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標題: | 類3號DNA甲基轉移酶 (Dnmt3L) 對小鼠精原幹細胞功能的影響 The role of Dnmt3L in maintaining the property of spermatogonial stem cells during mouse germ cell development |
作者: | Wendy Shou-Cheng Chen 陳守真 |
指導教授: | 林劭品(Shau-Ping Lin),吳信志(Shinn-Chih Wu) |
關鍵字: | 類3號DNA甲基轉移酶,精原幹細胞,小鼠,後生遺傳,組蛋白修飾,睪丸移植, Dnmt3L,spermatogonial stem cells,mice,epigenetics,histone modification,testis transplantation, |
出版年 : | 2011 |
學位: | 碩士 |
摘要: | 基因表現的後生遺傳調控是維持細胞正常功能之重要因素。部分體內之幹細胞被維持在一種「不活躍」的「休眠」狀態,並等待其適合自我複製或向下分化的時機。類3號DNA甲基轉移酶 (Dnmt3L) 是一個表現在哺乳動物生殖細胞發育過程中不可或缺的後生遺傳調控者,缺少此基因會對內源反轉錄病毒反轉錄子序列造成過低的DNA甲基化的情形,造成進入減數分裂之生殖細胞的凋亡,並隨著小鼠年齡增長,逐漸造成完全喪失生殖細胞之性狀(Sertoli cell-only syndrome),無法繁殖後代。然而Dnmt3L對於精原幹細胞功能之影響目前尚不清楚,因此本研究針對精原幹細胞發育之時期探討Dnmt3L對於維持精原幹細胞之功能之影響。
第一部分之研究顯示Dnmt3L對精原幹細胞之休眠狀態之建立或維持有重要的影響。此結果主要從Dnmt3L-/-之初生幼鼠表達Plzf的細胞數減少與兩個異染色質標記H3K9me3和H4K20me3之環狀染色特徵減少結果得知。另經過生殖細胞移植之功能性測試實驗發現Dnmt3L-/-之生殖細胞無法至宿主之睪丸中增生與分化。因此,此部分試驗顯示出Dnmt3L除了對DNA甲基化很重要外,也參與在異染色質之建立與/或維持之過程中。第二部分試驗顯示缺乏Dnmt3L至少會間接造成支持精原幹細胞正常功能之微環境受到影響。從VASA與STRA8之染色中,看見異常的生殖細胞與微環境細胞分離結果顯示,在Dnmt3L-/-五週大之小鼠睪丸中,精原幹細胞與其體細胞微環境 (niche) 間之交互作用出現問題。另外此部分之功能性測試的預先試驗顯示Dnmt3L-/-之睪丸無法支持野生型之精原細胞重新在生精細管中增生與分化。此試驗結果意味著Dnmt3L不僅在生殖細胞中佔有重要的角色,並可能對於支持精原幹細胞正常功能之微環境亦有其影響。 綜合以上所述,此研究利用了精原幹細胞的模式將Dnmt3L之角色從調控DNA甲基化聯結至影響組蛋白修飾之功能上,並且在功能性測試試驗中顯示Dnmt3L不僅是維持精原幹細胞功能之重要因子,而且也可能直接或間接性影響精原幹細胞之微環境。 Epigenetic regulation of gene activities is crucial for proper cellular function. Some of the stem cells in vivo are kept in a quiescent status, waiting for their turn to proliferate or differentiate. DNA methyl- transferase 3-like (Dnmt3L) is a very important epigenetic regulator expressed during germ line development. Deficiency of Dnmt3L in male germ cells causes severe hypomethylation in retrotransposons. This leads to germ cell depletion after the pachytene stage and eventually sertoli-cell only syndrome in mice. However, how Dnmt3L affects the function of spermatogonial progenitor/stem cells (SSCs) is not well understood. This study focuses on the developmental stages of SSCs in mice, and investigates the role of Dnmt3L in maintaining SSCs property. The first part of the study showed that Dnmt3L was essential for the establishment and/or maintenance of the quiescent status of SSCs. It was concluded from the reduced Plzf positive cells and perinuclear staining pattern of two heterochromatin markers H3K9me3 and H4K20me3 in Dnmt3L-/- neonatal mice testes. Our preliminary functional transplantation study also demonstrated that Dnmt3L-/- spermatogonia could not colonize the wild-type host testes. Taken together, the first part of the study has demonstrated a new role of Dnmt3L on keeping the quiescent status of SSCs. Also, Dnmt3L seems to be an epigenetic regulator not only important for DNA methylation, but also involved in proper heterochromatin establishment and/or maintenance in SSCs. The second part of this study showed that deficiency of Dnmt3L may affect the niche environment essential for sustain in the normal function of SSCs. Based on Vasa and Stra8 staining patterns, we’ve observed an abnormal germ cell-niche/somatic cell segregation, the niche structure that was supposed to hold the spermatogonia seemed to be disrupted in Dnmt3L-/- 5 wks-old testes. Preliminary functional transplantation studies also demonstrated that Dnmt3L-/- testes failed to support the colonization repopulation of wild-type spermatogonia. The second part of our study gives a hint that Dnmt3L is not only essential for germ cell function, but may also affect normal niche environment that supports SSCs. In conclusion, this study has used SSCs as a model to connect the epigenetic modifier Dnmt3L from DNA methylation to histone modification. Also, functional analysis and molecular staining demonstrate that Dnmt3L is not only necessary for normal SSC function, but may also directly or indirectly maintain the niche environment of SSCs. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/10287 |
全文授權: | 同意授權(全球公開) |
顯示於系所單位: | 動物科學技術學系 |
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