组织蛋白酶对成年绵羊卵母细胞体外发育能力的影响  

The Effect of Cathepsin on in vitro Developmental Capacity of Oocytes from Adult Sheep

作　　者：刘志涛[1,2] 田树军[1,2] 孙树春[1,2] 陈晓勇[3] 张艳普[1,2] 胡媛媛[1,2] 郭军艾[4] 

机构地区：[1]河北农业大学动物科技学院,保定071001 [2]河北省牛、羊胚胎工程技术研究中心,保定071001 [3]河北省畜牧兽医研究所,保定071000 [4]廊坊市畜牧水产局,廊坊065000

出　　处：《畜牧兽医学报》2011年第12期1704-1711,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家科技支撑计划(2011BAD191302);教育部科学技术研究重点项目(211018)

摘　　要：本研究旨在观察绵羊卵丘细胞中组织蛋白酶(CTSS、CTSB)的mRNA在体外成熟过程中的表达规律,探明卵丘细胞中组织蛋白酶(CTSS、CTSB)mRNA的表达与卵母细胞体外发育能力之间的相关性。采用Rea-ltimePCR方法检测绵羊在体外成熟0、8、16和24h的卵丘细胞中组织蛋白酶(CTSB、CTSS)的mRNA表达水平,以及在成熟液中添加组织蛋白酶抑制剂(E-64)后卵丘细胞中CTSB和CTSS的mRNA表达量在体外成熟前及体外成熟后的表达水平,并测定在成熟液中添加1、5、10μmol.L-1 E-64对卵母细胞成熟率、受精率及囊胚发育率的影响。结果,卵丘细胞中的CTSS和CTSB mRNA表达均随着体外成熟时间的增加(IVM 0h、IVM 8h和IVM 16h)呈下降的趋势(P<0.01)。添加5和10μmol.L-1 E-64组的卵丘细胞中CTSS和CTSB mRNA表达水平较对照组和1μmol.L-1组均显著性降低(P<0.01),而5和10μmol.L-1组间的CTSS和CTSB mRNA表达水平无显著差异(P>0.05)。成熟液中添加5和10μmol.L-1的E-64组的囊胚发育率(26.82%和30.95%)显著高于1μmol.L-1组和对照组(18.23%和15.63%,P<0.05),但5和10μmol.L-1组对卵母细胞的成熟率及卵裂率与1μmol.L-1组和对照组间无显著性差异(P>0.05)。结果表明,绵羊卵丘细胞中CTSS和CTSB的mRNA表达水平在体外成熟过程中呈下降趋势,其mRNA表达水平与胚胎的发育能力呈负相关;组织蛋白酶特异性抑制剂E-64可下调绵羊卵丘细胞中CTSS和CTSB的mRNA表达水平,并提高卵母细胞随后的体外发育能力。This study was conducted to explore the mRNA expression of cathepsin（CTSB,CTSS） in cumulus cells during in vitro maturation（IVM） from sheep and to detect the correlation beween the mRNA expression of CTSB and CTSS in cumulus cells and in vitro developmental capacity of oocytes.The expression abundance of CTSB and CTSS in cumulus cells of sheep was detected at IVM 0 h,IVM 8 h,IVM 16 h and IVM 24 h through Real-time PCR.The expression abundance of CTSB and CTSS in cumulus cells was detected before and after IVM in the mature medium which contains cathepsin inhibitor E-64 with different concentration.The effect of E-64 with different concentration in mature medium on in vitro maturation,fertilization and developmental capacity of oocytes were also observed.The mRNA expression of CTSB and CTSS in cumulus cells was down-regulated continuously during in vitro maturation（IVM 0 h,IVM 8 h and IVM 16 h,P〈0.01）,the mRNA expression of CTSB and CTSS in the groups with 5 and 10 μmol·L^-1-64 for IVM 24 h were significantly lower than that of the control and 1 μmol·L^-1-64 groups,nevertheless,there were no significant difference between the two former groups（P〈0.05）.The blastocysts rate in the groups of 5 and 10 μmol·L^-1 E-64（26.82%,30.95%） were higher than those of the control and 1 μmol·L^-1 E-64 groups（15.63%,18.23%,P〈0.05）,but there were no significant difference in the percentage of oocytes mature and cleavage rate among all the groups（P〈0.05）.The result demonstrate that the mRNA expression abundance of CTSB and CTSS in cumulus cells from sheep is down-regulated continuously during IVM,there is negative correlation between the mRNA expression of CTSB and CTSS in cumulus cells and the following embryo developmental capacity;the cathepsin inhibitor E-64 could improve the capacity of oocytes developing into blastocysts after fertilization by down-regulating the mRNA expression abundance of CTSB and CTSS in cumulus cells.

关 键 词：E-64 卵丘细胞 组织蛋白酶 体外发育 REAL-TIMEPCR 

分 类 号：S826[农业科学—畜牧学] S814.6[农业科学—畜牧兽医]