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.     

GM130, a cis-Golgi protein,regulates meiotic spindle assembly and asymmetric division in mouse oocyte

GM130, a cis-Golgi protein, plays key roles in various mitotic events, but its function in mammalian oocyte meiosis remains unknown. In this study, we found that GM130 was localized to the spindle poles at both metaphase I and metaphase II stages and associated with the midbody at telophase I stage. The association of GM130 with spindle poles was further confirmed by its colocalization with the centrosome-associated proteins, MEK1/2. By nocodazole treatment, we clarified that GM130 localization was consistently dependent on spindle assembly. Then we investigated the possible function of GM130 by specific morpholino microinjection. This treatment caused abnormal spindle formation, and decreased first polar body extrusion. Our results showed that knockdown of GM130 impaired the localization of MTOCs proteins γ-tubulin and Plk1. Using live cell imaging we observed that depletion of GM130 affected spindle migration and resulted in elongated spindle and large polar body extrusion. We further found that depletion of GM130 blocked p-MEK1/2 accumulation at the spindle poles. And, it was shown that GM130 detached from the spindle poles in oocytes treated with MEK specific inhibitor U0126. Taken together, our results suggested that GM130 regulates microtubule organization and might cooperate with the MAPK pathway to play roles in spindle organization, migration and asymmetric division during mouse oocyte maturation.     

哺乳动物卵母细胞不对称分裂的研究进展

哺乳动物卵母细胞成熟过程需要进行两次连续的不对称分裂,最终形成体积差异巨大的子细胞：大体积的卵母细胞和两种体积较小的极体。不对称分裂现象是哺乳动物卵母细胞减数分裂的典型特征,不对称分裂后的卵母细胞是高度极化的细胞。精卵结合后,细胞重新恢复了对称分裂,但是在卵母细胞减数分裂过程中形成的极性特征却得以保留并影响早期胚胎的极性。本文对近年来在哺乳动物卵母细胞不对称分裂方面的相关研究展开综述,从细胞质不对称分裂和细胞核不对称分裂两个方面对染色体、细胞骨架在哺乳动物卵母细胞不对称分裂中的作用、细胞器在哺乳动物卵母细胞成熟过程中的重组分配、染色体非随机分离等过程进行介绍,旨在从细胞和分子水平阐述哺乳动物卵母细胞不对称分裂的主要机制。     

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.     

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.     

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.     

Age‐dependent aneuploidy in mammalian oocytes instigated at the second meiotic division

Ageing severely affects the chromosome segregation process in human oocytes resulting in aneuploidy, infertility and developmental disorders. A considerable amount of segregation errors in humans are introduced at the second meiotic division. We have here compared the chromosome segregation process in young adult and aged female mice during the second meiotic division. More than half of the oocytes in aged mice displayed chromosome segregation irregularities at anaphase II, resulting in dramatically increased level of aneuploidy in haploid gametes, from 4% in young adult mice to 30% in aged mice. We find that the post‐metaphase II process that efficiently corrects aberrant kinetochore‐microtubule attachments in oocytes in young adult mice is approximately 10‐fold less efficient in aged mice, in particular affecting chromosomes that show small inter‐centromere distances at the metaphase II stage in aged mice. Our results reveal that post‐metaphase II processes have critical impact on age‐dependent aneuploidy in mammalian eggs.     

GTPase-activating protein Elmod2 is essential for meiotic progression in mouse oocytes

Meiotic failure in oocytes is the major determinant of human zygote-originated reproductive diseases, the successful accomplishment of meiosis largely relay on the normal functions of many female fertility factors. Elmod2 is a member of the Elmod family with the strongest GAP (GTPase-activating protein) activity; although it was identified as a possible maternal protein, its actual physiologic role in mammalian oocytes has not been elucidated. Herein we reported that among Elmod family proteins, Elmod2 is the most abundant in mouse oocytes, and that inhibition of Elmod2 by specific siRNA caused severe meiotic delay and abnormal chromosomal segregation during anaphase. Elmod2 knockdown also significantly decreased the rate of oocyte maturation (to MII, with first polar body extrusion), and significantly greater numbers of Elmod2-knockdown MII oocytes were aneuploid. Correspondingly, Elmod2 knockdown dramatically decreased fertilization rate. To investigate the mechanism(s) involved, we found that Elmod2 knockdown caused significantly more abnormal mitochondrial aggregation and diminished cellular ATP levels; and we also found that Elmod2 co-localized and interacted with Arl2, a GTPase that is known to maintain mitochondrial dynamics and ATP levels in oocytes. In summary, we found that Elmod2 is the GAP essential to meiosis progression of mouse oocytes, most likely by regulating mitochondrial dynamics.     

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.     

Activated proline‐rich tyrosine kinase 2 regulates meiotic spindle assembly in the mouse oocyte

Proline‐rich tyrosine kinase 2 (PYK2), a member of the protein tyrosine kinase family, plays an important role in various cellular processes. PYK2 can be phosphorylated on tyrosine 402 by diverse stimuli at the cell surface, and recent studies have shown that this activated form of PYK2 is enriched in oocytes and required for fertilization. However, the subcellular localization and functions of activated PYK2 in oocytes remain elusive. In this study, we demonstrate that the localization of p‐PYK2 undergoes dynamic changes during in vitro maturation of mouse oocytes. The signal of p‐PYK2 is initially dispersed in the cytoplasm, but begins to decorate organized microtubules after the germinal vesicle breakdown and localizes to spindle poles at metaphase. Our data further show that p‐PYK2 colocalizes with γ‐tubulin from the germinal vesicle stage through the end of meiosis in mouse oocytes. Nocodazole treatment and washout experiments confirm that p‐PYK2 associates with the oocyte spindle and spindle poles. Moreover, pharmacological inhibition of PYK2 activity dramatically alters the morphology of the bipolar spindle and prevents oocyte maturation. Together, these data suggest that activated PYK2 may function as a component of the microtubule organizing center to regulate spindle assembly during the meiotic process of mouse oocytes.     

Abnormalities of spindle and cytokine behavior leading to the formation of meiotic restitution nuclei in intergeneric cereal hybrids

Mobile stages of meiosis have been analysed by visualizing the spindle in fertile cereal F1 hybrids. We describe four different mechanisms of the formation of restitution nuclei in meiotic division: (1) centripetal migration of telophase chromosome groups from the poles of a curved spindle at early telophase; (2) centripetal migration of the chromosome groups at late telophase when cell plate formation has failed; (3) preferable migration of univalents to one of the poles although spindle appearance is morphologically normal; and (4) in the absence of chromosome segregation where kinetochore fibers have failed to form.     

Microtubule organizing centers regulate spindle positioning in mouse oocytes

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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.     

Astrin regulates meiotic spindle organization,spindle pole tethering and cell cycle progression in mouse oocytes

Astrin has been described as a microtubule and kinetochore protein required for the maintenance of sister chromatid cohesion and centrosome integrity in human mitosis. However, its role in mammalian oocyte meiosis is unclear. In this study, we find that Astrin is mainly associated with the meiotic spindle microtubules and concentrated on spindle poles at metaphase I and metaphase II stages. Taxol treatment and immunoprecipitation show that Astrin may interact with the centrosomal proteins Aurora-A or Plk1 to regulate microtubule organization and spindle pole integrity. Loss-of-function of Astrin by RNAi and overexpression of Tof the coiled-coil domain results in spindle disorganization, chromosome misalignment and meiosis progression arrestT. Thr24, Ser66 or Ser447 may be the potential phosphorylated sites of Astrin by Plk1, as site-directed mutation of these sites causes oocyte meiotic arrest at HTmetaphaseTH I with highly disordered spindles and disorganized chromosomes, although mutant Astrin localizes to the spindle apparatus. Taken together, these data strongly suggest that Astrin is critical for meiotic spindle assembly and maturation in mouse oocytes.     

Checkpoint kinase 1 is essential for meiotic cell cycle regulation in mouse oocytes

Checkpoint kinase 1 (Chk1) plays key roles in all currently defined cell cycle checkpoints, but its functions in mouse oocyte meiosis remain unclear. In this study, we report the expression, localization and functions of Chk1 in mouse oocyte meiosis. Chk1 was expressed from germinal vesicle (GV) to metaphase II (MII) stages and localized to the spindle from pro-metaphase I (pro-MI) to MII stages in mouse oocytes. Chk1 depletion facilitated the G₂/M transition while Chk1 overexpression inhibited the G₂/M transition as indicated by germinal vesicle breakdown (GVBD), through regulation of Cdh1 and Cyclin B1. Chk1 depletion did not affect meiotic cell cycle progression after GVBD, but its overexpression after GVBD activated the spindle assembly checkpoint and prevented homologous chromosome segregation, thus arresting oocytes at pro-MI or metaphase I (MI) stages. These results suggest that Chk1 is indispensable for prophase I arrest and functions in G₂/M checkpoint regulation in meiotic oocytes. Moreover, Chk1 overexpression affects meiotic spindle assembly checkpoint regulation and thus chromosome segregation.     

RNA- binding protein Stau2 is important for spindle integrity and meiosis progression in mouse oocytes

Staufen2 (Stau2) is a double-stranded RNA-binding protein involved in cell fate decision by regulating mRNA transport, mRNA stability, translation, and ribonucleoprotein assembly. Little is known about Stau2 expression and function in mammalian oocytes during meiosis. Herein we report the sub-cellular distribution and function of Stau2 in mouse oocyte meiosis. Western blot analysis revealed high and stable expression of Stau2 in oocytes from germinal vesicle (GV) to metaphase II (MII). Immunofluorescence showed that Stau2 was evenly distributed in oocytes at GV stage, and assembled as filaments after germinal vesicle breakdown (GVBD), particularly, colocalized with spindle at MI and MII. Stau2 was disassembled when microtubules were disrupted with nocodazole, on the other hand, when MTs were stabilized with taxol, Stau2 was not colocalized with the stabilized microtubules, but aggregated around the chromosomes array, indicating Stau2 assembly and colocalization with microtubules require both microtubule integrity and its normal dynamics. During interphase and mitosis of BHK and MEF cells, Stau2 was not distributed on microtubules, but colocalized with cis-Golgi marker GM130, implying its association with Golgi complex but not the spindle in fully differentiated somatic cells. Specific morpholino oligo-mediated Stau2 knockdown disrupted spindle formation, chromosome alignment and microtubule-kinetochore attachment in oocytes. The majority oocytes were arrested at MI stage, with bright MAD1 at kinetochores, indicating activation of spindle assembly checkpoint (SAC). Some oocytes were stranded at telophase I (TI), implying suppressed first polar body extrution. Together these data demonstrate that Stau2 is required for spindle formation and timely meiotic progression in mouse oocytes.     

Lysophosphatidylserine inhibits completion of meiotic maturation of rat oocytes in vitro

Follicular oocytes collected prior to the expected time of the LH surge from PMSG-treated immature rats were incubated cummulus-intact (with or without LH) or cumulus-free (CF). Oocytes were incubated in the presence or absence of lysophosphatidlylserine (LS), a naturally occurring membrane phospholipid that has been previously shown to block sperm-related membrane fusion events. Fusion events occurring during oocyte maturation that might be affected by LS include maintenance of the intact germinal vesicle (GVI) and prevention of GV breakdown (GVBD) and first polar body formation (PBI). LS had only a slight effect upon GVI. The incidence of GVI was significantly increased in only one of the three oocyte culture conditions employed (CF). Exposure to LS from the outset of collection and washing did not increase the incidence of GVI, indicating the lack of effect by LS was not owing to the passage of a sensitive period during oocyte collection. In contrast, LS was not owing to the passage of a sensitive period during oocyte colection. In contrast, LS almost completely abolished PBI in all oocyte culture conditions at 100 μ in PBI and those sperm-related fusion processes previously found to be sensitive to LS. Finally, LS or similar agents may be responsible for the block to maturation (often at anaphase I) and even the retarded cleavage observed in vitro during oocyte maturation or embryo culture in some species.     

TP53BP1 regulates chromosome alignment and spindle bipolarity in mouse oocytes

Meiotic oocytes lack classic centrosomes; therefore, bipolar spindle assembly depends on the clustering of acentriolar microtubule‐organizing centers (MTOCs) into two poles. The bipolar spindle is an essential cellular component that ensures accurate chromosome segregation during anaphase. If the spindle does not form properly, it can result in aneuploidy or cell death. However, the molecular mechanism by which the bipolar spindle is established is not yet fully understood. Tumor suppressor p53‐binding protein 1 (TP53BP1) is known to mediate the DNA damage response. Several recent studies have indicated that TP53BP1 has noncanonical roles in processes, such as spindle formation; however, the role of TP53BP1 in oocyte meiosis is currently unclear. Our results show that TP53BP1 knockdown affects spindle bipolarity and chromatin alignment by altering MTOC stability during oocyte maturation. TP53BP1 was localized in the cytoplasm and displayed an irregular cloud pattern around the spindle/chromosome region. TP53BP1 was also required for the correct localization of MTOCs into the two spindle poles during pro‐meiosis I. TP53BP1 deletion altered the MTOC‐localized Aurora Kinase A. TP53BP1 knockdown caused the microtubules to detach from the kinetochores and increased the rate of aneuploidy. Taken together, our data show that TP53BP1 plays crucial roles in chromosome stability and spindle bipolarity during meiotic maturation.