Fertilization and Early Embryogenesis
(M286) OSMOTIC STRESS INDUCED p38 MAPK ACTIVATION IN PREIMPLANTATION MOUSE EMBRYOS DURING IN VITRO CULTURE.
Fong, Barry1, 2, Watson, Andrew1, 2, 1 University of Western Ontario, London, ON, Canada2 Lawson Health Research Institute, London, ON, Canada
ABSTRACT- Cultured embryos are exposed to potentially harmful factors and develop under conditions which are suboptimal compared to the in vivo environment of the female reproductive tract. Factors influencing in vitro embryo development include temperature, pH, and osmolarity. Sensing osmolarity is a necessary ability that all organisms have developed, thus cells are capable of adapting and responding to changes in environmental osmolarity. The principle aim of this study was to investigate the influence of culture medium osmolarity on p38 mitogen-activated protein kinase (MAPK) activation during early mouse development. The hypothesized model for p38 MAPK activation is via the upstream kinase MEKK3-MKK3-p38 phosphorylation relay, mediated by an osmolarity induced protein called osmosensing scaffold for MEKK3 (OSM). The first objective was to characterize the expression of mRNAs encoding MEKK3, MKK3, MKK6, MKK4, and OSM during mouse preimplantation development. Results from application of standard RT-PCR methods demonstrate that mRNAs encoding MEKK3, MKK3 and MKK4 are present throughout preimplantation development; while mRNAs encoding MKK6 and the osmosensing scaffold for MEKK3 (OSM) are primarily detected in later preimplantation embryo stages (morulae/blastocysts). Results employing whole-mount indirect immunofluorescence revealed that blastocyst stage embryos cultured in hyperosmotic medium (KSOMaa with 100mM sorbitol; 390mOsm) for up to 12h displayed an obvious increase in fluorescence of the phosphorylated form of MAPK activated protein kinase-2 (MAPKAPK-2) compared to blastocysts cultured in control medium (290mOsm). MAPKAPK-2 is a downstream substrate of p38 MAPK and is a reliable marker of p38 MAPK pathway activation. In conclusion, the p38 MAPK pathway is a sensitive monitor of environmental osmolarity that mediates embryo-medium interactions during mouse preimplantation development. Research supported by the Canadian Institutes of Health Research (CIHR).
KEY WORDS: p38 mitogen-activated protein kinase (MAPK), osmolarity, preimplantation embryo, in vitro culture