臺灣博碩士論文加值系統

Spz1(Spermatogenesis-specific leucine Zipper)是一個新發現含有bHLH 功能區及leuine zipper功能區的轉錄基因，其 mRNA 全長約1.5 kb 而ORF(open reading frame)約1134 bp，在細胞中所轉譯出來的蛋白約42 kDa，經由北方墨點法的分析，發現該基因的 mRNA在成鼠身體的不同組織中只有表現在睪丸及副睪中。但與睪丸相較下，副睪中的表現量約為睪丸表現量的6分之一。研究該基因在成鼠睪丸不同時期的表現，可發現自小鼠出生後20天即有其mRNA之表現，而在40天時可達到最大量。隨後其表現量逐漸遞減，直到180天，幾乎看不到Spz1基因的mRNA在睪丸的表現。小鼠胚胎的表現自9.5天即開始有大量mRNA表現，一直持續到出生。
經由原位雜交和免疫組織呈色法的分析發現，在睪丸中Spz1的表現位置依成熟時間的不同，表現的地點亦不盡相同。在40天，Spz1的表現主要集中在Leydig細胞及Sertoli細胞，精細胞亦有少量的表現。但在80天的睪丸Spz1則主要集中在精細胞以後的精子形成過程，由免疫組織呈色法亦得到類似的結果。此外，以RT-PCR 對Leydig細胞株(LFG 6)及Sertoli細胞株(SF7) 的全體RNA及全體細胞蛋白抽出物作西方墨點法，其結果亦發現在這些10天大的細胞株中，mRNA及蛋白均可被偵測出表現。經由整體原位雜交亦可發現Spz1在副睪中的表現。副睪在切片後經由免疫組織呈色法，則可發現Spz1蛋白主要表現在副睪的上皮細胞。經由細胞螢光免疫呈色法及共軛焦顯微鏡分析Spz1在細胞中表現的位置，發現Spz1蛋白在Leydig細胞株中主要的表現位置集中在細胞核中。
經由細胞外特定DNA結合實驗(In vitro DNA targeting sequence assay)發現Spz1在細胞外系統中，可與特定DNA序列5’-GGG/AGGG G/A A/T T-3’結合。另外Spz1亦可與具E-box的DNA序列結合，此為bHLH家族成員的特性，也因此可推斷Spz1應是一個新的bHLH轉錄因子。此轉錄因子在睪丸中的表現可受額外施打性荷爾蒙，如retinoic acid 及睪固酮的調節而減少其mRNA的表現。經由染色體原位雜交試驗同時發現Spz1基因是位於小鼠13號染色的D1位置。
在Spz1生理功能的分析方面，經由CMV的啟動子將Spz1表現在COS-7及NIH 3T3細胞株中，均可發現Spz1的表現可使細胞形態改變。在COS-7細胞中由原本不規則狀變成較圓且形狀固定，stress fiber的表現亦增加。同時在有Spz1表現的兩種細胞株中，均可發現與對照組載體轉染的細胞株相較其增殖的速度增加許多。DNA的合成速度亦可經由〔3H〕-胞嘧啶併入試驗發現增加了6-7倍，由西方墨點法亦發現，如PCNA 及p27等與細胞週期相關因子均會受影響。同樣以CMV為啟動子將Spz 1轉殖在小鼠體內表現，進一步分析Spz1在小鼠體內所扮演的生理角色。在Spz1的基因轉殖小鼠中，經由對不同時期睪丸所作的病理切片，可清楚發現早在出生後1個星期，轉殖小鼠睪丸會產生許多Symplast及多核細胞，細精小管中的細胞亦明顯減少。並且從出生第二個星期開始，轉殖小鼠的睪丸則已明顯出現細胞死亡所產生的巨大空泡及漲大細胞，這種現象使得出生後14天細精小管只有少數精胚細胞(spermatogonia)卻無其他正常之細胞生成。到出生後第四個星期，轉殖基因小鼠睪丸的細精管都充斥者空泡細胞，原本應有成熟精子的細精小管亦只看到精細胞，此一現象說明之Spz1過度表現嚴重影響精子形成的過程的進行。
經由分析轉殖小鼠的生育能力，發現從出生後2個月，轉殖小鼠的生育能力 即明顯低於正常小鼠，到第6個月即有不孕的現象。進一步分析基因轉殖小鼠的精子，發現在4個月時轉殖小鼠所產生的精子，60%~70%都有問題，有些頭部未成熟，有些則尾部結構發育不正常，不足而一。在電子顯微的觀察下亦發現出生後40天的睪丸存在許多雙核細胞及進行細胞分裂的細胞。進一步經由TUNEL反應偵測細胞狀態，每個細精管平均有20%-25%的細胞在進行細胞凋亡反應，推究其原因，可能與轉殖小鼠睪丸中表現過量的Fas/CD 95有關。經由免疫組織呈色法發現轉殖小鼠睪丸在出生後兩個星期，即開始表現比正常小鼠還多的Fas/CD 95並延續到出生後第四個星期，經EIA (enzyme immunoassay)及RIA (radioimmunoassay)分析血液中賀爾蒙的濃度，發現黃體激素及睪固酮濃度會受到Spz1基因的表現而不同。在黃體激素方面在出生後1至2個星期，血液中黃體激素濃度的變化不大，但自第四個星期開始則有明顯不同，轉殖小鼠血液中的黃體激素在第四個星期時，低於正常小鼠，但隨後從第8個星期開始其血液濃度即遠高於正常小鼠。在睪固酮方面，正常小鼠自第2週進入青春期開始，血液中的睪固酮即大量增加，並持續保持至成鼠以後，但轉殖小鼠血液中的睪固酮濃度，在8個星期前一直保持非常低直到8週後才緩慢增加。由體外給予黃體激素及睪固酮，則可發現黃體激素可減少Spz1表現所產生的睪丸空泡現象，但睪固酮的給予則不如預期。
在Spz1蛋白的活化及訊息傳遞方面，經32P標示及免疫沈澱的實驗，發現Spz1蛋白可經由培養基中有無添加血清而決定是否進行磷酸化。胎牛血清的添加會促使細胞中的Spz1蛋白被磷酸化。經由MEK抑制劑(PD98059)及p38 kinase抑制劑(SB203580)的添加，則發現Spz1在Leydig細胞株中的磷酸化會被PD98059所抑制。另外經由細胞外系統的蛋白質激酶磷酸化實驗亦發現MEK1可在細胞外直接的對Spz1蛋白進行磷酸化，這些結果說明Spz1蛋白質的活化，可能是經由MEK/ERK訊息傳導來進行。經由螢光免疫呈色法亦發現胎牛血清的添加可使Spz1蛋白由細胞質中轉位到細胞核中，同樣亦發現PD98059的添加會抑制Spz1的轉位作用，將Spz1表現在COS-7及NIH 3T3細胞株中，Spz1蛋白的表現不僅使細胞形態改變，亦同時改變一些細胞的特性。經由PD98059及LY294002的作用發現PD98059同樣的可抑制Spz1轉位到細胞核中同時使細胞的形態轉向不規則，但LY294002則對Spz1的蛋白轉位沒有影響，亦即不影響其磷酸化的進行。
經由以上的實驗結果，可說明Spz1 是一重要的轉錄因子並在小鼠體內精子形成的發育上扮演重要的角色，另外也証實MAPK 訊息傳遞在小鼠睪丸中精子形成過程也許經由調控Spz1 蛋白的活性，進而調控某些精子形成的過程。

In the present study, we isolated and characterized a novel bHLH-Zip gene, Spz1 (Spermatogenesis-specific leucine zipper 1). Spz1 mRNA is exclusively expressed in the adult mouse testis and epididymis but not in any other organs examined. The cell types responsible for the strong signal in testis on Northern blots were determined by in situ hybridization and immunohistochemistry on mouse testicular sections. Signals were found in the germ cells, Sertoli cells, and Leydig cells of day 20~60 testes. The expression of Spz1 in Sertoli and Leydig cells was also confirmed by Western blot analysis of cell extracts isolated from a Sertoli cell line (SF7) and a Leydig cell line (LFG6). By in situ hybridization and immunohistochemistry, Spz1 could be detected at different stages (20, 40 and 80 days after birth) in epidedymis. The expression of Spz1 was detected primarily in epithelium cells of 40 days-old mice. The nuclear localization of Spz1 was determined in the LFG 6 Leydig cell line, indicating that Spz1 is a nuclear transcription factor that may bind to specific sequences through its basic domain to regulate its trans-regulation of target genes. A nuclear localization signal (NLS) and nuclear transport sequence were identified in the Spz1 sequence using a computer search. To identify the target-binding sites of Spz1 protein, a random selection and amplification approach was conducted. The 9 bp consensus binding site, 5’-GG(G/A)GGG(G/A)(A/T)-3’ , determined from 30 selected oligonucleotides indicated that Spz1 prefers to bind to asymmetric G-rich sequences. The binding ability of Spz1 to the E-box and G-box was also confirmed by EMSA analysis. Results from our EMSA analysis showed that Spz1 has a higher affinity to the E-box than to the G-box and the 9 bp G-rich sequences. By using Northern blot analysis, we found that Spz1 expression in pubertal testis was not changed after gonadotropin treatment. On the other hand, retinoic acid and testosterone reduced the expression level of Spz1. Since both retinoic acid and testosterone have been suggested to be involved in the regulation of cellular differentiation during embryonic and organ development, the downregulation of Spz1 expression by these two hormones implies that Spz1 may play a repressor role in the differentiation process during spermatogenesis. By the chromosomal in situ hybridization, the locus of Spz1 gene was located in the D1 region of chromosome 13.
As shown by protein kinas assays in vitro and in vivo, the Spz1 protein can be phosphorylated by MEK1, resulting in a translocation of the Spz1 protein from the cytoplasm into the nucleus. When transfected into COS-7 cell line, Spz1 transformed the epithelial cells into a fibroblast-like morphology, increased the expression level of tiam and rac. The Spz1-induced COS-7 cell proliferation was shown by a shortened duplication time and an increased [H3]-thymidine incorporation. In addition, the overexpression of Spz1 in vivo results in germ cells swell and in the blocking of spermatogenesis in testes, which progressed germ cell to death. These results demonstrated that Spz1 might also play an important role in the cell cycle progress and in spermatogenesis during development. It was found that the expression of Spz1 alone in NIH 3T3 and COS-7 cell lines were enough to transform both cell lines. The MEK1 (MAPK kinase) is an important regulator involved in Spz1-induced cell transformation. In COS-7 cell line, after phosphorylation by MEK1, Spz1 could shift from cytoplasm into nucleus where it could regulate downstream response factors.
The overexpresssion of Spz1 resulted in both the physiological and morphological changes in the transgenic mouse testis as other examined organs. The predominant morphological change was first observed during the first week of postnatal life. Comparing to the wild type mice, the number of Sertoli cells and gonocytes in the seminiferous tubules were decreased, but symblasts and multinuclear Sertoli cells were increased. Spermatogenesis was delayed in the Spz1 transgenic mice by cell death progress. At the two-week-old stage, primary and type A spermatogonia were swelling and cellular depletion were formed in the center of seminiferous tubules, which caused a pause in differentiation of spermatogonia and spermatogenesis progression was fatally blocked at meiosis prophase. The Spz1 overexpression-induced spermatogenesis blockage reached a climax at the end of the first wave of spermatogenesis, when the meiosis of the primary spermatocyte was stopped at pachytene stage. The number of swollen cells decreased in adulthood, but earlier spermatogenesis arrest resulted in hypospermatogonia, and an early infertility of the transgenic mice. Analyzing the morphology of transgenic sperm of 4 months-old mice, two thirds of them were premature and there were many defects in their structure that would influence its mating ability and led to infertile. These morphological changes were similar to those found in hEGF-transgenic mice. In transgenic mice, the LH level in blood were low comparing to the wild type mice at fourth week, but increased fastly to 8~10 folds higher than normal from 8th week. The testosterone level in the Spz1 transgenic mice was not raised to the level of wild type mice until two months postnatal, and a low level of testosterone can be considered to be one of the reasons for a serious interference in the progress of spermatogenesis. In Spz1 transgenic mice, the swollen spermatogonia were found to progress to death through apoptosis, probably via a mechanism of increase in Fas/CD95.
In summary, Spz1, a novel bHLH-zip transcription factor, is expressed during spermatogenesis in postnatal mouse testis, and can be downregulated by retinoic acid and testosterone in pubertal testis. In additional, it also plays an important role in cell transformation and proliferation. In vitro, we found it could change cell morphology by the Raf/MEK pathway and increase cell proliferation. In transgenic mice, the proliferation of early developmental gametes could block the progress of spermatogenesis in testes though an interferences in hormone regulation and other factors. The results from both in vitro and in vivo studies correlated very well and confirm the importance of Spz1 during the testis development.

目 次 頁數
中文摘要……………………………………………………………………………………...3
英文摘要…………………………………………………………………………………...…5
第一章 緒論………………………………………………………………………………….7
壹.、雄性生殖器官之發育及精子的形成……………………………………….8
一 、與性腺發育相關的基因
二、精子生成(Spermatogenesis)
貳.、鹼基螺旋-環-螺旋(bHLH)基因之結構及生理功能………………………15
一、E-box site:許多具細胞特異性基因表現的控制因子
二、HLH 的結構
三、HLH 在發育過程中的角色
參.、Raf/MEK1/2之訊息傳導途徑及生理功能……………………………….19
一、Mitogen-activated protein kinase (MAPK)途徑組成物的結構
二、真核細胞中的MAPK路徑
肆.、細胞凋亡(Apoptosis)及Fas/FasL訊息傳導途徑及生理功能…………...23
一、細胞凋亡在發育上所扮演的角色
二、細胞凋亡，細胞壞死及細胞程式死亡
三、形態學上的特徵
四、在細胞凋亡的基因研究
五、細胞凋亡在生物化學上特徵
六、細胞凋亡過程中的訊息傳遞
伍.、實驗的目的與研究方向…………………………………………………..34
第二章 材料與方法………………………………………………………………………..35
壹、材料………………………………………………………………………...36
一、EST菌株的獲得
二、藥品、試劑
三、抗體
四、質體
貳、方法……………………………………………………………………….38
一、篩選互補DNA全長(Full length cDNA)
二、RNA的純化
三、北方墨點法(Northern blot)
四、蛋白質的表現及純化
五、多株及單株抗體的製作(Poly- and monoclonal antibody)
六、原位雜交法(Whole mount and In situ hybridization)
七、免疫組織化學呈色法(Immunohistochemical analysis)
八、免疫螢光呈色法(Immunofluorecence analysis)
九、西方墨點法(Western blot)
十、電泳移動改變分析法(Electrophoresis mobility shift assay; EMSA)
十一、染色體原位雜交法(Chromosomal in situ hybridization)
十二、細胞內蛋白質的磷酸化反應(In vivo phosphoryaltion assay)
十三、蛋白激脢活性分析(In vitro protein kinase assay)
十四、質體DNA的轉染(Transfection)
十五、流動細胞儀分析(Flow cytometry analysis)
十六、氚-胸嘧啶標定法(〔3H〕-Thymidine incorporation)
十七、TUNEL組織呈色法
十八、電子顯微鏡分析(Electronic microscopical observation)
十九、血清及組織黃體激素濃度之測量( rLH assay)
二十、血清及組織睪固酮濃度之測量(rTestosterone assay)
二十一、組織病理切片之製作及分析(Pathological analysis)
第三章 實驗結果…………………………………………………………………………..46
壹、Spz1之基因選殖及表現……………………………………………………47
一、Spz1基因之全長及蛋白質結構之特徵
二、Spz1基因在胚胎(embryo)及成體的表現時間
三、Spz1基因在胚胎(embryo)及成體的表現位置
四、Spz1蛋白在Leydig及Sertoli細胞株中的表現
五、Spz1基因在副睪的表現
六、Spz1蛋白在Leydig細胞株中的位置
七、Spz1蛋白與特定核甘酸序列結合
八、不同性腺荷爾蒙對Spz1基因表現的調控
九、Spz1基因在染色體上的位置
貳、Spz1之生理功能分析…………………………………………………..…51
一、Spz1蛋白的表現增加COS-7細胞株之增殖
二、Spz1基因在小鼠體內過度表現對睪丸的影響
三、Spz1 過度表現減少小鼠的生育能力
四、Spz1 蛋白過度表現對精子生成過程的影響
五、Spz1 蛋白的表現造成精細胞死亡
六、Spz1 蛋白的表現對血液中LH及睪固酮的影響
參、Spz1 蛋白在細胞中可能的訊息傳遞………………………….…………55
一、磷酸化對Spz1蛋白的影響
二、磷酸化對Spz1蛋白在細胞中表現位置的影響
三、Spz1蛋白過多的表現對其他種細胞的影響
第四章 實驗討論………………………………………………………………………..57
一、bHLH在精子形成過程中可能扮演的角色……………………………….59
二、性腺賀爾蒙對Spz1基因表現及睪丸發育上的影響……………………...60
三、細胞凋亡在精子形成過程中的角色…………...…………………………. 61
四、MAPK訊息傳遞系統在精子形成過程的影響……………………………63
圖表……………………………………………………………………………………….65
參考文獻………………………………………………………………………………...108
附錄(發表之論文)………………………………………………………………………115

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