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

Effects of UCHL1 Inhibition on Porcine Oocyte Matuation in vitro,Zona Pellucida Ubiquitination and Polyspermy

试验旨在研究抑制泛素羧基末端水解酶-1(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。利用DAPI染色、Hoechst染色及SDS-PAGE等方法检测猪卵母细胞体外成熟率、透明带泛素化水平及多精入卵等情况。结果表明,添加不同浓度UCHL1抑制剂(10、20、25、30μmol/L,二甲基亚砜(DMSO)及对照组)体外培养猪卵母细胞46h后,对照组成熟率为86.22%,而30μmol/L UCHL1抑制剂组的成熟率为15.30%,且各处理组间成熟率差异显著(P<0.05);经Western blotting检测,各处理组的透明带在约61、80、106ku处均发生不同程度的泛素标记,通过灰度值分析差异显著(P<0.05);进行体外受精试验后,发现对照组透明带精子黏附数最多,精子入卵数较少,添加30μmol/L UCHL1抑制剂组的透明带精子黏附数最少,几乎没有精子入卵。结果显示,UCHL1抑制剂对猪卵母细胞的体外成熟有一定影响,随着UCHL1抑制剂浓度的增加,透明带发生泛素化的程度逐渐降低,且UCHL1可调节透明带精子黏附及多精入卵。

This study was aimed to examine the effects of UCHL1inhibition on porcine oocyte maturation in vitro,zona pellucida(ZP)ubiquitination and polyspermy.DAPI staining,Hoechst staining and SDS-PAGE methods were used to detect the matuation rate of porcine oocytes in vitro,the level of ubiquitination of zona pellucida(ZP)and polyspermy.The results showed that after different concentrations UCHL1inhibitor(10,20,25and30μmol/L,DMSO and control group)were added into maturation medium for culturing46h in vitro,the mature rate of control group was86.22%,while the maturation rate of30μmol/L group was15.30%,and the maturation rate of every treatment group had significant difference(P<0.05).Western blotting result showed that every group generated ubiquitin markers of ZP were about61,80and106ku in different degree.According to the analysis of gray value,the result had significant difference(P<0.05).Conducting fertilization in vitro,the number of sperm adhered on oocyte ZP in control group was the most,the number of sperm running into oocyte was fewer,the number of sperm adhered on oocyte ZP with30μmol/L UCHL1inhibitor was the fewest,and there was almost no sperm running into the oocyte.The results showed that UCHL1inhibitor had an impact on maturation of porcine oocytes in vitro.With the higher concentration of UCHL1,the lower degree of ZP protein ubiquitinated,UCHL1could regulate sperm attachment and polyspermy.