Expression Of DDX25during Spermatogenesis And Its Roles In Apoptotic Spermatogenic Cells

Objective: Male infertility accounts for about half of all infertility casesand affects one-sixth of couples worldwide. A substantial proportion of maleinfertility is accompanied by azoospermia, most often presenting asnon-obstructive azoospermia (NOA), which occurs in~1%of all adult men.Approximately25%of infertile men of Chinese ancestry exhibitazoospermia.Some researchers have suggested that NOA may result fromgenetic factors. The past several years have witnessed an explosion in thenumber of reports of gene mutations or polymorphisms that cause or arelinked to male infertility. Identifying genetic factors that influence maleinfertility will provide valuable insights into the causes of idiopathic infertilityand will aid the development of targeted therapies.Gonadotropin-regulated testicular RNA helicase (DDX25/GRTH), amember of the Glu-Asp-Ala-Glu (DEAD)-box protein family, is atestis-specific gonadotropin/androgen-regulated RNA helicase that isregulated by androgen occurs through direct actions of androgen in Leydigcells, and presumably in germ cells indirectly by the participation ofandrogen-responsive genes from Sertoli cells. DDX25is present in germ cells(meiotic spermatocytes and round spermatids) and Leydig cells. DDX25isessential for completion of spermatogenesis as a posttranscriptional regulatorof relevant genes during spermatogenic cellsdevelopment. Male mice lackingDDX25are sterile with spermatogenic arrest due to failure of roundspermatids to elongate, where striking structural changes and severe apoptosisof germ cells was also observed in spermatocytes entering the metaphase ofmeiosis. Several findings indicated that gene mutation of DDX25may beinvolved in male infertility of some patients with NOA, suggesting thatvariations of DDX25gene detected by single-nucleotide polymorphisms (SNPs) analysis may contribute to susceptibility to spermatogenic impairmentin humans.Here, we summarize the current status of findings on the essentialrole of DDX25/DDX25in spermatogenic cells development, with emphasison its multifunctional control of spermatogenesis.Spermatogenesis of mammalian is a highly specialized and complicateddevelopmental process, depending on the integrated expression of a series ofgenes that operate in a sequence to generate spermatozoa and requiring thebalance between spermatogenic cellsproliferation, differentiation andapoptosis. Gene expression during spermatogenesis undergoes temporaluncoupling of transcription and translation.Genetic breakthroughs inunderstanding the regulators and the causes of infertility in mammalianhave involved the studies of signaling pathway.Investigations of genes thatregulate spermatogenesis are being carried out mainly via the production andstudy of mice carrying transgenes or targeted gene disruptions. Conditionalgene targeting is labor intensive and time consuming and knockouts usuallyproduce different abnormalities in different cells at the same stage in a singletestis. Within the past few years, double-strand RNA-mediatedposttranscriptional gene silencing, or RNA interference (RNAi), has becomean important tool for studying the functions of genes in many organisms. Asthe structure and function of testis are complex, the physiology andbiochemistry function of spermatogenic cell is difficult to further research.Establish of spermatogenic cell culture system in vitro can provide a powerfultool to determin regulation of gene in spermatogenesis and study therelationship between spermatogenic cells and other cells.So far， DDX25null mice are common animal models for research, theeffect of DDX25on mouse spermatogenesis in vitro and in vivo has not beenstudied by using RNAi. In our study, the expression of gene and protein ofDDX25in mouse testis at different stages of spermatogenesis was firstlyinvestigated respectively; secondly, DDX25was knocked down in vivo inadult male mice using intratesticular injection of siRNA; and then,spermatogenic cellscoculture system was set up; finally siRNA with oligofectamine reagent was used to silience the gene expression of DDX25inthe co-cultured germ cells. In this study, the RNAi was firstly carried out toknockdown DDX25gene in in vitro and in vivo.The present research providesa new method and technology to investigate the role and potential signalpathways of DDX25in spermatogenesis.Methods:1Developmental expression of DDX25in mouse testesMale C57BL/6J mice were used from5to120postnatal days.â‘´Paraffinsections were used with haematoxylin-eosin(H&E) staining and PAS stainingto show morphological structure of normal C57BL/6J mouse spermatogenesisat postnatal stages of the developing testis. Subsequently we undertook adetailed analysis of immunoreactive staining present in defferent types ofgerm cells during the spermatogenic cycle of the adult mouse testis.Testeswere individually collected from postnatal C57BL/6J mouse at different ages(P5,P15,P21,P35,P42,P60and P120).⑵Immunohistochemistry andimmunofluorescence were used to detect the expression and location ofDDX25in the testis of5,15,21,35,42,60,and120day-old C57BL/6J mouse toinvestigate the cellular expression patterns and localization of DDX25proteinin the seminiferous tubules during spermatogenesis.⑶Western blot wereused to observe the expression of DDX25protein in the testis of5,15,21,35,42,60and120day-old C57BL/6J mouse to prevent nonspecificstaining.â‘·The mRNA expression of DDX25in the testis of5,15,21,35,42,60,and120day-old C57BL/6J mouse was detected by RT-qPCR.2The effect of RNAi against DDX25in vivo on cellular apoptotic factorsexpression of mouse testis60days,126male C57BL/6J mice were randomly divided into7groups:independent control group(without siRNA and transfection reagent); negativecontrol group(control siRNA-A and transfection reagent); vacuity controlgroup(transfection reagent); positive control group(β-Actin siRNA andtransfection reagent); experimental group(0.125μg/μl siRNA,0.25μg/μlsiRNA,0.5μg/μl siRNA).Inject siRNA into the testicular tissue. At24,48,72 hours, respectively, the mice were killed and the testes were removed. Oneportion of the testis tissues was fixed in4%paraformaldehyde in forhistological and immunohistochemical examinations, another portion of thetesticular tissue was used to abstract total RNA and protein. The structure oftesticular tissue was detected by H&E staining. Testicular spermatogenic cellsapoptosis was detected by TUNEL. The expression of DDX25were detectedby immunocytochemistry,immunofluoresence and Western blot. The mRNAlevels of DDX25were detected by RT-qPCR. The expression of Bid,Bad,Bak,Smac,p38MAPK,p-p38MAPK,p53,Bcl-2,Bcl-xL,p-Bad,Erk1/2andp-Erk1/2were detected by Western blot.3The effect of DDX25inhibition on apoptosis and MAPK pathway ofspermatogenic cellsThe testis were removed from60-day C57BL/6J mice and decapsulated forthe purification of germ cells and Leydig cells.The germ cells and Leydig cellswere seeded in culture plates or bicameral chambers and were co-cultured.Thegrowth and morphology of co-culture cells were monitored daily undercontrast phase microscope and the survival rate was determined bytrypan-blue staining. For RNAi research, the germ cells were divided into7groups: independent control group(without siRNA and transfection reagent);negative control group(control siRNA-A and transfection reagent); vacuitycontrol group(transfection reagent); positive control group(β-Actin siRNAand transfection reagent); experimental group(12.5nM siRNA;25nMsiRNA;50nM siRNA). Transfection was performed with EntransterTM-invitro reagent following the manufacturer’s protocol.All groups were incubatedfor24h,48h and72h. RT-qPCR,Immunocytochemistry andimmunofluorescence were used to detecte the effect of DDX25siRNAinhibition. The survival rate of germ cells were detected by trypan-bluestaining. The spermatogenic cells apoptosis induced by DDX25siRNA wasdetected by flow cytometry. The protein levels of Erk1/2， p-Erk1/2，p38MAPKå'Œp-p38MAPK were measured by Western blot.Result: 1. Developmental expression of DDX25in mouse testesâ‘´The DDX25antigen was not detected in testis of5-day-old and15-day-old mouse. At P21, there is a low level of DDX25in Leydig cells. AtP35, DDX25staining was observed in Leydig cells, spermatocytes, and roundspermatids. Spermatogonia within the basal compartment were clearlynegative as the Sertoli cells. At P42,P60and P120, all types of germ cells andLeydig cells were immunopositive except sperm. Although cytoplasmic andplasma membrane-localized DDX25was evident in all spermatogeniccellssubtypes and Leydig cells, spermatogenic cells nuclear staining was notdetected at day21, but was present rarely at day35in pachytenespermatocytes，corresponding with the period in which they are meiotic.Nuclear signal was also observed in round spermatids. The Nuclearlocalization was not observed in some Leydig cells from5to120postnataldays.⑵Express level of DDX25mRNA situate at low relatively at postnatalP5, increased to a low level at P15, then increased obviously at P21andreached peak P35, then increase at P42and retained high afterwards.Expression of exhibited peak in testis of mice P35when round spermatidstransformed to elongate spermatids，which indicated DDX25may be involvedin spermiation and spermiogenesis.2The effect of RNAi against DDX25in vivo on cellular apoptotic factorsexpression of mouse testisâ‘´To evaluate the efficiency of RNA interference：①The level of DDX25mRNA were detected by real-time PCR. Compared with control groups, theexpression of DDX25mRNA were significantly decreased in0.25μg/μlsiRNA experimental group and0.5μg/μl siRNA experimental group at24h(p<0.05). The level reached0.29to independent control group at24h.â‘¡Histological examination by H&E staining showed that the structure ofseminiferous tubules was normal without damage.â‘¢Immunocytochemistryshowed that the expression of DDX25in testis tissue with DDX25siRNAtreatment was lower than control groups. Western blot indicated that theexpression level of DDX25was lowest in0.25μg/μl siRNA experimental group and0.5μg/μl siRNA experimental group at72h (p<0.05).⑵TUNELindicated that the apoptosis of germ cells in0.25μg/μl siRNA experimentalgroup and0.5μg/μl siRNA experimental group at72h were markedlyincreased after RNAi (p<0.05).⑶The result of western blot showed that,compare with control groups, the expression levels of DDX25proteins weresignificantly decreased in0.25μg/μl siRNA experimental group and0.5μg/μlsiRNA experimental group at72h (p<0.05). The levels ofBid,Bad,Bak,Smac,p38MAPK,p-p38MAPK and p53proteins weresignificantly increased after RNAi(p<0.05). The protein levels of Bcl-2,Bcl-xL, p-Bad，Erk1/2and p-Erk1/2were decreased after RNAi（p<0.05）.3The effect of DDX25inhibition on apoptosis and MAPK pathway ofspermatogenic cellsâ‘´In co-culture system, short flagella were seen emerging from one end ofsome spermatogenic cells after two weeks. After three weeks, a number ofgerm cells still attached on the surface of sertoli cell. From day5~10,Thesecells maintained an stable survival rate and growed well.Immunocytochemistry and immunofluoresence showed that the positivestaining for DDX25was observed in spermatogonia, spermatocytes andhaploid round spermatids, and the expression of DDX25was stronger inspermatocytes.â‘¡Western blot indicated that the expression of DDX25wasobserved in germ cells.⑶After comparison and analysis of the fluorescenceintensity,the effective transfection concentration was identified. Transfectionefficiency was reached0.95.â‘·To evaluate efficiency of RNAi:â‘ DDX25siRNA markedly reduced the expression of DDX25in spermatogenic cells.The result of RT-qPCR showed that, compare with control groups, theexpression levels of DDX25mRNA of spermatogenic cells were significantlydecreased in25nM siRNA experimental group and50nM siRNA experimentalgroup at24h,48h and72h(p<0.05). After24hours, the levels of DDX25mRNA of25nM siRNA experimental group and50nM siRNA experimentalgroup were significantly decreased, compared with12.5nM siRNAexperimental group (p<0.05).â‘¡The result of Immunocytochemistry, immunofluorescence and Western blot indicated that,compare with controlgroups, the expression levels of DDX25proteins were significantly decreasedin25nM siRNA experimental group and50nM siRNA experimental group at72h(p<0.05).⑸The effect of DDX25siRNA on apoptosis of spermatogeniccells：①The result of trypan-blue staining showed that, compare with controlgroups, the survival rate of spermatogenic cells were significantly decreased in25nM siRNA experimental group and50nM siRNA experimental group at48h and72h (p<0.05).â‘¡The results of flow cytometry showed that theapoptotic spermatogenic cells among transfected with DDX25siRNA in25nM siRNA experimental group and50nM siRNA experimental group at72h were markedly higher than that in untransfected spermatogenic cells andspermatogenic cells trasfected with12.5nM siRNA(p<0.05).⑹The proteinlevel of p38MAPK and p-p38MAPK significantly increased,but Erk1/2and p-Erk1/2significantly decreased in25nM siRNA experimental group at72h by Western blot（p<0.05）.Conclusions:1The mRNA and protein was expressed in the testis of mice at differentstage during spermatogenesis.It indicated that DDX25may be involved inspermiation and spermiogenesis.2DDX25siRNA transfer into mice testis can efficially inhibited theDDX25gene expression in mouse testis in vitro without toxic effect.3The expression of cellular apoptotic factors of testes and the apoptoic rateof spermatogenic cells were changed by RNAi in vivo.4In co-culture system, the spermatogenic cells survived nearly as long asthree weeks, and some of them generated flagella, which stated clearly that theprocess of spermiogenesis could taken place in this co-culture system.5DDX25siRNA can efficially inhibited the gene expression of DDX25inco-cultured spermatogenic cells in vitro and may be induced apoptosis ofspermatogenic cells.These results suggested that DDX25may be involved inthe regulation of spermatogenic cell apoptosis by MAPK pathway.