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During the past 25 years, the characterization of sperm-triggered calcium signals in eggs has progressed from the discovery of a single calcium increase at fertilization in the medaka fish to the observation of repetitive calcium waves initiated by multiple meiotic calcium wave pacemakers in the ascidian. In eggs of all animal species, sperm-triggered inositol (1,4,5)-trisphosphate [Ins(1,4,5)P(3)] production regulates the vast array of calcium wave patterns observed in the different species. The spatial organization of calcium waves is driven either by the intracellular distribution of the calcium release machinery or by the localized and dynamic production of calcium-releasing second messengers. In the highly polarized egg cell, cortical endoplasmic reticulum (ER)-rich clusters act as pacemaker sites dedicated to the initiation of global calcium waves. The extensive ER network made of interconnected ER-rich domains supports calcium wave propagation throughout the egg. Fertilization triggers two types of calcium wave pacemakers depending on the species: in mice, the pacemaker site in the vegetal cortex of the egg is probably a site that has enhanced sensitivity to Ins(1,4,5)P(3); in ascidians, the calcium wave pacemaker may rely on a local source of Ins(1,4,5)P(3) production apposed to a cluster of ER in the vegetal cortex.
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Egg cells from every animal and plant species studied to date
elicit single or repetitive Ca
transients in response to
fertilizing sperm. As an important second messenger, Ca
triggers a broad range of cellular reactions, including
contraction, secretion and gene expression (Berridge, 1997;
Bootman et al., 2001). In eukaryotic cells, a calcium wave
starts with an initial increase in Ca
concentration in a
restricted region of the cell (a pacemaker site), which then
propagates, leading to a global Ca
wave (Berridge, 1997;
Bootman et al., 2001; McDougall et al., 2000; Marchant and
Parker, 2001). Several factors are important in determining the
transition from non-propagated elementary Ca
release events
‘puffs’) to the initiation and propagation of a global Ca
wave. They include the magnitude and kinetics of Ca
during each elementary event, the Ca
sensitivity of the Ca
release channels, the spatial organization of release sites, Ca
sequestration and Ca
diffusion, as well as Ca
within the cytosol (Berridge, 1997; Marchant et al., 1999;
Marchant and Parker, 2001; Bootman et al., 2001). In somatic
cells such as hepatocytes, acinar cells or HeLa cells, all or some
of these factors contribute to create a subcellular region of
higher sensitivity to the Ca
-releasing second messengers,
which becomes the pacemaker site (Rooney et al., 1990; Lee
et al., 1997; Petersen et al., 1999; Thomas et al., 1999; Ito et
al., 1999). In eggs, global Ca
waves always initiate in the
cortex and then propagate through the cortex or the whole
cytoplasm (reviewed in Sardet et al., 1998; Stricker, 1999;
McDougall et al., 2000; Kline et al., 1999; Deguchi et al.,
2000). After initiation of the Ca
wave in the cortex,
propagation is due to the sequential activation by Ca
of Ca
release channels at the front of the wave (Berridge, 1997;
Bootman et al., 2001). The Ca
wave speed, which mainly
depends on the rate of passive Ca
diffusion between Ca
release sites, can be influenced by the spatial organization of
these Ca
release sites (Bugrim et al., 1997).
In eggs, Ca
waves triggered by sperm entry result mainly
from the release of Ca
from intracellular stores by inositol
1,4,5 trisphosphate [Ins(1,4,5)P
]-induced Ca
release (IICR)
(reviewed in Miyazaki et al., 1993; Stricker, 1999; McDougall
et al., 2000). The mechanism underlying Ins(1,4,5)P
production in eggs at the time of fertilization is still intensely
debated. Similarly, the nature of the sperm factor(s) inducing
release at fertilization remains elusive, although several
competing groups agree that it must be a protein, possibly a
form of phospholipase C or an activator of it (see Stricker,
1999; Swann and Parrington, 1999; Parrington et al., 2000;
McDougall et al., 2000; Nixon et al., 2000; Runft and Jaffe,
2000; Mehlmann et al., 2001; Jaffe et al., 2001; Carroll, 2001;
Runft et al., 2002). Very recently, a mammalian sperm factor
was characterized and found to be a new form of PLC [PLCζ
(Saunders et al., 2002)]. In most species, it seems reasonable
to assume that the entering sperm delivers a factor into the egg
and that this factor generates Ins(1,4,5)P
either directly or
The first wave, which we will refer to as the ‘fertilization
wave’, is generally the largest and longest-lasting wave,
and, in some species, it is followed by repetitive Ca
of lower amplitude and shorter duration. Ca
pacemakers elicit waves for minutes (20-30 minutes in
During the past 25 years, the characterization of sperm-
triggered calcium signals in eggs has progressed from the
discovery of a single calcium increase at fertilization in the
medaka fish to the observation of repetitive calcium waves
initiated by multiple meiotic calcium wave pacemakers in
the ascidian. In eggs of all animal species, sperm-triggered
inositol (1,4,5)-trisphosphate [Ins(1,4,5)P
] production
regulates the vast array of calcium wave patterns observed
in the different species. The spatial organization of calcium
waves is driven either by the intracellular distribution of
the calcium release machinery or by the localized and
dynamic production of calcium-releasing second
messengers. In the highly polarized egg cell, cortical
endoplasmic reticulum (ER)-rich clusters act as pacemaker
sites dedicated to the initiation of global calcium waves. The
extensive ER network made of interconnected ER-rich
domains supports calcium wave propagation throughout
the egg. Fertilization triggers two types of calcium wave
pacemakers depending on the species: in mice, the
pacemaker site in the vegetal cortex of the egg is probably
a site that has enhanced sensitivity to Ins(1,4,5)P
; in
ascidians, the calcium wave pacemaker may rely on a local
source of Ins(1,4,5)P
production apposed to a cluster of ER
in the vegetal cortex.
Key words: Fertilization, Egg, Calcium oscillations, Calcium wave
pacemaker, Ins(1,4,5)P
, Endoplasmic reticulum, Cortex
Calcium wave pacemakers in eggs
Rémi Dumollard
*, John Carroll
, Geneviève Dupont
and Christian Sardet
Bio Mar Cell, Unité de Biologie du Développement UMR 7009 CNRS/Paris VI, Observatoire, Station Zoologique, Villefranche sur Mer, 06230
Department of Physiology, University College London, Gower Street, London, WC1E 6BT, UK
Unité de Chronobiologie Théorique, Faculté des Sciences, Université Libre de Bruxelles, Brussels, Belgium
*Author for correspondence (e-mail:
Journal of Cell Science 115, 3557-3564 © 2002 The Company of Biologists Ltd
ascidians, 45-60 minutes in some molluscs, 90 minutes in
nemerteans) or hours (4 hours in mammals), and they stop
operating at the end of the meiotic cell cycles (except in
mammals, in which they stop several hours after completion of
meiosis, at the time of pronuclei formation). The pacemaker
site can be fixed in the cortex or undergo dramatic movements
as the cortex is reorganized in preparation for development
(Sardet et al., 2002). Ca
wave pacemakers are either located
in a region of enhanced sensitivity to Ins(1,4,5)P
or reside in
the vicinity of a local source of Ins(1,4,5)P
. Here, we briefly
examine how the subcellular organization of the Ca
machinery may create stable Ca
wave pacemakers in the egg.
We also discuss how spatially and temporally regulated
production of Ins(1,4,5)P
can give rise to multiple calcium
wave pacemakers in a single egg cell.
Different calcium wave pacemakers in different
Two main types of egg can be distinguished with regards to
their patterns of sperm-triggered Ca
signals. Eggs of sea
urchins, amphibians, cnidarians, nematode and fish display a
single Ca
increase upon fertilization. Conversely, eggs of
nemerteans, some molluscs, annelids, ascidians and mammals
display repetitive Ca
waves. In most of these eggs, the
oscillations following the fertilization Ca
wave all emanate
from cortical sites distinct from the initial sperm entry site
(Eckberg and Miller, 1995; Kline et al., 1999; Deguchi et al.,
2000) (reviewed in Sardet, 1998; Stricker, 1999). Perhaps the
most elaborate of these examples is the egg of ascidians
(urochordates at the base of the vertebrate line). The mature
ascidian egg is arrested in metaphase I before fertilization, and
sperm entry induces two series of Ca
waves, driving,
successively, the completion of meiosis I and meiosis II (Fig.
1). The first series originates from the mobile meiosis-I-
associated Ca
wave pacemaker [the MI pacemaker
(McDougall and Sardet, 1995)]. The second meiotic cycle is
entrained by a second pacemaker (the MII pacemaker) stably
located in the vegetal cortex (Speksnijder, 1990b; McDougall
and Sardet, 1995; Dumollard and Sardet, 2001) (Figs 1 and 2).
We do not completely understand why some eggs display
repetitive Ca
waves whereas others exhibit only a single
Journal of Cell Science 115 (18)
Fig. 1. Cortical Ca
wave pacemakers in the ascidian and mouse egg. (A) Sperm-triggered Ca
waves in ascidians: the meiotic Ca
composed of a fertilization wave (F) followed by repetitive Ca
waves, are initiated by two pacemakers (MI PM
and MII PM
). (B) An
artificial pacemaker (artPM
, red arrowhead) can be induced in the animal pole of the egg (a) by global UV photorelease of cgPtdIns(4,5)P
The Ca
waves emitted by this pacemaker are preceded by a pacemaker Ca
rise (red asterisks). (C) The mouse egg is fertilized at metaphase
II and thus possesses only a MII pacemaker (MII PM
). After the fertilization wave (F) starting from the point of sperm entry, repetitive
calcium waves emanate from the vegetal cortex of the egg. Each calcium wave is preceded by a pacemaker calcium rise (red asterisks). (D) The
drawings represent schematically the organization in the ascidian (MI, MII and artPM
) and mouse egg (MII PM
) of the ER network (in
red) and mitochondria (in green). Red arrows indicate the direction of the waves, whereas the postulated sites of Ins(1,4,5)P
production are
symbolized as purple dots. sa: sperm aster; cp: contraction pole. (E) Postulated temporal variations of [Ins(1,4,5)P
, which may underlie the
activity of the meiotic Ca
wave pacemakers. Two possibilities remain for the ascidian and mouse MII pacemakers: a sustained Ins(1,4,5)P
production (pink trace) or oscillatory Ins(1,4,5)P
production (purple trace).
3559Calcium wave pacemakers in eggs
wave. Recent work on ascidian and mouse eggs reveals that
arresting the egg in meiotic metaphase is both sufficient and
necessary to sustain sperm-triggered Ca
oscillations (for
details, see Jones, 1998; Nixon et al., 2000; Carroll, 2001).
Meiotic ‘M-phase’ thus favors repetitive Ca
waves (as in
nemerteans, some molluscs, annelids, ascidians and
mammals). By contrast, only a single large fertilization Ca
wave is observed when fertilization causes a rapid transition to
an interphasic cytoplasm (<20 minutes, as in fish or
amphibians) and when fertilization takes place during
interphase (as in cnidarians or sea urchins). It remains to be
seen whether eggs of fish or amphibians can be made to
undergo Ca
oscillations when blocked in meiotic M-phase
after fertilization. The data relating an M-phase stage of the
cell cycle to the ability to generate multiple Ca
transients is
compelling in ascidian and mouse eggs (reviewed in Nixon et
al., 2000; Carroll, 2001). In these eggs, regulation of the Ca
release machinery by cell cycle factors probably participates in
determining the temporal pattern of the fertilization Ca
signals. However, whether such regulation proves to be
universal requires further research.
The calcium signalling hardware in eggs
The major organelles contributing to the regulation of
intracellular Ca
levels are the endoplasmic reticulum (ER),
the plasma membrane and mitochondria. Eggs also possess
large numbers of specific vesicular organelles (yolk platelets,
pigmented vesicles and cortical granules) that contain Ca
(Gillot et al., 1991); however their role in Ca
homeostasis is
The egg cortex and cytoplasm are filled with an extensive
and continuous ER network (Speksnijder et al., 1993; Jaffe and
Terasaki, 1993; Terasaki et al., 1996; Terasaki et al., 2001;
Stricker et al., 1998; Kline et al., 1999). The ER network
contains the intracellular Ca
channels – the Ins(1,4,5)P
receptors (IP3Rs) and ryanodine receptors (RyRs) – as well as
the sarco-endoplasmic reticulum Ca
-ATPases (SERCAs) that
pump calcium back into the ER.
Among the three known isoforms of IP3R found in somatic
cells (Taylor et al., 1999), IP3R1 is the most prevalent and
functionally important isoform in the egg [Xenopus (Runft et
al., 1999); mammals (Miyazaki et al., 1993; Fissore et al.,
1999; Brind et al., 2000); ascidian (Kyozuka et al., 1998)]. Low
levels of IP3R2 and IP3R3 have been reported in mouse eggs
(Fissore et al., 1999), but their physiological roles remain
unclear (Brind et al., 2000; Jellerette et al., 2000). The other
family of Ca
release channels (RyRs) is present on the
cortical ER of sea urchin eggs (McPherson et al., 1992) and in
ascidian (Albrieux et al., 2000) and mouse eggs (Ayabe et al.,
1995). However, except for sea urchins, the involvement of
RyRs in the initiation and propagation of sperm-triggered
calcium waves appears to be minor, and their role remains
unclear (reviewed in McDougall et al., 2000).
Eggs from all animal phyla seem principally to use Ca
release from internal stores to generate single or repetitive Ca
waves at fertilization. In some species, external Ca
is also
used for the fertilization wave [in molluscs (Deguchi et al.,
1996) (reviewed in Sardet et al., 1998)] or contributes to the
maintenance of the repetitive Ca
waves [in mice (McGuiness
et al., 1996) (reviewed in Stricker, 1999)]. In many species,
voltage-operated Ca
channels [VOCC (Arnoult and Villaz,
1994; Leclerc et al., 2000)] and Ca
-release-activated Ca
(CRAC) channels that mediate so-called ‘capacitative Ca
entry’ (Arnoult et al., 1996; Jaconi et al., 1997; Csutora et al.,
1999; Machaca et al., 2000; Putney et al., 2001) are also
present, but their role at fertilization is still ill defined.
In the mature mouse egg, the physiological Ca
load is
primarily cleared via SERCAs and plasma membrane Ca
ATPases (PMCAs). A minor contribution may also be provided
by the plasma membrane Na
exchanger (Carroll, 2000).
PMCAs are probably responsible for Ca
efflux from ascidian
eggs after each Ca
wave (Kuthreiber et al., 1993) as well as
for the loss of total Ca
content after fertilization in sea urchin
eggs (Gillot et al., 1991).
In the past few years, mitochondria have been shown to be
major regulators of Ca
signals (reviewed in Rutter and
Rizzuto, 2000; Rizzuto et al., 2000; Duchen, 2000).
Sequestration of Ca
by mitochondria has two regulatory
effects on IICR, suppressing positive and negative Ca
feedback on the opening of the IP3R. In addition, ATP
production by mitochondria might provide a further means of
modulating Ca
signals: ATP
sensitizes the IP3R (Mak et
al., 1999; Mak et al., 2001), whereas Mg
-complexed ATP is
consumed to refill the ER Ca
stores. Mitochondria can thus
provide negative or positive feedback on Ins(1,4,5)P
signals. Such negative feedback has been reported in a
wide range of somatic cells. For example, initiation of global
waves in myocytes preferentially occurs in
mitochondrion-poor regions of the cell (Boitier et al., 1999). A
positive feedback effect of mitochondria on Ins(1,4,5)P
mediated signals has been reported only in oligodendrocytes,
in which Ca
wave initiation and amplification sites are found
in mitochondrion-rich regions of the cell (Simpson et al.,
Except in sea urchins, in which mitochondria are a sink for
cytosolic Ca
(Eisen and Reynolds, 1985; Girard et al., 1991),
the role mitochondria play in Ca
signalling in eggs remains
largely obscure. In mouse eggs, collapsing mitochondrial
potential impairs Ca
clearance from the cytosol (Liu et al.,
2001), but no picture of the regulation of Ca
oscillations by
mitochondria can be drawn from only this study. In ascidian
eggs, mitochondria contribute to the activity of the second Ca
wave pacemaker both by buffering cytosolic Ca
and by
locally providing ATP (R.D., unpublished). Nevertheless, an
understanding of the role of mitochondria in regulating Ca
wave pacemakers will require measurement of the local
intracellular Ca
concentration and the local mitochondrial
ATP production in the vicinity of the IP3Rs. The recent
development and subcellular targeting of GFP-based Ca
indicators as well as luciferase-based ATP
indicators should allow the direct measurement of
mitochondrial Ca
levels, intracellular ATP concentration and
the Ins(1,4,5)P
concentration in the living zygote (Hirose et
al., 1999; Rutter and Rizzuto, 2000).
wave pacemakers in eggs reside in cortical ER-
rich domains
Given the central role played by Ca
release from intracellular
stores, the organization of the ER in eggs has received much
attention. In eggs that display repetitive Ca
waves, the
interconnected network of ER sheets and tubes is organized
into ER-rich domains (also called ER clusters). The cytoplasm
also has ER-poor domains, which contain high densities of
mitochondria and/or other vesicular organelles (Speksnijder et
al., 1993; Stricker et al., 1998; Kline et al., 1999; Dumollard
and Sardet, 2001). The ER clusters are made of densely packed
tubes and sheets of ER membrane (Speksnijder et al., 1993;
Fissore et al., 1999; Terasaki et al., 2001; Dumollard and
Sardet, 2001). In oscillating eggs [nemertean (Stricker et al.,
1998); mouse (Kline et al., 1999); ascidian (Dumollard and
Sardet, 2001; Sardet et al., 2002)] as well as in Xenopus eggs
(Terasaki et al., 2001), ER-rich domains are concentrated in the
2-6 µm thick layer beneath the plasma membrane and are more
dispersed in the deeper cytoplasm (deeper than 5 µm). In
ascidian eggs, meiosis II Ca
waves initiate in a large cortical
disc of concentrated ER tubes and sheets (20 µm in diameter
and 2-5 µm thick) located in the vegetal contraction pole (Fig.
2B-F) (Speksnijder, 1992; McDougall and Sardet, 1995;
Dumollard and Sardet, 2001). In mouse and nemerteans eggs,
ER clusters also line the vegetal cortex of the egg where
meiotic Ca
waves are initiated (Stricker et al., 1998; Kline et
al., 1999).
In Xenopus and mouse eggs, at least, these ER-rich domains
are rich in IP3R1s (Terasaki et al., 2001; Mehlmann et al., 1996;
Fissore et al., 1999). The appearance of cortical ER-rich
domains during maturation correlates with an increase in
Journal of Cell Science 115 (18)
Fig. 2. Ca
wave pacemakers in
eggs. (A) A sequence showing a
wave initiated by the
meiosis II pacemaker (MII PM)
in the vegetal pole (v) of a mouse
egg injected with sperm extracts.
The second polar body is visible
in the animal pole (a). (B) A
sequence showing two examples
of fertilized ascidian eggs
displaying Ca
waves initiated
by the MII calcium wave
pacemaker located in the vegetal
contraction pole (MII PM).
(C) Two examples of a UV flash
releasing cgPtdIns(4,5)P
between two waves, the fertilized
ascidian eggs respond by
eliciting a wave from the animal
pole of the egg (artPM). (D) The
distribution of ER and
mitochondria in the contraction
pole (cp). A layer of cortical ER
in the cortical most layer can be
seen (in red) juxtaposed to the
mitochondria-rich sub-cortical
region (in green). (E) Schematic
representation of the contraction
pole showing the microvillated
plasma membrane,
microfilaments (in blue), as well
as the ER-rich domains in the
cortex (red) and the
mitochondria-rich subcortical
domain (green). (F) Calcium
Green/Texas Red ratiometric
image of [Ca
, showing the
initiation of a Ca
wave elicited
by the MII pacemaker in the
contraction pole.
3561Calcium wave pacemakers in eggs
sensitivity to Ins(1,4,5)P
and to sperm-induced Ca
(Chiba et al., 1990; Shiraishi et al., 1995; Mehlmann and Kline,
1994; Terasaki et al., 2001) (reviewed in Sardet et al., 2002).
Local injections of Ins(1,4,5)P
and sperm extracts in mouse
eggs have revealed that the egg cortex is a region of higher
sensitivity to Ins(1,4,5)P
and to sperm extracts (Oda et al.,
1999). Indeed, although the abundance of ER in the egg cortex
renders this region more sensitive to Ins(1,4,5)P
, it is also
exposed to the highest concentrations of Ins(1,4,5)P
as it is
closest to the source of PtdIns(4,5)P
in the plasma membrane
(Halet et al., 2002; Sardet et al., 2002).
The organization of the ER network may regulate the
wave pacemakers
In several somatic cells, the location of the Ca
pacemakers corresponds to the area of the cell that is most
sensitive to Ins(1,4,5)P
(Ito et al., 1999; Thomas et al., 1999;
Petersen et al., 1999), and Ins(1,4,5)P
, which diffuses rapidly
in the cytoplasm, is thought to act as a global messenger
(Albritton et al., 1992; Kasai and Petersen, 1994).
The mouse MII pacemaker appears to be an example of this
type of pacemaker. In the mature mouse egg, ER-rich domains
are larger in the vegetal cortex (Kline et al., 1999), whereas
mitochondria are more abundant in the animal hemisphere
(Calarco, 1995; Van Blerkom et al., 2002). The MII pacemaker
of the mouse egg resides in the ER-enriched vegetal cortex,
which is probably a site of enhanced sensitivity to Ins(1,4,5)P
Therefore, similarly to the somatic cell Ca
wave pacemakers,
the mouse MII pacemaker site appears to be determined by the
organization of the Ca
stores of the egg, with Ins(1,4,5)P
acting as global messenger (Fig. 1, Fig. 2A).
Interestingly, an artificial pacemaker can be induced in the
ascidian egg by global uncaging of caged Ins(1,4,5)P
) or its poorly metabolised analogue
[caged 1-(a-Glycerophosphoryl)-D-myo-
inositol 4,5-bisphosphate, P4(5)]. This artificial pacemaker,
localized in the animal pole of the ascidian egg, functions
under globally elevated Ins(1,4,5)P
levels and thus resides in
the region of highest sensitivity to Ins(1,4,5)P
(Dumollard and
Sardet, 2001) (Figs 1 and 2). In common with the mouse MII
pacemaker, the location of this artificial pacemaker can be
explained by asymmetries in the distribution of the ER along
the animal-vegetal axis of the ascidian egg. In these eggs, the
ER-rich domains invade the whole egg except for the vegetal
subcortex, where most mitochondria accumulate (Fig. 2)
(Dumollard and Sardet, 2001). The corollary of this is that the
sperm-triggered MII pacemaker in the ascidian egg, located in
the vegetal pole (the site opposite the artificial pacemaker), is
not at a site of enhanced Ins(1,4,5)P
sensitivity. This indicates
that the general organization of the ER stores in these eggs is
not sufficient to determine the pacemaker site. The pacemakers
in the ascidian egg may then rely on mechanisms other than a
global increase in Ins(1,4,5)P
Local and dynamic production of Ins(1,4,5)
defines the Ca
wave pacemaker site
In ascidians, the MI pacemaker stimulated by sperm entry
resides in a cortical ER-rich domain that forms rapidly around
the sperm nucleus and centrosome and moves with them
towards the vegetal pole (Fig. 1) (Dumollard and Sardet, 2001).
This suggests that the MI pacemaker of the ascidian relies on
a localised moving source of Ins(1,4,5)P
. The ascidian MII
wave pacemaker does not reside in a region of enhanced
sensitivity to Ins(1,4,5)P
(Fig. 2) (Dumollard and Sardet,
2001); it might thus require local production of Ins(1,4,5)P
the vegetal contraction pole. The contraction pole possesses
numerous microvilli and is thus rich in PtdIns(4,5)P
(Figs 1
and 2) (Sardet et al., 2002). Therefore, the ascidian MII
pacemaker may be different from the characterized pacemakers
of somatic cells and the mouse MII pacemaker, since it would
rely on the apposition of cortical ER-rich clusters to a local
source of Ins(1,4,5)P
(Fig. 1) (Dumollard and Sardet, 2001).
Hypothesizing local production of Ins(1,4,5)P
even in the
large egg cell raises the question of how such a gradient is
maintained. Indeed, as Ins(1,4,5)P
diffuses rapidly through
the cytosol, locally produced Ins(1,4,5)P
would quickly
invade the whole cell, making Ins(1,4,5)P
gradients energy
consuming to maintain without dynamic Ins(1,4,5)P
production. Theoretically, repetitive Ca
waves can result
from either a sustained increase in Ins(1,4,5)P
levels or an
oscillating production of Ins(1,4,5)P
(Jacob, 1990). In
ascidians, a single, large and sustained Ins(1,4,5)P
(achieved by uncaging cgPtdIns(4,5)P
in the whole egg, Fig.
1) mimics the first series of Ca
oscillations, indicating that
the ascidian MI pacemaker is driven by a continuous moving
source of Ins(1,4,5)P
induced by sperm entry (Dumollard and
Sardet, 2001). Similarly in mouse eggs, a slow and continuous
uncaging of cIns(1,4,5)P
can reproduce the sperm-triggered
oscillations (Jones and Nixon, 2000). Therefore, the
mouse MII Ca
wave pacemaker can also be regulated by a
single and sustained increase in Ins(1,4,5)P
levels (Fig. 1).
Furthermore, the Ca
transients triggered by the ascidian MI
and artificial pacemakers, as well as those triggered by the
mouse MII pacemaker, are all preceded by a characteristic slow
rise in Ca
levels called a ‘pacemaker Ca
rise’ (Fig. 1)
(Jones and Nixon, 2000; Dumollard and Sardet, 2001). This
‘pacemaker Ca
rise’ is a hallmark of low-frequency (period
>20 seconds) Ca
oscillations generated under constantly
elevated Ins(1,4,5)P
levels (Jacob, 1990; Marchant and
Parker, 2001), which further suggests that a single and
sustained Ins(1,4,5)P
increase regulates the ascidian MI
pacemaker and the mouse MII pacemaker.
By contrast, the ascidian MII pacemaker cannot be
reproduced by a long-lasting increase in Ins(1,4,5)P
levels in
the egg, and no ‘pacemaker Ca
rise’ precedes these Ca
transients (Fig. 1) (Dumollard and Sardet, 2001). An
oscillating production of Ins(1,4,5)P
from the contraction pole
might underlie the activity of the ascidian Ca
wave MII
pacemaker (Fig. 1). The recent finding that the mammalian
sperm factor is possibly a Ca
-activated phospholipase C
(PLC) (Rice et al., 2000; Saunders et al., 2002) argues in favor
of Ins(1,4,5)P
oscillations driving sperm-triggered Ca
oscillations in eggs. Indeed, the prolonged stimulation of a
-activated PLC can result in Ca
oscillations regulated by
an oscillating production of Ins(1,4,5)P
(for details, see Meyer
and Stryer, 1988). In addition, Ins(1,4,5)P
regulating repetitive Ca
waves during prolonged exposure to
agonists have now been observed in several types of somatic
cells (Hirose et al., 1999; Nash et al., 2001). The issue of
oscillations is an intensively debated topic in cell
physiology, and the Ca
wave pacemakers in eggs could
provide an invaluable experimental system to resolve such
questions in the future.
A role for vegetal Ca
wave pacemakers in
Even though the ascidian and the mouse Ca
pacemakers seem to rely on different mechanisms, they are
both located in the vegetal cortex of the egg (as is the
pacemaker in eggs of the primitive nemertean). This suggests
that the location the Ca
wave pacemaker may have
developmental significance.
The polarized nature of the calcium signals may in itself
influence embryonic patterning by regulating early embryonic
cleavages. In ascidians, nemerteans and mouse, the egg cortex
is polarized along the animal-vegetal axis and, in ascidians, this
polarity amplifies after fertilization through actomyosin-driven
cortical contractions (Sardet et al., 2002). Is the generation of
repetitive Ca
waves from the vegetal cortical pacemaker a
mechanism used to prime the vegetal pole region for later
developmental events such as cleavage or gastrulation, which,
in nemerteans and ascidians, takes place in the vegetal/dorsal
pole of the embryo? Mouse embryos were long thought to have
no significant polarity until the late cleavage stage, but recent
marking experiments show that in fact, as in ascidians and
nemerteans, although regulation can override this polarity,
there is a relationship between the animal-vegetal axis, the
sperm entry point and the developmental axes of pre- and post-
implantation embryos (reviewed in Lu et al., 2001).
Finding out whether Ca
wave patterns play a role in later
development will require studies that interfere with the normal
spatio-temporal pattern of Ca
waves without perturbing
mitosis and cleavage. The rather simple ascidian embryo,
which displays two different meiotic Ca
wave pacemakers
and develops into a swimming tadpole within a day, is
particularly suited to studies of the relationship between
meiotic Ca
waves and development (Fig. 2) (Dumollard and
Sardet, 2001). It should be possible in the future to relate
patterns of Ca
waves and phenotypic differences in embryos.
We thank Mark Larman and Karl Swann for their help with imaging
calcium waves in mouse eggs injected with sperm extracts. We are
also grateful to Christian Rouviere and Mohammed Khamla for
technical assistance.
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Journal of Cell Science 115 (18)
... Cellular Ca 2+ homeostasis is the result of Ca 2+ 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 Ca 2+ waves (reviewed by Sardet et al., 1998;Dumollard et al., 2002). The network hosts Ca 2+ channels [Ins(1,4,5)P3 receptors (IP3R)], which mediate intracellular Ca 2+ release, and Ca 2+ pumps [sarco-endoplasmic reticulum Ca 2+ ATPases (SERCAs)]. ...
... Our data suggest that inhibiting mitochondrial Ca 2+ accumulation during PM2 activity using CN -, while blocking mitochondrial ATP hydrolysis with oligomycin rapidly stops the ability of the pacemaker PM2 to generate Ca 2+ 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), Ca 2+ buffering by mitochondria is necessary to keep the local [Ca 2+ ]c in the activating part of the bell shape curve. The reason why PM1 is insensitive to inhibition of R. Dumollard and others Fig. 5. Interactions between mitochondria and ER in the ascidian egg and in the Ca 2+ and ATP microdomain. ...
... mitochondrial Ca 2+ 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 [Ca 2+ ]c must reach higher levels to inhibit IP3Rs opening and Ca 2+ buffering by mitochondria would not be so crucial. ...
Full-text available
Fertilization increases both cytosolic Ca²⁺ concentration and oxygen consumption in the egg but the relationship between these two phenomena remains largely obscure. We have measured mitochondrial oxygen consumption and the mitochondrial NADH concentration on single ascidian eggs and found that they increase in phase with each series of meiotic Ca²⁺ waves emitted by two pacemakers (PM1 and PM2). Oxygen consumption also increases in response to Ins(1,4,5)P3-induced Ca²⁺ transients. Using mitochondrial inhibitors we show that active mitochondria sequester cytosolic Ca²⁺ during sperm-triggered Ca²⁺ waves and that they are strictly necessary for triggering and sustaining the activity of the meiotic Ca²⁺ wave pacemaker PM2. Strikingly, the activity of the Ca²⁺ wave pacemaker PM2 can be restored or stimulated by flash photolysis of caged ATP. Taken together our observations provide the first evidence that, in addition to buffering cytosolic Ca²⁺, the egg's mitochondria are stimulated by Ins(1,4,5)P3-mediated Ca²⁺ signals. In turn, mitochondrial ATP production is required to sustain the activity of the meiotic Ca²⁺ wave pacemaker PM2.
... In a matter of a few years a sperm-triggered Ca2+ wave in the egg cytosol were first measured by Lionel Jaffe's group using medaka eggs [1]. These Ca2+ waves have now been recorded in mollusks, nemertean, sea urchins, tunicates and vertebrates eggs [69][70][71] and they are all thought to be induced by a sperm factor (or Loeb's ''catalytic substance''). However the sperm factor(s) in tunicates and sea urchins are different from the mammalian PLCzeta and the identity of the sperm factor(s) in marine deuterostome eggs remains unknown [76,72]. ...
... The pattern of Ca2+ signals observed at fertilization varies depending on the cell cycle point of arrest of the mature egg [73]. A close look at the relationship between CSF arrest point and Ca2+ signals reveals that eggs which remain in meiosis for more than 20 min will elicit repetitive Ca2+ transients (mammals, tunicates, nemerteans) whereas eggs arrested in G1 (sea urchin, starfish, jellyfish) or that remain in M phase for less than 20 min after fertilization (like fish or frog) elicit a single Ca2+ transient [71]. Given that a sperm factor is delivered to egg cytosol for generating Ca2+ oscillations, this raises the question of how is the sperm factor inactivated in egg cytosol? ...
... Своеобразно генерация осцилляторной активности формируется у многоклеточных животных. Уже после оплодотворения яйцеклетка сразу начинает продуцировать собственную (эндогенную) генерацию осцилляторной активности [1,2,4,7], с последующим активированием дробления зиготы. У части клеток нервной системы эндогенно возникающая ритмика интерпретируется как появление прообразов нейронов -носителей ритмов [1]. ...
рассмотрены ключевые вопросы постнеклассической природы возникновения и поддержания осцилляторных процессов, являющихся первоосновой в генерации ритмов любого живого вещества — от бактерий до активности мозга человека, которым в последнее время уделяется большое внимание. Данные особенности изучаются в рамках парадигмы числового поля, которому будет отвечать структура-аттрактор гомеостатического фрактально-голографического конструкта, в частности, аттрактор Плыкина, а также в рамках возникновения иерархии кластеров нейронов. При этом попытка описания генерации ритмов живого вещества осуществляется с привлечением наиболее адекватного как физического, так и математического инструментария структуры-аттрактора гомеостатического фрактально-голографического конструкта.Затронутые математические особенности данного конструкта будут способствовать объяснению природы осцилляций живого вещества и природы психического. Все это в рамках данной концепции открывает большой простор для дальнейшего исследования различных психических феноменов и природы сознания человека. the key aspects of the post-nonclassical emergence and maintenance of much studied oscillatory processes as the root of any living creature rhythms, from bacteria to human brain activity. These features are studied within the numerical field paradigm being, a structure-attractor of the homeostatic fractal-holographic construct, in particular the Plykin attractor, and as the emergence of a neuron cluster hierarchy. An attempt to describe the generation of living matter rhythms uses the most adequate physical and mathematical tools of the structure-attractor and homeostatic fractal-holographic construct.The mathematical features of the construct will contribute to explaining the nature of living matter oscillations and the nature of the psychic. This concept is highly promising for further research into various psychic phenomena and the nature of human consciousness.
... Ca 2+ oscillations are often triggered by sperm as is the case in many invertebrate species and in mammalian eggs (see reviews by Sardet et al. 1998;Dumollard et al. 2002). These Ca 2+ oscillations are associated with the meiotic cell cycle (see review by Sardet et al. 1998;Stricker 1999). ...
... The calcium wave EPP is associated with cytoplasmic reorganization and other molecular changes in several phyla. Drawing based on photographs inDumollard et al. (2002). (b) Ooplasmic reorganization in an ascidian egg between fertilization and the start of cleavage. ...
... The spatiotemporal complexity of Ca 2+ signaling at fertilization reflects the diversity of cellular processes that are orchestrated throughout the course of egg activation, and the adverse outcomes associated with deviations from the normal pattern of oscillations (Ducibella and Fissore 2008). The programming of the fertilized egg to generate a series of transient increases in the intracellular Ca 2+ concentration (Dumollard et al. 2002) provides an internal molecular clock that directs sequentially enfolding events of egg activation (Ducibella and Fissore 2008). Clearly, signaling downstream of Ca 2+ is a high-level process that can operate through Ca 2+binding proteins and downstream pathways in other dynamic developing systems. ...
The key, versatile role of intracellular Ca2+ signaling during egg activation after fertilization has been appreciated for several decades. More recently, evidence has accumulated supporting the concept that cytoplasmic Ca2+ is also a major signaling nexus during subsequent development of the fertilized ovum. This chapter will review the molecular reactions that regulate intracellular Ca2+ levels and cell function, the role of Ca2+ signaling during egg activation and specific examples of repetitive Ca2+ signaling found throughout pre- and peri-implantation development. Many of the upstream and downstream pathways utilized during egg activation are also critical for specific processes that take place during embryonic development. Much remains to be done to elucidate the full complexity of Ca2+ signalling mechanisms in preimplantation embryos to the level of detail accomplished for egg activation. However, an emerging concept is that because this second messenger can be modulated downstream of numerous receptors and is able to bind and activate multiple cytoplasmic signaling proteins, it can help the coordination of development through up- and downstream pathways that change with each embryonic stage.
... In 19/24 C. lacteus zygotes, the later calcium waves eventually arise vegetally, even if the sperm enters the animal hemisphere, since a gradual animal-to-vegetal shifting of calcium wave onsets typically occurs in such cases [26]. Subsequently, the final waves tend to originate from a ''vegetal pacemaker'' [38], and these spatiotemporal patterns of calcium signals are typically accompanied by normal embryogenesis, at least as monitored up to blastular hatching. ...
Metaphase-I-arrested eggs of marine protostome worms in the phylum Nemertea generate a series of point-source calcium waves during fertilization. Such calcium oscillations depend on inositol-1,4,5-trisphosphate-mediated calcium release from endoplasmic reticulum (ER) stores that undergo structural reorganizations prior to and after fertilization. This article reviews fertilization-induced calcium transients and ER dynamics in nemertean eggs and compares these topics to what has been reported for other animals in order to identify unifying characteristics and distinguishing features of calcium responses during fertilization across the animal kingdom.
... In somatic cells, the ER system is disrupted in aging and in several pathological conditions [10], and low Ca 2þ in the ER is an effective trigger of cell death by suppressing protein synthesis [11][12][13]. In mammalian oocytes, the presence of the specialized ER clusters in metaphase II (MII) oocytes has been showed to act as sensitive pacemaker sites for the generation of Ca 2þ oscillations at fertilization [14,15]. It has been reported that aging-related changes in Ca 2þ oscillations in aged mouse oocyte might be attributed to dysfunction of intracellular Ca 2þ regulation, presumably of the Ca 2þ pump in the ER [16][17][18]. ...
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Accumulating evidences indicate that cellular and molecular abnormalities occur during oocyte aging, including fragmentation, the increased intracellular reactive oxygen species (ROS) and the abnormal calcium oscillations. The objective of the present study was to characterize relationships among intracellular ROS, calcium homeostasis of endoplasmic reticulum (ER), and fragmentations in aged porcine MII oocytes. Prolonged culture (36 h) of porcine oocytes resulted in the elevated intracellular ROS level, impaired ER Ca(2+) homeostasis (Ca(2+) storage, Ca(2+) rising patterns after electro-activation and the clusters distributions of ER) and increased fragmentation rates. However, when the porcine oocytes were treated with BAPTA-AM, an intracellular calcium chelator, the fragmentations were significantly inhibited during in vitro aging. In order to pursue the underlying mechanisms, the H2O2 and cycloheximide (CHX) were used to artificially increase or inhibit the intracellular ROS levels in aged porcine oocytes during in vitro culture, respectively. The results demonstrated that incubation of porcine MII oocytes with H2O2 damaged the ER clusters and the Ca(2+) regulation of ER, leading to high proportion of fragmented oocytes. Nevertheless, the CHX, an endogenous intracellular ROS generated inhibitor, prevented both of increase of ROS level and damage of the ER Ca(2+) homeostasis in porcine oocytes during aging resulting in low fragmentation rate. We concluded that the increased intracellular ROS damaged the ER clusters and ER Ca(2+) homeostasis resulting in the disorder ooplasmic free Ca(2+), which caused the fragmentations of porcine MII oocytes during aging.
Fertilization encompasses an exquisitely orchestrated, well‐timed physiological, biophysical, and biochemical series of events. Humans undergo internal fertilization, where the sperm are deposited into the female genital tract. Sperm must then overcome a variety of structural and biochemical obstacles and undergo dynamic changes in order to fulfill their potential. The egg (oocyte) plays more of a passive role, in a physical sense, awaiting the arrival of the sperm. Moreover, there is accumulating evidence that the oocyte–cumulus mass releases a chemoattractant to facilitate sperm attraction towards the oocyte. Following activation, the fertilized oocyte will exit meiotic arrest and undergo mitotic cell division and embryo development. This chapter will review the journey the sperm takes in the female reproductive tract, the biophysical and biochemical changes the sperm must undergo to be able to fertilize the oocyte, and the events of oocyte activation.
In the sexual reproduction of animals, fertilization is indispensable for the initiation of diploid embryonic development. Most animals exhibit monospermy, in which only one sperm enters an egg during normal fertilization. In monospermic species, a fast, electrical block on the egg membrane is one of the most important blocks to polyspermy. A fertilizing primary sperm usually causes a positive-going fertilization potential to prevent the subsequent entry of excess sperm. An increase in intracellular Ca²⁺ ([Ca²⁺]i) in the egg cytoplasm induced by the fertilizing sperm is necessary for egg activation and blocks polyspermy. The mechanism of voltage-dependent fertilization in monospermic amphibians is presented as a model system of vertebrate fertilization. The electrical polyspermy blocks in various animals are reviewed and their universality and diversity across the animal kingdom are discussed. Relationships between the fast, electrical block and [Ca²⁺]i increases in egg cytoplasm are discussed, as well as their changes throughout the course of animal evolution.
At fertilization in mammals the sperm triggers a series of oscillations in intracellular Ca2+ within the egg. These Ca2+ oscillations activate the development of the egg into an embryo. It is not known how the sperm triggers these Ca2+ oscillations. There are currently three different theories for Ca2+ signaling in eggs at fertilization. One idea is that the sperm acts as a conduit for Ca2+ entry into the egg after membrane fusion. Another idea is that the sperm acts upon plasma membrane receptors to stimulate a phospholipase C (PLC) within the egg which generates inositol 1,4,5-trisphosphate (InsP3). We present a third idea that the sperm causes Ca2+ release by introducing a soluble protein factor into the egg after gamete membrane fusion. In mammals this sperm factor is also referred to as an oscillogen because, after microinjection, the factor causes sustained Ca2+ oscillations in eggs. Our recent data in sea urchin egg homogenates and intact eggs suggests that this sperm factor has phospholipase C activity that leads to the generation of InsP3. We then present a new version of the soluble sperm factor theory of signaling at fertilization. J. Exp. Zool. (Mol. Dev. Evol.) 285:267–275, 1999. © 1999 Wiley-Liss, Inc.
Although it has been known for over three decades that mitochondria are endowed with a complex array of Ca2+ transporters and that key enzymes of mitochondrial metabolism are regulated by Ca2+, the possibility that physiological stimuli that raise the [Ca2+] of the cytoplasm could trigger major mitochondrial Ca2+ uptake has long been considered unlikely, based on the low affinity of the mitochondrial transporters and the limited amplitude of the cytoplasmic [Ca2+] rises. The direct measurement of mitochondrial [Ca2+] with highly selective probes has led to a complete reversion of this view, by demonstrating that, after cell stimulation, the cytoplasmic Ca2+ signal is always paralleled by a much larger rise in [Ca2+] in the mitochondrial matrix. This observation has rejuvenated the study of mitochondrial Ca2+ transport and novel, unexpected results have altered long-standing dogmas in the field of calcium signalling. Here we focus on four main topics: (i) the current knowledge of the functional properties of the Ca2+ transporters and of the thermodynamic constraints under which they operate; (ii) the occurrence of mitochondrial Ca2+ uptake in living cells and the key role of local signalling routes between the mitochondria and the Ca2+ sources; (iii) the physiological consequences of Ca2+ transport for both mitochondrial function and the modulation of the cytoplasmic Ca2+ signal; and (iv) evidence that alterations of mitochondrial Ca2+ signalling may occur in pathophysiological conditions.