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  • 學位論文

小鼠鋅指蛋白基因-1在生精作用中所扮演的角色

The Role of Zinc Finger Protein-1 Gene in Spermatogenesis of Male Mouse

指導教授 : 鄭登貴
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摘要


本試驗旨在研究小鼠鋅指蛋白-1基因 (mouse zinc finger gene-1, mZFG-1) 在雄性可能扮演之生理功能。鑑於前人的研究證明短暫阻斷 mZFG-1S 之表現,即可嚴重影響小鼠胚之埋殖前發育,因而增加該基因調控機制相關研究之困難度;本研究乃先針對 mZFG-1L cDNA 全長設計一長度為 926bp 之反義去氧核醣核酸序列 (antisensesequence),並將之構築於接受蛻皮素 (ecdysone) 誘導調控基因表現之載體中,進一步將此表現載體及調節載體 (regulator vector) 截切後,經純化隨即進行小鼠原核顯微注射 (pronuclear microinjection),從而產製出可經由蛻皮素誘導該轉殖基因表現之雙轉基因小鼠,試驗結果證明共出生 66 隻仔小鼠,經應用聚合酶鏈鎖反應 (polymerase chain reaction, PCR) 及南方吸漬法 (Southern blot analysis) 皆證實有四隻係帶有雙轉基因者;此等雙轉基因小鼠經篩選及繁殖後,對施打誘導劑 ponasterone A 之成年雄小鼠進行 RNA、蛋白質萃取及冷凍切片,結果顯示經誘導表現反義核苷酸者之睪丸中,mZFG-1L 基因表現量的降低會造成生精細管中精子數目減少,此結果顯示 mZFG-1 鋅指蛋白在成年雄小鼠的生精 (spermatogenesis) 過程中可能扮演一重要的角色。 進一步試驗經由RNA干擾 (RNA interference, RNAi)、二維膠體電泳 (two-dimensional polyacryamide gel electrophoresis, 2D-PAGE) 及質譜分析策略,探討 mZFG-1 表現量遭受 siRNA 干擾後,對於睪丸賽透力氏細胞 (Sertoli cell) 及萊迪吉氏細胞 (Leydig cell) 中下游基因表現之影響;試驗結果發現在賽透力氏細胞株中,兩個和生殖內分泌及生精作用有關之蛋白 Crisp-1 (Cysteine-rich secretory protein-1) 及 NPY (Neuropeptide Y) 會受 mZFG-1 的正向調控,即 mZFG-1 的表現量降低時兩者的表現量亦隨之下降,由於已知 Crisp-1及 NPY 為維持雄性正常生殖作用所不可或缺者;故初步試驗結果顯示,mZFG-1在睪丸中可能透過直接或間接的調控 Crisp-1 及 NPY 的表現而維持雄性小鼠正常的生精作用;此蛋白質體分析得到的結果與轉基因鼠發生的現象頗相契合,惟詳細的機制仍待進一步的探討。

關鍵字

鋅指蛋白 小鼠 生精作用

並列摘要


The purpose of this study was to clarify the molecular mechanisms of mouse zinc finger protein-1 (mZFG-1) gene potentially involved in the regulation of spermatogenesis within the mouse testis. To meet this purpose, an initial attempt was made to generate transgenic (Tg) mice characterized by conditional knock-down of the mZFG-1 gene. Of these studies, a transgene named anti-mZFG-1L/pIND/V5-His-TOPO, containing antisense transcript against to nucleotide sequences complementary to the long form of mZFG-1 cDNA, was constructed. Generation of Tg mice was conducted by co-injection transgenes of anti-mZFG-1L/pIND/V5-His-TOPO and pVgRXR, into the pronucleus of newly fertilized eggs. These have resulted in 10 out of 66 newborn mice to be transgenic and four out of these 10 Tg mice were confirmed, as evidences shown by PCR and Southern-blot analyses, to be harboring both of the transgenes said above. Each of the four double-transgenic founder mice appeared to be fertile with 16.7 ~ 100% of their transgenes being germline-transmitted to the F1 progenies obtained. Evidences from RT-PCR analyses further confirmed that, in comparison with those of the control mice, much less of endogenous mZFG-1 mRNA was found in the double-transgenic founder mice when they had been subjected to the ponasterone-A induction. While no gross anomalies was found in any somatic tissues from those ponasterone-A induced double-transgenic mice, much less number of mature spermatozoa were found in testis-specimens from the double-transgenic mice when comparison was made to those of control mice after ponasterone-A induction. Moreover, the effect of mZFG-1 on spermatogenesis at molecular level was further verified using mouse Sertoli cell lines (TM4 cell lines) with the specific small interferon RNA (siRNA) strategy for knock-down the expression of endogenous mZFG-1 and the consequent expression profiles of its down-stream genes were investigated. Based on results from RT-PCR, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS) analyses appeared that both Crisp-1 (Cysteine-rich secretory protein-1) and NPY (Neuropeptide Y) genes were down-regulated within Sertoli cells when they had been transfected with siRNA designed against to mZFG-1 gene. Conclusions came to these present studies were that appropriate expression of mZFG-1 gene do play its physiological significances for ensuring normal spermatogenesis occurred within the testes and the potential effect of mZFG-1 gene on spermatogenesis may be mediated by modulation the expression of both Crisp-1 and NPY genes.

並列關鍵字

mouse spermatogenesis zinc finger

參考文獻


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