Hyperglycemia-induced apoptotic cell death in the mouse blastocyst is dependent on expression of p53.
Murine preimplantation embryos exposed to hyperglycemia experience decreased glucose transport, and overexpression of the proapoptotic protein BAX, leading to increased apoptosis. These changes may account for the increased rates of miscarriages and malformations seen in women with diabetes mellitus. 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 52 mM 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 showed that wildtype embryos had a significantly higher percentage of TUNEL-positive nuclei than p53+/- and -/- embryos. To test whether p53 is upstream of BAX, immunofluorescent confocal microscopy and immunoprecipitation/ immunoblotting were performed on blastocysts cultured in high vs. control 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 transport was measured in individual blastocysts from p53+/+ and +/- diabetic vs. 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.
Keim
A L
AL
Department of Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, Missouri, USA.
Chi
M M
MM
Moley
K H
KH
D000704:Analysis of Variance; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001755:Blastocyst / Q000166:cytology / Q000502:physiology*; D003921:Diabetes Mellitus, Experimental; D005260:Female; D005838:Genotype; D005947:Glucose / Q000378:metabolism*; D006801:Humans; D006943:Hyperglycemia / Q000503:physiopathology*; D020287:In Situ Nick-End Labeling; D051379:Mice; D018613:Microscopy, Confocal; D011247:Pregnancy; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D051028:bcl-2-Associated X Protein
C494826:BAX protein, human; C494827:Bax protein, mouse; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D005947:Glucose