冷、热应激对猪孤雌胚胎体外发育的影响

Effects of Cold or Heat Stress on the Development of Porcine Parthenogenetic Embryos in vitro

为研究持续不同时间的冷、热应激对猪孤雌胚胎体外发育的影响,本研究以猪孤雌胚胎为材料,采用免疫荧光染色、实时荧光定量PCR技术检测不同时间的冷(31℃)、热应激(41℃)处理对猪孤雌胚胎发育后囊胚发育率、细胞数、细胞凋亡率、自噬相关基因及细胞凋亡相关基因mRNA转录水平的影响。结果显示,热应激12h后囊胚发育率显著低于对照组(P<0.05),冷应激18h后囊胚发育率显著低于对照组(P<0.05),而冷应激组囊胚发育率高于热应激组。热应激12h和冷应激18h后均导致囊胚内细胞数显著低于对照组(P<0.05),细胞凋亡率显著高于对照组(P<0.05),且冷应激组的细胞凋亡率低于热应激组。冷、热应激组自噬相关蛋白LC3的表达均高于对照组;冷、热应激中自噬相关基因Atg6和Atg8的表达均极显著高于对照组(P<0.01),Lamp2基因的表达均显著高于对照组(P<0.05),热应激组中的Atg6和Atg8基因的表达高于冷应激组。通过检测细胞凋亡相关基因mRNA的转录水平发现,冷、热应激组中细胞凋亡相关基因Bak、Casp-3、Fas的表达均极显著高于对照组(P<0.01),Bcl-xl基因的表达均显著低于对照组(P<0.05)。综上,猪孤雌胚胎对冷应激(31℃)的耐受性比对热应激(41℃)强,且热应激可诱导体外培养的猪孤雌胚胎自噬及凋亡相关基因的表达,从而降低孤雌胚胎发育的能力。

In order to study the effects of cold and heat stress at different time on the development of porcine parthenogenetic embryos in vitro,the porcine parthenogenetic embryos were used as the material in this study,immunofluorescence staining and Real-time PCR were adopted to detect the rate of blastocyst,cell count,cell apoptosis,and expression of autophagy related gene and transcription level of apoptosis related gene mRNA.The results showed that compared with control group,the rate of blastocyst decreased significantly after 12 h heat stress or cold stress for 18 h(P<0.05),while the rate of blastocyst in cold stress group was higher than that in heat stress group.After 12 h heat stress or cold stress for 18 h,the number of cells in blastocyst decreased significantly(P<0.05),the apoptotic rate increased significantly(P<0.05),and the apoptotic rate of cold stress group was lower than that in heat stress group.The expression of autophagy related genes Atg 6 and Atg 8 in cold and heat stress groups was extremely significantly higher than that in control group(P<0.01),Lamp2 expression was significantly higher than that in control group(P<0.05),and Atg 6 and Atg 8 genes expression in heat stress group were higher than that of cold stress group.By detecting the transcription level of apoptosis-related gene mRNA,the expression of apoptosis-related genes Bak,Casp-3 and FAs in cold and heat stress group was extremely significantly higher than that in control group(P<0.01),and Bcl-xl expression was significantly lower than that in control group(P<0.05).In summary,the tolerance of porcine parthenogenetic embryos to cold stress(31℃)was stronger than that of heat stress(41℃),and heat stress could induce the expression of autophagy and apoptosis-related genes in porcine parthenogenetic embryos cultured in vitro,that could reduce the development ability of parthenogenetic embryos.