Fancd2os Gene Augments Apoptosis In TM3 Cells Derived From Mice

Objective:On the basis of the previous study,mouse testicular stromal cells(TM3)models with stably over-expressed fancd2 os gene were constructed.And the effects of fancd2 os on the apoptosis of TM3 cells induced by ultraviolet(UV)were analyzed.On the other hand,fancd2 os gene was silenced with RNAi and the apoptosis of TM3 cells were observed under UV-exposure.In order to explore the possible mechanism of fancd2 os,apoptosis inhibitors were added and the apoptosis status of TM3 were analyzed again.Testosterone levels in culture medium were detected under fancd2 os over-expressed TM3 and fancd2 os silenced TM3 cells separately.The main objective of the study is to provide detaied clues for further research of the gene in spermatogenesis.Methods:1.Lentiviral vectors with fancd2 os was transiently infected into HEK293 T cells.Expression of fancd2 os in HEK293 T cells was detected by RT-PCR and Western blotting.2.To establish the stable expression TM3 cell model with fancd2 os over-expressed lentiviral vector.RT-PCR and Western blotting were used separately to detect the stable expression of fancd2 os mRNA and protein.3.The localization of FANCD2 OS protein in TM3 cells was demonstrated by indirect immunofluorescence staining.4.Apoptosis of TM3 cells was induced by UV irradiation.Apoptosis-relatedproteins including Cleaved-Caspase-3,BAX,BCL-2 and BCL-XL were analyzed by Western blotting.5.TM3 cells were infected with fancd2os-silenced vector and the expression levels of fancd2 os were detected by RT-PCR and Western blotting.6.The expression of apoptosis-related proteins BAX,BCL-2 and BCL-XL were detected by Western blotting.7.Apoptosis inhibitors against Caspase-8/9 were added,and the expression of caspase-3 was analyzed by Western blotting.8.Testosterone levels in culture medium of TM3 were detected by ELISA.9.Changes in testosterone levels after addition of apoptosis inhibitors after overexpression by ELISA.Results:1.The results of RT-PCR and Western blotting showed that fancd2 os were over-expressed in HEK293 T cells,which indicated the lentiviral vector was successfully constructed.2.RT-PCR and Western blotting results showed that fancd2 os were over-expressed in TM3 cells and fancd2 os stably over-expressed TM3 cell model was established successfully.3.Immunofluorescence staining demonstrated that FANCD2 OS protein was mainly distributed in the cytoplasm of TM3 cells;.4.The results of Western blotting showed that the expression levels of Cleaved-Caspase-3 and BAX in fancd2 os over-expressed group were significantly higher than those in control group(P <0.01),while the expression levels of BCL-2 and BCL-XL protein were significantly lower than those in control group(P<0.01),the difference was statistically significant.5.RT-PCR and Western blotting results showed that the expression of fancd2 os was decreased after TM3 cells have been transfected with fancd2 os silenced vector.6.Western blotting results showed that the expression level of BAX in fancd2 os silenced group was significantly lower than that in the control group,while the expression levels of BCL-2 and BCL-XL protein were significantly higher than those in the control group.The difference was statistically significant.7.Western blotting results showed that the expression of Caspase-3 was decreased after Caspase-8 inhibitor was added.8.The results of ELISA showed that testosterone content in fancd2 os stably over-expressed group was significantly lower than that in the naked cell group and empty vector infected group(P < 0.01),while the testosterone content in fancd2 os silenced group was significantly higher than that in the naked cell group and empty vector infected group(P < 0.01).9.The results of ELISA showed that the testosterone content of fancd2 os overexpression was significantly higher than that of the control group and the Caspase-9inhibitor group after the addition of Caspase-8 inhibitor.Conclusions:1.fancd2 os stably over-expressed TM3 cell model were successfully established.2.FANCD2 OS protein could augment UV-induced apoptosis in TM3 cells and this effect is mainly dependented on death receptor pathway.3.fancd2 os gene might inhibit the secretion of testosterone in TM3 cells,And this inhibition of testosterone secretion is due to the promotion of apoptosis of Leydig cells.