| BACKGROUND: Hypospadias is one of the most common congenital abnormalities of the male external genitalia. The main symptoms are abnormally located urethral meatus, penile chordee and excess dorsal prepuce. Some of the patients could not urinate to stand with decresed reproductive activity in adult. The incidence of hypopadias is 1/300~1/200 in live births and the incidence is increasing in many countries. Di(2-ethylhexyl) phthalate (DEHP) is by far the most commonly used in medical work and daily life as the plasticizer, and is one of the most common"white pollution"in China. DEHP is not easy to be degradated and can cumulate in body to mimic estrogenic biological activity. Our group had found that DEHP can induce mouse hypospadias, but till now, the mechanism is obscure. In this study, we will focus on the molecular mechanism of hypospadias induced by DEHP in rat, and explore the antagonist on attenuating the urethral development toxicity induced by DEHP.OBJECTIVE: To explore the reproductive toxic effect of DEHP using rat model of hypospadias. Observed the development of penis, investigated TGF-β1, TGF-βR3 expression and apoptosis in rat genital tubercle (GT). To explore the mechanisms of vitamin E on attenuating the urethral development toxicity induced by DEHP.METHODS: 1. Pregnant SD rats were randomly divided into 2 groups with 20 in each group: DEHP(500 mg/kg·d)groups and vehicle control (corn oil). Rat were gavaged from gestation day 12 to 19(GDl2-19). 10 pregnant rats were let to delivery normally in each group. After the pregnant rats had delivered, the male offspring were counted and the anal genital distance (AGD)and body weight were measured. Hypospadias were also observed after postnatal seventy days of the male offspring. Fetal rat were harvested at GDl9 in the remaining pregnant rat. Semi-quantitive RT-PCR were used to measure mRNA of TGF-β1, TGF-βR3 expression in rat genital tubercles of above two groups on GD19.2. Pregnant SD rats were randomly divided into 4 groups with 20 in each group: vehicle control (corn oil) , DEHP(500mg/kg·d) ,DEHP (500 mg/kg·d) + vitamin E (200mg/kg·d) and vitamin E (200mg/kg·d) group. Rat were gavaged from gestation day 12 to 19(GDl2-19). 10 pregnant rats were let to delivery normally in each group. After the pregnant rats had delivered, the male offspring were counted and the AGD(anal genital distance)and body weight were measured. Hypospadias were also observed after postnatal seventy days of the male offspring. Fetal rat were harvested at GD19 in the remaining pregnant rat. Semi-quantitive RT-PCR were used to measure mRNA of TGF-β1, TGF-βR3 expression and TUNEL were used to measure cell apoptosis in rat genital tubercles of above four groups on GD19.RESULTS: 1. Hypospadic rat model was induced by DEHP exposure, hypospadias displayed a proximal urethral opening on the distal penile shaft and cleaved prepuce. The rates of hypospadias were 13.0% in DEHP group. The AGD and body weight in DEHP group was significantly shorter than those of control group (p<0.05). The levels of TGF-β1, TGF-βR3 mRNA were up-regulated in DEHP group (p<0.05).2. Hypospsdias was induced in male rat. The levels of TGF-β1, TGF-βR3 mRNA were up-regulated in DEHP group (p<0.05). Cell apoptosis in rat genital tubercles on GD19 was reduced in DEHP group (p<0.01). Supplementation of vitamin E (200 mg/kg·d) reduced hypospadias, decreased the TGF-β1, TGF-βR3 mRNA expression, increased cell apoptosis in rat genital tubercles on GD19.CONCLUSIONS: Murine model of hypospadias has been established successfully by treatment with DEHP, and up-regulated TGF-β1, TGF-βR3 in early development of GT may be one of the pathogenesis of hypospadias. Vitamin E cotreatment showed protective effects against DEHP-induced urethral development toxicity and the mechanisms maybe associated with TGFβs expression and cell apoptosis in rat genital tubercles on GD19.
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