Oocyte Mechanics and Morphogenesis
Principal Investigators: Marie-Emilie TERRET, DR2 Inserm & Marie-Hélène VERLHAC, DRCE1 Cnrs
Our goal aims at understanding how a female gamete turns into a viable embryo, using morphological and biophysical approaches of meiotic divisions.
Our group studies the last stages of murine oogenesis, consisting in two successive asymmetric divisions without intervening DNA replication (Figure 1). It produces functional female gametes, the oocytes, required for sexual reproduction. Meiotic divisions can be reproduced and followed in vitro on synchronized population of cells. They are extremely asymmetric in size of the daughter cells, which allows the preservation of maternal stores required for embryo development. For that, oocytes rely on off-center spindle positioning, challenged by the lack of canonical centers of microtubule nucleation, namely centrosomes. During the last 5 years, we have identified mechanisms regulating the positioning of meiotic chromosomes in the absence of canonical centrosomes, processes that might have their share in the innate susceptibility of the female gamete to produce errors in chromosome segregation.
Figure 1: End of oocyte growth, meiotic divisions and early development in mammals. During their growth in the ovary, oocytes are arrested in Prophase I of meiosis. Under a maturation-inducing hormone, they resume meiosis and undergo two successive asymmetric meiotic divisions, without intervening DNA replication. NEBD (Nuclear Envelope BreakDown) marks the entry into meiotic divisions. DNA is in blue (dark blue for nucleolus) and microtubules in green. The black arrows indicate the direction of chromosome motion.
1/ A pressure gradient to position the nucleus
In oocytes from many species, nucleus position defines the future axis of the embryo and of the adult. However, in mammals, the oocyte nucleus is centrally located and does not instruct any future embryo axis. Yet an off-center nucleus correlates with a poor outcome for mouse and human oocyte development, arguing that central positioning is important for later embryo development. This is surprising since oocytes further undergo two extremely asymmetric divisions in size, requiring an off-centering of their chromosomes (Figure 1). Since oocytes in most organisms lack centrosomes, they cannot use centrosome-based nucleus positioning. Thanks to a multidisciplinary approach, we recently discovered how the nucleus robustly localizes in a unique manner at the center of mouse oocytes via actin-based mechanisms (Almonacid NCB 2015).
Figure 2: Formin 2 (Fmn 2) -/- oocytes present an off-center nucleus and display no cytoplasmic F-actin mesh. The introduction of Formin 2 into Fmn2-/- oocytes rapidly rescues the F-actin cytoplasmic mesh and in about 5h the central position of the nucleus. The black arrows indicate the direction of the pressure gradient exerted by actin-positive vesicles on the nucleus.
Oocytes derived from Formin 2 (Fmn2) knockout mice, which lack microfilaments in their cytoplasm, present off-centered nuclei (Figure 2). Formin 2 is a straight microfilament nucleator and also an essential maternal gene. The re-introduction of Formin 2 into Fmn2-/- oocytes, that harbour initially off-centered nuclei, induces a directional motion of the nucleus toward the center in about 5h in 100 % of the cases. The oocyte cytoplasmic mesh of F-actin is organized from Rab11a positive vesicles where two types of nucleators, Spire 1/2 and Formin 2 cooperate. We showed that the motion of the nucleus from the periphery to the center depends on a gradient of pressure coming from a slight bias in the speed and the directionality of actin-positive vesicles toward the cortex. By directly measuring the viscosity of the mouse oocyte cytoplasm using optical tweezers, we demonstrated that the motion of actin-positive vesicles, via Myosin Vb activity, promotes the fluidization of the whole cytoplasm, essential to favor the movement of the nucleus, a very large object (30 µm wide), from the periphery to the center of the oocyte.
2/ Cortex softening promotes asymmetry, while cortex stiffening promotes symmetry
The lack of true centrosomes in oocytes imposes peculiar modes of spindle morphogenesis and positioning that we study in the lab. In particular, since mouse oocytes lack true centrosomes, they also lack their associated astral microtubules, essential in mitotic cells to position the spindle. We and others have shown that actin controls meiotic spindle off-centering in oocytes. F-actin is organized in two networks, each required for spindle migration since when one is missing spindle motion is abolished.
The first one is a cytoplasmic actin meshwork comparable to the one present in Prophase I (Figure 3), including an F-actin cage surrounding the microtubule spindle. This meshwork replaces astral microtubules and connects the spindle poles to the cortex. It requires a motor, myosin-II, located at both poles of the actin cage to generate the forces required for spindle migration.
Figure 3: Actin networks, microtubule spindle and myosin-II in mouse oocyte. A. Spinning disk images of a mouse oocyte in Prophase I and 7h after Nuclear Envelope Breakdown (NEBD). DNA is in blue, F-actin in white, myosin-II in black. Scale bar 10 µm. The orange dash highlights the cortical actin thickening. B. Images from (Azoury 2008) showing a meiotic spindle (microtubules are in red) embedded in F-actin (green). White arrows point at F-actin filaments running along or in the microtubule spindle. Scale bar 5 µm.
We have discovered a second actin mesh, a cortical F-actin thickening nucleated by the Arp2/3 complex, a branched actin nucleator (Figure 3). Using multidisciplinary approaches, we showed that the nucleation of the cortical F-actin thickening excludes myosin-II from the cortex (Figure 3), decreasing cortical tension. This change in cortex mechanics amplifies an initial imbalance of pulling forces exerted by myosin-II at the poles of the actin cage. The forces are stronger at the pole closest to the cortex because of the initial slight asymmetry of nuclear position. Although the drop in cortical tension is required for spindle migration in oocytes, as artificially stiffening the cortex impairs spindle off-centring, spindle migration is also prevented by a too low tension. Thus the geometry of the division of mouse oocytes depends on a narrow window of cortical tension, regulated by myosin-II cortical localization, itself fine-tuned by actin nucleation (Chaigne NCB 2013; Chaigne Nat Commun 2015). If cortex softening favors asymmetry of meiotic spindle positioning, cortex stiffening in early zygotes after fertilization promotes symmetry of the division with spindle centering (Chaigne Nat Commun 2016). The abrupt change in cortex properties between oocyte and early zygote is essential for the meiotic to mitotic transition and appears conserved in humans.
Our project aims at understanding the impact of actin meshes in the control of chromosome positioning and on the developmental potential of the oocyte. Interestingly, nucleus position as well as cortex properties constitute non-invasive predictors of viability of early embryo development that could be used easily in Assisted Reproduction Technics where up to now embryo selection consists of a highly subjective morphological assessment.
Terret Marie-Emilie, DR2 INSERM
Verlhac Marie-Hélène, DRCE1 CNRS
Almonacid Maria, CRCN CNRS
Postdoctoral fellows, PhD Students & Master student:
Al Jord Adel, Postdoctoral fellow
Letort Gaëlle, Postdoctoral fellow
Nikalayevich Elvira, Postdoctoral fellow
Crozet Flora, PhD student
Da Silva Christelle, TCN CNRS
Eichmuller Adrien, CDD AI INSERM
- Bennabi, I., Crozet, F., Nikalayevich, E., Chaigne, A., Letort, G., Manil-Ségalen, M., Campillo, C., Cadart, C., Othmani, A., Attia, R., et al. (2020). Artificially decreasing cortical tension generates aneuploidy in mouse oocytes. Nat Commun 11, 1649.
- Bennabi, I., Verlhac, M.-H., and Terret, M.-E. (2020). [Cortical tension of the oocyte and euploidy: the right balance]. Med Sci (Paris) 36, 965–968.
- Colin, A., Letort, G., Razin, N., Almonacid, M., Ahmed, W., Betz, T., Terret, M.-E., Gov, N.S., Voituriez, R., Gueroui, Z., Verlhac, M.-H. (2020). Active diffusion in oocytes nonspecifically centers large objects during prophase I and meiosis I. J. Cell Biol. 219.
- Almonacid, M., Al Jord, A., El-Hayek, S., Othmani, A., Coulpier, F., Lemoine, S., Miyamoto, K., Grosse, R., Klein, C., Piolot, T., Mailly, P., Voituriez, R., Genovesio, A., Verlhac, M.-H. (2019). Active Fluctuations of the Nuclear Envelope Shape the Transcriptional Dynamics in Oocytes. Developmental Cell 51, 145-157.e10.
Preview from : Mogessie, B. (2019). Conducting Chromatin Motion: Actin Dynamizes Contents of the Oocyte Nucleus. Developmental Cell 51, 133–134.
- Almonacid, M., Terret, M.-E., and Verlhac, M.-H. (2019). Nuclear positioning as an integrator of cell fate. Curr. Opin. Cell Biol. 56, 122–129.
- Letort, G., Bennabi, I., Dmitrieff, S., Nedelec, F., Verlhac, M.-H., and Terret, M.-E. (2019). A computational model of the early stages of acentriolar meiotic spindle assembly. MBoC mbc.E18-10-0644.
- Simerly, C., Manil-Ségalen, M., Castro, C., Hartnett, C., Kong, D., Verlhac, M.-H., Loncarek, J., and Schatten, G. (2018). Separation and Loss of Centrioles From Primordidal Germ Cells To Mature Oocytes In The Mouse. Sci Rep 8, 12791.
- Manil-Ségalen, M., Łuksza, M., Kanaan, J., Marthiens, V., Lane, S. I. R., Jones, K. T., Terret, M.-E., Basto, R. & Verlhac, M.-H. (2018). Chromosome structural anomalies due to aberrant spindle forces exerted at gene editing sites in meiosis. J. Cell Biol. 217: 3416-3430.
- Al Jord, A., and Verlhac, M.-H. (2018). Spindle Assembly: Two Spindles for Two Genomes in a Mammalian Zygote. Curr. Biol. 28, R948–R951.
- Verlhac, M.-H. (2018). An actin shell delays oocyte chromosome capture by microtubules. J. Cell Biol. 217, 2601–2603.
- Bennabi I., Quéguiner I., Kolano A., Boudier T., Mailly P., Verlhac M.-H.* & Terret M.-E.*. (2018). Shifting meiotic to mitotic spindle assembly in oocytes disrupts chromosome alignment. EMBO Rep 19, 368-381 (*co-senior authors).
- Almonacid, M., Terret, M.-E., and Verlhac, M.-H. (2018). Control of nucleus positioning in mouse oocytes. Semin. Cell Dev. Biol. 82, 34–40.
- Chaigne A., Terret M.-E. & Verlhac M.-H. (2017). Asymmetries and Symmetries in the Mouse Oocyte and Zygote. Results Probl Cell Differ 61, 285–299.
- Bennabi I., Terret M.-E. & Verlhac M.-H. (2016). Meiotic spindle assembly and chromosome segregation in oocytes. J Cell Biol 215, 611-619.
- Chaigne A. (2016). Mécanique de la cellule et œuf mollet. Pour la Science 470, 44–53.
- Terret M.-E. & Verlhac M.-H. (2016). Comment l'embryon se divise-t-il ? La Recherche 518, 42–46.
- Verlhac M.-H. (2016). Mother centrioles are kicked out so that starfish zygote can grow. J Cell Biol 212, 759–761.
- Verlhac M.-H. & Terret M.-E. (2016). Oocyte Maturation and Development. F1000 Research 5, 309-317.
- Chaigne A., Campillo C., Voituriez R., Gov N. S., Sykes C., Verlhac M.-H.* & Terret M.-E.*. (2016). F-actin mechanics control spindle centring in the mouse zygote. Nat Commun 7, 10253-10267 (*co-senior authors).
- Grey C., Espeut J., Ametsitsi R., Kumar R., Luksza M., Brun C., Verlhac M.-H., Suja JÁ & de Massy B. (2016). SKAP, an outer kinetochore protein, is required for mouse germ cell development. Reprod 151, 239–251.
- Li H, Moll ., Winkler A., Frappart L., Brunet S., Hamann J., Kroll T., Verlhac M.-H., Heuer H., Herrlich P. & Ploubidou A. (2015). RHAMM deficiency disrupts folliculogenesis resulting in female hypofertility. Biol Open 4, 562–571.
- Almonacid M., Ahmed W. W., Bussonnier M., Mailly P., Betz T., Voituriez R., Gov N. S. & Verlhac M.-H. (2015). Active diffusion positions the nucleus in mouse oocytes. Nat Cell Biol 17, 470–479.
- Chaigne A., Campillo C., Gov N. S., Voituriez R., Sykes C., Verlhac M.-H.* & Terret M.-E*. (2015). A narrow window of cortical tension guides asymmetric spindle positioning in mouse oocyte, Nat Commun 6, 6027-6037 (*co-senior authors).
- Chaigne A., Verlhac M.-H. & Terret M.-E. (2014). Ramollir le cortex : un prérequis à l’asymétrie de la division ovocytaire. Médecine/Sciences 30, 18-21.
- Almonacid M., Terret M.-E. & Verlhac M.-H. (2014). Actin-based spindle positioning : new insights from female gametes. J Cell Sci 127, 477–483.
- Chaigne A., Campillo C., Gov N. S., Voituriez R., Azoury J., Umana-Diaz C., Almonacid M., Queguiner I., Nassoy P., Sykes C., Verlhac M.-H.* & Terret M.-E*. (2013). A soft cortex is essential for asymmetric spindle positioning in mouse oocytes. Nat Cell Biol 15,958-66 (*co-senior authors).
- Dumont J. & Verlhac M.-H. (2013). Using FRET to study RanGTP gradients in live mouse oocytes. Methods Mol Biol 957, 107-120.
- Łuksza M., Queguiner I., Verlhac M.-H.* & Brunet S*. (2013). Rebuilding MTOCs upon centriole loss during mouse oogenesis. Dev Biol 382, 48-56 (*co-senior authors).
- Terret M.-E., Chaigne A., Verlhac M.-H. (2013). Mouse oocyte, a paradigm of cancer cell. Cell Cycle 12,3370-3376.
- Kolano A., Brunet S., Silk A. D., Cleveland D. W. & Verlhac M.-H. (2012). Error prone mammalian female meiosis from silencing the SAC without normal interkinetochore tension. PNAS 109, E1858-E1867.
- Verlhac M.-H. (2011), Spindle positioning: going against the actin flow. Nat Cell Biol 12, 1183-1185.
- Azoury J., Lee K. W., Georget V., Hikal P. & Verlhac M.-H. (2011). Symmetry breaking in mouse oocytes requires transient F-actin meshwork destabilization. Development 138,2903-2908.
- Brunet S. & Verlhac M.-H. (2011). Positioning to get out of Meiosis: the asymmetry of division. Hum Reprod Update 17, 68-75.