【作者】 赵贵民；

【导师】 雷安民；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2011， 硕士

【摘要】 在哺乳动物卵子减数分裂调控中,MPF(CDK1-Cyclin B1)的活性变化起着关键性作用。P21蛋白是最早被发现并证实具有CKIs作用的Cip/Kip家族成员,在低等动物卵子中已经证实P21可以直接的抑制CDK1激酶Thr-161位点的磷酸化,从而抑制MPF的活性,但是P21在哺乳动物卵母细胞减数分裂调控中的作用尚未报道,这就是本研究关注的核心问题。本研究以牛卵母细胞为试验材料,首先利用地衣红染色技术观察了牛卵母细胞体外成熟以及减数分裂到有丝分裂转换过程中各个时期染色体的形态学变化,确定了牛卵母细胞各时期的具体时间段;其次从牛成纤维细胞中克隆了p21基因,并构建了pVenus-P21真核表达载体;最后运用Q-PCR和免疫荧光染色技术检测了P21在牛卵中的表达与定位,通过超表达与干扰P21蛋白初步研究了P21在M-I期和M-II期中的功能。研究取得以下结果:1.通过地衣红染色系统地观察了牛卵母细胞体外成熟过程中染色体的形态学变化特征,据此确定不同培养时间段中卵母细胞所处的细胞周期时相,建立了牛卵母细胞体外培养过程中染色体变化规律的时间进程表,即在体外培养0~5.45 h,11.40 h,13.15 h,17.10 h,18.35~24 h,大部分卵母细胞分别处于GV期、GVBD~pre-M-I期、M-I期、A-I期、T-I期、M-II期。然后比较了三种化学激活方法(Ionomycin单独使用,Ionomycin联合6-DMAP,Ionomycin联合Roscovitine)对牛卵第二极体排出以及原核形成的影响,发现Ionomycin单独使用或联合Roscovitine使用时,2 h后就有大量第二极体排出,在孤雌激活8 h后就有原核形成,在孤雌激活后16~18 h原核形成数达到最高;而应用Ionomycin联合6-DMAP使用时,会抑制第二极体的的排出;Ionomycin联合Roscovitine同样能达到Ionomycin联合6-DMAP孤雌激活牛卵的原核数。2.利用RT-PCR技术成功从牛成纤维细胞中克隆得到p21基因的CDS全长序列,经与NCBI上公布的p21序列对比,同源性为100%。将其连接到pVenus真核表达载体上成功构建了pVenus-P21真核表达载体,转染Hela细胞及其体外转录的cRNA显微注射卵母细胞后均能正确表达和准确定位。3.在mRNA水平上分别检测了不同组织以及卵母细胞与胚胎发育过程中p21的表达,并对结果进行了分析。结果表明,p21在颗粒细胞中表达较高;在卵母细胞成熟过程中的M-II期表达相对较高;在胚胎发育的不同阶段,以囊胚中p21 mRNA表达量为最高。此外,P21蛋白在卵母细胞核内分布远较细胞质丰富,表明P21具有核定位特性。在卵母细胞成熟过程中P21可以阻止卵子进入M-I期,同时促进卵母细胞从M-II期退出。本研究得到如下结论:建立了牛卵母细胞体外成熟培养过程中染色体形态变化的时间进程表;P21在卵母细胞成熟各时期中均有表达,P21定位在染色体及周围结构上;P21阻止卵母细胞进入M-I期,但促进卵子从M-II期退出。更多还原

【Abstract】 Stability of maturation promoting factor (MPF; CDK1/cyclin B1) is needed for completing meiotic cell cycle arrest in mammalian oocytes. P21 is pivotal cyclin-dependent kinase inhibitors (CKIs) which belongs to a member of the Cip/Kip family, and is required for negative regulation of the cell cycle progression. P21 can directly inhibit Thr-161 phosphorylation of CDK1 in the frug oocytes, then inactivation of MPF. This study focues on effect of P21 on mammalian oocytes maturation.The various periods morphological changes of chromosomes during in vitro maturation and the transition from meiosis to mitosis were observed by aceto-orcein staining technique in bovine oocyte. The bovine p21 gene was cloned from cDNA simple of fibroblasts and pVenus-P21 eukaryotic expression vector was constructed. Last, the mRNA of P21 in the bovine oocytes were examined by the Q-PCR, and the protein of P21 were examined by immunofluorescence staining. The effects of P21 on exit from the M-I and M-II were preliminarily researched by over-expression and interference P21. The results were as follows:1. First, The various periods morphological changes of chromosomes during in vitro maturation and the transition from meiosis to mitosis were observed by aceto-orcein staining technique in bovine oocyte. The timetable of oocytes chromosome morphological events were established and the time spent on each step was determined. The germinal vesicle (GV) was present from 0 to 5.45 h, GVBD to chromatin condensation at 5.45 to 11.4 h, metaphase I at 11.4 to 13.15 h, anaphase I at 13.15 to 17.10, telophase I at 17.10 to 18.35 h, and metaphase II at 18.35 to 24 h. Then the influences of three chemical activation methods(Ionomycin alone, Ionomycin coupled with 6-DMAP, and Ionomycin coupled with Roscovitine)on the second polar body extrusion and pronuclear formation. Results show that there are a large number of second polar body after 2 h of parthenogenetic activation, and pronuclear formation in the 8 h and have the highest number of pronuclear in 16~18 h after parthenogenetic activation by ionomycin alone. But using Ionomycin combination 6-DMAP inhibited the extrusion of the second polar body, and Ionomycin combination Roscovitine also could reached such quantity that the Ionomycin combination 6-DMAP to parthenogenetic activation of bovine oocytes.2. Full-length sequence of p21 gene CDS was cloned and connected to pVenus eukaryotic expression vector for getting vector pVenus-P21. After transfection or microinjection by in vitro transcription cRNA, the fusion protein could be expressed efficiently and localized accurately both in Hela cells and bovine oocytes.3. The content of p21 mRNA were detected in different tissues, and different stage during oocytes maturation and embryo development. The mRNA of p21 in granulosa cells is relatively higher among different tissues; and the content of p21 mRNA at M II stage was higher during oocytes maturation; and the content of p21 mRNA at blastocyst during embryos development. The P21 protein located prather in nuclear than the oocyte cytoplasm. In the process of oocyte maturation, P21 can prevent the oocyte enter the M-I phase, but P21-dependent inactivation of MPF during egg activation and promote its exit from the M-II block.In this study, the chromosomal status of oocytes was evaluated, frequencies were computed, and the time spent on each step was determined; P21 were expressed in the period of oocyte maturation, P21 was located in the chromosome and the surrounding structure; P21 can prevent oocytes enter into the M-I phase, but it promote oocytes exit from the M-II block.更多还原