1 Department of Physiology, University College London, Gower Street, London,
WC1E 6BT, UK
2 Department of Anatomy and Developmental Biology, University College London,
Gower Street, London WC1E 6BT, UK
* Author for correspondence (e-mail: r.dumollard{at}ucl.ac.uk)
Accepted 19 March 2004
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SUMMARY |
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We have used fluorescence and luminescence imaging to investigate mitochondrial activity in single mouse eggs. Simultaneous imaging of mitochondrial redox state (NADH and flavoprotein autofluorescence) and [Ca2+]i revealed that sperm-triggered [Ca2+] oscillations are transmitted to the mitochondria where they directly stimulate mitochondrial activity. Inhibition of mitochondrial oxidative phosphorylation caused release of Ca2+ from the endoplasmic reticulum because of local ATP depletion. Mitochondrial ATP production is an absolute requirement for maintaining a low resting [Ca2+]i and for sustaining sperm-triggered [Ca2+] oscillations. Luminescence measurements of intracellular [ATP] from single eggs confirmed that mitochondrial oxidative phosphorylation is the major source of ATP synthesis in the dormant unfertilised egg. These observations show that a high local ATP consumption is balanced by mitochondrial ATP production, and that balance is critically poised. Mitochondrial ATP supply and demand are thus closely coupled in mouse eggs. As mitochondrial ATP generation is essential to sustain the [Ca2+] signals that are crucial to initiate development, mitochondrial integrity is clearly fundamental in sustaining fertility in mammalian eggs.
Key words: Mouse, Egg, Meiosis, Inositol 1,4,5-trisphosphate, Mitochondria, Autofluorescence
![]() |
Introduction |
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In mice, the fertilisation Ca2+ signal takes the form of a
series of Ca2+ transients that continue for about 4 hours, stopping
at pronuclei formation (Marangos et al.,
2003). These Ca2+ transients are caused by
Ins(1,4,5)P3-induced Ca2+ release (IICR) from
the endoplasmic reticulum (ER) Ca2+ stores. The cytosolic
Ca2+ is pumped back into the ER by sarco-endoplasmic reticulum
Ca2+ ATPases (SERCAs) or extruded out of the cell by plasma
membrane Ca2+ ATPases (PMCAs)
(Carroll, 2001
;
Brini et al., 2003
) and
Na+/Ca2+ exchangers
(Pepperell et al., 1999
;
Carroll, 2000
).
In recent years, mitochondria have been shown to be major regulators of
intracellular Ca2+ homeostasis
(Duchen, 2000;
Rizzuto et al., 2000
). In
cells such as sea urchin (Eisen and
Reynolds, 1985
) and ascidian eggs
(Dumollard et al., 2003
),
mitochondria sequester Ca2+ during the fertilisation
Ca2+ transients. Ca2+ sequestration by mitochondria has
two main consequences. First, mitochondria act as passive Ca2+
buffers that can regulate intracellular Ca2+ release (reviewed by
Rizzuto et al., 2000
;
Duchen, 2000
). The second
consequence is that Ca2+ in the mitochondrial matrix is a
multisite activator of oxidative phosphorylation (or
mitochondrial ATP synthesis, see Fig.
11): it activates the dehydrogenases of the Krebs cycle and
the electron transport chain (McCormack
and Denton, 1993
; Hansford,
1994
) and has a direct action on the F0/F1
ATP synthase (Territo et al.,
2000
). In somatic cells and in ascidian eggs, mitochondrial
Ca2+ uptake has been shown to stimulate mitochondrial respiration
by promoting the reduction of mitochondrial NAD+ to NADH
(Duchen, 1992
;
Pralong et al., 1994
;
Hajnoczky et al., 1995
;
Dumollard et al., 2003
).
Furthermore, mitochondrial ATP production may directly regulate intracellular
Ca2+ release: ATP4 sensitises the
Ins(1/4/5)P3 receptor to activation by Ca2+
(Mak et al., 1999
;
Mak et al., 2001
) while
Mg2+-complexed ATP is consumed to refill the ER Ca2+
stores.
|
The involvement of mitochondria in maintaining the energetic status of the
egg is substantiated by the fact that early development (one- to eight-cell
stage) in the mouse is supported by the oxidation of pyruvate and lactate,
especially pyruvate (Leese,
1995). Glucose is poorly used because of the block to the
regulatory glycolytic enzyme phosphosfructokinase
(Barbehenn et al., 1974
). Mouse
eggs and early embryos can also use glutamine as an energetical substrate that
will be oxidised in the mitochondria
(Gardner et al., 1989
). The
mouse egg also possesses sizeable amounts of the enzyme lactate dehydrogenase,
which catalyses the oxidation of lactate to pyruvate
(Lane and Gardner, 2000
).
Finally, ß-oxidation of intracellular lipids has only been studied in pig
embryos, where this pathway does not seem to be used during early development
(Sturmey and Leese, 2003
).
Despite heavy reliance on mitochondria, oxygen consumption of the mouse egg
and early embryo is very low compared with oxygen consumption of the
blastocyst and mitochondria are thought to be immature in the
mouse egg and early embryo (Houghton et
al., 1996
; Trimarchi et al.,
2000
).
Therefore, in the early mammalian embryo, the source of ATP generation is still a matter of debate. We have investigated the mechanism of coupling ATP supply and demand at fertilisation when demand is increased. Furthermore, we use mitochondrial inhibitors to investigate the importance of mitochondrial function in providing for ATP demand generated by the mechanisms of Ca2+ homeostasis and by sperm-triggered Ca2+ oscillations.
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Materials and methods |
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In vitro fertilisation
For in vitro fertilisation, epididymis were dissected from proven fertile
MF1 males that had been culled by cervical dislocation. The epididymis were
transferred into 1 ml drops of pre-equilibrated (5% CO2 in air) T6
medium containing 10 mg ml1 bovine serum albumin (BSA,
Fraction V Sigma). Sperm were released by puncturing the epididymis with a 27
gauge needle. The sperm were placed in the incubator for 20 minutes during
which time they dispersed into a suspension. This suspension was diluted 1:5
in drops of the same medium under oil and incubated for another 2-3 hours for
capacitation to occur. IVF was performed in drops of H-KSOM under paraffin
oil. The zona pellucida of MII stage oocytes were removed by a brief exposure
to acidified Tyrodes solution (Sigma). Sperm were added to the
zona-free MII oocytes at a 1:10 dilution of the capacitated sperm suspension.
For imaging experiments, zona-free oocytes were placed in a heated chamber
with a cover glass base containing 0.5 ml of H-KSOM without BSA for 3-5
minutes. 0.5 ml of complete H-KSOM was then added to the chamber and the media
was covered with oil to prevent evaporation. Capacitated sperm were then added
to the chamber at time t=0 except where mentioned.
Microinjection
Cells were pressure injected with a micropipette and Narishige manipulators
mounted on an inverted Leica microscope essentially as described previously
(FitzHarris et al., 2003).
Oocytes were placed in a drop of H-KSOM covered with mineral oil and
immobilised with a holding pipette. To penetrate the plasma membrane, a brief
over-compensation of negative capacitance was applied.
Measurement of intracellular Ca2+ and photolysis of caged Ins(1,4,5)P3
Intracellular Ca2+ was measured using Fura red, Fura 2, Indo 1
or Rhod2 (Molecular Probes). Oocytes were loaded in H-KSOM containing 2 µM
of the acetoxymethyl ester (AM) form of the Ca2+ dyes and 0.02%
pluronics for 10 minutes at 37°C. After loading, oocytes were placed in a
drop of H-KSOM under oil in a chamber with a coverslip. The chamber was placed
in a heated stage on a Zeiss axiovert microscope or on a Zeiss LSM 510
confocal microscope. Excitation light (Fura red, 490 nm; Indo 1, 365 nm; Fura
2, 340 and 380 nm; Rhod 2, 543 nm) was delivered by a monochromator, while
emission was collected using a filter (Fura red, 600 nm longpass; Rhod 2, 580
nm longpass; Indo 1, 390-430 nm and 435-480 nm bandpass; Fura 2, 520 nm
longpass) placed in front of a cooled CCD camera (MicroMax, Princeton
Instruments). Measurements where obtained by averaging the signal collected in
a region of interest (ROI) drawn in the centre of the egg (see Figs
1 and
3 for typical examples of such
ROIs).
|
|
The excitation wavelengths and camera exposure times were controlled using Metafluor software (epifluorescence microscope) or the LSM 510 software (confocal microscope).
Autofluorescence measurements
NADH fluorescence was excited with UV light (360 nm or the 364 nm line of
the UV laser of the confocal microscope) and emission was collected using a
470 nm longpass or a 435-485 bandpass filter. Oxidised flavoproteins
(FAD++) fluorescence was excited with the 458 nm line of an argon
laser or with a 440-490 nm bandpass filter while emitted fluorescence was
collected through either a 520 nm longpass filter or a 505-550 nm bandpass
filter. Measurements were obtained by averaging the signal collected in a
region of interest (ROI) drawn in the centre of the egg (see Figs
1 and
3 for typical examples of such
ROIs). The measurements displayed (F/F0) in the graph are normalised values
with regard to the first value of the experiment (F0).
Monitoring of mitochondrial potential
Eggs were incubated for 15 minutes in H-KSOM containing 10 µg/ml of the
lipophilic cationic dye Rhod 123 (exc: 488 nm, em: 520 nm). With this loading
protocol, internalisation of the dye in the mitochondria quenches the Rhod123
fluorescence and depolarisation can be seen as an increase in green
fluorescence, while a hyperpolarisation is seen as a decrease in Rhod 123
fluorescence (for details, see Duchen and
Biscoe, 1992). For imaging of the mitochondria, eggs were
incubated for 15 minutes in H-KSOM containing 1 µg/ml of Mitotracker Green
(Molecular Probes, Netherlands) or 100 nM of tetramethylrhodamine methyl ester
(TMRM, Molecular Probes) and then imaged on the confocal microscope.
Measurements were obtained by averaging the signal collected in a region of
interest (ROI) drawn in the centre of the egg (see Figs
1 and
3 for typical examples of such
ROIs).
Labelling of endoplasmic reticulum
To label the endoplasmic reticulum, DiI18 (Molecular Probes) was
microinjected as a saturated solution in soybean oil (Sigma) 30 minutes before
imaging. Imaging was performed using a Zeiss LSM 510 microscope.
DiI18 was excited using the 514 nm line of an Argon laser and the
emitted light collected using a 600 nm longpass filter.
Measurement of intracellular ATP in a single egg
The luminescent ATP indicator firefly luciferase (Calbiochem)
(Allue et al., 1996;
Jouaville et al., 1999
) was
injected into unfertilised mouse eggs as described above. The concentration of
recombinant luciferase protein in the injection pipette was 2 mg
ml1. The eggs were then incubated in H-KSOM containing 100
µM luciferin for 30 minutes before any measurements could be made. The
luminescence emanating from individual luciferase-injected eggs was monitored
with an imaging photon detector (Photek, UK, using software and a system
design provided by science wares,
www.sciencewares.com).
Perfusion of substrates and inhibitors
The mitochondrial uncoupler FCCP (1 µM, Sigma) as well as inhibitors of
the respiratory chain [complex I, rotenone (5 µM, Sigma); complex II,
malonate (20 mM, Sigma); complex IV, CN (1 mM, Merck);
complex V, F0/F1 synthase oligomycin (60 µM, Sigma)] were added to the
chamber as 10x solutions. The energetical substrate pyruvate (2 mM,
Sigma) was also added as 10x solution. Thapsigargin (Calbiochem) was
used at 20 µM final concentration.
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Results |
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Redox fluorometry based on intrinsic fluorescence of reduced pyridine
nucleotides (NADH and NADPH) and oxidised flavoproteins has been a useful tool
for studying cellular energy metabolism (reviewed by Master and Chance, 1993;
Duchen 2000;
Duchen et al., 2003
). We
imaged NADH, a reducing equivalent formed by the Krebs cycle that is
oxidised to NAD+ at complex I of the mitochondrial electron
transport chain (Fig. 1A), and
flavoproteins, which form an integral component of complex II of the electron
transport chain (Fig. 1A).
Prior to addition of sperm (at time t=0) the NADH and
FAD++ autofluorescence was constant. Within 10 minutes of addition
of sperm, synchronous oscillations in [NADH] and [FAD++] could be
observed (Fig. 1B;
n=6). These oscillations correspond to reduction of NAD+
and FAD++ as increases in NADH fluorescence are mirrored by
decreases in FAD++ fluorescence.
The redox state is readily manipulated by pharmacological inhibition or stimulation of the respiratory chain. Inhibition of respiration by the inhibitor of complex IV CN causes maximal reduction of NAD+ and FAD++, seen as an increase in NADH fluorescence and a decrease in FAD++ fluorescence (Fig. 1C, part i), while the mitochondrial uncoupler FCCP causes maximal oxidation of NADH and FAD++, seen as a decrease in NADH fluorescence and an increase in FAD++ fluorescence (Fig. 1C, part iv).
The distribution of FAD++ autofluorescence on confocal
microscopy colocalised with the distribution of the potentiometric dye, TMRM
(Fig. 2A-C), which selectively
stains energised mitochondria (see Duchen
et al., 2003), confirming the mitochondrial origin of the
FAD++ signal. When imaged simultaneously from a single egg, NADH
and FAD++ also had a similar mitochondrial distribution
(Fig. 2D,E), although the NADH
image showed relatively high fluorescence in the cytosol, indicating that a
significant component of the NADH signal is cytosolic. By contrast, the
FAD++ signal was more clearly restricted to mitochondria
(Fig. 2A-D).
|
Together, these results show that sperm-triggered [Ca2+] oscillations stimulate mitochondrial oxidative phosphorylation in the mouse egg, suggesting that the Ca2+ signal is transmitted directly to the mitochondrial matrix and that it serves to upregulate mitochondrial oxidative phosphorylation.
Mitochondrial ATP production is necessary to sustain sperm-triggered [Ca2+] oscillations
In order to characterise the role played by mitochondria in the regulation
of sperm-triggered [Ca2+] oscillations, we perfused mitochondrial
inhibitors onto fertilised eggs. FCCP, a protonophore that collapses the
mitochondrial potential, disrupted the [Ca2+] oscillations after a
couple of minutes (n=5, Fig.
4A). Ca2+ homeostasis was disrupted and the
[Ca2+]c failed to recover after a Ca2+
transient. CN, an inhibitor of complex IV of the
mitochondrial respiratory chain also disrupted [Ca2+] oscillations
(n=5, Fig. 4B).
Oligomycin, which inhibits mitochondrial ATP synthesis without depolarising
the mitochondria (see Fig. 8C) had a very similar effect on the [Ca2+] oscillations (n=7,
Fig. 4C). After adding
oligomycin, the frequency of the [Ca2+] oscillations first
increased and then the [Ca2+]c failed to recover after a
[Ca2+] transient.
|
|
|
|
|
Perfusion with either oligomycin (Fig. 8B, n=13) or FCCP (Fig. 5C, Fig. 6B) caused a decrease in [ATP]i. That oligomycin alone should cause a rapid loss of [ATP]i strongly suggests: (1) that mitochondrial oxidative phosphorylation provides the major mechanism to sustain ATP in the resting mature MII oocyte and (2) that there must be a high consumption of ATP in these oocytes to cause a rapid fall after synthesis is stopped.
Mitochondrial membrane potential and activity of the ATP synthase in mouse eggs
The mitochondrial potential is established by the translocation of protons
from the mitochondrial matrix to the intermembrane space via the activity of
the complexes I, III and IV of the respiratory chain residing in the inner
mitochondrial membrane. This gradient is to some extent dissipated by proton
influx through the ATP synthase (Fig.
1A) in the process of ATP generation.
By measuring mitochondrial potential with Rhod123 using a dequench protocol (see Materials and methods), we found that oligomycin caused hyperpolarisation of the mitochondrial potential concomitant with the early [Ca2+] increase (Fig. 8A, n=25; Fig. 9B, n=4). This observation demonstrates that the mitochondrial ATP synthase is active in the mature MII egg and that oligomycin application blocked a resting proton flux into the mitochondrial matrix (see Fig. 8C).
|
Therefore, even when the mitochondrial electron transport chain is inhibited, mitochondria of mature eggs can still maintain a potential. Maintenance of a mitochondrial potential during inhibition of the electron transport chain is probably caused by the reversal of the ATP synthase, which acts as a proton translocating ATPase, hydrolysing ATP and pumping protons from the mitochondrial matrix (Fig. 9D). Accordingly, when the ATP synthase was blocked by oligomycin prior to the perfusion of the complex I inhibitor rotenone, rotenone on its own completely collapsed the mitochondrial potential (Fig. 9B). A further indication that the ATP synthase was acting in reverse mode is shown in Fig. 9C (n=4). In this experiment oligomycin, perfused after rotenone, provoked a depolarisation of the mitochondria as opposed to the hyperpolarisation induced by oligomycin alone (Fig. 9B). In this case, inhibition of the reversed ATP synthase stopped the outward proton flux that was maintaining the potential.
Our protocol revealed that there was no measurable delay between the mitochondrial depolarisation (or hyperpolarisation for oligomycin perfusion) and the [Ca2+] increase. The collapse of mitochondrial potential or inhibition of ATP synthase was in all cases matched with an increase in intracellular [Ca2+], suggesting a strong link between ATP demand and Ca2+ homeostasis.
Together these observations reveal that the mitochondrial potential is tightly regulated in the mouse egg and that the ATP synthase is a major regulator of the mitochondrial potential.
Close apposition of ER and mitochondria in the mouse egg
The observations of Ca2+ driven oscillations in mitochondrial
activity as well as the finding of a strong link between ATP demand and
Ca2+ homeostasis both suggest that functional interactions exist
between intracellular ER stores and the mitochondria. We therefore imaged ER
and mitochondria simultaneously in a mouse egg to assess their relative
distribution. Simultaneous imaging of ER and mitochondria in a mature MII egg
revealed a close apposition of mitochondria and ER
(Fig. 10). Strikingly, at high
magnification it can be seen that an aggregation of mitochondria is embedded
in sheets of ER (Fig. 10B,C).
Such spatial organisation of ER and mitochondria provides a structural support
for the functional interactions between ER and mitochondria that we have shown
to participate in the maintenance of a low resting
[Ca2+]c.
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![]() |
Discussion |
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Mitochondria are metabolically active in mouse eggs
A number of observations show that mitochondria are active in mouse eggs.
First, blocking selectively the mitochondrial F0/F1 ATP synthase with
oligomycin causes the rapid depletion of intracellular ATP levels,
demonstrating that a steady level of ATP is maintained in the unfertilised egg
by mitochondrial oxidative phosphorylation. This supports the idea that ATP is
primarily supplied by oxidation of mitochondrial substrates (such as
pyruvate), which is consistent with the known requirement for pyruvate during
early development (Leese,
1995). The data appear to be inconsistent with the suggestion that
mitochondria are immature and inactive in the early embryo
(Trimarchi et al., 2000
).
Despite a low respiratory rate (Houghton
et al., 1996
; Trimarchi et
al., 2000
), we show that mitochondria are active and
phosphorylating in the mouse egg and the large number of mitochondria in
mammalian eggs (around 100,000) (Piko and
Taylor, 1987
; Barritt et al.,
2002
) make a crucial contribution to ATP supply. Most remarkably,
and in strong contrast to most cells studied, the rapid decrease in cellular
ATP to levels crucial for the regulation of [Ca2+] in response to
oligomycin suggests that the resting MII oocyte has a high turnover of ATP and
that mitochondrial ATP generation and local ATP consumption must be delicately
balanced.
We also observed that the F0/F1 synthase is a major
regulator of the mitochondrial potential as respiratory chain inhibitors can
completely depolarise the mitochondria only if the F0/F1
synthase is blocked. As a result, a decrease in mitochondrial potential can be
offset by reversal of the F0/F1 synthase with a
resulting extrusion of H+ from the mitochondrial matrix that
restores the mitochondrial potential. This tight regulation of mitochondrial
potential may explain why we failed to observe any changes in mitochondrial
potential during the sperm-triggered [Ca2+] oscillations (not
shown) even though mitochondrial respiratory activity is upregulated by these
Ca2+ transients (see next section). The mitochondrial potential
must be maintained to support ATP synthesis; however, a high mitochondrial
potential will favour production of deleterious reactive oxygen species (ROS)
(Merry, 2002). As mouse eggs
and embryos are sensitive to oxidative stress
(Liu et al., 2000
), the
mitochondrial potential may be maintained to a value that allows an efficient
phosphorylation of ADP while minimising ROS production.
Ca2+ transients at fertilisation stimulate mitochondrial activity
By imaging the mitochondrial autofluorescence, we demonstrate
Ca2+ driven oscillations in mitochondrial activity, each
Ca2+ transient entraining the transient reduction of
NAD+ and flavoproteins. Our observations are best explained by the
following scheme: Ca2+ released into the cytosol enters the
mitochondrial matrix to activate the Krebs cycle
(Fig. 11). Activation of the
Krebs cycle would provide more NADH and lead to the reduction of
flavoproteins. The increased provision of reducing equivalents (NADH and
FADH2) will stimulate mitochondrial respiration and increase ATP
generation, causing a progressive re-oxidation by the electron transport chain
until a subsequent Ca2+ transient stimulates further reduction and
restarts the cycle. This role of Ca2+ in the mitochondrial matrix
allows the close matching of ATP production to energy demand: after triggering
ATP-consuming reactions in the cytosol, Ca2+ upregulates the
synthesis of ATP in the mitochondria (Fig.
11). Indeed, after fertilisation, approximately 3 µM of free
Ca2+ is released into the cytosol and must then be cleared from the
cytosol by active transport by the SERCAs and by the PMCAs. This released
Ca2+ also stimulates ATP-dependent exocytosis of cortical granules
and the extrusion of the second polar body, which also requires ATP. Therefore
synthesis of ATP is required to maintain the ATP level after fertilisation.
Matching ATP supply and demand via Ca2+ regulation of the
Krebs cycle provides a mechanism of maintaining low oxidative
phosphorylation that, in turn, minimises the production of potentially
damaging ROS by the electron transport chain
(Harvey et al., 2002;
Merry, 2002
).
Role of mitochondria in Ca2+ homeostasis and sperm-triggered [Ca2+] oscillations
Contrasting with the study of Liu et al.
(Liu et al., 2001), the
Ca2+ probe Rhod2 AM used in somatic cells to monitor mitochondrial
Ca2+ concentration, [Ca2+]mito
(Duchen, 2000
;
Duchen et al., 2003
), in our
hands, did not partition into mitochondria of the mouse egg
(Fig. 3); therefore the changes
in [Ca2+]mito could not be measured directly.
Nevertheless, we investigated the role mitochondria play in Ca2+
homeostasis and in sperm-triggered [Ca2+] oscillations by applying
different mitochondrial inhibitors onto mouse eggs. We found that the
mitochondrial uncoupler FCCP and the complex I (rotenone) and complex IV
(CN) inhibitors but not the complex II inhibitor (malonate)
induce a Ca2+ leak from the intracellular ER stores. The appearance
of this Ca2+ leak is due to ATP depletion, as oligomycin alone also
provokes this leak. Therefore, in the mature egg, mitochondrial ATP production
provides the energy used by the SERCA pumps to maintain a low resting
[Ca2+]c (Fig.
11). The innocuity of the complex II inhibitor malonate suggests
that mitochondria mostly oxidise complex I-linked substrates (i.e. pyruvate)
for oxidative phosphorylation, consistent with the known preferred energetic
substrate of the mouse egg (Leese,
1995
; Leese,
2002
).
The first rise in Ca2+ induced by mitochondrial inhibitors is followed by a larger secondary increase. The source of Ca2+ of the latter increase, presumably arising after a more pronounced ATP depletion, lies in the extracellular medium. Thus, ATP depletion in the egg affects first the SERCAs then the PMCAs. The rapid inhibition of the SERCAs by ATP depletion suggests that the activity of the SERCAs strictly depends on mitochondrial ATP production. The delay between the apparent failure of the SERCAs and the PMCAs is best explained if inhibition of mitochondrial ATP synthesis affects first local ATP supply close to the sites of demand by SERCA, and while the global [ATP] only falls sufficiently to cause failure of the PMCA later. These observations strongly suggest a functional coupling between ER and mitochondria mediated by ATP (Fig. 11). Simultaneous imaging of ER and mitochondria revealed a close apposition of mitochondria organised in aggregates around sheets of ER that is consistent with a functional interplay between these organelles. It will be interesting to determine whether SERCA pumps are especially enriched in the sheets of ER surrounded by mitochondria.
The crucial role for mitochondria in sustaining sperm-triggered [Ca2+] oscillations is to provide ATP whereas buffering of cytosolic Ca2+ by mitochondria seems to have no major influence on the pattern of the [Ca2+] oscillations. First, [Ca2+] oscillations can continue for several minutes after FCCP-induced mitochondrial depolarisation (which prevents mitochondria from sequestering cytosolic Ca2+). Second, oligomycin perfusion that does not hamper Ca2+ sequestering by mitochondria is still able to inhibit the [Ca2+] oscillations. The importance of mitochondrial ATP production was further illustrated by fertilisation in a medium devoid of any energetical substrates. In such cases, [Ca2+] oscillations were disrupted. The return of pyruvate to the medium was able to provoke the resumption of the [Ca2+] oscillations. Together, these observations characterise a crucial role for mitochondrial oxidative phosphorylation at the activation of development.
Conclusion
The tight coupling of ATP supply and demand proposed in this study provides
a major advantage for early mammalian development. The maternal inheritance of
mitochondria requires that mitochondria are protected from potentially
damaging ROS. The maintenance of a low level of oxidative phosphorylation that
can be stimulated on an increase in ATP demand provides one means of lowering
exposure of mitochondria to damaging oxidative stress. Our data suggest that
Ca2+ is the functional link that provides a mechanism for coupling
ATP supply and demand. As maternal aging is associated with increased
oxidative stress in human eggs (Tarin,
1996) it will interesting to define whether mitochondrial
physiology and the coupling of ATP supply and demand are impaired in eggs from
aged women.
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ACKNOWLEDGMENTS |
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