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研究生： 陳皞因
Chen, Hau-Inh
論文名稱： 探討不孕男性中SEPTIN12的序列變異
Identification of sequence alterations for SEPTIN12 in infertile male
指導教授： 郭保麟
kuo, Pao-Lin
學位類別： 碩士
Master
系所名稱： 醫學院 - 生物化學暨分子生物學研究所
Department of Biochemistry and Molecular Biology
論文出版年： 2010
畢業學年度： 98
語文別： 中文
論文頁數： 64
中文關鍵詞： 不孕 、Septin12 、spermatogenesis 、異型合子核苷酸突變 、GTPase功能區
外文關鍵詞： infertility, Septin12, spermatogenesis, heterozygous mutations, GTPase
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    • SEPTINs家族是屬於有GTPase活性的細胞骨架蛋白，且參與在許多不同的胞內生理功能，例如胞內絲狀骨架維持，細胞質分裂，細胞型態變化，細胞區室化，神經細胞極性建立以及囊泡運輸等。先前的許多研究指出SEPTIN9是高度特異性地表現在神經組織的神經膠細胞中,並且SEPTIN9序列的變異會造成遺傳性的神經痛性肌萎縮症(hereditary neuralgic amyotrophy, HNA)。而在我們之前研究中指出SEPTIN12 (SEPT12)也是高度特異性地表現在睪丸組織中，並且證實SEPT12在造精過程(spermatogenesis)中扮演了重要的角色。藉由研究Septin12基因剔除嵌合公鼠，其睪丸的大小和重量都明顯地下降，而在精液的分析也發現其精子數目以及精子活動力比起正常型態的公鼠減少許多，並且有許多精子都呈現不正常的型態。因此我們假設SEPTIN12 的序列變異可能會造成男性不孕。我們針對160 不孕男性病人的檢體以及200個正常可以生育的男性檢體來做SEPTIN12 基因定序分析，分析的結果發現了來自兩個不同病人其SEPTIN12各攜帶一種異型合子核苷酸突變 (heterozygous nucleotides alterations)，分別為266C>T以及589G>A，266C>T 造成SEPT12的第89個胺基酸由Thr變成Met (T89M)，而589G>A則會造成SEPT12的第197個胺基酸由 Asp變成Asn (D197N)。T89M以及D197N都位於高度保留的 GTPase 功能區 (GTPase domain)，深入分析後，我們發現相較於SEPT12WT，SEPT12T89M以及SEPT12D197N 的蛋白穩定性都明顯地下降，並且利用免疫螢光染色觀察在NT2/D1細胞中SEPT12T89M以及SEPT12D197N都無法形成具功能性的絲狀結構(filament)。而在共同免疫沉澱法也證實了T89M以及D197N會影響 SEPT12以及其他SEPTINs之間的蛋白質結合，且確認了SEPT12T89M以及SEPT12D197N與SEPT7之間的蛋白結合作用喪失。進一步再將SEPT12T89M及SEPT12D197N分別與SEPT7同時在NT2/D1細胞中表現，免疫螢光染色觀察發現 SEPT12T89M及SEPT12D197N仍然無法形成絲狀物質，並且無法與SEPT7共同分佈在同一個位置上，結果與共同免疫沉澱法具一致性。我們的研究結果都指出了SEPT12T89M及SEPT12D197N這兩個高度保留性的胺基酸突變確實影響了SEPT12在細胞內的正常生理功能並對於未來的臨床研究提供相當重要的意義。

    Septins are a family of cytoskeletal proteins with GTPase activity which have been implicated in intracellular filaments scaffolds, cytokinesis, cellular morphogenesis, neural polarity and vesicle trafficking. Previous studies have suggested that Septin9 is highly expressed in glia cells in neuronal tissues and sequence alterations in SEPTIN9 are known to cause hereditary neuralgic amyotrophy (HNA). Our previous studies indicated that Septin12 is a tissue-specific gene expressed in testis, and it is critical for spermatogenesis. In Septin12+/- chimera mice, the testis is smaller and the sperm count and motility are significantly lower than wild type mice. Furthermore the sperm morphology is abnormal. We hypothesized that sequence alterations of the SEPTIN12 gene might result in male infertility. Sequence analysis of the SEPTIN12 gene was performed in 160 infertile men and 200 fertile men. Two out of 160 infertility men were found to carry independent heterozygous mutations (C266T and G589A refer to T89M and D197N, respectively). These two mutations are located at the highly conserved GTPase domain. The protein stabilities of SEPT12T89M and SEPT12D197N were decreased significantly than SEPT12WT. Transfection of SEPT12T89M and SEPT12D197N showed abnormal filamentous structure, and the interactions of SEPT12 with its partners were also disrupted in NT2/D1 cells. These results indicate that SEPT12T89M and SEPT12D197N proteins were lost their functions, disrupting their interactions with other septin partners. Consistent with this hypothesis, co-immunoprecipitation experiments showed that SEPT12T89M and SEPT12D197N could not interact with SEPT7. Moreover, immunofluorescence assay showed that SEPT12T89M or SEPT12D197N were not co-localized with SEPT7, nor could they form filamentous structure in NT2/D1 cells. Our findings provide clinical relevance of the SEPTIN12 gene in human spermatogenesis.

    摘要................................................................................................................i Abstract........................................................................................................ii ACKNOWLEDGEMENT.........................................................................iv TABLE OF CONTENTS............................................................................v LIST OF TABLES.....................................................................................vii LIST OF FIGURES..................................................................................viii LIST OF APPENDIXES............................................................................ix LIST OF ABBREVIATIONS.....................................................................x 1. INTRODUCTION...................................................................................1 1.1 Male infertility............................................................................1 1.2 Spermatogenesis.........................................................................1 1.3 Genes involved in male infertility.............................................2 1.4 The septin family........................................................................3 1.5 Septins-related human diseases.................................................4 1.6 Functions of septin12..................................................................5 1.7 Objectives of the study...............................................................5 2. METERIALS AND METHODS............................................................6 2.1 Patient’s DNA samples and control DNA sample sort.............6 2.2 Cell culture..................................................................................6 2.3 Luciferase reporter assay...........................................................7 2.4 Sequence.......................................................................................9 2.5 Total RNA is olation....................................................................9 2.6 Reverse Transcriptase-Polymerase Chain Reaction.............10 2.7 Polymerase Chain Reaction (PCR).........................................11 2.8 Site-directed mutagenesis........................................................12 2.9 Co-Immumoprecipitation (Co-IP) analysis............................13 2.10 Western Blot analysis...............................................................14 2.11 Immnuofluorescence (IF) assay..............................................16 2.12 His-tagged protein purification...............................................17 2.13 Silver staining............................................................................19 2.14 GTPase activity assay...............................................................20 3. RESULTS...............................................................................................22 3.1 Define the human SEPTIN12 core promoter elements..........22 3.2 Identification of SNP and mutations in the SEPTIN12 promoter region........................................................................22 3.3 Identification of SNPs and mutations in the coding region of SEPTIN12 gene.........................................................................23 3.4 Confirmation of mutations by the TaqMan assay..................23 vi 3.5 The two mutations are located in the GTPase domain, and these two sites are highly conserved in different species......23 3.6 SEPT12-T89M and SEPT12-D197N are less stable than SEPT12-WT, and may alter protein assembling...................24 3.7 SEPT12-T89M and SEPT12-D197N altered the interactions of SEPT12 with SEPT4, 6, and 7.............................................25 3.8 SEPT-T89M and SEPT12-D197N could not co-localize with SEPT7 and form filamentous structure.................................26 4. DISCUSSIONS......................................................................................27 4.1 The roles of SEPT12 in spermiogenesis...................................27 4.2 Mechanistic link between loss of function mutation, protein stability and filament formation.............................................27 4.3 The physiological implication of different subcellular patterns of SEPT12 in NT2/D1 cell........................................................28 4.4 SEPT12 utilize different domains to interact with other SEPTINs family ……………………………………………...29 4.5 Mutant protein degradation pathway.....................................30 4.6 The role of SEPT7 in spermatogenesis....................................31 References..................................................................................................33 Figures........................................................................................................40 Tables..........................................................................................................57 Appendixes.................................................................................................62 CURRICULUM VITAE...........................................................................64

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