褪黑激素对猪卵母细胞体外发育的影响

发布时间：2018-06-28 13:39

本文选题：褪黑激素 + 猪卵母细胞　；参考：《西南大学》2015年硕士论文

【摘要】：褪黑激素(MT)是一种吲哚类激素,在人及哺乳动物体内主要是由松果腺分泌,其他器官和细胞也有少量分泌,分泌量受光照影响比较大,且呈现昼夜节律性,一般夜晚分泌多,白天分泌少。MT能够透过细胞膜及各种膜结构进入细胞器来发挥抗氧化作用。目前MT的抗氧化、抗凋亡作用在小鼠、绵羊、鸡等动物卵母细胞体外成熟及早期胚胎中均有报道,但在猪卵母细胞体外成熟中报道较少。本研究以猪为试验对象,研究了褪黑激素对卵母细胞体外成熟及胚胎发育的影响。主要内容如下：1褪黑激素对猪卵母细胞体外发育的影响(1)褪黑激素对猪卵母细胞体外成熟的影响挑选合格的卵母细胞,然后随机分成6组,在添加褪黑激素(分别为0M、10-11M、10-9M、10-7M、10-5M、10-3M)的体外成熟培养液中培养46h,统计各组卵母细胞的极体排出率。结果发现,10-9M组(77.54%)和10-7M组(69.2%)的极体率显著高于对照组(67.81%)和其他试验组,该两组之间差异不显著。(2)褪黑激素对猪卵母细胞孤雌胚胎发育的影响挑选合格的卵母细胞置于未添加褪黑激素的体外成熟液中培养46h,接着进行孤雌激活并随机将卵母细胞放到6组添加褪黑激素(分别为0M、10-1M、10-9M、10-7M、10-5M、10-3M)的胚胎培养液中继续培养。胚胎培养的第2d统计卵裂率,第7d统计囊胚率。结果发现,褪黑激素未能提高卵母细胞孤雌发育的卵裂率,对照组和各试验组间差异不显著。10-SM组(20.31%)的囊胚率显著高于对照组(9.45%)和10-3M组(10.68%)。(3)褪黑激素对猪卵母细胞体外发育的影响成熟液和胚胎培养液分别添加10-9M、10-5M褪黑激素,卵母细胞体外培养46h后统计卵裂率。孤雌激活后继续胚胎培养,第2d统计卵裂率,第7d统计囊胚率。结果发现,对照组和试验组的卵裂率(77.68%和81.37%)和囊胚率(17.89%和19.27%)差异不显著,试验组的极体率(78.03%)显著高于对照组(64.55%)。2褪黑激素对猪卵母细胞体外发育过程中GPx4和Caspase-3表达的影响通过RT-PCR方法鉴定外源性褪黑激素是否影响猪体外胚胎的GPx4和Caspase-3基因在转录水平的表达,探寻影响猪体外胚胎发育的机理。猪卵母细胞体外成熟及孤雌激活后培养基中均添加褪黑激素,收集体外成熟16h、24h、46h的猪卵母细胞以及2-cell、4-cell和囊胚期的孤雌胚胎,RT-PCR方法鉴定以上基因在转录水平的表达情况。褪黑激素能显著上调猪卵母细胞体外培养中胚胎培养阶段抗凋亡基因GPx4的表达(P0.05),抑制培养全程中促凋亡基因Caspase-3的表达(P0.05)。
[Abstract]:Melatonin (MT) is an indole hormone, which is secreted mainly by pineal gland in human and mammalian body. Other organs and cells also secrete a small amount. During the day, less. MT can enter the organelle through cell membrane and various membrane structures to play the role of antioxidation. At present, the antioxidation and anti-apoptotic effects of MT have been reported in mouse, sheep, chicken and other animal oocytes in vitro maturation and early embryos, but in pig oocytes in vitro maturation is less reported. The effects of melatonin on maturation and embryo development of oocytes in vitro were studied in pigs. The effects of melatonin on the development of porcine oocytes in vitro. (1) the effects of melatonin on the maturation of porcine oocytes in vitro. In vitro maturation medium supplemented with melatonin (10-11 MN 10-9 M 10-7 M 10-7 M 10-5 M 10-3 M) for 46 h, the polar body efflux rate of oocytes in each group was calculated. The results showed that the polar body rates in 10-9M group (77.54%) and 10-7M group (69.2%) were significantly higher than those in control group (67.81%) and other experimental groups. (2) the effect of melatonin on the development of parthenogenetic embryos of porcine oocytes. (2) the effect of melatonin on the development of parthenogenetic embryos of pig oocytes. (2) the eligible oocytes were cultured in maturation medium without melatonin for 46 hours, then parthenogenetic activation was performed and randomly assigned The oocytes were cultured in 6 groups of embryos supplemented with melatonin (10 ~ (-9) M ~ (-1) M ~ (-10 ~ (-9) M ~ (-7) ~ (-7) M ~ (-10 ~ (-7) M ~ (-5) M ~ (-3) M). The cleavage rate was calculated on the 2nd day and blastocyst rate on the 7th day. The results showed that melatonin did not increase the cleavage rate of parthenogenetic oocytes. The blastocyst rate of 10-SM group (20.31%) was significantly higher than that of control group (9.45%) and 10-3M group (10.68%). (_ 3). Oocytes were cultured in vitro for 46 hours and the cleavage rate was counted. The blastocyst rate and blastocyst rate were calculated on the 2nd day and the 7th day after parthenogenetic activation. The results showed that there was no significant difference in cleavage rate (77.68%) and blastocyst rate (17.89% and 19.27%) between control group and experimental group. Effects of melatonin on the expression of GPx4 and Caspase-3 in Porcine oocytes during Development in Vitro, the effects of exogenous melatonin on the expression of GPx4 and Caspase-3 genes in Porcine embryos in Vitro by RT-PCR At the transcriptional level, To explore the mechanism of affecting the development of porcine embryos in vitro. In vitro maturation and parthenogenetic activation of porcine oocytes, melatonin was added to the culture medium. Porcine oocytes were collected for 16 h, 24 h or 46 h after maturation, and 2-cell-4-cell and blastocyst stage parthenogenetic embryos were detected by RT-PCR to identify the expression of the above genes at the transcriptional level. Melatonin could significantly up-regulate the expression of anti-apoptotic gene GPx4 in porcine oocytes in vitro (P0.05) and inhibit the expression of pro-apoptotic gene Caspase-3 during the whole process of culture (P0.05).
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.23

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