线粒体信号通路在热应激猪睾丸生精细胞凋亡中的作用机制研究

Mammalian spermatogenesis was regulated by a variety of factors, including hormones and growth factors. The heat stress had detrimental effect on semen quality and spermatogenesis in the boar, but the mechanism remains unclear. Study of experimental animal rat found that heat stress could lead to the apoptosis of spermatogenic cells. Mitochondria signal pathway was one important regultor of apoptosis. Whether or not heat stress starting spermatogenic cell apoptosis by the mitochondria signal pathway, thereby affecting pig spermatogenesis, remains unclear as well. In this study, heat stress porcine testis was used as material. The apoptotic cell type and quantity was detected by TUNEL method. Apoptotic body and mitochondrial was observed by transmission electron microscopy. The expression of mitochondrial signal pathway members bcl-2, bax, cyt-c, caspase-3 were detected by QRT-PCR technique. The expression of mitochondrial pathway related proteins was analysed by Western Blot techinque. Localization of apoptotic related protein of mitochondrial signal pathway members in boar testis were conducted as well. The present study was designed to prove up the function mechanism of mitochondrial pathway in heat stress induced germ cell apoptosis in the boar, and to provide theoretical basis and molecular target for the management of heat stress's detrimental effect on spermatogenesis in the boar.

哺乳动物精子发生受到包括激素和生长因子等多种因素的调控，热应激严重损害猪睾丸精子发生和精液品质，但其分子机制尚不清楚。对实验动物大鼠等的研究发现，热应激会导致生精细胞凋亡，线粒体信号通路是细胞凋亡的重要调节途径。热应激是否经由线粒体信号通路起始生精细胞凋亡，进而影响猪精子发生，未见研究报道。本研究以热应激猪睾丸组织为材料，用TUNEL法原位检测生精细胞凋亡的类型和数量；用透射电镜技术观察凋亡细胞凋亡小体和线粒体结构的变化；用QRT-PCR技术定量线粒体信号通路成员bcl-2, bax, cyt-c, caspase-3 mRNA的表达水平；用Western Blot技术分析线粒体信号通路凋亡相关蛋白的表达；用免疫组织化学技术确定线粒体信号通路凋亡相关蛋白在猪睾丸的组织细胞定位。以期探明线粒体信号通路在热应激致猪睾丸生精细胞凋亡中的作用机制，为热应激损伤猪精子发生提供分子靶标和理论依据。

热应激严重损害猪睾丸精子发生和精液品质，但其分子机制尚不清楚。本研究的目的是探索热应激是否经由线粒体信号通路起始生精细胞凋亡，进而影响猪精子发生。本研究构建了热应激损伤猪精子发生模型。性成熟长白公猪，经环境热应激处理（每天置37～40ºC的猪舍环境3h或40～42ºC的猪舍环境1 h，实验处理时间为7 d 和42 d）或睾丸局部42ºC热刺激处理1 h。对照组饲养在20-27ºC的猪舍环境。取材睾丸组织，做基因和蛋白表达分析。结果发现：1、当猪舍温度接近或超过体温时（37-40ºC），阴囊表面温度可接近体温（32.5-37.5 ºC）；2、环境热应激处理导致精液品质下降，表现为精子活力降低、畸形率增加、顶体完整性降低；3、TUNEL结果显示，热应激处理组生精细胞凋亡率增加，精母细胞、圆形和长形精子细胞对热刺激敏感；4、电镜结果显示，热处理导致生精细胞染色质聚集，线粒体扩张；5、QRT-PCR和Western Blotting结果显示，热应激处理不同程度的导致凋亡调节蛋白Bax、Bak、Cyt-C、Apaf-1、Caspase-9、Caspase-3表达水平的升高；细胞凋亡抑制蛋白Bcl-2和Bclw表达也呈上调变化，但未能抑制细胞凋亡的增加。6、免疫组织化学研究显示，热应激处理导致Bax在精原细胞、精母细胞和精子细胞的重新分配，从细胞质转移至核旁区。Bcl-2倾向于近管腔部位生殖细胞表达。热应激处理导致Cyt-C的胞质内定位更加弥散，精母细胞和部分支持细胞Apaf-1阳性反应增强，减数分裂以后的生精细胞和支持细胞Caspase-9表达量升高，大量生精细胞的细胞核呈Caspase-3阳性着色；7、QRT-PCR、Western Blotting和免疫组织化学结果显示，热应激处理导致TGF-β3mRNA和蛋白的相对表达量升高，而Claudin-11mRNA和蛋白质相对表达量下降；8、QRT-PCR和Western Blotting结果显示，热应激导致GLUT8mRNA和蛋白表达水平下降；免疫组织化学研究显示，各阶段生精细胞GLUT8的表达均下降。结果提示：环境高温热应激和睾丸局部热处理均诱导猪睾丸生殖细胞凋亡率增加，线粒体信号通路凋亡相关蛋白的表达发生改变，线粒体信号通路参与热应激猪睾丸生精细胞凋亡。