小鼠胚胎体外培养方法的优化及胚胎质量评估

The Evaluation and Optimization of the Early Embryos' Development of Mouse

实验采用输卵管收集获得的 2 细胞、原核受精卵和体外受精得到的胚胎。 2 细胞胚胎在所有培养浓度下均达到较高的囊胚发育率 ,而ISF和IVF受精卵在减低培养浓度后发育率显著降低 (p <0 .0 1 ) ,ISF受精卵从高密度 (1 /μl)下约 82 .5 %的发育率到低密度 (1 /1 0 0 0 μl)下 2 2 .3 %的发育率。而IVF胚胎的这两个值分别为 46 .3 %和5 .2 %。2 细胞胚胎与其他两种受精卵相比 ,每个囊胚所含的细胞数目差异显著 (p <0 .0 1 )。添加 1ng/ml(1 /1 0 μl)和 1 0ng/ml(1 /1 0 0 μl)的PAF显著增加了IVF胚胎的囊胚发育率 (p <0 .0 1 )。在胚胎密度为 1 /1 0 μl的培养密度下 ,添加 1 0ng/ml以上的IGF Ⅰ同样改善IVF受精卵的发育能力 (p <0 .0 1 )。培养液中添加EGF对囊胚的发育率没有影响。PAF和IGF Ⅰ协同作用的效果与IGF Ⅰ单独处理的结果并无差异 (p >0 .0 5) ,但高于PAF单独处理组 (p <0 .0 5)。结果表明 ,胚胎生长所需的生长因子在低密度培养条件下被稀释并影响胚胎的正常发育。PAF和IGF Ⅰ作为自分泌性胚胎营养因子在一定程度上补偿了由于低密度培养所带来的负效应。

Embryos were collected at the zygote or 2-cell.zygotes were produced by fertilization in situ(ISV)or in vitro(IVF).Two-cell-stage embryos had a high rate of development to the blastocyst stage across the embryo concentration range of 1/μl～1/1000*!μl.By contrast,zygotes produced by either ISF or IVF were adversely affected by reduced the embryo concentration over this range(p<0.01),with approximate 82.5% of ISF zygotes developing to blastocysts at highest concentration but only 22.3% at the lowest.For IVF zygotes the corresponding results were 46.3% and 5.2%.Compared 2-cell embryos with ISF and IVF groups,the number of cell in each blastocyst was significantly higher.Platelet-activating factor(PAF) supplymentation of media caused a significant increase in the rate of blastocyst development of IVF zygotes at embryo concentration of 1/10*!μl(10*!ng/ml) and 1/100*!μl(100*!ng/ml).Insulin-like growth factor(IGF)(10*!ng/ml)also stimulated development of IVF zyogtes when cultured at an embryo concentration of 1/10*!μl.Eodermal growth factor was without effect over range of 1～1000*!ng/ml.Supplymentation of media with both PAF and IGF-Ⅰ gave no additional benefit over that caused by IGF-Ⅰ along,but this treatment was marginally better(p<0.05) than PAF treatment along.The results show that factors necessary for normal embryo development are diluted to suboptimal levels during culture at low embryo concentration.The ability of PAF,IGF-Ⅰ to partially compensate for the adverse effects of low embryo concentration during culture is consistent with having roles as autocrine embryotrophic factors.