ILAR Journal Online, Volume 41(4) 2000: Cryobiology of Embryos, Germ Cells, and Ovaries

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ILAR Journal V41(4) 2000
Cryobiology of Embryos, Germ Cells, and Ovaries

Cryopreservation of Murine Spermatozoa

J. K. Critser and L. E. Mobraaten

J. K. Critser, Ph.D., is Director of the Cryobiology Research Institute and Professor in the Departments of Pediatrics and Immunology and Microbiology, Indiana University Medical School, Indianapolis, Indiana. L.E. Mobraaten, Ph.D., is a Senior Staff Scientist at The Jackson Laboratory, Bar Harbor, Maine.

Abstract

Cryopreservation of mouse sperm provides an economic option for preserving the large number of mouse strains now being generated by transgenic and targeted mutation methodologies. The ability of a spermatozoan cell to survive cryobiological preservation depends on general biophysical constraints that apply to all cells, such as the avoidance or minimization of the formation of intracellular ice during cooling. This action is typically achieved by use of cryoprotectant substances and by controlled, slow rates of cooling. Superimposed on those general constraints may be special characteristics of mouse spermatozoa, such as more narrow, osmotically driven volume tolerance limits and the fact that relatively successful freezing can be obtained without the use of a permeating cryoprotective agent. The lack of important information regarding sperm cells' fundamental cryobiological properties, including their osmotic and membrane permeability characteristics, has hindered progress in developing anything but empirically derived methods. Genetic differences between inbred mouse strains are reflected in motility and fertility characteristics of mouse sperm and contribute to the difficulty of developing successful cryopreservation methods. Recovery of live young from frozen sperm has been much more successful with sperm from hybrid mice than from most inbred strains. There have been no published reports of successful cryopreservation of rat sperm. Nevertheless, in mice, success in deriving live young from intracytoplasmic sperm injection using sperm frozen under suboptimal conditions raises the possibility of using this technique for the "ultimate rescue" of sperm regardless of the success of cryopreservation. This technique, however, requires additional development and verification of its efficacy before it will be suitable for general laboratory use. Although cryopreservation of mouse sperm is not yet universally successful, it can be used reliably to supplement cryopreservation of embryos and other germline cells or tissues for preserving biomedically important strains of mice for research.

Keywords: cryopreservation; intracellular water; intracytoplasmic sperm injection (ICSI); membrane permeability; mouse; osmotic tolerance; rat; spermatozoa

Importance of Mouse and Rat Sperm Preservation

It is now clear that revolutionary advances in genome research and the ability to create genetically specific strains of mice are resulting in an exponential increase in the number of strains available for biomedical research (Sharp and Mobraaten 1997). Actual and projected numbers of publications based on transgenic or targeted mutant mice since 1985 are shown in Figure 1. Although much smaller in magnitude, a similar trend appears in the literature for publications based on transgenic rats (Figure 2). Freezing and storing sperm could most efficiently preserve the majority of these newly created strains wherein the strain or mutation of interest can be adequately preserved by haploid germplasm (Mobraaten 1998). The basis for the economy of sperm cryopreservation is best demonstrated by comparing the average live-offspring recovery from female embryo donors and male sperm donors, respectively. From the average number of embryos collected and frozen from a single female of the widely used inbred strain C57BL/6J, an average of four live births will result (Sharp and Mobraaten 1997). This result can be contrasted to the several thousand offspring that can be obtained theoretically from the sperm of just a single male (Thornton et al. 1999).

Sperm collection is relatively simple, does not require gonadotropin administration or mating before collection, and requires only a few animals. It is therefore not necessary to establish a large breeding colony to provide germplasm donors, as is required for embryo cryopreservation. For these reasons, cryopreservation of spermatozoa offers an economic alternative to the cryopreservation of embryos, especially for strains such as transgenics and induced mutants, including those produced by chemicals, homologous recombination, or the disruption of gene function (e.g., knockouts). Cryopreservation of sperm for these strains is especially efficacious because thawed sperm can be used to fertilize oocytes from an inbred or hybrid strain using in vitro fertilization and subsequent embryo transfer to produce founder animals for a breeding colony. Transgene or mutant carriers can be identified among the progeny by relatively efficient genotyping methods, and homozygotes can then be produced, if desired, by mating two heterozygous carriers.

Fundamental Cryobiology of Mammalian Spermatozoa

The ability of a cell to survive cryobiological preservation depends on two classes of factors. First, there is a series of biophysical constraints that apply to all cells. Second, superimposed on those constraints may be special characteristics of the cell type in question. In this section, we briefly outline the general biophysical constraints.

The first general physical constraint is to avoid or minimize the formation of intracellular ice when cooling the cells to low subzero temperatures. This cooling can be accomplished in two ways. The usual approach depends on the fact that above certain temperatures, cell water remains unfrozen and supercooled even in the presence of external ice. Because supercooled water has a greater chemical potential than ice, the result is the establishment of a driving force for the efflux of cell water. Success with this approach requires cooling cells slowly enough that the freezable water within them has time to flow out and freeze extracellularly before they cool to a temperature (nucleation temperature) at which intracellular water can no longer remain supercooled. The numerical definition of "slowly enough" depends on the following fundamental properties of the cell: its permeability to water (L_p¹), the activation energy (E_a¹) of that permeability, and the surface area to volume ratio of the cell (S/V¹). It also depends on the nucleation temperature. In general, the higher the L_p, the lower the E_a, the higher the S/V; and the lower the nucleation temperature, the faster the cooling can be without inducing intracellular ice. Depending on the values of these four variables, critical cooling rates can vary 1000-fold from <1°C/min for mouse embryos to 1000°C/min for human red blood cells (Mazur 1984).

Another, opposite approach is to avoid intracellular ice formation. It involves cooling cells at high enough rates to form an intracellular glass, rather than ice, and to warm them rapidly enough so that the vitrified cytoplasm remains vitreous during warming. If warming is not sufficiently rapid, the vitrified cytoplasmic solution will crystallize or devitrify. In the absence of added solutes, the cooling rates required to induce vitrification and the warming rates required to prevent devitrification are unattainable for the relatively large volumes that are required for cells. However, the required rates become attainable if high enough concentrations of certain glass-inducing solutes are introduced. Generally, the concentrations must be >5 M, and such concentrations are close to or exceed the tolerance limits of most cells. Nevertheless, some cell systems have been successfully vitrified, most notably mouse embryos (Rall and Fahy 1985) and Drosophila embryos (Mazur et al. 1992; Steponkus et al. 1990).

Most sperm cryopreservation approaches currently used are in the "slow" or "equilibrium" category. Although this approach requires cooling cells at rates less than critical, this requirement by itself is generally not sufficient for success. For most cell types, success also requires two other ingredients: (1) the presence of permeating cryoprotective solutes, the most common of which are glycerol, dimethylsulfoxide (DMSO¹), and ethylene glycol, usually in -1 M concentrations; and (2) avoidance of a cooling rate that is too low because it will translate into long-damaging exposure times to the concentrating external solution---damage often referred to as solution effects (Gao and Critser 2000). The result of these two opposing cooling rate-dependent factors is the existence of an optimum cooling rate, usually a rate that is high enough to minimize solution effects but low enough to preclude intracellular freezing. The numerical value of that optimum for a given cell type depends on sometimes complex interactions between the type and concentration of cryoprotective solute and the water permeability characteristics of the cell that determine susceptibility to intracellular freezing. Generally, the greater the concentration of cryoprotectant the cell will tolerate, the less susceptible it is to the slow freezing solution effects. One problem with low tolerance is that a cooling rate high enough to minimize solution effects is also high enough to induce some intracellular ice. Consequently, even at the optimum rate, survival is less than 100%, which may explain the mediocre survivals obtained even with bovine and human sperm.

The use of permeating cryoprotectants introduces another complication, in that cells undergo osmotic shrinkage during their introduction and osmotic volume excursions during their removal. The magnitude of the volume excursions depends primarily on the permeability of the cell to the cryoprotectant and secondarily on the time over which the cryoprotectant is added and removed. Excursions that are too extensive will damage the cell. The definition of "too extensive'' depends on the susceptibility of the particular cell type, which is partly determined by its S/V and the degree of excess plasma membrane it possesses in the form of microvilli and infolding.

If one knows the fundamental permeability properties of the cell in question, its S/V, and its tolerance to osmotic volume excursions, one can compute the critical cooling rate for intracellular freezing and design protocols for the addition and removal of cryoprotectant that will minimize osmotic damage. Mazur (1984) has described examples of estimating critical cooling rates. Critser and colleagues have published examples of development of methods to add and remove cryoprotectant that minimize cell damage (Gao et al. 1995; Gilmore et al. 1997, 1998; Phelps et al. 1999). This general approach is now being extended to explore the use of other, nonglycerol cryoprotectant agents (CPAs¹) as well as optimization of cooling and warming rates in boar sperm (Gilmore et al. 1997, 1998). Although results have revealed that both murine and boar spermatozoa have exquisitely narrow osmotically driven volume tolerance limits (Figure 3), Gilmore and colleagues (1999) have also demonstrated that extender components (e.g., egg yolk and skim milk in the case of the mouse) will significantly expand those limits (Figure 4).

Fate of Intracellular Water during Freezing

The chief physical events occurring in cells during freezing are depicted schematically in Figure 4 (Mazur 1984). At a temperature as low as -5°C, the cells and the surrounding medium remain unfrozen both because of supercooling and because of the depression of the freezing point by the protective solutes that are frequently present. Between -5° and approximately -15°C, ice forms in the external medium (either spontaneously or as a result of seeding the solution with an ice crystal). However, the cell contents remain unfrozen and supercooled, presumably because the plasma membrane blocks the growth of ice crystals into the cytoplasm. The supercooled water in the cells has, by definition, a greater chemical potential than that of water in the partly frozen solution outside the cell. In response to this difference in potential, water flows out of the cell osmotically and freezes externally. The subsequent physical events in the cell depend on cooling velocity. If cooling is sufficiently slow (Figure 5, top right), the cell is able to lose water rapidly enough by exosmosis to concentrate the intracellular solutes sufficiently to eliminate supercooling and maintain the chemical potential of intracellular water in equilibrium with that of extracellular water. The result is that the cell dehydrates and does not freeze intracellularly. However, if the cell is cooled too rapidly (Figure 5, center right), it is not able to lose water fast enough to maintain equilibrium; it becomes increasingly supercooled and eventually attains equilibrium by freezing intracellularly. These statements were first expressed quantitatively by Mazur (1963).

The rate of exosmosis of water during freezing may be described by four simultaneous equations. The solution to these equations is usually expressed as a family of curves of the water content of a cell at different cooling rates as a function of temperature. In cells that can survive freezing, the usual finding is that plots of the percentage of survival versus cooling rate form inverted "U"s, as exemplified in Figure 6. Survival is maximal at some critical rate, but the numerical value of that critical rate can vary 1000-fold in different cell types. The reduced survival at supraoptimal rates results from intracellular freezing. The calculated dehydration curves can be used to predict the highest cooling rate that is compatible with no intracellular freezing and, therefore, survival. With few exceptions, a high rate of survival demands that the cooling rate be sufficiently low to avoid or minimize such internal freezing. Although rates low enough to prevent extensive internal freezing are necessary for a high rate of survival, they are not sufficient because cooling at suboptimal rates can also be very injurious (Figure 6).

Nature and Elimination of Slow Freezing Injury

In most cells, 1 to 2 molar concentrations of CPA are required for maximal protection, much greater concentrations than are commonly used in the freezing of sperm. In addition, indications are that CPAs must permeate most cell types to protect them from slow freezing injury (Souzu and Mazur 1978). The cause of slow freezing injury and the basis of the protection by solutes like glycerol and DMSO are the subjects of continuing debate. During freezing, cells are sequestered in unfrozen channels between the growing ice crystals. The ice crystals grow by pulling pure water from these channels. Consequently, the solute concentration in the channels increases, and the channels progressively shrink in size. The increase in solute concentration causes the cells to progressively shrink osmotically, as depicted in the bottom right branch of Figure 5. Cryobiologists have ascribed slow freezing injury to the increase in solute (electrolyte) concentrations, the consequent cell shrinkage, or the diminishing water space.

Warming and Thawing

A cell that has avoided lethal damage during cooling to low subzero temperatures must still survive the series of physical-chemical challenges associated with warming. The rate of warming can exert effects on survival comparable with those of the rate of cooling. The effects depend on whether the prior rate of cooling has induced intracellular freezing or dehydration. In the former case, if the cells are not killed outright, the ice crystals tend to be small. Small crystals fuse to form larger crystals during warming if the rate of warming is low enough. This process, called recrystallization, is often damaging. If cells are cooled slowly enough to preclude intracellular freezing, the response to warming rate is extremely variable. In some cells, the warming rate makes little or no difference; but in others, rapid warming appears mandatory (Mazur 1990).

Need for Developing a More Complete Understanding of Murine Sperm Cryobiology

There are several aspects of current methods used to cryopreserve mammalian spermatozoa in general and mouse sperm in particular that are in apparent contrast to approaches to cryopreserve other cell types developed based on basic principles of fundamental cryobiology. These differences point to various areas where improvements might be anticipated if a more complete understanding of murine sperm cryobiology were available. For example, permeating CPAs such as glycerol, ethylene glycol, or 1-2, propanediol at concentrations between 1 and 2 molar are typically used in procedures to cryopreserve most other cells types. However, with mammalian sperm such as human, boar, and bull, concentrations of these CPAs are most often far less than 1 molar. More strikingly, often no permeating CPA at all is used with mouse sperm. This difference between empirically derived methods to cryopreserve mammalian sperm and other cell types is likely due to a lack of important information regarding the sperm cell's fundamental cryobiological properties including their osmotic and membrane permeability characteristics.

For all species studied to date, mammalian sperm have clearly defined and often very limited ability to withstand changes in cell volume (either decreases or increases), which occur as the cells are exposed to the various changes in the chemical composition of the surrounding solution. These osmotically driven changes in cell volume are fairly complex to predict in the absence of an overall understanding of several basic biophysical properties, described below. It is important to note that the vast majority of the work on sperm freezing over the more than 50 yr since Polge and Lovelock (1952), Polge and Rowson (1952), and Polge and others (1949) first reported success with poultry and later bull sperm has focused not on true cryobiological factors, but rather on the suspending media composition and media effects.

As far behind as the field of cryobiology of spermatozoa is in general, it is lagging much farther behind in terms of mouse sperm, and it is essentially nonexistent in terms of rat sperm. This situation must be remedied for the laboratory animal research community to be able to realize the enormous benefits germplasm preservation can provide and that are so badly needed to manage the exploding numbers of newly created rodent models.

Characteristics of Cryobiology

Species Differences

Although sperm from a large number of mammalian species have been successfully frozen and thawed, there are distinct differences in the degree of success with particular species. How rodent sperm differ from other mammalian sperm has been attributed largely to differences in sperm membrane lipid content or composition (Darin-Bennet and White 1977; Hammerstedt et al. 1990; Parks and Graham 1992; Parks and Lynch 1992). Mouse spermatozoa are especially fragile (Katkov and Mazur 1998). Furthermore, in mouse sperm, the cytoskeleton anchors the plasma membrane to the cell's internal structure, creating additional strains on the membrane under osmotic stress (Noiles et al. 1997).

Genetic Background Differences

Success of sperm cryopreservation may be measured in different ways and may include sperm viability, sperm motility, sperm fertility in in vitro fertilization, and ultimately live births resulting from fertilization with frozen/thawed sperm. There is not always a linear correlation among these criteria. For example, the total number of motile sperm can be reduced drastically after freezing and thawing, but a very high degree of fertilization (>90%) can be achieved from this small number (Sztein et al. 1997). Even more extreme is the example of live born young that have been produced using intracytoplasmic sperm injection (ICSI¹), from nonviable sperm and even freeze-dried sperm (Wakayama and Yanagimachi 1998; Wakayama et al. 1998). In the case of spermatozoa, therefore, technology such as ICSI can overcome limitations in cryobiology. However, most laboratories are not in a position to perform ICSI, which is technically difficult to perform and expensive to establish. For this reason, continued focus on optimizing cryopreservation methods that retain full functional integrity remains the focus of current research and development.

The relation between cryobiological characteristics and fertilization methodology in reaching the final product of live births must be considered in evaluating the success of a sperm cryopreservation method. This relation must also be considered in attempting to ascribe genetic background differences to the success of sperm cryopreservation. Because in vitro fertilization rates differ between strains of mice (Carey and Olds-Clark 1980; Fraser and Drury 1976; Kaleta 1977; Krzanowska 1970; McLaren and Bowman 1973; Niwa et al. 1980; Parkening and Chang 1976; Whitten and Dagg 1961), outcomes of cryopreservation of spermatozoa from inbred mice as measured by fertility in vitro do not necessarily reflect genetic differences in cryobiological characteristics. Characteristics of spermatozoa populations from inbred strains of mice have been shown to differ in terms of cell concentration and percentage of motility (Krzanowska et al. 1995; Williams et al. 1970); however, minimal data have been published to demonstrate strict cryobiological differences between sperm from inbred strains in their susceptibility to freezing. Tada et al. (1990) reported motility and in vitro fertilization results for sperm frozen in a 1.75% glycerol and 18% raffinose solution from several strains of mice, and although each showed significant differences from unfrozen controls, tests of significance between strains were not carried out. However, from the data presented, it was clear that there were differences in fertilizing ability after freezing and thawing of sperm from different strains, regardless of whether the sperm were used to fertilize eggs from the same strain as the sperm or eggs from a B6C3F1 hybrid. Differences between strains in motility after freezing and thawing were not evident.

Songsasen and Leibo (1997a) observed significant in vitro fertilization differences of B6C3F1 oocytes fertilized by frozen/thawed sperm from mice of three genetically diverse genetic backgrounds, B6D2F1 hybrids, C57BL/6J, and 129/J. Although B6D2F1 frozen sperm resulted in good fertilization (61%), as would be expected for a hybrid, C57BL/6J and 129/J frozen sperm resulted in only 3% and 17%, respectively. This pattern is similar to the case of mouse embryos, wherein strain background differences in response to freezing have been observed (Dinnyes et al. 1995; Pomp and Eisen 1990; Schmidt et al. 1985, 1987).

Murine Spermatozoa

Mouse

The first known live born mice derived from frozen sperm were obtained by G. L. Rappatz, who described this result in an unpublished and undated, but circulated, final progress report that was mentioned in a 1978 review (Graham et al. 1978). Cryopreservation of mouse sperm neither drew much attention nor was it very successful in the next decade (Sherman and Liu 1982).

At a workshop on embryo freezing held at The Jackson Laboratory in 1988, M. Yokoyama and coworkers from Japan reported successful attempts to freeze mouse sperm (Yokoyama et al. 1990). In the same year, two other reports were published (Okuyama et al. 1990; Tada et al. 1990). The Yokoyama and Tada groups used cryoprotectant solutions containing glycerol or DMSO with raffinose, whereas Okuyama used a solution of 18% raffinose in 3% skim milk as a cryoprotectant.

The Yokoyama and Tada groups examined the effects of different concentrations of raffinose and glycerol on sperm cryopreservation, and Tada and coworkers further tested survival of frozen sperm from several different strains of mice. Using 5% glycerol in 10% raffinose, Yokoyama and colleagues achieved an average of 37% in vitro fertilization with frozen and thawed B6CF1 sperm. Having used 1.75% glycerol in 18% raffinose, Tada and coworkers reported 13 to 64%, depending on strain. In a subsequent report using postthaw centrifugation to remove the cryoprotectant solution, Tada et al. (1993) describe enhanced fertilization rates of 53 to 85% and survival from two-cell to fetal day 18 rates of 41 to 68% for frozen sperm from several strains of mice (ICR, C3H/HeN, BALB/c, C57BL/6N, and SAM-P/6). One laboratory reported unsuccessful attempts to repeat these results (Penfold and Moore 1993).

Takeshima and colleagues (1991) reported successful results in freezing mouse sperm with the raffinose/skim milk solution described by Okuyama. Nakagata continued to publish a number of papers using this method (Nakagata 1992, 1993, 1994, 1995, 1996; Nakagata and Takeshima 1992, 1993; Nakagata et al. 1992, 1997). Takeshima et al. (1991) reported obtaining 34% two-cell embryos from in vitro fertilization using thawed sperm, and 45% of those two-cell embryos resulted in live births after transfer.

At the annual meeting of the Society for Cryobiology held in Leuven, Belgium, June 1991, Linda Penfold, Harry Moore, and Bill Holt of the Zoological Institute in London first reported successful freezing of mouse sperm by using a cryoprotectant solution supernatant that included centrifuged, sodium dodecyl sulfate-treated egg yolk (Penfold et al. 1991). The egg yolk supernatant was used with glycerol in a modified TES/TRIS cryoprotectant with which they obtained 59% in vitro fertilization but reported no live births. Their work was fully published in 1993, when they reported that 17% of two-cell embryos derived from in vitro culture using frozen/ thawed sperm were recovered as live fetuses on day 15 of pregnancy (Penfold and Moore 1993).

More recently, variations of the methods described above have been published. Various aspects of sperm freezing using a glycerol/egg yolk cryoprotectant to optimize the method have been studied (Songsasen and Leibo 1997a,b, 1998; Songsasen et al. 1997). They found that seeding the sample before freezing had a positive effect on the fertility of thawed sperm and that mouse spermatozoa tolerate osmolalities in the range of 200 to 400 mOsm but are damaged by osmolalities exceeding this range. Sztein et al. (1997) have shown that greater than 90% fertility can be obtained with sperm frozen from hybrid (B6D2F1) mice using a raffinose/ skim milk cryoprotectant when spermatozoa are collected at 37°C before freezing.

Osmotic tolerance limits. Other mammalian sperm (e.g., human and boar) have been shown to have a limited ability to reduce or increase cell volume (i.e., to shrink or to swell) and maintain structural and functional integrity (Gao et al. 1995; Gilmore et al. 1998). These data indicate that although the osmotic tolerance limits (95% maintenance) for plasma membrane integrity are relatively wide, the osmotic limits are extremely narrow for maintenance of motility. The effects of osmolality on sperm can be directly translated into effects related to the corresponding change in cell volume. Therefore, if the cryoprotectant permeability coefficients (P_CPAs¹) and corresponding membrane permeability values for water in the presence of CPA (L_p^CPA)¹ are known and coupled with knowledge of the osmotic tolerance limits, one can predict the following: (1) changes in cell volume that will occur when the cells are exposed to anisosmotic conditions, and (2) the conditions (methods) that will allow the cells to be maintained within their tolerance limits. This combination has been applied to human sperm to predict optimal approaches to add and remove glycerol (Gao et al. 1995). The general approach is now being extended to explore use of other, nonglycerol CPAs as well as optimization of cooling and warming.

Mechanical fragility. Mouse spermatozoa are exquisitely sensitive to mechanical stress. They can be rendered immotile by mechanical steps routinely used during sample preparation for low temperature storage such as pipetting and mixing (Schreuders et al. 1996). In addition, mouse sperm are very easily damaged by centrifugation. Therefore, the use of relatively low g forces and time must be used to avoid irreversible damage (Katkov and Mazur 1998, 1999). These unusual sensitivities to routine practices, which are normally considered innocuous, must be carefully considered when developing practical methods for cryopreservation of mouse spermatozoa.

Membrane permeability coefficients. The fundamental cryobiology of mouse spermatozoa has begun to be defined (Phelps et al. 1999; Willoughby et al. 1996). In Table 1 are shown membrane permeabilities for water in the presence of CPA (L_p^CPA) and for CPA (P_CPA) for two different genetic backgrounds of mice (ICR and B6C3F1) as well as similar values for human and boar spermatozoa. As with all other mammalian species studies to date, mouse spermatozoa have a significantly reduced permeability to water when placed in a medium containing a permeable CPA (e.g., glycerol or ethylene glycol) (Phelps et al. 1999). In general, mouse spermatozoa in the presence of CPA have a lower L_p level than human, but a higher L_p level than boar (Table 1).

Determination of these membrane permeability coefficients will enable mathematical modeling of the cells' volumetric response to CPA addition and removal. This information, in combination with the osmotic tolerance limits (see above), will make it possible to design step-wise methods to ensure that the cell's volume does not decrease more than (during CPA addition) or exceed (during CPA removal) those limits. Shown in Figure 7, A and B, are examples of these theoretical additions for mouse ICR and B6C3F1 spermatozoa.

Rat

To date there appear to have been no published reports of successful cryopreservation of rat sperm. Unpublished data from one of our laboratories (J. K. C.) indicate that rat sperm cryopreserved using common methods developed for mouse sperm survive at rates similar to the mouse. In those preliminary experiments, outbred Sprague Dawley sperm were cryopreserved using a solution of sucrose (12.5% w/v) and skim milk (3% w/v) (SSM¹). Rat epididymides were placed directly into 0.5 ml of SSM, and sperm were allowed to swim out for 15 min. The SSM/sperm suspension was loaded into 0.5-ml plastic straws and placed into the neck of a liquid nitrogen tank for 2 min, plunged directly into liquid nitrogen thawed (40°C water bath), and washed. The resulting aver-' age postthaw motility was approximately 20%. These data suggest that application of methods commonly used for mouse sperm cryopreservation may result in similar survival rates for rat spermatozoa. However, current levels of sperm survival using current methods for cryopreservation are very low relative to other mammalian species. Therefore, continued attempts to improve these methods are of absolute importance.

Current Status of Mouse and Rat Sperm Cryopreservation

In spite of the several methods that have been reported to have moderate success in mouse sperm cryopreservation, they are still inadequate for safe preservation of most mouse strains. Published data reveal that both fertility and recovery levels of live offspring derived from frozen sperm are generally higher for hybrid or outbred animals. Current success of sperm cryopreservation for inbred strains of mice is extremely variable and therefore too unreliable as a means for preserving those strains on an inbred background. Considerable work is currently being carded out in several laboratories in attempts to develop more reliable methods of both freezing and fertilization of oocytes by thawed sperm. One approach to achieve more certain fertilization from frozen mouse sperm is the direct injection of a spermatozoon into oocytes. This approach is commonly used in human infertility clinics throughout the world, where it has proven effective; however, it has been extremely difficult to adapt to mice. Work emanating from Dr. Yanagimachi's laboratory at the University of Hawaii has shown that fertilization can be achieved with normal spermatozoa (Kimura and Yanagimachi 1995a) as well as from frozen and thawed nonviable spermatozoa (Wakayama et al. 1998). Even more remarkable was their achievement of obtaining live mice derived from freeze-dried sperm stored for up to 3 mo (Wakayama and Yanagimachi 1998) and from secondary spermatocytes (Kimura and Yanagimachi 1995b).

Future Directions for Murine Sperm Cryopreservation

The economic advantages of sperm preservation over other forms of germplasm preservation and the need to find methods for efficient management of the vast numbers of mouse and rat models being developed by genetic engineering methods will continue to be the driving force to develop more reliable sperm preservation methods. Although ICSI appears very promising, much work lies ahead before this approach can be used with confidence. Even if ICSI becomes routinely possible, it may prove too expensive and/or difficult to be applied practically. Successful development of freeze-dried storage of mouse sperm along with successful ICSI development would simplify storage requirements but might be limited by the economic and practical constraints of ICSI. Alternative strategies such as ovarian tissue cryopreservation (Gunasena et al. 1997a,b; Sztein et al. 1999) and cloning by nuclear transfer also must be considered as a means to recover biomedically important strains for research from the storage of tissue other than germplasm. In this regard, it may be most efficient in the near future to preserve a number of different cells and tissue to maximize the possible recovery of the strains. One such approach could include cryopreserving a combination of some embryos, sperm, and ovarian tissue, which could then be used in a number of different combinations to maximize recovery and minimize costs.

Acknowledgments

This work was supported by the Cryobiology Research Institute and by grants from the National Institutes of Health (R24-RR13195 and U42-RR14821 to J.K.C.; and P40-RR01262, U01-RR15012, and P30-CA34196 to L.E.M.).

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¹Abbreviations used in this article: CPA, cryoprotective agent; DMSO, dimethylsulfoxide; E_a, activation energy of permeability; ICSI, intracytoplasmic sperm injection; L_p, permeability to water; L_p^CPA, membrane permeability values for water in the presence of CPA; P_CPA, cryoprotectant permeability coefficient; SSM, solution of sucrose and skim milk; S/V, surface area to volume ratio of a cell.

Figure 1 Actual publications per year citing transgenic and targeted mutant mice through 1998. Values are extrapolated after 1998.

Figure 2 Literature trends of publications based on transgenic rats.

Figure 3 Estimated normalized percentage motilities of human, mouse, and boar sperm after exposure to anisomotic conditions and return to isosmotic condition. With permission from Gilmore JA, Liu J, Peter AT, Critser JK. 1998. The determination of plasma membrane characteristics of boar spermatozoa and their relevance to cryopreservation. Biol Reprod 58:28-36.

Figure 4 Comparison of normalized mouse sperm motilities after a 5-rain exposure to various hypo- and hyperosmotic NaC1, egg yolk, and skim milk solution and return to isosmotic conditions. With permission from Gilmore JA, Liu J, Critser JK. 1999. Osmotic tolerance limits of murine spermatozoa in the presence of extender media. Cryobiology 39:353-354.

Figure 5 Schematic of physical events in cells during freezing. Redrawn with permission from Mazur P. 1984. Freezing of living cells: Mechanisms and implications. Am J Physiol (Cell Physiol 16) 247:C125-C 142.

Figure 6 Survival versus cooling rate of five types of cells. Redrawn from Mazur P. 1976. Freezing and low temperature storage of living cells. In: Muhlbock O, ed. Proceedings of the 1974 Workshop on Basic Aspects of Freeze Preservation of Mouse Strains, The Jackson Laboratory, Bar Harbor ME. Stuttgart: Gustav Fisher Verlag. p 1-12.

Figure 7 Theoretical simulation of step-wise addition of 1 M glycerol (dashed/dotted line), ethylene glycol (solid line), and propylene glycol (dotted line) to spermatozoa from (A) ICR and (B) B6C3F1 mice at 22°C. With permission from Phelps MJ, Liu J, Benson J, Willoughby CE, Gilmore JA, Critser JK. 1999. Effects of Percoll separation, cryoprotective agents, and temperature on plasma membrane permeability characteristic s of murine spermatozoa and their relevance to cryopreservation. Biol Reprod 61: ! 031-1041.

Table 1 Comparison of membrane permeability coefficients and activation energies for mouse, human, and boar spermatozoa

Species	CPA_a	L_p^CPA^a (mm/min/atm)	E_AL_p^CPA^a (kcal/mol)	P_CPA^a x10^-3 (cm/min)	E_AP_CPA^a (kcal/mol)
Mouse^b
ICR^b	Glycerol Ethylene glycol	0.38 + 0.04 0.38 + 0.04	8.5 7.1	2.2 + 0.1 3.4-+ 0.5	10.7 9.2
B6C3F1^b	Glycerol Ethylene glycol	0.68 -+ 0.19 0.57 +- 0.10	14.3 14.9	2.4 + 0.2 1.9 -+ 0.5	8.2 13.2
Human^c	Glycerol Ethylene glycol	0.77 -+ 0.08 0.74 + 0.06	12.0 7.8	2.1 -+ 0.01 7.9 + 0.07	10.4 8.0
Boar^d	Glycerol Ethylene glycol	0.14 + 0.01 0.20 -+ 0.02	7.8 11.5	0.5 -+ 0.05 2.0 -+ 0.11	4.1 7.5

^aCPA, cryoprotective agent; L_p^CPA, membrane permeability coefficient for water in the presence of CPA; E_AL_p^CPA, activation energy for L_p^CPA; P_CPA, membrane permeability coefficient for the CPA; E_AP_CPA, activation energy for P_CPA.
^bData from Phelps M J, Liu J, Benson J, Willoughby CE, Gilmore JA, Critser JK. 1999. Effects of Percoll separation, cryoprotective agents, and temperature on plasma membrane permeability characteristics of murine spermatozoa and their relevance to cryopreservation. Biol Reprod 61:1031-1041.
^cData from Gilmore JA, McGann LE, Liu J, Gao DY, Peter AT, Kleinhans FW, Critser JK. 1995. Effect of cryoprotectant solutes on water permeability of human spermatozoa. Biol Reprod 53:985-995; and Gilmore JA, Liu J, Woods E J, Peter AT, Critser JK. 2000. Cryoprotective agent and temperature effects on human sperm membrane permeabilities: Convergence of theoretical and empirical approaches for optimal cryopreservation methods. Hum Reprod 15:335-343.
^dData from Gilmore JA, Liu J, Peter AT, Critser JK. 1998. The determination of plasma membrane characteristics of boar spermatozoa and their relevance to cryopreservation. Biol Reprod 58:28-36.