猪卵母细胞体外成熟过程中细胞骨架之变化与激活后之发...

猪卵母细胞体外成熟过程中细胞骨架之变化与激活后之发...猪卵母细胞体外成熟过程中细胞骨架之变化与激活后之发... 好风光好风光恢复供货才豬卵母細胞體外成熟過程中細胞骨架之變化與激活後之發育蔡摯周伯蒨葉盛伯陳建宏陳宗賢曾榮凱朱志成國立中興大學畜產學系本研究目的在於建立良好之豬卵母細胞體外成熟(In vitro maturation,IVM)、激活 (activation)及其後續發育之培養系統,並觀察卵母細胞於成熟過程中細胞骨架之變化。自卵巢之表面濾泡(2~8mm)收集卵丘卵母細胞複合體(COCs)後,置於 NCSU-23+PMSG+hC...

猪卵母细胞体外成熟过程中细胞骨架之变化与激活后之发... 好风光好风光恢复供货才豬卵母細胞體外成熟過程中細胞骨架之變化與激活後之發育蔡摯周伯蒨葉盛伯陳建宏陳宗賢曾榮凱朱志成國立中興大學畜產學系本研究目的在於建立良好之豬卵母細胞體外成熟(In vitro maturation,IVM)、激活 (activation)及其後續發育之培養系統,並觀察卵母細胞於成熟過程中細胞骨架之變化。自卵巢之表面濾泡(2~8mm)收集卵丘卵母細胞複合體(COCs)後,置於 NCSU-23+PMSG+hCG培養液中成熟培養,成熟之卵子予以激活後觀察其後續之發育。試驗一將COCs於體外經0、11、22、33或44 h成熟培養後,分別將卵母細胞以免疫化學染色法觀察細胞核及細胞骨架之變化。試驗二將成熟之卵母細胞以一或兩次電激處理(electrical pulse,EP,1.0 KV/cm,60μsec)或以化學處理 (Thimerosal+dithiothreitol)方式激活,以觀察其體外發育之能力。結果顯示甫自卵巢取出之卵母細胞皆處於原泡核期,germinal vesicle stage, GV,,其中以雙染色體期(diplotene)之後期為主要階段(72%),當卵母細胞培養11h之後開始其原泡核崩解(GVBD)、22~33h至MI/AI期,而33 h後約43%之卵母細胞可發育至M?期, 大部份卵子(75%)經IVM 44h已成熟達M?期。卵母細胞成熟過程中細胞質微管 (microtubules)與紡錘體微管相互消長,惟卵黃膜於各處理組間無明顯變化。卵子經各種不同方式激活後,以兩次電激處理組之分裂率顯著優於對照組及化學處理組者(73% vs. 8.3及1.2%,p<0.05)。由以上結果顯示,卵母細胞之細胞核與細胞骨架於體外成熟過程經歷明顯之重組,而電激可有效激活體外成熟之豬卵子,並可發育至類桑椹期胚之階段。關鍵語,卵母細胞、孤雌發育、細胞骨架 CYTOSKELETAL CHANGES DURING MATURATION AND PARTHENOGENESIS OF IN VITRO-DERIVED PIG OOCYTES C. Tsay, P. C. Chou, S. P. Yeh, C. H. Chen, T. H. Chen, J. K. Tseng, J. C. Ju Department of Animal Science, National Chung-Hsing University The purposes of this study were to optimize in vitro culture systems for pig oocytes and embryos and to examine the cytoskeletal changes during in vitro maturation (IVM). Cumulus-oocyte complexes (COCs) were collected from slaughterhouse ovaries by follicle (2-8 mm) aspiration and were matured in NCSU-23 medium for 0, 11, 22, 33, and 44 h. After removal of cumulus cells at each time point, oocytes were fixed and stained for microtubules and microfilaments using anti-tubulins and rhodamine- pholloidin. Matured oocytes were activated by either thimerosal (200,M,10min)+ Dithiothreitol (DTT, 8 mM, 30min) combined treatment or electrical pulses (1 or 2 consecutive EPs, 1.0kV/cm, 60 ,sec). Results showed that most oocytes stayed at germinal vesicle stage immediately after retrieval from the follicle. There were 43% and 75% oocytes progressed to MII stage at 33 and 44 h post-IVM, respectively. Dynamic shift of spindle and cytoplasmic microtubules was evident, but no significant changes were observed in the vitelline ring of maturing oocytes. Cleavage or developmental rate of double EP-activated oocytes was 73%, which was significantly higher than that in the control (9%) and the thimerosal/DTT (4%) treated groups (P<0.05). These results suggest that IVM pig oocytes subject to cytoskeletal reorganization in process of maturation and the electrical pulse appears to be a better activation treatment than the thimerosal/DTT in this study. Key Words: Oocyte, Parthenogenesis, Cytoskeleton.

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