doi:10.1006/scdb.2000.0215, available online at http://www.idealibrary.com onseminars in CELL & DEVELOPMENTAL BIOLOGY, Vol. 12, 2001: pp. 3743

The initiation and regulation of Ca2+ signalling at fertilizationin mammalsJohn Carroll

The transition from oocyte to embryo in mammals istriggered by a series of calcium transients. There aretwo distinguishing features of this signal transductionpathway. First, it appears to be triggered by a cellfusion event between egg and sperm that allows thedirect introduction of a factor that leads to the release ofintracellular Ca2+. Second, it features a slow-frequencycalcium oscillator (one transient every 1020 min) thatpersists for 34 h. In this review I report on recentdevelopments in our understanding of how the Ca2+oscillations are started and on the regulation of the overalltemporal organization. The review focuses on mammalianfertilization and (inevitably) it is fertilization in themouse that will be predominantly discussed. Relevantand topical contributions from the excellent body ofliterature available on other species will be utilizedwhere appropriate but extensive reviews can be foundelsewhere [Stricker S A (1999) Comparative biology ofcalcium signaling during fertilization and egg activationin animals Dev Biol 211: 5776; Jaffe et al., this issue].

c 2001 Academic Press

Ca2+2+2+ signalling at fertilization: getting it started

The first indication that fertilization has started is an in-crease in intracellular Ca2+ some 13 min after sperm andegg cytoplasm has become continuous.13 Understandinghow Ca2+ signalling is initiated at fertilization will re-quire unravelling the events taking place during these firstminutes. This time between spermegg fusion and the cal-

From the Department of Physiology, University College London,Gower Street, London, WC1E 6BT, UK.E-mail: j.carroll@ucl.ac.uk

c2001 Academic Press10849521 /01 /010037+ 07 /$35.00 /0

cium increase is known as the latent period. The analogyhas been made with a burning fuse prior to it initiating anexplosion of calcium release.4 It has long been debatedwhether the fuse is lit by the interaction of the sperm andegg plasma membranes or by the introduction of a fac-tor from the fertilizing sperm that triggers Ca2+ release ineggs.57 Although there are exceptions, in mammals thereis some consensus that the latter of these two mechanismsis the most likely.57 The question therefore becomes whatis the Ca2+ releasing activity and how does it work?

Some leads are provided by looking to the requirementsfor the explosion of calcium release that is initiatedat the end of the latent period. This explosion canbe inhibited by microinjecting antibodies that inhibitinositol 1,4,5-trisphosphate receptors (InsP3Rs),8, 9 orby depleting eggs of InsP3Rs.10 This requirement forInsP3Rs for the explosion of calcium release suggeststhat phosphoinositide (PI) turnover leading to InsP3production is an essential component of the latent period.

Unlike other species,1113 direct biochemical measure-ment of PI turnover is not possible in mammalian eggswhere sufficient numbers of synchronous fertilized eggsare not available. However, the inhibition of Ca2+ oscil-lations by the PLC inhibitor U73122 supports a role forPLC in Ca2+ signalling in mammalian fertilization.14 Un-fortunately preincubation in U73122 inhibited spermeggfusion so it was not possible to examine its effectivenessduring the latent period.14 Further complications in mea-suring PI turnover during the latent period may arise if it islocalized initially to the small volume of cytoplasm aroundthe site of spermegg fusion, from which the first Ca2+wave originates.3, 15 The development of optical probessuch as GFP-PLC1 PH domain for measuring PIP2 hy-drolysis in single living cells16 may provide the first real-istic opportunity to reveal the events of the latent period inmammals.

PLC activity from the sperm or the egg?

Assuming the absence of an agonistreceptor interac-tion,57 there are at least two routes available to the

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fertilizing sperm for increasing InsP3 production. One isthe introduction of a sperm-derived PLC and the otheris the introduction of a factor that leads to the activationof egg PLCs (Figure 1). Experimental evidence for asperm-derived PLC has been provided in experimentsutilizing boar sperm extracts and sea urchin egg ho-mogenates. Depletion of sea urchin egg homogenates ofPIP2 with a bacterial PLC prevents the ability of spermextracts, but not InsP3, to stimulate Ca2+ release in thehomogenate. The response to sperm extracts is recoveredon repletion of the homogenate with exogenous PIP2.17This experiment does not distinguish between a spermor egg-derived PLC activity but two further experimentssuggest the former is responsible. Addition of PIP2 tothe sperm extract also restores the ability to release Ca2+in a PIP2-depleted homogenate, presumably because thesperm extract hydrolyses the exogenous PIP2 and gener-ates InsP3.17 This response can be inhibited with U73122suggesting PLC activity is necessary. Although PLC ac-tivity may be expected of any tissue extract, using the egghomogenate assay, activity was not detectable in a rangeof other tissues.18 These data provide strong support forthe idea that the sperm directly introduces a PLC activity.However, many questions remain unanswered. It has yetto be demonstrated that there is sufficient of the putativePLC activity in a single sperm to trigger Ca2+ releaseat fertilization. The most pressing question is, of course,the precise nature of the putative PLC. Demonstratingrecombinant activity and immunological or geneticablation of the activity will provide the final answers.

The alternative strategy for stimulating PI turnover ineggs is to activate oocyte PLCs (Figure 1). In sea urchin,star fish and ascidian eggs, miocroinjection of PLCSH2 domains to inhibit activation of PLC abolishesthe fertilization Ca2+ transient1923 (see Jaffe et al., thisissue). Furthermore, the src-related kinase, fyn, may beresponsible for activating PLC since SH2 domains fromfyn also inhibit egg activation.24, 25 To date the main bodyof evidence for egg PLCs has been obtained by the use ofSH2 domains. Further independent lines of evidence willlend support to this hypothesis and, like the sperm-derivedPLC story, the putative PLC activator needs to be isolatedand shown to be necessary and sufficient for Ca2+ releaseat fertilization.

The same SH2 proteins used successfully in sea urchinsand ascidians have no effect when microinjected intomouse and Xenopus eggs.25, 26 These studies argueagainst a role for SH2-mediated activation of PLC inPIP2 hydrolysis in vertebrate fertilization. This does notpreclude alternative routes of activation or the involve-ment of different isoforms of PLCs.26 Tyrosine kinaseshave been implicated in Ca2+ release at fertilization in

Figure 1. The PLC that leads to an increase in InsP3 atfertilization may originate from the sperm (A) or the egg (B).In A a finite bolus of PLC is introduced by the fertilizing sperm.Ca2+ release in this model relies on positive feedback by Ca2+on Ca2+ release through the InsP3R and on the sperm-derivedPLC. This model is consistent with the presence of a PLC inmammalian sperm extracts (see text for discussion). In B theincrease in Ca2+ is brought about by the sperm introducingan activating stimulus for egg PLCs. This provides an extraamplification step as the activating molecule (circles) stimulatesa number of egg PLCs. This results in a large surge of InsP3production and Ca2+ release. This model is consistent withdata obtained in sea urchins, star fish and ascidians. The sperm-derived activating stimulus is thought to lead to the activationof egg PLC since Ca2+ release can be blocked by injection ofPLC SH2 domains (see text for details).

the mouse,27, 28 but tyrosine kinase inhibitors have noeffect on the initiation of Ca2+ transients at fertilization.14Thus, evidence for activation of egg PLCs is strong insea urchins, ascidians and starfish but not in Xenopus ormammals. It may be that invertebrates and vertebrateshave adopted different approaches during the latent periodto meet the final goal of generating InsP3.

Regulation of the fertilization-induced Ca2+2+2+oscillations

For many species (sea urchin, star fish, Xenopus) theproblem of Ca2+ signalling at fertilization ends with

38

Ca2+ signalling at fertilization in mammals

the explosion of Ca2+ release arising from the eventsof the latent period.29 However, for mammalian eggsand a handful of other organisms, it triggers a newseries of questions. This is because the Ca2+ signaldoes not stop at the first explosion but rather continuesin the form of a series of Ca2+ transients,29 which inmammals continue for 34 h.4, 2931 A number of recentdevelopments provide some insight into the mechanismscontrolling this distinct pattern of Ca2+ oscillations.

InsP3R downregulation: a role in modulating Ca2+release at fertilization?

There are a number of ways of switching off a signallingpathway. At fertilization, the apparent absence of aconventional hormone/growth-factorreceptor interactionprecludes possibilities such as receptor desensitizationand internalization. One recently discovered mechanismfor turning off Ca2+ signalling is proteolytic downregula-tion of the InsP3Rs.32, 33 The most abundant intracellularCa2+ channel in mouse eggs is the type I InsP3R34, 35 and,as described above, inhibition or depletion of InsP3Rsinhibits Ca2+ release at fertilization.810 Thus regulationof levels of InsP3R protein provides a prime targetfor modulation of Ca2+ release at fertilization. Recentexperiments have revealed that InsP3Rs are indeed down-regulated after fertilization34, 36 although the functionalconsequence of this downregulation is not yet clear.

The InsP3R protein is maximally downregulated to6070% of that present in MII oocytes at 4 h post-fertilization.10, 37 This correlates well with the time thatCa2+ transients stop.38, 39 However, it is not clear that thetwo events are directly related. Within 2 h of fertilizationonly 50% of the InsP3Rs remain10, 37 yet little effect isseen on the generation of Ca2+ transients. Furthermore,inhibition of oocytes in MII using nocodazole has noeffect on the ability to cause downregulation but Ca2+oscillations can continue (Brind and Carroll, unpublishedobservations). More extensive downregulation, to 10%of the levels present in MII-arrested mouse oocytes, canbe achieved by microinjecting adenophostin A duringmaturation. This results in inhibition of fertilization-induced Ca2+ transients10 suggesting that there is athreshold level of InsP3R required for Ca2+ release atfertilization.

One recent study provides evidence that fertilization-induced downregulation of InsP3Rs can affect the abilityto generate Ca2+ oscillations. In response to microinjec-tion of sperm factor in interphase or mitosis, partheno-genetic one-cell embryos release more Ca2+ than their fer-tilized counterparts40 (Figure 2). The most likely explana-tion for this difference is that InsP3R downregulation oc-

Figure 2. Differences in the sensitivity of Ca2+ release infertilized and strontium-activated embryos correlates with thelevels of InsP3Rs (adapted from Reference 40). The ability togenerate Ca2+ transients in response to injection of sperm ex-tracts is consistently less in fertilized embryos than partheno-genetic embryos. This difference in Ca2+-releasing activity cor-relates with the ability of the activating stimulus to downregu-late InsP3Rs. Fertilization leads to InsP3R downregulation (lightshading) while parthenogenetic activation with strontium doesnot11, 37 (dark shading). This experiment40 also demonstratesthe cell-cycle-dependent sensitivity of the ability to generateCa2+ transients. Embryos in interphase do not undergo Ca2+oscillations while mitotic embryos do.

curs in fertilized but not in parthenogenetic embryos10, 37(Figure 2). Thus the sensitivity of Ca2+ release in responseto sperm factor correlates with the ability of the activatingstimulus to downregulate InsP3Rs. It may be, therefore,that the so-called once-acting maternal machinery40 sug-gested to regulate sperm-factor-induced Ca2+ signalling isactually a reflection of InsP3R downregulation.10, 37 If so,it is feasible that InsP3R downregulation at fertilizationcontributes to the cessation of transients, although it ap-pears not to be acting alone.

Cell-cycle-dependent changes in the ability to generateCa2+ oscillations at fertilization

There is a convincing body of work suggesting a rolefor Ca2+ in regulating cell cycle transitions in eggs

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and embryos (for a review see Whitaker and Larman,this issue). What has been realized only recently is thatthere may be a reciprocal interaction where the mitotickinases regulate the ability to generate Ca2+ transients andthereby govern the temporal pattern of Ca2+ signalling atfertilization.

The mitotic kinases that control meiosis are MPF (mat-uration promoting factor), which consists of cdk1/cyclin Band the MAP kinase pathway which is responsible for sta-bilizing MPF activity.41, 42 At fertilization, sperm-inducedCa2+ transients trigger cyclin B destruction and the lossof MPF activity. This leads to the completion of meio-sis as indicated by extrusion of the second polar bodyabout 90120 min after fertilization. The Ca2+ transientsdo not cease at polar body formation but continue and stoparound the time of pronucleus formation some 2 h later38when the MAP kinase pathway is inactivated.43 This cor-relation suggests a possible interaction between the gen-eration of Ca2+ oscillations and the stage of the cell cy-cle.39, 44

A direct relationship between pronucleus formation andthe cessation of Ca2+ oscillations is provided by theobservation that inhibition of pronucleus formation usingnocodazole leads to the continuation of Ca2+ transientsfor hours beyond the time they would normally stop.38Conversely, Ca2+ release induced by InsP3, sperm factorand strontium in interphase one-cell embryos is markedlyreduced compared to unfertilized eggs.34, 3840, 45 Thesedata show that exit from meiosis II and entry into thefirst mitotic interphase is associated with a decrease inthe ability to generate Ca2+ transients. Monitoring Ca2+during the first cell cycle suggests that the relationshipbetween cell cycle and the ability to generate Ca2+transients is not restricted to the specialized case of exitfrom meiotic arrest. In fertilized embryos, a Ca2+ transientcan be detected around the time of nuclear envelopebreakdown (NEBD) of the first mitotic division some1618 h after fertilization.39, 46 Mitotic Ca2+ transientscan also be stimulated in parthenogenetic embryos bystrontium39 or injection of sperm factor.40 Thus, provideda suitable stimulus is present (strontium or Ca2+ releasingsperm factors) meiosis II and the first mitosis support thegeneration of Ca2+ transients while interphase does not(Figure 3).

The response of oocytes to sperm factor, strontium andInsP3 decreases from meiosis II to the first mitosis39, 40(unpublished observations). To determine whether sperm-derived Ca2+ transients persist until mitosis of the sec-ond mitotic division we monitored Ca2+ during transi-tion from the 2-cell to the 4-cell stage. Cell division oc-curred normally but no Ca2+ transients were detected (un-published observations). This would indicate that by the

Figure 3. A summary of the interacting events that are respon-sible for modulating Ca2+ release at fertilization in mammals.The top panel depicts the progression of the metaphase II oocyteto the two-cell stage embryo. Two h after fertilization the oocyteextrudes a second polar body and forms pronuclei after 4 h. At1618 h after fertilization NEBD takes place before cleavageto the two-cell stage 90 min later. Two main factors are neces-sary for the generation of Ca2+ transients. An activating stimu-lus such as the level of sperm-derived Ca2+ releasing factors ( ) and, a sensitive environment for Ca2+ release (grey shad-ing) which occurs during meiosis II and mitosis.39 A furtherfactor proposed to contribute to the time that oscillations ceaseis the level of InsP3Rs (darker shading of embryo representinghigher levels of InsP3Rs). The middle panel depicts the fertiliza-tion Ca2+ signal. Note that Ca2+ oscillations occur when bothstimulus and sensitive environment are present. The lower panelshows the changes in MPF and MAP kinase activity during eggactivation. In a speculative model it is proposed that the inter-action between MAP kinase, MPF and Ca2+ may lead to a lowlevel of MPF sufficient to support the generation of Ca2+ tran-sients until MAP kinase falls. In the absence of MAP kinase cy-clin B is no longer stabilized and MPF activity stays low (seetext). It is at this stage the oscillations stop and the embryo en-ters interphase.

late 2-cell stage either the stimulus provided at fertiliza-tion (Figure 3) or the sensitivity provided during mitosishas declined to a level unable to support global Ca2+ tran-sients. An interesting corollary exists with the sea urchinwhere, like the mammal, a Ca2+ transient is detected at thefirst mitosis but not in subsequent cell divisions.47 Thusit seems that sperm-derived Ca2+ releasing factor(s) havebeen inactivated with the result that amplification of cell-cycle-related changes in Ca2+ release is lost.39, 48 As such,the last paternal input into Ca2+ signalling appears to beat NEBD of the first mitotic division.

In addition to this insensitivity during interphase,pronuclear formation appears to lead to a nuclear asso-ciation, or perhaps even sequestration, of sperm-derivedCa2+ releasing factors. Pronuclei from fertilized one-cellembryos stimulate Ca2+ transients when transferred intounfertilized eggs.49 Such localization of sperm-derivedCa2+-releasing factors is not solely responsible for

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Ca2+ signalling at fertilization in mammals

determining the pattern of Ca2+ transients but is actingon the background of a cell-cycle-dependent change inCa2+ releasing activity39, 44 described above. It remainsto be determined if the sperm-derived Ca2+ releasingfactor(s) is released or simply reactivated at the firstmitotic division and is responsible for generating theglobal NEBD Ca2+ transient.39

The mitotic kinases: a role in regulating Ca2+ release?

In considering the factors responsible for supportingCa2+ signalling in MII oocytes, MPF and the MAPkinase activities are good candidates. The observationthat Ca2+ transients continue past polar body extrusionand stop at pronucleus formation38 suggests a tightcorrelation between the generation of Ca2+ oscillationsand MAP kinase activity.43 However, Ca2+ transientscan often cease before pronucleus formation50 suggestingthat oscillations will stop in the presence of active MAPkinase.43 Support for a role for MPF is provided bythe finding that the MPF inhibitor, roscovitine, inhibitsthe ability to generate sperm-induced Ca2+ transients.51Roscovitine also depleted intracellular stores51 suggestingthat MPF may promote store filling, having the possibleeffect of increasing the sensitivity of Ca2+ release.52, 53Although further work is needed to confirm the specificityof the roscovitine effects, these data suggest that MPFis important for generating Ca2+ oscillations in mouseeggs. Determining the relative roles of MPF and the MAPkinase pathway in the generation of Ca2+ transients wouldbe greatly aided by the ability to correlate Ca2+ releaseand kinase activities in individual living eggs.

Some progress has been made toward this goal inascidians, which, although a digression from the mammal,provide compelling evidence for a role for MPF activityin the maintenance of Ca2+ oscillations. In ascidians,fertilization takes place at MI and stimulates two burstsof Ca2+ oscillations that coincide with the high levels ofMPF during MI and MII.54 The MAP kinase pathwaydoes not correlate with Ca2+ transients because it is activethrough the MIMII transition54 (when there is a gap inthe Ca2+ transients). This relationship has been examinedfurther in a series of experiments utilizing GFP-coupledcyclin B1 and a non-degradable mutant to modulate thelevels of MPF.55 Maintaining high levels of MPF byexpressing cyclin B1-GFP or a mutant cyclin that isnot degraded by the proteasome caused the persistentgeneration of Ca2+ transients.55 In contrast, inhibition ofMAP kinase activity with U0126 had no effect on thepattern of Ca2+ transients at fertilization.55 These dataprovide an elegant case for MPF.

If MPF is the main player in determining when the os-

cillations stop, the continuation of Ca2+ transients beyondextrusion of the second polar body needs explaining. Theanswer may lie in the fact that MPF activity can returnafter completion of meiosis II, resulting in the formationof a so-called metaphase III.56 The return of MPF activityafter polar body formation is presumably driven by contin-ued cyclin synthesis under the safeguard of MAP kinase.57If, like the ascidian, MPF supports the generation of Ca2+transients, it may be possible that the increase in MPF ac-tivity provides support for further Ca2+ transients which,in turn, stimulates inactivation of MPF (Figure 3, lowerpanel). As pronucleus formation approaches, the MAP ki-nase activity will fall43 with the result that MPF is nolonger stabilized.57 As a result, MPF activity will decreaseand the Ca2+ transients will cease (Figure 3, lower panel).It should be stressed that this is a working model based onindirect evidence but it is readily testable. Pronucleus for-mation is a fitting time to stop, since the transition frommeiotic oocyte to mitotic embryo is complete. The nextchallenge will be to determine which component(s) of theCa2+ signalling pathway are regulated either, directly orindirectly, by the mitotic kinase activity.

Acknowledgements

Thanks to Karl Swann and Sophie Brind for discussionsand critical reading of the manuscript and to Max Fun forhelp with the diagrams. Our work is funded by the MedicalResearch Council.

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Ca2+ signalling at fertilization: getting it startedFig. 1

Regulation of the fertilization-induced Ca2+ oscillationsFig. 2Fig. 3

References