Actomyosin tube formation in polar body cytokinesis requires Anillin in C. elegans

Polar body extrusion (PBE) is the specialized asymmetric division by which oocytes accomplish reduction in ploidy and retention of cytoplasm. During maternal gametogenesis, as in male meiosis and mitosis, cytokinesis is accomplished by a ring rich in active Rho, myosin, and formin-nucleated F-actin [1-7]. However, unlike mitosis, wherein the contractile ring encircles the cell equator, the polar body ring assembles as a discoid cortical washer. Here we show that in Caenorhabditis elegans, the meiotic contractile ring transforms during closure from a disc above the spindle to a cylinder around the spindle midzone. The meiotic midbody tube comprises stacked cytoskeletal rings. This topological transition suggests a novel mechanism for constriction of an initially discoid cytokinetic ring. Analysis of mouse PBE indicates that midbody tube formation is a conserved process. Depletion of the scaffold protein anillin (ANI-1) from C. elegans results in large and unstable polar bodies that often fuse with the oocyte. Anillin is dispensable for contractile ring assembly, initiation, and closure but is required for the meiotic contractile ring to transform from a disc into a tube. We propose that cytoskeletal bundling by anillin promotes formation of the midbody tube, which ensures the fidelity of PBE.     

Sperm Chromatin-Induced Ectopic Polar Body Extrusion in Mouse Eggs after ICSI and Delayed Egg Activation

Meiotic chromosomes in an oocyte are not only a maternal genome carrier but also provide a positional signal to induce cortical polarization and define asymmetric meiotic division of the oocyte, resulting in polar body extrusion and haploidization of the maternal genome. The meiotic chromosomes play dual function in determination of meiosis: 1) organizing a bipolar spindle formation and 2) inducing cortical polarization and assembly of a distinct cortical cytoskeleton structure in the overlying cortex for polar body extrusion. At fertilization, a sperm brings exogenous paternal chromatin into the egg, which induces ectopic cortical polarization at the sperm entry site and leads to a cone formation, known as fertilization cone. Here we show that the sperm chromatin-induced fertilization cone formation is an abortive polar body extrusion due to lack of spindle induction by the sperm chromatin during fertilization. If experimentally manipulating the fertilization process to allow sperm chromatin to induce both cortical polarization and spindle formation, the fertilization cone can be converted into polar body extrusion. This suggests that sperm chromatin is also able to induce polar body extrusion, like its maternal counterpart. The usually observed cone formation instead of ectopic polar body extrusion induced by sperm chromatin during fertilization is due to special sperm chromatin compaction which restrains it from rapid spindle induction and therefore provides a protective mechanism to prevent a possible paternal genome loss during ectopic polar body extrusion.     

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.     

Mechanisms that prevent catastrophic interactions between paternal chromosomes and the oocyte meiotic spindle

Meiosis produces haploid gametes by accurately reducing chromosome ploidy through one round of DNA replication and two subsequent rounds of chromosome segregation and cell division. The cell divisions of female meiosis are highly asymmetric and give rise to a large egg and two very small polar bodies that do not contribute to development. These asymmetric divisions are driven by meiotic spindles that are small relative to the size of the egg and have one pole juxtaposed against the cell cortex to promote polar body extrusion. An additional unique feature of female meiosis is that fertilization occurs before extrusion of the second polar body in nearly all animal species. Thus sperm-derived chromosomes are present in the egg during female meiosis. Here, we explore the idea that the asymmetry of female meiosis spatially separates the sperm from the meiotic spindle to prevent detrimental interactions between the spindle and the paternal chromosomes.     

Cdc42 activation couples spindle positioning to first polar body formation in oocyte maturation

During vertebrate egg maturation, cytokinesis initiates after one pole of the bipolar metaphase I spindle attaches to the oocyte cortex, resulting in the formation of a polar body and the mature egg. It is not known what signal couples the spindle pole positioning to polar body formation. We approached this question by drawing an analogy to mitotic exit in budding yeast, as asymmetric spindle attachment to the appropriate cortical region is the common regulatory cue. In budding yeast, the small G protein Cdc42 plays an important role in mitotic exit following the spindle pole attachment . We show here that inhibition of Cdc42 activation blocks polar body formation. The oocytes initiate anaphase but fail to properly form and direct a contractile ring. Endogenous Cdc42 is activated at the spindle pole-cortical contact site immediately prior to polar body formation. The cortical Cdc42 activity zone, which directly overlays the spindle pole, is circumscribed by a cortical RhoA activity zone; the latter defines the cytokinetic contractile furrow . As the RhoA ring contracts during cytokinesis, the Cdc42 zone expands, maintaining its complementary relationship with the RhoA ring. Cdc42 signaling may thus be an evolutionarily conserved mechanism that couples spindle positioning to asymmetric cytokinesis.     

WAVE2 regulates meiotic spindle stability,peripheral positioning and polar body emission in mouse oocytes

During oocyte meiotic maturation, meiotic spindles form in the central cytoplasm and then migrate to the cortex to extrude a small polar body, forming a highly polarized cell through a process involving actin and actin-related molecules. The mechanisms underlying oocyte polarization are still unclear. The Arp2/3 complex regulates oocyte polarization but it is not known whether the WASP family of proteins, a known regulator of the Arp2/3 complex, is involved in this context. In the present study, the role of WASP family member WAVE2 in mouse oocyte asymmetric division was investigated. (1) WAVE2 mRNA and protein were detected during mouse oocyte meiosis. (2) siRNA-mediated and antibody-mediated disruption of WAVE2 resulted in the failure of chromosome congression, spindle formation, spindle positioning and polar body extrusion. (3) WAVE2 regulated actin-driven chromosome migration since chromosomes were arrested in the central cytoplasm by WAVE2 RNAi in the absence of microtubules. (4) Localization of γ-tubulin and MAPK was disrupted after RNAi, confirming the effect of WAVE2 on spindle formation. (5) Actin cap and cortical granule-free domain (CGFD) formation was also disrupted, further confirming the failure of oocyte polarization. Our data suggest that WAVE2 regulates oocyte polarization by regulating meiotic spindle, peripheral positioning, probably via an actin-mediated pathway, and is involved in polar body emission during mouse oocyte meiotic maturation.     

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.     

The spatial and mechanical challenges of female meiosis

Recent work shows that cytokinesis and other cellular morphogenesis events are tuned by an interplay among biochemical signals, cell shape, and cellular mechanics. In cytokinesis, this includes cross-talk between the cortical cytoskeleton and the mitotic spindle in coordination with cell cycle control, resulting in characteristic changes in cellular morphology and mechanics through metaphase and cytokinesis. The changes in cellular mechanics affect not just overall cell shape, but also mitotic spindle morphology and function. This review will address how these principles apply to oocytes undergoing the asymmetric cell divisions of meiosis I and II. The biochemical signals that regulate cell cycle timing during meiotic maturation and egg activation are crucial for temporal control of meiosis. Spatial control of the meiotic divisions is also important, ensuring that the chromosomes are segregated evenly and that meiotic division is clearly asymmetric, yielding two daughter cells - oocyte and polar body - with enormous volume differences. In contrast to mitotic cells, the oocyte does not undergo overt changes in cell shape with its progression through meiosis, but instead maintains a relatively round morphology with the exception of very localized changes at the time of polar body emission. Placement of the metaphase-I and -II spindles at the oocyte periphery is clearly important for normal polar body emission, although this is likely not the only control element. Here, consideration is given to how cellular mechanics could contribute to successful mammalian female meiosis, ultimately affecting egg quality and competence to form a healthy embryo.     

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.     

Centriolin,a centriole-appendage protein,regulates peripheral spindle migration and asymmetric division in mouse meiotic oocytes

Unlike somatic cells mitosis, germ cell meiosis consists of 2 consecutive rounds of division that segregate homologous chromosomes and sister chromatids, respectively. The meiotic oocyte is characterized by an absence of centrioles and asymmetric division. Centriolin is a relatively novel centriolar protein that functions in mitotic cell cycle progression and cytokinesis. Here, we explored the function of centriolin in meiosis and showed that it is localized to meiotic spindles and concentrated at the spindle poles and midbody during oocyte meiotic maturation. Unexpectedly, knockdown of centriolin in oocytes with either siRNA or Morpholino micro-injection, did not affect meiotic spindle organization, cell cycle progression, or cytokinesis (as indicated by polar body emission), but led to a failure of peripheral meiotic spindle migration, large polar body emission, and 2-cell like oocytes. These data suggest that, unlike in mitotic cells, the centriolar protein centriolin does not regulate cytokinesis, but plays an important role in regulating asymmetric division of meiotic oocytes.     

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.     

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.     

Initial diameter of the polar body contractile ring is minimized by the centralspindlin complex

Polar body formation is an essential step in forming haploid eggs from diploid oocytes. This process involves completion of a highly asymmetric cytokinesis that results in a large egg and two small polar bodies. Unlike mitotic contractile rings, polar body contractile rings assemble over one spindle pole so that the spindle must move through the contractile ring before cytokinesis. During time-lapse imaging of C. elegans meiosis, the contractile ring moved downward along the length of the spindle and completed scission at the midpoint of the spindle, even when spindle length or rate of ring movement was increased. Patches of myosin heavy chain and dynamic furrowing of the plasma membrane over the entire embryo suggested that global cortical contraction forces the meiotic spindle and overlying membrane out through the contractile ring center. Consistent with this model, depletion of myosin phosphatase increased the velocity of ring movement along the length of the spindle. Global dynamic furrowing, which was restricted to anaphase I and II, was dependent on myosin II, the anaphase promoting complex and separase, but did not require cortical contact by the spindle. Large cortical patches of myosin during metaphase I and II indicated that myosin was already in the active form before activation of separase. To identify the signal at the midpoint of the anaphase spindle that induces scission, we depleted two proteins that mark the exact midpoint of the spindle during late anaphase, CYK-4 and ZEN-4. Depletion of either protein resulted in the unexpected phenotype of initial ingression of a polar body ring with twice the diameter of wild type. This phenotype revealed a novel mechanism for minimizing polar body size. Proteins at the spindle midpoint are required for initial ring ingression to occur close to the membrane-proximal spindle pole.     

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.     

Anillin promotes astral microtubule-directed cortical myosin polarization

Assembly of a cytokinetic contractile ring is a form of cell polarization in which the equatorial cell cortex becomes differentiated from the polar regions. Microtubules direct cytokinetic polarization via the central spindle and astral microtubules. The mechanism of central spindle-directed furrow formation is reasonably well understood, but the aster-directed pathway is not. In aster-directed furrowing, cytoskeletal factors accumulate to high levels at sites distal to the asters and at reduced levels at cortical sites near the asters. In this paper, we demonstrate that the cytoskeletal organizing protein anillin (ANI-1) promotes the formation of an aster-directed furrow in Caenorhabditis elegans embryos. Microtubule-directed nonmuscle myosin II polarization is aberrant in embryos depleted of ANI-1. In contrast, microtubule-directed polarized ANI-1 localization is largely unaffected by myosin II depletion. Consistent with a role in the induction of cortical asymmetry, ANI-1 also contributes to the polarization of arrested oocytes. Anillin has an evolutionarily conserved capacity to associate with microtubules, possibly providing an inhibitory mechanism to promote polarization of the cell cortex.     

Septin 7 is required for orderly meiosis in mouse oocytes

Septin 7 is a conserved GTP-binding protein. In this study, we examined the localization and functions of Septin 7 during mouse oocyte meiotic maturation. Immunofluorescent analysis showed that intrinsic Septin 7 localized to the spindles from the pro-MI stage to the MII stage. Knockdown of Septin 7 by siRNA microinjection caused abnormal spindles and affected extrusion of the first polar body. Septin 7 mRNA tagged with myc was injected into GV stage oocytes to overexpress Septin 7. Overexpressed Myc-Septin 7 localized to the spindle and beneath the plasma membrane displaying long filaments. Fluorescence intensity of spindle α-tubulin in myc-Septin 7-injected oocytes was weaker than that of the control group, demonstrating that Septin 7 may influence recruitment of α-tubulin to spindles. MII oocytes injected with myc-Septin 7 exhibited abnormal chromosome alignment, and parthenogenetic activation failed to allow extrusion of the second polar body, suggesting that overexpression of Septin 7 may affect extrusion of the polar body by disturbing the alignment of chromosomes and regulating α-tubulin recruitment to spindles. In summary, Septin 7 may regulate meiotic cell cycle progression by affecting microtubule cytoskeletal dynamics in mouse oocytes.     

Septin 7 is required for orderly meiosis in mouse oocytes

Septin 7 is a conserved GTP-binding protein. In this study, we examined the localization and functions of Septin 7 during mouse oocyte meiotic maturation. Immunofluorescent analysis showed that intrinsic Septin 7 localized to the spindles from the pro-MI stage to the MII stage. Knockdown of Septin 7 by siRNA microinjection caused abnormal spindles and affected extrusion of the first polar body. Septin 7 mRNA tagged with myc was injected into GV stage oocytes to overexpress Septin 7. Overexpressed Myc-Septin 7 localized to the spindle and beneath the plasma membrane displaying long filaments. Fluorescence intensity of spindle α-tubulin in myc-Septin 7-injected oocytes was weaker than that of the control group, demonstrating that Septin 7 may influence recruitment of α-tubulin to spindles. MII oocytes injected with myc-Septin 7 exhibited abnormal chromosome alignment, and parthenogenetic activation failed to allow extrusion of the second polar body, suggesting that overexpression of Septin 7 may affect extrusion of the polar body by disturbing the alignment of chromosomes and regulating α-tubulin recruitment to spindles. In summary, Septin 7 may regulate meiotic cell cycle progression by affecting microtubule cytoskeletal dynamics in mouse oocytes.     

Symmetry breaking and polarity establishment during mouse oocyte maturation

Mammalian oocyte meiosis encompasses two rounds of asymmetric divisions to generate a totipotent haploid egg and, as by-products, two small polar bodies. Two intracellular events, asymmetric spindle positioning and cortical polarization, are critical to such asymmetric divisions. Actin but not microtubule cytoskeleton has been known to be directly involved in both events. Recent work has revealed a positive feedback loop between chromosome-mediated cortical activation and the Arp2/3-orchestrated cytoplasmic streaming that moves chromosomes. This feedback loop not only maintains meiotic II spindle position during metaphase II arrest, but also brings about symmetry breaking during meiosis I. Prior to an Arp2/3-dependent phase of fast movement, meiotic I spindle experiences a slow and non-directional first phase of migration driven by a pushing force from Fmn2-mediated actin polymerization. In addition to illustrating these molecular mechanisms, mathematical simulations are presented to elucidate mechanical properties of actin-dependent force generation in this system.     

Stage specific effects of carbendazim (MBC) on meiotic cell cycle progression in mouse oocytes

The effects of the pesticide carbendazim (MBC) on the in vitro meiotic maturation of mouse oocytes were evaluated using conventional and confocal fluorescence microscopy. The response of oocytes exposed to 0, 3, 10, or 30 μM MBC during meiotic maturation was analyzed with respect to chromosome organization, meiotic spindle microtubules, and cortical actin using fluorescent labels for each of these structures. Continuous exposure to MBC during the resumption of meiosis resulted in a dose-dependent inhibition of meiotic cell cycle progression at metaphase of meiosis-1. Drug exposure at the metaphase-anaphase transition of meiosis-1 did not interfere with cell cycle progression to metaphase-2 except at high concentrations (30 μM). At the level of spindle microtubule organization, MBC caused a loss of nonacetylated microtubules and a decrease in spindle size at 3 or 10 μM concentrations. Thirty μM MBC prevented spindle assembly when added at the beginning of meiotic maturation or caused spindle pole disruption and fragmentation when added to preformed spindles. Spindle disruption involved a loss of phosphoprotein epitopes, as monitored by MPM-2 staining, and resulted in the appearance of dispersed chromosomes that retained a metaphase-plate location on spindle fragments associated with the oocyte cortex. Polar body extrusion was impaired by MBC, and abnormal polar bodies were observed in most treated oocytes. The results suggest that MBC disrupts cell cycle progression in mouse oocytes by altering meiotic spindle microtubule stability and spindle pole integrity. Mol. Reprod. Dev. 46:351–362, 1997. © 1997 Wiley-Liss, Inc.