Study On The Mechanism Of DNA Damage Caused By Abnormal Methylation In The Promoter Region Of Rad54 Gene Caused By Folate Deficiency

Part 1 The effects of low folic acid status in human seminal plasam on DNA methylation in sperm[Purpose] In this study,the seminal plasma folate concentration and semen parameters of105 male infertility patients were measured,through the analysis we found them in the seminal plasma folate concentration negative correlation with sperm DFI.SAM,a metabolite of folic acid,provides a methyl donor for DNA methylation,which can be affected by folate deficiency,so human long-term lack of folic acid can also affect the sperm genome DNA methylation.The purpose of this part is to study the changes of DNA methylation in sperm genome of people with low folic acid in seminal plasma,and to explore the potential mechanism of the negative correlation between folic acid concentration in seminal plasma and sperm DFI.[Methods] In this studay,8 subjects with normal concentration of folate were divided into one group and 8 subjects with low concentration of folate in the seminal plasma were divided into one group,and there was no difference in age,body mass index(BMI),folic acid concentration in seminal plasma was [26.56(25.58-31.43)nmol/L](25%-75%)and[12.9(11.45-15.12)nmol/L](0-25%)respectively.The whole genome of sperm was methylated by Reduced Representation Bisulfite Sequencing(RRBS)technique,and then the DNA methylation regions of differences were analyzed by bioinformatics.[Results] Through bioinformatics analysis of the methylation sequencing results of the two groups found that there are a total of 8 DNA methylation regions with significant differences,involving neural development,DNA fracture repair,RNA synthesis pathways and so on.Among them,the methylation level of Rad54 gene related to DNA double-bond fracture repair pathway was significantly higher in the low folic acid group than in the normal group.[Conclusions] Folate deficiency affects the methylation of the sperm genome,increasing the methylation levels of some genes and decreasing the methylation levels of some genes,which may be involved in important life processes,such as Rad54,which is related to the DNA double-strand damage repair pathwayPart 2 Study on the mechanism of DNA damage caused by abnormal methylation in the promoter region of Rad54 gene caused by folate deficiency[Purpose] DNA damage in sperm is one of the common causes of male infertility.In vivo,folic acid deficiency can increase the methylation level in the promoter region of Rad54 gene,which is an important gene related to the DSB repair pathway.The abnormal expression of Rad54 gene increases the sensitivity to the stimulation of external damage and sperm DFI during spermatogenesis.The purpose of this part is to preliminarily study the mechanism by which abnormal methylation of Rad54 gene caused by folic acid deficiency affects the DSB repair pathway and leads to the increase of DNA damage in sperm.[Methods] To investigate the mechanism of sperm DNA damage caused by folic acid deficiency,animal experiments and cell experiments were designed for verification in this study.In the animal experiment,male and female mice of normal 8-week-old C57 were randomly divided into 3 groups,with 8 males and 8 females in each group.The female mice in the three groups were fed low folic acid(0.3 mg/kg),normal folic acid(2 mg/kg)and high folic acid(20 mg/kg)diet for 2 weeks,and then were cooped with male mice fed with normal diet.After cooped,feeding conditions of each group remained unchanged.Serum folic acid concentration was detected at 8 weeks in F1 generation to determine whether the model was successfully constructed.Epididymal semen parameters of F1 generation male mice in each group were detected,and sperm DFI was detected by SCSA method to determine DNA damage.Sperm DNA was extracted and the methylation level in the promoter region of Rad54 gene was detected by BSP method.The expressions of Rad54 protein and γ-H2 AX protein in the testes of each group were detected by WB and immunofluorescence.F1 generation male mice mated with normal female mice,and the fertility of F1 generation was counted.In the cell experiment,the GC-2 cell model were cultured with folate-free medium and medium with folate concentrations of 4ng/ml,20ng/ml and 200ng/ml were constructed,and extracted cell genome DNA,total protein and total RNA,and detected the methylation level of Rad54 gene promoter region in each group by BSP method.WB and RT-PCR were used to detect the expression levels of Rad54 andγ-H2 AX.Cell slides were made and treated with 5mmol/L H2O2 for 10 min.Then immunofluorescence was used to detect the sensitivity of each group to external injury stimulation.[Results]In the animal experiment section,serum folic acid concentration of 8-week-old F1 generation male mice was detected.The low folic acid group was significantly lower than the normal group and the folic acid supplementation group,while the low folic acid group had lower sperm concentration and sperm motility than the normal group and the folic acid supplementation group.Immunofluorescence and WB results showed that the expression of Rad54 in the low-folic acid group was lower than that in the normal group and the folic acid supplementation group,the expression ofγ-H2 AX in the low-folic acid group was higher than that in the normal group and the folic acid supplementation group,the sperm DFI in the low-folic acid group was significantly higher than that in the normal group and the folic acid supplementation group,and the methylation level of the Rad54 gene promoter in the low-folic acid group was higher than that in the normal group and the folic acid supplementation group.In the cell experiment section,WB immunofluorescence and RT-PCR results showed that the expression of Rad54 in the folate-free group was significantly lower than that in the 4ng/ml,20ng/ml and 200ng/ml folate groups.The expression of the γ-H2 AX was significantly higher in the folate-free group than that in the 4ng/ml,20ng/ml and 200ng/ml folate groups,and the methylation level of the Rad54 gene promoter was significantly higher in the folate-free group.[Conclusions] Folic acid deficiency can increase the methylation level in the promoter region of Rad54 gene related to the DSB pathway,and reduce the expression of Rad54 protein,and affect the DSB repair pathway.In the process of spermatogenesis,it is more susceptible to the influence of external damage and stimulation,which increases the DFI of mature sperm.