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.

The activity of retinoid receptors activity can be regulated by various extracellular stimuli. In an effort to understand the molecular basis for this phenomenon, the role of b-arrestins was investigated. b-Arrestins constitute a class of proteins involved in the internalization of agonistactivated receptors. They have also been linked to MAPK activation suggesting a direct involvement in signaling cascades. Here, we report that b-arrestin 2 stimulates the transcriptional activation of the retinoid RAR and RXR receptors. Of all the retinoid receptors, the RAR b2 subtype showed the strongest sensitivity to b-arrestin 2 action. Interestingly, this event requires the presence of the MAP kinase ERK2, but not that of JNK or P38. Sitedirected mutagenesis showed that Ser 22 and Leu 217 are critical residues of the RAR b2 receptor through which b-arrestin 2 effects are mediated. More importantly, we demonstrate that the induction of PC12 growth inhibition by Nerve Growth Factor is indeed dependent upon RAR b2 transcriptional activation in a b-arrestin 2-and ERK2-dependent manner.