Abstract
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.
Highlights
Suboptimal culture conditions during preimplantation period can result in long-term effects on embryo competence and pregnancy establishment
Blastocysts derived from zygotes supplemented with epidermal growth factor (EGF) + hyaluronic acid (HA) did not show a significant difference in reactive oxygen species (ROS) levels compared to control and HA groups (Fig 3)
Our previous results demonstrated that, in vivo and in vitro alternative culture conditions altered the expression and Deoxyribonucleic acid (DNA) methylation pattern of genes involved in focal adhesion pathway in the resulted blastocysts [2, 3]
Summary
Suboptimal culture conditions during preimplantation period can result in long-term effects on embryo competence and pregnancy establishment. Despite many attempts to modulate the in vitro culture conditions to mimic the in vivo environment, the quality of in vitro produced embryos remains low [1]. Suboptimal embryo culture condition decreases the quality and hinders the developmental competence of the embryo by altering the expression and DNA methylation patterns of developmentally related genes and pathways [2, 3]. Focal adhesion pathway was one of the top dysregulated pathways in bovine embryos due to suboptimal culture conditions [4]. The functional loss of these proteins during embryogenesis would affect the cell-extracellular matrix (ECM) adhesion, cytoskeletal organization, polarity, migration and survivability of the embryos [7]
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