進階搜尋


 
系統識別號 U0026-0812200915180244
論文名稱(中文) 探討SEPTIN 12在男性生殖細胞的蛋白結構複合物
論文名稱(英文) Identify the SEPTIN12 complex in spermatozoa
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系碩博士班
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 97
學期 2
出版年 98
研究生(中文) 林芳瑜
研究生(英文) Fang-Yu Lin
電子信箱 t3695104@mail.ncku.edu.tw
學號 t3695104
學位類別 碩士
語文別 中文
論文頁數 51頁
口試委員 口試委員-林翰佳
口試委員-陳逸然
指導教授-郭保麟
口試委員-張權發
口試委員-傅子芳
中文關鍵字 SEPTIN7  SEPTIN6  SEPTIN12  SEPTIN4  男性生殖細胞 
英文關鍵字 spermatozoa  SEPTIN7  SEPTIN6  SEPTIN12  SEPTIN4 
學科別分類
中文摘要 SEPTIN(SEPT)家族是細胞骨架蛋白的一種,具有GTPase的活性。目前研究發現,SEPT會參與細胞核分裂(nuclear division)、細胞膜運輸(membrane trafficking)以及細胞骨架的組織(organizing the cytoskeleton)。在本實驗室的研究中發現,SEPTIN12是專一表現於男性生殖細胞的結構蛋白,且在男性不孕睪丸檢體中發現,septin12 mRNA表現量有明顯下降。此外,並發現SEPT12表現於sperm頸部的annulus上。目前,在已建立的Septin12 +/- chimera mice中,大部分的chimera mice都不孕。在觀察chimera mice精子後發現,其有嚴重的結構缺陷,如彎曲狀的尾巴(bent tail)、斷尾(broken tail)及頭部呈圓型(round head)。在電子顯微鏡下也觀察到粒線體有缺陷(mitochondrial defect)和頂體缺失(acrosomal defect)。Masafumi Ihara和其研究室成員建立Septin4基因剔除小鼠系統,發現SEPT1/4/6/7會形成sperm annulus的主要結構基礎。而annulus的結構可以分界精子的中節和尾巴。從觀察Septin12 +/- chimera mice的實驗結果,我們能推論,Septin12也許是精子annulus上SEPTs蛋白結構複合物的主要核心蛋白。因此,本篇研究的主要目的是探討成熟精子上SEPTIN12蛋白結構複合物的蛋白組成物。首先,我們分別構築了Septin12、Septin6和Septin7的載體(vector),並將這些載體個別表現到NT2D1細胞上。免疫螢光染色實驗結果發現SEPT6會與SEPT12表現於相同位置上。而SEPT7也會與SEPT12表現於相同位置上。使用免疫沉澱法發現SEPT6會與SEPT12相互作用形成蛋白結構複合物,但是SEPT7不會與SEPT12相互作用。在小鼠睪丸組織萃取物上,以免疫沉澱法發現SEPT4和SEPT6可以個別和SEPT12相互作用。最後,觀察在Septin12高剔除率Septin12 +/- chimeric mice上,發現隨著Septin12剔除率越來越高,SEPT4和SEPT6表現量明顯下降。我們發現在Septin12 +/- chimeric mice的精子SEPT7染色上,原本位於annulus上的SEPT7消失了。由上述結果推測SEPT4、SEPT6和SEPT7也許是形成這SEPT12為核心之蛋白結構複合物的組成蛋白。
英文摘要 SEPTINs are a family of cytoskeletal proteins with GTPase activity. SEPTINs have been implicated in various cellular functions, including intercellular filaments scaffolds, cytokinasis, cellular morphogenesis, neural polarity and vesicle trafficking. Our lab has found SEPTIN12 as a novel germ-cell specific structural protein. Septin 12 transcript amount was significantly decreased in the testicular samples of men with spermatogenic defect. We also found septin12 was localized at the sperm annulus. To investigate the role of septin 12 in mammalian spermatogenesis, we generated septin 12 +/- chimera mice. Most chimera mice were infertile and only some subfertile mice fathered wild-type mice. All chimera mice showed sperm with severe structural defects, including bent tail, broken tail and round head. Electron microscopy showed there sperm also had mitochondrial defect and acrosomal defect. Previous studies based on septin 4 KO mice suggested that septin 1/4/6/7 may be structural basis of the annulus, a cortical ring which separates the middle and principal pieces in sperm. The phenotype of our KO mice suggests septin 12 may be a core component of septin complex in the sperm annulus. In this study, we would like to identify the components of septin complex in sperm.
First, we constructed pFLAG-septin12, pcDNA3.1-septin6, pFLAG-septin7 and pEGFP-septin12. After transfecting pEGFP-Septin12 and pcDNA3.1-septin6 vector into NT2D1 cells, SEPT12 can co-localize with SEPT6 by immunofluorescence staining assay (IFA). In immunoprecipitation assay, SEPT12 and SEPT6 can form complex, but SEPT7 not. In the similar method, SEPT12 can co-localize with SEPT7 by IFA. Next, we used mouse testis lysates to investigate the interaction between different septins by co- immunoprecipitation assay and immunofluorescence staining. In our data, SEPT12 can interact with SEPT4/6 in vivo. Finally, we will examine SEPT1, septin 4, septin 6, septin 7 and septin 11 protein levels in the septin 12 +/- chimera mice using Western blot analysis and immunofluorescence staining. The SEPT4 and SEPT6 expression levels are decreased in Septin12 +/- chimeric mice. SEPTIN7 is dis-localized in the sperm annulus of the septin 12 +/- chimera mice by IFA. Our finding infers that SEPT4/6/7 may be the components of SEPTIN12 complex in human sperm.
論文目次 中文摘要 I
ABSTRACT III
誌謝 V
圖目錄 VI
1.緒論 1
1.1 男性不孕症的病理原因 1
1.2 哺乳動物的造精過程(mammalian spermatogenesis) 1
1.3 選定Septin12基因作為主要研究對象 3
1.4 簡介Septin (SEPT)的發現 3
1.5 SEPT的功能 4
1.6 Septin家族演化關係(phylogenetic relationship) 5
1.7 相互作用的SEPT蛋白 5
1.7.1 SEPT2/SEPT6/SEPT7結合形成蛋白結構複合物 5
1.7.2 SEPT7/SEPT9b/SEPT11相互作用形成蛋白結構複合物 6
1.7.3 於活體內(in vivo)的實驗中發現SEPT1/SEPT4/SEPT6/SEPT7表現於相同位置 7
1.7.4 SEPT12可以和SEPT6相互作用 7
1.7.5 降低SEPT表現量影響其他SEPTs的表現量 8
2.研究動機與實驗設計 10
3.材料與方法 12
3.1 細胞培養(Cell Culture) 12
3.2 RNA萃取方法 12
3.3 Reverse transcription 13
3.4 質體DNA 的製備 13
3.5 質體的轉染 15
3.6 萃取細胞的總蛋白質 16
3.7 西方墨點法 (Western blot) 16
3.8 免疫螢光染色法(immunofluorescence assay,IFA)實驗 18
3.9 免疫沉澱法(immunoprecipitation assay,IP) 19
4.結果 21
4.1 在NT2D1(人類胚胎腫瘤細胞human embryonal carcinoma cells)細胞模式上,探討SEPT4、SEPT6和SEPT7是否會與SEPT12相互作用,形成蛋白結構複合物。 21
4.1.1 在NT2D1細胞模式上,SEPT6會與SEPT12表現於相同位置上。 21
4.1.2 在NT2D1細胞模式上,SEPT6會與SEPT12相互作用形成蛋白結構複合物。 22
4.1.3 在NT2D1細胞模式上,SEPT7會與SEPT12表現於相同位置上。 23
4.1.4 在NT2D1細胞模式上,SEPT7與SEPT12不會相互作用形成蛋白結構複合物。 24
4.2 在小鼠睪丸和成熟精子上,探討SEPT12蛋白結構複合物。 25
4.2.1 在小鼠睪丸組織萃取物上以co-immunoprecipitation assay研究發現SEPT12和SEPT4、SEPT6會相互作用,形成蛋白結構複合物。 25
4.2.2 觀察在Septin12高剔除率Septin12 +/- chimeric mice的其他SEPTs表現量。 26
4.2.3 以免疫螢光染色法觀察SEPT4、SEPT6和SEPT7在成熟精子的表現,並比較表現位置於Septin12 +/- chimeric mice和Wild type mice成熟精子之不同處。 26
5. 結論 28
6. 討論 30
6.1 SEPT12蛋白結構物的結合模式 30
6.2 在小鼠的睪丸組織上,SEPT4的表現型態。 30
6.3 在小鼠的睪丸組織上,SEPT6的表現型態。 31
參考文獻 33
圖附錄 37
自述 51
參考文獻 Brandon E. Kremer, Timothy Haystead, and Ian G. Macara. Mammalian Septins Regulate Microtubule Stability through Interaction with the Microtubule-binding Protein MAP4. Molecular Biology of the Cell 2005; 16: 4648–4659

Christine S. Weirich, Jan P. Erzberger and Yves Barral. The septin family of GTPases: architecture and dynamics. Nature Reviews Molecular Cell Biology 2008; 2407

Christine M. Field and Douglas Kellogg. Septins: cytoskeletal polymers or signaling GTPases? trends in CELL BIOLOGY 1999; 9: 387-394

Claudia Low1 and Ian G. Macara. Structural Analysis of Septin 2, 6, and 7 Complexes. THE JOURNAL OF BIOLOGICAL CHEMISTRY 2006; 281: 30697–30706

D. M. DE KRETSER AND H. W. G. BAKER. Infertility in Men: Recent Advances and Continuing Controversies. The Journal of Clinical Endocrinology & Metabolism 1999; 84: 3443-3450

Fangfang Pan, Russell L Malmberg and Michelle Momany. Analysis of septins across kingdoms reveals orthology and new motifs. BMC Evolutionary Biology 2007;7 :103

Holger Kissel, Maria-Magdalena Georgescu, Sarit Larisch, Katia Manova, Gary R. Hunnicutt and Hermann Steller1. The Sept4 Septin Locus Is Required for Sperm Terminal Differentiation in Mice. Developmental Cell 2005; 8: 353–364

Koh-ichi Nagata, Tomiko Asano, Yoshinori Nozawa , and Masaki Inagaki. Biochemical and Cell Biological Analyses of a Mammalian Septin Complex, Sept7/9b/11. THE JOURNAL OF BIOLOGICAL CHEMISTRY 2004; 279: 55895–55904

Masafumi Ihara, Ayae Kinoshita, Shuichi Yamada, Hiromitsu Tanaka, Ai Tanigaki, Ayumi Kitano, Motohito Goto, Kazutoshi Okubo, Hiroyuki Nishiyama, Osamu Ogawa, Chiaki Takahashi, Shigeyoshi Itohara, Yoshitake Nishimune, Makoto Noda, and Makoto Kinoshita. Cortical Organization by the Septin Cytoskeleton Is Essential for Structural and Mechanical Integrity of Mammalian Spermatozoa. Developmental Cell 2005; 8: 343–352

Makoto Kinoshita, Christine M. Field, Margaret L. Coughlin, Aaron F. Straight, and Timothy J. Mitchison. Self- and Actin-Templated Assembly of Mammalian Septins. Developmental Cell 2002; 3: 791–802

Makoto Kinoshita and Makoto Noda. Roles of Septins in the Mammalian Cytokinesis Machinery. Cell structure and function 2001; 26: 667-670

Minhajuddin Sirajuddin, Marian Farkasovsky, Florian Hauer, Dorothee Kuhlmann, Ian G. Macara, Michael Weyand, Holger Stark & Alfred Wittinghofer. Structural insight into filament formation by mammalian septins. Nature 2007;06052

Nobuhiro Hanaia, Koh-ichi Nagataa, Aie Kawajiria, Takashi Shiromizua, Noriko Saitoha, Yasuhisa Hasegawab, Shingo Murakamic, Masaki Inagakia. Biochemical and cell biological characterization of a mammalian septin, Sept11. FEBS Letters 2004; 568: 83–88

OSATO F. GIWA-OSAGIE. ART in developing countries with particular reference to sub-Saharan Africa. WTO: 22-27

Peter A Hall, Kenneth Jung, Kenneth J Hillan and SE Hilary Russell. Expression profiling the human septin gene family. Journal of Pathology 2005; 206: 269–278

Rebecca Lindsey and Michelle Momany. Septin localization across kingdoms: three themes with variations. Current Opinion in Microbiology 2006; 9: 559-565

Ryoichi Ono, Masafumi Ihara, Hideaki Nakajima, Katsutoshi Ozaki, Yuki Kataoka-Fujiwara, Tomohiko Taki, Koh-ichi Nagata, Masaki Inagaki, Nobuaki Yoshida, Toshio Kitamura, Yasuhide Hayashi, Makoto Kinoshita and Tetsuya Nosaka1. Disruption of Sept6, a Fusion Partner Gene of MLL, Does Not Affect Ontogeny, Leukemogenesis Induced by MLL-SEPT6, or Phenotype Induced by the Loss of Sept4. MOLECULAR AND CELLULAR BIOLOGY 2005; 25: 10965–10978

Xiangming Ding, Wenbo Yu, Ming Liu, Suqin Shen, Fang Chen, Bo Wan and Long Yu. SEPT12 Interacts with SEPT6 and This Interaction Alters the Filament Structure of SEPT6 in Hela Cells. Journal of Biochemistry and Molecular Biology 2007; 40: 973-978
論文全文使用權限
  • 同意授權校內瀏覽/列印電子全文服務,於2010-07-21起公開。
  • 同意授權校外瀏覽/列印電子全文服務,於2010-07-21起公開。


  • 如您有疑問,請聯絡圖書館
    聯絡電話:(06)2757575#65773
    聯絡E-mail:etds@email.ncku.edu.tw