Bernal-Ulloa Sandra Milena, Heinzmann Julia, Herrmann Doris, Hadeler Klaus-Gerd, Aldag Patrick, Winkler Sylke, Pache Dorit, Baulain Ulrich, Lucas-Hahn Andrea, Niemann Heiner
Institute of Farm Animal Genetics, Biotechnology, Friedrich-Loeffler-Institut, Mariensee, Germany.
Facultad de Ciencias Agropecuarias, Universidad de Ciencias Aplicadas y Ambientales, Bogotá, Colombia.
PLoS One. 2016 Feb 29;11(2):e0150264. doi: 10.1371/journal.pone.0150264. eCollection 2016.
High cAMP levels during in vitro maturation (IVM) have been related to improved blastocyst yields. Here, we employed the cAMP/cGMP modulators, forskolin, IBMX, and cilostamide, during IVM to unravel the role of high cAMP in early embryonic development produced from prepubertal and adult bovine oocytes. Oocytes were collected via transvaginal aspiration and randomly assigned to three experimental groups: TCM24 (24 h IVM/control), cAMP30 (2 h pre-IVM (forskolin-IBMX), 30 h IVM-cilostamide), and DMSO30 (Dimethyl Sulfoxide/vehicle control). After IVM, oocytes were fertilized in vitro and zygotes were cultured in vitro to blastocysts. Meiotic progression, cAMP levels, mRNA abundance of selected genes and DNA methylation were evaluated in oocytes. Blastocysts were used for gene expression or DNA methylation analyses. Blastocysts from the cAMP30 groups were transferred to recipients. The cAMP elevation delayed meiotic progression, but developmental rates were not increased. In immature oocytes, mRNA abundance of PRKACA was higher for cAMP30 protocol and no differences were found for PDE3A, SMAD2, ZAR1, PRDX1 and SLC2A8. EGR1 gene was up-regulated in prepubertal cAMP30 immature oocytes and down-regulated in blastocysts from all in vitro treatments. A similar gene expression profile was observed for DNMT3b, BCL2L1, PRDX1 and SLC2A8 in blastocysts. Satellite DNA methylation profiles were different between prepubertal and adult oocytes and blastocysts derived from the TCM24 and DMSO30 groups. Blastocysts obtained from prepubertal and adult oocytes in the cAMP30 treatment displayed normal methylation profiles and produced offspring. These data indicate that cAMP regulates IVM in prepubertal and adult oocytes in a similar manner, with impact on the establishment of epigenetic marks and acquisition of full developmental competency.
体外成熟（IVM）过程中高浓度的环磷酸腺苷（cAMP）与囊胚产量的提高有关。在此，我们在IVM期间使用cAMP/环磷酸鸟苷（cGMP）调节剂、福斯高林、异丁基甲基黄嘌呤（IBMX）和西洛酰胺，以阐明高浓度cAMP在青春期前和成年牛卵母细胞产生的早期胚胎发育中的作用。通过经阴道抽吸收集卵母细胞，并随机分配到三个实验组：TCM24（24小时IVM/对照）、cAMP30（IVM前2小时（福斯高林 - IBMX），30小时IVM - 西洛酰胺）和DMSO30（二甲基亚砜/溶剂对照）。IVM后，卵母细胞进行体外受精，受精卵体外培养至囊胚。对卵母细胞的减数分裂进程、cAMP水平、选定基因的mRNA丰度和DNA甲基化进行评估。囊胚用于基因表达或DNA甲基化分析。将cAMP30组的囊胚移植到受体中。cAMP升高延迟了减数分裂进程，但发育率并未增加。在未成熟卵母细胞中，cAMP30方案的蛋白激酶A催化亚基α（PRKACA）的mRNA丰度较高，而磷酸二酯酶3A（PDE3A）、SMAD2、合子阻滞相关1蛋白（ZAR1）、过氧化物还原酶1（PRDX1）和溶质载体家族2成员8（SLC2A8）未发现差异。早期生长反应蛋白1（EGR1）基因在青春期前cAMP30未成熟卵母细胞中上调，而在所有体外处理的囊胚中下调。在囊胚中观察到DNA甲基转移酶3b（DNMT3b）、凋亡调节蛋白Bcl - 2样蛋白1（BCL2L1）、PRDX1和SLC2A8有类似的基因表达谱。青春期前和成年卵母细胞以及源自TCM24和DMSO30组的囊胚之间的卫星DNA甲基化谱不同。在cAMP30处理中，从青春期前和成年卵母细胞获得的囊胚显示出正常的甲基化谱并产生了后代。这些数据表明，cAMP以类似的方式调节青春期前和成年卵母细胞的IVM，对表观遗传标记的建立和完全发育能力的获得有影响。
