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(529) SPONTANEOUS AND INDUCED CHRONOLOGICAL OCCURRENCE OF ACTIVE CASPASE 3 AND APOPTOSIS IN BOVINE IN VITRO PRODUCED PRE-IMPLANTATION EMBRYOS. Gjorret, Jakob1, Fabian, DuMISSING CHARACTER ENTITY: scaronan2, Avery, Birthe3, 1 Royal Veterinary and Agricultural University, Fredriksberg, Denmark2 Slovak Academy of Sciences, KoMISSING CHARACTER ENTITY: scaronice, Slovakia3 Royal Veterinary and Agricultural University, Frederiksberg, Denmark ABSTRACT- The aim of this study was to investigate the spontaneous occurrence of active Caspase 3, apoptotic nuclear morphology and TUNEL-labeling, as markers of (1) apoptosis in untreated in vitro produced (IVP) bovine embryos, and (2) induced apoptosis in similar embryos treated with Staurosporine (S). Pre-implantation stage embryos were sampled from a regular bovine IVP system (2-cell, 4-cell, 8-cell, 16-cell, morula and blastocyst; n=5 to 10 in each group) and cultured with either no supplementation for 12 h (untreated) or with 10 KEY WORDS: Apoptosis, Bovine, Caspase 3, Staurosporine |
M S for 24 h. Embryos were fixed and subjected to immunocytochemical staining for active Caspase 3, TUNEL reaction for detection of DNA degradation, and DAPI staining for detection of apoptotic nuclear morphology followed by fluorescence microscopy. In untreated embryos, no apoptotic markers were observed at the 2- and 4-cell stage. However, active Caspase 3 and apoptotic nuclear morphology were for the first time detected in a 6-cell stage blastomer, and TUNEL labeling was detected at the 16-cell stage. Blastomeres concurrently displaying all markers were only present in a few cells in embryos at the 16-cell and morula stages, whereas all blastocysts contained at least one cell displaying all these features. In S-treated embryos, active Caspase 3 and apoptotic nuclear morphology were observed at the 4-cell stage, and blastomeres displaying all apoptotic markers were observed from the 8-cell stage. Further, such blastomeres appeared more numerous in S-treated than in untreated morula stages, and virtually every cell in treated blastocysts displayed all three apoptotic features or combinations thereof. However, some treated embryos from the 4-cell to morula stage appeared completely unaffected by S-treatment. The results show that activation of Caspase 3 is likely to be involved in both spontaneous and S induced apoptosis in pre-implantation embryos. Further, sensitivity to such induction of apoptosis varies both with embryonic stage of development as well as between individual embryos at a particular stage.