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研究生:朱永寧
研究生(外文):Yung - Ning Chu
論文名稱:骨橋蛋白對精子游動力及受孕力的調節機制
論文名稱(外文):Identification of OPN-regulated sperm motility and fertilizing capacity
指導教授:高淑慧高淑慧引用關係
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:醫學檢驗生物技術學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:98
中文關鍵詞:骨橋蛋白受孕力游動力體外受精率
外文關鍵詞:motilityfertilizationosteopontinin vitro fertilization
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精子游動力是目前被認為是受孕能力的重要指標。臨床上如何選擇高游動力或改善精子游動力,藉以提高受孕能力是極為重要的課題。骨橋蛋白(osteopontin , OPN),在公牛被認為受孕的指標蛋白。OPN廣泛存在於壺腹部、副睾、儲精囊及輸卵管中, 此外OPN是一種磷酸蛋白質,主要調節許多細胞,包括巨噬細胞(macrophages)、蝕骨細胞(osteoclasts)及癌症細胞的發生和轉移有關。本研究主要為探討OPN是否具有調節精子細胞的游動能力及受孕力。在Western blotting的分析結果中,發現在25kD以及50kD處有OPN的表現,並且其表現量與精子的游動力有相關性,游動力佳的精子25kD OPN表現量較多(54.4%?b11.6% vs. 81.8%?b6.8%,p<0.05)。並且依精子的游動力細分樣本為Group 1 為0~30%,Group 2為30%~60%,Group 3為60%~100%,並以電腦輔助精液分析儀(computer-assisted semen analysis)測定各項游動參數。當我們以添加0.1µM OPN與精子作用30分鐘前後游動力變化,發現在Group 1的精子中,游動力有增加的情形(15.6%?b2.1% vs. 39.6%?b4.6%,p<0.01),Group 2亦有相同的增加情形(51.7%?b1.5% vs. 69.5%?b6.0%,p<0.05)。在CASA的分析結果中,共計8個精子的運動參數中,有4個參數,包括VCL、LIN、STR與BCF,在游動力較差的Group 1有明顯升高的情形,其中VCL提高為原始之175%?b18.0%倍、LIN為226.5%?b25.0%倍、STR為241.1%?b30.0%倍,以及BCF也成為原先的154.4%?b13.0% (p<0.05),不過在活動力較好的兩組(Group 2及Group 3),OPN並沒有顯著的提升。進一步分析OPN對於精子受孕能力的影響,包括精子移動至卵子能力的測定(in vitro migration test)、精子穿透力試驗(sperm penetration assay)、頂體反應試驗(acrosomal reaction),以及老鼠體外受精試驗等,發現OPN可以提高精子移動到卵子的能力(1.5%?b0.2% vs. 6.3%?b1.5%,p<0.05)、穿透卵子的能力(1.4?b0.1個 vs. 2.3%?b0.1個,p<0.05)以及增加體外受精率(62.0%?b3.6% vs. 78.0%?b2.1%,p<0.05)。不過在精子頂體反應試驗中,結果並沒有發現有增加的情形。最後我們利用螢光染色分析方法,觀察到OPN表現於精子的頭部(head)與中節(mid-piece),並且在加入OPN後會造成精子的細胞骨架細胞(cytoskeleton)的改變,在actin及tubulin有增加的情形,而spectrin螢光表現強度並無明顯增加。本研究證實OPN具有調控精子游動力及受孕力的能力。
The motility of the spermatozoa is reported as a critical indicator of fertilizing capacity。In clinical, to enhance the fertilization rate by selecting sperms with high motility or improving the sperm motility is an important issue. Osteopontin (OPN) was proposed as a fertility indexes protein of bulls. OPN is a secreted phosphoprotein, regulates biological functions of diverse cell types, such as macrophages and osteoclasts. It is also involved in onset and metastasis of cancer cells. OPN is expressed in the ampulla, seminal vesicles, epididymis, as well as oviduct. Aim of this study was to address the role of OPN in regulation of sperm motility and fertility potential. A significantly increase of the OPN expression by Western Blotting. A significant increased expression of 25 kD OPN was found in the sperm with good motility (54.4%?b11.6% vs. 81.8%?b6.8%, p<0.05). Then we divided all the sample into three groups, motility from 0% to 30% (Group 1), 30% to 60% (Group 2), and up to 60% (Group 3). Follow the supplementation of 0.1µM OPN for 30 minutes, the alteration of sperm motility and moving characteristics were examined by computer-assisted semen analysis (CASA).The sperm motility was significantly increased in Group 1(51.7?s?b???|???s?nvs. 69.5%?b6.0%, p<0.05). OPN caused a significant increase in the percentage of VCL (175.0%?b18.0%, p<0.05), LIN (226.5%?b?????s?z?np<0.05?w, STR (241.1%?b30.0%, p<0.05), and BCF (154.4%?b13.0%, p<0.05) in Group1 but not seen in Group 2 and 3. The effects of OPN on fertilizing capacity were evaluated by in vitro migration test, sperm penetration assay, acrosomal reaction, and mouse in vitro fertilization test. The result showed that the capability of sperm migration (1.5%?b0.2%vs. 6.3%?b1.5%, p<0.05), sperm penetration number (1.4?s?b?~?|???nspermatozoa vs. 2.3%?b0.1 spermatozoa, p<0.05), and in vitro fertilization rate (62.0%?b3.6% vs. 78.0%?b2.1%, p<0.05) was enhanced by OPN. However, there was no difference in terms of acrosomal reaction followed by OPN treatment. In addition, the distribution of actin, tubulin and spectrin was determined by immunofluorescent analysis. Sperm actin and tubulin increased display, but not in spectrin. In conclusion, OPN plays an important role in the regulation of sperm motility and fertilizing capacity.
中文摘要…………………………………………………………………1
英文摘要…………………………………………………………………3
目 錄 ……………………………………………………………………5
圖表目次…………………………………………………………………9
縮 寫 表………………………………………………………………12
第一章 緒論………………………………………………………14
第一節 研究背景………………………………………………………14
一、 不孕現況
二、 精子異常之原因
三、 現今提高游動力之方法
第二節 研究動機………………………………………………………19
第三節 研究目的………………………………………………………20
第二章 文獻探討…………………………………………………21
第一節 精卵受精的機制………………………………………. 22
一、 精子的構造
二、 受精的發生
三、 受精的過程
四、 實驗室評估男性精子之檢查項目
五、 受精失敗的原因
六、 參與精子受精能力之分子標識
第二節 OPN的生化特性……………………………………………30
一、 OPN的發現
二、 OPN的結構
三、 OPN的生理功能
四、 OPN在生殖系統中之表現
五、 OPN在受精時扮演之角色
六、 OPN對精子的作用
第三章 研究方法……………………………………………………36
第一節、實驗設計…………………………………………………….37
一、 研究目的(一)
探討OPN於精液中之表現情形
二、 研究目的(二)
分析OPN於精子中之表現情形
三、 研究目的(三)
觀察添加OPN後對精子游動力之影響
四、 研究目的(四)
探討OPN對於精子受孕能力之影響
五、 研究目的(五)
探討OPN提高精子受孕能力之機制
第二節、實驗方法與材料…………………………………………….39
一、 精液收集
二、 精液分析檢查
三、 西方墨點法(Western Blot)分析OPN蛋白表現亮
四、 以OPN處理精子觀察對精子生理活性的影響
1. 精子穿透力試驗
2. 精子移動至卵子能力測定
3. 頂體反應試驗
4. 老鼠體外受精試驗
5. 精子細胞骨架之螢光免液染色
五、 統計方法
六、 病人權利之保護
第四章 實驗結果……………………………………………46
第五章 討論…………………………………………………52
第六章 圖表…………………………………………………58
第七章 參考文獻……………………………………………93
表目錄
Table. 1 電腦輔助分析儀之各項精子運動參數定義 59
Table. 2 精液分析結果 60
Table. 3 HTF之組成成分 61
Table. 4 分析OPN添加對精子各項參數的影響 62
圖目錄
Fig. 1 頂體反應 63
Fig. 2 CASA運動參數分析圖 64
Fig. 3 OPN的結構 65
Fig. 4 OPN的功能性結構 66
Fig. 5 實驗設計流程 67
Fig. 6 老鼠精子穿透力試驗(Sperm penetration assay;SPA)之實驗過程 68
Fig. 7 精子移動能力試驗裝置圖 69
Fig. 8 精子頂體化試驗(acrosomal reaction) 70
Fig. 9 老鼠體外受精試驗(mouse in vitro fertilization assay 71
Fig. 10 Western Blot分析OPN於精液中之表現情形 72
Fig. 11 以Western Blot測定精液中OPN之含量 73
Fig. 12 OPN對精子游動力之影響 74
Fig. 13 OPN對精子Progressive motility之影響 75
Fig. 14 OPN對精子VCL( curvilinear velocity)之影響 76
Fig. 15 OPN對精子LIN( linearity)之影響 77
Fig. 16 OPN對精子STR(straightness)之影響 78
Fig. 17 OPN對精子BCF(beat-cross frequency)之影響 79
Fig. 18 OPN對精子VAP(velocity average path)之影響 80
Fig. 19 OPN對精子ALH(amplitude of lateral head displacement)之影響 81
Fig. 20 OPN對精子VSL(velocity straight line)之影響 82
Fig. 21 OPN對精子WOB(wobble)之影響 83
Fig. 22 評估OPN對精子移行結果並對老鼠精子移動力之影響 84
Fig. 23 老鼠精子穿透力試驗之結果(Sperm penetration assay;SPA) 85
Fig. 24 OPN對老鼠精子穿透卵子透明帶之影響 86
Fig. 25 OPN對老鼠精子頂體反應試驗(Acrosomal reaction assay)之影響 87
Fig. 26 OPN對老鼠體外授精之影響 88
Fig. 27 於200倍顯微鏡下,觀察精子表現OPN之位置 89
Fig. 28 以共軛焦顯微鏡,觀察精子細胞OPN之位置(localization) 90
Fig. 29 免疫螢光染色-Actin、??-tubulin與spectrin 91
Fig. 30 實驗結果圖 92
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