RhoG-ELMO1-RAC1 Is Involved In Phagocytosis Of Sertoli Cells Suppresssed By Di-n-butyl Phthalate

Di-n-butyl phthalate(DBP)is one of the most dominant endocrine disrupting chemicals and is ubiquitous in the environment.Mono-butyl phthalate(MBP)is the active metabolite of DBP and exhibiting disruption effects.Previous studies reported that DBP could inhibit steroidogenesis and damage the male reproductive function.There has been a significant rise in the incidence of poor semen quality,cryptorchidism,hypospadias and so on,similar to human "symptom of testicular dysgenesis syndrome".Epidemiological studies also found that DBP/MBP is associated with a decrease in human sperm concentration and a reduction in the total number of sperm.Therefore,DBP maybe one of the important causes of developmental disorders in the male reproductive system.Apoptotic germ cells and multinucleated germ cells(MNGs)may reflect altered Sertoli cells function in fetal life,which predetermines sperm count and fertility in adulthood.We explored whether DBP-exposure in utero could induce apopotic germ cells and/or MNGs increasing in vivo firstly.Thereafter,mouse Sertoli cell line(TM4 cells)was selected to address whether RhoG-ELMO1-RAC1 pathway is involved in the decreased phagocytosis of Sertoli cells induced by mono-butyl phthalate(MBP)in vitro.Objective1.To evaluate the effects of DBP on apoptotic germ cells and MNGs;2.To explore the role of RhoG-ELMO1-RAC1 signaling pathway in the suppresssed phagocytosis of Sertoli cells induced by MBP.Methods1.Pregnant rats as models in vivo:Adult male and female Sprague-Dawley rats were paired(lvl).After plug detection,pregnant females were separated from the males,divided into three groups(n=20 per group)by the weight randomly and treated with DBP(300 mg/kg/day,500 mg/kg/day)or olive oil as vehicle(1 ml/kg/day)from GD12 to GD18 by oral gavage.After euthanized at GD19,fetal testes were collected and snap-frozen for protein extraction or fixed in Bouin's solution for histopathology analysis and TUNEL assay.2.The viability of Sertoli cells(TM4)was identified by CCK-8 assay.3.Phagocytosic capacity of TM4 cells was detected by flow cytometry and immunofluorescence.4.Lipid droplet formation of TM4 cells was detected by Oil Red O staining.5.Expression of mRNA(RhoG,ELMO1 and RAC1)were analysed by qRT-PCR.6.Expression of proteins(RhoG,ELMO1 and RAC1)were analysed by Western Blot.7.RAC1 activity was measured by RAC1 pull-down assay.8.Location of active RAC1 were examined by immunofluorecent staining.8.After RhoG and ELMO1 were overexpression,TM4 cells were treated by MBP(100?M)for 24 h.RAC 1 activity was measured by RAC 1 pull-down assay.Results1.DBP increased the apoptosis of germ cells in fetal rat testes.TUNEL assay of the fetal rat testes exhibited increased the number of apoptotic germ cells after 300 and 500 mg/kg/day DBP treaments in pregnant rats.2.DBP promoted the formation of MNGs in fetal rat testes.Histological examinations of the fetal rat testes exhibited the formation of MNGs and remarkable vacuolar spaces of Sertoli cells at dose of 300 and 500 mg/kg/day DBP.3.DBP decreased the number of spermatogenic cells in fetal rat testes.Histological examinations of the fetal rat testes exhibited gradual detraction of germ cells per seminiferous tubule under treatment with the 300 mg/kg/day and 500 mg/kg/day doses of DBP.4.DBP inhibited expression of ELMO1 in fetal rat testes.After DBP exposure,decreased expression levels of ELMO1 were observed in a dose-response manner.5.MBP inhibited phagocytosis of Sertoli cells in vitro.To identify the effects of MBP on cell viability,TM4 cells were exposed to various concentrations(0?1?10?50?100?300?500 ?M MBP)of MBP for 24 h or 48 h.The CCK-8 assay showed that no significant differences were identified among doses ranging from 0 ?M to 500 ?M,while 1000 ?M MBP resulted in a significant decrease of viable cells for 24 h and 48 h when compared with the control group.The 0,1,10 and 100 ?M MBP doses were used for the subsequent experiments for 24 h treatments.It could be observed that phagocytosis of beads by TM4 cells was significantly decreased determined by flow cytometry at 100 ?M MBP.And the lipid droplet formation decreased by ORO staining at the same dosage.6.MBP affected GTP-RAC1 expression and movemen to membrane.qRT-PCR and Western Blot results shown that levels of mRNA and total protein expression of RAC 1 remained comparable between selected concentrations of MBP and control group.Moreover,GST pull-down assay turned out that GTP-RAC1 levels were reduced obviously after 10 and 100 ?M MBP exposures(P<0.05).As indicated in confocal immunofluorescence,fewer RAC1 recruited to protruding edge in MBP-treated cells.This result suggested that RAC1 gathering in the cell membrane was suppressed by MBP.7.ELMO1 was involved in the reduction of RAC1 activity induced by MBP.qRT-PCR and Western Blot results suggested that expression of ELMO 1 mRNA and protein were down-regulated markedly after exposure to MBP.After transfected with ELMO1 plasmids,ELMO1 and RAC1 activity in TM4 cells were increased,while restored after MBP treatments together with ELMO1 overexpression(P<0.05).8.MBP suppressed phagocytosis of TM4 through the RhoG-ELMO1-RAC1 pathway.As expected,levels of RhoG mRNA and protein expression were down-regulated markedly after exposure to MBP.In addition,protein levels of ELMO1 and GTP-RAC1 increased remarkably when RhoG was over expressed.On the contrary,these expressions were recovered with overexpression of RhoG and MBP treatments simultaneously(P<0.05).ConclusionsThis study suggested that increased apoptotic germ cells and MNGs induced by DBP might due to the disrupted phagocytosis of Sertoli cells through RhoG-ELMO1-RAC1 pathway.