臺灣博碩士論文加值系統

Part I. ESX1的階段性表現-作為一X精子的候選標識蛋白
中文摘要
胚外組織-生精作用-同源箱基因1（Esx1基因）會轉譯出一具有X-性聯鎖的同源箱蛋白質。過去對於Esx1基因之表現形態，除了其mRNA在睪丸中的表現已被研究外，對於Esx1蛋白質（ESX1）在睪丸中的分佈型式仍然未知。在本論文中，以過去本實驗室所製作的ESX1抗血清探究ESX1在小鼠中的階段性及組織專一性表現。西方點墨分析及免疫螢光分析結果顯示ESX1的分佈與RNA的表現形式一致，主要是在胎盤及睪丸組織中。免疫螢光分析顯示ESX1在三週齡後的小鼠睪丸中表現，而此時生精細管開始出現圓形精細胞。此外，在圓形精細胞形成精子的過程中，在生精細管的管腔部位中ESX1的表現增強。相反的，在隱睪手術處理後的睪丸中，ESX1的表現量會下降。由此結果得知，ESX1之表現主要是在生精作用中減數分裂後的階段。為了更進一步了解ESX1是否在X-精子中表現，本研究利用ESX1抗血清進行免疫螢光顯微鏡分析試驗，分析ESX1在小鼠精子中的表現，結果發現大約一半的精子能被ESX1抗血清所辨識。從本研究中，我們認為ESX1能做為X-精子之標幟蛋白質。

Part II. 熱緊迫誘導不同表現型的Esx1轉錄子在睪丸及胎盤中的轉換表現以及Esx1基因啟動子在雄性生殖細胞中之區域分析
中文摘要
Esx1是一性聯染色體的同源箱基因，已知其mRNA高度表現於成鼠睪丸（a-型Esx1）以及胎盤中（b-型Esx1）。根據過去文獻以及基因庫序列所分析的結果顯示還有另一個新的mRNA表現型存在（在此命名為x-型Esx1），但是過去對於此新的表現型在睪丸中的表現型態及重要性，並沒有任何相關研究被發表。本研究利用兩個能誘導產生熱緊迫的動物模式系統：分別是執行隱睪手術以及將小鼠暴露在高溫環境下，以探究不同型式的Esx1 mRNA之專一性表現。以異構型特殊性RT-PCR分析結果發現此新的Esx1轉錄子表現在隱睪睪丸中，並且顯示會受到熱緊迫誘導而高度表現。此x-型Esx1在隱睪睪丸中會取代a-型轉錄子的表現，以維持Esx1 mRNA及ESX1蛋白質的穩定表現量。然而在受到嚴重熱緊迫下，x-型Esx1表現量有延遲表現的情形。當小鼠在37oC熱緊迫處理時間延長，Esx1在睪丸中的表現量伴隨著減數分裂後期生殖細胞的受損而下降。另外，探究懷孕母鼠在受到熱緊迫傷害下，Esx1基因在胎盤中的轉錄表現，發現當懷孕母鼠受到37oC處理一小時後，胎盤中的主要表現型b-型會轉變為a-型Esx1轉錄子。組織學研究也發現受到熱緊迫傷害的胎盤組織損傷主要發生在迷路層細胞區域，然而免疫螢光分析結果顯示ESX1的表現訊號並沒有因此造成表現量下降。這些結果指出在睪丸及胎盤中，不同Esx1轉錄子的轉換表現，部分發生原因可能是由於不同啟動子的轉換使用。為了探討此論點的可能性，整個Esx1基因之5’-端的序列被選殖出，以探討在不同階段的睪丸生殖細胞中Esx1啟動子是否有不同的調節區域。短暫轉染分析結果顯示Esx1啟動子可能有二個主要的近端作用調節區域存在：一是從核酸序列-965至-438之間的DNA區域以及另一介於核酸序列+25至+227之間的近端作用區域。前者能充份調控報導基因在圓形精細胞和初級精母細胞的表現；後者則能於精原細胞啟動報導基因的表現。這些試驗結果將幫助我們了解Esx1基因啟動子區域在睪丸中可能的作用與功能。

英文摘要 I.
Extra-embryonic tissue-spermatogenesis-homeobox gene 1 (Esx1) encodes an X-linked homeobox protein. Despite the fact that the temporal and spatial mRNA expression pattern has been studied extensively in the testis, specific localization of the Esx1 protein (ESX1) in the testis remains to be determined. In my study, the ESX1 antiserum was used to investigate the stage- and tissue-specific expression of ESX1 in the mouse. Western blotting and immunofluorescent analyses revealed that general localizations of ESX1 were consistent with its RNA expression patterns; that is, it was restricted mainly to the placenta and testis. Immunofluorescent studies demonstrated that ESX1 existed in the testes after 3 weeks of age, coincident with the appearance of round spermatids in the seminiferous tubules. Moreover, ESX1 expression became more abundant in the luminal regions of the seminiferous tubules as the development of round spermatids progressed into spermatozoa. In contrast, reduced expression of ESX1 was observed in experimentally induced cryptorchid testes. The later expression of ESX1 suggests a role in post-meiotic germ cell development. To further understand ESX1 expression in sperm with respect to X chromosome-bearing sperm, we used ESX1 antiserum to immunostain sperm and examined the sperm by confocal laser microscopy. Approximately half the sperm population was recognized by the ESX1 antiserum. On the basis of results of the present study, we suggest that ESX1 could be recognized as a protein marker for X-sperm.

英文摘要 II.
Esx1, an X-linked homeobox gene, is highly expressed in adult testis (as a-form Esx1) and in placenta (as b-form Esx1). Surveys of literatures and GenBank sequences identified the other novel isoform (herein referred to as x-form Esx1), which has not been histologically defined. In the present study, two heat-induced stress models, experimentally induced cryptorchidism and heat incubation, were carried out to examine the isoform-specific expression of Esx1 mRNA. The novel Esx1 isoform was revealed without precedent in cryptorchid testes and showed highly stress-inducible. Isoform-specific RT-PCR results revealed that x-form Esx1 replaces the a-form transcript, and maintains comparatively stable transcriptional expressions of Esx1 RNA and ESX1 protein in cryptorchid testes. However, under severe heat challenge, expression of the x-form transcript was delayed. Reduced expression of Esx1 in testes of mice under prolonged incubation at 37oC coincided with deteriorating cellular damage in postmeiotic germ cells. In addition, the effects of thermal insult on the transcriptional regulation of the Esx1 gene in the placenta were studied in pregnant mice. The results demonstrated that exposure of pregnant mice to 37 oC for one hour caused the constitutive b-form in placenta to switch to the a-form transcript. Histological examination of heated placenta revealed that damage had occurred in labyrinthine layer; however, immunofluorescent analysis showed that ESX1 signals were not reduced. The results of isoform switching suggest that the existences of different Esx1 transcripts might be partially due to the usage of alternative promoters in testis and in placenta. To investigate this proposition, the entire 5’-flanking sequences of Esx1 gene were used to study the distinct promoter regions that would regulate the Esx1 expression in testicular germ cells. Transient transfection assays revealed that there were two putatively distinct promoter regions: a distal promoter region between nt -965 and -438 and a proximal promoter region between nt +54 and +227, which were sufficient to retain the promoter activities in round spermatids and primary spermatocytes, and in spermatogonia, respectively. These results will help us to understand the possible promoter regions essential for Esx1 altenative gene expression in testes.

目錄 ………………………………………………………………………1
Part I. ESX1的階段性表現-作為X精子的候選標識蛋白
中文摘要…………………………………………………………………6
英文摘要…………………………………………………………………7
前言………………………………………………………………………8
一、 生精作用與精子生成………………………………………8
二、 Esx1基因……………………………………………………9
三、 X-及Y-精子之辨識…………………………………………11
材料及方法………………………………………………………………14
一、 試驗動物（Assay animals）……………………………14
二、 隱睪手術（Induction of cryptorchidism）………15
三、 分離精細胞及附睪尾取精（The separation of testicular germ cell populations and collection of spermatozoa from the cauda epididymis）……………………………………………………15
四、 西方點墨分析法（Western blotting analysis）………………17
五、 免疫螢光顯微鏡分析法 （Immunofluorescent microscopy）…18
六、 統計分析（Statistical analysis）………………………………19
結果………………………………………………………………………20
一、 ESX1在小鼠胎盤及睪丸組織的空間表現（Spatial expressions of ESX1 in mouse placenta and testis）…………………………20
二、 ESX1在睪丸發育過程中的階段性表現區域（Stage-specific locations of ESX1 during testicular development）……………21
三、 SRY在睪丸中的專一性分佈位置（Specific localizations of SRY in testis）……………………………………………………22
四、 ESX1及SRY在小鼠精子之不同分佈表現（Differential localizations of ESX1 and SRY in mouse sperm populations）………………………22
五、 利用ESX1及SRY抗血清辨識以向上游動技術所分離之X-及Y-精子（Use of both ESX1 and SRY antisera to recognize the X- and Y- sperm separated by swim-up technique）……………………………23
討論………………………………………………………………………25
參考文獻…………………………………………………………………30

Part II. 熱緊迫誘導不同表現型的Esx1轉錄子在睪丸及胎盤中的轉換表現以及Esx1基因啟動子在雄性生殖細胞中之區域分析
中文摘要………………………………………………………………38
文摘要…………………………………………………………………41
前言……………………………………………………………………42
一、 熱緊迫對生精作用的影響………………………………42
二、 Esx1基因之文獻探討及基因序列分析…………………44
三、 熱緊迫對Esx1轉錄子表現在睪丸及胎盤組織的作用…45
材料及方法……………………………………………………………50
一、 試驗動物（Assay animals）…………………………50
二、 隱睪手術（Induction of cryptorchidism）……50
三、 誘發熱緊迫試驗模式（Experimentally-induced heat stress）…51
四、 反轉錄-PCR及定量rt-PCR（Reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real time (rt)-PCR analyses）………………………………………………………51
五、 西方點墨分析（Western blotting analysis）…………………54
六、 組織切片分析及免疫螢光顯微鏡分析法（Histology and immunofluorescent microscopy）………………………………54
七、 TUNEL試驗（Terminal deoxyribonucleotidyl transferase (TdT)-mediated deoxyl-UTP nick-labeling (TUNEL) analysis）.55
八、 小鼠Esx1基因體DNA之選殖（Isolation of the mouse Esx1 genomic DNA）…………………………………………………55
九、 質體建構與製備（Plasmid constructions and preparations）…59
十、 精細胞分離及附睪尾取精（The separation of testicular germ cell populations and collection of spermatozoa from the cauda epididymis）……………………………………………………60
十一、 短暫轉染分析作用與冷光素酶分析（Transient transfection and luciferase assays）…………………………………………60
十二、 統計分析（Statistical analysis）……………………………62
結果………………………………………………………………………63
一、 x-型Esx1轉錄子在隱睪手術後之睪丸中被誘導表現（Induced expression of x-form Esx1 transcript in testis with cryptorchidism）…………………………………………………63
二、 Esx1 mRNA在受到急性熱緊迫下的睪丸中異常的表現（Aberrant expression of Esx1 mRNA in testes under acute heat stress）…………………………………………………………65
三、 ESX1在受到嚴重熱損害的睪丸中之分佈表現（Spatial expression of ESX1 in severely heat-injured testes）……………66
四、 受到嚴重熱緊迫的睪丸生殖細胞發生計畫性細胞凋亡（Programmed cell death in the germ cell population of the testis suffered severe stress）………………………………………66
五、 Esx1轉錄子之特定表現型在胎盤中的獨特性表現（Isoform-specific expressions of Esx1 transcripts in placentae）.67
六、 觀察受熱緊迫處理的小鼠胎盤組織形態及ESX1的表現（Morphological observations of cross sections and expression of ESX1 in heat-stressed placenta）………………………………69
七、 獲取及定序小鼠Esx1基因之5’-端調節區域及5’-端非轉譯區序列（Obtaining and sequencing of the 5’-regulatory region and the 5’-untranslated region of mouse Esx1 gene）……………………69
八、 Esx1基因啟動子在分離後的睪丸生殖細胞中之活性（Esx1 promoter activities in fractionated testicular germ cell populations）…………………………………………………70
討論………………………………………………………………………74
參考文獻…………………………………………………………………83
表目錄……………………………………………………………………91
表說明……………………………………………………………………92
圖目錄……………………………………………………………………94
圖說明……………………………………………………………………96
附件目錄………………………………………………………………116
附件說明………………………………………………………………117
個人資料………………………………………………………………122
論文發表………………………………………………………………123

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