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HYPERGLYCEMIA-INDUCED APOPTOSIS IN THE MURINE BLASTOCYST REQUIRES P53 EXPRESSION.
Keim, April1, Chi, Maggie1, Moley, Kelle1, 2, 1 2
ABSTRACT- Murine preimplantation embryos exposed to hyperglycemia, in vitro or in vivo, experience decreased expression of the facilitative glucose transporters, GLUTs 1,2, and 3, leading to decreased glucose transport and decreased levels of intraembryonic free glucose. These embryos also experience overexpression of the proapoptotic protein BAX, leading to increased apoptosis. Loss of key progenitor cells due to this hyperglycemia-induced apoptosis may account for the increased rates of miscarriages and malformations seen in women with insulin-dependent diabetes mellitus. These studies examine p53 as an upstream regulator of BAX. In order to test whether p53 expression is necessary for hyperglycemia-induced apoptosis, p53+/+, +/-, -/- embryos were obtained by superovulation. Two-cell embryos were cultured to a blastocyst stage in 52mM D- or L-glucose. Apoptosis 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 studies demonstrated that wildtype embryos experienced a significantly higher percentage of TUNEL-positive nuclei than p53+/- and -/- embryos. To test whether p53 is upstream of BAX overexpression, immunofluorescent confocal microscopy and immunoprecipitation-immunoblotting were performed on blastocysts cultured in high versus control glucose conditions. Blastocysts from p53+/+ mice exhibited 4.7- and 7.9-fold higher BAX staining vs p53+/- and -/- embryos, respectively. Next, to determine whether a decrease in glucose transport was upstream or downstream of p53, deoxyglucose transport was measured in individual blastocysts from p53 +/+ and +/- diabetic versus nondiabetic mice. Embryos from diabetic p53 +/- mice exhibit a 44% decrease in glucose transport, similar to the 38% decrease seen in embryos from diabetic p53 +/+ mice. Taken together, these results strongly indicate that p53 plays a role in hyperglycemia-induced apoptosis, upstream of BAX overexpression and downstream of the decrease in glucose transport experienced by the mouse preimplantation embryo.
KEY WORDS: hyperglycemia, glucose transport, blastocyst