Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/24498
標題: 熱緊迫蛋白質與公豬精子活力關係之研究
A study on the relationship between heat shock proteins and sperm motility
作者: 黃三元
Huang, San-Yuan
關鍵字: heat shock proteins;熱緊迫蛋白質;sperm motility;boars;精子活力;公豬
出版社: 畜產學系
摘要: 
夏季熱緊迫與冷卻、冷凍處理均顯著地影響公豬精子活力,進而降低其生殖力。過去的研究已證實70kDa熱緊迫蛋白質(HSP70)對熱緊迫傷害具保護效果,而90kDa熱緊迫蛋白質(HSP90)與一些和精子活力有關的因子都會有交互作用,因此,進一步探討研究HSP70及HSP90與公豬精液性狀及精子活力間的可能關係,或可作為未來改善公豬繁殖力之基礎。本研究之目的包括(一)探討公豬在不同季節之精液品質與HSP70之關係及(二)探討HSP90與公豬精子活力之關係,並推論其在公豬精子活力調控所扮演之角色。
在探討HSP70含量與精液品質關係之研究中,共使用29頭(13頭杜洛克、9頭藍瑞斯及7頭約克夏)性成熟公豬(平均月齡25.2±2.2月),每一頭公豬均分別在涼季與熱季收集3-4個精液樣品進行分析,精液品質性狀包括精子活力、正常與不正常精子百分比、頸部與中片部含原生質滴精子百分比及精子濃度。不同季節與不同品種在精液品質方面均有顯著差異,藍瑞斯公豬之精液性狀顯著地優於約克夏與杜洛克公豬者(P<0.05),熱季之精液品質顯著地較涼季為差(P<0.05)。精子蛋白質樣式以聚丙醯膠體電泳(SDS-PAGE)進行分析,HSP70之定量以西方免疫吸漬法與雷射光密度掃瞄儀進行。以單向SDS-PAGE來看時,不同季節間與不同品種的蛋白質樣式並無顯著之差異;西方免疫吸漬分析的結果顯示,組成性與誘發性形式的HSP70均存在於公豬精子,同季節內品種間的HSP70量無顯著差異,但是三個品種公豬精子的HSP70在熱季時均顯著地低於涼季者(P<0.05)。當所有樣品根據HSP70含量進行分群時,精液品質有隨著HSP70含量減少而降低的趨勢。此等結果顯示,公豬精子內HSP70的含量在熱季時會顯著地減少,此減少可能與精液品質的降低有關。
公豬精液冷凍後的精子活力下降可能由細胞內蛋白質變化所引起,因此,進一步之研究在評估公豬精子在冷卻過程中與冷凍保存後蛋白質樣式之改變。九頭性成熟公豬(平均年齡25.5±12.3月)之精液樣品於冷卻前、冷卻至15℃、冷卻至5℃、冷凍至-100℃再解凍及-196℃冷凍保存一週後再解凍等各階段分別採樣評估精液性狀,並進行精子蛋白質分析。精液性狀包括精子活力與形態正常精子百分比;5×106個精子之總蛋白質以SDS-PAGE進行分析。結果顯示冷凍解凍後的精子內,其90kDa蛋白質有明顯的減少,西方免疫吸漬分析證實此蛋白質為HSP90;時間序列分析顯示HSP90的減少發生在5℃冷卻的第一小時內,與冷卻前的新鮮精子比較時,冷卻到5℃的精子內之HSP90減少64%;但是,精子活力與正常精子百分比在此處理期間則無顯著下降,兩者均要冷凍至-100℃再解凍後才有明顯的降低。此結果顯示HSP90的降低早於精液性狀的下降,HSP90降低與精液性狀下降間的時間落差估計約2-3小時。此部份之結果顯示公豬精子HSP90的明顯降低與冷卻過程中精子活力的降低有關。
哺乳動物精子活力的分子調控機制仍不清楚。而根據前述精液冷卻與冷凍之結果顯示HSP90可能與公豬精子活力有關,因此,接續的研究乃應用HSP90特定抑制藥物膠達納黴素(geldanamycin, GA),進一步探討HSP90之獨特功能對精子活力的影響。試驗用精液取自正常的性成熟公豬,且精子活力大於80%者。稀釋後的精液加入0.5、1.0、2.5或5.0 μg/ml之GA,在37℃下培養15、30、45或60分鐘,培養結束後以電腦輔助精液分析儀測定精子活力。結果顯示,精子活力隨著GA添加劑量的增加與處理時間的延長而顯著地降低;而且,精液添加5.0 μg/ml GA並培養15分鐘後其精子完全失去活力。為了進一步檢測受GA處理的精子活力之可回復性,精液在與GA培養30分鐘後,將GA移去再加入新鮮的稀釋液或含5 mM咖啡因的稀釋液,然後再培養15、30、45或60分鐘。結果顯示,只將GA移去並無法使精子活力回復;相對地,添加咖啡因可回復部份精子活力;然而,添加添加咖啡因並無法回復添加2.5或5.0 μg/ml GA對於精子活力的抑制效應。基於GA能與HSP90結合的特異性,本研究結果顯示HSP90在公豬精子活力的調控上可能扮演重要角色。

Heat stress in summer, cooling and freezing treatment can significantly affect porcine sperm motility and then fertility. Many investigations have shown that 70 kDa heat shock protein (HSP70) plays a protective role in heat stress response. The 90 kDa heat shock protein (HSP90) is reported to interact with factors associated with sperm motility. These results raised our interest in exploring the possible relationship of HSP70 and HSP90 with semen quality traits including sperm motility. These results may be served as fundamentals for improving boar fertility in the future. The purposes of this study included: 1. To evaluate the relationship between HSP70 and semen quality in boars in different seasons; and 2. To evaluate the relationship between HSP90 and sperm motility in boar semen and to reveal its role in the regulation of sperm motility.
To evaluate the relationship between HSP70 and semen quality, 29 (13 Duroc, 9 Landrace, and 7 Yorkshire) sexually matured boars (mean age=25.2±2.2 months) were examined. Three to four ejaculates per boar were collected in both cool and hot seasons. Semen quality traits included sperm motility, percent of normal and abnormal sperm, percent of sperm with proximal and distal plasma droplets, and sperm concentration. There were significant seasonal and breed effects on semen quality. Landrace boars showed better semen quality than that in Yorkshire and Duroc boars (P<0.05). Semen quality declined significantly in hot season (P<0.05). The protein profiles of spermatozoa were determined by using sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The level of HSP70 was quantitated by Western blot analysis and laser densitometry. When visualized with one-dimensional SDS-PAGE, there was no significant difference in protein profiles between seasons and among breeds. The results of Western blot analysis demonstrated that both constitutive and inducible forms of HSP70 were present in boar spermatozoa. The level of HSP70 in spermatozoa was significantly lower in hot season in all three breeds (P<0.05). There was no significant difference in the level of HSP70 among breeds within season. When the samples were grouped according to the level of HSP70, the semen quality tended to decline significantly in samples with lower levels of HSP70. Taken together, the results of this study suggested that the levels of HSP70 in boar spermatozoa were significantly lower in hot season and might be associated with semen quality.
The decline in boar sperm motility after cryopreservation may be attributed to changes in intracellular proteins. Thus, the following study was to evaluate the change of protein profiles in boar spermatozoa during the process of cooling and after cryopreservation. A total of 9 sexually matured boars (mean age=25.5±12.3 mo) was used. Samples for protein analysis were collected before chilling, after cooling to 15℃, after cooling to 5℃, following thawing after freezing to -100℃, and following thawing after 1 wk of cryopreservation at -196℃. Semen characteristics evaluated included progressive motility and the percentage of morphologically normal spermatozoa. Total proteins from 5×106 spermatozoa were separated and analyzed by SDS-PAGE. The results revealed that there was a substantial decrease of a 90 kDa protein in the frozen-thawed spermatozoa. Western blot analysis demonstrated that this protein was HSP90. Time course study showed that the decrease of HSP90 in spermatozoa initially occurred in the first hour during cooling to 5℃. When compared with the fresh spermatozoa before chilling, there was a 64% decrease of HSP90 in spermatozoa after cooling to 5℃. However, the motility and percentage of normal spermatozoa did not significantly decrease during this period of treatment. Both declined substantially as the semen was thawed after freezing from -100℃. The results indicated that the decrease of HSP90 precedes the decline of semen characteristics. The time gap between the decrease of HSP90 and the decline in sperm motility was estimated to be 2 to 3 h. Taken together, the above results suggested that a substantial decrease of HSP90 might be associated with a decline in sperm motility during cooling of boar spermatozoa.
The molecular regulatory mechanisms of mammalian sperm motility are still largely undefined. The results of the above study on chilling and freezing semen suggested that HSP90 may be associated with porcine sperm motility. Thus, the next study was to further characterize the plausible novel function of HSP90 on sperm motility by applying an HSP90 specific inhibitor, geldanamycin (GA). Semen from normal sexually matured boars with sperm motility higher than 80% was used. GA was added to diluted semen at 0.5, 1.0, 2.5, or 5.0 μg/ml and the semen was incubated at 37℃ for 15, 30, 45, or 60 min. Sperm motility was determined by computer-assisted semen analyzer at the end of incubation. The result indicated that GA significantly reduced sperm motility in a dose and time dependent manner. Moreover, incubation of semen with 5.0 μg/ml GA for 15 min completely abolished sperm motility. To test the reversibility of the GA effect on sperm motility, GA was removed after 30 min incubation and replaced with fresh extender, or with the extender plus 5 mM caffeine, then incubated for another 15, 30, 45, or 60 min. The result showed that simply removing GA did not reverse the inhibitory effect of sperm motility. In contrast, this inhibitory effect of GA on sperm motility was partially reversed by adding caffeine. However, the effect of 2.5 or 5.0 μg/ml GA was not reversed by caffeine. Considering the specificity of GA targeting to HSP90, the above observations suggested that HSP90 may play a crucial role in regulating porcine sperm motility.
URI: http://hdl.handle.net/11455/24498
Appears in Collections:動物科學系

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