BOLL基因是一個具遺傳保留特性的生殖基因，從低階生物如果蠅到高階生物如人類，BOLL基因都已被證實參與調控精子生成。我們與其他人過去的實驗發現，BOLL的蛋白質可表現在正常睪丸粗絲期精母細胞及圓形精細胞的細胞質，另外BOLL的核糖核酸和蛋白質在造精功能異常的睪丸表現量有下降的情形，加上過去其他實驗證實BOLL基因很少有突變或是多型性變異的發生，因而我們推測造精功能異常跟附基因調控有關。我們過去genome-wide methylation array 和 cDNA microarray 的實驗發現BOLL在精子生成機能低下的睪丸發現有高甲基化的表現，所以我們這篇研究目的在於探討BOLL基因高甲基化與造精功能異常之間的關聯性。
我們先上相關網站鍵入關鍵字找出BOLL基因的CpG island (CGI)，對照我們methylation array的結果，發現methylation array所找到BOLL基因高甲基化表現的區域剛好位在CGI，接著我們挑選造精功能正常的不孕病人以及精子生成功能低下的不孕病人參與接下來的實驗，我們用methylation pyrosequencing去定量出每個CpG的甲基化表現，單個CpG甲基化表現量我們以多少比率甲基化來呈現，代表在這個CpG上有多少比率的胞嘧啶在pyrosequencing過程中被轉為胸線嘧啶，我們用 quantitative real-time RT-PCR去定量出BOLL 的轉錄表現量，用 spermatogenic core去評估造精功能異常的嚴重程度。甲基化比率和轉錄表現量間的關聯性以及甲基化比率和spermatogenic score間的關聯性我們用皮爾遜積差相關係數去分析，最後我們用 luciferase reporter assay的實驗試著去證實BOLL的CGI 啟動子一旦被甲基化會去影響啟動子的活性。
我們實驗結果證實BOLL的轉錄表現量在精子生成功能低下的睪丸比較低。為了方便分析BOLL啟動子所要研究的區域內共三十四個CpGs，我們利用實驗的primer將這區域切分成七個研究區塊，實驗結果發現其中有八個CpGs在精子生成功能低下的病人有高甲基化的表現，又其中有三個CpGs它的甲基化表現量跟轉錄表現量呈現有意義的負相關，七個CpGs的甲基化表現量跟spermatogenic score呈現有意義的負相關。七個區塊我們將它命名為區塊一到七，其中區塊二、區塊三、區塊六以及區塊二加三在精子生成功能低下的病人有高平均甲基化的表現，又其中區塊二的甲基化表現量跟轉錄表現量呈現有意義的負相關，區塊二、區塊三、區塊二加三的甲基化表現量跟spermatogenic score呈現有意義的負相關。在in vitro的luciferase reporter assay實驗，我們發現啟動子冷光活性在被Methyltransferase SssI甲基化後有下降的情形，在被Methyltransferase HhaI甲基化後則是沒有改變。我們的研究發現BOLL基因CGI啟動子的高甲基化會造成BOLL基因表現量的下降，進而造成人類造精功能異常。

BOLL, a highly conserved sterility-related gene, has been shown to play a role in spermatogenesis from flies to humans. Our and other studies demonstrated that BOLL is expressed in the cytoplasm of pachytene spermatocytes and round spermatids in the human testes with normal spermatogenesis, that the expression of BOLL mRNA and protein were decreased in infertile patients with spermatogenic failure and that neither mutation nor polymorphism of BOLL gene was identified in infertile men. Therefore, epigenetic regulation of BOLL might be responsible for the decreased BOLL expression. 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.

In addition to the results from methylation array, the CpG island (CGI) of BOLL gene was predicted in silico, and the predicted CGI contained the hypermethylation site identified from methylation array. Azoospermic patients with normal spermatogenesis and hypospermatogenesis were enrolled. The methylation status of each CpG was determined by pyrosequencing analysis after bisulfate treatment. The degree of each methylation at each CpG position was determined by the ratio of C to T incorporated during pyrosequencing (percentage of DNA methylation). 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. In vitro luciferase reporter assay was used to verify the effects of hypermethylation of CGI promoter on promoter activity.

The mRNA transcript levels of BOLL were significantly lower in patients with hypospermatogenesis. The amplified fragment in the promoter region of the BOLL gene was sequenced by using seven pairs of primers to analyze a total of 34 CpG sites. Of the 34 CpGs, eight showed significantly higher % methylation in hypospermatogenesis group, and significantly inverse correlation was found between CpG % methylation and transcript levels in three GpGs. For individual sequencing segment (segment 1-7), only segment 2, 3, 6 and 2+3 showed significantly higher methylation index (MtI, mean of % methylation at all CpG positions) in hypospermatogenesis, and segment 2 significantly and inversely correlated with transcript levels. Similarly, of the eight CpGs showing significantly higher % methylation in hypospermatogenesis group, significantly inverse correlation was found between CpG % methylation and spermatogenic score in seven GpGs. Of the four segments showing significantly higher MtI in hypospermatogenesis, three segments significantly and inversely correlated with spermatogenic score. In vitro luciferase reporter assay revealed that significantly decreased luciferase activity were noted in methyltransferase SssI treated constructs but not in methyltransferase HhaI treated constructs. 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.

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