Study On The Mechanism Of Hypospadias

BACKGROUND:Hypospadias is one of the most common congenital anomalies defined by abortive development of the urethral meatus,the penile chordee and the ventral prepuce.Some of the patients could not urinate standly with decreased reproductive activity in adult. Surgical repare is the only therapeutic measure,but the incidence of postoperative complications is still high.Researching on the etiology of hypospadias has been received attention by researchers.The occurrence of hypopadias about 1 of 300 to 1 of 200 live births, and noted an unexplained increase in the occurrence.Hypospadias has been shown to be inducible in utero by exposure to estrogenic compounds. Di(2-ethylhexyl) phthalate(DEHP) is by far the most commonly used plasticizer,it is an ingredient of products such as some toys,medical tubing and blood storage bags,and is one of the most common "white pollution" in China.In inviroment,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 cryptorchidism with destroying the normal function of animal testis and epididymis.At the same time,we found hypospadias in mouse with cryptorchidism.Actually,researches had reported that DEHP could induce a wide range of developmental and reproductive toxicities,and significantly correlated with the occurrence of some congenital diseases,such as hypospadias and cryptorchidism,but till now,the mechanism is obscure.In this study,we will focus on the reproductive toxic effect of DEHP,and research on the relationship between DEHP and hypospadias.OBJECTIVE:To explore the reproductive toxic effect of DEHP using mouse model of hypospadias.Observed the development of genital tubercle(GT),investigated the apoptosis and TGF-β1,ATF3 expression in vivo and in vitro.Compared the difference of TGF-β1 between patients with hypospadias and circumcise.METHODS:1.GD12 mice were randomly divided into the following 4 groups(n=10):Vehicle Control,mice were gavaged with 0.1mL corn oil only;Treated,the mice were respectively gavaged with 100mg/Kg·d (DEHP₁₀₀),200mg/Kg·d(DEHP₂₀₀),and 500mg/Kg·d(DEHP₅₀₀) of DEHP suspended in 0.1mL com oil from GD12 to GD17.Compared the development of fetal mouse GT and urethra at GD19.Urethral cast was used to remodel the below urinary tract of fetal mouse,the rate of hypospadias was statisticsed by measuring the length of urethra.The expression of TGF-β1 mRNA in GT was detected by using whole mount in situ hybridization(WISH).2.By using RT-PCR,immunofluorescent staining and western blot, the expressions of TGF-β1 and ATF3 were detected in GT of fetal and immature mouse.Apoptosis was also detected by using TUNEL method and Apoptosis-related genes(P53,Bcl-2 and Bax) were investigated.3.GTs of GD12.5 fetal mice were harvested to culture in vitro.After treated with different dosese of MEHP,TGF-β1 cytokine and MEHP+anti-TGF-β1 monoclonal antibody,the development of GTs were compared and the expressions of TGF-β1 and ATF3 were detected by using RT-PCR.4.Prepuce of patients with hypospadias and circumcise were collected, the expression of TGF-β1 mRNA was compared by using immuohistochemistry,RT-PCR and real time PCR.RESULTS:1.Hypospadic mouse model was induced by DEHP exposure,hypospadias displayed a proximal urethral opening on the distal penile shaft,shorter penis and cleaved prepuce.The rates of hypospadias were 7.1%,14.0%and 75.7%in DEHP₁₀₀,DEHP₂₀₀ and DEHP₅₀₀ group respectively.In experiment groups,body wight and anogenital distance (AGD) were significantly lower than that of control group,and exhibited a dose response to DEHP(p＜0.05).Moreover,urethral casts from hypospadias in middle and high dose DEHP groups showed that anterior urethras became shorted than that of control group(p＜0.001).Serial histological sections of GT showed that hypospadias had abnormalities in the closure of the urethral seam and delay in preputial development. TGF-β1 mRNA was selectively expressed in urethral plate and epidermis.2.TGF-β1 and ATF3 expressions were detected by immunofluorescent staining,RT-PCR and western blot at GD19.The expressions increased significantly with the dose of DEHP.But in the penises of PND20 and PND40 mice,the TGF-β1 mRNA expressions were markedly lower than that of control.Apoptosis of fetal mouse GT cells had notably decreased after DEHP treated,and P53 was down-regulated and the ratio of Bcl-2/Bax was not significantly changed.3.The fetal mouse GT grew well in vitro.MEHP and TGF-β1 cytokine could inhibit the development of GT significantly.The levels of TGF-β1 and ATF3 mRNA were not increased in MEHP treated groups, ATF3 mRNA could not be enhanced or depressed after TGF-β1 cytokine or antibody treated.4.The levels of TGF-β1 of prepuce were decreased in patients with hypospadias comparing with the control.CONCLUSIONS:Mouse hypospadias which is induced by DEHP might be related to upregulated TGF-β1 and ATF3 expressions.The abnormal expressions of TGF-β1 and ATF3 may be one key point of the mechanism of hypospadias,but TGF-β1-ATF3 signal pathway may not be involved in this process.