Study On The Mechanism Of Hypospadiac Male Rats Induced By Di-n-butyl Phthalate

Prat I: The differential expression and activity of steroidogenic acute regulatory protein in testes of hypospadiac male rats with utero exposure to di-n-butyl phthalateObjective: To explore the pathogenesis in di-n-butyl phthalate(DBP)-induced hypospadias, we detect the differential expression and activity of steroidogenic acute regulatory(St AR)protein in the testis of fetal male and measure the testosterone concentration in serum.Methods:(1)Forty pregnant rats were randomly divided into two groups. Dams were treated by gavage daily from gestation day(GD) 13 to GD19 with corn oil vehicle(3ml/kg) or DBP in corn oil at 800 mg/kg(total DBP and corn oil at 3ml/kg).(2)On GD19, fetuses were immediately removed from the uterus, and sexed by internal examination of the reproductive organs. The hypospadiac rats and the n ormal rats were randomly divided into two groups(hypospadiac groups and control group). The gross images of genital tubercles(GT) were check. The anogenital dis tance(AGD) was measured.The incidence of hypospadias was calculated. GT was stained with haematoxylin and eosin(H&E).(3)The testosterone concentration was measured with the ELISA kits. The testes were removed from male fetuses. Quantitative Real-time PCR(q PCR)、western blot、ELISA and immunofluorescence analysis were used to quantify expression of St ARm RNA or St AR protein in testes. Pearson correlation analyzes the relationship between the r elative expression of St AR protein and the testosterone concentration. Western blot was used to quantify expression of T-ERK1/2 and P-ERK1/2 in testes. To determine the activity of St AR protein, we analyse the level of ERK1/2 phosphorylation by the ratio o f P-ERK1/2/T-ERK1/2.Results:(1)H&E staining showed that the urethral orifice of hypospadias locates on the ventral surface of the GT and the urethral orifice of normal rat was near the tip of the GT. AGD in the hypospadiac group(1.77±0.12mm) was significantly lower when compared with the control group(2.25±0.15 mm,P<0.05), and the incidence of hypospadias was 43.3%。(2)The testosterone concentration of hypospadiac group(1.45±0.62ng/ml) was also lower than control group(4.48±0.93ng/ml, P<0.05). Significantly decreased expression of St AR was also found in both m RNA and protein in the hypospadiac group(St ARm RNA:0.23±0.08; St AR:0.33±0.07), when compared with the control group(St ARm RNA:1.00±0.00; St AR:1.44±0.19, P<0.05). St AR protein was mainly located in interstitial cell of the testis and the staining intensity was obviously weaker in the hypospadiac group than control group. Pearson’s correlation coefficient of hypospadiac group(r=0.642, P<0.05) was also lower than control group(r=0.851, P<0.05). The ratio of P-ERK1/2/ERK1/2 of hypospadiac group(P-ERK1/ERK1:0.17±0.03; P-ERK2/ERK2: 0.19±0.07) significantly decreased compared with control group(P-ERK1/ERK1: 0.31±0.08; P-ERK2/ERK2: 0.43±0.14, P<0.05).Conclusion: DBP not only decrease the expression of St AR at the gene transcription and protein translation, but also inhibit activity of St AR protein by decreasing the level of ERK1/2 phosphorylation to affect the synthesis of testosterone, thereby inducing hypospadias formation.Prat II: The differential expression of androgen receptors isoforms protein in fibroblast cells of hypospadiac male rats with utero exposure to di-n-butyl phthalateObjective: To explore the pathogenesis in di-n-butyl phthalate(DBP)-induced hypospadias, we detect the differential expression of androgen receptors isoforms protein(AR-A、AR-B) in fibroblast cells of genital tubercle(GT) and assay fibroblast cells proliferation and migration.Methods:(1)Pregnant rats were randomly divided into two groups. Dams were treated by gavage daily from gestation day(GD) 13 to GD19 with corn oil vehicle(3ml/kg) or DBP in corn oil at 800 mg/kg(total DBP and corn oil at 3ml/kg).(2)On GD19, fetuses were immediately removed from the uterus, and sexed by internal examination of the reproductive organs. The hypospadiac rats and the normal rats were randomly divided into two groups(hypospadiac group and control group). The anogenital distance(AGD) was measured.The incidence of hypospadias was calculated.(3)Primary culture of fibroblast cells in genital tubercles were cultured under full aseptic precautions.(4)Western blot、q PCR were used to detect the expression of AR isoforms protein and ARm RNA in fibroblast cells with primary culture by tissue-pieces.(5)Fibroblast cells proliferation and migration were determined using MTT assay and scratch-wound experiment.Results:(1)The result of hypospadias is referred to part I.(2)Under an inverted microscope, we can find spindle or polygonal cells with elongated filopodia. Immunofluorescence results showed that vimentin protein was also found in the two groups.(3)Significantly decreased expression of AR-B was also found in both cytopl asm and nucleus of fibroblast cells in the hypospadiac group(the cytoplasmic AR-B:0.16±0.07; the nuclear AR-B:0.13±0.07, P<0.05), when compared with the control group(the cytoplasmic AR-B:3.46±0.98; the nuclear AR-B:2.17±0.58, P<0.05). The expression of AR-A protein was only located in nuclear of fibroblast cell and was not statistically different between hypospadias with control. The relative expression of ARm RNA in fibroblast cells was also not statistically different between hypospad ias with control.(4)Compared with the fibroblast cell of control group, the hypospadiac group showed significantly decreased in cells proliferation(96h: 0.43±0.04 VS 0.34±0.05; 120h: 0.50±0.04 VS 0.41±0.04; 144h: 0.57±0.06 VS 0.47±0.04, P<0.05), and cell migration(12h: 0.17±0.04 mm VS 0.14±0.03mm; 24h: 0.26±0.05 mm VS 0.20±0.04 mm, P<0.05).Conclusion: DBP not only decrease the expression of AR-B and Collagen Type I in fibroblast cells, but also inhibit fibroblast cells proliferation to affect the development of genital tubercle, thereby inducing hypospadias formation.