| [Objective] To investigate the effection of glucose, insulin, IGF-Ⅱand EGF on the development of ICR mouse preimplantation embryos in the CZB culture medium 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 calculating the proportion of blastocyst, hatched blastocyst and their total cells. Experiment 1:To observe the effect of glucose, insulin and IGF-Ⅱon the development of mouse embryos. Embryos were divided into eight groups:Control:groupl, embryos were cultured in CZB medium; Treatments: group 2, CZB+IGF-Ⅱ(0.1 ng/ml); group 3, CZB+Ins (0.05 ug/ml); group 4, CZB+IGF-Ⅱ(0.1 ng/ml)+Ins (0.05 ug/ml); group 5, CZB+Glu (5 mmol/l); group 6, CZB+Glu (5 mmol/l)+IGF-Ⅱ(0.1 ng/ml); group 7, CZB+Glu (5 mmol/l)+Ins (0.05 ug/ml); group 8, CZB+Glu (5 mmol/l)+IGF-Ⅱ(0.1 ng/ml)+Ins (0.05 ug/ml). Experiment 2:To observe the effect of different concentrations of IGF-Ⅱon the development of mouse embryos with glucose and insulin. Embryos were divided into six groups:Control:group 1, embryo were cultured in CZB medium; Treatments: group 2,3,4,5,6:embryos were cultured in CZB+Glu (5 mmol/l)+Ins (0.05 ug/ml) medium with 0,0.1,1,10,100 ng/ml IGF-Ⅱ. Experiment 3:To observe the effect of different concentrations of EGF on the development of mouse embryos with glucose and insulin. Embryos were divided into six groups:Control:group 1, embryos were cultured in CZB medium; Treatments:group 2,3,4,5,6:embryos were cultured in CZB+Glu (5 mmol/l)+Ins (0.05 ug/ml) medium with 0,0.1,1,10,100 ng/ml EGF. Experiment 4:To observe the effect of different concentrations of EGF on the development of mouse embryos with glucose, insulin and IGF-Ⅱ. Embryos were divided into six groups:Control:group 1, embryos were cultured in CZB medium; Treatments:group 2,3,4,5,6:embryos were cultured in CZB+Glu (5 mmol/l)+Ins (0.05 ug/ml)+IGF-Ⅱ(0.1 ng/ml) medium with 0,0.1,1,10,100 ng/ml EGF.[Results] Experiment 1:Comparing with the control group, there were no differences in the rate of blastocyst and hatched blastocyst between group 2,3,4 and control (P> 0.05). But the rate of blastocyst and hatched blastocyst of group 5,6,7,8were increased (P<0.05). And the total cell numbers of blastocyst of all treatment groups were increased (P<0.05). By comparing among the treatment groups, there were no differences in the rate of blastocyst and hatched blastocyst among group 2,3,4 (P> 0.05), the same results of group 6,7,8 (P>0.05). But the rate of blastocyst and hatched blastocyst of group 5 were increased comparing with group 2,3,4 (P<0.05); reduced comparing with group 6,7,8 (P<0.05). And also the rate of group 6,7,8 were increased comparing with group 2,3,4 (P<0.05). There were no differences in the total cell numbers of blastocyst among group 2,3,4,5 (P>0.05), the same results of group 7,8 (P>0.05). But the total cell numbers of blastocyst of group6 were increased comparing with group 2 (P<0.05). And also of group 7,8, they were increased comparing with other treatment groups (P<0.05). Experiment 2: Comparing with the control group, the rate of blastocyst, hatched blastocyst and the total cell numbers of blastocyst of all treatment groups were increased (P<0.05). By comparing among the treatment groups, there were no differences in the rate of blastocyst and hatched blastocyst among group 2,3,4,5 (P>0.05), but the rate of group 2,3,4,5 were increased comparing with group 6 (P<0.05). There were no differences in the total cell numbers of blastocyst among treatment groups (P>0.05). Experiment 3:Comparing with the control group, there were no differences in the rate of morula,blastocyst and hatched blastocyst between group 2,6 and control (P>0.05). But the rate of morula, blastocyst and hatched blastocyst of group 3,4,5 were increased comparing with control (P<0.05). The total cell numbers of blastocyst of all treatment groups were increased (P<0.05). By comparing among the treatment groups, there were no differences in the rate of morula, blastocyst and hatched blastocyst between group 2 and group 6 (P>0.05), the same results of group 3,4,5 (P>0.05). The rate of morula,blastocyst and hatched blastocyst of group 3,4,5 were increased comparing with other treatments (P<0.05). And the total cell numbers of blastocyst of group 2,3,4,5 were increased comparing with group 6 (P<0.05). Experiment 4: Comparing with the control group, there were no differences in the rate of morula of group 2,6 (P>0.05). But the rate of morula of group 3,4,5 were increased (P<0.05). The rate of blastocyst and hatched blastocyst, the total cell numbers of blastocyst of all treatment groups were increased (P<0.05). By comparing among the treatment groups, there were no differences in the rate of morula, blastocyst and hatched blastocyst between group 2 and group 6 (P>0.05). Also, there were no differences in the rate of morula and blastocyst of group 3,4,5 (P>0.05), which were highter than other treatments (P<0.05). The rate of hatched blastocyst of group 4,5 were greatly higher than group 2,6 (P<0.05); lower than group 3 (P<0.05). And the rate of the total cell numbers of blastocyst of group 2,3,4,5 were highter than group 6 (P<0.05).[Conclusion] 1. Glucose could increase the rate of blastocyst, hatched blastocyst and the total cell numbers of blastocyst and promote the development in vitro of the ICR mouse embryos.2. Insulin and IGF-Ⅱcould increase the rate of blastocyst and hatched blastocyst in vitro only when glucose is added in CZB medium.3. Insulin and IGF-Ⅱcould increase the total cell numbers of blastocyst without glucose. If glucose joins the process, they play more important role in it. And IGF-Ⅱis less effective than insulin in this process.4. In promoting the ICR mouse embryonic development, it is not obviously that IGF-Ⅱ(0.1,1,10 ng/ml), insulin and glucose could suppress or promote among. But high concentration of IGF-Ⅱ(100 ng/ml), insulin and glucose exist inhibition.5. Adding EGF (0.1-10 ng/ml) in CZB medium with glucose, insulin and IGF-Ⅱcould greatly increase the rate of morula,blastocyst and hatched blastocyst. It involves in the regulation of differentiation of embryonic cells to improve embryo quality, and does not significantly promote the the proliferation.6. EGF, that of the appropriate consistency is 0.1 ng/ml, with glucose, insulin and IGF-Ⅱcould greatly promote embryo hatching.7. High concentration of EGF (100 ng/ml) with glucose, insulin and IGF-Ⅱrestrains differentiation and proliferation of embryonic cells.
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