【作者】 贾宝瑜；

【导师】 朱士恩； 傅祥伟；

【作者基本信息】 中国农业大学 ， 动物遗传育种与繁殖， 2014， 博士

【摘要】 实验旨在探究反10顺12共轭亚油酸(t10c12CLA)在猪卵母细胞体外成熟、玻璃化冷冻保存及孤雌激活胚胎发育过程中的生理作用。首先,将不同浓度的t10c12CLA添加至猪卵母细胞体外成熟体系中。培养结果表明：添加50μmol/Ltl0c12CLA可显著提高卵母细胞体外成熟效率,与对照组相比,其孤雌激活后的卵裂率(89.5%±1.2vs.83.4%±1.6)、囊胚率(51.9%±1.5vs.39.6%±1.5)和囊胚细胞数(62.2±1.6vs.50.7±1.2)均有显著提高(P<0.05)。检测成熟培养22和36h卵母细胞核相,发现t10c12CLA可加快卵母细胞减数分裂进程。Western Blot结果显示,添加t10c12CLA可显著提高成熟过程中MAPK3/1的活性及COX-2蛋白的表达量：它还可以提高MII期卵母细胞中MAPK3/1的磷酸化水平。在U0126存在时,t10c12CLA可部分恢复U0126对卵母细胞成熟及卵丘扩展的抑制作用,且可完全恢复U0126对COX-2表达的抑制作用。此外,t10c12CLA可完全恢复NS398对卵丘细胞扩展和卵母细胞成熟的抑制作用。但当U0126和NS398同时存在时,添加t10c12CLA不再有恢复效果。对成熟培养44h的卵母细胞进行胞质成熟评定,结果显示t10c12CLA显著提高成熟卵母细胞中谷胱甘肽(Glutathione, GSH),降低活性氧(Reactive oxygen species, ROS),提高卵母细胞的氧化还原能力。其次,选用最适浓度的t10c12CLA (50μmol/L)添加在体外成熟培养体系中,卵母细胞成熟至MII期后,采用Cryotop法对其进行玻璃化冷冻保存。结果表明,添加t10c12CLA显著提高卵母细胞冷冻·解冻后孤雌激活早期胚胎的囊胚发育率(25.43%±2.14vs.11.76%±0.62),并可提高脂肪颗粒形态正常(N型)卵母细胞的比例。用透射电子显微镜观察冷冻·解冻卵母细胞内部超微结构,发现t10c12CLA可有效改善冷冻.解冻后卵母细胞内脂肪颗粒的破碎、溢出及融合等现象；还可缓解冷冻·解冻引起的线粒体“簇状”不均匀分布及线粒体极性降低等损伤。最后,于猪孤雌激活后早期胚胎的培养体系中添加不同浓度的t10c12CLA,发现50μmol/Lt10c12CLA提高培养第5天囊胚发育率(34.54%±0.95vs.26.48%±2.19)和囊胚细胞数(54.65±1.63vs.48.67±2.14)。对8-细胞期胚胎中E-Cadherin表达量和胚胎质量相关指标进行检测,结果表明,与对照组相比,t10c12CLA可显著提高E-Cadherin在8-细胞胚胎中的表达,且显著增加多细胞(n>60)囊胚在总囊胚中的比例(40.79%±1.61vs.28.43%±1.14)。综上所述,t10c12CLA通过调节MAPK活性及COX-2表达量促进猪卵母细胞核成熟,通过提高卵母细胞内MAPK活性及氧化还原能力促进猪卵母细胞的胞质成熟。经添加t10c12CLA成熟培养的卵母细胞,在冷冻保存过程中,胞内线粒体极性升高,维持线粒体的正确分布,并可保护脂肪颗粒经历低温打击后形态的完整性,进而提高冷冻·解冻后卵母细胞的发育潜力。在猪孤雌激活产生的早期胚胎发育过程中,t10c12CLA可提高8-细胞胚胎中E-Cadherin的表达,促进桑椹胚致密化,进而加速囊胚的形成,提高胚胎质量。更多还原

【Abstract】 The aim of this study was to determine the effects of trans-10, cis-12conjugated linoleic acid (t10c12CLA) supplementation on oocyte maturation, embryo development and cryopreservation in pigs.First, various concentration of tl0cl2CLA were supplemented in porcine in vitro maturation (IVM) medium. Compared with the control, supplementation of50μmol/L tl0cl2CLA to IVM medium significantly increased the proportion of oocytes at the metaphase-Ⅱ (MII) stage and subsequent parthenogenetic embryo development in terms of cleavage rate (89.5%±1.2vs.83.4%±1.6), blastocyst formation rate (51.9%±1.5vs.39.6%±1.5), and cell numbers in blastocysts (62.2±1.6vs.50.7±1.2). The state of nucleus was examined at22and36h maturation, the results showed that t10c12CLA treated oocytes resumed meiotic maturation and progressed to the MII stage significantly faster than those of control. The expression of phosphorylated mitogen-activated protein kinase3/1(p-MAPK3/1) and cyclooxygenase-2(COX2) in cumulus oocyte complexes (COCs) at5,10, and22h of IVM were significantly increased in the t10c12CLA treatment group. The level of p-MAPK3/1in t10c12CLA treated MII oocytes was also higher (p<0.05) than that of control. Moreover, t10c12CLA supplementation partially overcame the negative effects of U0126on cumulus expansion and nuclear maturation, and completely recovered COX-2protein levels in the presence of U0126. Treatment of COCs with NS398also significantly suppressed cumulus expansion and nuclear maturation, which was overcome by t10c12CLA. Yet, this stimulatory effect of t10c12CLA was blocked in the presence of both U0126and NS398. The maturation of cytoplasm was also evaluated; and founded that t10c12CLA treatment significantly reduced reactive oxygen species (ROS) level and increased glutathione (GSH) concentrations in MII oocyte.Second, the appropriate concentration (50μmol/L) of t10c12CLA was supplemented to IVM medium, and MII oocytes were subjected to Cryotop vitrification. The results showed that the formation rate of parthenogenetic blastocysts underwent vitrification was higher in t10c12CLA treated group (25.43%±2.14vs.11.76%±0.62, p<0.05), and t10c12CLA increased the percentage of normal oocytes (type N, lipid droplets with sharpen edge) significantly. Transmission electron microscope (TEM) was used to exam the ultrastructure of vitrified oocytes.t10c12CLA protected the lipid droplets from fusion and rupture. Moreover, the distribution and membrane potential of mitochondria was maintained in t10c12CLA treated group.Last, a serious of concentrations of t10c12CLA was added to in vitro culture (IVC) medium of porcine parthenogenetic embryos. Compared with the control, the blastocysts formation at5d IVC (34.54±0.95vs.26.48±2.19) and total cells in blastocysts (54.65±1.63vs.48.67±2.14) were increased in t10c12CLA treated group (50μmol/L). Based on the results mentioned above, the concentration of50μmol/L was used in the following experiments. Then, the expression of E-Cadherin in8-cell embryos was measured to analysis the effects of t10c12CLA on porcine early embryos development, and founded that t10c12CLA promoted the expression of E-Cadherin significantly as well as the percentage of blastocysts with more cell numbers (n>60)(40.79±1.61vs.28.43±1.14).In conclusion, an optimal concentration of t10c12CLA supplemented to IVM medium enhances the maturation and subsequent in vitro developmental competence of porcine oocytes. The beneficial effect of t10c12CLA on nuclear maturation in porcine oocytes appears to act indirectly through the cumulus cells by the regulation of both MAPK and COX2activity. Culture in the presence of t10c12CLA was also advantageous for cytoplasmic maturation of porcine oocytes, increasing intracellular MAPK activity and modulating the redox status of the cell. After treated with t10c12CLA, the distribution and membrane potential of mitochondria of vitrified MII oocytes was improved, and the lipid droplets were maintained regularly, which promoted the developmental competence of porcine oocytes underwent vitrification. Moreover, t10c12CLA supplemented during IVC affected the parthenogenetic embryos development posisitively through increasing expression of E-Cadherin, which leaded to the acceleration of morula compaction and blastocysts formation.更多还原