1 Cryobiology Research Institute, The Herman B Wells Center for Pediatric Research, Riley Hospital for Children, 1044 West Walnut Street, Indianapolis, IN 46202 and 2 Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Purdue University, West Lafayette, IN 47907, USA
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Abstract |
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Key words: cryobiology/membrane/permeability/spermatozoa/temperature
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Introduction |
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Characterization of the fundamental cryobiology of spermatozoa has recently been initiated. The hydraulic conductivity (Lp) of human spermatozoa was determined using a time-to-lysis technique (Noiles et al., 1993) and these data were later confirmed (Gilmore et al., 1995
) using an electronic particle counter (EPC) technique. Other studies (Gao et al., 1992
; Du et al., 1994
) determined the permeability (P) of human spermatozoa to glycerol and the associated activation energy (Ea). Gilmore et al. (Gilmore et al., 1995
) expanded those studies to include human spermatozoa permeability to glycerol, dimethyl sulphoxide (DMSO), propylene glycol (PG) and ethylene glycol (EG), and the associated Lp in the presence of these CPA (Lp) at room temperature. In combination with the osmotic tolerance limits of human spermatozoa, determined earlier (Gao et al., 1995
), these data have been used to identify the human spermatozoon's ability to shrink and swell within certain limits during CPA addition and removal and during cooling and warming while still maintaining cell viability (membrane integrity and motility). Collectively, these studies have indicated that human spermatozoa: (i) have a relatively high Lp; (ii) have an Lp which is decreased in the presence of CPA; (iii) have a relatively low Ea of Lp; and (iv) have the ability to swell to 1.1 times and shrink to 0.75 times their iso-osmotic cell volume and still maintain
90% viability.
However, the information detailing the fundamental cryobiological characteristics of human spermatozoa has, to date, resulted in a discrepancy between theoretically (mathematically modelled) predicted optimal approaches for sperm cryopreservation and empirical outcomes for cooling and warming (Henry et al., 1993; Noiles et al., 1993
; Curry et al., 1994
). For example, it has been proposed (Noiles et al., 1993
) that human spermatozoa should tolerate extremely high cooling rates (i.e. ~1000°C/min), according to data describing permeability characteristics of the cells. However, when these rates were empirically tested (Henry et al., 1993
; Curry et al., 1994
), the resulting survival rates were very low, indicating that the spermatozoa could not tolerate these relatively high cooling rates. Two hypotheses have been proposed to explain these discrepancies: (i) Lp decreases in the presence of CPA; this hypothesis has been tested and confirmed (Gilmore et al., 1995
), and (ii) the Ea of Lp increases in the presence of CPA. The purpose of this study is to test this second hypothesis. Specifically, the Ea of PCPA and LpCPA for human spermatozoa was determined and this information was used to choose an optimum cryoprotectant, and to develop optimal cooling and warming rates for the cryopreservation of human spermatozoa.
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Materials and methods |
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Human spermatozoa preparation
Semen samples were obtained from healthy donors by masturbation, after informed consent. The samples were obtained after a minimum of 48 h of sexual abstinence. A minimum concentration of 2x107 spermatozoa per ml, with at least 40% motility, was required for the samples to be included in the study. The ejaculates were allowed to liquefy in an incubator (5% CO2/95% air, 37°C, and high humidity) for 30 min. Samples were layered on a discontinuous (90 and 47%) Percoll gradient to select for motile cells and then washed with TL HEPES supplemented with pyruvate (0.01 mg/ml) and resuspended to ~2.5 ml final sample volume. A fraction of the sample (5 µl) was analysed using computer-assisted semen analysis (CASA) (Cell Soft, Version 3.2/C; CRYOResources, Ltd, Montgomery, NY, USA). Subsequent CASA analysis was done at the completion of each experiment.
Electronic particle counter
An EPC (ZM model; Coulter Electronics, Inc., Hialeah, FL, USA) with a standard 50 µm aperture tube was used for all measurements. Volumes (V) were calibrated via spherical styrene beads (Duke Scientific Corporation, Palo Alto, CA, USA) with a diameter of 3.98 ± 0.03 µm (V = 33.1 µm3) at 22, 11 and 0°C. The relationship between conductivity and styrene bead volume was assumed to be the same as the relationship between conductivity and sperm volume.
Temperature control
Experiments were conducted using a cooled methanol circulating bath. Experimental temperatures were measured using a thermocouple, directly before and after each experimental run and kept within ±2°C (room temperature at 22°C, and a cooling bath at 11 and 0°C). At each experimental temperature, the experimental media, the cell sample, the aperture tube, the calibration beads and the pipette tips were pre-equilibrated to the experimental temperature.
Data analysis
Statistical analysis
Data were analysed using standard analysis of variance and multiple range test approaches with the SAS® General Linear Models program (SAS Institute Inc., Cary, NC, USA).
Determination of membrane permeability coefficients
A pair of coupled non-linear equations (Kedem and Katchalsky, 1958) was used as the theoretical model for analysis of cell membrane permeability in a ternary solution consisting of a permeable solute (cryoprotectant, subscript `s') and an impermeable solute (NaCl, subscript `n') and water. The cell volume and amount of intracellular solute concentration as functions of time are presented as:
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Evaluation of fitting calculation
The curve-fitting procedure was used to find parameter values that made the theoretical curve most resemble the plot of experimental data using the least-sum-of-squares method. The sum-of-squares (S) can be considered a function of the parameters S(P1, P2....Pm) in an (m + 1)-dimensional space. For the transport parameters LpCPA and PCPA the sum-of-squares is given by:
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Activation energies for parameters LpCPA and PCPA The Arrhenius relationship was used to determine the Ea of the parameters LpCPA and PCPA (Levin et al., 1976). The permeability value (LpCPA or PCPA) at any temperature T can be plotted as ln[Pa(T)] versus 1/T (Arrhenius plot):
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Simulation of intracellular water volume during freezing and thawing.
The changes of intracellular water volume and the mol number of intracellular CPA during temperature change of rate B can be calculated using the following coupled equations (Liu et al., 1997):
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Data acquisition.
The EPC was interfaced to a microcomputer using a CSA-1 interface (The Great Canadian Computer Company, Edmonton, Alberta, Canada). Aliquots of 100 µl of 2 mol/l glycerol, 2 mol/l DMSO, 2 mol/l PG, or 4 mol/l EG were added dropwise over 60 s to 100 µl of cell suspension, yielding final CPA concentrations of 1 and 2 mol/l, respectively. Cells were allowed to equilibrate for ~3 min, at which time they return to near normal volumes, before all 200 µl were returned to isosmotic media. The cells abruptly swell upon return to isosmotic conditions due to the influx of water (determined predominantly by LpCPA) and then return at a lower rate to normal volumes as CPA diffuses out (determined predominantly by PCPA). The resulting changes in cell volume were measured over time. A total of three donors were used and the experiments were performed at 22, 11 and 0°C. Data were analysed using a two parameter fitting method as previously described (Gilmore et al., 1998).
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Results |
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Data from the current study were used systematically to model the effects of cooling rates and activation energies on the intracellular water loss kinetics of human spermatozoa in the presence of glycerol. In a series of simulations, two cooling rates were used: 10°C/min and 100°C/min and four Ea/hydraulic conductivity combinations were used: Ea(LpCPA), 2Ea(LpCPA), Ea(PCPA) and 2Ea(PCPA). The warming rate was 1400°C/min. This was a typical warming rate obtained by shaking the straws in a 37°C water bath for 10 s. These combinations created eight situations. The results of the simulations are presented in Table III. The results indicated that the discrepancy between theoretical and empirical data was greatly reduced and the value for Ea of LpCPA in the presence of glycerol was a dominant factor in intracellular water loss kinetics. The water volume curves indicated that the best cooling rate was between 10 to 20°C/min, which was very close to the standard cooling rate (10°C/min).
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Discussion |
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The purpose of this study was to test the hypothesis that these discrepancies are due to, Lp decreases and Ea of Lp increases in the presence of CPA. The results indicate that both the hydraulic conductivity and solute permeability decrease with decreasing temperature. These data are similar to those previously reported for human spermatozoa [Ea of PGlycerol = 11.6 kcal/mol (Gao et al., 1992); 11.8 kcal/mol (Du et al., 1994
)], as well as in other cell types such as human red blood cells [Ea of PGlycerol = 7.2 kcal/mol (Mazur, 1976)], fertilized and unfertilized mouse ova [Ea of PGlycerol = 28.4 kcal/mol (Jackowski et al., 1980
)], and human platelets [Ea of PGlycerol = 19.8 kcal/mol (Armitage, 1986
)]. These data support the possibility that human spermatozoa contain channel-forming proteins selective for water (Macy, 1984
), and perhaps these channels are becoming blocked in the presence of CPA, as indicated by the increased Ea and decreased LpCPA.
The present study indicates that human spermatozoa are least permeable to DMSO, which had both the lowest LpCPA and PCPA. Ethylene glycol permeates human spermatozoa at the fastest rate of those CPA studied, even at lower temperatures. This study also indicates that PDMSO has the highest Ea (12.1 kcal/mol) while PEG had the lowest Ea (8.0 kcal/mol). These data support previous conclusions that, of those CPA studied, EG permeates human spermatozoa most rapidly, resulting in the least amount of volume excursion (Gilmore et al., 1997).
Prior studies of intracellular water volume flux during cooling and warming of human spermatozoa have predicted that these cells should tolerate relatively high cooling rates and still maintain viability after thawing (Noiles et al., 1993). However, empirical data suggests that such high cooling rates are lethal to these cells (Henry et al., 1993
; Curry et al., 1994
). The present study, which has determined that hydraulic conductivity is decreased in the presence of CPA, and the associated Ea is higher in the presence of CPA, significantly decreases the discrepancy between theoretical and empirical data.
Collectively, the data reported here suggest that the increase in Ea of Lp in the presence of CPA at lower temperatures is the most probable reason for the observed discrepancy between empirically derived data and theoretical predictions. Simulation suggests that doubling Ea for LpCPA makes the theoretical and empirical data in actual agreement. This effect has been recently reported to occur in mouse spermatozoa (Devireddy et al., 1998).
McGrath summarized Ea data for many cell types and indicated that the average Ea for Lp, obtained from studies conducted in the presence of extracellular ice, is approximately twice as large as that in unfrozen solution at above freezing temperatures (McGrath, 1988). Two possible reasons could account for the changes of the values of Ea at lower temperatures. First, membrane phase changes or membrane condensation without phase changes might cause discontinuity in the Ea or require a function of temperature to describe the Ea over a large temperature range. The second reason could be the effects of extracellular ice on the transmembrane movement of solvent and solute.
Our previous experimental data (Gilmore et al., 1997) have suggested that EG is an optimal CPA, of those studied, for human spermatozoa cryopreservation when using standard cooling and warming rates (3°C/min from 25 to 5°C; hold at 5°C for 10 min; after 3 min at 5°C, samples were seeded; 10°C/min from 5°C down to 80°C; samples plunged into liquid nitrogen). Spermatozoa cryopreserved in the presence of EG resulted in higher post-thaw motility than those samples cryopreserved in the presence of glycerol. Data from the present study suggest that the higher hydraulic conductivity in the presence of EG and the lower associated Ea allow cells to assume a greater degree of dehydration, thereby maintaining an intracellular water volume which is closer to equilibrium (Mazur, 1977
). Even when the values of Ea(LpCPA) and Ea(PCPA) were simulated to be doubled, intracellular water content was much less than that shown in Figure 6
, in which glycerol was present as the CPA.
In addition to determining optimal protocols for cryopreservation, membrane permeability coefficients can be used to characterize the fundamental physical mechanisms of solute and water transport across a cell membrane, e.g. channels versus lipid bilayer transport (Kleinhans, 1998). Water channel proteins (aquaporins; AQP) facilitate water transport across cell membranes (Verkman et al., 1996
) and are members of the major intrinsic protein family which form pores highly selective for water transport. Traditionally, it has been proposed that cell types presenting high permeability to water and low activation energies for water permeability (<67 kcal/mol), have specific water channels present (Finklestein, 1984). Previous studies have shown that human spermatozoa have a high osmotic water permeability with a low Ea (Gilmore et al., 1995
), suggesting the presence of water channels (AQP) within their plasma membranes; however, their physiological mechanism remains unknown. Human spermatozoa were found to be mercury resistant (Liu et al., 1995
) and lacking the CHIP 28 protein (an integral membrane protein and one of the first water channel proteins identified). More recently, the abundant expression of AQP7 (Ishibashi et al., 1997a
) and AQP8 (Ishibashi et al., 1997b) have been reported in the testis. Studies suggest that AQP7 is transient, appears only in the late stages of spermatogenesis, is mercury-resistant, and is expressed only in small amounts in mature spermatozoa (Ishibashi et al., 1997b). Further reports suggest that AQP8 may be a good candidate for water transport in spermatozoa (Ishibashi et al., 1997b), although it is mercury-sensitive and its expression has not been measured in mature spermatozoa. Data from the present study, indicating a change in water permeability and its associated Ea, in response to the addition of CPA, supports the hypothesis that facilitated transport in human spermatozoa is modulated by these compounds. Future efforts should include the further investigation of AQP8 and potential identification and purification of other protein water channels in this cell type.
In conclusion, cryopreservation techniques have been available for nearly 50 years for mammalian spermatozoa. However, it has been difficult to determine why some approaches result in high survival and some result in cell death. In addition it has been enigmatic why some species' spermatozoa respond to current cryopreservation methods with relatively high survival (e.g. human and bull) while others respond with little or no survival (e.g. boar and rat). The low-temperature permeability data combined with the theoretically predicted water loss curves presented in this study better explain the fundamental cryobiology of human spermatozoa and help to resolve the discrepancies between theoretically and empirically derived methods for cryopreservation.
tbryopreservation processing is a common procedure routinely performed for many assisted reproduction techniques. Recent studies comparing some of the most common clinically adapted procedures showed significant loss of motile spermatozoa associated with all the methods evaluated (Srisombut et al., 1998). By combining data determined in the present study indicating the superiority of ethylene glycol with recent findings that non-linear cooling rates may be optimum (Morris et al., 1999
), new protocols could be developed and implemented which could greatly enhance recovery of motile spermatozoa following cryopreservation processing.
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Acknowledgments |
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Notes |
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References |
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Submitted on May 4, 1999; accepted on October 22, 1999.