Effects Of Folate Deficiency On Endometrium Decidualization And Embryo Development As Well As Its Regulation For Methylation Patterns Of Genome In Mice

Background: Pregnancy outcome is closely related to environmental factors, and maternal diet could affect embryo development and phenotype of offspring through epigenetic regulation. Folate, an essential nutrient of mammal, participates in a series of important physiological processes, such as DNA synthesis and methylation. Folate deficiency(FD) or its metabolic disorders would change methylation patterns of genome, and affect expression of key genes, thus result in disorders. Previous studies focus on the relation between folate metabolism enzyme and embryo development during pregnancy, while much less is known about its effect on intrauterine microenvironment during early pregnancy, and existing research data can not fully understood the molecular mechanism by which folate deficiency induced abnormal embryo development. Our previous study found that marternal low folate level didn’t impair endometrial receptivity, Thus, this study aimed to clarify the following questions: 1,The effect of FD on decidualization and embryo development; 2,The relation between adverse effects of FD and changes of genomic methylation; 3,The effect of FD on expression and function of PCP core genes which participate in neural tube closure. This study would explore the influences of FD on pregnancy as well as its regulation for genomic methylation both from maternal and embryo, to fully understand the role of folate during pregnancy, thus providing experimental basis for adjusting the timing of folate supplementation, and improving the molecular mechanisms of folate affects pregnancy.Methods:1. Establishment of animal model and sample collection: The mice were bred with folate-free diet for five weeks, and the control group with normal diet. The serum folate concentration was detected by electrochemical luminescence to validate the establishment of the animal model.Pregnancy mice: Adult female mice were mated with fertile males of the same strain by co-caging to induce pregnancy. The appearance of the vaginal plug was considered to indicate Day 1 of pregnancy(D1). The mice of the two groups were sacrificed on D6-D13, the uterus and embryo tissues were collected.Mice of artificially induced decidualization: Adult female mice were mated with vasectomized males of the same strain by co-caging to induce pseudopregnancy. The appearance of the vaginal plug was considered to indicate Day 1 of pseudopregnancy(PD1). Artificial induction decidua model was constructed by injection corn oil into one uterine horn on PD4, and the contralateral uterine horn was without injection. The mice were sacrificed on PD8,the uterus tissues were collected.2. Observation of FD effects on preganancy:1) Detection of endometrium decidualization: The appearance of the uterus in decidualization period from two groups was observed, the decidua bulge number and diameter were analyzed. To measure successful induction of decidual, typical appearance of the uterus, wet of uterus, observation of uterine section by HE and detection of decidual marker(Bmp2, dt PRP) by Real-time PCR were performed. The success rate of decidualization was analyzed. Primary endometrial stromal cells were isolated from mice in the two groups, and then induced to be decidualized by hormone. Decidual markers(Bmp2 and dt PRP) were detected by Real-time PCR, and desmin was detected by immunofluorescence.2) Detection of fertility and embryonic development state: Breeding experiments were carried out to compare the reproductive capacity of mice in the two groups. The appearance of the uterus and the embryo on D9-D13 were observed, HE staining was used to observe the embryo state in the uterus, wet of uterus and embryo resorption rate were analyzed.3. Analysis of genomic methylation: RRBS was employed to detect the genomic methylation patterns of endometrium on D6-D8 and embryos on D9-D11 of mice from the two groups, and the differences in DNA methylation were analyzed. DMRs between the two groups were counted, and function clustering of differentially methylated genes was predicted by Bioinformatics methods(GO analysis). The m RNA levels of differentially methylated genes were detected by Real-time PCR.Results:1. The effect of FD on endometrium decidualization: Compared with the control mice, the number of implantation sites reduced and diameter of decidua bulge became shorter. The success rate of induction in folate deficiency mice was only 15%. The wet of stimulated horn decreased in response to folate deficiency. HE staining results showed that, decidual cells of bigger size and dual or multi-nucleus were observed in stimulus horn section from normal mice, while no similar phenomenon emerged in folate deficiency mice. The m RNA levels of Bmp2 and dt PRP after folate deficiency treatment decreased significantly compared to that in normal mice. Consistently, primary stromal cells of normal mice became into polygonal from fusiform by hormone treatment for 72 h, while cells from folate deficiency mice remain fibroblast-like appearance. Expression of Bmp2 and dt PRP in decidualized cells from folate deficiency mice decreased significantly compared to that in normal mice. Positive signals of desmin became weaker following folate deficiency treatment.2. The effect of FD on embryo development: The mumber of pups and litters of FD mice reduced significantly. Uterine hemorrhage was observed in FD mice beginning at D10, then became more severe at D11. Embryo resorption emerged at D12 and D13. The uterine wet of mice reduced significantly and embryo absorption rate increased to 70% following FD treatment. Compared to the normal development of control mice, the embryos from the FD mice appeared tissue adhesion and differentiation disorders.3. Changes of genomic methylation following FD treatment: The RRBS results showed that, the distribution of different m C(including m CG,m CHG, and m CHH,H=A,G or T) in endometrium genome of FD mice on D6 and D7 was similar with that in normal mice, the proportion of m CG increased and the proportion of m CHH decreased on D8 after FD treatment;The distribution of different m C in embryo genome of FD mice on D9 and D10 was similar with that in normal mice, the proportion of m CG increased and the proportion of m CHH decreased on D11 after FD treatment. Compared with normal mice, the average methylation level of C in endometrium of FD mice decreased on D7, and declined more on D8, the average methylation level of m C in FD mice began to reduce on D8; The average methylation level of C in embryo of FD mice decreased on D10, and declined more on D11, the average methylation level of m C in FD mice was higher than that in normal mice on D9 to D11, while the average methylation level of m CG reduced following FD treatment; The differences in methylation levels focused on CG and m CG. There are 666 DMRs on D6, 646 DMRs on D7, 785 DMRs on D8, 795 DMRs on D9, 2480 DMRs on D10 and 1602 DMRs on D11 between normal and FD mice. GO analysis of DMR-related genes on D6-D8 revealed that they mainly participate in biological adhesion, biological regulation, cell proliferation, development, metabolism, and signaling; DMR-related genes on D9-D11 mainly participate in biological regulation, cellular process, developmental process, the metabolic process, response to stimulus and signaling. Expression of differentially methylated genes was also changed.4. The effect of FD on PCP pathway: In FD mice, expression of Vangl1 and Vangl2 detected by Real-time PCR and Western blot began to decrease on D9, and then declined more obviously with pregnancy. The results of IP showed that, FD would inhibit interaction between Vangl1 and Dvl1, while the combination of Vangl1 with Dvl2 or Dvl3 was not affected. The interaction between Vangl2 and Dvl1, Dvl2 or Dvl3 was inhibited in FD mice. There were no significantly differences of methylation rate of per Cp G site and average methylation rate between normal and FD mice. The match results of CDS sequencing and NCBI confirmed that there are three SNP locis of Vangl genes between normal and FD mice(Vangl1rs36584696, Vangl2 rs48000091 and rs31578570).Conclusions:1. Folate deficiency impaired the uterine decidualization, and changed the methylation profiles of the genome, including distribution of different m C and average methylation level, then affected the expression and function of key genes, that may be the potential molecular mechanisms by which FD inhibit decidualization. Maternal low folate level would have an impact on pregnancy during early stage, these data provided basic research evidence for adjusting the timing of folate supplementation.2. Folate deficiency caused fertility and embryonic dysplasia and changed the methylation profiles of the embryo genome, including distribution of different m C and average methylation level, affected expression of development related genes.3. Folate deficiency inhibited expression and function of Vangl which belongs to PCP core genes. The effect of FD on Vangl was independent with DNA methylation. FD may not cause mutation of Vangl. The changes of genomic methylation and expression of key genes, as well as inhibition of PCP pathway may be the potential molecular mechanism by which FD affect embryo development.