doi: 10.1242/10.1242/dev.00296
Mitochondrial respiration and Ca2+ waves are linked during fertilization and meiosis completion
Rémi Dumollard1,*,
,
Katherine Hammar2,
Marshall Porterfield2,
Peter J. Smith2,
Christian Cibert3,
Christian Rouvière1 and
Christian Sardet1
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
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Fig. 5. Interactions between mitochondria and ER in the ascidian egg and in the
Ca2+ and ATP microdomain. (A-C) Imaging of the ER network (red) and
of mitochondria (green) in the vegetal contraction pole (v) of a fertilized
Phallusia egg (A) shows the cortical ER-rich domain closely apposed
to the subcortical mitochondria-rich domain. At higher resolution (B),
rod-shaped mitochondria are observed densely packed in the vegetal subcortex
(0.5 µm under the surface of the egg). Mitochondria are in close proximity
to ER-rich domains and tubes of ER (C; reveals ER tubes between ER-rich
domains shown in B). a, animal side. (D) Probable Ca2+ fluxes in
the Ca2+ microdomain forming at the interface between a
mitochondria (green) and an ER tubule (red) with clustered IP3Rs.
In the same space an ATP microdomain is formed between mitochondria (producing
and exporting ATP) and Mg-ATP-consuming pumps (SERCAs) and
ATP4--using channels (IP3Rs). Calcium released in the
cytosol can be sequestered by the mitochondria where it stimulates oxidative
phosphorylation. ATP4- is exported from the mitochondria into the
cytosol where it stimulates Ca2+ release by sensitizing the
IP3Rs to Ca2+. Finally, Mg-ATP generated by mitochondria
can energize Ca2+ pumping back into the ER lumen and replenish
Ca2+ stores.
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Fig. 2. Variations of the mitochondrial concentration of NADH
([NADH]mito) in ascidian eggs (Phallusia). (A) Images of
NADH of an egg before fertilization (left, t=0 minutes) and at the
end of meiosis (right, t=21 minutes 40 seconds). The gray lines are
artefacts caused by the CCD camera. In the `time-image' (center), time
(minutes) is measured on the x-axis and the y-axis
corresponds to the extracted lines of the mitochondria-rich domain
(a-b,a'-b'; see Materials and Methods). The varying length of each
extracted line of mitochondrial NADH is due to actomyosin driven cortical
contractions traversing the egg during the activity of both Ca2+
wave pacemakers (Roegiers et al.,
1999 ). The periods of activity of the two pacemakers are indicated
by arrowheads; the scale goes from black for low [NADH] values to white for
high [NADH] values. (B) Typical [NADH]mito variations observed
after fertilization of an egg measured in the middle of the mitochondria-rich
domain [between the two white horizontal lines in A (center)].
[NADH]mito increases during the period of activity of each
Ca2+ wave pacemaker (PM1 and PM2) (n=8). (C) Variations of
[NADH]mito after perfusion of an unfertilized egg with the
mitochondrial inhibitors CN- (2 mM) and FCCP (1 µm).
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Fig. 1. (A) Sperm-triggered [Ca2+]c oscillations during
meiosis in an ascidian egg (Phallusia). The first series of
Ca2+ oscillations is composed of the fertilization Ca2+
wave followed by four waves (PM1), which leads to the extrusion of the first
polar body (pb1) about 5-7 minutes after sperm-egg fusion. After 2-4 minutes,
the second series of Ca2+ oscillations (PM2) are triggered. These
last 15-20 minutes, ending when the second polar body (pb2) is emitted. The
values for [Ca2+]c displayed on the right of the graph
are from aequorin measurements
(Speksnijder et al., 1989).
(B) Variations of oxygen consumption during meiosis in ascidian eggs
(Phallusia). Oxygen fluxes are measured on a single egg using the
oxygen-sensitive self-referencing vibrating probe (upper images taken at
t=0 and t=12 minutes). Sperm-egg fusion triggers a first
increase in oxygen consumption, which peaks during the period of activity of
Ca2+ wave pacemaker PM1 and lasts until the first polar body (pb1)
is emitted. A second increase in oxygen consumption occurs during the period
of activity of Ca2+ wave pacemaker PM2 and terminates when the
second polar body is emitted (pb2) (n=7). The vertical bars in the
graph represent average measurements of the oxygen consumption before
fertilization (100%), after meiosis completion (126±15%) and during the
activity of the pacemakers PM1 (167±18%) and PM2 (173±14%)
(average from eight single eggs, five Phallusia and three
Ciona). The transient increase in oxygen consumption observed between
PM1 and PM2 in the example shown was not seen in other experiments. By
contrast, two transient increases associated with the periods of pacemakers
PM1 and PM2 activity were observed for every egg recorded. (C) Variations of
oxygen consumption in an ascidian egg (Ascidiella) activated by caged
Ins(1,4,5)P3 (cIP3) photolysis. Simultaneous
measurements of [Ca2+]c (using CG) and oxygen
consumption (using the vibrating probe) show that each time intracellular
Ins(1,4,5)P3 is photoreleased by a UV flash (black
arrowhead), it induces a Ca2+ transient accompanied by a transient
activation of oxygen consumption (all 20 UV flashes applied to 6 different
eggs generated similar responses in oxygen consumption). (D) Increase in
oxygen consumption in an ascidian egg (Ascidiella) perfused with the
mitochondrial uncoupler FCCP. The oxygen electrode is first positioned far
from the egg (1) then brought close to the egg (2) to measure its basal oxygen
consumption. A large increase in oxygen consumption is observed when FCCP is
added to the dish to give a final concentration of 1 µM (bar under the
graph).
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Fig. 3. Effect of mitochondrial inhibitors on Ca2+ levels and
Ca2+ signals in ascidian eggs (Phallusia). Unfertilized
eggs are injected with CG/TR only (A) or with CG/TR + 5 µM cIP3
(B,C) are perfused with CN- (C) and FCCP (B) with or without
oligomycin. Ca2+ transients are induced every 3 minutes by a UV
flash photoreleasing Ins(1,4,5)P3 (black arrowheads).
Rates of [Ca2+]c increase induced by the three
mitochondrial inhibitors on the egg are compared in the bar graph shown in D
(n=17). (E-I) The actions of CN- (F,I) and FCCP (G,H) on
the mitochondria in the presence (H,I) or absence (F,G) of oligomycin,
compared with the untreated egg (E).
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Fig. 4. Differential sensitivity of the three Ca2+ wave pacemakers of
ascidian eggs (Phallusia) to mitochondrial inhibitors. (A-D) Effects
of FCCP applied before fertilization (A, n=3) and during the period
of activity of the Ca2+ wave pacemaker PM2 (B, n=4)
inhibits PM2 activity. Perfusion of FCCP after extrusion of the second polar
body (pb2) (C, n=3) produces a Ca2+ transient. Perfusion
of FCCP during the period of activity of the Ca2+ wave pacemaker
PM3 (D, n=4) affects pacemaker PM3 only slightly after 4 minutes.
Perfusion of CN- during the period of activity of PM1 (E) or PM2
(F,G) in eggs injected with CG/TR only (E, n=4) or with CG/TR and
cATP (in F). Artificial production of ATP by UV flash photolysis of cATP
(black arrowheads) restores partially PM2 activity (F, n=3). In eggs
preincubated for 20 minutes in oligomycin before fertilization (G,
n=3), Ca2+ wave pacemaker PM2 is still sensitive to
CN- perfusion. Artificial production of ATP by UV flash photolysis
of cATP (black arrowheads) increases the frequency of the repetitive waves
emitted by Ca2+ wave pacemaker PM2 (H, n=4). The periods
for waves 1 to 4 (i.e. before the photorelease of ATP) and for waves 5 to 8
(i.e. after the photorelease of ATP) are indicated in the inset.
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© The Company of Biologists Ltd 2003
