Effect And Mechanism Of CD14 Hypomethylation Signature In High DFI Sperm

Part ⅠSignatures of gene hypomethylation and screening of key genes and pathways in high DFI spermBackground and ObjectiveThe sperm DNA fragmentation index(DFI)serves as a widely accepted clinical indicator reflecting sperm integrity and predicting assisted reproductive outcomes.Elevated DFI negatively affects various aspects of sperm function,fertilization,and the success rate of assisted reproduction techniques.Levels of DNA methylation in the sperm genome are strongly associated with male fertility and sperm quality.According to multiple studies,hypomethylation in promoter regions of imprinted genes such as H19 and non-imprinted genes such as DAZL may be a key factor in reduced male fertility.Sperm with altered DNA methylation patterns can result in decreased male fertility,affect sperm quality,vitality,fertility,disrupt normal embryo development after fertilization,and cause a variety of developmental disorders and diseases.In a previous study,we observed for the first time a significant correlation between genome-wide DNA methylation profiles and sperm DFI levels in the human sperm genome.The group with DFI>30% exhibited lower genome-wide DNA methylation levels compared to DFI<15%.The study found that the top 300 genes with differentially methylated regions(DMRs)identified through cluster analysis,along with 27 differentially expressed mi RNAs discovered by principal component analysis,can be used as potential molecular biomarkers for distinguishing sperm from DFI>30% group and DFI<15% group.Furthermore,by constructing a sperm DNA methylation-related matrix,it was discovered that in the DFI>30% group,the sperm chromosomes became more loose and more sensitive to damage from reactive oxygen species.From this,we hypothesize that DNA hypomethylation changes in the human sperm genome may promote sperm DNA damage.Therefore,we got this hypothesis as a breakthrough point and combined the results of our previous research,as well as the correlation analysis between sperm DFI and various semen parameters,focusing on genes with significantly decreased methylation levels in promoter regions,aiming to screen key genes and pathways associated with high DFI sperm.This study aims to provide new direction and evidence for studying mechanisms of sperm DNA damage and exploring clinical treatment strategies.Methods1.We selected two dataset from our study of "Genome-wide DNA Methylation Profiles and snc RNAs Signatures Study of High DFI Sperm" which identified the top 300 DMRs genes,as well as 27 differentially expressed mi RNAs for genes screening.Firstly,we selected genes with DNA hypomethylation from the top 300 DMRs genes to form a geneset.Then further screening the geneset should meet one of the following conditions:(1)the gene with the most significant DMRs in the Promoter region in the geneset;(2)We used the Tarbase v.8 database to compare genes in the geneset related to the 27 differentially expressed mi RNAs,selecting the gene of the highest predicted score and the most mi RNAs regulation.2.By setting selection criteria for the study subjects,526 male patients were selected as study subjects from June 2022 to April 2023 at the Reproductive Medicine Center at the First Affiliated Hospital of Naval Medical University.All clinical test data for routine semen analysis,seminal plasma markers,sperm DNA integrity,sperm morphology,and other parameters were collected for intergroup comparison,Pearson analysis,and multivariate unconditional logistic regression analysis.The highest risk factor for sperm DFI was selected as the study condition for screening for related genes in high DFI sperm by bioinformatics analysis,and verified by human semen samples.3.By setting human semen sample collection conditions,sperm samples from DFI>30%group and DFI<15% group were collected from May to June 2023.DNA MethylTarget sequencing was used to analyze the methylation signatures of screened high DFI sperm related genes by designing their specific gene fragment primers.From July to August 2023,30 sperm samples from the DFI>30% group and 30 sperm samples from the DFI<15%group were collected for the detection of m RNA expression levels of high DFI sperm related genes.4.We used GO function and KEGG enrich to analysis key pathways in high DFI sperm.Results1.Based on the study of "Genome-wide DNA Methylation Profiles and snc RNAs Signatures Study of High DFI Sperm",CD14 and CDKN1 B were filtered as high DFI sperm related genes.2.The analysis of the correlation between sperm DFI and semen parameters was found that the sperm progressive motility(PR)percentage was the highest risk factor for high DFI sperm,and there was a significant negative correlation.SOX9 was filtered as high DFI sperm related gene.3.MethylTarget sequencing showed that the DNA methylation levels of specific gene fragments CD14,CDKN1 B,and SOX9 were significantly decreased in sperm with DFI>30% group,with CD14 showing the most significant decrease(P=0.018).The DNA methylation levels of the three genes were positively correlated with PR and negatively correlated with sperm DFI.4.Enrichment analysis revealed multiple pathways in sperm with DFI>30% group,such as Shigellosis,Fatty acid metabolism and Endocytosis.The effect of Shigellosis pathway was the most significant(P=0.0002),and CD14 enrichment was found in this pathway.ConclusionIn part Ⅰ,we found that the DNA methylation level of CD14 was most significantly decrease in high sperm,the Shigellosis pathway was the most significant pathway that affected high DFI sperm,and CD14 was enriched in this pathway.We selected CD14 and the Shigellosis pathway as the key gene and pathway of high DFI sperm.Part ⅡEffect of CD14 hypomethylation on mouse GC-2 spd spermatocyte injuryBackground and ObjectiveCD14 is expressed on the cell membrane of human sperm,with higher enrichment levels in the neck and tail regions compared to the head.CD14 expression has also been observed in human and rat testes expressing Toll-like receptors.Animal studies have found that CD14 is expressed in different stages of the gene expression profile in mouse spermatogenesis,including B-type spermatogonia,primary spermatocytes,round and elongated spermatids;CD14 is expressed in spermatogonia of porcine testes at different developmental stages,and serves as a cell surface biomarker in porcine spermatogonia stem cells and undifferentiated spermatogonia.After male vasectomy,CD14 levels in seminal plasma were significantly decreased to only 36% of pre-vasectomy levels,suggesting that most of the CD14 originates in the testes,seminiferous tubules,and epididymis above the vasectomy site.Currently,there have been limited studies of the function and epigenetics of CD14 during spermatogenesis in the field of reproductive medicine.Therefore,to further analyze the effect of CD14 hypomethylation on sperm function and quality,based on the signature of CD14 hypomethylation observed in high DFI sperm in the part Ⅰ,we utilized mouse GC-2spd spermatocyte(GC-2 cells)to construct a model of CD14 demethylation in part Ⅱ.By analysing the marker changes in cell proliferation and apoptosis,we aimed to observe the effect of CD14 hypomethylation and differential CD14 expression levels on cell function.Methods1.Construction of GC-2 cell CD14 demethylation model.GC-2 cells were cultured in high glucose DMEM with 5% fetal bovine serum and 1% penicillin-streptomycin.5-aza-2’-deoxycitydine(5-Aza-Cd R)was added to adjust its final concentration to 10μM.The culture medium was changed every 24 hours and 5-Aza-Cd R was added each time.Cells were collected after culture at 37℃ and 5% CO2 for 48 h and the modeling effect was evaluated.5-Aza-Cd R was dissolved in DMSO and the same volume was used as a negative control.2.Effective sequence screening of si-CD14 transfected into GC-2 cells.Three si-CD14 s were respectively transfected into GC-2 cells using si-CD14 transfection reagent [20μM si-CD14+ribo FECT CP buffer(1×)+ribo FECT CP reagent].After being cultured in 5%CO2 at 37℃ for 48 h,the cells were collected to evaluate the transfection effect.si-Ctrl was used as a negative control.3.Evaluation of efficacy of CD14 overexpression plasmid transfected into GC-2 cells.GC-2 cells were transfected with plasmid transfection reagent(plasmid+Lipofectamine3000+Opti-MEM).After being cultured in 5% CO2 at 37℃ for 48 h,the cells were collected to evaluate the transfection effect.Vector was used as a negative control.4.Cell experimental groups.DMSO group(DMSO was added to GC-2 cells for culture),5-Aza-Cd R group(5-Aza-Cd R was added to GC-2 cells for culture),si-Ctrl group(GC-2cells were transfected with si-Ctrl),5-Aza-Cd R+si-CD14 group(downregulation5-Aza-Cd R group CD14 expression,5-Aza-Cd R was added to GC-2 cells transfected with si-CD14 for culture),Vector group(Vector was added to GC-2 cells for culture),and CD14-OV group(CD14 overexpression plasmid was added to GC-2 cells for culture).5.Indicators of detection in cell experiments.CCK-8 level was used to evaluate cell proliferation.Flow cytometry and Bax,Bcl-2,Cleaved Caspase-3 protein expression levels were used to analyze apoptosis levels.Results1.Methylation-specific PCR primers designed to the Chr18: 36858802-36859063 fragment of mouse CD14 showed strong specificity.The 5-Aza-Cd R modeling scheme of10μM was suitable for the establishment of CD14 demethylation model in GC-2 cells,and the CD14 methylation level was significantly decreased in model cells.2.The effective sequence screening results of si-CD14 transfected into GC-2 cells showed that si-CD14 2 was able to knock down the level of CD14 expression in GC-2 cells,with the most significant transfection effect.3.After transfection with CD14 overexpression plasmid,the CD14 expression level of GC-2 cells was significantly increased,and the plasmid construction and transfection were effective.4.In the 5-Aza-Cd R group,the proliferation of GC-2 cells was significantly inhibited,and the proportion of apoptotic cells increased significantly.The proliferation and apoptosis of GC-2 cells in the 5-Aza-Cd R+si-CD14 group were reversed compared with those in the 5-Aza-Cd R group.The effects of proliferation inhibition and apoptosis in GC-2 cells in the CD14-OV group were consistent with those in the 5-Aza-Cd R group.ConclusionIn part Ⅱ,we found that the proliferation of GC-2 cells was significantly inhibited,and the apoptosis was significantly increased after CD14 demethylation.CD14 hypomethylation can promote GC-2 cells injury and negatively affect cell function.Part ⅢCD14 hypomethylation mediates LPS/TLR4-induced NLRP3 inflammasome activation and inflammatory cytokine excitation leading to cell injury effectsBackground and ObjectiveIn the classical immune response,the CD14 molecule recognizes the lipopolysaccharide(LPS)of G-bacteria and activates Toll-like receptor 4(TLR4),mediating the innate immune response to LPS,inducing the production of pro-inflammatory mediators and bacterial clearance.CD14 is highly expressed in the neck and tail of human sperm,while TLR4 is highly expressed in the acrosome and tail of human sperm.After treatment with LPS in mouse testes,CD14 m RNA expression was strongly induced.Simultaneously,upon binding of CD14 to LPS,it negatively affected male sperm quality by mediating activation of the TLR4 pathway in the sperm,thereby inducing sperm apoptosis.Toll-like receptors and Nucleotide-binding oligomerization domain(NOD)-like receptors play important roles in the immune response mechanism leading to inflammation in the Shigellosis pathway in the innate immune system.Sertoli cells in the mammalian testes activate NLRP3 through TLR4 and NOD1/2 interaction with NF-κB,leading to Caspase-1 activation and IL-1βsecretion.Studies have shown that the levels of inflammatory cytokines such as IL-1β and Caspase-1 are associated with male infertility,according to the molecular activation and regulation of NLRP3 inflammasome in spermatogenesis and male infertility,it is suggested that inhibiting the NLRP3/Caspase-1/IL-1β inflammasome pathway may reduce testicular inflammation and its effect on male sperm quality and fertility.Therefore,the study selected the immune response mechanism in the Shigellosis pathway as the breakthrough point in part Ⅲ.By collecting clinical semen samples and constructing a model of GC-2cells CD14 demethylation,the mechanism of CD14 hypomethylation in high DFI sperm in mediating the effects of sperm injury was analyzed.Methods1.Collection of human semen samples.By setting the conditions for human semen sample collection,sixty semen samples were collected between July to August 2023,including 30 samples of DFI<15% and 30 samples of DFI>30%.The m RNA and protein expression levels of NLRP3,Caspase-1,CD14,and TLR4 in sperm,as well as the levels of LPS,IL-1β,and IL-18 in the seminal plasma were detected.2.Cell experimental groups.DMSO group(same method as Part II),5-Aza-Cd R group(same method as Part II),Vector group(same method as Part II),and CD14-OV group(same method as Part II).3.LPS was added to GC-2 cells culture.LPS dissolved in DEPC water was added to each group of GC-2 cells for culture,and the final concentration of LPS in each well of the6-well cell culture dishes was adjusted to 0μg/m L,1μg/m L,5μg/m L,and 10μg/m L,respectively.After incubation under 5% CO2 at 37°C for 24 hours.4.Indicators of detection in cell experiments.CCK-8 level was used to evaluate cell viability.Cell apoptosis was analyzed using flow cytometry and Bax,Cleaved Caspase-3protein expression levels.The levels of NLRP3,Caspase-1,IL-1β,and IL-18 were detected to analyze NLRP3 inflammasome activation.Results1.In the sperm of DFI>30% group,the m RNA expression levels of CD14,TLR4,NLRP3,and Caspase-1 in sperm were significantly increased than those in the DFI<15%group,and their protein expression levels were consistent with the m RNA detection results.The levels of LPS,IL-1β,and IL-18 in the seminal plasma of the sperm of DFI>30% group were significantly increased than those in the DFI<15% group.2.At the different concentrations of LPS,the viability of GC-2 cells in DMSO,5-Aza-Cd R,Vector,and CD14-OV groups decreased significantly with increasing LPS concentration.However,the apoptosis rate of GC-2 cells,the m RNA expression levels of CD14 and TLR4,the protein expression levels of NLRP3 and Caspase-1,and the levels of IL-1β and IL-18 in the culture supernatant of GC-2 cells were significantly increased with the increase of LPS concentration.3.At the same concentration of LPS,the viability of GC-2 cells in 5-Aza-CdR and CD14-OV groups was lower than in the corresponding control groups.However,the apoptosis rate of GC-2 cells,the m RNA expression of CD14 and TLR4,the protein expression of NLRP3 and Caspase-1,and the levels of IL-1β and IL-18 in the cell culture supernatant were all higher than those in the corresponding control group.ConclusionIn part Ⅲ,we found through clinical semen sample testing that the expression levels of CD14 and TLR4 in high DFI sperm were increased,activation of NLRP3 inflammasome and the levels of inflammatory cytokines IL-1β and IL-18 in seminal plasma were increased,all of which were associated with elevated levels of LPS in seminal plasma.Through cell experiments,it was found that LPS induced the expression of CD14 and TLR4 in GC-2 cells,activated NLRP3 inflammasome,and stimulated the secretion of inflammatory cytokines IL-1β and IL-18,suppressed cell proliferation,and promoted cell apoptosis.This effect was significantly enhanced after demethylation of CD14.Therefore,sperm injury is related to the activation of NLRP3 inflammasome induced by CD14/LPS/TLR4,which promotes the activation of intracellular Caspase-1 and the secretion of pro-inflammatory cytokines IL-1β and IL-18,resulting in a mechanism of cell injury effects.The hypomethylation of CD14 in sperm mediates a significant enhancement of this injury effect,promoting the formation of sperm with high DFI.