Kim Ji-Su, Chae Jung-Il, Song Bong-Seok, Lee Kyu-Sun, Choo Young-Kug, Chang Kyu-Tae, Park Humdai, Koo Deog-Bon
Development and Differentiation Research Center, Korea Institute of Bioscience and Biotechnology, 111 Gwahangno, Yuseong, Daejeon 305-806, Korea.
Reprod Fertil Dev. 2010;22(2):437-47. doi: 10.1071/RD08287.
Oviduct fluid contains various cytokines and growth factors that enhance the embryo development during the preimplantation period. In hatched embryos, prostacyclin (PGI(2)) improves implantation, but its role during oocyte maturation and early embryo development remains contentious. Therefore, in the present study, we examined the effects of a PGI(2) analogue (iloprost) on meiotic maturation and early embryonic development in pigs, as well on the structural integrity, mitochondrial membrane potential and apoptosis in blastocysts. First, meiotic maturation in pig oocytes was examined in the presence of increasing concentrations of iloprost (1, 5 and 10 muM). After IVM, a higher proportion of iloprost-treated compared with untreated oocytes was in MII (90.0% v. 65.7%, respectively; P < 0.05). In addition, protein kinase A activity increased in iloprost-treated oocytes, indicating increased intracellular cAMP concentrations. After 22 h iloprost treatment (44 h total incubation time), western blotting demonstrated increased expression of extracellular signal-regulated kinase (ERK) 1/2, phosphorylated (p-) ERK1/2, cAMP response element-binding protein (CREB), p-CREB and cyclo-oxygenase-2, indicating activation of the mitogen-activated protein kinase and PGI(2) pathways. In addition, the frequency of polyspermy decreased in iloprost-treated oocytes (19.9%) compared with control (35.8%), whereas the rate of blastocyst formation increased (P < 0.05). Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) showed that the number of nuclei containing fragmented DNA at the blastocyst stage decreased in the iloprost-treated group compared with control (1.2% v. 3.6%, respectively). In conclusion, iloprost appears to play a direct role in porcine oocyte maturation by enhancing blastocyst structure and survival.
输卵管液含有多种细胞因子和生长因子，可在植入前阶段促进胚胎发育。在孵化后的胚胎中，前列环素（PGI₂）可改善着床，但其在卵母细胞成熟和早期胚胎发育过程中的作用仍存在争议。因此，在本研究中，我们研究了PGI₂类似物（伊洛前列素）对猪减数分裂成熟和早期胚胎发育的影响，以及对囊胚的结构完整性、线粒体膜电位和细胞凋亡的影响。首先，在存在不同浓度伊洛前列素（1、5和10 μM）的情况下，检测猪卵母细胞的减数分裂成熟情况。体外成熟后，与未处理的卵母细胞相比，经伊洛前列素处理的卵母细胞处于MII期的比例更高（分别为90.0%和65.7%；P < 0.05）。此外，经伊洛前列素处理的卵母细胞中蛋白激酶A活性增加，表明细胞内cAMP浓度升高。在伊洛前列素处理22小时后（总孵育时间44小时），蛋白质印迹法显示细胞外信号调节激酶（ERK）1/2、磷酸化（p-）ERK1/2、cAMP反应元件结合蛋白（CREB）、p-CREB和环氧化酶-2的表达增加，表明丝裂原活化蛋白激酶和PGI₂途径被激活。此外，与对照组（35.8%）相比（19.9%），经伊洛前列素处理的卵母细胞多精受精频率降低，而囊胚形成率增加（P < 0.05）。末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）显示，与对照组相比，伊洛前列素处理组囊胚阶段含有碎片化DNA的细胞核数量减少（分别为1.2%和3.6%）。总之，伊洛前列素似乎通过增强囊胚结构和存活率在猪卵母细胞成熟中发挥直接作用。
