Reduced O2 concentration during CAM development--its effect on angiogenesis and gene expression in the broiler embryo CAM

Abstract

Hypoxia during embryogenesis may induce changes in the development of some physiological regulatory systems, thereby causing permanent phenotypic changes in the embryo. Various levels of hypoxia at different time points during embryogenesis were found to affect both anatomical and physiological morphogenesis. These changes and adaptations depended on the timing, intensity, and duration of the hypoxic exposure and, moreover, were regulated by differential expression of developmentally important genes, mostly expressed in a stage- and time-dependent manner. Eggs incubated in a 17%-oxygen atmosphere for 12 h/d from E5 through E12 exhibited a clear and significant increase in the vascular area of the chorioallantoic membrane (CAM); an increase that was already significant within 12 h after the end of the 1st hypoxic exposures (E6). We used the combination of the genes, β-actin, RPLP0 and HPRT as a reference for gene expression profiling, in studying the expression levels of hypoxia-inducible factor 1-alpha (HIF1α), vascular endothelial growth factor alpha-2 (VEGF α 2), vascular endothelial growth factor receptor 2 (KDR), matrix metalloproteinase-2 (MMP2), and fibroblast growth factor 2 (FGF2), under normal and hypoxic conditions. In general, expression of all five investigated genes throughout the embryonic day of development had similar patterns of hypoxia-induced alterations. In E5.5 embryos, expression of HIF1α, MMP2, VEGFα2, and KDR was significantly higher in hypoxic embryos than in controls. In E6 embryos expression of HIF1α, VEGFα2, and FGF2 was significantly higher in hypoxic embryos than in controls. From E6.5 onward expression levels of the examined genes did not show any differences between hypoxic and control embryos. It can be concluded that in this experimental model, exposing broiler embryos to 17% O(2) from E5 to E7 induced significant angiogenesis, as expressed by the above genes. Further studies to examine whether this early exposure to hypoxic condition affects the chick's ability to withstand a post-hatch hypoxic environment is still required.