Hyperglycemia-induced apoptotic cell death in the mouse blastocyst is dependent on expression of p53

Murine preimplantation embryos exposed to hyperglycemia experience decrease d glucose transport, and overexpression of the proapoptotic protein BAX, le ading to increased apoptosis. These changes may account for the increased r ates of miscarriages and malformations seen in women with diabetes mellitus . To test whether p53 expression is necessary for hyperglycemia-induced apo ptosis, P53+/+, +/-, -/- embryos were obtained by superovulation. Two-cell embryos were cultured to a blastocyst stage in 52 mM D- or L-glucose. Apopt osis was detected using terminal dUTP nick end labeling (TUNEL) assays. In vivo studies were performed in the same manner using blastocysts recovered from streptozotocin-induced diabetic mothers. Both in vitro and in vivo stu dies showed that wildtype embryos had a significantly higher percentage of TUNEL-positive nuclei than p53+/- and -/- embryos. To test whether p53 is u pstream of BAX, immunofluorescent confocal microscopy and immunoprecipitati on/ immunoblotting were performed on blastocysts cultured in high vs. contr ol glucose conditions. Blastocysts from P53+/+ mice exhibited increased BAX staining vs. p53+/- and -/- embryos. Next, to determine whether a decrease in glucose transport was upstream or downstream of p53, deoxyglucose trans port was measured in individual blastocysts from p53+/+ and +/- diabetic vs . nondiabetic mice. Embryos from diabetic p53+/- mice exhibit a 44% decreas e in glucose transport, similar to the 38% decrease seen in embryos from di abetic p53+/+ mice. Taken together, these results strongly indicate that p5 3 plays a role in hyperglycemia-incluced apoptosis, upstream of BAX overexp ression and downstream of the decrease in glucose transport experienced by the mouse preimplantation embryo. Mol. Reprod. Dev. 60: 214-224, 2001. (C) 2001 Wiley-Liss, Inc.