Active caspase-3 and ultrastructural evidence of apoptosis in spontaneous and induced cell death in bovine in vitro produced pre-implantation embryos
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Active caspase-3 and ultrastructural evidence of apoptosis in spontaneous and induced cell death in bovine in vitro produced pre-implantation embryos. / Gjørret, Jakob O.; Fabian, Dusan; Avery, Birthe; Maddox-Hyttel, Poul.
In: Molecular Reproduction and Development, Vol. 74, No. 8, 2007, p. 961-971.Research output: Contribution to journal › Journal article › Research › peer-review
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T1 - Active caspase-3 and ultrastructural evidence of apoptosis in spontaneous and induced cell death in bovine in vitro produced pre-implantation embryos
AU - Gjørret, Jakob O.
AU - Fabian, Dusan
AU - Avery, Birthe
AU - Maddox-Hyttel, Poul
PY - 2007
Y1 - 2007
N2 - In this study we investigated chronological onset and involvement of active caspase-3, apoptotic nuclear morphology, and TUNEL-labeling, as well as ultrastructural evidence of apoptosis, in both spontaneous and induced cell death during pre-implantation development of bovine in vitro produced embryos. Pre-implantation embryos (2-cell to Day 8 blastocysts) were cultured with either no supplementation (untreated) or with 10 µM staurosporine for 24 hr (treated). Embryos were subjected to immunohistochemical staining of active caspase-3, TUNEL-reaction for detection of DNA degradation and DAPI staining for detection of apoptotic nuclear morphology, and subjected to fluorescence microscopy. Additionally, treated and untreated blastocysts were fixed and processed for ultrastructural identification of apoptosis. Untreated embryos revealed no apoptotic features at 2- and 4-cell stages. However, active caspase-3 and apoptotic nuclear morphology were observed in an untreated 8-cell stage, and TUNEL-labeling was observed from the 16-cell stage. Blastomeres concurrently displaying all apoptotic features were present in a few embryos at 16-cell and morula stages and in all blastocysts. All three features were observed from the 8-cell stage in treated embryos, and blastomeres with apoptotic features appeared more numerous in treated than in untreated embryos. Ultrastructural ev idence of apoptosis occurred with a comparable distribution pattern as apoptotic features detected by fluorescence microscopy in both treated and untreated blastocysts. Activation of caspase-3 is likely involved in both spontaneous and induced apoptosis in bovine pre-implantation embryos, and immunohistochemical staining of active caspase-3 may be used in combination with other markers to identify apoptosis in pre-implantation embryos.
AB - In this study we investigated chronological onset and involvement of active caspase-3, apoptotic nuclear morphology, and TUNEL-labeling, as well as ultrastructural evidence of apoptosis, in both spontaneous and induced cell death during pre-implantation development of bovine in vitro produced embryos. Pre-implantation embryos (2-cell to Day 8 blastocysts) were cultured with either no supplementation (untreated) or with 10 µM staurosporine for 24 hr (treated). Embryos were subjected to immunohistochemical staining of active caspase-3, TUNEL-reaction for detection of DNA degradation and DAPI staining for detection of apoptotic nuclear morphology, and subjected to fluorescence microscopy. Additionally, treated and untreated blastocysts were fixed and processed for ultrastructural identification of apoptosis. Untreated embryos revealed no apoptotic features at 2- and 4-cell stages. However, active caspase-3 and apoptotic nuclear morphology were observed in an untreated 8-cell stage, and TUNEL-labeling was observed from the 16-cell stage. Blastomeres concurrently displaying all apoptotic features were present in a few embryos at 16-cell and morula stages and in all blastocysts. All three features were observed from the 8-cell stage in treated embryos, and blastomeres with apoptotic features appeared more numerous in treated than in untreated embryos. Ultrastructural ev idence of apoptosis occurred with a comparable distribution pattern as apoptotic features detected by fluorescence microscopy in both treated and untreated blastocysts. Activation of caspase-3 is likely involved in both spontaneous and induced apoptosis in bovine pre-implantation embryos, and immunohistochemical staining of active caspase-3 may be used in combination with other markers to identify apoptosis in pre-implantation embryos.
KW - Former LIFE faculty
KW - apoptotic markers
KW - caspase activation
KW - TUNEL
KW - nuclear morphology
KW - ultrastructure
U2 - 10.1002/mrd.20714
DO - 10.1002/mrd.20714
M3 - Journal article
C2 - 17393434
VL - 74
SP - 961
EP - 971
JO - Molecular Reproduction and Development
JF - Molecular Reproduction and Development
SN - 1040-452X
IS - 8
ER -
ID: 8078398