CLSM analysis of bovine IVF embryos.

The embryos were fixed and mounted on coverslips in such a way that the three-dimensional structure was maintained. DNA staining with DAPI is shown in white, f-actin filaments (phalloidin-TRITC) in orange. Scale bars represent 100 µm (overviews) or 10 µm (details). A–F: Arrest and death of early blastomeres during the first four cleavage cycles. A–D: Maximum intensity z-projections of optical serial sections of embryos examined at day 4 (A, B) or day 5 (C, D). Many embryos show large early blastomeres that are arrested at interphase (asterisks) or prophase (arrow heads) or already show clear signs of cell death: DAPI staining reveals variably sized and irregularly shaped clumps of highly condensed chromatin (large arrows). Notably, frequent findings are remnants of mitotic chromosome structures (small arrows). E, F: Two day 7 embryos that initially survived the death of large early blastomeres. Remnants of early blastomere death can be seen until blastocyst hatching. Panel E shows an embryo in the form of a single maximum intensity z-projection of the entire confocal image stack (left) and as a sequence of six projections of 20 µm image sub-stacks (right). Panel F presents a z-projection (overlay image and separate channel images) through an entire embryo. G–L: Cell death in the inner cell mass of bovine IVF blastocysts examined at day 7: In expanding and hatching blastocysts, DAPI staining reveals highly condensed chromatin structures that are irregularly shaped and variable in size referring to different modes and stages of cell death (arrows). G–J: Four expanding blastocysts. G is a maximum intensity z-projection of a 20 µm image stack, H, I, J are single optical sections. Dying/dead cells are also shown at higher magnification. K, K′: Hatched blastocyst. Panel K presents a maximum intensity z-projection of a stack spanning the entire embryo (overlay image and DAPI alone). K′: Single optical section of the same blastocyst and enlarged view of the inner cell mass: Note a dying/dead cell (arrow head) in the interior of the inner cell mass as well as the extrusion of a dead cell into the blastocoel (insert: transmission light image).