BIOLOGY OF REPRODUCTION 51, 1014-1021 (1994)
Calculated Optimal Cooling Rates for Ram and Human Sperm Cryopreservation
Fail to Conform with Empirical Observations'
M.R. CURRY, J.D. MILLAR, and P.F. WATSON 2
Department of Veterinary Basic Sciences, The Royal Veterinary College, London NW1 OTU, United Kingdom
ABSTRACT
The permeability coefficient to water (p) and its associated activation energy (E.) were measured for ram (8.47 Lm/min/
atm at 25°C, 1.06 kcal/mol) and human (2.89 m/min/atm at 30 0C, 1.93 kcal/mol) spermatozoa. By use of these figures,
predictive water loss curves were calculated, from published equations, for different cooling rates from 100°C/min to 100 0000 C/
min. The calculated curves show that ram spermatozoa cooled at even the fastest rate would be in osmotic equilibrium by
-200 C, and human spermatozoa cooled at rates up to 10 000°C/min would be in equilibrium by - 150C. If the nucleation temperature for spermatozoa is taken to be between -20°C and -30°C, then ram and human spermatozoa cooled at these rates
would apparently not exhibit any intracellular freezing. There is a significant discrepancy between these calculated optimal
cooling rates and the published empirically derived optimal rates of 50°C/min for ram and 10°C/min for human. The failure of
ram and human spermatozoa to conform with the established and previously successful model for prediction of optimal cooling
rates suggests that damage sustained at high cooling rates may be unrelated to intracellular ice formation.
INTRODUCTION
Spermatozoa were first successfully frozen in the 1940's
and 50's. Techniques have been optimized on an empirical
basis over the ensuing 45 years, but often without proper
account of advances in cryotheory [1]. As cooling proceeds
and water is removed from the extracellular solution as ice,
the cell must lose water at a sufficient rate to remain in
equilibrium and avoid an accumulation of supercooled water.
Any supercooling will result in intracellular freezing once
the nucleation temperature is reached. The relationship between cooling rate and the extent of supercooling within
the cell, and hence the likelihood of lethal intracellular ice
formation, has been modelled by use of the cell surface
area-to-volume ratio, the membrane water permeability
coefficient (Lp), defined as the volume of water that will
cross unit area of membrane in unit time at atmospheric
pressure, and its activation energy (Ea) as a measure of the
change in permeability with temperature [2]. With this model
the occurrence of intracellular ice at above optimal cooling
rates has been successfully predicted in a number of different cell types including yeast, HeLa and Chinese hamster
tissue culture cells, human lymphocytes and red blood cells,
rye protoplasts, and mouse ova and embryos [3]. Ravie and
Lake [4] used Mazur's model with a number of assumed
values to examine fowl spermatozoa and calculated that
cooling rates up to 10 000°C/min produced little likelihood
of intracellular ice formation. This result was at variance
with the empirically derived optimal freezing rate of only
1°C/min. Duncan and Watson [5] calculated an optimal
freezing rate for ram spermatozoa having first estimated the
ram sperm membrane permeability coefficient (Lp, 0.22 uIm/
min/atm) and its activation energy (Ea, 7.425 Kcal/mol).
Duncan and Watson's [5] value for Lp was low compared
with Ravie and Lake's [4] value for fowl spermatozoa (1.94
ILm/min/atm) and an earlier estimate [6] for bull spermatozoa (10.4 ptm/min/atm), but although more in line
with Lp values calculated for other cell types, it still yielded
a theoretically optimal freezing rate (500°C/min) somewhat higher than that determined in practice (50°C/min)
[5]. Spermatozoa are difficult subjects for Lp estimation, and
the methods used by Duncan and Watson [5] probably led
to inaccuracies in some parameters. Watson et al. [7] developed an alternative methodology for measuring Lp and
Ea and produced estimates of 2.1 and 10.8 m/min/atm
for fowl and bull spermatozoa Lp, which corresponded closely
to the existing published values for these species; Ea values
were low (fowl 4.4 kcal/mol, bull 3.0 kcal/mol). Noiles et
al. [8] used a similar methodology for human spermatozoa
(Lp 2.4 m/min/atm, Ea 7.48 kcal/mol) and again found
that the calculated optimal cooling rate was considerably
faster than had been determined in practice.
In this paper we have used the improved methodology
for ram spermatozoa in an attempt to establish whether there
is a real discrepancy between calculated and empirically
established optimal cooling rates. We also compared ram
and human spermatozoa to see if there is a difference in
permeability that might parallel differences in cryosurvival.
MATERIALS AND METHODS
Accepted July 5, 1994.
Received March 9, 1994.
'Supported by the Medical Research Council (G8912737SB) and NATO
(CRG920170).
2
Correspondence: Dr. P.F. Watson, Department of Basic Veterinary Sciences, The
Royal Veterinary College, Royal College Street, London NW1 OTU, UK FAX: 071 388
2342.
Semen Collection and Processing
Ram semen was obtained by artificial vagina from Friesland rams held at the Royal Veterinary College (London,
UK). Only ejaculates with high wave motion and a high per1014
1015
OPTIMAL COOLING RATES FOR SPERMATOZOA
For ram semen, 5 Ril of sperm suspension, preloaded
with CFDA, was added to PBS (1 ml) at each tonicity on a
vortex mixer. For human semen, 10 1 of CFDA-loaded
sperm suspension was added to PBS (100 Rl) at each tonicity (final tonicities adjusted accordingly). Stock solution
of PI was added to each sample as above.
110
100
90
80
70
m
60
Lysis Time
U
50
,
40
CFDA-loaded ram (10 Al) or human (15 Rl) semen was
added to 30 mOsm PBS (ram 900 Il, human 450 Rl) on a
vortex mixer followed at a variable time interval by a 10strength concentration of PBS (ram 100 Rl, human 50 1l)
to return the spermatozoa rapidly to isotonic conditions.
30
20
10
0
0
50
100
150
200
250
300
Osmolarity (mOsm)
FIG. 1. Determination of critical osmolarity of ram (solid line) and human (dotted line) spermatozoa. Values represent means (±+ SEM) of replicates from different individuals (n = 5). Data are presented as percentage
of those cells intact under isosmotic conditions.
centage of motility were used. Human semen was obtained
by masturbation from healthy donors; all samples were
judged normo-spermic by World Health Organization criteria [9].
Ram semen was diluted 1:2 with PBS (NaCl 160 mM,
Na2HPO 4 8 mM, NaH 2PO4 2H 2O 2 mM, pH 7.4, 300 mOsm).
Human semen was washed with a discontinuous Percoll
gradient [10] and resuspended in PBS to a final concentration of approximately 100 x 10 6 /ml.
FluorescentMarker Preparation
A stock solution of carboxyfluorescein diacetate (5-CFDA;
Sigma Chemical Co., St. Louis, MO) was prepared in dimethyl sulfoxide (0.5 mg/ml). Propidium iodide (PI; Sigma
Chemical Co.) was dissolved in distilled water (0.5 mg/ml).
Stock solutions were stored frozen in 100-RlA aliquots. Spermatozoa were pre-loaded with CFDA by adding stock solution to a known volume of sperm suspension (10 pzg/ml)
and allowing the mixture to stand at room temperature for
15 min. Stock PI was added after treatment (see below) to
a final concentration of 5 pzg/ml.
CriticalHypotonicity
The critical hypotonicity is the tonicity at which 50% of
cells will swell beyond their maximum volume-to-surface
area ratio and hence rupture. Hypotonic solutions were
prepared by diluting PBS with distilled water; actual tonicities were measured with a freezing point depression osmometer (3MO; Advanced Instruments Inc., Needham
Heights, MA).
Time 0 controls consisted of the addition of the concentrated PBS before the addition of the sperm suspension.
Stock PI was added as above.
Temperature Dependence of Lysis Time
Lysis time experiments were performed with samples and
reagents pre-equilibrated to three different temperatures.
For the ram, temperatures of 40, 30, and 18°C were used,
and for the human, temperatures of 35, 25, and 15°C. For
temperatures below 25 0C, spermatozoa were cooled at a
rate of 0.25°C/min with use of a programmable cooling bath
to minimize cold-shock damage.
Assessment of Cell Survival
Cell survival was assessed on the basis of plasma membrane integrity: cells retaining their green CFDA staining
over the entire head and tail were classified as plasma
membrane-intact; cells having lost their CFDA staining and
having red PI-stained nuclei were designated membranelysed [7, 11]. Cells were assessed either by fluorescence microscopy (Olympus BH2, FITC/Rhodamine filter set, minimum of 200 cells) or by flow cytometer (EPICS® Profile
Analyzer, minimum of 10 000 cells).
RESULTS
Critical Osmolarity
Ram spermatozoa showed an initial decrease in the percentage of membrane-intact cells between 300 and 150
mOsm (Fig. 1). Membrane integrity was maintained at around
45% between 150 and 100 mOsm before undergoing a further steep decline; this second decrease in cell survival is
considered to be the result of cells exceeding their critical
volume (see Discussion). For the purpose of calculating
critical osmolarity, 50% lysis was taken to occur at the midpoint between the plateau region and 0% survival, which
gave a value for critical osmolarity of 46 mOsm (Fig. 1).
Human spermatozoa showed no initial decrease in percentage of membrane-intact cells and maintained a high level
of survival down to an osmolarity of approximately 100
mOsm (Fig. 1). Below 100 mOsm there was a precipitous
1016
CURRY ET AL.
1
100
90
80
70
.4
U
7;
Jc
1I
Z!,
0
50
60
50
40
cc
40
30
30
20
r
0
2
4
6
8
10
0
4
2
Time (sec)
FIG. 2. Time to lysis for ram spermatozoa exposed to 30 mOsm solution at 40°C (dotted line), 30°C (solid line), and 18°C (dashed line). Values
represent means (+ SEM) of replicates from different individuals (n = 5).
Data are presented as percentage of those cells intact under isosmotic conditions.
decline in membrane integrity, with 50% lysis occurring at
67 mOsm (Fig. 1).
Lysis Time
Ram spermatozoa. On exposure to 30 mOsm PBS at
40°C, survival fell within 1 sec to approximately 55% of the
total number of intact cells under isosmotic conditions (approximately 75% of total cell number were intact at Time
0, i.e., under isosmotic conditions, in all cases), and at 30°C,
to approximately 45% of cells intact under isosmotic conditions. Thereafter membrane integrity was maintained for
around 3 sec, when cell lysis began to occur. As with the
critical osmolarity measurements, the initial cell loss was
considered to be unrelated to cells exceeding their critical
volume, and 50% lysis was calculated by taking cell survival
after the immediate loss as 100%. After the initial difference
in survival, which was not significant, the two curves followed an identical pattern. The times for 50% lysis were
4.7 sec at 40°C and 4.9 sec at 30°C (Fig. 2). At 18 0C, the
immediate loss of cell integrity was much greater, with only
15% of the cells intact under isosmotic conditions surviving. Thereafter there was a gradual decline in cell survival
over the ensuing 7 sec. The time for 50% lysis in this case
was 5.4 sec (Fig. 2).
Human spermatozoa. At 350C and 25°C there was no
immediate loss of membrane integrity, and 100% survival
was maintained for 4 sec; thereafter there was a steady decline in survival, with 50% lysis at 4.8 sec at 350C and 5.6
sec at 25°C (Fig. 3). At 15 0C there was a decline in cell
survival to approximately 60% of cells intact under isos-
6
Time (sec.)
8
10
12
FIG. 3. Time to lysis for human spermatozoa exposed to 30 mOsm
solution at 35°C (dotted line), 25°C (solid line), and 15°C (dashed line). Values represent means (+ SEM) of replicates from different individuals (n =
5). Data are presented as percentage of those cells intact under isosmotic
conditions.
motic conditions, a much less severe effect than that seen
with the ram spermatozoa. The 50% lysis time was 6.0 sec
(Fig. 3).
Calculation of Lp
Lp was calculated by use of Equation 4 of Leibo [12]
e(Vi - V2)
LP=
+ V21n
[(V - V,)]
I (V v2)J
(tRNT)
The equilibrium water volume is calculated as
Isotonic osmolarity
eHypotonicosmolarit x Isosmotic water volume (V1)
Hypotonic osmolarity
However, equilibrium volume is never reached as the fixed
cell surface area cannot accommodate the necessary volume increase, and the cell lyses. Hence, for V2, the volume
at the critical osmolarity is calculated as
Isotonic osmolarity
V2 =
critical osmolarity
xV
N is the number of osmoles of solute within the cell calculated as
N = isotonic osmolarity (Osm) x 10-15 (liters/cell) x
cell volume (izm 3)
t is the time taken to reach critical volume or the lysis
time assuming the cells behave as perfect osmometers. Parameters used to calculate L. are listed in Table 1. From
1017
OPTIMAL COOLING RATES FOR SPERMATOZOA
TABLE 1. Parameters used to calculate L,.
Parameter
Symbol (unit)
Ve (m 3 )
V (m 3)
V2 (m3 )
t (min)
A (m2)
R (Rm3/atm/mol/deg)
(K)
N (Osmol/cell)
Equil. water volume
Iso. water volume
Crit. water volume
Lysis time
Surface area
Gas constant
Temperature
Osmoles of solute within cell
Ram
Human
172.8
17.28*
112.6
0.082
139.0*
82.06 x 1012
303
5.2 x 10-1
170
17.0**
76.1
0.094
120**
82.06 x 1012
298
5.1 x 10- 5
*Values taken from Duncan and Watson [5].
**Values taken from Noiles et al. [81.
these values, for ram spermatozoa Lp = 8.47 Izm/min/atm
(at 25°C); for human spermatozoa Lp = 2.89 um/min/atm
(at 300C).
Calculation of Ea
The change in hydraulic conductivity with temperature
was calculated by expressing relative hydraulic conductivity
as relative permeability (1/lysis time, taking lysis time at
30 0C for ram and 25 0C for human as unity) and plotting the
natural log of the relative permeability against temperature
(Fig. 4). The temperature coefficient of the hydraulic conductivity is given by the slope of the graph. To obtain a
value for Ea in Kcal/mol, the temperature coefficient is
multiplied by an appropriate constant 1.65 x 105 [13].
For ram spermatozoa, the temperature coefficient of hydraulic conductivity was 0.0064/ 0C, giving an Ea value of
1.06 Kcal/mol. For human spermatozoa, the temperature
coefficient was 0.0117/°C and Ea was 1.93 Kcal/mol.
.
0.1
Calculation of Predicted Water Loss Curves
Equation 10 of Fahy [14] was used to calculate the kinetics of intracellular water loss
dV
g-Kg
eb(T-T)AR(T
+ 273.15)dT
BVw
[
XLn
I
S1
(V/n5 V, + 1)
(V/n5
V. + 1)2
1 + 0.00966T + 4.1025 x 10-5 T2
Equation 12 of Fahy [14] was used to calculate the equilibrium curve
V= Vwns(Xs-
- 1)
The parameters used are listed in Table 2.
Figures 5 and 6 show the plotted equilibrium cooling
curves for ram and human spermatozoa together with the
effects on relative cell volume of cooling at 100, 1000, 10 000,
and 100 000°C/min. The nucleation temperature for spermatozoa is difficult to obtain, but a figure between -20°C
and -40°C has been proposed [15]. Figure 5 shows that
ram spermatozoa cooled at 10 000°C/min are in equilibrium by -7C so would avoid intracellular freezing. Even
at a cooling rate of 100 000°C/min, the cells are back in
equilibrium by -20C. As shown in Figure 6, human spermatozoa cooled at 10 000°C/min have reached equilibrium
by -15°C so should avoid any intracellular ice formation.
DISCUSSION
0.0-
I
-0.1
10
20
30
Temp (deg. C)
FIG. 4. Effect of temperature on the water permeability of ram (open
squares) and human (closed squares) spermatozoa. Regression analysis was
used to determine the line of best fit; R2 was 0.97 for ram and 0.94 for
human.
The value of 8.47 jum/atm/min for ram spermatozoa L,
is considerably larger than the previous estimate of 0.22
Ilm/atm/min [5]. The difference in values for the ram is a
result of large changes in two of the parameters used. Duncan and Watson [5] in calculating critical osmolarity took
the 50% lysis point as being in the middle of the plateau
region of their curve, thereby using the whole curve. We
now believe that the initial loss of cell integrity at relatively
high osmolarities is unrelated to cells exceeding their critical volume. Curry and Watson [16] have shown that ram
spermatozoa taken to the same final osmolarities in a series
of 25 mOsm steps rather than in a single step do not show
1018
CURRY ET AL.
TABLE 2. Parameters used to calculate the equilibrium curve.
Parameter
Cell water volume
Temperature
Moles solute in cell
Partial molar vol H20
Constants*
Gas constant
Cell surface area
Reference temperature
Temp. coeffic. of K
Hydraulic conduct.**
Cooling rate
Symbol (unit)
Ram
Human
V0 (m 3)
T (C)
ns (mol)
3
V, (m /mol)
I
S
3
R (Am /atm/{mol deg})
2
A (m )
T (C)
b (/°C)
Kg(lm/min/atm)
°C/min
17.28
30
2.7
x 10-15
18.015 x 1012
1
0
82.057 x 1012
139.0
30
0.0064
8.47
variable
17.0
25
2.55 x 10-15
18.015 x 1012
1
0
82.057 x 1012
120.0
25
0.0117
2.89
variable
*1 and S are constants determined by the identity of the cryoprotectant and its concentration before freezing. For
the purposes of this calculation all solutions are assumed to behave ideally [28].
**Fahy [14] uses the symbol K for hydraulic conductivity, which may be considered here as equivalent to LP.
the same cell loss at osmolarities between 300 and 150 mOsm
but exhibit a survival curve very similar to that of the human spermatozoa (Fig. 1). This loss of membrane integrity
appears to be related to the rate at which water enters the
cell rather than the final volume attained. These cells exhibiting loss of membrane integrity at relatively high osmolarities would appear to constitute a sensitive subpopulation within the ejaculate since repeated cycles of exposure
to a 120 mOsm stress does not result in further cell rupture
after the initial exposure [16]. The size of this subpopulation reflects the numbers of sperm that fail to survive cryopreservation even under optimal conditions [17]. The true
50% lysis point is therefore at the midpoint of the curve
after the plateau region. Using the data presented here, this
interpretation of the graph gives a figure for critical os-
molarity of approximately half that used by Duncan and
Watson [5], 46 mOsm rather than 109 mOsm. The curve of
Duncan and Watson [5] yields a comparable figure if the
present logic is applied. Critical tonicity was measured at
only a single temperature in these experiments. Noiles et
al. [8] observed a small increase in critical tonicity for human sperm between 22 and 8°C; however, the effect on Lp
calculation is relatively small, and other membrane temperature effects, evidenced by the rapid loss of membrane
integrity of ram sperm at 18°C, are probably more significant. In addition, Duncan and Watson [5] measured lysis
time from the time taken for sperm tails to coil and straighten
in distilled water. Only a relatively small proportion of the
cells were recorded as behaving in this way, and the time
of 15.3 sec obtained may represent a small and particularly
1
4
U
.
.)
60
50
40
30
20
10
I
0
0
5
10
15
20
25
Temp (deg C)
FIG. 5. Calculated water loss curves for ram spermatozoa cooled infinitely slowly so as to remain in osmotic equilibrium (E) and at rates of
0
100°C/min (1), 1000°C/min (2), 10 000 C/min (3), and 100 000°C/min (4).
0
5
10
15
Temp (deg C)
20
25
FIG. 6. Calculated water loss curves for human spermatozoa cooled
infinitely slowly so as to remain in osmotic equilibrium (El and at rates of
100°C/min (1), 1000°C/min (2), 10000°C/min (3), and 100 000°C/min (4).
OPTIMAL COOLING RATES FOR SPERMATOZOA
resistant subpopulation within the ejaculate. The figure of
4.9 sec used here still does not represent the entire ejaculate since around 50% of the cells appear to lyse immediately on mixing with the 30 mOsm solution, probably
the same cells that showed loss of membrane integrity under relatively mild hyposmotic conditions. The value for
human spermatozoa L obtained here, 2.89 pLm/atm/min,
is in good agreement with the previously published value
of Noiles et al. [8] of 2.4 ILm/atm/min. The similarity of the
human values gives a degree of confidence in our value for
ram spermatozoa.
Ea was calculated by using the temperature coefficient
rather than directly from an Arrhenius plot because temperature coefficient (b) is the value substituted into Fahy's
equation [14]. However, Mazur et al. [13] have shown that
above -20°C the two methods of calculation yield very similar results. The calculated Ea values are low, reflecting the
very small changes in lysis time recorded at different temperatures. The increasing percentage of cells undergoing
very rapid membrane rupture (within 1 sec) seen with ram
spermatozoa as temperature was decreased means that the
lysis time recorded at 18°C was based on only a small percentage of the cells in the ejaculate (18%). This increased
sensitivity prohibits the use of lower temperatures to extend the temperature range used to calculate Ea. The comparatively large standard errors associated with the human
lysis time values indicate that there is no significant difference in the 50% lysis times at the different temperatures.
While these considerations require the absolute quoted values for Ea to be treated with a degree of caution, it is clear
that the true value is small and that, over the given range,
temperature has little effect on the permeability coefficient.
Duncan and Watson [5] found a relatively small discrepancy between their calculated optimal freezing rate and their
experimental results, and proposed that a more accurate
calculation of Lp and Ea would reconcile the figures. In
practice, the optimal cooling rate for ram spermatozoa is
50-60°C/min [5] and for human spermatozoa 1-10°C/min
[18]. The results presented here for ram spermatozoa, far
from reconciling the differences between calculated and
empirical optimal cooling rates, greatly exacerbated the
problem, with a cooling rate of 100 000°C/min reaching
equilibrium by -20 0C. For human spermatozoa, the results
predict that a cooling rate of 10 000°C/min would regain
equilibrium by -15°C, an even greater discrepancy than
the 100-1000-fold difference reported by Noiles et al. [8].
A number of possible explanations have been proposed
to account for the observed discrepancy. The parameters
necessary to calculate L and Ea are difficult to obtain for
spermatozoa and involve exposing the cells to extremely
large osmotic stresses. It is possible that the derived values
may still be erroneous. However, as figures from different
laboratories and for different species accumulate in the literature [4, 5, 7, 8], we are forced to conclude that spermatozoa are extremely permeable cells with very low activa-
1019
tion energies, even if there are minor errors in the absolute
values calculated. We have calculated that in order to reconcile the predicted and observed optimal cooling rates, Lp
would need to be 400 times lower and Ea 10 times higher,
changes that greatly exceed any experimental error associated with calculation of the values.
Spermatozoa are frozen/thawed in the presence of permeating cryoprotectants that alter cell water volumes and
the number of moles of solute within the cell, and may also
significantly decrease Lp and increase Ea. There is some evidence for this with human red blood cells, where glycerol
lowers the cooling rate required to cause hemolysis by a
factor of 10 [19]. Moreover, in mammalian embryos, intracellular cryoprotectant reduces LP by a factor of 2 or more
[20], and in human spermatozoa 1 M glycerol reduces L
by 33% [21]. None of these recorded effects of cryoprotectant on Lp would be of sufficient magnitude to account for
the observed discrepancies. It should also be noted that
glycerol concentrations used to freeze spermatozoa are
comparatively low compared to those used for other cell
types.
Lp and Ea may change significantly at temperatures below
zero. Noiles et al. have shown a small decrease in L of
human spermatozoa at -7°C with a corresponding increase
in Ea [8]. There may be greater changes at lower temperatures, but this is difficult to test below the freezing point.
Putative changes in L below the freezing point of the extracellular medium may in fact be of little relevance since
the very high initial permeability means that the bulk of the
cell water will have left the cell before any decrease in permeability occurs.
Neither of these hypotheses, the effect of cryoprotectant
or the possibility of nonlinear abrupt changes in Lp, can be
conclusively discounted at this time, but the magnitude of
the changes required would need to be considerable to
account for a difference of four orders of magnitude in critical cooling rate of ram spermatozoa.
Frozen/thawed spermatozoa have been shown to exhibit the inverted "u"-shaped survival curve with cooling
rate [5, 18] previously observed for a number of other cell
types [22]. As cooling rate is increased, cell survival also
increases up to a maximum, after which any further increase in cooling rate results in a decline [22]. This curve
has been interpreted as resulting from two opposing factors affecting cell survival [22]. Increasing cell death at low
cooling rates has been attributed to so-called solution effects, while at high cooling rates there is an increasing tendency for lethal intracellular ice formation. However, it now
seems questionable that the decline in sperm survival with
increased cooling rate illustrated by the right-hand portion
of the graph is a result of intracellular ice formation, although there is at present no direct confirmation of the
absence of intracellular ice at high freezing rates.
In the absence of intracellular freezing, it is not clear
what the lethal mechanism might be. A possible mechanism
1020
CURRY ET AL.
of membrane damage as a consequence of exceeding a critical gradient in osmotic pressure across the membrane has
been suggested by Muldrew and McGann [23, 24] whereby
the osmotically driven water flux exerts a frictional force
on the plasma membrane that, if sufficiently large, will result in membrane rupture. This hypothesis is consistent with
our own observation [16] that a subpopulation of ram spermatozoa are sensitive to the rate of water passage into the
cell under relatively mild hyposmotic conditions.
Although water movements across the plasma membrane are of foremost importance in determining cell survival during a freeze/thaw cycle, it is by no means clear
how water actually traverses the membrane. There are two
possible routes for water passage, either through the bilayer or via polar transport sites (pores). The data available
from other cell types, particularly erythrocytes, kidney
proximal tubule, and vasopressin-sensitive tight epithelia,
have established criteria to distinguish between these two
routes [25]. Compared with pure phospholipid membranes,
porous membranes generally have a high osmotic water
permeability coefficient but a low activation energy. The
ratio of the osmotic water permeability coefficient to the
diffusional water permeability coefficient in pure phospholipid membranes is equal to one, but is greater than one
where membrane pores are present [25]. Membrane pores
are also subject to inhibition by mercurial compounds such
as mercuric chloride and p-chloromercuriphenylsulphonic
acid [25]. These are empirically derived characteristics for
proposed water channels, and there is still little information as to the molecular basis of water transport, although
it is generally agreed in the literature that water passage is
essentially a passive process [26]. Although we have been
unable to demonstrate any inhibitory effect of mercurial
compounds on the water permeability of ram spermatozoa
(unpublished observations), and similarly Liu et al. [27] have
shown no effect on human spermatozoa, the Lv and Ea values for spermatozoa are consistent with a porous membrane.
It is difficult to account for the high water permeability
exhibited by spermatozoa, in terms of their physiology or
their biological function. Other cells with comparably high
water permeabilities tend either to have functions concerned with water regulation in the urinary system, e.g.,
renal epithelial cells, or to be exposed to large fluctuations
in their osmotic environment, e.g., erythrocytes during their
passage through the kidney medulla. Spermatozoa do experience a change in osmolarity on leaving the relatively
hyperosmotic epididymis, at the time of ejaculation, but for
most species this change would hardly seem sufficient to
justify such a large L value. Whatever the reason for this
high water permeability, it has profound implications for
sperm cryopreservation as evidence increasingly points to
water movement across sperm membranes as an important
source of cryoinjury.
ACKNOWLEDGMENTS
The authors thank Dr. P. Mazur (Oak Ridge National Laboratory, Oak Ridge, TN)
and Dr. J.K. Critser (Cryobiology Research Institute, Methodist Hospital of Indiana
Inc.) for helpful discussion of these results.
REFERENCES
1. Watson PF. The preservation of semen in mammals. In: Finn CA (ed.), Oxford
Reviews of Reproductive Biology. Oxford: Oxford University Press; 1979: 283350.
2. Mazur P. Kinetics of water loss from cells at subzero temperatures. J Gen Physiol
1963; 47:347-369.
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