Regulation Of SET Protein On Proliferation And Apoptosis Of Spermatogonias And Spermatocytes

Background and Ojectives:Spermatogenesis is a unique and sophisticated division and differentiation process that mainly consists of three phases:self-renewal and differentiation of spermatogonia, meiotic division of spermatocytes and spermiogenesis. Spermatogenesis has to be strictly regulated to enable correct transmission of genetic and epigenetic information to subsequent generations, including hormones,proteins and miRNAs. Germ cell apoptosis is conspicuous during normal spermatogenesis and germ cell apoptosis during testicular development in the mouse has two peaks corresponding to the time of migration of primordial germ cells into the gonads and the beginning of the first round of spermatogenesis.More than half of the male germ cells undergo apoptosis during normal spermatogenesis. In addition to its importance as a common phenomenon to remove abnormal germ cells and control germ cell numbers, an increase or decrease germ cells apoptosis may result in defects in normal spermatogenesis and lead to reproductive damage.SET, an oncogene,can produce two kinds of proteins,including TAF-la and SET/TAF-1p. TAF-la and SET/TAF-1p have different N-terminal amino acids and the latter plays a major biological function.SET protein has many functions including cell cycle, cell proliferation and apoptosis, DNA repair, gene transcription and epigenetic. Several studies discovered that SET protein was expressed in oocytes andtheca cells suggesting its regulation of oogenesis and androgen production in the ovary.We recently discovered that SET protein was mainly expressed in spermatogonias and spermatocytes of the seminiferous epithelium, Leydig cells, as well as low expression in Sertoli cells,suggesting that it was involved in spermatogenesis and androgen production.In conclusion, we assume that SET protein, as a testicular local factor, participate in spermatogenesis.To test our hypothesis, we use spermatogonias and spermatocytes as the models and knockout the expression of SET protein to investigate the effects of SET protein on the proliferation and apoptosis of spermatogonias and spermatocytes and its mechanisms.Methods:(1)GC-1 spg and GC-2spd cells were cultured in DMEM/HIGH GLUCOSE with 10% fetal bovine serum in 5%CO2 at 37℃. The control group was transfected with AdHl-siRNA/NS and the experimental group was transfected with AdH1-siRNA/SET.(2)The expression and cellular location of SET protein was assessed by Immunofluorescence and Confocal laser scanning microscopy.(3) GC-1 spg and GC-2spd cells were transfected with AdH1-siRNA/NS and AdHl-siRNA/SET for 48h.Western Blot was used to detect the expression level of SET protein before and after transfection.(4) GC-1 spg and GC-2spd cells were transfected with AdH1-siRNA/NS and AdH1-siRNA/SET for 48h. CCK8 assay was used to test the cell viability.(5) GC-1 spg and GC-2spd cells were transfected with AdH1-siRNA/NS and AdHl-siRNA/SET for 48h. BrdU incorporation assay was used to detect the cell proliferation.(6) GC-1 spg and GC-2spd cells were transfected with AdH1-siRNA/NS and AdHl-siRNA/SET for 48h. Flow cytometry was used to measure cell cycle.(7) GC-1 spg and GC-2spd cells were transfected with AdH1-siRNA/NS and AdH1-siRNA/SET for 72h. Flow cytometry was used to measure cell apoptosis.(8) GC-1 spg and GC-2spd cells were transfected with AdH1-siRNA/NS and AdHl-siRNA/SET for 48h or 72h. Western Blot was used to test the expressions of caspase3、cleaved-caspase3、bax、bcl2、caspase9、cleaved-caspase9、caspase8、 cleaved-caspase8、AKT、p-AKT(T308)、 p-AKT(S473).(9)Statistical methods:Data of the two groups were statistically analyzed by independent samples t-test using SPSS version 20.0. The values were presented as mean ± SD. Differences were considered statistically significant at P< 0.05.Results:(1)In GC-lspg and GC-2spd cells, SET protein was found in both cytoplasm and nucleus, but its location was mainly in the cytoplasm.(2)GC-1 spg and GC-2spd cells were transfected with AdH1-siRNA/NS and AdHl-siRNA/SET for 48h.Expression of SET Protein in the GC-lspg and GC-2spd cells transfected with AdH1-siRNA/SET was lowered 55%(**P<0.01) and 50%(*P< 0.05) respectively.(3)Knockdown of SET protein inhibited the cell viability by 80%(**P<0.01)%and 50%(**P<0.01) respectively. (4)Knockdown of SET protein inhibited the cell proliferation by 11%(*P<0.05) and 15%(**P<0.01) respectively.(5)After knockdown of SET protein, the percentage of S phase was decreased in both GC-1 spg and GC-2 spd cells by 7%(**P<0.01) and 18%(*P<0.05) respectively and an increase of percentage of G2/M phase in GC-1 spg by 40%(**P<0.01) and G0/G1 and G2/M phase in GC-2 spd cells by 11%(*><0.01) and 65%(*P<0.05) respectively was observed.(6)Knockdown of SET protein remarkably increased GC-1 spg and GC-2 spd cells apoptosis index by 1.5 times (**P<0.01) and 5 times (**P<0.01) respectively.(7)In GC-1 spg and GC-2 spd cells,after knockdown of SET protein, the expression of cleaved-caspase3 increased2.3times (*P<0.05) and1.6times (*P<0.05) respectively, Bcl2/Bax decreased 60%(*P<0.05) and 50%(**P<0.01) respectively,cleaved-caspase9 increased 40%(*P<0.05) and 33%(**P<0.01) respectively, cleaved-caspase8 increased 2.2 times (*P<0.05) and1.8 times (*P< 0.05) respectively and the expression of p-AKT (S473) decreased 25%(*P<0.05) and 40%(**P<0.01) respectively but the expression of p-AKT (T308) was not changed.Conclusion:(1)Knockdown of SET protein inhibites proliferation and promoted apoptosis of spermatogonia. Its mechanism may be through selectively inhibiting AKT phosphorylation at Ser-473 and AKT signaling pathway and activating intrinsic pathway and extrinsic pathway.(2)Knockdown of SET protein inhibites proliferation and promoted apoptosis of spermatocyte. Its mechanism may be through selectively inhibiting AKT phosphorylation at Ser-473 and AKT signaling pathway and activating intrinsic pathway and extrinsic pathway.(3)SET protein plays an important role in the proliferation and apoptosis of spermatogonias and spermatocytes and is an important local regulation factor of spermatogenesis(4) This research proves that SET protein is an important local regulation factor of the spermatogonias and spermatocytes proliferation and apoptosis. Intensive study of the function of SET protein in spermatogenesis is benefit for us to know the mechanisms of oligospermia, asthenospermia and teratospermia,which is beneficial to explore male infertility in clinical practice.