Effect Of EGF And IGF-â…¡ On Development Of 1-cell Mouse Embryo In Vitro

【Objective】:To study the effects of epidermal growth factor(EGF) and it combining insulin-like growth factor-Ⅱ(IGF-Ⅱ) on the development of one-cell mouse embryos in vitro,investigate the mechanism of embryogenesis and optimize the culture medium for preimplantation embryos.To provide the theoretical and experimental bases for the optimization of the culture medium of embryos.【Methods】:The 1-cell embryos were cultured up to 120h at 37℃under 5%CO2 in a sealed culture chamber and observed every 24h under a Nikon inverted microscope.The culture efficiency was evaluated by determining the proportion of one-cell,two-cell,four-cell,morula,Blastocyst and hatched blastocyst and counting the total cell counts in the blastocyst rates and hatched blastocyst.Experiment 1: One-cell embryos of ICR mouse were cultured in the mKSOM media supplemented with EGF at different concentrations and divided into five groups:Control:mKSOM medium with no EGF or IGF-Ⅱ;Treatments:4 groups,mKSOM media with 0.1,1, 10,100 ng/ml EGF respectively.Experiment 2:One-cell embryos of ICR mouse were cultured in the with mKSOM medias with EGF at the definite concentrations or with IGF-Ⅱat the definite concentrations or with EGF combining IGF-Ⅱat the definite concentrations and divided into four groups:Control:mKSOM medium with no EGF or IGF-Ⅱ;Treatments:3 groups,Group1:mKSOM media with 10ng/ml EGF;Group2:mKSOM media with 10ng/ml IGF-Ⅱ;Group3:mKSOM media with 10ng/ml EGF and 10ng/ml IGF-Ⅱ.Experiment 3 One-cell embryos of ICR mouse were cultured in the with mKSOM medias with EGF at the indefinite concentrations or with IGF-Ⅱat the indefinite concentrations or with EGF combining IGF-Ⅱat the definite concentrations and divided into two steps:Step 1:divided into four groups: (1)Control:mKSOM medium with no EGF or IGF-Ⅱ;(2) Treatments:3 groups, Group1:mKSOM media with 1ng/ml EGF;Group2:mKSOM media with 1ng/ml EGF and 1ng/ml IGF-Ⅱ;Group3:mKSOM media with 1ng/ml EGF and 10ng/ml IGF-Ⅱ.Step 2:divided into five groups:(1)Control:mKSOM medium with no EGF or IGF-Ⅱ;(2) Treatments:4 groups,Group1:mKSOM media with 10ng/ml EGF; Group2:mKSOM media with 10ng/ml EGF and 1ng/ml IGF-Ⅱ;Group3:mKSOM media with 10ng/ml EGF and 10ng/ml IGF-Ⅱ,Group4:mKSOM media with 1ng/ml EGF and 10ng/ml IGF-Ⅱ.【Results】:Experiment 1 Addtion of 1,10 ng/ml EGF significantly increasing the the proportion of blastocyst rates and hatched blastocyst rates as compared with the control(P＜0.05).However,there were no significant differences in the proportion of Blastocyst rates and hatched blastocyst rates between EGF addition to mKSOM (0.1,100 ng/ml) and EGF-free mKSOM(P＞0.05).Experiment 2:Additions of EGF with IGF-Ⅱsignificantly increased the rates of blastocyst,hatched blastocyst and the total cell numbers of hatched blastocyst as compared with three controls(P＜0.05). Only additions of EGF or IGF-Ⅱsignificantly increased the rates of blastocyst, hatched blastocyst,But the total cell numbers don't significantly increase(P＞0.05). Experiment 3 Step1:Addtion of 1 ng/ml EGF,1ng/ml EGF with 1ng/ml IGF-Ⅱ,1ng/ml EGF with 10ng/ml IGF-Ⅱsignificantly increasing the the proportion of blastocyst rates and hatched blastocyst rates as compared with the control without EGF and IGF-Ⅱ(P＜0.05);In the three groups,except the group which only addtion of 1 ng/ml EGF,the other two groups significantly increasing the the total cell numbers(P＜0.05).The two groups significantly increasing the the proportion of blastocyst rates,hatched blastocyst rate and the the total cell numbers as compared with the group which only addtion of 1 ng/ml EGF(P＜0.05).there were no significant differences in hatched blastocyst rate between the two groups(P＞0.05). Step 2:Compare to the control without EGF and IGF-Ⅱ,the other groups significantly increasing the the proportion of blastocyst rates and hatched blastocyst rates;except the group which only addtion of 10 ng/ml EGF,the other three groups significantly increasing the the total cell numbers(P＜0.05).In the three groups,the groups that of addtion 10ng/ml EGF with 10ng/ml IGF-Ⅱsignificantly increasing the the proportion of hatched blastocyst rates and the the total cell numbers(P＜0.05).【Conclusion】(1)Among the range of the concentrations 1-10ng/ml:the EGF could increase blastocyst rates and hatched blastocyst rates of 1-cell mouse embryo in vitro,but no effect on increasing the total cell counts.(2) EGF and IGF-Ⅱhave coordinating advanced effect on the development of 1-cell mouse embryo.(3) 10ng/mlEGF combining 10ng/mlIGF-Ⅱis the best group significantly increasing the the proportion of Blastocyst rates and hatched blastocyst rates of 1-cell mouse embryo in vitro.