Caspase activity and expression of cell death genes during development of human preimplantation embryos.
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.
Spanos
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S
Department of Reproductive Science and Medicine, Institute of Reproductive and Developmental Biology, The Wolfson and Weston Research Centre for Family Health, Imperial College, Hammersmith Hospital, Du Cane Road, London W12 ONN, UK.
Rice
S
S
Karagiannis
P
P
Taylor
D
D
Becker
D L
DL
Winston
R M L
RM
Hardy
K
K
D017209:Apoptosis / Q000235:genetics*; D001755:Blastocyst / Q000166:cytology / Q000201:enzymology*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D020169:Caspases / Q000378:metabolism*; D004622:Embryo, Mammalian / Q000201:enzymology; D047108:Embryonic Development / Q000502:physiology*; D005314:Embryonic and Fetal Development / Q000502:physiology; D005260:Female; D015870:Gene Expression; D006801:Humans; D018613:Microscopy, Confocal; D009865:Oocytes / Q000201:enzymology; D011247:Pregnancy; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051028:bcl-2-Associated X Protein; D051025:bcl-Associated Death Protein
C494819:BAD protein, human; C494826:BAX protein, human; D002352:Carrier Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D051028:bcl-2-Associated X Protein; D051025:bcl-Associated Death Protein; D020169:Caspases