Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M704630200 on October 24, 2007

J. Biol. Chem., Vol. 282, Issue 52, 37730-37737, December 28, 2007
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
282/52/37730    most recent
M704630200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Morgan, A. J.
Right arrow Articles by Galione, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Morgan, A. J.
Right arrow Articles by Galione, A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Fertilization and Nicotinic Acid Adenine Dinucleotide Phosphate Induce pH Changes in Acidic Ca2+ Stores in Sea Urchin Eggs*Formula

Anthony J. Morgan1 and Antony Galione

From the Department of Pharmacology, University of Oxford, Oxford OX1 3QT, United Kingdom

Received for publication, June 5, 2007 , and in revised form, October 17, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) releases Ca2+ from the acidic Ca2+ stores of many organisms, including those of the sea urchin egg. We investigated whether the pH within the lumen of these acidic organelles changes in response to stimuli. Fertilization activates the egg by Ca2+ release dependent upon NAADP, and accordingly, we report that fertilization also alters organellar pH in a spatio-temporally complex manner. Upon sperm fusion, vesicles deep in the egg center slowly acidify, whereas cortical vesicles undergo a rapid alkalinization. The cortical vesicle alkalinization is independent of exocytosis and cytosolic pH but coincides with the NAADP-dependent fertilization Ca2+ wave. Microinjection of NAADP mimicked the fertilization cortical response, suggesting that it occurred within NAADP-sensitive acidic Ca2+ stores. Our data show that NAADP and physiological stimuli alter the pH within intracellular organelles and suggest that NAADP signals through pH as well as Ca2+.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The sea urchin egg is an invaluable cell model for studying Ca2+ signaling since the discovery of new second messengers and new Ca2+ stores in this system has subsequently proven to have a major impact on mammalian biology (1). The mechanism of activation of the egg upon fertilization encompasses the rapid increases in the cytosolic pH and intracellular Ca2+ concentration ([Ca2+]i) important for fertilization envelope formation, DNA and protein synthesis, and embryological development (2). The pattern of [Ca2+]i changes within the egg are highly organized spatio-temporally and reflect the precisely timed and placed recruitment of different families of Ca2+ channels resident upon either the plasma membrane or intracellular organelles (2, 3). After the initial transient "cortical flash" caused by Ca2+ influx across the plasma membrane (2-5), Ca2+ release from intracellular stores is manifest as a Ca2+ wave that propagates across the entire egg initiating from the point of sperm entry (2). These phasic elevations in [Ca2+]i are driven by a complex interplay between the second messengers inositol 1,4,5-trisphosphate, cyclic adenosine diphosphoribose, and nicotinic acid adenine dinucleotide phosphate (NAADP)2 (1). Importantly, the relative contribution of each messenger to each phase of the Ca2+ signal is different, e.g. NAADP is the only messenger implicated in the cortical flash (4, 5), whereas cyclic adenosine diphosphoribose probably plays a later role in prolonging the main Ca2+ spike (6).

NAADP not only evokes a cortical flash but, as first revealed in sea urchin egg, also releases Ca2+ from acidic Ca2+ stores that are lysosome-like organelles (possibly yolk platelets in eggs), whereas inositol 1,4,5-trisphosphate and cyclic adenosine diphosphoribose release Ca2+ from the neutral endoplasmic reticulum (1, 7). Recently in sea urchin egg homogenate, we revealed that the luminal pH (pHL) of these acidic stores is dynamic and increases upon NAADP-induced Ca2+ release, which is a direct effect of NAADP (and not cytosolic Ca2+) via changes in luminal proton buffering/transport (8). We have now confirmed that the pHL increases observed in homogenate are physiologically relevant in the intact egg upon fertilization. Unexpectedly, these changes are confined to the NAADP-sensitive stores in the egg cortex during a novel, early fertilization event, which may have ramifications for the pHL of NAADP-sensitive stores in mammalian cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—NAADP was enzymatically synthesized (9) or purchased from Sigma-Aldrich. Mastoparan (synthetic, Vespula lewisii) was also obtained from Sigma-Aldrich. Acridine Orange, Lysosensor Yellow/Blue DND-160, Lysotracker Red DND-99, Rhod Dextran (10 kDa, high affinity form), Fluo-4 Dextran (10 kDa), and Alexa Fluor 647 Dextran (10 kDa) were from Invitrogen.

Gamete Preparation—Sea urchin eggs from Lytechinus pictus were harvested by intracoelomic injection of 0.5 M KCl, collected in artificial sea water (ASW; 435 mM NaCl, 40 mM MgCl2, 15 mM MgSO4, 11 mM CaCl2, 10 mM KC1, 2.5 mM NaHCO3, 20 mM Tris base, pH 8.0) and de-jellied by passage through 100-µm nylon mesh (Millipore). Sperm, on the other hand, were collected "dry" and maintained at 4 °C until use.

Confocal Laser Scanning Microscopy—Eggs adhering to polylysine-coated glass coverslips were placed on the stage of a Zeiss LSM 510 Meta confocal microscope equipped with 10x air and 40x and 63x oil immersion objectives (NA 0.3, 1.3, and 1.4, respectively) and maintained at room temperature in ASW. Simultaneous monitoring of several fluorescent dyes was carried out using the Multitrack mode of the microscope, which rapidly alternates channel collection and thereby greatly reduces bleed-through. For UV excitation, the 364-nm line of a Coherent Enterprise laser was used. The green channel was excited using the 488-nm line of an argon laser (emission, 505-530 nm), whereas the red channel used a 543-nm HeNe laser (emission, >560 nm). In the experiments using an Alexa Fluor 647 Dextran, this third fluorophore was excited with a 633-nm HeNe laser and emission collected at 645-719 nm using the Meta head.

Microinjection—Micropipettes were pulled from capillary glass with an internal filament and backfilled. The pipettes were then mounted in the electrode holder of an Injectman pressure injection system (Eppendorf) used at typical pressures of 200 hPa for 2 s, which produces ~1% injection volumes. Injectate compounds were usually prepared in 0.5 M KCl at 100 times the final required concentration, and insoluble debris was removed by centrifugation through a Centricon filter (100,000 molecular weight cutoff). Rhod Dextran was first prepared as 2 mM aliquots in 100 mM Tris-HCl, pH 8.0, to assist solubility and then diluted 1:1 with 1 M KCl. The injectate concentration of Alexa Fluor 647 Dextran was 50-200 µM.

pHL Measurements—We measured pHL with two different approaches. First, eggs were loaded with 2-10 µM Lysosensor Yellow/Blue DND-160 for 15 min with qualitatively similar results at all dye concentrations. Lysosensor Yellow/Blue was imaged ratiometrically on the confocal microscope with excitation at 364 nm and dual emission recorded at 385-470 nm (channel 1) and >505 nm (channel 2). The results are expressed as the ratio of channel 1/channel 2, which is directly proportional to pHL. In the majority of experiments, however, acridine orange was used because of its superior signal-to-noise and its documented use to monitor pHL in both sea urchin egg homogenate (8) and intact eggs (10). However, we modified the acridine orange protocol to measure pHL ratiometrically to circumvent movement and cell volume artifacts. Therefore, intact eggs were simultaneously loaded with 10 µM acridine orange and 1 µM Lysotracker Red DND-99 for 15-20 min at room temperature, at which time the fluorescence reaches an equilibrium; the dyes were present throughout the rest of the experiment. Acridine orange responds rapidly and profoundly to changes in pHL, whereas Lysotracker Red responds only poorly (in agreement with Invitrogen) and remains essentially unchanged throughout the experiment until the addition of NH4Cl. The results are expressed as the ratios of the acridine orange/Lysotracker Red signals such that an increase in the ratio reflects an increase in pHL. At higher magnification (see Fig. 6), Lysotracker Red exhibited some photobleaching that resulted in an artifactual rise in the basal ratio in some eggs. This fall in fluorescence was corrected by using a fifth order polynomial curve fit.

In experiments investigating the effect of sodium removal, the eggs were dye-loaded in normal ASW prior to washes in Na+-free ASW (also containing the pHL dyes) immediately before recording. Na+-free ASW was prepared by replacing NaCl with 435 mM N-methyl-D-glucamine and replacing NaHCO3 with 2.5 mM KHCO3.

Measurement of [Ca2+]i—The eggs were microinjected with the Ca2+-sensitive dye fluo-4 dextran (10 kDa, injectate concentration 1 mM, plus 250 mM KCl, Tris pH 8). After a 15-min recovery period, a second micropipette containing 50-200 µM Alexa Fluor 647 Dextran with or without 100 µM NAADP was used for injection.

Simultaneous Measurement of pHL and [Ca2+]i—The eggs were first microinjected with Ca2+-sensitive Rhod Dextran (Kd 0.7 µM, 1 mM injectate) and then loaded with acridine orange. However, because Rhod Dextran is a much dimmer dye under basal conditions than Lysotracker Red, acridine orange was used at the lower concentration of 1 µM to reduce bleed-through into the necessarily more sensitive red channel. Qualitatively similar results were seen at both 1 and 10 µM acridine orange when eggs were fertilized, but the fluorescence change was smaller with 1 µM.

Simultaneous Measurement of pHL and Exocytosis—Exocytosis was assessed with two different fluorescent techniques. First, by monitoring the disappearance of cortical granules; acridine orange monomers aggregate in acidic vesicles as a function of pHL, and consequently their fluorescence undergoes a red shift as a function of aggregation (10, 11). When excited with the red laser, a selective staining of a narrow (1-2 µm) peripheral ring is observed that corresponds to the acidic cortical granules docked at the plasma membrane (12). The eggs were therefore labeled as before with 10 µM acridine orange for 15-20 min, but this time they were simultaneously viewed in the green and red channels using the Multitrack mode of the microscope (emission/excitation, 488 nm/505-530 nm (green); 543 nm/>560 nm (red)). Note that in the previous ratiometric experiments, the Lysotracker Red signal swamped the dimmer acridine orange red fluorescence.

We used another approach to validate the loss of red acridine orange fluorescence; the extracellular space was also fluorescently labeled with a long wavelength, cell-impermeant dye (10 µM Alexa Fluor 647 Dextran), whose signal was simultaneously collected with the red (cortical granule) and green (pHL) channels as indicated above. At high magnification (63x objective, 2x digital zoom), the exocytosis of individual cortical granules observed with the red acridine orange fluorescence was confirmed when the space vacated by the granule filled with extracellular dye (data not shown), as originally developed by Terasaki (13). The data were analyzed by using Student's t test, paired where appropriate, or by creating a 2 x 2 contingency table analyzed with Fisher's exact test. The significance was set at p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Fertilization-induced pHL Changes—Sperm activate eggs and evoke Ca2+ release from acidic stores via NAADP (5). Measuring pHL changes within these acidic Ca2+ stores revealed that fertilization altered the pHL after a short lag (Fig. 1). The acridine orange signal changed substantially more than did Lysotracker Red (which is less pHL-sensitive, see "Experimental Procedures"), consistent with an alteration in pHL rather than a morphological artifact (Fig. 1, B and C). Fig. 1D shows the calculated ratio, and unexpectedly, this was not a spatially homogeneous response; around the periphery (cortex) of the egg, pHL promptly increased in a narrow band, whereas in the center of the egg pHL decreased, albeit with slower kinetics (Fig. 1, D and F). The transient and spatially restricted nature of the pHL increase was highly reminiscent of the [Ca2+]i cortical flash (4, 5), for which reason we have termed this novel phenomenon a "pHLASH" (pronounced "flash"). This peripheral vesicular alkalinization did not reflect an apparent unresponsiveness of the egg center because NH4Cl evoked a profound alkalinization across the entire cell (Fig. 1, A, C, and F). The results were confirmed using the chemically and spectrally dissimilar ratiometric dye, Lysosensor Yellow/Blue DND-160, albeit with an inferior signal-to-noise (see supplemental Fig. S1). Fertilization therefore induces changes in organellar pH that are highly organized in time and space.


Figure 1
View larger version (37K):
[in this window]
[in a new window]

  		FIGURE 1.
Fertilization-induced changes in pHL in intact sea urchin eggs. The eggs were co-loaded 10 µM acridine orange (AO) and 1 µM Lysotracker Red (LTR) and fertilized with 0.1% (v/v) ejaculate where indicated. All of the images shown were captured using a 10x objective. A, pseudocolor images of the ratio of acridine orange/Lysotracker Red fluorescence where cool colors represent low pHL, and warmer colors represent higher pHL. The time in seconds is shown in the upper left corner of each image. B, raw fluorescence of acridine orange (solid traces) or Lysotracker Red (broken traces) taken from the egg periphery in accordance with the inset schematic of a single egg. Sperm added when indicated followed by 10 mM NH4Cl (C) as for B except that the raw data is taken from the egg center. D, peripheral (red) and central (green) ratiometric (acridine orange/Lysotracker Red) pHL changes derived from the traces plotted in B and C. The broken portion of the red trace corresponds to the artifactual ratio because of the loss of fluorescence from the peripheral ROI as the fertilized egg apparently changes shape. E, the ratiometric pHL response proceeds as a wave around the egg cortex. The traces correspond to their color-matched ROIs in the inset for the same egg in B and C. F, bar graph showing the change in ratio in the egg periphery (Peri) and center (means ± S.E. of 77 eggs).

 
In cells fortuitously inseminated in the right orientation, this pHLASH proceeded as an alkalinization "wave" around the cell cortex (Fig. 1E). It is worth noting that in many eggs, the exocytotic lifting of the fertilization envelope results in such a dramatic change in cell shape/focus that a parallel fall in the fluorescence of both dyes occurs that is unrelated to changes in pHL and is depicted as a broken line in the ratio plot (Fig. 1D). (Note that this also accounts for the artifactual differences in the ratiometric NH4Cl response in the periphery versus the center; Fig. 1F.)

When measuring pHL alone, cell-to-cell asynchrony (Fig. 1A) made it difficult to gauge how this pHLASH related temporally with the fertilization Ca2+ response, i.e. would the pHLASH coincide with the Ca2+ cortical flash (Ca2+ influx) or the main Ca2+ wave (intracellular Ca2+ release)? Therefore, we simultaneously measured pHL and [Ca2+]i using acridine orange and the Ca2+ dye Rhod Dextran. In these dual labeling experiments, the first detectable insemination response was the Ca2+ cortical flash (Fig. 2A, left panels), followed after a delay by the main Ca2+ wave (Fig. 2).

Temporally, the pHLASH overlapped with the main Ca2+ wave (Fig. 2, A and B), with the pHL upstroke occurring ostensibly with or just after the sharp [Ca2+]i upstroke; the bar graph in Fig. 2C shows that the lag between sperm addition and the cortical flash is <30 s, whereas the main Ca2+ wave and pHLASH both peak at ~65 s. In other words, despite their spatial overlap, the Ca2+ cortical flash and pHLASH are temporally divorced; the latter coincides more with the main wave. This timing is consistent with the pHLASH being associated with the intracellular Ca2+ release phase.

Spatially, the Ca2+ wave and pHLASH wave initiated from the same pole in 7/10 cells (Fisher's exact test, p < 0.02) and propagated through the egg periphery to the antipode (Fig. 2B). Despite a heterogeneous pHL response, the main Ca2+ wave amplitude was the same in the periphery and center (data not shown). Because the Ca2+ wave initiates at the point of sperm entry (2), we conclude that the pHL wave also starts at the point of sperm fusion.


Figure 2
View larger version (35K):
[in this window]
[in a new window]

  		FIGURE 2.
Simultaneous measurement of Ca2+ and pHL changes upon fertilization of intact sea urchin eggs. The eggs were microinjected with rhod dextran, visualized with a 40x objective (3-µm optical slice) and loaded with 1 µM acridine orange and inseminated with fresh sperm (0.1% ejaculate, v/v). A, pseudocolored, self-ratio images of Ca2+ (left-hand panel) and pHL (right-hand panel), with time indicated in seconds, where 108 s represents the first detectable Ca2+ cortical flash. Of 10 eggs which showed equatorial, single initiation site Ca2+ waves, 7 eggs initiated their pHL waves from the same site. B, equivalent fluorescence records from the peripheral regions of interest numbered 1-3 in A corresponding to the initiation, midpoint, and antipode, respectively. Upper Ca2+ and lower pHL traces were normalized to the maximum response in each region and illustrate the progress of the waves through the cortex. C, kinetics of the timing of responses expressed as the delay between sperm addition and either the peak of the cortical flash and main Ca2+ wave or the maximum peripheral pHLASH. These data were calculated from the fluorescence throughout the entire egg periphery and expressed as the means ± S.E. of 21 eggs.

 
pHL Events and Exocytosis—However, it was possible that the pHL change is secondary to Ca2+-stimulated exocytosis (and/or endocytosis), e.g. upon vesicle fusion with the plasma membrane, the alkaline extracellular medium exchanges with the acidic lumen of cortical granules, the primary egg exocytotic vesicles (12, 14). Therefore, we simultaneously recorded pHL changes and exocytosis to see whether there was any spatial or temporal overlap. Exocytosis was monitored as the disappearance of cortical granule labeling (12).

Measuring the fluorescence at the periphery, fertilization induced a prompt fall in the red fluorescence, which corresponded to the loss of cortical granules via exocytosis (Fig. 3, A and B). By altering the focus, we verified that this was a true loss of staining rather than egg movement in the focal plane (data not shown). The initiation of the pHLASH and the loss of cortical granules were, on average, simultaneous (Fig. 3A) with a lag of only 5 ± 3 s, which was not significant (n = 34, p > 0.05). However, this obscured the fact that the pHLASH and exocytosis were seldom coincident on an individual egg basis, and plotting the initiation times revealed that there was no significant correlation between these two parameters (Fig. 3B, r2 = 0.003, p > 0.7). These data indicate that the pHLASH and exocytosis can be divorced kinetically.

In addition to their different kinetics, the pHLASH and exocytosis could be spatially distinguished. Fig. 3C shows a profile plot across the cell as indicated in Fig. 3A (upper left) and shows the mean fluorescence changes in the green and red channels after fertilization (Note that the red signal has been inverted for clarity); whereas the red changes were limited to the narrow outer cortical granule layer, the pHLASH occurred in a broader band (up to 25 µm away from the plasma membrane at low magnification).

The final piece of evidence to discount exocytosis as a causal factor comes from experiments with the wasp venom mastoparan. Known to stimulate fertilization envelope formation in sea urchin eggs (15), mastoparan is thought to do so independently of changes in [Ca2+]i (16), presumably by interacting with the exocytotic protein machinery itself. Indeed, although mastoparan readily evoked cortical granule loss (76 ± 3% of sperm control, n = 37), it did so without any detectable pHLASH (—4 ± 6% sperm, p < 0.001; Fig. 3D). Moreover, the addition of sperm after mastoparan elicited no response compared with parallel fertilization controls (—8 ± 1%, p < 0.001). This is consistent with a physical barrier (i.e. the fertilization envelope) being induced by the venom.

The fact that exocytosis and pHLASH can be divorced under a variety of circumstances strongly supports an independent mechanism for the latter. Because it is well known that endocytosis takes place many minutes after the exocytotic burst (12, 17), we conclude that neither of these events is responsible for the pHLASH.

Unitary pHL Events—Not only did the higher resolution images confirm that the pHLASH occurred well away from the fusion with the plasma membrane, but pHLASH was revealed as a summation of smaller events (which we term "pHLARES," pronounced "flares"). When focused at the egg equator, pHLARES were seen to propagate as a wave along a 15-µm-wide cortical band (Fig. 4A). When plotted as a function of distance from the plasma membrane, both the rate of rise and amplitude of these events were well maintained up to ~15 µm from the cortical granules (Fig. 4A, red to yellow ROIs), but both parameters fell off dramatically at deeper loci.


Figure 3
View larger version (29K):
[in this window]
[in a new window]

  		FIGURE 3.
Comparison of pHL and exocytosis during stimulation by sperm or mastoparan. Sea urchin eggs labeled with 10 µM acridine orange were viewed under green (pHL) and red (cortical granule, CG) fluorescence. A, sequence of images of both channels showing the raw green acridine orange fluorescence (upper left), and pHLASH (a pseudo-colored self-ratio) and red cortical granule exocytosis, at times indicated in seconds (sperm added 58 s). The traces in A depict the mean peripheral fluorescence changes of a single egg. B, correlation plot of the initiation times of pHLASH versus exocytosis with the line of best fit. C, profile plot along the equatorial line drawn on the cell in the upper left of A. Maximum fluorescence changes (mean of two or three consecutive images) were calculated as the difference between basal and fertilized eggs and normalized to their maximum values (pHL, green; cortical granules, red). D, effect of 200 µM mastoparan (and subsequent sperm and 10 mM NH4Cl) upon pHL (green trace) and cortical granule exocytosis (red trace). The results are representative of 34-55 eggs.

 


Figure 4
View larger version (32K):
[in this window]
[in a new window]

  		FIGURE 4.
Sperm-induced pHLASH unitary events monitored at two focal planes. Shown is a series of high magnification images focused at either the equator of the egg (A) or at the bottom of the egg viewed side by side (B) monitoring pHL changes with 10 µM acridine orange. Time(s) of each image is indicated in white. Sperm were added at 56 s (A) or 60 s (B). The traces are fluorescence records underlying the color-matched squares indicated in the first panel of the series. The images for A and B are depicted at the same magnification. The results are representative of four eggs/condition.

 
Discrete pHLARES were more clearly observed when focused along the bottom of the egg in the plane of the egg cortex. Viewed en face (Fig. 4B), individual pHLARES with a ~2-µm diameter were more clearly resolved (Fig. 4B, white arrows) presumably because of reduced scatter and turbidity at the egg-substratum interface. Unlike the equatorial slice, the bottom of the egg shows pHLARES firing across the entire field of view that were invariant with distance from the cell edge (Fig. 4B, cf. red and black ROIs). Together, the data indicate that pHL changes can be resolved as small events that are spatially divorced from exocytosis.

pHL and Cytosolic pH—In a previous egg population study, the slow vesicular acidification upon fertilization was secondary to changes in cytosolic pH driven by the plasma membrane sodium-proton exchanger (NHE) (10). Therefore, we tested whether the NHE was also involved in the subplasmalemmal pHLASH by inhibiting the NHE with Na+-free medium. However, this had no effect upon the pHLASH amplitude (Fig. 5, p > 0.1), pHLASH kinetics (time to peak in s: control, 67 ± 2; Na+-free, 69 ± 2; p > 0.4), or fertilization envelope formation (data not shown). Nonetheless, it entirely blocked the acidification of granules deeper in the egg (Fig. 5, B and C, p < 0.001) in agreement with previous work (10). We conclude that the cortical pHL response was not secondary to NHE-driven changes in cytosolic pH.

NAADP-induced pHLash—It was unexpected that fertilization could evoke vesicular alkalinization in such a restricted subcellular locus when acidic vesicles are found throughout the egg (7, 10). Given that fertilization is NAADP-dependent, and NAADP is the only Ca2+-mobilizing second messenger that evokes an increase in pHL (8), we microinjected eggs with NAADP to map the spatial distribution of NAADP-sensitive acidic Ca2+ stores that respond with a change in pHL.

In control eggs microinjected with a fluorescent injection marker alone, no substantial change of the pHL (Fig. 6, A and C) or Ca2+ (Fig. 6, D and F) was observed in any part of the egg. By contrast, when NAADP was co-injected into the center of the egg (as judged by the fluorescent marker, left hand images), a rapid and profound alkalinization was evoked in the characteristic peripheral pattern of the pHLASH (Fig. 6, B and C). This was not due to a heterogeneous Ca2+ response because NAADP evoked a global increase (Fig. 6, E and F). In other words, the pHLASH response seen with sperm is entirely consistent with the intrinsic nature of a subpopulation of NAADP-sensitive stores rather than upstream signal compartmentation.

Closer examination revealed that the pHL response to NAADP exhibited aspects that were both similar to as well as different from those induced by sperm. The spatial aspects of the pHLASH were clearly well preserved and sometimes proceeded in a quasi-wave-like manner akin to fertilization (data not shown). The quantitative differences we observed between NAADP- and sperm-induced responses suggest that NAADP generation is limiting during fertilization; the onset of the NAADP-induced response occurred after much a shorter delay compared with that to sperm (5.0 ± 0.5 s after NAADP injection versus ~35 s delay after insemination; Fig. 2C) and was of a much faster upstroke (time to peak, seconds: sperm, 31 ± 1; NAADP, 7.6 ± 0.5). The slower fertilization pHLASH kinetics are entirely consistent with the time taken for the egg to generate NAADP. In summary, NAADP alone is sufficient to generate a pHLASH because of the resident cellular architecture.


Figure 5
View larger version (14K):
[in this window]
[in a new window]

  		FIGURE 5.
Effect of removal of extracellular sodium upon fertilization-induced pHL responses in intact eggs. Eggs co-loaded with acridine orange and Lysotracker Red were then washed in ASW with (A) or without Na+ (B) immediately before fertilization. Red traces depict the peripheral pHL, and green traces show the central (broken parts of the trace coincide with artifactual ratios (see legend to Fig. 1 for details). C, data expressed as the means ± S.E. of 56-74 eggs. The peripheral (Peri) responses equate to the maximal rapid pHL increase, the central responses to the slower maximal decrease.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The release of Ca2+ from internal stores has long been recognized as the primary drive for the fertilization-induced Ca2+ signal (2) as well as other mammalian processes (1), but the involvement of non-endoplasmic reticulum Ca2+ stores was underscored when NAADP was shown to evoke Ca2+ release from acidic, lysosome-related organelles in both sea urchin egg and mammalian cells (1, 7). In sea urchin egg, NAADP-induced Ca2+ release seems to contribute to all phases of the fertilization Ca2+ spike from the initial cortical flash through to the falling phase of the main Ca2+ transient (5). Recently we demonstrated that the pHL of these acidic Ca2+ stores is increased by NAADP in sea urchin egg homogenate by a mechanism coupled to Ca2+ release via the NAADP receptor on acidic Ca2+ stores (8). Interestingly, this pHL change is not secondary to an increase in [Ca2+] at the cytosolic face, nor can it be mimicked by Ca2+ release from the endoplasmic reticulum but rather is a direct effect of NAADP upon the vesicles themselves (8). However, this is not at the level of the H+-ATPase or the vesicle membrane potential but may be due to changes in luminal H+ buffering (8). In view of this, we have investigated NAADP-dependent pHL events under physiological conditions, i.e. at fertilization.

The pHL of acidic vesicles in unfertilized eggs has been reported to be approximately pH 5.5 for both cortical granules (14) and yolk platelets (18), and it has been proposed that the pH within these vesicles serves to regulate luminal enzyme activity (14, 19-21) and/or vesicular integrity (18). That the pHL within egg acidic vesicles can slowly acidify has been suggested during oogenesis (11, 20, 21) and post-fertilization (10), but with the exception of the rapid alkalinization of cortical granules upon fusion and exocytosis (12, 14), rapid increases in the pHL of acidic vesicles have not been reported.

Corroborating our egg homogenate studies (8), the luminal pH of acidic organelles in intact eggs is coupled to both fertilization and NAADP-induced Ca2+ release with a prompt alkalinization. We would argue that our assays of pHL are reliable because similar results were ratiometrically recorded with both acridine orange, which has been well characterized in this system (8, 10), as well as with Lysosensor Yellow/Blue DND-160. Importantly, pHL changes were clearly independent of exocytosis/endocytosis and cytosolic pH changes. Moreover, acridine orange is unlikely to be responding to the oxidative burst at fertilization because this initiates much later, 2-3 min after insemination (19, 22-25), and direct addition of H2O2 (10 µM to 1 mM) to eggs did not induce a pHLASH (data not shown). Taken together, our data strongly support an alkalinization of cortical vesicles.

Spatial Characterization of the pHL Response—Fertilization evoked a spatially heterogeneous change in organellar pH, with a rapid, cortical alkalinization followed by a central, slow acidification. Only the latter was previously detected in asynchronous populations of eggs (10), hardly surprising when the transient pHLASH only extends 15-25 µm into the cortex (in an egg ~120 µm in diameter). Moreover, alkalinization propagated as a wave around the egg cortex at ~5 µm/s, a velocity similar to the main [Ca2+]i wave and the exocytotic lifting of the fertilization envelope (26, 27). At higher magnification, the alkalinization wave was manifest as the summation of smaller events (pHLARES, 2-3 µm in diameter) that appear to represent alkalinization at the individual vesicle level.


Figure 6
View larger version (36K):
[in this window]
[in a new window]

  		FIGURE 6.
Effect of NAADP upon pHL and [Ca2+]i in intact eggs. Acridine orange/Lysotracker Red was used to monitor pHL changes (A-C) with injectate containing 200 µM Alexa Fluor 647 Dextran alone (A) or together with 100 µM NAADP (B). To measure [Ca2+]i changes, unloaded eggs were injected with fluo-4 dextran prior to a second injection with or without NAADP (D-F). Images (A, B, D, and E) show the time in seconds in the upper left corners. The left-hand image of each trio shows the raw fluorescence of the location (and time) of Alexa Fluor 647 Dextran injection (bright center), along with the peripheral (Peri) (red) and central (green) ROIs used to derive the fluorescence traces. The central image shows a preinjection basal acridine orange/Lysotracker Red ratio (A and B) or F/F0 image (D and E), whereas the right-hand image depicts the ratio image at the peak of each response. Broken portions of the traces in B and E correspond to the artifactual fall because of the change in cell shape. Summary of maximal peak responses to injection measuring pHL (C) or Ca2+ (F). The data are the means ± S.E. of 12-16 eggs for pHL and for 3 (Control) and 9 (NAADP) eggs for Ca2+.

 


Figure 7
View larger version (34K):
[in this window]
[in a new window]

  		FIGURE 7.
Working model of pHL changes in cortical acidic Ca2+ stores at fertilization. The upper images show pHL and Ca2+ changes corresponding to the lower schematics of eggs and cortical acidic Ca2+ stores. For the upper images, blue represents basal levels, whereas warmer colors reflect increases in either pHL or Ca2+. Similarly, in the egg schematic a rainbow scheme reflects the concentration of Ca2+. Cortical granules and acidic vesicles are red when acidic, and green when alkalinized during the pHLASH. The red arrows correspond to Ca2+ influx across the plasma membrane (the cortical flash) or release from acidic stores through NAADP receptors (NAADP R).

 
From which vesicles these pHLARES emanate is presently unclear. From electron micrographs the 1-3-µm diameter yolk platelets (20, 28, 29) and other candidate "acidic vesicles" (28) correlate well with the size of a pHLARES, particularly if one allows for diffusion of acridine orange lost from the vesicle upon alkalinization. Deeper exocytotic cortical vesicle classes have been described (30) and the so-called "basal laminar" and "apical" vesicles are both in the right place to host the pHLARES. Defining the vesicle population will be crucial to understanding the ramifications of this novel phenomenon.

Why the fertilization pHLASH is restricted to the egg cortex is not entirely clear but might reflect either localized messenger accumulation or a unique property of the acidic stores themselves. Our data are more consistent with the latter because global NAADP microinjection evoked a local pHLASH, and it is known that the cortical Ca2+ stores are the most sensitive to NAADP (31, 32). Nonetheless, we cannot formally exclude local messenger production as a contributory factor at fertilization.

Temporal Characterization of the pHLash—Despite the spatial similarity of the pHLASH and Ca2+ cortical flash, there was no temporal overlap, and the pHLASH occurred with or just after the main Ca2+ wave, ~35 s later. However, this timing makes sense because it coincides with NAADP-induced Ca2+ release from intracellular stores (5). Indeed, both the pHLASH and main Ca2+ response propagate as waves, which is additional evidence for their being interrelated phenomena. Note that the difference in the upstroke kinetics of the Ca2+ and pHL changes in intact eggs is consistent with those observed in homogenate (8).

We were concerned that the pHLASH may be related to Ca2+-stimulated exocytosis, which occurs 6-8 s after the main [Ca2+]i rise in urchin eggs (13, 33) and which alkalinizes cortical granules upon plasmalemmal fusion (12, 14). Our conclusion is that the pHLASH is not due to exocytosis based on the following evidence: (a) Deeper pHL events are too remote to be due to fusion with the plasma membrane, although it might be argued that these deeper pHLARES artifactually represents the "tail" of exocytotic events emanating from out of the plane of focus, recording from thin optical slices (2-3% of the egg diameter) at the egg equator means that the curvature of the plasma membrane is minimal, and out-of-focus cortical granules will not overlap this in-focus region. In addition, the amplitude and upstroke kinetics of the pHLASH are well maintained over 15-25 µm, which is inconsistent with the tail of out-of-focus events, which would be blunted; (b) Exocytosis and pHL changes can be divorced, at the level of individual eggs, as well as by mastoparan, which stimulated cortical granule exocytosis but no pHLASH. Therefore, the approximate post-fertilization sequence of events is Ca2+ cortical flash >> main Ca2+ wave {approx} pHLASH > exocytosis (Fig. 7).

Not only do we describe a novel fertilization pHL event, but we also offer a mechanism whereby sperm elevate NAADP levels in the egg (5), which in turn raise pHL via activation of the Ca2+-mobilizing NAADP receptor on acidic vesicles (8) (Fig. 7). Moreover, we reveal that the unique spatial profile of the pHLASH reflects the intrinsic nature of the cortical stores themselves rather than some upstream mechanism. We also conclude that vesicle alkalinization is independent of changes in cytosolic Ca2+, vesicular membrane potential, H+-ATPase activity (8), cytosolic pH, and exocytosis and appears to be at the level of NAADP-induced changes in H+ buffering (8).

The importance of the egg cortex for egg function is underscored by many cell biological aspects (reviewed in Ref. 34). In addition to supporting layers of exocytotic vesicle populations (30), it also hosts the [Ca2+]i cortical flash (4, 5), a unique tubular endoplasmic reticulum morphology with an exclusive complement of luminal Ca2+-binding proteins (35, 36), as well as a greater density of ryanodine receptors (37). To this and other properties, we add the fertilization-dependent alkalinization of NAADP-sensitive acidic Ca2+ stores. The regulation of organelle pH by activation of the NAADP receptor on the vesicle membrane poses exciting questions relating not just to sea urchin biology but also to mammalian biology. Certainly, key enzymes in the lumen of egg vesicles are regulated by pH changes at fertilization (14, 38), but in the acidic organelles of mammalian cells, luminal pH influences processes as diverse as secretory vesicle fusion (39), autophagy (40), and proteolysis (41). Our results may have far-reaching implications for NAADP regulating various cellular pathways via pH and not just by Ca2+.


   FOOTNOTES
 
* This work was supported by a senior fellowship from the Wellcome Trust (to A. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1. Back

1 To whom correspondence should be addressed: Dept. of Pharmacology, University of Oxford, Mansfield Rd., Oxford OX1 3QT, UK. Tel.: 44-1865-271606; Fax: 44-1865-271853; E-mail: Anthony.morgan{at}pharm.ox.ac.uk.

2 The abbreviations used are: NAADP, nicotinic acid adenine dinucleotide phosphate; ASW, artificial sea water; pHL, luminal pH; pHLASH, cortical pHL response; pHLARES, elemental pHL events; ROI, region of interest; NHE, sodium-proton exchanger. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Katja Rietdorf and Dr. Grant Churchill and members of his laboratory (University of Oxford) for invaluable suggestions during the preparation of this manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Galione, A., and Ruas, M. (2005) Cell Calcium 38, 273-280[CrossRef][Medline] [Order article via Infotrieve]
  2. Whitaker, M. (2006) Physiol. Rev. 86, 25-88[Abstract/Free Full Text]
  3. Stricker, S. A. (1999) Dev. Biol. 211, 157-176[CrossRef][Medline] [Order article via Infotrieve]
  4. Lim, D., Kyozuka, K., Gragnaniello, G., Carafoli, E., and Santella, L. (2001) FASEB J. 15, 2257-2267[Abstract/Free Full Text]
  5. Churchill, G. C., O'Neill, J. S., Masgrau, R., Patel, S., Thomas, J. M., Genazzani, A. A., and Galione, A. (2003) Curr. Biol. 13, 125-128[CrossRef][Medline] [Order article via Infotrieve]
  6. Leckie, C., Empson, R., Becchetti, A., Thomas, J., Galione, A., and Whitaker, M. (2003) J. Biol. Chem. 278, 12247-12254[Abstract/Free Full Text]
  7. Churchill, G. C., Okada, Y., Thomas, J. M., Armando, A., Genazzani, Patel, S., and Galione, A. (2002) Cell 111, 703-708[CrossRef][Medline] [Order article via Infotrieve]
  8. Morgan, A. J., and Galione, A. (2007) Biochem. J. 402, 301-310[CrossRef][Medline] [Order article via Infotrieve]
  9. Morgan, A. J., Churchill, G. C., Masgrau, R., Ruas, M., Davis, L. C., Billington, R. A., Patel, S., Yamasaki, M., Thomas, J. M., Genazzani, A. A., and Galione, A. (2006) in Methods in Calcium Signalling (Putney, J. W., Jr., ed) 2nd Ed., pp. 265-334 CRC Press, Boca Raton, FL
  10. Lee, H. C., and Epel, D. (1983) Dev. Biol. 98, 446-454[CrossRef][Medline] [Order article via Infotrieve]
  11. Fagotto, F., and Maxfield, F. R. (1994) J. Cell Sci. 107, 3325-3337[Abstract]
  12. Smith, R. M., Baibakov, B., Lambert, N. A., and Vogel, S. S. (2002) Traffic 3, 397-406[CrossRef][Medline] [Order article via Infotrieve]
  13. Terasaki, M. (1995) J. Cell Sci. 108, 2293-2300[Abstract]
  14. Haley, S. A., and Wessel, G. M. (2004) Mol. Biol. Cell 15, 2084-2092[Abstract/Free Full Text]
  15. Saito, K., Imoto, M., Nagano, N., Toriyama, M., and Nakajima, T. (1995) Biochem. Biophys. Res. Commun. 215, 828-834[CrossRef][Medline] [Order article via Infotrieve]
  16. Lopez-Godinez, J., Garambullo, T. I., Martinez-Cadena, G., and Garcia-Soto, J. (2003) Biochem. Biophys. Res. Commun. 301, 13-16[CrossRef][Medline] [Order article via Infotrieve]
  17. Whalley, T., Terasaki, M., Cho, M. S., and Vogel, S. S. (1995) J. Cell Biol. 131, 1183-1192[Abstract/Free Full Text]
  18. Fagotto, F., and Maxfield, F. R. (1994) J. Cell Biol. 125, 1047-1056[Abstract/Free Full Text]
  19. Shapiro, B. M. (1991) Science 252, 533-536[Abstract/Free Full Text]
  20. Mallya, S. K., Partin, J. S., Valdizan, M. C., and Lennarz, W. J. (1992) J. Cell Biol. 117, 1211-1221[Abstract/Free Full Text]
  21. Song, J. L., Wong, J. L., and Wessel, G. M. (2006) Dev. Biol. 300, 385-405[CrossRef][Medline] [Order article via Infotrieve]
  22. Schomer, B., and Epel, D. (1998) Dev. Biol. 203, 1-11[CrossRef][Medline] [Order article via Infotrieve]
  23. Foerder, C. A., Klebanoff, S. J., and Shapiro, B. M. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 3183-3187[Abstract/Free Full Text]
  24. Heinecke, J. W., and Shapiro, B. M. (1992) J. Biol. Chem. 267, 7959-7962[Free Full Text]
  25. Wong, J. L., Creton, R., and Wessel, G. M. (2004) Dev. Cell 7, 801-814[CrossRef][Medline] [Order article via Infotrieve]
  26. Swann, K., and Whitaker, M. (1986) J. Cell Biol. 103, 2333-2342[Abstract/Free Full Text]
  27. Mohri, T., and Hamaguchi, Y. (1990) Cell Struct. Funct. 15, 309-315[Medline] [Order article via Infotrieve]
  28. Fishkind, D. J., Bonder, E. M., and Begg, D. A. (1990) Dev. Biol. 142, 439-452[CrossRef][Medline] [Order article via Infotrieve]
  29. Wessel, G. M., Zaydfudim, V., Hsu, Y. J., Laidlaw, M., and Brooks, J. M. (2000) Dev. Growth Differ. 42, 507-517[CrossRef][Medline] [Order article via Infotrieve]
  30. Matese, J. C., Black, S., and McClay, D. R. (1997) Dev. Biol. 186, 16-26[Medline] [Order article via Infotrieve]
  31. Churchill, G. C., and Galione, A. (2000) J. Biol. Chem. 275, 38687-38692[Abstract/Free Full Text]
  32. Churchill, G. C., and Galione, A. (2001) J. Biol. Chem. 276, 11223-11225[Abstract/Free Full Text]
  33. Matese, J. C., and McClay, D. R. (1998) Zygote 6, 55-64, 65a[Medline] [Order article via Infotrieve]
  34. Sardet, C., Prodon, F., Dumollard, R., Chang, P., and Chenevert, J. (2002) Dev. Biol. 241, 1-23[CrossRef][Medline] [Order article via Infotrieve]
  35. Terasaki, M., and Jaffe, L. A. (1991) J. Cell Biol. 114, 929-940[Abstract/Free Full Text]
  36. Papp, S., Dziak, E., Michalak, M., and Opas, M. (2003) J. Cell Biol. 160, 475-479[Abstract/Free Full Text]
  37. McPherson, S. M., McPherson, P. S., Mathews, L., Campbell, K. P., and Longo, F. J. (1992) J. Cell Biol. 116, 1111-1121[Abstract/Free Full Text]
  38. Deits, T., and Shapiro, B. M. (1985) J. Biol. Chem. 260, 7882-7888[Abstract/Free Full Text]
  39. Renstrom, E., Ivarsson, R., and Shears, S. B. (2002) J. Biol. Chem. 277, 26717-26720[Abstract/Free Full Text]
  40. Kawai, A., Uchiyama, H., Takano, S., Nakamura, N., and Ohkuma, S. (2007) Autophagy 3, 154-157[Medline] [Order article via Infotrieve]
  41. Busch, G. L., Lang, H. J., and Lang, F. (1996) Pfluegers Arch. Eur. J. Physiol. 431, 690-696[Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 DevelopmentHome page
J. L. Wong and G. M. Wessel
Extracellular matrix modifications at fertilization: regulation of dityrosine crosslinking by transamidation
Development, June 1, 2009; 136(11): 1835 - 1847.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
282/52/37730    most recent
M704630200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Morgan, A. J.
Right arrow Articles by Galione, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Morgan, A. J.
Right arrow Articles by Galione, A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2007 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement