measurements of Ca2+ and DAF-FM
fluorescence demonstrated that DAF-FM was not directly sensing
Ca2+ under our experimental conditions. In P. miliaris eggs loaded with DAF-FM and subsequently fertilized, an
increase in fluorescence was found to occur after the onset of the
Ca2+ wave (Fig. 1,
A and B). On average, DAF-FM fluorescence started to increase 68.4 ± 5.4 s post-fertilization (PF) or 5.3 ± 4.2 s after peak Ca2+ levels were reached
(n = 16). Increases in DAF-FM fluorescence were not
attenuated when eggs were fertilized in low sodium sea water in the
presence of 100 µM
5-(N,N-dimethyl)amiloride hydrochloride (DMA, Fig. 1B) to block the Na+/H+
antiporter and thus prevent an elevation of the pH (29, 30). We
observed differences in the shape of the fertilization Ca2+
transient when DMA-treated, DAF-FM-loaded eggs were compared with those
loaded with DAF-FM only (Fig. 1B, panel
i). The duration of the Ca2+ transient was
significantly prolonged. The time taken for
[Ca2+]i to fall to 25% of the peak level was
189.8 ± 5.5 s (n = 16) in control eggs
compared with 263.6 ± 6 s in DMA-treated (n = 14, p = < 0.001, t test). The response of
DAF-FM was also affected (Fig. 1B, panel ii). The
magnitude of the response was significantly greater (the DAF-FM
fluorescence levels, 200 s PF, were an average of 6.9 ± 0.9% greater than resting in DMA-treated eggs and 3.9 ± 0.4%
greater than resting in untreated eggs; p = <0.01,
t test). These results confirm that NO levels increase in
sea urchin eggs at fertilization and that this rise occurs during the
period of sustained Ca2+ elevation after the
Ca2+ wave has been generated. Interestingly, the enhanced
levels of NO production in fertilized eggs in which the pH change is
inhibited correlate with a Ca2+ transient of increased
duration.
It has been reported that the fluorescence levels of the NO indicator
DAF-2 increase very early during the fertilization response and the
consequent suggestion is that NO is a very early player in the
fertilization signaling cascade (23). In this study we analyzed whole
egg average fluorescence intensity immediately after sperm-egg fusion,
in DAF-2-loaded eggs of P. miliaris (50 µM,
applied externally) fertilized in normal sea water (n = 11), and DAF-FM loaded eggs with and without the addition of DMA
(n = 30) and failed to find the small increases in
fluorescence previously observed. Careful inspection of both DAF-2
(Fig. 1C, panel i) and DAF-FM fluorescence images
(Fig. 1C, panel ii) at the point of initiation of
the fertilization Ca2+ transient again offered no
confirmation of these earlier observations. Analysis of a 30-µm
diameter region (average egg diameter, 120 µm) at the point of sperm
egg fusion (as defined by the initial Ca2+ increase) in 17 eggs did not reveal any local increase in fluorescence. We estimate
that, given the noise in our measurements, if a local NO increase
occurs, the fluorescence increase locally must be <0.3% above mean
resting values for DAF-FM-labeled eggs and <0.8% for those labeled
with DAF-2.
Scavenging NO Shortens the Duration of the Fertilization
Ca2+ Transient--
Oxyhemoglobin scavenges NO (39-41).
To determine whether prolongation of the Ca2+ transient
that we observed in DMA-treated eggs was due to NO production, we
microinjected oxyhemoglobin (12-28 µM) into DMA-treated P. miliaris eggs. Oxyhemoglobin both delayed and
very markedly attenuated the post-fertilization increase in DAF-FM
fluorescence (Fig. 1D). In control eggs, the start of the
DAF-FM fluorescence increase was observed 60.6 ± 8 s after
peak [Ca2+]i (108.2 ± 8.1 s PF,
n = 10), whereas in oxyhemoglobin loaded eggs, where an
increase occurred, it was significantly later, at 128.6 ± 12.6 s after the [Ca2+]i peak (171.7 ± 13 s PF, n = 8, p < 0.001, t test). In oxyhemoglobin-loaded eggs DAF-FM increased on
average only 1.0 ± 0.4% above resting levels 200 s PF
(n = 10), whereas control eggs showed a 7.3 ± 0.8% increase (n = 10, p < 0.001, t test). For two out of ten oxyhemoglobin-loaded eggs, no
increase in DAF-FM fluorescence was detected during fertilization, but
Ca2+ transients leading to egg activation did occur. In the
presence of oxyhemoglobin, the latency, magnitude, and rise time of the fertilization Ca2+ transient was unaffected. The average
control latency, rise time, and amplitude were 27.3 ± 1.5 s,
21.6 ± 0.8 s, and 1.04 ± 0.1 µM,
respectively, in control eggs (n = 10), and the
corresponding values for 10 oxyhemoglobin-loaded eggs were 24.1 ± 1.3 s, 22.5 ± 2.7 s, and 1.1 ± 0.06 µM. However, the rate at which calcium decreases after
the [Ca2+]i peak was significantly increased. The
time taken for a fall to 25% of peak [Ca2+]i was
243.1 ± 14.6 s (n = 10) in control eggs,
compared with 192.9 ± 9.9 s in oxyhemoglobin-loaded eggs
(n = 10, p < 0.01, t test).
These observations show that inhibiting the NO rise in sea urchin eggs
does not abolish the fertilization Ca2+ transient and has
no effect on the initiation or rate of rise of the transient. However,
inhibiting the NO rise shortens the transient. Because enhanced NO
levels lengthen the transient (Fig. 1B), these data imply a
causal relationship between NO production and Ca2+
transient duration.
NO Production at Fertilization Is
Ca2+-dependent--
It has been demonstrated
that the treatment of unfertilized eggs with the calcium ionophore
ionomycin causes NO production as measured by the accumulation of
nitrite (23). The Ca2+ chelator dibromo-BAPTA is very
effective at suppressing Ca2+ transients and gradients (42,
43). Microinjection of 2.5-7 mM dibromo-BAPTA into
P. miliaris eggs before insemination prevents both the
fertilization Ca2+ wave (Fig.
2B) and the DAF-FM increase
(Fig. 2D). To control for the possibility that BAPTA itself
might scavenge NO, we added sodium nitroprusside to BAPTA-injected eggs
labeled with DAF-FM and noted NO increases equivalent to control eggs
(not shown). This finding demonstrates that the post-fertilization NO
increase is dependent on an increase in
[Ca2+]i.
cGMP Effects Ca2+ Release via the Ryanodine Receptor
Pathway and Regulates the Duration of the Ca2+ Transient at
Fertilization--
NO has been shown to trigger Ca2+
release in intact eggs and egg homogenates and to promote cGMP
synthesis (25). The inhibition of NO- and cGMP-mediated
Ca2+ release by nicotinamide, an ADP-ribosyl cyclase
inhibitor, implicates cADPR as the downstream effector of this pathway
(24, 25). We confirm and extend these findings. Fig.
3 shows that the Ca2+
transient induced by cGMP (100 µM, intracellular) in
unfertilized eggs of L. pictus was attenuated by
nicotinamide (200 µM, intracellular) and by the
competitive inhibitor 8-bromo-cADPR (30 µM), antagonists of the cADPR signaling pathway. These inhibitors caused a marked attenuation of fertilization envelope formation (Table
I). Moreover, the activation response to
lower doses of cGMP was potentiated by pre-injection of
cADPR Levels Increase after the Rise of the Fertilization
Ca2+ Transient--
No imaging method yet exists to
measure cADPR in single eggs, so cADPR levels in a population of
fertilized L. pictus eggs were measured to establish the
temporal relationship between NO production, cADPR synthesis, and the
fertilization Ca2+ transient. cADPR levels have been
previously shown to increase by 30 s after insemination in egg
populations of the sea urchin species Anthocidaris
crassispina and Hemicentrotus pulcherrimus (18). We
have previously shown (16) that at appropriate sperm densities a peak
of around 80% of eggs in a population are undergoing a
Ca2+ transient at 25 s after insemination. Fig.
4A shows that levels of
endogenous cADPR increase by 25 s after fertilization and reach a
peak 50 s after insemination of eggs of L. pictus.
Levels of cADPR then decline to resting levels by ~150 s. cADPR
production appears to lag the fertilization Ca2+ transient.
This is in contrast to previously measured InsP3
production, which precedes, and coincides with, the fertilization
Ca2+ transient (16; reproduced in this report as Fig.
4B).
cADPR Regulates the Later Phase of the Fertilization
Ca2+ Transient--
The timing of cADPR production
suggests it may have a role during the later phase of the
Ca2+ transient. We tested this idea by using 8-amino-cADPR,
a competitive inhibitor (Fig. 5). After
microinjection of 10 µM 8-amino-cADPR into eggs of
L. pictus, the duration of the fertilization
Ca2+ transient (the time taken for
[Ca2+]i levels to fall to 500 nM
post-maximum, or ~25% of peak values) was decreased significantly to
119 ± 19 s from 279 ± 55 s in the control
(p < 0.05, t test, n = 12).
This result demonstrates that cADPR plays a major part in sustaining
the Ca2+ transient.
Both InsP3- and ryanodine-receptor channels
participate in the sea urchin fertilization Ca2+ response
(10, 11). Previous work has uncovered a signaling pathway that could
regulate ryanodine receptors at fertilization. Data are consistent with
the notion that NO leads to cGMP production via guanylate cyclase
activation, and elevated cGMP promotes the production of cADPR by
ADP-ribosyl cyclase (24, 25), leading to ryanodine receptor activation
and Ca2+ release. Because fertilization-associated
increases in both cGMP (18, 26), cADPR (18), and NO levels (23) have
been measured, it is clear that the NO/cGMP/cADPR pathway is present in
eggs and activated at fertilization. What is less clear is the
contribution that the pathway makes to the physiology of
Ca2+ release at fertilization. On the one hand, it is
suggested that an NO-mediated pathway is the primary mechanism for egg
activation (23), while on the other there are abundant data that
suggest that the InsP3 signaling pathway plays the primary
role (12, 14, 17, 19-22). In this study we have used a physiological approach to dissect the contribution that the NO/cGMP/cADPR pathway makes in generating the fertilization calcium signal and show that the
pathway acts late in the response.
Nitric Oxide Is Generated after the Fertilization Ca2+
Transient Is Initiated--
We have used a recently available, and
pH-stable, NO indicator dye, DAF-FM, to show that NO levels do indeed
rise at fertilization. For the first time in the sea urchin egg we have
measured NO and Ca2+ levels simultaneously. We have been
unable to detect any increase in NO prior to the initiation of the
Ca2+ wave after sperm-egg fusion using DAF-FM, either
locally at the point of sperm-egg fusion, or globally throughout the
egg. When we performed the same analysis using the indicator DAF-2 used in a previous study (23), we again failed to detect an increase in NO
prior to the initiation of the wave.
Nitric Oxide Production Depends on the Fertilization
Ca2+ Transient and Is Sensitive to pH--
The two major
ionic signals at fertilization in sea urchins are the Ca2+
transient and the pH change (2). As we have seen, preventing the pH
increase significantly enhances NO production as measured by DAF-FM,
and microinjection of the Ca2+ chelator BAPTA blocks both
the fertilization Ca2+ transient and the NO increase. This
demonstrates that NO production is dependent on the Ca2+
increase at fertilization and may be regulated by changes in pH.
Nitric Oxide Regulates the Later Phase of the Ca2+
Transient--
The temporal characteristics of the NO rise may give us
clues to its physiological role. A lack of a detectable early increase using either DAF-2 or DAF-FM is not readily reconciled with a role in
initiation of the Ca2+ transient. This is supported by the
absence of any alteration in rise time or latency (Table
II) of the fertilization
Ca2+ transient by concentrations of an NO scavenger,
oxyhemoglobin, sufficient to suppress significantly the NO increase we
observed later during fertilization. These results contrast markedly
with those of Kuo et al. (23). It could be argued that, due
to the impurity of commercially available oxyhemoglobin, we were unable to inject concentrations of oxyhemoglobin previously reported to
abolish egg activation (23). Although this is a possibility, we have
found that abolition of the NO rise does not coincide with an abolition
of the fertilization Ca2+ transient. Because NO levels rise
during the period of sustained Ca2+ elevation after
the peak, a role for regulating this phase of the Ca2+
signal is possible. There are two pieces of evidence for this: inhibiting the pH change at fertilization results in enhanced NO
production linked to Ca2+ transients of longer duration,
and shorter duration Ca2+ transients are seen in cells
where NO increases are inhibited.
The NO/cGMP/cADPR/RyR Pathway Acts
Late at Fertilization--
We have extended previous findings using
agonists and antagonists of cGMP and cADPR signaling to show that a
cGMP/cADPR/RyR pathway, previously demonstrated in homogenates,
operates in a very similar manner in intact, unfertilized eggs.
However, the use of such inhibitors at fertilization has been limited
to demonstrating that the ryanodine receptor pathway played a role at
fertilization (10, 11). Here we show that the effect of an inhibitor of cGMP-dependent protein kinase or a competitive cADPR
antagonist is to curtail the fertilization Ca2+ transient.
It is also clear from this work that the same inhibitors have no effect
on the latency of the fertilization Ca2+ transient (Table
II). These observations, together with those using the NO scavenger
oxyhemoglobin, indicate that a major function of the NO/cGMP/cADPR/RyR
pathway is to sustain and prolong the sea urchin fertilization
Ca2+ signal.
Measurements of cADPR Levels at Fertilization Suggest cADPR Is a
Regulator, Not an Initiator, of the Ca2+
Transient--
Measurements of cGMP production at fertilization
indicate that levels of this messenger rise quickly after fertilization
to a peak at around 30 s (18, 26). Here we show a similar time course for cADPR production in L. pictus, which is in
agreement with the time course for cADPR production at fertilization in two other sea urchin species, A. crassispina and H. pulcherrimus (18). It is very difficult to draw conclusions about
the temporal sequence of cGMP and cADPR production, because, with the
resolution available to us, both seem to peak at about the same time,
roughly coincident with the peak of the population Ca2+
transient. What we can say is that the timing of cADPR production at
fertilization appears to be later than that of InsP3
measured in a previous study (16) in L. pictus and suggests
that cADPR regulates the later phase of the transient. Our data
indicate cADPR levels peak at 200 nM at fertilization;
cADPR levels of 40-150 nM are reported to release
Ca2+ in intact sea urchin eggs on microinjection (5, 10,
11), so the increases we observe are physiologically relevant. One paradox is that the previously measured levels of cGMP in A. crassispina and H. pulcherrimus (18) are orders of
magnitude lower than the concentration required to activate intact eggs
on microinjection (46, and the present study). However, this is not
unexpected if the role of the NO/cGMP/cADPR/RyR pathway is to enhance
an existing Ca2+ transient, rather than initiate it. On
this interpretation, the activation of eggs by NO, cGMP, and cADPR is
not a physiological response, although it reveals the presence of the
signaling pathway.
The Stimulation of the
NO/cGMP/cADPR/RyR Pathway by
Ca2+--
In direct contrast to our observation that the
NO increase is Ca2+-dependent, it has been
found that artificial Ca2+ increases do not stimulate the
production of cGMP (18, 26). This is surprising given that sea urchin
eggs are known to contain the
Ca2+/calmodulin-dependent neuronal NOS
isoform and can generate NO when Ca2+ is artificially
increased (23). The resolution of this paradox will depend upon a
robust and readily available method of measuring cGMP with good time resolution.
Conclusions--
The prevalent hypothesis of egg activation is
that InsP3 is generated by a protein that diffuses from
sperm to egg when sperm and egg fuse at fertilization (47, 48). In some
species this protein may be a phospholipase C (22) and in others a
component of tyrosine kinase signaling pathways (49-51). It is
generally accepted that the cGMP/cADPR pathway does not initiate
activation, because blockers of the pathway cannot by themselves block
fertilization, in marked contrast to antagonists of the
phosphoinositide signaling pathways (12, 14, 17, 19-21). Our data are
consistent with this majority view and assign a late, sustaining role
to the NO/cGMP/cADPR/RyR pathway at fertilization in sea urchin eggs.
Finally, it may be that, although the NO pathway itself is widely
distributed (52-54), its involvement at fertilization is confined to
echinoderms or perhaps echinoids. Neither frog (55), ascidian (56), nor
mammalian oocytes (57, 58) rely on the RyR receptor at fertilization, and neither mouse nor ascidian oocytes showed any evidence of increases
in NO at fertilization (27). One obvious difference between echinoids
and most other animals is that their eggs are arrested at interphase of
the cell cycle rather than in the meiotic metaphase. Whether this
correlation between the stage of meiotic arrest and the presence of an
NO signaling cascade is physiologically significant remains to be determined.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
SH2 domain
constructs, show that sperm-activated Ca2+ release and egg
activation are markedly delayed, then blocked, when activation of this
enzyme is inhibited (19-21). In mouse eggs, diffusion of a newly
characterized PLC, PLC
, from sperm to egg triggers Ca2+
oscillations and embryo development (22). These data strongly suggest
an InsP3-based initiation mechanism for the fertilization calcium wave.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-NAD+ or inhibitors, and then with
cGMP or injection buffer (control). For inhibitor treatments, control
responses to cGMP were performed after each experiment on eggs from the
same batch to exclude the possibility of a reduction in responsiveness
to cGMP with time. Ca2+ levels were measured using ratio
photometry and calibrated as described previously (3).
(Rp)-cAMP-S (final concentration, 2-4
mM) was co-loaded into eggs of L. pictus with
fura-2 dextran (final concentration, ~100 µM) and
Ca2+ levels measured and calibrated as for the simultaneous
Ca2 + and NO experiments, replacing the polychroic
beamsplitter and emission filter with a 400-nm dichroic and 510-nm
band-pass emission filter (Nikon).
20 °C prior to use in the
radioreceptor assay. The binding assay was performed as previously
described (35). Briefly, tracer [32P]cADPR was
synthesized from a high specific activity (1000 Ci/nmol) radiolabeled
form of its precursor [32P]NAD+. The
cyclization reaction was carried out for 2 h at room temperature using 250 mCi of [32P]NAD+, 100 ng
ml
1 of ADP-ribosyl cyclase, and 5 mM
Tris-HCl, pH 7.4. cADPR was purified using high-performance liquid
chromatography, and fractions containing cADPR were neutralized with
Tris base. The binding assay consisted of sea urchin egg homogenate
made up to a final concentration of 0.5 mg/ml protein in IM buffer (333 mM N-methylglucamine, 333 mM
potassium acetate, 27 mM HEPES, 1.3 mM
MgCl2; pH titrated to 7.2 with acetic acid). Approximately
22.5 fmol of [32P]cADPR was incubated with homogenate in
a total volume of 250 ml for 10 min at room temperature. The binding
reaction was terminated by rapid filtration with a Brandel cell
harvester using GF/B filters washed immediately before filtration with
ice-cold IM and washed twice with 2-4 ml of ice-cold IM immediately
after filtration. Radioactivity retained on the filters was determined
using standard scintillation counting techniques. Inhibitory effects of
acid extracts on [32P]cADPR binding were compared with
standard curves constructed using authentic cADPR. As a control, each
sample was heat treated at 85 °C for 45 min to hydrolyze the cADPR
to ADPR. The inhibitory effect on [32P]cADPR binding of
all samples was abolished by heat treatment.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (45K):
[in a new window]
Fig. 1.
Levels of NO rise after the initiation of the
Ca2+ wave and attenuation of this rise reduces the duration
of the Ca2+ transient. A, representative
example of an egg loaded with fura-2 (upper series)
and the NO indicator DAF-FM DA (50 µM, applied
externally, lower series) fertilized in ASW.
Numbers on images correspond to numbered points
on the adjacent plot showing whole cell [Ca2+]i
(red plot) and DAF-FM fluorescence intensity (blue
plot, expressed as a ratio with respect to fluorescence at
time = 0). Note the later rise in DAF-FM fluorescence relative to
the fertilization Ca2+ transient. The arrow
indicates the point of sperm addition. B, i,
average [Ca2+]i levels in a population of
DAF-FM-loaded eggs fertilized in the presence (blue plot,
n = 14) or absence (red plot,
n = 16) of 100 µM DMA. ii,
corresponding average DAF-FM fluorescence values, expressed as ratios
with respect to the fluorescence at t = 0, for eggs
represented in panel i, fertilized in the presence
(blue plot) or absence (red plot) of DMA.
Bars = S.E. of means. Arrows represent the
point of sperm-egg fusion (t = 100 s). Data are
the results of 10 experiments, using four and three separate batches of
eggs for DMA-treated and control eggs, respectively. C,
close examination of the sperm-egg fusion event at the point of sperm
entry reveals no increase in: i, DAF-2 fluorescence
(fertilized in normal ASW); ii, DAF-FM fluorescence
(fertilized in the presence of 100 µM DMA).
Representative images of [Ca2+]i (lower
series) and corresponding NO indicator fluorescence (upper
series) are shown for each treatment. The arrow
indicates the point of sperm entry. Numbered time points
are; 1, pre-fusion; 2, start of cortical
Ca2+ transient; 3, approximate mid point of
cortical Ca2+ transient; 4, start of
[Ca2+]i wave. D, the NO scavenger
oxyhemoglobin attenuates the DAF-FM response at fertilization and
reduces the duration of the fertilization induced Ca2+
transient. Pseudocolor images showing a representative
example of fertilization in eggs loaded with the Ca2+
indicator fura-2 dextran (50-100 µM, upper
series) and the NO indicator DAF-FM DA (50 µM
external application, lower series) as well as oxyhemoglobin
(12-28 µM), prior to treatment with DMA (100 µM). Numbers on the images
correspond to numbered points on the adjacent plot showing average
whole cell [Ca2+]i (red plot) and
DAF-FM (blue plot, fluorescence expressed as a ratio with
respect to fluorescence at time = 0 s).
View larger version (38K):
[in a new window]
Fig. 2.
The rise in DAF-FM fluorescence at
fertilization and, therefore, the increase in levels of endogenous NO
are dependent on the elevation of [Ca2+]i.
In contrast to control cells, which were not loaded with dibromo-BAPTA
(but were treated with DMA, A), the dibromo-BAPTA-loaded
cells (B) showed a marked reduction in levels of
[Ca2+]i at fertilization. This was reflected in
an almost complete abolition of the subsequent rise in DAF-FM
fluorescence in dibromo-BAPTA-loaded eggs (D) in contrast to
the controls (C). Pseudocolor images show a
representative cell for the population of control eggs (A
and C) and dibromo-BAPTA-loaded eggs (B and
D). Numbers on the images correspond
approximately with the time points on the plots beneath, with
t = 0, the point of sperm-egg fusion. The upper
plot represents the average [Ca2+]i levels
in a population of control eggs (blue plot,
n = 5) plotted against the average in a population of
dibromo-BAPTA-loaded eggs (red plot, n = 6)
after fertilization. The lower plot shows average whole cell
DAF-FM fluorescence (expressed as a ratio with respect to fluorescence
at the start of the experiment) in the same control and
dibromo-BAPTA-loaded eggs. Bars = S.E. of means.
-NAD+ (100 µM), the cADPR precursor, and
enabled concentrations of cGMP as low as 1 µM to activate
eggs (Table I). The role of cGMP at fertilization was examined by
injecting eggs of L. pictus with 2-4 mM
(Rp)-cAMP-S, which inhibits
cGMP-dependent protein kinase (44) and is effective in sea
urchin egg homogenates (45). The presence of
(Rp)-cAMP-S reduced the duration of the
fertilization Ca2+ transient (Fig. 3C). The mean
time of a fall to 50% of peak [Ca2+]i was
146.5 ± 13 s in control eggs (n = 8)
compared with 111 ± 9.2 s in the presence of
(Rp)-cAMP-S (n = 7, p = <0.05, t test). The latencies and rise
times were unaffected (latencies of 26.25 ± 0.7 s and
25.4 ± 0.5 s, and rise times of 21.1 ± 0.8 s and
20 ± 1.2 s, for control and
(Rp)-cAMP-S, respectively). There was a small
but significant increase in the amplitude of the Ca2+
transient with (Rp)-cAMP-S, up to 1.19 ± 0.03 µM compared with a control value of 1.03 ± 0.04 µM (p = <0.01, t test).
These findings suggest cGMP regulates Ca2+ release during
the later phase of the fertilization Ca2+ transient via the
activation of cGMP-dependent protein kinase, which leads to
cADPR production.
View larger version (14K):
[in a new window]
Fig. 3.
cGMP mobilizes [Ca2+]i
in sea urchin eggs via cADPR, and the activity of
cGMP-dependent protein kinases is required for a full
fertilization response. A and B, cGMP (100 µM) induced a large Ca2+ transient when
injected into unfertilized eggs, resulting in egg activation. Prior
injection of eggs with the cADPR signaling inhibitors nicotinamide (200 µM) and 8-bromo-cADPR (30 µM) inhibited the
Ca2+ transient induced by the subsequent injection of cGMP.
Plots are representative examples from each treatment.
[Ca2+]i was measured using fura-2 dextran and
ratio photometry. C, inhibition of
cGMP-dependent protein kinases using
(Rp)-cAMP-S (2-4 mM) significantly
reduced the duration of the fertilization Ca2+ transient.
Plots are of whole cell [Ca2+]i calculated from
images of a representative example of a fura-2 dextran-loaded egg
treated with (Rp)-cAMP-S (2.7 mM,
boldface line) and a control (thin line). All
quoted concentrations are intracellular. Note that the small, immediate
increase in [Ca2+]i after microinjection is an
artifact of microinjection (13).
Release of the fertilization envelope by cGMP and its inhibition
-NAD+, and
8-bromo-cADPR, at the indicated intracellular concentrations, were
injected 2-5 min before cGMP injection. Previous injection with KCl
did not affect the cGMP response.
View larger version (13K):
[in a new window]
Fig. 4.
cADPR is produced after the latent period of
the fertilization-induced Ca2+ transient.
A, average cADPR levels in cell extracts isolated from
populations of sea urchin eggs, inseminated at t = 0 s and then sampled at the specified time points. Levels of cADPR
were calculated using a radioreceptor assay. Each time point is the
mean (±S.E.) of the combined data from four experiments. B,
changes in [3H]inositol polyphosphate with time after
insemination, reproduced from Ciapa and Whitaker (16). Combined data
from five experiments are given. Mean ± S.E. are shown. ( )
n = 4, (
) n = 3. The dashed
curve in each figure indicates the population time course of an
event associated with the fertilization wave (16).
View larger version (33K):
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Fig. 5.
The competitive cADPR inhibitor 8-amino-cADPR
effectively reduced the duration of the fertilization-induced
Ca2+ transient compared with controls.
Pseudocolor images show the effects of 8-amino-cADPR (10 µM, intracellular, B) on the
fertilization-induced Ca2+ transient, compared with a
control cell (A). The plots below the
pseudocolor images show the average
[Ca2+]i change in the egg with time after sperm
addition (~t = 0) for the control (open
squares) and 8-amino-cADPR-treated (filled squares).
[Ca2+]i was determined from fura-2 dextran (~10
µM, intracellular) ratio values. Numbers on
the pseudocolor images relate to the corresponding number on
the plots beneath.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
The latency of the fertilization Ca2+ transient is
unaffected by modulators of the NO, cGMP, and cADPR signalling
pathways
-NAD+ (200 µM).
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ACKNOWLEDGEMENTS |
---|
We thank David Epel for advice about DAF-FM and the pH sensitivity of DAF-2 and Michael Aitchison for help with image processing and in the preparation of the figures.
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FOOTNOTES |
---|
* This work was supported by a grant from the Wellcome Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Biococca, Milan I-20132, Italy.
To whom correspondence should be addressed. Tel.:
44-191-222-6707; Fax: 44-191-222-6706; E-mail:
michael.whitaker@ncl.ac.uk.
Published, JBC Papers in Press, January 22, 2003, DOI 10.1074/jbc.M210770200
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ABBREVIATIONS |
---|
The abbreviations used are: InsP3, D-myo-inositol 1,4,5-trisphosphate; (Rp)-cAMP-S, adenosine cyclic 3',5'-monophosphorothioate, Rp isomer; PLC, phosphatidylinositol phospholipase C; NOS, nitric-oxide synthase; DAF-2 DA, 4,5-diaminofluorescein diacetate; DAF-FM DA, 3-amino-4-aminomethyl-2',7'-difluorofluorescein diacetate; ASW, artificial sea water; DMA, dimethylamiloride; PIPES, 1,4-piperazinediethanesulfonic acid; PF, post-fertilization; RyR, ryanodine receptor; cADPR, cyclic ADP-ribose; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid.
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