Foetal-neonatal Exposure Of Di-(2-ethylhexyl) Phthalate Disrupts Development In Mice By Inducing Autophagy

Objective: Phthalates(PAEs)are a class of endocrine disrupting chemicals(EDCs)that are widely used as plasticizers,including di(2-ethylhexyl)phthalate(DEHP)the most widely used.Existing studies have shown that DEHP has potential female reproductive toxicity,and the serious mechanism of female reproductive toxicity caused by increasingly DEHP contamination should concerned.Furthermore,ovary is the core organ of female reproductive function and the number of oocytes is fixed before birth.Obviously,if the pre-pubescent primordial follicle is damaged,it will directly affect the female fertility.Our previous research revealed that neonatal exposure of DEHP significantly inhibits the formation of primordial follicles,but its mechanism still needs further investigation.Then,autophagy is involved in maintaining the stability of the intracellular environment.Previous studies found that DEHP exposure significantly enhanced the expression of autophagy markers p62 and ATG5,but whether autophagy plays regulatory affection during the development of follicles caused by DEHP is worth further study.Therefore,this study aims to explore the following issues:(1)To fully understand the effects of DEHP exposure on primordial folliculogenesis during early period.(2)Determine whether autophagy is involved in the abnormal primordial follicle development caused by DEHP.(3)To investigate the molecular mechanism of DEHP-induced autophagy leading to early abnormal primordial folliculogenesis,providing experimental and scientific evidence for the prevention and control of plasticizer contamination and protection of female reproductive health.Methods1.Animal model establishment and specimen collection: BALB/C mice were randomly divided into control group,DEHP group,3-MA group,Compound C group,DEHP and 3-MA combined treatment group,DEHP and Compound C combined treatment group.The 16.5 dpc ovaries were cultured for 6 days and the neonatal mice were continuously injected intraperitoneally for 5 days.2.Investigated the effect of DEHP on the development of primordial follicles: H&E staining and oocyte counting were used to analyze the follicular development at embryonic/neonatal stage.3.Studied the effect of DEHP on autophagy: TME detected autophagosomes in oocytes after exposure to DEHP.Western-blot,qRT-PCR and immunofluorescence tested autophagy markers LC3,ATG5,Cathepsin B,Cathepsin L and LAMP2 expression levels.4.Investigated whether autophagy is involved in the process of abnormal primordial folliculogenesis caused by DEHP: Use 3-MA to interfere with autophagy and investigate the effect of DEHP on primordial folliculogenesis.Then statistically analyze whether the development of DEHP-inhibited primordial follicles can be restored.5.Investigated the signal pathway of DEHP-regulated autophagy: detect the expression levels of AMPK markers after DEHP exposure,use the AMPK inhibitor Compound C to block AMPK signal,and then study the expression of key autophagy proteins and the development of primordial follicle.Results1.DEHP impairs primordial folliculogenesis: H&E staining and oocyte counting results showed a severe disruption of the primordial follicle formation and an increased number of cyst-oocytes in DEHP group.2.DEHP activates autophagy in ovary: TME detected recognizable autophagosomes in DEHP group.Fuethermore,western-blot,qRT-PCR and immunofluorescence results showed autophagy markers LC3,ATG5,Cathepsin B,Cathepsin L and LAMP2 expression levels increased prominently.3.Activated-autophagy mediates DEHP-impaired primordial folliculogenesis: Inhibition of autophagy by 3-MA attenuated the adverse impact of DEHP on primordial folliculogenesis.4.DEHP induces autophagy by activating AMPK-SKP2-CARM1 signalling which results in disrupted primordial folliculogenesis: The key components of AMPK-SKP2-CARM1 signalling were up-regulated by DEHP,and AMPK inhibitor Compound C reduced autophagy-related gene expression and partially recovered primordial follicle assembly.Conclusion: Collectively,this study demonstrates that DEHP induces autophagy by activating AMPK-SKP2-CARM1 signalling in mice perinatal ovaries,which results in disrupted primordial folliculogenesis.