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1.  A Single Bivalent Efficiently Inhibits Cyclin B1 Degradation and Polar Body Extrusion in Mouse Oocytes Indicating Robust SAC during Female Meiosis I 
PLoS ONE  2011;6(11):e27143.
The Spindle Assembly Checkpoint (SAC) inhibits anaphase until microtubule-to-kinetochore attachments are formed, thus securing correct chromosome separation and preventing aneuploidy. Whereas in mitosis even a single unattached chromosome keeps the SAC active, the high incidence of aneuploidy related to maternal meiotic errors raises a concern about the lower efficiency of SAC in oocytes. Recently it was suggested that in mouse oocytes, contrary to somatic cells, not a single chromosome but a critical mass of chromosomes triggers efficient SAC pointing to the necessity of evaluating the robustness of SAC in oocytes. Two types of errors in chromosome segregation upon meiosis I related to SAC were envisaged: (1) SAC escape, when kinetochores emit SAC-activating signal unable to stop anaphase I; and (2) SAC deceive, when kinetochores do not emit the signal. Using micromanipulations and live imaging of the first polar body extrusion, as well as the dynamics of cyclin B1 degradation, here we show that in mouse oocytes a single bivalent keeps the SAC active. This is the first direct evaluation of SAC efficiency in mouse oocytes, which provides strong evidence that the robustness of SAC in mammalian oocytes is comparable to other cell types. Our data do not contradict the hypothesis of the critical mass of chromosomes necessary for SAC activation, but suggest that the same rule may govern SAC activity also in other cell types. We postulate that the innate susceptibility of oocytes to errors in chromosome segregation during the first meiotic division may not be caused by lower efficiency of SAC itself, but could be linked to high critical chromosome mass necessary to keep SAC active in oocyte of large size.
PMCID: PMC3220673  PMID: 22125605
2.  Orientation of Mitotic Spindles during the 8- to 16-Cell Stage Transition in Mouse Embryos 
PLoS ONE  2009;4(12):e8171.
Asymmetric cell divisions are involved in the divergence of the first two lineages of the pre-implantation mouse embryo. They first take place after cell polarization (during compaction) at the 8-cell stage. It is thought that, in contrast to many species, spindle orientation is random, although there is no direct evidence for this.
Methodology/Principal Findings
Tubulin-GFP and live imaging with a spinning disk confocal microscope were used to directly study spindle orientation in whole embryos undergoing the 8- to 16-cell stage transition. This approach allowed us to determine that there is no predetermined cleavage pattern in 8-cell compacted mouse embryos and that mitotic spindle orientation in live embryo is only modulated by the extent of cell rounding up during mitosis.
These results clearly demonstrate that spindle orientation is not controlled at the 8- to 16-cell transition, but influenced by cell bulging during mitosis, thus reinforcing the idea that pre-implantation development is highly regulative and not pre-patterned.
PMCID: PMC2781390  PMID: 19997595
3.  Inactivation of aPKCλ Reveals a Context Dependent Allocation of Cell Lineages in Preimplantation Mouse Embryos 
PLoS ONE  2009;4(9):e7117.
During mammalian preimplantation development, lineage divergence seems to be controlled by the interplay between asymmetric cell division (once cells are polarized) and positional information. In the mouse embryo, two distinct cell populations are first observed at the 16-cell stage and can be distinguished by both their position (outside or inside) and their phenotype (polarized or non-polarized). Many efforts have been made during the last decade to characterize the molecular mechanisms driving lineage divergence.
Methodology/Principal Findings
In order to evaluate the importance of cell polarity in the determination of cell fate we have disturbed the activity of the apical complex aPKC/PAR6 using siRNA to down-regulate aPKCλ expression. Here we show that depletion of aPKCλ results in an absence of tight junctions and in severe polarity defects at the 16-cell stage. Importantly, we found that, in absence of aPKCλ, cell fate depends on the cellular context: depletion of aPKCλ in all cells results in a strong reduction of inner cells at the 16-cell stage, while inhibition of aPKCλ in only half of the embryo biases the progeny of aPKCλ defective blastomeres towards the inner cell mass. Finally, our study points to a role of cell shape in controlling cell position and thus lineage allocation.
Our data show that aPKCλ is dispensable for the establishment of polarity at the 8-cell stage but is essential for the stabilization of cell polarity at the 16-cell stage and for cell positioning. Moreover, this study reveals that in addition to positional information and asymmetric cell divisions, cell shape plays an important role for the control of lineage divergence during mouse preimplantation development. Cell shape is able to influence both the type of division (symmetric or asymmetric) and the position of the blastomeres within the embryo.
PMCID: PMC2741596  PMID: 19768116
4.  Meiotic Regulation of TPX2 Protein Levels Governs Cell Cycle Progression in Mouse Oocytes 
PLoS ONE  2008;3(10):e3338.
Formation of female gametes requires acentriolar spindle assembly during meiosis. Mitotic spindles organize from centrosomes and via local activation of the RanGTPase on chromosomes. Vertebrate oocytes present a RanGTP gradient centred on chromatin at all stages of meiotic maturation. However, this gradient is dispensable for assembly of the first meiotic spindle. To understand this meiosis I peculiarity, we studied TPX2, a Ran target, in mouse oocytes. Strikingly, TPX2 activity is controlled at the protein level through its accumulation from meiosis I to II. By RNAi depletion and live imaging, we show that TPX2 is required for spindle assembly via two distinct functions. It controls microtubule assembly and spindle pole integrity via the phosphorylation of TACC3, a regulator of MTOCs activity. We show that meiotic spindle formation in vivo depends on the regulation of at least a target of Ran, TPX2, rather than on the regulation of the RanGTP gradient itself.
PMCID: PMC2556383  PMID: 18833336
5.  Murine Endogenous Retrovirus MuERV-L Is the Progenitor of the “Orphan” Epsilon Viruslike Particles of the Early Mouse Embryo▿  
Journal of Virology  2007;82(3):1622-1625.
Viruslike particles which displayed a peculiar wheellike appearance that distinguished them from A-, B- or C-type particles had previously been described in the early mouse embryo. The maximum expression of these so-called epsilon particles was observed in two-cell-stage embryos, followed by their rapid decline at later stages of development and no particles detected at the zygote one-cell stage. Here, we show that these particles are in fact produced by a newly discovered murine endogenous retrovirus (ERV) belonging to the widespread family of mammalian ERV-L elements and named MuERV-L. Using antibodies that we raised against the Gag protein of these elements, Western blot analysis and in toto immunofluorescence studies of the embryos at various stages disclosed the same developmental expression profile as that observed for epsilon particles. Using expression vectors for cloned, full-length, entirely coding MuERV-L copies and cell transfection, direct identification of the epsilon particles was finally achieved by high-resolution electron microscopy.
PMCID: PMC2224431  PMID: 18045933
6.  Changing Mad2 Levels Affects Chromosome Segregation and Spindle Assembly Checkpoint Control in Female Mouse Meiosis I 
PLoS ONE  2007;2(11):e1165.
The spindle assembly checkpoint (SAC) ensures correct separation of sister chromatids in somatic cells and provokes a cell cycle arrest in metaphase if one chromatid is not correctly attached to the bipolar spindle. Prolonged metaphase arrest due to overexpression of Mad2 has been shown to be deleterious to the ensuing anaphase, leading to the generation of aneuploidies and tumorigenesis. Additionally, some SAC components are essential for correct timing of prometaphase. In meiosis, we and others have shown previously that the Mad2-dependent SAC is functional during the first meiotic division in mouse oocytes. Expression of a dominant-negative form of Mad2 interferes with the SAC in metaphase I, and a knock-down approach using RNA interference accelerates anaphase onset in meiosis I. To prove unambigiously the importance of SAC control for mammalian female meiosis I we analyzed oocyte maturation in Mad2 heterozygote mice, and in oocytes overexpressing a GFP-tagged version of Mad2. In this study we show for the first time that loss of one Mad2 allele, as well as overexpression of Mad2 lead to chromosome missegregation events in meiosis I, and therefore the generation of aneuploid metaphase II oocytes. Furthermore, SAC control is impaired in mad2+/− oocytes, also leading to the generation of aneuploidies in meiosis I.
PMCID: PMC2082075  PMID: 18043727
7.  Meiotic spindle stability depends on MAPK-interacting and spindle-stabilizing protein (MISS), a new MAPK substrate 
The Journal of Cell Biology  2002;157(4):603-613.
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).
PMCID: PMC2173866  PMID: 12011110
MISS; MAP kinase; spindle stability; morpholino; mouse
8.  Kinetochore Fibers Are Not Involved in the Formation of the First Meiotic Spindle in Mouse Oocytes, but Control the Exit from the First Meiotic M Phase 
The Journal of Cell Biology  1999;146(1):1-12.
During meiosis, two successive divisions occur without any intermediate S phase to produce haploid gametes. The first meiotic division is unique in that homologous chromosomes are segregated while the cohesion between sister chromatids is maintained, resulting in a reductional division. Moreover, the duration of the first meiotic M phase is usually prolonged when compared with mitotic M phases lasting 8 h in mouse oocytes.
We investigated the spindle assembly pathway and its role in the progression of the first meiotic M phase in mouse oocytes. During the first 4 h, a bipolar spindle forms and the chromosomes congress near the equatorial plane of the spindle without stable kinetochore– microtubule end interactions. This late prometaphase spindle is then maintained for 4 h with chromosomes oscillating in the central region of the spindle. The kinetochore–microtubule end interactions are set up at the end of the first meiotic M phase (8 h after entry into M phase). This event allows the final alignment of the chromosomes and exit from metaphase. The continuous presence of the prometaphase spindle is not required for progression of the first meiotic M phase. Finally, the ability of kinetochores to interact with microtubules is acquired at the end of the first meiotic M phase and determines the timing of polar body extrusion.
PMCID: PMC2199729  PMID: 10402455
meiosis; microtubule; kinetochore; chromosome; spindle assembly checkpoint

Results 1-8 (8)