LDL对玻璃化冷冻解冻培养MⅡ期猪卵母细胞线粒体膜电位和透明带泛素化水平的影响

doi:10.16431/j.cnki.1671-7236.2017.07.009

摘要/Abstract

摘要：

本试验旨在探究添加低密度脂蛋白（low density lipoprotein，LDL）对玻璃化冷冻解冻后MⅡ期猪卵母细胞发育率、线粒体膜电位及透明带泛素化水平的影响。采用线粒体膜电位特异性探针JC-1检测各处理组线粒体膜电位，并采用SDS-PAGE及Western blotting方法分析不同处理组MⅡ期猪卵母细胞透明带蛋白泛素化水平。结果显示，玻璃化冷冻法处理中，10 mg/mL LDL处理组MⅡ期卵母细胞正常率和成熟率（71.92%和69.86%）显著高于对照组（58.26%和54.55%）（P<0.05），最接近未冷冻组。10 mg/mL LDL处理组MⅡ期卵母细胞线粒体膜电位显著高于对照组、1和20 mg/mL LDL组（P<0.05）。免疫印迹结果显示，未冷冻组和LDL处理组MⅡ期卵母细胞ZP1、ZP2及ZP3蛋白分别在61、80、106 ku发生不同程度的泛素化标记；10 mg/mL LDL处理组ZPs（ZP1、ZP2、ZP3）蛋白泛素化水平显著高于1和20 mg/mL LDL组（P<0.05），但显著低于非冷冻组（P<0.05）。结果表明LDL可改善玻璃化冷冻解冻培养后MⅡ期猪卵母细胞成熟率、线粒体膜电位及透明带泛素化水平。

关键词: 卵母细胞; 玻璃化冷冻; 低密度脂蛋白; 线粒体膜电位; 透明带泛素化

Abstract:

The purpose of this study was to determine the effect of LDL addition on oocyte developmental rate,mitochondrial membrane potential and ZP protein ubiquitination level in MⅡ oocytes from vitrified-warmed GV oocytes.Mitochondrial membrane potentials were labeled with specific probe JC-1,and ZP protein samples of different treatment groups in MⅡ oocytes were analyzed by SDS-PAGE and Western blotting methods. The results showed that normal rate and in vitro maturation of MⅡ oocytes in the 10 mg/mL LDL group (71.92% and 69.86%) were significantly higher than those in control group (58.26% and 54.55%)(P<0.05),that were nearest to non-vitrified group.The mitochondrial membrane potential of MⅡ oocytes in the 10 mg/mL LDL group was significantly higher than those in control group and 1,20 mg/mL LDL groups (P<0.05).The ubiquitinated ZP1,ZP2 and ZP3 of MⅡ oocytes in non-vitrified group and LDL treated groups were labeled at 61,80 and 106 ku,respectively.The ubiquitinated ZPs (ZP1,ZP2 and ZP3) level of MⅡ oocytes in the 10 mg/mL LDL group was significantly higher than those in 1 and 20 mg/mL LDL groups (P<0.05),while significantly lower than that of non-vitrified group (P<0.05). In conlusion, LDL could improve the maturation rate, mitochondrial membrane potential and ubiquitinated ZPs level of MⅡ oocytes from vitrified-warmed GV oocytes.

Key words: oocytes; vitrification; low density lipoprotein (LDL); mitochondrial membrane potential; ZP protein ubiquitination

中图分类号:

S814

姜泽, 玄彪, 李作臣, 王轶男, 金一. LDL对玻璃化冷冻解冻培养MⅡ期猪卵母细胞线粒体膜电位和透明带泛素化水平的影响[J]. 《中国畜牧兽医》, 2017, 44(7): 1961-1966.

JIANG Ze, XUAN Biao, LI Zuo-chen, WANG Yi-nan, JIN Yi. Effect of LDL on the Mitochondrial Transmembrane Potential and ZP Protein Ubiquitination in Vitrified-warmed MⅡ Oocytes[J]. , 2017, 44(7): 1961-1966.

参考文献

[1] Kim S,Lee Y J,Kim J. Changes in sperm membrane and ROS following cryopreservation of liquid boar semen stored at 15℃[J].Anim Reprod Sci,2011,124(1-2):118-124.
[2] Oldenhof H,Gojowsky M,Wang S,et al. Osmotic stress and membrane phase changes during freezing of stallion sperm:Mode of action of cryoprotective agents[J].Biol Reprod,2013,88(3):1-11.
[3] Horvath G,Seidel G E Jr. Vitrification of bovine oocytes after treatment with cholesterol-loaded methyl-beta-cyclodextrin[J].Theriogenology,2006,66(4):1026-1033.
[4] Moraes E A,Matos W C,Graham J K,et al. Cholestanol-loaded cyclodextrin improves the quality of stallion spermatozoa after cryopreservation[J]. Anim Reprod Sci,2015,158:19-24.
[5] Cobo A,Diaz C. Clinical application of oocyte vitrification:A systematic review and meta-analysis of randomized controlled trials[J].Fertil Steril,2011,96(2):277-285.
[6] Trokoudes K M, Pavlides C, Zhang X. Comparison outcome of fresh and vitrified donor oocytes in an egg-sharing donation program[J].Fertil Steril,2011,95(6):1996-2000.
[7] Fragouli E,Wells D. Mitochondrial DNA assessment to determine oocyte and embryo viability[J].Semin Reprod Med,2015,33(6):401-409.
[8] Aitken R J,Nixon B. Sperm capacitation:A distant landscape glimpsed but unexplored[J].Mol Hum Reprod,2013,19(12):785-793.
[9] Yonezawa N. Posttranslational modifications of zona pellucida proteins[J].Adv Exp Med Biol,2014,759:111-140.
[10] Bogliolo L,Ledda S,Innocenzi P,et al.Raman microspectroscopy as a non-invasive tool to assess the vitrification-induced changes of ovine oocyte zona pellucida[J].Cryobiology,2012,64(3):267-272.
[11] Schmidt M,Hyttel P,Greve T,et al.Ultrastructure of frozen-thawed bovine in vitro matured oocytes[J].Theriogenology,1993,39:304.
[12] 毋状元,罗永明,董红,等.不同卵黄成分、大豆卵磷脂及β-环糊精胆固醇对马精子冷冻效果的影响[J].中国畜牧兽医,2016,43(9):2435-2440.
[13] Seidel G E Jr.Modifying oocytes and embryos to improve their cryopreservation[J].Theriogenology,2006,65(1):228-235.
[14] Zeron Y,Sklan D,Arav A. Effect of polyunsaturated fatty acids upplementation on biophysical parameters and chilling sensitivity of ewe oocytes[J].Mol Reprod Dev,2002,61(2):271-278.
[15] Moore A I,Squires E L,Graham J K.Adding cholesterol to the stallion sperm plasma membrane improves cryosurvival[J].Cryobiology,2005,51(3):241-249.
[16] Hochi S,Kato M,Ito K,et al. Nuclear transfer in cattle:Effect of linoleic acid-albumin on freezing sensitivity of enucleated oocytes[J].Vet Med Sci,2000,62(10):1111-1113.
[17] Buschiazzo J,Ialy-Radio C,Auer J,et al. Cholesterol depletion disorganizes oocyte membrane rafts altering mouse fertilization[J].PLoS One,2013,8(4):e62919.
[18] Choi Y H,Velez I C,Macías-García B,et al. Effect of clinically-related factors on in vitro blastocyst development after equine ICSI[J].Theriogenology,2016,85(7):1289-1296.
[19] 朱淑文,张菁,华修国,等.玻璃化冷冻对猪卵母细胞超微结构的影响[J].上海交通大学学报(农业科学版),2006,24(2):121-126.
[20] 戴建军,吴彩凤,张廷宇,等.3种冷冻方法对猪MⅡ期卵母细胞线粒体分布和损伤的影响[J].畜牧兽医学报,2012,43(10):1525-1530.
[21] Huang W T,Wallenhorst C K,Wallenhorst S,et al. Transfer of porcine embryos through mini-laparotomy[J]. Theriogenology,2002,57(5):1533-1537.
[22] Bielańska-Osuchowska Z.Oogenesis in pig ovaries duringthe prenatal period:Ultrastructure and morphometry[J].Reprod Biol,2006,6(2):161-193.
[23] Tarazona A M, Rodríguez J I, Restrepo L F, et al. Mitochondrial activity,distribution and segregation in bovine oocytes and in embryos produced in vitro[J].Reprod Domest Anim,2006,41(4):5-11.
[24] Hales K G. The machinery of mitochondrial fusion,division,and distribution, and emerging connections to apoptosis[J].Mitochondrion,2004,4(4):285-308.
[25] Song W,Peng Z,Chen X,et al. Effects of vitrification on outcomes of in vivo mature,in vitro mature and immature human oocytes[J].Cell Physiol Biochem,2016,38:2053-2062.