Addition of ascorbate during cryopreservation stimulates subsequent embryo development

Michelle Lane1, Jeffery M. Maybach and David K. Gardner

Research Department, Colorado Center for Reproductive Medicine, 799 East Hampden Ave, Suite 520, Englewood, CO 80110, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Embryo development following cryopreservation is reduced compared with fresh embryos. One of the traumas that cryopreservation imparts on embryos is an increase in oxidative stress. Therefore, this study investigated the effects of the addition of the antioxidant ascorbate to the cryopreservation solutions on subsequent embryo development. METHODS: Mouse embryos at the 2-cell and blastocyst stages were either slow-frozen or vitrified in solutions containing either no ascorbate or 0.1 or 0.5 mmol/l ascorbate. The effects on the levels of hydrogen peroxide and subsequent embryo development and physiology were assessed. RESULTS: Addition of ascorbate to the cryopreservation solutions reduced the levels of hydrogen peroxide in embryos. Furthermore, addition of 0.1 mmol/l ascorbate significantly enhanced inner cell mass development in blastocysts. Embryos cryopreserved with ascorbate had significantly lower levels of lactate dehydrogenase leakage, and increased rates of metabolism compared with those cryopreserved in the absence of ascorbate. The benefits of ascorbate were significantly greater in embryos that were slow-frozen compared with those that were vitrified. CONCLUSIONS: These data indicate that the addition of 0.1 mmol/l ascorbate to the cryopreservation solutions for the mammalian embryo would be of significant value.

Key words: antioxidant/blastocyst/metabolism/slow freezing/vitrification


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The ability to successfully cryopreserve embryos with limited loss in viability is essential for the success of assisted reproductive technologies. Currently, however, irrespective of the stage of development (pronuclear, cleavage stages or blastocyst), embryos that have been cryopreserved exhibit substantially reduced viability following transfer compared with embryos that have not been cryopreserved. Therefore, cryopreservation exerts significant stress on the embryo, resulting in this loss in developmental competence. These stresses include osmotic stress due to dehydration, solution effects due to toxicity of the cryoprotectants and also damage to cell membranes. Loss of function of cell membranes interferes with transport systems such as pH regulatory systems on the cell membrane. Disruption of organelle membranes affects transport systems such as mitochondrial transport systems essential for oxidative phosphorylation, the major energy-generating pathway of the early embryo. One of the mechanisms by which the properties of a membrane can be damaged is by lipid peroxidation. Oxygen radicals such as hydroxyl radicals and superoxide anions are known to increase rates of lipid peroxidation in cells (Halliwell and Gutteridge, 1989Go, 1995Go; Gutteridge and Halliwell, 2000Go). It is plausible that cryopreservation may increase the rates of lipid peroxidation to cell membranes due to an increase in the levels of oxygen radicals.

Cells contain antioxidants such as glutathione and superoxide dismutase to protect against the production of oxygen radicals. However, it has been shown in sperm that the levels of these antioxidants are reduced by >50% following cryopreservation (Bilodeau et al., 2000Go). It has previously been demonstrated that the addition of antioxidant solutions to the in-vitro culture systems for embryos stimulates development (Nasr-Esfahani et al., 1990aGo; Noda et al., 1991Go; Nonogaki et al., 1991Go; Goto et al., 1992Go; Nasr-Esfahani et al., 1992Go; Payne et al., 1992Go; Umaoka et al., 1992Go; Orsi and Leese, 2001Go) and also results in a significantly improved ability of the embryos to subsequently survive cryopreservation (Tarin and Trounson, 1993Go). However, it is plausible that inclusion of an antioxidant in the cryopreservation solutions themselves may help in maintaining embryo viability after cryopreservation by reducing the effects of harmful oxygen radicals during the cryopreservation procedure itself. One antioxidant, ascorbate, is a very potent hydrophilic antioxidant that is able to scavenge hydrogen peroxide, superoxide anions, hydroxyl free radicals and singlet oxygens (Halliwell and Gutteridge, 1989Go; Meister, 1992Go). Additionally, ascorbate is found in follicular fluid (Paszkowski and Clarke, 1999Go), indicating that it may have a physiological role as an antioxidant in oocyte and embryo development. Therefore, the aim of this study was to examine the effects of the addition of ascorbate to cryopreservation solutions on the survival, intracellular peroxide levels and physiology of mouse 2-cell embryos and blastocysts. The effects of ascorbate were examined in traditional slow-freezing procedures and also during ultra-rapid vitrification to determine the potential benefits in both cryopreservation systems.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Media
The medium for embryo collection and the base medium for cryopreservation was a 3-(N-morpholino) propanesulphonic acid (MOPS)-buffered modification of medium G1.2 (MOPS–G1). Medium G1.2 was modified by omitting EDTA and replacing 20 mmol/l NaHCO3 with 20 mmol/l MOPS and adjusting to pH 7.35. Media for embryo culture were G1.2 and G2.2 supplemented with 5 mg/ml human serum albumin (Gardner and Lane, 1997Go; Gardner et al., 1998Go). All salts were Analar grade and purchased from BDH (Poole, Dorset, UK). Pyruvate, lactate, taurine, alanyl-glutamine and MOPS were purchased from Sigma Chemical Co. (St Louis, MO, USA). Amino acid and vitamin solutions were obtained from ICN (Aurora, OH, USA). EDTA and HSA were obtained from Vitrolife AB (Gothenburg, Sweden). Ascorbate (Sigma Chemical Co.) was added to the media at concentrations of 0.1 and 0.5 mmol/l.

Embryo collection
Embryos were collected from 4 week old F1 female mice (C57Bl/6xCBA). Multiple ovulations were induced by an i.p. injection of 5 IU pregnant mare’s serum gonadotrophin (Sigma Chemical Co.) followed 48 h later by 5 IU of hCG (Pregnyl; Organon Inc., West Orange, NJ, USA). After the second injection, females were placed with males of the same strain. Zygotes were collected at 21 h post-hCG in MOPS–G1 and denuded by incubation with hyaluronidase [0.5 mg/ml (Sigma Chemical Co.), bovine testes, type IV] for <1 min. Zygotes were washed twice in MOPS–G1 and once in medium G1.2 before placement in culture.

In-vivo developed 2-cell embryos were collected at 46 h post-hCG injection by flushing oviducts with MOPS–G1.

Embryo culture
Embryos were cultured in groups of 10 in 20 µl drops of medium under paraffin oil (BDH) (Lane and Gardner, 1992Go). All embryos were cultured in medium G1.2 at 37°C in 6% CO2/5% O2/89% N2. At the 2-cell stage, embryos were allocated to one of the cryopreservation protocols. The remaining embryos were cultured for a further 24 h in medium G1.2. Following warming/thawing, 2-cell embryos were cultured for a further 24 h to the 4–8-cell stage. After a total of 48 h of culture in G1.2, all embryos were washed twice in medium G2.2 and then cultured in G2.2 for a further 48 h to the blastocyst stage.

Slow-freezing procedure for 2-cell embryos
A published slow-freezing procedure was used (Emiliani et al., 2000Go). All freezing and thawing procedures were performed at room temperature. For freezing, 2-cell stage embryos were first exposed to 0.75 mol/l propanediol (Sigma Chemical Co.) in medium MOPS–G1 for 10 min, then to 1.5 mol/l propanediol for 10 min, and finally MOPS–G1 containing 1.5 mol/l propanediol and 0.1 mol/l sucrose (Sigma Chemical Co.) for 7 min. Between 15 and 20 2-cell embryos were loaded into 0.25 ml straws (Institute Medicine Vetinaire, Bicef, L’Aigle, France) and placed into the freezing machine (Freeze Control CL2000; CryoLogic, Napa, CA, USA). Embryos were cooled as follows: placed in the freezing machine at 20°C, cooled at 1.0°C/min to –6°C, manually seed at –6°C, held at –6°C for 10 min, and then cooled at 0.3°C/min to –37°C, before being plunged into liquid nitrogen. Embryos were stored in liquid nitrogen between 1 week and 2 months.

For thawing, straws were held in air for 15 s and then plunged into a 30°C water bath for 30 s. Two-cell stage embryos were thawed in a four-step procedure to remove the cryoprotectants as follows: 10 min in 1.5 mol/l propanediol and 0.2 mol/l sucrose, 10 min in 0.75 mol/l propanediol and 0.2 mol/l sucrose, 10 min in 0.2 mol/l sucrose and 10 min in MOPS–G1. Finally, the 2-cell embryos were washed in MOPS–G1 at 37°C for 10 min and placed into culture.

Slow-freezing procedure for blastocysts
Blastocyst freezing and thawing procedures were performed at room temperature. The base medium for blastocyst freezing was a MOPS-buffered version of G2.2 without essential amino acids and vitamins (MOPS–G2). For freezing, blastocysts were placed initially in MOPS–G2 with 5% glycerol and 0.1 mol/l sucrose for 10 min followed by a 7 min incubation in MOPS–G2 with 10% glycerol and 0.2 mol/l sucrose. Ten blastocysts were then loaded into a 0.25 ml straw (Institute Medicine Vetinaire) and placed into the freezing machine (Freeze Control CL2000) at –6°C. After 2 min the straws were manually seeded and then were held at –6°C for a further 10 min. Blastocysts were then cooled at 0.5°C/min to –32°C, before being plunged into liquid nitrogen. Embryos were stored in liquid nitrogen between 1 week and 2 months.

For thawing, straws were removed from the liquid nitrogen and air-thawed for 10 s. Straws were then placed into a 30°C water bath for 30 s. Blastocysts were expelled from the straw into MOPS–G2 containing 10% glycerol and 0.2 mol/l sucrose. Blastocysts were then transferred to MOPS–G2 containing 5% glycerol and 0.1 mol/l sucrose for 5 min, then incubated in MOPS–G2 with 0.1 mol/l sucrose for a further 5 min followed by a 5 min incubation in MOPS–G2. Blastocysts were then incubated in MOPS–G2 at 37°C for 5 min before being placed into culture.

Vitrification of 2-cell and blastocysts
All vitrification and warming procedures were performed at 37°C. For vitrification, embryos were vitrified in a two-step loading with cryoprotectants dimethylsulphoxide (DMSO) and ethylene glycol (Lane and Gardner, 2001aGo). First, embryos were placed in cryoprotectant solution MOPS–G1 containing 8% DMSO and 8% ethylene glycol for 1 min for 2 cells and 1:45 min for blastocysts. Embryos at both stages were then transferred into medium MOPS–G1 containing 16% DMSO, 16% ethylene glycol, 10 mg/ml Ficoll and 0.65 mol/l sucrose for ~20 s. Embryos were transferred to a nylon loop that had previously been dipped into the second vitrification solution to create a thin film, and plunged directly into a cryovial containing liquid nitrogen (Lane et al., 1999aGo,bGo). The vials were stored on standard canes. Embryos were stored in liquid nitrogen between 1 week and 2 months.

For warming, embryos were warmed in a two-step dilution with sucrose. The loop was removed from the vial under liquid nitrogen and placed directly into a well of MOPS–G2 containing 0.25 mol/l sucrose. The embryos fell off the loop and after 2 min in this solution were moved to MOPS–G2 containing 0.125 mol/l sucrose for 3 min. Finally, the embryos were washed in medium MOPS–G1 for 5 min and placed into culture.

Allocation of cells to the inner cell mass and trophectoderm
Allocation of cells in blastocysts to the inner cell mass (ICM) and trophectoderm (TE) was assessed using a published technique (Hardy et al., 1989Go) as modified and described previously (Gardner et al., 2000Go).

Pyruvate metabolism
Tricarboxylic acid cycle activity by individual embryos was assessed by incubation with radiolabelled [2-14C]pyruvate (0.002 µCi/µl, 0.32 mmol/l; New England Nuclear, Beverly, MA, USA). Individual embryos were placed in 3 µl drops of medium G1.2 containing labelled pyruvate on the lid of a microcentrifuge vial (Rieger et al., 1992Go; Lane and Gardner, 2001bGo). The lid was replaced onto the vial containing 1.5 ml of NaHCO3 equilibrated at 6% CO2/5% O2/89% N2 and the gas phase filled with 6% CO2/5% O2/89% N2. Embryos were incubated for 3 h at 37°C. Sham preparations containing the medium with the radiolabelled substrate, but without an embryo, were included to account for background counts and non-specific breakdown of the label. To enable quantification, 3 µl drops of medium were added directly to the 1.5 ml of NaHCO3 to obtain total counts. At the end of the 3 h incubation, 1.0 ml of the NaHCO3 was added to a scintillation vial containing 200 µl of 1 mol/l NaOH and kept at 4°C overnight. The following morning, 10 ml of scintillation fluid was added and the vials vortexed and counted in a liquid scintillation counter. Embryo metabolism was calculated based on the recovery efficiencies of the radiolabelled substrates as previously described (Rieger et al., 1992Go) and expressed as pmol/embryo/h.

Hydrogen peroxide
Levels of hydrogen peroxide in embryos were measured using a published procedure (Nasr-Esfahani et al., 1990bGo). Two-cell embryos were stained with either 2.0x10-5 mol/l 6-carboxy-2',7’-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) (CDCFDA) or 5-(and-6)-carboxy-2',7’-dichlorofluorescein diacetate (DCFDA) for 30 min to measure esterase and hydrogen peroxide levels respectively. As the levels of fluorescence of the dye detecting hydrogen peroxide is dependent on esterase activity, the ratio of hydrogen peroxide to esterase activity is a more accurate measure of relative levels of hydrogen peroxide in embryos. However, as these two parameters cannot be detected on the same embryo, a single ratio of an average of all embryos is presented. After loading of the dyes, embryos were washed in MOPS–G1 and immediately placed in a 2 µl drop of MOPS–G1 and the fluorescence determined. All readings occurred within 5 min of removal from the dye. Fluorescence of the dyes was quantified using a microfluorimeter with photometer attachments controlled by an optical switch (Solamere Technology Group, Salt Lake City, UT, USA). Embryos exposed to UV light for 30 min were used as positive controls for measuring levels of hydrogen peroxide. The levels of hydrogen peroxide were expressed as a percentage of in-vivo developed embryos.

Assessment of ICM and TE outgrowth
Blastocyst outgrowth was assessed as a measure of viability. Zygotes were cultured to the blastocyst stage in media G1.2/G2.2 and then cryopreserved by either slow freezing or vitrification in solutions either lacking ascorbate or containing 0.1 mmol/l ascorbate. Following thawing/warming, blastocysts were placed in 200 µl of medium Tissue Culture Medium-199 with 10% fetal calf serum (Hyclone, Logan, UT, USA) in gelatin-coated 96-well plates. Blastocysts were cultured at 37°C in 6% CO2/5% O2/89% N2 for 72 h. After 24 h, blastocyst hatching and attachment was assessed and after 72 h outgrowth of the ICM and TE assessed on a scale of 0–3 where 0 represents no growth and 3 represents extensive growth (Spindle and Pedersen, 1973Go).

Assessment of glucose uptakes by individual blastocysts
Glucose uptakes by individual blastocysts was assessed by an ultramicrofluorescence assay as previously described (Gardner and Leese, 1990Go, 1999Go). Using this procedure, concentrations of substrates in the femtomole range can be accurately measured in nanolitre volumes (Gardner and Leese, 1999Go). Blastocysts were incubated in 40 nl drops of medium G2.2 modified to contain 0.5 mmol/l glucose compared with 3.15 mmol/l in the original formulation. Embryos were incubated at 37°C in 6% CO2/5% O2/89% N2 for 3 h after thawing/warming. At the end of the incubation, blastocysts were removed from the drops and the media were frozen for subsequent analysis.

Glucose levels in the medium was assessed using a miniaturization of fluorometric assays. Assays were performed in 30 nl drops of reagent cocktail with 3 nl samples of medium. Fluorescence from the production of the pyridine nucleotide NADPH was detected using a microfluorimeter with photometer attachments controlled by an optical switch (Solamere Technology Group, Salt Lake City, UT, USA). Fluorescence was calibrated by a glucose standard curve run daily with each set of samples.

Determination of lactate dehydrogenase activity
Lactate dehydrogenase (LDH) activity was determined using quantitative microfluorescence based on the following reaction:

The composition of the reaction buffer was 50 mmol/l K2HPO4, 0.8 mmol/l KH2PO4, 0.63 mmol/l pyruvate, 0.116 mmol/l NaHCO3 and 0.057 mmol/l NADH. Reactions were performed at 37°C and all reagents were at the concentrations required for measurement of maximal activity (Martin et al., 1993Go; Lane and Gardner, 2000Go).

For the assay, a 50 nl drop of the reagents was placed on a siliconized slide, warmed to 37°C and its fluorescence quantified in the presence of UV light. A 15 nl drop of sample was warmed to 37°C and then added to the reagent drop. The change in fluorescence was assessed continuously for 5 min. The change in fluorescence was calculated from a NADH standard curve run each day. A control drop of cocktail without sample was warmed to 37°C and the fluorescence determined to establish the amount of photo-oxidation of NADH by exposure to UV light. A further control drop of sample and reagents in the absence of substrate (pyruvate) was also performed to control for non-specific oxidation. The activity of the enzyme was then calculated from the gradient and has been expressed as pmol NADH oxidized/embryo/h.

Statistics
Data for embryo morphology were analysed using linear logistic regression using the Software package GLIM. Data for embryo metabolism, cell numbers, outgrowth, LDH activity and hydrogen peroxide levels were assessed by ANOVA. No between-replicate differences were detected and data from all experiments were subsequently pooled for analysis. Between-treatment differences were determined using Bonferroni’s multiple comparison procedures. P < 0.05 was considered significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Effect of ascorbate in cryopreservation solutions on pyruvate oxidation of 2-cell embryos
Addition of ascorbate to the cryopreservation solutions at a concentration of 0.1 mmol/l significantly increased pyruvate oxidation by 2-cell embryos that were cryopreserved by either slow freezing or vitrification (P < 0.05; Table IGo). Increasing the concentration of ascorbate to 0.5 mmol/l did not affect the levels of pyruvate oxidation (Table I). For all concentrations of ascorbate, 2-cell embryos that were cryopreserved by vitrification had significantly higher levels of pyruvate oxidation compared with embryos cryopreserved using slow-freezing procedures (P < 0.05; Table IGo).


View this table:
[in this window]
[in a new window]
 
Table I. Effect of ascorbate in the cryopreservation solutions on pyruvate oxidation of 2-cell embryos
 
Effect of ascorbate in cryopreservation solutions on levels of hydrogen peroxide in 2-cell embryos
In the absence of ascorbate, cryopreservation of 2-cell embryos by the slow-freezing procedure significantly increased the levels of hydrogen peroxide (Table IIGo). Compared with in-vivo developed 2-cells, embryos that were cryopreserved by the slow-freezing procedure had significantly reduced levels of intracellular esterase activity and a 50% increase in the levels of hydrogen peroxide (P < 0.05). Addition of ascorbate to the slow-freezing solutions at either 0.1 or 0.5 mmol/l significantly increased endogenous esterase activity and also significantly reduced the levels of intracellular peroxide (P < 0.05; Table IIGo). When expressed as a ratio of esterase activity to hydrogen peroxide levels, the addition of ascorbate resulted in a larger decrease in hydrogen peroxide levels (Table IIGo). Two-cell embryos that were vitrified had significantly reduced levels of hydrogen peroxide compared with embryos that were slow-frozen (P < 0.05; Table IIGo). Vitrified 2-cell embryos did, however, have an ~20% increase in hydrogen peroxide levels compared with in-vivo developed embryos. Addition of ascorbate to the vitrification and warming solutions resulted in a significant reduction in hydrogen peroxide levels compared with embryos vitrified in the absence of ascorbate (P < 0.05; Table IIGo).


View this table:
[in this window]
[in a new window]
 
Table II. Effect of addition of ascorbate to the cryopreservation solutions on intracellular esterase and hydrogen peroxide levels of 2-cell embryos
 
Effect of ascorbate in the cryopreservation solutions on LDH leakage
Two-cell embryos that were cryopreserved using slow-freezing procedures had significantly higher levels of LDH leakage from the embryos compared with those that were vitrified (P < 0.05; Figure 1Go). Addition of 0.1 mmol/l ascorbate to the medium for slow freezing resulted in a significant decrease in the levels of LDH leakage (P < 0.05; Figure 1Go). Addition of ascorbate did not affect LDH leakage by vitrified embryos (Figure 1Go).



View larger version (11K):
[in this window]
[in a new window]
 
Figure 1. Lactate dehydrogenase (LDH) leakage by 2-cell embryos following cryopreservation in the presence or absence of ascorbate. n = at least 40 embryos per treatment (two replicate experiments). Different letters are significantly different (P < 0.05).

 
Effect of ascorbate in cryopreservation solutions on subsequent embryo development in culture
Two-cell embryos that were cryopreserved using either slow-freezing or vitrification procedures developed to the blastocyst stage at high rates (Table IIIGo). There was no difference in the numbers of blastocysts that were obtained between either cryopreservation procedure or when ascorbate was added to the cryopreservation solutions. Addition of ascorbate did not affect the total cell numbers of the blastocysts obtained. Blastocysts resulting from 2-cell embryos that were vitrified had significantly higher cell numbers than those that were cryopreserved using the slow-freezing procedure (P < 0.05; Table IIIGo). Addition of 0.1 mmol/l ascorbate to the cryopreservation solutions significantly increased the number of ICM cells as well as the percentage of ICM cells in the blastocysts following both cryopreservation procedures (P < 0.05; Table IIIGo). Addition of ascorbate did not affect the number of trophectoderm cells in the resultant blastocysts (Table IIIGo).


View this table:
[in this window]
[in a new window]
 
Table III. Effect of ascorbate in the cryopreservation solutions on 2-cell embryo development in culture
 
As a negative control, 2-cell embryos that were not cryopreserved were exposed to ascorbate for the length of the cryopreservation procedures (slow freezing, 77 min; vitrifica tion, 11.5 min). This short exposure to ascorbate did not affect development to the blastocyst stage or allocation of cells to the ICM and TE (data not shown).

Effect of ascorbate in cryopreservation solutions on blastocyst survival and development
Following thawing/warming in the presence or absence of 0.1 mmol/l ascorbate, blastocyst re-expansion, attachment and outgrowth of the ICM and TE were determined. Addition of 0.1 mmol/l ascorbate to the media for slow freezing significantly increased blastocyst re-expansion and attachment compared with embryos frozen in the absence of ascorbate (P < 0.05; Table IVGo). The addition of ascorbate did not affect blastocyst re-expansion or attachment when blastocysts were vitrified. Addition of ascorbate to the slow-freezing solutions for blastocysts resulted in a significant increase in the levels of both ICM and TE outgrowth compared with blastocysts cryopreserved in the absence of ascorbate (P < 0.05). Addition of ascorbate to the vitrification solutions also stimulated ICM outgrowth, but TE outgrowth was not affected (Table IVGo).


View this table:
[in this window]
[in a new window]
 
Table IV. Effect of ascorbate during blastocyst cryopreservation on subsequent attachment and outgrowth
 
The effect of ascorbate in the cryopreservation solutions was also assessed by determining the levels of glucose uptake by the resultant blastocysts. Glucose uptake by mouse blastocysts has previously been shown to be correlated with viability after transfer (Gardner and Leese, 1987Go; Lane and Gardner, 1996Go). Blastocysts cryopreserved by the slow-freezing procedure in the presence of ascorbate had significantly higher levels of glucose uptake compared with blastocysts that were cryopreserved in the absence of ascorbate (Figure 2Go). There was no significant effect on glucose uptakes by blastocysts following the addition of ascorbate to the vitrification solutions (Figure 2Go). Blastocysts that were vitrified had significantly higher glucose uptakes compared with those that were cryopreserved by slow freezing (P < 0.05).



View larger version (12K):
[in this window]
[in a new window]
 
Figure 2. Effect of ascorbate in the cryopreservation solutions on glucose uptakes by blastocysts. n = 20 embryos per treatment (two replicate experiments). *Significantly different from all other treatments (P < 0.05).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Addition of ascorbate to the cryopreservation solutions significantly enhanced the ability of mouse embryos both at the cleavage and blastocyst stages to survive cryopreservation. The major effect of ascorbate on embryo development was to stimulate the development of the ICM following cryopreservation. However, mouse embryos that were cryopreserved with ascorbate were also better able to maintain normal cell function. The beneficial effects of ascorbate were most evident in mouse embryos that were slow-frozen compared with those that were vitrified, indicating that the damage as a result of oxygen radicals is greater after slow freezing. A direct effect of ascorbate was to reduce the levels of hydrogen peroxide that were elevated by cryopreservation. The results from this study indicate that it would be beneficial for all solutions for the cryopreservation of mammalian embryos to contain 0.1 mmol/l ascorbate.

Cryopreservation of mouse 2-cell embryos by slow freezing significantly increased the levels of hydrogen peroxide in the blastomeres immediately after thawing. This increase in hydrogen peroxide levels is indicative of an increase in the levels of free oxygen radicals in these embryos. Addition of ascorbate to the cryopreservation solutions resulted in a significant reduction in the levels of hydrogen peroxide. This decrease in levels of hydrogen peroxide was correlated with increased developmental competence and an increased maintenance of normal embryo physiology. Increased levels of hydrogen peroxide in the embryos are significant as reactive oxygen species such as hydrogen peroxide can damage cells by enabling the formation of very damaging hydroxyl radials via the Fenton and Haber–Weiss reactions. This formation of hydroxyl radicals can be accelerated by the presence of metal ions such as iron or copper (Halliwell and Gutteridge, 1989Go). Several studies have demonstrated a beneficial effect of adding either antioxidants or metal ion chelators such as EDTA to culture media for mammalian embryos (Nasr-Esfahani et al., 1990aGo, 1992Go; Noda et al., 1991Go; Nonogaki et al., 1991Go; Goto et al., 1992Go; Payne et al., 1992Go; Umaoka et al., 1992Go; Gardner and Lane, 1996Go; Orsi and Leese, 2001Go) indicating that reactive oxygen species such as the hydroxyl radicals are very damaging to embryos. Even a transient rise in the levels of hydrogen peroxide has detrimental effects on embryo development in culture (Nasr-Esfahani et al., 1990bGo). Ascorbate has also been shown to reduce the production of reactive oxygen species in sperm (Donnelly et al., 1999Go). It is clear from the data presented in our study that the presence of ascorbate during the cryopreservation procedure had a significant beneficial effect on subsequent embryo development.

The levels of hydrogen peroxide observed were significantly lower in the mouse embryos that were vitrified compared with those that were cryopreserved by slow freezing. It therefore appears that the slow-freezing procedure results in the production of significantly more hydrogen peroxide than the ultra-rapid vitrification procedure used. The levels of esterase activity in the embryos cryopreserved using the slow-freezing procedure were also significantly reduced compared with those embryos that were vitrified. This suggests that the slow-frozen embryos were metabolically impaired compared with those that were vitrified. These differences may in part explain previous observations that the ultra-rapid vitrification procedure is more successful than the slow-freezing procedures for the cryopreservation of embryos (Lane and Gardner, 2001bGo).

Interestingly, the addition of ascorbate to the cryopreservation solutions for 2-cell mouse embryos resulted in blastocysts with significantly increased development of the ICM compared with embryos cryopreserved in the absence of ascorbate. Similarly, ascorbate in the freezing solutions for blastocysts also significantly increased the rates of ICM outgrowth. It therefore appears that the ICM is particularly sensitive to the production and presence of hydrogen peroxide. Cryopreservation of bovine blastocysts is known to result in an increased death of ICM cells (Iwasaki et al., 1994Go) and reduced rates of proliferation of ICM cells following cryopreservation (Takagi et al., 1996Go). Furthermore, culture of embryos from several species in elevated levels of oxygen (~20%) results in blastocysts with significantly reduced numbers of ICM cells compared with embryos grown in low (5%) physiological levels of oxygen (Gardner et al., 1999Go; Lim et al., 1999Go). This reduction in the development of the ICM is probably due to the increased production of oxygen radicals resulting from the high levels of oxygen. The data presented in this study indicate that the elevated levels of oxygen radicals may persist for some time following the cryopreservation procedure, affecting the later development of the ICM.

In our study, the detrimental effect of cryopreservation on ICM development was significantly greater when embryos were slow-frozen compared with vitrification. This is in contrast to the results of Kaidi et al. who found no difference in the survival of the ICM between slow freezing and vitrification for bovine blastocysts (Kaidi et al., 2001Go). However, similar to our results, these authors did observe a significant decrease in glucose uptakes by bovine blastocysts after slow freezing compared with vitrification (Kaidi et al., 2001Go). The differences in the sensitivity of the ICM following vitrification between the two studies may be explained in part by the different vitrification procedures used. In our study, we used an ultra-rapid vitrification using the cryoloop, whereas Kaidi et al. (2001) used a traditional vitrification procedure with a straw. Ultra-rapid vitrification procedures have reported superior results compared with more traditional vitrification procedures due to significant increases in the cooling rates which reduce the toxicity of the cryoprotectants (Vajta et al., 1998Go; Lane et al., 1999aGo,bGo; Chen et al., 2001Go; Nowshari and Brem, 2001Go). Alternatively, there may be species differences between the sensitivity of the ICM between mice and cows to cryopreservation.

In addition to the formation of the hydroxyl radicals, hydrogen peroxide can combine with other molecules to form alkoxyl and peroxyl radicals (Halliwell and Gutteridge, 1989Go). These oxygen species damage lipids in the cell membranes and also in the organelle membranes within a cell. In this study, determining the levels of leakage of the cytoplasmic enzyme LDH indirectly assessed damage to the cell membrane. The levels of LDH leakage from bovine embryos have previously been used to assess the viability of bovine embryos (Johnson et al., 1991Go). In normal healthy cells this enzyme does not cross the membrane and in this study LDH could not be detected in control embryos that were not cryopreserved. However, 2-cell mouse embryos that were cryopreserved by slow freezing had significant levels of LDH leakage, indicating an increase in the permeability of the cell membrane. The addition of ascorbate during cryopreservation reduced the amount of LDH leakage, indicating that a significant proportion of the damage to the membrane resulted from the damage due to oxygen radicals. Interestingly, the levels of LDH leakage were significantly reduced following vitrification compared with slow freezing. Therefore, it appears that the ultra-rapid vitrification procedure used in this study causes substantially less damage to the cell membrane.

Pyruvate oxidation was also reduced in embryos that were cryopreserved by slow freezing. The oxidation of pyruvate is a major energy-generating pathway of the early embryo (Leese, 1991Go). Oxidative phosphorylation of pyruvate relies upon transfer of pyruvate across the inner mitochondrial membrane by a specific carrier (Newsholme and Leech, 1985Go). Oxygen radicals can disrupt the function of the organelle membranes such as those of the mitochondria, resulting in a disruption in normal oxidative metabolism. This may be the case for the 2-cell embryos that were cryopreserved using the slow-freezing procedure, as the addition of ascorbate increased the rates of pyruvate oxidation.

Although the higher concentration of ascorbate used (0.5 mmol/l) would have been expected to act as an equivalent or better antioxidant compared with 0.1 mmol/l, the beneficial effects on embryo development were either significantly reduced or not evident. The concentration of ascorbate found in bodily fluids is reported to be between 30 and 150 µmol/l (Halliwell and Gutteridge, 1989Go; Seis et al., 1992Go) and the amount in follicular fluid has been reported to be 1/68th of the serum concentration (Paszkowski and Clarke, 1999Go). Therefore, it is plausible that the concentration of 500 µmol/l used in this study, which is significantly elevated compared with the physiological levels, resulted in a detrimental effect on embryo development. The mechanism for this inhibition is unknown; however, it has been demonstrated that ascorbate can modulate phosphorylation–dephosphorylation events in cultured cell lines (Monteiro et al., 1993Go).

In conclusion, the data presented in this study demonstrate that the inclusion of 0.1 mmol/l ascorbate for the cryopreservation of both mouse cleavage stage and blastocyst stage embryos is beneficial for subsequent embryo development. Therefore, it may be beneficial to include 0.1 mmol/l ascorbate in all solutions for the cryopreservation of embryos whether the procedure of choice is slow freezing or vitrification.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This work was supported by a research grant from Vitrolife AB to the Colorado Center for Reproductive Medicine. Pregnyl used for this study was kindly donated by Organon, Inc.


    Notes
 
1 To whom correspondence should be addressed. E-mail: mlane{at}colocrm.com Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Bilodeau, J.F., Chatterjee, S., Sirard, M.A. and Gagnon, C. (2000) Levels of antioxidant defenses are decreased in bovine spermatozoa after a cycle of freezing and thawing. Mol. Reprod. Dev., 55, 282–288.[ISI][Medline]

Chen, S.U., Lien, Y.R., Cheng, Y.Y., Chen, H.F., Ho, H.N. and Yang, Y.S. (2001) Vitrification of mouse oocytes using closed pulled straws (CPS) achieves a high survival and preserves good patterns of meiotic spindles, compared with conventional straws, open pulled straws (OPS) and grids. Hum. Reprod., 16, 2350–2356.[Abstract/Free Full Text]

Donnelly, E.T., McClure, N. and Lewis, S.E.M. (1999) Antioxidant supplementation in vitro does not improve human sperm motility. Fertil. Steril., 72, 484–495.[ISI][Medline]

Emiliani, S., Van den Bergh, M., Vannin, A.S., Biramane, J. and Englert, Y. (2000) Comparison of ethylene glycol, 1,2-propanediol and glycerol for cryopreservation of slow-cooled mouse zygotes, 4-cell embryos and blastocysts. Hum. Reprod., 15, 905–910.[Abstract/Free Full Text]

Gardner, D.K. and Lane, M. (1996) Alleviation of the ‘2-cell block’ and development to the blastocyst of CF1 mouse embryos: role of amino acids, EDTA and physical parameters. Hum. Reprod., 11, 2703–2712.[Abstract]

Gardner, D.K. and Lane, M. (1997) Culture and selection of viable blastocysts: a feasible proposition for human IVF? Hum. Reprod. Update, 3, 367–382.[Abstract/Free Full Text]

Gardner, D.K. and Leese, H.J. (1987) Assessment of embryo viability prior to transfer by the noninvasive measurement of glucose uptake. J. Exp. Zool, 242, 103–105.[ISI][Medline]

Gardner, D.K. and Leese, H.J. (1990) Concentrations of nutrients in mouse oviduct fluid and their effects on embryo development and metabolism in vitro. J. Reprod. Fertil, 88, 361–368.[Abstract]

Gardner, D.K. and Leese, H.J. (1999) Assessment of embryo metabolism and viability. In Trounson, A.O. and Gardner, D.K. (eds), Handbook of In Vitro Fertilization, 2nd edn. CRC Press, Boca Raton, pp. 347–372.

Gardner, D.K., Schoolcraft, W.B., Wagley, L., Schlenker, T., Stevens, J. and Hesla, J. (1998) A prospective randomized trial of blastocyst culture and transfer in in-vitro fertilization. Hum. Reprod., 13, 3434–3440.[Abstract]

Gardner, D.K., Lane, M., Johnson, J., Wagley, L., Stevens, J., and Schoolcraft, W.B. (1999) Reduced oxygen tension increases blastocyst development, differentiation, and viability. Fertil. Steril., 72 (Suppl. 1), S30–S31.

Gardner, D.K., Lane, M.W. and Lane, M. (2000) EDTA stimulates cleavage stage bovine embryo development in culture but inhibits blastocyst development and differentiation. Mol. Reprod. Dev., 57, 256–261.[ISI][Medline]

Goto, Y., Noda, Y., Narimoto, K., Umaoka, Y. and Mori, Y. (1992) Oxidative stress on mouse embryo development in vitro. Free Rad. Biol. Med., 13, 47–53.[ISI][Medline]

Gutteridge, J.M. and Halliwell, B. (2000) Free radicals and antioxidants in the year 2000. A historical look to the future. Ann. NY Acad. Sci., 899, 136–147.[Abstract/Free Full Text]

Halliwell, B. and Gutteridge, J.M. (1989) The chemistry of oxygen radicals and other derived species. In Halliwell, B. and Gutteridge, J.M. (eds), Free Radicals in Biology and Medicine. Clarendon Press, Oxford, pp. 22–85.

Halliwell, B. and Gutteridge, J.M. (1995) The definition and measurement of antioxidants in biological systems. Free Rad. Biol. Med., 18, 125–126.[ISI][Medline]

Hardy, K., Handyside, A.H. and Winston, R.M. (1989) The human blastocyst: cell number, death and allocation during late preimplantation development in vitro. Development, 107, 597–604.[Abstract]

Iwasaki, S., Yoshikane, Y., Li, X., Watanabe, S. and Nakahara, T. (1994) Effects of freezing of bovine preimplantation embryos derived from oocytes fertilized in vitro on survival of their inner cell mass cells. Mol. Reprod. Dev., 37, 272–275.[ISI][Medline]

Johnson, S.K., Jordan, J.E., Dean, R.G. and Page, R.D. (1991) The quantification of bovine embryo viability using a bioluminescent assay for lactate dehydrogenase. Theriogenology, 35, 425–433.[ISI]

Kaidi, S., Bernard, S., Lambert, P., Massip, A., Dessy, F. and Donnay, I. (2001) Effect of conventional controlled-rate freezing and vitrification on morphology and metabolism of bovine blastocysts produced in vitro. Biol. Reprod., 65, 1127–1134.[Abstract/Free Full Text]

Lane, M. and Gardner, D.K. (1992) Effect of incubation volume and embryo density on the development and viability of mouse embryos in vitro. Hum. Reprod., 7, 558–562.[Abstract]

Lane, M. and Gardner, D.K. (1996) Selection of viable mouse blastocysts prior to transfer using a metabolic criterion. Hum. Reprod., 11, 1975–1978.[Abstract]

Lane, M. and Gardner, D.K. (2000) Lactate regulates pyruvate uptake and metabolism in the preimplantation mouse embryo. Biol. Reprod., 62, 16–22.[Abstract/Free Full Text]

Lane, M. and Gardner, D.K. (2001a) Vitrification of mouse oocytes using a nylon loop. Mol. Reprod. Dev., 58, 342–347.[ISI][Medline]

Lane, M. and Gardner, D.K. (2001b) Inhibiting 3-phosphoglycerate kinase by EDTA stimulates the development of the cleavage stage mouse embryo. Mol. Reprod. Dev., 60, 233–240.[ISI][Medline]

Lane, M., Bavister, B.D., Lyons, E.A. and Forest, K.T. (1999a) Containerless vitrification of mammalian oocytes and embryos. Nat. Biotechnol., 17, 1234–1236.[ISI][Medline]

Lane, M., Schoolcraft, W.B. and Gardner, D.K. (1999b) Vitrification of mouse and human blastocysts using a novel cryoloop container-less technique. Fertil. Steril., 72, 1073–1078.[ISI][Medline]

Leese, H.J. (1991) Metabolism of the preimplantation mammalian embryo. Oxf. Rev. Reprod. Biol., 13, 35–72.[ISI][Medline]

Lim, J.M., Reggio, B.C., Godke, R.A. and Hansel, W. (1999) Development of in-vitro-derived bovine embryos cultured in 5% CO2 in air or in 5% O2, 5% CO2 and 90% N2. Hum. Reprod., 14, 458–464.[Abstract/Free Full Text]

Martin, K.L., Hardy, K., Winston, R.M. and Leese, H.J. (1993) Activity of enzymes of energy metabolism in single human preimplantation embryos. J. Reprod. Fertil., 99, 259–266.[Abstract]

Meister, A. (1992) On the antioxidant effects of ascorbic acid and glutathione. Biochem. Pharmacol., 44, 1905–1915.[ISI][Medline]

Monteiro, H.P., Ivaschenko, Y., Fischer, R. and Stern, A. (1993) Ascorbic acid inhibits protein tyrosine phosphatases in NIH 3T3 cells expressing human epidermal growth factor receptors. Int. J. Biochem., 25, 1859–1864.[ISI][Medline]

Nasr-Esfahani, M., Johnson, M.H. and Aitken, R.J. (1990a) The effect of iron and iron chelators on the in-vitro block to development of the mouse preimplantation embryo: BAT6 a new medium for improved culture of mouse embryos in vitro. Hum. Reprod., 5, 997–1003.[Abstract]

Nasr-Esfahani, M.H., Aitken, J.R. and Johnson, M.H. (1990b) Hydrogen peroxide levels in mouse oocytes and early cleavage stage embryos developed in vitro or in vivo. Development, 109, 501–507.[Abstract]

Nasr-Esfahani, M.H., Winston, N.J. and Johnson, M.H. (1992) Effects of glucose, glutamine, ethylenediaminetetraacetic acid and oxygen tension on the concentration of reactive oxygen species and on development of the mouse preimplantation embryo in vitro. J. Reprod. Fertil., 96, 219–231.[Abstract]

Newsholme, E.A. and Leech, A.R. (1985) Biochemistry for the medical sciences. Wiley, Chichester, pp. 167–245.

Noda, Y., Matsumoto, H., Umaoka, Y., Tatsumi, K., Kishi, J. and Mori, T. (1991) Involvement of superoxide radicals in the mouse two-cell block. Mol. Reprod. Dev., 28, 356–360.[ISI][Medline]

Nonogaki, T., Noda, Y., Narimoto, K., Umaoka, Y. and Mori, T. (1991) Protection from oxidative stress by thioredoxin and superoxide dismutase of mouse embryos fertilized in vitro. Hum. Reprod., 6, 1305–1310.[Abstract]

Nowshari, M.A. and Brem, G. (2001) Effect of freezing rate and exposure time to cryoprotectant on the development of mouse pronuclear stage embryos. Hum. Reprod., 16, 2368–2373.[Abstract/Free Full Text]

Orsi, N.M. and Leese, H.J. (2001) Protection against reactive oxygen species during mouse preimplantation embryo development: role of EDTA, oxygen tension, catalase, superoxide dismutase and pyruvate. Mol. Reprod. Dev., 59, 44–53.[ISI][Medline]

Paszkowski, T. and Clarke, R.N. (1999) The Graafian follicle is a site of L-ascorbate accumulation. J. Assist. Reprod. Genet., 16, 41–45.[ISI][Medline]

Payne, S.R., Munday, R. and Thompson, J.G. (1992) Addition of superoxide dismutase and catalase does not necessarily overcome developmental retardation of one-cell mouse embryos during in-vitro culture. Reprod. Fertil. Dev., 4, 167–174.[ISI][Medline]

Rieger, D., Loskutoff, N.M. and Betteridge, K.J. (1992) Developmentally related changes in the uptake and metabolism of glucose, glutamine and pyruvate by cattle embryos produced in vitro. Reprod. Fertil. Dev., 4, 547–557.[ISI][Medline]

Seis, S., Stahl, W. and Sundquist, A.R. (1992) Antioxidant functions of vitamins. Ann. NY Acad. Sci., 669, 7–20.[Abstract]

Spindle, A.I. and Pedersen, R.A. (1973) Hatching, attachment, and outgrowth of mouse blastocysts in vitro: fixed nitrogen requirements. J. Exp. Zool., 186, 305–318.[ISI][Medline]

Takagi, M., Sakonju, I. and Suzuki, T. (1996) Effects of cryopreservation of DNA synthesis in the inner cell mass of in vitro matured/in vitro fertilized bovine embryos frozen in various cryoprotectants. J. Vet. Med. Sci., 58, 1237–1238.[ISI][Medline]

Tarin, J.J. and Trounson, A.O. (1993) Effects of stimulation or inhibition of lipid peroxidation on freezing–thawing of mouse embryos. Biol. Reprod., 49, 1362–1368.[Abstract]

Umaoka, Y., Noda, Y., Narimoto, K. and Mori, T. (1992) Effects of oxygen toxicity on early development of mouse embryos. Mol. Reprod. Dev., 31, 28–33.[ISI][Medline]

Vajta, G., Holm, P., Kuwayama, M., Booth, P.J., Jacobsen, H., Greve, T. and Callesen, H. (1998) Open Pulled Straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Mol. Reprod. Dev., 51, 53–58.[ISI][Medline]

Submitted on April 2, 2002; accepted on May 30, 2002.





This Article
Abstract
FREE Full Text (PDF )
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (3)
Request Permissions
Google Scholar
Articles by Lane, M.
Articles by Gardner, D. K.
PubMed
PubMed Citation
Articles by Lane, M.
Articles by Gardner, D. K.