請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/24938
標題: 高溫對豬卵母細胞p38 MAPK訊息傳遞徑路之影響
The 38 MAPK signaling pathways in porcine oocytes after in vitro thermal stress
作者: 顏詩穎
Yen, Shih-Ying
關鍵字: heat shock
胞裂素活化蛋白質激酶卵母細胞
MAPK
oocyte
出版社: 動物科學系所
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摘要: 哺乳動物卵母細胞之成熟除內、外源性激素之影響外,其胞內分子訊息傳遞亦扮演著關鍵角色。高溫環境為造成家畜於熱季低繁殖率重要因素之一,而不同程度之溫度變化亦可能導致卵母細胞內訊息傳遞徑路之改變。因此本研究目的為探討熱緊迫(heat shock, HS)對豬卵母細胞MAPK(mitogen-activated protein kinase)superfamily蛋白質表現之影響,並針對p38/MAPKAPK2徑路之活性變化以期進一步釐清卵子抗熱機制。試驗一,將成熟卵子逢機分配至對照組(Control, 39℃經0或4 h)與熱處理組(HS, 41.5℃經1, 2或4 h)後以西方吸漬法分析卵子內ERK、p90rsk、JNK與p38活性之變化。結果顯示磷酸化ERK(p-ERK)之活性於各組間雖無顯著差異但隨熱處理時間延長而有微上升之趨勢,磷酸化p38(p-p38)之活性在熱處理1 h(HS1)顯著下降(53% vs. 100%)後其活性隨熱處裡時間延長至4 h(HS4,84%)顯著上升(P < 0.05),p38總蛋白(total p38)之表現量則隨熱處理時間延長至2 h或4 h時顯著上升(P < 0.05)。試驗二,進一步以免疫細胞化學染色後,發現p-p38之活性隨體外成熟培養期間延長而增加(P < 0.05),並於熱緊迫1 h後其活性顯著下降,且隨熱處理延長至2 h或4 h而顯著上升(P < 0.05)。試驗三,以添加10 μM之p38專一性抑制劑SB203580於培養液中,結果顯示其雖不影響卵母細胞GVBD(germinal vesicle breakdown),但可顯著抑制卵子成熟(41.9% vs. 79.7%;P < 0.05)。另外,發現成熟卵子在熱處理過程中添加10 μM SB203580無法抑制p38下游MAPKAPK2之活性。根據以上結果顯示,p38除調控緊迫反應外亦參與豬卵之成熟過程,且p38與ERK活性之變化可能為調控豬卵母細胞於高溫環境下走向凋亡或存活之重要角色。
Maturation of mammalian oocytes depends on both the internal and external hormonal milieu, as well as the intracellular molecular signaling. Elevated ambient temperature have been one of the major factors responsible for the reduced fertility in farm animals. It has also been suggested that oocytes either tend to survive or undergo apoptosis depending on the altered signal transduction pathway. The purposes of this study were to elucidate the the potential role of MAPK (mitogen-activated protein kinase) superfamily changes of oocytes under the influence of heat shock (HS), and focused on the activity of p38/MAPKAPK2 pathway in relation to the thermal resistance of porcine oocytes. In Experiment 1, matured porcine oocytes were selected randomly allocated to control groups (39℃ for 0 or 4 h) and HS groups (41.5℃ for 1, 2 or 4 h) and investigated the effect of different treatment groups on the ERK, p90rsk, JNK and p38 expressions. The activity of phosphorylated ERK (p-ERK) in matured oocytes was not significantly different among all time points, however, the expression increased gradually as time periods of heat shock prolonged. In contrast, phosphorylated p38 (p-p38) activity was significantly decreased after exposed to heat shock 1 h (HS1) compared with control 0 h (C0, 53% vs. 100%) and increased again in 4 h (HS4, 84%; P < 0.05). Total p38 protein level was significantly increased in HS2 and HS4 (P < 0.05) compared with C0. In Experiment 2, the results of immunocytochemical staining showed that the activity of p-p38 were significantly increased (P < 0.05) depending on the stage of nuclear maturation, and significantly decreased in HS1 (P < 0.05) then increased again when the heat shock is prolonged, especially in HS2 or HS4 (P < 0.05). In Experiment 3, supplement with a specific inhibitor of p38, SB203508, at 10 μM for maturation did not alter the proportion of oocytes undergoing GVBD (germinal vesicle breakdown), but it reduced the proportion of MII oocytes was reduced from 79.7% (Control) to 41.9% (P < 0.05). When matured oocytes were further cocultured with 10 μM SB203580 at different HS conditions, the expression of p-MAPKAPK2 (a downstream molecule of p38) was not inhibited under the prolonged HS. These results suggest that p38 might involve porcine oocyte maturation and the activities of p38 and ERK played a role in regulation of oocyte apoptosis or survival under HS condition.
URI: http://hdl.handle.net/11455/24938
其他識別: U0005-0902200911384800
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-0902200911384800
顯示於類別:動物科學系

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