Pipetting causes shear stress and elevation of phosphorylated stress-activated protein kinase/jun kinase in preimplantation embryos

Shear stress at 1.2 dynes/cm(2) induces stress-activated protein kinase/jun kinase phosphorylation that precedes and causes apoptosis in embryos (Xie et al., 2006b, Biol Reprod). Pipetting embryos is necessary for many protocols, from in vitro fertilization to collecting embryos prior to analyzing gene expression by microarrays. We sought to determine if pipetting upregulates phosphorylated MAPK8/ 9 (formerly known as stress-activated protein kinase/ jun kinase/SAPK/JNK1, 2). We found that phosphorylated MAPK8/9, a marker of MAPK8/9 activation, is upregulated in a dose-dependent manner by pipetting. Whereas embryos with the zona pellucida removed were more sensitive to stress-induced lethality mediated by 1.2 dynes/cm(2) shear force, phosphorylated MAPK8/9 was induced at lower numbers of pipet triturations in hatched embryos at E4.5. E4.5 embryos were more sensitive to induction of MAPK8/9 than unhatched embryos at E2.5 or E3.5. E3.5 embryos also showed a pipetting dose-dependent induction of FOS protein (formerly known as c-fos), a marker of shear stress in many cell types. Phosphorylated MAPK8/9 measured in ex vivo embryos from E1.5 to E4.5 were expressed at low levels. Embryos that had been pipetted sufficiently to induce phosphorylated MAPK8/9 and FOS had the same number of cells as untreated embryos 24 hr later. This suggests that rapid phosphorylation of MAPK8/9 due to transient shear stress does not mediate long-term negative biological outcomes. But, it is possible that techniques requiring multiple handling events would induce MAPK8/9 and cause biological outcomes or that other biological outcomes are affected by low amounts of transient shear stress. This study suggests that embryo handling prior to experimental measurement of signal transduction phosphoproteins, proteins and mRNA should be performed with care. Indeed, it is likely that shear stress may cause rapid transient changes in hundreds of proteins and mRNA.