Modifying oocytes and embryos to improve their cryopreservation
Introduction
A number of procedures have been developed to cryopreserve bovine embryos collected from donors 6–8 days after estrus with quite good success; in most cases, with reasonable management, pregnancy rates of thawed embryos are 80–90% of those with uncryopreseved controls [1]. Similarly successful cryopreservation occurs with in vivo-produced embryos at similar stages from other species of ruminants. However, in vitro-produced ruminant embryos are much more difficult to cryopreserve successfully, and in vivo-produced embryos from some breeds survive cryopreservation less successfully than those of other breeds [2].
Bovine oocytes are particularly difficult to cryopreserve successfully, and result in low rates of blastocyst production after thawing, in vitro fertilization and culture of resulting embryos [3], [4]. This may be in part due to the large size of oocytes, with consequent low surface to volume ratios, making it more difficult for water and cryoprotectants to move across cell plasma membranes [5].
The most common approach to deal with the lower success rates of cryopreservation of certain kinds of cells is to modify cryopreservation procedures [5], for example, by varying concentration and types of cryoprotectants, studying different times and temperatures of procedures, or using additives, such as sugars or surfactants. While this approach to optimizing procedures usually results in improvements, they often are quite limited. An alternative approach, modifying the cells themselves to make them more cryopreservable is the subject of this review. Information from a variety of sources and cells types that bear on this approach will be examined, including some recent work on modifying oocytes and embryos.
Cells undergo considerable physical stress during cryopreservation procedures; some kinds of cells tolerate these stresses better than others [5], and modifications to cell membranes can result in changing tolerance of such stress. One major stress is osmotic in nature; cells and cellular compartments undergo huge changes in volume due to movements of water and intracellular cryoprotectants [5]. Thus, cells with more flexible membranes will usually have less membrane damage than those with more rigid membranes. Also, there will be less osmotic stress if permeabilities of cell membranes to water and cryoprotectants are high. Such cellular properties are highly dependent on membrane composition.
The major components of cell membranes are phospholipids, cholesterol, other lipids and proteins. Except for the proteins, these components can be manipulated fairly readily in a variety of ways including nutrition of donor animals and composition of culture media. One other relevant property of cell membranes is that for normal function, they need to be in a relatively fluid, sol state rather than a gel state. Certain regions of some cell membranes readily change fluidity to gel at decreased temperatures [6]. Often the changes that occur in this phase transition are not reversible, and upon warming, the cellular components are not reassembled correctly [7]. It would seem to be best to eliminate the fluid to gel transition completely during cooling; other strategies include having the transition occur at lower temperatures or to speed the transition between these states with rapid cooling rates, to limit the opportunity for damaging changes to occur. As with osmotic properties, the tendency for phase changes in membranes during cooling is highly dependent on membrane composition [8].
One clue as to how cells might be changed specifically is the relationship between the ratio of cholesterol to phospholipids in cell membranes, and the success of cryopreservation of sperm from species to species [9]. Low ratios, such as those found in boars and stallions are associated with less successful cryopreservation, while high ratios in men and bulls result in more successful cryopreservation of sperm. Whether genetic differences in freezability of sperm from male to male within mammalian species, or breed differences in freezability of blastocysts have a similar basis is unknown [2], although such a relationship has been clearly demonstrated in poultry sperm [10]. Other differences in lipid content or lipid composition of the cells and/or cell membranes also are likely to be important. Studies demonstrating more successful cryopreservation after microsurgical delipidation of porcine embryos [11] provide clear evidence of the benefit of reducing cytoplasmic lipid content.
There also are several studies on changing the cryotolerance of cells by manipulating the diet of the donor animal [12], [13], or culturing the cells for a variable period (from minutes to days) in media that change freezability of cells. This latter approach is particularly attractive because one can control the process to improve cryopreservation success.
Changing the chemical composition of cells and cell membranes can be accomplished acutely by modifying the composition of cryopreservation media by adding egg yolk and extracellular cryoprotectants [13]. Additives, such as egg yolk, sugars, or serum albumin are beneficial for cryoprotection [14], and the mechanisms for these benefits have been elucidated in some cases [15]; however, it is not even entirely clear how most cryoprotectants, intracellular or extracellular, exert their beneficial effects. While the study of additives to cryopreservation media has been extremely important and productive, this review will concern modification of cells prior to the process of cryopreservation. I will concentrate on oocytes and embryos, but will include information on other cell types when instructive.
Section snippets
Thought-provoking observations
Anecdotal observations frequently provide a basis for formulating hypotheses to test, particularly when large effects are observed. For example, dismal success rates for cryopreserving porcine embryos at certain stages characterized by high lipid content prompted development of various approaches to circumvent the high lipid content, such as delipidation or delaying cryopreservation until the expanded blastocyst stage [11], [16]. Jersey embryos result in lower pregnancy rates after
Information from experiments with sperm
Recent studies in which addition of selected phospholipids [23] as well as cholesterol [24] to sperm cell membranes improved freezability have been encouraging. While added cholesterol decreases membrane fluidity at ambient temperatures, added cholesterol increases membrane fluidity at lower temperatures [8]. Cholesterol appears to accomplish this effect by modifying how membrane phospholipids interact with each other, making membranes less contracted at lower temperatures. Added cholesterol
Oocytes
Horvath and Seidel [4] added cholesterol to bovine oocytes in an attempt to increase success rates for vitrification. They found that it is quite important to have the correct conditions for transferring cholesterol to cell membranes as was also true for sperm [24]. Removing serum albumin (fatty acid-free) from the medium during transfer of cholesterol to oocytes improved success rates, likely because cholesterol was preferentially transferred to the fatty acid-free albumin if present. Horvath
In vitro-produced embryos
Culture of embryos in vitro results in fundamentally different embryos from those produced in vivo, particularly for ruminants [27]. In general, the longer the in vitro culture, the more deviation occurs relative to embryos recovered in vivo; this phenomenon appears to be exacerbated when in vitro oocyte maturation and in vitro fertilization procedures are added on to embryo culture procedures [19].
One of the clearest demonstrations that different culture conditions can result in embryos with
Future studies
In most cases, modifying cryopreservation methods to fit the cells to be cryopreserved is likely to be preferable to modifying cells to fit procedures for cryopreservation. Nevertheless, there are opportunities to do the latter, and such modifications (for example, removing serum from media) may also result in improved success of oocyte maturation and culture of embryos and perhaps even in healthier offspring [28]. Various studies with reproductive and non-reproductive cells provide numerous
Acknowledgements
Several students and colleagues assisted with preparation of this review. Related research was supported in part by National Research Initiative Grant No. 2003-35203-13705 from the USDA CSREES and by the Colorado State University Experiment Station via USDA Regional Project W-171.
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