1 BioMarCell, Unité de Biologie du Développement UMR 7009
CNRS/Paris VI, Observatoire, Station Zoologique, Villefranche sur Mer, 06230
France
2 National Vibrating Probe Facility, Marine Biological Laboratory, Woods Hole,
MA 02543-1015, USA
3 Laboratoire de Biologie du Développement, Institut Jacques Monod, CNRS,
Universités Paris 6 et Paris 7, 2, place Jussieu, F-75251 Paris,
France
* Present address: Department of Physiology, University College London, Gower
Street, London WC1E 6BT, UK
Author for correspondence (e-mail:
r.dumollard{at}ucl.ac.uk)
Accepted 15 November 2002
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SUMMARY |
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Key words: Fertilization, Respiration, Ca2+ waves, Mitochondria, Endoplasmic reticulum, ATP, Ca2+ wave pacemakers, Ascidian
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INTRODUCTION |
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Cellular Ca2+ homeostasis is the result of Ca2+
fluxes through the plasma membrane and the intracellular organelles. In every
egg studied so far, an extensive and continuous ER network plays a major role
in the sperm-triggered Ca2+ waves (reviewed by
Sardet et al., 1998;
Dumollard et al., 2002
). The
network hosts Ca2+ channels [Ins(1,4,5)P3
receptors (IP3R)], which mediate intracellular Ca2+
release, and Ca2+ pumps [sarco-endoplasmic reticulum
Ca2+ ATPases (SERCAs)]. Little is known about the role of
mitochondria in the regulation of sperm-triggered Ca2+ signals in
eggs. In sea urchins, mitochondria were suggested as a sink for
Ca2+ released during fertilization
(Eisen and Reynolds, 1985
;
Girard et al., 1991
). A role
for active mitochondria in the regulation of sperm triggered Ca2+
oscillations has been suggested in the mouse egg
(Liu et al., 2001
), but it
cannot be inferred from this study what the interplay is between mitochondria
and sperm-triggered Ca2+ oscillations.
In somatic cells, mitochondria can sequester and release Ca2+
during Ca2+ waves (Boitier et
al., 1999; Rizzuto et al.,
2000
; Hajnoczky et al.,
2000
). Mitochondrial Ca2+ sequestering generally
provides a negative feedback on Ins(1,4,5)P3-induced
Ca2+ release (IICR) (reviewed by
Rizzuto et al., 2000
;
Duchen, 2000
). Calcium is also
a pivotal `multisite' activator of oxidative phosphorylation in the
mitochondria (Hansford, 1994
).
Calcium activates the dehydrogenases of the Krebs cycle
(McCormack et al., 1990
) and
the electron transport chain (Gunter et
al., 1994
), and has a direct action on the F0-F1 ATP synthase
(Territo et al., 2000
). In
somatic cells, mitochondrial Ca2+ uptake can increase the ratio of
NADH/NAD+ (i.e. NAD+ is reduced to NADH) in the
mitochondria (Pralong et al.,
1994
; Hajnoczky et al.,
1995
) (reviewed by Duchen,
2000
) as well as oxygen consumption
(Duchen, 2000
). Theoretically,
mitochondrial ATP production can regulate IICR through ATP4-, an
allosteric regulator of IP3R opening
(Mak et al., 1999
;
Mak et al., 2001
). Therefore
active mitochondria may modulate Ca2+ signaling at two different
levels either via preferential Ca2+ fluxes (reviewed by
Duchen, 2000
;
Rizzuto et al., 2000
;
Hajnoczky et al., 2000
) or via
local ATP generation (Landolfi et al.,
1998
; Kennedy et al.,
1999
).
The first report of an increase in oxygen consumption after fertilization
was a study on sea urchins (Warburg,
1908), which led to the common belief that fertilization is also
accompanied by the metabolic activation of the egg. It is now recognized that
respiration in sea urchin eggs and embryos depends solely on electron
transport through the mitochondrial respiratory chain, except for a short
period after fertilization when secreted ovoperoxidases consume O2
to synthesize H2O2
(Perry and Epel, 1985
;
Heinecke and Shapiro, 1992
;
Yasumasu, 2000
). In contrast
to sea urchins and molluscs, this non-mitochondrial respiratory burst does not
occur in asteroid, echiuroid or ascidian eggs
(Schomer and Epel, 1998
).
Given the role of Ca2+ waves in somatic cells (reviewed by
Rizzuto et al., 2000;
Duchen, 2000
), sperm-triggered
Ca2+ waves may mediate the metabolic activation of the fertilized
egg, but this remains to be demonstrated. Fertilized ascidian eggs provide a
particularly favorable model to investigate the relationships between meiotic
Ca2+ waves and mitochondrial activity for several reasons.
(1) Fertilization does not involve a non-mitochondrial respiratory burst
(Schomer and Epel, 1998).
(2) Thousands of rod-shaped mitochondria form a dense 7 µm subcortical
layer lining the vegetal and equatorial regions of the egg
(Roegiers et al., 1995;
Roegiers et al., 1999
).
(3) Fertilization triggers two series of very stereotyped Ca2+
waves which are generated by two distinct Ca2+ wave pacemakers (PM1
and PM2) (Sardet et al., 1998;
Yoshida et al., 1998
;
Nixon et al., 2000
;
Dumollard and Sardet, 2001
).
Pacemakers PM1 and PM2 are located in the cortex of the egg (pacemaker PM2 is
at the vegetal pole of the egg) and they are known to rely mostly if not only
on a sperm-triggered Ins(1,4,5)P3 production
(McDougall and Sardet, 1995
;
Yoshida et al., 1998
;
Runft and Jaffe, 2000
).
(4) A third artificial pacemaker (PM3) can be generated opposite to
pacemaker PM2 (Dumollard and Sardet,
2001).
In this study, we have recorded two parameters of mitochondrial activity (oxygen consumption and mitochondrial NADH levels) during the period of activity of the Ca2+ wave pacemakers PM1, PM2 and PM3 of the ascidian egg. We have also examined the effects of mitochondrial inhibitors on the patterns of Ca2+ waves initiated by these three pacemakers. Our studies reveal that mitochondria are stimulated by cytosolic Ca2+ signals. In turn, the Ca2+ wave pacemaker PM2 strictly depends upon Ca2+ buffering and ATP production by mitochondria.
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MATERIALS AND METHODS |
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Microinjection
Unfertilized eggs were introduced into a wedge and injected as described
previously (McDougall and Sardet,
1995; Dumollard and Sardet,
2001
). The cytosolic dyes Calcium Green dextran (10 kDa, CG) and
Texas Red dextran (10 kDa, TR, Molecular Probes) as well as caged
Ins(1,4,5)P3 (cIP3, Calbiochem), caged
PtdIns(4,5)P2 (cgPIP2, Calbiochem) and NPE
caged ATP (cATP, Calbiochem) were dissolved in injection buffer consisting of
180 mM KCl, 100 µM EGTA, 30 mM BES pH 7.1. Approximately 1% of the egg
volume was injected to give final concentrations of 10-20 µM for CG and TR
dextran; 5 µM for cIP3; 35 µM for cgPIP2; and 1 mM
for cATP.
Photorelease of caged compounds and Ca2+ imaging
Injected oocytes were mounted in a perfusion chamber and imaged with a
Leica Wild Leitz CLSM or TCS SP2 confocal microscope
(McDougall and Sardet, 1995;
Dumollard and Sardet, 2001
).
For UV photorelease of cIP3, cgPIP2 or cATP oocytes were
co-injected with a mixture of CG and TR and the caged compound
(Dumollard and Sardet, 2001
).
UV flashes were produced on the Leica confocal microscope using
epifluorescence illumination (75 W mercury lamp). Some photorelease was also
performed on an inverted Zeiss microscope (Axiovert 100 TV) essentially as
described previously (Dumollard and Sardet,
2001
).
NADH imaging
NADH fluoresces at 470 nm with peak absorption at 356 nm
(Masters and Chance, 1993). A
470±20 nm bandpass filter (excitation: bandpass 360±10 nm,
Chroma Opticals) was used to collect images of intracellular NADH
concentration ([NADH]). The mitochondria-rich vegetal subcortex containing
mostly mitochondria (Roegiers et al.,
1995
; Roegiers et al.,
1999
) (Fig. 5A,B)
displayed a higher fluorescence (Fig.
2A). For each NADH image, the one pixel line running in the center
of the entire mitochondria-rich domain (lines ab and a'b' in
Fig. 2A) was extracted by image
analysis using Visilog software [details about the segmentation method can be
obtained by contacting C. C.
(cibert{at}ijm.jussieu.fr)].
To prevent motion artifacts, each extracted line of a NADH image (ab and
a'b' in Fig. 2A)
was divided by the same line extracted in an essentially NADH-insensitive
reference image [exc, bandpass 380± 10 nm; em, 470nm (Chroma Opticals)]
acquired quasi simultaneously with the NADH image. These divided extracted
lines (which are ratiometric measurements of [NADH]mito) were
stacked together to display the variations of [NADH]mito with time
(running from left to right in Fig.
2A `time image').
|
|
Measurement of oxygen fluxes with a self-referencing oxygen-sensitive
vibrating microelectrode
Oxygen fluxes in a single egg were measured using the oxygen-sensitive
self-referencing vibrating probe in the NVPF facility at the Marine Biological
Laboratory in Woods Hole (MA, USA) (Land
et al., 1999). The vibrating electrode was positioned within 5
µm of a denuded egg adhering to a coverslip in a petri dish
(Fig. 1B). For fertilization
experiments, activated sperm were added to the dish, while the egg was
observed using time-lapse recording. The presence of Ca2+ waves was
monitored by recording the wave of contraction that accompanies each
Ca2+ wave (Roegiers et al.,
1999
). When indicated, FCCP was added to give a final
concentration of 1 µM. In some experiments cIP3 was
photoreleased by UV illumination and [Ca2+]c was
monitored by recording the Calcium Green fluorescence using a Zeiss Atto Arc
imaging system while simultaneously recording the oxygen fluxes with the
oxygen probe.
|
Perfusion of mitochondrial inhibitors
The eggs were held under the microscope in a perfusion chamber and either
FCCP (1 µM in sea water, Sigma), cyanide anion (CN-; 2 mM in sea
water, Sigma) or oligomycin (100 µM in sea water, Sigma) perfused. Washing
of the inhibitors was carried out by performing several perfusions of sea
water. The perfusions lasted less than 1 minute. During that period no
recording could be made.
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RESULTS |
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To measure the oxygen fluxes around a single ascidian egg, we used an
oxygen-sensitive, self-referencing microelectrode
(Fig. 1B). This technique has
been successfully used to measure local oxygen consumption in living cells and
embryos (Land et al., 1999;
Trimarchi et al., 2000
). We
found that the basal oxygen consumption in the vegetal pole region of the
unfertilized ascidian egg was 38±10% higher than the oxygen consumption
measured near the animal pole region of the same egg (data not shown,
n=7). This is consistent with the presence of the bulk of
mitochondria in a subcortical location in the vegetal hemisphere of the egg
(see Fig. 5A).
Maximum oxygen consumption was measured by uncoupling mitochondria with the permeant protonophore FCCP that induces mitochondria to consume oxygen but also to hydrolyze ATP (see Fig. 3). Upon addition of 1 µM FCCP, the oxygen consumption of the egg increased more than six times in the example shown, reflecting the maximum rate of oxygen reduction by mitochondria (Fig. 1D). The graph shown in Fig. 1B represents a measurement made with the oxygen electrode positioned near the vegetal pole of the egg. Although there was some variability due to interspecimen differences or alternative positioning of the electrode, a general pattern of oxygen consumption was observed in the eight eggs examined (five Phallusia and three Ciona) (Fig. 1B). With the basal level of oxygen consumption before fertilization assigned a value of 100%, we observed that, on average, oxygen consumption rose during the activity of Ca2+ wave pacemaker PM1 to 167±18%, and then decreased during extrusion of polar body 1 to 140±15%. Oxygen consumption rose again to reach a maximum value during the period of Ca2+ wave pacemaker PM2 activity (173±14%). After the cessation of the meiotic Ca2+ waves, the oxygen consumption then stabilized to 126±11% (Fig. 1B).
|
To measure the impact of the rise in cytosolic Ca2+
concentration ([Ca2+]c) on oxygen consumption, we
artificially raised [Ca2+]c by photoreleasing injected
cIP3 with a global UV flash
(Dumollard and Sardet, 2001).
Increased oxygen consumption was associated with each Ca2+
transient caused by the photorelease of cIP3
(Fig. 1C). There was no
measurable delay between the onset of the Ca2+ transient and the
onset of the oxygen consumption transient measured with the vibrating
electrode. The decay of Ca2+ signal preceded the decrease in oxygen
consumption (Fig. 1C).
Calcium can directly or indirectly activate several mitochondrial
dehydrogenases of the Krebs cycle, thereby reducing the NAD+/NADH
pool (i.e. increasing the amount of reduced NADH and decreasing the oxidized
NAD+, Fig. 5D)
(Duchen, 2000;
Rizzuto et al., 2000
). As NADH
is endogenously fluorescent (Masters and
Chance, 1993
), we imaged NADH in a single ascidian egg
(Fig. 2).
The vegetal subcortex of the egg contains mostly mitochondria
(Fig. 5A,B) (Roegiers et al., 1995;
Roegiers et al., 1999
); we
therefore chose to measure the variations of [NADH] in this mitochondria-rich
domain to monitor the variations of [NADH]mito. This domain is
easily distinguished as a cortical crescent displaying higher NADH
fluorescence (Fig. 2A, see
Materials and Methods). As with oxygen consumption
(Fig. 1B), variations of
[NADH]mito measured in the middle of the mitochondria-rich region
(corresponding to the vegetal contraction pole)
(Roegiers et al., 1995
) showed
two main increases during the period of operation of the Ca2+ wave
pacemakers PM1 and PM2 (Fig.
2B). Blocking the reduction of oxygen by the electron-transport
chain with CN- led to an increase in [NADH]mito
(Fig. 2C). By contrast,
perfusion of the uncoupler FCCP, caused a decrease in [NADH]mito in
accordance with its presumed effect on the mitochondria (see Figs
3,
5). These observations imply
that there is a strong positive correlation between meiotic Ca2+
waves and the activation of mitochondrial respiration.
Mitochondrial depolarization induces a Ca2+ leak in
artificially activated or fertilized eggs
In order to examine the role of active mitochondria on the pattern,
frequency and amplitude of Ca2+ waves in the ascidian egg, we used
an array of mitochondrial inhibitors, which inhibit Ca2+ influx
into mitochondria. Energized mitochondria are characterized by an
electrochemical potential that is mainly powered by a pH gradient generated by
the electron transport chain (Fig.
3E). This electrochemical gradient (around -150 mV) is the driving
force for Ca2+ influx into the mitochondria via a Ca2+
uniporter (Gunter et al.,
1994; Duchen,
2000
). Calcium influx can be abolished by simply collapsing the pH
gradient across the inner mitochondrial membrane (with CN- or FCCP
see Fig. 3G,H). Perfusion of
unfertilized eggs with FCCP or CN- for 10-20 minutes caused only a
slight elevation of [Ca2+]c
(Fig. 3A, 0.03±0.02%,
n=6, Fig. 3D). By
contrast, when mitochondrial inhibitors were perfused after eggs have been
activated by cIP3 injection, they caused a long-lasting increase in
the resting [Ca2+]c
(Fig. 3B). Upon washout of
CN-, [Ca2+]c returned rapidly to its basal
level (Fig. 3B). The
FCCP-induced increase of resting [Ca2+]c was faster
(0.25±0.05%, n=7, Fig.
3D) than the CN--induced Ca2+ increase
(0.10±0.04%, n=9). Interestingly, when Ca2+
transients were generated by cIP3 photolysis, the continuous
presence of CN- or FCCP (for more than 10 minutes in the example
shown in Fig. 3B) apparently
did not affect the rate of the Ins(1,4,5)P3-mediated
Ca2+ rise or its decay (Fig.
3B is representative of 10 experiments).
The FCCP- or CN--induced Ca2+ increase described above could be inhibited by preincubating the egg in seawater containing 100 µM oligomycin (which blocks the F0-F1 ATP synthase) for 20 minutes (Fig. 3C,D). This strongly suggests that the FCCP/CN--induced Ca2+ increase was due to ATP depletion after the reversal of the mitochondrial ATP synthase when mitochondria were depolarized. The ATP depletion provoked by uncoupled mitochondria may well be responsible for an increased Ca2+ leak into the cytosol. Under these new net Ca2+ fluxes, the resting [Ca2+]c equilibrates to a new, slightly higher level (Fig. 3B, Fig. 4C,E). The resting [Ca2+]c did not increase further after 3 minutes of mitochondrial inhibitor perfusion, indicating that some Ca2+ extrusion from the cytosol could still operate (see Discussion).
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Inhibition of mitochondrial activity has different effects on the
three Ca2+ wave pacemakers
Fig. 4A,B,D illustrates the
differential sensitivity of the two physiological pacemakers (PM1 and PM2) and
of the artificial pacemaker PM3 (Dumollard
and Sardet, 2001) to FCCP perfusion. Calcium wave pacemaker PM1
triggered by sperm entry was not affected by the presence of FCCP before or
during fertilization (Fig. 4A,
n=3), while the artificial pacemaker PM3 created by global
photorelease of cgPIP2 in the unfertilized egg was only slightly
affected (Fig. 4D). We observed
a gradual decrease of peak [Ca2+]c and a gradual
increase of inter-spike [Ca2+]c about 4 minutes after
the perfusion of FCCP (n=4, Fig.
4D). In contrast, the continuous presence of FCCP inhibited the
pacemaker PM2 (n=3, Fig.
4A), which stopped emitting Ca2+ waves within seconds
after the perfusion of FCCP (n=4,
Fig. 4B). In addition,
perfusing FCCP after the meiotic Ca2+ waves had ceased induced a
transient elevation of [Ca2+]c superimposed to the slow
and sustained Ca2+ rise due to mitochondrial ATP hydrolysis
(Fig. 4C, n=3;
Fig. 4E, n=4).
Importantly, this transient rise in [Ca2+]c was
localized to the vegetal hemisphere of the egg where the subcortical
mitochondria-rich layer is located (data not shown). This FCCP-releasable
Ca2+ pool was not present in the unfertilized egg
(Fig. 3A) and it is different
from the FCCP- or CN--induced Ca2+ leak observed in eggs
activated by Ins(1,4,5)P3 or fertilized eggs. The most
likely interpretation is that this Ca2+ pool resides in
mitochondria that have sequestered and accumulated Ca2+ during the
passage of the multiple Ca2+ waves triggered by PM1 and PM2.
CN- had a similar effect to FCCP on the sperm-triggered pacemakers PM1 and PM2: pacemaker PM2 was inhibited, but PM1 was not perturbed (Fig. 4E, n=4; Fig. 4F, n=4). As with FCCP, PM2 activity recovered rapidly and completely upon washout of CN- (Fig. 4E,F). The similar effects of the two different mitochondrial inhibitors on the different pacemakers demonstrate that active mitochondria are necessary for the maintenance of Ca2+ wave pacemaker PM2 but that they only play minor roles in sustaining the activities of pacemakers PM1 or PM3.
In order to examine the role played by the decreased level of ATP in the
inhibition of pacemaker PM2, we used UV photolysis of NPE-caged ATP
(He et al., 1998). Fertilized
eggs were first exposed to CN- and then flashed with UV light to
increase [ATP]c globally. Increasing [ATP]c in
fertilized eggs perfused with CN- partially restored
Ca2+ wave pacemaker PM2 activity
(Fig. 4F). Global photorelease
of ATP caused Ca2+ waves to initiate in the contraction pole where
PM2 is located (data not shown). However recovery of PM2 activity was only
partial compared with the rapid and complete recovery observed after washout
of CN- (Fig. 4F).
These observations suggest that mitochondrial ATP hydrolysis and the resultant
decrease in [ATP]c accounts in part for the inhibition of the
Ca2+ wave pacemaker PM2 by mitochondrial inhibitors.
The role of mitochondrial Ca2+ influx on the activity of the Ca2+ wave pacemaker PM2 was then investigated by perfusing CN- on fertilized eggs in the presence of oligomycin. When incubated for 20 minutes in the presence of oligomycin, eggs were fertilized normally and the Ca2+ wave pacemakers PM1 and PM2 were not profoundly affected (Fig. 4G, n=3). CN- perfusion during the period of Ca2+ wave pacemaker PM2 activity in these eggs still drastically inhibited PM2 (Fig. 4G) even though no ATP hydrolysis by mitochondria occurs under these conditions (see drawings in Fig. 3H,I). Therefore, the rapid inhibition of pacemaker PM2 is probably due to a rapid mitochondrial depolarization that blocks mitochondrial Ca2+ influx. Cytosolic Ca2+ buffering by the mitochondria may thus be crucial for Ca2+ wave pacemaker PM2 activity, particularly in the presence of oligomycin a situation during which no mitochondrial ATP synthesis can occur (see Discussion). Finally we photoreleased NPE caged ATP during PM2 activity, in order to characterize a potential regulation of PM2 activity by intracellular ATP during the normal operation of PM2 (i.e. without any mitochondrial inhibitors, Fig. 4H, n=4). The most striking effect of adding exogenous ATP during Ca2+ wave pacemaker PM2 activity was to decrease the period between successive waves elicited by pacemaker PM2 (bars in Fig. 4H show this decrease is of 21% in this experiment).
Together, these observations suggest that complex functional interactions between mitochondria and surrounding Ca2+-release sites on the ER network occur during the operation of Ca2+ wave pacemaker PM2 and that they involve both local Ca2+ fluxes and mitochondrial ATP production.
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DISCUSSION |
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Our experiments with mitochondrial inhibitors and caged ATP also show that
the stable vegetal Ca2+ wave pacemaker PM2, which operates in
ascidian zygotes during completion of meiosis II, strictly depends on
mitochondrial activity in contrast to pacemakers PM1 and PM3. Because
Ca2+ wave pacemaker PM2 is located in a cortical domain of ER
accumulation sandwiched between the plasma membrane and the subcortical
mitochondria-rich domain (McDougall and
Sardet, 1995; Roegiers et al.,
1999
; Dumollard and Sardet,
2001
), we discuss our results in the light of possible functional
relationships between ER and mitochondria.
Ca2+ waves activate mitochondrial respiration in ascidian
eggs
Using two non-invasive recording techniques, we have been able to measure
oxygen consumption and mitochondrial NADH levels of individual ascidian eggs.
Basal oxygen consumption of the egg was highest in the vegetal pole region
where the bulk of subcortical mitochondria are located
(Roegiers et al., 1995;
Roegiers et al., 1999
;
Dumollard and Sardet, 2001
),
suggesting that the oxygen consumption we measured was mainly due to
mitochondrial respiration. The increases in both O2 consumption and
[NADH]mito observed at fertilization indicate that mitochondrial
activity is stimulated by sperm entry.
Ca2+ appears to be the stimulus for increased mitochondrial respiration as both O2 consumption and [NADH]mito increase during the activity of the Ca2+ wave pacemakers PM1 and PM2. Most remarkably, Ca2+ itself is sufficient to produce the increase in O2 consumption as demonstrated by the transient burst of O2 consumption in response to Ins(1,4,5)P3. These data suggest that the repetitive Ca2+ waves emitted by the two pacemakers (PM1 and PM2) are responsible for the sperm-triggered activation of the energetic metabolism in the egg.
The activation of mitochondrial metabolism by the sperm-triggered Ca2+ waves suggests that, in these eggs, mitochondria take up cytosolic Ca2+ during the passage of a Ca2+ wave. The appearance of a FCCP-releasable Ca2+ pool after the activity of the pacemakers PM1 and PM2 further supports such hypothesis. Indeed after meiotic Ca2+ oscillations have occurred, FCCP perfusion induced a Ca2+ transient in the vegetal region of the egg (superimposed on the slow and sustained increase in [Ca2+]c induced by FCCP or CN- perfusion in artificially activated or fertilized eggs) that most probably reflected release of Ca2+ from the mitochondria upon mitochondrial depolarization.
Although mitochondria were suggested to be a sink for the Ca2+
released during the fertilization wave in sea urchin eggs
(Eisen and Reynolds, 1985;
Girard et al., 1991
), no link
to mitochondrial respiration could be established because of the concomitant
non-mitochondrial respiratory burst occurring in these eggs at fertilization
(Schomer and Epel, 1998
;
Yasumasu, 2000
). In a very
recent study, mitochondrial Ca2+ has been suggested to play a role
in the control of mitochondrial respiration in sea urchin eggs
(Fujiwara et al., 2001
).
However this study predicts that a high [Ca2+]mito in
the unfertilized sea urchin egg would inhibit mitochondrial respiration
(Fujiwara et al., 2001
), which
is in complete contradiction with the previous studies on somatic cells, on
sea urchins (Eisen and Reynolds,
1985
; Girard et al.,
1991
) and with our findings in ascidian eggs.
Inhibition of the Ca2+ wave pacemaker PM2 by mitochondrial
inhibitors is not due to a global depletion in ATP levels
The presence in ascidian eggs of three Ca2+ wave pacemakers with
distinct locations and Ca2+ wave patterns provides an ideal model
system with which to study the influence of mitochondrial activity on the
regulation of intracellular Ca2+ waves. Perfusions of mitochondrial
inhibitors during the period of activity of the different pacemakers revealed
that, whereas Ca2+ wave pacemaker PM2 was immediately and
completely blocked upon FCCP or CN- perfusion, the pacemakers PM1
and PM3 were only slightly altered after lengthy perfusions of these
mitochondrial inhibitors. Several observations suggest that the rapid
inhibition of the Ca2+ wave pacemaker PM2 is not caused by emptying
Ca2+ stores due to a global depletion in ATP levels. First, the
activated egg could still respond to Ins(1,4,5)P3 after
more than 20 minutes of FCCP perfusion and the fertilized egg could respond to
Ins(1,4,5)P3 during PM2 inhibition by FCCP or
CN- (R. D., unpublished). Second, the resting
[Ca2+]c remained relatively low for long periods of time
during metabolic inhibition of the pacemaker PM2, implying that ATP-dependent
Ca2+ extrusion from the cytosol was still operating, with a part of
it probably refilling the ER Ca2+ stores. Therefore, mechanisms
other than global ATP depletion must account for the inhibition of
Ca2+ wave pacemaker PM2 by mitochondrial inhibitors.
Mitochondrial modulation of the Ca2+ wave pacemaker
PM2
In most eggs studied so far, Ins(1,4,5)P3-induced
Ca2+ release from the ER Ca2+ stores supports the
repetitive Ca2+ waves observed at fertilization. Specifically in
ascidians, it is known that injecting heparin
(McDougall and Sardet, 1995)
or the function blocking antibody specific to the IP3R1 (18A10)
(Yoshida et al., 1998
) both
inhibit the pacemaker PM2. Moreover, blocking specifically PLC
activity
inhibits both pacemakers (Runft and Jaffe,
2000
) further illustrating that the pacemakers PM1 and PM2 rely
mostly if not only on a sperm-triggered Ins(1,4,5)P3
production. Given the central role of Ins(1,4,5)P3
mediated Ca2+ release in PM2 activity, IP3Rs may be a
key target for the regulation by mitochondria. There are at least two
mechanisms by which mitochondria can regulate the opening of the
IP3R channels: first, by locally buffering
[Ca2+]c; second, by controlling the local concentration
of ATP (as MgATP2- and ATP4- ions).
In recent years it has been established that mitochondria sense the high
[Ca2+]c in the vicinity of the IP3Rs and take
up significant amount of Ca2+ during the passage of Ca2+
waves (Hajnoczky et al., 2000;
Duchen, 2000
,
Rizzuto et al., 2000
). In such
`Ca2+ microdomains' created between clusters of IP3Rs
and juxtaposed mitochondria (Fig.
5D), the local Ca2+ buffering provided by mitochondria
can promote IP3R opening or closure depending on a bell shape
sensitivity to Ca2+ at a given
[Ins(1,4,5)P3]c (i.e. Ca2+ above a
low threshold level promotes opening, while higher Ca2+ levels
inhibit IP3R opening). At low Ins(1,4,5)P3
levels, moderate Ca2+ levels inhibit IP3R opening, while
under a higher concentration of Ins(1,4,5)P3, higher
levels of Ca2+ are required to inhibit IP3R opening
(Bootman and Lipp, 1999
;
Mak et al., 1999
;
Mak et al., 2001
).
Our data suggest that inhibiting mitochondrial Ca2+ accumulation
during PM2 activity using CN-, while blocking mitochondrial ATP
hydrolysis with oligomycin rapidly stops the ability of the pacemaker PM2 to
generate Ca2+ waves. This finding is consistent with the hypothesis
that at the low Ins(1,4,5)P3 levels driving the pacemaker
PM2 (Dumollard and Sardet,
2001; Dumollard et al.,
2002
), Ca2+ buffering by mitochondria is necessary to
keep the local [Ca2+]c in the activating part of the
bell shape curve. The reason why PM1 is insensitive to inhibition of
mitochondrial Ca2+ accumulation is that it is driven by a larger
increase in Ins(1,4,5)P3 levels induced at the activation
wave (Dumollard and Sardet,
2001
; Dumollard et al.,
2002
). Under these conditions of high
Ins(1,4,5)P3 levels, the [Ca2+]c
must reach higher levels to inhibit IP3Rs opening and
Ca2+ buffering by mitochondria would not be so crucial.
A role for local control of ATP is indicated by the finding that
photoreleasing ATP4- in eggs in which PM2 was inhibited by
CN- perfusion caused a reinitiation of Ca2+ waves from
the contraction pole. Mitochondrial ATP production in an `ATP microdomain' (as
shown in Fig. 5D) can modulate
intracellular Ca2+ release
(Yang and Steele, 2000).
ATP4- is an allosteric regulator of IP3R1 opening
(Mak et al., 1999
;
Mak et al., 2001
) the
predominant IP3R in the egg cell
(Fissore et al., 1999
; Brind et
al., 2001) and also the most sensitive to ATP
(Miyakawa et al., 1999
;
Maes et al., 2000
).
Electrophysiological studies have revealed that ATP4- (but not the
MgATP2- complex) sensitizes the IP3R to activation by
Ca2+ (Mak et al.,
1999
; Mak et al.,
2001
). Moreover when
[Ins(1,4,5)P3]c is low, ATP4- can
also suppress Ca2+-dependent inhibition of the channel opening
(Mak et al., 2001
). During
mitochondrial inhibition of the pacemaker PM2, some of the photoreleased
ATP4- will directly bind to the IP3Rs, thereby
suppressing Ca2+-dependent inhibition of the channels and rendering
the vegetal contraction pole region excitable for the initiation of global
Ca2+ waves. In addition, the remaining ATP4- ions will
bind Mg2+ ions and form MgATP2- complexes that can be
used by SERCAs to refill the ER Ca2+ stores
(Fig. 5D). Such a model does
explain why, while PM2 is completely blocked by mitochondrial inhibition, the
egg can still respond to the rather high Ins(1,4,5)P3
increases achieved by uncaging cIP3 (between 100 and 500 nM after
each UV flash) (Dumollard and Sardet,
2001
) and PM1 (which functions under high
Ins(1,4,5)P3 levels) is not altered by mitochondrial
inhibition. This model can also account for the increase in frequency of
Ca2+ oscillations generated by PM2 after the photorelease of
ATP4-. As the threshold [Ca2+]c for
triggering a wave can be lowered by increased ATP4-
(Mak et al., 1999
;
Mak et al., 2001
) this
threshold can be reached more quickly after a wave.
Taken together, our observations and the above considerations indicate that functional interactions between ER and mitochondria occur during sperm-triggered Ca2+ waves via two mechanisms: local Ca2+ fluxes between the two organelles and mitochondrial production of ATP. While local Ca2+ buffering in the `Ca2+ microdomain' may suppress the Ca2+ inhibition of the IP3R, local ATP production in the `ATP microdomain' would sensitize and potentiate IICR (Fig. 5D). Even though this work raises the possibility that both modes of communication between ER and mitochondria are at work during the generation of sperm-triggered Ca2+ waves, only precise measurement of [ATP4-]c, [MgATP2-]c, [Ca2+]c in the `Ca2+ and ATP microdomain' in the living zygote will allow us to define the complex roles mitochondria play in the regulation of the meiotic Ca2+ wave pacemakers.
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ACKNOWLEDGMENTS |
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