The effect of collection temperature, cooling rate and warming chilling injury and cryopreservation of mouse spermatozoa

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1 The effect of collection temperature, cooling rate and warming chilling injury and cryopreservation of mouse spermatozoa Jun Tao, Junying Du, F. W. Kleinhans, E. S. Critser, P. Mazur and J. K. Critser 1Cryobiology Research Institute, Methodist Hospital of Indiana Inc., Indianapolis, IN 46202, USA; 2Department of Physics, Indiana University Purdue University Indianapolis, Indianapolis, IN 46202, USA; 3Department of Physiology and Biophysics and Department of Obstetrics and Gynecology, Indiana University School of Medicine, Indianapolis, IN 46202, USA; Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA; and 5Department of Veterinary Clinical Science, School of Veterinary Medicine, Purdue University, West Lafayette, IN 47907, USA The experiments presented here identify several factors that affect survival (motility) of cryopreserved mouse spermatozoa after freezing and thawing. Among these factors are: (i) the temperature at which spermatozoa are collected, (ii) the cooling rate to 0\s=deg\Cand (iii) the warming rate from \m=\196\s=deg\cto ambient. When excised epididymides were cooled to near 0\s=deg\(1\p=n\4\s=deg\C)and spermatozoa collected and mixed with cryoprotectant at that temperature, motilities after subsequent freezing and thawing were 8\p=n\10times higher than when the spermatozoa were collected from the epididymides at 22\s=deg\C. In addition, the survival rates of spermatozoa warmed at rates ranging from 150 to 2000\s=deg\Cmin\m=\1 were about five times higher than those in suspensions warmed at about 7500\s=deg\Cmin\m=\1. The combination of a low collection temperature and the lower warming rates resulted in approximately 50% motility relative to unfrozen controls. Motility was reduced to 6\p=n\8%when the collection temperature was 22\s=deg\C,and to approximately 10% when frozen suspensions of spermatozoa collected in the cold were rapidly warmed from \m=\196\s=deg\c.when spermatozoa collected at 22\s=deg\Cwere abruptly cooled to 0\s=deg\C,40\p=n\80%of the cells suffered an irreversible loss of motility after warming. In contrast, when spermatozoa were cooled to 0\s=deg\Cat 1\s=deg\C min\m=\1 and warmed (either rapidly or slowly), motilities were similar to those of uncooled controls (75\p=n\90%).These findings indicate sensitivity to cold shock. Finally, the addition of raffinose or sucrose to the suspending medium did not affect the survival of the spermatozoa cooled slowly, but it did increase the survival of spermatozoa that were rapidly cooled to 0\s=deg\C (55\p=n\60% versus 25\p=n\30%). rate on Introduction The mouse is an excellent model for studying mammalian gene function because of its wellcharacterized genetic makeup and its accessibility to experimental manipulation. A common method to accomplish this is to generate transgenic animals. Because the number of these lines is rapidly increasing, their preservation places an increasing strain on the resources of laboratories. Maintenance of standard breeding colonies is costly, labourintensive and space demanding, and can lead to genetic drift. An alternative way to preserve transgenic lines is to cryopreserve embryos or spermatozoa. Compared with the cryopreservation of embryos, there are a number of potential advantages to cryopreserving spermatozoa. Males would not be treated with gonadotrophins, and collection and manipu lation of spermatozoa would be faster and less demanding. In Received 21 December addition, fewer donor males would be required to produce an adequate genome resource bank. Human and bull spermatozoa can be cryopreserved with reasonably high functional survival (Sherman, 1973; Critser et al, 1988; Hammerstedt et al, 1990). However, until recently, attempts to cryopreserve mouse spermatozoa had failed. Sherman and Liu (1982) were the first to attempt this, but failed to obtain any surviving (motile) spermatozoa. Tada et al (1990) reported the successful preservation of mouse spermatozoa using a mixture of 18% (w/v) rafflnose and 1.75% (v/v) glycerol, with fast cooling and warming rates. Yokoyama et al (1990) reported similar results. However, Penfold and Moore (1993) were unable to obtain viable spermatozoa using these pro cedures. Mobraaten et al (1991) reported modest success using very different procedures, including slow cooling; procedures similar to those that Tada et al (1990) reported had failed. The experiments reported here represent an initial study of the cryobiology of mouse spermatozoa. The experiments were

2 . 5 C,, designed to test the hypotheses that: (i) the motility of cryopreserved mouse spermatozoa is affected by the collection temperature and by the warming rate; (ii) contrary to previous reports, mouse spermatozoa are sensitive to cold shock (that is, they are damaged by rapid, but not by slow, cooling to 0 C); and (iii) that cold shock is reduced by the presence of the same nonpermeating solutes (raffinose and sucrose) that protect against cryoinjury. Materials and Methods Collection of spermatozoa Sperm collection medium. A supplemented Dulbecco's PBS (DPBS) as described by Tao et al. (1993) was used. This consisted of DPBS (GIBCO Laboratories, Grand Island, NY), pyruvic acid (18.15 µg ml ; Sigma Chemical Co., St Louis, MO), DLlactic acid (2 µ ml"~, 60% aqueous solution; Sigma), and 2% (w/v) BSA (Sigma). The collection medium also contained 4% (v/v) glycerol (Aldrich Chemical Company, Inc., Milwaukee, WI). Sperm preparation. B6C3 mice (Harlan, SpragueDawley, Indianapolis, IN), 2025 weeks old, were used. The animals were killed by cervical dislocation and spermatozoa were obtained from the cauda epididymides at two different tem peratures, 22 C (room temperature) or 04 C (on ice). The cauda epididymides were removed and immediately immersed into the sperm collection medium at either 22 or 04 C. Spermatozoa were released by mincing the epididymides in the collection medium. The sperm suspensions were then gently mixed with the cryopreservation medium (see below) at a ratio of 1:1. The mixed sperm suspension was then aspirated into 250 µ plastic straws and sealed. For collection at 04 C, the collection medium and cryopreservation medium were cooled on ice before use. The epididymides were placed in cold medium during mincing, and the loaded and sealed straws were kept on ice until further treatment. Two experiments were performed. Experiment 1 compared the motilities of spermatozoa collected at 0 C and spermatozoa collected at 22 C with subsequent slow freezing to 196 C and warming at various rates. Experiment 2 examined the effect of cooling rate, warming rate, and cryoprotectant on the motility of spermatozoa cooled to 0 C and rewarmed (that is, no freezing). Expt 1: effect of collection temperature and warming rate Cryopreservation medium. The cryopreservation medium was prepared by mixing the sperm suspension in the collection medium at 04 or 22 C in a 1:1 ratio with a solution at 0 C or 22 C. The medium was the same as the collection medium with the addition of 1% (w/v) glycine, 0.1% (w/v) glucose, 15% (w/v) raffinose, and 4% (w/v) glycerol (Aldrich Chemical). (Unless indicated otherwise, all chemicals were obtained from Sigma Chemical Company (St Louis, MO).) The solution was stored at 80 C with the latter two components added just before use. The resulting medium thus contained 7.5% (0.13 mol 1 *) raffinose and 4% (0.54 mol 1 ') glycerol. Freezing was initiated 510 min later. Treezing procedure. Spermatozoa were cooled at a relatively low rate using a programmable freezer (Planer Products Ltd, SunburyonThames, Middlesex). For spermatozoa collected at room temperature, cells were cooled from 22 to 4 C at 3 C ~ min, held at 4 C for 2 min, and then cooled to 5 C at 3 C min From the cells were cooled without seeding (survival was better when seeding was omitted) to 30 C at 1 C min1, from 30 to 80 C at 3 C min1; and after 2 min the straws were plunged into liquid nitrogen ( 196 C). For samples collected in the cold, the cooling started from 4 C at a rate of 3 C min to 5 C. The remaining steps were the same as for samples collected at room temperature. All straws were stored in liquid nitrogen for periods between 1 h and 1 week. Thawing procedure. Four procedures were used to warm the samples from 196 C. The warming rates were measured with a 36 gauge bare copper constantan Type thermocouple, a reference ice junction, and an HP 3478A digital microvoltmeter. Data were logged on an HP9825 microcomputer at rates up to 20 measurements per second, and the thermal voltages converted to temperature versus time plots using a computer program. For warming rate 1, after removing the straws from liquid nitrogen, samples were thawed in air at room temperature. The maximum thawing rate was C min1 (Fig. la). For warming rate 2, the straws were taken out of liquid nitrogen and directly plunged into a 80 C ethanol bath for 510 min, and then transferred to a 20 C ethanol bath, and finally to a 04 C water bath. The maximum warming rates from 196 to 80 C and from 80 to 20 C were 1 " C min and C min " \ respectively (Fig. lb). For warming rate 3, the straws were first transferred from liquid nitrogen to a 80 C air chamber for 510 min, and then to a 20 C air chamber, and finally to air at room tempera ture. The maximum warming rates from 196 C to 80 C 1 and from 80 C to 20 C were C min and " C min respectively (Fig. 1c). For warming rate 4, the straws were transferred directly from liquid nitrogen to a water bath at 30 C. The maximum rate of warming was C min ": (Fig. id). After thawing, samples were diluted 20:1 at room tempera ture by dropwise addition of supplemented DPBS over 510 min, and the motility was determined after 15 min. Controls consisted of samples subjected to all the above treatments except subzero temperature exposure. Expt 2: effect of abrupt cooling above zero and the protective effects of cryoprotectant solutes Spermatozoa were collected at room temperature. In the first series of these experiments, aliquots were suspended in four solutions for 510 min. Each suspension was cooled to 0 C and allowed to reach thermal equilibrium (35 min) and rewarmed to room temperature using four combinations of cooling and

3 30 i (b) Time (s) Fig. 1. Representative thermocouple traces of the four warming methods used for cryopreservation of mouse spermatozoa, (a) Straws transferred from liquid nitrogen to air at room temperature (warming rate: C min~ '). (b) Stepwise warming from liquid nitrogen in liquid baths at 80 C, 20 C and 0 C; (c) stepwise warming from liquid nitrogen to air at 80 C, 20 C and room temperature; and (d) straws transferred abruptly from liquid nitrogen to a 30 C water bath. warming rates (16 treatments in all). One solution (1) was the collection medium without cryoprotectant. The other solutions were the collection medium with (2) 4% (v/v) glycerol, (3) 10% (w/v) raffinose, and (4) a mixture of 4% glycerol and 10% raffinose. The four cooling and warming treatments were (i) no cooling, (ii) rapid cooling to 0 C and rapid warming back to 22 C, (iii) slow cooling to 0 C and rapid warming to 22 C, and (iv) slow cooling and slow warming. Rapid cooling and warming were achieved by abruptly immersing the straws in ice and in 22 C water baths, respectively. Slow cooling and warming were at 1 C min A second series of experiments was a repeat of the first series, except that 4% (v/v) dimethylsulfoxide (DMSO; Sigma) and 10% (w/v) sucrose (Sigma) were substituted for glycerol and raffinose, respectively. After the 22 C to 0 C to 22 C temperature cycle, the suspensions were diluted in a stepwise manner with DPBS, as above, and the motility of spermatozoa was determined after 15 min. Motility assays The percentages of motility before and after treatment were assessed under a light microscope ( 20; Optiphot; Nikon, Tokyo) at room temperature by two independent researchers each counting at least 200 spermatozoa. Normalized motility was calculated as (posttreatment motility)/(prefreeze or control motility) 100. Statistical analyses Data are presented as means ± sem and were analysed using a standard analysis of variance approach, using the compu tational procedures of the Statistic Analyses System (SAS; Spector et al, 1986). Results Expt 1: effect of collection temperature and warming rate Effect of collection temperature on motility. The motility of spermatozoa collected at 04 C was significantly lower than that of spermatozoa collected at 22 C ( % («=11) versus 64.6 ± 2.2% (n 8); = means ± sem; < 0.01). Effect of collection temperature and warming rate on survival after freezing to 196 C. The absolute percentage motility values for the mouse spermatozoa collected at low temperature (04 C) were: 20.5 ± 2.4, 16.9 ±1.5, 20.2 ± 2.4 and 4.0 ±0.7 for warming rates (1)(4), respectively. The corresponding absolute percentage motility values for spermatozoa collected at 22 C were: 3.9 ±1.0, 5.7 ± 3.1, 5.5 ± 1.9 and 1.1 ±0.3,

4 '. '; m 60 > 50 c g CO fi 40 ~ 30 E 20 cd CL w 10 a a I Warming method Fig. 2. Effect of collection temperature and rate of warming from 196 C on the motility of mouse spermatozoa cooled slowly to 80 C then transferred abruptly to liquid nitrogen. Spermatozoa were collected at (D) 04 C (n 11) = or at ( ) 22 C (n 8). The = maximum warming rates with the four methods were (1) C min l; ' (2) C min (3) C min and (4) C ~ " min Different letters above the bars (mean ± sem) indicate signifi ~ cant differences (P<0.01) in percentage motility. Motilities are normalized to those of controls. respectively. The normalized motilities ((absolute postthaw motility)/(absolute prefreeze motility) 100) after the various treatments are shown (Fig. 2). In spite of the results obtained before freezing, the motilities after thawing of spermatozoa collected in the cold were significantly higher than those of spermatozoa collected at 22 C. Superimposed on the effect of collection temperature was an adverse effect of the highest warming rate (warming rate (4)). With the three lower warming rates, the normalized postthaw motilities of spermatozoa collected in the cold were 50.4 ± 5.1, 45.2 ± 7.2, and 50.3 ± 7.2% (means ± sem), six to ten times higher than the normalized motilities of spermatozoa collected at 22 C (58%). The motilities of spermatozoa collected in the cold and thawed at the highest rate (warming rate (4)) were reduced to 10.1%, but were still five times higher than those of spermatozoa collected at 22 C (1.8%). Expt 2: effect of abrupt cooling above zero and the protective effects of cryoprotectant solutes The results of subjecting spermatozoa to rapid or slow cooling to 0 C and rapid or slow rewarming to 22 C while suspended in DPBS only (control), or in DPBS with 4% glycerol, 10% raffinose, or both are shown (Table 1). The combination of rapid cooling and rapid warming produced significant a reduction in motility, although when raffinose was present motilities were about twice the values obtained when raffinose was absent. In contrast, when spermatozoa were cooled slowly (1 C min I) to 0 C, motilities were about equal to those of the uncooled, control set, irrespective of the suspending medium and the warming rate and ranged from 82 to 90%. Results were similar when 4% DMSO was substituted for glycerol and 10% sucrose was substituted for raffinose (Table 2); that is, rapid cooling was damaging, although less so, when the medium contained sucrose than when it did not. In addition, when cooling was slow, motilities were approxi mately equal to those of uncooled samples regardless of whether warming was rapid or slow and regardless of the suspending medium. Discussion In the present study, spermatozoa collected at low tempera tures (04 C) exhibited significantly higher motility after thawing than did samples collected at room temperature (22 C). Cooling the epididymides to 04 C and collecting spermatozoa at this temperature prevented the high frequency of progressive motility before exposure to subzero tempera tures. This contrasts with previously published studies, in which spermatozoa collected at room temperature became progressively motile before subsequent cooling (Tada el al, 1990; Sztein et al, 1992). These results suggest that either mouse spermatozoa are more resistant to cooling below 0 C if they have not developed full motility, or that maintaining spermatozoa at near 0 C prevents the development of some Table 1. Percentages of motile mouse spermatozoa after exposure to the cryoprotectants glycerol, raffinose or both, in combination with different cooling and warming rates Cooling and warming treatment Cryoprotectant treatment No 4% (v/v) 10% (w/v) 4% (v/v) glycerol cryoprotectant glycerol raffinose and 10% (w/v) raffinose No cooling Rapid cooling and warming Slow cooling and rapid warming Slow cooling and slow warming 94.6 ± 2.8a 26.7 ± 4.8a a a' 90.9 ± 4.4a 29.6 ± 5.8a " b 83.1 ±3.8" 59.1±4.1b 86.6 ± 3.0a 89.7 ± 2.0a 85.6 ±2.8" 56.8: 5.2D 87.3: 2.5a 86.7 ± 2.1a bwithin rows, means ( ± sem) with different superscripts are significantly different (P < 0.05).

5 ).. Table 2. Percentages of motile mouse spermatozoa after exposure to the cryoprotectants 4% (v/v) dimethyl sulfoxide (DMSO), or 10% (w/v) sucrose or both, in combination with different cooling and warming rates Cooling and warming treatment No cryoprotectant DMSO Sucrose DMSO and sucrose No cooling Rapid cooling and warming Slow cooling and rapid warming Slow cooling and slow warming 85.0 ± 4.8a 19.0 ± 5.2a 66.8 ± 3.4a 62.8 ± 2.7b 65.8 ± 7.6b 25.8 ± 5.2a 65.0 ± 4.8a 72.6 ± 4.3a " b a 72.8 ± 4.6a 53.6 ± 6.8" 49.6 ± 6.5b a 67.8 ± 4.8ab bwithin rows, means ( ± sem) with different superscripts are significantly different (P < 0.05). other characteristic that sensitizes them to subsequent cooling and warming. Although collection of spermatozoa at near 0 C substan tially increased their survival after cooling and warming, the motility before freezing was only twothirds of that of sper matozoa isolated at 22 C. The abrupt cooling of mouse spermatozoa from 22 C to near 0 C resulted in a significant, irreversible loss in motility. This result indicates that these cells are sensitive to cold shock, a finding that differs from previous reports (Watson, 1981). There are large interspecies differences with respect to the sensitivity of spermatozoa to cold shock; for example, ram and boar spermatozoa are very susceptible, whereas human spermatozoa are relatively resistant (Watson, 1981; Holt and North, 1984; De Leeuw et al, 1990; Drobnis et al, 1993). Human and bovine spermatozoa have been shown to be impermeable to sucrose (Mr 342) (Du et al, 1991, 1994). It is likely that mouse spermatozoa are also impermeable to sucrose and to raffinose (Mr 594), although this has not yet been tested. These solutes provided substantial protection against cold shock, and they also enhance cryosurvival of mouse spermatozoa (Tada et al, 1990; Yokoyama et al, 1990; Mobraaten et al, 1991; Sztein et al, 1992). In other cell types, nonpermeating solutes alone cannot protect against slow freezing injury. This is because they cannot prevent either the extreme concentrations of intracellular solutes or the extreme osmotic cell shrinkage that accom panies extracellular freezing (Lovelock, 1954). Mixtures of a nonpermeating solute (sucrose) and a permeating solute (glyc erol) have been shown to yield high survival rates of mouse embryos after cooling and warming at high rates (Széll and Shelton, 1986; Mazur, 1990), but only when the combined concentrations of glycerol and sucrose exceed 2 mol 1 One physical effect of nonpermeating solutes would be to cause an osmotic loss of about 35% of the intracellular water before cooling. Placed in the collection medium, the interior of the spermatozoa would be approximately 900 mosmol kg (0.54 mol glycerol I and 300 mosmol salts kg ). In the cryoprotectant medium, it would be approximately 1100 mosmol kg : (0.13 mol raffinose l, 0.54 mol glycerol 1, and 300 mosmol salts kg Because mouse spermatozoa behave as ideal osmometers (Du et al, 1994), water loss is proportional to the osmolality. It is unclear whether the amount of cell dehydration caused by these conditions would have a significant cryoprotective effect. The fact that these solutes do ameliorate chilling injury may, nevertheless, be one reason for their ability to yield viable cryopreserved spermatozoa obtained in studies like those of Tada et al. (1990). In contrast to raffinose, human and bovine (and presumably mouse) spermatozoa are permeable to glycerol (Du et al, 1991, 1994; Gao et al, 1992). However, Tada et al. (1990) reported that although glycerol is the most widely used cryoprotectant for other mammalian spermatozoa, it does not protect mouse spermatozoa against freezing injury. One major difference between the experiments presented here and previous reports, is that here glycerol was present both in the collection and the cryopreservation media. As a consequence, spermatozoa were exposed to, and probably fully permeated by, glycerol before exposure to raffinose. Because of this, the cells shrink much less when exposed to raffinose than when exposed to the two solutes simultaneously. Avoiding excessive variations in sperm volume has been associated with higher survival rates of human spermatozoa (Gao et al, 1995). The rate at which frozen cells are warmed often has profound effects on survival (Mazur, 1984). In the present studies, mouse spermatozoa were cooled slowly, and the lower warming rates (approximately C min1) yielded significantly higher survival than did the highest warming rate (approximately 7000 C min *). This is consistent with pre vious reports on other cell types, for example human red blood cells (Miller and Mazur, 1976), mouse embryos (Whittingham et al, 1972; Leibo et al, 1974; Critser et al, 1987) and human spermatozoa (Henry et al, 1993). In this regard, it should be noted that there can also be interactions between the type and concentration of cryoprotectant and the cooling and warm ing rates. For example, mouse embryos cooled slowly in DMSO are injured by rapid warming (Whittingham et al, 1972), but mouse embryos frozen in glycerol are not (Rail and Polge, 1984). Previous reports regarding cryopreservation of mouse spermatozoa have used a variety of cooling rates, warming rates and cryoprotective solutions with various and sometimes conflicting results. These variations and conflicts will probably not be eliminated until these interactions are elucidated. Although these and other questions remain to be resolved, the data presented here suggest methods which yield motilities for cryopreserved mouse spermatozoa that approach those obtained for human and bovine spermatozoa. This information provides the basis for further investigation of other critical endpoints of sperm function including acrosomal integrity and the ability to participate in fertilization.

6 This work was supported by Methodist Hosptial of Indiana, Inc., grants from the NIH (KO4HD00980 to J. Critser; 1R01 HD A2 to P. Mazur) and DOE contract DEAC05840R21400 with MartinMarietta Energy Systems. The authors thank K. L. Vemon for assistance in preparing the manuscript, and J. Williams for assistance in preparing the graphs. This work was carried out under a US Government contract No. DEAC05840R Accordingly, the US Government retains a nonexclusive, royaltyfree license to publish or reproduce the published form of this contribution, to do so, for US Government purposes. References or allow others Critser JK, Arneson BW, Aaker DV and Ball GD (1987) Factors affecting the cryosurvival of mouse twocell embryos Journal of Reproduction and Fertility Critser JK, Arneson BW, HuseBenda AR, Ball GD and Aaker DV (1988) Cryopreservation of human spermatozoa. III. 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