The Mechanism Research Of The Effect Of HBx Protein On Sperm And The Development Of Embryo

Backgroud and ObjectivesHepatitis B is a contagious disease caused by hepatitis B virus infection,It is a high incidence of hepatitis B in China.Hepatitis B virus can pass through the blood testis barrier,enter into the sperm nucleus and integrate on chromatin that the virus can be transmitted through germ cells.HBV infection is accompanied by a large number of epigenetic modification changes,but the influence on sperm quality has not yet been fully elucidated.With the progress of assisted reproductive technology,the requirement of sperm quality is greatly reduced in fertilization.However,whether the HBV virus can affect the development of embryos by sperm need to be further researched.Studying the effect of HBV on human sperm function is the cross field of hepatitis B virus infection molecular biology and reproduction.Studying the influence of sperm histone epigenetic modification on spermatozoa and embryo development will help clarify how HBV induce the male infertility.It also provides a theoretical basis for research and development to improve the treatment of male fertility and to block the vertical transmission of HBV.MethodsA retrospective analysis of 2359 couples for assisted reproductive technology(IVF/ICSI)in the reproductive center of our hospital from January 2014 to January 2016 was reviewed.The quality of the semen and the development of the progeny of the offspring were analyzed in groups according to whether it were infected with HBV.The semen samples of HBV patients were collected,and the expression spectrum was used to investigate the effect of HBV infection on sperm,and the differential expression genes were screened.It has been selected some differentially expressed genes.In order to select the proteins related to H3K9me3 and HBx,immunoprecipitation has been used to screen the proteins in sperm.And the we use RTq RCP and Western-blot to validate the microarray results.We construct Hbx stable expression in rat spermatogonial cells by using lentiviral transfection technology.Cell cycle experiment,apoptosis,si RNA interference expression of HBx protein were used for analyse the effect of HBx on sperm;Animal experiments were conducted to investigate the effects of HBx protein on the development of embryos.The mouse oocyte and sperm were collected for methylation analysis.The level of methylation on the imprinted genes and their effects on embryo development were observed.ResultThe sperm concentration,total sperm count,PR rate,sperm survival rate is low in HBV patients while the abnormal sperm rate is high.The outcome of ICSI is bad include lower normal fertilization rate and lower high quality embryo obtained rate.Hepatitis B virus high copy number decrease the quality of sperm and high quality embryo obtained rate.There are many differences in expression sperm gene in patients with HBV,including H3K9me3,histone methyltransferase(Suv39H1),DNA methyltransferase,HBx protein.The functions involved in histone modification,spermatogenesis,cell differentiation,chromatin formation;further study found that Hbx can decrease the expression of Suv39H1 in sperm accompanied the decrease of histone H3K9me3 in sperm histone,abnormal sperm DNA damage,spermatogonia cell cycle arrest and apoptosis.The expression of HBx may be regulated by UBP1,and HBx can interact with E2F5 to inhibit the expression of Suv39H1.Animal experiments showed that after the transfection of HBx,spermatozoa highly expressed H19 and KCNQ1OT1,and low expression of IGF2.In the in vitro culture system,the infection of paternal HBx had no effect on the development of preimplantation embryos.ConclusionHBV patients can express HBx protein in spermatogonia,and cause the abnormal histone-protamine alternation,sperm DNA damage,cell cycle arrest,apoptosis increased,and the decrease of the number and quality in sperm.In animal experiments,HBx could influence the methylation level of some imprinted genes in the control region.