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研究生:沈偉仁
研究生(外文):Wei-Jen Sheng
論文名稱:二硫蘇糖醇及電激活對精子顯微注射後豬卵母細胞的原核形成及胚發育的影響
論文名稱(外文):Effect of dithiothreitol and electrical activation on pronuclear formation and embryonic development following intracytoplasmic sperm injection in porcine oocytes
指導教授:陳銘正陳銘正引用關係
指導教授(外文):Ming-Cheng Chen
學位類別:碩士
校院名稱:國立宜蘭大學
系所名稱:生物技術研究所碩士班
學門:農業科學學門
學類:畜牧學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:130
中文關鍵詞:精子顯微注射豬卵母細胞二硫蘇糖醇電激活
外文關鍵詞:intracytoplasmic sperm injectionporcine oocytesdithiothreitolelectrical activation
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精子顯微注射 (intracytoplasmic sperm injection, ICSI) 是一項將精子直接注射進入卵母細胞細胞質而使其受精的重要技術,但是將精子顯微注射應用在豬上,卻有雄原核 (male pronuclear, MPN) 形成率偏低的問題存在。本試驗之目的乃應用二硫蘇糖醇 (dithiothreitol, DTT) 處理公豬精子並聯合豬卵母細胞的電激活 (electrical activation, EA),搭配壓電脈衝顯微操作儀 (piezo micro manipulator, PMM),改善ICSI後豬卵母細胞的雄原核形成及胚發育。以5 mM DTT處理公豬精子10或30分鐘,隨後將公豬精子注射到體外成熟(in vitro matured, IVM)的豬卵母細胞中,精子顯微注射後1.5小時,再利用電刺激 (1 x 30 μs pulse of 1.2 or 2.2 kV/cm DC) 來激活受精卵。精子顯微注射後經過16-18 hr的培養,以DTT處理公豬精子30分鐘且電激活強度為2.2 kV/cm的組別,其雄原核形成率 (60.7 ± 6.2 %) 是最高的,且顯著高於注射無前處理的精子至無電刺激卵母細胞的對照組 (41.9 ± 3.2 %, P < 0.05)。比較DTT與電激活的聯合處理或單一處理對ICSI後豬卵母細胞雄原核形成與胚發育的影響。DTT與電激活聯合處理組 (54.8 ± 12.7 %) 的雄原核形成率顯著高於 (P < 0.05) 注射無前處理的精子至無電刺激卵母細胞的對照組 (24.3 ± 9.0 %);DTT與電激活聯合處理組 (75.8 ± 12.1 %) 的卵裂率顯著高於 (P < 0.05) 注射無前處理的精子至無電刺激卵母細胞的對照組 (42.5 ± 11.5 %);DTT與電激活聯合處理組 (37.3 ± 12.8 %) 的桑椹胚發育率顯著高於 (P < 0.05) 注射無前處理的精子至無電刺激卵母細胞的對照組 (15.2 ± 8.4 %)。結論,以DTT處理豬精子與電激活的聯合處理能改善ICSI後豬卵母細胞的致活反應、精子的去濃縮作用、雄原核形成、正常受精作用、卵裂及桑椹胚的發育,且理想的電激活條件及精子暴露在DTT的時間對雄原核形成與胚發育來說是非常重要的。此外,需進一步研究去找尋適當的處理方式,改善豬ICSI的囊胚發育率。
Intracytoplasmic sperm injection (ICSI) is a procedure where the oocyte is fertilized by a direct injection of sperm into the cytoplasm. In porcine, the low rate of male pronuclear (MPN) formation has been reported after ICSI. The objective of this study was to improve male pronuclear formation and embryonic development following ICSI of dithiothreitol (DTT)–treated boar spermatozoa and electrical activation (EA) of porcine oocytes after ICSI with piezo micro manipulator (PMM). The in vitro matured (IVM) porcine oocytes were injected boar spermatozoa treated with 5 mM DTT for 10 and 30 min, then EA with 1 x 30 μs pulse of 1.2 or 2.2 kV/cm DC at 1.5 hr after ICSI. After 16-18 hr of culture, the male pronuclear formation rate in the group of porcine oocytes injected boar spermatozoa treated with DTT for 30 min combined with EA with 2.2 kV/cm (60.7 ± 6.2 %) was the best and higher than that in the control group (41.9 ± 3.2 %, P < 0.05) of unstimulated oocytes injected with untreated spermatozoa. We compared the effects of ICSI with and without a combination of DTT and EA on male pronuclear formation and embryonic development of porcine oocytes. The male pronuclear formation rate in the group treated with a combination of DTT and EA (54.8 ± 12.7 %) was higher (P < 0.05) than that in the control group (24.3 ± 9.0 %) of unstimulated oocytes injected with untreated spermatozoa. The oocytes cleavage rate in the group treated with a combination of DTT and EA (75.8 ± 12.1 %) was higher (P < 0.05) than that in the control group (42.5 ± 11.5 %) of unstimulated oocytes injected with untreated spermatozoa. The morula development rate in the group treated with a combination of DTT and EA (37.3 ± 12.8 %) was higher (P < 0.05) than that in the control group (15.2 ± 8.4 %) of unstimulated oocytes injected with untreated spermatozoa. In conclusion, pretreating sperm heads with DTT, and EA of oocytes after sperm head injection improved oocytes activation, sperm decondensation, male pronuclear formation, normal fertilization, oocytes cleavage, and morula development. The optimized electrical activation and an optimal time exposure of the spermatozoa in the DTT were very important for successful male pronuclear formation and embryos development. Further research is needed to find the more suitable treatment and to improve the rate of blastocyst formation in porcine ICSI.
中文摘要.............................................................I
英文摘要...........................................................III
誌謝................................................................V
表次...............................................................XII
圖次...............................................................XIV
壹、前言.............................................................1
貳、文獻檢討..........................................................3
一、卵母細胞的成熟作用與受精作用........................................3
(一)、卵母細胞的成熟作用...............................................3
(二)、卵母細胞的受精作用...............................................4
1、卵母細胞...........................................................5
2、精子..............................................................7
(1)、精子獲能作用.....................................................7
(2)、頭巾反應.........................................................8
3、受精作用...........................................................9
(1)、精卵融合.........................................................9
(2)、卵母細胞的致活機制...............................................10
(3)、防止多精入卵的機制...............................................11
(4)、原核的形成......................................................12
二、卵母細胞的體外成熟與體外受精.......................................13
(一)、卵母細胞的體外成熟..............................................13
(二)、卵母細胞的體外受精..............................................14
1、體外受精的發展與應用...............................................14
2、影響體外受精的主要因素.............................................15
(1)、卵母細胞........................................................15
(2)、精子...........................................................17
(3)、培養液的組成....................................................18
(4)、精卵共培養的比例與時間...........................................18
3、改善體外受精的新技術...............................................19
三、精子顯微注射.....................................................20
(一)、精子顯微注射的發展與應用.........................................20
(二)、精子顯微注射的過程與處理.........................................20
(三)、精子顯微注射後的觀察指標.........................................22
四、精子顯微注射在豬的應用與問題.......................................23
(一)、精子顯微注射在豬的應用與發展.....................................23
(二)、體外成熟豬卵母細胞的問題與改善....................................24
1、體外成熟培養系統的改善.............................................25
(1)、含FCS與pFF的IVM medium.........................................25
(2)、Defined IVM medium.............................................26
A、Defined IVM medium的應用.........................................26
B、Defined IVM medium的改善.........................................26
2、致活卵母細胞......................................................29
(1)、鈣離子濃度的提高.................................................30
A、受精作用對卵母細胞內鈣離子濃度的影響.................................30
B、電激活的應用與理想條件.............................................31
(2)、使用化學藥劑致活卵母細胞..........................................32
A、DMAP與CHX........................................................32
B、InsP3............................................................33
(三)、雄原核形成率偏低的問題與改善.....................................33
1、精子細胞膜的阻礙...................................................34
(1)、精子細胞膜的特性.................................................34
(2)、瓦解精子細胞膜的方法.............................................35
2、精子頭巾的阻礙....................................................36
(1)、精子頭巾的特性..................................................36
(2)、移除精子頭巾的方法...............................................37
3、魚精蛋白的阻礙....................................................38
(1)、魚精蛋白的特性..................................................38
(2)、還原魚精蛋白的方法...............................................39
(四)、公豬精子品質對卵母細胞的受精與發育的影響...........................39
(五)、化學藥劑對卵母細胞的傷害.........................................40
五、總結............................................................42
參、材料與方法.......................................................43
一、卵母細胞的來源與體外成熟...........................................43
二、精子的製備.......................................................43
(一)、精子的來源與性狀檢測............................................43
(二)、精子的體外獲能與處理............................................47
三、卵母細胞的體外受精................................................50
四、精子顯微注射.....................................................52
(一)、顯微注射針的製作與架針步驟.......................................52
(二)、精子顯微注射的步驟..............................................53
五、電激活的處理.....................................................54
六、體外培養.........................................................57
七、卵母細胞或胚的形態觀察.............................................57
八、評估指標.........................................................58
(一)、成熟評估.......................................................58
(二)、受精評估.......................................................58
(三)、發育評估.......................................................60
九、統計分析.........................................................62
十、試驗設計.........................................................62
(一)、豬卵母細胞的體外成熟、體外受精與精子顯微注射.......................62
(二)、不同的顯微注射針內徑對ICSI後豬卵母細胞存活與雄原核形成的影響.........62
(三)、DTT處理公豬精子的時間聯合電激活的電場強度對ICSI後豬卵母細胞的影響....63
(四)、DTT及電激活的聯合處理或單一處理對ICSI後豬卵母細胞原核形成的影響......63
(五)、DTT及電激活的聯合處理或單一處理對ICSI後豬卵母細胞卵裂的影響.........64
(六)、DTT及電激活的聯合處理或單一處理對ICSI後豬胚發育的影響...............64
肆、結果............................................................65
一、豬卵母細胞的體外成熟、體外受精與精子顯微注射.........................65
二、不同的顯微注射針內徑對ICSI後豬卵母細胞存活與雄原核形成的影響...........65
三、DTT處理公豬精子的時間聯合電激活的電場強度對ICSI後豬卵母細胞的影響......71
四、DTT及電激活的聯合處理或單一處理對ICSI後豬卵母細胞原核形成的影響........75
五、DTT及電激活的聯合處理或單一處理對ICSI後豬卵母細胞卵裂的影響...........79
六、DTT及電激活的聯合處理或單一處理對ICSI後豬胚發育的影響.................85
伍、討論............................................................93
陸、結論...........................................................100
柒、參考文獻........................................................101

表次
表1. 含抗生素生理鹽水.................................................44
表2. Dulbecco磷酸緩衝鹽溶液(D-PBS)...................................45
表3. North Carolina State University-23 (NCSU-23)培養液.............46
表4. 精子洗滌液......................................................48
表5. 豬精子體外獲能培養液.............................................49
表6. 豬精子體外受精培養液.............................................51
表7. Hepes-buffered Tyrode’ s (HbT)................................56
表8. 經體外成熟培養48小時的豬卵母細胞成熟率.............................66
表9. 分析IVF後16-18小時豬卵母細胞存活率、穿透率及多精入卵率..............67
表10. 分析ICSI後16-18小時的豬卵母細胞存活率及雄原核形成率................69
表11. 不同的顯微注射針內徑對ICSI後16-18小時的豬卵母細胞存活與雄原核形成的影響....70
表12. DTT與電激活聯合處理對豬卵母細胞經ICSI後16-18小時的存活與正常受精的影響....73
表13. DTT與電激活聯合處理對豬卵母細胞經ICSI後16-18小時的致活反應與雄原核形成的影響..74
表14. DTT及電激活對豬卵母細胞經ICSI後16-18小時的存活與正常受精的影響......76
表15. DTT及電激活對豬卵母細胞經ICSI後16-18小時精子形態的影響.............78
表16. DTT及電激活對豬卵母細胞經ICSI後16-18小時致活反應的影響.............80
表17. DTT及電激活對豬卵母細胞經ICSI後48小時卵裂的影響...................84
表18. DTT及電激活對豬卵母細胞經ICSI後48小時精卵形態的影響................86
表19. DTT及電激活對豬胚經ICSI後168小時發育的影響........................89

圖次
圖1. 使用piezo pulses來操作ICSI......................................55
圖2. 經IVM後44-48小時,成熟豬卵母細胞的形態............................59
圖3. ICSI後的豬卵母細胞在非正常受精情況下,完整精子頭部(ISH)及去濃縮精子頭部(DSH)的形態...61
圖4. 經IVF後16-18小時,豬卵母細胞多精入卵的形態.........................68
圖5. 經ICSI後16-18小時,豬卵母細胞正常受精的形態........................72
圖6. 經ICSI後16-18小時,豬卵母細胞非正常受精的形態......................77
圖7. 經ICSI後48小時,豬胚在2細胞期的形態...............................81
圖8. 經ICSI後48小時,豬胚在3細胞期的形態...............................82
圖9. 經ICSI後48小時,豬胚在4細胞期的形態...............................83
圖10. 經ICSI後48小時,豬卵母細胞在非正常卵裂(1-cell)的情況下,完整精子頭部(ISH)的形態...87
圖11. 經ICSI後48小時,豬卵母細胞在非正常卵裂(1-cell)的情況下,去濃縮精子頭部(DSH)的形態...88
圖12. 經ICSI後168小時,豬胚在桑椹胚期的形態............................91
圖13. 經ICSI後168小時,脆裂的豬胚形態.................................92
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