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| 研究生： |
汪雅雲 Wang, Ya-yung |
|---|---|
| 論文名稱： |
探討Septin12基因於精細胞末期分化之角色 The role of Septin12 in terminal differentiation of male germ cells |
| 指導教授： |
湯銘哲
Tang, Ming-Jer 郭保麟 Kuo, Pao-Lin |
| 學位類別： |
碩士 Master |
| 系所名稱： |
醫學院 - 分子醫學研究所 Institute of Molecular Medicine |
| 論文出版年： | 2009 |
| 畢業學年度： | 97 |
| 語文別： | 英文 |
| 論文頁數： | 82 |
| 中文關鍵詞： | γ-tubulin 、Cdc42 、microtubules 、不孕症 、spermiogenesis 、septin12 |
| 外文關鍵詞： | microtubules, γ-tubulin, spermiogenesis, Cdc42, septin12, infertility |
| 相關次數： | 點閱：92 下載：1 |
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不孕症是一個重要的全球性健康議題。近年來，科學家們致力於找出可能與不孕症的產生相關之基因缺陷或是多型性。我們發現了對男性生殖細胞具有表現專一性的基因，Septin12。SEPT家族屬於細胞骨架蛋白且具有GTPase活性，在哺乳動物中目前發現至少有13種Septin，其參與在許多細胞的生理功能，例如細胞質分裂、細胞型態生成、細胞骨架重整以及細胞區室化…等等。Septin具有獨特特性，可形成絲狀結構物質，精確地調控Septin聚合對於其行使正常生理功能是十分重要的。已有研究指出Septin聚合過程是受到Cdc42的負向調控。而Septin所形成的絲狀物質會和細胞骨架進行交互作用，例如：microtubule。目前已知microtubules在精細胞末期分化型態上產生大幅改變過程中扮演重要的角色。然而，在Spermiogenesis過程中Septin12 會表現在精子頭部、頸部以及尾巴。因此我們推測在精原細胞尾部形成之過程中，SEPT12所形成之絲狀物質會影響microtubules的分佈並且此過程是經由Cdc42調控。
首先經由更精細的觀察發現在小鼠生殖細胞末期分化過程中，SEPT12主要表現在減數分裂後的生殖細胞中並參與在多個不同細胞區間之形成，如精子頂體、頸部與精子中節和尾巴。藉由研究Septin12基因剃除小鼠，我們進一步瞭解SEPT12之功能。多數Septin12基因剃除嵌合公鼠為不孕，其睪丸重量明顯下降，組織切片染色後發現精子成熟過程停在round spermatid之階段。於精液分析發現，精子數目、活動力與正常型態精子數目明顯減少。此外，利用螢光免疫染色也發現在人類成熟精子上，SEPT12主要位於精子中節和尾巴交接的環狀結構，型態異常的精子其SEPT12表現量有明顯下降的情形，此結果在精子無力症患者更為明顯。接著在探討SEPT12 與tubulin之關係，首先利用免疫螢光染色以及共同免疫沈澱法證實SEPT12和α, β及γ-tubulin之間有交互作用。藉由觀察Septin12基因剃除嵌合公鼠睪丸切片之免疫螢光染色，發現在SEPT12表現量下降時，α以及β-tubulin失去正常分佈位置，此結果可能導致精原細胞無法形成尾部。更進一步地利用RNAi降低NT2/D1細胞中內生性SEPT12表現量，其細胞螢光染色與睪丸組織切片結果相似，α以及β-tubulin在細胞內分佈受到SEPT12表現量下降影響。總括以上研究結果，我們認為SEPT12與microtubules之間的交互作用於精子末期分化過程中扮演重要的角色。
During the past years, tremendous attempts have been made to identify genetic defects or polymorphisms that are associated with human infertility, which is a major healthy problem worldwide. We have identified a sterility related and male germ-cell specific gene, Septin12. Mammalian septins constitute a family of at least 13 cytoskeletal proteins with GTPase activity, and are involved in many cellular process such as cytokinesis, cellular morphogenesis, vesicle trafficking and cytoskeleton remodeling. SEPTINS can form homo- or heterooligomeric filaments, and the accurate regulation of septin assembly is necessary for physiological functions of septins. Previous studies showed that SEPT organization is negatively regulated by Cdc42. septin filaments can form network with microtubules, which are actively involved in tail formation and other morphologic changes during terminal differentiation of male germ cells. Considering SEPT12 is expressed at the sperm neck and tail during spermiogenesis, we proposed that SEPT12 filaments formation affects microtubules organization during sperm elongation and this process is regulated by CDC42.
Through more detailed observation, we found that SEPT12 is involved in the formation of multiple subcellular compartments, such as acrosome, neck, middle piece and tail, during terminal differentiation. We also generated Septin12 knock-out mice. Most of the chimeric mice were infertile and the testicular phenotypes appeared maturation arrest at the round spermatid stage. Semen analysis of the chimeras showed the decrease of sperm counts and motility, and defects involving all subcellular compartments. In human, SEPT12 signals were significantly decreased in sperm with abnormal morphology. We tested the effect of CDC42 on SEPT12 filament formation, and found that the filament formation of septin12 was negatively regulated by Cdc42. We also evaluated the relation between SEPT12 and microtubules by immunofluorescence staining (IFA) and co-immunoprecipitation. Our results support the interaction between SEPT12 and α-, β- or γ-tubulin. We also found microtubules disorganization which may lead to maturation arrest in the testicular sections of Septin12+/- chimeras. We further verified the effect of SEPT12 on the microtubule organization. RNAi was used to deplete the expression of endogenous SEPT12 in NT2/D1 cells. The organizations of α- and β-tubulin of setpin12-depleted cells were altered. Our results inferred that the cooperation of SEPT12 and tubulin is critical during mammalian sperm elongation.
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校內：2013-07-09公開校外：2013-07-31公開