Review
TRENDS in Biochemical Sciences
Vol.29 No.8 August 2004
Calcium and fertilization: the beginning
of life
Luigia Santella1, Dmitri Lim1,2 and Francesco Moccia1
1
2
Laboratory of Cell Biology, Stazione Zoologica ‘A. Dohrn’, Villa Comunale I-80121, Napoli, Italy
Department of Biochemistry, University of Padova, Viale G. Colombo 3, 35131, Padova, Italy
The explosive increase in Ca2C that occurs in the cytosol
at fertilization is brought about by the activation of
Ca2C-release channels in the intracellular stores. Inositol
1,4,5-trisphosphate (InsP3) is traditionally considered to
be the messenger that initiates the increase and
spreading of the activating Ca2C wave. In line with this
hypothesis, recent evidence suggests that the penetrating sperm delivers into mammalian eggs a novel isoform
of phospholipase C (PLC), which promotes the formation of InsP3. By contrast, data from echinoderms
studies indicate that the newly discovered second
messenger nicotinic adenine dinucleotide phosphate
(NAADP) promotes an initial, localized increase in Ca2C,
which is then followed by the InsP3-mediated globalization of the Ca2C wave. The mechanism by which the
interacting sperm triggers the production of NAADP and
subsequently that of InsP3 remains obscure.
After fertilization, oocytes or eggs from plants to humans
experience an increase in Ca2C that can occur in the form
of either a single transient or repetitive spikes at the point
of sperm entry (Figure 1). This increase in Ca2C then
propagates across the egg as a global wave [1–3]. It could
be preceded by a sudden rise in Ca2C in the cortex
(the ‘cortical flash’), which is produced by an influx of
Ca2C through voltage-gated Ca2C channels that are
activated during the fertilization potential, which is the
first detectable response of an egg to the sperm [4–6]
(Box 1). The rise in intracellular Ca2C triggers the
quiescent egg into metabolic activity and starts embryonic development: one might say that it marks the
beginning of new life.
The importance of Ca2C in egg activation was realized
in the 1920 s and 1930 s: the finding that various marine
eggs could be activated by exposure to solutions enriched
in Ca2C, or by inducing damage to the plasma membrane
(e.g. by UV or radium rays) in Ca2C-containing media, led
to the ‘Ca2C theory of activation’ [7], which proposed that
an influx of Ca2C was required to initiate development
of the egg. According to this theory, Ca2C induced a
complete structural reorganization of the egg in a process
that was defined as the ‘cortical reaction’: namely,
exocytosis of the cortical granules, which makes the egg
refractory to further insemination, and resumption of
the cell cycle.
Corresponding author: Luigia Santella ([email protected]).
The final proof that Ca2C had a crucial role, and the
precise definition of the chain of events that linked the
Ca2C increase to the cortical reaction, had to wait for
the development of reliable quantitative methods for
measuring intracellular Ca2C [8]. Once these methods
became available, it was conclusively established that the
cortical reaction was indeed caused by the intracellular
increase in Ca2C. It then became necessary to identify
the targets of this Ca2C increase. Gradually, the search
became intensive, eventually leading to the recent general
consensus that the targets are calmodulin-dependent
kinases. One of these kinases, Ca2C/calmodulin-dependent
kinase II, becomes activated after Ca2C oscillations in
Figure 1. Fertilization-induced Ca2C signals. Ca2C is monitored with the Ca2C
indicator Oregon Green 488 BAPTA-1. (a) A plot of the relative fluorescence of the
Ca2C indicator over time offers a numerical equivalent of the Ca2C increase induced
by sperm in a starfish oocyte (Astropecten auranciacus). Arrow indicates the first
detectable Ca2C signal induced by the sperm in the cortical region of the oocyte
(the ‘cortical flash’). (b) By contrast, the Ca2C oscillations associated with
fertilization of an ascidian oocyte (Ciona intestinalis) consist of two oscillatory
phases separated by a time gap of w3 min.
www.sciencedirect.com 0968-0004/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2004.06.009
Review
TRENDS in Biochemical Sciences
Vol.29 No.8 August 2004
401
Box 1. The fertilization potential
mammalian oocytes, and inhibitors that act on it (or on
calmodulin itself ) block cortical granule exocytosis and
delay formation of the second polar body [9,10].
The other process that the Ca2C theory of activation
traced back to Ca2C was resumption of the cell cycle. Here,
an important step forward occurred in 1971 with the
discovery of M-phase promoting factor (MPF) – a
CDK1/cyclin B kinase that drives mitosis and meiosis
exit [11–13]. In vertebrate oocytes arrested at metaphase
of meiosis II, the increase in Ca2C at fertilization is
translated into inactivation of the cytostatic factor – an
endogenous inhibitor of meiotic division responsible for
stabilizing the activity of MPF [14]. The consequent
reduction in MPF permits resumption of the cell cycle.
It is now known that in amphibians and mammals, the
sperm-induced Ca2C oscillations can trigger degradation
of cyclin B by activating the anaphase-promoting complex/cyclosome [15,16]. In echinoderms, Ca2C can inactivate mitogen-activated protein kinase by activating a
Ca2C-responsive phosphatase. Inactivation of this kinase
is both sufficient and necessary to initiate DNA synthesis
[17]. Here, we summarize the most recent findings on the
molecular mechanisms underlying the onset and propagation of the rise of intracellular Ca2C at fertilization.
Specifically, we highlight the part played by a novel
isoform of phospholipase C (PLC), namely PLCz, and the
recently discovered second messenger NAADP in the
sperm-induced Ca2C wave.
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+20
0
V (mV)
In numerous species, the first known response of oocytes or eggs to
the sperm is a change in the membrane potential (Vm) – the so-called
‘fertilization’ or ‘activation’ potential. Its main function is to provide a
fast block to polyspermy, which protects the oocytes or eggs during
the first minutes after insemination while a permanent block is being
established by modifications to their extracellular coat [4].
In most invertebrates, sperm induces a membrane depolarization
that shifts Vm to the threshold of activation of the voltage-gated Ca2C
channels, triggering a regenerative process, after which there is a
plateau or periodic oscillations, depending on the species. The mechanism responsible for the initial depolarization has been identified
in the ascidian Ciona intestinalis as a cationic current activated by
ADP-ribose [5]. Information is scarce on the trigger of the fertilization
potential in other invertebrate oocytes [4]. The prolonged plateau after
the Ca2C action potential is due to the entry of NaC, which is dependent on an intracellular release of Ca2C in echinoderms and nemerteans, but not in echiurans; by contrast, the second series of
oscillations in Vm recorded in the ascidian C. intestinalis requires the
contribution of a Ca2C -release-activated current [6].
Among vertebrates, only frogs show the electrical block to polyspermy, which is mediated by a ClK current triggered by an inositol
1,4,5-trisphosphate (InsP 3)-dependent intracellular Ca 2C wave.
Indeed, the electrophysiological response of mammalian oocytes to
fertilization consists of periodic membrane hyperpolarizations caused
by the activation of Ca2C-dependent KC channels. These hyperpolarizations have no role in preventing sperm entry.
In invertebrates, the Ca2C spike that accompanies the fertilization
potential contributes to intracellular Ca2C signaling, where it appears
as a spherically symmetric subcortical Ca2C increase – the so-called
‘cortical flash’ – preceding the point-source Ca2C wave (Figure I). In
bivalves and echiurans, however, the cortical flash spreads centripetally towards the center of the oocyte, with no contribution from
intracellular Ca2C-release channels [2]. In these species, therefore, the
fertilization also accomplishes the function of activating the oocytes.
−20
−40
−60
−80
30
60
90
120
Time (sec)
Figure I. Fertilization potential of Asterina pectinifera oocytes. The electrophysiological response of an echinoderm oocyte to the sperm. The oocyte is
first matured in vitro with 1-methyladenine (1-MA) and then exposed to the
sperm. The graph shows the Ca2C spike (arrow) recorded w70 s after the
addition of sperm. This is the Ca2C action potential that is responsible for
establishing the fast block to polyspermy. Approximately 5 s after the Ca2C
spike, the membrane potential reaches approximately K40 mV and then slowly
increases again reaching a peak of approximately C15 mV, after which it
recovers to the baseline within w25 min (not shown). During the recovery,
the elevation of the fertilization envelope gives rise to the slow block to
polyspermy.
Furthermore, in C. intestinalis, Ca2C influx during the second series of
oscillations in Vm, is required to refill internal Ca2C stores and to
maintain the intracellular Ca2C spikes.
Preparing the egg for the sperm: development of Ca2Crelease systems
Before the egg encounters the sperm, the systems that will
generate the Ca2C response in the egg must be prepared
for the event: in other words, the ability of eggs to produce
a proper Ca2C response and to undergo normal exocytosis
of cortical granules at fertilization must be developed.
This occurs during the so-called ‘maturation process’
[18,19]. A prominent aspect of this process is an increase
in the sensitivity of the endoplasmic reticulum (ER) Ca2Creleasing system to the gating ligand (mainly InsP3). This
increase in sensitivity is linked to reorganization of the
ER, which in several species forms discrete aggregates
(clusters) and domains that correlate with ability to
generate the normal Ca2C response at fertilization [20,21].
The maturation-associated changes in the Ca2C-release
mechanism in oocytes are now well documented [22,23].
Time-lapse visualization in Xenopus has shown that the
ER of maturing or mature oocytes moves rapidly,
suggesting that ER mobility has a role in the dynamic
redistribution of InsP3 receptors (InsP3Rs) to the cortical
region [24]. InsP3Rs have been also shown to increase and
to redistribute to the cortex in maturing mouse oocytes,
thereby enabling optimal Ca2C spiking at fertilization
[25]. In experiments where RNA interference was used
to prevent the increase in type 1 InsP3Rs, a 50%
decrease in the number of sperm-induced Ca2C spikes
was observed [26].
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Review
10 min
TRENDS in Biochemical Sciences
12 min
Vol.29 No.8 August 2004
14 min
18 min
Time after 1-MA
22 min
30 min
Figure 2. Spatio-temporal changes of the inositol 1,4,5-trisphosphate (InsP3)-induced Ca2C release during oocyte maturation. Starfish oocytes are coinjected with the Ca2C
dye Oregon Green 488 BAPTA-1 and InsP3, which is caged to inhibit its activity before photoliberation. The agonist is liberated by photoactivation at different times after
application of the maturing hormone 1-methyladenine (1-MA). Shown are pseudocolored relative fluorescence images of the InsP3-induced increase in Ca2C. Blue
corresponds to low Ca2C levels, whereas green and yellow correspond to higher Ca2C levels. After global photoactivation of InsP3, a Ca2C increase is detected in the animal
hemisphere containing the nucleus of an oocyte matured for 12 min with 1-MA. A slightly higher Ca2C response after uncaging of InsP3 is detected in a different oocyte
matured for 14 min with 1-MA. Note that at this time of maturation (14 min), the animal cortical region of the oocyte is much more sensitive to InsP3, which is liberated
throughout the oocyte after UV. The area of higher InsP3 sensitivity then spreads towards the vegetal hemisphere and becomes global 30 min after the hormonal stimulation.
These findings have been extended to starfish oocytes,
in which the Ca2C stores are well developed in the
immature stage: sensitivity to injected InsP3 increases in
oocytes challenged with the maturing hormone 1-methyladenine (1-MA) [18]. The ER of immature starfish oocytes
is composed of interconnected membrane sheets, which
form spherical shells after associating with the yolk
granule in response to 1-MA [27]. The increased sensitivity of Ca2C stores to InsP3 starts in the perinuclear area
at the animal hemisphere and propagates to the whole
oocyte along the animal–vegetal axis (Figure 2). Notably,
the change in response to InsP3 in starfish oocytes is not
linked to the redistribution of InsP3Rs or to the increase in
their expression [28], but to modulation of their sensitivity
to the ligand by the actin cytoskeleton [29].
The sperm meets the egg: Ca2C on move
Decades after the proposal of the Ca2C theory of
activation, a series of experiments was done in which
the activation of sea urchin eggs was induced with a Ca2C
ionophore in the absence of external Ca2C. The findings
shifted the emphasis to the liberation of Ca2C from
intracellular stores as the factor that promotes egg
activation [30], adding fresh interest to the debate on
the role of Ca2C influx in sea urchin fertilization [31].
Studies on the latent period between the time of gamete
fusion and the initiation of the activating Ca2C wave, in
which Ca2C influx was inhibited by lanthanum or by
buffering external Ca2C, supported the ‘sperm conduit
model of egg activation’ in sea urchin. In this model, Ca2C
flows from the seawater through the fused sperm
acromosal process into the cortical region of the egg [32].
In fact, recent work has partially revitalized this
proposal by showing that the increase in Ca2C in the
cortical region (the cortical flash), which in many species
precedes the propagating wave, spreads centripetally
towards the center of the cytoplasm in mollusc and
echiuran oocytes, originating a Ca2C wave that is dependent on Ca2C influx [4,33,34]. In starfish oocytes, an initial
release of Ca2C at a circumscribed point in the cortex
expands to generate the cortical flash and then a Ca2C
wave initiates. Injection of the InsP3R antagonist heparin
before fertilization inhibits globalization of the wave, but
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it does not affect the cortical flash, indicating that the
cortical flash is not related to the liberation of Ca2C from
InsP3-sensitive stores [35].
When the Ca2C theory of activation was proposed, no
direct quantitative estimate of the cytosolic increase in Ca2C
had been made. This measurement was first achieved much
later in medaka eggs, where a transient rise in the
luminescence of injected aequorin was observed and quantified after fertilization or after activation by a Ca2C
ionophore [8]. These experiments were rapidly extended to
several other species by using the full panoply of fluorescent dyes that had, in the meantime, become available.
InsP3 soon became a favorite object of study: the
involvement of its receptors in the onset of the Ca2C
response was suggested by the observation that the
injection of InsP3 triggered an intracellular release of
Ca2C in all species studied. Moreover, injection of the
antagonist heparin, monoclonal antibodies directed
against InsP3Rs or an ‘InsP3 sponge’ that sequesters
InsP3 strongly inhibited the Ca2C wave at fertilization in
most species including starfish [28,36,37]. Coupled with
experiments using the Src homology domain 2 (SH2)
domain of PLCg, these findings have provided compelling
support for the hypothesis that InsP3 has a dominant role
in generating the Ca2C signal at fertilization.
The mechanism that links the egg–sperm interaction to
the Ca2C increase rapidly became the topic of intensive
research and soon led to two main hypotheses. According
to the first, binding of the sperm to externally located
receptors initiates a signal transduction cascade that is
transduced in the activation of PLCs and in the intracellular increase in Ca2C through the production of InsP3
[38]. According to the second, the sperm delivers an
activating factor into the egg on fusion of the gametes.
This factor was initially proposed to be Ca2C itself, which
would open (gate) the InsP3-sensitive Ca2C channels once
liberated in the cytosol; however, the intracellular microinjection of Ca2C does not reproduce the pattern of Ca2C
increase observed at fertilization. As a result, the proposal
that the sperm delivers into the egg a factor other than
Ca2C that can stimulate the turnover of phosphoinositides
has gained popularity (Figure 3).
Review
TRENDS in Biochemical Sciences
(a) The receptor hypothesis
Vol.29 No.8 August 2004
403
(b) The sperm factor hypothesis
R
G proteins
Integrins Tyr K
PLC
PIP2
NAADP
PLCζ
NAADP channel
Ca2+
PIP2
Ca2+
InsP3 channel
ER
InsP3
Ca2+
InsP3
ER
Ca2+
Ca2+
Figure 3. Signal transduction pathways at fertilization. (a) According to the receptor hypothesis, generation of the Ca2C signal at fertilization might occur via several routes,
leading to activation of phospholipase C (PLC). The increase in PLC activity could be modulated by signaling cascades involving integrins, tyrosine kinases (Tyr K) and
G proteins. In this model, the PLC that cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) and forms inositol 1,4,5- trisphosphate (InsP3) originates from the egg. InsP3 binds
to corresponding InsP3 receptors and induces Ca2C release from the endoplasmic reticulum (ER) internal stores. (b) The sperm factor hypothesis is consistent with the
introduction of a finite bolus of PLCz by mammalian sperm, leading to an increase in InsP3. Alternatively, in echinoderms the fertilization-induced initial increase in Ca2C
could be triggered by the sperm injecting nicotinic acid adenine dinucleotide phosphate (NAADP), and the subsequent wave would be propagated by the InsP3 receptors.
Generation of Ca2C signals upon egg–sperm interaction
Option one: the receptor hypothesis
In sea urchin egg, effective contact of the sperm was
thought to occur through bindin, the main protein exposed
on the acrosomal process of the sperm. Bindin was purified
from sea urchin sperm and characterized as a factor that
could interact with a specific egg receptor [39]. The latter
was identified as a glycoprotein of 350 kDa with a short
C-terminal cytoplasmic domain and an extracellular
domain homologous to the Hsp70 heat-shock proteins
[40]. Later re-examination of the sequence of this receptor
in sea urchin egg indicated, however, that it was an
extrinsic binding protein of the vitelline layer rather than
an integral plasma membrane protein [41].
At present, therefore, the proposal of a receptor on
the egg for sperm is based on indirect evidence: for
example, on experiments of the overexpression of
G-protein-linked receptors in oocytes and on the
finding that the application of their ligands mimicks
activation. Naturally, these heterologous receptor
expression data do not conclusively prove that
G proteins function at fertilization, but collateral
evidence supports the suggestion that a G protein
can activate PLCb in response to the sperm interaction: the sperm-induced Ca2C transients are inhibited by injection of the G-protein antagonist guanosine
5 0 -thiodiphosphate (GDP-bS) in hamster oocytes [42].
Furthermore, injection of the hydrolysis-resistant GTP
analogue, GTP-gS, in sea urchin, frog and mammalian
eggs causes Ca2C release [43]. At variance with these
findings, other experiments have shown that pertussis
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toxin and the microinjection of inhibitory Gq do not
block the sperm-induced activation in mouse and
Xenopus eggs [44].
A variant of the receptor/G protein proposal, which is
gaining increasing attention, suggests that the increase in
Ca2C might be caused by activation of a tyrosine kinase
signaling pathway that targets PLCg. The recombinant
expression of membrane receptors known to release Ca2C
by a tyrosine kinase/PLCg pathway, such as the receptors
for epidermal growth factor or platelet-derived growth
factor, in frog and starfish oocytes indeed triggers Ca2C
release in response to the respective agonists, indicating
that a tyrosine kinase might be an upstream regulator of
PLCg at fertilization [44]. In line with this suggestion, an
increase in tyrosine kinase activity and in the amount of
tyrosine-phosphorylated proteins has been observed at
fertilization [45].
The PLCg hypothesis has generated several interesting
experiments. PLCg contains an SH2 domain, whose
microinjection into sea urchin eggs abolishes the Ca2C
transient at fertilization – a clear indication that the latter
might be initiated by the production of InsP3 by activated
PLCg [46]. In Xenopus oocytes, PLCg is tyrosine-phosphorylated and activated within a few minutes of
fertilization, and subsequently becomes associated with
and upregulated by a Src-related protein-tyrosine kinase
named Xyk [47]. The role of ‘non-receptor’ Src kinases is
supported by experiments on several species [48,49]. In
starfish oocytes, injection of an active Src kinase initiates
the Ca2C wave, resumption of meiosis and replication of
DNA in the absence of the fertilizing sperm [44].
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TRENDS in Biochemical Sciences
The production of InsP3 on the activation of tyrosine
kinases coupled to PLCg activation could be also caused by
integrin binding, an event that has been shown to have a
role in mammalian fertilization. Over the past decade,
several integrins (a family of transmembrane glycoprotein
receptors) have been identified and localized on the cell
surface of the cells of many animal species, including
humans [50]. Several signaling tyrosine kinases (e.g. FAK,
Src kinases) are stimulated in response to the activation of
integrin receptors.
One of the best-characterized molecules involved in
sperm–egg adhesion and fusion is fertilin-b (also known
as ADAM2), a sperm ligand belonging to the ADAMs
(a disintegrin and a metalloprotease domain) family.
Results from the inhibition of mouse fertilization with
an antibody specific for a6 integrin (GoH3) had previously
suggested that a6b1 integrin functions on mouse egg as
the receptor for fertilin-b [51]; however, a6b1 integrin is
apparently not essential for sperm–egg fusion because
normal fertilization occurs in the eggs of mice lacking the
a6 integrin subunit [52]. Recently, another egg surface
protein, CD9, has been proposed to function in sperm–egg
binding and fusion, either directly or by interacting with
egg proteins other than a6b1 [53]. But even if CD9 has an
essential role in promoting fusion, it does not initiate
signaling events per se.
Option two: the sperm factor hypothesis
The proposal that the sperm triggers Ca2C release
through a sperm factor that enters the egg after gamete
fusion to stimulate InsP3 synthesis is currently the most
popular hypothesis. The proposal was first advanced in
1985 by Dale et al. [54], on the basis of their finding that
the microinjection of an extract from sea urchin sperm
triggered the cortical reaction in sea urchin eggs. These
pioneering experiments opened a new avenue of investigation, and convincing evidence in favor of the hypothesis
has been produced in many other species. Curiously, however, it has not been confirmed in sea urchin. Notably,
sperm extracts have been found to promote a Ca2C
signaling cascade in oocytes that show Ca2C oscillations,
but not in oocytes or eggs that are characterized by a
single Ca2C transient, such as those of starfish, sea
urchin, fish and frog (Figure 1).
The nature of the sperm factor has been mysterious
for long time, but data from several laboratories have
suggested that it might be a protein [55–57]. For example,
the addition of sperm protein extracts to homogenates of
sea urchin eggs has been shown to induce the Ca2C
response via InsP3Rs and ryanodine receptors (RyRs), and
to do so by a mechanism independent of low molecular
weight messengers such as InsP3 or cyclic ADP-ribose
(cADPr). Oscillin, a 33-kDa protein homolog of glucosamine 6-phosphate isomerase or glucosamine 6-phosphate
deaminase, was detected and cloned in hamster spermatozoa and proposed to modulate the Ca2C oscillations [58];
however, the injection of human recombinant oscillin into
mouse metaphase II oocytes does not elicit a Ca2C
response, whereas the injection of soluble sperm extracts
does [59]. Later studies have shown that oscillin might
have a role in the acrosome reaction [60].
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Vol.29 No.8 August 2004
Very recent work has now significantly advanced our
knowledge of the nature of the sperm factor protein,
which, at least in mammalian sperm, might be a new type
of PLC named PLCz. The suggestion was prompted by
the finding that recombinant PLCz expressed in mouse
eggs not only triggers Ca2C oscillations similar to those
induced by the sperm, but also promotes subsequent
embryonic development [61]. A similar response is
obtained by microinjecting purified mouse PLCz protein
into mouse eggs [62]. These are exciting results, although
at present they are confined to mammals because a nonmammalian form of PLCz has not been identified.
A soluble factor from the sperm cytosol has been recently
shown to activate ascidian oocytes through the same
signal transduction molecules that are active at fertilization. The possibility that PLCg is recruited in response to
the liberation of a sperm factor that might directly or
indirectly regulate a Src kinase cannot be discounted [44].
NAADP: the initial Ca2C messenger at fertilization in
echinoderms?
The results described above have dealt with the role of
InsP3Rs in initiation of the Ca2C response at fertilization.
However, the injection of several other small molecules,
including cGMP, nitric oxide (NO), cADPr and NAADP,
into the eggs of various species can also mobilize Ca2C.
Specifically, the increase in Ca2C caused by cGMP is due to
mobilization of Ca2C through a route that is independent
of InsP3Rs [63]. NO synthase is present at high concentrations in activated sea urchin eggs: an increase in
nitrosation occurs seconds after insemination and ahead
of the Ca2C response, suggesting that NO might be a
universal activator of eggs [64]. Simultaneous measurements of intracellular NO and Ca2C in the same species
have established, however, that the rise in NO occurs only
after initiation of the Ca2C wave and thus acts as a
regulator of the duration of the Ca2C response [65].
The second messengers cADPr and NAADP have been
both found to release Ca2C from sea urchin microsomes
and intact eggs and from starfish oocytes [66–71] (Box 2).
Because cADPr was found to increase the Ca2C sensitivity
of the Ca2C -induced Ca2C-release mechanism in sea urchin
eggs, it was suggested that the cADPr/RyRs pathway
contributed to propagation of the Ca2C wave at fertilization, whereas the InsP3Rs initially triggered it [72].
When starfish oocytes are injected with the specific cADPr
antagonist 8-NH2-cADPr, however, no inhibition of the
Ca2C wave propagation is observed [68].
At least in echinoderms, novel data now point to a
specific role of NAADP receptors in initiation of the Ca2C
response. Early studies on sea urchin had shown that
NAADP mobilizes Ca2C from stores that are insensitive to
InsP3 and cADPr, in line with the observation that
NAADP-responsive sea urchin microsomes migrate differently from InsP3- and cADPr-sensitive pools in percol
gradients [69]. The NAADP-induced release of Ca2C
differs from the other two Ca2C-release systems by its
insensitivity to cytosolic Ca2C and pH – a feature that
would make NAADP more suitable for triggering the
Ca2C signal than for propagating it [73]. Notably, although
the inhibition of both InsP3Rs and cADPr/RyRs blocks the
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Box 2. The synthesis of cADPr and NAADP
The adenine nucleotides cyclic ADP-ribose (cADPr) and nicotinic
adenine dinucleotide phosphate (NAADP) are intracellular messengers that have recently joined inositol 1,4,5-trisphosphate (InsP3) in the
regulation of intracellular Ca2C [69]. cADPr and NAADP are synthesized by the same family of enzymes, namely the ADP-ribosyl
cyclases. This family includes enzymes purified from Aplysia ovotestis
and the protozoan Euglena, as well as the CD38 lymphocyte cellsurface antigen and the bone marrow stromal cell antigen 1 (CD157).
These proteins share 25–30% sequence identity and have different
intracellular localizations.
In Aplysia, the cyclase has been purified from the cytosolic fraction,
whereas CD38, CD157 and the Euglena cyclase are membrane-bound
enzymes. The largest amount of information is available on CD38,
which has been found in the plasma membrane, mitochondria,
endoplasmic reticulum and nuclei. cADPr originates from the cyclization of b-nicotinamide adenine dinucleotide (NAD) after displacement
(a)
of the nicotinamide moiety and is degraded by cADPr hydrolases
to ADPr (Figure I).
Both CD38 and CD157 are bifunctional enzymes, being also able to
hydrolyze cADPr, whereas the Aplysia cyclase lacks this hydrolyzing
activity. In addition to regulating cADPr metabolism, CD38 and the
Aplysia cyclase catalyze the exchange of the nicotinamide moiety of
b-NADP with nicotinic acid to produce NAADP. This base-exchange
reaction is dominant over the cyclization reaction at acidic pH and
undergoes differential regulation by cyclic nucleotides (Figure I).
Indeed, NAADP production might be potentiated by cAMP, whereas
cGMP mainly enhances the synthesis of cADPr.
It has been recently reported that the levels of cADPr increase at
fertilization [70] and that NAADP is synthesized in sea urchin sperm in
micromolar concentrations. NAADP might be released into the eggs on
sperm–egg interaction [71]. These results point to a role of cADPr and
NAADP in both onset and propagation of the sperm-induced Ca2C wave.
O
NH2
O
NH2
P
N1
P
NAD
NAD
Cyclization
P
(b)
P
O
O
NH2
NH2
O
O
NH2
N–
P
P
NAD
P
NAD
Base exchange
P
P
P
Figure I. Reactions catalyzed by the ADP-ribosyl cyclase. (a) cADPr is derived from NAD by a cyclization reaction. The site of cyclization is at the N1 position (green) of the
adenine ring, which liberates the nicotinamide group of the precursor NAD. (b) In the base-exchange reaction, ADP-ribosyl cyclase converts NADP to NAADP by replacing
nicotinamide with nicotinic acid.
fertilization-induced Ca2C wave, a small rise in Ca2C can
still be detected, which could be due to NAADP [72].
Observations in starfish oocytes have now strongly
reinforced the suggestion that NAADP has a triggering
role in the echinoderm Ca2C response at fertilization. The
Ca2C signal detected on the photoactivation of caged
NAADP uniformly distributed in the oocyte consists of a
cortical Ca2C flash, which spreads throughout the cytoplasm as a wave, leading to elevation of the fertilization
envelope, as seen by light microscopy (Figure 4). Notably, the
response is absent in Ca2C-free sea water and is selectively
inhibited by blockers of L-type and store-operated Ca2C
channels. Thus, an influx of Ca2C from the extracellular
space seems to be involved in the NAADP response [74]. In
agreement with this, NAADP activates a Ca2C-mediated
inward current that shows biophysical properties similar to
those of other Ca2C-mediated currents, namely, those activated by Ca2C store depletion and by arachidonic acid [75].
www.sciencedirect.com
A role for the still unknown NAADP receptor in
initiation of the Ca2C response at fertilization has been
recently supported by experiments on starfish oocytes
from which the germinal vesicle (nucleus) had been
removed before the initiation of maturation with 1-MA.
Removal of the germinal vesicle does not affect the Ca2C
response or the cortical exocytotic process induced by
NAADP, or the initial cortical flash or the cortical reaction
elicited by the sperm, but it abolishes cortical granule
exocytosis and globalization of the wave induced by InsP3
[74]. These results suggest that NAADP triggers the Ca2C
signal at fertilization in starfish oocytes, whereas InsP3Rs
propagate the wave from the sperm entry point to the
antipode.
Subsequent work has shown that NAADP also activates a cortical flash dependent on Ca2C influx in sea
urchin eggs. The desensitization of NAADP receptors
strongly reduces both the cortical flash and the Ca2C wave
Review
406
TRENDS in Biochemical Sciences
Transmitted light Relative fluorescence
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Vol.29 No.8 August 2004
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Seconds after UV irradiation
Figure 4. Increase in Ca2C induced by the global uncaging of NAADP in a starfish oocyte. Spatio-temporal changes after the photoactivation of NAADP are pseudocolored as
in Figure 2. The Ca2C dye is injected into the cytoplasm of an oocyte, which is then exposed for 50 min to 1-methyladenine (1-MA). After germinal vesicle breakdown, NAADP
– which is caged to maintain inactivity of the oocyte – is injected into the mature oocyte and allowed to spread throughout the whole cytoplasm for 10 min before the
uncaging reaction. The photoactivation of NAADP triggers a Ca2C response that occurs mainly in the cortical region (second pseudocolored relative fluorescence image),
even though the agonist is uniformly liberated throughout the cell after UV irradiation. The Ca2C increase then spreads centripetally to the center of the oocyte. The elevation
of the fertilization envelope occurs as a result of the increase in Ca2C (arrow). The removal of external Ca2C inhibits the NAADP-induced increase in Ca2C.
elicited by the sperm, indicating that NAADP is involved
in the activation of sea urchin eggs by the sperm [71]. The
origin of the NAADP that might intervene in this activation remains obscure, but recent experiments have shed
some light on this issue. Of particular significance is the
finding that a marked increase in NAADP occurs in the
sea urchin sperm after its contact with the egg jelly and
that NAADP is subsequently transferred into the egg [76].
Concluding remarks
The Ca2C theory of fertilization has been with us for a long
time. We owe to it the origins of a very fertile area of
research, which has accumulated a large body of data.
Until recently, the mechanism by which the sperm
generates the Ca2C signals had essentially remained
unknown; now, at long last, this situation seems to be
changing.
On the one hand, recent data have led to the identification of a credible candidate – a new isoform of PLC –
as the ‘sperm factor’, the nature of which had been
mysterious for a long time. Even if this candidate turns
out not to function in all species, this finding is of the
utmost interest. On the other hand, the emergence of
NAADP as a very plausible initiator of the Ca2C signal
offers a completely new perspective on the spatio-temporal
hierarchy of the Ca2C-linked messengers that have a role
in the process. Here again, future work will have to clarify
whether NAADP functions at fertilization in non-echinoderms, but the findings so far are certainly promising.
Elucidation of the Ca2C signaling at fertilization is thus
finally coming of age: some issues remain unresolved, such
as the relationship of PLCz to the generation and action of
NAADP, but we can confidently hope that important
solutions to this and other problems are now at hand.
Acknowledgements
We thank Ernesto Carafoli for helpful comments and for critically reading
the manuscript; Giovanni Gragnaniello for the image analysis and for
preparing the figures; and Gilda A. Nusco and Emanuela Ercolano for
their participation in some of the experimental work described.
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