BOLL啟動子甲基化失調跟精子生成功能之間的關聯性
林宗彥、鄭裕生
國立成功大學附設醫院泌尿部
The association of dysregulated methylation of BOLL promoter in human spermatogenesis
Tsung-Yen Lin、Yu-Sheng Cheng
Department of Urology, Medical College and Hospital, National Cheng-Kung University, Tainan, Taiwan
Abstract
Purpose: In our previous study, by using genome-wide methylation array and cDNA microarray, we demonstrated the hypermethylation status of BOLL in the testes with hypospermatogenesis compared to the testes with normal spermatogenesis. The aim of this project was set out to explore the association of BOLL gene hypermethylation and spermatogenic failure.
Materials and Methods: The possible regulated region of methylation at the CGI (CpG island) promter of BOLL was predicted based on previous result of methylation array. In vitro luciferase reporter assay was used to verify the effects of hypermethylation of selected CGI promoter on promoter activity. Azoospermic patients with normal spermatogenesis and hypospermatogenesis were enrolled. The methylation status of each CpG was determined by pyrosequencing analysis after bisulfate treatment. The mRNA transcript levels were determined by quantitative real-time RT-PCR. The severity of spermatogenic failure was determined by spermatogenic score. Pearson product moment correlation coefficients were calculated to determine the correlation between the percentage of methylation (% methylation) and transcript level and spermatogenic score.
Results: The segment (-1225 to -626) on CGI promoter was selected and inserted into construct for luciferase survey. In vitro luciferase reporter assay revealed that significantly decreased luciferase activity was noted in methyltransferase HhaII or SssI treated constructs but not in methyltransferase HhaI treated constructs. The mRNA transcript levels of BOLL were significantly lower in patients with hypospermatogenesis. Of the 33 CpGs in selected segment, seven showed significantly higher % methylation in hypospermatogenesis group, and significantly inverse correlation was found between CpG % methylation and transcript levels in six GpGs. Significant inverse correlation was found between CpG % methylation and spermatogenic score in seven CpGs.
Conclusions: Our results, for the first time, demonstrate that hypermethylation of CGI promoter of BOLL gene contributes to one of the causes of low expression of BOLL, which may lead to spermatogenic failure in humans.