| Objective: To induce hypospadiasand anorectal malformations(ARMs)with systemic developmental abnormalitiesin male rat offspringtreated by maternal exposure to di-n-butyl phthalate(DBP)during late pregnancy,investigate the impact on reproductive system and development of male rat offspring exposed to DBP.Methods: Hypospadias and ARMs rat models induced by DBP were established.The body weight(BW),the anogenital distance(AGD)and the organ/BW ratios of the important organs(kidney,lung,spleen,heart and liver)were measured and the incidence of hypospadias and ARMswas evaluated.The toxicityof DBP onpregnant rats exposed to DBP during late gestation was also evaluated.In hypospadias rat models induced by DBP,Real-time polymerase chain reaction(Real-time PCR)was used to quantify m RNA levels of sonic hedgehog(Shh),fibroblast growth factor 8(Fgf8),bone morphogenetic protein 4(Bmp4),fibroblast growth factor 10(Fgf10)and fibroblast growth factor receptor(Fgfr2)in the genital tubercle(GT)of control,undeformed and hypospadias male rats.The m RNA expressions of cytochrome P450,family 11,subfamily A,polypeptide 1(Cyp11a1),3?-hydroxysteroid dehydrogenase(Hsd3b),scavenger receptorclass B-1(Scarb1),steroidogenic acute regulatory protein(Star),steroid-5-alpha-reductase,steroid 5 alpha-reductase 2(Srd5a2)and androgen receptor(AR)in the testes of control,undeformed and hypospadias male rats were detected by Real-time PCR.We examined the m RNA expression profiles oftwo groups by Affymetrix Rat 230 2.0 Array: control untreated male ratsand hypospadias male rats induced by DBP.We selected 10 key genes from the differentially expressed genes(DEGs)for furtherquantitative real-time PCR(q PCR)verification.Five persistently upregulated and five persistently downregulated DEGs showed results consistent with the microarray data.In ARMs rat models induced by DBP,real-time PCR was used to quantify m RNA levels of Shh,Bmp4,Fgf8,Fgf10 and Fgfr2 in the TR of control and ARMs rats.We examined the m RNA and protein expression of AR,Fgf10 and Fgfr2 by q PCR,Western blot and immunohistochemistry(IHC),not only in terminal rectum(TR),but also in heart,liver,spleen,lung and kidney.Results:(1)Repeatable and higher incidencehypospadiasand ARMs rat models were established treated by maternal exposure to DBP during late pregnancy.The incidence of hypospadiasand ARMs in male offspring was 42.1% and 39.5%,respectively.The number of live pups in the DBP-treatment group was significantly lower compared with controls.Thematernal BW gain in the DBPtreatment group was significantly decreased.Inaddition,the gestation period of the pregnant rats exposed to DBP was clearly delayed.BW and relative AGD weregreatly decreased in hypospadias and ARM male rats compared to controls.The organ/BW ratios of the important organs(kidney,lung,spleen,heart and liver)in hypospadiasand ARMsrats were also greatly decreased as compared to DBP-untreated controls.The urethra opening at the head of the external genitalia in normal male rat,and the urethra opening ventrally at the base of the external genitaliain hpospadiac male rat,were observed by histological analysis.The serum testosterone concentration was significantly decreased inhypospadias and ARMs male rats as compared to controls.(2)In hypospadias rat models induced by DBP,the m RNA expression levels of ARand Srd5a2 in testes of undeformed rat pups were similar to those in controls.In contrast,both undeformed and hypospadiac rats had reduced m RNA expression of Cyp11a1,Hsd3 b,Scarb1 and Star in the testes,and ablated m RNA expression of Shh,Bmp4,Fgf8,Fgf10 and Fgfr2 in the GT as compared to those in DBP-unexposed controls.We examined the m RNA expression profiles of two groups by Affymetrix Rat 230 2.0 Array: control untreated male rats and hypospadias male rats induced by DBP.We identified 163 DEGs among the two groups,including 57 upregulated genesand 106 downregulated genes.We selected 10 key genes from the DEGs for further q PCR verification.Five persistently upregulated and five persistently downregulated DEGs showed results consistent with the microarray data.Gene Ontology(GO)pathway analyses revealed that the most DEGs were significantly enriched in embryonicdevelopment or organ morphogenesis or hormone metabolism or cell junction or cell proliferation or calcium ion metabolism.Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analyses revealed that the most DEGs were significantly enriched in metabolic or cell-cell junction or apoptosis.(3)In ARMs rat models induced by DBP,with the reduced expression of AR,the m RNA and protein expressions of Fgf10/Fgfr2 were significantly decreased.ARMs rats had reduced m RNA expression of Shh,Bmp4,Fgf8,Fgf10 and Fgfr2 in the TR as compared to those in DBP-unexposed controls.(4)The reduced expression of Fgf10/Fgfr2 m RNA and protein was seen in kidney,spleen,liver,heart in ARM male rats,whereas the reduced expression of AR was only observed in the kidney.Conclusion: DBP not only may inducehypospadias and ARMs in male rat offspring,but also hasan impact on pregnant rats.The expressions of some genes in the GT of hypospadias rats were similar to that in the TR of ARMs rats,which suggests us that DBP may disturb the development of embryonic cloaca and cause the occurrence of combined ARMs and hypospadias.Relatively normal levels of AR and Srd5a2 genes in genital tubercle of undeformed male rats may help us to find valuable clues to relieve the toxicity of DBP.We propose that DBP suppresses androgen synthesis and androgen-related signaling pathways to disturb the development of embryonic cloaca.DBP may also induce deformity byimpact on cell-cell junction,metabolism and apoptosis.Except androgen-related signaling pathways,there shouldbe another mechanism in the DBP induced systemic developmental abnormalities.The toxicity of DBP on reproductive system and development is complicated and not elucidatedcompletely.Further study on the mechanism of hypospadiasand ARMs may provide effective prevention to the deformity and valuable clues to reduce the incidence of hypospadiasand ARMs. |
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