RESUMO
Translation is critical for development as transcription in the oocyte and early embryo is silenced. To illustrate the translational changes during meiosis and consecutive two mitoses of the oocyte and early embryo, we performed a genome-wide translatome analysis. Acquired data showed significant and uniform activation of key translational initiation and elongation axes specific to M-phases. Although global protein synthesis decreases in M-phases, translation initiation and elongation activity increases in a uniformly fluctuating manner, leading to qualitative changes in translation regulation via the mTOR1/4F/eEF2 axis. Overall, we have uncovered a highly dynamic and oscillatory pattern of translational reprogramming that contributes to the translational regulation of specific mRNAs with different modes of polysomal occupancy/translation that are important for oocyte and embryo developmental competence. Our results provide new insights into the regulation of gene expression during oocyte meiosis as well as the first two embryonic mitoses and show how temporal translation can be optimized. This study is the first step towards a comprehensive analysis of the molecular mechanisms that not only control translation during early development, but also regulate translation-related networks employed in the oocyte-to-embryo transition and embryonic genome activation.
Assuntos
Desenvolvimento Embrionário , Oócitos , Biossíntese de Proteínas , Regulação da Expressão Gênica no Desenvolvimento , Meiose , Oócitos/citologia , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Animais , CamundongosRESUMO
Culture media used in assisted reproduction are commonly supplemented with gonadotropin hormones to support the nuclear and cytoplasmic maturation of in vitro matured oocytes. However, the effect of gonadotropins on protein synthesis in oocytes is yet to be fully understood. As published data have previously documented a positive in vitro effect of follicle-stimulating hormone (FSH) on cytoplasmic maturation, we exposed mouse denuded oocytes to FSH in order to evaluate the changes in global protein synthesis. We found that dose-dependent administration of FSH resulted in a decrease of methionine incorporation into de novo synthesized proteins in denuded mouse oocytes and oocytes cultured in cumulus-oocyte complexes. Similarly, FSH influenced methionine incorporation in additional mammalian species including human. Furthermore, we showed the expression of FSH-receptor protein in oocytes. We found that major translational regulators were not affected by FSH treatment; however, the amino acid uptake became impaired. We propose that the effect of FSH treatment on amino acid uptake is influenced by FSH receptor with the effect on oocyte metabolism and physiology.
Assuntos
Aminoácidos/metabolismo , Hormônio Foliculoestimulante/farmacologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Animais , Bovinos , Células Cultivadas , Fase de Clivagem do Zigoto/efeitos dos fármacos , Fase de Clivagem do Zigoto/metabolismo , Meios de Cultura/química , Meios de Cultura/farmacologia , Feminino , Humanos , Técnicas de Maturação in Vitro de Oócitos/métodos , Mamíferos , Camundongos , SuínosRESUMO
The rate of chromosome segregation errors that emerge during meiosis I in the mammalian female germ line are known to increase with maternal age; however, little is known about the underlying molecular mechanism. The objective of this study was to analyze meiotic progression of mouse oocytes in relation to maternal age. Using the mouse as a model system, we analyzed the timing of nuclear envelope breakdown and the morphology of the nuclear lamina of oocytes obtained from young (2 months old) and aged females (12 months old). Oocytes obtained from older females display a significantly faster progression through meiosis I compared to the ones obtained from younger females. Furthermore, in oocytes from aged females, lamin A/C structures exhibit rapid phosphorylation and dissociation. Additionally, we also found an increased abundance of MPF components and increased translation of factors controlling translational activity in the oocytes of aged females. In conclusion, the elevated MPF activity observed in aged female oocytes affects precocious meiotic processes that can multifactorially contribute to chromosomal errors in meiosis I.
Assuntos
Envelhecimento/metabolismo , Fator Promotor de Maturação/metabolismo , Meiose , Oócitos/metabolismo , Envelhecimento/genética , Animais , Feminino , Fator Promotor de Maturação/genética , Mesotelina , Camundongos , Membrana Nuclear/metabolismo , Membrana Nuclear/ultraestrutura , Oócitos/citologia , Fosforilação , Processamento de Proteína Pós-TraducionalRESUMO
A hallmark of oocyte development in mammals is the dependence on the translation and utilization of stored RNA and proteins rather than the de novo transcription of genes in order to sustain meiotic progression and early embryo development. In the absence of transcription, the completion of meiosis and early embryo development in mammals relies significantly on maternally synthesized RNAs. Post-transcriptional control of gene expression at the translational level has emerged as an important cellular function in normal development. Therefore, the regulation of gene expression in oocytes is controlled almost exclusively at the level of mRNA and protein stabilization and protein synthesis. This current review is focused on the recently emerged findings on RNA distribution related to the temporal and spatial translational control of the meiotic progression of the mammalian oocyte.
Assuntos
Oócitos/citologia , Oócitos/metabolismo , Biossíntese de Proteínas , Animais , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Meiose , Oogênese , RNA/análise , RNA/genética , TranscriptomaRESUMO
Mammalian oocyte development depends on the temporally controlled translation of maternal transcripts, particularly in the coordination of meiotic and early embryonic development when transcription has ceased. The translation of mRNA is regulated by various RNA-binding proteins. We show that the absence of cytoplasmic polyadenylation element-binding protein 3 (CPEB3) negatively affects female reproductive fitness. CPEB3-depleted oocytes undergo meiosis normally but experience early embryonic arrest due to a disrupted transcriptome, leading to aberrant protein expression and the subsequent failure of embryonic transcription initiation. We found that CPEB3 stabilizes a subset of mRNAs with a significantly longer 3'UTR that is enriched in its distal region with cytoplasmic polyadenylation elements. Overall, our results suggest that CPEB3 is an important maternal factor that regulates the stability and translation of a subclass of mRNAs that are essential for the initiation of embryonic transcription and thus for embryonic development.
Assuntos
Oócitos , Proteínas de Ligação a RNA , Oócitos/metabolismo , Animais , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Feminino , Camundongos , Meiose/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Regiões 3' não Traduzidas/genética , Poliadenilação , Estabilidade de RNA/genéticaRESUMO
During oocyte growth the cell accumulates RNAs to contribute to oocyte and embryo development which progresses with ceased transcription. To investigate the subcellular distribution of specific RNAs and their translation we developed a technique revealing several instances of localized translation with distinctive regulatory implications. We analyzed the localization and expression of candidate non-coding and mRNAs in the mouse oocyte and embryo. Furthermore, we established simultaneous visualization of mRNA and in situ translation events validated with polysomal occupancy. We discovered that translationally dormant and abundant mRNAs CyclinB1 and Mos are localized in the cytoplasm of the fully grown GV oocyte forming cloud-like structures with consequent abundant translation at the center of the MII oocyte. Coupling detection of the localization of specific single mRNA molecules with their translation at the subcellular context is a valuable tool to quantitatively study temporal and spatial translation of specific target mRNAs to understand molecular processes in the developing cell.
Assuntos
Ciclina B1/genética , Embrião de Mamíferos/química , Oócitos/crescimento & desenvolvimento , Proteínas Proto-Oncogênicas c-mos/genética , Imagem Individual de Molécula/métodos , Animais , Citoplasma/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Hibridização in Situ Fluorescente , Camundongos , Oócitos/química , Polirribossomos/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA não Traduzido/genéticaRESUMO
Successful specification of the two mouse blastocyst inner cell mass (ICM) lineages (the primitive endoderm (PrE) and epiblast) is a prerequisite for continued development and requires active fibroblast growth factor 4 (FGF4) signaling. Previously, we identified a role for p38 mitogen-activated protein kinases (p38-MAPKs) during PrE differentiation, but the underlying mechanisms have remained unresolved. Here, we report an early blastocyst window of p38-MAPK activity that is required to regulate ribosome-related gene expression, rRNA precursor processing, polysome formation and protein translation. We show that p38-MAPK inhibition-induced PrE phenotypes can be partially rescued by activating the translational regulator mTOR. However, similar PrE phenotypes associated with extracellular signal-regulated kinase (ERK) pathway inhibition targeting active FGF4 signaling are not affected by mTOR activation. These data indicate a specific role for p38-MAPKs in providing a permissive translational environment during mouse blastocyst PrE differentiation that is distinct from classically reported FGF4-based mechanisms.
Assuntos
Blastocisto/fisiologia , Endoderma/citologia , Biossíntese de Proteínas , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia , Animais , Diferenciação Celular , Linhagem da Célula , Proteínas de Ligação a DNA/fisiologia , Desenvolvimento Embrionário , Camundongos , Proteínas de Ligação a RNA/fisiologia , Serina-Treonina Quinases TOR/fisiologia , Fatores de Transcrição/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Cyclin dependent kinase 1 (CDK1) has been primarily identified as a key cell cycle regulator in both mitosis and meiosis. Recently, an extramitotic function of CDK1 emerged when evidence was found that CDK1 is involved in many cellular events that are essential for cell proliferation and survival. In this review we summarize the involvement of CDK1 in the initiation and elongation steps of protein synthesis in the cell. During its activation, CDK1 influences the initiation of protein synthesis, promotes the activity of specific translational initiation factors and affects the functioning of a subset of elongation factors. Our review provides insights into gene expression regulation during the transcriptionally silent M-phase and describes quantitative and qualitative translational changes based on the extramitotic role of the cell cycle master regulator CDK1 to optimize temporal synthesis of proteins to sustain the division-related processes: mitosis and cytokinesis.
Assuntos
Proteína Quinase CDC2/metabolismo , Animais , Proteína Quinase CDC2/genética , Ciclo Celular/genética , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
In several species, including Xenopus, mouse and human, two members of cyclin A family were identified. Cyclin A2, which is ubiquitously expressed in dividing cells and plays role in DNA replication, entry into mitosis and spindle assembly, and cyclin A1, whose function is less clear and which is expressed in spermatocytes, leukemia cells and in postmitotic multiciliated cells. Deletion of the gene showed that cyclin A1 is essential for male meiosis, but nonessential for female meiosis. Our results revealed, that the cyclin A1 is not only dispensable in oocytes, we show here that its expression is in fact undesirable in these cells. Our data demonstrate that the APC/C and proteasome in oocytes are unable to target sufficiently cyclin A1 before anaphase, which leads into anaphase arrest and direct inhibition of separase. The cyclin A1-induced cell cycle arrest is oocyte-specific and the presence of cyclin A1 in early embryos has no effect on cell cycle progression or chromosome division. Cyclin A1 is therefore not only an important cell cycle regulator with biased expression in germline, being essential for male and damaging for female meiosis, its persistent expression during anaphase in oocytes shows fundamental differences between APC/C function in oocytes and in early embryos.
Assuntos
Anáfase , Segregação de Cromossomos , Ciclina A1/fisiologia , Oócitos/citologia , Animais , Ciclina A2/fisiologia , Feminino , Masculino , Meiose , Metáfase , Camundongos , Microinjeções , Microscopia de Fluorescência , Complexo de Endopeptidases do Proteassoma/fisiologiaRESUMO
Increasing maternal age in mammals is associated with poorer oocyte quality, involving higher aneuploidy rates and decreased developmental competence. Prior to resumption of meiosis, fully developed mammalian oocytes become transcriptionally silent until the onset of zygotic genome activation. Therefore, meiotic progression and early embryogenesis are driven largely by translational utilization of previously synthesized mRNAs. We report that genome-wide translatome profiling reveals considerable numbers of transcripts that are differentially translated in oocytes obtained from aged compared to young females. Additionally, we show that a number of aberrantly translated mRNAs in oocytes from aged females are associated with cell cycle. Indeed, we demonstrate that four specific maternal age-related transcripts (Sgk1, Castor1, Aire and Eg5) with differential translation rates encode factors that are associated with the newly forming meiotic spindle. Moreover, we report substantial defects in chromosome alignment and cytokinesis in the oocytes of young females, in which candidate CASTOR1 and SGK1 protein levels or activity are experimentally altered. Our findings indicate that improper translation of specific proteins at the onset of meiosis contributes to increased chromosome segregation problems associated with female ageing.
Assuntos
Oócitos/metabolismo , Fatores Etários , Animais , Feminino , Humanos , MamíferosRESUMO
In the absence of transcription, the regulation of gene expression in oocytes is controlled almost exclusively at the level of transcriptome and proteome stabilization, and translation. A subset of maternal transcripts is stored in a translationally dormant state in the oocyte, and temporally driven translation of specific mRNAs propel meiotic progression, oocyte-to-embryo transition and early embryo development. We identified Ank2.3 as the only transcript variant present in the mouse oocyte and discovered that it is translated after nuclear envelope breakdown. Here we show that Ank2.3 mRNA is localized in higher concentration in the oocyte nucleoplasm and, after nuclear envelope breakdown, in the newly forming spindle where its translation occurs. Furthermore, we reveal that Ank2.3 mRNA contains an oligo-pyrimidine motif at 5'UTR that predetermines its translation through a cap-dependent pathway. Lastly, we show that prevention of ANK2 translation leads to abnormalities in oocyte cytokinesis.
Assuntos
Anquirinas/metabolismo , Citocinese , Embrião de Mamíferos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Meiose , Oócitos/fisiologia , Análise Espaço-Temporal , Animais , Anquirinas/genética , Embrião de Mamíferos/citologia , Feminino , Camundongos , Oócitos/citologia , Oogênese , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The tight correlation between mRNA distribution and subsequent protein localization and function indicate a major role for mRNA localization within the cell. RNA localization, followed by local translation, presents a mechanism for spatial and temporal gene expression regulation utilized by various cell types. However, little is known about mRNA localization and translation in the mammalian oocyte and early embryo. Importantly, fully-grown oocyte becomes transcriptionally inactive and only utilizes transcripts previously synthesized and stored during earlier development. We discovered an abundant RNA population in the oocyte and early embryo nucleus together with RNA binding proteins. We also characterized specific ribosomal proteins, which contribute to translation in the oocyte and embryo. By applying selected markers to mouse and human oocytes, we found that there might be a similar mechanism of RNA metabolism in both species. In conclusion, we visualized the localization of RNAs and translation machinery in the oocyte, that could shed light on this terra incognita of these unique cell types in mouse and human.
Assuntos
Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Oócitos/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/genética , Animais , Células Cultivadas , Embrião de Mamíferos/ultraestrutura , Feminino , Humanos , Camundongos , Oócitos/ultraestrutura , RNA Mensageiro/análise , Proteínas de Ligação a RNA/análise , Proteínas de Ligação a RNA/genética , TranscriptomaRESUMO
Fully grown mammalian oocytes utilize transcripts synthetized and stored during earlier development. RNA localization followed by a local translation is a mechanism responsible for the regulation of spatial and temporal gene expression. Here we show that the mouse oocyte contains 3 forms of cap-dependent translational repressor expressed on the mRNA level: 4E-BP1, 4E-BP2 and 4E-BP3. However, only 4E-BP1 is present as a protein in oocytes, it becomes inactivated by phosphorylation after nuclear envelope breakdown and as such it promotes cap-dependent translation after NEBD. Phosphorylation of 4E-BP1 can be seen in the oocytes after resumption of meiosis but it is not detected in the surrounding cumulus cells, indicating that 4E-BP1 promotes translation at a specific cell cycle stage. Our immunofluorescence analyses of 4E-BP1 in oocytes during meiosis I showed an even localization of global 4E-BP1, as well as of its 4E-BP1 (Thr37/46) phosphorylated form. On the other hand, 4E-BP1 phosphorylated on Ser65 is localized at the spindle poles, and 4E-BP1 phosphorylated on Thr70 localizes on the spindle. We further show that the main positive regulators of 4E-BP1 phosphorylation after NEBD are mTOR and CDK1 kinases, but not PLK1 kinase. CDK1 exerts its activity toward 4E-BP1 phosphorylation via phosphorylation and activation of mTOR. Moreover, both CDK1 and phosphorylated mTOR co-localize with 4E-BP1 phosphorylated on Thr70 on the spindle at the onset of meiotic resumption. Expression of the dominant negative 4E-BP1 mutant adversely affects translation and results in spindle abnormality. Taken together, our results show that the phosphorylation of 4E-BP1 promotes translation at the onset of meiosis to support the spindle assembly and suggest an important role of CDK1 and mTOR kinases in this process. We also show that the mTOR regulatory pathway is present in human oocytes and is likely to function in a similar way as in mouse oocytes.
Assuntos
Proteína Quinase CDC2/genética , Proteínas de Transporte/genética , Oócitos/crescimento & desenvolvimento , Fosfoproteínas/genética , Serina-Treonina Quinases TOR/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/metabolismo , Ciclo Celular/genética , Proteínas de Ciclo Celular , Fatores de Iniciação em Eucariotos , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Oócitos/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Biossíntese de Proteínas , Fuso Acromático/genéticaRESUMO
The developmental competence of oocytes is acquired progressively during folliculogenesis and is linked to follicular size. It has been documented that oocytes originating from larger follicles exhibit a greater ability to develop to the blastocyst stage. The differences in cytoplasmic factors such as mRNA transcripts could explain the differences in oocyte developmental potential. We used bovine oligonucleotide microarrays to characterize differences between the gene expression profiles of germinal vesicle stage (GV) oocytes with greater developmental competence from medium follicles (MF) and those with less developmental competence from small follicles (SF). After normalizing the microarray data, our analysis found differences in the level of 60 transcripts (≥1.4 fold), corresponding to 49 upregulated and 11 downregulated transcripts in MF oocytes compared to SF oocytes. The gene expression data were classified according to gene ontology, the majority of the genes were associated with the regulation of transcription, translation, the cell cycle, and mitochondrial activity. A subset of 16 selected genes was validated for GV oocytes by quantitative real-time RT-PCR; significant differences (PË0.01) were found in the level of TAF1A, MTRF1L, ATP5C1, UBL5 and MAP3K13 between the MF and SF oocytes. After maturation the transcript level remained stable for ATP5F1, BRD7, and UBL5 in both oocyte categories. The transcript level of another 13 genes substantially dropped in the MF and/or SF oocytes. It can be concluded that the developmental competence of bovine oocytes and embryos may be a quantitative trait dependent on small changes in the transcription profiles of many genes.
Assuntos
Bovinos/genética , Desenvolvimento Embrionário/genética , Oócitos/metabolismo , Oogênese/genética , Animais , Bovinos/fisiologia , Feminino , Fertilização in vitro , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Análise de Sequência com Séries de Oligonucleotídeos , Oócitos/fisiologia , Folículo Ovariano/metabolismoRESUMO
The fully grown mammalian oocyte is transcriptionally quiescent and utilizes only transcripts synthesized and stored during early development. However, we find that an abundant RNA population is retained in the oocyte nucleus and contains specific mRNAs important for meiotic progression. Here we show that during the first meiotic division, shortly after nuclear envelope breakdown, translational hotspots develop in the chromosomal area and in a region that was previously surrounded the nucleus. These distinct translational hotspots are separated by endoplasmic reticulum and Lamin, and disappear following polar body extrusion. Chromosomal translational hotspots are controlled by the activity of the mTOR-eIF4F pathway. Here we reveal a mechanism that-following the resumption of meiosis-controls the temporal and spatial translation of a specific set of transcripts required for normal spindle assembly, chromosome alignment and segregation.