化学激活在小鼠圆形精子细胞体外受精中的应用  

Application of chemical activation to in vitro fertilization by round spermatid injection in mice

作　　者：黄静[1] 姜宏[1] 汪存利[1] 宋小敏[1] 

机构地区：[1]解放军105医院生殖医学中心,安徽合肥230031

出　　处：《中华男科学杂志》2014年第2期111-116,共6页National Journal of Andrology

基　　金：南京军区医学科技创新项目(09MA040);安徽省自然科学基金(090413271X)~~

摘　　要：目的:通过对生精细胞体外培养所获圆形精子细胞显微注射(ROSI)后的小鼠卵母细胞进行单一和联合化学激活,寻找小鼠圆形精子细胞体外受精的最佳激活方案。方法:采用不同浓度的乙醇(EH)、离子霉素(Ion)、钙离子载体A23187(CIA)、氯化锶(SrCl2)、6-二甲基苯胺嘌呤(6-DMAP)及放线菌酮(CHX)对ROSI后的卵母细胞进行不同作用时间的激活,并根据不同的激活途径,联合两种最佳激活浓度和时间的激活剂对ROSI后的卵母细胞行联合激活,以受精率、卵裂率和桑囊胚形成率为观察指标,比较单一及联合方案对卵母细胞的激活效果。结果:①单一激活时,各激活剂的最佳方案分别为7%EH作用6 min,5μmol/L CIA作用5 min,5μmol/L Ion作用5 min,2 mmol/L 6-DMAP作用2 h,10 mmol/L SrCl2作用1.5 h,10μg/ml CHX作用1.5 h,其中10 mmol/L SrCl2作用1.5 h桑囊胚率最高,显著优于CHX方案(P<0.05),但与其它各组无显著性差异;②EH+6-DMAP组桑囊胚率(29.63%)显著高于其它联合激活组以及除SrCl2以外所有单一激活方案(P<0.05),且正常受精率、卵裂率及桑囊胚率均高于SrCl2组,但无显著性差异。结论:单一激活方案中10 mmol/L SrCl2作用1.5 h,联合方案中7%EH作用6 min+2 mmol/L 6-DMAP作用2 h对体外培养所获圆形精子细胞ROSI后的卵母细胞激活效果最佳。EH+6-DMAP的联合激活方案优于单一激活。Objective： To search for an optimal activation protocol by comparing the chemical activation effects of single-activator and combined activation protocols on mouse oocytes following injection of round spermatids （ROSI） from spermatogenic cells cultured in vitro. Methods ： Using different concentrations of ethanol, ionomycin （Ion） , calcium ionophore A23187 （CIA） , strontium chloride （SrCl2 ）, cycloheximide （CHX）, and 6-dimethylaminopurine （6-DMAP）, we activated post-ROSI oocytes for different times, and activated them by combined protocols at optimal concentrations and action times according to different activation channels. We compared the activation effects of single-activator and combined activation protocols by comparing the rates of fertilization, cleavages, and morulas and blastocysts. Results： With a single activator, the optimal protocols of different activators were as follows： 7% etha- nol for6 min, 5 μmol/L CIA for 5 min, 5μmol/L Ion for 5 min, 2 mmol/L 6-DMAP for 2 h, 10 mmol/L SrCl2 for 1.5 h, and 10 μg/ml CHX for 1.5 h, among which 10 mmol/L SrCl2 for 1.5 h achieved the highest rate of morulas and blastocysts, significantly better than CHX （ P 〈 0.05 ） but with no remarkable difference from other activators. The ethanol ＋ 6-DMAP group showed a significantly higher rate of morulas and blastocysts （29.63%） than all other combined activation groups and single-activator groups except SrCl2 （ P 〈 0.05 ） , and it also exhibited higher rates of normal fertilization, cleavages and morula than the SrCl2 group, but with no significant difference. Conclusion ： The single-activator 10 mmol/L SrC12 for 1.5 h and the combined activation of 7% ethanol for 6 min ＋ 2 mmol/L 6-DMAP for 2 h are the optimal protocols for chemical activation of mouse oocytes following ROSI, and the combined activation of ethanol ＋ 6-DMAP is even superior to the single-activator protocol.

关 键 词：圆形精子细胞 化学激活 卵母细胞 体外受精 显微注射 小鼠 

分 类 号：R321[医药卫生—人体解剖和组织胚胎学]