Multinucleated cells resulted from mitosis defect have been noted in pathophysiological states of the cells such as inflammation, senescence and cancer. Since oxidative stress has been known to correlate with these pathophysiological conditions, we tested the effect of H2O2 on the cell cycle progression and formation of multinucleated cells. H2O2 induced a significant delay in cell cycle progression in Chang liver cells. Interestingly, H2O2 actively induced hyperamplification of centrosomes (> or =3) and multipolar spindle formation during mitosis and subsequently increased the generation of multinucleated cells. A significant increase of the phospho-ERK level was observed upon H2O2 treatment but PD98059, an MEK1/2 inhibitor, didn't reduce the frequency of cells with hyperamplified centrosomes. On the other hand, treatment of either H2O2 or adriamycin increased intracellular ROS levels and multinucleated cells, which were significantly suppressed by antioxidants, N-acetylcysteine and PDTC. Thus, our results suggest that oxidative stress can trigger centrosome hyperamplification and multinucleated cell formation, which may promote pathophysiological progression.
Cell Line ; Cell Nucleus/*drug effects/*metabolism ; Centrosome/*drug effects/*metabolism ; Gene Amplification ; Humans ; Hydrogen Peroxide/*pharmacology ; MAP Kinase Signaling System ; Mitotic Spindle Apparatus/drug effects ; Phenotype ; Reactive Oxygen Species/metabolism ; Research Support, Non-U.S. Gov't

OBJECTIVE: To investigate the extent of meiotic spindle damages in cryopreserved-thawed mouse mature oocytes. METHODS: After slow freezing and ultra-rapid thawing using 1.5M dimethylsulfoxide(DMSO), mouse mature oocytes were stained by anti-alpha tubulin monoclonal antibody. The meiotic spindle and chromosomes configuration were assessed using confocal microscope. The influence of time to post-hCG oocytes retrieval (i.e. 12 hrs vs 17 hrs) was also evaluated. RESULTS: The normal meiotic spindles were observed in 89.8% of post-hCG 12 hrs group, and 80.1% of post-hCG 17 hrs group, and these were significantly lower than that of each unfreezed control. Post-hCG 12 hrs group showed a significantly higher incidence of normal meiotic spindles, compared with post-hCG 17 hrs group. CONCLUSION: The extent of meiotic spindle damages was significantly increased after cryopreservation in mouse mature oocytes. We proposed that 12 hrs interval of post-hCG oocytes retrieval may be more beneficial.
Animals ; Cryopreservation ; Dimethyl Sulfoxide ; Freezing ; Incidence ; Mice* ; Oocytes* ; Spindle Apparatus* ; Tubulin

OBJECTIVETo investigate the influence of two different vitrification cryopreservation methods on the spindles of mouse M II oocytes.

METHODSThree groups were included in the experiment, Group A, Group B and the control ( fresh oocytes). Mouse oocytes were vitrified by using cryoloop, with ethylene glycol( EG) in Group A and with EG + dimethyl sulphoxide ( DMSO) in Group B as cryoprotectants, and then the oocytes were placed directly into liquid nitrogen. Three hours after the frozen oocytes were thawed they were fixed, and the microtubule and chromosome were stained by indirect immunofluorescent method.

RESULTSThe survival rates of the oocytes after treated by the two vitrification cryopreservation methods had no difference ( 80. 3% vs 87. 5% , P > 0. 05) . The rate of the intact spindles in Group A was much lower than that of the control and Group B ( 15. 2% vs 78.7% , 15. 2% vs 77. 5% , P < 0. 05). But there was no difference between the latter two groups (78. 7% vs 77. 5% , P >0. 05). The oocytes with normal chromosome in Group A were much less than in the control and Group B (17.4% vs 76. 6% , 17. 4% vs 72. 5% , P <0. 05) , with no difference between the latter two groups(76. 6% vs 72. 5% , P >0. 05) ; The oocytes with abnormal chromosome were more in Group A than in the control and Group B (82. 6% vs 19. 1% , 82. 6% vs 27. 5% , P <0. 05) , with no difference between the latter two groups (19.1% vs 27.5% , P >0.05).

CONCLUSIONThe changed vitrification cryopreservation method helps conserve the intact spindle configuration of mouse oocytes.

Animals ; Cryopreservation ; methods ; Cryoprotective Agents ; Female ; Fertilization in Vitro ; Mice ; Mice, Inbred ICR ; Oocytes ; cytology ; Spindle Apparatus

In association with microtubules, a variety of kinesins play important roles in cellular functions such as intracellular transport of organelles or vesicles, signal transduction, and cell division. In a previous study we revealed that human kinesin superfamily protein member 4 (KIF4) is a chromokinesin that binds to chromosomes. Since localization of several kinds of kinesin at midzone called central spindle, or midbody that connects two daughter cells, or both, suggests their implication in cell division, we investigated KIF4 localization of during mitosis and cytokinesis in Hela cells. In addition to association with segregating chromosomes through entire mitosis, it also localized to the midzone and to midbody at ana/telophase through cytokinesis. Especially in cells at cytokinesis, KIF4 appeared as a doublet facing each other at the apical ends of two daughter cells. Three- dimensional analysis of architectural relationship between microtubule bundles and KIF4 indicated that KIF4 forms a ring structure wrapping around the microtubule bundles. These results suggest that KIF4 is involved in cytokinesis, although direct evidence was not provided in this study.
Animals ; Cell Division/*physiology ; Hela Cells ; Humans ; Immunohistochemistry ; Kinesin/*metabolism ; Mitotic Spindle Apparatus/*metabolism ; Research Support, Non-U.S. Gov't

In assisted reproductive techniques, the operator attempts to select morphologically best embryos to predict embryo viability. Development of polarized light microscope, which evaluates the oocytes' spindles according to birefringence of living cells, had been helpful in oocyte selection. The aim of this study is evaluating the relationship between meiotic spindles visualization and intracytoplasmic sperm injection (ICSI) outcomes in human oocytes. In this study, 264 oocytes from 24 patients with an average age of 30.5±7.5 years with infertility duration of 1 to 10 years were collected. The oocytes were randomly allocated to the control injection group (n=126) and the oocyte imaging group (spindle-aligned group) (n=138). In the spindle-aligned group, the meiotic spindle was identified by means of polarized light microscope to align the spindle at 6 or 12 o'clock. Then the spindle-aligned group was divided into three sub-groups based on spindle morphology: fine, average, and (poor). After ICSI, embryos were checked every 24 hours and scored; 72 hours later, high-grade embryos were transferred intravaginally to uterus. This study showed that the fertilization rate in the spindle-aligned group was higher than the control group (P<0.05). After cleavage, a positive correlation was observed between spindle morphology and embryo morphology. Among the sub-groups of spindle-aligned group, the embryos' morphology of the fine group was better than the other subgroups and embryos of the poor group had lower quality and more fragmentation. The results revealed that the selection of oocytes based on meiotic spindle imaging can significantly improve the rate of fertilization and embryo cleavage and certainly increase the rate of implantation.
Birefringence ; Embryonic Structures ; Fertilization ; Humans* ; Infertility ; Microscopy, Polarization ; Oocytes* ; Reproductive Techniques, Assisted ; Sperm Injections, Intracytoplasmic* ; Spindle Apparatus* ; Uterus

The Spindle-view, a specialized instrument for observing spindle image, was applied to observe the meiotic spindles of vitro matured porcine oocytes at 36, 42, 44, 48h, and enucleation from porcine, comparing to the previously methods (McGrath-Solter's method and two-step-squeezing method) in the enucleated. The results showed that: (1) there was no noticeable differences at vicinity of spindle images and 1st polar body among in vitro matured porcine oocytes at 40-48 h under the instrument; (2) Spindle-view is suitable for the observation of meiotic spindles of matured oocytes and enucleation from porcine; the modified Spindle-view method for enucleation is significantly better than McGrath-Solter' s method and two-step-squeezing method in the enucleated rates (95.5%, 42.1%, 74.2%, P < 0.0l) of absolutely removing nuclei matter; (3) the spindle images could be used to monitor the oocyte qualities.
Animals ; Cell Nucleus ; ultrastructure ; Cells, Cultured ; Cytological Techniques ; Female ; Nuclear Transfer Techniques ; Oocytes ; cytology ; Spindle Apparatus ; ultrastructure ; Swine

Tubulin is the major protein of microtubule. alpha- and beta- tubulins form heterodimers, while gamma-tubulin regulates microtubule organization. The present study aimed to observe the dynamic changes of gamma-tubulin in preimplantation development of parthenogenetic mouse embryos. Immunofluorescence and laser confocal microscopy were used to detect the location of gamma-tubulin in preimplantation parthenogenetic embryos activated by SrCl2. The oocytes were collected at 13-14 h after hCG injection, and then activated with 10 mmol/L SrCl2 in Ca(2+)-free CZB medium with 5 mmol/L cytochalasin B (CB), fixed at 1 h intervals until 6 h after activation. The results showed that spindle was paralleled with the cell membrane all the time, when the meiosis of MII mouse oocytes resumed. The rotation of spindle was inhibited, but karyokinesis was not influenced. At 0 h after activation, i.e. at metaphase, gamma-tubulin was distributed mainly on the two poles of spindle. At 1 h after activation, i.e. at anaphase, following the separation of chromosomes, gamma-tubulin was transformed from dense to disperse. At 2 h after activation, gamma-tubulin was localized between the segregated sister chromatids at telophase. However, at 3-6 h after activation, gamma-tubulin concentrated around the two female pronuclei during their formation and juxtaposition. Moreover, another group of MII oocytes were activated for 6 h and cultured in droplets of KSOM medium under mineral oil in 5% CO2 in air at 37 degrees C to permit parthenogenetic development. The embryos were collected and fixed at 3 h, 14 h, 16 h, and 18 h of culture. At 3 h after culture, i.e. at mitotic interphase, it was shown that amorphous gamma-tubulin distributed around the nuclei of early parthenogenetic embryos. At 24 h after culture, i.e. at prometaphase, gamma-tubulin migrated along the spindle microtubule to the two poles. Our results showed that gamma-tubulin had similar location patterns at metaphase, anaphase and telophase in meiosis and mitosis. It was concluded that gamma-tubulin assembly in parthenogenetically activated oocytes facilitated the formation of negative pole cap and the stabilization of microtubule, thus promoting the spindle formation at meiosis and mitosis. The relocation of gamma-tubulin at anaphase and telophase might be induced by the event of segregation of homologous chromosome being pulled away by the spindle. gamma-tubulin might contribute to the migration and juxtaposition of the two female pronuclei as well.
Animals ; Embryo, Mammalian ; Embryonic Development ; Female ; Meiosis ; Mice ; Mitosis ; Oocytes ; cytology ; Parthenogenesis ; Spindle Apparatus ; physiology ; Tubulin ; physiology

BACKGROUND: Mutations in centrosomal protein genes have been identified in a number of genetic diseases in brain development, including microcephaly. Centrosomal P4.1-associated protein (CPAP) is one of the causal genes implicated in primary microcephaly. We previously proposed that CPAP is essential for mother centriole maturation during mitosis. METHODS: We immunostained CPAP-depleted cells using the ninein antibody, which selectively detects subdistal appendages in mature mother centrioles. RESULTS: Ninein signals were significantly impaired in CPAP-depleted cells. CONCLUSION: The results suggest that CPAP is required for mother centriole maturation in mammalian cells. The selective absence of centriolar appendages in young mother centrioles may be responsible for asymmetric spindle pole formation in CPAP-depleted cells.
Brain ; Cell Cycle ; Centrioles* ; Centrosome ; Humans ; Microcephaly ; Mitosis ; Mothers* ; Spindle Poles

RSK2, also known as RPS6KA3 (ribosomal protein S6 kinase, 90 kDa, polypeptide 3), is a downstream kinase of the mitogen-activated protein kinase (MAPK) pathway, which is important in regulating survival, transcription, growth and proliferation. However, its biological role in mitotic progression is not well understood. In this study, we examined the potential involvement of RSK2 in the regulation of mitotic progression. Interestingly, depletion of RSK2, but not RSK1, caused the accumulation of mitotic cells. Time-lapse analysis revealed that mitotic duration, particularly the duration for metaphase-to-anaphase transition was prolonged in RSK2-depleted cells, suggesting activation of spindle assembly checkpoint (SAC). Indeed, more BubR1 (Bub1-related kinase) was present on metaphase plate kinetochores in RSK2-depleted cells, and depletion of BubR1 abolished the mitotic accumulation caused by RSK2 depletion, confirming BubR1-dependent SAC activation. Along with the shortening of inter-kinetochore distance, these data suggested that weakening of the tension across sister kinetochores by RSK2 depletion led to the activation of SAC. To test this, we analyzed the RSK2 effects on the stability of kinetochore–microtubule interactions, and found that RSK2-depleted cells formed less kinetochore–microtubule fibers. Moreover, RSK2 depletion resulted in the decrease of basal level of microtubule as well as an irregular distribution of mitotic spindles, which might lead to observed several mitotic progression defects such as increase in unaligned chromosomes, defects in chromosome congression and a decrease in pole-to-pole distance in these cells. Taken together, our data reveal that RSK2 affects mitotic progression by regulating the distribution, basal level and the stability of mitotic spindles.
Humans ; Kinetochores ; M Phase Cell Cycle Checkpoints ; Metaphase ; Microtubules ; Phosphotransferases ; Protein Kinases ; Ribosomal Protein S6 Kinases ; Ribosomal Protein S6 Kinases, 90-kDa* ; Siblings ; Spindle Apparatus*

Stem cells use asymmetric and symmetric cell division to generate progeny. Symmetric cell division is defined as the generation of daughter cells that are destined to acquire the same fate. Stem cells divide asymmetrically to generate one daughter with a stem-cell fate and one daughter with different fate. Disruption of the machinery that regulates asymmetric division may be a reason for the generation of cancer. The asymmetric mechanism is maintained by cell polarity factors, cell fate determinants, and the spindle apparatus. The mutation or dysregulation of these factors may change stem cells from asymmetric to symmetric cell division, then leading to tumorigenesis. Therefore, further study is needed on the mechanisms of stem cell control between asymmetric and symmetric cell division, as well as the relationships among stem cells, cancer stem cells, and tumor cells. It may bring us a new approach for the resistance, recurrence, and metastasis of tumors.
Animals ; Cell Division ; physiology ; Cell Polarity ; Cell Transformation, Neoplastic ; Drosophila ; cytology ; Humans ; Neoplasms ; pathology ; Neoplastic Stem Cells ; pathology ; Neurons ; cytology ; Spindle Apparatus ; metabolism ; Tumor Suppressor Proteins ; metabolism