猪卵丘卵母细胞复合体和裸卵中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

【目的】研究探讨卵丘细胞的完整性在支持和促进猪卵母细胞成熟过程中发挥的重要作用以及卵丘与卵母细胞互作的分子机理。【方法】对具有完整致密卵丘细胞包裹的和无卵丘细胞包裹的猪卵母细胞进行了体外成熟培养试验,应用mRNA差异显示(DDRT-PCR)和半定量反转录PCR技术(RT-PCR),对这2类猪卵母细胞中mRNA的差异表达进行研究,得到了14条差异表达条带,经过回收、克隆、测序后,再运用半定量(RT-PCR)进行检测。【结果】体外培养的裸卵细胞的各项成熟指标均低于卵丘-卵母细胞复合体。3个基因(PELP1,Myo5b and CAST)和一条新EST序列在有卵丘和无卵丘的猪卵母细胞中表现出差异表达。分离得到的差异表达基因在雌激素受体调节、细胞膜间的信息传导和细胞器物质运输等过程中发挥重要的作用。【结论】这些差异基因可能参与了卵丘细胞与卵母细胞间的相互作用,对卵母细胞的成熟具有重要作用。

[Objective] Several layers of cumulus cells 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. The studies were conducted to investigate the function of cumulus cells during in vitro maturation of porcine oocytes and the gene expression profiles between cumulus-oocyte complexes （COCs） and naked oocytes （NOs）. [Method] Using differential display reverse transcriptase polymerase chain reaction （DD-RT-PCR） analysis of differential mRNA expression and semi-quantitative reverse transcriptase-polymerase chain reaction, the mRNA expression in porcine oocytes from two sources, COCs and NOs, was compared. Fourteen differentially expressed cDNAs were cloned, sequenced, and their expression patterns were verified by semi-quantitative RT-PCR. [Result] Four cDNAs, including three genes （PELP1, Myo5b and CAST） and a new EST were confirmed preferentially expressed in the oocytes of COCs than NOs. The studies of three differentially expressed genes suggested that they mainly involved in estrogen receptor regulation, membrane trafficking, organelle transport, cellular signaling and a variety of other cellular processes. These results suggest that the aberrant transcription patterns detected in the oocytes of NOs compared with COCs may explain their reduced qualities in terms of development and maturation. [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.