RESUMO
Allorecognition-the ability of an organism to discriminate between self and nonself-is crucial to colonial marine animals to avoid invasion by other individuals in the same habitat. The cnidarian hydroid Hydractinia has long been a major research model in studying invertebrate allorecognition, establishing a rich knowledge foundation. In this study, we introduce a new cnidarian model Cladonema radiatum (C. radiatum). C. radiatum is a hydroid jellyfish which also forms polyp colonies interconnected with stolons. Allorecognition responses-fusion or regression of stolons-are observed when stolons encounter each other. By transmission electron microscopy, we observe rapid tissue remodeling contributing to gastrovascular system connection in fusion. Meanwhile, rejection responses are regulated by reconstruction of the chitinous exoskeleton perisarc, and induction of necrotic and autophagic cellular responses at cells in contact with the opponent. Genetic analysis identifies allorecognition genes: six Alr genes located on the putative allorecognition complex and four immunoglobulin superfamily genes on a separate genome region. C. radiatum allorecognition genes show notable conservation with the Hydractinia Alr family. Remarkedly, stolon encounter assays of inbred lines reveal that genotypes of Alr1 solely determine allorecognition outcomes in C. radiatum.
RESUMO
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.
Assuntos
Hidrozoários/fisiologia , Endogamia , Fenótipo , Animais , Cruzamento , Comportamento AlimentarRESUMO
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.
Assuntos
Hidrozoários/crescimento & desenvolvimento , Hidrozoários/fisiologia , Neuropeptídeos/fisiologia , Oócitos/crescimento & desenvolvimento , Oogênese/fisiologia , Sequência de Aminoácidos , Animais , Escuridão , Feminino , Perfilação da Expressão Gênica , Hormônios Esteroides Gonadais/genética , Hormônios Esteroides Gonadais/farmacologia , Hormônios Esteroides Gonadais/fisiologia , Hidrozoários/genética , Luz , Masculino , Neuropeptídeos/genética , Neuropeptídeos/farmacologia , Sistemas Neurossecretores/citologia , Oligopeptídeos/genética , Oligopeptídeos/farmacologia , Oligopeptídeos/fisiologia , Oócitos/efeitos dos fármacos , Oogênese/efeitos dos fármacos , Oogênese/genética , Especificidade da EspécieRESUMO
Over the course of evolution, the acquisition of novel structures has ultimately led to wide variation in morphology among extant multicellular organisms. Thus, the origins of genetic systems for new morphological structures are a subject of great interest in evolutionary biology. The larval skeleton is a novel structure acquired in some echinoderm lineages via the activation of the adult skeletogenic machinery. Previously, VEGF signaling was suggested to have played an important role in the acquisition of the larval skeleton. In the present study, we compared expression patterns of Alx genes among echinoderm classes to further explore the factors involved in the acquisition of a larval skeleton. We found that the alx1 gene, originally described as crucial for sea urchin skeletogenesis, may have also played an essential role in the evolution of the larval skeleton. Unlike those echinoderms that have a larval skeleton, we found that alx1 of starfish was barely expressed in early larvae that have no skeleton. When alx1 overexpression was induced via injection of alx1 mRNA into starfish eggs, the expression patterns of certain genes, including those possibly involved in skeletogenesis, were altered. This suggested that a portion of the skeletogenic program was induced solely by alx1. However, we observed no obvious external phenotype or skeleton. We concluded that alx1 was necessary but not sufficient for the acquisition of the larval skeleton, which, in fact, requires several genetic events. Based on these results, we discuss how the larval expression of alx1 contributed to the acquisition of the larval skeleton in the putative ancestral lineage of echinoderms.
Assuntos
Equinodermos/fisiologia , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/fisiologia , Estrelas-do-Mar/genética , Estrelas-do-Mar/fisiologia , Animais , Evolução Biológica , Equinodermos/genética , Perfilação da Expressão Gênica , Imuno-Histoquímica , Hibridização In Situ , Larva/genética , Larva/fisiologia , Fenótipo , Filogenia , Pepinos-do-Mar/genética , Pepinos-do-Mar/fisiologia , Análise de Sequência de RNA , Strongylocentrotus purpuratus/genética , Strongylocentrotus purpuratus/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Meiotic progression requires the translation of maternal mRNAs in a strict temporal order. In isolated animal oocytes, translation of maternal mRNAs containing a cytoplasmic polyadenylation element (CPE), such as cyclin B, is activated by in vitro stimulation of meiotic resumption which induces phosphorylation of CPEB (CPE-binding protein) and elongation of their polyadenosine (poly(A)) tails; whether or not this model can be applied in vivo to oocytes arrested at metaphase of meiosis I in ovaries is unknown. In this study, we found that active CDK1 (cyclin-dependent kinase 1) phosphorylated CPEB in ovarian oocytes arrested at metphase I in the starfish body cavity, but phosphorylation of CPEB was not sufficient for elongation of cyclin B poly(A) tails. Immediately after spawning, however, mRNA was polyadenylated, suggesting that an increase in intracellular pH (pHi ) upon spawning triggers the elongation of poly(A) tails. Using a cell-free system made from maturing oocytes at metaphase I, we demonstrated that polyadenylation was indeed suppressed at pH below 7.0. These results suggest that a pH-sensitive process, functioning after CPEB phosphorylation, is blocked under physiologically low pHi (<7.0) in metaphase-I-arrested oocytes. The increase in pHi (>7.0) that occurs after spawning triggers polyadenylation of cyclin B mRNA and progression into meiosis II.
Assuntos
Proteína Quinase CDC2/fisiologia , Ciclina B/genética , Metáfase/fisiologia , Oócitos/fisiologia , Poliadenilação , Estrelas-do-Mar , Adenosina/metabolismo , Animais , Ciclina B/metabolismo , Feminino , Concentração de Íons de Hidrogênio , Espaço Intracelular/metabolismo , Estágios do Ciclo de Vida , Meiose/fisiologia , Oócitos/citologia , Oogênese/fisiologia , Poliadenilação/genética , Polímeros/metabolismo , RNA Mensageiro/metabolismo , Estrelas-do-Mar/fisiologiaRESUMO
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.
Assuntos
Cálcio/metabolismo , Fertilização/fisiologia , Hidrozoários/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Óvulo/fisiologia , Interações Espermatozoide-Óvulo/efeitos dos fármacos , Animais , Cálcio/farmacologia , Hidrozoários/metabolismo , Inositol 1,4,5-Trifosfato/farmacologia , Fosforilação , Potássio/farmacologia , Proteínas Proto-Oncogênicas c-mos/genética , RNA Mensageiro/genéticaRESUMO
Maintenance of spindle attachment to the cortex and formation of the cleavage furrow around the protruded spindle are essential for polar body extrusion (PBE) during meiotic maturation of oocytes. Although spindle movement to the cortex has been well-studied, how the spindle is maintained at the cortex during PBE is unknown. Here, we show that activation of Diaphanous-related formin mediated by mitogen-activated protein kinase (MAPK) is required for tight spindle attachment to the cortex and cleavage furrow closure during PBE in starfish (Asterina pectinifera) oocytes. A. pectinifera Diaphanous-related formin (ApDia) had a distinct localization in immature oocytes and was localized to the cleavage furrow during PBE. Inhibition of the Mos-MAPK pathway or the actin nucleating activity of formin homology 2 domain prevented cleavage furrow closure and resulted in PBE failure. In MEK/MAPK-inhibited oocytes, activation of ApDia by relief of its intramolecular inhibition restored PBE. In summary, this study elucidates a link between the Mos-MAPK pathway and Diaphanous-related formins, that is responsible for maintaining tight spindle attachment to the cortex and cleavage furrow closure during PBE.
Assuntos
Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Proteínas do Tecido Nervoso/genética , Proteínas Proto-Oncogênicas c-mos/metabolismo , Fuso Acromático/genética , Animais , Meiose , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Oócitos/citologia , Oócitos/metabolismo , Fosforilação , Corpos Polares/citologia , Corpos Polares/metabolismo , Proteínas Proto-Oncogênicas c-mos/genética , Estrelas-do-MarRESUMO
The evolution of the echinoderm larval skeleton was examined from the aspect of interactions between skeletogenic mesenchyme cells and surrounding epithelium. We focused on vascular endothelial growth factor (VEGF) signaling, which was reported to be essential for skeletogenesis in sea urchin larvae. Here, we examined the expression patterns of vegf and vegfr in starfish and brittle stars. During starfish embryogenesis, no expression of either vegfr or vegf was detected, which contrast with previous reports on the expression of starfish homologs of sea urchin skeletogenic genes, including Ets, Tbr, and Dri. In later stages, when adult skeletogenesis commenced, vegfr and vegf expression were upregulated in skeletogenic cells and in the adjacent epidermis, respectively. These expression patterns suggest that heterochronic activation of VEGF signaling is one of the key molecular evolutionary steps in the evolution of the larval skeleton. The absence of vegf or vegfr expression during early embryogenesis in starfish suggests that the evolution of the larval skeleton requires distinct evolutionary changes, both in mesoderm cells (activation of vegfr expression) and in epidermal cells (activation of vegf expression). In brittle stars, which have well-organized skeletons like the sea urchin, vegfr and vegf were expressed in the skeletogenic mesenchyme and the overlying epidermis, respectively, in the same manner as in sea urchins. Therefore, the distinct activation of vegfr and vegf may have occurred in two lineages, sea urchins and brittle stars.
Assuntos
Evolução Biológica , Equinodermos/crescimento & desenvolvimento , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Asterina/embriologia , Asterina/crescimento & desenvolvimento , Asterina/metabolismo , Equinodermos/embriologia , Equinodermos/metabolismo , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Epitélio/embriologia , Epitélio/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Mesoderma/embriologia , Mesoderma/metabolismo , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas Proto-Oncogênicas c-ets/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Transdução de Sinais , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Transcrição Gênica , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Intracellular calcium ion concentration ([Ca(2+)](i)) transients are observed in the fertilized eggs of all species investigated so far, and are critical for initiating several events related to egg activation and cell cycle control. Here, we investigated the role of the Mos/MEK/ERK cascade and Cdk1 on Ca(2+) oscillations in fertilized ascidian eggs. The egg of the ascidian Phallusia nigra shows [Ca(2+)](i) oscillations after fertilization: Ca(2+) waves immediately following fertilization (phase I), and [Ca(2+)](i) oscillations between the first and second polar body extrusions (phase II). Our results show that in P. nigra eggs, ERK activity peaked just before the extrusion of the first polar body, and decreased gradually, eventually disappearing at the extrusion of the second polar body. Cyclin-dependent protein kinase 1(Cdk1) activity decreased to undetectable levels immediately after fertilization, and then periodically increased according to the meiotic and mitotic cell cycle. When the unfertilized eggs were incubated with U0126, an inhibitor of MEK, before insemination, ERK was immediately inactivated, and the phase II [Ca(2+)](i) oscillations disappeared. Alternatively, when the constitutively active Mos protein (GST-Mos) was injected into the unfertilized eggs, ERK activity was preserved for at least 120 min after fertilization, and the phase II [Ca(2+)](i) oscillations lasted for more than 120 min after the second polar body extrusion. These results suggest that ERK activity is necessary for maintaining [Ca(2+)](i) oscillations. GST-ΔN85-cyclin, which maintains Cdk1 activity, caused ERK activity in the eggs to persist for over 120 min after fertilization, and prolonged [Ca(2+)](i) oscillations. Moreover, the effects of GST-ΔN85-cyclin on the egg were abrogated by the application of U0126. Thus, Cdk1-mediated [Ca(2+)](i) oscillations seem to require ERK activity. However, GST-Mos triggered [Ca(2+)](i) oscillations after the second polar body extrusion, whereas GST-ΔN85-cyclin did not, although it prolongs the duration of [Ca(2+)](i) oscillations. Interestingly, GST-ΔN85-cyclin increased the frequency of [Ca(2+)](i) transients in the Mos-induced [Ca(2+)](i) oscillations after the extrusion of the second polar body. Thus, Cdk1 could maintain, but not activate, ERK and [Ca(2+)](i) oscillations. ERK activity and [Ca(2+)](i) oscillations seem to form a negative feedback loop which may be responsible for maintaining the meiotic period.
Assuntos
Urocordados/citologia , Urocordados/metabolismo , Zigoto/metabolismo , Animais , Butadienos/farmacologia , Proteína Quinase CDC2/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Ciclina B/metabolismo , Retroalimentação Fisiológica , Feminino , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Meiose , Nitrilas/farmacologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-mos/metabolismo , Urocordados/efeitos dos fármacos , Zigoto/efeitos dos fármacosRESUMO
Initiation of DNA replication in eukaryotic cells is controlled through an ordered assembly of protein complexes at replication origins. The molecules involved in this process are well conserved but diversely regulated. Typically, initiation of DNA replication is regulated in response to developmental events in multicellular organisms. Here, we elucidate the regulation of the first S phase of the embryonic cell cycle after fertilization. Unless fertilization occurs, the Mos-MAPK-p90Rsk pathway causes the G1-phase arrest after completion of meiosis in starfish eggs. Fertilization shuts down this pathway, leading to the first S phase with no requirement of new protein synthesis. However, how and in which stage the initiation complex for DNA replication is arrested by p90Rsk remains unclear. We find that in G1-arrested eggs, chromatin is loaded with the Mcm complex to form the prereplicative complex (pre-RC). Inactivation of p90Rsk is necessary and sufficient for further loading of Cdc45 onto chromatin to form the preinitiation complex (pre-IC) and the subsequent initiation of DNA replication. However, cyclin A-, B-, and E-Cdk's activity and Cdc7 accumulation are dispensable for these processes. These observations define the stage of G1 arrest in unfertilized eggs at transition point from pre-RC to pre-IC, and reveal a unique role of p90Rsk for a negative regulator of this transition. Thus, initiation of DNA replication in the meiosis-to-mitosis transition is regulated at the pre-RC stage as like in the G1 checkpoint, but in a manner different from the checkpoint.
Assuntos
Replicação do DNA , Fertilização/fisiologia , Óvulo/enzimologia , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Estrelas-do-Mar/citologia , Estrelas-do-Mar/enzimologia , Animais , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Ativação Enzimática , Feminino , Fase G1 , Meiose , Dados de Sequência Molecular , Óvulo/citologia , Origem de ReplicaçãoRESUMO
A key event in the oocyte-to-embryo transition is the start of the embryonic mitotic cell cycle. Prior to this start, the cell cycle in oocytes is generally arrested at a particular stage during meiosis, and the meiotic arrest is released by fertilization. However, it remains unclear how release from the meiotic arrest is implicated in the start of the embryonic cell cycle. To elucidate this link, we have used starfish eggs, in which G1 phase arrest occurs after completion of meiosis if the mature oocytes are not fertilized, and fertilization simply directs the start of the embryonic cell cycle. The starfish G1 arrest is known to rely on the Mos-MAPK-Rsk (p90 ribosomal S6 kinase) pathway, and inactivation of Rsk induces S phase in the absence of fertilization. However, here we show that this S phase is not followed by M phase when MAPK remains active, owing to poly(A)-independent repression of cyclin A and B synthesis. By contrast, inactivation of MAPK alone induces M phase, even when S phase is inhibited by constitutively active Rsk. Thus, there is a divergence of separate pathways downstream of MAPK that together block the start of the embryonic mitotic cycle. One is the previously known Rsk-dependent pathway that prevents S phase, and the other is a novel pathway that is not mediated by Rsk and that leads to prevention of the first mitotic M phase through suppression of protein synthesis of M phase cyclins. Release from such a 'dual-lock' by fertilization results in the start of the embryonic cell cycle.
Assuntos
Ciclo Celular/fisiologia , Óvulo/citologia , Estrelas-do-Mar/embriologia , Animais , Ciclina A/metabolismo , Ciclina B/metabolismo , Fase G1/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Mitose/fisiologia , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Fase S/fisiologia , Transdução de Sinais/fisiologia , Estrelas-do-Mar/citologiaRESUMO
Extensive survey of meiotic metaphase II arrest during oocyte maturation in vertebrates revealed that the mitogen-activated protein kinase (MAPK) pathway regulated by the c-mos proto-oncogene product, Mos, has an essential role in cytostatic activity, termed cytostatic factor (CSF). In contrast, little is known in invertebrates in which meiotic arrest occurs in most cases at metaphase I (MI arrest). A parthenogenetic insect, the sawfly Athalia rosae, in which artificial egg activation is practicable, has advantages to investigate the mechanisms of MI arrest. Both the MAPK/extracellular signal-regulated protein kinase kinase (MEK) and MAPK were phosphorylated and maintained active in MI-arrested sawfly eggs, whereas they were dephosphorylated soon after egg activation. Treatment of MI-arrested eggs with U0126, an inhibitor of MEK, resulted in dephosphorylation of MAPK and MI arrest was resumed. The sawfly c-mos gene orthologue encoding a serine/threonine kinase was cloned and analyzed. It was expressed in nurse cells in the ovaries. To examine CSF activity of the sawfly Mos, synthesized glutathione S-transferase (GST)-fusion sawfly Mos protein was injected into MI-resumed eggs in which MEK and MAPK were dephosphorylated. Both MEK and MAPK were phosphorylated again upon injection. In these GST-fusion sawfly Mos-injected eggs subsequent mitotic (syncytial) divisions were blocked and embryonic development was ceased. These results demonstrated that the MEK-MAPK pathway was involved in maintaining CSF arrest in sawfly eggs and Mos functioned as its upstream regulatory molecule.
Assuntos
Insetos/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas Proto-Oncogênicas c-mos/metabolismo , Sequência de Aminoácidos , Animais , Abelhas , Ciclo Celular , Inibidores Enzimáticos/farmacologia , Feminino , Dados de Sequência Molecular , Oócitos/metabolismo , Fosforilação , Homologia de Sequência de Aminoácidos , Xenopus laevisRESUMO
In sexual reproduction, the union of the male and female pronuclei occurs in fertilized eggs to mix genetic materials derived from both parents, thereby creating a new genome for the next generation [1-4]. The process leading to pronuclear union consists of pronuclear congression, which depends on astral microtubules derived from sperm centrosome [5-8], and the subsequent pronuclear fusion or karyogamy. The union process progresses in parallel with the first embryonic cell cycle, but the molecular mechanisms involved are poorly understood. Here, we devise a labeling method with Dendra2 to track both pronuclei individually in living starfish eggs. Although pronuclear union naturally proceeds while G1 arrest is released by fertilization and S phase progresses [9], we show that the cell-cycle resumption and progression are not prerequisites for pronuclear union. However, low levels of cyclin B- (but not cyclin A-) Cdk1 activity are detectable even in interphase, and are indispensable for pronuclear union, by contributing at least to pronuclear congression through formation of sperm aster. Pronuclear congression thus requires the activity of M-phase cell-cycle regulator in interphase, independently of the cell-cycle regulation. These findings not only provide a clue to the regulatory aspect of creation of new genome with fertilization, but also reveal a novel role for the M-phase Cdk1 during interphase.
Assuntos
Proteína Quinase CDC2/fisiologia , Núcleo Celular/enzimologia , Ciclina B/metabolismo , Interfase/fisiologia , Estrelas-do-Mar/citologia , Animais , Proteína Quinase CDC2/metabolismo , Núcleo Celular/ultraestrutura , Feminino , Masculino , Óvulo/citologia , Óvulo/enzimologia , Óvulo/ultraestrutura , Espermatozoides/citologia , Espermatozoides/enzimologia , Espermatozoides/ultraestrutura , Estrelas-do-Mar/enzimologiaRESUMO
The cell cycle in oocytes generally arrests at a particular meiotic stage to await fertilization. This arrest occurs at metaphase of meiosis II (meta-II) in frog and mouse, and at G1 phase after completion of meiosis II in starfish. Despite this difference in the arrest phase, both arrests depend on the same Mos-MAPK (mitogen-activated protein kinase) pathway, indicating that the difference relies on particular downstream effectors. Immediately downstream of MAPK, Rsk (p90 ribosomal S6 kinase, p90(Rsk)) is required for the frog meta-II arrest. However, the mouse meta-II arrest challenges this requirement, and no downstream effector has been identified in the starfish G1 arrest. To investigate the downstream effector of MAPK in the starfish G1 arrest, we used a neutralizing antibody against Rsk and a constitutively active form of Rsk. Rsk was activated downstream of the Mos-MAPK pathway during meiosis. In G1 eggs, inhibition of Rsk activity released the arrest and initiated DNA replication without fertilization. Conversely, maintenance of Rsk activity prevented DNA replication following fertilization. In early embryos, injection of Mos activated the MAPK-Rsk pathway, resulting in G1 arrest. Moreover, inhibition of Rsk activity during meiosis I led to parthenogenetic activation without meiosis II. We conclude that immediately downstream of MAPK, Rsk is necessary and sufficient for the starfish G1 arrest. Although CSF (cytostatic factor) was originally defined for meta-II arrest in frog eggs, we propose to distinguish ;G1-CSF' for starfish from ;meta-II-CSF' for frog and mouse. The present study thus reveals a novel role of Rsk for G1-CSF.
Assuntos
Asterina/fisiologia , Fase G1 , Óvulo/enzimologia , Óvulo/fisiologia , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Animais , Blastômeros/citologia , Blastômeros/enzimologia , Ativação Enzimática , Feminino , Glutationa Transferase/metabolismo , Histidina/química , Cinética , Meiose , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Óvulo/citologia , Partenogênese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/análise , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 90-kDa/química , Proteínas Quinases S6 Ribossômicas 90-kDa/genéticaRESUMO
Naturally spawned eggs of the hydrozoan jellyfish Cladonema pacificum are arrested at G1-like pronuclear stage until fertilization. Fertilized eggs of Cladonema undergo a series of post-fertilization events, including loss of sperm-attracting ability, expression of adhesive materials on the egg surface, and initiation of cell cycle leading to DNA synthesis and cleavage. Here, we investigate whether these events are regulated by changes in intracellular Ca2+ concentration and mitogen-activated protein kinase (MAP kinase) activity in Cladonema eggs. We found that MAP kinase is maintained in the phosphorylated form in unfertilized eggs. Initiation of sperm-induced Ca2+ increase, which is the first sign of fertilization, was immediately followed by MAP kinase dephosphorylation within a few minutes of fertilization. The fertilized eggs typically stopped sperm attraction by an additional 5 min and became sticky around this time. They further underwent cytokinesis yielding 2-cell embryos at approximately 1 h post-fertilization, which was preceded by DNA synthesis evidenced by BrdU incorporation into the nuclei. Injection of inositol 1,4,5-trisphosphate (IP3) into unfertilized eggs, which produced a Ca2+ increase similar to that seen at fertilization, triggered MAP kinase dephosphorylation and the above post-fertilization events without insemination. Conversely, injection of BAPTA/Ca2+ into fertilized eggs at approximately 10 s after the initiation of Ca2+ increase immediately lowered the elevating Ca2+ level and inhibited the subsequent post-fertilization events. Treatment with U0126, an inhibitor of MAP kinase kinase (MEK), triggered the post-fertilization events in unfertilized eggs, where MAP kinase dephosphorylation but not Ca2+ increase was generated. Conversely, preinjection of the glutathione S-transferase (GST) fusion protein of MAP kinase kinase kinase (Mos), which maintained the phosphorylated state of MAP kinase, blocked the post-fertilization events in fertilized eggs without preventing a Ca2+ increase. These results strongly suggest that all of the three post-fertilization events, cessation of sperm attraction, expression of surface adhesion, and progression of cell cycle, lie downstream of MAP kinase dephosphorylation that is triggered by a Ca2+ increase.
Assuntos
Cálcio/fisiologia , Fertilização/fisiologia , Hidrozoários/enzimologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Óvulo/enzimologia , Animais , Butadienos/farmacologia , Adesão Celular/fisiologia , Ciclo Celular/fisiologia , Quelantes/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Inositol 1,4,5-Trifosfato/fisiologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Nitrilas/farmacologia , Óvulo/efeitos dos fármacos , FosforilaçãoRESUMO
Meiotic resumption is generally under the control of an extracellular maturation-inducing hormone. It is equivalent to the G2-M phase transition in somatic cell mitosis and is regulated by cyclin B-Cdc2 kinase. However, the complete signaling pathway from the hormone to cyclin B-Cdc2 is yet unclear in any organism. A model system to analyze meiotic resumption is the starfish oocyte, in which Akt/protein kinase B (PKB) plays a key mediator in hormonal signaling that leads to cyclin B-Cdc2 activation. Here we show in starfish oocytes that when PDK1 activity is inhibited by a neutralizing antibody, maturation-inducing hormone fails to induce cyclin B-Cdc2 activation at the meiotic G2-M phase transition, even though PDK2 activity becomes detectable. These observations assign a novel role to PDK1 for a hormonal signaling intermediate toward meiotic resumption. They further support that PDK2 is a molecule distinct from PDK1 and Akt, and that PDK2 activity is not sufficient for the full activation of Akt in the absence of PDK1 activity.
Assuntos
Substâncias de Crescimento/metabolismo , Meiose/fisiologia , Oócitos/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Estrelas-do-Mar , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Sequência de Aminoácidos , Animais , Anticorpos/metabolismo , Proteína Quinase CDC2/metabolismo , Ciclina B/metabolismo , Ativação Enzimática , Dados de Sequência Molecular , Oócitos/citologia , Fosforilação , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Alinhamento de SequênciaRESUMO
The Polo-like kinase, Plk, has multiple roles in regulating mitosis. In particular, Plk1 has been postulated to function as a trigger kinase that phosphorylates and activates Cdc25C prior to the activation of cyclin B-Cdc2 and thereby initiates its activation. However, the upstream regulation of Plk1 activation remains unclear. Here we have studied the interplay between Plk1 and Cdc2 through meiotic and early embryonic cycles in starfish. Distinct kinases, cyclin B-Cdc2, MAPK along with cyclin B- and/or cyclin A-Cdc2 and cyclin A-Cdc2, were unique upstream regulators for Plk1 activation at meiosis I, meiosis II and embryonic M-phase, respectively, indicating that Plk1 is not the trigger kinase at meiotic reinitiation. When Plk1 was required for cyclin B-Cdc2 activation, the action of Plk1 was mediated primarily through suppression of Myt1 rather than through activation of Cdc25. We propose that Plk1 can be activated by either cyclin A- or cyclin B-Cdc2, and its primary target is Myt1.
Assuntos
Embrião não Mamífero/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Quinases/genética , Estrelas-do-Mar/embriologia , Animais , Proteína Quinase CDC2/fisiologia , Proteínas de Ciclo Celular , Divisão Celular , Ciclina B/metabolismo , DNA Complementar/genética , Embrião não Mamífero/citologia , Meiose , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas , Estrelas-do-Mar/citologia , Quinase 1 Polo-LikeRESUMO
In eukaryotes, entry into M-phase of the cell cycle is induced by activation of cyclin B-Cdc2 kinase. At G2-phase, the activity of its inactivator, a member of the Wee1 family of protein kinases, exceeds that of its activator, Cdc25C phosphatase. However, at M-phase entry the situation is reversed, such that the activity of Cdc25C exceeds that of the Wee1 family. The mechanism of this reversal is unclear. Here we show that in oocytes from the starfish Asterina pectinifera, the kinase Akt (or protein kinase B (PKB)) phosphorylates and downregulates Myt1, a member of the Wee1 family. This switches the balance of regulator activities and causes the initial activation of cyclin B-Cdc2 at the meiotic G2/M-phase transition. These findings identify Myt1 as a new target of Akt, and demonstrate that Akt functions as an M-phase initiator.