Risk And Mechanism Of Growth And Glucose Metabolism Disorder Involed In The Offsprings Conceived By Vitrified-Warmed Mouse Ovarian Orthotopic Transplantation

Parts 1 Effect of the process of vitrified-warmed ovarian orthotopic transplantation on mouse growth and glucose metabolismObsjective To explore the effect of vitrified-warmed and orthotopic transplantation process on mouse growth and glucose metabolism.Methods To establish mouse model of vitrified-warmed ovarian orthotopic transplantation and fresh ovarian orthotopic transplantation.The offsprings were as the research object,and the experiment was divided into three groups: fresh ovarian orthotopic transplantation offsprings group,vitrified-warmed ovarian orthotopic transplantation offsprings group,natural mating birth offsprings group was normal control group(Hereinafter referred to as the Fresh group,Vitrification group,Normal group).The growth weight from both to six month,fasting blood glucose level and glucose tolerance in six-month mice were detected in three groups.Serum insulin level was determined using Rat/Mouse Insulin ELISA kit.Results At 1week,2week and 3week,the female body weight of vitrification group was significantly lower than that in fresh and normal groups(P<0.05),but at 3 month,the body weight of vitrification group was only significantly lower than that in fresh group(P<0.05).At 1 day,3day,1week,2week and 3week,the male body weight of vitrification group was significantly lower than that in normal group(P<0.05).At 1 week and 3 week,the body weight of vitrification group was significantly lower than that in fresh group(P<0.05).At 1 week,the body weight of fresh group was also significantly lower than that in normalgroup(P<0.05).The results of fasting glucose level and glucose tolerance in six-month female mice showed that fasting blood glucose levels in fresh group and vitrification group were higher than that in normal group(P<0.05).The male fasting glucose level in fresh group was only higher than that in normal group(P<0.05),there was no significant difference between vitrification and fresh groups in fale and female mice(P> 0.05).Only 120 minute after glucose injection,the famel serum glucose level in fresh group was significantly higher than that in normal group(P<0.05),and the male serum glucose level in fresh group and vitrification group were significantly higher than that in normal group(P<0.05),there was no significant difference between vitrification and fresh groups in fale and female mice(P>0.05).The result of ELISA showed that higher serum insulin level was found in fresh group and vitrification group,there was no significant difference between vitrification and fresh groups(P> 0.05).Conclusion Both vitrified-warmed ovarian and orthotopic transplantation can adversely affect the growth and glucose metabolism in offsprings,and it can cause offsprings prepubertal growth retardation and adult glucose metabolism disorder.Parts 2 Studies on the mechanism of growth retardation and glucose metabolism disorder in vitrified-warmed ovarian allotransplantation offspringObsjective To study whether the process of infiltration,oxidative stress and ischemia reperfusion injury produced by external environment,vitrified-warmed ovarian and orthotopic transplantation can change the epigenetic information of the gametes in ovarian,which may affect the expression of Grb10 gene related to growth and glucose metabolism.To clarify the mechanism of growth retardation and glucose metabolism disorder in vitrified-warmed ovarian orthotopic transplantation offsprings produced by epigenetic regulation of Grb10 expression.Methods The six-month mice liver tissues were obtained from offsprings in part 1.The protein expressions of Dnmt1,Grb10 and insulin signaling pathway factors such asP-IGF1R、P-IRS2、P-AKT、P-MAPK regulatated by Grb10 were detected by Western blot technology.The mRNA expressions of Dnmt1,Dnmt3 a,Dnmt3b,Uhrf1,Grb10 and miR-145 b were detected by qRT-PCR.The wild type(psiCHECK-2-mGRB10-3U-W)and mutan(psiCHECK-2-mGRB10-3U-M)luciferase reporter gene carriers were constructed.The relevant plasmids(mmiR-145 b mimics,mmiR-145 b mimics NTC)and luciferase reporter gene carriers were cotransfected into 293 T cells to verify the relationship between miR-145 b and Grb10.The DNA methylation status of Grb10 promoter region neighbouring fragment was determined by Sequenom DNA sequencing.Results Compared with normal group,the protein and mRNA expressions of Grb10 increased significantly in vitrification group and fresh group(P<0.05).The protein expressions of P-IGF1 R,P-IRS2,P-AKT and P-MAPK decreased significantly in vitrification group and fresh group(P<0.05),there was no significant difference between vitrification and fresh groups(P> 0.05).The protein expression of Dnmt1 in vitrification group remarkably increased than that in normal group(P<0.05).The mRNA expressions of Dnmt1 and Dnmt3 a in vitrification group and fresh group remarkably increased than that in normal group(P<0.05),and higher mRNA expressions were found in fresh group than that in vitrification group(P<0.05).The mRNA expressions of Dnmt3 b and Uhrf1 in vitrification group and fresh group remarkably increased than that in normal group(P<0.05).Compared with normal group,the expression of miR-145 b decreased significantly in vitrification group and fresh group(P<0.05),but the expression of miR-145 b in vitrification group is higher than that in fresh group,there was no significant difference between vitrification and fresh groups(P> 0.05).The result of dual luciferase report gene assay showed that miR-145 b significantly diminished luciferase activity from wild type(P<0.05),whereas no suppression of luciferase activity was found in the mutant.The result of Sequenom showed that DNA methylation status on 4 methylation sites among the 11 sites of Grb10 promoter region fragment we have checked remarkably decreased in vitrification group(P<0.05),comparedwith normal group,there was no significant difference between vitrification and fresh groups(P> 0.05).Conclusion Vitrified-warmed ovarian orthotopic transplantation could induce the decreased DNA methylation status on some methylation sites of Grb10 promoter region fragment and high expressions of Uhrf1/Dnmts which could induce decreased expression of miR-145 b in offsprings from the level before and after transcription.These two together regulate a high expression of Grb10 gene.The high expression of Grb10 can cause offsprings growth retardation and glucose metabolism disorder by inhibiting the expressions of phosphorylation factors of insulin pathway signaling.It suggests that Ischemia-reperfusion may cause offsprings prepubertal growth retardation and adult glucose metabolism disorder rather than vitrification.