Commonly used fertility drugs, a diet supplement, and stress force AMPK-dependent block of stemness and development in cultured mammalian embryos.

Abstract

The purpose of the present study is to test whether metformin, aspirin, or diet supplement (DS) BioResponse-3,3'-Diindolylmethane (BR-DIM) can induce AMP-activated protein kinase (AMPK)-dependent potency loss in cultured embryos and whether metformin (Met) + Aspirin (Asa) or BR-DIM causes an AMPK-dependent decrease in embryonic development. The methods used were as follows: culture post-thaw mouse zygotes to the two-cell embryo stage and test effects after 1-h AMPK agonists' (e.g., Met, Asa, BR-DIM, control hyperosmotic stress) exposure on AMPK-dependent loss of Oct4 and/or Rex1 nuclear potency factors, confirm AMPK dependence by reversing potency loss in two-cell-stage embryos with AMPK inhibitor compound C (CC), test whether Met + Asa (i.e., co-added) or DS BR-DIM decreases development of two-cell to blastocyst stage in an AMPK-dependent (CC-sensitive) manner, and evaluate the level of Rex1 and Oct4 nuclear fluorescence in two-cell-stage embryos and rate of two-cell-stage embryo development to blastocysts. Met, Asa, BR-DIM, or hyperosmotic sorbitol stress induces rapid ~50-85% Rex1 and/or Oct4 protein loss in two-cell embryos. This loss is ~60-90% reversible by co-culture with AMPK inhibitor CC. Embryo development from two-cell to blastocyst stage is decreased in culture with either Met + Asa or BR-DIM, and this is either >90 or ~60% reversible with CC, respectively. These experimental designs here showed that Met-, Asa-, BR-DIM-, or sorbitol stress-induced rapid potency loss in two-cell embryos is AMPK dependent as suggested by inhibition of Rex1 and/or Oct4 protein loss with an AMPK inhibitor. The DS BR-DIM or fertility drugs (e.g., Met + Asa) that are used to enhance maternal metabolism to support fertility can also chronically slow embryo growth and block development in an AMPK-dependent manner.