| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Regular Article |
a Fundamental and Applied Cryobiology Group, Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, Tennessee 37932-2575
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
assisted reproductive technology, in vitro fertilization, male reproductive tract, sperm, sperm motility and transport
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Many of the media used for freezing mouse sperm contain high concentrations of the trisaccharide raffinose (commonly 18%) and low concentrations of the permeating cryoprotectant glycerol. In a recent study [8], we examined the survival of mouse sperm frozen to -70°C at approximately 20°C/min in various concentrations of raffinose, various concentrations of glycerol, various strengths of supplemented Dulbecco phosphate-buffered saline (SD-PBS) used to prepare the solutions, and in the presence and absence of Oxyrase. In that study, a minimum temperature of -70°C was selected, because most cryobiologically relevant events have occurred by that temperature and because we wished to study the effect of these events without introducing other possible sources of damage from an additional 130°C cooling with liquid nitrogen (LN2). A cooling rate of 
20°C/min was selected, both because it is commonly used and because evidence exists in both mouse sperm and sperm of other species that cooling rates approximately 10-fold below or above this rate are damaging [4, 9–13]. The best motilities (nearly 70% normalized to untreated controls) were obtained with 18% raffinose, 0% glycerol, and 3.8% Oxyrase in 0.25x SD-PBS. The 0.25x SD-PBS was efficacious because it kept the total osmolality of the raffinose/PBS combination from rising to damaging levels to mouse spermatozoa. In the present study, using this optimum set of conditions, we examined the effect of the cooling rate to -70°C, the effect of the warming rate from -70°C, and the effect of further cooling from -70 to -196°C. The third item was important, because sperm in banks are almost always maintained at -196°C. The finding in the previous study that the best survivals occurred in the complete absence of glycerol was surprising. Because the detrimental effect of glycerol addition had been defined only at single cooling rate, we also examined the dependence of survival on the cooling rate of samples suspended in a glycerol-containing medium.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Preparation of Sperm Suspension
The sperm suspensions were prepared as detailed by Koshimoto et al. [8] with some minor modifications. Male ICR mice (Sprague-Dawley, Inc., Harlem, IN), 15–25 wk old, were killed by asphyxiation with CO2 gas. Sample sperm were collected from each of the two cauda epididymides and vas deferentia and were suspended in 750 µl of 0.5x SD-PBS [14], which was supplemented with 7.5% (w/v) raffinose pentahydrate to maintain the total osmolality of the collection medium at 290 mOsm (isotonic). The sperm suspension was separated from most of the minced tissues by filtration through a nylon strainer (catalog no. 2350; Falcon, Becton-Dickinson Labware, Franklin Lakes, NJ) into a 250-µl drop of medium, resulting in approximately 850 µl of filtrate. Two or three 250-µl aliquots of that filtrate were transferred by a 1-ml syringe to individual tubes. To each tube, 250 µl of cryoprotective stock solution (CSS) were added in three steps to achieve a 1:1 (w/v) dilution.
The rationale for the stepwise dilution (25-, 60-, and 165-µl volumes at 15-sec intervals [8, 15]) was to avoid excessive and possibly damaging osmotic dehydration of the sperm during addition of the hyperosmotic permeating solute glycerol. When we are dealing with solutions that contain only (nonpermeating) raffinose and no glycerol as cryoprotective additives (CPA), that rationale disappears, because the extent of shrinkage is independent of the number of steps. However, each addition adds to the possibility of mechanical damage. To minimize such mechanical damage, we restricted the mixing by inversion until after the last addition, assuming that some diffusional and convective mixing would have occured with each previous addition. Even though the need for stepwise addition was not present in the absence of glycerol, we wanted to keep the mechanical aspects associated with stepwise addition the same for both glycerol-containing and non-glycerol media. The same considerations applied to the stepwise dilution after thawing.
The resulting suspension was then gently mixed twice by inversion and immediately loaded into straws. The remainder of the filtrate was used for controls. The basic solvent for CSS was 0.005 M phosphate buffer containing 0.5% BSA (15 mOsm, pH 7.2). To that, 29.5% (w/v) raffinose was added as a cryoprotectant. After mixing with the sperm suspension, this produced a 400 mOsm medium containing 18% (w/v) raffinose in 0.25x SD-PBS. In other experiments, a second CSS that contained glycerol was prepared with 29.5% raffinose and 1.6 M glycerol in the same 0.005 M phosphate buffer. In this case, the final suspension after 1:1 mixing with the sperm suspension resulted in 18% (w/v) raffinose and 0.8 M glycerol in 0.25x SD-PBS. The concentration of sperm at this point was approximately 6 x 10-6 sperm/ml.
To load the straws, approximately 10 µl of SD-PBS was drawn into 0.25-ml plastic straws (catalog no. AAA201; IMV, l'Aigle, France) to wet the resin at the end. This was followed by an approximately 1-cm air bubble and then 100 µl of the sperm suspension. The other end of the straw was heat-sealed. Freezing was initiated within 5 min after mixing to minimize a possibly negative effect of the relatively high osmotic strength of the medium and the high concentration of glycerol (when present).
Achieving Various Cooling Rates to -70°C
Sperm suspensions in the straws were cooled to -70°C at seven different cooling rates achieved by the procedures summarized in Table 1. The measured mean cooling rates from -10 to -65°C with these procedures in repeated runs were 0.29 ± 0.02, 2.0 ± 0.06, 27.0 ± 1.2, 44.0 ± 4.5, 130 ± 5.8, 261 ± 19, and 530 ± 41°C/min. These cooling rates were monitored in each freezing run by a dummy straw containing 100 µl of the suspending medium into which a 36-gauge, copper-constantan thermocouple attached to a Fluke 2190-A Digital Thermometer (John Fluke Manufacturing Co., Seattle, WA) was inserted. For the four lowest cooling rates, the sample and dummy straws were inserted coaxially into Pyrex test tubes (length, 125 mm; outer diameter, 8 mm), capped with a perforated rubber seal, and mounted on a plastic holder during cooling. To achieve a cooling rate of 130°C/min, the sample and dummy 0.25-ml straws were slightly reduced in length and inserted within 0.5-ml-larger straws (catalog no. AAA101; IMV) with the cotton plugs and resin removed. The larger straws containing the sample straws were then heat-sealed at both ends. The larger straw containing the small thermocouple dummy straw was heat-sealed at one end. To achieve the two highest cooling rates, the 0.25-ml naked sample straws were frozen by direct immersion into ethanol baths precooled to -42 and -70°C (Table 1).
|
Achieving Various Warming Rates from -70°C
Sample straws in all freezing procedures were transferred into a Dry-Ice ethanol bath at less than -70°C, held for more than 5 min, and then thawed at three different rates. These procedures, which produced mean warming rates from repeated runs of 11.5, 126, and 1875°C/min (between -70 and -15°C), are summarized in Table 2. The thawed sample suspensions were then expelled into 5-ml Falcon no. 2054 round-bottom tubes, immediately diluted 15-fold with SD-PBS in five steps (46, 81, 168, 515, and 590 µl at 15- to 30-sec intervals), and then gently mixed twice by inversion. Again, the total period that the sperm were in contact with the full-strength cryoprotectant solution after thawing was less than 5 min to minimize their exposure to relatively high osmotic stress.
|
The diluted samples were then centrifuged simultaneously at 400 x g for 5 min (model PR-2; IEC, Boston, MA) [2], and 1400 µl of the resulting supernatant in each sample were gently withdrawn. The soft sperm pellet in the bottom of the remaining 100 µl of solution was then resuspended by very gentle aspiration with a Pasteur pipette. Five to 20 min after centrifugation, the motility of the sperm in each straw was assessed on a microscope slide with a cover glass. Five microscope fields were counted per sample. Any spermatozoa exhibiting flagellar motion were scored as motile. Most of these exhibited progressive motility, but that was not scored. The motilities reported in the figures were normalized to those of unfrozen controls using procedures reported previously [8]. Three unfrozen controls were used: 1) aliquots of sperm obtained just after filtration; 2) an aliquot subjected to loading and unloading into and out of the straws, subsequent dilution, and centrifugation; and 3) an aliquot subjected to these steps in the presence of cryoprotectant. Details are given elsewhere [8]. The motility of frozen samples was normalized to the mean of the two highest values of the unfrozen controls, because the control value immediately after filtration was quite often lower (and apparently randomly so) than that in one or both of the two succeeding controls. The mean absolute motility of these unfrozen controls was 49.3 ± 0.7% (n = 189). This is lower than that reported by most other investigators, probably because our procedure involved no enrichment in motile sperm by swim-out or swim-up procedures.
Procedures for Cooling from -70 to -196°C
In almost all cases of cryopreservation, cells including mammalian gametes are frozen not to -70°C but rather to -196°C in LN2. Consequently, we needed to determine the effect of lowering the minimum freezing temperature from -70 to -196°C. In this series of experiments, sperm samples were suspended in 0.25x SD-PBS containing 18% raffinose (with no glycerol present) as described above and then frozen to -196°C in the three ways summarized in Table 3. In procedures A and B of this table and in a -70°C control, the sample straws surrounded by the glass tubes were frozen in two steps to -70°C at an average rate of 27°C/min (the third procedure in Table 1). The -70°C control remained at that temperature. In procedure A, the coaxial tube assembly was then transferred from the Dry-Ice ethanol bath into LN2. After approximately 2.5 min, the straw was removed from the outer glass tube and immersed directly into LN2. In procedure B, the naked straws were removed from the outer glass tubes held in the Dry-Ice ethanol bath and were placed in LN2 vapor (10 mm above the LN2 surface) for 2.5 min by putting them on a Styrofoam sheet that was floating on LN2. The straws were then plunged directly into LN2 to complete freezing. Procedure C was somewhat different from the above. In this case, the sample straws after preparation were transferred from room-temperature air directly into LN2 vapor by placing them on a Styrofoam sheet floating on LN2. Five minutes later, they were immersed directly in LN2. This procedure, which has been commonly used in many previous reports for freezing mouse sperm [9, 15, 16], produced an approximately sevenfold higher cooling rate from -10 to -70°C than did procedures A and B (Table 3). In all these procedures in this series of experiments, the frozen samples were thawed rapidly by transferring them from LN2 to a room-temperature water bath for approximately 20 sec after first holding them in air for 2 sec to minimize thermal mechanical damage. The frozen and thawed samples were then immediately diluted, centrifuged, and their motilities evaluated as described above.
|
Oxyrase
In the experiments designed to test lower oxygen tensions, the mouse sperm were handled in the presence of Oxyrase as described previously [8, 17]. Briefly, the samples were isolated, processed, and maintained in media that contained 3.8% Oxyrase (EC-Oxyrase Suspension; Oxyrase Corp., Mansfield, OH). Just before initial use, the oxygen concentration in these media was less than 1% of ambient, as indicated by the decoloration of methylene blue in test samples. We have reported elsewhere [7] that, except for one brief spike during filtration, the oxygen tension of the sperm suspensions remains at 3% of ambient or lower throughout the manipulations.
Statistics
The data in Figures 1–4 were analyzed by two-way ANOVA. Tables 4 and 5 give the number of replicate straws and animals for each treatment. In Figures 1–3, one factor was cooling rate; the other factor was either the presence or absence of Oxyrase (Fig. 1), the presence or absence of glycerol (Fig. 2), or warming rate (Fig. 3). In Figure 4, one factor was freezing procedure, and the other was the presence or absence of Oxyrase. If no significant interactions occurred between the two factors and the overall variance among the means within the main effects was significant, then the significance of differences between each pair of treatment means was assessed by the Bonferroni post hoc test. When significant interactions occurred between the two factors, the significance of the differences among the means in individual factors was assessed by one-way ANOVA. These analyses were performed on InStat version 3.0 (GraphPad Software, Inc., San Diego, CA) and SPSS version 9.0 (SPSS, Inc., Chicago, IL). Error bars in the figures and ± values in the tables depict the SEM.
|
|
|
|
|
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
In the first experiment, sperm were frozen to -70°C in 0.25x SD-PBS plus 18% raffinose (without glycerol) at average cooling rates ranging from 0.3 to 530°C/min in the presence or absence of Oxyrase. Frozen samples were then thawed at 1875°C/min by immersing them in a water bath. As shown in Figure 1, the shape of the survival curve at these cooling rates is an inverted "U". The shapes of the curves are similar both in the presence and in the absence of Oxyrase; the difference is that the curve is shifted upward in the presence of Oxyrase. In both cases, the optimum cooling rate lies between 27 and 130°C/min. Higher and lower cooling rates were both highly detrimental. The highest normalized motility of more than 60% was obtained at a cooling rate of 27°C/min in the presence of Oxyrase.
Effect of Cooling Rate to -70°C in the Presence and Absence of Glycerol
We previously demonstrated that the addition of glycerol as a cryoprotectant exerted a negative effect on the survival of mouse sperm frozen at approximately 20°C/min, especially in the absence of Oxyrase [8]. However, we had no information regarding the effect of glycerol at other cooling rates. Figure 2 compares the effect of the same range of cooling rates to -70°C on the survival both in the presence (lower curve) and in the absence (upper curve) of 0.8 M glycerol. The basic suspending medium was 0.25x SD-PBS, which contained 18% raffinose and 3.8% Oxyrase. Samples were thawed at an average rate of 1875°C/min. In both cases, the curves are again in the shape of an inverted "U", and the optimal cooling rate was 27°C/min. However, survival rates in the presence of glycerol were substantially lower than in its absence at all cooling rates between 27 and 530°C/min. At the two lowest rates (0.3 and 2.0°C/min), no difference was observed.
Effect of Warming Rate from -70°C after Freezing at Various Cooling Rates
Warming rate can be another contributor to the survival of frozen cells. Figure 3 describes the effect of warming rates of 1875, 126, and 11.5°C/min from -70°C on the motility of mouse sperm samples frozen at various cooling rates between 0.3 and 530°C/min. All samples were frozen in a medium composed of 0.25x SD-PBS, 18% raffinose (without glycerol), and 3.8% Oxyrase. The overall effect was that the lower the warming rate, the lower the resulting motilities. This reduction was especially evident in cells cooled at 27, 44, and 130°C/min (i.e., in cells cooled at what were the optimal rates when rapid warming was used). When cells were cooled at the supraoptimal rate of 261°C/min, motilities were only 7.0%, even with warming at 1875°C/min. Consequently, there can be little further obvious decline in survival rate at lower warming rates, although survival appeared to drop to 3% when the warming rate was lowered 15-fold. When cells were cooled at the suboptimal rate of 2°C/min, the same 15-fold reduction in warming rate to 126°C/min was without effect on survival (24.1 and 23.7%, respectively), but a further 10-fold reduction in warming rate to 11.5°C/min was damaging (8.5%).
Effect of Further Cooling from -70 to -196°C by Several Cooling Procedures in the Presence and Absence of Oxyrase
The effect of further cooling of mouse sperm to LN2 temperature was evaluated during the experiments depicted in Figure 4. As summarized in Table 3, the sperm suspensions were frozen to -196°C in three ways (procedures A, B, and C) that either varied the cooling rate from room temperature to -70°C or from -70 to -196°C. Procedures A and B involved an initial cooling to -70°C at the same moderately slow rate of 27°C/min found to be optimum in Figures 1–3. The two procedures differed in the rate of subsequent cooling between -70 and -196°C. In procedure A, the measured rate from -70 to -120°C was approximately 200°C/min; in procedure B, it was approximately 60°C/min. Procedure C was like procedure B with respect to the cooling rate from -70 to -120°C, but it produced an approximately sevenfold higher cooling rate between -10 and -70°C (180°C/min) than that in both procedures A and B. In each case, comparisons were performed between samples containing and not containing Oxyrase.
The results of these experiments are given in Figure 4. (The number of animals and straws are summarized in Table 5.) In the case of samples frozen in Oxyrase, no significant differences were observed between the percentage motilities of the -70°C control and those cooled to -196°C by procedures A, B, and C (P > 0.45). In the case of samples frozen in the absence of Oxyrase, the same was true of procedures A and B (P > 0.24), but this was not true of procedure C, the method that produced the sevenfold higher cooling rate from -10 to -70°C. In that case, the normalized motility of 10% is very significantly lower than the other values (P < 0.0001). The other overall finding was that the percentage motility of spermatozoa processed and frozen to -196°C in the presence of Oxyrase was consistently and significantly higher (P < 0.001) than that of spermatozoa processed in the absence of Oxyrase.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
With respect to the dependence of motility on cooling rate, the pattern both in the presence and in the absence of Oxyrase was that of an inverted "U" (Fig. 1). When subsequent warming was rapid enough, maximum survivals occurred over a cooling rate range of 27–130°C/min (52–62% normalized to unfrozen controls). Survival dropped abruptly to less than 10% when the cooling rate was raised to 261°C/min or higher. Survival dropped more gradually (to 24%) when the cooling rate was lowered to 2°C/min; however, a cooling rate of 0.3°C/min was lethal (1%). Although the patterns were similar, the survival in the absence of Oxyrase were significantly lower than those in its presence. Also, in the absence of Oxyrase, a cooling rate of 2°C/min was lethal (1%), whereas in the presence of Oxyrase, it was not (24%).
Other investigators have reported data regarding the survival of mammalian sperm versus cooling rates over at least a portion of the range reported here. Henry et al. [18] also found an inverted "U" relation between the survival of human sperm and cooling rates that ranged from 0.1 to 800°C/min. However, the optimum cooling rate was 1–10°C/min, which is substantially lower than that found here. They found a cooling rate of 800°C/min to be significantly more damaging, but not as damaging as that resulting from the similarly high rates reported here in the mouse. Woelders et al. [12] obtained results for bull sperm after cooling at rates of 50–300°C/min that were very similar to those obtained here over the same range. The optimum cooling range was 50–150°C/min, with a major decrease in viability after cooling at 300°C/min. They did not, however, examine rates less than 50°C/min, but Fiser and Fairfull [19] did so for ram sperm. They thoroughly examined the effects of cooling rates between 1 and 100°C/min and found, similar to our findings, that motility began to drop at cooling rates less than 10°C/min and dropped more sharply at those less than 5°C/min. Cooling rates of 30–100°C/min were optimal, with the latter actually being the best. They did not examine the consequences of cooling more rapidly than 100°C/min. Duncan and Watson [20] obtained similar results for ram sperm, in that a cooling rate of 0.5°C/min was highly deleterious and cooling rates of 40–60°C/min were optimal. Unlike the results of Fiser and Fairfull [19], however, a slightly higher cooling rate of 100°C/min was decidedly damaging. That rate is less than half the rate that we found to be damaging to mouse sperm. Fiser and Fairfull [21] also reported the effects of cooling rates from 1 to 100°C/min on boar sperm after rapid warming. These results were quite similar to their results for the ram: A cooling rate of 100°C/min was optimal, and cooling rates of 5 and 2°C/min were decidedly deleterious.
Other data regarding the effects of cooling rate on mouse sperm are fragmentary. Songsasen and Leibo [10] found, as we did, that far fewer mouse sperm survived cooling at 2°C/min than survived cooling at approximately 20°C/min. Dewitt et al. [13] determined the motilities of sperm frozen in three different media (1.75% glycerol and 18% raffinose in SD-PBS, 3% skim milk and 18% raffinose in water, and a Tes/Tris egg yolk-glycerol medium) at three rates (3, 10, and 50°C/min). The warming rate was unspecified (37°C air). The curve of motility versus cooling rate was a rather flat, inverted "U" shape, with the optimum rate being 10°C/min, or approximately one-third of that found here.
Our results showed that the survival of mouse sperm cooled at rates in the optimum range of 27–130°C/min was highly sensitive to the subsequent rates of warming. They further indicated that the sensitivity to slow warming increases after cooling at rates in the upper portion of that range. Thus, dropping the warming rate from 1875 to 126°C/min caused motility to drop from 62% to 34% for cells cooled at 27°C/min, and it caused motility to drop from 55% to 17% for cells cooled at 44°C/min. It should be noted that when the warming rate was 126°C/min, less than 30 sec elapsed between the onset of warming at -70°C and the onset of significant melting at greater than approximately -20°C when the temperature rises well above the eutectic point of the solution. The rapidity with which the damage occurred suggests a physical rather than a chemical basis. When the cooling rate was increased to 261°C/min, more than 90% of the sperm were killed, regardless of the warming rate. When the cooling rate was decreased to 2°C/min, a decrease in warming rate from 1875 to 126°C/min was again without effect; in both these cases, the survivals were approximately 25%. A further 10-fold decrease in warming rate was, however, decidedly damaging to sperm cooled at this low rate.
To our knowledge, few other data regarding the effects of warming rate on the motility of sperm are available. Fiser et al. [22] reported that porcine sperm generally responded similarly to mouse sperm in showing lower survival rates after slow warming than after rapid warming. They found, like us, that cells cooled at 30°C/min exhibited considerably lower survival after warming at rates of 10–200°C/min than after warming at 1200°C/min. On the other hand, they found that warming at 1800°C/min was more damaging than warming at 1200°C/min. The report of Tao et al. [14] is the only one that we have found dealing with the effect of warming rate on mouse sperm. Like Fiser et al. [22], Tao et al. [14] found that the highest warming rates (7000–8000°C/min) resulted in substantially lower motilities than did warming at approximately 200–2000°C/min, but they were dealing with sperm that had been cooled at 1–3°C/min, not at 20–30°C/min as in our study. Henry et al. [18] came to a similar conclusion for human sperm, in that cells cooled at less than 1°C/min survived better when warming was slow than when warming was rapid, whereas cells cooled at supraoptimal rates responded like our mouse sperm, in that slow warming at 1°C/min was more damaging than rapid warming at 400°C/min. Although this is by no means the general rule, several similar instances have been reported in other cell types (mouse embryos [23], human red cells [24]), in which very rapid warming is more damaging to slowly cooled cells than is slower warming whereas the reverse is true for rapidly cooled cells. An explanation offered for the damaging effects of very rapid warming on slowly cooled cells is that extracellular solutes are driven into cells during slow cooling and cannot escape fast enough during rapid warming to prevent osmotic shock. The explanation of the damaging effects of the slow warming of rapidly cooled cells is generally believed to be the recrystallization of intracellular or extracellular ice or the devitrification of intracellular or extracellular glassy solution.
With respect to our specific results for sperm frozen in raffinose solutions lacking glycerol, our interpretation regarding the effects of cooling rates between 27 and 500°C/min and the interactions with the warming rate is the following: When the sperm are frozen at 250 or 500°C/min, we suggest that more than 90% of them undergo lethal intracellular freezing. Internal freezing occurs because water is not able to flow out of the cells fast enough to maintain the cytoplasmic solution in thermodynamic equilibrium with the external ice and solution [25]. Cells that have undergone minimal intracellular freezing can sometimes survive if they are subjected to subsequent rapid warming. Rapid warming suppresses crystal growth by recrystallization. However, at these high cooling rates, we find that survival is poor, regardless of the warming rate, in mouse sperm. Consequently, we suggest that enough ice forms in the cells to kill them immediately after ice crystal formation. We return to the matter of higher cooling rate and intracellular freezing in a companion paper [26].
The picture is different at cooling rates of 27–130°C/min. In this case, survival is highly dependent on the warming rate. Survival is high when warming is rapid, but it becomes substantially lower when the warming rate is reduced. One possible explanation for the beneficial effect of rapid warming is that a cooling rate in this range induces a small, noninjurious amount of ice in the cells. The amount is sufficiently small to allow the cells to be rescued if warming is sufficiently rapid to prevent recrystallization, the conversion of small into large ice crystals [27]. A second possibility is that no intracellular ice forms during freezing at these intermediate cooling rates but, on the contrary, that the rates are sufficiently high to cause some of the external raffinose solution to form a glass rather than freezing. If subsequent warming is rapid enough, that portion remains a glass, but if it is slow, the glassy portion becomes converted to ice (i.e., devitrifies), with damaging mechanical consequences. It is well-documented [28] that the cooling rate required to form a glass is, at least in theory, lower than the warming rate required to prevent devitrification of that glass. One attractive feature of this latter hypothesis is that it can explain why a nonpermeating cryoprotectant, such as raffinose, can protect mouse sperm against freezing damage. As discussed by Koshimoto et al. [8], partial vitrification may occur because of a combination of the moderately high cooling rates and a large rise in the viscosity of the raffinose solution as it concentrates during progressive freezing. High viscosity is one of the important factors leading to vitrification [29, 30].
When sperm are cooled at 2°C/min, a 15-fold reduction in warming rate from 1875 to 126°C/min is without effect. Based on the above hypothesis, this suggests that, at this cooling rate, intracellular ice formation does not occur and the raffinose solution is converted into a stable, frozen solution rather than into a metastable glass. However, the motility is greatly reduced if the warming rate is lowered an additional 10-fold to 11.5°C/min. Perhaps this is a consequence of the longer exposure times to concentrated solutions resulting form the slower warming and thawing (i.e., what has been referred to as solution-effect injury). However, that explanation requires asymmetry between the effects of time during cooling and time during warming. That is, it requires an explanation of why sperm that spend 24 min between -10 and -50°C when cooled at 2°C/min and warmed at 12°C/min are so much more damaged than those that spend 20 min in that range when cooled at 2°C/min and warmed at 125°C/min.
One puzzling aspect of the present results and of those reported by Koshimoto et al. [8] is that glycerol not only is almost completely ineffective by itself in protecting frozen mouse sperm, but that it also interferes with the protection conferred by raffinose when the two are simultaneously present during cooling at 27–130°C/min (Fig. 2). With respect to the first point, Koshimoto et al. [8] obtained less than 1% motility after the sperm were frozen to -70°C at 20°C/min in 0.8 M glycerol and no raffinose. With respect to the second point, although we see from Figure 2 that the presence or absence of glycerol has no effect on the motility of sperm frozen at 0.3 or 2°C/min in the presence of 18% raffinose, it has a substantial detrimental effect on cells that are cooled at higher rates. If we are correct in suggesting that the high survival rates in that range of cooling rates are due to the ability of raffinose to produce a glassy state, then the poorer survival in the presence of glycerol suggests that it somehow suppresses or interferes with this glass-forming ability of raffinose.
The events that determine whether the mouse sperm cells do or do not survive freezing and thawing appear to have occurred (or to have not occurred) by -70°C. Additional cooling from -70 to -196°C caused no additional damage. One exception was observed; namely, procedure C in Fig. 4 did cause appreciable damage to cells frozen to -196°C in suspensions lacking Oxyrase. Procedure C involved cells that had been cooled from -10 to -70°C at 180°C/min, a sevenfold higher rate than those in procedures A and B. One sees from Figures 1–3 that this rate is on the edge of those causing appreciable damage, which we suggest is due to intracellular freezing. Perhaps survival with procedure C was higher in the presence of Oxyrase than in its absence because the Oxyrase permitted the recovery of cells that had been sublethally injured by freezing at the borderline rate. Oxyrase is known to enhance the recovery of cells that have been injured by a variety of agents [31].
Confirming what we have reported previously [8, 17], Oxyrase confers consistent protection. First, it raises the overall motilities by 10–20% (Fig. 1). Second, it somewhat broadens the conditions under which cells survive. In other words, it permits cells to survive under conditions in which they otherwise would not. Examples are the data at the cooling rate of 2°C/min in Figure 1 and the procedure C bars in Figure 4.
We believe it is increasingly clear why the consistent cryopreservation of mouse sperm has been such a formidable problem. Their survival after freezing and thawing is subject to a number of simultaneous—and sometimes conflicting—constraints. Most of these constraints stem from their high susceptibility to mechanical forces and their high sensitivity to osmotic forces, and the latter may be a subset of the former. Sensitivity to mechanical stresses mandates extreme care in their manipulation and centrifugation. They appear to be highly susceptible to mechanical damage from external ice crystals. High concentrations of raffinose minimize that damage, but constraints are placed on the useable concentrations of that solute by the fact these concentrations must not exceed the osmotic pressures that are tolerated by the sperm. The impermeability to raffinose necessitates the use of moderately high cooling rates to minimize solution-effect injury, but the use of these moderately high rates produces metastable ice or glass in the external medium, which in turn requires the use of high warming rates to prevent damage from the recrystallization of ice or the devitrification of glass. The cooling rates, however, cannot be so high as to produce lethal intracellular ice. When the procedures appropriately balance these constraints, the resulting motilities are 60–70% of those in unfrozen and untreated cells.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Supported by NIH grant R24-RR13194 (J. Critser, PI) under subcontract with Indiana University. A preliminary report was presented at the 37th Annual Meeting of the Society of Cryobiology; Cambridge, MA; 30 July to 1 August 2000. 
2 Correspondence: Peter Mazur, Fundamental and Applied Cryobiology Group, Dept. of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, 10515 Research Dr., Suite 300/10, Knoxville, TN 37932-2575. FAX: 865 974 8027; pmazur{at}utk.edu
3 Current address: Experimental Animal Center, 5200 Miyazaki Medical College, Kiyotake, Miyazaki 889-1692, Japan
Accepted: December 10, 2001.
Received: September 10, 2001.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  P. Mazur, S.P Leibo, and G. E Seidel Jr. Cryopreservation of the Germplasm of Animals Used in Biological and Medical Research: Importance, Impact, Status, and Future Directions Biol Reprod, January 1, 2008; 78(1): 2 - 12. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Yamauchi, A. Ajduk, J. M Riel, and M. A Ward Ejaculated and Epididymal Mouse Spermatozoa Are Different in Their Susceptibility to Nuclease-Dependent DNA Damage and in Their Nuclease Activity Biol Reprod, October 1, 2007; 77(4): 636 - 647. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Fonseca, M. Marin, and G. J. Morris Stabilization of Frozen Lactobacillus delbrueckii subsp. bulgaricus in Glycerol Suspensions: Freezing Kinetics and Storage Temperature Effects. Appl. Envir. Microbiol., October 1, 2006; 72(10): 6474 - 6482. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Ajduk, Y. Yamauchi, and M. A Ward Sperm Chromatin Remodeling after Intracytoplasmic Sperm Injection Differs from That of In Vitro Fertilization Biol Reprod, September 1, 2006; 75(3): 442 - 451. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Frederickx, A. Michiels, E. Goossens, G. De Block, A.C. Van Steirteghem, and H. Tournaye Recovery, survival and functional evaluation by transplantation of frozen-thawed mouse germ cells Hum. Reprod., April 1, 2004; 19(4): 948 - 953. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Ward, T. Kaneko, H. Kusakabe, J. D. Biggers, D. G. Whittingham, and R. Yanagimachi Long-Term Preservation of Mouse Spermatozoa after Freeze-Drying and Freezing Without Cryoprotection Biol Reprod, December 1, 2003; 69(6): 2100 - 2108. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Mazur and C. Koshimoto Is Intracellular Ice Formation the Cause of Death of Mouse Sperm Frozen at High Cooling Rates? Biol Reprod, May 1, 2002; 66(5): 1485 - 1490. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
