Detrimental Effects of Sodium during Mouse Oocyte Cryopreservation

^a Institute of Reproductive Medicine and Science of Saint Barnabas Medical Center, West Orange, New Jersey 07052

	ABSTRACT

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Cryopreservation is an established way of storing embryos, but effective methods are not available for freezing eggs. Most freezing damage is caused by high solute concentration (solution effects) and intracellular ice. Sodium salts are the major components of cryopreservation media, and the main contributor to the solution effects. The present experiments examine the effect of substituting choline for sodium as the major extracellular cation in the cryopreservation of mouse eggs. The effects of serum and various cryoprotectants were also examined. Survival, fertilization, and development were inversely related to the concentration of sodium in the freezing medium. Oocytes frozen in a choline-based medium had the highest (p < 0.001) survival and development rates. The absence of serum during thawing inhibited fertilization, whereas exposure to serum or opening the zona allowed fertilization to reach the control level. Dimethyl sulfoxide was as effective as 1,2 propanediol for obtaining high survival and fertilization rates. These results support the hypothesis that the high concentration of sodium in conventional freezing media is detrimental to cells and show that choline is a promising replacement for sodium. Reducing or eliminating sodium may allow oocytes and other cells to be frozen more efficiently.

	INTRODUCTION

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Cryopreservation is now an effective and established way of storing embryos from a number of mammalian species, notably those of cattle, laboratory mice, and humans. However, there are a number of situations both for assisted human reproduction and for animal breeding in which the option of storing embryos does not exist or in which it would be preferable to store unfertilized eggs. For instance, the ability to freeze unfertilized oocytes could be valuable for long-term storage of genetically important strains of mice and would serve as a model for oocyte cryopreservation in humans, commercial livestock, and endangered species. Although offspring have been produced from frozen-thawed oocytes in mice, humans, cattle, and rabbits [1–5], results have been variable and not sufficiently successful for routine use.

Current cryopreservation protocols have evolved from methods developed for freezing mouse, cattle, and sheep embryos [6–8]. The design of cryopreservation methods for living cells and tissues, including embryos and eggs, usually disregard the biological complexity of such material and focus instead on the more immediate physical consequences of cooling and rewarming as potential causes of cellular damage. This may explain the difficulty and relatively poor success of oocyte cryopreservation [9–14]. According to the most widely accepted explanation, high solute concentrations, known as "solution effects," and intracellular ice are responsible for most, if not all, damage to cells during cooling and rewarming. While both factors are often at work simultaneously, intracellular ice is most likely to occur during rapid cooling and relatively slow rewarming, whereas solution effects are more pronounced with slow cooling. Successful cryopreservation protocols for mammalian embryos depend to a large extent on slow cooling rates, and at slow rates, solution effects can be expected to pose particular problems.

The principle behind conventional freezing procedures is to induce ice formation extracellularly, thereby raising the solute concentration. In response, water is drawn out of the cells, preventing ice crystals from forming intracellularly [15]. However, the cooling rate must be sufficiently slow to allow water to flow out of the cells before they solidify. As a consequence of slow cooling, the cells are exposed to high solute concentrations for a prolonged period of time.

Permeating cryoprotectants, including glycerol, dimethyl sulfoxide (DMSO), and 1,2 propanediol (PrOH), interact with and partially replace water molecules in such a way that the freezing point of the solution is lowered during cooling as water is transformed into ice and the concentration of cryoprotectant increases. Upon solidification of the cryoprotectant/water mixture, a glass-like structure is formed, thereby preventing crystal formation and growth. Freezing damage may therefore occur when the cell is exposed to elevated concentrations of electrolytes or intracellular ice. Intracellular ice formation can be catalyzed by the presence of extracellular ice surrounding the cells as a result of seeding, or heterogeneously by intracellular structures. In the presence of cryoprotectants, however, intracellular ice formation resulting from seeding and/or heterogeneous ice nucleation does not occur [16]. Therefore, electrolytes, including sodium, would appear to impart the majority of the damage during cryopreservation. Lovelock [17] hypothesized that cellular damage is caused by an increase in electrolyte concentration, thereby destabilizing membranes. Mazur et al. [18] further demonstrated that the cell surface is a major site of freezing damage by showing that the nonpermeating solute sucrose was as effective in protecting erythrocytes from freeze/thaw damage as the permeable cryoprotectant glycerol.

Sodium salts are the major components of virtually all cell-handling media including those used for cryopreservation of mammalian embryos. Sodium ions therefore contribute significantly to the solution effects during cooling and rewarming of embryos. High intracellular sodium concentrations that may result from freezing are incompatible with normal cell function. Moreover, sodium toxicity has been specifically suggested to be a major factor in cryopreservation-related cell damage. The present experiments were undertaken to examine the effect of substituting choline for sodium as the major extracellular cation in the cryopreservation of unfertilized mouse eggs. Unlike the sodium ion, the choline ion is thought not to cross the cell membrane and therefore would not be expected to contribute to the intracellular cation load. A previous study also suggested a possible membrane-protective role for choline [19].

	MATERIALS AND METHODS

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Collection and Culture of Oocytes

Five- to 8-wk-old female C57BL/6 x SJL F1 mice (Jackson Laboratories, Bar Harbor, ME) were superovulated, and cumulus masses were removed from the oviducts 13 h post-hCG (Sigma Chemical Co., St. Louis, MO). Oocytes were collected after the cumulus masses were treated with 120 U/ml hyaluronidase (Sigma) for 10 min. The oocytes were then washed in Flushing Holding Medium (potassium simplex optimized medium [KSOM] buffered with HEPES; Specialty Media, Lavallette, NJ) and held at 23°C (room temperature [RT]) until cryopreservation or insemination. Insemination and embryo culture were carried out in a 5% CO₂ incubator at 37°C, using microdrops (30–50 µl) of Ham's F-10 (Sigma) and KSOM (Specialty Media), respectively, in Nunc dishes (VWR Scientific, Piscataway, NJ) under twice-washed mineral oil (Squibb; Park Avenue Chemists Ltd., New York, NY).

Oocyte Freezing and Thawing

Oocytes that were translucent and round, having extruded the first polar body, and that appeared normal were selected for cryopreservation. A portion of these unfertilized eggs was set aside to be used as unfrozen controls. The unfertilized eggs were cryopreserved in one of several different freezing media, all supplemented with 10% fetal bovine serum (FBS), unless otherwise noted. Incubation in freezing media before cryopreservation and after thawing was done at RT. For cryopreservation, oocytes were washed in freezing medium containing 1.5 M PrOH for 10 min and then transferred to freezing medium containing 1.5 M cryoprotectant and 0.1 M sucrose for 10–15 min. During this time, the oocytes were loaded into 0.25-ml French straws (IMV International, Minneapolis, MN). The straws were heat-sealed at both ends and placed in a BioCool III programmable freezer (FTS Systems, Stone Ridge, NY), cooled to -7°C at a rate of -2°C per minute, seeded by touching the side of each straw with forceps that had been cooled in liquid nitrogen (LN₂), held at -7°C for 10 min, and cooled to -33°C at a rate of -0.3°C per minute before being plunged into LN₂. After storage at -196°C for at least 5 days, the unfertilized eggs were thawed by exposure to RT air for 30 sec and then submersed into a 30°C water bath for an additional 10 sec. The eggs were subjected to 6 successive rinses at 5-min intervals to remove the cryoprotectant and sucrose. These thaw-step rinses, in the order of use, comprised freezing medium supplemented with 1) 0.2 M sucrose and 1.0 M cryoprotectant, 2) 0.2 M sucrose and 0.5 M cryoprotectant, 3) 0.2 M sucrose, 4) 0.1 M sucrose, 5) freezing medium alone, and 6) modified Chatot, Ziomek, and Bavister medium (mCZB) [20, 21]. The oocytes, while still in the mCZB, were placed on a slide warmer set at 37°C for an additional 5 min before being inseminated.

Fertilization and Embryo Culture

Spermatozoa from male B6SJL mice 3 to 9 mo old were released by epididymal puncture into Ham's F-10 medium (Sigma) supplemented with 4 mg/ml BSA (Sigma). The sperm were allowed to capacitate for 1–2 h before being used for insemination. Nonfrozen control and cryopreserved oocytes were cultured in Ham's F-10 medium containing approximately 5 x 10⁵ motile sperm for 5–9 h, then washed through three drops of KSOM, and cultured in KSOM until the 8-cell stage, at which point they were moved to a fresh drop of KSOM for an additional 2 days. Unless otherwise noted, zonae from thawed oocytes were opened by using a Fertilase 670-nm diode laser aiming beam and a collimated 1.48-µm laser beam (Zander Medical Supplies, Vero Beach, FL) at a power of 47 mW and irradiation time of 15 msec to cut a 30- to 40-µm opening in the zona pellucida before insemination [22]. Oocytes were judged to be fertilized by the presence of 2 pronuclei or 2 uniform blastomeres 25 h after insemination. The resulting embryos were examined daily for the extent of developmental progression. The number of embryos developing to the 2-cell, morulae, and blastocyst stages were recorded after 24, 72, and 96 h post-insemination, respectively.

Experiment 1: Effect of Altering the Sodium Concentration in the Cryopreservation Medium

The effect of reducing the sodium concentration in freezing medium on oocyte cryopreservation was examined. A typical sodium-based freezing medium (Embryo Transfer Freezing Medium; Gibco BRL, Gaithersburg, MD) was modified by substituting an equal molar amount of NaCl to yield solutions containing either 0, 34.2, 68.5, 102.7, or 137.0 mM choline chloride. It should be noted that at 137.0 mM choline chloride, all the NaCl was replaced with choline chloride, although sodium ions were still present in the medium because of the inclusion of Na₂HPO₄ and serum (see Table 2). This newly formulated cryopreservation medium, containing choline chloride and no NaCl, was named CJ1 (Table 1). Another freezing medium was also made without sodium by substituting equal molar amounts of choline chloride for NaCl and of K₂HPO₄ for Na₂HPO₄. This medium was essentially free of sodium ions, except those from the serum used as a supplement. Freshly ovulated mouse oocytes were frozen using these media and, after thawing, analyzed for their ability to survive freezing, become fertilized, and develop to the blastocyst stage.

Experiment 2: Effect of Serum on Oocyte Cryopreservation

Hardening of the zona pellucida caused by the premature release of cortical granules occurs during thawing and inhibits subsequent fertilization [13, 23–25]. Zona hardening has been shown to be reduced or prevented when FBS is included in the freezing and thawing media [14, 26]. To determine the effect of FBS on oocyte survival and subsequent ability to be fertilized, CJ1 was supplemented with 0, 10, 15, or 20% FBS for use in both the freezing and thawing procedures.

Experiment 3: Effect of Cryoprotectant Type on Oocyte Cryopreservation

All commonly used cryoprotectants used for the storage of embryos and sperm, such as PrOH, DMSO, glycerol, and ethylene glycol, are to some extent toxic to cells. Current embryo-freezing protocols use 1 to 2 M PrOH, DMSO, glycerol, or ethylene glycol [27–30]. The toxicity of these cryoprotectants varies with concentration and exposure time; the lower the concentration and the shorter the exposure time, the less toxic they are to embryo development (for review see [31]). The ability of mouse oocytes to survive freezing in CJ1 was examined using 1.5 M PrOH, 1.5 M DMSO, and 1.5 M ethylene glycol (Sigma).

Statistical Analysis

The data were analyzed using a general linear modeling procedure whereby the proportions were transformed onto the logistic scale, and treatment effects were estimated by the method of maximum likelihood. The algorithm used was GENSTAT [32]. This technique is an extension of the ANOVA test, but it allows different error structures, weighting, and variable transformations. In addition to the "portmanteau" tests on the homogeneity of the responses to the factor levels viewed collectively, individual comparisons among the levels were carried out by the same analytical method. Although all tests and analyses were carried out on the transformed scale, for purposes of clarity the tables display the estimated mean values on the original scale of proportions. This analysis was used throughout, except for comparison of blastocyst formation rates in Table 4. Because of the multiple zero responses for ethylene glycol at the blastocyst stage, comparisons among the three cryoprotectants were carried out by means of a distribution-free test (Fisher's randomization test) and using the algorithm described by Walters [33]. The experiments represented in the present manuscript were not always balanced because sometimes only a few oocytes were collected for freezing, and thus not every group in a particular experiment could be represented during that particular freeze. Whenever possible, all groups were represented during each freeze. Those groups that were not represented in a prior freeze were represented in a subsequent freeze. Because oocyte numbers were unevenly distributed among the groups, several groups were repeated more than others to increase the number of oocytes analyzed. The statistical analysis used took into account the variability between the experiments.

	RESULTS

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Experiment 1

A dramatic increase in oocyte survival, fertilization, and development was observed when NaCl was replaced by choline chloride (Table 2). There was no difference in survival, fertilization, or development when the freezing medium contained NaCl or 34.2 mM choline chloride. In contrast, when the NaCl in the freezing medium was replaced by 137.0 mM choline chloride, as in CJ1 medium, or when both NaCl and Na₂HPO₄ were removed, oocyte survival, fertilization, and blastocyst development were substantially improved (p < 0.001). Because the freezing medium was supplemented with 10% FBS, approximately 14 mM sodium was present during freezing and thawing. When oocytes were frozen in medium containing only 14 mM sodium, a high percentage of the eggs survived thawing (84.4%) and, after fertilization, developed to the blastocyst stage (Table 2). These percentages were similar (p > 0.05) to those obtained when oocytes were frozen in CJ1, and greater (p < 0.001) when compared with oocytes frozen in medium containing NaCl.

Experiment 2

The presence of FBS in the freezing medium had a significant effect on oocyte survival and fertilization (Tables 3 and 4). However, the 3 different FBS concentrations used all provided a significantly greater degree of protection compared to medium without serum (Table 3). Because media containing 10%, 15%, or 20% FBS were highly effective in maintaining oocyte integrity after thawing, the fertilization and development results were pooled (Table 4). Oocytes thawed in medium in which FBS was omitted from the last dilution (step 6) had a lower (p < 0.001) fertilization rate than oocytes that had their zona opened by laser manipulation or than zona-intact nonfrozen control oocytes. By contrast, zona-intact oocytes that were thawed in the presence of FBS throughout the entire step-out procedure exhibited fertilization rates similar to either frozen oocytes undergoing laser micromanipulation or nonfrozen zona-intact control oocytes.

Experiment 3

The cryoprotectant used for freezing affected oocyte cryopreservation (Table 5). The survival, fertilization, and developmental rates were similar for oocytes frozen with PrOH and DMSO as cryoprotectants. Oocytes frozen with ethylene glycol as the cryoprotectant exhibited significantly lower survival, fertilization, and developmental rates.

	DISCUSSION

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While attempting to focus on a single factor, the present study demonstrates both the difficulties and the shortcomings of such an approach, given the complexity of living cells and current cryotechnology. The results indicate that terms such as survival and viability are relative in the context of cryobiology, at least in the case of unfertilized eggs, but also, one suspects, in the case of other cells and tissues. Nonetheless, the present results lend support to the hypothesis that the high concentration of sodium ions that is an inevitable consequence of using conventional cell-handling media for cryopreservation poses a specific threat to the cells, and that choline is a promising candidate to replace sodium.

Although cells require sodium to function normally, they apparently not only tolerate substantially lower concentrations during freezing but even appear to benefit from a reduced sodium environment. Although some degree of membrane protection can be obtained in sodium-based freezing media, membrane and/or intracellular damage has occurred to such an extent that fertilization and development are impaired.

Under physiological conditions, sodium ions diffuse freely into the cell, but the excess is removed by sodium pumps, accounting for a substantial proportion of the cell's energy expenditure [34]. During freezing, the rise in the extracellular solute concentration brought about by ice formation favors not only the flow of water out of the cell but also an increase in diffusion of sodium into the cell. At the same time, the sodium pump may be expected to become increasingly disabled as the temperature decreases. It is therefore likely that the intracellular sodium load will have increased by the time the cell is transferred to LN₂. If so, this situation will still exist immediately after thawing and could lead to post-thaw damage. Substituting choline for sodium in the freezing medium would help overcome this problem.

It is possible that the positive effect of choline is not due solely to altering the sodium balance but also to a more direct stabilizing effect on the cell membrane, as choline is closely associated with phospholipids that make up the plasma membrane. While such an effect is speculative at this time, it may be an oversimplification to think that any nonpermeating, nontoxic compound could be used to mimic the effect observed with choline chloride, although good results have been obtained using a simple 0.3 M mannitol solution supplemented with 10% FBS and 1.5 M PrOH for cryopreservation of mouse oocytes (unpublished results).

Our results have again demonstrated that cryopreservation of oocytes may lead to hardening of the zona pellucida, and that FBS can protect the mouse zona from hardening as long as it is present throughout the thawing procedure [14, 26, 35]. Oocytes were noticeably susceptible to zona modifications (Table 4) if exposed, even briefly, to media that did not contain FBS before insemination. By using a laser to make a slit in the zona, we avoided cryopreservation-induced modifications of the zona pellucida and determined that oocytes frozen in a choline-based medium could be fertilized (> 65%) and develop normally [22]. By way of comparison, almost all nonfrozen control oocytes undergoing laser-assisted zona drilling (n = 44) before insemination were fertilized and cleaved to the 2-cell stage (93.2%), demonstrating that cryopreservation in some way alters the fertilization potential of the oocyte, even though the oocytes appear to be morphologically normal. Since laser-assisted zona drilling leads to other problems associated with fertilization and early development, including polyspermia and "trapping" of the blastocyst at hatching, FBS-supplemented medium was used throughout the cryopreservation procedure.

PrOH and DMSO are the most frequently used cryoprotectants for freezing oocytes and embryos. Although the majority of oocyte freezing studies in the mouse have used DMSO, PrOH is the cryoprotectant of choice for freezing human eggs because of its greater permeability, reduced toxicity, and improved success in embryo storage [28, 36–39]. Mouse oocytes have been frozen using PrOH, but with limited success [12, 40]. Todorow et al. [40] reported survival and fertilization rates of 63% and 27%, respectively, using PrOH. Freezing mouse oocytes using DMSO as the cryoprotectant yielded slightly better results [24–26, 35, 40–42]. In a recent study, Carroll et al. [14] published some of the highest survival, fertilization, and blastocyst formation rates reported to date for unfertilized mouse eggs frozen in DMSO. In order to obtain optimal survival and fertilization rates, the mouse oocytes used in their study were exposed to freezing medium at 5°C before they were quickly loaded into straws and the freezing process was begun. It was observed that exposure to higher temperatures and/or longer equilibration times before initiating the freeze resulted in reduced oocyte viability. However, in the present experiments, similar success rates were obtained with relatively prolonged (up to 25-min) exposure to the freezing medium at room temperature. Furthermore, PrOH was as effective as DMSO for oocyte freezing, despite the poor results reported in other studies using this cryoprotectant in a conventional sodium-based medium [12, 40].

The relative lack of success with cryopreservation of unfertilized eggs is not surprising. Rather, it is amazing that some embryos and indeed a host of other mammalian cells and tissues tolerate the drastic changes in extra- and intracellular conditions that accompany freezing and thawing. While physical considerations have been very helpful in elucidating the major causes of cellular damage during freezing and thawing and in determining optimal cooling and rewarming rates, more subtle biological factors may have to be taken into account to overcome the problems associated with freezing unfertilized eggs and certain other cells. This, in turn, may lead to overall improvements in cryopreservation procedures. We believe that the present experiments represent a step in that direction.

Using unfertilized mouse eggs as a model for a cell type that has been historically difficult to cryopreserve, we have gained insight into the often-overlooked phenomena of solution effects that are associated with cellular cryopreservation and have revealed the beneficial action of reducing the sodium ion concentration in the freezing medium. In light of our recent finding, it is interesting that other cells, including embryos, survive cryopreservation in a sodium-based medium as well as they do. This is not to say that sodium ions are not, in some way, detrimental to embryo freezing. On the contrary, sodium loading will most likely occur during embryo freezing as well, but embryos can apparently handle this stress much better than can oocytes. Therefore, alterations in cryopreservation media, such as reducing or eliminating sodium, may allow mouse oocytes to be easily and reproducibly frozen and may also have far-reaching applications for the storage of oocytes from other species, as well as other cells including embryos, sperm, erythrocytes, and tissues. It is difficult to determine whether the use of choline, the removal of sodium, or the combination of these had the greatest impact on improving egg freezing. We hypothesize that the removal of sodium is of primary importance and that choline worked as an excellent substitute.

	ACKNOWLEDGMENTS

 
We thank Giles Tomkin for his critical review of the manuscript. We also thank Dr. D.E. Walters for his comments and statistical analysis of the data.

	FOOTNOTES

 
¹ Correspondence: James J. Stachecki, 101 Old Short Hills Road, Suite 501, West Orange, NJ 07052. FAX: (973) 243–6235; james.stachecki{at}embryos.net

Accepted: March 31, 1998.

Received: December 2, 1997.

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