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

doi:10.16431/j.cnki.1671-7236.2017.12.024

摘要/Abstract

摘要：

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、30 μmol/L，二甲基亚砜（DMSO）及对照组）体外培养猪卵母细胞46 h后，对照组成熟率为86.22%，而30 μmol/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、106 ku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30 μmol/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。

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

Abstract:

This study was aimed to examine the effects of UCHL1 inhibition 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 UCHL1 inhibitor (10, 20, 25 and 30 μmol/L, DMSO and control group) were added into maturation medium for culturing 46 h in vitro, the mature rate of control group was 86.22%, while the maturation rate of 30 μmol/L group was 15.30%, 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 about 61, 80 and 106 ku 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 with 30 μmol/L UCHL1 inhibitor was the fewest, and there was almost no sperm running into the oocyte. The results showed that UCHL1 inhibitor had an impact on maturation of porcine oocytes in vitro. With the higher concentration of UCHL1, the lower degree of ZP protein ubiquitinated, UCHL1 could regulate sperm attachment and polyspermy.

Key words: porcine oocyte; UCHL1 inhibitor; IVM; ZP ubiquitination; polyspermy

中图分类号:

S814

史文姝, 金一. 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[J]. 《中国畜牧兽医》, 2017, 44(12): 3563-3569.

SHI Wen-shu, JIN Yi. Effects of UCHL1 Inhibition on Porcine Oocyte Matuation in vitro, Zona Pellucida Ubiquitination and Polyspermy[J]. , 2017, 44(12): 3563-3569.

参考文献

[1] Barrera S P, Vicente C T, Margarita T,et al. PKC-dependent GlyT1 ubiquitination occurs independent of phosphorylation:Inespecificity in lysine selection for ubiquitination[J]. PLoS One, 2015,10(9):e0138897.
[2] Nijman S M, Luna-Vargas M P, Velds A,et al. A genomic and functional inventory of deubiquitinating enzymes[J]. Cell, 2005, 123(5):773-786.
[3] Töpfer-Petersen E, Ekhlasi-Hundrieser M, Tsolova M,et al. Glycobiology of fertilization in the pig[J]. International Journal of Developmental Biology, 2008, 52(5-6):717-736.
[4] Naoto Y. Posttranslational modifications of zona pellucida proteins[J]. Advances in Experimental Medicine and Biology, 2014,759:111-140.
[5] Sekiguchi S, Kwon J, Yoshida E,et al. Localization of ubiquitin C-terminal hydrolase L1 in mouse ova and its function in the plasma membrane to block polyspermy[J]. The American Journal of Pathology, 2006, 169(5):1722-1729.
[6] Yi Y J, Manandhar G, Sutovsky M,et al. Ubiquitin C-terminal hydrolase-activity is involved in sperm acrosomal function and anti-polyspermy defense during porcine fertilization[J]. Biology of Reproduction, 2007, 77:780-793.
[7] Glickman M H, Ciechanover A. The ubiquitin-proteasome proteolytic pathway:Destruction for the sake of construction[J]. Physiological Review, 2002, 82(2):373-428.
[8] Park S J, So K H, Hyun S H. Effect of zeaxanthin on porcine embryonic development during in vitro maturation[J]. The Journal of Biomedical Research, 2017, 31(2):154-161.
[9] Riesenbeck A. Review on international trade with boar semen[J]. Reproduction in Domestic Animals, 2011, 46(2):1-3.
[10] 林筱璐,林秀娇,王秀爱,等. 不同因素对猪卵母细胞体外成熟培养的影响[J].中国畜牧兽医, 2014, 41(10):205-209.
[11] 崔晶晶,李蒙蒙,董海涛,等. 猪精子获能、顶体反应及冷休克对顶体酶抑制剂的影响[J]. 畜牧与兽医, 2014, 47(4):40-43.
[12] 谭玉燕,王志全,周海燕,等. 内质网应激在UCH-L1抑制剂诱导细胞凋亡中的作用[J].上海交通大学学报(医学版),2008,28(2):130-151.
[13] Xie M, Wang S H, Lu Z M,et al. UCH-L1 inhibition involved in CREB dephosphorylation in hippocampal slices[J]. Journal of Molecular Neuroscience, 2014,53(1):59-68.
[14] Tan Y Y, Zhou H Y, Wang Z Q,et al. Endoplasmic reticulum stress contributes to the cell death induced by UCH-L1 inhibitor[J]. Molecular and Cellular Biochemistry, 2008, 318(1-2):109-115.
[15] Burkart A D, Xiong B, Baibakov B,et al. Ovastacin, a cortical granule protease, cleaves ZP2 in the zona pellucida to prevent polyspermy[J]. Journal of Cell Science, 2012, 197(1):37-44.
[16] 安铁洙,潭建华,张学明.小鼠去透明带裸卵的体外受精及其胚胎发育[J].中国兽医学报,2002, 22(4):332-333.
[17] Okudaira Y, Wakai T, Funahashi H,et al. Levels of cyclic-AMP and cyclic-GMP in porcine oocyte-cumulus complexes and cumulus-free oocytes derived from small and middle follicles during the first 24-hour period of in vitro maturation[J]. Journal of Reproduction and Development, 2017, 63(2):191-197.
[18] Liu S, Liu X, Huang H,et al. Factors affecting efficiency of introducing foreign DNA and RNA into parthenogenetic or in vitro-fertilized porcine eggs by cytoplasmic microinjection[J]. In Vitro Cellular & Developmental Biology-animal, 2016, 52(7):13-22.
[19] Batista R I, Moro L N, Corbin E,et al. Combination of oviduct fluid and heparin to improve monospermic zygotes production duringporcine in vitro fertilization[J]. Theriogenology, 2016, 86(2):495-502.
[20] Xiong B, Zhao Y, Beall S,et al. A unique egg cortical granule localization motif is required for ovastacin sequestration to prevent premature ZP2 cleavage and ensure female fertility in mice[J]. PLoS Genetics, 2017, 13(1):1-18.
[21] Susor A, Liskova L, Toralova T,et al. Role of ubiquitin C-terminal hydrolase-L1 in antipolyspermy defense of mammalian oocytes[J]. Biology of Reproduction, 2010, 82(6):1151-1161.