Modification of in vitro mouse embryogenesis by X-rays and fluorochromes

Abstract

In view of planned studies using single particle irradiation at the Institute for Medical Radiobiology (IMR), confocal microscopy will become an important tool to visualise subtle changes in embryo morphology. Therefore, the influence of X-rays and that of three different fluorochromes on the in vitro development of murine 2-cell stage embryos was investigated. Embryos of B₆C₃F₁ mice were cultured at 32 h post-conception (pc) and treated with X-rays, acridine orange (AO), ethidium bromide (EB) or propidium iodide (PI), respectively, in various doses (0.0–3.0 Gy) or concentrations (AO: 0.05–2.00 µg/ml; EB and PI: 0.50–10.00 µg/ml). Additional experiments using combinations of AO and irradiation were performed. The embryos were cultivated for a total of 7 days and checked for their vital status by morphological endpoints such as the percentage of embryos that reached the blastocyst stage and the hatching rate. After treatment of the 2-cell embryos with AO, EB and PI, the dose-effect curves with the endpoint ‘hatching rate’ showed a 23-fold reduction of the ED₅₀ for AO (0.23 µg/ ml) compared with EB (5.30 µg/ml). Despite the higher toxicity, AO had much better staining qualities in subtoxic concentrations than EB. PI showed no toxicity in these experiments and was used as an inverse control for embryo vitality. No synergistic modification of the radiogenic effects could be seen in combination experiments (AO+X-rays).