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RESEARCH |
Faculty of Veterinary Medicine, Department of Equine Sciences, Utrecht University, Yalelaan 12, 3584 CM, Utrecht, The Netherlands
Correspondence should be addressed to T Tharasanit; Email: T.Tharasanit{at}vet.uu.nl
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Abstract |
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300 µm) than in large embryos when neither pretreatment was used (P > 0.05); the effect of embryo size was, however, not significant after pretreatment with trypsin or cyto-B, and trypsin improved the likelihood of an intact cytoskeleton post thaw. However, trypsin treatment also resulted in a ‘sticky’ capsule that complicated embryo handling, and cyto-B-induced actin-depolymerisation was not reversed during a 6-h post-thaw incubation. Thus, while trypsin pretreatment improved cytoskeleton preservation and both trypsin and cyto-B may reduce cell death during cryopreservation of large embryos, both treatments induced other changes likely to compromise embryo survival. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Acceptable pregnancy rates can be achieved with frozen-thawed horse embryos (50–60%: Skidmore et al. 1991, Lascombes & Pashen 2000) only if the embryos are frozen at an early developmental stage (day 6–6.5; morula to early blastocyst) when they are less than 250 µm in diameter (Slade et al. 1985), i.e. shortly after their arrival in the uterus at around day 6 after ovulation (Battut et al. 1997). Unfortunately, the precise time of uterine entry and rate of embryo development differ depending on the time of year, type of semen used (fresh vs frozen) and mare age (Meadows et al. 1999).
The reason why larger equine blastocysts do not tolerate cryopreservation is not clear, but it has been proposed that it involves the acellular capsule that forms around the embryo during blastulation (Betteridge et al. 1982, Flood et al. 1982). A negative correlation between capsule thickness and freezability has led to the suggestion that the capsule may impede access of cryoprotectants to the embryo proper (Legrand et al. 1999, Bruyas et al. 2000). Indeed, partially digesting the capsule by immersing expanded blastocysts in a trypsin solution prior to freezing resulted in an unusually high pregnancy rate after subsequent thawing and transfer to recipient mares in one report (6/8: Legrand et al. 2000), but not others (3/11: Legrand et al. 2002, 0/14: Maclellan et al. 2002). The probable cause of reduced embryo viability during freezing and thawing is the disruption of cell organelles, in particular the cytoskeleton, as a result of intracellular ice formation (Wilson et al. 1987, Dobrinsky 1996). Cytochalasin B (cyto-B) is a reversible inhibitor of actin polymerisation which, by temporarily deconstructing the actin filaments, has been shown to reduce cytoskeleton damage during cryopreservation and, thereby, improve the viability of frozen-thawed porcine embryos (Dobrinsky et al. 2000). Maclellan et al.(2002) reported that expanded horse blastocysts treated with cyto-B prior to cryopreservation subsequently yielded pregnancies (3/7) at a similar rate to embryos frozen using a conventional glycerol protocol (4/7), and contained similar levels of dead cells; they did not examine the effect of cyto-B on embryonic cytoarchitecture. Moreover, while the effects of cryo-preserving equine embryos on the microstructure of some cellular organelles (e.g. mitochondria; Wilson et al. 1987, Bruyas et al. 1993, Ferreira et al. 1997) has been examined, the conclusions with regard to the degree of damage and proportion attributable to cryoprotectant are conflicting, and the effect on the cytoskeleton has not been reported. The aims of the current study were to investigate the level of cytoskeleton disruption suffered by horse embryos during freezing and thawing using glycerol as cryoprotectant, and to examine whether additional pre-freezing treatment with trypsin to thin the capsule or cyto-B to temporarily depolymerise the cytoskeleton would improve post-thaw cell viability and actin cytoskeleton integrity.
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Materials and Methods |
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Experiment I: The effect of pre-freezing treatments on cellular and cytoskeletal integrity
Seventeen embryos were used to study the effect on cell viability and actin cytoskeleton quality of treatment with cryoprotectant (glycerol) alone or in combination with trypsin or cyto-B, without subsequent freezing and thawing. Four embryos were subjected to a standard pre-freezing, four-step, 10% glycerol equilibration procedure, i.e. 10 min each in 2.5, 5, 7.5 and 10% glycerol in PBS at room temperature (25 °C). A further five embryos were immersed in 0.2% (w/v) trypsin in PBS (Sigma-Aldrich Chemicals BV) for 15 min at 37 °C (as described by Legrand et al. 2000) before equilibration in the glycerol solutions, and the remaining eight embryos were equilibrated in glycerol solutions containing 7.5 µg/ml cyto-B (dissolved in dimethylsulphoxide; both Sigma-Aldrich Chemicals BV). After the 10-min incubation in 10% glycerol, the cryoprotectant and cyto-B were washed out using a reversed four-step equilibration in PBS containing decreasing glycerol concentrations (10 min each at 7.5, 5, 2.5 and 0%). After the final wash, the embryos were restained with DAPI so that dead cells could again be counted. Thereafter, embryos were fixed overnight in 4% paraformaldehyde and stored in PBS at 4 °C until further analysis. Of the eight cyto-B-treated embryos, two were stained and fixed immediately after removal of the cryoprotectant while the remaining six were first incubated in OCM at 37 °C for either 2 h (n = 2) or 4 h (n = 4). An additional ten embryos were fixed without either freezing or any other treatment, to establish ‘normal’ percentages of dead cells and patterns of actin distribution.
Experiment II: Effect of cryopreservation on cell viability and cytoarchitecture
After DAPI staining to count the number of dead cells, 42 embryos were assigned randomly to one of the three freezing protocols outlined above: 14 embryos using the glycerol-alone protocol, 11 after pretreatment with trypsin and 17 in glycerol solutions containing cyto-B. Six of the cyto-B embryos were assigned to a further 6-h culture post thaw to examine whether the effects of cyto-B on the actin cytoskeleton could be reversed after freezing and thawing. The ten untreated, unfrozen embryos described in experiment I were again used as the control group.
Freezing and thawing protocols
Cryopreservation was performed essentially as described by Czlonkowska et al.(1985). Briefly, the equilibrated embryo was loaded into a 0.25 ml polyvinyl chloride straw (IMV Technologies, L’Aigle, France) in a droplet of 10% glycerol solution sandwiched between two air bubbles. The straw was sealed with a straw plug and placed vertically in a programmable controlled-rate freezing machine (Planer Kryo 10 Series II, Sunbury-on-Thames, UK). Initial cooling was at 1 °C/min from ambient temperature down to –6 °C, at which temperature the straws were held for 5 min and ‘seeded’ by running metal forceps precooled in liquid nitrogen along them. After seeding, cooling was continued at 0.3 °C/min down to –33 °C, where the straws were held for 10 min before being plunged into liquid nitrogen. All embryos were stored in liquid nitrogen for at least 1 week before thawing for further examination.
The straws were thawed by holding them in the air for 10 s and then submerging them in 35 °C water for 1 min. Next, the embryo was expelled into a PBS solution containing 10% glycerol and 0.25 M sucrose (Sigma-Aldrich Chemicals BV). The cryoprotectant was then washed out by incubating the embryos for 10 min each in solutions of 0.25 M sucrose in PBS containing decreasing concentrations of glycerol (7.5, 5, 2.5 and 0%). Finally, the embryos were restained with DAPI to allow the number of dead cells to be counted, fixed overnight in 4% paraformaldehyde and stored in PBS at 4 °C until analysis of total cell number and cytoskeleton quality.
Assessing actin cytoskeleton integrity
To visualise the actin cytoskeleton, embryos were permeabilised by immersion in 0.1% Triton X-100 in PBS for 10 min, and then stained for 1 h at room temperature with a 15 µg/ml solution of Alexa Fluor 488-phalloidin (Molecular Probes Europe BV) in PBS. After washing in PBS, the embryos were counterstained with 0.1 µg/ml DAPI to count cell nuclei. To localise the cell plasma membrane, a number of embryos were labelled for 1.5 h at room temperature with a 5 µg/ml solution of a wheat germ agglutinin (WGA)-Alexa Fluor 594 conjugate (Molecular Probes Europe BV) in PBS. Labelled embryos were mounted on glass microscope slides with an antifade medium to retard photobleaching (vectashield; Vector Laboratories, Burlingame, CA, USA), sealed under a coverslip using nail polish and stored in the dark at room temperature until they could be analysed using either a Leica TCS SP confocal laser-scanning microscope or a Bio-Rad Radiance 2100MP confocal and multiphoton system (Bio-Rad, Hemel-Hempstead, UK) mounted on a Nikon TE300 inverted microscope (Uvikon, Bunnik, The Netherlands). A combination of a 488-argon ion and a 543-helium neon laser was used to produce sequential optical scans of the Alexa Fluor 488-phalloidin-labelled actin cytoskeleton and the WGA-Alexa Fluor 594-labelled plasma membrane respectively. The DAPI-stained nuclear material was excited using a 100 fs pulsed 780 nm excitation laser source (a mode-locked titanium:sapphire laser; Tsunami; Spectra Physics, Mountain View, CA, USA). The images produced by sequential scans via the different colour channels were then merged and recorded in digital format. Subsequently, the images were displayed using Adobe Photoshop 7 software (Adobe Systems Inc., Mountain View, CA, USA) and the appearance of the actin cytoskeleton was assessed. Following cytoskeleton assessment, the embryos were flattened by gently pressing on the coverslip and the total number of cells in each embryo was counted, using a conventional fluorescent microscope equipped with an eyepiece counting grid, for calculation of the percentage of dead cells.
Statistical analysis
The percentage of cells that died during freezing and thawing was calculated as the increase in the number of DAPI-positive cells divided by the total cell number. Pre-freezing embryo diameter and the extent of freezing-induced cell death were compared between experimental groups using one-way ANOVAs with a Bonferroni correction. Non-parametric tests (Kruskal–Wallis and Mann–Whitney U) were used to compare post-thaw actin cytoskeleton quality. In all cases, the statistics were performed using SPSS 11.0 for Windows (SPSS Inc., Chicago, IL, USA), and differences were considered significant when P < 0.05.
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Results |
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). The appearance of the actin cytoskeleton changed when embryos were treated with cyto-B or frozen and thawed, and the extent of this cytoskeleton disruption was accounted for by the creation of a further two quality grades. A grade II actin cytoskeleton was characterised by large areas where the cell borders, although grossly maintained, were indistinct and stained only patchily, and where small clumps of actin were visible within the cytoplasm (Fig. 1B). A grade III cytoskeleton had large areas where the cell borders lacked obvious actin staining and where, instead, most of the visible actin was in the form of intracytoplasmic clumps (Fig. 1C).
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). All of the embryos that had been exposed to glycerol alone or trypsin followed by glycerol were recorded to have a grade I actin cytoskeleton following washing to remove the glycerol (Figs 2 and 3A and B). By contrast, the actin cytoskeleton changed dramatically in embryos incubated in glycerol solutions containing 7.5 µg/ml cyto-B. In general, cyto-B treatment resulted in a lower intensity of actin labelling at the cell cortex and the appearance of patches of actin staining within the cytoplasm (i.e. a grade II cytoskeleton; Fig. 3C). With regard to the reversibility of the cyto-B effect, the actin cytoskeleton did not repolymerise during a subsequent 2-h incubation in cyto-B-free culture medium (Fig. 3D), but incubation for 4 h was sufficient to allow recovery of a ‘normal’ grade I actin cytoskeleton pattern (Figs 2 and 3E).
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The mean percentage of dead cells following freezing and thawing did not differ significantly between the conventional, trypsin- and cyto-B-treated groups (14.6 ± 2.4, 9.6 ± 2.8 and 9.7 ± 2.4% respectively; Table 2). However, the percentages of dead cells post thaw were significantly higher than in control, non-frozen embryos (0.15 ± 0.6%; P < 0.001).
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300 µm if they were frozen without additional trypsin or cyto-B treatment (19.0 ± 2.9 vs 8.5 ± 2.1%; P < 0.05).
On the other hand, the effect of blastocyst size on freezing-induced cell death was not statistically significant if embryos were pretreated with either trypsin (13.8 ± 4.9 vs 5.4 ± 0.7%; P = 0.14) or cyto-B (12.3 ± 5.5 vs 8.1 ± 2.3%; P = 0.32; Table 2
), primarily because the percentage of dead cells in larger embryos tended to be lower in these groups. As for the actin cytoskeleton, eight of the ten (80%) non-frozen control embryos had a grade I cytoskeleton (Figs 1A and 4), and freezing and thawing led to a dramatic drop in mean cytoskeleton quality. Mean cytoskelton quality also tended to differ between freezing protocols (P = 0.058); in particular, the number of embryos that retained a grade 1 cytoskeleton post thaw was significantly higher in trypsin-treated (4 of 11) than in the conventionally frozen (0 of 14) or cyto-B-treated (0 of 17) groups (P = 0.002; Fig. 3J). Immediately post thaw, all of the frozen-thawed cyto-B-treated embryos had a grade II actin cytoskeleton pattern (Fig. 3K), similar to that described in experiment I for embryos treated with cyto-B without freezing and thawing. In this case, however, a 6-h post-thaw incubation in cyto-B-free medium was not sufficient to return the actin cytoskeleton to a grade I appearance (Fig. 3L).
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) and to determine whether disruption of the latter during cryopreservation necessarily correlated with loss of plasma membrane integrity. The dual staining revealed that actin cytoskeleton disruption, typified by dispersal of actin labelling from the cell borders, often occurred in the absence of any disruption of the adjacent plasma membrane (Fig. 3I). |
Discussion |
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In the current study, cell damage was predominantly an effect of freezing and thawing per se rather than of glycerol toxicity; this broadly supports the findings of Wilson et al.(1987) who reported consistent mitochondrial damage in embryos cryopreserved in 10% glycerol but only sporadic abnormalities in embryos exposed to glycerol without freezing. That other authors arrived at the contradictory conclusion that glycerol toxicity is the more significant factor (Rieger et al. 1991, Bruyas et al. 1993, 1995) appears to be primarily a result of marked between-study differences in the duration of incubation post thaw and the indices of cell death employed. Bruyas and co-workers categorised cells with electron microscopically detectable nuclear pycnosis after 6-h incubation in vitro as dead (22– 54% of cells; Rieger et al. 1991, Bruyas et al. 1993, 1995, 2000); we counted cells with a permeable plasma membrane soon after glycerol removal as dead (5–19% of cells). Since a shorter culture (1 h) resulted in lower rates of cells classified as dead via electron microscopy (Wilson et al. 1987, Bruyas et al. 1993), and improved culture conditions dramatically reduced pycnosis (to 2–3%; Bruyas et al. 2000), it appears that the toxic effects of glycerol were exaggerated by the culture system used in earlier studies. Overall, it appears that glycerol may induce mild damage at the level of mitochondria that progresses to affect cell viability only if the embryo is forced to develop further in a suboptimal environment. Other factors likely to affect the apparent severity of glycerol toxicity include the equilibration procedure and the size/developmental stage of the embryo. In particular, relatively slow addition of glycerol (e.g. 4 x 10-min steps) is thought to increase the final intra-cellular glycerol concentration and thereby reduce osmotic cell damage (Bruyas et al. 2000); the fewer, shorter glycerol equilibration steps used in earlier studies may thus have exacerbated cell damage. With regard to embryo size, while most studies examined glycerol toxicity in relatively small day-6 embryos, we concentrated on day-7 expanded blastocysts more likely to sustain damage during freezing. This may have influenced the susceptibility to glycerol toxicity since Legrand et al.(2000) recently reported that blastocysts with a thick capsule suffer little cell death during equilibration in glycerol, possibly because the capsule completely blocks glycerol and fluid movements and thereby prevents cryoprotectant-induced osmotic cell damage. However, since these authors also report that a moderately thick capsule leads to the highest rates of cryoprotectant-induced cell death (they propose that in this situation water can exit but glycerol cannot enter), it must be assumed that trypsin treatment increases the permeability of the capsule to glycerol more dramatically than would be possible by a straightforward partial reduction in thickness.
The only pre-freezing treatment that noticeably affected embryo quality in the current study was cyto-B which, as expected, led to dispersal of actin from the cell cortex. Unexpectedly, the pattern of actin depolymerisation observed in cyto-B-treated horse embryos was very different from the complete disappearance of staining reported for similarly treated pig embryos (Dobrinsky et al. 2000). This suggests either that the cyto-B treatment used in the current study did not induce complete actin depolymerisation, or that depolymerisation takes different forms in these two species. Nevertheless, culturing cyto-B-treated embryos for 4 h in cyto-B-free medium allowed recovery of a grade I cytoskeleton, thereby demonstrating that the depolymerisation was reversible.
Freezing and thawing disrupted the actin cytoskeleton and induced cell death in all treatment groups, but small embryos (< 300 µm) tended to suffer less damage. In fact, the effect of size only reached statistical significance in embryos frozen using the standard glycerol protocol, primarily because large embryos in this group tended to suffer more cell death than in others; this suggests that trypsin and cyto-B may preferentially protect larger embryos. In the case of trypsin, this supports the finding of Legrand et al.(1999) that partial capsule digestion reduces cell death during cryopreservation. In addition, because in the current study only embryos pretreated with trypsin maintained a grade I cytoskeleton post thaw, it appears that thinning the capsule prevents cytoskeleton disruption during freezing. However, trypsin treatment also resulted in a ‘stickier’, more difficult to handle capsule and, since the capsule is essential for conceptus survival in vivo (Stout et al. 2005), it is possible that the poor pregnancy rates recorded in recent studies with trypsin-treated frozen-thawed blastocysts (Legrand et al. 2002, Maclellan et al. 2002) were a result of loss of the ‘sticky’ capsule during ET. Cyto-B has been proposed to reduce cell damage during embryo freezing or manipulation by depolymerising actin filaments and thereby increasing the flexibility of cell plasma membranes (Huhtinen et al. 1995); this may be why cyto-B-treated embryos in the current study tended to be less likely to fracture during thawing. In the case of pig embryos, cyto-B treatment prior to vitrification reduced cytoskeleton disruption and improved survival of expanded blastocysts, but not morulae or early blastocysts (Dobrinsky et al. 2000). The results of the current study similarly suggest that any protective effects of cyto-B during the cryopreservation of horse embryos are preferential to large blastocysts. On the other hand, the effects of cyto-B on the cytoskeleton could not be reversed during a 6-h post-thaw incubation, whereas in non-frozen embryos the cytoskeLeton repolymerised within 4 h. The failure, or delay, of cytoskeleton regeneration post thaw could be because freezing critically damaged the cytoskeleton or its reassembly mechanism. In this respect, cytoskeleton regeneration is powered by mitochondria (Ahn et al. 2002), and mitochondrial damage during freezing (Wilson et al. 1987) might have delayed or prevented this process. Nevertheless, since Maclellan et al.(2002) were able to establish pregnancies with frozen-thawed cyto-B-treated horse embryos, cyto-skeleton recovery must be possible after freezing and thawing and, as proposed for recovery from glycerol toxicity, it could be that any damage is more readily compensated in vivo than in vitro.
In conclusion, the current study used fluorescent stains to demonstrate that cryopreserving horse embryos leads to cytoskeleton disruption and cell death. Equilibration in glycerol, with or without cyto-B or trypsin, had no detectable lasting detrimental effects on cell architecture or viability. Trypsin pretreatment significantly improved the likelihood of an embryo maintaining an intact cytoskeleton during freezing, but further evidence that trypsin or cyto-B protected expanded blastocysts during cryopreservation was marginal. Moreover, questions remain about the suitability of these treatments because trypsin alters the physical characteristics of the blastocyst capsule, and cyto-B-induced actin depolymerisation is difficult to reverse after thawing. Nevertheless, cytoskeleton structure post thaw may be a valuable parameter for embryo quality and, as more fluorescent markers for intracellular structure and function become available, it is proposed that this approach will become an increasingly useful and sensitive adjunct to ET for evaluating the suitability of various freezing techniques.
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Acknowledgements |
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Footnotes |
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References |
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