Biochemical and Biophysical Research Communications 425 (2012) 588–594

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Biochemical and Biophysical Research Communications

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Fertilization in echinoderms
Luigia Santella ⇑, Filip Vasilev, Jong T. Chun
Laboratory of Cellular and Developmental Biology Stazione Zoologica Anton Dohrn, Villa Comunale 1, Napoli 80121, Italy

a r t i c l e i n f o a b s t r a c t

Article history: For more than 150 years, echinoderm eggs have served as overly favored experimental model systems in
Received 26 July 2012 which to study fertilization. Sea urchin and starfish belong to the same phylum and thus share many sim-
ilarities in their fertilization patterns. However, several subtle but fundamental differences do exist in the
This article is dedicated to Professor William
J. Lennarz fertilization of sea urchin and starfish, reflecting their phylogenetic bifurcation approximately 500 mil-
lion years ago. In this article we review some of the seminal and recent findings that feature similarities
and differences in sea urchin and starfish at fertilization.
Keywords: Ó 2012 Elsevier Inc. All rights reserved.
Sea urchin
Starfish
Fertilization
Calcium
Actin
InsP3
NAADP
cADPr
Polyspermy

1. Introduction ization. This review will discuss the information presently avail-
able on the process of fertilization in sea urchin and starfish eggs.
Owing to the availability of a large number of gametes that can
be easily fertilized in vitro, echinoderm eggs have been widely used
for the study of oocyte maturation and fertilization. At spawning, 2. Sperm activation
sea urchin eggs have already completed meiosis and contain a hap-
loid nucleus. In contrast, starfish oocytes are fertilized at the dip- The chain of reactions culminating in fertilization initiates in
loid stage, and the meiotic division is completed after the sperm the spermatozoon that undergoes physiological changes upon con-
entry. Because of this readiness of the eggs in the gonad, sea urchin tacting the eggs. In the testis, sea urchin spermatozoa are immotile
eggs have been favored in early studies showing the separation of due to the high CO2 pressure that keeps the intracellular pH (pHi)
the vitelline layer from the egg plasma membrane, which is the below the levels needed to activate dynein ATPase in the flagella.
hallmark of echinoderm fertilization. On the other hand, starfish When exposed to seawater, protons are released and the sperma-
represent synchronized oocytes arrested at the first prophase of tozoa begin to swim. Hyperpolarization of its plasma membrane
meiosis, which can be induced to undergo meiotic maturation. induced by the low K+ in the seawater contributes to the pHi rise
Thus, these larger and more transparent oocytes have provided and to the concomitant activation of adenylyl cyclase. This acti-
an opportunity to study the regulation of meiotic cell cycle as well vates cAMP-dependent protein kinase (PKA) that phosphorylates
as fertilization. Starfish sperm extend a long and slender acrosomal proteins essential for flagella motility [1]. Small diffusible activat-
process to reach the egg surface across the deep layer of the jelly ing peptides (SAPs) present in the egg jelly induce changes in
coat. Much has been learned about the morphological and bio- sperm motility and straighter swimming patterns towards the
chemical changes that happen in both gametes, and it has become egg. Speract, a decapeptide first purified from Strongylocentrotus
clear that Ca2+ is an essential actor in the processes. However, rel- purpuratus eggs, cross-links to a 77 kDa membrane receptor which
atively little is known about the precise mechanisms that regulate stimulates the activation of a membrane-bound guanylyl cyclase
the Ca2+ release and the changes that lead to monospermic fertil- resulting in a transient increase of cGMP. This opens a novel tetra-
meric K+-selective cyclic nucleotide-gated ion channel (Sp-tetra-
KCNG) and induces hyperpolarization of the sperm plasma
⇑ Corresponding author. Fax: +39 081 583 3289. membrane, which in turn rapidly increases pHi and concomitantly
E-mail address: santella@szn.it (L. Santella). decreases intracellular Ca2+ due to the concerted activity of two

0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bbrc.2012.07.159
 L. Santella et al. / Biochemical and Biophysical Research Communications 425 (2012) 588–594 589

exchangers, a Na+/H+ and a flagellar K+-dependent Na+/Ca2+ ex- a group of sulphated steroidal saponins and asterosap. The latter
changer [2]. The termination of the cGMP is ensured by a phospho- chemotactic agents bind to a guanylyl cyclase receptor in the fla-
diesterase in sperm flagella (suPDE5) [3]. The sperm gellar plasma membrane and hyperpolarize the sperm plasma
hyperpolarization is followed by a sustained depolarization. Ca2+ membrane to induce a transient increase of cGMP and Ca2+. Astero-
influx initiates at the onset of recovery from hyperpolarization sap transiently increases Ca2+ by activating the reverse-operation
then enhances itself during the depolarization phase due to the of a K+-dependent Na+/Ca2+ exchanger and decrease in pHi. The
voltage-dependent Ca2+ channels and the production of cAMP sustained increase of Ca2+ induced by the simultaneous treatment
which stimulates the opening of a Ca2+ channel. Thus, cycles of of sperm with ARIS and asterosap may involve a store operated
hyperpolarization and depolarization of the membrane potential Ca2+ channel [12,13].
induce changes in the curvature of the sperm swimming trajectory
through the generation of trains of Ca2+ fluctuations with Ca2+
reuptake by way of Ca2+ ATPases in the sperm head and Na+/ 4. Gametes interaction and fast block to polyspermy
Ca2+/K+ exchangers in the flagellum [4,5].
Fertilization thus depends on the activation of spermatozoa that
allows their binding and fusion to the egg plasma membrane. Spe-
3. Acrosome reaction cies-specific gamete interaction is of particular importance to the
species having external fertilization, and its molecular mechanism
The significance and the kinetics of the sperm acrosome reac- is relatively well-studied in sea urchin (see the review by Vacquier
tion (AR) were discovered by Dan [6] in starfish sperm. She found on bindin and sperm receptors in the same issue). On the other
that increased permeability to Ca2+ was the triggering event lead- hand, a fertilized egg requires a mechanism to prevent the forma-
ing to exocytosis of the acrosomal vesicle and to actin polymeriza- tion and development of the polyspermic zygotes. In echinoderm,
tion at the tip of the sperm head to prolong a 25 lm long filament while elevation of the fertilization envelope may serve as a
process (Fig. 1). In sea urchin, the acrosomal filament extends for mechanical block to polyspermy, whether or not a fast block mech-
only 1 lm from the head, and is covered with bindin, the adhesive anism exists remains controversial. As the rate of re-fertilization of
protein responsible for the species-specific attachment of sperm to sea urchin eggs is much lower than that of the initial fertilization, a
the egg’s vitelline layer [7]. The K+ and H+ efflux accompanying the block to polyspermy must be rapidly established (in about two sec-
extension of the acrosomal filament results in changes in the mem- onds) after the interaction of the first successful sperm [14]. Intra-
brane potential, and the Na+-dependent increase in pHi by a Na+/H+ cellular recordings from sea urchin eggs have shown that the first
exchange correlates temporally with Ca2+ uptake [8]. The compo- detectable electrical event at fertilization is the step-like depolar-
nents of the jelly coat that induce AR in S. purpuratus are fucose ization, which is accompanied by an increase in voltage noise
sulfate polymers (FSP) [9]. Three receptors for the egg jelly (suREJ1, about 13 s (the latent period) before the long depolarization of
suREJ2 and suREJ3) present in sea urchin sperm are presumably in- the membrane potential (fertilization potential). Jaffe [15] first
volved in the calcium signaling pathways eliciting the AR [10]. suggested that this rapid shift of the membrane potential to a po-
Upon binding of FSP to suREJ1, the biphasic Ca2+ influx and the sitive level causes the fast block to polyspermy, which renders the
activation of InsP3Rs at the acrosomal membrane maintain a sus- egg plasma membrane refractory to supernumerary spermatozoa.
tained intracellular Ca2+ increase in sperm [5]. The hypothesis of electrical fast block to polyspermy has been re-
Starfish spermatozoa undergo AR upon encountering three ported in several other species, but was not supported unani-
components of the jelly coat. Lillie [11] first suggested that the jelly mously [14]. No change in egg receptivity to sperm was observed
coat may represent the egg receptor for sperm, which induces the in S. purpuratus during the latent period [16]. Electrophysiological
AR in many echinoid species. A large sulphated proteoglycan-like measurements on eggs from two different sea urchin species at fer-
molecule called ARIS (AR-inducing substance) works with Co-ARIS, tilization in high sperm density showed that creation of multiple

Fig. 1. Morphology of sea urchin and starfish sperm before and after acrosome reaction. While sea urchin spermatozoon (S. purpuratus) generally appears cone-shaped (A),
starfish spermatozoon (A. pectinifera) bears more spherical outline (B). Acrosome-reacted sperm of starfish extends a long (ca 25 lm) rigid acrosomal process that is cored by
actin filaments.
590 L. Santella et al. / Biochemical and Biophysical Research Communications 425 (2012) 588–594

small step-depolarizations (polyspermy) were mainly dependent 6. cADPr and NAADP-mediated Ca2+ release at fertilization of
on sperm density, and not on the change of the membrane poten- sea urchin and starfish eggs
tial [17], which was rather interpreted as an electrical result of cor-
tical granules exocytosis [14]. Thus, whether membrane In analogy to the Ca2+ transients in other cell types, a CICR
depolarization is responsible for the fast block to polyspermy re- mechanism distinct from the InsP3-mediated Ca2+ release has been
mains controversial [18]. shown in sea urchin eggs. cADPr, a metabolite of NAD+, mobilized
Ca2+ by a CICR mechanism acting on ryanodine receptors (RyRs)
that were shown to be active in sea urchin eggs. Thus, this mode
of Ca2+ release in concert with the InsP3-mediated pathway may
5. Ca2+ signaling at fertilization of sea urchin eggs modulate the spatiotemporal pattern of the Ca2+ wave at fertiliza-
tion [37]. Three ADP-ribosyl cyclase isoforms (ARCs) that produce
A turning point in the studies on the egg activation was the pio- cADPr have been characterized in sea urchin eggs. While ARCa
neering observation of a Ca2+ increase in the eggs of sea urchin fol- has an extracellular localization, ARCb and ARCc are present in
lowing fertilization [19]. However, it took almost 40 years to the acidic cortical granules that generate the fertilization envelope
conclusively demonstrate that Ca2+ was indeed responsible for upon exocytosis. The active ARCb and ARCc might provide cADPr
egg activation of Lytechinus pictus and S. purpuratus eggs with the to induce Ca2+ rise to drive cortical granules exocytosis [38].
use of Ca2+-ionophore A23187 that elicited cortical granules exocy- One report suggested NO as the initiator of the Ca2+ release at
tosis, membrane conductance changes, the respiratory burst, and fertilization in sea urchin. NO production occurred in both acro-
the increases of protein and DNA synthesis, all hallmarks of egg some-reacted sperm and in activated eggs. Binding of NO to a gua-
activation [20]. The ionophore-induced Ca2+ increase and mem- nylyl cyclase could induce a cGMP-dependent activation of ARC
brane elevation still occurred in Ca2+-free seawater, indicating that and produce cADPr [34]. The level of cGMP increases at fertilization
liberation of Ca2+ from the intracellular stores is mainly account- [31,39], and thus could stimulate the synthesis of cADPr, support-
able for egg activation. The Ca2+-specific luminescent protein ing the hypothesis that cADPr may control intracellular Ca2+ signal-
aequorin visualized the explosive rise of free Ca2+ during fertiliza- ing in fertilized sea urchin eggs [40]. However, other investigators
tion of medaka and sea urchin eggs [21,22]. The Ca2+ signals in fer- showed that the NO increase occurred after the intracellular Ca2+
tilized eggs initiated at the point of sperm entry and spread over rise. It was thus concluded that the NO production was dependent
like a wave, reaching the opposite pole in about 20 s [23]. Two on the Ca2+ increase initiated by InsP3 at fertilization, and that its
phases of Ca2+ increase at fertilization have been demonstrated role was to sustain the second phase of Ca2+ response in a cAD-
in sea urchin eggs: (i) a synchronized Ca2+ increase at the egg Pr-regulated RyRs pathway [41].
periphery (cortical flash) due to a rapid influx, (ii) Ca2+ liberated The sea urchin egg is responsive to yet another Ca2+-linked sec-
from the intracellular stores starting from the sperm-interaction ond messenger: NAADP. In sea urchin eggs with centrifugation-
site [24]. stratified organelles, the patterns of Ca2+ release by uncaged
The search for the molecular mechanisms by which the sperm- NAADP was clearly distinct from those of cADPr and InsP3, indicat-
egg interaction liberates Ca2+ came across the contemporary find- ing the existence of a Ca2+ store that may be distinct from the ER. In
ing that an increase in the Ca2+-inducing second messenger 1,4,5 sea urchin eggs, NAADP appeared to mobilize Ca2+ from a lyso-
inositol trisphosphate (InsP3) correlated with the hydrolysis of some-related organelles, as judged by the loss of the Ca2+ signals
phosphatidylinositol 4,5-bisphosphate (PIP2) in the plasma mem- in the eggs treated with Glycyl-L-phenylalanine-ß-naphthylamide
brane by phospholipase C (PLC) [25]. The injection of InsP3 into (GPN) and bafilomycin A1 [42].
sea urchin eggs indeed caused a Ca2+ rise and the exocytosis of Recent findings have provided evidence that NAADP activates a
the cortical granules [26]. InsP3 formed at the sperm interaction two pore channel (TPC) located on the acidic Ca2+ stores in the
site thus appeared to bind to the receptor on the egg endoplasmic endo-lysosomal system [43]. S. purpuratus eggs express three TPCs
reticulum (ER), and the local release of Ca2+ may further propagate (SpTPCs). Immunostaining of SpTPC3 revealed a punctate cortical
the Ca2+ wave by a Ca2+-induced Ca2+ release (CICR, see below) and cytoplasmic staining pattern. Heterologous expression of
[27]. SpTPCs in starfish oocytes has shown that all three isoforms have
Studies on three species of sea urchin showed that microin- a cortical distribution [44], in line with the finding that the greatest
jected GTP-cS caused Ca2+ release and cortical granules exocytosis, NAADP sensitivity resides in the cortical region of starfish eggs
indicating that G-protein-linked phospholipase C (PLCb) may [45–47]. As NAADP largely increases in sea urchin sperm at AR, a
mediate egg activation [28]. Alternatively, the gametes interaction bolus of messenger might trigger the cortical flash preceding the
may activate SRC family kinases (SFKs), which in turn activates Ca2+ wave [35]. Thus, the current hypothesis in sea urchin eggs is
PLCc to produce InsP3 [29]. Indeed, echinoderm eggs contain both that NAADP, probably delivered into the egg by the sperm, induces
PLCb and PLCc [30], and tyrosine kinase activity becomes stimu- the cortical flash and the regenerative Ca2+ wave by acting on
lated in sea urchin eggs before the initiation of Ca2+ increase NAADP receptors (TPCs) localized on cortical acidic stores that
[31]. Inhibiting the endogenous PLCc by injecting the SH2 domain are functionally coupled to the ER Ca2+ channels through a CICR
of PLCc into sea urchin and starfish eggs have shown that fertiliza- mechanism.
tion Ca2+ wave may be mediated by a SFK-PLCc signaling pathway
[29].
Another line of thoughts regarding sperm-induced Ca2+ increase 7. The role of Ca2+ in cortical granules exocytosis
in fertilized egg is the conduit theory pioneered by Loeb in 1899
that has been elaborated in various model systems [32]. In this The autocatalytic cortical reaction that spreads from the sperm–
view, the Ca2+ increase in eggs at fertilization could be induced egg interaction site was first discovered by Derbès (1847) and has
by sperm through a direct infusion of Ca2+ itself [8,33] or other been most extensively studied in sea urchin eggs [48]. The fusion
‘sperm factors’. Among other candidates, InsP3, nitric oxide (NO), of the cortical granules with the plasma membrane extrudes their
nicotinic acid adenine dinucleotide phosphate (NAADP), and sev- contents into the perivitelline space due to the intracellular Ca2+
eral proteins were considered [26,34,35], and in mammalian fertil- rise [49]. Isolated cortical granules of sea urchin eggs (cortical
ization, the sperm-specific PLCf has recently earned its support as a ‘lawns’) could be induced to undergo exocytosis upon addition of
sperm factor [36]. Ca2+ [50], whereas the microinjection of EGTA prevented the eleva-
 L. Santella et al. / Biochemical and Biophysical Research Communications 425 (2012) 588–594 591

Fig. 2. Comparison of sea urchin and starfish eggs. (A) In photomicrographs displayed on the same scale, a sea urchin egg (Paracentrotus lividus) was apposed to the immature
and mature eggs of starfish (Astropecten aranciacus). The average diameter of P. lividus eggs (80 lm) is about the size of the germinal vesicle (GV) of the starfish oocytes. Due to
its large size (250–350 lm), even the nucleolus (n) is easily recognized in starfish oocytes. (B) Sea urchin and starfish eggs are fertilized at different meiotic stages (see the
text). The first (2n) and second (n) polar bodies are depicted with small circles in green and orange, respectively.

tion of the fertilization envelope [51]. At fertilization, the elevation tiple sperm enter the oocytes (Fig. 3A). Thus, the exposure to 1-MA
of the vitelline layer is initiated by a trypsin-like protease released drastically decreases the ability of the mature eggs to be pene-
from the cortical granules that frees the vitelline layer from the trated by supernumerary sperm (Fig. 3B). On the other hand, over-
plasma membrane. This may help to prevent supernumerary matured eggs beyond extrusion of the first polar body exhibit
sperm binding. The secreted material self-aggregates in the pres- polyspermy and abnormal development [58]. It is noteworthy that
ence of Ca2+, and this process is strongly inhibited by reducing overmatured eggs in human display regional loss of microvilli [59].
agents, as the hardening of the fertilization envelope is a conse- It is conceivable that the mature eggs at the optimal time frame for
quence of transglutaminase and peroxidise activities [52,53]. In fertilization present the adequate structural organization of the
addition, two bursts of microvillar elongation occur, which are cortex for ensuring both monospermic sperm entry and the normal
essential for sperm incorporation and the subsequent cleavage elevation of the fertilization envelope [60–62]. Likewise, in sea
[54]. urchin, immature oocytes also fail to undergo cortical granule exo-
cytosis at fertilization, and lead to polyspermy. These oocytes exhi-
8. Starfish oocytes as a model system for studying cortical and bit the first electrical change about 5 s after sperm addition, as
nuclear maturation at meiosis opposed to the 13 s period observed in the mature eggs. Each
sperm entry is marked by a step depolarization and by the forma-
The importance of the cytoplasmic maturation of the egg has tion of large cytoplasmic protrusions on the oocyte surface [63].
been emphasized for more than a century [55]. Immature oocytes Cortical maturation also includes cytoskeletal changes. Exposed
extracted from the starfish gonad are arrested at the first prophase to 1-MA, immature oocytes of starfish immediately reorganize
of meiosis, and contain a large nucleus named the germinal vesicle microvilli on the surface, caused by the rapid assembly and disas-
(GV) (Fig. 2A). They are transparent and very large (about 300 lm), sembly of the filamentous actin (F-actin) [64]. An equally rapid
and thus suitable for imaging experiments. By adding the hormone change that takes place is the fast release of intracellular Ca2+ in
1-methyladenine (1-MA), the oocytes can be induced to resume the cytoplasm and nucleus [65]. The Ca2+ wave stimulated by 1-
meiosis and to undergo the physiological and morphological MA starts at the vegetal hemisphere and propagates to the oppo-
changes necessary for normal fertilization, the most prominent site pole exclusively through the cortical layer. Interestingly, the
sign of which is the breakdown of the nuclear envelope (GVBD). dynamics of the 1-MA-induced Ca2+ signal is spatially and tempo-
Thus, starfish oocytes have provided an excellent system in which rally influenced by the architecture of the cortical actin cytoskele-
to study biochemical events of meiotic maturation, optimization of ton [66,67]. The surface of the mature eggs, however, shows a
the Ca2+-release systems, the establishment of polyspermy-pre- decrease in microvillar length, along with F-actin-based relocation
venting mechanisms, and the changes of the egg-dependent sperm of the cortical granules which are now seen intimately apposed to
pronucleus [56,57]. the plasma membrane [60,68].
At variance with sea urchin eggs, the optimum period to fertil- At meiotic maturation of starfish oocytes, the electrical proper-
ize starfish eggs is at the interval between GVBD and extrusion of ties of the plasma membrane abruptly shifts at GVBD due to the
the first polar body (Fig. 2B). When the immature oocytes at the GV loss of K+ permeability, and the resting potential switches from
stage are fertilized, several fertilization cones are formed and mul- about 90 to 20 mV [17,69], and the transition process may re-
592 L. Santella et al. / Biochemical and Biophysical Research Communications 425 (2012) 588–594

Fig. 3. Fertilization of the immature oocyte and mature eggs of starfish (A. aranciacus). (A) At fertilization, the GV-stage oocyte forms numerous cytoplasmic protrusions
(arrows) (A), while the mature eggs form a single fertilization cone (arrowhead) and elevation of the fertilization envelope (FE) (B). These differences are attributed to the
changes of the egg surface and the ectoplasm that take place during meiotic maturation.

quire contribution of nuclear components [70]. The mechanism by deo camera [81]. Such contact via the tip of the acrosomal process
which 1-MA alters the electrical properties of the plasma mem- was respectively manifested by the changes of the membrane po-
brane is unknown, but it may reflect the structural reorganization tential and the generation of the cortical flash and the initial Ca2+
of the cortex and the changes in the nucleus. These changes collec- wave at the site of sperm interaction, and both electrical and
tively optimize the Ca2+ response and ensure monospermic fertil- Ca2+ changes took place while the sperm head was still on the
ization [46,57,71]. rim of the jelly coat [80–82].
Starfish have another edge over sea urchin in studying the
9. Optimization of the intracellular Ca2+-release system during kinetics of Ca2+ rise. The starfish oocytes are much larger
maturation of starfish oocytes (Fig. 2A), and it take longer time for the Ca2+ wave to propagate
to the opposite side of the egg (Fig. 4). However, the planar veloc-
Compared with the immature oocytes at GV stage, matured ities of the Ca2+ waves in the two eggs are virtually the same,
eggs of starfish respond to the same dose of InsP3 with much high- implying that the ‘irritable matrix’ of the starfish and sea urchin
er Ca2+ release [61,62,71]. This might be in part due to the reorga- eggs might be the same. Nonetheless, the starfish and sea urchin
nization of the endoplasmic reticulum (ER) that takes place during eggs might have subtly different way of initiating the Ca2+ waves
the maturation process [72]. The increased sensitivity of the Ca2+ at fertilization. It was originally suggested that NAADP may have
stores to InsP3 starts at the animal hemisphere of the maturing oo- a triggering role in initiating the sperm-induced Ca2+ response at
cyte and spreads along the animal-vegetal axis to the opposite side fertilization [83,84]. Injection of NAADP or photoliberation of the
[62,73]. The development of the increased sensitivity to InsP3 is caged NAADP in the starfish eggs induced a Ca2+ response only in
linked to the nuclear activation of the maturation-promoting factor the cortical region. Unlike InsP3, Ca2+ response to NAADP was not
(MPF), a cyclin-dependent kinase controlling the entry of eukary- affected by the removal of the nucleus [45,46]. At variance with
otic cells into the M phase [62,74]. The higher Ca2+ response to sea urchin eggs, the NAADP-induced Ca2+ response in starfish eggs
InsP3 at the end of maturation was not linked to the redistribution required external Ca2+ and was selectively inhibited by blockers of
or to the increase of InsP3Rs, but rather correlated with the eggs’ L-type and store operated Ca2+ channels, which bears a striking dif-
development of an unusual ability to respond to latrunculin-A ference from the pathway involving NAADP-responsive Ca2+ stores
(LAT-A) with a Ca2+ release [75]. The spatiotemporal modifications in the acidic organelles of sea urchin eggs [35]. In line with a role in
of the actin cytoskeleton during maturation might thus be impor- initiating egg activation, NAADP mimicked the sperm-evoked
tant for optimizing the Ca2+ response to InsP3 and fertilizing sperm. depolarization by activating a Ca2+-mediated inward current which
was blocked by BAPTA but was not affected by the impairment of
lysosomes with Bafilomycin A1 or GPN, nor by agents blocking
10. Gametes interaction and activation in starfish

The fertilization of starfish eggs is of special interest because of

the long and slender sperm acrosomal process (Fig. 1). The ques-
tion of where it originates, i.e. from the egg or from the spermato-
zoon, has stimulated intense debates in the literature. The
observed acrosomal filament was initially thought to be an exten-
sion of the egg’s fertilization cone or the tubular protrusion of the
ectoplasm reaching the outer edge of the jelly layer to capture the
spermatozoon [76–78]. Just [79], rejected this early interpretation
by stating that ‘‘the cone forms after the attachment of the sperma-
tozoön to the vitelline membrane Moreover, this strand is a prolon-
gation of the spermatozoön, the tip of which is fixed within the
cone.’’ Dan [6] finally settled the argument by demonstrating that
sperm can form acrosomal process in the ‘‘egg water’’ without Fig. 4. Calcium signals in sea urchin and starfish eggs at fertilization. The
propagation of the Ca2+ waves in the A. aranciacus and P. lividus eggs were
eggs. The sperm’s functional contact on egg plasma membrane visualized by Calcium Green and displayed in pseudocolors on the same scale.
via acrosomal process was nicely demonstrated by electrophysiol- While it takes about 20 s for the wave front to reach the other end of the P. lividus
ogy [80] and calcium imaging coupled with digitally enhanced vi- eggs, the large eggs of A. aranciacus takes much longer time, proportional to its size.
 L. Santella et al. / Biochemical and Biophysical Research Communications 425 (2012) 588–594 593

RyRs and InsPRs [47,85,86]. To date, there is no evidence for an in- within the cytoplasm and that by redistribution of its structure
crease in NAADP in the activated starfish sperm nor in the egg at and relocalization of its activity, it establishes new cell-surface;
fertilization, and therefore it is still puzzling how NAADP could ini- and that during differentiation the ectoplasm increases in amount
tiate the Ca2+ response in this species. The possibility that a cADPr- and reveals a differential activity.’’
sensitive RyRs may sustain the fertilization Ca2+ response initiated
by NAADP in starfish is less likely in view of the finding that the References
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