Medium components and developmental arrest in preimplantation mouse embryos

Mary Kathryn Dienhart, Marquette University

Abstract

Explanted one-cell stage mouse embryos are extremely sensitive to in vitro conditions and often fail to complete the preimplantation period of development in culture, arresting after first or second cleavage. In this study, I have examined the effect of several medium components on the in vitro development of early mouse embryos to gain an understanding of the mechanism(s) behind the 2-cell block exhibited by some strains and the developmental arrest imposed by the purine, hypoxanthine. Culture of embryos in glucose-containing medium inhibits development, but the degree of inhibition varies depending on mouse strain and culture medium used. The results of this study suggest that different mechanisms are involved in the 2-cell and the hypoxanthine-induced blocks. First, embryos that exhibited the 2-cell block failed to develop to blastocyst whether or not glucose was present, whereas the hypoxanthine-induced arrest was glucose-dependent. In addition, EDTA, in combination with cAMP-elevating agents, reversed the hypoxanthine block but these same agents were without effect on the 2-cell block. Hypoxanthine uptake was increased in embryos cultured in glucose as were phosphoribosylpyrophosphate (PRPP) levels. The addition of hypoxanthine decreased PRPP levels which is consistent with consumption of PRPP via the salvage reaction. HPLC analysis of hypoxanthine metabolism by embryos revealed that hypoxanthine is converted to nucleotides and this increased significantly in glucose-containing medium. Reversal of the hypoxanthine-induced block by cAMP-elevating agents was EDTA-dependent and resulted in decreased uptake of glucose and hypoxanthine and changes in the relative metabolism of this purine base. In addition, hypoxanthine and low concentrations of adenosine or AICA riboside reversed the azaserine-induced arrest in glucose-free medium. These results support the idea that the inhibitory action of hypoxanthine on mouse embryos is mediated via negative feedback of purine nucleotides on PRPP synthetase, resulting in decreased PRPP availability and arrest of other PRPP-dependent pathways; while lower levels of nucleotide salvage maintains PRPP levels and embryo viability.

Recommended Citation

Mary Kathryn Dienhart, "Medium components and developmental arrest in preimplantation mouse embryos" (January 1, 1996). Dissertations (1962 - 2010) Access via Proquest Digital Dissertations. Paper AAI9719072.
http://epublications.marquette.edu/dissertations/AAI9719072

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