Wei-na Yuan1, Hong Jiang, Wen-xiang Zhang, Cun-lli Wang. 1. Reproductive Medicine Center, Clinical College of PLA Affiliated to Anhui Medical University, Hefei, Anhui 230031, P.R. China. jiangh105@sina.com.
Abstract
OBJECTIVE: To compare the methylation patterns of imprinting control region (ICR) of H19 gene and differentially methylated region (DMR) of IGF2r gene in mature sperms derived from epididymis of Kunming mice and in vitro cultured haploid spermatids. METHODS: The H19 ICR and IGF2r DMR2 were detected by bisulfite sequencing PCR (BSP). The results were compared with standard sequence derived from GenBank using a DNAman software. RESULTS: 96.67% (15 CpG sites) of H19 ICR was found to be methylated, and 94.29% IGF2r DMR2 was found to be unmethylated in mature sperms. By contrast, 69.33% of H19 ICR and 44.29% of IGF2r DMR2 were found to be methylated in the haploid spermatids cultured in vitro. A significant difference was detected in the methylation patterns between the two types of cells (P<0.01). CONCLUSION: The H19 ICR in mature sperm of Kunming mice was essentially methylated, while the IGF2r DMR2 was essentially unmethylated. Partial methylation loss in H19 ICR and abnormal methylation in IGF2r DMR2 were found in the haploid spermatids cultured in vitro.
OBJECTIVE: To compare the methylation patterns of imprinting control region (ICR) of H19 gene and differentially methylated region (DMR) of IGF2r gene in mature sperms derived from epididymis of Kunming mice and in vitro cultured haploid spermatids. METHODS: The H19 ICR and IGF2r DMR2 were detected by bisulfite sequencing PCR (BSP). The results were compared with standard sequence derived from GenBank using a DNAman software. RESULTS: 96.67% (15 CpG sites) of H19 ICR was found to be methylated, and 94.29% IGF2r DMR2 was found to be unmethylated in mature sperms. By contrast, 69.33% of H19 ICR and 44.29% of IGF2r DMR2 were found to be methylated in the haploid spermatids cultured in vitro. A significant difference was detected in the methylation patterns between the two types of cells (P<0.01). CONCLUSION: The H19 ICR in mature sperm of Kunming mice was essentially methylated, while the IGF2r DMR2 was essentially unmethylated. Partial methylation loss in H19 ICR and abnormal methylation in IGF2r DMR2 were found in the haploid spermatids cultured in vitro.