The Functional Analysis Of Asymmetric Factors During Mouse Oocyte Meiosis

ABSTRACTⅠ:NUMB REGULATES MEIOTICS PINDLE ORGANIZATION IN MOUSE OOCYTESBackground and Purpose:Asymmetric cell division refers to a mother cell dividing into two daughter cells with unequal sizes and different developmental potentials. It is one of the main developmental mechanisms that generate cell diversity in multicellular organisms and is usually achieved through asymmetric distribution of cell fate determinants, which is often accompanied by unequal cytokinesis. numb was originally discovered as a cell fate determinant in Drosophila. In the central nervous system of Drosophila, numb protein accumulates in cortical crescents at the basal neuroblast side, and as division ensues it is preferentially segregated into one daughter cell. The two sibling cells have different functions. The mouse Numb is the functional homologue of Drosophila numb. However, the expression of Numb is widespread in mouse embryos, and it is also expressed in most adult tissues, so it is likely that function of Numb is not limited to neurogenesis. In recent years, a large number of studies about Numb have been made in diverse fields, spanning asymmetric cell division and related areas in developmental neurobiology, to distant fields such as human cancer biology and neurodegenerative disease. Although, much has been learned during the past decade about the functions of Numb in mitosis, little is known about Numb in meiosis. The meiotic divisions in mouse oocytes is typically asymmetric, giving rise to two daughter cells of different size:a huge oocyte and a tiny polar body. This kind of division is essential for preserving most of the maternal resources to support further embryonic development. The present study was designed to investigate the expression, subcellular localisation and functional roles of Numb during mouse oocyte meiotic maturation.Materials and Methods:1. Total RNA and protein were collected from GV oocytes and MⅡeggs. Then, we detected the level of Numb expression by RT-PCR and Western-blot.2. GV/proMI/MI/AI/TI/MII oocytes were recovered. We used Immunofluorescent staining to demonstrate the localization of Numb at different stages of mouse oocytes.3. We disclosed the relationship between the distribution of Numb and cytoskeleton by adding nocodazole to the maturation medium at different time.4. Numb siRNA or control negative siRNA was microinjected into the cytoplasm of GV stage oocytes to reveal the function of Numb on mouse oocyte meiosis.Results:1. Using real-time polymerase chain reaction and western blotting, we found that the expression of Numb increased from the germinal vesicle (GV) to MⅡstages. 2. Immunofluorescent staining revealed that Numb was mainly concentrated in the GV before meiosis resumption. After GVBD Numb concentrated as several foci surround the clustered chromosomes. At prometaphaseⅠ, chromosomes began to arrange themselves, several Numb aggregates formed a broad plate at the poles of spindle. By metaphase I (MI), chromosomes aligned at the equatorial plate regularly, and Numb concentrated congregated as one dot at each pole of the first meiotic spindle. When oocytes progressed to anaphaseⅠ, homologous chromosomes began to separate and Numb was present as several discrete foci at the poles of the spindle. At telophaseⅠ, Numb was accumulated in small dots on the chromosomes or in the vicinity of the chromosomes. After the formation of the second meiotic spindle at metaphaseⅡ(MⅡ) stage, Numb again appeared at the spindle poles at the same pattern of MI stage (as one dot). Besides the staining at the spindle poles, Numb was also labeled as discrete dots distributed throughout the cytoplasm.3. After GV occytes being treated with nocodazole for 12 h, spindle was not detectable and chromosome formed a number of small aggregates scattering in the cytoplasm. Numb signal no longer appeared around the chromosome or at the spindle poles but diffused in the cytoplasm However, Numb appeared at the spindle poles again once the spindles had formed when nocodazole-treated oocytes were washed and cultured for spindle recovery.4. The targeting efficiency of RNAi was evaluated by quantitative real-time PCR. The mRNA level of Numb was significantly decreased to 32% of the control level, in which negative control siRNA was injected. The MⅡdevelopment rate and GVBD rate of oocytes were not evidently inhibited by microinjection with Numb siRNA compared with the control group. The Numb siRNA injected oocytes exhibited obvious abnormal spindles, for example, spindles lacking well-organized poles and the microtubule minus ends are splayed outward; the shortened spindles with broad poles; spindles disorganized and microtubules irregularly surround the chromosomes; disorganized spindle structures with reduced microtubule density; the double spindles. In addition, Numb-depleted oocytes displayed a severe defect in chromosome alignment, The rate of spindle aberrations and chromosomes displacement in the Numb siRNA injected group were significantly higher than that in control group.Conclusions:Our results suggest that Numb is critical for spindle organization during mouse oocytes meiosis. The present study provides evidence of a new function for Numb in addition to its action as a cell fate-determining factor.ABSTRACTⅡ:THE OVER-EXPRESSION OF DLG1 PERTURBS MEIOTIC MATURATION IN MOUSE OOCYTESBackground and Purpose:Asymmetric division is one of the primary mechanisms for regulating cell development in multicellular organisms, in which a cell produces two cell types that differ in size, shape and fate. In recent years, the studies of several model systems (for example, drosophila, threadworm) have revealed a general feacture of asymmetric cell division:the polarity cue is a prerequisite for asymmetric cell division. Cell polarity is defined as asymmetry in cell shape, protein distributions and cell functions. Analysis of evolutionarily diverse cell types reveals that cell-surface landmarks adapt core pathways for cytoskeleton assembly and protein transport to generate cell polarity. A polarity cue is first set up in the mother cell, the cell fate determinants (the rmRNAs or proteins) are then localized with respect to this cue, this localization requires actin filaments and certain myosin motors, and the axis of division is lined up with respect to the same cue so that the localized determinants are partitioned preferentially in to one of the two progeny cells. Mouse oocyte meiotic division is typically asymmetric. The organization and eccentric positioning of the spindle before cytokinesis is a prerequisite for asymmetric division during meiotic maturation. Immature mouse oocytes (oocytes at the germinal vesicle stage) exhibit no apparent polarity. After meiosis resumption, microtubules reorganized into a bipolar metaphase I spindle. The spindle initially forms in the center of the oocyte and then migrates to the nearest part of the cortex along the spindle axis. During spindle migration, an area enriched in actin microfilaments and devoid of microvilli begins to form in the cortex overlying the spindle, and prominent cortical polarity develops. After the spindle reaches the cortex, the polar body is extruded from this site and the first asymmetric division is completed. The Dlgl (Discs Large Homolog 1) gene was originally identified based on its ability to act as a tumor suppressor in Drosophila. Its product, the Dlgl protein, is the prototypic member of a growing family of proteins termed membrane-associated guanylate kinase (MAGUKs). Dlgl is an evolutionarily conserved polarity protein that is involved in the establishment of epithelial polarity from Drosophila to mammals. It has been detected in cell-cell junctions. Loss of Drosophila Dlgl results in a loss of cell polarity and some cell overproliferation Although, much has been learned during the past decade about the functions of Dlgl in mitosis, little is known about Dlgl in meiosis. We have reported that mouse oocytes meiotic maturation induced the polarization of Dlgl and this polarized action is highly related with cell cytoskeleton. Therefore, we used the methods of RNAi and over-expression to further investigate the function of Dlgl during meiotic maturation. This part is about the method of vector construction and the result of over-expresiion of Dlgl during mouse oocytes maturation.Materials and Methods:1. Total RNA was collected from mouse ovary. Dlgl was synthesis by RT-PCR.2. Insert the Dlgl DNA fragment into PMD-18T DNA. Transform DH5a competent cells with the generated recombinant. The DNA of the cells was collected and was sequenced.3. Human globin 3'UTR/5'UTR were synthesized.4. The above-mentioned components and IRES-EGFP were linked to pcglobin eukaryotic expression vector.5. For in vitro transcription, the vectors were linearized with restrictive endonuclease.6. After in vitro transcription, the mRNAs were purified.7. mRNAs were microinjected to the cytoplasm of GV mouse oocytes. EGFP mRNA transcribed from an empty pcGlobin-EGFP vector was microinjected for control. The microinjected oocytes were cultured and examined.Results:After in vitro transcription, the mRNAs were purified. The concentration is about 1.5μg/μl. Oocytes were microinjected with 7 p1 of the RNA solution. The microinjected oocytes were cultured in medium and a distinct EGFP signal was detected. It means that the vector was functional and the mRNA was correctly expressed in mouse oocytes. Then the oocytes were washed and transferred to IBMX-free medium to allow them to resume meiosis. The rate of GVBD and Pbl emission of Dlgl mRNA injected were significantly lower than the negative control group.Conclusions:our results suggest that the over-expression of dlgl perturbs meiotic maturation. Dlgl controls meiosis progression in mouse oocytes.