Study On The Epithelial-mesenchymal Transition In Urethral Development And Hypospadias

Background:Hypospadias is a common congenital genitourinary malformation in children,with high incidence and serious harm.However,the pathogenesis of hypospadias is still unclear.The process of embryonic development and the key step of the urethra is still controversial,and whether there are epithelial-mesenchymal transitions in the urethral plate fusion process remains to be study further.Objective:The aim of our study was to elucidate the urethral development dynamics,the mechanism of urethral plate fusion,with a special focus on the role of epithelial-mesenchymal transition in urethral seam formation,and the mechanism of hypospadias in male rats,providing a scientific foundation for prevention and treatment of hypospadias.Methods:1.Time-mated Sprague–Dawley rats were killed and the genital tubercles of male pups harvested on embryonic day?ED?15,16,18,and 19.External morphology of penis was observed under scanning electron microscope.Serial transverse sections were prepared to examine dynamic morphology changes of the urethral seam with hematoxylin–eosin staining.The biochemical and morphological changes of urethral epithelial cells during the formation of urethral seam were marked by double immunofluorescence and transmission electron microscopy.2.In vivo,time mated Sprague-Dawley rats were randomly dividedd into three groups:control,DEHP₅₀₀ and DEHP₇₅₀ group.On ED 12-ED 18,rats were intragastric administrated with corn oil,DEHP at a dosage of500mg/kg/day and 750mg/kg/day,respectively.Urethral seam formation was observed by scanning electron microscopy and HE staining,EMT was detected by immunofluorescence,elements in TGF-?/Smad signaling pathway?TGF-?1,Smad2/Smad3,Snail,Slug,EMT marker E-cadherin,?-catenin,occludin,ZO-1,?-SMA,N-cadherin,Vimentin?changes were evaluated by polymerase chain reaction and western blot.3.In vitro,penises of male pups were collected at ED16 and ramdomly diviede into control,DEHP and TGF-?group,cultured and treated with PBS,MEHP or MEHP+TGF-?1 for 48h,respectively.Histomorphology of penis and elements in TGF-?/Smad signaling pathway?the same as in vivo?changes were evaluated by immunofluorescence and western blot.4.ED16 fetal rat penises were randomly divided into control group,DEHP group,TGF-?group and DEHP+TGF-?group,treated with PBS,MEHP,MEHP+TGF-?1 for 24 h,and TGF-?1 24h after MEHP treatment24h,respectively.Morphology of cultured penises was observed.Results:1.Bilateral outgrowth of urethral swelling followed by urethral plate fusion in the midline to form urethral seam from the proximal to the distal of penis was observed from ED 16 onwards,urethra formed.2.In the process of urethral seam remodling,coexpression of epithelial and mesenchymal markers was observed in several cells at the urethral seam;a few cells with coexpression of epithelial and apoptotic markers were also observed.Under transmission electron microscope,several cells had lost their intercellular junctions,exhibited a filopodial appearance,transformed into mesenchymal cells with large nucleus and scarce cytoplasm,showing characteristics of mesenchymal cell.Several cells exhibited cell membrane shrinkage,chromatin margination,degeneration and disappearance of cell junctions,displaying apoptosis appearance.3.Compared with control group,hypopadias could be induced and urethral seam fusion were delayed after exposure to DEHP.TGF-?1,p-Smad2/Smad3,Snail,Slug,mesenchymal cell markers including?-SMA,N-cadherin and Vimentin were decreased,epithelial cell markers including E-cadherin,?-catenin,occludin and ZO-1 were increased?p<0.05?.In vitro,TGF-?1,p-Smad2/Smad3,Snail,Slug,?-SMA,N-cadherin and Vimentin were increased,E-cadherin,?-catenin,occludin and ZO-1 were decreased after treated with TGF-?1?p<0.05?.4.MEHP could lead to the development of penile growth disorders in vitro,and the opening of the urethra was closer to the root of the penis.The TGF-?1 factor could partially improve the developmental disorders of the urethra.Conclusion:Rat urethral development involves tubulogenesis from proximal to distal to form urethral seam.During remodeling,EMT and epithelial cell apoptosis have a role in urethral seam fusion.Decreased EMT in urethral seam formation would lead to urethral plate fusion retardation,hypospadias occurs.TGF-?1 can prevent this process and reduce the incidence of hypospadias.