Investigation of programmed cell death in the developing inner ear of mouse embryos *
The study of programmed cell death has flourished for some time, but there are many unknown matters regarding its control mechanism. Although the TUNEL method has been previously used in the detection of apoptosis, we reported that there was autophagic cell death, in addition to apoptosis, in the TUNEL positive cells in the 12-day-old mouse embryo inner ear. We administered CDDP to pregnant mice to create inner ear toxicity and analyzed the change. As a result, we found that the total cell death in the fetal inner ear was unchanged and that there was a decrease in the apoptosis fraction and an increase in the autophagic cell death fraction. We hypothesized that this change was a defense mechanism of fetal mouse inner ear. Therefore, we analyzed the 12-day-old mouse embryo inner ear by administering CDDP to mother mice to elucidate this defense mechanism by using the real-time PCR method, the western blotting, and immunohistochemistry. Real-time PCR showed the increase in the autophagy-related gene beclin1 and the apoptosisrelated gene caspase3 in the CDDP group. Immunohistochemistry revealed cleaved caspase3 protein only in apoptotic cells. In addition, the western blotting revealed the increase in beclin1 and caspase3 but the decrease in cleaved caspase3 in the CDDP group. The increase in beclin1 gene expression was compatible with the increase in the autophagic cell death ratio in the CDDP group as reported previously. Although the increase in mRNA level of caspase3 was inconsistent with the decrease in the ratio of apoptosis, there was no increase in protein levels. The decrease in cleaved caspase3, which is a cleaved-activity type protein, suggests the suppression of activation process of cleaving caspase3. (Accepted on October 8, 2009)
