油封M2与M16培养液及二甲基亚砜对小鼠双线期阻滞释放期间卵母细胞的影响
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摘要
目的探讨矿物油封闭(油封) M2液滴培养与传统M16液滴培养以及二甲基亚砜(DMSO)对双线期阻滞释放期间(即第一次减数分裂的恢复)小鼠卵母细胞形态及生存率的影响。方法提取卵母细胞,随机分成3组,分别在30μL油封M2液滴、油封过夜M16液滴和M16液滴中培养4 h至胚泡破裂期,镜下观察每组卵母细胞的形态改变及生存率。将卵母细胞随机分成3组,分别在0%(对照组)、1%、2%DMSO中培养4 h,观察不同浓度DMSO在双线期阻滞释放期间对卵母细胞的影响。结果与M16相比,油封M2培养的卵母细胞生存率更高(P <0.05),细胞形态结构更完好,对卵母细胞双线期阻滞释放的影响无统计学差异(P> 0.05)。对照组、1%、2%DMSO组卵母细胞存活率无统计学差异(P> 0.05)。2%DMSO对卵母细胞的双线期阻滞释放具有促进作用,差异有统计学意义(P <0.01)。结论在小鼠卵母细胞双线期阻滞释放期间,油封M2培养卵母细胞较M16生存率更高、形态更好。0%、1%和2%DMSO对卵母细胞的存活率无影响,而2%DMSO能更有效地促进卵母细胞双线期阻滞的释放。
Objective To study the effects of mineral oil covered M2 culture medium droplet culture,M16 droplet culture,and dimethyl sulfoxide(DMSO) on the morphology and survival rates of mouse oocytes during the release from diplotene arrest. Methods Oocytes were randomly divided into 3 groups and individually cultured for 4 h in M2 covered with mineral oil,M16 covered with mineral oil,and/or M16 only to cause germinal vesicle breakdown(GVBD). The morphological changes and survival rates of oocytes in each group were observed under the microscope. Oocytes were randomly divided into 3 groups and cultured in the medium with 0%,1%,and 2% DMSO. The effect of DMSO on oocytes was also observed during the release from diplotene arrest. Results The survival rates of oocytes in M2 covered with mineral oil were higher than those in M16(P < 0.05). There was no statistical difference with respect to release of mouse oocytes from diplotene arrest between the oocytes in M2 covered with mineral oil and oocytes in M16. The shape of oocytes in M2 with mineral oil was better than that of oocytes in M16. The effect of DMSO on the survival rate of oocytes was similar in the medium with 0%,1% and 2%DMSO. But the effect of 2% DMSO on the release of oocytes was statistically significant(P < 0.01). Conclusion During the release of mouse oocytes from diplotene arrest,oocytes in M2 covered with mineral oil have much better morphology and higher survival rate than those in M16. DMSO(0%,1% and 2%) has no effect on the survival rate of oocytes. However,2% DMSO is more effective in promoting the release of mouse oocytes from diplotene arrest.
Objective To study the effects of mineral oil covered M2 culture medium droplet culture,M16 droplet culture,and dimethyl sulfoxide(DMSO) on the morphology and survival rates of mouse oocytes during the release from diplotene arrest. Methods Oocytes were randomly divided into 3 groups and individually cultured for 4 h in M2 covered with mineral oil,M16 covered with mineral oil,and/or M16 only to cause germinal vesicle breakdown(GVBD). The morphological changes and survival rates of oocytes in each group were observed under the microscope. Oocytes were randomly divided into 3 groups and cultured in the medium with 0%,1%,and 2% DMSO. The effect of DMSO on oocytes was also observed during the release from diplotene arrest. Results The survival rates of oocytes in M2 covered with mineral oil were higher than those in M16(P < 0.05). There was no statistical difference with respect to release of mouse oocytes from diplotene arrest between the oocytes in M2 covered with mineral oil and oocytes in M16. The shape of oocytes in M2 with mineral oil was better than that of oocytes in M16. The effect of DMSO on the survival rate of oocytes was similar in the medium with 0%,1% and 2%DMSO. But the effect of 2% DMSO on the release of oocytes was statistically significant(P < 0.01). Conclusion During the release of mouse oocytes from diplotene arrest,oocytes in M2 covered with mineral oil have much better morphology and higher survival rate than those in M16. DMSO(0%,1% and 2%) has no effect on the survival rate of oocytes. However,2% DMSO is more effective in promoting the release of mouse oocytes from diplotene arrest.
引文
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[2]ZHENG P,BAIBAKOV B,WANG XH,et al.PtdIns(3,4,5)P3 is constitutively synthesized and required for spindle translocation during meiosis in mouse oocytes[J].Cell Sci,2013,126(Pt 3):715-721.DOI:10.1242/jcs.118042.
[3]MENG J,CUI C,LIU Y,et al.The role of 14-3-3εinteraction with phosphorylated Cdc25B at its Ser321 in the release of the mouse oocyte from prophaseⅠarrest[J].PLos One,2013,8(1):e53633.DOI:10.1371/journal.pone.0053633.
[4]李俊东,裴晓川,白春强,等.二甲基亚砜(DMSO)对骨髓瘤细胞RPMI8226影响的研究[J].吉林医学,2017,38(1):83-84.
[5]NAGY A,GERTSENSTEIN M,VINTERSTEN K,等.小鼠胚胎操作实验手册[M].第3版.北京:化学工业出版社,2006:1-537.
[6]LETE MG,BYRNE RD,ALONSO A,et al.Vesicular PtdIns(3,4,5)P3 and Rab7 are key effectors of sea urchin zygote nuclear membrane fusion[J].Cell Sci,2017,130(2):444-452.DOI:10.1242/jcs.193771.
[7]ZHAO MH,KIM NH,CUI XS.Zinc depletion activates porcine metaphaseⅡoocytes independently of the protein kinase C pathway[J].In Vitro Cell Dev Biol Anim,2014,50(10):945-951.DOI:10.1007/s11626-014-9784-8.
[8]DA ROSA PRA,DE CESARO MP,PEREIRA DAU AM,et al.Reversible meiotic arrest of bovine oocytes by EGFR inhibition and follicular hemisections[J].Theriogenology,2017,99:53-62.DOI:10.1016/j.theriogenology.2017.05.014.
[9]ABOUZARIPOUR M,FATHI F,DANESHI E,et al.Combined effect of retinoic acid and basic fibroblast growth factor on maturation of mouse oocyte and subsequent fertilization and development[J].Int JFertil Steril,2018,12(1):68.DOI:10.22074/ijfs.2018.5293.