Meiotic Maturation of the Mouse Oocyte Requires an Equilibrium between Cyclin B Synthesis and Degradation

AbstractAmong the proteins whose synthesis and/or degradation is necessary for a proper progression through meiotic maturation, cyclin B appears to be one of the most important. Here, we attempted to modulate the level of cyclin B1 and B2 synthesis during meiotic maturation of the mouse oocyte. We used cyclin B1 or B2 mRNAs with poly(A) tails of different sizes and cyclin B1 or B2 antisense RNAs. Oocytes microinjected with cyclin B1 mRNA showed two phenotypes: most were blocked in MI, while the others extruded the first polar body in advance when compared to controls. Moreover, these effects were correlated with the length of the poly(A) tail. Thus it seems that the rate of cyclin B1 translation controls the timing of the first meiotic M phase and the transition to anaphase I. Moreover, overexpression of cyclin B1 or B2 was able to bypass the dbcAMP-induced germinal vesicle block, but only the cyclin B1 mRNA-microinjected oocytes did not extrude their first polar body. Oocytes injected with the cyclin B1 antisense progressed through the first meiotic M phase but extruded the first polar body in advance and were unable to enter metaphase II. This suggested that inhibition of cyclin B1 synthesis only took place at the end of the first meiotic M phase, most likely because the cyclin B1 mRNA was protected. The injection of cyclin B2 antisense RNA had no effect. The life observation of the synthesis and degradation of a cyclin B1–GFP chimera during meiotic maturation of the mouse oocyte demonstrated that degradation can only occur during a given period of time once it has started. Taken together, our data demonstrate that the rates of cyclin B synthesis and degradation determine the timing of the major events taking place during meiotic maturation of the mouse oocyte

Meiotic spindle stability depends on MAPK-interacting and spindle-stabilizing protein (MISS), a new MAPK substrate

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)

A computational model of the early stages of acentriolar meiotic spindle assembly.

The mitotic spindle is an ensemble of microtubules responsible for the repartition of the chromosomal content between the two daughter cells during division. In metazoans, spindle assembly is a gradual process involving dynamic microtubules and recruitment of numerous associated proteins and motors. During mitosis, centrosomes organize and nucleate the majority of spindle microtubules. In contrast, oocytes lack canonical centrosomes but are still able to form bipolar spindles, starting from an initial ball that self-organizes in several hours. Interfering with early steps of meiotic spindle assembly can lead to erroneous chromosome segregation. Although not fully elucidated, this process is known to rely on antagonistic activities of plus end- and minus end-directed motors. We developed a model of early meiotic spindle assembly in mouse oocytes, including key factors such as microtubule dynamics and chromosome movement. We explored how the balance between plus end- and minus end-directed motors, as well as the influence of microtubule nucleation, impacts spindle morphology. In a refined model, we added spatial regulation of microtubule stability and minus-end clustering. We could reproduce the features of early stages of spindle assembly from 12 different experimental perturbations and predict eight additional perturbations. With its ability to characterize and predict chromosome individualization, this model can help deepen our understanding of spindle assembly

F-actin mechanics control spindle centring in the mouse zygote

International audienceMitotic spindle position relies on interactions between astral microtubules nucleated by centrosomes and a rigid cortex. Some cells, such as mouse oocytes, do not possess centrosomes and astral microtubules. These cells rely only on actin and on a soft cortex to position their spindle off-centre and undergo asymmetric divisions. While the first mouse embryonic division also occurs in the absence of centrosomes, it is symmetric and not much is known on how the spindle is positioned at the exact cell centre. Using interdisciplinary approaches, we demonstrate that zygotic spindle positioning follows a three-step process: (1) coarse centring of pronuclei relying on the dynamics of an F-actin/Myosin-Vb meshwork; (2) fine centring of the metaphase plate depending on a high cortical tension; (3) passive maintenance at the cell centre. Altogether, we show that F-actin-dependent mechanics operate the switch between asymmetric to symmetric division required at the oocyte to embryo transition

A centriole- and RanGTP-independent spindle assembly pathway in meiosis I of vertebrate oocytes

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 β. 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

Mps1 directs the assembly of Cdc20 inhibitory complexes during interphase and mitosis to control M phase timing and spindle checkpoint signaling

Cdc20 and Mad2 or Bub1 don’t come together in Mps1-null cells, resulting in a dramatic acceleration of anaphase onset (see also related papers by Hewitt et al. and Santaguida et al. in this issue)

controle de la transition meiose I/meiose II et role de DOC1R au cours de l'arret CSF lors de la maturation meiotique chez la souris

jury: president Dr Bernard Maro, rapporteurs Dr Claude Prigent et Dr Jacek Kubiak, examinateurs Dr Isabelle Vernos et Dr Thierry Lorca, Directrice de these Dr Marie-Helene Verlhac.Meiotic maturation of vertebrate oocytes differs from mitosis on many aspects. I was interested in two characteristics. 1) In meiosis I, homologous chromosomes are segregated, in mitosis sister chromatids are separated. In mitosis, a checkpoint blocks the cell in metaphase via APC/C inhibition until all chromosomes are properly aligned on the spindle. In meiosis, contradictory results exist, depending on the species, about the requirement of such a checkpoint in meiosis I. I have shown that separase activity (an activity indirectly regulated by APC/C) is required for metaphase to anaphase transition in meiosis I, suggesting that such a checkpoint is required in the mouse, an organism close of human. 2) At the end of meiotic maturation, oocytes are blocked in metaphase of meiosis II waiting for fertilization, whereas mitosis always ends. This block is due to a CSF activity and requires the Mos/.../MAPK pathway. I have shown that DOC1R, a new MAPK substrate, controls microtubule organization during the CSF arrest. These results establish a new view of the CSF arrest which was considered as a linear pathway responsible for MPF stabilization. The CSF arrest is a non linear pathway which also controls oocyte morphology.La maturation méiotique des vertébrés diffère de la mitose par plusieurs aspects. J'ai étudié deux de ces particularités. 1) En méiose I, les chromosomes homologues sont ségrégés, en mitose, les chromatides sœurs sont séparées. En mitose, un mécanisme de contrôle bloque la cellule en métaphase en inhibant l'APC/C tant que tous les chromosomes ne sont pas correctement alignés sur le fuseau. En méiose I, des résultats contradictoires existent selon les espèces quant à l'existence d'un mécanisme de contrôle de ce type. J'ai montré que l'activité séparase (activité indirectement régulée par l'APC/C) est requise pour effectuer la transition métaphase/anaphase en méiose I, suggérant qu'un mécanisme de contrôle de ce type est requis chez la souris, organisme proche de l'homme. 2) A l'issue de la maturation méiotique, l'ovocyte reste bloqué en métaphase de méiose II en attendant la fécondation, alors que la mitose s'achève toujours. Ce blocage est dû à l'activité CSF et requiert la voie Mos/.../MAPK. J'ai montré que DOC1R, un nouveau substrat des MAPK, contrôle l'organisation des microtubules au cours de l'arrêt CSF. Ces résultats font évoluer la vision de l'arrêt CSF qui était considéré comme une voie linéaire aboutissant à la stabilisation du MPF. L'arrêt CSF est une voie non linéaire contrôlant aussi la morphologie de l'ovocyte

L’acétylation des cohésines

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Contrôle de la transition méiose I/méiose II et rôle de Doc1R au cours de l'arrêt CSF lors de la maturation méiotique chez la souris

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Oocyte Maturation and Development

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