Choi Jiho, Park Seon-Mi, Lee Eugine, Kim Ji-Hye, Jeong Yeon-Ik, Lee Jong-Yun, Park Sun-Woo, Kim Huen-Suk, Hossein Mohammad Shamim, Jeong Yeon-Woo, Kim Sue, Hyun Sang-Hwan, Hwang Woo-Suk
SooAm Biotech Research Foundation, Bangbae3-dong, Seocho-gu, Seoul, Korea.
Mol Reprod Dev. 2008 Jul;75(7):1127-35. doi: 10.1002/mrd.20861.
In the present study, we investigated the effect of melatonin on the preimplantation development of porcine parthenogenetic and somatic cell nuclear transfer (SCNT) embryos. Parthenogenetic embryos were cultured in mNCSU-23 supplemented with various concentrations of melatonin for 7 days. The results revealed that 100 pM was the optimal concentration, which resulted in significantly increased cleavage and blastocyst formation rates. Additionally, 100 pM melatonin provided the highest increase in total cell number of blastocysts. Therefore, the subsequent experiments were performed with 100 pM melatonin. ROS level in 2-8 cell stage embryos in the presence or absence of melatonin was evaluated. Embryos cultured with melatonin showed significantly decreased ROS. Blastocysts cultured with melatonin for 7 days were analyzed by the TUNEL assay. It was observed that melatonin not only increased (P < 0.05) the total cell number but also decreased (P < 0.05) the rate of apoptotic nuclei. Blastocysts cultured with melatonin were assessed for the expression of apoptosis-related genes Bcl-xl and Bax, and of pluripotency marker gene Oct-4 by real-time quantitative PCR. Analysis of data showed that the expression of Bcl-xl was higher (1.7-fold) compared to the control while the expression of Bax was significantly decreased relative to the control (0.7-fold) (P < 0.05). Moreover, the expression of Oct-4 was 1.7-fold higher than the control. These results indicated that melatonin had beneficial effects on the development of porcine parthenogenetic embryos. Based on the findings of parthenogenetic embryos, we investigated the effect of melatonin on the development of porcine SCNT embryos. The results also demonstrated increased cleavage and blastocyst formation rates, and the total cell numbers in blastocysts were significantly higher when the embryos were cultured with melatonin. Therefore, these data suggested that melatonin may have important implications for improving porcine preimplantation SCNT embryo development.
在本研究中，我们探究了褪黑素对猪孤雌胚胎和体细胞核移植（SCNT）胚胎植入前发育的影响。将孤雌胚胎在添加了不同浓度褪黑素的mNCSU-23培养基中培养7天。结果显示，100 pM是最佳浓度，该浓度可显著提高卵裂率和囊胚形成率。此外，100 pM褪黑素使囊胚总细胞数增加最多。因此，后续实验采用100 pM褪黑素进行。评估了有无褪黑素存在时2-8细胞期胚胎中的活性氧（ROS）水平。用褪黑素培养的胚胎显示ROS显著降低。对用褪黑素培养7天的囊胚进行TUNEL检测分析。观察到褪黑素不仅增加了（P < 0.05）总细胞数，还降低了（P < 0.05）凋亡细胞核率。通过实时定量PCR评估用褪黑素培养的囊胚中凋亡相关基因Bcl-xl和Bax以及多能性标记基因Oct-4的表达。数据分析表明，与对照组相比，Bcl-xl的表达更高（1.7倍），而Bax的表达相对于对照组显著降低（0.7倍）（P < 0.05）。此外，Oct-4的表达比对照组高1.7倍。这些结果表明褪黑素对猪孤雌胚胎发育具有有益作用。基于孤雌胚胎的研究结果，我们探究了褪黑素对猪SCNT胚胎发育的影响。结果还表明，当胚胎用褪黑素培养时，卵裂率和囊胚形成率增加，且囊胚中的总细胞数显著更高。因此，这些数据表明褪黑素可能对改善猪植入前SCNT胚胎发育具有重要意义。
