The Study For The Function Of Attractin In Sertoli Cells Of Male Mouse Testis

PartⅠReproductive phenotype analysis in Attractin genedeficiency male mouseObjective: The purpose of this study was to primarily explore the effect of loss-of-function of Attractin (Atrn) in the male mouse reproduction system.Methods: Firstly, the morphological changes of testis and epdidymis between Atrnmutant (Atrn^mg-3J) and wild-type mice at 3months and 5months were studied. Also, theweight difference in testis and epdidymis, as well as the sperm dencity and motilitybetween mutants and controls were investigated. Further, the testicular lactatedehydrogenase (LDH), succinate dehydrogenase (SDH) among these animals weremeasured, and the fertility of their sperm in vivo and vitro were observed.Results: In comparison with the control group, no obviously pathological modify wasdetected in 3-month-old Atrn^mg-3J mouse testis except reduced SDH activity. In 5-month-oldmutants, the morphology of seminiferous tubule was disorder, the density and motility ofsperm were decreased in epididymis, the sperm fertility was impaired and testicular enzymeactivity were also descent.Conclusion: The data showed the age-related Atrn gene progressively loss-of-functionmight result in the degeneration in male Atrn^mg-3Jmouse, representated as the disordermorphology of seminiferous tubule and impaired sperm function, which would be one ofmain reasons to damage male reproductive ability (P＜0.05). PartⅡThe establishing of mouse sertoli cells targeting gene Atrnby RNA interferenceObjective: To stable suppress the Atrn gene expression in mouse Sertoli cells usingsiRNA eukaryotic expression vector and explore the function of Atrn gene in these cells.Methods: The expression of Atrn in TM4 was examined by Q-PCR, Western blot andimmunocytochemistry.The siRNAs were designed according to the coding the cDNAsequence of Atrn gene, and cloned into the downstream of H1 promoter of psiRNA-hH1neo.Then, the vector was cloned by transforming into DH5αstrain and identified byrestriction endonuclease digestion and DNA sequencing. Three recombinant vectors and thepsiRNA-hH1 were transfected into TM4 cells by Lipofectamine^TM2000. The mRNA andprotein expression of Atrn in TM4 cells were investigated by Q-PCR and Western blot afer72h. Then, the TM4 cells were transfected with the most effective Atrn-specific siRNAexpression vectors and psiRNA-hH1 respectively, selection media with G418 was addedon the third day after transfection. Cultures were maintained in selective media for two tothree weeks until the mostly cells of the blank control group were dead. The G418-selectedtransformed cells were grown and expanded further. At last, the analysis of inhibin alphamRNA and inbibin B expression in G418-selected transformed cells was carried out byRT-PCR and ELISA.Results: The Atrn mRNA and protein were both expressed in TM4 cells. Theconstructed psiAtrn-770, psiAtrn-2273, psiAtrn-2908 digested with restrictionendonuclease Ase I was linearized. The sequencing results confirmed that the sequence ofinserted fragment was correct. After transient transfection, the effect of suppression of theexpression of Atrn in the cells with the psiAtrn-2273 and psiAtrn-2908 were obviously (P＜0.05). Especially, the expression of ATRN protein of psiAtrn-2273 was more lower thanthe psiAtrn-2908 (P＜0.05). Then the psiAtrn-TM4 and psiRNA-TM4 cells wereestablished by stable transformed by psiAtrn-2273 and psiRNA-hH1. Compared with the psiRNA-TM4 cells, the expression of Atrn was suppressed obviously and the level of InhamRNA and Inh B was significantly higher in the psiAtrn-TM4 cells (P＜0.05)Conclusion: The eukaryotic expression vector of siRNA targeting Atrn gene wassuccessfully constructed, and the Atrn knock-down in mouse sertoli cells TM4(psiAtrn-TM4) was successfully established by stable RNA interference. When theendogenous expression of Atrn was suppressed, the level of inhibin secreted by Sertoli cellswas increased.PartⅢIdentification of differential proteins in TM4 mouse Sertoli cellswith Atrn silence by proteome analysisObjective: To separate and identify the differential proteins in TM4 mouse Sertoli cellswith Atrn partly or completely loss-of-function by proteome analysis, and provide ascientific basis to clarify the effort of Atrn in mouse Sertoli cells.Methods: The Atrn^mg-3J mouse primary sertoli cells was isolated and cultured byenzymatic treatments and identified through several methods. Atrn knock-down in mousesertoli cells TM4 (psiAtrn-TM4) was established by stable RNA interference. Comparedproteomic changes among the psiAtrn-TM4, primary cultures of testis Sertoli cells ofAtrn^mg-3J (mu-Sc) and control gourp (psiRNA-TM4) using two-dimensional gelelectrophoresis (2-DE) and Matrix-assisted laser desorption/ionization time of flight massspectrometry (MALDI-TOF-MS). At last, the expression levels of some of these differentialproteins were further measured by Q-PCR and Western blot. Results: Atrn^mg3-J mouse primary sertoli cells, with 95% purity,was established. Fifteendifferentially expression protein spots between the three cells were identified byMALDI-TOF-MS and the NCBI proteins database. Except the decreased expression ofSOD, others may be novel proteins associated with ATRN function such as down regulatedof ATP SYNTHASE, PEROXIREDOXINⅡand upregulated CASPASE6,KETOHEXOKINASE in psiAtrn-TM4 and mu-Sc. Futher, the mRNA and proteinexpression levels of SOD and CASPASE6 in the these cells were confirmed.Conclusion: These dates suggested that these differential protein may be associated withthe function of Atrn in Sertoli cells involving multi-pathways,including antioxidative stressand antiapoptosis pathway, which provided new clue to interpret the the function of Atrngene in mouse Sertoli cells and the mechanism of degeneration of testis in Atrn mutants.