1 Bioheat and Mass Transfer Laboratory, Department of Mechanical Engineering, 2 Department of Urologic Surgery and 3 Department of Obstetrics and Gynecology, University of Minnesota, Minneapolis, MN 55455, USA
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Abstract |
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Key words: cryopreservation/differential scanning calorimetry/human spermatozoa/membrane permeability/water transport
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Introduction |
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During freezing of cell suspensions, ice nucleates first in the extracellular space causing an osmotic gradient to be set up across the membrane, i.e. between the intracellular isotonic solution and the freeze-concentrated extracellular solution (Mazur, 1963). Depending on whether the cooling rate is `low' or `high', the intracellular water moves across the cell membrane and joins the extracellular ice phase, or freezes and forms ice inside the cell, respectively. In most cases, cells undergoing intracellular ice formation (IIF) are rendered osmotically inactive (lysed), due to the loss of cell membrane integrity (Mazur, 1984
). Similarly, cells which experience a severe loss of intracellular water are also rendered osmotically inactive (Lovelock, 1953
). Both IIF and long exposures to high solute concentrations are lethal to cells; thus, cooling rates which are either `too high' or `too low' can kill cells. Therefore, the `optimal' cooling rate should and does exist between the `high' and `low' freezing rates. This has been confirmed experimentally for a variety of cells, and the curve of cell survival plotted as a function of the cooling rate has a characteristic inverted U-shape (Mazur et al., 1972
). Whether a prescribed cooling rate is too `low' or too `high', is a function of cell membrane permeability to water and the probability that any water remaining trapped within the cell at any given subzero temperature will nucleate and turn to ice. Differences in membrane permeability to water and probability of IIF result in different `optimal' cooling rates for different cells. In general, the `optimal' cooling rate is defined as that cooling rate which minimizes both the slow cooling and IIF injury, i.e. cool as fast as possible without forming damaging intracellular ice. Therefore, to optimize a cryopreservation protocol it is important to measure the permeability of the cell membrane to water. There are currently no experimental techniques which yield data on how sperm cells either dehydrate or form IIF during freezing in the presence of extracellular ice (Gao et al., 1997
). Currently available cryomicroscopy techniques are limited: (i) by the resolution of the light microscope, which is close to or equal to some of the morphological dimensions of the sperm cell; and (ii) by the fact that they rely on the translation of projected two-dimensional area measurements to infer three-dimensional volumetric changes. Thus, the small radial dimensions and irregular (non-spherical) shapes of the sperm cells do not allow the observation and reduction of meaningful biophysical data through the cryomicroscope.
This study reports the use of a shape-independent differential scanning calorimeter (DSC) technique to measure the membrane permeability parameters of human sperm cells during freezing. The principle of the DSC technique can be stated as follows. During dehydration (water transport) of intact cells in the absence of intracellular freezing, the concentrations of intracellular impermeant electrolytes, substrates, cofactors, proteins, etc. increase as essentially pure water is transported across the plasma membrane into the medium where it freezes, releasing heat of fusion proportional to the amount of water removed from the cell. The cells are then lysed, and the DSC measurement then repeated exactly as before; the resulting heat released is lower because the water transport no longer functions; the difference in heat release so measured (qdsc) is used to calculate the cell water that has permeated across the plasma membrane, from which one can deduce the cell volume (V) as a function of subzero temperature (T). The DSC technique has been applied to measure the membrane permeability parameters in a variety of biological systems, including EpsteinBarr virus transformed (EBVT) human lymphocyte cell suspensions (Devireddy et al., 1998
), in a normal mammalian (rat) liver tissue system (Devireddy and Bischof, 1998
), in Dunning AT-1 rat prostate tumour tissue (Devireddy et al., 1999a
), in liver tissue slices of a freeze-tolerant wood frog, Rana sylvatica (Barratt et al., 1998
; Devireddy et al., 1999b
,c
), as well as in mouse sperm cell suspensions (Devireddy et al., 1999d
). The DSC technique is independent of the size and shape of the cells being observed, and is used in this study to measure the membrane permeability parameters of human sperm cells at two different cooling rates (5 and 10°C/min; the reasons for the choice of these cooling rates will be discussed later) in the presence of extracellular ice and cryoprotective agents (CPA). Numerical simulations of water transport in human sperm cells are then performed under a variety of cooling rates (5 to 100°C/min) using the experimentally determined membrane permeability parameters. The simulation results were analysed to predict the amount of water left in the cell after dehydration ceases, in the absence of IIF, and the `optimal cooling rates' for human sperm cryopreservation.
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Materials and methods |
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Isolation of human spermatozoa
Human semen samples were obtained from men seeking initial or follow-up evaluation for infertility at the Men's Reproductive Health Center at the University of Minnesota. Semen was collected by masturbation after 23 days abstinence and standard semen analyses were performed (WHO, 1999). Ejaculates with total progressive motility counts of <106 cells, or ejaculates that were positive for antisperm antibodies, were not used in this study. Additional semen samples were obtained from donors of unknown fertility status, as well as known fertile men. Within 4 h of collection, samples (usually two to four) were pooled and concentrated to 0.51 ml by centrifugation at 400 g for 10 min at room temperature. The concentrated sperm suspension was then re-centrifuged at 300 g for 25 min at room temperature through 2 ml of Isolate, a 90% density gradient medium (Irvine Scientific, Santa Ana, CA, USA), which removed most non-motile cells and seminal fluid debris. The resulting sperm pellet was resuspended to a volume of 300500 µl in modified Biggers, Whitten and Whittingham media (modified BWW media, 282 mOsm; Irvine Scientific) and aliquots were assayed for percent viability.
Viability assay
Subsamples (~25 µl) were assayed for plasma membrane integrity by dual fluorescent dye labelling using a LIVE/DEAD Sperm Viability Kit (Molecular Probes, Eugene, OR, USA) according to the instructions provided. Briefly, SYBR-14 (live cell stain) and propidium iodide (dead cell stain) were prepared fresh daily in HEPES-buffered saline and used at final concentrations of 100 nmol/l and 600 nmol/l respectively (Garner and Johnson, 1995). Cells were first exposed to SYBR-14 for 1530 min at 37°C followed by a 5- to 10-min incubation with propidium iodide. Between 100300 cells from each sample were counted at 200x magnification using a BX-50 fluorescence-equipped light microscope (Olympus, Tokyo, Japan) and the appropriate filters. The mean (± SD) percentage of spermatozoa with intact plasma membranes in the pools (n = 9) was 84.0 ± 14.9.
DSC dynamic cooling experiments
For DSC experiments in the absence of CPA, sperm suspensions were concentrated a final time by centrifugation at 300 g for 5 min, resuspended to 1200 µl in modified BWW media and placed on ice. Sperm cell concentrations were determined using a haemocytometer. All samples used in the DSC experiments had concentrations of viable cells ranging from 80x107 to 500x107 cells/ml. For DSC experiments in the presence of CPA, cells were treated in an identical manner as above except that stock CPA (48% TESTris, 30% citrate solution with 20% egg yolk/12% glycerol; Irvine Scientific) was added dropwise to achieve a 1:1 v/v CPA:modified BWW ratio just before the final centrifugation step (Graham et al., 1972; Weidel and Prins, 1987
). Dropwise addition of CPA was performed to minimize osmotic injury effects (Gao et al., 1995
; Gilmore et al., 1997
). Thus the final concentration of CPA media was 10% egg yolk and 6% glycerol in TESTriscitrate solution and modified BWW media. Osmolarity of the CPA media was calculated to be 1113 mOsm, which includes ~790 mOsm of glycerol. This value is supported by the osmolarity of the CPA media obtained using a vapour pressure osmometer, ~1134 ± 57 mOsm.
To ensure the accuracy and repeatability of the experimental data, the limitations of the DSC-Pyris 1 (Perkin Elmer Corporation, Norwalk, CT, USA) machine were studied, and a set of calibration and control experiments were performed as detailed previously for a DSC-7 machine (Devireddy et al., 1998). These included: (i) calibration and minimization of the thermal lag; (ii) baseline determination of the thermogram (a sigmoidal baseline was used); (iii) effect of Pseudomonas syringae on DSC heat release readings; (iv) experiments with osmotically inactive (lysed) sperm cells (
qdsc = 0); and (v) error due to noise in the DSC readings (less than 5% of
qdsc). The DSC technique presented in this study was relatively insensitive to the choice of baseline used since the heat releases were `differences' and not `absolute' values. In addition, the measured value of
qdsc ( in Figure 1
) was shown to be proportional to the initial concentration of viable cells in the DSC sample and also agreed quite closely with the predicted value of
qdsc (---- in Figure 1
).
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Step 1: The sample (sperm cell suspension either in modified BWW media or in the CPA media) + 0.51 mg of P.syringae bacteria initially at 4°C, was cooled at 5 (or 10) °C/min until the extracellular ice nucleated (~3.5°C in modified BWW media and ~4.5°C in the CPA media). Step 2: After nucleation, the sample was thawed at a warming rate (10°C/min) such that phase change temperature, Tph (0.53°C and 2.1°C for the isotonic modified BWW media and CPA media respectively) was reached (but not overshot) and ice remained in the extracellular solution. Equilibrium at Tph will permit the ice nuclei to exist but not grow into ice crystals, minimizing the osmotic shrinkage of the cells. Step 3: The sample was then cooled to 50°C at 5 (or 10) °C/min, which caused the sperm cells to undergo cellular dehydration. The lower curve in Figure 2A corresponded to the heat release associated with dehydration, and the total area is represented by qinitial. Step 4: The sample was re-equilibrated at Tph by thawing at 50°C/min. Step 5: To differentiate between the heat released by the media and the intracellular fluid in Step 3, the sample was cooled at a high cooling rate (200°C/min) down to 100°C. This caused all the sperm cells to lyse and become osmotically inactive. Step 6: Step 4 was repeated. Since all the sperm cells had compromised membranes, the intracellular water, proteins and salts were now continuous (no membrane barriers existed) as previously suggested (Körber et al., 1991
).Step 7: The sample was then cooled to 50°C at 5 (or 10) °C/min to measure the final heat release due to lysed or osmotically inactive sperm cells mixed with media. The upper curve in Figure 2A
corresponded to this heat release, and the total area was represented by qfinal.
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Translation of DSC-measured heat release to dynamic cell volume data
The heat release measurements of interest were qdsc and
q(T)dsc, which were the total and fractional differences between the heat releases measured by integration of the heat flows in Steps 3 and 7 respectively using the DSC Pyris 1 software (Perkin-Elmer Corp., Norwalk, CT, USA). The total difference in the integrated heat release between the baseline (constant and sigmoidal) and the actual thermogram in the two cooling runs (Step 3 and Step 7) was denoted as
qdsc (= qinitial qfinal) and is shown in Figure 2A
. The fractional difference in the integrated heat release from Tph down to a subzero temperature, T, is denoted by
q(T)dsc (= q(T)initial q(T)final) and is shown in Figure 2B
. This difference in heat release has been shown to be related to cell volume changes in cell suspensions (Devireddy et al., 1998
), in several tissue systems (Devireddy and Bischof, 1998
; Devireddy et al., 1999a
,b
), and in mouse sperm cell suspensions (Devireddy et al., 1999d
) as:
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This equation can be rearranged to measure water transport data from the DSC measured heat releases q(T)dsc and
qdsc as:
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Note that the DSC-measured heat release readings q(T)dsc and
qdsc are obtained separately at 5°C/min (shown in Figure 2
for sperm cells suspended in modified BWW media) and at 10°C/min (data not shown) both in the absence and presence of CPA. The DSC water transport data obtained using equation [4]
were shown to generate statistically similar data [statistically insignificant differences (P > 0.05) by Student's t-test] to those obtained using a standard cellular cryomicroscopy technique with a spherical cell system (Devireddy et al., 1998
), and also to the water transport data obtained using the low-temperature microscopy method in a normal rat liver tissue system (Devireddy and Bischof, 1998
). Since the use of equation [4]
to generate water transport data from DSC-measured heat release readings has been validated in both cell and tissue systems, it is reasonable to presume that it can also be used to generate the water transport data in a sperm cell suspension system. The unknowns needed in equation [4]
, apart from the DSC heat release readings, are Vi (the initial cell volume) and Vb (the osmotically inactive cell volume). The initial volume of the sperm cells in both modified BWW media and the CPA media was assumed to be the isotonic cell volume, Vo (~22.2 µm3) as reported earlier (Curry et al., 1996
). As stated above, the osmotically inactive cell volume, Vb, was assumed to be 0.23Vo in modified BWW media, while in the CPA media the presence of glycerol in the intracellular compartment increased the osmotically inactive cell volume to ~0.32Vo.
Numerical methods
A non-linear least squares curve fitting technique was implemented using a computer program to calculate the membrane permeability parameters (Lpg and ELp) that best fit the volumetric shrinkage data as previously described (Bevington and Robinson, 1992). The optimal fit of equation [1]
to the experimental data was obtained by selecting a set of parameters which minimized the residual variance,
2, and maximized a goodness of fit parameter, R2 (Montgomery and Runger, 1994
). In order to predict the membrane permeability parameters that produced a `combined best fit' to the experimental water transport data at two or more cooling rates, the non-linear curve fitting code was slightly modified such that R2 was minimized by one set of parameters for all cooling rates as described previously (Smith et al., 1998
). All the curve fitting results presented have an R2 value greater than or equal to 0.97, indicating that there was a good agreement between the experimental data points and the fit calculated using the estimated membrane permeability parameters. To simulate the biophysical response of a sperm cell under a variety of cooling rates the best fit parameters were substituted in the water transport equation (equations [1] and [2]
), which was then numerically solved using a fourth-order RungeKutta method using a FORTRAN code on a SGI (SGI, Mountain View, CA, USA) workstation (Pazhayannur and Bischof, 1997
).
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Results |
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Statistical analysis
The differences in the water transport data between the 5 and 10°C/min rates shown in Figure 3A and B were not statistically significant. However, the differences between the data obtained in the presence and absence of CPA were statistically significant in the dynamic part of the cooling curve (>80% confidence level using Student's t-test). The 10°C/min dynamic data were statistically significant from the equilibrium cooling response, both in the presence and absence of CPA (with >90% confidence level). However, the differences between the 5°C/min data and the equilibrium cooling response were not statistically significant.
Combined best fit parameters
A new set of membrane permeability parameters (Lpg and ELp or Lpg[cpa] and ELp[cpa]) were obtained that produced a `combined best fit' to the experimentally determined water transport data in modified BWW media and in the CPA media, as shown in Table I. The `combined best fit' membrane permeability parameters maximized the goodness of fit parameter, R2, for the 5 and 10°C/min water transport data concurrently. The contour plots of the goodness of fit parameter, R2 (= 0.98) in the Lpg and ELp (or Lpg[cpa] and ELp[cpa]) space that `fit' the water transport data at 5 and 10°C/min in modified BWW media and in the CPA media, are shown in Figure 4A and B
respectively. Any combination of Lpg and ELp (or Lpg[cpa] and ELp[cpa]) shown to be within the contour will `fit' the water transport data at that cooling rate, with an R2 value >0.98. An interesting observation was the `serrations' on the y-axis in Figure 4A
, which corresponded to the contour space that fitted the water transport data at 5°C/min. These `serrations' showed that combinations of ELp ~0.0 and Lpg ranging from 3.5 to 8x1014 m3/Ns (~0.2 to 0.5 µm/min-atm) can also simulate the 5°C/min data in the absence of CPA with a goodness of fit parameter, R2 >0.98. Note that the contour space that fitted the water transport data in the presence of CPA (Figure 4B
) was significantly smaller than in the absence of CPA (Figure 4A
), suggesting that a smaller set of parameters could predict the water transport response in the presence of CPA.
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Simulations of water transport
Water transport simulations obtained using the `combined best fit' parameters in equation [1] are shown for a variety of cooling rates (5100°C/min) in Figure 5
. In Figure 5A and B
, the numerically simulated non-dimensional cellular volume (V/Vo) obtained using these `combined best fit' parameters is shown for a variety of cooling rates (at 5, 10, 20, 40, 50 and 100°C/min) in modified BWW media and in the CPA media respectively. The non-dimensional cellular volume (V/Vo), which decreased due to dehydration during freezing, was plotted on the y-axis, while the subzero temperatures were plotted on the x-axis. From the simulations, the amount of trapped water (or a lower bound on the intracellular ice) was computed as a ratio of the volume of the water trapped inside the sperm cell at the temperature, T (~30°C) where intracellular ice formation can occur by a homogeneous or volume-catalysed nucleation (Toner, 1993
) to the initial sperm water volume, [(VVb)/(VoVb)] as described earlier for a rat liver tissue system (Pazhayannur and Bischof, 1997
). Note that V was the end volume after water transport ceased (at ~30°C), and Vo and Vb were the initial (isotonic) and final (osmotically inactive) sperm cell volumes respectively. In modified BWW media, for cooling rates of
10, 20, 40, 50 and 100°C/min, the trapped water volume was
1.2, 22.2, 52.7, 60.1 and 79.1% of initial osmotically active water volume respectively, and the corresponding end volumes were
0.24Vo, 0.4Vo, 0.64Vo, 0.7Vo and 0.84Vo respectively (Figure 5A
). In the CPA media (Figure 5B
), for cooling rates of
10, 20, 40, 50 and 100°C/min, the trapped water volume was
2.4, 6.7, 30.2, 40.3 and 66.2% of initial osmotically active water volume respectively. Therefore, depending on the concentrations of the CPA, the water transport simulations (Figure 5
) showed that cooling rates as low as 20°C/min could cause intracellular water to be trapped within the human sperm cells. With sufficient supercooling, this trapped water would ultimately form intracellular ice.
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Discussion |
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Experiments at higher cooling rates
As noted earlier and in other studies, it is essential that water transport parameters be obtained at the highest possible cooling rate at which dehydration occurs exclusively (Smith et al., 1998; Devireddy et al., 1999a
,d
). One of the main disadvantages of the DSC technique is that it cannot distinguish between the heat releases obtained during water transport and IIF. So, a correlative method is needed to determine that the biophysical response is in fact water transport and not IIF (Devireddy and Bischof, 1998
; Devireddy et al., 1998
). Since standard cellular cryomicroscopy cannot be used to determine the biophysical response during freezing in human spermatozoa, an indirect methodology was used to determine the maximum cooling rate at which water transport occurred exclusively in human spermatozoa. It has been reported (Henry et al., 1993
) that the plot of motility, plasma membrane integrity and mitochondrial function versus cooling rate resembles an inverted `U' curve with an optimal cooling rate of 10°C/min (with rapid thawing rate) in the presence of 0.85 mol/l glycerol. These data suggest that the loss of motility, plasma membrane integrity and mitochondrial function at cooling rates >10°C/min are due to the formation of IIF. Thus, the highest cooling rate which causes water transport to occur exclusively in human spermatozoa in the presence of 0.85 mol/l glycerol (which is very close to the concentration of glycerol used in this study, 0.79 mol/l) is ~10°C/min, and hence DSC experiments were not performed at higher cooling rates. It is possible that in the absence of CPA, some IIF may occur at 10°C/min. However, for the purpose of this study this possibility was considered negligible.
Membrane transport parameters in the absence of extracellular ice
As mentioned earlier, there are currently no experimental techniques which yield data on how sperm cells either dehydrate or form intracellular ice during freezing in the presence of extracellular ice (Gao et al., 1997). However, there exist a few techniques such as the `time to lysis' method (Noiles et al., 1993
; Watson, 1995
) and the `Coulter counter' technique (Gilmore et al., 1995
) to measure the volumetric response of sperm cells to external changes in osmolarity at suprazero temperatures. The parameters obtained using these techniques, suprazero membrane permeability, Lp ~0.855.1x1013m3/Ns (0.53 µm/min-atm) and activation energy at suprazero temperatures, Ea ~1230 kJ/mol (37 kcal/mol), serve to provide a good understanding of suprazero water transport response for human sperm cells (Gao et al., 1997
). The suprazero membrane permeability, Lp of human sperm cells is higher than that of other mammalian cells [with the possible exception of red blood cells (RBC) and hepatocytes] and activation energies, Ea are low in comparison with other mammalian cells (Curry et al., 1994
; Gao et al., 1997
). One reason for the high permeability values might be the presence of channel-forming proteins selective to water in the sperm plasma cell membrane. For example, the high membrane permeability value reported for human RBC is due to the presence of CHIP28, a channel-forming protein. It has been shown (Liu et al., 1995
) that CHIP28 does not mediate the water transport process in human sperm cells. However, the presence of an analogue to CHIP28 in the sperm cell plasma membrane cannot be ruled out (Hasegawa et al., 1994
; Gilmore et al., 1995
). It should also be noted that water-transporting proteins (water channels, aquaporins) were identified in several parts of the reproductive system, including the testis efferent ductules, seminal vesicles, prostate, uterus and placenta (see review by Verkman et al., 1996).
Suprazero permeability parameters cannot predict subzero response
The extrapolation of the suprazero permeability data obtained in the absence of extracellular ice using the techniques described above to subzero temperatures in the presence of extracellular ice has not been successful (Curry et al., 1994). This conclusion is based on the fact that water permeabilities predicted on the basis of the above-mentioned techniques suggest that human sperm cells should be able to dehydrate at cooling rates up to 7000°C/min during freezing, when in fact experiments show that the `optimal cooling rate' for human sperm cells is <100°C/min depending on the concentrations of CPA in the extracellular milieu (Henry et al., 1993
; Curry et al., 1994
). One reason for this discrepancy may be that the values of membrane permeability parameters at subzero temperatures in the presence of extracellular ice are markedly different than those reported in the literature at suprazero temperatures. In particular, if Lpg at subzero temperatures is lower by at least an order of magnitude than Lp at suprazero temperatures, and ELp at subzero temperatures is higher by at least an order of magnitude than the corresponding Ea at suprazero temperatures, then the discrepancy between numerical simulations and experimental data can be reconciled (Gao et al., 1997
). The best fit parameters obtained in this study using the DSC water transport data (shown in Tables I and II
) during freezing of human spermatozoa, confirm that this is indeed the case; Lpg ~3.70.5x1014 m3/Ns (0.220.03 µm/min-atm) and ELp ~130503 kJ/mol (31120 kcal/mol).
The discrepancy between the membrane permeabilities determined in this study and the suprazero permeabilities reported in previous studies may be associated with possible changes in the sperm cell plasma membrane during cooling. These changes could include either a lipid phase transition between 0 and 4°C and/or a cold shock damage or `chilling' injury during cooling (Watson, 1981; Drobnis et al., 1993
). The presence of extracellular ice further alters the cell membrane transport properties, as measured using cryomicroscopy for a variety of cells. In general, for mammalian cells the average activation energy obtained in the presence of extracellular ice is approximately twice as large as that for studies conducted in unfrozen solutions at higher temperatures (McGrath, 1988
). It has been found that for human granulocytes the activation energy is dramatically higher (~3 times) and membrane permeability is dramatically lower (~1015 times) at subzero temperatures in the presence of extracellular ice, when compared with activation energy at suprazero temperatures in the absence of extracellular ice (Schwartz and Diller, 1983
). This trend is consistent with the results obtained in this study for human sperm cells and in a previous study of mouse sperm cells (Devireddy et al., 1999d
). These changes in membrane transport properties at lower temperatures might be associated with a variety of thermotropic (temperature-dependent) phase phenomena. For example, the temperature reduction which induces solidification in the extracellular medium may lead to lyotropic (i.e. independent of cooling rate) membrane phase changes and corresponding alterations of membrane permeability (Steponkus, 1984
; Caffrey, 1987
). The relative importance of temperature and the effect of extracellular ice on the membrane permeability Lp, is dependent on the cell type. The present study shows that human sperm cells have dramatically different membrane permeabilities at subzero temperatures in the presence of extracellular ice as compared with previously published values at suprazero temperatures.
Membrane transport parameters in the presence of CPA
The DSC technique was used to obtain water transport data and water permeability parameters (Lpg[cpa] and ELp[cpa]) of human sperm cells in the presence of extracellular ice and CPA. Although the exact mechanism by which the presence of CPA modifies the water permeability parameters is as yet unknown, several studies have shown that the presence of CPA tends to reduce the membrane permeability parameters, Lpg[cpa] and ELp[cpa]. A similar trend is seen in the data shown in Tables I and II, where an increase in the concentration of solutes in the extracellular medium is shown to decrease the predicted value of reference membrane permeability of human sperm cells, Lpg from 2.4x1014 m3/Ns (0.14 µm/min-atm) in modified BWW media to Lpg[cpa] of 0.67x1014m3/Ns (0.04 µm/min-atm) in CPA media. This trend is consistent with results reported in previous studies on membrane permeability parameters of ova and embryos (Mazur, 1990
). It was demonstrated that the value of Lp[cpa] obtained in the presence of various CPA [1 mol/l glycerol; 1 mol/l propylene glycol; 1 mol/l dimethylsulphoxide (DMSO) and 2 mol/l ethylene glycol] was lower by 3060% than that obtained in their absence for human sperm cells at suprazero temperatures (i.e. Lp[cpa] < Lp) (Gilmore et al., 1995
). A similar decrease was also reported in the value Lpg[cpa] in isolated rat hepatocytes in the presence of 1 mol/l and 2 mol/l DMSO using standard cellular cryomicroscopy (Smith et al., 1998
). Earlier, it was stated (Mazur, 1990
) that ELp remains unaltered due to changes in the extracellular concentration (i.e. ELp[cpa] ~ ELp), while others (Gilmore et al., 1995
) measured an increase (by a factor of 2 to 3) in the value of the Ea[cpa] for human sperm cells (i.e. Ea[cpa] > Ea) using a Coulter counter technique. A reduction (by ~4060%) was also found in the value of ELp[cpa] with increasing concentrations of DMSO for isolated rat hepatocytes (i.e. ELp[cpa] < ELp) (Smith et al., 1998
).
To conclude, a new shape-independent differential scanning calorimeter (DSC) technique was used in this study to obtain the volumetric shrinkage during freezing of human sperm cell suspensions in the presence of extracellular ice and CPA at two different cooling rates (5 and 10°C/min). By fitting a model of water transport to the experimentally obtained volumetric shrinkage data, the best fit membrane permeability parameters (Lpg and ELp) were determined. The parameters obtained in this study (Lpg ~0.22 to 0.03 µm/min-atm and ELp ~30 to 120 kcal/mol, with and without the presence of CPA respectively) were significantly different from the membrane permeability parameters reported in the literature for human spermatozoa in the absence of extracellular ice at suprazero (and subzero) temperatures (Lp ~0.53.0 µm/min-atm and Ea ~3.07.0 kcal/mol, with and without the presence of CPA respectively). The new parameters obtained in this study help to explain the discrepancy in the predicted optimal cooling rates based on suprazero permeability parameters (~7000°C/min) and the experimentally determined optimal cooling rates (<100°C/min). It is hoped that the experimentally determined water transport data and modelling made available by the new DSC technique will lead to an understanding of how to optimize human sperm cryopreservation protocols on a firm biophysical basis.
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Acknowledgments |
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Notes |
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References |
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Submitted on November 8, 1999; accepted on February 10, 2000.