Preliminary Study Of Stra8 Inhibits Apoptosis In Spermatogenesis And Its Interaction With Setd8

Stra8,one of genes induced by retinoic acid(RA),is required for the meiotic initiation of male reproduction.Stra8 is a vertebrate-specific gene without significant homologous proteins expressed in the cytoplasm and nuclei of germ cells from mitosis to meiosis and presents exclusively in the embryonic and postnatal gonads of mammals.Stra8 shuttles between nucleus and cytoplasm and displays transcriptional activity.Stra8 knockout mice are sterile.But the mechanism of Stra8 during spermatogenesis is still unclear.The thesis divided into two parts:(1)Anti-apoptotic effect of Stra8 in spermatogenesis.(2)The analysis of interaction of Stra8 and Setd8 in vitro.Part 1.Stra8 inhibits apoptosis during spermatogenesis1.Stra8 inhibits apoptosis in mouse testis both in vivo and in vitro.It was first found that Stra8 might inhibit apoptosis in the Stra8.KO,vitamin A deficiency and recovery(VAD and VAR)mice.By using TUNEL assay,we found the number of apoptotic cells of 11-days-old Stra8KO mice testes was increased when compared with WT mice.The phenotype was also verified in vitamin A deficient and vitamin A recovery mice.We found that in the 45-day and 61-day VAD mouse models,apoptotic cells increased significantly.To further test the anti-apoptosis effect of Stra8 in virto,flow cytometric analysis and TUNEL were performed in Stra8-overexpressed GC1spg.The results of flow cytometric analysis demonstrate that the proportion of early apoptosis cells in overexpressed Stra8-GC1 spg decreased compared with the control-GC1 spg.In additionally,the results of the TUNEL assay confirmed the anti-apoptosis effect of Stra8 in germ cells.The number of apoptotic germ cells was significantly decreased when stra8 was overexpressed.2.The anti-apoptosis effect of Stra8 was further explored with microarray analysis.We found 5 up-regulated genes and 4 down-regulated differentially expressed genes(DEGs)related with apoptosis.The DEGs were as follows:PDK1(a key gene upstream of AKT);ANG2(a BCL2-inhibited gene);TCF4,GSTP1,and USP33(MAPK-related genes);OSGIN1(related to the P53 pathway);BCL2,P53,and ERK(MAPK1/3);JNK(MAPK8/9);and P38(MAPK14),all of which are key genes involved in the AKT signaling pathway.3.Stra8 may inhibit apoptosis through AKT pathway.We further analyzed the expression of these genes and their corresponding proteins.The results showed that both the mRNA and protein expression of PDK1,AKT,BCL2,and ERK was increased.Although the level of P53 mRNA expression was decreased,there was no significant difference in the level of protein.Moreover,Caspase 3,one of the executioner caspases,exhibited decreased in Stra8 overexpressed GC1 spg cells compared with the Control groups.Therefore,we assumed that Stra8 may directly or indirectly inhibit caspases through the AKT signaling pathway and ultimately exert an anti-apoptotic effect in testes.Part 2.The analysis of interaction of Stra8 and Setd8 in vitro1.Expression of Stra8,Setd8 and H4K20me1 in Stra8-overexpressed GC1spg and P19 cells.Compared with the control group,the expression of Set8 was decreased,but the expression of H4K20 me1 was increased,suggesting that three genes might interact with each other.We then detected the expression of Set8,H4K20me1 in P19 cells after inducing for 36-48 hr by 4μM RA.The results suggested the expression of Set8 was decreased,while H4K20me1 was increased when compared with uninduced P19 cells.These results were consistent with the phenotype of Stra8-overexpressed GC1 spg.2.Establishment of synchronization in Stra8 overexpressed GC1 spg.According the synchronization method in Hela cells,we developed the methods of synchronization in Stra8 overexpressed GC1 spg.Firstly,we found that the percentage of G1 phase cells of stra8-GC1 spg was 60-70%after serum deprivation for 72h,which was significantly higher than that of the control group of 31.73%(P<0.05).Secondly,the "double thymidine(2.5 mM)block method"was used to synchronize Stra8-GC1 spg to S phase.It was found that double thymidine blocking and second releasing for 3h,the percentage of S phase cells of Stra8-GC1 spg was 60-70%,which was significantly higher than that of the control group of 38.50%(P<0.05).Finally,The percentage of M phase cells of Stra8-GC1 was more than 90%when treated with nocodazole for 8h,which was significantly higher than that of the control group of 13.85%(P<0.05).3.Expression of Stra8,Setd8 and H4K20me1 in different stages of Stra8 overexpressed GC1 spg.By using immunofluorescence and Western Bloting methods,we detected the expression of Stra8,Setd8 and H4K20mel in different stage of Stra8 overexpressed GC1 spg.It was found that in Stra8 overexpressed GC1 spg,the expression of Stra8 was low in G1 phase,peaked in S phase,and Stra8 positive signal was found both in nuclear and cytoplasmic.The expression decreased in G2/M phase.The expression of Setd8 was not detected in G1 phase and began to express inlate S phase,and peaked in G2/M phase.The Setd8 positive signal was mainly in the nucleus in S phase.The expression pattern of H4K20mel was similar to that of Setd8.After phenotypic analysis and literature review,we hypothesized that Stra8 may inhibit the expression of E3 ubiquitin ligase-related genes,for example,CRL4Cdt2,SCFSkp2,etc,weakening its degradation function to Set8 in the S phase of the cell cycle,leading to a decrease of Set8 expression in Stra8 overexpressed GC1 spg.Instead,the overall expression level of H4K20me1 increased,due to the early expression of H4K20me1 in the S phase of Stra8 overexpressed GC1 spg.