- 學位論文
精蟲體粒線體DNA複製數及DNA甲基化與精蟲活動力之相關性
Association between Sperm Mitochondria DNA Copy Number and Global DNA Methylation on Sperm Motility
摘要
在精蟲發育過程中，成熟精蟲會脫去大部分細胞質裡的物質與胞器，僅保留遺傳物質並停止基因的轉錄與轉譯，另一方面會保留部分提供能量的粒線體排列在精蟲的中段 (midpiece)，多餘的粒線體會被轉移至細胞外，形成殘留體(residual body)。因此，目前研究顯示精蟲細胞裡殘留的粒線體以其 DNA 複製數(mitochondria DNA copy number)越多，暗示著精蟲的活動力較差。DNA 甲基化是細胞調控基因表現的機制之一，並在精蟲生成過程中扮演重要的角色。目前已經證實特定基因的 DNA 甲基化程度升高，會導致男性精蟲生成的缺陷，例如活動能力較差的精蟲，甚至導致不孕症的發生。因此我們想了解，DNA 甲基化與精蟲中粒線體 DNA 複製數以及精蟲活動力的相關性。 在此研究中，我們蒐集了 56 位臨床受試者之精液，透過梯度離心的方式將精液檢體裡的具有活動力精蟲與不具有活動力精蟲分離出來並萃取核酸。利用即時定量聚合酶連鎖反應 (Real-time PCR)，偵測樣本裡精蟲的粒線體 DNA 複製數。本研究以酵素免疫吸附法 (Enzyme-Linked Immunosorbent Assay，ELISA) 定量精蟲全域 DNA 甲基化 (global methylation)的程度，進而比較精蟲活動力、粒線體 DNA 複製數以及 DNA 全域甲基化之相關性。 結果顯示，不具活動力精蟲的粒線體 DNA 複製數 (118.99 ± 274.83)與全域甲基化程度 (13.58 ± 8.73 %) 皆高於具活動力的精蟲(粒線體 DNA 複製數：18.12 ± 44.69，全域甲基化程度：9.98 ± 6.50 %)，且有顯著性 (P < 0.05)。皮爾森相關性分析粒線體 DNA 複製數與全域 DNA 甲基化的相關性，不具有活動力之精蟲的粒線體 DNA 複製數與全域甲基化低度正相關性(R = 0.276，P < 0.05)。 綜合以上結果，我們確認了活動能力較差的精蟲，其粒線體 DNA 複製數的量也較高，並有較高的全域甲基化程度。因此我們認為，精蟲甲基化程度或許會調控粒線體 DNA 複製數的數量，進而影響精蟲的活動能力，詳細的機轉與因果關係仍需要釐清。
並列摘要
Sperm concentration, progressive motility, local motility and normal morphology are common in clinical assessment for sperm quality. The process of spermatogenesis directly effects the maturation and parameter above. During spermatogenesis, spermatids remove cytoplasm and part of mitochondria as residual body, and remains others arrange around sperm midpiece. Recent research suggest that more residual mitochondrial DNA copy number indicate worse sperm quality. DNA methylation is a mechanism to regulate gene expression. Hypermethylation at promotor can silence gene expression downstream. Some research show that, differential of DNA methylation pattern would influence male infertility and offspring heath. In this study, we tried to clarify whether sperm mitochondrial DNA copy number related with global DNA methylation in sperm genome and sperm quality. In this study we collected 56 semen sample from adult male. We used gradient centrifuge method to separate motile and immotile sperm. Then we extracted DNA from each sperm sample and quantified mitochondrial DNA copy number and global DNA methylation by real-time PCR and ELISA methods individually. Our results show that immotile sperm contain more mitochondrial DNA copy number (118.99 ± 274.83) than motile sperm (18.12 ± 44.69) (P < 0.05). Otherwise, immotile sperm perform more global DNA methylation (13.58% ± 8.73) than motile sperm (9.98 ± 6.50) (P < 0.05). Mitochondrial DNA copy number have positive correlation with global methylation. We confirm that higher mitochondrial DNA copy number indicate worse sperm motility. Global methylation shows the same tendency and correlated with mitochondrial DNA copy number. However, the mechanism between is still unknown.