绵羊卵泡中抗氧化酶表达的研究

Characterization of the Expression Pattern of Antioxidant Enzymes in Sheep Ovarian Follicle

[目的]分析绵羊不同直径有腔卵泡中抗氧化酶的表达情况,为明晰卵泡的抗氧化功能调控机制提供参考。[方法]收集绵羊不同直径(大腔≥6.0 mm,中腔2.0~6.0 mm,小腔≤2.0 mm)有腔卵泡的卵泡液,利用试剂盒检测卵泡液中的活性氧(reactive oxygen species,ROS)含量、总抗氧化能力(total antioxidant capacity,TAC)以及超氧化物歧化酶(superoxide dismutase,SOD)、过氧化氢酶(catalase,CAT)、谷胱甘肽过氧化物酶(glutathione peroxidase,GPX)、过氧化物酶(peroxidase,PRDX)活性;采用实时荧光定量PCR(qPCR)分别检测小腔、中腔、大腔卵泡的卵泡膜细胞、壁层颗粒细胞、卵丘细胞中Cu/Zn-SOD、CAT、GPX3和PRDX6基因mRNA相对表达量。[结果]不同直径有腔卵泡的卵泡液中TAC和ROS含量均无显著(P>0.05)差异。卵泡液中SOD、GPX、PRDX活性随着卵泡直径的增大呈下降趋势,小腔卵泡的卵泡液中SOD、GPX、PRDX活性极显著(P<0.01)高于大腔卵泡;卵泡液中CAT活性随着卵泡直径的增大呈上升趋势,大腔卵泡和中腔卵泡的卵泡液中CAT活性极显著(P<0.01)高于小腔卵泡。小腔卵泡的卵泡膜细胞和壁层颗粒细胞中Cu/Zn-SOD、GPX3和PRDX6基因mRNA相对表达水平极显著(P<0.01)高于中腔卵泡和大腔卵泡,而中腔卵泡卵丘细胞中Cu/Zn-SOD、GPX3和PRDX6基因的mRNA相对表达水平极显著(P<0.01)高于小腔卵泡和大腔卵泡;大腔卵泡的卵泡膜细胞和壁层颗粒细胞中CAT基因mRNA相对表达水平极显著(P<0.01)高于小腔卵泡和中腔卵泡,大腔卵泡和中腔卵泡卵丘细胞中CAT基因mRNA相对表达水平极显著(P<0.01)高于小腔卵泡。[结论]绵羊不同直径有腔卵泡的TAC和ROS含量无显著变化,但抗氧化酶SOD、CAT、GPX和PRDX在不同直径卵泡中的基因表达水平和活性具有空间特异性,说明绵羊有腔卵泡抗氧化功能的调节是多因素协同的过程。

[Objective]This study was conducted to characterize the expression pattern of antioxidant enzymes in sheep antral ovarian follicles with different diameters,so as to provide references for clarifying the regulatory mechanism of antioxidant function in ovarian follicles.[Method]The follicular fluids were sampled from sheep antral ovarian follicles with different diameters(large antra≥6.0 mm,medium antra 2.0-6.0 mm,small antra≤2.0 mm).The contents of reactive oxygen species(ROS)and total antioxidant capacity(TAC),as well as the activities of superoxide dismutase(SOD),catalase(CAT),glutathione peroxidase(GPX)and peroxidase(PRDX)were tested using the commercially available reagent kits.Real time fluorescence quantitative PCR(qPCR)assay was used to detect the relative mRNA expression levels of Cu/Zn-SOD,CAT,GPX3 and PRDX6 genes in the theca cells,mural granulosa cells and cumulus cells of small,medium and large antral ovarian follicles.[Result]There were no significant(P>0.05)differences in the contents of TAC and ROS in the follicular fluids of antral ovarian follicles with different diameters.The activities of SOD,GPX and PRDX in the follicular fluids decreased with the increase of ovarian follicle diameters.The small antral ovarian follicles had extremely significantly(P<0.01)higher activities of SOD,GPX and PRDX in the follicular fluids compared to the large antral ovarian follicles.The CAT activity in the follicular fluids elevated with the increase of ovarian follicle diameters.The large and medium antral ovarian follicles had extremely significantly(P<0.01)higher CAT activities in the follicular fluids than the small antral ovarian follicles.The relative mRNA expression levels of Cu/Zn-SOD,GPX3 and PRDX6 genes in the theca cells and mural granulosa cells of the small antral ovarian follicles were extremely significantly(P<0.01)higher than those of the medium and large antral ovarian follicles,while the relative mRNA expression levels of Cu/Zn-SOD,GPX3 and PRDX6 genes in the cumulus cells of the medium antral ovarian follicles were extremely significantly(P<0.01)higher than those of the small and large antral ovarian follicles.The large antral ovarian follicles had extremely significantly(P<0.01)higher relative mRNA expression levels of CAT gene in the theca cells and mural granulosa cells than the small and medium antral ovarian follicles,while the large and medium antral ovarian follicles had extremely significantly(P<0.01)higher relative mRNA expression levels of CAT gene in the cumulus cells than the small antral ovarian follicles.[Conclusion]There was no significant variations in contents of TAC and ROS in sheep antral ovarian follicles with different diameters,but the gene expression levels and the activities of antioxidant enzymes of SOD,CAT,GPX and PRDX in ovarian follicles with different diameters exhibited spatial specificity,indicating that the regulation of antioxidant function in sheep antral ovarian follicles was a synergistic process of multiple factors.