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A study on the relationship between heat shock proteins and sperm motility
|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:||
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.
|Appears in Collections:||動物科學系|
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