Expression Of Rap1during Germ Cell Development Of Rat And Its Roles In Apoptotic Spermatogenic Cells

ObjectiveRap1is one of Ras superfamily members, which is widely expressed ineucaryon organs, and regulates multiple biological activities. The most specialcharacter of this GTP binding protein is nuclear transfer activation in somecancer or human embryo kidney cell lines. We have previously found that Rap1expressed higher in infertile men’s germcells than that of fertile men by in situhybridization. So we supposed that it may regulate proliferation anddifferentiation of germ cells. In order to testify our assumption, we studied theexpression of Rap1in testicular development as well as in apoptotic germ cellsinduced by MAA, and explored its interaction with NF-κB.Methods 1. Immunohistochemistry and western blot were used to observe theexpression and location of Rap1in the rat testis at different developmentalstages of (1,7,15,24,37,50and90days). We used different fixation solutionsto make the comprehensive study of Rap1expression profile.2. Adult SD rats were treated with MAA (650mg/kg) intraperitoneally toinduce spermatocyte apoptosis. To further explore the relationship betweenRap1and the apoptosis, the expression of Rap1in apoptotic spermatocyte wasobserved by immunohistochemistry, western blotting and TUNEL. Two-labeling indirect immunofluorescence and co-immunoprecipitation wereapplied to study the co-expressions and interections between Rap1and NF-κB.Results1. In immunohistochemistry study using slides of Bouin’s solution fixation,Rap1expressions were found in nuclear area of germ cells of P1and P7. AfterP15, Rap1expressed in the paranuclear golgi apparatus of spermatocyte, andthis expression was strengthened with testis development. In the adult testis, theexpression can also be found in round spermatids, and its expressionstrengthened accompanied with Golgi growth in spermatocyte.2. In immunohistochemistry using slides of Paraformaldehyde solutionfixation, highly Rap1nuclear expressions were found in germ cells of P1and P7,and the positive cells in P1was significantly more than that of P7. By P15, Rap1had transferred out of nuclear to cytoplasm in spermatogonia, but it appearedagain in late-pachytene spermatocyte in P24-50. In testis of P90, no specificstaining of Rap1was found. Other cells in testis had not showed Rap1expressions.3. In western blot analysis, Rap1expression in P1testis was significantlyhigher than that of P7. 4. In spermatocyte apoptosis model driven by MAA (650mg/kg), Rap1,NF-κB and TUNEL positive spermatocytes were found in stage (VIII-XIV) after6h of injection. By12and24h, the positive cells increased. Western blotshowed no significant differences of Rap1and NF-κB expressions betweenexperimental groups and control groups.5. Rap1indirect immunofluorescence co-localized with TUNEL positivecells. And the number of Rap1+TUNEL positive cells occupied19.1%、57.7%、67.4%of the total Rap1nuclear positive cells. Rap1+TUNEL double stainingcells of MAA treated group were significantly higher than that of control group(P <0.01).6. Rap1also co-localized with NF-κB in apoptotic cells in stage VIII-XIVby indirect immunofluorescence analysis. Co-immunoprecipitation showedhigher co-expression rate than Rap1+TUNEL:75.6%、91.8%、94.6%. Thelysate from MAA treated group pulled down by NF-κB monoclonal antibodyhad significant higher expression of Rap1than that of control group.Conclusions1. Rap1expressed in part of germ cell nuclear and golgi apparatus, It maybe involved in the testicular development as well as gene regulations. Fixationsolutions had significant impact on Rap1staining patterns.2. Rap1and NF-κB was highly expressed in the nuclear of apoptosisspermatogenic cells induced by MAA, and their co-immunoprecipitationindicated their interactions. Rap1and NF-κB may be involved in the regulationof spermatogenic cell apoptosis.