Bernard Maro’s research while affiliated with Tel Aviv University and other places

La meiose, qui permet la production des cellules germinales haploides, comporte deux divisions, la premiere separant les chromosomes homologues, et la seconde, les chromatides sœurs. Les etudes effectuees sur les ovocytes de differentes especes, en particulier la souris, ont permis de determiner les specificites des divisons meiotiques, notamment les mecanismes gouvernant la maturation meiotique, la formation du fuseau de premiere division de meiose, le caractere asymetrique de ces divisions ainsi que le processus d'arret de la meiose jusqu'a la fecondation. Nombre de ces processus distinguent clairement la meiose de la mitose.

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.

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.

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.

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.

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. 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.

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. 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. 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.

Stack of confocal sections of siRNA-aPKCλ injected embryos stained for actin (red) and chromatin (green). (8.65 MB MOV)

Video-microscope analysis of control-siRNA (left panel) and siRNA-aPKCλ (right panel) injected embryos. Two-cell stage embryos were microinjected with siRNA (siRNA-aPKCλ or siRNA-Ctrl) in both blastomeres and visualized under a spinning-disk videomicroscope during the 8- and 16-cell stages. Acquisitions were performed every 30 minutes. (2.52 MB MOV)