發(fā)布時(shí)間：2018-02-22 02:08

  本文關(guān)鍵詞： 豬卵母細(xì)胞 UCHL1抑制劑 體外成熟 透明帶泛素化 多精入卵　出處：《延邊大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：哺乳動(dòng)物的受精過(guò)程中,多精入卵被視為最普遍的異常受精,而近年來(lái)的許多研究表明,透明帶(ZP)的泛素化異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)是一種主要的去泛素化酶,研究顯示UCHL1與透明帶蛋白的泛素化水平相關(guān)。本試驗(yàn)的主要目的是探究抑制UCHL1對(duì)豬卵母細(xì)胞體外成熟、透明帶泛素化及多精入卵的影響。試驗(yàn)主要分三部分進(jìn)行:(1)UCHL1抑制劑處理GV期卵母細(xì)胞后,用DAPI染色觀察極體排放情況,從而驗(yàn)證抑制UCHL1對(duì)豬卵母細(xì)胞體外成熟率的影響;(2)UCHL1抑制劑處理GV期卵母細(xì)胞后,用SDS-PAGE和Western blot等方法檢測(cè)豬卵母細(xì)胞透明帶泛素化水平的影響;(3)UCHL1抑制劑處理GV期卵母細(xì)胞后,用Hoechst染色觀察精卵粘附及多精入卵情況。試驗(yàn)得到以下結(jié)果:1.在成熟培養(yǎng)液中添加不同濃度UCHL1抑制劑(10μM,20μM,25μM,30μM,DMSO及對(duì)照組)體外培養(yǎng)豬卵母細(xì)胞46 h后,對(duì)照組成熟率為66.22%,而30μM組的成熟率為5.30%,且各處理組成熟率差異性顯著(P0.05),結(jié)果表明抑制UCHL1對(duì)豬卵母細(xì)胞的體外成熟有影響。2.在成熟培養(yǎng)液中添加不同濃度UCHL1抑制劑(1OμM,20μM,25μM,30μM,DMSO及對(duì)照組)體外培養(yǎng)豬卵母細(xì)胞46h后,經(jīng)Westernblot檢測(cè)結(jié)果顯示各處理組的ZP在約65kDa,85kDa,120kDa處均發(fā)生不同程度的泛素標(biāo)記,通過(guò)灰度值分析差異顯著(P0.05),結(jié)果表明隨UCHL1抑制劑濃度的增加,ZP發(fā)生泛素化的程度逐漸降低。3.在成熟培養(yǎng)液中添加不同濃度UCHL1抑制劑(10μM,20μM,25μM,30μM,DMSO及對(duì)照組)體外培養(yǎng)豬卵母細(xì)胞46 h后,進(jìn)行體外受精,經(jīng)Hoechst染色顯示對(duì)照組ZP精子粘附數(shù)最多,精子入卵數(shù)最少;隨著UCHL1抑制劑濃度逐漸增大,ZP精子粘附數(shù)逐漸減少,精子入卵數(shù)逐漸增多;添加30μM UCHL1抑制劑組的ZP精子粘附數(shù)少,沒(méi)有精子入卵。結(jié)果表明UCHL1調(diào)節(jié)ZP精子粘附及多精入卵。結(jié)果顯示,UCHL1抑制劑對(duì)豬卵母細(xì)胞的體外成熟有影響。隨著UCHL1抑制劑濃度的增加,ZP發(fā)生泛素化的程度逐漸降低。UCHL1調(diào)節(jié)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.
【學(xué)位授予單位】：延邊大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S828

【相似文獻(xiàn)】

相關(guān)期刊論文 前1條

1 高島良弘 ,史榮仙;關(guān)于牛染色體異常的最新研究(下)[J];國(guó)外畜牧學(xué)(草食家畜);1987年04期

，

本文編號(hào)：1523391