RESUMO
Faithful DNA replication is essential for genome integrity1-4. Under-replicated DNA leads to defects in chromosome segregation, which are common during embryogenesis5-8. However, the regulation of DNA replication remains poorly understood in early mammalian embryos. Here we constructed a single-cell genome-wide DNA replication atlas of pre-implantation mouse embryos and identified an abrupt replication program switch accompanied by a transient period of genomic instability. In 1- and 2-cell embryos, we observed the complete absence of a replication timing program, and the entire genome replicated gradually and uniformly using extremely slow-moving replication forks. In 4-cell embryos, a somatic-cell-like replication timing program commenced abruptly. However, the fork speed was still slow, S phase was extended, and markers of replication stress, DNA damage and repair increased. This was followed by an increase in break-type chromosome segregation errors specifically during the 4-to-8-cell division with breakpoints enriched in late-replicating regions. These errors were rescued by nucleoside supplementation, which accelerated fork speed and reduced the replication stress. By the 8-cell stage, forks gained speed, S phase was no longer extended and chromosome aberrations decreased. Thus, a transient period of genomic instability exists during normal mouse development, preceded by an S phase lacking coordination between replisome-level regulation and megabase-scale replication timing regulation, implicating a link between their coordination and genome stability.
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Sex chromosome disorders severely compromise gametogenesis in both males and females. In oogenesis, the presence of an additional Y chromosome or the loss of an X chromosome disturbs the robust production of oocytes1-5. Here we efficiently converted the XY chromosome set to XX without an additional Y chromosome in mouse pluripotent stem (PS) cells. In addition, this chromosomal alteration successfully eradicated trisomy 16, a model of Down's syndrome, in PS cells. Artificially produced euploid XX PS cells differentiated into mature oocytes in culture with similar efficiency to native XX PS cells. Using this method, we differentiated induced pluripotent stem cells from the tail of a sexually mature male mouse into fully potent oocytes, which gave rise to offspring after fertilization. This study provides insights that could ameliorate infertility caused by sex chromosome or autosomal disorders, and opens the possibility of bipaternal reproduction.
Assuntos
Engenharia Genética , Técnicas In Vitro , Oócitos , Cromossomo X , Animais , Feminino , Masculino , Camundongos , Oócitos/metabolismo , Oócitos/fisiologia , Cromossomo X/genética , Cromossomo Y/genética , Células-Tronco Pluripotentes/metabolismo , Síndrome de Down/genética , Síndrome de Down/terapia , Fertilização , Infertilidade/terapia , Homossexualidade Masculina , Transtornos dos Cromossomos Sexuais/complicações , Transtornos dos Cromossomos Sexuais/genética , Transtornos dos Cromossomos Sexuais/terapia , Engenharia Genética/métodosRESUMO
Recent studies have reported the differentiation of pluripotent cells into oocytes in vitro. However, the developmental competence of in vitro-generated oocytes remains low. Here, we perform a comprehensive comparison of mouse germ cell development in vitro over all culture steps versus in vivo with the goal to understand mechanisms underlying poor oocyte quality. We show that the in vitro differentiation of primordial germ cells to growing oocytes and subsequent follicle growth is critical for competence for preimplantation development. Systematic transcriptome analysis of single oocytes that were subjected to different culture steps identifies genes that are normally upregulated during oocyte growth to be susceptible for misregulation during in vitro oogenesis. Many misregulated genes are Polycomb targets. Deregulation of Polycomb repression is therefore a key cause and the earliest defect known in in vitro oocyte differentiation. Conversely, structurally normal in vitro-derived oocytes fail at zygotic genome activation and show abnormal acquisition of 5-hydroxymethylcytosine on maternal chromosomes. Our data identify epigenetic regulation at an early stage of oogenesis limiting developmental competence and suggest opportunities for future improvements.
Assuntos
Epigênese Genética , Oócitos , Feminino , Animais , Camundongos , Folículo Ovariano , Oogênese/genética , Células GerminativasRESUMO
During female germline development, oocytes become a highly specialized cell type and form a maternal cytoplasmic store of crucial factors. Oocyte growth is triggered at the transition from primordial to primary follicle and is accompanied by dynamic changes in gene expression1, but the gene regulatory network that controls oocyte growth remains unknown. Here we identify a set of transcription factors that are sufficient to trigger oocyte growth. By investigation of the changes in gene expression and functional screening using an in vitro mouse oocyte development system, we identified eight transcription factors, each of which was essential for the transition from primordial to primary follicle. Notably, enforced expression of these transcription factors swiftly converted pluripotent stem cells into oocyte-like cells that were competent for fertilization and subsequent cleavage. These transcription-factor-induced oocyte-like cells were formed without specification of primordial germ cells, epigenetic reprogramming or meiosis, and demonstrate that oocyte growth and lineage-specific de novo DNA methylation are separable from the preceding epigenetic reprogramming in primordial germ cells. This study identifies a core set of transcription factors for orchestrating oocyte growth, and provides an alternative source of ooplasm, which is a unique material for reproductive biology and medicine.
Assuntos
Oócitos/metabolismo , Oogênese/genética , Fatores de Transcrição/metabolismo , Animais , Linhagem da Célula , Epigênese Genética , Feminino , Fertilização , Meiose , Metilação , Camundongos , Oócitos/citologia , Folículo Ovariano/citologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismoRESUMO
Chromosomes must establish stable biorientation prior to anaphase to achieve faithful segregation during cell division. The detailed process by which chromosomes are bioriented and how biorientation is coordinated with spindle assembly and chromosome congression remain unclear. Here, we provide complete 3D kinetochore-tracking datasets throughout cell division by high-resolution imaging of meiosis I in live mouse oocytes. We show that in acentrosomal oocytes, chromosome congression forms an intermediate chromosome configuration, the prometaphase belt, which precedes biorientation. Chromosomes then invade the elongating spindle center to form the metaphase plate and start biorienting. Close to 90% of all chromosomes undergo one or more rounds of error correction of their kinetochore-microtubule attachments before achieving correct biorientation. This process depends on Aurora kinase activity. Our analysis reveals the error-prone nature of homologous chromosome biorientation, providing a possible explanation for the high incidence of aneuploid eggs observed in mammals, including humans.
Assuntos
Segregação de Cromossomos , Cinetocoros/metabolismo , Oócitos/citologia , Animais , Cromossomos/metabolismo , Humanos , Meiose , Camundongos , Microtúbulos/metabolismoRESUMO
Mammalian oocytes undergo a long-term meiotic arrest that can last for almost the entire reproductive lifespan. This arrest occurs after DNA replication and is prolonged with age, which poses a challenge to oocytes in maintaining replication-dependent chromosomal proteins required for the completion of meiosis. In this study, we show that chromosomal histones are reduced with age in mouse oocytes. Both types of histone H3 variants, replication-dependent H3.1/H3.2 and replication-independent H3.3, decrease with age. Aging-associated histone reduction is associated with transcriptomic features that are caused by genetic depletion of histone H3.3. Neither the genetic reduction of chromosomal H3.1/H3.2 nor H3.3 accelerates the aging-associated increase in premature chromosome separation that causes meiotic segregation errors. We suggest that aging-associated reduction of chromosomal histones is linked to several transcriptomic abnormalities but does not significantly contribute to errors in meiotic chromosome segregation during the reproductive lifespan of mice.
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Microinjection of spermatozoa or spermatids into oocytes is a major choice for infertility treatment. However, the use of premeiotic spermatocytes has never been considered because of its technical problems. Here, we show that the efficiency of spermatocyte injection in mice can be improved greatly by reducing the size of the recipient oocytes. Live imaging showed that the underlying mechanism involves reduced premature separation of the spermatocyte's meiotic chromosomes, which produced much greater (19% vs. 1%) birth rates in smaller oocytes. Application of this technique to spermatocyte arrest caused by STX2 deficiency, an azoospermia factor also found in humans, resulted in the production of live offspring. Thus, the microinjection of primary spermatocytes into oocytes may be a potential treatment for overcoming a form of nonobstructive azoospermia caused by meiotic failure.
Assuntos
Azoospermia , Espermatócitos , Animais , Humanos , Masculino , Meiose , Camundongos , Oócitos , EspermátidesRESUMO
An Amendment to this Article has been published and is linked from the HTML version of this paper.
RESUMO
In mouse oocytes, acentriolar MTOCs functionally replace centrosomes and act as microtubule nucleation sites. Microtubules nucleated from MTOCs initially assemble into an unorganized ball-like structure, which then transforms into a bipolar spindle carrying MTOCs at its poles, a process called spindle bipolarization. In mouse oocytes, spindle bipolarization is promoted by kinetochores but the mechanism by which kinetochore-microtubule attachments contribute to spindle bipolarity remains unclear. This study demonstrates that the stability of kinetochore-microtubule attachment is essential for confining MTOC positions at the spindle poles and for limiting spindle elongation. MTOC sorting is gradual and continues even in the metaphase spindle. When stable kinetochore-microtubule attachments are disrupted, the spindle is unable to restrict MTOCs at its poles and fails to terminate its elongation. Stable kinetochore fibers are directly connected to MTOCs and to the spindle poles. These findings suggest a role for stable kinetochore-microtubule attachments in fine-tuning acentrosomal spindle bipolarity.
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Cinetocoros , Fuso Acromático , Animais , Camundongos , Centro Organizador dos Microtúbulos , Microtúbulos , OócitosRESUMO
The study of the size of cells and organelles has a long history, dating back to the 1600s when cells were defined. In particular, various methods have elucidated the size of the nucleus and the mitotic spindle in several species. However, little research has been conducted on oocyte size and organelles in mammals, and many questions remain to be answered. The appropriate size is essential to cell function properly. Oocytes have a very large cytoplasm, which is more than 100 times larger than that of general somatic cells in mammals. In this review, we discuss how oocytes acquire an enormous cytoplasmic size and the adverse effects of a large cytoplasmic size on cellular functions.
Assuntos
Meiose , Oócitos , Animais , Citoplasma , Fuso Acromático/fisiologia , MamíferosRESUMO
Chromosome segregation requires the formation of a bipolar spindle. The timely bipolarization of the acentrosomal spindle during meiosis I in mouse oocytes depends on the antiparallel microtubule crosslinker Prc1. How Prc1 is regulated in oocytes remains poorly understood. In this study, we show that the kinase Cdk1 negatively regulates the spindle localization of Prc1 in mouse oocytes. The acute inhibition of Cdk1 activity led to excessive localization of Prc1 at the spindle and kinetochores, whereas the overactivation of Cdk1 had opposite effects. The overexpression of Prc1 carrying mutations at Cdk1-mediated phosphorylation sites increased its localization to the spindle, accelerated spindle bipolarization and caused spindle-checkpoint-dependent arrest at metaphase I. Overactivation of Cdk1 delayed spindle bipolarization, which was reversed by the overexpression of a phospho-mutant form but not the wild-type form of Prc1. These results suggest that Cdk1-mediated phosphorylation negatively regulates Prc1 localization to ensure the timely bipolarization of the acentrosomal spindle during meiosis I in mammalian oocytes.
Assuntos
Proteína Quinase CDC2/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Animais , Proteína Quinase CDC2/genética , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos , Feminino , Cinetocoros/metabolismo , Masculino , Metáfase , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Microtúbulos/metabolismo , Oócitos/metabolismo , Fuso Acromático/genéticaRESUMO
The accuracy of the two sequential meiotic divisions in oocytes is essential for creating a haploid gamete with a normal chromosomal content. Here, we have analysed the 3D dynamics of chromosomes during the second meiotic division in live mouse oocytes. We find that chromosomes form stable kinetochore-microtubule attachments at the end of prometaphase II stage that are retained until anaphase II onset. Remarkably, we observe that more than 20% of the kinetochore-microtubule attachments at the metaphase II stage are merotelic or lateral. However, < 1% of all chromosomes at onset of anaphase II are found to lag at the spindle equator and < 10% of the laggards missegregate and give rise to aneuploid gametes. Our results demonstrate that aberrant kinetochore-microtubule attachments are not corrected at the metaphase stage of the second meiotic division. Thus, the accuracy of the chromosome segregation process in mouse oocytes during meiosis II is ensured by an efficient correction process acting at the anaphase stage.
Assuntos
Anáfase , Cinetocoros/ultraestrutura , Metáfase , Microtúbulos/ultraestrutura , Oócitos/ultraestrutura , Sequência de Aminoácidos , Animais , Cromátides/metabolismo , Cromátides/ultraestrutura , Segregação de Cromossomos , Feminino , Humanos , Cinetocoros/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microtúbulos/metabolismo , Oócitos/metabolismo , Espermatócitos/metabolismo , Espermatócitos/ultraestrutura , Fuso Acromático/metabolismo , Fuso Acromático/ultraestrutura , Imagem com Lapso de TempoRESUMO
The kinetochore is the crucial apparatus regulating chromosome segregation in mitosis and meiosis. Particularly in meiosis I, unlike in mitosis, sister kinetochores are captured by microtubules emanating from the same spindle pole (mono-orientation) and centromeric cohesion mediated by cohesin is protected in the following anaphase. Although meiotic kinetochore factors have been identified only in budding and fission yeasts, these molecules and their functions are thought to have diverged earlier. Therefore, a conserved mechanism for meiotic kinetochore regulation remains elusive. Here we have identified in mouse a meiosis-specific kinetochore factor that we termed MEIKIN, which functions in meiosis I but not in meiosis II or mitosis. MEIKIN plays a crucial role in both mono-orientation and centromeric cohesion protection, partly by stabilizing the localization of the cohesin protector shugoshin. These functions are mediated mainly by the activity of Polo-like kinase PLK1, which is enriched to kinetochores in a MEIKIN-dependent manner. Our integrative analysis indicates that the long-awaited key regulator of meiotic kinetochore function is Meikin, which is conserved from yeasts to humans.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Sequência Conservada , Cinetocoros/metabolismo , Meiose , Animais , Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/deficiência , Proteínas Cromossômicas não Histona/genética , Feminino , Humanos , Infertilidade/genética , Infertilidade/metabolismo , Masculino , Camundongos , Dados de Sequência Molecular , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Quinase 1 Polo-LikeRESUMO
Proper kinetochore-microtubule attachment is essential for correct chromosome segregation. Therefore, cells normally possess multiple mechanisms for the prevention of errors in kinetochore-microtubule attachments and for selective stabilization of correct attachments. However, the oocyte, a cell that produces an egg through meiosis, exhibits a high frequency of errors in kinetochore-microtubule attachments. These attachment errors predispose oocytes to chromosome segregation errors, resulting in aneuploidy in eggs. This review aims to provide possible explanations for the error-prone nature of oocytes by examining key differences among other cell types in the mechanisms for the establishment of kinetochore-microtubule attachments.
Assuntos
Aneuploidia , Cinetocoros/fisiologia , Meiose , Microtúbulos/fisiologia , Oócitos/citologia , Oócitos/fisiologia , Animais , Feminino , Humanos , MamíferosRESUMO
Shugoshin (Sgo) is a conserved centromeric protein. Mammalian Sgo1 collaborates with protein phosphatase 2A (PP2A) to protect mitotic cohesin from the prophase dissociation pathway. Although another shugoshin-like protein, Sgo2, is required for the centromeric protection of cohesion in germ cells, its precise molecular function remains largely elusive. We demonstrate that hSgo2 plays a dual role in chromosome congression and centromeric protection of cohesion in HeLa cells, while the latter function is exposed only in perturbed mitosis. These functions partly overlap with those of Aurora B, a kinase setting faithful chromosome segregation. Accordingly, we identified the phosphorylation of hSgo2 by Aurora B at the N-terminal coiled-coil region and the middle region, and showed that these phosphorylations separately promote binding of hSgo2 to PP2A and MCAK, factors required for centromeric protection and chromosome congression, respectively. Furthermore, these phosphorylations are essential for localizing PP2A and MCAK to centromeres. This mechanism seems applicable to germ cells as well. Thus, our study identifies Sgo2 as a hitherto unknown crucial cellular substrate of Aurora B in mammalian cells.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Cinesinas/metabolismo , Proteína Fosfatase 2/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Aurora Quinase B , Aurora Quinases , Células Cultivadas , Células HeLa , Humanos , Fosforilação , Transporte ProteicoRESUMO
Meiotic errors of relatively small chromosomes in oocytes result in egg aneuploidies that cause miscarriages and congenital diseases. Unlike somatic cells, which preferentially mis-segregate larger chromosomes, aged oocytes preferentially mis-segregate smaller chromosomes through unclear processes. Here, we provide a comprehensive three-dimensional chromosome identifying-and-tracking dataset throughout meiosis I in live mouse oocytes. This analysis reveals a prometaphase pathway that actively moves smaller chromosomes to the inner region of the metaphase plate. In the inner region, chromosomes are pulled by stronger bipolar microtubule forces, which facilitates premature chromosome separation, a major cause of segregation errors in aged oocytes. This study reveals a spatial pathway that facilitates aneuploidy of small chromosomes preferentially in aged eggs and implicates the role of the M phase in creating a chromosome size-based spatial arrangement.
Assuntos
Aneuploidia , Segregação de Cromossomos , Meiose , Microtúbulos , Oócitos , Animais , Feminino , Camundongos , Cromossomos de Mamíferos/genética , Metáfase , Microtúbulos/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Conjuntos de Dados como AssuntoRESUMO
Sister chromatid cohesion, mediated by a complex called cohesin, is crucial--particularly at centromeres--for proper chromosome segregation in mitosis and meiosis. In animal mitotic cells, phosphorylation of cohesin promotes its dissociation from chromosomes, but centromeric cohesin is protected by shugoshin until kinetochores are properly captured by the spindle microtubules. However, the mechanism of shugoshin-dependent protection of cohesin is unknown. Here we find a specific subtype of serine/threonine protein phosphatase 2A (PP2A) associating with human shugoshin. PP2A colocalizes with shugoshin at centromeres and is required for centromeric protection. Purified shugoshin complex has an ability to reverse the phosphorylation of cohesin in vitro, suggesting that dephosphorylation of cohesin is the mechanism of protection at centromeres. Meiotic shugoshin of fission yeast also associates with PP2A, with both proteins collaboratively protecting Rec8-containing cohesin at centromeres. Thus, we have revealed a conserved mechanism of centromeric protection of eukaryotic chromosomes in mitosis and meiosis.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Pareamento Cromossômico , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas de Ciclo Celular/genética , Células HeLa , Humanos , Meiose , Mitose , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Fosfoproteínas Fosfatases/classificação , Fosfoproteínas Fosfatases/genética , Fosfoproteínas/metabolismo , Fosforilação , Ligação Proteica , Proteína Fosfatase 2 , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , CoesinasRESUMO
Chromosome segregation errors in oocytes lead to the production of aneuploid eggs, which are the leading cause of pregnancy loss and of several congenital diseases such as Down syndrome. The frequency of chromosome segregation errors in oocytes increases with maternal age, especially at a late stage of reproductive life. How aging at various life stages affects oocytes differently remains poorly understood. In this study, we describe aging-associated changes in the transcriptome profile of mouse oocytes throughout reproductive life. Our single-oocyte comprehensive RNA sequencing using RamDA-seq revealed that oocytes undergo transcriptome changes at a late reproductive stage, whereas their surrounding cumulus cells exhibit transcriptome changes at an earlier stage. Calorie restriction, a paradigm that reportedly prevents aging-associated egg aneuploidy, promotes a transcriptome shift in oocytes with the up-regulation of genes involved in chromosome segregation. This shift is accompanied by the improved maintenance of chromosomal cohesin, the loss of which is a hallmark of oocyte aging and causes chromosome segregation errors. These findings have implications for understanding how oocytes undergo aging-associated functional decline throughout their reproductive life in a context-dependent manner.
Assuntos
Envelhecimento/genética , Restrição Calórica/métodos , Perfilação da Expressão Gênica/métodos , Oócitos/metabolismo , Animais , Feminino , Humanos , CamundongosRESUMO
Zygotes require two accurate sets of parental chromosomes, one each from the mother and the father, to undergo normal embryogenesis. However, upon egg-sperm fusion in vertebrates, the zygote has three sets of chromosomes, one from the sperm and two from the egg. The zygote therefore eliminates one set of maternal chromosomes (but not the paternal chromosomes) into the polar body through meiosis, but how the paternal chromosomes are protected from maternal meiosis has been unclear. Here we report that RanGTP and F-actin dynamics prevent egg-sperm fusion in proximity to maternal chromosomes. RanGTP prevents the localization of Juno and CD9, egg membrane proteins that mediate sperm fusion, at the cell surface in proximity to maternal chromosomes. Following egg-sperm fusion, F-actin keeps paternal chromosomes away from maternal chromosomes. Disruption of these mechanisms causes the elimination of paternal chromosomes during maternal meiosis. This study reveals a novel critical mechanism that prevents aneuploidy in zygotes.
Assuntos
Citoesqueleto de Actina/metabolismo , Cromossomos/metabolismo , Fertilização , Proteína ran de Ligação ao GTP/metabolismo , Animais , Células Cultivadas , Feminino , Humanos , Camundongos , Camundongos EndogâmicosRESUMO
Meiosis comprises a pair of specialized nuclear divisions that produce haploid germ cells. To accomplish this, sister chromatids must segregate together during the first meiotic division (meiosis I), which requires that sister chromatid cohesion persists at centromeres. The factors that protect centromeric cohesion during meiosis I have remained elusive. Here we identify Sgo1 (shugoshin), a protector of the centromeric cohesin Rec8 in fission yeast. We also identify a homologue of Sgo1 in budding yeast. We provide evidence that shugoshin is widely conserved among eukaryotes. Moreover, we identify Sgo2, a paralogue of shugoshin in fission yeast, which is required for faithful mitotic chromosome segregation. Localization of Sgo1 and Sgo2 at centromeres requires the kinase Bub1, identifying shugoshin as a crucial target for the kinetochore function of Bub1. These findings provide insights into the evolution of meiosis and kinetochore regulation during mitosis and meiosis.