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Institute of Cell and Molecular Biosciences, Faculty of Medical Sciences, University of Newcastle, Newcastle upon Tyne, United Kingdom
ABSTRACT I. INTRODUCTION A. Context II. FERTILIZATION A. The Basic Model: Calcium Waves B. How a Sperm Activates an Egg: Survey of Experimental Approaches 1. Electrophysiology 2. Fluorescence imaging 3. Microinjection 4. Biochemical analysis A) PHOSPHOINOSITIDES. B) INSP3. C) CADPR. D) NAADP. E) CGMP. F) TYROSINE PHOSPHORYLATION. C. How a Sperm Activates an Egg: Survey of Hypotheses 1. Transduction via a sperm receptor 2. Transduction as a consequence of sperm-egg fusion D. How a Sperm Activates an Egg: An Evidence-Based Perspective E. What We Mean by Egg Activation F. Mechanism of Repetitive Calcium Spiking at Fertilization III. CALCIUM AND THE EMBRYONIC CELL CYCLE A. Separation of Chromosomes: Metaphase/Anaphase Transition 1. Sea urchin 2. Drosophila 3. Xenopus 4. Zebrafish B. Separation of Daughter Cells: Cytokinesis 1. Frog and fish 2. Echinoderm and mammalian embryos 3. Drosophila embryos 4. Targets of cleavage calcium signals C. Getting Into Mitosis: Nuclear Envelope Breakdown and Chromatin Condensation D. Genetic Approaches to Calcium Signaling in Early Embryos: C. elegans E. The Provenance and Targets of Embryonic Cell Cycle Calcium Signals IV. A NATURAL BREAK V. CALCIUM AND THE MEIOTIC CELL CYCLE OF OOCYTES A. Calcium Signals During Oocyte Maturation B. Calcium and Control of the Meiotic Cell Cycle C. Priming the Fertilization Calcium Wave VI. ENDOPLASMIC RETICULUM IN OOCYTES AND EMBRYOS VII. CALCIUM, AXES, AND PATTERN FORMATION IN EMBRYOS A. Calcium and Dorsoventral Axis Formation 1. Ascidian embryos 2. Frog embryos A) FGF SIGNALS AND PLC-{gamma}. B) THE NIEUWKOOP CENTER: THE WINGLESS (WNT) PATHWAY AND CALCIUM SIGNALING. C) GASTRULATION AND CONVERGENT EXTENSION OF THE NOTOCHORD, SOMITES, AND NEURAL PLATE. D) INDUCTION OF NEURAL TISSUE AND SOMITES. E) CALCIUM AND NEURAL INDUCTION: PUTTING ONE AND TWO TOGETHER. F) CALCIUM AND ORGANOGENESIS. G) CALCIUM AND SPECIFICATION OF NEUROTRANSMISSION IN THE NEURAL PLATE. H) CALCIUM AND DORSAL PATTERNING IN XENOPUS. 3. Zebrafish A) CYTOPLASMIC MOVEMENTS. B) CELL MIGRATION IN ZEBRAFISH AND THE DEVELOPMENT OF THE BODY PLAN. C) AXIS FORMATION. D) THE VIRTUES OF THE LUMINESCENCE METHOD FOR DETECTING EMBRYONIC CALCIUM SIGNALS. E) AEQUORIN IMAGING IN THE BLASTODISC. F) AEQUORIN AND EPIBOLY AND GASTRULATION. G) AEQUORIN AND SEGMENTATION AND ORGANOGENESIS. H) CALCIUM SIGNALS IN ZEBRAFISH EMBRYOS. B. Calcium and Left-Right Axis Formation 1. Mouse embryos and the role of ciliary calcium signals 2. Chick embryos and the role of calcium signals in gene expression 3. Calcium and left-right symmetry breaking VIII. CALCIUM SIGNALS IN EGGS AND EMBRYOS A. Development as a Showcase for Calcium Signaling 1. The fertilization showcase 2. The cell cycle showcase of the early blastula 3. The axis determination showcase of the late blastula 4. The zebrafish gastrula as a showcase for intercellular calcium waves B. What Calcium Signals Do IX. GLOSSARY GRANTS ACKNOWLEDGMENTS REFERENCES
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ABSTRACT |
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I. INTRODUCTION |
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But as it turns out, most cell types contain a very similar portfolio of calcium signaling elements. Calcium signals are apparently ubiquitous (41, 96). They are present in somatic cells, and also in the germline in both sperm (110) and eggs (522). This review takes as its theme the calcium signals that occur in oocytes, eggs, and embryos. Progress from oocyte to early embryo takes the form of a linear, irreversible program of events. Each set of events must occur in strict sequence at an appropriate time. There is no repetition. The context of each calcium signal is different. As will become apparent, calcium signals in oocytes and embryos mark changes in cell state and are the milestones of the transitions that form the developmental program.
Our understanding of the functional significance of these calcium signals ranges from very good to indifferent. A large, readily measured calcium increase like the fertilization calcium signal (509, 540, 603, 608) is secure in its functional importance, thanks to the large body of data that now is gathered. At the other extreme lie the small, sporadic calcium transients that occur as the early embryo moves towards and through gastrulation (596, 597); we can guess that they may be important in pattern formation, but data are scanty. In the middle of this continuum lie the calcium signals that have been correlated with cell cycle events in oocytes and early embryos. We start with the fertilization calcium signal, then discuss cell cycle-related calcium signals, and finish with the sporadic calcium signals that mark later development.
It is also useful at the outset to consider the ways in which questions about cell regulation can be answered in these large germline cells. Genetic approaches are, on the whole, very difficult because only mutations that spare the mother but compromise the oocyte or early embryo are useful as analytic tools in oocytes, eggs, and early embryos; widespread transcription of the zygote genome starts at about the time that the scope of this review begins to end. Such mutations, it appears, are rare. An example is a deletion of the mos gene. Mos protein is responsible for the mitotic arrest before fertilization in mammalian oocytes, its only role in development and beyond (580). Mos –/– mice have proven very useful, but mos is unusual in making such a crucial but brief appearance. Size, on the other hand, offers advantages. Oocytes are very large. Most work relies on microinjection methods and imaging, techniques that are more conveniently done in large cells. Calcium signals are now mostly measured using fluorescent calcium dyes. The development of laser scanning confocal microscopy has had a very big impact on the field. Dissecting the signaling pathways has been made much easier as recombinant constructs that perturb elements of each pathway have been made. Green fluorescent protein-tagged constructs have also been used very effectively. It will be possible eventually to understand the role of calcium signals in development without making extensive direct use of genetics, though of course genome sequence information will be essential in conceiving and designing molecular probes.
The view that calcium might be essential for egg activation stretches back to Lillie (327) and Heilbrunn (204). It took its present form as a result of two crucial observations. The luminescent calcium sensor aequorin was used to demonstrate a striking calcium wave in a fish egg (170); a calcium increase was also recorded in sea urchin eggs at fertilization (509), and the calcium ionophore A23187 was shown to activate both vertebrate and invertebrate eggs and oocytes (72, 512, 513). These two sets of experiments established that in most eggs the fertilizing sperm triggered a propagating calcium wave (228) that Jaffe has called a calcium explosion (233) and that this increase in intracellular calcium was sufficient to cause all the concomitants of egg activation (608).
The calcium responsible for egg activation was shown to be released from stores within the egg (69, 104, 512). As it emerged that phosphoinositide lipids might be involved in a signaling pathway that led to release of calcium from internal stores (35, 37), it was found that there was a marked increase in turnover of the phosphoinositides at fertilization of sea urchin eggs (572). Berridge and colleagues (520) demonstrated that the product of polyphosphoinositide (PPI) hydrolysis, inositol 1,4,5-trisphosphate (InsP3), causes calcium release from permeabilized pancreatic cells; this led to the demonstration that microinjection of InsP3 into sea urchin eggs caused their activation (605). A model of fertilization emerged in which the fertilizing sperm triggered production of InsP3 that then generated a propagating calcium wave (reviewed in Ref. 603). The debate at that time turned around whether the sperm activated a receptor cascade within the egg, or whether an activating agent was introduced into the egg cytoplasm when sperm and egg fused. This debate continues more than 10 years later and has been most recently reviewed in the context of the role of src-like tyrosine kinases in generating InsP3 at fertilization (231). The basic model remains intact. Here I intend to review in outline the various activation hypotheses and, taking a different perspective, link what is known about egg calcium channels to the various proposed mechanisms for calcium release at fertilization and consider some of the possible targets of fertilization calcium signals.
There has also been an increasing amount of data gathered about fertilization calcium signals in eggs and oocytes of taxa other than echinoderms and mammals and of calcium signaling early in oocyte maturation. The comparative biology of calcium signaling in eggs and oocytes has been comprehensively summarized and critically reviewed (522). I will therefore not discuss these data exhaustively, instead making only crucial comparisons where they can help build a general framework for understanding the causes and consequences of the calcium signals from a perspective of cell cycle progression and control in oocytes as they pass through meiosis and into mitosis. Key model organisms have made a major contribution to our understanding of calcium and the causal mechanisms of development: frog, zebrafish, ascidian, sea urchin, and starfish.
The third element of this article, the calcium signals that occur during early development, has not been comprehensively reviewed, although there are useful reviews and hypotheses in articles on calcium signals in development (36), in frog dorsoventral axis formation (388), and in frog and zebrafish pattern formation (595–597).
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II. FERTILIZATION |
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Sperm are small and eggs are large. Although calcium does not increase as a wave in all species (516), adaptation to this disparity of size has in most deuterostome species produced the fertilization calcium wave. Calcium first increases at the point of sperm-egg interaction and crosses the egg to the antipode (Fig. 1). In mammalian oocytes and echinoderm eggs, which are 
0.1 mm in diameter, the wave crosses the egg in 2 s in mammals and 20 s in echinoderms (116, 196, 234, 246, 540, 603) and Caenorhabditis elegans (456); in 1-mm-diameter frog eggs, it takes several minutes (55, 157, 164, 602). Calcium wave velocities are constant at 5–50 µm/s (238). Fertilization calcium waves are a subset of the class of calcium waves of this velocity that are found widely in both germline and somatic cells (234). Jaffe and co-workers (237, 503) present the evidence that these are, in terms of chemistry, reaction-diffusion waves analogous to the well-known Belusov-Zhabotinsky reaction.
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In general, however, the fertilization calcium wave is carried by the InsP3 receptor (InsP3R) (522); only in fish and echinoderm eggs does there appear to be a substantial contribution from the RyR (154, 164, 377, 435, 458). While the InsP3R is sensitive to calcium and can undergo CICR, production and diffusion of InsP3 can also participate in the reaction diffusion mechanism underlying InsP3R-mediated calcium waves (16, 255). There is evidence of calcium-dependent InsP3 production during the fertilization calcium wave (88, 94, 517, 606). Support for the idea from inhibition experiments is mixed. The InsP3 antagonist heparin delays the onset of the wave, without appearing to alter its velocity (105), while a dominant negative PH domain inhibitor of phospholipase C (PLC)-
not only delays the onset, but also appears to diminish its propagation (482). I will discuss this disparity further when we come to consider how the wave is initiated.
It is not merely the participation of the echinoderm RyR (491) in the propagation of the fertilization calcium wave that marks out the egg as different from frog and mammals, although similar to medaka and sea bream oocytes (154, 435). Study of the calcium release mechanisms of unfertilized eggs has uncovered two novel calcium-releasing messengers (13, 82, 121, 161, 165, 408, 464, 611), in part perhaps because of the availability of readily prepared egg homogenates that both sequester and release calcium in a reproducible and apparently physiological way (163). The first to be identified was cyclic ADP ribose (cADPr), the product of a ADP-ribosyl cyclase acting on 
-NAD+ (307, 313). cADPr releases calcium quite independently of InsP3, with its pharmacology indicating that it stimulates release via the RyR (161). The receptor for cADPr has not yet been identified; the only additional information we have is that its action requires calmodulin, which appears to increase affinity for cADPr and to increase the sensitivity of the RyR to CICR by several orders of magnitude (310, 311, 558). cADPr has been shown to mediate calcium signaling in pancreatic acinar cells, -cells and in heart (159, 223, 307, 560). Antagonists of cADPr do not alter the propagation of the fertilization calcium wave in sea urchin eggs (316) but appear to curtail the long falling tail of the transient (301), indicating that cADPr acts late during the fertilization calcium response. In eggs of species in which RyR do not make a large contribution to the initial fertilization calcium wave, cADPr does not cause global calcium release when microinjected (22, 276), despite the presence of the ryanodine receptor (22, 534). In these mammalian eggs, calcium release from RyR at the egg periphery may nonetheless contribute to cortical granule exocytosis (276) and to sustaining the multiple calcium oscillations that follow fertilization (534).
The same enzyme that generates cADPr from -NAD+ working in a different (base exchange) mode can generate nicotinic acid adenine dinucleotide phosphate (NAADP) from NADP and nicotinic acid in vitro (3, 308). NAADP is a very interesting calcium releasing messenger on two counts. First, its desensitization and activation curves in sea urchin eggs barely overlap: nanomolar concentrations of NAADP can desensitize the receptor to the tens of nanomolar concentrations that usually produce rapid and complete calcium release; its binding receptor in sea urchin eggs does not appear to be the RyR or InsP3R (34), although it has also been shown that physiological concentrations of NAADP can activate purified skeletal muscle RyR (214) and that NAADP can release calcium from the nuclear envelope of pancreatic acinar cells (168). Second, NAADP releases calcium from an internal store, but in sea urchin eggs, this is not the ER. There is every indication in sea urchin eggs that its target is a channel on a lysosomal class membrane compartment (87). This calcium store is filled by a Ca/H antiporter, driven by the compartment's low pH. Also curious is the observation that the store retains calcium for long periods even once the antiporter is inhibited, in some contrast to the rapid depletion of ER calcium when the ER SERCA pump is inhibited (87). Lee and co-workers have shown that uncaging NAADP in sea urchin eggs leads to calcium oscillations (2) and have suggested that calcium oscillations may reflect transfer between InsP3- and NAADP-sensitive stores (306). Galione and co-workers (85, 165, 223) put forward the similar idea that NAADP stores may be used by cells to enhance and reinforce InsP3- and RyR-mediated calcium signals. NAADP-induced calcium release is densensitized after fertilization in sea urchin (425), and prior uncaging of caged NAADP reduces the rate of rise of a subsequent fertilization calcium transient (165). However, NAADP-induced calcium release does not itself induce a regenerative, propagating calcium wave in sea urchin eggs (85, 165), nor does inactivating the NAADP pathway prevent initiation of the fertilization calcium transient and propagation of the calcium wave (86). Similarly in starfish oocytes at fertilization, photorelease of NAADP results in a cortical calcium increase that sometimes fails to spread (408) and sometimes propagates as a high velocity wave at 150 µm/s, an order of magnitude faster than a fertilization calcium wave (329, 381). In sea urchin, the rapid, transient cortical calcium increase at fertilization is known to be due to activation of voltage-activated L-type calcium channels (70, 111). This cortical flash is absent when the NAADP pathway is desensitized (86). Similarly in starfish, desensitization of the NAADP pathway leads to loss of the activation current carried by calcium (380). These observations suggest that NAADP may play an important role in regulating membrane currents at fertilization.
The idea is borne out by experiments that show that in mature starfish oocytes the NAADP-induced calcium increase requires extracellular calcium and is blocked by the calcium channel blockers nifedipine, verapamil, and SKF96365 (329, 464). cADPr also triggers local cortical calcium increases that are blocked by nifedipine and require extracellular calcium and which contribute to cortical granule exocytosis (408, 464). cADPr has also been reported to activate the channels that carry starfish fertilization cation currents independently of calcium (611). In ascidian oocytes at fertilization, NAADP signaling downregulates a plasma membrane calcium channel, cADPr signaling causes cortical granule exocytosis, while InsP3 generates the fertilization calcium wave, with NAADP necessary for subsequent calcium oscillations in conjunction with InsP3 (13).
There are observations that suggest that NAADP may initiate the fertilization wave in starfish. Enucleated mature oocytes have a much reduced sensitivity to InsP3 while retaining sensitivity to NAADP (329). When fertilized, these oocytes show a much attenuated propagation of the calcium wave and do not undergo cortical granule exocytosis, but the calcium increase close to the point of sperm entry remains strong. In sea urchin eggs, however, initiation and propagation of the calcium wave is unaffected by inactivation of NAADP signaling, although the duration of the transient is shortened and, interestingly, the rapid cortical calcium increase due to voltage-gated calcium influx is abolished (86). This last observation has an echo in the finding that NAADP-induced calcium release from internal calcium stores can be prevented by a dihydropyridine blocker of voltage-gated calcium channels (306).
The sea urchin egg finds itself in the vanguard of calcium signaling; study of its calcium signaling pathways has helped uncover three messengers: InsP3, cADPr, and NAADP. Ironically, despite this cornucopia, only InsP3 appears to contribute to the initiation of the fertilization calcium wave in sea urchin eggs, the other two messengers giving it a boost and longevity.
B. How a Sperm Activates an Egg: Survey of Experimental Approaches
Fertilization calcium waves are autonomous and a property of eggs and oocytes; they can be triggered by microinjection of calcium or InsP3 or, indeed, by a needle prick. The answer to how a sperm activates an egg is a simple one in outline: interaction of the sperm with the egg triggers a signal transduction pathway that initiates the autonomous calcium wave in the egg. What about the molecular detail? After almost 20 years of research, much of the detail is still missing. It also begins to look as though the detail will differ from one species to another, despite the strong conservation of the calcium wave mechanism itself. Before going on to the detail, it is worth asking why it has been so difficult to uncover.
The difficulty turns on the size disparities of egg and sperm. The area of interaction between egg and sperm can be as little as 100 nm in diameter, which is 0.000025% of the surface area of a 100-µm egg; similarly, if the initial signaling cascade begins in a 1-µm3 volume just beneath the point of sperm egg interaction, this represents 0.0002% of the egg volume. Detecting the early biochemical changes during sperm-egg interaction is beyond the limits of grind and measure approaches: a messenger would have to increase 0.5 million-fold in the 1-µm3 volume to represent a doubling over background concentration when measured by analyzing whole eggs. Not surprisingly, physiological approaches—electrophysiology, fluorescence imaging, and microinjection—in single eggs, have yielded the best insights into the detail of the early stages of sperm-egg interaction.
Echinoderm eggs depolarize at fertilization (514) (Fig. 2). The depolarization is a physiological response to the first interacting sperm that reduces the probability of interaction with sperm that engage subsequently (229, 230), the so-called fast block to polyspermy. It is fast, compared with the other time scales at fertilization, the membrane potential reaching 20 mV positive within 20 ms (70, 111, 229, 341). This rapid depolarization is due to activation of voltage-gated L-type calcium channels (70, 111, 607), the only further mention, incidentally, to voltage-gated ion channels that will be made in this review, save for the brief discussion of their role at fertilization in the ascidian and in starfish below, until we come to discuss neural induction in the embryo. The opening of these channels is triggered by a small monovalent cation current (71, 107, 342, 352, 537). Very elegant work by Chambers and his colleagues (334, 352) showed that the onset of this current is simultaneous with a capacitance increase in the small region of egg membrane with which the sperm is interacting, a capacitance increase due to the fusion of sperm and egg. Sperm-egg fusion is reversible in some circumstances (385), and current and capacitance always went hand in hand. The model that emerges from these experiments is of sperm-egg fusion providing a conduction pathway from egg to sperm that allows inward current through existing channels in the sperm membrane to depolarize the egg membrane, triggering a calcium action potential and thus the fast polyspermy block. As a consequence, once a single sperm has fused, it is difficult for a second sperm to do so, suggesting that sperm-egg fusion is fertilization's Rubicon in the sea urchin. This mechanism neatly and within the space of 20 ms ensures fertilization but prevents polyspermy, which is fatal developmentally in the sea urchin.
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20 (24). The absolute block to polyspermy is provided by the calcium-dependent exocytosis of cortical granules that cause elevation of the fertilization envelope, a mechanical barrier to polyspermy. In contrast, neomycin, a blocker of PPI metabolism, while abolishing the fertilization calcium wave as might be expected, also causes reversible sperm-egg fusion and prevents sperm incorporation (537), raising the possibility that PPI metabolism is required for sperm-egg incorporation. The production of PPI lipids may be necessary for the membrane anchoring of actin filaments (224, 287). Electrophysiology has uncovered two important roles for the calcium channel-dependent depolarization at fertilization: the polyspermy block and the facilitation of sperm incorporation. Equally important, it has provided key information about the timing of sperm-egg fusion relative to calcium influx. We shall return to this later.
Eggs and oocytes of other phyla have less informative electrophysiology. The depolarization polyspermy block is found in Rana, Xenopus, and ascidian (75, 183, 296, 593), but there is no information from electrophysiology about sperm-egg fusion in these species. Mouse oocytes show insignificant (<5 mV) membrane potential variation at fertilization, and there is no evidence of an electrical block to polyspermy (222, 232), although the block due to calcium-dependent cortical granule exocytosis operates (351). Hamster oocytes have very marked episodic hyperpolarizations (376) after fertilization due to calcium-dependent potassium channel activation by the calcium oscillations we shall later discuss; ascidian oocytes similarly show episodic depolarizations (184); their physiological function, if any, is unclear.
One can visualize sperm-egg fusion using confocal fluorescence imaging. A fluorescent dye microinjected into an egg will diffuse into the sperm when it fuses (Fig. 3). A fortunate confocal section will reveal the fertilizing sperm. If a calcium-sensitive fluorescent dye is used, the relative timings of sperm-egg fusion and the initiation of the fertilization calcium wave can be determined. In sea urchin eggs, dye transfer precedes the initiation of the calcium signal by 15–20 s (360, 538); in mice, dye transfer into the sperm head and tail antecedes the generation of the first fertilization calcium wave by a minute or more (246, 299, 538), and transfer of a 200-kDa protein also takes place long before the calcium wave is initiated (246).
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Confocal fluorescence imaging has defined the characteristics of fertilization calcium waves; they originate at the point of sperm entry and cross the egg with a spherical wave front whose geometry is modified by the boundary curvature of the egg as they propagate (157, 330, 359, 397, 408, 485, 524, 526, 527). Confocal imaging has also revealed the substructure of the repetitive fertilization calcium responses (375, 502, 505) of ascidians and mammalian eggs. In ascidians, the wave originator moves from the point of sperm entry laterally towards the vegetal pole with each successive wave (130, 358). In hamster oocytes, the first few calcium transients take the form of waves originating at the point of sperm entry; subsequent transients were reported to rise uniformly throughout the cytoplasm (375). Later it was found in mice that the subsequent transients tended to arise in the cortex of the vegetal pole of the egg (116), as in the ascidian, where clusters of endoplasmic reticulum are denser (274). The same pattern is seen in a nemertean worm (524). We will return to consider the significance of these patterns later.
In sea urchin eggs, calcium indicator dyes detect the calcium influx that occurs when the egg depolarizes (355, 485) (Fig. 2). This, as we have seen, is coincident with sperm-egg fusion. Although in starfish oocytes calcium influx and wave initiation occur within a few seconds of one another and each may precede the other (380), in sea urchin eggs, a remarkably long time elapses between sperm-egg fusion as defined by calcium influx and the initiation of the fertilization calcium wave: 15 s in Lytechinus pictus, equivalent to the time it then takes for the fertilization wave to cross the egg (359, 485, 527, 603). This latent period, first defined from kinetic analyses of fertilization rate defined by fertilization envelope elevation (17), is a characteristic feature of fertilization. The time from sperm-egg fusion to the initiation of the first fertilization wave in mouse is a minute or more. Response latencies of this degree imply a high amount of cooperativity in the signaling response. At fertilization, cooperativity is inherent in the CICR component of the fertilization calcium wave (256), which goes some way to help our understanding. However, even now there are scant data on changes in second messengers during the latent period; it remains dark as ever (603).
One approach to measuring phosphoinositides and InsP3 has been to use appropriate fluorescent PH domains from, for example, PLC-
(Fig. 4). The PH domain shows affinity for both phosphatidylinositol 4,5-bisphosphate (PtdInsP2) and InsP3 (318) and, coupled to green fluorescent protein (GFP), has been identified as an InsP3 indicator, moving from plasma membrane to cytoplasm as InsP3 increases (213, 576). In mouse eggs the PH-GFP indicator shows a slow and steady increase in localization to the plasma membrane throughout the period after fertilization during which repetitive calcium pulses occur (197). Release of exogenous InsP3 into the cytoplasm by photorelease of caged InsP3 was able to strip the indicator from the plasma membrane only at doses far higher than required to cause the calcium oscillations themselves. The indicator thus has a far higher apparent affinity for plasma membrane PtdInsP2 than for cytoplasmic InsP3 at its active concentration. The absence of periodic changes in plasma membrane PtdInsP2 as revealed by the PH domain indicator implies that the calcium pulses are not accompanied by episodes of PtdInsP2 hydrolysis, favoring a mechanism in which periodic calcium oscillations are generated by constant, though enhanced, concentrations of InsP3 (150, 245, 255, 386, 473). The sustained increase in plasma membrane PH domain-GFP fluorescence could be abrogated by blocking cortical granule exocytosis using a toxin directed against the fusion machinery. The significance of this latter observation is unclear. One possibility is that the addition of the granule membrane leads to lateral diffusion of PtdInsP2 and dequenching of the GFP fluorescence. Our unpublished observations using an identical PH-GFP in sea urchin eggs at fertilization show local increases in fluorescence coincident with cortical granule fusion events, supporting this interpretation. Similarly, a steady increase of PH-GFP is seen in ascidian oocytes after fertilization. There is an accumulation of fluorescence at the contraction pole, but no evidence of oscillatory behavior (65). One disadvantage of the PH-GFP probe is that its temporal resolution is limited by its slow diffusion relative to the much smaller InsP3 molecule. The slow rise of cytoplasmic PH-GFP fluorescence after fertilization in the sea urchin egg has led to the conclusion that the InsP3 increase may be very slow, indeed much slower than the onset of the fertilization transient (557). However, our own unpublished diffusion modeling suggests that the indicator cannot track a rapid increase in InsP3.
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Analysis of the effects of agonists and inhibitors is most readily achieved in eggs and oocytes by microinjection. A list of agonists that activate eggs includes the following: InsP3 (47, 358, 396, 430, 447, 464, 536, 540, 571, 605), cADPr (97, 159, 312, 408, 464), NAADP (13, 81, 97, 380, 381, 408, 425, 464, 559), guanosine 5'-O-(3-thiotriphosphate) (GTPS; Refs. 105, 273, 374, 571), botulinum C3 toxin (a rho GTPase activator) (565), src kinase (175, 563), cGMP (601), NO (189, 221, 617), latrunculin A (330), and an extract of sperm from various species (42, 108, 120, 218, 295, 325, 357, 412, 418, 523, 529, 533, 577, 609, 627). This class of experiment illustrates that eggs and oocytes possess signaling pathways that can be stimulated by these agonists to generate a calcium signal. That such a pathway exists is not, of course, evidence that it operates at fertilization. As an illustration, we can take GTPS, a G protein agonist. Although GTPS activates sea urchin eggs (571), the G protein antagonist guanosine 5'-O-(2-thiodiphosphate) (GDPS) does not prevent the initiation of the fertilization calcium transient by sperm (105) (it does, however, prevent cortical granule exocytosis). Echinoderm eggs clearly possess a trimeric G protein/PLC signaling pathway; indeed, if an exogenous G protein-linked receptor is expressed in starfish eggs by microinjection of the appropriate mRNA, then the oocytes, once mature, can be activated by exogenous 5-hydroxytryptamine (52, 489). Mouse oocytes can be activated in a similar way by expression of the muscarinic acetylcholine receptor (615). Nonetheless, this signaling pathway does not operate at fertilization (453, 614). Eggs and oocytes are promiscuous in their responses to agonists, perhaps because several latent signaling pathways are present in the unfertilized egg, not for use at fertilization, but for use at later stages of early development. It should be remembered that early development of embryos takes place largely in the context of stored maternal proteins and mRNA; significant transcription of zygotic genes takes place only at the mid-blastula stage (112, 194, 195).
A list of antagonists that block or delay the initiation of the fertilization calcium transient includes the following: BAPTA (192, 270, 271, 359, 516, 537, 611), EGTA (376, 540, 568, 639), oxyhemoglobin (an NO scavenger; yes, Ref. 292; no, Ref. 301), heparin (105, 164, 276, 312, 358, 384, 407, 458, 525, 559), an InsP3 chelator (227), U73122 (a PLC antagonist; Refs. 17, 131, 317), dominant negative domains of PLC- (60, 61, 452, 453, 482) (but not mammalian oocytes, Ref. 363) and of the src kinase family (4, 174, 264, 452), antibodies to a src kinase (also an agonist, Ref. 176), genistein and tyrphostin (tyrosine kinase antagonists) (18, 131, 178, 486) and neomycin (244, 537, 540, 568). Antagonists that fail to block the fertilization calcium signal include (in addition to GDPS): 8-bromo- and 8-amino-cADPr (13, 312, 316, 458), RcAMP-S (a cGMP antagonist, Ref. 316), and ryanodine (164, 276, 359, 407, 485, 524).
This broad survey (Table 1) suggests a pattern in which in invertebrates (and possibly frog) a src family tyrosine kinase pathway (89, 469, 563) may activate PLC- at fertilization to initiate the fertilization calcium wave (452). The exception is the mouse oocyte (363) and likely mammalian oocytes in general.
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I have already pointed out the difficulties inherent in biochemical measurements of the potentially very localized production of signaling molecules at the site of sperm-egg interaction. Nonetheless, it is logical to measure during fertilization the concentrations or turnover of signaling molecules known to activate eggs and oocytes. In fact, most of these measurements have been made in sea urchin eggs, some in frog eggs, because mammalian oocytes are very hard to obtain in sufficient quantity for biochemistry.
A) PHOSPHOINOSITIDES. Phosphoinositide signaling involves a cycle of synthesis and hydrolysis (94). The major signaling substrate PtdInsP2 is made by successive phosphorylation of phosphatidylinositol lipid. Hydrolysis of PtdInsP2 by a PLC generates InsP3. InsP3 is degraded by phosphatases, generating inositol that is then used to resynthesize phosphatidylinositol. The components of the cycle can be radioactively labeled using [3H]inositol or [32P]ATP. Turnover through the cycle increases markedly during fertilization and early development in the sea urchin (88, 94, 449, 572). Within 20 s of fertilization, turnover has increased 1,000-fold (88), with a net doubling of labeled PtdInsP2 (88, 572) and a concomitant halving of its precursor, phosphatidylinositol (251).
B) INSP3. [3H]InsP3 rises within 20 s of insemination and then falls at 60 s before rising again to a new plateau (94). This temporal pattern coincides with the timing of the fertilization calcium transient in the egg population, although it appears that the initial increase in [3H]InsP3 precedes the calcium transient peak in the egg population (88). A proportion of the response may be due to calcium stimulation of a PLC, since labeled sea urchin egg plasma membrane generates [3H]InsP3 when physiological micromolar concentrations of calcium are added (604). Labeling in these experiments did not reach equilibrium, so it is difficult to convert these data into estimates of concentration.
Competition assays using exogenous radiolabel are a better way to estimate concentration changes. In sea urchin, InsP3 concentrations rise to 0.2–0.3 µM in 20–30 s (293), further increasing to 1 µM at 120 s (293, 317), while in Xenopus, concentrations rise much more slowly to 0.5 µM (517, 518), declining over 10 min, as befits the much longer calcium wave in this large egg. No measurements of InsP3 have been made in mammalian oocytes.
C) CADPR. cADPr increases to a peak of 50–200 nM at 30–60 s after insemination of sea urchin eggs, falling back to resting levels again by 150 s after insemination (293, 301).
D) NAADP. NAADP rises in sperm to supramicromolar levels when sperm interact with the egg jelly coat (45, 86), and it is estimated that locally, these levels are attained at the point of sperm-egg fusion, while diffusion through the cytoplasm will result in a final concentration of 1 µM (86).
E) CGMP.
cGMP rises rapidly to a peak of 20–100 nM at 25 s after insemination of sea urchin eggs (90, 293).
F) TYROSINE PHOSPHORYLATION. Fyn kinase activity is reported to increase within 2 min after insemination in sea urchin (262); tyrosine kinase activity identified by phosphotyrosine antibodies increases with a similar time course (91); there is also a slow, long-lasting increase in tyrosine kinase activity after fertilization (263).
It is very difficult to draw conclusions from any temporal precedence of messengers in the sea urchin egg, as they all increase at comparable times, slightly preceding the fertilization calcium transient. However, a comparison of the reported concentrations with the efficacy of each messenger when microinjected is interesting: for cGMP, measured concentrations are 20–100 nM, while around 5–10 µM are needed to activate eggs (293, 601); for cADPr, the figures are 150 nM and 1–200 nM (160, 312); for NAADP, 1 µM and 50 nM (86, 165); and for InsP3, 1–100 µM and 2 nM (605). cGMP seems to be the most unlikely candidate for the activating messenger at fertilization on this purely quantitative argument.
C. How a Sperm Activates an Egg: Survey of Hypotheses
The title gives the game away. We do not yet have a definitive answer to the question of how a sperm activates the fertilization calcium wave. Nil desperandum; we are close to the answer. There are two classes of hypothesis (603). The first class imagines that the sperm activates a signal transduction receptor, much as a hormone might; the second class is based around the idea that sperm-egg fusion is the event that initiates the fertilization calcium wave.
1. Transduction via a sperm receptor
Eggs must capture sperm if they are to be fertilized. In species that are external fertilizers, they must capture the right sperm to prevent cross-species hybrids. This cell-cell recognition process is a key element in evolutionary speciation (542, 575). Cell-cell recognition processes at fertilization are likely to be a subset of the cell-cell recognition mechanisms that sort cells in an organism (147). These mechanisms generalize into cell-matrix and cell-cell interactions (12, 109). In the context of fertilization, the matrix is the egg coat, proteoglycan polymers (66, 191, 480, 481, 490, 592). One important component of the species specificity of fertilization is the response of the sperm to this egg coat. Homologous sperm interact with the egg coat and undergo the acrosome reaction (6, 46, 592, 625). The acrosome reaction is a specific response to the coat with its own signal transduction pathways (110) and involves the exocytosis of the acrosomal vesicle, which contains proteins and enzymes that dissolve the egg coat, allowing the sperm to pass through it. In broad terms, this is analogous to matrix remodeling (143, 521). Once the sperm reaches the egg, a cell-cell recognition process occurs.
In mammals, the cell-cell recognition process is mediated by an integrin/disintegrin interaction involving ADAM proteases and the CD9 tetraspannin fusion protein (83, 143, 144, 146, 248, 369, 638). An ADAM-based activation mechanism has also been proposed in the frog (406). In the sea urchin, the acrosome reaction unmasks an adhesion protein bindin, a very hydrophobic protein with affinity for carbohydrate (177) whose receptor has recently been identified (250). Cell-cell recognition is the second species-specific interaction at fertilization (592). One class of hypotheses of egg activation postulates that this sperm-egg receptor interaction includes a signal transduction component that activates the egg.
Certainly, integrin receptors can transduce a transmembrane signal to generate a calcium response in general (12). Peptides containing the RDG integrin recognition motif can induce activation of frog and bovine oocytes (57, 226, 581) and inhibit sperm binding and fusion (57). It has also been reported that application of bindin to sea urchin eggs can induce activation; however, the bindin receptor has no obvious signal transduction motifs (C. G. Glabe, personal communication). An earlier report of an egg membrane receptor for sperm (5) that possessed signal transduction motifs has not been confirmed. Nonetheless, it remains a possibility in mammalian oocytes that integrin signaling may activate tyrosine kinase pathways (623), presumably distinct from the PLC- pathway that has been shown not to operate at fertilization (363).
2. Transduction as a consequence of sperm-egg fusion
The second class of hypotheses that attempt to account for egg activation takes as a premise that activation requires fusion of sperm and egg. Whatever the merit of these hypotheses per se, they beg the question of how sperm-egg fusion occurs. The only indication of a mechanism lies in the observation that the hydrophobic sea urchin egg acrosomal protein bindin can induce fusion of lipid vesicles (177) and that the CD9 fusion protein of mammalian oocytes is involved in, for example, myocyte fusion (248). The implication of this observation is that once sperm and egg plasma membrane are glued together with bindin or integrin, then fusion will occur. There are no comparable data in other species.
Although we are ignorant of the mechanism of sperm-egg fusion, it doubtless occurs. The distinguishing feature of the second class of hypotheses is that activation occurs as a consequence of sperm-egg fusion. Does the sperm act as a conduit or a vehicle?
The conduit hypothesis is exemplified by the idea that, once fusion has occurred, calcium enters the egg by way of calcium channels in the sperm membrane (235, 626). This local calcium entry then sets off the calcium wave through a CICR mechanism. The idea is attractive in its simplicity. CICR would then be responsible both for the initiation and propagation of the fertilization calcium wave. It is a pity that this straightforward idea faces certain difficulties. The most telling is that acrosome-reacted sperm can activate sea urchin eggs in seawater that lacks calcium (68, 69, 139); it is hard to envisage how a calcium flux through the sperm could be sustained in seawater where calcium concentrations are lower or comparable to resting concentrations in the egg (246). Another argument against the conjecture is that in sea urchin eggs no local increase in calcium concentration in the region of sperm-egg contact is apparent until the calcium wave initiates (538). It could reasonably be argued that the calcium required to initiate the fertilization calcium wave may not be readily detectable. However, as we have seen, the cortical calcium increase that results from activation of voltage-dependent calcium channels at fertilization is certainly detectable (485, 538). Moreover, it takes many such action potentials and the CICR sensitizer thimerosal (355) to initiate a calcium transient via CICR.
The idea that the sperm is the vehicle that transmits an activating messenger to the egg once sperm egg fusion occurs has been a recurrent theme in the field (99, 108, 162, 219, 277, 325, 451, 452, 465, 523, 533, 609, 610, 624, 627), based on the observations that extracts of sperm cytoplasm will induce calcium transients when microinjected into eggs. Although the phenomenon can be demonstrated in echinoderm, ascidian, and mammalian eggs, most of the effort to identify the factor involved has been directed towards mammalian eggs. The main reason for this is the characteristic signature of the repetitive fertilization calcium transients in mammalian eggs. As we have seen, the promiscuous response of echinoderm eggs to activating agents makes them less attractive a model for isolating a calcium-releasing factor.
Heat sensitivity and molecular mass indicated that the factor was a protein (533). With the use of the mammalian fertilization calcium signature as a bioassay to test fractions, the factor was originally identified as a hexose phosphate isomerase (421). That this enzyme activity could generate calcium signals would have provided a novel slant on calcium signaling pathways, but the identification turned out to be mistaken (622). The well-characterized sea urchin egg homogenate was used to demonstrate that the factor possessed a PLC activity (242, 244, 443), and it is now reported that the factor is a testis-specific PLC of novel class designated PLC-
(100, 474). PLC- has been identified as tracking the active fraction from sperm, PLC- mRNA will induce the characteristic calcium signature when introduced into mammalian eggs, and PLC- antibodies can be used to immunodeplete the calcium releasing activity from extracts. Recombinant PLC- protein also produces the characteristic calcium signature when microinjected into eggs (280). It is calculated that a single sperm (474) contains sufficient enzyme to activate an egg. Inhibition of fertilization by inhibition or knockout of PLC- remains to be demonstrated. It is interesting to note that this phospholipase activity appears much more potent than PLC- (364), which, as we have said, does not appear to play a major role in mammalian fertilization (363).
Ascidian oocytes resemble those of mammals in having a characteristic calcium signature at fertilization. A start has been made on isolating and characterizing an ascidian sperm factor (295, 357, 609) (K. Jones, A. McDougall, and T. O'Sullivan, personal communication). The ascidian sperm factor appears to operate via the src/PLC- signaling pathway, as its action is blocked by the appropriate dominant negative SH2 domain construct (451).
D. How a Sperm Activates an Egg: An Evidence-Based Perspective
Having set the stage, we now have to test the notions of sperm-egg activation against each other.
The first thing to point out is that there may not be a single, universal biochemical mechanism that operates at fertilization. For example, SH2 domains inhibitory to PLC- and src-family kinases will block fertilization in sea urchin, starfish, zebrafish, and ascidian (60, 61, 265, 448, 452, 453, 482), but not in mammals or frogs (362, 363). Nonetheless, there is other evidence that the src/PLC- pathway may be central to fertilization in these species. A src-related tyrosine kinase in Xenopus coimmunoprecipitates with PLC- after fertilization; the association is blocked by the protein tyrosine kinase inhibitor PP1 (471). Methyl -cyclodextrin treatment of Xenopus oocytes, intended to deplete cholesterol from lipid signaling rafts, caused a marked decrease in tyrosine kinase activity and blocked the fertilization calcium transient (470). The tyrosine kinase inhibitors lavedustin A and tyrphostin B46 prevented the fertilization calcium wave, as did a 20-amino acid truncation of the src SH2 domain (178), inhibitions overcome by calcium injections. In rat oocytes, the tyrosine kinase inhibitors PP2 and SU 6656 blocked resumption of meiosis, but not other manifestations of the fertilization response (549). If there is a consensus, it is that activity of one or other PLC may be the common element (452, 474). To test this consensus, we will have to examine the suggestions that cGMP, NO, or NAADP may be the activating messengers at fertilization in echinoderms.
The suggestions around NO and cGMP are based on the observations that both NO and cGMP can trigger a calcium transient in sea urchin eggs and that both increase early during fertilization. cGMP has been measured in egg suspensions and shown to rise by 20 s after insemination, the earliest time point measured (90, 293). The timing in egg populations can be related to timings measured in single eggs by convolving the fertilization rate with the known kinetics of the latent period and the rise of the fertilization calcium transient (94). The peak of the calcium transients in an egg population (that is, the time at which the maximum number of eggs are undergoing a calcium transient) occurs at 30 s after insemination; however, since the latent period varies considerably in length from one egg to another, as many as 95% of eggs in a population will have initiated a calcium signal by 15 s after insemination (94). From timing alone, it is difficult to argue that any increase in cGMP precedes the calcium transient. Peak concentrations of cGMP have been measured as 20–100 nM (90, 293). A cytoplasmic concentration of 5–10 µM is required to activate an egg after microinjection (293, 601). This is a large discrepancy. In contrast, InsP3 concentrations reach 0.2–0.3 µM at their peak at 20 s after insemination (293), and eggs activate at cytoplasmic concentrations of 2 nM (605). The only known target of cGMP is the G-kinase, which can be inhibited by RcAMPS. At concentrations demonstrated to block cGMP-induced calcium release in both sea urchin homogenates and intact eggs, inhibitors of this pathway do not prevent the initiation of the fertilization calcium transient (164, 312, 316). On these grounds, I think it unlikely that cGMP is an initiating messenger at fertilization.
The early increase in nitric oxide at fertilization (292) was not detected in a subsequent study (301) in which the initiation of the calcium transient was measured simultaneously using fluorescence imaging. The later study was unable to reproduce the supporting observation that oxyhemoglobin, the NO scavenger, blocked the initiation of the fertilization calcium transient. It is clear that NO activates eggs by stimulating the cGMP/cADPr pathway (617), and it is known that blocking the later elements of this pathway does not prevent the fertilization calcium transient from occurring (164, 301, 312, 316) and that the NO pathway does not operate at fertilization in ascidian or mouse eggs (221). It is logical therefore to circumvent this anomaly in sea urchin by suggesting that NO may act through a nitrosylation mechanism (292), although there is no evidence that such a pathway exists in eggs. It is equally logical to suggest that since activation of eggs by NO (but not fertilization) is blocked completely by cGMP/cADPr antagonists, this pathway cannot be present.
The suggestion that NAADP may be the activating messenger is also based on the observation that NAADP triggers calcium release in unfertilized eggs (86, 307, 465) and that NAADP is found at activating concentrations in sperm (45, 86). However, inactivating the NAADP pathway does not prevent the initiation of the fertilization calcium wave in sea urchin eggs (86). Instead, it inhibits the calcium currents of the fertilization action and activation potentials (86, 380). Nonetheless, downregulation of InsP3 signaling in starfish does not prevent initiation of the fertilization calcium wave (329), implying that in starfish NAADP is an activating messenger. Set against this observation is the finding that microinjection of a protein domain that binds InsP3 with very high affinity completely abolishes the fertilization calcium transient at fertilization in starfish (227), as does microinjection of dominant negative SH2 domains that antagonize src kinase and PLC-
