[Show abstract][Hide abstract]ABSTRACT: 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 G 2/M transition while Chk1 overexpression inhibited the G 2/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 G 2/M checkpoint regulation in meiotic oocytes. Moreover, Chk1 overexpression affects meiotic spindle assembly checkpoint regulation and thus chromosome segregation.
Article · May 2012 · Cell cycle (Georgetown, Tex.)
[Show abstract][Hide abstract]ABSTRACT: Synaptotagmin1, a calcium sensor for exocytosis, forms the 7S complex, or so-called SNARE protein complex, together with SNAP -25, syntaxin and synaptobrevin to mediate docking and fusion of synaptic vesicles to the plasma membrane of the nerve terminal. Here, we identified the unique localization, expression and function of Syt1 during mouse oocyte meiotic maturation by using confocal microscopy, western blotting, Morpholino-based knockdown and time-lapse live cell imaging. We showed that Syt1 expression was gradually increased during oocyte maturation. Syt1 was localized at the oocyte cortex from GV to MII stages and at the spindle poles in MI and MII phases, with one third of a signal-free zone at the oocyte cortex, where the chromosomes are located, which is similar to the distribution pattern of CGs from the pro-MI to MII stages. Knockdown of Syt1 resulted in pro-MI/MI arrest and PB1 extrusion decrease, with severely disrupted spindles and misaligned chromosomes. Knockdown of Syt1 also caused abnormal localization of γ-tubulin, which became redistributed into the cytoplasm. Chromosome spreading showed failure of homologous chromosome segregation. The spindle assembly checkpoint protein Bub3 was detected at the kinetochores even after 10 h of oocyte culture. Live cell imaging analysis revealed that knockdown of Syt1 resulted in abnormal spindles with various morphologies and chromosomes arrested at the pro-MI/MI stage. Defective spindles failed to support chromosome alignment along microtubules, which led to repetitive unsuccessful metaphase-anaphase transitions and failure of PB1 extrusion after extended culture. Taken together, we suggest that Syt1 may act as a MTOC-associated protein to play important roles in mouse oocyte spindle organization/stability, and that it is indispensable for the metaphase-anaphase transition to promote mouse oocyte meiotic maturation.
Article · Feb 2012 · Cell cycle (Georgetown, Tex.)
[Show abstract][Hide abstract]ABSTRACT: Objective To explore the effects and mechanism of gene F10 over-expression on the tumorigenicity of A549 cells in nude mice. Methods Eighteen SPF nude mice (4-5weeks of age) were randomly equally divided into the three groups: A549-WT (vaccination with wild-type strain A549), Mock-A549 (vaccination with controlled cells Mock-A549 transfected by blank vectors) and F10+A549 (vaccination with F10+A549 cells which overexpressed F10 gene) according to their vaccination and then revaccinated into the subcutaneous tissue of the back with cell suspension (0.1ml with 5×106 cells in each mouse). After vaccination, the mice were observed and weighed once every 3-4 days, then the tumor formation time was recorded, the tumor growth curve drawn and tumor formation rate calculated. The mice were sacrificed and the subcutaneous tumor tissues were paraffin embedded and sectioned, and stained with HE for histopathological examinati on 5 weeks after the vaccination. Meanwhile, the expressions of Caspase-3, Bax and Bcl-2 in the tumor tissue were detected by immunohistochemistry. Results The tumor formation time was significantly different (F=13.523, P=0.000) between the groups of A549-WT (12.0±1.4d), Mock-A549 (11.7±1.0d) and F10+A549 (8.5±1.4d), and it was longer in the groups A549-WT and Mock-A549 than that in F10+A549 group (P < 0.05), while there was no significant difference between A549-WT and Mock-A549 groups (P=0.660). General observation of subcutaneous tumor tissues revealed that the tumor volume was increased more significantly in the F10+A549 group compared with that of the groups A549-WT and Mock-A549. The growth rate of subcutaneous tumor in the F10+A549 group was increased more significantly compared with groups A549-WT and Mock-A549 (P < 0.05), while there was no significant difference between A549-WT and Mock-A549 (P > 0.05). Pathological examination revealed that the tumor tissues in the groups A549-WT and Mock-A549 harbored more necrotic cells, with structure of homogeneously red stained and amorphous material. In F10+A549 group, the number of necrotic cell was less, and cell proliferation was obvious in the edge of tumor tissues, in which, microvessels were found. In groups A549-WT and Mock-A549, the expression of Bcl-2 was rarely observed, while it was obvious in F10+A549 group, with positive cells diffusely distributed. In groups A549-WT and Mock-A549, there was strong the expression of both Bax and Caspase-3, especially in the A549-WT group. But the expressions of Bax and Caspase-3 were weak in F10+A549 group. Conclusion F10 gene may down-regulate expressions of Caspase-3 and Bax, and up-regulate expression of Bcl-2, which further enhance the tumorigenicity of lung cancer cell line A549 in nude mice.
Article · Jan 2012 · Medical Journal of Chinese People's Liberation Army