Abnormal development of pre-implantation mouse embryos grown in vitro with [3H]thymidine

Abstract

Mouse embryos were grown in vitro from the 2-cell stage to blastocysts in the presence of [³H]thymidine. Methyl-T-thymidine and thymidine-ó-T(n) were used and both forms found to be lethal at concentrations above 0 · 1 μ Ci/ml. Both forms of [³H]Tdr at concentrations between 0 · 01 and 0 · 1 μ Ci/ml caused a highly significant (P < 0·001) reduction in blastocyst cell number. The reduction in cell number, which was positively correlated with specific activity and tritium concentration, was associated with cell damage typical of radiation damage caused by tritium disintegration. Thymidine-6-T(n) also significantly reduced the number of 2-cell embryos forming blastocysts whereas methyl-T-Tdr did not. This difference in effect is assumed to be caused by contamination of one form of [³H]Tdr with a by-product of the tritiation process. A study of the cleavage stages showed that almost all the reduction in cell numbers could be accounted for by selective cell death occurring at the 16-cell stage. Cells which survive that stage cleave at a normal rate. The cells that are most susceptible to [³H]Tdr damage were found to normally contribute to the inner cell mass. The [³H]Tdr-resistant cells form the trophoblast. It is possible to grow blastocysts in [³H]Tdr such that they contain no inner cell mass but are composed entirely of trophoblast. Comparatively short (12 h) incubation with [³H]Tdr at any stage prior to the 16-cell stage will cause this damage. Possible reasons for this differential effect are discussed, and also compared with damage caused by X-irradiation.