Ultrastructure and cell death of in vivo derived and vitrified porcine blastocysts

Abstract

Morphological and molecular signs of injury and cell death and subsequent regeneration following vitrification of porcine blastocysts were evaluated by light (LM) and transmission electron microscopy (TEM) as well as TUNEL/propidium iodide (PI) nuclear staining followed by confocal microscopy (CSM). In vivo derived blastocysts were assigned to one of the following four groups: Controls-(1) fixed immediately after collection (C0h) and (2) after 24 hr culture in vitro (C24h) and vitrified embryos-(3) fixed immediately after vitrification and warming (V0h), and (4) after 24 hr of culture upon warming after vitrification (V24h). Observation by LM and TEM showed that the V0h embryos displayed collapse of the blastocoele cavity (BC) and cell swelling, a general distension or shrinkage of mitochondria and massive increase in the amount of vesicles, vacuoles, and secondary lysosomes (SLs). Approximately 2/3 of the V24h embryos had recovered, whereas the remaining 1/3 were degenerated. Recovered embryos displayed almost normal blastocyst morphology, except for a widening of the perivitelline space, accumulation of debris and partial distension of mitochondria, whereas degenerated embryos were disintegrated into a poorly defined mass of cells and debris including cells with abundant degeneration of mitochondria and other organelles. Both recovered and degenerated embryos displayed a persistent abundance of presence of small membrane bounded vesicles, vacuoles, and SLs. Evaluation of TUNEL/PI stained embryos showed only occasional appearance of TUNEL positive nuclei with typical apoptotic morphology in controls (C0h 0.67%, C24h 1.22%) and in the V0h embryos (0.93%). The percentage of apoptotic nuclei in embryos at V24h was significantly higher than in all other groups (2.64%). Vitrified embryos showed significantly increased appearance of DNA fragmented nuclei without typical morphological features of apoptosis (V0h 1.43%, V24h 4.30%) compared with controls (C0h 0.26%, C24h 0.45%). The observed morphological changes and increased DNA fragmentation observed immediately after vitrification and warming probably reflects a direct damaging effect of vitrification. During 24 hr of culture a portion of the embryos was able to regenerate and along with the regenerative process, apoptosis--a possible pathway for elimination of damaged cells--became evident.

Evaluation of post-thaw DNA integrity of mouse blastocysts after ultrarapid and slow freezing

“…They reported an increase in both apoptotic index and apoptosis-related gene expressions. In a similar study, Fabian et al studied the effect of vitrification in inducing apoptosis in porcine blastocysts (37). Those authors demonstrated an increase in the DNA fragmentation index immediately after thawing, which increased after 24 h of incubation.…”

The value of fast blastocoele re-expansion in the selection of a viable thawed blastocyst for transfer

“…A study on porcine blastocysts has demonstrated that the junctional contacts between the TE cells were disintegrated immediately after warming of the vitrified blastocysts, leading to collapse of the blastocoele. Junctional contacts between TE cells are to some degree restored after in vitro culture (16); a blastocoele is then reformed. Because blastocoele fluid accumulation during cavitation is the result of the development of a permeable seal that mediates the transport of water across the TE (17), it is plausible that blastocoele re-expansion is a result of re-sealing of the TE cells after cryopreservation (18).…”

Artificial Collapse of Blastocysts Before Vitrification: Mechanical vs. Laser Technique and Effect on Survival, Cell Number, and Cell Death in Early and Expanded Blastocysts

“…This method has been used by various investigators to assess cell death in frozen embryos subjected to varying culture conditions and growth factor additives. 18,[28][29][30] Use of DNA damage as an early marker for cell death was a very valuable tool in elucidating differences between treatment groups even as early as 3 h after warming. Minimal damage was seen with either method of collapse (3-5%) in contrast to the non-collapsed control blastocysts (13%).…”