Abnormal Hypermethylation Of Vdac2 Promoter Is Associated With Male Idiopathic Asthenospermia

The most apparently characteristic of IAS is that all sperm parameters are normal except the value of sperm motility according to the World Health Organization.According to the studies,sperm is maturation in the testis and epididymis from spermatogonium to primary spermatocyte,secondary spermatocyte,sperm cells and functional sperm,which contains a series of complex regulation including gene expression,histone methylation and acetylatio and microRNAs,promoting the sperm differentiation and maturation.Abnormal expression of some proteins and the changes of epigenetic modification may have a bad effect on the sperm motility,sperm morphology,causing male infertility.Abnormal regulation in spermatogenesis process is a common aetiological agent of IAS.As is known to all,epigenetic modification,also known as genetics or epigenetics,play an important role in the spermatogenesis.Epigenetic modification is a discipline that there is no change in the sequence of DNA but the gene function is changed to be inheritance steady.In our previously study,we collected the normal adult sperm samples and detected the expression of VDAC protein in the spermatic flagellum.Besides that,we compared the mRNA expression of vdac subtype vdacl,vdac2 and vdac3 between the normal adults and IAS sperm samples and found that the vdac2 mRNA expression was associated with the sperm motility.Hence,we hypothesis that whether abnormal epigenetic modification in the IAS lead to the abnormal expression of VDAC2,which decrease the sperm motility and cause the male infertility.Objective:To further explore the difference methylation status of VDAC2 gene between normal spermatozoa and the asthenospermia,discuss the relationship between sperm parameters and the level of methylation,look for the potential pathogeny and clinical therapy of IAS.Methods:1.25 idiopathic asthenospermia and 27 fertile normozoospermic men were involved in this study,and recorded detail information including age,diet custom and all parameters of spermatozoa.Meanwhile the object were excluded with clear causes like varicocele,cryptorchidism and reproduction dysplasia.2.GC-2spd cell line was treated with different concentration(5?mol/L,10?mol/L,15?mol/L)of 5-aza-2'-deoxycytidine for 24h and 48h respectively,extract the total RNA of GC-2spd cell line and examine whether the gene vdac2 was regulated by methylated modification through RT-PCR.3.After predicting the promoter region with the Promoter Scan,construct plasmid containing vdac2 gene promoter region carrier and verify the active of vdac2 gene promoter in GC-2spd cell line by using a dual-luciferase activity detection.4.At last?extract the 52 sperm DNA and bisulfite sequencing PCR was used to analyze DNA methylation of the vdace2 promoter in these subjects.Results:1.We found that a significant increased expression of vdac2 mRNA after 48 hours with a drug concentration of 15?mol/L 5-aza-2'-deoxycytidine compared with other groups(P<0.05).2.A strong relative light unit was detected in the psi_CHECK-2-VDAC2 by a dual-luciferase activity detection compared with which in psi_CHECK-2-NC(P<0.05).3.After bisulphite genomic sequencing,the percentage of uncompleted methylation,mild methylation and moderate methylation in normozoospernic men were 83.65%±5.51%?8.73%±1.38%?7.61%±5.68%respectively;while the percentage of uncompleted methylation,mild methylation and moderate methylation in idiopathic asthenospermia were 76.01%±6.94%?7.14%±1.86%16.62%±8.27%respectively with P=0.005?P=0.02?P?0.003.Correlation analysis shows that a positive relationship between the percentage of uncompleted methylation and PR,while a negative relationship between percentage of moderate methylation and PR.Conclusion:1.Expression of vdac2 mRNA is associated with the status of methylation.An increasing expression of vdac2 mRNA after demethylation with the 5'-AZA-CdR.2.Sperm motility is associated with the status of the CpG island(-1337?-1059bp)in the vdac2 promoter region.3.High methylation ofvdac2 gene promoter region might be a potential cause of IAS by inhibits the expression of vdac2 mRNA?which decrease the exchange of energetic contents and weaken the sperm flagella vitality.