Study On Autophagy And Apoptosis In The Development Of Genital Tubercles Affected By Dibutyl Phthalate

Part 1 The effect of DBP on the development of genital tubercles in the fetal SD ratsObjective: Din-butyl phthalate(DBP) is an industrial chemical widely used as plasticizers which are distributed environmental contaminants. The present study is to examine the effect of prenatal exposure to DBP on the development of external genitals,the incidence of hypospadias and androgen level in male fetus.Methods: Time-dated pregnant Sprague-Dawley rats were randomly divided into 2groups: control group and DBP group. Rats in the DBP group were administered 750mg/kg/day of DBP by gavage from gestation days(GD) 13 to GD 18, whereas control group received placebo. On GD 19, pregnant rats were anesthetized with sodium pentobarbital. The embryos were quickly removed from the uterus, weighed and counted,and the gonads and penis were carefully examined to identify male embryos and embryos with hypospadias. The anogenital distance(AGD), diameter and length of genital tubercle(GT) were measured using a digital micrometer. Blood samples were collected from male embryos by decapitation for measurement of serum testosterone concentrations by Enzyme-linked immunosorbent assay(ELISA).Results: The ratio of male/female embryos in DBP treated pregnant rats was similar to the control rats while the number of embryos per pregnant rat was significantly less in the DBP treatment group compared to the control. The AGD, AGD/bodyweight, in the DBP exposed group were significantly decreased compared to those in the control group.GT volume in the DBP group was significantly smaller compared to the control group and it was still significantly lower than the control after adjusting for body weight.Hypospadias was observed in 43.64% male embryos exposed to DBP in utero, whereas no embryos presented with hypospadias in the control group. There was an approximate 80%reduction in serum testosterone level in the embryos with hypospadias compared to the control group.Conclusion: Prenatal exposure to DBP can induce hypospadias and affect the development of genital tubercle. Besides, exposure to DBP during pregnancy can significantly decrease the synthesis and the level of serum testosterone in male springs.Part 2 The effects of DBP on autophagy and apoptosis of genital tubercles in the fetal SD rats and the associated signaling pathways Objective: To study the autophagy and apoptosis affected by prenatal exposure to DBP in genital tubercles of male SD rats; to investigate the the associated autophagy signaling pathways regulated by prenatal exposure to DBP in genital tubercles of male rats.Methods: Establishing the animal model of hypospadias(the same as the first part). On GD 19, Frozen GT tissues from rats in control and hypospadias group were sectioned into thick slides. The apoptosis in the GT was determined by terminal deoxynucleotidyl transferase d UTP nick end labeling(TUNEL) assay. The ultrathin sections of GTs were made and autophagosomes and autolysosomes were examined under transmission electron microscope. Autophagy related proteins(light chain 3(LC3-I, LC3-II), and sequestosome(SQSTM1/p62)) were evaluated in the GT with Western blotting. The protein expressions of Akt, Beclin 1, phosphorylated Akt(p-Akt), p-S6, and phosphorylated mammalian target of rapamycin(p-m TOR) in the GT were analyzed by Western blotting.Results: In hypospadiac male rats, apoptotic cell number was significantly decreased in the GT, whereas the number of autophagosome was increased. The protein expressions of p-62, p-S6, p-m TOR, and phosphorylated Akt were significantly decreased in the GT from hypospadiac rats. LC3-II protein levels and the ratio of LC3-II/LC3-I were significantly increased in the GT.Conclusions: DBP-induced hypospadias might be associated with apoptosis and autophagy mediated by the PI3K/Akt/m TOR signaling pathway in the GT.