Kisspeptin在牛卵母细胞体外成熟过程中的表达和功能研究

发布时间：2018-07-26 17:49

【摘要】：Kisspeptin是KiSS-1基因编码的一种多肽,其在下丘脑表达,对哺乳动物生殖活动起中枢调控作用已被广泛证实。研究表明,Kisspeptin除在下丘脑表达外,在卵巢也有表达,但有关其功能和机制的研究极少。对猪和鼠的研究发现,Kisspeptin参与卵母细胞成熟,但其作用存在种属差异。牛卵母细胞上是否有Kisspeptin表达,其功能和机制如何,目前还不清楚。本文首先利用免疫荧光和荧光定量PCR技术研究了体外成熟过程Kisspeptin及其受体GPR54在牛卵母细胞中的分布定位及表达规律,随后研究了添加外源Kisspeptin对牛卵母细胞体外成熟的影响及其相关机制,研究结果主要分以下2部分:1.收集不同培养时间(0、6、12、18、24h)的牛卵母细胞进行免疫荧光染色,随后于倒置荧光显微镜下观察。研究结果表明:Kisspeptin在卵母细胞整个体外成熟过程中均有表达,且表达位置相同。但由细胞的荧光强度可知,不同时间点的Kisspeptin表达量存在差异。收集不同培养时间(0、6、12、18、24h)的卵母细胞,利用荧光定量PCR,检测KiSS-1、GPR54基因表达量,研究结果表明,KiSS-1及GPR54基因的表达量均在0h最高,随体外培养过程出现波动性变化,且变化曲线较为相似。2.按照添加外源Kisspeptin的浓度不同将培养液分为5组,分别为TCM-199对照组、添加1nmol/L、10nmol/L、102nmol/L、103nmol/L Kisspeptin的处理组,用5种培养液进行卵母细胞体外培养并统计成熟率,成熟率最高组的Kisspeptin浓度为添加Kisspeptin的最适浓度。结果显示,添加10nmol/L Kisspeptin组的细胞成熟率极显著高于其他4组(P0.01),确定10nmol/L为添加外源Kisspeptin的最适浓度。按照培养液的不同处理方式将其分为8组,分别为①TCM-199②TCM-199+Kisspeptin③TCM-199+FSH④TCM-199+FSH+Kisspeptin⑤TCM-199+Kisspeptin+FBS⑥TCM-199+FBS⑦TCM-199+Kisspeptin+p234⑧TCM-199+p234+FSH,用8种培养液进行卵母细胞体外培养并统计成熟率。结果显示,Kisspeptin组与Kisspeptin+FSH组的卵母细胞成熟率较高,两组间差异不显著,但均极显著高于FBS组(P0.01);同时添加FBS和Kisspeptin时,成熟率较低,与Kisspeptin组差异极显著(P0.01);同时添加Kisspeptin和p234,成熟率显著低于Kisspeptin组(P0.05);同时添加FSH和p234,成熟率与FSH组无显著差异。利用荧光定量PCR检测体外成熟过程中(12、24h)卵母细胞发育关键基因BMP15和GDF9的表达量。结果显示,BMP15和GDF9在12、24h表达量较低,但用添加Kisspeptin的培养液进行体外培养时,BMP15和GDF9的表达量显著上升,说明Kisspeptin可以促进成熟调控关键基因的表达。
[Abstract]:Kisspeptin is a polypeptide encoded by KiSS-1 gene. It is expressed in hypothalamus and plays a central role in the regulation of mammalian reproductive activity. The results showed that Kisspeptin was expressed not only in the hypothalamus but also in the ovary, but the function and mechanism of Kisspeptin were seldom studied. It was found that Kisspeptin was involved in oocyte maturation in pigs and mice, but its effects were different in species. It is unclear whether there is Kisspeptin expression in bovine oocytes and its function and mechanism. The distribution, localization and expression of Kisspeptin and its receptor GPR54 in bovine oocytes during maturation in vitro were studied by immunofluorescence and fluorescence quantitative PCR. The effect of exogenous Kisspeptin on bovine oocyte maturation in vitro and its related mechanism were studied. The results were divided into two parts: 1. Bovine oocytes were collected and stained with immunofluorescence for different culture time (0 ~ 6 ~ 12 ~ 1 ~ 18 ~ 24 h), and then observed under inverted fluorescence microscope. The results showed that: Kisspeptin was expressed in the whole process of oocyte maturation in vitro, and expressed in the same position. However, according to the fluorescence intensity of the cells, there were differences in the expression of Kisspeptin at different time points. The expression of KiSS-1GPR54 gene was detected by fluorescence quantitative PCR. the results showed that the expression of KiSS-1 and GPR54 genes were the highest in 0 h, and fluctuated with the process of culture in vitro, and the curve was similar. According to the concentration of exogenous Kisspeptin, the culture medium was divided into five groups: TCM-199 control group, treated with 1nmol / L 102nmol / L nmol / L Kisspeptin. The oocytes were cultured in vitro with five kinds of culture medium and the maturation rate was calculated. The Kisspeptin concentration of the highest maturation rate group was the optimum concentration of adding Kisspeptin. The results showed that the maturation rate of cells in the 10nmol/L Kisspeptin group was significantly higher than that in the other four groups (P0.01). The optimum concentration of 10nmol/L was determined as the addition of exogenous Kisspeptin. They were divided into 8 groups according to the different treatment methods of culture medium, 1TCM-1992TCM-199 Kisspeptin3TCM-199 FSH4TCM-199 FSH Kisspeptin5TCM-199 Kisspeptin FBS6TCM-199 FBS7TCM-199 Kisspeptin p2348TCM-199 p234FSH.The oocytes were cultured in vitro with 8 kinds of culture medium and the maturation rate was calculated. The results showed that the maturation rate of oocytes in Kisspeptin group and Kisspeptin FSH group was higher than that in FBS group (P0.01), and the maturation rate was lower when FBS and Kisspeptin were added at the same time. Compared with the Kisspeptin group, the maturation rate was significantly lower than that of the Kisspeptin group (P0.05), and the maturation rate of the FSH group was not significantly different from that of the FSH group with the addition of FSH and p234 (P0.01), and the addition of Kisspeptin and p234 was significantly lower than that of the Kisspeptin group (P0.05). Fluorescence quantitative PCR was used to detect the expression of BMP15 and GDF9, the key genes of oocyte development during in vitro maturation (1224 h). The results showed that the expression of BMP15 and GDF9 was low at 1224 h, but the expression of BMP15 and GDF9 in cultured medium supplemented with Kisspeptin increased significantly, indicating that Kisspeptin could promote the expression of key genes in maturation.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S823

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