热应激通过钙信号调控湖羊卵巢颗粒细胞凋亡和雌激素合成

Regulation of heat stress on ovarian granulosa cell apoptosis and estrogen synthesis in Hu sheep through calcium signal

热应激严重影响动物的繁殖性能,但热应激是如何通过调控绵羊卵巢颗粒细胞功能进而调控绵羊繁殖的相关研究较少。本研究以湖羊卵巢颗粒细胞为研究对象,利用Real-time PCR、流式细胞技术、Western blot、ELISA等方法探讨热应激对湖羊卵巢颗粒细胞钙信号通路、细胞凋亡和雌激素合成的影响。结果表明,热应激后,湖羊卵巢颗粒细胞中钙离子浓度显著上升(P<0.05),钙离子信号通路中相关基因CACNA1B、CACNA1C、RYR1、CAMK2表达量显著升高,线粒体分裂蛋白FIS1表达量显著上升,促凋亡蛋白家族代表基因BAX和抗凋亡蛋白代表基因BCL2 mRNA表达量比值(BAX/BCL2)显著上升,细胞凋亡率增加;雌二醇合成关键酶CYP19A1表达量显著下调,雌二醇含量显著下降(P<0.05)。热应激卵巢颗粒细胞中加入钙离子螯合剂(BAPTA-AM)明显降低钙离子浓度,同时减少细胞凋亡,缓解热应激造成的卵巢颗粒细胞雌二醇含量下调。综上表明,热应激打开了湖羊卵巢颗粒细胞膜电压门控钙离子通道和内质网膜上钙离子释放通道,导致细胞质中钙离子浓度上升,促进卵巢颗粒细胞凋亡,引起雌二醇含量下调。

Heat stress seriously affects the reproductive performance of animals,but there are few studies on the effects of heat stress on sheep reproduction by regulating the function of sheep ovarian granulosa cells.In this study,Hu sheep ovarian granulosa cells were used as the research objects,and real-time PCR,flow cytometry,Western blot,ELISA and other methods were used to investigate the effects of heat stress on calcium signaling pathway,apoptosis and estrogen synthesis in Hu sheep ovarian granulosa cells.The results showed that after heat stress,the concentration of Ca^(2+)in Hu sheep ovarian granulosa cells increased significantly(P<0.05),the expression levels of calcium signaling pathway-related genes CACNA1B,CACNA1C,RYR1 and CAMK2 and mitochondrial fission protein FIS1 were significantly increased,the mRNA expression ratio of pro-apoptotic protein family representative gene BAX and anti-apoptotic protein family representative gene BCL2(BAX/BCL2)increased significantly,and the apoptosis rate increased.The expression of CYP19A1,the key enzyme in estradiol synthesis,was down-regulated,and the content of estradiol was significantly decreased(P<0.05).The addition of calcium chelator BAPTA-AM to heat-stressed ovarian granulosa cells significantly reduced Ca^(2+)concentration,decreased cell apoptosis,and alleviated the down-regulation of estradiol content in ovarian granulosa cells caused by heat stress.In conclusion,heat stress opens the voltage-gated calcium ion channel on the cell membrane of Hu sheep ovarian granulosa cells and the calcium ion release channel on the endoplasmic reticulum membrane,which leads to the increase of calcium ion concentration in the cytoplasm,promotes the apoptosis of ovarian granulosa cells,and causes the down-regulation of estradiol content.