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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  • Jakob O. Gjørret
  • Dusan Fabian
  • Birthe Avery
  • Poul Maddox-Hyttel
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.
Original languageEnglish
JournalMolecular Reproduction and Development
Volume74
Issue number8
Pages (from-to)961-971
Number of pages11
ISSN1040-452X
DOIs
Publication statusPublished - 2007

    Research areas

  • Former LIFE faculty - apoptotic markers, caspase activation, TUNEL, nuclear morphology, ultrastructure

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