褪黑素对水貂GV期卵母细胞冷冻保存效率的影响  

Effect of melatonin on cryopreservation of GV stage oocytes in mink

作　　者：曹俊国 李文 韩玉萍 杨镒峰[1] 魏海军[1] 许保增[1] CAO Jun-guo;LI Wen;HAN Yu-ping;YANG Yi-feng;WEI Hai-jun;XU Bao-zeng(State Key Laboratory for Molecular Biology of Special Economic Animals,Institute of Special Animal and Plant Sciences,Chinese Academy of Agricultural Science,Changchun 130112,China)

机构地区：[1]中国农业科学院特产研究所、特种经济动物分子生物学重点实验室,吉林长春130112

出　　处：《中国兽医学报》2020年第4期816-822,共7页Chinese Journal of Veterinary Science

基　　金：吉林省国际科技合作资助项目(20170414049GH);中国农业科学院科技创新工程资助项目(CAAS-ASTIP-2018-ISAPS);中国农业科学院科技创新工程协同创新资助项目(CAAS-XTCX2016010-02).

摘　　要：旨在探究水貂生发泡期(GV期)卵丘-卵母细胞复合体(COCs)玻璃化冷冻、解冻及培养过程中添加褪黑素(melatonin,MT)对水貂卵母细胞解冻后存活率、体外培养COCs恢复减数分裂比例(GVBD率)、体外培养卵母细胞活性线粒体的分布以及体外培养COCs颗粒细胞糖酵解相关基因表达(卵母细胞与颗粒细胞缝隙连接好坏的标志分子)的影响。试验分为3组:无冷冻处理的对照组(C组)、冷冻组(V组)和添加MT(10-9 mol/L)的冷冻处理组(MT组)。结果显示:MT组卵母细胞在解冻后3 h的存活率(91%±4.8%,n=91)与V组的存活率(90%±2.7%,n=75)差异不显著(P>0.05),但两组存活率显著低于C组(100%,n=80)(P<0.05);C组(81%±1.9%,n=64)和MT组卵母细胞的GVBD率(70%±2.7%,n=80)显著高于V组(55%±4.1%,n=80)(P<0.05),但C组与MT组相比差异不显著(P>0.05);免疫荧光结果显示,MT组的卵母细胞线粒体正常分布率(34%±3.8%,n=75)与V组(30%±2.3%,n=60)相比差异不显著(P>0.05),但MT和V组的卵母细胞线粒体正常分布率显著低于C组(79%±3.4%,n=90)(P<0.05);qRT-PCR结果显示,MT组颗粒细胞糖酵解基因Ldh1、Pkm2及Pfkp的表达量显著高于V组(P<0.05),但前两者糖酵解基因的表达量显著低于C组(P<0.05)。结果表明,添加MT可以提高冻融后水貂卵母细胞的GVBD率,降低冷冻对卵母细胞和颗粒细胞连接的冷冻损失,但要建立一个高效的水貂GV期卵母细胞玻璃化冷冻保存体系仍需进一步研究。To investigate the effects of melatonin(MT)supplement during vitrification,thawing and maturation in vitro of mink cumulus-oocyte complexes(COCs)at germinal vesicle stage(GV stage)on theirs survival rates,the percentages of meiosis recovery(GVBD rate),and the distribution of active mitochondria in oocytes and the expression levels of glycolysis-related genes(markers of gap junction between oocytes and granulosa cells)in granulosa cells.The samples were divided into three groups:a control group without vitrification(C group),vitrification group(V group),and vitrification with MT(10-9 mol/L)supplement group(MT group).The results showed that the survival rate of oocytes in MT group(91%±4.8%,n=91)at 3 hours after thawing was comparable to that in V group(90%±2.7%,n=75),but both of them were lower than that in C group(100%,n=80)(P<0.05).The GVBD rate of oocytes in C group(81%±1.9%,n=64)and MT group(70%±2.7%,n=80)was significantly higher than that in V group(55%±4.1%,n=80),but there was no significant difference between C and MT groups(P>0.05).Immunofluorescence data demonstrated that the rate of normal mitochondria distribution in MT group(34%±3.8%,n=75)was not significantly different with that in V group(30%±2.3%,n=60),but both of them were significantly lower than that in C group(79%±3.4%,n=90)(P<0.05).The results of qRT-PCR showed that the expression levels of Ldh1,Pkm2 and Pfkp in granulosa cells of MT group were significantly higher than those in V group,while these genes expression levels in this both groups were significantly lower than these in the C group(P<0.05).The results suggested that the addition of MT increases the GVBD rate of mink oocytes after vitrification,and reduced the freezing loss of the connection between oocytes and granulosa cells.However,the establishment of an efficient vitrification in mink oocytes still needs further studies.

关 键 词：褪黑素 水貂 卵母细胞 玻璃化冷冻 

分 类 号：S865.2[农业科学—野生动物驯养] S814.6[农业科学—畜牧兽医]