| Part I Effects of IVF on growth and expression of Igf2/H19and their epigenetic mechanisms in the liver and skeletal muscleObjective:To investigate the growth, gene expression of Igf2, H19, Igf2r, miRNA, and methylation status of Igf2/H19in the liver and skeletal muscle by establishing mouse model of IVF, and to illuminate the short-term and long-term effects of IVF and mechanism involved.Materials and methods:1. To establish models of IVF and in vivo fertilization by using C57BL/6J mice. The in vivo group was used as the control group, and the effects of IVF on birth outcome were detected by comparing the pregnancy rate, gestation, birth number, sex ratio, litter size, and death ratio.2. The effects of IVF on development were detected by checking the growth weight from birth to the sex matured stage (10wk), and the specific gravity of organs at3wk,10wk, and1.5yr.3. The effects of IVF on gene expression of Igf2, H19, Igf2r, and miRNA-483were detected at birth,3wk,10wk, and1.5yr by real-time quantitative PCR.4. The methylation status of Igf2/H19DMRs in mouse liver and skeletal muscle was first determined by bisulfite PCR and clone sequencing. Then, the changed CpG sites of Igf2/H19DMR were verified by pyrosequencing. 5. The IGF2protein expression in the liver and skeletal muscle of IVF mice was detected at birth,3wk,10wk, and1.5yr by western blotting.Result (s):1. A total of51IVF and51in vivo mice were delivered. There were no significant differences between the IVF and in vivo groups in the gestation, litter size, birth death ratio, sex ratio, and pregnancy rate.2. At birth, the body weight of IVF mice was significantly higher than that of in vivo mice. At2wk and3wk, the body weight of IVF mice was significantly lower than that of in vivo mice. After3wk, the difference in body weight between the two groups disappeared. There were no significant differences in the weight of organs at all the time points between the two groups. However, the weight of spleen in IVF mice was significantly lower than that of in vivo mice at10wk.3. Effects of IVF on the expression levels of Igf2, H19, Igf2r, and miRNA-483(1) Expression levels of Igf2, H19, Igf2r, and miRNA-483in liverAt birth and3wk, there were significant differences in gene expression of Igf2, H19and miRNA-483between IVF mice and in vivo mice. At10wk, the difference in gene expression between IVF and in vivo mice disappeared. However, when the age reached to1.5yr, the significant differences in gene expression of Igf2, H19and miRNA-483were detected again between the two group mice. In addition, there were no differences in gene expression of Igf2r between the two groups from birth to old age.(2) Expression levels of Igf2, H19, Igf2r, and miRNA-483in skeletal muscleAt3wk, there were significant differences in gene expression of Igf2, H19, and miRNA-483. At10wk, no significant differences in gene expression of Igf2, H19and miRNA-483were found between two groups. However, when the age reached to1.5yr, the significant differences in gene expression of Igf2, H19and miRNA-483were detected again between the two group mice. At3wk and10wk, there were no differences in gene expression of Igf2r between the two groups. However, at1.5yr, gene expression of Igf2r was significantly lower in IVF mice than in control mice.4. Effects of IVF on the DNA methylation status of Igf2/H19and Igf2r DMRs Given the disrupted expression levels of Igf2/H19, the DNA methylation rate of the H19DMR (21lbp,12CpG sites) and Igf2DMR2(238bp,13CpG sites) were first calculated by cloning and sequencing. Among all the CpG sites of H19DMR, the CpG6, CpG7, and CpG8showed differences between the two groups. Also, the CpG4and CpG5sites of Igf2DMR2showed differences. Thus, the DNA methylation rate of the H19DMR (CpG6-8sites) and Igf2DMR2(CpG1-5sites) were chosen to verify by pyrosequencing. Further, we added Igf2r DMR2(4CpG sites) to analyze the DNA methylation status.(1) DNA methylation status of Igf2/H19and Igf2r DMRs in liver1)At birth, IVF mice showed significant difference in the methylation rate of H19DMR. At3wk and10wk, there was no significant difference in the methylation rate of H19DMR between the two groups. However, when the age reached to1.5yr, the significant differences in the methylation rate of H19DMR was detected again between the two group mice.2) At birth and3wk, there was significant difference in the methylation rate of Igf2DMR2between IVF and control mice. At10wk, there was no significant difference in the methylation rate of Igf2DMR2between the two groups. However, when the age reached to1.5yr, the significant differences in the methylation rate of H19DMR was detected again between the two group mice.3) There was no difference in the methlyation rate of Igf2r DMR2between the two groups of mice from birth to old age.(2) DNA methylation status of Igf2/Hl9DMRs and Igf2r in skeletal muscle1) At3wk, IVF mice showed significantly lower methylation rate of H19DMR when compared with in vivo mice. There was no significant difference in H19DMR between the two groups at10wk and old age.2) At3wk, there was significant difference in the methylation rate of Igf2DMR2between IVF mice and in vivo mice. The difference of methylation rate of Igf2DMR2in IVF mice disappeared at10wk. However, when the age reached to1.5yr, the significant difference in the methylation rate of Igf2DMR2was detected again between the two group mice. 3) At3wk and10wk, the methlyation rate of Igf2r DMR2of IVF mice was similar to that of in vivo mice. However, at old age, the methylation rate of Igf2r DMR was significantly higher in IVF mice than in control mice.5. Effects of IVF on the IGF protein expression in the liver and skeletal muscleAt3wk, both in the liver and skeletal muscle, the expression of IGF2protein was significantly lower in IVF mice than in control mice. However, there was no statistically significant difference in IGF2protein expression between IVF and in vivo groups at other stages.Conclusion (s):1. IVF could affect the early body growth, and the aberrant growth may be restored with age.2. The effects of IVF on gene expression of Igf2, H19and miRNA-483in the liver and skeletal muscle not only in newborn mice but also in elder mice.3. The aberrant growth that was noted along with Igf2/H19differences in IVF offspring may be associated with epigenetic mechanisms such as DNA methylation and miR-483. Part II Effects of ART on blood lipid metabolism in elder mice and mechanism involvedObjective:To investigate the effects of ART on blood lipid metabolism and mechanism involved, to determine the risk of ART induced adult disease, and to illuminate the main etiological factor and etiological time by ART.Materials and methods:1. Investigation on the phenotype and blood lipid metabolism of elder mice(1) Mice of the C57BL/6J were used as oocyte and sperm donors. Superovulated mouse oocytes were fertilized in vivo constituted the control group. The ART groups were established containing IVF, ICSI, and IVM conceived mice. In total,16controls,12IVF-conceived,12ICSI-conceived,10IVM-conceived mice were examined at1.5years of age.(2) The spatial learning and memory capability were detected by using the water maze and fear condition tests at1.5yr.(3) The blood pressure (BP) and heart ratio (HR) were analyzed by using a programmed sphygmomanometer.(4) Intraperitoneal glucose tolerance testing (IPGTT) was used to test the fasting glucose level and glucose tolerance.(5) Plasma chemistry was detected by Clinical Chemistry Analysers. Serum insulin level was determined using Rat/Mouse Insulin ELISA kit.(6) At1.5yr, some viscera were excised and weighed. In addition, histopathology analyses were done. Samples were fixed in4%paraform and embedded in paraffin wax. Sections were stained with hematoxilyn-eosin (HE) stain.2. Investigation on the involved mechanism Based on the results of blood lipid metabolism and pathologic changes in organs, the regulative pathway of INSIG-SCAP-SREBP and miRNA were analyzed in the liver and lung of elder mice conceived by ART and in the IVM oocytes.(1) mRNA expression of INSIG-SCAP-SREBP in elder miceGene expressions of Insigl, Insig2, Scap, Srebfl, Srebf2, miR-33and miR-122were analyzed by real-time quantitative PCR.(2) DNA methylation status of INSIG-SCAP-SREBP in elder miceThe methylation status of CpG inslands of Insig-Scap-Srebp was determined by BSP and pyrosequencing.(3) Protein expression of INSIG-SCAP-SREBP in elder miceThe protein expressions of INSIG-SCAP-SREBP were detected by western blotting or immunohistochemistry.(4) Expression of INSIG-SCAP-SREBP in the Mâ…¡ oocytesExpressions of INSIG-SCAP-SREBP in the Mâ…¡ oocytes were analyzed by real-time quantitative PCR and fluorescence immunocytochemistry.Result (s):1. Effects of ART on the phenotype and blood lipid metabolism(1) No differences were detected in the learning and memory ability including incubation period, the times of crossing, and freezing percent between ART and in vivo groups.(2) In the IVM treatment group, the BP and HR were significantly higher than other three groups at old age. No differences of BP and HR were found in ICSI and IVF groups as compared with the control group.(3) As the IPGTT shown, only30min after glucose injection, the serum glucose level in the ICSI group was significantly lower than that of in vivo group.(4) The results showed that when compared with the in vivo group, IVM or ICSI-conceived elder mice revealed significant differences in total cholesterol, LDL-C, HDL-C, apolipoprotein-A1, AST/ALT, and CRP. In addition, higher serum insulin level was found in IVM conceived mice.(5) In the ICSI and IVM groups, the heart and kidney weight were significantly higher than IVF group and in vivo group. Moreover, in the IVM group, the pancreas and liver weight were significantly higher than other three groups.(6) As HE stains shown, fatty degeneration of a kidney in IVM mice was observed. In addition, the pathological changes were found in the lung of IVF and ICSI conceived mice.2. Expression levels of INSIG-SC AP-SREBP in the liver of aged ART miceGiven the aberrant blood lipid metabolism in the IVM and ICSI aged mice, ART mice containing IVF, ICSI, and IVM were chosen to analyze. The in vivo fertilization constituted the control group.(1) Gene expressions of Insigl, Scap, Srebfl, and Srebf2were significantly higher in IVM and ICSI groups than in IVF and control groups (2-4folds, P<0.01). In addition, gene expressions of miR-33and miR-122were significantly lower in IVM and ICSI groups when compared with IVF and control groups.(2)Both IVM and ICSI groups showed significant differences in the DNA methylation rate of Insigl, Scap, Srebfl, and Srebf2.(3) The protein expression of SCAP was significantly higher in IVM and ICSI groups as compared with IVF and in vivo groups.3. Expression levels of INSIG-SCAP-SREBP in the lung of aged ART miceGiven the pathologic changes in the lung of IVF and ICSI aged mice, ART mice containing IVF and ICSI were chosen to analyze. The in vivo fertilization constituted the control group.(1) Gene expressions of Insigl, Insig2, Scap, Srebfl, and Srebf2were significantly lower in IVF and ICSI groups than in control group.(2) Both IVF and ICSI groups showed significant differences in the DNA methylation rate of Insigl, Scap, Srebf1, and Srebf2. Moreover, there are significant differences in the DNA methylation rate of Insig-Scap-Srebf between abnormal lung and normal lung. (3) No differences in protein expressions of INSIG-SCAP-SREBP were detected between ART groups and control group.4. Expression levels of INSIG-SCAP-SREBP in the Mâ…¡ oocytesGiven the abnormal blood pressure and blood fat in the IVM aged mice, the IVM oocytes (n=400) were chosen as the experimental group, and in vivo maturation oocytes (n=400) were chosen as the control group.(1) Gene expressions of Insigl, Scap, and Srebf1were significantly higher in IVM oocytes than in control oocytes. In addition, gene expressions of miR-33and miR-122were significantly lower in IVM oocytes than in control oocytes.(2) There are significant differences in protein expressions of INSIG1and SCAP between IVM oocytes and in vivo oocytes.Conclusion (s):1. IVM may induce higher blood pressure.2. Both IVM and ICSI could result in the changes of blood lipid metabolism, serum insulin, and organ morphous structure.3. ART can result in a higher risk of lipid metabolic abnormality in elder mice, which may be associated with alterations in gene expression of Insig-Scap-Srebf.4. The aberrant blood lipid metabolism that was noted along with Insig-Scap-Srebf differences in ART offspring may be linked with epigenetic mechanisms such as DNA methylation and miRNA.5. The effects of IVM on the expression of INSIG-SCAP-SREBP in oocytes, which verified the theory of embryo-fetal diseases. |
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