【作者】 王洪锋；

【导师】 窦忠英；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2006， 硕士

【摘要】 卵母细胞体外成熟技术是体外生产胚胎的基础,获得大量体外成熟卵母细胞将为体外受精及胚胎移植提供更多优质胚胎。虽然牛卵母细胞体外成熟培养技术已趋于成熟和完善,但卵母细胞成熟机理的研究还有待深入。本文对比了添加尿嘧啶和三价因子等试剂对牛卵母细胞成熟及后期发育的影响、观察了不同组之间成熟牛卵母细胞和孤雌激活胚胎显微结构变化和不同组之间颗粒细胞凋亡情况。旨在探讨成熟液中添加尿嘧啶和三价因子等试剂对牛卵母细胞成熟的影响。1、牛卵母细胞体外成熟实验分为3组:基础成熟液为对照组,基础成熟液添加50μg/mL尿嘧啶和10μL/mL三价因子为第一组,基础成熟液添加0.67 mg/mL ATP为第二组。结果显示第一组颗粒细胞完全扩散的卵母细胞率(87.31%)显著高于第二组(70.54%)(P<0.05),极显著高于对照组(68.67%)(P<0.01)。第二组颗粒细胞完全扩散的卵母细胞率(70.54%)高于对照组(68.67%),但差异不显著(P>0.05)。第一组卵母细胞成熟率(72.65%)、卵裂率(55.56%)和8～16细胞率(35.56%)极显著高于对照组(57.92%、43.88%和24.59%)(P<0.01);第二组卵母细胞成熟率(60.39%)与对照组(57.92%)差异不显著(P>0.05),但卵裂率和8～16细胞率差异显著(P<0.05)。第一组和第二组之间成熟率和卵裂率差异显著(P<0.05);但8～16细胞率差异不显著(P>0.05)。通过JC-1染色,第一组具有JC-1荧光的牛卵母细胞百分率(51.11%)显著高于第二组(40.00%),极显著高于对照组(33.33%)。第二组具有JC-1荧光的牛卵母细胞百分率高于对照组但差异不显著(P>0.05)。尿嘧啶和三价因子能够促进卵丘颗粒细胞的扩散,提高牛卵母细胞成熟率、卵裂率和8～16细胞率,并能提高线粒体活性,促进胚胎发育。2、电子显微镜观察成熟后牛卵母细胞及孤雌激活胚胎超微结构。结果显示:三组颗粒细胞变化不明显,卵母细胞和孤雌激活胚胎线粒体双层膜都不明显,空泡变性较多。第一组成熟卵母细胞线粒体电子密度最接近卵母细胞胞质,且比对照组和第二组丰富。第一组孤雌激活胚胎可见卵黄颗粒向细胞表面靠近,伸入透明带内的微绒毛基本消退,透明带出现纵向裂痕。第二组成熟卵母细胞线粒体量比对照组丰富,可见颗粒细胞突起伸进透明带。成熟卵母细胞线粒体电子密度增大,细胞表面微绒毛丰富。8～16细胞孤雌激活胚胎中可见细胞间连接。尿嘧啶和三价因子能够保护卵母细胞和孤雌激活胚胎的线粒体,提高线粒体的活性。3、地衣红染色结果显示,三组的颗粒细胞都存在凋亡现象。DNA电泳时梯度条带并不明显,结合流式细胞仪检测结果显示,第一组和第二组颗粒细胞凋亡率分别为30.46%和44.76%,对照组细胞凋亡率为33.28%。细胞周期结果显示:第一组G1期为65.23%,G2期为14.66%,S期为20.11%;第二组G1期为66.22%,G2期为10.54%,S期为更多还原

【Abstract】 In Vitro Maturation of oocytes is the basic of in vitro culture embryos, which can guarantee us more quality oocytes for in vitro fertilization and embryos transfer. Although the technology of In Vitro Maturation is more and more maturated, the mechanism of maturation needs further study. This thesis compared the effect of Uracil, ITS, and ATP on the maturation of bovine oocytes and further development, observed the microstructure changes of oocytes and parthenogenetic embryos among different groups, and granule cell apoptosis among different groups. The main point is to illustrate the effect of Uracil, ITS, and ATP on bovine oocytes’maturation.1. In bovine oocytes’IVM research the solution was divided into three groups: basic solution as the control group (CG), basic solution supplied with 50μg/mL Uracil and 10μL/mL ITS is the first group (FG), and basic solution added by 0.67 mg/mL ATP is the second group (SG). The result shows the rate of oocytes with granule cells full amplified in FG (87.31%) is significant higher than SG (70.54%) (P<0.05), and CG (68.67%) (P<0.01). This rate of SG is higher than CG, but not significant higher (P>0.05). The rate of maturation, cleavage, and 8~16 cell of FG (72.65%, 55.56%, and 35.56%) are significant higher than CG (57.92%, 43.88%, and 24.59%) (P<0.01). Although the maturation rate of SG (60.39%) is not significant higher than CG (57.92%) (P>0.05), the cleavage and 8~16 cell rate is significant higher than CG (P<0.05). The rate of maturation and cleavage are significant difference between FG and SG (P<0.05), however, there is not significant difference in 8~16 cell rate (P>0.05). The rate of bovine oocytes stained by JC-1 in FG (51.11%) is significant higher than SG (40.00%) (P<0.05), and CG (33.33%) (P<0.01). There is no significant difference between FG and SG. Uracil and ITS can proliferiate the granule cells, improve the maturation rate, cleavage rate, and 8~16 cells rate, and enhance the mitochondria’s activity.2. The results of electron microscope which observes maturated oocytes and pathenogenetic embryos show the changes of granule cells are not significant among three groups, the mitochondria’double deck of oocytes and pethnogenetic embryos are not clearly, and full of the vacuole denaturalization. The mitochondria are more abundant and its electron desity in FG is more closed to cytoplasm than other groups. We abserve the yalk granules are更多还原