BIOLOGY OF REPRODUCTION 48, 99-109 (1993)
Determination of Water Permeability Coefficient for Human Spermatozoa
and Its Activation Energy'
E.E. NOILES,3 4' P. MAZUR, 5 P.F. WATSON,6 F.W. KLEINHANS, 3 '7 and J.K. CRITSER 2 '3'4
Centerfor Reproduction and TransplantationImmunology,3 Methodist Hospital of Indiana, Inc.
Indianapolis, Indiana 46202
Departments of Physiology and Biophysics and Obstetrics and Gynecology4
Indiana University School Medicine, Indianapolis, Indiana 46202
Biology Division,5 Oak Ridge NationalLaboratory, Oak Ridge, Tennessee 37831
Royal Veterinary College,6 University of London, London, United Kingdom
Departmentof Physics,7 IUPUI, Indianapolis, Indiana 46205
ABSTRACT
Four experiments were conducted to determine the permeability coefficient of human sperm to water (,) and its activation
energy (E,). Critical tonicity (tonicity at which 50% of the cells swell and lyse) was determined by equilibrating sperm to 22°C
(experiments la and Ib), 30, 22, 8, or O0C (experiment 2a), and 0, -1, -3, -5, or -7°C (experiment 2b) and then exposing
them to various hypotonic media (215-3 mOsm). For Iv determination, sperm were equilibrated to 30, 22, 8, or O0C (experiment
3a), 8, 0, or -3°C (experiment 3b), and -1, -3, -5, or -7°C (experiment 3c), and then were exposed for increasing times
to hypotonic (40 mOsm) media. Activation energies were calculated from the results of the latter experiments (experiment 4).
Results indicate a temperature-dependent (p < 0.05) critical tonicity, with sperm exhibiting an increased membrane fragility at
8, 0, and -7°C, relative to 30, 22, -1, -3, or -5°C (67.5 ± 2.4, [mean ± SEM], 62.7 + 2.3, and 61.9 ± 3.7 mOsm vs. 57.4 ±
3.4, 57 ± 1.2, 54.8 ± 3.4, 60.1 ± 5.3, and 59.8 ± 5.2 mOsm, respectively). Human sperm have an L, of 2.40 + 0.20 Rpm/min/
atm at 22°C and an E, of 3.92 ± 0.59 kcal/mol between 30 and -7°C. The E, for cells incubated at temperatures above O°C
(3.92 kcal/mol) show an apparent discontinuity (p < 0.004) in water permeability in supercooled conditions (7.48 kcal/mol).
These data suggest that 1) human sperm have a high L, and low E,, relative to other cell types, above O0C; and 2) this high L,
and its low E, change significantly below O0C.
INTRODUCTION
and many aspects of the techniques used to freeze sperm
were established early, relative to the development of a more
complete understanding of the general principles of cryobiology [6].
Cryopreservation subjects cells to a sequence of steps
during freezing and thawing that are strongly anisosmotic.
They initially shrink when hypertonic cryoprotectants are
added; swell back to near isotonic volumes as the cryoprotectant permeates; shrink again as water leaves the cell during slow freezing, or alternatively, they undergo intracellular freezing if cooling rates are not sufficiently slow. The
cells return to isotonic volumes during thawing, and finally
undergo a major osmotic change in volume as the cryoprotectant is removed and cells are returned to physiological
medium. Since these events are osmotic in nature, knowledge of the permeability of a cell to water and cryoprotectants are powerful tools in predicting the likely optimum
values for the major steps involved in freezing. From the
value of the permeability coefficient of the cell to water,
and its temperature coefficient, one can predict the cooling
rate likely to be slow enough to preclude lethal intracellular freezing.
Water permeability is a fundamental biophysical property of all living cells. When measured in response to an
osmotic pressure differential, the specific coefficient is referred to as the hydraulic conductivity (Lp). This aspect of
cell membrane physiology can be determined by monitor-
The possibility of transmission of the human immunosuppressive virus (HIV) resulting in Acquired Immune Deficiency Syndrome (AIDS) from use of contaminated donor
semen has resulted in the requirement that only frozen semen be used in artificial insemination by donor programs
(AID). The ability to store frozen semen may allow sufficient time for seroconversion of donors so that possible
carriers of the HIV can be detected [1]. The imperative to
use only frozen sperm has highlighted a specific concern
that cryosurvival of sperm is low and a general concern
(although controversial) that conception rates may be lower
with frozen-thawed human sperm than with nonfrozen sperm
[2-4]. The low cryosurvival rate and the putative lower conception rate may be due to the fact that procedures for
cryopreservation of many mammalian cell types, including
sperm, have evolved empirically, often resulting in procedures that deviate from fundamental concepts of cryobiology [5]. Cryopreservation of spermatozoa has a long history,
Accepted August 19, 1992.
Received June 9, 1992.
'Supported by NIH grant HD25949, Office of Health and Environmental Research, U.S. Department of Energy, under contract DE-AC05-840R21400 with Martin
Marietta System, and Fulbright Travel Scholarship.
2Correspondence: Dr. J.K. Critser, Center for Reproduction and Transplantation
Immunology, Methodist Hospital of Indiana, Inc., 1701 North Senate Blvd., Indianapolis, IN 46202. FAX: (317) 929-2039.
99
100
NOILES ET AL.
ing cell volume changes when cells are subjected to anisosmotic conditions [7]. Water permeability assumes widely
different values among different cell types; even within a
given cell type, it may differ in different species [7-10]. It
has been determined in many cell types including sea urchin eggs [12], amphibian eggs [13], mammalian red cells
[14-20], tumor cells [21, 22], lymphocytes [23, 24], proximal
straight tubule epithelial cells [25], and oocytes [26-30]. The
water permeability characteristics of bovine and fowl spermatozoa have been studied [31-33], but those of human
sperm have not been examined.
A primary consideration during cryopreservation of any
cell type is to avoid intracellular ice crystal formation with
its lethal consequences to the cell [34]. Current techniques
usually achieve this by controlling the cooling rate. Initially
upon cooling, the sample often supercools to variable extents between -5 and -15°C before ice nucleates in the
extracellular medium, either spontaneously or induced as
a result of "seeding" [34]. The intracellular water remains
liquid and supercooled, putatively due to the plasma membrane-blocking nucleation of intracellular water. As a result
of the increased chemical potential of supercooled intracellular water, there is an exosmosis of water with subsequent dehydration of the cell [34]. If cooling rates are sufficiently low, intracellular water moves out of the cell to
maintain equilibrium. If cooling is too rapid, the cytoplasm
will be unable to dehydrate fast enough to maintain equilibrium, resulting in intracellular ice formation once the
cell reaches its ice nucleation temperature [34].
Mazur [35, see 34 for review] has proposed, and subsequently demonstrated, that several measurable fundamental biophysical cell characteristics are needed to predict the relation between cooling rate and intracellular
freezing: 1) the L and its activation energy, 2) the surfaceto-volume ratio, and 3) the temperature at which intracellular freezing occurs. The purpose of this study was to determine the first of the parameters, Lp, and its activation
energy (Ea) for human spermatozoa as part of an ongoing
investigation of the response of human sperm to freezing
and thawing [36-38].
A common procedure to determine L in, for example,
ova is to place cells in hypertonic concentrations of nonpermeating solutes (salts, sucrose) and follow their shrinkage with time under a microscope. The L can be determined from the kinetics of that shrinkage provided one has
demonstrated that the cell behaves as an ideal osmometer
(i.e., its volume is reciprocally related to the external osmolality) and that the volume fraction of the cell occupied
by solids and the cell's surface area is known. This approach is not feasible for sperm, however, because their
small volume, highly nonspherical shape, and high solid
contents make it difficult to observe shrinkage.
Some cells, such as human red cells and sperm, have
plasma membranes that are devoid of microvilli or other
forms of membrane reserve and are therefore fixed in area.
If such cells are placed in sufficiently low concentrations
of nonpermeating solutes, they will swell until their fixed
area of membrane encompasses the maximum volume (a
sphere or a nearly spherical shape). If the attainment of
osmotic equilibrium requires that they swell beyond that
critical volume, they will lyse [39]. The L can be determined by measuring the time required for the cells to attain that critical volume in hypotonic media (i.e., the time
to lysis). To determine the critical or lytic volume of intracellular water, one places the cells in a graded series of
hypotonic solutions of nonpermeating solutes to determine
the critical osmolality at which 50% of the cells lyse [40].
If the cell behaves as an ideal osmometer, which Du et al.
[41] have shown by electron spin resonance (ESR) techniques to be the case in human sperm, the critical volume
of water relative to the volume of water in the isotonic cell
is equal to the isotonic osmolality divided by the critical
osmolality.
In red cells, lysis can be assessed by the release of hemoglobin [42]. In sperm, one can measure the loss of membrane integrity by the fluorophores carboxyfluoroscein diacetate and propidium iodide, as detected using flow
cytometry [43]. Therefore, a series of experiments was performed using loss of membrane integrity to determine the
following: 1) osmolality at which 50% of the spermatozoa
swell and lyse (critical tonicity), and its temperature dependence; 2) Lp using the time to spermolysis in medium
with an osmolality below the critical value for sperm; and
3) Ea of Lp by determining L at various temperatures between 30 and -7°C.
MATERIALS AND METHODS
Samples
Ejaculates were obtained by masturbation from healthy
men, aged 20-40 yr. The inclusion criteria used for ejaculates were a minimum concentration of 20 x 106 spermatozoa/ml and 40% motility. Semen samples were allowed to liquify for a minimum of 20 min, at which time
they were subjected to analysis using CellSoft (Version 3.2/
C, CRYOResources, LTD., New York, NY), as described by
Jequier and Crich [44] and modified by Critser et al. [37].
Ejaculates were subjected to a swim-up procedure [45]
for 90 min. Following this, samples were centrifuged at 400
x g for 7 min. The resulting sperm pellet was then resuspended in isotonic Mann's Ringer solution (286 mOsm) [46].
Fluorescent Stains
The stock solutions for the fluorescence microscopy were
prepared by dissolving 1 mg carboxyfluorescein (CFDA) in
1 ml dimethyl sulfoxide (DMSO; Aldrich Chemical Company, Inc., Milwaukee, WI) and 1 mg propidium iodide (PI)
in 1 ml reagent grade water (MilliQ; Millipore System, Ionpure Technologies Corp., Bedford, MA). The stock CFDA
DETERMINATION OF HUMAN SPERM WATER PERMEABILITY
solutions for the flow cytometer were prepared by dissolving 0.25 mg CFDA in 1 ml DMSO.
FluorescenceMicroscopy
Sperm cells were stained by adding 10 R.1 (10 jig/ml)
CFDA stock solution and 10 l (10 pg/ml) PI to each 1 ml
of sperm suspension and mixing thoroughly. A minimum
of 100 cells per treatment were counted within 1 h of staining to determine the percentage of cells with intact and
damaged plasma membranes, using a Nikon Optiphot microscope (Scientific Instruments, Greenwood, IN).
Flow Cytometry
Sperm cells were stained by adding 10 1 (2.5 RIg/ml)
CFDA and 10 il (10 ig/ml) PI to each 1-ml sperm suspension and mixing thoroughly. The data reported here are
in terms of percentage of cells staining CFDA-positive/PInegative, as less than 10% dual staining (cells staining CFDApositive/PI-positive) was observed. These dual-stained cells
were excluded from analyses, as the presence of PI fluorescence indicates an ambiguity as to plasma membrane
integrity. Data were obtained using a FACStar Plus Analyzer
(Becton Dickinson, Rutherford, NJ) and FACStar Plus Research Software. A minimum of 1 x 10 4 spermatozoa/treatment were counted within 1 h of staining, using a 4-W Argon laser operated at 488 nm and 200 mW. The percentage
of cells exhibiting CFDA and PI fluorescence was measured
for each treatment, as well as forward and 90 ° light scatter.
Experiment 1: Determination of the Critical Tonicity
Experiment la: determine the critical tonicity at 22 0C.
To determine the osmolality at which 50% of a human
spermatozoa population attain critical volume of intracellular water and lyse, spermatozoa were exposed for 5 min
at 220C to isotonic (286 mOsm) or hypotonic conditions
(215, 163, 121, 92, 68, 52, 39, 30, 21, 17, and 3 mOsm, respectively). Hypotonic media were prepared by diluting
Mann's Ringer solution with reagent grade water to the required extent, as determined by a freezing point depression osmometer (Model 3D2; Advanced Instruments, Needham Hts., MA). Cells were returned to isotonic conditions
by the addition of appropriate concentrations and volumes
of NaCl solutions, stained with CFDA/PI, and analyzed for
percentage CFDA-positive/PI-negative staining by flow cytometry and fluorescence microscopy.
Experiment lb: refinement of the value of the critical
tonicity at 220C. To more clearly delineate the osmolality
at which 50% of a human sperm population attain the critical volume of intracellular water and lyse, spermatozoa were
exposed for 5 min at 22 0C to isotonic (286 mOsm) or hypotonic conditions (90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40,
and 3 mOsm, respectively). Cells were returned to isotonic
conditions and stained; data were collected as in experiment la.
101
Experiment 2: Determination of the Temperature
Dependence of the Critical Tonicity
Experiment2a: determine the suprazero temperaturedependance of the critical tonicity. To ascertain a possible
temperature dependence of the ability of human sperm to
swell and lyse at 0°C or above, aliquots of the sperm suspension were equilibrated to 0, 8, 22, and 30°C. Spermatozoa were equilibrated to the four temperatures, then exposed for 5 min to the broad range of hypotonicities used
in experiment la. Cells were then returned to isotonic conditions and stained; and data were collected as in experiment la.
Experiment 2b: determine the subzero temperature dependance of the critical tonicity. To establish a possible
temperature dependence of the ability of human sperm to
swell and lyse under supercooled conditions, aliquots of
the sperm suspension were equilibrated to 0, -1, -3, -5,
and -7°C. Spermatozoa were then exposed for 5 min to
the broad range of hypotonicities used in experiment la,
equilibrated to the five temperatures. Any samples inadvertently seeded (forming extracellular ice) at supercooled
conditions were discarded. Cells were returned to isotonic
conditions and stained; data were collected as in experiment la.
Experiment 3: Determination of the Lp
Experiment 3a: determine the Lp above O0C. Samples
of a sperm suspension in isotonic Mann's Ringer solution
were equilibrated to 0, 8, 22, and 30°C. Successive aliquots
of each sample were then abruptly exposed, with constant
vortexing, for increasing times (5, 7, 9, 11, 13, 15, 17, or 19
sec) to 286 (positive control), 40 (test solution), and 3 (negative control) mOsm media equilibrated to these temperatures. Cells were abruptly returned to isotonic conditions,
during constant vortexing, by the addition of appropriate
concentrations of NaCl solutions previously equilibrated at
those temperatures, stained with CFDA/PI, and analyzed for
percentage of CFDA-positive/PI-negative staining by flow
cytometry.
Calculationof Lp. The equation relating cell water volume (V) to time in hypotonic media is [47]:
dV
dt = Lp A(Ti
-
T,)
(1)
where L is the hydraulic conductivity (m/min/atm), A is
the surface area of the cell (m 2), and ri is the internal
osmotic pressure of the cells in atm at time t; i7e is osmotic
pressure in the cell when it reaches osmotic equilibrium
with the external medium, and it is also, therefore, the osmotic pressure of the external medium.
Expressing equation 1 in terms of the concentrations N/
V (number of osmoles in the cell, assumed to be fixed),
using r = RTN/V, where R is the gas constant (82.06 x
102
NOILES ET AL.
1012 ptm 3 atm/mol/deg), and T is absolute temperature, the
following equation is obtained [48]:
dt
LpARTN
(2)
V
where V is the osmotically active cell water volume at time
t and Ve is the cell water volume at equilibrium. Integrating
from VI, when t = 0 to V2 when t = t, the final equation
is obtained:
Ve(V - V2) + V n(
Lp =
Experiment 4a-c: Determination of the Activation
Energy for Lp
v)]
(tARNT)
For this study, the volume of osmotically available water in
the sperm at time = 0 is 17 pxm 3 (V1 = 17 Km3 ), assuming
a total isotonic water volume of 20 im 3 [49], and an osmotically active component of 85% [41], V2 is the volume
at time t, and N has the value 0.286 osmol/L H 20 x 17
pLm 3
10 - 15 liters/cell, or 4.86 x 10-15 osmol/cell, and
A, the surface area, has the value of 120 im 2. The equilibrium volume V is the isotonic osmolality divided by the
test hypotonic osmolality (40 mOsm) multiplied by V,:
17
Ve= 0.286 X -- = 122 m
0.04
3
during constant vortexing, to 286 (positive control), 40 (test
solution), and 3 (negative control) mOsm equilibrated to
-1, -3, -5, and -7°C. Any samples inadvertently seeded
at supercooled conditions were discarded. Cells were
abruptly returned to isotonic conditions at the above temperatures and stained; data were collected as in experiment
3a. The equations described in experiment 3a were applied
to determine Lp at the various temperatures.
(4)
The cells lyse long before they attain that volume (i.e., lysis
occurs when the cells attain the critical volume of water
[Vcrit], which is 0.286 X 17/critical tonicity). The L is calculated using equation 3 where V2 equals Vcrit and t is the
observed time for 50% spermolysis. To use this approach,
however, one must know 1) the tonicity that produces lysis
and 2) the volume of water in the cell at that critical tonicity
relative to the volume of water in the cell under isotonic
conditions. A previous study has demonstrated that human
sperm behave as "ideal osmometers" [41]. Therefore, the
relative volume of osmotically active sperm water at the
critical tonicity is the ratio of the isotonic osmolality to the
critical tonicities, as determined in experiment 1.
Experiment 3b: determine the Lp from 8 to -3 0 C. Aliquots of sperm suspensions were equilibrated to 8, 0, and
-3 0C. Spermatozoa were then abruptly exposed for increasing times (15, 19, 23, 27, 31, 35, 39, and 43 sec), during
constant vortexing, to 286 (positive control), 40 (test solution), and 3 (negative control) mOsm, equilibrated to 8,
0, and -3 0C. Any samples inadvertently seeded at supercooled conditions were discarded. Cells were abruptly returned to isotonic conditions and stained; data were collected as in experiment 3a. The equations described in
experiment 3a were applied to determine L at the various
temperatures.
Experiment 3c: determine the Lp below O°C Aliquots
of the sperm suspension were supercooled to -1, -3, -5,
and -7°C. Spermatozoa were then abruptly exposed for
increasing times (15, 19, 23, 27, 31, 35, 39, and 43 sec),
Activation energies of LP for the three temperature ranges
studied (above, surrounding, and below 0°C) were obtained from the slope of the linear relationship between
In(Lp) and 1/°K using the equation [26]:
E = -R x Slope
(5)
where R is the universal gas constant (1.987 cal/deg/mol).
StatisticalAnalyses
Data of critical tonicity determination were subjected to
arc sine transformation and normalized to isotonic values
([hypotonic staining %/isotonic staining %] x 100). Critical
tonicity data were analyzed using standard analysis of variance procedures and protected least significant difference
multiple range tests to compare membrane integrity [50]
for both the range and the interpolated 50% cell lysis tonicities. Comparison of flow cytometry to fluorescence microscopy data were performed by analysis of variance and
correlation procedures [50]. Differences among Lp and Ea
estimations for different temperatures were analyzed using
standard analysis of variance procedures and protected least
significant difference multiple range tests [50]. All analyses
were performed using SAS procedures [51].
RESULTS
Experiment 1: Determination of the Critical Tonicity
Experiment la: determine the critical tonicity at 22°C.
The percentages of human spermatozoa with intact plasma
membranes, as determined by CFDA staining using both
fluorescence microscopy and flow cytometry after exposure to various hypotonicities, are shown in Figure 1. Both
techniques indicate a sigmoidal response, in which the percentage of CFDA-positive cells decreased (p < 0.01) with
decreasing tonicity of the medium. The definition chosen
for the critical tonicity in this study was the tonicity that
produced 50% cell lysis. On that basis, the critical tonicity
for human spermatozoa is between 68 and 52 mOsm at
22°C (68 mOsm: 65.3 + 5.4%, [mean CFDA-positive cells +
SEM]; 52 mOsm: 23.2 + 4.5%).
Experiment lb: refinement of the criticaltonicityat 22°C.
Flow cytometric and fluorescence microscopy results for a
DETERMINATION OF HUMAN SPERM WATER PERMEABILITY
100
100
90
90
80
80
70
70
c
U,
<)
U
0
103
U
c
o
60
0
50
LL
0
r,U0
L-
0
<
b-
40
U
50
40
©
30
50
20
20
10
10
0
0
25
50
75 100 125 150 175 200 225 250 275 300
0
0
25
50
75
mOsm
FIG. 1. Percentage of human spermatozoa with intact plasma membranes, as determined by CFDA staining, after exposure to various hypotonicities (mean
SEM, n = 10).
narrower range of tonicities, decremented in 5 mOsm steps
(95-40 mOsm), are similar to results obtained in experiment la, as 50% of human sperm lysed when placed in a
medium of between 60 and 65 mOsm at 22 0C (65 mOsm:
54.3
8.9%; 60 mOsm: 41.8 + 9.0%).
The data from fluorescence microscopy and flow cytometry correlated well (r = 0.97, p > 0.04). Consequently,
subsequent data of human spermatozoal membrane integrity were collected using flow cytometric techniques.
Experiment 2: Determinationof the Critical Tonicities at
Various Temperatures
Experiment 2a-b: Determine the criticaltonicity at various temperatures. Flow cytometric results of human
spermatozoa with intact plasma membranes after exposure
to various hypotonicities at 30, 22, 8, 0, -1, -3, -5, and
-7 0C are shown in Figure 2. Estimated critical tonicity values are shown in Table 1. Analysis of variance indicated no
difference (p > 0.05) in estimated critical tonicity values at
0°C in experiments 2a and 2b between the two temperature
ranges (above and below O0C); therefore, the data were
pooled. Sperm incubated in 8, 0 and -7 ° C showed decreased (p < 0.0001) ability to swell in hypotonic solution
prior to lysing, relative to cells incubated at 30, 22, -1, -3,
and -5°C. Donors showed a high degree of variability (p
< 0.0001) in their ability to maintain membrane integrity
between the subzero temperatures studied.
Experiment 3: Determination of Lp
Experiment 3a-c: determination of Lp above, surrounding, and below O0C. From the critical tonicity data ob-
100 125 150 175 200 225 250 275 300
rOsm
FIG. 2. Percentage of human spermatozoa with intact plasma membranes, as determined by CFDA staining, after exposure to various hypotonicities at temperatures ranging from 30 to -7'C (mean - SEM, n = 10).
tained in experiment 2, a test solution of 40 mOsm was
chosen for L determinations. Placing cells in a 40 mOsm
medium, which is below the critical osmolality, causes the
cells to swell to volumes above the critical volume and to
lyse. The observed times to lysis of human sperm exposed
to 40 mOsm at 22°C are shown in Figure 3B. Data were
normalized to the percent CFDA-positive cells in the 286
mOsm control for each time, as depicted in Figure 3A. It
is evident from these isotonic control data that the duration
of constant vortexing in an isotonic medium did not affect
the ability of the plasma membrane to remain intact.
The computed kinetics of human sperm swelling in 40
mOsm medium at 22 0C are shown in Figure 3C. At room
temperature, 50% spermolysis occurs in 13.0 0.9 sec. From
the critical tonicity of 57
1.2 mOsm, the critical volume
of water is calculated to be (286/57), 5.02 times the volume
TABLE 1. Effect of temperature on critical tonicity, time to 50% cell lysis
in 40 mOsm medium, and the resulting L estimation.*
Temp °C
30
Critical tonicity
n
Time to 50% lysis
(sec)
L,
(ILm/min/atm)
n
57.4 + 3.4
10
12.0 + 0.5
2.5 + 0.1
10
10
13.0 + 0.98
13.4 0.9 ab
14.0 + 0.8 b
2.4 + 0.28
1.5 + 0.1
1.8+ 0.1 b
10
14
14
1.5+
22
8
0
57.0 - 1.2a
67.5 + 2.4b
62.7 + 2.3 b
20
-1
54.8 + 3.4
10
26.3 + 1.1
-3
60.1 + 5.3
10
28.9 + 1.6'
1.0 + 0.1C
14
10
27.6 + 2.3
1.1 -+0.1
10
10
27.2
-5
-7
59.8 + 5.2
a
a
61.9 + 3.7b
10
c
C
2.1'
*Columns with different superscripts differ (p < 0.05).
0.1 b
1.0 + 0.1c
10
10
104
NOILES ET AL.
100
FI
fI
I
I
100
- - T
I
A
A
80
80
o
c
r)
r
C)
o
0
Q
I\p
60
-
I
z
0
/
V
=-5
o
=-1
I, I
100
I
I
I
I
I
I
(
/
I
I I
/
0
o
r
U
r)
V
B
o
W
C)
a.
80
100
B
r'.
rc
=
O
0
60
G
80
A
=22
o =30
60
40
I
C
20
100
40
V
.f
80
0
..
20
60
40
I
0
0
C)
;>
10
20
30
40
50
Time (seconds)
20
'
N
-5
I
0
I
5
I
10
15
I
I
20
25
FIG. 4. Mean time (in sec) to lysis for human spermatozoa placed in
30
35
Time ((seconds)
......
/
FIG. 3. Mean time (in sec) to lysis for human spermatozoa placed in
a hypotonic solution. A) 286 mOsm control, indicating no increase in spermolysis over time. B) Percentage spermolysis in 40 mOsm solution, normalized to 286 control (mean - SEM, n = 10). C) Volume swelling curve
of a human spermatozoon exposed to 40 mOsm.
of water in the isotonic cell, or 85 pm3. To compute L,
equation 3 was used to determine what value of L will
generate this critical volume at the observed time for 50%
spermolysis. The required value of Lp is 2.4 Ipm/min/atm,
the value used to generate the curve in Figure 3C.
With the procedures described, Lp estimations were calculated for each temperature based upon the corresponding times to 50% lysis and critical tonicities for that temperature. The computed values of Lp are given in the next
to the last column of Table 1. Analysis of variance indicated
no difference (p > 0.05) in calculated Lpvalues at 8, 0, and
a nypotonlc solu[Ion
"^
'"" -t ^' """
-"-- --- '-^' ;^";~~''~~
ZO musm conrol, IIu.a;.llly
at ,3u to --r.
IIu
spermolysis over time. B) Percentage CFDA-positive sperm in 40 mOsm
solution, normalized to 286 mOsm control (mean - SEM, n = 10).
-3 0C in experiments 3a, b, and c, thus allowing pooling of
the data for given temperatures in different experiments.
Observed time to lysis of human sperm exposed to 40 mOsm
at 30, 22, 8, 0, -1, -3, -5, and -7 0C are shown in Figure 4B. As with Figure 3, data were normalized to the 286
mOsm controls for each respective time and temperature,
as depicted in Figure 4A. Sperm incubated at 0° C and
above exhibited shorter (p < 0.05) times to lysis than did
cells incubated in supercooled conditions (Table 1 and Fig.
4B).
The L calculations for the corresponding temperatures
also showed a difference (p < 0.0001) with temperature.
The estimated L is increased (p < 0.05) for spermatozoa
incubated at 220 and 30°C (2.4 pm/min/atm), relative to
°
cells at 8, °0 , -10 (1.6 Rm/min/atm), or cells at -3, -5
,
and -7C (1.0 Cpm/min/atm).
105
DETERMINATION OF HUMAN SPERM WATER PERMEABILITY
high that common methods of determining water perme-
°C
ahilitv hv mPeasrino cell volume changes over time in an-
isosmotic media, as reported for oocytes [26], are not applicable. The method used for this study involved two steps:
first, determining the volume of water in sperm when they
have swollen in hypotonic nonpermeating media to the point
where membrane leakiness or cell lysis occurs; and second,
measuring the time required for the cell to reach that critical volume, as previously reported for glycerol permeability in red blood cells [39].
1.0
0.8
0.6
a
0.4
Using Critical Tonicity to Determine the Critical Volume
of Sperm Water
0.2
0.0
-0.2
3.2
3.3
3.4
3.5
3.6
3.7
3.8
3.9
1/K (X 1000)
FIG. 5. Arrhenious plot of hydraulic conductivity of hL
tozoa between 30 and -7°C (mean + SEM; r = 0.90; n 11)mayieldinman
activation energy of 3.92 kcal/mol.
Experiment 4a-c: Determinationof the Activation Energy
for Lp
Figure 5 shows an Arrhenius plot, in which the natural
log of Lp is plotted against the reciprocal of the absolute
temperature (K) for all the temperatures studied. This plot
is in the Arrhenius form, with the full regression yielding
an r value equal to 0.90, indicating that the data are fairly
linear when plotted in this form. The slope of this line is
-1.97, which, from equation 5, yields an activation energy
of 3.92 kcal/mol. However, a comparison of the two partial
regressions (Fig. 6) reveal an interaction (p < 0.004) between the two slopes, indicating a lack of linearity or a discontinuity in water permeability above and below 0°C. The
activation energies (Fig. 6) obtained showed a marked (p
< 0.03) increase at temperatures below 0° (7.48 kcal/mol),
relative to above 0°C (2.40 kcal/mol), as obtained from the
slopes of -3.77 (r = 0.75) and -1.25 (r = 0.83), respectively.
DISCUSSION
The value of 2.4 FIm/min/atm at 22 0C for the water permeability (hydraulic conductivity) of human sperm obtained in this study is among the highest reported for mammalian cells (see [8], for review). To date, it is exceeded
only by that of human red blood cells (5 m/min/atm [52])
and it is similar to values reported for sperm in other species (bovine: 10.8 .lm/min/atm [33]; fowl: 1.9 pIm/min/
atm [53]; 2.1 I.m/min/atm [33]). The value is sufficiently
When a cell behaves as an ideal osmometer, the volume
of osmotically available water contained within the plasma
membrane will be reciprocally related to the osmolality of
nonpermeating solutes in the external medium [54]. Du et
al. [41] have shown by ESR procedures that human sperm
do behave as ideal osmometers over the range of 250 to
1500 mOsm. The plasma membrane of sperm, like that of
mammalian red blood cells, appears to be fixed in area
[32]. This fixed area would result in membrane leakiness
or lysis should a cell be placed in a medium sufficiently
hypotonic to result in the tendency to swell beyond the
maximum volume that can be encompassed by the plasma
membrane. Therefore, one can use the hypotonicity at which
1.0
4
0.8
0.6
a
0.4
0.2
0.0
-0.2
3.2
3.3
3.4
3.5
3.6
3.7
3.8
3.9
1/K (X 1000)
FIG. 6. Arrhenius plot of hydraulic conductivity of human spermatozoa between 30 and -7°C, showing a discontinuity above and below 0°C
(mean - SEM; n 10), yielding activation energies of 2.40 (r = 0.83) and
7.48 kcal/mol (r = 0.75), respectively.
106
NOILES ET AL.
membrane disruption occurs as a measure of the maximum
or critical volume of water. The method used to detect
membrane disruption in this study was the loss of CFDA
fluorescence. The fluorophore, CFDA, which is nonfluorescent, readily crosses the intact plasma membrane. Intracellular esterases hydrolyze CFDA to 6-carboxyfluoroscein, a
fluorescent, membrane-impermeable fluorophore. Consequently, spermatozoa with intact plasma membranes fluoresce bright green, but those with non-intact membranes
do not [55].
The mean critical tonicity of 57 mOsm that was determined at 22°C is somewhat higher than that reported in
other species (fowl: 17 mOsm; bull: 36 mOsm [33]; stallion:
47 mOsm [56]). Since sperm respond as ideal osmometers,
the volume of intracellular water at the critical tonicity relative to the volume of water in an isotonic sperm is 286/
57, or 5.02. The values at the other temperatures studied
(30 to -7°C) are similar (4.2 to 5.6). A critical, relative water
volume of 5 means that the water volume of human sperm
can increase 5-fold over isotonic before the available membrane area is exceeded and lysis occurs. A 5-fold increase
in water volume translates into a 3-fold increase in overall
cell volume. The ability of a cell with a fixed area of membrane to tolerate such an increase is a result of the highly
nonspherical shape of the isotonic sperm cell. The volume
of a sphere encompassing the 120-p1m 2 surface area of a
human sperm is 124 pzm 3 . The volume of the sperm cell at
the critical tonicity determined from the fragility measurements is close to that (3 x 34 [pm 3 [isotonic net cell volume] or 102 ptm 3).
Determination of the Time to Lysis and the Lp
Calculation at 22°C
The time to lysis was determined by abruptly exposing
sperm in isotonic Mann's Ringer solution to a 40 mOsm
solution of diluted Mann's Ringer and, after predetermined
times measured in seconds, abruptly returning the cells to
isotonic Mann's Ringer followed by flow cytometric determination of fluorescent staining of CFDA and PI. The 40
mOsm test solution was below the critical tonicity of 57
mOsm; therefore, as shown in the middle panel of Figure
3, a progressively increasing fraction of the sperm underwent membrane disruption. A value of 50% lysis is defined
as the time to lysis. The top panel of Figure 3 shows the
negligible effect of vortexing and other manipulations involved in the time-dependent procedure on membrane integrity.
The L is calculated by determining the value it has to
assume so that the water volume of the cell calculated by
equation 3 reaches the critical water volume at the observed time for 50% spermolysis. The calculated swelling
curve meeting these requirements at 22°C is shown in the
bottom panel of Figure 3. There are two primary sources
of error in these determinations of Lp. One is the time, and
variation in time, required to achieve complete mixing of
the solutions representing the initiation and completion of
the osmotically induced volume excursion. The other stems
from the fact that the difference between the critical tonicity (57 mOsm) and the test tonicity [40] is small, resulting
in a decreased rate of swelling as the critical volume is approached. Consequently, errors in the assumed critical tonicity or critical volume will magnify errors in the calculated
Lp. For example, if the critical volume was 4.21 (critical tonicity = 68 mOsm), the Lp calculated to produce that volume
at 13 sec would decrease by 40% from 2.4 to 1.5 pzm/min/
atm. The sensitivity of the calculated Lp to the value of the
critical volume could be decreased by decreasing the test
osmolality to increase the driving force for water entry, but
this would have the effect of decreasing the time to lysis
and magnifying the consequences of variations in mixing
times.
There are two lines of evidence that the time to lysis in
hypotonic salt is a measure of the rate of water influx, and
not a chemical consequence of increasing exposure to a
low ionic strength medium. One is that sperm do not
undergo time-dependent lysis in a near isotonic medium
in which the predominant solute is sucrose [41, 57]. Second, essentially the same procedure of time to spermolysis
in hypotonic salt was used to determine water entry in sperm
driven by the influx of glycerol [58]. The water entry in that
case was substantially slower than in the absence of glycerol because the sperm's permeability to glycerol is the ratelimiting step. The entry of water was sufficiently slowed that
it may be followed in near isotonic salt medium by an ESR
technique [59]. The finding that two methods, one using
sperm in hypotonic salt, the other using sperm in isotonic
salt, yield similar values for the kinetics of water flux in the
presence of glycerol lends credence to the view that decreased ionic strength is not a confounding factor in the
present study.
Effect of Temperature on the Critical Tonicity
The critical tonicity at 8 and 0°C was increased (p <
0.05) compared to that observed at 30 and 22 0C (Table 1).
Assuming ideal osmotic behavior, this difference means that
the sperm lyse at smaller critical volumes of water at the
lower temperatures. However, the trend did not continue
in the subzero temperature measurements on supercooled
solutions (the 5% of samples that froze were discarded).
They reversed somewhat and became more variable. One
possibility is that the differences were not due to temperature, but to some other unknown and uncontrolled variable. There is at least one reason, however, for believing
that the increase in critical tonicity between 22 and 8C is
due to the reduction in temperature: the human red blood
cell exhibits a shift in critical tonicity between 35 and 5C,
in similar magnitude to that observed here with human
sperm [60].
However, Seeman and colleagues [60] point out that, although the critical tonicity in the human red blood cell is
107
DETERMINATION OF HUMAN SPERM WATER PERMEABILITY
temperature dependent, direct hematocrit measurements
indicate that the maximum cell volume in the hypotonic
medium is actually invariant with temperature. The difference they show is due to nonmetabolic physical prelytic
release of K+ at the increased temperatures (i.e., it is due
to a departure from ideal osmometric behavior). It is possible that the seemingly anomalous critical tonicities of sperm
at subzero temperatures may reflect similar prelytic leaking. Mazur and Miller [39] argue, however, that because the
same discrepancies occur in both the osmotic fragility measurements and the time to lysis measurements, they cancel
out when the permeability coefficient is computed and,
consequently, introduce no error.
Temperature Dependence of Time to Spermolysis
and of Lp
In all cell types studied to date, Lp has been reported to
decrease with decreasing temperature. This is to be expected, as water itself shows a temperature dependence for
self diffusion of 4 to 5 kcal/mol [61]. If Lp decreases, then
the time to spermolysis in hypotonic media should increase, provided that the Vcri, remains constant. However,
as seen in Table 1, the time to lysis remained essentially
constant from 30 to 0°C (12-14 sec), and remained constant at approximately twice that value from -1 to -7°C.
However, as discussed above, Vcrit does not remain constant
with decreasing temperature. As shown in equation 3, Lp
depends on both time to lysis and Vcrit, with the net effect
of the variation of Vcrt, and lysis time being a somewhat
halting decrease in the value of Lp as the temperature is
lowered.
In the Arrhenius plots of ln(Lp) vs. 1/T in Figures 5 and
6, the data have been fitted in two ways. In Figure 5, a single line was fitted through all the data. In Figure 6, one
regression was performed on the L values between 30 and
0°C, and another, resulting in greater negative slope, on the
data between - 1 and -7°C. If the partial regressions reflect
reality, they indicate that a discontinuity exists at approximately -1C, and that below that temperature, L has a 3fold greater activation energy than at warmer temperatures.
This 3-fold increase in activation energy with decreased
temperature agrees with studies on liposomes (9.5 ± 1.28
and 26.4 ± 0.9 kcal/mol above and below 40°C [59]) and
leukocytes [23]. McGrath [11] suggests that the increase in
activation energy with decreased temperature may be solely
dependent upon temperature or may also depend on the
presence of extracellular ice. In the present study, however,
any tubes containing visible ice were excluded from analyses.
In spite of the degree of uncertainty as to the precise
value of the activation energy of L, in this study, and the
uncertainty as to whether one or two activation energies
apply between 30 and -7°C, the fact remains that the activation energy is low. The single value across all temperatures is similar to the value of approximately 4 kcal/mol
1 00
90
80
E
o
70
60
o
I
o
50
40
30
20
10
0
0
-5
-10
-15
-20
-25
-30
Temperature (C)
FIG. 7. Computed kinetics of water loss from human spermatozoa
cooled at -100, -1000, and -10 000°C/min without presence of cryoprotectants.
in human and bovine red cells [52] and is similar to the
value obtained by Watson et al. [33] of 3.1 kcal/mol in bovine sperm.
CryobiologicalImplications of the Value of Lp and Its
Activation Energy
To avoid intracellular freezing, a cell must dehydrate close
to the equilibrium value before it has cooled to its nucleation temperature (intracellular freezing temperature). For
human sperm, inferential evidence based on the temperature at which the cells succumb to rapid cooling suggest
that nucleation in rapidly cooled human sperm occurs at
approximately -30°C [38]. A major reason for determining
Lp and its activation energy is to compute the extent of dehydration on human sperm as a function of cooling rate at
subzero temperatures so as to be able to predict the relation between cooling rate and the probability of intracellular freezing-almost always a lethal event. Figure 7
shows the computed kinetics of the dehydration of human
sperm in saline without cryoprotectant as a function of the
cooling rate to -30 0C. The curves are based on equations
developed by Mazur [35] and modified by Mazur et al. [34],
using the maximum Ea of 7.48 kcal/mol obtained in the
present study and assuming that the Ea remains constant
from the freezing point of the solution to -30 0C. The computed curves indicate that even at cooling rates of 10 000°C/
min, human sperm will dehydrate to equilibrium before
cooling to -25°C and, therefore, should not undergo intracellular freezing. Experimental inferential evidence based
on the survival of sperm as a function of the rate at which
108
NOILES ET AL.
they are cooled to low subzero temperatures suggests that
human sperm begin to undergo intracellular freezing when
cooled more rapidly than 10°C/min [63], and the sperm of
other species do so when cooled at rates above 10-200°C/
min [5, 64]. The inference is based on the fact that these
rates are optimal and that survivals drop at higher rates. In
a number of cells, such as mouse embryos, the drop in
survival at supraoptimal rates has been shown to be closely
coupled with intracellular freezing [34]. If the inferences
are correct, there is a 100- to 1000-fold discrepancy between the cooling rates predicted by the approach in Figure 7 to induce intracellular freezing and those experimentally observed to do so.
This 100- to 1000-fold discrepancy between theory and
experiment may be due to several factors. First, is possible
that L decreases and its corresponding activation energy
increases significantly in the presence of cryoprotective
agents. In human red blood cells, the cooling rate that causes
hemolysis, presumably as a result of intracellular freezing,
is lowered by a factor of 10 in the presence of glycerol [42].
The presence of intracellular cryoprotectants is also known
to reduce Lp by a factor of 2 or more in mammalian embryos [48], and a recent study has reported a 33% reduction
of Lp in human sperm pre-equilibrated with 1 M glycerol
[65]. A second hypothesis is that there are further, perhaps
°
larger, decreases in Lpand/or increases in Ea below -7
C
due to the presence of extracellular ice. A third hypothesis
stems from the fact that the measurements reported in this
study yield values for the hydraulic conductivity of the plasma
membrane, whereas the experimentally derived estimates
of internal freezing commonly base survival on the retention of motility. Motility requires intact functioning subcellular components, such as mitochondria, and it is possible
that the L and its E of these components may be far lower
than the values for the plasma membrane. If so, a cooling
rate of 1000C/min might cause intra-organelle freezing even
though it is far too low to cause ice formation in the bulk
cytoplasm. These hypotheses are not mutually exclusive.
Testing the second is difficult, although technically possible, but current studies are underway to test the first and
third hypotheses.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the flow cytometric technical expertise of
H.D. Boldt and N.G. Higgins, without whose help this work would not have been
possible.
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