Vitrification is widely used to cryopreserve supernumerary embryos following in vitro fertilization (IVF). The mouse model was used to investigate the effects of vitrification on blastomere viability, using viability markers, and on the cytoskeleton, by analysing spindle/chromosome configurations, using confocal scanning microscopy. Ninety cleavage and morula stage dimethyl sulphoxide (DMSO)/EG vitrified mouse embryos were either processed immediately following warming for viability assessment by labelling with the fluorescent markers carboxyfluorescein-diacetate succinimidylester (CFSE) and propidium iodide (PI) or were cultured to the blastocyst stage and immunostained with α-tubulin antibody to visualize microtubules and DAPI or PI to visualize DNA. Sixty-five fresh embryos were also used as the control. Vitrified embryos showed high survival rates following warming, but they had a higher incidence of damaged blastomeres compared with fresh embryos. Most mitotic spindles examined in all groups were normal, but multivariable analysis revealed that the proportion of abnormal spindles was significantly higher in vitrified/warmed embryos (P < 0.05). This study is the first to examine the immediate effects of vitrification on blastomere viability, using fluorescent markers and shows that although vitrification results in a higher incidence of damaged blastomeres, vitrified embryos may compensate for this limited number of damaged/abnormal cells, as development to the blastocyst stage was not compromised.

The capacity of sister blastomeres of mouse embryos for induced fusion changed during the 2-cell stage. It was at low level (24%) at the early 2-cell stage, increased and reached 98.5% at the middle 2-cell stage and fell sharply to 31% at the late 2-cell stage. At the time corresponding to the G2/Mphase of the cell cycle the blastomeres fused in only 8% of cases. Vital staining of 2-cell embryos by rhodamine 123 showed that the mitochondria were dispersed throughout the cytoplasm with a ringlike (around the nucleus) or spot-like (over the metaphase plate) concentration in the centre of each blastomere. At the periphery of blastomeres the mitochondrial content was low. The behaviour of the mitochondria reflected the subsequent events of structural and functional integration of the sister blastomeres under induced fusion: a discernible boundary between partners during 30 min after electrofusion or 1 h after fusion with polyethylene glycol; movement of the two ‘rings’ to the centre of the blastomere fusion products (BFP) to form one large bright ‘spot’ over the common metaphase plate; mitochondria outlining the shape of the spindle and connection between sister blastomeres until completion of the first mitosis of BFP The data obtained suggest that fusion of the blastomeres does not lead to extensive changes in the hybrid cytoplasm and integration of nuclear material is taking place only at metaphase stage Cytogenetic examination of BFP at the 2-cell stage confirmed reconstruction of the tetraploid embryos and found that sister blastomeres of such embryos could asynchronously enter the next cleavage division similarly to normal diploid 2-cell embryos.

Under our culture conditions, mouse embryos from the BALB/c inbred mouse strain develop successfully in culture only from the late 2-cell stage onwards (so-called 2-cell block), whether or not EDTA is added to the culture medium. (CBA × C57BL) F2 embryos do not exhibit a 2-cell block. Medium conditioned by culture of non-blocking embryos from the 2-cell to the 8-cell stage did not improve the development of blocking embryos, nor did co-culture of blocking and non-blocking embryos, with or without conditioned medium. On the other hand phytohaemagglutinin (PHA)-assisted aggregation of an early 2-cell BALB/c embryo with five surrounding non-blocking F2 embryos (2-cell or 8-cell) or five BALB/c 8-cell embryos allowed the early 2-cell BALB/c embryos to develop into blastocysts within 72 h. Aggregation of blocking BALB/c 2-cell embryos with each other had no ‘rescue’ effect. When blocking and non-blocking 2-cell embryos were aggregated together, an integrated blastocyst was formed; but when the early 2-cell BALB/c embryos were aggregated with non-blocking 8-cell embryos, the blocking embryos formed a separate small blastocyst, which nonetheless retained adherent contact with the non-blocking embryos throughout the culture period. Ultrastructural analysis showed that 2-cell embryos aggregated with the aid of PHA form close adherent cell contacts up to several micrometres in length.

We report the use of a simple, reproducible, photocytometric method for measuring nuclear DNA content of DAPI-stained cells, using a computerised image analysis system: Seescan. As this technique is non-destructive and uses very short exposure to ultraviolet light, it can be used for either fixed or vital material. After correcting for any background cytoplasmic staining, the intensity of nuclear stain was measured by the Seescan and compared with that of control cells of known ploidy. Fixed material was found to stain more intensely than live material initially, but demonstrated a rapid loss of nuclear intensity over the first 90 min following removal from DAPI, after which the level plateaued. In contrast, live cells showed no change in nuclear intensity with time. The system was validated by measuring the DNA content of carefully timed mouse blastomeres, human fetal lung fibroblasts and parthenogenetically activated human oocytes. The results obtained were appropriate for the developmental stage or phenotypic appearance of each of the cell types measured.