黃火鍊臺灣大學：分子與細胞生物學研究所朱蔡豈Ju, Tsai-KaiTsai-KaiJu2007-11-252018-07-062007-11-252018-07-062004http://ntur.lib.ntu.edu.tw//handle/246246/49888為瞭解精蟲生成之分子機制，在此以扣除性篩選的方法來挑選鯉魚精巢特有之cDNA並加以選殖，選出的cDNA再以北方雜合實驗來驗證其為睪丸所特有，然後深入研究其特性。本實驗針對一個精巢特有的cDNA加以研究，其推衍出之氨基酸序列（218個氨基酸）與小鼠Tsga2 （或meichroacidin）蛋白非常相似：兩者皆具酸性特徵，且皆有七個保守的MORN （membrane occupation and recognition nexus）序列，顯示該cDNA很可能是小鼠Tsga2 （testis-specific gene A2）的同源基因。儘管此鯉魚基因與Tsga2之序列有著極高相似度，但兩基因之表現與蛋白產物在細胞內之分佈卻大異其趣：Tsga2之表現雖然大多集中於睪丸，於小鼠卵巢中仍可測到微量的mRNA及蛋白質，但鯉魚基因之表現卻僅侷限於精巢。其次，以免疫組織化學法觀察小鼠睪丸則發現Tsga2的表現時期是介於粗絲期精母細胞到初期精細胞之間，且Tsga2會附著到減數分裂中期之染色體與紡綞絲上；而鯉魚對應基因之蛋白產物卻侷限於分化末期之精細胞，並且大量存在於精蟲鞭毛及基底體。後續的鯉魚精蟲蛋白萃取及鞭毛微管再組合實驗則顯示該蛋白可能是軸絲的成分之一。由於與小鼠Tsga2有著這些差異，故將此鯉魚Tsga2同源基因另定名為MORN motif-containing sperm-specific axonemal or acidic protein（MSAP）。基因資料庫之搜尋顯示MSAP/Tsga2之同源基因也可能廣佈於各生物門中，詳加比對各物種的同源基因所衍生之蛋白質發現它們的N端皆有高度保守的MORN區域，但其C端序列變異很大，而且長度也參差不齊。利用MSAP的抗體偵測各種脊椎動物之可能同源蛋白證實各物種MSAP同源蛋白間的確有分子量差異的情形。除了細胞分佈與分子量之差異外，二維膠體電泳分析顯示鯉魚MSAP與小鼠Tsga2也可能有修飾上的差別：MSAP具有多種不同等電點但幾乎等分子量的蛋白形式，而Tsga2卻僅有單一等電點的形式存在。這些差異的存在不僅指出各物種MSAP/Tsga2同源蛋白於精蟲生成中扮演不同的功能，也暗示著與雄性生殖有關的基因能快速變異演化的趨勢。此外，為釐清鯉魚MSAP於精蟲內的生理功能，於是利用重組的MSAP表現蛋白來尋找可能的MSAP結合蛋白。其中一個MSAP結合蛋白，經質譜分析及免疫偵測確認為鯉魚septin7，它為一群保守的GTP水解酶家族成員之一。免疫螢光定位分析顯示septin7與MSAP共同位於精蟲鞭毛的基底體上。septin蛋白家族可能參與細胞質分裂及細胞形態生成等事件，因此推測MSAP/septin複合體的功能可能涉及鯉魚精細胞之極性建立及鞭毛形成。In order to explore the molecular mechanisms underlying spermatogenesis, genes from a subtractive screen of testicular cDNA library of common carp (Cyprinus carpio) were scrutinized. In this study, one of the positive cDNA clones, denoted as p196, which has shown to be expressed exclusively in adult testis of carp, is further characterized. The open reading frame of p196 encodes a 218-aa protein that has highly acidic and hydrophilic properties, as well as seven copies of the MORN (membrane occupation and recognition nexus) motif spanning more than half of N-terminal sequences of the protein. The intact sequences share high identity to the two-third segment of the murine Tsga2 (testis-specific gene A2) product (also called meichroacidin) N-terminally, a protein that may play roles in meiotic stages of spermatogenesis in mouse, indicating that the p196 cDNA could be a carp homologue of Tsga2 (or TSGA2 in human). However, the carp homologous protein is specifically expressed during late spermiogenesis rather than meiosis as murine Tsga2 reported previously and especially enriched in mature spermatozoa, in which the protein is localized to the basal bodies and flagella. Fractionation of sperm proteins and in vitro polymerization of flagellar tubulins revealed that the protein might be one of the axonemal components of carp sperm. Because of this distinctive distribution, the cyprinid gene is thus called MSAP (MORN motif-containing sperm-specific axonemal protein) to discriminate it from murine Tsga2. Database search and immunoblot analyses suggested that the MSAP/Tsga2 homologues were widespread in a variety of phyla but highly divergent in their C-terminal length and sequences. In contrast to the 2-DIGE (two-dimenaional gel electrophoresis) profile of Tsga2, existence of multiple pI variants of MSAP also displayed the difference in posttranslational regulation between the murine and cyprinid homologues. Divergence in the cellular distribution, C-terminal region and protein modification between the conserved molecules implicates an adaptive radiation of the MSAP/Tsga2 homologues during vertebrate phylogenesis and their distinct roles in male germline development among various vertebrates. In addition, possible MSAP-interacting proteins were examined by using blot overlay assay and tandem mass spectrometry. One of the interacting candidates was identified as the carp septin7, a member of a conserved GTPase family that may participate in cellular morphogenesis and cytokinesis. Immunoflourescence localization suggested that septin7 was associated with a subset of MSAP within the basal body of carp sperm. These findings lead to an MSAP/septin-mediated model for flagellar differentiation during spermiogenesis.TABLE OF CONTENTS 頁數 ACKNOWLEDGEMENTS……………………………………………... I ABSTRACT……………………………………………………………... II 中文摘要…………………………………………………………….. IV TABLE OF CONTENTS……………………………………………….. VI INTRODUCTION……………………………………………………..… 1 Comparative Morphology of Testis and Sperm between Vertebrates………………….….. 1 Advantages of Teleost Models for Gamete Researches………………………………….….. 5 Isolation of Testis-Specific Genes from A Teleost……………….………………………….… 6 MATERIALS AND METHODS……………………………………….. 9 Subtractive Screen of Testicular cDNA Library………………………………………………... 9 Cloning of Testis-Specific cDNA………………………………………………………………….. 10 Rapid Amplification of cDNA Ends……………………………………………………………….. 10 Reverse Transcriptase-Polymerase Chain Reaction………………………………………….. 11 Northern Hybridization……………………………………………………………………………... 11 Sequence Analysis………………………………………………………………………………….. 12 Production of Recombinant Proteins……………………………………………………………. 12 Antibody Production and Purification…………………………………………………………… 13 Preparation of Protein Extracts…………………………………………………………………… 14 Immunoblotting…………………………………………………………………………………….… 14 Immunofluorescence Microscopy………………………………………………………………... 15 Two-Dimensional Gel Electrophoresis……………………………………………………….….. 16 In Vitro Assembly of Flagellar Microtubules………………………………………………….… 17 2-DIGE/Blot Overlay Analysis……………………………………………………………………... 18 Tandem Mass Spectrometry……………………………………………………………………..… 18 RESULTS……………………………………………………………….. 20 Homology of MSAP to Mammalian Tsga2……………………….…………………………….... 20 Tissue Specificity of MSAP Transcription…………………………………………………….… 21 Cellular Distribution of MSAP Protein………………..………………………………………….. 22 Size Variations between MSAP Homologues……………………………………………….….. 24 Subcellular Localization of MSAP Protein………………………………………....…………… 25 Extraction and Characterization of Sperm MSAP………………….………………………….. 27 Spontaneous Association of MSAP with Reassembled Flagellar Microtubule…………… 29 Identification of An MSAP-Interacting Partner from Sperm………………………..…….….. 31 CONCLUSION AND DISCUSSION………………………………….. 33 Regulation of Post-Meiotic Expression of Carp MSAP Gene…………………………….….. 33 Roles of Sperm MSAP in Flagellar Differentiation and Activity……………………………... 34 Genetic Implications of MSAP/Tsga2 Genes………………………………………………….... 39 Evolutionary Significance of MSAP/Tsga2 Homologue…………………………………….… 42 TABLES……………………………………………………………….… 44 FIGURES AND LEGENDS………………………………………….… 46 ABREVIATIONS………………………………………………………... 61 REFERENCES……………………………………………………….… 623347798 bytesapplication/pdfen-US質譜分析軸絲精蟲生成二維膠體電泳精巢鞭毛形成MSAPspermiogenesisspermatogenesisTsga2axonemeotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/49888/1/ntu-93-D86225005-1.pdf