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.     

Nucleation Promoting Factors Regulate the Expression and Localization of Arp2/3 Complex during Meiosis of Mouse Oocytes

The actin nucleation factor Arp2/3 complex is a main regulator of actin assembly and is involved in multiple processes like cell migration and adhesion, endocytosis, and the establishment of cell polarity in mitosis. Our previous work showed that the Arp2/3 complex was involved in the actin-mediated mammalian oocyte asymmetric division. However, the regulatory mechanisms and signaling pathway of Arp2/3 complex in meiosis is still unclear. In the present work, we identified that the nucleation promoting factors (NPFs) JMY and WAVE2 were necessary for the expression and localization of Arp2/3 complex in mouse oocytes. RNAi of both caused the degradation of actin cap intensity, indicating the roles of NPFs in the formation of actin cap. Moreover, JMY and WAVE2 RNAi decreased the expression of ARP2, a key component of Arp2/3 complex. However, knock down of Arp2/3 complex by Arpc2 and Arpc3 siRNA microinjection did not affect the expression and localization of JMY and WAVE2. Our results indicate that the NPFs, JMY and WAVE2, are upstream regulators of Arp2/3 complex in mammalian oocyte asymmetric division.     

Small Molecule Inhibitor of Formin Homology 2 Domains (SMIFH2) Reveals the Roles of the Formin Family of Proteins in Spindle Assembly and Asymmetric Division in Mouse Oocytes

Dynamic actin reorganization is the main driving force for spindle migration and asymmetric cell division in mammalian oocytes. It has been reported that various actin nucleators including Formin-2 are involved in the polarization of the spindle and in asymmetric cell division. In mammals, the formin family is comprised of 15 proteins. However, their individual roles in spindle migration and/or asymmetric division have not been elucidated yet. In this study, we employed a newly developed inhibitor for formin family proteins, small molecule inhibitor of formin homology 2 domains (SMIFH2), to assess the functions of the formin family in mouse oocyte maturation. Treatment with SMIFH2 during in vitro maturation of mouse oocytes inhibited maturation by decreasing cytoplasmic and cortical actin levels. In addition, treatment with SMIFH2, especially at higher concentrations (500 μM), impaired the proper formation of meiotic spindles, indicating that formins play a role in meiotic spindle formation. Knockdown of the mDia2 formins caused a similar decrease in oocyte maturation and abnormal spindle morphology, mimicking the phenotype of SMIFH2-treated cells. Collectively, these results suggested that besides Formin-2, the other proteins of the formin, including mDia family play a role in asymmetric division and meiotic spindle formation in mammalian oocytes.     

Sequential actin-based pushing forces drive meiosis I chromosome migration and symmetry breaking in oocytes

Polar body extrusion during oocyte maturation is critically dependent on asymmetric positioning of the meiotic spindle, which is established through migration of the meiosis I (MI) spindle/chromosomes from the oocyte interior to a subcortical location. In this study, we show that MI chromosome migration is biphasic and driven by consecutive actin-based pushing forces regulated by two actin nucleators, Fmn2, a formin family protein, and the Arp2/3 complex. Fmn2 was recruited to endoplasmic reticulum structures surrounding the MI spindle, where it nucleated actin filaments to initiate an initially slow and poorly directed motion of the spindle away from the cell center. A fast and highly directed second migration phase was driven by actin-mediated cytoplasmic streaming and occurred as the chromosomes reach a sufficient proximity to the cortex to activate the Arp2/3 complex. We propose that decisive symmetry breaking in mouse oocytes results from Fmn2-mediated perturbation of spindle position and the positive feedback loop between chromosome signal-induced Arp2/3 activation and Arp2/3-orchestrated cytoplasmic streaming that transports the chromosomes.     

SKAP2 regulates Arp2/3 complex for actin-mediated asymmetric cytokinesis by interacting with WAVE2 in mouse oocytes

SKAP2 (Src kinase-associated phosphoprotein 2), a substrate of Src family kinases, has been suggested to be involved in actin-mediated cellular processes. However, little is known about its role in mouse oocyte maturation. In this study, we thus investigated the expression, localization, and functions of SKAP2 during mouse oocyte asymmetric division. SKAP2 protein expression was detected at all developmental stages in mouse oocytes. Immunofluorescent staining showed that SKAP2 was mainly distributed at the cortex of the oocytes during maturation. Treatment with cytochalasin B in oocytes confirmed that SKAP2 was co-localized with actin. Depletion of SKAP2 by injection with specific short interfering RNA caused failure of spindle migration, polar body extrusion, and cytokinesis defects. Meanwhile, the staining of actin filaments at the oocyte membrane and in the cytoplasm was significantly reduced after these treatments. SKAP2 depletion also disrupted actin cap and cortical granule-free domain formation, and arrested a large proportion of oocytes at the telophase stage. Moreover, Arp2/3 complex and WAVE2 expression was decreased after the depletion of SKAP2 activity. Our results indicate that SKAP2 regulates the Arp2/3 complex and is essential for actin-mediated asymmetric cytokinesis by interacting with WAVE2 in mouse oocytes.     

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.     

Arp2/3 Complex Inhibition Prevents Meiotic Maturation in Porcine Oocytes

The Arp2/3 complex regulates actin nucleation, which is critical for a wide range of cellular processes, such as cell polarity, cell locomotion, and endocytosis. In the present study, we investigated the possible roles of the Arp2/3 complex in porcine oocytes during meiotic maturation. Immunofluorescent staining showed the Arp2/3 complex to localize mainly to the cortex of porcine oocytes, colocalizing with actin. Treatment with an Arp2/3 complex specific inhibitor, CK666, resulted in a decrease in Arp2/3 complex localization at the oocyte cortex. The maturation rate of porcine oocytes decreased significantly after CK666 treatment, concomitant with the failure of cumulus cell expansion and oocyte polar body extrusion. The fluorescence intensity of F-actin decreased in the cytoplasm, and CK666 also disrupted actin cap formation. In summary, our results illustrate that the Arp2/3 complex is required for the meiotic maturation of porcine oocytes and that actin nucleation is critical for meiotic maturation.     

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.     

WHAMM is required for meiotic spindle migration and asymmetric cytokinesis in mouse oocytes

WASP homolog associated with actin, membranes and microtubules (WHAMM) is a newly discovered nucleation-promoting factor that links actin and microtubule cytoskeleton and regulates transport from the endoplasmic reticulum to the Golgi apparatus. However, knowledge of WHAMM is limited to interphase somatic cells. In this study, we examined its localization and function in mouse oocytes during meiosis. Immunostaining showed that in the germinal vesicle (GV) stage, there was no WHAMM signal; after meiosis resumption, WHAMM was associated with the spindle at prometaphase I (Pro MI), metaphase I (MI), telophase I (TI) and metaphase II (MII) stages. Nocodazole and taxol treatments showed that WHAMM was localized around the MI spindle. Depletion of WHAMM by microinjection of specific short interfering (si)RNA into the oocyte cytoplasm resulted in failure of spindle migration, disruption of asymmetric cytokinesis and a decrease in the first polar body extrusion rate during meiotic maturation. Moreover, actin cap formation was also disrupted after WHAMM depletion, confirming the failure of spindle migration. Taken together, our data suggest that WHAMM is required for peripheral spindle migration and asymmetric cytokinesis during mouse oocyte maturation.     

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.     

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.     

mTOR is required for asymmetric division through small GTPases in mouse oocytes

Mammalian target of rapamycin (mTOR) is central to the control of cell proliferation, growth, and survival in mammalian cells. Prolonged treatment with rapamycin inhibits mTOR complex 2 (mTORC2) activity, and both the mTORC1-mediated S6K1 and 4E-BP1/eIF4E pathways are essential for TORC2-mediated RhoA, Cdc42, and Rac1 expression during cell motility and F-actin reorganization. The functions of mTOR in the mouse oocyte remain unclear, however. The present study shows that rapamycin affects mTOR expression and cytoskeleton reorganization during meiotic maturation of mouse oocytes. mTOR mRNA was expressed in germinal vesicles (GV) until metaphase I (MI), and increased during metaphase II (MII). Immunostaining showed that mTOR localized around the spindle and in the cytoplasm of oocytes. Treatment of oocytes with rapamycin decreased mTOR at the RNA and protein level, and altered asymmetric division. Formation of the actin cap and the cortical granule-free domain were also disrupted after rapamycin treatment, indicating the failure of spindle migration. Injection of an anti-mTOR antibody yielded results consistent with those obtained for rapamycin treatment, further confirming the involvement of mTOR in oocyte polarity. Furthermore, rapamycin treatment reduced the mRNA expression of small GTPases (RhoA, Cdc42, and Rac1), which are crucial regulatory factors for cytoskeleton reorganization. Taken together, these results suggest that rapamycin inhibits spindle migration and asymmetric division during mouse oocyte maturation via mTOR-mediated small GTPase signaling pathways.     

Sleeping beauty transposon-based phenotypic analysis of mice: lack of Arpc3 results in defective trophoblast outgrowth

The Sleeping Beauty (SB) transposon system has generated many transposon-insertional mutant mouse lines, some of which have resulted in embryonic lethality when bred to homozygosity. Here we report one such insertion mapped to the mouse actin-related protein complex subunit 3 gene (Arpc3). Arpc3 is a component of the Arp2/3 complex, which plays a major role in actin nucleation with Y-shaped branching from the mother actin filament in response to migration signaling. Arpc3 transposon-inserted mutants developed only to the blastocyst stage. In vitro blastocyst culture of Arpc3 mutants exhibited severe spreading impairment of trophoblasts. This phenotype was also observed in compound heterozygotes generated using conventional gene-targeted and transposon-inserted alleles. Arpc3-deficient mutants were shown to lack actin-rich structures in the spreading trophoblast. Electron microscopic analysis demonstrated the lack of mesh-like structures at the cell periphery, suggesting a role of Arpc3 in Y-shaped branching formation. These data indicate the importance of Arpc3 in the Arp2/3 complex for trophoblast outgrowth and suggest that Arpc3 may be indispensable for implantation.     

Non-muscle tropomyosin (Tpm3) is crucial for asymmetric cell division and maintenance of cortical integrity in mouse oocytes

Tropomyosins are actin-binding cytoskeletal proteins that play a pivotal role in regulating the function of actin filaments in muscle and non-muscle cells; however, the roles of non-muscle tropomyosins in mouse oocytes are unknown. This study investigated the expression and functions of non-muscle tropomyosin (Tpm3) during meiotic maturation of mouse oocytes. Tpm3 mRNA was detected at all developmental stages in mouse oocytes. Tpm3 protein was localized at the cortex during the germinal vesicle and germinal vesicle breakdown stages. However, the overall fluorescence intensity of Tpm3 immunostaining was markedly decreased in metaphase II oocytes. Knockdown of Tpm3 impaired asymmetric division of oocytes and spindle migration, considerably reduced the amount of cortical actin, and caused membrane blebbing during cytokinesis. Expression of a constitutively active cofilin mutant and Tpm3 overexpression confirmed that Tpm3 protects cortical actin from depolymerization by cofilin. The data indicate that Tpm3 plays crucial roles in maintaining cortical actin integrity and asymmetric cell division during oocyte maturation, and that dynamic regulation of cortical actin by Tpm3 is critical to ensure proper polar body protrusion.     

Vesicular transport protein Arf6 modulates cytoskeleton dynamics for polar body extrusion in mouse oocyte meiosis

Arf6 (ADP-ribosylation factor 6) is known to play important roles in membrane dynamics through the regulation of actin filament reorganization for multiple cellular processes such as cytokinesis, phagocytosis, cell migration and tumor cell invasion. However, the functions of Arf6 in mammalian oocyte meiosis have not been clarified. In present study we showed that Arf6 expressed in mouse oocytes and was mainly distributed around the spindle during meiosis. Depletion of Arf6 by morpholino microinjection caused oocytes failing to extrude first polar body. Further analysis indicated that Arf6 knock down caused the aberrant actin distribution, which further induced the failure of meiotic spindle movement. And the loss of oocyte polarity also confirmed this. The regulation of Arf6 on actin filaments in mouse oocytes might be due to its effects on the phosphorylation level of cofilin and the expression of Arp2/3 complex. Moreover, we found that the decrease of Arf6 caused the disruption of spindle formation, indicating the multiple roles of Arf6 on cytoskeleton dynamics in meiosis. In summary, our results indicated that Arf6 was involved in mouse oocyte meiosis through its functional roles in actin-mediated spindle movement and spindle organization.     

Spire-type actin nucleators cooperate with Formin-2 to drive asymmetric oocyte division

Oocytes mature into eggs by extruding half of their chromosomes in a small cell termed the polar body. Asymmetric oocyte division is essential for fertility [1], but despite its importance, little is known about its mechanism. In mammals, the meiotic spindle initially forms close to the center of the oocyte. Thus, two steps are required for asymmetric meiotic division: first, asymmetric spindle positioning and second, polar body extrusion. Here, we identify Spire1 and Spire2 as new key factors in asymmetric division of mouse oocytes. Spire proteins are novel types of actin nucleators that drive nucleation of actin filaments with their four WH2 actin-binding domains [2-6]. We show that Spire1 and Spire2 first mediate asymmetric spindle positioning by assembling an actin network that serves as a substrate for spindle movement. Second, they drive polar body extrusion by promoting assembly of the cleavage furrow. Our data suggest that Spire1 and Spire2 cooperate with Formin-2 (Fmn2) to nucleate actin filaments in mouse oocytes and that both types of nucleators act as a functional unit. This study not only reveals how Spire1 and Spire2 drive two critical steps of asymmetric oocyte division, but it also uncovers the first physiological function of Spire-type actin nucleators in vertebrates.     

RhoA-mediated MLC2 regulates actin dynamics for cytokinesis in meiosis

During oocyte meiosis, the bipolar spindle forms in the central cytoplasm and then migrates to the cortex. Subsequently, the oocyte extrudes the polar body through two successive asymmetric divisions, which are regulated primarily by actin filaments. Myosin light chain2 (MLC2) phosphorylation plays pivotal roles in smooth muscle contraction, stress fiber formation, cell motility and cytokinesis. However, whether MLC2 phosphorylation participates in the oocyte polarization and asymmetric division has not been clarified. The present study investigated the expression and functions of MLC2 during mouse oocyte meiosis. Our result showed that p-MLC2 was localized in the oocyte cortex, with a thickened cap above the chromosomes. Meanwhile, p-MLC2 was also localized in the poles of spindle. Disruption of MLC2 activity by MLC2 knock down (K_D) caused the failure of polar body extrusion. Immunofluorescent staining showed that a large proportion of oocytes arrested in telophase stage and failed to undergo cytokinesis after culturing for 12 hours. In the meantime, actin filament staining at oocyte membrane and cytoplasm were reduced in MLC2 K_D oocytes. Finally, we found that the phosphorylation of MLC2 protein levels was decreased after disruption of RhoA activity. Above all, our data indicated that the RhoA-mediated MLC2 regulates the actin organization for cytokinesis during mouse oocyte maturation.     

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.     

Dynamic maintenance of asymmetric meiotic spindle position through Arp2/3-complex-driven cytoplasmic streaming in mouse oocytes

Mature?mammalian?oocytes?are?poised?for?completing?meiosis?II (MII) on fertilization by positioning the spindle close to an actomyosin-rich cortical cap. Here, we show that the Arp2/3 complex localizes to the cortical cap in a Ran-GTPase-dependent manner and nucleates actin filaments in the cortical cap and a cytoplasmic actin network. Inhibition of Arp2/3 activity leads to rapid dissociation of the spindle from the cortex. Live-cell imaging and spatiotemporal image correlation spectroscopy analysis reveal that actin filaments flow continuously away from the Arp2/3-rich cortex, driving a cytoplasmic streaming expected to exert a net pushing force on the spindle towards the cortex. Arp2/3 inhibition not only diminishes this actin flow and cytoplasmic streaming but also enables a reverse streaming driven by myosin-II-based cortical contraction, moving the spindle away from the cortex. Thus, the asymmetric MII spindle position is dynamically maintained as a result of balanced forces governed by the Arp2/3 complex.     

Arp2/3-dependent pseudocleavage [correction of psuedocleavage] furrow assembly in syncytial Drosophila embryos

BACKGROUND: In syncytial blastoderm Drosophila embryos, actin caps assemble during telophase. As the cell cycle progresses through interphase, these small caps expand and fuse to form pseudocleavage furrows that are structurally related to the cleavage furrows that assemble during somatic cell division. The molecular mechanism driving cell cycle coordinated actin reorganization from the caps to the furrows is not understood. RESULTS: We show that Drosophila embryos contain a typical Arp2/3 complex and that components of this complex localize to the margins of the expanding caps, to mature pseudocleavage furrows, and to somatic cell cleavage furrows during the postcellularization embryonic divisions. A mutation that disrupts the arpc1 subunit of Arp2/3 leads to spindle fusions that are characteristic of pseudocleavage furrow disruption. By contrast, this mutation does not significantly affect nuclear positioning during interphase, which is dependent on actin cap function. In vivo analysis of actin reorganization demonstrates that the arpc1 mutation does not prevent assembly of small actin caps but blocks cap expansion and furrow assembly as the cell cycle progresses through interphase. The scrambled gene is also required for cap expansion and furrow assembly, and Scrambled is required for Arp2/3 localization to the cap margins. CONCLUSIONS: The Drosophila Arp2/3 complex and Scrambled protein are required for actin cap expansion and pseudocleavage furrow formation during the syncytial blastoderm divisions. We propose that Scrambled-dependent localization of Arp2/3 to the margins of the expanding caps triggers local actin polymerization that drives cap expansion and pseudocleavage furrow assembly.