Hajian M, Kiani M, Hosseini M S, Ostadhosseini S, Forouzanfar M, Afrough M, Nasr-Esfahani M H
Department of Reproduction and Development, Royan Institute for Biotechnology, ACECR, Isfahan, Iran.
Cell Reprogram. 2013 Jun;15(3):247-57. doi: 10.1089/cell.2012.0089.
In this study, the effect of the steps involved in zona-free somatic cell nuclear transfer (SCNT) on oocyte transcripts was investigated in sheep. To establish the reliable combined electrical-chemical activation for zona-free oocytes, oocytes were first exposed to an electrical pulse and then treated with 18 chemical activation regimens designed through modifying duration and concentration of ionomycin and 6-dimethyl aminopurine (6-DMAP), which is routinely used for SCNT. Electrofusion-mediated nuclear transfer significantly reduced transcript abundances of CCNB1, POU5F1, NPM2, GMMN, and CX43 compared to intact oocytes. Maximum parthenogenetic blastocyst development was obtained when oocytes were submitted to electric pulse and then to (1) 5 μM ionomycin for 5 or 2.5 min, both followed by 2 h of incubation with 6-DMAP (41.7±1.1, and 42.4±1.4%, respectively), (2) 5 μM ionomycin for 1 min+6-DMAP for 4 h (43.1±1.4%), and (3) 2.5 μM ionomycin for 1 min+6-DMAP for 2 h (42.4±1.4%), with significant differences compared to all the other groups. Statistical assessment of interactions between duration and concentration of ionomycin and duration of 6-DMAP exposure revealed that (1) concentration of ionomycin may be a more important factor than its duration, (2) both a long exposure period and a low concentration of ionomycin had marked decreasing effects on parthenogenetic development of zona-free oocytes, and (3) high duration of exposure to 6-DMAP can reduce parthenogenetic development. Despite an activation preference of parthenogenetic oocytes, a significantly higher rate of cloned blastocyst development was observed when reconstructed oocytes were activated with 5 μM ionomycin for 5 min rather than 2.5 μM ionomycin for 1 min (8.8±2.5 vs. 1.25±2.2%). These results suggested that SCNT steps have determining effects on oocyte transcripts and activation preferences of the reconstituted oocytes compared to intact counterparts. In this sense, reconstituted oocytes may need a higher concentration of ionomycin for a longer period than intact oocytes.
在本研究中，我们在绵羊中研究了无透明带体细胞核移植（SCNT）所涉及的步骤对卵母细胞转录本的影响。为了建立可靠的无透明带卵母细胞电化学联合激活方法，首先对卵母细胞施加电脉冲，然后用18种通过改变离子霉素和6 - 二甲基氨基嘌呤（6 - DMAP，常用于SCNT）的持续时间和浓度设计的化学激活方案进行处理。与完整卵母细胞相比，电融合介导的核移植显著降低了CCNB1、POU5F1、NPM2、GMMN和CX43的转录本丰度。当卵母细胞先接受电脉冲，然后接受以下处理时，孤雌胚泡发育达到最大值：（1）5 μM离子霉素处理5分钟或2.5分钟，随后均用6 - DMAP孵育2小时（分别为41.7±1.1%和42.4±1.4%）；（2）5 μM离子霉素处理1分钟 + 6 - DMAP处理4小时（43.1±1.4%）；（3）2.5 μM离子霉素处理1分钟 + 6 - DMAP处理2小时（42.4±1.4%），与所有其他组相比有显著差异。对离子霉素的持续时间和浓度以及6 - DMAP暴露持续时间之间的相互作用进行统计评估发现：（1）离子霉素的浓度可能比其持续时间更重要；（2）离子霉素的长时间暴露和低浓度对无透明带卵母细胞的孤雌发育均有显著的降低作用；（3）6 - DMAP的高暴露持续时间会降低孤雌发育。尽管孤雌卵母细胞有激活偏好，但当用5 μM离子霉素处理5分钟而不是2.5 μM离子霉素处理1分钟激活重构卵母细胞时，观察到克隆胚泡发育率显著更高（8.8±2.5%对1.25±2.2%）。这些结果表明，与完整卵母细胞相比，SCNT步骤对卵母细胞转录本和重构卵母细胞的激活偏好有决定性影响。从这个意义上说，重构卵母细胞可能比完整卵母细胞需要更高浓度的离子霉素处理更长时间。
