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Ca²⁺ rises during fertilization

In both amniote and anamniote vertebrates, physiological polyspermy is found in species with internal fertilization and yolky eggs, such as cartilaginous fishes, urodele amphibians, reptiles, and birds (Elinson 1986,Iwao 2000a,Wong & Wessel 2006,Snooket al. 2011). Fertilizing sperm must provide a signal to trigger the initiation of development, i.e. egg activation. An increase in free Ca²⁺ concentration in the egg cytoplasm ([Ca²⁺]_i) induced by the fertilizing sperm is the most important factor for egg activation in both monospermic and physiologically polyspermic species (Fig. 1;Whitaker 2006). In the monospermic frog,Xenopus laevis, a transient and large increase in [Ca²⁺]_i induced by a single sperm spread into the entire egg cytoplasm as a Ca²⁺ wave (Fig. 1A and D;Nuccitelliet al. 1993,Fontanilla & Nuccitelli 1998), whereas a slow rise in [Ca²⁺]_i is reported in the polyspermy of the newt,Cynops pyrrhogaster, as detected by Ca²⁺-sensitive photoprotein (Yoshimoto & Hiramoto 1991,Yamamotoet al. 1999) and a locally propagative change in [Ca²⁺]_i was observed by a Ca²⁺-sensitive electrode inPleurodeles waltl (Grandin & Charbonneau 1992). The detailed changes in [Ca²⁺]_i at polyspermy have been investigated inCynops eggs by a Ca²⁺-sensitive fluorescence dye (Haradaet al. 2011). An initial Ca²⁺ rise at a sperm entry site propagates as a Ca²⁺ wave in the egg cytoplasm (Fig. 1B). The peak level of the Ca²⁺ rise is estimated to be 0.15 μM inPleurodeles (Grandin & Charbonneau 1992). Although the precise level of [Ca²⁺]_i has yet to be determined inCynops eggs, the peak level is much lower than that inXenopus eggs: about 1.2 μM in the cortex (Fontanilla & Nuccitelli 1998). The Ca²⁺ wave atCynops fertilization, in some cases, is preceded by an initial spike-like Ca²⁺ rise (Fig. 1E). Each Ca²⁺ wave initiated from the sperm entry site spreads in the egg cytoplasm but does not reach the opposite side of the egg. The observed velocity of 5.1 μm/s for the Ca²⁺ wave inCynops eggs is slightly slower than that observed in the cortex ofXenopus eggs (8.9 μm/s) but similar to that in the center (5.7 μm/s;Fontanilla & Nuccitelli 1998). The slower Ca²⁺ waves are probably due to a lack of endoplasmic reticulum (ER) as intracellular Ca²⁺ stores in the cortex of theCynops eggs (Fig. 2;Haradaet al. 2011). The Ca²⁺ wave induced by a single sperm propagates in one-eighth to one-quarter of the egg surface and multiple Ca²⁺ waves occur 10–15 min after the first sperm entry. The relatively high [Ca²⁺]_i is maintained for 30–40 min after fertilization. Thus, several sperm must enter to increase [Ca²⁺]_i over the entire egg. Usually, 2–20 sperm enter aCynops egg and then initiate egg activation (Iwaoet al. 1985,1993). The multiple Ca²⁺ waves induced by all fertilizing sperm are probably necessary for complete activation of physiologically polyspermic eggs. In the polyspermy of the frog,Discoglossus, an increase in [Ca²⁺]_i (0.4–1.3 μM) lasts for 50 min after fertilization, and a Ca²⁺ wave probably propagates toward the entire egg cortex from the sperm entry sites restricted in an animal dimple (Nuccitelliet al. 1988). Several spike-like depolarizations in response to each sperm entry are, however, preceded before a long-lasting depolarization mediated by the opening of Ca²⁺-activated Cl⁻ channels, as described below (Talevi 1989). This suggests that a nonpropagative small Ca²⁺ rise induced by each sperm entry occurs in advance of the major Ca²⁺ wave. The changes in [Ca²⁺]_i at fertilization of other polyspermic species remain unknown.

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Ca²⁺ rises and formation of zygote nucleus in vertebrate eggs. (A) A Ca²⁺ wave in an egg of the monospermic frog,Xenopus, spreading in the entire egg cytoplasm from the entry site of a single sperm. The transient Ca²⁺ increase peaks 1–3 min after fertilization and continued for about 15 min (D). The sperm pronucleus develops a large aster and makes contact with an egg pronucleus to form a zygote nucleus. (B) Multiple Ca²⁺ waves in an egg of the polyspermic newt,Cynops, showing each Ca²⁺ wave propagating in about one-eighth to one-quarter of the egg cytoplasm. Some Ca²⁺ waves are preceded by a spike-like increase (arrow). As each Ca²⁺ wave propagates for about 15–20 min, the total Ca²⁺ rise continues for about 40 min (E). The sperm pronucleus in the animal hemisphere develops larger asters and the nucleus nearest to the egg pronucleus likely makes contact with it to form a zygote nucleus. Other accessory sperm pronuclei undergo degeneration without participating in cell division. (C) Ca²⁺ oscillations in a monospermic mouse egg. The first Ca²⁺ wave is induced at the sperm entry site followed by repetitive Ca²⁺ waves. The Ca²⁺ rises continue every 10–15 min for 2 h after fertilization (F). The sperm pronucleus develops an aster to form a zygote nucleus with an egg pronucleus.

Citation: REPRODUCTION 144, 1;10.1530/REP-12-0104

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Schematic potential models of the signaling in egg activation of the monospermic frog,Xenopus (A), or of the polyspermic newt,Cynops (B). InXenopus, the protease activity on the sperm, in association with sperm surface glycoprotein (SGP) and ADAM16, may cleave an egg receptor, Uroplakin III (UPIII), and then the activated Src kinase (Src) stimulates phospholipase Cγ (PLCγ). Inositol 1,4,5-trisphosphate (IP₃) from phosphatidylinositol 4,5-bisphosphate (PIP2) induces a local Ca²⁺ increase in the egg cortex. The cortical Ca²⁺ increase seems to propagate through the cortical endoplasmic reticulum (ER), which is abundant in theXenopus egg, as a Ca²⁺ wave. InCynops, the sperm protease activity induces a small nonpropagative Ca²⁺ rise and then sperm-specific citrate synthase introduced from each sperm induces each Ca²⁺ wave. While the Ca²⁺ increase from the inner ER with cytoskeletons, or from mitochondria, may be caused by oxaloacetate, or by acetyl-CoA, respectively, another molecule in association with cytoskeletons may be involved in the course of Ca²⁺ rise induced by sperm citrate synthase.

Citation: REPRODUCTION 144, 1;10.1530/REP-12-0104

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Mechanisms of sperm-triggered Ca²⁺ release in polyspermic eggs

The rise in [Ca²⁺]_i induced by the fertilizing sperm is both necessary and sufficient for egg activation in physiologically polyspermic urodeles. Prevention of the Ca²⁺ rise at fertilization by a Ca²⁺ chelator inhibits resumption of meiosis (Yamamotoet al. 1999), and an artificial increase in [Ca²⁺]_i from intracellular Ca²⁺ stores caused egg activation (Charbonneau & Picheral 1983,Iwao & Masui 1995,Yamamotoet al. 1999). Although the eggs of both monospermic frogs and the monospermicHynobius salamander are activated by pricking with a fine needle to bring about a small Ca²⁺ influx, the eggs of most polyspermic urodeles, except forPleurodeles (Aimar & Labrousse 1975), are insensitive to pricking and less sensitive to a Ca²⁺ ionophore (Iwao & Masui 1995,Iwao 2000b), corresponding to a lack of ER as a Ca²⁺-propagating system in the egg cortex of newt eggs. Inositol-1,4,5-trisphosphate (IP₃) receptors on the ER are closely involved in the Ca²⁺ rises at polyspermy. Injection of IP₃ into the eggs or the isolated egg cytoplasm induces a Ca²⁺ rise, and injection of heparin to inhibit IP₃ receptors prevents Ca²⁺ waves at fertilization (Yamamotoet al. 2001,Haradaet al. 2011). The expression of exogenous phospholipase C (PLC) activatesCynops eggs with an accompanying Ca²⁺ rise (Haradaet al. 2007). Thus, the Ca²⁺ waves inCynops eggs are probably induced by the propagative Ca²⁺ release acting on IP₃ receptors directly or through IP₃ production (Fig. 2), but ryanodine receptors are unlikely to be involved in the Ca²⁺ rise (Yamamotoet al. 2001). However, the mechanism of Ca²⁺ rise is probably different from that in monospermicXenopus eggs with a single Ca²⁺ wave propagating whole egg cytoplasm.

Responses in egg activation

The Ca²⁺ rise at fertilization causes egg activation, which is characterized by a series of morphological and biochemical changes in the egg. In monospermic species, frogs, andHynobius salamanders, the Ca rise opens Ca²⁺-activated Cl⁻ channels on the egg plasma membrane to produce a rapid (<1 s) depolarization of the membrane and a positive-going fertilization potential, which prevents the entry of other sperm, as a fast block to polyspermy (Cross & Elinson 1980,Iwao 1989,Iwao & Jaffe 1989). In polyspermic urodeles, the eggs ofPleurodeles andAmbystoma mexicanum display no electrical responses to sperm entry (Charbonneauet al. 1983), butCynops eggs elicit small hyperpolarizations probably mediated by Na⁺ channels in response to each sperm entry (Iwao 1985). The polyspermic urodele eggs lack the ability to produce a positive-going fertilization potential, i.e. Ca²⁺-activated Cl⁻ channels. In addition, the entry ofCynops sperm into the eggs is not affected by the positive membrane potential, indicating a lack of the fast electrical block to polyspermy on the egg plasma membrane (Iwao & Jaffe 1989). On the other hand, polyspermicDiscoglossus eggs elicit a positive-going fertilization potential mediated by Cl⁻ channels (Taleviet al. 1985,Nuccitelliet al. 1988), but they do not block subsequent sperm entries, as sperm penetration is independent of the egg's membrane potential (Talevi 1989). Brief depolarizations occur in response to each sperm entry into the periphery of the dimple (Talevi & Campanella 1988), indicating a small Ca²⁺ rise induced by each sperm entry, as described above. Although there have been few studies on the electrical responses in other polyspermic vertebrate species, in the polyspermy of the ctenophore,Beroe ovata, each sperm induces a Na⁺-dependent depolarization, lasting about 60 s, preceded by an action potential (Goudeau & Goudeau 1993), suggesting that there is no electrical regulation of sperm–egg fusion that prevents polyspermy.

Monospermic frog eggs undergo cortical granule exocytosis to transform the vitelline envelope into the fertilization envelope through which the extra sperm cannot penetrate (Hedrick 2008). The eggs of urodele amphibian, including the monospermicHynobius (Iwao 1989), have no cortical granules, indicating the absence of a formed fertilization envelope. Cortical contraction, characterized by apparent movement of cortical pigments toward the animal pole, does not occur in response to the Ca²⁺ rise at newt egg activation, but small accumulations of cortical pigments at sperm entry sites are visible on the animal hemisphere (Iwao 2000a). The cell cycle of unfertilized newt eggs is arrested at the second meiotic metaphase with a high activity of M phase-promoting factor (MPF; cdc2 kinase (cdk1), and cyclin B) maintained by c-Mos (Iwao & Masui 1995,Sakamotoet al. 1998,Vauret al. 2004,Pelczaret al. 2007). The amount of cyclin B in the unfertilized egg is approximately one-fourth in cleaving eggs but is primarily distributed in the cortex of the animal hemisphere and chromosomes (Sakamotoet al. 1998,Iwaoet al. 2002). Cyclin B as well as c-Mos disappeared soon after fertilization when the sperm asters expand through the egg cytoplasm (Yamamotoet al. 2001,Iwaoet al. 2002,Pelczaret al. 2007) and then the activity of both cdc2 kinase and MAPK decreases (Iwaoet al. 1993,Sakamotoet al. 1998,Pelczaret al. 2007). Degradation of MPF might occur downstream of the Ca²⁺ rises at fertilization through a calcineurin/CaMKII/APC cascade alike inXenopus eggs (Nishiyamaet al. 2007). Inhibition of protein synthesis causes resumption of meiosis inCynops eggs (Iwao & Masui 1995), indicating that inhibition of short-lived proteins such as cyclin B, for example, is involved in egg activation downstream of the Ca²⁺ rises. Following the emission of the second polar body at the animal pole, the fertilized egg undergo S phase of the first mitotic cell cycle.

Comparison of the mechanisms of Ca²⁺ release in mono vs polyspermic eggs

There must be a specific mechanism by which a fertilizing sperm transmits the initial signal for the Ca²⁺ rise at physiological polyspermy. As mentioned earlier, faster egg activation is probably characteristic of monospermic species, but not of physiologically polyspermic species, so that the mechanisms underlying egg activation must be different between those species. To elucidate this process, we compared the signaling mechanisms in egg activation between monospermic and polyspermic species in vertebrates. There are at least two major models to account for the [Ca²⁺]_i increase at vertebrate fertilization (Fig. 2;Iwao 2000b,Nomikoset al. 2012). One is that a sperm binds to a receptor on the egg plasma membrane and then stimulates a signal transduction pathway causing the Ca²⁺ rise. Another is that a sperm introduces factors into the egg, such as Ca²⁺, PLC, or other agents, that can induce the Ca²⁺ rise. In any case, a fertilizing sperm must stimulate a propagative [Ca²⁺]_i increase in the egg cytoplasm to produce the Ca²⁺ wave.

In monospermicXenopus eggs, a Ca²⁺ rise is induced by external application of peptides containing RGD residues acting as ligands for integrins (Iwao & Fujimura 1996) or peptides from the disintegrin domain of xMDC16 (Shillinget al. 1998). Protease activity against a peptide containing GRR residues also induces activation inXenopus eggs (Iwaoet al. 1994,Mizoteet al. 1999). InXenopus fertilization, a protein tyrosine kinase, Src kinase, localized in membrane microdomains (membrane rafts) of unfertilized eggs, is phosphorylated and then the activated Src kinase stimulates IP₃ production through PLCγ (Fig. 2A;Satoet al. 1999,2003). Sperm induces transient phosphorylation of Uroplakin III (UPIII) on the egg membrane dependent on Src kinase (Mahbub Hasanet al. 2005,2007,Sakakibaraet al. 2005). It has been postulated that the sperm protease associated with sperm surface glycoprotein (SGP,Nagaiet al. 2009) cleaves UPIII to activate Src kinase. UPIII seems to serve as a primary sperm receptor for the Ca²⁺ release from cortical ER (Fig. 2A). In the monospermy of the primitive jawless fish lamprey, a positive-going fertilization potential blocks sperm–egg fusion, but not egg activation (Kobayashiet al. 1994), indicating the involvement of a receptor on the egg membrane in the signaling pathway for egg activation. Monospermic species without the fast polyspermy block, however, seem to possess a different system for egg activation. Bony fishes exhibit monospermy without the fast electrical block to polyspermy on the egg membrane (Nuccitelli 1980). Their monospermy is ensured by the micropyle on the chorion (vitelline envelope) to limit the number of sperm reaching the egg plasma membrane (Hart 1990,Iwamatsu 2000). Bony fish sperm contain a factor for activation of homologous eggs (Iwamatsu & Ohta 1974) or for inducing a Ca²⁺ rise in mouse eggs or sea urchin egg homogenates (Cowardet al. 2003), but PLCζ is unlikely involved in the sperm-induced activation of fish eggs (Cowardet al. 2011). Monospermic mammalian eggs also lack a fast electrical block to polyspermy (Jaffeet al. 1983,Gianaroliet al. 1994,Kline & Stewart-Savage 1994). Ca²⁺ oscillations induced by sperm, however, appear to play a role in the slow membrane block to polyspermy in mammalian fertilization (Gardneret al. 2007). A soluble sperm factor for egg activation enters into the egg cytoplasm after sperm–egg fusion in mammals (Swann 1990,Parringtonet al. 1996,Odaet al. 1999). A sperm-specific PLCζ generates Ca²⁺ oscillation in mouse eggs (Fig. 1C and F;Saunderset al. 2002,Kouchiet al. 2004) and a single sperm contains sufficient PLCζ protein to induce Ca²⁺ oscillation (Saunderset al. 2002,Yodaet al. 2004), indicating that the major active component for Ca²⁺ oscillations is PLCζ in mammalian fertilization. Thus, the signal transduction for egg activation around the sperm binding to the egg membrane may play a role in the fast electrical block to polyspermy in monospermic species.

Citrate synthase as a novel sperm factor for newt egg activation

In polyspermicCynops eggs, a small and nonpropagative Ca²⁺ rise is induced by the tryptic acrosomal protease (Haradaet al. 2011), but only a small number of eggs are activated by treatment with the sperm protease (Iwaoet al. 1994). The initial spike-like Ca²⁺ rise atCynops fertilization seems to be induced by the sperm protease at the binding of the sperm on the egg surface but is insufficient for inducing the propagative Ca²⁺ wave necessary for egg activation (Fig. 2B). The injection of sperm soluble components, a sperm factor, into the egg causes a Ca²⁺ wave and egg activation (Yamamotoet al. 2001). The Ca²⁺ wave induced by the sperm factor has a velocity of 6.2 μm/s, which is similar to those induced by the fertilizing sperm, and it triggers a complete activation, including resumption of meiosis, degradation of cyclin B and c-Mos, and DNA replication followed by abortive cleavage due to the lack of sperm centrioles. The sperm factor for egg activation is highly purified and characterized (Haradaet al. 2007), which reveals that citrate synthase in the sperm cytoplasm induces the Ca²⁺ wave that causes the egg activation (Fig. 2B). The sperm lack sufficient PLC activity to induce egg activation because no Ca²⁺ rise occurs inXenopus eggs by injection of theCynops sperm extract. The sperm-specific citrate synthase (45 kDa) is slightly heavier than those observed in heart tissue and in unfertilized eggs (43 kDa). The Ca²⁺ rise is induced by the injection of not only porcine citrate synthase but also citrate synthase mRNA into unfertilized eggs (Haradaet al. 2007). The egg-activating activity in the sperm extract was reduced by the treatment with anti-citrate synthase antibody. Most citrate synthase is distributed as a fibrous structure from the neck to the middle piece outside the mitochondria. The sperm citrate synthase can be exposed to egg cytoplasm soon after sperm entry because all sperm components, including the middle piece and the tail, are incorporated into the egg (Picheral 1977,Iwao 2000a). It is unknown whether a single newt sperm can induce sufficient egg activation activity in the newt egg. InCynops fertilization, 2–20 sperm enter an egg (Iwaoet al. 1985,1993), indicating that at least a double sperm entry is required for full activation. A sperm extract containing the cytoplasm equivalent of a single sperm is able to activate about 20% of the egg, corresponding well to the proportionately low level of citrate synthase (2 pg) and its enzymatic activity in a single sperm (Yamamotoet al. 2001,Haradaet al. 2007,2011). The multiple Ca²⁺ increases induced by all fertilizing sperm must be necessary for complete activation of polyspermicCynops eggs. The lower level of egg activation activity induced by a single sperm will delay the initiation of egg activation until several sperm enter the same egg. Thus, the sperm-specific citrate synthase appears to represent a novel and major sperm factor for egg activation in physiologically polyspermic newt eggs.

A Ca²⁺ rise by citrate synthase

It is important to understand the mechanism by which citrate synthase, derived from fertilizing sperm, can induce a Ca²⁺ rise in the egg cytoplasm. The ER containing IP₃ receptor in unfertilizedCynops eggs has been observed to form large clusters, probably with cytoskeletons, in the inner egg cytoplasm (Fig. 2B;Haradaet al. 2011). A local and spike-like Ca²⁺ rise is induced by each injection of the sperm extract into an isolated ER-rich fraction. Although adequate conformation of the ER must be necessary for the formation of Ca²⁺ waves, Ca²⁺ is mainly released from the ER rather than from mitochondria in response to sperm citrate synthase. The ability to induce multiple Ca²⁺ rises in the heavy ER clusters corresponds well with the multiple Ca²⁺ waves through the inner ER at egg activation and is somewhat analogous to that induced by the mouse sperm factor, a PLCζ-induced Ca²⁺ mobilization by hydrolyzing internal phospholipid stores (Yuet al. 2012). The purified sperm factor shows high enzymatic activity for citrate synthase and the inhibition of its activity prevents egg activation not only by the sperm extract but also by the fertilizing sperm (Haradaet al. 2011), indicating a central role for the enzymatic activity in the Ca²⁺ rises (Fig. 2B). Citrate synthase produces citrate from acetyl-CoA and oxaloacetate in the mitochondrial tricarboxylic acid (TCA) cycle but might inversely cleave the citrate, which is abundant in the egg cytoplasm, to produce acetyl-CoA and oxaloacetate (Srere 1992). Both acetyl-CoA and oxaloacetate have sufficient activity to induce Ca²⁺ waves and egg activation inCynops eggs, while citrate has not (Haradaet al. 2011). It is reported that acetyl-CoA sensitizes the IP₃ receptors on the ER to induce Ca²⁺ releases (Missiaenet al. 1997) and that oxaloacetate induces the Ca²⁺ release from mitochondria (Leikinet al. 1993). AsCynops eggs are more sensitive to acetyl-CoA than to oxaloacetate, the acetyl-CoA associated with the sperm citrate synthase seems to be a major signal for the Ca²⁺ release from IP₃ receptors on the ER at fertilization (Fig. 2B). However, the detailed mechanisms remain to be investigated. On the other hand, it is possible that sperm citrate synthase interacts with some other molecules involved in Ca²⁺ signaling in the egg cytoplasm. In this connection, the treatment ofCynops eggs with D₂O enhances microtubule polymerization to form numerous small cytoasters in the egg cortex and causes egg activation (Iwao & Masui 1995), suggesting at least some role for cytoskeletal filaments in the egg activation process. Interestingly, in the protozoaTetrahymena, citrate synthase displays dual functions: as an active enzyme in mitochondria and as a cytoskeleton protein for 14 nm filaments (Numataet al. 1985,Numata 1996). The 14 nm filament protein, a dephosphorylated form of citrate synthase, is involved in oral morphogenesis and pronuclear behavior during fertilization (Numataet al. 1985,Kojima & Numata 2002) and associated with the HSP60 protein (Takedaet al. 2001). Citrate synthase in newts may also play dual roles: as a mitochondrial enzyme and as a sperm factor for egg activation. Indeed, it is well known that some active enzymes, such as lactate dehydrogenase, have been recruited, unchanged, to an extra role as structural protein crystallins in the lens of the eye (Wistowet al. 1987,Tomarev & Piatigorsky 1996). We have also recently demonstrated that SGP on the sperm membrane has a bifunctional role in sperm binding to both the vitelline envelope and the egg plasma membrane atXenopus fertilization (Nagaiet al. 2009,Kuboet al. 2010). Further investigation of the function of citrate synthase, not only in fertilization but also in early embryonic development, will provide insight into Ca²⁺ signaling and cell cycle regulation.

Specificity and variations in the sperm factor in vertebrates

Egg activation by theCynops sperm factor, citrate synthase, is specific for homologous eggs.Xenopus eggs are insensitive not only to theCynops sperm factor but also to the homologous sperm extract without citrate synthase activity (Haradaet al. 2011). These results indicating lack of Ca²⁺ release-inducing factor inXenopus sperm cytoplasm supports the notion that the activation ofXenopus eggs is mediated by the membrane receptor.Xenopus sperm, however, contain a small amount of heat-stable activity to activateCynops eggs, but which is quite different from that induced by citrate synthase. In this connection, it is reported that aXenopus sperm extract contained a factor for triggering Ca²⁺ oscillations in mouse eggs (Donget al. 2000) and that the injection of severalXenopus sperm into a homologous egg or a sperm-borne protein (PAWP) caused egg activation (Aarabiet al. 2010). The sperm of the monospermic frog,Bufo arenarum, contained two different types of activity, causing activation by injection into the egg or by external treatment (Bonillaet al. 2008). Further characterization of those factors will be necessary to clarify their roles in egg activation at frog fertilization.

The changes in [Ca²⁺]_i have not been determined at polyspermy in other vertebrates, but an artificial increase in [Ca²⁺]_i in a bird blastodisc causes egg activation accompanied by pronucleus formation (Mizushimaet al. 2007,2009). The injection of a sperm extract, but not of somatic cell extracts, induces egg activation dependent on intracellular Ca²⁺ activity (Mizushimaet al. 2009). In addition, the injection of chicken sperm extract into mouse eggs induces Ca²⁺ rises and initiates embryo development (Donget al. 2000,Kim & Gye 2003). As bird PLCζ causes activation in quail eggs (Mizushimaet al. 2009) and triggers Ca²⁺ oscillations in mouse eggs (Cowardet al. 2005), PLCζ seems to be a potent sperm factor for egg activation in polyspermic birds, but further investigation for the factor like citrate synthase will be important.

A single Ca²⁺ rise in monospermy

A review of the variations in egg activation systems, and particularly the Ca²⁺ rise at fertilization in living vertebrates (Fig. 3), may elucidate the evolutionary history of egg activation concomitant with the acquisition of polyspermy. In vertebrates, most fishes exhibit monospermy, except for cartilaginous fishes (elasmobranchs) such as sharks and chimaera (Hart 1990). In the primitive jawless fishes (Agnathans), the lampreys exhibit monospermy with a positive-going fertilization potential mediated by Ca²⁺-activated Cl⁻ channels (Kobayashiet al. 1994). The pattern of fertilization potential alike in anuran eggs indicates a transient Ca²⁺ rise at lamprey fertilization. In the bony fish (Teleosts), Medaka (Oryzias latipes), a transient Ca²⁺ wave is induced by a fertilizing sperm from its entry site (Gilkeyet al. 1978). In frogs, monospermy is ensured by a positive-going fertilization potential mediated by the propagative opening of Ca²⁺-activated Cl⁻ channels (Kline & Nuccitelli 1985) preceded by a transient Ca²⁺ wave. In monospermic salamanders (urodeles),Hynobius eggs open Ca²⁺-activated Cl⁻ channels to produce a positive-going fertilization potential (Iwao 1989), indicating a transient Ca²⁺ wave at egg activation. Thus, the transient and large Ca²⁺ wave induced by a single sperm entry is probably characteristic of monospermic eggs in vertebrates, implying that the ancestor of vertebrates exhibited monospermy with a single and long-lasting Ca²⁺ wave.

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Variations in the mode of fertilization and Ca²⁺ rises in egg activation among vertebrates. poly, polyspermy; occ. poly, occasional polyspermy; mono, monospermy; single, a single Ca²⁺ rise at egg activation; multiple, multiple Ca²⁺ rises at egg activation; *Ca²⁺ rises presumed due to opening of Ca²⁺-activated Cl⁻ channels.

Citation: REPRODUCTION 144, 1;10.1530/REP-12-0104

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Multiple Ca²⁺ rises in polyspermy

In anamniotes, multiple Ca²⁺ waves (rises) are required for egg activation in the polyspermic eggs of physiological polyspermicCynops (urodeles). Multiple Ca²⁺ rises appear to occur in occasionally polyspermic eggs of the frogDiscoglossus (anurans). The multiple Ca²⁺ waves caused by a sperm cytosolic factor, e.g. citrate synthase, may reflect an evolutionary adaptation related to a change in mode of fertilization, e.g. the acquisition of physiological polyspermy. The close relationships in the acquisition of polyspermy between internal fertilization, increased egg size, centrosome dynamics, etc. have been the subject of much discussion (Elinson 1986,Wong & Wessel 2006,Snooket al. 2011). Physiological polyspermy might arise in large eggs with external fertilization, as in the Japanese giant salamander,Andrias japonicus, the large eggs (5–8 mm in diameter) inseminated after oviposition undergo polyspermic fertilization (Iwao 2000a). The extra space in the egg cytoplasm of large eggs is one of the most important factors for eliminating accessory sperm nuclei in the same egg cytoplasm, according to the ‘large egg model’ (Elinson 1986). Although physiological polyspermy is required to ensure fertilization in large eggs (Harper 1904), a single sperm does not contain sufficient sperm factor to induce the Ca²⁺ rise required for activation in a large egg. Multiple Ca²⁺ rises appear to be necessary for complete activation of large eggs. It has been hypothesized that the cytological polyspermy block in physiological polyspermy represents a more ancient type (Wong & Wessel 2006) and that urodeles and anurans may have arisen from different origins (Elinson 1986). As monospermic anurans may have branched from urodeles during the beginning of the Mesozoic period (240 million years ago;Feller & Hedges 1998), around the period when the ancestor of mammals appeared, multiple Ca²⁺ rises capable of activating large polyspermic eggs might be a characteristic shared with species that possess polyspermic amniotic eggs. It will be important to determine whether multiple Ca²⁺ rises occur at physiological polyspermy in living amniotes, reptiles, and birds.

Ca²⁺ oscillations in monospermic mammals

Higher eutherian mammals, however, exhibit monospermy and their sperm-specific PLCζ induce repetitive Ca²⁺ rises (oscillations) at egg activation (Fig. 1C and F;Swannet al. 2006,Nomikoset al. 2012). The fertilization of primitive mammals can be very instructive. The monotrematous platypus,Ornithorhynchus anatinus, lays large yolky eggs (about 4 mm in diameter) that undergo meroblastic cleavage in blastodiscs (Hughes & Hall 1998). The platypus eggs are polyspermic and several sperm probably enter into a blastodisc (41×368 μm;Gatenby & Hill 1924). In the small marsupial mammal,Sminthopsis crassicaudata, the relatively small egg (about 120 μm in diameter) contained a yolk mass in the center and some eggs are polyspermic (Breed & Leigh 1990). Thus, the decrease in egg size and yolk content is closely associated with the change in the mode of fertilization, from polyspermy to monospermy, in mammals. Although the changes in [Ca²⁺]_i at fertilization have not been reported in those primitive mammals, it may be that the ancestor of mammals exhibited polyspermy, which is required for repetitive Ca²⁺ waves and the induction of egg activation. Higher eutherians have small eggs (about 100 μm in diameter) without a yolk in the egg cytoplasm, but multiple Ca²⁺ rises lasting more than 1 h are still necessary for complete activation of mouse eggs (Ozil 1990,Ducibellaet al. 2002), indicating that the first Ca²⁺ rise by the entry of a single sperm is insufficient for egg activation. It may be the case that Ca²⁺ oscillations in the relatively small eutherian eggs might be functioning in place of multiple Ca²⁺ waves of the ancestral polyspermic eggs. It should be, however, mentioned that the monospermic eggs in invertebrates exhibit Ca²⁺ oscillations during fertilization (Stricker 1999,Dumollardet al. 2002) and multiple Ca²⁺ rises might be associated with longer period in completion of meiosis after fertilization.