Cheng Wen-Min, Sun Xu-Lei, An Lei, Zhu Shi-En, Li Xi-He, Li Ying, Tian Jian-Hui
Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, College of Animal Sciences and Technology, China Agricultural University, Beijing, People's Republic of China.
Anim Biotechnol. 2007;18(2):131-41. doi: 10.1080/10495390601096148.
The aim of this study was to investigate the effect of electrical pulse, ethanol, and ionomycin combined with cycloheximide (CHX), cytochalasin B (CB), and 6-dimethylaminopurine (6-DMAP) on parthenogenetic developmental competence of in vitro matured porcine oocytes. In experiment 1, oocytes were treated with direct current electrical pulse (DC pulse) and then incubated in the NCSU-23 medium supplemented with CHX, 6-DMAP, CB + CHX, and CB + 6-DMAP for 6 h, respectively. The rate of blastocyst development in DC pulse + CB + 6-DMAP group was significantly higher than those in other groups (42.4% vs 23.9% approximately 35.8%; P < 0.05); however, there were no differences in both of the cleavage rate and the cell number of blastocysts among four groups. In experiment 2, oocytes were treated with NCSU-23 medium containing 20 muM ionomycin for 40 min and then incubated in the NCSU-23 medium supplemented with CHX, 6-DMAP, CB + CHX and CB + 6-DMAP for 6 h, respectively. The rates of cleavage and blastocyst development in ionomycin + 6-DMAP group were higher than those obtained in other groups (66.2% vs 46.3% approximately 57.3%; 22.3% vs 7.4% approximately 16.1%; P < 0.05). In experiment 3, the activation effects of ethanol combined with 6-DMAP, CHX, CB + 6-DMAP and CB + CHX were investigated. The rates of cleavage and blastocyst development in ethanol + CB + 6-DMAP group were significantly higher than those in other groups (55.5% vs 42% approximately 46.2%; 18.0% vs 7.1% approximately 11.9%; P < 0.05). In experiment 4, the optimal activation protocols in each group plus DC pulse + ionomycin + 6-DMAP were compared. The results showed the rates of cleavage in DC pulse + CB + 6-DMAP group and ionomycin + 6-DMAP were higher than those in ethanol + CB + 6-DMAP and DC pulse + ionomycin + 6-DMAP (73.8-74.4% vs 56.5-57.5%; P < 0.05), but the blastocyst development only in DC pulse + CB + 6-DMAP group was significantly higher than that in other groups (34.1% vs 13.4% approximately 22.3%; P < 0.05). Total cell number of blastocysts in the group of DC pulse + ionomycin + 6-DMAP was higher than that in other groups (34.1 vs 25.3-27.2; P < 0.05). In conclusion, DC pulse, ethanol, CB, and 6-DMAP all affected the parthenogenesis of porcine oocytes matured in vitro, but their combination of DC pulse + CB + 6-DMAP showed the best result in both of cleavage and blastocyst development.
本研究旨在探讨电脉冲、乙醇、离子霉素分别与放线菌酮（CHX）、细胞松弛素B（CB）和6-二甲基氨基嘌呤（6-DMAP）联合使用对体外成熟猪卵母细胞孤雌发育能力的影响。实验1中，用直流脉冲（DC脉冲）处理卵母细胞，然后分别在添加CHX、6-DMAP、CB+CHX和CB+6-DMAP的NCSU-23培养基中孵育6小时。DC脉冲+CB+6-DMAP组的囊胚发育率显著高于其他组（42.4%对约23.9%和35.8%；P<0.05）；然而，四组之间的卵裂率和囊胚细胞数均无差异。实验2中，用含20μM离子霉素的NCSU-23培养基处理卵母细胞40分钟，然后分别在添加CHX、6-DMAP、CB+CHX和CB+6-DMAP的NCSU-23培养基中孵育6小时。离子霉素+6-DMAP组的卵裂率和囊胚发育率高于其他组（66.2%对约46.3%和57.3%；22.3%对7.4%和约16.1%；P<0.05）。实验3中，研究了乙醇分别与6-DMAP、CHX、CB+6-DMAP和CB+CHX联合使用的激活效果。乙醇+CB+6-DMAP组的卵裂率和囊胚发育率显著高于其他组（55.5%对约42%和46.2%；18.0%对7.1%和约11.9%；P<0.05）。实验4中，比较了每组加上DC脉冲+离子霉素+6-DMAP后的最佳激活方案。结果显示DC脉冲+CB+6-DMAP组和离子霉素+6-DMAP组的卵裂率高于乙醇+CB+6-DMAP组和DC脉冲+离子霉素+6-DMAP组（73.8 - 74.4%对56.5 - 57.5%；P<0.05），但仅DC脉冲+CB+6-DMAP组的囊胚发育率显著高于其他组（34.1%对约13.4%和22.3%；P<0.05）。DC脉冲+离子霉素+6-DMAP组的囊胚总细胞数高于其他组（34.1对25.3 - 27.2；P<0.05）。总之，DC脉冲、乙醇、CB和6-DMAP均影响体外成熟猪卵母细胞的孤雌生殖，但DC脉冲+CB+6-DMAP组合在卵裂和囊胚发育方面均显示出最佳结果。
