猪卵丘卵母细胞复合体和裸卵的卵母细胞发育及基因表达差异的研究  

Comparison of the oocyte development and gene expression patterns in porcine oocytes between cumulus-oocytes complex and naked oocytes

作　　者：朱桂玉[1] 冯书堂[1] 李吉涛[1] 牟玉莲[1] 潘登科[1] 郭丙冉[2] 

机构地区：[1]中国农业科学院北京畜牧研究所,北京100094 [2]曲阜师范大学生命科学学院,山东曲阜273165

出　　处：《广西农业生物科学》2006年第B09期186-187,共2页Journal of Guangxi Agricultural and Biological Science

基　　金：国家863计划项目[2003AA205100];国家"十五"重大攻关项目[2004BA717B-01]

摘　　要：卵丘细胞是指包裹在卵母细胞外周并与之进行代谢联系的颗粒细胞群,是一类与生殖细胞密切相关的体细胞。大量研究表明,卵丘细胞在卵母细胞的发育、成熟、排卵、受精以及受精卵发育过程中发挥了重要作用,卵母细胞与卵丘细胞之间的相互作用已成为关注的焦点。由于卵丘与卵母细胞之间的相互作用十分复杂,人们对参与这一细胞间互作的信号通路和反应过程相关基因的表达还缺乏深刻了解。本研究对比了有致密完整卵丘细胞包裹的卵母细胞和无卵丘细胞包裹裸卵的体外培养成熟过程中的成熟率、孤雌激活卵裂率、囊胚率的差异,并运用mRNA差异显示技术(DDRT-PCR)和半定量RT-PCR技术,比较了两者在基因表达上的差异,目的是进一步认识卵母细胞的发育机制,并推测卵母细胞和卵丘细胞间的相互作用的分子机理。材料与方法:采集大量猪的卵母细胞,捡出A级卵(胞质均匀且卵丘细胞层完整紧密,包裹五层以上的卵)和C级卵(有极少或无卵丘细胞包裹的卵)分别进行了体外培养、孤雌激活。同时对两类卵母细胞抽提RNA,进行DDRT-PCR,筛选出差异条带进行克隆测序,最后根据测序结果设计特异引物,最后通过半定量RT-PCR对筛选出的差异条带进行验证。结果与讨论:(1)在体外培养和成熟实验中,卵丘卵母细胞复合体(COC)中的卵母细胞的成熟率和卵裂率均显著高于裸卵的,而裸卵的囊胚率为0,表明卵丘细胞在卵母细胞的发育和成熟过程中起到了关键作用。(2)利用了3对锚定引物和随机引物,共发现了16条差异表达的片段,经过克隆测序最终得到了14条差异表达的序列,经过比对发现,其中3个片段分别与人的PELP1,Myo5b,calpastatin基因高度同源,另外5条序列与猪的EST同源,但功能未知,其余6条序列未找到同源片段。(3)经过半定量RT-PCR验证表明,大约有一半的条带在差异显示和半定量RT-Several layers of cumulus ceils surround the oocyte in the antral follicle and play an important support and regulation role in oocytes development and maturation via the intercellular communications and interactions between oocytes and cumulus cells. By using differential display reverse transcriptase polymerase chain reaction （DDRT-PCR） analysis of differential mRNA expression and semi-quantitative reverse transcriptase-polymerase chain reaction, mRNA expression was compared in porcine oocytes from two sources： cumulus-oocyte complexes （COCs） and naked oocytes （NOs）. Fourteen differentially expressed cDNAs were cloned, sequenced, and their expression patterns were verified by semi-quantitative RT-PCR. Four cDNAs, including three genes （PELP1, Myo5b and CAST） and a new EST were confirmed preferentially expressed in the oocytes of COCs, which expressions were more than those of NOs. The studies of three differentially expressed genes suggested that they mainly involved in estrogen receptor regulation, membrane trafficking, organdie transport, cellular signaling and a variety of other cellular processes. These results suggested that the aberrant transcription patterns detected in the oocytes of NOs compared with COCs may explain their reduced qualities in term of development and maturation. In conclusion, these differentially transcripted cDNAs may involved in the cellular interactions between oocytes and cumulus cells and thus to be considered as essential genes for the competence of oocytes.

关 键 词：猪卵母细胞 体外培养 差异表达 卵丘卵细胞复合体 

分 类 号：Q813[生物学—生物工程]