Differential expression and activity of steroidogenic acute regulatory protein in testes of hypospadiac male rats with in utero exposure to di-n-butyl phthalate

Abstract

Objective To explore the pathogenesis of di-n-butyl phthalate (DBP)-induced hypospadias, detect the differential expression and activity of steroidogenic acute regulatory (StAR) protein in fetal testes and measure the testosterone concentration in sera. Methods Forty pregnant Sprague Dawley rats were randomly divided into two groups (n=20 each). Dams were treated by gavage at 9 a. m daily from gestation day (GD) 13 to GD19 with corn oil vehicle (3 ml/kg) or DBP in corn oil at 800 mg/kg (total DBP & corn oil at 3 ml/kg). On GD19 at 12 a. m, fetuses were immediately removed from uterus and sexed by internal examination of reproductive organs. Hypospadiac and normal rats were randomly divided into hypospadiac and control groups. Outer appearance of genital tubercles(GT) was examined. And anogenital distance (AGD) was measured. The incidence of hypospadias was calculated. And GT was stained with hematoxylin & eosin (H&E). The concentration of testosterone was measured with enzyme-linked immunosorbent assay (ELISA) kits. Testes were removed from male fetuses. Real-time quantitative polymerase chain reaction (PCR), Western blot, ELISA and immunofluorescence were used for measuring the expression of StAR mRNA or StAR protein in testes. Pearson's correlation was used for analyzing the relationship between relative expression of StAR protein and testosterone concentration. Western blot was utilized for quantifying the expression of T-ERK1/2 and P-ERK1/2 in testes. To determine the activity of StAR protein, ERK1/2 phosphorylation was analyzed by the ratio of P-ERK1/2/T-ERK1/2. Results HE incidence of hypospadias at 43.3%(46/106). The testosterone concentration was also lower in hypospadiac group than that in control group [(1.45±0.62) ng/ml vs. (4.48±0.93) ng/ml, P<0.05]. Significant down-regulation of StAR was also found in both mRNA and protein in hypospadiac group (StAR mRNA: 0.23±0.08; StAR: 0.33±0.07) when compared with control group (StAR mRNA: 1.00±0.00; StAR: 1.44±0.19, P<0.05). StAR protein was located predominantly in interstitial cell of tests and staining intensity was obviously weaker in hypospadiac group than that in control group. Pearson’s correlation coefficient was also lower in hypospadiac group (r=0.642, P<0.05) than that in control group (r=0.851, P<0.05). The ratio of P-ERK1/2/ERK1/2 (P-ERK1/ERK1: 0.17±0.03; P-ERK2/ERK2: 0.19±0.07) significantly decreased in hypospadiac group as compared with control group (P-ERK1/ERK1: 0.31±0.08; P-ERK2/ERK2: 0.43±0.14, P<0.05). Conclusions DBP not only decreases the expression of StAR at the levels of gene and protein translation, but also inhibits the activity of StAR protein by decreasing the level of ERK1/2 phosphorylation to affect the synthesis of testosterone, thereby inducing hypospadias. Key words: Hypospadias; Laboratory animal; Phthalate; Testosterone