Identification of Caspase-6 in Rat Blastocysts and Its Implication in the Induction of Apoptosis by High Glucose1

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Abstract
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 caspase-6 is expressed in rat blastocysts, using reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry. Caspase-6 is detected in all cells of the blastocyst and is excluded from the nucleus. To assess the role of caspase-6 in the glucose-induced apoptosis, rat blastocysts were incubated for 24 h in either 6 or 28 mM glucose in the presence or absence of a specific inhibitor of caspase-6 (VEID-CHO, 100 nM). After incubation, blastocysts were examined for the proportion of nuclei showing signs of chromatin degradation and nuclear fragmentation. Addition of VEID-CHO was found to inhibit nuclear fragmentation, but did not prevent the increase in chromatin degradation 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. Furthermore, nuclear fragmentation in rat blastocysts is apparently mediated by the activation of caspase-6.