Formin-2 is required for spindle migration and for the late steps of cytokinesis in mouse oocytes

Female meiotic divisions in higher organisms are asymmetric and lead to the formation of a large oocyte and small polar bodies. These asymmetric divisions are due to eccentric spindle positioning which, in the mouse, requires actin filaments. Recently Formin-2, a straight actin filaments nucleator, has been proposed to control spindle positioning, chromosome segregation as well as first polar body extrusion in mouse oocytes. We reexamine here the possible role of Formin-2 during mouse meiotic maturation by live videomicroscopy. We show that Formin-2 controls first meiotic spindle migration to the cortex but not chromosome congression or segregation. We also show that the lack of first polar body extrusion in fmn2(-/-) oocytes is not due to a lack of cortical differentiation or central spindle formation but to a defect in the late steps of cytokinesis. Indeed, Survivin, a component of the passenger protein complex, is correctly localized on the central spindle at anaphase in fmn2(-/-) oocytes. We show here that attempts of cytokinesis in these oocytes abort due to phospho-myosin II mislocalization.     

Specific deletion of Cdc42 does not affect meiotic spindle organization/migration and homologous chromosome segregation but disrupts polarity establishment and cytokinesis in mouse oocytes

Mammalian oocyte maturation is distinguished by highly asymmetric meiotic divisions during which a haploid female gamete is produced and almost all the cytoplasm is maintained in the egg for embryo development. Actin-dependent meiosis I spindle positioning to the cortex induces the formation of a polarized actin cap and oocyte polarity, and it determines asymmetric divisions resulting in two polar bodies. Here we investigate the functions of Cdc42 in oocyte meiotic maturation by oocyte-specific deletion of Cdc42 through Cre-loxP conditional knockout technology. We find that Cdc42 deletion causes female infertility in mice. Cdc42 deletion has little effect on meiotic spindle organization and migration to the cortex but inhibits polar body emission, although homologous chromosome segregation occurs. The failure of cytokinesis is due to the loss of polarized Arp2/3 accumulation and actin cap formation; thus the defective contract ring. In addition, we correlate active Cdc42 dynamics with its function during polar body emission and find a relationship between Cdc42 and polarity, as well as polar body emission, in mouse oocytes.     

Translational control of cyclins

Background

An oocyte undergoes two rounds of asymmetric division to generate a haploid gamete and two small polar bodies designed for apoptosis. Chromosomes play important roles in specifying the asymmetric meiotic divisions in the oocytes but the underlying mechanism is poorly understood.

Results

Chromosomes independently induce spindle formation and cortical actomyosin assembly into special cap and ring structures in the cortex of the oocyte. The spindle and the cortical cap/ring interact to generate mechanical forces, leading to polar body extrusion. Two distinct force-driven membrane changes were observed during 2^nd polar body extrusion: a protrusion of the cortical cap and a membrane invagination induced by an anaphase spindle midzone. The cortical cap protrusion and invagination help rotate the spindle perpendicularly so that the spindle midzone can induce bilateral furrows at the shoulder of the protruding cap, leading to an abscission of the polar body. It is interesting to note that while the mitotic spindle midzone induces bilateral furrowing, leading to efficient symmetric division in the zygote, the meiotic spindle midzone induced cytokinetic furrowing only locally.

Conclusions

Distinct forces driving cortical cap protrusion and membrane invagination are involved in spindle rotation and polar body extrusion during meiosis II in mouse oocytes.     

Spindle positioning in mouse oocytes relies on a dynamic meshwork of actin filaments

Female meiosis in higher organisms consists of highly asymmetric divisions, which retain most maternal stores in the oocyte for embryo development. Asymmetric partitioning of the cytoplasm results from the spindle's "off-center" positioning, which, in mouse oocytes, depends mainly on actin filaments [1, 2]. This is a unique situation compared to most systems, in which spindle positioning requires interactions between astral microtubules and cortical actin filaments [3]. Formin 2, a straight-actin-filament nucleator, is required for the first meiotic spindle migration to the cortex and cytokinesis in mouse oocytes [4, 5]. Although the requirement for actin filaments in the control of spindle positioning is well established in this model, no one has been able to detect them in the cytoplasm [6]. Through the expression of an F-actin-specific probe and live confocal microscopy, we show the presence of a cytoplasmic actin meshwork, organized by Formin 2, that controls spindle migration. In late meiosis I, these filaments organize into a spindle-like F-actin structure, which is connected to the cortex. At anaphase, global reorganization of this meshwork allows polar-body extrusion. In addition, using actin-YFP, our FRAP analysis confirms the presence of a highly dynamic cytoplasmic actin meshwork that is tightly regulated in time and space.     

Actin filaments: key players in the control of asymmetric divisions in mouse oocytes

Meiotic maturation is characterized by the succession of two asymmetric divisions each giving rise to a small polar body and a large oocyte. These highly asymmetric divisions are characteristic of meiosis in higher organisms. They allow most of the maternal stores to be retained in the oocyte, a vital property for further embryo development. In mouse oocytes, the asymmetry is ensured by the migration and the anchoring of the division spindle to the cortex in meiosis I and by its anchoring to the cortex in meiosis II. In addition, and subsequent to this off‐centre positioning of the spindle, a differentiation of the cortex overhanging the chromosomes takes place and is necessary for the extrusion of small polar bodies. In the present review, we will emphasize the role of the actin cytoskeleton in the control of spindle positioning, spindle anchoring to the cortex and cortical differentiation.     

RhoA- and Cdc42-induced antagonistic forces underlie symmetry breaking and spindle rotation in mouse oocytes

Mammalian oocyte meiotic divisions are highly asymmetric and produce a large haploid gamete and 2 small polar bodies. This relies on the ability of the cell to break symmetry and position its spindle close to the cortex before anaphase occurs. In metaphase II–arrested mouse oocytes, the spindle is actively maintained close and parallel to the cortex, until fertilization triggers sister chromatid segregation and the rotation of the spindle. The latter must indeed reorient perpendicular to the cortex to enable cytokinesis ring closure at the base of the polar body. However, the mechanisms underlying symmetry breaking and spindle rotation have remained elusive. In this study, we show that spindle rotation results from 2 antagonistic forces. First, an inward contraction of the cytokinesis furrow dependent on RhoA signaling, and second, an outward attraction exerted on both sets of chromatids by a Ran/Cdc42-dependent polarization of the actomyosin cortex. By combining live segmentation and tracking with numerical modeling, we demonstrate that this configuration becomes unstable as the ingression progresses. This leads to spontaneous symmetry breaking, which implies that neither the rotation direction nor the set of chromatids that eventually gets discarded are biologically predetermined.

Mammalian oocyte meiotic divisions are highly asymmetric and produce a large haploid gamete and two small polar bodies, but the mechanisms underlying the required symmetry breaking and spindle rotation have remained elusive. This study shows that spindle rotation in activated mouse oocytes relies on spontaneous symmetry breaking resulting from an unstable configuration generated by cleavage furrow ingression and cortical chromosome attraction.     

Symmetry breaking in mouse oocytes requires transient F-actin meshwork destabilization

Female meiotic divisions are extremely asymmetric, giving rise to a large oocyte and small degenerating polar bodies, keeping the maternal stores for further embryo development. This asymmetry is achieved via off-center positioning of the division spindle. Mouse oocytes have developed a formin-2-dependent actin-based spindle positioning mechanism that allows the meiotic spindle to migrate towards the closest cortex. Using spinning disk microscopy and FRAP analysis, we studied the changes in the organization of the cytoplasmic F-actin meshwork during the first meiotic division. It is very dense in prophase I, undergoes a significant density drop upon meiosis resumption and reforms progressively later on. This meshwork remodeling correlates with endogenous formin 2 regulation. High formin 2 levels at meiosis I entry induce meshwork maintenance, leading to equal forces being exerted on the chromosomes, preventing spindle migration. Hence, the meshwork density drop at meiosis resumption is germane to the symmetry-breaking event required for successful asymmetric meiotic divisions.     

Anillin localization suggests distinct mechanisms of division plane specification in mouse oogenic meiosis I and II

Anillin is a conserved cytokinetic ring protein implicated in actomyosin cytoskeletal organization and cytoskeletal-membrane linkage. Here we explored anillin localization in the highly asymmetric divisions of the mouse oocyte that lead to the extrusion of two polar bodies. The purposes of polar body extrusion are to reduce the chromosome complement within the egg to haploid, and to retain the majority of the egg cytoplasm for embryonic development. Anillin's proposed roles in cytokinetic ring organization suggest that it plays important roles in achieving this asymmetric division. We report that during meiotic maturation, anillin mRNA is expressed and protein levels steadily rise. In meiosis I, anillin localizes to a cortical cap overlying metaphase I spindles, and a broad ring over anaphase spindles that are perpendicular to the cortex. Anillin is excluded from the cortex of the prospective first polar body, and highly enriched in the cytokinetic ring that severs the polar body from the oocyte. In meiosis II, anillin is enriched in a cortical stripe precisely coincident with and overlying the meiotic spindle midzone. These results suggest a model in which this cortical structure contributes to spindle re-alignment in meiosis II. Thus, localization of anillin as a conserved cytokinetic ring marker illustrates that the geometry of the cytokinetic ring is distinct between the two oogenic meiotic cytokineses in mammals.     

Cytostatic Activity Develops during Meiosis I in Oocytes of LT/Sv Mice

Oocytes of wild-type mice are ovulated as the secondary oocytes arrested at metaphase of the second meiotic division. Their fertilization or parthenogenetic activation triggers the completion of the second meiotic division followed by the first embryonic interphase. Oocytes of the LT/Sv strain of mice are ovulated either at the first meiotic metaphase (M I) as primary oocytes or in the second meiotic metaphase (M II) as secondary oocytes. We show here that duringin vitromaturation a high proportion of LT/Sv oocytes progresses normally only until metaphase I. In these oocytes MAP kinase activates shortly after histone H1 kinase (MPF) activation and germinal vesicle breakdown. However, MAP kinase activation is slightly earlier than in oocytes from wild-type F1 (CBA/H × C57Bl/10) mice. The first meiotic spindle of these oocytes forms similarly to wild-type oocytes. During aging, however, it increases in size and finally degenerates. In those oocytes which do not remain in metaphase I the extrusion of first polar bodies is highly delayed and starts about 15 h after germinal vesicle breakdown. Most of the oocytes enter interphase directly after first polar body extrusion. Fusion between metaphase I LT/Sv oocytes and wild-type mitotic one-cell embryos results in prolonged M-phase arrest of hybrids in a proportion similar to control LT/Sv oocytes and control hybrids made by fusion of two M I LT/Sv oocytes. This indicates that LT/Sv oocytes develop cytostatic factor during metaphase I. Eventually, anaphase occurs spontaneously and the hybrids extrude the polar body and form pronuclei in a proportion similar as in controls. In hybrids between LT/Sv metaphase I oocytes and wild-type metaphase II oocytes (which contain cytostatic factor) anaphase I proceeds at the time observed in control LT/Sv oocytes and hybrids between two M I LT/Sv oocytes, and is followed by the parthenogenetic activation and formation of interphase nuclei. Also the great majority of hybrids between M I and M II wild-type oocytes undergoes the anaphase but further arrests in a subsequent M-phase. These observations suggest that an internally triggered anaphase I occurs despite the presence of the cytostatic activity both in LT/Sv and wild-type M I oocytes. Anaphase I triggering mechanism must therefore either inactivate or override the CSF activity. The comparison between spontaneous and induced activation of metaphase I LT/Sv oocytes shows that mechanisms involved in anaphase I triggering are altered in these oocytes. Thus, the prolongation of metaphase I in LT/Sv oocytes seems to be determined by delayed anaphase I triggering and not provoked directly by the cytostatic activity.     

TGN38 is required for the metaphase I/anaphase I transition and asymmetric cell division during mouse oocyte meiotic maturation

The cellular functions of the trans-Golgi network protein TGN38 remain unknown. In this research, we studied the expression, localization and functions of TGN38 in the meiotic maturation of mouse oocytes. TGN38 was expressed at every stage of oocyte meiotic maturation and colocalized with γ-tubulin at metaphase I and metaphase II. The spindle microtubule disturbing agents nocodazole and taxol did not affect the colocalization of TGN38 and γ-tubulin. Depletion of TGN38 with specific siRNAs resulted in increased metaphase I arrest, accompanied with spindle assembly checkpoint activation and decreased first polar extrusion (PB1). In the oocytes that had extruded the PB1 after the depletion of TGN38, symmetric division occurred, leading to the production of 2 similarly sized cells. Moreover, the peripheral migration of metaphase I spindle and actin cap formation were impaired in TGN38-depleted oocytes. Our data suggest that TGN38 may regulate the metaphase I/anaphase I transition and asymmetric cell division in mouse oocytes.     

Cdc42 protein acts upstream of IQGAP1 and regulates cytokinesis in mouse oocytes and embryos

Cdc42 and Rac1 Rho family GTPases, and their interacting protein IQGAP1 are the key regulators of cell polarity. We examined the role of Cdc42 and IQGAP1 in establishing the polarity of mouse oocyte and regulation of meiotic and mitotic divisions. We showed that Cdc42 was localized on the microtubules of meiotic and mitotic spindle and in the cortex of mouse oocytes and cleaving embryos. IQGAP1 was present in the cytoplasm and cortex of growing and fully-grown oocytes. During maturation it disappeared from the cortex and during meiotic and mitotic cytokinesis it concentrated in the contractile ring. Toxin B inhibition of the binding activity of Cdc42 changed the localization of IQGAP1, inhibited emission of the first polar body, and caused disappearance of the cortical actin without affecting the migration of meiotic spindle. This indicates, that in maturing oocytes accumulation of cortical actin is not indispensable for spindle migration. In zygotes treated with toxin B actin cytoskeleton was rearranged and the first and/or subsequent cytokinesis were inhibited. Our results indicate that Cdc42 acts upstream of IQGAP1 and is involved in regulation of cytokinesis in mouse oocytes and cleaving embryos, rather than in establishing the polarity of the oocyte.     

Brefeldin A disrupts asymmetric spindle positioning in mouse oocytes

Polar body formation in oocytes is an extreme form of asymmetric cell division, but what regulates the asymmetric spindle positioning and cytokinesis is poorly understood. During mouse oocyte maturation, the metaphase I spindle forms at the center but then moves to the cortex prior to anaphase I and first polar body emission. We show here that treating denuded mouse oocytes with brefeldin A, an inhibitor of Golgi-based membrane fusion, abolished the asymmetric positioning of the metaphase I spindle and resulted in the formation of two half-size metaphase II eggs, instead of a full-sized egg and a polar body. The normal metaphase II spindle is similarly asymmetrically positioned in the mature egg, where the spindle lies with its axis parallel to the cortex but becomes perpendicular before anaphase II and emission of the second polar body. When ovulated eggs were activated with strontium in the presence of brefeldin A, the metaphase II spindle failed to assume perpendicular position, and the chromosomes separated without the extrusion of the second polar body. Remarkably, symmetric cytokinesis began following a 3 h delay, forming two half-size eggs each containing a pronucleus. BFA-sensitive intracellular vesicular transport is therefore required for spindle positioning in both MI and MII.     

Rac activity is polarized and regulates meiotic spindle stability and anchoring in mammalian oocytes

Mammalian meiotic divisions are asymmetrical and generate a large oocyte and two small polar bodies. This asymmetry results from the anchoring of the meiotic spindle to the oocyte cortex and subsequent cortical reorganization, but the mechanisms involved are poorly understood. We investigated the role of Rac in oocyte meiosis by using a fluorescent reporter for Rac-GTP. We find that Rac-GTP is polarized in the cortex overlying the meiotic spindle. Polarization of Rac activation occurs during spindle migration and is promoted by the proximity of chromatin to the cortex. Inhibition of Rac during oocyte maturation caused a permanent block at prometaphase I and spindle elongation. In metaphase II-arrested oocytes, Rac inhibition caused the spindle to detach from the cortex and prevented polar body emission after activation. These results demonstrate that Rac-GTP plays a major role in oocyte meiosis, via the regulation of spindle stability and anchoring to the cortex.     

Rotation of meiotic spindle is controlled by microfilaments in mouse oocytes

The completion of meiosis requires the spatial and temporal coordination of cytokinesis and karyokinesis. During meiotic maturation, many events, such as formation, location, and rotation of the meiotic spindle as well as chromosomal movement, polar body extrusion, and pronuclear migration, are dependent on regulation of the cytoskeleton system. To study functions of microfilaments in meiosis, we induced metaphase II (MII) mouse oocytes to resume meiosis by in vitro fertilization or parthenogenetic activation, and we treated such oocytes with cytochalasin B (CB). The changes of the meiotic spindle, as visualized in preparations stained for beta-tubulin and chromatin, were observed by fluorescent confocal microscopy. The meiotic spindle of MII oocytes was observed to be parallel to the plasmalemma. After meiosis had resumed, the spindle rotated to the vertical position so that the second polar body could be extruded into the perivitelline space. When meiosis resumed and oocytes were treated with 10 micro g/ml of CB, the spindle rotation was inhibited. Consequently, the oocyte formed an extra pronucleus instead of extruding a second polar body. These results indicate that spindle rotation is essential for polar body extrusion; it is the microfilaments that play a crucial role in regulating rotation of the meiotic spindle.     

MEI-1/katanin is required for translocation of the meiosis I spindle to the oocyte cortex in C elegans

In most animals, successful segregation of female meiotic chromosomes involves sequential associations of the meiosis I and meiosis II spindles with the cell cortex so that extra chromosomes can be deposited in polar bodies. The resulting reduction in chromosome number is essential to prevent the generation of polyploid embryos after fertilization. Using time-lapse imaging of living Caenorhabditis elegans oocytes containing fluorescently labeled chromosomes or microtubules, we have characterized the movements of meiotic spindles relative to the cell cortex. Spindle assembly initiated several microns from the cortex. After formation of a bipolar structure, the meiosis I spindle translocated to the cortex. When microtubules were partially depleted, translocation of the bivalent chromosomes to the cortex was blocked without affecting cell cycle timing. In oocytes depleted of the microtubule-severing enzyme, MEI-1, spindles moved to the cortex, but association with the cortex was unstable. Unlike translocation of wild-type spindles, movement of MEI-1-depleted spindles was dependent on FZY-1/CDC20, a regulator of the metaphase/anaphase transition. We observed a microtubule and FZY-1/CDC20-dependent circular cytoplasmic streaming in wild-type and mei-1 mutant embryos during meiosis. We propose that, in mei-1 mutant oocytes, this cytoplasmic streaming is sufficient to drive the spindle into the cortex. Cytoplasmic streaming is not the normal spindle translocation mechanism because translocation occurred in the absence of cytoplasmic streaming in embryos depleted of either the orbit/CLASP homolog, CLS-2, or FZY-1. These results indicate a direct role of microtubule severing in translocation of the meiotic spindle to the cortex.     

Mouse polo-like kinase 1 associates with the acentriolar spindle poles, meiotic chromosomes and spindle midzone during oocyte maturation

We have examined the dynamics of the localisation of the polo-like kinase 1 (Plk1) during maturation of the mouse oocyte. Levels of Plk1 protein increase following germinal vesicle breakdown, at which time the enzyme begins to accumulate at discrete positions on the condensing chromosomes and, subsequently, at the poles of the meiotic spindle, which moves towards the cortex of the egg. Interestingly, at metaphase in both meiotic divisions, Plk1 shows a punctate localisation along the broad spindle poles. Moreover, the punctate distribution of Plk1 on the meiotic chromosomes appears at early anaphase to correspond to the centromeric regions. The protein relocates to the spindle midzone during late anaphase and then associates with the midbody at telophase. We have confirmed the specific pattern of immuno-localisation seen in fixed preparations by observing the distribution of Plk1 tagged with green fluorescent protein in living oocytes. We discuss the localisation of the enzyme in light of the structure of the spindle poles, which are known to lack centrioles, and the highly asymmetric nature of the meiotic divisions. Received: 8 August 1998 / Accepted: 13 September 1998     

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

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.     

Asymmetric positioning and organization of the meiotic spindle of mouse oocytes requires CDC42 function

The mature mammalian oocyte is highly polarized because asymmetrical spindle migration to the oocyte cortex ensures extrusion of small polar bodies in the two meiotic divisions, essential for generation of the large egg. Actin filaments, myosin motors, and formin-2, but not microtubules, are required for spindle migration. Here, we show that Cdc42, a key regulator of cytoskeleton and cell polarity in other systems , is essential for meiotic maturation and oocyte asymmetry. Disrupting CDC42 function by ectopic expression of its GTPase-defective mutants causes both halves of the first meiotic spindle to extend symmetrically toward opposing cortical regions and prevents an asymmetrical division. The elongated spindle has numerous astral-like microtubules, and aPKCzeta, normally associated with the spindle poles, is distributed along its length. Dynactin is displaced from kinetochores, consistently homologous chromosomes do not segregate, and polar body extrusion is prevented. Perturbing the function of aPKCzeta also causes elongation of the meiotic spindle but still permits spindle migration and polar body extrusion. Thus, at least two pathways appear to be downstream of CDC42: one affecting the actin cytoskeleton and required for migration of the meiotic spindle, and a second affecting the spindle microtubules in which aPKCzeta plays a role.     

Heteroplasmic conjugates formed by the fusion of starfish oocyte pairs with a 12 minute time difference in the maturation phase

Two starfish oocytes with a 12 min time difference in the maturation phase were fused together with electric pulses to make a heteroplasmic conjugate. The starfish used were Asterina pectinifera. The emergence of the first meiotic spindle and the extrusion of the polar bodies in the conjugate were timed. Under polarization microscopy two meiotic spindles emerged with a time difference of 10-11 min, which is close to the time difference in the maturation phase between the original oocytes before fusion. In contrast, subsequent formation of the first two polar bodies occurred successively with a short time lag of 1-3 min between them. Times for the formation of both polar bodies were midway between the anticipated times for polar body formation in respective non-fused control oocytes. Thus, in one nucleus the meiotic division was delayed, while in another nucleus it was accelerated, in a single heteroplasmic conjugate. These two sets of observations indicate the presence of a certain control system that regulates progression of the cell cycle at a point during the period from the entry into metaphase through to late anaphase of meiosis I in starfish oocytes. This type of cell cycle control in starfish oocytes is obviously distinct from the currently accepted view of the cell cycle control by the spindle assembly checkpoint that monitors unattached kinetochores of mitotic chromosomes.     

The maturation divisions of the parthenogenetic stick insect Carausius morosus Br. (Orthoptera,Phasmidae)

The meiotic stages in the maturation of the egg of the parthenogenetic stick insect Carausius morosus Br. is described. The meiosis consists of two equational divisions and parthenogenesis is thus apomictic. The first prometaphase is formed between 5.8–5.5 days before oviposition; the first metaphase lasts until oviposition; the meiosis ends between 14 and 24 hours after oviposition. An extra chromosome doubling occurs before first anaphase, causing the first metaphase chromosomes to resemble bivalents and requiring that meiosis consists of two divisions instead of one. A terminal affinity between the daughter chromosomes influences chromosome movement during first and second metaphase and anaphase. The first and second polar bodies degenerate. The first polar body divides amitotically during pycnosis. Meiosis takes place ventrally in the egg, the germ anlage development dorsally. The pronucleus divides mitotically in the ventral part of the egg during its migration from ventral to dorsal, enabling blastoderm development to take place both ventrally and dorsally.