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標題: 熱緊迫對豬卵母細胞發育潛能及其細胞生理之影響
Alterations in developmental competence and cellular parameters of heat-shocked porcine oocytes
作者: 曾榮凱
Tseng, Jung-Kai
關鍵字: apoptosis;細胞凋亡;calcium;heat shock;hsp 70;pig oocyte;鈣離子;熱緊迫;熱誘蛋白70;豬卵母細胞
出版社: 動物科學系所
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本研究目的在於探討熱緊迫(heat shock)對成熟豬卵母細胞熱誘蛋白(heat shock protein, hsp)70之表現、細胞凋亡(apoptosis)及細胞內鈣離子濃度與其後續發育潛能等變化之生理機制。自屠宰場取得之女豬卵巢表面濾泡回收包被完整之卵丘卵母細胞複合體(cumulus-oocyte complexes, COCs),經NCSU-23培養液體外成熟42 h後,選取成熟卵母細胞逢機分配至對照組(Control, 39 ℃, 4 h)與熱處理組(HS, 41.5 ℃, 1, 2或4 h)。在試驗一中,各處理組以西方吸漬法分析hsp 70之表現,發現於熱處理組間並無顯著之差異,但以2 h熱處理(HS2h)組有較其他組高之趨勢;利用Annexin V與TUNEL偵測卵子發生細胞凋亡(apoptosis)之情形,其中以TUNEL方式並未偵測出成熟卵子之凋亡現象,推測可能與其genomic DNA尚未被致活(activation)及DNA之緻密結構有關。反之,以Annexin V偵測細胞凋亡則發現卵子細胞膜之螢光強度隨著培養時間延長而增加。當成熟卵子經熱處理後以2.2 kv/cm、30 µsec電刺激加上蛋白質激酶抑制劑6-dimethyaminopurine(2.5 µM)共激活4 h,其分裂率、囊胚率與囊胚細胞數顯著低於對照組(P<0.05),且隨加熱時間增長熱處理之效應更趨明顯。試驗二探討熱緊迫對卵子細胞內鈣離子釋放能力之影響。利用thimerosal (200 µM)或inositol 1,4,5-trisphosphate (0.5 mM)刺激卵子內鈣離子之釋放。試驗結果顯示,HS2h組之卵子經thimerosal或IP3刺激其鈣離子之釋放效果顯著高於其他各組者(P<0.05),但延長培養時間顯著降低鈣離子之釋放能力。試驗三乃探討細胞質與細胞核對熱緊迫之敏感性。分別將Control組與HS2h組卵子之染色體交換後再以thimerosal處理後,測定其鈣離子之釋放能力。結果發現細胞核或細胞質經熱處理後之重組胚,其鈣離子之釋放能力較未經熱處理者間互換之對照組為低,但其囊胚率卻顯著較佳(43-50% vs. 31%; P<0.05)。根據上述結果可知適度之熱緊迫可能提高胚之發育能力,但嚴重之高溫刺激可能經由造成細胞骨架、染色質之變化、細胞凋亡之發生或改變鈣離子釋放能力等變化而造成家畜之卵子或胚不同程度之傷害,可能因而影響其後續之發育能力。

The objectives of this study were to determine the effect of a short-term heat shock (HS) on the developmental competence and cellular physiology including expression of heat shock protein 70 (hsp 70), apoptosis and alteration of intracellular calcium concentration ([Ca2+]i) of the in vitro matured (IVM) porcine oocytes. Cumulus-oocyte-complexes (COCs) were collected from local abattoirs and were IVM in NCSU-23 medium for 42 h. Matured oocytes were selected and randomly allocated to different treatment groups. The COCs were cultured at 39 ℃ for 0 or 4 h in the control groups (without HS) and at 41.5 ℃ for 1, 2, or 4 h in the HS groups. The expression of hsp70 was not significantly different among all groups. TUNEL-positive signals (apoptosis) was not observed in the heated oocytes compared to the Controls, but the intensity of Annexin V-FITC signals increased with the duration of HS and in vitro culture. The cleavage and blastocyst rates were declined after more than 2h HS. In addition, The Ca2+ releasing ability of matured oocytes was enhanced by a shorter duration (2h) of HS, but it declined after prolonged heat exposure and in vitro culture. After spindles/chromosomes were exchanged between non-HS oocytes and HS2h oocyets, different sensitivity of the nucleus and the ooplasma was not clearly observed. The physiologic effects and mechanisms of HS on oocytes are complex processes. HS causes multiple changes of the oocyte including enzymatic reactions, ionic influxes, DNA structure and cytoskeleton, etc. Changes in the [Ca2+] of oocytes in response to signaling molecules after different intensities of HS may be important to evaluate their developmental competence. The delicate equilibrium between the deleterious effects and thermotolerance of oocytes or embryos in response to HS is a decisive factor determining their developmental destiny. This study provides clues for further investigations to clarify the mechanism of thermal resistance in oocytes. Further investigations will be focused on regulation of Ca2+-related kinases and looking into their encoding genes responsible for regulation of thermotolerance.
其他識別: U0005-2306200709241800
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