The Effect of Ethanol Exposure on Extraembryonic Vascular Development in the Chick Area Vasculosa

Abstract

The effect of ethanol (EtOH) exposure on extraembryonic vascular development was examined using the chick embryo area vasculosa (AV) in shell-less culture. Embryos were placed in cultures at Hamburger Hamilton (HH) stage 11/12 and a single dose of EtOH (10, 30 or 50%) was applied to the center of the blastodisc. Untreated/sodium-chloride-treated controls showed normal embryonic growth and well-developed extraembryonic vessels at 24/48 h of treatment. At doses of 30 and 50%, the mortality rate was significantly increased, and survivors demonstrated significant growth retardation and inhibition of normal vascular development in a dose-dependent manner. Immunostaining for vascular endothelial growth factor (VEGF) showed that mesenchymal cells continued to differentiate into angioblasts to form blood islands, but their assembly into primitive vessels was perturbed in a dose-dependent manner. Northern blot analyses of basic fibroblast growth factor, VEGF, Flt-1 and Flk-1 mRNA expression supported these findings and showed a dose-dependent decrease in EtOH-treated cultures compared to controls. Co-treatment with α-tocopherol (0.05 M) or all-trans-retinoic acid (10^–8M) significantly decreased the mortality rate and improved both embryonic growth and extraembryonic vascular development in the cultures. On the other hand, almost all embryos treated with 10% EtOH survived the first 48 h after treatment. However, the complexity of the vascular tree measured as the relative vasculogenesis index, the surface area of the AV and the mRNA expression of vasculogenic molecules were increased during the first 24 h. This acute effect disappeared 48 h after treatment and the vascular tree continued to develop parallel to the controls. No significant growth retardation was observed in this group. These results suggest that, in terms of extraembryonic vascular development, an early, single, low-dose EtOH exposure may have an acute, short-term positive effect, whereas moderate- or high-dose EtOH exposure may severely perturb this process disabling the necessary absorption of the nutrients for the embryo to develop properly. The mechanisms of action of EtOH on extraembryonic vascular development may involve the establishment of reactive oxygen species, resulting in the initiation of oxidative stress and perturbation of retinoic acid signaling and alterations in the expression of growth-regulatory vasculogenic factors and their receptors.

Disruption of circulation by ethanol promotes fetal alcohol spectrum disorder (FASD) in medaka (Oryzias latipes) embryogenesis

“…In chick embryo, ethanol altered the early vascular development in area vasculosa in a concentration-dependent manner (Tufan and SatirogluTufan, 2003). The mechanisms proposed for the disruption were the generation of reactive oxygen species, perturbation of retinoic acid signaling, and alteration in the expression of growth regulatory vasculogenic factors and their receptors (Tufan and Satiroglu-Tufan, 2003). Reduction in cellular protein and RNA contents in medaka embryogenesis by ethanol (an index of oxidative stress) thus prompted us to extend our investigation to determine the status of oxidative stress and alcohol metabolizing enzyme mRNA levels in medaka embryos.…”

The Chick Embryo Chorioallantoic Membrane as a Model System for the Study of Tumor Angiogenesis, Invasion and Development of Anti-Angiogenic Agents

“…However, there is no clear evidence that there is any significant difference between data derived using in ovo or shell-less culture method [103]. In order for the chick embryos to develop, eggs must be maintained at 37 o C, in a humidified atmosphere [103][104][105][106][107][108]. The first day in these conditions is considered to be day 1 of embryonic development.…”

Ethanol exposure leads to disorder of blood island formation in early chick embryo

“…Satiroglu-Tufan found that the exposure of (10, 30 or 50%) ethanol could increase the embryonic mortality rate and inhibit the vascular development in a dose-dependent manner during latter developmental stages of chick embryos [41]. In this study, gastrula chick embryo (earlier stages) is used as a model to investigate the effects of 2% ethanol exposure on early vascular development.…”