Preimplantation development is the "free-living" period of mammalian development that extends from insemination in the fallopian tube, to embryo implantation in the uterus. Blastocyst formation is required for implantation, establishment of pregnancy, and is a principal determinant of embryo quality prior to embryo transfer. Studies from our laboratory have revealed that the responses of preimplantation embryos to culture medium hyperosmolarity include increased p38 mitogen activated protein kinase (MAPK) activation and increased expression of Osmosensing scaffold for MeKK3. We have also reported that fluid movement across the trophectoderm is facilitated by the presence of Aquaporins (AQP) including AQP 3 and 9. The best current preimplantation embryo culture protocols employ media with osmolarities around 260 mOsm in contrast to the up to 350 mOsm reported for reproductive tract fluid. The rationale for this practice lies in that preimplantation embryo do not survive in media with osmolarities greater than 300 mOsm unless osmolytes are present when placed into culture. It is important to investigate and define the mechanisms and pathways that embryos employ to offset the deleterious effects that culture environments impose on preimplantation embryos. We have investigated the influences of hyperosmotic culture environments on preimplantation embryos and explored mechanisms and pathways embryos employ to offset these effects, all of which are crucial for a better understanding of embryo development and culture media optimization. We hypothesized that MAPK pathways mediate embryonic responses to hyperosmotic stress, by regulating AQP 3 and 9 expression as well as embryonic apoptosis. Real-Time PCR and whole-mount immunofluorescence were used to investigate AQP3 and 9 mRNA levels and protein distribution in embryos cultured in hyperosmotic media at 3h, 6h, and 24h. AQP3 and 9 mRNA did not vary significantly following 3 hours of exposure to sucrose hyperosmotic medium (300-460 mOsm) compared to control and glycerol hyperosmotic media. Following 6 hours we detected a significant increase in AQP3 and 9 mRNA in the sucrose 350mOsm treatment group. After 24 hours in sucrose hyperosmotic medium AQP3 and 9 mRNA levels remained significantly elevated compared to controls. When cultured in the presence of sucrose hyperosmotic medium and a p38 inhibitor, the increase in AQP3 and 9 mRNA levels were not observed. Whole-mount immunofluorescence revealed a dramatic shift in AQP9 protein from its predominantly cortical distribution observed in control embryos to become primarily localized to cytoplasmic/peri-nuclear regions in embryos cultured for 6 hours under hyperosmotic conditions (300mOsm-350mOsm). Apoptosis was investigated at 6h by using terminal deoxynucleotidyl transferase-mediated dUDP nick-end labeling (TUNEL) and Caspase-3 assays. Following hyperosmotic treatment in 350mOsm sucrose, we observed a significant increase in apoptosis levels while embryos exposed to 350mOsm glycerol did not vary from control apoptosis levels. In the presence of JNK inhibitor, but not p38 or ERK inhibitors, the 350mOsm sucrose-treated embryos displayed a significant decrease in apoptosis. Our results demonstrate that AQP 3 and 9 mRNA levels and protein localization along with apoptosis are responsive to hyperosmotic culture stress. Furthermore, the use of MAPK inhibitors has determined that regulation of AQP expression and localization is primarily regulated by the p38 MAPK pathway while embryonic apoptosis is primarily regulated by the JNK/SAPK pathway. Research supported by operating funds from the Canadian Institute of Health Research.