In vitro studies of mouse embryos bearing mutations in the T complex: effects of culture in suboptimal medium upon t 6 /t 6 and normal embryos

Abstract

We have compared the behaviour of normal and t6/t6 embryos in PCMF, a suboptimal 'delay' medium which arrests normal development. Morphologically, the two types of embryos are indistinguishable in this medium. Although beta-glucuronidase activity in embryos does not rise as quickly in delay medium as in cNCTC, a nutritive medium, the rate of increase is similar in t6/t6 and normal embryos. We conclude from these observations that the lethality of t6/t6 embryos is not a consequence of their reaching a given absolute age. Together with previous studies, our data suggest that embryo lethality correlates more closely with metabolic state than with morphological stage. Blastocysts maintained in PCMF are unable to give rise to trophoblast outgrowths but do so upon transfer into cNCTC medium. When a mixture of fourth-day t6/t6 and normal embryos are transferred to cNCTC medium after lengthy pre-incubation periods in PCMF, trophoblast outgrowth is initiated from both types of embryos at approximately the same time. However, when embryos are removed from the genital tract on the second or third day of gestation, t6/t6 embryos are slower to produce trophoblast outgrowths than are normal embryos upon transfer from PCMF to cNCTC medium. Although the reason for this differential behaviour is not yet clear, it is hypothesized that some product(s) required for the outgrowth process is (are) more unstable in third-day t6/t6 embryos than in normal third-day embryos or fourth-day t6/t6 embryos. Our ability to separate t6/t6 from normal embryos by their delayed initiation of trophoblast outgrowth provides us with a convenient way to identify, and to isolate for analysis, enriched populations of homozygous mutant embryos prior to the time at which they show gross morphological abnormalities.