Ryusaku Deguchi

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 (Ca2+) in all animals that have been examined, and such Ca2+ elevations, in turn, provide key activating signals that are required for non‐parthenogenetic development. Several lines of evidence indicate that the Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ signals are typically propagated as global waves that depend on Ca2+ 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 Ca2+ transients are also examined. In addition, the importance of fertilization‐induced Ca2+ signals for activating development is underscored by noting some major downstream effects of these signals in various animals. Mol. Reprod. Dev. 80: 787–815, 2013.

External Ca2+ is predominantly used for cytoplasmic and nuclear Ca2+ increases in fertilized oocytes of the marine bivalve Mactra chinensis.

Oocytes of the marine bivalve Mactra chinensis are spawned and arrested at the germinal vesicle stage (first meiotic prophase) until fertilization, without undergoing a process called oocyte maturation. As is the case of other animals, a fertilized oocyte of the bivalve displays increases in intracellular free Ca2+. We have clarified here the spatiotemporal patterns and sources of the intracellular Ca2+ changes at fertilization. Shortly after insemination, increased Ca2+ simultaneously appeared at the whole cortical region of the oocyte and spread inwardly to the center, attaining the maximal Ca2+ levels throughout the oocyte, including the cytoplasm and nucleus. The initial maximal Ca2+ peak was followed by a submaximal plateau phase of cytoplasmic and nuclear Ca2+ elevations, which persisted for several minutes. The nuclear envelope began to break down shortly before the termination of the plateau phase. These sperm-induced Ca2+ changes were inhibited by suppression of the influx of external Ca2+ from seawater but not by disturbance of the release of internal Ca2+ from inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]-sensitive stores, suggesting that the increased Ca2+ is from an external source. In contrast to the situation observed at fertilization, an oocyte artificially stimulated with serotonin (5-hydroxytryptamine, 5-HT) displayed repetitive Ca2+ transients, each of which started from one cortical region and propagated across the oocyte as a Ca2+ wave. The 5-HT-induced Ca2+ transients persisted even in the absence of external Ca2+. Experiments with caged Ins(1,4,5)P3 revealed that Ca2+ release from Ins(1,4,5)P3-sensitive stores is another pathway that is sufficient to trigger meiosis reinitiation from the first prophase. These results demonstrate that Mactra oocytes can potentially use two different Ca2+-mobilizing pathways: Ca2+ influx producing a centripetal Ca2+ wave from the whole cortex and Ca2+ release from Ins(1,4,5)P3-sensitive stores producing a point-source propagating Ca2+ wave. However, it seems likely that the Ca2+ influx pathway is predominantly activated at fertilization.

Serotonergic Ligands Induce Spawning but not Oocyte Maturation in the Bivalve Mactra chinensis from Central Japan

We examined the spawning sensitivity to serotonin and serotonergic ligands in the Japanese bivalve Mactra chinensis. Spawning was induced by both injected and externally applied scrotonin (5-hydroxytryptamine. 5-HT). The vertebrate 5-HT2 receptor agonist alpha-methyl 5-HT and the selective 5HT1A agonist 8-OH-DPAT were also effective at inducing spawning. However TFMPP (m-trifluoromethylphenylpiperazine, a verterbrate 5-HT1 receptor agonist) and 1-methyl-chlorophenyl biguanide (a vertebrate 5-HT3 agonist) were not effective spawning inducers. The 5-HT-induced spawning was blocked by mianserin (a vertebrate 5-HT2 antagonist). The rank order of potency of the agonists was: 5-HT > alpha-methyl 5-HT > 8-OH-DPAT > TFMPP > 1-methyl-chlorophenyl biguanide; these data support a growing body of literature invoking a mixed 5-HT1/5-HT2 pharmacological profile for serotonin receptors mediating reproductive processes in bivalves. However, neither 5-HT nor 8-OH-DPAT induced germinal vesicle breakdown (GVBD) in Mactra oocytes. Sperm induced GVBD in a high percentage of oocytes. This is the first report of a bivalve in which spawning, but not GVBD, can be induced by 5-HT. This result might be expected because Mactra spawns germinal vesicle oocytes that normally undergo GVBD upon fertilization, but is in contrast to the case of the closely related Spisula spp. in which serotonin induces both processes. The ability of 5-HT to induce spawning but not GVBD makes Mactra chinensis a model organism for studying spawning and meiotic mechanisms in bivalves.

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. Mol. Reprod. Dev. 80: 223–232, 2013.

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. Mol. Reprod. Dev. 78:708–725, 2011.

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.

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 µm 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.

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.

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.

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

ABSTRACT 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. Highlighted Article: W/RPRPamide-related tetrapeptides released from neural-type gonad ectoderm cells in response to light cues cause oocyte maturation and spawning in male and female hydrozoan jellyfish.