TNF-α对牦牛卵母细胞HIF-1α和HSP70的表达及后续胚胎发育能力的影响

The Effects of Tumor Necrosis Factor-α (TNF-α) on the Expression of HIF-1α and HSP70 in Yak Oocytes and the Subsequent Embryo Development

旨在研究肿瘤坏死因子(TNF-α)对牦牛卵母细胞体外成熟和对低氧诱导因子1α(HIF-1α)、热休克蛋白70(HSP70)的表达以及后续胚胎发育能力的影响。在牦牛卵母细胞体外成熟培养液中添加不同浓度的TNF-α(最终浓度分别为0、10、25、50 ng·mL^-1),牦牛卵丘-卵母细胞复合体(cumulus-oocyte complexes,COCs)培养成熟后,统计其卵母细胞成熟率及体外受精后早期胚胎的发育率,采用RT-PCR扩增牦牛 HIF-1α、 HSP 70基因,采用实时荧光定量PCR(qRT-RCR)、蛋白免疫印迹法(Western blotting)和免疫荧光染色技术检测HIF-1α、HSP70在基因和蛋白水平的表达情况。结果发现:1)成熟液中加入TNF-α,卵母细胞的成熟率提高,且随着TNF-α浓度的升高,卵母细胞的成熟率、卵裂率和囊胚率逐渐升高,当TNF-α浓度为 25 ng·mL^-1 时,卵母细胞的成熟率和卵裂率达到最高,分别为84.98%和66.85%,而囊胚率也达到了21.48%,均显著高于对照组( P <0.05);2)随着TNF-α浓度的升高, HIF-1α的表达量逐渐降低,对照组 HIF-1α的表达量极显著高于其他组(P <0.01),50 ng·mL^-1 TNF-α组 HIF-1α的表达量极显著低于其他组(P <0.01),HIF-1α在卵丘细胞和卵母细胞上均有表达;3)25 ng·mL^-1 TNF-α组的HSP 70表达量最高,极显著高于其他组(P <0.01),而当TNF-α达到50 ng·mL^-1 时,HSP 70的表达量最低。综上表明,在牦牛卵母细胞体外成熟过程中,TNF-α明显提高了卵母细胞的发育能力,同时还诱导了HSP 70的表达,抑制了HIF-1α的表达,为今后探讨TNF-α、 HIF-1α和HSP70在牦牛生殖过程中发挥的作用以及对胚胎发育的影响提供理论依据。

The aim of this study was to investigate the effects of tumor necrosis factor-α(TNF-α) on yak oocytes maturation in vitro ,the expression of epidermal growth factor (HIF-1α) and heat shock protein receptor (HSP70) in yak oocytes and the subsequent embryo development. In this experiment,TNF-α with different concentrations (0,10,25,50 ng·mL^-1 ) were added to in vitro maturation medium of yak oocytes. After the cumulus-oocyte complexes (COCs) matured,the oocyte maturation rate and the development rate of early embryos after in vitro fertilization were counted. The HIF -1α and HSP 70 genes of yak were amplified by RT-PCR. The mRNA and protein expression of HIF-1α and HSP70 were detected by real-time PCR (qRT-RCR),Western blotting and immunofluorescence staining. The results showed that: 1) The addition of TNF-α to the oocyte maturation medium promoted the maturation rate of oocytes. With the increase of TNF-α concentration,the maturation rate,cleavage rate and blastocyst rate of oocytes increased gradually. When the concentration of TNF-α was 25 ng·mL^-1 ,the maturation rate and cleavage rate of oocytes reached the highest,84.98% and 66.85%,respectively,and the blastocyst rate also reached 21.48%,which were significantly higher than that in the control group( P <0.05). 2) With the increase of TNF-α concentration,the expression level of HIF-1α decreased gradually . When the concentration of TNF-α was 0 ng·mL^-1 ,the expression level of HIF -1α was the highest, which was significantly higher than that in other groups ( P <0.01). The expression level of HIF-1α was the lowest when the concentration of TNF-α was 50 ng·mL^-1 ,which was sig- nificantly lower than that in other groups ( P <0.01). Immunofluorescence assay showed that HIF-1α was expressed in both cumulus cells and oocytes. 3) When the concentration of TNF-α was 25 ng·mL^-1 , the expression level of HSP 70 was the highest,which was significantly higher than that in other groups ( P <0.01),and when the concentration of TNF-α reached 50 ng·mL^-1 , the expression level of HSP 70 was the lowest. The results showed that TNF-α significantly increased the developmental capacity of oocytes during maturation of yak oocytes in vitro ,and also induced the expression of HSP 70 and inhibited the expression of HIF -1α. These would provide a theoretical basis for exploring the role of TNF-α,HIF-1α and HSP70 in the reproductive process of yak and the impact on embryo development.