Effect Of The Circadian Rhythm Disorder Before Pregnancy For Lipid Metabolism Of Offspring And Mechanism

Part I Establishment of pre-pregnancy circadian rhythm disorder rat model and phenotypic verificationObjective:To establish a pre-pregnancy circadian rhythm disorder model through photoperiod interference and determine the reproductive phenotype by fertility assessment,oocyte fertilizing ability evaluation and ovarian rhythm.Materials and methods:The common photoperiod of female SD rats about 6-8 weeks old was disturbed by 18 hours of light/6 hours of darkness for 3 months.Blood samples were collected to detect the levels of serum reproductive hormones(FSH,LH,E2,P,T AMH)and melatonin.Fresh ovaries were collected for weighing and HE section staining to observe the ovarian structure.The pregnancy rate and litter size were compared with control group by mating experiment.The number of retrieved oocytes,the proportion of MII oocytes and the cleavage rate after IVF with normal male rat sperm were compared as well.The oxidative stress level of MII oocytes was determined by ROS staining,and the distribution and oxidation of mitochondria were determined by mitochondrial probe staining.The expression levels of clock genes(Arntl,Clock,Cry 1/2,Per 1/2,Rora,Aanat)in the ovaries were determined by fluorescent quantitative PCR.Results:In the experimental group,the estrous cycle was significantly prolonged after circadian disorder,especially the estrous period.The ovary weight decreased with expanded follicles and reduced corpus luteum,suggesting that the ovulation function of the rats in the experimental group was abnormal.In the experimental group,serum AMH increased and the level of melatonin decreased with no change of serum FSH,LH,E2,T and P.The pregnancy rate and litter size of the experimental group were significantly lower than those of the control group.The number of oocytes retrieved after ovarian hyperstimulation was lower than its counterpart with no significant change of MII oocytes proportion or cleavage rate after subsequent fertilization.The level of ROS,the distribution of mitochondria and the degree of mitochondrial oxidation did not change significantly.The expression level of Rora,Aanat decreased significantly,and the expression of Clock and Per1/2 presented decreasing tendency in the experimental group.Conclusion:Interfering female rats with the photoperiod of 18 hours light/6 hours darkness,increasing the duration of light,induced significant reproductive phenotypes,including decreased ovulation,the disorder of estrous cycle,increased AMH level,and decreased fertility.Part Ⅱ Pre-pregnancy circadian disorder interferes offspring lipid metabolismMaterials and methods:Female SD rats of 6-8 weeks old were exposed to abnormal photoperiod(18 hours of light/6 hours of darkness)for 3 months to establish the animal model of circadian disorder,and then housed in the normal photoperiod(12 hours of light/12 hours of darkness).The offspring rats of circadian disorder model group and control group were bred by mating with normal adult male rats.The growth curve of the offspring was monitored from 1 week to 4 months.The body fat rate of the offspring at 4 months old was measured by magnetic resonance imaging.The serum lipid levels(TG,TC,HDL-c and LDL-c)were detected.The liver was stained with oil red O and the TG level was quantitatively detected.Liver tissues were collected every 4 hours for transcriptome sequencing,KEGG analysis,GO analysis and JTK_Cycle analysis.Fluorescent quantitative PCR was used to verify the expression level of clock gene and target gene.The expression level of the target protein was verified by Western blot.Results:The body weight and body fat rate increased in the offspring in the circadian disorder group,especially in the male offspring.At the age of 4 months,the blood lipid level of male offspring was disorder,mainly in TG level.The liver lipid deposition of male offspring in circadian disorder group was significantly higher than that in control group tested by oil red O staining and TG quantitative detection.In the circadian rhythm disorder group,the expression amplitude of Arntl,Per 1/2 and Cry 1/2 in the liver of male offspring decreased.The number of genes rhythmically expressed in transcriptome decreased with phase changed.The expression of Acsl4,a key gene regulating lipid metabolism,was up-regulated.Conclusion:Maternal circadian rhythm disorder before pregnancy induced lipid metabolism disorder phenotype in male offspring rats,including increased body weight,body fat rate,dyslipidemia,and increased liver TG content,may be caused by the decreased amplitude of liver clock gene expression and increased expression of Acsl4.Part III Oocytes DNA methylation patterns reprograming induced by maternal pre-pregnancy circadian disorder results in offspring liver lipid metabolism disorderObjective:To explore the epigenetic mechanism by which maternal circadian rhythm disorder exerts on offspring liver.Materials and methods:Female SD rats aged 6-8 weeks were exposed to abnormal photoperiod(18 hours light/6 hours dark)for 3 months to establish the prepregnancy circadian disorder animal model.MII oocytes were collected after ovulation induction for 5mC and 5hmC staining and methylation sequencing analysis.The methylation status of Mecp2 promoter was verified by pyrosequencing.Real-time PCR and Western blot were used to verify the expression level of Mecp2 in offspring liver.The effect of MECP2 on the expression of liver clock gene and Acsl4 was confirmed by siRNA knockdown of MECP2 in rat liver cell line BRL.Results:106 differentially methylated genes were screened by methylation sequencing analysis of MII oocytes between experimental group and control group,which were mainly enriched in neural development related pathways.There was no significant difference in DNA methylation level by 5mC and 5hmC staining between the two groups.Compared with male offspring of control group,Mecp2 decreased and the methylation level of promoter region increased in the liver of male offspring of experimental group.After Mecp2 knockdown,the expression of Acsl4 decreased.Conclusion:The increased methylation level of Mecp2 in the oocyte of rats with circadian rhythm disorder can be inherited to the liver of offspring male rats,resulting in the decrease of Mecp2 expression,and regulating lipid metabolism through Acls4.