Di-(2-ethylhexyl) Phthalate Induces Testicular Endoplasmic Reticulum Stress And Germ Cell Apoptosis In Adolescent Mice

Background Di-(2-ethylhexyl)phthalate(DEHP)is the most common phthalate,and it is also an environmental endocrine disruptor.DEHP has some estrogenic activity and can lead to testicular atrophy in males.It has been proved that male exposure to DEHP can lead to apoptosis of testicular cells,however,studies on which pathways have been used to induce apoptosis are not comprehensive.Previous in vitro cell experiments showed that DEHP exposure can cause endoplasmic reticulum stress in cells,and in vivo experiments in animals have demonstrated that DEHP exposure can cause endoplasmic reticulum stress in the kidneys,so we speculate that male DEHP exposure can cause endoplasmic reticulum stress and induce apoptosis in testicular tissue,causing testis atrophy.Previously,our research team found that exposure to a certain metal in adolescent mice can lead to testicular toxicity and reduce sperm quality,and indirectly affect male fertility and intrauterine growth and development of their offspring.So we explored whether this phenomenon is equally applicable to adolescent male mice exposed to DEHP.Objective To investigate the potential mechanism of testicular damage in adolescent male DEHP exposure and its effect on fertility and intrauterine growth and development of their offspring.Methods 6-week-old male mice were randomly divided into four groups,and were administered with DEHP at 0,0.5,50,and 500mg/kg/d for 35 consecutive days.After mating,male mice were fed for 9 days,then the testis and epididymis were weighed,and the epididymis was cut for sperm count.Testis tissue was stained with hematoxylin and eosin,and its morphology was observed under a microscope to establish a model of reproductive toxicity.The TUNEL kit was used to detect the apoptosis of testicular cells,and Western-blotting was used to detect the expression of related proteins in the endoplasmic reticulum-induced apoptosis pathway.After administration,the male were mated with the female mice at a ratio of 1: 1 for 5 days continuously.The pregnant mice were fed to GD18,and the situation of empty pregnancy and abortion during the feeding period were recorded.Calculate the mating success rate,fertility success rate,and abortion rate of pregnant mice.Pregnant mice were sacrificed in GD18,recorded resorptions,dead fetuses and live fetuses.Recorded the weight and the crown-rump length of live fetuses,the diameter and weight of placenta.The placental tissue was stained with hematoxylin and eosin,and the effects of male DEHP exposure on the growth and development of placenta and fetal mice were observed.Results(1)Compared with the control group,adolescent male mice exposed to500mg/kg/d of DEHP significantly affected the weight of testes and epididymis and the number of sperm(p <0.05),but did not have indicators for the 0.5 and 50mg/kg/d groups(p> 0.05).The proportion of seminiferous tubules in the IX-XII stage of the500mg/kg/d group increased significantly(p <0.05),while the proportion of seminiferous tubules in the VII-VIII stage decreased significantly(p <0.05).(2)The proportion of apoptotic positive cells in the 500mg/kg/d group was significantly higher than that in the control group(p <0.05).Indicators related to apoptosis in testis such as lysing Caspase-3 in testis was significantly increased(p <0.05),the protein expression of Bcl-2 was significantly decreased(p <0.05).DEHP had no effect on protein expression of testicular Bax(p> 0.05).(3)The expression of testicular GRP78 was significantly decreased in the 500mg/kg/d group(p <0.05).The expressions of p-IRE1?,p-JNK and CHOP in the testis increased significantly in the 500mg/kg/d group(p<0.05).(4)The DEHP exposure had no significant effect on the mating success rate of male and female mice,and the female pregnancy success rate(p> 0.05).There was no significant difference in the abortion rate of pregnant mice in each dose group compared with the control group(p> 0.05).(5)There were no significant differences between the number the numbers of resorptions,dead fetuses and live fetuses in each dose group compared with the control group(p> 0.05).There was no significant difference in the weight and the crown-rump length of live fetuses,the diameter and weight of placenta in each dose group compared with the control group(p> 0.05).Male mice DEHP exposed had no effect on the structure of the placenta of pregnant mice.Conclusion DEHP can cause endoplasmic reticulum stress in testis and induce subsequent apoptosis pathway,which makes testis atrophy and sperm quantity decrease.However,adolescent DEHP exposure had no effect on male fertility.It has no significant effect on the birth outcomes and intrauterine growth and development of the offspring.These findings clarify the impact of DEHP treatment on male reproduction and its potential mechanisms,and provide new ideas and scientific basis for further research on the effects of DEHP on reproduction.