The NO Pathway Acts Late during the Fertilization Response in Sea Urchin Eggs*

Calum LeckieDagger , Ruth Empson§, Andrea BecchettiDagger , Justyn Thomas§, Antony Galione§, and Michael WhitakerDagger ||

From the Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 PLCgamma 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, PLCzeta , 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.

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -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).

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 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 -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.

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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


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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).

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.


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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.

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 beta -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.


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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).


                              
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Table I
Release of the fertilization envelope by cGMP and its inhibition
The percentage of activated eggs, and the latency between injection and the start of envelope elevation, are shown for the indicated treatments. cGMP intracellular concentration ranged from 1 µM (null effect) to 100 µM (approximately 90% of eggs activated). Nicotinamide, beta -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.

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).


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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. (diamond ) n = 4, (triangle ) n = 3. The dashed curve in each figure indicates the population time course of an event associated with the fertilization wave (16).

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.


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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

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.


                              
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Table II
The latency of the fertilization Ca2+ transient is unaffected by modulators of the NO, cGMP, and cADPR signalling pathways
The mean latent period (±S.E.), from the start of the "cortical flash" to the start of the spike rise, was measured. The NO inhibitor oxyhemoglobin (12-28 µM), the cGMP-kinase inhibitor (Rp)-cAMP-S (2-4 mM), and the cADPR inhibitors nicotinamide (10 mM, external) and 8-bromo-cADPR (10-30 µM) had no significant effect on the length of the latent period. This was also true in eggs treated with the cADPR precursor beta -NAD+ (200 µM).

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.

    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.

    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

    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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RESULTS
DISCUSSION
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