Calcium wave pacemakers in eggs

Abstract

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.

Full text

Introduction
Egg cells from every animal and plant species studied to date
elicit single or repetitive Ca
2+
transients in response to
fertilizing sperm. As an important second messenger, Ca
2+
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
2+
concentration in a
restricted region of the cell (a pacemaker site), which then
propagates, leading to a global Ca
2+
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
2+
release events
(Ca
2+
‘puffs’) to the initiation and propagation of a global Ca
2+
wave. They include the magnitude and kinetics of Ca
2+
release
during each elementary event, the Ca
2+
sensitivity of the Ca
2+
release channels, the spatial organization of release sites, Ca
2+
sequestration and Ca
2+
diffusion, as well as Ca
2+
buffering
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
2+
-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
2+
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
2+
wave in the cortex,
propagation is due to the sequential activation by Ca
2+
of Ca
2+
-
release channels at the front of the wave (Berridge, 1997;
Bootman et al., 2001). The Ca
2+
wave speed, which mainly
depends on the rate of passive Ca
2+
diffusion between Ca
2+
release sites, can be influenced by the spatial organization of
these Ca
2+
release sites (Bugrim et al., 1997).
In eggs, Ca
2+
waves triggered by sperm entry result mainly
from the release of Ca
2+
from intracellular stores by inositol
1,4,5 trisphosphate [Ins(1,4,5)P
3
]-induced Ca
2+
release (IICR)
(reviewed in Miyazaki et al., 1993; Stricker, 1999; McDougall
et al., 2000). The mechanism underlying Ins(1,4,5)P
3
production in eggs at the time of fertilization is still intensely
debated. Similarly, the nature of the sperm factor(s) inducing
Ca
2+
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
3
either directly or
indirectly.
The first wave, which we will refer to as the ‘fertilization
Ca
2+
wave’, is generally the largest and longest-lasting wave,
and, in some species, it is followed by repetitive Ca
2+
waves
of lower amplitude and shorter duration. Ca
2+
wave
pacemakers elicit waves for minutes (20-30 minutes in
3557
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.
Key words: Fertilization, Egg, Calcium oscillations, Calcium wave
pacemaker, Ins(1,4,5)P
3
, Endoplasmic reticulum, Cortex
Summary
Calcium wave pacemakers in eggs
Rémi Dumollard
1,2,
*, John Carroll
2
, Geneviève Dupont
3
and Christian Sardet
1
1
Bio Mar Cell, Unité de Biologie du Développement UMR 7009 CNRS/Paris VI, Observatoire, Station Zoologique, Villefranche sur Mer, 06230
France
2
Department of Physiology, University College London, Gower Street, London, WC1E 6BT, UK
3
Unité de Chronobiologie Théorique, Faculté des Sciences, Université Libre de Bruxelles, Brussels, Belgium
*Author for correspondence (e-mail: r.dumollard@ucl.ac.uk)
Journal of Cell Science 115, 3557-3564 © 2002 The Company of Biologists Ltd
doi:10.1242/jcs.00056
Commentary

3558
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
2+
wave pacemakers are either located
in a region of enhanced sensitivity to Ins(1,4,5)P
3
or reside in
the vicinity of a local source of Ins(1,4,5)P
3
. Here, we briefly
examine how the subcellular organization of the Ca
2+
release
machinery may create stable Ca
2+
wave pacemakers in the egg.
We also discuss how spatially and temporally regulated
production of Ins(1,4,5)P
3
can give rise to multiple calcium
wave pacemakers in a single egg cell.
Different calcium wave pacemakers in different
species
Two main types of egg can be distinguished with regards to
their patterns of sperm-triggered Ca
2+
signals. Eggs of sea
urchins, amphibians, cnidarians, nematode and fish display a
single Ca
2+
increase upon fertilization. Conversely, eggs of
nemerteans, some molluscs, annelids, ascidians and mammals
display repetitive Ca
2+
waves. In most of these eggs, the
oscillations following the fertilization Ca
2+
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
2+
waves, driving,
successively, the completion of meiosis I and meiosis II (Fig.
1). The first series originates from the mobile meiosis-I-
associated Ca
2+
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
2+
waves whereas others exhibit only a single
Journal of Cell Science 115 (18)
Fig. 1. Cortical Ca
2+
wave pacemakers in the ascidian and mouse egg. (A) Sperm-triggered Ca
2+
waves in ascidians: the meiotic Ca
2+
waves,
composed of a fertilization wave (F) followed by repetitive Ca
2+
waves, are initiated by two pacemakers (MI PM
asc
and MII PM
asc
). (B) An
artificial pacemaker (artPM
asc
, red arrowhead) can be induced in the animal pole of the egg (a) by global UV photorelease of cgPtdIns(4,5)P
2
.
The Ca
2+
waves emitted by this pacemaker are preceded by a pacemaker Ca
2+
rise (red asterisks). (C) The mouse egg is fertilized at metaphase
II and thus possesses only a MII pacemaker (MII PM
mouse
). 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
asc
) and mouse egg (MII PM
mouse
) 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
3
production are
symbolized as purple dots. sa: sperm aster; cp: contraction pole. (E) Postulated temporal variations of [Ins(1,4,5)P
3
]
c
, which may underlie the
activity of the meiotic Ca
2+
wave pacemakers. Two possibilities remain for the ascidian and mouse MII pacemakers: a sustained Ins(1,4,5)P
3
production (pink trace) or oscillatory Ins(1,4,5)P
3
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
2+
oscillations (for
details, see Jones, 1998; Nixon et al., 2000; Carroll, 2001).
Meiotic ‘M-phase’ thus favors repetitive Ca
2+
waves (as in
nemerteans, some molluscs, annelids, ascidians and
mammals). By contrast, only a single large fertilization Ca
2+
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
2+
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
2+
transients is
compelling in ascidian and mouse eggs (reviewed in Nixon et
al., 2000; Carroll, 2001). In these eggs, regulation of the Ca
2+
release machinery by cell cycle factors probably participates in
determining the temporal pattern of the fertilization Ca
2+
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
2+
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
2+
(Gillot et al., 1991); however their role in Ca
2+
homeostasis is
unknown.
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
2+
channels – the Ins(1,4,5)P
3
receptors (IP3Rs) and ryanodine receptors (RyRs) – as well as
the sarco-endoplasmic reticulum Ca
2+
-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
2+
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
2+
release from internal stores to generate single or repetitive Ca
2+
waves at fertilization. In some species, external Ca
2+
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
2+
waves [in mice (McGuiness
et al., 1996) (reviewed in Stricker, 1999)]. In many species,
voltage-operated Ca
2+
channels [VOCC (Arnoult and Villaz,
1994; Leclerc et al., 2000)] and Ca
2+
-release-activated Ca
2+
(CRAC) channels that mediate so-called ‘capacitative Ca
2+
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
2+
load is
primarily cleared via SERCAs and plasma membrane Ca
2+
ATPases (PMCAs). A minor contribution may also be provided
by the plasma membrane Na
+
/Ca
2+
exchanger (Carroll, 2000).
PMCAs are probably responsible for Ca
2+
efflux from ascidian
eggs after each Ca
2+
wave (Kuthreiber et al., 1993) as well as
for the loss of total Ca
2+
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
2+
signals (reviewed in Rutter and
Rizzuto, 2000; Rizzuto et al., 2000; Duchen, 2000).
Sequestration of Ca
2+
by mitochondria has two regulatory
effects on IICR, suppressing positive and negative Ca
2+
feedback on the opening of the IP3R. In addition, ATP
production by mitochondria might provide a further means of
modulating Ca
2+
signals: ATP
4–
sensitizes the IP3R (Mak et
al., 1999; Mak et al., 2001), whereas Mg
2+
-complexed ATP is
consumed to refill the ER Ca
2+
stores. Mitochondria can thus
provide negative or positive feedback on Ins(1,4,5)P
3
-mediated
Ca
2+
signals. Such negative feedback has been reported in a
wide range of somatic cells. For example, initiation of global
Ca
2+
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
3
-
mediated signals has been reported only in oligodendrocytes,
in which Ca
2+
wave initiation and amplification sites are found
in mitochondrion-rich regions of the cell (Simpson et al.,
1997).
Except in sea urchins, in which mitochondria are a sink for
cytosolic Ca
2+
(Eisen and Reynolds, 1985; Girard et al., 1991),
the role mitochondria play in Ca
2+
signalling in eggs remains
largely obscure. In mouse eggs, collapsing mitochondrial
potential impairs Ca
2+
clearance from the cytosol (Liu et al.,
2001), but no picture of the regulation of Ca
2+
oscillations by
mitochondria can be drawn from only this study. In ascidian
eggs, mitochondria contribute to the activity of the second Ca
2+
wave pacemaker both by buffering cytosolic Ca
2+
and by
locally providing ATP (R.D., unpublished). Nevertheless, an
understanding of the role of mitochondria in regulating Ca
2+
wave pacemakers will require measurement of the local
intracellular Ca
2+
concentration and the local mitochondrial
ATP production in the vicinity of the IP3Rs. The recent
development and subcellular targeting of GFP-based Ca
2+
and
Ins(1,4,5)P
3
indicators as well as luciferase-based ATP
indicators should allow the direct measurement of
mitochondrial Ca
2+
levels, intracellular ATP concentration and
the Ins(1,4,5)P
3
concentration in the living zygote (Hirose et
al., 1999; Rutter and Rizzuto, 2000).
Ca
2+
wave pacemakers in eggs reside in cortical ER-
rich domains
Given the central role played by Ca
2+
release from intracellular
stores, the organization of the ER in eggs has received much
attention. In eggs that display repetitive Ca
2+
waves, the

3560
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
2+
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
2+
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
2+
wave pacemakers in
eggs. (A) A sequence showing a
Ca
2+
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
2+
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
2
applied
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
2+
]
c
, showing the
initiation of a Ca
2+
wave elicited
by the MII pacemaker in the
contraction pole.

3561Calcium wave pacemakers in eggs
sensitivity to Ins(1,4,5)P
3
and to sperm-induced Ca
2+
release
(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
3
and sperm extracts in mouse
eggs have revealed that the egg cortex is a region of higher
sensitivity to Ins(1,4,5)P
3
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
3
, it is also
exposed to the highest concentrations of Ins(1,4,5)P
3
as it is
closest to the source of PtdIns(4,5)P
2
in the plasma membrane
(Halet et al., 2002; Sardet et al., 2002).
The organization of the ER network may regulate the
Ca
2+
wave pacemakers
In several somatic cells, the location of the Ca
2+
wave
pacemakers corresponds to the area of the cell that is most
sensitive to Ins(1,4,5)P
3
(Ito et al., 1999; Thomas et al., 1999;
Petersen et al., 1999), and Ins(1,4,5)P
3
, 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
3
.
Therefore, similarly to the somatic cell Ca
2+
wave pacemakers,
the mouse MII pacemaker site appears to be determined by the
organization of the Ca
2+
stores of the egg, with Ins(1,4,5)P
3
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
3
(cIns(1,4,5)P
3
) or its poorly metabolised analogue
cgPtdIns(4,5)P
2
[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
3
levels and thus resides in
the region of highest sensitivity to Ins(1,4,5)P
3
(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
3
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
3
levels.
Local and dynamic production of Ins(1,4,5)
P
3
defines the Ca
2+
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
3
. The ascidian MII
Ca
2+
wave pacemaker does not reside in a region of enhanced
sensitivity to Ins(1,4,5)P
3
(Fig. 2) (Dumollard and Sardet,
2001); it might thus require local production of Ins(1,4,5)P
3
in
the vegetal contraction pole. The contraction pole possesses
numerous microvilli and is thus rich in PtdIns(4,5)P
2
(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
3
(Fig. 1) (Dumollard and Sardet, 2001).
Hypothesizing local production of Ins(1,4,5)P
3
even in the
large egg cell raises the question of how such a gradient is
maintained. Indeed, as Ins(1,4,5)P
3
diffuses rapidly through
the cytosol, locally produced Ins(1,4,5)P
3
would quickly
invade the whole cell, making Ins(1,4,5)P
3
gradients energy
consuming to maintain without dynamic Ins(1,4,5)P
3
production. Theoretically, repetitive Ca
2+
waves can result
from either a sustained increase in Ins(1,4,5)P
3
levels or an
oscillating production of Ins(1,4,5)P
3
(Jacob, 1990). In
ascidians, a single, large and sustained Ins(1,4,5)P
3
increase
(achieved by uncaging cgPtdIns(4,5)P
2
in the whole egg, Fig.
1) mimics the first series of Ca
2+
oscillations, indicating that
the ascidian MI pacemaker is driven by a continuous moving
source of Ins(1,4,5)P
3
induced by sperm entry (Dumollard and
Sardet, 2001). Similarly in mouse eggs, a slow and continuous
uncaging of cIns(1,4,5)P
3
can reproduce the sperm-triggered
Ca
2+
oscillations (Jones and Nixon, 2000). Therefore, the
mouse MII Ca
2+
wave pacemaker can also be regulated by a
single and sustained increase in Ins(1,4,5)P
3
levels (Fig. 1).
Furthermore, the Ca
2+
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
2+
levels called a ‘pacemaker Ca
2+
rise’ (Fig. 1)
(Jones and Nixon, 2000; Dumollard and Sardet, 2001). This
‘pacemaker Ca
2+
rise’ is a hallmark of low-frequency (period
>20 seconds) Ca
2+
oscillations generated under constantly
elevated Ins(1,4,5)P
3
levels (Jacob, 1990; Marchant and
Parker, 2001), which further suggests that a single and
sustained Ins(1,4,5)P
3
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
3
levels in
the egg, and no ‘pacemaker Ca
2+
rise’ precedes these Ca
2+
transients (Fig. 1) (Dumollard and Sardet, 2001). An
oscillating production of Ins(1,4,5)P
3
from the contraction pole
might underlie the activity of the ascidian Ca
2+
wave MII
pacemaker (Fig. 1). The recent finding that the mammalian
sperm factor is possibly a Ca
2+
-activated phospholipase C
(PLC) (Rice et al., 2000; Saunders et al., 2002) argues in favor
of Ins(1,4,5)P
3
oscillations driving sperm-triggered Ca
2+
oscillations in eggs. Indeed, the prolonged stimulation of a
Ca
2+
-activated PLC can result in Ca
2+
oscillations regulated by
an oscillating production of Ins(1,4,5)P
3
(for details, see Meyer
and Stryer, 1988). In addition, Ins(1,4,5)P
3
oscillations
regulating repetitive Ca
2+
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
Ins(1,4,5)P
3
oscillations is an intensively debated topic in cell

3562
physiology, and the Ca
2+
wave pacemakers in eggs could
provide an invaluable experimental system to resolve such
questions in the future.
A role for vegetal Ca
2+
wave pacemakers in
development
Even though the ascidian and the mouse Ca
2+
wave
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
2+
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
2+
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
2+
wave patterns play a role in later
development will require studies that interfere with the normal
spatio-temporal pattern of Ca
2+
waves without perturbing
mitosis and cleavage. The rather simple ascidian embryo,
which displays two different meiotic Ca
2+
wave pacemakers
and develops into a swimming tadpole within a day, is
particularly suited to studies of the relationship between
meiotic Ca
2+
waves and development (Fig. 2) (Dumollard and
Sardet, 2001). It should be possible in the future to relate
patterns of Ca
2+
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.
References
Albrieux, M., Moutin, M. J., Grunwald, D. and Villaz, M. (2000).
Calmodulin and immunophilin are required as functional partners of a
ryanodine receptor in ascidian oocytes at fertilization. Dev. Biol. 225, 101-
111.
Albritton, N. L., Meyer, T. and Stryer, L. (1992). Range of messenger action
of calcium ions and IP3. Science 258, 1812-1815.
Arnoult, C. and Villaz, M. (1994). Differential developmental fates of the
two calcium currents in early embryos of the ascidian Ciona intestinalis. J.
Membr. Biol. 137, 127-135.
Arnoult, C., Grunwald, D. and Villaz, M. (1996). Novel postfertilization
inward Ca2+ current in ascidian eggs ensuring a calcium entry throughout
meiosis. Dev. Biol. 174, 322-334.
Ayabe, T., Kopf, G. S. and Schultz, R. M. (1995). Regulation of mouse egg
activation: presence of ryanodine receptors and effects of microinjected
ryanodine and cyclic ADP ribose on uninseminated and inseminated eggs.
Development 121, 2233-2244.
Berridge, M. J. (1997). Elementary and global aspects of calcium signalling.
J. Physiol. 499, 291-306.
Boitier, E., Rea, R. and Duchen, M. (1999). Mitochondria exert a negative
feedback on the propagation of intracellular Ca
2+
waves in rat cortical
astrocytes. J. Cell Biol. 145, 795-808.
Bootman, M. D., Lipp, P. and Berridge, M. J. (2001). The organisation and
functions of local Ca
2+
signals. J. Cell Sci. 114, 2213-2222.
Brind, S., Swann, K. and Carroll, J. (2000). Inositol 1,4,5-trisphosphate
receptors are downregulated in mouse oocytes in response to sperm or
adenophostin A but not to increases in intracellular Ca(2+) or egg activation.
Dev. Biol. 223, 251-265.
Bugrim, A. E., Zhabotinsky, A. M. and Epstein, I. R. (1997). Calcium waves
in a model with a random spatially discrete distribution of Ca
2+
release sites.
Biophys. J. 73, 2897-2906.
Calarco, P. G. (1995). Polarization of mitochondria in the unfertilised mouse
oocyte. Dev. Genet. 16, 36-43.
Carroll, J. (2000). Na
+
-Ca
2+
exchange in mouse oocytes: modifications in the
regulation of intracellular free Ca
2+
during oocyte maturation. J. Reprod.
Fertil. 118, 337-342.
Carroll, J. (2001). The initiation and regulation of Ca
2+
signalling at
fertilization in mammals. Semin. Cell Dev. Biol. 12, 37-43.
Chiba, K., Kado, R. T. and Jaffe, L. A. (1990). Development of calcium
release mechanisms during starfish oocyte maturation. Dev. Biol. 140, 300-
306.
Csutora, P., Su, Z., Kim, H. Y., Bugrim, A. and Cunningham, K. W.
(1999). Calcium influx factor is synthesized by yeast and mammalian
cells depleted of organellar calcium stores. Proc. Natl. Acad. Sci. USA
96, 121-126.
Deguchi, R., Osanai, K. and Morisawa, M. (1996). Extracellular Ca
2+
entry
and Ca
2+
release from inositol 1,4,5- trisphosphate-sensitive stores function
at fertilization in oocytes of the marine bivalve Mytilus edulis. Development
122, 3651-3660.
Deguchi, R., Shirakawa, H., Oda, S., Mohri, T. and Miyazaki, S. (2000).
Spatiotemporal analysis of Ca
2+
waves in relation to the sperm entry site
and animal-vegetal axis during Ca
2+
oscillations in the fertilised mouse eggs.
Dev. Biol. 218, 299-313.
Duchen, M. (2000). Mitochondria and calcium: from cell signalling to cell
death. J. Physiol. 529, 57-68.
Dumollard, R. and Sardet, C. (2001). Three different calcium wave
pacemakers in ascidian eggs. J. Cell Sci. 114, 2471-2481.
Eisen, A. and Reynolds, G. T. (1985). Source and sinks for the calcium
released during fertilization of single sea urchin eggs. J. Cell Biol. 100,
1522-1527.
Eckberg, W. R. and Miller, A. L. (1995). Propagated and non propagated
calcium transients during egg activation in the annelid, Chaetopterus. Dev.
Biol. 172, 654-664.
Fissore, R. A., Longo, F. J., Anderson, E., Parys, J. B. and Ducibella, T.
(1999). Differential distribution of inositol trisphosphate receptor isoforms
in mouse oocytes. Biol. Reprod. 60, 49-57.
Gillot, I., Ciapa, B., Payan, P. and Sardet, C. (1991). The calcium content
of cortical granules and the loss of calcium from sea urchin eggs at
fertilization. Dev. Biol. 146, 396-405.
Girard, J., Gillot, I., Renzis, G. D. and Payan, P. (1991). Calcium pools in
sea urchin eggs: roles of endoplasmic reticulum mitochondria in relation to
fertilization. Cell. Calcium 12, 289-299.
Halet, G., Tunwell, R., Balla, T., Swann, K. and Carroll, J. (2002). The
dynamics of plasma membrane PtdIns(4,5)P(2) at fertilization of mouse
eggs. J. Cell Sci. 115, 2139-2149.
Hirose, K., Kadowaki, S., Tanabe, M., Takeshima, H. and Iino, M. (1999).
Spatiotemporal dynamics of inositol trisphosphate that underlies complex
calcium mobilization patterns. Science 284, 1527-1530.
Ito, K., Miyashita, Y. and Kasai, H. (1999). Kinetic control of multiple forms
of Ca2+ spikes by inositol trisphosphate in pancreatic acinar cells. J. Cell
Biol. 146, 405-413.
Jacob, R. (1990). Calcium oscillations in electrically non-excitable cells.
Biochem. Biophys. Acta 1052, 427-428.
Jaconi, M., Pyle, J., Bortolon, R., Ou, J. and Clapham, D. (1997). Calcium
release and influx colocalize to the endoplasmic reticulum. Curr. Biol. 7,
599-602.
Jaffe, L. F. (1991). The path of calcium in cytosolic calcium oscillations: a
unifying hypothesis. Proc. Natl. Acad. Sci. USA 88, 9883-9887.
Jaffe, L. A. and Terasaki, M. (1993). Structural changes of the endoplasmic
reticulum of sea urchin eggs during fertilization. Dev. Biol. 156, 566-573.
Jaffe, L. A., Giusti, A. F., Carroll, D. J. and Foltz, K. R. (2001). Ca
2+
Journal of Cell Science 115 (18)

3563Calcium wave pacemakers in eggs
signalling during fertilization of echinoderm eggs. Semin. Cell. Dev. Biol.
12, 45-51.
Jellerette, T., He, C. L., Wu, H., Parys, J. B. and Fissore, R. A. (2000).
Down-regulation of the inositol 1,4,5-trisphosphate receptor in mouse eggs
following fertilization or parthenogenetic activation. Dev. Biol. 223, 238-
250.
Jones, K. (1998). Ca
2+
oscillations in the activation of the egg and
development of the embryo in mammals. Int. J. Dev. Biol. 42, 1-10.
Jones, K. and Nixon, V. (2000). Sperm-induced Ca(2+) oscillations in mouse
oocytes and eggs can be mimicked by photolysis of caged inositol 1,4,5-
trisphosphate: evidence to support a continuous low level production of
inositol 1, 4,5-trisphosphate during mammalian fertilization. Dev. Biol. 225,
1-12.
Kasai, H. and Petersen, O. H. (1994). Spatial dynamics of second
messengers: IP3 and cAMP as long-range and associative messengers.
Trends Neurosci. 17, 95-101.
Kline, D., Mehlmann, L., Fox, C. and Terasaki, M. (1999). The cortical
endoplasmic reticulum (ER) of the mouse egg: localization of ER clusters
in relation to the generation of repetitive calcium waves. Dev. Biol. 215, 431-
442.
Kuthreiber, W. M., Gillot, I., Sardet, C. and Jaffe, L. F. (1993). Net calcium
and acid release at fertilization in eggs of sea urchins and ascidians. Cell.
Calcium 14, 73-86.
Kyozuka, K., Deguchi, R., Mohri, T. and Miyazaki, S. (1998). Injection of
sperm extract mimics spatiotemporal dynamics of Ca
2+
responses and
progression of meiosis at fertilization of ascidian oocytes. Development 125,
4099-4105.
Leclerc, C., Guerrier, P. and Moreau, M. (2000). Role of dihydropyridine-
sensitive calcium channels in meiosis and fertilization in the bivalve molluscs
Ruditapes philippinarum and crassostrea gigas. Biol. Cell 92, 285-299.
Lee, M. G., Xu, X., Zheng, W., Diaz, J. and Wojcikiewicz, R. J. (1997).
Polarized expression of Ca2+ channels in pancreatic and salivary gland cells.
J. Biol. Chem. 272, 15765-15770.
Liu, L., Hammar, K., Smith, P. J., Inoue, S. and Keefe, D. L. (2001).
Mitochondrial modulation of calcium signaling at the initiation of
development. Cell. Calcium 30, 423-433.
Lu, C. C., Brennan, J. and Robertson, E. J. (2001). From fertilization to
gastrulation: axis formation in the mouse embryo. Curr. Opin. Genet. Dev.
11, 384-392.
Machaca, K. and Haun, S. (2000). Store-operated calcium entry inactivates
at the germinal vesicle breakdown stage of Xenopus meiosis. J. Biol. Chem.
275, 38710-38715.
Mak, D., McBride, S. and Foskett, J. (1999). ATP regulation of type 1
inositol 1,4,5-trisphosphate receptor channel gating by allosteric tuning of
Ca
2+
activation. J. Biol. Chem. 274, 22231-22237.
Mak, D., McBride, S. and Foskett, J. (2001). ATP regulation of recombinant
type 3 inositol 1,4,5-trisphosphate receptor gating. J. Gen. Physiol. 117,
447-456.
Marchant, J. S. and Parker, I. (2001). Role of elementary Ca
2+
puff in
generating repetitive Ca
2+
oscillations. EMBO J. 20, 65-76.
Marchant, J. S., Callamaras, N. and Parker, I. (1999). Initiation of InsP
3
-
mediated calcium waves in Xenopus oocytes. EMBO J. 18, 5285-5299.
McDougall, A. and Sardet, C. (1995). Function and characteristics of
repetitive calcium waves associated with meiosis. Curr. Biol. 5, 318-328.
McDougall, A., Shearer, J. and Whitaker, M. (2000). The initiation and
propagation of the fertilization wave in sea urchin eggs. Biol. Cell 92, 205-
214.
McGuinness, O. M., Moreton, R. B., Johnson, M. H. and Berridge, M. J.
(1996). A direct measurement of increased divalent cation influx in fertilised
mouse oocytes. Development 122, 2199-2206.
McPherson, S. M., McPherson, P. S., Mathews, L., Campbell, K. P. and
Longo, F. J. (1992). Cortical localization of a calcium release channel in
sea urchin eggs. J. Cell Biol. 116, 1111-1121.
Mehlmann, L. M. and Kline, D. (1994). Regulation of intracellular calcium
in the mouse egg: calcium release in response to sperm or inositol
trisphosphate is enhanced after meiotic maturation. Biol. Reprod. 51, 1088-
1098.
Mehlmann, L. M., Mikoshiba, K. and Kline, D. (1996). Redistribution and
increase in cortical inositol 1,4,5-trisphosphate receptors after meiotic
maturation of the mouse oocyte. Dev. Biol. 180, 489-498.
Mehlmann, L. M., Chattopadhyay, A., Carpenter, G. and Jaffe, L. A.
(2001). Evidence that phospholipase C from the sperm is not responsible
for initiating Ca
2+
release at fertilization in mouse eggs. Dev. Biol. 236, 492-
501.
Meyer, T. and Stryer, L. (1988). Molecular model for receptor-stimulated
calcium spiking. Proc. Natl. Acad. Sci. USA 85, 5051-5055.
Miyazaki, S., Shirakawa, H., Nakada, K. and Honda, Y. (1993). Essential
role of the inositol trisphosphate receptor/Ca
2+
release channel in Ca
2+
waves and Ca
2+
oscillations at fertilization of mammalian eggs. Dev. Biol.
158, 62-78.
Nash, M. S., Young, K. W., Willars, G. B., Challiss, R. A. J. and
Nahorski, S. R. (2001). Single-cell imaging and graded Ins(1,4,5)P3
production following G-protein-coupled-receptor activation. Biochem. J.
356, 137-142.
Nixon, V. L., McDougall, A. and Jones, K. T. (2000). Calcium oscillations
and the cell cycle at fertilization of mammalian and ascidian eggs. Biol. Cell
92, 187-196.
Oda, S., Deguchi, R., Mohri, T., Shikano, T., Nakanishi, S. and Miyazaki,
S. (1999). Spatiotemporal dynamics of the [Ca
2+
]i rise induced by
microinjection of sperm extracts into mouse eggs: preferential induction of
a Ca
2+
wave from the cortex mediated by the inositol 1,4,5-trisphosphate
receptor. Dev. Biol. 209, 172-185.
Parrington, J., Lai, F. A. and Swann, K. (2000). The soluble mammalian
sperm factor protein that triggers Ca2+ oscillations in eggs: evidence for
expression of mRNA(s) coding for sperm factor protein(s) in spermatogenic
cells. Biol. Cell 92, 267-275.
Petersen, O. H., Burdakov, D. and Tepikin, A. V. (1999). Polarity in
intracellular calcium signalling. BioEssays 21, 851-860.
Putney, J. W., Jr, Broad, L. M., Braun, F. J., Lievremont, J. P. and Bird,
G. S. (2001). Mechanisms of capacitative calcium entry. J. Cell Sci. 114,
2223-2239.
Rice, A., Parrington, J., Jones, K. T. and Swann, K. (2000). Mammalian
sperm contain a Ca(2+)-sensitive phospholipase C activity that can generate
InsP(3) from PIP(2) associated with intracellular organelles. Dev. Biol. 228,
125-135.
Rizzuto, R., Bernardi, P. and Pozzan, T. (2000). Mitochondria as all-round
players of the calcium game. J. Physiol. 529, 37-47.
Rooney, T. A., Sass, E. J. and Thomas, A. P. (1990). Agonist-induced
cytosolic calcium oscillations originate from a specific locus in single
hepatocytes. J. Biol. Chem. 265, 10792-70796.
Runft, L. L. and Jaffe, L. A. (2000). Sperm extract injection into ascidian
eggs signals Ca(2+) release by the same pathway as fertilization.
Development 127, 3227-3236.
Runft, L. L., Watras, J. and Jaffe, L. A. (1999). Calcium release at
fertilization of Xenopus eggs requires type I IP(3) receptors, but not SH2
domain-mediated activation of PLCgamma or G(q)-mediated activation of
PLCbeta. Dev. Biol. 214, 399-411.
Runft, L. L., Jaffe, L. A. and Mehlmann, L. M. (2002). Egg activation at
fertilization: where it all begins. Dev. Biol. 245, 237-254.
Rutter, G. A. and Rizzuto, R. (2000). Regulation of mitochondrial
metabolism by ER Ca2+ release: an intimate connection. Trends Biochem.
Sci. 25, 215-221.
Sardet, C., Roegiers, F., Dumollard, R., Rouviere, C. and McDougall, A.
(1998). Calcium waves and oscillations in eggs. Biophys. Chem. 72, 131-
140.
Sardet, C., Prodon, F., Dumollard, R., Chang, P. and Chenevert, J. (2002).
Structure and function of the egg cortex. Dev. Biol. 241, 1-23.
Saunders, C. M., Larman, M. G., Parrington, J., Cox, L. J., Royse, J.,
Blayney, L. M., Swann, K. and Lai, F. A. (2002). PLCζ: a sperm-specific
trigger of Ca
2+
oscillations in eggs and embryo development. Development
129, 3533-3544.
Shiraishi, K., Okada, A., Shirakawa, H., Nakanishi, S., Mikoshiba, K. and
Miyazaki, S. (1995). Developmental changes in the distribution of the
endoplasmic reticulum and inositol 1,4,5-trisphosphate receptors and the
spatial pattern of Ca
2+
release during maturation of hamster oocytes. Dev.
Biol. 170, 594-606.
Simpson, P. B., Mehotra, S., Lange, G. D. and Russell, J. T. (1997). High
density distribution of endoplasmic reticulum proteins and mitochondria at
specialized Ca2+ release sites in oligodendrocyte processes. J. Biol. Chem.
272, 22654-22661.
Speksnijder, J. (1992). The repetitive calcium waves in the fertilised ascidian
egg are initiated near the vegetal pole by a cortical pacemaker. Dev. Biol.
153, 259-271.
Speksnijder, J., Sardet, C. and Jaffe, L. (1990a). The activation wave of
calcium in the ascidian egg and its role in ooplasmic segregation. J. Cell
Biol. 110, 1589-1598.
Speksnijder, J., Sardet, C. and Jaffe, L. (1990b). Periodic calcium waves
cross ascidian eggs after fertilization. Dev. Biol. 142, 246-249.

3564
Speksnijder, J., Terasaki, M., Hage, W., Jaffe, L. and Sardet, C. (1993).
Polarity and reorganisation of the endoplasmic reticulum during
fertilization and ooplasmic segregation in the ascidian egg. J. Cell Biol.
120, 1337-1346.
Stricker, S. (1999). Comparative biology of calcium signaling during
fertilization and egg activation in animals. Dev. Biol. 211, 157-176.
Stricker, S. A., Silva, R. and Smythe, T. (1998). Calcium and endoplasmic
reticulum dynamics during oocyte maturation and fertilization in the marine
worm Cerebratulus lacteus. Dev. Biol. 203, 305-322.
Swann, K. and Parrington, J. (1999). Mechanism of Ca
2+
release at
fertilization in mammals. J. Exp. Zool. 285, 267-275.
Taylor, C. W., Genazzani, A. A. and Morris, S. A. (1999). Expression of
inotistol trisphosphate receptors. Cell. Calcium 26, 237-251.
Terasaki, M., Jaffe, L. A., Hunnicutt, G. R. and Hammer, J. A., 3rd (1996).
Structural change of the endoplasmic reticulum during fertilization:
evidence for loss of membrane continuity using the green fluorescent
protein. Dev. Biol. 179, 320-328.
Terasaki, M., Runft, L. L. and Hand, A. R. (2001). Changes in organisation
of the endoplasmic reticulum during Xenopus oocyte maturation and
activation. Mol. Biol. Cell 12, 1103-1116.
Thomas, D., Lipp, P., Tovey, S. C., Berridge, M. J. and Li, W. (1999).
Microscopic properties of elementary calcium release sites in non-excitable
cells. Curr. Biol. 18, 8-15
Van Blerkom, J., Davis, P., Mathwig, V. and Alexander, S. (2002). Domains
of high-polarized and low-polarized mitochondria may occur in mouse and
human oocytes and early embryos. Hum. Reprod. 17, 393-406.
Journal of Cell Science 115 (18)