Cryopreservation of mammalian embryos is an important tool for the application of reproductive biotechnology. Recent evidence indicates that apoptosis may be important in determining the viability of cryopreserved embryos. Our goal was to detect apoptosis and characterize and quantify the embryonic cell death caused by cryopreservation. Mouse morulae were collected, selected, and separated into three groups: fresh, slow-freezing, and vitrification. In the slow-freezing procedure, embryos were exposed to 10% ethylene glycol (EG) for 10 min. After loading, the straws were placed into methanol at -7°C for 5 min, seeded and after 5 min cooled at 0.5°C/minute. After 10 minutes at -31°C, straws were plunged into and stored in liquid nitrogen. Slow-frozen straws were thawed in air for 10 s, and then immersed in a 25°C water bath for 20 s. Embryos were vitrified by exposing them to 10% and 20% EG for 5 min followed by 40% EG + 18% Ficoll + 10% sucrose (EFS) for 30 s and the 0.25-mL straws then plunged into and stored in liquid nitrogen. The vitrified straws were warmed by immersing them in 25°C water for 20 s. Cell membrane integrity was assessed by Hoechst and propidium iodide double staining (H/PI). Fresh and thawed embryos were scored (following IETS recommendations) and then fixed after 30 min in PBS + 10% FCS. Morphology and apoptosis were assessed with Haematoxylin-Eosin staining (HE) and by electron microscopy (MET). The number of Grade I embryos recovered after thawing was higher for slow-frozen embryos (61.5%) than vitrified embryos (29.5%). H/PI detected more membrane permeability in the vitrified embryos (69.7%), than in the slow-frozen (48.4%) or non-frozen (13.8%) groups (P < 0,05, Wilcoxon's test). Nuclear evaluation by HE revealed that vitrification and slow-freezing induced pyknosis and chromatin condensation. Mitotic pattern was observed in the fresh and slow-frozen group, but not in vitrification group suggesting that the embryos were either not randomly allocated to the groups or not-treated and fixed at the same age, or that vitrification changed the nuclear status of the embryos. HE staining revealed weakly staining cytoplasm and degenerated cells in the vitrification group (indicating oncosis), while in the slow-frozen group the presence of cytoplasmic condensation and eosinophilic structures indicated apoptosis. The ultrastructure examination confirmed the HE observations. In conclusion, the results demonstrated that staining with HE allows detection of oncosis and apoptosis in cryopreserved embryos. According to these data, vitrification caused more cellular injuries than slow-freezing, and oncosis was the predominant injury. It is important to point that specific molecular apoptosis tests must be performed to confirm these results.

This work was supported by FAPESP 04/01252-4.