RESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
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.
Asunto(s)
Ancirinas/metabolismo , Citocinesis , Embrión de Mamíferos/fisiología , Regulación del Desarrollo de la Expresión Génica , Meiosis , Oocitos/fisiología , Análisis Espacio-Temporal , Animales , Ancirinas/genética , Embrión de Mamíferos/citología , Femenino , Ratones , Oocitos/citología , Oogénesis , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
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.
Asunto(s)
Aminoácidos/metabolismo , Hormona Folículo Estimulante/farmacología , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Animales , Bovinos , Células Cultivadas , Fase de Segmentación del Huevo/efectos de los fármacos , Fase de Segmentación del Huevo/metabolismo , Medios de Cultivo/química , Medios de Cultivo/farmacología , Femenino , Humanos , Técnicas de Maduración In Vitro de los Oocitos/métodos , Mamíferos , Ratones , PorcinosRESUMEN
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.
Asunto(s)
Envejecimiento/metabolismo , Factor Promotor de Maduración/metabolismo , Meiosis , Oocitos/metabolismo , Envejecimiento/genética , Animales , Femenino , Factor Promotor de Maduración/genética , Mesotelina , Ratones , Membrana Nuclear/metabolismo , Membrana Nuclear/ultraestructura , Oocitos/citología , Fosforilación , Procesamiento Proteico-PostraduccionalRESUMEN
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.
Asunto(s)
Embrión de Mamíferos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Oocitos/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/genética , Animales , Células Cultivadas , Embrión de Mamíferos/ultraestructura , Femenino , Humanos , Ratones , Oocitos/ultraestructura , ARN Mensajero/análisis , Proteínas de Unión al ARN/análisis , Proteínas de Unión al ARN/genética , TranscriptomaRESUMEN
Although the involvement of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathway in the regulation of cytostatic factor (CSF) activity; as well as in microtubules organization during meiotic maturation of oocytes; has already been described in detail; rather less attention has been paid to the role of ERK1/2 in the regulation of mRNA translation. However; important data on the role of ERK1/2 in translation during oocyte meiosis have been documented. This review focuses on recent findings regarding the regulation of translation and the role of ERK1/2 in this process in the meiotic cycle of mammalian oocytes. The specific role of ERK1/2 in the regulation of mammalian target of rapamycin (mTOR); eukaryotic translation initiation factor 4E (eIF4E) and cytoplasmic polyadenylation element binding protein 1 (CPEB1) activity is addressed along with additional focus on the other key players involved in protein translation.
Asunto(s)
Meiosis , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Oocitos/metabolismo , Biosíntesis de Proteínas , Animales , Citoplasma/genética , Citoplasma/metabolismo , Factor 4E Eucariótico de Iniciación/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Poliadenilación , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Factores de Escisión y Poliadenilación de ARNm/metabolismoRESUMEN
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.