Caspase activity and expression of cell death genes during development of human preimplantation embryos

Abstract

It has been observed that apoptosis occurs in human blastocysts. In other types of cell, the characteristic morphological changes seen in apoptotic cells are executed by caspases, which are regulated by the BCL-2 family of proteins. This study investigated whether these components of the apoptotic cascade are present throughout human preimplantation development. Developing and arrested two pronucleate embryos at all stages were incubated with a fluorescently tagged caspase inhibitor that binds only to active caspases, fixed, counterstained with 4,6-diamidino-2-phenylindole (DAPI) to assess nuclear morphology and examined using confocal microscopy. Active caspases were detected only after compaction, at the morula and blastocyst stages, and were frequently associated with apoptotic nuclei. Occasional labelling was seen in arrested embryos. Expression of proapoptotic BAX and BAD and anti-apoptotic BCL-2 was examined in single embryos using RT-PCR and immunohistochemistry. BAX and BCL-2 mRNAs were expressed throughout development, whereas BAD mRNA was expressed mainly after compaction. Simultaneous expression of BAX and BCL-2 proteins within individual embryos was confirmed using immunohistochemistry. The onset of caspase activity and BAD expression after compaction correlates with the previously reported appearance of apoptotic nuclei. As in other types of cell, human embryos express common molecular components of the apoptotic cascade, although apoptosis appears to be suppressed before compaction and differentiation.

Temporal detection of caspase-3 and -7 in bovine in vitro produced embryos of different developmental capacity

“…Some of the two-cell embryos which stained positive for caspases and belonged to the slow developing group may have been in developmental arrest, which is in accordance with the previous studies where apoptosis has been demonstrated in arrested two-to four-cell human and pig embryos (Spanos et al 2002, Mateusen et al 2005. Notwithstanding the presence of the full apoptotic machinery in normal developing early stage embryos, in humans, caspase could only be detected from the compacted morula stage onwards (Spanos et al 2002). A family of proteins called inhibitors of apoptosis proteins (IAPs) could play a pioneering role in this respect by binding directly to and inhibiting active caspases (Deveraux & Reed 1999, Goyal 2001, Spanos et al 2002.…”

“…The fluorescent detection of active caspases as early as the two-cell stage in our study has confirmed this possibility. Some of the two-cell embryos which stained positive for caspases and belonged to the slow developing group may have been in developmental arrest, which is in accordance with the previous studies where apoptosis has been demonstrated in arrested two-to four-cell human and pig embryos (Spanos et al 2002, Mateusen et al 2005. Notwithstanding the presence of the full apoptotic machinery in normal developing early stage embryos, in humans, caspase could only be detected from the compacted morula stage onwards (Spanos et al 2002).…”

“…Notwithstanding the presence of the full apoptotic machinery in normal developing early stage embryos, in humans, caspase could only be detected from the compacted morula stage onwards (Spanos et al 2002). A family of proteins called inhibitors of apoptosis proteins (IAPs) could play a pioneering role in this respect by binding directly to and inhibiting active caspases (Deveraux & Reed 1999, Goyal 2001, Spanos et al 2002. In this respect, it could be interesting to focus on immunofluorescent or other detection methods of IAPs in early arrested and non-arrested embryos.…”

“…Interestingly, recent research has discovered a third pathway of apoptosis in which endonuclease G, released from mitochondria and translocated to the nucleus, can fragmentize DNA independently of active caspases (Li et al 2001). Nevertheless, the importance of caspases in the apoptotic mechanism cannot be neglected, since in several species, such as human, mouse, and rat, detection of caspase mRNAs and/or active caspase proteins has been associated with apoptosis during embryo development, mostly in a stage-specific manner (Exley et al 1999, Hinck et al 2001, Spanos et al 2002, Jurisicova et al 2003, Metcalfe et al 2004. From the results of both our experiments, it was obvious that the percentage of caspase-positive cells declined in advanced developmental stages.…”

Apoptosis Inhibition by Insulin-Like Growth Factor (IGF)-I During In Vitro Maturation of Bovine Oocytes

“…Although little is known about possible signals that may trigger apoptosis in embryos and the precise mechanisms by which the apoptosis program is executed and controlled, all apoptotic pathways appear to terminate with activation of the caspase family of proteases [10][11][12]. The caspase family of cysteine proteases mediates proteolytic cleavage of a large number of proteins important for cell integrity and survival and finally activates the endonucleases that are responsible for DNA fragmentation [13][14][15].…”