The SUMO pathway functions in mouse oocyte maturation

Sumoylation is an important post-translational modification in which SUMO (small ubiquitin-related modifier) proteins are bonded covalently to their substrates. Studies on the roles of sumoylation in cell cycle regulation have been emerging in both mitosis from yeast to mammals and meiosis in budding yeast, but the functions of sumoylation in mammalian meiosis, especially in oocyte meiotic maturation are not well known. Here, we examined the localization and expression of SUMO-1 and SUMO-2/3, the two basic proteins in the sumoylation pathway and investigated their roles through over-expression of Senp2 during mouse oocyte maturation. Immunofluorescent staining revealed differential patterns of SUMO-1 and SUMO-2/3 localization: SUMO-1 was localized to the spindle poles in prometaphase I, MI and MII stages, around the separating homologues in anaphase I and telophase I stages of first meiosis, while SUMO-2/3 was mainly concentrated near centromeres during mouse oocyte maturation. Immunoblot analysis uncovered the different expression profiles of SUMO-1 and SUMO-2/3 modified proteins during mouse oocyte maturation. Over-expression of Senp2, a SUMO-specific isopeptidase, caused changes of SUMO-modified proteins and led to defects in MII spindle organization in mature eggs. These results suggest that the SUMO pathway may play an indispensable role during mouse oocyte meiotic maturation.     

Perturbation of Spc25 expression affects meiotic spindle organization,chromosome alignment and spindle assembly checkpoint in mouse oocytes.

Spc25 is a component of the Ndc80 complex which consists of Ndc80, Nuf2, Spc24, and Spc25. Previous work has shown that Spc25 is involved in regulation of kinetochore microtubule attachment and the spindle assembly checkpoint in mitosis. The roles of Spc25 in meiosis remain unknown. Here, we report its expression, localization and functions in mouse oocyte meiosis. The Spc25 mRNA level gradually increased from the GV to MI stage, but decreased by MII during mouse oocyte meiotic maturation. Immunofluorescent staining showed that Spc25 was restricted to the germinal vesicle, and associated with chromosomes during all stages after GVBD. Overexpression of Spc25 by mRNA injection resulted in oocyte meiotic arrest, chromosome misalignment and spindle disruption. Conversely, Spc25 RNAi by siRNA injection resulted in precocious polar body extrusion and caused severe chromosome misalignment and aberrant spindle formation. Our data suggest that Spc25 is required for chromosome alignment, spindle formation, and proper spindle checkpoint signaling during meiosis.     

The SUMO pathway functions in mouse oocyte maturation

Sumoylation is an important posttranslational modification in which SUMO (small ubiquitin-related modifier) proteins are bonded covalently to their substrates. Studies on the roles of sumoylation in cell cycle regulation have been emerging in both mitosis from yeast to mammals and meiosis in budding yeast, but the functions of sumoylation in mammalian meiosis, especially in oocyte meiotic maturation are not well known. Here, we examined the localization and expression of SUMO-1 and SUMO-2/3, the two basic proteins in the sumoylation pathway and investigated their roles through overexpression of Senp2 during mouse oocyte maturation. Immunofluorescent staining revealed differential patterns of SUMO-1 and SUMO-2/3 localization: SUMO-1 was localized to the spindle poles in prometaphase I, MI and MII stages, around the separating homologues in anaphase I and telophase I stages of first meiosis, while SUMO-2/3 was mainly concentrated near centromeres during mouse oocyte maturation. Immunoblot analysis uncovered the different expression profiles of SUMO-1 and SUMO-2/3 modified proteins during mouse oocyte maturation. Overexpression of Senp2, a SUMO-specific isopeptidase, caused changes of SUMO-modified proteins and led to defects in MII spindle organization in mature eggs. These results suggest that the SUMO pathway may play an indispensable role during mouse oocyte meiotic maturation.Key words: sumoylation, mouse oocyte maturation, overexpression, Senp2, MII spindle     

Nuf2 is required for chromosome segregation during mouse oocyte meiotic maturation

Nuf2 plays an important role in kinetochore-microtubule attachment and thus is involved in regulation of the spindle assembly checkpoint in mitosis. In this study, we examined the localization and function of Nuf2 during mouse oocyte meiotic maturation. Myc₆-Nuf2 mRNA injection and immunofluorescent staining showed that Nuf2 localized to kinetochores from germinal vesicle breakdown to metaphase I stages, while it disappeared from the kinetochores at the anaphase I stage, but relocated to kinetochores at the MII stage. Overexpression of Nuf2 caused defective spindles, misaligned chromosomes, and activated spindle assembly checkpoint, and thus inhibited chromosome segregation and metaphase-anaphase transition in oocyte meiosis. Conversely, precocious polar body extrusion was observed in the presence of misaligned chromosomes and abnormal spindle formation in Nuf2 knock-down oocytes, causing aneuploidy. Our data suggest that Nuf2 is a critical regulator of meiotic cell cycle progression in mammalian oocytes.     

BubR1 is a spindle assembly checkpoint protein regulating meiotic cell cycle progression of mouse oocyte

BubR1 (Bub1-related kinase or MAD3/Bub1b) is an essential component of the spindle assembly checkpoint (SAC) and plays an important role in kinetochore localization of other spindle checkpoint proteins in mitosis. But its roles in mammalian oocyte meiosis are unclear. In the present study, we examined the expression, localization and function of BubR1 during mouse oocyte meiotic maturation. The expression level of BubR1 increased progressively from germinal vesicle to metaphase II stages. Immunofluorescent analysis showed that BubR1 localized to kinetochores from the germinal vesicle breakdown to the prometaphase I stages, co-localizing with polo-like kinase 1, while it disappeared from the kinetochores at the metaphase I stage. Spindle disruption by nocodazole treatment caused relocation of BubR1 to kinetochores at metaphase I, anaphase I and metaphase II stages; spindle microtubules were disrupted by low temperature treatment in the BubR1-depleted oocytes in meiosis I, suggesting that BubR1 monitors kinetochore-microtubule (K-MT) attachments. Over-expression of exogenous BubR1 arrested oocyte meiosis maturation at the M I stage or earlier; in contrast, dominant-negative BubR1 and BubR1 depletion accelerated meiotic progression. In the BubR1-depleted oocytes, higher percentage of chromosome misalignment was observed and more oocytes overrode the M I stage arrest induced by low concentration of nocodazole. Our data suggest that BubR1 is a spindle assembly checkpoint protein regulating meiotic progression of oocytes.     

Hec1 inhibition alters spindle morphology and chromosome alignment in porcine oocytes

Aneuploidy is caused by incorrect chromosome segregation and can result in cancer or birth defects. The spindle assembly checkpoint (SAC) guarantees proper cell cycle progression. Highly Expressed in Cancer protein 1 (Hec1, also called Ndc80) is the core component of the Ndc80 complex and is involved in regulating both kinetochore-microtubule interactions and the SAC during mitosis in multiple cell types. However, its involvement in pig oocyte meiotic maturation remains uncertain. Thus, we investigated Hec1 expression, localization, and possible functions during porcine oocyte meiosis. Immunofluorescent staining showed that Hec1 was expressed in porcine oocytes and was associated with centromeres at both the metaphase I and metaphase II stages. Disrupting Hec1 function with its inhibitor INH1 resulted in polar body extrusion defects in porcine oocytes. Moreover, inhibiting Hec1 activity also resulted in severe chromosome misalignments and aberrant spindle morphology. Our results showed a unique localization pattern for Hec1 in porcine oocytes and suggested that Hec1 was required for chromosome alignment and spindle organization. Thus, Hec1 might regulate spindle checkpoint activity during mammalian oocyte meiosis.     

Nek9 regulates spindle organization and cell cycle progression during mouse oocyte meiosis and its location in early embryo mitosis

Nek9 (also known as Nercc1), a member of the NIMA (never in mitosis A) family of protein kinases, regulates spindle formation, chromosome alignment and segregation in mitosis. Here, we showed that Nek9 protein was expressed from germinal vesicle (GV) to metaphase II (MII) stages in mouse oocytes with no detectable changes. Confocal microscopy identified that Nek9 was localized to the spindle poles at the metaphase stages and associated with the midbody at anaphase or telophase stage in both meiotic oocytes and the first mitotic embyros. Depletion of Nek9 by specific morpholino injection resulted in severely defective spindles and misaligned chromosomes with significant pro-MI/MI arrest and failure of first polar body (PB1) extrusion. Knockdown of Nek9 also impaired the spindle-pole localization of γ-tubulin and resulted in retention of the spindle assembly checkpoint protein Bub3 at the kinetochores even after 10 h of culture. Live-cell imaging analysis also confirmed that knockdown of Nek9 resulted in oocyte arrest at the pro-MI/MI stage with abnormal spindles, misaligned chromosomes and failed polar body emission. Taken together, our results suggest that Nek9 may act as a MTOC-associated protein regulating microtubule nucleation, spindle organization and, thus, cell cycle progression during mouse oocyte meiotic maturation, fertilization and early embryo cleavage.     

Microinjected centromere [corrected] kinetochore antibodies interfere with chromosome movement in meiotic and mitotic mouse oocytes [published erratum appears in J Cell Biol 1990 Dec;111(6 Pt 1):following 2800]

Kinetochores may perform several functions at mitosis and meiosis including: (a) directing anaphase chromosome separation, (b) regulating prometaphase alignment of the chromosomes at the spindle equator (congression), and/or (c) capturing and stabilizing microtubules. To explore these functions in vivo, autoimmune sera against the centromere/kinetochore complex are microinjected into mouse oocytes during specific phases of first or second meiosis, or first mitosis. Serum E.K. crossreacts with an 80-kD protein in mouse cells and detects the centromere/kinetochore complex in permeabilized cells or when microinjected into living oocytes. Chromosome separation at anaphase is not blocked when these antibodies are microinjected into unfertilized oocytes naturally arrested at second meiotic metaphase, into eggs at first mitotic metaphase, or into immature oocytes at first meiotic metaphase. Microtubule capture and spindle reformation occur normally in microinjected unfertilized oocytes recovering from cold or microtubule disrupting drugs; the chromosomes segregate correctly after parthenogenetic activation. Prometaphase congression is dramatically influenced when antikinetochore/centromere antibodies are introduced during interphase or in prometaphase-stage meiotic or mitotic eggs. At metaphase, these oocytes have unaligned chromosomes scattered throughout the spindle with several remaining at the poles; anaphase is aberrant and, after division, karyomeres are found in the polar body and oocyte or daughter blastomeres. Neither nonimmune sera, diffuse scleroderma sera, nor sham microinjections affect either meiosis or mitosis. These results suggest that antikinetochore/centromere antibodies produced by CREST patients interfere with chromosome congression at prometaphase in vivo.     

Perturbation of survivin expression affects chromosome alignment and spindle checkpoint in mouse oocyte meiotic maturation

Survivin is a member of inhibitors of apoptosis proteins (IAPs), which have multiple regulatory functions in mitosis, but its roles in meiosis remain unknown. Here, we report its expression, localization and functions in mouse oocyte meiosis. Survivin displayed maximal expression levels in GV stages, and then gradually decreased from Pro-MI to MII stages. Immunofluorescent staining showed that survivin was restricted to the germinal vesicle, associated with centromeres from pro-metaphase I to metaphase I stages, distributed at the midzone and midbody of anaphase and telophase spindles, and located to centromeres at metaphase II stages. Depletion of survivin by antibody injection and morpholino injection resulted in severe chromosome misalignment, precocious polar body extrusion, and larger-than-normal polar bodies. Overexpression of survivin resulted in severe chromosome misalignment and prometaphase I or metaphase I arrest in a large proportion of oocytes. Our data suggest that survivin is required for chromosome alignment and that it may regulate spindle checkpoint activity during mouse oocyte meiosis.     

Septin2 is modified by SUMOylation and required for chromosome congression in mouse oocytes

In mitosis, centrosomes nucleate microtubules that capture the sister kinetochores of each chromosome to facilitate chromosome congression. In contrast, during meiosis chromosome congression on the acentrosomal spindle is driven primarily by movement of chromosomes along laterally associated microtubule bundles. Previous studies have indicated that septin2 is required for chromosome congression and cytokinesis in mitosis, we therefore asked whether perturbation of septin2 would impair chromosome congression and cytokinesis in meiosis. We have investigated its expression, localization and function during mouse oocyte meiotic maturation. Septin2 was modified by SUMO-1 and its levels remained constant from GVBD to metaphase II stages. Septin2 was localized along the entire spindle at metaphase and at the midbody in cytokinesis. Disruption of septins function with an inhibitor and siRNA caused failure of the metaphase I /anaphase I transition and chromosome misalignment but inhibition of septins after the metaphase I stage did not affect cytokinesis. BubR1, a core component of the spindle checkpoint, was labeled on misaligned chromosomes and on chromosomes aligned at the metaphase plate in inhibitor-treated oocytes that were arrested in prometaphase I/metaphase I, suggesting activation of the spindle assembly checkpoint. Taken together, our results demonstrate that septin2 plays an important role in chromosome congression and meiotic cell cycle progression but not cytokinesis in mouse oocytes.     

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.     

Nek9 regulates spindle organization and cell cycle progression during mouse oocyte meiosis and its location in early embryo mitosis

Nek9 (also known as Nercc1), a member of the NIMA (never in mitosis A) family of protein kinases, regulates spindle formation, chromosome alignment and segregation in mitosis. Here, we showed that Nek9 protein was expressed from germinal vesicle (GV) to metaphase II (MII) stages in mouse oocytes with no detectable changes. Confocal microscopy identified that Nek9 was localized to the spindle poles at the metaphase stages and associated with the midbody at anaphase or telophase stage in both meiotic oocytes and the first mitotic embyros. Depletion of Nek9 by specific morpholino injection resulted in severely defective spindles and misaligned chromosomes with significant pro-MI/MI arrest and failure of first polar body (PB1) extrusion. Knockdown of Nek9 also impaired the spindle-pole localization of γ-tubulin and resulted in retention of the spindle assembly checkpoint protein Bub3 at the kinetochores even after 10 h of culture. Live-cell imaging analysis also confirmed that knockdown of Nek9 resulted in oocyte arrest at the pro-MI/MI stage with abnormal spindles, misaligned chromosomes and failed polar body emission. Taken together, our results suggest that Nek9 may act as a MTOC-associated protein regulating microtubule nucleation, spindle organization and, thus, cell cycle progression during mouse oocyte meiotic maturation, fertilization and early embryo cleavage.     

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.     

Characterization of Polo-like kinase-1 in rat oocytes and early embryos implies its functional roles in the regulation of meiotic maturation,fertilization, and cleavage

Polo-like kinase 1 (Plk1) is a family of serine/threonine protein kinases that play important regulatory roles during mitotic cell cycle progression. In this study, Plk1 expression, subcellular localization, and possible functions during rat oocyte meiotic maturation, fertilization, and embryonic cleavages were studied by using RT-PCR, Western blot, confocal microscopy, drug-treatments, and antibody microinjection. Both the mRNA and protein of this kinase were detected in rat maturing oocytes and developing embryos. Confocal microscopy revealed that Plk1 distributed abundantly in the nucleus at the germinal vesicle (GV) stage, was associated with spindle poles during the formation of M-phase spindle, and was translocated to the spindle mid-zone at anaphase. In fertilized eggs, Plk1 was strongly stained in the cytoplasm between the apposing male and female pronuclei, from where microtubules radiated. Throughout cytokinesis, Plk1 was localized to the division plane, both during oocyte meiosis and embryonic mitosis. The specific subcellular distribution of Plk1 was distorted after disrupting the M-phase spindle, while additional aggregation dots could be induced in the cytoplasm by taxol, suggesting its intimate association with active microtubule assembly. Plk1 antibody microinjection delayed the meiotic resumption and blocked the emission of polar bodies. In conclusion, Plk1 may be a multifunctional kinase that plays pivotal regulatory roles in microtubule assembly during rat oocyte meiotic maturation, fertilization, and early embryonic mitosis.     

PP2A regulates kinetochore-microtubule attachment during meiosis I in oocyte

Studies using in vitro cultured oocytes have indicated that the protein phosphatase 2A (PP2A), a major serine/threonine protein phosphatase, participates in multiple steps of meiosis. Details of oocyte maturation regulation by PP2A remain unclear and an in vivo model can provide more convincing information. Here, we inactivated PP2A by mutating genes encoding for its catalytic subunits (PP2Acs) in mouse oocytes. We found that eliminating both PP2Acs caused female infertility. Oocytes lacking PP2Acs failed to complete 1^st meiotic division due to chromosome misalignment and abnormal spindle assembly. In mitosis, PP2A counteracts Aurora kinase B/C (AurkB/C) to facilitate correct kinetochore-microtubule (KT-MT) attachment. In meiosis I in oocyte, we found that PP2Ac deficiency destabilized KT-MT attachments. Chemical inhibition of AurkB/C in PP2Ac-null oocytes partly restored the formation of lateral/merotelic KT-MT attachments but not correct KT-MT attachments. Taken together, our findings demonstrate that PP2Acs are essential for chromosome alignments and regulate the formation of correct KT-MT attachments in meiosis I in oocytes.     

A comparative study of the distribution of fluorescently labeled calmodulin and tubulin in the meiotic apparatus of the mouse oocyte

The localizations of tubulin and calmodulin were investigated in the mouse oocyte during the second meiosis by fluorescently labeling and microinjecting these proteins prepared from porcine brain tissue. When injected, both tubulin and calmodulin were quickly incorporated into the preformed meiotic apparatus of the oocyte at metaphase. The localization of labeled tubulin was coincident with that of birefringence. However, the localization of labeled calmodulin was somewhat different: the fluorescence of calmodulin was intense in the polar regions of the spindle. After the chromosomes began to move, followed by parthenogenetic activation upon microinjection of a calcium buffer, these two fluorescent proteins, localized in the meiotic apparatus, moved to the interzonal region of the spindle during anaphase. At late anaphase and throughout telophase, calmodulin was excluded from the mid-bodylike structures in the interzonal region, whereas tubulin did accumulate in these structures.     

The Microtubule-Associated Protein ASPM Regulates Spindle Assembly and Meiotic Progression in Mouse Oocytes

The microtubule-associated protein ASPM (abnormal spindle-like microcephaly-associated) plays an important role in spindle organization and cell division in mitosis and meiosis in lower animals, but its function in mouse oocyte meiosis has not been investigated. In this study, we characterized the localization and expression dynamics of ASPM during mouse oocyte meiotic maturation and analyzed the effects of the downregulation of ASPM expression on meiotic spindle assembly and meiotic progression. Immunofluorescence analysis showed that ASPM localized to the entire spindle at metaphase I (MI) and metaphase II (MII), colocalizing with the spindle microtubule protein acetylated tubulin (Ac-tubulin). In taxol-treated oocytes, ASPM colocalized with Ac-tubulin on the excessively polymerized microtubule fibers of enlarged spindles and the numerous asters in the cytoplasm. Nocodazole treatment induced the gradual disassembly of microtubule fibers, during which ASPM remained colocalized with the dynamic Ac-tubulin. The downregulation of ASPM expression by a gene-specific morpholino resulted in an abnormal meiotic spindle and inhibited meiotic progression; most of the treated oocytes were blocked in the MI stage with elongated meiotic spindles. Furthermore, coimmunoprecipitation combined with mass spectrometry and western blot analysis revealed that ASPM interacted with calmodulin in MI oocytes and that these proteins colocalized at the spindle. Our results provide strong evidence that ASPM plays a critical role in meiotic spindle assembly and meiotic progression in mouse oocytes.     

Stable kinetochore–microtubule interactions depend on the Ska complex and its new component Ska3/C13Orf3

Ska1 and Ska2 form a complex at the kinetochore–microtubule (KT–MT) interface and are required for timely progression from metaphase to anaphase. Here, we use mass spectrometry to search for additional components of the Ska complex. We identify C13Orf3 (now termed Ska3) as a novel member of this complex and map the interaction domains among the three known components. Ska3 displays similar characteristics as Ska1 and Ska2: it localizes to the spindle and KT throughout mitosis and its depletion markedly delays anaphase transition. Interestingly, a more complete removal of the Ska complex by concomitant depletion of Ska1 and Ska3 results in a chromosome congression failure followed by cell death. This severe phenotype reflects a destabilization of KT–MT interactions, as demonstrated by reduced cold stability of KT fibres. Yet, the depletion of the Ska complex only marginally impairs KT localization of the KMN network responsible for MT attachment. We propose that the Ska complex functionally complements the KMN, providing an additional layer of stability to KT–MT attachment and possibly signalling completion of attachment to the spindle checkpoint.     

Bub3 Is a Spindle Assembly Checkpoint Protein Regulating Chromosome Segregation during Mouse Oocyte Meiosis

In mitosis, the spindle assembly checkpoint (SAC) prevents anaphase onset until all chromosomes have been attached to the spindle microtubules and aligned correctly at the equatorial metaphase plate. The major checkpoint proteins in mitosis consist of mitotic arrest-deficient (Mad)1–3, budding uninhibited by benzimidazole (Bub)1, Bub3, and monopolar spindle 1(Mps1). During meiosis, for the formation of a haploid gamete, two consecutive rounds of chromosome segregation occur with only one round of DNA replication. To pull homologous chromosomes to opposite spindle poles during meiosis I, both sister kinetochores of a homologue must face toward the same pole which is very different from mitosis and meiosis II. As a core member of checkpoint proteins, the individual role of Bub3 in mammalian oocyte meiosis is unclear. In this study, using overexpression and RNA interference (RNAi) approaches, we analyzed the role of Bub3 in mouse oocyte meiosis. Our data showed that overexpressed Bub3 inhibited meiotic metaphase-anaphase transition by preventing homologous chromosome and sister chromatid segregations in meiosis I and II, respectively. Misaligned chromosomes, abnormal polar body and double polar bodies were observed in Bub3 knock-down oocytes, causing aneuploidy. Furthermore, through cold treatment combined with Bub3 overexpression, we found that overexpressed Bub3 affected the attachments of microtubules and kinetochores during metaphase-anaphase transition. We propose that as a member of SAC, Bub3 is required for regulation of both meiosis I and II, and is potentially involved in kinetochore-microtubule attachment in mammalian oocytes.