RESUMO
Homologous chromosome segregation during meiosis I (MI) in mammalian oocytes is carried out by the acentrosomal MI spindles. Whereas studies in human oocytes identified Ran GTPase as a crucial regulator of the MI spindle function, experiments in mouse oocytes questioned the generality of this notion. Here, we use live-cell imaging with fluorescent probes and Förster resonance energy transfer (FRET) biosensors to monitor the changes in Ran and importin ß signaling induced by perturbations of Ran in mouse oocytes while examining the MI spindle dynamics. We show that unlike RanT24N employed in previous studies, a RanT24N, T42A double mutant inhibits RanGEF without perturbing cargo binding to importin ß and disrupts MI spindle function in chromosome segregation. Roles of Ran and importin ß in the coalescence of microtubule organizing centers (MTOCs) and MI spindle assembly are further supported by the use of the chemical inhibitor importazole, whose effects are partially rescued by the GTP hydrolysis-resistant RanQ69L mutant. These results indicate that RanGTP is essential for MI spindle assembly and function both in humans and mice.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Meiose/fisiologia , Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Oócitos/metabolismo , Fuso Acromático/fisiologia , beta Carioferinas/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Segregação de Cromossomos , Feminino , Fatores de Troca do Nucleotídeo Guanina/genética , Camundongos , Mutação , Proteínas Nucleares/genética , Oócitos/citologia , beta Carioferinas/genética , Proteína ran de Ligação ao GTP/genéticaRESUMO
Mammalian oocytes are arrested at meiotic prophase I. The dual-specificity phosphatase CDC25B is essential for cyclin-dependent kinase 1 (CDK1) activation that drives resumption of meiosis. CDC25B reverses the inhibitory effect of the protein kinases WEE1 and MYT1 on CDK1 activation. Cdc25b-/- female mice are infertile because oocytes cannot activate CDK1. To identify a role for CDC25B following resumption of meiosis, we restored CDK1 activation in Cdc25b-/- oocytes by inhibiting WEE1 and MYT1, or expressing EGFP-CDC25A or constitutively active EGFP-CDK1 from microinjected complementary RNAs. Forced CDK1 activation in Cdc25b-/- oocytes allowed resumption of meiosis, but oocytes mostly arrested at metaphase I (MI) with intact spindles. Similarly, approximately a third of Cdc25b+/- oocytes with a reduced amount of CDC25B arrested in MI. MI-arrested Cdc25b-/- oocytes also displayed a transient decrease in CDK1 activity similar to Cdc25b+/+ oocytes during the MI-MII transition, whereas Cdc25b+/- oocytes exhibited only a partial anaphase-promoting complex/cyclosome activation and anaphase I entry. Thus, CDC25B is necessary for the resumption of meiosis and the MI-MII transition.
Assuntos
Meiose , Oócitos , Anáfase , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Animais , Feminino , Mamíferos , Metáfase , Camundongos , Oócitos/metabolismo , Fosfatases cdc25RESUMO
The Aurora protein kinases are well-established regulators of spindle building and chromosome segregation in mitotic and meiotic cells. In mouse oocytes, there is significant Aurora kinase A (AURKA) compensatory abilities when the other Aurora kinase homologs are deleted. Whether the other homologs, AURKB or AURKC can compensate for loss of AURKA is not known. Using a conditional mouse oocyte knockout model, we demonstrate that this compensation is not reciprocal because female oocyte-specific knockout mice are sterile, and their oocytes fail to complete meiosis I. In determining AURKA-specific functions, we demonstrate that its first meiotic requirement is to activate Polo-like kinase 1 at acentriolar microtubule organizing centers (aMTOCs; meiotic spindle poles). This activation induces fragmentation of the aMTOCs, a step essential for building a bipolar spindle. We also show that AURKA is required for regulating localization of TACC3, another protein required for spindle building. We conclude that AURKA has multiple functions essential to completing MI that are distinct from AURKB and AURKC.
Assuntos
Aurora Quinase A/genética , Proteínas de Ciclo Celular/genética , Proteínas Fetais/genética , Meiose/genética , Proteínas Associadas aos Microtúbulos/genética , Oócitos/crescimento & desenvolvimento , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Animais , Aurora Quinase B/genética , Aurora Quinase C/genética , Divisão do Núcleo Celular/genética , Segregação de Cromossomos/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Camundongos , Centro Organizador dos Microtúbulos/metabolismo , Oócitos/metabolismo , Fuso Acromático/genética , Polos do Fuso/genética , Quinase 1 Polo-LikeRESUMO
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/genéticaRESUMO
Meiotic oocytes lack classic centrosomes and, therefore, bipolar spindle assembly depends on clustering of acentriolar microtubule-organizing centers (MTOCs) into two poles. However, the molecular mechanism regulating MTOC assembly into two poles is not fully understood. The kinase haspin (also known as GSG2) is required to regulate Aurora kinase C (AURKC) localization at chromosomes during meiosis I. Here, we show that inhibition of haspin perturbed MTOC clustering into two poles and the stability of the clustered MTOCs. Furthermore, we show that AURKC localizes to MTOCs in mouse oocytes. Inhibition of haspin perturbed the localization of AURKC at MTOCs, and overexpression of AURKC rescued the MTOC-clustering defects in haspin-inhibited oocytes. Taken together, our data uncover a role for haspin as a regulator of bipolar spindle assembly by regulating AURKC function at acentriolar MTOCs in oocytes.
Assuntos
Aurora Quinase C/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Centro Organizador dos Microtúbulos/metabolismo , Oócitos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Metáfase , Camundongos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Transporte Proteico , Fuso Acromático/metabolismoRESUMO
Polo-like kinase 1 (PLK1) is involved in essential events of cell cycle including mitosis in which it participates in centrosomal microtubule nucleation, spindle bipolarity establishment and cytokinesis. Although PLK1 function has been studied in cycling cancer cells, only limited data are known about its role in the first mitosis of mammalian zygotes. During the 1-cell stage of mouse embryo development, the acentriolar spindle is formed and the shift from acentriolar to centrosomal spindle formation progresses gradually throughout the preimplantation stage, thus providing a unique possibility to study acentriolar spindle formation. We have shown previously that PLK1 activity is not essential for entry into first mitosis, but is required for correct spindle formation and anaphase onset in 1-cell mouse embryos. In the present study, we extend this knowledge by employing quantitative confocal live cell imaging to determine spindle formation kinetics in the absence of PLK1 activity and answer the question whether metaphase arrest at PLK1-inhibited embryos is associated with low anaphase-promoting complex/cyclosome (APC/C) activity and consequently high securin level. We have shown that inhibition of PLK1 activity induces a delay in onset of acentriolar spindle formation during first mitosis. Although these PLK1-inhibited 1-cell embryos were finally able to form a bipolar spindle, not all chromosomes were aligned at the metaphase equator. PLK1-inhibited embryos were arrested in metaphase without any sign of APC/C activation with high securin levels. Our results document that PLK1 controls the onset of spindle assembly and spindle formation, and is essential for APC/C activation before anaphase onset in mouse zygotes.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fuso Acromático/metabolismo , Zigoto/metabolismo , Anáfase , Animais , Blastocisto , Proteínas de Ciclo Celular/antagonistas & inibidores , Centrossomo/metabolismo , Feminino , Cinética , Cinetocoros/metabolismo , Masculino , Camundongos , Microscopia Confocal , Mitose , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Pteridinas/farmacologia , Imagem com Lapso de Tempo , Zigoto/efeitos dos fármacos , Quinase 1 Polo-LikeRESUMO
Aurora-A kinase (AURKA), a member of the serine/threonine protein kinase family, is involved in multiple steps of mitotic progression. It regulates centrosome maturation, mitotic spindle formation, and cytokinesis. While studied extensively in somatic cells, little information is known about AURKA in the early cleavage mouse embryo with respect to acentrosomal spindle assembly. In vitro experiments in which AURKA was inactivated with specific inhibitor MLN8237 during the early stages of embryogenesis documented gradual arrest in the cleavage ability of the mouse embryo. In the AURKA-inhibited 1-cell embryos, spindle formation and anaphase onset were delayed and chromosome segregation was defective. AURKA inhibition increased apoptosis during early embryonic development. In conclusion these data suggest that AURKA is essential for the correct chromosome segregation in the first mitosis as a prerequisite for normal later development after first cleavage.
Assuntos
Aurora Quinase A/metabolismo , Segregação de Cromossomos/fisiologia , Mitose/fisiologia , Zigoto/fisiologia , Animais , Aurora Quinase A/antagonistas & inibidores , Azepinas/farmacologia , Segregação de Cromossomos/efeitos dos fármacos , Técnicas de Cultura Embrionária , Feminino , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microscopia Confocal , Microscopia de Fluorescência , Mitose/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Imagem com Lapso de Tempo , Zigoto/efeitos dos fármacosRESUMO
Aurora kinase A (AURKA) is an important mitotic kinase involved in the G2/M transition, centrosome maturation and separation, and spindle formation in somatic cells. We used transgenic models that specifically overexpress in mouse oocytes either wild-type (WT-AURKA) or a catalytically inactive (kinase-dead) (KD-AURKA) AURKA to gain new insights regarding the role of AURKA during oocyte maturation. AURKA activation occurs shortly after hCG administration that initiates maturation in vivo. Although AURKA activity is increased in WT-AURKA oocytes, resumption of meiosis is not observed in the absence of hCG administration. Control oocytes contain one to three microtubule organizing centers (MTOCs; centrosome equivalent) at prophase I. At the time of germinal vesicle breakdown (GVBD), the first visible marker of resumption of meiosis, the MTOC number increases. In WT-AURKA oocytes, the increase in MTOC number occurs prematurely but transiently without GVBD, whereas the increase in MTOC number does not occur in control and KD-AURKA oocytes. AURKA activation is biphasic with the initial activation not requiring CDC25B-CDK1 activity, whereas full activation, which is essential for the increase in MTOCs number, depends on CDK1 activity. AURKA activity also influences spindle length and regulates, independent of its protein kinase activity, the amount of MTOC associated with gamma-tubulin. Both WT-AURKA and KD-AURKA transgenic mice have normal fertility during first 6 mo of life. These results suggest that although AURKA is not a trigger kinase for G2/M transition in mouse oocytes, it regulates MTOC number and spindle length, and, independent of its protein kinase activity, gamma-tubulin recruitment to MTOCs.
Assuntos
Meiose/genética , Centro Organizador dos Microtúbulos/metabolismo , Oócitos/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Aurora Quinase A , Aurora Quinases , Células Cultivadas , Gonadotropina Coriônica/farmacologia , Ativação Enzimática/efeitos dos fármacos , Feminino , Células HeLa , Humanos , Masculino , Meiose/efeitos dos fármacos , Meiose/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Centro Organizador dos Microtúbulos/efeitos dos fármacos , Oócitos/enzimologia , Oócitos/metabolismo , Oogênese/efeitos dos fármacos , Oogênese/genética , Oogênese/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Mammalian oocytes are arrested at prophase I until puberty when luteinizing hormone (LH) induces resumption of meiosis of follicle-enclosed oocytes. Resumption of meiosis is tightly coupled with regulating cyclin-dependent kinase 1 (CDK1) activity. Prophase I arrest depends on inhibitory phosphorylation of CDK1 and anaphase-promoting complex-(APC-CDH1)-mediated regulation of cyclin B levels. Prophase I arrest is maintained by endogenously produced cyclic adenosine monophosphate (cAMP), which activates protein kinase A (PKA) that in turn phosphorylates (and activates) the nuclear kinase WEE2. In addition, PKA-mediated phosphorylation of the phosphatase CDC25B results in its cytoplasmic retention. The combined effect maintains low levels of CDK1 activity that are not sufficient to initiate resumption of meiosis. LH triggers synthesis of epidermal growth factor-like factors in mural granulosa cells and leads to reduced cGMP transfer from cumulus cells to oocytes via gap junctions that couple the two cell types. cGMP inhibits oocyte phosphodiesterase 3A (PDE3A) and a decline in oocyte cGMP results in increased PDE3A activity. The ensuing decrease in oocyte cAMP triggers maturation by alleviating the aforementioned phosphorylations of WEE2 and CDC25B. As a direct consequence CDC25B translocates into the nucleus. The resulting activation of CDK1 also promotes extrusion of WEE2 from the nucleus thereby providing a positive amplification mechanism for CDK1 activation. Other kinases, e.g. protein kinase B, Aurora kinase A and polo-like kinase 1, also participate in resumption of meiosis. Mechanisms governing meiotic prophase I arrest and resumption of meiosis share common features with DNA damage-induced mitotic G2-checkpoint arrest and checkpoint recovery, respectively. These common features include CDC14B-dependent activation of APC-CDH1 in prophase I arrested oocytes or G2-arrested somatic cells, and CDC25B-dependent cell cycle resumption in both oocytes and somatic cells.
Assuntos
Proliferação de Células , Fase G2 , Meiose , Oócitos/metabolismo , Oogênese , Transdução de Sinais , Fatores Etários , Ciclossomo-Complexo Promotor de Anáfase , Animais , Proteínas Cdh1 , Proteínas de Ciclo Celular/metabolismo , AMP Cíclico/metabolismo , Ciclina B1/metabolismo , Proteínas Inibidoras de Quinase Dependente de Ciclina/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Feminino , Prófase Meiótica I , Metáfase , Camundongos , Complexos Ubiquitina-Proteína Ligase/metabolismoRESUMO
CDK1 is a pivotal regulator of resumption of meiosis and meiotic maturation of oocytes. CDC25A/B/C are dual-specificity phosphatases and activate cyclin-dependent kinases (CDKs). Although CDC25C is not essential for either mitotic or meiotic cell cycle regulation, CDC25B is essential for CDK1 activation during resumption of meiosis. Cdc25a -/- mice are embryonic lethal and therefore a role for CDC25A in meiosis is unknown. We report that activation of CDK1 results in a maturation-associated decrease in the amount of CDC25A protein, but not Cdc25a mRNA, such that little CDC25A is present by metaphase I. In addition, expression of exogenous CDC25A overcomes cAMP-mediated maintenance of meiotic arrest. Microinjection of Gfp-Cdc25a and Gpf-Cdc25b mRNAs constructs reveals that CDC25A is exclusively localized to the nucleus prior to nuclear envelope breakdown (NEBD). In contrast, CDC25B localizes to cytoplasm in GV-intact oocytes and translocates to the nucleus shortly before NEBD. Over-expressing GFP-CDC25A, which compensates for the normal maturation-associated decrease in CDC25A, blocks meiotic maturation at MI. This MI block is characterized by defects in chromosome congression and spindle formation and a transient reduction in both CDK1 and MAPK activities. Lastly, RNAi-mediated reduction of CDC25A results in fewer oocytes resuming meiosis and reaching MII. These data demonstrate that CDC25A behaves differently during female meiosis than during mitosis, and moreover, that CDC25A has a function in resumption of meiosis, MI spindle formation and the MI-MII transition. Thus, both CDC25A and CDC25B are critical for meiotic maturation of oocytes.
Assuntos
Meiose , Oócitos/citologia , Oócitos/enzimologia , Fosfatases cdc25/metabolismo , Animais , AMP Cíclico/metabolismo , Feminino , Expressão Gênica , Camundongos , Oócitos/química , Fosfatases cdc25/análiseRESUMO
The aim of this study was to investigate the involvement of the serine/threonine protein kinase AKT (also called protein kinase B) in the control of meiosis of porcine denuded oocytes (DOs) matured in vitro. Western blot analysis revealed that the two principal AKT phosphorylation sites, Ser473 and Thr308, are phosphorylated at different stages of meiosis. In freshly isolated germinal vesicle (GV)-stage DOs, Ser473 was already phosphorylated. After the onset of oocyte maturation, the intensity of the Ser473 phosphorylation increased, however, which declined sharply when DOs underwent GV breakdown (GVBD) and remained at low levels in metaphase I- and II-stage (MI- and MII-stage). In contrast, phosphorylation of Thr308 was increased by the time of GVBD and reached maximum at MI-stage. A peak of AKT activity was noticed around GVBD and activity of AKT declined at MI-stage. To assess the role of AKT during meiosis, porcine DOs were cultured in 50 microM SH-6, a specific inhibitor of AKT. In SH-6-treated DOs, GVBD was not inhibited; on the contrary, a significant acceleration of meiosis resumption was observed. The dynamics of the Ser473 phosphorylation was not affected; however, phosphorylation of Thr308 was reduced, AKT activity was diminished at the time of GVBD, and meiotic progression was arrested in early MI-stage. Moreover, the activity of the cyclin-dependent kinase 1 (CDK1) and MAP kinase declined when SH-6-treated DOs underwent GVBD, indicating that AKT activity is involved in the regulation of CDK1 and MAP kinase. These results suggest that activity of AKT is not essential for induction of GVBD in porcine oocytes but plays a substantial role during progression of meiosis to MI/MII-stage.
Assuntos
Meiose , Proteína Oncogênica v-akt/fisiologia , Oócitos/fisiologia , Suínos , Animais , Bucladesina/farmacologia , Técnicas de Cultura de Células , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Feminino , Meiose/fisiologia , Proteína Oncogênica v-akt/antagonistas & inibidores , Proteína Oncogênica v-akt/metabolismo , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Oogênese/efeitos dos fármacos , Oogênese/fisiologia , Fosfatidilinositóis/farmacologia , Fosforilação/efeitos dos fármacos , Suínos/metabolismoRESUMO
Meiotic division is a dynamic process that exhibits active interactive behaviors amongst different intracellular structures and components for spindle assembly and chromosome segregation. Understanding the mechanisms of meiotic spindle assembly and chromosome segregation therefore requires a quantitative analysis of spatiotemporal relationships among different structures and components. In this chapter, we describe a method for triple-color live imaging of meiotic division in mouse oocytes. This approach combines the microinjection of RNAs encoding proteins tagged with green and red fluorescent proteins and the visualization of microtubules with the fluorogenic far-red probe SiR-Tubulin. This method enables the simultaneous spatiotemporal mapping of three different components of the spindle and chromosomes, which opens the way to quantitative analysis of their interactive behaviors.
Assuntos
Meiose , Microscopia de Fluorescência/métodos , Oócitos/citologia , Animais , Células Cultivadas , Segregação de Cromossomos , Feminino , Proteínas de Fluorescência Verde/metabolismo , Processamento de Imagem Assistida por Computador , Proteínas Luminescentes/metabolismo , Camundongos , Microtúbulos/metabolismo , Oócitos/fisiologia , Fuso Acromático/metabolismo , Tubulina (Proteína)/metabolismo , Proteína Vermelha FluorescenteRESUMO
Errors in chromosome segregation during female meiosis I occur frequently, and aneuploid embryos account for 1/3 of all miscarriages in humans [1]. Unlike mitotic cells that require two Aurora kinase (AURK) homologs to help prevent aneuploidy (AURKA and AURKB), mammalian germ cells also require a third (AURKC) [2, 3]. AURKA is the spindle-pole-associated homolog, and AURKB/C are the chromosome-localized homologs. In mitosis, AURKB has essential roles as the catalytic subunit of the chromosomal passenger complex (CPC), regulating chromosome alignment, kinetochore-microtubule attachments, cohesion, the spindle assembly checkpoint, and cytokinesis [4, 5]. In mouse oocyte meiosis, AURKC takes over as the predominant CPC kinase [6], although the requirement for AURKB remains elusive [7]. In the absence of AURKC, AURKB compensates, making defining potential non-overlapping functions difficult [6, 8]. To investigate the role(s) of AURKB and AURKC in oocytes, we analyzed oocyte-specific Aurkb and Aurkc single- and double-knockout (KO) mice. Surprisingly, we find that double KO female mice are fertile. We demonstrate that, in the absence of AURKC, AURKA localizes to chromosomes in a CPC-dependent manner. These data suggest that AURKC prevents AURKA from localizing to chromosomes by competing for CPC binding. This competition is important for adequate spindle length to support meiosis I. We also describe a unique requirement for AURKB to negatively regulate AURKC to prevent aneuploidy. Together, our work reveals oocyte-specific roles for the AURKs in regulating each other's localization and activity. This inter-kinase regulation is critical to support wild-type levels of fecundity in female mice.
Assuntos
Aurora Quinase A/genética , Aurora Quinase B/genética , Aurora Quinase C/genética , Meiose , Oócitos/metabolismo , Aneuploidia , Animais , Aurora Quinase A/metabolismo , Aurora Quinase B/metabolismo , Aurora Quinase C/metabolismo , Segregação de Cromossomos/genética , Feminino , Fertilidade/genética , CamundongosRESUMO
Because low levels of DNA double strand breaks (DSBs) appear not to activate the ATM-mediated prophase I checkpoint in full-grown oocytes, there may exist mechanisms to protect chromosome integrity during meiotic maturation. Using live imaging we demonstrate that low levels of DSBs induced by the radiomimetic drug Neocarzinostatin (NCS) increase the incidence of chromosome fragments and lagging chromosomes but do not lead to APC/C activation and anaphase onset delay. The number of DSBs, represented by γH2AX foci, significantly decreases between prophase I and metaphase II in both control and NCS-treated oocytes. Transient treatment with NCS increases >2-fold the number of DSBs in prophase I oocytes, but less than 30% of these oocytes enter anaphase with segregation errors. MRE11, but not ATM, is essential to detect DSBs in prophase I and is involved in H2AX phosphorylation during metaphase I. Inhibiting MRE11 by mirin during meiotic maturation results in anaphase bridges and also increases the number of γH2AX foci in metaphase II. Compromised DNA integrity in mirin-treated oocytes indicates a role for MRE11 in chromosome integrity during meiotic maturation.
Assuntos
Dano ao DNA/genética , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Meiose/genética , Oócitos/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Histonas/genética , Proteína Homóloga a MRE11 , Metáfase/genética , Camundongos , Oócitos/crescimento & desenvolvimento , Zinostatina/administração & dosagemRESUMO
Polo-like kinase 1 (PLK1) orchestrates multiple events of cell division. Although PLK1 function has been intensively studied in centriole-containing and rapidly cycling somatic cells, much less is known about its function in the meiotic divisions of mammalian oocytes, which arrest for a long period of time in prophase before meiotic resumption and lack centrioles for spindle assembly. Here, using specific small molecule inhibition combined with live mouse oocyte imaging, we comprehensively characterize meiotic PLK1's functions. We show that PLK1 becomes activated at meiotic resumption on microtubule organizing centers (MTOCs) and later at kinetochores. PLK1 is required for efficient meiotic resumption by promoting nuclear envelope breakdown. PLK1 is also needed to recruit centrosomal proteins to acentriolar MTOCs to promote normal spindle formation, as well as for stable kinetochore-microtubule attachment. Consequently, PLK1 inhibition leads to metaphase I arrest with misaligned chromosomes activating the spindle assembly checkpoint (SAC). Unlike in mitosis, the metaphase I arrest is not bypassed by the inactivation of the SAC. We show that PLK1 is required for the full activation of the anaphase promoting complex/cyclosome (APC/C) by promoting the degradation of the APC/C inhibitor EMI1 and is therefore essential for entry into anaphase I. Moreover, our data suggest that PLK1 is required for proper chromosome segregation and the maintenance of chromosome condensation during the meiosis I-II transition, independently of the APC/C. Thus, our results define the meiotic roles of PLK1 in oocytes and reveal interesting differential requirements of PLK1 between mitosis and oocyte meiosis in mammals.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Meiose/fisiologia , Oócitos/crescimento & desenvolvimento , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Animais , Western Blotting , Segregação de Cromossomos/fisiologia , Feminino , Processamento de Imagem Assistida por Computador , Cinetocoros/metabolismo , Camundongos , Microscopia Confocal , Centro Organizador dos Microtúbulos/metabolismo , Membrana Nuclear/metabolismo , Quinase 1 Polo-LikeRESUMO
At the end of the growth phase, mouse antral follicle oocytes acquire full developmental competence. In the mouse, this event is marked by the transition from the so-called non-surrounded nucleolus (NSN) chromatin configuration into the transcriptionally quiescent surrounded nucleolus (SN) configuration, which is named after a prominent perinucleolar condensed chromatin ring. However, the SN chromatin configuration alone is not sufficient for determining the developmental competence of the SN oocyte. There are additional nuclear and cytoplamic factors involved, while a little is known about the changes occurring in the cytoplasm during the NSN/SN transition. Here, we report functional analysis of maternal ELAVL2 an AU-rich element binding protein. Elavl2 gene encodes an oocyte-specific protein isoform (denoted ELAVL2°), which acts as a translational repressor. ELAVL2° is abundant in fully grown NSN oocytes, is ablated during the NSN/SN transition and remains low during the oocyte-to-embryo transition (OET). ELAVL2° overexpression during meiotic maturation causes errors in chromosome segregation, indicating the significance of naturally reduced ELAVL2° levels in SN oocytes. On the other hand, during oocyte growth, prematurely reduced Elavl2 expression results in lower yields of fully grown and meiotically matured oocytes, suggesting that Elavl2 is necessary for proper oocyte maturation. Moreover, Elavl2 knockdown showed stimulating effects on translation in fully grown oocytes. We propose that ELAVL2 has an ambivalent role in oocytes: it functions as a pleiotropic translational repressor in efficient production of fully grown oocytes, while its disposal during the NSN/SN transition contributes to the acquisition of full developmental competence.
Assuntos
Proteína Semelhante a ELAV 2/metabolismo , Meiose/fisiologia , Oócitos/metabolismo , Animais , Linhagem Celular , Proteína Semelhante a ELAV 2/genética , Feminino , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oócitos/citologia , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
The maturation of vertebrate oocyte into haploid gamete, the egg, consists of two specialized asymmetric cell divisions with no intervening S-phase. Ran GTPase has an essential role in relaying the active role of chromosomes in their own segregation by the meiotic process. In addition to its conserved role as a key regulator of macromolecular transport between nucleus and cytoplasm, Ran has important functions during cell division, including in mitotic spindle assembly and in the assembly of nuclear envelope at the exit from mitosis. The cellular functions of Ran are mediated by RanGTP interactions with nuclear transport receptors (NTRs) related to importin ß and depend on the existence of chromosome-centered RanGTP gradient. Live imaging with FRET biosensors indeed revealed the existence of RanGTP gradient throughout mouse oocyte maturation. NTR-dependent transport of cell cycle regulators including cyclin B1, Wee2, and Cdc25B between the oocyte cytoplasm and germinal vesicle (GV) is required for normal resumption of meiosis. After GVBD in mouse oocytes, RanGTP gradient is required for timely meiosis I (MI) spindle assembly and provides long-range signal directing egg cortex differentiation. However, RanGTP gradient is not required for MI spindle migration and may be dispensable for MI spindle function in chromosome segregation. In contrast, MII spindle assembly and function in maturing mouse and Xenopus laevis eggs depend on RanGTP gradient, similar to X. laevis MII-derived egg extracts.
Assuntos
Diferenciação Celular/fisiologia , Meiose/fisiologia , Oócitos/citologia , Oócitos/fisiologia , Oogênese/fisiologia , Vertebrados/embriologia , Proteína ran de Ligação ao GTP/fisiologia , Animais , Diferenciação Celular/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/fisiologia , Feminino , Humanos , Meiose/genética , Oócitos/metabolismo , Oogênese/genética , Vertebrados/genética , Vertebrados/metabolismo , beta Carioferinas/fisiologia , Proteína ran de Ligação ao GTP/genética , Proteína ran de Ligação ao GTP/metabolismoRESUMO
Aurora kinase A (AURKA), which is a centrosome-localized serine/threonine kinase crucial for cell cycle control, is critically involved in centrosome maturation and spindle assembly in somatic cells. Active T288 phosphorylated AURKA localizes to the centrosome in the late G(2) and also spreads to the minus ends of mitotic spindle microtubules. AURKA activates centrosomal CDC25B and recruits cyclin B1 to centrosomes. We report here functions for AURKA in meiotic maturation of mouse oocytes, which is a model system to study the G(2) to M transition. Whereas AURKA is present throughout the entire GV-stage oocyte with a clear accumulation on microtubule organizing centers (MTOC), active AURKA becomes entirely localized to MTOCs shortly before germinal vesicle breakdown. In contrast to somatic cells in which active AURKA is present at the centrosomes and minus ends of microtubules, active AURKA is mainly located on MTOCs at metaphase I (MI) in oocytes. Inhibitor studies using Roscovitine (CDK1 inhibitor), LY-294002 (PI3K inhibitor) and SH-6 (PKB inhibitor) reveal that activation of AURKA localized on MTOCs is independent on PI3K-PKB and CDK1 signaling pathways and MOTC amplification is observed in roscovitine- and SH-6-treated oocytes that fail to undergo nuclear envelope breakdown. Moreover, microinjection of Aurka mRNA into GV-stage oocytes cultured in 3-isobutyl-1-methyl xanthine (IBMX)-containing medium to prevent maturation also results in MOTC amplification in the absence of CDK1 activation. Overexpression of AURKA also leads to formation of an abnormal MI spindle, whereas RNAi-mediated reduction of AURKA interferes with resumption of meiosis and spindle assembly. Results of these experiments indicate that AURKA is a critical MTOC-associated component involved in resumption of meiosis, MTOC multiplication, proper spindle formation and the metaphase I-metaphase II transition.
Assuntos
Meiose/genética , Oócitos/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Aurora Quinase A , Aurora Quinases , Blastodisco/metabolismo , Ciclo Celular/genética , Ciclo Celular/fisiologia , Quinases Ciclina-Dependentes/metabolismo , Quinases Ciclina-Dependentes/fisiologia , Feminino , Células HeLa , Humanos , Meiose/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Centro Organizador dos Microtúbulos/metabolismo , Células NIH 3T3 , Oócitos/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinases/fisiologia , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/fisiologia , Fuso Acromático/metabolismoRESUMO
BACKGROUND INFORMATION: In fully grown mouse oocytes, a decrease in cAMP concentration precedes and is linked to CDK1 (cyclin-dependent kinase 1) activation. The molecular mechanism for this coupling, however, is not defined. PKB (protein kinase B, also called AKT) is implicated in CDK1 activation in lower species. During resumption of meiosis in starfish oocytes, MYT1, a negative regulator of CDK1, is phosphorylated by PKB in an inhibitory manner. It can imply that PKB is also involved in CDK1 activation in mammalian oocytes. RESULTS: We monitored activation of PKB and CDK1 during maturation of mouse oocytes. PKB phosphorylation and activation preceded GVBD (germinal vesicle breakdown) in oocytes maturing either in vitro or in vivo. Activation was transient and PKB activity was markedly reduced when virtually all of the oocytes had undergone GVBD. PKB activation was independent of CDK1 activity, because although butyrolactone I prevented CDK1 activation and GVBD, PKB was nevertheless transiently phosphorylated and activated. LY-294002, an inhibitor of phosphoinositide 3-kinase-PKB signalling, suppressed activation of PKB and CDK1 as well as resumption of meiosis. OA (okadaic acid)-sensitive phosphatases are involved in PKB-activity regulation, because OA induced PKB hyperphosphorylation. During resumption of meiosis, PKB phosphorylated on Ser(473) is associated with nuclear membrane and centrosome, whereas PKB phosphorylated on Thr(308) is localized on centrosome only. CONCLUSIONS: The results of the present paper indicate that PKB is involved in CDK1 activation and resumption of meiosis in mouse oocytes. The presence of phosphorylated PKB on centrosome at the time of GVBD suggests its important role for an initial CDK1 activation.