Kim J S, Cho Y S, Song B S, Wee G, Park J S, Choo Y K, Yu K, Lee K K, Han Y M, Koo D B
Center for Regenerative Medicine, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-806, Republic of Korea.
Theriogenology. 2008 Feb;69(3):290-301. doi: 10.1016/j.theriogenology.2007.09.024. Epub 2007 Oct 30.
High concentrations of cyclic AMP in germinal vesicle oocytes generally inhibit GVBD. Thus, maintaining the GV stage in growing oocytes is essential for the developmental competence of the eggs. In this study, we traced the effects of dibutyryl cyclic AMP on meiotic maturation and early embryonic development in pigs. We also investigated several blastocyst qualities, including structural integrity, mitochondrial membrane potential, and apoptosis, which are affected by dbcAMP. To determine whether increased concentrations of cAMP inhibit GVBD, we explored the meiotic patterns and during maturation of pig oocytes. When treated with dbcAMP for 22h, 91.1% of the oocytes were arrested in the GV stage compared to only 38.8% of the oocytes in the control group (P<0.05). After completion of IVM, a higher proportion of the dbcAMP-treated oocytes were in metaphase II than the untreated ones (91.3% vs. 72.8%, P<0.05). Western blot analysis showed a reduction (at 22h) and/or increase (at 44h) in MPF and MAP kinase activities in porcine oocytes treated with dbcAMP for the first 22h of IVM compared to the untreated control. We also confirmed that protein kinase A activity increased in dbcAMP-treated oocytes, indicating an elevated intracellular concentration of cAMP. After IVF, the frequency of polyspermy in the dbcAMP-treated group decreased compared to that in the control group (22.4% vs. 47.4%, P<0.05). Furthermore, blastocyst formation, the blastocyst cell number, mitochondrial membrane potential, and apoptosis were enhanced and/or reduced by dbcAMP in both IVF and SCNT embryos. We concluded that synchronizing meiotic resumption by dbcAMP treatment improved the developmental capacity and embryonic qualities of IVF and SCNT embryos by increasing the mitochondrial membrane potential and decreasing the incidence of apoptosis in preimplantation-stage porcine embryos.
生发泡期卵母细胞中高浓度的环磷酸腺苷（cAMP）通常会抑制生发泡破裂（GVBD）。因此，使生长中的卵母细胞维持在GV期对卵子的发育能力至关重要。在本研究中，我们追踪了二丁酰环磷酸腺苷（dbcAMP）对猪减数分裂成熟和早期胚胎发育的影响。我们还研究了几种受dbcAMP影响的囊胚质量，包括结构完整性、线粒体膜电位和细胞凋亡。为了确定cAMP浓度升高是否会抑制GVBD，我们探究了猪卵母细胞在成熟过程中的减数分裂模式。用dbcAMP处理22小时后，91.1%的卵母细胞停滞在GV期，而对照组只有38.8%的卵母细胞处于该阶段（P<0.05）。体外成熟（IVM）完成后，经dbcAMP处理的卵母细胞处于中期II的比例高于未处理的卵母细胞（91.3%对72.8%，P<0.05）。蛋白质免疫印迹分析显示，与未处理的对照组相比，在IVM的前22小时用dbcAMP处理的猪卵母细胞中，成熟促进因子（MPF）和丝裂原活化蛋白激酶（MAP激酶）活性在22小时时降低，在44小时时升高和/或降低。我们还证实，经dbcAMP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。体外受精（IVF）后，与对照组相比，dbcAMP处理组的多精受精频率降低（22.4%对47.4%，P<0.05）。此外，在IVF和体细胞核移植（SCNT）胚胎中，dbcAMP均增强和/或降低了囊胚形成、囊胚细胞数量、线粒体膜电位和细胞凋亡。我们得出结论，通过dbcAMP处理使减数分裂恢复同步，可通过增加植入前阶段猪胚胎的线粒体膜电位和降低细胞凋亡发生率，提高IVF和SCNT胚胎的发育能力和胚胎质量。
