Mechanism Of DBP Disrupting Proliferation Of Rat Sertoli Cells During Prepuberty

Sertoli cells play a critical role in supporting normal spermatogenesis in male mammalian.Proliferation of Sertoli cell during prepuberty determines testicular spermatogenesis capability in adults.Peak of Sertoli cells do not proliferate any longer after puberty.So once proliferation of Sertoli cells before puberty is affcted by exogenous injury,spermatogenesis will be abnormal in adult.We found that Sertoli cells exposed to plasticizer dibutyl phthalate(DBP)in vitro could decrease cell proliferation,damaged membrane and increased apoptosis.DBP exposed to puberty male rats could decreased testis organ coefficient,reduced testosterone level,damaged tight junction,and lead to testicular digenesis.Previous study confirmed that low dose treatment of MBP in Sertoli cells promoted cell proliferation in vitro,but the mechanism is not clear.The current study was designed to explore molecular mechanism of Sertoli cells treated with low dose DBP.We detected the alteration miRNA and its target genes in DBP exposed Sertoli cells through microarray and analyzed genes of differential RNA expression by bioinformatics.Moreover,we observed the effects of intrauterine exposure to DBP on the developmental condition of F1 male rats and their reproductive system.Part 1 Comparison of the proliferative potential of 9-day-and 21-day-old Sertoli cells through mRNA profilingObjectiveTo compare the proliferative potential of 9-day-old and 21-day-old Sertoli cells and screening the mRNA associated with cell proliferation.Method1.Primary culture and identification of rat testicular Sertoli cell2.Place Sertoli cells on 96-well culture plate and incubate them for 1,2,3,4 and 5 days.Cell viability was evaluated by the CCK-8 test.3.We screened the cells from 9-day-old and 21-day-old rats for mRNA expression by microarray and analyzed genes ontology by bioinformatics.Results1.After 48 h primary culture,Sertoli cells demonstrated a spindle-shaped morphology and strong adhension.Immunofluorescent confirmed that all cultured cells were highly positive for FSHR and AR,suggesting a high purity of culture.2.The 9-day-old Sertoli cells showed a significantly stronger proliferative potential compared with 21-day-old Sertoli cells,especially in the 2.5×104 cells/ml group.3.In the microarray analysis,9-day-old Sertoli cells were viewed as the control and 21-day-old cells as the treatment group.mRNA profiling revealed that a total of 540 genes were significantly altered between 9-day-old and 21-day-old Sertoli cells.Of the 540 genes,256 were significantly up-regulated while 284 were evidently down-regulated in 21-day-old Sertoli cells.Transferrin(Tf)showed the largest up-regulation(12.05-fold)relative to 9-day-old Sertoli cells,whereas thrombospondin 2(Thbs2)showed the greatest down-regulation(-6.66-fold).Conclusion9-day-old Sertoli cells have a stronger proliferative activity compared with 21-day-old Sertoli cells,and 21-day-old cells have more perfect function.Part 2 Mechanism of DBP disrupting proliferation of rat Sertoli cells during prepuberty ObjectiveTo investigate the mechanism of low dose MBP promoting Sertoli cells proliferation in vitro and observe the effects of intrauterine exposure to DBP on F1 generation rats.Method1.In vitro(1)Cells were divided into 4 groups:Control,0.1 mM,1 mM,10 mM MBP exposure group and detected after 24 h,48 h culture.(2)Cell morphology were observed after exposed to various dose of MBP.(3)Use joint miRNA-mRNA analyze to screen the alteration miRNA and its target mRNA.(4)Real-time PCR was used to validate the array data.(5)We detected Rasdl and the phosphorylation of MEK in both mRNA and protein level.2.In vivo(1)16 pregnant rats were administered DBP in 4 doses(Control,50mg/(kg·day),250mg/(kg·day),500mg/(kg-day))by gavage during the critical period of Sertoli cell differentiation in rats(from GD12.5 to GD 21.5).AGD of F1 generation male rats were measured at postnatal days 9.At day 9,21 and 90,blood sample were collected by removing eyeball,respectively.(2)Radioimmunoassay was applied to detected the hormones level in serum,including FSH,LH,E2 and T.(3)H&E staining were used to observed testis tissue alteration under optical microscope.The expression of Rasd1 and MEK in testis were observed using immunohistochemistry.Results1.In vitro(1)Cell viability was significantly increased after 24 hours MBP exposure at a concentration of 0.1mM,and was decreased at 10mM.(2)There were no significant changes in Sertoli cells morphology after exposing up to 10mM.However,cells number were decreased significantly in 10mM group.(3)RNA expression profiling revealed 9 miRNAs and 304 genes were substantial changes in Sertoli cells.There were 47 genes were overlapped with miRNA predicted target genes.In the 9 miRNAs,7 miRNAs were evidently up-regulated while 2 miRNAs were significantly down-regulated.Among the altered miRNAs,miRNA-199a-3p,miRNA-301b-3p and miRNA-3584-5p were directly associated with cell proliferation according to miRNA target database.(4)Q-PCR suggest the reliability of array data.(5)With the increase dose of MBP,the expression of Rasd1 were decreased.And the expression of all selected miRNAs were consistant with the array data.2.In vivo(1)AGD of F1 generation male rats were significantly decreased in 500 mg/(kg·day).(2)The results show that experimental doses of DBP exerted significant effect on serum hormone:serum testosterone in groups treated with 500 mg/(kg·day)were significantly increased,and E2 level in 500 mg/(kg·day)were increased,ecept the day 35 group.(3)The testicular structure of F1 generation rats was damaged in 500 mg/(kg·day)group.Interstitial tissue were reduced at PND9 and PND21 in 500 mg/(kg·day)group,while there were no significant change at PND90.At PND21,Rasd1 were significantly decreased in 500 mg/(kg·day)group.ConclusionLow-dose MBP could promote Sertoli cells proliferation in vitro through up-regulating the expression of miRNA-199a-3p.miRNA-301b-3p,miRNA-3584-5p and down-regulating Rasd1.Intrauterine exposure to DBP reveal significant effect on the development of reproductive system in F1 male rats,and can lead to TDS.