Di-butyl Phthalate Induces Hypospadias By Affecting The Expression Of Sex Hormones In Pregnant Rat And Their Offspring

Part ? Effects of Di-butyl Phthalate on Testosterone,Estrogen and Progesterone Levels in Pregnant Rats and Their OffspringObjective:The development of extra-embryonic genitalia depends on the induction of sex hormones.As a potent environmental endocrine disruptor(EED),di-butyl phthalate has an important impact on the occurrence and development of extra-embryonic genitalia.This section explored changes in sex hormone levels in the pregnant SD rats and their offspring upon DBP exposure.Methods:Pregnant rats during pregnancy were randomly divided into three groups:(1)800 mg/kg corn oil gavage group(control group),(2)400 mg/kg DBP gavage group,(3)800 mg/kg DBP gavage group.During the 13-18 days of pregnancy,the gavage was administered,and the blood samples of the pregnant rats were collected.The sex hormonal contents were detected by ELISA.Fetal rats were harvested by cesarean section at day 19 of pregnancy.The genital tubercles of both male and female fetus,and fetal testes were collected for morphological studies on H&E staining section.Blood samples were collected from pregnant rats and fetal rats,and sex hormonal levels were detected by ELISA.Results:1.Hypospadias were induced in male fetal rats at an incidence of 35.1%in 800 mg/kg DBP gavage group,which was characterized by absence of ventral foreskin of male genital tubercles,incomplete fusion of urethral plates,and ventral fissure-like malformation.2.In the 13-18 days of pregnancy,the levels of testosterone,estrogen and progesterone in the three groups of pregnant rat continued to rise,but the levels of sex hormones suddenly decreased at 19 days of pregnancy.3.Compared with the control group,the testosterone and estrogen levels in the DBP gavage group increased,especially in the low-dose DBP gavage group,while the progesterone level continued to be low,and the rising trend was not obvious.4.Compared with the control group,DBP exposed pregnant rats had relatively higher testosterone levels and relatively lower progesterone levels in the late pregnancy,while relatively lower testosterone levels and relatively higher progesterone levels in male offspring.Conclusion:800 mg/kg DBP may induce hypospadias in male pups,there were opposite differences between maternal sex hormone levels and fetal sex hormone levels,and this effect is more pronounced at lower doses.Part ? Effects of Di-butyl Phthalate on the Expression of PR,ATF4 and p-eIF2 a in the Reproductive Organs of RatsObjective:Ingestion of progesterone during pregnancy may increase the risk of hypospadias in male offspring,which indicates that progesterone may play an important role in the development of external reproductive organs of the male offspring,and the physiological effects are mainly regulated by interaction with the progesterone receptor(PR).In addition,cell apoptosis and autophagy may also be involved in the occurrence of hypospadias.This section explored the effects of di-butyl phthalate on the expression of PR,ATF4 and p-eIF2a in the reproductive organs of fetal rats.Methods:Pregnant rats were randomly divided into three groups:(1)800 mg/kg corn oil gavage group(control group),(2)400 mg/kg DBP gavage group,(3)800 mg/kg DBP gavage group.The reproductive nodules and testicular tissues of their offspring were collected.The distribution of progesterone receptors in the external reproductive system of the offspring was observed by immunohistochemistry.PCR and Western bolt experiments were used to observe the differences of the PR and ATF4 and p-eIF2a expression in the gene and molecular level.Results:1.Observed by immunohistochemistry,progesterone receptors are mainly distributed in the urethral plate and glandular cells in the genital tubercles of fetal rats.2.Compared with the control group,the progesterone receptor(PR)gene level,protein level,and immunohistochemical mean optical density(AO)values of the male and female rats' genital tubercles and male testosterone in the DBP gavage group increased,especially in 400 mg/kg DBP gavage group,which was statistically significant(P<0.05).3.Apoptosis and autophagy-related factors ATF4 and p-eIF2a increased with the increase of DBP intragastric dose,and their protein levels increased gradually.Conclusion:Progesterone receptors play a role in the development of reproductive nodules,especially in the development of urethral plates.Low doses of DBP stimulate significant proliferation of progesterone receptors in the reproductive system.The increase in ATF4 and p-eIF2a protein content in the DBP exposed group demonstrated that apoptosis and autophagy are likely to be involved in the development of male hypospadias.