Mechanism Of DEHP-Induced Developmental Retardation Of Quail Ovary And Oviduct

Di-2-ethylhexyl phthalate（DEHP）is one of the most widely used plasticizers all over the world that has been found in the atmosphere,water,and soil and has been classified as a potential environmental endocrine-disrupting chemical（EDC）by World Health Organization.DEHP can cause reproductive dysfunction,result in abnormal development of ovary and oviduct,interfere with follicular development.However,the exact molecular mechanism of DEHP-induced ovarian and oviductal toxicity remains to be elucidated.In this study,the functionary mechanisms and the effective action target of DEHP-induced ovarian and oviductal toxicity were explored in female quail during reproductive development.150 female Japanese quail were randomly divided into 5 groups（30quail/group）and treated as follows:Con group（control group）,Vcon group（vehicle control group）,low-dose poisoning group（250 mg/kg DEHP group）,the middle-dose poisoning group（500 mg/kg DEHP group）and the high-dose poisoning group（750 mg/kg DEHP group）by oral gavage administration for 45 days.The alterations of ovarian and oviductal morphology and ultrastructure of ovarian granulosa cells and theca cells were observed,residue detection of DEHP and its metabolites in the ovary and oviduct,serum sex hormone levels,sex hormone secretion related regulatory factors,antioxidant function,PPAR signaling pathway-related regulatory factors,Wnt/Ctnnb1 signaling pathway,and Hoxa family-related regulatory factors were detected.Furthermore,to verify the molecular mechanism of the toxicity of DEHP and its major metabolite MEHP（mono-（2-ethylhexy）phthalate）,primary quail ovarian granulosa cells as models were used in vitro experiment.After screening the concentration and reaction time of MEHP,PPAR activator rosiglitazone,and PPAR inhibitor GW9662,ovarian granulosa cells were divided into 8 groups:Con group（control group）,GW9662 group（10μM GW9662）,Rosi group（50μM rosiglitazone）,MEHP group（50,100,200μM MEHP）,MEHP+GW9662 group（200μM MEHP+10μM GW9662）,MEHP+Rosi group（200μM MEHP+50μM rosiglitazone）.After 24 hours of treatment,cell activity,cell morphology,ultrastructure,sex hormone secretion related regulatory factors,and PPAR signaling pathway-related regulatory factors were examined.The results show that:（1）At high doses of DEHP,the residual value of MEHP in the ovary and oviduct are 1.8 times and2.3 times greater than that of DEHP,indicating that MEHP is the major metabolite which induced ovarian and oviductal toxicity;DEHP exposure induces decreased T-AOC capacity,GST,and T-SOD activity and increased MDA content in the ovary and oviduct,which induces oxidative stress in the ovary and oviduct.（2）Exposure to DEHP results in a significant reduction in the number and volume of mature follicles in the ovary,the unclear border between theca cells and granulosa cells,lightly stained of the nuclear,the disordered arrangement of theca cells,decreased thickness of ovarian granulosa cell layer.DEHP induces significantly increased degeneration of the epithelial cell layer in the oviduct,the unclear border of interstitial nuclei,increased vacuolation,decreased percentage of ciliary cells in the epithelial cell layer.DEHP induced mitochondrial damage（decreased mitochondrial volume density,decreased mitochondrial cristae,increased mitochondrial vacuolation,increased percentage of damaged mitochondria）in the ovary and oviduct.It indicates that DEHP exposure results in the pathological changes of the ovary and oviduct and abnormal development of follicles.（3）Exposure to DEHP inhibits the serum levels of FSH,PRL,and E₂,increases the serum levels of T,LH,and P,decreases the expression of sex hormone secretion related factors（Gn RH,17β-HSD,P450arom,ERβ,and FSH）,and increases the expression of sex hormone secretion related factors（PRLH,LH,PRL,PGR,St AR,3β-HSD,P450scc,and ERα）,indicating that DEHP exposure results in sex hormone secretion-related regulatory factors expression dysregulation and sexual hormone synthesis disorders.（4）DEHP exposure enhances the immunofluorescence intensity of PPAR signaling pathway-related regulatory factors（PPARαand PPARγ）,weakens the immunofluorescence intensity of P450arom,activates PPAR signaling pathway,inhibits the expression of P450arom and the production of E₂,results in the damage to ovarian granulosa cells,and affects follicular development.（5）MEHP results in significant cytotoxicity of ovarian granulosa cells,including decreased cell activity,changes of cell morphology and ultrastructure,and disordered expression of sex hormone secretion-related regulatory factors;PPARγagonist rosiglitazone exacerbates MEHP-induced ovarian granulosa cells proliferation inhibition and mitochondrial damage,inhibits the expression of P450arom;PPARγantagonist GW9662 antagonizes MEHP-induced ovarian granulosa cell proliferation inhibition and mitochondrial damage,increases the expression of P450arom,and then antagonizes MEHP-induced expression dysregulation of sex hormone secretion-related regulatory factors by activating the production of E₂,indicating that MEHP exposure leads to dysfunction of quail ovarian granulosa cells by activating PPAR-mediated sex hormone regulation.（6）Exposure to DEHP increases the expression of Wnt/Ctnnb1 signaling pathway-related regulatory factors（Wnt5a,Wnt7b,Fzd6,Dvl1,Axin1,Ctnnb1,Lef1,Tcf7l2,Hoxa9,and Hoxa10）,decreases the expression of Wnt/Ctnnb1 signaling pathway-related regulatory factors（Gsk3β）,indicating that DEHP exposure causes disordered expression of Wnt/Ctnnb1 signaling pathway-related regulatory factors in the oviduct,which may regulate the transcription of the target gene by activating the Wnt/Ctnnb1 signaling pathway,leading to changes in the morphology and function of the oviduct,and eventually causing developmental retardation of quail oviduct.In summary,DEHP exposure causes developmental retardation of quail ovary and oviduct,decreased egg production and eggshell weight,oxidative stress in ovary and oviduct,ovarian granulosa cell proliferation inhibition and mitochondrial damage,decreased activity of P450arom and E₂ synthesis,and abnormal follicle development through activating the PPAR signaling pathway and Wnt/Ctnnb1signaling pathway,and eventually causing developmental retardation of quail ovary and oviduct.This study provides new evidence for the molecular mechanism of DEHP-induced ovarian and oviductal developmental toxicity.