RESUMO
Cyclins associate with cyclin-dependent serine/threonine kinase 1 (CDK1) to generate the M phase-promoting factor (MPF) activity essential for progression through mitosis and meiosis. Although cyclin B1 (CCNB1) is required for embryo development, previous studies concluded that CCNB2 is dispensable for cell cycle progression. Given previous findings of high Ccnb2 mRNA translation rates in prophase-arrested oocytes, we re-evaluated the role of this cyclin during meiosis. Ccnb2-/- oocytes underwent delayed germinal vesicle breakdown and showed defects during the metaphase-to-anaphase transition. This defective maturation was associated with compromised Ccnb1 and Moloney sarcoma oncogene (Mos) mRNA translation, delayed spindle assembly and increased errors in chromosome segregation. Given these defects, a significant percentage of oocytes failed to complete meiosis I because the spindle assembly checkpoint remained active and anaphase-promoting complex/cyclosome function was inhibited. In vivo, CCNB2 depletion caused ovulation of immature oocytes, premature ovarian failure, and compromised female fecundity. These findings demonstrate that CCNB2 is required to assemble sufficient pre-MPF for timely meiosis re-entry and progression. Although endogenous cyclins cannot compensate, overexpression of CCNB1/2 rescues the meiotic phenotypes, indicating similar molecular properties but divergent modes of regulation of these cyclins.
Assuntos
Ciclina B2/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Animais , Ciclina B1/genética , Ciclina B1/metabolismo , Ciclina B2/genética , Feminino , Masculino , Meiose/genética , Meiose/fisiologia , Mesotelina , Camundongos , Camundongos Mutantes , Proteínas Proto-Oncogênicas c-mos/genética , Proteínas Proto-Oncogênicas c-mos/metabolismo , RNA Mensageiro/metabolismoRESUMO
The Moloney sarcoma oncogene (MOS) encodes a protein serine/threonine kinase and MOS is expressed at high levels in oocytes undergoing meiotic maturation. The MOS/MAPK pathway is normally required for the maintenance of microtubules and chromatin in a metaphasic state during the meiotic divisions. To determine the pathogenic genes in a female infertile patient due to large polar body oocytes, whole-exome sequencing was performed on the patient and available family members. We identified a novel homozygous missense mutation c.591T > G in MOS. Bioinformatics analysis showed that the mutation is harmful. These findings suggest that MOS mutation results in oocytes with a large polar body and poor embryonic development in patients. The MOS variant may regulate oocyte asymmetric division by MAPK/WAVE2/Arp2/3/actin signaling pathway. This will help to understand the comprehensive role of MOS in early human reproductive process and provide genetic markers for future genetic counseling for more individualized treatments.
Assuntos
Infertilidade Feminina , Sarcoma , Humanos , Feminino , Corpos Polares , Meiose , Infertilidade Feminina/metabolismo , Proteínas Proto-Oncogênicas c-mos/genética , Proteínas Proto-Oncogênicas c-mos/metabolismo , Oócitos/fisiologia , Mutação , Sarcoma/metabolismoRESUMO
The objective of this study was to determine the effects of TNF-α and IL-1ß on development and survival of bovine secondary follicle culture in vitro for 18 days. Secondary follicles (~0.2 mm) were isolated from ovarian cortex and individually cultured at 38.5°C, with 5% CO2 in air, for 18 days, in TCM-199+ alone (cultured control) or supplemented with 10 ng/ml IL-1ß, 10 ng/ml TNF-α or both TNF-α and IL-1ß. The effects of these treatments on growth, follicular survival, antrum formation, viability, ultrastructure and mRNA levels for GDF-9, c-MOS, H1foo and Cyclin B1 were evaluated. The results showed that addition of TNF-α to culture medium increased follicular diameter and rate of antrum formation, whereas that of IL-1ß and a mixture of IL-1ß and TNF-α did not do so. Ultrastructural analysis showed that, among the tested cytokine treatments, follicles cultured in the presence of TNF-α had the best-preserved oocytes and granulosa cells. The presence of TNF-α, IL-1ß or both did not influence the expression of mRNAs analysed. In conclusion, in contrast to IL-1ß, TNF-α promotes growth of and antrum formation in in vitro cultured bovine secondary follicles, while their ultrastructure and viability were maintained.
Assuntos
Bovinos/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-1beta/farmacologia , Folículo Ovariano/crescimento & desenvolvimento , Fator de Necrose Tumoral alfa/farmacologia , Animais , Ciclina B1/genética , Ciclina B1/metabolismo , Feminino , Fator 9 de Diferenciação de Crescimento/genética , Fator 9 de Diferenciação de Crescimento/metabolismo , Histonas/genética , Histonas/metabolismo , Interleucina-1beta/administração & dosagem , Folículo Ovariano/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mos/genética , Proteínas Proto-Oncogênicas c-mos/metabolismo , Técnicas de Cultura de Tecidos/veterinária , Fator de Necrose Tumoral alfa/administração & dosagemRESUMO
Identification of genetic variants associated with feet and legs disorders (FLD) will aid in the genetic improvement of these traits by providing knowledge on genes that influence trait variations. In Denmark, FLD in cattle has been recorded since the 1990s. In this report, we used deregressed breeding values as response variables for a genome-wide association study. Bulls (5,334 Danish Holstein, 4,237 Nordic Red Dairy Cattle, and 1,180 Danish Jersey) with deregressed estimated breeding values were genotyped with the Illumina Bovine 54k single nucleotide polymorphism (SNP) genotyping array. Genotypes were imputed to whole-genome sequence variants, and then 22,751,039 SNP on 29 autosomes were used for an association analysis. A modified linear mixed-model approach (efficient mixed-model association eXpedited, EMMAX) and a linear mixed model were used for association analysis. We identified 5 (3,854 SNP), 3 (13,642 SNP), and 0 quantitative trait locus (QTL) regions associated with the FLD index in Danish Holstein, Nordic Red Dairy Cattle, and Danish Jersey populations, respectively. We did not identify any QTL that were common among the 3 breeds. In a meta-analysis of the 3 breeds, 4 QTL regions were significant, but no additional QTL region was identified compared with within-breed analyses. Comparison between top SNP locations within these QTL regions and known genes suggested that RASGRP1, LCORL, MOS, and MITF may be candidate genes for FLD in dairy cattle.
Assuntos
Doenças dos Bovinos/genética , Bovinos/genética , Doenças do Pé/genética , Pé/anatomia & histologia , Polimorfismo de Nucleotídeo Único , Animais , Cruzamento , Dinamarca , Feminino , Doenças do Pé/veterinária , Estudo de Associação Genômica Ampla , Técnicas de Genotipagem , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Modelos Lineares , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Modelos Biológicos , Fenótipo , Proteínas Proto-Oncogênicas c-mos/genética , Proteínas Proto-Oncogênicas c-mos/metabolismo , Locos de Características QuantitativasRESUMO
Mammalian oocytes are arrested in metaphase of second meiosis (MII) until fertilization. This arrest is enforced by the cytostatic factor (CSF), which maintains the M-phase promoting factor (MPF) in a highly active state. Although the continuous synthesis and degradation of cyclin B to maintain the CSF-mediated MII arrest is well established, it is unknown whether cyclin-dependent kinase 1 (Cdk1) phosphorylations are involved in this arrest in mouse oocytes. Here, we show that a dynamic equilibrium of Cdk1 phosphorylation is required to maintain MII arrest. When the Cdc25A phosphatase is downregulated, mouse oocytes are released from MII arrest and MPF becomes inactivated. This inactivation occurs in the absence of cyclin B degradation and is dependent on Wee1B-mediated phosphorylation of Cdk1. Thus, our data demonstrate that Cdk1 activity is maintained during MII arrest not only by cyclin turnover but also by steady state phosphorylation.
Assuntos
Metáfase/fisiologia , Oócitos/citologia , Fosfatases cdc25/metabolismo , Células Cultivadas , Humanos , Immunoblotting , Fator Promotor de Maturação/metabolismo , Meiose/genética , Meiose/fisiologia , Mesotelina , Metáfase/genética , Oócitos/metabolismo , Proteínas Proto-Oncogênicas c-mos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fosfatases cdc25/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
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
The oocytes of vertebrates are typically arrested at metaphase II (mII) by the cytostatic factor Emi2 until fertilization. Regulatory mechanisms in Xenopus Emi2 (xEmi2) are understood in detail but contrastingly little is known about the corresponding mechanisms in mammals. Here, we analyze Emi2 and its regulatory neighbours at the molecular level in intact mouse oocytes. Emi2, but not xEmi2, exhibited nuclear targeting. Unlike xEmi2, separable N- and C-terminal domains of mouse Emi2 modulated metaphase establishment and maintenance, respectively, through indirect and direct mechanisms. The C-terminal activity was mapped to the potential phosphorylation target Tx(5)SxS, a destruction box (D-box), a lattice of Zn(2+)-coordinating residues and an RL domain. The minimal region of Emi2 required for its cytostatic activity was mapped to a region containing these motifs, from residue 491 to the C terminus. The cytostatic factor Mos-MAPK promoted Emi2-dependent metaphase establishment, but Mos autonomously disappeared from meiotically competent mII oocytes. The N-terminal Plx1-interacting phosphodegron of xEmi2 was apparently shifted to within a minimal fragment (residues 51-300) of mouse Emi2 that also contained a calmodulin kinase II (CaMKII) phosphorylation motif and which was efficiently degraded during mII exit. Two equimolar CaMKII gamma isoform variants were present in mII oocytes, neither of which phosphorylated Emi2 in vitro, consistent with the involvement of additional factors. No evidence was found that calcineurin is required for mouse mII exit. These data support a model in which mammalian meiotic establishment, maintenance and exit converge upon a modular Emi2 hub via evolutionarily conserved and divergent mechanisms.
Assuntos
Proteínas F-Box/metabolismo , Metáfase , Sequência de Aminoácidos , Animais , Proteínas F-Box/química , Proteínas F-Box/genética , Feminino , Masculino , Camundongos , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas c-mos/genética , Proteínas Proto-Oncogênicas c-mos/metabolismo , Alinhamento de Sequência , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMO
Until fertilization, the meiotic cell cycle of vertebrate eggs is arrested at metaphase of meiosis II by a cytoplasmic activity termed cytostatic factor (CSF), which causes inhibition of the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that targets mitotic cyclins-regulatory proteins of meiosis and mitosis-for degradation. Recent studies indicate that Erp1/Emi2, an inhibitor protein for the APC/C, has an essential role in establishing and maintaining CSF arrest, but its relationship to Mos, a mitogen-activated protein kinase (MAPK) kinase kinase that also has an essential role in establishing CSF arrest through activation of p90 ribosomal S6 kinase (p90rsk), is unclear. Here we report that in Xenopus eggs Erp1 is a substrate of p90rsk, and that Mos-dependent phosphorylation of Erp1 by p90rsk at Thr 336, Ser 342 and Ser 344 is crucial for both stabilizing Erp1 and establishing CSF arrest in meiosis II oocytes. Semi-quantitative analysis with CSF-arrested egg extracts reveals that the Mos-dependent phosphorylation of Erp1 enhances, but does not generate, the activity of Erp1 that maintains metaphase arrest. Our results also suggest that Erp1 inhibits cyclin B degradation by binding the APC/C at its carboxy-terminal destruction box, and this binding is also enhanced by the Mos-dependent phosphorylation. Thus, Mos and Erp1 collaboratively establish and maintain metaphase II arrest in Xenopus eggs. The link between Mos and Erp1 provides a molecular explanation for the integral mechanism of CSF arrest in unfertilized vertebrate eggs.
Assuntos
Proteínas F-Box/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Proteínas Proto-Oncogênicas c-mos/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis , Ciclossomo-Complexo Promotor de Anáfase , Animais , Proteínas F-Box/química , Meiose , Metáfase , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oócitos/enzimologia , Fosforilação/efeitos dos fármacos , Complexos Ubiquitina-Proteína Ligase/metabolismo , Proteínas de Xenopus/química , Xenopus laevis/embriologiaRESUMO
In vertebrates, unfertilized eggs (or mature oocytes) are arrested at metaphase of meiosis II by a cytoplasmic activity called cytostatic factor (CSF). The classical Mos-MAPK pathway has long been implicated in CSF arrest of vertebrate eggs, but exactly how it exerts CSF activity remains unclear. Recently, Erp1 (also called Emi2), an inhibitor of the anaphase-promoting complex/cyclosome (APC/C) required for degradation of the mitotic regulator cyclin B (ref. 5), has also been shown to be a component of CSF in both Xenopus and mice. Erp1 is destroyed on fertilization or egg activation, like Mos. However, despite these similarities the Mos-MAPK (mitogen-activated protein kinase) pathway and Erp1 are thought to act rather independently in CSF arrest. Here, we show that p90rsk, the kinase immediately downstream from Mos-MAPK, directly targets Erp1 for CSF arrest in Xenopus oocytes. Erp1 is synthesized immediately after meiosis I, and the Mos-MAPK pathway or p90rsk is essential for CSF arrest by Erp1. p90rsk can directly phosphorylate Erp1 on Ser 335/Thr 336 both in vivo and in vitro, and upregulates both Erp1 stability and activity. Erp1 is also present in early embryos, but has little CSF activity owing, at least in part, to the absence of p90rsk activity. These results clarify the direct link of the classical Mos-MAPK pathway to Erp1 in meiotic arrest of vertebrate oocytes.
Assuntos
Proteínas F-Box/metabolismo , Sistema de Sinalização das MAP Quinases , Meiose , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oócitos/citologia , Proteínas Proto-Oncogênicas c-mos/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis , Animais , Proteínas F-Box/química , Proteínas F-Box/genética , Oócitos/enzimologia , Oócitos/metabolismo , Fosforilação , Fosfosserina/metabolismo , Fosfotreonina/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Proteínas de Xenopus/química , Proteínas de Xenopus/genéticaRESUMO
Meiosis in mammalian females is marked by two arrest points, at prophase I and metaphase II, which must be tightly regulated in order to produce a haploid gamete at the time of fertilization. The transition metal zinc has emerged as a necessary and dynamic regulator of the establishment, maintenance, and exit from metaphase II arrest, but the roles of zinc during prophase I arrest are largely unknown. In this study, we investigate the mechanisms of zinc regulation during the first meiotic arrest. Disrupting zinc availability in the prophase I arrested oocyte by treatment with the heavy metal chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) causes meiotic resumption even in the presence of pharmacological inhibitors of meiosis. We further show that the MOS-MAPK pathway mediates zinc-dependent prophase I arrest, as the pathway prematurely activates during TPEN-induced meiotic resumption. Conversely, inhibition of the MOS-MAPK pathway maintains prophase I arrest. While prolonged zinc insufficiency ultimately results in telophase I arrest, early and transient exposure of oocytes to TPEN is sufficient to induce meiotic resumption and bypass the telophase I block, allowing the formation of developmentally competent eggs upon parthenogenetic activation. These results establish zinc as a crucial regulator of meiosis throughout the entirety of oocyte maturation, including the maintenance of and release from the first and second meiotic arrest points.
Assuntos
Prófase Meiótica I/fisiologia , Oócitos/citologia , Oócitos/metabolismo , Zinco/metabolismo , Animais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/fisiologia , Quelantes/farmacologia , Etilenodiaminas/farmacologia , Feminino , Técnicas In Vitro , Sistema de Sinalização das MAP Quinases , Prófase Meiótica I/efeitos dos fármacos , Camundongos , Oócitos/efeitos dos fármacos , Oogênese/efeitos dos fármacos , Oogênese/fisiologia , Partenogênese , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Proto-Oncogênicas c-mos/metabolismo , Telófase/efeitos dos fármacos , Telófase/fisiologia , Zinco/deficiênciaRESUMO
Zinc is essential for many biological processes, including proper functioning of gametes. We recently reported that zinc levels rise by over 50% during oocyte maturation and that attenuation of zinc availability during this period could be achieved using the membrane-permeable heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). This zinc insufficiency resulted in formation of large polar bodies, failure to establish metaphase II arrest, and impaired establishment of cortical polarity. As these phenotypes resemble those of MOS null oocytes, we examined the impact of zinc insufficiency on the MOS-MAPK pathway. Reduced levels of both MOS protein and phosphorylation of MAP2K1/2 are observed in zinc-insufficient oocytes; however, these differences appear only after completion of the first meiotic division. In addition, activation of the downstream effector of the MOS pathway, MAPK3/1, is not affected by zinc insufficiency, and reduced MOS levels are observed only with the presence of TPEN after the first polar body extrusion. These data are inconsistent with the hypothesis that reduced MOS mediates the observed phenotype. Finally, MOS overexpression does not rescue the phenotype of zinc-insufficient oocytes, confirming that the observed disruption of asymmetric division and spindle abnormalities cannot be attributed to impaired MOS signaling. Zinc-insufficient oocytes do not increase maturation promoting factor (MPF) activity following the first meiotic division, and increasing MPF activity through expression of nondegradable cyclin B1 partially rescues the ability of zinc-insufficient oocytes to enter metaphase II. Although we have shown that zinc has a novel role in the meiotic cell cycle, it is not mediated through the MOS-MAPK pathway.
Assuntos
Divisão Celular , Sistema de Sinalização das MAP Quinases/fisiologia , Meiose , Oócitos/citologia , Proteínas Proto-Oncogênicas c-mos/fisiologia , Zinco/fisiologia , Proteínas de Capeamento de Actina/metabolismo , Animais , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Divisão Celular/fisiologia , Cromatina/fisiologia , Feminino , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Meiose/efeitos dos fármacos , Meiose/genética , Meiose/fisiologia , Mesotelina , Camundongos , Modelos Biológicos , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Oogênese/efeitos dos fármacos , Oogênese/fisiologia , Fosforilação , Proteínas Proto-Oncogênicas c-mos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Zinco/deficiência , Zinco/metabolismo , Zinco/farmacologiaRESUMO
The mitotic and meiotic cell cycle share many regulators, but there are also important differences between the two processes. The meiotic maturation of Xenopus oocytes has proved useful for understanding the regulation of Cdc2-cyclin-B, a key activator of G2/M progression. New insights have been made recently into the signalling mechanisms that induce G2-arrested oocytes to resume and complete the meiotic cell cycle.
Assuntos
Ciclo Celular/fisiologia , Meiose/fisiologia , Oócitos/metabolismo , Proteínas de Xenopus , Animais , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclina B/metabolismo , Ciclinas/metabolismo , Fase G2/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Mitose/fisiologia , Oócitos/citologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-mos/metabolismo , Fase S/fisiologia , XenopusRESUMO
Vertebrate eggs awaiting fertilization are arrested at metaphase of meiosis II by a biochemical activity termed cytostatic factor (CSF). This activity inhibits the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that triggers anaphase onset and mitotic/meiotic exit by targeting securin and M-phase cyclins for destruction. On fertilization a transient rise in free intracellular calcium causes release from CSF arrest and thus APC/C activation. Although it has previously been shown that calcium induces the release of APC/C from CSF inhibition through calmodulin-dependent protein kinase II (CaMKII), the relevant substrates of this kinase have not been identified. Recently, we characterized XErp1 (Emi2), an inhibitor of the APC/C and key component of CSF activity in Xenopus egg extract. Here we show that calcium-activated CaMKII triggers exit from meiosis II by sensitizing the APC/C inhibitor XErp1 for polo-like kinase 1 (Plx1)-dependent degradation. Phosphorylation of XErp1 by CaMKII leads to the recruitment of Plx1 that in turn triggers the destruction of XErp1 by phosphorylating a site known to serve as a phosphorylation-dependent degradation signal. These results provide a molecular explanation for how the fertilization-induced calcium increase triggers exit from meiosis II.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Cálcio/farmacologia , Proteínas F-Box/metabolismo , Meiose/efeitos dos fármacos , Complexos Ubiquitina-Proteína Ligase/antagonistas & inibidores , Proteínas de Xenopus/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Animais , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas de Ciclo Celular/metabolismo , Fertilização/fisiologia , Modelos Biológicos , Óvulo/citologia , Óvulo/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-mos/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mos/metabolismo , Proteínas Proto-Oncogênicas c-mos/farmacologia , Especificidade por Substrato , Complexos Ubiquitina-Proteína Ligase/metabolismo , XenopusRESUMO
Mitogen-activated protein kinase (MAPK) and maturation/M phase promoting factor (MPF) play crucial roles in oocyte meiotic maturation in mammals. However, the underlying molecular mechanisms have not been addressed. In this study, the effects of the MEK/MAPK pathway inhibitor U0126 and the MPF inhibitor roscovitine on meiotic maturation and maternal gene expression in pig cumulus-oocyte complexes (COCs) and denuded oocytes (DOs) were investigated. Both inhibitors can reversibly block the resumption of meiosis in pig oocytes. COCs or DOs initially cultured in drug-free medium for 24 h and then transferred to medium containing U0126 showed normal progress to the Metaphase II stage (MII); (90.38 vs. 92.16% control group). In contrast, roscovitine treatment from 24 to 44 h significantly inhibited maturation of COCs and DOs. To explore the underlying molecular mechanisms, expression patterns and polyadenylation states of five representative maternal transcripts, cyclin B1, Cdc2, C-mos, GDF9 and BMP15, were examined by real-time PCR and poly(A)-test PCR (PAT assay). U0126 treatment resulted in aberrant expression of Cdc2 and GDF9, while roscovitine significantly maintained all five maternal transcripts at very high levels in treated COCs compared with the control group. The polyadenylation of these mRNAs increased as well. Furthermore, the experiments were repeated in DOs, and the results also indicated that both Cdc2 and GDF9 showed significantly higher expression in both mRNA and polyadenylation levels in the drug treatment groups. Together, these results provide the first demonstration in a mammalian system that MAPK and MPF play important roles in regulation of maternal gene expression during oocyte maturation.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Sistema de Sinalização das MAP Quinases , Fator Promotor de Maturação/metabolismo , Mitose , Oócitos/metabolismo , Oogênese , Animais , Proteína Morfogenética Óssea 15/genética , Proteína Morfogenética Óssea 15/metabolismo , Butadienos/farmacologia , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Células do Cúmulo/fisiologia , Ciclina B1/genética , Ciclina B1/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Fator 9 de Diferenciação de Crescimento/genética , Fator 9 de Diferenciação de Crescimento/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fator Promotor de Maturação/antagonistas & inibidores , Mitose/efeitos dos fármacos , Moduladores de Mitose/farmacologia , Nitrilas/farmacologia , Oócitos/efeitos dos fármacos , Oogênese/efeitos dos fármacos , Poliadenilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-mos/genética , Proteínas Proto-Oncogênicas c-mos/metabolismo , Purinas/farmacologia , RNA Mensageiro/metabolismo , Roscovitina , Sus scrofaRESUMO
BACKGROUND: Vertebrate oocytes are arrested in metaphase II of meiosis prior to fertilization by cytostatic factor (CSF). CSF enforces a cell-cycle arrest by inhibiting the anaphase-promoting complex (APC), an E3 ubiquitin ligase that targets Cyclin B for degradation. Although Cyclin B synthesis is ongoing during CSF arrest, constant Cyclin B levels are maintained. To achieve this, oocytes allow continuous slow Cyclin B degradation, without eliminating the bulk of Cyclin B, which would induce release from CSF arrest. However, the mechanism that controls this continuous degradation is not understood. RESULTS: We report here the molecular details of a negative feedback loop wherein Cyclin B promotes its own destruction through Cdc2/Cyclin B-mediated phosphorylation and inhibition of the APC inhibitor Emi2. Emi2 bound to the core APC, and this binding was disrupted by Cdc2/Cyclin B, without affecting Emi2 protein stability. Cdc2-mediated phosphorylation of Emi2 was antagonized by PP2A, which could bind to Emi2 and promote Emi2-APC interactions. CONCLUSIONS: Constant Cyclin B levels are maintained during a CSF arrest through the regulation of Emi2 activity. A balance between Cdc2 and PP2A controls Emi2 phosphorylation, which in turn controls the ability of Emi2 to bind to and inhibit the APC. This balance allows proper maintenance of Cyclin B levels and Cdc2 kinase activity during CSF arrest.
Assuntos
Proteína Quinase CDC2/metabolismo , Proteínas F-Box/metabolismo , Oócitos/citologia , Fosfoproteínas Fosfatases/metabolismo , Proteínas Proto-Oncogênicas c-mos/metabolismo , Proteínas de Xenopus/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Animais , Proteínas Cdc20 , Proteínas de Ciclo Celular/metabolismo , Ciclina B/metabolismo , DNA Complementar , Inibidores Enzimáticos/farmacologia , Biblioteca Gênica , Humanos , Meiose , Ácido Okadáico/farmacologia , Oócitos/metabolismo , Fosforilação , Ligação Proteica/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , XenopusRESUMO
Vertebrate eggs prevent parthenogenetic development by producing cytostatic factor (CSF), which blocks exit from metaphase of meiosis II until fertilization. CSF was never purified but recently suspected to inhibit the anaphase-promoting complex (APC), an ubiquitin ligase required for entry into anaphase. In a recent paper in Genes & Development, Schmidt et al. describe the Xenopus APC inhibitor Erp1, which seems to be the best candidate yet for the downstream effector of CSF activity.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/metabolismo , Meiose/fisiologia , Oócitos/citologia , Complexos Ubiquitina-Proteína Ligase/metabolismo , Proteínas de Xenopus/metabolismo , Anáfase/fisiologia , Ciclossomo-Complexo Promotor de Anáfase , Animais , Cálcio/metabolismo , Feminino , Fertilização/fisiologia , Oócitos/metabolismo , Proteínas Proto-Oncogênicas c-mos/metabolismo , Xenopus laevis/metabolismoRESUMO
Vertebrate oocytes arrest in metaphase of the second meiotic division (MII), where they maintain a high cdc2/cyclin B activity and a stable, bipolar spindle because of cytostatic factor (CSF) activity. The Mos-MAPK pathway is essential for establishing CSF. Indeed, oocytes from the mos-/- strain do not arrest in MII and activate without fertilization, as do Xenopus laevis oocytes injected with morpholino oligonucleotides directed against Mos. In Xenopus oocytes, p90Rsk (ribosomal S6 kinase), a MAPK substrate, is the main mediator of CSF activity. We show here that this is not the case in mouse oocytes. The injection of constitutively active mutant forms of Rsk1 and Rsk2 does not induce a cell cycle arrest in two-cell mouse embryos. Moreover, these two mutant forms do not restore MII arrest after their injection into mos-/- oocytes. Eventually, oocytes from the triple Rsk (1, 2, 3) knockout present a normal CSF arrest. We demonstrate that p90Rsk is not involved in the MII arrest of mouse oocytes.
Assuntos
Blastômeros/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Meiose/fisiologia , Oócitos/fisiologia , Proteínas Proto-Oncogênicas c-mos/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Animais , Feminino , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Knockout , Gravidez , Proteínas Proto-Oncogênicas c-mos/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Xenopus laevisRESUMO
BACKGROUND: RNA interference (RNAi) is a powerful approach to study a gene function. Transgenic RNAi is an adaptation of this approach where suppression of a specific gene is achieved by expression of an RNA hairpin from a transgene. In somatic cells, where a long double-stranded RNA (dsRNA) longer than 30 base-pairs can induce a sequence-independent interferon response, short hairpin RNA (shRNA) expression is used to induce RNAi. In contrast, transgenic RNAi in the oocyte routinely employs a long RNA hairpin. Transgenic RNAi based on long hairpin RNA, although robust and successful, is restricted to a few cell types, where long double-stranded RNA does not induce sequence-independent responses. Transgenic RNAi in mouse oocytes based on a shRNA offers several potential advantages, including simple cloning of the transgenic vector and an ability to use the same targeting construct in any cell type. RESULTS: Here we report our experience with shRNA-based transgenic RNAi in mouse oocytes. Despite optimal starting conditions for this experiment, we experienced several setbacks, which outweigh potential benefits of the shRNA system. First, obtaining an efficient shRNA is potentially a time-consuming and expensive task. Second, we observed that our transgene, which was based on a common commercial vector, was readily silenced in transgenic animals. CONCLUSIONS: We conclude that, the long RNA hairpin-based RNAi is more reliable and cost-effective and we recommend it as a method-of-choice when a gene is studied selectively in the oocyte.
Assuntos
Técnicas de Silenciamento de Genes/métodos , Oócitos/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transgenes/genética , Animais , Clonagem Molecular , Cruzamentos Genéticos , Feminino , Técnicas de Silenciamento de Genes/economia , Vetores Genéticos/genética , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Plasmídeos/genética , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas c-mos/genética , Proteínas Proto-Oncogênicas c-mos/metabolismoRESUMO
Spindle assembly and accurate chromosome segregation require the proper regulation of microtubule dynamics. MCAK, a Kinesin-13, catalytically depolymerizes microtubules, regulates physiological microtubule dynamics, and is the major catastrophe factor in egg extracts. Purified GFP-tagged MCAK domain mutants were assayed to address how the different MCAK domains contribute to in vitro microtubule depolymerization activity and physiological spindle assembly activity in egg extracts. Our biochemical results demonstrate that both the neck and the C-terminal domain are necessary for robust in vitro microtubule depolymerization activity. In particular, the neck is essential for microtubule end binding, and the C-terminal domain is essential for tight microtubule binding in the presence of excess tubulin heterodimer. Our physiological results illustrate that the N-terminal domain is essential for regulating microtubule dynamics, stimulating spindle bipolarity, and kinetochore targeting; whereas the C-terminal domain is necessary for robust microtubule depolymerization activity, limiting spindle bipolarity, and enhancing kinetochore targeting. Unexpectedly, robust MCAK microtubule (MT) depolymerization activity is not needed for sperm-induced spindle assembly. However, high activity is necessary for proper physiological MT dynamics as assayed by Ran-induced aster assembly. We propose that MCAK activity is spatially controlled by an interplay between the N- and C-terminal domains during spindle assembly.