热应激对猪睾丸死亡受体Fas及其配体FasL表达与定位的影响

Effect of Heat Stress on the Expression and Location of Death Receptor Fas and Its Ligand FasL in Boar Testis

猪睾丸内精子发生与精液质量受高温影响,但相关分子机制的报道尚未给出完整定论。利用9头14月龄性成熟长白公猪随机建立3组热应激模型,每组3头,分别为对照组、环境热应激组、局部热应激组,其中,对照组不作任何处理,室温(20～25℃)圈养在猪舍中,每天正常给水、喂食;环境热应激组每日将猪饲养在37～40℃猪舍中3 h,结束后赶回室温猪舍,连续处理7 d;局部热应激组的猪用自制的可控温加热垫覆盖在左侧睾丸阴囊表面(42℃加热1 h)。各组猪睾丸处理结束后,迅速将猪睾丸组织摘取,一部分组织切成1.5 cm3的3小块置于Bouin液固定,用于石蜡组织切片的制作;剩余部分冻存于液氮,用于总RNA、总蛋白的提取。Fas,FasL通过qRT-PCR与Western Blotting检测m RNA与蛋白的相对表达量,通过免疫组织化学染色观察生殖细胞的定位。研究旨在探索环境高温和阴囊局部热刺激导致的死亡受体Fas及其配体FasL表达与定位的影响。结果表明,qRT-PCR检验结果显示,FasmRNA相对表达量与对照组相比,环境热应激组升高、局部加热组显著升高;与对照组相比,环境加热组中FasLm RNA相对表达量显著降低、局部加热组显著升高。Western Blotting检测结果显示,与对照组相比,Fas蛋白的相对表达量环境加热组与局部加热组均升高,其中,局部加热组显著升高;与对照组比较,FasL蛋白在环境加热组的相对表达量显著降低,局部加热组显著升高。免疫组织化学结果显示,Fas免疫反应阳性物主要定位在各级生精细胞,热应激导致其阳性表达增强,且局部加热组表达量最高;FasL主要定位在支持细胞、精母细胞以及圆形精子细胞,与对照组相比,局部加热导致其表达量升高,环境加热导致其表达量降低。结果说明,局部热刺激条件下,支持细胞FasL的表达量升高,以旁分泌的方式作用于邻近生精细胞膜上的膜受体Fas,引发细胞凋亡;环境热应激组未见FasL的升高,可能是因为以不依赖于细胞凋亡通路Fas/FasL的方式进行的。

It is have not been reported as integrated that the related molecular mechanism of high temperature heat stress seriously affects spermatogenesis and quality of semen in pigs. The aims of present study was to investigate the effects of high environmental temperature and local heat stress on the expression and localization of Fas and its ligand FasL in boar testis. 9 healthy boars were divided into 3 groups, randomly. 3 boars were kept in a controlled temperature house as an environmental heat stress group(EHS,37-40 ℃,3 h,7 d). 3 boars left side testis were wrapped thermostatic heating pad as a local heat stress group(LHS,42 ℃,1 h), then drive the boars back to a room temperature house(20-27 ℃). 3 boars were kept in a room temperature house(20-27 ℃) as a control group. The expression and localization of Fas/FasL were detected by qRT-PCR, Western blotting and immunohistochemical staining. qRT-PCR showed that the relative expression of FasmRNA was higher than EHS and significantly higher in LHS compared with control group. The relative expression of FasLmRNA in EHS significantly decreased, and LHS significantly increased. Western blotting showed that the relative expression level of Fas protein increased in EHS and LHS compared with the control, while the LHS increased significantly. The relative expression of FasL protein in EHS significantly reduced, and in LHS significantly increased compared with control group. Immunohistochemistry found that Fas postive immunoreactivity was observed in all stages of germ cells. Heat stress resulted in the postive immunoreactivity of Fas increased. And the expression of Fas in LHS group was the highest. FasL was localized in the Sertoli cells, spermatocyte and round spermatid. The expression levels of FasL was increased in LHS group, while decreased in EHS group compared with the control. The results indicated that the expression of FasL in Sertoli cells increased under local heat stress, and the cell apoptosis was induced by paracrine acting on the membrane receptor Fas on the adjacent spermatogenic cell membrane. The increase of FasL was not observed in the group of environmental heat stress, which might be independent of the pathway Fas/FasL of apoptosis.