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TIMING OF CASPASE ACTIVITY AND EXPRESSION OF APOPTOSIS-RELATED GENES IN HUMAN PREIMPLANTATION EMBRYOS.
Spanos, Sophie1, Rice, Suman1, Becker, David2, Winston, Robert1, Hardy, Kate1, 1 2
ABSTRACT- Human preimplantation embryos exhibit a range of cellular abnormalities caused by cell cycle defects, including chromosomal and nuclear abnormalities and cell cycle arrest. The genesis of these anomalies is not well understood. In addition, the majority of human blastocysts in vitro have one or more dying cells with the characteristic morphological features of apoptosis, including fragmenting nuclei and DNA. It has been suggested that apoptosis plays a significant role in developmental arrest. The characteristic morphological changes seen in apoptotic cells are executed by a family of cysteine proteases known as caspases. Their activity is regulated by the BCL-2 family of proteins, which are thought to regulate the release of pro-apoptotic factors (e.g. cytochrome c) from mitochondria. Here we have investigated whether components of the apoptotic machinery are expressed in the human embryo and whether there is a correlation between the appearance of active caspases and the presence of apoptotic nuclei. We have examined the timing of caspase activity during human preimplantation development using a fluorescently tagged caspase inhibitor that only binds to active caspases. Human oocytes (n=10) and embryos (n=16) at all stages were incubated with CaspaTag-TM, fixed in 4% paraformaldehyde, counterstained with 4,6-diamidino-2-phenylindole (DAPI) and assessed using confocal microscopy. Caspase activity was detected in aged oocytes, blastomeres with fragmented nuclei and in post-compaction embryos. The timing of caspase activity parallels that of nuclear and DNA fragmentation observed during preimplantation development. We have also examined the expression profile of pro-apototic BAX and BAD and anti-apoptotic BCL-2 in single embryos of all stages using RT-PCR and immunohistochemistry. BAX (n=35) and BCL-2 (n=8) mRNA was expressed throughout development whilst BAD (n=30) was found to be expressed in oocytes and in blastocysts. The presence of BAX and BCL-2 proteins within the same blastocyst (n=7) was confirmed using immunohistochemistry on serial sections. It appears that, in common with other cell types, human embryos express common molecular components of the apoptotic cascade. However, the developmental role and triggers of apoptosis during preimplantation development remain unclear.
KEY WORDS: Programmed cell death, Apoptosis, Caspases, Blastocyst