Maria Almonacid
email: maria-elsa.almonacid@college-de-france.fr

Marie-Emilie Terret

Marie-Hélène Verlhac

Control of nucleus positioning in mouse oocytes

Keywords:

The position of the nucleus in a cell can instruct morphogenesis in some cases, conveying spatial and temporal information and abnormal nuclear positioning can lead to disease. In oocytes from worm, sea urchin, frog and some fish, nucleus position regulates embryo development, it marks the animal pole and in Drosophila it defines the future dorso-ventral axis of the embryo and of the adult body plan. However, in mammals, the oocyte nucleus is centrally located and does not instruct any future embryo axis. Yet an off-center nucleus correlates with poor outcome for mouse and human oocyte development. This is surprising since oocytes further undergo two extremely asymmetric divisions in terms of the size of the daughter cells (enabling polar body extrusion), requiring an off-centering of their chromosomes. In this review we address not only the bio-physical mechanism controlling nucleus positioning via an actinmediated pressure gradient, but we also speculate on potential biological relevance of nuclear positioning in mammalian oocytes and early embryos.

Introduction

Regulation of nuclear position is essential for the achievement of a variety of cellular and developmental functions. In the female gamete of most species, nuclear positioning close to the oocyte cortex prepares for the very asymmetric divisions allowing the maintenance of maternal stores supporting embryo development. Indeed, meiotic spindles form where the nucleus was, and thus a cortically-associated spindle will promote asymmetric partitioning of the cytoplasm. In most oocyte models, nucleus positioning relies on microtubules, and, depending whether or not oocytes have kept mother centrioles, involves or not centrosomes. In echinoderms, the nucleus is maintained close to the cortex by centrosomemicrotubules asters [START_REF] Miyazaki | Premeiotic aster as a device to anchor the germinal vesicle to the cell surface of the presumptive animal pole in starfish oocytes[END_REF][START_REF] Miyazaki | Role of microtubules and centrosomes in the eccentric relocation of the germinal vesicle upon meiosis reinitiation in sea-cucumber oocytes[END_REF]. In Drosophila oocytes, the nucleus is pushed towards the antero-dorsal cortex by microtubules, following two redundant pathways: a centrosomal-microtubules pathway and a pathway involving the microtubule-associated protein Mud/NuMa [START_REF] Tissot | Distinct molecular cues ensure a robust microtubule-dependent nuclear positioning in the Drosophila oocyte[END_REF]. In C.elegans oocytes, that are devoid of centrosomes, nuclear positioning is Kinesin 1 dependent [START_REF] Mcnally | Kinesin-dependent transport results in polarized migration of the nucleus in oocytes and inward movement of yolk granules in meiotic embryos[END_REF]. In mammals, oocyte growth takes place in the ovary. Fully-grown oocytes arrested in Prophase I resume meiosis and undergo two successive asymmetric divisions. Fertilization triggers the second meiotic division. The zygote then achieves the very first embryonic division. Nuclear centering takes place during the growth phase. Asymmetric meiotic divisions occur with spindle and chromosomes off-centering. Fertilization brings the paternal genome into the egg. Then both maternal and paternal genomes, packed into pronuclei, merge together near the center of the zygote to prepare for the first embryonic division. Blue: nuclei, chromosomes and sperm. Dark blue: nucleoli. Green: spindle microtubules. Fig. 2. Nuclear centering by a cytoplasmic actin mesh. A: In fully-grown mouse oocytes, the straight actin nucleator Formin 2 nucleates a cytoplasmic actin mesh involved in nuclear centering. Left: Fmn2 -/oocytes lack a cytoplasmic actin mesh and also display abnormally off-centered nuclei. Right: Reintroducing Formin 2 back into Fmn2 -/oocytes restores the cytoplasmic actin mesh that exerts a pressure gradient on the nucleus, inducing its repositioning from the cortex to the oocyte center, as for control Fmn2 +/-oocytes. Single plane confocal microscopy images: F-actin is in white and DNA is in blue. DNA forms a ring around the nucleolus in fully-grown oocytes. For Fmn2 -/oocytes, the only pools of actin are cortical actin and small seeds in the cytoplasm corresponding to actin-coated vesicles. For Fmn2 +/-oocytes, two pools of actin coexist: cortical actin and the cytoplasmic actin mesh. Scale bar is 10 m. Figurative images: nucleus is in blue, nucleolus in dark blue and actin filaments are in red. Black arrows represent the pressure gradient. B: Model for nucleus positioning in mouse oocytes. The nucleus experiences a propulsion force Fp = -␣ <v 2 > (␣ being constant) resulting from a gradient of Myosin Vb-dependent activity of actin vesicles <v 2 >. This propulsion force is balanced by a friction force Ff = -V (V nuclear velocity and proportional to cytoplasmic viscosity). Thus, nuclear velocity V = -(␣/) <v 2 > is proportional to the activity gradient of actin vesicles and inversely proportional to the viscosity. The gradient of activity <v 2 > vanishes close to the center, thus keeping the nucleus in a central position. Blue: nucleus, red dots: actin vesicles, red lines: actin filaments, purple: Myosin Vb.

In mouse oocytes, nuclear positioning occurs during oocyte growth, which takes place in the ovary. During embryonic life, a population of germ cells synchronously progress into meiosis until becoming arrested in Prophase I. Then, periodically, all along the reproductive life, some oocytes enter into a growth phase upon hormonal stimulation, increasing in size up to 80 m diameter and accumulating the reserves necessary for further embryonic development (for a review, see [START_REF] El-Hayek | Control of oocyte growth and development by intercellular communication within the follicular niche[END_REF]). This growth phase prepares the female gamete for the following steps of meiotic divisions, fertilization and early embryonic divisions (Fig. 1). In particular, the production of a pool of dormant mRNAs is very important since transcription is strongly reduced at the end of oocyte growth, so that the following steps of meiotic divisions and early embryonic divisions take place without transcription, until the activation of the zygotic genome at the two-cell stage. During the growth phase, the nucleus moves from an off-centered position to the center of the oocyte. At the end of growth, meiosis resumes and the oocyte undergoes two successive asymmetric divisions, with off-centering of its genome. Thus, contrary to most oocyte models, nuclear centering in mouse and human does not position the genome in preparation for future asymmetric divisions. However, surprisingly, nuclear centering correlates with the success of meiotic divisions in mouse and human oocytes [START_REF] Brunet | Germinal vesicle position and meiotic maturation in mouse oocyte[END_REF][START_REF] Levi | Morphological and molecular markers are correlated with maturation-competence of human oocytes[END_REF]. Another interesting feature of nuclear positioning in mouse oocytes is its very original mechanism, that does not involve centrosomes and microtubules, as mouse oocytes lose their centrioles early during oocyte growth [START_REF] Szollosi | Absence of centrioles in the first and second meiotic spindles of mouse oocytes[END_REF].

An original mechanism for nuclear positioning

A cytoplasmic actin mesh involved in nuclear centering

The fully-grown mouse oocyte is filled with a cytoplasmic mesh of actin filaments, which is involved in spindle off-centering during asymmetric divisions [START_REF] Azoury | Spindle positioning in mouse oocytes relies on a dynamic meshwork of actin filaments[END_REF][START_REF] Schuh | A new model for asymmetric spindle positioning in mouse oocytes[END_REF]. This actin mesh is highly dynamic and is made out of actin-coated vesicles and actin filaments nucleated from the surface of these vesicles by the actin nucleator Formin 2 [START_REF] Schuh | An actin-dependent mechanism for long-range vesicle transport[END_REF] (Fig. 2A). Formin 2 is a straight microfilament nucleator, which permits the elongation of the barbed-end via conserved FH1-FH2 domains. In mouse oocytes, Formin 2 cooperates with Spire 1/2 in a functional unit to nucleate actin filaments at the surface of actin vesicles [START_REF] Schuh | An actin-dependent mechanism for long-range vesicle transport[END_REF][START_REF] Pfender | Spire-type actin nucleators cooperate with Formin-2 to drive asymmetric oocyte division[END_REF]. Formin 2 and Spire 1/2 are known to interact with each other [START_REF] Pechlivanis | Identification of a short Spir interaction sequence at the C-terminal end of formin subgroup proteins[END_REF]. In Drosophila oocytes, the formin Cappuccino and Spire also work together, as suggested by their genetic and physical interaction [START_REF] Quinlan | Regulatory interactions between two actin nucleators, Spire and Cappuccino[END_REF][START_REF] Rosales-Nieves | Coordination of microtubule and microfilament dynamics by Drosophila Rho1, Spire and Cappuccino[END_REF]. Recently, it has been proposed that Formin 2 and Spire 1/2 alternately kick-off each other from the barbed end of the filament, leading to alternating phases of processive assembly and arrested growth, in a kind of "ping-pong" mechanism [START_REF] Montaville | Spire and Formin 2 synergize and antagonize in regulating actin assembly in meiosis by a ping-pong mechanism[END_REF].

Oocytes invalidated for Formin 2 (Fmn2 -/-), lacking a cytoplasmic actin mesh, display also abnormally off-centered nuclei (Fig. 2A). Reintroducing Formin 2 back into Fmn2 -/oocytes allows for nucleus repositioning from the cortex to the oocyte center within 4-6 h. The mouse oocyte nucleus, about 30 m wide, moves a 20 m distance in about 5 h, with a global mean velocity of 0.07 m/min. This is slower than the movement of the meiotic spindle towards the cortex, which occurs within 2-3 h at a mean velocity of 0.12 m/min [START_REF] Verlhac | Asymmetric division in mouse oocytes: with or without Mos[END_REF], maybe because spindle movement depends on Myosin II contractility [START_REF] Schuh | A new model for asymmetric spindle positioning in mouse oocytes[END_REF][START_REF] Chaigne | A soft cortex is essential for asymmetric spindle positioning in mouse oocytes[END_REF][START_REF] Chaigne | A narrow window of cortical tension guides asymmetric spindle positioning in the mouse oocyte[END_REF] and because the spindle moving along its long axis provides less viscous friction than a round nucleus [START_REF] Verlhac | Asymmetric division in mouse oocytes: with or without Mos[END_REF].

This process is also extremely slow compared to the velocity of nucleus positioning mediated by microtubules in other model systems. For example in starfish oocytes, the sperm aster, to which the sperm nucleus is attached, reaches the oocyte center with a mean velocity of about 5 m/min [START_REF] Tanimoto | Shape-motion relationships of centering microtubule asters[END_REF]. The difference in efficiency of nucleus centration might reside in the 100 fold difference in the persistence length of actin filaments versus microtubules [START_REF] Gittes | Flexural rigidity of microtubules and actin filaments measured from thermal fluctuations in shape[END_REF].

The mouse nucleus centering process is very robust, as it occurs in all Fmn2 -/rescued oocytes. It is also not reversible, because the nucleus remains perfectly centered after disassembly of the actin cytoplasmic mesh following Cytochalasin D treatment in wild-type oocytes.

Dynamics of the cytoplasmic actin mesh and creation of an activity gradient

High temporal resolution movies of the cytoplasmic actin mesh during the process of nuclear repositioning, using the filamentous actin probe GFP-UtrCH [START_REF] Burkel | Versatile fluorescent probes for actin filaments based on the actin-binding domain of utrophin[END_REF], highlight the important dynamics of actin vesicles within the meshwork.

Tracking of these actin positive vesicles revealed that they establish a velocity gradient from the cortex, where they move faster, towards the oocyte center, where they move slower. This gradient could be explained by an attractive effect of the cortex enriched in actin filaments (Fig. 2A). Accordingly, the activity of actin vesicles, represented by their squared velocity v 2 , which is a measure of thermal agitation of particles in a fluid, follows a gradient distribution from the cortex to the oocyte center (Fig. 2B). By using an analogy of a perfect gas, this activity gradient of actin vesicles <v 2 > generates a propulsion force Fp = -␣ <v 2 >. This propulsion force is proportional to the gradient value and is thereby the trigger of the nuclear movement towards the oocyte center. In the zygote, the activity of the cytoplasmic mesh is homogeneous within the cell, whereas the density of the meshwork varies. The meshwork is denser between the pronuclei and the closest cortex (black arrows). This local actin enrichment may be due to the cortical differentiated zone above the second meiotic spindle for the female pronucleus and to the fertilization cone for the male pronucleus. This heterogeneity disappears around pronuclei that have migrated towards the center. Therefore, in the zygote, the force moving the pronuclei is generated by a density gradient rather than an activity gradient. Blue: pronuclei, dark blue: nucleoli, red lines: actin filaments.

The cytoplasmic actin mesh propels the cytoplasm by advection

The centripetal movement of the nucleus is concomitant with the appearance of a strong cytoplasmic streaming, corresponding to random movement of fluids and intracellular compartments. Remarkably, injection into wild-type oocytes of a dominantnegative of Myosin Vb, the molecular motor driving the dynamics of the cytoplasmic actin mesh [START_REF] Schuh | An actin-dependent mechanism for long-range vesicle transport[END_REF], fully abolishes both cytoplasmic streaming and nuclear positioning [START_REF] Almonacid | Active diffusion positions the nucleus in mouse oocytes[END_REF]. Cytoplasmic streaming is generated by the hydrodynamic drag of fluid and cytoplasmic components around Myosin Vb-driven actin vesicles. Therefore, the whole cytoplasm is subjected to advection, a transport mode where objects are moved with a flow. Advection has been reported in many developmental contexts. For example, in the Drosophila oocyte, two successive types of streaming occur during oogenesis (for a review, see [START_REF] Quinlan | Cytoplasmic streaming in the drosophila oocyte[END_REF]): a slow streaming at stages 8 and 9, followed by the appearance of a fast rotational streaming from stage 11, which are both driven by microtubules and Kinesin 1. During C.elegans oogenesis, an actin-dependent cytoplasmic streaming flows through the gonad [START_REF] Wolke | Actin-dependent cytoplasmic streaming in C. elegans oogenesis[END_REF] and in the oocyte/zygote, a circular cytoplasmic flow, driven by microtubules and Kinesin 1, reverses occasionally [START_REF] Mcnally | Kinesin-dependent transport results in polarized migration of the nucleus in oocytes and inward movement of yolk granules in meiotic embryos[END_REF][START_REF] Yang | MEI-1/katanin is required for translocation of the meiosis I spindle to the oocyte cortex in C. elegans[END_REF]. In the alga Chara corallina, an helical streaming is generated by Myosin XI sliding on actin filaments that are aligned along cylindrical cells [START_REF] Woodhouse | Cytoplasmic streaming in plant cells emerges naturally by microfilament self-organization[END_REF]. The role of such cytoplasmic streams is not fully understood but several studies suggest they may be particularly relevant during development. In Meiosis II arrested mouse oocytes, an Arp2/3-dependent fountain flow maintains the spindle beneath the oocyte cortex [START_REF] Yi | Dynamic maintenance of asymmetric meiotic spindle position through Arp2/3-complex-driven cytoplasmic streaming in mouse oocytes[END_REF]. In Drosophila oocytes, the slow cytoplasmic streaming during mid-oogenesis allows the transport of the body axes determinant, mRNA oskar, to the posterior cortex [START_REF] Ganguly | Cytoplasmic streaming in Drosophila oocytes varies with kinesin activity and correlates with the microtubule cytoskeleton architecture[END_REF], whereas the role of the later fast cytoplasmic streaming could be the mixing of cytoplasmic components provided by nurse cells [START_REF] Quinlan | Cytoplasmic streaming in the drosophila oocyte[END_REF]. In Arabidopsis, actin-Myosin XI dependent cytoplasmic streaming could be involved in plant size regulation [START_REF] Tominaga | Cytoplasmic streaming velocity as a plant size determinant[END_REF].

Dynamics of the cytoplasmic actin mesh and active diffusion

Addressing the mechanical properties of the mouse oocyte cytoplasm has been very informative in order to better understand the whole process of nucleus positioning. One way to gain access to the mechanical properties of a material is to track some particles embedded in it. If the particles move a lot, then the material is probably more fluid than if they move less. Calculating the Mean Squared Displacement (or MSD) of a particle, which represents the space explored by the particle in a given amount of time, allows having a measure of the particle motion. The MSD = <r 2 > = 2dDt, where d is the dimension of the system, D is the diffusion coefficient and t is the time. D is inversely proportional to the viscosity, due to the Stokes-Einstein equation D = k b T/(6R), where k b is the Boltzmann's constant, T is the temperature, is the viscosity and R is the radius of the particle. Thus, the extent of space explored by a particle is inversely proportional to the viscosity of the surrounding material. This is the case for diffusive (or Brownian) motion, which describes the motion of a particle only driven by thermal agitation around it. In a purely viscous material, a diffusive motion is characterized by a line of slope 1 when plotting the MSD against time in a LOG-LOG representation (Fig. 3A). Brownian motion is a particular case of a more general theorem of statistical physics, the Fluctuation Dissipation Theorem (or FDT). According to the FDT, the movements of a particle provide information about the mechanical properties of the surrounding environment [START_REF] Kubo | The fluctuation-dissipation theorem[END_REF]. However, in some cases, this relationship between particle movements and environmental mechanical properties is not verified, what physicists call a "violation of the Fluctuation Dissipation Theorem". One example is precisely the cell cytoplasm, where the movement of particles is the result of both a passive (or Brownian) contribution and an active one resulting from the activity of molecular motors, as in mouse oocytes. Thus, the cell cytoplasm undergoes a unique regime of "active diffusion", with the characteristics of a diffusive motion, but which is driven by motors (for reviews on active diffusion, see [START_REF] Brangwynne | Cytoplasmic diffusion: molecular motors mix it up[END_REF][START_REF] Brangwynne | Intracellular transport by active diffusion[END_REF]).

How to explain such an apparent paradox? In a visco-elastic material like the cytoplasm, a motion purely driven by thermal agitation would appear as constrained, with a slope <1 on a LOG-LOG representation of MSD (Fig. 3A), whereas an active process can result in a slope of 1, otherwise characteristic of diffusive motion. This is exactly the case for actin vesicles in the mouse oocyte, that are characterized by an MSD plot presenting a slope of 1 (Fig. 3A). Such a discrepancy between the mechanical properties of the surrounding environment and the observed motion of a particle requires to directly measuring the mechanical properties inside the cytoplasm. This is the principle of Force Spectrum Microscopy, which combines direct measurement by optical tweezers in the cytoplasm with the tracking of particles [START_REF] Almonacid | Active diffusion positions the nucleus in mouse oocytes[END_REF][START_REF] Guo | Probing the stochastic, motor-driven properties of the cytoplasm using force spectrum microscopy[END_REF]. It allows to separate the contributions of thermal activity and motor activity in the motion of cytoplasmic particles and thus to highlight the violation of FDT as well as to detect active diffusion.

Therefore, we took the challenging approach to probe directly the mechanical properties of the mouse oocyte cytoplasm by direct measurements using optical tweezers in wild-type oocytes, in Fmn2 -/oocytes and in wild-type oocytes expressing the Myosin Vb dominant negative (Fig. 3B) [START_REF] Almonacid | Active diffusion positions the nucleus in mouse oocytes[END_REF]. The cytoplasm of oocytes expressing Myosin Vb is 2-3 times stiffer than in other conditions. Thus, by promoting active diffusion, the dynamics of the cytoplasmic actin meshwork fluidizes the cytoplasm, thereby favoring the movement of such a big object like the mouse oocyte nucleus.

A dual model for nuclear positioning

All these data contribute to elaborate a mathematical model (Fig. 2B). Nuclear movement results from an equilibrium between the propulsion force Fp and a friction force Ff = -V (V nuclear velocity and proportional to cytoplasmic viscosity). Thus, nuclear velocity V = -(␣/) <v 2 > is proportional to the activity gradient of actin vesicles and inversely proportional to the viscosity. In this context, Myosin Vb plays a dual role:

-It generates the activity gradient ( <v 2 >), which is the source of the propulsion force Fp. -It generates advection, fluidizing the cytoplasm and thus ensuring a viscosity compatible with the movement of a large organelle like the nucleus.

The model also predicts that the propulsion force Fp vanishes at the oocyte center, as does the gradient (Fig. 2B), thus keeping the nucleus in a central position. Interestingly, this mechanism is necessary to move the nucleus to the center, but not for maintain-ing it. Indeed, removing both the actin mesh and the microtubules has no effect on nuclear positioning: in wild-type oocytes treated with Cytochalasin D and Nocodazole, the nucleus stays at the center [START_REF] Almonacid | Active diffusion positions the nucleus in mouse oocytes[END_REF]. This suggests a high intrinsic viscosity of the cytoplasm, independent on actin and microtubules. Fully-grown oocytes have accumulated a high quantity of dormant RNAs engaged into RNPs (RiboNucleoProteins) that are necessary for the further steps of meiotic divisions and the first steps of embryonic development. These dormant RNAs/RNPs could provide the viscosity of this matrix, as it has been observed for C. elegans oocytes [START_REF] Hubstenberger | Translation repressors an RNA helicase, and developmental cues control RNP phase transitions during early development[END_REF].

Another interesting feature of this mechanism is its nonspecificity. Due to the physical nature of the gradient, we suspect that any object placed within the gradient would find its way to the center. In agreement with this, injected fluorescent latex beads were found at the center 18 h after injection instead of 5 h for the nucleus [START_REF] Almonacid | Active diffusion positions the nucleus in mouse oocytes[END_REF]. The size of the object may be a critical parameter and further work will be required to fully characterize this gradient and its properties.

A conserved mechanism: pronuclear centering in the mouse zygote

Later during development, in the mouse zygote, the migration of the male and female pronuclei shares striking similarities with nuclear positioning in fully-grown oocytes [START_REF] Chaigne | F-actin mechanics control spindle centring in the mouse zygote[END_REF]. Following fertilization, the two pronuclei assemble at the periphery of the embryo. Then, they migrate towards the embryo center in 12-15 h where they remain roughly positioned until the merging of the two sets of chromosomes into the metaphase plate [START_REF] Chaigne | F-actin mechanics control spindle centring in the mouse zygote[END_REF]. Pronuclear migration also occurs in the absence of true centrosomes and astral microtubules since the centrioles brought by the sperm are rapidly degraded [START_REF] Manandhar | Centrosome reduction during gametogenesis and its significance[END_REF] and rebuilt de novo at the 64-cell stage [START_REF] Gueth-Hallonet | Gamma-tubulin is present in acentriolar MTOCs during early mouse development[END_REF].

Interestingly, as for oocyte nuclear positioning, this coarse pronuclear centering in the mouse zygote depends on F-actin-and Myosin Vb driven cytoplasmic flows [START_REF] Chaigne | F-actin mechanics control spindle centring in the mouse zygote[END_REF]. However, some differences can be pinpointed. In the case of oocyte nuclear centering, the trigger of nuclear movement is the anisotropy in actin vesicles activity, whereas the density of actin vesicles remains constant across the cytoplasm [START_REF] Almonacid | Active diffusion positions the nucleus in mouse oocytes[END_REF]. Conversely, in the zygote, the activity of the cytoplasmic mesh is homogeneous within the cell; the anisotropy triggering nuclear movement comes from the heterogeneous density of the actin mesh. Indeed, the meshwork between the pronuclei and the closest cortex is denser than the meshwork on the other side of the pronuclei (Fig. 4). This probably corresponds to a local actin enrichment due to the presence of maternal and paternal chromosomes underneath the cortex (for the female pronucleus, the cortical differentiated zone above the second meiotic spindle and for the male pronucleus, the fertilization cone formed upon sperm entry [START_REF] Maro | Changes in actin distribution during fertilization of the mouse egg[END_REF]). This heterogeneity is lost around pronuclei that have migrated towards the center (Fig. 4).

Accordingly, the model describing pronuclear movement is different from the one describing oocyte nuclear movement. Here again, by keeping the analogy of the cytoplasm as a perfect gas, the pressure resulting from the agitation of n particles in the gas is P = nk b T e , where k b is the Boltzmann's constant and T e is the effective temperature of the particle. k b T e represents the activity of particles, like <v 2 > in the previous oocyte nuclear migration model. But here, <v 2 > and so k b T e remain constant throughout the cytoplasm, so that the gradient in the number n of actinpositive foci directly results in a pressure gradient. In other words, in the zygote, the force moving the pronuclei stems from a density gradient whereas in the Prophase I arrested oocyte, the force moving the nucleus comes from a velocity gradient. Interestingly, the model predicts that the force moving the pronuclei depends both on the pressure gradient P and on the volume (and thus the size) of the object. This could explain why the pronuclei move slower towards the center than the bigger Prophase I nucleus, and raises the question of object size dependence also for the nuclear centering mechanism in fully-grown oocytes. An alternative explanation could be that an activity gradient, as in the case of Prophase I nuclear centering, is more efficient than a density gradient.

Biological significance of nuclear centering

The robustness of the nuclear centering machinery in the mouse oocyte is in apparent contradiction with the following steps of meiotic divisions, which are highly asymmetric, with chromosome off-centering. This could explain why the cytoplasmic mesh is dismantled prior meiosis resumption, to favor symmetry breaking preceding spindle migration and asymmetric division [START_REF] Azoury | Symmetry breaking in mouse oocytes requires transient F-actin meshwork destabilization[END_REF]. One can wonder about the biological significance of nuclear centering in this context.

In most types of oocytes, nuclear positioning acts as a developmental cue that predefines the future developmental axes of the embryo (Fig. 5). For example, in the Drosophila oocyte, the nucleus moves from the posterior to the antero-dorsal cortex. This localization initiates the establishment of the dorso-ventral polarity of the egg chamber and the future embryo. In echinoderm oocytes, the position of the nucleus marks the animal pole [START_REF] Miyazaki | Premeiotic aster as a device to anchor the germinal vesicle to the cell surface of the presumptive animal pole in starfish oocytes[END_REF][START_REF] Miyazaki | Role of microtubules and centrosomes in the eccentric relocation of the germinal vesicle upon meiosis reinitiation in sea-cucumber oocytes[END_REF]. In the C.elegans oocyte, the nucleus undergoes migration to the future site of spindle attachment at the anterior cortex and thus defines the antero-posterior axis of the future embryo [START_REF] Mcnally | Kinesin-dependent transport results in polarized migration of the nucleus in oocytes and inward movement of yolk granules in meiotic embryos[END_REF].

However, in mammals, the position of the nucleus does not prefigure the adult body plan. Indeed, developmental axes are established later during development, after embryo implantation [START_REF] Wennekamp | A self-organization framework for symmetry breaking in the mammalian embryo[END_REF]. Yet the correlation between centering and the success of meiotic divisions [START_REF] Brunet | Germinal vesicle position and meiotic maturation in mouse oocyte[END_REF][START_REF] Levi | Morphological and molecular markers are correlated with maturation-competence of human oocytes[END_REF] suggests a possible developmental role for nuclear centering.

In this context, an attractive hypothesis is that nuclear positioning in the mouse oocyte, and more precisely its force generated mechanism, could regulate the "competence" of the oocyte, i.e its ability to be fit for fertilization and for later embryonic development. Random cytoplasmic mixing concomitant to nuclear centering could contribute to the homeostasis of the oocyte by avoiding unphysiological compartmentalization of the cytoplasm [START_REF] Dalton | Biased inheritance of mitochondria during asymmetric cell division in the mouse oocyte[END_REF][START_REF] Fitzharris | Changes in endoplasmic reticulum structure during mouse oocyte maturation are controlled by the cytoskeleton and cytoplasmic dynein[END_REF]. Indeed, it has been shown that the transport of small molecules or nutrients is enhanced by active diffusion within the cell [START_REF] Guo | Probing the stochastic, motor-driven properties of the cytoplasm using force spectrum microscopy[END_REF]. Altogether, this could maintain the oocyte in a nonpolarized state, not only at the cortical level [START_REF] Halet | Rac activity is polarized and regulates meiotic spindle stability and anchoring in mammalian oocytes[END_REF], but also in the cytoplasm, where all subcellular compartments are micronized [START_REF] Dalton | Biased inheritance of mitochondria during asymmetric cell division in the mouse oocyte[END_REF][START_REF] Fitzharris | Changes in endoplasmic reticulum structure during mouse oocyte maturation are controlled by the cytoskeleton and cytoplasmic dynein[END_REF]. As a consequence, all the cytoplasmic components would be homogeneously distributed, and nuclear central position would contribute to the creation of such an isotropic landscape. Homogenization of the cytoplasm could be vital for mammalian eggs, which compared to Xenopus oocytes do not possess a huge amount of vitellus, a major source of energy required to sustain the long Metaphase II arrest as well as early stages of embryo development. For example, homogenization of mitochondria in Prophase I, coupled to their specific retention in the oocyte cytoplasm during the two asymmetric divisions [START_REF] Dalton | Biased inheritance of mitochondria during asymmetric cell division in the mouse oocyte[END_REF] might be essential elements to ensure sufficient energy supplies for mouse oocytes. This is totally different from other model systems, like the Drosophila oocyte. These oocytes also possess a cytoplasmic actin mesh nucleated by the formin Cappuccino, but differently from the mouse oocyte, this mesh prevents the appearance of premature fast microtubule-dependent cytoplasmic streaming that would lead to the dispersion of important developmental determinants like the bicoid and oskar mRNAs that define the anterior-posterior axis of the embryo [START_REF] Quinlan | Cytoplasmic streaming in the drosophila oocyte[END_REF][START_REF] Dahlgaard | Capu and Spire assemble a cytoplasmic actin mesh that maintains microtubule organization in the Drosophila oocyte[END_REF]. It is intriguing that apparently similar cytoskeletal structures, but in two different organisms, serve such opposite developmental purposes.

Conclusion

Nuclear positioning in the mouse oocyte involves a novel mechanism that relies on actin vesicles dynamics and is independent from centrosomes and associated microtubules.

It is the very first description of an activity gradient of actin vesicles producing a force triggering the movement of the oocyte nucleus, a very large organelle, the size of a somatic cell. Differently from directional transport by molecular motors along microtubules or microfilaments, the source of motion here is the activity of actin vesicles induced by Myosin Vb, that generates advection by dragging the fluid and other cytoplasmic components around moving vesicles, a hallmark of active diffusion.

Such a mechanism is particularly important in large cells devoid of centrosomes, where dynamic actin meshes generate forces and motion across long distances, as an alternative to microtubulebased transport.

Beyond these implications in cell biology, nuclear positioning in the mouse oocyte could have potential developmental roles, by keeping the oocyte in a non-polarized state, which, unlike in other organisms, may favor early embryonic development.

Fig. 1 .

 1 Fig. 1. Mammalian meiosis and early development. In mammals, oocyte growth takes place in the ovary. Fully-grown oocytes arrested in Prophase I resume meiosis and undergo two successive asymmetric divisions. Fertilization triggers the second meiotic division. The zygote then achieves the very first embryonic division. Nuclear centering takes place during the growth phase. Asymmetric meiotic divisions occur with spindle and chromosomes off-centering. Fertilization brings the paternal genome into the egg. Then both maternal and paternal genomes, packed into pronuclei, merge together near the center of the zygote to prepare for the first embryonic division. Blue: nuclei, chromosomes and sperm. Dark blue: nucleoli. Green: spindle microtubules.

Fig. 3 .

 3 Fig. 3. Addressing active diffusion and mechanical properties within the mouse oocyte cytoplasm using optical tweezers. A: Diffusive-like motion of actin vesicles. LOG-LOG representation of the Mean Squared Displacement (MSD) of actin vesicles fitted as a function of the delay in seconds to a power-law model f(t) = at b , where t is the time and b = 1.03. The power-law is the slope of the line of the LOG-LOG plot. A slope of 1 is the signature of a diffusive-like motion. B: Optical tweezer set-up to measure micro-rheology of the oocyte cytoplasm. The optical trap catches a vesicle in the cytoplasm. The mechanical response is measured by applying a sinusoidal force to the trapped vesicle and observing the subsequent displacement.

Fig. 4 .

 4 Fig.4. Actin-dependent pronuclear centering in the zygote. In the zygote, the activity of the cytoplasmic mesh is homogeneous within the cell, whereas the density of the meshwork varies. The meshwork is denser between the pronuclei and the closest cortex (black arrows). This local actin enrichment may be due to the cortical differentiated zone above the second meiotic spindle for the female pronucleus and to the fertilization cone for the male pronucleus. This heterogeneity disappears around pronuclei that have migrated towards the center. Therefore, in the zygote, the force moving the pronuclei is generated by a density gradient rather than an activity gradient. Blue: pronuclei, dark blue: nucleoli, red lines: actin filaments.

Fig. 5 .

 5 Fig. 5. Nuclear positioning and determination of the body axes. Differently from the Drosophila oocyte, nuclear positioning in the mouse oocyte does not act as a morphogen for the future body plans. Left: Determination of the body axes in Drosophila (from top to bottom, egg chamber with oocyte, cellular blastoderm with pole cells at the posterior, adult). In the oocyte, the nucleus marks the antero-dorsal cortex and establishes the dorso-ventral polarity of the egg chamber and the future embryo. Right: Determination of the body axes in mouse (from top to bottom, fully-grown oocyte, blastocyst, adult). The central nucleus in the oocyte does not predefine the future developmental axes. Nuclei are drawn in blue, nucleoli in dark blue.

Acknowledgments

We thank Nature Cell Biology for the permission to reproduce parts of figures from [23] in Fig. 3.

Funding sources

This work was supported by grants from the Fondation pour la Recherche Médicale (FRM Label to M-H Verlhac-Please cite this article in press as: M. Almonacid, et al., Control of nucleus positioning in mouse oocytes, Semin Cell Dev Biol (2017), http://dx.doi.org/10.1016/j.semcdb.2017.08.010 ARTICLE IN PRESS G Model YSCDB-2319; No. of Pages 7 M. Almonacid et al. / Seminars in Cell & Developmental Biology xxx (2017) xxx-xxx

DEQ20150331758), from the ANR (ANR-14-CE11 to M-H Verlhac) and from the Labex Memolife (to M-H Verlhac). This work has received support under the program « Investissements d'Avenir » launched by the French Government and implemented by the ANR, with the references: ANR-10-LABX-54 MEMO LIFE, ANR-11-IDEX-0001-02 PSL* Research University.

Declaration of interests statement

The authors declare no competing financial interests.