The Correlation And Regulation Mechanism Study Between Smad/c-Jun-NH(2)-terminal Kinase And Hypospadias Formation

Objective: Hypospadias is one of the most common congenitalanomalies occurring in the pediatric urology population, approximately1:(200～300) newborn boys, with significant variations according to ethnicorigins, and it showed a increasing trends. Currently, the efficient treatmentoptional is surgery. However, any kinds of corrections will leave a series ofcomplications and it possibly causes psychological shadow to them.Therefore, this study carried out was aim to investigate the correlationbetween Smad/JNK and hypospadias formation in two sides: hypospadiaspatients and hypospadias animal experiment study so as to clarify themechanism of hypospadias formation and lay a scientific foundation for itsfurther prevention.Methods:1. Clinical study: preputial tissues were obtained from32males withhypospadias at surgical repair in the Children’s Hospital of ChongqingMedical University. Degrees of hypospadias were classified as mild (meatus at or distal to mid shaft of penis,16cases) or severe (meatus proximal to midshaft, and scrotum or perineum,16cases). Normal preputial skin wasobtained from16subjects undergoing elective circumcision during the sameperiod and with age-matched. The expressions of Smad and JNK in preputialtissues were detected by real-time quantitative polymerase chain reaction(q-PCR), immunohistochemistry (IHC) and western blotting, respectively.2. Animal experiments1). The establishment of the hypospadias rat model: forty pregnant SDrats were divided into four groups randomly: soybean oil control group andthree doses groups of di-(2-ethylhexyl) phthalate (DEHP),500,750and1000mg/kg bw/day, respectively. On gestation day (GD)12～19, thepregnant rats were given DEHP once daily by gastric intubation. Onpostnatal day (PND)1/8/16/20/30, respectively, live pups and live malepups were counted, weighted, and their anogenital distance were measured.On PND30, penial length were measured and the external genitalia wereexamined to identify hypospadias.2). Twenty pregnant SD rats were divided into two groups randomly:soybean oil control group and the suitable DEHP modeling doses group,which obtained from the result of above section. On GD20, fetal rat obtainedby surgical cesarean and penis were cut. The expressions of Smad and JNKin fetal penial tissues were detected by real-time quantitative polymerasechain reaction (q-PCR), immunohistochemistry (IHC) and western blotting, respectively.Results:1. Compared to controls, the expressions of Smad7, Smad3and Smad2in preputial tissues were increased97%,22.8%and59.9%, respectively, andall showed statistical differences (P<0.05). Likewise, the expressions ofJNK1mRNA and p-JNK1/2protein showed statistical differences betweencontrols and subjects with mild and severe hypospadias (both P<0.05); therewere statistical significant differences in expressiones of phosphorylationJNK2protein between subjects with mild and severe hypospadias (P<0.05).Immunohistochemistry analysis revealed that Smad7, phosphorylationSmad2/3and JNk1/2proteins were expressed in the subcutaneousmesenchymal cell layer, and with a high expression in hypospadias preputialtissues.2. Compared to controls, with the increasing dosage of DEHP exposure,live pups and live male pups per litter were reduced significantly(P<0.05),duration of pregnancy were prolonged, and numbers of dystocia and stillbirth rats were increased, pups body weight, AGD, body weight/AGD andpenis longth were reduced (P<0.05). The incidence of male offspringhypospadias induced by above three different doses DEHP were10.7%,30.6%and37.0%, respectively. And different severity hypospadias wereidentified, of which, the incidence of severe hypospadias were0%,8.3%and22.2%, respectively, and there were significant differences among them (all P<0.05). The suitable dose for establishing hypospadias rat model was750mg/kg bw/day.3. Compared to controls, fetal rats body weight, AGD and their ratiowere reduced significantly following DEHP exposure (P<0.05). Theexpressions of Smad7, Smad3and Smad2mRNA in DEHP exposure groupwere increased87.0%,61.9%and30.7%respectively, and they all showedstatistical differences (P<0.05). Immunohistochemistry analysis revealedthat the protein expressions of Smad7and p-Smad2/3were rich expressed inDEHP exposure group, but there was not any expression in controls. Whilethe expressions of JNK1/2mRNA and p-JNK1/2protein in DEHP exposuregroup increased significantly (P<0.05), but the expressions of T-JNK proteinbetween them showed no statistical difference.Conclusions: Similar to human hypospadias rats were induced afterexposure to DEHP maternally. The Smad and JNK signal were up-regulatedin both hypospadias patients foreskin and hypospadias rat penial tissues. Weproposed that, under the estrogen-like effects of DEHP-kinds EEDs, theexpressions and balance among AR, ER and Smad protein in vivo wereinterfered, Smad, as intercell signaling protein, activated JNK signalingabnormally, and further interfered cells migration of the foreskin and urethra,and resulting in the occurrence of hypospadias.