RESUMO
Most human embryos are aneuploid. Aneuploidy frequently arises during the early mitotic divisions of the embryo, but its origin remains elusive. Human zygotes that cluster their nucleoli at the pronuclear interface are thought to be more likely to develop into healthy euploid embryos. Here, we show that the parental genomes cluster with nucleoli in each pronucleus within human and bovine zygotes, and clustering is required for the reliable unification of the parental genomes after fertilization. During migration of intact pronuclei, the parental genomes polarize toward each other in a process driven by centrosomes, dynein, microtubules, and nuclear pore complexes. The maternal and paternal chromosomes eventually cluster at the pronuclear interface, in direct proximity to each other, yet separated. Parental genome clustering ensures the rapid unification of the parental genomes on nuclear envelope breakdown. However, clustering often fails, leading to chromosome segregation errors and micronuclei, incompatible with healthy embryo development.
Assuntos
Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Aneuploidia , Animais , Bovinos , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Centrossomo/metabolismo , Segregação de Cromossomos/fisiologia , Cromossomos/metabolismo , Fertilização/genética , Humanos , Masculino , Microtúbulos/metabolismo , Mitose , Oócitos/metabolismo , Espermatozoides/metabolismo , Zigoto/metabolismoRESUMO
Accurate chromosome segregation during meiosis is crucial for reproduction. Human and porcine oocytes transiently cluster their chromosomes before the onset of spindle assembly and subsequent chromosome segregation. The mechanism and function of chromosome clustering are unknown. Here we show that chromosome clustering is required to prevent chromosome losses in the long gap phase between nuclear envelope breakdown and the onset of spindle assembly, and to promote the rapid capture of all chromosomes by the acentrosomal spindle. The initial phase of chromosome clustering is driven by a dynamic network of Formin-2- and Spire-nucleated actin cables. The actin cables form in the disassembling nucleus and migrate towards the nuclear centre, moving the chromosomes centripetally by interacting with their arms and kinetochores as they migrate. A cage of stable microtubule loops drives the late stages of chromosome clustering. Together, our data establish a crucial role for chromosome clustering in accurate progression through meiosis.
Assuntos
Actinas , Oócitos , Humanos , Animais , Suínos , Actinas/genética , Actinas/metabolismo , Oócitos/metabolismo , Meiose/genética , Microtúbulos/metabolismo , Cinetocoros/metabolismo , Segregação de Cromossomos , Fuso Acromático/genética , Fuso Acromático/metabolismo , Mamíferos/metabolismoRESUMO
Full-grown oocytes are transcriptionally silent and must stably maintain the messenger RNAs (mRNAs) needed for oocyte meiotic maturation and early embryonic development. However, where and how mammalian oocytes store maternal mRNAs is unclear. Here, we report that mammalian oocytes accumulate mRNAs in a mitochondria-associated ribonucleoprotein domain (MARDO). MARDO assembly around mitochondria was promoted by the RNA-binding protein ZAR1 and directed by an increase in mitochondrial membrane potential during oocyte growth. MARDO foci coalesced into hydrogel-like matrices that clustered mitochondria. Maternal mRNAs stored in the MARDO were translationally repressed. Loss of ZAR1 disrupted the MARDO, dispersed mitochondria, and caused a premature loss of MARDO-localized mRNAs. Thus, a mitochondria-associated membraneless compartment controls mitochondrial distribution and regulates maternal mRNA storage, translation, and decay to ensure fertility in mammals.
Assuntos
Mitocôndrias , Oócitos , RNA Mensageiro Estocado , Animais , Feminino , Hidrogéis , Mitocôndrias/genética , Mitocôndrias/metabolismo , Oócitos/metabolismo , RNA Mensageiro Estocado/genética , RNA Mensageiro Estocado/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Humanos , Camundongos , Suínos , Bovinos , Proteínas do Ovo/genética , Proteínas do Ovo/metabolismoRESUMO
Human oocytes are prone to assembling meiotic spindles with unstable poles, which can favor aneuploidy in human eggs. The underlying causes of spindle instability are unknown. We found that NUMA (nuclear mitotic apparatus protein)-mediated clustering of microtubule minus ends focused the spindle poles in human, bovine, and porcine oocytes and in mouse oocytes depleted of acentriolar microtubule-organizing centers (aMTOCs). However, unlike human oocytes, bovine, porcine, and aMTOC-free mouse oocytes have stable spindles. We identified the molecular motor KIFC1 (kinesin superfamily protein C1) as a spindle-stabilizing protein that is deficient in human oocytes. Depletion of KIFC1 recapitulated spindle instability in bovine and aMTOC-free mouse oocytes, and the introduction of exogenous KIFC1 rescued spindle instability in human oocytes. Thus, the deficiency of KIFC1 contributes to spindle instability in human oocytes.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cinesinas/deficiência , Oócitos/fisiologia , Oócitos/ultraestrutura , Fuso Acromático/fisiologia , Polos do Fuso/fisiologia , 1-Alquil-2-acetilglicerofosfocolina Esterase/metabolismo , Animais , Bovinos , Complexo Dinactina/metabolismo , Dineínas/metabolismo , Feminino , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Centro Organizador dos Microtúbulos/fisiologia , Centro Organizador dos Microtúbulos/ultraestrutura , Microtúbulos/metabolismo , Proteínas Recombinantes/metabolismo , Fuso Acromático/ultraestrutura , Polos do Fuso/ultraestrutura , SuínosRESUMO
Chromosome segregation errors during female meiosis are a leading cause of pregnancy loss and human infertility. The segregation of chromosomes is driven by interactions between spindle microtubules and kinetochores. Kinetochores in mammalian oocytes are subjected to special challenges: they need to withstand microtubule pulling forces over multiple hours and are built on centromeric chromatin that in humans is decades old. In meiosis I, sister kinetochores are paired and oriented toward the same spindle pole. It is well established that they progressively separate from each other with advancing female age. However, whether aging also affects the internal architecture of centromeres and kinetochores is currently unclear. Here, we used super-resolution microscopy to study meiotic centromere and kinetochore organization in metaphase-II-arrested eggs from three mammalian species, including humans. We found that centromeric chromatin decompacts with advancing maternal age. Kinetochores built on decompacted centromeres frequently lost their integrity and fragmented into multiple lobes. Fragmentation extended across inner and outer kinetochore regions and affected over 30% of metaphase-II-arrested (MII) kinetochores in aged women and mice, making the lobular architecture a prominent feature of the female meiotic kinetochore. We demonstrate that a partial cohesin loss, as is known to occur in oocytes with advancing maternal age, is sufficient to trigger centromere decompaction and kinetochore fragmentation. Microtubule pulling forces further enhanced the fragmentation and shaped the arrangement of kinetochore lobes. Fragmented kinetochores were frequently abnormally attached to spindle microtubules, suggesting that kinetochore fragmentation could contribute to the maternal age effect in mammalian eggs.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Proteínas Cromossômicas não Histona/fisiologia , Cinetocoros/metabolismo , Oócitos/metabolismo , Envelhecimento , Animais , Proteínas de Ciclo Celular/metabolismo , Centrômero/fisiologia , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos/fisiologia , Feminino , Células HEK293 , Humanos , Cinetocoros/fisiologia , Meiose/fisiologia , Camundongos , Microtúbulos/metabolismo , Células NIH 3T3 , Oócitos/fisiologia , Óvulo/metabolismo , Óvulo/fisiologia , Fuso Acromático/fisiologia , Suínos , CoesinasRESUMO
Chromosome errors, or aneuploidy, affect an exceptionally high number of human conceptions, causing pregnancy loss and congenital disorders. Here, we have followed chromosome segregation in human oocytes from females aged 9 to 43 years and report that aneuploidy follows a U-curve. Specific segregation error types show different age dependencies, providing a quantitative explanation for the U-curve. Whole-chromosome nondisjunction events are preferentially associated with increased aneuploidy in young girls, whereas centromeric and more extensive cohesion loss limit fertility as women age. Our findings suggest that chromosomal errors originating in oocytes determine the curve of natural fertility in humans.
Assuntos
Envelhecimento , Aneuploidia , Segregação de Cromossomos , Fertilidade , Oócitos/citologia , Adolescente , Adulto , Criança , Feminino , Humanos , Meiose , Não Disjunção Genética , Adulto JovemRESUMO
Aneuploidy in human eggs is the leading cause of pregnancy loss and several genetic disorders such as Down syndrome. Most aneuploidy results from chromosome segregation errors during the meiotic divisions of an oocyte, the egg's progenitor cell. The basis for particularly error-prone chromosome segregation in human oocytes is not known. We analyzed meiosis in more than 100 live human oocytes and identified an error-prone chromosome-mediated spindle assembly mechanism as a major contributor to chromosome segregation defects. Human oocytes assembled a meiotic spindle independently of either centrosomes or other microtubule organizing centers. Instead, spindle assembly was mediated by chromosomes and the small guanosine triphosphatase Ran in a process requiring ~16 hours. This unusually long spindle assembly period was marked by intrinsic spindle instability and abnormal kinetochore-microtubule attachments, which favor chromosome segregation errors and provide a possible explanation for high rates of aneuploidy in human eggs.
Assuntos
Aneuploidia , Segregação de Cromossomos , Meiose , Oócitos/patologia , Fuso Acromático/metabolismo , Anáfase , Animais , Células Cultivadas , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Cinetocoros/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Centro Organizador dos Microtúbulos/metabolismo , Proteína ran de Ligação ao GTP/metabolismoRESUMO
Aneuploidy in human eggs is the leading cause of pregnancy loss and Down's syndrome. Aneuploid eggs result from chromosome segregation errors when an egg develops from a progenitor cell, called an oocyte. The mechanisms that lead to an increase in aneuploidy with advanced maternal age are largely unclear. Here, we show that many sister kinetochores in human oocytes are separated and do not behave as a single functional unit during the first meiotic division. Having separated sister kinetochores allowed bivalents to rotate by 90 degrees on the spindle and increased the risk of merotelic kinetochore-microtubule attachments. Advanced maternal age led to an increase in sister kinetochore separation, rotated bivalents and merotelic attachments. Chromosome arm cohesion was weakened, and the fraction of bivalents that precociously dissociated into univalents was increased. Together, our data reveal multiple age-related changes in chromosome architecture that could explain why oocyte aneuploidy increases with advanced maternal age.
Assuntos
Aneuploidia , Segregação de Cromossomos , Cinetocoros/metabolismo , Meiose , Oócitos/fisiologia , Humanos , Idade MaternaRESUMO
The position of the meiotic spindle in relation to the injection site has been a matter of concern since intracytoplasmic sperm injection (ICSI) was developed. Disruption of the spindle may result in cell death or aneuploidy, and it is therefore essential to avoid injecting directly into the area of the spindle. Before the development of the PolScope, it was only possible to visualize the spindle after fixing and staining oocytes. The PolScope allows the spindle to be visualized in living oocytes, and thus their subsequent development can be followed. Studies have shown that the spindle is not, as had been assumed, always adjacent to the polar body. However, the proportion of oocytes in which the spindle is significantly displaced is low, and the displacement is not necessarily associated with poor oocyte and embryo development. Even oocytes in which no spindle is visualized may go on to fertilize and develop normally, and the evidence indicates that disruption of the spindle is not a significant factor in determining the likelihood of aneuploidy and developmental anomalies. Indeed, it has been suggested that injecting spermatozoa closer to the site of the spindle may in fact improve the outcome.
Assuntos
Citoesqueleto/ultraestrutura , Meiose , Oócitos/ultraestrutura , Injeções de Esperma Intracitoplásmicas , Feminino , Humanos , Masculino , Microscopia Eletrônica , Injeções de Esperma Intracitoplásmicas/métodosRESUMO
A major puzzle in biology is how mammalian sperm maintain the correct swimming direction during various phases of the sexual reproduction process. Whilst chemotaxis may dominate near the ovum, it is unclear which cues guide spermatozoa on their long journey towards the egg. Hypothesized mechanisms range from peristaltic pumping to temperature sensing and response to fluid flow variations (rheotaxis), but little is known quantitatively about them. We report the first quantitative study of mammalian sperm rheotaxis, using microfluidic devices to investigate systematically swimming of human and bull sperm over a range of physiologically relevant shear rates and viscosities. Our measurements show that the interplay of fluid shear, steric surface-interactions, and chirality of the flagellar beat leads to stable upstream spiralling motion of sperm cells, thus providing a generic and robust rectification mechanism to support mammalian fertilisation. A minimal mathematical model is presented that accounts quantitatively for the experimental observations.DOI: http://dx.doi.org/10.7554/eLife.02403.001.
Assuntos
Mamíferos , Espermatozoides/fisiologia , Animais , Bovinos , Quimiotaxia , Fertilização/fisiologia , Humanos , Dispositivos Lab-On-A-Chip , Masculino , Modelos Teóricos , Óvulo/fisiologia , Motilidade dos Espermatozoides/fisiologiaRESUMO
Meiotic recombination between highly similar duplicated sequences (nonallelic homologous recombination, NAHR) generates deletions, duplications, inversions and translocations, and it is responsible for genetic diseases known as 'genomic disorders', most of which are caused by altered copy number of dosage-sensitive genes. NAHR hot spots have been identified within some duplicated sequences. We have developed sperm-based assays to measure the de novo rate of reciprocal deletions and duplications at four NAHR hot spots. We used these assays to dissect the relative rates of NAHR between different pairs of duplicated sequences. We show that (i) these NAHR hot spots are specific to meiosis, (ii) deletions are generated at a higher rate than their reciprocal duplications in the male germline and (iii) some of these genomic disorders are likely to have been underascertained clinically, most notably that resulting from the duplication of 7q11, the reciprocal of the deletion causing Williams-Beuren syndrome.