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目的探讨体外培养阶段(IVC)添加褪黑素(MT)对卵母细胞老化引起发育受损的改善效果。方法采用不同浓度的过氧化氢(H_2O2)处理小鼠MII期卵母细胞诱导老化,浓度分别为0(对照组)、10、50、100、150μmol/L,体外受精(IVF)后统计各组二细胞率、囊胚形成率,检测老化卵母细胞的线粒体活性及丰度(Mitotracker Red、JC-1)、活性氧(ROS)水平、线粒体拷贝数等指标;在100μmol/L H_2O2处理条件下,老化卵母细胞在IVF后的培养阶段分别添加不同浓度褪黑素(10-5、10-7、10-9 mol/L),统计二细胞率、囊胚率,并且分别检测各组获得囊胚的细胞数及凋亡率。结果不同浓度H_2O2诱导卵母细胞老化后,囊胚发育率随H_2O2浓度的升高而降低,50μmol/L和100μmol/L H_2O2组囊胚形成率分别为(26.27±0.06)%和(28.46±3.45)%,相比对照组的(34.90±1.77)%显著降低,差异有统计学意义(P<0.05);在H_2O2诱导老化卵母细胞中,100μmol/L、150μmol/L H_2O2浓度时,ROS水平相比对照组显著升高,差异具有统计学意义(P<0.05);而活性线粒体的丰度及拷贝数呈现下降趋势,膜电位呈上升趋势,但相比对照组无显著性差异(P>0.05);100μmol/L H_2O2处理诱导卵母细胞老化后,在培养液中添加10-9 mol/L褪黑素组与老化对照组相比,囊胚率[(29.42±2.39)%vs.(20.87±4.12)%]、囊胚细胞数(39.36±9.78vs.37.91±4.25)均显著升高,凋亡率显著下降(2.57%vs.3.18%),差异均有统计学意义(P<0.05);并且这些指标均达到与未经老化处理组的相似发育水平。结论老化卵母细胞体外受精后发育率和发育质量偏低的现象,可以通过在受精后体外培养阶段添加褪黑素得到改善。
Abstract：Objective: To explore effect of supplementing melatonin into medium of in vitro culture on development potential of aging oocytes.Methods:The MII oocytes retrieved from ovaries were incubated with M2 culture medium without(as control)or with H_2O2 at different concentrations(10,50,100,150μmol/L)for 3hours to induce oocyte aging.The 2-cell rate and blastocyst rate were counted after in vitro fertilization(IVF).The mitochondrial mass and activity(Mitotracker Red and JC-1),reactive oxygen species(ROS)levels,as well as mitochondrial DNA(mtDNA)copy number were also detected in control and aging oocytes.After treated with 100μmol/L H_2O2,the aging oocytes were in vitro fertilized and cultured in medium without or with melatonin at different concentrations(10-5,10-7,10-9 mol/L).Then,the 2-cell rateand blastocyst rate were counted,and the cell number of blastocysts and apoptotic rate were detected in the each groups.Results:Different concentrations of H_2O2 induced aging of oocyte showed that the blastocyst development rate was decreased along with the increase of H_2O2 concentration.The blastocyst rate of aging oocytes induced by 50μmol/L and 100μmol/L H_2O2 was(26.27±0.06)% and(28.46±3.45)%,respectively,which were significantly lower than that in the control group[(34.90±1.77)%](P<0.05).H_2O2 at higher concentration(100or 150μmol/L)induced significantly higher intracellular reactive oxygen species(ROS)in aging oocytes(P<0.05),as well as a tendency of reduced mitochondrial mass and increased mitochondrial membrane potential(MMP),but the difference was not significant(P>0.05).The blastocyst rate[(29.42±2.39)% vs.(20.87±4.12)%,P<0.05)and the cell number of blastocysts from aging oocytes treated with 100μmol/L H_2O2 were significantly increased[(39.36±9.78)vs.(37.91±4.25),P<0.05)and the apoptotic rate was significantly decreased(2.57% vs.3.18%,P<0.05)by supplementing 10-9 mol/L melatonin into culture medium.And these indicators reached a similar level of development without aging treatment group.Conclusions: The lower development rate and development quality in aging oocytes in vitro fertilization can be reversed and improved by supplementing melatonin into culture medium.
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基本信息:
中图分类号:R714.8
引用信息:
[1]王伟周,任立坤,沈玉良,等.褪黑素对老化卵母细胞体外发育能力的改善效果评价[J].生殖医学杂志,2017,26(05):470-476.
基金信息:
海军总医院创新培育基金(CXPY201408)
2017-05-15
2017-05-15