Research Of The Interaction Between Spermatogenesis-related Protein Stra8 And Setd8

Stra8 (stimulated by retinoic acid gene8) was a RA-responsive gene.Stra8 expression was restrictetd to the male developing gonad during embryogeneis,and in adult,the Stra8 protein was expressed in testis premeiotic germ cell.Stra8-/- male mice were infertile,due to severe spermatogenesis impairment.In the previous studies,we screened interacting proteins of Stra8 from mouse spermatogonial stem cells cDNA library though the yeast two-hybrid technique,As a result,we found Setd8 could interact with Stra8.Setd8 was the sole enzyme known to specically catalyze monomethylation of histone H4 lysine 20,Setd8 played an important role in many physiological processes,But so far its function in spermatogenesis was still not reported. In this study,we mainly studied the interaction mechanism between spermatogenesis-related protein Stra8 and Setd8.Our results lay a foundation for further studies on the interactional mechanism for Stra8 and Setd8 during spermatogenesis.Firstly,Stra8 and Setd8 could transcriptional regulated each other.On one hand,we construct the luciferase reporter recombinant plasmid pGL3-Stra8Pro.Then,transfectied it into 293T cells with pRL-CMV and detected luciferase activity.The result showed that the Stra8 promoter possesses transcriptional activity.Then We co-transfected the pCMV-HA,pCMV-HA-Setd8 with pGL3-Stra8Pro plasminds into 293T cells respectively and analyzed luciferase activity.The results suggested that Setd8 protein can suppress the activity of Stra8 promoter.In addtion,We added 0,0.0625μg,0.125μg,0.25μg and 0.5μg Setd8 protein. The results showed that different doses of Setd8 protein have no affects on Stra8 promoter activity.On the other hand,we construct the Setd8 promoter report plasmids containing different fragments, co-transfected them with pRL-CMV into 293T cells and detected luciferase activity. The result showed that all of these Setd8 promoters possess activities,Further more, the promoter in upstream of Setd8 mRNA-1499～+1bp has the strongest transcriptional activity.So we use this promoter in the following experiments.We co--transfected pCMV-Myc, pCMV-Myc-Stra8 with pGL3-Setd8ProF2R into 293T cells respectively.The results suggested that Stra8 protein can up-regulate the activity of Setd8 promoter.In addtion,We added 0,0.0625μg,0.125μg,0.25μg and 0.5μg Stra8 protein.The results showed that when amount of protein reached to 0.25μg, activity of Setd8 promoter enhanced obviously.Secondly, we analyzed the interactional domains between Stra8 and Setd8.We constructed five recombinant plasmids pGADT7-Setd8 containing different fragments of Setd8.Then co-translated Setd8 full length and five recombinant plasmids and Setd8NO.50 (81-286aa) with pGBKT7 - Stra8 fragments respectively into yeast AH 109 and cultured in SD/auxotroph medium - LTAH/X-α-gal.We found that Setd8 full length and five recombinant plasmids could not interact with Stra8 fragments, while Setd8NO.50 (81-286aa) could interact with Stra8 fragments.Then, we constructed eukaryotic expression recombinant vectors containing different Setd8 fragments. Then these recombinants were transfected into 293T cells and expression of proteins was identified by Western blot. The results showed that all recombinants can express protein correctly. We co-transfected these five recombinant plasmids, pCMV-HA-Setd8NO.50 with pCMV-Myc-Stra8 into 293T cells respectively and co-immunoprecipitation experiments were performed. As a result, only Setd8NO.50 could interact with Stra8, while the rest of the other recombinants could not interact with Stra8.Thirdly,interactional domain between Setd8 and Stra8 was determined by co-immunoprecipitation.We constructed eukaryotic expression vectors containing different mouse Stra8 fragments.Then the recombinants were transfected into 293T cells by using liposome transfection method and expression of proteins was identified by Western blot.But the results showed that only one recombinant can express protein correctly, the other two recombinants can not express corresponding proteins. This expressed recombinant plasmid and pCMV-HA-Setd8 were co-transfected into 293T and co-immunoprecipitation experiment was performed. As a result, the recombinant can not interact with Setd8.Fourthly,the effect of Stra8 over-expression on the proliferation and apoptosis was observed in spermatogenic cell line.SRB experiment was used to study the effect of overexpression of Stra8 in spermatogenic cells. The results showed that we find that the number of cells have no statistical differences when compared with control cells. We deduced that overexpression of Stra8 has no obvious influence on the proliferation of cultured spermatogenic cells. We then used flow cytometry to detect the cell cycle, overexpression of Stra8 have no obvious impact on cell cycle. The results was in conformity with the SRB proliferation experiments.To sum up the Stra8 have no obvious influence on the proliferation of spermatogenic cells.PI/Annexin Ⅴ double-tagging methods was used to detect the rat of the apoptosis between the Stra8-GCl and Control-GC1,The results showed that the early apoptosis rate of Stra8-GC1 is obviously lower than Control-GC1.Finally,we synchronized Stra8-GC1 and detected the Stra8 and Setd8 mRNA level in different periods of cell cycle.After the synchronization, expression of Stra8 and Setd8 mRNA in different periods of cell cycle was detected by using RT-PCR.The result showed that Stra8 and Setd8 were the lowest in G0/G1 phase, and gradually rise to S phase. Setd8 mRNA maintained the same level, while Stra8 mRNA decreased in G2/M phase.In summary.we found that Stra8 and Setd8 could transcriptional regulated by each other. Stra8 and Setd8 can interact with each other. Overexpression of Stra8 had no obvious influence on the proliferation of spermatogenic cells, while it could decrease the early apoptosis rate. We detected the Stra8 and Setd8 mRNA level in different periods of cell cycle.These results lay a foundation for further studies on interaction mechanism of Stra8 and Setd8 during spermatogenesis.