Keywords

1 Introduction

Fertilization is indispensable for sexual reproduction in animals. Both sperm and eggs are highly specialized to ensure development with the diploid genome. We have focused on the molecular mechanism of fertilization in amphibians, which are one of the best models for studying fertilization, in particular, for understanding the evolution in egg activation and polyspermy blocks in vertebrates. There are two different types of fertilization in vertebrates: monospermy, in which only one sperm penetrates into the egg, and physiological polyspermy, in which several sperm enter the egg at normal fertilization. Fertilization in the ancestral vertebrates seems to be monospermic, because not only deuterostome invertebrates such as sea urchins and ascidians, but also most fishes, including a primitive fish, the lamprey, exhibit monospermy (Iwao 2012). Amphibians consist of three groups: anurans (frogs and toads), urodeles (newts and salamanders), and caecilians (limbless amphibians) (Iwao 2000a). Although there is little information on fertilization of caecilians, most anurans exhibit external and monospermic fertilization (Table 15.1). Only one sperm is incorporated into the egg, and other sperm are prevented from entering the fertilized egg. Several blocks to polyspermy operate to exclude the extra sperm outside the egg plasma membrane. In contrast, most urodeles exhibit internal fertilization and the female stores the sperm in a spermatheca near the cloaca (Akiyama et al. 2011). The eggs are inseminated by a small number of sperm released from the spermatheca just before oviposition. Although several sperm enter a physiologically polyspermic urodele egg, development with the diploid genome is ensured by the intracellular block to polyspermy in egg cytoplasm (Fankhauser 1948; Iwao and Elinson 1990; Iwao et al. 1993, 2002). Only one sperm nucleus forms a zygote nucleus with the egg nucleus, and the other extra sperm nuclei degenerate before cleavage. However, the most primitive Hynobius salamanders exhibit external fertilization and monospermy as with anurans (Iwao 1989). Thus, comparative studies in fertilization among amphibians will provide better understanding of the role of egg activation in establishment of polyspermy during vertebrate evolution.

Table 15.1 Characteristics of fertilization in amphibians
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2 Egg Activation at Physiologically Polyspermic Fertilization

The egg nucleus in unfertilized eggs of vertebrates is arrested at metaphase of the second meiotic division until the sperm breaks the attest at fertilization. Fertilization provides the sperm nucleus into the egg, as well as initiates its embryonic development, which is called egg activation. An increase in [Ca2+] i in the egg cytoplasm induced by the fertilizing sperm is essential for egg activation in vertebrate fertilization (Iwao 2000b). In monospermic anurans, the fertilizing sperm induces a transient and single [Ca2+] i increase that propagates from a sperm entry site toward the opposite site on the whole egg surface as a [Ca2+] i wave (Fontanilla and Nuccitelli 1998). The [Ca2+] i increase causes the opening of Cl− channels on the egg plasma membrane to produce a positive fertilization potential within 1 s (Kline and Nuccitelli 1985), which prevents the entry of another sperm as a fast, but temporal, block to polyspermy (Cross and Elinson 1980; Iwao 1989; Iwao and Jaffe 1989). The Ca2+ wave then induces exocytosis of cortical granules, resulting in transformation of the vitelline coat into a fertilization coat (Hedrick 2008). The fertilization coat prevents extra sperm from reaching the fertilized egg, as a slow and permanent block to polyspermy. In the monospermic salamander Hynobius nebulosus, the eggs exhibit a large positive fertilization potential mediated by opening of Ca2+-activated Cl− channels (Iwao 1989). The fast and transient opening of Cl− channels indicates a single Ca2+ wave at fertilization. Polyspermy is also prevented by a positive fertilization potential without formation of a fertilization coat in monospermic salamanders. Thus, the fast generation of a single Ca2+ wave soon after the entry of the first sperm is important for the accomplishment of the fast polyspermy block in monospermic species.

In contrast, in physiologically polyspermic newts, small and multiple increases of [Ca2+] i occur at fertilization (Grandin and Charbonneau 1992; Yamamoto et al. 1999; Harada et al. 2011). The [Ca2+] i increase at the sperm entry site propagates as a Ca2+ wave (Harada et al. 2011). The small Ca2+ wave is induced by each fertilizing sperm in the polyspermic egg. Because 2 to 20 sperm enter an egg at normal newt fertilization (Iwao et al. 1985), multiple Ca2+ waves induce [Ca2+] i increase, lasting 30–40 min after the first sperm entry (Harada et al. 2011). The [Ca2+] i increase induces no change (Charbonneau et al. 1983) or a very small hyperpolarization in response to each sperm entry (Iwao 1985) in the egg membrane potential. Sperm entry into newt eggs is not sensitive to the positive membrane potential (Iwao and Jaffe 1989). No cortical granule is observed in urodele eggs, indicating lack of fertilization envelope formation (Iwao 2000a).

3 The Signaling Mechanism of [Ca2+] i Increase Induced by the Fertilizing Sperm

The Ca2+ increase at fertilization is caused by the release of Ca2+ ions from the endoplasmic reticulum (ER), a major intracellular Ca2+ store in the egg cytoplasm (Fig. 15.1). Inositol 1,4,5-trisphosphate (IP3) generated by phospholipase C (PLC) opens Ca2+ channels of IP3 receptors on the ER. However, the mechanism for induction of [Ca2+] i increase by the fertilizing sperm is quite different between monospermic and physiologically polyspermic eggs. In monospermic anurans, a sperm agonist (ligand) probably binds an egg receptor at contact between the sperm and egg membranes, and then a signal for stimulating IP3 production is transmitted into the egg cytoplasm. Indeed, Xenopus eggs are activated by external treatment with tryptic sperm protease (Iwao et al. 1994; Mizote et al. 1999), which can hydrolyze one of the candidates of egg receptors, uroplakin III (UP III), on the egg plasma membrane (Sakakibara et al. 2005; Sato et al. 2003). The cleavage of UP III induces activation of Src kinase and PLCγ to produce IP3 in egg cytoplasm (Mahbub Hasan et al. 2005, 2007; Ijiri et al. 2012). In addition, the [Ca2+] i increase in Xenopus eggs is induced by external treatment of the egg with RGD-containing peptides (Iwao and Fujimura 1996) or KTE-containing peptides (Shilling et al. 1998), which binds integrins on the plasma membrane accompanied by activation of Src kinase (Sato et al. 1999). RGDS peptide also activates the eggs of monosperrmic Hynobius salamanders (Iwao, unpublished observations, 2012). Although the precise interaction between those molecules remains to be investigated, the initial Ca2+ release induced at the sperm entry site is propagated through further activation of PLCγ or direct sensitization of IP3 receptors on ER abundant in egg cortex, resulting in the formation of a single Ca2+ wave.

Fig. 15.1
figure 1

Model of signaling for Ca2+ increase in a physiologically polyspermic newt egg. The sperm-specific citrate synthase is introduced from the sperm cytoplasm into the egg cytoplasm after sperm–egg fusion. Citrate synthase, in association with cytoskeletons, sensitizes the inositol-1,4,5-trisphosphate (IP3) receptor on the inner endoplasmic reticulum (ER) to release Ca2+ ions. The local Ca2+ increase propagates through the inner ER with cytoskeletons as a Ca2+ wave by the activation of phospholipase C (PLC) to produce IP3 or stimulation of IP3 receptors

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In polyspermic newt eggs, the signal for egg activation is provided from sperm cytoplasm after sperm and egg fusion (Fig. 15.1). Injection of an extract containing newt sperm cytoplasm into unfertilized eggs induces egg activation accompanied by a Ca2+ wave (Yamamoto et al. 2001; Harada et al. 2007, 2011). A sperm-specific form of citrate synthase is purified from the sperm extract as one of the major components of the sperm factor for egg activation (Harada et al. 2007). A large amount of citrate synthase is localized in the neck to the midpiece of newt sperm (Fig. 15.2A), but a smaller amount is also distributed under the plasma membrane around the nucleus (Fig. 15.2B). Injection of not only purified citrate synthase protein, but also mRNA of citrate synthase, induces egg activation with a Ca2+ increase (Harada et al. 2007). A single newt sperm contains about 2 pg citrate synthase, but injection of sperm cytoplasm equivalent to one sperm activates about 20 % of the eggs, indicating that the entry of at least two sperm is necessary for activating the egg. This estimation corresponds well to the observation that a small Ca2+ wave is induced by each sperm entry in the polyspermic newt egg (Harada et al. 2011). How does the sperm-specific citrate synthase induce the Ca2+ wave in the egg cytoplasm? In some cases, the Ca2+ wave is preceded by a small spike-like Ca2+ increase (Harada et al. 2011). The sperm tryptic protease seems to be involved in the small and nonpropagative Ca2+ increase, but this is insufficient for inducing the Ca2+ increase to cause egg activation, probably because of the lack of cortical ER in newt eggs. The inner ER forms a larger complex with some cytoskeletons and is required to trigger a Ca2+ wave by the sperm factor (Harada et al. 2011). Egg activation not only by injection of the sperm factor, but also by fertilizing sperm, is probably mediated by the enzymatic activity of sperm citrate synthase (Harada et al. 2011). Reactive substrates of citrate synthase, acetyl CoA and oxaloacetate, induce Ca2+ increase to cause egg activation, but citrate does not. The reverse reaction might occur in egg cytoplasm containing a large amount of citrate, and acetyl CoA might then sensitize IP3 receptors to release Ca2+. Further investigation is, however, necessary for determining the exact changes of those substances at fertilization. Furthermore, it is possible that citrate synthase interacts with other molecules such as cytoskeletons (see Fig. 15.1; Iwao and Masui 1995). Investigations into the role of microtubules and microfilaments are important for clarifying the Ca2+-signaling cascade by the sperm factor.

Fig. 15.2
figure 2

(A), (B) Newt Cynops pyrrhogaster sperm show localization of citrate synthase (red) on left and merge with the differential interference contrast (DIC) image on right. (C), (D) Salamander Hynobius nebulosus sperm show citrate synthase (red) on left, α-tubulin (green) in middle, and merge with DIC image on right. A acrosome, H head region, M midpiece

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4 Evolution of a Sperm Factor in Vertebrate Fertilization

It is worth discussing the species specificity of sperm factors to understand the evolution of egg activation in vertebrates. Although it is reported that extract of the monospermic Xenopus sperm induces Ca2+ oscillation when injected into mouse eggs (Dong et al. 2000), and the injection of several sperm into a Xenopus egg causes egg activation (Aarabi et al. 2010), no activity to activate Xenopus eggs is detected in homologous sperm extract (Harada et al. 2011). Xenopus eggs do not respond to the newt sperm factor, and no citrate synthase is detected in Xenopus sperm (Table 15.1). Not only polyspermic newt sperm, but also monospermic Hynobius sperm, contain a large amount of citrate synthase under the plasma membrane in the head region, except for the acrosomal region (Fig. 15.2C). Citrate synthase is distributed in close association with microtubules (Fig. 15.2D). A large amount of sperm citrate synthase is observed in mammalian mouse sperm, but not in fish carp sperm (Iwao and Harada, unpublished observations, 2011). Thus, the extramitochondrial localization of citrate synthase in the sperm appears to be acquired in the transition between monospermy and physiological polyspermy in urodele amphibians.

Taken together, the large and single Ca2+ wave induced by the first sperm entry is necessary for ensuring monospermy to elicit the positive fertilization potential mediated by Ca2+-activated Cl− channels in monospermic vertebrates, such as lampreys (Kobayashi et al. 1994), frogs, and Hynobius salamanders (Table 15.1). In the bony fishes, a single Ca2+ wave is induced by a fertilizing sperm (Gilkey et al. 1978; Webb and Miller 2013), but monospermy is ensured by a micropyle (canal) on the hard chorion, through which only one sperm approaches the egg (Iwamatsu 2000). Thus, the single Ca2+ wave at egg activation is characteristic of monospermic vertebrates (Iwao 2012). In this connection, it is interesting to know the Ca2+ increase at the physiological polyspermy of large eggs in sharks and chimera (Hart 1990). In contrast, multiple Ca2+ waves are necessary for egg activation in physiological polyspermy because a single newt sperm does not have a sufficient amount of sperm factor to induce egg activation and multiple Ca2+ increases are necessary for complete activation of the large eggs (Iwao 2012). Some transitional characteristics are, however, observed in occasionally polyspermic eggs of the frog Discoglossus picutus with multiple Ca2+ increases (Talevi 1989), or in external and polyspermic fertilization in the Japanese giant salamander Andrias japonicus (Table 15.1) (Iwao 2000a). Physiological polyspermy probably appeared in species whose egg size was more than about 2 mm in diameter (Table 15.1). Reptiles and birds lay larger and yolky eggs, but their Ca2+ increase at polyspermy remains to be investigated. In primitive mammals, the monotrematous platypus laying big eggs exhibits physiological polyspermy (Gatenby and Hill 1924). Although a small and yolkless egg of the higher eutherian mouse exhibits monospermy, it elicits multiple Ca2+ increase to ensure sufficient egg activation (Ozil 1990; Ducibella et al. 2002). Sperm-specific PLCζ is known as a potent sperm factor for egg activation in mammals (Saunders et al. 2002; Kouchi et al. 2004) and birds (Mizushima et al. 2009). In mammalian egg activation, the role of sperm citrate synthase remains unknown.

5 Perspective

Thus, comparative studies in fertilization among vertebrates provide better understanding of the role of egg activation in establishment of polyspermy during evolution. Because egg activation by sperm citrate synthase is tightly linked to slow egg activation in physiological polyspermy, investigations in polyspermic birds and reptiles are important to clarify the evolution of egg activation in vertebrates. It is also interesting to know the mechanisms of egg activation in bony fishes that exhibit monospermy but lack the fast electrical block to polyspermy. In addition, investigations in invertebrates, such as ascidians and sea urchins, may provide us with the ancestral and universal mechanisms of egg activation during the evolution of animal reproduction.