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1.
Development ; 149(5)2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35112132

RESUMO

Successful reproduction requires an oocyte competent to sustain early embryo development. By the end of oogenesis, the oocyte has entered a transcriptionally silenced state, the mechanisms and significance of which remain poorly understood. Histone H3.3, a histone H3 variant, has unique cell cycle-independent functions in chromatin structure and gene expression. Here, we have characterised the H3.3 chaperone Hira/Cabin1/Ubn1 complex, showing that loss of function of any of these subunits causes early embryogenesis failure in mouse. Transcriptome and nascent RNA analyses revealed that transcription is aberrantly silenced in mutant oocytes. Histone marks, including H3K4me3 and H3K9me3, are reduced and chromatin accessibility is impaired in Hira/Cabin1 mutants. Misregulated genes in mutant oocytes include Zscan4d, a two-cell specific gene involved in zygote genome activation. Overexpression of Zscan4 in the oocyte partially recapitulates the phenotypes of Hira mutants and Zscan4 knockdown in Cabin1 mutant oocytes partially restored their developmental potential, illustrating that temporal and spatial expression of Zscan4 is fine-tuned at the oocyte-to-embryo transition. Thus, the H3.3 chaperone Hira complex has a maternal effect function in oocyte developmental competence and embryogenesis, through modulating chromatin condensation and transcriptional quiescence.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Desenvolvimento Embrionário/genética , Feminino , Técnicas de Silenciamento de Genes , Chaperonas de Histonas/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oogênese/genética , Fatores de Transcrição/genética , Zigoto/metabolismo
2.
Development ; 149(21)2022 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-36227586

RESUMO

High-resolution ribosome fractionation and low-input ribosome profiling of bovine oocytes and preimplantation embryos has enabled us to define the translational landscapes of early embryo development at an unprecedented level. We analyzed the transcriptome and the polysome- and non-polysome-bound RNA profiles of bovine oocytes (germinal vesicle and metaphase II stages) and early embryos at the two-cell, eight-cell, morula and blastocyst stages, and revealed four modes of translational selectivity: (1) selective translation of non-abundant mRNAs; (2) active, but modest translation of a selection of highly expressed mRNAs; (3) translationally suppressed abundant to moderately abundant mRNAs; and (4) mRNAs associated specifically with monosomes. A strong translational selection of low-abundance transcripts involved in metabolic pathways and lysosomes was found throughout bovine embryonic development. Notably, genes involved in mitochondrial function were prioritized for translation. We found that translation largely reflected transcription in oocytes and two-cell embryos, but observed a marked shift in the translational control in eight-cell embryos that was associated with the main phase of embryonic genome activation. Subsequently, transcription and translation become more synchronized in morulae and blastocysts. Taken together, these data reveal a unique spatiotemporal translational regulation that accompanies bovine preimplantation development.


Assuntos
Blastocisto , Desenvolvimento Embrionário , Gravidez , Feminino , Bovinos , Animais , Desenvolvimento Embrionário/genética , Mórula/metabolismo , Blastocisto/metabolismo , Oócitos/metabolismo , Ribossomos/genética , Regulação da Expressão Gênica no Desenvolvimento
3.
Nucleic Acids Res ; 51(22): 12076-12091, 2023 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-37950888

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 , Camundongos
4.
Nucleic Acids Res ; 48(6): 3211-3227, 2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-31956907

RESUMO

Tens of thousands of rapidly evolving long non-coding RNA (lncRNA) genes have been identified, but functions were assigned to relatively few of them. The lncRNA contribution to the mouse oocyte physiology remains unknown. We report the evolutionary history and functional analysis of Sirena1, the most expressed lncRNA and the 10th most abundant poly(A) transcript in mouse oocytes. Sirena1 appeared in the common ancestor of mouse and rat and became engaged in two different post-transcriptional regulations. First, antisense oriented Elob pseudogene insertion into Sirena1 exon 1 is a source of small RNAs targeting Elob mRNA via RNA interference. Second, Sirena1 evolved functional cytoplasmic polyadenylation elements, an unexpected feature borrowed from translation control of specific maternal mRNAs. Sirena1 knock-out does not affect fertility, but causes minor dysregulation of the maternal transcriptome. This includes increased levels of Elob and mitochondrial mRNAs. Mitochondria in Sirena1-/- oocytes disperse from the perinuclear compartment, but do not change in number or ultrastructure. Taken together, Sirena1 contributes to RNA interference and mitochondrial aggregation in mouse oocytes. Sirena1 exemplifies how lncRNAs stochastically engage or even repurpose molecular mechanisms during evolution. Simultaneously, Sirena1 expression levels and unique functional features contrast with the lack of functional importance assessed under laboratory conditions.


Assuntos
Mitocôndrias/genética , Oócitos/metabolismo , RNA Longo não Codificante/genética , RNA Mensageiro/genética , RNA Mitocondrial/genética , Animais , Técnicas de Inativação de Genes , Camundongos , Mitocôndrias/ultraestrutura , Oócitos/crescimento & desenvolvimento , Oócitos/ultraestrutura , Poliadenilação/genética , Ratos , Transcriptoma/genética
5.
Histochem Cell Biol ; 154(6): 609-620, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32930837

RESUMO

Protein syntheses at appropriate timings are important for promoting diverse biological processes and are controlled at the levels of transcription and translation. Pou5f1/Oct4 is a transcription factor that is essential for vertebrate embryonic development. However, the precise timings when the mRNA and protein of Pou5f1/Oct4 are expressed during oogenesis and early stages of embryogenesis remain unclear. We analyzed the expression patterns of mRNA and protein of Pou5f1/Oct4 in mouse oocytes and embryos by using a highly sensitive in situ hybridization method and a monoclonal antibody specific to Pou5f1/Oct4, respectively. Pou5f1/Oct4 mRNA was detected in growing oocytes from the primary follicle stage to the fully grown GV stage during oogenesis. In contrast, Pou5f1/Oct4 protein was undetectable during oogenesis, oocyte maturation and the first cleavage stage but subsequently became detectable in the nuclei of early 2-cell-stage embryos. Pou5f1/Oct4 protein at this stage was synthesized from maternal mRNAs stored in oocytes. The amount of Pou5f1/Oct4 mRNA in the polysomal fraction was small in GV-stage oocytes but was significantly increased in fertilized eggs. Taken together, our results indicate that the synthesis of Pou5f1/Oct4 protein during oogenesis and early stages of embryogenesis is controlled at the level of translation and suggest that precise control of the amount of this protein by translational regulation is important for oocyte development and early embryonic development.


Assuntos
Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Fator 3 de Transcrição de Octâmero/genética , Oogênese/genética , Animais , Feminino , Camundongos , Camundongos Endogâmicos ICR , Fator 3 de Transcrição de Octâmero/metabolismo , Gravidez
6.
Int J Mol Sci ; 21(4)2020 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-32070012

RESUMO

Meiotic maturation of oocyte relies on pre-synthesised maternal mRNA, the translation of which is highly coordinated in space and time. Here, we provide a detailed polysome profiling protocol that demonstrates a combination of the sucrose gradient ultracentrifugation in small SW55Ti tubes with the qRT-PCR-based quantification of 18S and 28S rRNAs in fractionated polysome profile. This newly optimised method, named Scarce Sample Polysome Profiling (SSP-profiling), is suitable for both scarce and conventional sample sizes and is compatible with downstream RNA-seq to identify polysome associated transcripts. Utilising SSP-profiling we have assayed the translatome of mouse oocytes at the onset of nuclear envelope breakdown (NEBD)-a developmental point, the study of which is important for furthering our understanding of the molecular mechanisms leading to oocyte aneuploidy. Our analyses identified 1847 transcripts with moderate to strong polysome occupancy, including abundantly represented mRNAs encoding mitochondrial and ribosomal proteins, proteasomal components, glycolytic and amino acids synthetic enzymes, proteins involved in cytoskeleton organization plus RNA-binding and translation initiation factors. In addition to transcripts encoding known players of meiotic progression, we also identified several mRNAs encoding proteins of unknown function. Polysome profiles generated using SSP-profiling were more than comparable to those developed using existing conventional approaches, being demonstrably superior in their resolution, reproducibility, versatility, speed of derivation and downstream protocol applicability.


Assuntos
Membrana Nuclear/genética , Oócitos/crescimento & desenvolvimento , Polirribossomos/genética , Proteínas de Ligação a RNA/genética , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Meiose/genética , Camundongos , Membrana Nuclear/metabolismo , Oócitos/metabolismo , RNA Mensageiro Estocado/genética , RNA Ribossômico 18S/genética , RNA Ribossômico 28S/genética , RNA-Seq
7.
Biol Reprod ; 101(4): 719-732, 2019 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-31290535

RESUMO

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 , Suínos
8.
Int J Mol Sci ; 19(3)2018 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-29494492

RESUMO

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.


Assuntos
Meiose , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Oócitos/metabolismo , Biossíntese de Proteínas , Animais , Citoplasma/genética , Citoplasma/metabolismo , Fator de Iniciação 4E em Eucariotos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Poliadenilação , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo
9.
Int J Mol Sci ; 19(9)2018 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-30235877

RESUMO

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-Traducional
10.
Cell Tissue Res ; 363(1): 69-84, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26340983

RESUMO

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 , Transcriptoma
11.
J Cell Sci ; 126(Pt 5): 1081-5, 2013 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-23345398

RESUMO

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/genética
12.
Cells ; 13(10)2024 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-38786074

RESUMO

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/genética
13.
Open Biol ; 13(8): 230081, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37553074

RESUMO

Preimplantation mouse embryo development involves temporal-spatial specification and segregation of three blastocyst cell lineages: trophectoderm, primitive endoderm and epiblast. Spatial separation of the outer-trophectoderm lineage from the two other inner-cell-mass (ICM) lineages starts with the 8- to 16-cell transition and concludes at the 32-cell stages. Accordingly, the ICM is derived from primary and secondary contributed cells; with debated relative EPI versus PrE potencies. We report generation of primary but not secondary ICM populations is highly dependent on temporal activation of mammalian target of Rapamycin (mTOR) during 8-cell stage M-phase entry, mediated via regulation of the 7-methylguanosine-cap (m7G-cap)-binding initiation complex (EIF4F) and linked to translation of mRNAs containing 5' UTR terminal oligopyrimidine (TOP-) sequence motifs, as knockdown of identified TOP-like motif transcripts impairs generation of primary ICM founders. However, mTOR inhibition-induced ICM cell number deficits in early blastocysts can be compensated by the late blastocyst stage, after inhibitor withdrawal; compensation likely initiated at the 32-cell stage when supernumerary outer cells exhibit molecular characteristics of inner cells. These data identify a novel mechanism specifically governing initial spatial segregation of mouse embryo blastomeres, that is distinct from those directing subsequent inner cell formation, contributing to germane segregation of late blastocyst lineages.


Assuntos
Blastocisto , Embrião de Mamíferos , Camundongos , Animais , Diferenciação Celular/fisiologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Linhagem da Célula , Mamíferos
14.
J Cell Physiol ; 227(4): 1592-603, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21678411

RESUMO

Post-translational protein modification by ubiquitination, a signal for lysosomal or proteasomal proteolysis, can be regulated and reversed by deubiquitinating enzymes (DUBs). This study examined the roles of UCHL1 and UCHL3, two members of ubiquitin C-terminal hydrolase (UCH) family of DUBs, in murine fertilization and preimplantation development. Before fertilization, these proteins were associated with the oocyte cortex (UCHL1) and meiotic spindle (UCHL3). Intracytoplasmic injection of the general UCH-family inhibitor ubiquitin-aldehyde (UBAL) or antibodies against UCHL3 into mature metaphase II oocytes blocked fertilization by reducing sperm penetration of the zona pellucida and incorporation into the ooplasm, suggesting a role for cortical UCHL1 in sperm incorporation. Both UBAL and antibodies against UCHL1 injected at the onset of oocyte maturation (germinal vesicle stage) reduced the fertilizing ability of oocytes. The subfertile Uchl1(gad-/-) mutant mice showed an intriguing pattern of switched UCH localization, with UCHL3 replacing UCHL1 in the oocyte cortex. While fertilization defects were not observed, the embryos from homozygous Uchl1(gad-/-) mutant females failed to undergo morula compaction and did not form blastocysts in vivo, indicating a maternal effect related to UCHL1 deficiency. We conclude that the activity of oocyte UCHs contributes to fertilization and embryogenesis by regulating the physiology of the oocyte and blastomere cortex.


Assuntos
Desenvolvimento Embrionário/fisiologia , Fertilização/fisiologia , Ubiquitina Tiolesterase/fisiologia , Animais , Desenvolvimento Embrionário/genética , Feminino , Fertilização/efeitos dos fármacos , Fertilização/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Oócitos/efeitos dos fármacos , Oócitos/enzimologia , Oogênese/fisiologia , Gravidez , Ubiquitina Tiolesterase/antagonistas & inibidores , Ubiquitina Tiolesterase/deficiência , Ubiquitina Tiolesterase/genética , Ubiquitinas/farmacologia
15.
Reproduction ; 144(3): 349-59, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22733802

RESUMO

This study was conducted to investigate the effect of silencing nucleophosmin in the development of in vitro-produced bovine embryos. Nucleophosmin is an abundant multifunctional nucleolar phosphoprotein that participates, for example, in ribosome biogenesis or centrosome duplication control. We showed that although the transcription of embryonic nucleophosmin started already at late eight-cell stage, maternal protein was stored throughout the whole preimplantation development and was sufficient for the progression to the blastocyst stage. At the beginning of embryogenesis, translation occurs on maternally derived ribosomes, the functionally active nucleoli emerge during the fourth cell cycle in bovines. We found that nucleophosmin localisation reflected the nucleolar formation during bovine preimplantation development. The protein was detectable from the beginning of embryonic development. Before embryonic genome activation, it was dispersed throughout the nucleoplasm. The typical nucleolar localisation emerged with the formation of active nucleoli. At the blastocyst stage, nucleophosmin tended to localise especially to the trophectoderm. To see for how long is maternal nucleophosmin preserved, we silenced the nucleophosmin mRNA using RNA interference approach. Although a large portion of nucleophosmin was degraded in embryos with silenced nucleophosmin mRNA, an amount sufficient for normal development was preserved and we detected only a temporal delay in nucleophosmin relocalisation to nucleoli. Moreover, we observed no defects in nuclear shape or cytoskeleton previously found in somatic cells and only a non-significant decrease in embryonic developmental competence. Thus, our results show that the preserved amount of maternal nucleophosmin is sufficient for preimplantation development of bovine embryo.


Assuntos
Blastocisto/fisiologia , Bovinos/embriologia , Desenvolvimento Embrionário/fisiologia , Inativação Gênica , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Animais , Blastocisto/ultraestrutura , Nucléolo Celular/química , Expressão Gênica , Proteínas Nucleares/análise , Nucleofosmina , RNA Mensageiro/genética
16.
Eur J Cell Biol ; 101(2): 151210, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35240557

RESUMO

In mammalian females, oocytes are stored in the ovary and meiosis is arrested at the diplotene stage of prophase I. When females reach puberty oocytes are selectively recruited in cycles to grow, overcome the meiotic arrest, complete the first meiotic division and become mature (ready for fertilization). At a molecular level, the master regulator of prophase I arrest and meiotic resumption is the maturation-promoting factor (MPF) complex, formed by the active form of cyclin dependent kinase 1 (CDK1) and Cyclin B1. However, we still do not have complete information regarding the factors implicated in MPF activation. In this study we document that out of three mammalian serum-glucocorticoid kinase proteins (SGK1, SGK2, SGK3), mouse oocytes express only SGK1 with a phosphorylated (active) form dominantly localized in the nucleoplasm. Further, suppression of SGK1 activity in oocytes results in decreased CDK1 activation via the phosphatase cell division cycle 25B (CDC25B), consequently delaying or inhibiting nuclear envelope breakdown. Expression of exogenous constitutively active CDK1 can rescue the phenotype induced by SGK1 inhibition. These findings bring new insights into the molecular pathways acting upstream of MPF and a better understanding of meiotic resumption control by presenting a new key player SGK1 in mammalian oocytes.


Assuntos
Proteínas Imediatamente Precoces , Fator Promotor de Maturação , Animais , Pontos de Checagem do Ciclo Celular , Feminino , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Mamíferos/metabolismo , Fator Promotor de Maturação/metabolismo , Meiose , Prófase Meiótica I , Camundongos , Oócitos/metabolismo , Proteínas Serina-Treonina Quinases/genética
17.
Cell Tissue Res ; 346(1): 1-9, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21969023

RESUMO

Gametogenesis and fertilization are the key events in sexual reproduction. In the female, meiosis results in a large oocyte that is competent for fertilization and fundamental for the success of early embryonic development. Progression through meiosis is monitored by fine regulatory mechanisms. In this review, we focus on one of the most well-known regulatory elements, the E3 ligase APC/C, which mediates proteolytic degradation of a number of important substrates via the ubiquitin proteasome pathway (UPP). The UPP also indirectly regulates protein synthesis by affecting proteins involved in RNA metabolism, a process that is paramount for the transcriptionally silent oocyte. During the past few years, more evidence has accumulated to suggest that the UPP has an important role in zona pellucida penetration and gamete fusion in mammals. This review focuses on the function of the UPP in regulating oocyte meiotic maturation in mammals, with special attention to its role in chromosome segregation and polar body extrusion, its role in the acquisition of meiotic/developmental competence and recent advances in our understanding of the UPP role in fertilization.


Assuntos
Meiose/fisiologia , Oócitos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Interações Espermatozoide-Óvulo/fisiologia , Proteínas Ubiquitinadas/metabolismo , Animais , Feminino , Humanos , Masculino , Mamíferos , Oócitos/citologia , RNA , Ubiquitina-Proteína Ligases/metabolismo , Zona Pelúcida/metabolismo
18.
Noncoding RNA Res ; 6(2): 107-113, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34278057

RESUMO

Fully grown mammalian oocytes store a large amount of RNA synthesized during the transcriptionally active growth stage. A large part of the stored RNA belongs to the long non-coding class which contain either transcriptional noise or important contributors to cellular physiology. Despite the expanding number of studies related to lncRNAs, their influence on oocyte physiology remains enigmatic. We found an oocyte specific antisense, long non-coding RNA, "Rose" (lncRNA in Oocyte Specifically Expressed) expressed in two variants containing two and three non-coding exons, respectively. Rose is localized in the nucleus of transcriptionally active oocyte and in embryo with polysomal occupancy in the cytoplasm. Experimental overexpression of Rose in fully grown oocyte did not show any differences in meiotic maturation. However, knocking down Rose resulted in abnormalities in oocyte cytokinesis and impaired preimplantation embryo development. In conclusion, we have identified an oocyte-specific maternal lncRNA that is essential for successful mammalian oocyte and embryo development.

19.
J Mol Biol ; 433(19): 167166, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34293340

RESUMO

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/genética
20.
Biol Open ; 10(8)2021 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-34357391

RESUMO

Human multipotent neural stem cells could effectively be used for the treatment of a variety of neurological disorders. However, a defining signature of neural stem cell lines that would be expandable, non-tumorigenic, and differentiate into desirable neuronal/glial phenotype after in vivo grafting is not yet defined. Employing a mass spectrometry approach, based on selected reaction monitoring, we tested a panel of well-described culture conditions, and measured levels of protein markers routinely used to probe neural differentiation, i.e. POU5F1 (OCT4), SOX2, NES, DCX, TUBB3, MAP2, S100B, GFAP, GALC, and OLIG1. Our multiplexed assay enabled us to simultaneously identify the presence of pluripotent, multipotent, and lineage-committed neural cells, thus representing a powerful tool to optimize novel and highly specific propagation and differentiation protocols. The multiplexing capacity of this method permits the addition of other newly identified cell type-specific markers to further increase the specificity and quantitative accuracy in detecting targeted cell populations. Such an expandable assay may gain the advantage over traditional antibody-based assays, and represents a method of choice for quality control of neural stem cell lines intended for clinical use.


Assuntos
Diferenciação Celular , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Biomarcadores , Linhagem Celular , Linhagem da Célula/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Imuno-Histoquímica , Espectrometria de Massas , Neuroglia , Neurônios
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