Calcium and fertilization: the beginning of life

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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.

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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. www.sciencedirect.com

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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.

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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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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 www.sciencedirect.com

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).

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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 www.sciencedirect.com

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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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]. www.sciencedirect.com

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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

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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.

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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

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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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