This study was undertaken to obtain information about characteristics of different types of induced apoptosis in preimplantation embryos. Freshly isolated mouse blastocysts were cultured in vitro with the addition of two apoptotic inductors – TNFα and actinomycin D – at various doses and times. The average number of nuclei and the percentage of dead cells were evaluated in treated embryos. Classification of dead cells was based on morphological assessment of their nuclei evaluated by fluorescence microscopy, the detection of specific DNA degradation (TUNEL assay), the detection of active caspase-3 and cell viability assessed by propidium iodide staining. The addition of both apoptotic inductors into culture media significantly increased cell death incidence in blastocysts. Their effects were dose and time dependent. Lower concentrations of inductors increased cell death incidence, usually without affecting embryo growth after 24 h culture. Higher concentrations of inductors caused wider cell damage and also retarded embryo development. In all experiments, the negative effect of actinomycin D on blastomere survival and blastocyst growth was greater than the effect of TNFα. Furthermore, the addition of actinomycin D into culture media increased cell death incidence even after 6 h culture. Differences resulted probably from diverse specificity of apoptotic inductors. The majority of dead cells in treated blastocysts were of apoptotic origin. Morphological and biochemical features of apoptotic cell death induced by both TNFα and actinomycin D were similar and had homologous profile. In blastomeres, similarly to somatic cells, the biochemical pathways of induced apoptosis included activation of caspase-3 and internucleosomal DNA fragmentation.

Introns constitute most of the length of typical pre-mRNAs in vertebrate cells. Thus, the turnover rate of introns may significantly influence the availability of ribonucleotides and splicing factors for further rounds of transcription and RNA splicing, respectively. Given the importance of intron turnover, it is surprising that there have been no reports on the half-life of introns from higher eukaryotic cells. Here, we determined the stability of IVS1Cβ1, the first intron from the constant region of the mouse T-cell receptor-β (TCR-β) gene. Using a tetracycline (tet)-regulated promoter, we demonstrate that spliced IVS1Cβ1 and its pre-mRNA had half-lives of 6.0 ± 1.4 min and 3.7 ± 1.0 min, respectively. We also examined the half-lives of these transcripts by using actinomycin D (Act.D). Act.D significantly stabilized IVS1Cβ1 and its pre-mRNA, suggesting that Act.D not only blocks transcription but exerts rapid and direct posttranscriptional effects in the nucleus. We observed that in vivo spliced IVS1Cβ1 accumulated predominantly as lariat molecules that use a consensus branchpoint nucleotide. The accumulation of IVS1Cβ1 as a lariat did not result from an intrinsic inability to be debranched, as it could be debranched in vitro, albeit somewhat less efficiently than an adenovirus intron. Subcellular-fractionation and sucrose-gradient analyses showed that most spliced IVS1Cβ1 lariats cofractionated with pre-mRNA, but not always with mRNA in the nucleus. Some IVS1Cβ1 also appeared to be selectively exported to the cytoplasm, whereas TCR-β pre-mRNA remained in the nucleus. This study constitutes the first detailed analysis of the stability and fate of a spliced nuclear intron in vivo.