热应激对猪睾丸Cyt-C和Caspase-3表达的影响

doi:10.3864/j.issn.0578-1752.2017.05.015

摘要/Abstract

摘要： 【目的】环境高温影响猪精子发生和精液品质，但其分子机制尚不清楚。为此探索环境温度升高导致的热应激对猪睾丸细胞凋亡调节蛋白Cyt-C和Caspase-3表达的影响。【方法】性成熟长白公猪9头，对照组3头，饲养在20—27℃的猪舍环境；短期热应激处理组（heat stress 7 d，HS7d）3头，每天置37—40℃的猪舍环境3 h，连续7 d；一个精子发生周期热应激处理组（heat stress 42 d，HS42d）3头，每天置37—40℃的猪舍环境3 h，连续42 d；每天于热应激处理后将猪驱赶回20—27℃的猪舍。手术摘取睾丸组织，用QRT-PCR、Western blot和免疫组织化学方法检测Cyt-C和Caspase-3表达与定位的变化。【结果】 QPT-PCR结果显示，与对照组相比，热应激处理7 d组和42 d组，Cyt-C和Caspase-3mRNA的相对表达量均显著升高；热应激处理7 d组Cyt-C和Caspase-3mRNA的相对表达量最高。Western blot结果显示，热应激处理7 d组和42 d组Cyt-C和Caspase-3蛋白水平与对照组相比均显著升高；热应激处理7 d组Cyt-C和Caspase-3的蛋白表达水平最高。免疫组织化学研究发现，Cyt-C在猪睾丸组织中免疫反应阳性物定位于间质细胞、支持细胞和各个发育阶段生精细胞的胞质中，在生精细胞Cyt-C低表达于精原细胞，高表达于减数分裂后精母细胞和精子细胞。热应激处理导致Cyt-C的胞质内定位更加弥散，提示Cyt-C从线粒体到胞质的释放。Caspase-3在间质细胞和精原细胞低表达，大量减数分裂后的生精细胞呈Caspase-3阳性。与对照组相比，热应激处理导致部分支持细胞Caspase-3的表达，大量生精细胞的细胞核呈Caspase-3阳性着色。【结论】高温热应激处理导致猪睾丸Cyt-C和Caspase-3的蛋白和mRNA表达水平升高、细胞定位改变，提示Cyt-C和Caspase-3可能与热应激导致的猪精液品质下降存在关联。

关键词: 热应激, 公猪, 睾丸, Cyt-C, Caspase-3

Abstract: 【Objective】 Elevated ambient temperature has detrimental effects on sperm quality in mammalian species including pig. But the molecular mechanism has not yet well understood. The aims of present study were to investigate the effect of heat stress on the expression of Cyt-C and Caspase-3 in boar testis. 【Method】 Nine mature boars (Landrace, 18 months of age) were used in the study. Three boars were subjected to 37-40℃ for 3 h daily for 7 days (heat stress 7 d, HS7d). Three boars were subjected to 37-40℃ for 3 h daily for 42 days (heat stress 42 d, HS42d). Following daily heat treatment the boars were driven back to normal house (20-27℃). The other 3 boars were kept in the normal house as control group (20-27℃). All boars were castrated and the testes were harvested. Western blot, QRT-PCR, and immunohistochemistry were used to analyze the expression of Cyt-C and Caspase-3. 【Result】 QRT-PCR showed that mRNA expression of Cyt-C and Caspase-3 were all increased in HS7d group and HS42d group compared with the control. And the relative expression of Cyt-C and Caspase-3mRNA in HS7d group was the highest. Western blot showed that the protein expression of Cyt-C and Caspase-3 was all increased in HS7d and HS42d as well, compared with the control group. And the protein expression of Cyt-C and Caspase-3 in HS7d group was the highest. Immunohistochemistry found that Cyt-C localized in the cytoplasm of Leydig cells, Sertoli cells and all stages of germ cells. In spermatogonia, the Cyt-C expression was low, while in postmeiosis germ cells, the expression of Cyt-C was higher. Heat treatment resulted in a more diffuse cytoplasmic localization of Cyt-C, which suggested that Cyt-C was released from mitochondria to the cytoplasm. In Leydig cells and spermatogonia, the Caspase-3 expression was low. A large number of postmeiosis germ cells showed Caspase-3 positive immunoreactive staining. Heat stress resulted in some Sertoli cells Caspase-3 positive staining and Caspase-3 positive with nucleus staining increased.【Conclusion】Elevated ambient temperature induced the increase of the expression of Cyt-C and Caspase-3, and the changes of cellular location, which indicated that the expression change of Cyt-C and Caspase-3 may be associated with the sperm quality decrease induced by heat stress.

Key words: heat stress, boar, testis, Cyt-C, Caspase-3

范小瑞，张禛，席华明，梁亚俊，贺俊平. 热应激对猪睾丸Cyt-C和Caspase-3表达的影响[J]. 中国农业科学, 2017, 50(5): 924-931.

FAN XiaoRui, ZHANG Zhen, XI HuaMing, LIANG YaJun, HE JunPing. Effect of Heat Stress on the Expression of Cyt-C and Caspase-3 in Boar Testis[J]. Scientia Agricultura Sinica, 2017, 50(5): 924-931.

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