臺灣博碩士論文加值系統

本研究之目的在於探討成熟階段之牛與豬卵母細胞，經高溫或熱緊迫後其染色體（質）與細胞骨架（cytoskeleton）形態之變化，並探討高溫處理後之恢復能力。由屠宰場取得牛或女豬卵巢，自其表面濾泡取出包被完整之卵丘卵母細胞複合體（cumulus oocyte complexes, COCs），分別經M199或NCSU#23培養液體外成熟20或42 h後，將含第一極體之成熟卵母細胞逢機分配至對照組（Control, 39℃, 經0或4 h）與熱處理組（HS, 41.5℃, 經1, 2,或4 h）；另外則將豬卵母細胞經熱處理（41.5℃）4 h後，恢復至39℃繼續培養1、2、4、8或16 h；各組經熱處理後以免疫細胞化學染色法觀察染色體與細胞骨架形態之變化。結果顯示，經體外成熟培養後之牛與豬卵母細胞成熟率分別為42%（n= 300）與60%（n= 1570）；成熟卵子經HS 1 h、2 h或4 h者其染色體、紡錘體及細胞質出現微管之比例明顯較對照組高，且隨熱處理時間延長其百分率亦隨之增加。至於豬卵母細胞經熱處理4 h後恢復正常溫度繼續培養8和16 h後，兩組均僅有20.6%（7/34）之染色體恢復至第二次減數分裂中期（Metaphase II, MII），並重新排列於紡錘體中央赤道板（equatorial plane）上，而兩組分別有32.4%（11/34）與23.5%（8/34）紡錘體重新出現於細胞質中，但細胞質出現微管（cytoplasmic microtubules, cyto. MT）之比例仍高達91.2%（31/34）與97.1%（33/34）。以上結果證明卵母細胞於成熟後遭受熱緊迫，會造成染色體、紡錘體及卵胞質微管分佈之變化；而當溫度恢復後，僅有少數卵子之染色體（20.6%, n=34）或紡錘體（23.5%~32.4%, n=34）能恢復成正常形態。故熱緊迫明顯造成卵子生理上之變化，而此可能為導致其後續受精率下降與其發育能力減低之主要原因之一。此類卵子於受精後，是否因紡錘體與染色體之異常，而導致胚無法正常發育，則有待進一步之研究。

The objective of this study was to determine the effect of a short-term heat shock (HS) on the chromatin/chromosome and microtubular structures (MT) of the in vitro matured (IVM) pig and cattle oocytes. Cumulus-oocyte-complexes (COCs) were collected from local abattoirs and were IVM in NCSU#23 or M199 media for 42 h. Matured oocytes were selected and randomly allocated to different treatment groups. The COCs were cultured at 39 °C for 0 or 4 h in the control groups (without HS) and at 41.5°C for 1, 2, or 4 h in the HS groups. The IVM-derived oocytes were HS for 4 h and were fixed in MTSB-XF at 1, 2, 4, 8 and 16 h following HS for immunocytochemical staining or confocal microscopy. The average maturation rates of the oocytes were 60% (n=1570) and 42% (n=300) in pig and cattle oocytes, respectively. Some oocytes revealed chromatin abnormality in 1 h (34/113) or 2 h (53/104) of HS treatment. For the oocytes subjected to 4 h of HS, dispersed chromatin clusters with severe condensation occurred in both pig and cattle oocytes. Disassembly or complete disappearance of spindle MT (85%, n=111) and formation of cytoplasmic MT (87%, n=111) were also clearly observed 4 h after HS. Eight to sixteen hours after HS, resumption of MII chromosome and spindle morphology was only observed in 21% (7/34) and 24-32% (n=34) of oocytes, respectively. Around 91-97% of the HS oocytes showed cytoplasmic MT. In conclusion, a short-term or acute HS condenses the chromatins and disassembles spindle MT in the IVM-derived pig and cattle oocytes. Only a small proportion of HS oocytes were capable of recovering from the altered chromosomal and cytoskeletal configurations after prolonged culture at 39°C. These alterations may contribute to the reduced fertility in domestic species during the hot season.

目錄
頁次
摘要 ------------------------------------------------------------------------------------------1
前言 ----------------------------------------------------------------------------------------3
文獻檢討 --------------------------------------------------------------------------------------5
壹、 卵母細胞之生長與成熟 -------------------------------------------------------------5
一、卵巢濾泡之發育 ----------------------------------------------------------------------5
二、卵母細胞之成熟 -------------------------------------------------7
三、 調控卵母細胞成熟之因子 ----------------------------------------------------8
貳、細胞骨架（cytoskeleton）---------------------------------------------------------------10
一、 細胞骨架之組成與功能-------------------------------------------------------------10
（一） 微管（microtubule）-------------------------------------------------------------10
（二） 微絲（microfilament）----------------------------------------------------------13
（三） 中間絲（intermediate filament）----------------------------------------------14
參、 紡錘體微管之組成與分離--------------------------------------------------------------15
肆、 卵母細胞成熟、受精及後續發育其細胞骨架之變化- -----------------------------19
伍、熱緊迫對卵母細胞與其細胞骨架之影響----------------------------------------------21
研究試驗
試驗壹、熱處理對牛卵母細胞染色體與細胞骨架之影響-----------------------------23
一、前言 -------------------------------------------------------------------------------23
二、材料與方法 ----------------------------------------------------------------------24
三、結果 -------------------------------------------------------------------------------35
四、討論 -------------------------------------------------------------------------------46
試驗貳、豬卵母細胞經熱處理後染色體與細胞骨架形態之變化--------------------48
一、前言 -------------------------------------------------------------------------------48
二、材料與方法 ----------------------------------------------------------------------49
三、結果 -------------------------------------------------------------------------------53
四、討論 -------------------------------------------------------------------------------64
試驗參、熱緊迫造成豬卵母細胞之染色質（體）與細胞骨架變化之可逆性----67
一、前言 --------------------------------------------------------------67
二、材料與方法-------------------------------------------------------68
三、結果 -------------------------------------------------------------------------------70
四、討論 -------------------------------------------------------------------------------79
試驗肆、利用雷射掃瞄共軛焦顯微鏡觀察高溫對豬卵母細胞染色體與細胞骨架
形態之影響----------------------------------------------------------------------- 81
一、前言 -----------------------------------------------------------------------------81
二、材料與方法 ----------------------------------------------------------------------82
三、結果 -------------------------------------------------------------------------------84
四、討論 -------------------------------------------------------------------------------88
結論 -------------------------------------------------------------------------------------------90
參考文獻 -------------------------------------------------------------------------------------91
英文摘要 ------------------------------------------------------------------------------------101
圖次
頁次
圖 1. 微管及微絲之結構------------------------------------------------------------11
圖 2. 染色體利用微管排列於赤道板之過程-------------------------------------16
圖 3. 動物細胞紡錘體之構造-----------------------------------------------------17
圖 4. 成熟卵母細胞紡錘體微管形態之分類----------------------------------------------33
圖 5. 不同紡錘體分類之影像分析圖-------------------------------------------------------34
圖 6. 熱緊迫對牛卵母細胞染色質（體）之影響----------------------------------36
圖 7. 熱緊迫對牛卵紡錘體微管之影響--------------------------------------------------38
圖 8. 熱緊迫對牛卵透明帶肌動蛋白、卵膜環及胞質微絲之影響--------------------42
圖 9. 熱緊迫對牛卵母細胞透明帶肌動蛋白之變化-------------------------------------43
圖 10. 熱緊迫對牛卵母細胞卵膜環之變化-----------------------------------------------44
圖 11. 熱緊迫對牛卵母細胞胞質微絲之變化--------------------------------------------45
圖 12. 熱緊迫對豬卵母細胞染色質（體）之影響-----------------------------------------54
圖 13. 熱緊迫對豬卵母細胞紡錘體微管之影響-----------------------------------------55
圖 14. 熱緊迫對豬卵母細胞透明帶肌動蛋白、卵膜環及胞質微絲之影響----------60
圖 15. 熱緊迫對豬卵母細胞透明帶肌動蛋白之變化-----------------------------------61
圖 16. 熱緊迫對豬卵母細胞卵膜環之變化-----------------------------------------------62
圖 17. 熱緊迫對豬卵母細胞胞質微絲之變化--------------------------------------------63
圖 18. 熱緊迫對豬卵母細胞染色體（質）與紡錘體微管影響之可逆性--------------72
圖 19. 熱緊迫對豬卵母細胞透明帶肌動蛋白、卵膜環及胞質微絲影響之可逆性--------------------------------------------------------------------------------------------75
圖 20. 熱緊迫對豬卵母細胞透明帶肌動蛋白影響之可逆性--------------------------76
圖 21. 熱緊迫對豬卵母細胞卵膜環影響之可逆性--------------------------------------77
圖 22. 熱緊迫對豬卵母細胞胞質微絲影響之可逆性-----------------------------------78
圖 23. 以共軛焦顯微鏡觀察熱緊迫對豬卵子細胞核與細胞骨架形態之影響-----85
圖 24. 熱緊迫對豬卵母細胞胞質微絲密度之影響--------------------------------------87
表次
頁次
表1. 牛卵母細胞體外成熟培養液之組成分-------------------------------------25
表 2. 熱緊迫對牛卵母細胞染色質（體）、紡錘體與胞質微管形態之影響-----37
表 3. 熱緊迫對牛卵母細胞紡錘體形態與螢光強度之變化----------------------------40
表 4. NCSU#23基礎培養液之組成分---------------------------------------------50
表 5. 熱緊迫對豬卵母細胞染色質（體）、紡錘體與胞質微管形態之影響-----56
表 6. 熱緊迫對豬卵母細胞紡錘體形態與螢光強度之變化----------------------------58
表 7. 熱緊迫對豬卵母細胞核與紡錘體微管形態之影響-------------------------------74

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