臺灣博碩士論文加值系統

哺乳類動物之生殖細胞發育，從生殖母細胞經由減數分裂變成單倍
體配子的過程相當複雜，其中生殖細胞專一性表達基因對於生殖細胞的發育扮演重要的角色。而為了瞭解調控生殖細胞發育的分子機制，實驗室鑑定出生殖細胞專一性表達基因－Gcse。Gcse 位於小鼠14 號染色體A3 區塊，轉錄出兩種主要的mRNA 剪輯型式: Gcse-l（1589 bp）與Gcse-s（906 bp），分別可轉譯出215 與152 個胺基酸序列。Gcse-l 與Gcse-s 蛋白的N 端118 個氨基酸組成一致，而Gcse-s 蛋白的C 端有56 個胺基酸與人類AKT2 （Protein kinase B）的催化區域高達50%的相似度。從Northern blots 與RT-PCR 實驗得知，Gcse-l 表現於睪丸與卵巢之中，Gcse-s專一地存在於睪丸組織。以RT-PCR，原位雜交與組織化學免疫染色分析不同發育時期的卵巢組織，發現Gcse-l 專一表現於卵細胞，從進入減數分裂的初級卵母細胞（primary oocyte）到成熟卵細胞（mature oocyte）皆有Gcse-l mRNA 表現，而Gcse- l 蛋白則從初級濾泡時期明顯增加，然而在受精卵中並未偵測到Gcse-l mRNA 的表現。在睪丸組織中，Gcse-l mRNA 訊號從粗絲期晚期精母細胞（pachytene spermatocytes）表現到早期單倍體圓精細胞（early round spermatids）中，而Gcse-s 只在早期的單倍體圓精細胞偵測到訊號。然而Gcse-l 與Gcse-s 的蛋白除了表現於睪丸外，其蛋白亦表現於副睪組織（成熟精子）中。藉由分離生殖細胞螢光免疫染色分析，發現Gcse-l 蛋白存在於精母細胞核內，而在減數分裂完成的單倍體精細胞中，則發現Gcse 蛋白會轉移頂體構造，與頂體標記蛋白－Lectin-PNA 訊號位置重疊。進一步利用可以進行減數分裂產生單倍體生殖細胞的精母細胞株－GC2 進行Gcse-l-EGFP 與Gcse-s-EGFP的轉殖實驗，細胞免疫螢光染色結果顯示，在初級精母細胞GC2 中，Gcse-l-EGFP 存在於細胞核之中，到了次級精母細胞GC2 階段，Gcse-l-EGFP 會由細胞核往細胞質運輸，而圓精細胞GC2 中，Gcse-l-EGFP存在於前頂體構造中，並與高基氏體、Acrosin 訊號位置重疊，而Gcse-s-EGFP 蛋白則存在圓精細胞GC2 細胞核。同時，藉由共同免疫沉澱法與免疫螢光共同染色，發現在次級精母細胞GC2 中，Gcse-l 會與磷酸化ERK1/2 結合並由細胞核往細胞質運輸，但到了成熟精子，Gcse-l 與磷酸化ERK1/2 不再有交互作用。由蛋白結構的分析發現，Gcse 蛋白具有RNA 結合區，我們推測在生殖細胞減數分裂過程中，初級精母細胞時期，Gcse-l 蛋白在核內與RNA 結合，次級精母細胞時期， 磷酸化ERK1/2協助Gcse-l 由核運輸至細胞質。待完成減數分裂後，細胞質中的Gcse-l成為成熟精細胞頂體蛋白的一部分，而Gcse-s 蛋白在圓精細胞核中除了可能與RNA 結合外，可能也具有磷酸激酶的功能。因此，Gcse 在精子形成過程之生殖細胞減數分裂及頂體生成過程可能扮演相當重要的角色。

Gametogenesis is a complicated developmental process of germ cells driving from primordial diploid cells. To understand the mechanisms controlling gametogenesis, a novel germ cell-specific gene, Gcse, which is located in mouse chromosome 14A3 with two major transcripts of Gcse-l (1589 bp) and Gcse-s (906 bp) in length, was identified. Gcse-l and Gcse-s encode 215 and 152 amino-acid proteins, respectively. The N-terminal 118 amino acids of Gcse-l and Gcse-s proteins are identical and the C-terminal 56 amino acids of Gcse-s show the maximum (50%) similarity to catalytic domain of human AKT2 protein (protein kinase B). Results of Northern blots and RT-PCR analysis revealed that Gcse-l expressed in both testes and ovaries, but Gcse-s expressed in testes only. During female gonad development, Gcse-l was detected from embryonic 13.5-day to adult and exclusively in oocytes. Results of in situ hybridization and immunohistochemistry both confirmed that Gcse-l specifically expressed in oocytes from primordial follicles to mature follicles. However, there was no Gcse-l signal in fertilized eggs. During male gonad development, Gcse-l and Gcse-s transcripts became detectable from postnatal day 15 (spermatocytes) and 21 (spermatids) and onwards, respectively. Strong Gcse-l signal started to express from late pachytene spermatocytes to early round spermatids and Gcse-s only existed in early round spermatids. However, both Gcse-l and Gcse-s proteins were existed not only in testes but in epididymis (spermatozoa). The transcription and translation of Gcse became temporally uncoupled since the elongated spermatids were transcriptional incompetently at the onset of spermiogenesis. Gcse-l proteins were consistently present in the nuclei of isolated spermatocytes by immunofluorescence analysis. Following meiosis, Gcse proteins were transferred to the Golgi complex of round spermatids, then combined to the acrosomal caps of elongated spermatids or spermatozoa by co-localized with the acrosomal marker （Lectin-PNA）. Results of transfection and immunofluorescence studies indicate that Gcse-l existed in the nuclei of GC2 cells（primary spermatocyte-like）. Following first meiosis, Gcse-l was translocated from nucleus to cytoplasm. In postmeiotic cells, Gcse-l proteins were detected in the proacrosomal vesicles with Golgi complex marker and proacrosomal marker（Acrosin）, and Gcse-s proteins expressed in cell nucleus of spermatid. Gcse-l was associated with activated-ERK1/2 in GC2 (secondary spermatocyte-like) cells but not in spermatozoa by co-immunoprecipitation and immunocytofluorescence assay. Prediction of protein structure revealed that Gcse contains RNA binding sites, and ERK1/2 was reported to be required for translocation of RNA binding protein by phosphorylation during two meiosis divisions. We suggest that Gcse-l protein acts as RNA-binding protein in the nucleus of primary spermatocyte. Then, activated-ERK1/2 assists Gcse-l to transport form nucleus to cytoplasm in secondary spermatocyte. Finally, Gcse-l became the acrosome protein of haploid sperm cells. However, Gcse-s protein was predicted to bind to RNA in nucleus of spermatid and possess the role of protein kinase. In short, Gcse may play an important role in meiosis and be involved in acrosome biogenesis during spermiogenesis.

摘要---------------------------------------------------P.1-4
中文摘要-----------------------------------------------P.1-2
Abstract-----------------------------------------------P.3-4
序論（Introduction）----------------------------------P.5-18
材料與方法（Material and method）--------------------P.19-60
結果（Result）---------------------------------------P.61-80
討論（Discussion）-----------------------------------P.81-89
圖表（Figures）-----------------------------------P.90-P.125
參考文獻（References）---------------------------P.126-P.130
附圖-------------------------------------------- P.131-P.134
附錄圖------------------------------------------ P.135-P.139
附錄表------------------------------------------ P.140-P.151
引子附表-----------------------------------------P.152-P.153

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