RESUMEN

Female meiosis in higher organisms consists of highly asymmetric divisions, which retain most maternal stores in the oocyte for embryo development. Asymmetric partitioning of the cytoplasm results from the spindle's "off-center" positioning, which, in mouse oocytes, depends mainly on actin filaments [1, 2]. This is a unique situation compared to most systems, in which spindle positioning requires interactions between astral microtubules and cortical actin filaments [3]. Formin 2, a straight-actin-filament nucleator, is required for the first meiotic spindle migration to the cortex and cytokinesis in mouse oocytes [4, 5]. Although the requirement for actin filaments in the control of spindle positioning is well established in this model, no one has been able to detect them in the cytoplasm [6]. Through the expression of an F-actin-specific probe and live confocal microscopy, we show the presence of a cytoplasmic actin meshwork, organized by Formin 2, that controls spindle migration. In late meiosis I, these filaments organize into a spindle-like F-actin structure, which is connected to the cortex. At anaphase, global reorganization of this meshwork allows polar-body extrusion. In addition, using actin-YFP, our FRAP analysis confirms the presence of a highly dynamic cytoplasmic actin meshwork that is tightly regulated in time and space.

Asunto(s)

Citoesqueleto de Actina/fisiología , Oocitos/fisiología , Huso Acromático/fisiología , Actinas/metabolismo , Animales , Femenino , Ratones , Proteínas de Microfilamentos/metabolismo

RESUMEN

Spindle formation is essential for stable inheritance of genetic material. Experiments in various systems indicate that Ran GTPase is crucial for meiotic and mitotic spindle assembly. Such an important role for Ran in chromatin-induced spindle assembly was initially demonstrated in Xenopus laevis egg extracts. However, the requirement of RanGTP in living meiotic cells has not been shown. In this study, we used a fluorescence resonance energy transfer probe to measure RanGTP-regulated release of importin beta. A RanGTP-regulated gradient was established during meiosis I and was centered on chromosomes throughout mouse meiotic maturation. Manipulating levels of RanGTP in mice and X. laevis oocytes did not inhibit assembly of functional meiosis I spindles. However, meiosis II spindle assembly did not tolerate changes in the level of RanGTP in both species. These findings suggest that a mechanism common to vertebrates promotes meiosis I spindle formation in the absence of chromatin-induced microtubule production and centriole-based microtubule organizing centers.

Asunto(s)

Centriolos/metabolismo , Meiosis/fisiología , Proteínas de Unión al GTP Monoméricas/metabolismo , Oocitos/citología , Huso Acromático/metabolismo , Proteína de Unión al GTP ran/metabolismo , Animales , Cromosomas de los Mamíferos/metabolismo , Femenino , Transferencia Resonante de Energía de Fluorescencia , Guanosina Trifosfato/metabolismo , Ratones , Ratones Endogámicos , Proteínas de Unión al GTP Monoméricas/genética , Oligonucleótidos Antisentido , Oocitos/metabolismo , Vertebrados , Xenopus laevis , beta Carioferinas/metabolismo , Proteína de Unión al GTP ran/genética

RESUMEN

Female meiotic divisions in higher organisms are asymmetric and lead to the formation of a large oocyte and small polar bodies. These asymmetric divisions are due to eccentric spindle positioning which, in the mouse, requires actin filaments. Recently Formin-2, a straight actin filaments nucleator, has been proposed to control spindle positioning, chromosome segregation as well as first polar body extrusion in mouse oocytes. We reexamine here the possible role of Formin-2 during mouse meiotic maturation by live videomicroscopy. We show that Formin-2 controls first meiotic spindle migration to the cortex but not chromosome congression or segregation. We also show that the lack of first polar body extrusion in fmn2(-/-) oocytes is not due to a lack of cortical differentiation or central spindle formation but to a defect in the late steps of cytokinesis. Indeed, Survivin, a component of the passenger protein complex, is correctly localized on the central spindle at anaphase in fmn2(-/-) oocytes. We show here that attempts of cytokinesis in these oocytes abort due to phospho-myosin II mislocalization.

Asunto(s)

Citocinesis/fisiología , Proteínas del Tejido Nervioso/fisiología , Oocitos/citología , Huso Acromático , Animales , Cromosomas , Ratones

RESUMEN

Vertebrate oocytes arrest in metaphase of the second meiotic division (MII), where they maintain a high cdc2/cyclin B activity and a stable, bipolar spindle because of cytostatic factor (CSF) activity. The Mos-MAPK pathway is essential for establishing CSF. Indeed, oocytes from the mos-/- strain do not arrest in MII and activate without fertilization, as do Xenopus laevis oocytes injected with morpholino oligonucleotides directed against Mos. In Xenopus oocytes, p90Rsk (ribosomal S6 kinase), a MAPK substrate, is the main mediator of CSF activity. We show here that this is not the case in mouse oocytes. The injection of constitutively active mutant forms of Rsk1 and Rsk2 does not induce a cell cycle arrest in two-cell mouse embryos. Moreover, these two mutant forms do not restore MII arrest after their injection into mos-/- oocytes. Eventually, oocytes from the triple Rsk (1, 2, 3) knockout present a normal CSF arrest. We demonstrate that p90Rsk is not involved in the MII arrest of mouse oocytes.

Asunto(s)

Blastómeros/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Meiosis/fisiología , Oocitos/fisiología , Proteínas Proto-Oncogénicas c-mos/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Animales , Femenino , Sistema de Señalización de MAP Quinasas/genética , Ratones , Ratones Noqueados , Embarazo , Proteínas Proto-Oncogénicas c-mos/genética , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , Xenopus laevis

RESUMEN

For the success of fertilization, spindles of vertebrate oocytes must remain stable and correctly organized during the arrest in metaphase II of meiosis. Using a two-hybrid screen with MAPK as a bait, we have recently identified MISS (MAPK interacting and spindle stabilizing) which controls mouse oocyte metaphase II spindle stability. Using the same screen, we identify another MAPK partner, DOC1R (Deleted in oral cancer one related), a murine homologue of a potential human tumor suppressor gene. We characterize DOC1R during mouse oocyte meiosis resumption. DOC1R is regulated by phosphorylation during meiotic maturation by MPF (M-phase promoting factor) and by the MOS/./MAPK pathway. DOC1R and a DOC1R-GFP fusion localize to microtubules during meiotic maturation. Consistent with this microtubular localization, we show, by antisense and double-stranded RNA injection, that depletion of DOC1R induces microtubule defects in metaphase II oocytes. These defects are rescued by overexpressing a Xenopus DOC1R, showing that they are specific to DOC1R. Thus, the discovery of DOC1R, a substrate of MAPK that regulates microtubule organization of metaphase II mouse oocytes, reinforces the importance of this pathway in the control of spindle stability during the metaphase II arrest.

Asunto(s)

Meiosis/fisiología , Metafase/fisiología , Microtúbulos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Oncogénicas/metabolismo , Oocitos/fisiología , Secuencia de Aminoácidos , Animales , Ciclina B/metabolismo , Genes Supresores de Tumor , Humanos , Mesotelina , Ratones , Datos de Secuencia Molecular , Proteínas Oncogénicas/genética , Oocitos/citología , Fosforilación , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Huso Acromático/metabolismo , Técnicas del Sistema de Dos Híbridos , Xenopus laevis

RESUMEN

Vertebrate oocytes arrest in the second metaphase of meiosis (metaphase II [MII]) by an activity called cytostatic factor (CSF), with aligned chromosomes and stable spindles. Segregation of chromosomes occurs after fertilization. The Mos/.../MAPK (mitogen-activated protein kinases) pathway mediates this MII arrest. Using a two-hybrid screen, we identified a new MAPK partner from a mouse oocyte cDNA library. This protein is unstable during the first meiotic division and accumulates only in MII, where it localizes to the spindle. It is a substrate of the Mos/.../MAPK pathway. The depletion of endogenous RNA coding for this protein by three different means (antisense RNA, double-stranded [ds] RNA, or morpholino oligonucleotides) induces severe spindle defects specific to MII oocytes. Overexpressing the protein from an RNA not targeted by the morpholino rescues spindle destabilization. However, dsRNA has no effect on the first two mitotic divisions. We therefore have discovered a new MAPK substrate involved in maintaining spindle integrity during the CSF arrest of mouse oocytes, called MISS (for MAP kinase-interacting and spindle-stabilizing protein).

Asunto(s)

Proteínas Portadoras/aislamiento & purificación , Proteínas de Ciclo Celular/aislamiento & purificación , Péptidos y Proteínas de Señalización Intracelular , Sistema de Señalización de MAP Quinasas/fisiología , Meiosis/fisiología , Proteínas Nucleares/aislamiento & purificación , Oocitos/metabolismo , Huso Acromático/metabolismo , Secuencia de Aminoácidos/genética , Animales , Secuencia de Bases/genética , Proteínas Portadoras/genética , Proteínas de Ciclo Celular/genética , Clonación Molecular , Embrión de Mamíferos/efectos de los fármacos , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Femenino , Genes myc/genética , Ratones , Ratones Noqueados , Mitosis/genética , Datos de Secuencia Molecular , Mutación/genética , Proteínas Nucleares/genética , Sondas de Oligonucleótidos/farmacología , Proteínas Oncogénicas v-mos/genética , Oocitos/citología , Fenotipo , Estructura Terciaria de Proteína/genética , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación