| The embryo cryopreservation is a very important technology for establishing embryo bank,which is a very important part of assisted reproductive technology(ART).Embryo vitrification is the most advanced method for embryo cryopreservation at present,it use cryoprotectants to protect embryos without form ice intracellular through ultra-rapidly cooling.Embryo vitrification is convenient operation,time saving and little frozen damage compared to the slow freezing procedure,therefore,it became research hotspots in embryo cryopreservation.But the cryoprotectants for vitrification always have high concentration which have toxic at room temperature,and it can inevitably harm embryos in frozen procedures,offsprings came from embryo vitrification may have changes in phenotypes and molecular characteristics.In this research,we compared the differences of the offsprings between normal breeding and frozen-thawed tranfer in phenotypes and molecular characteristics,the objective of this research is to investigate the influences of embryo vitrification on the offsprings,which can give some advices for establishing human embryo bank.Experiment 1.Measuration and analysis of partial biological characteristics of micederived from vitrified-warmed embryo transfer and their offspringTo investigate the average weight gain and serum biochemical indexes changes of C57BL/6J mice(B6 mouse)and their offsping after frozen-thawed embryo transfer of B6 mice.The mice were divided into three groups in this study.First generation of frozen-thawed embryos tranfer(F-Ⅰ,30 males and 20 females),collected 2-cell embryos after in-vitro fertilization,and cryopreserved by EFS method,then obtained the offspring after transplanted the recovered embryos to oviduct of recipient mice(ICR mouse);Second generation of frozen-thawed embryos tranfer(F-Ⅱ,26 males and 17 females),when the mice from F-Ⅰ grew to maturity,to obtain the offspring from natural mating of mice from F-Ⅰ;Control group(20 males and 20 females),offspring came from conventional feeding and natural mating.Three groups of mice were raised to 16 weeks old,weighing the mousebody weight at a regular time,measuring glucose tolerence of 8 weeks old mice,the serum biochemical indexes could be obtained from serums of 16 weeks old mice,then the average body weight,the glucose tolerence and serum biochemical indexes changes of the mice could be analysed.The results shows that the average body weight of F-Ⅰ mice is significantly higher than control group in every weeks(P<0.01).The average body weight of F-Ⅱ female mice is significantly higher than control group in 12～16 weeks old(P<0.01),but the average body weight of F-Ⅱ male mice has no signifcant differences compared to control group except few weeks.The glucose level of F-Ⅰ and F-Ⅱ mice is significantly higher than control group in every time points,but the situation of F-Ⅱ mice is better than F-Ⅰ mice.The serum biochemical indexes of F-Ⅰ and F-Ⅱ mice were changed in every items except for AST,TP and Ca.We can know that there are some effects on the average weight gain,glucose tolerences and serum biochemical indexes of B6 mice and their offsping after frozen-thawed embryo transfer of B6 mice.Experiment 2.Molecular characterictics of fetus derived from vitrified-warmed embryo transfer and expression and analysis of fetal and placental imprinting gene H19,Igf2 and Ube3aIn order to investigate the effect of embryo cryopreservation on embryos molecular characteristics and imprinting genes,we made embryo genechips and measurate H19/Igf2 and Ube3a expression changes of embryos and placentas after frozen-thawed embryo tranfer of B6 mice.Firstly,collected 2-cell embryos after in-vitro fertilization,cryopreserved by EFS method,then obtained the embryos and placentas after transplanted the recovered embryos to oviduct of recipient mice(ICR mouse)on 14 days(three replicates each group,collected 10 embryos or placentas from 3 to 5 mice for every replicates).First of all,made genechips of the embryos to know the gene expression changes on the whole,then measurate the mRNA level of H19/Igf2 and Ube3a using qPCR method and obtained the methylation level of CpG islands from imprinting control region(ICR)of H19/Igf2 by bisulfite method,then measurate mRNA level of Dnmts(Dnmt1,Dnmt3a,Dnmt3b)using qPCR method.The embryos and placentas of control group came from conventional feeding and natural mating,then compared and analysed the results between experimental group and control group.After frozen-thawed embryo tranfer of B6 mice,the molecular characteristics of the embryos have changed,compared to control group,595 genes were significantly up-regulated and 760 genes were significantly down-regulated(p<0.05).The H19/Igf2 and Ube3a mRNA level of experimental group were significantly lower than control group(P<0.01),the methylation level of H19/Igf2 ICR were significantly lower than control group(P<0.01),the Dnmts mRNA level of experimental group were also significantly lower than control group(P<0.01).The results shows that frozen-thawed embryo tranfer of B6 mice affected the molecular characteristics of embryos,the H19/Igf2 and Ube3a mRNA level of embryos and placentas also had changed.the methylation level of H19/Igf2 ICR had significantly down-regulated,which could affected the expression of H19 and Igf2.In addition,the expression of Dnmts were significantly down-regulated compared to control group,which could brought down the methylation level of the genome indirectly.Generally,embryo vitrification could affect the expression of H19,Igf2 and Ube3a,which could affect the body weight gain,serum biochemistry value and glucose tolerance subsequently,and all the influences might still exist in adult stage and eventhe offsprings. |
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