Institute of Animal Science, Department of Animal Breeding and Husbandry, University of Bonn, Bonn, Germany.
Animal and Poultry Physiology Department, Animal and Poultry Production Division, Desert Research Center, Mataria, Cairo, Egypt.
PLoS One. 2019 Oct 29;14(10):e0223753. doi: 10.1371/journal.pone.0223753. eCollection 2019.
Focal adhesion pathway is one of the key molecular pathways affected by suboptimal culture conditions during embryonic development. The epidermal growth factor (EGF) and hyaluronic acid (HA) are believed to be involved in the focal adhesion pathway function by regulating the adherence of the molecules to the extracellular matrix. However, regulatory and molecular mechanisms through which the EGF and HA could influence the embryo development is not clear. Therefore, this study aimed to investigate the effect of continued or stage specific supplementation of EGF and/or HA on the developmental competence and quality of bovine preimplantation embryos and the subsequent consequences on the expression and DNA methylation patterns of genes involved in the focal adhesion pathway. The results revealed that, the supplementation of EGF or HA from zygote to the blastocysts stage reduced the level of reactive oxygen species and increased hatching rate after thawing. On the other hand, HA decreased the apoptotic nuclei and increased blastocyst compared to EGF supplemented group. Gene expression and DNA methylation analysis in the resulting blastocysts indicated that, combined supplementation of EGF and HA increased the expression of genes involved in focal adhesion pathway while supplementation of EGF, HA or a combination of EGF and HA during the entire preimplantation period changed the DNA methylation patterns of genes involved in focal adhesion pathway. On the other hand, blastocysts developed in culture media supplemented with EGF + HA until the 16-cell stage exhibited higher expression level of genes involved in focal adhesion pathway compared to those supplemented after the 16-cell stage. Conversely, the DNA methylation level of candidate genes was increased in the blastocysts obtained from embryos cultured in media supplemented with EGF + HA after 16-cell stage. In conclusion, supplementation of bovine embryos with EGF and/or HA during the entire preimplantation period or in a stage specific manner altered the DNA methylation and expression patterns of candidate genes involved in the focal adhesion pathway which was in turn associated with the observed embryonic developmental competence and quality.
焦点黏附路径是胚胎发育过程中因培养条件不佳而受影响的关键分子路径之一。表皮生长因子（EGF）和透明质酸（HA）被认为通过调节分子与细胞外基质的黏附参与焦点黏附路径的功能。然而，EGF 和 HA 影响胚胎发育的调节和分子机制尚不清楚。因此，本研究旨在探讨持续或阶段特异性补充 EGF 和/或 HA 对牛胚胎早期发育能力和质量的影响，以及随后对参与焦点黏附路径的基因的表达和 DNA 甲基化模式的影响。研究结果表明，从受精卵到囊胚阶段补充 EGF 或 HA 可降低解冻后活性氧的水平并提高孵化率。另一方面，HA 减少了凋亡核并增加了囊胚数，与补充 EGF 的组相比。对产生的囊胚进行基因表达和 DNA 甲基化分析表明，EGF 和 HA 的联合补充增加了参与焦点黏附路径的基因的表达，而在整个胚胎着床前阶段补充 EGF、HA 或 EGF 和 HA 的组合改变了参与焦点黏附路径的基因的 DNA 甲基化模式。另一方面，在补充 EGF + HA 的培养基中培养至 16 细胞阶段的胚胎中，参与焦点黏附路径的基因表达水平高于补充 EGF + HA 后培养的胚胎。相反，在补充 EGF + HA 后的培养基中培养的胚胎中获得的囊胚中候选基因的 DNA 甲基化水平增加。总之，在整个胚胎着床前阶段或在特定阶段补充牛胚胎 EGF 和/或 HA 改变了参与焦点黏附路径的候选基因的 DNA 甲基化和表达模式，这反过来又与观察到的胚胎发育能力和质量有关。
