Effect Of Glucose On Development Of Mouse Embryos In Vitro

[Objective]:Try to study the effects and the roles of glucose on the development of mouse embryos in vitro. To provide the theoretical and experimental bases for the optimization of the culture medium of embryos.[Methods]:Experiment 1:To observe the effects of different concentrations of glucose on the development of one-cell mouse embryos in vitro. Embryos were divided into six groups: Control: group 1, embyos were cultured in CZB medium with no glucose; Treatments: group 2,3,4,5,6, embyos were cultured in CZB media with 0．5,1．0, 3．0, 5．0 or 10mmol/L glucose respectively. The embryos were cultured up to 120h at 37°c 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 two-cell, four-cell, morula, blastocyst, or hatched blastocyst, counting the total cell counts in the blastocysts and hatched blastocysts. Experiment 2: To observe the effects of glucose on differently developmental stages of mouse embryos. Embryos were divided into six. Control:group 1, continuous culture in glucose-free CZB. Treatments:group:2,3,4,5,6．Group2,3,4,5: embryos were removed from glucose-free CZB to CZB containing 3．0mmol/L glucose respectively at one-cell, two-cell, four-cell or morula stage and removed to glucose-free CZB 24h later. Group 6: embryos were continuously cultured in CZB containing 3．0mmol/L glucose. The embryos of each group were shifted twice in vitro. Similarly, measuring the percentages of two-cell, four-cell, morula, blastocysts, or hatched blastocysts and counting the total cell counts in the blastocysts and hatched blastocysts. [Results] : Experiment 1: There were no significant differences in the proportions of two-cell, four-cell or morula embryos between glucose addition to CZB (0．5,1．0,3．0,5．0or10mmol/L) and glucose-free CZB(P>0．05) . However, addition of 0．5, 1．0, 3．0, 5．0 or10．0mmol/L glucose significantly increased blastocyst/hatched blastocyst development and the total cell counts as compared with controls(P<0．05) . But there were no significant differences in the rates of four-cell, blastocyst or hatched blstocyst among glucose-containing groups(P > 0．05) . Culture with 3．0mmol/L glucose significantly increased the total cell numbers as compared with other groups(P<0．05) . Experiment 2: Addition of glucose at two-cell, four-cell stages and continuous culture in glucose-containing CZB significantly increased the rate of blastocyst as compared with control(P<0．05) . However, only addition of glucose at 4-cell stages significantly increased the rate of hatched blastocyst as compared with control(P<0．05) ,while addition of glucose at one-cell or morula stages did not increase the rates of blastocyst and hatched blastocyst(P>0．05) . Compared among the treatments, the blstotocyst rate of addition of glucose at two-cell, four-cell stages or continuous culture in glucose-containing CZB were significantly higher than that of addition of glucose at one-cell or morula stages(P< 0．05) , and the hatched blastocyst rate of addition of glucose at four-cell stages was significantly higher than that of addition of glucose at one-cell or morula stages(P< 0．05) .[Conclusion] :(1) The is no inhibitory effect on the development of one-cell mouse embryos in vitro when glucose is presented in CZB medium for the first 48 hour culture.(2) In our study range of the concentration(0．5mmol/L-10mmol/L), glucose has no toxic effect on development of the mouse embryos in vitro.(3) The optimal concentration of glucose added in medium could be 3．0mmol/L.(4) Addition of glucose, beginning at 2-cell and extending to morula before, is necessary to development of mouse embryos in vitro.