Environmental pH signals the release of monosaccharides from cell wall in coral symbiotic alga.

Reef-building corals thrive in oligotrophic environments due to their possession of endosymbiotic algae. Confined to the low pH interior of the symbiosome within the cell, the algal symbiont provides the coral host with photosynthetically fixed carbon. However, it remains unknown how carbon is released from the algal symbiont for uptake by the host. Here we show, using cultured symbiotic dinoflagellate, Breviolum sp., that decreases in pH directly accelerates the release of monosaccharides, that is, glucose and galactose, into the ambient environment. Under low pH conditions, the cell surface structures were deformed and genes related to cellulase were significantly upregulated in Breviolum. Importantly, the release of monosaccharides was suppressed by the cellulase inhibitor, glucopyranoside, linking the release of carbon to degradation of the agal cell wall. Our results suggest that the low pH signals the cellulase-mediated release of monosaccharides from the algal cell wall as an environmental response in coral reef ecosystems.

Coral reefs are known as 'treasure troves of biodiversity' because of the enormous variety of different fish, crustaceans and other marine life they support. Colonies of marine animals, known as corals, which are anchored to rocks on the sea bed, form the main structures of a coral reef. Many corals rely on partnerships with microscopic algae known as dinoflagellates for most of their energy needs. The dinoflagellates use sunlight to make sugars and other carbohydrates and they give some of these to the coral. In exchange, the coral provides a home for the dinoflagellates inside its body. The algae live inside special compartments within coral cells known as symbiosomes. These compartments have a lower pH (that is, they are more acidic) than the rest of the coral cell. Previous studies have shown that the algae release sugars into the symbiosome but it remains unclear what triggers this release and whether it only occurs when the algae are in a partnership. Ishii et al. studied a type of dinoflagellate known as Breviolum sp. that had been grown in sea water-like liquid in a laboratory. The experiments found that the alga released two sugar molecules known as glucose and galactose into its surroundings even in the absence of a host coral. Increasing the acidity of the liquid caused the alga to release more sugars and resulted in changes to some of the structures on the surface of its cells. The alga also produced an enzyme, called cellulase, to degrade the wall that normally surrounds the cell of an alga. Treating the alga with a drug that inhibits the activity of cellulase also suppressed the release of sugars from the cells. These findings suggest that when dinoflagellates enter acidic environments, like the guts of marine animals or symbiosomes inside coral cells, the decrease in pH can activate the algal cellulase enzyme, which in turn triggers the release of sugars for the coral. This research will provide a new viewpoint to those interested in how partnerships between animals and algae are sustained in marine environments. It also highlights the importance of the alga cell wall in establishing partnerships with corals. Further work will seek to clarify the precise biological mechanisms involved.

On the origin of appetite: GLWamide in jellyfish represents an ancestral satiety neuropeptide.

Food intake is regulated by internal state. This function is mediated by hormones and neuropeptides, which are best characterized in popular model species. However, the evolutionary origins of such feeding-regulating neuropeptides are poorly understood. We used the jellyfish Cladonema to address this question. Our combined transcriptomic, behavioral, and anatomical approaches identified GLWamide as a feeding-suppressing peptide that selectively inhibits tentacle contraction in this jellyfish. In the fruit fly Drosophila, myoinhibitory peptide (MIP) is a related satiety peptide. Surprisingly, we found that GLWamide and MIP were fully interchangeable in these evolutionarily distant species for feeding suppression. Our results suggest that the satiety signaling systems of diverse animals share an ancient origin.

A highly photostable and bright green fluorescent protein.

The low photostability of fluorescent proteins is a limiting factor in many applications of fluorescence microscopy. Here we present StayGold, a green fluorescent protein (GFP) derived from the jellyfish Cytaeis uchidae. StayGold is over one order of magnitude more photostable than any currently available fluorescent protein and has a cellular brightness similar to mNeonGreen. We used StayGold to image the dynamics of the endoplasmic reticulum (ER) with high spatiotemporal resolution over several minutes using structured illumination microscopy (SIM) and observed substantially less photobleaching than with a GFP variant optimized for stability in the ER. Using StayGold fusions and SIM, we also imaged the dynamics of mitochondrial fusion and fission and mapped the viral spike proteins in fixed cells infected with severe acute respiratory syndrome coronavirus 2. As StayGold is a dimer, we created a tandem dimer version that allowed us to observe the dynamics of microtubules and the excitatory post-synaptic density in neurons. StayGold will substantially reduce the limitations imposed by photobleaching, especially in live cell or volumetric imaging.

Gene expression alterations from reversible to irreversible stages during coral metamorphosis.

For corals, metamorphosis from planktonic larvae to sedentary polyps is an important life event, as it determines the environment in which they live for a lifetime. Although previous studies on the reef-building coral Acropora have clarified a critical time point during metamorphosis when cells are committed to their fates, as defined by an inability to revert back to their previous states as swimming larvae (here referred to as the "point of no return"), the molecular mechanisms of this commitment to a fate remain unclear. To address this issue, we analyzed the transcriptomic changes before and after the point of no return by inducing metamorphosis of Acropora tenuis with Hym-248, a metamorphosis-inducing neuropeptide. Gene Ontology and pathway enrichment analysis of the 5893 differentially expressed genes revealed that G protein-coupled receptors (GPCRs) were enriched, including GABA receptor and Frizzled gene subfamilies, which showed characteristic temporal expression patterns. The GPCRs were then classified by comparison with those of Homo sapiens, Nematostella vectensis and Platynereis dumerilii. Classification of the differentially expressed genes into modules based on expression patterns showed that some modules with large fluctuations after the point of no return were biased toward functions such as protein metabolism and transport. This result suggests that in precommitted larvae, different types of GPCR genes function to ensure a proper environment, whereas in committed larvae, intracellular protein transport and proteolysis may cause a loss of the reversibility of metamorphosis as a result of cell differentiation.

Handling and Manipulation of Gametes and Embryos of the Annelidan Worm Pseudopotamilla occelata.

Pseudopotamilla occelata is a polychaete worm distributed widely in the northern part of the Pacific coast, having value as fishing bait as well as biological material for some basic research areas, including reproduction. Here we describe methods for handling the gametes and embryos of this worm, focusing on such topics as maintenance of adults, induction of oocyte maturation and fertilization, culture of embryos and larvae, microinjection into oocytes, and calcium (Ca2+) imaging.

Isolation and Characterization of Feeding-Deficient Strains in Inbred Lines of the Hydrozoan Jellyfish Cladonema pacificum.

Feeding behavior in cnidarians has been studied as a model experimental system in physiology and neurobiology. Although the feeding response in cnidarians, such as Hydra, is triggered by chemical signals, the underlying molecular mechanisms that ensure their precise execution are not well understood. It could be largely due to the lack of genetic analysis in cnidarian experimental systems. Cladonema pacificum is a hydrozoan jellyfish that is easy to maintain and cross for genetic analysis in the laboratory. To establish C. pacificum as a model experimental animal in cnidarians, we have been inbreeding strains of jellyfish. Here, we document our progress in developing C. pacificum inbred lines and feeding-defective strains that we isolated in the course of inbreeding. In the inbred lines, an increasing number of feeding-defective strains appeared as descending generations and finally all the F5 progeny showed a feeding-deficient phenotype presumably owing to inbreeding depression. Feeding behaviors of these strains were analyzed by video microscopy and we found that the feeding-defective strains captured prey, but could not kill them. After trapping prey, wild-type medusae contracted their tentacles tightly and then bent the tentacles to bring the prey to the mouth; however, feeding-defective medusae rarely contracted their tentacles and did not bend. These feeding-defective phenotypes are caused by lack of stinging nematocytes in their tentacle batteries. These findings furnish a clue to the regulatory aspects of feeding behavior, but also reveal the mechanisms of stinging nematocyte transport in tentacles.

Identification of jellyfish neuropeptides that act directly as oocyte maturation-inducing hormones.

Oocyte meiotic maturation is crucial for sexually reproducing animals, and its core cytoplasmic regulators are highly conserved between species. By contrast, the few known maturation-inducing hormones (MIHs) that act on oocytes to initiate this process are highly variable in their molecular nature. Using the hydrozoan jellyfish species Clytia and Cladonema, which undergo oocyte maturation in response to dark-light and light-dark transitions, respectively, we deduced amidated tetrapeptide sequences from gonad transcriptome data and found that synthetic peptides could induce maturation of isolated oocytes at nanomolar concentrations. Antibody preabsorption experiments conclusively demonstrated that these W/RPRPamide-related neuropeptides account for endogenous MIH activity produced by isolated gonads. We show that the MIH peptides are synthesised by neural-type cells in the gonad, are released following dark-light/light-dark transitions, and probably act on the oocyte surface. They are produced by male as well as female jellyfish and can trigger both sperm and egg release, suggesting a role in spawning coordination. We propose an evolutionary link between hydrozoan MIHs and the neuropeptide hormones that regulate reproduction upstream of MIHs in bilaterian species.

A gonad-expressed opsin mediates light-induced spawning in the jellyfish Clytia.

Across the animal kingdom, environmental light cues are widely involved in regulating gamete release, but the molecular and cellular bases of the photoresponsive mechanisms are poorly understood. In hydrozoan jellyfish, spawning is triggered by dark-light or light-dark transitions acting on the gonad, and is mediated by oocyte maturation-inducing neuropeptide hormones (MIHs) released from the ectoderm. We determined in Clytia hemisphaerica that blue-cyan light triggers spawning in isolated gonads. A candidate opsin (Opsin9) was found co-expressed with MIH within specialised ectodermal cells. Opsin9 knockout jellyfish generated by CRISPR/Cas9 failed to undergo oocyte maturation and spawning, a phenotype reversible by synthetic MIH. Gamete maturation and release in Clytia is thus regulated by gonadal photosensory-neurosecretory cells that secrete MIH in response to light via Opsin9. Similar cells in ancestral eumetazoans may have allowed tissue-level photo-regulation of diverse behaviours, a feature elaborated in cnidarians in parallel with expansion of the opsin gene family.

Surfing the vegetal pole in a small population: extracellular vertical transmission of an 'intracellular' deep-sea clam symbiont.

Symbiont transmission is a key event for understanding the processes underlying symbiotic associations and their evolution. However, our understanding of the mechanisms of symbiont transmission remains still fragmentary. The deep-sea clam Calyptogena okutanii harbours obligate sulfur-oxidizing intracellular symbiotic bacteria in the gill epithelial cells. In this study, we determined the localization of their symbiont associating with the spawned eggs, and the population size of the symbiont transmitted via the eggs. We show that the symbionts are located on the outer surface of the egg plasma membrane at the vegetal pole, and that each egg carries approximately 400 symbiont cells, each of which contains close to 10 genomic copies. The very small population size of the symbiont transmitted via the eggs might narrow the bottleneck and increase genetic drift, while polyploidy and its transient extracellular lifestyle might slow the rate of genome reduction. Additionally, the extracellular localization of the symbiont on the egg surface may increase the chance of symbiont exchange. This new type of extracellular transovarial transmission provides insights into complex interactions between the host and symbiont, development of both host and symbiont, as well as the population dynamics underlying genetic drift and genome evolution in microorganisms.

Calcium signals and oocyte maturation in marine invertebrates.

In various oocytes and eggs of animals, transient elevations in cytoplasmic calcium ion concentrations are known to regulate key processes during fertilization and the completion of meiosis. However, whether or not calcium transients also help to reinitiate meiotic progression at the onset of oocyte maturation remains controversial. This article summarizes reports of calcium signals playing essential roles during maturation onset (=germinal vesicle breakdown, GVBD) in several kinds of marine invertebrate oocytes. Conversely, other data from the literature, as well as previously unpublished findings for jellyfish oocytes, fail to support the view that calcium signals are required for GVBD. In addition to assessing the effects of calcium transients on GVBD in marine invertebrate oocytes, the ability of maturing oocytes to enhance their calcium-releasing capabilities after GVBD is also reviewed. Furthermore, possible explanations are proposed for the contradictory results that have been obtained regarding calcium signals during oocyte maturation in marine invertebrates.

Evidence for participation of GCS1 in fertilization of the starlet sea anemone Nematostella vectensis: implication of a common mechanism of sperm-egg fusion in plants and animals.

It has been reported that GCS1 (Generative Cell Specific 1) is a transmembrane protein that is exclusively expressed in sperm cells and is essential for gamete fusion in flowering plants. The GCS1 gene is present not only in angiosperms but also in unicellular organisms and animals, implying the occurrence of a common or ancestral mechanism of GCS1-mediated gamete fusion. In order to elucidate the common mechanism, we investigated the role of GCS1 in animal fertilization using a sea anemone (Cnidaria), Nematostella vectensis. Although the existence of the GCS1 gene in N. vectensis has been reported, the expression of GCS1 in sperm and the role of GCS1 in fertilization are not known. In this study, we showed that the GCS1 gene is expressed in the testis and that GCS1 protein exists in sperm by in situ hybridization and proteomic analysis, respectively. Then we made four peptide antibodies against the N-terminal extracellular region of NvGCS1. These antibodies specifically reacted to NvGCS1 among sperm proteins on the basis of Western analysis and potently inhibited fertilization in a concentration-dependent manner. These results indicate that sperm GCS1 plays a pivotal role in fertilization, most probably in sperm-egg fusion, in a starlet sea anemone, suggesting a common gamete-fusion mechanism shared by eukaryotic organisms.

Intracellular calcium signaling in the fertilized eggs of Annelida.

Fertilization is such a universal and indispensable step in sexual reproduction, but a high degree of variability exists in the way it takes place in the animal kingdom. As discussed in other reviews in this issue, recent works on this subject clarified many points. However, important results on the mechanisms of fertilization are obtained mainly from a few restricted model organisms. In this sense, it is utterly important to collect more information from various phyla. In this review, we have re-introduced Annelida as one of the most suitable models for the analysis of fertilization process. We have briefly reviewed the historical works on the fertilization of Annelida. Then, we have described recent findings on the two independent Ca(2+) increases in the fertilized eggs of Annelida, which arise from two different mechanisms and may have distinct physiological roles toward sperm entry and egg activation. We propose that the Ca(2+) increase in the fertilized eggs reflect the specific needs of the zygote in a given species.

Polyspermy block in jellyfish eggs: collaborative controls by Ca(2+) and MAPK.

Jellyfish eggs neither undergo apparent cortical reaction nor show any significant change in the membrane potential at fertilization, but nevertheless show monospermy. Utilizing the perfectly transparent eggs of the hydrozoan jellyfish Cytaeis uchidae, here we show that the polyspermy block is accomplished via a novel mechanism: a collaboration between Ca(2+) and mitogen-activated protein kinase (MAPK). In Cytaeis, adhesion of a sperm to the animal pole surface of an egg was immediately followed by sperm-egg fusion and initiation of an intracellular Ca(2+) rise from this site. The elevated Ca(2+) levels lasted for several minutes following the sperm-egg fusion. The Ca(2+) rise proved to be necessary and sufficient for a polyspermy block, as inhibiting a Ca(2+) rise with EGTA promoted polyspermy, and conversely, triggering a Ca(2+) rise by inositol 1,4,5-trisphosphate (IP3) or excess K(+) immediately abolished the egg's capacity for sperm-egg fusion. A Ca(2+) rise at fertilization or by artificial stimulations evoked dephosphorylation of MAPK in eggs. The eggs in which phosphorylated MAPK was maintained by injection of mRNA for MAPK kinase kinase (Mos), like intact eggs, exhibited a Ca(2+) rise at fertilization or by IP3 injection, and shut down the subsequent sperm-egg fusion. However, the Mos-expressing eggs became capable of accepting sperm following the arrest of Ca(2+) rise. In contrast, addition of inhibitors of MAPK kinase (MEK) to unfertilized eggs caused MAPK dephosphorylation without elevating Ca(2+) levels, and prevented sperm-egg fusion. Rephosphorylation of MAPK by injecting Mos mRNA after fertilization recovered sperm attraction, which is known to be another MAPK-dependent event, but did not permit subsequent sperm-egg fusion. Thus, it is possible that MAPK dephosphorylation irreversibly blocks sperm-egg fusion and reversibly suppresses sperm attraction. Collectively, our data suggest that both the fast and late mechanisms dependent on Ca(2+) and MAPK, respectively, ensure a polyspermy block in jellyfish eggs.

Comparative biology of sperm factors and fertilization-induced calcium signals across the animal kingdom.

Fertilization causes mature oocytes or eggs to increase their concentrations of intracellular calcium ions (Ca²âº) in all animals that have been examined, and such Ca²âº elevations, in turn, provide key activating signals that are required for non-parthenogenetic development. Several lines of evidence indicate that the Ca²âº transients produced during fertilization in mammals and other taxa are triggered by soluble factors that sperm deliver into oocytes after gamete fusion. Thus, for a broad-based analysis of Ca²âº dynamics during fertilization in animals, this article begins by summarizing data on soluble sperm factors in non-mammalian species, and subsequently reviews various topics related to a sperm-specific phospholipase C, called PLCζ, which is believed to be the predominant activator of mammalian oocytes. After characterizing initiation processes that involve sperm factors or alternative triggering mechanisms, the spatiotemporal patterns of Ca²âº signals in fertilized oocytes or eggs are compared in a taxon-by-taxon manner, and broadly classified as either a single major transient or a series of repetitive oscillations. Both solitary and oscillatory types of fertilization-induced Ca²âº signals are typically propagated as global waves that depend on Ca²âº release from the endoplasmic reticulum in response to increased concentrations of inositol 1,4,5-trisphosphate (IP3). Thus, for taxa where relevant data are available, upstream pathways that elevate intraoocytic IP3 levels during fertilization are described, while other less-common modes of producing Ca²âº transients are also examined. In addition, the importance of fertilization-induced Ca²âº signals for activating development is underscored by noting some major downstream effects of these signals in various animals.

Neuropeptides trigger oocyte maturation and subsequent spawning in the hydrozoan jellyfish Cytaeis uchidae.

Oocyte maturation and subsequent spawning in hydrozoan jellyfish are generally triggered by light-dark cycles. To examine if the initiation of the maturation process after light stimulus is mediated by neurotransmitters, neuropeptides isolated originally from Hydra magnipapillata were applied to sexually mature female medusae of the hydrozoan jellyfish Cytaeis uchidae. Among the Hydra neuropeptides tested, Hym-53 (NPYPGLW-NH2 ), as well as a nonphysiological peptide, CGLWamide (CGLW-NH2 ), were most effective in inducing oocyte maturation and spawning. Hym-355 (FPQSFLPRG-NH2 ) also triggered these events, but the stimulatory effect was weaker. Since Hym-53-OH (NPYPGLW) and Hym-355-OH (FPQSFLPRG) had no effect, amidation at the C-terminus may be critical for the stimulatory activities of the peptides. Exposure to Hym-53 for 2 min was sufficient to trigger of oocyte maturation, and the spawned eggs were able to be fertilized and to develop normally. Transmission electron microscopy confirmed that bundles of axon-like structures that contain dense-core synaptic vesicles and microtubules are present in the ovarian ectodermal epithelium overlying the oocytes. In addition, immunohistological analyses revealed that some of the neurons in the ectodermal epithelium are GLWamide- and PRGamide-positive. These results suggest that a neuropeptide signal transduction pathway is involved in mediating the induction of oocyte maturation and spawning in this jellyfish.

Comparative biology of cAMP-induced germinal vesicle breakdown in marine invertebrate oocytes.

During maturation, oocytes must undergo a process of nuclear disassembly, or "germinal vesicle breakdown" (GVBD), that is regulated by signaling pathways involving cyclic AMP (cAMP). In vertebrate and starfish oocytes, cAMP elevation typically prevents GVBD. Alternatively, increased concentrations of intra-oocytic cAMP trigger, rather than inhibit, GVBD in several groups of marine invertebrates. To integrate what is known about the stimulation of GVBD by intra-oocytic cAMP, this article reviews published data for ascidian, bivalve, brittle star, jellyfish, and nemertean oocytes. The bulk of the review concentrates on the three most intensively analyzed groups known to display cAMP-induced GVBD-nemerteans, ascidians, and jellyfish. In addition, this synopsis also presents some previously unpublished findings regarding the stimulatory effects of intra-oocytic cAMP on GVBD in jellyfish and the annelid worm Pseudopotamilla occelata. Finally, factors that may account for the currently known distribution of cAMP-induced GVBD across animal groups are discussed.

Early development and cleavage pattern of the Japanese purple mussel, Septifer virgatus.

Despite easy access to bivalves, few studies have examined the development of these animals, at least in part because most bivalve eggs are very small. In addition, annotating cells of the early bivalve embryo is difficult because few landmarks are present. We conducted detailed cell annotations of the Japanese purple mussel, Septifer virgatus, during early embryogenesis because of its relatively large eggs (ca. 130 microm in diameter). Septifer virgatus underwent the unique cell division profile reported for four other bivalve species, suggesting that the cleavage pattern itself is important for bivalve morphogenesis. The shell field invagination was led by 2d (X) lineage cells, supporting the hypothesis that lineage cells differentiate into cells excreting the shell matrix. The large egg size enabled us to trace cell movements in the early gastrulation phase, during which the invagination of the archenteron was initiated by 4d (M) lineage cells. These observations will serve as a basis for future analyses of S. virgatus embryogenesis and will contribute to understanding the evolution of the molluscan body plan, which is achieved by modification of early embryogenesis.

Novel two-step Ca2+ increase and its mechanisms and functions at fertilization in oocytes of the annelidan worm Pseudopotamilla occelata.

Mature oocytes of the annelidan worm Pseudopotamilla occelata have a wide perivitelline space between the oocyte surface and the vitelline envelope and are arrested at the first metaphase (MI). We found a novel two-step Ca2+ increase in normally fertilized oocytes. The first Ca2+ increase originated at a cortex situated underneath a fertilizing sperm on the vitelline envelope, but failed to propagate beyond the center of the oocyte. The first localized Ca2+ increase was then followed by a larger Ca2+ increase starting from the whole oocyte cortex and spreading inwardly to the center. The first localized Ca2+ increase at fertilization was suppressed by the phospholipase C inhibitor U73122, and a similar Ca2+ change was induced by inositol 1,4,5-trisphosphate (IP3). On the other hand, the second global Ca2+ increase in fertilized oocytes was blocked by removal of external Ca2+ or the voltage-gated Ca2+ channel blocker D-600, and a similar Ca2+ change could be mimicked by addition of excess K+ only when external Ca2+ was present. These results suggest that the first localized Ca2+ increase and the second global Ca2+ increase at fertilization are regulated by Ca2+ release from IP3-sensitive stores and Ca2+ influx via voltage-gated Ca2+ channels, respectively. Our data also demonstrated that the localized Ca2+ increase induces the formation of large cytoplasmic protrusion, which helps the fertilizing sperm to enter the oocyte, whereas the following global Ca2+ increase is a prerequisite for the retraction of the cytoplasmic protrusion and the resumption of meiosis from MI.

Fertilization causes a single Ca2+ increase that fully depends on Ca2+ influx in oocytes of limpets (Phylum Mollusca, Class Gastropoda).

Mature limpet oocytes arrested at the first metaphase (MI) of meiosis are activated by the stimulation of fertilizing sperm. The aim of the present study was to clarify the spatiotemporal property and mechanism of intracellular Ca2+ increase in limpet oocytes, which is a prerequisite signal for initiation of development at fertilization. In all of the five limpet species tested, the initial Ca2+ rising phase just after fertilization took the form of a centripetal Ca2+ wave spreading from the whole cortex to the center (cortical flash), yielding a homogeneous Ca2+ elevation throughout the oocyte. The Ca2+ level remained high during the subsequent plateau phase lasting for several minutes and then returned nearly to the original value. No additional Ca2+ increase followed the plateau phase at least by the time of first cleavage. Both rising and plateau phases of Ca2+ increase at fertilization were inhibited by removal of external Ca2+, suggesting that continuous Ca2+ entry occurs throughout the Ca2+ increase. Injection of inositol 1,4,5-trisphosphate (IP3) was effective in generating a Ca2+ increase in mature limpet oocytes arrested at MI; however, their ability to show an IP3-induced Ca2+ increase was extremely low, as compared with other animals. Responsiveness to IP3 injection in immature oocytes arrested at the first prophase (PI) was similar to that in the mature oocytes, suggesting that the IP3-induced Ca2+ release system does not develop during the process of meiotic maturation in limpet oocytes. Caffeine, cyclic adenosine diphosphate ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP), the agents known to stimulate internal Ca2+ release mechanisms distinct from an IP3-dependent pathway, had no effect on intracellular Ca2+ changes in mature limpet oocytes. Labeling of the endoplasmic reticulum (ER) with DiI revealed that cortical ER clusters are only present in the localized region around meiotic chromosomes in mature oocytes. These data strongly suggest that Ca2+ release and its propagating mechanisms are undeveloped in limpet oocytes and that Ca2+ influx is the only Ca2+-mobilizing system available and functioning at fertilization.