【作者】 罗茜；

【导师】 陆阳清； 卢克焕；

【作者基本信息】 广西大学 ， 动物遗传育种与繁殖， 2012， 硕士

【摘要】 活性氧基团(ROS)被认为是不利于卵母细胞体外成熟和胚胎体外发育的因素。在体外培养条件下,胚胎遇到比体内更多的氧化因子。褪黑素可以提高卵母细胞质量,使卵母细胞免于氧化危害。褪黑素和其代谢物组成了清除ROS的高效的级联反应,褪黑素有别于其他的抗氧化剂就是因为这个特性。本研究探讨在水牛卵母细胞体外成熟液和胚胎培养液中添加褪黑素对卵母细胞体外成熟和胚胎发育的影响。试验一,在水牛卵母细胞体外成熟培养液中,分别添加三种浓度的褪黑素,即10-10mol/L,10-9mol/L,10-8mol/L,不添加为对照组。目的在于探讨添加不同浓度褪黑素的成熟液水牛卵母细胞的成熟情况。Ⅳmol/L24h后,以第一极体的排出作为成熟标准。试验结果表明,10-9mol/L试验组成熟率与其他试验组相比,差异不显著(46.28±1.74%vs43.15±5.24%,43.40+3.12%,P>0.05),但显著高于对照组(46.28±1.74%vs32.90±2.21%,P<0.05)。试验二,仅在胚胎培养液中添加褪黑素情况下,10-8mol/L组分裂率与其他组相比,差异不显著(59.6%vs53.6%,49.5%,48.0%,P>0.05),然而,其囊胚率显著高于对照组(34.3%vs20.6%,P<0.05)。根据试验一结果,在成熟培养阶段添加最优浓度(10-9mol/L)褪黑素的同时,在胚胎培养液中也添加不同浓度的褪黑素(10-8mol/L,10-9mol/L,10-10mol/L, Omol/L)。结果表明,10"9mol/L组分裂率与其他组相比无显著差异(70.0%vs64.8%,65.2%,60.9%,P>0.05),囊胚率与其他试验组相比也没有显著差异(28.9%vs15.4%,26.1%,25.3%,P>0.05),但显著高于对照组(28.9%vs15.4%,P<0.05)。试验三,在水牛卵母细胞成熟培养液中分为添加褪黑素(试验组)和不加褪黑素(对照组),IVM24h后,通过DCHFDA荧光染色方法测定水牛卵母细胞中的ROS含量。结果表明,与对照组的荧光强度相比,褪黑素处理组的ROS水平显著低于对照组。试验四,通过实时定量PCR测定线粒体拷贝数。在水牛卵母细胞成熟液中添加10-9mol/L褪黑素和不加褪黑素,IVM24h。试验组和对照组的拷贝数没有差别。这说明褪黑素对线粒体拷贝数没有影响。本研究结果表明,褪黑素可以提高水牛卵母细胞体外成熟率和早期胚胎体外发育率,同时还参与清除活性氧的过程。更多还原

【Abstract】 The reactive oxygen species (ROS) were considered as aggressive molecules against oocyte maturation and embryo development in vitro. Embryos cultured in vitro encountered higher oxygen toxicity elicited by ROS than their in vivo counterparts. Melatonin could improve oocyte quality and protects oocyte from oxidative stress. The process of melatonin and its metabolites (?) successively scavenging ROS is referred as the free radical scavenging cascade. Melatonin differs from other conventional antioxidants because the cascade reaction is a novel property. Mitochondria are an important target of melatonin.This study was carried out to investigate the effect of different concentrations of melatonin supplemented in vitro maturation and culture media on buffalo oocyte maturation and embryo development in vitro.In experiment1, immature oocytes were cultured for24h in vitro in maturation medium supplemented with different concentrations of melatonin (10-10mol/L,10-9mol/L,10-8mol/L) and without melatonin as control. After24hr, oocytes with first polar body were counted as matured ones. The result showed that supplementation of melatonin at10-9mol/L concentration in the maturation medium resulted in similar maturation rate to other treatments (46.28±1.74%vs 43.15±5.24%,43.40±3.12%, P>0.05), but significantly higher than the control (46.28±1.74%vs32.90±2.21%, P<0.05).In experiment2, only culture medium was supplemented with various concentrations of melatonnin (10-8mol/L,10-9mol/L,10-10mol/L), the cleavage rate of10-8mol/L treatment was similar to the other groups (59.6%vs53.6%,49.5%,48.0%, P>0.05), but the blastocyst rate was significantly higher than the control (34.3%vs20.6%, P<0.05). Based on the results of experiments1, the maturation medium was supplemented with the most optimal concentration of melatonin (10-9mol/L) and the culture medium was supplemented with three threatments (10-8mol/L,10-9mol/L,10-10mol/L) and control. The cleavage rate of10"9mol/L treatment was similar to the other groups (70.0%vs64.8%,65.2%,60.9%, P>0.05). And the blastocyst rate of10-9mol/L treatments was similar to the other groups (28.9%vs15.4%,26.1%,25.3%, P>0.05), however, only significantly higher than control (28.9%vs15.4%, P<0.05).In experiment3, buffalo oocytes were matured in the presence or absence of melatonin for24hr. The quantity of ROS produced by the oocytes was measured using2’,7’-dichlorodihydrofluorescein diacetate. The oocytes without the first polar body that matured in the absence of melatonin was the control. Compared with the control, melatonin-treated (10-9mol/L) oocytes had significantly lower levels of ROS than did the control oocytes.In experiment4, Mitochondria copies were measured by real-time polymerase chain reaction (RT-PCR). Buffalo oocytes were matured with 10"9mol/L melatonin and without melatonin for24hr. There is no difference in the melatonin-treatment group and control. The result indicated that melatonin had no effects on the Mitochondria copies.These data suggest that melatonin could increase the maturation and blastocyst rates of buffalo oocyte in vitro and may have important implications for scavenging ROS.更多还原