Effects Of Folic Acid Deficiency On Genomic Stability And Spindle-assembly Checkpoint Related Protein Gene Expression In Spermatogonial Cells Of Mice

Folic acid(FA)is an important methyl donor in the body's carbon metabolic cycle and plays a key role in DNA synthesis,stability and repair.FA deficiency leads to genomic instability(GIN)such as DNA base mismatch,deletion,DNA strand and chromosome breakage.As a safety supervision mechanism between metaphase and anaphase of mitosis,spindle assembly checkpoint(SAC)ensures the accurate distribution of chromosomes to progeny cells and prevents the generation of aneuploid cells.The quality and density of sperm are crucial to male reproduction.Infertile patients have poor sperm quality and low density,and the number of aneuploid sperm significantly increases.Aneuploid sperm promotes the development of GIN.Little has been reported on whether folic acid deficiency affects SAC and what role it plays in folate deficiency leading to genomic instability.This research aimed to investigate the effects of FA deficiency on genomic stability and gene expression of SAC related proteins in mouse spermatogonial cells(GC-1spg).CCK-8,cytokinesis blocking micronucleus group analysis(CBMN-cyt),flow cytometry,fluorescence quantitative PCR,Western bolt and other experimental techniques were used.Folic acid concentration to 2000 nmol/L as normal control group,the comparative analysis of the concentration of FA 200 nmol/L,20 nmol/L and 0nmol/L,effects on genomic stability of mouse spermatogonial cells and sensitivity to folate deficiency,to study the different concentrations of FA affects the quantity of SAC related protein gene expression,discusses the FA deficiency of germ cell genome instability induced potential mechanism.The results showed that(1)After continuous culture of GC-1spg cells for two weeks,cell morphology changes and growth rate significantly decreased,suggesting that the experimental model could be achieved after continuous culture for two weeks.(2)FA concentration of 200 nmol/L significantly inhibited cell proliferation(P<0.01),and the lower the FA concentration,the more obvious the inhibition of proliferation,indicating that the inhibition of FA on cell proliferation is concentration-dependent.(3)Major genetic damage markers(MN,micronucleus;NPB,nuclear plasma bridge;NBud,nuclear bud)was higher than normal control group(P<0.05),genetic damage was significantly increased in the 20 nmol/L group and the 0 nmol/L group compared withthe normal control group(P<0.01),and the dominant signal was micronucleus(MN)and abnormal DNA amplification(P <0.05).Therefore,when FA was 200 nmol/L,the stability of cell genome could not be maintained.(4)When FA concentration was 200nmol/L,the apoptotic rate was not significantly different from that of the normal control group.When FA concentration was 20 nmol/L and 0 nmol/L,apoptosis occurred(P<0.05),and apoptosis was more obvious in the 0 nmol/L group(P <0.01),suggesting that FA concentration below 200 nmol/L would induce apoptosis.(5)Fluorescence quantitative PCR results showed that with the decrease of FA concentration,the expression levels of Mad 1,Mad2,Bub1,Bub 3,BubR1,CDC20 and c-myc mRNA in GC-1spg cells increased(P<0.05),and the lower the concentration,the higher the expression level.The Western bolt experiment results showed that the protein expression levels of Mad2,Bub1 and BubR1 significantly increased(P<0.01)there was no statistically significant difference in the expression levels of Bub3 mRNA and protein.The results showed that not all SAC protein genes were highly expressed when FA was deficient,and the highly expressed protein genes were concentration-dependent,and FA deficiency might regulate SAC through the c-myc pathway.(6)Prediction of methylation and experimental results of 5-AZA suggest that SAC may be regulated by methylation.The reserch the different concentrations of FA on GC-1spg protein gene expression related to cell GIN and SAC,confirmed: in the case of FA concentration of 200 nmol/L will be induced by GC-1spg cell genetic damage occurs,the condition suppresses cell proliferation,cell apoptosis,not occurring FA concentration below 200 nmol/L,the GC-1spg cell genetic damage is aggravating inhibits cell proliferation,inducing apoptosis,prompt FA concentration for 200 nmol/L,GC-1spg cells has reached the lack of state.We conclued that FA deficiency causes the high expression of SAC gene,c-myc mRNA and SAC protein,and it shows the dependence of FA concentration.Combined with the methylation prediction results,it suggests that SAC may be regulated by c-myc pathway or methylation when FA deficiency occurs.