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.

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

“…We used hyperosmotic sorbitol at 200 mM because this dose is not toxic to embryos, TSCs, or ESCs [41,55,56,65,79] but slows their proliferation and has significant effects in causing AMPK-dependent Cdx2 and Id2 loss [41,45], PL1 increase in TSCs [80], Oct4 and Rex1 loss and first lineage Dab2 and LRP2 markers in ESCs [65,81,82], and significant AMPK-dependent Id2 loss in blastocysts [41]. We had previously shown that 200 mM sorbitol causes Cdx2 and Id2 loss in two-cell embryos in an AMPK-dependent manner [45], and this report adds AMPK-dependent Rex1 and Oct4 loss to make up a group of four AMPK-regulated potency factors expressed in both ESC and TSC lineages by the blastocyst stage.…”

“…For inhibitor studies (except where indicated), the inhibitors were preloaded with embryos 3 h before the stress and continued during the stress. In the text, the level of sorbitol (w/v) added is used to produce the given molarity of sorbitol [55,56]. For inhibitor studies, the inhibitors were incubated with embryos for 2 h before stress was added and during stress.…”

Stress Clamp Experiments on Multicellular Tumor Spheroids

“…Osmotic stress is known to have direct effects on cell growth and apoptosis in particular through the mitogen activated protein kinase (MAPK) pathway [12][13][14][15]. However, in all these studies, the effect of an osmotic shock is only measured for an osmotic stress two orders of magnitude larger than the one applied in our experiments (1M P a compared to 10kP a).…”

Cell volume regulation in mammalian oocytes and preimplantation embryos

“…In blastocysts, hypertonicity can initiate both p38 MAPK and SAPK/JNK signaling (Xie et al, 2007;Bell et al, 2009). Neither of these appears to be required to activate NHE1 in 2-cell embryos (Zhou and Baltz, 2012), although it remains to be determined if they have any other role in cell volume homeostasis.…”