摘要
The embryonic developmental block occurs at the 8-cell stage in cattle and is characterized by a lengthening of the cell cycle and an increased number of embryos that stop development. The maternal-embryonic transition arises at the same stage resulting in the transcription of many genes. Gene expression studies during this stage may contribute to the understanding of the physiological mechanisms involved in the maternal-embryonic transition. Herein we identified genes differentially expressed between embryos with high or low developmental competence to reach the blastocyst stage using differential display PCR. Embryos were analysed according to developmental kinetics: fast cleavage embryos showing 8 cells at 48 h post insemination (hpi) with high potential of development (F8), and embryos with slow cleavage presenting 4 cells at 48 hpi (S4) and 8 cells at 90 hpi (S8), both with reduced rates of development to blastocyst. The fluorescence DDPCR method was applied and allowed the recovery of 176 differentially expressed bands with similar proportion between high and low development potential groups (52% to F8 and 48% in S4 and S8 groups). A total of 27 isolated fragments were cloned and sequenced, confirming the expected primer sequences and allowing the identification of 27 gene transcripts. PI3KCA and ITM2B were chosen for relative quantification of mRNA using real-time PCR and showed a kinetic and a time-related pattern of expression respectively. The observed results suggest the existence of two different embryonic genome activation mechanisms: fast-developing embryos activate genes related to embryonic development, and slow-developing embryos activate genes related to cellular survival and/or death.
摘要译文
胚胎发育阻滞发生在牛的8细胞阶段，其特征在于细胞周期的延长和停止发育的胚胎数量的增加。母胚 - 胚胎转变发生在同一阶段，导致许多基因的转录。在该阶段的基因表达研究可以有助于理解母体 - 胚胎转变中涉及的生理机制。在这里，我们鉴定了具有高或低发育能力的胚胎之间差异表达的基因，以使用差异显示PCR达到胚泡阶段。根据发育动力学分析胚胎：快速切割胚胎在授精后48小时显示8个细胞（hpi），具有高发育潜力（F8），具有缓慢切割的胚胎在48hpi（S4）呈现4个细胞，在90个细胞处呈现8个细胞。 hpi（S8），均降低胚泡发育速度。应用荧光DDPCR方法并允许回收176个差异表达的条带，在高和低发育潜力组之间具有相似的比例（52％至F8，S4和S8组为48％）。克隆并测序总共27个分离的片段，证实了预期的引物序列并允许鉴定27个基因转录物。选择PI3KCA和ITM2B用于使用实时PCR对mRNA进行相对定量，并分别显示动力学和时间相关的表达模式。观察到的结果表明存在两种不同的胚胎基因组激活机制：快速发育的胚胎激活与胚胎发育相关的基因，而缓慢发育的胚胎激活与细胞存活和/或死亡相关的基因。
Paula Ripamonte (a1); L铆gia Garcia Mesquita (a1); Sylvia Sanches Cortezzi (a1); J煤lio C茅sar de Carvalho Balieiro (a1); Giovana Krempel Fonseca Merighe (a1); Yeda Fumie Watanabe (a1); Alexandre Rodrigues Caetano (a2);Fl谩vio Vieira Meirelles (a3).... Differential gene expression and developmental competence in in vitro produced bovine embryos[J]. Zygote, 2012,20(3): 281-290