【作者】 吕鸿；

【导师】 陈子江；

【作者基本信息】 山东大学 ， 妇产科学， 2010， 博士

【摘要】 第一部分：Numb调节小鼠卵母细胞减数分裂过程中纺锤体的组装研究背景和目的：不对称分裂是指一个细胞分裂成两个从细胞形态到发育潜能都不相同的子代细胞,这是多细胞器官产生细胞多样性的一种基本方式。这种不对称分裂通常是通过细胞命运决定蛋白的不对称分配来实现的,往往伴有细胞质的不对称分裂。Numb作为细胞命运决定蛋白最早在果蝇中被发现。在果蝇中枢神经系统神经母细胞分裂过程中,Numb呈新月型不对称分布于神经母细胞基底侧,在随后的分裂过程中不对称分配,一个子代细胞优先获得,从而形成不同功能的子代细胞。Numb在进化过程中功能保守。Numb不但在小鼠胚胎中广泛表达,在成体细胞中也可见Numb分布,说明Numb的作用不仅仅限于神经系统的发生。近些年关于Numb的研究日益增多,对Numb的了解也越来越深入,涵盖了从不对称分裂及其相关的发育神经生物学到人类癌症生物学及神经变性疾病等多个领域。但是这些研究局限于有丝分裂中,Numb在减数分裂中的作用尚未见报道。小鼠卵母细胞成熟是一种典型的不对称分裂,通过两次减数分裂产生较大的卵母细胞和较小的极体,这种分裂形式保证了卵母细胞获得绝大部分的遗传物质,以维持后续的胚胎发育进程,极体逐渐退化。本实验中我们探讨了Numb在小鼠卵细胞发育过程中的表达、亚细胞分布及其在减数分裂过程中的作用。材料和方法：1.提取GV期、MII期卵细胞的蛋白质,通过western blot方法检测Numb基因在GV期、MII期卵母细胞中的表达水平。2.收集GV期、proMI期、MI期、AI期、TI期、MII期卵母细胞,固定,透化,免疫荧光染色,通过免疫荧光共聚焦显微镜观察Numb蛋白在小鼠卵母细胞发育过程中的亚细胞分布变化。3.在小鼠卵母细胞发育过程的不同时期,通过在培养基中添加微管解聚药物nocodazole,处理细胞,观察Numb的分布变化,研究Numb与微管之间的关系。4.通过Numb siRNA以及control siRNA显微注射GV期卵母细胞,下调Numb基因表达水平,干扰Numb基因的功能,研究其对小鼠卵母细胞减数分裂的影响。结果：1. western blot方法证实Numb在GV期、MII期卵母细胞中均有表达,且随着卵母细胞发育Numb在蛋白水平上表达增加。2.免疫荧光染色,共聚焦显微镜观察显示在GV期卵母细胞中,Numb局限于生发泡中;生发泡破裂后Numb形成散在的点状,围绕在浓缩的染色质周围；prometaphase I期,染色体开始排列,Numb聚集呈板状位于纺锤体两端；至MI期,染色体整齐排列于纺锤体中板,Numb在纺锤体两端聚集成点状；AI期,染色体开始分离,Numb形成多个点状位于纺锤体两端；TI期,染色体到达纺锤体两端,Numb位于染色体上或染色体周围；至MII期,染色体再次整齐排列于赤道板,Numb重新聚集成点状位于纺锤体两端,同时可见Numb呈多个散在点状分布于细胞质中。3.GV期卵细胞在含有nocodazole的培养液中培养12小时,微管被破坏,纺锤体不再形成,染色体聚集成团,散在分布,Numb的特异性分布消失,弥散于细胞质中。Nocodazole处理过的卵细胞在不含药物的培养液中继续培养1.5小时,围绕散在的染色体团,细胞质中形成多个纺锤体,Numb聚集到每个纺锤体的两极。4. Numb基因干扰后,通过real-time PCR验证干扰效率,处理组中Numb mRNA表达水平为对照组的32%。统计两组中GVBD率、MII形成率,其差异无统计学意义。免疫荧光染色观察发现Numb siRNA注射组纺锤体形态出现明显异常,如：纺锤体两极形态异常,微管呈放射状向外分布；纺锤体变短,两极变宽；纺锤体染色变弱；甚至出现双纺锤体。此外,染色体排列散乱,对齐异常。与对照组相比,Numb基因干扰组中纺锤体形态异常率、染色体排列异常率均升高,且差异有统计学意义。结论：Numb基因参与小鼠减数分裂过程中纺锤体的组装,这是Numb基因作为细胞命运决定因子之外又一功能。第二部分：Dlg1基因过表达阻碍小鼠卵母细胞成熟研究背景和目的：多细胞器官中细胞多样性是由于细胞不对称分裂造成的。不对称分裂是指一个细胞分裂产生的子细胞在大小,形态和／或包含的遗传物质上互不相同。近些年,细胞不对称分裂的重要性逐渐为人们所认识,通过对一系列生物模型(比如：果蝇,线虫等)的研究揭示了不对称分裂的共同特点——细胞不对称分裂是以细胞极性的形成为前提。细胞极性是指细胞在形态、蛋白质分布和细胞功能上的不对称。通过对不同种类细胞的研究分析表明细胞表面标志性分子通过关键的分子通路调节细胞骨架的组装和蛋白质的运输,从而产生细胞极性。母细胞首先建立极性基础,细胞命运决定分子(包括mRNA和蛋白质)在这个基础上进行极性分布,分布过程需要肌动蛋白丝和某些肌动球蛋白马达的参与,通过形成细胞分裂的轴,细胞命运决定蛋白优先分配到一个子代细胞,最终导致两种不同命运的细胞产生。小鼠卵母细胞减数分裂是典型的不对称分裂。减数分裂过程中纺锤体的组装以及其偏离中心的位置分布是不对称分裂产生的基础。未成熟卵母细胞停滞于第一次减数分裂前期,即生发泡期,此时卵母细胞无明显极性。随着生发泡破裂(germinal vesicle breakdown, GVBD),染色体浓缩,微管组装,减数分裂特有的双极纺锤体形成,该纺锤体最初形成于卵母细胞胞质相对中间的位置,然后沿着纺锤体轴方向迁移到最近的皮质区域。在纺锤体迁移的过程中,一个无微绒毛但富含微丝的区域形成于其上方皮质区域。至此,卵母细胞的极性形成。当纺锤体到达皮质区域下方,极体在该位置排出,第一次减数分裂完成。果蝇discs-large (Dig)基因是一种肿瘤抑制基因,同时也是膜相关鸟苷酸激酶(MAGUK)家族的一员,Dlg基因具有多个蛋白-蛋白结合区,通过与不同蛋白结合,在调控细胞增殖,上皮细胞极性,神经突触形成等方面发挥重要作用,果蝇中Dlg突变时会导致细胞极性的丧失和细胞的过度增殖。Dlgl基因是果蝇Dlg基因哺乳动物同系物,Dlg1基因通过PDZ区与其他分子相互作用参与微管极化,中心体再定向以及细胞迁移。哺乳动物存在Dlg1突变时,会出现微丝和微管的异常组装而导致的细胞极性异常,柱状上皮细胞去极化和细胞粘附分子的异常分布。但这些研究局限于细胞有丝分裂中,到目前为止未见关于Dlg1基因在细胞减数分裂中作用的报道。本实验室前期的研究工作证实小鼠卵母细胞减数分裂过程诱导了Dlg1的极性分布,并且Dlg1的这种极性分布与细胞骨架微丝微管之间存在着密切关系。在此基础上我们通过RNA干扰技术及构建Dlg1表达载体,引入外源性mRNA,进一步研究Dlg1在小鼠卵母细胞减数分裂过程中的作用。本节主要介绍Dlg1表达载体构建方法以及Dlg1蛋白过表达后的实验结果。材料和方法：1.提取小鼠卵巢总RNA,通过RT-PCR方法合成Dlg1 2.将Dlgl与PMD-18T载体连接,转化感受态细胞,扩增,测序3.合成human globin 3’ UTR、5’UTR 4.将测序正确的组件及IRES-EGFP与pcGlobin真核表达空载体连接5.连有Dlg1的pcGlobin-EGFP载体和pcGlobin-EGFP空载体通过限制性内切酶酶切,使其线性化6.将线性化的载体DNA体外转录为mRNA并纯化7.收集小鼠GV期卵母细胞,利用显微操作系统将mRNA注射到GV期卵母细胞胞浆中。实验组注射Dlg1-EGFP mRNA,对照组注射EGFP RNA。显微注射后培养,观察,计数并进行统计分析。结果：体外转录的mRNA,纯化后,测定浓度为1.5μg/μl,每个小鼠卵母细胞显微注射约7p1 RNA,显微注射后,在含有dbCAMP的培养液中培养,荧光显微镜下观察,可见由EGFP发出的绿色荧光。证明pcGlobin-EGFP-Dlg1载体建成功,mRNA显微注射后在小鼠卵母细胞中正常表达。转入不含dbCAMP的培养液中继续培养,进一步统计发现实验组中GVBD率和第一极体排出率均低于阴性对照组,且差别有统计学意义。结论：过度表达Dlg1蛋白阻碍了小鼠卵母细胞成熟,Dlg1基因参与调控小鼠卵母细胞减数分裂的进程。更多还原

【Abstract】 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.更多还原