| Background: Hypospadias,one of congenital malformations in boys,defines as ectopic displacement of the urethral meatus from the head of the penis to the proximal penis and the scrotum,often along with a defect in the ventral foreskin and penile curvature.The incidence rate is 1/200 to 1/300,which continues to rise.Patients with severe hypospadias are at risks of complications leading to lifelong difficulties with urination,sexual function,and psychological problems,and surgery is the main treatment.Although the etiology of hypospadias remains obscure,it is thought to be multifactorial,including factors such as genetic predisposition,environmental influences,inadequate hormonal level.Dihexyl phthalate(DEHP),a member of phthalates(PEs),is by far the most commonly used plasticizer in China.As a kind of typical environmental endocrine disruptor,it has been identified to induce a wide range of developmental disorders and reproductive toxicities.However,only limited genes have been reported to be related to hypospadias and the mechanism of DEHP-induced hypospadias remains obscure.As a link between genetic information carrier genes and biological functional proteomes,the transcriptome can comprehensively and quickly obtain almost all transcripts and gene sequences of a specific tissue in a specific state,providing the most comprehensive transcriptome information.Hence,it has been widely used in the medical field.However,transcriptome sequencing of penile tissue in fetal rat haven’t been reported yet.Androgens and androgen receptors play important roles in the virilization of the urethra,which are also related to the occurrence of hypospadias.V-mafmusculoaponeurotic fibrosarcoma oncogene family protein B(Mafb)belongs to the Maf family.Studies have found that it is related to male genitourinary development although its mechanism remains unclear.Objective: To screen the key genes and important signal pathways during normal urethral development through transcriptome sequencing of fetal penis on(embryonal day)ED16 and ED19.After the differentially expressed genes(DEGs)were screened out,the androgen-related differential gene Mafb was selected to verify.And expression of Mafb and its related proteins were detected in the human foreskin and in the DEHP-induced hypospadiac offspring of rats.Methods: Forty time-pregnant SD rats were randomly divided into four groups,namely GD16 group and GD19 group(in which the male genital tubercles(GTs)were collected on gestation day [GD] 16 and GD19 for RNA-seq,respectively),control group and DEHP exposure group(with administration of oil and 750 mg/kg DEHP by gavage from GD12 to GD19,respectively).The DEGs in the GTs were screened using lllumina HiSeq 2000 followed by GO and KEGG enrichment analyses to characterize the transcriptome profile.Immunofluorescence assay was performed to verify the DEGs(Mafb)according to the RNA-seq results.After collecting GTs from male fetuses of control group and DEHP exposure group on GD 19.5,Scanning Electron Microscope and hematoxylin eosin staining were performed to observe GTs changes.Immunofluorescence assay and Western blotting were used to examine the expression levels of Mafb and β-catenin in the penile tissue.Besides,Mafb and its related proteins were detected in the prepuce of normal children,mild hypospadiac paitients,and severe hypospadiac paitients.Results: A total of 1360 DEGs in the GTs were detected between GD16 group and GD19 group by RNA-seq.Among these genes,797 were up-regulated and 563 were down-regulated.DEGs were mainly enriched in the cell adhesion plaque signaling pathway,axon guidance signaling pathway,and extracellular matrix receptor signaling pathway.Compared with the GD16 group,Mafb was significantly up regulated in the GD19 group,which were consistent with the sequencing results.However,Mafb and β-catenin was significantly down-regulated in the DEHP exposed group compared with the control group.In addition,the expression of Mafb in the prepuce of patients with hypospadias was significantly down-regulated compared with normal foreskin tissue,while the expression of protein c-Fos,which is closely associated with Mafb,was significantly increased.Conclusion: Mafb increased gradually with the development of GTs of male SD rats.On the contrary,Mafb was significantly down-regulated in the DEHP exposed group and in the penile tissue of hypospadias patients compared with the control group.c-Fos,a gene relative to the Mafb,increased in both the genital tubercles of DEHP-induced hypospadiac rats and the penile tissue of hypospadias patients.Thus,the down regulation of Mafb and overexpression of c-Fos might contribute to hypospadias. |
PDF Full Text Request