Using hyperosmolar stress to measure biologic and stress-activated protein kinase responses in preimplantation embryos

Abstract

We used hyperosmolar stress to test blastocysts for their biologic and enzymatic responses to culture stress. Embryos mount dose- and time-dependent responses to hyperosmolar stress. Biological responses included slowed cavitation and cell accumulation and increased apoptosis at increasing doses. These responses were preceded by stress-activated protein kinase (SAPK) phosphorylation and nuclear translocation consistent with its causal role. For cavitation and new cell cycle initiation, 200 mM sorbitol caused stasis. Above 200 mM, sorbitol was ultimately lethal and below 200 mM, its embryos had milder effects. Phosphorylated SAPK was induced rapidly in embryos at 0.5 h in a dose-dependent manner from 0 to 600 mM sorbitol. Higher hyperosmolarity caused a biphasic peak of phosphorylated SAPK, but there was no return to baseline through 3 h. At 24 h, a dose-dependent response persisted that was linear from 0 to 200 mM sorbitol. Hyperosmolar stress rapidly induced, within 0.5 h, phosphorylated, nuclear c-Jun and decreased phosphorylated, nuclear c-Myc in a SAPK-dependent manner. The data suggest that SAPK is induced and functions on down-stream effector molecules in a temporal and quantitative manner consistent with its function in the embryonic homeostatic response to stress. The remarkable resistance of embryos to high concentrations of sorbitol suggests that part of its homeostatic response is different from that of somatic cells.