Maturation and fertilization of echinoderm eggs: Role of actin cytoskeleton dynamics

Biochemical and Biophysical Research Communications xxx (2018) 1e11

Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc

Maturation and fertilization of echinoderm eggs: Role of actin cytoskeleton dynamics Luigia Santella*, Nunzia Limatola, Filip Vasilev, Jong Tai Chun Department of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, Villa Comunale, 80121, Napoli, Italy

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 September 2018 Accepted 13 September 2018 Available online xxx

Starfish and sea urchin are excellent models to study the mechanisms that regulate oocyte maturation and egg activation. Hormonal stimulation of starfish oocytes and their following interaction with spermatozoa induce rapid changes of F-actin and Ca2þ increases which are prerequisites for normal fertilization and development. Fully grown oocytes isolated from the gonads of starfish contain a large nucleus (~60e70 mm) (termed germinal vesicle, GV), which is arrested at the first prophase of meiosis. If inseminated, these immature oocytes are penetrated by additional spermatozoa. However, starfish oocytes naturally shed into the sea have already initiated the (meiotic) maturation and are normally fertilized between GV breakdown and the extrusion of the first polar body. This is considered the optimum period to ensure monospermic instead of polyspermic fertilization. By contrast, sea urchin eggs are fertilized only after being fully matured, i.e., at the end of the two meiotic divisions. Here, we provide a comparative review of the role of the actin cytoskeleton in oocyte maturation and fertilization in starfish and sea urchin. It has become increasingly evident that the exquisite regulation of the cortical Factin is involved in nearly all aspects of the molecular events taking place during the progression of meiotic maturation and fertilization. © 2018 Elsevier Inc. All rights reserved.

Keywords: Starfish Sea urchin Actin Calcium Maturation Fertilization

1. Meiotic maturation of starfish oocytes involves rearrangement of actin filaments and intracellular Ca2þ increases Classical in vitro studies on starfish have provided a large body of information on the morphological and biochemical changes required to produce competent eggs ensuring successful monospermic fertilization that occur in the oocytes during maturation. The hormone inducing oocyte maturation in starfish is 1methyladenine (1-MA), which can be applied in vitro to immature oocytes. The stimulating hormone acts on the oocyte surface (Fig. 1 A) and promotes resumption of the meiotic cycle through a series of cortical and nuclear events [1,2]. The breakdown of the envelope of GV (GVBD) represents the most characteristic sign that the meiotic cycle has been reinitiated (Fig. 1 B), and allows the intermixing of the nucleoplasm and cytoplasm (Fig. 1 C). As a result of two rounds of meiotic divisions, two polar bodies are sequentially formed [3]. In starfish, fertilization is known to take place when the meiotic

* Corresponding author. E-mail address: [email protected] (L. Santella).

stages of the oocytes are between GVBD and the emission of the first polar body. In this review, oocytes at this phase are referred to as ‘mature eggs’. At fertilization, the mature eggs fully elevate the fertilization envelope (FE) following cortical granules (CG) exocytosis, which does not normally occur in immature oocytes upon insemination (Fig. 1 D). This difference is due to the profound morphological changes taking place on the surface and the cortex of the oocytes during the maturation process [4,5]. As shown in the scanning electron microscopy (SEM) micrograph, the plasma membrane of an immature oocyte of Asterina pectinifera (Fig. 2 A) is surrounded by a layer of follicle cells that make numerous contacts with it through cytoplasmic projections penetrating the vitelline coat of the oocyte (Fig. 2 B). Following 1-MA treatment, the follicle cells cluster on a side of the mature egg surface (Fig. 1C and D) and eventually detach from the egg (Fig. 2 C). The cortical changes of the maturing oocytes also include shortening of microvilli, the fingershaped processes on their surface [6,7]. As shown in SEM, a large number of microvilli protruding underneath the vitelline coat of the immature oocytes (Fig. 2 D) become significantly retracted after the oocytes were stimulated with 1-MA (Fig. 2 E). The shortening of microvilli is also evident in the transmission electron microscope images; compare Fig. 2 F and G.

https://doi.org/10.1016/j.bbrc.2018.09.084 0006-291X/© 2018 Elsevier Inc. All rights reserved.

Please cite this article in press as: L. Santella, et al., Maturation and fertilization of echinoderm eggs: Role of actin cytoskeleton dynamics, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.084

2

L. Santella et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e11

Fig. 1. Morphological changes of the starfish oocyte during meiotic maturation. A) Fully grown immature oocytes (A. pectinifera) enclosed in the layer of follicle cells (FC). With the large nucleus, (germinal vesicle, GV), these oocytes are arrested at the first prophase of meiosis. B) Maturing oocyte 25 min after the stimulation with 1-MA: the nuclear envelope is breaking down. C) Mature eggs stimulated for 50 min with the hormone. At this stage of the maturation process, the FC are now clustered on one side of the eggs. This time of treatment represents the optimal period (i.e., before the extrusion of the first polar body) for monospermic fertilization and physiological Ca2þ

As for the 1-MA signal transduction pathway, it has been suggested that the maturation-inducing hormone interacts with a G protein-coupled receptor that spans the plasma membrane of the immature oocytes, yet the receptor itself has not been identified [8e10]. The passage of the oocyte through the G2/M transition of meiosis is mediated by activation of the maturation-promoting factor (MPF), which is known to be a complex of cyclin B and cyclin-dependent kinase 1 (CDK1). MPF can directly phosphorylate and suppress the antagonistic activity of protein phosphatase PP2A [11]. Alternatively, recent work in A. pectinifera has shown that the kinase Greatwall, which is exclusively present in the nucleus of immature oocytes, participates in the autoregulatory activation of CDK1 by suppressing the protein phosphatase 2A-B55 [12]. 1-MA triggers a Ca2þ wave in the immature oocytes of A. pectinifera that occurs a few minutes after its addition. This wave is confined to the cortical region, but is followed by a Ca2þ increase in the nucleus about 20 s later [13e15]. This delay of the Ca2þ increase in the nucleus might be peculiar to starfish oocytes because it is either nonexistent or insignificant in mammalian cells [16,17]. More recent results have indicated that the 1-MA-induced Ca2þ increase is very fast and spatially restricted. It always initiates at the vegetal hemisphere to propagate exclusively through the oocyte cortex, and it reaches the opposite side where the GV is located in about 20 s [18]. The 1-MA-induced Ca2þ wave can take place even in Ca2þ-free seawater (CaFSW), strongly suggesting that Ca2þ originates exclusively from the intracellular stores at the cortex. Interestingly, the state of the structural organization of the actin cytoskeleton of the immature oocyte cortex was found to play a critical role in generating the 1-MA-induced Ca2þ increase, as its disassembly by latrunculin-A (LAT-A) or reinforcement by jasplakinolide (JAS) significantly inhibited the 1-MA-triggered Ca2þ signal [18]. Recently, a novel Ca2þ increase has been observed at the time of GVBD: 20e40 min after 1-MA addition. A train of Ca2þ influxes of short duration was detected in the cortex of maturing A. pectinifera oocytes [19]. At variance with the 1-MA induced Ca2þ wave mentioned above, these Ca2þ spikes were entirely dependent on the presence of external Ca2þ. During the 1-MAeinduced maturation, the morphological modifications of the cortex include the formation of transient actin-filled spikes on the oocyte surface and changes in the microvillar F-actin [20]. While maturing oocytes of starfish normally exhibit microvilli shortening (Fig. 2 E) and intimate positioning of CG underneath the plasma membrane in a Factin-dependent manner [6,7,21], the oocytes stimulated to undergo maturation in CaFSW displayed even more retracted microvilli, CG and clear vesicles and yolk platelets appreciably dislodged away from the plasma membrane [19]. These results underline the influence of extracellular Ca2þ on the F-actin remodeling that normally occurs in the cortical layer of maturing oocytes. This Factin remodeling plays a key role in the acquisition of the fertilization competence of the egg as revealed by the observation that eggs matured in CaFSW give rise to an altered Ca2þ response at fertilization and to early embryos that often fail to divide correctly [19]. These results highlight the strong association between the surface structural modification during fertilization with the stages of embryonic development [22]. Interestingly, the eggs matured in CaFSW prior to fertilization in the artificial seawater containing 10 mM Ca2þ displayed faster expulsion of the two polar bodies, which represents nuclear divisions [19,23]. In turn, the changes of the cortical actin cytoskeleton manifested by the retraction of microvilli can modulate the Ca2þ fluxes across ion channels, which

responses. D) Eggs fertilized 50 min after the stimulation with 1-MA. They show a full elevation of the fertilization envelope (FE) 5 min after insemination.

Please cite this article in press as: L. Santella, et al., Maturation and fertilization of echinoderm eggs: Role of actin cytoskeleton dynamics, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.084

L. Santella et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e11

3

Fig. 2. Scanning and transmission electron microscopy examination of the surface of starfish oocytes before and after hormonal treatment. A) A scanning electron micrograph of a GV-stage oocyte (A. pectinifera) surrounded by a layer of follicle cells (FC). B) A higher magnification of the oocyte surface showing the projections of the FC (arrow) penetrating the vitelline coat (VC). C) The surrounding FC are no longer visible in the mature egg treated with 1-MA for 50 min. D) An intact immature oocyte fractured during fixation to expose the microvilli (MV) beneath the VC. Unstimulated GV-stage oocytes contain longer MV than mature eggs. Same magnification as in panel E. E) A fractured mature egg treated with 1-MA for 50 min shows shorter MV under the VC. F) A transmission electron microscope image of an immature starfish oocyte confirming the presence of longer MV on the surface. Same magnification as in panel G. G) The short MV on the surface of mature eggs. At this stage of the meiotic maturation, cortical granules (CG) are closer to the plasma membrane and MV.

has been widely reported in other cellular systems [24e26]. The morphological modifications of microvilli and the cortical actin filaments parallel the progressive decrease of Kþ current in maturing oocytes. Thus, at the time of GVBD, the membrane potential abruptly shifts to a less negative value [27,28]. While this transition requires involvement of some nuclear components [29e31], the shortening of microvilli might contribute to the selective loss of the ion currents [6,28], as the retraction of microvilli translates into reduced presence of ion channels (see also Fig. 2 E and G). When fertilized, competent eggs in their optimum meiotic phase display a normal fertilization potential and Ca2þ signaling that reflect the structural and functional integrity of the egg cortex that are required for the quality of embryo development [31e34]. The cortical changes initiated by the fertilizing sperm include

thickening of the subplasmalemmal F-actin layer and translocation of actin filaments from the cortex towards the center of the zygote [35]. This F-actin redistribution is essential for embryonic development [36]. During the maturation process, cytoskeletal modifications are also reflected by the changes in the rigidity of the oocyte cortex during GVBD and around the extrusion site of the two polar bodies [37e39]. 2. The actin cytoskeleton plays a role in optimizing the Ca2þ release mechanisms during oocyte maturation The ability of an egg to generate a proper Ca2þ response to a fertilizing sperm develops during maturation. According to the literature, this is achieved through the reorganization of the major Ca2þ storage organelle, the endoplasmic reticulum (ER). Its

Please cite this article in press as: L. Santella, et al., Maturation and fertilization of echinoderm eggs: Role of actin cytoskeleton dynamics, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.084

4

L. Santella et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e11

remodeling may enhance the sensitivity of inositol 1,4,5trisphosphate (InsP3) receptors to InsP3, which is synthesized in the fertilized eggs [40e44]. Indeed, the same amount of InsP3 injected into mature eggs of starfish released much higher levels of Ca2þ than in immature oocytes [45e47]. In mature eggs of Astropecten aranciacus, photoliberation of a subthreshold dose of preinjected caged InsP3 first started to release Ca2þ in the animal pole [48] where the GV is anchored to the cell surface by microtubules and actin filaments [3,49]. As the oocyte maturation proceeded, the InsP3-dependent Ca2þ release occurred from a larger portion of the ER. One hour after 1-MA treatment, the fertilizationcompetent eggs displayed uniform Ca2þ release in the entire cytoplasm in response to globally photo-liberated InsP3 [48]. This increased sensitivity of the InsP3-modulated Ca2þ stores developed along the animal-vegetal axis, in analogy with the spatiotemporal pattern of MPF activation [50]. It was thus of interest to investigate whether the increased sensitivity of the InsP3-linked Ca2þ stores was related to the activation of MPF in the cytoplasm and nucleus [15]. Oocytes were either enucleated or cut into halves prior to 1-MA stimulation so that only one of them contained the nucleus. When stimulated, the increased sensitivity to InsP3 nicely correlated with the sequential activation of MPF in the cytoplasm and its transfer into the nucleus, which induced structural modifications of the actin cytoskeleton [48]. In support of the idea that F-actin rearrangement plays a role in fine-tuning the intracellular Ca2þ release mechanisms during maturation, visualization of F-actin before and during the maturation of living starfish oocytes with fluorescent phalloidin has shown dramatic changes in actin filaments (Fig. 3). The disappearance of the dense network of F-actin in the cytoplasm, which is characteristic of immature oocytes (Fig. 3 A), is evident in A. aranciacus oocytes treated with 1-MA for 70 min, i.e. the optimum period to generate a normal Ca2þ signal and to achieve a monospermic fertilization. In this window of time, actin filaments become oriented perpendicular to the plasma membrane of the egg (Fig. 3 B). By contrast, a clear derangement and reduction of F-actin are observed in the overripe eggs that were incubated in seawater for 6 h after the hormonal treatment (Fig. 3 C). Related to that, a unique phenomenon in which F-actin disassembly by LAT-A resulted in long lasting Ca2þ oscillations in mature eggs of A. aranciacus starfish [51] suggested that F-actin may play a direct or indirect role in releasing Ca2þ [52,53]. Interestingly, LAT-A failed to induce a similar Ca2þ increase in immature oocytes, indicating that this effect may be temporally and spatially correlated with the sensitization of the Ca2þ stores to InsP3, which is acquired during maturation [48]. Our earlier suggestion of the InsP3dependent nature of the LAT-A-induced Ca2þ oscillations [51] has been rendered more complex by recent findings that heparin, a conventional inhibitor of the InsP3 receptors, induces hyperpolymerization of actin in the cortex of starfish oocytes and eggs [18,54,55]. More recently, it has been found that depolymerization of F-actin induced by LAT-A actually increases the intracellular contents of InsP3 [56]. These results add weight to the idea that Factin could have an important role in Ca2þ signaling also by stimulating the activity of phospholipase C that generates InsP3 [56]. In starfish oocytes, two additional Ca2þ-linked second messengers nicotinic acid adenine dinucleotide phosphate (NAADP) and cyclic ADP-ribose (cADPr) also appear to be functional in evoking intracellular Ca2þ release, albeit in different ways [57,58]. In A. aranciacus oocytes, the “maturation” of the cADPr-sensitive Ca2þ stores also takes place after 1-MA stimulation. The global photoactivation of pre-injected caged cADPr in the cytoplasm of immature oocytes promotes only scattered Ca2þ increases that start simultaneously to converge into a wave that spreads to the center of the cell. In contrast, the Ca2þ response of a competent mature egg

Fig. 3. Visualization of F-actin in live oocytes of starfish during maturation. A) A confocal image of the F-actin network in a living immature oocyte (A. aranciacus). B) The F-actin network is no longer visible in the egg cytoplasm 70 min after 1-MA stimulation. At this time of maturation, which represents the optimum period for normal fertilization of this species, the thick actin filaments are oriented perpendicular to the egg surface. C) F-actin of a mature starfish egg staled for 6 h in seawater, showing the complete derangement and reduction of actin filaments in the cortex.

to the exogenous cADPr starts from one cortical region and is followed by a synchronized Ca2þ increase in the entire surface of the egg. The sites of Ca2þ release in the inner cortex are also nearly continuous in contrast to the immature oocytes [59]. This cortical Ca2þ release closely resembles the cortical flash (CF) that is induced by the fertilizing sperm prior to the Ca2þ wave [60]. Interestingly, the removal of the external Ca2þ, which drastically affects the morphology of the egg surface and the Ca2þ response upon cADPr uncaging, abolished the CF and led to a failed elevation of the vitelline layer [59]. In intact sea urchin eggs, the direct demonstration of the release of Ca2þ from cADPr and NAADP-sensitive Ca2þ stores using the specific Ca2þ-linked second messengers has suggested that the two processes are separate but interacting [61]. It has been recently reported that NAADP may be delivered by the sperm at fertilization to induce a local Ca2þ increase by acting on two-pore channels (TPC) located on acidic lysosome-like vesicles in the sea urchin eggs [62,63]. These acidic vesicles would accumulate Ca2þ in their lumen by a Ca2þ pump, which is different from the sarco/endoplasmic reticulum Ca2þ ATPase in a mechanism that is dependent on the low luminal pH of the vesicles [64]. The close apposition of ER regions to these vesicles could be responsible for the globalization of the Ca2þ increase initiated by NAADP on the vesicles [65,66]. In A. pectinifera immature oocytes and mature eggs, at variance

Please cite this article in press as: L. Santella, et al., Maturation and fertilization of echinoderm eggs: Role of actin cytoskeleton dynamics, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.084

L. Santella et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e11

with sea urchin, the NAADP-linked Ca2þ response is strongly dependent upon external Ca2þ [67]. As uncaging of NAADP mimics the fertilization potential in the mature eggs of starfish, this finding suggests that NAADP could initiate the sperm-induced cortical Ca2þ response by evoking Ca2þ influx from the surrounding seawater [60,68]. Interestingly, the NAADP-dependent Ca2þ mobilization mechanism may undergo subtle changes during the maturation process of starfish oocytes [67]. It has been reported that removal of external Ca2þ effects stronger inhibition on the Ca2þ increase following the uncaging of NAADP in mature eggs than in immature oocytes, possibly reflecting the changes of the structural organization of the cortex in the maturing oocytes such as shortening of microvilli (Fig. 2) [6,7]. The finding that the NAADP-induced Ca2þ current was reduced after disassembly or stabilization of the actin cytoskeleton is in line with the idea that the cortical actin cytoskeleton modulates the activity of NAADP [69]. Indeed, the Ca2þ increase induced by photo-liberation of caged NAADP was augmented by pre-injection of the eggs with human actinsevering/depolymerizing protein cofilin [70]. Thus, while the exact molecular mechanism of how NAADP contributes to intracellular Ca2þ increase is still largely unknown, it appears that the cortical actin filaments play a modulatory role in the vicinity of the cognate NAADP receptor ion channels in the NAADP-sensitive Ca2þ stores. 3. The state of cortical F-actin affects the sperm-induced Ca2þ response and sperm penetration Reorganization of F-actin in the maturing oocytes of starfish and sea urchin plays a critical role in the translocation and reorientation of the CG beneath the plasma membrane, which is thought to facilitate their exocytosis at fertilization [21,71]. The positioning of CG underneath the plasma membrane may be regulated or restricted by the membrane-associated actin [21,72,73]. Indeed, dislodgement of CG from the sea urchin egg plasma membrane by procaine or urethane has been shown to be, in part, due to the increase of the filamentous actin in the egg cortex caused by these two anesthetics [74,75]. The requirement of a Ca2þ increase for CG exocytosis was clearly demonstrated in vitro by the disappearance of CG following the addition of Ca2þ to the ‘cortical lawns’ isolated from sea urchin eggs [76]. In line with this, direct microinjection of Ca2þ or Ca2þ chelators into the eggs respectively triggered or prevented CG exocytosis [77,78]. These results have shown that CG exocytosis in fertilized eggs is triggered by a transient intracellular Ca2þ increase [79,80]. However, the precise relationship between Ca2þ increase and CG exocytosis is still not clear [81]. The possibility that these granules may contribute to the generation of the Ca2þ signal at fertilization is not excluded since CG in sea urchin eggs may contain from 30 to 95 mM of total calcium [82]. They are also the site where cADPr is synthesized by ADP-ribosyl cyclase enzyme, which is localized to their lumen [83]. According to the prevailing view, CG exocytosis and FE elevation serve as a mechanism to prevent the entry of supernumerary spermatozoa besides protecting the embryo during development [84]. However, even if it usually takes 30 s to 1 min after the attachment of the fertilizing sperm for the elevation of the FE to occur all over the egg surface, only monospermic entry can be seen in normal fertilization. By contrast, multiple spermatozoa enter overripe starfish eggs which do elevate FE with the same time as in control eggs [85,86]. These results indicate that mono- or polyspermic entry is independent of whether or not the FE is elevated, but might be determined by other physical parameters such as changes in cortical actin cytoskeleton, the morphology of which is characteristically distinct in immature oocytes, mature eggs, and

5

overripe eggs (Fig. 3). Thus, the transition of the structural organization of the oocyte surface and cortical actin cytoskeleton taking place during meiotic maturation may play a decisive role in determining single or multiple sperm interactions, as well as in modulating the sperm-induced Ca2þ signals. In line with this, the pattern of the Ca2þ release at fertilization in the immature oocytes of starfish displays a typical polyspermic feature: multiple Ca2þ releasing spots in the cortex that spread towards the center of the oocyte (Fig. 4 A) at a propagation speed notably slower than that in the mature eggs (Fig. 4 B). By contrast, in the eggs inseminated at their optimum maturation period, i.e. between GVBD and the extrusion of the first polar body, a quick CF usually precedes the Ca2þ wave that traverses the egg from the sperm-egg interaction site to the opposite pole (Fig. 4 B). The fast and normal CF elicited by the fertilizing sperm, which in control eggs is strictly dependent on the integrity of the cortical actin cytoskeleton [19], may be an indication that the surface and the cortical actin cytoskeleton are properly organized and highly reactive to the stimulation by the first sperm. In line with the changes in the structural organization of the actin cytoskeleton, an actin polymerization wave has been visualized in sea urchin eggs to initiate at the sperm entry site and to propagate to the entire cortex [87]. However, since it takes about 2 min for the completion of the propagation, its role in the establishment of the fast structural block to polyspermy can be ruled out. Initial changes in the cortical actin cytoskeleton, which are probably difficult to detect, may thus occur much earlier. In line with this, in the eggs incubated in seawater for 6 h after 1-MA addition (overripe eggs), the CF exhibits higher amplitude at fertilization, and the Ca2þ wave initiates from multiple origins reflecting polyspermic gamete interaction. Thus, the perturbed structural organization of the cortical F-actin in these overripe eggs (Fig. 3 C) suggests that the compromised fertilization process (Fig. 4 C) might be attributable to the cytoskeletal changes in the egg cortex. In sea urchin and starfish eggs, a few minutes after insemination, the dramatic actin polymerization in the egg cortex is accompanied by accumulation of alpha-actinin, an F-actinbundling protein, which may facilitate formation of the fertilization cone for sperm incorporation [88e90]. The remarkable differences in the F-actin structure at the sperm entry sites displayed by polyspermic immature oocytes and monospermic mature eggs (Fig. 5) support the idea that the integrity of the actin cytoskeleton is dependent upon structural changes that are utterly important for sperm incorporation in normal fertilization [54]. The entry of additional spermatozoa in the immature oocytes involves F-actin at the oocyte cortex (Fig. 5 A’) that lacks the formation of F-actin bundles required for incorporating the sperm into deeper cytoplasm, which is typically observed in the mature eggs (Fig. 5 B0 ). Indeed, 28 min after insemination, spermatozoa still remain in the outer region of the immature oocyte cortex. It is interesting that the structural organization of the cortical F-actin is perturbed also in the overripe eggs (Fig. 3 C), and that these eggs produce polyspermic fertilization and an altered Ca2þ signal pattern (Fig. 4 C). Furthermore, migration of the spermatozoa in the cytoplasm of the overripe eggs is also significantly impeded (Fig. 5 B0 and C0 , compare the positions of the egg-incorporated sperm heads). Finally, it is remarkable that the formation of the FE itself is not sufficient to prevent the entry of supernumerary spermatozoa in the overripe eggs (Fig. 5C’) [86]. In this regard, it is conceivable that eggs are penetrated by multiple spermatozoa when their microvilli and cortical actin cytoskeleton are compromised, highlighting the importance of their structural and functional integrity, which are known to participate in sperm-egg fusion ensuring monospermic fertilization [91,92]. We suggest that the microvillar and cortical actin cytoskeleton in these eggs respond slowly to the stimulation of the fertilizing sperm, and that the delayed structural response in

Please cite this article in press as: L. Santella, et al., Maturation and fertilization of echinoderm eggs: Role of actin cytoskeleton dynamics, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.084

6

L. Santella et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e11

Fig. 4. The sperm-induced Ca2þ increases in the oocytes before, during, and after maturation. A) The insemination of GV-stage oocytes (A. aranciacus) leads to a series of cortical releases of Ca2þ (arrows) that eventually merge to form a Ca2þ wave in the cytoplasm. B) A mature egg treated with 1-MA for 70 min responds to the fertilizing sperm with a simultaneous release of Ca2þ in the egg cortex (arrow), which is followed by a Ca2þ wave that starts from the sperm-egg interaction site and propagates to the opposite pole. C) The sperm-induced Ca2þ signals in a mature egg staled for 6 h in seawater. Note the higher amplitude of the Ca2þ release in the egg cortex (arrow) and the faster propagation of Ca2þ in the egg that respond to multiple spermatozoa (arrows).

Fig. 5. F-actin changes in the cortex of oocyte/egg upon insemination. A) Epifluorescence image of F-actin in a starfish immature oocyte (A. aranciacus) before insemination. A0 ) F-actin changes at the multiple sperm entry sites of the same oocyte in A. The merged view in the lower panel represents spermatozoa stained with Hoechst 33342 (arrows) 28 min after insemination. B) Epifluorescence image of F-actin of a mature egg treated for 70 min with 1-MA. B0 ) F-actin changes of the same egg in B upon fertilization. Note the bundles of actin filaments (arrow, upper panel) being attached to the head of sperm in the cytoplasm of the activated egg and the position sperm in the cytoplasm (arrow, lower panel). C) Epifluorescence image of F-actin of a mature egg staled for 6 h in seawater. C0 ) F-actin changes and multiple sperm incorporation of the same egg in C upon insemination. Note the lack of the characteristic actin bundles (arrows) being found during sperm incorporation in the control mature eggs of B’. The transmitted light shows the entry of supernumerary spermatozoa in the presence of the elevated fertilization envelope (FE). These spermatozoa remain localized at the egg cortex (arrows).

the cortex may allow the attachment and penetration of additional spermatozoa. Alternatively, other components that regulate gamete interaction ensuring monospermic fertilization (e.g. sperm receptors and/or lipids on the egg plasma membrane) could change in the overripe eggs to increase the rate of polyspermy. Furthermore, the observation that not only one but multiple spermatozoa are able to enter the eggs with full elevation of the FE may also indicate that the structure of the latter is not functioning properly by the aging of the eggs. At fertilization, sea urchin eggs undergo extensive surface reorganization called ‘cortical reaction’. Within a few seconds after

sperm-egg interaction in several species, a transient partial flattening of the egg takes place at the region of initial FE elevation [93]. The elevation of the FE propagates around the egg cortex to the opposite pole as a result of the CG exocytosis [94e96]. According to Ernest Everett Just, ….“With membrane-separation the eggs undergo some change and it is this changednot its result, membrane separationdwhich constitutes the block to the entrance of additional spermatozoa. Thus this block, which is more subtle than the mechanical obstacle interposed by the presence of a separated membrane, is established before membrane-separation occurs” …. [32, p. 199].

Please cite this article in press as: L. Santella, et al., Maturation and fertilization of echinoderm eggs: Role of actin cytoskeleton dynamics, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.084

L. Santella et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e11

In 1976, evidence was presented that the abrupt change in the membrane potential displayed by sea urchin eggs upon insemination establishes a fast altered receptivity of the egg towards spermatozoa [97,98]. Intense investigations on sea urchin eggs produced a large amount of data on the kinetics and ionic fluxes characterizing the fertilization (activation) potential. In most cases, however, electrophysiological studies on polyspermy necessitated rather invasive manipulation of the eggs and the use of excess spermatozoa. Upon insemination of sea urchin and starfish eggs, the small initial depolarization event (a voltage change towards positive values) is followed approximately 12 s later (or several seconds in starfish) by a slower but longer-lasting depolarization, the so-called ‘fertilization potential’ [99,100]. These two electrophysiological events parallel the Ca2þ changes measured in starfish and sea urchin eggs at fertilization [3]. In line with a role of a positive shift in the egg's membrane potential in blocking supernumerary sperm entry, nicotine, which promotes polyspermy in sea urchin [101e103], has been known to subtly alter the electrical property of the plasma membrane and to reduce the amplitude of the fertilization potential [104]. However, it is important to note that ultrastructural studies have indicated that nicotine, which affects the formation of FE and the hyaline layer, also induced profound changes on the structure of the CG [105]. One source of the controversy surrounding the notion of a fast electrical block to polyspermy is the fact that the experimental conditions used did not reflect the natural environment in the sea, which normally permits successful fertilization [106e109]. E. E. Just related polyspermic fertilization to the egg quality, which was considered the decisive experimental condition (how well it matched the ones in nature), and assessed by examining the timing and quality of FE elevation. He wrote …. ”Normally monospermic eggs can be rendered polyspermic by experimental treatments …, in short, by methods for inducing injury or weakness. The eggs also tend to be polyspermic when below optimum condition as, for example, those obtained toward the end of the breeding season or from moribund animals. Polyspermy in them thus is a sign of weakness and hence pathological. In all conditions favorable to poly-sperm-entry the ectoplasmic response differs from that in normal fertilization” …. [32, p. 202]. Consistent with Just's observations, starfish and sea urchin eggs underwent polyspermic fertilization and showed deregulated Ca2þ responses if the integrity of the actin cytoskeleton of their cortex had been altered by JAS and phalloidin which promoted actin polymerization, and by LAT-A and cytochalasin B (CYT-B) which induced its depolymerization [54,110]. Finally, the microvilli containing F-actin bundles that emanate from the fertilized eggs of starfish and sea urchin to traverse the perivitelline space [54,111,112] may serve to ensure equidistant lifting of the FE from the egg surface, showing that the actin cytoskeleton plays a role in this process. This jibes with the observations of Just, who wrote: …. ”Membranes that separate incompletely and are not equidistant from the egg at all points and are slow in rate of separation mean eggs of poor fertilizability that subsequently develop abnormally” …. [32, p. 205]. Thus, our results, which are consistent with those of E. E. Just reported nearly a century ago, suggest that the rapid block to polyspermy seen in starfish and sea urchin is a structural one mediated by rapid changes in the cortical actin cytoskeleton. 4. Intracellular Ca2þ increase and the physiological role of Factin rearrangements in fertilized eggs The pathway leading to egg activation has been the subject of intense debate. The suggestion that Ca2þ release is implicated in CG breakdown and fertilization envelope or vitelline layer elevation in the sea urchin eggs activated by spermatozoa or a variety of chemicals and physical agents goes back to the colloid chemistry of

7

‘protoplasm hypothesis’ suggested by Heilbrunn [113] 80 years ago, according to which the mechanism by which the protoplasm is stimulated would be dependent upon the liberation of previously bound intracellular Ca2þ. Mazia [114] supported the idea by measuring the amount of Ca2þ released at fertilization in Arbacia eggs. Later work on sea urchin eggs showing that the Ca2þ response at fertilization is initiated by a Ca2þ influx triggered by plasma membrane depolarization [115,116] was long neglected due to the conflicting results of experiments assessing the role of external Ca2þ in egg activation [117]. It was claimed that fertilization could still occur in CaFSW if acrosome-reacted spermatozoa were employed to activate the eggs, and that InsP3 could induce the elevation of the vitelline envelope following Ca2þ increase in eggs incubated in CaFSW [118,119]. However, later studies indicated that acrosome-reacted spermatozoa failed to ensure successful fertilization when the eggs were inseminated in seawater containing reduced amounts of Ca2þ [120], probably as a result of their decreased fertilizability. Exposure of sea urchin eggs to micromolar amounts of the Ca2þ ionophore A23187 resulted in activation of the eggs with a rapid discharge of CG and the elevation of vitelline envelopes independently of the external Ca2þ [121]. The observation that the Ca2þ ionophore could stimulate Ca2þ increase and related physiological changes in CaFSW argued against the necessity of Ca2þ influx for a normal egg activation. However, even if the results supported the idea that intracellular Ca2þ release is necessary and sufficient for egg activation, it was later shown that Ca2þ ionophores, added to eggs of several animal species, produced striking alterations in cell surface morphology [122,123]. Moreover, human oocytes activated by A23187 to overcome difficulties after intracytoplasmic sperm injection produce a single Ca2þ transient instead of the characteristic Ca2þ oscillations observed in mammalian eggs at fertilization [124e126]. The different pattern of Ca2þ mobilization elicited by Ca2þ ionophores in human oocytes may be due to the fact that this transmembrane ion carrier increases intracellular Ca2þ in a mechanism distinct from that of Ca2þ-linked second messengers, and it cannot be ruled out that Ca2þ ionophores might act on an intracellular store that is not utilized in the normal fertilization process [127]. In the immature oocytes of starfish, application of another Ca2þ ionophore (ionomycin) promotes smaller intracellular Ca2þ increases when the cells are suspended in CaFSW. Furthermore, ionomycin induces a drastic rearrangement of the actin cytoskeleton in the cortex and cytoplasm of the oocytes, as well as the fusion of CG with other vesicles [36]. At the ultrastructural level, the elongated microvilli in the perivitelline space, which are normally observed in fertilized eggs presumably as a result of microvilli extension [3,111,112,128], were conspicuously absent when mature eggs were artificially activated by ionomycin (Fig. 6). Thus, the full and equidistant elevation of the FE constitutes an easily visible indicator of the quality of the underlying cortical changes initiated by the fertilization reaction [32]. More often than not, the vitelline layer elevated by ionomycin is not as distant from the surface of the cell as in the fertilized eggs. This might imply that the nature and pattern of the Ca2þ increase stimulated by ionomycin is not adequate to subsequently induce the proper remodeling of the underlying cortical F-actin, especially in the perivitelline space (Fig. 6). Thus, the intracytoplasmic sperm injection, and the subsequent ionophore application that is used for the treatment of male infertility, might have a negative impact on the later events of development; these treatments bypass the natural selection of the sperm and may thereby eliminate some aspects of the cortical reaction which are essential for a normal embryo development [129].

Please cite this article in press as: L. Santella, et al., Maturation and fertilization of echinoderm eggs: Role of actin cytoskeleton dynamics, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.084

8

L. Santella et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e11

Fig. 6. Transmission electron microscopy observation of microvilli elongation following activation of A. aranciacus starfish eggs. Mature eggs were activated by the addition of spermatozoa or by the Ca2þ ionophore ionomycin. Both treatments induced the elevation of the fertilization envelope (A) and of the vitelline layer (B). However, note that the elongation of microvilli (arrows) in the perivitelline space (PS) beneath the fertilization envelope (arrow) of inseminated eggs is missing in those activated by ionomycin.

5. Successful fertilization requires fine regulation of actin dynamics Starfish spermatozoa also undergo a drastic acrosome reaction [130], which is known to be triggered by the contact with the egg jelly coat [131]. The fertilizing sperm thus touches the egg surface with its long acrosomal process containing F-actin bundles [132,133], and the egg responds with the CF which is followed by the global Ca2þ wave [54]. For the eggs, the rearranging of F-actin in the cortex also plays a role in shaping the Ca2þ signals at fertilization. Microinjection of the eggs with the actin-severing/depolymerizing protein cofilin enhanced the sperm-induced Ca2þ signal [70]. Conversely, microinjection of the specific antibodies against starfish oocyte ADF/ cofilin depactin promoted F-actin changes and the failure of CG exocytosis, as well as attenuated the sperm-triggered Ca2þ waves which often exhibited signs of polyspermic interaction [134,135]. Furthermore, the sequestration of PIP2, which is known to regulate actin cytoskeleton dynamics [136], by microinjecting the pleckstrin homology domain of PLC- d1, caused similar results [111]. The importance of the structural and functional integrity of the cortical actin cytoskeleton in properly responding to the fertilizing sperm has also been confirmed in sea urchin eggs [137]. Their treatment with CYT-B and LAT-A, which promoted actin depolymerization, induced prolongation of the time between the CF and the beginning of the Ca2þ wave, multiple Ca2þ waves due to supernumerary sperm interaction, and the reduction of the Ca2þ peak. Different effects were observed with JAS or phalloidin that induced F-actin stabilization. While JAS repressed the CF without affecting the global Ca2þ wave, phalloidin microinjected into the eggs enhanced both the CF and the following Ca2þ wave. Interestingly, CYT-B, LAT-A, JAS and phalloidin all attenuated the speed of the Ca2þ waves induced by spermatozoa [110]. Experiments to test the dose-dependent effects of these actin drugs on sperm incorporation and elevation of the FE confirmed the findings in starfish; Ca2þ increase alone could not trigger cortical granules exocytosis if the cortical actin cytoskeleton had been perturbed with these drugs. Furthermore, despite the multiple sperm-interactions that generated as many Ca2þ waves, the eggs pretreated with LAT-A in most cases did not incorporate sperm [110]. These experiments suggest that the Ca2þ wave at fertilization is not essential for sperm entry. Alternatively, they may indicate that a proper pattern of Ca2þ release is necessary to produce the subsequent actin rearrangement [138] which is required for the subsequent embryonic development. The importance of the fine regulation of the actin

cytoskeleton in guiding monospermic fertilization and CG exocytosis is supported by the demonstration that numerous spermatozoa could enter sea urchin eggs when cortical F-actin was altered by CYT-B even when the FE was fully elevated. Conversely, the eggs pretreated with LAT-A, displaying no elevation of the FE, often failed to allow sperm entry despite the absence of the presumed mechanical barrier to polyspermy [110]. In summary, the results described in this review suggest that the structural and functional integrity of the actin filaments in the cortex of starfish and sea urchin eggs is crucial in regulating the spatiotemporal events that take place in the surface of fertilized eggs from gamete interaction to the first few rounds of cleavages. The eggs are electrically excitable and highly sensitive to a mechanical stress. The rapid cortical F-actin modifications following a successful collision and union of the gametes may produce a fast alteration of the egg's surface so as to prevent polyspermic fertilization. For successful fertilization to occur, the oolemmal actin cytoskeleton must be aptly rearranged to serve as the master event ensuring proper Ca2þ signaling and sperm entry. Certainly, the integrity of the actin cytoskeleton is one of the fundamental parameters of oocyte quality in assisted human in vitro fertilization and reproductive technology. Thus, the deciphering of the molecular events accounting for the deregulated cytoskeletal changes in the oocytes and eggs of echinoderms may provide invaluable insights into other areas of cell biology. Acknowledgements LS is indebted to Dr. Gaku Kumano for his kind hospitability at the Center for Marine Biology of Asamushi of Japan, and to Dr. Keiichiro Kyozuka for stimulating discussion and for supplying Asterina pectinifera. We thank D. Caramiello for the maintenance of the animals, and R. Graziano, F. Iamunno, and G. Lanzotti of the AMOBIO Unit of the Stazione Zoologica Anton Dohrn (SZN) for transmission and scanning electron microscopy services. We also thank G. Gragnaniello for preparing the Figures. N. Limatola has been financially supported by the SZN postdoctoral fellowship. The authors are grateful to Drs. Issei Mabuchi and W. Malcolm Byrnes for their valuable and insightful comments on the manuscript. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.09.084.

Please cite this article in press as: L. Santella, et al., Maturation and fertilization of echinoderm eggs: Role of actin cytoskeleton dynamics, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.084

L. Santella et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e11

References [1] K. Kishimoto, H. Kanatani, Cytoplasmic factor responsible for germinal vesicle breakdown and meiotic maturation in starfish oocyte, Nature 260 (1976) 321e322. [2] L. Meijer, P. Guerrier, Maturation and fertilization in starfish oocytes, Int. Rev. Cytol. 86 (1984) 129e196. [3] L. Santella, N. Limatola, J.T. Chun, Calcium and actin in the saga of awakening oocytes, Biochem. Biophys. Res. Commun. 460 (2015) 104e113. [4] T.E. Schroeder, Microfilament-mediated surface change in starfish oocytes in response to 1-methyladenine: implications for identifying the pathway and receptor sites for maturation-inducing hormones, J. Cell Biol. 90 (1981) 362e371. [5] T.E. Schroeder, S.A. Stricker, Morphological changes during maturation of starfish oocytes: surface ultrastructure and cortical actin, Dev. Biol. 98 (1983) 373e384. [6] S. Hirai, H. Shida, Shortening of microvilli during the maturation of starfish oocyte from which the vitelline coat was removed, Bull. Mar. Biol. St. Asamushi, Tohoku Univ. 16 (1979) 161e167. [7] F.J. Longo, M. Woerner, K. Chiba, M. Hoshi, Cortical changes in starfish (Asterina pectinifera) oocytes during 1-methyladenine-induced maturation and fertilisation/activation, Zygote 3 (1995) 225e239. [8] L.A. Jaffe, C.J. Gallo, R.H. Lee, et al., Oocyte maturation in starfish is mediated by the bg-subunit complex of a G-protein, J. Cell Biol. 121 (1993) 775e783. [9] K. Chiba, K. Kontani, H. Tadenuma, et al., Induction of starfish oocyte maturation by the bg subunit of starfish G protein and possible existence of the subsequent effector in cytoplasm, Mol. Biol. Cell 4 (1993) 1027e1034. [10] K. Kyozuka, J.T. Chun, A. Puppo, et al., Guanine nucleotides in the meiotic maturation of starfish oocytes: regulation of the actin cytoskeleton and of Ca2þ signaling, PloS One 4 (2008), e6296. [11] T. Kishimoto, Entry into mitosis: a solution to the decades-long enigma of MPF, Chromosoma 124 (2015) 417e428. [12] M. Hara, Y. Abe, T. Tanaka, et al., Greatwall kinase and cyclin B-Cdk1 are both critical constituents of M-phase-promoting factor, Nat. Commun. 3 (2012) 1059. [13] L. Santella, K. Kyozuka, Reinitiation of meiosis in starfish oocytes requires an increase in nuclear Ca2þ, Biochem. Biophys. Res. Commun. 203 (1994) 674e680. [14] L. Santella, K. Kyozuka, L. De Riso, E. Carafoli, Calcium, protease action, and the regulation of the cell cycle, Cell Calcium 23 (1998) 123e130. [15] L. Santella, E. Ercolano, D. Lim, et al., Activated M-phase-promoting factor (MPF) is exported from the nucleus of starfish oocytes to increase the sensitivity of the Ins(1,4,5)P3 receptors, Biochem. Soc. Trans. 31 (2003) 79e82. [16] M. Brini, M. Murgia, L. Pasti, et al., Nuclear Ca2þ concentration measured with specifically targeted recombinant aequorin, EMBO J. 12 (1993) 4813e4819. [17] L. Santella, E. Carafoli, Calcium signaling in the cell nucleus, Faseb. J. 11 (1997) 1091e1109. [18] K. Kyozuka, J.T. Chun, A. Puppo, et al., Actin cytoskeleton modulates calcium signaling during maturation of starfish oocytes, Dev. Biol. 320 (2008) 426e435. [19] N. Limatola, J.T. Chun, K. Kyozuka, L. Santella, Novel Ca2þ increases in the maturing oocytes of starfish during the germinal vesicle breakdown, Cell Calcium 58 (2015) 500e510. [20] J.J. Otto, T.E. Schroeder, Assembly-disassembly of actin bundles in starfish oocytes: an analysis of actin-associated proteins in the isolated cortex, Dev. Biol. 101 (1984) 263e273. [21] L. Santella, L. De Riso, G. Gragnaniello, K. Kyozuka, Cortical granule translocation during maturation of starfish oocytes requires cytoskeletal rearrangement triggered by InsP3-mediated Ca2þ release, Exp. Cell Res. 248 (1989) 567e574. [22] W.M. Byrnes, S.A. Newman, Ernest Everett just: egg and embryo as excitable systems, J. Exp. Zool. B Mol. Dev. Evol. 322 (2014) 191e201. [23] R.D. Allen, Fertilization and artificial activation of the egg of the surf-clam Spisula solidissima, Biol. Bull. 105 (1953) 213e239. [24] T. Schubert, A. Akopian, Actin filaments regulate voltage-gated ion channels in salamander retinal ganglion cells, Neuroscience 125 (2004) 583e590. [25] K. Fukatsu, H. Bannai, S. Zhang, et al., Lateral diffusion of inositol 1,4,5trisphosphate receptor type 1 is regulated by actin filaments and 4.1N in neuronal dendrites, J. Biol. Chem. 279 (2004) 48976e48982. [26] M.G. Dalghi, M. Ferreira-Gomes, J.P. Rossi, Regulation of the plasma membrane calcium ATPases by the actin cytoskeleton, Biochem. Biophys. Res. Commun. Nov 24 (2017), https://doi.org/10.1016/j.bbrc.2017. [27] S.I. Miyazaki, H. Ohmori, S. Sasaki, Potassium rectifications of the starfish oocyte membrane and their changes during oocyte maturation, J. Physiol. 246 (1975) 55e78. [28] W.J. Moody, J.B. Lansman, Developmental regulation of Ca2þ and Kþ currents during hormone-induced maturation of starfish oocytes, Proc. Natl. Acad. Sci. U. S. A. 80 (1983) 3096e3100. [29] B. Dale, A. de Santis, M. Hoshi, Membrane response to 1-methyladenine requires the presence of the nucleus, Nature 282 (1979) 89e90. [30] M. Terasaki, Redistribution of cytoplasmic components during germinal vesicle breakdown in starfish oocytes, J. Cell Sci. 107 (1994) 1797e1805. [31] D. Lim, K. Kyozuka, G. Gragnaniello, et al., NAADPþ initiates the Ca2þ

[32] [33] [34] [35] [36] [37] [38] [39]

[40]

[41]

[42] [43]

[44] [45] [46]

[47]

[48]

[49]

[50]

[51] [52] [53] [54]

[55]

[56]

[57]

[58]

[59]

[60] [61]

[62]

[63] [64]

9

response during fertilization of starfish oocytes, Faseb. J. 15 (2001) 2257e2267. E.E. Just, The Biology of the Cell Surface, P. Blakiston's Son & Co., Inc., Philadelphia, 1939. J. Sapp, “Just” in time: gene theory and the biology of the cell surface, Mol. Reprod. Dev. 76 (2009) 903e911. L. Santella, F. Vasilev, J.T. Chun, Fertilization in echinoderms, Biochem. Biophys. Res. Commun. 425 (2012) 588e594. M. Terasaki, Actin filament translocations in sea urchin eggs, Cell Motil. Cytoskelet. 34 (1996) 48e56. F. Vasilev, J.T. Chun, G. Gragnaniello, et al., Effects of ionomycin on egg activation and early development in starfish, PloS One 7 (2012), e39231. ^ji, M.S. Hamaguchi, Y. Hiramoto, Mechanical properties of the endoY. Sho plasm in starfish oocytes, Exp. Cell Res. 117 (1978) 79e87. S.K. Satoh, A. Tsuchi, R. Satoh, et al., The tension at the top of the animal pole decreases during meiotic cell division, PloS One 2013 (8) (2013), e79389. Y. Hamaguchi, T. Numata, S.K. Satoh, Quantitative analysis of cortical actin filaments during polar body formation in starfish oocytes, Cell Struct. Funct. 32 (2007) 29e40. S.A. Stricker, T.L. Smythe, Endoplasmic reticulum reorganizations and Ca2þ signaling in maturing and fertilized oocytes of marine protostome worms: the roles of MAPKs and MPF, Development 130 (2003) 2867e2879. J.S. Mann, K.M. Lowther, L.M. Mehlmann, Reorganization of the endoplasmic reticulum and development of Ca2þ release mechanisms during meiotic maturation of human oocytes, Biol. Reprod. 83 (2010) 578e583. L. Sun, F. Yu, A. Ullah, et al., Endoplasmic reticulum remodeling tunes IP₃dependent Ca2þ release sensitivity, PloS One 6 (2011), e27928. T. Wakai, V. Vanderheyden, S.Y. Yoon, et al., Regulation of inositol 1,4,5trisphosphate receptor function during mouse oocyte maturation, J. Cell. Physiol. 227 (2012) 705e717. R. Deguchi, N. Takeda, S.A. Stricker, Calcium signals and oocyte maturation in marine invertebrates, Int. J. Dev. Biol. 59 (2015) 271e280. K. Chiba, R.T. Kado, L.A. Jaffe, Development of calcium release mechanisms during starfish oocyte maturation, Dev. Biol. 140 (1990) 300e306. J.A. Jaffe, M. Terasaki, Structural changes in the endoplasmic reticulum of starfish oocytes during meiotic maturation and fertilization, Dev. Biol. 164 (1994) 579e587. H. Iwasaki, K. Chiba, T. Uchiyama, et al., Molecular characterization of the starfish inositol 1,4,5-trisphosphate receptor and its role during oocyte maturation and fertilization, J. Biol. Chem. 277 (2002) 2763e2772. D. Lim, E. Ercolano, K. Kyozuka, et al., The M-phase-promoting factor modulates the sensitivity of the Ca2þ stores to inositol 1,4,5-trisphosphate via the actin cytoskeleton, J. Biol. Chem. 278 (2003) 42505e42514. A. Miyazaki, E. Kamitsubo, S.I. Nemoto, Premeiotic aster as a device to anchor the germinal vesicle to the cell surface of the presumptive animal pole in starfish oocytes, Dev. Biol. 218 (2000) 161e171. K. Ookata, S. Hisanaga, T. Okano, et al., Relocation and distinct subcellular localization of p34cdc2-cyclin B complex at meiosis reinitiation in starfish oocytes, EMBO J. 11 (1992) 1763e1772. D. Lim, K. Lange, L. Santella, Activation of oocytes by latrunculin A, Faseb. J. 16 (2002) 1050e1056. K. Lange, Microvillar Caþþ signaling: a new view of an old problem, J. Cell. Physiol. 180 (1999) 19e34. K. Lange, J. Gartzke, F-actin-based Ca signaling-a critical comparison with the current concept of Ca signaling, J. Cell. Physiol. 209 (2006) 270e287. A. Puppo, J.T. Chun, G. Gragnaniello, et al., Alteration of the cortical actin cytoskeleton deregulates Ca2þ signaling, monospermic fertilization, and sperm entry, PloS One 3 (2008), e3588. J.T. Chun, L. Santella, The actin cytoskeleton in meiotic maturation and fertilization of starfish eggs, Biochem. Biophys. Res. Commun. 384 (2009) 141e143. F. Vasilev, N. Limatola, D.R. Park, et al., Disassembly of subplasmalemmal actin filaments induces cytosolic Ca2þ increases in Astropecten aranciacus eggs, Cell. Physiol. Biochem. 48 (2018) 2011e2034. D.L. Clapper, T.F. Walseth, P.J. Dargie, H.C. Lee, Pyridine nucleotide metabolites stimulate calcium release from sea urchin egg microsomes desensitized to inositol trisphosphate, J. Biol. Chem. 262 (1987) 9561e9568. H.C. Lee, T.F. Walseth, G.T. Bratt, et al., Structural determination of a cyclic metabolite of NADþ with intracellular Ca2þ-mobilizing activity, J. Biol. Chem. 264 (1989) 1608e1615. G.A. Nusco, D. Lim, P. Sabala, L. Santella, Ca2þ response to cADPr during maturation and fertilization of starfish oocytes, Biochem. Biophys. Res. Commun. 290 (2002) 1015e1021. F. Moccia, D. Lim, K. Kyozuka, L. Santella, NAADP triggers the fertilization potential in starfish oocytes, Cell Calcium 36 (2004) 515e524. H.C. Lee, R. Aarhus, Functional visualization of the separate but interacting calcium stores sensitive to NAADP and cyclic ADP-ribose, J. Cell Sci. 24 (2000) 4413e4420. G.C. Churchill, Y. Okada, J.M, et al., NAADP mobilizes Ca2þ from reserve granules, lysosome-related organelles, in sea urchin eggs, Cell 111 (2002) 703e708. P.J. Calcraft, M. Ruas, Z. Pan, et al., NAADP mobilizes calcium from acidic organelles through two-pore channels, Nature 459 (2009) 596e600. S. Patel, S. Muallem, Acidic Ca2þ stores come to the fore, Cell Calcium 50 (2011) 109e112.

Please cite this article in press as: L. Santella, et al., Maturation and fertilization of echinoderm eggs: Role of actin cytoskeleton dynamics, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.084

10

L. Santella et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e11

[65] A.J. Morgan, L.C. Davis, S.K. Wagner, et al., Bidirectional Ca2⁺ signaling occurs between the endoplasmic reticulum and acidic organelles, J. Cell Biol. 200 (2013) 789e805. [66] J.V. Gerasimenko, R.M. Charlesworth, M.W. Sherwood, et al., Both RyRs and TPCs are required for NAADP-induced intracellular Ca2⁺ release, Cell Calcium 58 (2015) 237e245. [67] L. Santella, K. Kyozuka, A.A. Genazzani, et al., Nicotinic acid adenine dinucleotide phosphate-induced Ca(2þ) release. Interactions among distinct Ca(2þ) mobilizing mechanisms in starfish oocytes, J. Biol. Chem. 275 (2000) 8301e8306. [68] L. Santella, D. Lim, F. Moccia, Calcium and fertilization: the beginning of life, Trends Biochem. Sci. 29 (2004) 400e408. [69] F. Moccia, D. Lim, G.A. Nusco, et al., NAADP activates a Ca2þ current that is dependent on F-actin cytoskeleton, Faseb. J. 17 (2003) 1907e1909. [70] G.A. Nusco, J.T. Chun, E. Ercolano, et al., Modulation of calcium signalling by the actin-binding protein cofilin, Biochem. Biophys. Res. Commun. 348 (2006) 109e114. [71] G.M. Wessel, S.D. Conner, L. Berg, Cortical granule translocation is microfilament mediated and linked to meiotic maturation in the sea urchin oocyte, Development 129 (2002) 4315e4525. [72] J.H. Henson, D.A. Begg, Filamentous actin organization in the unfertilized sea urchin egg cortex, Dev. Biol. 127 (1988) 338e348. [73] A. Spudich, J.T. Wrenn, N.K. Wessells, Unfertilized sea urchin eggs contain a discrete cortical shell of actin that is subdivided into two organizational states, Cell Motil. Cytoskelet. 9 (1988) 85e96. [74] B.L. Hylander, R.G. Summers, The effect of local anesthetics and ammonia on cortical granule-plasma membrane attachment in the sea urchin egg, Dev. Biol. 86 (1981) 1e11. [75] D.A. Begg, G.K. Wong, D.H. Hoyle, J.M. Baltz, Stimulation of cortical actin polymerization in the sea urchin egg cortex by NH4Cl, procaine and urethane: elevation of cytoplasmic pH is not the common mechanism of action, Cell Motil. Cytoskelet. 35 (1996) 210e224. [76] V.D. Vacquier, The isolation of intact cortical granules from sea urchin eggs: calcium ions trigger granule discharge, Dev. Biol. 43 (1975) 62e74. [77] T.G. Hollinger, A.W. Schuetz, “Cleavage” and cortical granule breakdown in Rana pipiens oocytes induced by direct microinjection of calcium, J. Cell Biol. 71 (1976) (1976) 395e401. [78] T. Mohri, Y. Hamaguchi, Propagation of transient Ca2þ increase in sea urchin eggs upon fertilization and its regulation by microinjecting EGTA solution, Cell Struct. Funct. 16 (1991) 157e165. [79] R. Steinhardt, R. Zucker, G. Schatten, Intracellular calcium release at fertilization in the sea urchin egg, Dev. Biol. 58 (1977) 185e196. [80] Y. Hamaguchi, M.S. Hamaguchi, Simultaneous investigation of intracellular Ca2þ increase and morphological events upon fertilization in the sand dollar egg, Cell Struct. Funct. 15 (1990) 159e162. [81] M. Terasaki, Visualization of exocytosis during sea urchin egg fertilization using confocal microscopy, J. Cell Sci. 108 (1995) 2293e2300. [82] I. Gillot, B. Ciapa, P. Payan, C. Sardet, The calcium content of cortical granules and the loss of calcium from sea urchin eggs at fertilization, Dev. Biol. 146 (1991) 396e405. [83] L.C. Davis, A.J. Morgan, M. Ruas, et al., Ca2þ signaling occurs via second messenger release from intraorganelle synthesis sites, Curr. Biol. 18 (2008) 1612e1678. [84] H. Schuel, The prevention of polyspermic fertilization in sea urchin eggs, Biol. Bull. 167 (1984) 271e309. [85] T. Fujimori, S. Hirai, Differences in starfish oocyte susceptibility to polyspermy during the course of maturation, Biol. Bull. 157 (1979) 249e257. [86] L. Santella, N. Limatola, J.T. Chun, Actin cytoskeleton and fertilization in starfish eggs, in: H. Sawada, N. Inoue, M. Iwano (Eds.), Sexual Reproduction in Animals and Plants, Springer Verlag, 2014, pp. 141e155. [87] S. Yonemura, I. Mabuchi, Wave of cortical actin polymerization in the sea urchin egg, Cell Motil. Cytoskelet. 7 (1987) 46e53. [88] Y. Hamaguchi, I. Mabuchi, Alpha-actinin accumulation in the cortex of echinoderm eggs during fertilization, Cell Motil. Cytoskelet. 6 (1986) 549e559. [89] L.G. Tilney, L.A. Jaffe, Actin, microvilli, and the fertilization cone of sea urchin eggs, J. Cell Biol. 87 (1980) 771e782. [90] K. Kyozuka, K. Osanai, Fertilization cone formation in starfish oocytes: the role of the egg cortex actin microfilaments in sperm incorporation, Gamete Res. 20 (1988) 275e285. [91] H.T. McMahon, J.L. Gallop, Membrane curvature and mechanisms of dynamic cell membrane remodeling, Nature 438 (2005) 590e596. [92] K.E. Runge, J.E. Evans, Z.Y. He, et al., Oocyte CD9 is enriched on the microvillar membrane and required for normal microvillar shape and distribution, Dev. Biol. 304 (2007) 317e325. [93] J.D. Green, R.G. Summers, Formation of the cortical concavity at fertilization in the sea urchin egg, Dev. Growth Differ. 22 (1980) 821e829. [94] E.E. Just, The fertilization reaction in Echinarachnius parma, I. Cortical response of the egg to insemination, Biol. Bull. 36 (1919) 1e10. [95] L.H. Hyman, Some notes on the fertilization reaction in echinoderm eggs, Biol. Bull. 5 (1923) 254e278. [96] F. Moser, Studies on a cortical layer response to stimulating agents in the Arbacia egg. I. Response to insemination, J. Exp. Biol. 80 (1939) 423e446. [97] L.A. Jaffe, Fast block to polyspermy in sea urchin eggs is electrically mediated, Nature 261 (1976) 68e71.

[98] L.A. Jaffe, Electrical polyspermy block in sea urchin eggs: nicotine and low sodium experiments, Dev. Growth Differ. 22 (1980) 503e507. [99] B. Dale, L.J. De Felice, V. Taglietti, Membrane noise and conductance increase during single spermatozoon-egg interactions, Nature 275 (1978) 217e219. [100] B. Dale, M. Dan-Sohkawa, A. De Santis, M. Hoshi, Fertilization of the starfish Astropecten aurantiacus, Exp. Cell Res. 132 (1981) 505e510. [101] O. Hertwig, R. Hertwig, Über den Befruchtungs und Teilungsvorgang des €usserer Agentien, Jenaische Zeit 20 tierischen Eies unter dem Einfluss a (1887) 120e243. €nning, Electron microscopic studies on the block to polyspermy. The [102] S. Lo influence of nicotine, Sarsia 18 (1965) 17e23. [103] F.J. Longo, E. Anderson, The effects of nicotine on fertilization in the sea urchin, Arbacia punctulata, J. Cell Biol. 46 (1970) 308e325. [104] P.I. Ivonnet, E.L. Chambers, Nicotinic acetylcholine receptors of the neuronal type occur in the plasma membrane of sea urchin eggs, Zygote 5 (1997) 277e287. €m, R.D. Allen, The mechanism of nicotine-induced polyspermy, [105] B.E. Hagstro Exp. Cell Res. 10 (1956) 14e23. [106] B. Dale, A. Monroy, How is polyspermy prevented? Gamete Res. 4 (1981) 151e169. [107] B. Dale, L.J. DeFelice, Polyspermy prevention: facts and artifacts? J. Assist. Reprod. Genet. 28 (2011) 199e207. [108] B. Dale, Is the idea of a fast block to polyspermy based on artifact? Biochem. Biophys. Res. Commun. 450 (2014) 1159e1165. [109] L. Santella, J.T. Chun, Actin, more than just a housekeeping protein at the scene of fertilization, Sci. China Life Sci. 54 (2011) 733e743. [110] J.T. Chun, N. Limatola, F. Vasilev, L. Santella, Early events of fertilization in sea urchin eggs are sensitive to actin-binding organic molecules, Biochem. Biophys. Res. Commun. 450 (2014) 1166e1174. [111] J.T. Chun, A. Puppo, F. Vasilev, et al., The biphasic increase of PIP2 in the fertilized eggs of starfish: new roles in actin polymerization and Ca2þ signaling, PloS One 5 (2010), e14100. [112] D.R. Burgess, T.E. Schroeder, Polarized bundles of actin filaments within microvilli of fertilized sea urchin eggs, J. Cell Biol. 74 (1977) 1032e1037. [113] L.V. Heilbrunn, An Outline of General Physiology, W. B. Sauders Co., Philadelphia and London, 1937, p. 748. [114] D. Mazia, The release of calcium in Arbacia eggs, J. Cell. Physiol. 10 (1937) 291e304. [115] M. Paul, R.N. Johnston, Uptake of Ca2þ is one of the earliest responses to fertilization of sea urchin eggs, J. Exp. Zool. 203 (1978) 143e149. [116] E.L. Chambers, J. de Armendi, Membrane potential, action potential and activation potential of eggs of the sea urchin, Lytechinus variegatus, Exp. Cell Res. 122 (1979) 203e218. [117] T. Schmidt, C. Patton, D. Epel, Is there a role for the Ca2þ influx during fertilization of the sea urchin egg? Dev. Biol. 90 (1982) 284e290. [118] Y.M. Takahashi, M. Sugiyama, Relation between the acrosome reaction and fertilization in the sea urchin: I. Fertilization in Ca-free sea water with eggwater-treated spermatozoa, Dev. Growth Differ. 15 (1973) 261e267. [119] I. Crossley, K. Swann, E. Chambers, M. Whitaker, Activation of sea urchin eggs by inositol phosphates is independent of external calcium, Biochem. J. 252 (1988) 257e262. [120] K. Sano, H. Kanatani, External calcium ions are requisite for fertilization of sea urchin eggs by spermatozoa with reacted acrosomes, Dev. Biol. 78 (1980) 242e246. [121] R.A. Steinhardt, D. Epel, Activation of sea-urchin eggs by a calcium ionophore, Proc. Natl. Acad. Sci. U.S.A. 71 (1974) 1915e1919. [122] N.D. Holland, Effects of ionophore A23187 on oocytes of Comanthus Japonica (Echinodermata:Crinoidea), Dev. Growth Differ. 22 (1980) 203e207. [123] M.E. Schalkoff, N.H. Hart, Effect of A23187 upon cortical granule exocytosis in eggs of Brachydanio, Roux’s Arch. Dev. Biol. 195 (1986) 39e48. [124] C.T. Taylor, Y.M. Lawrence, C.R. Kingsland, et al., Oscillations in intracellular free calcium induced by spermatozoa in human oocytes at fertilization, Hum. Reprod. 8 (1993) 2174e2179. [125] P. Rinaudo, M. Massobrio, J.R. Pepperell, D.L. Keefe, S. Buradgunta, Dissociation between intracellular calcium elevation and development of human oocytes treated with calcium ionophore, Fertil. Steril. 68 (1997) 1086e1092. [126] T. Ebner, M. Montag, Artificial oocyte activation: evidence for clinical readiness, Reprod. Biomed. Online 32 (2016) 271e273. [127] L. Santella, B. Dale, Assisted yes, but where do we draw the line? Reprod. Biomed. Online 31 (2015) 476e478. [128] T.E. Schroeder, Microvilli on sea urchin eggs: a second burst of elongation, Dev. Biol. 1978 (64) (1978) 342e346. [129] B. Ciapa, C. Arnoult, Could modifications of signalling pathways activated after ICSI induce a potential risk of epigenetic defects? Int. J. Dev. Biol. 55 (2011) 143e152. [130] J.C. Dan, Studies on the acrosome. I. Acrosome reaction in starfish spermatozoa, Biol. Bull. 107 (1954) 203e218. [131] M. Hoshi, H. Moriyama, M. Matsumoto, Structure of acrosome reactioninducing substance in the jelly coat of starfish eggs: a mini review, Biochem. Biophys. Res. Commun. 425 (2012) 595e598. [132] L.G. Tilney, S. Hatano, H. Ishikawa, M.S. Mooseker, The polymerization of actin: its role in the generation of the acrosomal process of certain echinoderm sperm, J. Cell Biol. 59 (1973) 109e126. [133] K. Niikura, M.S. Alam, M. Naruse, et al., Protein kinase A activity leads to the extension of the acrosomal process in starfish sperm, Mol. Reprod. Dev. 84

Please cite this article in press as: L. Santella, et al., Maturation and fertilization of echinoderm eggs: Role of actin cytoskeleton dynamics, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.084

L. Santella et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e11 (2017) 614e625. [134] I. Mabuchi, An actin-depolymerizing protein (depactin) from starfish oocytes: properties and interaction with actin, J. Cell Biol. 97 (1983) 1612e1621. [135] J.T. Chun, F. Vasilev, L. Santella, Antibody against the actin-binding protein depactin attenuates Ca2þ signaling in starfish eggs, Biochem. Biophys. Res. Commun. 441 (2013) 301e307. [136] G. Di Paolo, P. de Camilli, Phosphoinositides in cell regulation and membrane

11

dynamics, Nature 443 (2006) 651e657. [137] J.T. Chun, F. Vasilev, N. Limatola, L. Santella, Fertilization in starfish and sea urchin: roles of actin, in: M. Kloc, J.Z. Kubiak (Eds.), Marine Organisms as Model Systems, Series Title: Results and Problems in Cell Differentiation, Springer Nature, 2018, pp. 33e47. [138] Y. Hamaguchi, I. Mabuchi, Accumulation of fluorescently labeled actin in the cortical layer in sea urchin eggs after fertilization, Cell Motil. Cytoskelet. 9 (1988) 153e163.

Please cite this article in press as: L. Santella, et al., Maturation and fertilization of echinoderm eggs: Role of actin cytoskeleton dynamics, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.084