TNF? Stimulates The Proliferation Of Immature Sertoli Cells By Attenuating UPS-degration Of Cyclin D1 And Leads To The Delay Of BTB Maturation

Objective: Sertoli cells(SCs)play a key role in the development of germ cells in the mammals' testis.The ability of sperm production is determined by the final number of SCs.SCs proliferation is mainly regulated by follicle stimulating hormone and paracrine factors,which only occurs in fetal and prepubertal periods.During these periods,immature SCs may become an important target for pathogenic factors affecting testicular development.TNF?(tumor necrosis factor ?)is involved in many biological activities and multiple processes of spermatogenic cell development in the testis.SCs are a major target for TNF? to play a pathological or physiological role.TNF? participates in BTB(blood-testis barrier)remodeling,apoptosis,and inflammatory responses by recognizing receptors on the SCs.Studies have found that TNF? can induce the proliferation of immature SCs in vitro,but lacking of its regulatory pathways,mechanism of action,and validation experiments in vivo.This study attempts to reveal the effect of TNF? on the mechanism of immature SCs proliferation and the influence on the maturation process of BTB.Methods: 1.The Effect of TNF? on the proliferation of immature SCs and the differential genes screened by the ubiquitination PCR array.SCs isolated from testis of 10 dpp SD rats were cultured for 1-7 d in vitro and treated with different concentrations of TNF? on the second day.Following experiments were carried out at different time.(1)Cell proliferation rate was measured by CCK-8 and EdU.(2)The differential genes were screened by the ubiquitination PCR array.Then,qPCR was used to verify the expression of Fbxo4 after SCs were dealed by TNF?(10 ng/ml).(3)Western blot was used to detect the expression of PCNA,Fbxo4 and cyclin D1.(4)Immunoprecipitation was used to detect the ubiquitination of cyclin D1 and the interaction between Fbxo4 and cyclin D1.2.The effect of Fbxo4 overexpression on TNF? promoting SCs proliferation.The adenoviral recombinant plasmid containing rat Fbxo4 gene was constructed.The virus particles were packaged and the suitable MOI value was selected.The primary immature SCs were infected 24 h on the second day and TNF? treatment was performed on the fourth day to detect the cell proliferation rate and relative protein expression level.3.The effect of inhibiting NF?B activity on TNF? promoting SCs proliferation.Immunoblotting detected relevant signaling pathway protein activity.SCs isolated from 10 dpp SD rats were cultured in vitro and the optimal concentration of PDTC was determined.The PDTC(10 ?M)was pretreated for 2 h.After that,PDTC and TNF? were treated for 48 h.Subsequently,the proliferation rate and related protein expression were detected.4.The effect of TNF? on the establishment of BTB function in immature rats in vivo.12 dpp immature male SD rats were selected as vivo experimental subjects.Intraperitoneal injection of TNF?(10 ?g/kg b w)was given every day for 3 consecutive days.The control group was injected with equal dose of dd H2 O at different time(16 dpp,20 dpp,24 dpp,26 dpp,30 dpp,32 dpp,and 35 dpp).Following experiments were performed.(1)Biotinlabeled method was used to detect the permeability of BTB in rats,and comparing the time of the maturity of BTB in each group.(2)The distribution of BTB connexin was detected by immunofluorescence.(3)The levels of PCNA,Fbxo4,cyclin D1 and connexin were detected by western blot.Results: 1.TNF? inhibits ubiquitination of cyclin D1 and promotes proliferation of immature SCs.CCK-8,EdU,and PCNA expression level show that TNF? significantly promotes proliferation of immature SCs.Five related differential genes(>3 fold)are screened by ubiquitination PCR Array.Western blot shows that TNF? significantly downregulates the E3 ubiquitin ligase Fbxo4,while cyclin D1(as substrate of Fbxo4)level increases.Co-immunoprecipitation shows that TNF? inhibites the interaction of cyclin D1 with Fbxo4 and the level of cyclin D1' ubiquitination.2.Overexpression of Fbxo4 inhibits the proliferation of immature SCs by TNF?.Adenovirus Ade-Fbxo4 infects primary immature SCs in vitro.Fbxo4 was successfully overexpressed and detected by western blot.CCK-8 and EdU tests show that Adv-Fbxo4+TNF? group has no significant change compared with Adv-Fbxo4 group in cell proliferation.Immunoblotting and immunoprecipitation results show that the expression level of PCNA and cyclin D1,and the ubiquitination level of cyclin D1 in Adv-Fbxo4+TNF? group have no significant change compared with Adv-Fbxo4 group.3.TNF? promotes the proliferation of immature SCs by activating NF?B.Western blot shows that the ratios of p-p38/p38,pI?B ?/I?B ? and p-NF?B p65/NF?B p65 increase after TNF? treatment,indicating that TNF? can activate the p38 MAPK and NF?B pathways in immature SCs.CCK-8 and EdU tests show that TNF? could not promote immature SCs proliferation after PDTC treatment(p38 MAPK inhibitors do not have this effect).Western blot and immunoprecipitation also show that after PDTC treatment TNF? no longer causes significantc hanges in the expression levels of PCNA,Fbxo4,and cyclin D1,and the ubiquitination level of cyclin D1.Besides,TNF? can still activate NF?B after overexpression of Fbxo4.4.TNF? induces the delays of BTB maturation in rats.BTB function test reveals in vivo that BTB treated with TNF? matures on the 35 th day later than the control group(20 dpp)obviously.Immunofluorescence shows expression levels of occludin and JAM-A decease after TNF? treatment.The distribution is abnormal.However,with the increase of the age of rats,the changes of TNF? induced occludin and JAM-A diminish,and they get right after 26 dpp(JAM-A)and 32 dpp(occludin).Conclusion: TNF? inhibits the ubiquitinated degradation of cyclin D1 through the NF?B pathway.Furthermore,the proliferation of immature SCs is promoted and the rat BTB maturation delays.