The Role Of Endoplasmic Reticulum Stress On Testis Injury Induced By DEHP In Male Mice

Di(2-ethylhexyl)phthalate(DEHP)is the most widely used phthalate.As a widely existed endocrine disruptor,DEHP caused reproductive,developmental,neural,and immunological toxicities in rodent and human body.Among these effects,damage to the male reproductive system was the most serious.It has been reported that DEHP exposured during the gestation could causeadverse effects to the offspring.ICR females(6 week old)were continuously paired with ICR males(9 week old)for generating offspring.Females were examined for the presence of vaginal plugs,the presence of which was set as pregnant mice.The pregnant mice were randomly assigned to one of the following groups:(1)olive oil as vehicle control;(2)675 mg/kg/day DEHP;(3)675 mg/kg/day DEHP and 100 mg/kg/day 4-PBA(the inhibitor of the endoplasmic reticulum stress).Olive oil and DEHP administered by oral gavage from E 9.5 to E 15.5.4-PBA was injected for the pregnant mouse from E 9.5 to E 18.5 and re-injected for the male offsprings from PND 10 until the the offsprings were sacrificed by cervical dislocation.The testis were excised from each group and weighted on Embryo days(ED)18.5 and postnatal day(PND)14,28 and 35.In this study,we evaluated the reproductive effects of DEHP from the level of histomorphological and ultrastructural changes on the testis of F1 offspring by hematoxylin and eosinstaining and transmission electron microscopy(TEM).Futher,we also explored the contaction between endoplasmic reticulum stress and testis injury induced by DEHP from the point of endoplasmic reticulum stress and adherin junction through RNA-seq,immunohistochemical staining,quantitative real-time PCR,and western blotting.The results showed testis were damaged by DEHP.1.The number of seminiferous tubules were significantly reduced and the endoplasmic reticulum expanded at ED 18.5d.2.In adolescence,the anogenital distance(AGD)and testis weight were significantiy decreased.In addition,we also observed that DEHP administration led to the interstitial space expansion between spermatogenic cells,the seminiferous tubules atrophy,the sloughed cell,vacuoles and the swelling ER.3.Transcriptomic analysis also revealed the differentially expressed genes were decreased significantly at PND 35 d.4.Immunohistochemistry and western bloting showed that the expression of N-cadherin was reduced in the Leydig cells,Sertoli cell and the perimeter of seminiferous tubules in DEHP groups at PND 28 and 35 d.5.DEHP activated ER stress in F1 offspring and also started up ER stress-mediated apoptosis signaling.7.The phenomenon of seminiferous tubules atrophy,the swelling ER and the expression of N-cadherin were all restored after treatment with 4-PBA.In conclusion,this study demonstrated that DEHP exposure led to reproductive damages of male mice testis which caused the abnormal histomorphometry during the early development and adolescence.In addition,we also demonstrated UPR were activated and the ER stress-mediated apoptosis signaling in testis of male mice offsprings after in uterus exposure of DEHP which gave rise to the decreasing of protein synthesis including the adhesion molecular N-cadhesion.The using of ER stress inhibitor further proved seminiferous tubules atrophy and the swelling ER were closely related to ER stress.Our findings will be helpful for better understanding the basic mechanism with DEHP expoure.