DDX25在小鼠精子发生过程中的表达及其与生精细胞凋亡的关系
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摘要
目的：目前，全世界有六分之一的夫妇受到不育症的困扰，其中一半以上是男性不育。相当比例的男性不育症患者的睾丸不能产生精子，通常被称为非梗阻性无精子症（NOA），此类患者的数量在成年男性中占1%左右。在中国，25%的男性不育症患者的长辈中有无精子症患者。多项研究也显示NOA与遗传有关。近年来与男性不育密切相关的基因突变或多态现象相继被发现并日益成为研究热点。因此，对调控精子发生的相关基因的深入研究，不仅有助于阐明特发性不育症的病因，还对男性不育症靶向治疗的发展和男性避孕有重要的理论及应用价值。
促性腺激素调节的睾丸RNA解旋酶（DDX25/GRTH）属于DEAD-box家族成员，是目前唯一明确由激素调控的RNA解螺旋酶，由雄激素通过雄激素受体在不同发育阶段调节其转录和翻译水平，其调控途径分别是睾丸间质细胞分泌的雄激素直接作用或睾丸支持细胞中雄激素易感基因介导的间接作用。DDX25在睾丸中特异表达，主要高表达于精母细胞、圆形精子细胞和间质细胞中。DDX25基因敲除的雄性小鼠表现为不育，其形态学表现是精子形成过程被完全阻断在精子发生的第8/9阶段，圆形精子细胞无法变长，造成无精子症，同时还发生了严重的精母细胞凋亡。不同发育阶段及不同生殖细胞表达的差异表明DDX25可能在精子发生过程中的特定阶段发挥不可或缺的作用。也有研究发现，在NOA患者中发现DDX25的单核苷酸多态性，说明DDX25是导致男性不育的重要因素。
哺乳动物的精子发生是一个高度精细和复杂的过程，依赖一系列相关基因在特定阶段的顺序表达，在维持生精细胞的增殖、分化和凋亡平衡的过程中伴随多种基因的短暂转录和翻译。目前，针对不育症相关基因的研究已经深入至信号分子水平，主要研究工具是转基因或基因敲除鼠。但是，条件基因的定向敲除不仅容易导致同一睾丸组织中相同细胞的不同表现，而且历时长、耗费巨大、动物易死亡。因此，RNA干扰技术已经成为研究基因功能的重要工具。同时，由于睾丸组织结构与功能的复杂性和特殊性，在整体情况下很难对生殖细胞的功能和与其他细胞间的相互作用进行深入研究。生精细胞体外培养技术的建立使深入阐明精子发生过程和细胞间的关系成为可能。
目前为止，应用RNA干扰技术在体内外沉默DDX25基因进行作用的研究尚未见报道。本研究首先检测生后不同日龄即不同发育阶段小鼠睾丸组织中DDX25基因和蛋白表达的情况；其次应用RNA干扰技术在小鼠活体体内抑制DDX25的表达，检测睾丸组织中凋亡相关蛋白的表达变化；再次建立生精细胞的体外培养体系，应用siRNA沉默体外培养生精细胞中DDX25基因，检测沉默DDX25基因对生精细胞凋亡的影响和可能途径，为研究DDX25在精子发生中的功能和可能机制提供新的研究手段和技术方法。
方法：
1DDX25在正常小鼠睾丸发育中的表达
以出生当天为d0，取出生后d5、d15、d21、d35、d42、d60和d120的C57BL/6J小鼠睾丸组织，4%多聚甲醛固定，制作石蜡切片进行HE染色，观察小鼠睾丸发育的形态结构特点；取60日龄小鼠睾丸组织石蜡切片进行PAS染色，观察精子发生过程中生精上皮的各个时相的形态特点和细胞组成；免疫组化检测DDX25蛋白在精子发生过程中的表达特性和细胞内的定位；免疫印迹检测DDX25在小鼠睾丸组织中的表达，明确DDX25蛋白的表达是否与免疫组织化学结果一致，以避免免疫组织化学产生非特异性着色影响实验结果；RT-qPCR检测DDX25的mRNA在不同日龄小鼠睾丸组织中的表达，明确DDX25基因在小鼠睾丸发育和精子发生过程中的表达特点。
2体内抑制DDX25对生精细胞凋亡相关蛋白表达的影响
将12周龄雄性C57BL/6J小鼠随机分为7组，⑴空白对照组：只加转染试剂；⑵阴性对照组：转染试剂和control siRNA-A；⑶无关对照组：未加siRNA和转染试剂；⑷阳性对照组：转染试剂和β-actin siRNA；⑸实验组：A组：0.125μg/μl siRNA转染组；B组：0.25μg/μl siRNA转染组；C组：0.5μg/μl siRNA转染组。每组18只小鼠，应用纳米级转染试剂溶解DDX25siRNA进行睾丸局部注射，于干扰后24、48和72h各取6只小鼠处死取双侧睾丸，一侧的睾丸固定于4%多聚甲醛中做石蜡切片，用于形态学观察；另一侧的睾丸快速放入液氮中冻存用于RNA和蛋白的提取。RT-qPCR检测干扰后各组小鼠睾丸组织中DDX25mRNA的表达变化，确定干扰效果与有效浓度；HE染色观察干扰后小鼠睾丸组织是否有改变；免疫组织化学、免疫荧光和免疫印迹检测转染前后DDX25蛋白在睾丸组织中的表达情况，确定最佳干扰浓度和作用时间；TUNEL检测睾丸组织中生精细胞的凋亡情况；免疫印迹检测0.25μg/μl siRNA转染组于转染72h后小鼠睾丸组织中Bid, Bad, Bak, Smac， p38MAPK，p-p38MAPK，p53，Bcl-2, Bcl-xL, p-Bad，Erk1/2和p-Erk1/2蛋白表达情况。
3体外抑制DDX25对生精细胞凋亡及MAPK信号通路的影响
取12周龄雄性C57BL/6J小鼠睾丸组织分别分离纯化睾丸间质细胞和支持细胞-生精细胞混合细胞团，将二者分别接种于Transwell小室和六孔板底进行双室培养，相差显微镜下观察细胞的生长情况；台盼蓝染色法检测生精细胞存活率；应用DDX25siRNA转染共培养的生精细胞，分组：无关对照组（未加siRNA和转染试剂）；空白对照组（只加转染试剂）；阴性对照组（control siRNA-A和转染试剂）；阳性对照组：（β-actin siRNA和转染试剂）；实验组（DDX25siRNA（12.5nM、25nM、50nM）和转染试剂）。倒置荧光显微镜检测有效干扰浓度；RT-qPCR、免疫组化、免疫荧光与免疫印迹检测评价干扰效能；台盼蓝染色法、流式细胞术、TUNEL法检测转染后生精细胞的凋亡情况；免疫印迹检测干扰前后生精细胞中Erk1/2，p-Erk1/2，p38MAPK和p-p38MAPK蛋白的表达情况。
结果：
1DDX25在正常小鼠睾丸发育中的表达
⑴DDX25蛋白的阳性表达首先出现于21日龄小鼠睾丸间质细胞的胞质中，呈弱阳性表达；在35日龄小鼠睾丸组织中DDX25蛋白则分别在睾丸间质细胞、精原细胞、精母细胞和圆形精子细胞的胞质中表达，其中间质细胞和精母细胞中的阳性表达较强（p<0.05）；42日龄小鼠睾丸组织中DDX25蛋白表达于间质细胞的胞质，精原细胞、精母细胞和圆形精子细胞的胞核和胞质，其中精母细胞和圆形精子细胞的阳性表达更强（p<0.05）；60日龄和120日龄小鼠睾丸组织中DDX25蛋白的表达与42日龄小鼠睾丸组织中的表达一致，无显著差异（p＞0.05）。⑵对60日龄成年小鼠睾丸生精上皮的生精周期的各个时相进行免疫组织化学检测，结果显示，处于生精周期的12个时相的生精上皮中均有DDX25的阳性表达，其中以Ⅰ、Ⅲ、Ⅴ、Ⅵ、Ⅶ期的阳性表达程度较高。⑶免疫印迹检测结果显示，5日龄和15日龄小鼠睾丸组织总蛋白中无DDX25蛋白条带出现；21日龄睾丸组织总蛋白中分子量61KD处有条带出现；35日龄睾丸组织总蛋白中分子量61KD处有较21日龄灰度明显的单一条带（p<0.05）；42、60和120日龄睾丸组织总蛋白中可见分子量61KD和56KD处两条条带，灰度值没有明显差异（p＞0.05）。⑷RT-qPCR结果显示DDX25基因在不同日龄小鼠组织中均有表达，表达水平从21日龄开始升高，到35日龄表达水平显著上调（p<0.05），42日龄后表达上调缓慢，但各日龄小鼠睾丸中DDX25mRNA的表达水平无明显差异（p＞0.05）。
2体内抑制DDX25对生精细胞凋亡相关蛋白表达的影响
⑴DDX25siRNA干扰的效能评价：①RT-qPCR检测结果显示，0.125μg/μl siRNA实验组干扰48h和72h后DDX25mRNA表达低于各对照组，但差异不显著（p>0.05）；干扰24h、48h和72h的0.25μg/μl siRNA实验组和0.5μg/μl siRNA实验组睾丸组织中DDX25mRNA表达均明显低于各对照组（p<0.05），但干扰24h,48h和72h的0.25μg/μl siRNA实验组DDX25mRNA的表达与0.5μg/μl siRNA实验组无明显差异（p>0.05）。②形态学观察结果显示：各组小鼠睾丸组织结构基本正常。③免疫组化与免疫印迹结果显示，0.125μg/μl siRNA实验组干扰72h后DDX25表达降低较无关对照组显著（p＜0.05）；干扰48h和72h的0.25μg/μl siRNA实验组和0.5μg/μl siRNA实验组DDX25蛋白的阳性表达均显著低于各对照组（p＜0.05）；干扰72h的50μg/μl siRNA实验组生精细胞内几乎没有DDX25蛋白的表达。⑵干扰DDX25表达对生精细胞凋亡的影响：TUNEL检测结果显示，干扰72h的0.5μg/μl siRNA实验组生精细胞凋亡率较无关对照组和其他实验组明显增高（p<0.05）。⑶Western blot检测结果显示，0.5μg/μl siRNA干扰72h后，Bid, Bad, Bak, Smac，p38MAPK，p-p38MAPK和p53的表达在干扰后显著增高（p<0.05）；Bcl-2, Bcl-xL, p-Bad，Erk1/2和p-Erk1/2的表达在干扰后显著降低（p<0.05）。
3体外抑制DDX25对生精细胞凋亡及MAPK信号通路的影响
⑴倒置相差显微镜观察，双室培养第5~10天，生精细胞生长状态较稳定，细胞存活率无明显降低（p>0.05）。⑵分离纯化的生精细胞中DDX25的表达：①免疫组化和免疫荧光结果显示，分离纯化的成年小鼠睾丸的生精细胞中可见DDX25蛋白的阳性表达，主要在精原细胞、精母细胞和圆形精子细胞中表达，其中精母细胞中的表达强度最高（p<0.05）。②免疫印迹结果显示，分离纯化的成年小鼠睾丸的生精细胞中有DDX25蛋白的阳性表达。⑶siRNA有效转染浓度的筛选：倒置荧光显微镜观察，与12.5nM转染组相比，转染24h后25nM转染组与50nM转染组绿色荧光强度均显著增强（p<0.05），但二者强度差别不大（p>0.05），转染效率可达95%以上。⑷DDX25siRNA干扰的效能评价：①RT-qPCR检测结果显示，干扰24h、48h和72h的25nM siRNA实验组DDX25mRNA表达均明显低于各对照组，（p<0.05），其中干扰24h和48h的25nM siRNA实验组DDX25mRNA的表达较干扰72h组降低更明显（p<0.05）。②免疫组化、免疫荧光与免疫印迹结果显示，干扰72h的25nM siRNA实验组和50nM siRNA实验组生精细胞中DDX25蛋白的阳性表达均显著低于各对照组（p<0.05）。⑸干扰DDX25表达对生精细胞凋亡的影响：①台盼蓝染色法检测结果显示，与无关对照组相比，干扰72h的50nM siRNA实验组和25nM siRNA实验组生精细胞存活率明显降低（p<0.05），但二者之间无显著差异（p>0.05）。②流式检测结果显示，参照无关对照组，空白对照组、阴性对照组和阳性对照组的生精细胞凋亡率无明显变化；干扰72h的25nM siRNA实验组和50nM siRNA实验组生精细胞凋亡率率明显增高（p<0.05），但二者之间无显著差异（p>0.05）。⑹Western blot检测结果显示，25nM siRNA实验组干扰72h后生精细胞中Erk1/2和p-Erk1/2的表达较无关对照组显著降低（p<0.05），p38MAPK和p-p38MAPK的表达较无关对照组显著升高（p<0.05）。
结论：
1DDX25基因和蛋白在小鼠精子发生过程中呈阶段特异性表达；DDX25主要在成年小鼠的精子发生过程中发挥转录后调控作用，主要作用于精母细胞和圆形精子细胞。
2应用纳米材料通过睾丸局部直接注射方法将DDX25siRNA转染至小鼠体内，能有效抑制DDX25mRNA的表达，干扰效果良好且无毒性作用。可成为体内研究基因功能的有效方手段。
3活体体内DDX25基因沉默后，小鼠睾丸组织中生精细胞的凋亡增多，多种凋亡相关蛋白表达发生改变。
4将睾丸间质细胞与支持细胞-生精细胞进行双室培养，生精细胞可以存活达20天以上，可见部分生精细胞尾部出现鞭毛。
5应用DDX25siRNA体外转染生精细胞抑制生精细胞中DDX25的表达使生精细胞凋亡增加； DDX25可能分别通过ERK1/2与p38MAPK途径抑制生精细胞分化和诱导生精细胞凋亡。
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 testes
Male C57BL/6J mice were used from5to120postnatal days.⑴Paraffinsections 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.⑷The 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 testis
60days,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 cells
The 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，p38MAPK和p-p38MAPK were measured by Western blot.
Result:
1. Developmental expression of DDX25in mouse testes
⑴The 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 testis
⑴To 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.②Histological examination by H&E staining showed that the structure ofseminiferous tubules was normal without damage.③Immunocytochemistryshowed 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 cells
⑴In 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.②Western 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.⑷To evaluate efficiency of RNAi:①DDX25siRNA 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).②The 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).②The 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.
促性腺激素调节的睾丸RNA解旋酶（DDX25/GRTH）属于DEAD-box家族成员，是目前唯一明确由激素调控的RNA解螺旋酶，由雄激素通过雄激素受体在不同发育阶段调节其转录和翻译水平，其调控途径分别是睾丸间质细胞分泌的雄激素直接作用或睾丸支持细胞中雄激素易感基因介导的间接作用。DDX25在睾丸中特异表达，主要高表达于精母细胞、圆形精子细胞和间质细胞中。DDX25基因敲除的雄性小鼠表现为不育，其形态学表现是精子形成过程被完全阻断在精子发生的第8/9阶段，圆形精子细胞无法变长，造成无精子症，同时还发生了严重的精母细胞凋亡。不同发育阶段及不同生殖细胞表达的差异表明DDX25可能在精子发生过程中的特定阶段发挥不可或缺的作用。也有研究发现，在NOA患者中发现DDX25的单核苷酸多态性，说明DDX25是导致男性不育的重要因素。
哺乳动物的精子发生是一个高度精细和复杂的过程，依赖一系列相关基因在特定阶段的顺序表达，在维持生精细胞的增殖、分化和凋亡平衡的过程中伴随多种基因的短暂转录和翻译。目前，针对不育症相关基因的研究已经深入至信号分子水平，主要研究工具是转基因或基因敲除鼠。但是，条件基因的定向敲除不仅容易导致同一睾丸组织中相同细胞的不同表现，而且历时长、耗费巨大、动物易死亡。因此，RNA干扰技术已经成为研究基因功能的重要工具。同时，由于睾丸组织结构与功能的复杂性和特殊性，在整体情况下很难对生殖细胞的功能和与其他细胞间的相互作用进行深入研究。生精细胞体外培养技术的建立使深入阐明精子发生过程和细胞间的关系成为可能。
目前为止，应用RNA干扰技术在体内外沉默DDX25基因进行作用的研究尚未见报道。本研究首先检测生后不同日龄即不同发育阶段小鼠睾丸组织中DDX25基因和蛋白表达的情况；其次应用RNA干扰技术在小鼠活体体内抑制DDX25的表达，检测睾丸组织中凋亡相关蛋白的表达变化；再次建立生精细胞的体外培养体系，应用siRNA沉默体外培养生精细胞中DDX25基因，检测沉默DDX25基因对生精细胞凋亡的影响和可能途径，为研究DDX25在精子发生中的功能和可能机制提供新的研究手段和技术方法。
方法：
1DDX25在正常小鼠睾丸发育中的表达
以出生当天为d0，取出生后d5、d15、d21、d35、d42、d60和d120的C57BL/6J小鼠睾丸组织，4%多聚甲醛固定，制作石蜡切片进行HE染色，观察小鼠睾丸发育的形态结构特点；取60日龄小鼠睾丸组织石蜡切片进行PAS染色，观察精子发生过程中生精上皮的各个时相的形态特点和细胞组成；免疫组化检测DDX25蛋白在精子发生过程中的表达特性和细胞内的定位；免疫印迹检测DDX25在小鼠睾丸组织中的表达，明确DDX25蛋白的表达是否与免疫组织化学结果一致，以避免免疫组织化学产生非特异性着色影响实验结果；RT-qPCR检测DDX25的mRNA在不同日龄小鼠睾丸组织中的表达，明确DDX25基因在小鼠睾丸发育和精子发生过程中的表达特点。
2体内抑制DDX25对生精细胞凋亡相关蛋白表达的影响
将12周龄雄性C57BL/6J小鼠随机分为7组，⑴空白对照组：只加转染试剂；⑵阴性对照组：转染试剂和control siRNA-A；⑶无关对照组：未加siRNA和转染试剂；⑷阳性对照组：转染试剂和β-actin siRNA；⑸实验组：A组：0.125μg/μl siRNA转染组；B组：0.25μg/μl siRNA转染组；C组：0.5μg/μl siRNA转染组。每组18只小鼠，应用纳米级转染试剂溶解DDX25siRNA进行睾丸局部注射，于干扰后24、48和72h各取6只小鼠处死取双侧睾丸，一侧的睾丸固定于4%多聚甲醛中做石蜡切片，用于形态学观察；另一侧的睾丸快速放入液氮中冻存用于RNA和蛋白的提取。RT-qPCR检测干扰后各组小鼠睾丸组织中DDX25mRNA的表达变化，确定干扰效果与有效浓度；HE染色观察干扰后小鼠睾丸组织是否有改变；免疫组织化学、免疫荧光和免疫印迹检测转染前后DDX25蛋白在睾丸组织中的表达情况，确定最佳干扰浓度和作用时间；TUNEL检测睾丸组织中生精细胞的凋亡情况；免疫印迹检测0.25μg/μl siRNA转染组于转染72h后小鼠睾丸组织中Bid, Bad, Bak, Smac， p38MAPK，p-p38MAPK，p53，Bcl-2, Bcl-xL, p-Bad，Erk1/2和p-Erk1/2蛋白表达情况。
3体外抑制DDX25对生精细胞凋亡及MAPK信号通路的影响
取12周龄雄性C57BL/6J小鼠睾丸组织分别分离纯化睾丸间质细胞和支持细胞-生精细胞混合细胞团，将二者分别接种于Transwell小室和六孔板底进行双室培养，相差显微镜下观察细胞的生长情况；台盼蓝染色法检测生精细胞存活率；应用DDX25siRNA转染共培养的生精细胞，分组：无关对照组（未加siRNA和转染试剂）；空白对照组（只加转染试剂）；阴性对照组（control siRNA-A和转染试剂）；阳性对照组：（β-actin siRNA和转染试剂）；实验组（DDX25siRNA（12.5nM、25nM、50nM）和转染试剂）。倒置荧光显微镜检测有效干扰浓度；RT-qPCR、免疫组化、免疫荧光与免疫印迹检测评价干扰效能；台盼蓝染色法、流式细胞术、TUNEL法检测转染后生精细胞的凋亡情况；免疫印迹检测干扰前后生精细胞中Erk1/2，p-Erk1/2，p38MAPK和p-p38MAPK蛋白的表达情况。
结果：
1DDX25在正常小鼠睾丸发育中的表达
⑴DDX25蛋白的阳性表达首先出现于21日龄小鼠睾丸间质细胞的胞质中，呈弱阳性表达；在35日龄小鼠睾丸组织中DDX25蛋白则分别在睾丸间质细胞、精原细胞、精母细胞和圆形精子细胞的胞质中表达，其中间质细胞和精母细胞中的阳性表达较强（p<0.05）；42日龄小鼠睾丸组织中DDX25蛋白表达于间质细胞的胞质，精原细胞、精母细胞和圆形精子细胞的胞核和胞质，其中精母细胞和圆形精子细胞的阳性表达更强（p<0.05）；60日龄和120日龄小鼠睾丸组织中DDX25蛋白的表达与42日龄小鼠睾丸组织中的表达一致，无显著差异（p＞0.05）。⑵对60日龄成年小鼠睾丸生精上皮的生精周期的各个时相进行免疫组织化学检测，结果显示，处于生精周期的12个时相的生精上皮中均有DDX25的阳性表达，其中以Ⅰ、Ⅲ、Ⅴ、Ⅵ、Ⅶ期的阳性表达程度较高。⑶免疫印迹检测结果显示，5日龄和15日龄小鼠睾丸组织总蛋白中无DDX25蛋白条带出现；21日龄睾丸组织总蛋白中分子量61KD处有条带出现；35日龄睾丸组织总蛋白中分子量61KD处有较21日龄灰度明显的单一条带（p<0.05）；42、60和120日龄睾丸组织总蛋白中可见分子量61KD和56KD处两条条带，灰度值没有明显差异（p＞0.05）。⑷RT-qPCR结果显示DDX25基因在不同日龄小鼠组织中均有表达，表达水平从21日龄开始升高，到35日龄表达水平显著上调（p<0.05），42日龄后表达上调缓慢，但各日龄小鼠睾丸中DDX25mRNA的表达水平无明显差异（p＞0.05）。
2体内抑制DDX25对生精细胞凋亡相关蛋白表达的影响
⑴DDX25siRNA干扰的效能评价：①RT-qPCR检测结果显示，0.125μg/μl siRNA实验组干扰48h和72h后DDX25mRNA表达低于各对照组，但差异不显著（p>0.05）；干扰24h、48h和72h的0.25μg/μl siRNA实验组和0.5μg/μl siRNA实验组睾丸组织中DDX25mRNA表达均明显低于各对照组（p<0.05），但干扰24h,48h和72h的0.25μg/μl siRNA实验组DDX25mRNA的表达与0.5μg/μl siRNA实验组无明显差异（p>0.05）。②形态学观察结果显示：各组小鼠睾丸组织结构基本正常。③免疫组化与免疫印迹结果显示，0.125μg/μl siRNA实验组干扰72h后DDX25表达降低较无关对照组显著（p＜0.05）；干扰48h和72h的0.25μg/μl siRNA实验组和0.5μg/μl siRNA实验组DDX25蛋白的阳性表达均显著低于各对照组（p＜0.05）；干扰72h的50μg/μl siRNA实验组生精细胞内几乎没有DDX25蛋白的表达。⑵干扰DDX25表达对生精细胞凋亡的影响：TUNEL检测结果显示，干扰72h的0.5μg/μl siRNA实验组生精细胞凋亡率较无关对照组和其他实验组明显增高（p<0.05）。⑶Western blot检测结果显示，0.5μg/μl siRNA干扰72h后，Bid, Bad, Bak, Smac，p38MAPK，p-p38MAPK和p53的表达在干扰后显著增高（p<0.05）；Bcl-2, Bcl-xL, p-Bad，Erk1/2和p-Erk1/2的表达在干扰后显著降低（p<0.05）。
3体外抑制DDX25对生精细胞凋亡及MAPK信号通路的影响
⑴倒置相差显微镜观察，双室培养第5~10天，生精细胞生长状态较稳定，细胞存活率无明显降低（p>0.05）。⑵分离纯化的生精细胞中DDX25的表达：①免疫组化和免疫荧光结果显示，分离纯化的成年小鼠睾丸的生精细胞中可见DDX25蛋白的阳性表达，主要在精原细胞、精母细胞和圆形精子细胞中表达，其中精母细胞中的表达强度最高（p<0.05）。②免疫印迹结果显示，分离纯化的成年小鼠睾丸的生精细胞中有DDX25蛋白的阳性表达。⑶siRNA有效转染浓度的筛选：倒置荧光显微镜观察，与12.5nM转染组相比，转染24h后25nM转染组与50nM转染组绿色荧光强度均显著增强（p<0.05），但二者强度差别不大（p>0.05），转染效率可达95%以上。⑷DDX25siRNA干扰的效能评价：①RT-qPCR检测结果显示，干扰24h、48h和72h的25nM siRNA实验组DDX25mRNA表达均明显低于各对照组，（p<0.05），其中干扰24h和48h的25nM siRNA实验组DDX25mRNA的表达较干扰72h组降低更明显（p<0.05）。②免疫组化、免疫荧光与免疫印迹结果显示，干扰72h的25nM siRNA实验组和50nM siRNA实验组生精细胞中DDX25蛋白的阳性表达均显著低于各对照组（p<0.05）。⑸干扰DDX25表达对生精细胞凋亡的影响：①台盼蓝染色法检测结果显示，与无关对照组相比，干扰72h的50nM siRNA实验组和25nM siRNA实验组生精细胞存活率明显降低（p<0.05），但二者之间无显著差异（p>0.05）。②流式检测结果显示，参照无关对照组，空白对照组、阴性对照组和阳性对照组的生精细胞凋亡率无明显变化；干扰72h的25nM siRNA实验组和50nM siRNA实验组生精细胞凋亡率率明显增高（p<0.05），但二者之间无显著差异（p>0.05）。⑹Western blot检测结果显示，25nM siRNA实验组干扰72h后生精细胞中Erk1/2和p-Erk1/2的表达较无关对照组显著降低（p<0.05），p38MAPK和p-p38MAPK的表达较无关对照组显著升高（p<0.05）。
结论：
1DDX25基因和蛋白在小鼠精子发生过程中呈阶段特异性表达；DDX25主要在成年小鼠的精子发生过程中发挥转录后调控作用，主要作用于精母细胞和圆形精子细胞。
2应用纳米材料通过睾丸局部直接注射方法将DDX25siRNA转染至小鼠体内，能有效抑制DDX25mRNA的表达，干扰效果良好且无毒性作用。可成为体内研究基因功能的有效方手段。
3活体体内DDX25基因沉默后，小鼠睾丸组织中生精细胞的凋亡增多，多种凋亡相关蛋白表达发生改变。
4将睾丸间质细胞与支持细胞-生精细胞进行双室培养，生精细胞可以存活达20天以上，可见部分生精细胞尾部出现鞭毛。
5应用DDX25siRNA体外转染生精细胞抑制生精细胞中DDX25的表达使生精细胞凋亡增加； DDX25可能分别通过ERK1/2与p38MAPK途径抑制生精细胞分化和诱导生精细胞凋亡。
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 testes
Male C57BL/6J mice were used from5to120postnatal days.⑴Paraffinsections 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.⑷The 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 testis
60days,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 cells
The 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，p38MAPK和p-p38MAPK were measured by Western blot.
Result:
1. Developmental expression of DDX25in mouse testes
⑴The 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 testis
⑴To 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.②Histological examination by H&E staining showed that the structure ofseminiferous tubules was normal without damage.③Immunocytochemistryshowed 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 cells
⑴In 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.②Western 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.⑷To evaluate efficiency of RNAi:①DDX25siRNA 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).②The 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).②The 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.
引文
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