Influences Of DEHP On Testis Early Development Of Males Mice After In Uterus Exposure

Di-(2-ethylhexyl)phthalate(DEHP),a widely existed endocrine disruptor,has been concerned for many years because of its toxicity in nerve,thyroid,kidney,heart,reproductive development and other aspects.It was also observed that testis was more sensitive than ovarian to DEHP.DEHP exposure can significantly decrease the weights of epididymides,testis,prostates and anogenital distance and cause cryptorchidism.The mechanism of DEHP injury on male reproduction involved changes of DNA methylation,apoptosis of spermatogenic cells,inhibition of spermatogenic cell proliferation,decreases of androgen synthesis and damage of DNA.In this study,DEHP was administered at 75,225 or 675 mg/kg/d body weight by oral gavage in the olive oil vehicle from day 9.5 to 15.5 of ICR mice pregnancy.Olive oil alone was administered to the control mice.Male offsprings were sacrificed by cervical dislocation at Embryo days(ED)18.5,postnatal day(PND)14,28,and 35,and testes were excised as soon as possible.The pathologic damage of F1 mice testis after in uterus DEHP exposure were observed using HE staining and transmission electron microscopy.Then we also explored endoplasmic reticulum stress,apoptosis mediated by endoplasmic reticulum stress and the expression levels of genes participated in cell proliferation and testosterone synthesis by using western blotting TUNEL and immunohistochemistry.The results showed testis were damaged by DEHP.1.The number of seminiferous tubules were significantly reduced at ED18.5,and the gaps between seminiferous tubules or spermatogenic cells were enlarged.vacuolization,fault,shedding cells and cells with abnormal staining were observed in DEHP treatment mice testis after birth.2.Compared with the control group mices,endoplasmic reticulum was swollen in spermatogenic cells after DEHP treatment.The protein levels of an ER chaperones,GRP78 was changed according in a dose-dependent manner.GRP78 was downregulated in male mices at PND14 and PND28,and upregulated at PND35.3.Western blotting analysis showed Cleaved Caspase-12,a maker of the endoplasmic reticulum stress-mediated appotosis,were dose-dependently upregulated in testis of male mices at PND28 and PND35 after prenatal exposures to 675 mg/kg/d DEHP.The number of apoptotic testis cells was increased at PND35 detected by TUNEL.In addition,the proportion of tubules with positive PCNA expression was significantly down-regulated analyzed by immunohistochemistry.4.The number of lipid droplets was decreased and the structure-abnormal mitochondria was observed in leydig cells under the transmission electron microscope.STARD10,a cholesterol transporter,were decreased in male mices at ED18.5 after prenatal exposures to 675 mg/kg/d DEHP.Furthermore,the expression of androgen-binding protein(ABP1)was decreased by analysis of immunohistochemistry.These resulte suggested that DEHP exposure led to reproductive damages of male mice testis during the early development,which might arise from the changes of structure of endoplasmic reticulum and the accordingly apoptosis mediated by the endoplasmic reticulum stress.Furthermore,both of the inhibition of the spermatogenic cell proliferation and the testosterone synthesis contributed to the testis injury arosed by DEHP exposure.In this study,we investigated the injury and its mechanism of DEHP exposure on the early stage of testis development of F1 male mice.For the first time we found the endoplasmic reticulum stress-mediated spermatogenic cell apoptosis was involved in DEHP-induced male reproductive injury.This provided a theoretical basis for further understanding of the relationship between DEHP exposure during human pregnancy and male infertility in adults.