Effect Of Insulin On The Development Of ICR Mouse Embryos In Vitro

【Objective】:By additions of insulin in the culture medium of ICR mouse embryos investigate the effection of insulin on the development of mouse preimplantation embryos in vitro,which could provide the theoretical and practice basements for 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 embryos reaching blastocyst,hatched blastocyst and its total cells.Experiment 1:Embryos were divided into six groups:Control:group 1,embyos were cultured in medium without insulin;Treatments:group 2,3,4,5,6,embyos were cultured in medium with 0.005,0.05,0.5,5 and 10 ug/ml insulin respectively.Experiment 2:Embryos were divided into six groups.Control group 1,continuous culture in insulin-free medium. Treatments:Group2,3,4,5:embryos were removed from insulin-free medium to medium containing 0.05 ug/ml insulin respectively at one-cell,two-cell,four-cell or morula stage and 24h later removed to insulin-free medium.Group 6:embryos were continuously cultured in medium containing 0.05 ug/ml insulin until the hatched blastocyst stage.The embryos of each group were shifted twice in vitro.Similarly. Experiment 3:Embryos were divided into six groups:Control group 1,Embryos were cultured in medium without insulin;Treatments:group:2-6.Embryos were cultured containing different concentrations(0.005,0.05,0.5,5 and 10 ug/ml)insulin at two-cell or four-cell culture respectively.【Results】Experiment 1:Additions of 0.005,0.05,0.5,5 and 10 ug/ml insulin significantly increased the rates of blastocyst,hatched blastocyst and the total cell numbers of hatched blastocyst as compared with the control(P＜0.05).But there were no significant differences in the rate of blastocyst and hatched blstocyst among insulin-containing groups(P＞0.05).The Culture with 0.005,0.05 ug/ml insulin significantly increased the total cell numbers of hatched blastocyst as compared with other groups(P＜0.05).But There were no significant differences with the total cell numbers between two groups.Experiment 2:Additions of insulin at 2-cell,4-cell stages and continuous culture in insulin-containing medium significantly increased the rate of blastocyst and its total cell numbers as compared with the control(P＜0.05). However,the addition of insulin at 4-cell stages significantly increased the rate of hatched blastocyst as compared with the control(P＜0.05),while additions of insulin at 1-cell or morula stages did not change the rates of blastocyst,hatched blastocyst and its total cell numbers(P＞0.05).Experiment3:Additions of different concentrations of insulin significantly increased the rates of blastocyst,hatched blastocyst and its total cell numbers as compared with the control at 2-cell(P＜0.05).But there were no significant differences in the rates of blastocyst and hatched blstocyst among insulin-containing groups(P＞0.05).The culture with 0.005 ug/ml and 0.05 ug/ml insulin significantly increased the total cell numbers of hatched blstocyst as compared with other groups(P＜0.05).There were no significant differences with the total cell numbers of hatched blstocyst between 0.005 ug/ml and 0.05 ug/ml insulin groups. Additions of different concentrations of insulin significantly increased the rates of blastocyst,hatched blastocyst and its total cell numbers as compared with the control at 4-cell(P＜0.05).But there were no significant differences in the rates of blastocyst and hatched blstocyst among insulin-containing groups(P＞0.05).Tne culture with 0.005 ug/ml and 0.05 ug/ml insulin significantly increased the total cell numbers as compared with other groups(P＜0.05).There were significantly differences with the total cell numbers between 0.005 ug/ml and 0.05 ug/ml groups(P＜0.05).【Conclusion】1,Insulin is presented in mKSOM medium could promote the development in vitro of the ICR embryos.2,The addition of insulin,from at 2-cell stage to before morula,is better than any other stages.3,There were different concentrations demand of insulin at different development stages of mouse embryos in vitro,the appropriate concentration of insulin added in the culture medium could be 0.05 ug/ml.4,In our study the range of the concentrations(0.005-10 ug/ml),insulin has no toxic effect on the development of ICR mouse embryos in vitro.