抑制UCHL1对猪卵母细胞体外成熟、透明带泛素化及多精入卵的影响

发布时间：2018-02-22 02:08

本文关键词： 猪卵母细胞 UCHL1抑制剂体外成熟透明带泛素化多精入卵　出处：《延边大学》2017年硕士论文　论文类型：学位论文

【摘要】：哺乳动物的受精过程中,多精入卵被视为最普遍的异常受精,而近年来的许多研究表明,透明带(ZP)的泛素化异常是造成多精入卵的主要原因之一。泛素羧基末端水解酶-1(UCHL1)是一种主要的去泛素化酶,研究显示UCHL1与透明带蛋白的泛素化水平相关。本试验的主要目的是探究抑制UCHL1对猪卵母细胞体外成熟、透明带泛素化及多精入卵的影响。试验主要分三部分进行:(1)UCHL1抑制剂处理GV期卵母细胞后,用DAPI染色观察极体排放情况,从而验证抑制UCHL1对猪卵母细胞体外成熟率的影响;(2)UCHL1抑制剂处理GV期卵母细胞后,用SDS-PAGE和Western blot等方法检测猪卵母细胞透明带泛素化水平的影响;(3)UCHL1抑制剂处理GV期卵母细胞后,用Hoechst染色观察精卵粘附及多精入卵情况。试验得到以下结果:1.在成熟培养液中添加不同浓度UCHL1抑制剂(10μM,20μM,25μM,30μM,DMSO及对照组)体外培养猪卵母细胞46 h后,对照组成熟率为66.22%,而30μM组的成熟率为5.30%,且各处理组成熟率差异性显著(P0.05),结果表明抑制UCHL1对猪卵母细胞的体外成熟有影响。2.在成熟培养液中添加不同浓度UCHL1抑制剂(1OμM,20μM,25μM,30μM,DMSO及对照组)体外培养猪卵母细胞46h后,经Westernblot检测结果显示各处理组的ZP在约65kDa,85kDa,120kDa处均发生不同程度的泛素标记,通过灰度值分析差异显著(P0.05),结果表明随UCHL1抑制剂浓度的增加,ZP发生泛素化的程度逐渐降低。3.在成熟培养液中添加不同浓度UCHL1抑制剂(10μM,20μM,25μM,30μM,DMSO及对照组)体外培养猪卵母细胞46 h后,进行体外受精,经Hoechst染色显示对照组ZP精子粘附数最多,精子入卵数最少;随着UCHL1抑制剂浓度逐渐增大,ZP精子粘附数逐渐减少,精子入卵数逐渐增多;添加30μM UCHL1抑制剂组的ZP精子粘附数少,没有精子入卵。结果表明UCHL1调节ZP精子粘附及多精入卵。结果显示,UCHL1抑制剂对猪卵母细胞的体外成熟有影响。随着UCHL1抑制剂浓度的增加,ZP发生泛素化的程度逐渐降低。UCHL1调节ZP精子粘附以及多精入卵。
[Abstract]:During mammalian fertilization, polyospermia is considered to be the most common abnormal fertilization, and many studies in recent years have shown that, The abnormal ubiquification of ZP is one of the main causes of multiple spermatozoa. UCHL1) is a major deubiquitin enzyme. The main purpose of this study was to investigate the inhibition of UCHL1 on the maturation of porcine oocytes in vitro. Effects of pellucida ubiquitization and polyospermia into oocytes. The experiment was divided into three parts. After treated with the inhibitor of 1: 1 and UCHL1, the DAPI staining was used to observe the emission of polar bodies. The effect of inhibition of UCHL1 on the maturation rate of porcine oocytes in vitro was verified. After the treatment of GV phase oocytes with UCHL1 inhibitor, the effect of SDS-PAGE and Western blot on the level of ubiquitin in the pellucida of porcine oocytes was detected after the treatment of GV phase oocytes with UCHL1 inhibitor. The following results were obtained: 1. Porcine oocytes were cultured in vitro for 46 h with different concentrations of UCHL1 inhibitor 10 渭 M ~ 20 渭 m ~ (20 渭 m) and 30 渭 m ~ (30 渭 m) DMSO, respectively. The maturation rate of control group was 66.222.The maturation rate of 30 渭 M group was 5.30, and the difference of maturation rate of each treatment group was significant (P 0.05). The results showed that inhibition of UCHL1 had an effect on the maturation of porcine oocytes in vitro. 2. Add different concentrations of UCHL1 inhibitor to maturation medium. Porcine oocytes were cultured in vitro for 46 h. The results of Westernblot showed that Ubiquitin labeling occurred at about 65kDa, 85kDa and 120kDa in all treatment groups. The results of gray-scale analysis showed that the degree of ubiquification of ZP decreased gradually with the increase of UCHL1 inhibitor concentration. After cultured in vitro for 46 h, porcine oocytes were cultured with different concentrations of UCHL1 inhibitor (10 渭 m ~ 20 渭 m ~ (20) 渭 m ~ (20)) and control group (n = 30). In vitro fertilization, Hoechst staining showed that the number of ZP sperm adhesion was the highest and the number of sperm entering eggs was the least in the control group, and with the increase of the concentration of UCHL1 inhibitor, the adhesion number of ZP spermatozoa decreased gradually, and the number of sperm entered eggs increased gradually. The number of sperm adhesion in the 30 渭 M UCHL1 inhibitor group was less than that in the control group. The results showed that UCHL1 regulated ZP sperm adhesion and multiple spermatozoa. The results showed that UCHL1 inhibitor had an effect on the maturation of porcine oocytes in vitro. With the increase of UCHL1 inhibitor concentration, the degree of ubiquification of ZP was gradually increased. Decreased. UCHL1 regulated ZP sperm adhesion and multiple spermatozoa.
【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S828

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