For precise temporal activation of the egg during amphibian fertilization, the sperm must provide a signal for egg activation at the time of membrane binding or fusion between sperm and eggs. A fertilizing sperm causes a Ca2 wave which is both necessary and sufficient for egg activation at amphibian fertilization. The Ca2 wave seems to be mediated by IP3-receptors on the endoplasmic reticulum and by IP3 produced by hydrolysis of PLC activated by a Src-related protein tyrosine kinase (Xyk) in Xenopus eggs. We have proposed three different hypotheses for initiation of egg activation in amphibian eggs: the Ca2 -influx model, the membrane receptor model, and the soluble factor model. The membrane receptor model and the soluble factor model seems to be applied to the monospermic Xenopus fertilization and the physiologically polyspermic Cynops fertilization, respectively. The Ca2 wave at egg activation induces a positive fertilization potential which prevents entry of a second sperm in fertilization of monospermic species. In physiologically polyspermic urodele eggs, several sperm enter the egg at normal fertilization, but only one sperm nucleus with a centrosome participates in the embryonic development. The degeneration of accessory sperm nuclei is closely involved in differential distributions of both γ-tubulin and cyclin B in the egg cytoplasm, which causes developing a larger sperm aster and earlier entry into M phase in a zygote nucleus, respectively. We have discussed the molecular mechanisms of egg activation and polyspermy blocks in amphibians and make some comparisons with other vertebrates, such as fishes and mammals.
Fertilization brings about at least three distinct reactions: restoration of the diploid configuration with mixing of the male and female genomes, introduction of the centriole necessary for cell division into the egg in most vertebrates, and activation of the process of cell division for development. Precise temporal activation of the egg during fertilization is essential for normal development, since activation before entry of the sperm nucleus can cause parthenogenesis. Conversely, a delay in activation can cause pathological polyspermy. Thus, the sperm must provide a signal for egg activation at the time of membrane binding or fusion between sperm and eggs.
Amphibians contain two groups exhibiting very different blocks to polyspermy (Elinson, 1986; Iwao, 2000). One is a block before sperm-egg fusion, operating in monospermic eggs of anurans and some primitive urodeles, which is a species of the Hynobius genus (Iwao, 1989). Another is a block in egg cytoplasm after sperm entry, operating in physiologically polyspermic eggs in other urodeles. In monospermic species, development of an embryo with a diploid genome and a single centrosome (centriole) derived from a sperm is ensured by recruitment of a fast, electrical block to polyspermy on egg membrane, followed by a block at fertilization envelope formed by cortical granule exocytosis. The entry of more than two sperm causes abnormal development in monospermic species. In physiologically polyspermic species, several sperm enter an egg at normal fertilization, but only a single sperm nucleus with a single centrosome ultimately participates in embryonic development, while the other sperm nuclei and centrosomes degenerate before cleavage. Since faster activation seems to be necessary for the polyspermy block in monospermic species compared with physiologically polyspermic species, different mechanisms of egg activation may operate between these species. In this review we will discuss the molecular mechanisms of egg activation and polyspermy blocks in amphibians and will make comparisons with other vertebrates, such as fishes and mammals.
Primary role of Ca2+ in amphibian egg activation
A fertilizing sperm causes an increase in intracellular free Ca2+ ([Ca2+]i) in the eggs of both the anuran Xenopus laevis (Fig. 1A) (Busa and Nuccitelli, 1985; Nuccitelli et al., 1993; Iwao and Fujimura, 1996; Fontanilla and Nuccitelli, 1998), and the urodeles Pleurodeles waltl (Gradin and Charbonneau, 1992) and Cynops pyrrhogaster (Fig. 1B) (Yamamoto et al., 1999a), as well as in other vertebrates (Stricker, 1999). In Xenopus eggs, an initial Ca2+ increase occurs near the sperm entry site, followed by a propagative Ca2+ wave spreading towards the opposite side of the egg (Nuccitelli et al., 1993; Fontanilla and Nuccitelli, 1998). The peak level of [Ca2+]i is estimated to be about 1.2 μ M in the cortex and 0.7 μ M in the center of the egg (Fontanilla and Nuccitelli, 1998). The Ca2+ increase in Xenopus eggs is similar to that observed in Ca2+ oscillation of mammalian eggs (0.5–2.5 μ M, Miyazaki et al., 1993), but lower than that in the eggs of Oryzias latipes eggs (30 μ M, Gilkey et al., 1978), which is a fish. The Ca2+ increase (0.15 μ M) in Pleurodeles is less than those in other vertebrates (Gradin and Charbonneau, 1992). Since the velocity of the Ca2+ wave in Xenopus is somewhat greater in the cortex (8.9 μ m/sec) than in the center of the egg (5.7 μ m/sec) (Fontanilla and Nuccitelli, 1998), the Ca2+ wave reaches the opposite side of the egg about 10 min after initiation. At Cynops fertilization, after an initial spike-like Ca2+ increase lasting about 30 sec, a Ca2+ wave spreads with a velocity of 5.0–6.0 μ m/sec for about 40 min (Fig. 1B) (Yamamoto et al., 1999a,b). The velocity of Ca2+ waves in amphibians is somewhat slower than that in fishes (9–12 μ m/sec, Gilkey et al., 1978; Lee et al., 1999) or mammals (16–28 μ m/sec, Miyazaki et al., 1993). Since the single Ca2+ increase occurs at amphibian fertilization, a relatively high [Ca2+]i continues for 10–15 min in Xenopus eggs (Fig. 1A) and for 30 – 40 min in Cynops eggs (Fig. 1B). Oryzias eggs exhibit a single Ca2+ wave lasting about 15 min (Gilkey et al., 1978), but a repetitive Ca2+ increase (Ca2+ oscillation) occurs in mammalian eggs, each lasting 0.5–4 min for about 2 hor (Miyazaki et al., 1993).
The increase in [Ca2+]i is both necessary and sufficient for egg activation in amphibians. Prevention of this increase at fertilization by injection of the Ca2+ chelator, BAPTA, inhibits all events in egg activation, including elicitation of the fertilization potential, cortical granule exocytosis, cortical contraction in Xenopus (Kline, 1988), and resumption of meiosis in both Xenopus (Kline, 1988) and Cynops (Yamamoto et al., 1999a). A [Ca2+]i increase induced by the Ca2+ ionophore A23187 causes egg activation in both anurans (Steinhardt et al., 1974; Iwao, 1982) and urodeles (Charbonneau and Picheral, 1983; Iwao and Masui, 1995). Anurans eggs can be activated by introduction of Ca2+ into the egg cytoplasm by injection (Cross, 1981) or by pricking with a fine needle (Goldenberg and Elinson, 1980; Iwao et al., 1981). Longer treatment with a higher concentration of ionophore A23187 is necessary for activation of Cynops eggs in comparison with anuran eggs (Iwao and Masui, 1995). While pricking can cause egg activation in Pleurodeles (Aimar and Larousse, 1975) and Hynobius nebulosus (Iwao, 1989), the eggs of most urodeles are relatively insensitive to pricking (Fankhauser, 1967; Iwao and Masui, 1995). Injection of Ca2+ into Oryzias eggs induces egg activation with a Ca2+ wave (Iwamatsu et al., 1988a,b). The introduction of Ca2+ into mammalian eggs does not appear to be sufficient to induce sustained Ca2+ oscillation (Swann and Ozil, 1994).
One Ca2+ store in amphibian eggs seems to be the endoplasmic reticulum, which is abundant in the egg cortex (Gardiner and Grey, 1983; Campanella et al., 1988). Inositol-1,4,5-trisphosphate (IP3)-receptors preferentially localized in the egg cortex (Kume et al., 1993) are likely involved in the Ca2+ increase at amphibian fertilization. The amount of IP3 in Xenopus egg cytoplasm increases 3- to 5-fold at fertilization (Stith et al., 1993, 1994; Snow et al., 1996). Injection of IP3 into the eggs of both Xenopus (Busa et al., 1985; Larabell and Nuccitelli, 1992) and Cynops (Yamamoto et al., 1999b) causes an increase in Ca2+. Injection of heparin, an inhibitor of IP3-receptors, prevents Ca2+ waves at fertilization in both Xenopus (Nuccitelli et al., 1993) and Cynops (Yamamoto et al., 1999b) eggs. Injection of an antibody against type 1 IP3-receptor into Xenopus eggs reduces the Ca2+ increase at fertilization (Runft et al., 1999). The Ca2+ wave in Xenopus eggs seems to be induced by Ca2+ that promotes Ca2+-induced Ca2+ release (CICR) acting on IP3-receptors directly or through IP3 production (Nuccitelli et al., 1993). The faster Ca2+ wave in the cortex may be due to abundant endoplasmic reticulum with IP3-receptors in the cortex. However, local and transient Ca2+ increases, known as “hot spots”, have been observed at fertilization of Xenopus eggs injected with heparin (Nuccitelli et al., 1993; Fontanilla and Nuccitelli, 1998) or anti-IP3 receptor antibody (Runft et al., 1999). Since each hot spot probably represents a Ca2+ increase at each sperm entry site, another mechanism that is not mediated by IP3 receptors may operate in the initial phase of the Ca2+ increase in Xenopus eggs. IP3-injection causes a Ca2+ increase in Oryzias eggs (Nuccitelli et al., 1987; Iwamatsu et al., 1988a). Injection of an antibody against IP3-receptors into hamster eggs completely inhibits the Ca2+ increase at fertilization (Miyazaki et al., 1992). However, IP3 can not fully mimic a Ca2+ oscillation in mammalian eggs (Swann and Ozil, 1994). Although ryanodine receptors also seem to function in the Ca2+ oscillation in mammalian eggs (Stricker, 1999), they are unlikely to be involved in the Ca2+ increase in amphibian eggs, since they have not been observed in Xenopus eggs (Parys et al., 1992) and injection of cyclic-ADP ribose does not cause a Ca2+ increase in Xenopus egg homogenate (Whitaker and Swann, 1993) or in Cynops eggs (Yamamoto S and Iwao Y, unpublished data).
Signaling pathways in the Ca2+ increase at egg activation
IP3 is produced by hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) into IP3 and diacylglycerol (DG) with the phospholipase C (PLC) enzymes which include three subgroups: PLC-β, PLC-γ, and PLC-δ, (Fig. 2). Both a functional G-protein/PLC-β pathway (Kline et al., 1988) and a PLC-γ (Yim et al., 1994) are present in Xenopus eggs, which are responsive to exogenously expressed receptors for serotonin 1C and epidermal growth factor, respectively. However, the Ca2+ increase in Xenopus eggs is not inhibited by pertussis toxin, which inhibits a Gi family of G-proteins (Kline et al., 1991), or by an antibody against a Gq family G-protein (Runft et al., 1999). There is no direct evidence in favor of the involvement of a G-protein/PLC-β pathway in egg activation at amphibian fertilization. The Ca2+ increase is not inhibited by injection of the SH2-domain of PLC-γ1 in which tyrosine kinases bind to activate PLC-γ (Runft et al., 1999). No PLC-γ 2 is detected in Xenopus eggs (Runft et al., 1999), although some inhibitors of tyrosine kinases block a Ca2+ increase and egg activation in Xenopus eggs (Glahn et al., 1998; Sato et al., 1998; 1999). It has been shown that a Src-related protein tyrosine kinase (Xyk) localized in the egg cortex is activated and translocated to egg cytoplasm (a soluble fraction) at fertilization in Xenopus (Sato et al., 1996; 1999). Both activation and translocation are induced by a fertilizing sperm, but not by an artificial Ca2+ increase induce by an ionophore or electric shock (Sato et al., 1999), while injection of a peptide that inhibits Xyk does block egg activation in Xenopus (Sato et al., 1999). These results suggest that Xyk plays a role in the cascade between sperm-egg binding/fusion and the Ca2+ increase. Xyk seems to stimulate PLC-γ through a SH2-domain-independent mechanism, such as a partial proteolysis or noncatalytic interaction with other molecules, since Xyk is associated with PLC-γ and activation of PLC-γ is inhibited by a specific inhibitor of Src-related kinases (Sato K, et al., 2000). However, since Ca2+ hot spots are seen at fertilization of eggs in which the Ca2+ waves were inhibited by a tyrosine kinase inhibitor (Glahn et al., 1998), a pathway that is different from the Xyk cascade may be involved in the initial and local Ca2+ increase at the sperm entry site. The role of PLC-δ in the Ca2+ increase remains to be investigated in amphibian fertilization. Among mammals, injection of GDP-β-S inhibits G-proteins and blocks the Ca2+ increase in hamster eggs (Miyazaki et al., 1993), while injection of an antibody against the Gq family G-protein does not inhibit activation of mouse eggs (Williams et al., 1998). Furthermore, a Ca2+ increase in mouse eggs is not blocked by injection of the SH2-domain of PLC-γ (Mehlmann et al., 1998).
Mechanisms of sperm-induced initiation of Ca2+ increase
While it is unknown just how a fertilizing sperm transmits the initial signal for the Ca2+ increase at fertilization, there are at least three different hypotheses for initiation of egg activation in animal eggs: the Ca2+-influx model, the membrane receptor model, and the soluble factor model (Fig. 2). In any case, a fertilizing sperm must stimulate IP3 production in the egg cytoplasm to potentiate the Ca2+ wave.
(A) In the Ca2+ influx model (Fig. 2A), a fertilizing sperm induces an influx of external Ca2+ required for egg activation either through Ca2+ channels on the sperm membrane following sperm-egg fusion or on the egg membrane at the sperm-egg binding/fusion. Amphibian eggs can be artificially activated by a Ca2+ release from internal stores in the absence of external Ca2+ (Steinhardt et al., 1974; Yamamoto et al., 1999b). The progression of the Ca2+ wave is not affected by depletion of external Ca2+ (Fontanilla and Nuccitelli, 1998; Yamamoto, 1999b). No cortical flush of the Ca2+ increase is seen at the initial phase of the Ca2+ increase. The Ca2+ increase from internal stores seems to be sufficient for egg activation of amphibian eggs. Since CICR is induced by Ca2+ injection (Cross, 1981) and PLCs can be stimulated by 1–10 μ M Ca2+ (Hwang et al., 1996), it remains to be determined whether the initial Ca2+ increase around the sperm entry site is dependent upon external Ca2+. In order to determine a role of the Ca2+ influx at fertilization of amphibians, it should be determine whether amphibian eggs can be fertilized and are normally activated in the absence of external Ca2+ ions. Oryzias eggs can be fertilized, and the Ca2+ wave is not affected in the absence of external Ca2+ (Gilkey et al., 1978). In zebrafish eggs, the external spawning medium triggers an activating Ca2+ wave without sperm-egg fusion, but neither sperm nor external Ca2+ is required to initiate the Ca2+ wave (Lee et al., 1999). The exact mechanism of egg activation in fishes remains to be investigated. In mouse eggs, a fertilizing sperm can induce the Ca2+ increase even where there is a very low concentration of external Ca2+ (13 nM) (Jones et al., 1998b).
(B) The membrane receptor model proposes that an agonist (ligand) on the sperm membrane binds to a receptor on the egg membrane to cause IP3 production in the egg cytoplasm (Fig. 2B). We have suggested that a positively charged molecule(s) on the sperm membrane is involved in sperm-egg binding and fusion, based on the analysis of cross-fertilization between voltage-sensitive and voltage-insensitive species (see below). In Xenopus eggs, a Ca2+ increase is induced by external treatment with peptides containing an RGD sequence (Iwao and Fujimura, 1996), which is well known as a ligand for integrins. RGD-containing peptides can induce a Ca2+ increase in the absence of external Ca2+. The treatment with RGD-containing peptides causes activation in Hynobius eggs (Fujumura and Iwao, 1997), but does not in Cynops eggs (Iwao Y, unpublished data). Xenopus sperm contain a protein of the metalloprotease/disintegrin/cysteine-rich (MDC) family (xMDC16) (Shilling et al., 1997). Peptides containing a sequence (KTE) of its disintegrin domain inhibit fertilization (Shilling et al., 1997). Treatment with a high concentration of these peptides causes a Ca2+ increase and activation in Xenopus eggs (Shilling et al., 1998). These results indicate that the sperm protein binds to a receptor, probably an integrin(s), on the egg membrane and transmits a signal for the Ca2+ increase at fertilization. However, no receptor has been found for either the RGD-containing peptides or the xMDC16. Another potential candidate for the sperm agonist is a sperm acrosomal protease purified from Cynops sperm (Fig. 3) (Iwao et al., 1994; Mizote et al., 1999). Xenopus eggs can be fertilized by Cynops sperm and are completely activated by the external application of the protease purified from Cynops sperm. The sperm protease causing a Ca2+ wave in Xenopus eggs (Iwao et al., 1995) is a high-molecular weight complex with a unique tryptic protease that efficiently hydrolyzes the C-terminus of double arginine protein residues (Mizote et al., 1999). Homologous Xenopus fertilization is inhibited by inhibitors for the sperm protease (Mizote et al., 1999) and a similar protease is localized on the Xenopus sperm membrane (Mizote et al., 1999; Iwao Y, unpublished data). The sperm protease might cleave a receptor on the egg membrane, as does a protease-activating receptor for the thrombin system (Vu et al., 1991). Thus, there are strong evidences in support of the membrane receptor model for the Ca2+ increase in Xenopus eggs, although the target of the sperm protease on the egg membrane is still unknown, In mammals, sperm-egg binding is mediated by the interaction between the fertilin α/β MDC proteins on the sperm membrane and the integrin α6/β1 on the egg membrane (Almeida et al., 1995). Since mouse sperm that lack the fertilin subunit can fertilize the eggs and cause activation (Cho et al., 1998), fertilin is probably not involved in egg activation in mammals. Since CD9, which is a member of the transmembrane-4 superfamily, is found on the egg membrane and is required for membrane fusion (Miyado et al., 2000; Le Naour et al., 2000), its role in Ca2+ signaling in mammalian eggs should be investigated further.
(C) The soluble factor model proposes that a soluble component(s) in sperm cytoplasm is transmitted to egg cytoplasm after sperm-egg fusion, which then causes the Ca2+ increase. This model, based on the latent period between sperm-egg membrane fusion and the onset of the Ca2+ increase in egg cytoplasm (Whitaker and Swann, 1993), may apply to Cynops egg activation. Injection of sperm soluble components into Cynops eggs causes a Ca2+ increase and complete egg activation (Yamamoto et al., 1999b). This appears to be consistent with the finding that Cynops eggs are resistant to a transient Ca2+ increase by pricking and to the treatment with RGD-containing peptides, as discussed above. In support of this, only a small percentage of Cynops eggs are activated by external treatment with the sperm protease (Iwao et al., 1994). The sperm factor in Cynops is known to be a heat-labile and proteinous molecule(s). However, further investigation is necessary to determine whether Cynops sperm contains a sufficient amount of sperm factor to activate an egg. Recent studies on mammals strongly support the soluble factor model for Ca2+ oscillation (Swann and Parrington, 1999). Injection of a soluble sperm extract into mammalian eggs is known to trigger a Ca2+ oscillation (Parrington et al., 1996; Swann and Parrington, 1999). While a 33-kDa protein (oscillin) has been proposed as the sperm factor responsible for the Ca2+ oscillation (Parrington et al., 1996), other recent candidates include a PLC (Dupont et al., 1996; Jones et al., 1998a), a truncated c-kit (Sette et al., 1997), or various perinuclear substances (Perry et al., 2000).
Fast polyspermy block at the egg membrane in monospermic amphibians
The unfertilized amphibian eggs are surrounded with several jelly layers and a vitelline envelope which play important roles in polyspermy block (Iwao, 2000). In monospermic amphibian species, the eggs from which external coats have been removed and surrounded with only egg membrane exhibit monospermy, indicating a polyspermy block at the level of the sperm-egg binding or fusion (Elinson, 1973; Katagiri, 1974). The propagative Ca2+ wave at egg activation induces a propagative opening of Cl– channels (halide ion channels) on the egg membrane (Kline and Nuccitelli, 1985), which causes a positive shift in the potential of the egg membrane (fertilization potential) in conditions of low external Cl– such as fresh water (Fig. 1C). While the level of fertilization potential is species specific, the eggs of most species have positive potentials of about +10~+40 mV (Iwao, 2000). The positive fertilization potential prevents entry of a second sperm for 10–15 min after fertilization. When the membrane potential of unfertilized eggs remains higher than 0 mV under voltage-clamp conditions, both sperm entry and egg activation are blocked (Cross and Elinson, 1980; Charbonneau et al., 1983; Jaffe et al., 1983a; Iwao 1989; Iwao et al., 1994). In contrast, polyspermy occurs when the egg membrane potential remains below 0 mV under voltage-clamp (Cross and Elinson, 1980; Charbonneau et al., 1983; Jaffe et al., 1983a; Iwao and Jaffe, 1989; Iwao et al., 1994) or in the presence of concentrated external halide ions (Grey et al., 1982). The voltages that inhibit fertilization correspond well to those of fertilization potentials induced by sperm (Iwao, 2000). Thus, a positive fertilization potential functions as a fast, electrical block to polyspermy in voltage-sensitive species. In physiologically polyspermic urodeles, fertilization is not blocked by any positive potentials (Charbonneau et al., 1983; Iwao and Jaffe, 1989). Cross-fertilization between the eggs of voltage-sensitive species and the sperm of voltage-insensitive species is not affected by positive potentials, resulting in polyspermy (Jaffe et al., 1983a; Iwao and Jaffe, 1989), while a cross between the eggs of voltage-insensitive species and the sperm of voltage-sensitive species is sensitive to the voltage of the egg membrane (Iwao and Jaffe, 1989). These results indicate that the voltage-sensor for fertilization is localized on the membrane of the sperm, and not on the egg membrane. Potential candidates for the voltage sensor are the sperm protease and the xMDC16 protein, because egg activation by their molecules is voltage-dependent (Iwao et al., 1994; Shilling et al., 1998). While the fast block is transient, the eggs accomplish a complete polyspermy block by the formation of a fertilization envelope and by hydration of the jelly layers (Iwao, 2000).
Oryzias (bony fish, Osteichthyes) eggs do not elicit a positive fertilization potential and their fertilization is voltage-insensitive (Nuccitelli, 1980), indicating lack of a fast electrical block. Limitation of sperm entry through a narrow micropyle on the egg envelope (chorion) is necessary to ensure monopsermy in bony fishes (Kobayashi and Yamamoto, 1981). In contrast, monospermic fertilization of the lamprey (jawless fishes, Agantha) in fresh water is ensured by a fast electrical block (Kobayashi and Yamamoto, 1994; Kobayashi et al., 1994). Lamprey eggs elicit a large positive fertilization potential mediated by the opening of Cl– channels which mainly localize in the animal pole region (Kobayashi et al., 1994). The positive potential blocks sperm-oocyte fusion, but not egg activation. These results suggest that the membrane receptor model can be applied to lamprey fertilization and that molecules with different voltage-sensitivities are involved in sperm-egg membrane fusion and the signaling pathway for egg activation. Fertilized mammalian eggs elicit repetitive hyperpolarizations (negative-going potentials) mediated by the opening of K+ channels in response to the Ca2+ oscillation (Miyazaki and Igusa, 1981; 1982). A fast electrical block does not operate in mammalian eggs (Jaffe et al., 1983b), where monospermy is generally accomplished by a zona reaction mediated by cortical granule exocytosis (Wassarmann, 1999).
Behavior of sperm nuclei and centrosomes in physiologically polyspermic urodele eggs
Physiological polyspermy is seen in some invertebrates and in several species of vertebrates, including fishes, urodele amphibians, reptiles, and birds (Austin, 1965). The mechanism of a polyspermy block in physiologically polyspermic eggs is well understood in urodeles (Fankhauser, 1948; Elinson, 1986; Iwao, 2000). Several sperm (2–20 sperm/egg) enter the egg at normal fertilization of Cynops (Iwao et al., 1985; 1993). While most sperm enter animal hemispheres or equatorial regions, some enter at the vegetal hemispheres (Iwao et al., 1993). The number of fertilizing sperm is limited by the hydration of the jelly layers (McLaughlin and Humphries, 1978; Matsuda and Onitake, 1984). All incorporated sperm undergo nuclear decondensation and form sperm pronuclei with functional centrosomes (Fig. 4). Each sperm pronucleus is associated with each sperm aster, but the asters in animal hemispheres are larger than those in vegetal hemispheres (Iwao et al., 1997). The size of the asters is dependent upon the state of the egg cytoplasm (Iwao et al., 1997), and probably upon the amount of γ-tubulin in the centrosomes which is responsible for microtubule polymerization (Iwao Y, unpublished data). A single sperm pronucleus, the “principal sperm nucleus”, forms a zygote nucleus with an egg pronucleus in the animal hemisphere. Although the exact mechanism for selection of the principal sperm nucleus remains unclear, the sperm nucleus nearest the egg nucleus appears capable of contacting the egg nucleus. All sperm and egg nuclei enter the S phase, but both the onset and the completion of DNA synthesis are earlier in the zygote nucleus (the egg and the principal sperm nuclei) than they are in other accessory sperm nuclei (Iwao et al., 1993). When the zygote nucleus enters the pro-metaphase, its single centrosome divides and forms a bipolar spindle with a diploid set of condensed chromosomes (Fig. 4). When the zygote nucleus enters the anaphase, centrosomes in the accessory sperm nuclei do not separate, and the nuclear membranes of the sperm nuclei in the vegetal hemispheres remain distinct and enclose the decondensed chromatins. Accessory sperm nuclei in the equatorial region sometimes form mono-polar spindles with a haploid set of chromosomes. After the first cleavage, all accessory sperm nuclei undergo degeneration. Their chromatins undergo pycnosis and the materials of their centrosomes are dispersed in the egg cytoplasm. Thus, only one sperm nucleus with a centrosome (a centriole) participates in the embryonic development of urodele eggs (Fig. 4).
The degeneration of accessory sperm nuclei is closely related to their failure to enter the M-phase (Iwao, 2000). M-phase promoting factor (MPF), consisting of cdc2 kinase (cdk1) and cyclin B (Lohka et al., 1988; Gautier et al., 1990), is a key component involved in entering the M-phase in many animal cells (Masui, 1992). Injection of an MPF-rich cytoplasmic fraction prevents some accessory sperm nuclei from degenerating in animal hemispheres or equatorial regions (Iwao and Elinson, 1990). The rescued accessory sperm nuclei form extra bipolar spindles with haploid sets of chromosomes, and then induce multipolar cleavage. In addition to the increased MPF activity, there are greater amounts of both cdc2 kinase and cyclin B in animal hemispheres compared with vegetal hemispheres (Sakamoto et al., 1998). Cyclin B is mainly distributed in the egg cortex (Sakamoto et al., 1998), and a large amount of cyclin B is associated with the zygote nucleus (Fig. 5A). Microtubule structures, such as sperm asters and cortical microtubules (Iwao et al., 1997), probably regulate the localization of cyclins in egg cytoplasm (Fig. 5B). Since DNA synthesis and centrosome separation in Xenopus eggs is dependent upon cdc2/cdk2 kinases (Chevalier et al., 1995) and cdk2/cyclins (Hinchcliffe et al., 1999), respectively, earlier entry of the zygote nucleus into both the S-phase and the M-phase, as well as centrosome separation, is probably due to the abundant cdc2 or cdk2 around the zygote nucleus. Furthermore, earlier entry into the anaphase in the zygote nucleus is likely stimulated by anaphase-promoting factor (APF) (Peters et al., 1996), which inactivates MPF by destruction of cyclin B through ubiquitin-dependent proteolysis (Aizawa et al., 1996; Tokumoto et al., 1997). Earlier disappearance of cyclin B around the zygote nucleus is seen around the anaphase of the first cleavage in Cynops eggs (Iwao Y, unpublished data). These results indicate that the accessory sperm nuclei are exposed to APF without sufficient exposure of MPF, which causes degeneration of accessory sperm nuclei. However, the molecular mechanism of degradation of accessory sperm chromatins and centrosomes remains unknown.
There are few reports of the nuclear behavior in other physiologically polyspermic animals that are comparable with urodele amphibians. In the domestic fowl, some accessory sperm form bipolar spindles at the M-phase for the first cleavage, but they never cause the extra cleavage furrow (Perry, 1987). The accessory sperm nuclei that disperse slightly towards the margin of a germinal disc degenerate after one round of mitosis (Perry, 1987; Waddington et al., 1998). There may be some difference in the ability to induce cleavage in the egg cortex. In the polyspermic invertebrate ctenophore Bore ovata, one sperm nuclei is selected to form the zygote nucleus after an egg nucleus approaches different sperm nuclei along the microtubules in egg cytoplasm (Carré and Sardet, 1984; Rouvière et al., 1994). Although each sperm pronucleus is associated with a sperm aster, they do not migrate (Houliston et al., 1993). The egg nucleus which enters into the center of the sperm aster forms the zygote nucleus (Carré et al., 1991). The mechanisms for suppression of accessory sperm in these species remain to be investigated.
Two different models for egg activation may apply in amphibian fertilization: the membrane receptor model for the anuran Xenopus and the sperm factor model for the urodele Cynops. Although the molecular mechanisms of egg activation are not fully understood in amphibians, the observed voltage-sensitive and voltage-insensitive fertilization seems to correspond well to the membrane receptor model and the sperm factor model, respectively. Since it is estimated that the second sperm probably reaches the egg membrane within several seconds of the arrival of the first sperm (Iwao, 2000), the signal transmission through the membrane receptor seems to be suitable for faster egg activation (faster generation of a positive fertilization potential) to prevent polyspermy in monospermic species. From the phylogenetic perspective, voltage-insensitive fertilization was probably acquired concomitant with the emergence of physiological polyspermy in urodeles. Recent molecular studies indicate that the anuran group may have branched relatively early from the urodele/caecillian (limbless amphibians) group, perhaps during the beginning of the Mesozoic period (240 million years ago), while the urodele and caecillians groups probably branched relatively late, in the late Mesozoic period (160–190 million years ago) (Feller and Hedges, 1998). In this connection, monospermy with the fast electrical block, but without the cortical granule-mediated block, in urodeles belonging to the genus Hynobius (Iwao, 1989; 2000), apparently shows an intermediate mode between monospermic anurans, with both fast and cortical granule-mediated blocks, and physiologically polyspermic urodeles, which lack both blocks.
In this context, the mode of fertilization in the ancestor of amphibians may provide an important view of the relationship between the type of polyspermy block and the mode of egg activation. Amphibians are believed to share a common ancestor with the bony fishes of subclass Sarcopterygii (lobefinned fishes), which contains Crossopterigii (coelacanth, Latimeria) and Dipnoi (lung fishes) (Meyer and Dolven, 1992). The molecular analysis in extant animals suggests that lung-fishes comprise the closest sister group of tetrapods (amphibians) (Zardoya and Meyer, 1997). Latimera is ovoviviparous (Smith et al., 1975), but the mode of fertilization in sacropterygian fishes remains unknown. As is the case in monospermic amphibians, lampreys exhibit voltage-sensitive fertilization and their eggs generate a positive fertilization potential mediated by Cl– channels. A Cl–-dependent fertilization potential may be necessary for monospermic fertilization in vertebrates that live in fresh water. Since fish sperm contain cleavage-initiation activities (Iwamatsu and Ohta, 1974), molecular mechanisms of egg activation in voltage-insensitive monospermic bony fishes and polyspermic cartilaginous fishes merit further investigation. Mammalian eggs exhibit voltage-insensitive fertilization and appear to be activated by the sperm factor. However, the mechanisms of activation of polyspermic yolky eggs of birds and reptiles remain to be investigated.
I would like to express my sincere thanks to Dr. Chiaki Katagiri, Dr. Ken-ichi Sato and Mr. Satoshi Yamamoto for their valuable comments on the manuscript and for using unpublished data. This work was supported by the Grant-in-Aid for Scientific Research (C) in The Ministry of Education, Science, Sports and Culture to Yasuhiro Iwao.
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