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Shear Stress Induces Preimplantation Embryo Death That Is Delayed by the Zona Pellucida and Associated with Stress-Activated Protein Kinase-Mediated Apoptosis¹

CS Mott Center for Human Growth and Development of Ob/Gyn,³ Department of Anatomy and Cell Biology,⁴ Karmanos Cancer Institute,⁵ Institute of Environmental Health Sciences,⁶ Wayne State University School of Medicine, Detroit, Michigan 48201 Departments of Civil and Environmental Engineering,⁷ Clarkson University, Potsdam, New York 13699-5710

ABSTRACT

In this study, we discovered that embryos sense shear stress and sought to characterize the kinetics and the enzymatic mechanisms underlying induction of embryonic lethality by shear stress. Using a rotating wall vessel programmed to produce 1.2 dynes/cm² shear stress, it was found that shear stress caused lethality within 12 h for E3.5 blastocysts. Embryos developed an approximate 100% increase in mitogen-activated protein kinase 8/9 (formerly known as stress-activated protein kinase/junC kinase 1/2) phosphorylation by 6 h of shear stress that further increased to approximately 350% by 12 h. Terminal deoxynucleotidyltransferase dUTP nick end labeling/apoptosis was at baseline levels at 6 h and increased to approximately 500% of baseline at 12 h, when irreversible commitment to death occurred. A mitogen-activated protein kinase 8/9 phosphorylation inhibitor, D-JNKI1, was able to inhibit over 50% of the apoptosis, suggesting a causal role for mitogen-activated protein kinase 8/9 phosphorylation in the shear stress-induced lethality. The E2.5 (compacted eight-cell/early morula stage) embryo was more sensitive to shear stress than the E3.5 (early blastocyst stage) embryo. Additionally, zona pellucida removal significantly accelerated shear stress-induced lethality while having no lethal effect on embryos in the static control. In conclusion, preimplantation embryos sense shear stress, chronic shear stress is lethal, and the zona pellucida lessens the lethal and sublethal effects of shear stress. Embryos in vivo would not experience as high a sustained velocity or shear stress as induced experimentally here. Lower shear stresses might induce sufficient mitogen-activated protein kinase 8/9 phosphorylation that would slow growth or cause premature differentiation if the zona pellucida were not intact.

apoptosis, embryo, kinases, signal transduction, stress

¹ Supported by grants from NASA (NRA-NAG2–1503) and the National Institute of Child Health and Human Development, NIH (R01-HD40972).

² Correspondence: D.A. Rappolee, CS Mott Center for Human Growth, Wayne State University School of Medicine, 275 East Hancock, Detroit, MI 48201. FAX: 313 577 8554; drappole{at}med.wayne.edu