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1 Introduction

Conceptually, fertilization is a simple event: union of sperm and egg from the same species to produce an offspring. The actual fertilization process, however, involves a series of preparatory changes that have to be exquisitely coordinated in both gametes. For example, before reaching the egg, spermatozoa should gain their cytological competence by undergoing acrosome reaction and acquire apt motility for successful fertilization to occur. The substance on the egg surface may induce these changes and emit the cues for chemotaxis. On the other hand, the oocytes should be matured into fertilization-competent eggs before fertilization. The sequential programs that lead to gamete maturation, chemotaxis, acrosome reaction, and the binding of sperm to eggs may fundamentally vary depending on the phyla and species, as has been reviewed elsewhere (Trimmer and Vacquier 1986; Hirohashi et al. 2008; Santella et al. 2012). Upon sperm’s binding and fusion, the fertilized egg, which should be now called a zygote, undergoes a series of changes. The first detectable change is a swift shift of the membrane potential, which is accompanied by other cytological modifications such as cytoskeletal alteration and the increase of cytosolic pH and Ca2+ that are known to play important roles for the resumption of cell cycle and subsequent embryonic development. The intracellular Ca2+ increase in fertilized eggs is achieved mainly by two mechanisms, i.e., influx from the extracellular sources through voltage-gated ion channels and the ligand-gated release from the intracellular stores. The contribution and pattern of these two modes of Ca2+ increase may also differ widely depending on the species (Jaffe 1993; Miyazaki et al. 1975; Miyazaki 2006; Stricker 1999; Santella et al. 2004, 2012, 2015; Whitaker 2006). Hence, fertilization involves fine regulation of ion channel activities and cytoskeleton, as well as other cell signaling events that usher in a new life cycle in the zygote. In this chapter, we review how the basic cytoskeletal protein actin is utilized in the fertilization of starfish and sea urchin eggs.

2 Actin in Sperm Acrosome Reaction

In the research of fertilization, sea urchin and starfish have traditionally served as important animal models since sperm’s entry into eggs was first viewed in the echinoderm species: Echinus esculentus (Dufossé 1847), Paracentrotus lividus (Hertwig 1876), and Asterias glacialis (Fol 1879). While sperm in animal kingdom conspicuously display vast interspecific diversities in their morphology and the strategy to reach and fuse with the eggs (Pitnick et al. 2009), echinoderm spermatozoa generally tend to use filamentous projection from the head. Owing to this characteristic morphological feature and the timely development of phase-contrast microscopy, acrosome reaction was convincingly demonstrated in starfish sperm (Dan 1952). When exposed to the homologous egg water, sperm from diverse species of starfish (Asterina pectinifera, Asterias amurensis, and Astropecten scoparius) readily extended long, slender, and rigid filaments (Dan et al. 1954). This so-called acrosomal process of starfish sperm can reach 25 μm in length, which is much longer than the one produced by sea urchin sperm (Fig. 3.1), and may serve as a tether to draw the sperm into the fertilized egg (Dan et al. 1954; Puppo et al. 2008). The activated sperm of another echinoderm, sea cucumber can extend even longer acrosomal process than starfish sperm. In the case of Thyone briareus, the extending acrosomal process can reach over 70 μm in length within just 7 s. The exceedingly fast extension of the acrosomal process turned out to be due to the rapid polymerization of actin monomers that are reserved between the acrosomal vesicle and the nucleus of the sperm (Tilney et al. 1973; Tilney and Inoué 1982). This phenomenon was one of the first examples suggesting that actin polymerization per se can provide protrusive force to push the cell boundary (Footer et al. 2007) and rendered insights into the mechanisms of cell motility and plasticity through the changes in specialized actin-based subcellular structures such as lamellipodia, filopodia, microspikes, and growth cones (Welch et al. 1997; Luo 2002; Svitkina 2018). Owing to this long acrosomal process, which traverses the egg jelly coat, starfish spermatozoa can trigger the initial responses in the fertilized eggs, such as swift membrane depolarization, and Ca2+ increases while the sperm head still remains outside of the jelly coat (Puppo et al. 2008).

Fig. 3.1
figure 1

Fertilizing sperm on the egg surface viewed by scanning electron microscopy. (a) Starfish (A. pectinifera). Several well-like holes are open in the egg vitelline coat. Sperm acrosomal process (arrow) pierces into the hole and gains access to egg microvilli. (b) Sea urchin (Paracentrotus lividus). Note that the acrosomal process extending from the sea urchin sperm head (arrow) is much shorter than that of the starfish sperm. Image (b) adapted from Embryogenesis Explained (Gordon and Gordon 2016), Copyright@2016 World Scientific Publishing, Singapore

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In contrast to the sperm head, sperm tail (flagellum) is predominantly enriched with microtubules that are organized in the highly conserved “9 + 2” arrangement together with the molecular motor dyneins to constitute the axoneme. While the flagella of echinoderm sperm do not contain detectable level of actin (Tilney et al. 1973), F-actin has been intermittently visualized in the sperm of some other species including crane fly and mammals (Behnke et al. 1971; Dvoráková et al. 2005; Cohen et al. 2004; Correa et al. 2007). It has been reported that actin inhibitors such as cytochalasin B and D can deform the tail of macaque sperm and moderately diminish sperm motility in a dose-dependent manner (Correa et al. 2007). However, the presence of actin in the sperm tail has not been clearly demonstrated for echinoderms, and accordingly its contribution to sperm motility has not been much explored.

3 Actin in Oocyte Maturation

Toward the end of oogenesis, fully grown oocytes in the ovary are arrested in certain phases of meiotic cell cycle until they are utilized. For fertilization, these oocytes should be converted to fertilization-competent eggs. In starfish, this transition (release from the arrest at the first prophase) is initiated by the oocyte maturation hormone, which was identified as 1-methyladenine (1-MA) (Kanatani et al. 1969). Upon binding to its yet unidentified receptor, which is presumably present in the plasma membrane (Kanatani and Hiramoto 1970), 1-MA induces a series of biochemical and cytological modifications in the oocytes, such as intracellular Ca2+ increase (within 1–2 min), reorganization of the actin cytoskeleton, and changes in protein phosphorylation and membrane potential. With the progression of these changes, the nucleus of the oocyte (germinal vesicle, GV) loses its envelope (GV breakdown), and the nucleoplasm intermixes with cytoplasm eventually to render the cell physiologically suitable for fertilization (Moreau et al. 1978; Dale et al. 1979; Dorée et al. 1981; Schroeder and Stricker 1983; Meijer and Guerrier 1984; Lim et al. 2001). It has been shown that, following GV breakdown, the nuclear zone of the starfish oocyte forms a contractile actin network that captures the chromosomes and delivers them within the reach of microtubule asters (Lénárt et al. 2005). Thus, actin is expected to assist in the subsequent progression of meiotic progression.

The surface of oocytes is covered with countless projections of F-actin bundles called microvilli and with the meshwork of subplasmalemmal actin filaments. Both microvilli and the cortical actin meshwork undergo drastic reorganization during the meiotic maturation of oocytes. It is quite conspicuous that the 1-MA-induced reorganization of the actin cytoskeleton, visualized by the structural changes of the microvilli on the surface of oocyte, is as prompt as the Ca2+ increase (Schroeder and Stricker 1983; Santella and Kyozuka 1994; Kyozuka et al. 2008). Thus, in response to 1-MA, the number of microvilli inserting into vitelline coat becomes significantly reduced (Hirai et al. 1971), leading to the selective loss of microvilli-residing K+ channels and the consequent shift in membrane potential during the course of meiotic maturation (Dale et al. 1979; Moody and Bosma 1985).

One crucial cytoplasmic change that takes place during meiotic maturation is the translocation of cortical granules from the inner cytoplasm to the subplasmalemmal zone. This process appears to be heavily dependent upon actin dynamics both in starfish and sea urchin, as judged by its inhibition by the agents interfering with actin dynamics (Santella et al. 1999; Wessel et al. 2002). Cortical granules that are now apposed to the egg plasma membrane are more readily exocytosed and elevate the vitelline layer when the Ca2+ wave arrives during fertilization. In addition, both GV and endoplasmic reticulum (ER) appear to be bound and modulated by the actin cytoskeleton (Stricker and Schatten 1991; Terasaki 1994). ER is the major Ca2+ store in the egg, which is studded with ion channels such as InsP3 receptor and ryanodine receptor. The actin-based rearrangement of ER in the maturing oocytes may in part contribute to the progressive sensitization of the maturing oocytes to InsP3 (Chiba et al. 1990; Lim et al. 2003). This phenomenon, in which the same amount of exogenous InsP3 evokes a much larger Ca2+ increase in the matured eggs than in the immature oocytes, can be precluded by latrunculin A (LAT-A), a drug promoting actin depolymerization (Lim et al. 2003).

Starfish oocytes reentering meiotic cell cycle by the action of 1-MA manifest intracellular Ca2+ increases at two different time points: the early response readily induced by the hormone and the late Ca2+ increases at the time of GV breakdown. The earlier response takes place within 1–2 min after the hormone addition, and it comprises spatiotemporally coordinated Ca2+ release from internal stores that starts from the vegetal hemisphere and propagates as a single wave along the subplasmalemmal zone (Moreau et al. 1978; Santella and Kyozuka 1994; Dorée and Kishimoto 1981; Kyozuka et al. 2008). The few minutes’ time lag required for the Ca2+ wave to respond to 1-MA implies that the signal transduction involved in this pathway is quite efficient. Experimental evidence has indicated that 1-MA-induced meiotic resumption is mediated by ßɣ subunit of heterotrimeric G-protein (Chiba et al. 1993; Jaffe et al. 1993). Indeed, metabolically stable GTP (GTPɣS) microinjected into starfish oocytes mimicked 1-MA by readily inducing a similar Ca2+ wave, while GDPßS inhibited the GV breakdown, the hallmark of meiotic maturation of oocytes (Kyozuka et al. 2009). Interestingly, it has been shown that alteration of the actin cytoskeleton by LAT-A, jasplakinolide (JAS), or heparin suppresses the 1-MA-induced Ca2+ increase (Kyozuka et al. 2008, 2009). For example, pretreatment of A. pectinifera oocytes with 12 μM JAS nearly blocked the 1-MA-induced Ca2+ wave (Fig. 3.2). However, these oocytes retained intact activities of InsP3 receptor, as judged by the peak amplitude of the Ca2+ increase triggered by InsP3 uncaging. On the other hand, oocytes of A. pectinifera are known not to respond to cyclic ADP-ribose with a Ca2+ increase (Nusco et al. 2002). These and other observations led to the suggestion that the Ca2+ increase induced by 1-MA may be mediated by a mechanism distinct from InsP3 receptor or ryanodine receptor (Kyozuka et al. 2008). The late-stage Ca2+ increases in maturing oocytes of starfish are due to the influx from outside through the L-type Ca2+ channels, which is detected as a train of Ca2+ spikes taking place around the time of GV breakdown (Limatola et al. 2015) when the oocytes undergo continual depolarization and swift shift of membrane potential (Dale et al. 1979). This late stage of Ca2+ response to 1-MA, which had not been previously reported, was also shown to be dependent upon the status of cortical actin cytoskeleton. Exposure of the maturing oocytes to 1 μM LAT-A 8 min after the hormone addition (thus after the early Ca2+ wave has subsided) led to the significant reduction in the frequency of the Ca2+ spikes, while the average amplitude of the Ca2+ spikes was significantly enhanced by the LAT-A-induced actin depolymerization (Limatola et al. 2015). Taken together, these observations suggest that the reorganization of the cortical actin cytoskeleton and microvilli readily induced by 1-MA is instrumental in fine-tuning other structural and functional changes that take place in maturing eggs, in particular the optimization of the intracellular Ca2+ stores and ion channels.

Fig. 3.2
figure 2

The Ca2+ increase induced by 1-MA in the starfish (A. pectinifera) oocytes is suppressed by JAS. (a) Actin filaments visualized by Alexa Fluor 568 phalloidin in live oocytes with or without (control) preincubation with 12 μM JAS. Note that the subplasmalemmal actin is hyperpolymerized by the incubation with JAS (arrow). (b) Quantified Ca2+ responses after 1-MA stimulation. Adapted from Kyozuka et al. (2009) PLoS One 4: e6296

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4 Actin in Fertilized Eggs

Fertilization requires successful gamete recognition, adhesion, and fusion. As aforementioned, the egg and sperm influence each other to induce morphological and physiological changes in a sequential manner, which involves multiple bilateral receptor-ligand couplings (Ohlendieck and Lennarz 1996; Vacquier 2012). In starfish, the egg jelly coat provides at least three signaling substances for homologous spermatozoa to induce acrosome reaction: (1) acrosome reaction-inducing substance (ARIS), which is a sulfated proteoglycan-like molecule with >1000 repeating units; (2) Co-ARIS, sulfated steroidal saponins; and (3) asterosap, a peptide. The cognate receptors on sperm mediate a cascade of biochemical changes affecting acrosomal reaction (Matsumoto et al. 2008). Similarly, sea urchin spermatozoa possess the specific receptors (e.g., suREJ1) for egg jelly protein that are activated to trigger the acrosome reaction (Moy et al. 1996). Exocytosis of the acrosomal vesicle as a result of the Ca2+ increase in the sperm may now expose species-specific adhesion/fusion molecules such as bindin on the surface of the acrosomal process (Vacquier and Moy 1977). Two bindin receptors have been identified in sea urchin eggs, which might provide protease activity as well (Kamei and Glabe 2003; Mah et al. 2005). Because an earlier study showed that the 350-kDa bindin receptor is predominantly localized in the tip of microvilli (Ohlendieck et al. 1994), it seems probable that the microvilli may play a crucial role in intercellular fusion during fertilization, as well as in the pathogenic processes and in the immune responses (Wilson and Snell 1998). Indeed, lymphocytes are also covered with oocyte-like microvilli (Majstoravich et al. 2004). The actin-filled protrusions from both gametes, i.e., the acrosomal process of activated sperm and the microvilli of eggs, are likely to subserve sperm-egg fusion and the subsequent entrance of sperm into eggs. Analyses of the ultrastructure of sea urchin eggs at fertilization suggested that several microvilli at the sperm fusion site may undergo lateral association to produce a fertilized cone (Tilney and Jaffe 1980). Adjoining of the acrosomal process of incoming sperm with the fertilization cone apparently forms a thick “tether” that can be seen even in the light microscope due to its characteristic optical density (Fig. 3.3). After entering the egg, the sperm head is now attached to several thick bundles of actin filaments in the egg cytoplasm (Fig. 3.4, also see Santella et al. 2016).

Fig. 3.3
figure 3

A moment before sperm entry into the fertilized egg of starfish (A. aranciacus). The acrosomal process of sperm pierces the vitelline coat, which is about to elevate (arrow), and is adjoined by the egg microfilaments in the fertilization cone. Apparently, the connection continues to a tubular structure in the inner cytoplasm (arrowheads). Adapted from Puppo et al. 2008 PLoS One 3: e3588

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Fig. 3.4
figure 4

A moment after sperm entry into the fertilized egg of starfish (A. aranciacus). Actin filaments were visualized by Alexa Fluor 568 phalloidin in live eggs at fertilization. (a) Confocal image. (b) Bright field view. While microvilli of the fertilized egg prolong and traverse the perivitelline space, thick actin bundles are now formed and attached to the egg-incorporated sperm head (arrow)

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Following fertilization, starfish and sea urchin eggs elongate their microvilli as the fertilization envelope is formed. The elongated microvilli traversing the entire depth of the perivitelline space may mechanically contribute to the elevation of the vitelline layer or to the accommodation of the plasma membrane whose area is greatly increased as a result of cortical granule exocytosis and the membrane fusion (Carron and Longo 1982; Chun et al. 2010). Below microvilli, portions of subplasmalemmal actin filaments in the fertilized eggs undergo centripetal translocation (Terasaki 1996; Vasilev et al. 2012), while other filaments remain locally and become reorganized to form a new surface boundary of the zygote (Chun et al. 2010). Although precise roles of the inwardly translocating actin filaments are not known, it is conceivable that their centripetal migration might also assist in the concomitant sperm entry into the egg cortex. Although it is known that the migration of the male and female pronuclei toward each other is mediated by microtubule-based movement, the centrosome separation has been attributed to the actin filaments (Schatten et al. 1988; Holy and Schatten 1991).

Being heavily involved in gamete fusion and sperm entry, actin is thought to play an important role in controlling the number of sperm incorporated into the egg. In line with earlier observation that interference with actin dynamics using cytochalasin caused polyspermy in the fertilized eggs of clams (Ziomek and Epel 1975), starfish and sea urchin eggs treated with actin-inhibiting drugs manifested either increased polyspermy rates or failure of sperm entry. Hyperpolymerization of subplasmalemmal actin induced by JAS and heparin in starfish egg (A. aranciacus) led to supernumerary sperm-egg interactions that induced multiple initiations of Ca2+ release (Puppo et al. 2008). The same effect was observed when endogenous depactin, an actin-depolymerizing protein, was inhibited in the eggs of A. aranciacus by a specific function-blocking antibody (Chun et al. 2013). In sea urchin (P. lividus), pretreatment of eggs with moderate dose of cytochalasin B (400 nM) or phalloidin (3 μM) increased the rate of polyspermy, but sperm entry was suppressed by higher dose of the same drugs (10 and 30 μM, respectively). Likewise, 10 nM of LAT-A and 12 μM of JAS severely inhibited sperm entry, as judged by Hoechst-33342-stained spermatozoa (Chun et al. 2014). Taken together, these observations suggested that fine regulation of egg surface actin filaments is a prerequisite for correct gametes interaction and fusion.

The egg actin cytoskeleton also appears to contribute to the regulation of exocytosis. In the early studies utilizing the “cortical lawn” prepared from fractured sea urchin eggs, negative data showing no effect of actin or tubulin inhibitors on exocytosis led to the suggestion that Ca2+ is the sole modulator of cortical granule exocytosis (Whitaker and Baker 1983; Whitaker 1994). However, later studies with intact eggs led to a different conclusion. Pretreatment of starfish eggs with an actin drug JAS did not apparently affect the intracellular Ca2+ increases in response to InsP3 uncaging or fertilizing sperm, but the elevation of the vitelline layer was severely inhibited as a sign of impaired cortical granule exocytosis (Kyozuka et al. 2008; Puppo et al. 2008; Santella and Chun 2011). Similarly, sea urchin eggs pretreated with JAS or other actin drugs often failed to elevate the vitelline layer at fertilization, although the Ca2+ response at fertilization was unaffected or comparable with that in the control eggs (Chun et al. 2013). Hence, in live eggs, the subplasmalemmal actin cytoskeleton surrounding cortical granules also seem to serve as a decisive factor controlling exocytosis.

5 Modulation of Intracellular Ca2+ Signaling by the Actin Cytoskeleton

The subplasmalemmal actin filaments also affect the spatiotemporal pattern of intracellular Ca2+ signaling. Whereas Ca2+ signals affect structural organization and function of the actin cytoskeleton through the action of Ca2+-dependent actin-binding proteins such as gelsolin, the status of actin filaments can also influence the Ca2+ signals in return. This reciprocal relationship may comprise a feedback loop and contribute to Ca2+ homeostasis inside cells (Santella et al. 2008; Chun and Santella 2009a, b). Actin may influence intracellular levels of Ca2+ at least in two different ways. Firstly, actin is a highly abundant protein, and the monomeric actin displays an unusually high binding affinity to free Ca2+ with the Kd value being estimated around 2 × 10−9 M (Gershman et al. 1986; Carlier et al. 1986). This implied that actin can serve as a highly efficient Ca2+ buffer inside the cell and led to the hypothesis that polymerizing actin can absorb excess Ca2+ ions into the microfilament, which can serve as a different kind of Ca2+ store. At depolymerization, these filaments can now release free Ca2+ (Lange 1999). For the same reason, monomeric actin and the treadmilling actin filaments can serve as a diffusion barrier for free Ca2+. Indeed, the diffusion of individual Ca2+ ions is known to be conspicuously limited in cells like oocytes (Allbritton et al. 1992). All these physical parameters of actin may serve as a direct mechanism to regulate intracellular Ca2+ levels. Secondly, the subplasmalemmal and cortical actin filaments may set the tone to the microenvironment of the ion channels that are studded in the plasma membrane or on the membranes of organelles and vesicles such as ER. For these reasons, polymerization status of the actin filaments and the dynamics of their rearrangements were expected to influence the efficiency of ion channel activities. It has long been known that the InsP3 receptor binds to actin filaments, which may modulate its Ca2+-transmitting activities (Fujimoto et al. 1995; Fukatsu et al. 2004; Bose and Thomas 2009). In addition to the Ca2+ release through InsP3 receptor, it has been shown that Ca2+ uptake through the pump on ER is under the regulation of the actin cytoskeleton in neurons (Wang et al. 2002).

Starfish and sea urchin eggs are supposed to be suitable in addressing the questions regarding the causal relationship between the actin cytoskeleton and intracellular Ca2+ signaling. Certainly, large eggs with resilient surface and transparent cytoplasm have an advantage in tackling these questions in live cells by microinjection of fluorescent dyes and imaging technology. Indeed, the first experimental models in which intracellular Ca2+ wave was demonstrated were medaka fish and sea urchin eggs at fertilization (Ridgway et al. 1977; Steinhardt et al. 1977). The biochemical and electrical signals transmitted by a fertilizing sperm have to be transduced at the plasma membrane/cytoskeleton interface in the egg which is covered by microvilli and is crowded with actin filaments. With starfish eggs, several methodological strategies have been adopted to alter the F-actin structure and dynamics: microinjection of actin-binding protein cofilin (Nusco et al. 2006), metabolically stable guanidine nucleotides (Kyozuka et al. 2009), recombinant protein sequestering PIP2 (Chun et al. 2010), and so on. In each case, certain aspects of Ca2+ influx (cortical flash) and intracellular Ca2+ release have been observed to be significantly changed by the alteration of the actin cytoskeleton. For example, starfish eggs microinjected with human cofilin, which binds and severs actin filaments, led to enhancement of the Ca2+ wave at fertilization with the concomitant suppression of the cortical flash (Nusco et al. 2006). Corroborating this observation, inhibiting the endogenous activity of depactin (structural homologue of cofilin in starfish) with microinjection of the function-blocking antibody suppressed the Ca2+ wave in starfish eggs at fertilization (Chun et al. 2013). The modulation of intracellular Ca2+ signaling by the actin cytoskeleton is not a peculiar phenomenon restricted to starfish. When sea urchin eggs (P. lividus) were pretreated with actin drugs prior to fertilization, the spatiotemporal pattern of the Ca2+ increase was changed. For example, two actin drugs LAT-A and cytochalasin B, which promote actin depolymerization but by different mechanisms, displayed similar effects. In both cases, the fertilized eggs required longer time lag to generate the Ca2+ wave after firing cortical flashes (Chun et al. 2014). The exact physiological significance of this interval between the cortical flash (sperm-induced egg membrane depolarization) and the onset of the Ca2+ wave at fertilization is not clear (Dale and De Santis 1981; Santella et al. 2015). It might represent the time required for the egg to receive the sperm-borne signal and convert it into the synthesis of Ca2+-mobilizing second messengers. While the exact mechanism by which a fertilizing sperm initiates Ca2+ wave in the egg is not fully established and probably varies depending on the animal species (Parrington et al. 2007), it is noteworthy that disassembly of subplasmalemmal actin filaments with LAT-A or cytochalasin induces both fertilization-like Ca2+ waves and depolarization of the membrane potential in starfish (A. aranciacus) eggs (Lim et al. 2002; Moccia 2007). Our recent finding that the Ca2+ increase induced by LAT-A is caused by de novo synthesis of InsP3 by stimulating PLCɣ (Vasilev et al. 2018) suggests that the subplasmalemmal actin filaments also modulate the activity of a key enzyme involved in intracellular Ca2+ signaling.

6 Concluding Remarks

The old and new experimental data obtained from the oocytes and eggs of starfish and sea urchin that we have reviewed here suggest that actin is functionally involved in nearly all aspects of fertilization both in sperm and eggs: maturation, interaction, and fusion of the gametes. Actin provides both rigidity and plasticity for the membranes of sperm and eggs. The actin cytoskeleton is highly dynamic as exemplified by the elongating acrosomal process of sperm and the microvilli extending into the perivitelline space of the fertilized eggs. The actin pools in the sperm head or in the egg cortex maintain the spatiotemporal balance between F-actin and G-actin, which can be swiftly shifted as needed. In this review, our emphasis has been focused on the role of actin filaments in the subplasmalemmal space, which has also been referred to as “ectoplasm” (Just 1939). Conceptually, the actin filaments in the sperm acrosomal process and egg microvilli also fall into this category in a sense that they are subjacent to the plasma membrane. The actin filaments located in this tight zone of plasma membrane/cytoskeleton interface bear utmost importance not only for fertilization but also for all intercellular signaling. Although fertilization takes place on the egg surface, the subplasmalemmal zone is the locus where all the signal-transducing events take place. Whereas many aspects of fertilization may considerably vary among species, what we have reviewed here for echinoderms, and the other findings reported in the literature, support the idea that the actin meshwork in the subplasmalemmal zone may serve as a scaffold on which receptors, signaling molecules such as G-proteins, and protein kinases, as well as pertinent enzymes and ion channels, are functionally and perhaps physically interlinked together through the self-remodeling framework of the actin cytoskeleton.