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研究生:林靜怡
研究生(外文):Ching-YiLin
論文名稱:在缺氧情況下精子生成相關基因的轉錄調控
論文名稱(外文):Transcriptional regulation of spermatogenesis-related gene by hypoxia
指導教授:郭余民郭余民引用關係
指導教授(外文):Yu-Min Kuo
學位類別:碩士
校院名稱:國立成功大學
系所名稱:細胞生物及解剖學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:40
中文關鍵詞:精子發育缺氧缺氧誘導因子Miwi2
外文關鍵詞:SpermatogenesishypoxiaHIFMiwi2
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精子發育起源於睪丸中的曲細精管,過程中必須消耗大量氧氣。目前已知曲細精管中所需要的氧氣只能藉由擴散作用獲得,因此造精過程是處在低氧環境中;但對於精子發育如何適應在低氧環境的機制仍尚未清楚。缺氧誘導因子-1 (HIF-1)是受到氧氣含量調控的重要轉錄因子,並且已知廣泛的表現在睪丸當中。利用生物資訊的方法分析,我們得知在人類以及老鼠的睾丸中,Miwi2 基因之表現有可能會受到HIF-1的調控。HIF-1可能藉由與Miwi2 啟動子上的缺氧反應元件(hypoxia response element)序列結合進而調控Miwi2之表現。免疫組織化學染色的結果顯示HIF-1主要表現在小鼠的初級精母細胞、次級精母細胞、精細胞以及精蟲,而Miwi2 則表現在初級以及次級精母細胞。藉由細胞缺氧實驗,我們發現在缺氧環境下Miwi2的表現量會上升,但在抑制HIF-1表現後會減弱Miwi2的表現;相反的,在常氧環境下過度表現HIF-1時則會增加Miwi2的表現量。此外,藉由染色質免疫沉澱分析結果得知HIF-1能與Miwi2基因啟動子缺氧反應元件結合,進而增加Miwi2的表現。綜合我們的實驗結果證實在缺氧的情況下,Miwi2會受到HIF-1的正向調控。由於在生殖細胞中,Miwi2 能調控小型干擾核糖核酸透過甲基化作用而抑制反轉錄跳躍子(retrotransposon)的表現。因此我們推測,在缺氧的環境下,Miwi2的表現量上升為維持正常造精功能的重要機制。
Spermatogenesis occurs in the seminiferous tubule, and this complex process requires considerable oxygen consumption. It is known that oxygen reaches to the luminal compartment of tubules only by diffusion, which results in very low oxygen concentration within adluminal compartment. To date, the mechanism through which low oxygen environment regulates spermatogenesis remains largely unknown. Hypoxia-inducible factor-1α (HIF-1α) is one of the most important factors that response to the change of oxygen homeostasis; and it has been shown that HIF-1α is constitutively expressed in testis. Using bioinformatics search, we have identified that HIF-1α might regulate Miwi2 expression via binding to the hypoxia response element (HRE) in both mouse and human testes. HIF-1α immunopositive reaction was detected in spermatogonia, spermatocyte, spermatid and spermatozoa of mouse seminiferous tubules; while Miwi2 immunopositive reaction was detected in spermatogonia and spermatocyte. Miwi2 was found to be significantly up-regulated by hypoxia in mouse germ cell lines. Silencing of HIF-1 attenuated the Miwi2 expression under hypoxia and overexpression of HIF-1 increased Miwi2 expression under normoxia. Additionally, cross-linked chromatin of Miwi2 HRE region could be amplified in HIF-1 but not in IgG immunoprecipitated samples. These results suggest that the regulation of Miwi2 expression by hypoxia is mediated by HIF-1. Given that Miwi2 has been shown to be essential for piRNA-mediated retrotransposon silencing via methylation in male germline, the upregulation of Miwi2 by hypoxia demonstrated in our study suggests an important mechanism essential for normal male germline development.
第一章 緒論.......................................................................................1
1-1缺氧誘導因子 (Hypoxia-inducible factor)....................................1
1-2 HIF-1α之結構.................................................................................2
1-3 HIF-1α後轉譯機制.........................................................................3
1-4精子發育與 HIF-1α........................................................................4
1-5賀爾蒙調控與HIF-1α.....................................................................5
1-6假說與研究目標...............................................................................5

第二章 材料與方法..............................................................................7
2-1搜尋HIF-1之標的基因.................................................................7
2-2免疫組織化學染色..........................................................................7
2-3細胞株與細胞養..............................................................................8
2-4細胞缺氧培養..................................................................................9
2-5 RNA萃取及定量聚合酶連鎖反應.................................................9
2-6西方墨點法.....................................................................................11
2-7小片段干擾RNA (siRNA) ............................................................12
2-8 HIF-1α質體轉染...........................................................................12
2-9染色質免疫沉澱.............................................................................13
2-10統計分析......................................................................................14

第三章 結果.........................................................................................15
3-1搜尋在人類及小鼠睾丸中可能被HIF-1調控之標的基因........15
3-2缺氧環境誘導Miwi2 表現量增加...............................................15
3-3 HIF-1α與Miwi2共同表現於初級以及次級精母細胞.................................................................................16
3-4 Miwi2在缺氧環境下透過缺氧誘導因子-1α調控促使表現量增加.............................................................................17
3-5在缺氧中HIF-1α直接與Miwi2 啟動子區域的HRE 序列結合進而促進Miwi2的表現......................................17

第四章 討論..........................................................................................28
第五章 參考資料..................................................................................34
第六章 附錄..........................................................................................39





表目錄
表一. 以生物資訊方法搜尋HIF-1標的基因....................................19






圖目錄
圖一. 搜尋在造精過程中可能被HIF-1調控之基因之流程圖20
圖二. 缺氧培養對HIF-1標的基因表現量之影響21
圖三. HIF-1α以及Miwi2在成年小鼠睪丸之表現23
圖四. HIF-1α與Miwi2在小鼠曲精細管12週期之表現情形24
圖五. 在常氧環境下HIF-1α過度表現對Miwi2 mRNA表現量
之影響25
圖六. 在缺氧環境下抑制HIF-1α對Miwi2表現量之影響26
圖七. HIF-1α與Miwi2的HRE序列在常氧及缺氧中的交互作用27


Aravin, A. A., and Bourc'his, D. (2008). Small RNA guides for de novo DNA methylation in mammalian germ cells. Genes & Development 22, 970-975.

Aravin, A. A., van der Heijden, G. W., Castaneda, J., Vagin, V. V., Hannon, G. J., and Bortvin, A. (2009). Cytoplasmic compartmentalization of the fetal piRNA pathway in mice. PLoS Genetics 5, e1000764.

Belancio, V. P., Deininger, P. L., and Roy-Engel, A. M. (2009). LINE dancing in the human genome: transposable elements and disease. Genome Medicine 1, 97.

Bourc'his, D., and Bestor, T. H. (2004). Meiotic catastrophe and retrotransposon reactivation in male germ cells lacking Dnmt3L. Nature 431, 96-99.

Carmell, M. A., Girard, A., van de Kant, H. J., Bourc'his, D., Bestor, T. H., de Rooij, D. G., and Hannon, G. J. (2007). MIWI2 is essential for spermatogenesis and repression of transposons in the mouse male germline. Developmental Cell 12, 503-514.

Dang, C. V., and Semenza, G. L. (1999). Oncogenic alterations of metabolism. Trends in Biochemical Sciences 24, 68-72.

Gat, Y., Gornish, M., Perlow, A., Chakraborty, J., Levinger, U., Ben-Shlomo, I., and Pasqualotto, F. (2010). Azoospermia and Sertoli-cell-only syndrome: hypoxia in the sperm production site due to impairment in venous drainage of male reproductive system. Andrologia 42, 314-321.

Ghildiyal, M., and Zamore, P. D. (2009). Small silencing RNAs: an expanding universe. Nature Reviews Genetics 10, 94-108.

Grivna, S. T., Pyhtila, B., and Lin, H. (2006). MIWI associates with translational machinery and PIWI-interacting RNAs (piRNAs) in regulating spermatogenesis. Proceedings of the National Academy of Sciences of the United States of America 103, 13415-13420.

Hwang, G. S., Chen, S. T., Chen, T. J., and Wang, S. W. (2009). Effects of hypoxia on testosterone release in rat Leydig cells. Am J Physiol Endocrinol Metab 297, E1039-1045.

Hwang, G. S., Wang, S. W., Tseng, W. M., Yu, C. H., and Wang, P. S. (2007). Effect of hypoxia on the release of vascular endothelial growth factor and testosterone in mouse TM3 Leydig cells. Am J Physiol Endocrinol Metab 292, E1763-1769.

Iyer, N. V., Kotch, L. E., Agani, F., Leung, S. W., Laughner, E., Wenger, R. H., Gassmann, M., Gearhart, J. D., Lawler, A. M., Yu, A. Y., and Semenza, G. L. (1998). Cellular and developmental control of O2 homeostasis by hypoxia-inducible factor 1 alpha. Genes & Development 12, 149-162.

Jiang, B. H., Rue, E., Wang, G. L., Roe, R., and Semenza, G. L. (1996). Dimerization, DNA binding, and transactivation properties of hypoxia-inducible factor 1. Journal of Biological Chemistry 271, 17771-17778.

Ke, Q., and Costa, M. (2006). Hypoxia-inducible factor-1 (HIF-1). Molecular Pharmacology 70, 1469-1480.

Koh, M. Y., Darnay, B. G., and Powis, G. (2008). Hypoxia-associated factor, a novel E3-ubiquitin ligase, binds and ubiquitinates hypoxia-inducible factor 1alpha, leading to its oxygen-independent degradation. Molecular and Cellular Biology 28, 7081-7095.

Kotaja, N., and Sassone-Corsi, P. (2007). The chromatoid body: a germ-cell-specific RNA-processing centre. Nature Reviews Molecular Cell Biology 8, 85-90.

Kotch, L. E., Iyer, N. V., Laughner, E., and Semenza, G. L. (1999). Defective vascularization of HIF-1alpha-null embryos is not associated with VEGF deficiency but with mesenchymal cell death. Developmental Biology 209, 254-267.

Kuramochi-Miyagawa, S., Kimura, T., Yomogida, K., Kuroiwa, A., Tadokoro, Y., Fujita, Y., Sato, M., Matsuda, Y., and Nakano, T. (2001). Two mouse piwi-related genes: miwi and mili. Mechanisms of Development 108, 121-133.

Kuramochi-Miyagawa, S., Watanabe, T., Gotoh, K., Takamatsu, K., Chuma, S., Kojima-Kita, K., Shiromoto, Y., Asada, N., Toyoda, A., Fujiyama, A., et al. (2010). MVH in piRNA processing and gene silencing of retrotransposons. Genes & Development 24, 887-892.

Kuramochi-Miyagawa, S., Watanabe, T., Gotoh, K., Totoki, Y., Toyoda, A., Ikawa, M., Asada, N., Kojima, K., Yamaguchi, Y., Ijiri, T. W., et al. (2008). DNA methylation of retrotransposon genes is regulated by Piwi family members MILI and MIWI2 in murine fetal testes. Genes & Development 22, 908-917.

Leatherman, J. L., and Jongens, T. A. (2003). Transcriptional silencing and translational control: key features of early germline development. BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology 25, 326-335.

Liao, W., Cai, M., Chen, J., Huang, J., Liu, F., Jiang, C., and Gao, Y. (2010). Hypobaric hypoxia causes deleterious effects on spermatogenesis in rats. Reproduction 139, 1031-1038.

Lysiak, J. J., Kirby, J. L., Tremblay, J. J., Woodson, R. I., Reardon, M. A., Palmer, L. A., and Turner, T. T. (2009). Hypoxia-inducible factor-1alpha is constitutively expressed in murine Leydig cells and regulates 3beta-hydroxysteroid dehydrogenase type 1 promoter activity. Journal of Andrology 30, 146-156.

Lysiak, J. J., Nguyen, Q. A., and Turner, T. T. (2000). Fluctuations in rat testicular interstitial oxygen tensions are linked to testicular vasomotion: persistence after repair of torsion. Biology of Reproduction 63, 1383-1389.

Marti, H. H. (2002). Isoform-Specific Expression of Hypoxia-Inducible Factor-1 During the Late Stages of Mouse Spermiogenesis. Molecular Endocrinology 16, 234-243.

Maxwell, P., and Salnikow, K. (2004). HIF-1: an oxygen and metal responsive transcription factor. Cancer Biology & Therapy 3, 29-35.

Meikar, O., Da Ros, M., Korhonen, H., and Kotaja, N. (2011). Chromatoid body and small RNAs in male germ cells. Reproduction 142, 195-209.

O'Donnell, K. A., and Boeke, J. D. (2007). Mighty Piwis defend the germline against genome intruders. Cell 129, 37-44.

Paul, C., Teng, S., and Saunders, P. T. K. (2009). A Single, Mild, Transient Scrotal Heat Stress Causes Hypoxia and Oxidative Stress in Mouse Testes, Which Induces Germ Cell Death. Biology of Reproduction 80, 913-919.

Pillai, R. S., and Chuma, S. (2012). piRNAs and their involvement in male germline development in mice. Development, Growth & Differentiation.

Ryan, H. E., Lo, J., and Johnson, R. S. (1998). HIF-1 alpha is required for solid tumor formation and embryonic vascularization. The EMBO Journal 17, 3005-3015.

Salceda, S., and Caro, J. (1997). Hypoxia-inducible factor 1alpha (HIF-1alpha) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. Its stabilization by hypoxia depends on redox-induced changes. Journal of Biological Chemistry 272, 22642-22647.

Seagroves, T. N., Ryan, H. E., Lu, H., Wouters, B. G., Knapp, M., Thibault, P., Laderoute, K., and Johnson, R. S. (2001). Transcription factor HIF-1 is a necessary mediator of the pasteur effect in mammalian cells. Molecular and Cellular Biology 21, 3436-3444.

Semenza, G. L. (2012). Hypoxia-inducible factors in physiology and medicine. Cell 148, 399-408.

Sheth, U., and Parker, R. (2003). Decapping and decay of messenger RNA occur in cytoplasmic processing bodies. Science 300, 805-808.

Shoji, M., Tanaka, T., Hosokawa, M., Reuter, M., Stark, A., Kato, Y., Kondoh, G., Okawa, K., Chujo, T., Suzuki, T., et al. (2009). The TDRD9-MIWI2 complex is essential for piRNA-mediated retrotransposon silencing in the mouse male germline. Developmental Cell 17, 775-787.

Siomi, M. C., Mannen, T., and Siomi, H. (2010). How does the royal family of Tudor rule the PIWI-interacting RNA pathway? Genes & Development 24, 636-646.

Siomi, M. C., Sato, K., Pezic, D., and Aravin, A. A. (2011). PIWI-interacting small RNAs: the vanguard of genome defence. Nature Reviews Molecular Cell Biology 12, 246-258.

Vagin, V. V., Sigova, A., Li, C., Seitz, H., Gvozdev, V., and Zamore, P. D. (2006). A distinct small RNA pathway silences selfish genetic elements in the germline. Science 313, 320-324.

Volm, M., and Koomagi, R. (2000). Hypoxia-inducible factor (HIF-1) and its relationship to apoptosis and proliferation in lung cancer. Anticancer Research 20, 1527-1533.

Welch, J. E., Brown, P. L., O'Brien, D. A., Magyar, P. L., Bunch, D. O., Mori, C., and Eddy, E. M. (2000). Human glyceraldehyde 3-phosphate dehydrogenase-2 gene is expressed specifically in spermatogenic cells. Journal of Andrology 21, 328-338.

Wenger, R. H., and Katschinski, D. M. (2005). The hypoxic testis and post-meiotic expression of PAS domain proteins. Seminars in Cell & Developmental Biology 16, 547-553.

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