Chronic Shear Stress Is Lethal to Preimplantation Mouse Embryos Acting Through SAPK/JNK Phospho and TUNEL, But Acute Mouth Pipetting Also Increases SAPK/JNK and c-Fos

Abstract

To test for the effects of embryo movement creating shear stress. Observational laboratory study to test whether shear stress in a rotating wall vessel or during mouth pipetting can induce stress activated protein kinase/Jun kinase (SAPK/JNK) phosphorylation and mediate poor outcomes in the preimplantation embryo. Mouse embryos were shear stressed in laboratory experiments using a rotating wall vessel and a Synthecon rotational cell culture system (RCCS)-1 to produce shear stress estimated to be 1.2dynes/cm2 or by mouth pipetting and tested for induction of SAPK/JNK diphospho and c-Fos, and TUNEL/apoptosis assayed by immunocytochemical assays. Experiments were also done to test for the role of SAPK/JNK and the Zona pellucida in the shear stress pathway. Embryos developed time-dependent SAPK phosphorylation that preceded TUNEL/apoptosis and irreversible death occurring at 12hr. A marker of shear stress, c-Fos protein, was induced in the outer placental epithelial cells of the blastocyst within 0.5hr of the initiation of shear stress, and was greater than rotational control (RC), while static control (SC) embryos had only background levels of c-Fos protein. A SAPK inhibitor, SP600125 was able to inhibit a large fraction of the apoptosis, suggesting that SAPK is induced by shear stress and causal to increased TUNEL/apoptosis. In comparison with hyperosmolar stress, shear stress caused death faster than 2M sorbitol, a very high dose. Removal of the Zona pellucida also enhanced lethality (in speed and magnitude) in shear stress, but had no effect on embryos in SC. Mouth pipetting also induced SAPK/JNK phospho and c-Fos in a dose-dependent manner. Shear stress induces rapid c-Fos protein and SAPK phosphorylation, followed by TUNEL/apoptosis. The Zona pellucida delays the lethal effects of shear stress. Taken together, these data agree with an earlier report where the 8-cell stage embryo was also found to be most sensitive to lethality after removing the zone pellucida and re-implanting into the oviduct (Bronson and McLaren, 1970), conditions which would expose the embryo to low levels of shear stress. In this earlier report, removal of the Zona pellucida (an acellular protein coat without known function after fertilization), enhanced embryo lethality. This suggests that the function of the Zona after fertilization may be to prevent shear stress caused lethality. In addition, mouth pipetting E3.5 and E4.5 mouse embryos caused a rapid dose-dependent increase in c-Fos and SAPK/JNK phospho, suggesting that handling embryos can cause transient shear stress.