【作者】 顾晓龙；

【导师】 曾宪垠； 张德福；

【作者基本信息】 四川农业大学 ， 临床兽医学， 2013， 博士

【摘要】 首例体细胞核移植克隆猪虽已诞生多年,但核移植效率仍然很低。体细胞核移植多采用体外成熟的MⅡ期卵母细胞去核后作为受体卵。但由于猪卵母细胞体外成熟体系还不够完善,所以仍需进一步优化。同时,重构胚胎的不完全重编程会导致附植前胚胎不能正常发育。即使极少数胚胎能够发育存活并诞下后代,后代也常伴有不同程度的发育缺陷或表型异常。相比着床前的胚胎和克隆动物胎儿,目前对表型异常的克隆猪的甲基化模式研究仍然较少,其分子生物学机制仍不清楚。本文拟通过在卵母细胞成熟液中添加红景天多糖(rhodiola sachalinensis polysaccaride,RSP)来获得高质量的体外成熟卵母细胞,同时尝试应用新型甲基化转移酶抑制剂RG108来纠正附植前胚胎的异常甲基化修饰状态,以提高核移植效率。本实验室在随后的克隆猪的生产中一共诞下了4头克隆猪,其中一头出生后不久死亡,另有2头克隆猪发生隐睾。针对这两头隐睾克隆猪,我们研究了其睾丸发育相关基因表达及其甲基化的变化。主要研究结果如下：(1)在猪卵母细胞成熟液中添加0.6、6和60mg·L-1的RSP,以第一极体作为核成熟的标志,计算卵母细胞核成熟率；统计体外受精(IVF)和体细胞核移植(SCNT)后胚胎的卵裂率和囊胚率,并检测成熟卵母细胞内谷胱甘肽(GSH)的浓度及活性氧(ROS)水平。结果表明：成熟液中添加6和60mg·L-1RSP均能显著提高成熟后卵母细胞内GSH含量,且添加60mg·L-1RSP组成熟的卵母细胞用于IVF和SCNT后卵裂率和囊胚率都显著增高。(2)使用RG108处理猪胎儿成纤维细胞(Porcine fetal fibroblast,PFF),研究其对细胞甲基化水平和核移植效率的影响。使用0.05,0.5,5和50μmol·L-1RG108分别处理细胞24,48和72h后,用高效液相色谱和亚硫酸氢盐测序(Bisulfite sequencing PCR,BSP)法分析细胞整体甲基化水平和印迹基因H19和IGF2R的DMR区甲基化率,并检测RG108处理对细胞生长、凋亡、染色体变化。结果表明：RG108降低细胞整体甲基化水平呈时间依赖性,50μmol·L-1RG108处理PFF细胞72h后整体甲基化水平显著低于对照组；5和50μmol·L-1处理PFF72h后H19的DMR区域甲基化率显著降低；PFF经RG1080.5,5和50μmol·L-1处理72h后凋亡比例显著提高,但处理组对细胞生长和细胞染色体数目没有显著影响。(3)选用四种浓度的RG108处理时间为72h,研究单独处理供体细胞、单独处理融合后重构胚以及同时处理供体细胞和融合后重构胚三种不同处理方式对核移植效率的影响。结果发现,50μmol·L-1RG108处理供体细胞后的重构胚囊胚率显著提高。随后通过免疫荧光法比较IVF囊胚、SCNT囊胚和50μmol·L-1RG108处理供体细胞后获得的SCNT囊胚的甲基化水平发现,RG108处理组的囊胚的甲基化水平与IVF囊胚更接近。(4)为查明本课题组所获得的2头体细胞克隆小型猪猪隐睾发生的可能原因,本研究采用相对荧光定量PCR技术和亚硫酸氢盐测序法分析SOX9和INSL3基因在克隆猪隐睾中的表达量及其启动子区CpG位点甲基化状态。结果表明：除了SOX9基因在克隆猪C2中的表达量显著高于对照组N外,克隆猪SOX9和lNSL3基因的表达量都显著低于对照组N；亚硫酸氢盐测序结果显示SOX9基因启动子区在克隆猪C1和C2中的甲基化程度没有显著变化,而INSL3基因启动子区在克隆猪C1和C2中的甲基化程度均显著高于对照猪N。综上所述：成熟液中添加RSP能够提高IVM卵母细胞质量,60mg·L-1RSP组成熟的卵母细胞用于IVF和SCNT后能显著提高胚胎发育能力；RG108处理PFF72h后细胞整体甲基化水平降低且呈时间依赖性,同时H19的DMR区域甲基化率也显著降低；50μmol·L-1108处理PFF72h组还能显著提高核移植效率,其囊胚整体甲基化水平较对照组SCNT囊胚低,与IVF囊胚的水平更接近；INSL3基因在克隆猪隐睾中的DNA甲基化模式异常,影响其正常表达,这可能是导致克隆猪发生隐睾的重要因素之一。更多还原

【Abstract】 Although it has been years since the first smatic cell nuclear transfer cloned pig was born, the efficiency of the nuclear transfer still needs to be promoted. Currently, MⅡ-phase deuncleated oocytes matured in vitro are mostly used as receptor eggs in SCNT. Pig oocytes in vitro maturation system is still immature and has yet to be further improved. Meanwhile, incomplete reprogramming of the reconstructed embryos leads to the unnormal development of preimplantation embryos. Even if few survivals, some developmental defects and abnormal phenotypes are usually accompanied to different degrees in the offspring. Compared with preimplantation embryos and cloning animals fetus, the studies on the methylationpatterns in phenotypic abnormalities cloned pigs are still insufficient, the molecular biology mechanism remains unclear. In this study, we added rhodiola sachalinensis polysaccaride(RSP) in oocyte maturation medium to obtain high quality oocytes matured in vitro. And tried to apply the new methylation inhibitor RG108to correct abnormal preimplantation embryo methylation status, in order to improve the efficiency of nuclear transfer. Four cloned piglets were born in the subsequent production of cloning pigs in the laboratory, one died after it was born, and two cloned piglets occurred in cryptorchidism. We studied the related gene expressions in testis development and the changes of the methylation in the two cryptorchidism cloned pigs. The main results are as follows:(1) Immature oocytes were matured in vitro and supplemented with different concentrations of RSP, the extrusion of first polar body was treated as a symbol of nuclear maturation, then the nuclear maturation rates, oocyte intracellular GSH content and ROS level, Cleavage and blastocyst rates of IVF and SCNT were conducted. The results showed that treated with6and60mg·L-1RSP in the vitro maturation medium could significantly increase the GSH content (P<0.05), and60mg·L-1group of mature oocytes can significantly improve the cleavage and blastocyst rate of IVF and SCNT.(2) This experiment was conducted to study the effect of RG108treated PFF as donor cells on their methylation levels and nuclear transplantation efficiency. Four concentrations,0.05,0.5, Sand50μmol·L-1RG108were selected. PFF was treated for24,48,72h respectively, and the cells methylation levels was determined by HPLC and BSP method; Observed the situation of cell growth and apoptosis by using the MTT method and fluorescent microscope observation method; Using Giemsa staining detected cells’ chromosome and detected nuclear transfer efficiency after treated with four concentrations RG108for72h. The results showed that:The results indicated that PFF incubated with50μmol·L-1RG108RG108for72h reduced the cells’ overall methylation levels and it was time-dependent and5and50μmol·L-1RG108for72h reduced the regional rate of methylation of the imprinted gene DMR, And RG108had no significant impact on normal cell activity.(3) Four concentrations of RG108were selected to treat donor cell or/and SCNT embryon After fusion for72h,studied the effect of different treatments on the efficiency of nuclear transfer.The results showed that blastocyst rate of embryos obtained from donor cells which treated by5μmol·L-1RG108was significantly improved. Subsequently, immunofluorescence was used to detect the Methylation levels of IVF blastocysts, SCNT blastocysts and blastocysts obtained from donor cells which treated by5μmol·L-1RG108. The results showed that blastocyst of the RG108treated group and IVF group had similar methylation level.(4) In order to identify the possible causes of the cryptorchidism in cloning pigs which producted by our research group, The relative fluorescence quantitative PCR techniques and bisulfite salt sequencing analysis are used to analysis the expression and promoter methylation status of the promoter CpG sites of SOX9and INSL3genes in the cloned pig’s cryptorchidism. The results showed that, The expression of SOX9and INSL3genes in the cloned pigs cryptorchidism were abnormal.But analysis of the methylation status showed that INSL3gene was hypermethylation and the SOX9gene methylation status had no significant change.In summary, added RSP in IVM medium improved the quality of matured oocytes in vitro. Oocyte maturation in60mg·L-1RSP used to IVF and SCNT can significantly enhance the viability of the embryos. RG108reduced the levels of the cell methylation and it was Time-dependent. Meanwhile the DMR methylation of H19was also significantly reduced. Nuclear transfer efficiency was significantly improved by PFF treated with5μmol·L-1RG108for72h. The levels of the blastocyst methylation were lower than the control group, but closed to the IVF group.had an abnormal DNA methylation patterns, it causesed abnormal expression.The aberrant DNA methylation patterns occured in INSL3gene and infulenced gene expression, which could be one of the important factors leading to the occurrence of cryptorchidism in cloned pigs.更多还原