Alterated Expression And Epigenetic Modification Of Genes In Renal Renin-angiotensin System In Mice Conceived From In-vitro Oocyte Maturation

Objective:To illustrate the impacts of in-vitro oocyte maturation (IVM) on the expression and epigenetic modification of genes in renal renin-angiotensin system in miceMaterials and methods:1. Immature oocytes, used for IVM, were obtained after the injection of pregnant mare serum gonadotropin (PMSG) in C57BL/6J mice, which were cultured to MII stage oocytes (IVM oocytes) in vitro. Mature oocytes (CON oocytes) were acquired after the injection of PMSG and human chorionic gonadotrophin (HCG) in C57BL/6J mice. Then IVM oocytes and CON oocytes both were fertilized by ICSI and transferred to pseudopregnant mice, respectively. The kidney tissue were obtained after the offspring of IVM and CON were sacrificed at10w and1.5y respectively.2. By the use of real-time quantitative PCR, genes of renal RAS and miRNAs (miRNA69S, miRNA27a., miRNA155and miRNA143) were meassured in lOw and1.5y mice, respectively.3. The methylation status of renal Agt and Ace was deternined by pyrosequencing in 10w and1.5y mice of the above two groups, respectively.4. The AG1\ACE*ACE2and AGTR1protein in lOw and1.5y mouse kidneys of the above two groups were detected by Western blot.Result (s):1. Effects of IVM on the expression levels of renal RAS(1) Expression levels of renal RAS and miRNAs in10w mice At10w, when conpared to CON offspring, renal Agt, Ace, Renl, Ren2and Agtr-1b showed significantly up-regulated in IVM offspring, but the mRNA level of Agtr-1a and Ace2of IVM offspring were lower than CON offspring. And no significant differences were found in Agtr-2between the above two groups. While compared to CON offspring, the mRAN levels of renal miRNA-69%, miRNA-27a and miRNA-155of IVM offspring were increased significantly. And there were no significant differences in the miRNA-\43mRNA level between the above two groups.(2) Expression levels of renal RAS and miRNAs in1.5y mice At1.5y, when compared to CON offspring, renal Agt and Ace of IVM offspring were significantly increased, but renal Renl, Agtr-1a, Agtr-1b, Ace2and Agtr-2of IVM were significantly decreased. And there were no significant differences in renal Ren2between the above two groups. While compared to CON offspring, miRNA-69%, miRNA-27a and miRNA-143mRAN levels were significantly higher in IVM offspring, but the mRNA level ofmiRNA155of FVM offspring was lower significantly.2. Effects of IVM on the DNA methylation status of renal Agt and Ace Given the disrupted expression levels of Agt and Ace, the DNA methylation rate of Agt (3CpG sites) and Ace (4CpG sites) were determined by pyrosequencing. (1) DNA methylation status of renal Agt and Ace in10w mice When compared to CON offspring, Agt (CpG2) showed lower methylation rate in IVM offspring, but Ace was significanly higher methlated in IVM offspring. And no significant differences were found in other CpG sites of Agt and Ace between the above two groups.(2) DNA methylation status of renal Agt and Ace in1.5y mice Renal Ace (CpGl) of the IVM offspring showed lower methylation rate than CON offspring. And there were no significant differences in other CpG sites of Agt and Ace between the above two groups.3. Effects of IVM on the renal AGT, ACE, ACE2and AGTR-1protein At age of10w and1.5y, when compared with CON offspring, the AGT protein level was changing from lower level to higher level in IVM mouce kidney tissue and the differences were statistically significant. There were no significant differences were detected in the ACE, ACE2and AGTR-1proteins between the above two groups at both phases.Conclusion:1. IVM process can induce the alteration of RAS gene expression in kidney tissue of IVM offspring.2. The changes of RAS gene and miRNAs expression in IVM offspring were associated with DNA methylation modification and miRNAs regulation.