Role Of DNA Methylation In The Pathogenesis Of The Dehp-induced Cryptorchidism

OBJECTIVE: To investigate epigenetic changes (specifically, DNA methylation) and its impact on gene expression in cryptorchidism mice induced by maternal exposure to Di-2-(ethylhexyl) phthalate (DEHP).METHODS: Kunming mice (KM mice) were divided into 3 groups randomly with 20 mice in each group: normal control group, corn oil group and DEHP-exposed group. The last two group were administrated respectively with corn oil and DEHP [500 mg/(kg·d)] to the pregnant mice from gestation day 12.5 (GD12.5) to GD18.5, and normal group with nothing. High-performance liquid chromatography (HPLC) was performed to analyze DNA methylation status, genome-wide DNA methylation patterns of CCGG sites were detected by methylation-sensitive amplification polymorphism technique (MSAP), and the status of methylation of CpG islands in INSL3 gene was analyzed using methylation-specific PCR (MSP). Expression levels of INSL3 and the DNA methyltransferases were examined by quantitative real-time PCR (qPCR). RESULTS: DEHP significantly increased DNA methylation levels,and methylated the CpG islands in the first exon of INSL3. The average incidence of DNA methylation was approximately 34.03%±3.05% in DEHP-exposed mice, 28.37%±2.37% in normal control group, and 28.58%±2.45% in corn oil group. Testes were a main target for DEHP as evidenced by a reduction in INSL3 expression. INSL3 mRNA Expression in DEHP-exposed group was about one-third of that in normal group, which was significantly decreased (P <0.05). Similarly, INSL3 protein expression in DEHP-exposed group was about quarter of that in normal group, which was also significantly decreased (P <0.05). DEHP also significantly increased DNA methyltransferases'expression. For DNA methyltransferases, the expression was significantly up-regulated (Dnmt1, P<0.05; Dnmt3a, P<0.01; Dnmt3b, P<0.05).CONCLUSION: Hypermethylation of CpG islands in the first exon of INSL3 is probably responsible for low INSL3 expression in cryptorchid mice. Changes in DNA methylation may play an important role in the manifestation of cryptorchidism caused by environmental factors such as maternal exposure to DEHP, which may be one possible mechanism of DEHP-mediated testicular toxicity.