THE JOURNAL OF EXPERIMENTAL ZOOLOGY 263:382-397 (1992)

Signal Transduction by Calcium and Protein Kinase C During Egg Activation WILLIAM M. BEMENT Department of Biology, Yale University, New Haven, Connecticut 06511-81 12

Fertilization is one of the most dramatic and consequential transitions of early development. In the short term, union with the sperm triggers egg "activation," a complex series of phenomena which include establishment of polyspermy blocks, metabolic stimulation, and resumption of the cell cycle. In the long term, fertilization initiates pattern formation and results in an irrevocable commitment to ontogenesis. Because all of these events are induced by the fusion of egg and sperm, questions about fertilization have naturally been framed in terms of signal transduction: What, exactly, are the immediate signals elicited by sperm-egg fusion? What are the mechanisms which generate these signals? How are these signals transduced into the events of egg activation? The first of these questions has largely been answered. It is now evident that the fundamental signal initiated by sperm-egg union is a transient rise in intracellular free calcium ([Ca2+Ii;Fig. 1). In every species in which [Ca2+lihas been measured during fertilization, it has been demonstrated that an increase in [Ca2+Iiprecedes egg activation. Moreover, in many of these species it has been further shown that artificial elevation of [Ca2+li triggers egg activation in the absence of fertilization while blocking the rise in [Ca2+liduring fertilization prevents egg activation. In short, a transient rise in [Ca2+liis both necessary and sufficient t o initiate the events of egg activation in most, if not all, animal species (Epel, '90; Nuccitelli, '91). The identification of the [Ca2+liincreases as an essential signal evoked by fertilization has brought into focus the other two questions posed above: 1) How is the calcium signal generated? 2) How is the calcium signal transduced into the events of egg activation (Fig. 2)? It is the second of these questions that is the major concern of this review. For discussion pertaining to the mechanisms which underlie the generation of the calcium signal, see Nuccitelli ('91) for review. 01992 WILEY-LISS,INC.

CALCIUM AND FROG EGG ACTIVATION In anuran amphibians, egg activation is accompanied by two dramatic morphological events that foster normal development: cortical granule (CG) exocytosis and cortical contraction (Fig. 3). CG exocytosis occurs in a wave-like fashion beginning at the site of sperm-egg fusion (Grey et al., '74). Release of CG contents results in the formation of the fertilization envelope, a sperm-impermeant barrier which surrounds the egg and provides a mechanical block t o polyspermy (Grey et al., '76). Cortical contraction follows CG exocytosis, and is revealed by the movement of pigment granules toward the animal pole of the egg. Cortical contraction is thought to facilitate pronuclear union by moving the sperm pronucleus closer to the female pronucleus (Elinson, '77). Because of the large size of frog eggs (>1mm in diameter),both CG exocytosis and cortical contraction can be observed with the aid of a dissecting microscope. The former is revealed by the formation of the fertilization envelope around the egg, the latter by the apparent darkening and shrinking of the animal hemisphere (Figs. 1, 3). Another cortical event triggered by egg activation is an enlargement of the surface microvilli, which follows CG exocytosis (Charbonneau and Picheral, '83).Microvillar enlargement, which is reported t o follow CG exocytosis in a wave-like fashion (see Charbonneau and Grandin, '891, presumably allows the egg t o cope with the large-scale insertion of membrane into the cell surface resulting from CG exocytosis (Bement and Capco, '90a). Less obvious than CG exocytosis and cortical contraction, but no less important, are the cell cycle events which accompany egg activation (Fig. 3). Prior to fertilization, the amphibian egg is arrested in metaphase I1 of meiosis; consequently, the chromatin is condensed, the nuclear envelope and Golgi are disassembled (Brachet et al., '701, and endocytosis is relatively slight (Bernardini et al., '87). At the biochemical level, the activity of maturation promoting factor (MPF) is high (Gerhart et al., ,841,

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Activated Egg

+ [Ca2'Ii

Rise

e

Fig. 1. Schematic diagram of egg activation. Sperm-egg union initiates a rise in intracelM a r free calcium, which triggers egg activation.

as is the activity of cytostatic factor (CSF; Meyerhof and Masui, '79). MPF is a multisubunit complex possessing histone H1 kinase activity that is universally high during M-phase of the cell cycle and low during interphase of the cell cycle (Murray and Kirschner, '89). CSF contains or is equivalent to the c-mos gene product, a serinekhreonine kinase that appears t o be active specifically during vertebrate meiosis (Sagata et al., '89). Upon activation, cell cycle arrest is terminated, the egg completes its meiotic division, and then enters the first of a series of rapid mitotic divisions. Activation thus triggers transit of the egg into an interphase state (Fig. 3); consequently, the chromatin decondenses, the pronuclear envelopes and Golgi assemble (StewartSavage and Grey, '821, and endocytosis resumes (Bernardini et al., '87). These events result at least in part from the diminution of MPF activity, which occurs immediately after egg activation (Gerhart et al., '84;Lorca et al., '91). Egg activation also results in a dramatic decrease in CSF activity, although this occurs somewhat later (Lorca et al., '91).

The various morphological and biochemical changes initiated by fertilization are preceded by a series of fertilization-induced ionic changes. Fertilization triggers a transient depolarization of the plasma membrane referred to as the fertilization or activation potential. The fertilization potential is due to an efflux of C 1 ions, ~ and provides a shortterm block to polyspermy (Cross and Elison, '80). A second ionic event triggered by egg activation is an alkalinization of the egg cytoplasm of 0.3 pH units, from 7.5 to 7.8 (Webb and Nuccitelli, '81). Upon what common ground do these apparently diverse morphological,biochemical, and ionic phenomena rest? The available evidence indicates that all are triggered by a fertilization-induced rise in [Ca2+Ii.Prior to activation, egg [Ca2+Iiis approximately 400 nM. Fertilization results in a rise in [Ca2+liwhich reaches a peak of 1.2-1.8 p.M in the egg cortex and which travels in a wave-like manner away from the point of sperm entry (Busa and Nuccitelli, '85; Kubota et al., '87; Nuccitelli et al., '88; Larabell and Nuccitelli, '92). This [Ca2+Ii rise can be mimicked by pricking the egg with a

Activated Egg

Fig. 2. The "unknowns" of egg activation. The mechanisms that act upstream and downstream of the rise in intracellular free calcium during egg activation have yet to be determined.

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Egg

Cortical Contraction

Cortical Granule Exocyt0s is

Activation

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Act ivated

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., ,. . . . .., .. . .'.. . ._.. '

-

CGs

Transit Into lnterphase

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Fig. 3. Activation events in the frog egg. Activation entails contraction of the egg cortex, exocytosis of cortical granules, and the transit from metaphase I1 of meiosis into the first mitotic interphase.

needle in the presence of extracellular calcium (Kubota et al., '871, by microinjection of inositol1,4,5 trisphosphate (IP3)(Busa et al., '851, or by treatment with calcium ionophore (Busa and Nuccitelli, '85). The evidence that links this transient elevation of [Ca2+lito the other events of egg activation is compelling. First, it has been shown that the fertilizationinduced [Ca2+liwave is precisely correlated in space and time with cortical granule exocytosis and microvillar elongation (Busa and Nuccitelli, '85; Kubota et al., '87). Second, in the absence of fertilization, experimental elevation of [Ca2+Ijtriggers CG exocytosis (Wolf,'741, cortical contraction (Schroeder and Strickland, '741, microvillar elongation (Charbonneau and Picheral, '831, chromatin decondensation and pronuclear envelope assembly (Lohka

and Masui, '841, the resumption of endocytosis (Bement and Capco, '91a), MPF and CSF inactivation (Gerhart et al., '84; Lorca et al., ,911, the fertilization potential (Cross, '81; Webb and Nuccitelli, '85), and cytoplasmic alkalinization (Grandin and Charbonneau, '91). Moreover, experimental elevation of [Ca2+1iin eggs also results in "pseudocleavage,"the formation of abortive cleavage furrows, after approximately 90 minutes, which is about the same time that cleavage furrows form in fertilized eggs (Busa et al., '85). Third, experimental forestallment of the rise in [Ca2+liin fertilized eggs blocks cortical contraction, cortical granule exocytosis, chromatin decondensation, assembly of the pronuclear envelope, and the fertilization potential (Kline, '88). Experiments performed using egg lysates have further demonstrated that maintenance

EGG ACTIVATION AND CALCIUM SIGNAL TRANSDUCTION

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acterize egg activation. For example, the various isoforms have different sensitivities to calcium and diacylglycerol,which would allow them to become active under slightly different conditionswithin the cell (Kikkawa et al., '89). Moreover, the different isoforms also have different substrate affinities, and apparently control different intracellular events (e.g., Melloni et al., '90). Further, different isotypes are associated with different subcellular compartments, including the nuclear envelope (Leach et al., '89) and the cytoskeleton (Jaken et al., '89). Finally, following activation by calcium and diacylglycerol or phorbol esters, PKC becomes cleaved by calpain into domains representing the regulatory site and the catalytic site, with the latter being constitutively active and apparently having different substrate specificity than the intact enzyme (Pontremoli et al., '87; Nakabayashi et al., '91). Thus, PKCs have the potential for multiple levels of regulation, and, consequently,the potential to mediate a broad array of cellular events. PKC has been identified in oocytes and eggs of the frog Xenopus Zaevis by investigators using both biochemical (Laurent et al., '88; Otte et al., '90; Sahara et al., '92) and molecular cloning (Chen et al., '89) approaches. Xenopus oocytes and eggs express at least two isoforms of PKC, which, like PKC from somatic cells, are activated by calcium and DAG, and by the phorbol ester 4-beta phorbol

of low [Ca2+Iiprevents chromatin decondensation and nuclear envelope reassembly (Lohka and Masui, '84), as well as preventing inactivation of MPF and CSF (Lorca et al., '91).

PROTEIN KINASE C AND EGG ACTIVATION The foregoing discussion makes it clear that the wave-like elevation of [Ca2+Ii that follows fertilization in frog eggs is the essential signal provided by sperm-egg fusion. How, then, does the egg transduce this rise in [Ca2+liinto the divergent events of activation? While this question remains open, accumulating evidence indicates that one of the major transducers of the calcium signal is protein kinase C (PKC),a serinelthreonine kinase which is activated by calcium and diacylglycerol (DAG).PKC actually represents a highly conserved, ubiquitous family (with at least seven isoforms; Kikkawa et al., '89) of kinases, which have the intriguing property of becoming active upon translocation t o the plasma membrane and other intracellular membranes (Fig. 4). PKC can be activated experimentally by exogenous application of diacylglycerol or tumor-promoting phorbol esters; under these conditions, PKC becomes active even in the absence of a rise in [Ca2+Ii(Kikkawa and Nishizuka, '86). The known properties of PKC make it an ideal candidate for mediator of the diverse events that char-

PMA

csb

PM

Ctl

PhosDhorvlation

I'

Cel I u l a r Effect

Fig. 4. Schematic diagram of protein kinase C (PKC)regulation. PKC is activated naturally by increases in calcium (triangle) and diacylglycerol (DAG), which cause the kinase to translocate to the plasma membrane (PM). At the PM, PKC becomes fully active in association with phosphatidylserine, and elicits various cellular effects by phosphorylating protein

substrates. PKC can be activated artificially by application of phorbol esters (PMA) which mimic DAG. Both PMA and DAG greatly increase the affinity of PKC for calcium, allowing the kinase to become active at resting levels of intracellular free calcium.

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12-myristate 13-acetate (4-beta PMA), but not by biologically inactive 4-alpha phorbol esters, such as 4-alpha phorbol12,13 didecanoate (4-alpha PDD; Laurent et al., '88; Sahara et al., '92). Two lines of evidence demonstrate that activation of PKC in Xenopus oocytes and eggs triggers events characteristic of fertilization. The first is provided by studies that show that treatment of both oocytes and eggs with the PKC agonist PMA triggers CG exocytosis, cortical contraction, contractile ring formation, and later, pseudocleavage (Bement and Capco, '89, 'gob, '91b; Grandin and Charbonneau, ,911, even though oocytes are normally considered to be "activation incompetent" (see below). Exocytosis and contraction are also triggered by application of DAG, but not by treatment with 4-alpha PDD, which does not stimulate PKC (Bement and Capco, '89; Grandin and Charbonneau, '91; Bement, unpublished results). Moreover, PMA and DAG stimulate several other events characteristic of egg activation including endocytosis, reformation of the Golgi, assembly of the male pronucleus (Bement and Capco, '91a), and enlargement of the microvilli (Bement, unpublished results). The second line of evidence that implicates PKC in events of egg activation is provided by the study of Muramatsu et al. ('89). These workers expressed mammalian PKC in Xenopus oocytes by injecting oocytes with DNA constructs encoding full-length (normally regulated) and truncated (constitutively active) PKC. Following injection and expression, oocytes were examined to determine whether or not meiotic maturation had occurred. Although expression of these constructs did not induce meiotic maturation, the micrographs presented in this study clearly demonstrate that oocytes expressing both normal and constitutively active mammalian PKC have undergone cortical contraction (Muramatsu et al., '89; Fig. 3B,C). These results provide independent confirmation of the pharmacological studies discussed above and firmly establish that PKC activation does indeed trigger phenomena characteristic of egg activation in both oocytes and eggs. However, demonstrating that PKC stimulation induces activation phenomena is by no means clear evidence that PKC is the downstream transducer of the calcium signal during fertilization. Based on the evidencejust presented, it is equally likely the PKC activation somehow triggers an increase in [Ca2+Iias is known to occur in several cell types (e.g., Albert et al., '87). That is, PKC may be acting upstream of the calcium signal to induce egg activation. The followingfindings exclude this possibility. First, PMA triggers activation events in

oocytes (Bement and Capco, '891, which are refractory t o stimuli that raise [Ca2+Ii(see below). Second, PMA triggers CG exocytosis, cortical contraction, endocytosis, Golgi reformation, and pronucleus formation even under conditions in which [Ca2+lihas been clamped by microinjection with the calcium chelator BAPTA (Bement and Capco, 'gob, '91a). Third, treatment of Xenopus eggs with PMA triggers neither the activation potential nor cytoplasmic alkalinization as it would be expected to do if it acted by raising [Ca2+li(Grandin and Charbonneau, '91). Fourth, and most importantly, direct measurement of [(la2+Ii with either calciumsensitive electrodes or with calcium-sensitive fluorophores reveals that treatment of eggs with PMA does not trigger a measurable increase in [Ca2+li(Bement and Capco, '90b; Grandin and Charbonneau, '91; Larabell and Nuccitelli, personal communication). Rather, it appears that PKC activation attenuates the calcium signal, such that eggs treated with PMA are greatly reduced in their ability to generate an increase in [Ca2+Iiin response to calcium ionophore (Grandin and Charbonneau, '91). These latter results suggest that PKC activation may actually shut off the calcium signal. That is, following the initial rise in [Ca2+li,PKC becomes active and participates in a feedback loop that promotes shuttling of free calcium into stores that are not sensitive to the original stimulus. Thus, PKC does not trigger events of egg activation by acting upstream of the calcium signal. This does not, in and of itself, prove that PKC is a natural downstream effector of the calcium signal. Such proof requires demonstration that inhibition of PKC inhibits calcium-induced egg activation. This appears t o be the case, although the results are presently equivocal as several factors complicate interpretation of the results obtained with PKC antagonists. For example, the PKC antagonists H7 and sphingosine, while potent PKC inhibitors in mammalian systems, may be somewhat less effective in Xenopus, as suggested by their limited ability to inhibit CG exocytosis and cortical contraction in eggs treated with PMA (Bement and Capco, '90b). Nevertheless, H7 and sphingosine, as well as a staurosporine (a somewhat more potent PKC inhibitor; Tamoki et al., '86) do indeed partially inhibit calcium ionophore-induced activation in Xenopus eggs (Bement and Capco, '90b; Larabell and Nuccitelli, personal communication). However, sphingosine and staurosporine-mediated inhibition is apparent only when calcium is absent from the extracellular medium (calcium-induced egg activation does not require extracellular calcium; Wolf, '74; Schroeder

EGG ACTIVATION AND CALCIUM SIGNAL TRANSDUCTION

and Strickland, '74; Busa and Nuccitelli, '85). In contrast, when sphingosine or staurosporine are applied to Xenopus eggs in calcium-containing medium, they trigger a large increase in [Caz+li, which, in the case of sphingosine, is followed by CG exocytosis and cortical contraction. In the case of staurosporine, the increase in [Ca2+liis not followed by normal activation in that CG exocytosis occurs only very slowly and cortical contraction occurs not at all (Grandin and Charbonneau, '91). The situation is further complicated by the finding that treatment of eggs with sphingosine or staurosporine in calcium-free medium reduces or abrogates subsequent ionophore-induced rises in [CaZ'li (Larabell and Nuccitelli, personal communication). Thus, of the various putative PKC inhibitors, only staurosporine has been unambiguously shown to inhibit activation events under conditions

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where an increase in [Ca2+liwas known to occur (Grandin and Charbonneau, '91). Moreover, there are at present no published studies that have shown whether any of these agents actually inhibit Xenopus PKCs. Clearly, then, interpretation of any of these data must be undertaken with care. The final piece of evidence required to establish that PKC is a downstream transducer of the calcium signal is the demonstration that PKC activity increases during egg activation. Because PKC is DAG-dependent,an indirect means to demonstrate an increase in PKC activity is t o demonstrate increase in cellular levels of DAG upon egg activation. Increases in cellular DAG generally arise as a result of hydrolysis of two plasma membrane phospholipids, phosphatidylinositol 4,5-bisphosphate (PIP2)and phosphatidylcholine (PC) (Fig. 5). PIP2 hydrolysis is mediated by PIPz-specificphospholi-

Fer t i1 iz a t ion

P I P,

DAG

DAG

Fc

, I PKC,

Fig. 5. Hypothesized mechanism of PKC activation during frog egg fertilization. Arrows indicate interactions between signaling molecules; positive interactions are identified by circled plus signs, negative interactions by circled minus signs. Fertilization initiates a rise in intracellular free calcium ([Ca2+Ii; triangles). The rise in [Ca2+liactivates phospholipase C (PLC) which hydrolyses PIP2, thereby generating IP3 (solid oval) and diacylglycerol (DAG). IP3 causes release from IPSsensitive calcium stores (ISCS), which, in concert with calcium released from calcium-sensitive calcium stores (CSCS), maintains and propagates the rise in [Ca2 I,. The formation of DAG and the large increase in [Ca2+Iiresult in the translo+

cation of inactive PKC from the cytoplasm (PKCI) to the plasma membrane, whereupon it becomes fully active (PKCA).Once activated, PKC stimulates further DAG production by upregulating phospholipase D (PLD) which hydrolyses phosphatidylcholine (PO. PKC phosphorylates cellular substrates that regulate events of egg activation such as cortical granule exocytosis and cortical contraction. PKC also downregulates the rise in [Ca2+li,thereby contributing to the return of [Ca2+Iito resting levels. The three positive feedback loops discussed in the text are identified by numbers contained within ovals.

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pase C (which is itself activated by increases in [Ca2+li)and results in the stoichiometric production of DAG and IP,, a second messenger that acts by raising [Ca2+li(Berridge, '87). PC hydrolysis, on the other hand, is mediated by either PC-specific phospholipase C or by phospholipase D (Billah and Anthes, '90). Does egg activation entail hydrolysis of either PIP2 or PC? Given that both PI- and PC-specific phospholipases C are present in Xenopus oocytes (Tigyi et al., '90; Garcia de Herreros et al., '91), and given that fertilization triggers a large increase in [Ca2+Ii(Busa and Nuccitelli, '85), it would be expected that egg activation entails PIP2 or PC hydrolysis. Indeed, this appears to be the case. Le Peuch et al. ('85)have reported a 10-folddecrease in PIP2levels upon Xenopus egg activation triggered either by fertilization or pricking. These results have been directly confirmed by measurement of PIP2 levels in unactivated and activated eggs (see Nuccitelli, '91). Further confirmation is provided by the demonstration that inhibition of PIP2 hydrolysis (and the resultant formation of IP,) reduces the magnitude of the calcium wave by half (from 1.8 p,M to 0.9 FM), indicating that PIP2 hydrolysis is a natural component of egg activation (Larabell and Nuccitelli, '92). Presumably, the rest of the wave is accounted for and carried by calcium-induced calcium release, rather than IP,-induced calcium release (Busa and Nuccitelli, '85; Busa, '88). Thus, Xenopus egg activation entails at least PIP2 hydrolysis, a finding that strongly suggests that DAG, and hence PKC activity, increases upon egg activation. This supposition is firmly corroborated by the recent work of Stith and coworkers. These researchers have measured DAG and IP, levels duringxenopus meiosis (Stith et al., '91) and egg activation (Stith, personal communication).Their work indicates that Xenopus fertilization triggers a large increase in egg DAG (2- to 4-fold) and IP, (up to 7-fold).The DAG increase is rapid, occurring within a minute of sperm addition, as would be predicted if PKC activation controls events such as CG exocytosis and cortical contraction during fertilization. They also find that approximately 99% of the DAG increase is derived from non-PIP2 sources (Stith, personal communication).Independent demonstration of PKC stimulation during egg activation is provided by the finding that calcium-induced activation results in a pattern of protein phosphorylation in Xenopus eggs similar to that induced by PMA when analyzed by two-dimensional gel electrophoresis (Bement and Capco, unpublished results). Collectively, the results described above support

a role for PKC as the downstream mediator of the calcium signal during Xenopus egg activation. However, this hypothesis must be considered tentative until the following pieces of information are provided. First, it should be shown directly that PKC activity increases upon fertilization of amphibian eggs (e.g., by measurement of the amount of PKC associated with the plasma membrane before, during, and after egg activation); second, it should be shown that specific inhibition of PKC specifically inhibits egg activation (e.g., by microinjection of PKC "pseudosubstrate domain" or by antisensemediated ablation of endogenous PKC message in the oocyte). Notwithstanding these reservations, the following hypothesis for signal transduction during egg activation is proposed (Fig. 5).Fertilization induces a rise in [Ca2+li,which is sustained by both PIP2 hydrolysis and calcium-inducedcalcium release. The calcium serves as a cofactor for PKC, but also activates the calcium-sensitive phospholipases C, thereby providing the other cofactor necessary for PKC activation, namely DAG. This scheme contains within it three positive feedback loops. The first two, which are essentially identical with models proposed previously (Busa et al., '85; Busa, "9,result from the interplay between the calcium stimulus provided by PIP2 hydrolysis and calcium-induced calcium release. That is, both IP, formation and calcium release result in more IP, formation and calcium release: directly, by stimulating IP3-sensitive calcium stores and calcium-sensitive calcium stores, and indirectly, by stimulating PIP2-specific phospholipase C, which, in turn, generates IP, and, consequently, more calcium (Fig. 5).The third positive feedback loop results from the activation of PKC. PKC activation, itself dependent on DAG formation, triggers activation of phospholipase D (Billah and Anthes, '90). This results in PC hydrolysis and the resultant production of more DAG, and in turn, further PKC activation (Fig. 5).Upon activation, PKC initiates CG exocytosis, cortical contraction, and other events characteristic of egg activation. Three features make this model attractive. First, by virtue of the three positive-feedbackloops it accounts for both the explosive rise in [Ca2+lithat occurs during egg activation, as well as the dramatic rise in DAG measured by Stith. Second, if one assumes that PKC activity attenuates the initial calcium signal, a means for negative feedback is also provided by PKC-mediated attenuation of the calcium transient. Third, as discussed below, if it is assumed that PKC acts as a downstream transducer of the calcium signal, it becomes clear why PKC agonists

EGG ACTIVATION AND CALCIUM SIGNAL TRANSDUCTION

such as PMA can trigger activation events in “immature” amphibian oocytes, while agents that raise [Ca2 Ii cannot. How does PKC transduce the calcium signal into events characteristic of egg activation? PKC is a kinase, consequently, answering this question requires identification of PKC substrates within the egg. Activation phenomena that may be PKCmediated can be subsumed into two broad classes: membrane fusion events (e.g., CG exocytosis,endocytosis, pronuclear envelope assembly) and cytoskeletal events (e.g., cortical contraction, microvillar enlargement, cleavage furrow/contractile ring formation). In both cases, potential PKC substrates can be identified based on known PKC substrates and known protein constituents o f Xenopus eggs. For example, calpactin I is a PKC substrate that is thought to play a role in exocytosis in a variety of cell types (Klee, ’88). Because calpactin I has been identified in Xenopus oocytes and eggs by molecular cloning and immunoblot analysis (Izant and Bryson, ’911,phosphorylation of this protein by PKC may play a role in CG exocytosis. Similarly, previous work has indicated that myosin mediates cortical contraction at egg activation (Christensen et +

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al., ’84;Ezzell et al., ’85). Embryonic myosin light chain is a known PKC substrate (de Lanerolle and Nishikawa, ’88); consequently, cortical contraction may result from PKC-mediated phosphorylation of myosin light chain within the egg.

ACTIVATION INCOMPETENCE: A FAILURE TO COMMUNICATE Up t o this point, we have considered only the response of the mature, metaphase 11-arrestedegg to activating stimuli. One of the most useful features of the amphibian system, however, is the fact that one can easily obtain large numbers of prophase I-arrested, immature oocytes, the developmental precursors of eggs. Further, it is possible to induce the oocyte-to-egg transition (meiotic maturation) in vitro by simply treating oocytes with progesterone (Fig. 6). Using this approach, several investigators have shown that whereas the eggs ofXenopus and Rana pipiens, the leopard frog, undergo cortical contraction and CG exocytosis in response to sperm fusion, calcium ionophore treatment, or pricking with a needle in calcium-containing medium, the oocyte does not (Kemp and Istock, ’67;Belanger

Oocyte

Activated Egg

Fig. 6. Natural development of the activated frog egg from the oocyte. Progesterone initiates formation of the egg from the oocyte, a process referred to as meiotic maturation. Following meiotic maturation, the egg can be activated by fertilization.

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and Schuetz, '75; Elinson, '77; Campanella et al., '84; Charbonneau and Grey, '84). Similarly, the immature oocyte of the toad, Bufo bufo japonicus, requires exposure to a 10-fold greater concentration of calcium ionophore than does the egg in order to undergo CG exocytosis (Iwao, '82). Thus, anuran oocytes appear to be "activation incompetent" until the development of competence at some point during meiotic maturation. What is the basis of activation incompetence? It was previously believed that activation incompetence simply reflects incompletely developed calcium signaling machinery in the oocyte (Campanella et al., '84;Charbonneau and Grey, '84). That is, it was thought that the oocyte, unlike the egg, cannot transduce the stimulus provided by sperm, ionophore, or pricking into a calcium transient of the magnitude required to induce activation events. While it is clear that the oocyte lacks the ability to propagate a calcium wave, it is also clear that this hypothesis alone is inadequate to explain activation incompetence. For example, if this hypothesis were entirely sufficient, it would be predicted that simply raising oocyte [Ca2+liby experimental means to the level attained during fertilization should induce cortical contraction and cortical granule exocytosis. This, however, is not the case. First, treatment of Xenopus oocytes with calcium ionophore in either normal (Charbonneau and Grey, '84) or high-calcium medium induces neither CG exocytosis nor cortical contraction (Bement and Capco, '~OC), even though the latter has been shown to raise [Ca2+Iiwell above the resting level (Belle et al., '77). Second, direct microinjection of calcium into oocytes induces neither CG exocytosis nor cortical contraction until well after the start of meiotic maturation (Hollinger and Schuetz, '76). (While it was reported that calcium microinjection induced localized exocytosis in oocytes, micrographs published to support this claim reveal that the effect of calcium microinjection into oocytes was not exocytosis, but rather cell lysis, as demonstrated by the presence of intact pigment granules, mitochondria, and other organelles outside the plasma membrane; see Hollinger et al., '79, Figs. 1,5.) Third, microinjection of inositol 1,4,5trisphosphate into oocytes induces increases in [Cazfliof up to 4 pM (at least 10 times the resting level of [Ca2+Iiand more than twice the level achieved during fertilization), without inducing CG exocytosis or cortical contraction (Ferguson et al., '91; R. Nuccitelli, personal communication). Thus, raising [Ca2+Iiin oocytes to levels attained in the egg during fertilization triggers neither CG

exocytosis nor cortical contraction (Fig. 7). In stark contrast to these observations is the fact that both CG exocytosis and cortical contraction can be triggered in oocytes by treatment with PMA or synthetic diacylglycerol (Fig. 7; Bement and Capco, '89; Larabell and Nuccitelli, '92; Bement, unpublished results). If PKC is indeed a downstream transducer of the calcium signal during egg activation, it follows that the oocyte may lack the ability to completely couple a rise in [Ca2+lito PKC activation (Fig. 8). This hypothesis explains both why phorbol esters activate oocytes and why calcium ionophores or IP3 injection does not. Seen in this light, activation incompetence results from a failure t o communicate at, as a minimum, two steps during the signal transduction pathway (Fig. 8). First, fusion with sperm or pricking fails to trigger a propagated wave of calcium release in the oocyte. Second, even under conditions in which a rise in oocyte [Ca2+liis induced, such as ionophore treatment in high-calcium medium or injection of IP3, the calcium signal is not completely coupled to PKC activation in the oocyte. In addition to changes in the signal transduction circuitry, it is likely that reorganization of the physical machinery (e.g., the cytoskeleton)is also required for the acquisition of activation competence, at least as far as cortical contraction is concerned (Fig. 8). This is suggested by the fact that although PMA triggers both exocytosis and contraction in oocytes, PMAinduced cortical contraction is much slower in oocytes than eggs (Bement, unpublished results), whereas the rate of CG exocytosis in oocytes is comparable to that in eggs (Bement and Capco, '89). In other words, PMA triggers immediate CG exocytosis in both oocytes and eggs, but immediate cortical contraction only in eggs, indicating that, unlike the exocytotic machinery, which is fully competent in oocytes (assuming that PKC is activated), the oocyte cortical contractile network is not fully assembled. Presumably, the inability t o couple a rise in [Ca2+lito PKC reflects one or more underlying molecular deficiencies. If, as proposed above, the pathway that couples the calcium signal to PKC in eggs consists of [Ca2+Iirise ---t 1)phospholipase activation + 2) phospholipid hydrolysis and consequent DAG generation + 3) PKC activation, then the number of potential deficiencies is limited to three. Of these, a deficiency in PKC can be excluded since PMA, which acts by way of PKC, is fully capable of triggering activation events in oocytes. That leaves either a deficiency in the ability of the oocyte to couple the calcium signal t o phospholipase acti-

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Oocyte

Fig. 7. Oocytes exhibit a different response than eggs to activating stimuli. Prior to meiotic maturation, the oocyte cannot be induced to undergo cortical granule exocytosis or cortical contraction by stimuli that raise [Ca2+Ii,such as sperm, calcium ionophore A23187,IP3,or pricking. In contrast, contraction and

exocytosis are induced in oocytes by treatment with agents that activate PKC such as phorbol esters (PMA)and diacylglycerol (DAG).Following meiotic maturation, however, contraction and exocytosis are elicited by rises in ICa2+l, as well as PKC activation.

vation, the ability to couple phospholipase activation to phospholipid hydrolysis, or both. In other words, oocyte phospholipases may be too few, or too insensitive to rises in [Ca2+li,to generate a large increase in DAG. Alternatively, there may be insufficient stores of utilizable phospholipids (e.g., PIP2) to act as phospholipase substrates for DAG production. Both of these possibilities are tenable, and the latter has direct experimental support. Le Peuch et al. ('85) have reported that meiotic maturation entails a 10-fold increase in the oocyte PIP2 content. This is one of several reports that have shown that Xenopus cell cycle transitions are accompanied by changes in levels of cellular phosphoinositides and DAG (Lacal et al., '87; Dworkin and DworkinRastl, '89; Carrasco et al., '90; Varnold and Smith, '90; Wasserman et al., '90; Stith et al., ,911, raising the intriguing possibility that cell cycle transitions in general may be regulated by lipid metabolism and consequent PKC activation. How PKC activation interfaces with the cell cycle machinery at the transit into interphase is unknown, although it does not appear to inactivate MPF (Bement and Capco,

'91a; Lorca et al., ,911; rather, it appears that MPF inactivation is mediated by a calmodulin-dependent protein (Lorca et al., '91). Nonetheless, a growing body of evidence indicates that PKC regulates one or more cell cycle transitions (including M-phase exit) in a diverse array of organisms beside frogs including yeast (Levin et al., '901, plants (Larsen et al., '89; Larsen and Wolniak, ,901, and mammals (Huang and Ives, '871, which, given the known activity of PKC agonists as mitogens (Blumberg, '881, is not entirely surprising.

EGG ACTIVATION IN OTHER SYSTEMS The foregoing discussion centered upon the role of PKC during egg activation of amphibians in general, and Xenopus in particular. In contrast, studies conducted using sea urchin eggs, the most popular system for the study of fertilization, have led to somewhat different results. Specifically, several studies have shown that PKC does not participate in sea urchin egg exocytosis. In both intact eggs and isolated cortical "lawns," neither DAG nor phorbol esters stimulate exocytosis (Whitaker and

W.M. BEMENT

392

Oocyte

DeveloPment of Activatibn Competence

f

Activation Incompetent

Activation Competent

Cannot propagate Ca2+ wave Cannot couple Ca2+ rise to PKC activation

Can propagate Ca2+wave Can couple Ca2+ rise to PKC activation Has complete cortical contractile network

Lacks complete cortical contractile network

Fig. 8. Development of activation competence in frog eggs. to PKC activation, and a lack of a complete cortical contractile Prior to meiotic maturation, the oocyte is activation incompe- network. Meiotic maturation reverses these deficiencies,resulttent, due to at least three deficiencies: the inability to propa- ing in the formation of the activation-competent egg. gate a calcium wave, the inability to couple a rise in [Ca2+Ii

Atchison, '86; Ciapa et al., '88; Heinecke and Shapiro, '90). Moreover, PKC inhibitors have no apparent effect on calcium-induced exocytosis (Heinecke and Shapiro, '90). Because of the popularity of the sea urchin system as a paradigm for egg activation, these results have led t o the widespread conception that frog eggs are the odd cell out with respect to the role of PKC in exocytosis. A review of the literature, however, indicates that sea urchins may employ a mechanism of exocytosis that is fundamentally different from that in many other classes of animals, including mammals, fish, ascidians, and amphibians. For example, exocytosis in urchin eggs occurs much more rapidly in response to the calcium signal than exocytosis in eggs from other species. The time to exocytosis following microinjection of calcium or agents that elevate [Ca2+Iihas been measured as 10 times faster in sea urchins than in amphibians or fish (Table 1). In mammals and ascidians, exocytosis is even slower, occurring on the order of minutes after the fertilization-induced [Ca2+Ii rise (e.g., Yokosawa et al., '89). These data suggest that in sea urchins calcium acts directly on the exocytotic machinery, whereas chordates rely on some intermediary to transduce the calcium signal. This point is underscored by the fact that preparations that consist of isolated sea urchin cortices

(lawns) undergo exocytosis when stimulated by calcium concentrations identical with those achieved during fertilization (Whitaker and Baker, '831, whereas similar lawns prepared from frog eggs require calcium levels of 40 p M or greater (Goldenberg and Elinson, '801, a level 20 times greater than that achieved during frog egg fertilization (Busa and Nuccitelli, '85; Kubota et al., '87; Nuccitelli et al., '881, and 40 times greater than the minimum level required for exocytosis in intact Xenopus eggs (Larabell and Nuccitelli, '92). Analogous results were obtained in permeabilization studies of Xenopus eggs: calcium-sensitive cortical granule exocytosis is abolished by treatment of eggs with digitonin, a treatment that results in leakage of PKC from the cell cytoplasm (Schmaltzig and Kroner, '90). Thus, isolated frog egg cortices or penneabilized frog eggs lack one or more components required for exocytosis induced as a response to physiologically relevant levels of calcium, and a likely candidate TABLE 1 . Calculated lag time between [ C d + l irise and exocvtosis Organism Sea urchin Medaka fish Frog

Lag time, sec

Reference

0.3-0.6 7.9 4-6

Mohri and Hamaguchi ('89) Iwamatsu ('89) Charbonneau and Picheral('83)

EGG ACTIVATION AND CALCIUM SIGNAL TRANSDUCTION

as one of these components is PKC. The potential role for PKC as a mediator of calcium-induced exocytosis in chordate eggs is strengthened by the finding that phorbol esters induce exocytosis in eggs of both ascidians (Yokosawa et al., '89) and mammals (Endo et al., '87; Ducibella et al., '91). Besides a difference in the mechanisms of exocytosis, it is also becoming clear that sea urchin eggs differ from frog eggs in other important aspects of fertilization as well. For example, heparin, which prevents binding of IP3to its receptor, does not block the normal fertilization-induced calcium increase (Rakowand Shen, 'go), suggestingthat IP3-mediated calcium release is not required for the calcium wave at fertilization in sea urchin eggs. In Xenopus eggs, however, heparin blocks the regenerative calcium wave, and microinjection of anti-PIP2 antibodies, which block IP3formation, reduces the magnitude of the calcium-wave in Xenopus eggs by half (Larabell and Nuccitelli, '92). Furthermore, while it is now apparent that sea urchins utilize a caffeinesensitive calcium store during calcium release, apparently sensitive to cyclic ADP ribose (Dargie et al., '90; Galione et al., '91), this does not appear to be the case in frogs (Busa, '88; Larabell and Nuccitelli, '92). The foregoing discussion is not meant to imply that PKC has no role in sea urchin egg activation, as evidence indicates that PKC mediates a variety of fertilization-inducedevents in urchin eggs including oxygen consumption (Heinecke and Shapiro, '901, movement of intracellular organelles (Ciapa et al., '88), as well as a possible role in regulation of intracellular alkalinization (Swan and Whitaker, '85; Shen and Burgart, '86; Shen and Buck, '90). In keeping with these data, it has been directly demonstrated that both phosphoinositide turnover (Turner et al., '84) and DAG increases (Ciapa and Whitaker, '86) occur upon sea urchin egg activation. In addition to sea urchins, PKC has been implicated in the activation of other nonchordate eggs, particularly those of the surf clam, Spisula solidissima. In Spisula, fertilization or activation induced by increasing the extracellular K + concentration entails breakdown of the germinal vesicle (the Spisula oocyte is arrested in prophase I at the time of fertilization) and phosphorylation of a characteristic subset of cellular proteins as the fertilized oocyte resumes the cell cycle. Evidence implicating PKC in Spzsulu activation is at least as strong as that suggesting a role of PKC in amphibian egg activation. First, PMA triggers germinal vesicle breakdown and phosphorylation of the appropriate subset of proteins when applied to Spisula oocytes

393

(Dube et al., '87; Eckberg et al., '87). Second, PKC antagonists inhibit activation induced by fertilization, PMA, and raised K + (Eckberg et al., '87). Third, fertilization triggers a rapid, 100%decrease in PIP2, which would presumably result in a n increase in levels of oocyte diacylglycerol and thus PKC activity (Bloom et al., '88). Obviously, then, a role for PKC during fertilization is not restricted to amphibians. Nevertheless, it is difficult to directly compare results obtained in amphibians to nonchordate species, because eggs in different species are frequently in different stages of the cell cycle. Mammalian systems, however, are similar to those of amphibians in that eggs are fertilized at metaphase I1 of meiosis. Further, as discussed above for frogs (and other organisms), a rise in [Ca2+Iiis required for mammalian egg activation in response to fertilization in that 1)fertilization triggers a rise in egg [Ca2+1i(Cuthbertson and Cobbold, '85); 2) experimental elevation of egg [Ca2+Iitriggers activation (Steinhardt et al., '74; Cuthbertson et al., '81; Miyazaki et al., '86) and; 3) experimental arrest of the calcium transient blocks key activation events at fertilization including polar body emission, cortical granule exocytosis, and resumption of the cell cycle (Kline and Kline, '92). With respect to the role of PKC in mammalian egg activation, the parallels to the amphibian system are striking. As in frogs, treatment of both mouse oocytes and eggs with PMA induces cortical granule exocytosis (Endo et al., '87; Colonna et al., '89; Ducibella et al., ,911, whereas treatments that raise [Ca2+lionly trigger exocytosis after meiotic maturation has resumed (Ducibella et al., '90a,b). It is therefore probable that, as proposed above for amphibians, meiotic maturation in mammals results in the installment of a link between the calcium signal and PKC, and consequently, between the calcium signal and egg activation events. Moreover, evidence indicates that PMA or DAG treatment of mammalian eggs can lead to activation directly, bypassing the calcium signal (G.I. Gallicano,R.W. McGaughey, and D.G. Capco, manuscript in preparation) as it does in frogs.

MORE QUESTIONS The demonstration of a transient rise in [Ca2+Ii as the key signal of fertilization has opened a broad doorway of insight into the events of egg activation. Beyond this door, a number of parallel, and sequential, calcium-dependent pathways lead to the formation of the activated egg (Fig. 9). The challenges for future studies are many. For example, precisely how does fertilization trigger the calcium tran-

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W.M. BEMENT

pH Rise Fig. 9. Hypothesized signaling events downstream of the fertilization-induced rise in [Ca2+li.The rise in egg [Ca2+liis transduced into the various events of egg activation by pathways acting both in sequence and in parallel. Both PKC-dependent and calmodulin (CM)-dependent pathways are involved, as well as pathways that have yet to be determined.

sient? What other calcium-dependent entities besides PKC mediate events of egg activation? What are the substrates upon which these entities act? What is the role of tyrosine phosphorylation during egg activation (Ciapa and Epel, '91; Yim et al., '91)? What is the role of GTP-binding proteins (Lacal et al., '87; Johnson et al., '90; Kline et al., '91)? Does egg activation, like mitogenesis in somatic cells, result from the sequential interplay of tyrosine phosphorylation, increased [Ca2+Ii,GTP-binding protein stimulation, and PKC activation? While it appears likely that important parallels will exist between mitogenesis in somatic cells and fertilization, these and other questions remain open.

ACKNOWLEDGMENTS The author is extremely grateful to the following individuals for helpful discussions, critical reading of the manuscript, and/or communication of results prior to publication: Dr. William B. Busa, Johns Hopkins University; Dr. David G. Capco, Arizona State University; Dr. Thomas Ducibella, Tufts University; G. Ian Gallicano, Arizona State University; Dr. Tama B. Hasson, Yale University; Dr. Matthew B. Heintzelman, Yale University; Dr. Douglas Kline, Kent State University; Dr. Carolyn A. Larabell, University of California at Berkeley; Dr. Robert W. McGaughey, Arizona State University; Dr. Richard Nuccitelli, University of California at Davis; and Dr. Bradley J. Stith, University of Colorado at Denver. This work was supported by American Cancer Society Fellowship 03625 and is dedicated to Keye Luke.

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