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Cryobiology40, 187–209 (2000)doi:10.1006/cryo.2000.2238, available online at http://www.idealibrary.com on

The Enhancement of the Ability of Mouse Sperm to Survive Freezing andThawing by the Use of High Concentrations of Glycerol and the

Presence of an Escherichia coli Membrane Preparation(Oxyrase) to Lower the Oxygen Concentration1

Peter Mazur,*,†,2 Igor I. Katkov,*,3,4 Nadezhda Katkova,*,4 and John K. Critser‡

*Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, U.S.A.;†Department of Biochemistrand Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee, U.S.A.; and

‡Cryobiology Research Institute, Indiana University Medical School, Indianapolis, Indiana, U.S.A.

The cryobiological preservation of mouse spermatozoa has presented difficulties in the form of poor motilitiesirreproducibility. We have hypothesized several underlying problems. One is that published studies have used concentrof the cryoprotectant glycerol that are substantially lower (,0.3 M) than the;1 M concentrations that are optimal for mostmammalian cells. Another may arise from the known high susceptibility of mouse sperm to free radical damage. We hbeen able to obtain high motilities in 0.8 M glycerol provided that the exposure time is held to;5 min to minimize toxicityand provided that the glycerol is added and removed stepwise to minimize osmotic shock. Since free radical damamouse sperm is proportional to the oxygen concentrations, we have determined the consequences of reducing the oto,3% of atmospheric by maintaining the sperm in contact with anEscherichia colimembrane preparation, Oxyrase, fromthe moment of collection throughout the assessment of motility. Prior studies have shown that the procedure significreduces damage from centrifugation and osmotic shock. In the experiments reported here we obtained;50% motilityrelative to untreated controls when suspensions containing 3.8% Oxyrase were exposed;5 min to a solution of 0.8 Mglycerol and 0.17 M (10%) raffinose in a supplemented PBS and then frozen at;25°C/min to275°C. In the absence ofOxyrase, the normalized motility dropped to 31%. The protection by Oxyrase was in part a consequence of minimicentrifugation damage, but in part it reflected a reduction in freeze–thaw damage. Preliminary experiments indicate thanumber of motile sperm after cryopreservation in Oxyrase is higher when the sperm are collected without swim-upwhen they are collected by swim-up. This is in part due to the fact that more cells are collected in the absence of swimand in part due to a greater protective effect of Oxyrase on those cells. The minimum temperature in these initial experimwas limited to275°C to avoid the potential contribution of other injurious factors between275 and2196°C. © 2000

Academic Press

INTRODUCTION

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Received July 2, 1999; accepted February 2, 2000.Supported by NIH Grants RO1-HD30274 to the U

Department of Energy and 7R24- RR13194 by subconfrom Indiana University to the University of Tennessee

1 A preliminary report was presented at the 35th AnnMeeting of the Society for Cryobiology, Pittsburgh, PU.S.A., July 12–16, 1998.

2 Present address: Fundamental and Applied CryobioGroup, Department of Biochemistry and Cellular and Mlecular Biology, University of Tennessee, Knoxville, T37392-2575, U.S.A.

3 Dr. Katkov carried out this research under the auspof an ORNL Postdoctoral Fellowship administered byRidge Associated Universities, Oak Ridge, TN, U.S.A.

4 Present address: Universal Preservation Technologies11045 Roselle Street, Suite C, San Diego, CA 92121, U.S.

187

tthe use of molecular genetic techniques, is reing in the creation of accelerating numbersmutant lines. The maintenance of these linestandard breeding techniques is producingcreasing and often intolerable stresses on faciand budgets. Consequently, the need is criticdevelop reliable and cost-effective alternameans of germplasm preservation. One alternis to cryopreserve embryos, but there is a growbelief that the maintenance of lines by cryopserved sperm and their subsequent rederivwould be simpler and less costly, providedsufficient numbers of sperm survive freezingthawing in a reliable fashion.

Prior to 1990, efforts to cryobiologically prserve mouse sperm had failed (37). The pic

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changed that year when Tadaet al. (45) andce

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188 MAZUR ET AL.

Yokoyamaet al. (51) reported success. Sin990, there have been about a score of a

ional reports of “success” by minor and maariants of the Tada and Yokoyama proceduut the procedures have been troubling w

espect to repeatability. Two reports of inabio obtain survivals using the earlier proceduave been published (15, 34), and othersnecdotal. Although more recently publishrocedures appear to be yielding reproducesults for some strains of mice (e.g., Songsnd Leibo (41)) there remains the underlyroblem that the published procedures areirical and many aspects of them seem at oith fundamental cryobiological principles deloped from studies on other cell types. Ibscure, for example, why the cryopreservaf mouse sperm requires the presence of aermeating sugar like raffinose and the presf sizeable concentrations of macromolec

ike bovine serum albumin, egg yolk, or skilk, when such substances by themselves

neffective cryoprotectants for most cell typrimarily because they are nonpermeating.We have been attempting to dissect and id

ify the factors that underlie the ability of mouperm to survive or not survive freezing andontribute to the difficulties in reproducibryopreserving the cells. We have so far idified four. These are low tolerance to osmoolume excursions (48), sensitivity to mechcal stresses associated with pipetting and mng (36), sensitivity to centrifugation (24, 25nd osmotic damage and toxicity of glyceoncentrations.0.3 M (23). In the presetudy we have tested two other potential cributors, namely, freezing damage associith the use of too low a concentration ofermeating cryoprotectant glycerol and thecerbation of freezing damage by the sublections of free radicals.

smotic Sensitivity

The abrupt introduction and removal ofermeating cryoprotectant like glycerol puces transient osmotic volume excursioilloughbyet al.(48) and Songsasen and Le

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imited tolerance to both osmotic shrinkageypertonic nonpermeating solutions andotic swelling in hypotonic solutions. Katkot al. (23) have found that the abrupt introd

ion and removal of 0.8 M glycerol causesubstantial loss in motility.

echanical Sensitivity

We have determined that mouse spermighly sensitive to the manner in which theyipetted and mixed during collection and mipulation. Motility is substantially affected b

he type of pipette, the number and velocityipettings, and the vigor of the mixing (36).ipetting and mixing are carried out approptely gently in the course of collection aubsequent routine manipulation, motilitiese fivefold higher than if they are carried o

nappropriately. Appropriate conditions incluipetting by Pasteur pipette or capillary pipettached to a micrometer barrel and mixingentle inversion.

ensitivity to Centrifugation

Centrifugation is a necessary step in remng cryoprotectants from suspensions andurning the cells to physiological media. In vief their extreme mechanical sensitivity,ere concerned that the pelleting and the reension of pelleted cells could be damage have found that to be the case (24), as hzorneket al. (33). There are two importapposing factors: If the relative centrifugalelerations (RCF) or centrifugation timesoo high or too long, all the cells are pelletedhe motility after resuspension is adverselyected. If the RCF and centrifugation timeoo low or too short, the motility of the pelletells is high after resuspension, but more talf of the cells do not sediment and are24). In terms of banking sperm, the moield is more important than percentage of mility, and motile yield is maximized at an iermediate RCF/time combination such asin at 400g. Theory predicts (25), and expeents confirm (24), that the optimum RCF/ti

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189FREEZING OF MOUSE SPERM IN HIGH GLYCEROL AND LOW OXYGEN

the column being centrifuged. These dimsions affect the time taken for cells in differetube locations to reach the bottom of the tand the length of time the cells remain packethe pellet. Injury is proportional to that time

Toxicity and Osmotic Damage from the Useof Glycerol at Concentrations GreaterThan 0.3 M

The optimum cryopreservation of most mamalian cells by slow freezing requires the prence of a permeating cryoprotectant, commoglycerol, in concentrations of;1 M. Nearly allpublished procedures for mouse sperm, hever, have restricted the concentration of gerol to 0.17 to 0.24 M (1.25 to 1.75% (v/v)).other cell types such as marrow stem cellsand tissue culture cells (29), the consequencsuch suboptimal concentrations is that evecooling rates that yield maximum survival, thmaximum falls far short of 100%. We beliethat that partly explains the relatively poor svivals of slowly frozen mouse sperm.

Presumably, investigators have avoideduse of higher concentrations of glycerol becathey have found that mouse sperm can noterate them. The lack of tolerance could htwo causes. One is that the abrupt introducof higher concentrations of glycerol andsubsequent abrupt removal of that glycerol pduce osmotic shrinkage and swelling, resptively, that exceed that tolerated by the speThe other is that higher concentrations of gerol are toxic. The first seems to be the mimportant in human sperm, for Gaoet al. (16),or example, have shown that the spermetain motility in 1 M glycerol if the glycerol idded and removed in appropriate size stepestrict the osmotic volume excursions. Stot al. (44) have carried stepwise additionlycerol to its ultimate by the use of dialysnd with this procedure they found that 36%ouse sperm maintained intact membrane

er freezing in a medium of 6% glycerol a.5% raffinose. The percentage with intembranes was even higher when 7.5% tr

ose was substituted for raffinose. Using exp

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helpset al. (35) have computed and compahe volume changes experienced by moperm subjected to the addition and remova% glycerol in eight steps vs dialysis. Theetics, of course, are different but both methold volume excursions to within tolerated li

ts. Katkov et al. (23), however, have showhat both osmotic damage and toxicity open mouse sperm. If the former is reducedliminated by the addition and removal oflycerol in steps calculated to restrict the vme excursions, there remains a time-depenecrease in motility of sperm in glycerol.0.5. One can, however, obtain high motilities.8 M glycerol if the time of exposure to tholution does not exceed 5 min. [Althoueibo and co-workers have kept the time

ween the addition of glycerol and the initiatif freezing short (10 min) and constant, mthers have not. In most published procedu

he sperm are placed in the glycerol-containedia at the time of initial collection, so that txposure times could easily exceed 20 mspecially when time is allowed for swim-ou

voidance of Free Radical Damage byMaintaining Low O2 Tensions

Although low and controlled concentratioof reactive oxygen species including free racals appear to play an important role in prosperm physiology (10), oxygen-derived racals, paradoxically, have been proposed byvarez and Storey (6) to contribute to the losmotility of frozen–thawed sperm. The basisthat proposal is their finding (5) that the plasmembranes of human, rabbit, and mouse spare highly sensitive to spontaneous peroxidaby free radicals produced by exposure to2.This peroxidation causes the sperm’s plamembrane to become leaky and eventuallysults in a complete loss of motility. They hashown further that the rate of lipid peroxidatin these sperm and the rate of consequent lomotility is a linear function of the oxygen partpressure in the medium and the temperatur

Because of the central role of oxygen information of superoxide radicals and other

active oxygen species (ROS), Mazur and Mazuronighprotanre

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190 MAZUR ET AL.

et al. (30, 31) have suggested that contributiof radicals and ROS to freezing damage mbe lessened if the entire cryopreservationcedure (collection, processing, cryoprotecexposure, freezing, thawing, cryoprotectantmoval, and survival assay) were carriedunder reduced oxygen tension. To achieveoxygen tensions at all times, he proposedincorporate an Escherichia coli membranpreparation, Oxyrase, into all solutions. Thmembrane preparations contain the electransport systems and in the presence of aable hydrogen donor substrate (e.g., lactthey can reduce the oxygen concentrationsolutions to low levels even in rather open ctainers (1).

Before incorporating Oxyrase into crypreservation experiments, we needed to dmine the degree of oxygen depletion achieduring the prefreeze and postthaw procedsteps and whether either the Oxyrase oroxygen depletion has adverse effects onsperm (27). Measurements of the oxygen ccentration in aliquots of solutions after variosteps in the procedure showed that, excepabout a 2-min period after a filtration step,Oxyrase maintained the oxygen concentratat 3% or less of that in solutions equilibratedair. The presence of Oxyrase and these lowoxygen tensions has no effect on the motilitythe sperm even after 2.5 h at 23°C. Whenconditions are such as to produce an intermate concentration of oxygen (e.g., largerspace above the solution, low substrate contration), the Oxyrase, however, does causnear doubling of the beat frequency of thegella. The phenomenon, which we term hypmotility, is different from hyperactivation.

We have found that the presence of Oxyrthroughout the procedure does indeed cosubstantial protection on mouse sperm inregards. First, it nearly eliminates the damresulting from centrifugation. Second, itduces the osmotic damage associated withabrupt introduction and removal of hyprermotic concentrations of glycerol. Third, bcause of the second point and in combina

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it enhances one’s ability to use concentratof glycerol that are about three times higthan those generally used heretofore. Fourtcombination with the higher glycerol concetrations, it substantially enhances the motilityfrozen–thawed sperm. The first three powere the subject of the study by Katkovet al.(23). The last point is the main subject ofpresent paper.

MATERIALS AND METHODS

Unless otherwise noted, chemicals were fSigma (St. Louis, MO, U.S.A.), the water wdeionized and glass distilled, and osmolaliwere measured with a Wescor Model 55 vapressure osmometer (Wescor, Inc., Logan,U.S.A.).

Isolation of Spermatozoa and Preparation oSuspensions

Collection without swim-up.Male ICR mice(Sprague–Dawley, Inc., Indianapolis, IU.S.A.), 20–25 weeks old, were killed by cvical dislocation. The cauda epididymides avas deferentia were excised and the two pwere transferred to two 35-mm plastic pdishes (Falcon, Cat. No. 3001) containing 0ml of a modification of the medium used by Tet al. (47), namely Dulbecco’s PBS (GibBRL–Life Technologies, Rockville, MDU.S.A., Cat. No. 14040-026) supplemenwith 14 mM DL-lactate, 16.7mM pyruvate, and0.5% of bovine serum albumin (Bayer/MilPentex, Kanakee, IL, U.S.A., Cat. No. 8001-3) (SD-PBS). The SD-PBS in one ofdishes also contained Oxyrase (see below)ter removal of adventitious tissue, the two edidymides were transferred to two Columwatch glasses (A. H. Thomas, Cat. N979OR15) containing;1 ml of SD-PBS anhen minced. One of the watch glasses cained Oxyrase (see below). The released spere then transferred by Pasteur pipettelastic 1.5-ml Eppendorf centrifuge tube (Co

ng Costar Corp., Cambridge, MA, U.S.A., Co. 3620). Spermatozoa were also strip

rom the corresponding vas deferens and ad

to the same tube. The latter constituted aboutserve

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10% of the total sperm collected. [Songsaand Leibo (42) have reported that cryopreseejaculated sperm have similar propertiescryopreserved caudal epididymal sperm.]contents of the tube were then allowed to pthrough a nylon strainer (Falcon, Cat. No. 23with a mesh size of 70mm to remove mostissue. The filtration was achieved by touchthe bottom of the strainer to a drop of SD-Pin a 3001 Falcon petri dish. The resulting spension was then transferred to a second, tEppendorf tube. The final volume after filtratiwas adjusted to;1.5 ml with SD-PBS and th

xact volume was determined from measents of the weight assuming a density o/ml. A few microliters were removed to sers what we refer to as the “Raw” control. Toncentration of sperm at this point was;12 306/ml.Collection by swim-up.After mincing the ep

ididymides and squeezing out the contentthe vas deferentia, 0.7–0.9 ml of the spetissue suspension was pipetted into the cuend of a Kolmer centrifuge tube (Corning Sentific, Big Flats, NY, U.S.A., Cat. No. 8360This truncated tube was then placed in a 3incubator for 30 min. When Oxyrase wpresent (below), the truncated tip of the Kolmtube was filled to the top with suspensioncovered with Parafilm during the incubatioAfter the incubation, all except the bottomto 0.2 ml of the column of suspension (whcontained adventitious tissue) was removedtransferred to a tared Eppendorf tube. SufficSD-PBS was added to bring the volume to ab1.5 ml, and the exact volume was determineweighing the tube.

Preparation of suspensions in the presencOxyrase.As stated above, in each experimthe epididymis and vas deferens from oneof the mouse were isolated, processed,maintained in medium that contained OxyraThis bacterial membrane preparation wpresent from the moment of sperm extracthrough the determination of motility. Priorextraction of the sperm, some eight to1.5-ml Eppendorf tubes were nearly filled w

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ml of EC-Oxyrase suspension (Oxyrase CoMansfield, OH, U.S.A.), making the final cocentration of Oxyrase 3.8% (v/v), and leavminimal air space in the tubes. Lactate servethe substrate for the Oxyrase. Its concentrawas 17 mM [14 mM in the SD-PBS plus 3 mcontributed by the lactate that is included instock Oxyrase (EC-Oxyrase consists of aticulate fraction of E. coli membranes supended in 20 mM pH 7 phosphate buffer asupplemented with 100 mM lactate)]. The Ependorfs were tightly capped and placed i37°C incubator for at least 30 min prior to uIn about 10 to 15 min at 37°C, the oxygconcentration dropped to less than 1% of ament as indicated by the decoloration of test sples containing 0.001% methylene blue.

These anaerobic preparations of OxyrasePBS were used in each step of the collectionprocessing procedure in lieu of SD-PBS. Oan Eppendorf was uncapped, the desiredumes were used immediately, and any resiwas discarded. As mentioned, Kressinet al.(27) and Mazur and Cole (unpublished) hshown that this procedure keeps the oxyconcentration in the preparations at less thanof equilibrium, except for a brief spike durinthe nylon filtration when it rises to;10% ofequilibrium.

Mixing with Cryoprotective Solution

In the absence of Oxyrase.A 200-ml aliquotof the untreated sperm suspension was drinto a calibrated 0.25-ml straw (No. A20IMV, l’Aigle, France) with a 1-ml syringe antransferred into a 1.5-ml Eppendorf tube. [Tstraws were calibrated by placing colored maat intervals corresponding to 50, 100, 150,200ml. Gravimetric measurements indicate tthe volumes were accurate to 10ml.] At zerotime, a total of 200ml of a double-strengtcryoprotective solution was added in three sto the 200ml of sperm. The stepwise additiwas achieved by adding 20, 50, and 130ml ofthe stock solutions at 15-s intervals usinRanin Pipetteman. The sample was gemixed by inversion after the second and th

additions. The volumes added and the time in-eefteits

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192 MAZUR ET AL.

tervals between them were calculated to kthe maximum osmotic shrinkage constant aeach step of the addition and within the limtolerated by the sperm (22).

The double-strength cryoprotective solutconsisted of 20% (w/v) raffinose pentahydr1.6 M glycerol, and 2.0% (w/v) bovine serualbumin in the modified supplemented Dulbco’s phosphate-buffered saline (SD-PBS).cause 20% raffinose exceeds the solubilitroom temperature, the solution was warme47°C to achieve dissolution, cooled backroom temperature, and used within;2 h of

reparation. We have determined that thenose will remain supersaturated for at le–8 h, although with longer times it beginsrecipitate (Mazur and Cole, unpublished).reliminary test experiments, the motilityperm exposed to 7.5% raffinose and 0.8lycerol for 5 min at 0°C was unaffectedarying the BSA concentration from 0.5 to 4%s a result of the 1:1 dilution with the speuspension, the final sperm suspensionained 10% (w/v) raffinose pentahydrate (0), 0.8 M glycerol, and 1.25% BSA in SDBS. The osmolality exclusive of the glyceas 460 mosm.In the presence of Oxyrase.The paired sam

le of sperm collected in the presencexyrase was then immediately diluted 1:1 wouble-strength cryoprotective solution follo

ng the same procedure as under “In the absf Oxyrase” above, except that in this caseryoprotective solution contained 3.8% Oxyrnd had been rendered anaerobic by preinc

ion. In repeat experiments, the order of mixhe no-Oxyrase and Oxyrase samples wasersed.

oading into Straws

Immediately after mixing the sperm wryoprotective solution, 100ml of the resulting

suspensions was drawn by a 1-ml syringetwo 0.25-ml straws, one for the Oxyrase spension and one for the suspension lac

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An approximate 1-cm column of SD-PBS wdrawn into the straw, followed by a;0.5-cmolumn of air, followed by the 100ml of sperm

suspension, followed by a;1-cm column of airand finally followed by 30–40ml of PBS. Thisfirst column wetted the resin plug thereby sing that end of the straw. The opposite eabove the 30–40ml of PBS was heat sealeEach straw was then inserted approximacoaxially, heat-sealed end down, into a loglass tube (1253 8 mm o.d.) capped withrubber seal and mounted on a plastic holOnly about 3 mm of the top of the straw ptruded from the top of the rubber seal. Tsperm suspension was located about halfinto the tube. The plastic holder also heldglass tube containing a dummy straw in whthe junction of a calibrated 30-gauge coppconstantan thermocouple was inserted midinto a 100-ml column of cryoprotective solutioor a 100-ml column of SD-PBS. The thermcouple was attached to a Fluke 2190A digthermometer (John Fluke Mgf. Co., SeatWA, U.S.A.), and freezing was initiated with0 1 5 min (0 being the time of initial conta

etween the sperm and the cryoprotective sion).

The exact sequence was as follows: A nxyrase suspension was mixed 1:1 with cryp

ective solution and loaded into a straw asescribed, and the straw was placed in

mmediately thereafter, a suspension wxyrase was mixed 1:1 with cryoprotective

ution containing Oxyrase and loaded intoecond straw, which was placed in ice. Btraws, along with a dummy straw furnishith a thermocouple, were immediately froz

o and maintained at270°C by the procedureescribed under “Freezing” below. Five mtes or less elapsed between the initial mixith cryoprotectant and the initiation of free

ng. The procedure was then repeated foecond set of two straws, but with the mixnd loading order reversed; i.e., the Oxyruspension was handled first and then thexyrase suspension.

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Freezing

Freezing was initiated by abruptly immersthe three glass tubes containing the two eximental straws and the dummy straw withthermocouple to within 1–2 cm of their top ina stirred alcohol bath at2406 1°C (FTS Sysems, Stoneridge, NY, U.S.A., Model BC7A). They were held in that bath until the teerature indicated by the thermocouple fel35°C and were then abruptly transferred

arge 4-liter Dewar containing ethanol cooled75°C with dry ice. The temperature in tummy straw fell to270 to272°C. Two min-tes later, the two experimental straws wemoved from their glass tubes and quicmmersed to near their tops in a small Deontaining ethanol at270 to 275°C, wherehey were held while the freezing procedurehen repeated with the second pair of expental straws.The contents of the straws were not seede

nduce crystallization. As a consequence, tupercooled to210 to215°C before nucleatinpontaneously. Their temperature rebounde25°C and then fell again after a short platehe postplateau temperature and times woted. The mean cooling rate from210 (postlateau) to265°C was 276 2°C/min. A rep-esentative plot of temperature vs time is sho

FIG. 1. Representative cooling curve of a 0.10.25-ml straw subjected to the two-step cooling d

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o the275°C bath. The purpose of the cappong glass outer tubes was twofold. One walow the cooling rate of the straws. The otas to minimize vertical temperature gradieoth by the capping and by the fact that

ength of the tubes allowed nearly complmmersion of the straws in the bath. The rubap also served to hold the straws coaxiallhe glass tubes.

hawing and Dilution

Each of the four experimental straws whawed and diluted separately so as to minimhe length of contact with the 0.8 M glycerolhe cryoprotective solution. Thawing wasected by holding a straw for;2 s in air andhen placing it in room-temperature water.5 to 1 min. The straw was then cut on eitide of the column of suspension and the 30

ml column of SD-PBS, and the contents of bcolumns were expelled into an Eppendorf tuThe mixing of the two columns producedfirst step of a five-step dilution. To that 14150 ml of suspension was then added 81, 1515, and 590ml of SD-PBS at 15-, 15-, 30-, an30-s intervals for a total of 1400ml. The resulting 15-fold dilution thus quickly reduced t

column of SD-PBS/0.8 M glycerol/10% raffinose in aribed under Materials and Methods.

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concentrations of glycerol and raffinose to 0.05tepth

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194 MAZUR ET AL.

M and 0.7% (0.01 M), respectively. These ssizes and spacing were calculated to keepmaximum osmotic swelling constant after estep of the dilution of glycerol and within thlimits tolerated by the sperm (22). The secstraw was then subjected to the same thawand 15-fold dilution procedures. The total tithat the sperm were in contact with the fustrength 0.8 M glycerol and 10% raffinose.0°C was#5 min. The SD-PBS used to diluhe Oxyrase suspension also contained Oxynd had been rendered anaerobic prior to u

ontrols

Three controls were used. “Raw” contronsisted of aliquots of the Oxyrase and nxyrase sperm suspensions withdrawn after

on filtration and not subjected to any othreatment except a later 1:1 dilution with SBS to bring the counts into the proper ra

;80 cells/microscope field). A zero-glycerentrifuged control consisted of the “raw” corol subjected to a 1:1 dilution with SD-PBS aentrifugation (see below), but not loaded ir expelled from straws. A “prefreezing” co

rol consisted of samples exposed to cryoective solution, loaded and expelled frtraws, and diluted with the same procedused for the experimental straws, but not s

ected to cooling or freezing. This prefreezontrol was prepared either while the expental samples were at270°C or after thxperimental samples had been thawed an

uted 15-fold.

entrifugation

The four diluted experimental samples (tith Oxyrase; two without), the two diluterefreezing controls (one with Oxyrase; oithout), and the two 0-glycerol controls (oith Oxyrase; one without) were centrifugimultaneously at 2506 2 g for 8 min in anngle-head bench top centrifuge with an ef

ive radius of centrifugationR of 7 cm. Theentrifugal acceleration RCF (expressed ing’s)t the bottom of the tube was calculated us

he formula RCF. 11.2 * N2 * R, whereN is

e

g

t

se.

--

-

s-

-

i-

-

g

rpm’s) as measured by a stroboscope (MDS-303; The Pioneer Electric and ReseaCorp., Forrest Park, IL), andR is the effectiveradius of centrifugation (expressed in cm). TRCF and centrifugation time were selectedthe combination that we estimated would mimize both the total yield and the motility, bason the studies by Katkov and Mazur (24, 2After centrifugation,;1450ml of the supernatant was withdrawn with a 16-gauge cannattached to a 10-ml syringe and discarded.now emptied cannula was then used to repend the pellet in the remaining 50ml by verygentle aspiration. This procedure concentrthe sperm approximately 30-fold and returthem to the same concentration they had anylon filtration and prior to the addition of thglycerol. This was approximately 123 106/ml.

Determination of Motility

The motility was assessed essentially asscribed by Katkov and Mazur (24). The ondifferences were that the counts were musing 20-mm depth Microcells (ConceptioTechnologies, San Diego, CA, U.S.A.) ratthan Hamilton–Thorne chambers, andcounting chambers were loaded with 6ml ofsperm using a Pipetteman rather than a Drmond 100-ml Microcap attached to a barrel mcrometer. Based on Schreuderset al.(36) studywe had believed that the Drummond microcwould produce less mechanical injury thanPipetteman, but, when tested, we did notthat the case with respect to loading the coing chambers.

Motility counts were made;15-30 min afteentrifugation, except in one experiment, tere made;l hr later. Five or more fields we

counted at a magnification of 250 X.

Statistics

Homogeneity of nontreated (raw) contsperm data was assessed by Cochran’s andlett’s criteria. All statistical analyses were pformed on Microsoft Excel 5.0 with a setformulas developed earlier (26).

The results were analyzed in terms of th

values: (i) motility (the percentage of cells in atyusoferepeumars

tsceol-/v)ola

ndado-ina

lowhy

asge

he

totathetioenef

dialf-

mo-

toperedll-

strength SD-PBS 1:1 with water and then add-eir

SD-

rmr 5Mn-arsx-ub-hermof

ristA,the

aseven

atedirdionor

rs)heropari-se/iallyerefththe

taon-es-in

ingof

seetrol,of

theve


microscope field that are motile), (ii) motilinormalized to that in untreated raw sperm spension, and (iii) motility normalized to that0-glycerol centrifuged control. Estimates walso made of the numbers of motile spermmouse, both absolute and relative to the nbers of motile sperm in raw controls. Error bon the graphs are standard errors.

RESULTS

Effect of the Concentration of Raffinose andthe Total Osmolality of NonpermeatingSolutes on Motility

As a preliminary to the freezing experimenwe needed to decide on the appropriate contration of raffinose to include in the glycercontaining medium. The addition of 15% (wraffinose pentahydrate raises the total osmity of nonpermeating solutes (raffinose1 SD-PBS) to 575 mosm. Willoughbyet al. (48) haveshown that that degree of hypertonicity aconsequent cell shrinkage is damaging. Thedition of 0.8 M glycerol raises the total osmlality to 1430 mosm, so that if it were addedone abrupt step, the cells would experienceadditional transient shrinkage to volumes bethe nontransient shrinkage produced by theperosmolal nonpermeating solutes. In our chowever, the additional transient shrinkafrom the glycerol is kept small by adding tglycerol stepwise (22).

There are two approaches to keeping theosmolality of nonpermeating solutes withinbenign range. One is to reduce the concentraof raffinose. The other is to reduce the conctration of SD-PBS. We have compared thefects of 0, 7.5, and 15% raffinose in mecontaining full strength (290 mosm) or hastrength (145 mosm) SD-PBS. The total oslalities of raffinose1 SD-PBS in 0% raffinose/13 PBS, 7.5% raffinose/0.53 PBS, 7.5%raffinose/13 PBS, 15% raffinose/0.53 PBS,and 15% raffinose/13 PBS were determinedbe 290, 294, 417, 432, and 576 mosm, restively. The half-strength SD-PBS was prepaby diluting suspensions of sperm in fu

-

r-

,n-

l-

-

n

-e,

l

n--

-

c-

ing BSA, lactate, and pyruvate to keep thconcentrations the same as in full-strengthPBS.

Figure 2A compares the motility of spewith and without Oxyrase after suspension fomin at 0°C in these five media containing 0.8glycerol, followed by stepwise dilution and cetrifugation. In addition, the second set of bfrom the left shows the motilities of cells eposed to neither glycerol nor raffinose but sjected to centrifugation (0-Glyc-Cfg), and tright-hand set shows the motilities of spesimilarly exposed to a commercial medium10% egg yolk and 0.82 M glycerol in Tes/Tbuffer [TYGB (TEST yolk glycerol buffer), CaNo. 9971, Irvine Scientific, Santa Ana, CU.S.A.). In each of these seven cases and inuntreated control, Oxyrase and non-Oxyrwere compared in split samples from a gimouse.

As reported by Katkovet al. (23), there issignificant drop in absolute motility associawith the 5-min exposure to 0.8 M glycerol (thvs second set of bars in Fig. 2A). The additof 7.5% raffinose to either half-strength PBSfull strength PBS (fourth and fifth set of baproduced no further drop in motility, but taddition of 15% raffinose did produce a dthat was significant in several of the compsons relative to that of sperm in 7.5% raffinofull-strength PBS. The decrease was especsignificant when 7.5 and 15% raffinose wcompared in full-strength PBS (seventh vs fiset of bars) and was even more significant inabsence of Oxyrase (P 5 0.001) than in itspresence (P 5 0.03). On thebasis of these daand since we wished to keep the raffinose ccentration in the range reported by other invtigators, we decided to use 10% raffinosefull-strength SD-PBS in subsequent freezexperiments. This solution has an osmolality460 mosm.

With respect to the effects of Oxyrase, wethat in each case except the untreated conthe motilities were higher in the presenceOxyrase than in its absence. In three ofindividual sets (indicated by probabilities abo

sig-ocheonos

iesthe

thatol-re--se.of

tan-rol/

dirsbers

roltheingthe

. In

196 MAZUR ET AL.

the bars), the differences were statisticallynificant or marginally significant. All the protective effect of Oxyrase appears to be assated with its ability to protect the cells from tphysical manipulations involved in collectiand centrifugation. That can be seen mclearly in Fig. 2B where the absolute motilitin Fig. 2A have been normalized to that of

FIG. 2. Motilities of mouse sperm after a 5-min0, 7.5, and 15% (w/v) raffinose pentahydrate (bars3, 5, and 7) or half-strength SD-PBS (bar pairs 4 a:300, :450, and :600 are the nominal milliosmolaligiven in the text. The figure also shows data formedium (right-hand set of bars) and two controlsabsence of glycerol and raffinose (two left-hand sOxyrase were split samples from a given mouse.sperm were first isolated through the time that the(B), they are normalized to that of the 0-glycerol-

-

i-

t

0-glycerol-centrifuged samples. Expressedway, the protective effect of Oxyrase is abished. This finding is consistent with thatported by Katkovet al. (23) for sperm suspended in glycerol in the absence of raffino

We note parenthetically that the motilitiescells exposed to TYGB medium were substially higher than those exposed to the glyce

osure at 0°C to solutions containing 0.8 M glycerol anbeled 0R, 7.5R, and 15R)] in either full-strength (bar pa) and in the presence or absence of Oxyrase. The numof SD-PBS1 raffinose. The actual measured values are

ls exposed to a commercial Tes–Tris–egg yolk–glycereated sperm and sperm subjected to centrifugation inof bars)]. The suspensions containing and not containen Oxyrase was present, it was present from the timeotility was assessed. In (A), the motilities are absolutetrifuged controls (four mice;n 5 4).

expla

nd 6tiescel[untetsWhir mcen

2Aenc

exin-

dy/rafase

s tole

M

rmw s int the

u on-O splits otil-i thep ignif-i nceo ft)s ofn s ito by an rs( s inm d tot ng;i ndo ing


raffinose/SD-PBS medium and that in Fig.there was no difference between the presand absence of Oxyrase. We have not yetplored the effects of freezing in media containg egg yolk since our goal in this initial stuwas to investigate the response to glycerolfinose. However, we note again that Songsand Leibo (41) and Gilmoreet al. (19) havehown that egg yolk broadens the osmoticrance of mouse sperm.

otilites of Sperm after Freezing to270°Cand Thawing

Figure 3A compares the motility of speith and without Oxyrase after various step

he freezing procedure normalized to that of

FIG. 2—

e-

-n

-

ntreated raw controls. The Oxyrase and nxyrase suspensions were prepared fromamples from each mouse. The absolute mty of the raw controls was 53 and 56% inresence and absence of Oxyrase (a nons

cant difference). Centrifugation in the absef glycerol (second pair of bars from the leignificantly reduced the normalized motilityon-Oxyrase suspensions to 77%, whereanly reduced that of Oxyrase suspensionsonsignificant 4 to 96%. The third pair of baPFRZ) shows that there was additional losotility when the suspensions were subjecte

he full range of treatments except for freezi.e., the stepwise addition of 1.6 M glycerol ather components of the medium with mix

ntinued
Co

isgawaen

n be-t pen-s

inthenermpwised

eno

omas

198 MAZUR ET AL.

after each addition, a 5-min exposure, stepwdilution and mixing, and subsequent centrifution and resuspension. That additional losssignificant in the case of the Oxyrase susp

FIG. 3. Effect of the presence or absence of Othe cryopreservation process. Untreated or “raw”Nylon filter or after swim-up. 0-Gly-CFG refers toand resuspension of suspensions that contained nsubjected to the identical procedures as frozen–thaddition of cryoprotective solution, loading intoresuspension. FROZ refers to the motility of suspeIn (A), motilities are normalized to that of the untre0-Gly-CFG control. The data bars represent the rswim-up and two with swim-up). Suspensions cona given mouse frozen at the same time. At the coadded to a second set of plus and minus Oxyras10). Theerror bars are standard errors. Valuesdifferences between Oxyrase and no Oxyrase. Tdifference.

e-s-

sions (P , 0.02) but was notsignificant in theon-Oxyrase suspensions. The difference

ween the Oxyrase and non-Oxyrase susions was marginal (P , 0.1). Thefourth pair

se on the motility of mouse sperm after various stepsers to the motility of the sperm after passage through

motility of the untreated sperm after the centrifugatioryoprotective solution. PFRZ refers to the motility of spd samples except for the absence of freezing, i.e., steout of straws, stepwise dilution, centrifugation, an

ons after cooling at 25°C/min to275°C and rapid thawing.d control. In (B), motilities are normalized to that of th

lts of experiments on sperm from five mice (three withning and not containing Oxyrase were split samples frletion of the freezing of the first pair, cryoprotectant wspensions and this second set was frozen to275°C (n 5

ove the pairs of bars are thet test probabilities for theabsence of a value meansP . 0.1; i.e., a nonsignificant

xyrareftheo caweandnsiate

esutaimp

e suabhe

fterl-

i e oO , ah mO ft eO

byO ons omc rem the0 theu orm ro-

t or-m ses

omt d tos col-l woc om-p arsi thep litiesa ols.T hatO tec-t tedw


of bars (FROZ) compares the motilities afreezing to270°C and thawing. The normazed motilities in the presence and absencxyrase were 47 and 31%, respectivelyighly significant protective effect froxyrase (P , 0.001). Theabsolute motility o

he frozen and thawed sperm in the presencxyrase was 25%.A substantial amount of the protectionxyrase after freezing and thawing was a cequence of the protection it conferred frentrifugation. That is shown in Fig. 3B wheotilities have been normalized to that of-glycerol-centrifuged controls instead ofntreated raw controls. But even with that nalization, Oxyrase conferred significant p

FIG. 3—

f

of

-

-

ection from freezing and thawing (49% nalized motility vs 41% for non-Oxyra

uspensions,P , 0.01).The results in Fig. 3 are pooled data fr

hree experiments using sperm not subjectewim-up and two experiments using spermected after swim-up. The results for the tollection procedures are separated and cared in the center and right-hand pairs of b

n Fig. 4 for sperm collected and frozen inresence and absence of Oxyrase. The motire normalized to the respective raw contrwo conclusions are evident. One is txyrase exerted a substantially greater pro

ive effect on frozen–thawed sperm collecithout swim-up (54 vs 33%,P , 0.01) than

ntinued
Co

-upe

nt,in

ondof

ase

seanledelyfac

chhatin

re-n-mit ishe

tileeanm

edromre

tive

200 MAZUR ET AL.

on frozen–thawed sperm collected by swim(38 vs 28%). The difference between the formpair is highly significant (P , 0.01), but thedifference in the latter pair is not significaprobably becausen was only 2 for each setthe pair.

The second point is that the normalized mtilities of non-swim-up sperm after freezing athawing were actually higher than thatswim-up sperm. The difference in the Oxyrsuspensions (54 vs 38%) was significant (P ,0.01), but thedifference in the non-Oxyrasuspensions (33 vs 28%) was not significWith the Oxyrase and non-Oxyrase data poothe two values were 43 and 33%, respectivPart of that difference is ascribable to the

FIG. 4. A comparison between the motilities of fr6) swim-up and in the presence or absence of ORaw or unhandled controls.

r

-

t.,.t

that the motility in the raw controls on whithe normalization was based were somewhigher in the swim-up samples (61%) thanthe non-swim-up samples (48%).

If one assumes that sperm motility is a prequisite for full function, the important quatity from the point of view of mouse sperbanks is less the percentage of motility thanthe motile yield after freezing and thawing. Tabsolute motile yield is the number of mocells in a suspension after treatment. The mnumber of motile cells in the raw controls froa given mouse was 728 3 106. The meannumber of motile cells in the frozen–thawsuspensions from a given mouse varied f1.4 to 3.33 106. Both sets of numbers we

n–thawed sperm collected with (n 5 4) or without (n 5se. The motilities are normalized to that of the respec

ozexyra

subject to considerable variability because ofse

onn oasthefoenera

awt o

ash ofc thec taia n.

lityo

heumatnugelu-t o

sto

ateupanted

f Int vau ren wat then dsaa

rea nsih susp lity

after 75 and 150 min at room temperature. Theialo-forpec-

m-

n-othnce.es,

inof

tiallyionsverhese%.ose

om-e isgar;ofall,0.3oseons

o-gg-

tose.umf-m

wasob-entoododutes-bina--

tion


animal-to-animal variation, mouse-to-moudifferences in the efficiency of the collectiprocedure, and limitations as to the precisiothe volume in which the centrifuged pellet wresuspended. Because of these variations,were no significant differences in the valuessperm collected and processed in the presand absence of Oxyrase and between spprocessed with and without swim-up. There wa tendency for the absolute motile yield of rswim-up suspensions to be lower than thanon-swim-up suspensions (6.83 106 vs 8 3106) even though the percentage of motility w

igher in the former than the latter. Thisourse reflects the fact that in swim-up,ollected supernatant fraction does not conll the motile cells in the original suspensioThe animal-to-animal sources of variabi

are eliminated if one examines the relative mtile yield; i.e., the number of motile cells in tfrozen-thawed suspensions divided by the nber of motile cells in the raw control for thsuspension. These relative motile yields aremerically equal to the normalized percentaof motilities shown in Fig. 4, and the concsions are those given in connection with thaFig. 4.

The ability of Oxyrase to protect againdamage from centrifugation in the absenceglycerol and freezing also tended to be grefor non-swim-up sperm than for swim-sperm, but the differences were not significThe motilities of these 0-glycerol-centrifugcontrols in Oxyrase were 996 3 and 916 8%or no swim-up and swim-up, respectively.he absence of Oxyrase, the correspondinges were 816 4 and 716 1%. These values aormalized to the raw controls. The same

rue for the prefreeze controls. In Oxyrase,ormalized motilities for no swim-up anwim-up were 866 5 and 726 2%. In thebsence of Oxyrase, the values were 696 11nd 676 7%.The motilities shown in Figs. 3 and 4 we

ssessed some 10 to 30 min after the suspead been centrifuged and the pellets reended. Figure 5 shows the stability of moti

f

rercems

f

n

-

-

-s

f

fr

.

l-

s

on-

motilities are normalized to that of the initmotility of the raw controls. The absolute mtilities of those controls were 52 and 56%Oxyrase and non-Oxyrase suspensions restively. The sperm were collected without swiup. In agreement with the findings of Kressinetal (27), the motility of the untreated Raw cotrols remained stable over the 2.5-h period bin the presence of Oxyrase and in its abseThe motility of the frozen–thawed samplhowever, did decrease significantly with timeboth suspensions. As in Fig. 3, the motilitythe Oxyrase suspensions was substanhigher than that of the non-Oxyrase suspensat the first time point, and it remained so othe 2.5-h period. At the 15-min time point, tnormalized motility of the sperm in Oxyrawas 54%. At the 150-min point, it was 23The absolute motilities were about half thvalues.

DISCUSSION

The media used to freeze mouse sperm cmonly contain three major components. Onglycerol; the second is a nonpermeating suand the third is a macromolecule or complexmacromolecules. Glycerol, when present atis generally used in low concentrations (0.2–M). The usual nonpermeating sugar is raffinpentahydrate in 10–18% (w/v) concentrati(0.17–0.3 M). The macromolecule or macromlecular complex is bovine serum albumin, eyolk, or skim milk. The skim milk also contributes some nonpermeating sugar; i.e., lacTadaet al. (45) developed a successful mediusing a combination of low glycerol, high rafinose, and low BSA. It is basically the mediuused in our study. They found that the sugarmore important than the glycerol; i.e., theytained no motility when glycerol was presbut raffinose was absent. They obtained gmotility (43%) with the reverse, nearly as goas the 61% they reported when the two solwere combined. Songsasenet al. (39) developed a successful medium based on a comtion of a low molarity of glycerol, a high concentration of raffinose, and a high concentra

46omin

att inan

rvaolerosu

ionM,edses

inure

or-ingthe.25tes

)andnd

ofwing

202 MAZUR ET AL.

of egg yolk. Nakagata and co-workers (32,developed a successful medium that is cposed of 3% skim milk and 18% raffinosewater. It lacks glycerol.

A common thread in all the media is thglycerol is either not present or is presenconcentrations that are distinctly lower thhave been found optimal for the cryopresetion of most mammalian cells when it is the scryoprotectant. Mouse marrow stem cells pvide a nice example of the dependence ofvival on glycerol concentration. Leiboet al.

FIG. 5. Effect of the presence (– – –) or absenand frozen–thawed (bottom two curves) sperm asresuspension. Motilities are normalized to thatnon-Oxyrase controls. The sperm were initially coOxyrase and subsequently maintained either in thwere carried out in duplicate straws.

)-

-

-r-

(28) found that as the glycerol concentratwas increased from 0 to 0.4, 0.8, and 1.25survival at the optimal cooling rate increasfrom 3% to 20, 45, and 65%. Similar responto glycerol concentration have been foundother cells, including hamster tissue cultcells (29) and human red cells(43).

The stem cell study illustrated another imptant point, namely, that the optimum coolrate increased from 1° to 100°C/min asglycerol concentration was reduced from 1to 0.4 M. The need to use higher cooling ra

(—) of Oxyrase on the motility of Raw (top two curvesnction of the elapsed time at 23°C after centrifugationthe 15-min point for the respective raw Oxyrase ated without swim-up either in the presence or absenceesence or in the absence of Oxyrase. Freezing and tha

cea fuofllece pr

with suboptimal glycerol concentrations mayus- t

nlyeibllsthewan a°Cd

tri-rmnd,ofa

M)hawa

conith

boedw

ee1320edol%ol,f

topti-ei-fteiveel-rme-

ese

lyco

onee3)

in frozen–thawed ram and porcine sperm. Weonsun-the

23).en-valiedtic

thatuchthatmi-ex-lossarein-tyb-to

eshem-M

byef-u-

beap-iveuc-of

illt ro-z tes)i ed5

freer aves theo rad-i inf sultso genc inga tra-


explain why most published studies on mosperm have used cooling rates that are 1020-fold higher than the 1–2°C/min commoused for mammalian cells. Songsasen and L(38), for example, reported that with the cesuspended in raffinose plus 0.3 M glycerol,percentage of sperm with intact membranesmany fold higher when the sperm were froze20°C/min than when they were frozen at 2min. Similarly, Fiser and Fairful (12, 13) founthat lower cooling rates were distinctly demental to the motility of ram and porcine spefrozen in 3–4% glycerol. On the other haStoreyet al. (44) obtained high percentagesintact membranes in mouse sperm frozen1–3°C/min in the presence of 6% (0.65glycerol plus 7.5% raffinose (or, better, trelose), especially when the cryoprotectantadded slowly by dialysis.

The responses of mouse sperm to hightrolled rates have not been determined wmouse sperm, probably because rates a20°C/min are not obtainable with controllcooling rate freezers like the Planer (40). Hoever, cooling rates up to 100°C/min have binvestigated in ram and porcine sperm (12,and rates up to 800°C/min in human sperm (In the former studies, the highest rates yieldslightly higher motility when 2–4% glycerwas used but a slightly lower survival with 8glycerol. In human sperm in 0.85 M glycerHenry et al. (20) found that the motility osperm cooled at 800°C/min was reducedabout half of that of sperm cooled at the omum rate of 1–10°C/min. Interestingly, in nther case was the motility reduced to zero arapid cooling as would be expected if extensintracellular freezing had occurred. If intraclular freezing does not occur in human specooled at 800°C/min, that is a likely consquence of the high water permeability of thcells (18).

Presumably, investigators have kept the gerol concentrations for mouse sperm at 3%lower because they have found that higher ccentrations give inferior results. That has bwell documented by Fiser and Fairfull (12, 1

eo

o

st

t

-s

-

ve

-n)).a

r

-r-

n

have recently reported that concentratiabove 0.3 M are increasingly damaging tofrozen mouse sperm and have shown thatdamage is a consequence of two factors (One is that the introduction of higher conctrations of glycerol and its subsequent remois mechanically damaging if they are not carrout in appropriate steps to minimize osmovolume excursions. Willoughbyet al. (48) andSongsasen and Leibo (41) have shownmouse sperm are especially sensitive to sosmotic perturbations. The second factor ishigher concentrations of glycerol are checally toxic to mouse sperm; i.e., a 20-minposure to 0.8 M glycerol causes substantialin motility even when osmotic excursionsrestricted. Confirmatory evidence for thevolvement of toxicity is that the loss in motiliin 0.8 M glycerol is time dependent. It is sustantially reduced if the exposure time is held5 min or less. Yet this description oversimplifitoxicity. Storeyet al. (44) obtain a rather higprefreeze and postthaw percentage of mbrane intact cells in media containing 0.65glycerol when the glycerol is added slowlydialysis. The authors suggest that the toxicfects of glycerol may be reversible with incbation time.

Our hypothesis is that if mouse sperm canmade to tolerate concentrations of glycerolproaching 1 M, a higher percentage will survfreezing and thawing and do so more reprodibly. This picture is supported by the studyClulow et al. (9) who find that toad sperm wolerate 20% (v/v) glycerol and that when fen (apparently slowly, but at unspecified ran that concentration, the motilities exceed0%.Mouse sperm are also highly sensitive to

adical damage. Alvarez and Storey (5) hhown that the damage is proportional toxygen concentration. To test whether free-

cal damage or other ROS are implicatedreezing damage, we have compared the ref processing and freezing sperm at low oxyoncentrations with the results of processnd freezing them at atmospheric concen

tions of oxygen. We use the term “atmospheric”thens

os e os lowo thec ia.O ati

-m ass ng

dn nds het yc-e tiap ant erf thp stei icac n-o er-e arlt (b)i ives oticd e tf Mg –3 -t byK o-t ndm get

tedt t1 et tlyd ati mW em ityo o-t di-

tions. The osmolality of the raffinose and PBSi on-c heyf

hatw iti-a her-m inga rmt –3m oxice °C( psef ur-i rolw 40)h hens erolw ndp tedt nrr ol-i entr li sist and2 itri-fi -fi ghs ucha in-j edb

de-s nifi-c se;it hel en-t tot ossf o-t i-n( Rm teds sing

204 MAZUR ET AL.

rather than “normal” to emphasize thatsperm do not see atmospheric concentratioinvivo. Rather, they see about one-fifth of atmpheric (3) and have evolved in the presencuch reduced concentrations. To achievexygen we have introduced Oxyrase intoollection, freezing, and diluting medxyrase is anE. coli membrane fraction. Wh

s novel about its use is that it permits the O2 tobe kept low (,5% of ambient) from the mo

ent of isolation of the sperm through theessment of motility after freezing and thawiThe experiments reported here combine

umber of potentially beneficial factors aought to test the specific points of (i) whethe combination of a high concentration of glrol and short exposure would yield substanercentages of motile sperm after freezing

hawing and (ii) whether the motilities aftreezing and thawing would be enhanced byresence of Oxyrase. The combination te

ncluded (a) exposure to a more-or-less typryoprotective solution containing 10% raffise and 1.25% BSA, with the important diffnce that the glycerol concentration was ne

hreefold higher than usual (0.8 vs 0.3 M);ntroducing and removing the cryoprotectolution in steps designed to minimize osmamage (22); (c) restricting the total exposur

ull-strength cryoprotectant (and the 0.8lycerol) to 5 min or less; (d) cooling at 200°C/min to270°C; (e) rapid thawing; (f) cen

rifugation at an acceleration and time shownatkov and Mazur (24) to give maximum m

ile yield; and (g) carrying out all pipetting aixing gently to minimize mechanical dama

o the sperm (36).One comment about point (a): We restric

he concentration of raffinose pentahydrate0% (w/v) because the data in Fig. 2 show

hat 15% raffinose in SD-PBS is significanamaging. The osmolality of the (nonperme

ng) solutes in that latter solution is 576 mosilloughby et al. (48) have shown that thotility of ICR mouse sperm in that osmolalf NaCl/PBS is reduced to 30% of that in is

onic media even after return to isotonic con

-f

-.a

r

ld

edl

y

o

od

-.

n our freezing solution was 460 mosm, a centration of nonpermeating solutes that tound much less damaging.

Two comments about point (d): One is te purposefully avoided seeding prior to inting subzero cooling. The reason was that tal equilibration of the straws prior to seedt 24 to 25°C would have exposed the spe

o 0.8 M glycerol for about an additional 2in near zero, and we have shown that the tffects of 0.8 M glycerol are not reduced at 023). Furthermore, additional time would elaor the completion of the freezing plateau, dng which time the concentration of glyceill about double. Songsasen and Leibo (ave reported somewhat higher survivals wtraws are seeded, but they used 0.2 M glychere toxicity is not a problem. The secooint about (d) is that we purposefully restric

he minimum temperature to275°C rather thaapidly cooling them from275 to2196°C. Theeason was twofold. One is that the rapid cong by an additional 125°C and the subsequapid warming from2196 to275°C could welntroduce damage of much different genehan those factors operating between 075°C, factors such as thermal stresses, v

cation, and devitrification. Yet275°C is sufciently low to constitute genuine, althouhorter term, cryopreservation; i.e., factors ss intracellular freezing and solution effect

ury will already have occurred or not occurry that temperature.The combination of procedural steps

cribed three paragraphs earlier yielded sigant motility even in the absence of Oxyra.e., 176 1.0% absolute and 316 1.5% relativeo that of untreated raw controls (Fig. 3A). Tatter percentage included a 23% loss from crifugation, a further 9% loss from exposurehe cryoprotective medium, and a 37% lrom freezing and thawing. Although that mility is substantially lower than the 61% origally reported by Tadaet al. (45) using 1.75%v/v) (0.24 M) glycerol with sperm from ICice, it is substantially higher than that repor

ubsequently by some other investigators u

Tada-like media. Penfold and Moore (34) ando--

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Fuller and Whittingham (15) obtained 0% mtility. Songsasenet al. (41) obtained 3–5% moility for sperm from the B6D2F1 strain.

The motility of frozen–thawed sperm wncreased substantially (from 31 to 47% of rontrols) when Oxyrase was present throughe procedure. In Oxyrase, there was a 4%rom centrifugation, a further 15% loss upxposure to cryoprotective medium, and a 3

oss from freezing. Most of the protection whus conferred during the centrifugation. Thalso seen if we normalize the motilities to tf the controls centrifuged in the absencelycerol. In that case (Fig. 3B), much of trotective effect of Oxyrase in frozen–thawamples disappears. Nevertheless, signifirotection is still present (49 vs 41%). Theesults are consistent with our findings wespect to the damage of unfrozen sperm fxposure to high glycerol concentrations (2

n that case, Oxyrase primarily protects agahe mechanical stresses engendered by cegation and osmotic volume excursions.ote that freezing and thawing also prod

arge osmotic volume excursions, and theyainly impose other mechanical forces.

The protective effect of Oxyrase on frozehawed sperm was considerably greaterperm collected without swim-up thanperm collected by swim-up. The normalizotilities for the former were 54% (absolu6%) in the presence of Oxyrase vs 33% inbsence (Fig. 4). The enhanced protectioon-swim-up sperm by Oxyrase suggests

he non-swim-up population contains soperm that are more susceptible to reactivegen species and consequently are more sive to centrifugation and freezing unless pected by Oxyrase.

It might be useful to compare the motilitihat we have obtained for non swim-up spehat are processed and frozen to275°C andhawed in the presence of Oxyrase and 0.lycerol with some of the better survivals tave been reported by others after freezin196C using low glycerol concentrations.entioned, Tadaet al. (45) obtained 61% ab

ts

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ers have failed to achieve. Songsasen and L(40, 41) have reported absolute motilities ofto 29% using a low glycerol/raffinose mediuin which BSA is replaced by egg yolk. Wobtained 25% absolute motility in our higlycerol/BSA/raffinose medium. Several poishould be kept in mind in comparing our reswith those of others. One is that nearly all othstudies used sperm collected by swim-upswim-out. A second is that the other studsubjected the suspensions to rapid coolin2196°C after cooling them at controlled rato ;270°C, whereas, for the reasons statedlimited the cooling to275°C. We have not ayet determined what effect, if any, the adtional cooling will have with our procedureSecond, the mouse strains often differed indifferent reports, and Songsasen and Leibohave shown that strains differ in their surviafter freezing and thawing. Third, Songsaand colleagues maintained the thawed spand assessed their motility at 37°C whereas,most others, we maintained them at room tperature and assessed motility at 25°C. In geral, the decay rates that we observed at 225°C over 150 min (Fig. 5) are similar to thothey reported at 37°C.

Kressinet al. (27) have shown that Oxyraholds the oxygen concentration at,5% of am-bient throughout the procedure (except fobrief increase during nylon filtration). We blieve that its protective effect is due to tlowered oxygen, although it is conceivable tit could be due to some sort of chemical ptection from theE. coli membrane fragmenand other components in this complex mateIt is difficult to devise a definitive control thcan test the effect of the presence of Oxywithout its producing lowered oxygen tensioOne approach would be to exclude lactate fthe medium, since lactate or its equivalentrequired substrate for Oxyrase to remove ogen (1). One problem is that the removallactate might affect the physiology of the speand their freezing sensitivity. Another is thOxyrase will remove oxygen quite effective

even when the lactate concentrations are 10-fold

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206 MAZUR ET AL.

lower than those used here (1).If Oxyrase is indeed protecting the froze

thawed sperm by virtue of its lowering the oygen tension, the next question is whetherhypothesized, that protection is the result oconcomitant decrease in the concentrationoxygen-derived free radicals and other reacoxygen species. While we have no direct edence on this point, there is circumstantial sport for it. Alvarez and Storey (4) and Aitkenetal. (2) have shown that the plasma membraof mouse, rabbit, and human sperm undelipid peroxidation from radicals producedexposure to O2. This peroxidation causes tmembranes to become leaky and eventuallysults in a complete loss of motility. As metioned, Alvarez and Storey (5) have shownthe rate of peroxidation and loss in motilitymouse sperm is a linear function of the parpressure of oxygen in the medium. The ratefully oxygenated medium with a PO2 of 150mm Hg is five to six times that at a PO2 of 30mm Hg, the approximate partial pressurelymph (3) and presumably in the epididymis.our procedure, Oxyrase lowers the partial psure to,7 mm Hg, and it maintains these lotensions throughout the whole procedure (was noted, the exception of a 1- to 2-min perioSince plasma membranes are an importantperhaps the chief target of freezing injury, tinjury may be exacerbated if the membrahave undergone prior free radical damaMoreover, Alvarez and Storey (6) have shothat freezing and thawing lowers the concentions of superoxide dismutase (SOD) in humsperm and as a consequence it reduces theity of the sperm to inactivate the superoxradical which is derived from O2 and is a majocontributor to membrane peroxidation. If fewsuperoxide radicals are produced in the pence of Oxyrase because of the lowered oxytension, even an impaired level of SOD mayable to dismutate them to hydrogen peroxwhich is then converted to water by catalasethe other hand, in a subsequent paper, Alvand Storey (7) concluded that free-radicalduced lipid peroxidation was not a major fac

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motility after freezing and thawing was not iproved by the postthaw addition ofa-tocoph-erol, hypotaurine, or BSA, all known protectoagainst lipid peroxidation. Oxyrase is likelyhave two advantages over these other cpounds in reducing the concentration of reacoxygen species. One is that since oxygefreely diffusible across cell membranes, aduction in its extracellular concentration willquickly translated into a reduction in its intcellular concentration. In the case of adcompounds, limitations in their ability to pemeate the membrane would curtail their incellular effectiveness. Second, with Oxyrasecells see low oxygen concentrations before,ing, and after freezing and thawing and notafter thawing as was the case in the AlvarezStorey study.

Atmospheric concentrations of oxygen hbeen found to be detrimental to thein vitroculture of a number of cell types. That is stringly true of mammalian embryos, as has bcarefully reviewed by Bavister (8). That is phaps not surprising since as he points out, ogen tensions in the oviducts and uteri of hasters are only 50–60 mm Hg, i.e., about oquarter of that in air. In monkey uteri, it is lethan 20 mm Hg. As he documents, a numbestudies show that incubation under reducedygen (usually 5%) results in substantialprovement in thein vitro developmental chaacteristics of mammalian embryos. More recexamples of that beneficial effect have breported for rabbit (17) and bovine embry(14). Bavister discusses the evidence forhypothesis that the injury from contact watmospheric concentrations of oxygen is largdue to the generation of oxygen free radicMore recently, Fujitaniet al. (14) have obtainerather direct evidence that the improvementhe developmental rate to blastocyst of bovembryos in 5% oxygen is related to a decrein free radicals. In the presence of the fradical generator azobis (amidinopropane)drochloride, embryo development was reduin 5% oxygen but it was abolished in 20oxygen. If the free-radical scavengers hypo

rine or SOD were added to suspensions contain-tagbuorno

eno

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ing the free radical generator, the percendeveloping in 5% oxygen was increased,that in 20% oxygen was not. If hypotaurineSOD were added to suspensions that didcontain the free radical generator, the percage developing in 20% oxygen was doubledtripled, but the percentage developing inoxygen was unaffected.

As discussed, Songsasenet al. (39) havedemonstrated considerably higher motilitiesfrozen–thawed mouse sperm when the medcontains egg yolk and no BSA than whencontains BSA and no egg yolk. Others suchDe Leeuwet al. (11) have reported substanprotective effects of egg yolk on frozen–thawsperm, protection that some believe is a resumembrane stabilization (49). There is also soevidence that egg yolk may protect by inducchanges in the plasma membranes of spermlessen free radical damage. Katkov (21)shown that the presence of 20% (v/v) egg ydecreases the extent of peroxidation ofmembranes of frozen–thawed bull spermmay do so because it causes a shift inspectrum of long-chain fatty acids in the mebrane from unsaturated to saturated. For exple, the percentage of C-18-saturated moieincreases from 8 to 13% in its presence andof C-22 with six unsaturated bonds decreafrom 51 to 42%. Fatty acids with a reducnumber of unsaturated bonds will be less sject to peroxidation. Another effect of egg yopresumably unrelated to free radical damawas to increase the cholesterol content ofmembranes by about 21%. Presumably,consequence of these and perhaps other mular changes induced in the membranes, bosperm suspended in egg yolk showed greresistance to the electric fields involved in etroporation and they showed higher motilitafter freezing and thawing (26). Perhaps relato the above is the finding of SongsasenLeibo (41) and Gilmoreet al. (19) that egg yolalso substantially reduces the osmotic sensity of mouse sperm to anisosmotic media.

If egg yolk protects sperm against free raddamage, it would be instructive to kno

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to mouse sperm frozen and thawed in the cbination of egg yolk and Oxyrase. The dataFig. 2 show that it does not confer additioprotection to cells that have been subjectecentrifugation and other manipulations but hnot been frozen and thawed.

It needs to be emphasized that Oxyrasepears to protect more against the damage aciated with centrifugation and osmotic shothan it does against the damage producefreezing and thawing per se. As discussed ewhere (24), the damage from centrifugatcould also have a free radical genesis.

In summary, the work reported here suppor is consistent with our hypotheses that twothe problems with cryopreserving mouse spmay be the use of suboptimal concentrationglycerol and their high sensitivity to free racals. Although the motilities we obtained wthe combination of high glycerol and Oxyraapproach or equal the better of those reporteothers, we are not claiming this procedure aprotocol for cryopreserving mouse sperm. Ibut a first step in untangling the multiple vaables that appear to have been responsiblthe difficulties in their cryopreservation. Soof the variables that require further exploratto more fully understand the underlying cryoology and to optimize the procedure are sevident or have been implied. When the produre is optimized with respect to motility, oalso needs to determine whether it is optimathe full function of the sperm from sevestrains, i.e., for the ability to fertilize oocytand produce mouse pups.

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