Identification of caspase-3 and caspase-activated deoxyribonuclease in rat blastocysts and their implication in the induction of chromatin degradation (but not nuclear fragmentation) by high glucose.
Previous investigations have shown that maternal diabetes impairs rodent embryo development during the earliest phase of gestation. Exposure to high concentrations of glucose before implantation results in a decrease in the number of cells per embryo and in a concomitant increase in two nuclear markers of apoptosis, chromatin degradation and nuclear fragmentation. In the present study, we show that two intracellular effectors of apoptosis, caspase-3 and caspase-activated deoxyribonuclease (CAD), are involved in the embryotoxicity of high glucose. Using reverse transcription-polymerase chain reaction and immunocytochemistry, we first demonstrated that these two effectors were expressed in rat blastocysts. The two effectors were detected in all the cells of the blastocysts and the immuno-signals were excluded from the nuclei. Rat blastocysts were incubated for 24 h in either 6 mM or 28 mM glucose in the presence or absence of specific inhibitors (DEVD-CHO [10 microM] against caspase-3 and aurin [1 microM] against CAD). After incubation, blastocysts were examined for the proportion of nuclei showing signs of chromatin degradation or nuclear fragmentation. Addition of DEVD-CHO or aurin was found to inhibit the increase in chromatin degradation induced by high glucose. None of these two inhibitors prevented the increase in nuclear fragmentation triggered by excess glucose. Our data indicate that chromatin degradation and nuclear fragmentation are two nuclear damages that are induced separately by high glucose in rat blastocysts. Chromatin degradation is apparently mediated by the activation of caspase-3 and CAD.
Hinck
L
L
Physiology of Human Reproduction Research Unit, Université Catholique de Louvain, 1200 Brussels and 1348 Louvain-la-Neuve, Belgium.
Van Der Smissen
P
P
Heusterpreute
M
M
Donnay
I
I
De Hertogh
R
R
Pampfer
S
S
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001483:Base Sequence; D001755:Blastocyst / Q000201:enzymology*; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000032:analysis*; D002467:Cell Nucleus / Q000187:drug effects / Q000648:ultrastructure; D002843:Chromatin / Q000187:drug effects / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000032:analysis* / Q000037:antagonists & inhibitors; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005947:Glucose / Q000494:pharmacology*; D007150:Immunohistochemistry; D008297:Male; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction
D061945:Caspase Inhibitors; D002843:Chromatin; D004791:Enzyme Inhibitors; D003851:Deoxyribonucleases; C109663:caspase-activated deoxyribonuclease; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D005947:Glucose