Publications by authors named "Bernard Maro"

27 Publications

  • Page 1 of 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 18;6(11):e27143. Epub 2011 Nov 18.

Department of Developmental Biology, Max Planck Institute of Immunobiology, Freiburg, Germany.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0027143PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3220673PMC
April 2012

The conformation and activation of Fyn kinase in the oocyte determine its localisation to the spindle poles and cleavage furrow.

Reprod Fertil Dev 2011 ;23(7):846-57

Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, Tel-Aviv, Israel.

Several lines of evidence imply the involvement of Fyn, a Src family kinase, in cell-cycle control and cytoskeleton organisation in somatic cells. By live cell confocal imaging of immunostained or cRNA-microinjected mouse oocytes at metaphase of the second meiotic division, membrane localisation of active and non-active Fyn was demonstrated. However, Fyn with a disrupted membrane-binding domain at its N-terminus was targeted to the cytoplasm and spindle in its non-active form and concentrated at the spindle poles when active. During metaphase exit, the amount of phosphorylated Fyn and of spindle-poles Fyn decreased and it started appearing at the membrane area of the cleavage furrow surrounding the spindle midzone, either asymmetrically during polar body II extrusion or symmetrically during mitosis. These results demonstrate that post-translational modifications of Fyn, probably palmitoylation, determine its localisation and function; localisation of de-palmitoylated active Fyn to the spindle poles is involved in spindle pole integrity during metaphase, whereas the localisation of N-terminus palmitoylated Fyn at the membrane near the cleavage furrow indicates its participation in furrow ingression during cytokinesis.
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http://dx.doi.org/10.1071/RD11033DOI Listing
January 2012

Fyn kinase is involved in cleavage furrow ingression during meiosis and mitosis.

Reproduction 2010 Dec 14;140(6):827-34. Epub 2010 Sep 14.

Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, Ramat-Aviv, Tel-Aviv 69978, Israel.

Fertilization of mammalian oocytes triggers their exit from the second meiotic division metaphase arrest. The extrusion of the second polar body (PBII) that marks the completion of meiosis is followed by the first mitotic cleavage of the zygote. Several lines of evidence in somatic cells imply the involvement of Fyn, an Src family kinase (SFK), in cell cycle control and actin functions. In this study, we demonstrate, using live cell confocal imaging and microinjection of Fyn cRNAs, the recruitment of Fyn to the oocyte's cortical area overlying the chromosomes and its colocalization with filamentous actin (F-actin) during exit from the meiotic metaphase. Fyn concentrated asymmetrically at the cortical site designated for ingression of the PBII cleavage furrow, where F-actin had already been accumulated, and then redispersed throughout the entire cortex only to be recruited again to the cleavage furrow during the first mitotic division. Although microinjection of dominant negative Fyn did not affect initiation of the cleavage furrow, it prolonged the average duration of ingression, decreased the rates of PB extrusion and of the first cleavage, and led to the formation of bigger PBs and longer spindles. Extrusion of the PBII was blocked in oocytes exposed to SU6656, an SFK inhibitor. Our results demonstrate, for the first time, a continuous colocalization of Fyn and F-actin during meiosis and imply a role for the SFKs, in general, and for Fyn, in particular, in regulating pathways that involve actin cytoskeleton, during ingression of the meiotic and mitotic cleavage furrows.
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http://dx.doi.org/10.1530/REP-10-0312DOI Listing
December 2010

The involvement of Fyn kinase in resumption of the first meiotic division in mouse oocytes.

Cell Cycle 2010 Apr 15;9(8):1577-89. Epub 2010 Apr 15.

Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.

The process of resumption of the first meiotic division (RMI) in mammalian oocytes includes germinal vesicle breakdown (GVBD), spindle formation during first metaphase (MI), segregation of homologous chromosomes, extrusion of the first polar body (PBI) and an arrest at metaphase of the second meiotic division (MII). Previous studies suggest a role for Fyn, a non-receptor Src family tyrosine kinase, in the exit from MII arrest. In the current study we characterized the involvement of Fyn in RMI. Western blot analysis demonstrated a significant, proteasome independent, degradation of Fyn during GVBD. Immunostaining of fixed oocytes and confocal imaging of live oocytes microinjected with Fyn complementary RNA (cRNA) demonstrated Fyn localization to the oocyte cortex and to the spindle poles. Fyn was recruited during telophase to the cortical area surrounding the midzone of the spindle and was then translocated to the contractile ring during extrusion of PBI. GVBD, exit from MI and PBI extrusion were inhibited in oocytes exposed to the chemical inhibitor SU6656 or microinjected with dominant negative Fyn cRNA. None of the microinjected oocytes showed misaligned or lagging chromosomes during chromosomes segregation and the spindle migration and anchoring were not affected. However, the extruded PBI was of large size. Altogether, a role for Fyn in regulating several key pathways during the first meiotic division in mammalian oocytes is suggested, particularly at the GV and metaphase checkpoints and in signaling the ingression of the cleavage furrow.
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http://dx.doi.org/10.4161/cc.9.8.11299DOI Listing
April 2010

Orientation of mitotic spindles during the 8- to 16-cell stage transition in mouse embryos.

PLoS One 2009 Dec 4;4(12):e8171. Epub 2009 Dec 4.

CNRS, UMR7622-Laboratoire de Biologie Cellulaire du Développement, Paris, France.

Background: 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.

Conclusions: 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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0008171PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2781390PMC
December 2009

Inactivation of aPKClambda reveals a context dependent allocation of cell lineages in preimplantation mouse embryos.

PLoS One 2009 Sep 21;4(9):e7117. Epub 2009 Sep 21.

CNRS, UMR7622 - Laboratoire de Biologie Cellulaire du Développement, 9 Quai Saint-Bernard, Bâtiment C, Paris, France.

Background: 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 aPKClambda expression. Here we show that depletion of aPKClambda results in an absence of tight junctions and in severe polarity defects at the 16-cell stage. Importantly, we found that, in absence of aPKClambda, cell fate depends on the cellular context: depletion of aPKClambda in all cells results in a strong reduction of inner cells at the 16-cell stage, while inhibition of aPKClambda in only half of the embryo biases the progeny of aPKClambda 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.

Conclusion: Our data show that aPKClambda 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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0007117PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2741596PMC
September 2009

Meiotic regulation of TPX2 protein levels governs cell cycle progression in mouse oocytes.

PLoS One 2008 Oct 3;3(10):e3338. Epub 2008 Oct 3.

UMR7622, Université Pierre et Marie Curie/CNRS, Bat. C, 5e, 9 quai Saint Bernard, Paris, France.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0003338PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2556383PMC
October 2008

Morphogenesis of the mammalian blastocyst.

Mol Cell Endocrinol 2008 Jan 19;282(1-2):70-7. Epub 2007 Nov 19.

Laboratoire de Biologie Cellulaire du Développement, UMR 7622, CNRS, Université Pierre et Marie Curie, 9 Quai St Bernard, 75252 Paris Cedex 05, France.

The first 4 days of mouse pre-implantation development are characterized by a period of segmentation, including morphogenetic events that are required for the divergence of embryonic and extra-embryonic lineages. These extra-embryonic tissues are essential for the implantation into the maternal uterus and for the development of the foetus. In this review, we first discuss data showing unambiguously that no essential axis of development is set up before the late blastocyst stage, and explain why the pre-patterning described during the early phases (segmentation) of development in other vertebrates cannot apply to mammalian pre-implantation period. Then, we describe important cellular and molecular events that are required for the morphogenesis of the blastocyst.
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http://dx.doi.org/10.1016/j.mce.2007.11.004DOI Listing
January 2008

Murine endogenous retrovirus MuERV-L is the progenitor of the "orphan" epsilon viruslike particles of the early mouse embryo.

J Virol 2008 Feb 28;82(3):1622-5. Epub 2007 Nov 28.

Unité des Rétrovirus Endogènes et Eléments Rétroïdes des Eucaryotes Supérieurs, CNRS UMR 8122, Institut Gustave Roussy, 39 Rue Camille Desmoulins, F-94805 Villejuif, France.

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.
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http://dx.doi.org/10.1128/JVI.02097-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2224431PMC
February 2008

Changing Mad2 levels affects chromosome segregation and spindle assembly checkpoint control in female mouse meiosis I.

PLoS One 2007 Nov 28;2(11):e1165. Epub 2007 Nov 28.

CNRS UMR7622 Biologie du Développement, Paris, France.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0001165PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2082075PMC
November 2007

Germinal vesicle position and meiotic maturation in mouse oocyte.

Reproduction 2007 Jun;133(6):1069-72

UMR 7622 Biologie du Développement, CNRS-UPMC, 9 quai St Bernard, 75005 Paris, France.

During meiotic maturation, mammalian oocytes undergo an asymmetric division which is crucial for the formation of a functional gamete. In various organisms, accurate positioning of the nucleus before M-phase plays a major role in asymmetric cell divisions. However, the role of the position of the nucleus (or germinal vesicle, GV) during the prophase I arrest has not been investigated in mammalian oocytes. Here, we show that incompetent mouse oocytes possess a peripheral GV, while competent oocytes mainly exhibit a central position of the GV. At that time, the position of the GV correlates with the ability of the oocyte to complete meiotic maturation. Moreover, a lower efficiency in GV centering and a reduced ability to progress through meiosis are observed in oocytes from old mice. Thus, the position of the GV could be used as a simple morphological marker of oocyte quality.
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http://dx.doi.org/10.1530/REP-07-0036DOI Listing
June 2007

Conditional knock-out reveals that zygotic vezatin-null mouse embryos die at implantation.

Mech Dev 2007 Jul 23;124(6):449-62. Epub 2007 Mar 23.

Biologie cellulaire du Développement, CNRS UMR 7622, Université Pierre et Marie Curie, 9 Quai St Bernard, 75252 Paris cedex 05, France.

Vezatin, a protein associated to adherens junctions in epithelial cells, is already expressed in mouse oocytes and during pre-implantation development. Using a floxed strategy to generate a vezatin-null allele, we show that the lack of zygotic vezatin is embryonic lethal, indicating that vezatin is an essential gene. Homozygous null embryos are able to elicit a decidual response but as early as day 6.0 post-coitum mutant implantation sites are devoid of embryonic structures. Mutant blastocysts are morphologically normal, but only half of them are able to hatch upon in vitro culture and the blastocyst outgrowths formed after 3.5 days in culture exhibit severe abnormalities, in particular disrupted intercellular adhesion and clear signs of cellular degeneration. Notably, the junctional proteins E-cadherin and beta-catenin are delocalized and not observed at the plasma membrane anymore. These in vitro observations reinforce the idea that homozygous vezatin-null mutants die at the time of implantation because of a defect in intercellular adhesion. Together these results indicate that the absence of zygotic vezatin is deleterious for the implantation process, most likely because cadherin-dependent intercellular adhesion is impaired in late blastocysts when the maternal vezatin is lost.
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http://dx.doi.org/10.1016/j.mod.2007.03.004DOI Listing
July 2007

Vezatin, a ubiquitous protein of adherens cell-cell junctions, is exclusively expressed in germ cells in mouse testis.

Reproduction 2007 Mar;133(3):563-74

Biologie Cellulaire du Développement, UMR 7622, CNRS, Université Pierre et Marie Curie, 9 Quai St Bernard, 75252 Paris cedex 05, France.

In the male reproductive organs of mammals, the formation of spermatozoa takes place during two successive phases: differentiation (in the testis) and maturation (in the epididymis). The first phase, spermiogenesis, relies on a unique adherens junction, the apical ectoplasmic specialization linking the epithelial Sertoli cells to immature differentiating spermatids. Vezatin is a transmembrane protein associated with adherens junctions and the actin cytoskeleton in most epithelial cells. We report here the expression profile of vezatin during spermatogenesis. Vezatin is exclusively expressed in haploid germ cells. Immunocytochemical and ultrastructural analyses showed that vezatin intimately coincides, temporally and spatially, with acrosome formation. While vezatin is a transmembrane protein associated with adherens junctions in many epithelial cells, it is not seen at the ectoplasmic specializations, neither at the basal nor at the apical sites, in the seminiferous epithelium. In particular, vezatin does not colocalize with espin and myosin VIIa, two molecular markers of the ectoplasmic specialization. In differentiating spermatids, ultrastructural data indicate that vezatin localizes in the acrosome. In epididymal sperm, vezatin localizes also to the outer acrosomal membrane. Considering its developmental and molecular characteristics, vezatin may be involved in the assembly/stability of this spermatic membrane.
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http://dx.doi.org/10.1530/REP-06-0271DOI Listing
March 2007

Where do we stand now? Mouse early embryo patterning meeting in Freiburg, Germany (2005).

Int J Dev Biol 2006 ;50(7):581-6; discussion 586-7

Max-Planck Institute of Immunobiology, Freiburg, Germany.

Mechanism underlying mammalian preimplantation development has long been a subject of controversy and the central question has been if any "determinants" play a key role in a manner comparable to the non-mammalian "model" system. During the last decade, this issue has been revived (Pearson, 2002; Rossant and Tam, 2004) by claims that the axes of the mouse blastocyst are anticipated at the egg ("prepatterning model"; Gardner, 1997; Gardner, 2001; Piotrowska et al., 2001; Piotrowska and Zernicka-Goetz, 2001; Zernicka-Goetz, 2005), suggesting that a mechanism comparable to that operating in non-mammals may be at work. However, recent studies by other laboratories do not support these claims ("regulative model"; Alarcon and Marikawa, 2003; Chroscicka et al., 2004; Hiiragi and Solter, 2004; Alarcon and Marikawa, 2005; Louvet-Vallee et al., 2005; Motosugi et al., 2005) and the issue is currently under hot debate (Vogel, 2005). Deepening our knowledge of this issue will not only provide an essential basis for understanding mammalian development, but also directly apply to ongoing clinical practices such as intracytoplasmic sperm injection (ICSI) and preimplantation genetic diagnosis (PGD). These practices were originally supported by a classical premise that mammalian preimplantation embryos are highly regulative (Tarkowski, 1959; Tarkowski, 1961; Tarkowski and Wroblewska, 1967; Rossant, 1976), in keeping with the "regulative model". However, if the "prepatterning model" is correct, the latter will require critical reassessment.
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http://dx.doi.org/10.1387/ijdb.062181thDOI Listing
November 2006

Resolution of chiasmata in oocytes requires separase-mediated proteolysis.

Cell 2006 Jul;126(1):135-46

Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, A-1030 Vienna, Austria.

In yeast, resolution of chiasmata in meiosis I requires proteolytic cleavage along chromosome arms of cohesin's Rec8 subunit by separase. Since activation of separase by the anaphase-promoting complex (APC/C) is supposedly not required for meiosis I in Xenopus oocytes, it has been suggested that animal cells might resolve chiasmata by a separase-independent mechanism related to the so-called "prophase pathway" that removes cohesin from chromosome arms during mitosis. By expressing Cre recombinase from a zona pellucida promoter, we have deleted a floxed allele of separase specifically in mouse oocytes. This prevents removal of Rec8 from chromosome arms and resolution of chiasmata. It also hinders extrusion of the first polar body (PBE) and causes female sterility. mRNA encoding wild-type but not catalytically inactive separase restores chiasma resolution. Both types of mRNA restore PBE. Proteolytic activity of separase is therefore essential for Rec8's removal from chromosome arms and for chiasma resolution but not for PBE.
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http://dx.doi.org/10.1016/j.cell.2006.05.033DOI Listing
July 2006

Embryology: does prepatterning occur in the mouse egg?

Nature 2006 Jul;442(7099):E3-4; discussion E4

Max Planck Institute of Immunobiology, 79108 Freiburg, Germany.

A recurring question in developmental biology has been whether localized determinants play any role in mammalian preimplantation development. This is a controversial issue that brings back the idea of prepatterning and is explored further by Plusa et al., who claim it is the first cleavage of the mouse zygote that predicts the blastocyst axis, rather than the animal pole or sperm entry point, as previously suggested. However, other evidence indicates that the blasotcyst axis is not predetermined and there is no prepatterning in the mouse egg. Here we investigate the origin of these different views and conclude that they arise from differences in the data themselves and in their interpretation.
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http://dx.doi.org/10.1038/nature04907DOI Listing
July 2006

Cytoskeleton and cell cycle control during meiotic maturation of the mouse oocyte: integrating time and space.

Reproduction 2005 Dec;130(6):801-11

UMR 7622 Biologie du Développement, CNRS-UPMC, 9 Quai St Bernard, 75005 Paris, France.

During meiotic maturation of mammalian oocytes, two successive divisions occur without an intermediate phase of DNA replication, so that haploid gametes are produced. Moreover, these two divisions are asymmetric, to ensure that most of the maternal stores are retained within the oocyte. This leads to the formation of daughter cells with different sizes: the large oocyte and the small polar bodies. All these events are dependent upon the dynamic changes in the organization of the oocyte cytoskeleton (microtubules and microfilaments) and are highly regulated in time and space. We review here the current knowledge of the interplay between the cytoskeleton and the cell cycle machinery in mouse oocytes, with an emphasis on the two major activities that control meiotic maturation in vertebrates, MPF (Maturation promoting factor) and CSF (Cytostatic factor).
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http://dx.doi.org/10.1530/rep.1.00364DOI Listing
December 2005

Vezatin, a protein associated to adherens junctions, is required for mouse blastocyst morphogenesis.

Dev Biol 2005 Nov 29;287(1):180-91. Epub 2005 Sep 29.

Laboratoire de Biologie Cellulaire du Développement, UMR 7622, CNRS, Université Pierre et Marie Curie, 75252 Paris cedex 05, France.

Cell-cell interactions play a major role during preimplantation development of the mouse embryo. The formation of adherens junctions is a major feature of compaction, the first morphogenetic event that takes place at the 8-cell stage. Then, during the following two cell cycles, tight junctions form, and the outer layer of cells differentiate into a functional epithelium, leading to the formation of the blastocoel cavity. Until now, E-cadherin was the only transmembrane molecule localized in adherens junctions and required for early development. Vezatin is a transmembrane protein of adherens junctions, interacting with the E-cadherin-catenins complex. Here, we show that vezatin is expressed very early during mouse preimplantation development. It co-localizes with E-cadherin throughout development, being found all around the cell cortex before compaction and basolaterally in adherens junctions thereafter. In addition, vezatin is also detected in nuclei during most of the cell cycle. Finally, using a morpholino-oligonucleotide approach to inhibit vezatin function during preimplantation development, we observed that inhibition of vezatin synthesis leads to a cell cycle arrest with limited cell-cell interactions. This phenotype can be rescued when mRNAs coding for vezatin missing the 5'UTR are co-injected with the anti-vezatin morpholino-oligonucleotide. Cells derived from blastomeres injected with morpholino-oligonucleotide had a reduced amount of vezatin concomitantly with a decrease in the quantity of E-cadherin and beta-catenin localized in the areas of intercellular contact. Shift in E-cadherin cortical distribution was correlated with a strong decrease in E-cadherin mRNA and protein contents. Altogether, these observations demonstrate that vezatin is required for morphogenesis of the preimplantation mouse embryo.
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http://dx.doi.org/10.1016/j.ydbio.2005.09.004DOI Listing
November 2005

Asymmetric distribution of PAR proteins in the mouse embryo begins at the 8-cell stage during compaction.

Dev Biol 2005 Jun;282(2):307-19

Laboratoire de Biologie Cellulaire du Développement, UMR 7622, CNRS, Université Pierre et Marie Curie, 9 Quai St Bernard, 75252 Paris cedex 05, France.

In many organisms, like Caenorhabditis elegans and Drosophila melanogaster, establishment of spatial patterns and definition of cell fate are driven by the segregation of determinants in response to spatial cues, as early as oogenesis or fertilization. In these organisms, a family of conserved proteins, the PAR proteins, is involved in the asymmetric distribution of cytoplasmic determinants and in the control of asymmetric divisions. In the mouse embryo, it is only at the 8-cell stage during compaction that asymmetries, leading to cellular diversification and blastocyst morphogenesis, are first observed. However, it has been suggested that developmentally relevant asymmetries could be established already in the oocyte and during fertilization. This led us to study the PAR proteins during the early stages of mouse development. We observed that the homologues of the different members of the PAR/aPKC complex and PAR1 are expressed in the preimplantation mouse embryo. During the first embryonic cleavages, before compaction, PARD6b and EMK1 are observed on the spindle. The localization of these two proteins becomes asymmetric during compaction, when blastomeres flatten upon each other and polarize. PARD6b is targeted to the apical pole, whereas EMK1 is distributed along the baso-lateral domain. The targeting of EMK1 is dependent upon cell-cell interactions while the apical localization of PARD6b is independent of cell contacts. At the 16-cell stage, aPKCzeta colocalizes with PARD6b and a colocalization of the three proteins (PARD6b/PARD3/aPKCzeta can occur in blastocysts, only at tight junctions. This choreography suggests that proteins of the PAR family are involved in the setting up of blastomere polarity and blastocyst morphogenesis in the early mammalian embryo although the interactions between the different players differ from previously studied systems. Finally, they reinforce the idea that the first developmentally relevant asymmetries are set up during compaction.
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http://dx.doi.org/10.1016/j.ydbio.2005.03.001DOI Listing
June 2005

Mitotic spindles and cleavage planes are oriented randomly in the two-cell mouse embryo.

Curr Biol 2005 Mar;15(5):464-9

Laboratoire de Biologie Cellulaire du Développement, UMR 7622, Centre National de la Recherche Scientifique, Université Pierre et Marie Curie, 9 Quai St. Bernard, 75252 Paris cedex 05, France.

Most experimental embryological studies performed on the early mouse embryo have led to the conclusion that there are no mosaically distributed developmental determinants in the zygote and early embryo (for example see [1-6]). It has been suggested recently that "the cleavage pattern of the early mouse embryo is not random and that the three-dimensional body plan is pre-patterned in the egg" (in [7] for review see [8-10]). Two major spatial cues influencing the pattern of cleavage divisions have been proposed: the site of the second meiotic division [11, 12] and the sperm entry point [13-14], although the latter is controversial [15-17]. An implication of this hypothesis is that the orientations of the first few cleavage divisions are stereotyped. Such a define cleavage pattern, leading to the segregation of developmental determinants, is observed in many species [18]. Recently, it was shown that the first cleavage plane is not predetermined but defined by the topology of the two apposing pronuclei [19]. Because the position of the female pronucleus is dependent upon the site of polar body extrusion and the position of the male pronuclei is dependent upon the sperm entry point [19-20], this observation leaves open the possibility that the sperm may provide some kind of directionality [7]. But, even if asymmetries were set up only after fertilization, a stereotyped cleavage pattern should take place during the following cleavage divisions. Thus, we studied the cleavage pattern of two-cell embryos by videomicroscopy to distinguish between the two hypotheses. After the mitotic spindle formed, its orientation did not change until cleavage. During late metaphase and anaphase, the spindle poles appear to be anchored to the cortex through astral microtubules and PARD6a. Only at the time of cleavage, during late anaphase, do the forming daughter cells change their relative positions. These studies show that cleavage planes are oriented randomly in two-cell embryos. This argues against a prepatterning of the mouse embryo before compaction.
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http://dx.doi.org/10.1016/j.cub.2004.12.078DOI Listing
March 2005

Phosphorylation of ezrin on threonine T567 plays a crucial role during compaction in the mouse early embryo.

Dev Biol 2004 Jul;271(1):87-97

Laboratoire de Biologie Cellulaire du Développement, UMR7622, CNRS-Université Pierre et Marie Curie, 9 Quai St-Bernard, 75252 Paris cedex 05, France.

The preimplantation development of the mouse embryo leads to the divergence of the first two cell lineages, the inner cell mass and the trophectoderm. The formation of a microvillus pole during compaction at the eight-cell stage and its asymmetric inheritance during mitosis are key events in the emergence of these two cell populations. Ezrin, a member of the ERM protein family, seems to be involved in the formation and stabilization of this apical microvillus pole. To further characterize its function in early development, we mutated the key residue T567, which was reported to be essential for regulation of ezrin function through phosphorylation. Here, we show that expression of ezrin mutants in which the COOH-terminal threonine T567 was replaced by an aspartate (to mimic a phosphorylated residue; T567D) or by an alanine (to avoid phosphorylation; T567A) interferes with E-cadherin function and disrupts the first morphogenetic events of development: compaction and cavitation. The active mutant ezrin-T567D induces the formation of numerous and abnormally long microvilli at the surface of blastomeres. Moreover, it localizes all around the cell cortex and inhibits cell-cell adhesion and cell polarization at the eight-cell stage. During the following stages, only half of the embryos are able to compact and finally to cavitate. In those embryos, the amount of ezrin-T567D decreases in the basolateral areas, while the proportion of adherens junctions increases. The reverse inactive mutant ezrin-T567A is mainly cytoplasmic and does not perturb compaction at the eight-cell stage. However, at the 16-cell stage, it relocalizes at the basolateral cortex, leading to a strong decrease in the surface of adherens junctions, and finally, embryos abort development. Our results show that ezrin is directly involved in the formation of microvilli in the early mouse embryo. Moreover, they indicate that maintenance of ezrin in basolateral areas prevents microvilli breakdown and inhibits the formation of normal cell-cell contacts mediated by E-cadherin, thereby impairing blastomeres polarization and morphogenesis of the blastocyst.
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http://dx.doi.org/10.1016/j.ydbio.2004.03.024DOI Listing
July 2004

Two PAR6 proteins become asymmetrically localized during establishment of polarity in mouse oocytes.

Curr Biol 2004 Mar;14(6):520-5

Laboratoire de Biologie Cellulaire du Développement, Unité Mixte de Recherche 7622, Centre National de la Recherche Scientifique, Université Pierre et Marie Curie, 9 quai Saint-Bernard, 75252 Paris cedex 05, France.

Meiotic maturation in mammals is characterized by two asymmetric divisions, leading to the formation of two polar bodies and the female gamete. Whereas the mouse oocyte is a polarized cell, molecules implicated in the establishment of this polarity are still unknown. PAR proteins have been demonstrated to play an important role in cell polarity in many cell types, where they control spindle positioning and asymmetric distribution of determinants. Here we show that two PAR6-related proteins have distinct polarized distributions in mouse oocytes. mPARD6a is first localized on the spindle and then accumulates at the pole nearest the cortex during spindle migration. In the absence of microtubules, the chromosomes still migrate to the cortex, and mPARD6a was found associated with the chromosomes and was facing the cortex. mPARD6a is the first identified protein to associate with the spindle during spindle migration and to relocalize to the chromosomes in the absence of microtubule behavior, suggesting a role in spindle migration. The other protein, mPARD6b, was found on spindle microtubules until entry into meiosis II and relocalized to the cortex at the animal pole during metaphase II arrest. mPARD6b is the first identified protein to localize to the animal pole of the mouse oocyte and likely contributes to the polarization of the cortex.
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http://dx.doi.org/10.1016/j.cub.2004.02.061DOI Listing
March 2004

The meiosis I-to-meiosis II transition in mouse oocytes requires separase activity.

Curr Biol 2003 Oct;13(20):1797-802

Division Méiotiques, Centre National de la Recherche Scientifique, Université Pierre et Marie Curie, 75252 Paris, cedex 05, France.

Faithful segregation of homologous chromosomes during the first meiotic division is essential for further embryo development. The question at issue is whether the same mechanisms ensuring correct separation of sister chromatids in mitosis are at work during the first meiotic division. In mitosis, sister chromatids are linked by a cohesin complex holding them together until their disjunction at anaphase. Their disjunction is mediated by Separase, which cleaves the cohesin. The activation of Separase requires prior degradation of its associated inhibitor, called securin. Securin is a target of the APC/C (Anaphase Promoting Complex/Cyclosome), a cell cycle-regulated ubiquitin ligase that ubiquitinates securin at the metaphase-to-anaphase transition and thereby targets it for degradation by the 26S proteasome. After securin degradation, Separase cleaves the cohesins and triggers chromatid separation, a prerequisite for anaphase. In yeast and worms, the segregation of homologous chromosomes in meiosis I depends on the APC/C and Separase activity. Yet, it is unclear if Separase is required for the first meiotic division in vertebrates because APC/C activity is thought to be dispensable in frog oocytes. We therefore investigated if Separase activity is required for correct chromosome segregation in meiosis I in mouse oocytes.
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http://dx.doi.org/10.1016/j.cub.2003.09.032DOI Listing
October 2003

Metaphase I arrest upon activation of the Mad2-dependent spindle checkpoint in mouse oocytes.

Curr Biol 2003 Sep;13(18):1596-608

Laboratoire de Biologie Cellulaire du Développement, UMR7622, CNRS, Université Pierre et Marie Curie, 9 quai Saint Bernard, 75005 Paris, France.

Background: The importance of mitotic spindle checkpoint control has been well established during somatic cell divisions. The metaphase-to-anaphase transition takes place only when all sister chromatids have been properly attached to the bipolar spindle and are aligned at the metaphase plate. Failure of this checkpoint may lead to unequal separation of sister chromatids. On the contrary, the existence of such a checkpoint during the first meiotic division in mammalian oocytes when homologous chromosomes are segregated has remained controversial.

Results: Here, we show that mouse oocytes respond to spindle damage by a transient and reversible cell cycle arrest in metaphase I with high Maturation Promoting Factor (MPF) activity. Furthermore, the mitotic checkpoint protein Mad2 is present throughout meiotic maturation and is recruited to unattached kinetochores. Overexpression of Mad2 in meiosis I leads to a cell cycle arrest in metaphase I. Expression of a dominant-negative Mad2 protein interferes with proper spindle checkpoint arrest.

Conclusions: Errors in meiosis I cause missegregation of chromosomes and can result in the generation of aneuploid embryos with severe birth defects. In human oocytes, failures in spindle checkpoint control may be responsible for the generation of trisomies (e.g., Down Syndrome) due to chromosome missegregation in meiosis I. Up to now, the mechanisms ensuring correct separation of chromosomes in meiosis I remained unknown. Our study shows for the first time that a functional Mad2-dependent spindle checkpoint exists during the first meiotic division in mammalian oocytes.
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http://dx.doi.org/10.1016/j.cub.2003.08.052DOI Listing
September 2003

DOC1R: a MAP kinase substrate that control microtubule organization of metaphase II mouse oocytes.

Development 2003 Nov 27;130(21):5169-77. Epub 2003 Aug 27.

UMR 7622, CNRS, Université Paris VI, 9 quai Saint Bernard, Bat. C, 75252 Paris, cedex 05, France.

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.
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http://dx.doi.org/10.1242/dev.00731DOI Listing
November 2003

Polar body formation: new rules for asymmetric divisions.

Nat Cell Biol 2002 Dec;4(12):E281-3

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http://dx.doi.org/10.1038/ncb1202-e281DOI Listing
December 2002

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

J Cell Biol 2002 May 13;157(4):603-13. Epub 2002 May 13.

Biologie Cellulaire et Moléculaire du Developpement, Centre National de la Recherche Scientifique, Université Pierre et Marie Curie, Paris, France.

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).
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http://dx.doi.org/10.1083/jcb.200202052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2173866PMC
May 2002